Development of Novel PET Probes for Central 2-Amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic Acid Receptors

Article abstract of DOI:10.1021/acs.jmedchem.5b00712

We document the development of PET probes for central AMPA receptors and their application to in vivo animal imaging. An initial screening of perampanel derivatives was performed to identify probe candidates. Despite the high autoradiographic contrast yie

Full text of DOI:10.1021/acs.jmedchem.5b00712

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Article
Development of Novel PET Probes for Central 2-Amino-3-(3-
hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) Receptors
Norihito Oi, Masaki Tokunaga, Michiyuki Suzuki, Yuji Nagai, Yosuke Nakatani, Noboru Yamamoto,
Jun Maeda, Takafumi Minamimoto, Ming-Rong Zhang, Tetsuya Suhara, and Makoto Higuchi
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00712 • Publication Date (Web): 15 Oct 2015
Downloaded from http://pubs.acs.org on October 25, 2015
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Development of Novel PET Probes for
Central
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2
-Amino-3-(3-hydroxy-5-methyl-4-isoxazolyl
)
propionic acid (AMPA) Receptors
†
,‡
‡
†,‡
‡
Norihito Oi,
Masaki Tokunaga, Michiyuki Suzuki,
Yuji Nagai,
Yosuke
†
,‡
†
‡
‡
Nakatani,
Noboru Yamamoto, Jun Maeda, Takafumi Minamimoto, Ming-Rong
‡
‡
,‡
Zhang, Tetsuya Suhara, and Makoto Higuchi*
†
Tsukuba Research Laboratories, Eisai Co., Ltd., 5ꢀ1ꢀ3 Tokodai, Tsukuba, Ibaraki
‡
300ꢀ2635, Japan, Molecular Imaging Center, National Institute of Radiological
Sciences, 4ꢀ9ꢀ1 Anagawa, Inageꢀku, Chiba, Chiba 263ꢀ8555, Japan
*
To whom correspondence should be addressed: Phone: +81 43 206 4700. Fax: +81 43
53 0396. Eꢀmail: mhiguchi@nirs.go.jp. Address: Molecular Imaging Center, National
2
Institute of Radiological Sciences, 4ꢀ9ꢀ1 Anagawa, Inageꢀku, Chiba, Chiba 263ꢀ8555,
Japan.
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ABSTRACT
We document the development of PET probes for central AMPA receptors and their
application to in vivo imaging of animals. Initial screening of perampanel derivatives
was performed to identify probe candidates. Despite the high autoradiographic contrast
yielded by several radioligands, rat PET scans did not support their in vivo suitability.
Further focused derivatization and second screening by ex vivo LCꢀMS measurements
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led
to
the
selection
of
2ꢀ[1ꢀ(3ꢀmethylaminophenyl)ꢀ2ꢀoxoꢀ5ꢀ(pyrimidinꢀ2ꢀyl)ꢀ1,2ꢀdihydropyridinꢀ3ꢀyl]benzoni
11
trile, 21a, and its analogs as candidates. [ C]21a was shown by autoradiography to
specifically bind to the neocortex and hippocampus, consistent with AMPA receptor
11
localization. PET imaging with [ C]21a demonstrated moderate uptake of radioactivity
in rat and monkey brains, with the retention of radiosignals being consistent with
autoradiograms, and the uptake was blocked by pretreatment with unlabeled 21a in a
doseꢀdependent manner. The current approach has facilitated the discovery of a PET
probe potentially suitable for translational research and development focused on AMPA
receptors.
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INTRODUCTION
Glutamate is the primary excitatory neurotransmitter in the brain, and it exerts its
physiologic effects via interaction with two major families of receptor proteins: mGluRs
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1
and iGluRs. mGluRs allow glutamate to modulate cell excitability by activating second
messenger signaling pathways, while iGluRs are ligandꢀgated tetrameric ion channels
2
−4
that mediate fast synaptic responses to glutamate. Three classes of iGluRs have been
3
identified as AMPA, kainate, and NMDA receptors. iGluRs mediate the majority of
excitatory synaptic neurotransmission in CNS, and regulate synaptic plasticity
5
underlying memory/learning and differentiation/growth of the nervous system. AMPA
receptors have also been implicated in excitotoxic conditions exemplified by epilepsy
6
,7
and ischemia, highlighting the significance of these receptors as therapeutic targets.
