Selective antagonists at group I metabotropic glutamate receptors: Synthesis and molecular pharmacology of 4-aryl-3-isoxazolol amino acids

Article abstract of DOI:10.1021/jm010443t

Homologation of (S)-glutamic acid (Glu, 1) and Glu analogues has previously provided ligands with activity at metabotropic Glu receptors (mGluRs). The homologue of ibotenic acid (7), 2-amino-3-(3-hydroxy-5-isoxazolyl)propionic acid (HIBO, 8), and the 4-ph

Full text of DOI:10.1021/jm010443t

988
J . Med. Chem. 2002, 45, 988-991
Selective An ta gon ists a t Gr ou p I Meta botr op ic Glu ta m a te Recep tor s: Syn th esis
a n d Molecu la r P h a r m a cology of 4-Ar yl-3-isoxa zolol Am in o Acid s
Hasse Kromann, Frank A. Sløk, Tine B. Stensbøl, Hans Bra¨uner-Osborne, Ulf Madsen, and
Povl Krogsgaard-Larsen*
NeuroScience PharmaBiotec Research Center, Department of Medicinal Chemistry, The Royal Danish School of Pharmacy,
2 Universitetsparken, DK-2100 Copenhagen, Denmark
Received September 21, 2001
Homologation of (S)-glutamic acid (Glu, 1) and Glu analogues has previously provided ligands
with activity at metabotropic Glu receptors (mGluRs). The homologue of ibotenic acid (7),
2-amino-3-(3-hydroxy-5-isoxazolyl)propionic acid (HIBO, 8), and the 4-phenyl derivative of 8,
compound 9a , are both antagonists at group I mGluRs. Here we report the synthesis and
molecular pharmacology of HIBO analogues 9b-h containing different 4-aryl substituents.
All of these compounds possess antagonist activity at group I mGluRs but are inactive at group
II and III mGluRs.
Ch a r t 1
In tr od u ction
The central excitatory neurotransmitter (S)-glutamic
acid (Glu, 1) (Chart 1) operates through two classes of
receptors in the central nervous system (CNS): the
ionotropic receptors (iGluRs) and the metabotropic
receptors (mGluRs). iGluRs are ligand-gated ion chan-
nels and are subdivided into N-methyl-D-aspartic acid
(NMDA), (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isox-
azolyl)propionic acid (AMPA), and kainic acid recep-
tors.1,2 mGluRs belong to the family of G-protein coupled
receptors. Currently eight mGluR subtypes are known,
which have been classified into three groups on the basis
of similarities in pharmacology, sequence homology of
the receptor proteins, and second messenger pathways.
Group I mGluRs include mGluR1 and mGluR5; group
II mGluRs comprise mGluR2 and mGluR3; and mGluR4,
mGluR6, mGluR7, and mGluR8 constitute group III.2,3
It is generally agreed that both iGluRs and mGluRs play
important roles in the healthy as well as the diseased
CNS and that all subtypes of these receptors are
potential therapeutic targets.4,5 Thus, the design of
subtype-selective receptor ligands is of great interest
and is the first step in the development of drugs
selective for subtypes of iGluRs and mGluRs.
A number of heterocyclic analogues of 1, notably
quisqualic acid (2) and (S)-2-amino-3-(3-hydroxy-5-
methyl-4-isoxazolyl)propionic acid [(S)-AMPA, 3a ], are
very potent AMPA receptor agonists, 3a being highly
selective for this type of GluR.2 A large number of
analogues of 3a have been synthesized and pharmaco-
logically characterized,2 including (S)-2-amino-3-(3-hy-
droxy-5-phenyl-4-isoxazolyl)propionic acid [(S)-APPA,
3b]6 and (S)-2-amino-3-[3-hydroxy-5-(2-pyridyl)-4-isox-
azolyl)propionic acid [(S)-2-Py-AMPA, 3c].7 Whereas 3c
is approximately equipotent with 3a as an AMPA
receptor agonist,7 the isomeric 3′- and 4′-pyridyl ana-
logues are essentially inactive.8
We have previously shown that homologation of 1 as
well as of its heterocyclic analogues provided with potent
iGluR agonist activity, such as 2 and 3a , results in
compounds interacting predominantly with mGluRs.
