Discovery of Imidazo[1,2- a]pyrazines and Pyrazolo[1,5- c]pyrimidines as TARP γ-8 Selective AMPAR Negative Modulators

Article abstract of DOI:10.1021/acsmedchemlett.8b00599

This report discloses the discovery and characterization of imidazo[1,2-a]pyrazines and pyrazolo[1,5-c]pyrimidines as selective negative modulators of α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors (AMPARs) associated with transmembrane AMPAR regulatory protein γ-8. Imidazopyrazine 5 was initially identified as a promising γ-8 selective high-throughput screening hit, and subsequent structure-activity relationship optimization yielded subnanomolar, brain penetrant leads. Replacement of the imidazopyrazine core with an isosteric pyrazolopyrimidine scaffold improved microsomal stability and efflux liabilities to provide 26, JNJ-61432059. Following oral administration, 26 exhibited time- and dose-dependent AMPAR/γ-8 receptor occupancy in mouse hippocampus, which resulted in robust seizure protection in corneal kindling and pentylenetetrazole (PTZ) anticonvulsant models.

Full text of DOI:10.1021/acsmedchemlett.8b00599

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Letter
Discovery of Imidazo[1,2-a]pyrazines and Pyrazolo[1,5-
c]pyrimidines as TARP #-8 Selective AMPAR Negative Modulators
Brad M. Savall, Dongpei Wu, Devin Maun Swanson, Mark Seierstad, Nyantsz Wu, Jorge
Vives Martinez, Beatriz Garcia Olmos, Brian Lord, Kevin J Coe, Tatiana Koudriakova,
Timothy W Lovenberg, Nicholas I. Carruthers, Michael P. Maher, and Michael K. Ameriks
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00599 • Publication Date (Web): 26 Dec 2018
Downloaded from http://pubs.acs.org on December 27, 2018
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Discovery of Imidazo[1,2-a]pyrazines and Pyrazolo[1,5-c]pyrimidines
as TARP -8 Selective AMPAR Negative Modulators
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‡
†
†
†
†
Brad M. Savall , Dongpei Wu , Devin M. Swanson , Mark Seierstad , Nyantsz Wu , Jorge Vives


†
†
†
Martinez ,
Beatriz García Olmos , Brian Lord , Kevin Coe , Tatiana Koudriakova , Timothy
†
†
†
†
W. Lovenberg , Nicholas I. Carruthers , Michael P. Maher , and Michael K. Ameriks*
†Janssen Research & Development L.L.C., 3210 Merryfield Row, San Diego, CA 92121 United States
‡Sanford Burnham Prebys Medical Discovery Institute, 10901 N. Torrey Pines Rd., La Jolla, CA 92037 United States
Eurofins-Villapharma Research S.L., Parque Tecnológico de Fuente Alamo, Carretera El Estrecho-Lobosilo, Km. 2,5, E-
30320, Fuente Alamo (Murcia), Spain
KEYWORDS AMPA, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, AMPA, TARP -8, transmembrane AMPA
receptor regulatory protein, auxiliary subunit, glutamate, hippocampus, epilepsy, glutathione, autoradiography
ABSTRACT: This report discloses the discovery and characterization of imidazo[1,2-a]pyrazines and pyrazolo[1,5-c]pyrimidines
as selective negative modulators of -amino-3-hydroxy-5-methylisoxazole-4-propionate receptors (AMPARs) associated with
transmembrane AMPAR regulatory protein (TARP) -8. Imidazopyrazine 5 was initially identified as a promising -8 selective HTS
hit, and subsequent SAR optimization yielded sub-nanomolar, brain penetrant leads. Replacement of the imidazopyrazine core with
an isosteric pyrazolopyrimidine scaffold improved microsomal stability and efflux liabilities to provide 26, JNJ-61432059. Following
oral administration, 26 exhibited time- and dose-dependent AMPAR/-8 receptor occupancy in mouse hippocampus, which resulted
in robust seizure protection in corneal kindling and pentylenetetrazole (PTZ) anticonvulsant models.
