Synthesis and pharmacological evaluation of novel N-Mannich bases derived from 5,5-diphenyl and 5,5-di(propan-2-yl)imidazolidine-2,4-dione core

Article abstract of DOI:10.1016/j.bmcl.2019.06.001

The aim of this study was to design and synthesize two series of N-Mannich bases with imidazolidine-2,4-dione core as a potential anticonvulsant with reduced toxicity and broad antiseizure activity. Preliminary screening revealed that the majority of synt

Full text of DOI:10.1016/j.bmcl.2019.06.001

Accepted Manuscript
Synthesis and Pharmacological Evaluation of Novel N-Mannich Bases Derived
from 5,5–Diphenyl and 5,5-Di(propan-2-yl)imidazolidine-2,4-dione Core
Anna Czopek, Hanna Byrtus, Agnieszka Zagórska, Joanna Rychtyk, Małgorzata
Góra, Kinga Sałat, Anna Rapacz, Jolanta Obniska
PII:
S0960-894X(19)30368-3
https://doi.org/10.1016/j.bmcl.2019.06.001
BMCL 26479
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 March 2019
26 May 2019
1 June 2019
Please cite this article as: Czopek, A., Byrtus, H., Zagórska, A., Rychtyk, J., Góra, M., Sałat, K., Rapacz, A., Obniska,
J., Synthesis and Pharmacological Evaluation of Novel N-Mannich Bases Derived from 5,5–Diphenyl and 5,5-
Di(propan-2-yl)imidazolidine-2,4-dione Core, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://
doi.org/10.1016/j.bmcl.2019.06.001
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Synthesis and Pharmacological Evaluation of Novel N-Mannich Bases Derived from
5,5-Diphenyl and 5,5-Di(propan-2-yl)imidazolidine-2,4-dione Core
Anna Czopeka*, Hanna Byrtusa, Agnieszka Zagórskaa, Joanna Rychtyka, Małgorzata Góraa,
Kinga Sałatb, Anna Rapaczb, Jolanta Obniskaa
a
Department of Medicinal Chemistry, Faculty of Pharmacy, Jagiellonian University Medical
College, 9 Medyczna St., 30-688 Krakow, Poland
b
Department of Pharmacodynamics, Faculty of Pharmacy, Jagiellonian University Medical
College, 9 Medyczna St., 30-688 Krakow, Poland
Running head: Imidazolidine-2,4-diones - anticonvulsant and antinociceptive activity
*Corresponding author: Anna Czopek, Department of Medicinal Chemistry, Faculty of
Pharmacy, Jagiellonian University Medical College, 9 Medyczna, 30-688 Krakow, Poland,
email: anna.czopek@uj.edu.pl, phone: +48 12 6205450

Abstract
The aim of this study was to design and synthesize two series of N-Mannich bases with
imidazolidine-2,4-dione core as a potential anticonvulsant with reduced toxicity and broad
antiseizure activity. Preliminary screening revealed that the majority of synthesized
compounds were effective in the maximal electroshock seizure (MES) and/or subcutaneous
pentylenetetrazole (scPTZ) test. The most active in vivo compound, 18 (3-((4-
methylpiperazin-1-yl)methyl)-5,5-diphenylimidazolidine-2,4-dione), exhibited an ED50 value
comparable to that of phenytoin in the MES test (38.5 mg/kg vs 28.1 mg/kg), and more
importantly, it showed four times higher potency than phenytoin in the 6 Hz test (12.2 mg/kg
vs >60 mg/kg). Additionally, 18 exhibited antiallodynic properties in the von Frey test in
neuropathic (oxaliplatin-treated) mice. Compound 18 also demonstrated a broader spectrum
of anticonvulsant activity than phenytoin and showed statistically significant antinociceptive
properties in selected models of chronic pain.
Keywords: anticonvulsants, antinociceptive, imidazolidine-2,4-dione, hydantoin

