Kynurenic acid amides as novel NR2B selective NMDA receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2006.10.033

A novel series of kynurenic acid amides, ring-enlarged derivatives of indole-2-carboxamides, was prepared and identified as in vivo active NR2B subtype selective NMDA receptor antagonists. The synthesis and SAR studies are discussed.

Full text of DOI:10.1016/j.bmcl.2006.10.033

Bioorganic & Medicinal Chemistry Letters 17 (2007) 406–409
Kynurenic acid amides as novel NR2B selective NMDA
receptor antagonists
*
´
´
´
´
´
´
Istvan Borza, Sandor Kolok, Kornel Galgoczy, Aniko Gere, Csilla Horvath,
´
´
´
Sandor Farkas, Istvan Greiner and Gyo¨rgy Domany
Gedeon Richter Ltd, Budapest 10, PO Box 27, H-1475, Hungary
Received 5 September 2006; revised 11 October 2006; accepted 13 October 2006
Available online 17 October 2006
Abstract—A novel series of kynurenic acid amides, ring-enlarged derivatives of indole-2-carboxamides, was prepared and identified
as in vivo active NR2B subtype selective NMDA receptor antagonists. The synthesis and SAR studies are discussed.
Ó 2006 Elsevier Ltd. All rights reserved.
N-Methyl-D-aspartate (NMDA) receptors are ligand-
gated cation-channels embedded in the cell membrane
of neurones. Overactivation of NMDA receptors by glu-
tamate, their natural ligand, can lead to calcium over-
load of the cells. Antagonists of the NMDA receptors
may be used for treating many disorders that are accom-
panied with excess release of glutamate. The NMDA
receptors are heteromeric assemblies built up mostly
from NR1 and NR2 (NR2A-D) subunits. Particularly
interesting of these is the NR2B subunit due to its
restricted distribution (highest densities in the forebrain
and substantia gelatinosa of the spinal cord). This sub-
unit is the focus of increasing interest as therapeutic tar-
get in a wide range of CNS pathologies, including acute
and chronic pain, stroke and head trauma, drug-induced
dyskinesias, and dementias.1 Ifenprodil (1) is the proto-
typical selective antagonist of these ionotropic receptors,
a property that was discovered many years after its
launch as a vasodilator.2
time eliminating its original cardiovascular effects result-
ed in a diverse set of compounds.3–6
Previously we reported indole-2-carboxamides 2, 37 and
benzimidazole-2-carboxamide 4,8 as highly potent, selec-
tive and orally active NR2B antagonists.
X
Y
N
N
H
O
2 X = CH, Y = 5-OH
3 X = CH, Y = 6-OH
4 X = N, Y = 5(6)-OH
As continuation of these studies a series of kynurenic
acid amides, ring-enlarged derivatives of indole-2-carb-
oxamides, were prepared and tested.9
OH
N
Compounds 28–32 were synthesized as described in
Scheme 1. The key step involves a standard coupling
reaction10 between an appropriately substituted kynure-
nic acid and a piperidine. The kynurenic ester intermedi-
ates (15–18 and 20) were prepared by modified Conrad–
Limpach method, starting from the corresponding ani-
line derivatives.11 Alkaline hydrolysis of esters readily
produced the corresponding substituted kynurenic acids
(21–23, 26, and 27). The acetylamino-substituted
kynurenic ester (19) was prepared by catalytic reduction
of the nitro precursor compound 18 followed by acety-
lation of the amine. The hydroxy substituted kynurenic
CH3
HO
1
Modification of this structure with the aim of increasing
affinity towards the NMDA receptors and at the same
Keywords: NR2B antagonist; Kynurenic acid amide.
