Synthesis and Biological Evaluation of [18F]FECNT-d 4 as a Novel PET Agent for Dopamine Transporter Imaging

Article abstract of DOI:10.1007/s11307-021-01603-2

Purpose: The dopamine transporter (DAT) is a marker of the occurrence and development of Parkinson’s disease (PD) and other diseases with nigrostriatal degeneration. 2β-Carbomethoxy-3β-(4-chlorophenyl)-8-(2-[¹⁸F]-fluoroethyl)nortropane ([¹⁸F]FECNT), an ¹⁸F-labelled tropane derivative, was reported to be a useful positron-emitting probe for DAT. However, the rapid formation of brain-penetrating radioactive metabolites is an impediment to the proper quantitation of DAT in PET studies with [¹⁸F]FECNT. Deuterium-substituted analogues have presented better in vivo stability to reduce metabolites. This study aimed to synthesize a deuterium-substituted DAT radiotracer, [¹⁸F]FECNT-d₄, and to make a preliminary investigation of its properties as a DAT tracer in vivo. Procedures: The ligand [¹⁸F]FECNT-d₄ was obtained by one-step radiolabelling reaction. The lipophilicity was measured by the shake-flask method. Binding properties of [¹⁸F]FECNT-d₄ were estimated by in vitro binding assay, biodistribution, and microPET imaging in rats. In vivo stability of [¹⁸F]FECNT-d₄ was estimated by radio-HPLC. Results: [¹⁸F]FECNT-d₄ was synthesized at an average activity yield of 46 ± 17 % (n = 15) and the molar activity was 67 ± 12 GBq/μmol. The deuterated tracer showed suitable lipophilicity and the ability to penetrate the blood-brain barrier (brain uptake of 1.72 % ID at 5 min). [¹⁸F]FECNT-d₄ displayed a high binding affinity for DAT comparable to that of [¹⁸F]FECNT in rat striatum homogenates. Biodistribution results in normal rats showed that [¹⁸F]FECNT-d₄ exhibited a higher ratio of the target to non-target (striatum/cerebellum) at 15 min post administration (5.00 ± 0.44 vs 3.84 ± 0.24 for [¹⁸F]FECNT-d₄ vs [¹⁸F]FECNT). MicroPET imaging studies of [¹⁸F]FECNT-d₄ in normal rats showed that the ligand selectively localized to DAT-rich striatal regions and the accumulation could be blocked with DAT inhibitor. Furthermore, in the unilateral PD model rat, a significant reduction of the signal was found in the lesioned side relative to the unlesioned side. Striatal standardized uptake value of [¹⁸F]FECNT-d₄ remained ~4.02 in the striatum between 5 and 20 min, whereas that of [¹⁸F]FECNT fell rapidly from 4.11 to 2.95. Radio-HPLC analysis of the plasma demonstrated better in vivo stability of [¹⁸F]FECNT-d₄ than [¹⁸F]FECNT. Conclusion: The deuterated compound [¹⁸F]FECNT-d₄ may serve as a promising PET imaging agent to assess DAT-related disorders.

Full text of DOI:10.1007/s11307-021-01603-2

Mol Imaging Biol (2021)
DOI: 10.1007/s11307-021-01603-2
* World Molecular Imaging Society, 2021
RESEARCH ARTICLE
Synthesis and Biological Evaluation
of [18F]FECNT-d4 as a Novel PET Agent
for Dopamine Transporter Imaging
Shanshan Cao,1 Jie Tang,2 Chunyi Liu,2 Yi Fang,2 Linyang Ji,1 Yingjiao Xu,2
1,2
Zhengping Chen
1Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, School of Pharmacy, Xuzhou Medical University, Xuzhou,
221004, China
2NHC Key Laboratory of Nuclear Medicine, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear
Medicine, Wuxi, 214063, China
Abstract
Purpose: The dopamine transporter (DAT) is a marker of the occurrence and development of
Parkinson’s disease (PD) and other diseases with nigrostriatal degeneration. 2β-Carbomethoxy-3β-
(4-chlorophenyl)-8-(2-[18F]-fluoroethyl)nortropane ([18F]FECNT), an 18F-labelled tropane derivative,
was reported to be a useful positron-emitting probe for DAT. However, the rapid formation of brain-
penetrating radioactive metabolites is an impediment to the proper quantitation of DAT in PET
studies with [18F]FECNT. Deuterium-substituted analogues have presented better in vivo stability to
reduce metabolites. This study aimed to synthesize a deuterium-substituted DAT radiotracer,
[
18F]FECNT-d4, and to make a preliminary investigation of its properties as a DAT tracer in vivo.
Procedures: The ligand [18F]FECNT-d4 was obtained by one-step radiolabelling reaction. The
lipophilicity was measured by the shake-flask method. Binding properties of [18F]FECNT-d4 were
estimated by in vitro binding assay, biodistribution, and microPET imaging in rats. In vivo stability
of [18F]FECNT-d4 was estimated by radio-HPLC.
Results: [18F]FECNT-d4 was synthesized at an average activity yield of 46 17 % (n = 15) and the
molar activity was 67 12 GBq/μmol. The deuterated tracer showed suitable lipophilicity and the
ability to penetrate the blood-brain barrier (brain uptake of 1.72 % ID at 5 min). [18F]FECNT-d4
displayed a high binding affinity for DAT comparable to that of [18F]FECNT in rat striatum
homogenates. Biodistribution results in normal rats showed that [18F]FECNT-d4 exhibited a higher
ratio of the target to non-target (striatum/cerebellum) at 15 min post administration (5.00 0.44 vs
3.84 0.24 for [18F]FECNT-d4 vs [18F]FECNT). MicroPET imaging studies of [18F]FECNT-d4 in
normal rats showed that the ligand selectively localized to DAT-rich striatal regions and the
accumulation could be blocked with DAT inhibitor. Furthermore, in the unilateral PD model rat, a
significant reduction of the signal was found in the lesioned side relative to the unlesioned side.
