Design, Synthesis, and Anticonvulsant Activity of New Hybrid Compounds Derived from 2-(2,5-Dioxopyrrolidin-1-yl)propanamides and 2-(2,5-Dioxopyrrolidin-1-yl)butanamides

Article abstract of DOI:10.1021/acs.jmedchem.5b00578

The library of 27 new 1-(4-phenylpiperazin-1-yl)- or 1-(morpholin-4-yl)-(2,5-dioxopyrrolidin-1-yl)propanamides and (2,5-dioxopyrrolidin-1-yl)butanamides as potential new hybrid anticonvulsant agents was synthesized. These hybrid molecules join the chemica

Full text of DOI:10.1021/acs.jmedchem.5b00578

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Article
Design, synthesis and anticonvulsant activity of new
hybrid compounds derived from 2-(2,5-dioxopyrrolidin-1-
yl)propanamides and 2-(2,5-dioxopyrrolidin-1-yl)butanamides
Krzysztof Kami#ski, Miros#aw Zagaja, Jarogniew J. #uszczki, Anna Rapacz,
Marta Andres-Mach, Gniewomir Latacz, and Katarzyna Kie#-Kononowicz
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00578 • Publication Date (Web): 08 Jun 2015
Downloaded from http://pubs.acs.org on June 11, 2015
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Design, Synthesis and Anticonvulsant Activity of New Hybrid Compounds
Derived from 2-(2,5-Dioxopyrrolidin-1-yl)propanamides and 2-(2,5-
Dioxopyrrolidin-1-yl)butanamides
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†
,
‡
‡,§
#
Krzysztof Kamiński *, Mirosław Zagaja , Jarogniew J. Łuszczki , Anna Rapacz , Marta
‡
⊥
⊥
AndresꢀMach , Gniewomir Latacz , Katarzyna KiećꢀKononowicz
†
Department of Medicinal Chemistry, Faculty of Pharmacy, Jagiellonian University Medical
College, Medyczna 9, 30ꢀ688 Kraków, Poland
‡
Isobolographic Analysis Laboratory, Institute of Rural Health, Jaczewskiego 2, 20ꢀ950
Lublin, Poland
§
Department of Pathophysiology, Medical University of Lublin, Ceramiczna 1, 20ꢀ150
Lublin, Poland
#
Department of Pharmacodynamics, Faculty of Pharmacy, Jagiellonian University Medical
College, Medyczna 9, 30ꢀ688 Kraków, Poland
⊥ꢀDepartment of Technology and Biotechnology of Drugs, Faculty of Pharmacy, Jagiellonian
University Medical College, Medyczna 9, 30ꢀ688 Kraków, Poland
*
To whom correspondence should be addressed. Phone: +48 12 620 54 59.
Fax: +48 12 620 54 58. Eꢀmail: k.kaminski@uj.edu.pl
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ABSTRACT
The library of 27 new 1ꢀ(4ꢀphenylpiperazinꢀ1ꢀyl)ꢀ or 1ꢀ(morpholinꢀ4ꢀyl)ꢀ(2,5ꢀ
dioxopyrrolidinꢀ1ꢀyl)propanamides and (2,5ꢀdioxopyrrolidinꢀ1ꢀyl)butanamides as potential
new hybrid anticonvulsant agents was synthesized. These hybrid molecules join the chemical
fragments of wellꢀknown antiepileptic drugs (AEDs) such as ethosuximide, levetiracetam and
lacosamide. Compounds 5, 10, 11, and 24 displayed the broad spectra of activity across the
preclinical seizure models, namely the maximal electroshock (MES) test, the subcutaneous
pentylenetetrazole (scPTZ) test, and the sixꢀhertz (6 Hz) model of pharmacoresistant limbic
seizures. The highest protection demonstrated 11 (ED50 MES = 88.4 mg/kg, ED50 scPTZ =
59.9 mg/kg, ED50 6 Hz = 21.0 mg/kg). This molecule did not impair the motor coordination of
animals in the chimney test even at high doses (TD50 >1500 mg/kg) that yielded superb
protective indexes (PI MES >16.97, PI PTZ >25.04, PI 6 Hz >71.43). As a result, 11
displayed distinctly better safety profile than clinically relevant AEDs ethosuximide,
lacosamide or valproic acid.
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INTRODUCTION
Epilepsy affects approximately 50 million people worldwide, making it the second
1,2
most common neurological disorder after stroke. For many years epilepsy has been
recognized as a disease of young people, as the first symptoms occur usually before the age of
10. However, current data show that the prevalence increases with the age, being 0.7% in
3,4
range of 55–64 years and 1.2% in population over age of 85. This fact becomes more and
more serious owing to the demographic changes related to the aging of societies in
industrialized countries. Despite the significant advances in epilepsy research, convulsions in
5
,6
30% of epileptics are still inadequately controlled by standard drug therapy. Furthermore,
compliance is often limited by adverse side effects most notably related to the central nervous
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system (CNS) exposure like diminished attention, executive function, intelligence, language
7
skills, memory, and processing speed. Data collected from eight of the biggest markets show
8
that therapyꢀresistant epilepsy affects about 1.8 million people. In recent times, several new
drugs such as levetiracetam, felbamate, lamotrigine, gabapentin, and topiramate have been
implemented for the treatment of epilepsy. Although these drugs have been shown to be
effective in epileptic syndromes in a number of patients, their efficacy does not appear to be
superior to that of the established AEDs. Therefore, the ideal AED should prevent different
types of seizures without producing side effects that adversely affect patients’ quality of life.
Taking into consideration the aforementioned facts, the continued search for safer and more
effective antiepileptic drugs is both urgent and necessary.
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The incomplete information on the pathogenesis of human epilepsy and the complex
mechanism of action of majority AEDs make it difficult to use the rational drug design
technique that is based on the threeꢀdimensional structure of the biological target.
Conceptually, there are two different methods of obtaining new AEDs, namely, ligandꢀbased
9,10
approach and screening approach.
The ligandꢀbased approach relies on the use of existing
biological data for old and new drugs or other anticonvulsant active compounds. This method
is applied mainly for structural modifications of the currently available AEDs, with the aim of
obtaining more efficacious substances that will suppress different types of seizures and/or
drugs with minimal or no adverse effects compared to original AEDs. It was successfully
used in the discovery of several thirdꢀgeneration AEDs (e.g., eslicarbazepine,
fluorofelbamate, pregabalin) as well as compounds that are currently in Phase 3 of clinical
trials (e.g. brivaracetam or seletracetam). The screening approach involves a comprehensive
screening process of either diverse or focused compound libraries and utilizes rodent models
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of human epilepsies.
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Previous research from our laboratory identified pyrrolidineꢀ2,5ꢀdiones differently
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substituted at positionꢀ1 and ꢀ3 as candidates for new AEDs.
were effective in the maximal electroshock (MES) and subcutaneous pentylenetetrazole
scPTZ) tests. Studies on the structure–activity relationship (SAR) demonstrated the potent
Many of these compounds
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and wide spectrum of anticonvulsant activity for the 2ꢀ(2,5ꢀdioxopyrrolidinꢀ1ꢀyl)acetamides
containing phenylpiperazines with highly electronegative chlorine, fluorine, or
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trifluoromethyl substituents at the amine function.
Considering the aforementioned facts, the aim of the study was to obtain
anticonvulsants with a broad spectrum of activity in “classic” animal models of epilepsy,
MES and scPTZ, as well as in the 6 Hz model of pharmacoresistant limbic seizures. Thus, the
hypothesis assumes that the combination of chemical fragments of AEDs active in the MES
test (lacosamide), PTZ test (ethosuximide), and 6 Hz seizures (levetiracetam, lacosamide) in
one hybrid molecule may yield substances effective in all aforementioned preclinical animal
seizure models.
In view of the above hypothesis in the current studies, the library of new 1ꢀ(4ꢀ
phenylpiperazinꢀ1ꢀyl)ꢀ or 1ꢀ(morpholinꢀ4ꢀyl)ꢀ(2,5ꢀdioxopyrrolidinꢀ1ꢀyl)propanamides and
(
2,5ꢀdioxopyrrolidinꢀ1ꢀyl)butanamides was synthesized. The alkylamide moiety, namely,
propanamide or butanamide, connected with the fiveꢀmembered pyrrolidineꢀ2,5ꢀdione ring
approximated the structures of new hybrid molecules to first generation AED ꢀ ethosuximide
and levetiracetam which is one of the newest AEDs (Figure 1 A).