Glutamate is the primary excitatory neurotransmitter in the
central nervous system (CNS), where it functions as an agonist
at several metabotropic (mGluR) and ionotropic (iGluR)
receptors. The -amino-3-hydroxy-5-methyl-4-isoxazole
propionic acid (AMPA) subtype of ionotropic glutamate
HO
O
O
Ph
N
OCF3
N
CN
H
N
H
N
N
N
receptors mediates fast synaptic transmission within the CNS
O
O
N
Cl
and plays a critical role in modulating synaptic plasticity.1
N
H
S
AMPA receptors (AMPARs) are assembled as tetramers of
pore-forming subunits (GluA1-GluA4) together with a host of
accessory proteins that work in concert to regulate receptor
trafficking and pharmacology. Most, if not all, AMPARs are
associated with transmembrane AMPAR regulatory proteins
2
3
1
perampanel
no TARP selectivity
JNJ-55511118
TARP -8 selective
LY3130481(CERC-611)
TARP -8 selective
Figure 1. Representative AMPAR negative modulators
(
TARPs), which are classified by sequence homology and
2
function as Type I (-2, -3, -4, -8) or Type II (-5, -7).
Whereas GluA subunits are widely distributed as heteromeric
complexes throughout the brain, several TARPs exhibit more
discrete expression patterns. In particular, TARP -8 is highly
enriched in the hippocampus as well as certain subregions of
the forebrain, with minimal localization in the cerebellum or
Several classes of AMPAR negative allosteric modulators
have been disclosed previously (Figure 1). These include non-
competitive AMPAR antagonists, such as perampanel (1),
which bind at the interface between the ion-channel and the
8
ligand binding domain. Although perampanel is an FDA-
approved adjunctive treatment for uncontrolled partial-onset
seizures, CNS-related side-effects such as dizziness, sedation,
and falling have been reported at therapeutically relevant
3
brainstem. Accordingly, in rodents, selective negative
modulation of AMPARs associated with -8 has been shown to
9
attenuate hyperexcitability in the hippocampus without direct
doses. Perampanel is not a TARP-selective antagonist, and
4
-6
effects on the midbrain or hind brain. Since excitatory
neurotransmission plays a key role in seizure generation and
propagation, selective AMPAR/TARP -8 negative modulators
are expected to function as effective anticonvulsants with an
improved therapeutic index relative to current standards of care,
inhibition of AMPARs associated with stargazin (-2), the
primary TARP expressed in cerebellum, may contribute to the
1
0
motor impairment observed in patients. Recently, several
AMPAR negative modulators selective for -8 have been
5
,11
reported,
including
JNJ-55511118
(2)
and
7
12
which affect synaptic transmission throughout the brain.
LY3130481/CERC-611 (3). These compounds appear to
partially disrupt an important protein-protein interaction
between the TARP and the pore-forming subunit of the ion-
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channel.4-6,13 Herein, we describe the discovery, optimization,
and in vivo characterization of a structurally distinct class of
substituted imidazo[1,2-a]pyrazine and pyrazolo[1,5-
1
2
3
4
5
6
7
8
9
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
Table 1. Initial imidazo[1,2-a]pyrazine SAR
c]pyrimidine-based selective AMPAR/-8 negative modulators.
O
N
A high-throughput screening (HTS) campaign identified
1
2
+
N
N
compounds that blocked glutamate-induced Ca flux in HEK-
X
N
2
93 cells expressing a fusion protein between -8 and the
3
5
GluA1o “flop” splice variant. Confirmed hits were counter-
screened in heterologous cells co-transfected with GluA1o and
R
Cmpd
R
X
GluA1/-8
GluA1/-2
LLE

-2. From this effort, benzimidazole 4 and imidazo[1,2-
a
b
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
pIC50
pIC50
a]pyrazine 5 emerged as preliminary leads due to their
encouraging potency and selectivity for -8 (Figure 2).
