Studies on an (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor antagonist IKM-159: Asymmetric synthesis, neuroactivity, and structural characterization

Article abstract of DOI:10.1021/jm301590z

IKM-159 was developed and identified as a member of a new class of heterotricyclic glutamate analogues that act as AMPA receptor-selective antagonists. However, it was not known which enantiomer of IKM-159 was responsible for its pharmacological activities. Here, we report in vivo and in vitro neuronal activities of both enantiomers of IKM-159 prepared by enantioselective asymmetric synthesis. By employment of (R)-2-amino-2-(4- methoxyphenyl)ethanol as a chiral auxiliary, (2R)-IKM-159 and the (2S)-counterpart were successfully synthesized in 0.70% and 1.5% yields, respectively, over a total of 18 steps. Both behavioral and electrophysiological assays showed that the biological activity observed for the racemic mixture was reproduced only with (2R)-IKM-159, whereas the (2S)-counterpart was inactive in both assays. Racemic IKM-159 was crystallized with the ligand-binding domain of GluA2, and the structure revealed a complex containing (2R)-IKM-159 at the glutamate binding site. (2R)-IKM-159 locks the GluA2 in an open form, consistent with a pharmacological action as competitive antagonist of AMPA receptors.

Full text of DOI:10.1021/jm301590z

Article
pubs.acs.org/jmc
Studies on an (S)‑2-Amino-3-(3-hydroxy-5-methyl-4-
isoxazolyl)propionic Acid (AMPA) Receptor Antagonist IKM-159:
Asymmetric Synthesis, Neuroactivity, and Structural Characterization
Lina Juknaite,∥ Yutaro Sugamata,† Kazuya Tokiwa,† Yuichi Ishikawa,† Satoshi Takamizawa,† Andrew Eng,‡
̇
Ryuichi Sakai,§ Darryl S. Pickering,∥ Karla Frydenvang,∥ Geoffrey T. Swanson,‡ Jette S. Kastrup,∥
and Masato Oikawa*,†
†Graduate School of Nanobioscience, Yokohama City University, Seto 22-2, Kanazawa-ku, Yokohama 236-0027, Japan
‡Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Feinberg School of Medicine, 303 East
Chicago Avenue, Chicago, Illinois 60611, United States
§Faculty of Fisheries Sciences, Hokkaido University, Hakodate 041-8611, Japan
∥Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2100
Copenhagen Ø, Denmark
S
* Supporting Information
ABSTRACT: IKM-159 was developed and identified as a
member of a new class of heterotricyclic glutamate analogues
that act as AMPA receptor-selective antagonists. However, it
was not known which enantiomer of IKM-159 was responsible
for its pharmacological activities. Here, we report in vivo and in
vitro neuronal activities of both enantiomers of IKM-159
prepared by enantioselective asymmetric synthesis. By employ-
ment of (R)-2-amino-2-(4-methoxyphenyl)ethanol as a chiral
auxiliary, (2R)-IKM-159 and the (2S)-counterpart were
successfully synthesized in 0.70% and 1.5% yields, respectively,
over a total of 18 steps. Both behavioral and electro-
physiological assays showed that the biological activity observed for the racemic mixture was reproduced only with (2R)-
IKM-159, whereas the (2S)-counterpart was inactive in both assays. Racemic IKM-159 was crystallized with the ligand-binding
domain of GluA2, and the structure revealed a complex containing (2R)-IKM-159 at the glutamate binding site. (2R)-IKM-159
locks the GluA2 in an open form, consistent with a pharmacological action as competitive antagonist of AMPA receptors.
inspired by the marine natural products kainic acid (KA, 1,
Figure 1)11 and neodysiherbaine,2 and we further characterized
the pharmacological profile of the most potent molecule, IKM-
159.9 The activities of natural templates and the synthetic
INTRODUCTION
■
Ionotropic glutamate receptors (iGluRs) mediate the majority
of fast excitatory neurotransmission in the mammalian central
nervous system (CNS) and play an important role in higher
brain functions such as learning and memory.1 iGluRs are also
thought to be involved, fully or partly, in nociception and
closely related to several brain disorders such as epilepsy,
ischemia-induced excitotoxicity, and Alzheimer, Huntington,
and Parkinson diseases.2−5 Structurally, iGluRs are assembled
as homomers or heteromers composed of four subunits that
belong to same subunit classes. A total of 18 structurally diverse
iGluR subunit superfamily proteins have been identified in the
mammalian CNS.6 One of the major interests in neuro-
chemistry is, therefore, to discover specific ligands to control
biological functions of iGluRs selectively, thereby establishing
the molecular basis of structurally diverse iGluRs.
Figure 1. Excitatory glutamate analogue kainic acid (1)11 and (2R)-
IKM-159 (2). The (2R)-enantiomer was found to be responsible for
the antagonist activity of IKM-159 in the present study.
