Lead Optimization of 5-Aryl Benzimidazolone- and Oxindole-Based AMPA Receptor Modulators Selective for TARP γ-8

Article abstract of DOI:10.1021/acsmedchemlett.8b00215

Glutamate mediates fast excitatory neurotransmission via ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. The trafficking and gating properties of AMPA receptors (AMPARs) can be amplified by transmembrane AMPAR regulatory

Full text of DOI:10.1021/acsmedchemlett.8b00215

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Letter
Lead Optimization of 5-Aryl Benzimidazolone and Oxindole-
Based AMPA Receptor Modulators Selective for TARP #-8
Suchitra Ravula, Brad M. Savall, Nyantsz Wu, Brian Lord, Kevin J Coe, Kai Wang, Mark Seierstad, Devin
Maun Swanson, Jeannie Ziff, Minh Nguyen, Perry Leung, Ray Rynberg, David La, Daniel J. Pippel, 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.8b00215 • Publication Date (Web): 13 Jul 2018
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ACS Medicinal Chemistry Letters
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Lead Optimization of 5-Aryl Benzimidazolone and Oxindole-
Based AMPA Receptor Modulators Selective for TARP γ-8
Suchitra Ravula, Brad M. Savall†, Nyantsz Wu, Brian Lord, Kevin Coe, Kai Wang, Mark Seierstad,
Devin M. Swanson, Jeannie Ziff, Minh Nguyen, Perry Leung, Ray Rynberg, David La, Daniel J. Pippel,
Tatiana Koudriakova, Timothy W. Lovenberg, Nicholas I. Carruthers, Michael P. Maher, and
Michael K. Ameriks*
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Janssen Research & Development L.L.C., 3210 Merryfield Row, San Diego, CA 92121 United States
KEYWORDS AMPA, α-amino-3-hydroxy-5-methy-4-isoxazolepropionic acid, AMPA, TARP γ-8, transmembrane AMPA
receptor regulatory protein, auxiliary subunit, glutamate, hippocampus, epilepsy, glutathione, autoradiography
ABSTRACT: Glutamate mediates fast excitatory neurotransmission via ionotropic α-amino-3-hydroxy-5-methyl-4-
isoxazolepropionic acid (AMPA) receptors. The trafficking and gating properties of AMPA receptors (AMPARs) can be amplified
by transmembrane AMPAR regulatory proteins (TARPs), which are often expressed in localized brain regions. Herein, we describe
the discovery, lead optimization, and preclinical characterization of 5-arylbenzimidazolone and oxindole-based negative modulators
of AMPARs associated with TARP γ-8, the primary TARP found in hippocampus. HTS lead 4 was optimized for potency and brain
penetration to provide benzimidazolone 3, JNJ-55511118.1 Replacement of the benzimidazolone core in 3 with an oxindole
mitigated reactive metabolite formation and led to the identification of 18 (GluA1/γ-8 pIC50 = 9.7). Following oral dosing in rats,
18 demonstrated robust target engagement in hippocampus as assessed by ex vivo autoradiography (ED50 = 0.6 mg/kg, plasma EC50
=
9
ng/mL).
