Preparation and characterization of tetrabenazine enantiomers against vesicular monoamine transporter 2

Article abstract of DOI:10.1021/ml1000189

As a clinical medication for the treatment of hyperkinetic movement disorders, in conditions such as Huntington's disease, tetrabenazine (TBZ) has always been used in its racemic form. To establish whether or not its beneficial therapeutic actions are enantiospecific, a practical total synthetic route was developed to yield each enantiomeric form to allow their chemical and pharmacological characterization. We briefly summarize the total synthesis of TBZ and report a detailed procedure for resolution of TBZ into its enantiomers, (+)-TBZ and (-)-TBZ. This allowed determination of the optical rotation and absolute configurations of each TBZ enantiomer, based on X-ray crystallographic analysis, together with characterization of their inhibitory action at the vesicular monoamine transporter 2, where (+)-TBZ proved 3-fold more active than (-)-TBZ.

Full text of DOI:10.1021/ml1000189

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Preparation and Characterization of Tetrabenazine
Enantiomers against Vesicular Monoamine Transporter 2
Qian-sheng Yu,† Weiming Luo,† Jeffery Deschamps,‡ Harold W. Holloway,† Theresa Kopajtic,§
Jonathan L. Katz,§ Arnold Brossi, and Nigel H. Greig*,†
†Drug Design & Development Section, Laboratory of Neurosciences, Intramural Research Program, National Institute on Aging,
National Institutes of Health, 251 Bayview Boulevard, Baltimore, Maryland 21224, ‡Laboratory for the Structure of Matter,
Department of the Navy, Naval Research Laboratory, Washington, D.C. 20375, §Psychobiology Section, Intramural Research
Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, Maryland 21224, and School of Pharmacy,
University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
ABSTRACT As a clinical medication for the treatment of hyperkinetic movement
disorders, in conditions such as Huntington's disease, tetrabenazine (TBZ) has
always been used in its racemic form. To establish whether or not its beneficial
therapeutic actions are enantiospecific, a practical total synthetic route was
developed to yield each enantiomeric form to allow their chemical and pharma-
cological characterization. We briefly summarize the total synthesis of TBZ and
report a detailed procedure for resolution of TBZ into its enantiomers, (þ)-TBZ and
(-)-TBZ. This allowed determination of the optical rotation and absolute config-
urations of each TBZ enantiomer, based on X-ray crystallographic analysis,
together with characterization of their inhibitory action at the vesicular mono-
amine transporter 2, where (þ)-TBZ proved 3-fold more active than (-)-TBZ.
KEYWORDS Tetrabenazine enantiomers, vesicular monoamine transporter 2
(VMAT2), Huntington's chorea, hyperkinesias
etrabenazine (TBZ) (8), also known as Ro 1-9569,1
Nitoman (Hoffmann-La Roche, Nutley, NJ), and Xena-
approach to reducing the risk of potential side effects has
been to combine TBZ with other neuroleptic medications.3
T
zine (Lundbeck, Deerfield, IL), is a benzoquinolizine
The structure of TBZ utilized as a medication, with the
exception of some of its derivatives, is a racemic form.3,7-9
The medicinally active enantiomeric form of TBZ remains to
be determined, and synthesis and characterization of each
may allow more effective use of this increasingly used drug.
An approach to obtain optically active TBZ can be initiated
by catalytic asymmetric condensation of dihydro-isoquino-
line (2) and desired malonate to give an enantiomeric
excessive intermediate as described in the literature.10 Be-
cause our objective was to characterize both enantiomers,
the classical resolution of racemic TBZ seemed more effi-
cient for our study. Moreover, Openshaw and Whittaker11
have described the racemization of benzo[R]quinolizine, the
primary ring structure of TBZ enantiomers 12 or 13, via
isoquinolinium structure 9. Hence, asymmetric synthesis
may encounter racemization midway within the syn-
thetic pathway and may provide a final product that lacks
optical purity.
derivative with the chemical name 1,3,4,6,7,11b-hexahydro-
9,10-dimethoxy-3-(2-methylpropyl)-2H-benzo[a]quinolizin-
2-one. Originally developed as an antipsychotic agent in the
1950s,2 it has proved more valuable for other indications, as
in the treatment of disorders characterized by excessive
involuntary movement.3,4 These hyperkinesias have a largely
unknown pathophysiology, appear to involve neurotransmit-
ter dysfunction, and include tremor, dystonia, ballism, tics,
akathisia, stereotypies, chorea, myoclonus, and athetosis.
