Synthesis and Determination of Lipophilicity, Anticonvulsant Activity, and Preliminary Safety of 3-Substituted and 3-Unsubstituted N-[(4-Arylpiperazin-1-yl)alkyl]pyrrolidine-2,5-dione Derivatives

Article abstract of DOI:10.1002/cmdc.201700539

A new series of 1,3-substituted pyrrolidine-2,5-dione derivatives as potential anticonvulsant agents are described. Initial pharmacological screening of these compounds was performed by using acute models of seizures (MES and scPTZ tests) in mice after in

Full text of DOI:10.1002/cmdc.201700539

Accepted Article
Title: Synthesis, determination of the lipophilicity, anticonvulsant
activity and preliminary safety of new derivatives of N-[(4-
arylpiperazin-1-yl)-alkyl]-pyrrolidine-2,5-dione
Authors: Sabina Rybka, Jolanta Obniska, Paweł Żmudzki, Paulina
Koczurkiewicz, Katarzyna Wójcik-Pszczoła, Elżbieta Pękala,
Adrian Bryła, and Anna Rapacz
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ChemMedChem
Synthesis, determination of the lipophilicity, anticonvulsant activity and
preliminary safety of new 3-substituted and 3-unsubstituted derivatives of
N-[(4-arylpiperazin-1-yl)-alkyl]-pyrrolidine-2,5-dione
Sabina Rybka,1* Prof. Jolanta Obniska,1 Dr. Paweł Żmudzki,1 Dr. Paulina Koczurkiewicz,2 Dr.
Katarzyna Wójcik-Pszczoła,2 Prof. Elżbieta Pękala,2 Adrian Bryła,3 Dr. Anna Rapacz3
1Department of Medicinal Chemistry, Faculty of Pharmacy, Jagiellonian University, Medical
College, 9 Medyczna Street, 30-688 Kraków, Poland
2Department of Pharmaceutical Biochemistry, Faculty of Pharmacy, Jagiellonian University
Medical College, 9 Medyczna Street, 30-688 Kraków, Poland
3Department of Pharmacodynamics, Faculty of Pharmacy, Jagiellonian University, Medical
College, 9 Medyczna Street, 30-688 Kraków, Poland
Corresponding Author
Phone: + 48 12 620 54 55; Fax: +48 12 657 02 62; E-mail: rybka.sabina@gmail.com
1
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Abstract
A new series of derivatives of 1,3-substituted pyrrolidine-2,5-dione as potential anticonvulsant
agents are described in this paper. Initial pharmacological screening for these compounds was
performed using acute models of seizures (MES and scPTZ tests) in mice after intraperitoneal
administration. A quantitative pharmacological research revealed that the most promising
compound
were
N-[{4-(3-trifluoromethylphenyl)-piperazin-1-yl}-propyl]-3-benzhydryl-
pyrrolidine-2,5-dione monohydrochloride (11) with ED50=75.9 mg/kg (MES test) and N-[{4-
(3,4-dichlorophenyl)-piperazin-1-yl}-ethyl]-3-methyl-pyrrolidine-2,5-dione monohydrochloride
(18) with ED50=88.2 mg/kg (MES test) and ED50=65.7 kg/mg (scPTZ test). These compounds
displayed a more beneficial protective index than well-known antiepileptic drugs. The plausible
mechanism of the action of the compounds 11 and 18 (molecule 11 blocked sodium channel (site
2) and 18 both sodium (site 2) and L-type calcium channels) and their preliminary safety in vitro
were evaluated. Besides, the lipophilicity of all synthesized compounds was determined using the
UPLC/MS method.
Keywords: Pyrrolidine-2,5-diones; Anticonvulsant activity; Lipophilicity; UPLC/MS
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Introduction
About 50 million people worldwide suffer from epilepsy, which makes it one of the most
common neurological diseases globally. Epilepsy is defined as a chronic disorder of the brain
involving recurrent seizures, which are brief episodes of involuntary tremors.[1] Despite the fact
that throughout the recent decades a new generation of antiepileptic drugs (AEDs) has been
introduced to clinical use, seizures are controlled inadequately in up to 30 % of patients with
epilepsy. Moreover, currently available medicaments can trigger a number of serious adverse
effects such as diminished attention span, impaired memory function, intelligence, and language
skills, which significantly reduce the quality of patients’ life.[2] Consequently, the need to identify
more effective and less toxic antiepileptic drugs still exists.
Although intensive research concerning the understanding of the molecular basis of
epileptogenesis have been conducted, the pathophysiology of epilepsy has been unknown, which
makes it difficult for a rational design of new antiepileptic drugs. Therefore, the ligand based
pharmacophore approach, which relies on using modifications of the structures of currently
known AEDs with a view to obtaining new, more efficient, and safer anticonvulsant agents, is
gaining importance.[3] Validity of this fact can be confirmed by studies that provided the third-
generation AEDs such as eslicarbamazepine – an analog of carbamazepine, fluorofelbamate – an
analog of felbamate as well as molecules being presently tested in clinical trials like brivaracetam
and beprodone (VLB-001).[4] Moreover, in this way new promising chemical molecules as
potential anticonvulsants are obtained in many laboratories.[5]
Heterocyclic structures are considered to be relevant in drug designing of new potential
medicaments.[6] A scaffold consisting of a nitrogen heteroatomic system with at least one
carbonyl group and phenyl ring was established as meaningful for anticonvulsant activity.[3] The
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10.1002/cmdc.201700539
ChemMedChem
mentioned core can be observed in both first generation AEDs (phenytoin, ethosuximide) and
novel drugs (levetiracetam). Furthermore, our previous research revealed that 1,3-substituted
pyrrolidine-2,5-dione proved to be essential to design new anticonvulsant molecules. The most
promising properties showed N-Mannich bases derivatives containing phenylpiperazine with
electronegative substituents (especially with chlorine atoms and trifluoromethyl group) at
position-1 and alkyl or aryl groups at position-3 of the imide ring.[7]
Bearing in mind all the above mentioned facts, with a view to search for new active
compounds and to continue systematic SAR (structure-active relationship) discussion among
variously substituted succinimide derivatives, the present study describes the results of the
following modifications. First, benzhydryl, isopropyl, and methyl groups were introduced at
position-3 of the imide ring, unsubstituted derivatives were synthesized, as well. Moreover, 3-
trifluoromethyl- and 3,4-dichloro-phenylpiperazine were connected by ethylene or propylene
linker with pyrrolidine-2,5-dione at position-1 (Fig. 1). The proposed modifications enable us to
establish the influence of mentioned structures on the anticonvulsant activity, which enriches our
knowledge concerning the structure-activity relationship in a group of pyrrolidine-2,5-dione
derivatives.