In the next step, the synthesis of the 3ꢀmethoxypropanamide analog of the most
effective compound from the series of 2ꢀ(2,5ꢀdioxopyrrolidinꢀ1ꢀyl)alkylamides was carried
out. The introduction of the methoxy functional group at the alkylamide linker allowed the
imitation of the mode of substitution to that observed for lacosamide. This AED, chemically
1
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classified as functionalized amino acid (FAA),
has been recently approved for the
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treatment of partialꢀonset seizures with or without secondary generalization as monotherapy
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or adjunctive therapy in humans.
B.
The proposed structural modification is shown in Figure
1
The final compounds were tested in vivo for anticonvulsant activity using the maximal
electroshockꢀinduced seizure test (MES), which is the primary animal model of epilepsy used
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for the discovery and development of new AEDs. In addition to the MES screening, the
most potent substances were tested in the subcutaneous pentylenetetrazole (scPTZ) and the
sixꢀhertz (6 Hz) seizure models. To determine the plausible mechanism of action for the most
effective compounds in vitro binding assays were carried out. Considering drug safety
evaluation, which is important in the preclinical identification of new drug candidates, the
antiproliferative potential of the most promising molecule, and its influence on function of
recombinant human CYP3A4 cytochrome isoenzyme were studied in vitro. Furthermore, the
metabolic stability was examined using the in silico and in vitro methods.
RESULTS AND DISCUSSION
Chemistry. Compounds 3–28 were synthesized in a twoꢀstep reaction according to
Scheme 1. First, the condensation reaction of commercially purchased succinic anhydride,
with DLꢀαꢀalanine or DLꢀ2ꢀaminobutyric acid, yielded corresponding intermediates 1 and 2.
In the next step, 1 or 2 was converted to 3–28 by coupling with the appropriate
phenylpiperazine derivative or morpholin in the presence of carbonyldiimidazole (CDI),
which is a commonly used reagent for the synthesis of amides from carboxylic acids and
2
7,28
amines through the acyl imidazole intermediate.
The reaction was carried out in dry
tetrahydrofuran (THF) at room temperature. The progress of the reaction was monitored using
HPLC chromatography (completion at approx. 24 h). Compounds 3–28 were obtained with
yields ranging between 73% and 86%. The synthesis of 3ꢀmethoxypropanamide derivative 30
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was accomplished as shown in Scheme 2. First, the coupling reaction of 2ꢀchloroꢀ3ꢀ
methoxypropanoic acid and 1ꢀ[3ꢀ(trifluoromethyl)phenyl]piperazine in the presence of CDI
yielded intermediate 29, which was then used for alkylation of pyrrolidineꢀ2,5ꢀdione to form
the desired compound, 30. Alkylation in a biphasic system was carried out in dry acetone at
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60 °C for approx. 24 h in the presence of anhydrous potassium carbonate and potassium
iodide. Due to the high reactivity of the chlorine atom at the αꢀposition of 3ꢀ
methoxypropanoic amide 29, the target compound 30 was obtained in high yield–84%. All
compounds were isolated as racemic mixtures. The final substances were fully characterized
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by elemental analyses (C, H, N), H NMR, C NMR, F NMR, and LC/MS spectra (details
are shown in the Supporting Information).
Pharmacology. The anticonvulsant activity profile of final molecules 3–28 and 30
was determined using the MES test, a mechanismꢀindependent animal seizure model which
enables identification of compounds preventing seizure spread. This test is thought to be an
experimental model of tonic–clonic epilepsy and of partial convulsions with or without
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secondary generalization in humans.
It should be noted that despite significant advances
in epilepsy research in the past several years, the MES model still persists as the most useful
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tool for the identification of new anticonvulsants. Compounds 3–28 and 30 were
administered in mice intraperitoneally (i.p.) at the fixed dose of 300 mg/kg, and the
anticonvulsant protection was observed at four pretreatment times–0.25, 0.5, 1 and 2 h. The
method applied here allowed the determination of the number of animals (in a group
consisting of four mice) protected against electrically induced seizures as well as the
estimation of the time course of anticonvulsant activity together with time of peak effect
(TPE). The protection profiles for 2ꢀ(2,5ꢀdioxopyrrolidinꢀ1ꢀyl)propanamides (3–15, 30) and
2ꢀ(2,5ꢀdioxopyrrolidinꢀ1ꢀyl)butanamides (16–28) are shown in Table 1.
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The preliminary pharmacological screening revealed that only two compounds (15,
8), representing morpholine derivatives, were completely devoid of anticonvulsant activity.
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In the whole series, 10 molecules 5, 7–11, 16, 18, 24, and 30 revealed rapid onset and longꢀ
lasting anticonvulsant protection showing activity up to 2 h. Except for weak active molecules
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, 12, 19, 22, 23, 25, and 27, all other compounds showed 50–100% protection lasting from
0.25 h up to 2 h. In the whole series, the most potent was 11 with three points of maximal
protection at 0.5, 1, and 2 h. Slightly weaker efficacy was observed for its, 3ꢀchlorine (5), 3ꢀ
fluorine (8), 2ꢀtrifloromethyl (10), and butanamide (24) analogs. With the exclusion of highly
active compound 26, the exchange of electronegative trifluoromethyl group or chlorine atom
in the metaꢀposition of the phenylpiperazine moiety with electronꢀdonating methyl or
methoxy substituents yielded less active molecules 13, 14, and 27. The analysis of
preliminary pharmacological data revealed that the presence of highly electronegative atoms
or groups in position 3 seems preferential for anticonvulsant properties. Their removal or
replacement into orthoꢀ or paraꢀposition caused a decrease in anticonvulsant activity. It is
worth noting that higher protection was observed in general for compounds representing 2ꢀ
(
2,5ꢀdioxopyrrolidinꢀ1ꢀyl)propanamides. Finally, slight weakening of activity was observed in
case of replacement of propanamide moiety of compound 11 into 3ꢀmethoxypropanamide of
0. It should be stressed, however, that anticonvulsant protection of the latter substance
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increased over time from 25% at 0.25 h to 100% at 1 h and 2 h. This observation may suggest
its delayed and prolonged activity.
On the basis of the above preliminary data, 17 active compounds were screened in
mice (i.p.) at the fixed dose of 100 mg/kg, at four pretreatment times 0.25, 0.5, 1, 2 h (Table
2).
The results obtained at the dose of 100 mg/kg revealed that 12 molecules 3, 5–11, 18,
24, 26, and 30 showed satisfactory activity (at least of 50% protection in MES seizures).
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Among these molecules, 5, 10, 11, and 30 protected mice starting from 0.25 h till 1 h with
different TPEs, i.e., 0.5 h for 5 and 11 (75% protection) or 0.25 h for 10 and 30 (100%
protection). In general, other compounds showed short duration of anticonvulsant activity
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In the aim of finding new hybrid anticonvulsants with wide spectrum of activity in the
preclinical studies, 5 the most potent compounds in the MES test 5, 10, 11, 24, and 30 (see
Table 2) were studied in the subcutaneous pentylenetetrazole (scPTZ) test and psychomotor 6
Hz seizure model.
The scPTZ test employs chemically induced myoclonic seizures and is proposed to
identify the agents raising the seizure threshold. This test is related to human generalized
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absence seizures. It should be stressed that ethosuximide being the chemical prototype for
compounds proposed herein is an antiꢀabsence medication effective in the PTZ seizures,
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however inactive in the MES test. The results after i.p. injection in mice (doses of 300 and
100 mg/kg) are summarized in Tables 3 and 4.
As is shown in Table 3, all molecules displayed potent activity at dose of 300 mg/kg,
protecting at least 75% of animals in all time points. It should be emphasized that compound
1 showed maximal protection starting from 0.25 till 2 h. This molecule displayed also the
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most potent activity at the dose of 100 mg/kg, with one peak of 100% protection at 0.5 h, and
points of 75% protection at 1 and 2 h (Table 4). Other compounds screened at the dose of 100
mg/kg revealed at least of 50% protection in different time intervals. Notably, 11 and 30
displayed prolonged and satisfactory activity in all the time points.
Traditionally, most screening programs assess efficacy of AED candidates against
either electrically (e.g. MES) or chemically (e.g. PTZ) 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
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would be uniquely effective in the therapyꢀresistant population. One example supporting this
hypothesis is provided by levetiracetam, which has demonstrated efficacy in refractory human
partial epilepsies. It was found to be inactive against MES and PTZ seizures even at high
doses, whereas showed high efficacy in the 6 Hz model of pharmacoresistant limbic
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seizures. Bearing in mind the aforementioned facts and the structural similarities of new
compounds with levetiracetam (see Figure 1), 5 molecules 5, 10, 11, 24, and 30 tested before
in the MES and PTZ seizures, were screened in the 6 Hz model. The results after i.p.
administration are summarized in Table 5.
