Structural evaluation and electrophysiological effects of some kynurenic acid analogs

Article abstract of DOI:10.3390/molecules24193502

Kynurenic acid (KYNA), a metabolite of tryptophan, as an excitatory amino acid receptor antagonist is an effective neuroprotective agent in case of excitotoxicity, which is the hallmark of brain ischemia and several neurodegenerative processes. Therefore, kynurenine pathway, KYNA itself, and its derivatives came into the focus of research. During the past fifteen years, our research group has developed several neuroactive KYNA derivatives, some of which proved to be neuroprotective in preclinical studies. In this study, the synthesis of these KYNA derivatives and their evaluation with divergent molecular characteristics are presented together with their most typical effects on the monosynaptic transmission in CA1 region of the hippocampus of the rat. Their effects on the basic neuronal activity (on the field excitatory postsynaptic potentials: fEPSP) were studied in in vitro hippocampal slices in 1 and 200 μM concentrations. KYNA and its derivative 4 in both 1 and 200 μM concentrations proved to be inhibitory, while derivative 8 only in 200 μM decreased the amplitudes of fEPSPs. Derivative 5 facilitated the fEPSPs in 200 μM concentration. This is the first comparative study which evaluates the structural and functional differences of formerly and newly developed KYNA analogs. Considerations on possible relations between molecular structures and their physiological effects are presented.

Full text of DOI:10.3390/molecules24193502

molecules
Article
Structural Evaluation and Electrophysiological Effects
of Some Kynurenic Acid Analogs
Evelin Fehér 1 , István Szatmári
,2,†
3,4,5,†
, Tamás Dudás , Anna Zalatnai , Tamás Farkas * ,
2
2
2,
Bálint L o˝ rinczi 3 , Ferenc Fülöp
,4
3,4,5
, László Vécsei
1,6
and József Toldi 2
1
Department of Neurology, Interdisciplinary Excellence Centre, Albert Szent-Györgyi Clinical Center, Faculty
of Medicine, University of Szeged, Semmelweis u. 6, H-6725 Szeged, Hungary;
feher.evelin23@gmail.com (E.F.); vecsei.laszlo@med.u-szeged.hu (L.V.)
Department of Physiology, Anatomy and Neuroscience, University of Szeged, Közép fasor 52,
H-6726 Szeged, Hungary; dudi.14t@gmail.com (T.D.); zalatnaianna@gmail.com (A.Z.);
toldi@bio.u-szeged.hu (J.T.)
Institute of Pharmaceutical Chemistry, University of Szeged, Eötvös u. 6, H-6720 Szeged, Hungary;
szatmari.istvan@pharm.u-szeged.hu (I.S.); lorinczi.balint@pharm.u-szeged.hu (B.L.);
fulop@pharm.u-szeged.hu (F.F.)
2
3
4
5
6
Stereochemistry Research Group of the Hungarian Academy of Sciences, Eötvös utca 6,
H-6720 Szeged, Hungary
Institute of Pharmaceutical Chemistry, Interdisciplinary Excellence Centre, University of Szeged, Eötvös u. 6,
H-6720 Szeged, Hungary
MTA-SZTE Neuroscience Research Group, Semmelweis u. 6, H-6725 Szeged, Hungary
Correspondence: tfarkas@bio.u-szeged.hu; Tel.: +36-(62)-544-381
These authors contributed equally to this work.
*
†
Academic Editor: Tomasz Tuzimski
Received: 12 September 2019; Accepted: 24 September 2019; Published: 26 September 2019
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: Kynurenic acid (KYNA), a metabolite of tryptophan, as an excitatory amino acid receptor
antagonist is an effective neuroprotective agent in case of excitotoxicity, which is the hallmark of brain
ischemia and several neurodegenerative processes. Therefore, kynurenine pathway, KYNA itself,
and its derivatives came into the focus of research. During the past fifteen years, our research group
has developed several neuroactive KYNA derivatives, some of which proved to be neuroprotective
in preclinical studies. In this study, the synthesis of these KYNA derivatives and their evaluation
with divergent molecular characteristics are presented together with their most typical effects on the
monosynaptic transmission in CA1 region of the hippocampus of the rat. Their effects on the basic
neuronal activity (on the field excitatory postsynaptic potentials: fEPSP) were studied in in vitro
hippocampal slices in 1 and 200
concentrations proved to be inhibitory, while derivative
fEPSPs. Derivative facilitated the fEPSPs in 200 M concentration. This is the first comparative study
µM concentrations. KYNA and its derivative
4 in both 1 and 200 µM
8
only in 200 M decreased the amplitudes of
µ
5
µ
which evaluates the structural and functional differences of formerly and newly developed KYNA
analogs. Considerations on possible relations between molecular structures and their physiological
effects are presented.