Figure 1. Proposed structural modifications.
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The activity of synthesized compounds was investigated in vivo, using the maximal
electroshock-induced seizures test (MES) and the subcutaneous pentylenetetrazole test (scPTZ),
which are commonly employed to identify new anticonvulsant candidates in the preclinical
stages. In addition, neurotoxicity was evaluated in the rotarod test (NT).[8] With the aim of
explaining the possible mechanism of action for selected molecules, the binding assays were
conducted in vitro.
Considering that lipophilicity is one of the most crucial physicochemical properties in drug
discovery and design, as it determines many properties such as absorption, distribution,
metabolism, passing through the blood-brain barrier and toxicity,[9] for the synthesized
compounds lipophilic properties were evaluated using the UPLC/MS method. Furthermore, for
the reason that, drugs’ safety assessment plays important role in the preclinical identification of
new medicaments, the selected promising compounds were evaluated in vitro toward their
potential hepatotoxicity and neurotoxicity. Besides, for these derivatives metabolic stability was
examined in vitro.
Results and discussion
Chemistry
Compounds 9–24 were synthesized according to Scheme 1. In the beginning, 2-benzhydryl- (1)
and 2-isopropyl- (2) succinic acids were prepared according to the method described by Miller
and Long,[10] whereas 2-metylsuccinic and succinic acids was purchased from Sigma–Aldrich
(USA). The corresponding 1-(2-aminoethyl)- or 1-(3-aminopropyl)-4-phenylpiperazines were
obtained by the reaction of 4-phenylpiperazines with N-(3-bromoethyl)- or N-(3-bromopropyl)-
phthalimide, which gave 1-ethyl- and 1-propyl-4-phenylpiperazine-phthalimide, respectively.
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ChemMedChem
Next, to obtain suitable 1-(2-aminoethyl)- or 1-(3-aminopropyl)-4-phenylpiperazines,
corresponding phenylpiperazine-alkylphthalimide derivatives were treated with hydrazine.[11]
Compounds 9-24 were obtained by the cyclocondensation reaction of dicarboxylic acid (1-4)
with appropriate substituted 1-(2-aminoethyl)- or 1-(3-aminopropyl)-4-arylpiperazines.
Thereafter, crude products were purified by using column chromatography and then converted
into hydrochloride salts, except for compounds 10, 22 and 24. The purity of all final compounds
was determined by using the HPLC method amounting to > 98 %. Chemical structures were
13
confirmed by both spectral (1H NMR, C NMR, 19 F NMR, LC/MS) and elemental (C, H, N)
analyzes. The synthetic procedure and physicochemical data are described in the experimental
section.
Scheme 1. Synthetic procedures of intermediates 5-8 and final compounds 9-24
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Anticonvulsant screening
Anticonvulsant activity of derivatives 9–24 was evaluated in the MES test after intraperitoneal
injection and scPTZ tests after subcutaneous injection into mice at doses of 100 mg/kg, and
observations were conducted at two different time intervals (0.5 and 4 h). Acute neurological
toxicity was determined in the minimal motor impairment-rotarod screening (NT) in the same
conditions.
The results of the MES, scPTZ and NT tests are presented in Table 1. It can be seen that
most of the synthesized compounds showed protection against the MES and/or scPTZ
convulsions, and the anticonvulsant activity was mainly determined by the presence of the
substituents at position-3 of the imide ring. Derivatives containing benzhydryl (10-12) or
isopropyl (14-16) group at position-3 revealed the most favourable protection in the scPTZ test at
a dose of 100 mg/kg especially after 4 h, whereas compounds devoid of any substituent (21-23)
or possessing methyl group (17-19) at this position tend to be effective in the MES test after 0.5
and/or 4 h. Unfortunately, compounds 15-20 showed neurotoxicity in the rotarod test.
Table 1. Anticonvulsant activity and neurotoxicity of compounds 9–24 (mice, i.p.).
MESa
scPTZb
NTc
Cmpd
R
n
R1
0.5 h 4 h 0.5h
4h
–
0.5 h 4 h
(C6H5)2CH
(C6H5)2CH
2
2
3-CF3
3,4-Cl
–
–
–
–
–
–
–
–
–
9
100
100
10
7
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ChemMedChem
(C6H5)2CH
(C6H5)2CH
(CH3)2CH
(CH3)2CH
(CH3)2CH
(CH3)2CH
CH3
3
3
2
2
3
3
2
2
3
3
2
2
3
3
3-CF3
3,4-Cl
3-CF3
3,4-Cl
3-CF3
3,4-Cl
3-CF3
3,4-Cl
3-CF3
3,4-Cl
3-CF3
3,4-Cl
3-CF3
3,4-Cl
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
100
100
–
–
–
–
–
–
–
–
–
11
12
13
14
15
16
17
18
19
20
21
22
23
24
–
–
100
100
100
–
100
–
100
100
100
100
100
100
–
–
–
–
–
–
–
–
–
–
–
100 100
CH3
–
100 100
100
–
CH3
100 100
–
–
CH3
–
–
–
H
100 100
100
100 100
–
H
–
–
–
–
100
–
–
H
–
H
–
–
–
–
The values in the table indicate the minimum dose whereby bioactivity or neurotoxicity was demonstrated in half or
more animals. A dash indicates the absence of activity or neurotoxicity at a dose of 100 mg/kg. a Maximal
electroshock test; b Subcutaneous pentylenetetrazole test; c Neurotoxicity screening - rotarod test.