Recently, we developed a series of synthetic heterotricyclic
glutamate analogues as subtype-selective antagonists for the
(S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid
(AMPA) type iGluR.7−10 The structures were originally
Received: October 28, 2012
Published: February 23, 2013
© 2013 American Chemical Society
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Scheme 1. Construction of Optically Active Oxanorbornenes by Tandem Ugi/Diels−Alder Reaction Using Chiral Amine 3
compounds toward iGluRs were found to be significantly
different; for example, IKM-159 selectively antagonized AMPA
receptors9 whereas kainic acid is an agonist for both AMPA and
KA-type iGluRs.12
acid (4)17 (Scheme 1). For the carboxylic acid component, the
(E)-isomer 4 was chosen because the product was expected to
be obtained as crystals from our previous study.13 In this
domino reaction, an inseparable mixture of diastereomers 7
(2R) and 7′ (2S) was obtained in 65% yield (IKM-159
numbering; see Figure 1). The ratio of 7 (2R) and 7′ (2S) was
36:64. The structures were determined as follows. Acetylation
(acetic anhydride (Ac2O), 4-dimethylaminopyridine (DMAP),
pyridine) of a mixture of 7 (2R) and 7′ (2S) furnished two
acetates 8 (2R) and 8′ (2S), which were fortunately separated
by carefully performing silica gel column chromatography, in
36% and 55% yields, respectively. The diastereomer that was
less polar on thin-layer chromatography was the minor product
and was further purified by recrystallization (Et2O, mp 88−90
°C). We successfully determined the crystal structure of the
minor product as 8 (2R) by single-crystal X-ray analysis at 183
K. The ORTEP drawing [Burnett, M. N.; Johnson, C. K.
ORTEP-III: Oak Ridge Thermal Ellipsoid Plot Program for
Crystal Structure Illustrations; Report ORNL-6895; Oak Ridge
National Laboratory: Oak Ridge, TN, 1996] is shown in Figure
2. Both isomers 8 (2R) and 8′ (2S) independently led to
optically active IKM-159 as follows.
To acquire additional insight into the mode of interaction
between IKM-159 and AMPA receptors, we studied the
structure−activity relationships of several synthetic analogues
of IKM-159.9,13 However, previous biological studies were
performed only on the racemates because no pathway was
available for asymmetric synthesis. Here we report the first
asymmetric synthesis and biological evaluation of both
enantiomers of IKM-159. We show that (2R)-IKM-159 (2) is
neuronally active, whereas the (2S)-counterpart (20) is inactive,
and further provide structural insight into the binding mode of
(2R)-IKM-159 with the GluA2 AMPA receptor ligand-binding
domain (LBD). The observation in the present structure−
activity relationship study of optical isomers of IKM-159 affords
new insight into an unconventional mode of interaction
between antagonists with this novel chemical template and
AMPA receptors.
RESULTS AND DISCUSSION
Scheme 2 shows a so-called ring-rearrangement metathesis18
of the oxanorbornene framework leading to optically active
heterotricycle 13. N-Ns amine (Ns = 2-nitrobenzenesulfon-
yl)19,20 10 was synthesized in 70% yield by reaction of iodide 8
(2R) with N-Ns-allylamine (9)21 and Cs2CO3 at 50 °C. The
■
Chemical Synthesis. The asymmetric synthesis was carried
out on the basis of our route for 12 glutamate analogues,7−10
which was thereafter extended to the racemic IKM-159.9,13
To synthesize both enantiomers, we screened chiral amines
used for tandem Ugi/Diels−Alder reaction. Among the
benzylic amines tested, 2-amino-2-(4-methoxyphenyl)ethanol
(3)14 was found to be the most efficient in terms of (1) the
stability throughout the synthesis, (2) the formation of
crystalline derivative used for structural analysis, and (3) high
reactivity toward oxidation for the removal at the final stage.
The commercially available desmethoxy analogue, 2-amino-2-
phenylethanol, was less practical because of its resistance to the
removal under a variety of conditions (see below).
3
stereochemistry was determined on the basis of JH4,H5 (3.6
Hz), which was consistent with that of the corresponding
intermediate for the synthesis of racemic IKM-159.13 Although
the reaction took place with a retention of configuration at C5,
the mechanism can be reasonably understood as a sequence of
elimination of hydrogen iodide followed by 1,4-conjugate
addition of 9, from a less-hindered endo face.22 The reaction
also produced a trace amount of aromatized product (structure
not shown). Oxanorbornene 10 was then converted efficiently
into heterotricycle 12 by domino metathesis reaction in one
step, using Hoveyda−Grubbs second-generation catalyst 11.23
Thus, the reaction was conducted with vinyl acetate in benzene
at 60 °C to provide heterotricycle 12 (E/Z = 6:4) in 71% yield
By use of (R)-amine 3 prepared in 48% yield over five steps
from (R)-(4-hydroxyphenyl)glycine,14 an oxanorbornene
framework was readily constructed by a tandem reaction with
benzyl isocyanide (5),15,16 2-furfural (6), and (E)-iodoacrylic
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Scheme 3. Alkaline Methanolysis Unexpectedly Provides a
Complex Mixture
Figure 2. Thermal ellipsoid drawing of the asymmetric molecular unit
at the 50% probability level of 8 (2R) at 183 K. Elements are color-
coded: C (gray), H (white), I (purple), N (blue), and O (red).
Scheme 2. Ring-Rearrangement Metathesis of
Oxanorbornene Framework Leading to Optically Active
Heterotricycle 13
vinyl carbonates (15A, 15B) could not be controlled, and only
a poor reproducibility was observed, while the substrate 13 was
rapidly consumed after 1.5 h.
The geometry of the vinyl group apparently indicated that
the (Z)-vinyl carbonate was produced by intramolecular
migration of the Boc group from the amide group to the
intimate enol group. The independence of the product
distribution on the E/Z ratio (6:4) of the reaction substrate
13 may indicate that the reaction proceeds via the intermediary
aldehyde 16, which is not detected yet. However, the
mechanism is likely complicated judging from the poor
reproducibility observed (see above). Although the methanol-
ysis provided a complex mixture of products, the synthetic
study was continued using the mixture without purification or
separation, as follows.
