Synthesis and biological evaluation of 10-11C- dihydrotetrabenazine as a vesicular monoamine transporter 2 radioligand

Article abstract of DOI:10.1002/ardp.201300307

In this study, we synthesized a new carbon-11-labeled radiotracer, 10- ¹¹C-dihydrotetrabenazine (10-¹¹C-DTBZ), and evaluated its potential as a vesicular monoamine transporter 2 (VMAT2) radioligand. The radiolabeled precursor 10-O-de

Full text of DOI:10.1002/ardp.201300307

Arch. Pharm. Chem. Life Sci. 2014, 347, 313–319
313
Full Paper
Synthesis and Biological Evaluation of
10-11C-Dihydrotetrabenazine as a Vesicular
Monoamine Transporter 2 Radioligand
Xiaomin Li, Zhengping Chen, Jie Tang, Chunyi Liu, Pei Zou, Hongbo Huang, Cheng Tan,
and Huixin Yu
Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine,
Jiangsu Institute of Nuclear Medicine, Wuxi, P. R. China
In this study, we synthesized a new carbon-11-labeled radiotracer, 10-11C-dihydrotetrabenazine (10-11C-
DTBZ), and evaluated its potential as a vesicular monoamine transporter 2 (VMAT2) radioligand. The
radiolabeled precursor 10-O-desmethyl-dihydrotetrabenazine (10-O-desmethyl-DTBZ) was prepared with
a six-step reaction using 3-methoxy-4-benzyloxybenzaldehyde as starting material. 10-11C-DTBZ was
synthesized by heating 1.0 mg of 10-hydroxy precursor and 11C-methyl iodide in the presence of 0.3 mL
of dimethyl sulfoxide and 4.0 mL of 3 N KOH at room temperature for 3 min. After purification by solid
phase extraction using an alumina Sep-Pak cartridge, the final 10-11C-DTBZ product was obtained with
a radiochemical purity of >99% and an uncorrected radiochemical yield of 18–26% (end of
bombardment (EOB), n ¼ 6). The overall synthesis time was approximately 20 min from the EOB to
release of the product for quality control. Using small-animal positron emission tomography
(microPET), the striatum of normal rats was found to exhibit symmetrical labeling (STR/STL ¼ 0.98
ꢀ 0.05, n ¼ 3) and the highest uptake of radioactivity (striatum/cerebellum, ST/CB ¼ 2.89 ꢀ 0.31 at
30–60 min, n ¼ 3). In contrast, rats with 6-hydroxydopamine unilateral lesions yielded asymmetrical
striatal images with a higher 10-11C-DTBZ concentration on the unlesioned side (STunlesioned
/
CB ¼ 2.53 ꢀ 0.18, at 30–60 min, n ¼ 3) compared with the lesioned side (STlesioned/CB ¼ 1.26 ꢀ 0.10,
n ¼ 3). These results suggest that 10-11C-DTBZ may represent a promising PET radiotracer for imaging
VMAT2.
Keywords: Carbon-11 / Dihydrotetrabenazine / Parkinson’s disease / Positron emission tomography / Vesicular
monoamine transporter
Received: August 15, 2013; Revised: November 14, 2013; Accepted: November 15, 2013
DOI 10.1002/ardp.201300307
Introduction
diagnose and monitor neurological and psychiatric disorders
such as Parkinson’s disease (PD), Huntington’s disease (HD),
Alzheimer’s disease (AD), and schizophrenia [1–4].
In the brain, vesicular monoamine transporter 2 (VMAT2) is
localized exclusively in presynaptic monoaminergic termi-
nals and is predominantly expressed in rodent and human
monoaminergic neurons of the central nervous system (CNS).
Therefore, the imaging of VMAT2 in the brain with positron
emission tomography (PET) enables the quantification of
neuronal densities, and this technique has been used to
In the past two decades, a wide variety of radioligands,
including tetrabenazine (TBZ) analogs, ketanserin analogs,
lobeline analogs, and 3-amine-2-phenylpropene analogs, have
been developed for in vitro and in vivo studies of VMAT2 in both
the animal and human brain [5–8]. Among these compounds,
analogs of TBZ (Nitoman1) are promising because of their
high, selective affinity for VMAT2, high cerebral accumula-
tion, suitable lipophilicity, short-lived pharmacological
effects, and low toxicity. Furthermore, these analogs have
been synthesized and used for in vivo brain imaging of
VMAT2 with PET, including [11C]tetrabenazine ([11C]TBZ),
Correspondence: Prof. Zhengping Chen, Key Laboratory of Nuclear
Medicine, Ministry of Health, Jiangsu Institute of Nuclear Medicine, No. 20
Qianrong Road, Wuxi 214063, P. R. China.
E-mail: chenzhengping@jsinm.org
Fax: þ86 0510 85513113
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

314
X. Li et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 313–319
Figure 1. Structures of DTBZ and 10-11C-DTBZ.