Striatal standardized uptake value of [18F]FECNT-d4 remained ~4.02 in the striatum between 5 and
20 min, whereas that of [18F]FECNT fell rapidly from 4.11 to 2.95. Radio-HPLC analysis of the
plasma demonstrated better in vivo stability of [18F]FECNT-d4 than [18F]FECNT.
Conclusion: The deuterated compound [18F]FECNT-d4 may serve as a promising PET imaging
agent to assess DAT-related disorders.
Key words: DAT, MicroPET imaging, 18F, Deuterium, In vivo stability
Correspondence to: Zhengping Chen; e-mail: chenzhengping@jsinm.org

Cao S. et al.: Synthesis and Biological Evaluation
structure, which overcame many defects of the original drug
such as short half-life and high clinical dosage [21].
Recently, the deuterium substitution strategy has also been
applied to radiopharmaceutical for PET imaging. For
example, [18F]FE-(+)-DTBZ-d4 [22] and [18F]MNI-659-d2
[23] were employed as radiotracers for vesicular monoamine
transporter 2 (VMAT2) and phosphodiesterase 10A (PDE
10A), respectively.
In this study, we designed and synthesized a deuterated
18F-labelled analogue [18F]FECNT-d4 (Fig. 1), aiming to
improve upon the in vivo metabolic stability of [18F]FECNT.
Herein, we performed binding affinity, lipophilicity, autora-
diography, biodistribution, and PET imaging of
[18F]FECNT-d4. Our findings of excellent stability in vivo
and high specific binding properties of [18F]FECNT-d4
imply that the deuterated tracer may be an attractive PET
imaging agent for DAT.
Introduction
The dopamine transporter (DAT) plays almost the sole
executor of dopamine reutilization which is vitally correlated
with Parkinson’s disease (PD), and other neurodegenerative
diseases [1, 2]. The dramatic loss of dopaminergic neurons
in early-stage PD, found in previous clinicopathologic
studies, suggests that the degeneration of this region begins
before motor symptoms [3, 4]. As widely expressed in
dopaminergic neurons, DAT is an important indicator for the
diagnosis of early PD. Therefore, potential radioligands for
image DAT with positron emission tomographic (PET) may
aid diagnosis and treatment of PD by providing probes to
estimate the density of DAT in specific brain regions.
Over the decades, researchers have developed several
DAT radiotracers for PET imaging, 11C-labelled ligands
such as [11C]PE2I [5] and [11C]CFT [6]. However, 11C has a
very short half-life (20 min); 18F-labelled ligands with a
longer half-life (109 min) would be more practical for
widespread use, such as [18F]FP-CMT [7], [18F]FE-PE2I [8],
[18F]LBT-999 [9], and [18F]FECNT (2β-carbomethoxy-3β-
(4-chlorophenyl)-8-(2-[18F]fluoroethyl)nortropane) [10].
[18F]FECNT has 25- and 156-fold selectivity for DAT (Ki
= 1.5 nM) more than for serotonin transporter and
noradrenaline transporter, respectively [10]. Davis et al.
reported that PET imaging with [18F]FECNT has favorable
kinetics in humans and could distinguish DAT deficits
between Parkinson’s patients and healthy individuals [11].
However, it is found that [18F]FECNT, which has the 18F on
the N-alkyl group, tends to be de-alkylated by cytochrome
P450 enzyme to form radioactive metabolites, namely
Materials and Methods
General
All of the organic solvents and chemical reagents were
obtained from Aladdin (China), J&K Scientific (China),
Sigma-Aldrich (China), and used directly without further
purification. FECNT and CFT were synthesized according to
previously published papers [24, 25]. Kryptfix222 (K222
,
98.0 %) was purchased from ABX (Germany). Sep-Pak C18
cartridges and Sep-Pak lightweight QMA (QMA) were
purchased from Waters (USA). Analytical high-
performance liquid chromatography (HPLC) system was
used to evaluate radiochemical purity (RCP) and the
radiochemical yield (RCY) of [18F]FECNT and
[18F]FECNT-d4. Analytical high-performance liquid chro-
matography (HPLC) system consists of Waters 1525 HPLC
pump, 2489 UV/Visible Detector (Waters, USA), and
radiometric 610TR detector (PerkinElmer, USA). The
analytical conditions were as follows: analytical column
(C18, 5 μm, 4.6 × 150 mm, Phenomenex, Gemini, USA),
1.0 mL/min, UV detection at 220 nm, method A: H2O/
CH3CN/Et3N (40/60/0.1, v/v/v); method B: H2O/CH3CN/
Et3N (38/62/0.1, v/v/v). Semi-prepared HPLC (semi-prep
HPLC) consists of Waters 1525 HPLC pump, 2489 UV/
Visible Detector (Waters, USA), and flow-count radioactiv-
ity detector (Biscan, USA). The semi-prep HPLC system
was used to separate and purify crude products. The
preparation conditions (method C) were as follows: semi-
prep column (C18, 5 μm, 10 × 250 mm, Waters, XBridge,
USA), 5.0 mL/min, UV detection at 220 nm, H2O/CH3CN/
Et3N (45/55/0.1, v/v/v). The chemical structure of
[18F]FECNT-d4 was identified by co-injected with the non-
labelled standard compound.
[
18F]fluoroacetaldehyde, [18F]fluoroethanol, and
[18F]fluoroacetatic acid [12]. These metabolites can cross
the blood-brain barrier (BBB) and distribute in the whole
brain, confounding quantitative measurement of DAT. To
accurately quantify the uptake and binding of [18F]FECNT
to DAT, Nye and colleagues raised an arterial input model of
[18F]FECNT uptake for DAT density quantification in
humans [13]. However, this protocol required a 180-min
PET scan and a series of arterial blood samples during
imaging, which might be not convenient enough for clinical
routine application. Nevertheless, this N-dealkylation me-
tabolism pathway is not unique to [18F]FECNT and has also
been observed in other ligands with high affinity and
selectivity for DAT such as [18F]FE-PE2I [14] and
[18F]LBT-999 [15].
In recent years, a great deal of work has revealed the
deuterium isotope effect on the biological reaction [16, 17].