According to epidemiological studies, epilepsy is one of the most common disorders of the
central nervous system (CNS) affecting more than 60 million people worldwide.1 A
characteristic feature of epilepsy is the occurrence of recurrent spontaneous seizures with or
without the loss of consciousness, which are accompanied by motor, sensory, or autonomic
disturbances.2 Despite an enhanced understanding of the pathogenesis of the disease and the
introduction of novel therapeutics whose mechanisms of action differ from commonly used
antiepileptic drugs (AEDs), drug resistance is a common phenomenon in one-third of
patients.3
The activity of most anticonvulsant drugs used clinically is related to neither one specific
mechanism of action nor any particular site of action.4 AEDs exhibit their activity via many
different mechanisms of action. Insufficient understanding of the pathogenesis of human
epilepsy and its complexity make it difficult to develop rational methodologies for the
discovery of candidates for new AEDs. Therefore, at present, researchers mainly focus on
investigations for new anticonvulsant agents through conventional ligand-based screening
approaches, which are based on structure of existing drugs or different pharmacophore
models. To obtain more efficacious compounds that will decrease seizures and will reduce
adverse effects compared to those of the maternal compound, a variety of structural
modifications are introduced.5 Many lines of well-documented evidence emphasize that
anticonvulsant activity is mainly attributed to the presence of the nitrogen heterocyclic
system, usually a cyclic imide, with at least one carbonyl group and hydrophobic aryl/alkyl
group attached to the heterocyclic system. These are essential requirements for substances to
possess a potential antiepileptic activity. This common pharmacophore is present in the
structures of well-known anticonvulsants, for example, ethosuximide and phenytoin (Figure
1).6
Figure 1. Structures of known antiepileptic drugs.

In addition to antiepileptic activity, AEDs exhibit other promising therapeutic activities. They
are used in the treatment of many CNS diseases such as migraine, anxiety, and bipolar
disorder, which appear to be linked to varying hyperexcitability within specific structures of
the CNS.7
AEDs also have applications in the treatment of pain; for example, gabapentin and pregabalin
(Figure 1), second-generation AEDs, are currently recommended as first-line pharmacological
treatment for neuropathic pain,8 and phenytoin (5,5-diphenylimidazolidine-2,4-dione), a first-
generation AED, shows analgesic properties.9 Other differently substituted imidazolidine-2,4-
dione (hydantoin) derivatives also exhibit antinociceptive properties; for instance, a recently
published series of amide derivatives with a hydantoin moiety exhibited analgesic properties
in an acute model of pain.10 Other research studies involving imidazolidin-2,4-dione
fragments have shown their antidepressant, antipsychotic,11,12 and antiarrhythmic properties.13
All these findings highlight that modifications in the heterocyclic core of imidazolidine-2,4-
dione create a variety of opportunities for designing and obtaining new promising therapeutic
agents. Considering the abovementioned information, our research focused on systematic
structural modifications of different heterocyclic systems to elucidate their pharmacological
properties.10,14–17 Therefore, as a continuation of chemical and pharmacological studies in the
designing of new anticonvulsant agents, in the present study, a new series of N-Mannich
bases with imidazolidine-2,4-dione core was obtained. The imidazolidine-2,4-dione ring,
present in phenytoin structure, was modified to obtain less toxic molecules than the maternal
compound. Furthermore, to evaluate the role of aromatic rings in the neurotoxic and
anticonvulsant activity, two phenyl groups present in the phenytoin structure were replaced by
alkyl groups.
Structure–activity relationship studies have demonstrated12,14–16 that anticonvulsant activity
may be enhanced by the presence of the arylpiperazine fragment in the compound structure,
and therefore, favorable, selected amines, for example, phenylpiperazines (unsubstituted or
substituted with electron-withdrawing atoms), benzylpiperazines, phenylethylpiperazines, and
methylpiperazines, have been introduced. To determine whether the introduced modifications
affect their pharmacological properties, the designed molecules were evaluated for their
anticonvulsant and neurotoxic properties in the Antiepileptic Drug Development (ADD)
Program in Epilepsy Branch, National Institutes of Health, National Institute of Neurological
Disorders and Stroke (NIH/NINDS), Rockville, MD. Further, the most active compound from
the MES test was chosen for the assessment of its antinociceptive activity by using the
formalin test and the oxaliplatin-induced neuropathic pain model.