TBZ depletes brain monoamine levels, including serotonin,
dopamine, and norepinephrine. It achieves this by reversibly
binding to the vesicular monoamine transporter 2 (VMAT2)5,6
to inhibit monoamine uptake into granular vesicles of pre-
synaptic neurons and, thereby, augment their degradation by
monoamine oxidases within the cytoplasm. Despite five
decades of medical history and approval for use in Britain in
1971, TBZ was only recently approved (August 15, 2008) by
the FDA in the United States as the first drug to treat chorea
associated with Huntington's disease.7
There are two known primary methods for the synthesis
of TBZ: the cyclization of tetrahydro-isoquinoline deriva-
tives1 or the condensation of a 3,4-dihydro-isoquinoline
Whereas TBZ has been shown to be efficacious for the
treatment of a variety of disabling hyperkinetic movement
disorders, it has potential side effects, such as dysphasia,
depression, sedation, parkinsonism, and others that are
common to neuroleptic and psychotic medications.8,9 One
Received Date: January 22, 2010
Accepted Date: March 20, 2010
Published on Web Date: March 31, 2010
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Scheme 1. Synthesis of TBZ Enantiomers
derivative with β-amino ketone (7).12 A brief description of
the synthesis of TBZ based on the second condensation
method is illustrated in Scheme 1.
6,7-Dimethoxy-3,4-dihydro-quinoline (2) was obtained by
catalytic oxidization of 6,7-dimethoxy-1,2,3,4-tertahydro-
quinoline (1) in almost quantitative yield, according to a
known procedure in the literature.13 Compound 2 was
separated from the reaction mixture by forming a solid
fumarate14 to avoid evaporation of the solvent, decane,
which has a high boiling point.
Ethyl R-isobutyl-acetoacetate (5) was generated by con-
densation of ethyl acetoacetate (3) with 1-iodo-2-methylpro-
pane (4) in a solution of Na/EtOH. The specific conditions
utilized were adapted from the literature,15 following their use
in the synthesis of a compound similar to 5. After work up,
final distillation of the reaction mixture by a short column
provided relatively pure compound 516 (97 °C/15 mg). Follow-
ing the procedure for the synthesis of 3-dialkylaminomethyl-
alkane-2-ones and their methiodides,12 compound 5 was
reacted with formaldehyde and dimethylamine hydrochlor-
ide; thereafter, decarbonylation gave compound 617 that then
was reacted with iodomethane to provide compound 7.18 In
accord with the literature,12 compounds 2 and 7 (1:1 mol)
were refluxed in EtOH overnight to generate a crude product
that was then chromatographed and crystallized to provide
pure racemic TBZ (8).19 Under such a condition, only thermo-
dynamically stable enantiomers (8) were formed, as shown in
the 1H NMR of compound 8. No other diastereomers, such as
epimers, were found to be present. Finally, adaptation of the
methodology developed to successfully resolve racemic 2-
oxo-3-ethyl-benzo[R]qinolizine11 allowed the generation of
(þ)-TBZ (12) and (-)-TBZ (13), as detailed below.
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Figure 1. Structure and absolute configuration of (1S)-(þ)-10-camphorsulfonate of (þ)-TBZ (10) provided by X-ray crystallography: The
absolute configuration was determined unambiguously from the anomalous data [Flack = -0.02(7)]. The absolute configuration as
reported by PLATON is C3 = R, N5 = S, C12 = R, C10 = S (from acid), and C40 = R (also from acid). For additional details, see the Supporting
Information.
A solution of TBZ (317.4 mg, 1 mmol) and (1S)-(þ)-10-
Table 1. VMAT2 Binding Affinity of TBZ Enantiomeric Forms
camphorsulfonic acid (232.3 mg, 1 mmol) in ethyl acetate
(10 mL), together with a small magnetic stirring bar, was placed
in a sealed tube. This was heated while stirring in an oil bath at
80 °C for 28 h, and the resulting solution was set aside at room
temperature overnight. Filtration provided crystalline (1S)-(þ)-
Ki value ( SEM
(nM)a
compound
11
10
8
(1R)-(-)-10-camphorsulfonate of (-)-TBZ
(1S)-(þ)-10-camphorsulfonate of (þ)-TBZ
(()-TBZ
11.20 ( 1.03
4.61 ( 0.31
8.07 ( 0.20
23
10-camphorsulfonate (400 mg, 0.7 mmol, 70%), [R]D þ20.0
(c 0.35, EtOH), which was then dissolved in acetone (10 mL)
and kept at room temperature for 2 days. Filtration gave the
crystalline (1S)-(þ)-10-camphorsulfonate of (þ)-TBZ (10) (200
a Mean ( standard error of the mean (N = 3); each is statistically
different from the others ( p < 0.05, Bonferonni t test).
