Synthesis and Anticonvulsant Properties of New 3,3-Diphenyl-2,5-dioxo-pyrrolidin-1-yl-acetamides and 3,3-Diphenyl-propionamides

Article abstract of DOI:10.1002/ardp.201600368

The focused library of new amides derived from 3,3-diphenyl-2,5-dioxo-pyrrolidin-1-yl-acetic acid (2a–t) and 3,3-diphenyl-propionic acid (3a–t) as potential anticonvulsant agents was synthesized. The final products were obtained in the amidation reaction

Full text of DOI:10.1002/ardp.201600368

Arch. Pharm. Chem. Life Sci. 2017, 350, e1600368
Full Paper
Synthesis and Anticonvulsant Properties of New 3,3-Diphenyl-2,5-
dioxo-pyrrolidin-1-yl-acetamides and 3,3-Diphenyl-propionamides
1
2
1
1
1
_
ꢀ
Jolanta Obniska , Anna Rapacz , Sabina Rybka , Małgorzata Gora , Paweł Zmudzki
,
1
ꢀ
and Krzysztof Kaminski
1
ꢀ
Department of Medicinal Chemistry, Jagiellonian University Medical College, Krakow, Poland
Faculty of Pharmacy, Department of Pharmacodynamics, Jagiellonian University Medical College,
Krakow, Poland
2
ꢀ
The focused library of new amides derived from 3,3-diphenyl-2,5-dioxo-pyrrolidin-1-yl-acetic acid (2a–t)
and 3,3-diphenyl-propionic acid (3a–t) as potential anticonvulsant agents was synthesized. The final
products were obtained in the amidation reaction of the given carboxylic acid (2, 3) with appropriate
secondary amines in the presence of carbonyldiimidazole (CDI) as a coupling reagent. The initial
anticonvulsant screening was performed in mice intraperitoneally (i.p.) using the “classical” maximal
electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) seizure models, whereas the acute
neurological toxicity was determined applying the rotarod test. Additionally, several compounds were
studied also in the 6-Hz seizures recognized as the animal model of human pharmacoresistant epilepsy.
In this series, compound 3q displayed a broad spectrum of activity across the preclinical seizure models
(ED50 MES ¼ 31.64 mg/kg; ED50 scPTZ ¼ 75.41 mg/kg, ED50 6-Hz (32 mA) ¼ 38.15 mg/kg). Consequently,
compound 3q revealed a wider spectrum of protection, higher activity or/and a better safety profile
than the commonly used antiepileptic drugs such as phenytoin, ethosuximide, valproic acid, or/and
levetiracetam. Notably, the in vitro studies showed that the most possible mechanism of action of 3q
may be connected to the interaction with neuronal voltage-sensitive sodium channels (site 2). Other
substances were active predominantly in the chemically induced seizures. The results of the current
studies indicate that the presence of the pyrrolidine-2,5-dione ring is important but not indispensable
for anticonvulsant activity.
Keywords: Anticonvulsant activity / In vitro studies / In vivo studies / Pyrrolidine-2,5-dione / Succinimide
Received: December 7, 2016; Revised: February 21, 2017; Accepted: February 22, 2017
DOI 10.1002/ardp.201600368
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
with epilepsy are likely to have drug-resistant epilepsy
despite the significant advancements made in epilepsy
Introduction
Approximately, 50 million people worldwide are affected by
epilepsy, making it the second most common neurological
disorder after stroke [1, 2]. As many as 20–40% of patients
research [3]. In case of pharmacotherapy of seizures,
compliance is often limited by adverse side effects most
notably related to the central nervous system (CNS) expo-
sure, such as diminished attention, executive function,
intelligence, language skills, memory, and processing
speed [4]. During the last 20 years, several new AEDs such
as levetiracetam, felbamate, lamotrigine, gabapentin, and
topiramate have been implemented for the treatment of
epilepsy. Although these medicines have been shown to be
effective in many patients with epileptic syndromes, their
efficacy does not appear to be superior to that of the
ꢀ
Correspondence: Dr. Krzysztof Kaminski, Department of
Medicinal Chemistry, Jagiellonian University Medical College, 9
ꢀ
Medyczna Street, 30-688 Krakow, Poland.
E-mail: k.kaminski@uj.edu.pl
Fax: þ48-12-6570262
ß Deutsche Pharmazeutische Gesellschaft
www.archpharm.com
(1 of 14) e1600368

Arch. Pharm. Chem. Life Sci. 2017, 350, e1600368
J. Obniska et al.
established AEDs. Therefore, the ideal AED should prevent
different types of seizures without producing side effects
that adversely affect patients’ quality of life. Therefore,
continued research on safer and more effective AEDs is
urgently necessary.
The variety of molecular targets, not always precisely
definite, makes it difficult to establish an unequivocal
mechanism of action of AEDs. This fact is also reflected in
the limited possibilities of designing new drugs based on the
structure of a given biological target [5]. In the past decades,
there have been many demonstrations attempting to
identify the structural features of compounds crucial for
anticonvulsant activity. Thus, the amide bond is one of the
important core fragments of anticonvulsants that may
appear in the structure in chain form or as a part of the
heterocyclic ring usually as lactam or imide (Fig. 1) [6].
Therefore, the previous research in our group has identified
the pyrrolidine-2,5-dione system differently substituted at
position-1 and -3 as targets for new AEDs. Many of these
compounds were effective in the MES and/or scPTZ screens
that are known as the most popular seizure models in the
early stages of search for the new anticonvulsants. Notably,
the most potent activity was observed for compounds
containing differently substituted piperazines as amine
function [7–12].
Bearing in mind the aforementioned facts, in the current
study we have focused on the synthesis of two series of
compounds; one is the acetamide derivatives with the 3,3-
diphenyl-pyrrolidine-2,5-dione fragment (series 1) and the
other is without the imide ring, that is, benzhydryl derivatives
(series 2). Thus, the main structural modification relied on the
presence or lack of succinimide core; hence, the aim of
the current study was to elucidate how the structure of imide/
amide fragment affects the anticonvulsant activity. The
general structures for compounds representing both series
are shown in Fig. 2.
Figure 2. The general structures for compounds from series 1
and 2.
Results and discussion
Chemistry
The intermediate 2 and the final compounds 2a–t, 3a–t were
synthesized according to Scheme 1. In the first step, the
condensation reaction of the 2,2-diphenyl-succinic acid (1)
(prepared as previously reported) [13], with amionoacetic
acid, yielded corresponding intermediate 2. 3,3-Diphenyl-
propionic acid (3) was commercially purchased from Sigma–
Aldrich (St. Louis, USA). In the next step, final substances (2a–t
and 3a–t) were obtained in the coupling reaction of
intermediate
2 or 3,3-diphenyl-propionic acid (3) with
equimolar amounts of appropriate 4-substituted piperazines,
benzyl-piperidine, or morpholine in the presence of CDI as
coupling reagent. The reaction was carried out at room
temperature in dry DMF for 24 h. The oily residues were
crystallized from 2-propanol after DMF’s evaporation. Their
purity and homogeneity were assessed by TLC and gradient
UPLC chromatography. The purity of all final compounds
determined by use of UPLC method was >99%. The final
Figure 1. Structures of model amide/imide/
lactam derived AEDs.
ß Deutsche Pharmazeutische Gesellschaft
www.archpharm.com
(2 of 14) e1600368

Arch. Pharm. Chem. Life Sci. 2017, 350, e1600368
Anticonvulsant Activity of Imide/Amide Derivatives
Scheme 1. Synthetic procedures for compounds 2a–t (series 1) and 3a–t (series 2).
substances were characterized by 1H NMR, 13C NMR, 19F NMR,
and LC-MS spectra.
or without the secondary generalization in humans. The scPTZ
test uses chemically induced myoclonic seizures and is
proposed to identify the agents raising the seizure threshold.
This test is related to the absence seizures in human
generalized epilepsies [14–18]. All the final compounds were
tested in vivo for anticonvulsant activity using the MES and
scPTZ models.
The anticonvulsant activity was connected closely with
chemical structures of compounds. Except for MES active 2c,
the other 3,3-diphenyl-2,5-dioxo-pyrrolidin-1-yl-acetamides
(series 1) showed protection exclusively it the scPTZ test or
were inactive (Table 1). In this series, the most potent activity
revealed the 4-phenylpiperazine derivatives with chlorine
atom(s) at the aromatic ring (2e–g). These substances were
effective in both time intervals 0.5 and 2 h, indicating the quick
onset and long in duration anticonvulsant protection. The
other 4-phenylpiperazin-amides active at dose of 100 mg/kg
Anticonvulsant activity
For the treatment of epilepsy, the pre-clinical development of
new chemical agents is based on the use of animal models
that imitate the given type of human seizures. Thus, empirical
screening has successfully led to the identification of all
clinically relevant AEDs. Despite the diversity of models that
could potentially be used to screen for anticonvulsant activity,
the MES model and the scPTZ model remain the “gold
standards” in the early stages of testing. The MES test is the
mechanism-independent animal seizure model that enables
to identify the compounds preventing seizure spread. This
test uses an electrical stimulus to produce generalized tonic–
clonic seizures, and thus it is thought to be an experimental
model of tonic–clonic epilepsy and of partial convulsions with
ß Deutsche Pharmazeutische Gesellschaft
www.archpharm.com
(3 of 14) e1600368

