Synthesis and Preliminary Evaluations of a Triazole-Cored Antagonist as a PET Imaging Probe ([18F]N2B-0518) for GluN2B Subunit in the Brain

Article abstract of DOI:10.1021/acschemneuro.8b00591

GluN2B is the most studied subunit of N-methyl-d-aspartate receptors (NMDARs) and implicated in the pathologies of various central nervous system disorders and neurodegenerative diseases. As pan NMDAR antagonists often produce debilitating side effects, new approaches in drug discovery have shifted to subtype-selective NMDAR modulators, especially GluN2B-selective antagonists. While positron emission tomography (PET) studies of GluN2B-selective NMDARs in the living brain would enable target engagement in drug development and improve our understanding in the NMDAR signaling pathways between normal and disease conditions, a suitable PET ligand is yet to be identified. Herein we developed an ¹⁸F-labeled potent antagonist, 2-((1-(4-[¹⁸F]fluoro-3-methylphenyl)-1H-1,2,3-triazol-4-yl)methoxy)-5-methoxypyrimidine ([¹⁸F]13; also called [¹⁸F]N2B-0518) as a PET tracer for imaging the GluN2B subunit. The radiofluorination of [¹⁸F]13 was efficiently achieved by our spirocyclic iodonium ylide (SCIDY) method. In in vitro autoradiography studies, [¹⁸F]13 displayed highly region-specific binding in brain sections of rat and nonhuman primate, which was in accordance with the expression of GluN2B subunit. Ex vivo biodistribution in mice revealed that [¹⁸F]13 could penetrate the blood-brain barrier with moderate brain uptake (3.60% ID/g at 2 min) and rapid washout. Altogether, this work provides a GluN2B-selective PET tracer bearing a new chemical scaffold and shows high specificity to GluN2B subunit in vitro, which may pave the way for the development of a new generation of GluN2B PET ligands.

Full text of DOI:10.1021/acschemneuro.8b00591

Subscriber access provided by Iowa State University | Library
Article
Synthesis and Preliminary Evaluations of a Triazole-cored Antagonist
([18F]N2B-0518) as PET Imaging Probe for GluN2B Subunit in the Brain
Hualong Fu, Weiting Tang, Zhen Chen, Vasily V. Belov, Genwei Zhang, Tuo Shao,
Xiaofei Zhang, Qingzhen Yu, Jian Rong, Xiaoyun Deng, Wei Han, Scott Myers, Pilar
Giffenig, Lu Wang, Lee Josephson, Yihan Shao, April Davenport, James Daunais,
Mikhail I Papisov, Hongjie Yuan, Zijing Li, Stephen F. Traynelis, and Steven H Liang
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00591 • Publication Date (Web): 30 Jan 2019
Downloaded from http://pubs.acs.org on February 1, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 33
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 2 of 33
1
2
3
4
5
6
Synthesis and Preliminary Evaluations of a Triazole-cored Antagonist ([18F]N2B-
0518) as PET Imaging Probe for GluN2B Subunit in the Brain
7
8
Hualong Fu,† Weiting Tang,§,○ Zhen Chen,† Vasily V. Belov,ǁ Genwei Zhang,┴ Tuo Shao,† Xiaofei
9
Zhang,† Qingzhen Yu,† Jian Rong,† Xiaoyun Deng,† Wei Han,§, Scott J. Myers,§ Pilar Giffenig,ǁ Lu
Wang, †,∆ Lee Josephson,† Yihan Shao,┴ April T. Davenport,# James B. Daunais,# Mikhail Papisov,ǁ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
‡
Hongjie Yuan,§ Zijing Li,*, Stephen F. Traynelis,*,§ and Steven H. Liang*,†
†Department of Radiology, Division of Nuclear Medicine and Molecular Imaging, Massachusetts General
Hospital and Harvard Medical School, 55 Fruit Street, Boston, Massachusetts 02114, United States
§Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322, United States
○Department of Neurology, Xiangya Hospital, Central South University, Changsha, Hunan, 410008, P. R.
China
ǁDepartment of Radiology, Massachusetts General Hospital, Harvard Medical School, and the Shriners Burns
Hospital, Boston, Massachusetts 02114, United States
┴Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019, United
States

Department of Neurology, Children’s Hospital of Chongqing Medical University, Chongqing, 400014, P. R.
China
#Department of Physiology and Pharmacology, Wake Forest School of Medicine, Winston Salem, North
Carolina 27157, United States
∆Department of Nuclear Medicine and PET/CT-MRI Center, the First Affiliated Hospital of Jinan University
& Institute of Molecular and Functional Imaging, Jinan University, Guangzhou 510630, P. R. China
‡State Key Laboratory of Molecular Vaccinology, Molecular Diagnosis & Center for Molecular Imaging and
Translational Medicine, School of Public Health, Xiamen University, Xiamen 361102, P. R. China
ABSTRACT
GluN2B is the most studied subunit of N-methyl-D-aspartate receptors (NMDARs) and implicated
in the pathologies of various central nervous system disorders and neurodegenerative diseases. As
ACS Paragon Plus Environment