Thus, (S)-aminoadipic acid (4) and compound 5, which
is the homologue of the nonselective iGluR agonist 2,
become nonselective mGluR ligands,9 and (S)-homo-
AMPA (6), derived from the specific AMPA agonist 3a ,
turns out to be a specific mGluR6 agonist.10,11 (S)-2-
* Correspondence: Professor Povl Krogsgaard-Larsen, Department
of Medicinal Chemistry, The Royal Danish School of Pharmacy, 2
Universitetsparken, DK-2100 Copenhagen, Denmark. Phone: (+45)
35 30 65 11. Fax: (+45) 35 30 60 40. E-mail: ano@dfh.dk.
10.1021/jm010443t CCC: $22.00 © 2002 American Chemical Society
Published on Web 01/11/2002

Brief Articles
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4 989
Ta ble 1. Receptor Binding Affinities, Agonist or Antagonist Effects in the Cortical Wedge Model, and Antagonist Effects at Group
I-III mGluR (pIC50, pEC50, and pKi Values Are ( SEM, n ) 3-4)
receptor binding
electropharmacology
EC50 (µM)/[pEC50 IC50 (µM)/[pIC50
agonism antagonism
cellular pharmacology
IC50 (µM)/[pIC50
]
]
]
Ki (µM)/[pKi] antagonism
compd
[3H]CPP
[3H]AMPA
[3H]kainic acid
mGluR1R
mGluR5a
490c
nd
45
mGluR4a mGluR2
8
9a
9b
>100a
0.8a
>100a
>100d
>100
329a,b
250c
>1000c
>1000d
>1000
>1000c
>1000d
>1000
>100d
>100
>100d
>100
590b,d
160d
125
>1000b
[3.92 ( 0.08] [4.40 ( 0.08]
9c
9d
9e
9f
42
62
>100
>100
>100
>100
>100
>100
990b
226
[3.65 ( 0.06]
71
nd
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
[4.41 ( 0.08] [4.22 ( 0.07]
[3.06 ( 0.10]
20
>100
∼1000e
65
[4.71 ( 0.07]
>100
[4.18 ( 0.12] [4.20 ( 0.02]
396 224
[3.40 ( 0.04] [3.70 ( 0.08]
119 62
[3.94 ( 0.08] [4.20 ( 0.04]
249 53
[3.60 ( 0.01] [4.30 ( 0.07]
92 70
[4.10 ( 0.16] [4.20 ( 0.04]
1.3
23b
[5.84 ( 0.07]
>100
[4.65 ( 0.06]
>100
∼1000f
9g
9h
14
>100
>100
715e
[3.15 ( 0.03]
>1000
[4.87 ( 0.04]
>100
a
b
d
Reference 12. AMPA agonist. c Reference 15. Reference 13. e NMDA antagonist. f AMPA and NMDA antagonist.
Amino-3-(3-hydroxy-5-isoxazolyl)propionic acid [(S)-
Sch em e 1a
HIBO, 8], related to the nonselective iGluR agonist
ibotenic acid (7), is a weak AMPA agonist and a group
I mGluR antagonist.12,13 In an attempt to eliminate the
AMPA agonist effect of 8 and to optimize the mGluR
antagonist effects, we have previously reported its
4-phenyl analogue 9a which is an mGluR antagonist but
almost devoid of effect at AMPA receptors (Table 1).13
This observation prompted us to synthesize and phar-
macologically characterize compounds 9b-h containing
aromatic substituents in the 4-position of the 3-isox-
azolol ring of 8.
