Synthesis and Preliminary Studies of a Novel Negative Allosteric Modulator, 7-((2,5-Dioxopyrrolidin-1-yl)methyl)-4-(2-fluoro-4-[11C]methoxyphenyl) quinoline-2-carboxamide, for Imaging of Metabotropic Glutamate Receptor 2

Article abstract of DOI:10.1021/acschemneuro.7b00098

Metabotropic glutamate 2 receptors (mGlu₂) are involved in the pathogenesis of several CNS disorders and neurodegenerative diseases. Pharmacological modulation of this target represents a potential disease-modifying approach for the treatment of substance abuse, depression, schizophrenia, and dementias. While quantification of mGlu₂ receptors in the living brain by positron emission tomography (PET) would help us better understand signaling pathways relevant to these conditions, few successful examples have been demonstrated to image mGlu₂ in vivo, and a suitable PET tracer is yet to be identified. Herein we report the design and synthesis of a radiolabeled negative allosteric modulator (NAM) for mGlu₂ PET tracer development based on a quinoline 2-carboxamide scaffold. The most promising candidate, 7-((2,5-dioxopyrrolidin-1-yl)methyl)-4-(2-fluoro-4-[¹¹C]methoxyphenyl) quinoline-2-carboxamide ([¹¹C]QCA) was prepared in 13% radiochemical yield (non-decay-corrected at the end of synthesis) with >99% radiochemical purity and >74 GBq/μmol (2 Ci/μmol) specific activity. While the tracer showed limited brain uptake (0.3 SUV), probably attributable to effects on PgP/Bcrp efflux pump, in vitro autoradiography studies demonstrated heterogeneous brain distribution and specific binding. Thus, [¹¹C]QCA is a chemical probe that provides the basis for the development of a new generation mGlu₂ PET tracers.

Full text of DOI:10.1021/acschemneuro.7b00098

Article
Synthesis and preliminary studies of a novel negative allosteric
11
modulator [C]QCA for imaging of metabotropic glutamate receptor 2
Xiaofei Zhang, Katsushi Kumata, Tomoteru Yamasaki, Ran Cheng, Akiko
Hatori, Longle Ma, Yiding Zhang, Lin Xie, Lu Wang, Hye Jin Kang, Douglas
J. Sheffler, Nicholas D.P. Cosford, Ming-Rong Zhang, and Steven H Liang
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00098 • Publication Date (Web): 31 May 2017
Downloaded from http://pubs.acs.org on June 12, 2017
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Synthesis and preliminary studies of a novel negative allosteric modulator
[11C]QCA for imaging of metabotropic glutamate receptor 2
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Xiaofei Zhang,†,¶,# Katsushi Kumata,‡,# Tomoteru Yamasaki,‡ Ran Cheng,† Akiko Hatori,‡
Longle Ma,† Yiding Zhang,‡ Lin Xie,‡ Lu Wang,† Hye Jin Kang,┴ Douglas J. Sheffler,§
Nicholas D. P. Cosford,§ MingꢀRong Zhang*,‡ and Steven H. Liang*,†
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†
Nuclear Medicine and Molecular Imaging, Massachusetts General Hospital & Department of
Radiology, Harvard Medical School, Boston, MA, 02114, USA.
¶State Key Laboratory and Institute of Elemento-Organic Chemistry, Collaborative Innovation
Center of Chemical Science and Engineering, Nankai Unviersity, Tianjin 300071, China
‡Department of Radiopharmaceutics Development, National Institute of Radiological Sciences,
National Institutes for Quantum and Radiological Science and Technology, Chiba, 263ꢀ8555, Japan.
┴Department of Pharmacology & National Institute of Mental Health Psychoactive Drug Screening
Program, University of North Carolina at Chapel Hill, North Carolina, 27515, USA.
§Cell Death and Survival Networks Program and Conrad Prebys Center for Chemical Genomics,
SanfordꢀBurnham Medical Research Institute, La Jolla, CA, 92037, USA.
#These authors contribute equally to this work.
Abstract: Metabotropic glutamate 2 receptors (mGlu2) are involved in the pathogenesis of several
CNS disorders and neurodegenerative diseases. Pharmacological modulation of this target represents a
potential diseaseꢀmodifying approach for the treatment of substance abuse, depression, schizophrenia
and dementias. While quantification of mGlu2 receptors in the living brain by positron emission
tomography (PET) would help us better understand signaling pathways relevant to these conditions,
few successful examples have been demonstrated to image mGlu2 in vivo and a suitable PET tracer is
yet to be identified. Herein we report the design and synthesis of a radiolabeled negative allosteric
modulator (NAM) for mGlu2 PET tracer
development based on a quinoline 2ꢀcarboxamide
scaffold. The most promising candidate,
7ꢀ((2,5ꢀdioxopyrrolidinꢀ1ꢀyl)methyl)ꢀ4ꢀ(2ꢀfluoroꢀ4ꢀ
[11C]methoxyphenyl)
quinolineꢀ2ꢀcarboxamide
([11C]QCA) was prepared in 13% radiochemical
yield (nonꢀdecay corrected at the end of synthesis) with >99% radiochemical purity and >74
GBq/µmol (2 Ci/µmol) specific activity. While the tracer showed limited brain uptake (0.3 SUV),
probably attributable to effects on PgP/Bcrp efflux pump, in vitro autoradiography studies
demonstrated heterogeneous brain distribution and specific binding. Thus, [11C]QCA is a
chemical probe that provides the basis for the development of a new generation mGlu2 PET
tracers.
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Keywords: positron emission tomography, metabotropic glutamate receptor 2, mGlu2, 11C,
negative allosteric modulator
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INTRODUCTION
Glutamate is the most abundant endogenous excitatory neurotransmitter and glutamate
receptors (Glus) play a pivotal role in modulating a wide scope of neurological functions in
the central nervous system (CNS).1ꢀ6 Glutamatergic signaling is primarily mediated via two
distinct groups, namely ionotropic receptors (iGlus) and the G proteinꢀcoupled metabotropic
receptors (mGlus). Based on different sequence homology, anatomical distribution and
pharmacology, the mGlus are typically divided into three subcategories. Group I mGlus
(mGlu1 and mGlu5), coupled to Gq/G11 proteins, mobilize calcium from intracellular stores
upon activation and increase protein kinase C activity. Group II (mGlu2 and mGlu3) and
Group III (mGlu4, mGlu6, mGlu7 and mGlu8) mGlus, coupled to Gi/o proteins, downregulate
cAMP formation through inhibition of adenylyl cyclase.7 In particular, mGlu2 receptors
negatively regulate endogenous glutamate release and consequently may be involved in the
protection of neurons against excitotoxicity. The receptors are predominantly localized on
presynaptic membranes although they are also found in peripheral regions of the synapse.8,9
Moderateꢀtoꢀhigh expression of mGlu2 can be found in many brain regions, including the
cerebral cortex, cerebellum, amygdala and hippocampus.10ꢀ14 It has been reported that mGlu2
is involved in the pathogenesis of numerous brain dysfunctions, including psychiatric
disorders and neurodegenerative diseases.7,15ꢀ19 Therefore pharmacological modulation of
mGlu2 represents a promising therapeutic approach for the treatment of several CNS
diseases,20 including drug dependence,21ꢀ23 chronic pain,24 anxiety,17 depression,25,26
schizophrenia,27 Parkinson’s disease28,29 and Alzheimer’s disease.30 Initial drug discovery
efforts focused on nonꢀselective mGlu2/3 agonists and antagonists that bind to the mGlu
orthosteric binding site (evolutionarily conserved glutamate binding site);31 however, in recent
years there has been a shift towards allosteric modulation strategies (consisting of positive
and negative allosteric modulators; abbreviated as PAM and NAM, respectively) that offer the
potential for improved selectivity for mGlu2 or mGlu3.32,33 While a wide range of highly
selective mGlu2 PAMs have been discovered and recently reviewed,33,34 the development of
selective mGlu2 NAMs has remained in its nascent stage with only one report based on a
dihydroquinoline 2ꢀcarboxamide scaffold in the primary literature.35
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Positron emission tomography (PET) is a nonꢀinvasive imaging technology that is
capable of quantifying biochemical processes in vivo,36ꢀ39 which would enable investigations
of mGlu2 based glutamatergic signaling under normal and disease conditions, and assessment
of distribution and testing target engagement and dose occupancy of mGlu2 drug candidates
for clinical trials. Unlike several mGlu1 or mGlu5ꢀtargeting PET radiotracers in human
use,40ꢀ45 there is an unmet need for probing mGlu2 in clinical research and drug development
towards this important receptor subtype in the glutamatergic pathway. As shown in Figure 1,
there are continuous research efforts in the development of mGlu2 PET tracers,40ꢀ43 including
[11C]CMGDE (1),46,47 [11C]JNJꢀ42491293 (2),48,49 [18F]FEꢀJNJ42491293 (3),50 [11C]CMDC
(4)51 and two tracers (18Fꢀcompound 5 and 11Cꢀcompound 6) in the patent literature.52,53
Among these studies [11C]CMGDE (1), the first mGlu2 radiotracer based on a prodrug of an
antagonist LY341495, provided a foundation for the further development of a specific mGlu2
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tracer.47 On the other hand [11C]CMDC (4), a derivative of JNJꢀ40068782,54 was not further
pursued due to limited brain penetration (peak brain uptake ca. 0.6 SUV).51 Preliminary
evaluation in PET imaging studies for compounds 3, 5, 6 are not yet disclosed for the
development of mGlu2 tracers.52,53 Only two PET tracers, namely [11C]JNJꢀ42491293 (2) and
a
11Cꢀcompound from Merck, have been advanced to firstꢀinꢀhuman studies.