Fast excitatory transmission in the central nervous system
(CNS) is mediated primarily by glutamate-gated ionotropic α-
amino-3-hydroxy-5-methyl-4-isoxazole propionic acid
as dizziness, sedation, and falling.9 Perampanel is not a TARP-
selective AMPAR antagonist, and motor impairment in
patients may result from blockade of AMPARs dependent on
stargazin (γ-2), the primary TARP expressed in cerebellum.10
(AMPA) receptors. In the endoplasmic reticulum, AMPA
receptors (AMPARs) are formed as tetrameric assemblies of
subunits (GluA1-GluA4) and then trafficked to the synaptic
membrane where they play an integral role in regulating
synaptic strength.2 Although AMPARs are abundantly
distributed throughout the brain, their activity can be
modulated by auxiliary proteins that often have more discrete
expression profiles. Specifically, transmembrane AMPAR
regulatory proteins (TARPs), which are classified by sequence
homology and function as Type I (γ-2, γ-3, γ-4, γ-8) or Type II
(γ-5, γ-7), enhance AMPAR pharmacology by altering gating
properties and amplifying receptor surface expression.3 In
mammals, TARP γ-8 is predominantly expressed in forebrain
regions and highly enriched in hippocampus.4 Consistent with
this restricted expression profile, selective pharmacological
disruption of AMPARs associated with γ-8 has been shown to
preferentially attenuate excitatory neurotransmission in rat
forebrain.1,5-6 Targeting this mechanism in human may
ultimately provide improved therapies for temporal lobe
epilepsy and other neurological disorders characterized by
hyperexcitability within the hippocampus.7
Recently,
Lilly
reported
the
discovery
of
2,
LY3130481/CERC-611, an AMPAR negative modulator
selective for γ-8.11 We have also disclosed a structurally
distinct compound 3, JNJ-55511118, which exhibited
comparable in vitro and in vivo pharmacology to 2.1 Notably,
both γ-8 selective negative modulators were orally efficacious
in rodent seizure models and did not impair motor function.
Herein, we describe the medicinal chemistry efforts that
resulted in the identification of 3, as well as a second-
generation series of oxindole analogs with improved in vivo
potency and reduced propensity for reactive metabolite
formation.
The non-competitive AMPAR antagonist 1 (perampanel,
FycompaTM) is approved by the FDA as an adjunctive
treatment for partial-onset and generalized tonic-clonic
seizures.8 However, like many antiepileptic drugs, perampanel
administration is associated with CNS-related side effects such
Figure 1. Perampanel and AMPAR/TARP γ-8 modulators
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A high-throughput screen (HTS) of the Janssen global
Page 2 of 7
Having established that benzimidazolone 6 was a CNS-
penetrant drug-like lead, we sought to optimize this molecule
into a tool compound suitable for evaluating AMPAR/γ-8
pharmacology in vivo. To this end, a library of ortho, ortho-
disubstituted 5-arylbenzimidazolones was prepared, and
promising compounds were initially profiled in a variety of in
vitro assays (Table 1). Replacement of a single methyl group
with a chlorine provided a 5-fold improvement in activity
(compare 6 and 7), and consequently this group was held
constant while scanning the remaining ortho-position. Further
gains in γ-8 potency were driven largely by lipophilicity, with
relatively small structural changes yielding dramatic
improvements (compare 7 and 8, as well as 3 and 13). The
introduction of heterocycles (10) and polar substituents (11,
13) reduced potency, except for benzyl nitrile 12 (JNJ-
56022486), which had the highest lipophilic ligand efficiency
(LLE = 5.4). In addition to the encouraging potency and
selectivity profile for 12, its auspicious physicochemical
properties (i.e. high free fraction and solubility) prompted us
to radiolabel the compound and establish binding and
autoradiography assays in native tissue.12 As described
previously, 12 was tritiated in the 6-position of the
benzimidazolone, and the resulting radioligand displayed high
specific binding to hippocampus in rodent and primate brain
slices.1 Furthermore, the regional brain binding profile of [3H]-
12 matched the γ-8 (CACNG8) mRNA expression pattern
1
2
3
4
5
6
7
8
screening deck was conducted to identify compounds that
blocked glutamate-induced Ca2+ flux in HEK-293 cells
expressing a fusion protein between γ-8 and the GluA1o
“flop” splice variant.1 Confirmed hits were counter-screened
against a similar cellular construct co-expressing GluA1o and
γ-2. From this effort, 5-aryl oxindole 4 was identified as a
preliminary lead due to its high ligand efficiency and
selectivity over γ-2 (Figure 2). A subsequent methyl scan
around the distal aromatic ring established that 2,6-
disubstitution was preferred for γ-8 potency and provided a
sub-micromolar analog 5 (pIC50 = 6.4). Replacement of the
oxindole core with an isosteric benzimidazolone scaffold
9
10
11
12
13
14
15
16
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18
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improved activity (6, pIC50
= 6.8) without sacrificing
selectivity against γ-2. Furthermore, when administered to
rats, 6 exhibited encouraging oral bioavailability (%F = 68),
low i.v. clearance (9 mL/min/kg), and acceptable brain
exposure (Kp u,u = 0.3).
