Structure-activity relationship and cardiac safety of 2-aryl-2-(pyridin-2-yl)acetamides as a new class of broad-spectrum anticonvulsants derived from Disopyramide

Article abstract of DOI:10.1016/j.bioorg.2020.103717

A series of 2-aryl-2-(pyridin-2-yl)acetamides were synthesized and screened for their anticonvulsant activity in animal models of epilepsy. The compounds were broadly active in the ‘classical’ maximal electroshock seizure (MES) and subcutaneous Metrazol (scMET) tests as well as in the 6 Hz and kindling models of pharmacoresistant seizures. Furthermore, the compounds showed good therapeutic indices between anticonvulsant activity and motor impairment. Structure-activity relationship (SAR) trends clearly showed the highest activity resides in unsubstituted phenyl derivatives or compounds having ortho- and meta- substituents on the phenyl ring. The 2-aryl-2-(pyridin-2-yl)acetamides were derived by redesign of the cardiotoxic sodium channel blocker Disopyramide (DISO). Our results show that the compounds preserve the capability of the parent compound to inhibit voltage gated sodium currents in patch-clamp experiments; however, in contrast to DISO, a representative compound from the series 1 displays high levels of cardiac safety in a panel of in vitro and in vivo experiments.

Full text of DOI:10.1016/j.bioorg.2020.103717

BioorganicChemistry98(2020)103717
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Structure-activity relationship and cardiac safety of 2-aryl-2-(pyridin-2-yl)
acetamides as a new class of broad-spectrum anticonvulsants derived from
Disopyramide
⁎
Maciej Dawidowskia, , Marek Króla, Bartłomiej Szulczyka,b, Andrzej Chodkowskia,
Piotr Podsadnia, Piotr Konopelskic, Marcin Ufnalc, Piotr Szuberskia, Martyna Zofia Wróbela,
Yihong Zhangd, Aziza El Harchid, Jules C. Hancoxd, Dagmar Jarkovskae, Eliska Mistrovae,
Jitka Sviglerovae, Milan Štengle, Grzegorz M. Popowiczf, Jadwiga Turłoa
a Department of Drug Technology and Pharmaceutical Biotechnology, Medical University of Warsaw, Banacha 1, 02-097 Warsaw, Poland
b Laboratory of Physiology and Pathophysiology, Centre for Preclinical Research, Medical University of Warsaw, Banacha 1B, 02-097 Warsaw, Poland
c Department of Experimental Physiology and Pathophysiology, Centre for Preclinical Research, Medical University of Warsaw, Banacha 1B, 02-097 Warsaw, Poland
d School of Physiology, Pharmacology and Neuroscience, Faculty of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom
e Department of Physiology, Biomedical Center, Faculty of Medicine in Pilsen, Charles University, Alej Svobody 1655/76, 323 00 Pilsen, Czech Republic
f Institute of Structural Biology, Helmholtz Zentrum München, Ingolstädter Landstrasse 1, 85764 Neuherberg, Germany
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of 2-aryl-2-(pyridin-2-yl)acetamides were synthesized and screened for their anticonvulsant activity in
animal models of epilepsy. The compounds were broadly active in the ‘classical’ maximal electroshock seizure
(MES) and subcutaneous Metrazol (scMET) tests as well as in the 6 Hz and kindling models of pharmacoresistant
seizures. Furthermore, the compounds showed good therapeutic indices between anticonvulsant activity and
motor impairment. Structure-activity relationship (SAR) trends clearly showed the highest activity resides in
unsubstituted phenyl derivatives or compounds having ortho- and meta- substituents on the phenyl ring. The 2-
aryl-2-(pyridin-2-yl)acetamides were derived by redesign of the cardiotoxic sodium channel blocker
Disopyramide (DISO). Our results show that the compounds preserve the capability of the parent compound to
inhibit voltage gated sodium currents in patch-clamp experiments; however, in contrast to DISO, a re-
presentative compound from the series 1 displays high levels of cardiac safety in a panel of in vitro and in vivo
experiments.
Voltage-gated sodium channel
Sodium channel blocker
Disopyramide
Drug discovery
Structure-activity relationships
Anticonvulsant agent
Refractory epilepsy
Drug repositioning
Medicinal Chemistry
Cardiac safety
1. Introduction
through structural modification of drugs that are already known to
possess desirable clinical profiles. This strategy primarily leads to
Epilepsy is a major neurological disorder that affects 65–70 million
people globally [1]. At present, the main way of treatment of the dis-
ease is the chronic administration of Antiepileptic Drugs (AEDs) that
provide symptomatic benefit by decreasing the number of seizure epi-
sodes. Since the introduction of potassium bromide in the mid-19th
century, nearly 40 clinically effective AEDs have been developed. De-
spite the significant improvement in efficacy and safety of pharma-
cotherapy of epilepsy, still an adequate seizure control can be achieved
in only 70% of patients. Given this failure rate and in view of physical,
social and psychological consequences of uncontrolled seizures, there is
a pressing need for new agents to treat refractory epilepsy.
‘evolutionary AEDs’ that display similar activity spectra but improved
pharmacokinetics and safety when compared with the parent ther-
apeutics (e.g. Oxcarbazepine, Eslicarbazepine, Fosphenytoin,
Brivaracetam) [2,3].
A similar approach has been proposed, that exploits agents used in
other indications, but which are known to act through potentially
beneficial mechanisms of action. For example, epileptic seizures and
cardiac arrhythmias share commonalities, one of which is the improper
electrophysiological activity of brain neurons and cardiomyocytes, re-
spectively [4]. A known drug, Phenytoin (PHE), exemplifies a direct use
of a single chemical entity for both indications. There are other data
suggesting that certain antiarrhythmic drugs (AADs) and their
One possible approach to the development of new AEDs leads is
⁎ Corresponding author.
E-mail address: maciej.dawidowski@wum.edu.pl (M. Dawidowski).
https://doi.org/10.1016/j.bioorg.2020.103717
Received 2 October 2019; Received in revised form 23 December 2019; Accepted 28 February 2020
Availableonline05March2020
0045-2068/©2020TheAuthors.PublishedbyElsevierInc.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).

M. Dawidowski, et al.
BioorganicChemistry98(2020)103717
derivatives are active in animal epilepsy models [4,5]; Examples are
presented in Public Access to Neuroactive & Anticonvulsant Chemical
Evaluations (PANAChE) database [6], a remarkable tool that provides
open access to nonproprietary chemical structures and biological data
for compounds previously screened in the Epilepsy Therapy Screening
Program (ETSP, formerly: Anticonvulsant Screening Program, ASP) [7]
of the National Institute of Neurological Disorders and Stroke (NINDS),
National Institutes of Health (NIH), USA. On the other hand, direct
repositioning of AADs to AEDs might bear a risk of causing severe side
effects in epileptic patients. Hence, we proposed that another strategy,
consisting of careful structural re-design of certain AADs might have the
potential to reposition them towards management of epileptic seizures.
This may be achieved through specifying those pharmacophoric fea-
tures of a template AAD that would be responsible for the desired ac-
tivity within the CNS and those that are expected to trigger the un-
wanted cardiac side-effects. Subsequently, the design strategy should be
validated by chemical synthesis and a thorough pharmacological eva-
luation of the re-designed template.
substitution patterns of the benzene ring or by replacing it with either
bioisosteric pyridine or a larger, more aromatic and hydrophobic
naphthalene. The planned derivatives 3a–q (Schemes 1 and 2) were
synthesized adopting the established synthetic pathway [16,17].
Briefly, the starting arylacetonitriles were deprotonated with KOH in
DMSO and the resulting carboanions were C-arylated with 2-bromo-
pyridine in a one-pot procedure. Subsequently, the obtained 2-aryl-2-
pyridilacetonitriles 2a–q were hydrolyzed to give the corresponding
amides 3a–q.
Next, we sought to investigate derivatives bearing small hydro-
phobic substituents (-F and –CH3) onto alpha-carbon of 2-aryl-2-pir-
idinylacetamide 3r and 3s. Deprotonation of 2a with LDA, followed by
methylation or electrophilic fluorination gave the respective 2-methyl-
and 2-fluoro-2-phenyl-2-pyridilacetonitriles 2r and 2s (Scheme 3). The
subsequent acidic hydrolysis provided the corresponding amides 3r and
3s.
Finally, by synthesizing derivatives 5a–e we aimed to examine
whether various (aryl)aliphatic residues on the amide functional group
are tolerated (Scheme 4). Briefly, 2a was subjected to Pinner reaction to
give the ester 4. The attempts to hydrolyze 4 to the corresponding
carboxylic acid failed and only the decarboxylation product, 2-benzyl-
pyridine, was detected by LC/MS analyses of the test reaction mixtures.
To circumvent this issue, 4 was converted to the secondary amides 5a–e
directly, through a TBD-catalyzed aminolysis [18] with the corre-
sponding amines.
We have chemically modified an old-generation antiarrhythmic,
disopyramide (DISO), to obtain 2-aryl-2-(piridin-2-yl)acetamide,
a
novel scaffold for centrally acting voltage-gated Na+ current inhibitors
with a broad spectrum activity across the in vivo animal models of
epilepsy [8]. Importantly, we have demonstrated, that a compound
from the class, 2-(2-chlorophenyl)-2-(pyridin-2-yl)acetamide (1,
ADD424042, Fig. 1A) was effective in suppressing seizures in six-hertz
(6 Hz) and kindling models of pharmacoresistant epilepsy and epi-
leptogenesis. In the present study, we present a thorough structure-
antiseizure activity relationship study of 2-aryl-2-piridinylacetamide
series of anticonvulsants derived from DISO.
2.2. Anticonvulsant testing
The synthesized compounds were evaluated in various in vivo rodent
models of epilepsy within the ETSP (formerly: ASP) of NINDS, USA,
according to the well-established experimental procedures and decision
schemes that have been described in details in a recent article [7] and in
the PANAChE database [6].
It has been shown that there exist pathophysiological similarities
between epilepsy and some forms of chronic pain and AEDs are among
the important medications for this indication [9,10]. One of the phy-
siological backgrounds by which some AEDs attenuate pain is mod-
ulation of abnormal neuronal activity by the blockade of voltage-gated
Na+ currents [11]. As this mechanism is also characteristic for 2-aryl-2-
(piridin-2-yl)acetamides, in the current study we investigated whether
this class of compounds is effective in a preliminary model of hyper-
algesia in mice, the formalin test [12].
Initially, the compounds were assessed qualitatively in three rodent
models of acute seizures: the MES, the scMET and the six-hertz (6 Hz)
tests. The MES and scMET tests are often described as the ‘classical
models’. The MES model employs an electrical stimulus to generate
generalized tonic-clonic seizures and is capable of identifying the
compounds that prevent seizure spread. The scMET test uses Metrazol,
a chemoconvulsant, to induce myoclonic seizures and is proposed to
recognize the agents raising the seizure threshold. The ‘classical’ MES
and scMET models are sensitive to a wide range of marketed antic-
onvulsant agents, and since decades they have played an important role
in discovery of new AEDs. On the other hand, they sometimes fail to
identify compounds with novel mechanisms of action that are efficient
in treatment of refractory epilepsy (eg. Levetiracetam, LEV)[19,20].
Therefore, another preliminary model, the six-hertz (6 Hz) test, is now
routinely performed by ETSP to raise the threshold for advancing
compounds for the management of pharmacoresistant seizures. The
model employs an electrical current of low intensity and long duration
to elicit ‘psychomotor’ seizures. The selected 2-aryl-2-(pyridin-2-yl)
acetamide derivatives were tested in two variants of the 6 Hz test, at 32
and 44 mA current intensities. In those variants, different stimuli are
DISO has a relatively narrow therapeutic index and is known to
produce cardiotoxicity by several possible mechanisms [13–15]. No-
tably, our preliminary studies in rats show that contrary to the parent
DISO, the 2-aryl-2-piridinylacetamide scaffold lacked apparent cardiac
side-effects in rats [8]. Here, we investigate this phenomenon in more
detail, at a molecular, cellular and in vivo level.
2. Results and discussion
2.1. Chemistry
We systematically exploited structure–activity relationship (SAR)
within 2-aryl-2-(pyridin-2-yl)acetamide series by dividing the scaffold
into three subunits (Fig. 1B).
First, we varied the aromatic portion of the molecule by changing
Fig. 1. (A) Chemical structures of DISO and 1; (B) the proposed derivatization sites for the structure–activity relationship (SAR) study.
2