Scheme 1. Chemical Structures of AMPA Receptor Antagonists
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The first reported competitive AMPA receptor antagonists were quinoxalinedione
8
derivatives such as 1 (NBQX), but their therapeutic utility was hampered by poor
water solubility and low BBB penetration. This finding triggered further development of
9
drugs with similar mechanism of action, including 2 (fanapanel, ZK200775), 3
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(
becampanel, AMP397), 4 (tezampanel, LY293558),
and 5 (selurampanel,
12
BGG492). 3 and 5 were evaluated in clinical studies, but no competitive
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AMPA/kainate antagonist has so far reached the market (Scheme 1).
Meanwhile, noncompetitive AMPA receptor antagonists have actively been explored,
expecting that their effectiveness will not significantly influence normal glutamatergic
1
3
activity as compared with competitive antagonists. A 2,3ꢀbenzodiazepine derivative,
GYKIꢀ53655, is one of the bestꢀknown compounds of this category, and 6 (talampanel,
14,15
GYKIꢀ537773) reached the Phase II clinical trial stage as an antiꢀepileptic agent.
Other structurally similar compounds with anticonvulsant activity, including 7
1
6
17
(
CP465022) and 8 (YM928), have also been identified as noncompetitive AMPA
receptor antagonists. Among additional drug candidates of this class, 9 (irampanel,
18
BIIR561) has been tested for treatment of stroke in Phase I/IIa trials, and 10
19
(perampanel, E2007) has been approved by the US Food and Drug Administration and
other regulatory agencies for treatment of epilepsy.
In vivo PET assays of central AMPA receptors and their occupancy by drugs would
help to gain insights into the molecular basis of neurospychiatric disorders and to obtain
20ꢀ22
proof of therapeutic concepts in clinical trials.
PET is a nonꢀinvasive, molecular
imaging modality that can be used for translational development of AMPA receptor
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ligands from animal models to humans. The relationship between AMPA receptor
occupancy and dose or plasma concentration of a drug may allow us to clarify relevant
dose settings and to minimize adverse events.
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PET assessments of treatments targeting AMPA receptors require a radioprobe
competing with the therapeutic agent on a binding pocket at the receptor. A small
number of PET probes for AMPA receptors have been published, including
11
Nꢀacetylꢀ1ꢀ(4ꢀchlorophenyl)ꢀ6ꢀmethoxyꢀ7ꢀ[ C]methoxyꢀ1,2,3,4ꢀtetrahydroisoquinoline
11
23,24
11
(
[ C]11),
but PET studies with [ C]11 demonstrated its rapid clearance from the
23
CNS and low specific binding in rats. Several radiolabeled talampanel derivatives
25
11
have been described in a patent application, and radiosynthesis of [ C]10 was recently
26
documented. However, their utility as PET probes is yet to be established. In addition,
transmembrane AMPA receptor regulatory proteins (TARPs) have been highlighted as
one of promising drug targets.Among such drugs, LY450295 is classified as a
2
7,28
potentiator of TARP that can label AMPA potentiator binding sites,
and
18
[
F]TARP252 has been evaluated in a clinical PET study by the National Institute of
Mental Health in the United States.
Here, we report a systematic approach to the discovery of suitable radioprobes that
can be used for PET imaging of AMPA receptors targeting the CNS. We identified
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candidate PET probes suitable for AMPA receptor imaging from perampanel derivatives,
which is our novel class of compounds with high binding affinity for this receptor. We
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9ꢀ31
initially radiolabeled several perampanel analogs
to determine a K value sufficient
i
for producing high autoradiographic contrasts of specific binding components in a rat
brain slice.A focused chemical library was then constructed from one of the initially
tested compounds in consideration of the permeability through BBB, which was
32
quantified in rats by cassette dosing of unlabeled compounds. Subsequent evaluation
of candidates by in vivo PET imaging in rats and monkeys demonstrated that a
compound, 21a, could be a potential PET probe for AMPA receptors.