After Pinnick oxidation (NaClO2, 2-methyl-2-butene,
NaH2PO4)24 of the methanolysis mixture (14A, 14B, 15A,
15B), the resulting carboxylic acid was esterified with
trimethylsilyldiazomethane (TMS-CHN2) to give dimethyl
ester (Scheme 4). Without purification, acetylation of the
Scheme 4. A Series of Functional Group Transformations,
Successfully Performed on the Methanolysis Mixture (14A,
14B, 15A, 15B), Provided Two Products 15A and 17
with quantitative recovery of unreacted oxanorbornene 10,
which was again used for the metathesis. Next, heterotricycle 12
was treated with Boc2O (Boc = tert-butoxycarbonyl), DMAP,
and triethylamine to afford N-Boc-imide 13 (E/Z = 6:4) in 93%
yield so that transformation to the methyl ester in the next step
could be performed under mild conditions.
Methanolysis of 13 was performed by K2CO3 in methanol at
−10 °C to generate aldehydes (14A, 14B) and (Z)-vinyl
carbonates (15A, 15B) as an inseparable mixture in the ratio of
26:11:31:32, as estimated from 1H NMR (Scheme 3). Alcohols
14B and 15B were produced by decomposition of the Ac group
under the alkaline conditions. Many attempts to suppress the
undesired byproducts (14B, 15A, 15B) were unsuccessful. For
example, the use of Li2CO3 for the base resulted in no reaction
even at elevated temperature. It is notable that methanolysis
also produced unexpected vinyl carbonates (15A, 15B), which
were generated solely as the thermodynamically unfavorable
hydroxy group on the chiral auxiliary, which had been partially
generated by methanolysis, was performed. These trans-
formations successfully culminated to give rise to two products,
the desired diester 17 and (Z)-vinyl carbonate (15A) in 20%
and 30% yields (from 13), respectively, after chromatographic
purification. No other notable product was observed. The
results indicate that Pinnick oxidation, TMS-CHN2 treatment,
and acetylation have proceeded with high chemoselectivity.
3
(Z)-isomer, as judged from the JH,H (7.2 Hz). The product
ratio between desired aldehyde (14A, 14B) and undesired (Z)-
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Transformation of 15A into the reusable aldehyde such as 16 is
under investigation.
Chiral auxiliary was then oxidatively removed by ceric
ammonium nitrate (CAN) at 0 °C to afford N-monoalkylamide
18 in 90% yield (Scheme 5). The reaction proceeded as
Scheme 5. Final Elaboration toward (2R)-IKM-159 (2)
Figure 3. (2S)-IKM-159 (20), synthesized from diastereomeric
oxanorbornene 8′ (2S).
up to 4 h. Occasional and transient stereotyped behaviors such
as scratching were observed. Flaccidity was produced in all mice
tested at higher doses (>35 nmol/mouse), and the latency was
proportional to the dose injected. A lower dose of (2R)-IKM-
159 (7 nmol/mouse) produced variable responses, with one
animal losing the righting reflex and two others exhibiting
catalepsy and ataxia during recovery from drug effects. These
characteristic behavioral phenotypes were scored as follows:
flaccidness lasting for >4 h (score 7), 2−4 h (score 6), 1−2 h
(score 5), <1 h (score 4); catalepsy (score 3); loss of voluntary
movement (200% of control, score 2; 150−200% of control,
score 1; same as control, score 0). A dose−response curve
generated from the fitted curve from five data points using
three mice at each data point yielded an ED50 for (2R)-IKM-
159 of 6.3 (3.5−11.4) nmol/mouse.
Racemic IKM-159 was characterized as an AMPA receptor
antagonist.9 This observation was consistent with its behavioral
activity because other AMPA receptor antagonists, including
the noncompetitive benzodiazepine GYKI52466 and its
analogues27 as well as the competitive quinoxaline
ZK200775,28 induce muscle relaxation and ataxia similar to
IKM-159. To confirm that (2R)-IKM-159 was the enantiomer
responsible for AMPA receptor antagonism, we tested both
(2R) and (2S) compounds in whole-cell patch clamp
recordings of AMPA receptor excitatory postsynaptic currents
(EPSCs) from cultured rat hippocampal neurons (Figure 4). N-
Methyl-D-aspartic acid (NMDA) and γ-aminobutyric acid type
A (GABAA) receptor currents were inhibited using respective
antagonists (see the Supporting Information). Under these
conditions, AMPA receptors mediated very-large-amplitude
bursts of synaptic currents. Representative traces in the figure
show that AMPA receptor-mediated EPSCs were profoundly
reduced by (2R)-IKM-159 but were unaffected by the (2S)-
enantiomer (both tested at 20 μM), which was confirmed by
quantitation of the mean charge transfer during spontaneous
EPSCs ((2R), 16 7% of control charge transfer; (2S), 96
13%; n = 4 recordings for each compound; ∗, p < 0.05 in a
paired t test). Thus, (2R)-IKM-159 is responsible for both the
behavioral and pharmacological activity of the racemate.