Furthermore, on the basis of the above preliminary data, the most active molecules were
selected for their quantification of pharmacological parameters (ED50 and TD50) in the MES
and/or scPTZ tests. The results are presented in Table 2. The quantitative data were examined at a
previously estimated time to peak effect (TPE). It can be noticed that the median effective doses
(ED50) and protection index (PI) of all compounds were significantly more beneficial than
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ChemMedChem
reference antiepileptic drugs (in the MES and scPTZ tests than valproic acid, and in the scPTZ
than ethosuximide). In addition, molecules 18 and 22 showed good anticonvulsant activity in
both the MES and scPTZ tests, which means that, they have the potentiality to prevent different
types of seizures such as generalized tonic-clonic and absence seizures.
Table 2. Quantitative anticonvulsant data in mice after i.p. administration.
ED50 (mg/kg)b
PIc
TD50
(mg/kg)b
Cmpd TPE (h)a
MES
scPTZ
MES
scPTZ
72.1
4
4
4
4
4
4
4
4
4
–
>300
>300
>300
–
>4.16
10
11
12
16
17
18
19
21
22
(49.7-104.7)
75.9
–
–
–
–
>3.95
>3.31
3.54
–
(61.8-93.3)
90.5
–
(72.0-113.8)
69.6
246.6
–
(54.9-88.3)
(236.2-257.4)
78.6
244.3
–
3.10
3.04
1.43
1.70
2.95
(66.2-93.3)
(234.1-254.9)
88.2
65.7
268.4
4.15
1.80
–
(69.3-112.4)
(46.1-93.7)
(251.5-286.4
81.4
116.8
–
–
(69.2-97.2)
(102.2-133.5)
95.4
162.4
(86.5-105.4)
(145.6-181.2)
101.5
59.7
>300
5.02
(70.9-145.4)
(36.3-98.3)
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92.1
4
–
<300
318.0
<3.26
–
2.26
–
23
(50.3-168.6)
140.4
ETXd
LCSd
VPAd
0.25
0.5
0.5
–
–
(115.8–170.2) (295.8-341.9)
9.24
46.3
–
5.0
1.7
(8.3–10.3)
(44.5-48.0)
252.7
239.5
430.8
1.8
(220.1–290.2) (209.2–274.1) (407.9-454.9)
a Time to peak effect; b Results are represented as mean ± SEM at 95% confidence limit (MES–maximal
c
electroshock test; scPTZ–subcutaneous pentylenetetrazole test; NT–neurotoxicity-rotarod screen); Protection index
(TD50/ED50); d Reference drug tested in the same conditions. Data from the previous study.[12] AEDs: ETX–
ethosuximide; LCS–lacosamide; VPA–valproic acid.
Structure-activity relationship
Based on conducted research (Tables 1 and 2), it is possible to conclude that anticonvulsant
activity is closely related to the sort of substituent at position-3 of pyrrolidine-2,5-dione. The
groups at this position determined effectiveness in protection against different types of seizures.
In the scPTZ test 3-benzhydryl (9-12) and 3-isopropyl (13-16) derivatives demonstrated the most
beneficial anti-seizures protection, whereas 3-methyl (17-20) or unsubstituted (21-24) molecules
were active in the MES test. Moreover, all obtained 3-benzhydryl derivatives did not show
neurotoxicity. With regard to the influence of phenylpiperazine moiety at position-1 on
anticonvulsant activity, both 3-trifluoromethyl- and 3,4-dichloro-phenylpiperazine derivatives
revealed effectiveness, however, 3-trifluoromethyl- (15, 17, 19, 21 and 23) derivatives had
tendency to be active mostly in MES test and 3,4-dichloro- (10, 12, 14, 16, 18 and 22) derivatives
in both the MES and scPTZ tests. In addition, it can be noticed that the length of alkyl chain
influenced the anticonvulsant activity, namely, compounds with benzhydryl (9) and isopropyl
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(13) groups at position-3 of the imide ring, and ethylene linker between imide and 3-
trifluoromethylphenylpiperazine as well as derivatives of pirolidine-2,5-dione and its 3-methyl
(20) and unsubstituted (24) analogs with a propylene linker and 3,4-dichlorophenylpiperazine did
not show any anticonvulsant activity.
Taking into account the previous research[7,13] and results described above, some general
conclusions about the relationship between the structure and the anticonvulsant activity in a
group of aminoalkyl derivatives of 1,3-substituted pyrrolidine-2,5-dione can be made. First, 3-
benzhydryl derivatives of imide revealed the most beneficial anticonvulsant effectives in the
scPTZ test as well as a lack of neurotoxicity. Then, introduction of 1-phenylethyl or benzyl
moieties at the position-3 caused a decline in anti-seizures protection, but removing the
substituents from this position or introduction of a small electro-donor methyl group led to obtain
structures showing a significant activity in both the MES and scPTZ tests. As mentioned above,
the most promising anticonvulsant activity showed derivatives containing 3-trifluoromethyl- and
3,4-dichloro-phenylpiperazine at position-1 of the imide ring (N-Mannich bases), consequently,
for these substances their analogs with ethylene or propylene linker between imide and piperazine
nitrogen atom have been synthesized. It enabled us to obtain compounds characterizing favorable
anticonvulsant activity. It can be noticed that lengthening of the alkyl chain resulted in
derivatives revealing anticonvulsant activity not only after 4 h, but also after 0.5 h, which means
that they displayed rapid onset and long-lasting anticonvulsant activity. Furthermore, the fact is
worth mentioning, that isopropyl derivatives possessing ethylene or propylene linker between
imide and piperazine nitrogen showed activity, contrary to their analogs, which in this place had
methylene group. To sum up, based on the described analysis of structure-activity relationship, it
is possible to design more effective new anticonvulsants.