RESULTS AND DISCUSSION
Workflow for Selection of PET Probe Candidates. An outline flow of our
strategy for the development of AMPA receptor PET probes is provided in Supporting
Information, Figure 1. We firstly selected a set of perampanel derivatives, 12a–20a,
11
from Eisai’s compound library according to the presence of a possible C radiolabeling
site, and evaluated K values of these compounds for AMPA receptors by in vitro
i
binding assays using rat brain homogenates. Compounds 12a–20a displayed a
considerable range of affinity for AMPA receptors (5 nM < K < 3 ꢀM) and lipophilicity
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2 < cLogP < 5). Subsequently, radiosyntheses of 12a–20a were performed, and their
specific binding to AMPA receptors was evaluated by in vitro autoradiography of rat
brain slices. Similar to previous reports on the development of other classes of PET
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probes,
a K value required for highꢀcontrast autoradiographic detection of the target
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component was determined in this experiment. We then found that [ C]19a and
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[
C]20a exhibited relatively high affinity and clear autoradiographic contrasts for the
target.
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[
C]20a yielded the highest contrast of specific binding sites in autoradiography, but
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the in vivo uptake of [ C]20a into the wildꢀtype rat brain was very low. By contrast, rat
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PET data demonstrated that a significant amount of [ C]19a was transferred to the
brain, but this did not produce a regional difference in radioactivity retention, implying
that there was insufficient binding affinity of this compound for AMPA receptors.
Based on these findings, we constructed a smallꢀscale focused chemical library
consisting of 80 compounds derived from 20a, and identified the second set of
candidates with K value < 30 nM and FR < 2.0. Cassette dosing of 12 unlabeled test
i
compounds followed by LCꢀMS measurements of these chemicals in tissue extracts was
conducted in rats. Four compounds, 21a–24a, showed moderate BBB penetration, and
were selected as test chemicals for PET assays. These compounds were radiolabeled,
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and were further characterized by in vitro autoradiography of brain slices and in vivo
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PET imaging of rats and a rhesus monkey, resulting in identification of [ C]21a as a
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potential radioprobe applicable to AMPA receptor PET imaging in animals and humans.
Table 1. Pharmacological Properties of Initially Tested PET Probe Candidates
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a
cLogP value was calculated by Daylight Software ver. 4.94 (Daylight Chemical
b
Information Systems, Inc., Niguel, CA). Log D values were quantified in
nꢀoctanol/phosphate buffer (pH 7.4) by the shakeꢀflask method (n = 3; maximum range,
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c
d
±
5%). Procedures for FR assays are provided in Experimental Section 6. Not tested.
Table 2. Pharmacological Properties of PET Probe Candidates Derived From 20a
a
cLogP value was calculated by Daylight Software ver. 4.94 (Daylight Chemical
b
Information Systems, Inc., Niguel, CA). Log D values were quantified in
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nꢀoctanol/phosphate buffer (pH 7.4) by the shakeꢀflask method (n = 3; maximum range,
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9–31
Chemistry.
precursors were classified into three groups by their central ring structure as
(1H)ꢀpyridone, 3(2H)ꢀpyridazinone, and 4,5ꢀdihydroꢀ1,2,4ꢀtriazinꢀ3(2H)ꢀone.
Preparation of all derivatives is illustrated in Schemes 2–4.
,3,5ꢀTrisubstituted 2(1H)ꢀpyridone derivatives were synthesized by halogenation
preferably iodination) of intermediate 26 followed by sequential couplings under
modified Ullmann conditions and SuzukiꢀMiyaura conditions. A typical procedure is
Compounds listed in Tables 1 and 2 and their radiosynthesis
2
1
(
2
3
shown in Scheme 2. The introduction of substituents Ar and Ar to 2(1H)ꢀpyridone
29,30
ring was achievable, and these procedures were described elsewhere in detail.