Due to the cleavage rate of a carbon-deuterium (C–D) bond
being about 6–7 times slower than that of a carbon-hydrogen
(C–H) bond, the deuterium-substitution at easily metaboliz-
able positions of the molecules can help to improve the
metabolic stability of active pharmaceuticals [18, 19].
Recently, a deuterated form of tetrabenazine, SD-809, has
been approved by the FDA to treat tardive dyskinesia [20].
Another example is CTP-656, a deuterated ivacaftor
The human neuroblastoma (SH-SY5Y) cell line was
purchased from the National Collection of Authenticated
Cell Cultures (Shanghai, China). The cells were cultured in
DMEM: F12 medium (containing 1 % penicillin-

Cao S. et al.: Synthesis and Biological Evaluation
Fig. 1. Chemical structures of [18F]FECNT ([18F]1) and [18F]FECNT-d4 ([18F]2).
streptomycin, 1 % gluta-max, 15 % fetal bovine serum) and
kept under an atmosphere of 5 % CO2 at 37 °C.
Methyl(1R,2S,3S,5S)-3-(4-Chlorophenyl)-8-(2-
Hydroxyethyl-1,1,2,2-d4)-8-
Male Sprague-Dawley (SD, 220−260 g) rats and Institute
of Cancer Research mice (ICR, 20−30 g) were provided by
Vital River Laboratory Animal Technology Co. Ltd.
(China). The striatal unilateral 6-hydroxydopamine (6-
OHDA) lesion model was performed with saline containing
16 μg (4 μg/μl) 6-OHDA, at the coordinates: AP: −4.0 mm
(Bregma), ML: −1.4 mm (midline), DV: −7.5 mm (hole)
[26]. All the animals were housed in clean cages with free
access to food and water with a 12-h light-dark cycle and
then tested. All animal experiment procedures were ap-
proved by the Animal Care and Ethics Committee of Jiangsu
Institute of Nuclear Medicine.
Azabicyclo[3.2.1]Octane-2-Carboxylate (7)
M e t h y l ( 2 S , 3 S ) - 3 - ( 4 - c h l o r o p h e n y l ) - 8 -
azabicyclo[3.2.1]octane-2-carboxylate (6) (1 mmol, 1.0
equiv.), 2-bromoethan-1,1,2,2-d4-1-ol (4 mmol, 4.0 equiv.),
and triethylamine (5 mmol, 5.0 equiv.) were added to 3 mL
of anhydrous acetonitrile. The solution was stirred at room
temperature for 15 min to completely dissolve the solid.
Subsequently, the reaction system was refluxed at 80 °C for
5 h. After the reaction was complete, the system turned
yellow and the solvent was evaporated to dryness under
reduced pressure. The residue was dissolved in dichloro-
methane and washed with 4 mL NaOH (1 M) and 4 mL
water, respectively. The organic phase was dried over
anhydrous Na2SO4 and filtered, and the filtrate was
concentrated under reduced pressure. The residue was
purified with silica gel chromatography with Et2O/Et3N
Ethane-1,2-diyl-d4 bis(4-Methylbenzenesulfonate)
(4)
1
(v/v = 95/5) to give (7) (279 mg, 85 %) as a white solid. H
The compound (4) was synthesized following the procedures
reported by Raaphorst et al. [27]. 1H NMR (400 MHz,
CDCl3) δ 7.73 (d, J = 8.0 Hz, 4H), 7.33 (d, J = 8.0 Hz, 4H),
2.45 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 145.40,
132.53, 130.10, 128.09, 66.12, 21.81. ESI-MS: m/z calcd for
C16H14D4O6S2 [M+Na]+ 397.08, found 397.17.
NMR (400 MHz, CDCl3) δ 7.24 (d, J = 8.6 Hz, 2H), 7.18 (d,
J = 9.5 Hz, 2H), 3.60 (q, J = 3.8 Hz, 1H), 3.49 (s, 3H), 3.44–
3.40 (m, 1H), 3.01 (dt, J = 12.7, 5.2 Hz, 1H), 2.92–2.88 (m,
1H), 2.60 (td, J = 12.7, 3.1 Hz, 1H), 2.14–1.95 (m, 2H),
1.82–1.74 (m, 1H), 1.74–1.66 (m, 2H), 1.64 (d, J = 11.8 Hz,
1H). 13C NMR (100 MHz, CDCl3) δ 172.23, 141.20,
131.93, 128.91, 128.27, 62.87, 61.09, 52.89, 51.43, 34.39,
34.21, 26.56, 26.22. ESI-MS: m/z calcd for C17H18D4ClNO3
[M+H]+ 328.15, found 328.21.
2-Fluoroethyl-1,1,2,2-d4 4-
Methylbenzenesulfonate (5)
A solution of (4) (1.06 mmol, 1.0 equiv.) in acetonitrile (5
mL) was added to a solution of tetrabutylammonium
fluoride (TBAF, 1.6 mmol, 1.5 equiv.) under nitrogen
atmosphere. Then, the reaction system was heated to reflux
for 1.5 h. The organic solvent was removed under reduced
pressure and the residue was extracted with hexane/ethyl
acetate (v/v = 5/1) and (5) was obtained as a colorless oil
Methyl(1R,2S,3S,5S)-3-(4-Chlorophenyl)-8-(2-
((Methylsulfonyl)oxy)Ethyl-1,1,2,2-d4)-8-
Azabicyclo[3.2.1]Octane-2-Carboxylate (8)
A mixture of (7) (0.5 mmol, 1.0 equiv.) and methanesulfonic
anhydride (2.2 mmol, 4.4 equiv.) in freshly dried dichloro-
methane (2 mL) was stirred at 30 °C for 3 days under
nitrogen. The chemical purity of (8) was monitored by the
analytical HPLC under the following condition (H2O/
CH3CN/TFA, 65/35/0.2, v/v/v). After the reaction was
complete, the solvent was removed under reduced pressure.
The residue was dissolved in 1 mL dichloromethane and
1
(101.3 mg, 43 %) after purification. H NMR (400 MHz,
CDCl3) δ 7.84–7.77 (m, 2H), 7.40–7.32 (m, 2H), 2.45 (s,
3H). 13C NMR (100 MHz, CDCl3) δ 145.15, 132.69,
129.94, 127.96, 77.24, 29.70, 21.65. ESI-MS: m/z calcd
for C9H7D4FO3S [M+Na]+ 245.07, found 245.13.

