Synthesis and biological effects of some kynurenic acid analogs

Article abstract of DOI:10.1016/j.bmc.2011.10.029

The overactivation of excitatory amino acid receptors plays a key role in the pathomechanism of several neurodegenerative disorders and in ischemic and post-ischemic events. Kynurenic acid (KYNA) is an endogenous product of the tryptophan metabolism and,

Full text of DOI:10.1016/j.bmc.2011.10.029

Bioorganic & Medicinal Chemistry 19 (2011) 7590–7596
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological effects of some kynurenic acid analogs
K. Nagy a,b, I. Plangár b, B. Tuka b, L. Gellért a, D. Varga a, I. Demeter a,b, T. Farkas a, Zs. Kis a, M. Marosi a,
D. Zádori b, P. Klivényi b, F. Fülöp c, I. Szatmári c, L. Vécsei b, , J. Toldi a, ,
⇑
a Department of Physiology, Anatomy and Neuroscience, University of Szeged, Közép fasor 52, H-6726 Szeged, Hungary
b Department of Neurology, University of Szeged, PO Box 427, H-6701 Szeged, Hungary
c Institute of Pharmaceutical Chemistry and Research Group for Stereochemistry, Hungarian Academy of Sciences, University of Szeged, H-6720 Szeged, Eotvos u. 6, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
The overactivation of excitatory amino acid receptors plays a key role in the pathomechanism of several
neurodegenerative disorders and in ischemic and post-ischemic events. Kynurenic acid (KYNA) is an
endogenous product of the tryptophan metabolism and, as a broad-spectrum antagonist of excitatory
amino acid receptors, may serve as a protective agent in neurological disorders. The use of KYNA is
excluded, however, because it hardly crosses the blood–brain barrier. Accordingly, new KYNA analogs
which can readily cross this barrier and exert their complex anti-excitatory activity are generally needed.
During the past 6 years, we have developed several KYNA derivatives, among others KYNA amides. These
new analogs included one, N-(2-N,N-dimethylaminoethyl)-4-oxo-1H-quinoline-2-carboxamide hydro-
chloride (KYNA-1), that has proved to be neuroprotective in several models.
This paper reports on the synthesis of 10 new KYNA amides (KYNA-1–KYNA-10) and on the effective-
ness of these molecules as inhibitors of excitatory synaptic transmission in the CA1 region of the hippo-
campus. The molecular structure and functional effects of KYNA-1 are compared with those of other
KYNA amides. Behavioral studies with these KYNA amides demonstrated that they do not exert signifi-
cant nonspecific general side-effects. KYNA-1 may therefore be considered a promising candidate for clin-
ical studies.
Received 27 July 2011
Accepted 10 October 2011
Available online 18 October 2011
Keywords:
Kynurenic acid
KYNA amides
Synthesis of KYNA amides
Neuroprotection
Hippocampus
Open-field study
Electrophysiology
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
limited clinical use. A complete NMDA receptor blockade is fre-
quently accompanied by side-effects such as psychosis, nausea,
It is well established that excitotoxicity is the hallmark of sev-
eral neurodegenerative processes that result from acute events,
such as stroke, traumatic brain injury or bacterial meningitis.1–4
The background of these processes includes an abnormally high
glutamate (Glu) level in the brain. In the course of the pathological
events, neurons are damaged or killed by the overactivation of
receptors of excitatory amino acids (EAAs) such as N-methyl-5-
vomiting and a memory impairment, which underlines the key role
of the NMDA receptors in normal neuronal processes.12 However,
glycine (Gly) and polyamine site agents, NR2B subunit-specific
antagonists and ion channel blockers with lower affinity may come
into consideration, as they exert attenuated side-effects.13 Kynure-
nic acid (KYNA), an intermediate of the tryptophan metabolism,14–
16
would be a good candidate, because it is a broad-spectrum
D-aspartate (NMDA),
a-amino-3-hydroxy-5-methyl-4-isoxazole-
endogenous antagonist at ionotropic EAA receptors.17,18 KYNA
inhibits NMDA receptors at the Gly-binding site19 and it can non-
propionic acid (AMPA) and kainate.5 Glu may also play important
roles in the pathogenesis of other brain diseases, for example, Alz-
heimer’s disease, Parkinson’s disease, Huntington’s disease, amyo-
trophic lateral sclerosis, multiple sclerosis, epilepsy and
migraine.6,7
competitively inhibit
a
7-nicotinic acetylcholine receptors,20
whereby it can also mediate the inhibition of Glu release in the
striatum.21
However, the use of KYNA as a neuroprotective agent must be
excluded because it is barely able to cross the blood–brain barrier
(BBB),22 which rules out its systemic use. This has stimulated the
A reduction of the excitotoxicity, which may be achieved by the
blockade of NMDA or other Glu receptors, attenuates some of the
pathological symptoms in experimental models of acute and
chronic neurodegenerative diseases.8–11
Although most of the NMDA receptor antagonists prevent
excitotoxicity in cellular and animal models, these drugs have only
design of several new KYNA analogs, prodrugs or derivatives.14
A
few years ago, we began to create new KYNA amides.23 These com-
pounds are of promise because they are potentially capable of
selective inhibition of the NR2B subunit containing NMDA recep-
tors.24 In the present work we have designed and synthesized
new KYNA amides which the following structural properties:
(1) containing a water-soluble side-chain; (2) containing a new
⇑
Corresponding author. Tel.: +36 62 544153; fax: +36 63 544291.