As radiolabeling precursors for compounds with a methoxy group, 12a, 13a, and 14a,
we generated hydroxy derivatives of these compounds, 12b, 13b, and 14b, by cleavage
of the methoxy group with acidic deprotection. Candidates with a cyano group, 15a–
23a, were also generated, and corresponding bromo derivatives, 15b–23b, were
prepared as radiolabeling precursors. Additionally, a protecting group was incorporated
in an amino group of 20a–23a and their precursors, 20b–23b. 24a and 24b were readily
prepared from the common starting material 24c. A fluorinated compound, 24a, was
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directly prepared from 24c by treatment with bis(2ꢀmethoxymethyl)aminosulfur
trifluoride (BAST), and its radiolabeling precursor, 24b, was derived from 24c by
tosylation.
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a
Scheme 2. Syntheses of Common Trisubstituted 2-(1H)-Pyridone Derivatives
a
1
Reagents and conditions: (a) i: Ar ꢀBr, Pd(OAc)
2
, PPh
3
2
, K CO
3
, DME–H O, 80°C, 1
2
2
day; ii: 4N–HCl, 80°C, 1 day; (b) NIS, CHCl
3
, 50°C, 2 h; (c) Ar ꢀboronic acid,
3
3
2 3
Cu(OAc) , TEA, THF or CHCl , 60°C, 2 h; (d) Ar ꢀborate or Ar ꢀboronic acid,
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Pd(PPh
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, Cs
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CO
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3 2 2
, DMF, 110°C, 2 h; (e) BBr , CH Cl , rt, 0.5 h; (f) TFA, neat or
CH
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2
Cl , rt, 0.5 h; (g) TsCl, TEA, CH
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Cl , 0°C, 1 h; (h) BAST, CH Cl , 0°C, 1 h.
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9
0
Synthesis of 4,5ꢀdihydroꢀ1,2,4ꢀtriazinꢀ3(2H)ꢀone derivatives is outlined in Scheme
3
1
3.
An initial intermediate, 28, was generated by bromination and azidation of an
aromatic ring with an acetyl moiety. A secondary intermediate aminohydrazone, 29,
was obtained by condensation of 28 with aryl hydrazines, followed by reduction.
Notably, triazinone ring closure resulting in 30 was afforded by modified cyclization
2
reaction of 29 with carbodiimidazole (CDI) with high yield. Ar group was then
introduced to 30 by the same procedure as described in Scheme 2, leading to
trisubstituted triazinones, 16b, and 18b. Subsequently, 16a and 18a were readily
prepared by cyanation of 16b and 18b, respectively.
Scheme
3.
Syntheses
of
Common
Trisubstituted
a
4,5-Dihydro-1,2,4-triazin-3(2H)-one Derivatives
a
Reagents and conditions: (a) i: Br
2 3
, AcOH, 80°C, 3 h; ii: NaN , DMF, rt, 1 h; (b) i:
3
Ar ꢀNHNH
2
, EtOH, rt, 1.5 h; ii: PPh
3
, THF–H
2
O, 80°C, 1 day; (c) i: CDI, THF, rt; ii:
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toluene, reflux, 2 h; (d) Ar ꢀboronic acid, Cu(OAc) , TEA, THF or CHCl , reflux, 3 h;
2
3
(e) CuCN, DMF, 120°C, 1 h.
