N1-Substituted Quinoxaline-2,3-diones as Kainate Receptor Antagonists: X-ray Crystallography, Structure-Affinity Relationships, and in Vitro Pharmacology

Article abstract of DOI:10.1021/acschemneuro.8b00726

Among the ionotropic glutamate receptors, the physiological role of kainate receptors is less well understood. Although ligands with selectivity toward the kainate receptor subtype GluK1 are available, tool compounds with selectivity at the remaining kain

Full text of DOI:10.1021/acschemneuro.8b00726

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Article
N1-Substituted Quinoxaline-2,3-diones as Kainate Receptor Antagonists: X-ray
Crystallography, Structure-Affinity Relationships and in vitro Pharmacology
Jakob S. Pallesen, Stine Møllerud, Karla Frydenvang, Darryl S. Pickering, Jan Bornholdt, Birgitte
Nielsen, Diletta Pasini, Liwei Han, Laura Marconi, Jette Sandholm Kastrup, and Tommy N. Johansen
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00726 • Publication Date (Web): 08 Jan 2019
Downloaded from http://pubs.acs.org on January 11, 2019
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N1-Substituted Quinoxaline-2,3-diones as Kainate
Receptor Antagonists: X-ray Crystallography,
Structure-Affinity Relationships and in vitro
Pharmacology
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Jakob Pallesen, Stine Møllerud, Karla Frydenvang,* Darryl S. Pickering,* Jan
†
Bornholdt, Birgitte Nielsen, Diletta Pasini, Liwei Han, Laura Marconi, Jette Sandholm
Kastrup and Tommy N. Johansen*
Department of Drug Design and Pharmacology, Faculty of Health and Medical
Sciences, University of Copenhagen, DK-2100 Copenhagen, Denmark.
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ABSTRACT
Among the ionotropic glutamate receptors, the physiological role of kainate receptors is
less well understood. Although ligands with selectivity towards the kainate receptor
subtype GluK1 are available, tool compounds with selectivity at the remaining kainate
receptor subtypes are sparse. Here, we have synthesized a series of quinoxaline-2,3-
diones with substitutions in the N1-, 6- and 7-position to investigate the structure-activity
relationship (SAR) at GluK1-3 and GluK5. Pharmacological characterization at native and
recombinant kainate and AMPA receptors revealed that compound 37 had a GluK3-
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binding affinity (K) of 0.142 M and 8-fold preference for GluK3 over GluK1. Despite
i
lower binding affinity of 22 at GluK3 (K = 2.91 M) its preference for GluK3 over GluK1
i
and GluK2 was >30-fold. Compound 37 was crystallized with the GluK1 ligand-binding
domain to understand the SAR. The X-ray structure showed that 37 stabilized the protein
in an open conformation, consistent with an antagonist binding mode.
Keywords: quinoxalinediones, kainate receptors, antagonists, structure-activity studies,
x-ray crystallography, binding affinities
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INTRODUCTION
(S)-Glutamate (Glu) activates two main classes of central nervous system receptors:
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metabotropic glutamate receptors (mGluRs) and ionotropic glutamate receptors (iGluRs).
The mGluRs are G-protein-coupled receptors that mediate a signaling cascade inside the
neurons, whereas the iGluRs are ligand-gated ion channels, which are essential for
mediating fast synaptic transmission. The iGluRs consist of three subfamilies, named
1
after their response to the small molecule agonists AMPA, NMDA, and kainate. AMPA
and NMDA receptors are well known for their fundamental involvement in several brain
disorders, such as epilepsy, pain and neurodegenerative disorders,1-4 but so far AMPA
and NMDA receptors have been shown to be difficult therapeutic targets to handle mainly
due to a deep involvement of both NMDA and AMPA receptors in many physiological
processes. In contrast to AMPA and NMDA receptors which are localized
postsynaptically, kainate receptors are found both pre- and postsynaptically and are
believed to have a modulatory function in central nervous system neurotransmission.1, 5,
6
Compared to AMPA and NMDA receptors, kainate receptors are less well characterized,
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mainly due to a lack of selective agonists and antagonists, and this fact has impeded the
understanding of the physiological and pharmacological potential of kainate receptors as
drug targets. Some of the first antagonists to be used in kainate receptor research were
the 1,4-dihydroquinoxaline-2,3-diones (QXs), such as CNQX, as well as the isoxazole-
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based acidic amino acid (S)-ATPO (Figure 1). Today, only one structure of the ligand-
binding domain (LBD) of GluK1 has been determined in complex with a quinoxalinedione
8
(
compound 2), containing one substituent different from hydrogen in the 6-position. So
far, it has not been possible to crystallize the LBDs of GluK2-5 with antagonists.