Page 3 of 33
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
pan NMDAR antagonists often produce debilitating side effects, new approaches in drug discovery
have shifted to subtype-selective NMDAR modulators, especially GluN2B-selective antagonists.
While positron emission tomography (PET) studies of GluN2B-selective NMDARs in the living
brain would enable target engagement in drug development and improve our understanding in the
NMDAR signaling pathways between normal and disease conditions, a suitable PET ligand is yet
to be identified. Herein we developed an 18F-labeled potent antagonist, 2-((1-(4-[18F]fluoro-3-
methylphenyl)-1H-1,2,3-triazol-4-yl)methoxy)-5-methoxypyrimidine ([18F]13; also called
[18F]N2B-0518) as a PET tracer for imaging the GluN2B subunit. The radiofluorination of [18F]13
was efficiently achieved by our spirocyclic iodonium ylide (SCIDY) method. In in vitro
autoradiography studies, [18F]13 displayed highly region-specific binding in brain sections of rat
and non-human primate, which was in accordance with the expression of GluN2B subunit. Ex vivo
biodistribution in mice revealed that [18F]13 could penetrate the blood-brain barrier with moderate
brain uptake (3.60% ID/g at 2 min) and rapid washout. Altogether, this work provides a GluN2B-
selective PET tracer bearing new chemical scaffold and shows high specificity to GluN2B subunit
in vitro, which may pave the way for the development of a new generation of GluN2B PET ligands.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
18
Keywords: subtype-selective, PET imaging, GluN2B subunit, F-labeling, spirocyclic iodonium
ylide, autoradiography
INTRODUCTION
N-Methyl-D-aspartate receptors (NMDARs) are ligand-gated ion channel ionotropic glutamate
receptors (iGluRs) that are fundamental to excitatory neurotransmission in the mammalian central
2
nervous system (CNS).1, NMDARs are involved in numerous neurological disorders and
neurodegenerative diseases such as ischemic insults, epilepsy, Parkinson’s disease and Alzheimer’s
disease (AD).3, 4 Several pan NMDAR antagonists including phencyclidine (PCP), ketamine, MK-
801, Memantine, and amantadine were developed and used in the clinical setting;5-8 however, these
drugs often showed debilitating adverse effects and thus had a very narrow therapeutic window.7, 9
Of note, NMDARs are typically assembled as tetramers containing two GluN1 subunits and two
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 4 of 33
1
2
3
4
5
6
7
8
9
GluN2 subunits.10 The GluN2 subunits, which include four subtypes of GluN2A−GluN2D, have
distinct developmental expression patterns in CNS and control a variety of functional properties of
NMDARs.11-13 For example, the GluN2B subunit is widespread throughout the brain with strong
expression levels around birth; but in adults, becomes progressively restricted to the forebrain while
barely detectable in cerebellum.11, 12, 14, 15 This subunit endows the NMDARs with various binding
sites for both positive allosteric modulators (PAM, e.g., polyamines) and subunit-selective negative
allosteric modulators (NAM, e.g., ifenprodil)8, 16-19 and thus attracts sufficient attention in drug
development as a therapeutic target of high interest for various CNS pathologies, such as cerebral
ischemia, acute and chronic pain, schizophrenia, and AD.5, 7, 20, 21 The discovery of the first GluN2B-
selective NMDAR antagonist, ifenprodil, provided an alternative solution for the treatment of
NMDAR-related diseases.22 Compared with the pan NMDAR antagonists, ifenprodil uses a unique
activity-dependent mechanism that avoids excessive blockade by leaving transiently activated
receptors relatively unaffected, which could mitigate its adverse effect at high drug doses.18, 23-25
The finding of ifenprodil has stimulated the continuous development of other potent GluN2B-
selective antagonists and several drug candidates have entered human trials, including CP-101606
(traxoprodil),26, 27 RGH-896 (radiprodil),8 MK-0657 (rislenemdaz),28, 29 and EVT-101.30 However,
despite multiple clinical trials, no GluN2B-selective antagonists have been approved for use in man
by FDA.4, 8 Thus, a targeted probe that enables the understanding of in vivo molecular progress of
GluN2B subunit is critical in assessing the future prospects for drug development, like facilitating
drug candidates by evaluating the receptor occupancy.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Page 5 of 33
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. Chemical structures of representative GluN2B-selective NMDAR PET tracers.
Positron emission tomography (PET) imaging of NMDAR GluN2B subunit could help to
diagnose and monitor related neurological disorders and further validate treatment intervention and
efficacy. In parallel with drug discovery, there is an unmet clinical need for GluN2B-selective
NMDAR PET tracers in basic and clinical research. There are continuous research efforts on the
development of 11C- or 18F-labeled probes targeting the GluN2B subunit of NMDAR, which have
been applied for PET imaging studies in rats or non-human primates (NHP).31-33 Among them,
ifenprodil-like ligands (±)[11C]methoxy-CP-101606 ((±)[11C]1)34 and [11C]EMD-95885 ([11C]2,
Figure 1)35 displayed low brain uptake and homogenous distribution in different brain regions,
which was inconsistent with GluN2B expression in the brain.11, 12, 14, 15 In 2004, a pyridine derivative
[11C]Ro-647312 ([11C]3, Figure 1) also showed homogenous brain distribution in rat brain and
exhibited high plasma/brain uptake ratio by PET.36 In 2006, a benzamidine derivative [11C]4 (Figure
1) demonstrated poor brain uptake and rapid metabolism in the rat brain, which impeded its further
application in PET imaging.37 Although in vitro studies revealed two 18F-labeled probes [18F]trans-
5 and [18F]cis-5 (Figure 1) had promising Bmax/Kd ratios (8–37) in different rat brain regions, PET
studies failed to observe significant difference of radioactivity signals in different brain regions.38,
39
More recently, [11C]Me-NB1 ([11C]6, Figure 1) was generated from a benzo[d]azepin GluN2B
subunit antagonist.40 [11C]6 had high affinity (Ki = 5.3 nM) to GluN2B subunit and was successfully
used in measurement of receptor occupancy by eliprodil (an NMDAR antagonist). It is worthy of
note that the specific uptake of [11C]6 in rat brain was also affected by (+)-pentazocine (a σ-1
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 6 of 33
1
2
3
4
5
6
7
8
9
receptor agonist) and haloperidol (an inverse agonist of the σ-1 receptor) blocking studies, which
was explained by the indirect effect of the σ-1 receptor in vivo. Furthermore, Cai et al. reported an
interesting sulfur analog of benzo[d]azepin PET tracer [11C]NR2B-SMe ([11C]7, Figure 1).41 In PET
imaging of rats, the brain uptake of [11C]7 was reduced by the pretreatment with eliprodil, ifenprodil,
and NR2B-SMe (self-blocking), as well as a σ-1 receptor agonist SA 4503 in a dose-dependent
manner. Despite continuous research efforts in the development of PET ligands for this target, to
date, no PET tracer of GluN2B subunit has been advanced to human study and PET imaging of
GluN2B subunit remains challenging.31-33
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Herein we developed a new series of fluorinated compounds that are amenable for 18F-labeling
based on a novel class of GluN2B antagonists recently disclosed from Janssen Pharmaceutical
patents,42 with the aim to discover new generation of GluN2B PET ligands with diverse chemotypes
and pharmacology. Starting from an efficient parallel synthesis of several methoxypyrimidine
compounds (11–14), we performed preliminary pharmacological evaluation and identified two
promising molecules (11 and 13) with high potency and selectivity to GluN2B subunit. Molecular
docking studies also indicated several key interactions including π-π stacking and hydrogen bonds
were found to support our pharmacology findings. As for radiochemistry, our spirocyclic iodonium
ylide (SCIDY) method43, 44 enables rapid, efficient and modular synthesis of [18F]11 and [18F]13 in
high radiochemistry yields (RCYs), which is otherwise difficult to access by traditional aromatic
nucleophilic substitution. The following in vitro autoradiography (ARG) in brain sections of both
rat and NHP demonstrated that [18F]13 could bind to GluN2B subunit with high specificity.
Furthermore, ex vivo biodistribution in mice revealed that [18F]13 possessed moderate brain uptake,
although short retention and fast clearance was also observed. As proof-of-concept, our work
provides a preliminary evaluation of new potent and selective triazole-based PET probes targeting
GluN2B subunit and may lead to new chemotypes to discover new chemical scaffolds for NMDAR
drug discovery and PET tracer development.
RESULTS AND DISCUSSION
ACS Paragon Plus Environment

Page 7 of 33
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
Chemistry. The synthesis of new GluN2B-selective NMDAR antagonists amenable for
radiolabeling (11−14) is illustrated in Scheme 1. The synthesis of 11 commenced with the
preparation of aromatic azide 9. In acidic conditions, aniline 8 reacted with sodium nitrite and
subsequently sodium azide, which resulted in diazonium salts in situ and then converted into azide
9 in a high yield of 89% over two steps. The key triazole intermediate 10 was prepared by copper(I)-
induced alkyne−azide cycloaddition ‘click’ reactions between azidobenzene 9 and propargyl
alcohol in a high yield of 71% as a white solid. Then the nucleophilic aromatic substitution (SNAr)
reaction between triazole 10 and 2-bromo-5-methoxypyrimidine afforded the target compound 11
in a moderate yield of 29%. In brief, the synthesis of 11 was efficiently achieved in three steps with
an overall yield of 18%. In an analogous manner, the other target compounds 12−14 were obtained
as white solids in overall yields of 13%−18%.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 1. Synthesis of GluN2B-selective NMDAR antagonists (11–14).
Reagents and conditions: (a) NaNO2, NaN3, HCl (6 N), 0–5 °C, 2 h, 89% yield. (b) Propargyl alcohol, DIPEA, CuI, THF,
40 °C, 2 h, 71% yield. (c) 2-Bromo-5-methoxypyrimidine, NaH, THF, 40 °C, 2–3 h, 29% yield.
Pharmacology and Physicochemical Properties.
In most cases, NMDARs are dimer of dimers containing two glycine-binding GluN1 and two
glutamate-binding GluN2 subunits, and their functionating relies on joint action of glycine and
glutamate.2, 10 The potencies of compounds 11−14 as GluN2B-selective antagonists were evaluated
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 8 of 33
1
2
3
4
5
6
7
8
9
via glutamate/glycine (100 μM/100 μM) assays with Xenopus oocytes expressing human
GluN1/GluN2B (GenBank NP_015566/GenBank NP_000825) receptors. The current responses of
GluN1/GluN2B receptors were inhibited by 11−14 in a dose-dependent manner (Figure 2A). As
shown in Table 1, 11 had the highest potency with the IC50 value of 19 nM, followed by 13 with
the value of 28 nM. However, the potencies of 12 and 14 (positional isomers of 11 and 13,
respectively) significantly decreased to 339 and 89 nM (IC50 values), respectively. We also
evaluated the subtype-selectivity of compounds 11 and 13 for GluN2B subunit over other GluN2
subunits. Xenopus oocytes expressing GluN1 with human GluN2A, rat GluN2C, or human GluN2D
subunit were used, and current responses to maximal agonists (glutamate/glycine, 100 μM/100 μM)
concentrations were recorded in the presence of 11 or 13 (1 μM). The activity of GluN1/GluN2B
receptors was substantially inhibited by 11 and 13 with the %current responses of 9.3% and 15.0%,
respectively (Table 1 and Figure 2B). In contrast, the current responses of other iGluRs including
GluN1/GluN2A, GluN1/GluN2C, GluN1/GluN2D, GluA1, and GluK2 were virtually not affected
by 11 or 13 (Table 1 and Figure 2B).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Two different GluN2 subunits, GluN2A and GluN2B, have been found in most NMDAR-
expressing cells and are assembled as triheteromers like GluN1/GluN2A/GluN2B (2A/2B) and
diheteromers like GluN1/GluN2A/GluN2A (2A/2A) and GluN1/GluN2B/GluN2B (2B/2B).2, 45
Thus, we further compared compounds 11 and 13 inhibition of triheteromeric receptors (2A/2B) to
that of diheterometric 2A/2A and 2B/2B receptors. Compounds 11 and 13 inhibited current
responses from wild-type 2B/2B with an IC50 of 11 ± 2.3 and 20 ± 3.4 nM, maximal inhibition of
86% ± 1.8% (n = 11) and 88% ± 1.8% (n = 11), respectively, whereas responses from wild-type
2A/2A were insensitive to 1 μM 11 (n = 10) and 13 (n = 11) (Figures 2C and 2D). The inhibition of
2A/2B triheteromeric receptors by 11 and 13 (1 μM) was reduced compared to that of 2B/2B, since
IC50 values increased to 141 ± 8.9 nM for 11 and 154 ± 28 nM for 13, and maximal inhibition was
23% ± 2.5% (n = 11) for 11 and 27% ± 2.0% for 13 (n = 13). Similar to the effects of ifenprodil,45
our data suggest that reduced sensitivity of triheteromers to these two compounds (11 and 13) is
mainly mediated by a diminished maximal inhibition, in addition to reduction in potency. As a
ACS Paragon Plus Environment