Compounds 3-18 were obtained according to Scheme 1. The starting materials, namely 5,5-
di(propan-2-yl) (1) and 5,5-diphenyl-imidazolidine-2,4-diones (2), were either purchased
from Merck or prepared in line with Bucherer-Bergs reaction.18 The final compounds 3–18
were obtained via aminoalkylation reaction from appropriately substituted imidazolidine-2,4-
diones (1,2), formaldehyde, and corresponding arylpiperazines. The reaction was carried out
in absolute ethanol at reflux for 24 h. The final products after recrystallization from absolute
ethanol were obtained as free bases in yields ranging from 60% to 95%. The purity of
compound and their structures were confirmed by both elemental and spectral (1H-NMR)
analyses (see Supplementary Materials).
Scheme 1. Reagents and reaction conditions: (a) KCN, (NH4)2CO3, 50% ethyl alcohol;
(b) 4-substituted piperazines, 37% formaldehyde, 96% ethyl alcohol.
The designed compounds were evaluated for their anticonvulsant activity in the Antiepileptic
Drug Development Program (ADD) developed by the NIH (Rockville). Initially, the
anticonvulsant activity was established by the maximal electroshock (MES)19 and
subcutaneous pentylenetetrazole (scPTZ) tests,20 while the neurotoxic properties were
measured by the minimal motor impairment rotarod screening test (NT). The preliminary
results are presented in Table 1. To identify entities with broad-spectrum anticonvulsant

activity, the most active compounds in the MES and scPTZ tests were chosen for the
psychomotor seizure (6 Hz) test. The experimental procedure used for pharmacological
studies are described in Supplementary Materials.
In the first step of anticonvulsant screening, the activity of titled compounds was determined
by the MES and scPTZ tests. In the MES test, the majority of compounds were effective at
doses of 100 or 300 mg/kg at 4 h. Three compounds (13, 17, and 18) exhibited anticonvulsant
activity in this test at the initial dose of 30 mg/kg, but only for compound 18, the initial dose
was gradually reduced to 10 mg/kg (Table 1). Furthermore, eight compounds (4, 5, 10, 11,
and 15–18) were also active in the MES test at doses of 100 and 300 mg/kg at 0.5 h. Based on
the aforementioned results in the MES test, the anticonvulsant activity of 13 compounds can
be classified as either long-lasting (4, 5, 11, and 15–18) or delayed (3, 6, 7, and 12–14)
(Table 1), while compound 10 exhibited a rapid-onset and short-lived activity in the same test.
It is worth noting that the most potent molecule in this test (18), representing
methylpiperazine derivative, showed a long-lasting activity up to 4 h at the fixed dose of
10 mg/kg. A slightly weaker effect was observed for its 2-fluorophenyl (13) and phenethyl
(17) analogues, which showed protective effect at 4 h at a dose of 30 mg/kg.
Table 1. The results of screening anticonvulsant activity after ip administration into mice
(3-18).
O
R1
R
N
N
R
N
N
H
Z
O
Intraperitoneal administration in
micea
ASP
class
MES b
scPTZ c
NT d
Compd R/R
Z
R1
0.5 h 4 h 0.5 h 4 h 0.5 h 4 h
iPr/iPr
iPr/iPr
iPr/iPr
iPr/iPr
iPr/iPr
iPr/iPr
-
H
3-Cl
2-F
4-F
3-CF3
H
-
300
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2
1
1
1
1
3
1
4
3
4
-
100 100
100 100
-
100 300 300
5
-
-
-
100
-
-
-
300 300
6
-
-
100
-
-
-
-
-
7
-CH2-
-
-
-
300
8
iPr/iPr -CH2CH2-
iPr/iPr -CH3
H
-
100 3001
9
-
300
-
300
10

