Synthesis and Preclinical Evaluation of an 18F-Labeled Synaptic Vesicle Glycoprotein 2A PET Imaging Probe: [18F]SynVesT-2

Article abstract of DOI:10.1021/acschemneuro.9b00618

Synaptic vesicle glycoprotein 2A (SV2A) is a 12-pass transmembrane glycoprotein ubiquitously expressed in presynaptic vesicles. In vivo imaging of SV2A using PET has potential applications in the diagnosis and prognosis of a variety of neuropsychiatric diseases, e.g., Alzheimer's disease, Parkinson's disease, schizophrenia, multiple sclerosis, autism, epilepsy, stroke, traumatic brain injury, post-traumatic stress disorder, depression, etc. Herein, we report the synthesis and evaluation of a new ¹⁸F-labeled SV2A PET imaging probe, [¹⁸F]SynVesT-2, which possesses fast in vivo binding kinetics and high specific binding signals in non-human primate brain.

Full text of DOI:10.1021/acschemneuro.9b00618

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Article
Synthesis and Preclinical Evaluation of an 18F-Labeled Synaptic
Vesicle Glycoprotein 2A PET Imaging Probe: [18F]SynVesT-2
Zhengxin Cai, Songye Li, Wenjie Zhang, Richard Pracitto, Xiaoai Wu, Evan Baum, Sjoerd
J. Finnema, Daniel Holden, Takuya Toyonaga, Shu-fei Lin, Marcel Lindemann, Anupama
Shirali, David C. Labaree, Jim Ropchan, Nabeel Nabulsi, Richard E. Carson, and Yiyun Huang
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.9b00618 • Publication Date (Web): 21 Jan 2020
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Synthesis and Preclinical Evaluation of an 18F-Labeled Synaptic Vesi-
cle Glycoprotein 2A PET Imaging Probe: [18F]SynVesT-2
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Zhengxin Cai,*,† Songye Li,*,† Wenjie Zhang,†,♯ Richard Pracitto,† Xiaoai Wu,†,♯ Evan Baum,†, Sjoerd J.
Finnema,†, Daniel Holden,† Takuya Toyonaga†, Shu-fei Lin,† Marcel Lindemann,† Anupama Shirali,†
David C. Labaree,† Jim Ropchan,† Nabeel Nabulsi,† Richard E. Carson,† Yiyun Huang†
†PET Center, Department of Radiology and Biomedical Imaging, Yale University, New Haven, CT 06520, USA.
♯Department of Nuclear Medicine, Laboratory of Clinical Nuclear Medicine, West China Hospital, Sichuan University,
Chengdu, Sichuan 610041, China.
Supporting Information Placeholder
and [18F]SDM-2/[18F]SynVesT-2 ([18F]7)39 as 18F-labeled candi-
ABSTRACT: Synaptic vesicle glycoprotein 2A (SV2A) is a 12-
date SV2A imaging probes with optimal imaging characteristics.
Radioligand 6 was initially synthesized by us and Invicro inde-
pendently, and given different names (SDM-8 by Yale, MNI-1126
by Invicro). To avoid future confusion in reference to the radiolig-
and, we and the Invicro group agreed to use SynVesT-1 (Synaptic
Vesicle Tracer-1) to refer to this radioligand. Accordingly, SDM-
2 will be referred to as SynVesT-2 in future publications. Herein,
we report the synthesis and preliminary evaluations of [18F]7 in
nonhuman primates.
pass transmembrane glycoprotein ubiquitously expressed in pre-
synaptic vesicles. In vivo imaging of SV2A using PET has potential
applications in the diagnosis and prognosis of a variety of neuro-
psychiatric diseases, e.g., Alzheimer’s disease, Parkinson’s dis-
ease, schizophrenia, multiple sclerosis, autism, epilepsy, stroke,
traumatic brain injury, post-traumatic stress disorder, depression,
etc. Herein, we report the synthesis and evaluation of a new 18F-
labeled SV2A PET imaging probe, [18F]SynVesT-2, which pos-
sesses fast in vivo binding kinetics and high specific binding signals
in nonhuman primate brain.
O
N
F
KEYWORDS: SV2A, PET, nonhuman primate, Alzheimer’s dis-
N
O Me
F
F
F
ease, Parkinson’s disease, fluorine-18.
11
CH
3
N
18
N
F
11
N
NH
C
2
O
INTRODUCTION
N
F
O
11
11
18
Synaptic vesicle glycoprotein 2A (SV2A) is an essential presyn-
aptic transmembrane protein1. SV2A is critical for neural system
functioning2, 3, and is dysregulated in epilepsy4-7. SV2A is also the
binding target of the antiepileptic drug, levetiracetam (Keppra®)8,
which has been shown to improve cognitive function in patients
with amnestic mild cognitive impairment (aMCI) and rodent mod-
els of memory loss and Alzheimer’s disease (AD)7, 9, 10, probably
through regulation of mitochondrial function11. Positron emission
tomography (PET) is a non-invasive quantitative imaging modal-
ity that provides functional information in living systems, and
SV2A-targeted PET imaging may provide potentially prognostic
information and elucidate the etiology and progression of a vari-
ety of neuropsychological diseases12.
[
C]Levetiracetam (1)
[
C]UCB-A (2)
[
F]UCB-H (3)
1
18
18
R
F
F
N
N
N
F
F
2
R
Me
F
Me
N
O
=
2
N
O
N
O
1
2
11
11
18
18
18
R
= F; R
18
CH : [ C]UCB-J(4)
[
F]SynVesT-1 (6)
[
[
F]SynVesT-2 ([ F]7)
3
1
18
18
18
R
=
F; R = CH : [ F]UCB-J (5)
[
[
F]MNI-1126
F]SDM-8
F]SDM-2
3
18
Figure 1. Current SV2A radioligands.
Several radioligands have been developed for imaging SV2A
(Figure 1), including [11C]levetiracetam (1)13, [11C]UCB-A (2)14,
[18F]UCB-H (3)15-18, [11C]UCB-J (4)19-22, [18F]UCB-J (5)23, and
[18F]SDM-8/[18F]MNI-1126/[18F]SynVesT-112, 24-26 (6). Among
them, [11C]UCB-J (4) has proved to be an excellent PET probe to
image and quantify synaptic density in living brain20, and has
been applied to the studies of a variety of neurodegenerative and
neuropsychiatric disorders22, 27, 28. However, the use of[11C]UCB-
J requires an on-site cyclotron, and its accessibility as a clinical
research tool is limited due to the short half-life of 11C (20.38
minutes). Thus, we set out to develop the 18F-labeled counterpart,
[18F]UCB-J23, 29, while exploring the chemical space around the
common pyrrolidinone pharmacophore, and synthesized analogs
of UCB-J that are readily radiofluorinated using recently devel-
oped radiofluorination methods30-37. Among the newly synthe-
sized analogs, we identified [18F]SDM-8/[18F]SynVesT-1 (6)24, 38
O
9
F
O
1.
F
Ph3P
OEt
THF/CH2Cl2
Pd/C, H2
MeOH
NH
F
CO2Et
NO2
CHO
11
2. MeNO2, DBU
8
10
N
F
F
N
N
Cl
1
12
Chiral HPLC
Me
Me
Me
NaH, TBAI, THF
N
N
O
O
Rac-7
7
Scheme 1. Synthesis of the standard compound for [18F]SDM-
2/[18F]SynVesT-2 ([18F]7).
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O
9
(S)-7, were 18 nM and 71 nM for rat and human SV2A, respec-
tively40. Although the Ki values of 7 were higher than those of
I
O
Ph3P
1.
OEt
I
1
THF/CH2Cl2
Raney-Ni, H2
MeOH
NH
I
40
2
CO2Et
NO2
UCB-J (Ki: 3.5 nM and 1.5 nM for rat and human SV2A)
an4d0
SynVesT-1 (Ki: 2.0 nM and 4.7 nM for rat and human SV2A) , 7
2. MeNO2, DBU
CHO
3
4
5
6
7
8
9
13
14
15
still holds promise as a suitable PET tracer candidate for brain
SV2A imaging, because of the high Bmax of SV2A in the brain.
N
O
O
I
I
Assuming the protein content in the brain tissue is 10% and tissue
Cl
N
O
1) TFA, Oxone, CHCl3
2)
12
density is 1 g/mL, the Bmax values are 4.5 pmol/mg protein41 (450
nM) and 3-4 pmol/mg protein41 (300-400 nM) in rat and human
brains, respectively.