As is shown in Table 5, all compounds tested displayed satisfactory activity in the 6
Hz seizures. The most beneficial anticonvulsant properties was observed for 11, which
displayed three points of 100% protection – 0.5, 1, and 2 h. Furthermore, high efficacy with
two points of the maximal activity revealed also 5, 24, and 30, whereas 10 protected 100% of
animals only at 0.25 h after i.p. administration.
Based on the preliminary results, in the next step of the pharmacological studies, the
median effective doses (ED50) were determined for compounds which showed minimum 50%
protection at the dose of 100 mg/kg (MES, PTZ or 6 Hz tests). Moreover, for compounds with
the best ED values (<120 mg/kg), the median neurotoxic doses (TD ,) were estimated in the
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chimney test. This data was used to calculate the protective indexes (PIs), which are the
measure of the benefitꢀtoꢀrisk ratio of the therapeutic agent. In parallel, the same studies were
performed for model AEDs active in the MES (lacosamide), PTZ (ethosuximide), 6 Hz
(
levetiracetam, lacosamide) tests as well as effective in all seizure models (valproic acid)
Table 6).
(
The quantitative i.p. data in mice confirmed the wide spectrum and potent
anticonvulsant activity of 5, 10, 11, and 24 containing the 3ꢀchloroꢀ (5), 2ꢀ(trifluoromethyl)ꢀ
(
10) or 3ꢀ(trifluoromethyl)phenylpiperazines (11, 24) as amine function. Among these
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compounds, the most promising was 11 which did not impair motor coordination of animals
even at high doses (TD >1500 mg/kg) that yielded superb protective indexes (>16.97, MES;
50
>25.04, PTZ; >71.43, 6 Hz) at time point of 0.5 h. As a result, 11 displayed distinctly better
safety profile than all model AEDs. Despite compound 11 showing lower protection in the
MES and 6 Hz seizures compared to the lacosamide, it showed better safety profile in the
chimney test that resulted in 4.7ꢀfold (MES) and 13.6ꢀfold (6 Hz) more beneficial PI values.
Notably, this molecule revealed comparable efficacy to levetiracetam (6 Hz test), and
distinctly higher activity in comparison with PTZꢀactive ethosuximide (2.5ꢀfold), as well as
MES/PTZ/6 Hzꢀactive valproic acid as follows: 2.4ꢀfold (MES), 4.6ꢀfold (PTZ), and 6.2ꢀfold
(6 Hz). Additionally, compound 11 displayed potent activity and markedly low neurotoxicity
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h after administration. High activity and wide spectrum of anticonvulsant protection was
observed also for other trifluoromethyl analogs 10, 24, and metaꢀchloro derivative 5;
however, these molecules were more neurotoxic in the chimney test, resulting in worsening of
PIs compared to model AEDs (ethosuximide, lacosamide, and levetiracetam). It should be
stressed that all aforementioned substances were more potent compared to valproic acid
(
MES, PTZ, 6 Hz seizures), as well as provided better protection in the scPTZ test than model
PTZꢀactive drug – ethosuximide. Notably, the metaꢀchloro derivative 5 displayed ED50 of
5.6 mg/kg in the 6 Hz test 0.25 h after i.p. injection (data not indicated in Table 6). The
1
quantitative data revealed that in case of the most effective substance 11, the introduction of
the methoxy group into the propanamide fragment (compound 30) increased activity in the
MES test, however decreased the protection in the 6 Hz seizures. Furthermore, it was not
possible to determine ED value in the scPTZ test up to dose of 120 mg/kg. This observation
5
0
may result from the presence of 3ꢀmethoxypropanamide fragment that is common for both 30
and lacosamide, which is known to be inactive in the PTZ seizures. Despite the lower PI of 30
compared to parent compound 11, the aforementioned structural modification may lead to
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highly effective substances and will be examined precisely in the next studies. In conclusion,
the results of the current studies as well as our previous research in a group of Nꢀbenzylꢀ2ꢀ
(2,5ꢀdioxopyrrolidinꢀ1ꢀyl)propanamide
and
2ꢀ(2,5ꢀdioxopyrrolidinꢀ1ꢀyl)butanamide
32
derivatives, highly support the proposed hypothesis of hybrid anticonvulsants based on the
10
11
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pyrrolidineꢀ2,5ꢀdione core fragment.
In vitro radioligand binding studies. Intensive research into the physiological and
biochemical events which occur during epileptic seizures has provided insight into the
molecular mechanisms by which these might be controlled. Thus, AEDs act on diverse
molecular targets to selectively modify the excitability of neurons so that seizureꢀrelated
firing is blocked without disturbing nonꢀepileptic activity. This occurs largely through effects
on voltageꢀgated sodium and calcium channels, or by promoting inhibition mediated by
GABA (γꢀaminobutyric acid, type A) receptors. The fundamental role in establishing and
A
regulating the excitability of CNS neurons as well as suppression of seizures is ascribed to
3
0
voltageꢀgated sodium channels (VGSCs), and voltageꢀdependent calcium channels (VDCC).
Thus, the brain voltageꢀgated sodium and calcium channels are the molecular targets of
33–36
numerous chemically diverse AEDs.
Using radioligand binding techniques, Willow and
associates have found an allosteric interaction between classical and clinically relevant AEDs
ꢀ
phenytoin and carbamazepine, and the batrachotoxinin (BTX) binding site of sodium
37,38
channels from rat brain.
Electrophysiological studies of neuroblastoma cells demonstrate a
frequencyꢀ and voltageꢀdependent blockade of sodium currents by aforementioned
39,40
anticonvulsants.
These findings suggest that blockade of sodium channel activity by these
agents underlies their anticonvulsant actions. Such mode of action is characteristic of
substances active in the MES test in animals. Due to the activity of the compounds reported in
this paper in the MES seizures, for 3 the most active molecules (11, 24, 30), and
+
carbamazepine (as a reference AED), the binding assays for Na channel (site 2) were
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performed using the [ H]BTX as radioligand. Compound binding was expressed as a
percentage of inhibition of the binding of a radioactively labeled ligand. The inhibition values
for 11, 24, and 30 were determined at four concentrations, 1 ꢁM, 10 ꢁM, 100 ꢁM, and 500
ꢁ
M (Table 7).
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Compounds 11, 24, and 30 were observed as relatively effective binders to the
+
neuronal voltageꢀsensitive Na channel at the highest concentration–500 ꢁM. The moderate
activity was observed also for 11, and 30 at a concentration of 100 ꢁM. Notably,
+
carbamazepine, which is known to be the anticonvulsant that acts as Na channel blocker,
showed a moderate effect only at 500 ꢁM, and was weaker binder compared to 11, 24, and 30.
The above results may suggest that the mechanism of anticonvulsant protection of compounds
tested is probably related to their influence on voltageꢀgated sodium channel. Furthermore,
this observation was strengthened by the in vivo quantitative data (Table 6), namely the more
+
potent compound in the electrically evoked seizures (MES test) the higher Na channel
affinity. Compound 11 has a broad spectrum of anticonvulsant activity (MES, scPTZ, 6 Hz
models), most likely reflecting its multiple sites of action. Thus, with the aim of investigating
the more precise mechanisms by which 11 is believed to exert its antiꢀseizure effects, the
2+
2+
binding assays for voltageꢀgated Nꢀtype Ca channel and Lꢀtype Ca channel were carried
out. In parallel, the same tests were performed for 30 which was inactive in the scPTZ
seizures (ED50 > 120 mg/g), and provided weaker protection in the 6 Hz model. As shown
2
+
in Table 8, 11 revealed distinctly stronger affinity to Lꢀtype Ca channel than 30, and both
2+
compounds did not bind to Nꢀtype Ca channel. This data indicate that interaction of 11 with
2
+
voltageꢀgated
Lꢀtype Ca channel may play an important role for its activity in the PTZ
seizures especially, however also in the 6 Hz test. It should be emphasized that a modulation
2
+
of neuronal
Lꢀtype Ca channel activity is an essential mechanism of action for topiramate
4
2,43
(
TPM), which is the novel AED with broad therapeutic spectrum.
Notably, compound 11
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proved to be a more effective binder to the voltageꢀgated
mentioned AED at a concentration of 100 ꢁM.
Lꢀtype Ca channel compared to the
In vitro and in silico assays. In the modern approach to the drug development
process, drugꢀlike properties of the new compounds should be evaluated in parallel with the
10
11
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4,45
study on their efficacy at early discovery phases.
In the present study, compound 11, as
the most promising in the pharmacological studies, was chosen for preliminary in vitro assays
of selected ADME–Tox parameters, such as antiproliferative activity and metabolic stability.
Additionally, the bioluminescence assay was used for the prediction of the influence of
compound 11 on the activity of recombinant cytochrome P450 3A4, considering the potential
drug–drug interactions (DDIs).
