Synthesis and in vivo evaluation of [18F]UCB-J for PET imaging of synaptic vesicle glycoprotein 2A (SV2A)

Article abstract of DOI:10.1007/s00259-019-04357-w

Purpose: Synaptic abnormalities have been implicated in a variety of neuropsychiatric disorders, including epilepsy, Alzheimer’s disease, and schizophrenia. Hence, PET imaging of the synaptic vesicle glycoprotein 2A (SV2A) may be a valuable in vivo biomarker for neurologic and psychiatric diseases. We previously developed [¹¹C]UCB-J, a PET radiotracer with high affinity and selectivity toward SV2A; however, the short radioactive half-life (20 min for ¹¹C) places some limitations on its broader application. Herein, we report the first synthesis of the longer-lived ¹⁸F-labeled counterpart (half-life: 110 min), [¹⁸F]UCB-J, and its evaluation in nonhuman primates. Methods: [¹⁸F]UCB-J was synthesized from the iodonium precursors. PET imaging experiments with [¹⁸F]UCB-J were conducted in rhesus monkeys to assess the pharmacokinetic and in vivo binding properties. Arterial samples were taken for analysis of radioactive metabolites and generation of input functions. Regional time–activity curves were analyzed using the one-tissue compartment model to derive regional distribution volumes and binding potentials for comparison with [¹¹C]UCB-J. Results: [¹⁸F]UCB-J was prepared in high radiochemical and enantiomeric purity, but low radiochemical yield. Evaluation in nonhuman primates indicated that the radiotracer displayed pharmacokinetic and imaging characteristics similar to those of [¹¹C]UCB-J, with moderate metabolism rate, high brain uptake, fast and reversible binding kinetics, and high specific binding signals. Conclusion: We have accomplished the first synthesis of the novel SV2A radiotracer [¹⁸F]UCB-J. [¹⁸F]UCB-J is demonstrated to be an excellent imaging agent and may prove to be useful for imaging and quantification of SV2A expression, and synaptic density, in humans.

Full text of DOI:10.1007/s00259-019-04357-w

European Journal of Nuclear Medicine and Molecular Imaging
https://doi.org/10.1007/s00259-019-04357-w
ORIGINAL ARTICLE
Synthesis and in vivo evaluation of [18F]UCB-J for PET imaging
of synaptic vesicle glycoprotein 2A (SV2A)
Songye Li1 & Zhengxin Cai1 & Wenjie Zhang2 & Daniel Holden1 & Shu-fei Lin1 & Sjoerd J. Finnema1 & Anupama Shirali1
Jim Ropchan1 & Stephane Carre3 & Joel Mercier3 & Richard E. Carson1 & Nabeel Nabulsi1 & Yiyun Huang1
&
Received: 25 January 2019 /Accepted: 2 May 2019
#
Springer-Verlag GmbH Germany, part of Springer Nature 2019
Abstract
Purpose Synaptic abnormalities have been implicated in a variety of neuropsychiatric disorders, including epilepsy, Alzheimer’s
disease, and schizophrenia. Hence, PET imaging of the synaptic vesicle glycoprotein 2A (SV2A) may be a valuable in vivo
biomarker for neurologic and psychiatric diseases. We previously developed [11C]UCB-J, a PET radiotracer with high affinity
and selectivity toward SV2A; however, the short radioactive half-life (20 min for 11C) places some limitations on its broader
application. Herein, we report the first synthesis of the longer-lived 18F-labeled counterpart (half-life: 110 min), [18F]UCB-J, and
its evaluation in nonhuman primates.
Methods [18F]UCB-J was synthesized from the iodonium precursors. PET imaging experiments with [18F]UCB-J were con-
ducted in rhesus monkeys to assess the pharmacokinetic and in vivo binding properties. Arterial samples were taken for analysis
of radioactive metabolites and generation of input functions. Regional time–activity curves were analyzed using the one-tissue
compartment model to derive regional distribution volumes and binding potentials for comparison with [11C]UCB-J.
Results [18F]UCB-J was prepared in high radiochemical and enantiomeric purity, but low radiochemical yield. Evaluation in
nonhuman primates indicated that the radiotracer displayed pharmacokinetic and imaging characteristics similar to those of
[11C]UCB-J, with moderate metabolism rate, high brain uptake, fast and reversible binding kinetics, and high specific binding
signals.
Conclusion We have accomplished the first synthesis of the novel SV2A radiotracer [18F]UCB-J. [18F]UCB-J is demonstrated to
be an excellent imaging agent and may prove to be useful for imaging and quantification of SV2A expression, and synaptic
density, in humans.
.
.
.
.
.
Keywords SV2A PET Radiotracer Fluorination Nonhuman primates Alzheimer’s disease
Introduction
The synaptic vesicle glycoprotein 2A (SV2A) is one of the
Songye Li and Zhengxin Cai contributed equally to this work.
three isoforms of synaptic vesicle glycoprotein 2 (SV2), and is
expressed ubiquitously in all neurons across the brain [1, 2].
As a vesicle protein, SV2A is essential for neural function and
can be used as a biomarker of synaptic density [3, 4]. Further,
changes in SV2A have been implicated in a variety of neuro-
logic and psychiatric disorders, including epilepsy [1, 5, 6],
Alzheimer’s disease (AD) [7, 8], Parkinson’s disease (PD) [9],
and schizophrenia [10]. For example, regional synaptic loss
has been shown to be a robust indicator of neurodegeneration
[11]. As a result, its measurement can potentially serve as a
diagnostic or prognostic tool for AD and other neurodegener-
ative diseases, and thus may allow the early detection of these
diseases at the preclinical or prodromal stage [12]. Positron
This article is part of the Topical Collection on Translational Research
* Songye Li
songye.li@yale.edu
* Zhengxin Cai
jason.cai@yale.edu
1
PET Center, Department of Radiology and Biomedical Imaging, Yale
University, New Haven, CT 06520, USA
2
Department of Nuclear Medicine, West China Hospital of Sichuan
University, Chengdu 610041, Sichuan, China
3
UCB Biopharma, Braine-l’Alleud, Belgium