9-[11C]dihydrotetrabenazine (9-11C-DTBZ), [11C]methoxytetra-
benazine ([11C]MTBZ), and 9-[18F]fluoropropyl-(þ)-desmethyl-
dihydrotetrabenazine (9-[18F]FP-(þ)-DTBZ) [9–12].
(see Fig. 1), labeled on the 10-O-methyl position of the aromatic
ring, was synthesized and imaged using small-animal PET
(microPET) in rats to evaluate its potential as an in vivo
radioligand for imaging VMAT2.
TBZ is a well-characterized antipsychotic drug that has been
used clinically for more than five decades. In vivo, TBZ is
rapidly and extensively metabolized to its reduced form, Results and discussion
dihydrotetrabenazine (DTBZ, see Fig. 1). DTBZ has been
labeled with 3H on the C-2 hydrogen and successfully used as a
selective radioligand in in vitro brain homogenate binding
studies and in autoradiographic studies [13, 14]. DTBZ has also
been labeled with 11C on the 9-O-methyl group of the
aromatic ring and applied in the diagnosis and monitoring
of VMAT2-related disorders such as PD [15, 16].
Continuing the active research in this area, we herein
report the synthesis of a novel carbon-11-labeled DTBZ
derivative in order to explore the effect of 10-O-[11C]methoxyl
group on the specific brain uptake and retention, and
metabolism.
In this study, 3-isobutyl-9-methoxy-10-[11C]-methoxy-2,3,4,
6,7,11b-hexahydro-1H-pyrido[2,1-a]isoquinoline-2-ol (10-11C-DTBZ)
Chemistry
The radiolabeled precursor 3-isobutyl-9-methoxy-2,3,4,
6,7,11b-hexahydro-1H-pyrido[2,1-a]isoquinoline-2,10-diol (10-
O-desmethyl-DTBZ) was synthesized in a six-step reaction
from the starting material 3-methoxy-4-benzyloxybenzalde-
hyde. The target compound was identified by IR, ESI-MS, and
1H NMR, and the results agreed well with the expected
chemical structure. The general synthetic procedure for 10-O-
desmethyl-DTBZ is shown in Scheme 1.
The starting compound (1) was prepared in good yield (75%)
via a Knoevenage reaction of 3-methoxy-4-benzyloxybenzal-
dehyde, ammonium acetate, and nitromethane in acetic acid
under refluxing. Then, compound 1 was reacted further by a
Scheme 1. Synthesis of 10-O-desmethyl-
DTBZ. Reactions and conditions: (a) CH3NO2,
NH4Ac, HAc, 130°C, 2 h; (b) LiAlH4, THF, Et2O,
60°C, 2 h; (c) HMTA, HAc, TFA, 80°C, 2 h;
(d) 3-dimethylaminomethyl-5-methyl-hexan-2-
one, TEBAC, H2O, 90°C, 4 h; (e) NaBH4, EtOH,
r.t., 16 h; (f) H2, Pd/C, EtOH, r.t., 16 h.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 313–319
10-11C-Dihydrotetrabenazine as a VMAT2 Radioligand
315
Scheme 2. Radiochemical synthesis of 10-11C-DTBZ. Reagents and conditions: (g) 11C-CH3I, DMSO, 3 N KOH, r.t., 3 min.
LiAlH4 reduction reaction to yield compound 2. Compound 3
was created via the reaction of Bischler–Napieralski in the
presence of HMTA. We synthesized 3-dimethylaminomethyl-5-
methyl-hexan-2-one using our previously described pro-
cess [17]. Compound 4 was obtained by Michael addition at
90°C, which was catalyzed using triethylbenzylammonium
chloride (TEBAC) in water. The reduction of compound 4 in
the presence of NaBH4 yielded compound 5. Finally,
deprotection of the benzyl group of compound 5 via
hydrogenolysis using a Pd/C catalyst resulted in target
compound 6.
admissible error (Fig. 2). The radiochemical purity (RCP)
exceeded 99%, and the product was radiochemically stable for
at least 1 h. The chemical purity of 10-11C-DTBZ after
purification was greater than 95%. The specific radioactivity
(SA) was 45 ꢀ 14 GBq/mmoL (end of synthesis, n ¼ 6). It is
expected that further optimization of the process would raise
the value of SA.
MicroPET imaging
10-11C-DTBZ binding to VMAT2 in the brain was demonstrated
in vivo by performing microPET imaging in normal and
6-hydroxydopamine (6-OHDA) unilaterally lesioned rats.
Radiochemistry
Radiosynthesis from the 10-hydroxy precursor was conducted
as shown in Scheme 2. 10-11C-DTBZ was prepared according to
the reported procedure with some modifications [18].
A simple, rapid, semi-automated method for the synthesis
of 10-11C-DTBZ was developed. 11CO2 was produced by a
14N(p,a)11C nuclear reaction on a target of nitrogen gas using
a HM-7 cyclotron (Sumitomo Heavy Industries, Ltd., Japan).
