Synthesis and preliminary evaluation of novel 11C-labeled GluN2B-selective NMDA receptor negative allosteric modulators

Article abstract of DOI:10.1038/s41401-020-0456-9

N-methyl-D-aspartate receptors (NMDARs) play critical roles in the physiological function of the mammalian central nervous system (CNS), including learning, memory, and synaptic plasticity, through modulating excitatory neurotransmission. Attributed to et

Full text of DOI:10.1038/s41401-020-0456-9

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BRIEF COMMUNICATION
11
Synthesis and preliminary evaluation of novel C-labeled
GluN2B-selective NMDA receptor negative allosteric
modulators
Ji-yun Sun1,2, Katsushi Kumata3, Zhen Chen2, Yi-ding Zhang3, Jia-hui Chen1,2, Akiko Hatori3, Hua-long Fu2, Jian Rong2, Xiao-yun Deng2,
Tomoteru Yamasaki3, Lin Xie3, Kuan Hu3, Masayuki Fujinaga3, Qing-zhen Yu2, Tuo Shao2, Thomas Lee Collier2, Lee Josephson2,
Yi-han Shao4, Yun-fei Du5, Lu Wang1,2, Hao Xu1, Ming-rong Zhang3 and Steven H Liang2
N-methyl-D-aspartate receptors (NMDARs) play critical roles in the physiological function of the mammalian central nervous system
(CNS), including learning, memory, and synaptic plasticity, through modulating excitatory neurotransmission. Attributed to
etiopathology of various CNS disorders and neurodegenerative diseases, GluN2B is one of the most well-studied subtypes in
preclinical and clinical studies on NMDARs. Herein, we report the synthesis and preclinical evaluation of two 11C-labeled GluN2B-
selective negative allosteric modulators (NAMs) containing N,N-dimethyl-2-(1H-pyrrolo[3,2-b]pyridin-1-yl)acetamides for positron
emission tomography (PET) imaging. Two PET ligands, namely [11C]31 and [11C]37 (also called N2B-1810 and N2B-1903,
respectively) were labeled with [11C]CH3I in good radiochemical yields (decay-corrected 28% and 32% relative to starting [11C]CO2,
respectively), high radiochemical purity (>99%) and high molar activity (>74 GBq/μmol). In particular, PET ligand [11C]31
demonstrated moderate specific binding to GluN2B subtype by in vitro autoradiography studies. However, because in vivo PET
imaging studies showed limited brain uptake of [11C]31 (up to 0.5 SUV), further medicinal chemistry and ADME optimization are
necessary for this chemotype attributed to low binding specificity and rapid metabolism in vivo.
Keywords: ionotropic glutamate receptors (iGluRs); NMDARs; GluN2B subunit; positron emission tomography (PET); carbon-11
Acta Pharmacologica Sinica (2020) 0:1–8; https://doi.org/10.1038/s41401-020-0456-9
INTRODUCTION
gradually restricted to the forebrain, including in the hippocam-
pus, cerebral cortex, striatum, and thalamus, and it is rarely
detected in the cerebellum in adult brains [11–14]. Although since
the 1990s, several NMDAR antagonists, such as phencyclidine
(PCP), ketamine, MK-801, memantine, and amantadine, have been
developed and used in clinical applications, chronic glutamate
stimulation to NMDARs may lead to excitotoxicity, along with
many adverse CNS effects, including hallucinations, increasing
blood pressure, and catatonia [15–18]. Therefore, GluN2B-selective
NMDAR antagonists have attracted increasing attention because
they have shown promising therapeutic potential by treating
NMDAR-related diseases, yet they lack the same adverse reactions
due to their unique activity [19–21].
Positron emission tomography (PET) is a noninvasive nuclear
imaging technique that can help profile in vivo biological targets
by detecting the uptake, distribution, and metabolism of radio-
tracers [22–27]. GluN2B-selective PET imaging radiotracers could
provide a powerful tool for understanding the biological role of
NMDAR subtypes in the pathophysiology of various brain diseases.