Resu lts
Ch em istr y. We have previously reported the pal-
ladium-catalyzed reaction of 3-ethoxy-4-iodo-5-methyl-
isoxazole with a number of arylboronic acids or aryl-
tributyltin analogues as a versatile way to synthesize
4-aryl or heteroaryl 3-alkoxy-5-methylisoxazoles.14 In
addition, Heck couplings using 3-ethoxy-4-iodo-5-meth-
ylisoxazole have proven useful in synthesizing 4-sub-
a
(a) NBS, (PhCO)2O2; (b) AcNHCH(COOCH3)2, NaH; (c) 48%
aq HBr; (d) H2, 5% Pd/C; (e) 37% aq HCl, AcOH.
stituted 3-ethoxy-5-methylisoxazole. The bromination of
compounds 10e-g,i-l with N-bromosuccinimide (NBS)
in the presence of benzoyl peroxide yielded 11e-g,i-l.
Compounds 11e-g,i-l were reacted with dimethyl
acetamidomalonate in the presence of NaH to give the
fully protected compounds 12e-g,i-l (Scheme 1). Re-
duction of the styryl doublebond of 12l was performed
at 1 atm and room temperature to obtain 13 (Scheme
1). Reflux of 12e-g,i-k with aqueous HBr yielded the
target compounds 9b-g in the zwitterionic form. Com-
pound 13 was refluxed in a 10 to 1 mixture of concen-
trated HCl and AcOH to give 9h .
In Vitr o P h a r m a cology. In this study we tested the
effects of the synthesized HIBO analogues at mGluR1R,
mGluR2, mGluR4a, and mGluR5a (Table 1), using
established second messenger assay systems.10 In agree-
ment with previous studies on HIBO analogues,13,15,16
compounds 9b-h were shown to exhibit antagonist
effect at the group I receptors, mGluR1R and mGluR5a,
while no effect was observed at mGluR2 or mGluR4a
(at 1 mM concentrations), representing group II and III
receptors, respectively. The most potent compounds at
mGluR1R proved to be 9d and 9h with Ki values of 71
µM and 92 µM, respectively, being 2-3 times more
potent than the (S)-form of HIBO (8). When tested at
mGluR5a, all new compounds but 9e were shown to be
8-10 times more potent than 8.
The synthesized 4-substituted HIBO analogues (9b-
h ) were studied in different receptor binding assays in
order to determine their affinity for the iGluRs. The
ligands [3H]-(RS)-3-(2-carboxy-4-piperazinyl)propyl-1-
phosphonic acid ([3H]CPP),17 [3H]AMPA,18 and [3H]-
kainic acid19 were used to determine affinity for NMDA,
AMPA, and kainic acid receptors, respectively (Table
1). None of the compounds showed detectable affinity
toward kainic acid receptors. Compounds 9c, 9d , and
9g possessed comparable although weak affinities in the
[3H]CPP binding assay. All other compounds were
devoid of affinity toward [3H]CPP labeled binding sites.
Compound 9c showed weak affinity toward AMPA
receptors whereas 9e was found to be a fairly potent
inhibitor of [3H]AMPA binding. The receptor binding
studies were supported by in vitro electrophysiological
experiments, using the rat cortical wedge model.20,21 In
accordance with the binding studies, 9e proved to be
an AMPA receptor agonist (EC50 ) 23 µM). Compounds
9d and 9g showed weak NMDA antagonism (IC50 values
> 1 mM and 715 µM, respectively) while compound 9f
proved to be a very weak NMDA and AMPA antagonist
(IC50 > 1 mM). Compound 9h was inactive when tested
as an agonist (EC50 > 1000 µM) or an antagonist (IC50
> 1000 µM) at the iGluRs.