[11C]JNJꢀ42491293 (2) was discontinued for mGlu2 imaging due to unexpected offꢀtarget
binding in vivo during clinical trials55,56 and only limited preliminary data on the Merck
compound (no structural information) were reported in abstracts.57,58 These mGlu2 imaging
efforts combined with pharmaceutical development and the potential of mGlu2ꢀmodulating
pharmacotherapy provide a strong impetus to advance PET tracer development for this
target.
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Figure 1. PET tracers targeting mGlu2. While the majority of compounds in this figure have
no selectivity data available in the primary literature, compound 2 showed mGlu2 selectivity
greater than 350 fold over the other mGlu receptors.49
Herein we describe the synthesis of a small array of potent and selective mGlu2 NAMs
that are amenable for radiolabeling and their preliminary evaluation in rodents by PET. In
vitro autoradiography studies confirm the specific binding of these new NAMs bearing a
quinoline 2ꢀcarboxamide moiety, which provides an excellent starting point for future mGlu2
PET tracer design.
RESULTS AND DISCUSSION
Chemistry. We designed a focused library of small molecule based on a series of NAMs
disclosed in the patent literature,59 with the goal to develop a selective mGlu2 PET tracer
amenable to radiolabeling with either 11C or 18F. In particular, the succinimidyl compound 16
was selected in our proofꢀofꢀconcept studies because of reported low EC50 value (8 nM),
reasonable cLogP value (2.95) and amenability for 11Cꢀlabeling from its corresponding
phenolic precursor. Thus a set of quinolone 2ꢀcarboxamides and their labeling precursors
were synthesized according to our synthetic strategy (see detailed retrosynthetic analysis in
Scheme S1 of Supporting Information, SI). As summarized in Scheme 1, oxidation of
7ꢀmethylquinoline (7) with mCPBA followed by cyanide addition provided
quinolineꢀ2ꢀcarbonitrile 8 in 74% yield over two steps, which was converted to
chloroquinoline 9 after mCPBA oxidation and POCl3 chlorination (60% yield). Siteꢀspecific
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bromination at the benzyl position of chloroquinoline 9 followed by succinimide substitution
gave key intermediate 11 in 80% yield. Several parallel syntheses were carried out to
introduce aryl groups at the ipso position of the chloride via palladium catalyzed Suzuki
crossꢀcoupling reactions. The coupling reaction with (2ꢀfluoroꢀ4ꢀmethoxyphenyl)boronic acid
gave quinoline nitrile 12, which was hydrolyzed in basic H2O2 solution to afford the final
standard
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7ꢀ((2,5ꢀdioxopyrrolidinꢀ1ꢀyl)methyl)ꢀ4ꢀ(2ꢀfluoroꢀ4ꢀmethoxyphenyl)quinolineꢀ2ꢀcarboxamide
(QCA; 16) in 55% yield over 2 steps. The cross coupling procedure was also applied to obtain
phenol 17 (51% yield), fluoroethyl (FEPAD (18), 60% yield) and fluoropropyl derivatives
(FPPAD (19), 53% yield). In brief, the synthesis of QCA (16), its precursor 17 and fluorinated
derivatives (18ꢀ19) were achieved in eight to nine steps with overall yields of 6% ꢀ 10%.
Scheme 1. Synthesis of quinoline 2ꢀcarboxamide analogs. (i) mCPBA, DCM, 1h; (ii)
TMSCN, dimethylcarbamic chloride, DCM, 12 h, 74% for two steps; (iii) mCPBA, DCM, 40
ºC, 4 h; (iv) POCl3,DMF, CHCl3, 70ºC, 6 h, 64% yield for two steps; (v) NBS, Benzoyl
peroxide, CCl4, 85ºC, 4 h, 60% yield; (vi) succinimide, Cs2CO3, DMF, 30 min, 80% yield;
(vii) arylboronic acid, Pd(PPh3)4, Na2CO3, 1,4ꢀdioxane, H2O, 100ºC; (viii) Cs2CO3, DMF, 12
h, IC2H4F for 14, 45% yield over 2 steps from 11; IC3H6F for 15, 53% yield over 2 steps from
11; (ix) sodium percarbonate, 55% yield for QCA (16) over 2 steps from 11, 51% yield for
precursor 17 over 2 steps from 11, 60% yield for FEQCA (18), and 53% yield for FPQCA
(19).
Pharmacology and physicochemical properties. QCA and its two fluorinated
derivatives (FEQCA and FPQCA) were subsequently screened for their in vitro activity
towards mGlu2 and mGlu3, and the results are shown in Figure 2. A thallium flux assay in
human embryonic kidney 293 (HEK) cells expressing heteromeric Gꢀprotein coupled
inwardly rectifying potassium (GIRK) channels60 and human mGlu2 or mGlu3, was utilized to
determine potency, efficacy and selectivity. The concentrationꢀresponse relationship that
antagonizes the effect of an EC80 concentration of glutamate was determined for each
candidate. Potency is expressed as the IC50 for inhibition of the glutamate EC80 response. All
three candidates showed NAM activity (IC50 values 45 ± 5 nM for QCA, 130 ± 10 nM for
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FEQCA, and 1080 ± 1300 nM for FPQCA) at human mGlu2 and excellent selectivity for
mGlu2 over mGlu3. We utilized MNIꢀ137, a mGlu2/3 NAM, as a positive control in our GIRK
assay,61 and found no evidence of mGlu3 potencies up to the highest concentration of 30 µM.
In addition, QCA was inactive towards other mGlu receptors at the test concentration of 30
µM, and showed no significant interaction with major CNS targets, which was conducted via
GPCRome assays62 developed by the NIH PDSP program. (see excel data sheet in the
associate content).
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Figure 2. In vitro evaluation of the potencies of QCA, FEQCA, FPQCA, and the control
mGlu2/3 NAM MNIꢀ137 in mGlu2 GIRK (A) or mGlu3 GIRK (B) functional assays.
We next evaluated whether QCA inhibits mGlu2 by a competitive or noncompetitive
mechanism of action in functional studies by performing a Schild analysis.63 For these studies,
the concentrationꢀresponse relationships of glutamateꢀinduced increases in thallium flux were
evaluated for both mGlu2 and mGlu3 in the absence or presence (30 µM, 10 µM, 3333 nM,
1111 nM, 370 nM, 123 nM, or 41 nM) of QCA (Figure 3). QCA doseꢀdependently
rightꢀshifted the concentrationꢀresponse of glutamate toward mGlu2 (Figure 3A) and
decreased the maximal glutamate response, consistent with a noncompetitive mode of action.
Alternatively, consistent with the previous in vitro studies (Figure 2B), QCA had no effect on
either the glutamate potency or glutamate maximal response toward mGlu3 (Figure 3B),
which also serves as an additional control demonstrating the mGlu2 selectivity of QCA.