L-Glutamate, a major endogenous excitatory neurotransmitter in
the mammalian central nervous system (CNS), regulates a wide
range of nervous system functions. The excitatory neurotransmit-
ter signal is mediated through two types of L-glutamate receptors:
ligand-gated ion channel ionotropic receptors (iGluRs) and G-
protein-coupled metabotropic receptors (mGluRs). NMDARs are
one class of iGluRs with three subunits (GluN1-GluN3), and the
dysregulation of NMDARs has been identified in the pathophy-
siology of various brain diseases, such as Parkinson’s disease (PD),
Alzheimer’s disease (AD), schizophrenia, and neuropathic pain
[1–6]. The GluN2B subunit, one of four subunits of GluN2, has
been found to play essential roles in these neurological and
psychiatric disorders. This subunit encompasses polyamines as
positive allosteric modulators (PAMs) and incorporates zinc ion-
binding sites for docking negative allosteric modulators (NAMs),
and these sites are also called ifenprodil-like sites [7–10]. The
GluN2B subunit has high and widespread expression throughout
the brain around birth; however, the distribution of this subunit is
1Center of Cyclotron and PET Radiopharmaceuticals, Department of Nuclear Medicine and PET/CT-MRI Center, the First Affiliated Hospital of Jinan University, Guangzhou 510630,
China; 2Department of Radiology, Division of Nuclear Medicine and Molecular Imaging Massachusetts General Hospital and Harvard Medical School, 55 Fruit Street, Boston, MA
02114, USA; 3Department of Advanced Nuclear Medicine Sciences, National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and
Technology, Chiba 263-8555, Japan; 4Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019, USA and 5Tianjin Key Laboratory for Modern Drug
Delivery & High-Efficiency, School of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, China
Correspondence: Hao Xu (txh@jnu.edu.cn) or Ming-rong Zhang (zhang.ming-rong@qst.go.jp) or Steven H Liang (liang.steven@mgh.harvard.edu)
Thease authors contributed equally: Ji-yun Sun, Katsushi Kumata
Received: 6 April 2020 Accepted: 3 June 2020
© CPS and SIMM 2020

Development of two novel GluN2B-selective PET radioligands
JY Sun et al.
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Fig. 1 Representative GluN2B-selective NMDAR PET radiotracers [28–41].
Since several excellent reviews [1, 19–21] have discussed the
current status of NMDAR radiotracers, we merely listed several
representative GluN2B-selective PET radiotracers in Fig. 1 [28–41].
[11C]( )-Methoxy-CP-101606 (1, traxoprodil) [28, 29] was the first
“prodil” PET radiotracer, and it was reported by Haradahira and
coworkers in 2002. MK-0657 (10, rislenemdaz) is a new class of
GluN2B-selected antagonists that has entered clinical trials, and
trans-[18F]10 and cis-[18F]10 have been studied for imaging the
GluN2B subunit in vitro and in vivo [31, 32]. In 2018, Kramer et al.
developed a new type of PET ligand derived from sigma receptor
modulators. For example, 11C-Me-NB1 ([11C]14) displayed high
affinity for the GluN2B-NTD-binding site and enabled target
engagement studies [35, 36]. Recently, we reported a novel
GluN2B-selective PET radiotracer containing a triazole core with
high in vitro-specific binding in brain sections [41]. However,
clinical applications of the aforementioned radiotracers have not
been demonstrated. Therefore, further studies on clinically
relevant GluN2B-selective PET radiotracers are necessary.