2+
6
7
8
Cl
F
8.0 ± 0.3
7.9 + 0.1
6.3 ± 0.4
< 5.0
< 5.0
< 5.0
3.7
4.2
2.1
Whereas 4 moderately inhibited Ca flux in cells expressing
OH
OH
GluA1/-2 (pIC50 = 5.9), 5 was inactive towards -2 at the
highest concentrations tested (100 M). Furthermore, whole
cell electrophysiology confirmed that 5 inhibited time-
dependent AMPA responses to glutamate in acutely dissociated
rat hippocampal neurons, but not rat cerebellar Purkinje
neurons (Supplementary Figure 1). Specifically, in
hippocampal neurons, a saturating concentration of 5 (10 M)
partially reduced peak currents (~50% inhibition) and
completely blocked steady-state currents evoked by 10 mM L-
glutamate, which is similar in magnitude to the effects observed
with previously disclosed AMPAR/-8 selective negative
F
OCH3
9
F
F
F
7.2 ± 0.3
< 5.0 (n = 1)
9.3 ± 0.1
< 5.0
< 5.0
< 5.0
4.1
<1.6
6.4
NH2
1
1
0
1
5
,6
modulators.
NHAc
O
N
O
N
H
N
N
F
N
N
Cl
N
1
2
F
9.2 ± 0.2
< 5.0
5.5
N
N
H
apIC50 measured in a FLIPR assay using HEK-293 cells expressing a human
GluA1o--8 fusion construct; unless noted, all data are the result of at least three
assays run in triplicate with the mean value and standard deviation reported.
N
OH
N
5
OCH3
b
pIC50 measured in a FLIPR assay using HEK-293 cells expressing human
4
GluA1o co-transfected with -2.
GluA1/-8 pIC50 = 6.7
GluA1/-2 pIC50 = 5.9
GluA1/-8 pIC50 = 6.9
GluA1/-2 pIC50 < 5.0
Although 11 and 12 displayed similar -8 potency, cross-
reactivity with numerous CYP isozymes (i.e. 2C9 IC50 = 500
nM) prevented further progression of indazole 12.
Consequently, the oxindole group was selected as a preferred
arene at C-3 and held fixed during further SAR optimization
around the C-8 position (Table 2). Several substituted cyclic
amines maintained the promising -8 potency of the parent
morpholine 11, and 4-fluoropiperidine 14 was especially
impressive in this regard (IC50 = 100 pM). In addition, these
morpholine replacements provided marginal improvements in
both human and rat liver microsome stability, although often at
the expense of increased Pgp-mediated efflux in a MDCK-
MDR1 permeability assay. Subsequent in vivo profiling in rat
revealed that brain penetration correlated well with in vitro
efflux ratios, as the compounds with the lowest predicted efflux
(11 and 14) also exhibited the highest unbound brain partition
Figure 2. AMPAR/-8 selective HTS hits
Comparison of the two HTS leads suggested merging
structural elements through replacement of the substituted
pyrazole in 5 with the phenol in 4 to generate hybrid analog 6
(Table 1). Gratifyingly, imidazopyrazine 6 was 10-fold more
potent than 5 and maintained exquisite selectivity over -2.
Incorporation of a para-fluorophenyl group in the 2-position (7)
retained potency and improved lipophilic ligand efficiency
relative to para-chlorophenyl 6. Consequently, the para-
fluorophenyl moiety was held constant at C-2 while scanning
aromatic groups in the 3-position. The dramatic difference in
potency between phenol 7 (pIC50 = 7.9) and methoxyphenyl 8
(pIC50 = 6.3) implicated the importance of a hydrogen-bond
donor in the para position. Relative to the phenol, aniline 9 was
less potent (pIC50 = 7.2), and acylation of the amine (10)
completely ablated activity. However, constraining the
hydrogen bond donor within a ring dramatically improved
potency, as both oxindole 11 and indazole 12 were sub-
nanomolar compounds. In all cases, no inhibition of -2 was
ever observed.
p
coefficients (K u,u > 0.3). Unfortunately, all imidazopyrazines
profiled in vivo suffered from low to moderate bioavailability
and high clearance, sometimes greater than hepatic blood flow
(i.e. 14, Cl = 86 mL/min/kg).