The 11CO2 produced was transported to an automated
synthesis system of 11C-methyl iodide. 10-11C-DTBZ was
synthesized by rapid 10-O-[11C]methylation of compound 6
with 11C-methyl iodide as a methylation agent.
After purification by solid-phase extraction using an
alumina Sep-Pak cartridge, the final 10-11C-DTBZ product
was obtained. The radiochemical yield of 10-11C-DTBZ was 18–
26% (n ¼ 6) without decay correction at the end of bombard-
ment (EOB). Further optimization of the process may
successfully increase the total yield. The overall synthesis
time was approximately 20 min from EOB to release of the
product for quality control.
Quality control
Figure 2. RCP of 10-11C-DTBZ analyzed by HPLC. The chromato-
grams of purified 10-11C-DTBZ were determined with a radiation
detector, and the co-injection of DTBZ with a UV detector was
performed under the same HPLC analytical conditions. 10-11C-
DTBZ exhibited the same retention time as cold DTBZ.
The identity of 10-11C-DTBZ was confirmed by comparing the
retention time of the radioactive product with that of the cold
compound, DTBZ, by HPLC. The retention time of 10-11C-DTBZ
was 7.8 min, which matched well with that of DTBZ within an
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

316
X. Li et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 313–319
Figure 3. In vivo 10-11C-DTBZ microPET studies of normal and 6-OHDA-lesioned PD rats. PET images of 10-11C-DTBZ in the brains of
normal (a) and 6-OHDA-lesioned PD (b) rats. PET data were collected using an animal PET scanner with a transaxial resolution of 1.4 mm.
Each PET image was generated by the summation of data collected 8–60 min after injection.
Coronal microPET images of normal and unilateral 6-OHDA-
lesioned rat brains at 8–60 min after tail vein injection of
10-11C-DTBZ are shown in Fig. 3a and b, respectively.
MicroPET imaging with 10-11C-DTBZ in normal rats
produced high-quality images, as shown in Fig. 3a. A high
accumulation of radioactivity was observed in the striatum,
the site with the highest concentrations of VMAT2. In contrast
to the symmetrical uptake of the radioligand in the brains of
normal rats, in unilateral 6-OHDA-lesioned rats (see Fig. 3b),
the radioactivity of the tracer was localized to the unlesioned
side in distinct levels, whereas almost no visible uptake of
10-11C-DTBZ was observed on the lesioned side in microPET
images. These in vivo microPET results confirmed the affinity
of 10-11C-DTBZ to VMAT2.
cerebellum exhibiting maximal activity at ꢁ1 min. The egress
of radioactivity from brain regions was also very rapid, with
good differentiation of the striatum from the cerebellum
visible between
8 and 60 min after injection. In the
cerebellum, the time–activity curve showed a peak within
1 min post-injection and then fell off rapidly. In the
meanwhile, the striatum exhibited the highest uptake level
and good retention of the radioligand. The striatum to
cerebellum ratio (ST/CB) of radioactivity reached a maximum
value of 2.89 ꢀ 0.31 at 30 min after injection. In the microPET
images, the striatum of normal rats exhibited symmetrical
labeling (STR/STL ¼ 0.98 ꢀ 0.05, n ¼ 3) and the highest uptake
of radioactivity (ST/CB ¼ 2.89 ꢀ 0.31 at 30–60 min, n ¼ 3). In
contrast, 6-OHDA unilateral lesioned rats yielded asymmetri-
cal striatal images with a higher 10-11C-DTBZ concentration
on the unlesioned side (STunlesioned/CB ¼ 2.53 ꢀ 0.18, at 30–
Figure 4 shows time–radioactivity curves for the striatum
and cerebellum following tail vein injection of 10-11C-DTBZ.
After the injection of 10-11C-DTBZ, the radioactivity was
rapidly taken up by the brain, with the striatum and
60 min, n ¼ 3) compared with the lesioned side (STlesioned
CB ¼ 1.26 ꢀ 0.10, n ¼ 3).
/
Figure 4. Time–activity curves of 10-11C-DTBZ
in the brains of normal and 6-OHDA-lesioned
PD rats, expressed as the ID%/cc, for the right
striatum (STR), left striatum (STL), and cerebel-
lum (CB). PET data were collected for 60 min.