As a continuation of our previous work on GluN2B-selective PET
radiotracers [41], we report two 11C-labeled compounds selected
from a recently reported novel class of GluN2B-negative allosteric
modulators based on their favorable pharmacological and
physicochemical properties [42]. Compounds 31 and 37 demon-
strated excellent in vitro GluN2B binding affinity (Ki = 11 nM and
4.3 nM, respectively) and possessed the desired GluN2B occu-
pancy in the brain (peak occupancy for 31 = 83%, peak occupancy
for 37 = 96%) in preliminary receptor occupancy studies. Pharma-
cological evaluation of 31 and 37 showed their potential for
evaluation as PET ligands for GluN2B subunits. In this work, with
an efficient N-methylation 11C-labeling strategy, radiosynthesis,
and preliminary evaluation of [11C]31 and [11C]37 in the rodent
brain were carried out. The brain permeability and binding
specificity were evaluated by PET imaging in vivo, and autoradio-
graphy studies were conducted in vitro.
MATERIALS AND METHODS
The general procedure for the experimental section was described
previously [43, 44] and was used with minor modifications in this
work. All chemicals were purchased from commercial vendors and
were used without further purification unless otherwise indicated.
Thin-layer chromatography (TLC) was conducted with 0.25-mm
silica gel plates (60F254) and visualized by exposure to UV light
(254 nm) or by staining with potassium permanganate. Column
chromatography separations were performed on silica gel
(SiliCycle Inc., 230–400 mesh, 40–63 μm). 1H, 13C, and 19F NMR
spectra were obtained at 300, 75, and 282 MHz, respectively, on a
Bruker spectrometer in CDCl3 or d6-DMSO solutions at rt, and the
chemical shifts are reported in δ values (parts per million, ppm)
downfield relative to the internal TMS. The multiplicities are
abbreviated as follows: s = singlet, d = doublet, t = triplet, q =
quartet, m = multiplet, br = broad signal, and dd = doublet of
doublets. For LC-MS, the ionization method was ESI using Agilent
6430 Triple Quad LC/MS. The animal experiments were approved
by the Institutional Animal Care and Use Committee of
Massachusetts General Hospital and National Institute of
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Development of two novel GluN2B-selective PET radioligands
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Radiological Sciences (Japan). CD1 (ICR) mice (female; 8 weeks,
20–25 g) and Sprague–Dawley rats (male; 7 weeks; 210–230 g)
were kept on a 12 h light/12 h dark cycle and were allowed food
and water ad libitum.
In vitro autoradiography
The general procedure for in vitro autoradiography was described
previously [43, 44] and was used with minor modifications in this
work. Briefly, rat brains were cut into 20 mm sections and stored at
−80 °C until they were used for the experiment. The rat brain
sections were preincubated with Tris·HCl buffer (pH 7.4, 50 mM),
MgCl2 (1.2 mM), and CaCl2 (2 mM) solution for 20 min at ambient
temperature and then incubated with [11C]31 (5.21 MBq/50 mL,
4.37 × 10−4 μg/mL) or [11C]37 (6.07 MBq/50 mL, 5.46 × 10−4 μg/
mL) for 30 min at ambient temperature. For blocking studies, the
blocking reagent (two known GluN2B inhibitors, BMT-108908
(10 μM) and (+)-CP101606 (10 μM), and unlabeled 31 or 37
(10 μM)) was added to the incubation solution in advance to
determine the specificity of the radioligand binding. After
incubation, brain sections were washed twice with Tris buffer for
2 min and dipped in cold distilled water for 10 s. The brain
sections were dried with cold air and then placed on imaging
plates (BASMS2025, GE Healthcare, NJ, USA) for 60 min. Auto-
radiograms were obtained, and ROIs were carefully drawn based
on observation by the naked eye. The radioactivity is expressed as
photostimulated luminescence values per unit area (PSL/mm2),
measured by a Bio-Imaging analyzer system (BAS5000, Fujifilm)
and normalized to a % of radioactivity vs the control.
General procedure A for the synthesis of 30 and 31
A mixture of compound 27 or 28 (1 mmol), (4-fluoro-3-methyl-
phenyl)boronic acid (29, 1.1 mmol, 1.1 equiv), Pd(dppf)Cl2
(0.2 mmol, 0.2 equiv) and Cs2CO3 (3 mmol, 3 equiv) in 11 mL of
a mixture of 1,4-dioxane/H2O = 10/1 (v/v) was heated to 90 °C
under an argon atmosphere for 4 h. After completion of the
reaction, the solvent was removed, and the residue was dissolved
in EtOAc (30 mL) and washed with water (20 mL) and brine
(20 mL). After removing the solvent, 30 and 31 were purified by
chromatography on silica gel (elution with EtOAc/n-hexane = 80/
20, v/v).
