In vitro and in vivo studies of benzisoquinoline ligands for the brain synaptic vesicle monoamine transporter

Article abstract of DOI:10.1021/jm950117b

Tetrabenazine is a high-affinity inhibitor of the vesicular monoamine transporter in mammalian brain. As part of a program to develop in vivo imaging agents for these transporters in human brain, a series of 2-alkylated dihydrototrabenazine ligands was synthesized and evaluated ia vitro and in vivo for binding to the brain vesicular monoamine transporter. Additions of organometallic reagents to tetrabenazine produced 2-methyl, 2-ethyl, 2-n- propyl, 2-isopropyl, and 2-isobutyl derivatives of dihydrotetrabenazine. The stereochemistry and conformation of the addition products were thoroughly verified by two-dimensional NMR techniques. All of these alkyl derivatives displayed in vitro affinity for the vesicular monoamine transporter binding site in rat brain using competitive assays with the radioligand [³H]methoxytetrabenazine. Except for the isopropyl derivative, all compounds when tested at 10 mg/kg iv showed an ability to inhibit in vivo accumulation of the radioligand [¹¹C]methoxytetrabenazine in the mouse brain striatum. Derivatives with small alkyl groups (methyl, ethyl) were more effective than those with large groups (propyl, isobutyl). These studies suggest that large groups in the 2-position of the benzisoquinoline structure will significantly diminish both in vitro and in vivo binding of these compounds to the vesicular monoamine transporter.

Full text of DOI:10.1021/jm950117b

J . Med. Chem. 1996, 39, 191-196
191
In Vitr o a n d in Vivo Stu d ies of Ben zisoqu in olin e Liga n d s for th e Br a in Syn a p tic
Vesicle Mon oa m in e Tr a n sp or ter
Lihsueh C. Lee, Thierry Vander Borght, Phillip S. Sherman, Kirk A. Frey, and Michael R. Kilbourn*
Division of Nuclear Medicine, Department of Internal Medicine, University of Michigan Medical School,
Ann Arbor, Michigan 48109
Received February 13, 1995X
Tetrabenazine is a high-affinity inhibitor of the vesicular monoamine transporter in mammalian
brain. As part of a program to develop in vivo imaging agents for these transporters in human
brain, a series of 2-alkylated dihydrotetrabenazine ligands was synthesized and evaluated in
vitro and in vivo for binding to the brain vesicular monoamine transporter. Additions of
organometallic reagents to tetrabenazine produced 2-methyl, 2-ethyl, 2-n-propyl, 2-isopropyl,
and 2-isobutyl derivatives of dihydrotetrabenazine. The stereochemistry and conformation of
the addition products were thoroughly verified by two-dimensional NMR techniques. All of
these alkyl derivatives displayed in vitro affinity for the vesicular monoamine transporter
binding site in rat brain using competitive assays with the radioligand [3H]methoxytetra-
benazine. Except for the isopropyl derivative, all compounds when tested at 10 mg/kg iv showed
an ability to inhibit in vivo accumulation of the radioligand [11C]methoxytetrabenazine in the
mouse brain striatum. Derivatives with small alkyl groups (methyl, ethyl) were more effective
than those with large groups (propyl, isobutyl). These studies suggest that large groups in
the 2-position of the benzisoquinoline structure will significantly diminish both in vitro and in
vivo binding of these compounds to the vesicular monoamine transporter.
In tr od u ction
The brain vesicular monoamine transporter (VMAT2)
is an integral component of monoaminergic neurons and
functions to transport newly synthesized or recovered
monoamine neurotransmitters from the cytosol into the
lumen of the synaptic storage vesicles.1 The synaptic
vesicular transporter of human brain has been recently
cloned and sequenced and its chromosomal location
identified.2,3 The ontogeny, pharmacology, and regional
brain distribution of VMAT2 in mammalian brain
including humans have been extensively studied using
the tritiated radioligand R-[3H]dihydrotetrabenazine
(2R-hydroxy-3-isobutyl-1,2,3,4,6,7-hexahydro-11bH-ben-
zo[a]quinolizine, 1a ).4 Determination of VMAT2 bind-
F igu r e 1.