Keywords: kynurenic acid; Mannich reaction; neuroprotection; excitatory amino acid receptors
1
. Introduction
Glutamate is the main excitatory neurotransmitter in the central nervous system (CNS) that acts
both on ionotropic and metabotropic receptors. The excessive glutamate level results in excitotoxicity
which is the hallmark of several neurodegenerative processes. Excitotoxicity may result from acute
Molecules 2019, 24, 3502; doi:10.3390/molecules24193502
www.mdpi.com/journal/molecules

Molecules 2019, 24, 3502
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events like stroke or traumatic brain injury [
neurological diseases [4].
1–
3]. On the other hand, it plays a role in several
In case of high glutamate level, the receptors of excitatory amino acids
(EAARs) such as N-methyl-d-aspartate ionotropic glutamate receptor (NMDAR),
alfa-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPAR), and kainate receptors
are overactivated, leading to neuronal damage. The overactivation of these receptors can lead
to dysregulation of Ca2 homeostasis, triggering the production of free radicals, oxidative stress,
+
mitochondrial dysfunctions, and finally cell death [5]. The NMDAR is a ligand-gated ion channel
that plays an outstanding role in these pathological processes; therefore, it is a potential therapeutic
target [6].
Several preclinical studies showed that the attenuation of excitotoxicity may be achieved
by blockage of EAARs such as NMDAR [
7–
10]. Although it is clear that NMDAR antagonists,
such as MK-801, prevents excitotoxicity [11
,12], their clinical use is very limited, as a complete
NMDAR blockade resulting from their usage can cause serious side effects such as nausea, vomiting,
and memory impairment [13]. Additionally, NMDAR blockers may produce psychosis and symptoms
of schizophrenia in humans, while in laboratory animals it can induce ataxia and stereotypy [14–16].
However, N-methyl-d-aspartate receptor (NR2B) subunit-specific antagonists glycine (Gly) and
polyamine site agents and ion channel blockers with low affinity got into the focus of research,
as they have attenuated the side effects [17]. This was the reason why kynurenic acid (KYNA),
an intermediate of the tryptophan metabolism [18
KYNA is the only known endogenous NMDAR blocker (beside Mg ) that also has broad-spectrum
targets: it inhibits NMDARs at Gly-binding site [20] and it can noncompetitively inhibit 7-nicotinic
,19], also came into the interest of researchers.
2
+
α
acetylcholine receptors [21], and through this, it can moderate Glu release presynaptically [22]. KYNA
has been proposed as the endogenous agonist of G protein-coupled receptor 35 (GPR35), a Gi-protein
receptor [23], and a nuclear receptor, called aryl hydrocarbon receptor (AHR) [24]. In addition to the
direct effects of KYNA on glutamatergic neurotransmission, an indirect inverse regulation between
striatal KYNA and dopamine (DA) levels has also been raised [25].
Reviewing the wide spectrum of effects of KYNA, it can be concluded that KYNA proved to be a
neuroprotective substance in most preclinical studies [26–28]. This has stimulated the intense study
of KYNA itself and of its derivatives, design, and development of new KYNA analogs, prodrugs,
and derivatives [18,29,30].
More than fifteen years ago, our research group also started to develop
new KYNA derivatives.
One of our molecules
(4),
a KYNA amide derivative
N-(N’,N’-dimethylaminoethyl)-4-hydroxy-4-quinoline-2-carboxamide hydrochloride), showed
electrophysiological properties similar to KYNA in in vitro studies [31], proved to be protective
in a transgenic animal model of Huntington’s disease [32] and also in many other preclinical
studies [33–36].
In this article, the synthesis of neuroactive KYNA derivatives and their basic electrophysiological
effects studied in in vitro are presented. The tests were carried out in normal artificial cerebrospinal
fluid (ACSF).