Me
N
NaH, TBAI, THF
Na CO , EtOH
O
2
3
Me
N
O
O
O
Me
N
O
O
O
16
As the synthesis of [18F]7 involved the radiofluorination of an un-
activated aromatic ring37,43, we reasoned that hypervalent io-
donium precursors, i.e., diaryliodonium salts32 and iodonium
ylides34, 44-48, and organostannane precursors36 would be suitable
for the radiosynthesis of [18F]7. Thus, we synthesized both the io-
donium ylide and organostannane precursors. Synthesis of the io-
donium ylide precursor (Scheme 2) started with a Wittig reaction
using 13 and 9, followed by Michael addition, Raney-Ni-cata-
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18
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Scheme 2. Synthesis of the iodonium ylide precursor (18).
O
Br
O
9
Br
Ph3P
NH
Fe, EtOH/H2O
Br
1.
2.
OEt
THF/CH2Cl2
CO2Et
NO2
CHO
MeNO2, DBU
21
20
19
lyzed reduction, and cyclization, yielding pyrrolidinone 15 in 30%
yield over four steps. N-alkylation of 15 with 12 generated 16 in
68% yield. The iodonium ylide 18 was prepared in 30% yield by
oxidation of the iodide 16 with Oxone, followed by reaction with
6,10-dioxaspiro[4.5]decane-7,9-dione (17). Next, we tested the ra-
diolabeling conditions by heating 18 in N,N-dimethylformamide
(DMF) with [18F]Et4NF and Et4NHCO3 at 150 °C for 10 min, and
obtained the formulated [18F]7 in less than 1% radiochemical
yield (RCY), after a reverse phase HPLC purification and a chiral
HPLC separation, with molar activity of 857 ± 770 MBq/nmol (n
= 9) at the end of synthesis (EOS) (Table S1). Lower radiochemi-
cal conversions were observed at lower radiolabeling tempera-
tures, based on the radio-HPLC analysis of the crude reaction
mixture.
N
SnMe3
Br
(SnMe3)2, Pd(PPh3)4, LiCl
toluene
Cl
12
N
N
Me
Me
Me
N
N
NaH, TBAI, THF
chiral separation
O
(R)-22
O
23
Scheme 3. Synthesis of the organostannane precursor (23).
O
O
O
I
18F
SnMe3
N
O
N
N
18F
18F
Me
N
O
Me
N
Chiral separation
Me
Cu catalyst
N
O
O
We then explored the radiofluorination using an organostannane
precursor, which was synthesized following the same synthetic
route as 18. Starting with 3-bromobenzaldehyde (19), the bromide
()-22 was synthesized in 67% yield over five steps (Scheme 3).
After separation of the two enantiomers of 22, enantiopure or-
ganostannane (R)-23 was prepared in 59% yield via palladium-
catalyzed stannylation (Scheme 3). Radiofluorination of 23 was
carried out in N,N-dimethylacetamide (DMA) at 110 °C for 20
min in the presence of [18F]KF, copper(II) triflate, and pyridine to
give [18F]7 in an average radiochemical yield of 6.8% (n = 9, de-
cay corrected) after semipreparative HPLC (Figure 2A), with an
average molar activity of 141 MBq/nmol (n=8) at EOS (Table
S2). The accrued synthesis, purification, and formulation time was
less than 90 min. The radiochemical purity of [18F]7 was greater
than 99% and [18F]7 coeluted with 7 under the same HPLC condi-
tions (Figure 2B). No racemization of the product was observed
under these radiolabeling conditions (Figure 2C). And the final
product in the formulated solution was tested to be stable for at
least 4 h at room temperature.
[
18F]7
18
23
Scheme 4. Radiosynthesis of [18F]SDM-2/[18F]SynVesT-2
([18F]7) via the iodonium ylide precursor (18) or organostannane
precursor (23).
RESULTS AND DISCUSSION
Chemistry. As radiofluorination of UCB-J was shown to be chal-
lenging, we decided to modify the structure of UCB-J to allow for
efficient radiofluorination using existing methodologies without
compromising the binding affinity to SV2A. The structural modi-
fications also provided us with the opportunity to study the struc-
ture-pharmacokinetics (PK) relationship of this series of SV2A
ligands. Compared with UCB-J, its monofluorinated analogs pos-
sess attractive characteristics as being amenable for radiofluorina-
tion, less lipophilic, and potentially less nonspecific binding in
vivo. Because even subtle structural changes of small organic
molecules could lead to dramatic changes in binding affinity, in
vivo stability and PK profile, we synthesized and screened a li-
brary of monofluorinated analogs and found that compound 7 pos-
sessed the highest binding affinity39. Analog 7 was synthesized
starting from a Wittig reaction using commercially available 3-
fluorobenzaldehyde (8) and (carbethoxymethylene)tri-
phenylphosphorane (9), followed by treatment with nitromethane
under basic conditions to give compound 10, which was reduced
and cyclized to yield pyrrolidinone 11. N-alkylation of 11 with 4-
(chloromethyl)-3-methylpyridine (12) generated racemic 7 in 78%
yield (Scheme 1). The enantiopure compound 7 and its enantio-
mer were isolated after chiral HPLC separation. The in vitro bind-
ing affinity of 7 was then tested through competition binding as-
says with [3H]UCB-J in rat and human brain tissue homoge-
nates40. The Ki values of 7 were 7.6 nM and 12 nM for rat and hu-
man SV2A respectively; while the Ki values of its enantiomer, the
Figure 2. Representative HPLC chromatograms of [18F]7 as in the
crude reaction mixture (A), co-injected with the standard com-
pound 7 on a Luna C18 (5휇m, 4.6 × 250 mm) column (B), and
co-injected with the racemic 7 on a ChiralCel ODRH (5 휇m, 4.6
× 150 mm) column, eluting with a mobile phase (pH 4.2) of
35% CH3CN and 65% 0.1 M ammonium formate solution with
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0.5% acetic acid at a flow rate of 1 mL/min (C). The red indicates
gamma peaks; the blue indicates UV peaks.
1
2
3
4
5
6
7
In Vitro Study. The partition coefficient logD was determined by
the ratio of decay-corrected radioactivity concentrations in n-oc-
tanol and PBS. LogD of [18F]7 is 2.17  0.02 (n = 3), which is
within the optimal range for blood-brain barrier penetration49, and
is lower than those of 4 and 6 (LogD: 2.53 and 2.32 for 4 and 6,
respectively).
PET Imaging in Nonhuman Primates. The injected activity
ranged from 163.7 to 185.9 MBq (n = 4), corresponding to 23.3 –
57.6 ng of 7. The plasma free fraction (fP) of [18F]7 was 41 ± 2%
(n = 3), which is slightly lower than that of 4 (46.2 ± 2.5%, n =
11). The total radioactivity in the plasma showed a sharp increase
within three minutes, a fast washout phase, followed by a slow
washout phase (Figure 3A). The metabolism rate of [18F]7 was
moderate and similar to that of 4, with 34 ± 0.1% of intact parent
radiotracer at 30 min post-injection (p.i.), which further decreased
to 24 ± 3% and 22 ± 4%, at 60 and 90 min respectively (Figure
3B). The radioactive metabolites in the plasma were shown to
have shorter retention times than the parent compound on the
HPLC chromatogram, indicating that they were more hydrophilic
and less likely to penetrate the BBB (Figure 3C).
High quality brain PET images were generated with [18F]7 in rhe-
sus monkey, with high tracer uptake in grey matter and very low
uptake in white matter (Figure 4A). The tracer entered brain
quickly after intravenous injection, with peak standardized uptake
value (SUV) around 8.5 within 10 min p.i. (Figure 4B). We ob-
served higher tracer uptake in the frontal cortex and putamen
(peak SUV > 8), and lowest uptake in the white matter region,
i.e., centrum semiovale (SUV < 2). [18F]7 displayed faster kinetics
than 4 and 6 (Figure S1), reaching apparent equilibrium within
60 min p.i. in most brain regions (Figure 4C). No tracer uptake in
skull was observed even from the late PET images, indicating the
lack of in vivo defluorination. When the animal was pretreated
with UCB-J (150 µg/kg, i.v.), the whole brain SUV decreased to
the same level as centrum semiovale, indicating the in vivo bind-
ing specificity of [18F]7 to SV2A (Figure4D).