ing sites has been proposed as a measure of mono-
aminergic neuronal density, based on demonstrated
2 have similar binding affinities for VMAT2.12 As part
of our development of radioligands for in vivo emission
tomographic imaging and quantification of VMAT2 in
the human brain, we have become interested in the
structure-activity relationship of benzisoquinoline bind-
ing to the VMAT2. Our goal is the preparation of
benzisoquinolines labeled with positron- and single-
photon-emitting radionuclides for in vivo imaging using
PET and SPECT (single-photon emission-computed
tomography). For development of SPECT radioligands
in particular, molecular modifications to accommodate
an iodine atom or a chelating group for a metal radio-
nuclide (e.g., 99mTc) are necessary; such substituents add
considerable steric bulk and lipophilicity to an existing
chemical structure. Previous attempts to modify the
structure of 1a to include a iodine-bearing group have
produced a radioligand suitable for in vitro studies of
the VMAT2 site but have not led to successful in vivo
radioligands.13 Development of new imaging agents for
the vesicular monoamine transporter will require a
better knowledge of the structural requirements and
limitations for high-affinity binding to the VMAT2. A
reductions in VMAT2 sites in animal models of neuro-
degenerative diseases and in postmortem samples of
patients with Parkinson’s disease.5 Recently, we pro-
posed the use of radiolabeled benzisoquinolines related
to tetrabenazine (2-oxo-3-isobutyl-1,2,3,4,6,7-hexahydro-
11bH-benzo[a]quinolizine, 2) as potential in vivo imag-
ing agents for quantification of VMAT2 sites in living
human brain using positron emission tomography (PET).6
Using the benzisoquinolines 1a , 2 and methoxytetra-
benazine (2R-methoxy-3-isobutyl-1,2,3,4,6,7-hexahydro-
11bH-benzo[a]quinolizine, 3) (Figure 1) isotopically
labeled with carbon-11 (â+, t1/2 ) 20.4 min), studies in
monkeys and humans have demonstrated the feasibility
of imaging and quantifying this transporter using
PET.7-10
Tetrabenazine (2) has a long history of clinical use,
and its in vivo effects on monoamine concentrations in
the brain have been extensively studied.11 In vivo, 2 is
rapidly and extensively metabolized to 1a , which is
likely the active pharmacological agent; in vitro, 1a and
X Abstract published in Advance ACS Abstracts, November 1, 1995.
0022-2623/96/1839-0191$12.00/0 © 1996 American Chemical Society

192 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1
Lee et al.
Sch em e 1. Synthesis of 2-Alkyldihydrotetrabenazine
Derivatives
the alcohol can be observed during Grignard reagent
additions to ketones.16
The reduction of 2 using NaBH4 at 0 °C produced
R-dihydrotetrabenazine (1a ) and â-dihydrotetraben-
azine (1b), in an 4:1 ratio. Two recrystallizations
provided 1a in 99% purity as determined by HPLC.
Flash chromatography was used to isolate the 1b isomer
from the residue.
St r u ct u r a l Assign m en t s a n d Con for m a t ion b y
NMR. A variety of NMR spectroscopic tools were
employed to unequivocally determine the relative con-
figurations and ring conformations for compounds 1-8.
These methods included acquisition of spectra using the
experimental techniques of HETCOR (1H-13C hetero-
nuclear shift-correlated spectroscopy), GATEDEC (gated
decoupled J -spectroscopy), J MODXH (heteronuclear
J -modulation spectroscopy), COSY (1H-1H chemical
shift-correlated spectroscopy), and NOESY (nuclear
Overhauser enhancement chemical shift-correlated spec-
troscopy). It was usually possible to assign the complete
1H NMR spectrum of these compounds based on HET-
COR experiments only, as most of the peaks were well
separated on the 13C NMR spectrum. In most cases,
however, 13C NMR J MODXH experiments were also
performed to eliminate problems related to the 13C NMR
signals that were too close to each other. COSY spectra
were then obtained to confirm spectral assignments.
Finally, NOESY experiments allowed the complete
assignment of the relative configurations and confirmed
the assigned conformation for the ring structure. Our
NMR studies were in agreement with previous inves-
tigations of such benzisoquinolines and related struc-
tures.17-24
number of benzisoquinolines have been prepared over
the years, including (a) analogs of 2 with different
3-alkyl substituents,11 (b) derivatives of 1a with alkyl
substituents at the 2-position,11,13 and (c) benzisoquino-
line analogs with amino, amido, and ester functional
groups at the 2-position.12,14 There has, however, never
been a systematic study of the in vitro or in vivo
structure-activity relationship of benzisoquinoline bind-
ing to the VMAT2 site (or, for that matter, any system-
atic study of the binding site requirement for VMAT2).
Alterations of the structure of 2 do have profound effects
on its biological activity; some modifications increase
pharmacologic activity,11 while others produce dif-
ferential depletions of certain monoamines.15 As an
initial investigation of the effects of adding substituents
to the benzisoquinoline structure, we report here the
synthesis, structural characterization, in vitro binding
affinities, and in vivo biological activities of a series of
alkylated derivatives of dihydrotetrabenazine.
