Synthesis and anticonvulsant activity of new N-phenyl-2-(4-phenylpiperazin-1-yl)acetamide derivatives

Article abstract of DOI:10.1007/s00044-015-1360-6

Twenty-two new N-phenyl-2-(4-phenylpiperazin-1-yl)acetamide derivatives have been synthesized and evaluated for their anticonvulsant activity in animal models of epilepsy. These molecules have been designed as analogs of previously obtained anticonvulsant

Full text of DOI:10.1007/s00044-015-1360-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-015-1360-6
ORIGINAL RESEARCH
Synthesis and anticonvulsant activity of new N-phenyl-2-
(4-phenylpiperazin-1-yl)acetamide derivatives
1
Krzysztof Kaminski Beata Wiklik1 Jolanta Obniska1
•
•
´
Received: 21 October 2014 / Accepted: 2 March 2015
Ó The Author(s) 2015. This article is published with open access at Springerlink.com
Abstract Twenty-two new N-phenyl-2-(4-phenylpiper-
azin-1-yl)acetamide derivatives have been synthesized and
evaluated for their anticonvulsant activity in animal models
of epilepsy. These molecules have been designed as analogs
of previously obtained anticonvulsant active pyrrolidine-
2,5-diones in which heterocyclic imide ring has been chan-
ged into chain amide bound. The final compounds were
synthesized in the alkylation reaction of the corresponding
amines with the previously obtained alkylating reagents
2-chloro-1-(3-chlorophenyl)ethanone (1) or 2-chloro-1-[3-
(trifluoromethyl)phenyl]ethanone (2). Initial anticonvulsant
screening was performed using standard maximal elec-
troshock (MES) and subcutaneous pentylenetetrazole
screens in mice and/or rats. Several compounds were tested
additionally in the psychomotor seizures (6-Hz model). The
acute neurological toxicity was determined applying the
rotarod test. The results of pharmacological studies showed
activity exclusively in the MES seizures especially for
3-(trifluoromethyl)anilide derivatives, whereas majority of
3-chloroanilide analogs were inactive. It should be empha-
size that several molecules showed also activity in the 6-Hz
screen which is an animal model of human partial and ther-
apy-resistant epilepsy. In the in vitro studies, the most potent
derivative 20 was observed as moderate binder to the neu-
ronal voltage-sensitive sodium channels (site 2). The SAR
studies for anticonvulsant activity confirmed the crucial role
of pyrrolidine-2,5-dione core fragment for anticonvulsant
activity.
Keywords Phenylacetamides Á Anticonvulsant activity Á
SAR studies Á In vivo studies Á In vitro studies Á
Epilepsy
Introduction
Epilepsy is one of the most prevalent neurological disor-
ders, affecting approximately 0.5–1 % people worldwide.
It has a chronic and progressive nature, characterized by
recurring seizures of various manifestations. The basic
method of the treatment of epilepsy is pharmacotherapy. It
is a symptomatic treatment which allows abolishing or
reducing the number of seizure episodes, however, does not
inhibit the pathophysiological processes of epileptogenesis
and does not eliminate organic changes in the central
nervous system (CNS), which are the underlying causes of
the disease. In about 25 % of patients, the desired results
cannot be achieved with the use of pharmacotherapy, this
form of disease is known as refractory epilepsy, the treat-
ment of which is based on surgical vagus nerve stimulation
(WHO, 2010; Bell and Sander, 2002; Chang and Lowen-
stein, 2003). The variety of molecular targets, not always
precisely definite, makes it difficult to establish an
unequivocal mechanism of action of anti-epileptic drugs
(AEDs). This fact is also reflected in the limited possi-
bilities of designing new drugs on the base of the structure
of a given biological target. Therefore, currently, there are
two different methods of search for new anticonvulsants:
modifications of clinically effective AEDs or synthesis of
entirely new structures (Khan et al., 2012). Taking into
consideration the above limitations, many authors con-
ducted attempts to identify the structural features crucial
for anticonvulsant activity. On the basis of these
´
& Krzysztof Kaminski
k.kaminski@uj.edu.pl
1
Department of Medicinal Chemistry, Faculty of Pharmacy,
Jagiellonian University Medical College, 9 Medyczna Street,
´
30-688 Krakow, Poland
123

Med Chem Res
researches, several pharmacophoric models, enabling a
more rational design of new anticonvulsants, have been
described. Thus, one of the important core fragments of
anticonvulsants is defined by nitrogen heterocyclic system,
usually imide or lactam and phenyl or alkyl groups at-
tached to the heterocyclic system (Wong et al., 1986;
Bruno-Blanch et al., 2003; Malawska, 2005). This common
template is present in the structures of old, however well-
established AEDs, such as ethosuximide and phenytoin as
well as among the newest drugs, e.g., levetiracetam, bri-
varacetam or seletracetam (Rogawski and Porter, 1990;
Bialer et al., 2007) (Fig. 1).
epilepsy (Obniska et al., 2010). The main modification
relied on the change of imide ring into chain amide bound
(Fig. 4). Therefore, the aim of the current studies was to
elucidate how the structure of imide/amide fragment
affects anticonvulsant activity.
Results and discussion
Chemistry
The synthesis of compounds 3–24 was accomplished as
shown in Scheme 1. The starting materials 2-chloro-1-(3-
Bearing in mind the aforementioned, in our laboratory,
since many years we have been conducting studies in the
group of pyrrolidine-2,5-dione derivatives differently sub-
stituted at the imide nitrogen atom as well as at the 3-po-
sition of the heterocyclic ring as candidates on new AEDs.
Many of these compounds were highly effective in the
chlorophenyl)ethanone
(1)
and
2-chloro-1-[3-(tri-
fluoromethyl)phenyl]ethanone (2) were prepared by acy-
lation of 3-chloroaniline or 3-trifluoromethylaniline with
2-chloroacetyl chloride. The reaction was carried out at
0 °C for 3 h in the mixture consisted of dichloromethane
(DCM) and 2 % aqueous sodium hydroxide solution. The
final compounds 3–24 were synthesized in the alkylation
reaction of the corresponding amines with the previously
obtained alkylating reagents 1 and 2. The reaction was
carried out at a temperature of 60 °C in a biphasic liquid–
solid system consisting of dry acetone, potassium carbon-
ate and catalytic amount of potassium iodide. The progress
of the reaction was monitored using HPLC chromatogra-
phy. Compounds 3–24 were obtained in yields ranging
from 44 to 78 %. Their purities were assessed by HPLC
chromatography (the HPLC chromatogram of 22 is shown
in Fig. 5). The final compounds were fully characterized by
´
animal models of epilepsy (Kaminski et al., 2014, 2013,
2011a, b; Obniska et al., 2013, 2010; Obniska and
´
Zagorska, 2003). Structures and designated median effec-
tive doses (ED50) in the maximal electroshock (MES) sei-
zure test for the model pyrrolidine-2,5-diones obtained in
former research (Obniska et al., 2013) are shown in Fig. 2.
Another important structural fragment of many anti-
convulsants as well as compounds being currently in
clinical trials is an amide moiety presented in a chain form
(Bruno-Blanch et al., 2003; Hadjipavlou-Litina, 1998;
Luszczki, 2009; Hen et al., 2010; Morieux et al., 2008;
Ghidini et al., 2006; Beguin et al., 2003; Masereel et al.,
2002). It should be stressed that this chemical structure
possessed several the newest AEDs such as lacosamide
(chemically the functionalized amino acid) or valrocemide
and valnoctamide which are the unteratogenic derivatives
of valproic acid (Fig. 3).
1
elemental analyses (C, H, N) and H NMR, 13C NMR, 19F
NMR, LC/MS spectra. The detailed physicochemical and
analytical data are listed in the experimental section.
The 1H NMR spectrum of compound 3 displayed several
proton chemical shifts consistent with the proposed struc-
ture. The eight protons of piperazine moiety were observed
as broad multiplet (d 3.06–3.93 ppm). The methylene
linker at the acetamide fragment occurred as singlet at d
4.33 ppm. The aromatic protons were observed at the ex-
pected chemical shifts as six multiplets within the range of
d 6.81–7.86 ppm. The amide (NH) proton appeared as
Taking into consideration the above findings in the
current studies, we have focused on synthesis of new
N-phenylacetamide derivatives as targets for new AEDs.
These compounds were designed as analogs of corre-
sponding pyrrolidine-2,5-dine derivatives, which showed
high anticonvulsant activity in the animal models of
Fig. 1 Structures of known AEDs containing nitrogen heterocyclic ring
123

Med Chem Res
Fig. 2 Structures of model
pyrrolidine-2,5-diones obtained
in the previous research
Fig. 3 Structures of known
AEDs containing chain amide
bound
Fig. 4 The exchange of the
pyrrolidine-2,5-dione core
fragment for chain amide bound
broad singlet at d 10.86 ppm. The proton of protonated
piperazine fragment (?NH) was observed as broad singlet
at d 11.55 ppm. The 13C NMR spectrum of 3 showed four
peaks at d 51.3, 51.9, 52.5 and 53.3 ppm which correspond
to carbon atoms of piperazine ring. The carbon atom of the
methylene linker at the acetamide fragment occurred as
peak at d 54.1 ppm. The peaks of carbon atoms of the
aromatic rings were detected at the expected chemical
shifts within the range d 111.7–146.3 ppm. The peak at
172.1 ppm was ascribed to a carbon in a carbonyl group.
singlet at d 10.05 ppm. The proton of protonated mor-
pholine fragment (?NH) was observed as broad singlet at d
11.41 ppm. The 13C NMR spectrum of 13 showed five
peaks at d 51.8, 52.6, 53.2, 67.8 and 68.6 ppm which
correspond to carbon atoms of morpholine ring and the
methylene linker at the acetamide fragment. The aromatic
carbons in the benzene ring were observed as six peaks
within the range d 117.8–138.1 ppm. The peak at
171.5 ppm was ascribed to a carbon in a carbonyl group.