Antiproliferative assay. The incubation of human embryonic kidney cell line (HEK–
2
93) in the presence of the most promising molecule 11 for 48 h showed the decreasing
viability of examined cells above the 10 µM concentration (Figure 2). The calculated IC50
value of 11 against HEKꢀ293 was 50.73 µM. However, in comparison with the IC50 value of
0.46 µM for the reference antiproliferative drug–doxorubicin (DX), compound 11 possesses
over 100ꢀfold lower antiproliferative activity. Thus, the results obtained qualify compound 11
as safe in relation to the antiproliferative effect.
Metabolic stability. The metabolic stability of compound 11 was examined first in
46
silico by using MetaSite software. The plot of MetaSite predictions for sites of metabolism
for 11 using the liver computational model is shown in Figure 3. The darker red color of
atoms marked with circles indicate their higher probability to be involved in the metabolism
pathway. The blue circle marks the site of compound 11 involved in metabolism with the
highest probability (100% score). According to in silico data, the highest probability of
metabolism will occur at the positionꢀ4 of phenyl moiety. Moreover, compound 11 will be
likely metabolized also at the alkylamide linker and at pyrrolidineꢀ2,5ꢀdione ring. The
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metabolic stability of 11 was also evaluated in vitro using human liver microsomes (HLMs).
A full scan chromatogram of compound 11 after 2 h of incubation with HLMs showed the
presence of four metabolites–M1, M2, M3, and M4 (Figure 4 A). The LC/MS analysis
+
provided the molecular masses of obtained metabolites: M1 [M+H] = 382.27 m/z, M2
0
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+
+
[
M+H] = 416.31 m/z, M3 [M+H] = 402.28 m/z, and M4 [M+H] = 400.29 m/z (Figure 4 B).
To confirm the structures of HLM metabolites of compound 11, the ion fragment analysis was
performed. The metabolite structures generated on the basis of in silico studies and the ion
fragment analysis considered the following metabolic pathways of 11: (i) the dehydrogenation
of piperazine moiety (M1); (ii) doubleꢀhydroxylation of the alkylamide linker and
pyrrolidineꢀ2,5ꢀdione moiety (M2); (iii) hydroxylation of the alkylamide linker with the
reduction of one carbonyl group of pyrrolidineꢀ2,5ꢀdione ring (M3); and (iv) the introduction
of the hydroxyl group next to the trifluoromethyl substituent in the phenylpiperazine moiety
(M4) (Figure 5).
Influence on recombinant human CYP3A4 P450 cytochrome. Many drug–drug
interactions are metabolism based and are mediated primarily via the microsomal cytochrome
P450 (CYP) family of enzymes. Ten CYP isoforms are expressed in a typical human liver and
six of them (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) appear to be
employed in the biotransformation of most drugs. Notably, among these isoforms, the
CYP3A4 is responsible for the metabolism of more than 50% of medicines and the associated
drug–drug interactions. The inhibition of these enzymes may decrease the metabolic clearance
of a coꢀadministered drug, resulting in elevated blood concentration, which may cause
adverse drug effects or toxicity. As detailed in the draft guidance document for drug–drug
47
interactions, the FDA has placed emphasis on evaluating the inhibition potential of a new
chemical entity (NCE) at an earlier stage in drug development to avoid the development of
compounds with the potential to yield adverse drug interactions.
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To evaluate the potential inhibition or induction of cytochrome CYP3A4 by
compound 11, we used the luminescence CYP3A4 P450ꢀGlo™ assay based on the conversion
of the luciferinꢀPPXE (the beetle
Dꢀluciferin derivative) into Dꢀluciferin by recombinant
human CYP3A4 isoenzyme. After the addition of firefly luciferase, the measured amount of
10
11
12
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4
8
light produced in the reaction was proportional to the concentration of
shown in the following (Figure 6), compound 11 inhibited slightly the cytochrome activity at
5 µM (68.4% of control activity). However, comparing with the effect of ketoconazole
Dꢀluciferin. As it is
2
which completely inhibited the CYP3A4 activity at 10 µM (calculated IC50=0.14 ꢁM),
compound 11 may be considered as a very weak CYP3A4 inhibitor. Thus, the data suggest
that compound 11 is unlikely to show inhibitory interactions with other CYP3A4 metabolized
drugs in vivo.
CONCLUSION
The purpose of this study was to design and synthetize the focused library of 27 new
1ꢀ(4ꢀphenylpiperazinꢀ1ꢀyl)ꢀ or 1ꢀ(morpholinꢀ4ꢀyl)ꢀ(2,5ꢀdioxopyrrolidinꢀ1ꢀyl)propanamides
and (2,5ꢀdioxopyrrolidinꢀ1ꢀyl)butanamides as potential new hybrid anticonvulsant agents.
These hybrid molecules join the chemical fragments of wellꢀknown AEDs such as
ethosuximide, levetiracetam, and lacosamide. The initial anticonvulsant screening was
performed in mice (i.p.) using the ‘classical’ maximal electroshock (MES) and subcutaneous
pentylenetetrazole (scPTZ) tests as well as in the sixꢀhertz (6 Hz) model of pharmacoresistant
limbic seizures. The acute neurological toxicity was determined applying the chimney test.
Four compounds 5, 10, 11, and 24 showed the broad spectra of activity across all the
preclinical seizure models. The quantitative pharmacological studies in mice i.p.
demonstrated the highest protection and a substantial safety profile in the chimney for 11.
This compound emerged as the most promising molecule with superb protective indexes (PI
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MES >16.97, PI PTZ >25.04, PI 6 Hz >71.43). The in vitro binding studies proved that the
most plausible mechanism of action for 11 was the influence on the neuronal voltageꢀ
sensitive sodium and Lꢀtype calcium channels. The results obtained in the current studies
support the proposed hypothesis of hybrid anticonvulsants based on the pyrrolidineꢀ2,5ꢀdione
core fragment.
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EXPERIMENTAL SECTION
Chemistry. General. All chemicals and solvents were purchased from SigmaꢀAldrich
(St. Louis, USA) and were used without further purification. Melting points (mp) were
determined in open capillaries on a Büchi 353 melting point apparatus (Büchi Labortechnik,
Flawil, Switzerland) and are uncorrected. The purity and homogeneity of the compounds were
assessed by TLC and gradient HPLC chromatography. The thin–layer chromatography (TLC)
was performed on Merck silica gel 60 F254 preꢀcoated aluminium sheets (Merck; Darmstadt,
1
Germany), using developing system consisting of: S – dichloromethane : methanol (9 : 1;
v/v), S – dichloromethane : methanol (9 : 0.3; v/v). Spots were detected by their absorption
2
under UV light (λ = 254 nm). HPLC analyses were run on the HPLC Waters 2695 Separation
Module (Waters, Milford, USA) equipped with photodiode array detector Waters 2998
(Waters, Milford, USA). The Chromolith RP–18 SpeedROD column (4.6 × 50 mm) was used.
Conditions applied were as follow: eluent A (water/0.1% TFA), eluent B (acetonitrile/0.1%
TFA); flow rate of 5 mL/min, gradient of 0–100% B over 3 min were used, injection volume
was 10 ꢁL. Standard solutions (1 mg/mL) of each compound were prepared in analytical
grade acetonitrile and analyzed at wave lengths 214 and 254 nm. Retention times (t ) are
R
given in minutes. The purity of all final compounds determined by use of chromatographic
HPLC method was >95%. Elemental analysis for C, H, and N were carried out by a micro
method using the elemental Vario EI III Elemental analyzer (Hanau, Germany). The results of
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elemental analyses were within ±0.4 % of the theoretical values. H NMR, C NMR and F
NMR spectra were obtained in a Varian Mercury spectrometer (Varian Inc., Palo Alto, CA,
1
13
19
USA), in CDCl
3
operating at 300 MHz ( H NMR), 75 MHz ( C NMR), and 282 MHz ( F
1
NMR). Chemical shifts are reported in δ values (ppm) relative to TMS δ = 0 ( H), as internal
standard. The J values are expressed in Hertz (Hz). Signal multiplicities are represented by
the following abbreviations: s (singlet), br s (broad singlet), d (doublet), dd (doublet of
doublets), ddd (double double doublet), t (triplet), td (triplet of doublets), q (quartet), m
10
11
12
13
14
15
16
17
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19
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43
44
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60
(
multiplet). The mass spectra (LCꢀMS) were obtained on Waters ACQUITY™ TQD system
with the TQ Detector (Waters, Milford, USA). The ACQUITY UPLC BEH C18, 1.7 ꢁm, 2.1
ꢀ 50 mm column was used (Waters, Milford, USA). Preparative column chromatography
was performed using silica gel 60 (particle size 0,063–0,200 mm; 70–230 Mesh ASTM)
purchased from Merck (Darmstadt, Germany).