10
11
12
13
14
15
16
17
18
19
20
21
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23
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60
Pyridazinone derivatives were synthesized according to reaction sequences delineated
31
in Scheme 4. Condensation of 2ꢀacetylpyridine with benzaldehyde followed by
conjugate addition of cyanohydrin gave γ−ketonitrile, 31. Compound 31 could be
readily hydrolyzed, and the following treatment with methanol resulted in the
generation of a common intermediate γ−ketoester, 32. Ring formation was carried out
by reaction of 2ꢀbromophenyl hydrazine with 32 to give a compound with a
4
,5ꢀdihydroꢀ3(2H)ꢀpyridazinone ring, 33a, which was readily oxidized to lead a
compound with a 3(2H)ꢀpyridazinone ring. As we used 2ꢀbromophenyl hydrazine as an
3
Ar substituent, 33a was easily converted to 14a by cyanation via production of 33b. A
radiolabeling precursor, 14b, was prepared from 14a by treatment with BBr3.
a
Scheme 4. Syntheses of Common Trisubstituted 3(2H)-Pyridazinone Derivatives
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a
2
Reagents and conditions: (a) i: Ar ꢀCHO, KOH, MeOH:H
2
O = 5:1 (v/v), 0°C, 3 h; ii:
cyanohydrine, K CO , MeOH, rt, 6 h; (b) i: HCl, reflux, 8 h; ii: MeI, K CO , DMF, rt, 1
2
3
2
3
0
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9
0
3
h; (c) i: Ar ꢀNHNH
2
, EtOH, rt, 3 h; ii: AcOH, reflux, 12 h; (d) CuCN, DMF, 120°C, 1
h; (e) BBr , CH Cl , rt, 0.5 h.
3
2
2
11
18
Radiosynthesis. Radiosynthesis of [ C]12a–23a and [ F]24a was performed using
11
11
an automated synthesis system with various units for [ C]methyliodide, [ C]HCN,
11
18
–
[
C]methyltriflate, and [ F]F . Radiosynthesis is outlined in Scheme 5.
11
11
O-[ C]methyl ligands, [ C]12a–14a, were prepared by reacting their precursors,
11
11
12b–14b, with [ C]methyl iodide ([ C]CH I) (Scheme 5), and specific activity of the
3
final products was 37–185 GBq/ꢀmol at the end of synthesis (EOS).
11
11
[ C]CH I was prepared by reducing cyclotronꢀproduced [ C]CO with LiAlH ,
3
2
4
11
followed by iodination with 57% hydroiodic acid. For generation of [ C]12a–14a,
11
[
C]CH I was trapped in a DMF solution containing 12b–14b with an appropriate
3
11
amount of base at –15°C. In [ C]methylation of precursors with a 2ꢀhydroxypyridine
ring, 12b and 13b, N-methylation was the primary process, and the required
11
11
11
Oꢀ[ C]methylated compounds, [ C]12a and [ C]13a, were produced with low yield.
11
Compounds with a cyano substituent, [ C]15a–23a, were radiolabeled by
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[
C]cyanation of their bromo precursors 15b–23b. Typically, [ C]CO was stepwise
2
1
1
11
33,35
converted to [ C]HCN via [ C]CH ,
absorbed in Cu(I) solution, and reacted with
4
10
11
12
13
14
15
16
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21
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1
1
15b–23b to give corresponding [ C]cyanated derivatives. Compounds with an amino
group protected by the BOC moiety, 20b–22b, were deprotected by treatment with TFA
in situ after cyanation.
18
18
–
[
F]24a was obtained from 24b by treatment with [ F]F in the presence of K CO /
2 3
4
,7,13,16,21,24ꢀhexaoxaꢀ1,10ꢀdiazabicyclo[8,8,8]hexacosane (Kryptofix 222) at 85°C
18
–
in CH CN. In this reaction, [ F]F eluted by K CO (66 mM) and Kryptofix 222
3
2
3
36
18
according to a routine procedure gave [ F]24a with a low yield (8%). By reducing the
1
8
amount of K CO to 10 mM, [ F]24a was produced with a high yield (34%).
2
3
a
Scheme 5. Radiosynthesis of All Candidates
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0
a
Reagents and conditions: (a) LiAlH , THF, –15°C, 2 min; (b) hydroiodic acid, 180°C,
4
2
min; (c) AgOTf, 180°C, 1 min; (d) K
2
CO
3
, DMF, 50°C, 3 min; (e) H , Ni, 400°C, 2
2
min; (f) NH
3 2 2 5 4 2
, Pt, 960°C, 2 min; (g) Na S O , CuSO , H O, 80°C, 2 min; (h) i: DMF,
1
2 3
65°C, 3 min; ii: TFA, 80°C, 2 min, in case of deprotection of BOC group; (i) K CO ,
CH CN, 85°C, 10 min.