1
The H NMR spectrum of 13 revealed two multiplets
Anticonvulsant activity
within the range of d 1.59–1.88 and 2.96–3.56 ppm which
were ascribed to eight protons of the morpholine ring. The
methylene linker at the acetamide fragment occurred as
singlet at d 4.15 ppm. Four aromatic protons gave three
multiplets and on triplet within the range of d
7.13–7.86 ppm. The amide (NH) proton appeared as broad
The preclinical development of new chemical agents for
the treatment of epilepsy is based on the use of predictable
animal seizure models, which correspond to different types
of human epilepsies. Despite the diversity of models that
123

Med Chem Res
Scheme 1 Synthetic pathways of intermediates 1, 2 and target compounds 3–24
could potentially be used to screen for anticonvulsant ac-
tivity, the MES model and the subcutaneous pentylenete-
trazole (scPTZ) model remain the ‘‘gold standards’’ in the
early stages of testing. The MES test is the mechanism-
independent animal seizure model which enables identifi-
cation of compounds preventing seizure spread. This test
uses an electrical stimulus to produce generalized tonic–
clonic seizures and thus is thought to be an experimental
model of tonic–clonic epilepsy and of partial convulsions
with or without secondary generalization in humans. The
scPTZ test employs chemically induced myoclonic sei-
zures and is proposed to identify the agents raising the
seizure threshold. This test is related to human generalized
absence seizures (Rogawski, 2006).
Considering the aforementioned facts, the profile of
anticonvulsant activity of final compounds 3–24 was
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Med Chem Res
Fig. 5 The HPLC chromatogram of compound 22
established in the MES and scPTZ tests, after intraperi-
toneal (i.p.) injection in mice at doses of 30, 100 and
300 mg/kg. An observation was carried out at two different
time intervals—0.5 and 4 h. Furthermore, in addition to the
primary anticonvulsant screening, the acute neurological
toxicity (NT) was determined in mice by the rotarod test.
The results of the preliminary pharmacological studies are
summarized in Table 1.
action. The anti-MES activity at dose of 100 mg/kg
showed also morpholine derivative 24 (0.5 h). The differ-
ences in times points in which the anticonvulsant protec-
tion was observed for compounds containing morpholine
(24) or phenylpiperazine moieties (14, 16, 18, 20) may
result from the lipophilic properties of these molecules. As
it is presented in Table 1, the more lipophilic molecules
(higher clog P values)—14, 16, 18, 20 showed activity
mainly at 4 h, whereas less lipophilic compound 24 was
effective only at time point of 0.5 h. These observations
may be connected with higher affinity of more lipophilic
compounds to the peripheral tissues that causes slower
distribution to the CNS. The further SAR analysis indicated
that exchange of phenylpiperazine moiety into ben-
zylpiperazine (22) or hydroxyethylpiperazine (23) de-
creased anticonvulsant activity. The screening data
revealed that all active compounds showed weaker anti-
MES protection than phenytoin that is still recognized as
model AED active in MES test. Furthermore, the anti-
convulsant protection was distinctly lower in comparison
with respective succinimide analogs described in advance
(Obniska et al., 2013). This observation confirms the role
of pyrrolidine-2,5-dione ring as a pharmacophore crucial
for anticonvulsant activity.
The compounds tested showed protection exclusively in
the MES seizures. Furthermore, in vivo data revealed that
anticonvulsant activity was closely connected with the type
of substituent at the 3-position of the anilide moiety. Ex-
cept for N-(3-chlorophenyl)-2-(4-methylpiperazin-1-yl)ac-
etamide (12)—effective in the MES test at 0.5 h (dose of
100 mg/kg) and N-(3-chlorophenyl)-2-morpholino-ac-
etamide (13) which showed protection in both time points
(100 mg/kg at 0.5 h, and 300 mg/kg at 4 h), all other
3-chloroanilides (3–11) were devoid of anticonvulsant ac-
tivity. As it can be seen, considerably higher anticonvulsant
protection was observed for 3-(trifluoromethyl)anilides
(14–24). Among these derivatives, all compounds, except
for those containing 4-(2-chlorophenyl)piperazine (15),
4-(2,3-dichlorophenyl)piperazine (17) and 4-pyrimidin-2-
yl-piperazine (21), revealed protection in the MES test at
dose of 100 mg/kg and/or 300 mg/kg in different pre-
treatment times. The highest anticonvulsant activity was
observed for 19 which protected animals in both time in-
tervals (300 mg/kg at 0.5 h, and 100 mg/kg at 4 h). Other
active compounds with phenylpiperazine fragment as
amine function (14, 16, 18, 20) revealed protection in the
MES seizures only 4 h after i.p. administration that means
delayed onset, however, long in duration anticonvulsant
The pharmacological results obtained in the current
studies proved that introduction of the fluorine atom or
trifluoromethyl group was essential for anticonvulsant ac-
tivity in this series of compounds. Furthermore, it is widely
accepted that incorporation of fluorine into biologically
active molecules provides analogs with increased
metabolic stability due to strength of C–F bond and further
reduction of cytochrome P450 oxidative metabolism.
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Med Chem Res
Table 1 Anticonvulsant and neurotoxicity screening after i.p. administration in mice (3–24)
No.
R1
R2
X
R3
Intraperitoneal administration in micea
clog Pe
MESb
0.5 h
scPTZc
NTd
4 h
0.5 h
4 h
0.5 h
4 h
3
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
–
–
–
–
–
–
–
N
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
3.15
3.71
3.71
4.26
3.31
3.31
4.07
1.72
4
2–Cl
4–Cl
2,3–Cl
2–F
4–F
3–CF3
–
5
6
7
8
9
N
N
10
11
Cl
–
N
–
–
–
–
300
–
2.80
12
13
14
15
16
17
18
19
20
21
Cl
–
N
O
–
–CH3
100
100
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
1.07
0.92
3.51
4.07
4.07
4.63
3.67
3.67
4.43
2.09
Cl
–
–
–
–
–
–
–
–
300
100
–
300
300Z
–
–
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
H
300
–
2–Cl
4–Cl
2,3–Cl
2–F
4–F
3–CF3
–
–
–
–
–
100
–
–
–
–
–
–
–
–
–
100
100
100
–
–
–
–
300
–
300
–
–
–
–
–
N
N
–
300
–
N
22
CF3
–
N
300
–
–
–
30033
–
3.17
23
CF3
CF3
–
–
N
O
–(CH2)2–OH
–
300
100
30
300
–
–
–
–
–
–
–
300
100
100
–
0.92
1.28
24
Phenytoinf
–
30
100
Z 33
Response comments: respiratory depression, tremors
a
Doses of 30, 100 or 300 mg/kg were administered intraperitoneally. The data indicate the minimum dose effective or neurotoxic in half or
more animals tested. A dash indicates the absence of anticonvulsant activity or neurotoxicity at the maximum dose administered
b
Maximal electroshock test
c
Subcutaneous pentylenetetrazole test
d
Neurotoxicity screening using rotarod test
e
clog P values calculated using a log P module of ChemDraw Ultra program, version 7.0.1 (Cambridge Soft Corporation, Cambridge, MA,
USA)
f
Phenytoin, reference antiepileptic drug, tested by use of ADD Program procedures in NIH/NINDS
Moreover, the unique features of fluorine, such as
relatively small size, combined with a very high elec-
tronegativity can specifically modulate the steric and
electronic properties of the fluorinated derivatives and
significantly affect biological activity. In addition, incor-
poration of fluorine residues into aromatic system leads to
increased lipophilicity and better distribution to CNS (Kirk,
2006; Song et al., 2005; Ismail, 2002; Smart, 2001). It
should be stressed here that beneficial influence of fluorine
substitution on anticonvulsant activity was observed in a
group of 2-azaspiro[4.4]nonane- and 2-azaspro[4.5]decane-
1,3-diones described in advance (Obniska et al., 2006).
In the rotarod test (NT) for acute neurological toxicity,
most of 3-chloro- as well as 3-(trifluoromethyl)anilides with
electron-withdrawing substituents (Cl, F, CF3) at the
phenylpiperazine fragment did not show neurotoxicity in the
123

Med Chem Res
maximum dose administered (300 mg/kg). The other
derivatives, namely 11, 13, 14, 19, 21–23 showed neuro-
toxicity at a dose of 300 mg/kg. At the same dose, mice
showed respiratory depression after administration of 14 and
revealed tremors after administration of 22. Only one
compound 24 revealed neurotoxicity at a dose of 100 mg/kg.
