Synthesis and X-ray analysis of dihydrotetrabenazine, a metabolite of tetrabenazine

Article abstract of DOI:10.1080/15421406.2012.661315

The dihydrotetrabenazine (DTBZ) was synthesized by reduction of tetrabenazine with sodium borohydride in ethanol. Crystals suitable for X-ray analysis were obtained from a mixed solution of dichloromethane and ethanol in a five-to-one volume ratio. The crystal is monoclinic, space group P ₂₁c with crystallographic parameters: a = 15.0129(14), b = 12.5677(12), c = 9.7715(9), = 98.556(2), = 0.078 mm¹, V = 1823.1(3) ³, Z = 4, Dc = 1.164 g/cm³, F(000) = 696, T = 296(2) K. The X-ray analysis and the chiral HPLC show that the DTBZ prepared by our method consists of (2R,3R,11bR) and (2S,3S,11bS) enantiomers. Taylor and Francis Group, LLC.

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Synthesis and X-ray Analysis of
Dihydrotetrabenazine, a Metabolite of
Tetrabenazine
a
a
a
a
Chunyi Liu , Zhengping Chen , Xiaomin Li & Jie Tang
a
Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu
Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of
Nuclear Medicine, Wuxi, China
Published online: 30 Jul 2012.
To cite this article: Chunyi Liu , Zhengping Chen , Xiaomin Li & Jie Tang (2012) Synthesis and X-
ray Analysis of Dihydrotetrabenazine, a Metabolite of Tetrabenazine, Molecular Crystals and Liquid
Crystals, 562:1, 191-199, DOI: 10.1080/15421406.2012.661315
To link to this article: http://dx.doi.org/10.1080/15421406.2012.661315
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Mol. Cryst. Liq. Cryst., Vol. 562: pp. 191–199, 2012
Copyright © Jiangsu Institute of Nuclear Medicine
ISSN: 1542-1406 print/1563-5287 online
DOI: 10.1080/15421406.2012.661315
Synthesis and X-ray Analysis of
Dihydrotetrabenazine, a Metabolite of Tetrabenazine
∗
CHUNYI LIU, ZHENGPING CHEN, XIAOMIN LI,
AND JIE TANG
Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key
Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear
Medicine, Wuxi, China
The dihydrotetrabenazine (DTBZ) was synthesized by reduction of tetrabenazine with
sodium borohydride in ethanol. Crystals suitable for X-ray analysis were obtained
from a mixed solution of dichloromethane and ethanol in a five-to-one volume ratio.
The crystal is monoclinic, space group P2
1
/c with crystallographic parameters: a =
3
´
´˚
´˚
◦
−1
,
1
5.0129(14) A, b = 12.5677(12) A, c = 9.7715(9) A, β = 98.556(2) , µ = 0.078 mm
´
3
V = 1823.1(3) A , Z = 4, Dc = 1.164 g/cm , F(000) = 696, T = 296(2) K. The X-ray
analysis and the chiral HPLC show that the DTBZ prepared by our method consists of
(2R,3R,11bR) and (2S,3S,11bS) enantiomers.
Keywords Chiral center; dihydrotetrabenazine; enantiomer; tetrabenazine
Introduction
Tetrabenazine (TBZ) is a clinically used drug and it is a relatively safe and effective treat-
ment for a wide variety of hyperkinetic movement disorders, particularly tardive dyskinesia
(TD) and chorea. Dihydrotetrabenazine (DTBZ) has been identified as the pharmacologi-
cally active metabolite of TBZ. In mammalian, TBZ is rapidly and extensively metabolized
by reduction of the 2-keto group, producing DTBZ [1,2]. DTBZ is a high-affinity ligand for
the vesicular monoamine transporter 2 (VMAT2) in rodent and human brain neurons [3].
Therefore, imaging VMAT2 in the brain with radiolabeled DTBZ or its derivatives provides
a measurement reflecting the integrity of monoaminergic neurons, and so as to monitor or
diagnose neurodegenerative disorders such as Parkinson’s, Alzheimer’s, Tourette’s, and
Huntington’s diseases [4]. In view of this, DTBZ and its derivatives have recently been la-
beled with carbon-11 and fluorine-18 radioisotopes and used for in vitro and in vivo studies
1
1
18
of the VMAT2 in animal and human brain. For example, C-DTBZ [5–7], F-FE-DTBZ
[
18
8], and F-FP-DTBZ [9–11] have been prepared as the positron emission tomography
PET) imaging agents targeting VMAT2. In order to synthesize and examine the novel
radiolabeled DTBZ’s derivatives, namely F-FB-DTBZ, gram quantities of DTBZ were
(
18
∗
Address correspondence to Zhengping Chen, Key Laboratory of Nuclear Medicine, Ministry
of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear
Medicine, 20 Qianrong Road, Wuxi, Jiangsu 214063, P.R. China. Tel.: +86-0510-85514482, Ext.
3523; Fax: +86-0510-85513113. E-mail: lcy seu@yahoo.com.cn
191