8
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Figure 4. (A) Summed SUV images of [18F]SynVesT-2 ([18F]7)
in the brain of a rhesus monkey from different imaging windows.
(B) Regional time-activity curves (TACs) of [18F]7 from baseline
PET scans (n = 3). (C) Time course of regional tracer uptake to
arterial input ratios. (D) TACs of pre-blocking scan with pre-in-
jected UCB-J (150 µg/kg, i.v., n = 1).
Table 1. Distribution volumes (VT/mL.cm-3)* of 4, 6, 7, and 7
with pre-blocking in monkey brain regions calculated with 1TCM.
[18F]Syn- [11C]UCB- [18F]Syn-
[18F]Syn-
VesT-1 (6) VesT-2 (7)
n = 3 Preblocking
Radiotracer
VesT-2 (7) J (4)
n = 3 n = 6
Brain regions
Cingulate cortex 18.2 ± 2.2 55.6 ± 10 48.5 ± 8.9 5.34
Frontal cortex 17.5 ± 2.2 55.4 ± 8.2 47.1 ± 9.1 4.9
Nucleus accum-
16.9 ± 2.7 54 ± 9.2 45.2 ± 7.9 4.82
bens
Insular cortex
16.4 ± 2.9 54.6 ± 6.7 46.1 ± 9.2 4.72
Occipital cortex 15.7 ± 2.8 52.9 ± 7.1 43.7 ± 10.7 4.53
Temporal cortex 14.7 ± 2.6 50.5 ± 6.9 42.2 ± 10.0 4.31
Caudate nucleus 13.6 ± 2.3 44.9 ± 4.6 35.0± 6.3 4.36
B
A
Thalamus
Putamen
13.3 ± 2.6 40.3 ± 6.5 34.2 ± 3.0 3.82
13.2 ± 2.2 45.2 ± 3.1 34.6 ± 4.9 4.35
Hippocampus 10.9 ± 2.7 34.5 ± 2.8 30.1 ± 5.6 3.47
Cerebellum 10.8 ± 2.8 36± 5.3 28.9 ± 6.8 3.79
Globus pallidus 8.8 ± 1.9 28 ± 3 21.6 ± 4.2 4.01
8.8 ± 2.5 24.8 ± 1.9 24.8± 8.5 2.63
6.6 ± 3 23.6 ± 2.9 16.3± 3.6 3.1
6.5 ± 3.5 23.4 ± 3.2 16.0± 3.4 3.08
Amygdala
Brainstem
C
Pons
Centrum semio-
4.4 ± 1.6 13.6 ± 2.9 8.9± 2.0
3.19
vale
*Data are single or multiple (n) measurements represented as sin-
gle value or mean ± SD, respectively. SD is standard deviation.
Pre-blocking scan was done with pre-injected UCB-J (150 µg/kg,
n = 1).
Figure 3. (A) Total plasma radioactivity (kBq/mL) over time. (B)
Unmetabolized parent fraction of [18F]7 (in green) and 4 (in red)
from baseline nonhuman primate PET scans. (C) Radio-HPLC
chromatograms of plasma metabolite analysis of [18F]7 (retention
time at 11 min) over time. Organic contents captured on a short
C18 column were backflushed onto a Phenomenex Luna C18(2)
column (5휇m, 4.6 × 250 mm) eluting with 37% acetonitrile/
63% 0.1 M ammonium formate (pH6.4) at flow rate of 2 mL/min.
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PET tracer with faster pharmacokinetics are expected to reach
1
2
3
4
5
6
7
8
equilibrium more quickly, and needs shorter scan time for reliable
VT estimation. Thus, we evaluated the time stability of the VT esti-
mation (1TCM) for 17 brain regions, by choosing different scan
durations for 1-tissue compartmental modelling, and compared
the minimum scan times required by [18F]7, 4, and 6. As expected,
a 30-min scan time was needed for [18F]7 to generate reliable VT
estimates (averaged VT values within 10% deviation from the 120-
min VT values); while 45 min and 60 min scan durations were
needed for 4 and 6, respectively (Figure 5). As early imaging pro-
vides K1 map, reflecting brain perfusion22, dynamic scans using
[18F]7 could provide information on both cerebral blood flow and
SV2A binding in shorter scan time than that required for 4 and6.
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The specific binding signals, as presented by the regional non-dis-
placeable binding potential (BPND) values, were highly similar to
those of [11C]UCB-J (4) (Table 2), due to the lower nonspecific
binding as evidenced by the lower uptake of [18F]7 in the white
matter, which was used as the reference region in BPND calcula-
tion. The BPND value was highest in the cingulate cortex, followed
by the frontal cortex, nucleus accumbens, insula, occipital, tem-
poral cortex, caudate putamen, thalamus, cerebellum, hippocam-
pus, globus pallidus, amygdala, and brainstem, consistent with the
regional distribution of SV2A19, 20. Due to partial volume effect in
the sentrum semiovale of nonhuman primates, the BPND calcu-
lated using sentrum semiovale could be biased. The issue of using
centrum semiovale as reference for quantitative data analysis has
Figure 5. Time stability of VT estimates (1TCM) of 4, 6, and
[18F]7 in 17 brain regions of nonhuman primates. The VT values
estimated using scan durations ranging from 30 to 120 min were
analysed in 15 min increments. The y-axis represents percentage
of VT value derived from 120 min scan datasets.
Kinetic Modelling. For [18F]7, the 1-tissue compartment model
(1TCM) produced suitable fits and reliable estimates of regional
distribution volume (VT), the equilibrium ratio of brain to plasma.
The VT values ranged from 4.4 ± 1.6 (n = 3) for centrum semio-
vale to 18.2 ± 2.2 (n = 3) for cingulate cortex, and were globally
lower than those of [11C]UCB-J (4) (Table 1). Note that [18F]7
has lower uptake than [11C]UCBJ (4) and[18F]SDM-8/SynVesT-1
(6) in the centrum semiovale, which is considered a region with
mainly non-specific binding and could be used as reference region
for noninvasive quantitative data analysis50. The lower VT values
of [18F]7 in centrum semiovale (4.4 ± 1.6 VT/mL.cm-3, compared
with 13.6 ± 2.9 VT/mL.cm-3 and 8.9 ± 2.0 VT/mL.cm-3 for 4 and 6,
respectively) (Table 1) is consistent with its slightly lower logD
value (2.17, compared with 2.53 and 2.32 for 4 and 6, respec-
tively).
been studied by several groups50-52
.
20
y    x - 
R  
15
10
5
p  
Table 2. Binding potential (BPND) values* of 4, 6, 7, and 7 with
pre-blocking in monkey brain regions calculated from the VT val-
ues using centrum semiovale as reference region.
[18F]Syn- [11C]UCB [18F]Syn- [18F]Syn-
Radiotracer
0
0
5
10
15
20
25
VesT-2 (7) -J(4)
VesT-1 (6) VesT-2 (7)
VT(baseline)
Brain regions
n = 3
n = 6
n = 3
Preblocking
0.68
Figure 6. Receptor occupancy plot (Lassen plot) of the difference
between the VT values of [18F]7 with and without pre-injected
UCB-J (150 µg/kg, i.v.) against the baseline VT values in the same
rhesus monkey. The slope of the linear regression is the estimated
SV2A occupancy by UCB-J (150 µg/kg, i.v.), and the x-intercept
is the estimated nondisplaceable volume of distribution (VND).
Cingulate cortex 3.5 ± 1.6 3.2 ± 0.9 4.5 ± 0.4
Frontal cortex
3.3 ± 1.5 3.2 ± 1
4.3 ± 0.6 0.54
Nucleus accum-
bens
3.2 ± 1.3 3.1 ± 0.7 4.1 ± 0.4
0.48
Insular cortex
3 ± 1.3
3.2 ± 0.9 4.2 ± 0.3 0.51
3 ± 0.7 3.9 ± 0.2 0.42
Based on Lassen plot analysis, the SV2A-specific ligand UCB-J
(150 µg/kg, preinjected intravenously) blocked 84% of tracer
binding, indicating high in vivo binding specificity of [18F]7 (Fig-
ure 6). The non-displaceable volume of distribution (VND) of
[18F]7 calculated from Lassen plot was 2.1 mL/cm3, which was
lower than the VND of 4 (6.27 mL/cm3), and 6 (2.97 mL/cm3). Un-
der the same blocking condition, similar SV2A occupancy was
achieved when 4 (87%) or 6 (79%, Figure S3) was used as the
PET ligand19.