However, neither of the early compounds are optimal for kainate receptor research as
they are better antagonists of AMPA receptors than of kainate receptors. Later on, GluK1
receptor-preferring amino acid-based antagonists have been reported, such as the
substituted willardiine UBP310 and the substituted tetrahydroisoquinoline LY 466195.
Using such GluK1 receptor-preferring antagonists, kainate receptors and especially
GluK1-containing kainate receptors have been demonstrated to be putative targets in
disorders such as epilepsy, pain and migraine.5, 7 However, due to lack of selective
antagonists for the remaining kainate receptors, the physiological role and therapeutic
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potential of these receptors are still unclear. Only a few GluK2- and/or GluK3-preferring
compounds have been reported. One interesting example is the GluK3-preferring N1-
substituted quinoxaline-2,3-dione LU 97175 (compound 1) reported to have a sub-
micromolar affinity at GluK3 receptors as well as a 4-fold and >10-fold preference for
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GluK3 over GluK1 and GluK2 receptors, respectively. Furthermore, compound 1 was
able to block AMPA-induced cell death in vitro and had anticonvulsant activities in rat
kindling studies.9 More recently, another series of new quinoxaline-2,3-diones
represented by compounds 2 and 3 has been reported (Figure 1).8, 10 Whereas
compound 2 showed mid-micromolar affinity with no selectivity among the recombinant
receptors tested (GluA2, GluK1–3), compound 3 showed a preference for GluK3 over
GluK1,2 and in particular over GluA2-receptors.
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S
H
N
H2O3P
COOH
NH2
H
HOOC
O
R1
R2
O
O
O
N
O
O
H2NO2S
O2N
COOH
NH2
N
N
N
N
H
N
H
O
O
QX: R1 = R2 = H
CNQX: R1 = CN, R2 = NO2
DNQX: R1 = R2 = NO2
NBQX
(S)-ATPO
UBP310
H
N
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R3
2: R3 =
N
H
HN
N
O
O
HOOC
H
H
H
H
COOH
N
COOH
N
NH2
COOH
NH
F3C
N
H
O
F
F
3
: R3 =
1
LY 466195
Figure 1. Chemical structures of selected AMPA/kainate receptor antagonists.
In the effort to identify tools useful for the pharmacological characterization of native
kainate receptors, we prepared a series of quinoxaline-2,3-diones substituted in the 1-
position, 6-position and 7-position, and characterized the compounds in radioligand
receptor binding assays using native and recombinant receptors. Besides at GluA2 and
GluK1-3, the new compounds were characterized with respect to binding affinity at the
kainate receptor GluK5 at which only a limited number of compounds have been tested.11
Furthermore, compound 1 was studied in a two-electrode voltage clamp (TEVC) assay
using recombinant GluK1, GluK2 and GluK3 receptors expressed in Xenopus laevis
oocytes. Finally, in order to understand the molecular interaction of this series of
compounds with kainate receptors, we report an X-ray structure of one of the new
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compounds, compound 37, co-crystallized together with the GluK1 ligand-binding domain
(LBD). Notably, four of the new compounds (19, 21, 22 and 37) showed preference for
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the GluK3 receptor, for which a need of potent and selective ligands exists.
RESULTS AND DISCUSSION
Chemistry. Compound (1, LU 97175) was synthesized as shown in Scheme 1 according
to the procedures described by Lubisch et al.12 The chlorine of 4 could easily be
substituted by benzhydrazide to give 5 which underwent acylation, reduction and ring
closure to form compound 6. Dinitration of 6 to 7 was followed by hydrolysis of the
benzamide bond to give 8. Compound 8 was acylated with various acylating agents to
give target compounds 9 – 12. Finally, the nitro group of compound 12 was reduced to
the corresponding amine, which was directly converted into the corresponding pyrrole 1.
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Scheme 1. Synthesis of target compounds 1 and 6 – 12.a
NO2
HN
N
O
O
HN
N
O
O
NH2
N
HN
NH
O
O2N
F3C
O2N
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O
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Cl
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d
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N
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N
H
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N
H
F3C
NO2
F3C
NO2
4
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f
g
O
O
S
(
i) 9: R =
NH
R
HN
N
O
O
HN
N
O
O
(
ii) 10: R =
O
N
O
O2N
F3C
N
O
O2N
F3C
N
b,h
N
H
O
N
H
O
F3C
N
H
O
(iii) 11: R =
HN
O
12
1
a
Reagents and conditions: (a) benzhydrazide, K CO , DMF, 110 °C, 4 h; (b) ethyl
2
3
chlorooxoacetate, TEA, dry THF; (c) iron powder, AcOH, reflux, 30 min; (d) KNO , H SO ,
3
2
4
0 °C, 1 h; (e) 90% H SO , 80 °C, 12 h; (f) (i) benzenesulfonyl chloride, N-methylimidazole,
2
4
dry DMSO, rt, 10 min; (ii) Ac O, reflux, 1 h; (iii) AcCl, N-methylimidazole, dry DMSO, rt,
2
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several days; (g) benzoyl chloride, N-methylimidazole, dry DMSO, rt, 10 min; (h) 2,5-
dimethoxytetrahydrofurane, AcOH, reflux, 10 min.