General procedure for the preparation of the 2-(2,5-dioxopyrrolidin-1-
yl)propanoic acid (1) and 2-(2,5-dioxopyrrolidin-1-yl)butanoic acid (2). Succinic
anhydride (5.0 g, 0.05 mol) was suspended in 20 mL of water and DLꢀαꢀalanine (4.4 g, 0.05
mol) or DLꢀ2ꢀaminobutyric acid (5.2 g, 0.05 mol) was gradually added. The mixture was
heated in a term–regulated 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 column
chromatography (dichloromethane : methanol, 9 : 1, v/v) to afford intermediates 1 and 2 as
yellow oils.
2
-(2,5-Dioxopyrrolidin-1-yl)propanoic acid (1). Yellow oil. Yield 75%; TLC: R = 0.61
f
+
(S
1
); HPLC (purity 100%): t
R
= 0.278 min.; ESI–MS: 172.1 (C
7
H
9
NO
4
[M+H] ). Anal calcd
for C
7
H
9
NO
4
(171.15): C, 49.12; H, 5.30; N, 8.18. Found: C, 49.20; H, 5.18; N, 8.25.
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2
2
2
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f 1
2-(2,5-Dioxopyrrolidin-1-yl)butanoic acid (2). Yellow oil. Yield 72%; TLC: R = 0.71 (S );
+
HPLC (purity 99.2%): t = 0.524 min.; ESI–MS: 185.1 (C H NO [M+H] ). Anal calcd for
R
8
11
4
C
8
H
11NO
4
(185.15): C, 51.89; H, 5.99; N, 7.56. Found: C, 51.95; H, 5.90; N, 7.66.
General method for the preparation of 2-(2,5-dioxopyrrolidin-1-yl)propanamides
3-15) and 2-(2,5-dioxopyrrolidin-1-yl)butanamides (16–28). Carbonyldiimidazole (0.97 g,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
0
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2
3
4
5
6
7
8
9
0
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2
3
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6
7
8
9
0
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2
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4
5
6
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9
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(
0.006 mol) in 5 mL of dry THF was added to a solution of intermediate 1 (1.02g, 0.006 mol)
or 2 (1.11g, 0.006 mol) dissolved in 10 mL of anhydrous THF while stirring. After the end of
gaseous (carbon dioxide) evolution (approx. 0.5 h), the secondary amine (0.006 mol)
dissolved in 5 mL of anhydrous THF was added dropwise. The mixture was stirred at room
temperature (approx. 24 h) and evaporated to dryness. The crude product was purified by
column chromatography (dichloromethane : methanol, 9 : 0.3, v/v). The final amides were
obtained as solid substances after concentration of organic solvents under reduced pressure.
1-[1-Oxo-1-(4-phenylpiperazin-1-yl)propan-2-yl]pyrrolidine-2,5-dione (3). White solid.
Yield: 77%; mp 129–130 ºC; TLC: R
f
= 0.45 (S
2
); HPLC (purity 100%): t
R
= 0.975 min.; ESI–
+
MS: 316.1 (C17H N O
21 3 3
[M+H] ). Anal. calcd for C17
H N O
21 3 3
(315.37): C: 64.74, H: 6.71,
N:13.32; Found C: 64.88, H: 6.62, N: 13.50.
-{1-[4-(2-Chlorophenyl)piperazin-1-yl]-1-oxopropan-2-yl}pyrrolidine-2,5-dione
R
White solid. Yield: 73%; mp 118–119 ºC; TLC: R = 0.52 (S ); HPLC (purity 100%): t =
1
(4).
f
2
+
1.247 min.; ESI–MS: 350.2 (C17
C: 58.37, H: 5.76, N: 12.01; Found C: 58.55, H: 5.85, N: 11.88.
-{1-[4-(3-Chlorophenyl)piperazin-1-yl]-1-oxopropan-2-yl}pyrrolidine-2,5-dione
H
20ClN
3
O
3
[M+H] ). Anal. calcd for C17
H
20ClN
3
O
3
(349.81):
1
(5).
White solid. Yield: 78%; mp 148–149 ºC; TLC: R = 0.56 (S ); HPLC (purity 100%): t =
f
2
R
+
1.380 min.; ESI–MS: 350.2 (C17
H
20ClN
3
O
3
[M+H] ). Anal. calcd for C17
H
20ClN
3
O
3
(349.81):
C: 58.37, H: 5.76, N: 12.01; Found C: 58.46, H: 5.80, N: 11.94.
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1-{1-[4-(4-Chlorophenyl)piperazin-1-yl]-1-oxopropan-2-yl}pyrrolidine-2,5-dione
(6).
White solid. Yield: 80%; mp 137–138 ºC; TLC: R = 0.57 (S ); HPLC (purity 100%): t =
f
2
R
+
1
.413 min.; ESI–MS: 350.2 (C17
C: 58.37, H: 5.76, N: 12.01; Found C: 58.22, H: 5.83, N: 12.08.
-{1-[4-(2-Fluorophenyl)piperazin-1-yl]-1-oxopropan-2-yl}pyrrolidine-2,5-dione
White solid. Yield: 77%; mp 122–123 ºC; TLC: R = 0.49 (S ); HPLC (purity 100%): t
.241 min.; ESI–MS: 334.1 (C H FN O [M+H] ). Anal. calcd for C H FN O (333.36):
H
20ClN
3
O
3
[M+H] ). Anal. calcd for C17
H
20ClN
3
O
3
(349.81):
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
(7).
f
2
R
=
+
1
1
7
20
3
3
17 20
3
3
C: 61.25, H: 6.05, N: 12.61; Found C: 61.19, H: 5.98, N: 12.68.
-{1-[4-(3-Fluorophenyl)piperazin-1-yl]-1-oxopropan-2-yl}pyrrolidine-2,5-dione
R
White solid. Yield: 79%; mp 121–122 ºC; TLC: R = 0.52 (S ); HPLC (purity 100%): t =
1
(8).
f
2
+
1
.235 min.; ESI–MS: 334.1 (C17
C: 61.25, H: 6.05, N: 12.61; Found C: 61.20, H: 5.92, N: 12.65.
-{1-[4-(4-Fluorophenyl)piperazin-1-yl]-1-oxopropan-2-yl}pyrrolidine-2,5-dione
H
20FN
3
O
3
[M+H] ). Anal. calcd for C17
H
20FN
3
O
3
(333.36):
1
(9).
White solid. Yield: 81%; mp 83–84 ºC; TLC: R
f
= 0.50 (S
2
); HPLC (purity 100%): t
R
= 1.104
+
min.; ESI–MS: 334.1 (C17
H20FN
3
O
3
[M+H] ). Anal. calcd for C17
H20FN
3
O
3
(333.36): C:
61.25, H: 6.05, N: 12.61; Found C: 61.18, H: 5.99, N: 12.59.
1-(1-Oxo-1-{4-[2-(trifluoromethyl)phenyl]piperazin-1-yl}propan-2-yl)pyrrolidine-2,5-
dione (10). White solid. Yield: 82%; mp 118–119 ºC; TLC: R
100%): t = 1.518 min.; ESI–MS: 384.2 (C18
f
2
= 0.62 (S ); HPLC (purity
+
R
H
20
F
3
N
3
O
3
[M+H] ). Anal. calcd for
C
18
H
20
F
3
N
3
O
3
(383.36): C: 56.39, H: 5.26, N: 10.96; Found C: 56.44, H: 5.29, N: 10.79.
-(1-Oxo-1-{4-[3-(trifluoromethyl)phenyl]piperazin-1-yl}propan-2-yl)pyrrolidine-2,5-
dione (11). White solid. Yield: 80%; mp 135–136 ºC; TLC: R = 0.68 (S ); HPLC (purity
1
f
2
+
1
R 20 3 3 3
00%): t = 1.590 min.; ESI–MS: 384.2 (C18H F N O [M+H] ). Anal. calcd for
C
18
H
20
F
3
N
O
3 3
(383.36): C: 56.39, H: 5.26, N: 10.96; Found C: 56.42, H: 5.33, N: 10.84.
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2
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2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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5
5
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1-(1-Oxo-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}propan-2-yl)pyrrolidine-2,5-
dione (12). White solid. Yield: 80%; mp 147–148 ºC; TLC: R = 0.67 (S ); HPLC (purity
f
2
+
1
00%): t
R
= 1.465 min.; ESI–MS: 384.2 (C18
(383.36): C: 56.39, H: 5.26, N: 10.96; Found C: 56.45, H: 5.27, N: 10.90.