Arch. Pharm. Chem. Life Sci. 2017, 350, e1600368
J. Obniska et al.
Table 1. Anticonvulsant activity (MES, scPTZ) and acute neurotoxicity (rotarod test) following i.p. administration in mice
(2a–t).
MESa)
scPTZb)
NTc)
Cmpd
R
X
R1
0.5 h
2 h
0.5 h
2 h
0.5 h
2 h
clog Pd)
2b
2c
2e
2f
2g
2i
2j
2l
2t
2-F
4-F
3-Cl
–
–
–
–
–
–
–
–
O
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
100
–
–
–
–
–
–
–
–
–
100
–
100
100
100
–
–
–
100
–
–
–
–
–
100
–
–
–
–
–
–
–
–
–
–
–
–
3.45
3.45
4.02
4.02
4.67
4.32
3.64
3.16
2.20
100
100
100
–
100
100
–
4-Cl
2,3-Cl
3-CF3
2-CH3
2-OCH3
–
Dose of 100 mg/kg was administered. The data indicate the minimum dose whereby anticonvulsant activity was demonstrated.
The animals were examined at 0.5 and 2 h. A dash indicates the absence of anticonvulsant activity or neurotoxicity at the
maximum dose administered (100 mg/kg).
a)Maximal electroshock test. b) Subcutaneous pentylenetetrazole test. c) Neurotoxicity screening À rotorod test. d) clog P values
calculated using a log P module of ChemDraw Ultra program, version 7.0.1 (Cambridge Soft Corporation, Cambridge, MA,
USA).
include 2-fluorine (2b), 2-methyl (2j), and 2-methoxy (2l)
derivatives. The aforementioned substances protected mice at
time point of 0.5 h (2j, 2l) or 2 h (2b). In series 1, only one
unaromatic compound 2t containing the morpholine ring
revealedtheactivityinthechemicallyinducedseizures2 hafter
i.p. administration. In the rotarod test for acute neurological
toxicityinactivecompound2iimpairedthemotorcoordination
in mice, whereas other substances were non-neurotoxic at the
dose tested – 100 mg/kg.
More diversified anticonvulsant activity was observed for
3,3-diphenyl-propanamides (Table 2). In series 2, six com-
pounds protected the mice in the MES test (3f, 3l, 3p, 3q, 3r, 3t)
and five substances were effective in scPTZ seizures (3h, 3i, 3m,
3p, 3q). The widest spectrum of anticonvulsant activity (MES/
scPTZ) was observed for cyclohexyl (3p) and hydroxyethyl (3q)
derivatives. Thus, these substances may be potentially
effective in different types of epilepsy in humans, including
tonic–clonic epilepsy, partial convulsions with or without
secondary generalization, and generalized absence seizures.
The other molecules were active in one seizure model. In this
series, only two compounds 3i and 3r showed acute
neurological toxicity at dose of 100 mg/kg. The comparison
of the in vivo data for series 1 and 2 revealed that the removal
of the pyrrolidine-2,5-dione ring decreased the activity in case
of 4-phenylpiperazin-amides, while it increased the protec-
tion for (hydroxyl)alkyl derivatives.
In the next step of the pharmacological investigations,
the median effective doses (ED50) were determined for
compounds showing minimum 50% protection at the dose
of 100 mg/kg in the preliminary studies (MES, scPTZ tests).
Moreover, at the same time the median neurotoxic doses
(TD50) were estimated in the rotarod test. These data were
used to calculate the protective indexes (PIs), which are the
measure of the benefit-to-risk ratio of the therapeutic agent.
Notably, with the aim of estimating the anticonvulsant
potency of compounds obtained, the same studies were
performed for model and clinically relevant AEDs such as
ethosuximide, phenytoin, and valproic acid. These studies
were carried out in mice after i.p. injection at the time of peak
activity taken from the screening data or literature in case of
model AEDs. Table 3 summarizes the obtained results.
The quantitative i.p. data in mice confirmed the broad-
spectrum and potent anticonvulsant activity for 3p and 3q.
These substances revealed approximately fivefold (3p) and
eightfold (3q) better activity in the MES test as well as 3.2-fold
(both compounds) more potent protection in the scPTZ
seizures compared to wide-spectrum AED – valproic acid.
Unfortunately, compared to the aforementioned AED, 3q
showed worse safety profile in the rotarod test that yielded
more beneficial PI value in case of MES test, and less favorable
PI for PTZ seizures. The cyclohexyl derivative 3p provided
better PIs in both seizure models. The rest of the derivatives
ß Deutsche Pharmazeutische Gesellschaft
www.archpharm.com
(4 of 14) e1600368

Arch. Pharm. Chem. Life Sci. 2017, 350, e1600368
Anticonvulsant Activity of Imide/Amide Derivatives
Table 2. Anticonvulsant activity (MES, scPTZ) and acute neurotoxicity (rotarod test) following i.p. administration in mice
(3a–t).
MESa)
scPTZb)
NTc)
Cmpd
R
X
R1
0.5 h
2 h
0.5 h
2 h
0.5 h
2 h
clog Pd)
3f
3h
3i
3l
3m
3p
3q
3r
4-Cl
3,4-Cl
3-CF3
2-OCH3
–
–
–
–
–
N
N
N
O
–
–
–
–
100
–
–
–
–
100
100
100
100
100
–
–
100
–
–
–
–
–
–
–
100
–
–
–
100
100
–
–
–
100
–
–
–
–
100
–
–
–
–
–
–
–
–
–
–
4.54
5.20
4.84
3.68
3.68
5.10
2.63
3.28
2.72
100
100
–
100
100
100
–
3-OCH3
–
C6H11
(CH2)2OH
CH3
–
–
–
–
3t
–
–
–
Dose of 100 mg/kg was administered. The data indicate the minimum dose whereby anticonvulsant activity was demonstrated.
The animals were examined at 0.5 and 2 h. A dash indicates the absence of anticonvulsant activity or neurotoxicity at the
maximum dose administered (100 mg/kg).
a)Maximal electroshock test. b) Subcutaneous pentylenetetrazole test. c) Neurotoxicity screening À rotorod test. d) clog P values
calculated using a log P module of ChemDraw Ultra program, version 7.0.1 (Cambridge Soft Corporation).
(except for 2c effective in MES test) showed satisfying
protection exclusively in the scPTZ test. Except for relatively
neurotoxic compound 3r and aforementioned 3q, other
substances were more potent and provided also more
favorable PI values than ethosuximide and valproic acid.
Surprisingly enough, despite of structural similarities to
phenytoin, namely the presence of biphenyl fragment, all
new compounds revealed weak activity in the MES test
or were devoid of protection in electrically induced seizures
(see Tables 1–3).
Traditionally, most screening programs assess efficacy of
AED candidates against either electrically (e.g., MES) or
chemically (e.g., scPTZ) induced seizures. The number of
new AEDs currently available, or in development, for the
management of epilepsy certainly attest to the success of
this approach. However, this method may overlook novel
compounds that would be uniquely effective in the
therapy-resistant population. One example supporting this
hypothesis is provided by levetiracetam, which has demon-
strated efficacy in refractory human partial epilepsies. It
was found to be inactive against MES and scPTZ seizures
even at high doses, whereas it showed high efficacy in the
6-Hz model of pharmacoresistant limbic seizures [19].
Bearing in mind the aforementioned facts with the aim
of finding substances active in the latter test, eight
structurally diversified molecules (phenylpiperazine or
(hydroxyl)alkylpiperazine derivatives) were screened in
the 6-Hz (32-mA) model. The results after i.p. administra-
tion are summarized in Table 4.
As is shown in Table 4, five compounds tested displayed
satisfactory activity in the 6-Hz seizure model. It should be
emphasized that except for 3i, the most beneficial anticon-
vulsant properties were observed for amides containing the
aliphatic amines; cyclohexylpiperazine (2p) and especially
2-hydroxyethylpiperazine (2q, 3q). The quantitative pharma-
cological parameters for 2p, 2q, 3i, and 3q revealed lower
activity and worse PIs compared to 6-Hz active AED:
levetiracetam. Nevertheless, 2p, 2q, and 3i were more potent
and safer than valproic acid (Table 5).
Intensive studies into the physiological and biochemical
events occurring during epileptic seizures have provided
insight into the molecular mechanisms by which these might
be controlled. The fundamental role in establishing and
regulating the excitability of CNS neurons as well as
suppression of seizures is ascribed to voltage sodium and
calcium channels [20, 21]. Bearing in mind the above-
mentioned facts for the most promising compound (3q) the
binding properties for sodium channel (site 2) and L-type
calcium channel (dihydropyridine site) were conducted. The
results obtained are presented in Table 6.
It can be noticed that 3q revealed the high affinity to sodium
channel at the lowest concentration of 1 mM (54.2% inhibition
of control specific binding). Notably, the affinity for this
molecular target increased distinctly with the concentration
ß Deutsche Pharmazeutische Gesellschaft
www.archpharm.com
(5 of 14) e1600368

Arch. Pharm. Chem. Life Sci. 2017, 350, e1600368
J. Obniska et al.
Table 3. The quantitative pharmacological parameters ED50 (MES, scPTZ), TD50 (rotarod test), and PI in mice i.p.
ED50 MES
(mg/kg)b)
ED50 scPTZ
c)
Cmpd
2b
TPE (h)a)
(mg/kg)b)
TD50 (mg/kg)b)
PI (TD50/ED50)
2.0
NT
70.32
(41.83–118.20)
NT
>400
>5.70
2c
2e
2f
2g
2j
2.0
2.0
0.5
0.5
0.5
0.5
2.0
85.55
(57.23–127.87)
NT
>300
>400
>400
>300
>300
>300
>400
>3.50
>4.90
>4.70
>3.90
>3.43
3.57
81.41
(68.21–97.16)
85.55
(57.23–127.87)
76.86
(55.64–106.18)
87.53
(56.11–136.55)
NT
NT
NT
NT
NT
2l
84.07
(63.04–112.13)
108.04
2t
>3.70
(75.27–155.18)
0.5
2.0
2.0
3f
>120
NT
NT
–
3h
NT
42.83
(24.47–74.99)
72.12
>400
>9.34
3i
0.5
NT
>400
>5.54
(61.06–85.19)
3l
3m
2.0
0.5
>120
NT
NT
86.51
NT
339.95
–
3.93
(62.81–119.16)
73.26
(57.41–93.48)
(292.49–383.69)
268.38
(251.54–286.35)
0.5 (MES)
2.0 (PTZ)
50.91
(36.61–70.79)
5.27 (MES)
3.66 (scPTZ)
3p
3q
31.64
(27.21–36.80)
75.41
(63.60–89.42)
116.82
(102.21–133.51)
3.70 (MES)
1.50 (scPTZ)
0.5
38.15
(33.97–42.84)
>120
6.65
(5.42–8.16)
78.30
(65.82–93.13)
NT
56.91
(48.53–66.74)
318.01
(295.80–341.89)
430.77
(407.92–454.90)
3r
0.5
0.5
1.0
NT
NT
NT
2.05 (MES)
–
3t
PHTd)
8.56 (MES)
ETXd)
VPAd)
0.25
0.5
NT
2.26 (scPTZ)
140.40
(115.81–170.21)
239.45
252.74
(220.10–290.22)
1.70 (MES)
1.80 (scPTZ)
(209.18–274.10)
NT, not tested.
a)Time to peak effect. b) Results are represented as mean Æ SEM at 95% confidence limit (MES, maximal electroshock test; scPTZ,
subcutaneous pentylenetetrazole; TD50, acute neurological toxicity determined in the rotarod test). c) Protection index (TD50
ED50). Reference AEDs: PHT (phenytoin), ETX (ethosuximide), and VPA (valproic acid) tested in the same conditions.
/
d)
tested. As far as binding to L-type calcium channel is concerned,
compound 3q also significantly interacted with this channel,
nevertheless at the highest concentration of 100 mM (97.0%).
On the whole, these results suggest that the mechanism of
action of compound 3q may be primarily related to interaction
with the sodium channel (site 2).
In summary, the results of the current study indicate that
the presence of the pyrrolidine-2,5-dione ring is important,
however, not indispensable for anticonvulsant activity.
Furthermore, the bioactivity found in the series of 3,3-
diphenyl-propanamides and importantly the wide spectrum
of protection in the MES, scPTZ, and 6-Hz seizures models for
ß Deutsche Pharmazeutische Gesellschaft
www.archpharm.com
(6 of 14) e1600368