CONCLUSIONS. In summary, we evaluated a new 18F-labeled
SV2A PET imaging probe, [18F]7, which was synthesized via io-
donium ylide and organostannane precursors in high molar activ-
ity and high radiochemical purity. PET imaging in monkeys
demonstrated that the new SV2A radioligand [18F]7 exhibited ex-
cellent image quality and suitable pharmacokinetics for quantita-
tive kinetic modelling and provided specific binding signals
highly similar to those of [11C]UCB-J (4) in nonhuman primates
using white matter as reference region, but with faster kinetics,
which could be advantageous in the clinical setting. The synthesis
Occipital cortex 3 ± 1.5
Temporal cortex 2.7 ± 1.4 2.8 ± 0.7 3.7 ± 0.3 0.35
Caudate nucleus 2.3 ± 0.9 2.4 ± 0.8 3.0 ± 0.4 0.36
Putamen
2.3 ± 0.9 2.5 ± 0.7 2.9 ± 0.4 0.37
2.2 ± 0.8 2.1 ± 0.7 2.9 ± 0.7 0.20
1.6 ± 0.8 1.7 ± 0.4 2.2 ± 0.2 0.19
1.6 ± 0.6 1.6 ± 0.4 2.4 ± 0.4 0.09
Thalamus
Cerebellum
Hippocampus
Globus pallidus 1.2 ± 0.7 1.1 ± 0.3 1.4 ± 0.3 0.26
Amygdala
Brainstem
Pons
1.1 ± 0.3 0.9 ± 0.4 1.9 ± 1.1 -0.18
0.6 ± 0.5 0.8 ± 0.3 0.8 ± 0.0 -0.03
0.5 ± 0.6 0.8 ± 0.3 0.8 ± 0.0 -0.03
*Data are single or multiple (n) measurements represented as sin-
gle value or mean ± SD, respectively. SD is standard deviation.
Pre-blocking scan was done with pre-injected UCB-J (150 µg/kg,
n = 1).
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ACS Chemical Neuroscience
process using the organostannane precursor (23) has been vali-
with Na2SO4, and concentrated in vacuo to give 9 as a yellow oil
dated for production of [18F]7 for human use. Translation of [18F]7
into human evaluation is underway at Yale PET Center and will
be reported in due course.
(5.7 g, 77%), which was used in the next step of synthesis without
further purification. 1H NMR (CDCl3, 400 MHz): δ 7.39 – 7.21
(m, 1H), 7.11 – 6.90 (m, 3H), 4.73 (dd, J = 12.8, 6.7 Hz, 1H),
4.63 (dd, J = 12.7, 8.2 Hz, 1H), 4.20 – 4.04 (m, 2H), 4.04 – 3.83
(m, 1H), 2.74 (dd, J = 7.4, 2.4 Hz, 2H), 1.18 (t, J = 7.1 Hz, 3H).
1
2
3
4
METHODS
5
4-(3-Fluorophenyl)pyrrolidin-2-one (11): A solution of ethyl 3-
(3-fluorophenyl)-4-nitrobutanoate (10, 0.56 g, 2.2 mmol) in
MeOH (5 mL) was degassed with argon for 5 min, Pd/C (10%,
0.36 g) was added, and the suspension stirred under an atmos-
phere of hydrogen at ambient temperature for 12 h. The reaction
mixture was filtered through a layer of Celite and the filtrate con-
centrated in vacuo. The crude product was purified by flash col-
umn chromatography on silica gel (0-10% EtOH/EtOAc) to pro-
vide 11 as a colorless oil (390 mg, 99%). 1H NMR (CDCl3, 400
MHz): δ 7.29 (q, J = 7.3, 6.89 Hz, 1H), 7.01 (d, J = 7.6 Hz, 1H),
6.94 (t, J = 8.2 Hz, 2H), 3.78 (t, J = 8.8 Hz, 1H), 3.68 (p, J = 8.2
Hz, 1H), 3.39 (t, J = 8.5 Hz, 1H), 2.73 (dd, J = 16.9, 8.9 Hz, 1H),
2.46 (dd, J = 16. 9, 8.6 Hz, 1H).
Chemistry. Materials, Instruments, and General Methods. All re-
agents and solvents were obtained from commercial sources
(Sigma-Aldrich, TCI, and Fisher Scientific) and used without fur-
ther purification unless noted otherwise. Nuclear magnetic reso-
nance (1H-, 13C-, and 19F-NMR) spectra were recorded on an Ag-
ilent DD2 400 MHz (A400a), Agilent DD2 500 MHz (A500a),
Varian 400 MHz, or AVANCE III 400 MHz UltraShield-Plus
Digital NMR Spectrometer. Chemical shifts are reported in parts
per million, with the solvent resonance as the internal standard
(CDCl3: 7.26 ppm; DMSO-d6: 2.49 ppm). Melting point was ex-
amined on an Electrothermal Mel-Temp instrument. High-perfor-
mance liquid chromatography-mass spectrometry (HPLC-MS)
was performed with an Agilent 1200RRLC/6110SQD system.
High resolution mass spectrometry (HRMS) was done with
Thermo LTQ Orbitrap spectrometer. [18F]Fluoride was produced
via the 18O(p, n) 18F nuclear reaction in a 16.5-MeV GE PETtrace
cyclotron (Uppsala, Sweden). H218O was obtained from Huayi
Isotopes (Toronto, Canada). Anion exchange Chromafix car-
tridges (PS-HCO3) were purchased from Macherey-Nagel
(Dueringen, Germany). Solid-phase extraction (SPE) cartridges
were purchased from Waters Associates (Milford, MA, USA). All
chemicals used in this study were of ≥ 95% purity, based on
HPLC, LC-MS, or NMR analysis. The semi-preparative HPLC
system comprises a Shimadzu LC-20A pump, a Knauer K200 UV
detector, and a Bioscan -flow detector, with a Luna C18(2) col-
umn (10 × 250 mm, 10 m, Phenomenex, Torrance, CA, USA).
The chiral semi-preparative HPLC system uses a ChiralCel OD-H
column (10 × 250 mm, 5 m, ChiralCel, West Chester, PA,
USA). For quality control, we used a Shimadzu LC-20A pump, a
Shimadzu SPD-M20A PDA or SPD-20A UV detector, aBioscan
-flow detector, with a Genesis C18 column (4.6 × 250 mm, 4
m, Hichrom, Berkshire, UK) eluting with a mobile phase (pH
4.2) of 34% CH3CN and 66% 0.1 M ammonium formate solution
with 0.5% acetic acid at a flow rate of 2 mL/min for purity check
as well as a ChiralCel OD-RH (4.6 × 150 mm, 5 m, ChiralCel,
West Chester, PA, USA) eluting with a mobile phase (pH 4.2) of
30% CH3CN and 70% 0.1 M ammonium formate solution with
0.5% acetic acid at a flow rate of 1 mL/min or a mobile phase (pH
4.2) of 35% CH3CN and 65% 0.1 M ammonium formate solution
with 0.5% acetic acid at a flow rate of 1 mL/min. Chemical iden-
tity of the tracer was confirmed by co-elution of the tracer co-in-
jected with its non-radioactive (R)-7.