Cao S. et al.: Synthesis and Biological Evaluation
washed with 2 mL ether 5 times. Finally, the precipitate was
obtained by 99 % yield as a white solid (8) under reduced
pressure. H NMR (400 MHz, CDCl3) δ 9.21 (s, 1H), 7.33–
Sep-Pak C18 light cartridge. Then, the product was eluted
with 1 mL of ethanol from the C18 cartridge. The product
was analyzed for quality control by radio-HPLC (method A).
1
7.28 (m, 2H), 7.18–7.10 (m, 2H), 4.49 (d, J = 5.0 Hz, 2H),
3.44 (s, 4H), 3.21 (s, 3H), 3.05 (dd, J = 6.6, 2.3 Hz, 1H),
2.95 (t, J = 14.1 Hz, 1H), 2.84 (s, 3H), 2.71–2.49 (m, 2H),
2.18 (q, J = 11.0, 10.4 Hz, 2H), 2.04–1.95 (m, 1H). 13C
NMR (100 MHz, CDCl3) δ 173.78, 136.36, 133.96, 129.14,
128.89, 64.31, 63.57, 52.96, 49.55, 39.55, 37.56, 34.08,
32.14, 24.79, 23.84. ESI-MS: m/z calcd for
C18H20D4ClNO5S [M+H]+ 406.13, found 406.48.
In vitro Stability Assays
The stability of [18F]FECNT-d4 was assayed in phosphate-
buffered solution (PBS) and fetal bovine serum (FBS).
[18F]FECNT-d4 (37 MBq) was added to PBS (pH 7.4, 500 μL)
and FBS (500 μL), respectively. The mixtures were incubated for
0, 1, 2, 4, and 6 h at 37 °C. The radiochemical purity was recorded
by analytical radio-HPLC (method A) at each time point. For
serum samples, acetonitrile (100 μL) was added to a serum
sample (100 μL) and the protein precipitate was removed by high-
speed centrifugation before purity analysis.
2β-Carbomethoxy-3β-(4-Chlorophenyl)-8-(2-
Fluoroethyl-1,1,2,2-d4)-Nortropane (FECNT-d4,
2)
A mixture of (6) (1 mmol, 1.0 equiv.), (5) (1 mmol, 1.0
equiv.), and N,N-diisopropylethylamine (DIPEA, 2 mmol,
2.0 equiv.) in methanol (8 mL) was heated at 100 °C for
24 h under nitrogen. After cooling to room temperature, the
reaction mixture was stirred for 1 h with NaOH (1 M). Then
the solution was concentrated with benzene under reduced
pressure. The residue was purified by silica gel chromatog-
raphy with PE/CH2Cl2 (4/1, v/v) to afford (2) as a white
In vivo Stability Assays
Following the injection of [18F]FECNT-d4 or [18F]FECNT
(20 MBq in 0.2 mL 10 % ethanol/saline) to the mice (n = 3), the
blood samples were obtained from the mice’s tail at 5, 30, and
60 min. Each sample was collected in a test tube containing
CH3CN (50 μL) with FECNT (20 μM). Male Sprague-Dawley
rats (n = 3) were anesthetized with 2.5 % isoflurane in oxygen
followed by intravenous injection of radioligand
([18F]FECNT-d4 or [18F]FECNT: 185 MBq in 0.5 mL 10 %
ethanol/saline). At 5, 30, and 60 min after injection, three rats
were sacrificed at each time point respectively. The striatum
and cerebellum were rapidly removed. Then, each sample of
isolated striatum, cerebellum, and plasma was homogenized in
0.5 mL acetonitrile containing 1.0 mg/ml FECNT, respec-
tively. The cold FECNT was added in samples to improve the
extraction efficiency. The homogenates were centrifuged at
12,000g for 5 min at 4 °C for deproteinization. After
centrifugation, the radioactivity counts of the supernatant and
the precipitate were measured with a γ-counter (2480 Wizard2,
PerkinElmer, USA) separately to calculate the extraction
efficiency of radioactive material. Finally, a portion of the
resulting supernatant was injected into the analytical radio-
HPLC system (method B). On the HPLC chromatogram, the
percent ratio was calculated as the peak area of the unchanged
form to total peak area (corrected for decay) × 100 %.
1
solid (128 mg, 38 %). H NMR (400 MHz, CDCl3) δ 7.25–
7.21 (m, 2H), 7.21–7.16 (m, 2H), 3.78 (dd, J = 7.3, 3.5 Hz,
1H), 3.51 (s, 3H), 3.43 (q, J = 3.7 Hz, 1H), 2.97 (dt, J =
12.7, 5.0 Hz, 1H), 2.92–2.86 (m, 1H), 2.58 (td, J = 12.5, 2.9
Hz, 1H), 2.16–2.08 (m, 1H), 2.00 (td, J = 12.5, 8.0 Hz, 1H),
1.81–1.71 (m, 1H), 1.70–1.63 (m, 2H). 13C NMR (100
MHz, CDCl3) δ 171.73, 141.61, 131.49, 128.71, 128.00,
63.57, 62.22, 52.66, 51.05, 34.00, 33.61, 26.20, 25.77. 19F
NMR (376 MHz, CDCl3) δ -222.10. MS calcd. for
C17H17D4ClFNO2 [M+H]+ 330.15, found: 330.42.
Radiosynthesis of [18F]FECNT-d4 ([18F]2)
The synthetic method of [18F]FECNT was referenced to the
previous article [24]. The automatic radiosynthesis of
[18F]FECNT-d4 was completed by the PET-MF-2 V-IT-1
(Beijing PET Technology, China) multifunctional synthesis
module. First, [18F]fluoride (10 GBq) was captured on the
QMA cartridge by the enriched [18O]H2O through cyclotron.