M. Dawidowski, et al.
BioorganicChemistry98(2020)103717
Scheme 1. i. 2-bromopyridine, KOH, DMSO, 60 °C; ii. H2SO4, CH3COOH, 50 °C.
Scheme 2. i. 2-bromopyridine, KOH,
DMSO, 60 °C; ii. H2SO4, CH3COOH, 50 °C.
Scheme 3. i. LDA, THF, −78 °C, then CH3I
(1r) or NFSI (1s), −78 °C to 25 °C; ii. H2SO4,
CH3COOH, 50 °C.
Scheme 4. i. HCl, MeOH, 25 °C; ii. RNH2,
TBD (30 mol%), THF, 60 °C.
employed for inducing seizures that are unequally susceptible to known
AEDs. For example, LEV and Ethosuximide (ESM) display high efficacy
in the 6 Hz test at 32 mA current intensity, while a significant loss in
activity is observed when 44 mA stimuli is used in the same model.
Thus, it is generally accepted that testing in both variants is useful for
pre-clinical differentiation of novel investigative AEDs [19]. In addition
to the primary anticonvulsant screening, the acute neurological im-
pairment (TOX) was evaluated in the rotorod test.
screening, respectively, after i.p. administration in mice (Table 1).
Analyzing the influence of the phenyl substitution pattern, we observed
a general trend that the highest anticonvulsant activity and lowest
neurotoxicity resides in the ortho- and meta-derivatives, whereas their
para- counterparts are significantly less efficient. Notable exceptions to
this tendency were 4–Cl and 4–F derivatives 3j and 3 m, respectively,
both providing seizure protection at a dose of 100 mg/kg, 0.5 h post-
administration. Nevertheless, both compounds were inactive at later
time points, and, similar to all other para-derivatives, they produced
pronounced neurotoxicity and mortality at a higher dose of 300 mg/kg.
Another general observation was the loss of antiseizure efficacy of
–OMe derivatives, only the o-OMe substituted compound 3c showed
activity, albeit it was more pronounced at dose of 300 mg/kg, at which
neurotoxicity occurred. Derivative 3a, lacking substituents in the ben-
zene ring, displayed full seizure protection, at 100 mg/kg, 0.5 h post-
administration. We did not observe acute toxic effects or motor im-
pairment at this dose. Mortality occurred at a significantly higher dose
of 300 mg/kg, still, preliminary screening indicated that a reasonable
therapeutic window could be expected for the compound in the quan-
tification studies. An important observation was that besides 3d, a de-
rivative of 3a containing a fluorine atom in the benzene ring, the ortho-
substituted compounds did not cause animal death even at a dose of
300 mg/kg, indicating good margins of tolerability and safety. On the
The preliminary screening was performed using small (1–8) groups
of animals per at least one dose of the test compound, at more than one
time point after its administration. The compounds showing activity in
the primary MES, scMET, 6 Hz (32 and 44 mA) and TOX tests were
subsequently quantified to determine the median effective doses
(ED50), toxic doses (TD50) and protective indices (PI = TD50/ED50) at
the previously estimated time of peak effects (TPEs). The experimental
ED50, TD50 and PI values were then compared with those of reference
AEDs. Finally, according to ETSP dispositions, selected agents were
chosen for screening in the Corneal Kindled Mouse (CKM) model and
Lamotrigine (LTG)-Resistant Amygdala Kindled Rat model of pharma-
coresistant, complex partial seizures. The results of the aforementioned
tests are summarized in Tables 1–8 and S1–5.
Most of the synthesized compounds showed anticonvulsant activity
and moderate neurological toxicity in the preliminary MES and TOX
3

M. Dawidowski, et al.
BioorganicChemistry98(2020)103717
Table 1
Anticonvulsant activity and neurotoxicity of compounds in the MES model following intraperitoneal (ip.) administration in mice.
Compound
Dose (mg/kg)
MESa
TOXb
R1
R2
R3
H
0.5 h
2.0 h
4.0 h
0.5 h
2.0 h
4.0 h
c
1
o-ClPh-
Ph-
CONH2
CN
100
300
100
300
100
100
300
100
300
100
300
100
300
100
100
100
100
100
100
300
100
300
100
300
100
300
100
300
100
300
100
300
100
300
100
300
100
300
100
300
100
300
100
300
100
300
100
300
100
300
3/3
1/1
4/4
4/4
4/4
3/3
–
–
2/3
1/1
–
0/8
0/4
0/8
8/8
1/8e
0/8
4/4f
0/8
2/8
0/8
3/4
0/8
3/4e
0/8
0/8
0/8
–
–
0/4
0/2
–
–
–
2a
H
2/4
–
0/8
8/8d
1/8
–
–
–
2d
3a
o-FPh-
CN
H
H
4/4
–
1/4
0/3
–
0/8
0/8
–
Ph-
CONH2
–
–
3b
3c
3d
o-MePh-
o-OMePh-
o-FPh-
CONH2
CONH2
CONH2
H
H
H
3/3
1/1
1/3
1/1
3/3
1/1
4/4
1/4
0/3
–
–
2/3
1/1
1/3
0/1
0/3
–
–
0/4
0/2
0/8
0/2
0/4
0/1
0/8
0/8
0/4
–
–
–
–
–
–
–
–
–
–
–
3e
3f
m-ClPh-
m-MePh-
m-OMePh-
m-FPh-
CONH2
CONH2
CONH2
CONH2
CONH2
CONH2
H
H
H
H
H
H
0/4g
0/4g
–
0/4
0/4
0/3
–
0/8
0/8
–
3g
3h
3i
–
–
m-CF3Ph-
p-ClPh-
4/4
2/3
1/1
0/3
0/1
0/3
–
0/4h
–
0/4
0/3
–
2/8
0/8
3/4i
0/8
2/4e
0/8
3/4i
0/8
4/4f
0/8
0/4
0/8
1/8
0/8
8/8
0/8
1/8
0/8
8/8
2/8
8/8
0/8
0/8
0/8
1/8
0/8
0/8
0/8
8/8
0/8
7/8
0/8
0/8
0/8
–
0/8
0/4
–
3j
–
–
3k
3l
p-MePh-
p-OMePh-
p-FPh-
m,p-diClPh-
2-pyridinyl-
1-naphthyl-
2-naphthyl-
Ph-
CONH2
CONH2
CONH2
CONH2
CONH2
CONH2
CONH2
CONH2
CONH2
COOMe
CONHMe
CONHEt
CONHnPr
CONHiPr
CONHBn
H
H
H
H
H
H
H
Me
F
–
0/3
–
–
0/4
0/1
0/4
0/0
0/4
–
–
–
–
0/3
–
–
–
–
3m
3n
3o
3p
3q
3r
3s
4
2/3
–
–
0/3
–
–
–
–
2/3
1/1
0/4
0/4
3/4
4/4
1/4
4/4
4/4
4/4
4/4
4/4
0/4
1/4
0/4
4/4
4/4
4/4
2/4
4/4
1/4
4/4
1/4
4/4
–
0/3
1/1
–
–
0/4
0/2
–
–
–
0/4
0/4
0/4
1/4
2/4
4/4
1/4
4/4
4/4
4/4
0/4
0/4
2/4
4/4
4/4
4/4
1/4
4/4
2/4
4/4
0/4
4/4
0/8
1/8
0/8
0/8
0/8
0/8
0/8
6/8
1/8
8/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
7/8
0/8
1/8
0/8
0/8
–
–
–
–
–
–
–
–
–
–
–
–
–
–
Ph-
–
–
–
–
Ph-
H
H
H
H
H
H
–
–
–
–
5a
5b
5c
5d
5e
Ph-
–
–
–
–
Ph-
–
–
–
–
Ph-
–
–
–
–
Ph-
–
–
–
–
Ph-
–
–
–
–
Ratios where at least one animal was protected or displayed neurotoxicity have been highlighted in bold for easier data interpretation.
a
b
c
d
e
f
g
h
Maximal Electroshock test (number of animals protected/number of animals tested).
Neurotoxicity test (number of animals exhibiting neurological toxicity/number of animals tested).
Not determined.
6 animals died.
1 animal died
4 animals died.
Active in 3/4, 1.0 h post administration.
Continuous seizure activity.
i
3 animals died.
contrary, para- derivatives that lacked anticonvulsant activity in the
MES model elicited high rates of mortality at the same time.
the MES model.
Introduction of small –Me and –F substituents at the alpha-carbon of
3a produced corresponding 3r and 3s that were active in the pre-
liminary MES screening and produced pronounced motor impairment
in the initial TOX testing.
Replacement of the phenyl moiety of 3a with 2-pyridinyl resulted in
3o, which lacked activity. On the contrary, more bulky and hydro-
phobic 1- and 2-naphthalenes 3p and 3q, respectively, were tolerated in
4