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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30
31
32
33
34
35
36
37
38
39
40
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43
44
45
46
47
48
49
50
51
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53
54
55
56
57
58
59
60
4-(3-Fluorophenyl)-1-((3-methylpyridin-4-yl)methyl)pyrroli-
din-2-one (7): To an oven-dried vial cooled at 0 C was added a
solution of 4-(3-fluorophenyl)pyrrolidin-2-one (11) (55 mg, 0.31
mmol) in anhydrous THF (2.0 mL) and sodium hydride (61 mg,
1.54 mmol). The corresponding chloromethyl pyridine (12, 0.37
mmol) was then added and the mixture stirred overnight at ambi-
ent temperature. The reaction was quenched with saturated Na-
HCO3 solution (6 mL) and extracted with EtOAc (3 mL × 3). The
organic extracts were combined, dried over Na2SO4 and concen-
trated in vacuo. The crude product was purified with flash column
chromatography on silica gel eluting with 0-10% EtOH/EtOAc to
afford the racemic 7 (69 mg, 78%) as a light yellow oil. 1H NMR
(CDCl3, 400 MHz): δ 8.40 (s, 2H), 7.29 (q, J = 7.4, 7.4 Hz, 1H),
7.03 (d, J = 4.8 Hz, 1H), 6.95 (d, J = 8.0 Hz, 2H), 6.88 (d, J =
10.0 Hz, 1H), 4.60 (d, J = 15.6 Hz, 1H), 4.40 (d, J = 15.5 Hz, 1H),
3.59 (m, 2H), 3.26 (m, 1H), 2.90 (dd, J = 17.1, 8.2 Hz, 1H), 2.62
(dd, J = 17.1, 7.6 Hz, 1H), 2.29 (s, 3H). 13C NMR (CDCl3, 100
MHz): δ 173.57, 151.05 (2C), 147.77 (2C), 142.96,131.65,
130.48 (split 2 peaks), 122.32 (split 2 peaks), 122.29, 114.15 (split
2 peaks), 113.71 (split 2 peaks), 53.76, 43.54, 38.21, 36.94, 15.93.
19F NMR (CDCl3, 376 MHz): δ -112.17. HRMS: calculated for
C17H17FN2O [M + H]+ 285.1398; found, 285.1393.
Chiral HPLC separation of 7: A solution of the racemic 7 (2
mg/mL, in n-hexane/EtOH, 70/30, v/v, with 0.1% Et3N) was
loaded on to a Daicel Chiralpak IA column (10 × 250 mm, 5 µm)
and eluted with n-hexane/EtOH (70/30, v/v, with 0.1% Et3N) at a
flow rate of 4 mL/min. (R)-7 was obtained after combination of
collected fractions and evaporation of solvents under reduced
pressure, with ee. > 98.6%. The stereochemistry assignment was
based on analogy with the assignment of the two enantiomers of
UCB-J on chiral HPLC eluting sequence, i.e., the first peak being
(S), the second peak (R).
Ethyl 3-(3-fluorophenyl)-4-nitrobutanoate (10): 3-Fluoroben-
zaldehyde (8, 4 g, 32.3 mmol) was dissolved in anhydrous THF (8
mL) in an oven-dried round bottom flask and the solution cooled
on ice to ca. 5 C. The solution of carbethoxymethylene triphenyl
phosphorene (9, 11.8 g, 33.9 mmol) in anhydrous CH2Cl2 (25 mL)
was then added dropwise at 5 C. The reaction mixture was stirred
for 30 min at 5 C, and concentrated to dryness in vacuo. A mix-
ture of n-hexanes and Et2O (200 mL, 10:1, v/v) was added to the
white residue and the suspension stirred for 30 min at ambient
temperature. The solid was filtered and the filtrate evaporated to
yield a white solid (6.5 g), which was dissolved in nitromethane
(4 mL, 74.8 mmol) and cooled on ice to 5 C. 1,8-Diazabicy-
clo(5.4.0)undec-7-ene (DBU, 4.39 g, 28.9 mmol) was then added
dropwise and the solution stirred for 1 h at 5 C. The reaction was
quenched with saturated NH4Cl solution (50 mL), acidified with
aqueous HCl (2 M), and extracted with EtOAc (50 mL × 3). The
combined organic phases were washed with brine (100 mL),dried
Ethyl 3-(3-iodophenyl)-4-nitrobutanoate (14): 3-Iodobenzalde-
hyde (13, 4 g, 17.2 mmol) was dissolved in anhydrous THF (10
mL) in an oven-dried round bottom flask and the solution was
cooled in an ice bath to ca. 5 C. A solution of carbethoxymeth-
ylene triphenyl phosphorene (6 g, 17.2 mmol) in anhydrous
CH2Cl2 (20 mL) was then added dropwise at 5 C. The reaction
mixture was stirred for 30 min at 5 C. When the reactants were
completely consumed based on TLC analysis, the reaction mix-
ture was evaporated in vacuo to near dryness. A mixture of hex-
anes and Et2O (150 mL, 10:1, v/v) was added, and the suspension
was stirred for 2 h at ambient temperature. The solution was fil-
tered, and the filtrate was evaporated to dryness in vacuo to yield
a white solid (4.5 g), which was dissolved in nitromethane (2.5
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16.4, 8.7 Hz, 1H), 2.50 (dd, overlap with DMSO solvent residue
peak, 1H), 2.23 (s, 3H), 1.94 (m, 4H), 1.63 (m, 4H).
mL, 46.7 mmol) and cooled on ice to ca. 5 C. Then, 1,8-diazabi-
cyclo(5.4.0)undec-7-ene (DBU, 2.62 g, 17.2 mmol) was added
dropwise, and the mixture was stirred at ca. 5 C for 1 h. The re-
action was quenched with saturated NH4Cl solution (20 mL), and
acidified with aqueous HCl (2 M). The mixture was extracted
with EtOAc (25 mL × 3). The combined organic extracts were
washed with brine (50 mL), dried with Na2SO4, filtered, and con-
centrated in vacuo to provide 14 as a yellow oil (6.7 g), which was
used in the next step of synthesis without further purification. 1H
NMR (CDCl3, 400 MHz): δ 7.70 – 7.53 (m, 2H), 7.22 (m,1H),
7.07 (td, J = 7.7, 1.8 Hz, 1H), 4.82 – 4.68 (m, 1H), 4.68 – 4.57 (m,
1H), 4.18 – 4.01 (m, 2H), 4.01 – 3.84 (m, 1H), 2.73 (ddd, J = 7.7,
3.6, 1.6 Hz, 2H), 1.18 (td, J = 7.1, 2.0 Hz, 3H).
1
2
3
4
5
6
7
8
Ethyl 3-(3-bromophenyl)-4-nitrobutanoate (20): To a solution
of compound 19 (5.00 mL, 31.2 mmol) in anhydrous THF (40.0
mL) under argon and cooled at 0 C was added dropwise a solu-
tion of phosphorus ylide 9 (10.9 g, 31.23 mmol) in anhydrous
CH2Cl2 (60.0 mL). The reaction mixture was stirred for 1 h at 5
oC, and then concentrated in vacuo. A mixture of hexanes and
Et2O (4:1, v/v, 40 mL) was added and the suspension was stirred
for 10 min at ambient temperature. Filtration of the mixture
through a silica gel plug and evaporation of the solvents afforded
a white solid intermediate ethyl (Z)-3-(3-bromophenyl)acrylate
(8.0 g, quantitative), which was used in the next step of synthesis
without further purification. 1H NMR (CDCl3, 400 MHz): δ 7.65
(s, 1H), 7.58 (d, J = 16.0 Hz, 1H), 7.48 (dt, d, J = 7.9, 2.0 Hz,
1H), 7.42 (dt, d, J = 8.0, 1.4 Hz, 1H), 7.27-7.20 (m, 1H), 6.41 (d,
J = 16.0 Hz, 1H), 4.25 (q, J = 7.1 Hz, 2H), 1.32 (t, J = 7.1 Hz,
3H).
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
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48
49
50
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58
59
60
4-(3-Iodophenyl)pyrrolidin-2-one (15): A solution of ethyl 3-
(3-iodo phenyl)-4-nitrobutanoate (14, 545 mg, 1.5 mmol) in EtOH
(5 mL) was degassed with argon for 5 min. Raney-Ni (50% in wa-
ter, 0.8 mL) was added in one portion. The suspension was then
stirred under an atmosphere of hydrogen at ambient temperature
for 2 days. The mixture was filtered through a layer of Celite and
the filtrate concentrated in vacuo. The crude product was dis-
solved in toluene (50 mL), heated to reflux for 3 h, and concen-
trated in vacuo. The crude product was purified by flash column
chromatography on silica gel (0-10% EtOH/EtOAc) to give 15 as
a yellow oil (430 mg, 30%). 1H NMR (CDCl3, 400 MHz): δ 7.59
(d, J = 6.4 Hz, 2H), 7.21 (d, J = 7.7 Hz, 1H), 7.07 (t, J = 8.0 Hz,
1H), 5.99 (s, 1H), 3.76 (t, J = 8.8 Hz, 1H), 3.62 (p, J = 8.3 Hz,
1H), 3.38 (t, J = 7.4 Hz, 1H), 2.71 (dd, J = 16.9, 8.9 Hz, 1H), 2.44
(dd, J = 16.9, 8.6 Hz, 1H).