11
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In vitro radioligand binding studies
Despite many years of intensive physiological and biochemical researches concerning the
explanation of epilepsy’s pathogenesis, still it is not exactly known in what way the
anticonvulsant drugs decline seizures. Nevertheless, it is known that seizures are provoked by the
hyperactivity of voltage-gated sodium channels (VGSCs) and voltage-gated calcium channels
(VDCCs) in CNS. The mentioned channels are molecular targets of a great number of chemically
diversified anticonvulsant drugs.[14] Hence, two different in terms of structure (N-[{4-(3-
trifluoromethylphenyl)-piperazin-1-yl}-propyl]-3-benzhydryl-pyrrolidine-2,5-dione (11) and N-
[{4-(3,4-dichlorophenyl)-piperazin-1-yl}-ethyl]-3-methyl-pyrrolidine-2,5-dione (18)) and activity
(in the MES and/or scPTZ tests) compounds were chosen, and their influence on sodium (site 2)
and calcium channels (L and N type) was evaluated in vitro. As shown in Table 3, both
derivatives 11 and 18 displayed prominent blocking of voltage-sensitive Na+ channel at a
concentration of 100 μM. It should be highlighted that these compounds exhibited a stronger
affinity to sodium channel than phenytoin – well-known blocker of voltage-sensitive sodium
channel. In terms of voltage-sensitive Ca2+ channel, molecule 18 exhibited similar binding to this
molecular target as phenytoin. Nonetheless, the tested compounds did not block the N-type
calcium channel. The conducted study proved that the mechanism of action of the synthesized
derivatives is substantially related to blocking of the sodium channel (site 2).
Table 3. In vitro binding assays (concentration 100 μM).
% Inhibition of control specific bindinga
Voltage-sensitive Na+ channel
Voltage-sensitive Ca2+ channel
No
(site 2) b
L-typec
72.3
4.2
11
18
82.7
54.4
57.8
53.9
Phenytoin
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a Results showing an inhibition higher than 50% are considered to represent significant effects of the test compounds;
results showing an inhibition between 25% and 50% are indicative of moderate effect; results showing an inhibition
lower than 25% are not considered significant and mostly attributable to variability of the signal around the control
level.
b Compound was evaluated in synaptoneurosomal preparations from the rat cerebral cortex as inhibitors of the
specific binding of [3H]BTX to the voltage-sensitive sodium channel.[15]
c
Compound was evaluated in synaptoneurosomal preparations from the rat cerebral cortex as inhibitors of the
specific binding of [3H]nitrendipine to the voltage-sensitive Ca2+ channel.[16]
Lipophilicity
Lipophilicity is one of the most relevant physicochemical properties, therefore, it was decided to
evaluate this parameter for the synthesized compounds. Determination of lipophilicity was
performed by Ultra Performance Liquid Chromatography coupled with Mass Spectrometry
(UPLC/MS) and it is worth emphasizing that new derivatives were examined alongside one
another. Moreover, in silico studies were performed by using the ACD/Labs and ChemBioDraw
to compare log P values obtained by the experimental method (UPLC/MS) and in silico. The
results are shown in Table 4. As expected, the values of lipophilicity were determined by the type
of substituents at position-3 of the imide ring, namely, the most lipophilic properties displayed
compounds with benzhydryl group at this position, and then isopropyl, methyl and unsubstituted
derivatives of pyrrolidine-2,5-dione. Moreover, compounds possessing propylene linker between
imide and piperazine nitrogen atom revealed slightly higher lipophilicity than their appropriate
analogs with ethylene linker.
Table 4. Log P values received for compounds 9-24.
Log P
ChemBioDraw ACD
Compound
UPLC/MS
5.268
5.64
5.84
5.75
5.94
3.27
3.46
5.64
6.22
6.20
6.44
3.64
3.87
9
5.267
5.329
5.425
3.985
10
11
12
13
14
4.125
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3.37
3.57
2.52
2.71
2.06
2.82
1.95
2.15
2.62
2.25
3.85
3.57
2.52
3.00
2.97
3.21
1.95
2.50
2.48
2.25
3.893
4.043
3.286
3.344
3.310
3.372
3.005
3.049
3.060
3.110
15
16
17
18
19
20
21
22
23
24
Based on presented results, it can be noticed that both log P values obtained by both the
experimental method (UPLC/MS) and in silico studies are similar. In addition, correlation
coefficients (r2) between log P designated in UPLC/MS and log P obtained via in silico methods
were calculated and their values amounted to 0.9693 and 0.975, respectively, (see Fig. 2)
whereby the UPLC/MS method seems to be appropriate to determinate lipophilicity for these
types of compounds. Moreover, using the UPLC/MS method many compounds can be examined
alongside one another, which makes this method quicker and cheaper (it is used considerably less
solvents) than others.
8
7
6
5
4
3
2
8
7
6
5
4
3
2
R² = 0,975
R² = 0,9693
1
2
3
4
5
6
7
1
2
3
4
5
6
7
log P UPLC
log P UPLC
Figure 2. Correlation between experimental method (UPLC/MS) and in silico studies.
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Furthermore, the relationship between lipophilicity (log P) and anticonvulsant activity
both in the MES and scPTZ was evaluated by using the non-parametric Mann-Whitney test. The
results are shown in Fig. 3. It can be observed that activity in the MES test showed compounds
which average value of log P was 3.276, while molecules with average value of log P 4.115
revealed effectiveness in the scPTZ test. Therefore, it can be concluded that in these series of
derivatives, lipophilicity determined a type of anticonvulsant activity. The obtained correlation in
terms of the MES test is statistically significant (p=0.005), however, when it comes to the scPTZ
test is not statistically significant (p=0.074).