reported
in
the
Allen
Mouse
Brain
Atlas.13
Figure 2. Evolution of benzimidazolone 6 from HTS lead 4
Table 1. SAR and in vitro ADME/DMPK data for disubstituted 5-arylbenzimidazolones
GluA1/γ-2 HLM/RLM
pIC50
MDCK-MDR1d
Solubility
PPB,fub
(h/r)f
GluA1/γ-8
Compound
X
R1
R2
cLogP LLE
a
b
stabilityc
(B-A)/(A-B) x10-6cm/s (SGF/FaSSIF)e ꢀM
pIC50
6
CH CH3
CH3
6.8 ± 0.1
< 5
0.3 / 0.5
34 / 42
5 / 276
2.1 / 3.7
3.4
3.4
7
8
CH
CH
CH
N
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
CH3
cPr
Cl
7.3 ± 0.3
8.2 ± 0.2
7.3 ± 0.2
6.6 ± 0.2
6.6 ± 0.3
< 5
< 5
<0.3 / 0.6
<0.3 / 0.5
<0.3 / 0.3
<0.3 / <0.2
<0.3 / <0.2
<0.3 / 0.6
0.3 / 0.6
32 / 25
18 / 10
49 / 26
83 / 16
70 / 14
70 / 6.6
50 / 26
24 / 22
<4 / 146
<4 / 90
<4 / 69
42 / 52
<4 / 5
0.9 / 1.4
0.2 / 1.5
0.5 / 0.6
2.9 / 2.7
5.1 / 7.1
2.7 / 7.6
3.9 / 6.5
0.3 / 1.5
3.7
4.1
3.9
2.6
2.9
2.6
3.0
4.1
3.6
4.1
3.4
3.9
3.7
5.4
3.8
4.2
9
< 5
10
11
12
13
3
Cl
< 5
CH
CH
CH
CH
CN
< 5
CH2CN 8.0 ± 0.4ref 1
< 5ref 1
< 5
26 / 230
19 / 142
<4 / 74
OCH3
6.9 ± 0.2
OCF3 8.3 ± 0.3 ref 1
< 5ref 1
<0.3 / 0.3
apIC50 measured in a FLIPR assay using HEK-293 cells expressing a human GluA1o-γ-8 fusion construct; all data are the result of at least 3 assays run in triplicate
with the mean and standard deviation reported. bpIC50 measured in a FLIPR assay using HEK-293 cells expressing human GluA1-flop cotransfected with γ-2.
d
cStability in human and rat liver microsomes at 1 ꢀM; data are reported as extraction ratios. Apparent permeability in Madin Darby canine kidney cells transfected
f
with the MDR1 (P-gp) gene. eThermodynamic solubility in simulated gastric fluid (pH=1.6) and fasted state simulated intestinal fluid (pH=6.5). Human and rat
plasma protein binding reported as fraction unbound (fub) at 1 ꢀM. Details for all assay conditions are provided in the supplemental information.
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Table 2. Pharmacokinetic parameters and ex vivo GluA/γ-8 receptor occupancy in rat brain for compounds 3 and 8
1
2
3
4
Compound
Rat PK Parametersa
Rat ex vivo GluA/γ-8 Receptor Occupancyb
Cl
Vss
(L/kg)
i.v.