E-mail address: toldi@bio.u-szeged.hu (J. Toldi).
MTA SZTE Neuroscience Research Group.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.10.029

K. Nagy et al. / Bioorg. Med. Chem. 19 (2011) 7590–7596
7591
A: KYNA-1
OH
HCl
H
N
N
H
N
O
C14H18ClN3O2
MW: 295.76
MW: 309.79
B: KYNA-2
OH
HCl
H
N
N
N
H
O
C15H20ClN3O2
C: KYNA-6
HCl
OH
H
N
N
H
N
O
C16H20ClN3O2
MW: 321.80
D: KYNA-11
O
NH2
O
O
S
OH
HO
O
H
O
NH2
C10H14N2O3 H2SO4 MW: 306.30
Figure 1. Kynurenic acid (KYNA) analogs: (A) KYNA-1, (B) KYNA-2, (C) KYNA-6. (D) KYNA-11 is
L-kynurenine sulfate.
O
O
O
H2N
H2N
R
N
N
R
n
X
H
N
H
N
R
OH
i
i
N
N
N
H
N
R
N
H
n
H
O
X
O
O
KYNA
KYNA-1
n = 1; R = Me:
1
KYNA-4
X = O:
n = 2; R = Me: KYNA-2
X = CH2: KYNA-5
KYNA-3
n = 1; R = Et:
NH2
i
N
N
RN
i
H2N
O
NH
i
O
N
O
O
NR
NR
H
N
H
N
R = H
ii
N
N
N
N
H
N
N
H
N
H
O
H
O
O
O
KYNA-8
R = Me:
R = H: KYNA-9
KYNA-6
KYNA-7
R = Bz: KYNA-10
Scheme 1. Reagents and conditions: (i) 1-Hydroxybenztriazole-hydrate, N,N0-di-isopropylcarbodiimide, DMF, rt, 48 h; (ii) Bz–Br, Na2CO3, H2O/toluene, rt, 10 h.
cationic center; (3) with side-chain substitution enhancing pene-
tration into the brain.
mice, and ex vivo electrophysiological experiments on both mice
and rats.
A further aim was to compare the effects of some of these ana-
logs in electrophysiological and behavioral experiments.
First of all, in vitro testing of the 10 new KYNA amides was car-
ried out on hippocampal slices of rats (n = 10). Six-week-old
C57Bl/6 male mice (n = 60) were used in the behavioral experi-
ments, while the ex vivo electrophysiological study involved the
use of adult Wistar rats (n = 47, 280–300 g) and mice (n = 12,
28–36 g). The animals were kept under controlled environmental
conditions at 22 2 °C under a 12-h light/dark cycle. Food and
water were available ad libitum. The local Animal Ethics Commit-
tee had approved all experiments. The care and use of the
2. Materials and Methods
2.1. Animals
Experiments were carried out on both mice and rats: in vitro
electrophysiological experiments on rats, behavioral studies on

7592
K. Nagy et al. / Bioorg. Med. Chem. 19 (2011) 7590–7596
experimental animals were in full accordance with the 86/609/EEC
directive.
2.4.1. N-(2-N,N-Dimethylaminoethyl)-4-oxo-1H-quinoline-2-
carboxamide hydrochloride (KYNA-1)
0.54 g (86%); mp 178–179 °C. 1H NMR (D2O); 3.00 (6H, s, N–
CH3); 3.47 (2H, t, J = 5.8 Hz, CH2); 3.87 (2H, t, J = 5.7 Hz, CH2);
6.90 (1H, s, C3-H); 7.58 (1H, t, J = 7.2 Hz, Ar-H); 7.81–7.86 (2H,
m, Ar-H); 8.18 (1H, d, J = 8.1 Hz, Ar-H)
2.2. Drugs
L-Kynurenine sulfate (KYNA-11; Fig. 1D) and probenecid (PROB)
were from Sigma–Aldrich (Steinheim, Germany), and were admin-
istered by the intraperitoneal (ip) route in dosages of 200 mg/kg.
New KYNA amide derivatives (KYNA-1–KYNA-10) were
prepared from KYNA and the appropriate amine, by using N,N0-
diisopropylcarbodiimide as coupling reagent according to Scheme
1 (for details, see Section 2.3).
