Anticonvulsant activity, crystal structures, and preliminary safety evaluation of N - trans -cinnamoyl derivatives of selected (un)modified aminoalkanols

Article abstract of DOI:10.1016/j.ejmech.2015.10.051

Adequate control of seizures remains an unmet need in epilepsy. In order to identify new anticonvulsant agents, a series of N-trans-cinnamoyl derivatives of selected aminoalkanols was synthetized. The compounds were obtained in the reaction of N-acylation carried out in a two-phase system. The substances were tested in animal models of seizures induced either electrically (maximal electroshock - MES; 6-Hz test) or chemically, by subcutaneous injection of pentetrazol (scPTZ). Neurotoxicity was determined by the rotarod test. Lipophilicity of the active compounds, expressed as R_M0, was determined by reversed-phase thin layer chromatography and it ranged from 1.390 to 2.219. From among the tested series of compounds, R,S-(E)-N-(1-hydroxypropan-2-yl)-3-phenylprop-2-enamide (1) and R,S-(E)-N-(2-hydroxypropyl)-3-phenylprop-2-enamide (3) exhibited the best anticonvulsant activity. Compound 1, when administered to mice by intraperitoneal (i.p.) injection, showed the ED₅₀ values of 86.6, 60.9, and 109.6 mg/kg in the MES, 6-Hz, and scPTZ tests, respectively. For compound 3, the ED₅₀ values were found to be 47.1 mg/kg in MES and 77.1 mg/kg in scPTZ (mice, i.p.). The distances measured in crystals of compound 1 were: 7.99 ? - from the phenyl ring to the hydroxyl group in the amide moiety, 5.729 ? - from the phenyl ring to the amide group, and 3.112 - from the amide group to the hydroxyl group in the amide moiety. The reported compounds did not exhibit mutagenic potential when assayed in the Ames test. Compounds 1 and 3 did not affect viability and morphology of human hepatocellular carcinoma cells (HepG2).

Full text of DOI:10.1016/j.ejmech.2015.10.051

European Journal of Medicinal Chemistry 107 (2016) 26e37
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Anticonvulsant activity, crystal structures, and preliminary safety
evaluation of N-trans-cinnamoyl derivatives of selected (un)modified
aminoalkanols
a
,
*
b
c
_
_
ꢀ
Agnieszka Gunia-Krzyzak , Ewa Zesławska , Karolina Słoczynska ,
c
d
a
a
_
Paulina Koczurkiewicz , Wojciech Nitek , Dorota Zelaszczyk , Natalia Szkaradek ,
Anna M. Waszkielewicz , Elzbieta Pe˛kala , Henryk Marona
a Department of Bioorganic Chemistry, Chair of Organic Chemistry, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, 30-688
a
c
a
_
ꢀ
Krakow, Poland
b
_
ꢀ
Departament of Chemistry, Institute of Biology, Pedagogical University, Podchora˛zych 2, 30-084 Krakow, Poland
c
ꢀ
Department of Pharmaceutical Biochemistry, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, 30-688 Krakow, Poland
Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krakow, Poland
d
ꢀ
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 July 2015
Received in revised form
26 September 2015
Accepted 28 October 2015
Available online 2 November 2015
Adequate control of seizures remains an unmet need in epilepsy. In order to identify new anticonvulsant
agents, a series of N-trans-cinnamoyl derivatives of selected aminoalkanols was synthetized. The com-
pounds were obtained in the reaction of N-acylation carried out in a two-phase system. The substances
were tested in animal models of seizures induced either electrically (maximal electroshock e MES; 6-Hz
test) or chemically, by subcutaneous injection of pentetrazol (scPTZ). Neurotoxicity was determined by
the rotarod test. Lipophilicity of the active compounds, expressed as RM0, was determined by reversed-
phase thin layer chromatography and it ranged from 1.390 to 2.219. From among the tested series of
compounds, R,S-(E)-N-(1-hydroxypropan-2-yl)-3-phenylprop-2-enamide (1) and R,S-(E)-N-(2-
hydroxypropyl)-3-phenylprop-2-enamide (3) exhibited the best anticonvulsant activity. Compound 1,
when administered to mice by intraperitoneal (i.p.) injection, showed the ED50 values of 86.6, 60.9, and
109.6 mg/kg in the MES, 6-Hz, and scPTZ tests, respectively. For compound 3, the ED50 values were found
to be 47.1 mg/kg in MES and 77.1 mg/kg in scPTZ (mice, i.p.). The distances measured in crystals of
compound 1 were: 7.99 Å e from the phenyl ring to the hydroxyl group in the amide moiety, 5.729 Å e
from the phenyl ring to the amide group, and 3.112 Å e from the amide group to the hydroxyl group in
the amide moiety. The reported compounds did not exhibit mutagenic potential when assayed in the
Ames test. Compounds 1 and 3 did not affect viability and morphology of human hepatocellular carci-
noma cells (HepG2).
Keywords:
Anticonvulsant activity
Cinnamamide
Crystal structure
Mutagenicity
Hepatotoxicity
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
effects [4]. Therefore, the search for new, more effective and better
tolerable antiepileptic drugs is a major challenge for medicinal
Epilepsy is a complex disorder of brain function that takes the
form of recurring seizures [1]. It is estimated that about 1% of
people suffer from epilepsy i.e. globally 50 million people are
affected [2]. About one third of patients have poor seizure control
despite proper pharmacotherapy [3]. Moreover, antiepileptic drugs
(AEDs) are known to cause multiple and often serious adverse
chemistry.
Cinnamamide derivatives constitute a very promising group of
compounds with central nervous system activity including anti-
convulsant properties [5]. A number of antiepileptic drugs contain
aryl ring and amidic function within their molecules [6]. In cinna-
mamides these two pharmacophoric features are linked with an
olefinic spacer which results in desired rigidity of the structure.
Dimmock et al. proposed simple N-(3-phenylprop-2-enoyl) moiety
as a pharmacophoric feature of anticonvulsant agents because it
contributed to the desired pharmacological properties [7]. On the
* Corresponding author.
_
E-mail address: agnieszka.gunia@uj.edu.pl (A. Gunia-Krzyzak).
http://dx.doi.org/10.1016/j.ejmech.2015.10.051
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

_
A. Gunia-Krzyzak et al. / European Journal of Medicinal Chemistry 107 (2016) 26e37
27
other hand, pharmacophore model of anticonvulsant agents, active
in maximal electroshock test proposed by Khan et al.,. consists of a
hydrophobic unit (e.g. aryl ring), a hydrogen bonding domain (HBD)
formed by an electron donor atom in close distance to NH group,
and an additional electron donor atom. These three pharmaco-
phoric features should be properly arranged in space [8]. Cinna-
mamide derivatives could fit into this model because they possess
phenyl ring and hydrogen bonding domain may be formed if the
amide group has only one substituent. Furthermore, not only
double bond may serve as an electron donor, but also an additional
substituent with electron donor atom, which has been introduced
in a particular position of the amide moiety (Fig. 1).
Anticonvulsant activity of cinnamamides has been continuously
reported over the past decades. For example, in the Chinese tradi-
tional medicine Piper species have been used to treat epileptic
seizures. Analytical methods made it possible to isolate the active
ingredients from plant material, alkaloids piperine and anti-
epilepsirine [9,10] (Fig. 2).
of spectral methods and elemental analysis. All synthetized com-
pounds had the trans configuration of the double bond.
2.2. Anticonvulsant activity
Anticonvulsant activity and neurotoxicity were evaluated under
the Anticonvulsant Screening Program (ASP) at the National Insti-
tute of Neurological Disorders and Stroke, National Institutes of
Health, Bethesda, USA [13,14]. Fifteen compounds (1e13, 15e16)
were subjected to standard protocols including three tests:
maximal electroshock (MES) and subcutaneous pentetrazol (scPTZ)
tests for anticonvulsant activity as well as rotarod test for neuro-
toxicity (TOX). The evaluations were performed in mice 0.5 and 4 h
after intraperitoneal (i.p.) administration of the studied com-
pounds. Additionally, compound 11 at the dose of 100 mg/kg was
tested 0.25 h after i.p. administration. Compound 14 was tested in
6-Hz test for anti-seizure activity and rotarod for neurotoxicity
while MES and scPTZ tests were omitted in this case, because the
compound was subjected to modified protocol of evaluation of
anticonvulsant activity. Compounds 1, 3, 8, 11, and 16 were tested in
6-Hz after successful screens in MES. Furthermore, compounds
1e4, 8, 11, and 15 were evaluated in rats after oral administration.