-{1-[4-(3-Methylphenyl)piperazin-1-yl]-1-oxopropan-2-yl}pyrrolidine-2,5-dione
20 3 3 3
H F N O [M+H] ). Anal. calcd for
18 20 3 3 3
C H F N O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
(13).
R
); HPLC (purity 100%): t =
White solid. Yield: 86%; mp 105–106 ºC; TLC: R
f
= 0.55 (S
2
+
0
6
1
.994 min.; ESI–MS: 330.20 (C H N O [M+H] ). Anal. calcd for C H N O (329.39): C:
18
23
3
3
18 23
3
3
5.63, H: 7.04, N: 12.76; Found C: 65.69, H: 7.15, N: 12.88.
-{1-[4-(3-Methoxyphenyl)piperazin-1-yl]-1-oxopropan-2-yl}pyrrolidine-2,5-dione (14).
White solid. Yield: 80%; mp 131–132 ºC; TLC: R
f
= 0.54 (S
2
); HPLC (purity 100%): t
R
=
+
1
6
1
7
.004 min.; ESI–MS: 346.1 (C18
H
23
N
3
O
4
[M+H] ). Anal. calcd for C18
H
23
N
3
O
4
(345.39): C:
2.59, H: 6.71, N: 12.17; Found C: 62.68, H: 6.80, N: 12.18.
-[1-(Morpholin-4-yl)-1-oxopropan-2-yl]pyrrolidine-2,5-dione (15). White solid. Yield:
5%; mp 124–125 ºC; TLC: R
f
= 0.44 (S
1
); HPLC (purity 100%): t
R
= 0.811 min.; ESI–MS:
+
241.1 (C11
H
16
N
2
O
4
[M+H] ). Anal. calcd for C11
H
16
N
2
O
4
(240.26): C: 54.99, H: 6.71, N:
11.66; Found C: 54.88, H: 6.82, N: 11.70.
1-[1-Oxo-1-(4-phenylpiperazin-1-yl)butan-2-yl]pyrrolidine-2,5-dione (16). White solid.
Yield: 77%; mp 116–117 ºC; TLC: R
f
= 0.53 (S
2
R
); HPLC (purity 100%): t = 1.039 min.; ESI–
+
MS: 330.1 (C18H N O
23 3 3
[M+H] ). Anal. calcd for C18
H N O
23 3 3
(329.39): C: 65.63, H: 7.04,
N:12.76; Found C: 65.73, H: 7.08, N: 12.88.
-{1-[4-(2-Chlorophenyl)piperazin-1-yl]-1-oxobutan-2-yl}pyrrolidine-2,5-dione
1
(17).
White solid. Yield: 73%; mp 129–130 ºC; TLC: R = 0.62 (S ); HPLC (purity 100%): t =
f
2
R
+
1.351 min.; ESI–MS: 364.3 (C18
H
22ClN
3
O
3
[M+H] ). Anal. calcd for C18
H
22ClN
3
O
3
(363.84):
C: 59.42, H: 6.09, N: 11.55; Found C: 59.55.88, H: 6.20, N: 11.60.
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1-{1-[4-(3-Chlorophenyl)piperazin-1-yl]-1-oxobutan-2-yl}pyrrolidine-2,5-dione
(18).
White solid. Yield: 78%; mp 108–109 ºC; TLC: R = 0.65 (S ); HPLC (purity 100%): t =
f
2
R
+
1
.545 min.; ESI–MS: 364.3 (C18
C: 59.42, H: 6.09, N: 11.55; Found C: 59.46, H: 6.34, N: 11.77.
-{1-[4-(4-Chlorophenyl)piperazin-1-yl]-1-oxobutan-2-yl}pyrrolidine-2,5-dione
White solid. Yield: 80%; mp 166–167 ºC; TLC: R = 0.63 (S
H
22ClN
3
O
3
[M+H] ). Anal. calcd for C18
H
22ClN
3
O
3
(363.84):
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
(19).
f
2
); HPLC (purity 99.8%): t
R
=
+
1
.415 min.; ESI–MS: 364.4 (C H ClN O [M+H] ). Anal. calcd for C H ClN O (363.84):
18
22
3
3
18 22
3
3
C: 59.42, H: 6.09, N: 11.55; Found C: 59.50, H: 6.29, N: 11.60.
-{1-[4-(2-Fluorophenyl)piperazin-1-yl]-1-oxobutan-2-yl}pyrrolidine-2,5-dione
R
White solid. Yield: 77%; mp 128ꢀ129 ºC; TLC: R = 0.58 (S ); HPLC (purity 100%): t =
1
(20).
f
2
+
1
.311 min.; ESI–MS: 348.1 (C18
C: 62.23, H: 6.38, N: 12.10; Found C: 62.19, H: 6.50, N: 12.11.
-{1-[4-(3-Fluorophenyl)piperazin-1-yl]-1-oxobutan-2-yl}pyrrolidine-2,5-dione
2 R
White solid. Yield: 79%; mp 147–148 ºC; TLC: R = 0.57 (S ); HPLC (purity 100%): t =
H
22FN
3
O
3
[M+H] ). Anal. calcd for C18
H
22FN
3
O
3
(347.38):
1
(21).
f
+
1.277 min.; ESI–MS: 348.1 (C18
C: 62.23, H: 6.38, N: 12.10; Found C: 62.28, H: 6.54, N: 12.16.
-{1-[4-(4-Fluorophenyl)piperazin-1-yl]-1-oxobutan-2-yl}pyrrolidine-2,5-dione
White solid. Yield: 81%; mp 154–155 ºC; TLC: R = 0.59 (S
H
22FN
3
O
3
[M+H] ). Anal. calcd for C18
H
22FN
3
O
3
(347.38):
1
(22).
f
2
); HPLC (purity 99.7%): t
[M+H] ). Anal. calcd for C18 (347.38):
C: 62.23, H: 6.38, N: 12.10; Found C: 62.11, H: 6.48, N: 12.04.
-(1-Oxo-1-{4-[2-(trifluoromethyl)phenyl]piperazin-1-yl}butan-2-yl)pyrrolidine-2,5-
dione (23). White solid. Yield: 82%; mp 148–149 ºC; TLC: R = 0.72 (S ); HPLC (purity
R
=
+
1.209 min.; ESI–MS: 348.1 (C18
H
22FN
3
O
3
3 3
H22FN O
1
f
2
+
1
R 22 3 3 3
00%): t = 1.589 min.; ESI–MS: 398.1 (C19H F N O [M+H] ). Anal. calcd for
C
19
H
22
F
3
N
3
O (397.39): C: 57.43, H: 5.58, N: 10.57; Found C: 57.30, H: 5.59, N: 10.79.
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1-(1-Oxo-1-{4-[3-(trifluoromethyl)phenyl]piperazin-1-yl}butan-2-yl)pyrrolidine-2,5-
dione (24). White solid. Yield: 80%; mp 128–129 ºC; TLC: R = 0.76 (S ); HPLC (purity
f
2
+
1
00%): t
R
= 1.646 min.; ESI–MS: 398.1 (C19
O (397.39): C: 57.43, H: 5.58, N: 10.57; Found C: 57.45, H: 5.68, N: 10.65.
-(1-Oxo-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butan-2-yl)pyrrolidine-2,5-
22 3 3 3
H F N O [M+H] ). Anal. calcd for
19 22 3 3
C H F N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
dione (25). White solid. Yield: 85%; mp 148–149 ºC; TLC: R
f
2
= 0.77 (S ); HPLC (purity
+
1
00%): tR = 1.537 min.; ESI–MS: 398.1 (C H F N O [M+H] ). Anal. calcd for
19
22
3
3
3
C
19 22 3 3
H F N O (397.39): C: 57.43, H: 5.58, N: 10.57; Found C: 57.50, H: 5.70, N: 10.60.
1-{1-[4-(3-Methylphenyl)piperazin-1-yl]-1-oxobutan-2-yl}pyrrolidine-2,5-dione
(26).
White solid. Yield: 75%; mp 95–96 ºC; TLC: R
f
= 0.60 (S
2
); HPLC (purity 99.8%): t
R
= 1.148
+
min.; ESI–MS: 344.3 (C19
H
25
N
3
O
3
[M+H] ). Anal. calcd for C19
H
25
N
3
O
3
(343.42): C: 66.45,
H: 7.34, N: 12.24; Found C: 66.35, H: 7.50, N: 12.30.
-{1-[4-(3-Methoxyphenyl)piperazin-1-yl]-1-oxobutan-2-yl}pyrrolidine-2,5-dione
1
(27).