General procedure B for the synthesis of 36 and 37
NaH (0.75 mmol, 1.5 equiv, 60 wt% in mineral oil) was carefully
added to a solution of compound 35 (0.5 mmol) in anhydrous
DMF (5 mL) in an ice bath. After stirring for 20 min, intermediate
24 or 25 (0.55 mmol, 1.1 equiv) was added to the reaction, and the
mixture was stirred at rt for another 10 min. The reaction was
quenched by cool water and extracted with EtOAc (20 mL × 3),
and the combined organic layers were washed with water
(20 mL × 2) and brine (20 mL). After removing the solvent, 36
and 37 were purified by chromatography on silica gel (elution
with EtOAc/n-hexane = 80/20, v/v).
PET imaging studies
The general procedure for PET studies was described previously
[43, 44] and was used with minor modifications in this work.
Briefly, PET scans were carried out with an Inveon PET scanner
(Siemens Medical Solutions, Knoxville, TN, USA). Sprague–Dawley
male rats were kept under anesthesia with 1%–2% (v/v) isoflurane
during the scan. [11C]31 or [11C]37 (37 MBq/100 µL,1.55 μg/mL for
[11C]31 and 1.66 μg/mL for [11C]37, respectively) was injected via a
preinstalled catheter via the tail vein. A dynamic scan was
acquired for 90 min in three-dimensional list mode. For pretreat-
ment studies, unlabeled 31 or 37 (1 mg/kg rat weight) dissolved in
300 μL of saline containing 10% ethanol and 5% Tween® 80 was
injected via the pre-embedded tail vein catheter 5 min before the
injection of [11C]31 or [11C]37. 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 hippocampus, thalamus, pons,
striatum, cerebral cortex, and cerebellum, were placed using
ASIPro VM. The radioactivity was decay-corrected, and is
expressed as the standardized uptake value. SUV = (radioactivity
per mL tissue)/(injected radioactivity) × body weight.
General procedure for 11C-methylation
[11C]CH3I was synthesized from cyclotron-produced [11C]CO2,
which was produced by 14N(p,α)11C nuclear reaction. Briefly,
[11C]CO2 was bubbled into a solution of LiAlH4 (0.4 M in THF,
300 μL). After evaporation, the remaining reaction mixture was
treated with hydroiodic acid (57% aqueous solution, 300 μL). The
resulting [11C]CH3I was transferred under helium gas with heating
into a reaction vessel containing a solution of the precursor
(1.0 mg) in presaturated anhydrous DMSO (300 μL) with NaOH
powder (6.0 mg, 85 wt% powder). After the radioactivity reached a
plateau during the transfer, the reaction vessel was warmed to
80 °C and maintained for 5 min. The mobile phase (2.5 mL) and
H2O (1.5 mL) were added to the reaction mixture, which was then
injected into a semipreparative HPLC system. HPLC purification
was completed on a Triart C18 ExRS column (10 mm i.d. ×
250 mm) using the appropriate mobile phases. The radioactive
fraction corresponding to the desired product was collected into a
flask containing polysorbate (80) (75 mL) and ethanol (150 mL),
evaporated to dryness in vacuo, and redissolved in 3 mL of sterile
normal saline. The radiochemical and chemical purities were
measured by analytical HPLC (Triart C18 column 4.6 mm i.d. ×
250 mm, UV at 254 nm; flow rate at 1.0 mL/min). The identities of
[11C]31 and [11C]37 were confirmed by coinjection with unlabeled
31 and 37, respectively.