Eur J Nucl Med Mol Imaging
emission tomography (PET) is a noninvasive and quantitative
imaging technique used to investigate physiological and bio-
chemical changes in living subjects. Hence, the development
and application of SV2A PET imaging agents can facilitate
the investigation and understanding of the structural disrup-
tion and alterations of synapses in diseases, and in the evalu-
ation of drug candidates targeted at synaptic repair and
restoration.
spectrometer (A400a, A400c, or A600a). Chemical shifts are
reported in parts per million (ppm), with the solvent resonance
as the internal standard (CDCl3: 7.26 ppm; DMSO-d6:
1
2.49 ppm in H NMR, and CDCl3: 77.0 ppm; DMSO-d6:
39.7 ppm in 13C NMR). Multiplicities are indicated as s (sin-
glet), d (doublet), t (triplet), q (quartet), quint (quintet), or m
(multiplet), with the coupling constant (J) given in hertz (Hz).
High-resolution mass spectrometry (HRMS) was performed
with a Thermo Scientific LTQ-Orbitrap XL Elite system.
Within the past few years, we have prepared three radio-
tracers, [11C]UCB-A (1), [18F]UCB-H (2), and [11C]UCB-J
(3), and evaluated their ability to image and quantify SV2A
in vivo (Fig. 1). Among these three ligands, [11C]UCB-A
suffers from slow kinetics [13] and [18F]UCB-H provides lim-
ited specific binding signals in monkeys and humans [14–17].
Replacement of the fluorine substituent on the pyridine ring in
UCB-H with a methyl group leads to the discovery of UCB-J
[18], with increased in vitro binding affinity and selectivity for
SV2A. In vivo, the radiotracer [11C]UCB-J demonstrated sev-
eral attractive pharmacokinetic and imaging properties, in-
cluding fast tissue kinetics, high brain uptake, and high spe-
cific binding signals in both nonhuman primates and humans,
and has been validated as an imaging biomarker for synaptic
density [19–21]. The application of SV2A PET has opened a
new avenue for investigating synaptic alterations in a large
variety of neurodegenerative and neuropsychiatric disorders
[22, 23], and for monitoring the clinical efficacy of disease-
modifying therapies [24, 25].
3,5-Difluoro-4-iodobenzonitrile (5) A solution of lithium
diisopropylamide (55 mL, 2 M in THF) was added dropwise
to a solution of 3,5-difluorobenzonitrile (4, 13.7 g, 98.5 mmol)
in anhydrous tetrahydrofuran (THF, 130 mL) under argon at
−78 °C, followed by a solution of iodine (26.3 g, 103.6 mmol)
in anhydrous THF (75 mL). The reaction was slowly warmed
to room temperature, stirred for 1 h, and then quenched with
10% sodium thiosulfate solution (100 mL). The mixture was
extracted with ethyl acetate (EtOAc)/hexanes (1:1, v/v,
50 mL × 3). The combined organic phase was dried over
Na2SO4 and concentrated in vacuo to afford compound 5 as
a brownish solid (10.7 g, 41%), which was used in the next
1
step of synthesis without further purification. H NMR
(CDCl3, 400 MHz): δ 7.16 (d, J = 5.10 Hz, 2H).
3,5-Difluoro-4-iodobenzaldehyde (6) A solution of
diisobutylaluminium anhydride (40.5 mL, 1 M in CH2Cl2)
was added dropwise to a solution of compound 5 (10.7 g,
40.4 mmol) in anhydrous CH2Cl2 (85 mL) under argon at
0 °C. The reaction was slowly warmed to room temperature,
stirred for 1 h, and then quenched with 6 N HCl solution
(85 mL). The mixture was extracted with CH2Cl2 (50 mL ×
3). The combined organic phase was dried over Na2SO4 and
concentrated in vacuo to afford compound 6 as a white solid
(9.7 g, 91%), which was used in the next step of synthesis
Despite the excellent imaging properties of [11C]UCB-J,
the short radioactive half-life (20.4 min) places some restric-
tions on its broader application, especially in multicenter clin-
ical trials, as its production requires an on-site cyclotron, and
distribution to other imaging sites is limited. Thus, we set out
to synthesize the longer-lived [18F]-labeled (half-life:
110 min) counterpart of [11C]UCB-J and carried out an
in vivo characterization of [18F]UCB-J in nonhuman primates.
1
without further purification. H NMR (CDCl3, 400 MHz): δ
9.91 (s, 1H), 7.37 (d, J = 5.51 Hz, 2H).
Materials and methods
Ethyl-3-(3,5-difluoro-4-iodophenyl)acrylate (7) A solution of
carbethoxymethylene triphenylphosphorane (11.0 g,
31.57 mmol) in anhydrous CH2Cl2 (50 mL) was added
dropwise to a solution of compound 6 (7.70 g, 28.73 mmol)
in anhydrous THF (30 mL) under argon at 0 °C. The reaction
mixture was stirred for 1 h at 5 °C, and then concentrated in
vacuo. A mixture of hexanes and diethyl ether (4:1, v/v,
150 mL) was added, and the suspension was stirred for
Chemistry
All reagents and solvents were purchased from commercial
sources (Sigma-Aldrich, VWR, and Fisher Scientific) and
used without further purification. Proton, carbon, and fluorine
nuclear magnetic resonance (1H, 13C, and 19F NMR) spectra
were recorded on an Agilent 400 MHz or 600 MHz
18F
F
F
F
Fig. 1 PET radiotracers for SV2A
O
O
O
O
N
N
N
N
F
F
N
N
N
N
N
F
F
F
F
11 CH3
18F
11 CH3
CH3
[
11 C]UCB-A (1)
[
18F]UCB-H (2)
[
11C]UCB-J (3)
[
18F]SDM-8