3
11
37
We also labeled 21a with C on two distinct sites (Scheme 6) to examine the
11
metabolic profile of this compound. [ C]21a was obtained from 20a by treatment with
11
38
[ C]methyl triflate in acetone at moderate yield with a trace amount of a dimethylated
11
product. [ CN]21a was synthesized by cyanation of 21b and subsequent deprotection in
situ by TFA. Autoradiographic and PET data in the present work were obtained by using
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[
C]21a radiolabeled on the methyl group.
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20
21
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a
Scheme 6. Two-way Radiosynthesis of 21a
a
Reagents and conditions: (a) acetone, rt, 1 min; (b) i: DMF, 165°C, 3 min; ii: TFA,
80°C, 2 min.
Identities of all labeled compounds were confirmed by coꢀinjection with the
corresponding unlabeled reference standard on reversedꢀphased analytical HPLC.
Radiochemical purities of these products in the final formulation were higher than 95%.
Additionally, specific activity of each product was calculated based on the UV
absorption area at 254 nm using standard curves with known concentrations of
unlabeled samples in a common ratio. The amount of carrier in the final product
solution was measured by the same analytical HPLC. Moreover, these radioprobes did
not show radiolysis at rt for 90 min after formulation, suggesting radiochemical stability
over the period of at least a single PET scan.
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In Vitro Autoradiography & Rat PET with Initially Selected Radioprobes.
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Representative in vitro autoradiographic images of sagittal rat brain sections with
11
initially examined radiocompounds, [ C]12a–20a, are displayed in Figure 1. Detectable
specific binding of these compounds that could be abolished by addition of
corresponding unlabeled compounds was primarily intensified in the cerebral cortex
(
CTX) and hippocampus (HIP) (red arrow heads), and was also present at varying levels
in other areas including the cerebellum, in agreement with the known distribution of
AMPA receptors, and the contrast of specific signals was consistent with in vitro Ki
values of these compounds in receptor binding assays. K values approximating 100 nM
i
or below were required for detectable autoradiographic contrast between
receptorꢀenriched target region (i.e. CTX and HIP) and reference region with minimum
receptor expression (i.e. brain stem [BS]). Notably, compounds with a pyridone ring,
1
1
11
11
[
C]17a, [ C]19a, and [ C]20a, yielded higher contrasts than triazinone ring
1
1
11
11
derivatives, [ C]16a and [ C]18a. Another pyridine derivative, [ C]15a, which was
1
1
structurally similar to [ C]17a, showed binding to the entire slice, presumably due to its
low affinity for target receptors relative to reactivity with offꢀtarget binding components.
11
Reactivity of a pyridazinone ring compound, [ C]14a, with AMPA receptors was not
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sufficient for conceiving this pharmacophore to be of realistic utility. Compound
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[ C]20a presented the highest affinity (K = 6 nM) and autoradiographic contrast
i
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among the series of chemicals listed in Table 1, indicating its potential applicability to
in vivo PET studies.