M. Dawidowski, et al.
BioorganicChemistry98(2020)103717
Table 2
Quantification studies of selected compounds in the MES, scMET and neurotoxicity tests in mice.
ED50 MESa
ED50 scMETb
TD50
PId
TPEe
(h)
c
Compound
(mg/kg)
(mg/kg)
(mg/kg)
1f
48 (38–58)
61 (51–72)g
56 (51–61)i
85 (66–114)
60 (53–67)
41 (34–45)
45 (36–57)g
58 (44–72)i
40 (32–47)
43 (38–50)
45 (40–50)
48 (38–64)
81 (70–93)
76 (66–85)
44 (36–52)
> 270
270 (252–266)
–
5.6
–
1.0
h
–
0.25
1.0
292 (224–366)
107 (86–144)
59 (47–73)
> 500
> 8.9
1.8
2.8
4.8
3.7
> 8.6
4.9
4.8
3.3
3.8
–
2a
2d
3a
156 (134–167)
168 (148–195)
198 (180–224)
166 (159–178)
> 500
0.5
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
2.0
140 (115–165)
–
160 (119–212)
> 190
3b
3d
3e
3i
196 (170–240)
207 (182–235)
148 (139–164)
183 (162–216)
–
132 (105–164)
127 (115–139)
113 (101–124)
261 (209–329)
196 (125–282)
174 (119–227)
3q
3ri
3si
> 500
> 6.6
417 (339–488)
9.5
0.5
Carbamazepinej
Phenytoinj
17 (14–21)
18 (16–28)i
7 (5–8)
> 45
41 (36–47)
136 (117–157)
51 (47–54)
89 (80–99)
30 (25–36)
27 (26–28)
2.4
7.6
7.3
11.1
4.3
6.0
0.25
0.5
1.0
4.0
1.0
0.5
122 (108–146)
> 50
8 (7–9)i
> 90
Lamotriginek
7 (6–9)
> 40
Lacosamidel
4.5 (3.7–5.5)
> 25
Unless otherwise stated, the compounds were administered i.p. in male mice. Values in parentheses are 95% confidence intervals determined by probit analysis.
a
b
c
d
e
f
g
h
i
j
k
l
Maximal Electroshock test.
Subcutaneous Metrazol test.
ED50 for the neurotoxicity test.
Protective index (TD50/ED50 MES).
Time to peak effect for the MES test.
Data from Ref. [8].
Tested in female mice.
Not tested.
Oral administration of the test compound.
Data from PANAChE database [1,6].
Data from Ref. [19].
Data from Ref. [34].
Table 3
Quantification studies of selected compounds in the MES, scMET and neurotoxicity tests in rats.
ED50 MESa
ED50 scMETb
TD50
PId
TPEe
(h)
c
Compound
(mg/kg)
(mg/kg)
(mg/kg)
1f
33 (23–43)
27 (16–49)g
14 (7–23)
82 (57–101)
> 250
175 (162–186)
> 500
5.3
0.25
2.0
> 18.5
14.8
3.0
h
2a
2d
3a
3b
3d
3i
–
207 (158–284)
103 (78–152)
150 (104–200)
139 (117–168)
144 (114–166)
158 (123–201)
> 500
2.0
34 (22–50)
35 (25–49)
16 (10–24)
23 (19–27)
28 (19–38)
41 (25–69)g
91 (66–122)
45 (31–68)g
34 (27–40)
51 (36–69)
> 125
73 (34–114)
–
0.5
4.3
0.5
8.7
0.25
0.5
6.3
5.6
0.25
0.25
0.5
> 250
–
> 12.2
> 2.7
> 11.1
3n
> 250
> 250
> 500
2.0
Carbamazepinei
Phenytoini
4 (3–6)
> 35
34 (28–45)
365 (223–500)
15 (12–19)
> 1000
> 8.5
0.25
5 (3–8)g
2 (1–4)
> 250
> 100
> 500
73.0
1.0
6.4
0.25
2.0
28 (20–35)g
> 35.7
Unless otherwise stated, the compounds were administered i.p. Values in parentheses are 95% confidence intervals determined by probit analysis.
a
b
c
d
e
f
g
h
Maximal Electroshock test.
Subcutaneous Metrazol test.
ED50 for the neurotoxicity test.
Protective index (TD50/ED50 MES).
Time to peak effect.
Data from Ref. [8].
Oral administration of the test compound.
Not tested.
i
Data from PANAChE database.
5

M. Dawidowski, et al.
BioorganicChemistry98(2020)103717
Table 4
Table 6
Effects of selected compounds on the threshold for minimal seizures induced by
the timed intravenous infusion of Metrazol (MET) in mice.
Quantification studies of selected compounds in the 6 Hz (44 mA) and neuro-
toxicity tests in mice.
METa (mg/kg
S.E.M.)
Compound
ED50 6 Hza
(mg/kg)
TD50
PIc 6 Hz
TPEd
(h)
b
Compound
Dose (mg/kg)
(mg/kg)
First twitch
Clonus
1e
82 (75–89)
270 (252–266)
> 500f
3.3
0.5
1b
0
27.1
32.1
51.6
32.5
36.1
73.3
27.2
38.6
59.8
26.1
32.6
55.2
1.21
29.3
38.8
62.4
35.9
39.6
112.6
29.3
46.5
84.2
28.4
35.9
72.6
1.29
104 (87–126)f
47 (42–52)
> 4.8
3.6
1.0
48
270
0
1.48c
2.98c
1.22
3.05c
3.56c
1.47
2d
3a
168 (148–195)
184 (75–300)
> 500f
0.25
0.25
0.5
78 (55–110)
100 (82–118)f
72 (63–80)g
75 (55–93)
2.4
2d
3a
3d
> 5.0
2.3
29
168
0
1.26d
2.39c
1.27
2.09e
6.61c
1.46
166 (159–178)g
183 (162–216)
138 (101–191)
> 500f
0.25
0.25
0.25
0.5
3i
2.4
3p
3r
77 (65–94)
1.8
41
198
0
1.64c
1.20c
0.89
2.95c
3.71c
1.05
96 (84–108)f
47 (40–85)
> 5.2
3.1
3 s
5b
145 (130–166)
0.5
163 (103–220)
305 (276–317)
1.9
1.0
43
207
0.93c
2.96c
0.84c
3.01c
Carbamazepineh
34 (28–37)
45 (43–47)
51 (47–54)
30 (25–36)
27 (26–28)
> 500
1.3
0.25
2.0
1.0
0.5
1.0
Phenytoini
> 60
< 1.0
< 1.0
2.1
Carbamazepinef
Phenytoinf
Mexiletinf
0
30.7
31.0
33.0
33.8
33.5
27.1
35.2
32.5
28.6
33.1
48.8
49.3
1.03
36.9
37.5
43.1
28.4
39.7
33.4
39.8
37.3
32.0
38.3
66.5
92.7
1.46
Lamotriginei
Lacosamidej
Levetiracetami
> 60
11
43
0
1.45e
1.04e
0.95
1.29e
2.13d
1.05
13 (10–18)
1089 (787–2650)
> 0.5
6
1.05e
1.48c
0.59
1.33e
1.28c
1.17
Unless otherwise stated, the compounds were administered i.p. in male mice.
Values in parentheses are 95% confidence intervals determined by probit
analysis.
41
0
10
30
0
0.85c
1.19c
1.32
1.02e
1.65c
1.55
a
b
c
d
e
f
g
h
6 Hz test at 44 mA.
ED50 for the neurotoxicity test
Valproatef
263
340
2.77c
3.39c
6.91c
11.72c
Protective index (TD50/ED50 6 Hz).
Time to peak effect for the 6 Hz test.
Data from Ref. [8].
The compound was administered orally (po.).
Data are expressed as means
S.E.M.
a
Tested in female mice.
Intravenous Metrazol Seizure Threshold test.
Data from Ref. [8].
b
c
d
e
f
Data from PANAChE database [1,6].
i
j
Data from Ref. [19].
p < 0.01, Dunnett’s test.
Data from Ref. [35].
p < 0.05, Dunnett’s test.
p > 0.05, Dunnett’s test.
Data from PANAChE database [1,6].
Table 7
Quantification studies of selected compounds in the Corneal Kindled Mouse
Table 5
model after intraperitoneal (i.p.) administration in mice.
Quantification studies of selected compounds in the 6 Hz (32 mA) and neuro-
Compound
ED50 CKMa
(mg/kg)
TPEb (h)
toxicity tests following intraperitoneal (i.p.) administration in mice.
ED50 6 Hza
(mg/kg)
TD50
PIc 6 Hz
TPEd
(h)
b
Compound
1c
3a
3i
59 (38–83)
53 (33–96)
62 (45–87)
0.5
(mg/kg)
0.25
0.25
1e
67 (58–75)
29 (19–42)
20 (9–49)
270 (252–266)
168 (148–195)
184 (75–300)
196 (170 – 240)
207 (182–235)
183 (162–216)
4.0
5.8
9.2
3.0
6.0
2.7
1.0
Carbamazepined
9 (5–11)
0.5
2d
3a
3b
3d
3i
0.25
0.25
0.25
0.5
Valproatee
174 (136–208)
16 (6–43)
0.25
0.5
Levetiracetamd
65 (45–83)
34 (25–43)
69 (66–72)
0.5
Values in parentheses are 95% confidence intervals determined by probit
analysis.
Carbamazepinef
24 (18–26)
45 (43–47)
51 (47–54)
30 (25–36)
27 (26–28)
> 500
1.9
0.25
2.0
1.0
0.5
1.0
Phenytoinf
> 60
< 1.0
< 1.0
2.7
a
b
c
d
Corneal Kindled Seizure model.
Lamotrigineg
Lacosamideh
Levetiracetamg
> 60
Time to peak effect.
10 (8–13)
19 (10–36)
Data from Ref. [8].
> 26.3
Data from Ref. [36].
e
Data from PANAChE database [1,6].
Values in parentheses are 95% confidence intervals determined by probit
analysis.
a
b
c
d
e
f
g
h
Table 8
6 Hz test at 32 mA.
Quantification study of 3a in the Lamotrigine (LTG)-Resistant Amygdala
Kindled Rat model after intraperitoneal (i.p.) administration in rat.
ED50 for the neurotoxicity test.
Protective index (TD50/ED50 6 Hz).
Compound
ED50 LTGKa
Time of test (h)
Time to peak effect for the 6 Hz test.
Data from Ref. [8]
(mg/kg)
Data from PANAChE database [1,6].
3a
50 (37–67)
169 (131–206)
0.5
1.5
Data from Ref. [19].
Felbamateb
Data from Ref. [34].
Values in parentheses are 95% confidence intervals determined by probit
analysis.
Switching the primary amide in 3a to nitrile resulted in 2a that was
equally efficient to the parent compound, whereas ester 4 was devoid of
in vivo activity. Modifications consisting in substitution of the primary
amide by small alkyl moieties led to 5a–e that were effective in primary
MES screening in mice. However, except for ethylamide 5b, that
a
Lamotrigine (LTG) Resistant Amygdala Kindled Seizure model.
b
Data from Ref. [21].
6