Eur J Nucl Med Mol Imaging
10 min at room temperature. Filtration of the mixture through
a silica gel plug and evaporation of the solvents afforded com-
pound 7 as a white solid (9.7 g, quantitative), which was used
light brown solid (128 mg, 65%), M.P. 158–160 °C. 1H NMR
(CDCl3, 400 MHz): δ 8.41 (s, 2H), 7.02 (d, J = 4.81 Hz, 1H),
6.72 (d, J = 7.11 Hz, 2H), 4.61 (d, J = 15.42 Hz, 1H), 4.39 (d,
J = 15.42 Hz, 1H), 3.70–3.50 (m, 2H), 3.25–3.17 (m, 1H),
2.91 (dd, J = 16.87, 8.61 Hz, 1H), 2.56 (dd, J = 16.86,
7.95 Hz, 1H), 2.30 (s, 3H).
1
in the next step of synthesis without further purification. H
NMR (CDCl3, 400 MHz): δ 7.54 (d, J = 15.94 Hz, 1H), 7.03
(d, J = 6.89 Hz, 2H), 6.45 (d, J = 15.96 Hz, 1H), 4.27 (q, J =
7.09 Hz, 2H), 1.33 (t, J = 7.12 Hz, 3H).
(R,S)-8-((2,6-Difluoro-4-(1-((3-methylpyridin-4-yl)methyl)-5-
oxopyrrolidin-3-yl)phenyl)-λ3-iodanylidene)-6,10-
dioxaspiro[4.5]decane-7,9-dione ((R,S)-11) Trifluoroacetic ac-
id (TFA, 0.28 mL, 3.63 mmol), followed by Oxone (75 mg,
0.24 mmol), was added to a solution of compound 10 (52 mg,
0.12 mmol) in CHCl3 (1.0 mL). The reaction mixture was
stirred at room temperature for 2 h until it turned to a white
suspension. The volatile contents were removed in vacuo. The
dried residue was suspended in EtOH (1.0 mL), and 6,10-
dioxaspiro[4.5]decane-7,9-dione (SPI-5, 24 mg, 0.14 mmol)
was added, followed by 10% Na2CO3 solution, until the pH
reached 10. The reaction mixture was stirred at room temper-
ature for 2 h, diluted with water (1.0 mL), and extracted with
CH2Cl2 (1.0 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 10–40%
EtOH/EtOAc. Recrystallization of the product in EtOAc/
hexanes afforded compound (R,S)-11 as a white solid
(22 mg, 31%), M.P. 122–125 °C (decomposition). 1H NMR
(DMSO-d6, 400 MHz): δ 8.37 (s, 2H), 7.27 (d, J = 8.29 Hz,
2H), 7.17 (s, 1H), 4.53 (d, J = 15.69 Hz, 1H), 4.33 (d, J =
15.90 Hz, 1H), 3.78–3.70 (m, 2H), 3.63–3.57 (m, 1H),
2.73 (dd, J = 16.41, 8.90 Hz, 1H), 2.60 (dd, J = 18.45,
11.34 Hz, 1H overlap with DMSO residue peak), 2.20 (s,
3H), 1.89–1.75 (m, 4H), 1.63–1.55 (m, 4H). 13C NMR
(DMSO-d6, 150 MHz): δ 173.2, 173.0 (2C), 163.3 (2C),
162.0, 160.4, 158.3, 158.1, 150.8, 147.9, 144.0, 112.3
(2C), 111.5, 59.7, 53.1, 43.2, 40.5, 37.6, 37.1 (2C), 23.2
(2C), 15.8. 19F NMR (DMSO-d6, 376 MHz): δ − 94.00.
HRMS: calculated for C25H23F2IN2O5 ([M + H]+),
597.0693; found, 597.0743.
Ethyl 3-(3,5-difluoro-4-iodophenyl)-4-nitrobutanoate (8)
Compound 7 (9.76 g, 28.87 mmol) was dissolved in nitro-
methane (5.0 mL, 93.38 mmol) under argon and cooled at
−20 °C. 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU, 4.8 mL,
32.10 mmol) was added, and the reaction mixture was stirred
continuously for 2 h at −20 °C. Water (20 mL) was added,
followed by 12 N HCl, until the pH of the mixture reached 1.
The mixture was extracted with EtOAc (50 mL × 3). The com-
bined organic phase was dried over Na2SO4 and concentrated
in vacuo. The crude product was purified on a silica gel col-
umn eluting with 0–15% EtOAc/hexanes to afford compound
1
8 as a white solid (8.3 g, 93%). M.P. 48–50 °C. H NMR
(CDCl3, 400 MHz): δ 6.80 (d, J = 6.63 Hz, 2H), 4.72 (dd,
J = 12.98, 8.40 Hz, 1H), 4.61 (dd, J = 12.98, 6.38 Hz, 1H),
4.12 (q, J = 7.14 Hz, 2H), 3.97 (quint., J = 7.34 Hz, 1H), 2.79–
2.65 (m, 2H), 1.21 (t, J = 7.14 Hz, 3H).
4-(3,5-Difluoro-4-iodophenyl)pyrrolidin-2-one (9) NH4Cl
(20.3 g, 386.75 mmol) was added to a suspension of com-
pound 8 (5.0 g, 12.50 mmol) and iron powder (7.0 g,
125.25 mmol) in a mixture of EtOH and water (120 mL,
2:1, v/v). After stirring for 16 h at room temperature, the re-
action mixture was adjusted to pH 14 with saturated NaOH
solution and extracted with EtOAc (50 mL × 3). The com-
bined organic phase was dried over Na2SO4 and concentrated
in vacuo to afford compound 9 as a white solid (1.88 g, 46%),
which was used in the next step of synthesis without further
1
purification. H NMR (CDCl3, 400 MHz): δ 6.80 (d, J =
7.60 Hz, 2H), 5.63 (s, 1H), 3.83–3.75 (m, 1H), 3.67 (quint.,
J = 7.62 Hz, 1H), 3.42–3.30 (m, 1H), 2.74 (dd, J = 17.02,
8.72 Hz, 1H), 2.41 (dd, J = 16.63, 8.10 Hz, 1H).
Ethyl 3-(3-bromo-4,5-difluorophenyl)acrylate (13) Compound
13 was prepared according to procedures similar to those de-
scribed above for compound 7. Yield: quantitative. 1H NMR
(CDCl3, 400 MHz): δ 7.89 (d, J = 15.90 Hz, 1H), 7.42 (m,
2H), 6.29 (d, J = 15.89 Hz, 1H), 4.27 (q, J = 7.12 Hz, 2H),
1.33 (t, J = 7.13 Hz, 3H).
4-(3,5-Difluoro-4-iodophenyl)-1-((3-methylpyridin-4-
yl)methyl)pyrrolidin-2-one (10) Sodium hydride (74 mg,
1.84 mmol) was added to a solution of compound 9
(150 mg, 0.46 mmol) in anhydrous THF (3.0 mL) under argon
cooled to 0 °C. Tetrabutylammonium iodide (TBAI, 9 mg,
0.02 mmol) and 4-(chloromethyl)-3-methylpyridine hydro-
chloride (98 mg, 0.55 mmol) were added after 30 min. The
reaction mixture was stirred continuously for 16 h at room
temperature, then quenched with saturated NaHCO3 solution
(6 mL) and extracted with EtOAc (3 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 10 as a
Ethyl 3-(3-bromo-4,5-difluorophenyl)-4-nitrobutanoate (14)
Compound 14 was prepared according to procedures similar
1
to those described above for compound 8. Yield: 80%. H
NMR (CDCl3, 400 MHz): δ 7.44 (t, J = 8.11 Hz, 1H), 7.10–
7.04 (m, 1H), 4.72 (d, J = 6.76 Hz, 2H), 4.37 (quint., J =
6.75 Hz, 1H), 4.10 (q, J = 6.74 Hz, 2H), 2.86–2.74 (m, 2H),
1.20 (t, J = 7.24 Hz, 3H).