The combined NMR experiments allowed assignment
of the 2-ethyl, n-propyl, isopropyl, and isobutyl groups
as being trans to the isobutyl group at the 3-position
and a B,C ring conformation unchanged from that of
the parent compounds 1 and 2. For the sterically
smaller methyl groups, both isomers are formed in the
addition reaction, again with no alterations in the
conformation of the ring system. For all compounds
synthesized, these NMR analyses indicated that the
relative configurations of substituents on the carbon
atoms at positions C-3 and C-11b were maintained
during the Grignard addition reactions.
Resu lts a n d Discu ssion
Syn th eses of 2-Alk yld ih yd r otetr a ben a zin es. The
2-alkyldihydrotetrabenazines 4-8 (Scheme 1) were
prepared by the addition of Grignard or organolithium
reagents (RMgX or RLi; R ) Me, Et, n-Pr, i-Pr, i-Bu; X
) Cl, Br) to the ketone group of 2. Crude products were
purified by flash column chromatography. As confirmed
by spectroscopic data (see following sections), these
derivatives retained the same relative configurations at
carbons C-3 and C-11b and the trans B/C ring junction
as found in the parent compounds 1 and 2. In the
addition of an organometallic reagent to the ketone of
2, two isomers can be formed, with the incoming alkyl
group assuming either the axial or equatorial position.
These isomers are designated the â- and R-isomers,
respectively. For such additions, the major product of
the reactions would be expected to have the R group in
the sterically less hindered, equatorial (R) position. For
addition of CH3Li, both the R- and the â-methyl isomers
were obtained with the major product being 3a in a ratio
of about 3:1. Increasing the chain length (ethyl, n-
propyl, i-propyl, i-butyl, and i-pentyl) resulted in the
isolation of only the R-alkyl-â-hydroxy isomers (only one
isomer apparent from TLC and NMR analyses); whether
the other isomers were formed in minor amounts during
the addition reactions was not determined. With in-
creasing size of the alkyl group, additional side products
were obtained which were identified as a mixture of the
two isomers of 1, the result of simple reduction of the
ketone of 2. This was not surprising, as reduction to
We have recently shown that R-dihydrotetrabenazine,
synthesized by reduction of commercially available
tetrabenazine, is a mixture of two enantiomers. Only
one of the two isomers is biologically active in either in
vitro or in vivo assays of radioligand binding to the
vesicular monoamine transporter of mouse or human
brain.25,26 All the benzisoquinolines we have synthe-
sized here from tetrabenazine are thus racemic mix-
tures.
In Vitr o Stu d ies. The in vitro Ki values for inhibi-
tion of [3H]methoxytetrabenazine binding to the VMAT2
of rat striatum27 are shown in Table 1 for 2 and the six
alkylated derivatives 4-8 prepared in this study. The
highest affinity was shown by the â-methyl compound
3a with a general decrease in binding affinity upon
either lengthening or branching of the carbon chain.
Stereoselectivity of binding was shown between the two
isomers 4a ,b, with a nearly 5-fold higher affinity found
for 4a . For comparison, literature IC50 values for
inhibition of [3H]dihydrotetrabenazine binding in rat

SAR of Ligands for Vesicular Monoamine Transporters
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1 193
Ta ble 1. In Vitro Ki Values of Benzisoquinoline Ligands for
the Binding of [3H]Methoxytetrabenazine to Rat Brain
Vesicular Monoamine Transportera
to determine if measures of in vitro properties, such as
binding affinities, lipophilicities, or serum protein bind-
ing, necessarily predict in vivo biological activities. In
the area of radiopharmaceutical design, Katzenellen-
bogen and co-workers have demonstrated the usefulness
of an in vitro measure, termed the binding selectivity
index (defined as the ratio of the measured in vitro
binding affinity to estimated nonspecific binding), as an
indicator of the in vivo behavior of steroid radio-
tracers.30,31 Similarly, Eckelman et al.32 have shown a
good correlation between in vitro binding affinities and
in vivo radioligand-binding inhibition for a series of
iodinated derivatives of 3-quiniclidinyl benzilate, a
muscarinic cholinergic receptor antagonist.
ligand
Ki (nM)
2
8.1 ( 0.4
2.6 ( 0.2
12 ( 1
4a
4b
5
6
7
42 ( 5
84 ( 14
136 ( 12
33 ( 2
8
a
Data are shown as mean ( SD (n ) 3).