Structure Determination. Racemic IKM-159 was postu-
lated to be a competitive AMPA receptor antagonist based on a
preliminary pharmacological analysis, although some uncer-
tainty in this conclusion existed because relatively high
concentrations (100 μM) of the racemate did not displace
radioligand from the orthosteric binding site on recombinant
AMPA receptors.9 In order to clarify the mechanism of action
and to gain insight into the molecular basis for the action of this
chemically novel antagonist, we used X-ray crystallography to
solve the structure of IKM-159 (from the racemic mixture)
expected from our previous synthetic study of artificial
glutamates,10 wherein the 4-methoxybenzyl group had been
removed at −10 °C in 71−80% yield. Of interesting note, we
found that the acetyl and methoxy groups in 17 were
indispensable for the reaction because the desmethoxy
counterpart of 17, derived from commercially available 2-
amino-2-phenylethanol, could not be deprotected under a
variety of conditions using reagents such as CAN, 2,3-dichloro-
5,6-dicyano-p-benzoquinone (DDQ), H2/Pd,25 O2/NaOH,26
and free radical. Similarly, the auxiliary could not be removed
with CAN before acetylation. Chromatographic and spectro-
scopic data of 18 were identical to those for the racemate.10,13
To purify thoroughly by chromatography, the Ns group in 18
was then replaced with a Boc group by two-step transformation
(PhSH, Cs2CO3, then Boc2O) to give N-Boc-amine 19 in 82%
yield.7,10 Finally, all protecting groups were simultaneously
removed by hydrolysis with 6 M hydrochloric acid at 65 °C to
give rise to (2R)-IKM-159 (2) in 92% yield. Chromatographic
and spectroscopic data of 2 were completely identical to those
of racemic IKM-159.13 The yield from 8 (2R) was 6.3% over 11
steps, and the total yield was 0.70% over 18 steps from (R)-(4-
hydroxyphenyl)glycine by way of 8 (2R). The enantiomeric
(2S)-IKM-159 (20) was also synthesized using the same
reaction sequence starting from (R)-(4-hydroxyphenyl)glycine
by way of 8′ (2S), in 1.5% yield over 18 steps (Figure 3). The
values of optical rotation for both enantiomers were found to
be consistent: [α]2D2.0 +19.8 (c 0.20, H2O) for (2R)-IKM-159
(2) and [α]2D0.9 −19.8 (c 0.16, H2O) for (2S)-IKM-159 (20).
Biological Evaluation. Biological activity of both (2R)- and
(2S)-IKM-159 was next tested in mice. The drug was injected
intracerebroventricularly at five doses ranging from 0.7 to 355
nmol/mouse. Injection of (2R)-IKM-159 (KT1-121-2, 355
nmol/mouse) caused flaccidness in mice, while the antipode
(2S)-IKM-159 (SG1-146-2, 355 nmol/mouse) did not show
significant activity. Though the behavioral phenotype observed
here was similar to that reported for the racemic mixture, a
dose-dependent profile of the compound was not previously
determined. The highest dose (355 nmol/mouse) caused mice
to become totally flaccid, and their righting reflex was lost for
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Figure 4. AMPA receptor excitatory synaptic currents (EPSCs) recorded from rat hippocampal neurons are inhibited by (2R)- but not (2S)-IKM-
159 (20 μM). Representative traces are shown on the left. Quantitation of the charge transfer in the presence of the IKM-159 compounds as a
percentage of their respective predrug activities is shown on the right. (2R)-IKM-159 reduced charge transfer significantly, whereas (2S) was inactive
(n = 4 for each condition; ∗, p < 0.05).
with the GluA2 LBD to 2.3 Å resolution (Table 1). Two dimers
(A/C and B/D) were found in the asymmetric unit of the
crystal, each dimer existing as a mixed dimer of one molecule
containing (2R)-IKM-159 in the binding site (molecules A and
B) and the other molecule a sulfate or phosphate ion (modeled
as sulfate; molecules C and D) (see Figure 5A). The D1−D2
domain opening in GluA2 with (2R)-IKM-159 relative to the
glutamate bound structure (PDB code 1FTJ,29 molB) is 20.1°
in molecule A and 20.0° in molecule B, similar to that seen in
the apo structure of GluA2 LBD (PDB code 1FTO).29 Partial
domain opening is seen in the two GluA2 molecules containing
a sulfate ion, i.e., 13.1° in molecule C and 11.9° in molecule D.
The distance between the two artificial Gly-Thr linker regions
connecting segments S1 and S2, as measured between Ile654 of
each molecule of the dimer, is 27.6 Å in the A/C dimer and
27.5 Å in the B/D dimer, which is comparable to the GluA2
LBD apo structure and other antagonist bound structures.30
The binding affinity (Ki) of racemic IKM-159 at GluA2 LBD,
which is predominantly a monomer in solution, was found to
be 0.21 0.02 mM (n = 3) with Hill coefficient nH = 1.03
0.09. Thus, the binding affinity of racemic IKM-159 at the
GluA2 glutamate binding site is low and comparable to that at
the GluA2(R)o full-length receptor (Ki = 0.56 0.07 mM, nH =
Ser675. Finally, the amide carbonyl oxygen forms a hydrogen
bond to the side chain hydroxyl group of Tyr753.