990 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4
Brief Articles
5-Br om om e t h yl-3-e t h oxy-4-(2-m e t h oxyp h e n yl)isox-
a zole (11i). CC (toluene, 1% AcOH) gave 11i (63%) as a yellow
oil. Recrystallization (diethyl ether/hexane) of a small sample
gave 11i as colorless crystals: mp 48.5-50 °C; 1H NMR
(CDCl3) δ 1.39 (t, 3H, J ) 7.0 Hz), 3.85 (s, 3H), 4.35 (s, 2H),
4.36 (dd, 2H, J ) 7.0 Hz), 6.99 (d, 1H, J ) 8.2 Hz), 7.04 (dt,
1H, J ) 7.6 Hz, J ) 1.1 Hz), 7.32-7.42 (m, 2H); 13C NMR
(CDCl3) δ 14.46, 19.80, 55.43, 65.91, 105.85, 111.25, 116.29,
Discu ssion
All of the phenolyl analogues 9b-d show antagonist
effects at group I mGluRs, but whereas 9c,d in addition
to the mGluR effect also show effects at iGluRs (Table
1), compound 9b, containing a 2-phenolyl substituent,
does not interact detectably with iGluRs. Thus com-
pound 9b is a selective group I mGluR antagonist
showing marginal preference for mGluR5. The presence
of a hydroxy group in the 2′- or 4′-position in compounds
9b and 9d seems favorable compared to 9a and 9c. This
may be due to the ability of the hydroxy group to
function as a hydrogen bond acceptor and/or donor. The
presence of a potential hydrogen bond acceptor in the
same position of the aromatic substituent in 9e does,
on the other hand, not facilitate an antagonist effect at
these receptors (Table 1). Compound 9e is, however, a
rather potent AMPA receptor agonist, analogous to the
potent AMPA agonist activity observed in (S)-2-Py-
AMPA (3c) containing the same heterocyclic substitu-
ent.7 The activity data of 9b and 9e emphasize the
different structural requirements necessary to activate
AMPA receptors and to block group I mGluRs.
Compounds 9f and 9g, containing in the 4-position a
1′-naphthyl and 2′-naphthyl substituent, respectively,
show additional weak iGluR antagonist effects (Table
1). The iGluR antagonist effects of 9f and 9g were
eliminated by replacing the naphthyl moiety with the
phenylethyl group to give 9h .
In conclusion, using the nonselective iGluR and
mGluR ligand HIBO (8) as a lead, we were able to
develop compounds 9b,f,h as selective group I mGluR
antagonists, showing some (2-5-fold) preference for
mGluR5 (Table 1). These compounds illustrate that
group I mGluRs can accommodate 3-isoxazolol amino
acid ligands containing relatively bulky and lipophilic
substituents and may be useful in the attempts to
develop therapeutically useful group I mGluR antago-
nists. These new compounds did not interact detectably
with mGluR2 or mGluR4a as representatives for group
II and group III mGluRs, respectively.
120.78, 130.08, 131.26, 156.99, 163.96, 169.78. Anal. (C13H14
NO3Br) C, H, Br, N.
-
Gen er a l P r oced u r e for th e P r ep a r a tion of Meth yl
2-Aceta m id o-2-(m eth oxyca r bon yl)-3-(4-a r yl-3-eth oxy-5-
isoxa zolyl)p r op ion a t es (12e-g,i-l). Dimethyl acetami-
domalonate (4.2 mmol) was added in small portions to a
suspension of NaH (60% suspension in mineral oil) (4.2 mmol)
in DMF (10 mL). The mixture was stirred at room temperature
for 30 min. A solution of 11e-g,i-l (4.2 mmol) in DMF (10
mL) was added, and the reaction was stirred overnight at room
temperature. The mixture was evaporated, and H2O was added
and extracted with diethyl ether. Drying (MgSO4) of the
organic phase, filtration, and concentration in vacuo gave the
crude product that was purified by CC.
Meth yl 2-Aceta m id o-2-(m eth oxyca r bon yl)-3-[3-eth oxy-
4-(2-m eth oxyp h en yl)-5-isoxa zolyl]p r op ion a te (12i). CC
(toluene/EtOAc 9:1, 1% AcOH) gave 12i as colorless crystals.