Page 9 of 33
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
result, 11 and 13 are identified as potent GluN2B-selective antagonists with high selectivity over
other GluN2 subunits and iGluR units, representing a promising chemical scaffold for GluN2B-
selective NMDAR PET ligands.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. Pharmacology studies of our GluN2B-selective NMDAR antagonists. (A) Concentration−response curves for
antagonists 11−14 (0.03−1.0 μM) on human GluN1/GluN2B were plotted as the percent of the maximal response to
glutamate/glycine (100 μM/100 μM) and fit by the Hill equation. (B) %Current responses to glutamate/glycine (100
μM/100 μM for NMDAR) or glutamate (100 μM for AMPAR and KAR) co-applied with compound solution (1 μM) of
11 or 13 were recorded in Xenopus oocytes expressing human GluN1/GluN2A receptors, human GluN1/GluN2B
receptors, human GluN1/GluN2D receptors, rat GluN1/GluN2C receptors, rat GluA1(flip) subunit or rat GluK2(Q)
subunit. The data were expressed as the percent of the maximal response to agonists. (C and D) Inhibition of
triheteromeric receptors by compounds 11 (C) and 13 (D), respectively. Concentration−response curves were generated
from the triheteromeric receptors including GluN1/GluN2A/GluN2A (2A/2A), GluN1/GluN2B/GluN2B (2B/2B), and
GluN1/GluN2A/GluN2B (2A/2B) upon activated by glutamate/glycine (100 μM/100 μM). Data are mean  SEM from
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 10 of 33
1
2
3
4
10–14 oocytes.
5
6
7
8
9
Table 1. Potency and selectivity of compounds 11−14
potencya
selectivityb
%control at 1 μM, mean
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
entry
IC50, mean (nM)
(99% CI)
pIC50, mean (95% CI)
GluN2A GluN2B GluN2C GluN2D
GluA1
97
GluK2
92
19
102
13
93
92
11
12
13
14
-1.72 (-1.75, -1.69)
339
(93, 111)
(7, 19)
(92, 94)
(88, 96)
(90, 104) (84, 100)
n.d.
-0.55 (-0.76, -0.34)
28
99
17
95
96
98
91
-1.57 (-1.63, -1.51)
89
(93, 105) (11, 23) (92, 98)
(94, 98)
(91, 105) (80, 102)
n.d.
-1.12 (-1.30, -0.94)
aDetermined with Xenopus oocytes expressing human GluN1/GluN2B receptors in 100 M glutamate/glycine
assay coapplied with increasing concentrations of 11−14 (n = 6−12). b%Control response was expressed as the percent
of the maximal response to 100 M glutamate/glycine (for GluN2a-GluN2D subunits), or to 100 M glutamate (for
GluA1 and GluK2 subunits). Xenopus oocytes coexpressing human GluN1/GluN2A receptors, human GluN1/GluN2B
receptors, human GluN1/GluN2D receptors, rat GluN1/GluN2C receptors, rat GluA1(flip) subunit or rat GluK2(Q)
subunit were used. n.d., not determined.The lipophilicity with preferred range of 2.0−3.5 in candidate
compounds is an empirical factor that could predict the ability of the blood-brain barrier (BBB)
penetration.46-48 The calculated logP (clogP) values of 11−14 are 3.32, 3.32, 2.90, and 2.90,
respectively (Table 2). The logD values of [18F]11 and [18F]13 (see radiochemistry below, Scheme
2) were determined to be 3.14 and 2.89 by “shake-flask” method,49 respectively, which are within
the optimal range for BBB permeability. In silico prediction gave a reasonable topological polar
surface area (tPSA) value of 71.4 Å (<90 Å) for all the compounds (Table 2), which indicated their
great potentials as brain penetrant leads.50 We also explored the CNS PET multiparameter
optimization (MPO) scores of these compounds. As reported, a higher MPO score of a compound
often leads to a higher probability in achieving optimal CNS drug-like properties such as
ACS Paragon Plus Environment

Page 11 of 33
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
permeability, clearance, and safety.51, 52 As shown in Table 2, 13 and 14 had preferred MPO
scores >4.9 while 11 and 12 exhibited a lower value of 4.05. It is worth mentioning that compound
13 has the highest MPO score of 5.16, which is situated in the optimal range (>5) that is
characteristic of successful PET ligands.53 These results led to further evaluations of [18F]13 in in
vitro ARG and ex vivo biodistribution studies.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 2. Physicochemical properties of compounds 11−14.
b
compound clogPa
logD7.4
tPSA (Å)a MPOc
11
12
13
14
3.32 3.14 ± 0.037
3.32 n. d.
2.90 2.89 ± 0.037
2.90 n. d.
71.4
71.4
71.4
71.4
4.05
4.05
5.16
4.91
aClogP and tPSA values were calculated by ChemBioDraw Ultra 14.0. bLogD was measured with “shake-flask” method
by using corresponding 18F-labeled tracers (18.5 MBq) and expressed as mean ± SD (n = 3). cMPO scores were calculated
using the method reported in Zhang, L.et al. 53. n. d., not determined.
Molecular Docking Studies
A 2.6-Å resolution crystal structure of the NMDA receptor subunit GluN1b and GluN2B
complex was downloaded (PDB ID: 3QEL)18 and the original ligand, ifenprodil, was removed
before performing the molecular docking studies. The goal is to identify possible and preliminary
molecular interaction between our GluN2B inhibitors 11 & 13 and binding domain based on the
fact that low nanomolar binding affinities were identified. First, we reproduced the binding pose of
the original ligand, ifenprodil, within 3QEL using AutoDock Vina, which yielded a nearly-perfect
overlapping between the docked pose and original pose (RMSD = 0.36 Å). Subsequently, candidate
compounds 11 and 13 were docked onto the 3QEL structure in the ifenprodil site. A consensus
binding pose with highest docking scores was found for these two compounds within the ifenprodil
binding channel, which is the interface between subunits GluN1b and GluN2B (Figure 3). Inside
the binding pocket, angled π-π stacking interactions were observed between the phenyl group of
either compound and nearby Phe429 and Tyr82 side chains (Figure 3). Multiple hydrogen bonds
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 12 of 33
1
2
3
4
5
6
7
8
9
were also found between the pyrimidine moiety of the compounds and Arg88 side chain. In addition,
the charged side chain of Glu421 was found ~3Å away from both compounds, indicating possible
electrostatic interactions. The high docking scores of 11 and 13 (-9.1 and -9.4, respectively)
indicated strong bindings between the compounds and NMDAR subunit, which agree well the
experimental IC50 data in Table 1. Overall, this preliminary docking study showed that compounds
11 and 13 interacted with NMDAR subunit at the ifenprodil site with π-π stacking interactions,
hydrogen bonds, and possibly electrostatic interactions.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3. Molecular docking structures of compounds 11 (purple in A) and 13 (orange in B) onto NMDAR subunit
GluN1b and GluN2B interface.
Radiochemistry. SNAr reactions with [18F]F- is one of the widely used and highly attractive
methods for the synthesis of 18F-labeled aromatic PET ligands.54 In our initial design of the labeling
strategy, we took advantage of SNAr reactions to incorporate fluorine-18 into the desired
compounds. As shown in Scheme 2A, compound 17 was prepared by the copper(I)-induced “click”
reaction between aromatic azide 15 and intermediate 16 (the synthesis of 15 and 16 is shown in
Scheme S1, Supporting Information) and used as the precursor for one-step 18F-labeling, in which
18
-NO2 serves as the leaving group. However, F-labeled compound 11 ([18F]11) was obtained in
poor radiochemistry yield (RCY) <3% (decay corrected) and difficult to purify by high-
performance liquid chromatography (HPLC) because of overlapping impurities. The low RCY of
ACS Paragon Plus Environment