11
In vivo performance of [ C]20a as an AMPA receptor probe was accordingly
evaluated by PET scans of rat brains. There was only minimal uptake of radioactivity in
the brains of wildꢀtype rats (Figure 2B), precluding in vivo assessments of specific
radioprobe binding. We postulated that a relatively high FR value of 20a (FR = 2.8; see
Table 1) accounted for its insufficient entry to the brain. Pꢀgp–knockout rats were then
employed for PET scans with this probe, and notably high retention of radioactivity in
AMPA receptorꢀrich brain regions such as HIP and CTX versus BS was observed in
these animals (Figure 2A, C). Compared to wildꢀtype controls, an increase in
radioactivity uptake was also observed in peripheral organs of Pꢀgp–knockout animals,
presumably because due to the lack of Pꢀgpꢀmediated excretion of the radioprobe into
11
urine, bile and intestinal lumen. Additional PET scans were conducted using [ C]20a in
a mouse doubly deficient in Pꢀgp and breast cancer resistance protein (BCRP). Peak
radioprobe uptake in these mice was nearly doubled as compared to wildꢀtype mice
(Supporting Information, Figure 3), and this combined genetic effect did not exceed the
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impact of single Pꢀgp deficiency in rats, implying that there was no overt contribution of
1
1
BCRP to the efflux of [ C]20a from the brain. We also conducted a supplementary
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PET experiment with [ C]19a (Supporting Information, Figure 2) in wildꢀtype rats, and
1
1
brain uptake of this probe was much higher than that of [ C]20a (Figure 2), in
11
agreement with its FR value (FR = 1.2; see Table 1). However, [ C]19a gave no
i
regional difference in radioactivity, providing further evidence that an in vitro K value
around 10 nM or below is required for detecting AMPA receptors.
Figure 1. Representative in vitro autoradiographic images of rat brain sections reacted
11
with initially selected radioprobes, [ C]12a–20a, in the absence (total binding; upper
radiographs) and presence (nonspecific binding; lower radiographs) of 10 ꢀM of
corresponding unlabeled compounds. All sagittal slices as illustrated in Nisslꢀstained
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brain atlas were collected approximately 2.0 mm lateral to the midline, and are aligned
i
from left to right in descending order of K values for AMPA receptors. Distribution of
10
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AMPA receptors is also indicated by displaying an autoradiogram with a reference
3
radioprobe, [ H]perampanel.
11
Figure 2. [ C]20aꢀPET imaging of rat brains. (A, B) Representative orthogonal PET
images of Pꢀgp–knockout (A) and wildꢀtype (B) rat brains generated by averaging
11
dynamic data at 0–60 min after intravenous injection of [ C]20a. PET images are
superimposed on an MRI template. (C) Time–activity curves (TACs) in CTX, HIP, and
11
BS of Pꢀgp–knockout and wildꢀtype rat brains after injection of [ C]20a. Radioactivity
is expressed as a percentage of injected radioligand dose per unit volume of tissue (%
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injection dose/mL; %ID/mL ).
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Cassette Dosing Study. Twelve compounds derived from 20a were evaluated by
cassette dosing to rats in order to investigate their transfer to the brain. These
compounds were selected according to the in vitro criteria for affinity (K value < 30
i
nM) and potential transꢀBBB permeability (FR < 2.0), and were divided into three test
groups. Each group was composed of compounds with different molecular weights, and
standard curves of these compounds were obtained by LCꢀMS in advance to ex vivo
quantification. A cocktail of four compounds was prepared and intravenously
administered to rats, followed by a collection of the brains at 5, 30, 60 min after
compound injection. Supernatants after centrifugation of brain homogenates were
deproteinized and used for quantification of intact peaks corresponding to test
compounds by LCꢀMS. We chose four compounds, 21a–24a, with acceptable in vitro Ki
value and concentration in the brain achieved at 5 min after injection for subsequent
autoradiographic and PET studies (Table 3). In this evaluation, we selected compounds
with a ratio of brain uptake (%ID/mL) to K (ꢀM) exceeding 5.0 for the following PET
i
evaluation. Overall, FR values of the compounds were correlated with their ex vivo
brain concentrations, but did not necessarily predict the efficacy of their transfer to the
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brain.
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21
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Table 3. Concentration of Compounds Quantified by Cassette Dosing
a
A cocktail of 50 ꢀg/kg of four compounds in each of three groups was administered
intravenously to SpragueꢀDawley (SD) rats (n = 2, duplicate). Concentrations of the
compounds in brain homogenates at 5 min after injection were calculated as % injection
b
dose (%ID) by LCꢀMS. Peak neocortical uptake at 5 min after intravenous injection of
11
[ C]20a in PET scans of wildꢀtype rats is displayed as reference.