Then 18F ion was transferred into the reaction tube from the
irradiated QMA with a stock solution ((K222 (40 μmol),
CH3CN (0.8 mL), K2CO3 (20 μmol), water (0.2 mL)). The
mixture was dried at 105 °C for 2 min under nitrogen, and
further dried twice at 105 °C with ultra-dry acetonitrile.
After drying, cool to 40 °C, the precursor (8) (5 mg)
dissolved in CH3CN (1 mL) was added to the reaction tube
and kept at 90 °C for 20 min. Next, the reaction was
quenched with H2O (0.5 mL) and the mixture was
transferred to the semi-prepared HPLC (method C). After
purification, the collected liquid was diluted with 10-fold
water and the tracer [18F]FECNT-d4 was trapped using a
Cellular Uptake and Blocking Assays
SH-SY5Y cells were seeded into 12-well plates (1.5 × 105
cells per well) and cultured at 37 °C overnight for cell
adherence. During the experiment, the cells were assigned
for the control group, CFT blocking group, and DTBZ
blocking group. The control group was added with
[18F]FECNT-d4 (18.5 KBq) in fresh medium (200 μL), the
other two groups were added with [18F]FECNT-d4 and CFT
(20 μM) or [18F]FECNT-d4 and DTBZ (20 μM). The cells
were separately incubated at 37 °C for 15, 30, 60, 90, and

Cao S. et al.: Synthesis and Biological Evaluation
120 min. At each time point, the basic medium was
extracted to a test tube, and the cells were rinsed once with
ice-cold PBS. Then, the cells were lysed and collected with
NaOH (1 M) resolution. The radioactivity counts of cell
lysate and supernatant were measured by a γ-counter and
calculated as % uptake. The cellular uptake values of
radioligand ([18F]FECNT-d4 or [18F]FECNT: 12 MBq in
0.5 ml 10 % ethanol/saline) through the tail vein, and they
were executed by cervical dislocation at 5, 15, 30, 60, and
120 min under anesthesia. The whole brain (including the
striatum, cerebellum (CB), hippocampus (HIP), cortex (CX))
and each organ (including the heart, liver, spleen, lung,
kidney, stomach, small intestines, testis, bone, muscle,
pancreas) were quickly dissected and weighed. In addition,
blood (200 μL) was obtained from the carotid artery. These
radioactive samples were counted by a γ-counter and the
results were expressed as a percentage of the injected dose
per organ (% ID) or a percentage of the injected dose per
gram of wet tissue (% ID/g) by calibrating with 1 % initial
dose counts (100-fold diluted of the injection).
[18F]FECNT-d4 (mean
SD, n = 3) were calculated as
following formula: [CPM (the cell lysate)] / [CPM (the cell
lysate) + CPM (the medium)] × 100 %. The blocking
percentage was calculated with [CPM (control) − CPM (with
inhibitor)] / CPM (control)] × 100 %.
Determination of Lipophilicity
The octanol-water partition coefficient of [18F]FECNT-d4
and [18F]FECNT was determined at room temperature by the
shake-flask method. [18F]FECNT-d4 or [18F]FECNT (18.5
KBq) was added into two-phase mixture of n-octanol (3 mL)
and PBS (pH 7.4, 3 mL). After shaking for 5 min, the PBS
and the n-octanol phase were separated via centrifugation.
Then, the PBS (1 mL) solution and n-octanol (1 mL) were
pipetted into test tubes, respectively. The radioactivity in n-
octanol and PBS were determined by a γ-counter. Subse-
quently, the remaining n-octanol (1 mL) was redistributed
into a mixture containing 2 mL n-octanol and 3 mL PBS.
The above experiment was repeated 5 times to obtain an
average logP value. The logP values were calculated by the
formula: logP = log(CPMn-octanol/CPMPBS). All measure-
ments were tested in triplicate.
Ex vivo Autoradiography
Normal and 6-OHDA model rats were respectively injected
with 37 MBq [18F]FECNT-d4 via the tail vein and sacrificed
at 30 min post injection. The brain of each rat was separated
and frozen quickly. Coronal sections (20 μm) were cut on a
cryostat and dried at room temperature. The dried sections
were exposed to the imager plate for 45 min, and images
were attained using Cyclone Plus Storage Phosphor System
(Perkin Elmer, USA).
MicroPET Imaging
A high-resolution microPET system (Siemens Medical
Solutions, Knoxville, TN, Germany) was used to monitor
0–120 min scanning of the rat brain in real time. The rat was
placed on the bed of microPET and the brain position was
fixed under isoflurane anesthesia (2.5 % in oxygen gas)
during the experiment. Each rat (n = 5) was injected with
12 MBq of [18F]FECNT-d4 or [18F]FECNT (0.5 ml, 10 %
ethanol/saline) through the tail vein. The scan results were
acquired in 3 min per frame sequences to obtain 40
consecutive frames, and then images were reconstructed
using the analytical filter back projection (FBP) method.
Time activity curves (TACs) for tissue uptake were
expressed as standard uptake values (SUVs). The
injected radioactivity was corrected by time decay. To
verify the specific binding of [18F]FECNT-d4 to DAT
in vivo, a DAT-specific ligand CFT (1.0 mg/kg) was co-
injected with [18F]FECNT-d4 into the tail vein and the
rats were scanned from 10 to 40 min. Furthermore, the
PD model rats were also scanned at 10–40 min after
administration of [18F]FECNT-d4. All obtained images
were processed and analyzed with ASIPro VM software;
the regions of interest (ROIs) were plotted on summa-
tional images for a sum of all frames [28]. The binding
potential (BPND) in the striatum was calculated by the
simplified reference tissue method [29] using the TAC
with the cerebellum as a reference region.
Competitive Binding Assays
The rats were decapitated under ether anesthesia. The
striatum (ST) was quickly pulled out and homogenized in
50 mM Tris-HCl buffer (pH 7.4) containing 120 mM NaCl,
2 mM KCl, 1 mM CaCl2, and 1 mM MgCl2 through a high
throughput group grinder. Each 100 μL of homogenate is
incubated with [18F]FECNT (0.5–0.8 nM in buffer) and 100
μL of various concentrations of the test compound (FECNT-
d4 or FECNT, 10−11–10−7 M) in a final volume of 0.5 mL of
buffer. Non-specific binding was determined by co-
incubation with 20 μM of CFT. These mixtures were
incubated at room temperature for 30 min and terminated
by rapid filtration using Whatman GF/C glass-fiber filters,
followed by washing four times with ice-cold PBS. The
radioactivity of the pellets bond with [18F]FECNT was
measured using an automatic γ-counter. The 50 % inhibitory
concentration (IC50) was calculated using the nonlinear
least-square curve fitting with GraphPad Prism.