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Figure 3. QCA noncompetitively rightꢀshifts the glutamate concentrationꢀresponse for mGlu2
and decreases the maximal glutamate response (A) but has no effect on the glutamate
concentrationꢀresponse for mGlu3 (B).
Lipophilicity of candidate compounds can be used as a predictive parameter for assessing
bloodꢀbrain barrier permeability, with preferred range of 1.0ꢀ3.5.64-66 The cLog
P values of
compounds QCA (16), FEQCA (18) and FPQCA (19) were predicted to be 2.95, 3.44 and
3.69, respectively, using Pallas 3.0 prediction software (Table 1). Using liquidꢀliquid partition
ꢀoctanol and water (“shake flask method”),67 the Log
between n P values for QCA, FEQCA,
and FPQCA were 1.27 ± 0.29, 1.75 ± 0.14 and 1.99 ± 0.12, respectively. Since in vitro
assessments of pH stability, plasma and microsomal stability are important to the initial
selection of candidate compounds,68 we carried out preliminary experiments to determine
these parameters. We first evaluated the stability of the compounds under physiological
conditions (pH = 5.0ꢀ9.4). All three compounds showed excellent stability at pH 5 and
diminished stability at pH 7.4 and 9.4 at 37°C, which may be attributed to hydrolysis of
succinimidyl group at higher pH. In addition, QCA and its fluorinated derivatives showed
reasonable plasma and microsomal stability. Based on the functional assay results (cf. Figs. 2
& 3) and physicochemical properties (cf. Table 1), QCA (16) exhibited the lowest EC50 value
among all derivatives and reasonable lipophilicity and plasma/microsomal stability, which
warrants further radiolabeling and subsequent evaluation by in vivo PET imaging and ex vivo
biodistribution studies.
lipophilicitya
pH stabilityb
7.4 9.4
plasma
stabilityc
microsomal
stabilityd
Entry
cLogP
LogP
5.0
2.95
1.27
16
18
19
97.9% 50.9% 46.7%
98.5% 53.6% 59.0%
91.5% 72.5% 76.3%
22.5%
46.7%
87.5%
64.4%
70.1%
64.6%
3.44
3.69
1.75
1.99
Table 1. Physicochemical properties of QCA (16), FEQCA (18) and FPQCA (19).
a.lipophilicity is calculated and measured by Pallas 3.0 software and the shake flask method,
respectively. bꢀd.percent (average, n ≥ 3) remaining of the parent molecule after incubation at
37 °C for 60 min. c.diltiazem and d.verapamil as positive controls, respectively.
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Radiochemistry. As shown in Scheme 2, there are three possible labeling strategies for
QCA (16), namely (1) 11Cꢀcyanation followed by H2O2 hydrolysis from 2ꢀchloroquinoline
11
precursor; (2) Cꢀcarbonylation via [11C]CO followed by aminolysis from 2ꢀchloroquinoline
11
precursor and (3) Cꢀmethylation from the the phenolic precursor 17. Although the first two
approaches may entail a general and unified strategy for the formation of 11Cꢀcarbonyl labeled
quinoline carboxamides, as proof of concept, we chose the most convenient phenolic site for
QCA labeling. The radiosynthesis of [11C]QCA was performed by the reaction of the phenolic
precursor 17 (0.5 mg) with [11C]CH3I in the presence of NaOH (1.25 µmol) in DMF (300 µL).
The reaction was carried out at 80 °C for 5 min, followed by purification using
semiꢀpreparative HPLC. Specifically, [11C]QCA was obtained from the reaction between its
phenolic precursor and [11C]CH3I. The radiochemical yield was 13 ± 4% nonꢀdecay corrected
(n = 3), calculated from starting [11C]CO2. The [11C]QCA was then reformulated in a saline
solution containing 100 µL of 25% ascorbic acid in sterile water and 100 µL of 20% Tween®
80 in ethanol at the end of synthesis (see details in Methods). The radiochemical and chemical
purity were greater than 99% and specific activity was greater than 74 GBq/µmol (2 Ci/µmol).
The overall synthesis time was ca. 30 min and no radiolysis was observed up to 90 min.
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O
*
O
O
A.
N
Cl
F
N
[
11C]CN or [11C]CO
N
N
NH2
R = CH3
O
O
F
B.
[
11C]CH3I, NaOH
*
R = H
OR
OCH3
* = site of 11
C
Scheme 2. Radiosynthesis of [11C]QCA (A) potential labeling methods; (B) 11CH3I labeling
method.
Whole body biodistribution studies in mice. The uptake, distribution and clearance of
[11C]QCA ([11C]16) were studied in mice at five time points (1, 5, 15, 30 and 60 min) post
tracer injection. The results are expressed as the percentage of the injected dose per gram of
wet tissue (%ID/g) in Figure 4 and Table S1 (SI) and standardized uptake value (SUV) in
Figure S1 and Table S2 (SI). High uptake (>3% ID/g) was observed in the heart, lungs, liver,
pancreas, kidneys and small intestine at 1 min post injection of [11C]QCA. After the initial
phase the radioactivity levels in most tissues decreased rapidly, while the signals in the liver
and small intestine continually increased until 30 min and then decreased slowly. The
radioactivity was efficiently cleared from blood (1 min/60 min ratio of 2.4) and high
radioactivity in the liver, kidneys and small intestine indicated urinary and hepatobiliary
excretion, as well as a possible intestinal reuptake pathway. The distribution of [11C]QCA in
the peripheral organs was similar to prior reports with other compounds,51,56,69,70 in which
rapid clearance of radioactivity from heart, lungs, spleen and muscle was observed. Limited
brain uptake (peak value 0.42% ID/g at 1 min post injection) was observed, and thus the
regional brain distribution was further studied by in vitro autoradiography.
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Figure 4. Ex vivo biodistribution in mice at five different time points (1, 5, 15, 30 and 60 min)
post [11C]QCA injection. Data are expressed as %ID/g.
In vitro Autoradiography. The binding specificity of [11C]QCA to mGlu2 was
confirmed by in vitro autoradiography. Representative in vitro autograms of [11C]QCA on
sagittal sections of rat brains are shown in Figure 5A. In the baseline study, the distribution of
bound radioactivity was heterogeneous with signal levels from high to low in the order of
cerebral cortex, striatum, hippocampus, cerebellum and pons/medulla (Figure 5B). These
autographic results are in agreement with both the previously published distribution of mGlu2
in rat brain,10,12 and with other autoradiography studies with the mGlu2 radioligands
[11C]CMDC,51 [3H]JNJꢀ40068782,54 [3H]LY34149571 and [3H]LY459477.14 As shown in
Figure 5C, quantitative analysis of radioactivity binding in the mGlu2ꢀrich regions (cerebral
cortex, striatum, hippocampus and cerebellum) with unlabeled QCA (1 µM) and a NAM
MNIꢀ13761 (1 µM) showed ca. 50ꢀ60% reduced binding compared with that of baseline. We
also observed marginal reductions (ca. 10ꢀ20%) of radioactivity binding when a mGlu2 PAM
LY48737972 was used for blocking study, which may indicate a possible shared, yet at a low
level, binding site between positive and negative allosteric modulators.73,74 These results
indicate that [11C]QCA has a moderateꢀtoꢀhigh level of in vitro specific binding to mGlu2 and
the binding mechanism is consistent with that of a negative allosteric modulator.
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Figure 5. In vitro autoradiography of [11C]QCA binding in rat brain sections. (A) Brain
sections were treated with [11C]QCA in the absence (baseline) or presence of QCA, MNIꢀ137,
LYꢀ487379 (1 µM each). Cer, cerebellum; Hip, hippocampus; Cx, cortex; Str, striatum. (B)
The radioactivity distribution was quantified in regional rat brain. The data are expressed as
radioactivity per mm2 (n = 4). (C) Blocking studies. The data are normalized to % of
radioactivity vs control (n = 4).