Regions of interest (ROIs) were identified
according to the stereotaxic atlas. The radio-
activities in the striatum and cerebellum were
plotted against the time post-injection.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 313–319
10-11C-Dihydrotetrabenazine as a VMAT2 Radioligand
317
These microPET images of rat brains indicated a high level
of uptake in the striatal regions rich in VMAT2 but low uptake
in the cerebellum. The time–activity curves of 10-11C-DTBZ
also revealed high-level accumulation in the striatum, with a
low level in the cerebellum of the rat brain. The rapid brain
uptake and clearance kinetics of 10-11C-DTBZ were similar to
those of many previously reported radioligands, such as
11C-TBZ, 11C-MTBZ, 9-11C-DTBZ, 9-11C-(þ)-DTBZ, 9-18F-FE-DTBZ,
and 9-18F-FP-DTBZ [19–23]. The striatum-to-cerebellum ratio
was maximal (2.89 ꢀ 0.31) at 30 min post-injection, which was
higher than that previously reported for 11C-MTBZ
(2.7 ꢀ 0.4) [20], 9-18F-FE-DTBZ (2.55) [24], and 11C-TBZ (2.85 ꢀ
0.52) [25] but lower than that for 9-11C-(þ)-DTBZ (5.24) [24] and
9-18F-FP-DTBZ (5.08 ꢀ 0.81) [26]. In addition, the uptake of
10-11C-DTBZ was sharply decreased in the striatum in the
lesioned side of 6-OHDA unilateral lesioned rats. These results
were consistent with many reports of VMAT2 imaging in
unilateral 6-OHDA-lesioned rats using 9-11C-(þ)-DTBZ, 9-18F-FP-
DTBZ, or a variety of radiolabeled VMAT2 ligands [26, 27].
Together, these results suggest that 10-11C-DTBZ may serve as
a radiotracer with favorable properties for quantifying brain
VAMT2 function with PET. Additional evaluations, such as
brain biodistribution studies, blocking experiments, and
metabolism profiles are in progress and will be reported in the
future.
(Bruker, Germany), and the chemical shift value was given
relative to the internal tetramethylsilane (TMS) standard.
Electron spray ion (ESI) mass spectra were determined using a
Waters Platform ZMD 4000 LC/MS (Waters, USA). IR data were
obtained using a Bruker Tensor 27 FT-IR infrared spectrophotom-
eter (Bruker, Germany). The high-performance liquid chromatog-
raphy (HPLC) analysis system for characterizing 10-11C-DTBZ
consisted of a binary HPLC pump (Waters 1525, USA), a UV
detector (Waters 2487, USA), and a flow scintillation analyzer
(Radiomatic 610TR, Perkin-Elmer, USA). All Sprague-Dawley rats
were supplied by the Shanghai SLAC Laboratory Animal Co., Ltd.
(Shanghai, China). The 6-OHDA was purchased from Sigma Co.,
and the left-sided 6-OHDA-lesioned PD rats were established
as previously described [29]. All the animal experiments were
carried out in compliance with the relevant national laws
associated with the conduct of animal experimentation.
Chemistry
3-Methoxy-4-benzyloxy-b-nitrostyrene (1)
Nitromethane (10.25 mL, 191 mmol) was added to a solution of 3-
methoxy-4-benzyloxybenzaldehyde (20.64 g, 85.3 mmol) and am-
monium acetate (6.45 g, 83.77 mmol) in acetic acid (66 mL) at
40°C. The reaction mixture was heated to reflux at 130°C for 2 h
and then cooled to room temperature. The crude product was
purified by crystallization with ethanol to produce compound 1
(17.9 g, 75%) as a yellow crystal with the following properties:
m.p.: 125–127°C; IR (KBr, cmꢂ1) n: 1624, 1595 (C C), 1513, 1334
–
–
(–NO2); 1H NMR (CDCl3, 400 MHz) d: 7.96–7.93 (d, 1H, J ¼ 13.6 Hz,
–
NO CH–), 7.53–7.49 (d, 1H, J ¼ 13.6 Hz, C CH–), 7.44–7.42 (d, 2H,
–
2
J ¼ 7.2 Hz, o-ArC–H), 7.40–7.37 (t, 2H, J ¼ 7.2 Hz, m-ArC–H), 7.34–
7.33 (d, 1H, J ¼ 7.2 Hz, p-ArC–H), 7.11–7.09 (dd, 1H, J ¼ 8.4, 2.0 Hz,
Ar–H), 7.03–7.02 (d, 1H, J ¼ 2.0 Hz, Ar–H), 6.93–6.91 (d, 1H, J ¼ 8.4
Hz, Ar–H), 5.22 (s, 2H, OCH2–), 3.93 (s, 3H, OCH3–); and MS (ESI)
m/z: 285.3, found: 286.1 [MþH]þ.
The tracer used in this study was a racemic mixture of the
(þ) and (ꢂ) enantiomers, and it was previously reported that
the in vitro binding of (ꢀ)-DTBZ was stereospecific, with a high
binding affinity (Ki ¼ 0.97 nM) only for (þ)-DTBZ [28]. Thus, an
improved target-to-nontarget ratio (ST/CB) of 10-11C-(þ)-DTBZ
should be expected, and further studies in the rodent brain
with 10-11C-(þ)-DTBZ are ongoing.