In the binding cavity of molecule A with (2R)-IKM-159
bound, three water molecules within 3.5 Å of (2R)-IKM-159
could be unambiguously modeled, one of which forms a direct
hydrogen bond to (2R)-IKM-159 (W1, Figure 5B). This water
molecule interacts with the distal carboxylate of (2R)-IKM-159
and is also seen in molecule B. The second water molecule
(W2) forms a close contact to the secondary amine of (2R)-
IKM-159. As only very weak density for this water molecule
was observed in molecule B, it was not modeled into the
binding site of molecule B. The third water molecule (W3) is
located in the vicinity of Glu423 and Thr707, which in agonist
bound structures form an important interdomain hydrogen
bond.30 In molecule B, this water molecule is not seen because
of a different conformation of Glu423 (Figure 6B). Five
additional water molecules were located within 3.5 Å of (2R)-
IKM-159 in molecule B, denoted W4−W8 (Figure 5C). As
only very weak density for these water molecules was observed
in molecule A, they were not modeled into the binding site of
molecule A. Water molecules W4−W6 form polar contacts to
(2R)-IKM-159: W4 to the distal carboxylate, W5 to the
carbonyl oxygen and the α-nitrogen, and W6 to the α-
carboxylate (last contact not shown in Figure 5C). Finally, W7
forms hydrogen bonds to Thr707 and Tyr723, and W8 forms
hydrogen bonds to Thr501 and Ser675 (contacts not shown).
A putative chloride ion is located in the binding site of
molecules A and B, ∼4 Å from (2R)-IKM-159 (not shown).
Finally, in the partially closed molecules C and D, the sulfate
ion forms a salt bridge to the side chain guanidinium group of
Arg506 in D1 and hydrogen-bonds to the backbone nitrogen
and side chain hydroxyl group of Ser675 in D2 (Figure 5E).
The structure of GluA2 LBD with (2R)-IKM-159 was
compared to other GluA2 LBD structures with antagonists and
was found to mostly resemble the GluA2 LBD structures with
(S)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-
isoxazolyl]propionic acid ((S)-ATPO)32 (PDB code 1N0T)
and ZK20077533 (PDB code 3KGC). Whereas the ring systems
1.01
0.07 (n = 3)). (2R)-IKM-159 makes several polar
contacts to GluA2 binding site residues (Figure 5B−D). These
contacts are the same in molecules A and B. The α-carboxylate
group of (2R)-IKM-159 forms contacts with the side chain
guanidinium group of Arg506 and the backbone nitrogen of
Thr501, similar to what has previously been seen for other
amino acid containing antagonists.30 The α-nitrogen atom in
(2R)-IKM-159 donates one hydrogen bond to the backbone
carbonyl oxygen of Pro499. The positively charged secondary
amine in the six-membered ring of (2R)-IKM-159 attracts the
γ-carboxylate group of Glu726 to form a salt bridge and also
forms an intramolecular salt bridge to the distal carboxylate of
(2R)-IKM-159. The distal carboxylate of (2R)-IKM-159 in
addition makes a hydrogen bond with the backbone nitrogen of
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different conformation of Glu423 in the structure with (2R)-
IKM-159 compared to the two other structures.
Table 1. Data Collection and Refinement Statistics of GluA2
LBD in Complex with (2R)-IKM-159
The observation that (2R)-IKM-159 binds to the GluA2
LBD structure supports the hypothesis that the compound acts
as a competitive AMPA receptor antagonist and is in
accordance with the observation that the (2R)-form is
responsible for both the behavioral and pharmacological activity
of IKM-159. Racemic IKM-159 inhibited both neuronal EPSCs
and rapid, glutamate-evoked whole-cell currents from recombi-
nant AMPA receptors containing GluA2 and GluA4 with
micromolar potency. The mechanism of action was suggested
to be competitive antagonism because equilibrium currents
were reduced in an agonist-concentration dependent manner,
but some uncertainty existed in this interpretation because a
physiologically active concentration of IKM-159 (100 μM)
failed to displace [3H]AMPA from GluA1, GluA2, or GluA4
AMPA receptors.9 Here we show that racemic IKM-159 has
quite a low binding affinity for full-length GluA2 (Ki of 0.56
mM), consistent with these earlier results. Similar divergences
between very low antagonist binding affinity and >10-fold
higher potency (for inhibition of receptor activation) have
previously been observed for other antagonists derived from
the natural product willardiine. For example, UBP282 exhibits a
binding affinity at the GluA2 LBD of 0.29 mM (the IC50 for
displacement of [3H]AMPA)34 but an IC50 of 10 μM for
reduction of the fast component of the dorsal-root-evoked
ventral root potential (fDR-VRP).35 UBP282 has a greater
number of contacts with the GluA2 binding site residues and is
more stable thermally compared to 6-cyano-7-nitroquinoxaline-
2,3-dione (CNQX) and 6,7-dinitroquinoxaline-2,3-dione
(DNQX), but secondary effects on the entropy of the system
as measured by isothermal titration calorimetry likely accounted
for the lower binding affinity.34 It is possible that similar effects
underlie the low apparent binding affinity of IKM-159. Finally,
we note that the structural and pharmacological data
supporting the competitive nature of antagonism by IKM-159
do not exclude the possibility that (2R)-IKM-159 has other
noncompetitive site(s) of action on AMPA receptors.