PET imaging studies in normal rat brain. Dynamic PET acquisitions were carried out
with [11C]QCA in SpragueꢀDawley rats for 60 min. Representative PET images (summed
0ꢀ60 min) in whole brain and timeꢀactivity curves are shown in Figure 6. The tracer
[11C]QCA showed limited brain uptake (ca. 0.3 SUV whole brain) in rat and no obvious
washout (ratio of SUV5 min / SUV90 min = 1.1). Pretreatment with unlabeled QCA (1 mg/kg)
failed to show significant reduction of brain uptake, likely attributed to low brain permeability
and possible in vivo nonꢀspecific binding (Figure 6C). We next carried out radiometabolite
analysis and PET imaging in PgP/Bcrp knockout mice to investigate possible reasons of
limited CNS penetration.
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Figure 6. PET/MRI fused images of [11C]QCA in rat brain: (A) baseline and (B) selfꢀblocking
with QCA (1 mg/kg). (C) Timeꢀactivity curves in whole brain under baseline and QCA
selfꢀblocking.
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9
Radiometabolite analysis. To evaluate the in vivo stability of [11C]QCA,
radiometabolites in the plasma and brain homogenate of SpragueꢀDawley rats were evaluated
postꢀtracer injection. The percentages of unchanged [11C]QCA and the corresponding
radiometabolites, as determined by radioꢀHPLC, are shown in Figure 7. The fraction
corresponding to unchanged [11C]QCA in plasma was 93% at 5 min, 88% at 20 min and 79%
at 60 min, respectively, with only one other more polar metabolite observed (Top three
possible sites for the metabolism are predicted by SMARTCyp75 and the results are listed as
Table S3 in the supporting information). Analysis of rat brain homogenates in the same time
interval showed unchanged [11C]QCA was 78%, 41% and 34%, respectively, with the same
radiometabolite as found in plasma. These results indicate that the resulting polar
radiometabolite may be brain penetrant.
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Figure 7. Percentages of unchanged [11C]QCA in rat brain tissue and plasma (n = 3) at 5, 20
and 60 min post injection.
PET imaging studies in PgP/Bcrp knockout mice. Based on the results of in vitro
autoradiography and in vivo PET studies on rat brain, we speculated that insufficient brain
permeability of [11C]QCA might be induced by ATPꢀbinding cassette efflux transporters
located at the bloodꢀbrain barrier,66,76ꢀ79 particularly, Pꢀglycoprotein (PgP, ABCB1) and breast
cancer resistance protein (Bcrp, ABCG2). To test this hypothesis, we carried out PET imaging
studies of [11C]QCA on wildꢀtype and PgP/Bcrp knockout (ABCB1a/1bꢀ/ꢀABCG2ꢀ/ꢀ) mice, and
compared pharmacokinetic profiles, particularly brain uptake and clearance.
As shown in Figure 8, peak brain uptake in whole brain was ca. 0.8 SUV in PgP/Bcrp
knockout mice, indicating a significant difference compared with that (ca. 0.3 SUV) of the
wildꢀtype mice. Whole brain uptake increased 130% in PgP/Bcrp knockout mice compared
with that of wildꢀtype mice (calculated based on area under curve). Therefore these results
indicated that [11C]QCA had intensive interactions with brain efflux pumps on the murine
bloodꢀbrain barrier and is likely a PgP/Bcrp substrate in rodents.
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Figure 8. PET/MRI fused images in the whole brain of (A) wildꢀtype and (B) Pgp/Bcrp
knockout mouse. (C) Timeꢀactivity curves of whole brain in wildtype and Pgp/Bcrp knockout
mouse after [11C]QCA injection.
CONCLUSION
We have efficiently synthesized a focused library of NAMs targeting mGlu2, and
radiolabeled the most promising ligand, namely [11C]QCA in good radiochemical yield, high
radiochemical purity and high specific activity. The pharmacokinetic profile (ex vivo
distribution, uptake and clearance), in vitro autoradiography, brain penetration, efflux pump
and metabolism studies were evaluated to determine the suitability of [11C]QCA as a mGlu2
tracer. While [11C]QCA is not likely pursued for in vivo mapping of mGlu2 due to limited
brain permeability, in vitro specific binding studies by autoradiography showed promise as a
new chemotype for mGlu2 tracer development. Further SAR studies of succinimidyl and/or
quinoline carboxamide functionality are necessary to test PgP/Bcrp efflux liability and to
facilitate next generation tracer design with improved brain permeability. Inꢀdepth
pharmacology evaluation is equally important to validate in vivo specificity using mGlu2
knockout mice and/or mGlu2ꢀspecific NAMs. Radiotracer’s binding will also be evaluated
under different agonist concentrations to characterize and select NAMꢀ or PAMꢀbased tracer
for future clinical translation.”
METHODS
Materials and Methods. General Consideration. All the chemicals employed in the
syntheses were purchased from commercial vendors and used without further purification.
Thinꢀlayer chromatography (TLC) was conducted with 0.25 mm silica gel plates (60F254) and
visualized by exposure to UV light (254 nm) or stained with potassium permanganate. Flash
column chromatography was performed using silica gel (particle size 0.040ꢀ0.063 mm).
HꢀNuclear magnetic resonance (NMR) spectra were obtained on a 300 MHz on Bruker
spectrometers and 13C NMR spectra were obtained at 75 MHz. Chemical shifts (δ) are
reported in ppm and coupling constants are reported in Hertz. The multiplicities are
abbreviated as follows: s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, sext =
sextet, sept = setpet, m = multiplet, br = broad signal, dd = doublet of doublets. For
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LCꢀMS/MS measurements, the ionization method is ESI using Agilent 6430 Triple Quad
LC/MS. Lipophilicity was calculated by Pallas 3.4 ADME prediction software (CompDrug
International, Inc., USA). The animal experiments were approved by the Institutional Animal
Care and Use Committee of Massachusetts General Hospital or the Animal Ethics Committee
at the National Institute of Radiological Sciences. DdY mice (male; 7 weeks, 34ꢀ36 g),
Pgp/Bcrp knockout (Abcb1a/1bꢀ/ꢀAbcg2ꢀ/ꢀ; male; 17–18 weeks old; 31–33 g), wildꢀtype (male;
17–18 weeks old; 30–32 g) FAB mice and SpragueꢀDawley rats (male; 7 weeks; 210ꢀ230 g)
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were kept on a 12 h light/12 h dark cycle and were allowed food and water ad libitum
.
Chemistry.
7ꢀmethylquinolineꢀ2ꢀcarbonitrile (8). 7ꢀmethylquinoline (10 g, 57.9 mmol) was
dissolved in dichloromethane (200 mL) in a round bottom flask with a stir bar.
3ꢀChloroperoxybenzoic acid (14.4 g, 75%, 75.33 mmol) was added in portions with ice bath.
The mixture was stirred at room temperature overnight, then quenched with 400 mL 1N
NaOH(aq.) and extracted with dichloromethane (200 mL x 3). The combined organic layers
were washed with saturated aqueous sodium bicarbonate, dried over MgSO4 and concentrated
to give 7ꢀmethylquineline
Nꢀoxide. The crude product was used without further purification.
To a solution of 7ꢀmethylquinoline
N
ꢀoxide in dichloromethane (200 mL) was added
trimethylsilyl cyanide (11.9g, 120.6 mmol) and dimethylcarbamoyl chloride (13.0 g 120.6
mmol). The mixture was stirred at room temperature overnight, then quenched with saturated
sodium bicarbonate and extracted with dichloromethane (200 mL x 3). The combined organic
layers were washed with saturated aqueous sodium chloride, dried over MgSO4 and
concentrated in vacuo. The residue was purified by flash chromatography on silica gel
(hexanes to ethyl acetate gradient column) to yield the compound 8 as white solid (7.3 g, 74%
over two steps). Rf = 0.3 (Hexanes/EtOAc = 20:1). 1H NMR (400 MHz, CDCl3)
δ
8.27 (d,
= 8.3 Hz, 1H), 7.58 – 7.52 (m,
δ 148.3, 142.1, 137.1, 133.3, 131.8, 128.6,
J =
8.3 Hz, 1H), 7.95 (s, 1H), 7.81 (d,
J = 8.4 Hz, 1H), 7.66 (d, J
1H), 2.62 (s, 3H). 13C NMR (100 MHz, CDCl3)
127.3, 126.8, 122.6, 117.5, 21.9.