3-Methoxy-4-benzyloxyphenethylamine (2)
A solution of compound 1 (9.03 g, 31.68 mmol) in tetrahydrofuran
(160 mL) was added dropwise to a suspension of lithium
aluminum hydride (3.37 g, 88.72 mmol) in ethyl ether (80 mL)
and tetrahydrofuran (80 mL) at 0°C over a period of 1 h. The
reaction mixture was heated to reflux at 60°C for 2 h and then
cooled to room temperature. Then, water (6 mL) and tetrahydro-
furan (12 mL) were added, followed by 10% sodium hydroxide
solution (6 mL). The salts were removed by filtration, and the
filtrate was dried over anhydrous sodium sulfate. The solvent was
removed under reduced pressure, and the crude product was
purified by column chromatography on silica gel eluted with
100:20:0.2 dichloromethane/ethanol/triethylamine to produce
compound 2 (5.26 g, 65%) as a yellow oil with the following
properties: IR (KBr, cmꢂ1) n: 3361 (–NH2); 1H NMR (CDCl3,
400 MHz) d: 7.44–7.42 (d, 2H, J ¼ 7.2 Hz, o-ArC–H), 7.37–7.34 (t, 2H,
J ¼ 7.2 Hz, m-ArC–H), 7.30–7.29 (d, 1H, J ¼ 7.2 Hz, p-ArC–H), 6.83–
6.81 (d, 1H, J ¼ 8.0 Hz, Ar–H), 6.75–6.74 (d, 1H, J ¼ 2.0 Hz, Ar–H),
6.68–6.66 (dd, 1H, J ¼ 8.0, 2.0 Hz, Ar–H), 5.13 (s, 2H, OCH2–), 3.88
(s, 3H, OCH3–), 2.95–2.92 (t, 2H, J ¼ 6.8 Hz, Ar–CH2–), 2.69–2.66 (t,
2H, J ¼ 6.8 Hz, NH2–CH2–); and MS (ESI) m/z: 257.3, found: 258.1
[MþH]þ.
Conclusions
In this study, we designed, synthesized, and evaluated the
biological characteristics of a novel carbon-11-labeled DTBZ
derivative, 10-11C-DTBZ. 10-11C-DTBZ was synthesized by rapid
10-O-[11C]methylation of desmethyl-DTBZ with 11C-methyl
iodide and obtained at a high RCP. In vivo microPET studies
demonstrated that 10-11C-DTBZ showed high brain uptake
and high affinity and specificity for brain VMAT2. Therefore,
10-11C-DTBZ may serve as a probe for the in vivo PET imaging of
monoaminergic terminal loss associated with neurological
disease.
Experimental
All chemicals were commercially obtained and used without
further purification unless otherwise noted. Melting points were
measured using a Yanaco MP-S3 melting point apparatus
(Shimadzu, Japan). Nuclear magnetic resonance (NMR) spectra
were recorded on a Bruker AVANCE-III 400 MHz spectrometer
6-Methoxy-7-benzyloxy-3,4-dihydroisoquinoline (3)
Compound 2 (4.33 g, 16.85 mmol) was dissolved in acetic acid
(24 mL) and trifluoroacetic acid (6 mL), and hexamethylenetetramine
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

318
X. Li et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 313–319
(4.72 g, 33.67 mmol) was then added and stirred at room
temperature for 30 min. The reaction mixture was heated to reflux
at 80°C for 2 h and then cooled to room temperature. The cooled
solution was diluted with water (50 mL), basified with 20% sodium
hydroxide solution (50 mL), and extracted with dichloromethane
(3 ꢃ 100 mL). Organic extracts were combined and concentrated in
a vacuum. The crude product was purified by column chromatog-
raphy on silica gel eluted with 100:3 dichloromethane/ethanol to
produce compound 3 (2.9 g, 65%) as a pale yellow oil with the
following properties: 1H NMR (CDCl3, 400 MHz) d: 8.15 (s, 1H,
N–CH–), 7.46–7.44 (d, 2H, J¼ 7.2 Hz, o-ArC–H), 7.40–7.36 (t, 2H,
J¼ 7.2 Hz, m-ArC–H), 7.33–7.32 (d, 1H, J¼ 7.2 Hz, p-ArC–H), 6.83
(s, 1H, ph, quinoline-H), 6.69 (s, 1H, ph, quinoline-H), 5.15 (s, 2H,
OCH2–), 3.92 (s, 3H, OCH3–), 3.74–3.69 (m, 2H, NCH2CH2–), 2.69–2.65
(t, 2H, J¼ 7.8 Hz, N–CH2–); and MS (ESI) m/z: 267.3, found: 268.1
[MþH]þ.