t1/2 (h)
p.o. Cmax
(ng/mL)
c
%F
ED50
EC50 [plasma]
EC50 [brain]
Kpu,u (brain)
(mL/min/kg)
5
6
7
0.5 mg/kgref 1
3.2 mg/kg
145 ng/mLref 1
486 ng/mL
545 ng/g
0.6
0.3
5.8 + 1.6
2.7 + 0.6
1027 + 70
502 + 111
133 + 31
121 + 17
3
8
3.3 + 0.3
4.6 + 0.2
6.9 + 1.2
21 + 4.0
1611 ng/g
8
9
aCompounds dosed as solutions in 20% HP-β-CD at 1 mg/kg (i.v.) and 5 mg/kg (p.o.) in Sprague Dawley rats (n = 3/group). bGluA/γ-8 receptor occupancy in rat
hippocampus at Tmax for each compound as measured by ex vivo autoradiography using [3H]12. cCompounds dosed p.o. in 0.5% HPMC (compound 3) or 20% HP-β-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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44
45
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57
58
59
60
CD
(compound
8)
in
Sprague
Dawley
rats
(8
doses,
n
=
3/dose
group).
Although 12 proved to be a valuable compound for
attributed to reactive metabolites (Figure 3). M3, a glutathione
(GSH) adduct formed through oxidative dechlorination in
mouse, implicated the 5-aryl ring as the site of bioactivation.
However, modifications to this region of the molecule did not
prevent glutathione trapping when compounds were incubated
in human liver microsomes supplemented with GSH and
NADPH (data not shown). Fortunately, a scan of isosteric
replacements for the benzimidazolone scaffold ultimately
mitigated this key issue (Table 3). While benzoxazolone 14
(pIC50 =5.6) and benzothiazolone 15 (pIC50 = 8.6) differed
greatly in γ-8 potency, both compounds trapped glutathione
following metabolic activation. In contrast, oxindole analogs
16-18 did not form GSH adducts under identical conditions.
Although oxindole 16 (pIC50 = 7.8) was 5-fold less active than
the corresponding benzimidazolone 3 (pIC50 = 8.3), the
introduction of small lipophilic substituents in the 7-position
improved γ-8 potency dramatically. Notably, the sub-
nanomolar 7-methyl derivative 18 (pIC50 = 9.7) was 25-times
more potent than 3.
validating AMPAR/γ-8 selectivity in vitro, poor brain
penetration limited its utility in vivo. However,
trifluoromethoxy 3 and cyclopropyl 8 exhibited comparable
potency and selectivity to 12, in addition to improved intrinsic
permeability and efflux in a MDCK-MDR1 cellular assay.
Since 3 and 8 achieved the best balance of potency and
ADME/DMPK properties, both compounds were profiled
extensively in vivo (Table 2). In rat pharmacokinetic studies, 3
and 8 displayed high bioavailability, as well as low to
moderate i.v. clearance that correlated with the rank order
predicted by in vitro liver microsomal stability. Furthermore,
following oral dosing, both compounds reached full γ-8
receptor occupancy in rat hippocampus with plasma EC50s of
145 ng/mL and 486 ng/mL for 3 and 8, respectively.
Due to its superior in vivo potency, long half-life, and
promising anticonvulsant profile in rodents1, compound 3 was
advanced to a 14-day repeat dose rat toleration study.
Although no CNS-related issues were noted at exposures that
corresponded to full γ-8 receptor occupancy, subsequent
histological analysis revealed hepatocellular necrosis in all
dose groups (20 and 60 mg/kg/day), which precluded further
development of 3.
Table 3. Reactive metabolite SAR for analogs of compound 3
Cmpd
3
X
NH
O
R
H
H
H
H
F
GSH Adductb
cLogP
4.1
a
GluA1/γ-8 pIC50
8.3 ± 0.3ref 1
5.6 (n = 1)
8.6 ± 0.3
Yes
Yes
Yes
No
14
4.1
15
S
4.7
16
CH2
CH2
7.8 ± 0.2
3.6
17
8.6 ± 0.3
No
4.1
18
CH2 CH3
9.7 ± 0.3
No
4.2
apIC50 measured in a FLIPR assay using HEK-293 cells expressing a human
GluA1o-γ-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. bCompounds were incubated for 1h in human liver microsomes
supplemented with glutathione (GSH) and NADPH.