The results of the MES, scPTZ, and TOX screening in mice after
intraperitoneal administration of the compounds are presented in
Table 1. Most of the compounds were proved to be active anti-
convulsants in MES at the dose of 100 and/or 300 mg/kg half an
hour after administration. Compound 11 was active in MES in three
out of three tested animals when determined 0.25 h after admin-
istration. What is interesting is that some of the tested compounds
at the dose of 300 mg/kg also prevented seizures evoked by pen-
tetrazol injected subcutaneously. The neurotoxic effects of the
tested derivatives were also noticed, especially at the dose of
300 mg/kg. In particular the results of 6-Hz test suggest that the
compounds have useful pharmacological properties (Table 2).
Compounds 1, 8, and 11 showed anticonvulsant activity 0.25 and
0.5 h after administration, while compound 3 was active up to 1.0 h
after administration. Moreover, some of the compounds exhibited
anticonvulsant activity in MES after oral administration in rats
(Table 3), which indicates that these compounds can be absorbed
from the gastrointestinal tract. In order to quantify their anticon-
vulsant activity (expressed as ED50) and neurotoxicity (expressed as
TD50), different doses of the active compounds were given i.p. to
mice and/or p.o. to rats. The results of quantification studies are
presented in Table 4 (mice) and Table 5 (rats).
Modifications of the structure of antiepilepsirine resulted in
discovery of several active compounds such as (E)-N-(2-
hydroxyethyl)-3-phenylprop-2-enamide
hydroxycyclohexyl)-3-phenylprop-2-enamide
[11],
[12],
(E)-N-(4-
1-[(E)-3-
phenylprop-2-enoyl]piperidin-4-one,
and
(E)-N,N-diethyl-3-
phenylprop-2-enamide [7], which have been presented in Fig. 3.
In the present study we report a series of trans-cinnamoyl de-
rivatives of selected (un)modified aminoalkanols. All these com-
pounds were trans-cinnamamide derivatives and contained
pharmacophoric features of anticonvulsant agents: hydrophobic
units, hydrogen bonding domains and additional electron donor
atoms in the amide moieties. We anticipated to calculate distances
between these features by means of crystallographic methods. The
compounds were tested for anticonvulsant activity in various ani-
mal models of seizures. We also evaluated lipophilicityeactivity
relationship, as log P is an important parameter of drugs acting on
the central nervous system. Moreover, we evaluated the studied
compounds for safety; they were tested for potential mutagenic
activity using the Ames test and for hepatotoxicity on human he-
patocellular carcinoma cell line.
2. Results and discussion
2.1. Chemistry
Synthetic route and chemical structures of the tested com-
pounds are shown in Scheme 1 (and Table 1A Supplementary
material). These compounds were obtained by N-acylation of
appropriate aminoalkanols or amino acid esters by means of trans-
cinnamoyl chloride. The reaction was carried out in a two-phase
system. Some of the obtained derivatives were subjected to
further modifications including acetylation, oxidation, and
ammonolysis followed by N-Mannich base formation. The struc-
tures and purity of the final compounds were confirmed by means
2.3. Crystal structures
2.3.1. Crystal structure of compound 1
An overview of the asymmetric unit of compound 1 with the
atom numbering is shown in Fig. 4. The crystal structure of 1
confirms its (R,S) configuration. 3-phenylprop-2-enamide moiety of
1 is almost planar. The angle between the plane of aromatic ring
and the plane of amide group is 5.3(1)ꢀ. The side chain is conju-
gated with aromatic ring, as shown by the bond distances C1eC2
1.485(1) Å, C2aC3 1.336(2) Å, C3eC4 1.465(2) Å. The methyl and
hydroxymethyl groups at C10 are directed in opposite side of planar
moiety of molecule.
From topological point of view, there are three possibilities in
the molecule of 1 to generate intermolecular hydrogen bonds: C]
O, NeH and OeH. All three groups are involved in hydrogen bonds
(Fig. 5). The packing of molecules in the unit cell can be charac-
terized by intermolecular interaction listed in Table 6. Two neigh-
bouring molecules are connected by two mutual hydrogen bounds
between hydroxyl and carbonyl groups. The amine group forms
hydrogen bond with the oxygen atom of hydroxyl group.
Fig. 1. Trans-cinnamamide derivative matched with pharmacophoric features of an-
ticonvulsants active in maximal electroshock test (R e hydrophobic ring, HBD
hydrogen bonding domain, D-electron donor atom).
e

_
28
A. Gunia-Krzyzak et al. / European Journal of Medicinal Chemistry 107 (2016) 26e37
Fig. 2. The structure of piperine (a) and antiepilepsirine (b).
Fig. 3. Cinnamamide derivatives with anticonvulsant activity: a) (E)-N-(2-hydroxyethyl)-3-phenylprop-2-enamide, b) (E)-N-(4-hydroxycyclohexyl)-3-phenylprop-2-enamide, c) 1-
[(E)-3-phenylprop-2-enoyl]piperidin-4-one, d) (E)-N,N-diethyl-3-phenylprop-2-enamide.
Scheme 1. Synthesis of the tested compounds 1e16. A: (R,S)-2-aminopropan-1-ol for 1, (R,S)-1-aminopropan-2-ol for 3, (R,S)-2-aminobutan-1-ol for 6, (R,S)-1-aminobutan-2-ol for
8, 2-amino-2-methylpropan-1-ol for 9, 5-aminopropan-1-ol for 10, (R,S)-2-amino-3-methylbutan-1-ol for 11, trans-D,L-2-aminocyclohexanol for 13, 4-hydroxypiperidine for 15, B:
methyl (R,S)-2-amino-3-methylbutanoate hydrochloride, C: methyl 2-aminopropanoate, D: methanol, 25% ammonia, E: formaldehyde, piperidine.

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A. Gunia-Krzyzak et al. / European Journal of Medicinal Chemistry 107 (2016) 26e37
29
Table 1
Table 3
Anticonvulsant activity and neurotoxicity data of the tested compounds (mice, i.p.).
Anticonvulsant activity of tested compounds in rats after oral administration at the
dose of 30 mg/kg.
Compd.
Dose [mg/kg]
MESa
scPTZa
0.5 h
Neurotoxicityb
Compd.
Test
Time/results
0.25 h
0.5 h
4.0 h
4.0 h
0.5 h
4.0 h
0.5 h
1.0 h
2.0 h
4.0 h
1
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
100
ec
3/3
1/1
e
e
e
1/1
e
e
e
e
e
e
e
e
1/1
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
1/1
e
e
e
e
1/3
e
e
e
ǀ
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
ǀ
e
e
e
e
e
e
e
e
e
1/2
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
4/4(4)
e
e
ǀ
e
e
1
MESa
TOXb
MES
TOX
MES
TOX
MES
TOX
MES
TOX
MES
TOX
MES
TOX
1/4
e
1/4
e
2/4
e
ec
e
e
1/1
e
4/4
e
e
2
2
e
e
1/4
e
e
e
3/3
1/1
e
e
e
e
e
e
e
e
4/4
e
3
e
2/4
e
2/4
e
e
e
3
e
e
e
e
2/3
1/1
e
e
1/8
4/4
e
4
e
e
e
e
e
1/1
e
e
e
e
e
e
4
8
4/4
e
3/4
e
1/4
e
1/4
e
1/4
e
2/3
1/1
e
e
e
5/5
e
2/4
e
11
15
e
e
e
e
1/4
e
5
e
e
e
e
e
e
e
e
1/4
e
2/4
e
e
e
1/1
e
e
4/4
e
e
e
e
6
e
The animals were tested at 0.25, 0.5 1.0, 2.0 and 4 h after oral administration of the
compounds. aThe results are presented as the number of animals protected/number
of animals used in MES test and bthe number of animals showing neurotoxicity/
number of animals used for neurotoxicity assessment. cThe dash (ꢁ) indicates an
absence of activity/neurotoxicity at the tested dose.
1/3
1/1
e
e
3/8
4/4
e
1/1
e
7
e
e
e
e
e
e
8
e
e
e
2/3
1/1
e
e
e
2.3.2. Crystal structure of compound 5
1/1
e
4/4
e
9
An overview of the asymmetric unit of compound 5 with the
atom numbering is shown in Fig. 6. The side chain is conjugated
with aromatic ring: C1eC2 1.487, C2]C3 1.332, C3eC4 1.469. 3-
phenylprop-2-enamide moiety is not so planar as in crystal struc-
ture of compound 1. The angle between the aromatic ring and
peptide moiety is 24ꢀ (in 1: 5.36ꢀ). Oxygen atom O2 has another
orientation in comparison to 1, the torsion angle N1eC10eC11eO2
is ꢁ3.57ꢀ whereas in 1e66.96ꢀ. This conformation prevents the
carbonyl group C11]O2 to make strong intermolecular hydrogen
bond.