The intermediate (8.0 g, 31.35 mmol) was dissolved in CH3NO2
(15 mL, 280 mmol) under argon and cooled at -20 C. DBU (4.5
mL, 30.2 mmol) was added and the reaction mixture was kept stir-
ring for 2 h at -20 C. Water (10 mL) was added, followed by 12
N HCl until the pH of the mixture reached 1. The mixture was ex-
tracted with EtOAc (15 mL × 3). The combined organic phase
was dried over Na2SO4 and concentrated in vacuo. The crude
product compound 20 was obtained as a clear oil (9.5 g, quantita-
tive) and used in the next step without further purification. 1H
NMR (CDCl3, 400 MHz): δ 7.41 (dt, J = 7.7, 1.6 Hz, 1H), 7.36(d,
J = 1.9 Hz, 1H), 7.22-7.13 (m, 2H), 4.71 (dd, J = 12.8, 6.8 Hz,
1H), 4.61 (dd, J = 12.8, 8.0 Hz, 1H), 4.08 (q, J = 7.1 Hz, 2H),
3.94 (quint., J = 7.4 Hz, 1H), 2.72 (m, 2H), 1.17 (t, J = 7.1 Hz,
3H).
4-(3-Iodophenyl)-1-((3-methylpyridin-4-yl)methyl)pyrrolidin-
2-one (16): To a solution of 15 (250 mg, 0.87 mmol) in anhy-
drous THF (3 mL) under argon and cooled at 0 C was added so-
dium hydride (110 mg, 2.75 mmol). Tetrabutylammonium iodide
(TBAI, 17 mg, 0.05 mmol) and 4-(chloromethyl)-3-methylpyri-
dine hydrochloride (11, 180 mg, 0.96 mmol) were added after 30
min. The reaction mixture was kept stirring for 16 h at ambient
temperature, then quenched with saturated NaHCO3 solution (6
mL) and extracted with EtOAc (3 mL × 3). The organic extracts
were combined, dried over Na2SO4 and concentrated in vacuo.
The crude product was purified on a silica gel column eluting with
0-10 % EtOH/EtOAc to afford compound 16 as a light brown oil
(256 mg, 68 %). 1H NMR (CDCl3, 400 MHz): δ 8.41 (s, 2H), 7.58
(dt, J = 7.8, 1.4 Hz, 1H), 7.51 (m, 1H), 7.13 (dt, J = 7.8, 1.4 Hz,
1H), 7.05 (d, J = 10.1 Hz, 1H), 7.03 (d, J = 7.5 Hz, 1H ), 4.57 (d,
J = 15.5 Hz, 1H), 4.42 (d, J = 15.5 Hz, 1H), 3.59 (m, 1H), 3.53 (p,
J = 8.6 Hz, 1H), 3.23 (m, 1H), 2.88 (dd, J = 17.0, 8.8 Hz, 1H),
2.60 (dd, J = 17.0, 8.1 Hz, 1H), 2.30 (s, 3H).
4-(3,-Bromophenyl)pyrrolidin-2-one (21): To a suspension of
compound 20 (9.5 g, 30.2 mmol) and iron powder (17 g, 304.4
mmol) in a mixture of EtOH and water (2:1, v/v, 300 mL) was
added NH4Cl (55 g, 1,028 mmol). After stirring for 16 h at ambi-
ent temperature, the reaction mixture was adjusted to pH 14 with
saturated NaOH solution and extracted with EtOAc (200 mL × 3).
The combined organic phase was dried over Na2SO4 and concen-
trated in vacuo to afford compound 21 as a white solid (6.4 g,
88%), M.P. 95 - 97 oC. 1H NMR (CDCl3, 400 MHz): δ 7.39 (m,
2H), 7.23-7.14 (m, 2H), 5.82 (s, 1H), 3.77 (t, J = 8.2 Hz, 1H),
3.71-3.56 (m, 1H), 3.39 (m, 1H), 2.72 (dd, J = 16.9, 8.9 Hz, 1H),
2.45 (dd, J = 16.9, 8.6 Hz, 1H).
4-(3-Bromophenyl)-1-((3-methylpyridin-4-yl)methyl) pyrroli-
din-2-one (22): To a solution of compound 21 (2 g, 8.3 mmol) in
anhydrous THF (40 mL) under argon and cooled to 0 C was
added NaH (0.9 g, 22.5 mmol). TBAI (0.13 g, 0.35 mmol) and
compound 12 (1.58 g, 8.87 mmol) were added after 30 min. The
reaction mixture was kept stirring for 16 h at ambient temperature,
then quenched with saturated NaHCO3 solution (50 mL) and ex-
tracted with EtOAc (30 mL × 3). The combined organic phase
was dried over Na2SO4 and concentrated in vacuo. The crude
product was purified on a silica gel column eluting with 0-10%
EtOH/EtOAc to afford compound 22 as a light brown oil (2.2 g,
76%). 1H NMR (CDCl3, 400 MHz): δ 8.40 (m, 2H), 7.38 (m,1H),
7.31 (s, 1H), 7.18 (t, J = 7.8 Hz, 1H), 7.10 (dt, J = 7.7, 1.4 Hz,
1H), 7.02 (d, J = 5.0 Hz, 1H), 4.58 (d, J = 15.5 Hz, 1H), 4.42 (d, J
= 15.5 Hz, 1H), 4.10 (q, J = 7.2 Hz, 1H), 3.65-3.58 (m, 1H), 3.28-
3.17 (m, 1H), 2.96-2.82 (m, 1H), 2.61 (dd, J = 17.0, 8.1 Hz, 1H),
2.30 (s, 3H).
8-((3-(1-((3-Methylpyridin-4-yl)methyl)-5-oxopyrrolidin-3-
yl)phenyl)-λ3-iodaneylidene)-6,10-dioxaspiro[4.5]decane-7,9-
dione (18): To a solution of 16 (300 mg, 1.04 mmol) in CHCl3
(1.5 mL) was added trifluoroacetic acid (TFA, 2.5 mL, 32.4
mmol) followed by Oxone (480 mg, 1.56 mmol). The reaction
mixture was stirred for 2 h until it turned to a white suspension.
The volatile contents were removed in vacuo. The residue was
suspended in EtOH (2 mL) and 6,10-dioxaspiro[4.5]decane-7,9-
dione (17, 220 mg, 1.29 mmol) was added, followed by 10%
Na2CO3 until the pH of the mixture reached 10. The reaction mix-
ture was stirred for 3 h, diluted with water, and extracted with
CH2Cl2 (1.0 mL × 3). The organic phases were combined, dried
over MgSO4, filtered, and concentrated in vacuo. The crude prod-
uct was purified on a silica gel column eluting with 10-40%
EtOH/EtOAc to afford 18 as a white solid (120 mg, 30%). 1H
NMR (DMSO-d6, 400 MHz): δ 8.35 (s, 2H), 7.72 (d, J = 1.8 Hz,
1H), 7.59 (dt, J = 8.0, 1.4 Hz, 1H), 7.47 (d, J = 7.8, 1.2 Hz, 1H),
7.38 (t, J = 7.8 Hz, 1H), 7.11 (d, J = 4.8 Hz, 1H), 4.51 (d, J = 16.1
Hz, 1H), 4.33 (d, J = 16.1 Hz, 1H), 3.70 (p, J = 8.3 Hz, 1H), 3.61
(t, J = 8.7 Hz, 1H), 3.20 (dd, J = 9.3, 7.3 Hz, 1H), 2.76 (dd, J =
Chiral HPLC separation: Chiral separation of two enantiomers
of 22 was done on a Chiralpak IA preparative column (10 × 250
mm, 5 µm), eluting with 100% MeOH. The enantiopurity was de-
termined using a Chiralpak IA analytic column (4.6 × 250 mm)
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solution of KOTf (10 mg/mL, 0.45 mL) and K2CO3 (1 mg/mL, 50
eluting with 100% MeOH at flow rate of 1 mL/min, with the first
1
2
3
4
5
6
7
8
peak (6.5 min) being (S)-22 and second peak (9.0 min) being (R)-
22 (ee. > 99.8%). The enantiopure (R)-22 was used in the next
step.