Ta ble 2. Competition of Unlabeled Benzisoquinolines for the
in Vivo Localization of [11C]Methoxytetrabenazine in Mouse
Striatum and Hypothalamusa
We have obtained both in vitro and in vivo data for a
series of benzisoquinoline ligands for the VMAT2 and
can examine whether the in vitro data would have
predicted the in vivo results. Although in vivo competi-
tion studies performed here do not control for confound-
ing factors of pharmacokinetics, plasma protein binding,
nonspecific distribution, and metabolism, it is interest-
ing that the in vitro binding affinities and in vivo data
are indeed correlated. Thus, the greatest in vivo
competition is demonstrated by the highest in vitro
affinity compounds, bearing either no (2) or small (4a ,b
and 5) alkyl groups. Addition of the larger alkyl groups
(propyl (6), isopropyl (7), and isobutyl (8)) reduces both
in vitro binding affinity as well as in vivo competition
for radioligand binding, although the in vivo results
certainly reflect the effects of increased lipophilicity and
possibly less drug delivery to the tissue. If the isobutyl
derivative 8 is excluded (see below), the correlation
between in vitro binding affinities and in vivo radio-
ligand competition is remarkably good (r2 ) 0.994;
correlation not shown).
The isobutyl compound 8 proves very interesting.
Despite a good binding affinity (Ki ) 33 nM), this
derivative shows relatively poor ability in vivo to
compete for radioligand binding to the vesicular mono-
amine transporter. This is likely due to greater protein
binding and partitioning into membranes due to the
higher lipophilicity. The binding affinity observed for
8 may, however, indicate an important option in the
future development of in vivo imaging agents. The
increase in binding affinity when moving from the
isopropyl to the isobutyl derivative may be due to new,
favorable interactions of the longer 2-alkyl group with
a hydrophobic site near the binding site for these
benzisoquinolines. This would explain the very high
binding affinity observed in vitro for one of the two
possible 2-iodovinyl derivatives of dihydrotetrabenazine
reported by Kung and co-workers;13 although the ster-
eochemistry of the high-affinity isomer remains un-
determined, if it proves to be the â-iodovinyl-R-hydroxy
derivative, then it would possibly present a large
lipophilic substituent into approximately the same space
as reached by the 2â-isobutyl derivative 8 tested here.
If such substituents contribute to a high binding affinity,
a successful in vivo radiotracer will then require modi-
fications of other structural features of the benziso-
quinolines to reduce overall lipophilicity of the molecule.
hypothalamus
competing ligand
striatum
(%ID/g)
none (controls, N ) 64)
6.38 ( 1.37
2.26 ( 0.42b
2.09 ( 0.27b
2.35 ( 0.32b
3.24 ( 0.58b
4.26 ( 0.37b
6.21 ( 1.4
3.87 ( 0.63
1.71 ( 0.25b
1.96 ( 0.39b
1.79 ( 0.27b
2.00 ( 0.27b
1.96 ( 0.35b
3.06 ( 0.7b
2.93 ( 0.51b
2
4a
4b
5
6
7
8
5.33 ( 0.86b
a
CD-1 mice were coinjected with 10 mg/kg blocking agent and
100-200 µCi of [11C]methoxytetrabenazine and regional brain
distributions determined at 15 min after injection. Control
animals were injected with radiotracer in saline but with no added
competing agent. Data are shown as mean ( SD of percent
injected dose/g (%ID/g). Statistical analyses were done using an
b
unpaired Student’s t-test. p < 0.005 as compared to controls.
brain12 by 1a ,b are 6 and 20 nM, respectively, and we
have in separate studies determined a Ki of 0.97 nM
for (+)-R-dihydrotetrabenazine.25
In Vivo Stu d ies. In the development of new organ-
or tissue-specific imaging radiopharmaceuticals, a dif-
ficult step is the selection of a candidate compound for
subsequent radiolabeling and in vivo evaluation. For
practical or economic reasons, it is not usually feasible
to radiolabel all compounds in a series (such as all of
the alkylated benzisoquinolines prepared here), and
thus there is a need for methods to reduce such lists to
a more manageable number of candidate compounds.
The in vivo biological activities of the new compounds
prepared here were assayed using their ability to
compete for the accumulation of [11C]methoxytetra-
benazine ([11C]-3) in mouse striatum and hypothalamus
after peripheral administration. We have previously
demonstrated that the retention of tritiated and carbon-
11-labeled forms of 1-3 in rodent brain tissues in vivo
is due to binding to the VMAT2 site6,28 and that the in
vivo regional brain distributions of these radioligands
determined 10-15 min after iv injection are highly
correlated with in vitro measures of [3H]dihydrotetra-
benazine binding sites (r ) 0.99) and regional in vitro
monoamine concentrations (r ) 0.98; correlations not
shown).29 Using this simple assay, we have demon-
strated (Table 2) that all of the new benzisoquinolines
with the exception of the isopropyl derivative are
capable of inhibition of radiotracer binding to the
vesicular monoamine transporter, although with differ-
ent efficiencies: Derivatives with smaller alkyl groups
(methyl, ethyl) are more potent than those incorporating
the larger groups (propyl, isopropyl, isobutyl).