The packing of molecules in the unit cell can be characterized by
intermolecular interaction listed in Table 6. Two neighbouring
molecules are connected by two mutual hydrogen bounds between
peptide moiety (Fig. 7). The amine group forms hydrogen bond
with the oxygen atom of carbonyl group. This interaction leads to
1/3
1/1
e
e
e
1/1
e
4/4
e
10
11
12
13
15
16
e
e
e
e
e
e
e
e
e
e
e
e
1/1
e
e
2/4
e
e
e
e
e
1/1
e
e
e
e
e
3/3
e
e
8/8
4/4
e
e
e
e
3/3
ǀd
1/4
e
e
1/1
ǀ
4/4(4)
e
1/4
ǀ
e
The animals were examined at 0.5 h and 4 h after injections of the compounds.
aNumber of animals protected/number of animals used in the MES or scPTZ test;
bNumber of animals displaying neurotoxicity/number of animals used in the rotarod
test, number in parentheses indicates number of observed deaths; cThe dash (ꢁ)
indicates an absence of activity/neurotoxicity at the tested dose dThe line (ǀ) in-
dicates that the compound was not tested in ceratin conditions.
Table 4
Results of quantitative evaluation of tested compounds and standard drugs in mice
after i.p. administration.
Comp.
Test
Time [h]
0.25
ED50 [mg/kg]
PIb
1
MES
scPTZ
6-Hzc
TOX
86.6 (69.7e105.1)a
109.6 (73.8e157.6)
60.9 (44.8e75.1)
223.2 (185.9e258.3)
47.1 (40.9e57.8)
77.1 (51.9e103.6)
136.1 (123.5e159.2)
105.9 (90.6e119.4)
190.1 (169.0e221.7)
263 (237e282)
220 (177e268)
126 (94.5e152)
398 (356e445)
7.81 (6.32e8.45)
>50
45.4 (32.9e54.4)
2.6
2.0
3.7
Table 2
3
MES
scPTZ
TOX
0.25
0.25
2.9
1.8
Anticonvulsant activity of tested compounds in 6-Hz test (32 mA) at the dose of
100 mg/kg (mice, i.p.).
Time/resultsa
0.25 h
11
6-Hzc
TOX
1.8
Compd.
0.5 h
1.0 h
2.0 h
4.0 h
Valproic acidd
MES
scPTZ
6-Hz
TOX
MES
scPTZ
TOX
1.5
1.8
3.2
1
3
8
11
14
16
3/4
4/4
4/4
4/4
e
2/4
4/4
2/4
4/4
1/4
1/4
eb
4/4
1/4
e
e
e
e
e
e
e
e
e
1/4
e
Carbamazepined
5.8
e
e
e
1/4
e
a
The animals were tested at 0.25, 0.5 1.0, 2.0 and 4 h after injections of the com-
pounds. aThe results are presented as the number of animals protected/number of
animals used in the 6-Hz test. bThe dash (ꢁ) indicates an absence of activity at the
tested dose.
95% confidence interval.
Protection Index (PI) calculated as TD50/ED50
6-Hz test with 32 mA.
b
c
.
d
Data from Ref. [14].

_
A. Gunia-Krzyzak et al. / European Journal of Medicinal Chemistry 107 (2016) 26e37
30
Table 5
Results of quantitative evaluation of tested compounds and standard drugs in rats
after p.o. administration.
Comp.
Test
Time [h]
1.0
ED50 [mg/kg]
PIb
>8.1
1
MES
scPTZ
TOX
31.0 (20.3e52.1)a
>250
>250
3
MES
scPTZ
TOX
MES
scPTZ
TOX
MES
scPTZ
TOX
MES
scPTZ
TOX
0.5
0.5
22.8 (16.5e30.5)
46.2 (21.0e108.2)
>500
41.5 (29.9e58.0)
>250
>11.0
>10.8
8
>12.1
Fig. 6. The molecular structure of compound 5 showing the atom numbering scheme.
Displacement ellipsoids are drawn at the 50% probability level.
>500
Valproic acidc
Carbamazepinec
485 (324e677)
646 (466e869)
784 (503e1176)
5.35 (3.26e7.62)
>250
1.6
1.2
68
364 (223e500)
a
95% confidence interval.
Protection Index (PI) calculated as TD50/ED50
Data from Ref. [14].
b
c
.
Fig. 7. Partial view of packing of 5 molecules projected along [010] direction. Dashed
lines indicate hydrogen bonds.
Fig. 4. The molecular structure of compound 1 showing the atom numbering scheme.
Displacement ellipsoids are drawn at the 50% probability level.
the layers in the crystal structure. Between the layers only weak
CeH/O interactions are observed.
2.4. Evaluation of lipohilicity
The relative lipophilicity (RM0) of the tested compounds was
determined using reversed-phase thin-layer chromatography (RP-
TLC) according to Boyce and Milborrow [15] and Pe˛kala and Marona
[16]. The investigations were carried out using solvent systems
containing methanol and water. The experimental data showed a
linear correlation between RM values and the concentration of
methanol in the mobile phase. The correlation coefficients ranged
from 0.970 to 0.998. (An example of the relationship obtained be-
tween RM and the concentration of methanol in the mobile phase
for compound 1 is shown in Fig. 1A Supplementary material). The
relative lipophilicity values (RM0) of the tested compounds, calcu-
lated as extrapolations of RM for 0% methanol concentrations,
ranged from 1.390 (compound 1) to 2.742 (compound 12). The
detailed data are presented in the Experimental section.
Fig. 5. Partial view of packing of molecules in the unit cell of the crystalline 1 projected
along [001] direction. Dashed lines indicate hydrogen bonds.
We determined the correlations between lipophilicity
expressed as RM0 and both anticonvulsant activity (in MES test) and
Table 6
Intermolecular interaction for compounds 1 and 5.
Compd.
D-H/A
H/A (Å)
D/A (Å)
D-H-A (ꢀ)
Symmetry codes
1
O2eH2O/O1
N1eH1/O2
N1eH1/O1
C3eH3/O2
C5eH5/O2
C10eH10A/O1
1.90(2)
2.09(2)
2.08(2)
2.52
2.72
2.58
2.711(2)
2.924(2)
2.939(2)
3.363(2)
3.553(2)
3.233(2)
162(2)
159(1)
173(2)
150.8
149.3
141.1
ꢁx, ꢁy þ 1, ꢁz
ꢁx þ 1/2, y ꢁ 1/2, z
x þ 1/2, ꢁy þ 1/2, ꢁz þ 1
xꢁ1/2, ꢁy þ 1/2, ꢁz þ 1
x ꢁ 1/2, ꢁy þ 1/2, ꢁz þ 1
ꢁx, ꢁy þ 1, ꢁz þ 1
5

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31
neurotoxicity in the tested series. A compound was classified as
active/neurotoxic when it showed a protective/neurotoxic effect in
more than a half of the tested animals at certain experimental
conditions. The groups were compared with the ManneWhitney
test (Fig. 8). The RM0 ranges were wider for not active/neurotoxic
compounds as compared to compounds active/not neurotoxic. The
mean value of RM0 was 1.844 for the active compounds and 2.129
for the compounds not active while the mean RM0 values of the
neurotoxic and not neurotoxic derivatives were 2.034, and 1.868,
respectively. Differences between the tested groups were, however,
not statistically significant (p for active/not active ¼ 0.0939, p for
neurotoxic/not neurotoxic ¼ 0.3307).
2.5. Evaluation of mutagenicity
Compounds 1, 3, 6, 8, 11, 13, and 15 were evaluated for their
mutagenic activity in the Ames (Salmonella) test. These compounds
were not mutagenic in the tested concentrations. The detailed re-
sults of the test are presented in Table 4A included in Supple-
mentary material.
2.6. Evaluation of hepatotoxicity
Compounds 1, 3, 8, and 11 were evaluated for their hepatotox-
icity. As control substances valproic acid and doxorubicin were
used. The compounds were tested in vitro on human hepatocellular
carcinoma cell line (HepG2) in two viability assays: MTT and trypan
blue exclusion. Morphology of cells incubated in the presence of
analyzed compounds was also evaluated. As showed in Fig. 9, the
tested compounds (1, 3, 8, and 11) did not affect viability of HepG2
Fig. 9. Results from MTT (a) and trypan blue exclusion (b) viability assays performed
on HepG2 cells after 24 h incubation with tested compounds. Results are presented as
mean SD calculated from at least three independent experiments. More data were
included in Supplementary material (Tables 5A and 6A).
cells at concentrations up to 100 mM after 24 h incubation. What is
more, the cells' morphology was not changed even after longer
incubation (48 h) in the presence of compounds 1, 3, 8 and 11
(appropriate pictures were included in Fig. 2A Supplementary
material).