Biodistribution Studies
The rats were divided into 10 groups (n = 5) for the
biodistribution studies. Each rat was injected with

Cao S. et al.: Synthesis and Biological Evaluation
Results
radioactive metabolites and 7.5 min for parent radiotracer.
For mice, after administration of [18F]FECNT-d4 or
[18F]FECNT, polar metabolites of [18F]FECNT began to
appear in plasma at 30 min post administration. More
metabolites of [18F]FECNT were observed at 60 min,
whereas only a little fraction of radiometabolites of
[18F]FECNT-d4 were detected (Suppl. Fig. 1, see ESM).
For rats, the parent tracer and radiolabelled metabolite
fractions in brain tissues and plasma were described in
Table 1.
Synthesis
The non-radioactive compound FECNT-d4 was synthesized
with (6) in 38 % yield. The mesylate precursor (8) was
obtained in 99 % yield by esterification (Scheme 1). Finally,
[18F]FECNT-d4 was synthesized from (8) by one step in a
multifunctional synthesis module. The crude product solu-
tion was clear and colorless, and the RCP was greater than
99.9 % after purification. The molar activity (Am) was 67
12 GBq/μmol and the RCY was 46 17 % (n = 15) at the
end of [18F]FECNT-d4 synthesis.
Cellular Uptake and Blocking Assays
The cell uptake of [18F]FECNT-d4 was evaluated in SH-
SY5Y cells. The accumulation of [18F]FECNT-d4 in SH-
SY5Y cells reached 1.75 % uptake at 120 min. The binding
specificity of [18F]FECNT was preliminarily evaluated using
CFT- and DTBZ-treated cells. Compared to the control
group, the accumulation of [18F]FECNT-d4 was inhibited
~66 % (0.60 % uptake) by an excess of CFT at 120 min
(Suppl. Fig. 2, see ESM). All the time, the uptake of
[18F]FECNT-d4 was not influenced by DTBZ.
In vitro Stability Assays
[18F]FECNT-d4 did not breakdown during its synthesis
procedure. The HPLC analysis results showed satisfactory
stability of [18F]FECNT-d4 for up to 6 h at 37 °C with RCP
9 98 % in PBS and 9 96 % in serum, respectively (Fig. 2).
In Vivo Stability Assays
To verify the in vivo stability of [18F]FECNT-d4 and
[18F]FECNT, it was necessary to analyze the parent tracer
content in mice or rats after injected with radiotracer using
radio-HPLC. The analysis showed that the retention times in
the HPLC diagram were approximately 2.2 min for polar
Lipophilicity and Binding Affinity
As shown in Table 2, the logP7.4 values of [18F]FECNT-d4
and [18F]FECNT were found to be 2.37 and 2.25, respec-
tively. The IC50 values of the two compounds exhibited in
Scheme 1. Synthesis of FECNT-d4 (2) and the precursor (8): (a) TsCl, rt, 24 h; (b) TBAF, CH3CN, 80 °C; (c) BrCD2CD2OH,
CH3CN, 90 °C; (d) Ms2O, CH2Cl2, 30 °C; (e) 5, DIPEA, CH3OH, 100 °C.

Cao S. et al.: Synthesis and Biological Evaluation
Fig. 2. a Radiolabelling of [18F]FECNT-d4 ([18F]2) by one-step. b Radio-HPLC analysis of [18F]FECNT-d4 before and after
purification. In vitro stability analysis of [18F]FECNT-d4 in PBS (c) or FBS (d) for different time (0, 1, 2, 4, 6 h).
the single-digit nanomolar range (FECNT-d4, IC50 = 2.17
nM and FECNT, IC50 = 1.99 nM).
activity of [18F]FECNT-d4 in the striatum reached 2.03
0.34 % ID/g at 5 min and retained at 2.06 0.07 % ID/g
until 30 min. The activity of [18F]FECNT decreased rapidly
in the striatum from 5 min (2.51 0.28 % ID/g) to 30 min
(1.58 0.19 % ID/g).
Biodistribution Studies
The biodistribution results of [18F]FECNT-d4 and
[18F]FECNT at each time point (5, 15, 30, 60, and 120
min) in rats were shown in Fig. 3. After the rats were
severally injected with [18F]FECNT-d4 and [18F]FECNT, the
tracers were distributed primarily to the liver (15.81 0.34
% ID vs 16.88 1.46 % ID), lung (1.12 0.17 % ID vs 1.93
0.59 % ID), kidney (1.94 0.08 % ID vs 1.82 0.18 %
ID), and brain (1.72 0.10 % ID vs 1.66 0.20 % ID), and
lower accumulation in the bone, heart, small intestine,
pancreas, and spleen. Brain distribution showed that the
Ex vivo Autoradiography
The regional distribution of [18F]FECNT-d4 in rat brain was
further assessed by ex vivo autoradiography. As shown in
Fig. 4, [18F]FECNT-d4 was located substantially in the
caudate putamen (CPu), substantial nigra (SN), and nucleus
accumbens (NAc), with low concentration in the cerebellum
and cortex.