Recrystallization (EtOAc/hexane) gave 12i (83%) as colorless
crystals: mp 115-116 °C; 1H NMR (CDCl3) δ 1.36 (t, 3H, J )
7.1 Hz), 1.60 (s, 3H), 3.66 (s, 6H), 3.83 (s, 3H), 3.86 (s, 2H),
4.30 (dd, 2H, J ) 7.1 Hz), 6.55 (bs, 1H), 6.95 (d, 1H, J ) 8.3
Hz), 6.99 (t, 1H, J ) 7.4 Hz), 7.24 (dd, 1H, J ) 7.4 Hz, J ) 1.8
Hz), 7.32 (dt, 1H, J ) 7.4 Hz, J ) 1.8 Hz); 13C NMR (CDCl3)
δ 14.42, 22.03, 30.73, 53.49, 55.32, 64.55, 65.61, 106.33, 111.31,
116.64, 120.55, 129.61, 131.44, 150.66, 164.64, 167.44, 169.28,
169.73. Anal. (C20H24N2O8) C, H, N.
Meth yl 2-Aceta m id o-2-(m eth oxyca r bon yl)-3-(3-eth oxy-
4-p h en yleth yl-5-isoxa zolyl)p r op ion a te (13). To a solution
of compound 12l (1.93 mmol) in absolute ethanol (20 mL) was
added 5% Pd/C (20 mg). The reaction was left under an
atmosphere of H2 for 3 h at room temperature, followed by
filtration through a pad of Celite and concentration in vacuo.
CC (toluene/EtOAc 9:1) gave 13 (93%) as colorless crystals:
1
mp 101-102 °C; H NMR (CDCl3) δ 1.40 (t, 3H, J ) 7.0 Hz),
1.88 (s, 3H), 2.45 (t, 2H, J ) 6.9 Hz), 2.72 (t, 2H, J ) 6.9 Hz),
3.53 (s, 2H), 3.81 (s, 6H), 4.25 (dd, 2H, J ) 7.0 Hz), 5.76 (bs,
1H), 7.09-7.26 (m, 5H). 13C NMR (CDCl3) δ 9.91, 18.72, 25.49,
31.82, 52.03, 63.82, 64.55, 110.06, 129.76, 132.19, 132.21,
145.47, 169.57, 173.99, 176.35, 177.67. Anal. (C21H26N2O7) C,
H, N.
Exp er im en ta l Section
Gen er a l P r oced u r e for th e P r ep a r a tion of Am in o Acid
9b-g. Compound 12e-g,i-k refluxed for 3 h in aqueous HBr
(48%). The reaction mixture was cooled and evaporated. The
residue was dissolved in H2O and evaporated three times.
(RS)-2-Am in o-3-[3-h yd r oxy-4-(2-p h en olyl)-5-isoxa zolyl]-
p r op ion ic Acid (9b). Compound 12i (3.2 mmol) was refluxed
for 3 h in aqueous HBr (48%, 20 mL). The reaction mixture
was cooled and evaporated. The residue was dissolved in H2O
and evaporated three times. The residue was washed several
times with hot EtOAc and H2O giving colorless crystals.
Recrystallization (water) gave 9b (22%) as colorless crystals:
Ch em istr y. Thin-layer chromatography (TLC) was per-
formed on silica gel F254 plates (Merck). All compounds were
detected using UV light and a KMnO4 spraying reagent.
Compounds containing amino groups were visualized using a
ninhydrin spraying reagent. 1H, 13C, and associated proton test
(APT) spectra were recorded on a 300 MHz Varian Gemini
spectrometer or a Bruker AC-200 F spectrometer, using CDCl3
or D2O/NaOD as the solvent. Chemical shifts are given in ppm
(δ) using TMS or dioxane (δ 3.70/67.3) as the internal
standard, and coupling constants (J ) are given in hertz.
Column chromatography (CC) was performed on Merck silica
gel 60 (0.063-0.200 mm). Melting points were determined in
open capillaries and are uncorrected. All solvents and reagents
were obtained from Fluka or Aldrich and used without further
purification, except DMF, which was stored over 3 Å molecular
sieves. Compounds 10e-g,i-l were prepared as previously
described.14 Elemental analyses were performed at the Ana-
lytical Research Department, H. Lundbeck A/S, Denmark, or
by J . Theiner, Microanalytical Laboratory, Institute of Physical
Chemistry, University of Vienna, Austria, and are within
(0.4% of the theoretical value unless otherwise stated.