2.4.2. N-(3-N,N-Dimethylaminopropyl)-4-oxo-1H-quinoline-2-
carboxamide hydrochloride (KYNA-2)
0.51 g (77%); mp 149–150 °C. 1H NMR (D2O); 2.10 (2H, t,
J = 8.2 Hz, CH2); 2.92 (6H, s, N–CH3); 3.25 (2H, t, J = 7.9 Hz, CH2);
3.55 (2H, t, J = 6.7 Hz, CH2); 6.87 (1H, s, C3-H); 7.57 (1H, t,
J = 7.4 Hz, Ar-H); 7.83–7.86 (2H, m, Ar-H); 8.18 (1H, d, J = 8.2 Hz,
Ar-H)
Among the new KYNA amides,23 N-(2-N,N-dimethylamino-
ethyl)-4-oxo-1H-quinoline-2-carboxamide hydrochloride (KYNA-
1) proved to be outstandingly effective as a neuroprotective
agent.
2.4.3. N-(2-N,N-Diethylaminoethyl)-4-oxo-1H-quinoline-2-
carboxamide hydrochloride (KYNA-3)
KYNA and the 10 new KYNA amides were subjected to electro-
physiological testing (in vitro studies). These studies revealed that
KYNA and only one of the new molecule (KYNA-1) displayed inhib-
itory effects, KYNA-1 and two other relatively non-effective KYNA
amides (KYNA-2 and KYNA-6) were chosen to carry out behavioral
experiments. The electrophysiological and neuroprotective effects
0.55 g (80%); mp 192–194 °C. 1H NMR (D2O); 1.34 (6H, t,
J = 7.3 Hz, N–CH2–CH3); 3.30–3.38 (4H, m, N–CH2–CH3); 3.47 (2H,
t, J = 6.4 Hz, CH2); 3.86 (2H, t, J = 6.4 Hz, CH2); 6.89 (1H, s, C3-H);
7.57 (1H, t, J = 7.6 Hz, Ar-H); 7.59–7.87 (2H, m, Ar-H); 8.16 (1H,
d, J = 8.3 Hz, Ar-H)
of L
-kynurenine sulfate (KYNA-11) were studied earlier,9–11,25 and
for purposes of comparison, KYNA-11 was therefore also included
in the present studies.
2.4.4. N-(2-N-Morpholinoethyl)-4-oxo-1H-quinoline-2-
carboxamide hydrochloride (KYNA-4)
0.54 g (75%); mp 230–232 °C. 1H NMR (D2O); 3.30 (2H, br s,
CH2), 3.51 (2H, t, J = 6.12 Hz, CH2); 3.67 (2H, br s, CH2); 3.90 (2H,
t, J = 5.9 Hz, CH2); 3.91 (2H, br s, CH2); 4.13 (2H, br s, CH2); 6.89
(1H, s, C3-H); 7.57 (1H, t, J = 7.4 Hz, Ar-H); 7.86 (1H, d, J = 8.4 Hz,
Ar-H); 8.19 (1H, t, J = 7.1 Hz, Ar-H)
The mice received ip injections of KYNA-1 (Fig. 1A; 200 mg/kg/
day, in a volume of 5 ml/kg, dissolved in distilled water, the pH of
which was adjusted to 6.5 with 1 N NaOH). For comparison, KYNA-
2, KYNA-6 (Fig. 1B,C) in equimolar dosages (likewise in a volume of
5 mL/kg, dissolved in distilled water, the pH of which was adjusted
to 6.5 with 1 N NaOH) or the vehicle (0.1 M PBS, in a volume of
5 ml/kg) was administered at the same time each day from
6 weeks of age.
In behavioral experiments, the mice were treated once (in acute
experiments) or for 9 days (in chronic experiments) according to
the regime detailed above. Acute electrophysiological experiments
were carried out on both rats and mice.
2.4.5. N-(2-N-Piperidinylethyl)-4-oxo-1H-quinoline-2-
carboxamide hydrochloride (KYNA-5)
0.58 g (81%); mp 206–208 °C. 1H NMR (D2O); 1.48–1.99 (6H, m),
3.02 (2H, t, J = 12.5 Hz, CH2); 3.41 (2H, t, J = 6.2 Hz, CH2); 3.63–3.67
(2H, m, CH2); 3.86 (2H, t, J = 6.2 Hz, CH2); 6.88 (1H, s, C3-H); 7.57
(1H, t, J = 7.4 Hz, Ar-H); 7.81–7.89 (2H, m, Ar-H); 8.19 (1H, d,
J = 8.2 Hz, Ar-H)
2.3. Experimental procedures
2.4.6. N-(2-N-Pyrrolidinylethyl)-4-oxo-1H-quinoline-2-
carboxamide hydrochloride (KYNA-6)
2.3.1. Synthesis
Melting points were determined on a Kofler micro melting
apparatus and are uncorrected. Elemental analyses were per-
formed with a Perkin-Elmer 2400 CHNS elemental analyzer. Merck
Kieselgel 60F254 plates were used for TLC. The 1H NMR spectra
were recorded in D2O solution in 5 mm tubes, at room tempera-
ture, on a Bruker Avance DRX400 spectrometer at 400.13 (1H)
and 100.61 (13C) MHz, with the deuterium signal of the solvent
as the lock and TMS as internal standard.