M. Dawidowski, et al.
BioorganicChemistry98(2020)103717
produced full seizure protection at 100 mg/kg, the activities of amide
nitrogen-substituted derivatives were more pronounced at a relatively
high dose of 300 mg/kg.
‘classical’ sodium channel blockers, the agents investigated here do
exhibit activity in the scMET model, which could be regarded a useful
feature in their further preclinical development.
Compounds 2a,d and 3a–n were screened in the preliminary scMET
screening in mice. Only 2d showed appreciable activity (Table S1),
hence, according to ETSP dispositions, the primary screening in this
model was not performed for the remaining compounds.
As an extension of the investigations in the scMET model, 1, 2d,
3a,d were assayed in a more discriminative timed i.v. infusion of MET
(ivMET) test in mice (Table 4). This model is used to differentiate those
agents that lower the seizure threshold and, as such, may be pro-
convulsant, from those compounds that elevate seizure threshold, and
are thus anticonvulsant. The results show, that contrary to the ‘classical’
sodium channel blockers, and, similarly to mechanistically unrelated
Valproic Acid (VPA), all tested arylacetamide derivatives significantly
and dose-dependently increased the length in time to first twitch and
sustained clonus following timed infusion of the convulsant MET.
The selected compounds were further tested in two variants of the
6 Hz test: at 32 and 44 mA current intensities, after intraperitoneal
administration in mice. The results of the initial qualitative screening
closely followed the SAR patterns previously observed during the pri-
mary assessments in the MES model (Tables S4–S5). When evaluated
quantitatively using a 32 mA stimuli, 1, 2d, 3a,b,d,i displayed high
seizure-suppressing efficacy (ED50 = 20–69 mg/kg) and favorable
safety margins (PI = 2.7–9.2, Table 5). Ortho-F nitrile 2d and un-
substituted phenyl 3a derivatives were most active in the series
(ED50 = 29 and 20 mg/kg; PI = 5.8 and 9.2, respectively). Increasing
the current intensity to 44 mA resulted in a slight drop of the antic-
onvulsant potency of most of the tested compounds 1, 2d, 3a,i,p,r,s, 5b
(ED50 = 47–82 mg/kg) and narrower safety indices (PI = 1.8–3.6,
Table 6). Similar to what we had observed during the quantification
studies in MES model, differentiation experiments in female mice per-
formed for 3a did not reveal significant changes in activity in 6 Hz test.
Likewise, the change in route of administration to oral greatly dimin-
ished levels of motor impairment produced by ADD424042 and 3a.
The significant activity of phenylacetamides in the 6 Hz model is an
important characteristic that differentiates them from ‘classical’ and
newer anticonvulsant sodium channel blockers - CBZ, PHT and LTG.
Further, though most of the investigated were less potent than LAC in
terms of ED50 values comparison, they revealed favorable (at 32 mA
stimuli) or at least similar (at 44 mA current intensities) PI values.
Finally, some of the phenylacetamide derivatives displayed ED50 values
comparable with mechanistically unrelated Levetiracetam (LEV) when
evaluated at 32 mA and outcompeted LEV in assays employing 44 mA
current intensities.
The selected derivatives showing high activity in the preliminary
screening in mice (2a,d, 3a,b,d,e,i,q,r,s), were tested quantitatively in
the MES, scMET and TOX models (Table 2). The ED50 values in the MES
quantification studies after i.p. administration in male mice were
roughly comparable for most compounds (ED50 = 40–48 mg/kg), only
the nitriles 2a,b and the naphthyl derivative 3q were slightly less active
(ED50 = 85, 60 and 81, respectively). Propitiously, the TD50 values in
the TOX test (TD50 = 148–270 mg/kg) indicated satisfactory PIs
(PI ~ 5 for most compounds) for the MES model. As expected on the
basis of preliminary screens, the potency of the compounds in the
scMET test was markedly lower than in the MES model, with ED50 al-
most reaching the TD50 values. A notable exception from this trend was
the nitrile 2d, which exhibited a high anticonvulsant activity and fair
safety margins (ED50 = 59 mg/kg, PI = 2.8 for this model). In addition
to the standard male mice quantification, 1 and 3a were tested in fe-
male mice. We did not observe considerable differences in MES and
TOX activities in both sexes. Selected compounds were evaluated
quantitatively using oral route of administration. The anticonvulsant
activities did not differ markedly as compared to the i.p. delivery.
However, we observed a significant decrease in motor impairment,
giving rise to higher PI values.
It has been shown in the past that seizures evoked in various rodent
species are unequally susceptible to treatment with AEDs and that this
feature can be useful for differentiation of novel investigative AEDs [6].
Therefore, according to ETSP dispositions, selected compounds
showing activity in preliminary MES mice screening were additionally
tested in rats. The qualitative results (Tables S2–S3) follow the similar
SAR patterns for both rodent species. However, we observed a generally
greater sensitivity of rats to antiseizure effects produced by adminis-
tration of the compounds, when compared to mice. Quantification
studies were performed for the selected compounds active in the qua-
litative rat screening (2a,d, 3a,b,d,i,n). The results show that the ED50
values in the MES were lower for most of the tested agents
(ED50 = 14–35 mg/kg) than those obtained in the similar mice study,
nitrile 2a and ortho-CH3 derivative 3b being most potent (ED50 = 14
and 16 mg/kg, respectively) in this model (Table 3). As a result, the PI
values for the MES test were considerably better (PI = 3.0–14.8) when
compared to mice results. Quantification results of the compounds in
the scMET model show a similar trend, the estimated ED50 values being
up to three-fold lower than those obtained from the corresponding mice
experiments. 1, 3i,n were quantified using oral dosage. In analogy to
what we had observed in the mice experiment, the anticonvulsant ac-
tivity in the MES model did not depend greatly on the way of admin-
istration and a marked loss of motor impairment occurred, as judged by
the TOX test. However, other than in mice, the oral administration of
compounds in rats ceased their effectiveness in the scMET model
completely.
According to ETSP dispositions, compounds 3a and 3i were further
evaluated in the CKM model of complex partial seizures (Table 7). Their
ED50 values closely matched the one reported by us [8] for 1 and were
in the ranges of potencies of CBZ, VPA and LEV used as reference AEDs.
We have recently demonstrated that 1 suppressed kindling devel-
opment, when assayed qualitatively in the LTG-Resistant Amygdala
Kindled Seizure (LKR) model of pharmacoresistant seizures and epi-
leptogenesis in rats [8]. In the current study, 3a was subjected to
quantitative evaluations in this model (Table 8). The compound dis-
played efficacy that surpassed this of Felbamate (FEB)[21], taken as the
reference AED. The activity of arylacetamides in the LKR model is an-
other important characteristics that distinguishes them from ‘classical’
anticonvulsant sodium channel blockers, such as CBZ [22].
Comparison of the potency of the investigated compounds in the
‘classical’ MES and scMET models with activities of marketed “old”-
(PHE and Carbamazepine, CBZ) and “new”-generation (LTG and
Lacosamide, LAC) anticonvulsant sodium channel blockers leads to
several conclusions. First, the ED50 values of arylacetamide derivatives
in the MES test are markedly lower than those of the aforementioned
reference AEDs. On the other hand, both display similar PI values,
suggesting the investigated arylacetamides may still have potential to
be clinically useful. Noteworthy, upon variation of basic experimental
conditions (ie. rodent species and routes of administration), the in-
vestigated compounds follow same qualitative changes in the activity
patterns as the reference AEDs. Importantly, other than the reference
2.3. Analgesic properties in the formalin test in mice
Many of the marketed AEDs, including ‘old’ and ‘new-generation’
sodium channel blockers, are effective in treatment of pain. Thus, in the
present study the selected arylacetamide derivatives were screened by
ETSP in the formalin test, which is a preliminary model of acute and
inflammatory pain. The compounds were tested after intraperitoneal
(i.p) administration in mice, at doses corresponding to their ED50 values
in the MES test (Table 9).
None of the tested derivatives 1, 3a,b,d,m were efficient in alle-
viating acute phase of pain in a statistically significant manner. On the
7