*
Corresponding author. Fax: +36
i.borza@richter.hu
1
432 6002; e-mail:
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.10.033

I. Borza et al. / Bioorg. Med. Chem. Lett. 17 (2007) 406–409
407
O
O
COOCH3
OCH3
OCH3
ii
i
R
R
R
+
OCH3
6-BnO
NH2
N
H
N
COOCH3
H
O
O
H
H
H
5 R =
15 R =
16 R =
10 R =
11 R =
13 R =
3-BnO,
4-NO2
O
4-BnO
7-BnO,
6-NO2
3-BnO,
4-NO2
O
4-BnO
6 R =
7 R =
17 R =
12 R =
H
N
H
N
R =
R =
R =
8
9
18
19
20
iii,
iv
6-AcNH
H
N
R = 3,4-
14
R = 3,4-
O
O
O
R = 6,7-
O
O
O
Y
v, vi
viii
R
R
OH
N
N
H
Z
N
H
O
O
H
H
28 R =
21 R =
22 R =
24 R =
7-BnO,
7-OH,
6-BnO
6-OH
7-OH,
6-OH
30 a-g R =
H
N
23 R =
25 R =
29 R =
31 R =
vii
6-AcNH
6-AcNH
H
N
O
R =
R = 6,7-
32
26
27
O
O
R = 6,7-
O
Scheme 1. Reagents and conditions: (i) MeOH, reflux, 2 h; (ii) Dowtherm, 240 °C, 10 min; (iii) H2, 10% Pd/C, MeOH, rt; (iv) Ac2O, reflux;
(v) NaOH, MeOH, H2O, rt; (vi) HCl; (vii) H2, 10% Pd/C, MeOH, rt; (viii) piperidine derivs., HBTU, Et3N, DMF, rt.
acids (24 and 25), however, were prepared from their
O-benzyl derivatives by catalytic hydrogenolysis. The
synthesis of quinazoline derivative 38 started from com-
mercially available 4-hydroxyanthranilic acid and pro-
ceeded according to Scheme 2. The anthranilamide 36
was prepared via standard procedures in six steps. The
quinazoline ring was obtained by thermal condensation
of the oxalic acid diamide 37 and catalytic hydrogenol-
ysis of O-benzyl protecting group afforded the end-prod-
uct. The IR, 1H NMR, 13C NMR and MS spectra for all
intermediates and final compounds were consistent with
the assigned structures. Moreover, the purity of the sam-
ples was checked by HPLC and HRMS analysis.
the analogous 5- and 6-hydroxyindole-2-carboxamides
(2 and 3), which were almost equiactive, 7-hydroxyky-
nurenic acid amid 29 was significantly less active than
its 6-hydroxy analogue 30a. Among the 6-hydroxyky-
nurenic acid amides 30a–g there were relatively slight
variations in the potency. It seemed that substituents
on the benzyl group or the replacement of the CH2 of
these benzyl groups for O did not affect the activity of
the compounds. The only exception was 30e which
showed about 5-fold weaker potency compared to its
congeners. In order to improve metabolic stability the
H-bond donor OH was substituted for an NH on the
left-hand side of the molecule. The acetamido analogue
31 lost its activity, while its heterotricyclic analogue 32,
bearing the NH at the same position, had slightly lower
potency compared to 6-hydroxy derivatives.
Biological activity of the prepared compounds was mea-
sured in a functional assay where the inhibition of
NMDA-evoked increase of intracellular Ca2+ level was
determined in rat cortical cell culture. Baseline and
NMDA-evoked changes of intracellular Ca2+ were mon-
itored with fluorimetry using a Ca2+-selective fluorescent
dye (Fluo-4/AM) and a plate reader fluorimeter.12 Selec-
tivity towards NR2A subunit containing NMDA recep-
tors was tested by the same functional assay using cells
expressing recombinant NR1/NR2A receptors. The re-
sults of these assays for selected compounds are summa-
rized in Table 1. Although functional data were sufficient
for lead optimisation the most active compound 30g was
also investigated in a binding assay on rat forebrain
membrane using tritiated Ro-25,6981 as radioligand.13–15
In vivo analgesic activity was tested in the mouse
formalin test,16,17 a model of persistent pain.
Compared to the indole-2-carboxamides (2 and 3), the
corresponding benzimidazole-2-carboxamide 4 had sig-
nificantly higher activity. Analogously the addition of
a further N to the kynurenic acid skeleton resulted in
the quinazoline 38. The activity of this compound, how-
ever, was much lower than that of the parent compound.