Valproic acid only slightly decreased viability of HepG2 cells
although its effect was stronger than compounds 1, 3, 8 and 11
pharmacoresistance in epilepsy remains at a constant level of 30%.
For this reason, testing new compounds in models of resistant
seizures was introduced at the early stages of drug development in
the expectation of discovering new effective antiepileptics [17]. One
of them is a 6-Hz psychomotor seizure model in mice [18]. The
compounds examined in the present study were subjected either to
standard anticonvulsant evaluation (MES, scPTZ, rotarod e com-
pounds 1e13, 15e16) or to modified procedures (6-Hz, rotarod e
compound 14).
(concentration 100
mM). Doxorubicin significantly decrease the
number of viable cells in tested conditions.
3. Discussion and SAR studies
Compounds 1e16 reported here were designed to contain
structural features considered to be essential for anticonvulsant
activity. Pharmacophoric features of the tested compounds
comprise a hydrophobic/aromatic ring and a hydrogen bonding
domain formed by secondary amide group (except for compound
15). Furthermore, a hydroxyl or an oxo group in the amide moiety,
Maximal electroshock test and seizures induced chemically by
subcutaneous injection of pentetrazol for decades have been
considered as ‘gold standards’ for development of new antiepileptic
drugs. These techniques made it possible to identify most of the
currently marketed antiepileptic drugs [2]. On the other hand,
although
many
antiepileptic
drugs
are
available,
Fig. 8. Correlation of anti-MES activity (a) and neurotoxicity (b) of the tested compounds with their lipophilicity expressed as RM0
.

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A. Gunia-Krzyzak et al. / European Journal of Medicinal Chemistry 107 (2016) 26e37
Table 7
acting as an electron donor, could increase the probability of
hydrogen bonding at the binding site. Our former works referred to
the N-(3-phenylprop-2-enyl) (N-cinnamyl) analogues of com-
pounds 1e16 [19]. It was observed that N-cinnamyl derivatives of
aminoalkanols showed some activity in MES but were devoid of
activity in scPTZ test. The results obtained in the present study led
to a conclusion that amide function contributed to anticonvulsant
activity, especially against seizures evoked by subcutaneous injec-
tion of pentetrazol.
The comparison of calculated distances between pharmacophoric features in com-
pounds 1 and 5 with reported pharmacophore model of anti-MES active agents.
Compd./reference
Ring-D
Ring-HBD
HBD-D
1
5
7.990
8.523
3.02e5.68
5.729
5.716
4.9e8.02
3.112
3.331
2.89e6.0
Reference [8]
Distances are presented in Å, abbreviations: D e electron donor atom in N-sub-
stituent, HBD e hydrogen bonding domain.
In order to investigate the structureeactivity relationship, we
designed the series in such a way that the compounds differed in N-
substituent in terms of the hydrocarbon chain properties and the
position of hydroxyl group. Additionally, we modified the N-
hydroxyalkyl moiety by O-acetylation to obtain the compounds 2,
4, 7, and 14, by oxidation to synthetize compound 5 or by synthesis
of N-Mannich base in compound 16.
Modifications of the simple aminoalkanol moiety generally
resulted in a decrease of anticonvulsant efficacy and none of such
derivatives was subjected to quantitative evaluation. Interesting
results were obtained for the acetyl derivatives of 1 and 3 e com-
pounds 2 and 4, respectively. We synthetized them as potential
pro-drugs and indeed they proved to be active in the MES and
scPTZ tests performed in mice after intraperitoneal administration.
Compound 4 showed especially promising activity in scPTZ test in
mice 0.5 h after i.p. administration. It protected five out of five
tested animals from seizures, which suggested that acetylation was
successful modification in increasing efficacy of this derivative.
However, compounds 2 and 4 did not prove to be effective in MES-
induced seizures in rats after oral administration, which was
probably caused by impaired absorption from the gastrointestinal
tract.
The analysis of the distances between two pharmacophoric
features: amide group and hydroxyl substituent in the amide
moiety led to a conclusion that the optimum length is determined
by two carbon atoms between nitrogen and oxygen in the side
chain. Extension of the distance up to five carbon atoms (compound
10), four [12] or three [20] led to a significant decrease of anti-
convulsant activity. Compounds with extended distance between
the amide group and the hydroxyl substituent were not active at
the dose of 100 mg/kg in MES test (mice, i.p.).
We also attempted to compare the distances between phar-
macophoric features in the structure of 1 with the proposed
pharmacophore model of anticonvulsants active in MES (Fig. 10,
Table 7). Two distances fitted into the model, but the third one,
between the aryl ring and electron donor atom in N-substituent,
was longer than the reference distance. The differences can be
explained by the fact that the compound was active in both MES
and scPTZ models of seizures.
the conversion of hydroxyalkyl group into an oxoalkyl, than the
differences in the distances between pharmacophoric features.
Compounds possessing cyclic substituents in the amide moiety,
regardless of the NeO distance, were either not active (compounds
14, 16) or active but also significantly neurotoxic (compounds 13,
15). Derivatives with non-cyclic N-alkyl chains showed more
beneficial pharmacological properties. In the group of the tested
compounds, the following aminoalkanols substituted with trans-
cinnamoyl moiety exhibited beneficial properties: 2-aminopropan-
1-ol (compound 1), 1-aminopropan-2-ol (compound 3), 1-
aminobutan-2-ol (compound 8), and 2-amino-3-metylbutan-1-ol
(compound 11).
Lipophilicity has been characterized as one the most important
physicochemical parameters affecting CNS activity because only
appropriate partition coefficient value of the molecule enables
sufficient penetration of the blood-brain barrier [21]. The lip-
ophilicity parameters RM0 of the reported compounds were found
to range from 1.390 to 2.742, while RM0 of the most active com-
pounds (protection of more than 50% of mice in MES test, i.p.),
ranged from 1.390 to 2.219 with the mean value of 1.844. Earlier
studies on other group of cinnamamide derivatives showed that the
optimum lipophilicity for anti-MES activity was rather high (logP in
the range of 3.8e4.3) [10]. These differences may suggest that lip-
ohilicity, although important, is not crucial for anticonvulsant ac-
tivity of cinnamamide derivatives.
4. Conclusion
We identified potent anticonvulsant agents among series of N-
trans-(3-phenyl-2-propenoyl) derivatives of selected amino-
alkanols. The most active compounds were found among the de-
rivatives of 2-aminopropan-1-ol, 1-aminopropan-2-ol, 1-
aminobutan-2-ol, and 2-amino-3-metylbutan-1-ol. They were
active both in electrically (MES, 6-Hz) and chemically (subcutane-
ous pentetrazol) induced seizures in animal models. Our results
showed that RM0 (measure of lipophilicity) of the most active
compounds ranged from 1.390 to 2.219. The most active com-
pounds were found to be non-mutagenic in the Ames test and non-
hepatotoxic while tested on human hepatocellular carcinoma cell
line (HepG2).
The distances in compound 5 were similar to those in com-
pound 1 except for ring-D, which was longer. These findings might
suggest that the decreased activity of compound 5 was due more to
5. Experimental section
Some of the compounds reported in the article were previously
known. Compounds 1 and 3 were tested for myorelaxant activity in
rodents [22], however, the stereochemistry of a double bound and a
asymmetric carbon atom were not stated as well as the NMR
spectra were not published. For one of the stereoisomer of 13 (CAS:
154235-47-9) the antinociceptive activity was reported by Pau et al.
[23]. Compounds 6 and 8 were known in Chemical Abstract Data-
base but also without stereochemical descriptions and moreover no
biological activity was reported for them. Compound 10 possesses
CAS number 113297-90-8 and its synthesis was published by Ihara
et al. [24]. Compound 15 possesses CAS number 1666120-10-0 and
Fig. 10. Distances between pharmacophoric features of R,S-(E)-N-(1-hydroxypropan-
2-yl)-3-phenylprop-2-enamide (1).

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33
its synthesis was published by Wessig et al. [25]. Other CAS
numbers indicated below (sections 5.3.1e5.3.16) were previously
published in our patent claim [26].
respectively. 0.015 mol of the substrate was dissolved in 20 mL
acetic anhydride and stirred in room temperature. The reactions
were controlled by means of TLC (mobile phase: chloroform/
methanol 9/1 v/v). After the reaction was completed, the mixture
was poured on ice. In case of compound 14, the precipitate was
obtained, filtered, and crystallized. For compounds 2, 4, and 7 the
products were extracted with methylene chloride (50 ml þ 30 ml).