2
. Results
2
.1. Design and Development of KYNA Derivatives
The synthesis of the precursor ethyl 4-hydroxyquinoline-2-carboxylate (3) has already been
published [37]. During that process, aniline was reacted with diethyl acetylenedicarboxylate, forming
◦
the intermediate enamine. In the second ring-closing reaction in diphenyl ether at 250 C led to the
formation of the ethyl ester in an overall yield of 58%. In our case, the following optimizations were
applied: (i) After the formation of the enamine
2, column chromatography was used to purify the
intermediate; (ii) Diphenyl-ether was replaced by 1,2-dichlorobenzene that has lower boiling point

Molecules 2019, 24, 3502
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◦
(
190 C) allowing an easier work-up procedure. These latter conditions led to the formation of
3
in an
overall yield of 67% (Scheme 1).
Scheme 1. Syntheses of KYNA ethyl ester.
Compounds 4, 5 were synthesized by direct amidation of the ethyl ester of KYNA (3)
1
1
1
1
with N ,N -dimethylethane-1,2-diamine (to achieve
to achieve 5) (Scheme 2).
4) or with N ,N -dimethylpropane-1,3-diamine
(
Scheme 2. Syntheses of KYNA amide derivatives.
As previous studies indicated, the addition of a proton acceptable nitrogen is preferable for better
biological activity [38]. This can be achieved either through amidation using amines containing tertiary
nitrogen (Scheme 2), or via modified Mannich reaction (mMR) by using secondary amine derivatives.
It has been suspected that the presence of multiple cationic centers together will contribute to the
biological effect with greater magnitude. Thus, the syntheses of
groups were planned in a step by step synthetic pathway (Scheme 3). First, the synthesis of
carried out. During the aminoalkylation of , morpholine as a representative secondary cyclic amine
with formaldehyde was used to form the iminium salt that would attack the C-3 position on the
KYNA skeleton. Afterward, the amidations of were achieved in dimethylformamide through the
7
and
8
containing two such functional
6
was
3
6
0
activation of the free carboxylic group with N,N -diisopropylcarbodiimide (DIC). During the reactions,
-hydroxybenzotriazole hydrate (HOBt) was also used to achieve an active-ester intermediate and
further improve the yield (Scheme 3).
1
Scheme 3. Syntheses of aminoalkylated KYNA amide derivatives.

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2
.2. Electrophysiology
Schaffer collateral-stimulation results in field excitatory postsynaptic potentials (fEPSPs) in the
pyramidal layer of region CA1 of the hippocampus with 0.5–0.8 mV amplitudes (Figure 1A, inset).
Administration of KYNA or the presented KYNA derivatives in 200 M concentration resulted in
µ
either decrease or increase in the amplitudes of the fEPSPs. KYNA derivatives (except 4 and 5) applied
in 1 µM concentration induced less unequivocal and significant changes.
Figure 1. Effect of KYNA and
recorded in CA1 region of hippocampus.
4
(one of its derivatives) on the field excitatory evoked potentials (fEPSPs)
: KYNA administration (in 200 M concentration) for 30 min
A
µ
resulted in decrease of the fEPSPs’ amplitudes reversibly. During wash-out, the amplitudes not only
recovered but also were facilitated in the recorded period (arrow) *: samples for the baseline were
taken during the last 5 min of control period. #: samples for calculation of the effect were taken during
the last 5 min of wash-in period. Inset: an original fEPSP. Amplitudes were measured from ‘a’ to ‘b’.
Calibration: 20 ms and 0.5 mV.
M, and 200 M, respectively. In case of KYNA, n = 7 at 1
In case of 4 n = 10 at 1 µM and 16 µM and n = 8 at 200 µM.
B
: dose-dependent responses to KYNA and
4
in concentrations 1
µ
M,
M.
16
µ
µ
µM and 16
µ
M and n = 10 at 200 µ
Following the control period, an immediate decrease in fEPSP amplitudes could be detected at
the wash-in period of KYNA (200 M) (Figure 1A). The maximal reduction was 25%–30% compared
µ
to controls, which lasted until the end of wash-in (Figure 1A). During wash-out period, amplitudes
started to rise immediately, and after 20–25 min, they became stable, showing a 6%–8% increase
compared to the controls (arrow in Figure 1A). KYNA and
concentrations, resulted in significant decrease in the amplitude (Figure 2 KYNA and compound
To show the effect of in comparison with KYNA in more detailed, dose–effect relations are presented
Figure 1B). The effect of is fundamentally different. Its administration at 200 M concentration
M concentration its effect was
4 (a KYNA amide), both in 1 µM and 200 µM
4).