0.56 g (82%); mp 185–187 °C. 1H NMR (D2O); 2.02–2.19 (4H, m),
3.15–3.20 (2H, m CH2); 3.52 (2H, t, J = 6.0 Hz, CH2); 3.74–3.77 (2H,
m, CH2); 3.85 (2H, t, J = 6.0 Hz, CH2); 6.89 (1H, s, C3-H); 7.56 (1H, t,
J = 7.4 Hz, Ar-H); 7.79–7.86 (2H, m, Ar-H); 8.17 (1H, d, J = 8.2 Hz,
Ar-H)
2.4.7. N-[2-(2-N-Methylpiperidinyl)methyl]-4-oxo-1H-
quinoline-2-carboxamide hydrochloride (KYNA-7)
0.55 g (77%); mp 218–220 °C. 1H NMR (D2O); 1.55–2.32 (6H, m),
3.02 (3H, s); 3.11–3.95 (5H, m); 6.85 (1H, s, C3-H); 7.53 (1H, t,
J = 7.4 Hz, Ar-H); 7.77–7.82 (2H, m); 8.17 (1H, d, J = 8.2 Hz, Ar-H)
2.4. General method for the synthesis of KYNA amides (KYNA-
1–KYNA-10)
To a stirred solution of KYNA (0.40 g; 2.13 mmol) in 30 mL of
DMF, 1-hydroxy-benztriazole hydrate (0.28 g; 2.13 mmol) and
the appropriate amine (2.13 mmol) were added. The mixture was
stirred at 0 °C for 30 min and 0.4 mL (2.34 mmol) of N,N0-diisopro-
pylcarbodiimide was then added. The reaction mixture was stirred
for an additional 48 h at room temperature. After evaporation of
the solvent, the crude product was purified by column chromatog-
raphy, with MeOH as eluent. The free base was suspended in EtOH
(10 mL) and 1 mL of ethanolic HCl solution (22%) was next added.
The mixture was stirred for 0.5 h, after which Et2O (30 mL) was
added. The precipitate was filtered off and washed with Et2O
(2 ꢀ 20 mL).
2.4.8. N-(4-Methylpiperazinyl)-4-oxo-1H-quinoline-2-
carboxamide hydrochloride (KYNA-8)
0.60 g (82%); mp 192–194 °C. 1H NMR (D2O); 2.92 (3H, s), 3.12–
3.97 (8H, m); 6.65 (1H, s, C3-H); 7.51 (1H, t, J = 7.4 Hz, Ar-H); 7.67
(1H, d, J = 8.4 Hz, Ar-H); 7.80 (1H, t, J = 7.1 Hz, Ar-H); 8.12 (1H, d,
J = 8.2 Hz, Ar-H)
2.4.9. N-(Piperazinyl)-4-oxo-1H-quinoline-2-carboxamide
hydrochloride (KYNA-9)
0.47 g (67%); mp >350 °C. 1H NMR (D2O); 3.21–3.98 (8H, m),
6.51 (1H, s, C3-H); 7.51 (1H, t, J = 7.4 Hz, Ar-H); 7.67 (1H, d,

K. Nagy et al. / Bioorg. Med. Chem. 19 (2011) 7590–7596
7593
J = 8.4 Hz, Ar-H); 7.80 (1H, t, J = 7.1 Hz, Ar-H); 8.15 (1H, d,
J = 8.2 Hz, Ar-H)
2.6.2. Ex vivo electrophysiology
The rats were anesthetized with urethane (1.25 g/kg, ip). In some
cases, the tail vein was catheterized. In most of the experiments, the
drugs were administered ip, through a syringe implanted at the
beginning of the experiments. For recordings in area CA1, a
2–3-mm diameter hole was drilled over the dorsal hippocampus
(3.0–3.8 mm posterior to the bregma and 1.8–2.3 mm lateral to
the sagittal suture) and the recording electrode was lowered 2.2–
2.8 mm from the cortical surface. Contralaterally, a 1–2-mm hole
was drilled for the CA3 stimulating electrode (3.7 mm posterior to
the bregma, and 3.3 mm lateral to the sagittal suture: the final elec-
trode depth was 3.8 mm below the dura). The electrodes were low-
ered and the final position was adjusted so that the maximal CA1
population spike was obtained in response to contralateral CA3
stimulation (Fig. 3, inset). The sites in areas CA1 and CA3 were con-
firmed histologically. Responses to a range of stimulus intensities
were recorded under control conditions so as to produce an input-
2.4.10. N-(4-Benzylpiperazinyl)-4-oxo-1H-quinoline-2-
carboxamide hydrochloride (KYNA-10)
To a stirred solution of KYNA-9 (0.165 g (0.5 mmol) and Et3N
(0.126 g, 1.25 mmol) in 10 mL of EtOH, benzyl bromide (0.073 g,
0.55 mmol) dissolved in 5 mL of EtOH was added. The reaction
mixture was stirred for 10 h at room temperature. After the evap-
oration of the solvent, the crude product was purified by column
chromatography, with MeOH as eluent. The process to obtain the
hydrochloride salt of KYNA-10 was as described above.