23
mg, 0.35 mmol, 35%): prism, mp 132-133 °C, [R]D þ36.00
(c 0.36, EtOH). Anal. (C29H43NO7S.H2O) C, H, N. These trans-
parent prism crystals were recrystallized two times from
acetone, and the [R] value was determined to be constant.
X-ray crystallography analysis of the optically pure compound
gave an unambiguous absolute configuration of 10, which is
shown in Figure 1. The salt, 10, was transferred to base, 12, bya
routine procedure: dissolved in water, basified by aqueous Na2-
CO3, extracted by ethyl ether, and dried over Na2SO4. The
resulting ether solution was evaporated to remove ether and
provide the crystalline base of (þ)-TBZ (12): prism, mp
macologically relevant nanomolar range, the (þ)-form (10)
proved to be 3-fold more potent than the (-)-enantiomer
(11) (p < 0.05, Bonferonni t test). As expected, the Ki value
of the racemic form, 8, was midway between those of the
pure enantiomers and was in accord with the literature.5,6
The efficacy of TBZ in a variety of hyperkinetic movement
disorders demonstrates that the pharmacological regulation
of VMAT2 function in humans can have clinical benefits and
can be well-tolerated.3,4,7-9 Additionally, studies of VMAT2
heterozygote knockout mice in stimulant place conditioning
(e.g., reward) are attenuated, suggesting a role of VMAT2 in
mediating the behavioral effects of abused psychostimulant
drugs20-23 and furthering interest in VMAT2 as a drug target.
In vivo, TBZ is rapidly and extensively hepatically meta-
bolized to its reduced form, DHTBZ, in both humans
and rodents.24,25 DHTBZ, like TBZ, has a high affinity for
VMAT2,5,6,22 and its concentrations over time following TBZ
administration to humans have been reported to be 148-fold
greater than that of TBZ.24 As the affinity of DHTBZ is similar
to that of (()-TBZ (8), the actions of TBZ are likely mediated
primarily by its metabolite(s). Consequent to the chiral
centers within clinically available (()-TBZ (8), at C-3 and
C-11b, several stereoisomers can be generated in vivo, each
of which may then give rise to further metabolites.24,25
Synthesis, separation, and characterization of several of
these have determined that for R-DHTBZ, VMAT2 affinity is
highly enantioselective, with the (þ)-isomer possessing high
affinity (0.97 (0.48 nM)26,27 and the (-)-isomer low affinity
23
108-110 °C (mp 126 °C, lit.10), [R]D þ71.02 (c 0.5, EtOH),
23
23
[R]D þ67.60 (c 0.4, CH2Cl2) {[R]D þ37.2 (c 0.4, CH2Cl2),
lit.10}. 1H NMRand MS (CI) are identical with that of compound
8. Anal. (C19H27NO3) C, H, N.
Using (1R)-(-)-10-camphorsulfonic acid, instead of (1S)-
(þ)-10-camphorsulfonic acid, the same procedures gave the
(1R)-(-)-10-camphorsulfonate of (-)-TBZ (11): prism, mp
23
132-133 °C, [R]D -36.01 (c 0.35, EtOH). Anal. (C29H43
-
NO7S.H2O) C, H, N. The base of (-)-TBZ (13): prism, mp
108-110 °C, [R]D23 -71.12 (c 0.5, EtOH), [R]D23 -67.75 (c
0.3, CH2Cl2). 1H NMR and MS (CI) are identical with that of
compound 8. Anal. (C19H27NO3) C, H, N.
Assessment of VMAT2 binding affinity was undertaken
by quantifying displacement of [3H]dihydrotetrabenazine
([3H]DHTBZ) from rat striatum by compounds (Supporting
Information) and is shown as a dissociation constant (Ki)
value in Table 1. Whereas both enantiomeric forms of TBZ
possessed high affinities for VMAT2 that were in the phar-
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(2.2 ( 0.3 μM).26,27 The VMAT2 affinity of (þ)-R-DHTBZ is
both in the range of (þ)-TBZ, reported herein, and shows
similar chiral preference with a far greater (2000-fold)
selectivity. The use of enantiomerically pure (þ)-TBZ in the
clinic may reduce the reported high individual variability in
the pharmacokinetics and metabolism associated with (()-
TBZ in humans24,25 and optimize its benefits in hyperkinetic
movement disorders as well as other conditions in which
inhibition of VMAT2 is efficacious.
(11) Openshaw, H. T.; Whittaker, N. The synthesis of emetine and
related compounds. Part V. A stereochemically favourable
synthesis of emetine. J. Chem. Soc. 1963, 1461–1471.