4
(
5
µ
resulted in 15%–25% facilitation of fEPSPs amplitudes, while in 1
µ
slightly inhibitory (Figure 2, compound 5).
Though the structures of
in 200 M concentration induced a slight increase in the amplitudes of fEPSPs during the wash-in
period, while in 1 M, it had a weak inhibitory effect, but none of them proved to be significant
Figure 2, compound ). In case of , the results acquired during the administration were not stable.
In most cases it (in 200 M) resulted in a serious decrease in amplitudes (to 60% of controls, e.g.,
Figure 2, compound ), and in few cases, the inhibitory effect of was smaller. In 1 M concentration,
it had hardly any effect.
7 and 8 are similar to each other, their effects are different. Administration
of
7
µ
µ
(
7
8
µ
8
8
µ

Molecules 2019, 24, 3502
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Figure 2. Structure and molecular weight of KYNA and of its derivatives (4, 5, 7, 8), respectively,
together with their effects on the field excitatory postsynaptic potentials (fEPSPs) recorded in CA1
region of hippocampus. Control (c) was observed during the last 5 min of resting period, before
administration of a compound (* in Figure 1A). Effect on the amplitudes of fEPSPs was observed during
the last 5 min of wash-in periods, respectively (# in Figure 1A). Each slice was subjected to only one
measurement. Changes in fEPSP amplitudes (mean
analogs are shown in the bar graphs. The numbers of measurements (N) are indicated in the columns;
±
SEM) due to application of KYNA or of its
n.s. = p > 0.05; **p < 0.01; ***p < 0.001).

Molecules 2019, 24, 3502
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3
. Discussion
A mass of early results from other laboratories [39
–
41] and our own previous studies demonstrated
that neuroprotection can be achieved not only with KYNA but also by administration of either
kynurenine (KYN, the prodrug of KYNA) or KYNA derivatives [10 26 42 45]. During the past
,
, –
one and half decade, we have made great efforts to develop new KYNA derivatives which are
neuroactive (and hopefully protective in such pathological states as ischemia or in the models of
different neurological diseases) [28,30,46].
In this study, we focused on four neuroactive KYNA derivatives; keeping in mind the structure of
molecules and the effects of substances on the neuronal transmission in a relatively simple monosynaptic
preparation (Schaffer collateral/commissural pathway - pyramidal cells). The induced fEPSPs well
demonstrate the neural signaling, in which mainly NMDA and AMPA receptors are involved [47
The electrophysiological studies were carried out both in lower and higher concentrations (1 and
M, respectively). While KYNA and decreased the amplitudes effectively in both concentrations,
the effect of derivatives , and in 1 M concentration was insignificant. In this study, we focused on
the 200 M concentration because in another series of experiments, aiming to study the phenomenon
,48].
2
00
µ
4
5,
7
8
µ
µ
of neuroprotection, that concentration was used. KYNA administration resulted in reduction in
amplitudes of fEPSPs, which can be well explained by its binding to the strychnine-insensitive glycine
binding site of the NMDA receptor [20]. After KYNA application, during the wash-out period,
the fEPSPs not only recovered but they were also augmented. Looking for explanation, one can think of
the results of Prescott et al., who found that KYNA has a dual action on AMPA receptors [49]. Similarly,
we found that KYNA has a Janus-faced effect on fEPSPs; in micromolar range it has inhibitory, while in
nanomolar range it has facilitatory effect [50]. This might be based on that during the wash-out period
the KYNA concentration gradually decreased to nanomolar range.
The phenomenon was true for
observed during its administration in all studied concentrations. To demonstrate the similar inhibitory
properties of KYNA and , their dose-dependent responses were presented (Figure 1B). Similar result
was published by Marosi et al. [31]. was our firstly synthesized KYNA amide, effective as an
inhibitor of fEPSPs in in vitro studies, and it proved to be outstandingly effective in earlier studies of
neuroprotection [31 32 43 51]. It was effective also in systemic administration, which suggests that
might penetrate the blood-brain barrier (BBB) much more effectively than KYNA [30]. It can be
explained by the molecular structure of as it has a side chain containing cationic center that may
4 as well; significant decrease in amplitudes of fEPSPs was
4
4
,
, ,
4
4
help the penetration through the BBB and through the creation of hydrochloride salt it can grant
water solubility.