Gen er a l P r oced u r e for th e Br om in a tion of 4-Su bsti-
tu ted 3-Eth oxy-5-m eth ylisoxa zoles (11e-g,i-l). To a solu-
tion of compound 10e-g,i-l (8.9 mmol) in CCl4 (40 mL) was
added NBS (1.7 g, 9.8 mmol) and dibenzoyl peroxide (50 mg).
The reaction was heated at reflux for 6 h, followed by cooling
and filtration. Concentration in vacuo gave the crude product
that was purified by CC.
1
mp 233-235 °C dec; H NMR (D2O/NaOD) δ 2.48 (dd, 1H, J
) 15.0 Hz, J ) 8.3 Hz), 2.69 (dd, 1H, J ) 15.0 Hz, J ) 4.9
Hz), 3.24 (dd, 1H, J ) 8.3 Hz, J ) 4.9 Hz), 6.85-6.93 (m, 2H),
7.05 (dd, 1H, J ) 7.5 Hz, J ) 1.8 Hz), 7.21 (ddd, 1H, J ) 8.3
Hz, J ) 7.5 Hz, J ) 1.8 Hz). Anal. (C12H12N2O ) C, N; H: calcd,
4.58; found, H, 5.01.
(RS)-2-Am in o-3-(3-h ydr oxy-4-ph en yleth yl-5-isoxazolyl)-
p r op ion ic Acid (9h ). Compound 13 (1.9 mmol) was refluxed
overnight in HCl (37%, 10 mL) and AcOH (1 mL). Subse-
quently the mixture was evaporated to dryness, and the
residue was washed several times with hot EtOAc and H2O
giving compound 9h (70%) as colorless crystals. Recrystalli-
zation (water) of a small sample gave 9h as colorless crystals:
mp 205-207 °C (dec); 1H NMR (D2O/NaOD) δ 2.06 (dd, 1H, J
) 15.0 Hz, J ) 8.4 Hz), 2.24 (dd, 1H, J ) 15.0 Hz, J ) 5.1
Hz), 2.33 (dt, 2H, J ) 6.6 Hz, J ) 2.7 Hz) 2.61 (dt, 2H, J ) 6.6
Hz, J ) 2.7 Hz), 2.92 (dd, 1H, J ) 8.4 Hz, J ) 5.1 Hz), 6.99-

Brief Articles
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4 991
7.16 (m, 5H); 13C NMR (D2O/NaOD) δ 23.89, 31.92, 34.97,
55.02, 108.85, 126.89, 129.31, 128.96, 142.83, 166.56, 178.57,
182.24. Anal. (C14H16N2O4) C, H, N.
(7) J ohansen, T. N.; Ebert, B.; Falch, E.; Krogsgaard-Larsen, P.
AMPA receptor agonists: Resolution, configurational assign-
ment, and pharmacology of (+)-(S)- and (-)-(R)-2-amino-3-[3-
hydroxy-5-(2-pyridyl)isoxazol-4-yl]propionic acid (2-Py-AMPA).
Chirality 1997, 9, 274-280.
(8) Falch, E.; Brehm, L.; Mikkelsen, I.; J ohansen, T. N.; Skjærbæk,
N.; Nielsen, B.; Stensbøl, T. B.; Ebert, B.; Krogsgaard-Larsen,
P. Heteroaryl analogues of AMPA. 2. Synthesis, absolute ster-
eochemistry, photochemistry, and structure-activity relation-
ships. J . Med. Chem. 1998, 41, 2513-2523.
(9) Bra¨uner-Osborne, H.; Krogsgaard-Larsen, P. Pharmacology of
(S)-homoquisqualic acid and (S)-2-amino-5-phosphonopentanoic
acid [(S)-AP5] at cloned metabotropic glutamate receptors. Br.
J . Pharmacol. 1998, 123, 269-274.
(10) Bra¨uner-Osborne, H.; Sløk, F. A.; Skjærbæk, N.; Ebert, B.;
Sekiyama, N.; Nakanishi, S.; Krogsgaard-Larsen, P. A new
highly selective metabotropic excitatory amino acid agonist:
2-amino-4-(3-hydroxy-5-methylisoxazol-4-yl)butyric acid. J . Med
Chem. 1996, 39, 3188-3194.