Whole-body ex vivo biodistribution studies
The general procedure for the ex vivo biodistribution studies was
described previously [43, 44] and was used with minor modifica-
tions in this work. Briefly, a solution of [11C]31 or [11C]37 (1.85
MBq/100 µL) was injected into CD1 mice via the tail vein. These
mice (each time point n = 3–4) were sacrificed at 5, 15, 30, and
60 min post radiotracer injection. The major organs, including the
whole brain, heart, liver, lungs, spleen, kidneys, small intestine
(including contents), muscles, pancreas, stomach, and blood, were
quickly harvested and weighed. The radioactivity present in these
tissues was measured using a Cobra Model 5002/5003 auto-
gamma counter, and all the radioactivity measurements were
automatically decay-corrected based on the half-life of carbon-11.
The results are expressed as the percentage of the injected dose
per gram of wet tissue (% ID/g).
Measurement of lipophilicity
The general procedure for the lipophilicity measurement was
described previously [43–45] and was used with minor modifica-
tions in this work. Briefly, the LogD values were measured by
mixing [11C]31 or [11C]37 (radiochemical purity >99%) with n-
octanol (3.0 g) and PBS (0.1 M, 3.0 g) in a test tube. Both n-octanol
and PBS were presaturated with each other prior to use. The
sample was first vortexed for 5 min and then centrifuged
(~3500–4000 rpm) for an additional 5 min. PBS and n-octanol
were aliquoted and weighed, and the radioactivity in each
component was measured using a Cobra Model 5002/5003
autogamma counter. The LogD was determined from the Log of
the ratio of radioactivity between the n-octanol and PBS solutions
(n = 3).
Radiometabolite analysis
After intravenous injection of [11C]31 through the tail vein, mice
(n = 2) were sacrificed at 30 min post injection. Blood samples
were collected and centrifuged at 14,000 rpm for 3 min at 4 °C to
separate the plasma. The supernatant was collected and added to
an ice-cooled test tube containing 100 μL of CH3CN. After
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Development of two novel GluN2B-selective PET radioligands
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vortexing for 10 s, the mixture was centrifuged at 14,000 rpm for
3 min at 4 °C for deproteinization. The supernatant was collected,
and the process was repeated until no precipitate was observed
when CH3CN was added. The mouse brains were immediately
dissected, homogenized with 400 μL of ice-cooled CH3CN, and
then centrifuged at 14,000 rpm for 5 min at 4 °C. The supernatant
was collected into a test tube containing 100 μL of ice-cooled
CH3CN, and the process (vortex and centrifuge) was repeated until
no precipitate was observed when CH3CN was added. The
resulting supernatants of the plasma and brain samples were
mixed with 50 μL of a solution of cold compound 31 and then
analyzed on a radio-HPLC system using a Phenomenex C18
column (5 μm, 10 × 250 mm, 5 mL/min, elution: CH3CN/50 mM
AcONH4 = 55%/45%). The radioactivity was collected and counted
using a 1480 Wizard gamma counter (PerkinElmer, USA). The
radioactivity overlapping with 31 in the UV signal corresponded to
that of [11C]31 (tR = 6.50 min). The percentage of [11C]31 to the
total radioactivity was calculated as (counts for [11C]31/total
counts) × 100.
Statistical analysis
Statistical analysis was performed by Student’s two-tailed t test,
and asterisks are used to indicate statistical significance: *P < 0.05,
**P ≤ 0.01, ***P ≤ 0.001, and ****P < 0.0001.
RESULTS AND DISCUSSION
Chemistry
Two GluN2B-selective NAMs, compounds 31 and 37, and their
corresponding 11C-labeling precursors were obtained following
the synthetic routes in Scheme 1. Amides 24 and 25 were
obtained through amidation of 2-bromoacetic acid (23) with the
corresponding amines. The SN2 reactions between amides 24 or
25 and 6-bromo-1H-pyrrolo[3,2-b]pyridine (26) in the presence of
NaH gave intermediates 27 and 28 in 52% and 61% yields,
respectively. Desired standard compound 31 and the correspond-
ing precursor 30 were obtained via palladium-catalyzed cross-
coupling reactions between 28 or 27 and substituted aromatic
boronic acid 29 in 79% and 61% yields, respectively (Scheme 1a).