0.118 g (62%); mp 213–215 °C. 1H NMR (D2O); 3.15–4.17 (8H,
m); 4.38 (2H, s); 6.55 (1H, s, C3-H); 7.42–7.61 (6H, m, Ar-H);
7.70 (1H, d, J = 8.4 Hz, Ar-H); 7.82 (1H, t, J = 7.1 Hz, Ar-H); 8.17
(1H, d, J = 8.2 Hz, Ar-H)
2.5. Behavioral studies
output curve by changing the duration (10–100
ls), using current
(up to 50 A) square pulses. Stimuli were triggered at low frequency
l
2.5.1. Open-field test
(0.05 Hz). Response stability was monitored for 30 min prior to drug
administration. KYNA derivatives were administered in the same
dosage as in the behavioral experiments. The mice were anesthe-
tized with urethane (2.4 g/kg, ip).
We earlier carried out a number of electrophysiologial experi-
ments on rats.11,25,26 Accordingly, for the present results to be fully
comparable with the previous ones, we detail here only those ob-
tained in rats; otherwise, the results obtained in mice were quite
similar to those in rats.
The spontaneous locomotor and exploration activities of the
mice were examined 2 h after the first treatment (acute), and then
on the last day (day 9) of treatment (chronic). The tests were per-
formed at the same time of day so as to avoid possible changes due
to the diurnal rhythm. Each mouse was placed at the center of a
black box (48 ꢀ 48 ꢀ 40 cm) and its behavior during a 5-min peri-
od was recorded with the aid of Conducta 1.0 software (Experime-
tria Ltd., Budapest, Hungary). The ambulation time, the mean
velocity, the local time and the number of rearings were evaluated.
For a few rats, the corresponding parameters were also tested
under acute conditions at the same dosages, but the complete
experiments were performed on mice, as described above.
2.7. Statistical analysis
Population spikes evoked by CA3 stimulation were measured
from peak to peak (see Scharfman and Goodman, 1998, and
Fig. 3, inset). To test the statistical significance of the differences
in amplitudes of the responses recorded during the control and
drug-treatment periods, one-way ANOVA following the post hoc
Bonferroni test was applied. Values of p <0.05 were considered sig-
nificant. For statistical evaluation of the data in the behavioral
tests, one-way ANOVA was used, followed by Fisher’s LSD post
hoc test with StatView 4.53 for Windows software (Abacus Concept
Inc., Berkeley, CA, USA). These data were expressed as
means standard error of the mean (S.E.M.). Statistical signifi-
cances in the behavioral tests were taken as ⁄p 6 0.5 and ⁄⁄p 6 0.01.
2.6. Electrophysiology
2.6.1. In vitro electrophysiology
Coronal slices (400 lm; 4 slices/animal), prepared from the
middle part of the rats’ hippocampi with a vibratome (Campden
Instruments, UK), were kept in an ice-cold artificial cerebrospinal
solution (ACSF) composed of (in mM): 130 NaCl, 3.5 KCl, 1 NaH2-
PO4, 24 NaHCO3, 1 CaCl2, 3 MgSO4 and 10 D-glucose (all from
Sigma, Germany), saturated with 95% O2 + 5% CO2. The slices then
were transferred to a Haas recording chamber and incubated at
room temperature for 1 h to allow the slices to recover in the solu-
tion used for recording (differing only in that it contained 3 mM
CaCl2 and 1.5 mM MgSO4). The flow rate of the recording solution
was 1.5–2 mL/min and the experiments were performed at 34 °C.
In order to achieve orthodromic stimulation of the Schaffer collat-
eral/commissural pathway, the stimulating electrode (either a
micropipette pulled from a theta glass or a bipolar concentric
stainless steel electrode developed by Neuronelektrod Ltd, Hun-
gary) was placed in the stratum radiatum between areas CA1
and CA2. The stimulus intensity was adjusted to between 30 and
3. Results
Following the in vitro electrophysiological studies on KYNA and
the 10 new KYNA amides, 4 molecules were further studied in
ex vivo and behavioral experiments. They were chosen for the fol-
lowing reasons: KYNA-1 was the only new amide effective as an
inhibitor in the in vitro study, and it had proved to be outstand-
ingly effective in earlier studies of neuroprotection;27–30 KYNA-2
is structurally very similar to KYNA-1, differing only in containing
an additional –CH2- group in the side-chain; KYNA-6 and KYNA-11
60 lA (constant current, 0.2-ms pulses delivered at 0.05 Hz) to
evoke the half-maximal response.