GluA2 LBD with
parameter
(2R)-IKM-159
Data
space group
P212121
unit cell dimensions
a (Å)
b (Å)
c (Å)
62.5
88.8
194.5
a
molecules (au)
4
b
resolution (Å)
29.2−2.3 (2.42−2.30)
48090 (6547)
3.9 (2.9)
98.3 (93.2)
5.6 (12.8)
9.5 (5.2)
no. of unique reflections
average redundancy
completeness (%)
c
Rmerge (%)
I/σ(I)
Refinement
amino acid residues
1036
2
(2R)-IKM-159
water/sulfate ions/chloride ions
446/6/8
18.5
d
Rwork (%)
e
Rfree (%)
25.8
rmsd on bond lengths (Å)/angles (deg)
0.007/1.0
100
residues in allowed regions of Ramachandran
f
plot (%)
B (Å2)
Wilson
21
protein (MolA/MolB/MolC/MolD)
(2R)-IKM-159
23/23/24/22
22/21
water/sulfate ions/chloride ions
23/46/49
a
b
au: asymmetric unit of the crystal. Values in parentheses correspond
c
to the outermost resolution shell. Rmerge = ∑h∑i|Ii(h) − ⟨I(h)⟩|/
d
∑h∑i(h), where Ii(h) is the ith measurement. Rwork = ∑hkl(||Fo,hkl| − |
Fc,hkl||)/|Fo,hkl|, where |Fo,hkl| and |Fc,hkl| are the observed and calculated
e
structure factor amplitudes. Rfree is equivalent to Rwork but calculated
with reflections omitted from the refinement process (5% of
reflections omitted). The Ramachandran plot was calculated
f
according to PROCHECK.31
Mixed dimers of GluA2 LBD have previously been seen in
which glutamate is present in one molecule of the dimer and an
antagonist33,36 or an agonist37 is found in the other molecule of
the dimer. To our knowledge, this is the first time that the
structure of a mixed dimer of GluA2 has been solved with an
antagonist molecule located in one molecule of the dimer
(exhibiting an open domain structure similar to the apo
structure and other antagonist structures) whereas the other
molecule of the dimer occurs in a partially closed domain
structure in which a sulfate ion bridges lobes D1 and D2. As a
result, the sulfate-bound molecule assumes a structure slightly
more open than that containing the partial agonist kainate.29
The mixed dimer structure shows the same Ile654−Ile654
linker−linker distance as the GluA2 apo structure (27.6 Å).
Thus, binding of only one (2R)-IKM-159 molecule within the
dimer seems to be enough to capture the dimer in a closed ion
channel state. For agonists, the ion channel pore opens when
two sites are occupied and the current increases as more
binding sites are occupied with agonist.38 By analogy, the mixed
GluA2 dimer with (2R)-IKM-159 suggests that in some cases
only two subunits need to be occupied by antagonist to block
receptor function.
in the three antagonists are located differently, the distal
carboxylate of (2R)-IKM-159 is located in the same region of
lobe D2 (Ser675 and Thr676) as the phosphonate groups of
(S)-ATPO and ZK200775 (Figure 6). However, the
phosphonate groups of (S)-ATPO and ZK200775 form a
more extensive network of interactions than the distal
carboxylate of (2R)-IKM-159. In (S)-ATPO (Figure 6C) and
ZK200775 (Figure 6D) the phosphonate group forms a close
contact to the γ-carboxylate of Glu726, whereas in the structure
with (2R)-IKM-159 (Figure 6B) Glu726 interacts with the
secondary amine of (2R)-IKM-159. The side chain of Glu726
in the structure with (S)-ATPO adopts a conformation
resembling the conformation in structures with agonists30 and
thereby forms polar contacts to the ammonium group of (S)-
ATPO and to Tyr753. As Glu726 points away from Tyr753 in
the structures with (2R)-IKM-159 and ZK200775, these
hydrogen bonds are not present in these structures. Instead,
the carbonyl oxygen of (2R)-IKM-159 makes a hydrogen bond
to Tyr753, and in the structure with ZK200775 a contact is
formed between Tyr753 and a water molecule. In addition, the
binding of (2R)-IKM-159 sequesters the water molecule (W3)
bridging Glu423 and Thr707, which is not present in the
structures with (S)-ATPO and ZK200775. This leads to a
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Figure 5. Crystal structure of a mixed dimer of GluA2 LBD containing the antagonist (2R)-IKM-159 in one molecule of the dimer and a potential
sulfate ion in the other molecule of the dimer. (A) Cartoon structure of GluA2 LBD, illustrating the different domain openings induced by (2R)-
IKM-159 and a sulfate ion, respectively. Also, the Ile654−Ile654 linker−linker distance is indicated. GluA2 LBD with (2R)-IKM-159 (molecule A) is
shown in violet and with the sulfate ion (molecule C) in cyan. (2R)-IKM-159 and the sulfate ion are shown in spheres representation with carbon
atoms colored dark gray, nitrogen atoms blue, oxygen atoms red, and sulfur atom yellow. Lobe D1 is located at the top of the figure and lobe D2 at
the bottom. (B, C) Zoom on the ligand-binding site of GluA2 LBD with (2R)-IKM-159 and 2Fo − Fc omit map at 1σ carved around the ligand at 1.4
Å radius: (B) molecule A; (C) molecule B. Residues and water molecules within 3.5 Å from (2R)-IKM-159 are included. GluA2 is shown in violet
and (2R)-IKM-159 in dark gray. Heteroatoms are colored as in (A). Potential hydrogen bonds within 3.1 Å from (2R)-IKM-159 to water molecules
(red spheres) and surrounding residues are shown as black dashed lines. (D) 2D ligand−receptor interaction plot between GluA2 LBD (molecule A)
and (2R)-IKM-159. The GluA2 residues are shown as violet circles, and contacts from (2R)-IKM-159 to the receptor are shown as dotted arrows as
calculated by the program MOE [Molecular Operating Environment (MOE), version 2011.10; Chemical Computing Group Inc. (1010 Sherbooke
Street West, Suite No. 910, Montreal, Quebec, H3A 2R7, Canada), 2011]. The water molecule in contact with (2R)-IKM-159 is shown as a white
circle and its contact as a dotted line. (E) 2D ligand−receptor interaction plot between GluA2 LBD (molecule C) and the sulfate ion. The GluA2
residues are shown as cyan circles and contacts between the sulfate ion and the receptor as dotted arrows.
compound. Final purity of >95% was confirmed by HPLC. For
details, see the Supporting Information.