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The aryl bromide 13 and the aryl iodide 16 were used as starting material for the
synthesis of the 7-bromo and the 7-iodo analogues (15 and 18, respectively) (Scheme 2).
In both cases the fluorides were selectively substituted by benzhydrazide to give 14 and
17, respectively, which underwent acylation, reduction and ring closure to compounds 15
and 18, respectively. Compound 18 was also used as starting material in Sonogashira
cross coupling reactions to give the 7-alkynes 19 – 22. The two target quinoxaline-2,3-
diones (24 and 27) were successfully synthesized starting out from arylhalides 4 and
commercial available 1-fluoro-2-nitro-4-(trifluoromethyl)benzene (25), respectively,
following a procedure similar to that of the synthesis of target compound 15 from
arylhalide 13.
Scheme 2. Synthesis of target compounds 15, 18 – 22, 24 and 27.a
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HN
N
O
O
HN
N
O
O
HN
NH
O
R1
NO2
HN
NH
O
a
e
b,c
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b,c
R2
F3C
N
H
O
R2
N
H
O
R2
NO2
F3C
NO2
13: R1 = F, R2 = Br
16: R1 = F, R2 = I
: R1 = Cl, R2 = CF3
23
24
1
5: R2 = Br
18: R2 = I
1
1
4: R2 = Br
7: R2 = I
4
25: R1 = F, R2 = CF3
d
f
HO
HO
(
i) 19; R3 =
(ii) 20; R3 =
HN
O
O
b,c
N
(
iii) 21; R3 =
HO
N
O
O
NH
N
H
O
(iv)
22; R3 =
F3C
N
H
F3C
NO2
R3
2
6
27
a
Reagents and conditions: (a) benzhydrazide, DMA, microwave, 160 °C, 30 min; (b)
ethyl chlorooxoacetate, TEA, dry THF; (c) iron powder, AcOH, reflux; (d) DMF, Et NH,
2
.
Pd(dppf)Cl DCM, CuI, (i) propargyl alcohol, (ii) 2-methyl-3-butyn-2-ol, (iii) 3-butynol, (iv)
2
phenylacetylene; (e) acetohydrazide, DMF, 120 °C, 2 h; (f) 2-phenylethan-1-amine,
DIPEA, MeCN, rt, 2 h.
Target compounds 33 – 39 were synthesized via the N1-amino substituted key
intermediate and target compound 32 as shown in Scheme 3. Commercially available 28
was selectively nitrated to give 29, which was converted to the corresponding
difluorosubstituted compound, 30, to increase the reactivity in the following substitution
reaction. Using Boc-protected hydrazine it was possible to selectively substitute one of
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the two fluoro substituents to give compound 31. Attempts to acylate the aromatic
nitrogen of compound 31 and then to reduce the nitro group using iron in acetic acid
failed. However, the key quinoxalinedione 32 was prepared using a one-pot procedure,
first reducing the nitro group of 31 with hydrogen and Pd/C and then converting the amino
intermediate into compound 32 using oxalyl chloride.
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0
Scheme 3. Synthesis of target compounds 32 – 39.a
R4
R3
NH2
N
HN
N
O
O
HN
N
O
O
R1
R2
F
O
O
F
Cl
Cl
F
a
d,e
f
g
F3C
N
H
O
F3C
F3C
NO2
F3C
N
H
F3C
N
H
O
28
29: R1 = R2 = Cl
30: R1 = R2 = F
32
(i) 33: R3 = H
(i) 37: R4 = o-OH
(ii) 38: R4 = m-OH
(iii) 39: R4 = p-OH
b
c
(
ii) 34: R3 = o-OCH3
(
iii) 35: R3 = m-OCH3
31: R1 = F, R2 = NHNHBoc
(iv) 36: R3 = p-OCH3
a
Reagents and conditions: (a) KNO , conc. H SO , rt, 18 h; (b) KF, DMF, 160 °C, 2.5
3
2
4
h; (c) tert-butyl hydrazinecarboxylate, dry DMSO, rt, 30 min; (d) 5% Pd/C, H (60 psi),
2
EtOH, rt, 17 h; (e) oxalyl chloride, 1,2-dichlorobenzene 90 °C, 1h; (f) (i) benzoyl chloride,
chlorobenzene, 135 °C, 24 h; (ii) 2-methoxybenzoyl chloride, chlorobenzene, 135 °C, 3
days; (iii) 3-methoxybenzoyl chloride, chlorobenzene, 135 °C, 24 h; (iv) 4-
methoxybenzoyl chloride, chlorobenzene, 135 °C, 3 days; (g) (i) piperazine, dimethyl
acetamide, 150 °C, 8 h; (ii) and (iii) boron tribromide, dry DCM, rt, 48 h.