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Autoradiography & Rat PET with Focused Library Compounds Derived from
20a. We autoradiographically analyzed the second set of radiolabeled compounds,
0
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[ C]21a–23a and [ F]24a, which were derived from 20a and were selected by LCꢀMS
11
measurements of cassetteꢀdosed compounds. As illustrated in Figure 3, [ C]21a–23a
produced a high contrast of CTX and HIP versus BS in rat brain slices similar to
1
1
[
C]20a, and radiolabeling in other brain regions was also consistent with known
18
AMPA receptor distribution. In contrast, autoradiographic signals yielded by [ F]24a
were mostly unrelated to the distribution of AMPA receptors, indicating substantial
nonspecific binding possibly arising from the high lipophilicity of the compound.
Autoradiographic binding of these radioprobes was also assessed in rhesus monkey
11
brain slices (Supporting Information, Figure 4). [ C]20a–23a exhibited abundant
specific binding in CTX and HIP in a manner similar to the labeling of rat brains.
We next assessed the in vivo performance of these radioprobes by conducting rat
PET assays. Among these chemicals, 21a showed a K value for AMPA receptors
i
comparable with 20a, and its FR value was lower than 20a, supporting applicability of
11
this compound to in vivo imaging. Indeed, peak uptake of [ C]21a into the brains of
11
wildꢀtype rats was nearly twoꢀfold higher than that of [ C]20a (compare Figures 2C
11
and 4C). Notable retention of [ C]21a in HIP and CTX also contrasted with the rapid
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clearance of radioactivity in BS (Figure 4), in distinction from the lack of regionality in
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the kinetics of [ C]19a (Supporting Information, Figure 2).
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Unlike [ C]21a, PET scans indicated that there was minimal uptake of [ C]22a into
the rat brain due to relatively low BBB penetration, which was in good agreement with
the cassette dosing study (Supporting Information, Figure 5). Meanwhile, entry of
11
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[
C]23a to the brain was even higher than the level observed in [ C]21aꢀPET, but
there were subtle differences in radioactivity retention between AMPA receptorꢀrich
and reference regions (Supporting Information, Figure 6). Furthermore, PET scans of
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wildꢀtype rats with [ F]24a demonstrated its efficient transfer through BBB, followed
by prompt clearance of radioactivity from all brain regions in a fashion unrelated to the
localization of AMPA receptors (Supporting Information, Figure 7). This finding again
supports the notion that K ≤ 10 nM is a requisite for producing detectable AMPA
i
receptor signals. It is also noteworthy that the radioactivity in the brain was gradually
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elevated beyond 10 min after injection of [ F]24a (Supporting Information, Figure 7B),
presumably due to spillover of radioactivity from defluorination products accumulating
in the skull.
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Page 28 of 76
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Figure 3. In vitro autoradiographic images of rat brain slices with [ C]20a and its
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derivatives, [ C]21a–23a and [ F]24a. Brain sections were reacted with indicated
concentrations of labeled componds in the absence (total binding; upper images) and
presence (nonspecific binding; lower images) of 10 ꢀM of corresponding unlabeled
compounds. All sagittal brain slices were collected approximately 2.0 mm lateral to the
midline.
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Journal of Medicinal Chemistry
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Figure 4. [ C]21aꢀPET imaging of rat brains. (A) Representative orthogonal PET
images of wildꢀtype rat brain generated by averaging dynamic data at 0–90 min after
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intravenous injection of [ C]21a. PET images are superimposed on an MRI template.
(
B) TargetꢀtoꢀBS ratio of AUC (0–40 min) of wildꢀtype rat brain in CTX (closed
column) and HIP (open column). (n = 3). Radioactivity is expressed as percentage of
injected radioligand dose per unit volume of tissue (% ID/mL). Vertical axis begins at
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0.8. (C) TACs in CTX, HIP and BS of wildꢀtype rat brains after injection of [ C]21a.
Vertical bars in graphs represent SD.
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