(12) Openshaw, H. T.; Whittaker, N. The synthesis of emetine and
related compounds. Part IV. A new synthesis of 3-substituted
1,2,3,4,6,7-hexahydro-9,10-dimethoxy-2-oxo-11bH-benzo[a]-
quinolizines. J. Chem. Soc. 1963, 1449–1460.
(13) Murahashi, S.; Naota, T.; Taki, H. Ruthenium-catalysed oxida-
tion of secondary amines to imines using t-butyl hydroper-
oxide. J. Chem. Soc. Chem. Commun. 1985, 613.
(14) Fumarate of compound 2: 1H NMR (300 MHz, DMSO): δ =
2.55 (t, 2H, -CH2-), 2.65 (t, 2H, -CH2-Nd) 3.82 (s, 3H,
-OCH3), 3.85 (s,3H, -OCH3), 6.60 (s, 1H, -CHdN), 6.85 (s,
1H, Ar-H), 7.05 (s, 1H, Ar-H), 8.20 (s, 1H, NHþ). Base of
compound 2: MS (CI) m/z 192 (M þ 1)þ.
SUPPORTING INFORMATION AVAILABLE Elemental ana-
lysis, X-ray crystallography of 10, and VMAT2 binding. This material
is available free of charge via the Internet at http://pubs.acs.org.
(15) Organic Syntheses; Wiley & Sons: New York, 1941; Collect.
Vol. I, p 248.
AUTHOR INFORMATION
Corresponding Author: *To whom correspondence should be
addressed. E-mail: greign@mail.nih.gov.
(16) Compound 5: 1H NMR (300 MHz, CDCl3): δ = 0.90 (d, 6H,
2-CH3), 1.30 (t, 3H, -CH3), 1.50-1.80 (m, 3H, -CH2-CH<),
2.20 (s, 3H, -CO-CH3), 3.45 (t, 1H, -CO-CH-CO-), 4.16
(q, 2H, -OCH2-). MS (CI) m/z 187 (M þ 1)þ.
1
(17) Compound 6: H NMR (300 MHz, CDCl3): δ = 0.89 (d, 3H,
Funding Sources: This work was supported in part by the
Intramural Research Programs of the National Institute on
Aging and National Institute on Drug Abuse, National Institutes of
Health.
-CH3), 0.92 (d, 3H, -CH3), 1.12-1.55 (m, 3H, -CH2-CH<),
2.13 (s, 3H, -CO-CH3), 2.16 [s, 6H, -N(CH3)2], 2.12-2.75
(m, 3H, -CO-CH-CH2-N<). MS (CI) m/z 172 (M þ 1)þ.
(18) Compound 7: mp 170-171 °C. 1H NMR (300 MHz, DMSO):
δ = 0.92 (d, 3H, -CH3), 0.98 (d, 3H, -CH3), 1.10-1.70 (m,
3H, -CH2-CH<), 2.28 (s, 3H, -CO-CH3), 3.02 [s, 9H,
-Nþ(CH3)3I-], 2.12-2.75 (m, 3H, -CO-CH-CH2-N).
(19) Compound 8: mp 128 °C. 1H NMR (300 MHz, DMSO): δ =
0.90 (d, J = 5.0 Hz, 3H, -CH3), 0.92 (d, J = 5.0 Hz, 3H,
-CH3), 1.05 (m, 1H, -CH<), 1.65 (m, 1H, -CH-), 1.80 (m,
1H, -CH-), 2.35 (dd, J = 12.0, 12.0 Hz, 1H, -CH-), 2.54
(m, 1H, -CH-), 2.58 (m, 1H, -CH-), 2.71 (m, 1H, -CH-),
2.74 (m, 1H, -CH-), 2.90 (dd, J = 15.0, 3.0 Hz, 1H, -CH-),
3.09 (m, 1H, -CH-), 3.14 (m, 1H, -CH-), 3.28 (dd, J =
12.0, 6.0 Hz, 1H, -CH-), 3.50 (d, J = 12.0 Hz, 1H, -CH-),
3.82 (s, 3H, -CO-CH3), 3.88 (s, 3H, -CO-CH3), 6.54 (s, 1H,
Ar-H), 6.61 (s, 1H, Ar-H). MS (CI) m/z 318 (M þ 1)þ.
(20) Takahashi, N.; Miner, L. L.; Sora, I.; Ujike, H.; Revay, R. S.;
Kostic, V.; Jackson-Lewis, V.; Prezedborski, S.; Uhl, G. R.
VMAT2 knockout mice: Heterozygotes display reduced am-
phetamine conditioned reward, enhanced amphetamine
locomotion and enhanced MPTP toxicity. Proc. Natl. Acad.
Sci. U.S.A. 1997, 94, 9938–9943.
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