M. Dawidowski, et al.
BioorganicChemistry98(2020)103717
Table 9
inhibition of maximal amplitudes of sodium currents (62
3%,
Activity of selected compounds in the formalin model of hyperalgesia after
intraperitoneal (i.p.) administration in mice.
56
5%, 84
5%, 80
6%) at the tested concentration. One
explanation of this would be the possible differences in their ADME/PK
characteristics. On the other hand, the existence of another mechanism
of action that contributes to the compounds’ in vivo anticonvulsant
activity cannot be ruled out. Importantly, 5e inhibited sodium currents
Compound
Dose (mg/kg)
% of control
Acute phase
Inflammatory phase
significantly more weakly than 3a (88
8% vs 56
5%, respec-
1
48
41
40
43
52
79
66
69
94
85
78
49
66
74
41
17%a
16%a
13%a
17%a
11%a
73
15%a
11%b
14%b
15%a
23%a
tively, Fig. 2, p < 0.05), which is in agreement with their unequal
potencies in the primary anticonvulsant testing. In addition, we found
that the parent DISO shares the same potency of blocking neuronal
sodium currents as the anticonvulsant arylacetamides. We have pre-
viously reported that administration of this drug does not produce in
vivo antiseizure effects in mice, which could result from different tissue
distribution caused by the presence of the dialkylamino chain [8].
3a
3b
3d
3m
56
32
118
94
Carbamazepinec
10
17%a
74
36
20
48
20
29%a
19%d
5%d
36
5%d
Phenytoinc
6
21%a
5%b
Valproatec
100
300
4%d
6%d
6%d
2.5. In vitro and in vivo cardiac safety.
Data are expressed as means
S.E.M.
a
b
c
n = 8, p > 0.05 vs control group, Student’s t-test.
n = 8, p < 0.05 vs control group, Student’s t-test.
Data from PANAChE database.
Clinical use of DISO is associated with some incidence of acquired
long QT syndrome and torsades de pointes arrhythmia [23–25] and
the drug has been established to be an inhibitor of the human
Ether-à-go-go-Related Gene (hERG) potassium channels, which mediate
repolarizing “IKr” potassium current, at clinically relevant concentra-
d
n = 8, p < 0.01 vs control group, Student’s t-test
other hand, 3a and 3b attenuated the inflammatory phase in a manner
similar to the reference CBZ, PHE and VPA. Overall, they showed si-
milar profile to CBZ in this model, justifying further investigations of
their possible anti-pain properties.
tions [13,26].We have shown that in
a contrast to DISO, a
representative 2-aryl-2-(piridin-2-yl)acetamide 1 produced no apparent
cardiotoxic effects in rats [8], although this species does not rely on
hERG for ventricular repolarization. Therefore, in order to fully
evaluate the risk of potential hERG-related cardiotoxicity of 2-aryl-2-
(piridin-2-yacetamides), in the present study 1 and DISO were applied
2.4. Inhibition of voltage-gated sodium currents
to hERG-expressing HEK293 cells and hERG current (IhERG
) was
measured under whole-cell voltage clamp, using the protocol shown
in Fig. 3A. At a concentration of 100 µM, DISO nearly abolished
IhERG (Fig. 3Ai); in contrast the current was only little altered by 1.
Fig. 3B shows mean concentration–response plots for 3 concentrations
of each of DISO and 1. The derived IC50 for DISO inhibition of IhERG was
We have shown that 1 is a potent sodium channel blocker in a
whole-cell patch-clamp experiment [8]. To further validate this me-
chanism of anticonvulsant action, we investigated the influence of a
broader set of arylacetamide derivatives on voltage-gated sodium
channels (Table 10). Maximal sodium currents were evoked using
rectangular voltage-steps from the holding potential of −65 mV. We
established previously that at this holding potential sodium currents are
more sensitive to 1 as compared to −90 mV [8]. Maximal sodium
currents were recorded in control and in the presence of a tested
compound in a fixed concentration of 1 µM. Even at the relatively low
concentration, all of the tested compounds significantly inhibited so-
dium currents, except 5e, which exerted a statistically insignificant
effect. Although 1, 3a,b,s have a similar anticonvulsant profiles and
potencies in the in vivo epilepsy models, they produced notably unequal
7.56
0.54 µM, close to previously reported values [13,26] and
within therapeutic plasma levels of 6–8 µM [27]. By contrast, at con-
centrations between 10 µM and 1 mM, 1 produced comparatively little
I
hERG block; even at 1 mM the compound produced less than 30% mean
IhERG inhibition. Higher concentrations could not be tested, and as
no tested concentration produced > 50% inhibition an accurate
experimentally derived IC50 could not be obtained, though it is likely to
approximate or exceed 10 mM.
Our IhERG data indicate that at micromolar concentrations 1 pro-
duces little IhERG inhibition, unlike the parent compound. Mutagenesis
and in silico docking data have located the DISO binding site on hERG
low within the channel’s inner cavity; the drug is able to make aromatic
stacking interactions with aromatic residues on the S6 helix that line
the inner cavity [26], whilst the protonated tertiary amino group can
make cation-π interactions [26]. 2-Aryl-2-(piridin-2-yl)acetamides lack
an alkylamino chain and this appears radically to reduce the inhibition
of hERG; presumably the smaller molecule is less well able to make
contacts to key binding residues, as has been observed previously for
structurally similar Ranolazine and (smaller) Lidocaine [28].
Table 10
Influence of the selected compounds (1 μM) on
averaged, normalized maximal amplitudes of so-
dium currents in rat prefrontal cortex pyramidal
neurons, evoked from
a
holding potential of
−65 mV.
Compound
% inhibitiona
1b
62
80
56
84
80
88
3%
3%
5%
5%
6%
8%
DISOc
3ab
3bd
3sd
The dialkylaminoethyl moiety attached to biarylmethyl system
present in DISO resembles common pharmacophore patterns not only
for hERG inhibitors, but is also a characteristic structural motif present
in several muscarinic cholinergic (M) receptor ligands [29,30]. More-
over, it has been shown that DISO binds this receptor and produces
anticholinergic effects in heart tissue [31], which might contribute to
its cardiac effects. Therefore, we compared inhibitory activities of DISO
and a representative 2-Aryl-2-(piridin-2-yl)acetamide 1 on binding of
[3H]-Quinuclidinyl benzilate ([3H]-QNB) to Wistar Rat M receptors, at
clinically relevant concentration of 10 µM (Table 11). We found out
that, in contrast to DISO, 1 did not inhibit binding of [3H]-QNB to M
receptors, which might be another feature determining its increased
cardiac safety. Indeed, in addition to its high in vivo cardiac safety in
5ee
a
b
Data are expressed as means
S.E.M.
n = 5, p < 0.01, one-way ANOVA followed by
the Dunnett’s post hoc test.
c
n = 4, p < 0.05, one-way ANOVA followed by
the Dunnett’s post hoc test.
d
n = 5, p < 0.05, one-way ANOVA followed by
the Dunnett’s post hoc test.
e
n = 6, p > 0.05, one-way ANOVA followed by
the Dunnett’s post hoc test.
8

M. Dawidowski, et al.
BioorganicChemistry98(2020)103717
Fig. 2. A: Compound 3a potently inhibits voltage-gated Na+ currents in prefrontal cortex pyramidal neurons. Aa: Averaged normalized maximal Na+ currents
amplitudes are shown. Ab: Example recordings of Na+ currents in control and in the presence of 3a (1 μM). B: 5e slightly inhibits voltage-gated Na+ currents. Ba:
Averaged normalized maximal Na+ currents amplitudes are shown. Bb: Example recordings of Na+ currents in control and in the presence of 5e (1 μM).
rats [8], the compound did not trigger cardiotoxicity in guinea pigs or
rabbits (see: Supplementary Material).
activity pattern of the compounds than it would have been expected for
the sodium current inhibition alone.
The 2-aryl-2-(pyridin-2-yl)acetamides have been derived from
DISO, a known hERG potassium channel blocker and M receptor binder.
Here, we found that a representative compound from the series, 1, lacks
these undesirable, cardiotoxicity-triggering features, most likely due to
the absence of the alkylamino chain of the parent compound. As a re-
sult, the compound is characterized by an excellent in vivo cardiac
safety, which, along with its broad anticonvulsant activity in rodent
models and a proper ADME/PK profile (see: Supplementary Material),
may merit further investigations on 2-aryl-2-(pyridin-2-yl)acetamide
scaffold.
3. Conclusions
We have synthesized a number of 2-aryl-2-(pyridin-2-yl)acetamide
derivatives and subjected them for screening in animal models of epi-
lepsy within the ETPS of NIH. The SAR of the phenyl substituents
showed that the para- position was clearly disfavored. Analyzing the
type of substituents we observed a general trend that –OMe derivatives
were inactive. Compounds bearing a larger naphthalene ring were
slightly less active to the benzene derivatives, whereas the pyridine
bioisoster completely lacked the desired anticonvulsant activity.
Modifications of the amide functional group yielded highly active ni-
triles or secondary amides that were generally less potent in antiseizure
models.
4. Experimental section
4.1. Chemistry
Notwithstanding distinctive SAR trends within the series, the in-
vestigated arylacetamides display a markedly broader spectrum of ac-
tivity in animal models of epilepsy than the reference ‘classical’ sodium
channel blocking AEDs. Above all, this enhancement is manifested by
higher activities of the compounds in the ‘classical’ scMET model, as
well as in the 6 Hz and kindling models of pharmacoresistant seizures.
The results of the patch-clamp experiments show that the com-
pounds block voltage-gated sodium channel. Intriguingly, besides the
3a–5e molecular pair, there is no distinct correlation between the po-
tency of inhibition of the normalized maximal amplitudes of neuronal
sodium currents and the ED50 values of the compounds in the in vivo
epilepsy models. On one hand, this could be due to the possible var-
iations in their ADME/PK profiles. Nevertheless, additional mechanisms
of action cannot be ruled out, which is supported by the broader in vivo
Melting points were determined on an Electrothermal 9100 appa-
ratus in open capillary tubes and were uncorrected. Elemental analyses
were performed using Elementar Vario EL III. The NMR spectra were
obtained on a Varian Inova 500 MHz, a Bruker AVHD400 or a Bruker
AVHD300 spectrometer. Chemical shifts (δ) were expressed in parts per
million (ppm) relative to tetramethylsilane (TMS) or solvent used as the
internal reference. The following abbreviations were used to describe
the peak patterns: s (singlet), d (doublet), t (triplet), q (quartet), qt
(quintet), m (multiplet), p (pseudo-) and b (broad-). Coupling constants
(J) were in hertz (Hz). Thin-layer chromatography (TLC) was run on
Merck Silica gel-60 F254 plates. The spots were visualised by ultraviolet
light (254 nm) or iodine vapors. Flash column chromatography (FC)
was carried out on Merck Silica gel 60 (particle size: 0.040–0.063 mm).
9