Procedures for all chemical syntheses are described in the
Supporting Information.
CONCLUSION
■
Here we provide a route to the synthesis of (2R)-IKM-159, the
first characterized member of a novel type of AMPA receptor
antagonist. The (2R)-IKM-159 was shown to be responsible for
the biological activity of the compound, and X-ray structure
determination supported that (2R)-IKM-159 acts as a
competitive antagonist at GluA2. Molecules with this
pharmacological activity represent critical tools in neuroscience
and continue to be explored as potential therapeutic agents.
Generation of new routes of synthesis for IKM-159 and related
isomers will facilitate structure−activity relationships that could
lead to antagonists with novel activity profiles in future
structural and biological studies.
Behavioral Assays. Mice assay was performed under approval by
the Ethical Committee of Experimental Animal Care at Hokkaido
University, Japan. An aqueous solution (20 μL) of sample was injected
intracerebroventricularly in male ddY mice of 3−4 weeks (22.4 0.96
g, Japan SLC Inc., Hamamatsu) as described previously.3 Behaviors
were observed for up to 8 h, and dose-dependent characteristic
behavioral phenotypes were scored as follows: flaccidness (complete
loss of righting reflex) lasting for >4 h (score 7), 2−4 h (score 6), 1−2
h (score 5), <1 h (score 4); catalepsy (score 3); loss of voluntary
movement as measured by time that a mouse stays still on a stage 6.5
cm diameter and 4.5 cm height (200% of control, score 2; 150−200%
of control, score 1; less than 150% of control, score 0). A dose−
response curve was generated from the fitted curve from five data
points (355, 71, 35, 7, 0.7 nmol/mouse, n = 3 at each dose). Data were
analyzed by using the computer software GraphPad Prism.
EXPERIMENTAL SECTION
■
(2R)-IKM-159 (2, KT1-121-2) and (2S)-IKM-159 (20, SG1-146-2)
were purified by HPLC using the following conditions: Cosmosyl 20
mm × 250 mm, water 90%, CH3CN 10% with 0.05% TFA. The peak
eluting at 15 min was collected and lyophilized to give pure
Electrophysiology. Whole-cell patch clamp recordings were
carried out from hippocampal neurons isolated from E18 rat pups
and cultured for 2−3 weeks under standard conditions, as described
previously.9 To isolate AMPA receptor EPSCs, 50 μM D-2-amino-5-
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Figure 6. Comparison of the structures of GluA2 LBD with (2R)-IKM-159, (S)-ATPO, and ZK200775, with zoom on the ligand-binding site. The
structures have been superimposed on lobe D1 residues. (A) Overlay of (2R)-IKM-159 (molecule A, dark gray), (S)-ATPO (PDB code 1N0T,
molecule A, beige) and ZK200775 (PDB code 3KGC, molecule B, cyan). Nitrogen atoms are blue, oxygen atoms red, and phosphor atoms orange.
(B) Structure of GluA2 LBD with (2R)-IKM-159 (molecule A). Selected protein residues of lobe D1 are shown in violet and of lobe D2 in dark
violet. Glu423 for which the side chain conformation differs in molecules A and B is also shown for molecule B (dark gray). (2R)-IKM-159 is shown
in dark gray sticks representation, water molecules as red spheres, and potential hydrogen bonds within 3.1 Å as black dashed lines. (C) Structure of
GluA2 LBD with (S)-ATPO (molecule A). The representation mode is the same as in (B) except that (S)-ATPO is shown in beige. The contacts
between Glu726 and (S)-ATPO are slightly longer than 3.1 Å (3.2−3.3 Å). (D) Structure of GluA2 LBD with ZK200775 (molecule B). The
representation mode is the same as in (B) except that ZK200775 is shown in cyan.
phosphonovaleric acid (D-APV), 10 μM bicuculline methiodide, and
50 μM picrotoxin were included in the extracellular bathing solution.
CNQX (50 μM) was applied at the conclusion of all recordings to
verify that the recorded currents arose from AMPA receptors. Charge
transfer during AMPA receptor activations, which in most cases
occurred as compound bursts of events, was analyzed in Mini-Analysis,
version 6.03 (Synaptosoft) and Clampfit 10 (MDS Software). IKM-
159 molecules were bath-applied for 5 min after establishing a stable
control recording. Effects of the compounds on AMPA receptor
EPSCs were calculated as the charge transfer during the last minute of
IKM-159 application relative to that during the last minute of the
control period before compound application, expressed as a
percentage. A paired Student’s t test was used to test statistical
significance.
Binding Affinity. Radioligand binding assays were conducted
using (RS)-[5-methyl-3H]AMPA (45.8 Ci/mmol) (PerkinElmer,
MA), as previously detailed.39 Recombinant baculovirus containing
full-length rat GluA2(R)o was used for infection of Sf9 insect cells, and
the membranes were utilized for radioligand binding assays. Non-
specific binding at receptors was determined in the presence of 1 mM
(S)-glutamate. IKM-159 affinity at full-length GluA2(R)o and GluA2
LBD was determined from competition studies using 1 μM to 2 mM
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ligand. Competition data were analyzed using Grafit, version 3.00
(Erithacus Software Ltd., Horley, U.K.), as previously detailed.40
Crystallization. The GluA2 LBD (GluR2-S1S2J)29 comprises a N-
terminal Gly-Ala cloning remnant, amino acid residues 413−527 from
segment S1 of the membrane-bound receptor, a Gly-Thr linker, and
residues 653−796 from segment S2 (numbering with signal peptide).