Con clu sion s
In Vivo-in Vitr o Cor r ela tion s. In the design of
new drugs and radiopharmaceuticals, it is of interest
Tetrabenazine and related benzisoquinolines are high-
affinity ligands for the VMAT2 in mammalian brain. A

194 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1
Lee et al.
147.10 (C-9), 129.82 (C-11a), 126.64 (C-7a), 111.46 (C-8), 107.90
(C-11), 71.07 (C-2), 57.89 (C-11b), 57.48 (C-4), 55.96 and 55.79
(2 × OCH3), 52.15 (C-6), 41.93 (C-3), 45.56 (C-1), 36.03 (C-1′),
29.05 (C-7), 25.90 (C-2′), 24.12 (C-3′), 21.69 (C-3′), 28.43 (C-
1′′). Anal. (C20H31NO3) C,H,N.
methyl group in either the R- or â-configuration at the
2-position of dihydrotetrabenazine is well tolerated, and
the highest in vitro affinity for the VMAT2 site of rat
brain was found for the 2â-methyl-R-hydroxy isomer 4a .
Addition of alkyl groups larger than a methyl group in
the 2R-position of 1b reduces both in vitro binding
affinity as well as the ability to occupy VMAT2 sites in
vivo.
2â-E t h y l-2r-h y d r o x y -3-i s o b u t y l-9,10-d i m e t h o x y -
1,2,3,4,6,7-h exa h yd r o-11bH-ben zo[a ]qu in olizin e (5): yield
1
620 mg (38%); mp 122-123 °C; H NMR (CDCl3) δ 6.70 (1H,
s, H-8), 6.61 (1H, s, H-11), 3.82 (3H, s, CH3-9), 3.87 (3H, s,
CH3-10), 3.48 (1H, d, J ) 12 Hz, H-11b), 3.00 (1H, m, H-7),
3.01 (1H, m, H-6), 2.88 (1H, dd, J ) 11.6, 4.3 Hz, H-4), 2.37
(1H, dd, J ) 11.6, 11.6 Hz, H-4), 2.67 (1H, m, H-7), 2.56 (1H,
ddd, J ) 11.4, 11.4, 4 Hz, H-6), 2.16 (1H, dd, J ) 14.1, 2.6 Hz,
H-1), 1.88 (1H, m, H-3), 1.67 (1H, m, H-1), 1.65 (2H, m, H-2,
1′′), 1.29 (1H, ddd, J ) 12.9, 10.5, 2.5 Hz, H-1′), 1.12 (1H, ddd,
J ) 13.9, 9.8, 4 Hz, H-1′), 0.96 (3H, t, J ) 8.4 Hz, H-2′′), 0.98
(3H, d, J ) 6.4 Hz, CH3-3′), 0.95 (3H, d, J ) 6.4 Hz, CH3-3′);
13C NMR (CDCl3) δ 147.31 (C-10), 147.04 (C-9), 130.09 (C-11a),
126.75 (C-7a), 111.48 (C-8), 108.04 (C-11), 73.25 (C-2), 57.64
(C-11b), 57.10 (C-4), 55.99 and 55.76 (2 × OCH3), 52.10 (C-6),
38.93 (C-3), 41.45 (C-1), 35.51 (C-1′), 29.08 (C-7), 25.57 (C-2′),
24.22(C-3′), 21.67 (C-3′), 33.03 (C-1′′), 8.03 (C-2′′); HRMS calcd
347.2460, found 347.2468. Anal. (C21H33NO3) C,H,N.
Exp er im en ta l Section
Gen er a l. Chemicals were purchased from Aldrich Chemi-
cal Co. (St. Louis, MO) and are reagent grade unless otherwise
noted. Kieselgel (silica gel 60, 6-300 µm, 70-230 mesh) and
thin layer chromatographic plastic sheets (silica gel 60 F254
,
layer thickness 0.2 mm) were purchased from Merck Co.
Melting points were determinated in a capillary tube on a Mel-
Temp apparatus and are uncorrected. 1H and 13C NMR
spectra were obtained on a Bruker AC-360 spectrometer and
recorded in CDCl3 (7.24 ppm for 1H NMR and 77.00 ppm for
13C NMR as a reference). Mass spectra were measured with
a VG 70-250s spectrometer. High-resolution mass spectra
were measured with a Fisons magnetic sector mass spectrom-
eter. Elemental analyses were performed by the University
of Michigan CHN/AA Laboraotry (Ann Arbor, MI) and are
within 0.4% of the calculated values, unless otherwise noted.