M. Dawidowski, et al.
BioorganicChemistry98(2020)103717
Fig. 3. A: IhERG traces elicited in control
conditions and following exposure to
100 µM of DISO (Ai) and 1 (Aii). In each
panel the voltage protocol is shown as the
lower trace. 100 µM DISO nearly completely
abolished IhERG (Ai), but at the same con-
centration 1 produced little IhERG Inhibition.
Inset to Aii shows expanded IhERG tails, un-
derscoring the small degree of inhibition
seen with 1. B: Concentration-response plots
for effects of 100 µM DISO and 1 on IhERG
tails (n ≥ 6 for each concentration for both
compounds). IhERG tail amplitude was mea-
sured as difference between peak outward
tail current and the current elicited by the
brief prepulse to −40 mV that preceded the
test depolarisation. Mean
SEM fractional
block is plotted for each concentration of the
two compounds. A fit to the DISO data with
a standard Hill equation gave an IC50 of
7.56
0.98
0.54 µM, with a Hill slope (nH) of
0.07. The highest concentration of 1
tested was 1 mM, which produced < 30%
mean fractional inhibition. Higher con-
centrations were not possible to obtain due
to poor solubility at such concentrations. A
complete dose-response fit was not possible,
but the IC50 is likely to approximate or ex-
ceed 10 mM.
Lithium N,N-Diisopropylamide (25.4 mL of 1 M solution in THF,
25.4 mmol, 1.2 eq.), dropwise. The solution was stirred for 1 h and
Methyl Iodide (1.45 mL, 23.2 mmol, 1.1 eq.) was added dropwise. The
mixture was brought to room temperature and stirred overnight. The
reaction was quenched with saturated aqueous solution of NH4Cl
(75 mL). The mixture was extracted with AcOEt (2 × 75 mL). The
organic extracts were washed with saturated aqueous solution of NaCl
(2 × 50 mL), dried over anhydrous Na2SO4, filtered and concentrated.
The residue was purified by FC (hexane/AcOEt 6:1 to 2:1). Colorless oil
(3.67 g, 76%); 1H NMR (400 MHz, CDCl3): δ = 8.65 (ddd, J = 5.0, 2.0,
1.0, 1H), 7.71 (td, J = 8.0, 2.0, 1H), 7.51–7.44 (m, 3H), 7.42–7.36 (m,
2H), 7.35–7.30 (m, 1H), 7.26 (ddd, J = 7.5, 5.0, 1.0, 1H), 2.21 (s, 3H);
13C NMR (101 MHz, CDCl3): δ = 159.1, 149.3, 140.4, 137.3, 129.0,
128.0, 126.4, 123.0, 122.9, 121.8, 48.7, 26.9; MS (ESI+): m/z 209 [M
+H]+
Table 11
Inhibition of binding of [3H]-Quinuclidinyl benzi-
late ([3H]-QNB) to Wistar Rat cerebral cortex mus-
carinic cholinergic receptors by DISO and
ADD424042.
Compound
% inhibitiona
DISO
1
52%
−14%
a
10 μM of a test compound, n = 3.
Solvents were dried and purified by standard methods. All reagents
were purchased from commercial sources and used as received.
Solvents for chromatography were distilled prior use. The purities of
final compounds were ≥95%, (LC/MS and/or elemental analysis), ac-
cording to ETSP dispositions. Compounds 1, 2a, 2d, 3a, 3b, 3c, 3d, 3j,
3k, 3l, 3m were synthesized as described previously [32].
4.1.2. 2-fluoro-2-phenyl-2-(pyridin-2-yl)acetonitrile (2 s)
To a stirred, cooled (-78 °C) solution of 2-phenyl-2-(pyridin-2-yl)
acetonitrile 2a (2.80 g, 14.4 mmol) in dry THF (50 mL) was added
Lithium N,N-Diisopropylamide (17.3 mL of 1 M solution in THF,
17.3 mmol, 1.2 eq.), dropwise. The resulting solution was stirred for 1 h
4.1.1. 2-methyl-2-phenyl-2-(pyridin-2-yl)acetonitrile (2r)
To a stirred, cooled (-78 °C) solution of 2-phenyl-2-(pyridin-2-yl)
acetonitrile 2a (4.10 g, 21.1 mmol) in dry THF (75 mL) was added
10