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Table 2. SAR and profile of 8-substituted imidazo[1,2-a]pyrazine derivatives
1
2
3
4
5
6
7
8
9
NR2
8
1
N
N
F
N
3
N
O
H
rat PK parameterse
rat BBBf
p
[brain]/[plasma] K u,u
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
d
GluA1/-8 GluA1/-2 HLM/RLM
MDCK-MDR1
Cl
V
ss
%F
Cmpd NR
2
a
b
c
-6
pIC50
pIC50
stability
ratio/(A-B) x10 cm/s
(mL/min/kg)
(L/kg)
(ng/g) / (ng/mL)
(brain)
O
1
1
1
3
9.3 ± 0.1
9.4 ± 0.4
< 5.0
< 5.0
0.8 / 0.8
0.7 / 0.4
2.8 / 13
59 + 12
3.2 + 0.5 35 + 15
2.4 + 0.2 25 + 3
205 / 105
0.33
N
OH
86 / 1.0
1.6 / 0.5
59 + 11
86 + 13
39 / 270
0.03
0.56
N
F
14
10.0 ± 0.2
< 5.0
0.7 / 0.7
5.9 + 0.8 28 + 9
231 / 137
N
H
N
O
1
1
5
6
9.3 ± 0.2
9.4 ± 0.2
< 5.0
< 5.0
0.7 / 0.7
0.6 / 0.5
72 / 0.6
78 / 0.7
62 + 11
64 + 4
2.2 + 0.2
8 + 2
11 / 214
25 / 130
0.01
0.06
N
Ac
N
2.5 + 0.5 37 + 7
N
apIC50 measured in FLIPR assay using HEK-293 cells expressing a human GluA1o--8 fusion construct; all data are the result of at least three assays run in triplicate
with the mean value and standard deviation reported. pIC50 measured in a FLIPR assay using HEK-293 cells transiently expressing human GluA1o co-transfected with
2 (n = 1, run in triplicate). Stability in human and rat liver microsomes at 1 M; data are reported as extraction ratios. Apparent permeability, reported as ratio of B to
2
A direction/ A to B direction. Compounds dosed as solutions in 20% HP--CD (cmpds 13,15,16), 30% SBE-CD (cmpd 11), or 50% PEG400/H O (cmpd 14) at 1 mg/kg
b
c
d

e
f
(
i.v.) and 5 mg/kg (p.o.) in Sprague Dawley rats (n=3/group). Compounds dosed as solutions in 20% HP--CD at 10 mg/kg (p.o) Sprague Dawley rats (n=2/group).
Details for all assay conditions are provided in the supplemental information.
Scheme 1. Synthesis of imidazo[1,2-a]pyrazines 5-16a
NR2
O
Cl
NR2
Cl
Cl
N
N
N
d
N
NH2
N
c
N
a,b
X
N
X
X
N
+
N
N
N
X
R1
Br
Br
X = Cl or F
5-16
18
1
7
a
o
Reagents and conditions: (a) Et NPh, CH CN or CH CH CN, reflux, 50-54%; (b) NBS, CH Cl , 69-85%; (c) R NH, Et N or iPrNEt , CH CN, 80 C, 75-87%;
2
3
3
2
2
2
2
3
2
3
1
o
(
d) R B(OH) , Pd(PPh ) , 1:1 dioxane/1M Na CO , wave, 110 C, 57-86%.