Eur J Nucl Med Mol Imaging
4-(3-Bromo-4,5-difluorophenyl)pyrrolidin-2-one (15)
Compound 15 was prepared according to procedures similar
to those described above for compound 9. Yield: 64%. H
NMR (CDCl3, 400 MHz): δ 7.42 (t, J = 8.51 Hz, 1H), 7.19–
7.13 (m, 1H), 5.66 (s, 1H), 4.06 (quint., J = 7.25 Hz, 1H),
3.86–3.80 (m, 1H), 3.33–3.28 (m, 1H), 2.78 (dd, J = 17.07,
9.03 Hz, 1H), 2.36 (dd, J = 17.09, 6.68 Hz, 1H).
concentrated in vacuo. The residue was purified on a basic
alumina column eluting with 0–10% MeOH/CH2Cl2 to pro-
vide the mono-triflate salt. Recrystallization of the product in
CH2Cl2/Et2O afforded compound (R,S)-18 as a light brown
1
1
solid (50 mg, 27%), M.P. 150–152 °C (decomposition). H
NMR (DMSO-d6, 600 MHz): δ 8.68 (dd, J = 9.52, 7.80 Hz,
1H), 8.8.41–8.34 (m, 2H), 8.16 (d, J = 9.05 Hz, 2H), 7.92 (dd,
J = 12.05, 7.90 Hz, 1H), 7.21(d, J = 4.99 Hz, 1H), 7.00(d, J =
9.09 Hz, 2H), 4.55 (d, J = 16.00 Hz, 1H), 4.30 (d, J =
15.97 Hz, 1H), 4.10 (quint, J = 8.55 Hz, 1H), 3.74 (s, 3H),
3.41 (dd, J = 9.71, 8.34 Hz, 1H), 3.18 (dd, J = 9.66, 7.68 Hz,
1H), 2.72 (dd, J = 16.44, 9.00 Hz, 1H), 2.52 (dd, J = 16.43,
8.93 Hz, 1H), 2.24 (s, 3H). 13C NMR (DMSO-d6, 150 MHz):
δ 172.5, 162.5, 150.9, 147.9, 143.9, 143.1, 137.4, 131.9 (2C),
126.6, 126.4, 122.3, 122.2, 118.3, 118.2, 118.0 (2C), 115.9,
106.5, 55.2, 54.0, 43.3, 40.5, 39.2, 15.8; 19F NMR (DMSO-
d6, 564 MHz): δ 77.8 (3F), −129.8 (1F), −135.1 (1F). HRMS:
calculated for C25H22F5IN2O5S ([M + H]+), 685.0287; found,
685.0279.
4-(3-Bromo-4,5-difluorophenyl)-1-((3-methylpyridin-4-
yl)methyl)pyrrolidin-2-one (16) Compound 16 was prepared
according to procedures similar to those described above for
1
compound 10. Yield: 33%. M.P. 120–121 °C. H NMR
(CDCl3, 400 MHz): δ 8.41–8.38 (m, 2H), 7.40 (t, J =
8.48 Hz, 1H), 7.02–6.97 (m, 2H), 4.58–4.46 (m, 2H), 3.94
(quint., J = 7.40 Hz, 1H), 3.70–3.66 (m, 1H), 3.15–3.12 (m,
1H), 2.93 (dd, J = 17.18, 9.13 Hz, 1H), 2.54 (dd, J = 17.16,
6.57 Hz, 1H), 2.29 (s, 3H).
4-(3,4-Difluoro-5-iodophenyl)-1-((3-methylpyridin-4-
yl)methyl)pyrrolidin-2-one (17) Copper(I) iodide (27 mg,
0.14 mmol), compound 16 (1.07 g, 2.82 mmol), and sodium
iodide (1.69 g, 11.27 mmol) were mixed, and the reaction
vessel was evacuated and backfilled with argon three times.
A solution of racemic trans-N,N′-dimethyl-1,2-
cyclohexanediamine (45 μL, 0.29 mmol) in anhydrous 1,4-
dioxane (10.0 mL) was added. The reaction mixture was
Radiochemistry
H218O was obtained from Huayi Isotopes (Toronto, Canada).
Anion exchange Chromafix cartridges (PS-HCO3) were pur-
chased from Macherey-Nagel (Düren, Germany). Solid-phase
extraction (SPE) C18 Sep-Pak cartridges were purchased from
Waters Corporation (Milford, MA, USA).
1
stirred at 120 °C, and conversion was monitored by H
NMR. One addition of each chemical reagent in 20% of the
original amount was added every 24 h. After 72 h of heating,
the resulting suspension was cooled to room temperature, di-
luted with 30% ammonia solution (10 mL), poured into water
(30 mL), and extracted with CH2Cl2 (15 mL × 3). The com-
bined organic phase was dried over Na2SO4 and concentrated
in vacuo. The crude product was purified on a silica gel col-
umn eluting with 0–15% EtOH/EtOAc to afford compound
17 as a beige solid (0.47 g, 39%), M.P. 115–117 °C. 1H NMR
(CDCl3, 400 MHz): δ 8.43–8.40 (m, 2H), 7.64 (t, J = 8.18 Hz,
1H), 7.01 (d, J = 4.89 Hz, 1H), 6.97 (dd, J = 11.40, 7.88 Hz,
1H), 4.58–4.46 (m, 2H), 3.84–3.76 (m, 1H), 3.71–3.65 (m,
1H), 3.13–3.04 (m, 1H), 2.94 (dd, J = 17.26, 9.02 Hz, 1H),
2.49 (dd, J = 17.21, 6.29 Hz, 1H), 2.30 (s, 3H).
The high-performance liquid chromatography (HPLC) sys-
tem used for purification of crude product included a
Shimadzu LC-20A pump, a Knauer K-200 UV detector, and
a Bioscan gamma-flow detector, with a ChiralCel OD-H semi-
preparative column (10 × 250 mm, 5 μm, Chiral
Technologies, West Chester, PA, USA) eluting with a mobile
phase of 35% CH3CN and 65% 20 mM ammonium bicarbon-
ate solution (pH 8.6) at a flow rate of 5 mL/min. The HPLC
system used for quality control tests comprised a Shimadzu
LC-20A pump, a Shimadzu SPD-M20A PDA or SPD-20A
UV detector, and a Bioscan gamma-flow detector, with a
Genesis C18 column (4.6 × 250 mm, 5 μm) eluting with a
mobile phase of 38% CH3CN and 62% 0.1 M ammonium
formate with 0.5% acetic acid (pH 4.2) at a flow rate of
2 mL/min. Identity was confirmed by co-injection of the prod-
uct with UCB-J (3) and detection of a single UV peak on the
chromatogram. Enantiomeric purity of the radiolabeled com-
pounds was determined on a ChiralCel OD-H analytical col-
umn (4 × 150 mm, 5 μm) eluting with a mobile phase of 35%
CH3CN and 65% 0.1 M ammonium formate with 0.5% acetic
acid (pH 4.2) at a flow rate of 1 mL/min.
(R,S)-(2,3-Difluoro-5-(1-((3-methylpyridin-4-yl)methyl)-5-
oxopyrrolidin-3-yl)phenyl)(4-methoxyphenyl)iodonium
trifluoromethanesulfonate ((R,S)-18) A solution of compound
17 (150 mg, 0.35 mmol) and anisole (50 μL, 0.46 mmol) in
C H 3 C N ( 1 m L ) w a s a d d e d t o a s o l u t i o n o f
trifluoromethanesulfonic acid (125 μL, 1.41 mmol) and
Oxone (213 mg, 0.69 mmol) in CH2Cl2 (1 mL) over 2 h,
followed by another portion of Oxone (107 mg, 0.35 mmol).
The reaction mixture was stirred at room temperature for 16 h,
diluted with H2O (2 mL), and extracted with CH2Cl2 (2 mL ×
3). The combined organic phase was dried over Na2SO4 and
18F-Fluoride was produced via the 18O(p, n)18F nuclear
reaction in a 16.5-MeV PETtrace cyclotron (GE Healthcare,
Waukesha, WI, USA). The aqueous 18F-fluoride solution in
18O-water was trapped with a resin cartridge (PS-HCO3,
Macherey-Nagel, Düren, Germany) in a lead-shielded hot cell.