Ph/Ph
Ph/Ph
Ph/Ph
Ph/Ph
Ph/Ph
Ph/Ph
-
H
300 100
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
1
1
1
1
1
1
1
1
11
12
-
3-Cl
2-F
4-F
-
-
-
100
30
-
13
-
-
100
14
3-CF3 300 100
15
-CH2-
H
H
-
300 100
16
Ph/Ph -CH2CH2-
100
10
30
10
30
17
Ph/Ph
-CH3
-
100 100
100 100
18
PHTe
-
30
a Doses of 30, 100, and 300 mg/kg were administered. The data indicate the minimum dose whereby
bioactivity was demonstrated. The animals were examined at 0.5 and 4.0 h. A dash indicates the absence of
anticonvulsant activity and neurotoxicity at the maximum dose administered (300 mg/kg). iPr – isopropyl;
Ph – phenyl; Response comments: 1 death.
b Maximal electroshock test
c Subcutaneous pentylenetetrazole test
d Neurotoxicity screening using rotarod test
eReference drugs, data for phenytoin (PHT) from Ref.33
In the scPTZ test, anticonvulsant activity was observed only for compounds 5 and 9 at the
dose of 100 mg/kg (Table 1). Thus, both compounds exhibited delayed anticonvulsant activity
at 4 h in the scPTZ test. It should be emphasized that only compound 5 was active in both
seizure models (MES and scPTZ), whereas the other molecules showed anticonvulsant
activity exclusively in one test. In the whole series, only one compound (8), representing
benzylpiperazine derivative, was completely devoid of any anticonvulsant activity.
Generally, compounds containing 5,5-di(propan-2-yl)imidazolidine-2,4-dione ring (3–10)
were less active and more toxic than their 5,5-diphenyl analogues (11–18). Only six of the
tested compounds (5, 6, 8–10, and 18) showed neurotoxic activity in the NT test; compounds
5, 6, 8–10, and 18 exhibited toxicity at doses of 300 or 100 mg/kg at 0.5 h, and compounds 5,
6, and 18 were also neurotoxic at 4 h (Table 1). It is worth noting that the dose at which
anticonvulsant activity was observed was 3- to 6-fold higher than that at which neurotoxicity
appeared.
Oral bioavailability is one of the important properties of clinical candidates as it allows to
inhibit convulsions after administration by the oral route. Therefore, the selected compounds
that showed the highest activity in mice in the MES test (3, 4, 7, and 11–18) were examined
for their anticonvulsant activity after oral (po) administration in rats at a fixed dose of 30
mg/kg. All tested compounds, namely 3, 4, 7, and 11–18, exhibited satisfactory activity (at
least in 50% of tested animals) in the MES test after oral administration (Table 2). Among
them, two compounds were active at all time points (11 and 18), but only compound 18

afforded protection against convulsions in up to 100% of the examined rats during this period.
Both compounds 7 and 13 protected rats against convulsions starting from 0.5 h until 4 h, and
compound 17 was active for a shorter time until 2 h. Unexpectedly, compound 3 showed
delayed onset of anticonvulsant activity after 6 h, probably due to the action of its metabolites.
Table 2. Anticonvulsant activity and toxicity of selected compounds 3, 4, 7 and 11-18
administrated orally (30 mg/kg, po) into rats.
MES test a
NT b
1h
0
Compd
0.25 h 0.5 h
1h
0
0
1
1
2
3
0
4
0
1
4
3
2h 4h
6h
2
-
0.25 h 0.5 h
2h 4h 6h
0
0
0
2
1
0
0
0
1
0
1
1
0
0
1
2
0
2
2
1
2
4
3
4
0
1
2
1
1
3
0
4
0
3
3
3
0
2
3
3
1
1
2
4
1
0
1
3
0
0
0
0
0
0
0
0
0
0
0
-
0
0
0
0
0
0
0
0
0
0
0
-
0
0
0
0
0
0
0
0
0
0
0
-
0
0
0
0
0
0
0
0
0
0
0
-
0
-
-
-
-
-
-
-
-
-
-
-
3
4
0
-
0
7
-
0
11
12
13
14
15
16
17
18
PHTc
-
0
-
0
-
0
-
0
-
0
-
0
-
0
-
-
aThe data indicate the number of rats of four that were protected at a dose of 30 mg/kg.
bNeurotoxicity screening using rotarod test. The data indicate the number of rats of four in which neurotoxicity
was observed at a dose of 30 mg/kg.
cPHT – phenytoin reference drug, data from Ref.34
Subsequently, four compounds (13, 15, 17, and 18) were chosen for the psychomotor seizure
test (6 Hz, Table 3). All tested compounds showed satisfactory activity at a dose of 100 mg/kg
for various time intervals (compounds 7 and 17 from 1 to 4 h, compounds 13 and 15 only at 2
h, and compound 18 from 0.25 to 4 h). The most promising anticonvulsant properties were
detected for compound 18 containing 4-methylpiperazine moiety, which showed up to 100%
protection against convulsions at lower doses of 25 and 50 mg/kg, starting from 0.25 to 1 h.
Table 3. Anticonvulsant activity 6 Hz test of selected compounds 7, 13, 15, 17 and 18, after
administrated intraperitoneally into mice (current 32 mA).
6 Hz testa
NTb
Dose
Compd
(mg/kg) 0.25 h 0.5 h 1h
2h
4h
0.25 h 0.5 h
1h
2h
4h