[3H]Methoxytetrabenazine (82 Ci/mmol) was prepared by
custom tritiation (Amersham Corp.). [11C]Methoxytetra-
benazine (>500 Ci/mmol) was prepared as previously de-
scribed.33
Syn th eses of 2-Alk yld ih yd r otetr a ben a zin e Der iva -
t ives. 2r- a n d 2â-H yd r oxy-2-m et h yl-3-isob u t yl-9,10-
d im et h oxy-1,2,3,4,6,7-h exa h yd r o-11bH -b en zo[a ]q u in o-
lizin e (4a ,b). Syntheses of alkylated dihydrotetrabenazines
4-8 were done using the general procedure described here for
the synthesis of compound 4. To a stirred solution of 2 (1.621
g, 5.106 mmol) in dry tetrahydrofuran (30 mL) at 0 °C was
added methyllithium (4.0 mL, 1.4 M solution in THF) dropwise
over 10 min. The reaction mixture was stirred at 0 °C for
another 2.5 h and then allowed to warm to room temperature.
After 12 h, the reaction was quenched by addition of water (2
mL). The organic layer was separated, and the aqueous layer
was extracted with dichloromethane (3 × 20 mL). The
combined organic layers were dried over sodium sulfate and
filtered. Removal of the solvent from the filtrate gave an oily
solid. Column chromatographic purification using a methanol
in dichloromethane gradient yielded two compounds, 4a (189
mg, 0.567 mmol) and 4b (533 mg, 1.60 mmol), in 11% and 31%
yields, respectively (total 42%).
4a : mp 76-78 °C; 1H NMR (CDCl3) δ 6.56 (1H, s, H-8), 6.62
(1H, s, H-11), 3.83 (3H, s, CH3-9), 3.82 (3H, s, CH3-10), 3.17
(1H, d, J ) 12 Hz, H-11b), 3.08 (1H, m, H-7), 3.00 (1H, m,
H-6), 2.93 (1H, dd, J ) 11.9, 4.1 Hz, H-4), 2.06 (1H, dd, J )
11.6, 11.6 Hz, H-4), 2.62 (1H, m, H-7), 2.47 (1H, ddd, J ) 10.9,
10.9, 4.7 Hz, H-6), 2.29 (1H, dd, J ) 12.4, 4.2 Hz, H-1), 1.82
(1H, m, H-3), 1.59 (2H, m, H-1,2′), 1.38 (1H, ddd, J ) 13, 10.2,
2.5 Hz, H-1′), 1.22 (3H, s, CH3-1′′), 0.99 (1H, ddd, J ) 13, 9.8,
3.2 Hz, H-1′), 0.93 (3H, d, J ) 6.3 Hz, CH3-3′), 0.90 (3H, d, J
) 6.3 Hz, CH3-3′); 13C NMR (CDCl3) δ 147.45 (C-10), 147.13
(C-9), 129.49 (C-11a), 126.44 (C-7a), 111.52 (C-8), 107.86 (C-
11), 72.36 (C-2), 59.99 (C-11b), 59.05 (C-4), 55.96 and 55.81 (2
× OCH3), 51.78 (C-6), 44.50 (C-3), 47.46 (C-1), 36.21 (C-1′),
29.11 (C-7), 25.74 (C-2′), 24.24 (C-3′), 21.79 (C-3′), 20.64 (C-
2â-n -P r op yl-2r-h yd r oxy-3-isob u t yl-9,10-d im e t h oxy-
1,2,3,4,6,7-h exa h yd r o-11bH-ben zo[a ]qu in olizin e (6): yield
1
227 mg (33%); mp 147-148 °C; H NMR (CDCl3) δ 6.62 (1H,
s, H-8), 6.53 (1H, s, H-11), 3.80 (3H, s, CH3-9), 3.81 (3H, s,
CH3-10), 3.42 (1H, d, J ) 11.2 Hz, H-11b), 3.08 (1H, m, H-7),
2.93 (1H, m, H-6), 2.80 (1H, dd, J ) 11.6, 4.3 Hz, H-4), 2.31
(1H, dd, J ) 11.6, 11.6 Hz, H-4), 2.60 (1H, m, H-7), 2.49 (1H,
ddd, J ) 11.4, 11.4, 4 Hz, H-6), 2.12 (1H, dd, J ) 13.5, 2.6 Hz,
H-1), 1.80 (1H, m, H-3), 1.58 (2H, m, H-1,2′), 1.52 (2H, m,
H-1′′,2′′), 1.34 (2H, m, H-1′′,2′′), 1.22 (1H, ddd, J ) 12.9, 10.5,
2.5 Hz, H-1′), 1.05 (1H, ddd, J ) 16, 11.8, 4.4 Hz, H-1′), 0.90
(3H, t, J ) 7.1 Hz, H-3′′), 0.90 (3H, d, J ) 6.4 Hz, CH3-3′),
0.87 (3H, d, J ) 6.4 Hz, CH3-3′); 13C NMR (CDCl3) δ 147.02
(C-9), 147.30 (C-10), 129.95 (C-11a), 126.65 (C-7a), 111.45 (C-
8), 108.00 (C-11), 72.95 (C-2), 57.64 (C-11b), 57.05 (C-4), 55.98
and 55.73 (2 × OCH3), 52.05 (C-6), 39.41 (C-3), 42.07 (C-1),
35.53 (C-1′), 28.99 (C-7), 25.56 (C-2′), 24.21 (C-3′), 21.65 (C-
3′), 43.13 (C-1′′), 16.81 (C-2′′), 14.67 (C-3′′); HRMS calcd
361.2617, found 361.2607. Anal. (C22H35NO3) C, H, N.