Eur J Nucl Med Mol Imaging
The 18F-fluoride was then eluted with a solution of
tetraethylammonium bicarbonate (TEAB, 2 mg) in CH3CN/
H2O (1 mL, 7:3, v/v) into a 5 mL V-Vial (clear borosilicate
glass vial sealed with phenolic screw cap; Wheaton Industries/
DWK Life Sciences, Millville, NJ, USA) or a NanoTek appa-
ratus (Advion, Louisville, TN). The solvents were
azeotropically evaporated at 100 °C, with the addition of
two more portions of anhydrous CH3CN (1 mL). Dried 18F-
fluoride was re-dissolved in anhydrous DMF for
radiosynthesis. The starting activity was 13.0–22.2 GBq. All
the radioactivity and radiochemical yields were reported as
measured and not corrected for decay.
(0.5 mL) through a microreactor (2 m coiled glass silica tube
with 100 μm internal diameter; 15.7 μL) at a set flow rate and
temperature. Optimization was performed by changing the
precursor concentration, reactor temperature, and flow rates
to obtain the best incorporation of 18F-fluoride.
Incorporation yield was calculated based on radio-HPLC anal-
ysis using a Luna C18 column (4.6 × 250 mm, 5 μm;
Phenomenex, Torrance, CA). The best reaction parameters
for the NanoTek reactor were found to be a flow rate of
50 μL/min for both the solutions of compound (R,S)-18 and
18F-fluoride at a reaction temperature of 200 °C. Purification
and formulation procedures were the same as those described
above for 4-[18F]UCB-J.
(R)- and (S)-4-(3,5-Difluoro-4-(fluoro-18F)phenyl)-1-((3-
methylpyridin-4-yl)methyl)pyrrolidin-2-one (4-[18F]UCB-J
and (S)-4-[18F]3) A solution of compound (R,S)-11 (2.5 mg)
in DMF (0.5 mL) was added to the solution of dried 18F-
fluoride-TEAB in DMF (0.5 mL) and the reaction mixture
was heated at 170 °C for 10 min. The reaction mixture was
diluted with deionized (DI) water (10 mL) and passed through
a Waters C18 Sep-Pak cartridge. The cartridge was washed
with DI water (10 mL). The crude products were eluted off the
cartridge with EtOH (1 mL), diluted with DI water (1 mL),
and loaded onto the semi-preparative ChiralCel OD-H column
(typical residual activity in the reaction vessel was about 10%
of the total after loading of the contents to the HPLC column).
The radioactive peak eluting at 19.4 min was the (S)-enantio-
mer ((S)-4-[18F]3), and the following radioactive peak, eluting
at 21.8 min, was the (R)-enantiomer (4-[18F]UCB-J). The ab-
solute configuration of UCB-J was previously determined by
UCB Pharma [18], and the configuration of the radiotracer
[18F]UCB-J and its optically opposite enantiomer was deter-
mined by co-injection in HPLC with enantiomeric pure UCB-
J reference standard and its other enantiomer provided by
UCB.
The HPLC fraction with the desired enantiomer was col-
lected, diluted with DI water (50 mL), and passed through a
Waters C18 Sep-Pak cartridge. The cartridge was washed with
DI water (10 mL) and dried. The radioactive product, trapped
on the cartridge, was recovered by elution with US
Pharmacopeial Convention (USP) ethanol (1 mL), followed
by USP saline (3 mL). The resulting mixture was then passed
through a sterile membrane filter (0.2 μm) for terminal steril-
ization, and collected in a sterile vial pre-charged with 7 mL of
USP saline to afford a formulated solution ready for adminis-
tration. The radiotracers were stable for at least 1 h after the
end of synthesis.
PET imaging experiments in rhesus monkeys
PET scan procedures PET imaging experiments were per-
formed in rhesus monkeys (Macaca mulatta) according to a
protocol approved by the Yale University Institutional Animal
Care and Use Committee. Three monkeys (2 male, 1 female)
were used in a total of five 120-min scans, including two
baseline scans with 5-[18F]UCB-J (female monkey and male
monkey A), one baseline scan with 4-[18F]UCB-J (male mon-
key B), one baseline scan with the inactive enantiomer (S)-
5-[18F]3 (female monkey), and one self-blocking scan with
5-[18F]UCB-J using UCB-J (3, 0.15 mg/kg, male monkey
A) administered as a 10-min infusion starting 10 min before
radiotracer injection to verify the binding specificity. One ad-
ditional 180-min scan with 5-[18F]UCB-J (male monkey A)
was carried out to evaluate the displacement effect, in which
SV2A specific ligand levetiracetam (30 mg/kg) was given as a
5-min infusion at 90 min post-radiotracer injection. In addi-
tion, four baseline scans with [11C]UCB-J were conducted in
these three monkeys for comparison purpose.
In preparation for each scan, the monkey was subjected to
fasting overnight, and immobilized with ketamine (10 mg/kg,
intramuscularly) at least 2 h prior to the PET scan. A venous
line was inserted for administration of radiotracer and
blocking/displacement drug in one limb. A catheter was
placed in the femoral artery in the other limb for blood sam-
pling. Endotracheal intubation was performed to enable ad-
ministration of isoflurane (1.5–2.5% in oxygen). A water-
jacket heating pad was used to maintain body temperature.
The animal was attached to a physiological monitor, and vital
signs (heart rate, blood pressure, respiration, SPO2, EKG,
ETCO2, and body temperature) were monitored continuously.
Dynamic PET scans were performed on a Focus 220 scan-
ner (Siemens Medical Solutions, Knoxville, TN, USA) with a
reconstructed image resolution of approximately 1.5 mm.
Before radiotracer injection, a 9-min transmission scan was
obtained for attenuation correction. The radiotracer was ad-
ministered by an infusion pump over 3 min. Emission data
were collected in list mode for 120 min and reformatted into
(R)- and (S)-4-(3,4-Difluoro-5-(fluoro-18F)phenyl)-1-((3-
methylpyridin-4-yl)methyl)pyrrolidin-2-one (5-[18F]UCB-J
and (S)-5-[18F]3) The solution of dried 18F-fluoride-TEAB in
DMF (0.5 mL) was loaded into a loop on the NanoTek device
and co-infused with compound (R,S)-18 (2.5 mg) in DMF

Eur J Nucl Med Mol Imaging
33 successive frames of increasing duration (6 × 30 s, 3 ×
1 min, 2 × 2 min, and 22 × 5 min). For the 180-min scan,
additional frames (12 × 5 min) were acquired.
anatomical localization of regions of interest (ROIs). The
MR image was registered to an atlas and to the PET images,
as previously described [28].
PET data were corrected for attenuation, scanner normali-
zation, scatter, and random events, and dynamic images were
reconstructed using a Fourier rebinning and filtered back-
projection algorithm. Using the MR images, the following
ROIs were defined: amygdala, brainstem, caudate nucleus,
centrum semiovale, cerebellum, cingulate cortex, frontal cor-
tex, globus pallidus, hippocampus, insula, nucleus accum-
bens, occipital cortex, pons, putamen, temporal cortex, and
thalamus. For each PET scan, radiotracer concentrations over
time, i.e., time–activity curves (TACs), were generated for the
ROIs.
Regional TACs were fitted and analyzed with the one-
tissue compartment (1TC) model [29], which has been shown
to be a reliable model with low standard error (SE), as de-
scribed previously [19]. Regional distribution volume (VT,
mL/cm−3) was calculated from kinetic analysis of regional
TACs using the metabolite-corrected arterial plasma concen-
tration as the input function [30].
Plasma metabolite analysis and input function measurement
Arterial blood samples were collected at preselected time
points and assayed for radioactivity in whole blood and plas-
ma with cross-calibrated well-type gamma counters (Wizard
1480/2480, Perkin Elmer, Waltham, MA, USA). Seven sam-
ples, drawn at 0, 5, 15, 30, 60, 90 and 120 min, were processed
and analyzed to measure radioactive metabolites by HPLC
analysis using the column-switching method [26]. Whole
blood samples in EDTA tubes were centrifuged at 2930×g at
4 °C for 5 min to separate the plasma. Supernatant plasma was
collected, and activity in 0.2 mL aliquots was counted on a
gamma counter. Plasma samples were then mixed with urea
(8 M) to denature the plasma proteins, filtered through a
1.0 μm Whatman 13 mm CD/X syringe filter, and loaded onto
an automatic column-switching HPLC system connecting a
capture column (4.6 × 19 mm) self-packed with
Phenomenex Strata-X polymeric SPE sorbent and eluting
with 1% CH3CN/water at 2 mL/min for 4 min. The trapped
activity in the capture column was then back-flushed and elut-
ed through a Gemini-NX column (4.6 × 250 mm, 5 μm) with
40% CH3CN in 0.1 M ammonium formate (pH = 6.4) solution
at a flow rate of 1.55 mL/min. The eluent fractions were col-
lected with an automated fraction collector (Spectrum
Chromatography CF-1). Radioactivity in the whole blood,
plasma, filtered plasma–urea mix, filter, and HPLC eluent
fractions was all counted with the automatic gamma counters.
The sample recovery rate, extraction efficiency, and HPLC
fraction recovery were monitored. The un-metabolized parent
fraction was determined as the ratio of the sum of radioactivity
in fractions containing the parent compound to the total
amount of radioactivity collected, and fitted with inverted
gamma function and corrected for filtration efficiency. The
arterial plasma input function was then calculated as the prod-
uct of the total counts in the plasma and the interpolated parent
fraction at each time point, and was used in the analysis of
brain time–activity curves and the calculation of binding pa-
rameters with kinetic models.
Non-displaceable binding potential (BPND) was calculated
from regional VT values using centrum semiovale as the ref-
erence region, i.e., BPND = (VT, ROI – VT, semiovale)/VT, semiovale
.
SV2A occupancy by UCB-J was obtained from the occu-
pancy plot using the regional VT from the baseline scan and VT
difference between baseline and blocking scans [31].
Results
Chemistry
The presence of three fluorine atoms on the benzene ring of
UCB-J (3) allows the placement of an 18F-label on the mole-
cule without changing the chemical structure. Our initial ef-
forts to effect radiofluorination through traditional nucleophil-
ic substitution of iodo or chloro leaving groups, however,
were not successful. Instead, they resulted in isotopically ex-
changed products exclusively, likely due to the mutual activa-
tion of the fluorine atoms (Scheme 1). Not surprisingly, treat-
ment of UCB-J with 18F-fluoride under the same radiolabeling
conditions also led to an isotopically exchanged product, pre-
sumably with 18F on the 4-position of the benzene ring, in its
racemic form and with low molar activity.
We then turned to a new radiofluorination method using
hypervalent iodonium precursors for the radiosynthesis of
[18F]UCB-J [32–34]. Two precursors (compounds 11 and
18) were prepared for radiofluorination. Iodonium ylide pre-
cursor 11 was prepared from a seven-step synthesis, with
~3.2% overall yield (Scheme 2). 3,5-Difluorobenzonitrile (4)
was iodinated and reduced to form 3,5-difluoro-4-
iodobenzaldehyde (6), which underwent a Wittig reaction to
Measurement of radiotracer free fraction (fp) in plasma An
ultrafiltration method was used for measuring the unbound
portion (free fraction) of [18F]UCB-J in plasma, as previously
described [27]. The fP was determined as the ratio of the ra-
dioactivity concentration in the filtrate to the total activity in
plasma. Measurements of fP were performed in triplicate for
each scan.
Imaging analysis and kinetic modeling High-resolution mag-
netic resonance (MR) images were acquired with a Siemens
3T Trio scanner to assist with image co-registration and