µL), together with MeCN (0.5 mL). After three cycles of azeo-
tropic trying as described above, a solution of the precursor 23 (2-
3 mg) in anhydrous N,N-dimethylacetamide (DMA, 0.4 mL) was
added to the reaction vial, followed by the solution of pyridine (1
M in DMA, 0.1 mL) and copper(II) triflate (0.2 M in DMA, 67
µL). The reaction mixture was agitated and then heated at 110° C
for 20 min. The formulated [18F]7 solution was obtained follow-
ing the purification and formulation protocol described above,
without the use of chiral HPLC.
Measurement of lipophilicity. The logD of [18F]7 was deter-
mined by the modified method from previously published proce-
dures53. Briefly, an aliquot of 0.74 MBq of the radioligand is
added to a 2.0 mL microtube containing 0.8 mL 1-octanol and 0.8
mL 1X Dulbecco’s phosphate buffered saline (1X D-PBS, pH
7.4). The mixture is subject to vigorous vortex for 20 s and centri-
fuged at 2,000 g for 2 min. A subsample of the octanol (0.2 mL)
and 1X D-PBS (0.7 mL) layers was evaluated with a gamma
counter. The major portion of the octanol layer (0.5 mL) was di-
luted with another 0.3 mL of octanol, mixed with a fresh portion
of 0.8 mL PBS, vortexed, centrifuged, and analyzed as described
above, till the logD value stabilized.
(R)-1-((3-Methylpyridin-4-yl)methyl)-4-(3-(trime-
thylstannyl)phenyl)pyrrolidin-2-one (23): To a solution of com-
pound (R)-22 (0.15 g, 0.43 mmol) in anhydrous toluene (1.5 mL)
was added LiCl (0.11 g, 2.60 mmol), Pd(PPh3)4 (0.1 g, 0.09
mmol) and hexamethylditin (0.14 mL, 0.65 mmol) under argon.
The reaction mixture was degassed with argon for 3 min and kept
stirring at 105 °C for 1 h, then diluted with EtOAc (8 mL), passed
through Celite and rinsed with EtOAc (4 mL  2). The filtrate was
concentrated in vacuo. The crude product was purified on a silica
gel column eluting with 0-20% EtOH/EtOAc, followed by the trit-
uration with CHCl3 and hexane to afford the product 23 as a white
solid (110 mg, 59%), M.P. 102-103 C. 1H NMR (CDCl3,400
MHz): δ 8.40-8.35 (m, 2H), 7.36 (dt, J = 7.2, 1.1 Hz, 1H), 7.31-
7.23 (m, 2H), 7.13 (dt, J = 7.8, 1.7 Hz, 1H), 7.03 (d, J = 5.0 Hz,
1H), 4.60 (d, J = 15.6 Hz, 1H), 4.40 (d, J = 15.6 Hz, 1H), 3.65-
3.53 (m, 2H), 3.28 (dd, J = 5.4, 2.4 Hz, 1H), 2.90 (dd, J = 17.0,
8.9 Hz, 1H), 2.67 (dd, J = 17.0, 8.4 Hz, 1H), 2.29 (s, 3H), 0.26 (s,
9H). 13C NMR (CDCl3, 100 MHz): δ 174.04, 151.14, 147.91,
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
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60
143.37, 143.07, 141.24, 134.73, 134.26, 131.59, 128.48, 126.36,
122.22, 54.28, 43.58, 38.52, 37.31, 15.94, -9.51 (3C). HRMS: cal-
culated for C20H26N2OSn ([M + H]+) 431.1140; found, 431.1133.
Measurement of Plasma Free Fraction (fp). The unbound frac-
tion of [18F]7 in the plasma (fp) was measured in triplicate using
the ultrafiltration method24. Briefly, [18F]7 (5.55 MBq, 0.1 mL)
was added to 3.0 mL of whole nonhuman primate blood. After 10
min incubation at ambient temperature, the blood sample was cen-
trifuged at 2,930 g for 5 min. The supernatant plasma (0.3 mL)
was loaded onto the reservoir of a micropartition device (Milli-
poreSigma Centrifree Ultrafiltration, Burlington, MA) in triplicate
and centrifuged at 1,228 g for 20 min. The fp value was calculated
as the ratio of radioactivity in the filtrate to that in the plasma.
Radiochemistry.
(R)-4-(3-(fluoro-18F)phenyl)-1-((3-methylpyridin-4-yl)me-
thyl)pyrrolidin-2-one ([18F]7): Preparation from precursor 18:
The cyclotron produced aqueous [18F]fluoride solution in 18O-H2O
was transferred to a lead-shielded hot cell, where the [18F]fluoride
anion was trapped on a Chromafix® 30-PS-HCO3 cartridge. The
[18F]fluoride was then eluted with a solution of tetraethyl ammo-
nium bicarbonate (TEAB, 2 mg) in CH3CN/water (7:3, v/v) into a
reaction vessel. The solvent was azeotropically dried at 110 °C for
15 min, and dried further with anhydrous MeCN (1.0 mL × 2) at
110 C. Then, a solution of about 2 mg of the radiolabeling pre-
cursor in 0.5 mL DMF was added. The reaction vessel was sealed,
and heated at 150 °C for 10 min. The crude reaction mixture was
diluted with HPLC mobile phase (1.5 mL) and purified by a re-
verse phase HPLC column [Luna C18(2), 10 µm, 100 Å, 10 × 250
mm]. The mobile phase was constituted with 25% CH3CN and
75% ammonium formate solution (0.1 M, with 0.5 % AcOH, pH
4.2) at a flow rate of 5 mL/min. The eluent was monitored by a
UV detector and a radioactivity detector. The fraction containing
the racemic [18F]7 was collected, diluted with deionized (DI) wa-
ter (50 mL), trapped on a C18 SepPak, washed with DI water (10
mL). The crude product was eluted off with ethanol (1 mL), di-
luted with DI water (1 mL), and loaded onto a ChiralCel OD-H
column (10 × 250 mm, 5 m) eluting with a mobile phase of 30%
CH3CN and 70% ammonium formate solution (0.1 M, with 0.5 %
AcOH, pH 4.2) at a flow rate of 5 mL/min for separation of the
two enantiomers. The fraction containing the (R)-enantiomer,
[18F]7, at 27.5 min, was collected (the other enantiomer eluted out
at 24.2 min), diluted with DI water (50 mL), loaded on a C18 Sep-
Pak. The SepPak was washed with DI water (10 mL), and dried.
The product was eluted off with EtOH (1 mL), diluted with USP
grade saline (3 mL), passed through a sterile membrane filter
(0.22 m), and collected in a sterile vial pre-charged with 7 mL of
USP saline and 20 μL of 8.4% NaHCO3 solution to afford a for-
mulated solution ready foradministration.
Nonhuman Primate PET Imaging. The study in this project was
performed under a protocol approved by the Yale University Insti-
tutional Animal Care and Use Committee (IACUC). Rhesus mon-
keys (Macaca mulatta) were fasted overnight prior to the imaging
study. The animals were sedated using intramuscular (i.m.) injec-
tion of ketamine and glycopyrrolate approximately 2 h before the
PET scan, and anesthesia was subsequently maintained with
isoflurane (1.5%–2.5%) for the duration of the imaging experi-
ments. A water-jacket heating pad was used to maintain body
temperature. The animal was attached to a physiological monitor,
and vital signs (pulse rate, blood pressure, respirations, EKG,
ETCO2 and body temperature) were continuously monitored. Af-
ter a transmission scan, the radioligand was injected i.v. as a 3 min
slow bolus by an infusion pump (PHD 22/2000; Harvard Appa-
ratus). Dynamic PET scans were performed for two hours with a
Focus-220 PET scanner (Siemens Preclinical Solutions) with a re-
constructed image resolution of approximately 1.5 mm. Arterial
blood samples were collected during the PET scans to measure
the radioactivity in plasma for generation of the metabolite-cor-
rected arterial plasma input function. PET images were recon-
structed with built-in corrections for attenuation, normalization,
scatter, randoms, and deadtime. PET images were registered to
the animal’s MR image, which was subsequently registered to a
brain atlas to define theregions-of-interest.