Figure 3. Cross-species hepatocyte metabolism for 3
From metabolite identification studies in hepatocytes with
3, multiple thiol adducts were observed by LC-MS/MS
analysis as the primary metabolites in all species except
monkey, which suggested that the liver toxicity in rat may be
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Table 4. SAR and in vitro ADME/DMPK data for disubstituted 5-aryloxindoles
1
2
3
4
5
6
7
8
GluA1/γ-2 GSH
pIC50
HLM/RLM
stabilityd
MDCK-MDR1e
(B-A)/(A-B) (SGF/FaSSIF)f
Solubility
PPB,fub BTB,fub
GluA1/γ-8
Cmpd
X
R1
R2
cLogP LLE
a
b
adductc
(h/r)g
rath
pIC50
9
18
CH Cl
OCF3
OCF3
9.7 ± 0.3
5.0i
No
0.5 / 0.4
NDj
<4 / 55
0.3 / 0.6
0.4
4.2
5.5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
19
20
N
N
Cl
8.6 ± 0.3
8.1 ± 0.2
< 5
< 5
No
No
<0.3 / 0.5
<0.3 / 0.4
51 / 44
66 / 34
11 / 45
4.4 / 6.0
11 / 8.6
1.4
4.8
3.2
2.6
5.4
5.4
Cl OCF2H
140 / 64
apIC50 measured in FLIPR assay using HEK-293 cells expressing a human GluA1o-γ-8 fusion construct; all data are the result of at least 3 assays run in triplicate with
the mean value and standard deviation reported. bpIC50 measured in a FLIPR assay using HEK-293 cells transiently expressing human GluA1o co-transfected with γ2
(n = 1, run in triplicate). cCompounds were incubated for 1h in human liver microsomes supplemented with glutathione (GSH) and NADPH. dStability in human and
rat liver microsomes at 1 ꢀM; data are reported as extraction ratios, where the predicted hepatic clearance is divided by the species-specific hepatic blood flow.
eApparent permeability (x 10-6cm/s) in Madin Darby canine kidney cells transfected with the MDR1 (P-gp) gene. fThermodynamic solubility (ꢀM) in simulated gastric
fluid (pH=1.6) and fasted state simulated intestinal fluid (pH=6.5). gHuman and rat plasma protein binding reported as fraction unbound (fub) at 1 ꢀM. hRat brain tissue
i
j
binding reported as fraction unbound (fub) at 5 ꢀM. Calcium-flux data for 18 with related GluA/TARP constructs are shown in Supplementary Table 1. Not
determined due to low compound recovery. Details of all assay conditions are provided in the supplemental information.
Scheme 1. Synthesis of compounds 16-20 as a representative route to 5-aryl oxindoles/benzimidazolones.a
To offset the increased lipophilicity introduced in the 7-
position of the oxindole, pyridines were investigated as phenyl
replacements in the 5-position (Table 4). Consistent with the
SAR established for the benzimidazolone core, 4-pyridyl 19
(pIC50 = 8.6) was an order of magnitude less potent than the
corresponding phenyl analog 18 (pIC50 = 9.7), but had
comparable lipophilic ligand efficiency. The reduced
hydrophobicity translated into improved physicochemical
properties as pyridines 19-20 displayed lower plasma protein
and brain tissue binding, as well as higher aqueous solubility
at acidic pH compared to 18. Difluoromethoxypyridine 20, the
compound with the lowest cLogP, was especially impressive
in this regard. Furthermore, both pyridines exhibited minimal
turnover in human microsomes, high intrinsic permeability,
and low efflux. This contrasts with the large efflux ratios
observed for related derivatives from the benzimidazolone
series (i.e. see 10 Table 1), presumably due to the differing
number of hydrogen bond donors between the two cores. In
addition to their favorable in vitro ADME profiles, oxindoles
18-20 retained exquisite selectivity for γ-8 over γ-2 (>1000x)
and did not form glutathione adducts in the presence of human
liver microsomes.