Eur J Nucl Med Mol Imaging
18F
F
F
F
O
O
O
18F
X
N
N
N
X
X
N
N
N
F
F
X = Cl or I
Racemic [18F]
3
X = Cl or I
Isotopic exchange products
Scheme 1 Attempted radiosynthesis of [18F]UCB-J ([18F]-3) from chloro or iodo precursor.
form the α,β-unsaturated ester (7). Michael addition with ni-
tromethane then provided compound 8, followed by reduction
with iron powder under mildly acidic conditions. The reduc-
tion with iron powder can be completed either at elevated
temperature in 3 h or at room temperature for an extended
reaction time. The iodine at the 4-position in compound 8,
however, did not survive the reaction at elevated temperature.
Therefore, this reduction was carried out at room temperature
for 16 h, followed by in situ cyclization with the addition of
saturated NaOH solution to afford compound 9. Interestingly,
this cyclization can only be achieved with saturated NaOH,
and the use of less concentrated NaOH solution or other bases
to quench the reaction resulted in only trace amounts of the
product 9. Coupling with 4-(chloromethyl)-3-methylpyridine
then gave compound 10. Finally, the iodonium ylide 11 was
prepared from compound 10 following previously reported
procedures [33]. Trituration with EtOAc and hexanes afforded
the iodonium ylide precursor 11 as a white powder.
Synthesis of the iodonium salt precursor 18 (Scheme 3)
started with 3-bromo-4,5-difluorobenzaldehyde (12) and
followed similar steps as those described above to prepare
compound 16. Compound 16 was converted to the iodo com-
pound 17 using the copper-catalyzed aromatic Finkelstein re-
action [35]. Formation of the iodonium salt 18 was achieved
using procedures modified from Bielawski et al. [36], which
indicated that the purification of iodonium salt on Al2O3
would lead to a mono-triflate iodonium salt. The prepared
iodonium salt was further tested with 19F-NMR analysis and
determined as the mono-triflate using the integration of fluo-
rine peaks on the 19F-NMR spectrum.
[37] and copper-catalyzed fluorination with arylstannanes
through single electron transfer [38], and to compare with
the hypervalent iodonium precursors.
Radiochemistry
With conventional heating, three radiofluorination tempera-
tures (120, 150 and 170 °C) were tested with the 4-iodonium
ylide precursor 11, and the radiochemical yield gradually in-
creased with increasing temperature (Scheme 4). The same
results were obtained with the 5-iodonium salt precursor 18.
We further optimized the radiofluorination conditions with
precursor 18 using the NanoTek microfluidic reactor. A
screening of radiolabeling conditions indicated DMF as the
preferred reaction solvent for the radiosynthesis of
5-[18F]UCB-J from the iodonium salt precursor 18. Similar
to reaction with conventional heating, the rate of 18F incorpo-
ration gradually increased when the reaction temperature was
increased from 120 °C to 200 °C with different flow rates and
precursor-to-fluoride ratios in the microfluidic reactor. The
best conditions for 18F incorporation were found to be reaction
at 200 °C, with flow rate of 50 μL/min for both the precursor
and 18F-fluoride solutions (78% incorporation based on radio-
HPLC). The HPLC yield was calculated from the radiochro-
matogram. It is worth noting, though, that this yield was ap-
parently overestimated when compared with the isolated
yield, as there was most likely significant retention of radio-
activity on the HPLC column, and recovery of activity was not
checked during these test runs.
Radiofluorination simulation with UCB-J was conducted
under these established optimal microfluidic flow rates to
check for racemization, and showed complete racemization
of the compound at temperatures higher than 120 °C in the
presence of the base TEAB (1 mg/mg of UCB-J). As a result,
We also prepared a few other precursors (19–21) to test the
radiofluorination methodologies and conditions that have
been reported recently, for example PhenoFluor-mediated
deoxyfluorination through concerted nucleophilic substitution
F
F
H
O
F
F
O
F
F
F
F
F
F
F
F
O
O
H
b
c
d
a
e
I
OEt
I
OEt
CN
I
CN
I
I
NH
H
O2N
4
5
6
7
8
9
F
F
O
O
F
O
O
O
O
f
g
N
N
I
I
N
N
F
10
11
Scheme 2 Synthesis of racemic 4-iodonium ylide precursor (11).

Eur J Nucl Med Mol Imaging
Scheme 3 Synthesis of racemic 5-iodonium salt precursor (18).
18F-fluorination was performed with racemic iodonium pre-
cursors on the Nanotek microfluidic reactor, or manually in a
hot cell at elevated temperatures (200 °C for the
microfluidic reactor; and 170 °C for manual synthesis),
followed by purification on a semi-preparative chiral
HPLC column to resolve the two enantiomers. Total syn-
thesis time was ~120 min including purification and for-
mulation. The radiotracers were prepared in >99% radio-
chemical purity and enantiomeric purity (Fig. 2). Isolated
radiochemical yield (RCY) for the desired enantiomer was
low, at 1–2%, and molar activity at the end of synthesis
was moderate (59.0 35.7 MBq/nmol, n = 5).
Employing the procedures reported in the literature
(Scheme 5), the racemic hydroxy-complex precursor (19) pro-
duced the racemic product (R,S)-[18F]3 in ~ 5% RCY (HPLC
incorporation yield, decay-uncorrected), similar to the results
from hypervalent iodonium precursors, while the trimethyltin
precursors (20 and 21) did not produce (R,S)-[18F]3 in detect-
able RCY.
Plasma analysis The results from plasma analysis are shown in
Fig. 3. [18F]UCB-J displayed a moderate rate of change over
time in plasma, with 51 8% of intact parent radiotracer at
30 min after injection, which decreased to 35 2% and 27
2% at 60 and 90 min, respectively (n = 4). After a bolus injec-
tion, the parent radioactivity level in plasma exhibited a quick
increase to peak and a sharp decline phase within 5 min, then a
slow decrease afterwards. On the reversed-phase HPLC chro-
matograms (Fig. 3c), the observed metabolite peaks were
more polar than the parent (retention time of ~ 7 min for
metabolites vs. ~ 11 min for the parent radiotracer). The fp of
the radiotracer in plasma was 42 6% (n = 4), similar to that
measured with [11C]UCB-J (43 5%, n = 4).
Brain analysis Representative PET images summed from 20 to
40 min post-injection are shown in Fig. 4. In the baseline scan
(Fig. 4c), high uptake was seen throughout the gray matter
regions, and lower uptake in the centrum semiovale (white
matter) region, similar to that of [11C]UCB-J (Fig. 4b). A clear
reduction in radiotracer uptake was observed in the self-
blocking scan (Fig. 4d).
PET imaging experiments in rhesus monkeys
Brain kinetics of [18F]UCB-J were fast and reversible.
Regional TACs for [18F]UCB-J indicated excellent radiotracer
uptake in the monkey brain, with a peak standard uptake value
(SUV) of ~12 at 20–30 min post-injection (Fig. 5a). The
highest radioactivity concentrations were observed in the
Injection parameters A total of six PETscans were performed
in three different monkeys. Mean injected radioactivity was
57.3 MBq, with mean injected mass of 0.31 μg.
Scheme 4 Radiosynthesis and chiral separation of [18F]UCB-J and (S)-[18F]3.