Unfortunately, an analogous synthetic strategy was not suitable
for the preparation of 36 and 37, which bear a thiophene
fragment. The existence of N–H functional groups in 27 and 26
hampered their cross-coupling reactions with thiophene borate
33 (Scheme 1c). To our delight, palladium-catalyzed cross-
coupling reactions between Boc-protected intermediate 32 and
thiophene borate 33 provided key intermediate 35 and Boc-
protected product 34 (converted to 35 under the treatment with
3 N aqueous HCl) in high efficiency. Specifically, compound 35
was synthesized from 32 in 66% overall yield over two steps. In all,
the final synthesis was completed with the SN2 reactions between
35 and α-bromo amide 25 or 24 to give standard compound 37
and the corresponding precursor 36 in 36% and 29% yields,
respectively (Scheme 1b).
Scheme 1 Synthesis of GluN2B-selective NMDAR antagonists and
corresponding precursors. Reagents and conditions: a (i) (1) 1.5
equiv (COCl)2, DCM, DMF, rt, 30 min; (2) 1.2 equiv H2NMe (2 M in
THF) for 24 or HNMe2 (2 M in THF) for 25, DCM, 0 °C to rt, 4 h, 23%
for 24, 43% for 25; (ii) 1 equiv of 26, 1.5 equiv NaH, DMF, rt, 2 h, 52%
for 27, 61% for 28; (iii) 1.1 equiv of 29, 0.2 equiv Pd(dppf)Cl2, 3 equiv
Cs2CO3, 1,4-dioxane/H2O = 10/1, 90 °C, 4 h, 61% for 30, 79% for 31;
b (iv) 1.2 equiv Boc2O, 1 equiv DMAP, 1 equiv Et3N, DCM, rt,
overnight, 82%; (v) 1 equiv 33, 0.2 equiv Pd(dppf)Cl2, 3 equiv
Cs2CO3, 1,4-dioxane/H2O = 5/1, 90 °C, overnight; (vi) 20 equiv 3 N
HCl, THF, reflux, 1 h, 66% overall for 35 from 32 and 34; (vii) 1.1 equiv
24 or 25, 1.5 equiv NaH, DMF, 0 °C to rt, 10 min, 29% for 36, 36% for
37; c (viii) 1.1 equiv of 33, 0.2 equiv Pd(dppf)Cl2, 3 equiv Cs2CO3, 1,4-
dioxane/H2O = 10/1, 90°C, 4 h, 0% for 35; (ix) 1.1 equiv of 33, 0.2
equiv Pd(dppf)Cl2, 3 equiv Cs2CO3, 1,4-dioxane/H2O = 10/1, 90°C,
4 h, 0% for 36; DCM dichloromethane, DMF N,N-dimethylformamide,
THF tetrahydrofuran, Pd(dppf)Cl2 [1,1′-bis(diphenylphosphino)ferro-
cene]dichloropalladium(II), Boc2O di-tert-butyl dicarbonate, DMAP 4-
dimethylaminopyridine.
Radiochemistry
As shown in Scheme 2, the N-methyl amide moieties of precursors
30 and 36 were selected as the most convenient sites for labeling
with [11C]CH3I [46]. Initially, [11C]CH3I was transferred into a
reaction vessel containing precursor 30 in a mixed solvent of
DMSO and aqueous KOH (45 wt%), followed by heating at 130 °C
for 5 min. However, only trace amounts of [11C]31 were obtained
by analytic radio-HPLC (radiochemical conversion, RCC < 5%). In
our modified method, a mixture of DMSO and aqueous KOH was
replaced with presaturated anhydrous DMSO with NaOH powder
at rt. The reaction mixture was heated to 80 °C and maintained for
5 min after the completion of the [11C]CH3I transfer (Scheme 2a).