2 3 4 2 3
Imidazo[1,2-a]pyrazines 5-16 were prepared using the
an isosteric pyrazolo[1,5-c]pyrimidine scaffold showed the
most promise (Scheme 2). This 6,5-heterocycle was assembled
through an initial condensation between 4-methyl-2-
(methylthio)pyrimidine and para-fluorophenyl ethyl benzoate
to give ketone 19, followed by conversion to oxime 20 and an
iron-mediated ring closure to form the core 21. Bromination
at the 3-position (22) preceded sulfide oxidation to provide an
important sulfone intermediate (23). Functionalization at C-7
synthetic sequence shown in Scheme 1.14 Specifically,
condensation of 2-amino-3-chloropyrazine with -chloro-para-
fluoro-acetophenone, followed by bromination, afforded a key
dihalo
imidazopyrazine
intermediate
17.
Selective
1
5
displacement of the 8-chloro group was achieved by heating
with an amine to deliver 18, which was then functionalized at
the 3-position through Suzuki couplings.
To mitigate the poor PK properties of the imidazopyrazine
series, several core replacements were investigated. Ultimately,
N
was accomplished using S Ar displacements with a variety of
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amines (24), and the C-3 oxindole was installed via a dppf-
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1
2
3
4
5
6
7
8
9
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
mediated Suzuki reaction to afford the final products 25-29.
Scheme 2. Synthesis of pyrazolo[1,5-c]pyrimidines 25-29a
SCH3
SCH3
SCH3
SCH3
O
OH
N
N
N
O
b
N
N
N
c
N
N
d
N
N
a
EtO
F
+
CH3
F
F
F
21
20
19
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
NR2
Pin B
2
SCH3
N
SO CH3
NR2
O
N
2
N
N
N
F
N
e
N
N
f
N
N
N
N
N
H
F
F
F
g
Br
Br
Br
23
24
2
2
N
O
H
5-29
2
a
o
o
o
Reagents and conditions: (a) LiHMDS, THF, rt -> 45 C, 85%; (b) H NOH-HCl, 3M NaOH, MeOH, 65 C, 50%; (c) Et N, TFAA, 0.3 eq FeCl , DME, 80 C, 53%;
2
3
2
o
o
o
(
d) NBS, CH CN, rt, 82%; (e) mCPBA, NaHCO , CH Cl , 0 C, 100%; (f) R NH, DMA, 100 C, 40-67%; (g) cat Pd(dtbpf)Cl , NaHCO , dioxane/water, 110 C,
3 3 2 2 2 2 3
1
7-76%.
Table 3. SAR and profile of pyrazolo[1,5-c]pyrimidines 25-29
SAR and select in vitro data for pyrazolopyrimidines 25-29
are shown in Table 3. Although switching from the
imidazopyrazine to pyrazolopyrimidine core resulted in a loss
of potency for several compounds (see Table 2; compare 14/27,
as well as 15/28), other homologs retained activity (compare
11/25, 13/26, and 16/29). Furthermore, a majority of the
pyrazolopyrimidine analogs displayed lower efflux ratios, as
well as improved stability in human liver microsomes, relative
to their imidazopyrazine matched pairs. Hydroxypiperidine 26
NR
2
7
1
N
N
N
F
3
N
O
H
Cmpd
NR
2
GluA1/-8 HLM/RLM
MDCK-MDR1d
efflux ratio/(A-B)
(
JNJ-61432059) appeared especially promising, and further
a,b
c
pIC50
stability
characterization confirmed that this compound was highly
selective for AMPAR/-8. When tested at 10 M, 26 did not
inhibit glutamate-induced calcium-flux in heterologous cells
that co-expressed AMPARs with any TARP other than -8
O
N
2
5
9.3 ± 0.3
9.7 ± 0.5
0.8 / 0.6
0.6 / 0.5
1.9 / 8.5
13 / 3.3
(
Supplementary Table 1). In addition, no cross-reactivity was
OH
noted when 26 was screened against a panel of 52 receptors, ion
channels, and transporters using radioligand displacement
assays (<50% inh @ 1 M; Eurofins/Cerep, Poitiers, France).
Furthermore, at concentrations as high as 10 M, 26 did not
displace [ H]dofetilide in a hERG binding assay, although
inhibition of CYPs 2C8 and 2C9 were noted at lower
concentrations (IC50s = 3.0 and 1.9 M, respectively).