3-Isobutyl-9-methoxy-2,3,4,6,7,11b-hexahydro-1H-
pyrido[2,1-a]isoquinoline-2,10-diol (10-O-desmethyl-
DTBZ) (6)
Compound 5 (0.33 g, 0.84 mmol) was dissolved in ethanol (50 mL),
followed by the addition of 10% palladium on carbon (0.1 g) and
the mixture was hydrogenated at room temperature for 16 h. The
reaction mixture was filtered, and the filtrate was dried over
anhydrous sodium sulfate. The solvent was removed under
reduced pressure, and the crude product was purified by column
chromatography on silica gel eluted with 100:4 dichloromethane/
methanol to produce compound 6 (0.16 g, 63%) as a pink solid
with the following properties: IR (KBr, cmꢂ1) n: 3099 (–OH), 2949
(–CH3); 1H NMR (CDCl3, 400 MHz) d: 6.74 (s, 1H, ph, quinolizine-H),
6.56 (s, 1H, ph, quinolizine-H), 3.85 (s, 3H, OCH3–), 3.41–3.34
(m, 1H, –CH–), 3.13–3.10 (m, 2H, –CH–), 3.06–2.99 (m, 2H, –CH–),
2.68–2.61 (m, 2H, –CH–), 2.56–2.43 (m, 2H, –CH–), 2.00–1.95 (t, 1H,
J ¼ 11.4 Hz, –CH–), 1.63–1.59 (m, 1H, –CH–), 1.52–1.46 (t, 2H,
J ¼ 11.8 Hz, –CH–), 1.08–1.01 (m, 1H, –CH(CH3)), 0.95–0.91 (dd, 6H,
J ¼ 6.4, 6.4 Hz, –CH3); and MS (ESI) m/z: 305.4, found: 306.1
[MþH]þ.
3-Isobutyl-9-methoxy-10-benzyloxy-2,3,4,6,7,11b-
hexahydro-1H-pyrido[2,1-a]isoquinoline-2-one
(10-BnO-TBZ) (4)
3-Dimethylaminomethyl-5-methyl-hexan-2-one (1.2 g, 6.55 mmol),
compound 3 (1.45 g, 5.4 mmol), and TEBAC (0.38 g, 1.65 mmol)
were dissolved in water (25 mL). The mixture was stirred and
heated under reflux at 90°C for 4 h. The solvent was then removed
under reduced pressure, and the crude product was purified
by crystallization with ethanol, which yielded compound 4
(1.04 g, 49%) as a yellow crystal with the following properties:
m.p.: 133–135°C; 1H NMR (CDCl3, 400 MHz) d: 7.43–7.41 (d, 2H,
J ¼ 7.2 Hz, o-ArC–H), 7.37–7.34 (t, 2H, J ¼ 7.2 Hz, m-ArC–H), 7.30–
7.28 (d, 1H, J ¼ 7.2 Hz, p-ArC–H), 6.64 (s, 1H, ph, quinolizine-H),
6.57 (s, 1H, ph, quinolizine-H), 5.09 (s, 2H, OCH2–), 3.86 (s, 3H,
OCH3–), 3.45–3.42 (m, 1H, –CH–), 3.28–3.24 (dd, J ¼ 6.4, 6.4 Hz,
1H, –CH–), 3.14–3.06 (m, 2H, –CH–), 2.77–2.65 (m, 3H, –CH–),
2.62–2.54 (m, 1H, –CH–), 2.46–2.40 (t, 1H, J ¼ 12.4 Hz, –CH–), 2.35–
2.29 (t, 1H, J ¼ 11.8 Hz, –CH–), 1.82–1.75 (m, 1H, –CH–), 1.70–1.67
(m, 1H, –CH–), 1.05–0.98 (m, 1H, –CH(CH3)), 0.92–0.89 (dd, 6H,
J ¼ 5.2, 5.2 Hz, –CH3); and MS (ESI) m/z: 393.5, found: 394.2
[MþH]þ.
3-Isobutyl-9-methoxy-10-[11C]-methoxy -2,3,4,6,7,11b-
hexahydro-1H-pyrido[2,1-a]isoquinoline-2-ol
(10-11C-DTBZ) (7)
Precursor product 6 (1.0 mg) was dissolved in dimethyl sulfoxide
(0.3 mL) with 3 N potassium hydroxide (4.0 mL). The 11C-methyl
iodide synthesized was transferred to the reaction vessel via a
nitrogen stream. After reacting at room temperature for 3 min,
the reaction mixture was transferred and loaded onto an alumina
Sep-Pak cartridge (previously activated with 7 mL of ethyl ether
with 1% ethanol). The Sep-Pak cartridge was washed with 1 mL of
ethyl ether with 1% ethanol to remove the radioactive impurities
and then eluted with 4 mL of ethyl ether with 1% ethanol. The
solution was blown dry with N2 for 2 min to yield 10-11C-DTBZ.
Product 7 was diluted with saline and stored for further use.
Quality control
The RCP of 10-11C-DTBZ was determined by radio-HPLC using a
Waters 600-type HPLC system with a Symmetry C18 reversed-
phase column (5 mm, 4.6 mm ꢃ 150 mm, Waters). 10-11C-DTBZ
was isocratically eluted with acetonitrile and 0.05 M ammonium
acetate (20:80 v/v, pH 4.0) at a flow rate of 0.8 mL/min and then
identified by co-injection with the unlabeled reference compound
DTBZ. DTBZ was monitored with a UV detector at 280 nm. The
chemical purity of 10-11C-DTBZ after purification was determined
by HPLC with UV detection at 280 nm.