Figure 3. Relationship between lipophilicity and anticonvulsant activity in MES and scPTZ tests.
Hepatotoxicity and neurotoxicity
Safety profile of drugs’ candidates for preclinical development continue to be a challenge for the
pharmaceutical science.[17] Hepatotoxicity and neurotoxicity remain the main reasons for
compound termination. Considering that assessment of preliminary safety of new compounds is
an important issue in drug discovery, compounds 11 and 18 were evaluated in vitro for their
hepatotoxicity and neurotoxicity. Preliminary screening of compounds’ safety can be conducted
using standardized in vitro methods.[18]
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Results obtained from a viability test clearly showed that both the analyzed compounds
(11, 18) did not show any cytotoxic effect on hepatoma as well as neuroblastoma cell lines. The
performed in vitro analyzes proved that tested compounds are safe and do not affect cellular
metabolism (MTT). In addition, the cytotoxicity effect of chemotherapeutic agents – doxorubicin
(anthracycline antibiotics) and valproic acid (primarily used to treat epilepsy and bipolar
disorder) were evaluated as positive controls. Doxorubicin and valproic acid used in the same
concentrations as evaluated compounds (11, 18) possessed stronger cytotoxic effects (Fig. 4).
Figure 4. Result from MTT viability assays performed on HepG2 and SH-SY5Y cells after 24 h
incubation with tested and references compounds. Results are presented as mean±SEM calculated
from at least three independent experiments.
In vitro metabolic stability
Metabolism as one of the drugs’ pharmacokinetic characteristics can be evaluated during the
early preclinical stage to assess the degree of drug candidate conversion into a set of metabolites.
Metabolic stability can be assessed by the incubation of a potent drug in the presence of liver
microsomes and NADPH to gain an insight into metabolic properties.[19] Metabolic stability is
usually quantified by monitoring the disappearance over time of the parent compound in liver
preparations. The disappearance of the parent compound was monitored by calculating the
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percentage of each compound remaining relative to 0 ꢀmin and this was plotted against the
incubation time. Currently, commercially available mouse liver microsomes (MLM) have been
the most widely used in vitro models for metabolic stability studies.[20]
Compounds 11 and 18 were selected for in vitro metabolic stability assays. In terms of the
conditions of our study, the mentioned compounds are extensively biotransformed in mouse
microsomes model. They are unstable because they exhibit a relatively high clearance of 136 and
255 μl/mg/min, respectively. Moreover, during the biotransformation process these derivatives
generated six metabolites involving reaction of hydroxylation and N-dealkylation. (see
Supporting Information Table 1S and Figure 1S).
Conclusion
A library of 16 new aminoalkyl derivatives of pyrrolidine-2,5-dione was synthesized and
evaluated for anticonvulsant activity in the MES and scPTZ seizure tests in mice. The obtained
results revealed that most of the new molecules displayed more favourable anticonvulsant
activity in the MES and/or scPTZ test than the well-known anticonvulsant drugs such as valproic
acid, ethosuximide or lacosamide. Furthermore, in vitro binding studies provided essential
evidence that the possible mechanism of action of new compounds is related to blocking of
voltage-sensitive sodium (site 2) and L-type calcium channels. Moreover, for the most promising
compounds hepatotoxicity and neurotoxicity was performed in vitro, and none of the tested
derivatives revealed cytotoxic effect on hepatoma and neuroblastoma cell lines. Although the
chosen compounds did not show metabolic stability, this research provided us considerably
significant information concerning possible ways of metabolism, which will contribute to
designing new more stable and effective anticonvulsant molecules.
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Moreover, the lipophilicity using the UPLC/MS method was established for all the newly
synthesised compounds. The derivatives showed diversity in lipophilic properties. Comparing
obtained lipophilic values (the UPLC/MS method) with theoretical lipophilic values (in silico
methods), it can be noticed that the UPLC/MS method is suitable for the determination of
lipophilicity of derivatives of 1,3-substituted pyrrolidine-2,5-dione. It should be also emphasized
that UPLC/MS method enabled us to examine alongside one another many compounds, which
makes this method quicker and cheaper than others. Furthermore, it is worth mentioning that it
was possible to find relationship between log P and anticonvulsant activity, namely compounds
with lower (3.0-3.4) log P value tended to be active in the MES test, while derivatives with
higher (3.5-5.4) log P value showed protection in the scPTZ test.
Experimental section
Chemistry
All the chemicals and solvents were purchased from Sigma Aldrich (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 with thin-layer
chromatography (TLC) and HPLC. The TLC was performed on Merck silica gel 60 F254
aluminum sheets (Merck; Darmstadt, Germany), using developing systems consisted of S1
dichloromethane: methanol (9 : 0.5, v/v). Spots were detected by their absorption under UV light
(λ = 254 nm). HPLC analyses were run on a HPLC Waters 2695 Separation Module equipped
with photodiode array detector (Waters 2698). A Chromolith RP-18 SpeedROD column (4.6 x 50
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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 μL. Standard solutions (1 mg/ml) of each compound were prepared in
analytical grade acetonitrile and analysed at wavelengths 214 nm and 254 nm. Retention times
1
13
19
(tR) are given in minutes. H NMR, C NMR and F NMR spectra were obtained in a Varian
Mercury spectrometer (Varian Inc., Palo Alto, CA, USA), in CDCl3 or DMSO, operating at 300
MHz ,75 MHz, or 282 MHz, respectively. Chemical shifts are reported in δ values (ppm) relative
to TMS δ = 0 (1H), as internal standard. The J values are expressed in Hertz (Hz). Signal
multiplicities are represented by the following abbreviations: s (singlet), brs (broad singlet), d
(doublet), dd (doublet of doublets), t (triplet), m (multiplet). The mass spectra for compounds 5-
24 were obtained on Waters ACQUITY™ TQD system with the TQ Detector (Waters, Milford,
USA). The ACQUITY UPLC BEH C18, 1.7 μm, 2.1  100 mm column was used (Waters,
1
13
Milford, USA). H NMR, C NMR, 19F NMR and LC/MS spectra for selected final compounds
were shown in Supporting Information. Preparative column chromatography was performed
using silica gel 60 (particle size 0.063-0.200; 70-230 Mesh ATM) purchased from Merck
(Darmstadt, Germany).