); HPLC (purity 100%): t
(359.42): C:
White solid. Yield: 80%; mp 144–145 ºC; TLC: R
f
= 0.58 (S
2
R
=
+
1.109 min.; ESI–MS: 360.3 (C19
25
H N
3
O
4
[M+H] ). Anal. calcd for C19
H
25
N
3
O
4
6
1
7
3.49, H: 7.01, N: 11.69; Found C: 63.52, H: 7.20, N: 11.75.
-[1-(Morpholin-4-yl)-1-oxobutan-2-yl]pyrrolidine-2,5-dione (28). White solid. Yield:
5%; mp 117–118 ºC; TLC: R
f
= 0.56 (S
1
); HPLC (purity 100%): t
R
= 0.923 min.; ESI–MS:
+
255.1 (C12
H
18
N
2
O
4
[M+H] ). Anal. calcd for C12
H
18
N
2
O
4
(254.28): C: 56.68, H: 7.13, N:
11.02; Found C: 56.72, H: 7.25, N: 11.07.
Synthesis of 2-chloro-3-methoxy-1-{4-[3-(trifluoromethyl)phenyl]piperazin-1-
yl}propan-1-one (29). Carbonyldiimidazole (0.97 g, 0.006 mol) in 5 mL of dry THF was
added to a solution of 2ꢀchloroꢀ3ꢀmethoxypropanoic acid (0.83 g, 006 mol) dissolved in 10
mL of anhydrous THF while stirring. After the end of gaseous (carbon dioxide) evolution
(approx. 0.5 h), 1–[3 (trifluoromethyl)phenyl]piperazine (1.38 g, 0.006 mol) dissolved in 5
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mL of anhydrous THF was added dropwise. The mixture was stirred at room temperature
(
approx. 24 h), and evaporated to dryness. The crude product was purified by column
chromatography (dichloromethane : methanol, 9 : 0.5, v/v). After concentration of organic
solvents under reduced pressure compound 29 was obtained as light oil. Yield: 66%; TLC: R
f
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
=
0.77 (S
2
); HPLC(purity 98.5%): t
R
= 1.688 min.; ESI–MS: 351.3 (C15
3 2 2
H18ClF N O
+
[M+H] ). Anal. calcd for C15
H18ClF
3
N
2
O
2
(350.76): C: 51.36, H: 5.17, N: 7.99; Found C:
5
1.50, H: 5.20, N: 8.05.
-{1-[4-(3-Trifluoromethylphenyl)piperazin-1-yl]-3-methoxy-1-oxopropan-2-
yl}pyrrolidine-2,5-dione (30). A mixture of pyrrolidineꢀ2,5ꢀdione (0.0075 mol), 2ꢀchloroꢀ3ꢀ
methoxyꢀ1ꢀ{4ꢀ[3ꢀ(trifluoromethyl)phenyl]piperazinꢀ1ꢀyl}propanꢀ1ꢀone (29), anhydrous
1
potassium carbonate (0.0375 mol) and potassium iodide (0.2 g) in 15 mL of acetone was
stirred at 60 °C for approx. 24 h. Then, the inorganic solid was filtered off and acetone was
evaporated to dryness. The oily residue obtained was purified by column chromatography
using a dichloromethane : methanol, 9 : 0.7 (v/v) mixture as a solvent system. Compound 30
was isolated as white solid after concentration of organic solvents under reduced pressure.
f 1 R
Yield: 84%; mp 164–166 ºC; TLC: R = 0.80 (S ); HPLC (purity 100%): t = 1.576 min.; ESI–
+
MS: 414.3 (C H F N O [M+H] ). Anal. calcd for C H F N O (413.39): C: 55.20, H:
1
9
22
3
3
4
19 22
3
3
4
5.36, N: 10.16; Found C: 55.25, H: 5.20, N: 10.30.
ASSOCIATED CONTENT
Supporting Information Available: Molecular Formula Strings Spreadsheet (CSV), H
1
1
3
19
NMR, C NMR, and F NMR spectra description, animal procedures, in vitro and in silico
studies reports are available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author: *K.K.: phone, +48 12 620 54 59; fax, +48 12 620 54 58; eꢀmail,
k.kaminski@uj.edu.pl.
Author Contributions: The manuscript includes contributions from all authors. All authors
have approved the final version of the manuscript.
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Notes: The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The studies were supported by the Polish National Scientific Centre grant DEC–
2012/05/D/NZ7/02328.
ABBREVIATIONS USED
ADMEꢀTox,
absorption,
distribution,
metabolism,
excretion,
toxicity;
CDI,
carbonyldiimidazole; DX, doxorubicin; FDA, Food and Drug Administration; HLM, human
liver microsomes; MES, maximal electroshock seizure test; PI, protective index (TD50/ED50);
scPTZ, subcutaneous pentylenetetrazole seizure test; 6 Hz, sixꢀhertz test; TPE, time of peak
effect.
REFERENCES
(1)
Nadkarni, S.; LaJoie, J.; Devinsky, O. Current treatments of epilepsy. Neurology 2005,
4, S2ꢀS11.
6
(
2)
3)
http://www.who.int/mediacentre/factsheets/fs999/en/ (WHO, Epilepsy).
(
Cloyd, J.; Hauser, W.; Towne, A.; Ramsay, R.; Mattson, R.; Gilliam, F.; Walczak, T.
Epidemiological and medical aspects of epilepsy in the elderly. Epilepsy Res. 2006,
68, S39ꢀ48.
ACS Paragon Plus Environment

Page 25 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(4)
Craig, I.; Tallis, R. General practice management of adult–onset epilepsy analyzed.
Care Elderly 1991, 3, 69ꢀ72.
(5)
(6)
(7)
(8)
Schmidt, D.; Löscher, W. Drug resistance in epilepsy: putative neurobiologic and
clinical mechanisms. Epilepsia 2005, 46, 858ꢀ877.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Perucca, E.; French, J.; Bialer, M. Development of new antiepileptic drugs:
challenges, incentives, and recent advances. Lancet Neurol. 2007, 6, 793ꢀ804.
Cramer, J.A.; Mintzer, S.; Wheless, J.; Mattson, R. H. Adverse effects of antiepileptic
drugs: a brief overview of important issues. Expert Rev. Neurother. 2010, 10, 885ꢀ891.
Pipeline Insight: Epilepsy © Datamonitor (Published 10/2006).
(9)
Morphy, R.; Rankovic, Z. Designed multiple ligands. An emerging drug discovery
paradigm. J. Med. Chem. 2005, 48, 6523ꢀ6543.
(10) Khan, H. N.; Kulsoom, S.; Rashid, H. Ligand based pharmacophore model
development for the identification of novel antiepileptic compounds. Epilepsy Res.
2012, 98, 62ꢀ71.
(11) Simonato, M.; BrooksꢀKayal, A. R.; Engel, J.; Galanopoulou, A. S.; Jensen, F. E.;
Moshé, S. L.; O'Brien, T. J.; Pitkanen, A.; Wilcox, K. S.; French, J. A. The challenge
and promise of antiꢀepileptic therapy development in animal models. Lancet Neurol.
2014, 13, 949ꢀ960.
(12) Rogawski, M.A. Molecular targets versus models for new antiepileptic drug discovery.
Epilepsy Res. 2006, 68, 22ꢀ28.
(13) Kamiński, K.; Obniska, J.; Chlebek, I.; Wiklik, B.; Rzepka, S. Design, synthesis and
anticonvulsant properties of new NꢀMannich bases derived from 3ꢀphenylpyrrolidineꢀ
2,5ꢀdiones. Bioorg. Med. Chem. 2013, 21, 6821ꢀ6830.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 50
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(14) Kamiński, K.; Obniska, J.; Chlebek, I.; Liana, P.; Pękala, E. Synthesis and biological
properties of new NꢀMannich bases derived from 3ꢀmethylꢀ3ꢀphenylꢀ and 3,3ꢀ
dimethylꢀsuccinimides. Part V. Eur. J. Med. Chem. 2013, 66, 12ꢀ21.
(
15) Obniska, J.; Rzepka, S.; Kamiński, K. Synthesis and anticonvulsant activity of new Nꢀ
Mannich bases derived from 3ꢀ(2ꢀfluorophenyl)ꢀ and 3ꢀ(2ꢀbromophenyl)ꢀpyrrolidineꢀ
2,5ꢀdiones. Part II. Bioorg. Med. Chem. 2012, 20, 4872ꢀ4780.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
16) Kamiński, K.; Obniska, J.; Wiklik, B.; Atamanyuk D. Synthesis and anticonvulsant
properties of new acetamide derivatives of phthalimide, and its saturated cyclohexane
and norbornene analogs. Eur. J. Med. Chem. 2011, 46, 4634ꢀ4641.