In order to selectively acylate the exocyclic nitrogen atom in compound 32, acylation
was tried under basic condition using TEA. However, this approach resulted exclusively
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
4
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5
5
5
5
5
5
5
5
6
in acylation at the N4-position of the quinoxalinedione. By performing the acylation without
base, but at higher temperature, selective exocyclic acylation was achieved, even though
long reaction times were needed. As illustrated in Scheme 3, compound 32 was
successfully acylated to 33 – 36 using the appropriate acyl chlorides in refluxing
chlorobenzene. Cleavage of the methyl ethers 34 – 36 was initially attempted using boron
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
tribromide in DCM. The method was feasible only for the synthesis of compound 38 and
39. In case of the ortho-substituted compound 37, the final demethylation was
successfully carried out under basic conditions using piperazine in dimethyl acetamide at
1
50 °C.14
Pharmacology. The N1-substituted quinoxaline-2,3-diones and the reference
compounds investigated in the present study were characterized in receptor binding
studies at native and recombinant iGluRs. Binding affinities are summarized in Table 1.
Table 1. Receptor binding affinities (mean ± SEM) at native and recombinant rat iGluRs.
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Native rat
iGluR
Recombinant homomeric rat iGluR
Com-
K (M)
i
K (M)
i
pound
AMPA
n.d.a
GluK1
GluK2
GluK3
GluK5
GluA2
467 ± 82
0.333 ±
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
QX
422 ± 146
187 ± 14
129 ± 10
> 1,000
0
.637 ±
1.28 ± 0.30b
1.49 ± 0.10b
8.40 ± 0.88c
CNQX
n.d.
n.d.
n.d.
b
b
0.050
0.028
0.652 ±
0.362 ±
0.033
0.254 ±
DNQX
2.10 ± 0.32b
5.38 ± 1.24
7.12 ± 0.91
152 ± 23
b
b
b
0.028
0.014
0.0773 ±
NBQX
2.60 ± 0.14
0.697 ± 0.123
1.53 ± 0.17
1.42 ± 0.14
19.0 ± 2.1
3.36 ± 0.61
0
.0102
0
0
.51 ±
.07
0.488 ±
0.058
1
6
7
8
9
0.187 ± 0.021 23.9 ± 4.0
1.52 ± 0.25
8.34 ± 0.64
3.51 ± 0.68
11.2 ± 2.4
1.55 ± 0.30
2
0
.24 ±
.43
2.64 ± 0.81 0.726 ± 0.077
2.33 ± 0.34 0.458 ± 0.085
103 ± 32
107 ± 39
> 100
1
0
.90 ±
.39
2
.74 ±
27.4 ± 3.5
14.0 ± 2.6
0.30
0.53 ±
0.06
1.40 ± 0.42
1.51 ± 0.13 0.916 ± 0.135
78 ± 16
1
1
0
1
n.d.
n.d.
36.8 ± 4.7
20.3 ± 2.7
22.6 ± 3.6
5.97 ± 0.56
7.06 ± 0.64
> 100
> 100
44.6 ± 11.1
27.4 ± 2.7
11.3 ± 0.30
1
0
.91 ±
.29
1
2
1.08 ± 0.11
2.54 ± 0.67
1.31 ± 0.30
2.02 ± 0.09 0.526 ± 0.150 43.4 ± 10.9 4.93 ± 0.59
1
0
.63 ±
.25
15
18
3.33 ± 0.20 0.983 ± 0.059
77 ± 5
8.93 ± 1.18
7.83 ± 0.99
1
0
.36 ±
.06
1.96 ± 0.32 0.379 ± 0.020 42.3 ± 7.9
1
2
9
0
n.d.
n.d.
15.9 ± 1.7
> 100
9.78 ± 0.45
> 100
1.19 ± 0.10
 100
37.4 ± 4.6
> 100
 100
> 100
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2
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2
2
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3
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5
5
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6
2
2
2
2
1
2
4
7
n.d.
n.d.
n.d.
n.d.
.17 ±
12.7 ± 0.8
> 100
n.d
1.10 ± 0.14
n.d.
> 100
 100
2.91 ± 0.29
8.83 ± 0.78
5.86 ± 0.20
> 100
> 100
> 100
23.6 ± 6.0
32.4 ± 5.0
15.3 ± 4.2
17.4 ± 1.2
10.7 ± 0.7
22.4 ± 2.0
11.1 ± 2.3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
3
3
3
3
2
3
4
5
12.6 ± 1.8
0.802 ± 0.111
4.35 ± 0.92
1.76 ± 0.45
4.74 ± 0.33
1.12 ± 0.14
0.80 ± 0.18
2.15 ± 0.36
14.0 ± 1.3
5.24 ± 0.55
> 100
22.7 ± 7.3
6.15 ± 1.40
8.96 ± 1.42
4.56 ± 0.76
9.87 ± 1.26
4.12 ± 0.61
6.02 ± 0.10
5.90 ± 0.40
0
.30
n.d.