General procedure for the synthesis of 2-benzhydryl- and 2-isopropyl-succinic acids (1, 2):
The starting 2-benzhydryl- (1) and 2-isopropyl-succinic (2) acids have been obtained by using
procedure described elsewhere,[10] (2-metyl or succinic acids was purchased from Sigma–Aldrich
(USA)).
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General procedure for the preparation of the 1-(2-aminoethyl)- and 1-(3-aminopropyl)-4-
arylpiperazines (5–8): 3-trifluoromethyl- (2.3 g, 0.01 mol) or 3,4-dichlorophenylpiperazine (2.3
g, 0.01 mol) was dissolved in 1-butanol (25 mL) and N-(3-bromoethyl)- (3.8 g, 0.015 mol) or N-
(3-bromopropyl)-phthalimide (4.0 g, 0.015 mol ) was added. The mixture was refluxed for 20 h.
Thereafter, the reaction mixture was quenched with NH4OH and extracted with methylene
chloride (3 x 20 mL). The organic layer was evaporated and then hydrazine monohydrate (1.5g,
0.03 mol) in EtOH was added and heating for 1 h. Subsequently, 33% HCl (5 mL) was added and
the mixture was heated for 4 h. After cooling it, n-hexane and NH4OH were added and the
mixture was extracted with methylene chloride. The crude products were purified by
crystallisation with ethanol.
2-[4-(3-trifluoromethylphenyl)-piperazin-1-yl]-ethan-1-amine (5): Yellow oil; yield: 2.1 g, 77
%; HPLC (purity 96 %): tR = 3.36 min; ESI-MS m/z: 274.14 (C13H18N3F3 [M+H]+); Anal. calcd
for C13H18N3F3 (273.15): C: 57.13, H: 6.64, N: 15.38; Found C: 57.20, H: 6.72, N: 15.42.
3-[4-(3-trifluoromethylphenyl)-piperazin-1-yl]-propan-1-amine (6): Yellow oil; yield: 2.3 g,
80 %; HPLC (purity 95 %): tR = 2.49 min; ESI-MS m/z: 288.09 (C14H20N3F3 [M+H]+); Anal.
calcd for C14H20N3F3 (287.16): C: 58.52, H: 7.02, N: 14.62; Found C: 58.68, H: 7.13, N: 14.68.
2-[4-(3,4-dichlorophenyl)-piperazin-1-yl]-ethan-1-amine (7): Yellow oil; yield: 2.1 g, 78 %;
HPLC (purity 95 %): tR = 3.34 min; ESI-MS m/z: 274.24 (C12H17N3Cl2 [M+H]+); Anal. calcd for
C12H17N3Cl2 (274.19) C: 52.57, H: 6.25, N: 15.33; Found C: 52.61, H: 6.32, N: 15.41.
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3-[4-(3,4-dichlorophenyl)-piperazin-1-yl]-propan-1-amine (8): Yellow oil; yield: 2.2 g, 76 %;
HPLC (purity 96 %): tR = 2.74 min; ESI-MS m/z: 287.14 (C13H19N3Cl2 [M+H]+); Anal. calcd for
C13H19N3Cl2 (288.22) C: 54.17, H: 6.64, N: 14.58; Found C: 54.23, H: 6.75, N: 14.65.
General procedure for the preparation of the N-[(4-arylpiperazinyl-1-yl)-alkyl]-pyrrolidine-
2,5-dione derivatives (9–24): 0.001 mol of the appropriately succinic acid (1-4) was dissolved in
10 mL of water and 0.001 mol of substituted 1-aminoethyl- (5, 6) or 1-aminopropyl-4-
arylpiperazine (7, 8) was added. The mixture was heated in termperature-regulated sand bath (ST
72 Roth, Karlsruhe, Germany) with simultaneous distillation of water. After complete removal of
water, the temperature of the reaction mixture was raised up to 180°C and maintained for 1.5 h.
The crude products were purified by column chromatography (dichloromethane: methanol 9 :
0.3, v/v (9-16) or dichloromethane: methanol 9 : 0.5, v/v (17-24). The compounds (9-21 and 23)
were converted into hydrochloride salts, while the other derivatives (10, 22 and 24) were
obtained as free bases.
N-[{4-(3-trifluoromethylphenyl)-piperazin-1-yl}-ethyl]-3-benzhydryl-pyrrolidine-2,5-dione
monohydrochloride (9): White powdery crystals; yield: 351 mg, 61 %; mp 238–240°C;
TLC: Rf = 0.63 (S1); HPLC (purity 98.68 %): tR = 6.20 min; ESI-MS m/z: 522.48 (C30H30N3O2F3
[M+H]+); Anal. calcd for C30H31N3O2F3Cl (558.03) C: 64.57, H: 5.60, N: 7.53; Found C: 64.68,
H: 5.70, N: 7.58
N-[{4-(3,4-dichlorophenyl)-piperazin-1-yl}-ethyl]-3-benzhydryl-pyrrolidine-2,5-dione (10):
White powdery crystals; yield: 328 mg, 63 %; mp 220–222°C; TLC: Rf = 0.62 (S1); HPLC (purity
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100 %): tR = 6.36 min; ESI-MS m/z: 522.24 (C29H29N3O2Cl2 [M+H]+); Anal. calcd for
C29H29N3O2Cl2 (522.47): C: 66.67, H: 5.59, N: 8.04; Found C: 66.75, H: 5.63, N: 8.10.