Page 13 of 33
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
this method may be caused by the lack of an appropriate leaving group in 17, i.e., an electron-
withdrawing group at its ortho or para position.55, 56 As our alternative strategy, we explored a step-
wise synthesis of [18F]11 and [18F]13 using SCIDY method43, 44 (Scheme 2B). The corresponding
precursors 20 and 21 were prepared from the oxidation of iodinated azidobenzenes (18 and 19, the
synthesis is shown in Scheme S1, Supporting Information) with mCPBA, followed by ligand
exchange with SPIAd44 in the same overall yields of 36%. As illustrated in Scheme 2B, the
radiolabeling commenced with the generation of [18F]9 and [18F]22, and the subsequent step was
accomplished by “click” reaction between 18F-labeled azidobenzenes ([18F]9 and [18F]22) and 16,
which afforded [18F]11 and [18F]13 in high overall isolated RCYs of 35% and 39% (n = 5, decay
corrected), respectively. The products of [18F]11 and [18F]13 were generated in high radiochemistry
purity >99% and good molar activity of ~19 GBq/μmol (n = 5) for [18F]13 and ~6 GBq/μmol for
[18F]11 (n = 5). Furthermore, since the 1,2,3-triazole linker showed good biostability in vivo and is
used as an isosteric surrogate for peptide bond,57 our radiolabeling method may provide a highly-
efficient modular synthesis route for the preparation of new 18F-arene click agents.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 2. Synthesis of the precursors (17, 20, and 21) and radiosynthesis of [18F]11 and [18F]13.
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 14 of 33
1
2
3
4
5
6
7
8
9
Reagents and conditions: (a) DIPEA, CuI, THF, 40 °C, 4 h, 25% yield. (b) [18F]F-, Kryptofix222/K2CO3 (7.5/1.5 mg),
anhydrous DMF, 140 °C, 10 min, <3% yield (decay corrected). (c) 1) mCPBA, CHCl3, rt, overnight; 2) SPIAd, Na2CO3
(10%), ethanol, pH = 10, rt, 6 h, 36% yield over two steps. (d) [18F]F-, TEAB, anhydrous DMF, 120 °C, 10 min. (e)
DIPEA, CuI, anhydrous DMF, 100 °C, 10 min, 35% and 39% yield for [18F]11 and [18F]13, respectively, over two steps
(decay corrected).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In Vitro Autoradiography. The GluN2B subunit is widespread throughout the CNS with high
expression in the forebrain including cortex, hippocampus, and thalamus, while barely detectable
in the cerebellum.11, 12, 14, 15 To assess the specificity of the new tracer to GluN2B subunit, we used
[18F]13 (with higher MPO score and lower lipophilicity compared to [18F]11) to perform in vitro
ARG on the brain cryosections (20 μm) of both rat and NHP. As shown in Figure 3A, the
radioactivity of [18F]13 in rat brain sections showed brain region-specific localization
(heterogeneous distribution), in which the uptake was the highest in hippocampus and cortex
(especially orbital, prelimbic, and somatomotor areas), moderate in striatum and thalamus, and low
in the cerebellum. This distribution pattern was in accordance with the in vitro autoradiograms of
[3H]CP-101606,58 (±)[11C]1,34 and [18F]cis-5,38 as well as with mRNA distribution of GluN2B
subunit.59 In self-blocking experiments, the uptake of [18F]13 was substantially reduced (~90%) in
the presence of cold compound 13 (10 μM, Figure 3C, also see Figure S3 in SI for self-blocking
results at 1 μM) and the specific binding of [18F]13 was 94%, 94%, 92%, and 88% (n = 3, p <0.001,
T-test, Figure 3E) in brain regions of hippocampus, frontal cortex, striatum, and thalamus,
respectively. The specificity of [18F]13 to GluN2B subunit was further confirmed by using a
GluN2B-selective NAM, the ifenprodil derivative BMT-108908 (Ki = 1.4 nM),60, 61 and the results
showed that the binding of [18F]13 was strongly inhibited by this drug (Figure 3D and 3E) in a
similar pattern of self-blocking. This result also indicated that [18F]13 associated with GluN2B
subunit in the same binding site as BMT-108908, which was also confirmed by the molecular
docking results (Figure 3).
ACS Paragon Plus Environment

Page 15 of 33
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3. In vitro autoradiography results of [18F]13 (37 kBq/mL, 1.58 nM) in rat brain sections. Representative
autoradiograms in rat brain sagittal sections: [18F]13 alone (A, baseline), and pre-blocked by cold compound 13 (C, self-
blocking, 10 µM) and BMT-108908 (D, a NAM drug blocking, 10 µM). (B) GluN2B mRNA expression in mouse brain
and the data were retrieved from mouse.brain-map.org (experiment: 69257725). (E) Quantitative analysis of baseline and
blocking experiments. The value is expressed as the ratio of the radioactivity of different brain regions to that of the
cerebellum (reference region). HIP, hippocampus; FCx, frontal cortex; STR, striatum; THM, thalamus; Cb, cerebellum;
OLF, olfactory bulb. Asterisks indicate statistical significance: ***p <0.001, and **p <0.01 vs control.
Given the high specificity of [18F]13 to GluN2B subunit, we further explored the distribution
pattern of GluN2B subunit in the NHP brain. Figure 4A showed that the highest bound activity of
[18F]13 was found in brain regions of the hippocampus, entorhinal cortex, and insular cortex. The
activity was moderate in caudate nucleus and putamen, low in the thalamus and claustrum, and no
appreciable binding in white matter of forebrain. The nonspecific binding was determined by self-
blocking studies using non-radioactive 13 (10 μM, Figure 4B) and the results showed that the
radioactivity of [18F]13 distributed to several brain regions with high specificity including
hippocampus (87%, n = 3, p <0.001, T-test, Figure 4D), entorhinal cortex (89%, p <0.01), insular
cortex (82%, p <0.001), caudate nucleus (79%, p <0.01), and putamen (73%, p <0.01), but lower
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 16 of 33
1
2
3
4
5
6
7
8
9
in thalamus (53%, p <0.01). The specific binding to NMDAR GluN2B subtype was further
confirmed by the blocking experiments with BMT-108908 (10 μM)60, 61 and the %blockade results
were similar to those of self-blocking studies (Figure 4C and 4D). In summary, [18F]13 showed
high specific binding to GluN2B subunit in the NHP brain sections, which showed characteristic
and regional-specific GluN2B subunit expression and target specificity (validated by blocking
studies).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4. In vitro autoradiography results of [18F]13 (37 kBq/mL, 1.58 nM) in NHP brain sections. Representative
autoradiograms in NHP brain sections: [18F]13 alone (A, baseline), and pre-blocked by cold compound 13 (B, self-
blocking, 10 µM) and BMT-108908 (C, a NAM drug blocking, 10 µM). (D) Quantitative analysis of baseline and
blocking experiments. The value is expressed as the ratio of the radioactivity of different brain regions to that of the white
matter of forebrain. HIP, hippocampus; ECx, entorhinal cortex; ICx, insular cortex; CdN, caudate nucleus; CLA,
claustrum; PUT, putamen; THM, thalamus; FWM, white matter of forebrain. Asterisks indicate statistical significance:
ACS Paragon Plus Environment