According to the Anticonvulsant Screening Project dis-
positions, three compounds 13, 20 and 22 which showed
protection in mice were selected and examined for their
anticonvulsant activity (MES screen) and neurotoxicity
after oral (p.o.) administration in rats at fixed dose 30 mg/
kg. This screen discloses the time of onset, the approximate
time of peak effect (TPE) and the duration of anticonvul-
sant activity or neurotoxicity. The results are shown in
Table 2.
parameters (ED50 and TD50) after p.o. application. The
quantitative evaluation of the MES median effective dose
(ED50) and toxic dose (TD50) was performed at previously
estimated TPE—2 h. The results of the quantitative tests
along with data for the standard AEDs—valproic acid,
ethosuximide and phenytoin—are listed in Table 3.
The quantitative data showed that 20 had higher ED50
than phenytoin (MES test), however, was distinctly more
potent than valproic acid in this seizure model. It should be
emphasized that in both cases 20 revealed more favorable
protective indexes in comparison with mentioned model
AEDs. Furthermore, substance 20 was almost twice weaker
in the MES test than model succinimides presented in
Fig. 2. These results support strongly crucial role of suc-
cinimide ring for anticonvulsant activity.
Among derivatives tested, only 20 was active in 50 % of
animals at time point 2 h. This compound was also active
in 25 % of animals at 0.5 h. The other molecules revealed
25 % protection at 0.5 h (13) and in all five time intervals
(25). All compounds given orally did not cause motor
impairment in the rotarod screen (data not indicated in
Table 2). The compounds tested showed lower activity
than phenytoin which protected from 25 to 100 % of mice
in different time intervals.
In the next step of pharmacological investigations, four
compounds 12, 19, 20 and 24 were chosen for the
evaluation of anticonvulsant activity in the psychomotor
6-Hz test (Table 4). The selection was made randomly as a
part of the search of molecules providing anti-6-Hz pro-
tection among chemically diversified compounds pursued
in the NIH/NINDS. It should be noticed that 6-Hz screen
has been validated as a model of therapy-resistant epilepsy
(Barton et al., 2001). Furthermore, the 6-Hz stimulation is
suggested to be capable for identifying anti-seizure agents
with a novel spectrum of activity and unknown mechanism
On the basis of the preliminary data in rats, compound
20 was chosen for quantification of the pharmacological
Table 2 Anticonvulsant activity of selected compounds administrated orally in rats (MES screen)
No.
Oral administration in ratsa
0.25 h
0.5 h
1 h
2 h
4 h
13
0/4
0/4
1/4
1/4
1/4
1/4
1/4
4/4
0/4
0/4
1/4
3/4
0/4
2/4
1/4
3/4
0/4
0/4
1/4
3/4
20
22
Phenytoinb
a
Dose of 30 mg/kg was administrated orally. The data indicate: number of rats protected/number of rats tested
Phenytoin, reference antiepileptic drug, tested by use of ADD Program procedures in NIH/NINDS
b
Table 3 Quantification data in rats after p.o. administration
c
No.
TPE (h)a
ED50 MESb (mg/kg)
ED50 scPTZb (mg/kg)
TD50 NTb (mg/kg)
PI (TD50/ED50)
20
Valproic acidd
Phenytoind
2
1
1
52.30 (46.38–60.22)
485 (324–677)
ND
[500
[9.56 (MES)
646 (466–869)
[500
784 (503–1176)
[100
1.6 (MES) 1.2 (scPTZ)
[3.6 (MES)
28.10 (20.7–35.2)
ND not determined
a
Time to peak effect
b
Results are presented as mean SEM at 95 % confidence limit (MES maximal electroshock test, scPTZ subcutaneous pentylenetetrazole test,
NT neurotoxicity, rotarod test)
c
Protective index (TD50/ED50
)
d
Reference antiepileptic drugs tested by use of ADD Program procedures in NIH/NINDS
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Med Chem Res
Table 4 Anticonvulsant activity—i.p. psychomotor seizure test in mice (6-Hz)
No.
Intraperitoneal injection into micea
0.25 h
0.5 h
1 h
2 h
4 h
12
19
20
24
3/4
0/4
1/4
4/4
3/4
0/4
0/4
2/4
0/4
0/4
0/4
3/4
0/4
0/4
1/4
0/4
0/4
1/4
1/4
0/4
Current 32 mA
a
Dose of 100 mg/kg was administrated intraperitoneally. The data indicate: number of mice protected/number of mice tested
of anticonvulsant action. One example supporting this
hypothesis is provided by levetiracetam, which has
demonstrated efficacy in refractory human partial epilep-
sies. It was found to be inactive against classical MES and
PTZ seizures even at high doses, whereas showed high
effectiveness in the 6-Hz psychomotor seizure model of
partial epilepsy (Rogawski, 2006). Thus, it is suggested
that the 6-Hz model might be capable for identifying anti-
seizure agents with a novel spectrum of activity and un-
known mechanism of anticonvulsant action.
demonstrate a frequency- and voltage-dependent blockade
of sodium currents by both of these anticonvulsants (Wil-
low et al., 1985; Matsuki et al., 1984). These findings
suggest that blockade of sodium channel activity by these
agents underlies their anticonvulsant actions. It should be
stressed here that such mode of action is characteristic for
compounds active in the MES seizure test in animals. Due
to activity of reported compounds in the MES seizures,
for the most active molecule 20, the binding assays
for Na? channel (site 2) were performed using the [3H]
batrachotoxin as radioligand (Brown, 1986). Compound
binding was calculated as a percent inhibition of the
binding of a radioactively labeled ligand. The inhibition
values for 20 were obtained in four concentrations, 1, 10,
100, and 500 lM. All experiments were performed in du-
plicate. The results are given in Table 5.
The results obtained showed the most potent activity for
morpholine derivative 24 which protected 100 % of mice
at 0.25 h, 75 % of animals at 1 h and 50 % of animals at
0.5 h at a dose of 100 mg/kg after (i.p.). Compound 12
protected 75 % of animals at 0.25 and 0.5 h. The other
compounds 19 and 20 were less active and protected 25 %
of animals at 4 h (19) as well as at 0.25, 2 and 4 h (20).
None of these compounds caused motor impairment during
mentioned studies.
As it can be seen, compound 20 was observed as a
moderate binder to the neuronal voltage-sensitive sodium
channel at the highest concentration—500 lM. This result
may suggest different mechanism of anticonvulsant action
than only influence on VGSCs.
In vitro sodium channels radioligand binding studies
Intensive studies into the physiological and biochemical
events taking place during epileptic seizures have provided
insight into the molecular mechanisms by which these
might be controlled. The fundamental role in establishing
and regulating excitability of CNS neurons as well as
suppression of seizures is ascribed to voltage-gated sodium
channels (VGSCs) (Meldrum and Rogawski, 2007). Thus,
the brain sodium channels are the molecular targets of
numerous chemically diverse AEDs from which phenytoin
and carbamazepine are the most representative (Rogawski
Experimental
Chemistry
All the chemicals and solvents were purchased from Sig-
ma-Aldrich (St. Louis, USA) and were used without further
purification. Melting points (mp.) were determined in open
capillaries on a Bu¨chi 353 melting point apparatus (Bu¨chi
Labortechnik, Flawil, Switzerland) and are uncorrected.
HPLC analyses were run on a HPLC WatersÒ 2695
Separation Module equipped with photodiode array de-
tector (WatersÒ 2998). A Chromolith RP–18 SpeedROD
column (4.6 9 50 mm) was used. Conditions applied were
as follows: eluent A (water/0.1 % TFA), eluent B (ace-
tonitrile/0.1 % TFA), flow rate 5 ml/min, gradient of
0–100 % B over 3 min were used, and injection volume
was 10 ll. Standard solutions (1 mg/ml) of each compound
were prepared in analytical grade acetonitrile and analyzed
¨
and Loscher, 2004; Liu et al., 2003; Bialer et al., 2009).
Using radioligand binding techniques, Catterall and asso-
ciates have found an allosteric interaction between these
drugs and the batrachotoxin (BTX) binding site of sodium
channels from rat brain (Willow and Catterall, 1982; Cat-
terall et al., 1981). Phenytoin and carbamazepine block the
influx of sodium into rat brain synaptosomes elicited by
BTX, which activates sodium channels (Willow et al.,
1984). Electrophysiological studies of neuroblastoma cells
123

Med Chem Res
2-Chloro-N-(3-chlorophenyl)acetamide (1) White pow-
1
Table 5 In vitro Na? channel (site 2) binding assays
dery crystals; mp 84–85 °C; HPLC: Rt = 1.089 min; H
No.