Field excitatory postsynaptic potentials (fEPSPs) were recorded
with glass micropipettes with a resistance of 1.5 M
(KYNA-11 is identical with L-kynurenine sulfate, which was al-
ready tested before9,25) differ appreciably in structure not only
from each other, but also from KYNA-1 and KYNA-2 (see Fig. 1).
X filled with
ACSF. The recordings were amplified with a neutralized, high-in-
put-impedance preamplifier and filtered (1 Hz–3 kHz). The fEPSPs
were digitized (AIF-03, Experimetria Ltd. Hungary), acquired at a
sampling rate of 10 kHz, saved to a PC and analyzed off-line with
Origin 6.0 software (OriginLab Corporation, USA).
3.1. Electrophysiology
3.1.1. In vitro study
When the Schaffer collaterals were stimulated, and the fEPSPs
and their amplitudes were recorded in the pyramidal layer of
region CA1 of the hippocampus before, during and after the admin-
istration of KYNA or KYNA-1, micromolar concentrations of these
KYNA and the new KYNA amides were dissolved in ACSF and
presented at 16
30 min.
lM to the hippocampal slices via perfusion for

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K. Nagy et al. / Bioorg. Med. Chem. 19 (2011) 7590–7596
compounds resulted in decreases in the amplitudes of the fEPSPs.
KYNA-1 proved to be an even more effective inhibitor than KYNA
itself (Fig. 2). However, non of the other 9 new KYNA amides
displayed a similar effect. None of them reduced the amplitudes
of the fEPSPs. Indeed, KYNA-6 slightly increased the amplitudes
of the fEPSPs, while KYN-2 had no effect at all (Fig. 2). The same
was true for the other new KYNA amides (data not presented).
A
b
%
KYNA-11 + PROB
130
120
110
100
90
0.5 mV
a
5 ms
3.1.2. Ex vivo study
The blocking effects of KYNA analogs on NMDA receptors can be
tested adequately also via the amplitudes of population spikes
recorded in area CA1 of the hippocampus.
80
The responses of the pyramidal cells in area CA1 to contralateral
CA3 stimulation were tested before and after the injection of the
vehicle or the KYNA analog. For comparison, KYNA-11 (L-kynuren-
70
60
ine sulfate) (200 mg/kg) was administered ip together with PROB
(200 mg/kg) to one group of animals; this combination is well
known to result in a gradual decrease in amplitude of the popula-
tion spikes25 (Fig. 3A).
0
30
60
90
120
150
180
210min
B
The electrophysiological effects of these new KYNA analogs on
the responses of the area CA1 pyramidal cells to contralateral
CA3 stimulation have not been studied previously. In comparison
with KYNA-11 + PROB, KYNA-1 proved similarly or even more
effective in reducing the amplitude of the population spikes.
KYNA-1 started to reduce the amplitude immediately after its
administration, without a latency period, The amplitude decreased
to 75% of the control level within 60 min, and to 70% within 90 min
(Fig. 3B), whereas in the case of KYNA-11 + PROB it had not reached
this level even after 180 min (Fig. 3A).
In contrast with KYNA-1, the other analogs did not reduce the
population spike amplitudes significantly, as illustrated by the data
on KYNA-2 (Fig. 3C), despite its similar structure to that of KYNA-1.
Similarly, negative results were observed after KYNA-6 administra-
tion (not shown).
control
KYNA - 1
%
130
Saline
120
110
100
90
80
KYNA - 1
70
60
*
0
30
60
90
120
150
180
210 min
3.2. Behavioral studies
C
%
KYNA - 2
Open-field observations were carried out on five groups of ani-
mals: a control group (injected with saline), and KYNA-1, KYNA-2,
130
120
110
100
90
80
70
60
0
30
60
90
120
150
180
210
min
Figure 3. Examples of the effects of the administered compounds on the population
spike amplitudes recorded in area CA1. (A) Examples of the inhibitory effect of
KYNA-11 + PROB, which resulted in decreases in amplitude with a long delay. Lines
with circles, squares or triangles relate to individual experiments carried out on 3
rats. Each circle, triangle and square represents an average of 10 potentials. Inset:
an example of evoked spikes whose amplitude was measured between peaks a and
b. Calibration: 0.5 mV; 5 ms. (B) KYNA-1 injection resulted in an immediate
decrease in amplitude of the CA1 population spikes (open circles), while the saline
injection did not induce any significant change in amplitude (filled squares). Each
square and circle denotes the mean S.D. for 5 ꢀ 10 potentials observed in 5
animals. After the 30-min recordings, the animals received a saline or KYNA-1
injection ip (arrows). ⁄The horizontal line indicates the time range in which the
differences between the saline and KYNA-1 effects were significant (p <0.005). (C)
Following KYNA-2 injection, hardly any significant change in amplitude was
observed. Each square denotes the mean S.D. for 3 ꢀ 10 potentials observed in 3
animals.