Whereas compounds 3-15 were synthesized using Suzuki
reactions between commercially available or readily accessible
aryl halides and boronic acids/esters, convergent multi-step
routes were often required to construct oxindoles 16-20. As a
representative example, the reaction sequences used to prepare
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perfluoroalkoxy analogs 16-20 are shown in Scheme 1. Final
for 19 (5.4 mg/kg) was significantly right-shifted relative to 18
(0.6 mg/kg). The corresponding plasma and brain EC50s for 18
were 9 ng/mL and 71 ng/g, respectively, which represents a
16-fold improvement in efficacious plasma concentration
relative to benzimidazolone 3 (see Table 2).
1
2
3
4
5
6
7
8
compounds were assembled from Pd-catalyzed couplings
between functionalized phenyl or pyridyl iodides 25-27 and
oxindole boronic esters 31-33, which were either
commercially available or derived from
a sequential
bromination/borylation sequence starting with 7-substituted
oxindoles (i.e. 28, R2 = F). Intermediate 25 (X=CH) was
prepared
in
one
pot
through
a
regioselective
lithiation/iodination of 3-chloro-5-trifluoromethoxy benzene
21, while the perfluoroalkoxy groups in pyridines 26 (X = N,
R1 = H) and 27 (X = N, R1 = F) were installed after the
iodination step. Specifically, alkylation of hydroxypyridine 24
with sodium chlorodifluoroacetate14 provided difluoromethoxy
26, whereas trifluoromethoxy 27 was assembled in two steps
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
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from
24
via
fluoride
displacement
of
a
bromodifluoroalkoxypyridine intermediate.15
Table 5. PK/PD profiles of oxindoles 18-20 in rat
Cmpd
PK parametersa
ex vivo ARGb
Vss
(L/kg)
Cl
t1/2
(h)
[brain] /
GluA/γ-8
R.O.c
(mL/min/kg)
[plasma]d
Figure 4. Ex vivo GluA/γ-8 receptor occupancy in rat
hippocampus for compounds 18 and 19: dose dependency
following p.o. administration (n=3/dose + SEM). Receptor
occupancy was measured at Tmax for each compound using [3H]-
12 as the radiotracer.
18
19
20
3.5+1.6
2.7+0.6
1.9+0.1
28 + 0.5
37 + 4.0
19 + 2.0
1.9+0.4
1.4+0.1
1.2+0.1
82+10%
86+3%
72+6%
524/135
983/788
2276/2367
In summary, we have described the discovery and
aCompounds dosed as solutions in 1:1 PEG400/H2O at 1 mg/kg (i.v.) in
optimization of
oxindole based AMPAR modulators selective for TARP γ-8.
Starting from micromolar benzimidazolone lead 4,
systematic modification of the 5-aryl region to optimize
potency and brain penetration afforded single-digit
a series of 5-arylbenzimidazolone and
b
Sprague Dawley rats (n = 3/group). Compounds dosed p.o. as suspensions in
0.5% HPMC at 3 mg/kg (compound 18) or 10 mg/kg (compounds 19-21) in
Sprague Dawley rats (n = 2/dose group). cGluA/γ-8 receptor occupancy in rat
hippocampus at Tmax for each compound as measured by ex vivo
a
d
autoradiography using [3H]12. Brain and plasma concentrations in ng/g and
a
ng/mL, respectively, at Tmax.e ND, not determined.
nanomolar tool compound 3. Although hepatotoxicity in rats
prevented development of 3, this compound established that
selective negative modulation of AMPAR/γ-8 can provide
robust seizure protection in rodents without causing sedation
or motor impairment. A benzimidazolone to oxindole scaffold
switch mitigated the reactive metabolite liabilities associated
with 3, and the introduction of small lipophilic groups in the 7-
position of the oxindole dramatically improved potency.
Accordingly, in rats, the lead oxindole 18 exhibited an order of
magnitude lower efficacious plasma concentration than
benzimidazolone 3. Related series with favorable PK profiles
in higher species have been identified and will be disclosed in
due course.
Based on their promising in vitro ADME/DMPK profiles,
oxindoles 18-20 were further evaluated in vivo (Table 5).