The resulting mixture was purified by semiprep radio-HPLC to
provide desired ligand [11C]31 in an average of 28% (n = 2) decay-
corrected radiochemical yield (RCY) relative to starting [11C]CO2 at
the end of the synthesis, and the obtained material showed high
radiochemical purity (>99%) and high molar activity (>74 GBq/
μmol). Following a similar protocol, [11C]37 was obtained in 32%
(n = 2) decay-corrected RCY with a high radiochemical purity
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(>99%) and high molar activity (>74 GBq/μmol). No radiolysis was
observed within 90 min of formulation. The lipophilicities of [11C]
31 and [11C]37 were measured through the “shake flask” method
[45], and the results indicated that both radiotracers possessed
favorable logD values (2.81 0.01 and 3.18 0.03, respectively) for
brain penetration [47].
2-(6-(4-Fluoro-3-methylphenyl)-1H-pyrrolo[3,2-b]pyridin-1-
yl)-N-(λ1-methyl-11C)-N-methylacetamide ([11C]31): According
to the general procedure for 11C-methylation, the retention time
for [11C]31 was 10.0 min. The synthesis time was ∼40 min from the
end of the bombardment. HPLC purification was completed on a
Triart C18 ExRS column (10 mm i.d. × 250 mm) using a mobile
phase of CH3CN/50 mM AcONH4 = 45/55 at a flow rate of 4.0 mL/
min. The radiochemical and chemical purities were measured by
analytical HPLC (Triart C18 column, 4.6 mm i.d. × 250 mm, UV at
254 nm; CH3CN/50 mM AcONH4 = 50/50 at a flow rate of 1.0 mL/
min). The decay-corrected radiochemical yield was 27.8% 7.2%
(n = 2) based on [11C]CO2 with >99% radiochemical purity and
greater than 74 GBq/μmol molar activity.
2-(6-(5-Chloro-4-methylthiophen-2-yl)-1H-pyrrolo[3,2-b]pyri-
din-1-yl)-N-(λ1-methyl-11C)-N-methylacetamide ([11C]37): Accord-
ing to the general procedure for 11C-methylation, the retention time
for [11C]37 was 10.2 min. The synthesis time was ∼40 min from the
end of the bombardment. HPLC purification was completed on a
Triart C18 ExRS column (10 mm i.d. × 250 mm) using a mobile phase
of CH3CN/50 mM AcONH4 = 50/50 at a flow rate of 5.0 mL/min. The
radiochemical and chemical purities were measured by analytical
HPLC (Triart C18 column, 4.6 mm i.d. × 250 mm, UV at 254 nm;
CH3CN/50 mM AcONH4 = 50/50 at a flow rate of 1.0 mL/min). The
decay-corrected radiochemical yield was 31.6% 4.7% (n = 2) based
on [11C]CO2 with >99% radiochemical purity and greater than 74
GBq/μmol molar activity.
Autoradiography
With two 11C-labeled radioligands in hand, in vitro autoradio-
graphy studies were carried out in brain sections of Sprague–
Dawley rats to evaluate the binding specificity of the radiotracers
towards the GluN2B subunit (Fig. 2). As shown in Fig. 2a, [11C]31
exhibited a heterogeneous distribution of radioactivity in rat brain
sections at the baseline, in which the uptakes decreased in the
order of hippocampus, striatum, thalamus, and cortex. The
weakest signal was detected in the cerebellum, which is in
accordance with the expression pattern of GluN2B subunits in
rodents [11–14]. In the blocking studies (Fig. 2b), pretreatment
with “cold” (nonradioactive) compound 31 substantially reduced
the uptake of [11C]31. Blocking studies with commercially
available GluN2B inhibitors, BMT-108908 and (+)-CP101606,
showed a similar reduction in bound activity, which confirmed
the specific binding of [11C]31 to GluN2B. In in vitro ARG studies of
[11C]37 (Fig. 2c, d) showed 34%, 30%, and 37% reduced binding in
the hippocampus by pretreatment with 37, BMT-108908 and
(+)-CP101606, respectively. In addition, up to a 30% reduced
uptake of [11C]37 in the cerebral cortex compared with that of the
baseline was detected in the blocking studies. Encouraged by our
in vitro experimental results, we performed an in vivo evaluation
of these radiotracers by PET imaging studies.