Concentration (lM)
% Inhibition
of control specific bindinga
NMR (CDCl3): d 4.21 (s, 2H, –CH2–), 7.41–7.50 (m, 2H,
ArH), 7.58–7.78 (d, 1H, ArH, J = 7.70 Hz), 7.85 (s, 1H,
ArH), 8.31 (brs, 1H, NH); 13C NMR (75 MHz, CDCl3): d
43.3 (–CH2–), 115.6, 119.2, 124.2, 126.7, 131.6, 139.2
(6Caromatic), 168.8 (–CO–). ESI–MS: 204.1 (C8H7Cl2NO
[M?H]?). Anal. Calcd for C8H7Cl2NO (204.05): C, 47.09;
H, 3.46; N, 6.85. Found: C, 47.19; H, 3.55, N, 6.70.
20
1
10
4.2
7.5
100
500
10.8
46.7
a
Compounds were each evaluated in synaptoneurosomal prepara-
tions from rat cerebral cortex as inhibitors of the specific binding of
[3H]BTX to the voltage-sensitive sodium channel. 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 weak to moderate effects; results
showing an inhibition lower than 25 % are not considered significant
and mostly attributable to variability of the signal around the control
level
2-Chloro-N-[3-(trifluoromethyl)phenyl]acetamide (2) White
powdery crystals; mp 72–73 °C; HPLC: Rt = 1.201 min;
1H NMR (300 MHz, CDCl3): d 4.21 (s, 2H, –CH2–),
7.42–7.52 (m, 2H, ArH), 7.57–7.78 (m, 1H, ArH,
J = 7.70 Hz), 7.85 (s, 1H, ArH), 8.33 (brs, 1H, NH); 13C
NMR (75 MHz, CDCl3): d 45.6 (–CH2–), 107.2 (–CF3),
117.1, 118.6, 120.5, 123.3, 126.4, 130.8, 135.6 (6Caromatic),
168.8 (–CO–). ESI–MS: 238.10 (C9H7ClF3NO [M?H]?).
Anal. Calcd for C9H7ClF3NO (237.61): C, 45.49; H, 2.98;
N, 5.89. Found: C, 45.55; H, 3.15, N, 5.88.
at wavelengths 214 and 254 nm. Retention times (Rt) are
given in minutes. Elemental analysis for C, H, and N was
carried out by a micro method using the elemental Vario EI
III Elemental analyzer (Hanau, Germany). The results of
elemental analyses were within 0.4 % of the theoretical
values. 1H NMR,13C NMR and 19F NMR spectra were
obtained in a Varian Mercury spectrometer (Varian Inc.,
Palo Alto, CA, USA), in CDCl3 operating at 300 MHz (1H
NMR), 75 MHz (13C NMR) and 282 MHz (19F NMR).
Chemical shifts are reported in d values (ppm) relative to
TMS d = 0 (1H), as internal standard. The J values are
expressed in Hertz (Hz). Signal multiplicities are repre-
sented by the following abbreviations: s (singlet), brs
(broad singlet), d (doublet), ddd (double double doublet), t
(triplet) and m (multiplet). The mass spectra were obtained
on Waters ACQUITYTM TQD system with the TQ detector
(Waters, Milford, USA). The ACQUITY UPLC BEH C18,
1.7 lm, 2.1 9 50 mm column was used (Waters, Milford,
USA).
General procedure for the synthesis of final compounds
3–24
A total of 0.002 mol of intermediate 1 or 2 and equimolar
amount of corresponding secondary amine were dissolved
in 10 ml of dry acetone. 0.006 mol of dry potassium car-
bonate and catalytic amount of potassium iodide were
added, and the reaction mixture was stirred for about 24 h
at a temperature of 60 °C (HPLC control). After this time,
inorganic ingredients were filtered off and acetone was
distilled off on a rotary evaporator. The crude products
were obtained as colored oils. Compounds 3–22 and 24
were purified by column chromatography on Silica gel 60
(Merck, Darmstadt, Germany) using a dichloromethane–
methanol (9: 0.7 v/v) mixture as a solvent system. Due to
oily form, compounds 3–13, 15, 18–21, 23 and 24 were
converted into hydrochloride salts in anhydrous ethanol
saturated with HCl gas. They were crystallized from an-
hydrous ethanol.
General procedure for preparation of intermediates 1
and 2
A total of 0.05 mol of the 3-chloroaniline or 3-tri-
fluoromethylaniline was dissolved in 30 ml of DCM and
mixed with 5 ml 2 % aqueous solution of sodium hy-
droxide. Then, 0.05 mol of 2-chloroacetyl chloride dis-
solved in DCM (10 ml) was added dropwise over an hour.
During that time, the reaction mixture was cooled with ice.
After the acylating agent was added, stirring was continued
for 2 h. Subsequently, the reaction mixture was extracted
with saturated potassium bisulfate solution, water and
brine. The organic layer was dried over the anhydrous
sodium sulfate an distilled of on a rotary evaporator to
obtain white crystalline powders.
N-(3-chlorophenyl)-2-(4-phenylpiperazin-1-yl)acetamide
monohydrochloride (3) White powdery crystals; mp.
199–201 °C; HPLC: Rt = 1.357 min; 1H NMR (300 MHz,
DMSO–d6): d 3.06–3.93 (m, 8H, piperazine), 4.33 (s, 2H,
–CH2–), 6.81–6.89 (m, 1H, ArH), 6.96–7.05 (m, 1H, ArH),
7.12–7.29 (m, 4H, ArH), 7.32–7.41 (m, 1H, ArH),
7.53–7.60 (m, 1H, ArH), 7.76–7.86 (m, 1H, ArH), 10.86
(brs, 1H, NH), 11.55 (brs, 1H, ?NH); 13C NMR (75 MHz,
DMSO–d6): d 51.3 (–CH2–), 51.9 (–CH2–), 52.5 (–CH2–),
53.3 (–CH2–), 54.1 (–CH2–), 111.7, 117.0, 118.5, 123.6,
130.2, 130.9, 135.4, 140.6, 146.3 (12Caromatic), 172.1
(–CO–). ESI–MS: 330.4 (C18H20ClN3O [M?H]?).
123

Med Chem Res
Anal. Calcd for C18H21Cl2N3O (366.28): C, 59.02; H, 5.78;
N, 11.47. Found: C, 59.12; H, 5.90, N, 11.50.
2.03, 1.03 Hz), 7.85 (t, 1H, ArH, J = 1.97 Hz), 10.84 (brs,
1H, NH), 11.45 (brs, 1H, ?NH); 13C NMR (75 MHz,
CDCl3): d 50.8 (–CH2–), 51.9 (–CH2–), 53.2 (–CH2–), 54.4
(–CH2–), 55.8 (–CH2–), 114.2, 115.3, 116.5, 117.0, 117.8,
122.9, 125.4, 129.1, 129.6, 134.4, 138.5, 143.2 (12Caromatic),
171.9 (–CO–); 19F NMR (282 MHz, DMSO–d6): d –122.92–
(–122.83) (m, 1F). ESI–MS: 348.8 (C18H19ClFN3O
[M?H]?). Anal. Calcd for C18H20Cl2FN3O (384.28): C,
56.26; H, 5.25; N, 10.93. Found: C, 56.30; H, 5.32; N, 11.04.
N-(3-chlorophenyl)-2-[4-(2-chlorophenyl)piperazin-1-yl]
acetamide monohydrochloride (4) White powdery
crystals; mp. 238–240 °C; HPLC: Rt = 1.456 min; 1H
NMR (300 MHz, DMSO–d6): d 3.09–3.50 (m, 8H, piper-
azine), 4.33 (s, 2H, –CH2–), 7.06–7.22 (m, 3H, ArH),
7.29–7.45 (m, 3H, ArH), 7.56 (ddd, 1H, ArH, J = 8.23,
1.92, 0.91 Hz), 7.82–7.88 (m, 1H, ArH), 10.87 (s, 1H, NH),
11.43 (brs, 1H, ?NH); 13C NMR (75 MHz, DMSO–d6): d
51.8 (–CH2–), 52.3 (–CH2–), 53.1 (–CH2–), 54.3 (–CH2–),
54.9 (–CH2–), 110.6, 116.8, 117.5, 118.3, 118.8, 124.5,
126.5, 130.3, 131.1, 136.8, 140.1, 144.2 (12Caromatic), 173.2
(–CO–). ESI–MS: 365.2 (C18H19Cl2N3O [M?H]?). Anal.
Calcd for C18H20N3OCl3 (400.73): C, 53.95; H, 5.03; N,
10.49. Found: C, 54.03; H, 5.12; N, 10.55.
N-(3-chlorophenyl)-2-[4-(4-fluorophenyl)piperazin-1-yl]
acetamide monohydrochloride (8) White powdery
crystals; mp. 214–216 °C; HPLC: Rt = 1.392 min; 1H
NMR (300 MHz, DMSO–d6): d 2.95–3.89 (m, 8H, piper-
azine), 4.32 (s, 2H, –CH2–), 6.96–7.19 (m, 4H, ArH), 7.36
(t, 2H, ArH, J = 8.11 Hz), 7.54–7.60 (m, 1H, ArH),
7.84–7.87 (m, 1H, ArH), 10.86 (brs, 1H, NH), 11.55 (brs,
1H, ?NH); 13C NMR (75 MHz, DMSO–d6): d 52.5
(–CH2–), 53.5 (–CH2–), 54.6 (–CH2–), 55.5 (–CH2–), 56.3
(–CH2–), 115.2, 116.4, 117.2, 117.9, 123.9, 125.8, 129.0,
134.6, 140.1, 145.1 (12Caromatic), 173.2 (–CO–); 19F NMR
(282 MHz, DMSO–d6): d –123.98 (s, 1F). ESI–MS: 348.7
(C18H19ClFN3O [M?H]?). Anal. Calcd for C18H20Cl2-
FN3O (384.28): C, 56.26; H, 5.25; N, 10.93. Found: C,
56.35; H, 5.34; N, 10.99.