100
100
100
50
0
1
0
1
3
4
3
1
1
0
1
3
4
4
2
1
1
3
4
4
4
3
4
4
3
0
2
4
3
1
3
4
0
2
4
0
0
0
0
0
0
0
0
0
0
7
13
15
17
18
18
18
0
0
0
0
0
0
0
0
0
0
25
0
0
0
0
0
50
0
3c
0
4c
0
4c
0
4c
0
4c
100
aThe data indicate the number of mice of four that were protected at a respective dose (mg/kg).
bNeurotoxicity screening using rotarod test. The data indicate the number of mice of four in which neurotoxicity
was observed at a respective dose (mg/kg).
cNumber of animals of four which were unable to grasp rotarod or had muscle spasms.
Based on the above preliminary results, in the next step, two of the most potent compounds (9
and 18) were chosen for further quantification studies (ED50 and TD50) in the MES, scPTZ,
and 6 Hz tests, after intraperitoneal into mice and for the most active one after oral
administration into rats. The aforementioned parameters were then compared to the data
obtained in the same studies for well-known reference anticonvulsant such as phenytoin
(Table 4). In the MES test, compound 18 showed slightly lower ED50 and TD50 values than
reference drug phenytoin (e.g. ED50 po = 38.5 mg/kg vs 28.1 mg/kg, respectively), but its
duration of anticonvulsant activity lasted longer than that of phenytoin (TPE: 2 h po or 4 h ip
vs. 1 h po/ip). Additionally, compound 9 in scPTZ test displayed 5.5-fold higher activity than
phenytoin (ED50 values: 89.36 mg/kg vs > 500 mg/kg). It is worth mentioning that compound
18 in the 6 Hz test showed the median effective dose (ED50) of 12.2 mg/kg and a toxic dose of
80.6 mg/kg at a TPE of 1 h. In quantitative studies, compound 18 showed 5-fold lower ED50
value (ED50 = 12.2 mg/kg) than that obtained for the reference drug, phenytoin (Table 4).
Table 4. Quantification studies of selected compounds 9 and 18 in the MES, scPTZ, 6 Hz and
neurotoxicity (NT) tests, after intraperitoneal and oral administration into mice (ip) or rats
(po), respectively.
b
b
b
b
MES ED50 scPTZ ED50
6 Hz ED50
NT TD50
PIc
TPE (h)a
Compd
(mg/kg)
-
(mg/kg)
89.36
(mg/kg)
(mg/kg)
9 (ip)
4
2
(66.39 –
120.3)
> 200
> 500
>2.23(scPTZ)
38.5
18 (po)
> 250
>12.99(MES)
(30.6 – 51.7)