Eur J Nucl Med Mol Imaging
regional uptake similar to that of the self-blocking scan with
5-[18F]UCB-J (compare Fig. 5d to b).
Regional TACs were analyzed with the 1TC model using
the metabolite-corrected plasma radioactivity as input func-
tion to calculate binding parameters. The 1TC model pro-
duced good fits of the TACs and reliable estimates of regional
VT. Values of VT were highest in cortical regions, followed by
caudate, putamen, and thalamus, and lowest in white matter
(Table 1). An apparent reduction in regional VT values was
seen in the self-blocking scan, while the inactive enantiomer
exhibited VT values largely indistinguishable among the brain
regions, reflecting non-specific binding. The rank order and
magnitude of the calculated BPND values (Table 2) in various
brain regions were similar between [18F]UCB-J and
[11C]UCB-J in the same monkeys.
In the self-blocking study, pretreatment with 0.15 mg/kg of
UCB-J at 10 min before 5-[18F]UCB-J injection greatly re-
duced regional VT and BPND values across the monkey brain
regions (Tables 1 and 2). Occupancy by UCB-J was calculated
at 73%, similar to that produced by the same dose of UCB-J
with [11C]UCB-J (87%) [19].
Fig. 2 HPLC chromatograms for (R,S)-3 (A), UCB-J (B) and [18F]UCB-
J (C)
Discussion
We previously reported [11C]UCB-J as a PET radiotracer for
SV2A with high specific binding signals in nonhuman pri-
mates and humans [19, 20], and have demonstrated its use
as a valid biomarker for quantification of synaptic density
[20]. In this study, we describe the synthesis and in vivo eval-
uation of [18F]UCB-J in rhesus monkeys, and its comparison
with [11C]UCB-J.
frontal cortex and putamen, while the lowest uptake was seen
in the centrum semiovale. Self-blocking with UCB-J at a dose
of 0.15 mg/kg resulted in earlier peak uptake within 10 min
and a rapid decrease thereafter, with greatly reduced differ-
ences in regional activity levels (Fig. 5b). Displacement with
the SV2A specific ligand levetiracetam at 90 min after radio-
tracer administration resulted in a rapid reduction in radiotrac-
er binding, leading to fairly homogeneous uptake across the
brain regions (Fig. 5c). As expected, a baseline scan with the
other enantiomer, (S)-5-[18F]3, displayed homogeneous
The radiosynthesis of [18F]UCB-J appeared to be challeng-
ing, and initial attempts using the chloro/iodo precursors failed
to produce [18F]UCB-J. Instead, a product resulting from iso-
topic exchange was formed.
Scheme 5 Radiosynthesis of racemic [18F]3 with boronic ester, phenol-complex, and arylstannanes precursors.

Eur J Nucl Med Mol Imaging
a
b
c
Metabolites
UCB-J parent peak
1.0
0.8
0.6
0.4
0.2
0.0
5
4
3
2
1
0
11
[
[
[
[
[
C]UCB-J 60 min
18F]UCB-J 60 min
18
F]UCB-J 30 min
18F]UCB-J 15 min
18
F]UCB-J 5 min
0
2
4
6
8
10 12 14
0
30
60
90
120
0
30
60
90
120
Time (min)
Time (min)
Time (min)
Fig. 3 Parent fraction of [18F]UCB-J in plasma (a), metabolite-corrected plasma radioactivity (b) over time (n = 4, mean SD), and metabolite analysis
of 5-[18F]UCB-J at selected time points in comparison with [11C]UCB-J (C)
Recent developments in radiofluorination methodologies
using boron, iodonium, sulfonium, or tin precursors [37–40]
provide new ways to synthesize radiotracers that cannot be
readily accessed through conventional radiofluorination ap-
proaches. Thus, we carried out a preliminary investigation of
radiofluorination (Scheme 5) with the boronic ester t-butyl
4-[3,4-difluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)phenyl]-2-oxo-pyrrolidine-1-carboxylate (22) using the
copper-mediated 18F-labeling conditions described by
Tredwell et al. [41]. However, no 18F-labeled product was
observed, indicating that [18F]UCB-J may not be accessible
through boron precursors.
We next turned to the use of hypervalent iodonium precur-
sors. A few iodonium precursors were thus synthesized, with
good overall yield and purity. In the end, [18F]UCB-J, labeled
at either the 4- or 5-position, was successfully produced using
iodonium precursors under conventional heating or
microfluidic reaction conditions. Nonetheless, the isolated ra-
diochemical yield was still low, at 1–2%. In this initial study,
we did not perform further optimization to improve the label-
Fig. 4 Template MRI (a) and
180.0
a) MRI
representative summed PET
images (20–40 min post-
injection) from a [11C]UCB-J
baseline scan (b), a [18F]UCB-J
baseline scan (c), and a self-
blocking scan (with 0.15 mg/kg
UCB-J, d)
140.0
100.0
60.0
20.0
b) [11C]UCB-J
12.0
8.0
4.0
0.0
c) [18F]UCB-J
12.0
8.0
4.0
0.0
d) [18F]UCB-J self-blocking
12.0
8.0
4.0