192
C. Liu et al.
H CO
3
H
CO
3
N
NaBH4
EtOH
N
H CO
3
H CO
3
O
OH
TBZ
DTBZ
Scheme 1. Synthesis of DTBZ.
required. Reported herein is the synthesis of DTBZ as shown in Scheme 1. The objective
1
product was characterized by H NMR, IR, and MS.
The DTBZ has three chiral centers at C-2, C-3, and C-11b as shown in Fig. 1. Therefore,
in theory, there are eight isomers of the DTBZ. X-ray diffraction and chiral HPLC were
employed to investigate the structural groups of the isomers of the DTBZ prepared by our
group.
Experimental
Materials and Physical Measurements
The TBZ was synthesized according to literature methods [12,13]. All other solvents and
reagents were of analytical grade and were used without further purification. IR spectra in
−1
the range 4000–400 cm were obtained from samples in the form of KBr tablets using a
Bruker Tensor27 infrared spectrometer. MS spectra were recorded with a Waters Platform
ZMD 4000 mass spectrometer. Proton NMR spectra were obtained on a Bruker Avance
III 400 MHz Digital NMR Spectrometer in CDCl3. A Bruker CCD APEX2 diffractometer
was used for the X-ray structure study.
Synthesis of Dihydrotetrabenazine (DTBZ)
Tetrabenazine (2.92 g, 9.20 mmol) was dissolved in anhydrous ethanol (240 mL) with
heating. The resulting solution was stirred and cooled to room temperature; then sodium
borohydride (3.50 g, 92 mmol) was added. The reaction mixture was stirred at room
temperature for 12 h under nitrogen. The reaction solvent was removed under reduced
8
7
H CO9
3
6
1
0
N
H CO
3
4
3
1
1
11b
1
2
OH
Figure 1. Structure of numbered DTBZ.

Synthesis and X-ray Analysis of DTBZ
193
pressure. The residue was partitioned between dichloromethane (50 mL) and water (50 mL).
The layers were separated, and the aqueous layer was extracted with dichloromethane (2 ×
25 mL). Organic extracts were combined and concentrated in vacuum. The crude product
was purified by crystallization with methanol giving DTBZ (1.50 g) as colorless crystals in
+
+
−1
a moderate yield (51%). MS, m/z: 320.1 [M+H] ; 342.1 [M+Na] . IR (KBr, cm ): 3138;
1
2
3
1
952; 2911; 1609; 1512; 1463. H NMR (CDCl3, 400 MHz), δ: 6.70 (s, 1H), 6.60 (s, 1H),
.86 (s, 6H), 3.45–3.37 (m, 1H), 3.16–2.99 (m, 4H), 2.68–2.42 (m, 3H), 2.03–1.97 (t, 1H),
.81–1.68 (m, 2H), 1.64–1.47 (m, 3H), 1.12–1.05 (m, 1H), 0.97–0.93 (dd, 6H).
The colorless single crystal of DTBZ was grown from a mixed solution of
dichloromethane and ethanol in a five-to-one volume ratio by slow evaporation at room
temperature.
X-ray Crystallography
A single crystal of the DTBZ with dimensions 0.23 × 0.20 × 0.17 mm was chosen for
X-ray diffraction study. The data were collected on a Bruker APEX2 diffractometer
equipped with graphite-monochromatic Mo-Kα radiation (λ = 0.71073 Å) by using ψ and
◦
◦
ω scan mode at 296(2) K. In the range of 1.37 < θ < 25.00 , a total of 9900 reflections were
collected, of which 3201 were independent (Rint = 0.0380) and 2699 were observed with
I > 2σ(I).
The structure was solved by direct and difference Fourier map methods with SHELXS-
2
97 [14]. Non-hydrogen atoms were refined by full-matrix least-squares techniques on F
with anisotropic thermal parameters, using SHELXL-97 [15]. All H atoms were allowed to
´
´
˚
˚
ride on their parent atoms at distances of 0.93 A (C H aromatic), 0.96 A (C H methyl),
0
´
´
˚
˚
.97 A (C H methylene), and 0.98 A (C H methine) with Uiso(H) values of 1.2–1.5 times
Ueq of the parent atoms.
Results and Discussion
Synthesis of Dihydrotetrabenazine (DTBZ)
The DTBZ was synthesized through procedures described in the literature [16]. However,
the synthesis steps were improved on the basis of the literature. The literature method was
◦
◦
as follows. The reaction mixture was stirred at 0 C–3 C for 3 h under N2. Our approach was
that the mixed solution was stirred at room temperature for 12 h under nitrogen. Therefore,
convenient operation was achieved through the slight optimization of reaction conditions.
Spectroscopic Characterization
−1
The infrared spectra of the DTBZ exhibits a broad band of large intensity at 3138 cm ,
which is attributed to the stretching vibration of the hydroxyl radical ( OH). The DTBZ
−
1
shows strong absorption bands at 2952 and 2911 cm , with a shoulder at approximately
2
−1
900 cm due mainly to the C H stretching vibration of the methyl and methylene groups
present in the DTBZ molecule. The C C skeletal vibration of the benzene ring in the DTBZ
−
1
molecule appears at 1609, 1512, and 1463 cm .
1
The H NMR spectrum of the DTBZ in CDCl3 solution shows two singlet peaks for
the benzene ring hydrogen atoms ( PhH) at δ 6.70 and 6.60 ppm. The hydrogen atoms
of the methoxy groups ( OCH3) bonded to the aromatic ring in the DTBZ is observed in