N-(3-chlorophenyl)-2-[4-(4-chlorophenyl)piperazin-1-yl]
acetamide monohydrochloride (5) White powdery
crystals; mp. 217–219 °C; HPLC: Rt = 0.747 min; 1H
NMR (300 MHz, DMSO–d6): d 3.06–4.12 (m, 8H, piper-
azine), 4.30 (s, 2H, –CH2–), 6.96–7.04 (m, 2H, ArH),
7.15–7.20 (m, 1H, ArH), 7.24–7.31 (m, 2H, ArH), 7.37 (t,
1H, ArH, J = 8.11 Hz), 7.54 (d, 1H, ArH, J = 8.46 Hz),
7.83–7.84 (m, 1H, ArH), 10.71 (s, 1H, NH), 11.37 (brs, 1H,
?NH); 13C NMR (75 MHz, DMSO–d6): d 52.0 (–CH2–),
52.8 (–CH2–), 53.2 (–CH2–), 54.1 (–CH2–), 54.8 (–CH2–),
112.6, 114.5, 118.5, 119.3, 124.8, 126.6, 129.5, 135.7,
141.5, 146.4 (12Caromatic), 174.1 (–CO–). ESI–MS: 365.2
(C18H19Cl2N3O [M?H]?). Anal. Calcd for C18H20Cl3N3O
(400.73): C, 53.95; H, 5.03; N, 10.49. Found: C, 53.98; H,
5.09; N, 10.53.
N-(3-chlorophenyl)-2-[4-[3-(trifluoromethyl)phenyl]piper-
azin-1-yl]acetamide monohydrochloride (9) White pow-
dery crystals; mp. 199–200 °C; HPLC: Rt = 1.602 min; 1H
NMR (300 MHz, DMSO–d6): d 3.15–3.49 (m, 4H, piper-
azine), 3.57–3.70 (m, 2H, piperazine), 3.87–4.01 (m, 2H,
piperazine), 4.33 (s, 2H, –CH2–), 7.10–7.18 (m, 2H, ArH),
7.21–7.30 (m, 2H, ArH), 7.32–7.49 (m, 2H, ArH),
7.54–7.60 (m, 1H, ArH), 7.85 (t, 1H, ArH, J = 2.00 Hz),
10.95 (brs, 1H, NH), 11.56 (brs, 1H, ?NH); 13C NMR
(75 MHz, DMSO–d6): d 49.8 (–CH2–), 51.2 (–CH2–), 52.8
(–CH2–), 54.5 (–CH2–), 55.7 (–CH2–), 116.6, 118.0, 118.5,
119.1, 119.9, 121.2, 123.9, 125.6, 129.1, 131.2, 137.4,
139.9, 141.8 (–CF3, 12Caromatic), 170.5 (–CO–); 19F NMR
(282 MHz, DMSO–d6): d –61.07 (s, 3F). ESI–MS: 398.4
(C19H19ClF3N3O [M?H]?). Anal. Calcd for C19H20Cl2
F3N3O (434.28): C, 52.55; H, 4.64; N, 9.68. Found: C,
52.68; H, 4.77; N, 9.80.
N-(3-chlorophenyl)-2-[4-(2,3-dichlorophenyl)piperazin-1-yl]
acetamide monohydrochloride (6) White powdery crys-
1
tals; mp. 241–242 °C; HPLC: Rt = 1.601 min; H NMR
(300 MHz, DMSO–d6): d 3.14–3.78 (m, 8H, piperazine),
4.31 (s, 2H, –CH2–), 7.14–7.25 (m, 2H, ArH), 7.30–7.42
(m, 3H, ArH), 7.51–7.57 (m, 1H, ArH), 7.82–7.86 (m, 1H,
ArH), 10.79 (brs, 1H, NH), 11.32 (brs, 1H, ?NH); 13C
NMR (75 MHz, DMSO–d6): d 52.2 (–CH2–), 53.3 (–CH2–
), 53.9 (–CH2–), 54.5 (–CH2–), 55.6 (–CH2–), 116.6, 117.1,
117.5, 118.7, 119.8, 124.7, 126.6, 129.3, 130.1, 136.5,
140.8, 147.1 (12Caromatic), 174.6 (–CO–). ESI–MS: 399.3
(C18H18Cl3N3O [M?H]?). Anal. Calcd for C18H19Cl4N3O
(435.17): C, 49.68; H, 4.40; N, 9.66. Found: C, 49.75; H,
4.48; N, 9.74.
N-(3-chlorophenyl)-2-(4-pyrimidin-2-ylpiperazin-1-yl)ac-
etamide monohydrochloride (10) White powdery crys-
1
tals; mp. 248–249 °C; HPLC: Rt = 1.138 min; H NMR
(300 MHz, DMSO–d6): d 3.11–3.74 (m, 8H, piperazine),
4.28 (s, 2H, –CH2–), 6.76 (t, 1H, ArH, J = 4.79 Hz), 7.16
(ddd, 1H, ArH, J = 8.02, 2.12, 1.01 Hz), 7.36 (t, 1H, ArH,
J = 8.09 Hz), 7.52–7.58 (m, 1H, ArH), 7.84 (t, 1H, ArH,
J = 1.97 Hz), 8.40–8.48 (m, 2H, ArH), 10.91 (brs, 1H,
NH), 11.50 (brs, 1H, HCl); 13C NMR (75 MHz, DMSO–
d6): d 50.2 (–CH2–), 52.1 (–CH2–), 54.1 (–CH2–), 55.2
N-(3-chlorophenyl)-2-[4-(2-fluorophenyl)piperazin-1-yl]
acetamide monohydrochloride (7) White powdery
crystals; mp. 224–225 °C; HPLC: Rt = 1.393 min; 1H
NMR (300 MHz, DMSO–d6): d 3.02–3.79 (m, 8H, piper-
azine), 4.32 (s, 2H, –CH2–), 6.98–7.21 (m, 5H, ArH),
7.33–7.42 (m, 1H, ArH), 7.56 (ddd, 1H, ArH, J = 8.23,
123

Med Chem Res
(–CH2–), 56.1 (–CH2–), 115.2, 117.9, 120.1, 123.3, 129.4,
133.1, 137.8, 158.5, 164.9 (10Caromatic), 170.8 (–CO–).
ESI–MS: 332.7 (C16H18ClN5O [M?H]?). Anal. Calcd for
C16H19Cl2N5O (368.26): C, 52.18; H, 5.20; N, 19.02.
Found: C, 52.23; H, 5.29; N, 19.08.
2-(4-Phenylpiperazin-1-yl)-N-[3-(trifluoromethyl)phenyl]
acetamide (14) White powdery crystals; mp. 152–153 °C;
HPLC: Rt = 1.204 min; 1H NMR (300 MHz, CDCl3): d 2.81
(t, 4H, piperazine, J = 5.0 Hz), 3.23 (s, 2H, –CH2–), 3.29
(t, 4H, piperazine, J = 5.0 Hz), 6.87–6.97 (m, 3H, ArH),
7.25–7.32 (m, 2H, ArH), 7.37 (d, 1H, ArH, J = 7.44 Hz),
7.46 (t, 1H, ArH, J = 8.6 Hz), 7.82 (s, 2H, ArH), 9.27 (s, 1H,
NH); 13C NMR (75 MHz, CDCl3): d 52.5 (–CH2–), 53.2
(–CH2–), 53.8 (–CH2–), 54.7 (–CH2–), 55.3 (–CH2–), 109.7,
116.1, 117.5, 118.3, 123.5, 128.2, 129.7, 136.4, 139.9, 147.4
(–CF3, 12Caromatic), 175.3 (–CO–); 19F NMR (282 MHz,
CDCl3): d –62.74 (s, 3F). ESI–MS: 364.0 (C19H20F3N3O
[M?H]?). Anal. Calcd for C19H20F3N3O (363.38): C, 62.80;
H, 5.55; N, 11.56. Found: C, 62.86; H, 5.62; N, 11.63.
2-(4-Benzylpiperazin-1-yl)-N-(3-chlorophenyl)acetamide
dihydrochloride (11) White powdery crystals; mp.