Contrary to the small structural difference (
the effect of is fundamentally different; its administration (in 200
fEPSPs but facilitated the amplitudes (Figure 2, compound ). Similar results were also found in the
early tests of these molecules made on anesthetized animals [30].
The difference between the molecular structures of and
unsubstituted at position 3, while and are aminoalkylated at C-3 of the KYNA skeleton. Though
and have similar structure (the only difference between the two molecules is the cationic center
of the amide side chain), their effects (in 200 M) were fundamentally different; resulted in a slight
but insignificant facilitation while in most cases induced a marked inhibition of fEPSP amplitudes
Figure 2, compounds 7 and 8).
Although it is hard to discuss comprehensive correlations between structure and biological effects,
we could determine certain structural changes leading to different biological activity. Compound
as previously investigated, shows similar effect as KYNA; however, the insertion of an additional
CH -group in the amide sidechain (compound ) resulted in an antagonist effect. Similar results were
that bears the amide sidechain of and an additional
5 differs from 4 only in a -CH - in the side chain),
2
5
µM) did not induce inhibition of
5
7
,
8
4, 5 is that the latter compounds are
7
8
7
8
µ
7
8
(
4,
-
5
2
observed during the evaluation of compound
7
4
aminoalkyl group at C-3. These results mean that the insertion of an additional cationic center at
position 3 has a characteristic influence on the electrophysiological effects.

Molecules 2019, 24, 3502
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The explanation of the phenomenon is not clear. Probably, it cannot be elucidated simply with
the binding to the synaptic AMPA- and/or NMDA receptors. It is known that beside the synaptic
glutamatergic receptors, perisynaptic and extrasynaptic receptors exist, which are distinct from each
other [52–55]. Basically, according to the subunit composition, in adults, GluN2A subunits of NMDA
receptors are predominantly synaptic, GluN2B-containing receptors are mostly extrasynaptic, though
it is not true in all cases, for example, GluN2B subunits are also located in synapses in several
brain regions [56]. Activation of these extrasynaptic receptors leads to changes in excitability of
neurons and take part in several pathological processes like excitotoxicity, neurodegeneration, or
neuroinflammation [57]. It might be that the amplitudes’ changes involve different mechanisms.
Our most effective molecules are KYNA amides, which are known to be capable of the selective
inhibition of the NR2B subunit-containing NMDA receptors [58]. However, the effects of different
neuroactive KYNA derivatives have never been compared keeping in mind their structure. This is the
first comparative study which evaluates the structural and functional differences of formerly and newly
developed KYNA analogs. Unfortunately, at this moment, we do not know more details about the
binding of these molecules to the different synaptic and extrasynaptic glutamatergic receptors, but the
results of these preclinical studies are promising, as these KYNA derivatives might be promising
candidates for clinical trials.
4
. Materials and Methods
4
.1. Drugs
KYNA was from Sigma-Aldrich (Steinheim, Germany). Compounds (
used at final concentration of 1 and 200 µM, respectively, while KYNA and 4
and 200
at 7.4.
5
,
7
,
8
) in these studies were
in concentrations of 1, 16,
µM, dissolved in normal ACSF. The pH of the solutions was continuously monitored and kept
4
.2. Synthesis
1
13
The H and C spectra were recorded in DMSO or D O solutions in 5 mm tubes, at room
2
1
13
temperature, with a Bruker spectrometer (Karlsruhe, Germany) at 500 ( H) and 125 ( C) MHz, with
the deuterium signal of the solvent as the lock and TMS as internal standard.
Melting points were determined on a Hinotek X-4 melting point apparatus (Ningbo, China).
Elemental analyses were performed with a Perkin-Elmer 2400 CHNS elemental analyzer
(Waltham, MA, USA). Merck Kieselgel 60F254 plates (Budapest, Hungary) were used for TLC.
The microwave reactions were performed with a CEM Discover SP microwave reactor (Matthwes,
NC, USA).