Scheme 2 Radiosynthesis of GluN2B-selective radiotracers [11C]31 (a)
and [11C]37 (b).
Fig. 2 In vitro autoradiography results of [11C]31 and [11C]37 in rat brain sections. a [11C]31 (baseline), pretreatment with 31 (self-blocking,
10 μM), BMT-108908 (10 μM) and (+)-CP101606 (10 μM). b Quantitative analysis of the baseline and blocking experiments (n = 4, see
quantitative data in Supporting Information Table). c [11C]37 (baseline), pretreatment with 37 (self-blocking, 10 μM), BMT-108908 (10 μM) and
(+)-CP101606 (10 μM). d Quantitative analysis of the baseline and blocking experiments (n = 4, see quantitative data in Supporting
Information Table). The value is expressed as the ratio of the radioactivity of different brain regions to that of the cerebellum (reference
region). Hi hippocampus, Cx cerebral cortex, St striatum, Th thalamus, Cb cerebellum. Asterisks indicate statistical significance: ****P < 0.0001,
***P < 0.001, **P < 0.01, *P < 0.05, and N.S. = no statistics.
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Fig. 3 Representative PET images and time–activity curves of [11C]31 and [11C]37 in rat brain. a Baseline PET images (summed 0–20 min,
20-40 min, 40–60 min, and 60–90 min) of [11C]31. b Time–activity curves in different brain regions at baseline. c Baseline PET images (summed
0–20 min, 20-40 min, 40–60 min, and 60–90 min) of [11C]37. d Time–activity curves in different brain regions at baseline.
Fig. 4 Whole-body ex vivo biodistribution in mice at four different time points (5, 15, 30, and 60 min, n = 3–4) post injection. a [11C]31
and b [11C]37. Asterisks indicate statistical significance: ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05.
PET study
d, respectively. Unfortunately, both ligands displayed homoge-
nous distributions, inconsistent with the expression pattern of
GluN2B subunits in previously reported works [11–14], and the
uptakes of [11C]31 and [11C]37 in rat brain were limited to a peak
SUV of 0.5–0.7, respectively, at ca. 2 min. Although [11C]31 and
[11C]37 possess favorable logD values (2.81 0.01 and 3.18 0.03,
Dynamic PET scans (0-90 min) were carried out with [11C]31 and
[11C]37 in Sprague–Dawley rats. Representative PET images
(coronal and sagittal, summed images for [11C]31 in Fig. 3a and
[11C]37 in Fig. 3c) in the whole brain and time–activity curves of
six representative brain regions (baseline) are shown in Fig. 3b and
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Development of two novel GluN2B-selective PET radioligands
JY Sun et al.
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respectively) for brain penetration, high plasma protein binding
(reported as 95.53% for 31 and 96.68% for 37 in rats [42]) could
potentially reduce the available radiotracers in the plasma that
can traverse the BBB. The self-blocking studies by pretreatment
with the corresponding “cold” compounds showed no substantial
reductions in brain uptake, indicating low levels of binding
specificity (Supporting Information).
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ACKNOWLEDGEMENTS
We thank Drs. Thomas J. Brady and Lei Zhang for their helpful discussion. This work
was financially supported by NSFC (No. 81901802, No. 81701751, No. 81871383),
Postdoctoral Fund of the First Affiliated Hospital, Ji-nan University (No. 801328) to
JYS, the Science and Technology Program of Guangzhou (201804010440), and the
Project of Innovative Team for the Guangdong Universities (2018KCXTD001).The
characterization of small molecules in this work was performed (in part) using the
JEOL 500 MHz NMR Spectrometer that was purchased with funding from a National
Institutes of Health SIG grant (S10OD025234).
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