Arch. Pharm. Chem. Life Sci. 2017, 350, e1600368
Anticonvulsant Activity of Imide/Amide Derivatives
Table 4. Anticonvulsant activity, 6-Hz (32 mA) test in mice i.p.
6 Hz
Cmpd
R
X
R1
0.5 h
2 h
2h
2i
2p
2q
3h
3i
3,4-Cl
3-CF3
–
–
3,4-Cl
3-CF3
–
–
–
N
N
–
–
N
–
–
–
–
–
–
–
–
–
–
–
–
C6H11
(CH2)2OH
–
100
100
–
100
100
100
–
3p
3q
C6H11
(CH2)2OH
–
N
Dose of 100 mg/kg was administered. The data indicate the minimum dose whereby anticonvulsant activity was demonstrated.
The animals were examined at 0.5 and 2 h. A dash indicates the absence of anticonvulsant activity or at the maximum dose
administered (100 mg/kg).
3q, provides an opportunity for the development of highly
effective substances in this chemical group and will be
examined more precisely in the next studies.
The in vitro studies showed that the most possible mechanism
of action of compound 3q may be connected to the
interaction with neuronal voltage-sensitive sodium channel
(site 2).7
Conclusions
Experimental
The library of 40 3,3-diphenyl-2,5-dioxo-pyrrolidin-1-
yl-acetamides and 3,3-diphenyl-propanamides as potential
new anticonvulsant agents was synthesized. The initial
anticonvulsant screening was performed in mice (i.p.) using
the “classical” MES and scPTZ tests. Several compounds were
studied also in the six-Hertz (6-Hz) seizures recognized as the
animal model of human pharmacoresistant epilepsy.
The acute neurological toxicity was determined applying
the rotarod test. The pharmacological data revealed predom-
inant activity in the pentylenetetrazole seizures. The broadest
spectrum of efficacy across the preclinical seizure models
displayed 3q. This substance revealed wider spectrum of
protection, higher activity or/and better safety profile than
the commonly used antiepileptic drugs (AEDs) such as
phenytoin, ethosuximide, valproic acid, or/and levetiracetam.
Chemistry
General
The reagents and solvents were purchased from Sigma–Aldrich.
Melting points (mp) were determined in open capillaries on a
€
€
Buchi 353 melting point apparatus (Buchi Labortechnik, Flawil,
Switzerland). The purity and homogeneity of the compounds
were confirmed by use of the thin-layer chromatography (TLC)
and high performance liquid chromatography (HPLC). The TLC
was performed on Merck silica gel 60 F254 aluminum sheets
(Merck; Darmstadt, Germany), using subsequent developing
systems: S1 ¼ chloroform/acetone (9:1 v/v), S2 ¼ methylene chlo-
ride/methanol (9:0.2 v/v), S3 ¼ methylene chloride/methanol
(9:1 v/v), S4 ¼ ethylacetate/hexane (8:5 v/v). Spotsweredetected
1
by their absorption under UV light (l ¼ 254 nm). H NMR and
Table 5. The quantitative pharmacological parameters ED50 (6 Hz, 32 mA), TD50 (rotarod test) and PI in mice i.p.
c)
Cmpd
TPE (h)a)
ED50 6 Hz (mg/kg)b)
TD50 (mg/kg)b)
PI (TD50/ED50)
2p
2q
3i
0.5
0.5
0.5
0.5
0.5
0.5
88.24 (69.25–112.43)
39.23 (31.14–49.41)
42.83 (24.47–74.99)
38.15 (33.97–42.84)
130.64 (117.61–145.19) 430.77 (407.92–454.90)
15.73 (10.45–23.69) >500
>300
>300
>400
>3.40
>7.65
>9.34
3.06
3.30
>31.79
3q
116.82 (102.21–133.51)
VPAd)
LEVd)
a) Time to peak effect. b) Results are represented as mean Æ SEM at 95% confidence limit (6 Hz, psychomotor seizure model; TD50
,
c)
d)
acute neurological toxicity determined in the rotarod test). Protection index (TD50/ED50). Reference AEDs: VPA (valproic
acid), levetiracetam (LEV) tested in the same conditions.
ß Deutsche Pharmazeutische Gesellschaft
www.archpharm.com
(7 of 14) e1600368

Arch. Pharm. Chem. Life Sci. 2017, 350, e1600368
J. Obniska et al.
Table 6. In vitro binding assays for compound 3q.
% Inhibition of control specific bindinga)
Voltage-sensitive Naþ Voltage-sensitive Ca2þ
Cmpd
3q
Concn (mM)
channel (site 2)b) channel L-typec)
1
10
100
54.2
80.4
99.6
–
19.0
97.4
a) Results showing an inhibition higher than 50% are considered to represent significant effects of the test compounds; results
showing an inhibition between 25 and 50% are indicative of moderate effect; results showing an inhibition lower than 25%
are not considered significant and mostly attributable to variability of the signal around the control level. b) Compound was
evaluated in synaptoneurosomal preparations from rat cerebral cortex as inhibitors of the specific binding of [3H]BTX to the
voltage-sensitive sodium channel [28]. c) Compound was evaluated in synaptoneurosomal preparations from rat cerebral cortex
as inhibitors of the specific binding of [3H]nitrendipine to the voltage-sensitive Ca2þ channel [29].
13C NMR spectra were obtained in a Varian Mercury spectrom-
eter (Varian Inc., Palo Alto, CA, USA), in CDCl3 operating at
300 MHz (1H NMR) or 75 MHz (13C NMR). 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
imide), 4.36 (s, 2H, CH2), 7.29–7.40 (m, 10H, ArH); C18H15NO4
(309.32) monoisotopic mass 309.10; [MÀ H]À ¼ 308.1;
[MþH]þ ¼ 309.1.
General procedure for the synthesis of compounds 2a–t
and 3a–t
multiplicities
abbreviations: s (singlet), br s (broad singlet), d (doublet), dd
(doublet of doublets), (triplet), td (triplet of
are
represented
by
the
following
Carbonyldiimidazole (0.97 g, 0.006 mol, 1.2 eq) in 5 mL of dry
DMF was added under stirring to a solution of 3,3-diphenyl-
2,5-dioxo-pyrrolidin-1-yl-acetic acid (2) (1.55 g, 0.005 mol,
1 eq) or 3,3-diphenyl-propionic acid (3) (1.13 g, 0.005 mol,
1 eq) dissolved in 10 mL of anhydrous DMF. After the end of
gaseous (carbon dioxide) evolution (ca. 0.5 h), the respective
secondary amine derivative (0.006 mol, 1.2 eq) in 5 mL of
anhydrous DMF was added dropwise in 5 min. The mixture
was stirred overnight at room temperature. After concen-
tration of organic solvent under reduced pressure the oil
residue was crystallized from 2-propanole. The final amides
were obtained as solid substances. Compounds 3i, 3q, and 3r
due to oily form were converted into solid hydrochloride
salts by stirring in anhydrous ethanol saturated with
gaseous HCl.
t
doublets), m (multiplet). The UPLC analyses and mass spectra
(LC–MS) were obtained on a Waters ACQUITYTM TQD system
(Waters, Milford, USA) with the MS–TQ detector and
UV-Vis–DAD el detector. The ACQUITY UPLC BEH C18, 1.7 mm
(2.1 Â 100 mm) column was used with the VanGuard Acquity
UPLC BEH C18, 1.7 mm (2.1 Â 5 mm) (Waters, Milford, USA).
Standard solutions (1 mg/mL) of each compound were prepared
in analytical grade acetonitrile/water mixture (1:1 v/v). Con-
ditions applied were as follow: eluent A (water/0.1% HCOOH),
eluent B (acetonitrile/0.1% HCOOH); flow rate of 0.3 mL/min,
gradient of 5–100% B over 10 min were used, injection volume
was 10 mL. Preparative column chromatography was performed
using silica gel 60 (particle size 0.063–0.200; 70–230 Mesh ATM)
purchased from Merck (Darmstadt, Germany).
The InChI codes of the investigated compounds together
with some biological activity data are provided as Supporting
Information.
3,3-Diphenyl-1-[2-oxo-2-(4-phenyl-piperazin-1-yl)-ethyl]-
pyrrolidine-2,5-dione (2a)
White powdery crystals. Yield: 58%; mp 123–125°C; TLC:
Rf ¼ 0.61 (S1), Rf ¼ 0.36 (S2); UPLC: tR ¼ 7.63 min; 1H NMR
(300 MHz, CDCl3): d 3.15–3.23 (m, 4H, piperazine), 3.57 (s, 2H,
imide), 3.61–3.68 (m, 2H, piperazine), 3.74–3.83 (m, 2H, pipera-
zine), 4.42 (s, 2H, CH2), 6.93 (d, 3H, ArH, J ¼ 7.2 Hz), 7.26–7.40 (m,
12H, ArH); 13C NMR (75 MHz, CDCl3): d 40.1, 42.2, 44.7, 45.5, 49.4,
57.3, 116.8, 120.8, 127.6, 128.6, 129.3, 141.5, 163.2, 174.8,
178.4; C28H27N3O3 (453.53); monoisotopic mass 453.20;
[MþH]þ ¼ 454.1.
General procedure for the synthesis of 3,3-diphenyl-2,5-
dioxo-pyrrolidin-1-yl-acetic acid (2)
2,2-Diphenyl-succinic acid (5.0 g, 0.018 mol, 1 eq) was sus-
pended in 20 mL of water and aminoacetic acid (1.39 g, 0.018,
1 eq) was gradually added. The mixture was heated in a
thermoregulated sand bath (ST 72 Roth, Karlsruhe, Germany),
with simultaneous distillation of water. After the water was
completely removed, the temperature of the reaction was
maintained at 180°C for 1 h. The crude products were purified
by crystallization from 2-propanole.
1-{2-[4-(2-Fluorophenyl)-piperazin-1-yl]-2-oxo-ethyl}-3,3-
diphenyl-pyrrolidine-2,5-dione (2b)
White solid (yield 68%), mp 86–88°C; TLC: Rf ¼ 0.58 (S2);
White powdery crystals. Yield: 65%; mp 140–143°C; TLC:
UPLC: tR ¼ 5.86 min; 1H NMR (300 MHz, CDCl3): d 3.54 (s, 2H,
Rf ¼ 0.69 (S1), Rf ¼ 0.47 (S2); UPLC: tR ¼ 7.84 min; 1H NMR
ß Deutsche Pharmazeutische Gesellschaft
www.archpharm.com
(8 of 14) e1600368