4
.2.1. General Procedure for the Synthesis of Amides (4, 5)
Ethyl 4-hydroxyquinoline-2-carboxylate ( ) (1.0 mmol), N ,N -dimethylethane-1,2-diamine
), or N ,N -dimethylpropane-1,3-diamine (2.0 mmol, to achieve ) were
placed in a 50 mL round bottom flask. The mixture was refluxed in 30 mL EtOH for 7–8 h. After
the evaporation of the solvent, the residues were crystallized with Et O (10 mL). The products were
1
1
3
1
1
(2.0 mmol, to achieve
4
5
2
dissolved in 15 mL of EtOH and HCl/EtOH (22%) was added until reaching pH = 1. Et O (10 mL) was
2
added to the solution until precipitation. The crystals were filtered and washed with 2 × 15 mL Et O
2
yielding 4 or 5, respectively.
N-(2-(dimethylamino)ethyl)-4-hydroxyquinoline-2-carboxamide hydrochloride (
4
). Yield: 241 mg (82%); m.p.
◦
1
1
50–153 C. H-NMR (D O); 3.03 (6H, s); 3.50 (2H, t, J = 6.0 Hz); 3.88 (2H, t, J = 6.1 Hz); 6.80 (1H, s); 7.54
2
13
(
1H, t, J = 7.4 Hz); 7.76 (1H, d, J = 8.3 Hz); 7.83 (1H, t, J = 7.9 Hz); 8.12 (1H, d, J = 8.3 Hz); C-NMR (D O);
2
3
5.3; 43.2; 53.4; 106.8; 119.4; 124.3; 124.7; 125.8; 134.0; 139.4; 141.7; 163.7; 180.3; ppm. C H ClN O
14 18 3 2
(295.76): calcd. C, 56.80; H, 6.09; N, 14.20; found C, 56.73; H, 6.08; N, 14.16.

Molecules 2019, 24, 3502
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◦
N-(3-(dimethylamino)propyl)-4-hydroxyquinoline-2-carboxamide (
5
). Yield: 238 mg (77%); m.p. 222–226 C.
1
H-NMR (D O); 2.09–2.17 (2H, m); 2.95 (6H, s); 3.28 (2H, t, J = 8.6 Hz); 3.57 (2H, t, J = 6.7 Hz); 6.86
2
(
1H, s); 7.58 (1H, t, J = 7.2 Hz); 7.80 (1H, d, J = 8.0 Hz); 7.86 (1H, t, J = 7.1 Hz); 8.16 (1H, d, J = 8,3 Hz);
1
3
C-NMR (D O); 24.0; 37.1; 42.9; 55.4; 106.1; 119.5; 124.1; 124.2; 126.3; 134.3; 139.3; 142.5; 162.9; 179.0
2
ppm. C H ClN O (309.79): calcd. C, 58.10; H, 6.46; N, 13.56; found C, 58.14; H, 6.42; N, 13.53.
15
20
3
2
4-hydroxy-3-(morpholinomethyl)quinoline-2-carboxylic acid (
6). Morpholine (174 mg, 2.0 mmol), ethyl
4
-hydroxyquinoline-2-carboxylate ( ; 217 mg, 1.0 mmol), 22% aqueous formaldehyde (410 mg, 3.0
3
mmol), and 30 mL of 1,4-dioxane were placed in a 50 mL round bottom flask. The mixture was refluxed
for 6 h. After the evaporation of the solvent, the residue was dissolved in 40 mL water and extracted
with 3
×
30 mL of DCM. The collected organic phases were dried (Na SO ), the solvent was removed
2 4
by evaporation, and the residue was crystallized from 15 mL n-hexane:EtOAc (4:1) to yield
6
. Yield:
◦
1
196 mg (68%); m.p. 253–256 C. H-NMR (DMSO); 3.16–3.40 (4H, m); 3.66–3.84 (2H, m); 3.84–4.01 (2H,
m); 4.56 (2H, s); 7.43 (1H, t, J = 7.4 Hz); 7.75 (1H, t, J = 7,9 Hz); 8.04 (1H, d, J = 8.0 Hz); 8.14 (1H, d, J = 7.9
Hz); 10.39 (1H, brs); 12.18 (1H, brs); 13C-NMR (DMSO); 52.0; 52.2; 64.1; 110.7; 120.5; 125.2; 125.5; 125.9;
1
33.8; 139.9; 143.3; 164.6; 178.2 ppm. C H N O (288.30): calcd. C, 62.43; H, 5.55; N, 9.71; found C,
15 16 2 4
6
2.38; H, 5.56; N, 9.75.