2â-Isop r op yl-2r-h yd r oxy-3-isob u t yl-9,10-d im et h oxy-
1,2,3,4,6,7-h exa h yd r o-11bH-ben zo[a ]qu in olizin e (7): yield
1
340 mg (26%); mp 167-168; H NMR (CDCl3) δ 6.54 (1H, s,
H-8), 6.63 (1H, s, H-11), 3.81 (3H, s, CH3-9), 3.80 (3H, s, CH3-
10), 3.41 (1H, d, J ) 12 Hz, H-11b), 3.08 (1H, m, H-7), 2.96
(1H, m, H-6), 2.83 (1H, dd, J ) 11.6, 4.3 Hz, H-4), 2.33 (1H,
dd, J ) 11.6, 11.6 Hz, H-4), 2.61 (1H, m, H-7), 2.59 (1H, ddd,
J ) 11.4, 11.4, 4 Hz, H-6), 2.06 (1H, dd, J ) 13.6, 2.8 Hz, H-1),
2.02 (3H, m, H-3,1′′), 1.44 (1H, dd, J ) 13.4, 12 Hz, H-1), 1.60
(1H, m, H-2′), 1.22 (1H, ddd, J ) 13.2, 10.5, 2.6 Hz, H-1′), 1.03
(1H, ddd, J ) 15.2, 10.8, 4 Hz, H-1′), 1.01 (3H, d , J ) 6.9 Hz,
H-2′′), 0.84 (3H, d, J ) 6.9 Hz, H-2′′), 0.90 (3H, d, J ) 6.6 Hz,
CH3-3′), 0.88 (3H, d, J ) 6.6 Hz, CH3-3′); 13C NMR (CDCl3) δ
146.86 (C-9), 147.20 (C-10), 130.18 (C-11a), 126.68 (C-7a),
111.36 (C-8), 108.02 (C-11), 74.96 (C-2), 57.28 (C-11b), 56.73
(C-4), 55.92 and 55.61 (2 × OCH3), 51.87 (C-6), 37.41 (C-3),
35.58 (C-1′), 35.08 (C-1), 28.92 (C-7), 25.07 (C-2′), 24.22 (C-
3′), 21.39 (C-3′), 33.94 (C-1′′), 17.26 (C-2′′), 16.23 (C-2′′); HRMS
calcd 361.2617, found 361.2607. Anal. (C22H35NO3) C,H,N.
2â-Isob u t yl-2r-h yd r oxy-3-isob u t yl-9,10-d im e t h oxy-
1,2,3,4,6,7-h exa h yd r o-11bH-ben zo[a ]qu in olizin e (8): yield
1
750 mg (39%); mp 175-177 °C; H NMR (CDCl3) δ 6.55 (1H,
1′′); HRMS calcd 333.2304, found 333.2292. Anal. (C20H31
-
s, H-8), 6.51 (1H, s, H-11), 3.81 (3H, s, CH3-9), 3.82 (3H, s,
CH3-10), 3.40 (1H, d, J ) 11.4 Hz, H-11b), 2.94 (1H, m, H-7),
3.04 (1H, m, H-6), 2.81 (1H, dd, J ) 11.6, 4.3 Hz, H-4), 2.28
(1H, dd, J ) 11.6, 11.6 Hz, H-4), 2.60 (1H, m, H-7), 2.49 (1H,
ddd, J ) 11.4, 11.4, 4 Hz, H-6), 2.24 (1H, m, H-1), 1.87 (1H,
m, H-2′′), 1.66 (1H, m, H-3), 1.59 (1H, m, H-2′), 1.56 (1H, m,
H-1), 1.50 (1H, m, H-1′′), 1.38 (1H, m, H-1′′), 1.25 (1H, ddd, J
) 12.9, 10.5, 2.5 Hz, H-1′), 1.02 (1H, ddd, J ) 14.2, 10.4, 4.7
Hz, H-1′), 1.01 (3H, d, J ) 6.7 Hz, H-3′′), 0.94 (3H, d, J ) 6.7
Hz, H-3′′), 0.92 (3H, d, J ) 6.4 Hz, CH3-3′), 0.89 (3H, d, J )
6.4 Hz, CH3-3′); 13C NMR (CDCl3) δ 146.89 (C-9), 147.21 (C-
10), 129.91 (C-11a), 126.64 (C-7a), 111.37 (C-8), 108.07 (C-11),
NO3) C,H,N.