In this case increasing the number of heteroatoms in the
central ring-system decreased the potency.
All the kynurenic acid amides were found to be inactive
on the NR2A subtype of the NMDA receptors.
In the Ro-25,6981 binding assay compound 30g was
active, it showed high affinity (Ki: 4.2 nM (n = 3))
towards the ifenprodil binding site.
Similarly to many known NR2B antagonists the pres-
ence of an OH group, as a H-bond donor moiety, on
the quinolinone ring-system was a prerequisite of high
potency. The position of this OH, however, significantly
influenced the activity of the compounds. In contrast to
Compounds 30a–g and 32 were tested in the mouse for-
malin test as well (Table 1). Several members of this series
(30b, 30d, 30e and 30g) showed good oral activity, howev-
er others with similarly good in vitro potency (30a, 30c,

408
I. Borza et al. / Bioorg. Med. Chem. Lett. 17 (2007) 406–409
O
C
O
O
C
HO
OCH3
Ac
BnO
OCH3
C
HO
OH
i, ii
iii, iv
N
H
NH2
NH2
33
34
35
O
O
C
BnO
C
NH2
BnO
NH2
vii
v, vi
N
H
C
O
C
O
N
NH2
36
37
O
HO
N
viii
N
N
H
O
38
Scheme 2. Reagents and conditions: (i) SOCl2, MeOH, À20 °C to rt; (ii) 1—Ac2O, 50 °C; 2—aq Na2CO3, rt; (iii) benzylbromide, acetone, 4 h; (iv)
HCl, MeOH, reflux, 1 h; (v) 1—10% NaOH, MeOH, reflux, 1 h; 2—HCl; (vi) 1—SOCl2, CHCl3, 1 drop DMF, reflux, 2 h; 2—NH3, water, 0 °C; (vii)
(4-benzylpiperidin-1-yl)(oxo)-acetic acid, HBTU, Et3N, DMF; (viii) 1—250 °C, 1.5 h; 2—H2, 10% Pd/C, THF, rt.
Table 1. Biological assay results for compounds 28, 29, 30a–g, 31, 32 and 38
O
Y
R6
R7
X
N
Z
N
H
O
Compound R6
R7
X
Y
Z
Mp (°C) NMDA-evoked D[Ca2+
]
n
NR1/NR2A Inhib Formalin test po
(%) in 15 lM
a,b
i
Inhib (%)c IC50 (nM)
ED50 (mg/kg)
28
29
30a
30b
30c
30d
30e
30f
30g
31
32
38
1
H
H
OH
H
H
H
H
H
H
H
H
CH CH2
CH CH2
CH CH2
CH CH2
H
H
H
F
226–227 4.5%
1
1
5
3
3
3
4
3
3
3
3
6
9
3
1.1
9.1
H
174–175 27.9%
126–127 9.1 0. 8
125–126 8.7 1.0
190–192 9.1 0.58
OH
OH
OH
OH
OH
OH
OH
AcNH
8.9
Inactived
9.5
À0.9
À5.1
À2.4
0.7
CH CH2 Cl
Inactived
10.6
CH CH2 CH3 151–152 11.0 0.8
CH
CH
CH
O
O
O
H
Cl
268–270 49.6 9.9
191–193 7.1 0. 8
8.5
Inactived
8.2
5.1
CH3 258–260 6.2 0.78
À3.5
12.4
À2.1
13.9
À2.7
17.2
CH CH2
CH CH2
NH CH2
H
H
H
153–154 2.6%
290–292 21.7 2.8
–HN–C(O)–O–
OH
>20
H
240–242 317 8
470 51
2
25
18
8
4
3
13 15.6
21.0
4
2.2 0.4
7
a Values represent means SEM. The number of experiments (n) is indicated.
b NMDA-evoked changes of intracellular Ca2+
.
c Inhib (%) were obtained using 0.1 lM concentration of compound.
d No effect at 20 mg/kg.
30f and 32) did not show activity at 20 mg/kg oral dose.
One can assume that the differences in the ADME param-
eters (not measured) reflected in the in vivo activities.
In summary, we have developed a new class of NMDA
subtype-selective antagonists designed from indole and
benzimidazole derivatives.