Arch. Pharm. Chem. Life Sci. 2017, 350, e1600368
Anticonvulsant Activity of Imide/Amide Derivatives
(300 MHz, CDCl3): d 3.06–3.17 (m, 4H, piperazine), 3.57 (s, 2H,
imide), 3.62–3.69 (m, 2H, piperazine), 3.76–3.84 (m, 2H,
piperazine), 4.42 (s, 2H, CH2), 6.92–7.09 (m, 4H, ArH),
7.26–7.39 (m, 10H, ArH); 13C NMR (75 MHz, CDCl3): d 40.1,
42.4, 44.9, 45.5, 50.1, 50.6, 57.3, 116.2, 119.3, 123.3, 124.6, 127.6,
128.8, 141.5, 163.1, 174.8, 178.4; C28H26N3O3F (471.52); mono-
isotopic mass 471.20; [MþH]þ ¼ 472.1.
imide), 3.63–3.68 (m, 2H, piperazine), 3.77–3.85 (m, 2H
piperazine), 4.42 (s, 2H CH2), 7.13–7.24 (m, 3H, ArH),
7.27–7.39 (m, 10H, ArH); 13C NMR (75 MHz, CDCl3): d 40.1,
42.5, 45.0, 45.5, 50.9, 51.3, 57.3, 118.8, 125.3, 127.6, 128.8,
141.5, 150.3, 163.2, 174.8, 178.4; C28H26N3O3Cl2 (522.42);
monoisotopic mass 521.13; [MþH]þ ¼ 522.0.
1-{2-[4-(3,4-Dichlorophenyl)-piperazin-1-yl]-2-oxo-ethyl}-
3,3-diphenyl-pyrrolidine-2,5-dione (2h)
1-{2-[4-(4-Fluorophenyl)-piperazin-1-yl]-2-oxo-ethyl}-3,3-
diphenyl-pyrrolidine-2,5-dione (2c)
White powdery crystals. Yield: 57%; mp 198–200°C; TLC:
Rf ¼ 0.72 (S1), Rf ¼ 0.84 (S2); UPLC: tR ¼ 8.53 min; 1H NMR
(300 MHz, CDCl3): d 3.15–3.26 (m, 4H, piperazine), 3.57 (s, 2H,
imide), 3.59–3.66 (m, 2H, piperazine), 3.74–3.81 (m, 2H,
piperazine), 4.41 (s, 2H, CH2), 6.71–6.77 (m, 1H, ArH),
6.95–6.98 (m, 1H, ArH), 7.26–7.40 (m, 11H, ArH); 13C NMR
(75 MHz, CDCl3): d 40.0, 41.9, 44.4, 45.5, 48.7, 57.3, 115.9,
118.0, 123.6, 127.6, 128.8, 130.6, 141.5, 150.1, 163.2, 174.7,
178.4; C28H26N3O3Cl2 (522.42); monoisotopic mass 521.13;
[MþH]þ ¼ 522.0.
White powdery crystals. Yield: 62%; mp 82–85°C; TLC: Rf ¼ 0.91
(S1), Rf ¼ 0.82 (S2); UPLC: tR ¼ 7.62 min; 1H NMR (300 MHz,
CDCl3): d 3.11 (t, 4H, piperazine, J ¼ 5.3 Hz), 3.56 (s, 2H, imide),
3.77 (d, 4H, piperazine, J ¼ 5.4 Hz), 4.41 (s, 2H, CH2), 6.86–7.01
(m, 4H, ArH), 7.26–7.37 (m, 10H, ArH); C28H26N3O3F (471.52);
monoisotopic mass 471.20; [MþH]þ ¼ 473.1.
1-{2-[4-(2-Chlorophenyl)-piperazin-1-yl]-2-oxo-ethyl}-3,3-
diphenyl-pyrrolidine-2,5-dione (2d)
White powdery crystals. Yield: 68%; mp 174–176°C; TLC:
Rf ¼ 0.83 (S1), Rf ¼ 0.87 (S2); UPLC: tR ¼ 8.19 min; 1H NMR
(300 MHz, CDCl3): d 3.01–3.14 (m, 4H, piperazine), 3.57 (s, 2H,
imide), 3.62–3.68 (m, 2H, piperazine), 3.77–3.84 (m, 2H,
piperazine), 4.42 (s, 2H, CH2), 6.97–7.07 (m, 2H, ArH),
7.20–7.25 (m, 1H, ArH), 7.31–7.37 (m, 11H, ArH); 13C NMR
(75 MHz, CDCl3): d 40.1, 42.6, 45.1, 45.5, 50.8, 51.2, 57.3,
120.6, 124.4, 127.6, 128.7, 130.7, 141.6, 148.4, 163.2, 174.8,
178.4; C28H26N3O3Cl (487.98); monoisotopic mass 487.17;
[MþH]þ ¼ 488.1.
1-{2-[4-(3-Trifluoromethylphenyl)-piperazin-1-yl]-2-oxo-
ethyl}-3,3-diphenyl-pyrrolidine-2,5-dione (2i)
White powdery crystals. Yield: 57%; mp 188–190°C; TLC:
Rf ¼ 0.70 (S1), Rf ¼ 0.82 (S2); UPLC: tR ¼ 8.38 min; 1H NMR
(300 MHz, CDCl3): d 3.21–3.32 (m, 4H, piperazine), 3.57 (s, 2H,
imide), 3.62–3.69 (m, 2H, piperazine), 3.77–3.84 (m, 2H,
piperazine), 4.42 (s, 2H, CH2), 7.02–7.19 (m, 4H, ArH),
7.26–7.39 (m, 10H, ArH); 13C NMR (75 MHz, CDCl3):
d 40.08, 42.0, 44.5, 45.5, 48.8, 57.3, 112.9, 116.9, 119.4,
127.8, 128.6, 129.7, 141.5, 150.8, 163.3, 174.7, 178.4;
C29H26N3O3F3 (521.53); monoisotopic mass 521.20;
[MþH]þ ¼ 522.1.
1-{2-[4-(3-Chlorophenyl)-piperazin-1-yl]-2-oxo-ethyl}-3,3-
diphenyl-pyrrolidine-2,5-dione (2e)
White powdery crystals. Yield: 70%; mp 154–156°C; TLC:
Rf ¼ 0.73 (S1), Rf ¼ 0.87 (S2); UPLC: tR ¼ 8.16 min; 1H NMR
(300 MHz, CDCl3): d 3.20 (t, 4H, piperazine, J ¼ 4.8 Hz), 3.57
(s, 2H, imide), 3.63 (t, 2H, piperazine, J ¼ 4.7 Hz), 3.77 (t, 2H,
piperazine, J ¼ 5.0 Hz), 4.41 (s, 2H, CH2), 6.76–6.88 (m, 3H,
ArH), 7.16–7.37 (m, 11H, ArH); C28H26N3O3Cl (487.98); mono-
isotopic mass 487.17; [MþH]þ ¼ 488.1.
1-{2-[4-(2-Methylphenyl)-piperazin-1-yl]-2-oxo-ethyl}-3,3-
diphenyl-pyrrolidine-2,5-dione (2j)
White powdery crystals. Yield: 85%; mp 175–177°C; TLC:
Rf ¼ 0.85 (S1), Rf ¼ 0.87 (S2); UPLC: tR ¼ 8.29 min; 1H NMR
(300 MHz, CDCl3): d 2.32 (s, 3H, CH3), 2.88–2.98 (m, 4H,
piperazine), 3.57 (s, 2H, imide), 3.59–3.65 (m, 2H, piperazine),
3.73–3.81 (m, 2H, piperazine), 4.42 (s, 2H, CH2), 6.97–7.06
(m, 2H, ArH), 7.14–7.23 (m, 2H, ArH), 7.25–7.39 (m, 10H, ArH);
13C NMR (75 MHz, CDCl3): d 17.8, 40.1, 42.91, 45.4, 51.5, 57.3,
119.3, 123.9, 126.7, 127.6, 128.8, 131.1, 132.7, 141.6, 150.6,
163.2, 174.8, 178.4; C29H29N3O3 (467.56); monoisotopic mass
467.22; [MþH]þ ¼ 468.2.
1-{2-[4-(4-Chlorophenyl)-piperazin-1-yl]-2-oxo-ethyl}-3,3-
diphenyl-pyrrolidine-2,5-dione (2f)
White powdery crystals. Yield: 62%; mp 145–147°C; TLC:
Rf ¼ 0.66 (S1), Rf ¼ 0.82 (S2); UPLC: tR ¼ 8.11 min; 1H NMR
(300 MHz, CDCl3): d 3.12–3.23 (m, 4H, piperazine), 3.56 (s, 2H,
imide), 3.60–3.67 (m, 2H, piperazine), 3.74–3.81 (m, 2H,
piperazine), 4.41 (s, 2H, CH2), 6.84 (d, 2H, ArH, J ¼ 9.2 Hz),
7.23 (d, 2H, ArH, J ¼ 8.9 Hz), 7.31–7.37 (m, 10H, ArH);
1-{2-[4-(3-Methylphenyl)-piperazin-1-yl]-2-oxo-ethyl}-3,3-
diphenyl-pyrrolidine-2,5-dione (2k)
C28H26N3O3Cl
(487.98);
monoisotopic
mass
487.17;
White powdery crystals. Yield: 66%; mp 123–125°C; TLC:
Rf ¼ 0.64 (S1), Rf ¼ 0.87 (S2); UPLC: tR ¼ 7.93 min; 1H NMR
(300 MHz, CDCl3): d 2.32 (s, 3H, CH3), 3.14–3.27 (m, 4H,
piperazine), 3.57 (s, 2H, imide), 3.59–3.67 (m, 2H, piperazine),
3.74–3.82 (m, 2H, piperazine), 4.42 (s, 2H, CH2), 6.75 (s, 3H,
ArH), 7.14–7.21 (m, 1H, ArH), 7.27–7.39 (m, 10H, ArH);
[MþH]þ ¼ 488.1.
1-{2-[4-(2,3-Dichlorophenyl)-piperazin-1-yl]-2-oxo-ethyl}-
3,3-diphenyl-pyrrolidine-2,5-dione (2g)
White powdery crystals. Yield: 49%; mp 203–205°C; TLC:
Rf ¼ 0.83 (S1), Rf ¼ 0.91 (S2); UPLC: tR ¼ 8.61 min; 1H NMR
(300 MHz, CDCl3): d 3.01–3.11 (m, 4H, piperazine), 3.57 (s, 2H,
C29H29N3O3
(467.56);
monoisotopic
mass
467.22;
[MþH]þ ¼ 468.1.
ß Deutsche Pharmazeutische Gesellschaft
www.archpharm.com
(9 of 14) e1600368