Due to its encouraging in vitro profile, 26 was further
evaluated in vivo. After oral dosing at 10 mg/kg in rats, 26
distributed into the brain (K u,u = 0.4) despite low plasma
exposures (Cmax = 26 ng/mL) and high clearance (Cl = 57
mL/min/kg). The high in vivo clearance was unexpected based
on the extraction ratio estimated from rat liver microsomes. A
subsequent cross-species metabolite ID study revealed that the
higher than anticipated clearance was likely due to a rat-specific
UGT-mediated glucuronidation. Specifically, when 26 was
26
N
F
3
e
2
2
7
8
8.3 ± 1.2
0.6 / 0.6
ND / 1.2
N
H
N
O
8.3 ± 0.7
9.6 ± 0.3
0.7 / 0.6
0.6 / 0.5
135 / 0.3
34 / 2.1
p
N
Ac
N
29
N
apIC50 measured in a FLIPR assay using HEK-293 cells expressing a human
GluA1o--8 fusion construct; all data are the result of at least three assays run in
triplicate with the mean value and standard deviation reported. All compounds
tested had pIC50 <5 at GluA1/-2. Stability in human and rat liver microsomes
at 1 M; data are reported as extraction ratios. Apparent permeability (x10
cm/s) reported as ratio of B to A direction/ A to B direction. Not determined
incubated with hepatocytes for 4h at 37 °C, the O-glucuronide
was detected as the major metabolite in rat, but only as a minor
metabolite in human, mouse, dog, and monkey hepatocytes.
This species-specific metabolism was further supported by
mouse PK studies, in which 26 displayed improved clearance
(40 mL/min/kg) and ~80-fold higher plasma exposures (Cmax
037 ng/mL) compared to an equivalent dose in rat.
Furthermore, when administered orally at 10 mg/kg, 26 showed
b
c
d
-
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e
due to poor compound recovery in the B to A direction. Details for all assay
conditions are provided in the supplemental information.
=
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high target engagement in mouse hippocampus, as measured by
ex vivo autoradiography , with maximal receptor occupancy
Based on the robust target engagement observed in vivo, 26
was evaluated in several mouse seizure models. Protection in
the corneal kindling model was nearly complete at saturating
doses, with an ED50 = 1.3 + 0.1 mg/kg (Figure 4, left panel,
black curve). Seizure protection did not attenuate after 5 days
of continued oral dosing at 5 mg/kg/day, indicating that there
was no toleration to drug treatment (Figure 4, middle panel, red
line). To assess potential compound-related effects on motor
function, all animals were subjected to the rotarod test
immediately prior to seizure challenge. At all doses tested, no
ataxia was observed (Figure 4, left and middle panels, blue
lines). In the i.v. pentylenetetrazole (PTZ) test, the threshold
amounts of PTZ required to generate twitch and clonus were
increased after a single 5 mg/kg oral dose of 26 (Figure 4, right
panel). As with the corneal kindling model, anticonvulsant
efficacy was maintained after chronic oral administration as
well (5 mg/kg/day).
In summary, we have described the discovery, optimization,
and in vivo characterization of imidazo[1,2-a]pyrazines and
pyrazolo[1,5-c]pyrimidines as AMPAR modulators selective
for TARP -8. Starting from HTS hits 4 and 5, a focused
medicinal chemistry effort provided a series of potent and
selective imidazopyrazine leads. Although brain penetrant
compounds from this chemotype could be identified, high in
vivo clearance prevented further development. Replacement of
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exceeding 90% at one hour (Figure 3, top panel). The plasma
and brain exposures at Tmax were highly linear over a wide dose
range, and the receptor occupancy was well-described by a Hill
function with ED50 = 2.9 + 0.7 mg/kg (Figure 3, bottom panel).
Fitting the occupancy as a function of exposure, this
corresponds to an EC50 = 77 + 7 ng/g in brain and 362 + 59
ng/mL in plasma.