Although 20 exhibited the lowest plasma clearance, this
compound also had the shortest i.v. half-life due to its reduced
volume of distribution relative to 18 and 19. To assess target
engagement in rat hippocampus, compounds were dosed orally
at 3 mg/kg (for 18) or 10 mg/kg (for 19-20), and receptor
occupancy was determined by ex vivo autoradiography. All
three compounds reached micromolar brain concentrations,
which resulted in robust target engagement (>70% GluA/γ-8
receptor occupancy).
Since oxindoles 18 and 19 had the lowest efficacious
plasma concentrations in the rat ex vivo autoradiography
screen, full dose responses were obtained (Figure 4). Whereas
18 displayed dose-proportional plasma and brain exposures,
the plasma levels for pyridyl 19 were below the lower limit of
quantification at low doses (<0.3 mg/kg) and increased in a
non-linear manner with ascending doses (i.e. 87 ng/mL at 3
mg/kg and 2049 ng/mL at 10 mg/kg). Consequently, the ED50
ASSOCIATED CONTENT
Supporting
Information.
Experimental
procedures,
characterization, and assay conditions. This material is available
free of charge via the Internet at http://pubs.acs.org.
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(5) Maher, M.P.; Matta, J.A.; Gu, S.; Seierstad, M.; Bredt, D.S.
Getting a handle on neuropharmacology by targeting receptor-
AUTHOR INFORMATION
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2
3
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7
associated proteins. Neuron, 2017, 96, 989-998.
Corresponding Author
* mameriks@its.jnj.com
(6) Kato, A.S.; Burris, K.D.; Gardinier, K.M.; Gernert, D.L.;
Porter, W.J.; Reel, J.; Ding, C.; Tu, Y.; Schober, D.A.; Lee, M.R.,
et al. Forebrain-selective AMPA-receptor antagonism guided by
TARP γ-8 as an antiepileptic mechanism. Nat. Med., 2016, 22,
1496-1501.
Present Address
† Sanford Burnham Prebys Medical Discovery Institute, 10901 N.
Torrey Pines Rd., La Jolla, CA 92037 United States
8
9
Author Contributions
(7) Small, S.A.; Schobel, S.A.; Buxton, R.B.; Witter, M.P.;
Barnes, C.A. A pathophysiological framework of hippocampal
dysfunction in ageing and disease. Nat. Rev. Neurosci., 2011, 12,
585-601.
All authors have given approval to the final version of the
manuscript.
Notes
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The authors declare no competing financial interest.
(8) Hibi, S.; Ueno, K.; Nagato, S.; Kawano, K.; Ito, K.; Norimine,
Y.; Takenaka, O.; Hanada, T.; Yonaga, M. Discovery of 2-(2-oxo-
1-phenyl-5-pyridin-2-yl-1,2-dihydropyridin-3-yl)benzonitrile
(perampanel): a novel, noncompetitive α-amino-3-hydroxy-5-
methyl-4-isoxazolepropanoic acid (AMPA) receptor antagonist. J.
Med. Chem., 2012, 55, 10584-10600.
ACKNOWLEDGMENT
The authors wish to thank Moravek, Inc. (Brea, CA) for the
synthesis of [3H]12, NovAliX (Strasbourg, France) and WuXi
AppTec (Shanghai, China) for the resynthesis of compounds 3
and 8, and BioBlocks, Inc. (San Diego, CA) for the resynthesis of
compound 20.
(9) Greenwood, J.; Valdes, J. Perampanel (Fycompa) a review of
clinical efficacy and safety in epilepsy. P T, 2016, 41(11), 683-
688.