M. Dawidowski, et al.
BioorganicChemistry98(2020)103717
and N-Fluorodi(benzenesulfonyl)amine (5.00 g, 15.9 mmol, 1.1 eq.)
was added portionwise, over 10 min. The mixture was brought to room
temperature and stirred overnight. The reaction was quenched with
saturated aqueous solution of NH4Cl (50 mL). The mixture was ex-
tracted with AcOEt (2 × 50 mL). The organic extracts were washed
with saturated aqueous solution of NaCl (2 × 25 mL), dried over an-
hydrous Na2SO4, filtered and concentrated. The residue was purified by
FC (hexane/AcOEt 8:1 to 5:1). Pale-yellow oil (2.15 g, 70%); 1H NMR
(300 MHz, CDCl3): δ = 8.70 (ddt, J = 5.0, 2.0, 1.0, 1H), 7.85 (td,
J = 8.0, 2.0, 1H), 7.63 (dq, J = 8.0, 1.0, 1H), 7.61–7.56 (m, 2H),
7.53–7.42 (m, 3H), 7.38 (ddd, J = 7.5, 5.0, 1.5, 1H); 13C NMR
(75 MHz, CDCl3): δ = 155.2 (d, J = 28.5), 149.8 (d, J = 2.0), 137.7,
135.9 (d, J = 24.0), 130.3 (d, J = 2.0), 129.0, 125.87 (d, J = 5.5),
124.6 (d, J = 1.5), 119.7 (d, J = 5.5), 116.7 (d, J = 33.5), 92.1 (d,
J = 185.5); MS (ESI+): m/z 234 [M+Na]+
δ = 173.1, 159.8, 158.5, 149.0, 140.1, 137.3, 129.7, 124.4, 122.3,
120.7, 114.2, 112.8, 60.5, 55.2; MS (ESI+): m/z 265 [M+Na]+; Anal.
calcd for C11H14N2O2: C 69.41, H 5.82, N 11.56, found: C 69.49, H 5.99,
N 11.52.
4.1.7. 2-(3-fluorophenyl)-2-(pyridin-2-yl)acetamide (3h)
3-fluorophenylacetonitrile (10.00 g, 74.0 mmol), 2-bromopyridine
(9.79 mL, 111.0 mmol) and KOH (20.78 g, 370.0 mmol), re-
crystallization from n-hexane/AcOEt 1:1 (v/v). White powder (6.53 g,
35%); mp: 95–96 °C; 1H NMR (500 MHz, CDCl3): δ = 8.60 (d, J = 4.0,
1H), 7.82 (bs, 1H), 7.67 (td, J = 7.5, 2.0, 1H), 7.32–7.14 (m, 5H), 6.94
(dt, J = 8.5, 1.5, 1H), 6.18 (bs, 1H), 5.01 (s, 1H); 13C NMR (125 MHz,
CDCl3): δ = 172.7, 162.8 (d, J = 246.3), 158.0, 141.0 (d, J = 7.5),
137.4, 130.1 (d, J = 8.5), 124.4, 124.1 (d, J = 3.0), 122.5, 115.3 (d,
J = 22.5), 114.3 (d, J = 21.0), 60.0; MS (ESI+): m/z 253 [M+Na]+
;
Anal. calcd for C13H11FN2O: C 67.82, H 4.82, N 12.17, found: C 67.90,
H 5.01, N 11.98.
4.1.3. General procedure for synthesis of 3e–i and 3n–q
Appropriate starting arylacetonitrile (1 eq.) was added to a stirred
suspension of KOH (5 eq.) in DMSO (200 mL per 1 mol of KOH). The
mixture was brought to 60 °C. Bromopyridine (1.5 eq.) was then added
dropwise, at 60–70 °C, followed by further stirring at this temperature
for 4 h. The reaction mixture was cooled and poured onto water
(500 mL) and the resulting cloudy solution was extracted with AcOEt
(2 × 150 mL), washed with water (100 mL) and brine (100 mL). The
organic extract was dried over anhydrous MgSO4, filtered and con-
centrated under reduced pressure. The residue was dissolved in AcOH
(100 mL) and concentrated H2SO4 (35 mL). The solution was heated at
100 °C until TLC showed complete reaction (1–3 h). The solution was
cooled and poured onto excess of ice-25% aqueous ammonia. The re-
sulting mixture was extracted with CH2Cl2 (3 × 75 mL). The combined
organic extracts were washed with water (2 × 75 mL), brine (75 mL),
dried over anhydrous Na2SO4, filtered and evaporated. The title com-
pounds were purified by recrystallization or by FC.
4.1.8. 2-(3-trifluoromethylphenyl)-2-(pyridin-2-yl)acetamide (3i)
3-trifluorophenylacetonitrile (10.00 g, 54.0 mmol), 2-bromopyr-
idine (9.79 mL, 81.0 mmol) and KOH (15.15 g, 270.0 mmol), re-
crystallization from n-hexane/AcOEt 1:1 (v/v). White powder (7.26 g,
48%); mp: 116–118 °C; 1H NMR (500 MHz, CDCl3): δ = 8.62 (d,
J = 4.0, 1H), 7.91 (bs, 1H), 7.74–7.62 (m, 3H), 7.51 (d, J = 8.0, 1H),
7.42 (t, J = 8.0, 1H), 7.30 (d, J = 8.0, 1H), 7.27–7.21 (m, 1H), 6.12
(bs, 1H), 5.05 (s, 1H); 13C NMR (125 MHz, CDCl3): δ = 172.5, 157.8,
149.2, 139.6, 137.6, 131.8, 131.0 (q, J = 32.0), 129.1, 125.1 (q,
J = 3.0), 124.4, 124.3 (q, J = 4.0), 124.0 (q, J = 272.5), 122.7, 60.0;
MS (ESI+): m/z 281 [M+H]+; Anal. calcd for C14H11F3N2O: C 60.00,
H 3.96, N 9.99, found: C 59.72, H 4.12, N 9.97.
4.1.9. 2-(3,4-dichlorophenyl)-2-(pyridin-2-yl)acetamide (3n)
From 3,4-dichlorophenylacetonitrile (10.00 g, 53.8 mmol), 2-bro-
mopyridine (7.70 mL, 80.6 mmol) and KOH (15.00 g, 269.0 mmol),
recrystallization from DCM. White powder (7.07 g, 47%); mp:
146–147 °C; 1H NMR (500 MHz, CDCl3): δ = 8.62 (d, J = 4.5, 1H),
7.90 (bs, 1H), 7.70 (td, J = 7.5, 1.5, 1H), 7.55 (d, J = 2.0, 1H), 7.38 (d,
J = 8.5, 1H), 7.33–7.23 (m, 3H), 5.82 (bs, 1H), 4.93 (s, 1H); 13C NMR
(125 MHz, CDCl3): δ = 172.3, 157.8, 149.5, 139.0, 137.9, 133.0,
131.9, 130.8, 130.5, 128.0, 124.7, 123.0, 59.6; Anal. calcd for
4.1.4. 2-(3-chlorophenyl)-2-(pyridin-2-yl)acetamide (3e)
3-chlorophenylacetonitrile (15.00 g, 98.9 mmol), 2-bromopyridine
(14.16 mL, 160.42 mmol) and KOH (25.52 g, 494.5 mmol), re-
crystallization from n-hexane/AcOEt 1:1 (v/v). White powder (9.81 g,
37%); mp: 96–97 °C; 1H NMR (500 MHz, CDCl3): δ = 8.60 (d, J = 4.5,
1H), 7.85 (bs, 1H), 7.67 (td, J = 7.5, 1.5, 1H), 7.43 (s, 1H), 7.32–7.34
(m, 1H), 7.29 (d, J = 8.0, 1H), 7.26–7.20 (m, 3H), 6.12 (bs, 1H), 4.98
(s, 1H); 13C NMR (125 MHz, CDCl3): δ = 172.6, 157.9, 149.1, 140.5,
137.5, 134.5, 129.9, 128.4, 127.6, 126.6, 124.4, 122.5, 59.9; MS (ESI
+): m/z 269 [M+Na]+; Anal. calcd for C13H11ClN2O: C 63.29, H 4.49,
N 11.36, found: C 63.40, H 4.55, N 11.28.
C14H14N2O: C 55.54, H 3.59, N 9.96, found: C 55.49, H 3.64, N 10.12.
4.1.10. 2,2-di(pyridin-2-yl)acetamide (3o)
From 2-pyridylacetonitrile (25.00 g, 211.6 mmol), 2-bromopyridine
(30.32 mL, 317.4 mmol) and KOH (59.35 g, 1058.0 mmol), re-
crystallization from n-hexanes/AcOEt 2:3 (v/v). White powder
(10.79 g, 24%); mp: 186–187 °C; 1H NMR (500 MHz, CDCl3): δ = 8.58
(ddd, J = 5.0, 2.0, 1.0, 2H), 8.23 (bs, 1H), 7.67 (td, J = 7.5, 1.5, 2H),
7.44 (d, J = 7.5, 2H), 7.19 (ddd, J = 6.0, 5.0, 1.0, 2H), 5.95 (bs, 1H),
5.25 (s, 1H); 13C NMR (125 MHz, CDCl3): δ = 172.0, 157.9, 149.3,
137.2, 123.8, 122.4, 62.8; MS (ESI+): m/z 214 [M+H]+; Anal. calcd
for C12H11N3O: C 67.58, H 5.21, N 19.71, found: C 67.56, H 5.20, N
19.61.
4.1.5. 2-(3-methylphenyl)-2-(pyridin-2-yl)acetamide (3f)
3-methylphenylacetonitrile (15.00 g, 114.3 mmol), 2-bromopyr-
idine (15.13 mL, 171.5 mmol) and KOH (32.06 g, 571.5 mmol), re-
crystallization from n-hexane/AcOEt 1:1 (v/v). White powder (15.02 g,
53%); mp: 96–97 °C; 1H NMR (500 MHz, CDCl3): δ = 8.59 (d, J = 4.0,
1H), 7.67 (bs, 1H), 7.64 (td, J = 7.5, 2.0, 1H), 7.28 (d, J = 8.0, 1H),
7.23 (s, 1H), 7.22–7.16 (m, 3H), 7.06 (d, J = 6.5, 1H), 6.00 (bs, 1H),
4.98 (s, 1H), 2.31 (s, 3H); 13C NMR (125 MHz, CDCl3): δ = 173.4,
158.8, 149.0, 138.6, 138.4, 137.2, 129.0, 128.6, 128.2, 125.3, 124.4,
122.2, 60.5, 21.4; MS (ESI+): m/z 249 [M+Na]+; Anal. calcd for
4.1.11. 2-(1-naphthyl)-2-(pyridin-2-yl)acetamide (3p)
From 1-naphthylacetonitrile (19.00 g, 113.6 mmol), 2-bromopyr-
idine (16.28 mL, 170.4 mmol) and KOH (31.67 g, 568.0 mmol), re-
crystallization from n-hexanes/AcOEt 2:3 (v/v). White powder (6.02 g,
20%); mp: 103–106 °C; 1H NMR (500 MHz, CDCl3): δ = 8.61 (ddd,
J = 5.0, 2.0, 1.0, 1H), 8.14 (pd, J = 8.0, 1H), 7.86 (dd, J = 7.5, 2.0,
1H), 7.80 (d, J = 8.5, 1H), 7.64 (td , J = 7.5, 2.0, 1H), 7.58 (d, J = 6.5,
1H), 7.53–7.46 (m, 2H), 7.44 (dd, J = 8.5, 7.0, 1H), 7.26 dt, J = 8.0,
1.0), 7.22 (ddd, J = 7.5, 5.0, 1.5, 1H), 6.92 (bs, 1H), 5.96 (s, 1H), 5.94
(bs, 1H); 13C NMR (125 MHz, CDCl3): δ = 173.1, 158.4, 148.2, 137.8,
134.1, 131.7, 128.9, 128.6, 126.8, 126.3, 125.9, 125.5, 124.8, 123.6,
122.5, 56.3; MS (ESI+): m/z 263 [M+H]+; Anal. calcd for C17H14N2O:
C
14H14N2O: C 74.31, H 6.24, N 12.38, found: C 74.25, H 6.37, N 12.30.
4.1.6. 2-(3-methoxyphenyl)-2-(pyridin-2-yl)acetamide (3g)
3-methoxylphenylacetonitrile (12.00 g, 81.5 mmol), 2-bromopyr-
idine (10.79 mL, 122.3 mmol) and KOH (22.86 g, 407.5 mmol), re-
crystallization from n-hexane/AcOEt 1:1 (v/v). White powder (8.82 g,
41%); mp: 62–64 °C; 1H NMR (500 MHz, CDCl3): δ = 8.59 (d, J = 4.0,
1H), 7.69 (bs, 1H), 7.64 (td, J = 8.0, 2.0, 1H), 7.29 (d, J = 8.0, 1H),
7.25–7.17 (m, 2H), 7.02–6.97 (m, 2H), 6.79 (dt, J = 8.0, 2.0, 1H), 5.84
(bs, 1H), 4.98 (s, 1H), 3.76 (s, 3H); 13C NMR (125 MHz, CDCl3):
11