Arch. Pharm. Chem. Life Sci. 2017, 350, e1600368
J. Obniska et al.
1-{2-[4-(2-Methoxyphenyl)-piperazin-1-yl]-2-oxo-ethyl}-
3,3-diphenyl-pyrrolidine-2,5-dione (2l)
(300 MHz, CDCl3): d 2.52 (t, 2H, CH2, J ¼ 5.0 Hz), 2.57–2.60
(m, 4H, piperazine), 3.48–3.51 (m, 2H, CH2), 3.55 (s, 2H, imide),
3.64 (t, 4H, piperazine, J ¼ 5.2 Hz), 4.36 (s, 2H, CH2), 7.26–7.36
(m, 10H, ArH); C24H27N3O4 (421.49); monoisotopic mass
421.20; [MþH]þ ¼ 422.2.
Whitepowderycrystals.Yield:55%;mp173–176°C;TLC:Rf ¼ 0.56
(S1), Rf ¼ 0.24 (S2); UPLC: tR ¼ 7.41 min; 1H NMR (300 MHz, CDCl3):
d 3.27–3.46 (m, 2H, piperazine), 3.57 (s, 2H, imide), 3.64–3.77 (m,
1H, piperazine), 3.79–3.88 (m, 1H, piperazine), 3.91 (s, 3H, OCH3),
3.95–4.22 (m, 4H, piperazine), 4.42 (s, 2H, CH2), 6.93–7.05 (m, 2H,
ArH), 7.14–7.43 (m, 12H, ArH); C29H29N3O4 (483.56); monoiso-
topic mass 483.22; [MþH]þ ¼ 484.2.
1-[2-(4-Methyl-piperazin-1-yl)-2-oxo-ethyl]-3,3-diphenyl-
pyrrolidine-2,5-dione (2r)
White powdery crystals. Yield: 57%; mp 140–142°C; TLC:
Rf ¼ 0.52 (S1), Rf ¼ 0.56 (S3); UPLC: tR ¼ 4.59 min; 1H NMR
(300 MHz, CDCl3): d 2.32 (s, 3H, CH3), 2.43 (dt, 4H, piperazine,
J ¼ 15.1 Hz, J ¼ 5.0 Hz), 3.46–3.50 (m, 2H, piperazine), 3.55 (s,
2H, imide), 3.64 (d, 2H, piperazine, J ¼ 4.9 Hz), 4.36 (s, 2H, CH2),
7.25–7.39 (m, 10H, ArH); C23H25N3O3 (391.46); monoisotopic
mass 391.20; [MþH]þ ¼ 392.1.
1-{2-[4-(3-Methoxyphenyl)-piperazin-1-yl]-2-oxo-ethyl}-
3,3-diphenyl-pyrrolidine-2,5-dione (2m)
White powdery crystals. Yield: 65%; mp 71–73°C; TLC:
Rf ¼ 0.22 (S1), Rf ¼ 0.83 (S2); UPLC: tR ¼ 7.56 min; 1H NMR
(300 MHz, CDCl3): d 3.15–3.26 (m, 4H, piperazine), 3.56 (s, 2H,
imide), 3.58–3.65 (m, 2H, piperazine), 3.76 (d, 2H, piperazine,
J ¼ 5.6 Hz), 3.79 (s, 3H, OCH3), 4.41 (s, 2H, CH2), 6.44–6.56
(m, 3H, ArH), 7.16–7.37 (m, 11H, ArH); C29H29N3O4 (483.56);
monoisotopic mass 483.22; [MþH]þ ¼ 484.2.
1-(2-Morpholine-2-oxyethyl)-3,3-diphenyl-pyrrolidine-2,5-
dione (2s)
White powdery crystals. Yield: 58%; mp 168–170°C; TLC:
Rf ¼ 0.82 (S1), Rf ¼ 0.37 (S3); UPLC: tR ¼ 6.05 min; 1H NMR
(300 MHz, CDCl3): d 3.56 (s, 2H, imide), 3.64 (t, 4H, morpholine,
J ¼ 5.2 Hz), 3.70–3.74 (m, 4H, morpholine), 4.36 (s, 2H, CH2),
7.26–7.37 (m, 10H, ArH); C22H22N2O4 (378.42); monoisotopic
mass 378.16; [MþH]þ ¼ 379.1.
1-[2-(4-Benzyl-piperazin-1-yl)-2-oxo-ethyl]-3,3-diphenyl-
pyrrolidine-2,5-dione (2n)
White powdery crystals. Yield: 52%; mp 133–135°C; TLC:
Rf ¼ 0.22 (S1), Rf ¼ 0.16 (S2); UPLC: tR ¼ 5.40 min; 1H NMR
(300 MHz, CDCl3): d 2.44–2.50 (m, 4H, piperazine), 3.43–3.48
(m, 2H, piperazine), 3.53 (s, 2H, imide), 3.55 (s, 2H, CH2),
3.60–3.67 (m, 2H, piperazine), 4.35 (s, 2H, CH2), 7.23–7.39
(m, 15H, ArH); C29H29N3O3 (467.56); monoisotopic mass
467.22; [MþH]þ ¼ 468.1.
1-[2-(4-Benzyl-piperidin-1-yl)-2-oxoethyl]-3,3-diphenyl-
pyrrolidine-2,5-dione (2t)
White powdery crystals. Yield: 68%; mp 126–128°C; TLC:
Rf ¼ 0.80 (S1), Rf ¼ 0.84 (S3); UPLC: tR ¼ 8.46 min; 1H NMR
(300 MHz, CDCl3): d 1.10–1.37 (m, 2H, piperidine), 1.64–1.86
(m, 4H, piperidine), 2.47–2.66 (m, 2H, CH2), 2.94–3.10 (m, 2H,
piperidine), 3.55 (s, 2H, imide), 3.66–3.80 (m, 1H, piperidine),
4.35 (s, 2H, CH2), 7.09–7.42 (m, 15H, ArH); C30H30N2O3 (466.57);
monoisotopic mass 466.23; [MþH]þ ¼ 467.1.
1-[2-(4-Pyrimidin-piperazin-1-yl)-2-oxo-ethyl]-3,3-
diphenyl-pyrrolidine-2,5-dione (2o)
White powdery crystals. Yield: 72%; mp 182–184°C; TLC:
Rf ¼ 0.44 (S1), Rf ¼ 0.22 (S2); UPLC: tR ¼ 6.61 min; 1H NMR
(300 MHz, CDCl3): d 3.53–3.55 (m, 2H, piperazine), 3.57 (s, 2H,
imide), 3.70–3.73 (m, 2H, piperazine), 3.90 (dt, 4H, piperazine,
J ¼ 5.2 Hz, J ¼ 19.9 Hz), 4.42 (s, 2H, CH2), 6.55 (t, 1H, pyrimi-
dine, J ¼ 4.7 Hz), 7.26–7.37 (m, 10H, ArH), 8.33 (d, 2H,
pyrimidine, J ¼ 4.6 Hz); C26H25N5O3 (455.52); monoisotopic
mass 455.19; [MþH]þ ¼ 456.1.
3,3-Diphenyl-1-(4-phenylpiperazin-1-yl)-propan-1-one
(3a)
White powdery crystals. Yield: 55%; mp 130–132°C; TLC:
Rf ¼ 0.72 (S1), Rf ¼ 0.62 (S4); UPLC: tR ¼ 7.69 min; 1H NMR
(300 MHz, CDCl3): d 2.86 (t, 2H, piperazine, J ¼ 5.2 Hz), 3.03
(t, 2H, piperazine, J ¼ 5.2 Hz), 3.10 (d, 2H, CH2, J ¼ 7.7 Hz), 3.48
(t, 2H, piperazine, J ¼ 5.0 Hz), 3.70 (t, 2H, piperazine, J ¼ 5.3
Hz), 4.69 (t, 1H, CH, J ¼ 7.4 Hz), 6.84–6.92 (m, 3H, ArH),
7.14–7.31 (m, 12H, ArH); C25H26N2O (370.49); monoisotopic
mass 370.20; [MþH]þ ¼ 371.4.
1-[2-(4-Cyclohexyl-piperazin-1-yl)-2-oxo-ethyl]-3,3-
diphenyl-pyrrolidine-2,5-dione (2p)
White powdery crystals. Yield: 52%; mp 80–82°C; TLC:
Rf ¼ 0.23 (S1), Rf ¼ 0.36 (S2); UPLC: tR ¼ 5.32 min; 1H NMR
(300 MHz, CDCl3): d 1.19–1.26 (m, 6H, cyclohexane), 1.78–1.83
(m, 4H, cyclohexane), 2.54–2.62 (m, 4H piperazine, 1H
cyclohexane), 3.45 (d, 2H, piperazine, J ¼ 4.6 Hz), 3.55 (s, 2H,
imide), 3.59–3.67 (m, 2H, piperazine), 4.36 (s, 2H, CH2),
7.29–7.36 (m, 10H, ArH); C28H33N3O3 (459.58); monoisotopic
mass 459.25; [MþH]þ ¼ 460.2.
1-[4-(2-Fluorophenyl)-piperazin-1-yl]-3,3-diphenylpropan-
1-one (3b)
White powdery crystals. Yield: 62%; mp 111–113°C; TLC:
Rf ¼ 0.87 (S1), Rf ¼ 0.71 (S4); UPLC: tR ¼ 7.92 min; 1H NMR
(300 MHz, CDCl3): d 2.73 (t, 2H, piperazine, J ¼ 5.0 Hz), 2.91
(t, 2H, piperazine, J ¼ 5.1 Hz), 3.10 (d, 2H, CH2, J ¼ 7.4 Hz), 3.50
(t, 2H, piperazine, J ¼ 5.1 Hz), 3.72 (t, 2H, piperazine, J ¼ 5.0
Hz), 4.69 (t, 1H, CH, J ¼ 7.6 Hz), 6.99 (m, 1H, ArH), 7.02–7.06
(m, 3H, ArH), 7.24–7.32 (m, 10H, ArH); C25H25N2OF (388.48);
monoisotopic mass 388.20; [MþH]þ ¼ 389.4.
1-[2-(2-Hydroxyethyl-piperazin-1-yl)-2-oxo-ethyl]-3,3-
diphenyl-pyrrolidine-2,5-dione (2q)
White powdery crystals. Yield: 50%; mp 113–115°C; TLC:
Rf ¼ 0.47 (S1), Rf ¼ 0.54 (S3); UPLC: tR ¼ 4.51 min; 1H NMR
ß Deutsche Pharmazeutische Gesellschaft
www.archpharm.com
(10 of 14) e1600368