N-[{4-(3-trifluoromethylphenyl)-piperazin-1-yl}-propyl]-3-benzhydryl-pyrrolidine-2,5-
dione monohydrochloride (11): White powdery crystals; yield: 343 mg, 60 %; mp 144–146°C;
TLC: Rf = 0.58 (S1); HPLC (purity 99.47 %): tR = 6.14 min; ESI-MS m/z: 536.27 (C31H32N3O2F3
[M+H]+); Anal. calcd for C31H33N3O2F3Cl (572.06): C: 65.09, H: 5.81, N: 7.35; Found C: 65.13,
H: 5.89, N: 7.46
N-[{4-(3,4-dichlorophenyl)-piperazin-1-yl}-propyl]-3-benzhydryl-pyrrolidine-2,5-dione
monohydrochloride (12): White powdery crystals; yield: 371 mg, 65%; mp 146–148°C; TLC:
Rf = 0.52 (S1); HPLC (purity 98.49 %): tR = 6.20 min; ESI-MS m/z: 536.20 (C30H31N3O2Cl2
[M+H]+); Anal. calcd for C30H32N3O2Cl3 (572.95): C: 62.89, H: 5.63, N: 7.33; Found C: 62.95,
H: 5.72, N: 7.41.
N-[{4-(3-trifluoromethylphenyl)-piperazin-1-yl}-ethyl]-3-isopropyl-pyrrolidine-2,5-dione
monohydrochloride (13): White powdery crystals; yield: 329 mg, 76%; mp 216–218°C; TLC:
Rf = 0.46 (S1); HPLC (purity 99.42 %): tR = 4.78 min; ESI-MS m/z: 398.22 (C20H26N3O2F3
[M+H]+); Anal. calcd for C20H27N3O2 F3Cl (433.90): C: 55.36, H: 6.27, N: 9.68; Found C: 55.41,
H: 6.37, N: 9.73
N-[{4-(3,4-dichlorophenyl)-piperazin-1-yl}-ethyl]-3-isopropyl-pyrrolidine-2,5-dione mono-
hydrochloride (14): White powdery crystals; yield: 338 mg, 78 %; mp 228–230°C; TLC:
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Rf = 0.51 (S1); HPLC (purity 98.52 %): tR = 4.94 min; ESI-MS m/z: 398.09 (C19H25N3O2Cl2
[M+H]+); Anal. calcd for C19H26N3O2Cl3 (434.79): C: 52.49, H: 6.03, N: 9.66; Found C: 52.52,
H: 6.11, N: 9.72.
N-[{4-(3-trifluoromethylphenyl)-piperazin-1-yl}-propyl]-3-isopropyl-pyrrolidine-2,5-dione
monohydrochloride (15): White powdery crystals; yield: 358 mg, 80 %; mp 196–198°C; TLC:
Rf = 0.42 (S1); HPLC (purity 98.95 %): tR = 4.82 min; ESI-MS m/z: 412.18 (C21H28N3O2F3
[M+H]+); Anal. calcd for C21H29N3O2F3Cl (447.92): C: 56.31, H: 6.53, N: 9.38; Found C: 56.38,
H: 6.59, N: 9.45
N-[{4-(3,4-dichlorophenyl)-piperazin-1-yl}-propyl]-3-isopropyl-pyrrolidine-2,5-dione
monohydrochloride (16): White powdery crystals; yield: 340 mg, 76 %; mp 197–199°C; TLC:
Rf = 0.35 (S1); HPLC (purity 99.52 %): tR = 4.86 min; ESI-MS m/z: 412.11 (C20H27N3O2Cl2
[M+H]+); Anal. calcd for C20H28N3O2Cl3 (448.82): C: 53.52, H: 6.29, N: 9.36; Found C: 53.58,
H: 6.36, N: 9.47.
N-[{4-(3-trifluoromethylphenyl)-piperazin-1-yl}-ethyl]-3-methyl-pyrrolidine-2,5-dione
monohydrochloride (17): White powdery crystals; yield: 304 mg, 75%; mp 230–232°C; TLC:
Rf = 0.40 (S1); HPLC (purity 99.64 %): tR = 4.08 min; ESI-MS m/z: 370.17 (C18H22N3O2F3
[M+H]+); Anal. calcd for C18H23N3O2F3Cl (405.84): C: 53.27, H: 5.71, N: 10.35; Found C: 53.35,
H: 5.79, N: 10.39.
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N-[{4-(3,4-dichlorophenyl)-piperazin-1-yl}-ethyl]-3-methyl-pyrrolidine-2,5-dione
mono-
hydrochloride (18): White powdery crystals; yield: 316 mg, 78%; mp 254–256°C; TLC:
Rf = 0.44 (S1); HPLC (purity 100 %): tR = 4.33 min; ESI-MS m/z: 370.11 (C17H21N3O2Cl2
[M+H]+); Anal. calcd for C17H22N3O2Cl3 (406.73): C: 50.20, H: 5.45, N: 10.33; Found C: 50.28,
H: 5.53, N: 10.38.
N-[{4-(3-trifluoromethylphenyl)-piperazin-1-yl}-propyl]-3-methyl-pyrrolidine-2,5-dione
monohydrochloride (19): White powdery crystals; yield: 320 mg, 80 %; mp 201–203°C; TLC:
Rf = 0.41 (S1); HPLC (purity 100 %): tR = 4.40 min; ESI-MS m/z: 384.20 (C19H24N3O2F [M+H]+);
Anal. calcd for C19H25N3O2F3Cl (419.87): C: 54.35, H: 6.00, N: 10.01; Found C: 54.40, H: 6.09,
N: 10.06.