Figure 2. Comparison of suppressor effects of KYNA and some of its new analogs.
KYNA (16
fEPSPs. However, KYNA-1 (16
l
M) induced a definitive decrease (to 70–75%) in the amplitudes of the
M) proved to be even more effective in decreasing
l
the amplitudes, to 50–55% of the control level. None of the other new KYNA amides
showed a similar effect at this concentration. As examples, the lack of an inhibitory
effect of KYNA-2 and KYNA-6 is presented. In this Figure, the amplitudes are
normalized to the amplitudes in the control period (10 min). The illustrated results
are representative examples; similar findings were observed for all four slices
tested for each compound. The gray-striped area indicates the period of adminis-
tration of KYNA or KYNA amide.

K. Nagy et al. / Bioorg. Med. Chem. 19 (2011) 7590–7596
7595
only in the numbers of rearings. Both KYNA-1 and KYNA-11
decreased the number of rearings in the acute experiments, and
KYNA-11 did so in the chronic experiments too (Fig. 4C).
A
B
Similar behavioral observations were made on rats, but those
results are not detailed here.
4. Discussion
KYNA is one of the few known endogenous NMDA receptor
inhibitors.31 It can influence important physiological and patholog-
ical processes. The available experimental data and theoretical
considerations suggest that KYNA, as an EAA receptor antagonist,
could exert therapeutic effects in neurological disorders,32–37 but
its use as a neuroprotective agent is rather restricted because it
has only limited ability to cross the BBB.
During the past decade, we have made great efforts to develop
new neuroprotective agents and strategies based on kynurenines.
We initially tested L-kynurenine sulfate (KYNA-11) as a neuropro-
tective compound which crosses the BBB and which can be trans-
formed to the EAA receptor antagonist KYNA by the enzyme
kynurenine aminotransferase, located primarily in the glia,38
which has the ability to take up KYNA-11 and to release KYNA.39,40
We achieved good results as concerns neuroprotection with
KYNA-11,9–11,25 but its use (even in preclinical studies) is limited,
primarily because it is converted to the EAA receptor agonist quin-
olic acid (QUIN) in high proportion after systemic administration.
QUIN may cross the BBB, especially in a diseased organism, and
it may induce an opposite effect. We further found that the admin-
istration of KYNA-11 in a dosage of 300 mg/kg ip, together with
PROB, induces a transient facilitation of the population spike
amplitudes, and its inhibitory effect commences only 60–80 min
after administration.25 Similarly, the inhibitory effect of KYNA-11
+ PROB was observed only after a long delay when it was adminis-
tered in a dosage of 200 mg/kg ip. The long delay of its inhibitory
effect and its relatively moderate effectiveness are disadvanta-
geous if the aim is an immediate reduction of the excitotoxicity in-
duced by excess Glu, for example, after a stroke or a traumatic
brain injury.
C
*
**
*
**
**
This situation turned our interest to KYNA derivatives,23 one
of which (KYNA-1) turned out to be effective as a neuroprotec-
tive agent. Its effects have already been tested in several
models,27–30,35,41 but its influence on the NMDA-mediated
synaptical function has not been studied previously.
Figure 4. Observations in the open-field arena. (A) Time spent in ambulation. No
difference was found between the control and treated groups in either the acute or
the chronic experiments. (B) Velocity of the movements of the mice. No difference
was found between the control and treated groups in either the acute or the chronic
experiments. (C) Number of rearings during 5-min observation periods. KYNA-1
and KYNA-11 significantly reduced the number of rearings in the acute exper-
iments, as did KYNA-11 in the chronic experiments. If not labeled, the comparisons
were made with the controls. CO: control.
As the first step, KYNA-1 was studied in an in vitro electrophys-
iological experiment together with the other 9 newly synthesized
KYNA amides and with KYNA itself. The molecular structure of
KYNA-2 is similar to that of KYNA-1 (differing in a –CH2- group),
while the others are structurally quite different. KYNA-1 was the
only KYNA amide which reduced the amplitude of the fEPSPs,
which suggests that KYNA-1 exerts an inhibitory effect on the syn-
aptic transmission mediated by NMDA receptors.
KYNA-6 or KYNA-11-treated groups. In acute behavioral experi-
ments, the animals were treated with saline (CO) or KYNA amide
analog 2 h prior to the behavioral observations, while chronic
treatment was administered on nine successive days, with obser-
vations on day 9, 2 h after the final injection.
The animals treated with one or other KYNA amide analog did
not differ greatly in behavior from those that received the saline
vehicle. The posture and activity were quite similar in each group.