(11) Ahmadian, H.; Nielsen, B.; Bra¨uner-Osborne, H.; J ohansen, T.
N.; Stensbøl, T. B.; Sløk, F. A.; Sekiyama, N.; Nakanishi, S.;
Krogsgaard-Larsen, P.; Madsen, U. (S)-Homo-AMPA, a specific
agonist at the mGlu6 subtype of metabotropic glutamic acid
receptors. J . Med. Chem. 1997, 40, 3700-3705.
(12) Bishoff, F.; J ohansen, T. N.; Ebert, B.; Krogsgaard-Larsen, P.;
Madsen, U. Excitatory amino acid receptor ligands: Asymmetric
synthesis, absolute stereochemistry and pharmacology of (R)-
and (S)-homoibotenic acid. Bioorg. Med. Chem. 1995, 3, 553-
558.
(13) Madsen, U.; Bra¨uner-Osborne, H.; Frydenvang, K.; Hvene, L.;
J ohansen, T. N.; Nielsen, B.; Sa´nchez, C.; Stensbøl, T. B.;
Bischoff, F.; Krogsgaard-Larsen, P. Synthesis and pharmacology
of 3-isoxazolol amino acids as selective antagonists at group I
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(14) Kromann, H.; Sløk, F. A.; J ohansen, T. N.; Krogsgaard-Larsen,
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dron 2001, 57, 2195-2201.
Cell Cu ltu r e. The Chinese hamster ovary (CHO) cell line
expressing mGluR1R, mGluR2, mGluR4, and mGluR5a were
maintained as described previously.22-24 The cell lines were
grown in a humidified 5% CO2/95% air atmosphere at 37 °C
in DMEM containing a reduced concentration of (S)-glutamine
(100 mg/mL) and 10% dialyzed fetal calf serum (all GIBCO,
Paisley, Scotland). Two days before the inositol phosphate
assay, 1.8 × 106 cells were divided into the wells of 48-well
plates; and 2 days before the cyclic AMP-assay, 1.0 × 106 cells
were divided into the wells of 96-well plates.
Mea su r em en t of P I Hyd r olysis a n d Cyclic AMP F or -
m a tion . The mGluR subtypes mGluR1R, mGluR2, mGluR4a,
and mGluR5a were expressed in CHO cell lines. All compounds
were tested for agonist and antagonist activity at 1 mM
concentrations unless otherwise stated, by the method previ-
ously described.15 Ki values were calculated from IC50 values
by use of the Cheng-Prusoff equation.25
Recep tor Bin d in g Assa ys. Affinities for NMDA, AMPA,
and kainic acid receptors were determined using [3H]CPP,17
[3H]AMPA,18 and [3H]kainic acid19 with the modifications
previously described.6 The membrane preparation used in all
the receptor binding experiments were prepared according to
the method described by Ransom and Stec.26 The amount of
bound radioactivity was determined using a Packard TOP-
COUNT microplate scintillation counter. Data were analyzed
using Grafit 3.0 Leatherbarrow software. Data were fitted to
the equation, B ) 100 - (100 × [inhibitor]n)/(IC50 + [inhibi-
n
tor]n), where B is the binding as a percentage of total specific
binding and n the Hill coefficient.
In Vitr o Electr op h ysiology. A rat cortical preparation20
in a modified version21 was used for the determination of the
depolarizing effects of the excitatory amino acid analogues
under study. Agonists were applied for 90 s. Receptor selectiv-
ity was determined by antagonizing responses, approximately
corresponding to the EC50 values of the compounds in question,
with 5 µM CPP or 5 µM NBQX for NMDA and AMPA
receptors, respectively. Antagonists were applied for 90 s,
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×
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Ack n ow led gm en t. The technical assistance of Ms.
Heidi Petersen and Ms. Lisbeth Eriksen and the sec-
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University) is gratefully acknowledged for providing the
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