3-Isobutyl-9-methoxy-10-benzyloxy-2,3,4,6,7,11b-
hexahydro-1H-pyrido[2,1-a]isoquinoline-2-ol
(10-BnO-DTBZ) (5)
Compound 4 (1.0 g, 2.54 mmol) was dissolved in ethanol (60 mL),
and sodium borohydride (0.97 g, 25.4 mmol) was then added and
stirred at room temperature for 16 h. The mixture was diluted
with water (50 mL) and dichloromethane (50 mL) and then
extracted with dichloromethane (2 ꢃ 50 mL). Organic extracts
were combined and concentrated in a vacuum. The crude product
was purified by crystallization with methanol to produce
compound 5 (0.67 g, 67%) as a white crystal with the following
properties: m.p.: 178–180°C; 1H NMR (CDCl3, 400 MHz) d: 7.44–
7.42 (d, 2H, J ¼ 7.2 Hz, o-ArC–H), 7.38–7.34 (t, 2H, J ¼ 7.2 Hz,
m-ArC–H), 7.31–7.29 (d, 1H, J ¼ 7.2 Hz, p-ArC–H), 6.71 (s, 1H, ph,
quinolizine-H), 6.60 (s, 1H, ph, quinolizine-H), 5.10 (s, 2H, OCH2–),
3.85 (s, 3H, OCH3–), 3.38–3.32 (m, 1H, –CH–), 3.13–2.93 (m, 4H,
–CH–), 2.65–2.61 (m, 1H, –CH–), 2.47–2.40 (m, 1H, –CH–), 1.98–
1.92 (t, 1H, J ¼ 11.4 Hz, –CH–), 1.74–1.66 (m, 2H, –CH–), 1.58–1.36
(m, 3H, –CH–), 1.08–1.01 (m, 1H, –CH(CH3)), 0.94–0.90 (dd, 6H,
J ¼ 6.4, 6.4 Hz, –CH3); and MS (ESI) m/z: 395.5, found: 396.2
[MþH]þ.
MicroPET imaging
Imaging was performed using a Siemens Inveon 5000 microPET
system (Siemens, Germany). The microPET scanner had an
effective axial field of view (FOV) of 12.7 cm. The resolution at
the center of the FOV was 1.4 mm. Three normal rats and three 6-
OHDA unilaterally lesioned rats were used for PET image
acquisition. Rats were anesthetized with 1.5% isoflurane in
oxygen, positioned on the bed of the microPET gantry, and fixed
near the center of the scanner. Isoflurane anesthesia was
continued throughout the study. Each rat was injected
intravenously via the tail vein with an average of 29.4 MBq
(0.8 mCi) of 10-11C-DTBZ in saline. The scan duration was 60 min
from the time of injection.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 313–319
10-11C-Dihydrotetrabenazine as a VMAT2 Radioligand
319
Scans were obtained in the frame sequence 6 ꢃ 10 s, 6 ꢃ 60 s,
4 ꢃ 120 s, and 9 ꢃ 300 s. The data were histogrammed into 25
consecutive frames, and the images were reconstructed using a
three-dimensional ordered subsets expectation maximum (OSEM)
algorithm. Normalization and scatter correction were performed
during the reconstructions. Images were analyzed with ASIProVM
(Concorde Microsystems, USA). Frames from 8 to 60 min were
summed to manually draw regions of interest (ROIs). ROIs were
drawn manually from two brain regions (striatum and cerebel-
lum) on coronal slices guided by a mouse brain stereotaxic atlas,
and time–activity curves (TACS) for the ROIs were obtained.
Radioactivity concentrations (nCi/cc) were normalized to the
injected dose of the radiotracer.
[10] R. A. Koeppe, K. A. Frey, A. Kume, R. Albin, M. R. Kilbourn,
D. E. Kuhl, J. Cereb. Blood Flow Metab. 1997, 17, 919–931.
[11] M. R. Kilbourn, P. S. Sherman, L. C. Abbott, Nucl. Med. Biol.
1995, 22, 565–567.
[12] L. Zhu, Y. J. Liu, K. Plössl, B. Lieberman, J. Liu, H. F. Kung, Nucl.
Med. Biol. 2010, 37, 133–141.
[13] D. Scherman, R. Raisman, A. Ploska, Y. Agid, J. Neurochem.
1988, 50, 1131–1136.
[14] M. R. Kilbourn, P. S. Sherman, Eur. J. Pharmacol. 1997, 331,
161–168.
[15] M. R. Kilbourn, E. R. Butch, T. Desmond, P. Sherman,
P. E. Harris, K. A. Frey, Nucl. Med. Biol. 2010, 37, 3–8.
[16] R. A. Koeppe, S. Gilman, L. Junck, K. Wernette, K. A. Frey,
The authors are very grateful to the National Natural Science
Foundation of China (30970844), Natural Science Foundation of
Jiangsu Province (BK2010155), Outstanding Professionals Foundation
of Jiangsu Health Bureau (RC2011096) and Public Service Platform for
Science and Technology Infrastructure Construction Project of Jiangsu
Province (BM2012066) for their financial support.