Page 17 of 33
ACS Chemical Neuroscience
1
2
3
4
***p <0.001, and **p <0.01 vs control.
5
6
7
8
9
Whole Body Ex Vivo Biodistribution Studies and Radiometabolite Analysis. GluN2B
antagonist 13 exhibited a favorable MPO score (5.16), proper lipophilicity (logD7.4 2.89) and
reasonable polar surface area (71.4 Å), indicating a high probability to be a successful PET tracer.
We next performed whole body ex vivo biodistribution studies to examine the uptake, distribution
and clearance of [18F]13 in organs of interest. The studies were conducted in mice at four different
time points (2, 10, 30, and 60 min) after the injection of [18F]13. As shown in Figure 5A, several
organs including heart, lung, kidney, liver, and small intestine showed high initial uptakes (> 5%
ID/g) of [18F]13 at 2 min postinjection. After initial uptake, the radioactivity in organs/tissues of
heart, lung, kidney, spleen, pancreas, and muscle displayed rapid washout with the uptake <0.6%
ID/g at 60 min postinjection, while the levels in liver (20.84% ID/g) and small intestine (39.15%
ID/g) remained high until 10 min post injection and then decreased rapidly (Figure 5A and Table
S1 in Supporting Information). These results indicated that [18F]13 probably underwent
hepatobiliary elimination. Of note, [18F]13 displayed reasonable initial brain uptake (brain2min) with
the value of 3.60 %ID/g, followed by a very rapid washout to 0.72 %ID/g at 10 min post injection,
then to 0.018% ID/g at 60 min post injection (Figure 5B). The uptake in blood was higher than that
in the brain at all time points (Figure 5). As a result, the observed abnormal rapid clearance of
[18F]13 in the brain (brain2min/brain10min = 5, and brain2min/brain60min = 200) diminished our
enthusiasm in pursuing in vivo PET evaluation in rodents. Notably, no substantial bone uptake of
[18F]13 was observed (<2 %ID/g, Figure 4B), indicating little defluorination occurred in vivo. In
addition, probe [18F]11 with higher lipophilicity (logD7.4 3.14) displayed similar biodistribution
results as [18F]13 (Table S1) in different organs. In particular, [18F]11 showed lower initial brain
uptake (brain2min = 3.27% ID/g) and slower brain washout (brain2min/brain60min = 55).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 18 of 33
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 5. (A) Ex vivo biodistribution in mice at four different time points (2, 10, 30, and 60 min) post injection of [18F]13.
The results are expressed as the percentage of the injected dose per gram of wet tissue (% ID/g) with the exception that
stomach uptake was expressed as % ID/organ. (B) The values of the brain, blood, and bone uptakes of [18F]13. The values
for remaining organs of interest are included in Table S1 of Supporting Information.
To further understand the pharmacokinetics of [18F]13, the metabolism studies in ICR mice (n
= 2) were carried out by radioHPLC analysis of plasma, brain, and liver extracts at 15 min post
injection. The results showed that [18F]13 displayed moderate stability in the brain with 56.6%
parent fraction left. In contrast, this probe degraded to a greater extent in plasma and liver with
11.8% and 5.1% parent fraction remained, respectively. Only polar radiometabolites of [18F]13 were
found in plasma, brain, and liver. In addition, [18F]11 exhibited better stability than [18F]13 in mice
with 84.8%, 32.0%, and 17.7% parent tracer remained in brain, plasma, and liver, respectively, at
15 min post injection. The results indicated that both [18F]11 and [18F]13 underwent moderate
metabolism in the brain but rapid degradation in plasma and liver, which may contribute to the
moderate brain uptake seen in the ex vivo biodistribution studies.
Conclusion
In this report, we successfully designed and synthesized a series of GluN2B-selective NMDAR
antagonists (11–14) with the chemical scaffold containing triazole and methoxypyrimidine groups.
ACS Paragon Plus Environment

Page 19 of 33
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
Two compounds, 11 and 13, showed high potency and high selectivity to GluN2B subunit in in
vitro glutamate/glycine assays with Xenopus oocytes, which identified them as potent candidates
for PET ligand development. Molecular docking studies also identified the binding of both
compounds to the interface between GluN1 and GluN2B subunits. In silico prediction showed that
11 and 13 had optimal clogP and tPSA values for CNS drug discovery and 13 exhibited the highest
MPO score (5.16), indicating its great potential for PET imaging studies. Our SCIDY method
enables an efficient radiosynthesis of [18F]11 and [18F]13 (also called [18F]N2B-0518) in high RCYs
(>35%). The following in vitro ARG in rat brain sections unveiled the specific binding of [18F]13
in brain regions of the hippocampus, frontal cortex, striatum, and thalamus, but not cerebellum,
which is in accordance with the expression of GluN2B subunit. Furthermore, autoradiograms of
[18F]13 in NHP brain sections demonstrated that the GluN2B subunit expressed strongly in the
hippocampus, entorhinal cortex, insular cortex, caudate nucleus, and putamen, moderately in
claustrum and thalamus. Ex vivo biodistribution in mice showed that [18F]13 penetrated the brain
with moderate initial uptake and rapid washout, which may be attributed to poor biostability of
[18F]13 in the plasma. Further PET imaging work of [18F]13 in higher species will be planned to
investigate if inter-species differences exist in the brain uptake and metabolic rate in the plasma. In
summary, we provided a potent and selective PET ligand for in vitro detection of GluN2B subunit,
which not only widens the landscape of PET probes targeting GluN2B subunit but also offers a
new structural framework for medicinal chemistry design.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
METHODS
General Information. All chemicals were purchased from commercial vendors and 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 purification was performed using
silica gel (SiliCycle Inc., 230−400 mesh, 40−63 μm). 1H, 13C, and 19F NMR were obtained at 300,
75, and 282 MHz, respectively, on a Bruker spectrometer in CDCl3 or d6-DMSO solutions at room
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 20 of 33
1
2
3
4
5
6
7
8
9
temperature, and the chemical shifts were quoted 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 is ESI using Agilent 6430 Triple Quad LC/MS. The compounds 11–14 did
not show any promiscuous moieties in the Pan Assay Interference Compounds Assay (PAINS)
using the in silico filter (http://www.swissadme.ch/index.php).62, 63 The animal experiments were
approved by the Institutional Animal Care and Use Committee of Massachusetts General Hospital.
CD1 (ICR) mice (female; 8 weeks, 23-27 g) were kept on a 12 h light/12 h dark cycle and were
allowed food and water adlibitum.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Chemistry.
4-Azido-2-bromo-1-fluorobenzene (9). Compound 9 was prepared by a previously reported
procedure.64 Dark brown oil, yield 89%. 1H NMR (300 MHz, CDCl3) δ 7.22 (d, J = 2.6 Hz, 1H),
7.10 (t, J = 8.2 Hz, 1H), 6.95 – 6.93 (m, 1H). 13C NMR (75 MHz, CDCl3) δ 156.38 (d, J = 245.6
Hz), 136.85, 123.70, 119.29 (d, J = 7.1 Hz), 117.28 (d, J = 24.0 Hz), 109.97 (d, J = 22.6 Hz). 19F
NMR (282 MHz, CDCl3) δ -108.03 – -108.05 (m).
(1-(3-Bromo-4-fluorophenyl)-1H-1,2,3-triazol-4-yl)methanol (10). To a solution of compound 9
(2.0 g, 9.3 mmol) in THF (20 mL) was added propargyl alcohol (1.0 g, 18.0 mmol) and DIPEA
(244 mg, 1.9 mmol), and then CuI (88 mg, 0.5 mmol) was added under stirring. The reaction
mixture was stirred at 40 °C for 2 h. Upon completion the reaction mixture was filtered, and the
solvent was removed under vacuum. The residue was purified via silica gel column
chromatography using a gradient mixture of hexane–ethyl acetate (50%–100%) to obtain the pure
compound of 10 as white solid (1.8 g, 6.6 mmol, 71%). 1H NMR (300 MHz, d6-DMSO) δ 8.73 (s,
1H), 8.30 (d, J = 2.7 Hz, 1H), 7.99 (d, J = 4.2 Hz, 1H), 7.61 (t, J = 8.7 Hz, 1H), 5.34 (s, 1H), 4.60
(s, 2H). 13C NMR (75 MHz, d6-DMSO) δ 158.26 (d, J = 246.1 Hz), 149.76, 134.39 (d, J = 3.1 Hz),
19
125.24, 121.79, 121.69, 118.26 (d, J = 24.0 Hz), 109.54 (d, J = 22.6 Hz), 55.34. F NMR (282
MHz, d6-DMSO) δ -104.77 – -104.80 (m).
2-((1-(3-Bromo-4-fluorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-5-methoxypyrimidine
(11).
ACS Paragon Plus Environment