194
C. Liu et al.
Figure 2. The molecular structures of DTBZ enantiomers; 30% probability ellipsoids are shown.
1
the H NMR spectrum as a singlet, exhibiting at δ 3.86 ppm. The two doublets at δ 0.97∼
0.93 ppm is assigned to the hydrogen atoms of the methyl groups ( CH3). The hydroxyl
radical ( OH) is at δ 2.03–1.97 ppm. On the other hand, the signals referred to the hydrogen
atoms of the methylene and methine groups ( CH2R; CHR2) appear at 3.45–3.37,
3.16–2.99, 2.68–2.42, 1.81–1.68, 1.64–1.47, and 1.12–1.05 ppm respectively.
Crystal Structure
The DTBZ crystallized in the monoclinic system, space group P21/c with crystallographic
´
´
´
˚
◦
˚
˚
parameters: a = 15.0129(14) A, b = 12.5677(12) A, c = 9.7715(9) A, β = 98.556(2) ,
−1
´˚ 3
3
µ = 0.078 mm , V = 1823.1(3) A , Z = 4, Dc = 1.164 g/cm , F(000) = 696, T = 296(2)
K. The molecular structure of DTBZ is shown in Fig. 2 and a perspective view of the crystal
packing in the unit cell is shown in Fig. 3.
There are three rings in the structure of DTBZ, namely benzene ring and two nitrogen-
containing six-membered rings. They do not share a common plane. The benzene ring is a
plane conformation while the other two rings are chair conformations.
The DTBZ synthesized by our team is a racemic form. According to the molecular
ellipsoid graph of DTBZ in Fig. 2, the single crystal of DTBZ consists of (2R,3R,11bR)
and (2S,3S,11bS) enantiomers. The crystal data and refinement details are listed in Table 1.

Synthesis and X-ray Analysis of DTBZ
195
Figure 3. Packing of the DTBZ molecules viewed along the c axis.
The selected bond lengths and bond angles are listed in Table 2. The interatomic distances
´
´
˚
˚
of conjugated C C bonds in the benzene ring vary from 1.368(3) A to 1.407(3) A. Those
´
˚
´
˚
of other C C bonds range from 1.441(7) A to 1.531(3) A. The bond lengths of C N and
C O single bonds vary in the range of 1.467(2) to 1.481(2) A and 1.364(3) to 1.425(3)
A, respectively. These data are in accordance with the typical bond lengths in organic
´
˚
´
˚
compounds [17].
There are two different patterns of the stacked arrangements in general aromatic
systems for π–π interactions: offset face-to-face and edge-to-face packing. The distance
´
˚
between aromatic ring centroids with the range of 3.3–3.8 A is the valid value of π–π
interactions in both the patterns [18]. The shortest distance between two benzene ring
´
˚
centroids is 5.891 A in the crystal stacking of DTBZ as shown in Fig. 4. Obviously, in
the crystal packing, there is no π–π interaction, and the molecules of DTBZ are mainly
stabilized by Van der Waals forces and hydrogen bonds.