Alzheimers Dement. 2008, 4, S67–S76.
[17] X. M. Li, Z. P. Chen, C. Y. Liu, J. Tang, J. Chem. Ind. Eng. 2012, 63,
567–571.
[18] G. Quincoces, M. Collantes, R. Catalan, M. Ecay, E. Prieto,
E. Martino, F. J. Blesa, L. Álvarez-Erviti, P. Areses, J. Arbizu,
J. A. Obeso, J. M. Martí-Climent, J. A. Richter, I. Peñuelas, Rev.
Esp. Med. Nucl. 2008, 27, 13–21.
[19] J. N. DaSilva, M. R. Kilbourn, E. F. Domino, Synapse 1993, 14,
128–131.
The authors have declared no conflict of interest.
[20] T. M. Vanden Borght, M. R. Kilbourn, R. A. Koeppe,
J. N. DaSilva, J. E. Carey, D. E. Kuhl, K. A. Frey, J. Nucl. Med.
1995, 36, 2252–2260.
References
[21] G. L. Chan, J. E. Holden, A. J. Stoessl, A. Samii, D. J. Doudet,
T. Dobko, K. S. Morrison, M. Adam, M. Schulzer, D. B. Calne,
T. J. Ruth, J. Nucl. Med. 1999, 40, 283–289.
[1] D. C. German, P. K. Sonsalla, Adv. Behav. Biol. 2003, 54, 131–
137.
[22] J. M. Zhang, X. J. Zhang, Y. G. Li, J. H. Tian, Molecules 2012, 17,
6697–6704.
[2] N. I. Bohnen, R. A. Koeppe, P. Meyer, E. Ficaro, K. Wernette,
M. R. Kilbourn, D. E. Kuhl, K. A. Frey, R. L. Albin, Neurology
2000, 54, 1753–1759.
[23] R. Goswami, D. E. Ponde, M. P. Kung, C. Hou, M. R. Kilbourn,
H. F. Kung, Nucl. Med. Biol. 2006, 33, 685–694.
[3] S. Gilman, R. A. Koeppe, R. Little, H. An, L. Junck, B. Giordani,
C. Persad, M. Heumann, K. Wernette, Ann. Neurol. 2004, 55,
774–780.
[24] M. R. Kilbourn, B. Hockley, L. C. Lee, C. Hou, R. Goswami,
D. E. Ponde, M. P. Kung, H. F. Kung, Nucl. Med. Biol. 2007, 34,
233–237.
[4] J. K. Zubieta, S. F. Taylor, P. Huguelet, R. A. Koeppe,
M. R. Kilbourn, K. A. Frey, Biol. Psychiatry 2001, 49, 110–116.
[25] J. N. DaSilva, M. R. Kilbourn, Life Sci. 1992, 51, 593–600.
[5] J. N. DaSilva, J. E. Carey, P. S. Sherman, T. J. Pisani,
[26] Z. P. Chen, C. Y. Liu, X. M. Li, J. Tang, C. Tan, H. B. Huang,
M. R. Kilbourn, Nucl. Med. Biol. 1994, 21, 151–156.
H. X. Yu, S. N. Luo, Nucl. Sci. Technol. 2012, 23, 40–46.
[6] J. P. Henry, B. Gasnier, M. F. Isambert, F. Darchen,
[27] M. Collantes, I. Penuelas, L. Alvarez-Erviti, J. Blesa, J. M. Marti-
Climent, G. Quincoces, M. Delgado, M. Ecay, A. Martinez,
J. Arbizu, M. C. Rodriguez-Oroz, J. Obeso, J. A. Richter, Rev. Esp.
Med. Nucl. 2008, 27, 103–111.
D. Scherman, Adv. Biosci. 1991, 82, 147–150.
[7] D. K. Miller, P. A. Crooks, G. Zheng, V. P. Grinevich,
S. Norrholm, L. P. Dwoskin, J. Pharmacol. Exp. Ther. 2004, 310,
1035–1045.
[28] M. R. Kilbourn, L. C. Lee, T. M. Vanden Borght, D. M. Jewett,
K. A. Frey, Eur. J. Pharmacol. 1995, 278, 249–252.
[8] R. P. Perera, D. S. Wimalasena, K. Wimalasena, J. Med. Chem.
2003, 46, 2599–2605.
[29] S. P. Wang, Z. P. Chen, X. M. Li, J. Tang, C. Y. Liu, M. F. Zou,
D. H. Pan, C. X. Lu, Y. P. Xu, X. J. Xu, X. Q. Zhou, J. Jin, Nucl. Sci.
Technol. 2009, 20, 11–16.
[9] M. R. Kilbourn, J. N. DaSilva, K. A. Frey, R. A. Koeppe,
D. E. Kuhl, J. Neurochem. 1993, 60, 2315–2318.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com