75.55
(66.07 –
89.75)
4 (MES)
11.14
12.2
> 80
6.78(MES)
6.19 (6 Hz)
18 (ip)
1 (6 Hz, (9.4 -12.57)
NT)
(7.6 – 17.8)
28.1
PHTd
1
(27.7 –
35.20)
> 500
> 500
> 1000
>35.58(MES)
7.27(MES)
(po)
5.64
41.00
PHTd (ip)
1
> 60e
(4.74-6.45)
(39.4 - 43)
a Time to peak effect.
b Results are represented as mean ± SEM at 95% confidence limit (MES-maximal electroshock test;
scPTZ-subcutaneous pentylenetetrazole test; 6 Hz test; NT-neurotoxicity).
c Protection index (TD50/ED50).
d PHT – Phenytoin, reference drug, MES test data from Ref.34
e 6 Hz test data for PHT from Ref.32
Apart from epilepsy, AEDs are widely used for many nonepileptic conditions, including
neuropathic pain and other chronic pain types.21 Hence, in the next stage of our experiments,
following the ethical “3R rule,”22 we selected the most promising compound, that is,
compound 18, to assess its ability to attenuate symptoms of chronic pain. This compound was
chosen for further extended in vivo studies in mice considering its: (1) high anticonvulsant
activity (equal or superior to the reference drugs) in the MES and 6 Hz tests, (2) broad
therapeutic index, (3) anticonvulsant activity in both mice and rats, and (4) biological activity
after oral administration at doses that were not neurotoxic to experimental animals. We used
two mouse pain models reflecting distinct mechanisms of pain formation, namely the formalin
test–a model of neurogenic and inflammatory pain,23 and chemotherapy (oxaliplatin)-induced
neuropathic pain model. Because the latter is thought to involve distinct mechanisms of pain
development during the early phase (e.g., the overexpression of voltage-gated sodium and
calcium channels, altered neuronal excitability, TRPA1 channel activation, and impaired
calcium homeostasis) and the late phase (mitochondrial dysfunction, glial cell activation,
oxidative stress, axon degeneration, and neuroinflammation),24,25 the efficacy of analgesics
used for pain relief in these two phases of chemotherapy-induced neuropathic pain might also
be different.26–28
In the formalin test, in both the acute and inflammatory phases, compound 18 did not show a
statistically significant activity. However, some nonsignificant effects may be observed in the
late phase, 35 min after injection of formalin (Figure 2 and Supplementary Materials). This

discovery is likely to prove indirectly that the test compound 18 is devoid of anti-
inflammatory properties and it does not affect nociceptors. This in turn excludes such an anti-
inflammatory activity as a mechanism underlying this compound’s effect observed in vivo.23
Figure 2. Antinociceptive activity of compound 18 in formalin test, after intraperitoneal
administration into mice.
Subsequently, compound 18 was tested in the oxaliplatin-induced neuropathic pain model
(Figure 3). In the von Frey test, an overall effect of treatment was observed (F[4,36] = 21.49;
p < 0.0001). In animals not treated with oxaliplatin, the mean force that caused paw
withdrawal in the von Frey test was 3.08 ± 0.3 g. Three hours after oxaliplatin injection, a
significant reduction of pain sensitivity threshold was observed in mice (p < 0.0001 vs before
oxaliplatin treatment). In this acute phase in neuropathic animals, the test compound 18
significantly elevated pain sensitivity threshold for mechanical stimulation by 53% (p < 0.001
vs pre-drug paw withdrawal force, Figure 3).