1
4b: mp 108-111 °C; H NMR (CDCl3) δ 6.64 (1H, s, H-8),
6.54 (1H, s, H-11), 3.82 (3H, s, CH3-9), 3.81 (3H, s, CH3-10),
3.39 (1H, d, J ) 12 Hz, H-11b), 3.08 (1H, m, H-7), 2.95 (1H,
m, H-6), 2.80 (1H, dd, J ) 11.6, 4.3 Hz, H-4), 2.27 (1H, dd, J
) 11.6, 11.6 Hz, H-4), 2.61 (1H, m, H-7), 2.50 (1H, ddd, J )
11.4, 11.4, 4 Hz, H-6), 2.22 (1H, dd, J ) 14.1, 2.6 Hz, H-1),
1.82 (1H, m, H-3), 1.57 (2H, m, H-1,2′), 1.30 (1H, ddd, J )
13.4, 11, 2.4 Hz, H-1′), 1.06 (1H, ddd, J ) 13.4, 9.7, 4 Hz, H-1′),
1.27 (3H, s, CH3-1′′), 0.92 (3H, d, J ) 6.6 Hz, CH3-3′), 0.88
(3H, d, J ) 6.6 Hz, CH3-3′); 13C NMR (CDCl3) δ 147.34 (C-10),

SAR of Ligands for Vesicular Monoamine Transporters
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1 195
73.24 (C-2), 57.50 (C-11b), 57.02 (C-4), 55.90, 55.58 (2 × OCH3),
51.81 (C-6), 40.75 (C-3), 42.24 (C-1), 35.54 (C-1′), 28.89 (C-7),
23.24 (C-2′), 25.10 (C-3′), 21.45 (C-3′), 49.13 (C-1′′), 25.49 (C-
2′′), 24.56 (C-3′′); HRMS calcd 375.2773, found 375.2773. Anal.
(C23H37NO3) C,H,N.
removed and dissected. Samples of striatum (left and right
combined) and hypothalamus (whole) were quickly counted for
carbon-11 (automatic γ-counter) and then weighed. Data were
calculated as percent injected dose/g (%ID/g) for each tissue.
2r- a n d 2â-Hyd r oxy-3-isobu tyl-1,2,3,4,6,7-h exa h yd r o-
11bH-ben zo[a ]qu in olizin e (1a ,b). The two isomers of di-
hydrotetrabenazine were isolated both as side products in the
synthesis of the alkylated derivatives, above, and from NaBH4
reduction of the ketone in ethanol solution.
Ack n ow led gm en t. This work was supported by
National Institutes of Health Grant NS 15655, MH
47611, AG 08671, and T-32-CA09015 (to L.C.L.) and the
Department of Energy Grant DE-FG021-87ER60561.
We thank Mr. Frank Parker, Dr. Christopher Kojiro,
and Dr. Peter Toogood (Department of Chemistry,
University of Michigan) for their assistance in obtaining
and interpreting the NMR spectra.
1a : 1H NMR (CDCl3) δ 6.62 (1H, s, H-8), 6.71 (1H, s, H-11),
3.88 (6H, s, OCH3-9,10), 3.42 (1H, m, H-2), 3.16 (1H, d, J )
12 Hz, H-11b), 3.09 (2H, m, H-4,7), 3.03 (1H, m, H-6), 2.69
(1H, m, H-7), 2.63 (1H, m, H-1), 2.49 (1H, ddd, J ) 10.9, 10.9,
4.7 Hz, H-6), 2.01 (1H, dd, J ) 11.6, 11.6 Hz, H-4), 1.75 (2H,
m, H-3, 2′), 1.62 (1H, ddd, J )13, 10.2, 2.5 Hz, H-1′), 1.53 (1H,
dd, J ) 13.6, 12 Hz, H-1), 1.09 (1H, ddd, J ) 13, 9.8, 3.2 Hz,
H-1′), 0.98 (3H, d, J ) 6.6 Hz, CH3-3′), 0.96 (3H, d, J ) 6.6
Hz, CH3-3′); 13C NMR (CDCl3) δ 147.24 (C-9), 147.52 (C-10),
129.39 (C-11a), 126.44 (C-7a), 111.49 (C-8), 107.96 (C-11), 74.60
(C-2), 60.96 (C-11b), 60.11 (C-4), 55.99 and 55.89 (2 × OCH3),
51.96 (C-6), 41.63 (C-3), 40.60 (C-1), 39.74 (C-1′), 29.22 (C-7),
25.38 (C-2′), 24.24 (C-3′), 21.82 (C-3′).
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