The organic phase was separated, dried over anh. Na2SO4, and
filtered. The solvent was evaporated and the residue was
crystallized.
5.1. Chemistry
Reagents were manufactured by Alfa Aesar (Karlsruhe, Ger-
many). Solvents were commercially available materials of reagent
grade. Melting points (m.p.) are uncorrected and were determined
using a Büchi SMP-20 apparatus (Büchi Labortechnik, Flawil,
Switzerland). The 1H NMR spectra were obtained in CDCl3 or
DMSO-d6 by means of a Varian Mercury-VX 300 NMR spectrometer
(Varian Inc., Palo Alto, USA) with TMS or DMSO as internal stan-
dard, respectively. Results of 1H NMR are presented in the following
5.2.3. Preparation of compound 5
Compound 5 was obtained from 3 in the reaction of oxidation by
means of Dess-Martin periodinane (DMP, 1,1,1-tris(acetyloxy)-1,1-
dihydro-1,2-benziodoxol-3-(1H)-one). The reaction was carried
out in methylene chloride and controlled with TLC (mobile phase:
chloroform/acetone/methanol 5/1/0.1 v/v). When the reaction was
completed, solution of sodium thiosulfate in saturated sodium
hydrocarbonate was added and mixed until two clear phases were
obtained. The organic phase was separated, dried over anh. Na2SO4,
and filtered. The solvent was evaporated and the residue was
crystallized.
format: chemical shift
d in ppm, multiplicity, coupling constant J in
Hertz (Hz), number of protons, protons' position. Multiplicities are
showed as the abbreviations: s (singlet), bs (broad singlet),
d (doublet), dd (doublet of doublets), t (triplet), m (multiplet).
Elemental analyses were performed on a Vario EI III elemental
analyzer (Hanau, Germany). The IR spectra were recorded on a
Jasco FT/IR 410 spectrometer (Gross-Umstadt, Germany) using KBr
pellets and are reported in cmꢁ1. The mass spectra for compounds
1e16 were obtained on Waters ACQUITYTM TQD system with the
TQ detector (Waters, Milford, USA). The ACQUITY UPLC BEH C18,
1.7 mm 2.1 mm ꢂ 50 mm column was used (Waters). Preparative
column chromatography was performed using silica gel 60 (particle
size 0.063e0.200; 70e230 Mesh ATM) purchased from Merck. The
purity of obtained compounds was confirmed by the thin-layer
chromatography (TLC), carried out on pre-coated aluminum
sheets (silica gel 60 F-254, Merck, Darmstadt, Germany) using
mixture of chloroform/methanol (9/1 v/v). Spots were visualized by
UV light. Theoretical values of the partition coefficient of the tested
compounds were calculated by means of on-line molinspitation
tool (miLogP) [27].
5.2.4. Preparation of compound 16
Compound 16 was obtained in the reaction of trans-cinnamoyl
chloride with methyl (R,S)-2-aminopropanoate. The crude product
was subjected to ammonolysis by means of 25% ammonia in
methanol. The obtained trans-(R,S)-2-[(3-phenylprop-2-enoyl)
amino]propanamide (reported in Ref. [12]) was refluxed in ethanol
with formaldehyde and piperidine for 12 h. Then, the solvent was
evaporated, toluene was added and the mixture was heated under
DeaneStark apparatus for 2 h. Then the solvent was evaporated and
the residue was crystallized.
5.3. Analytical data for compounds 1e16
5.2. General procedure of preparation of tested compounds
5.3.1. R,S-(2E)-N-(1-hydroxypropan-2-yl)-3-phenylprop-2-
enamide (1)
CAS: 1609486-29-4 [26]. White solid (yield 82%), m.p.
144e146 ꢀC. M ¼ 205.26. Anal. C12H15NO2 calcd: C, 70.22; H, 7.37; N,
6.82. Found: C, 70.32; H, 7.41; N, 6.78. 1H-NMR (CDCl3, 300 MHz)
Syntheses of the tested compounds were carried out according
to Scheme 1.
5.2.1. Preparation of compounds 1, 3, 6, 8e13, and 15
Compounds 1, 3, 6, 8e13, and 15 were obtained by means of N-
acylation [12,28]. In the reaction 0.015 mol of appropriate amino-
alkanol such as (R,S)-2-aminopropan-1-ol for 1, (R,S)-1-
aminopropan-2-ol for 3, (R,S)-2-aminobutan-1-ol for 6, (R,S)-1-
aminobutan-2-ol for 8, 2-amino-2-methylpropan-1-ol for 9, 5-
aminopropan-1-ol for 10, (R,S)-2-amino-3-methylbutan-1-ol for
11, trans-D,L-2-aminocyclohexanol for 13, 4-hydroxypiperidine for
15, or amino acid ester e methyl (R,S)-2-amino-3-methylbutanoate
hydrochloride (for 12) was dissolved in 50 ml 5.5% solution of
K2CO3. Then 10 mL toluene was added and the solution of 2.5 g
(0.015 mol) trans-cinnamoyl chloride in 30 ml of toluene was being
added dropwise for about 20 min while the reaction mixture was
mixed on magnetic stirrer. The crude products were filtered form
the reaction mixture and washed with 20 ml of 5% K2CO3 and 50 ml
of distilled water. The obtained compounds were dried and crys-
tallized with a mixture of n-hexane and toluene (3:1 v/v). Trans-
D,L-2-aminocyclohexanol for preparation of compound 13 was
synthesized in reaction of cyclohexene oxide with 25% ammonia
[29]. Other aminoalkanols and amino acid ester were purchased
from the commercial supplier.
ppm
d
1.25 (d, J ¼ 6.9; 3H, CH3); 2.86 (bs,1H, OH); 3.58e3.65 (m,1H,
CHHeOH); 3.72e3.79 (m, 1H, CHHeOH); 4.18e4.26 (m, 1H,
CHe(CH2OH)); 5.81 (bs,1H, NH); 6.40 (d, J ¼ 15.6; 1H, AreCH]CH);
7.34e7.51 (m, 5H, AreH); 7.64 (d, J ¼ 15.4; 1H, AreCH]CH). IR:
3430; 3280; 2976; 1661; 1609; 1563. ESI-MS: 205.92 (C12H16NO2
[M þ H]þ). Rf ¼ 0.63; RM0 ¼ 1.390; miLogP ¼ 1.468.
5.3.2. R,S-2-{[(2E)-3-phenylprop-2-enoyl]amino}propyl acetate (2)
White solid (yield 62%), m.p. 74e76 ꢀC. M ¼ 247.30. Anal.
C
14H17NO3 calcd: C, 68.00; H, 6.93; N, 5.66. Found: C, 67.49; H, 6.69;
N, 5.63. 1H-NMR (CDCl3, 300 MHz) ppm
d
1.24 (d, J ¼ 6.7; 3H,
CH(CH3)); 2.09 (s, 3H, COeCH3); 4.07 (dd, J ¼ 4.1; J ¼ 11.3; 1H,
CHHeOeCO); 4.20 (dd, J ¼ 5.6; J ¼ 11.3; 1H, CHHeOeCO);
4.39e4.47 (m, 1H, CH(CH3)); 5.82 (d, J ¼ 7.7; 1H, NH); 6.37 (d,
J ¼ 15.6; 1H, AreCH]CH); 7.34e7.40 (m, 3H, AreH); 7.48e7.51 (m,
2H, AreH); 7.80 (d, J ¼ 15.6; 1H, AreCH]CH). IR: 3282; 3069;
2986; 1726; 1652; 1644; 1617. ESI-MS: 248.09 (C14H18NO3
[M þ H]þ). Rf ¼ 0.85; RM0 ¼ 1.880; miLogP ¼ 2.172.
5.3.3. R,S-(2E)-N-(2-hydroxypropyl)-3-phenylprop-2-enamide (3)
CAS: 1609486-30-7 [26]. White solid (yield 80%), m.p.
131e133 ꢀC. M ¼ 205.26. Anal. C12H15NO2 calcd: C, 70.22; H, 7.37; N,
6.82. Found: C, 70.11; H, 7.61; N, 6.82. 1H-NMR (CDCl3, 300 MHz)
5.2.2. Preparation of compounds 2, 4, 7 and 14
Compounds 2, 4, 7, and 14 were obtained by means of acetyla-
tion with acetic anhydride of compounds 1, 3, 6, and 13,
ppm
d
1.24 (d, J ¼ 6.4; 3H, CH3); 2.80 (d, J ¼ 4.4; 1H, OH); 3.21e3.30

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34
(m, 1H, NHeCHH); 3.56e3.63 (m, 1H, NHeCHH); 3.97e4.04 (m, 1H,
CH(OH)eCH3); 6.12 (bs, 1H, NH); 6.43 (d, J ¼ 15.4; 1H, AreCH]CH);
7.34e7.52 (m, 5H, AreH); 7.64 (d, J ¼ 15.6; 1H, AreCH]CH). IR:
3299; 2980; 2934; 1652; 1605; 1560. ESI-MS: 206.18 (C12H16NO2
[M þ H]þ). Rf ¼ 0.63; RM0 ¼ 1.512; miLogP ¼ 1.501.