Table 1. Parent tracer and radiolabelled metabolite fractions in brain tissues and plasma of rats (% of total radioactivity, mean SD, n = 3)
Time
(min)
[
18F]FECNT-d4
[
18F]FECNT
Striatum
Cerebellum
Plasma
Striatum
Cerebellum
Plasma
Parent tracer
Metabolites
5
98 0.8 %
95 2 %
91 1 %
95 3 %
77 8 %
46 8 %
76 9 %
31 7 %
13 5 %
24 9 %
69 7 %
87 5 %
96 2 %
82 1 %
70 4 %
89 5 %
41 9 %
27 4 %
11 5 %
59 9 %
73 4 %
35 15 %
16 3 %
30
60
5
30
60
7
1 %
2
5
9
0.8 %
2 %
1 %
6
3 %
4
2 %
65 15 %
84 3 %
93 1 %
23 8 %
54 8 %
18 1 %
30 4 %

Cao S. et al.: Synthesis and Biological Evaluation
Table 2. In vitro binding affinity and lipophilicity of compounds FECNT-
d4 and FECNT
cross the BBB [30]. This argument was also supported by
the biodistribution of brain uptake. Biodistribution results
showed that [18F]FECNT-d4 was rapidly distributed to the
striatum but not retained in the regions without DAT. The
ratio of target to non-target (ST/CB) at equilibrium can be
used to evaluate specific binding in the target region of the
brain. A comparative study of the biodistribution of
[18F]FECNT-d4 and [18F]FECNT in rats displayed that the
two tracers have different brain metabolic trends (Fig. 3a, b).
The ST to CB ratio of [18F]FECNT-d4 was higher than
[18F]FECNT at each point except at 5 min (Suppl. Table 2
and 4, see ESM). A slower elimination rate of [18F]FECNT-
d4 than [18F]FECNT was also found in other high uptake
organs (Fig. 3c, d). These results indicated that
[18F]FECNT-d4 improved the ratio of target to non-target
and decelerated the drug’s degradation rate in vivo compared
to [18F]FECNT. Besides, we found that the bone uptakes in
biodistribution results were very low from 0 to 120 min for
both [18F]FECNT-d4 and [18F]FECNT, suggesting the
stability of [18F]fluoroethyl group with negligible in vivo
defluorination [18, 31].
Compound
IC50 (nM)
LogP7.4
FECNT-d4
2.17
2.37
(1.87–2.54)
1.99
(1.98–2.51)
2.25
FECNT
(1.70–2.34)
(2.12–2.39)
MicroPET Imaging
To confirm in vivo biological activity of [18F]FECNT-d4,
microPET imaging was performed with normal and PD
model rats. As shown in Fig. 5, in normal rats, prominent
radioactivity was found in the striatum and the concentration
could be inhibited to the background level by CFT.
Meanwhile, in 6-OHDA unilaterally lesioned brains, the
concentration of [18F]FECNT-d4 was noticeably reduced in
the lesioned side relative to the unlesioned side. Dynamic
TACs showed that the radioactivity of [18F]FECNT-d4 in the
striatum maintained high uptake with SUVs of 4.02 0.45 at
5–20 min, and the SUVs of [18F]FECNT reached 4.11
0.61 at 5 min and then declined rapidly. The volume of
interest analysis by the Logan reference tissue method
Then, the specific binding of [18F]FECNT-d4 to DAT
in vivo was confirmed using ex vivo autoradiography and
microPET imaging. Autoradiography in normal rats showed
high accumulation of [18F]FECNT-d4 in the CPu, NAc, and
SN, with abundant radioactivity in the symmetrical striata
and low uptake in the CB and cortex. The binding specificity
of [18F]FECNT-d4 was confirmed to be consistent with the
known localization of DAT in rodent brains [32]. Besides, in
unilateral PD model rats, an obvious reduction of
[18F]FECNT-d4 uptake was found in the lesioned hemi-
sphere relative to the unlesioned side (Fig. 4).
showed BPND of 1.95
0.22 (mean
SD, n = 5) for
[18F]FECNT-d4 and 1.65 0.51 (n = 5) for [18F]FECNT,
respectively.
Discussion
In this study, we synthesized [18F]FECNT-d4 as a PET
ligand for DAT based on the chemical structure of FECNT.
The novel agent [18F]FECNT-d4 presents a better target to
non-target ratio and in vivo stability than the previously used
contrast agent [18F]FECNT. However, its application as a
DAT radioligand in humans still needs further investigation.
The present study provides the basis for future research.
To preliminarily assess [18F]FECNT-d4 bioactivity
in vitro, we applied the radiotracer to the cellular uptake
assay. The cellular uptake of [18F]FECNT-d4 showed a
remarkable climbing tendency in 2 h, and the uptake of
[18F]FECNT-d4 after pretreatment with the DAT inhibitor
showed a 66 % tracer binding inhibition. However, the
radioactivity did not decline when incubated with VMAT2
ligand. The results initially demonstrated its bioactivity and
high selectivity for DAT (Suppl. Fig. 2, see ESM). Then, the
binding affinity of FECNT-d4 or FECNT to DAT was
determined using [18F]FECNT and rat striatum membrane
homogenates. The results showed that FECNT-d4 exhibited
a comparable affinity for DAT to FECNT in the nanomolar
range [10], which implied that binding affinity was almost
unaffected by hydrogen-deuterium substitution.
MicroPET imaging was performed on normal and PD
model rats to further evaluate the potential of [18F]FECNT-
d4 for DAT tracing (Fig. 5a). The resulting images were well
aligned with autoradiography which showed high tracer
uptake in regions of DAT-rich striata. The sufficient brain
uptake and rapid washout of non-target tissue of the tracer
showed in dynamic TAC were in agreement with
biodistribution results of brain tissue. The uptake of
[18F]FECNT-d4 in the striatum remained a relatively stable
high-plateau period at 5–20 min, whereas the uptake of
[18F]FECNT in the striatum began to decrease from 4 min
after injection (Fig. 5b). While co-injected with the well-
characterized specific DAT ligand, the uptake of
[18F]FECNT-d4 was mostly inhibited in the target, resulting
in the target to non-target ratio close to unity (ST/CB =
1.04). The BPND values of both radioligands were 1.95
0.22 for [18F]FECNT-d4 and 1.65 0.51 for [18F]FECNT.