4
.2.2. General Procedure for the Syntheses of Aminoalkylated Amides 7, 8
The 4-hydroxy-3-(morpholinomethyl)quinoline-2-carboxylic acid
6
(1.0 mmol) with
1
1
N ,N -dimethylethane-1,2-diamine (2.0 mmol to achieve
mmol to achieve
7) or 2-(pyrrolidin-1-yl)ethanamine (2.0
◦
8
) were dissolved in DMF (20 mL). The reaction was cooled to
−
10 C while HOBt
0
(2.0 mmol) was added and the solution was stirred for 10 min. N,N -diisopropylcarbodiimide (2.0
mmol) was added. After reaching room temperature, the mixture was stirred for additional 7–10
h. After the evaporation of the solvent, the residue was dissolved in 20 mL DCM and was washed
with 2
×
20 mL H O. The organic phase was dried with Na SO and the solvent was removed under
2 2 4
reduced pressure. The residue was crystallized using Et O (10 mL) yielding 7 or 8, respectively.
2
N-(2-(dimethylamino)ethyl)-4-hydroxy-3-(morpholinomethyl)quinoline-2-carboxamide (
7
). Yield: 161 mg
◦
(
45%); m.p. > 350 C. 2.19 (6H, s); 2.41–2.48 (6H, m); 3.45–3.52 (2H, m); 3.53–3.59 (4H, m); 3.60 (2H, s)
7
.31 (1H, t, J = 7.3 Hz); 7.64 (1H, t, J = 7.7 Hz); 7.86 (1H, d, J = 8.3 Hz); 8.08 (1H, d, J = 8.1 Hz); 10.63 (1H,
13
brs); C-NMR (DMSO); 38.4; 45.8; 51.0; 52.4; 59.1; 67.0; 113.7; 120.4; 124.4; 124.8; 126.0; 131.0; 132.8;
1
40.5; 163.9; 177.8; ppm; C H N O (358.43): calcd. C, 63.61; H, 7.25; N, 15.62; found C, 63,66; H, 7.26;
19 26 4 3
N, 15.65.
4
(
3
-hydroxy-3-(morpholinomethyl)-N-(2-(pyrrolidin-1-yl)ethyl)quinoline-2-carboxamide (
8
). Yield: 165 mg
43%); m.p. > 350 C. H-NMR (DMSO); 1.63–1.73 (4H, m); 2.46–2.52 (6H, m); 2.64 (2H, t, J = 6.2 Hz);
.35–3.41 (2H, m); 3.48–3.63 (8H, m); 7.34 (1H, t, J = 7.1 Hz); 7.66 (1H, t, J = 7.2 Hz); 7.88 (1H, d, J = 8.0
◦
1
13
Hz); 8.09 (1H, d, J = 8.0 Hz); 10.82 (1H, brs); 11.78 (1H, brs); C-NMR (DMSO); 23.6; 50.6; 52.2; 54.1;
5
5.4; 66.6; 113.4; 119.6; 124.1; 124.3; 125.7; 132.6; 139.7; 144.2; 163.2; 177.6 ppm; C H N O (384.47):
21 28 4 3
calcd. C, 65.54; H, 7.28; N, 14.56; found C, 65.50; H, 7.27; N, 14.55.
4
.3. Animals and Housing Conditions
Recordings were made from brain slices taken from 20- to 28-day-old male Wistar rats (N = 62)
supplied by Charles River Laboratories (Wilmington, MA, USA). Animals were kept under constant
environmental conditions (23 Celsius degree; humidity 55 5%; a 12-h/12-h light/dark cycle) and were
±
housed individually in standard plastic cages, where they had free access to food and water. Every
effort was made to minimize the number of animals used and their suffering. The principles of animal
care (NIH publication No. 85–23) and the protocol for animal care approved both by the Hungarian
Health Committee (1998) and by the European Communities Council Directive (2010/63/EU) were
followed. Ethical license: XX./842/2016. Before the experimental procedures, all the rats were in normal
health and had no neurological deficits.