245–247 °C; HPLC: Rt = 1.254 min; 1H NMR (300 MHz,
DMSO–d6): d 3.21–3.78 (m, 8H, piperazine), 4.15 (s, 2H,
–CH2–), 4.40 (s, 2H, –CH2–), 7.15 (ddd, 1H, ArH,
J = 7.95, 2.05, 1.03 Hz), 7.35 (t, 1H, ArH, J = 8.08 Hz),
7.41–7.48 (m, 3H, ArH), 7.48–7.55 (m, 1H, ArH),
7.60–7.70 (m, 2H, ArH), 7.82 (t, 1H, ArH, J = 2.05 Hz),
9.94 (s, 1H, NH), 11.17 (brs, 1H, ?NH), 12.11 (brs, 1H,
?NH); 13C NMR (75 MHz, DMSO–d6): d 50.3 (–CH2–),
51.5 (–CH2–), 52.7 (–CH2–), 53.8 (–CH2–), 54.4 (–CH2–),
65.7 (–CH2–, benzyl), 115.2, 116.9, 118.2, 124.8, 129.2,
130.4, 134.8, 136.5, 139.4 (12Caromatic), 174.6 (–CO–).
ESI–MS: 344.8 (C19H22ClN3O [M?H]?). Anal. Calcd for
C19H24Cl3N3O (416.77): C, 54.75; H, 5.80; N, 10.08.
Found: C, 54.90; H, 5.94; N, 10.11.
2-[4-(2-Chlorophenyl)piperazin-1-yl]-N-[3-(trifluoromethyl)
phenyl]acetamide monohydrochloride (15) White pow-
dery crystals; mp. 239–241 °C; HPLC: Rt = 1.298 min; 1H
NMR (300 MHz, DMSO–d6): d 3.14–3.65 (m, 8H, piper-
azine), 4.33 (s, 2H, –CH2–), 7.10 (t, 1H, ArH,
J = 7.59 Hz), 7.25 (d, 1H, ArH, J = 7.95 Hz), 7.33 (t, 1H,
ArH, J = 7,71 Hz), 7.45 (t, 2H, ArH, J = 8.46 Hz), 7.62
(t, 1H, ArH, J = 7.95 Hz), 7.85 (d, 1H, ArH,
J = 8.46 Hz), 8.14 (s, 1H, ArH), 10.78 (brs, 1H, NH),
11.47 (brs, 1H, ?NH); 13C NMR (75 MHz, DMSO–d6): d
52.6 (–CH2–), 52.9 (–CH2–), 53.5 (–CH2–), 54.3 (–CH2–),
54.9 (–CH2–), 109.8, 116.4, 117.6, 118.5, 119.8, 120.4,
123.2, 126.0, 130.6, 131.2, 135.7, 139.8, 143.9 (–CF3,
12Caromatic), 175.1 (–CO–); 19F NMR (282 MHz, DMSO–
d6): d –61.43 (s, 3F). ESI–MS: 397.8 (C19H19ClF3N3O
[M?H]?). Anal. Calcd for C19H20Cl2F3N3O (434.28): C,
52.55; H, 4.64; N, 9.68. Found: C, 52.61; H, 4.79; N, 9.79.
N-(3-chlorophenyl)-2-(4-methylpiperazin-1-yl)acetamide
dihydrochloride (12) White powdery crystals; mp.
237–238 °C; HPLC: Rt = 0.958 min; 1H NMR (300 MHz,
DMSO–d6) d 2.80 (s, 3H, CH3), 3.25–3.79 (m, 8H, piper-
azine), 4.17 (s, 2H, –CH2–), 7.15 (ddd, 1H, ArH, J = 8.01,
2.12, 0.90 Hz), 7.35 (t, 1H, ArH, J = 8.09 Hz), 7.53 (ddd,
1H, ArH, J = 8.22, 2.04, 1.03 Hz), 7.83 (t, 1H, ArH,
J = 1.92 Hz), 10.91 (brs, 1H, NH), 11.22 (brs, 1H, ?NH),
11.89 (brs, 1H, ?NH); 13C NMR (75 MHz, DMSO–d6): d
32.8 (–CH3), 49.6 (–CH2–), 50.6 (–CH2–), 51.7 (–CH2–),
52.6 (–CH2–), 53.4 (–CH2–), 118.0, 120.5, 123.2, 125.6,
129.8, 137.9 (6Caromatic), 169.8 (–CO–). ESI–MS: 268.7
(C13H18ClN3O [M?H]?). Anal. Calcd for C13H20Cl3N3O
(340.68): C, 45.83; H, 5.92; N, 12.33. Found: C, 45.88; H,
5.99; N, 12.41.
2-[4-(4-Chlorophenyl)piperazin-1-yl]-N-[3-(trifluoromethyl)
phenyl]acetamide (16) White powdery crystals; mp.
170–171 °C; HPLC: Rt = 1.365 min; 1H NMR (300 MHz,
CDCl3): d 2.79 (t, 4H, piperazine, J = 5.00 Hz), 3.23 (s,
2H, –CH2–), 3.25 (t, 4H, piperazine, J = 4.87 Hz)
6.83–6.89 (m, 2H, ArH), 7.20–7.26 (m, 2H, ArH), 7.37 (d,
1H, ArH, J = 7.44 Hz), 7.46 (t, 1H, ArH, J = 8.6 Hz),
7.81 (s, 2H, ArH), 9.23 (s, 1H, NH); 13C NMR (75 MHz,
CDCl3): d 51.5 (–CH2–), 52.5 (–CH2–), 53.5 (–CH2–), 54.
0 (–CH2–), 55.3 (–CH2–), 111.3, 116.5, 117.9, 118.6,
119.8, 124.5, 126.8, 129.9, 136.8, 141.7, 144.4 (–CF3,
12Caromatic), 173.5 (–CO–); 19F NMR (282 MHz, CDCl3):
d –62.74 (s, 3F). ESI–MS: 398.3 (C19H19ClF3N3O
[M?H]?). Anal. Calcd for C19H19ClF3N3O (397.82):
C, 57.36; H, 4.81; N, 10.56. Found: C, 57.49; H, 4.89; N,
10.63.
N-(3-chlorophenyl)-2-morpholino-acetamide monohydrochlo-
ride (13) White powdery crystals; mp. 189–190 °C;
HPLC: Rt = 1.067 min; H NMR (300 MHz, DMSO–d6):
1
d 1.59–1.88 (m, 4H, morpholine), 2.96–3.56 (m, 4H,
morpholine), 4.15 (s, 2H, –CH2–), 7.13–7.19 (m, 1H, ArH),
7.37 (t, 1H, ArH, J = 8.08 Hz), 7.51–7.57 (m, 1H, ArH),
7.82–7.86 (m, 1H, ArH), 10.05 (brs, 1H, NH), 11.41 (brs,
1H, ?NH); 13C NMR (75 MHz, DMSO–d6): d 51.8
(–CH2–), 52.6 (–CH2–), 53.2 (–CH2–), 67.8 (–CH2–), 68.6
(–CH2–), 117.8, 120.3, 122.9, 125.8, 128.4, 138.1 (6Caromatic),
171.5 (–CO–). ESI–MS: 255.7 (C12H15Cl N2O2 [M?H]?).
Anal. Calcd for C12H16 Cl2N2O2 (291.17): C, 49.50; H, 5.54;
N, 9.62. Found: C, 49.54; H, 5.61; N, 9.68.
2-[4-(2,3-Dichlorophenyl)piperazin-1-yl]-N-[3-(trifluoromethyl)
phenyl]acetamide (17) White powdery crystals; mp.
174–175 °C; HPLC: Rt = 1.434 min; 1H NMR (300 MHz,
123

Med Chem Res
CDCl3): d 2.84 (t, 4H, piperazine, J = 4.74 Hz), 3.15 (t,
4H, piperazine, J = 4.48 Hz), 3.23 (s, 2H, –CH2–),
6.98–7.02 (m, 1H, ArH), 7.14–7.21 (m, 2H, ArH), 7.37 (d,
1H, ArH, J = 7.69 Hz), 7.46 (t, 1H, ArH, J = 7.95 Hz),
7.79 (s, 1H, ArH), 7.87 (d, 1H, ArH, J = 8.2 Hz), 9.28
(brs, 1H, NH); 13C NMR (75 MHz, CDCl3): d 53.2 (–CH2–
), 54.0 (–CH2–), 54.8 (–CH2–), 55.5 (–CH2–), 56.7 (–CH2–
), 115.8, 117.0, 117.8, 118.5, 119.5, 120.6, 124.3, 126.2,
129.5, 131.8, 137.5, 141.1, 146.2 (–CF3, 12Caromatic), 175.8
(–CO–); 19F NMR (282 MHz, CDCl3): d –62.70 (s, 3F).
ESI–MS: 432.1 (C19H18Cl2F3N3O [M?H]?). Anal. Calcd
for C19H18Cl2F3N3O (432.27): C, 52.79; H, 4.20; N, 9.72.
Found: C, 52.90; H, 4.28; N, 9.83.
7.32–7.49 (m, 2H, ArH), 7.54–7.60 (m, 1H, ArH), 7.85 (t,
1H, ArH, J = 2.00 Hz), 10.61 (brs, 1H, NH), 11.46 (brs,
1H, ?NH); 13C NMR (75 MHz, DMSO–d6): 52.2 (–CH2–
), 52.9 (–CH2–), 53.8 (–CH2–), 55.7 (–CH2–), 56.8 (–CH2–
), 115.2, 117.8, 118.6, 119.2, 119.9, 120.5, 121.6, 124.0,
126.3, 129.8, 131.7, 139.4, 142.2, 148.8 (–CF3, –CF3,
12Caromatic), 177.2 (–CO–); 19F NMR (282 MHz, DMSO–
d6): d –61.41 (s, 3F), –61.35 (s, 3F). ESI–MS: 431.4
(C20H19F6N3O [M?H]?). Anal. Calcd for C20H20ClF6N3O
(467.84): C, 51.35; H, 4.31; N, 8.98. Found: C, 51.49; H,
4.44; N, 9.04.