Page 21 of 33
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
Compound 10 (108.7 mg, 0.4 mmol) was dissolved in 10 mL THF, along with 2-bromo-5-
methoxypyrimidine (50 mg, 0.3 mmol), and then NaH (70 mg, 1.7 mmol, 60% dispersion in mineral
oil) was added in small portion under stirring. The reaction mixture was stirred at 40 °C for 3 h,
and subsequently quenched with saturated solution of ammonium chloride. After that, the mixture
was extracted with dichloromethane, and the combined organic extracts were dried over anhydrous
magnesium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel
column chromatography using a gradient mixture of hexane–ethyl acetate (30%–80%), and the
product was obtained as white solid (29 mg, 0.08 mmol, 29%). 1H NMR (300 MHz, CDCl3) δ 8.23
(s, 2H), 8.08 (s, 1H), 7.98 (d, J = 2.5 Hz, 1H), 7.66 (d, J = 4.8 Hz, 1H), 7.30 – 7.27 (m, 1H), 5.59
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
13
(s, 2H), 3.87 (s, 3H). C NMR (75 MHz, CDCl3) δ 159.23, 158.95 (d, J = 250.2 Hz), 149.80,
145.46, 145.17, 133.76 (d, J = 3.6 Hz), 125.84, 121.28, 121.07 (d, J = 7.7 Hz), 117.40 (d, J = 24.1
Hz), 110.22 (d, J = 22.8 Hz), 61.07, 56.52. 19F NMR (282 MHz, CDCl3) δ -102.37. HRMS (m/z):
[M + Na]+ calculated for C14H11BrFN5O2Na+, 401.9972, found 401.9978.
Compounds 12–14 were synthesized by following the same procedure for 11 described above.
2-((1-(4-Bromo-3-fluorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-5-methoxypyrimidine (12). White
solid, yield 30%. 1H NMR (300 MHz, d6-DMSO) δ 8.96 (s, 1H), 8.40 (s, 2H), 8.06 (d, J = 9.7 Hz,
13
1H), 7.95 (t, J = 7.5 Hz, 1H), 7.79 (d, J = 8.7 Hz, 1H), 5.46 (s, 2H), 3.84 (s, 3H). C NMR (75
MHz, d6-DMSO) δ 159.15, 159.00 (d, J = 245.4 Hz), 150.02, 146.14, 144.54, 137.46 (d, J = 10.0
Hz), 135.14, 123.50, 117.86 (d, J = 3.5 Hz), 109.37 (d, J = 27.5 Hz), 108.25 (d, J = 20.7 Hz), 60.48,
56.97.19F NMR (282 MHz, d6-DMSO) δ -100.92 – -101.00 (m). HRMS (m/z): [M + Na]+ calculated
for C14H11BrFN5O2Na+, 401.9972, found 401.9975.
2-((1-(4-Fluoro-3-methylphenyl)-1H-1,2,3-triazol-4-yl)methoxy)-5-methoxypyrimidine (13).White
1
solid, yield 30%. H NMR (300 MHz, CDCl3) δ 8.23 (s, 2H), 8.05 (s, 1H), 7.58 (s, 1H), 7.46 (s,
1H), 7.13 (t, J = 8.3 Hz, 1H), 5.59 (s, 2H), 3.87 (s, 3H), 2.35 (s, 3H). 13C NMR (75 MHz, CDCl3)
δ 160.98 (d, J = 249.8 Hz), 159.32, 149.77, 145.45, 144.74, 132.93, 126.78 (d, J = 19.0 Hz), 123.89
(d, J = 5.5 Hz), 121.42, 119.64 (d, J = 8.6 Hz), 116.10 (d, J = 24.3 Hz), 61.22, 56.53, 14.66 (d, J =
3.3 Hz). 19F NMR (282 MHz, CDCl3) δ -112.34. HRMS (m/z): [M + Na]+ calculated for
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 22 of 33
1
2
3
4
5
6
C15H14FN5O2Na+, 338.1024, found 338.1029.
2-((1-(3-Fluoro-4-methylphenyl)-1H-1,2,3-triazol-4-yl)methoxy)-5-methoxypyrimidine
(14).
7
8
9
White solid, yield 24%. 1H NMR (300 MHz, d6-DMSO) δ 8.89 (s, 1H), 8.39 (s, 2H), 7.73 (dd, J =
26.9, 9.4 Hz, 2H), 7.49 (t, J = 8.2 Hz, 1H), 5.44 (s, 2H), 3.84 (s, 3H), 2.28 (s, 3H). 13C NMR (75
MHz, d6-DMSO) δ 161.04 (d, J = 244.4 Hz), 159.18, 150.00, 146.13, 144.23, 136.00 (d, J = 10.3
Hz), 133.13 (d, J = 6.0 Hz), 125.29 (d, J = 17.2 Hz), 123.33, 116.14 (d, J = 3.3 Hz), 107.74 (d, J =
27.5 Hz), 60.53, 56.95, 14.29. 19F NMR (282 MHz, d6-DMSO) δ -110.48 – -110.55 (m). HRMS
(m/z): [M + Na]+ calculated for C15H14FN5O2Na+, 338.1024, found 338.1025.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Pharmacology study. Recombinant receptors were expressed from cDNAs encoding human
NMDA receptor subunits GluN1-1a (hereafter GluN1; GenBank NP_015566), GluN2A (GenBank
NP_000824), GluN2B (GenBank NP_000825), and GluN2D (GenBankNP_000827.1)65 Rat
GluN1-1a (U11418, U08261) and GluN2C (M91563), GluA1-flip (P19490), and GluK2(Q)
(P42260) were used for the study of subunit selectivity. cRNA was transcribed in vitro from
plasmids containing NMDAR cDNAs.66 Xenopus laevis stage VI oocytes were prepared from
commercially available ovaries (Xenopus one Inc, Dexter, MI, USA). The ovary was placed in a
petri plate at room temperature, gently pulled apart with two fine forceps and transferred into
Collagenase Type 4 (Worthington-Biochem, Lakewood, NJ, USA) solution (850 μg/ml, 15 ml for
a half ovary) in Ca2+-free Barth’s solution (see below). The ovary was digested on a mixer at room
temperature (23 C) for 2 h. If the oocytes were under digested, they were kept on a mixer for an
additional 15 min. The oocytes were then rinsed 5 times with Ca2+-free Barth’s on a mixer for 10
min (with 35–40 mL of fresh solution each time), and further rinsed for 4 times with normal Barth’s
on the mixer for 10 min (35–40 mL of fresh solution each time). The oocytes were placed in petri
plates for sorting and kept in a 16°C incubator afterward. The oocytes were injected with 5–10 ng
cRNA and stored at 15 C in media containing (Barth’s, in mM) 88 NaCl, 2.4 NaHCO3, 1 KCl,
0.33 Ca(NO3)2, 0.41 CaCl2, 0.82 MgSO4, 5 HEPES, 1 U/mL penicillin, 0.1 mg/mL gentamicin
sulfate, and 1 μg/mL streptomycin (pH 7.4, adjusted with NaOH). Two to seven days after injection,
two-electrode voltage-clamp recordings66 were performed at room temperature in an extracellular
ACS Paragon Plus Environment