The protein was expressed and purified as described previously41,42
except that (S)-aspartate was present during purification instead of
(S)-glutamate. GluA2 LBD in complex with racemic IKM-159 was
crystallized using the hanging drop vapor diffusion method at 7 °C.
The drop contained 1 μL of protein solution (5.9 mg/mL GluA2 LBD
and 6 mM racemic IKM-159 in 10 mM N-(2-hydroxyethyl)piperazine-
N′-ethanesulfonic acid (HEPES), pH 7.0, 20 mM NaCl, and 1 mM
ethylenediaminetetraacetic acid (EDTA)) and 1 μL of reservoir
solution of 20% PEG4000, 0.1 M lithium sulfate, and 0.1 M
phosphate−citrate, pH 4.5. The reservoir volume was 500 μL. The
crystals appeared within 1 week, and they were flash cooled with liquid
nitrogen after immersion in the reservoir solution containing 20%
glycerol.
IKM-159 on mice. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +81-45-787-2403. Fax: +81-45-787-2403. E-mail:
moikawa@yokohama-cu.ac.jp.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank Prof. S. Nishiyama (Keio University, Japan)
for access to the HRMS facility. Heidi Peterson is thanked for
help with production of the GluA2 protein. The MAX-Lab,
Lund, Sweden, is thanked for providing beam time, and
GluTarget and Danscatt are thanked for financial support (to
L.J., D.S.P., K.F., and J.S.K.). G.T.S. acknowledges support from
the National Institute of Neurological Diseases and Stroke
(Grant 2R01NS44322). This work was partly supported by the
grant for 2010−2012 Strategic Research Promotion (Nos.
T2202, T2309, T2401) of Yokohama City University, Japan.
Financial support (a Grant-in-Aid for Scientific Research
(Grant 21603004 to M.O, Grants 2238011401 and
231850301 to R.S.)) from the Ministry of Education, Science,
Sports, Culture and Technology, Japan, is also gratefully
acknowledged.
X-ray Structure Determination. Single-crystal X-ray analysis of 8
(2R) was performed on a Bruker SMART APEX CCD area (graphite-
monochromated Mo Kα radiation (λ = 0.710 73 Å) with a nitrogen
flow temperature controller. Data collection was performed at 183 K.
Empirical absorption corrections were applied using the SADABS
program [Sheldrick, G. M. SADABS; University of Gottingen:
̈
Gottingen, Germany, 1996]. The structure was solved by direct
̈
methods (SHELXS-97)43 and refined by full-matrix least-squares
calculations on F2 (SHELXL-97)43 using the SHELX-TL program
package. Non-hydrogen atoms were refined anisotropically. Hydrogen
atoms were fixed at calculated positions and refined using a riding
model. Crystallographic data of the structure are summarized in the
Supporting Information. CCDC-900962 contains the supplementary
crystallographic data for this paper. The data can be obtained free of
charge from The Cambridge Crystallographic Data Centre via http://
www.ccdc.cam.ac.uk/data_request/cif.
ABBREVIATIONS USED
■
Ac2O, acetic anhydride; AMPA, (S)-2-amino-3-(3-hydroxy-5-
methyl-4-isoxazolyl)propionic acid; D-APV, D-2-amino-5-phos-
phonovaleric acid; ATPO, 2-amino-3-[5-tert-butyl-3-(phospho-
nomethoxy)-4-isoxazolyl]propionic acid; Boc, tert-butoxycar-
bonyl; CAN, ceric ammonium nitrate; CNQX, 6-cyano-7-
nitroquinoxaline-2,3-dione; CNS, central nervous system;
DDQ, 2,3-dichloro-5,6-dicyano-p-benzoquinone; DMAP, 4-
dimethylaminopyridine; DNQX, 6,7-dinitroquinoxaline-2,3-
dione; EDTA, ethylenediaminetetraacetic acid; EPSC, excita-
tory postsynaptic current; GABA, γ-aminobutyric acid; HEPES,
N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid; iGluR,
ionotropic glutamate receptor; KA, kainic acid; LBD, ligand-
binding domain; NMDA, N-methyl-D-aspartic acid; Ns, 2-
nitrobenzenesulfonyl; TMS-CHN2, trimethylsilyldiazomethane
Data for the crystal structure of GluA2 LBD in complex with (2R)-
IKM-159 were collected at the I911-3 beamline (MAX-Lab, Lund,
Sweden) and processed using XDS44 and the CCP4 suite of
programs.45 The structure was solved by molecular replacement
using PHASER46 within CCP4 using the structure of GluA2 LBD with
(S)-ATPO [PDB code 1N0T, molA]32 as a search model. A clear
solution comprising four molecules forming two mixed dimers (A/C
and B/D, respectively) was obtained. Molecules A and B contain (2R)-
IKM-159, and molecules C and D contain sulfate or phosphate at the
glutamate binding site (has been modeled as sulfate). Afterward, the
amino acid residues of GluA2 were automatically modeled into the
electron density using ARP/wARP47 within CCP4 except for a few
residues that were manually built using COOT.48 The output model
was further refined in PHENIX.49 Between every refinement step the
structure was checked and modified in COOT. The ligand coordinates
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