2-(4-Pyrimidin-2-ylpiperazin-1-yl)-N-[3-(trifluoromethyl)
phenyl]acetamide monohydrochloride (21) White pow-
dery crystals; mp. 149–150 °C; HPLC: Rt = 1.147 min; 1H
NMR (300 MHz, DMSO–d6): d 3.14–3.80 (m, 8H, piper-
azine), 4.26 (s, 2H, –CH2–), 6.75 (t, 1H, ArH, J =
4.87 Hz), 7.48 (d, 1H, ArH, J = 7.18 Hz), 7.61 (t, 1H,
ArH, J = 8.08 Hz), 7.78–7.82 (m, 1H, ArH), 8.12 (s, 1H,
ArH), 8.44 (d, 2H, ArH, J = 4.88 Hz), 10.59 (s, 1H, NH),
11.26 (brs, 1H, ?NH); 13C NMR (75 MHz, DMSO–d6): d
51.4 (–CH2–), 52.5 (–CH2–), 53.3 (–CH2–), 54.1 (–CH2–),
55.2 (–CH2–), 113.1, 118.8, 120.1, 121.3, 124.5, 129.9,
132.6, 139.8, 157.9, 167.8 (–CF3, 10Caromatic), 174.2
(–CO–); 19F NMR (282 MHz, DMSO–d6): d –61.42 (s,
3F). ESI–MS: 366.0 (C17H18F3N5O [M?H]?). Anal. Calcd
for C17H19ClF3N5O (401.81): C, 50.82; H, 4.77; N, 17.43.
Found: C, 50.97; H, 4.82; N, 17.55.
2-[4-(2-Fluorophenyl)piperazin-1-yl]-N-[3-(trifluoromethyl)
phenyl]acetamide monohydrochloride (18) White pow-
dery crystals; mp. 228–231 °C; HPLC: Rt = 1.247 min; 1H
NMR (300 MHz, DMSO–d6): d 3.21–3.63 (m, 8H, piper-
azine), 4.30 (s, 2H, –CH2–), 7.00–7.20 (m, 5H, ArH), 7.48
(d, 1H, ArH, J = 7.69 Hz), 7.61 (t, 1H, ArH,
J = 8.07 Hz), 7.83 (d, 1H, ArH, J = 8.46 Hz), 10.61 (brs,
1H, NH), 11,35 (brs, 1H, ?NH); 13C NMR (75 MHz,
DMSO–d6): d 51.7 (–CH2–), 52.9 (–CH2–), 53.5 (–CH2–),
54.4 (–CH2–), 55.5 (–CH2–), 111.0, 114.2, 115.9, 116.3,
117.0, 119.8, 121.7, 125.1, 128.8, 129.9, 133.8, 140.5,
147.1 (–CF3, 12Caromatic), 176.6 (–CO–); 19F NMR
(282 MHz, DMSO–d6): d –122.93–(–122.84) (m, 1F),
–61.43 (s, 3F). ESI–MS: 381.9 (C19H19F4N3O [M?H]?).
Anal. Calcd for C19H20ClF4N3O (417.82): C, 54.62; H,
4.82; N. 10.06. Found: C, 54.67; H, 4.92; N, 10.15.
2-(4-Benzylpiperazin-1-yl)-N-[3-(trifluoromethyl)phenyl]
acetamide
(22) White
powdery
crystals;
mp.
2-[4-(4-Fluorophenyl)piperazin-1-yl]-N-[3-(trifluoromethyl)
phenyl]acetamide monohydrochloride (19) White pow-
dery crystals; mp. 204–206 °C; HPLC: Rt = 1.219 min; 1H
NMR (300 MHz, DMSO–d6): d 3.13–3.69 (m, 8H, piper-
azine), 4.32 (s, 2H, –CH2–), 6.98–7.12 (m, 4H, ArH), 7.48
(d, 1H, ArH, J = 7.69 Hz), 7.60 (t, 1H, ArH,
J = 7.95 Hz), 7.84 (d, 1H, ArH, J = 8.46 Hz), 8.14 (s, 1H,
ArH), 10,72 (brs, 1H, NH), 11,48 (s, 1H, ?NH); 13C NMR
(75 MHz, DMSO–d6): d 52.1 (–CH2–), 53.0 (–CH2–), 53.9
(–CH2–), 54.5 (–CH2–), 55.3 (–CH2–), 112.2, 115.8, 117.5,
118.6, 120.7, 124.4, 125.9, 129.2, 135.7, 141.9, 146.8
(–CF3, 12Caromatic), 175.3 (–CO–); 19F NMR (282 MHz,
DMSO–d6): d –124.07 (s, 1F), –61.42 (s, 3F). ESI–MS:
381.8 (C19H19F4N3O [M?H]?). Anal. Calcd for C19H20
ClF4N3O (417.82): C, 54.62; H, 4.82; N, 10.06. Found: C,
54.67;, 4.93; N, 10.13.
115–116 °C; HPLC: Rt = 1.147 min; 1H NMR (300 MHz,
CDCl3): d 2.55–2.67 (m, 8H, piperazine), 3.15 (s, 2H,
–CH2–), 3.56 (s, 2H, –CH2–), 7.24–7.37 (m, 6H, ArH),
7.45 (t, 1H, ArH, J = 7.95 Hz), 7.79–7.83 (m, 2H, ArH),
9.29 (s, 1H, NH); 13C NMR (75 MHz, CDCl3): d 49.2
(–CH2–), 50.1 (–CH2–), 51.2 (–CH2–), 52.3 (–CH2–), 53.5
(–CH2–), 65.8 (–CH2–), 114.8, 117.5, 118.6, 120.2, 125.3,
128.9, 130.9, 133.7, 135.8, 138.3 (–CF3, 12Caromatic), 175.5
(–CO–); 19F NMR (282 MHz, CDCl3): d –62.70 (s, 3F).
ESI–MS: 378.4 (C20H22F3N3O [M?H]?). Anal. Calcd for
C20H22F3N3O (377.40): C, 63.65; H, 5.88; N, 11.13.
Found: C, 63.73; H, 5.96; N, 11.28.
2-[4-(2-Hydroxyethyl)piperazin-1-yl]-N-[3-(trifluoromethyl)
phenyl]acetamide dihydrochloride (23) White powdery
crystals; mp. 210–212 °C; HPLC: Rt = 0.841 min; 1H
NMR (300 MHz, DMSO–d6): d 3.23–3.94 (m, 15H; 8H:
piperazine, 4H: –C2H4–, 2H: –CH2–, 1H: –OH), 7.55 (d,
2H, ArH, J = 7.42 Hz), 7.58 (t, 1H, ArH, J = 7.95 Hz),
7.84 (d, 1H, ArH, J = 8,2 Hz), 9.75 (brs, 1H, NH), 10.97
(brs, 1H, ?NH), 11.89 (brs, 1H, ?NH); 13C NMR
(75 MHz, DMSO–d6): d 47.5 (–CH2–), 48.6 (–CH2–), 49.9
(–CH2–), 51.1 (–CH2–), 52.4 (–CH2–), 63.4 (–CH2–),
N-[3-(Trifluoromethyl)phenyl]-2-[4-[3-(trifluoromethyl)
phenyl]piperazin-1-yl]acetamide
monohydrochloride
(20) White powdery crystals; mp. 189–190 °C; HPLC:
Rt = 1.437 min; 1H NMR (300 MHz, DMSO–d6): d
3.13–3.69 (m, 8H, piperazine), 4.33 (s, 2H, –CH2–),
7.10–7.18 (m, 2H, ArH), 7.21–7.30 (m, 2H, ArH),
123

Med Chem Res
118.7, 119.6, 120.8, 123.1, 125.8, 131.6, 138.8 (–CF3,
6Caromatic), 170.9 (–CO–); 19F NMR (282 MHz, DMSO–
d6): d –61.41 (s, 3F). ESI–MS: 332.2 (C15H20F3N3O2
[M?H]?). Anal. Calcd for C15H22Cl2F3N3O2 (404.26): C,
44.57; H, 5.49; N, 10.39. Found: C, 44.65; H, 5.57; N,
10.48.
corneal electrodes primed with an electrolyte solution
containing an anesthetic agent. The endpoint is the tonic
extension of the hind limbs. In the control groups, the
procedures cause immediate hind limb tonic extension.
Mice not displaying hind limb tonic extension are consid-
ered to be protected from seizures.
2-Morpholino-N-[3-(trifluoromethyl)phenyl]acetamide
monohydrochloride (24) White powdery crystals; mp.