Page 23 of 33
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
solution containing (in mM) 90 NaCl, 1 KCl, 10 HEPES, 0.5 BaCl2, and 0.01 EDTA (pH 7.4,
adjusted with NaOH). NMDAR current responses from oocytes were recorded at a holding
potential of -40 mV. Concentration–response curves for GluN2B antagonists were generated by co-
applying 100 M glutamate and 100 M glycine with variable concentrations of test compounds
up to 1 μM. Test compounds were prepared as 20 mM stock solutions in DMSO and diluted to the
final concentration in recording solution. DMSO content was 0.05–0.5% (v/v). The response to test
compounds was given as a percentage of the initial response to glutamate and glycine alone. Data
for individual cells were fitted in OriginPro (v 9.0) with the Hill equation:
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Response (%) = (100 - minimum)/(1 + ([concentration]/IC50)N) + minimum
where the minimum is the residual response in saturating inhibitor, IC50 is the concentration that
reduces the response by half, and N is the Hill slope. Mean fitted IC50 values from individual cells
are given with the 95% confidence interval (CI) determined from the log(IC50) was used to
determine the CI, which were then transformed back to concentration. For the graphical
representation, the data were normalized to the current response to agonist alone, averaged across
all cells, and fitted to the Hill equation.
The triheteromeric receptors (2A/2B) and diheterometric receptors (2A/2A and 2B/2B) used here
were developed by the method previously reported,45 and their current amplitude evoked by
agonists glutamate/glycine (100 M/100 M) was showed in Figure S2 in SI.
Molecular Docking Studies. Candidate compounds 11 and 13 were docked onto the selected
protein structure (PDB ID: 3QEL) using the AutoDock Vina module in the UCSF Chimera software.
Briefly, the mol2 file of protein was prepared by deleting the solvent, adding hydrogen atoms and
charges using the default settings. Standard residues were determined according to the AMBER 14
force field, while non-standard residues were ignored. The predicted free energy of binding was
calculated in AutoDock Vina using a hybrid scoring function (combined knowledge-based and
empirical approaches).67
Radiochemistry. One-step radiolabeling of [18F]11: [18F]F- on the QMA cartridge was eluted into
a 5 mL reaction vial with 1 mL of Kryptofix222/K2CO3 solution (7.5 mg of Kryptofix222 and 1.5 mg
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 24 of 33
1
2
3
4
5
6
7
8
9
of K2CO3 in CH3CN/H2O, 4/1, v/v). The solvent was removed, and then the residue was dried
azeotropically with anhydrous acetonitrile (1 mL × 3) under a stream of nitrogen at 120 ºC. The
vessel was cooled to room temperature, and a solution of nitro precursor 17 (2.5 – 3.5 mg) in
anhydrous DMF (1 mL) was added. The reaction mixture was heated to 140 ºC and kept for 10 min.
After that, the reaction mixture was purified with HPLC (Waters) using a YMC C18 reverse phase
column (5 μm, 10 × 250 mm, elution: CH3CN/H2O = 45%/55%, 4 mL/min). To the separated
solution of [18F]11, 20 mL of water was added, and then passed through a preconditioned Sep-Pak
Plus-C18 cartridge (Waters). The cartridge was washed with 10 mL of water, and the labeled
compound [18F]11 was eluted to a glass vial with 1 mL of ethanol. The ethanol eluent was
concentrated under nitrogen flow at 60 ºC and then formulated with saline (containing 5%–8%
ethanol) for further studies. The radiochemical yield is ~3% (decay corrected), and the
radiochemical purity is > 99%.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Two-step radiolabeling of [18F]11 and [18F]13: [18F]F- on the QMA cartridge was eluted into a 5
mL reaction vial with 1 mL of TEAB solution (3.5 mg, CH3CN/H2O, 7/3, v/v). After [18F]F- was
dried under a nitrogen stream and cooled to room temperature (same procedure as above), a solution
of SCIDY precursor 20 or 21 (~5 mg) in anhydrous DMF (400 μL) was added. After the reaction
at 120 ºC for 10 min, 16 (2–3 mg), CuI (2–3 mg), and DIPEA (5 μL) were added and the reaction
mixture was kept at 100 ºC for another 10 min. Then the reaction mixture was quenched with 10
mL water, passed through a preconditioned Sep-Pak Plus-C18 cartridge (Waters), washed with 10
mL water and eluted with 1–1.5 mL acetonitrile. The eluent was purified with HPLC (Waters) using
a YMC C18 reverse phase column (5 μm, 10 × 250 mm, 5 mL/min, elution: CH3CN/H2O = 45%/55%
and 40%/60% for [18F]11 and [18F]13, respectively). The fraction containing [18F]11 (or [18F]13)
was mixed with 20 mL of water and then passed through a preconditioned Sep-Pak Plus-C18
cartridge (Waters). The cartridge was washed with 10 mL of water, and the labeled compound
[18F]11 (or [18F]13) was eluted to a reaction vial with 1 mL of ethanol. The ethanol eluent was
concentrated under nitrogen flow at 60 ºC and then formulated with saline (containing 5%–8%
ethanol) for further studies. The identities of [18F]11 and [18F]13 were confirmed with HPLC by the
ACS Paragon Plus Environment

Page 25 of 33
ACS Chemical Neuroscience
1
2
3
4
5
6
co-injection with corresponding cold compounds of 11 or 13, respectively (Figure S1 in Supporting
Information).
7
8
9
In Vitro Autoradiography of [18F]13 in Brain Sections of Rat and NHP. Rat brain cryosections
(20 μm) were preincubated for 20 min in 50 mM Tris-HCl buffer (pH 7.4) at room temperature.
These sections were incubated for 60 min at room temperature with Tris-HCl buffer containing
[18F]13 (37 kBq/mL, 1.58 nM). After incubation, the brain sections were washed with cold buffer
(0.5 mL) and dried with cold air. Then the sections were opposed to imaging plates (GE) overnight
and autoradiograms were obtained using an Amersham Typhoon 5 (GE) analyzer system with the
resolution of 10 μm and sensitivity of 4000. Self-blocking experiment with cold compound of 13
(10 μM, 0.1% DMSO, 1% ethanol in Tris-HCl buffer) was used to determine the nonspecific
binding of [18F]13: before incubation with [18F]13, the sections were incubated with the solution of
13 for 30 min and then washed with cold Tris-HCl buffer (1 mL). In blocking experiments with
BMT-108908 (10 μM, 0.1% DMSO, 1% ethanol in Tris-HCl buffer), the same procedure as self-
blocking was used. The in vitro ARG in NHP brain sections including baseline, self-blocking, and
BMT-108908 blocking experiments were carried out by following the same procedure as described
above.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Distribution of radioactivity on the plates was analyzed by ImageJ, in which the regions of
interest (ROIs) were placed on the hippocampus, frontal cortex, striatum, thalamus, and cerebellum
in rat brain sections, and hippocampus, entorhinal cortex, insular cortex, caudate nucleus, putamen,
thalamus and white matter of forebrain in NHP brain sections. The radioactivity in these regions
was expressed as gray value/unit area, which is in linear with the radioactivity. The relative
radioactivity was represented as the ratio of gray value/unit area (radioactivity) of different brain
regions to that of the cerebellum (for rat) or white matter of forebrain (for NHP), where only slight
radioactivity was observed.
Whole Body Biodistribution Study and Radiometabolite Analysis in Mice. The CD1 (ICR)
mice (female, 8 weeks, 23–27 g) were intravenously injected with [18F]13 (740 kBq/100 μL in
saline containing 5% ethanol) through the tail vein. The mice were sacrificed at various time points
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 26 of 33
1
2
3
4
5
6
7
8
9
of 2, 10, 30, and 60 min post-injection. The organs of interest were removed and weighed, and the
radioactivity was measured with an automatic γ-counter (WALLAC/Wizard 1470, USA). The
percent dose per gram (% ID/g) or percent dose per organ (% ID/organ) of wet tissue values were
calculated by relating the tissue radioactivity counts to that of suitably diluted aliquots of the
injected dose. All radioactivity measurements were decay-corrected.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
After intravenous injection of [18F]13 through tail vein, the mice (n = 2) were sacrificed at 15 min.
Blood sample was collected and centrifuged at 14000 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 vortex for 10 s, the mixture was centrifuged at 14000 rpm for 3 min at 4 °C for
deproteinization. The supernatant was collected, and the process was repeated until no
precipitations were observed when CH3CN was added. The mice brain was immediately dissected,
homogenized with 400 μL of ice-cooled CH3CN, and then centrifuged at 14000 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 precipitations were observed when
CH3CN was added. The resulting supernatants of plasma and brain were mixed with 50 μL of a
solution of cold compound [18F]13 and then analyzed on a radioHPLC system using a Phenomenex
C18 column (5 μm, 10 × 250 mm, 5 mL/min, elution: CH3CN/H2O = 55%/45%). The radioactivity
was collected and counted using 1480 Wizard gamma counter (PerkinElmer, USA). The
radioactivity overlapped with [18F]13 in the UV signal corresponded to that of [18F]13 (tR = 6.90
min). The percentage of [18F]13 to total radioactivity was calculated as (counts for [18F]13/total
counts) × 100. The whole body biodistribution study and radiometabolite analysis in mice of [18F]11
were conducted by following the same procedures for [18F]13.
ASSOCIATED CONTENT
Characterization of all new compounds and NMR spectra; ex vivo biodistribution data. This
material is available free of charge via the Internet at http://pubs.acs.org.
ACS Paragon Plus Environment