1.41 ±
0
.813 ±
0.084
0.277 ± 0.031 30.9 ± 3.7
1.20 ± 0.25
1.35 ± 0.19
> 100
0
.17
0
.95 ±
1.85 ± 0.57 0.487 ± 0.078 46.8 ± 5.3
0
.12
2
.91 ±
36
37
2.93 ± 0.25 0.970 ± 0.177
0.908 ±
 100
0
.44
0
.96 ±
.02
0.142 ± 0.015 26.4 ± 2.2
0.329 ± 0.010 45.2 ± 3.6
0
0.167
0
.72 ±
.05
0.842 ±
0.185
3
3
8
9
0
2.07 ±
0.43
2.94 ± 0.09
1.79 ± 0.56
> 100
a
b
c
n.d.: not determined. Ref. 9. Ref 14.
Whereas the N1-unsubstituted reference quinoxaline-2,3-diones QX, CNQX and DNQX
showed no appreciable preference among the AMPA and the kainate receptors GluK1-3,
NBQX had a marked preference for GluA2 receptors as compared to kainate receptors
GluK1-3 (34-70-fold preference). The N1-substituted reference quinoxaline-2,3-dione
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(
compound 1, LU 97175) showed 2-8-fold preference for GluK1-3 over GluA2. Among the
recombinant kainate receptors, compound 1 showed a 4-fold preference for GluK3 over
GluK1 in the present assays, which was less pronounced than previously reported.9
Furthermore, it is interesting to notice that the unsubstituted QX showed weak affinity
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
4
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4
4
4
4
4
4
5
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5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(high micromolar K) at all recombinant receptors tested.
i
In the present study, three categories of analogues of compound 1 have been included
Table 1). Among the first series of 6-substituted analogues (6, 15 and 18 – 22), it is clear
(
that smaller-sized substituents, such as CF (6), Br (15) and I (18), all are well accepted
3
at GluK1-3 receptors, with 18 showing similar affinity at GluK3 receptors as compared to
compound 1. The GluK1/GluK2- and GluK1/GluK3-affinity ratios for compounds 6, 15 and
1
8 were close to those of compound 1, indicating a similar trend towards selectivity.
However, introduction of alkyne substituents in the 6-position (compounds 19 – 22)
revealed that some of these compounds (except 20) are well tolerated at GluK3 receptors
(K’s in the range 1-3 M) and far better tolerated than at GluA2, GluK1 and GluK2
i
receptors. Interestingly, compound 22 with a phenylethynyl substituent showed a marked
GluK3-preference among kainate receptors (GluK1/GluK3- and GluK2/GluK3-affinity
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ratios of at least 30), and a GluA2/GluK3-affinity ratio of 8. On the other hand, compound
19 showed a marked 31-fold preference for GluK3 over GluA2, combined with a GluK3
0
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9
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2
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9
0
preference over GluK1 (13-fold) and GluK2 (8-fold). Similarly, compound 21 had at least
a 100-fold preference for GluK3 over GluA2. The observed affinity ratios indicate that the
GluK3 LBD can accept lipophilic and larger-sized ethynyl substituents in the 6-position of
the N1-substituted quinoxaline-2,3-dione better than GluA2, GluK1 and GluK2.
Secondly, we have synthesized a series of analogues with variation in position 7,
including pyrrolyl (1), nitro (12) and fluoro substituents (33) as well as the unsubstituted
analogue (6). Binding affinities (Table 1) demonstrated that these four compounds have
similar preference among the kainate receptors tested and low-micromolar affinities (K’s
i
in the range 0.2-2.6 M). Among these compounds, 33 showed the largest GluK3 over
GluA2 preference (22-fold).
The major efforts in the present study have been focused on variations at the N1-
position. Target compounds include one group of compounds (7 – 12) with a nitro
substituent in position 7, one group (6, 24, 27) without any substituent in position 7, and
finally, a series of target compounds (32 – 39) with a fluoro substituent in position 7. The
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binding affinity data (Table 1) showed that 7-nitro analogues containing aromatic amido
substituents (12, benzamido; 7, m-nitrobenzamido and 9, benzenesulfonamido) all were
potent ligands at GluK1, GluK2 and GluK3 receptors with affinities close to those of
compound 1, whereas analogues containing smaller and nonaromatic substituents (8, 10,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
3
3
4
4
4
4
4
4
4
4
4
4
5
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5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
11) were less potent. Of the tested 7-nitro analogues, a similar affinity rank order for
compounds 6, 7, 9, 11 and 12 was observed (GluK3 > GluK1 > GluK2 > GluA2).