4ꢀchloroꢀ7ꢀmethylquinolineꢀ2ꢀcarbonitrile (9). Compound 8 (7.0 g, 41.6 mmol) was
dissolved in dichloromethane (300 mL) in a round bottom flask with a stir bar.
3ꢀChloroperoxybenzoic acid (28.7 g, 75%, 124.8 mmol) was added in portions with ice bath.
The mixture was stirred at 40ºC for 3 h, then additional 3ꢀchloroperoxybenzoic acid (28.7 g,
75%, 124.8 mmol) was added. The mixture was stirred at room temperature overnight, then
quenched with 400 mL 1N NaOH(aq.) and extracted with dichloromethane (200 mL x 3). The
combined organic layers were washed with saturated aqueous sodium bicarbonate, dried over
MgSO4 and concentrated to give 4ꢀchloroꢀ7ꢀmethylquinolineꢀ2ꢀcarbonitrile
crude product was used without further purification. To
4ꢀchloroꢀ7ꢀmethylquinolineꢀ2ꢀcarbonitrile ꢀoxide in chloroform (200 mL) was added DMF
Nꢀoxide. The
a
solution of
N
(3.0 g, 41.3 mmol). Then POCl3 (38.0 g, 247.6 mmol) was added in three portions at 70ºC in
4 h. The mixture was stirred at 70 ºC for 2 h before pour into iced water and extracted with
dichloromethane (200 mL x 3). The combined organic layers were washed with saturated
aqueous sodium chloride, dried over MgSO4 and concentrated in vacuo. The residue was
purified by flash chromatography on silica gel (hexanes to ethyl acetate gradient column) to
yield the compound 9 as white solid (5.4 g, 64% over two steps). Rf = 0.3 (Hexanes/EtOAc =
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20:1). H NMR (400 MHz, CDCl3)
1H), 7.64 (dd,
= 8.7, 1.7 Hz, 1H), 2.62 (s, 3H). 13C NMR (100 MHz, CDCl3)
143.0, 133.1, 132.8, 129.2, 125.3, 123.8, 122.5, 116.7, 21.8.
δ
8.17 (d,
J
= 8.6 Hz, 1H), 8.00 – 7.91 (m, 1H), 7.72 (s,
4
5
6
J
δ
149.0, 143.7,
7
8
9
7ꢀ(bromomethyl)ꢀ4ꢀchloroquinolineꢀ2ꢀcarbonitrile (10
)
. To a solution of the compound 9
(1.17 g, 5.76 mmol) in CCl4 (110 mL) under Ar was added
Nꢀbromosuccinimide (1.13 g, 6.33
mmol) and benzoyl peroxide (139.50 mg, 0.579 mmol). The mixture was stirred at 85°C for 4
h, then quenched with water (50 mL) and extracted with dichloromethane (100 mL x 3). The
combined organic layers were washed with saturated aqueous sodium chloride, dried over
MgSO4 and concentrated in vacuo. The residue was purified by flash chromatography on
silica gel (hexanes to ethyl acetate gradient column) to yield the compound 10 as white solid
10
11
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(60%, 967 mg). Rf = 0.2 (Hexanes/EtOAc = 20:1). 1H NMR (400 MHz, CDCl3)
8.6 Hz, 1H), 8.17 (s, 1H), 7.84 (d,
= 8.6 Hz, 1H), 7.80 (s, 1H), 4.68 (s, 2H). 13C NMR (100
MHz, CDCl3) 148.7, 144.0, 142.1, 133.8, 131.4, 129.8, 126.8, 125.1, 123.7, 116.4, 31.5.
4ꢀchloroꢀ7ꢀ((2,5ꢀdioxopyrrolidinꢀ1ꢀyl)methyl)quinolineꢀ2ꢀcarbonitrile (11 . To a solution
δ 8.29 (d, J =
J
δ
)
of the compound 10 (719 mg, 2.55 mmol) and succinimide (303 mg, 3.06 mmol) in DMF (15
mL) was added Cs2CO3 (1.66 g, 5.11 mmol). The mixture was stirred at room temperature for
0.5 h, then quenched with saturated aqueous KH2PO4 (40 mL) and extracted with ethyl acetate
(40 mL x 3). The combined organic layers were washed with saturated aqueous sodium
chloride, dried over MgSO4 and concentrated in vacuo. The residue was purified by flash
chromatography on silica gel (hexanes to ethyl acetate gradient column) to yield the
compound 11 as white solid (80%, 612 mg). Rf = 0.2 (Hexanes/EtOAc = 1:1). 1H NMR (400
MHz, CDCl3)
δ
8.25 (d,
J
= 8.7 Hz, 1H), 8.09 (s, 1H), 7.80 (d, J = 8.8 Hz, 1H), 7.77 (s, 1H),
13
4.92 (s, 2H), 2.81 (s, 4H). C NMR (100 MHz, CDCl3)
130.9, 129.3, 126.6, 124.8, 123.4, 116.4, 41.8, 28.2.
δ 176.5, 148.7, 144.0, 140.0, 133.6,
7ꢀ((2,5ꢀdioxopyrrolidinꢀ1ꢀyl)methyl)ꢀ4ꢀ(2ꢀfluoroꢀ4ꢀmethoxyphenyl)quinolineꢀ2ꢀcarboxam
ide (QCA; 16 . To a solution of the 2ꢀfluoroꢀ4ꢀmethoxyphenylboronic acid (54.4 mg, 0.320
)
mmol) and Na2CO3 (56.6 mg, 0.534 mmol) in 1,4ꢀdioxane : water (v/v, 10/1, 1.8 mL) was
added compound 11 (80.0 mg, 0.267 mmol) and Pd(PPh3)4 (30.9 mg, 0.0267 mmol) under Ar.
The mixture was stirred at 100ºC for 4 h, then quenched with water (3 mL) and extracted with
ethyl acetate (5 mL x 3). The combined organic layers were washed with saturated aqueous
sodium chloride, dried over MgSO4 and concentrated in vacuo. The residue was used without
further purification. To the residue solution in acetone : water (v/v, 2/1, 15 mL) was added
sodium percarbonate (0.251 g, 1.60 mmol). The mixture was stirred at room temperature for
4h, then added ethyl acetate (5 mL) and extracted with ethyl acetate (5 mL x 3). The residue
was purified by flash chromatography on silica gel (hexanes to ethyl acetate gradient column)
to yield QCA (16) as white solid (42% for two steps, 55 mg). Rf = 0.2 (Hexanes/EtOAc = 1:3).
1H NMR (300 MHz, DMSOꢀd6
7.56 (m, 2H), 7.46 (t, = 8.6 Hz, 1H), 6.86 – 6.72 (m, 2H), 4.78 (s, 2H), 3.87 (s, 3H), 2.73 (s,
4H). 13C NMR (75 MHz, DMSOꢀd6
178.1, 166.4, 161.9 (d, = 11.0 Hz), 160.0 (d,
244.1 Hz), 150.8, 146.8, 143.7, 139.2, 132.6 (d, J = 4.9 Hz), 128.9, 128.3, 126.8, 126.3, 120.0,
116.7 (d, = 15.9 Hz), 111.6, 102.4 (d, = 25.4 Hz), 56.3, 41.6, 28.7.