The ambulation time, the mean velocity and the number of rear-
ings did not exhibit highly significant differences in most cases
(Fig. 4). Although the ambulation time was somewhat decreased
after KYNA-11 administration, the changes were not significant
(Fig. 4A). The mean velocities values were nearly the same in each
group (Fig. 4B). Significant changes in performance were observed
In the ex vivo experiments, KYNA-1 (200 mg/kg ip) displayed
inhibition of the population spike amplitude without any delay,
without any transient facilitation. Its inhibitory effect attained its
maximum within 1 h and surpassed that of KYNA-11. It was inter-
esting that, despite its structure being similar to that of KYNA-1,
KYNA-2 did not exhibit any significant effect on the population
spike amplitudes.
It was also important that KYNA-1, which had proved neuropro-
tective in several models (cited above), and partially inhibited
NMDA-mediated synaptical transmission in the hippocampus,
did not induce significant changes in the behavior of the tested ani-
mals. This was true for both rats and mice, but we present here
only the data observed on mice. The results confirmed that none
of the studied KYNA derivatives induced major changes in the

7596
K. Nagy et al. / Bioorg. Med. Chem. 19 (2011) 7590–7596
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There is accumulating evidence that Glu-induced excitotoxicity
is mainly mediated by NMDA receptor channels.42,43 From clinical
aspects, the massive blockade of NMDA receptors would result in
unacceptable symptoms.12 Accordingly, Gly and polyamine site
agents, NR2B subunit specific antagonists and ion channel blockers
with lower affinity may come into consideration, as they exert
acceptable side-effects.13 KYNA would theoretically be a good can-
didate, because it is a broad-spectrum endogenous antagonist on
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at the Gly-binding sites19 and it can non-competitively inhibit
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a
7-nicotinic acetylcholine receptors.20 Blockade of these nicotinic
receptors can also mediate the inhibition of Glu release in the stri-
atum.21 In addition, in the past few years it has been proposed that
KYNA is the endogenous ligand of the G-protein coupled receptor
35 (GPR35) too, which is highly expressed in the gastrointestinal
tract, but is also present in the central nervous system.44 It has
recently been suggested that KYNA effects in the brain are medi-
ated, at least partially, by GRP35 activation and reduced Glu
release.45
23. Fülöp, F.; Szatmári, I.; Vámos, E.; Zádori, D.; Toldi, J.; Vécsei, L. Curr. Med. Chem.
2009, 16, 4828.
However, from pharmacological considerations, KYNA itself has
several disadvantages, which rule out its systemic use. Accord-
ingly, several new KYNA analogs or prodrugs have been designed.14
An important group of these compounds comprises the KYNA
amides, which are excellent candidates,23 because they are known
to be capable of the selective inhibition of the NR2B subunit con-
taining NMDA receptors.24 The most prominent of these KYNA
amides is KYNA-1, the beneficial effects of which were reported
earlier,27–30,40 and whose partial inhibitory effect on the hippocam-
pal pyramidal neurons in area CA1 was demonstrated in the pres-
ent study. Moreover, as KYNA-1 did not significantly influence the
behavioral performance in the open-field arena, KYNA-1 treatment
does not appear to have any appreciable side-effects.
24. Borza, I.; Kolok, S.; Galgóczy, K.; Gere, A.; Horváth, C.; Farkas, S.; Greiner, I.;
Domany, G. Bioorg. Med. Chem. Lett. 2007, 17, 406.
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Neuropharmacology 2009, 57, 425.
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L.; Toldi, J. J. Neural. Transm. 2010, 117, 183.
}
30. Zádori, D.; Nyiri, G.; Szonyi, A.; Szatmári, I.; Fülöp, F.; Toldi, J.; Freund, T. F.;
Vécsei, L.; Klivényi, P. J. Neural. Transm. 2010. PMID: 21194001.
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2009, 116, 1403.
In summary, we believe that KYNA-1 is an effective neuropro-
tectant and may therefore be a promising candidate for clinical
trials.
37. Kincses, Z. T.; Toldi, J.; Vécsei, L. J. Cell. Mol. Med. 2010, 14, 2045.
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Acknowledgments
The present study was supported by an OTKA Grant (K 75628),
TÁMOP-4.2.1/B-09/1/KONV-2010-0005, ETT(02-64), and the Teller
Ede Foundation (NAP-BIO-06-BAYBIOSZ). T.F. and I.S. are Bolyai
Fellows of the Hungarian Academy of Sciences, K. N. is supported
by Richter Gedeon Nyrt. Talentum Foundation.
43. Wee, X. K.; Ng, K. S.; Leung, H. W.; Cheong, Y. P.; Kong, K. H.; Ng, F. M.; Soh, W.;
Lam, Y.; Low, C. M. Br. J. Pharmacol. 2010, 159, 449.
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