Compound 8 showed a similar rank order at kainate receptors but was found to be weakly
GluA2 preferring, whereas the rank order for 10 was GluK3 > GluK2 > GluK1 > GluA2.
Compound 24 (with a non-aromatic methylamido substituent in the N1-position, but
without substituent in position 7) also showed lower affinity at all receptors tested as
compared to compound 6, supporting the observation that aromatic amido substituents
are essential in the N1-position. Interestingly, compound 27, having a more flexible phenyl
ethyl substituent in the N1-position but no amido group, showed a 4-8-fold lower affinity
at GluK1, GluK2 and GluK3 receptors as compared to compound 6, illustrating the
importance of having an amido substituent in the N1-position. The 7-fluoro analogues (33
– 39) were all potent ligands at GluK1, GluK2 and GluK3 receptors with sub- or low
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6
micromolar affinities (K’s in the range 0.14-4.7 M), supporting the observation seen
i
above for compound 7 that substitution in the benzamido substituent is allowed. Among
the group of compounds (33 – 39), compound 37 showed the highest affinity at GluK3
receptors (K = 0.142 M) and similar to that of compound 1 (K = 0.187 M). In addition,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
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3
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1
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0
i
i
compound 37 showed GluK1/GluK3-, GluK2/GluK3- and GluA2/GluK3-affinity ratios of 8,
6
and 29, respectively.
The compounds were also tested in our newly established GluK5 receptor binding
14
assay. CNQX and DNQX possessed the greatest binding affinity at GluK5 (K of 8.40
i
and 7.12 M, respectively), indicating that GluK5 prefers small antagonists. Among the
novel N1-substituted quinoxalinediones, compounds 1, 33 and 37 were the most potent,
showing K values of 23.9, 30.9 and 26.4 µM, respectively. No distinct structure-affinity
i
relationship could be identified among the novel N1-substituted quinoxalinediones when
tested at GluK5 homomeric receptors.
Binding affinities for the target compounds were also determined at native NMDA
3
receptors (rat brain synaptosomes) using [ H]-CGP-39653. In the NMDA receptor binding
assay, none of the compounds showed substantial affinity (K > 10 µM), except for
i
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compound 1 (K = 2.53 ± 0.35 µM). Furthermore, the binding affinity of compound 1 to rat
i
3
brain synaptosomes was also determined using [ H]-kainate. In this assay, compound 1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
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4
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4
4
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4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
showed a K value of 0.42 ± 0.05 µM. Finally, the binding affinity (K) of compound 37 was
i
i
determined at the ligand-binding domain of GluK1 (GluK1-LBD) to be 113 ± 23 nM, which
is 10-fold higher affinity than seen at full-length GluK1. Similar differences between the
affinity at full-length GluK1 and GluK1-LBD have previously been observed for other
ligands.15
In functional pharmacological studies using homomeric GluK1, GluK2 and GluK3
receptors expressed in X. laevis oocytes, antagonist affinities (K ) were measured by
b
TEVC electrophysiology (Figure 2). Compound 1 was confirmed to be an antagonist at
GluK1–3 with the calculated K values (mean ± SEM, nM): GluK1 = 115 ± 19; GluK2 =
b
323 ± 27; GluK3 = 50 ± 4.
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0
1
2
3
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0
1
2
3
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9
0
Figure 2. Antagonism by compound 1 (LU 97175) of homomeric kainate receptors. The
weakly desensitizing (Cys-Cys)GluK1–3 mutants were expressed in X. laevis oocytes and
stimulated in duplicate with Glu in the absence or presence of increasing concentrations
of 1. Shown is the mean ± SEM of pooled values from 3–10 oocytes normalized to the
control response (absence of antagonist) of each oocyte. The top and bottom of each
curve are fixed at 100% and 0%, respectively. K (mean ± SEM, nM) = 115 ± 19 (,
b
GluK1, 10 oocytes); 323 ± 27 (, GluK2, 6 oocytes); 50 ± 4 (, GluK3, 3 oocytes). The
K of GluK2 is statistically significantly different from that of GluK1 and GluK3 (p < 0.05,
b
Kruskal-Wallis One Way ANOVA on Ranks with Dunn’s post-test). Hill slopes were unity
for all curves.
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Structural analysis. To obtain information on the detailed binding mode and interactions
of this series of compounds, we crystallized GluK1-LBD with 37 as a representative. The
reason for selecting 37 was its high affinity at GluK1 combined with its acceptable
solubility for crystallization. The X-ray crystal structure of the GluK1-LBD in complex with
compound 37 was determined at 1.85 Å resolution and contained one molecule in the
asymmetric unit of the crystal (Table 2). This molecule forms a dimer with a symmetry-
related molecule (Figure 3A). Binding of 37 led to a domain opening of 33–35° relative to
the structure of GluK1-LBD with Glu, which is in the range seen for other antagonists.15
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. Crystal data, data collection and structure refinement of GluK1-LBD in complex
with compound 37.