7ꢀ((2,5ꢀdioxopyrrolidinꢀ1ꢀyl)methyl)ꢀ4ꢀ(2ꢀfluoroꢀ4ꢀhydroxyphenyl)quinolineꢀ2ꢀcarboxami
de (17 . To a solution of the 2ꢀfluoroꢀ4ꢀhydroxyphenylboronic acid (50 mg, 0.320 mmol) and
) δ 8.02 (s, 1H), 8.01 (s, 1H), 7.97 (s, 1H), 7.82 (s, 1H), 7.69 –
J
)
δ
J
J =
J
J
)
Na2CO3 (56.6 mg, 0.534 mmol) in 1,4ꢀdioxane : water (v/v, 10/1, 1.8 mL) was added
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compound 11 (80.0 mg, 0.267 mmol) and Pd(PPh3)4 (30.9 mg, 0.0267 mmol) under Ar. The
mixture was stirred at 100ºC for 4 h, then quenched with water (3 mL) and extracted with
ethyl acetate (5 mL x 3). The combined organic layers were washed with saturated aqueous
sodium chloride, dried over MgSO4 and concentrated in vacuo. The residue was used without
further purification. To the residue solution in acetone : water ( v/v, 2/1, 1.5 mL) was added
sodium percarbonate (0.251 g, 1.60 mmol). The mixture was stirred at room temperature for 4
h, then added ethyl acetate (5mL) and extracted with ethyl acetate (5 mL x 3). The residue
was purified by flash chromatography on silica gel (hexanes to ethyl acetate gradient column)
to yield the compound 13 as white solid (39% for two steps, 49 mg). Rf = 0.2
10
11
12
13
14
15
16
17
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(Hexanes/EtOAc = 1:3). 1H NMR (300 MHz, DMSOꢀd6
)
δ
10.32 (s, 1H), 8.31 (s, 1H), 8.01 (s,
= 8.6 Hz, 1H), 6.86 – 6.72 (m,
177.6, 166.0, 159.6 (d,
1H), 7.96 (s, 1H), 7.81 (s, 1H), 7.71 – 7.66 (m, 2H), 7.34 (t,
J
13
2H), 4.78 (s, 2H), 2.73 (s, 4H). C NMR (75 MHz, DMSOꢀd6
)
δ
J =
243.7 Hz), 160.0 (d,
127.9, 126.5, 126.0, 119.5, 114.7 (d,
7ꢀ((2,5ꢀdioxopyrrolidinꢀ1ꢀyl)methyl)ꢀ4ꢀ(2ꢀfluoroꢀ4ꢀ(2ꢀfluoroethoxy)phenyl)quinolineꢀ2ꢀca
rboxamide (FEQCA; 18 . To a solution of the 2ꢀfluoroꢀ4ꢀhydroxyphenylboronic acid (50 mg,
J
= 11.7 Hz), 150.3, 146.4, 143.6, 138.6, 132.2 (d, J = 5.0 Hz), 128.4,
J
= 15.7 Hz), 112.3, 103.0 (d, = 24.1 Hz), 41.2, 28.2.
J
)
0.320 mmol) and Na2CO3 (56.6 mg, 0.534 mmol) in 1,4ꢀdioxane : water (v/v, 10/1, 1.8 mL)
was added compound 11 (80.0 mg, 0.267 mmol) and Pd(PPh3)4 (30.9 mg, 0.0267 mmol)
under Ar. The mixture was stirred at 100ºC for 4 h, then quenched with water (3 mL) and
extracted with ethyl acetate (5 mL x 3). The combined organic layers were washed with
saturated aqueous sodium chloride, dried over MgSO4 and concentrated in vacuo. The residue
was used without further purification. To the residue solution in DMF (1.5 mL) was added
1ꢀfluoroꢀ2ꢀiodoethane (112 mg, 0.640 mmol) and Cs2CO3 (209 mg, 0.640 mmol). The
mixture was stirred at room temperature overnight, then quenched with saturated aqueous
KH2PO4 (5 mL). The combined organic layers were washed with saturated aqueous sodium
chloride, dried over MgSO4 and concentrated in vacuo. The residue was purified by flash
chromatography on silica gel (hexanes to ethyl acetate gradient column) to yield the
compound 14 as white solid (45% for two steps, 50.6 mg) and used directly in the next step.
To the solution of compound 14 (42.1 mg, 0.1 mmol) in acetone : water (v/v, 2/1, 2 mL) was
added sodium percarbonate (78 mg, 0.50 mmol). The mixture was stirred at room temperature
for 4h, then added ethyl acetate (5 mL) and extracted with ethyl acetate (5 mL x 3). The
residue was purified by flash chromatography on silica gel (hexanes to ethyl acetate gradient
column) to yield FEQCA (18) as white solid (60%, 26.3 mg). Rf = 0.2 (Hexanes/EtOAc =
1:3). 1H NMR (300 MHz, DMSOꢀd6
7.69 – 7.59 (m, 2H), 7.47 (t, = 8.6 Hz, 1H), 7.45 – 7.02 (m, 2H), 4.79 (dt,
4.78 (s, 2H), 4.37 (dt,
=30.0, 3.5 Hz), 2.73 (s, 4H). 13C NMR (75 MHz, DMSOꢀd6
166.4, 160.0 (d, = 243.8 Hz), 160.6 (d,
4.7 Hz), 128.9, 128.4, 126.7, 126.3, 120.0, 117.1 (d,
)
δ
8.34 (s, 1H), 8.03 (s, 1H), 7.99 (s, 1H), 7.83 (s, 1H),
=47.9, 3.5 Hz),
J
J
J
)
δ
178.1,
J
J
= 11.1 Hz), 150.7, 146.8, 143.6, 139.2, 134.7 (d,
J
=
J
= 15.8 Hz), 112.0, 103.0 (d,
J
= 25.7
Hz), 82.4 (d, J = 165.6 Hz), 68.2 (d, J = 18.8 Hz), 41.6, 28.6.
7ꢀ((2,5ꢀdioxopyrrolidinꢀ1ꢀyl)methyl)ꢀ4ꢀ(2ꢀfluoroꢀ4ꢀ(3ꢀfluoropropoxy)phenyl)quinolineꢀ2ꢀ
carboxamide (FPQCA; 19 . To a solution of the 2ꢀfluoroꢀ4ꢀhydroxyphenylboronic acid (50
)
mg, 0.320 mmol) and Na2CO3 (56.6 mg, 0.534 mmol) in 1,4ꢀdioxane : water (v/v, 10/1, 1.8
mL) was added compound 11 (80.0 mg, 0.267 mmol) and Pd(PPh3)4 (30.9 mg, 0.0267 mmol)
under Ar. The mixture was stirred at 100ºC for 4 h, then quenched with water (3 mL) and
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extracted with ethyl acetate (5 mL x 3). The combined organic layers were washed with
saturated aqueous sodium chloride, dried over MgSO4 and concentrated in vacuo. The residue
was used without further purification. To the residue solution in DMF (1.5 mL) was added
1ꢀfluoroꢀ3ꢀiodopropane (120 mg, 0.640 mmol) and Cs2CO3 (209 mg, 0.640 mmol). The
mixture was stirred at room temperature overnight, then quenched with saturated aqueous
KH2PO4 (5 mL). The combined organic layers were washed with saturated aqueous sodium
chloride, dried over MgSO4 and concentrated in vacuo. The residue was purified by flash
chromatography on silica gel (hexanes to ethyl acetate gradient column) to yield the
compound 15 as white solid (47% for two steps, 54.6 mg) and used directly in the next step.
To the solution of compound 15 (43.6 mg, 0.1 mmol) in acetone : water (v/v, 2/1, 2 mL) was
added sodium percarbonate (78 mg, 0.50 mmol). The mixture was stirred at room temperature
for 4 h, then added ethyl acetate (5 mL) and extracted with ethyl acetate (5 mL x 3). The
residue was purified by flash chromatography on silica gel (hexanes to ethyl acetate gradient
column) to yield FPQCA (19) as white solid (53%, 24 mg). Rf = 0.2 (Hexanes/EtOAc = 1:3).
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1H NMR (300 MHz, DMSOꢀd6
7.57 (m, 2H), 7.46 (t, =8.6 Hz, 1H), 7.45 – 7.02 (m, 2H), 4.78 (s, 2H), 4.63 (dt,
Hz), 4.19 (t,
= 6.3 Hz), 2.73 (s, 4H), 2.22 – 2.07 (m, 2H). 13C NMR (75 MHz, DMSOꢀd6
178.1, 166.4, 160.0(d, = 244.4 Hz), 161.0 (d, = 12.5 Hz), 150.7, 146.8, 143.7, 139.2, 132.6
(d, J = 5.0 Hz), 128.9, 128.3, 126.8, 126.3, 120.0, 116.8 (d, = 16.0 Hz), 112.0, 102.9 (d,
25.6 Hz), 81.2 (d, = 160.7 Hz), 64.7 (d, = 5.3 Hz), 41.6, 64.7 (d, = 19.6 Hz), 28.6.
)
δ
8.33 (s, 1H), 8.02 (s, 1H), 7.97 (s, 1H), 7.82 (s, 1H), 7.69 –
= 47.3, 5.9
J
J
J
) δ
J
J
J
J =
J
J
J
Pharmacology.