The results indicated that the binding potential of
[18F]FECNT-d4 in the rat striatum was higher and more
stable than that of [18F]FECNT. The binding potential of
[18F]FECNT-d4 in rat striatum was comparable to that of
Lipophilicity is one of the factors limiting a drug’s ability
to cross the BBB. In agreement with our hypothesis, the
results of lipophilicity displayed that [18F]FECNT-d4 meets
the criteria of appropriate logP value (2 G logP G 3.5) to
another DAT imaging agent, [18F]FP-CMT (2.50
0.25)
[7]. A radiopharmaceutical of another tropane analogue,
[18F]LBT999, has a significantly higher BPND (4.39 0.31)

Cao S. et al.: Synthesis and Biological Evaluation
Fig. 3. Biodistribution of [18F]FECNT-d4 and [18F]FECNT in rats. a Brain data of [18F]FECNT-d4 and [18F]FECNT were
expressed as the percentage of injected dose per gram of tissue (% ID/g, n = 5, mean SD). b Organ data of [18F]FECNT-d4
and [18F]FECNT were expressed as the percentage of the injected dose per organ (% ID, n = 5, mean SD).
in rats [33] and investigated in patients with dopaminergic
impairment [34, 35]. However, the effects of metabolites on
BPND during prolonged scanning (4 h) are appreciable [36].
Although the binding potential was lower than some of other
DAT tracers, [18F]FECNT-d4 still showed good brain uptake
and target to non-target ratio, allowing good visualization of
DAT in striatal and extra-striatal regions.
After in vitro and in vivo biological studies, we analyzed
the metabolism of [18F]FECNT-d4 in vivo. A competent
radiotracer should be stable in vivo to retain its biological
activity. Similar to cocaine [37], the tropane derivatives
would undergo oxidative cleavage of the N-substituent and
other metabolic pathways in vivo. For tropane analogues,
CYP3A4 exhibited the highest catalytic activity for N-
dealkylation [38]. Indeed, in vivo stability studies in rats
showed that [18F]FECNT-d4 also transformed to polar
metabolite. The major metabolite was issued from N-
dealkylation, concomitantly with the departure of the
fluoroalkyl chain (formation of fluorine ethanol or its
oxidation product). Thirty minutes after the injection, around
69 % of the parent [18F]FECNT-d4 in plasma was
transformed, and such polar metabolites of [18F]FECNT
have been demonstrated in human and non-human primate
plasma [12]. Nevertheless, the metabolic results showed that
the parent fraction of [18F]FECNT-d4 in striatum (91 %) was
predominant up to 60 min post injection. At 30 min, there
were only 16 % of the unchanged [18F]FECNT in plasma
and 82 % in striatum. The metabolic stability of
Fig. 4. Representative ex vivo autoradiography of DAT binding sites in normal or PD model rats’ brains. The intensity of
18F]FECNT-d4 binding was well matched with the DAT expression in the brain. CPu, caudate putamen; NAc, nucleus
accumbens; SN, substantia nigra; CB, cerebellum.
[

Cao S. et al.: Synthesis and Biological Evaluation
Fig. 5. a Representative PET images of [18F]FECNT-d4 in brains of rats. Each PET image was generated by the summation of
data collected 10–40 min after injection. The white arrows indicate the striatum. b TACs of [18F]FECNT-d4 and [18F]FECNT were
obtained from regions of interest located on the striatum and cerebellum during 0–120 min and the TACs of [18F]FECNT-d4
were blocked by DAT ligand CFT. Values were generated at fixed times after injection during PET scan and shown as mean
SD, n = 5.
[18F]FECNT-d4 observed in rats was superior to that of
[18F]FECNT. Interestingly, we also found a more in vivo
stability of the tracers in mice than in rats, probably due to
the different kinetics of drug metabolism between the
species.
specific binding in the brain [41]. These results might confound
the quantitative analysis. The radiopharmaceutical of the same
chemical class, [18F]LBT999, appears more stable with a slow
transformation rate in vivo. There was 60 % unmetabolized
plasmatic fraction in humans at 30 min post injection [35],
which seems to facilitate clinical imaging. However, the ligand
[18F]LBT999 has the same behavior as the biotransformation
of PE2I, suggesting that its metabolites also affect PET images
and subsequent quantification [42]. Furthermore, the high bone
accumulation of the radioactivity implicated high
defluorination of [18F]LBT999 [15]. In recent years, in order
to overcome these limitations, great efforts have been made to
search for better DAT imaging agents. Although the tropane
derivative still undergoes metabolism after deuterium substitu-
tion, the present study showed that replacing the N-fluoroethyl
group of [18F]FECNT with an N-fluoroethyl-d4 could reduce
the oxidative cleavage of the N-substituent and led to a
significant improvement in the pharmacological profile of this
ligand.
Some other 18F-labelled tropane analogs targeting DAT,
such as [18F]FP-CMT, [18F]FE-PE2I and [18F]LBP999, have
been reported and their in vivo metabolism have been
investigated. Metabolite analysis of [18F]FP-CMT produced
only one hydrophilic polar metabolite in vivo [7]. The longer
carbon chain at the N-substituent seems to produce less
oxidative dealkylation in vivo [39]. However, the metabolic
analysis in rats displayed that [18F]FP-CMT has a fast
metabolic rate with ~88 % parent molecule transformed at
30 min post injection [7]. The polar metabolite was supposed to
be a 4-carboxy analogue of [18F]FP-CMT, but the metabolite’s
brain penetration ability was not be ascertained. Besides N-
dealkylation, [18F]FE-PE2I also suffers from hydroxylation
and benzylic hydroxylation to yield a series of brain-
penetrating metabolites [40]. The in vivo metabolism rate of
[18F]FE-PE2I appears rapid kinetics in humans, with 86 % of
the parent molecule transformed at 30 min post injection [41].
The prominent accumulation of major radiometabolite 4-
hydroxymethyl analogue in the striatum suggests a high
affinity for DAT as well as 4-carboxy analogue presents non-
Conclusion
In summary, we prepared and characterized a novel
deuterated compound [18F]FECNT-d4 based on the tropane
structure which was confirmed as a good candidate for DAT

Cao S. et al.: Synthesis and Biological Evaluation
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Foundation of China (81671723, 81801742), the Natural Science Founda-
tion of Jiangsu Province (BK20201133), Wuxi Municipal Science and
Technology Development Fund (WX18IIAN048, N20192015), Program
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Conflict of Interest
The authors declare that they have no conflict of interest.
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