ABBREVIATIONS
AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid;
TARP, transmembrane AMPA receptor regulatory protein; CNS,
central nervous system; FDA, Food and Drug Administration; LE,
ligand efficiency; LLE, ligand-lipophilicity efficiency; ADME,
absorption, distribution, metabolism, excretion; DMPK, drug
metabolism and pharmacokinetics; SAR, structure-activity
relationship; CYP, cytochrome P450; MW, molecular weight;
FLIPR, fluorescence imaging plate reader; p.o., per os; i.v.,
intravenous; PK, pharmacokinetics; CL, clearance; Vss, volume of
(10) Chen, L.; Chetkovich, D.M.; Petralia, R.S.; Sweeney, N.T.;
Kawasaki, Y.; Wenthold, R.J.; Bredt, D.S.; Nicoll, R.A. Stargazin
regulates synaptic targeting of AMPA receptors by two distinct
mechanisms. Nature, 2000, 408, 936-943.
(11) Gardinier, K.M.; Gernert, D.L.; Porter, W.J.; Reel, J.K.;
Ornstein, P.L.; Spinazze, P.; Stevens, F.C.; Hahn, P.; Hollinshead,
S.P.; Mayhugh, D., et al. Discovery of the first α-amino-3-
hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor
antagonist dependent upon transmembrane AMPA receptor
regulatory protein (TARP) γ-8. J. Med. Chem., 2016, 59, 4753-
4768.
distribution at steady state; t1/2
, half-life; Cmax, maximum
concentration; F, bioavailability; RLM, rat liver microsome;
HLM, human liver microsome; PPB, plasma protein binding; fub,
fraction unbound; MDR1, multi-drug resistance gene; MDCK-
MDR1, Madin Darby canine kidney cell line transfected with
MDR1; SGF, simulated gastric fluid; FaSSIF, fasted state
simulated intestinal fluid; GSH, glutathione; NADPH,
nicotinamide adenine dinculeotide phosphate; RO, receptor
occupancy; HPMC, hydroxypropyl methylcellulose; PEG,
polyethylene glycol; SEM, standard error of the mean
(12) Langlois, X.; te Riele, P.; Wintmolders, C.; Leysen, J.E.;
Jurzak, M. Use of the β-imager for rapid ex vivo autoradiography
exemplified with central nervous system penetrating neurokinin 3
antagonists. J. Pharmacol. Exp. Ther., 2001, 299, 712-717.
(13) Lein, E.S.; Hawrylycz, M.J.; Ao, N.; Ayres, M.; Bensinger,
A.; Bernard, A.; Boe, A.F.; Boguski, M.S.; Brockway, K.S.;
Byrnes, E.J., et al. Genome-wide atlas of gene expression in the
adult mouse brain. Nature, 2007, 445, 168-176.
REFERENCES
(1) Maher, M.P.; Wu, N.; Ravula, S.; Ameriks, M.K.; Savall,
B.M.; Liu, C.; Lord, B.; Wyatt, R.M.; Matta, J.A.; Dugovic, C.; et
al. Discovery and characterization of AMPA receptor modulators
selective for TARP-γ8. J. Pharmacol. Exp. Ther., 2016, 357, 394-
414.
(14) Sperry, J.B.; Sutherland, K. A safe and practical procedure
for difluoromethylation of methyl 4-hydroxy-3-iodobenzoate.
Org. Process Res. Dev., 2011, 15, 721-725.
(2) Huganir, R.L.; Nicoll, R.A. AMPARs and synaptic plasticity:
the last 25 years. Neuron, 2013, 80, 704-717.
(15) (a) Xingya, L.; Heqi. P.; Xikui, J. Spontaneous reactions of
potassium phenoxide with dibromoperfluoroalkanes, first
evidence for bromophilic attack on C-Br bonds by the phenoxide
anion. Tetrahedron Lett., 1984, 4937-4940.
(3) Jackson, A.C.; Nicoll, R.A. The expanding social network of
ionotropic glutamate receptors: TARPs and other transmembrane
auxiliary subunits. Neuron, 2011, 70, 178-199.
(4) Tomita, S.; Chen, L.; Kawasaki, Y.; Petralia, R.S.; Wenthold,
R.J.; Nicoll, R.A.; Bredt, D.S. Functional studies and distribution
define a family of transmembrane AMPA receptor regulatory
proteins. J. Cell Biol., 2003, 161(4), 805-815.
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