185–186 °C; HPLC: Rt = 0.985 min; 1H NMR (300 MHz,
DMSO–d6): d 1.65–1.77 (m, 4H, morpholine), 3.14–3.48
(m, 4H, morpholine), 4.16 (s, 2H, –CH2–), 7.47 (d, 1H,
ArH, J = 7.70 Hz), 7.59 (t, 1H, ArH, J = 7.94 Hz), 7.83
(d, 1H, ArH, J = 8.20 Hz), 8.12 (s, 1H, ArH), 9.96 (brs,
1H, NH), 11.42 (brs, 1H, ?NH); 13C NMR (75 MHz,
DMSO–d6): d 50.3 (–CH2–), 51.5 (–CH2–), 52.2 (–CH2–),
67.5 (–CH2–), 67.6 (–CH2–), 117.6, 119.2, 121.0, 123.3,
125.8, 133.4, 137.9 (–CF3, 6Caromatic), 172.1 (–CO–); 19F
NMR (282 MHz, DMSO–d6): d –61.44 (s, 3F). ESI–MS:
289.2 (C13H15F3N2O2 [M?H]?). Anal. Calcd for C13H16
ClF3N2O2 (324.72): C, 48.08; H, 4.97; N, 8.63. Found: C,
48.20; H, 5.02; N, 8.75.
Subcutaneous pentylenetetrazole seizure test (scPTZ)
The scPTZ test utilizes a dose of pentylenetetrazole
(85 mg/kg in mice) that produces clonic seizures lasting for
a period of at least 5 s in 97 % (CD97) of animals tested.
PTZ is administered 0.5 and 4 h after injections of tested
compounds, and observation is carried out for 30 min. In
the control groups, the first episode of clonic convulsions is
observed between 6 and 15 min of observation. The ab-
sence of clonic convulsions in the observed time period of
0.5 and 4 h is interpreted as the compound’s ability to
protect against PTZ-induced seizures.
Neurotoxicity screening
The neurological toxicity test (NT) induced by a compound
was detected in mice or rats using standardized rotarod test
(Dunham and Miya, 1957). Untreated control mice or rats,
when placed on the rod, can maintained their equilibrium for
a prolonged time period. The acute motor impairment can be
demonstrated by the inability of the animal to maintain
equilibrium for 1 min in each of three successive trials.
Pharmacology
In vivo studies
Compounds 3–24 were pharmacologically preevaluated
within the Antiepileptic Drug Development (ADD) Pro-
gram in Epilepsy Branch, Neurological Disorders Program,
National Institute of the Neurological and Communicative
Disorders and Stroke (NIH/NINDS), Rockville, MD, USA,
by using procedures described elsewhere (Krall et al.,
1978; Kupferberg, 1989).
The psychomotor 6-Hz model
The 6-Hz model is an alternative electroshock paradigm
that uses low-frequency (6 Hz), long-duration (3 s) elec-
trical stimulation. Corneal stimulation (0.2 ms duration
monopolar rectangular pulses at 6-Hz for 3 s) was deliv-
ered by a constant-current device. During the stimulation,
mice were manually restrained and released into the ob-
servation cage immediately after the current application.
The seizures manifest in ‘‘stunned’’ posture associated with
rearing, forelimb, automatic movements and clonus,
twitching the vibrissae and Straub–tail. The duration of the
seizure activity ranges from 60 to 120 s in untreated ani-
mals. At the end of the seizure, animals resume their nor-
mal exploratory behavior. The experimental end point is
protection against the seizure. The animal is considered to
be protected if it resumes its normal exploratory behavior
within 10 s from the stimulation (Brown et al., 1953).
Phase I of the in vivo studies involved three tests: MES,
scPTZ androtarodtest for NT. Malealbinomice (CF#1 strain,
weighing 18–25 g) and male albino rats (Sprague–Dawley)
were used as experimental animals. The animals were housed
in metabolic cages and allowed free access to food and water.
The compounds were injected intraperitoneally to mice as a
suspension in 0.5 % methylcellulose/water mixture at a dose
level 30, 100 or 300 mg/kg with anticonvulsant activity and
neurotoxicity assessment at 0.5 and 4 h after administration.
Promising derivatives from phase I underwent phase VIa in
which they were administrated orally into rats at a fixed dose
of 30 mg/kg for both MES and the rotarod toxicity determi-
nationatfivepretreatmenttimes: 0.25, 0.5,1,2, 4 h. Groupsof
eight mice or four rats are employed.
Maximal electroshock seizure test (MES)
In vitro studies–sodium channel binding assay
In the MES screen, an electrical stimulus of 0.2 s in du-
ration (50 mA in mice and 150 mA in rats) is delivered via
The radioligand binding studies were performed commer-
cially in Cerep Laboratories (Poitiers, France) using testing
123

Med Chem Res
Table 6 Experimental conditions—Na? channel (site 2) binding assays
Source
Ligand
Concentration
10 nM
Kd
Nonspecific binding
Incubation
Detection method
Rat cerebral cortex
[3H] Batrachotoxin
91 nM
Veratridine (300 lM)
60 min 37 °C
Scintillation counting
Brown GB (1986) 3H-batrachotoxinin-A benzoate binding to voltage-
sensitive sodium channels: inhibition by the channel blockers
tetrodotoxin and saxitoxin. J Neurosci 6(7):2064–2070
Brown WC, Schiffman DO, Swinyard EA, Goodman LS (1953)
Comparative assay of an antiepileptic drugs by psychomotor
seizure test and minimal electroshock threshold test. J Pharmacol
Exp Ther 107(3):273–283
procedures described in detail elsewhere (Brown, 1986).
The general information is listed in Table 6.
Conclusion
´
Bruno-Blanch L, Galvez J, Garcia-Domenach R (2003) Topological
In the current studies, the library of twenty-two new
N-phenyl-2-(4-phenylpiperazin-1-yl)acetamide derivatives
has been synthesized and evaluated for their anticonvulsant
activity in the maximum electroshock (MES) and scPTZ
seizure tests. The results of in vivo pharmacological studies
showed activity exclusively in the MES seizures especially
for 3-(trifluoromethyl)anilide derivatives, whereas majority
of 3-chloroanilide analogs were inactive. Several mole-
cules showed activity in the 6-Hz screen that may indicate
their potential usefulness in partial and therapy-resistant
epilepsy. The pharmacophoric role of the pyrrolidine-2,5-
dione ring for anticonvulsant properties was proved.
virtual screening: a way to find new anticonvulsant drugs from
chemical diversity. Bioorg Med Chem Lett 13(16):2749–2754
Catterall WA, Morrow CS, Daly JW, Brown GB (1981) Binding of
batrachotoxinin A 20-alpha-benzoate to a receptor site associ-
ated with sodium channels in synaptic nerve ending particles.
J Biol Chem 256(17):8922–8927
Chang BS, Lowenstein DH (2003) Epilepsy.
349(13):1257–1266
Dunham NW, Miya TA (1957) A note on a simple apparatus for
detecting neurological deficit in rats and mice. J Am Pharm
Assoc 46(3):208–209 (Baltim.)
Ghidini E, Delcanale M, De Fanti R, Rizzi A, Mazzuferi M, Rodi D,
Simonato M, Lipreri M, Bassani F, Battipaglia L, Bergamaschi
M, Villetti G (2006) Synthesis and anticonvulsant activity of a
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N Eng J Med
Acknowledgments The authors wish to thank the Antiepileptic
Drug Development Program (Epilepsy Branch, National Institute of
Neurological Disorders and Stroke, National Institute of Health,
Rockville, MD, USA), for pharmacological data. We are also grateful
Hen H, Bialer M, Wlodarczyk B, Finnell RF, Yagen B (2010) Syntheses
and evaluation of anticonvulsant profile and teratogenicity of
novel amide derivatives of branched aliphatic carboxylic acids
with 4-aminobenzensulfonamide. J Med Chem 53(10):4177–4186
Ismail FMD (2002) Important fluorinated drugs in experimental and
clinical use. J Fluorine Chem 118(1–2):27–1033
´
to Professor Katarzyna Kiec-Kononowicz (Department of Tech-
nology and Biotechnology of Drugs, Jagiellonian University Medical
´
College, Krakow, Poland) the coordinator of the ADD Program from
Polish site and Mr. Janusz Gorecki for the synthesis of several
´
compounds.
´
Kaminski K, Rzepka S, Obniska J (2011a) Synthesis and anticonvulsant
Conflict of interest The authors declare no conflict of interest.
activity of new 1-[2-oxo-2-(4-phenylpiperazin-1-yl)ethyl]pyrrolidine-
2,5-diones. Bioorg Med Chem Lett 21(19):5800–5803
Open Access This article is distributed under the terms of the
Creative Commons Attribution License which permits any use,
distribution, and reproduction in any medium, provided the original
author(s) and the source are credited.
´
Kaminski K, Obniska J, Wiklik B, Atamanyuk D (2011b) Synthesis
and anticonvulsant properties of new acetamide derivatives of
phthalimide, and its saturated cyclohexane and norbornene
analogs. Eur J Med Chem 46(9):4634–4641
´
Kaminski K, Obniska J, Chlebek I, Liana P, Pe˛kala E (2013)
Synthesis and biological properties of new N-Mannich bases
derived from 3-methyl-3-phenyl- and 3,3-dimethyl-succinimides
Part V. Eur J Med Chem 66:12–21
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