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the
imidazopyrazine
scaffold
with
an
isosteric
pyrazolopyrimidine core improved microsomal stability and
efflux liabilities, ultimately delivering compound 26 (JNJ-
6
1432059). Following oral administration, 26 exhibited time-
and dose-dependent receptor occupancy in mouse
hippocampus. In addition, after acute and chronic dosing, 26
provided robust protection in rodent seizure models without
adversely affecting motor function. This preclinical profile
provides further support for the development of selective
AMPAR/-8 negative modulators as novel, differentiated
antiepileptics.
Figure 3. Exposures and GluA/-8 receptor occupancy for
compound 26 in mouse. (Top panel) Time dependency following
single p.o. dose (10 mg/kg, n = 3/time point + SEM). (Bottom
panel) Dose dependency following p.o. administration (n=3/dose +
SEM). Receptor occupancy was measured by ex vivo ARG as
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described previously using [ H] JNJ-56022486 as the radiotracer.
Figure 4. Anticonvulsant models. (Left panel) Protection as a function of dose in the mouse corneal kindling model for compound 26. N
=
8-11 mice/cohort. Animals were tested at t = 1h following oral dosing, and data show fraction of animals with Racine scores of 3 or lower
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(black curve). Rotarod failure data (blue curve) represent the fraction of animals in each cohort that failed a rotarod test immediately prior
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to seizure challenge. (Middle panel) Fraction of animals protected in the corneal kindling model (red line) tested at t = 1h following
once-daily oral dosing of compound 26 (5 mg/kg/day; N = 12-14 animals per cohort). (Right panel) Intravenous PTZ test at t = 2h
following a single (acute) or 5 days of once-daily (day 5) oral dosing with 5 mg/kg of compound 26 (N = 9-11 per cohort).
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ASSOCIATED CONTENT
ABBREVIATIONS
Supporting
Information.
Experimental
procedures,
AMPA, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid;
TARP, transmembrane AMPA receptor regulatory protein; CNS,
central nervous system; FDA, Food and Drug Administration;
LLE, ligand-lipophilicity efficiency; ADME, absorption,
distribution, metabolism, excretion; DMPK, drug metabolism and
pharmacokinetics; SAR, structure-activity relationship; CYP,
cytochrome P450; FLIPR, fluorescence imaging plate reader; p.o.,
per os; i.v., intravenous; PK, pharmacokinetics; CL, clearance; Vss,
volume of distribution at steady state; t1/2, half-life; Cmax, maximum
concentration; F, bioavailability; RLM, rat liver microsome; HLM,
characterization, and assay conditions. This material is available
free of charge via the Internet at http://pubs.acs.org
AUTHOR INFORMATION
Corresponding Author
*
mameriks@its.jnj.com
Author Contributions
All authors have given approval to the final version of the
manuscript.
Notes
p
human liver microsome; K u,u, partition coefficient for unbound
compound concentration in tissue of interest relative to unbound
concentration in plasma; BBB, blood-brain-barrier; MDR1, multi-
drug resistance gene; MDCK-MDR1, Madin Darby canine kidney
cell line transfected with MDR1; RO, receptor occupancy; HP--
CD, (2-hydroxypropyl)--cyclodextrin; SBE-CD, sulfobutylether-
The authors declare no competing financial interest.
ACKNOWLEDGMENT

-cyclodextrin; PEG, polyethylene glycol; SEM, standard error of
the mean; PTZ, pentylenetetrazole
The authors acknowledge the contributions of the following
individuals associated with Janssen Research and Development for
their contributions to the work described here: Ning Qin cloned
several of the GluA and TARP genes and generated the expression
constructs. Hong Yu and Jose Matta conducted electrophysiology
experiments in neurons. Raymond Rynberg developed
formulations for several in vivo experiments. Leslie Nguyen,
Minerva Batugo, Xiaohui Jiang, and Brian Scott performed the
bioanalytical studies. H. Steve White and Cameron Metcalf at
NeuroAdjuvants, Inc. performed the anticonvulsant studies.
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