M. Dawidowski, et al.
BioorganicChemistry98(2020)103717
C 77.54, H 5.38, N 10.68, found: C 77.32, H 5.39, N 10.40.
4.1.16. General procedure for synthesis of 5a–e
A mixture of methyl 2-phenyl-2-(piridin-2yl)acetate 3 (1.0 eq.),
amine (10.0 eq.), TBD (0.3 eq.) in THF (2 mL per 1 mmol of 3) was
refluxed for 3 h. The resulting solution was cooled, concentrated under
reduced pressure and purified by FC (CH2Cl2/MeOH 99:1 to 95:5, v/v).
4.1.12. 2-(2-naphthyl)-2-(pyridin-2-yl)acetamide (3q)
From 1-naphthylacetonitrile (10.00 g, 59.8 mmol), 2-bromopyr-
idine (8.57 mL, 89,68 mmol) and KOH (16.77 g, 299.0 mmol), FC
(CH2Cl2/MeOH 99:1 to 95:5, v/v). White powder (4.20 g, 27%); mp:
136–137 °C; 1H NMR (500 MHz, CDCl3): δ = 8.61 (ddd, J = 5.0, 1.5,
1.0, 1H), 8.86 (d, J = 1.0, 1H), 7.80–7.75 (m, 3H), 7.73 (bs, 1H), 7.66
(td, J = 8.0, 2.0, 1H), 7.55 (dd, J = 8.5, 2.0, 1H), 7.46–7.41 (m, 2H),
7.34 (dt, J = 8.0, 1.0, 1H), 7.21 (ddd, J = 8.0, 5.0, 1.0, 1H), 6.06 (bs,
1H), 5.24 (s, 1H); 13C NMR (125 MHz, CDCl3): δ = 173.1, 158.5, 148.7,
137.5, 136.0, 133.4, 132.6, 128.5, 128.0, 127.5, 127.2, 126.3, 126.2,
126.1, 124.6, 122.4, 60.3; MS (ESI+): m/z 263 [M+H]+; Anal. calcd
for C17H14N2O: C 77.54, H 5.38, N 10.68, found: C 77.71, H 5.44, N
10.68.
4.1.17. N-methyl-2-phenyl-2-(piridin-2yl)acetamide (5a)
White powder (1.85 g, 93%); mp: 129–130 °C; 1H NMR (500 MHz,
CDCl3): δ = 2.83 (d, J = 4.5 Hz, 3H), 5.02 (s, 1H), 7.19 (ddd, J = 7.5,
J = 5.0, J = 1.0, 1H), 7.26 (tt, J = 6.0, J = 1.0, 1H), 7.23–7.31 (m,
3H), 7.36–7.40 (m, 2H), 7.64 (td, J = 7.5, J = 2.0, 1H), 7.78 (bs, 1H),
8.58 (ddd, J = 5.0, J = 2.0, J = 1.0, 1H); 13C NMR (125 MHz, CDCl3):
δ = 20.4, 60.5, 122.1, 124.4, 127.2, 128.2, 128.6, 137.2, 139.0, 148.8,
158.9, 171.2; MS (ESI+): m/z 227 [M+H]+
; Anal. calcd for
C14H14N2O: C 74.31, H 6.24, N 12.38, found: C 74.04, H 6.24, N 12.24.
General procedure for synthesis of 3r,s. 2r or 2s was dissolved in
AcOH (50 mL) and concentrated H2SO4 (18 mL). The solution was
heated at 100 °C until TLC showed complete reaction (1–3 h). The so-
lution was cooled and poured onto excess of ice-25% aqueous am-
monia. The resulting mixture was extracted with CH2Cl2 (3 × 35 mL).
The combined organic extracts were washed with water (2 × 35 mL),
brine (35 mL), dried over anhydrous Na2SO4, filtered and evaporated.
The title compounds were purified by FC.
4.1.18. N-ethyl-2-phenyl-2-(piridin-2yl)acetamide (5b)
White powder (2.01 g, 94%); mp: 120–122 °C; 1H NMR (500 MHz,
CDCl3): δ = 1.01 (t, J = 7.0, 3H), 3.12 (dq, J = 7.0 Hz, J = 1.5, 2H),
5.10 (s, 1H), 7.21–7.26 (m, 2H), 7.29–7.33 (m, 2H), 7.35–7.39 (m, 2H),
7.71 (td, J = 7.5, J = 2.0, 1H), 8.35 (bt, J = 5.5), 8.49 (ddd, J = 7.5,
J = 2.0, 1H); 13C NMR (125 MHz, CDCl3): δ = 14.6, 33.7 50.5, 121.9,
122.9, 126.7, 128.3, 128.6, 136.5, 139.3, 148.7, 159.6, 169.8; MS (ESI
+): m/z 241 [M+H]+; Anal. calcd for C15H16N2O: C 74.97, H 6.71, N
11.66, found: C 74.84, H 6.88, N 11.91.
4.1.13. 2-methyl-2-phenyl-2-(pyridin-2-yl)acetamide (3r)
From 2r (3.10 g, 14.9 mmol), FC (CH2Cl2/MeOH 99:1 to 95:5, v/v).
White powder (2.37 g, 70%); mp: 141–143 °C; 1H NMR (300 MHz,
CDCl3): δ = 8.63 (ddd, J = 5.0, 2.0, 1.0, 1H), 7.81–7.69 (m, 1H),
7.54–7.37 (m, 2H), 7.38–7.22 (m, 4H), 7.20–7.14 (m, 2H), 6.00 (bs,
1H), 2.01 (s, 3H); 13C NMR (75 MHz, CDCl3): δ = 176.4, 163.1, 148.4,
145.7, 137.3, 128.5, 127.6, 126.9, 122.8, 122.1, 58.4, 26.2; MS (ESI+):
m/z 227 [M+H]+; Anal. calcd for C14H14N2O: C 74.31, H 6.24, N
12.38, found: C 74.47, H 6.26, N 12.32.
4.1.19. N-propyl-2-phenyl-2-(piridin-2yl)acetamide (5c)
White powder (1.03 g, 92%); mp: 79–80 °C; 1H NMR (500 MHz,
(CD3)2CO): δ = 0.85 (t, J = 7.5, 3H), 1.84 (sx, J = 7.5, 2H), 3.18 (t,
J = 7.0, 1H), 3.19 (t, J = 7.0, 1H), 5.10 (s, 1H), 7.20–7.25 (m, 2H),
7.27–7.32 (m, 2H), 7.43–7.48 (m, 3H), 7.71 (td, J = 7.5, J = 2.0, 1H),
7.91 (bs, 1H), 8.52 (ddd, J = 5.0, J = 2.0, J = 1.0, 1H); 13C NMR
(125 MHz, (CD3)2CO): δ = 11.7, 23.5, 41.7, 61.5, 122.8, 124.4, 127.6,
129.1, 129.4, 137.4, 140.7, 149.6, 160.8, 170.9; MS (ESI+): m/z 255
[M+H]+; Anal. calcd for C16H18N2O: C 75.56, H 7.13, N 11.01, found:
C 75.78, H 7.02, N 10.76.
4.1.14. 2-fluoro-2-phenyl-2-(pyridin-2-yl)acetamide (3s)
From 2 s (1.90 g, 8.95 mmol), FC (CH2Cl2/MeOH 99:1 to 97:3, v/v).
White powder (1.52 g, 74%); mp: 168–169 °C; 1H NMR (400 MHz,
CDCl3): δ = 8.62–8.59 (m, 1H), 7.66 (td, J = 8.0, 2.0, 1H), 7.51 (m,
2H), 7.41 (dt, J = 8.0, 1.0, 1H), 7.33–7.29 (m, 3H), 7.26–7.21 (m, 1H),
7.17 (bs, 1H), 6.28 (bs, 1H); 13C NMR (101 MHz, CDCl3): δ = 171.0 (d,
J = 25.0), 157.3 (d, J = 24.0 Hz), 148.9, 137.4 (d, J = 22.0), 137.2,
129.2 (d, J = 2.0), 128.4, 126.6 (d, J = 7.5), 123.8 (d, J = 2.0 Hz),
122.3 (d, J = 6.5 Hz), 97.8 (d, J = 188.5 Hz); MS (ESI+): m/z 231 [M
+H]+; Anal. calcd for C13H11N2FO: C 67.82, H 4.82, N 12.17, found: C
67.61, H 4.79, N 12.04.
4.1.20. N-isopropyl-2-phenyl-2-(piridin-2yl)acetamide (5d)
White powder (1.01 g, 90%); mp: 111–114 °C; 1H NMR (500 MHz,
CDCl3): δ = 1.01 (dd, J = 6.5, 6H), 4.09 (m, 1H), 4.96 (s, 1H), 7.19
(ddd, J = 8.0, J = 5.0, J = 1.0, 1H), 7.27–7.32 (m, 3H), 7.23 (tt,
J = 7.5, J = 2.0, 1H), 7.36–7.40 (m, 2H), 7.51 (bd, 1H), 7.65 (td,
J = 7.5, J = 2.0, 1H), 8.49 (ddd, J = 5.0, J = 2.0, J = 1.0, 1H); 13C
NMR (125 MHz, CDCl3): δ = 22.5, 22.6, 41.5, 60.7, 122.1, 124.4,
127.2, 128.1, 128.6, 137.2, 139.2, 148.8, 159.1, 169.6; MS (ESI+): m/z
255 [M+H]+; Anal. calcd for C16H18N2O: C 75.56, H 7.13, N 11.01,
found: C 75.43, H 7.05, N 10.56.
4.1.15. Methyl 2-phenyl-2-(piridin-2yl)acetate (4)
2-phenyl-2-(pyridin-2-yl)acetonitrile 2a (5.40 g, 27.8 mmol) was
dissolved in saturated methanolic solution of HCl (60 mL), at 4 °C. The
mixture was left overnight at room temperature. The resulting clear
solution was diluted with water (150 mL) and brought to pH 10 with
3 N aqueous solution of NaOH. The resulting mixture was extracted
with AcOEt (3 × 100 mL). The combined organic extracts were washed
with brine (100 mL), dried over anhydrous MgSO4, filtered and con-
centrated under reduced pressure. The crude product was recrystallized
from EtOH. White powder (3.90 g, 62%); mp: 74–76 °C; 1H NMR
(500 MHz, CDCl3): δ = 3.76 (s, 3H), 5.24 (s, 1H), 7.16 (ddd, J = 7.5,
J = 5.0, J = 1.0, 1H), 7.22 (dt, J = 8.0, J = 1.0, 1H), 7.28 (tt, J = 7.0,
J = 1.5, 1H), 7.32–7.37 (m, 2H), 7.37–7.41 (m, 2H), 7.60 (td, J = 7.5,
J = 2.0, 1H), 8.57 (ddd, J = 5.0, J = 2.0, J = 1.0, 1H); 13C NMR
(125 MHz, CDCl3): δ = 52.4, 59.6, 122.1, 123.0, 127.6, 128.7 20.4,
60.5, 122.1, 124.4, 127.2, 128.2, 128.6, 128.9, 136.7, 137.1, 149.3,
4.1.21. N-benzyl-2-phenyl-2-(piridin-2yl)acetamide (4e)
White powder (1.22 g, 91%); mp: 112–115 °C; 1H NMR (500 MHz,
CDCl3): δ = 4.48 (dd, J = 15.0, J = 6.0, 1H), 4.52 (dd, J = 15.0,
J = 6.0, 1H), 5.07 (s, 1H), 7.18–7.32 (m, 9H), 7.39–7.42 (m, 2H), 7.65
(td, J = 8.0, J = 2.0, 1H), 8.24 (bt, J = 6.0, 1H), 8.55 (ddd, J = 5.0,
J = 2.0, J = 1.0, 1H); 13C NMR (125 MHz, CDCl3): δ = 43.5, 60.5,
122.2, 124.5, 127.2, 127.3, 127.4, 128.2, 128.5, 128.7, 137.2, 138.3,
139.0, 148.8, 158.8, 170.6; MS (ESI+): m/z 303 [M+H]+; Anal. calcd
for C20H18N2O: 79.44, H 6.00, N 9.26, found: C 79.54, H 5.95, N 8.91.
4.2. Anticonvulsant testing
The compounds were evaluated in the in vivo rodent models of
epilepsy within the ETSP, according to the well-established experi-
mental procedures and decision schemes described in the PANAChE
database [1,6].
158.6, 172.2; MS (ESI+): m/z 228 [M+H]+
; Anal. calcd for
C
14H13N1O2: C 73.98, H 5.78, N 6.16, found: C 74.09, H 5.75, N 6.33.
12

M. Dawidowski, et al.
BioorganicChemistry98(2020)103717
4.3. Sodium current recordings
University (program for the Development of Scientific Fields, Progress
Q39). YZ, AEH and JCH acknowledge support from the British Heart
Foundation (PG/12/69/29784; PG/14/61/31015) and Heart Research
UK (RG2640).
The experimental procedures used in this study adhered to the in-
stitutional and international guidelines on the ethical use of animals.
The methodology of current recordings was the same as in our previous
studies [8]. Briefly, 3-week-old rats were decapitated under ethyl-
chloride anaesthesia and their brains were removed. Parts of the slices
containing the prefrontal cortex were mechanically and enzymatically
dispersed. Pipette solution and extracellular solutions were the same as
in our previous study [8]. Currents were recorded using an Axopatch
1D amplifier and analysed with pClamp software (Axon Instruments
and Molecular Devices, CA, USA). Patch-pipettes had resistances be-
tween 4 and 5 MΩ. The access resistance was between 5 and 7 MΩ. A
series resistance compensation of 80% was applied. The sodium cur-
rents were leak subtracted. Recordings were performed at room tem-
perature. Voltage-gated potassium currents were not recorded because
they were blocked by TEA-CL in the extracellular solution. Voltage-
gated calcium currents were blocked by cadmium and lanthanum ions
in the extracellular solution. Compounds were applied to the whole-
bath.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.103717.
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