5.3.9. (2E)-N-(1-hydroxy-2-methylpropan-2-yl)-3-phenylprop-2-
enamide (9)
White solid (yield 85%), m.p. 133e135 ꢀC. M ¼ 219.29. Anal.
C
13H17NO2 calcd: C, 71.21; H, 7.81; N, 6.39. Found: C, 70.82; H, 7.77;
N, 6.35. 1H-NMR (CDCl3, 300 MHz) ppm
d 1.37 (s, 6H, (CH3)2); 3.66
(s, 2H, CH2eOH); 4.89 (bs, 1H, OH); 5.69 (bs, 1H, NH); 6.37 (d,
J ¼ 15.6; 1H, AreCH]CH); 7.34e7.41 (m, 3H, AreH); 7.45e7.53 (m,
2H, AreH); 7.61 (d, J ¼ 15.4; 1H, AreCH]CH). IR: 3270; 3078; 3331;
2970; 1655; 1616; 1564; 1450. ESI-MS: 220.11 (C13H18NO2
[M þ H]þ). Rf ¼ 0.71; RM0 ¼ 2.151; miLogP ¼ 1.947.
5.3.4. R,S-1-{[(2E)-3-phenylprop-2-enoyl]amino}propan-2-yl
acetate (4)
White solid (yield 60%), m.p. 106e108 ꢀC. M ¼ 247.30. Anal.
C
14H17NO3 calcd: C, 68.00; H, 6.93; N, 5.66. Found: C, 68.00; H, 7.03;
N, 5.69. 1H-NMR (CDCl3, 300 MHz) ppm
d
1.28 (d, J ¼ 6.4; CHeCH3);
5.3.10. (2E)-N-(5-hydroxypentyl)-3-phenylprop-2-enamide (10)
CAS: 113297-90-8 [24]. White solid (yield 75%), m.p. 68e70 ꢀC.
M ¼ 233.31. Anal. C14H19NO2 calcd: C, 72.07; H, 8.21; N, 6.00. Found:
C, 71.80; H, 8.28; N, 5.98. 1H-NMR (CDCl3, 300 MHz) ppm
2.07 (s, 3H, COeCH3); 3.46e3.64 (m, 2H, NHeCH2); 5.00e5.10 (m,
1H, COOeCH); 5.98 (bs, 1H, NH); 6.40 (d, J ¼ 15.6; 1H, AreCH]CH);
7.25e7.38 (m, 3H, AreH); 7.47e7.52 (m, 2H, AreH); 7.63 (d, J ¼ 15.4;
1H, AreCH]CH). IR: 3324; 3080; 2991; 2921; 1732; 1651; 1611.
ESI-MS: 248.09 (C14H18NO3 [M þ H]þ). Rf ¼ 0.84; RM0 ¼ 2.003;
miLogP ¼ 2.205.
d
1.43e1.50 (m, 2H, CH2eCH2eCH2eCH2eCH2); 1.56e1.68 (m, 4H,
CH2eCH2eCH2eCH2eCH2); 3.40 (q, J ¼ 6.6; 2H, CH2eOH); 3.66 (t,
J ¼ 6.4; 2H, NHeCH2); 5.73 (b2, 1H, NH); 6.58 (d, J ¼ 15.6; 1H,
AreCH]CH); 7.32e7.52 (m, 5H, AreH); 7.62 (d, J ¼ 15.6; 1H,
AreCH]CH). IR: 3291; 2941; 2860; 1652; 1621; 1542. ESI-MS:
5.3.5. (2E)-N-(2-oxopropan-1-yl)-3-phenylprop-2-enamide (5)
White solid (yield 59%), m.p. 125e127 ꢀC. M ¼ 203.24. Anal.
234.13 (C14H20NO2 [M þ H]þ). Rf
miLogP ¼ 2.184.
¼
0.55; RM0
¼
1.983;
C
12H13NO2 calcd: C, 70.92; H, 6.45; N, 6.89. Found: C, 70.65; H, 6.70;
N, 6.83. 1H-NMR (CDCl3, 300 MHz) ppm
d 2.26 (s, 3H, COeCH3);
4.31 (d, J ¼ 4.4; 2H, NHeCH2eCO); 6.39 (bs, 1H, NH); 6.48 (d,
J ¼ 15.6; 1H, AreCH]CH); 7.36e7.41 (m, 3H, AreH); 7.49e7.53 (m,
2H, AreH); 7.64 (d, J ¼ 15.6; AreCH]CH). IR: 3294; 3058; 3027;
1723; 1658; 1617; 1560. ESI-MS: 204.09 (C12H14NO2 [M þ H]þ).
Rf ¼ 0.73; RM0 ¼ 1.478; miLogP ¼ 1.315.
5.3.11. R,S-(2E)-N-(1-hydroxy-3-methylbutan-2-yl)-3-phenylprop-
2-enamide (11)
CAS: 1609486-26-1 [26]. White solid (yield 82%), m.p.
98e100 ꢀC. M ¼ 233.31. Anal. C14H19NO2 calcd: C, 72.07; H, 8.21; N,
6.00. Found: C, 71.99; H, 8.05; N, 5.84. 1H-NMR (CDCl3, 300 MHz)
ppm
d
0.99 (d, J ¼ 5.4; 3H, CH3); 1.01 (d, J ¼ 5.4; 3H, CH3); 1.97 (dq,
J ¼ 13.7; J ¼ 6.9; CH(CH3)2); 2.53 (bs, 1H, OH); 3.70e3.83 (m, 2H,
CH2eOH); 3.83e3.94 (m, 1H, NHeCH); 5.84 (d, J ¼ 8.0; NH); 6.45 (d,
J ¼ 15.6; 1H, AreCH]CH); 7.31e7.42 (m, 3H, AreH); 7.45e7.56 (m,
2H, AreH); 7.65 (d, J ¼ 15.6; 1H, AreCH]CH). IR: 3258; 3059; 2960;
1655; 1615; 1551. ESI-MS: 234.24 (C14H20NO2 [M þ H]þ). Rf ¼ 0.60;
RM0 ¼ 2.219; miLogP ¼ 2.247.
5.3.6. R,S-(2E)-N-(1-hydroxybutan-2-yl)-3-phenylprop-2-enamide
(6)
White solid (yield 79%), m.p. 116e118 ꢀC. M ¼ 219.29. Anal.
C
13H17NO2 calcd: C, 71.21; H, 7.81; N, 6.39. Found: C, 70.77; H, 7.91;
N, 6.54. 1H-NMR (CDCl3, 300 MHz) ppm
d
0.99 (t, J ¼ 7.4; 3H,
CH2eCH3); 1.52e1.72 (m, 2H, CH2eCH3); 2.48 (bs, 1H, OH); 3.66
(dd, J ¼ 5.9; J ¼ 11.2; 1H, CHHeOH); 3.77 (dd, J ¼ 3.6; J ¼ 11.2; 1H,
CHHeOH); 3.97e4.04 (m, 1H, NHeCH); 5.87 (d, J ¼ 7.4; 1H, NH);
6.44 (d, J ¼ 15.4; 1H, AreCH]CH); 7.26e7.40 (m, 3H, AreH);
7.47e7.51 (m, 2H, AreH); 7.64 (d, J ¼ 15.6; 1H, AreCH]CH). IR:
3411; 3270; 3077, 2964; 1648; 1588; 1554. ESI-MS: 220.21
(C13H18NO2 [M þ H]þ). Rf ¼ 0.60; RM0 ¼ 2.093; miLogP ¼ 2.003.
5.3.12. R,S-Methyl 3-methyl-2-{[(2E)-3-phenylprop-2-enoyl]
amino}butanoate (12)
White solid (yield 86%), m.p. 134e136 ꢀC. M ¼ 261.32. Anal.