Crystal data
Space group
H32
Unit cell: a, b, c (Å)
88.87, 88.87, 157.15,
90.0, 90.0, 120.0

   (°)
Molecules in a.u.a
1
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Data collection
Wavelength (Å)
0.97625
0
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7
8
9
0
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8
9
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9
0
3
7.38-1.85 (1.95-
Resolution (Å)
1.85)b
No. of unique reflections
Average redundancy
Completeness (%)
Rmergec (%)
20,688 (2,971)
10.0 (9.6)
100 (100)
8.1 (44.1)
6.1 (1.6)
I/σ(I)
CC1/2
1.00 (0.93)
20.3
Wilson B (Å2)
Refinement
Rwork (%) /R (%)e
d
18.7/21.6
250/1
free
Amino acid residues/37
Glycerol/sulfate/chloride/water
R.m.s. deviation bond length
5/1/1/110
0.013/1.1
(Å)/angles (°)
2
Average B-values (Å ) for:
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Amino acid residues/37
32.8/21.1
Glycerol/sulfate/chloride/water
77.0/65.2/76.6/33.2
1
1
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4
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5
5
5
5
5
5
5
6
0
1
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9
0
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9
0
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9
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8
9
0
Non-glycine residues in
allowed regions of the
Ramachandran plot (%)f
100
a
b
A.u.: asymmetric unit of the crystal. Values in parentheses correspond to the outermost
resolution shell. c Rmerge is calculated as follows: I(hkl) is the intensity of an individual
i
measurement of the reflection with Miller indices hkl, and I(hkl) is the intensity from
d
multiple observations. Rmerge = ∑ ∑|I(hkl) - I(hkl)| /∑ ∑ |I(hkl)|. R
=∑ |F
obs
- Fcalc
|
hkl
i
i
hkl
i
i
work
hkl
/
∑hkl |F | where F
and Fcalc are the observed and calculated structure factor
obs
obs
e
amplitudes, respectively, for reflection hkl. R is equivalent to Rwork, but calculated with
free
f
5% of reflections omitted from the refinement process. The Ramachandran plot was
calculated according to MolProbity.16
In complex with 37, the GluK1-LBD forms a compact dimer not previously observed
(
Figure 3A). Besides the usual interactions between D1 lobes of each subunit comprising
the dimer, in the present dimer we also observe tight packing between D2 lobes. This
2
results in a buried surface area at the dimer interface of 1339 Å , using the PISA server.17
The Gly-Thr linker in D2 that substitutes the parts of the receptor protruding to the
membrane is in close proximity in the GluK1-LBD dimer with 37. The Pro667-Pro667
linker-linker distance between the two subunits is 9.7 Å (Figure 3A). The segment Lys544,
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Gly-Thr linker and Pro667-Asp673, as well as Thr753-Gly757, is engaged in D2-lobe
contacts in the dimer, resulting in a salt bridge being formed between Lys554 of one
subunit and Asp673 of the other subunit, and vice versa (Figure S1). For comparison, the
0
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18
dimer of GluK1-LBD with the antagonist (S)-ATPO (pdb-code 1VSO ) has a more
common, less compact structure (Figure 3B), resulting in a buried surface area of 1021
2
Å and a linker-linker distance of 20.5 Å. On the other hand, the apex distance measured
between Gly771 of each subunit is similar in the GluK1-LBD dimer with 37 (13.7 Å, Figure
3A) and (S)-ATPO (12.3 Å; Figure 3B). It seems that this new dimer arrangement of
GluK1-LBD is also possible in full-length GluK1, but will require rearrangement of the
LBD-transmembrane domain (TMD) linker region (Figure S2). We cannot exclude that
the dimer arrangement is an artifact of considering an LBD-construct. However, previous
studies on agonists and antagonists have shown that there is a good agreement between
the dimer arrangement in the full-length GluA2 receptor and the ligand-binding domain of
GluA2 (e.g.19, 20). Also, the dimer arrangement could be due to crystal packing contacts,
although from comparing crystal packing in different GluK1-LBD structures this is not
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obvious as similar regions are predominantly involved in crystal packing despite different
surroundings (Figure S3).