Arch. Pharm. Chem. Life Sci. 2017, 350, e1600368
Anticonvulsant Activity of Imide/Amide Derivatives
1-[4-(4-Fluorophenyl)-piperazin-1-yl]-3,3-diphenylpropan-
1-one (3c)
(300 MHz, CDCl3): d 2.64 (t, 2H, piperazine, J ¼ 4.9 Hz), 2.85
(t, 2H, piperazine, J ¼ 5.0 Hz), 3.10 (d, 2H, CH2, J ¼ 7.7 Hz), 3.52
(t, 2H, piperazine, J ¼ 5.0 Hz), 3.73 (t, 2H, piperazine, J ¼ 4.7
Hz), 4.69 (t, 1H, CH, J ¼ 7.4 Hz), 6.78–6.82 (dd, 1H, ArH,
J ¼ 2.1 Hz, J ¼ 5.4 Hz), 7.11–7.33 (m, 12H, ArH); C25H24N2OCl2
(439.38); monoisotopic mass 438.13; [MþH]þ ¼ 439.3.
White powdery crystals. Yield: 72%; mp 140–142°C; TLC:
Rf ¼ 0.62 (S1), Rf ¼ 0.76 (S2); UPLC: tR ¼ 7.72 min; 1H NMR
(300 MHz, CDCl3): d 2.74 (t, 2H, piperazine, J ¼ 5.1 Hz), 2.93
(t, 2H, piperazine, J ¼ 5.1 Hz), 3.09 (d, 2H, CH2, J ¼ 7.4 Hz),
3.47 (t, 2H, piperazine, J ¼ 5.1 Hz), 3.69 (t, 2H, piperazine,
J ¼ 5.1 Hz), 4.68 (t, 1H, CH, J ¼ 7.6 Hz), 6.78–6.84 (m, 2H,
ArH), 6.92–6.99 (m, 2H, ArH), 7.18–7.31 (m, 10H, ArH);
13C NMR (75 MHz, CDCl3): d 38.7, 41.6, 45.7, 47.4, 50.4, 115.5,
115.8, 118.5, 126.3, 126.5, 127.8, 128.5, 144.0,
169.7; C25H25N2OF (388.48); monoisotopic mass 388.20;
[MþH]þ ¼ 389.4.
1-[4-(3,4-Dichlorophenyl)-piperazin-1-yl]-3,3-
diphenylpropan-1-one (3h)
White powdery crystals. Yield: 83%; mp 113–115°C; TLC:
Rf ¼ 0.74 (S1), Rf ¼ 0.66 (S4); UPLC: tR ¼ 8.76 min; 1H NMR
(300 MHz, CDCl3): d 2.78 (t, 2H, piperazine, J ¼ 5.0 Hz), 2.99
(t, 2H, piperazine, J ¼ 5.1 Hz), 3.09 (d, 2H, CH2, J ¼ 7.4 Hz), 3.45
(t, 2H, piperazine, J ¼ 5.1 Hz), 3.68 (t, 2H, piperazine, J ¼ 5.1
Hz), 4.67 (t, 1H, CH, J ¼ 7.6 Hz), 6.64–6.68 (dd, 1H, ArH,
J ¼ 2.8 Hz, J ¼ 6.2 Hz), 6.87 (d, 1H, ArH, J ¼ 2.8 Hz), 7.15–7.31
(m, 11H, ArH); C25H24N2OCl2 (439.38); monoisotopic mass
438.13; [MþH]þ ¼ 439.3.
1-[4-(2-Chlorophenyl)-piperazin-1-yl]-3,3-
diphenylpropan-1-one (3d)
White powdery crystals. Yield: 62%; mp 130–132°C; TLC:
Rf ¼ 0.43 (S1), Rf ¼ 0.57 (S2); UPLC: tR ¼ 8.30 min; 1H NMR
(300 MHz, CDCl3): d 2.68 (t, 2H, piperazine, J ¼ 4.9 Hz), 2.87
(t, 2H, piperazine, J ¼ 5.0 Hz), 3.11 (d, 2H, CH2, J ¼ 7.7 Hz), 3.52
(t, 2H, piperazine, J ¼ 5.0 Hz), 3.73 (t, 2H, piperazine, J ¼ 5.0
Hz), 4.69 (t, 1H, CH, J ¼ 7.6 Hz), 6.88–6.91 (dd, 1H, ArH,
J ¼ 1.5 Hz, J ¼ 6.4 Hz), 6.96–7.02 (td, 1H, ArH, J ¼ 1.5 Hz, J ¼ 6.1
Hz), 7.16–7.34 (m, 12H, ArH); 13C NMR (75 MHz, CDCl3): d 38.2,
41.9, 46.1, 47.4, 51.1, 120.4, 124.2, 126.4, 127.6, 127.9, 128.5,
128.9, 130.6, 144.0, 169.8; C25H25N2OCl (404.94); monoiso-
topic mass 404.17; [MþH]þ ¼ 405.3.
Monohydrochloride 1-[4-(3-trifluoromethylphenyl)-
piperazin-1-yl]-3,3-diphenylpropan-1-one (3i)
White powdery crystals. Yield: 73%; mp 175–176°C; TLC:
Rf ¼ 0.16 (S1), Rf ¼ 0.12 (S4); UPLC: tR ¼ 8.15 min; 1H NMR
(300 MHz, DMSO-d6): d 3.08 (t, 4H, piperazine, J ¼ 4.9 Hz),
3.16 (d, 2H, CH2, J ¼ 7.6 Hz), 3.52 (t, 2H, piperazine,
J ¼ 4.7 Hz), 3.64 (br s, 2H, piperazine), 4.49 (t, 1H, CH,
J ¼ 7.6 Hz), 7.09–7.18 (m, 3H, ArH), 7.21–7.33 (m, 10H, ArH),
7.42 (t, 1H, ArH, J ¼ 7.9 Hz); C26H26N2OClF3 (474.94); amine:
1-[4-(3-Chlorophenyl)-piperazin-1-yl]-3,3-
diphenylpropan-1-one (3e)
C26H25N2OF3 (438.50); monoisotopic
mass 438.19;
[MþH]þ ¼ 438.2.
White powdery crystals. Yield: 58%; mp 107–108°C; TLC:
Rf ¼ 0.80 (S1), Rf ¼ 0.64 (S4); UPLC: tR ¼ 8.33 min; 1H NMR
(300 MHz, CDCl3): d 2.83 (t, 2H, piperazine, J ¼ 5.1 Hz), 3.03
(t, 2H, piperazine, J ¼ 5.1 Hz), 3.09 (d, 2H, CH2, J ¼ 7.7 Hz), 3.45
(t, 2H, piperazine, J ¼ 5.0 Hz), 3.68 (t, 2H, piperazine, J ¼ 5.1
Hz), 4.68 (t, 1H, CH, J ¼ 7.6 Hz), 6.69–6.73 (m, 1H, ArH),
6.79–6.86 (m, 3H, ArH), 7.13–7.31 (m, 10H, ArH); C25H25N2OCl
(404.94); monoisotopic mass 404.17; [MþH]þ ¼ 405.3.
1-[4-(2-Methylphenyl)-piperazin-1-yl]-3,3-
diphenylpropan-1-one (3j)
White powdery crystals. Yield: 78%; mp 84–86°C; TLC:
Rf ¼ 0.83 (S1), Rf ¼ 0.77 (S4); UPLC: tR ¼ 8.47 min; 1H NMR
(300 MHz, CDCl3): d 2.28 (s, 3H, CH3), 2.57 (t, 2H, piperazine,
J ¼ 4.9 Hz), 2.73 (t, 2H, piperazine, J ¼ 4.9 Hz), 3.11 (d, 2H, CH2,
J ¼ 7.4 Hz), 3.48 (t, 2H, piperazine, J ¼ 4.9 Hz), 3.69 (t, 2H,
piperazine, J ¼ 4.7 Hz), 4.70 (t, 1H, CH, J ¼ 7.4 Hz), 6.88 (d, 1H,
ArH, J ¼ 7.9 Hz), 6.97–7.03 (m, 1H, ArH), 7.14–7.33 (m, 12H,
ArH); 13C NMR (75 MHz, CDCl3): d 17.7, 38.7, 42.2, 46.3, 46.7,
51.6, 119.1, 123.7, 126.0, 127.3, 128.5, 131.1, 132.7, 144.1,
169.8; C26H28N2O (384.51); monoisotopic mass 384.22;
[MþH]þ ¼ 385.4.
1-[4-(4-Chlorophenyl)-piperazin-1-yl]-3,3-
diphenylpropan-1-one (3f)
White powdery crystals. Yield: 78%; mp 157–159°C; TLC:
Rf ¼ 0.74 (S1), Rf ¼ 0.58 (S4); UPLC: tR ¼ 8.28 min; 1H NMR
(300 MHz, CDCl3): d 2.79 (t, 2H, piperazine, J ¼ 5.0 Hz), 2.98
(t, 2H, piperazine, J ¼ 5.1 Hz), 3.09 (d, 2H, CH2, J ¼ 7.4 Hz), 3.46
(t, 2H, piperazine, J ¼ 5.0 Hz), 3.69 (t, 2H, piperazine, J ¼ 5.1
Hz), 4.68 (t, 1H, CH, J ¼ 7.6 Hz), 6.74–6.79 (m, 2H, ArH),
7.15–7.31 (m, 12H, ArH); 13C NMR (75 MHz, CDCl3): d 38.7, 41.4,
45.5, 47.4, 49.3, 117.7, 126.5, 127.8, 128.3, 129.0, 143.9, 169.8;
1-[4-(3-Methylphenyl)-piperazin-1-yl]-3,3-
diphenylpropan-1-one (3k)
White powdery crystals. Yield: 82%; mp 121–123°C; TLC:
Rf ¼ 0.77 (S1), Rf ¼ 0.62 (S4); UPLC: tR ¼ 8.07 min; 1H NMR
(300 MHz, CDCl3): d 2.31 (s, 3H, CH3), 2.85 (t, 2H, piperazine,
J ¼ 5.1 Hz), 3.02 (t, 2H, piperazine, J ¼ 5.1 Hz), 3.10 (d, 2H,
CH2, J ¼ 7.4 Hz), 3.47 (t, 2H, piperazine, J ¼ 5.1 Hz), 3.69
(t, 2H, piperazine, J ¼ 5.1 Hz), 4.68 (t, 1H, CH, J ¼ 7.6 Hz),
6.64–6.73 (m, 3H, ArH), 7.12–7.31 (m, 11H, ArH);
C25H25N2OCl
(404.94);
monoisotopic
mass
404.17;
[MþH]þ ¼ 405.3.
1-[4-(2,3-Dichlorophenyl)-piperazin-1-yl]-3,3-
diphenylpropan-1-one (3g)
White powdery crystals. Yield: 57%; mp 119–121°C; TLC:
C26H28N2O
(384.51);
monoisotopic
mass
384.22;
Rf ¼ 0.80 (S1), Rf ¼ 0.74 (S4); UPLC: tR ¼ 8.29 min; 1H NMR
[MþH]þ ¼ 385.4.
ß Deutsche Pharmazeutische Gesellschaft
www.archpharm.com
(11 of 14) e1600368