C
15H19NO3 calcd: C, 68.94; H, 7.33; N, 5.36. Found: C, 68.59; H, 7.38;
N, 5.37. 1H-NMR (CDCl3, 300 MHz) ppm
d
0.96 (d, J ¼ 6.9; 3H, CH3);
0.99 (d, J ¼ 6.9; 3H, CH3); 2.20e2.26 (m, 1H, CH(CH3)2); 3.77 (s, 3H,
OeCH3); 4.71e4.76 (m, 1H, NHeCH); 6.10 (d, J ¼ 8.7; 1H, NH); 6.47
(d, J ¼ 15.6; 1H, AreCH]CH); 7.35e7.40 (m, 3H, AreH); 7.50e7.53
(m, 2H, AreH); 7.65 (d, J ¼ 15.6; 1H, AreCH]CH). IR: 3469; 3244;
3060; 2970; 1743; 1654; 1618; 1557. ESI-MS: 262.11 (C15H20NO3
[M þ H]þ). Rf ¼ 0.91; RM0 ¼ 2.742; miLogP ¼ 2.741.
5.3.7. 2-{[(2E)-3-phenylprop-2-enoyl]amino}butyl acetate (7)
White solid (yield 62%), m.p. 81e83 ꢀC. M ¼ 261.32. Anal.
C
15H19NO3 calcd: C, 68.94; H, 7.33; N, 5.36. Found: C, 68.90; H, 7.22;
N, 5.30. 1H-NMR (CDCl3, 300 MHz) ppm
d
0.99 (t, J ¼ 7.7; 3H,
CH2eCH3); 1.53e1.65 (m, 2H, CH2eCH3); 2.10 (s, 3H, COeCH3);
4.23e4.29 (m, 2H, CHeCH2eO); 5.66 (bs, 1H, NH); 6.39 (d, J ¼ 15.6;
1H, AreCH]CH); 7.35e7.53 (m, 5H, AreH); 7.64 (d, J ¼ 15.6;
AreCH]CH). IR: 3292; 2970; 1726; 1655; 1627; 1542. ESI-MS:
5.3.13. Trans, D,L-(2E)-N-(2-hydroxycyclohexyl)-3-phenylprop-2-
enamide (13)
White solid (yield 64%), m.p. 154e156 ꢀC. M ¼ 245.31. Anal.
262.11 (C15H20NO3 [M þ H]þ). Rf
miLogP ¼ 2.008.
¼
0.88; RM0
¼
2.118;
C
15H19NO2 calcd: C, 73.44; H, 7.81; N, 5.71. Found: C, 73.36; H, 7.87;
N, 5.66. 1H-NMR (DMSO-d6, 300 MHz) ppm
d 1.08e1.85 (m, 8H,
cyclohex.); 3.21e3.30 (m, 1H, cyclohex.); 3.47e3.56 (m, 1H,
NHeCH); 4.62 (d, J ¼ 5.1; 1H, OH); 6.66 (d, J ¼ 15.6; 1H, AreCH]
CH); 7.32e7.55 (m, 6H, AreH þ AreCH]CH); 7.92 (d, J ¼ 7.7; 1H,
COeNH). IR: 3410; 3050; 2910; 1648; 1620; 1545. ESI-MS: 246.16
(C15H20NO3 [M þ H]þ). Rf ¼ 0.68; RM0 ¼ 2.054; miLogP ¼ 2.525.
5.3.8. R,S-(2E)-N-(2-hydroxybutyl)-3-phenylprop-2-enamide (8)
CAS: 1609486-32-9 [26]. White solid (yield 85%), m.p. 90e92 ꢀC.
M ¼ 219.29. Anal. C13H17NO2 calcd: C, 71.21; H, 7.81; N, 6.39. Found:
C, 71.22; H, 7.63; N, 6.34. 1H-NMR (CDCl3, 300 MHz) ppm
d 0.99 (t,
J ¼ 7.4; 3H, CH2eCH3); 1.49e1.60 (m, 2H, CH2eCH3); 2.20 (bs, 1H,
OH); 3.23e3.32 (m, 1H, CH(OH)eCH2); 3.60e3.76 (m, 2H,
NHeCH2); 6.16 (bs, 1H, NH); 6.43 (d, J ¼ 15.6; 1H, AreCH]CH);
7.35e7.40 (m, 3H, AreH); 7.47e7.51 (m, 2H, AreH); 7.64 (d, J ¼ 15.6;
1H, AreCH]CH). IR: 3412; 3272; 3067, 2962; 1638; 1560. ESI-MS:
5.3.14. Trans, D,L 2-{[(2E)-3-phenylprop-2-enoyl]amino}cyclohexyl
acetate (14)
White solid (yield 52%), m.p. 146e148 ꢀC. M ¼ 287.36. Anal.
C
17H21NO3 calcd: C, 71.06; H, 7.37; N, 4.87. Found: C, 71.20; H, 7.34;
220.21 (C13H18NO2 [M þ H]þ). Rf
miLogP ¼ 2.004.
¼
0.65; RM0
¼
1.937;
N, 4.82. 1H-NMR (CDCl3, 300 MHz) ppm
cyclohex.); 1.67e1.87 (m, 2H, cyclohex.); 1.93e2.01 (m, 1H,
d 1.17e1.64 (m, 4H,

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35
cyclohex.); 2.03 (s, 3H, COeCH3); 2.13e2.26 (m, 1H, cyclohex.);
5.5.1. MES test
3.91e4.09 (m, 1H, NHeCH]); 4.74 (td, J ¼ 10.7; J ¼ 4.5; 1H,
CH(OCOCH3); 5.81 (d, J ¼ 7.9; 1H, COeNH); 6.31 (d, J ¼ 15.6; 1H,
AreCH]CH); 7.32e7.42 (m, 3H, AreH); 7.46e7.53 (m, 2H, AreH);
7.58 (d, J ¼ 15.6; 1H, AreCH]CH). IR: 3435; 3236; 3062; 2936;
2864; 1725; 1653; 1616; 1551. ESI-MS: 288.16 (C17H22NO3
[M þ H]þ). Rf ¼ 0.91; RM0 ¼ 2.579; miLogP ¼ 3.229.
Seizures were induced by delivering for a 0.2 s alternating cur-
rent at 60 Hz and 50 mA (mice) or 150 mA (rats) via corneal elec-
trodes. Protection was defined as the abolition of the hindlimb tonic
extension of the seizures.
5.5.2. scPTZ test
5.3.15. (2E)-1-(4-hydroxypiperidin-1-yl)-3-phenylprop-2-en-1-one
(15)
CAS: 1666120-10-0 [25]. White solid (yield 85%), m.p.
120e122 ꢀC. M ¼ 231.30. Anal. C14H17NO2 calcd: C, 72.70; H, 7.41; N,
6.06. Found: C, 72.59; H, 7.47; N, 6.08. 1H-NMR (CDCl3, 300 MHz)
Seizures were elicited by subcutaneous injection of pentetrazol
dissolved in 0.9% NaCl at the dose of 85 mg/kg (mice) or 70 mg/kg
(rats). Protection was scored if there was not observed episode of
clonic spasm of at least 5 s.
ppm
d 1.45e2.07 (m, 4H, CH2eCH(OH)eCH2); 2.17 (bs, 1H, OH);
3.30e4.12 (m, 5H, CH2eNeCH2, CHeOH); 6.91 (d, J ¼ 15.4; 1H,
AreCH]CH); 7.30e7.42 (m, 3H, AreH); 7.47e7.56 (m, 2H, AreH);
7.66 (d, J ¼ 15.4; 1H, AreCH]CH). IR: 3408; 2932; 2862; 1644;
1592; 1457; 1441. ESI-MS: 232.14 (C14H18NO2 [M þ H]þ). Rf ¼ 0.53;
RM0 ¼ 1.759; miLogP ¼ 1.040.
5.5.3. Neurotoxicity (TOX)
In mice the neurological deficit was measured by rotarod test in
which an 1 inch diameter knurled plastic rod rotating at 6 rpm was
used. Each mouse was placed on the rod. If it was not able to
maintain on the rod for at least 1 min in each of three trials the
neurotoxicity was scored. In rats neurological deficit was measured
in behavioral tests and scored when observed ataxia and loss of
placing response and muscle tone.
5.3.16. R,S-(2E)-N-{1-oxo-1-[(piperidin-1-ylmethyl)amino]propan-
2-yl}-3-phenylprop-2-enamide (16)
White solid (yield 45%), m.p. 147e149 ꢀC. M ¼ 315.42. Anal.
C
18H25N3O2 calcd: C, 68.54; H, 7.99; N, 13.32. Found: C, 68.73; H,
7.92; N, 13.27. 1H-NMR (CDCl3, 300 MHz) ppm
d
1.39e1.48 (m, 2H,
5.5.4. 6-Hz test
pip.); 1.46 (d, J ¼ 7.2; 3H, CHeCH3); 1.53e1.60 (m, 4H, pip.); 2.50 (t,
J ¼ 5.2; 4H, pip.); 4.09e4.20 (m, 2H, NHeCH2eN); 6.46 (d, J ¼ 15.6;
1H, AreCH]CH); 6.54 (d, J ¼ 7.7; 1H, COeNHeCH2); 7.35e7.37 (m,
3H, AreH); 7.48e7.51 (m, 2H, AreH); 7.64 (d, J ¼ 15.6; 1H, AreCH]
CH). IR: 3276; 2939; 1653; 1615; 1535. ESI-MS: 316.21 (C18H26N3O2
[M þ H]þ). Rf ¼ 0.06; RM0 ¼ 2.338; miLogP ¼ 2.131.