Figure 3. Antiallodynic effect of the compound 18 used at the dose of 11 mg/kg in the von
Frey test in oxaliplatin-induced neuropathic pain model in mice. Results are shown as the
mean force (±SEM) that caused paw withdrawal. Statistical analysis: repeated measures
ANOVA followed by Bonferroni’s multiple comparison. Significance vs. paw withdrawal
force before oxaliplatin administration (‘pre-ox’): #### p < 0.0001, and vs. pre-drug
(baseline) paw withdrawal force: *** p < 0.001.
In the late phase, that is, 7 days after oxaliplatin injection, a statistically significant (p <
0.0001) reduction of pain sensitivity threshold was observed in oxaliplatin-treated mice as
compared to that in nontreated animals (Figure 3). Treatment with compound 18 resulted in a
statistically significant antiallodynic effect (61%, p < 0.001 vs pre-drug paw withdrawal
force).
Summarizing, in this experiment, high antiallodynic activity of compound 18 was observed in
the oxaliplatin-induced neuropathic pain model. Oxaliplatin is a third-generation platinum-
based antitumor drug that induces the formation of DNA crosslinks, thus causing apoptotic
death of dividing cells, but it has also affinity for the peripheral nervous system. In rodents, a
single injection of oxaliplatin induces painful peripheral neuropathy accompanied by
mechanical (tactile) allodynia.29,30
In neuropathic animals treated with oxaliplatin, the tested compound 18 elevated the
nociceptive threshold in the acute and late phases of tactile allodynia. This in vivo study
result, together with the results obtained in tests assessing anticonvulsant activity, might
suggest potential mechanisms of action of compound 18. It is thought that drugs that act by
blockade of voltage-gated sodium and to a lesser degree calcium channels (with the exception
of ethosuximide) are effective in the MES test, and they show no anticonvulsant activity in
the PTZ model. In contrast, numerous GABAergic AEDs and the T-type calcium channel
blocker ethosuximide are effective in the PTZ model. Mixed AEDs are active in both tests,
which is probably due to their multitarget profile of the anticonvulsant activity.20,31 Bearing
that in mind, it can be concluded that the influence of the test compound 18 on ion channels as
a mechanism underlying its anticonvulsant and antiallodynic activities should be considered.
In terms of the oxaliplatin model of chronic pain, such ion channel-related mechanisms seem
to have an impact on the activity of compound 18 mainly in the early-phase oxaliplatin-
induced tactile allodynia. The mechanisms underlying the delayed antiallodynic effects of
compound 18 remain unclear, but again–considering results from the formalin test, they do
not seem to involve attenuation of inflammation. It should be therefore concluded that the

elucidation of the precise mechanism of action of compound 18 requires further extended
studies (electrophysiological, biochemical, etc.), and the results obtained in the present
research might be helpful for the establishment of directions for mechanistic studies.
The aim of this study was to synthesize a library of new 5,5-diphenyl- and 5,5-di(propan-2-
yl)imidazolidine-2,4-dione derivatives and to evaluate their anticonvulsant properties in the
“classical” animal models of seizures, namely MES and scPTZ tests. Acute neurotoxicity of
these compounds was determined by the rotarod test. After the initial screening, four most
promising compounds were tested using the focal seizure model (6 Hz test). In the
aforementioned screening evaluation, two compounds, namely 9 and 18, were selected for
further quantitative pharmacological studies. Among them, compound 9 showed significant
ED50 values and protective index in the scPTZ test, but compound 18 emerged as the most
promising molecule with ED50 values similar to that of the reference drug phenytoin in the
MES test and 4-fold lower ED50 dose than phenytoin in the 6 Hz test. Additionally, compound
18 was tested in a formalin model of tonic pain and in an oxaliplatin-induced neuropathic pain
model. In the oxaliplatin-induced pain model, this molecule exhibited antiallodynic activity.
Taken together, this research identified compound 18 (3-((4-methylpiperazin-1-yl)methyl)-
5,5-diphenylimidazolidine-2,4-dione) to show very promising anticonvulsant activity, that is,
a broad spectrum of protection against different type of seizures, namely generalized tonic–
clonic type (MES) and focal (6 Hz) seizures and to have favorable antiallodynic properties.
However, further extensive studies need to be carried out to explain the precise mechanism of
action and toxicological effects of this molecule.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
The authors thank Professor James Stables from National Institute of Neurological Disorders
and Stroke (NIH, Rockville, USA) for providing the results of anticonvulsant and
antinociceptive activity (formalin test) as well Professor Katarzyna Kieć-Kononowicz
(Department of Technology and Biotechnology of Drugs, Jagiellonian University Medical
College, Kraków, Poland) the coordinator of the ADD Project from Polish site. This study
was co-founded by Funds for Statutory Activity of Jagiellonian University Medical College
(No K/ZDS/006208) and National Science Centre (NSC) Poland grants No DEC-
2013/11/B/NZ7/02081 and 2017/25/B/NZ7/01048.
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