Cell line generation and thallium flux assays. In order to generate human mGlu2 and
mGlu3 stable cell lines to be used for thallium flux assays, human mGlu2 and mGlu3 were
prepared by PCR amplification of the entire coding sequence of each receptor and cloning
into pIRES puro 3 (Invitrogen). For mGlu2 and mGlu3, the cloning sites were NheI/NotI.
HEK GIRK cells, generously provided by Lily Jan (University of California San Francisco,
San Francisco, CA), were transfected with 24 ꢁg of DNA using Fugene6 (Promega), stable
transfectants were selected with 1000 ng/mL puromycin dihydrochloride (SigmaꢀAldrich, St.
Louis, MO), and polyclonal human mGlu2 GIRK and mGlu3 GIRK cell lines were established.
Cells were maintained following selection in 45% DMEM, 45% Ham's F12, 10% FBS, 100
units/mL penicillin/streptomycin, 20 mM HEPES, pH 7.3, 1 mM sodium pyruvate, 2 mM
glutamine, 700 ꢁg/mL G418 (Mediatech, Inc., Herndon, VA), and 600 ꢁg/mL puromycin
(growth media) at 37oC in the presence of 5% CO2. All cell culture reagents were purchased
from Invitrogen Corp. (Carlsbad, CA) unless otherwise noted.
Human mGlu2 and mGlu3 thallium flux in vitro assays. Compound activity at mGlu2 and
mGlu3 was assessed using thallium flux through GIRK channels, a method that has been
described in detail.80,81 Briefly, cells were plated into 384ꢀwell, blackꢀwalled, clearꢀbottomed
polyꢀDꢀlysineꢀcoated plates at a density of 15,000 cells/20 µL/well in DMEM containing 10%
dialyzed FBS, 20 mM HEPES, and 100 units/mL penicillin/streptomycin (assay media).
Plated cells were incubated overnight at 37°C in the presence of 5% CO2. The following day,
the medium was exchanged from the cells to assay buffer [Hanks’ balanced salt solution
(Invitrogen) containing 20 mM HEPES, pH 7.3] using an ELX405 microplate washer
(BioTek), leaving 20 µL/well, followed by the addition of 20 µL/well FluoZin2ꢀAM (330 nM
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final concentration) indicator dye (Invitrogen; prepared as a stock in DMSO and mixed in a
1:1 ratio with Pluronic acid Fꢀ127) in assay buffer. Cells were incubated for 1 h at room
temperature, and the dye exchanged to assay buffer using an ELX405, leaving 20 µL/well.
For concentrationꢀresponse curve experiments, compounds were serially diluted 1:3 into 10
point concentration response curves and were transferred to daughter plates using an Echo
acoustic plate reformatter (Labcyte, Sunnyvale, CA). Test compounds were diluted to 2 times
their final desired concentration in assay buffer (0.3% DMSO final concentration). Agonists
were diluted in thallium buffer [125 mM sodium bicarbonate (added fresh the morning of the
experiment), 1 mM magnesium sulfate, 1.8 mM calcium sulfate, 5 mM glucose, 12 mM
thallium sulfate, and 10 mM HEPES, pH 7.3] at 5 times the final concentration to be assayed.
Cell plates and compound plates were loaded onto a kinetic imaging plate reader (FDSS 6000
or 7000; Hamamatsu Corporation, Bridgewater, NJ). Appropriate baseline readings were
taken (10 images at 1 Hz; excitation, 470 ± 20 nm; emission, 540 ± 30 nm) and test
compounds were added in a 20 µL volume and incubated for approximately 1 hour at room
temperature before the addition of 10 µL of thallium buffer with or without an EC80
concentration of the agonist glutamate for potency evaluation experiments or with a full
concentrationꢀresponse of glutamate for Schild analysis experiments. After the addition of
agonist, data were collected for approximately an additional 2.5 min. Data were analyzed
using Excel (Microsoft Corp, Redmond, WA). The slope of the fluorescence increase
beginning 5 s after thallium/agonist addition and ending 15 s after thallium/agonist addition
was calculated, corrected to vehicle and maximal agonist control slope values, and plotted in
using either XLfit (ID Business Solutions Ltd) or Prism software (GraphPad Software, San
Diego, CA) to generate concentrationꢀresponse curves. Potencies were calculated from fits
using a fourꢀpoint parameter logistic equation.
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Measurement of physicochemical properties.
Measurement of partition coefficient (LogP) (“shake flask method”). The measurement
of Log
ꢀoctanol (475 µL) and water (475 µL) in a test tube. The
preꢀsaturated with each other before use. The tube was vortexed for 1 min before shaken at
37°C overnight. Water phase and ꢀoctanol phase (200 µL each) were aliquoted. The amount
of the test compound in each phase was determined by LCꢀMS/MS (Agilent 6430 Triple
Quad LC/MS). The Log was calculated by Log [ratio between the amount of test compound
in ꢀoctanol and water solution]. The procedure was repeated triplicate and the value was
shown in Table 1.
P
value was performed by mixing test compound (50µL, 20 µM in DMSO) with
n
nꢀoctanol and water were
n
P
n
Measurement of pH stability. The stability of compounds in buffer solutions was measured
using a HPLC method adapted from our previous protocol.82 Briefly, testing compounds 16,
18ꢀ19 (0.25 µmol) were each dissolved in 1 mL DMSO to make a stock solution. An aliquot (50
µL stock solution) was mixed with phosphate buffer (950 µL, 20 mM, pH 7.4), boric
acidꢀKClꢀNaOH buffer (950 µL, 20 mM, pH 9.4) or sodium acetateꢀKClꢀHCl buffer (950 µL,
20 mM, pH 5.0) and incubated for 1 h at 37 ºC. The percentage of the unchanged compound
was monitored by HPLC (Luna analytical column, 4.6 x 250 mm, 5 µm, CH3CN / H2O + 0.1%
TFA).
Measurement of plasma stability. The stability of candidate compounds in rat serum was
measured using a literature method.83 Briefly, the test was performed by mixing a candidate
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compound (10 µL, 10 µM in DMSO stock solution) with 250 µL aliquot of rat serum (Abcam,
Inc. No. ab7488) in a test tube. The tube was vortexed before incubated at 37 oC for 60 min.
The reaction was quenched by the addition of 250 µL iceꢀcold CH3CN, followed by
centrifuge at 10,000 x g for 10 min. The amount of the test compound was quantified by
LCꢀMS/MS (Agilent 6430 Triple Quad LC/MS). The percentage remaining was calculated by
(peak area at 60 min) / (peak area at 0 min) x 100%. The procedure was repeated at least
triplicate and diltiazem was used as a positive control.
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Measurement of liver microsomal stability. The stability of candidate compounds in liver
microsomes was measured using a literature method.84 Briefly, the test was performed by
mixing a candidate compound (0.5 µL, 2 mM in DMSO stock solution) with PBS (432 µL)
and 13 µL aliquot of SpragueꢀDawley rat liver microsomes (SigmaꢀAldrich, No. M9066) in a
test tube. The tube was vortexed before shaken at 37oC for 5 min, followed by the addition of
NADPH (50 µL, 10 mM in PBS stock solution). The mixture was incubated at 37oC for 60
min, and quenched by the addition of 250µL iceꢀcold CH3CN and centrifuge at 10,000 x g for
10 min. The amount of the test compound was quantified by LCꢀMS/MS (Agilent 6430 Triple
Quad LC/MS). The percentage remaining was calculated by (peak area at 60 min) / (peak area
at 0 min) x 100%. The procedure was repeated at least triplicate and verapamil was used as a
positive control.
Radiochemistry. Radiolabeling of [11C]QCA. [11C]Methyl iodide ([11C]CH3I) was
14
synthesized from cyclotronꢀproduced [11C]CO2, which was produced by N(p, α)11C nuclear
reaction. Briefly, [11C]CO2 was bubbled into a solution of LiAlH4 (0.4 M in THF, 300 ꢁL).