Seizures were induced in mice by delivering 0.2 ms pulses of
electric current at 6 Hz and 32 mA for 3 s via corneal electrodes. The
mouse was released into an observation cage immediately after
stimulation. The protection was scored if the mouse resumed its
normal activity within 10 s from the stimulation.
5.4. Evaluation of lipophilicity parameter RM0
5.6. Crystallography
Thin-layer chromatography was performed on silica gel RP-18
F254 plates (Merck, Darmstadt, Germany). The mobile phases
constituted mixtures of methanol and water, with the methanol
content between 95 and 65% (v/v). The tested compounds were
dissolved separately in methanol (1 mg/mL). Before developing the
plates, chromatographic chambers were saturated with mobile
phase for 1 h. The spots were observed in UV light. For each con-
centration of methanol Rf values were calculated (Table 2A Sup-
plementary material). RM values were calculated from the
experimental Rf using equation: RM ¼ log(1/Rf ꢁ 1) [15,16] (Table 3A
Supplementary material). The correlation coefficients between RM
values and the concentration of methanol in the mobile phase are
presented in Table 3A Supplementary material. The calculated
values of RM were extrapolated for 0% methanol concentration
using equation: RM ¼ RM0 þ bC where C is the concentration of
methanol in the mobile phase and RM0 is the value of relative lip-
ophilicity parameter. Statistical analysis of correlation of activity
and RM0 parameter were performed by means of GraphPad Prism.
Crystals suitable for an X-ray structure analysis were obtained
from an acetonitrile solution for 1 or a propyl acetate solution for 5
by slow evaporation of the solvent at room temperature.
Data for single crystal were collected using the Oxford Diffrac-
tion SuperNova four circle diffractometer, equipped with the Mo
(0.71069 Å) Ka radiation source and graphite monochromator. The
structure was solved by direct methods using SIR-97 [31] and all
non-hydrogen atoms were refined anisotropically using weighted
full-matrix least-squares on F2. Refinement and further calculations
were carried out using SHELXL-97 [32]. The hydrogen atoms
bonded to carbons were included in the structure at idealized po-
sitions and were refined using a riding model with Uiso(H) fixed at
1.2 Ueq of C and 1.5 Ueq for methyl groups.
Hydrogen atoms attached to oxygen and nitrogen atoms were
found from the difference Fourier map and refined without any
restraints. For molecular graphics ORTEP [33] and MERCURY [34]
programs were used.
Compound 1: C12H15NO2, Mr ¼ 205.25, crystal size ¼ 0.162
ꢂ
0.315
ꢂ
0.463 mm3, orthorhombic, space group Pbca,
5.5. Pharmacology
a ¼ 12.143(5) Å, b ¼ 7.578(5) Å, c ¼ 23.374(5) Å, V ¼ 2150.9(17) Å3,
Z ¼ 8, T ¼ 293(2)K, 27768 reflections collected, 2673 unique re-
Pharmacological tests were performed at Epilepsy Branch, Na-
tional Institute of Neurological Disorders and Stroke, National In-
stitutes of Health (NIH, Bethesda, USA) within Anticonvulsant
Screening Program (ASP). Pharmacological evaluation was accepted
by Institutional Animal Care and Use Committee. All procedures
were previously published [13,14] as well as they are available for
public in NIH website [30]. In the tests, albino mice Carworth Farms
No.1 or rats SpragueeDawley were used. The tested compounds
were dissolved or suspended in 0.5% methycellulose. Brief
description of the experiments are presented below.
flections (Rint ¼ 0.0309), R1 ¼ 0.0373, wR2 ¼ 0.0952 [I > 2
s
(I)].
Compound
5:
C12H13NO2,
Mr
¼
203.23,
crystal
size ¼ 0.385 ꢂ 0.528 ꢂ 0.562 mm3, orthorhombic, space group
Pbca, a ¼ 9.843(5) Å, b ¼ 7.422(5) Å, c ¼ 28.468(5) Å, 2079.7(18) Å3,
Z ¼ 8, T ¼ 120(2) K, 24405 reflections collected, 2581 unique re-
flections (Rint ¼ 0.0339), R1 ¼ 0.0387, wR2 ¼ 0.0926 [I > 2
s(I)].
CCDC 1410352 and 1410353 contain the supplementary crys-
tallographic data. These data can be obtained free of charge from
The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.
uk/data_request/cif.

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A. Gunia-Krzyzak et al. / European Journal of Medicinal Chemistry 107 (2016) 26e37
36
5.7. Evaluation of mutagenicity
(Ortophan2000). For each measured value, 200 cells were analyzed
from six replicates and the result was expressed as a mean
value standard deviation [39].
5.7.1. Bacterial tester strains
Salmonella typhimurium tester strains TA100, TA1535 and TA98
used for the assay were obtained from Dr. T. Nohmi, Division of
Genetics and Mutagenesis, National Institute of Hygienic Sciences,
Tokyo, Japan. TA100 and TA1535 strains detect base-pair sub-
stitutions, whereas TA98 strain is used to identify compounds
leading to frameshift mutations.
5.8.3. Cell morphology
Cells were seeded into 12-well plates (Corning) at a density of
5 ꢂ 104 cells/cm2. After 24 h the medium was replaced with a fresh
portion either without compounds (control) or with analyzed
compounds. After 48 h of incubation pictures of cells were taken.
Cell morphology was recorded with a Leica DMI6000B system
equipped with a temperature/CO2 chamber (37 ꢀC, 5% CO2), inter-
ference modulation contrast optics and a cooled, digital DFC360FX
CCD camera.
5.7.2. Mutagenicity assay
The Ames mutagenicity assay was performed using a pre-
incubation method with TA100, TA1535 and TA98 S. typhimurium
strains as described previously [35e37]. Salmonella cultures of each
bacterial strain were prepared by inoculation form the stock cul-
tures into nutrient broth and incubation for 12 h at 37 ꢀC with
Acknowledgments
shaking. Subsequently, 100
pounds at different concentrations (final concentrations were: 4,
20, 100, 500 and 1000 g/plate in DMSO), and 500 l of buffer so-
ml of the culture, 50 ml of test com-
The authors would like to thank prof. James P. Stables, prof. Jeff
Jiang, and Tracy Chen PhD for providing the results of pharmaco-
logical assays within the ASP program at National Institute of
Neurological Disorders and Stroke, National Institutes of Health
m
m
lution were added to 2 ml of the top agar containing trace amount
of histidine. Next, the mixture was mixed and poured onto the
surface of a glucose minimal agar plates. After 48 h incubation at
37 ꢀC revertant S. typhimurium colonies were counted. Positive
control (sodium azide in TA100 and TA1535 strains and 4-nitro-O-
phenylenediamine in TA98 strain) and negative control (DMSO)
were performed alongside. Three independent repetitions were
performed for each compound. Additionally, the mutagenic index
(MI) that is the ratio between the number of revertants per plate
with the test compound and the number of revertants observed in
the negative control was calculated. A sample was considered
mutagenic when a doseeresponse relationship was stated and at
least two-fold increase in the number of revertants (MI ꢃ 2) was
observed with at least one tested concentration [38].
ꢀ
(Bethesda, USA), as well as prof. Katarzyna Kiec-Kononowicz for
coordination of the cooperation between the NIH and Faculty of
Pharmacy Jagiellonian University Medical College. The work was
supported by the European Regional Development Fund through
the Innovative Economy Operational Program (contracts no:
POIG.01.01.02-12-012/09-00, and POIG.02.01.00-12-023/08 - the
equipment used for X-ray data collection) as well as Jagiellonian
University Medical College (Grants K/ZDS/005487, K/DSC/001954,
K/ZDS/005488) and Pedagogical University in Krakow.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.10.051. These data include MOL
file and InChiKeys of the most important compounds described in
this article.
5.8. Evaluation of hepatotoxicity
5.8.1. Cells culture
Human hepatocellular carcinoma cell line (HepG2) ATCC®
59195™ were used in the study. The cells were cultured in standard
conditions (37 ꢀC, 5% CO2), in EMEM medium (Sigma Aldrich),
supplemented with 10% FBS (Sigma Aldrich) and antibiotics (Sigma
Aldrich).
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