I. Borza et al. / Bioorg. Med. Chem. Lett. 17 (2007) 406–409
409
12. Nagy, J.; Boros, A.; Dezso}, P.; Kolok, S.; Fodor, L.
Neurochem. Intern. 2003, 43, 19.
References and notes
13. Fischer, G.; Mutel, V.; Trube, G.; Malherbe, P.; Kew, J.
N.; Mohacsi, E.; Heitz, M. P.; Kemp, J. A. J. Pharmacol.
Exp. Ther. 1997, 283, 1285.
14. Mutel, V.; Buchy, D.; Klingelschmidt, A.; Messer, J.;
Bleuel, Z.; Kemp, J. A.; Richards, J. G. J. Neurochem.
1998, 70, 2147.
15. Richards, J. G.; Messer, J.; Buchy, D.; Klingel schmidt,
A.; Mutel V. Br. J. Pharmacol. 1997, 120, Proc. Suppl., p
39.
16. Peak effect of pain-related behaviour (licking/biting of
paw) elicited by formalin injection in mice 20–25 min post-
formalin was measured. Inhibition of this late phase
response is considered as analgesic effect against chemi-
cally induced persistent pain. Fasted male NMRI mice
(20–25 g, Charles River Hungary) were po treated with
vehicle (5% Tween 80) or test drugs (in dose-range:
6a = 0.5–8 mg/kg, 6c = 2–32 mg/kg) 15 min pre-formalin.
Then 20 ml of 1% formalin was sc injected into the right
hindpaw. Animals were visually observed in an open glass
cylinder. Percent decrease in licking time was determined
for each dose in comparison with the daily vehicle-treated
groups. Statistical significance was tested by Mann–
Whitney U-test. ED50 values were calculated by Boltz-
mann’s sigmoidal fitting.
1. Chazot, P. L. Curr. Med. Chem. 2004, 11, 389.
2. Williams, K. Mol. Pharmacol. 1993, 44, 851.
3. Chenard, B. L.; Menniti, F. S. Curr. Pharm. Design 1999,
5, 381.
4. Nikam, S. S.; Meltzer, L. Curr. Pharm. Design 2002, 8, 845.
5. McCauley, J. A. Expert Opin. Ther. Patents 2005, 15, 389.
´
6. Borza, I.; Domany, Gy. Curr. Top. Med. Chem. 2006, 6,
687.
7. Borza, I.; Kolok, S.; Gere, A.; Agai-Csongor, E.; Agai, B.;
´
´
´
´
Tarkanyi, G.; Horvath, Cs.; Barta-Szalai, G.; Bozo, E.;
´
´
´
´
Kiss, Cs.; Bielik, A.; Nagy, J.; Farkas, S.; Domany, Gy.
´
Bioorg. Med. Chem. Lett. 2003, 13, 3859.
8. Borza, I.; Kolok, S.; Gere, A.; Nagy, J.; Fodor, L.;
´
´
Galgoczy, K.; Fetter, J.; Bertha, F.; Agai, B.; Horvath,
Cs.; Farkas, S.; Domany, Gy. Bioorg. Med. Chem. Lett.
´
´
2006, 16, 4638.
9. Borza, I.; Horvath, Cs.; Farkas, S.; Nagy, J.; Kolok, S.
´
PCT Int. Appl. WO 2006/010967 A1. 2006; Chem. Abstr.
2006, 144, 170893.
10. Nicolaou, K. C.; Ramanjulu, J. M.; Natarajan, S.; Braese,
S.; Li, H.; Boddy, C. N. C.; Rubsam, F. Chem. Commun.
1997, 19, 1899.
¨
11. Jaen, J. C.; Laborde, E.; Bucsh, R. A.; Caprathe, B. W.;
Sorenson, R. J.; Fergus, J.; Spiegel, K.; Marks, J.;
Dickerson, M. R.; Davis, R. E. J. Med. Chem. 1995, 38,
4439.
17. Hunskaar, S.; Fasmer, O. B.; Hole, K. J. Neurosci.
Methods 1985, 14, 69.