After evaporation, the remaining reaction mixture was treated with hydroiodic acid (57%
aqueous solution, 300 ꢁL). The resulting [11C]CH3I was transferred under helium gas with
heating into a preꢀcooled (ꢀ15 to ꢀ20 °C) reaction vessel containing precursor 17 (0.5 mg),
NaOH (2.5 ꢁL, 0.5 M) and anhydrous DMF (300 ꢁL). After the radioactivity reached a
plateau during transfer, the reaction vessel was warmed to 80 °C and maintained for 5 min.
CH3CN / H2O + 0.1% Et3N (v/v, 4/6, 0.5 mL) was added to the reaction mixture, which was
then injected to a semiꢀpreparative HPLC system. HPLC purification was completed on a
Capcell Pak UG80 C18 column (10 mm ID × 250 mm) using a mobile phase of CH3CN / H2O
+ 0.1% Et3N (v/v, 4/6) at a flowrate of 6.0 mL/min. The retention time for [11C]QCA was 9.5
min. The radioactive fraction corresponding to the desired product was collected in a sterile
flask, evaporated to dryness in vacuo, and reformulated in a saline solution (3 mL) containing
100 µL of 25% ascorbic acid in sterile water and 100 µL of 20% Tween® 80 in ethanol. (Note:
We added ascorbic acid to prevent potential radiolysis and Tween® 80 to improve aqueous
solubility.) The synthesis time was ca. 30 min from endꢀofꢀbombardment. Radiochemical and
chemical purity were measured by analytical HPLC (Capcell Pak UG80 C18, 4.6 mm ID ×
250 mm, UV at 254 nm; CH3CN / H2O + 0.1% Et3N (v/v, 4/6) at a flowrate of 1.2 mL/min).
The identity of [11C]QCA was confirmed by the coꢀinjection with unlabeled QCA.
Radiochemical yield was 46% decayꢀcorrected based on [11C]CO2 with >99% radiochemical
purity and greater than 2 Ci/ꢁmol specific activity.
Ex vivo biodistribution in mice. A solution of [11C]QCA (50 ꢁCi / 150ꢀ200 µL) was
injected into DdY mice via the tail vein. These mice (n = 5, each time point) were sacrificed
at 1, 5, 15, 30 and 60 min post tracer injection. Major organs, including whole brain, heart,
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liver, lung, spleen, kidneys, small intestine (including contents), muscle, testes, and blood
samples were quickly harvested and weighted. The radioactivity present in these tissues was
measured using a gamma counter (PerkinElmer, USA), and all radioactivity measurements
were automatically decay corrected based on halfꢀlife of 11C. The results are expressed as the
percentage of injected dose per gram of wet tissue (% ID/g) or standardized uptake value
(SUV).
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In vitro autoradiography. Rat brain was cut into 20 µM sections and stored at ꢀ80ºC
until they were used for experiment. The rat brain sections were preꢀincubated with TrisꢀHCl
buffer (50 mM), MgCl2 (1.2 mM) and CaCl2 (2 mM) solution for 20 min at ambient
temperature, followed by incubation with [11C]QCA (0.48 nM). For blocking studies,
unlabeled QCA (1 µM) was added to incubation solution in advance to determine the
specificity of radioligand binding. After incubation, brain sections were rinsed with iceꢀcold
buffer three times for 2 min, dipped in cold distilled water for 10 sec. The brain sections were
dried with cold air, then placed on imaging plates (BASꢀMS2025, GE Healthcare, NJ, USA)
for optimized contact periods. Autoradiograms were obtained and ROIs were carefully drawn
with the reference of nakedꢀeye observation. Radioactivity was expressed as photostimulated
luminescene values per unit area (PSL/mm2) and measured by a BioꢀImaging analyzer system
(BAS5000, Fujifilm)
Small-animal PET imaging studies. PET scans were acquired by an Inveon PET
scanner (Siemens Medical Solutions, Knoxville, TN, USA). SpragueꢀDawley rats were kept
under anesthesia with 1ꢀ2% (v/v) isoflurane during the scan. The radiotracer (ca. 1 mCi /
150ꢀ200 µL) was injected via a preinstalled catheter via tail vein. A dynamic scan in 3D list
mode was acquired for 60 min. For pretreatment studies, QCA (1 mg/kg) preꢀdissolved in 300
µL saline containing 10% ethanol and 5% Tween® 80 was injected at 30 min via the tail vein
catheter before the injection of [11C]QCA.
As we previously reported,78,79 the PET dynamic images were reconstructed using
ASIPro VW software (Analysis Tools and System Setup/Diagnostics Tool, Siemens Medical
Solutions). Volumes of interest, including the whole brain, cerebral cortex, cerebellum,
striatum, thalamus and pons were placed using ASIPro software. The radioactivity was
decayꢀcorrected and expressed as the standardized uptake value. SUV = (radioactivity per mL
tissue / injected radioactivity) x body weight.
Radiometabolite analysis. Following the intravenous injection of [11C]QCA,
SpragueꢀDawley rats were sacrificed at 5, 20 and 60 min (n = 3 each time point). Blood and
whole brain samples were quickly removed and the blood samples were centrifuged at 15,000
x g for 2 min at 4 °C to separate the plasma. The supernatant (0.5 mL) was then collected in a
test tube containing CH3CN (0.5 mL) and the resulting mixture was vortexed for 15s and
centrifuged at 15,000 x g for 2 min for deproteinization. The rat brain was homogenized in an
iceꢀcooled CH3CN/H2O (1 mL, 1/1, v/v) solution. The homogenate was centrifuged at
150,000 rpm for 2 min at 4 °C and the supernatant was collected. The recovery of
radioactivity into the supernatant was > 90% based on the total radioactivity in the brain
homogenate.
An aliquot of the supernatant (100 µL) obtained from the plasma or brain homogenate
was injected into the radioꢀHPLC system, and analyzed using a Capcell Pak UG80 C18
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column (4.6 mm ID × 250 mm) in a mobile phase of CH3CN / H2O + 0.1% Et3N (v/v, 45/55)
at a flowrate of 1.0 mL/min. The retention time of [11C]QCA is 7.8 min. The percentage of
[11C]QCA to total radioactivity (corrected for decay) on the HPLC charts was calculated as
(peak area for [11C]QCA/total peak area) × 100.
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Retrosynthetic analysis; Characterization of all new compounds and NMR spectra; assay
methods; GPCRome data sheet. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
* Tel: +81 433 823 709. Fax: +81ꢀ43ꢀ206ꢀ3261. Eꢀmail: zhang.mingꢀrong@qst.go.jp.
* Tel: +1 617 726 6107. Fax: +1 617 726 6165. Eꢀmail: liang.steven@mgh.harvard.edu.
Author Contributions
The manuscript was written through contributions of all authors. All authors have given
approval to the final version of the manuscript. #X. Zhang and K. Kumata contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
We would like to thank the staff at the radiochemistry program, Massachusetts General
Hospital, MA, USA and National Institutes for Quantum and Radiological Science and
Technology, National Institute of Radiological Sciences, Chiba, Japan for their support. We
thank the National Institute of Mental Health's Psychoactive Drug Screening Program (NIMH
PDSP) for the compound screening. The NIMH PDSP is directed by Bryan L. Roth MD, PhD
at the University of North Carolina at Chapel Hill and Project Officer Jamie Driscoll at NIMH,
Bethesda MD, USA. We also thank Drs. Thomas J. Brady and Lei Zhang for helpful
discussion. X.Z. is supported by China Scholarship Council Fellowship (201606200041).
N.D.P.C. is supported by a National Institute of Mental Health grant (R01ꢀMH106865). S.H.L
is a recipient of NIH career development award from the National Institute on Drug Abuse
(DA038000).
ABBREVIATIONS
PET, positron emission tomography; mGlu2, metabotropic glutamate receptor 2; PAM,
positive allosteric modulator; NAM, negative allosteric modulator; NBS,
Nꢀbromosuccinimide;
AIBN,
2,2ꢀazobis(2ꢀmethylpropionitrile);
mCPBA,
metaꢀchloroperbenzoic acid; DCM, dichloromethane; DMF, dimethylformamide; ADME,
absorption, distribution, metabolism and excretion; MPO, multiparameter optimization; SUV,
standardized uptake value; TAC, timeꢀactivity curve; %ID/g, percentage of injected dose per
gram of wet tissue; KO, knockout; PgP, Pꢀglycoprotein; Bcrp, breast cancer resistance
protein.
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