Arch. Pharm. Chem. Life Sci. 2017, 350, e1600368
J. Obniska et al.
1-[4-(2-Methoxyphenyl)-piperazin-1-yl]-3,3-
diphenylpropan-1-one (3l)
Hz), 3.32 (t, 2H, piperazine, J ¼ 5.1 Hz), 3.53 (t, 2H, pipera-
zine, J ¼ 5.0 Hz), 4.66 (t, 1H, CH, J ¼ 7.4 Hz), 7.13–7.30 (m, 10H,
ArH); C25H32N2O (376.54); monoisotopic mass 376.25;
[MþH]þ ¼ 377.4.
White powdery crystals. Yield: 59%; mp 116–118°C; TLC:
Rf ¼ 0.67 (S1), Rf ¼ 0.52 (S4); UPLC: tR ¼ 7.44 min; 1H NMR
(300 MHz, CDCl3): d 2.72 (t, 2H, piperazine, J ¼ 5.0 Hz), 2.89
(t, 2H, piperazine, J ¼ 5.1 Hz), 3.10 (d, 2H, CH2, J ¼ 7.4 Hz),
3.53 (t, 2H, piperazine, J ¼ 5.0 Hz), 3.73 (t, 2H, piperazine,
J ¼ 5.1 Hz), 3.86 (s, 3H, OCH3), 4.69 (t, 1H, CH, J ¼ 7.6 Hz),
6.79–6.94 (m, 3H, ArH), 6.99–7.05 (m, 1H, ArH), 7.15–7.31
(m, 10H, ArH); C26H28N2O2 (400.51); monoisotopic mass
400.22; [MþH]þ ¼ 401.4.
Monohydrochloride 1-[4-(2-hydroxyethyl)-piperazin-1-yl]-
3,3-diphenylpropan-1-one (3q)
White powdery crystals. Yield: 60%; mp 215–216°C; TLC:
Rf ¼ 0.14 (S1), Rf ¼ 0.11 (S4); UPLC: tR ¼ 4.04 min; 1H NMR
(300 MHz, DMSO-d6): d 3.11 (t, 4H, piperazine, J ¼ 4.9 Hz), 3.17
(d, 2H, CH2, J ¼ 7.6 Hz), 3.34–3.46 (m, 5H, hydroxyethyl), 3.75
(t, 4H, piperazine, J ¼ 5.0 Hz), 4.47 (t, 1H, CH, J ¼ 7.3 Hz),
7.09–7.17 (m, 2H, ArH), 7.20–7.34 (m, 8H, ArH), 10.61–10.92
(m, 1H, HCl); C21H27N2O2Cl (374.90); amine: C21H26N2O2
(338.45); monoisotopic mass 338.20; [MþH]þ ¼ 339.4.
1-[4-(3-Methoxyphenyl)-piperazin-1-yl]-3,3-
diphenylpropan-1-one (3m)
White powdery crystals. Yield: 58%; mp 110–112°C; TLC:
Rf ¼ 0.71 (S1), Rf ¼ 0.63 (S4); UPLC: tR ¼ 7.63 min; 1H NMR
(300 MHz, CDCl3): d 2.84 (t, 2H, piperazine, J ¼ 5.1 Hz), 3.03
(t, 2H, piperazine, J ¼ 5.1 Hz), 3.09 (d, 2H, CH2, J ¼ 7.4 Hz), 3.46
(t, 2H, piperazine, J ¼ 5.1 Hz), 3.69 (t, 2H, piperazine, J ¼ 5.1
Hz), 3.78 (s, 3H, OCH3), 4.68 (t, 1H, CH, J ¼ 7.6 Hz), 6.38 (t, 1H,
ArH, J ¼ 2.3 Hz), 6.43–6.48 (m, 2H, ArH), 7.14–7.31 (m, 11H,
ArH); C26H28N2O2 (400.51); monoisotopic mass 400.22;
[MþH]þ ¼ 401.4.
Monohydrohloride 1-(4-methyl-piperazin-1-yl)-3,3-
diphenylpropan-1-one (3r)
White powdery crystals. Yield: 65,8%; mp 264–266°C; TLC:
Rf ¼ 0.16 (S1), Rf ¼ 0.12 (S4); UPLC: tR ¼ 4.09 min; 1H NMR
(300 MHz, DMSO-d6): d 2.68 (s, 3H, CH3), 3.17 (d, 2H, CH2), 3.33
(br s, 8H, piperazine), 4.46 (t, 1H, CH, J ¼ 7.3 Hz), 7.09–7.17
(m, 2H, ArH), 7.20–7.34 (m, 8H, ArH), 11.02–11.62 (m, 1H, HCl);
C20H25N2OCl (344.88); amine: C20H24N2O (308.43); monoiso-
topic mass 308.20; [MþH]þ ¼ 309.4.
1-(4-Benzyl-piperazin-1-yl)-3,3-diphenylpropan-1-one (3n)
White powdery crystals. Yield: 71%; mp 130–132°C; TLC:
Rf ¼ 0.28 (S1), Rf ¼ 0.18 (S4); UPLC: tR ¼ 5.34 min; 1H NMR
(300 MHz, CDCl3): d 2.14 (br s, 2H, piperazine), 2.30 (t, 2H,
piperazine, J ¼ 5.0 Hz), 3.03 (d, 2H, CH2, J ¼ 7.4 Hz), 3.34
(t, 2H, piperazine, J ¼ 4.9 Hz), 3.43 (s, 2H, CH2), 3.55 (br s, 2H,
piperazine), 4.65 (t, 1H, CH, J ¼ 7.4 Hz), 7.14–7.35 (m, 15H,
ArH); 13C NMR (75 MHz, CDCl3): d 38.7, 41.6, 45.7, 47.3, 52.6,
52.8, 62.7, 126.4, 127.2, 127.8, 128.3, 128.5, 129.1, 144.1,
169.6; C26H28N2O (384.51); monoisotopic mass 384.22;
[MþH]þ ¼ 385.4.
1-Morpholine-3,3-diphenylpropan-1-one (3s)
White powdery crystals. Yield: 81%; mp 134–136°C; TLC:
Rf ¼ 0.84 (S1), Rf ¼ 0.61 (S4); UPLC: tR ¼ 5.97 min; 1H NMR
(300 MHz, CDCl3): d 3.04 (d, 2H, CH2, J ¼ 7.7 Hz), 3.31 (s, 4H,
morpholine), 3.53 (s, 4H, morpholine), 4.66 (t, 1H, CH,
J ¼ 7.6 Hz), 7.16–7.31 (m, 10H, ArH); 13C NMR (75 MHz, CDCl3):
d 38.5, 42.0, 46.2, 47.4, 66.3, 66.7, 126.5, 127.8, 128.3, 143.9,
169.9; C19H21NO2 (295.38); monoisotopic mass 295.16;
[MþH]þ ¼ 296.3.
3,3-Diphenyl-[4-(pyrimidin-2-yl)-piperazin-1-yl]-propan-1-
one (3o)
1-(4-Benzylpiperidin-1-yl)-3,3-diphenylpropan-1-one (3t)
White powdery crystals. Yield: 52%; mp 95–97°C; TLC:
Rf ¼ 0.87 (S1), Rf ¼ 0.70 (S4); UPLC: tR ¼ 8.65 min; 1H NMR
(300 MHz, CDCl3): d 0.97–1.01 (m, 1H, piperidine), 1.47–1.69
(m, 4H, piperidine), 2.35–2.52 (m, 2H, CH2, 1H, piperidine),
2.70–2.80 (m, 1H, piperidine), 2.95–3.12 (m, 2H, CH2), 3.77 (d,
1H, piperidine, J ¼ 13.6 Hz), 4.55 (d, 1H, piperidine, J ¼ 13.1
Hz), 4.67 (t, 1H, CH, J ¼ 7.6 Hz), 7.08–7.31 (m, 15H, ArH);
13C NMR (75 MHz, CDCl3): d 31.7, 32.2, 38.1, 38.7, 42.1, 42.9,
46.0, 47.4, 126.0, 126.3, 128.2, 129.0, 139.9, 144.2, 144.3,
169.4; C27H29NO (383.53); monoisotopic mass 383.22;
[MþH]þ ¼ 384.4.
White powdery crystals. Yield: 63%; mp 193–194°C; TLC:
Rf ¼ 0.42 (S1), Rf ¼ 0.53 (S4); UPLC: tR ¼ 6.44 min; 1H NMR
(300 MHz, CDCl3): d 3.10 (d, 2H, CH2, J ¼ 7.60 Hz), 3.38 (dd, 2H,
piperazine, J ¼ 6.5 Hz, J ¼ 4.1 Hz), 3.49–3.56 (m, 2H, pipera-
zine), 3.59–3.65 (m, 2H, piperazine), 3.66–3.72 (m, 2H,
piperazine), 4.70 (t, 1H, CH, J ¼ 7.7 Hz), 6.51 (t, 1H, ArH,
J ¼ 4.7 Hz), 7.15–7.21 (m, 2H, ArH), 7.23–7.31 (m, 8H, ArH), 8.30
(d, 2H, ArH, J ¼ 4.7 Hz); C23H24N4O (372.47); monoisotopic
mass 372.20; [MþH]þ ¼ 373.4.
1-(4-Cyclohexylpiperazin-1-yl)-3,3-diphenylpropan-1-one
(3p)
Anticonvulsant activity
General
Male albino mice (CD-1 strain) weighing 16–26 g were used as
experimental animals. The animals were housed in cages at
room temperature of 22 Æ 2°C, under a light/dark (12/12) cycle
and had free access to food and water. Ambient temperature
of the room and humidity were kept consistent throughout all
White powdery crystals. Yield: 72%; mp 100–101°C; TLC:
Rf ¼ 0.18 (S1), Rf ¼ 0.06 (S4); UPLC: tR ¼ 5.25 min; 1H NMR
(300 MHz, CDCl3): d 1.05–1.28 (m, 5H, cyclohexane), 1.62 (d,
1H, cyclohexane, J ¼ 11.3 Hz), 1.77 (d, 2H, cyclohexane,
J ¼ 6.4 Hz), 2.16–2.25 (m, 3H, 1H cyclohexane, 2H piperazine),
2.39 (t, 2H, piperazine, J ¼ 5.1 Hz), 3.04 (d, 2H, CH2, J ¼ 7.4
ß Deutsche Pharmazeutische Gesellschaft
www.archpharm.com
(12 of 14) e1600368