Page 27 of 33
ACS Chemical Neuroscience
1
2
3
4
5
6
AUTHOR INFORMATION
Corresponding Author
7
8
9
*S.H.L.: tel: +1 617 726 6107; fax, +1 617 726 6165; e-mail, liang.steven@mgh.harvard.edu.
*S.F.T.: tel, +1 404-727-1375; fax, +1 404-727-0365; e-mail, strayne@emory.edu.
*Z.L.: tel, +86 592 2880645; fax, +86 592 2880645; e-mail, zijing.li@xmu.edu.cn.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Author Contributions
The manuscript was written through contributions of all authors. All authors have read and
approved the final version.
Notes
S.F.T. is a consultant for Janssen Pharmaceuticals Inc., is PI on a research grant from Janssen to
Emory University School of Medicine, is a member of the SAB for Sage Therapeutics, is co-
founder of NeurOp Inc, and receives royalties for software. S.F.T. is co-inventor on Emory-owned
Intellectual Property that includes allosteric modulators of NMDA receptor function. H.Y. is PI on
a research grant from Sage Therapeutics to Emory University School of Medicine. The other
authors declare no competing financial interest.
ACKNOWLEDGMENT
We thank Dr. Lei Zhang (Medicine Design, Pfizer, Inc) and Professor Thomas J. Brady (Nuclear
Medicine and Molecular Imaging, Radiology, MGH and Harvard Medical School) for helpful
discussion. Z.L. acknowledges financial supports from the National Natural Science Foundation of
China (81501534), Fourth Round Fujian Health Education Joint Research Projects (WKJ2016-2-
08), Fundamental Research Funds for the Central Universities (20720180050), Fujian Province
Young Teacher Research Program (JA15010) and Scientific Research Foundation of State Key
Laboratory of Molecular Vaccinology and Molecular Diagnostics (2016ZY002). Financial support
from the NIH grants (NINDS NS065371 to S.F.T.; NICHD HD082373 to H.Y.; NIAAAAA019431
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 28 of 33
1
2
3
4
to J.B.D. and NIAAAAA014106 to David P. Friedman) is gratefully acknowledged.
5
6
7
ABBREVIATIONS
8
9
AD, Alzheimer’s disease; ARG, autoradiography; BBB, blood-brain barrier; CI, confidence
interval; CNS, central nervous system; HPLC, high-performance liquid chromatography; iGluRs,
ionotropic glutamate receptors; MPO, multiparameter optimization; NAM, negative allosteric
modulators; NMDAR, N-methyl-D-aspartate receptors; NHP, non-human primates; PAINS, Pan
Assay Interference Compounds Assay; PAM, positive allosteric modulators; PCP, phencyclidine;
PET, positron emission tomography; RCY, radiochemistry yield; ROI, region of interest; SCIDY,
spirocyclic iodonium ylide; SNAr, nucleophilic aromatic substitution; TLC, thin-layer
chromatography; tPSA, topological polar surface area.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
REFERENCES
1. Paoletti, P., Bellone, C., and Zhou, Q. (2013) NMDA receptor subunit diversity: impact on
receptor properties, synaptic plasticity and disease, Nat. Rev. Neurosci. 14, 383-400.
2. Traynelis, S. F., Wollmuth, L. P., McBain, C. J., Menniti, F. S., Vance, K. M., Ogden, K. K.,
Hansen, K. B., Yuan, H., Myers, S. J., and Dingledine, R. (2010) Glutamate receptor ion channels:
structure, regulation, and function, Pharmacol. Rev. 62, 405-496.
3. Parsons, M. P., and Raymond, L. A. (2014) Extrasynaptic NMDAreceptor involvement in central
nervous system disorders, Neuron 82, 279-293.
4. Gonzalez, J., Jurado-Coronel, J. C., Avila, M. F., Sabogal, A., Capani, F., and Barreto, G. E.
(2015) NMDARs in neurological diseases: a potential therapeutic target, Int. J. Neurosci. 125, 315-
327.
5. Chazot, P. L. (2004) The NMDA receptor NR2B subunit: a valid therapeutic target for multiple
CNS pathologies, Curr. Med. Chem. 11, 389-396.
6. Ogden, K. K., and Traynelis, S. F. (2011) New advances in NMDA receptor pharmacology,
Trends Pharmacol. Sci. 32, 726-733.
7. Kemp, J. A., and McKernan, R. M. (2002) NMDA receptor pathways as drug targets, Nat.
Neurosci. 5, 1039-1042.
8. Mony, L., Kew, J. N., Gunthorpe, M. J., and Paoletti, P. (2009) Allosteric modulators of NR2B-
containing NMDA receptors: molecular mechanisms and therapeutic potential, Br. J. Pharmacol.
157, 1301-1317.
9. Kemp, J.A., Kew, J.N.C., Gill, R. (1999) NMDA Receptor Antagonists and Their Potential as
ACS Paragon Plus Environment

Page 29 of 33
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
Neuroprotective Agents. In Ionotropic Glutamate Receptors in the CNS. Handbook of Experimental
Pharmacology (Jonas, P., and Monyer, H., Ed.), pp 495–527, Springer, Berlin.
10. Madden, D. R. (2002) The structure and function of glutamate receptor ion channels, Nat. Rev.
Neurosci. 3, 91-101.
11. Monyer, H., Burnashev, N., Laurie, D. J., Sakmann, B., and Seeburg, P. H. (1994)
Developmental and regional expression in the rat brain and functional properties of four NMDA
receptors, Neuron 12, 529-540.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
12. Sheng, M., Cummings, J., Roldan, L. A., Jan, Y. N., and Jan, L. Y. (1994) Changing subunit
composition of heteromeric NMDA receptors during development of rat cortex, Nature 368, 144-
147.
13. Paoletti, P. (2011) Molecular basis of NMDA receptor functional diversity, Eur. J. Neurosci. 33,
1351-1365.
14. Watanabe, M., Inoue, Y., Sakimura, K., and Mishina, M. (1992) Developmental changes in
distribution of NMDA receptor channel subunit mRNAs, Neuroreport 3, 1138-1140.
15. Akazawa, C., Shigemoto, R., Bessho, Y., Nakanishi, S., and Mizuno, N. (1994) Differential
expression of five N-methyl-D-aspartate receptor subunit mRNAs in the cerebellum of developing
and adult rats, J. Comp. Neurol. 347, 150-160.
16. Zhu, S., and Paoletti, P. (2015) Allosteric modulators of NMDA receptors: multiple sites and
mechanisms, Curr. Opin. Pharmacol. 20, 14-23.
17. Karakas, E., Simorowski, N., and Furukawa, H. (2009) Structure of the zinc-bound amino-
terminal domain of the NMDA receptor NR2B subunit, EMBO J. 28, 3910-3920.
18. Karakas, E., Simorowski, N., and Furukawa, H. (2011) Subunit arrangement and
phenylethanolamine binding in GluN1/GluN2B NMDA receptors, Nature 475, 249-253.
19. Mony, L., Zhu, S., Carvalho, S., and Paoletti, P. (2011) Molecular basis of positive allosteric
modulation of GluN2B NMDA receptors by polyamines, EMBO J. 30, 3134-3146.
20. Loftis, J. M., and Janowsky, A. (2003) The N-methyl-D-aspartate receptor subunit NR2B:
localization, functional properties, regulation, and clinical implications, Pharmacol. Ther. 97, 55-
85.
21. Hu, C., Chen, W., Myers, S. J., Yuan, H., and Traynelis, S. F. (2016) Human GRIN2B variants
in neurodevelopmental disorders, J. Pharmacol. Sci. 132, 115-121.
22. Chenard, B. L., and Menniti, F. S. (1999) Antagonists selective for NMDA receptors containing
the NR2B subunit, Curr. Pharm. Des. 5, 381-404.
23. Kew, J. N., Trube, G., and Kemp, J. A. (1996) Anovel mechanism of activity-dependent NMDA
receptor antagonism describes the effect of ifenprodil in rat cultured cortical neurones, J. Physiol.
497 ( Pt 3), 761-772.
24. Stroebel, D., Buhl, D. L., Knafels, J. D., Chanda, P. K., Green, M., Sciabola, S., Mony, L.,
Paoletti, P., and Pandit, J. (2016) A Novel Binding Mode Reveals Two Distinct Classes of NMDA
Receptor GluN2B-selective Antagonists, Mol. Pharmacol. 89, 541-551.
25. Burger, P. B., Yuan, H., Karakas, E., Geballe, M., Furukawa, H., Liotta, D. C., Snyder, J. P., and
Traynelis, S. F. (2012) Mapping the binding of GluN2B-selective N-methyl-D-aspartate receptor
negative allosteric modulators, Mol. Pharmacol. 82, 344-359.
ACS Paragon Plus Environment