Molecules 2019, 24, 3502
9 of 13
4
.4. Slice Preparation
Coronal slices (350
µm), prepared from the middle part of their hippocampi with a vibratome
(Leica VT1200S, Wetzlar, Germany), were kept in an ice-cold artificial cerebrospinal solution (ACSF)
composed of (in mM) 130 NaCl, 3.5 KCl, 1 NaH PO , 24 NaHCO , 1 CaCl , 3 MgSO , and 10 D-glucose
2
4
3
2
4
(
all from Sigma, Germany), saturated with 95% O + 5% CO . 3 slices were made from the brain of an
2 2
animal. The slices were then transferred to a slice holder chamber, where they were warmed up to
◦
◦
3
2 C shortly, and then kept 19–20 C until usage to allow the slices to recover in the solution used for
recording (differing only in that it contained 3 mM CaCl and 1.5 mM MgSO ). The flow rate in the
2
4
◦
Haas-type recording chamber was 1.5–2 mL/min and the experiments were performed at 34 C.
4
.5. Electrophysiology
In order to obtain orthodromic stimulation of the Schaffer collateral/commissural pathway,
the stimulating electrode (a bipolar concentric stainless steel electrode developed by Neuronelektrod
Ltd., Hungary) was placed in the stratum radiatum between the CA1 and CA2 regions. The stimulus
intensity was adjusted between 20 and 50
evoke the half-maximal response. Field excitatory postsynaptic potentials (fEPSPs) were recorded with
glass micropipettes with a resistance of 1.5–2.5 M filled with ACSF. The recordings were amplified
µA (constant current, 0.2-ms pulses delivered at 0.05 Hz) to
Ω
with a neutralized, high-input-impedance preamplifier and filtered (1 Hz–3 kHz). The fEPSPs were
digitized (AIF-03, Experimetria Ltd., Budapest, Hungary), acquired at a sampling rate of 10 kHz,
saved to a PC, and analyzed off-line with Origin 8.0 software (OriginLab Corporation, Northampton,
MA, USA). The fEPSPs were monitored for 15–20 min until the amplitudes were stable. After having
stabilized the amplitudes, the last 5 min of resting period (before administration of a compound) were
taken as baseline (control, see * in Figure 1A). After baseline recording, KYNA or its analogs were
bath-applied for 30 min, and a wash-out occurred for another 30-min-period. The effect of a compound
was calculated based on the amplitudes obtained during the last 5 min of wash-in period (see # in
Figure 1). The properties of the basal glutamatergic synaptic transmission were also tested by means of
fEPSP recordings; in IO (input-output) graphs the fEPSP amplitudes are expressed against gradually
increasing stimulating currents (0–60
µA, in 5-
µA steps) in certain groups (data not shown). Each slice
was subjected to only one measurement [59
,60]. 7–10 measurements (N) were carried out in case of
each concentration of each compound, respectively. The actual numbers of measurements (N) are in
the bars of graphs in Figure 2. Altogether, 117 slices were used to test the effects of KYNA and of
its analogs.
5
. Statistical Analysis
Field excitatory postsynaptic potentials (fEPSPs) evoked by Schaffer collateral stimulation
were measured from ‘a’ to ‘b’ (see Figure 1A, inset). Origin 8.0 software (OriginLab Corporation,
Northampton, MA, USA) was used for the analyses. Results are expressed as mean SEM. One-way
±
repeated-measures ANOVA with Dunnett post hoc tests was used for assessing the differences between
amplitudes of controls and of KYNA and of its analogs during wash-in.
Author Contributions: F.F. and I.S. planned the derivatives and developed the synthesis; L.V. and J.T. conceived
and designed the experiments; I.S. and B.L. created the KYNA derivatives; E.F., T.D., and A.Z. performed the
electrophysiological experiments; T.F. analyzed the data and wrote the paper.
Funding: This work was supported by the Economic Development and Innovation Operational Program
(
Gazdaságfejlesztési és Innovációs Operatív Programs; GINOP 2.3.2-15-2016-00048, STAY ALIVE and
GINOP-2.3.2-15-2016-00034), financed by the European Union and by the MTA-SZTE Neuroscience Research
Group of the Hungarian Academy of Sciences as well as by the University of Szeged, Ministry of Human Capacities,
Hungary grant (20391-3/2018/FEKUSTRAT). This work was supported by the University of Szeged Open Access
Fund, grant number: 4299.
Acknowledgments: We are grateful to Jennifer Tusz, native speaker (Edmonton, Canada), for valuable contribution
in proofreading of the manuscript.

Molecules 2019, 24, 3502
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Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).