Eur J Nucl Med Mol Imaging
Fig. 5 Regional time–activity
curves: (a) [18F]UCB-J baseline
scan; (b) [18F]UCB-J self-
blocking scan (with 0.15 mg/kg
of UCB-J); (c) [18F]UCB-J base-
line scan followed by displace-
ment with levetiracetam
a
b
14
12
10
8
14
12
10
8
Cerebellum
Frontal cortex
Hippocampus
Putamen
Centrum Semiovale
6
6
4
4
(30 mg/kg, given i.v. at 90 min);
(d) baseline scan with (S)-5-
[18F]3
2
2
0
0
0
30
60
90
120
0
30
60
90
120
Time (min)
Time (min)
c 14
12
10
8
d 14
12
10
8
6
6
4
4
2
2
0
0
0
30 60 90 120 150 180
Time (min)
0
30
60
90
120
Time (min)
ing yield, as the current yield of [18F]UCB-J was sufficient for
PET imaging studies in nonhuman primates to assess its
in vivo binding and pharmacokinetic properties for compari-
son with [11C]UCB-J.
Further radiolabeling studies with the phenol-complex pre-
cursor as well as the arylstannane precursors did not improve
the radiochemical yield of [18F]UCB-J.
In rhesus monkeys, [18F]UCB-J displayed a moderate rate
of change in plasma, similar to that of [11C]UCB-J. Plasma fp
of [18F]UCB-J was high and can be reliably measured. The
radiotracer readily entered the monkey brain and accumulated
with high concentrations throughout the gray matter regions.
Regional TACs demonstrated fast and reversible kinetics, with
peak uptake reached within 30 min post-injection in all brain
Table 1 Regional distribution
Brain region
VT (mL/cm−3
)
volume (VT) from baseline scans
of [18F]UCB-J (n = 4)* and (S)-5-
[18F]3 (n = 1), as well as a self-
blocking scan of [18F]UCB-J with
0.15 mg/kg UCB-J (n = 1), in
comparison with those from
[18F]UCB-J
baseline
[18F]UCB-J
self-blocking
(S)-5-[18F]3
baseline
[11C]-UCB-J
baseline
Amygdala
24.1 6.4
17.3 2.1
33.1 3.4
26.9 1.7
47.9 7.9
46.2 6.1
19.2 4.3
27.6 3.8
42.5 5.5
45.7 5.9
41.3 6.6
17.0 2.6
34.3 5.3
9.5 1.7
10.6
8.7
5.0
5.7
6.6
5.7
7.2
7.2
6.3
5.7
6.8
6.3
6.6
6.1
6.6
6.9
6.5
6.3
24.1 2.2
20.1 2.8
40.3 6.1
30.5 3.5
47.3 7.5
48.9 7.4
24.5 3.1
31.3 2.3
47.7 7.5
45.7 6.2
45.1 6.9
19.8 2.7
40.1 6.8
11.1 1.6
42.8 6.7
36.2 5.2
[11C]UCB-J baseline scans (n = 4)
Brainstem
Caudate
13.5
11.6
16.0
16.2
10.6
11.3
14.8
15.8
13.4
8.5
Cerebellum
Cingulate cortex
Frontal cortex
Globus pallidus
Hippocampus
Insula
Nucleus accumbens
Occipital cortex
Pons
Putamen
12.3
6.9
Centrum semiovale
Temporal cortex
Thalamus
39.2 5.6
32.3 7.3
12.8
14.0
*Data are from three baseline scans with 5-[18 F]UCB-J including the first 90 min of data in the displacement scan,
and one baseline scan with 4-[18 F]UCB-J

Eur J Nucl Med Mol Imaging
Table 2 Regional non-
Brain region
BPND
displaceable binding potential
(BPND) values from [18F]UCB-J
baseline scans (n = 4)* and a self-
blocking scan of [18F]UCB-J with
0.15 mg/kg UCB-J (n = 1), in
comparison with those from
[11C]UCB-J baseline scans (n = 4)
[18F]UCB-J
baseline
[11C]UCB-J
baseline
[18F]UCB-J
self-blocking
Amygdala
1.7 0.9
0.8 0.2
2.5 0.5
1.9 0.4
4.0 0.3
3.9 0.5
1.1 0.5
2.0 0.7
3.5 0.5
3.9 0.5
3.4 0.2
0.8 0.2
2.6 0.4
3.1 0.4
2.5 1.0
1.2 0.4
0.8 0.2
2.7 0.7
1.8 0.3
3.3 0.9
3.5 0.9
1.2 0.2
1.9 0.3
3.3 0.8
3.2 0.7
3.1 0.7
0.8 0.2
2.6 0.5
2.9 0.6
2.3 0.7
0.5
0.3
1.0
0.7
1.3
1.3
0.5
0.6
1.1
1.3
0.9
0.2
0.8
0.9
1.0
Brainstem
Caudate
Cerebellum
Cingulate cortex
Frontal cortex
Globus pallidus
Hippocampus
Insula
Nucleus accumbens
Occipital cortex
Pons
Putamen
Temporal cortex
Thalamus
*Data are from three baseline scans with 5-[18 F]UCB-J including the first 90 min of data in the displacement scan,
and one baseline scan with 4-[18 F]UCB-J
regions. A baseline scan of the inactive enantiomer (S)-5-[18F]3
indicated a lack of specific binding for the compound and thus
the chiral selectivity of radiotracer binding to SV2A, consistent
with its low binding affinity to human SV2A protein (pIC50 of
7.1 for the (S)-enantiomer versus 8.2 for the (R)-enantiomer
UCB-J) [18]. In the self-blocking study, pretreatment of the
animal with UCB-J (0.15 mg/kg) greatly reduced the binding
of [18F]UCB-J across all brain regions, thus demonstrating the
radiotracer’s binding specificity in vivo. In the displacement
study, administration of the SV2A-selective anticonvulsant le-
vetiracetam at 90 min post-injection resulted in a rapid reduc-
tion in [18F]UCB-J binding across gray matter regions, indicat-
ing competition of the displacing drug for the same binding
target. These results confirmed the saturable, reversible, and
specific nature of [18F]UCB-J binding for SV2A in vivo.
Previous studies with [11C]UCB-J indicated that the 1TC
model was an appropriate model for the estimation of binding
parameters. This was also true for [18F]UCB-J, with the 1TC
model producing good fits of regional TACs and stable VT
estimates. As expected, binding of [18F]UCB-J was seen in
gray matter areas, with high VT values in cortical areas and the
lowest VT in the centrum semiovale (white matter), consistent
with autoradiography and in vitro binding results [42].
Given the equivalent chemical structure of [18F]UCB-J and
[11C]UCB-J, the two radiotracers behaved very similarly
when evaluated in the same monkeys, in terms of metabolism
and clearance (parent fraction of 51 8% for [18F]UCB-J vs.
47 12% for [11C]UCB-J at 30 min post-injection), plasma
free fraction (42 6% vs. 43 5%), brain uptake kinetics,
regional distribution, and specific binding signals as measured
by BPND (Fig. 6 and Table 2). Both [18F]UCB-J and
[11C]UCB-J provide BPND values of >0.75 in all brain re-
gions, levels of specific binding signals that can be reliably
and accurately estimated by quantitative kinetic modeling
analysis, and one of the important characteristics for an effec-
tive neuroimaging radiotracer [43].
Fig. 6 Regional time–activity
Cerebellum
a
b
14
curves of [18F]UCB-J (a) and
12
14
Frontal cortex
12
[11C]UCB-J (b) in the same
rhesus monkey brain (n = 1)
Hippocampus
Putamen
10
10
Centrum semiovale
8
6
4
2
0
8
6
4
2
0
0
30
60
90
120
0
30
60
90
120
Time (min)
Time (min)

Eur J Nucl Med Mol Imaging
Although [18F]UCB-J was proved to be an excellent radio-
tracer, its radiosynthesis is low-yielding. Thus, we conclude that
clinical translation of [18F]UCB-J would be challenging unless
alternative approaches to its radiosynthesis in high radiochem-
ical yield could ultimately be found. As a result, we have turned
to look for alternative [18F]-labeled SV2A radiotracers.
Removal of one or two fluorine atoms on the phenyl moiety
of the UCB-J structure led to the discovery of SDM-8 and
SDM-2, both of which displayed good in vitro binding affinity
for SV2A [44]. We recently reported the successful synthesis of
[18F]SDM-8 (Fig. 1) with greatly improved radiochemical yield
using the organotin precursor [45]. Imaging evaluation in non-
human primates indicated that [18F]SDM-8 exhibited pharma-
cokinetic and binding profiles similar to those of [18F]UCB-J,
and with higher specific binding signals (BPND). The same
radiolabeled compound, but with a different name (MNI-
1126), was reported by another group [46]. Based on the results
in nonhuman primates, we anticipate that [18F]SDM-8 will be
an excellent [18F]-labeled radiotracer for imaging of SV2A, and
have advanced it to evaluation in humans.
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