196
C. Liu et al.
Table 1. Crystallographic data and structure refinement details
CCDC deposit number
Compound name
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal size (mm)
Crystal system
Space group
831592
Dihydrotetrabenazine
C19H29NO3
319.43
296 (2)
0.71073
0.23 × 0.20 × 0.17
Monoclinic
P21/c
a (Å)
15.0129(14)
b (Å)
12.5677(12)
c (Å)
9.7715(9)
90.00
98.556(2)
90.00
◦
α ( )
◦
β ( )
◦
γ ( )
V (Å )
3
1823.1(3)
Z
4
−3
Dca.(g·cm )
1.164
696
0.078
F(000)
−1
Absorption coeff. (mm )
θ range ( )
◦
1.37–25.00
Index ranges
−17 ≤ h ≤ 14; −14 ≤ k ≤ 14; −8 ≤ l ≤ 11
Reflections collected
Independent reflections
Observed reflections
Data/restraints/parameters
Goodness-of-fit on F2
R, wR indices [I > 2σ(I)]
R, wR indices (all data)
9900
3201 [Rint = 0.0380]
2699
3201/18/214
1.255
0.0603, 0.1799
0.0740, 0.2024
0.734, −0.377
−3
Largest diff. peak and hole (e·Å )
◦
Table 2. Selected bond lengths (Å) and angles ( )
Bond lengths
C2–N1
C16–O1
C2–C3
1.481(2)
1.379(2)
1.521(3)
1.531(3)
C11–O3
C3–C4
C2–C6
1.415(2)
1.396(3)
1.523(3)
1.497(5)
C12–C18
C20–C22
Bond angles
C16–O1–C21
C16–C4–C3
C8–C17–C10
C12–C18–C20
114.08(18)
122.51(19)
112.95(17)
117.5(2)
C7–N1–C2
O3–C11–C12
C6–C11–C12
C22–C20–C23
109.60(14)
109.15(16)
110.55(15)
109.5(4)

Synthesis and X-ray Analysis of DTBZ
197
Figure 4. The shortest distance between two benzene ring centroids in the crystal stacking of DTBZ.
Configuration of Dihydrotetrabenazine
In theory, there are eight isomers of the DTBZ, namely (2R,3R,11bR),
(2S,3S,11bS), (2R,3R,11bS), (2S,3S,11bR), (2R,3S,11bR), (2S,3R,11bS), (2S,3R,11bR),
and (2R,3S,11bS). The DTBZ was synthesized by using racemic TBZ as raw material, which
was a racemic mixture of (3R,11bR)-TBZ and (3S,11bS)-TBZ. Therefore, the DTBZ pre-
pared by our method contains a maximum of four isomers. The X-ray diffraction of the sin-
gle crystal indicated that the precipitated crystal contains (2R,3R,11bR) and (2S,3S,11bS)
enantiomers. As the single crystal X-ray diffraction analysis of the sample is only in a
crystal, the result is not universal. Therefore, the analysis of DTBZ was investigated on a
chiral HPLC column (Phenomenex Chirex (S)-Val and (R)-NEA; 250 × 4.6 mm) using
0
0
0
.30
.25
.20
AU 0.15
0
0
0
.10
.05
.00
5
.00
10.00
15.00
20.00
Minutes
Figure 5. The chiral HPLC analysis of DTBZ.

198
C. Liu et al.
isocratic 95% A/5% B at 1.0 mL/min with ultraviolet (UV) detection at 280 nm with sol-
vent A being hexane/1,2-dichloroethane (2:1) and solvent B being 0.1% trifluoroacetic acid
(TFA) ethanol solution. The result is shown in Fig. 5. By this means, we can determine that
our DTBZ consists of two isomers. Finally, we can infer that the DTBZ obtained from our
group consists of (2R,3R,11bR) and (2S,3S,11bS) enantiomers.
Conclusions
1
The DTBZ was characterized by HNMR, IR, MS, and X-ray diffraction. By means of
X-ray diffraction and chiral HPLC, we confirmed that the DTBZ prepared by our method
consists of (2R,3R,11bR) and (2S,3S,11bS) enantiomers.
Supplementary Material
CCDC-831592 contains the supplementary crystallographic data for this paper. These data
can be obtained free of charge via www.ccdc.cam.ac.uk/data request/cif, by e-mailing
at data request@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic Data
Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44(0)1223−336033.
Acknowledgment
We are grateful for the financial support provided by the National Natural Science Founda-
tion of China (30970844) and the Outstanding Professionals Foundation of Jiangsu Health
Bureau (RC2011096). This work was also supported in part by the Natural Science Foun-
dation of Jiangsu Province (BK2010155) and the Youth Research Foundation of Jiangsu
Institute of Nuclear Medicine (QN201109).
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