(17) Obniska, J.; Byrtus, H.; Kamiński, K.; Pawłowski, M.; Szczesio, M.; Karolak–
Wojciechowska, J. Design, synthesis, and anticonvulsant activity of new NꢀMannich
bases derived from spirosuccinimides and spirohydantoins. Bioorg. Med. Chem. 2010,
18, 6134ꢀ6142.
(
18) Kamiński, K.; Rzepka, S.; Obniska, J. Synthesis and anticonvulsant activity of new 1ꢀ
[2ꢀoxoꢀ2ꢀ(4ꢀphenylpiperazinꢀ1ꢀyl)ethyl]pyrrolidineꢀ2,5ꢀdiones. Bioorg. Med. Chem.
Lett. 2011, 21, 5800ꢀ5003.
(19) Choi, D.; Stables, J. P.; Kohn, H. Synthesis and anticonvulsant activities of Nꢀbenzylꢀ
2ꢀacetamidopropionamide derivatives. J. Med. Chem. 1996, 39, 1907ꢀ1916.
(20) Stoehr, T.; Kupferberg, H. J.; Stables, J. P.; Choi, D.; Harris, R. H.; Kohn, H.; Walton,
N.; White, H. S. Lacosamide, a novel anticonvulsant drug, shows efficacy with a wide
safety margin in rodent models for epilepsy. Epilepsy Res. 2007, 74, 147ꢀ154.
(
21) Morieux, P.; Stables, J.P.; Kohn, H. Synthesis and anticonvulsant activities of Nꢀ
benzyl (2R)ꢀ2ꢀacetamidoꢀ3ꢀoxysubstituted propionamide derivatives. Bioorg. Med.
Chem. 2008, 16, 8968ꢀ8975.
ACS Paragon Plus Environment

Page 27 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(22) Salomé, C.; SaloméꢀGrosjean, E.; Park, K. D.; Morieux, P.; Swendiman, R.; DeMarco,
E.; Stables, J. P.; Kohn, H. Synthesis and anticonvulsant activities of (R)ꢀNꢀ(4’ꢀ
substituted)benzyl 2ꢀacetamidoꢀ3ꢀmethoxypropionamides. J. Med. Chem. 2010, 53,
1288ꢀ1305.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(
23) King, A. M.; Salomé, C.; SaloméꢀGrosjean, E.; De Ryck, M.; Kaminski, R.; Valade,
A.; Stables, J. P.; Kohn, H. Primary amino acid derivatives: compounds with
anticonvulsant and neuropathic pain protection activities. J. Med. Chem. 2011, 54,
4815ꢀ4830.
(
24) Lattanzi, S.; Cagnetti, C.; Foschi, N.; Provinciali, L.; Silvestrini, M. Lacosamide
monotherapy for partial onset seizures. Seizure 2015, 27, 71ꢀ74.
(25) http://www.ucb.com/ourꢀproducts/productꢀlist/cns/vimpat
(
26) CastelꢀBranco, M. M.; Alves, G. L.; Figueiredo, I. V.; Falcão, A. C.; Caramona, M.
M. The maximal electroshock seizure (MES) model in the preclinical assessment of
potential new antiepileptic drugs. Methods Find. Exp. Clin. Pharmacol. 2009, 31, 101ꢀ
106.
(
(
(
(
27) Anderson, G. W., Paul, R. N,N’ꢀcarbonyldiimidazole, a new reagent for peptide
synthesis, J. Am. Chem. Soc. 1958, 80, 4423.
28) Paul, R.; Anderson, G. W. N,N'ꢀcarbonyldiimidazole, a new peptide forming reagent.
J. Am. Chem. Soc. 1960, 82, 4596ꢀ4600.
29) Löscher, W. Critical review of current animal models of seizures and epilepsy used in
the discovery and development of new antiepileptic drugs. Seizure 2011, 20, 359ꢀ368.
30) Rogawski, M.A.; Löscher, W. The neurobiology of antiepileptic drugs. Nat. Rev.
Neurosci. 2004, 5, 553ꢀ564.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 50
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(31) Barton, M. E.; Klein, B. D.; Wolf, H. H.; White, H. S. Pharmacological
characterization of the 6 Hz psychomotor seizure model of partial epilepsy. Epilepsy
Res. 2001, 47, 217ꢀ227.
(
32) Kamiński, K.; Rapacz, A.; Łuszczki, J. J.; Latacz, G.; Obniska, J.; KiećꢀKononowicz,
K.; Filipek, B. Design, synthesis and biological evaluation of new hybrid
anticonvulsants derived from Nꢀbenzylꢀ2ꢀ(2,5ꢀdioxopyrrolidinꢀ1ꢀyl)propanamide and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
ꢀ(2,5ꢀdioxopyrrolidinꢀ1ꢀyl)butanamide derivatives. Bioorg. Med. Chem. 2015, 23,
548ꢀ2561.
(
33) Meldrum, B. S.; Rogawski, M. A. Molecular targets for antiepileptic drug
development. Neurotherapeutics 2007, 4, 18ꢀ61.
(
34) Liu, G.; YarovꢀYarovoy, V.; Nobbs, M.; Clare, J .J.; Scheuer, T.; Catterall, W. A.
Differential interactions of lamotrigine and related drugs with transmembrane segment
IVS6 of voltageꢀgated sodium channels. Neuropharmacology 2003, 44, 413ꢀ422.
35) Yogeeswari, P.; Ragavendran, J. V.; Thirumurugan, R.; Saxena, A.; Sriram, D. Ion
channels as important targets for antiepileptic drug design. Curr. Drug Targets 2004,
(
(
(
(
5
, 589ꢀ602.
36) Rogawski, M. A.; Tofighy, A.; White, H. S.; Matagne, A.; Wolff, C. Current
understanding of the mechanism of action of the antiepileptic drug lacosamide.
Epilepsy Res. 2015, 110, 189ꢀ205.
37) Willow, M.; Catterall, W.A. Inhibition of binding of [3H]batrachotoxinin A 20ꢀalphaꢀ
benzoate to sodium channels by the anticonvulsant drugs diphenylhydantoin and
carbamazepine. Mol. Pharmacol. 1982, 22, 627ꢀ635.
38) Willow, M.; Kuenzel, E. A.; Catterall, W. A. Inhibition of voltageꢀsensitive sodium
channels in neuroblastoma cells and synaptosomes by the anticonvulsant drugs
diphenylhydantoin and carbamazepine. Mol. Pharmacol. 1984, 25, 228ꢀ234.
ACS Paragon Plus Environment

Page 29 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(39) Willow, M.; Gonoi, T.; Catterall, W.A. Voltage clamp analysis of the inhibitory
actions of diphenylhydantoin and carbamazepine on voltageꢀsensitive sodium channels
in neuroblastoma cells. Mol. Pharmacol. 1985, 27, 549ꢀ558.
(
40) Matsuki, N.; Quandt, F. N.; Ten Eick, R. E.; Yeh, J. Z. Characterization of the block
of sodium channels by phenytoin in mouse neuroblastoma cells. J. Pharmacol. Exp.
Ther. 1984, 228, 523ꢀ530.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(
41) Brown, G. B. 3HꢀbatrachotoxininꢀA benzoate binding to voltageꢀsensitive sodium
channels: inhibition by the channel blockers tetrodotoxin and saxitoxin. J. Neurosci.
1986, 6, 2064ꢀ2070.
(42) Shank, R. P.; Gardocki, J. F.; Streeter, A. J.; Maryanoff, B. E. An overview of the
preclinical aspects of Topiramate: pharmacology, pharmacokinetics, and mechanism
of action. Epilepsia 2000, 41, S3ꢀS9.
(
43) Russo, E.; Constanti, A.; Ferreri, G.; Citraro, R.; De Sarro, G. Nifedipine affects the
anticonvulsant activity of Topiramate in various animal models of epilepsy.
Neuropharmacology 2004, 46, 865ꢀ878.
(
44) Wang, J.; Urban, L. The impact of early ADME profiling on drug discovery and
development. Drug Disc. World 2004, 5, 73–86.
(
45) Kerns, E. H.; Di L. Drug-like properties: concepts, structure design and methods from
ADME to toxicity optimization, Academic Press, Elsevier Inc.: Burlington, MA, 2008;
pp 6ꢀ17.
(
46) Cruciani, G.; Carosati, E.; De Boeck, B.; Ethirajulu, K.; Mackie, C.; Howe, T.;
Vianello, R. MetaSite: understanding metabolism in human cytochromes from the
perspective of the chemist. J. Med. Chem. 2005, 48, 6970-6979.
(47) USFDA Draft Guidance for Industry: Drug Interaction Studies-Study Design, Data
Analysis, and Implications for Dosing and Labeling; Department of Health and
ACS Paragon Plus Environment