1
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2
2
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6
0
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0
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0
Compound 37 could unambiguously be located at the ligand-binding site (Figure 3C). It
interacts with residues of lobe D1 (Figure 3C), forming direct hydrogen bonds as
15
previously observed for agonists and antagonists, whereas water-mediated hydrogen
bonds only are observed to lobe D2 residues. The 2,3-quinoxazolinedione moiety of 37
interacts with the essential Arg523 in the same manner as previously seen in the structure
8
of GluK1-LBD with compound 2. Both oxygen atoms of the 2,3-quinoxazolinedione in 37
make hydrogen bonds with Arg523. Furthermore, the 3-one forms a hydrogen bond to
the backbone nitrogen atom of Thr518 as well as a dipole-dipole interaction to the side-
chain hydroxy group of Thr518. Additionally, the 2-one interacts with a water molecule.
The N4-nitrogen atom of the 2,3-quinoxazolinedione forms a hydrogen bond to the
backbone oxygen atom of Pro516. Pi-pi stacking is observed between the 2,3-
quinoxazolinedione moiety and the residue Tyr489 (Figure 3C; distance ~3.7 Å; not
shown).
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Figure 3. X-ray crystal structure of 37 in GluK1-LBD. (A) The overall conformation of
GluK1-LBD dimer (molA grey; symmetry molA dark salmon) is shown in surface
representation as well as 37, sulfate and chloride in spheres representation with carbon
atoms in green. The Pro667-Pro667 linker-linker distance between the two subunits is
shown as well as the apex Gly771–Gly771 distance and domain opening (arrow). (B) For
comparison, the dimer of GluK1-LBD in complex with (S)-ATPO (PDB-code 1VSO) is
shown as in (A), with (S)-ATPO in spheres representation with carbon atoms in cyan. The
lobes D1 and D2 are indicated on the figure. (C) Residues forming hydrogen bonds to 37
as well as residues discussed in text are shown as lines with carbon atoms in grey.
Potential hydrogen bonds within 3.2 Å are shown as dashed, black lines as well as the
distance between Glu441 in lobe D1 and Ser721 in lobe D2 as dashed, red lines.
Furthermore, a standard 2F -F omit map (grey) contoured at 1 sigma and carved around
1
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8
9
0
o
c
the ligand at 2.0 Å radius is shown. The protein backbone is shown as cartoon. Alternative
conformation of a part of D2 is colored cyan. Oxygen atoms are red, nitrogen atoms blue
and sulfur is yellow. Water molecules are displayed as red spheres and the sulfate ion
located in the binding site is shown in sticks representation. (D). Superimposition on D1
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of all 14 available antagonist structures of rat GluK1-LBD (green sticks: 37 and the binding
site sulfate ion; grey sticks: (S)-ATPO (1VSO); thin grey lines: PDB-codes 2F34, 2F35,
0
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2OJT, 2QS1, 2QS2, 2QS3, 2QS4, 3S2V, 4DLD, 4QF9, 4YMB, 5M2V). (E). B-factors of
the protein reveal a highly dynamic lobe D2. Color range is from blue (10.5 Å) to red
2
(
110.8 Å ). One D2 helix is located in two conformations and therefore appears as less
flexible. (F) Surface representation to illustrate binding cavity. Residues discussed in text
are shown in sticks representation.
The o-hydroxy-benzamido substituent at the N1 nitrogen atom of 37 has no direct
hydrogen bonds to GluK1. In 37, the phenol ring is positioned in plane with the amide
moiety, which allows for an intramolecular hydrogen bond between the carbonyl oxygen
of the amide and the hydroxy group. This interaction might reduce the entropy penalty
upon binding of 37 at the ligand-binding site. In addition, the amide carbonyl oxygen and
the amide nitrogen atom both form a hydrogen bond to a water molecule (Figure 3C).
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Pi-pi stacking is observed between the o-hydroxy-benzamido substituent and the protein
backbone of Gly688–Ser689 (Figure 3C; distance ~3.4 Å).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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0
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1
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9
0
A sulfate ion is located within 4 Å from the aromatic fluorine atom in 37 (Figure 3C),
which forms direct hydrogen bonds to Ser689, Thr690 and Glu738 as well as water
molecules. A sulfate ion was also seen at this position in the GluK1-LBD co-crystal
8
structure with compound 2 reported by Demmer et al, but not in any of the remaining
GluK1 antagonist co-crystal structures.15 A superposition of 37 and all 13 previously
15
reported rat GluK1-LBD structures with antagonists reveals that 37 occupies a region
in the binding site similar to that of (S)-ATPO (Figure 3D). Through this binding mode, the
o-hydroxy-benzamido substituent and the sulfate ion are positioned in the same region of
the binding pocket as the phosphonic acid substituent in (S)-ATPO, with the phosphonic
acid substituent of (S)-ATPO positioned in between the hydroxy-benzamido substituent
in 37 and the sulfate ion. The phosphonic acid group in (S)-ATPO forms hydrogen bonds
to the side-chain hydroxy group and backbone nitrogen atom of Ser689 and four water
molecules, whereas the hydroxy-benzamido substituent of 37 has no hydrogen bonding
interactions with the protein.
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