Arch. Pharm. Chem. Life Sci. 2017, 350, e1600368
Anticonvulsant Activity of Imide/Amide Derivatives
tests. For the experiments, the animals were randomly
selected. Each group consisted of three to four animals
(anticonvulsant screening) and each mouse was used only
once. The compounds were suspended in 0.5% methylcellu-
lose/water mixture. All the compounds were injected intra-
peritoneally into mice at the screening dose of 100 mg/kg with
anticonvulsant activity and neurotoxicity assessment at
0.5 and 2 h after administration. Procedures involving animals
and their care were conducted in accordance with current
European Community (EU Directive 2010/63/EU) and Polish
legislation on animal experimentation. The experimental
protocols and procedures described in this manuscript were
approved by the Local Ethics Committee at the Jagiellonian
University Medical College in Cracow (Approval Nos. 100/2014
and 47/2015).
twitching of the vibrissae, stun, and Straub-tail. The main
seizure end-point was the duration of the immobility. Mice
resuming normal behavior within 10 s from the stimulation
were considered as protected [26].
Quantification studies
The ED50 is defined as the dose of a drug protecting 50% of
animals against the MES, scPTZ, and 6-Hz seizures. Groups of
six animals received various doses of the compound until at
least three points were established in the range of 10–90%
seizure protection or minimal motor impairment. Both ED50
and TD50 values with 95% confidence limits were calculated
by probit analysis [27]. The PI (protective index) value was
calculated as the ratio of TD50 to ED50 (TD50/ED50 ¼ PI).
Radioligand binding assays
The maximal electroshock test (MES)
Sodium and calcium channels binding studies were performed
commercially in Cerep Laboratories (Poitiers, France) using
testing procedures described elsewhere (Naþ channel À site
2 [28], L-type Ca2þ [29]).
In the MES screen, electroconvulsions were produced by an
electrical stimulus of 0.2 s in duration at a frequency of 50 Hz
delivered via standard auricular electrodes by an electroshock
generator (Hugo Sachs rodent shocker, Germany). The tonic
hind-limb extension was taken as the end-point. Mice not
displaying hind-limb tonic extension were considered pro-
tected from seizure [22].
We are pleased to acknowledge the generous financial
support of this work by the grant of the Polish National
Scientific Centre, Poland (Grant No. DEC-2013/11/B/NZ7/
02081).
Subcutaneous pentylenetetrazole seizure test (scPTZ)
scPTZ-induced seizure was performed by subcutaneously
injection of pentylenetetrazole (Sigma–Aldrich, Poland) at a
dose of 85 mg/kg. This produced clonic convulsions lasting for
at least five seconds in 97% of animal tested. The absence of
clonic convulsions in the observed time period of 30 min was
interpreted as the compound’s ability to protect against PTZ-
induced seizure [23].
The authors have declared no conflict of interest.
References
[1] S. Nadkarni, J. LaJoie, O. Devinsky, Neurology 2005, 64,
S2–S11.
[2] http://www.who.int/mediacentre/factsheets/fs999/en/
(WHO, Epilepsy).
The neurological toxicity (NT)
Neurological toxicity induced by compound was detected in
mice using standardized rotarod test. Untreated control mice
when placed on the rod, can maintain their equilibrium for a
prolonged time period. The acute motor impairment can be
demonstrated by the inability of animal to maintain
equilibrium on the rod revolting at 10 rpm for at least
1 min [24].
[3] P. Kwan, S. C. Schachter, M. J. Brodie, N. Engl. J. Med.
2011, 365, 919–926.
[4] J. A. Cramer, S. Mintzer, J. Wheless, R. H. Mattson, Expert
Rev. Neurother. 2010, 10, 885–891.
[5] H. N. Khan, S. Kulsoom, H. Rashid, Epilepsy Res. 2012, 98,
62–71.
ꢀ
[6] L. Bruno-Blanch, J. Galvez, R. Garcia-Domenach, Bioorg.
Med. Chem. Lett. 2003, 13, 2749–2754.
ꢀ
6-Hz psychomotor seizures
[7] S. Rybka, J. Obniska, A. Rapacz, B. Filipek, K. Kaminski,
The 6-Hz seizure model was performed according to the
protocol originally described by Brown et al. [25] and more
recently by Barton et al. [19]. Psychomotor seizures were
induced via corneal stimulation (6 Hz, 32 mA, 0.2 ms rectan-
gular pulse width, 3 s duration) using a constant-current
device (ECT Unit 57800, Ugo Basile, Italy). A drop of 0.5%
solution of lidocaine hydrochloride was applied to the mouse
corneas before stimulation to provide local anesthesia and
ensure optimal current conductivity. After the electrical
stimulation, mice were gently released and observed for
the presence or absence of seizure activity, being character-
ized by immobility associated with rearing, forelimb clonus,
Arch. Pharm. Chem. Life Sci. 2014, 347, 768–776.
ꢀ
[8] J. Obniska, A. Rapacz, S. Rybka, B. Powroznik, E. Pe
̨
_
ꢀ
B. Filipek, P. Zmudzki, K. Kaminski, Eur. J. Med. Chem.
2015, 102, 14–25.
ꢀ
[9] K. Kaminski, M. Zagaja, J. J. Łuszczki, A. Rapacz,
ꢀ
M. Andres-Mach, G. Latacz, K. Kiec-Kononowicz, J.
Med. Chem 2015, 58, 5274–5286.
ꢀ
[10] J. Obniska, A. Rapacz, S. Rybka, M. Gora, K. Kaminski,
K. Sałat, Bioorg. Med. Chem. 2016, 24, 1598–1607.
ꢀ
[11] K. Kaminski, M. Zagaja, A. Rapacz, J. J. Łuszczki,
M. Andres-Mach, M. Abram, J. Obniska, Bioorg. Med.
Chem. 2016, 24, 606–618.
ß Deutsche Pharmazeutische Gesellschaft
www.archpharm.com
(13 of 14) e1600368

Arch. Pharm. Chem. Life Sci. 2017, 350, e1600368
J. Obniska et al.
ꢀ
[12] K. Kaminski, A. Rapacz, B. Filipek, J. Obniska, Bioorg.
[20] M. R. Kaminski, M. A. Rogawski, H. Klitgaard, Neuro-
therapeutics 2014, 11, 385–400.
Med. Chem. 2016, 24, 2938–2946.
[13] A. R. Katritzky, K. N. Satheesh, R. M. Witek,
S. M. Hutchins, ARKIVOC 2003, V, 9–18.
[21] B. S. Meldrum, M. A. Rogawski, Neurotherapeutics 2007,
4, 18–61.
[14] M. Smith, K. S. Wilcox, H. S. White, Neurotherapeutics
2007, 4, 12–17.
[22] J. E. P. Toman, E. A. Swinyard, L. S. Goodman, J.
Neurophysiol. 1946, 9, 231–239.
[15] M. M. Castel-Branco, G. L. Alves, I. V. Figueiredo,
A. C. Falcao, M. M. Caramona, Methods Find. Exp. Clin.
Pharmacol. 2009, 3, 101–106.
[23] A. Rapacz, J. Obniska, B. Wiklik-Poudel, S. Rybka,
K. Sałat, B. Filipek, Eur. J. Pharmacol. 2016, 781, 239–249.
[24] J. R. Boissier, J. Tardy, J. C. Diverres, Med. Exp. 1960, 3,
81–84.
[25] W. C. Brown, D. O. Schiffman, E. A. Swinyard, L. S.
Goodman, J. Pharmacol. Exp. Ther. 1953, 107, 273–283.
[26] K. Leclercq, R. M. Kaminski, Epilepsia 2015, 56, 310–318.
[27] J. T. Litchfield, F. Wilcoxon, J. Pharmacol. Exp. Ther. 1949,
96, 99–113.
[16] W. Loscher, Seizure 2011, 20, 359–368.
[17] S. H. White, J. H. Woodhead, K. S. Wilcox, J. P. Stables,
H. J. Kupferberg, H. H. Wolf, in Antiepileptic Drugs. (Eds.:
R. H. Levy, R. H. Mattson, B. S. Meldrum, E. Perucca,
Lippincott, Philadelphia, PA 2002, chapter 3.
[18] R. Thirumurugan, D. Sriram, A. Saxena, J. Stables,
P. Yogeeswari, Bioorg. Med. Chem. 2006, 14, 3106–3112.
[19] M. E. Barton, B. D. Klein, H. H. Wolf, H. S. White, Epilepsy
Res. 2001, 47, 217–227.
[28] G. B. Brown, J. Neurosci. 1986, 6, 2064–2070.
[29] R. J. Gould, K. M. Murphy, S. H. Snyder, Proc. Natl. Acad.
Sci. USA 1982, 79, 3656–3660.
ß Deutsche Pharmazeutische Gesellschaft
www.archpharm.com
(14 of 14) e1600368