Plasma Radiometabolite Analysis. Plasma radiometabolite anal-
ysis was performed using the column switching method, follow-
ing published protocol54. Briefly, arterial blood samples were col-
lected at 5, 15, 30, 60, and 90 min p.i., treated with urea (8 M), fil-
tered, trapped on a capture column (4.6 × 19 mm), back flashed to
a Phenomenex Luna C18(2) column (5 휇m, 4.6 × 250 mm),
eluted with 37% acetonitrile/63% 0.1 M ammonium formate (pH
6.4) at flow rate of 2 mL/min, and analyzed by radio-HPLC to de-
termine the fraction of intact [18F]7 in theplasma.
Preparation from precursor 23: [18F]fluoride solution in 18O-H2O
was trapped on a Chromafix cartridge pre-washed with EtOH (5
mL), an aqueous solution of potassium triflate (KOTf) (90
mg/mL, 5 mL) and DI water (5 mL). The potassium [18F]fluoride
was eluted off the cartridge into a 2 mL V-vial with an aqueous
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Kinetic Modeling. A rhesus monkey brain atlas was used for gen-
1. Bajjalieh, S.; Peterson, K.; Shinghal, R.; Scheller, R., SV2, a
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2. Crowder, K. M.; Gunther, J. M.; Jones, T. A.; Hale, B. D.;
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Bajjalieh, S. M., Abnormal neurotransmission in mice lacking
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U.S.A. 1999, 96 (26), 15268-15273.
3. Mendoza-Torreblanca, J. G.; Vanoye-Carlo, A.; Phillips-
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Synaptic vesicle protein 2A: basic facts and role in synaptic
function. Eur. J. Neurosci. 2013, 38 (11),3529-3539.
4. Feng, G.; Xiao, F.; Lu, Y.; Huang, Z.; Yuan, J.; Xiao, Z.;
Xi, Z.; Wang, X., Down-regulation synaptic vesicle protein
2A in the anterior temporal neocortex of patients with
intractable epilepsy. J. Mol. Neurosci. 2009, 39 (3),354-359.
5. Loscher, W.; Gillard, M.; Sands, Z. A.; Kaminski, R. M.;
Klitgaard, H., Synaptic Vesicle Glycoprotein 2A Ligands in
the Treatment of Epilepsy and Beyond. CNS Drugs 2016, 30
(11), 1055-1077.
6. Tokudome, K.; Okumura, T.; Shimizu, S.; Mashimo, T.;
Takizawa, A.; Serikawa, T.; Terada, R.; Ishihara, S.;
Kunisawa, N.; Sasa, M.; Ohno, Y., Synaptic vesicle
glycoprotein 2A (SV2A) regulates kindling epileptogenesis
via GABAergic neurotransmission. Sci. Rep. 2016, 6,27420.
7. Sanchez, P. E.; Zhu, L.; Verret, L.; Vossel, K. A.; Orr, A.
G.; Cirrito, J. R.; Devidze, N.; Ho, K.; Yu, G. Q.; Palop, J.
J.; Mucke, L., Levetiracetam suppresses neuronal network
dysfunction and reverses synaptic and cognitive deficits in an
Alzheimer’s disease model. Proc. Natl. Acad. Sci. U.S.A.
2012, 109 (42), E2895-903.
8. Lynch, B. A.; Lambeng, N.; Nocka, K.; Kensel-Hammes, P.;
Bajjalieh, S. M.; Matagne, A.; Fuks, B., The synaptic vesicle
protein SV2A is the binding site for the antiepileptic drug
levetiracetam. Proc. Natl. Acad. Sci. U.S.A. 2004, 101 (20),
9861-9866.
9. Bakker, A.; Krauss, Gregory L.; Albert, Marilyn S.; Speck,
Caroline L.; Jones, Lauren R.; Stark, Craig E.; Yassa,
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M., Reduction of Hippocampal Hyperactivity Improves
Cognition in Amnestic Mild Cognitive Impairment. Neuron
2012, 74 (3), 467-474.
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Gallagher, M., Treatment Strategies Targeting Excess
Hippocampal Activity Benefit Aged Rats with Cognitive
Impairment. Neuropsychopharmacology 2010, 35 (4), 1016-
1025.
11. Stockburger, C. M., Davide; Baeumlisberger, Marion; Pallas,
Thea; Arrey, Tabiwang N.; Karas, Michael; Friedland,
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Protein in Aging and Alzheimer’s Disease: Studies with
Levetiracetam. J. Alzheimer's Dis. 2016, 50 (1),201-215.
12. Cai, Z.; Li, S.; Matuskey, D.; Nabulsi, N.; Huang, Y., PET
imaging of synaptic density: A new tool for investigation of
neuropsychiatric diseases. Neurosci. Lett. 2019, 691,44-50.
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A Potential Marker for PET Imaging of SV2A Expression.
ACS Med. Chem. Lett. 2014, 5 (10), 1152-1155.
1
2
3
4
5
6
7
8
eration of regions-of-interest (ROIs) and time-activity curves
(TACs). Volume of distribution (VT, mL·cm-3) values were de-
rived through 1-tissue (1T) compartment kinetic modeling with
the metabolite-corrected arterial plasma input function, which was
calculated as the product of the fitted total plasma curve and the
parent radiotracer fraction curve. Non-displaceable binding poten-
tial (BPND) values were calculated from VT values using centrum
semiovale (CS) as reference region, i.e., BPND = (VT ROI – VT
CS)/VT CS. Specific binding of the radioligands to SV2A was eval-
uated by one pre-blocking study with unlabeled UCB-J(150
µg/kg, i.v., given 10 min before tracer injection, as used in previ-
ous studies19) in one of the three rhesus monkeys. SV2A occu-
pancy by UCB-J was calculated using the Lassen plot55. For time
stability of VT estimates (1TCM) of [18F]7, 4, and 6, the VT esti-
mates of 17 brain regions (amygdala, brainstem, caudate, cerebel-
lum, cingulate cortex, frontal cortex, globus pallidus, hippocam-
pus, insula, nucleus accumbens, occipital, pons, putamen, centrum
semiovale, substantia nigra, temporal cortex, and thalamus) were
generated using scan durations ranging from 30 to 120 min in 15
min increments. The VT estimates were normalized by VT esti-
mates using 120 min scan duration. Within 10% deviation of the
averaged VT estimates from VT estimates using 120 min scan du-
ration was considered acceptable.
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ASSOCIATED CONTENT
Supporting Information
NMR spectra of compounds 7, 10, 11, 14, 15, 16, 18, 20, 21, 22,
and 23. HPLC profiles of 7 and 22, table of quality control results,
and table of radiosynthesis results of [18F]7. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: jason.cai@yale.edu; songye.li@yale.edu; Fax: +1-
(203)785-2994; Tel: +1-(203)785-7691.
Author Contributions
Z.C., S.L. contributed equally. Z.C., S.L., R.P., W.Z., X.W., E.B.,
D.H, S.J.F., D.L., J.R., and N.B.N. performed the experiments.
Z.C., S.L., R.E.C., and Y.H. designed the experiments. Z.C. wrote
the first draft of the manuscript. All authors contributed to and ap-
proved the final version of the manuscript.
Funding
This research was supported by NIH K01EB023312,
R01AG058773, and R01AG052560. Z.C. is an Archer Foundation
Research Scientist.
Notes
The authors declare no competing financial interest.
♯Current address: Department of Nuclear Medicine, West China
Hospital, Sichuan University, Chengdu, China.
Current address: Brown University, Providence, RI. USA.
Current address: AbbVie, Chicago, IL. USA.
ACKNOWLEDGMENT
14. Estrada, S.; Lubberink, M.; Thibblin, A.; Sprycha, M.;
Buchanan, T.; Mestdagh, N.; Kenda, B.; Mercier, J.;
Provins, L.; Gillard, M.; Tytgat, D.; Antoni, G., [11C]UCB-
A, a novel PET tracer for synaptic vesicle protein 2 A. Nucl.
Med. Biol. 2016, 43 (6), 325-332.
15. Warnock, G. I.; Aerts, J.; Bahri, M. A.; Bretin, F.; Lemaire,
C.; Giacomelli, F.; Mievis, F.; Mestdagh, N.; Buchanan, T.;
Valade, A.; Mercier, J.; Wood, M.; Gillard, M.; Seret,A.;
The authors thank Yale PET center staff for the production of 18
and the expert support of nonhuman primate PET imaging.
F
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