N-[{4-(3,4-dichlorophenyl)-piperazin-1-yl}-propyl]-3-methyl-pyrrolidine-2,5-dione
monohydrochloride (20): White powdery crystals; yield: 344 mg, 82%; mp 222–224°C; TLC:
Rf = 0.38 (S1); HPLC (purity 100 %): tR = 4.43 min; ESI-MS m/z: 384.34 (C18H23N3O2Cl2
[M+H]+); Anal. calcd for C18H24N3O2Cl3 (420.76): C: 51.38, H: 5.75, N: 9.99; Found C: 51.45,
H: 5.83, N: 10.08.
N-[{4-(3-trifluoromethylphenyl)-piperazin-1-yl}-ethyl]-pyrrolidine-2,5-dione monohydro-
chloride (21): White powdery crystals; yield: 293 mg, 75%; mp 234–236°C; TLC: Rf = 0.48 (S1);
HPLC (purity 98.76 %): tR = 3.96 min; ESI-MS m/z: 356.15 (C17H20N3O2F [M+H]+); Anal. calcd
for C17H21N3O2F3Cl (391.82) C: 52.11, H: 5.40, N: 10.72; Found C: 52.20, H: 5.46, N: 10.80.
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N-[{4-(3,4-dichlorophenyl)-piperazin-1-yl}-ethyl]-pyrrolidine-2,5-dione (22): White powdery
crystals; yield: 267 mg, 70%; mp 160–162°C; TLC: Rf = 0.36 (S1); HPLC (purity 99.06 %):
tR = 4.05 min; ESI-MS m/z: 356.08 (C16H19N3O2Cl2 [M+H]+); Anal. calcd for C16H19N3O2Cl2
(356.25) C: 53.94, H: 5.38, N: 11.80; Found C: 54.00, H: 5.46, N: 11.85
N-[{4-(3-trifluoromethylphenyl)-piperazin-1-yl}-propyl]-pyrrolidine-2,5-dione monohydro-
chloride (23): White powdery crystals; yield: 324 mg, 80%; mp 229–231°C; TLC: Rf = 0.33 (S1);
HPLC (purity 98.23 %): tR = 3.97 min; ESI-MS m/z: 370.17 (C18H22N3O2F3 [M+H]+); Anal. calcd
for C18H23N3O2F3Cl (405.84) C: 53.27, H: 5.71, N: 10.35; Found C: 53.32, H: 5.79, N: 10.42
N-[{4-(3,4-dichlorophenyl)-piperazin-1-yl}-propyl]-pyrrolidine-2,5-dione
(24):
White
powdery crystals; yield: 284 mg, 77 %; mp 121–123°C; TLC: Rf = 0.40 (S1); HPLC (purity 98.76
%): tR = 3.98 min; ESI-MS m/z: 370.04 (C17H21N3O2Cl2 [M+H]+); Anal. calcd for C17H21N3O2Cl2
(370.27) C: 55.14, H: 5.72, N: 11.35; Found C: 55.20, H: 5.79, N: 11.43
Lipophilicity
Reagents: LC-MS grade methanol and 99.99% ammonium acetate were purchased from Sigma-
Aldrich (Saint Louis, MO, USA). LC-MS grade 25% ammonium hydroxide solution was
purchased from Fluka. HPLC grade water was obtained from an HLP 5 (HYDROLAB Poland)
apparatus and was filtered through a 0.2µm filter before use.
Separation conditions: The UPLC/MS system consisted of a Waters ACQUITY UPLC (Waters
Corporation, Milford, MA, USA) coupled to a Waters TQD mass spectrometer (electrospray
ionization mode ESI – tandem quadrupole). Chromatographic separations were carried out using
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the Acquity UPLC BEH (bridged ethyl hybrid) C18 column; 2.1 × 100 mm, and 1.7 µm particle
size, equipped with Acquity UPLC BEH C18 VanGuard pre-column; 2.1 × 5 mm, and 1.7 µm
particle size. The column was maintained at 40°C, and eluted under isocratic conditions using
from 10% to 70% in 5% intervals of eluent A over 30 min, at a flow rate of 0.3 mL min-1. Eluent
A: water/ammonium acetate (10 mM, pH = 7.4); eluent B: methanol. Chromatograms were
recorded using Waters eλ PDA detector. Spectra were analyzed in 200-700 nm range with 1.2 nm
resolution and sampling rate 20 points/s. 1 µL of each of the solutions of the investigated
compounds was injected. Data acquisition software was MassLynx V 4.1 (Waters).
Preparation of samples: 1 mg of each compound was weighed and dissolved in 1 mL of water-
methanol mixture (1:1, v/v) making 1 mg mL-1 stock solutions. Samples were prepared by mixing
of 20 µL of the stock solutions and diluting the mixture to 1 mL making solution containing the
investigated compounds in the concentration of 20 µg mL-1. Compounds were grouped in order
to avoid preparation of mixture containing several compounds possessing similar m/z and
isotopic peaks pattern.
Acknowledgements
We are pleased to acknowledge the generous financial support of this work by the grant of the
Polish National Scientific Centre, Poland (Grant No. DEC-2013/11/N/NZ7/00426).
Declaration of Interest Statement
The authors report no declarations of interest.
Supplementary material
Supplementary data associated with this article can be found in the online version.
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Abbreviations used:
AEDs, antiepileptic drugs; DOX, Doxorubicin; ETX, ethosuximide; LCS, lacosamide; MES,
maximal electroshock test; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium
Bromide; NT, neurotoxicity; PI, protection index; scPTZ, subcutaneous pentylenetetrazole test;
TPE, time to pick effect; UPLC, Ultra Performance Liquid Chromatography coupled with Mass
Spectrometry; VPA, valproic acid.
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