Vesamicol receptor mapping of brain cholinergic neurons with radioiodine-labeled positional isomers of benzovesamicol

Article abstract of DOI:10.1021/jm9507486

Alzheimer's disease is characterized by progressive cerebral cholinergic neuronal degeneration. Radiotracer analogs of benzovesamicol, which bind with high affinity to the vesamicol receptor located on the uptake transporter of acetylcholine storage vesic

Full text of DOI:10.1021/jm9507486

J . Med. Chem. 1996, 39, 3331-3342
3331
Vesa m icol Recep tor Ma p p in g of Br a in Ch olin er gic Neu r on s w ith Ra d ioiod in e-
La beled P osition a l Isom er s of Ben zovesa m icol1
Yong-Woon J ung, Kirk A. Frey, G. Keith Mulholland,† Renato del Rosario, Phillip S. Sherman, David M. Raffel,
Marcian E. Van Dort, David E. Kuhl, David L. Gildersleeve, and Donald M. Wieland*
Division of Nuclear Medicine, Department of Internal Medicine, University of Michigan Medical Center,
Ann Arbor, Michigan 48109-0552
Received October 10, 1995X
Alzheimer’s disease is characterized by progressive cerebral cholinergic neuronal degeneration.
Radiotracer analogs of benzovesamicol, which bind with high affinity to the vesamicol receptor
located on the uptake transporter of acetylcholine storage vesicles, may provide an in vivo
marker of cholinergic neuronal integrity. Five positional isomers of racemic iodobenzovesamicol
(4′-, 5-, 6-, 7-, and 8-IBVM) were synthesized, exchange-labeled with iodine-125, and evaluated
as possible in vivo markers for central cholinergic neurons. Only two isomers, 5-IBVM (5)
and 6-IBVM (10), gave distribution patterns in mouse brain consistent with cholinergic
innervation: striatum . hippocampus g cortex > hypothalamus . cerebellum. The 24-h tissue-
to-cerebellum concentration ratios for 5-IBVM (5) were 3-4-fold higher for striatum, cortex,
and hippocampus than the respective ratios for 6-IBVM (10). Neither 8-IBVM (16) nor 4′-
IBVM (17) exhibited selective retention in any of the brain regions examined. In the heart,
only 5-IBVM (5) exhibited an atria-to-ventricles concentration ratio consistent with high
peripheral cholinergic neuronal selectivity. The 7-IBVM (14) isomer exhibited an anomalous
brain distribution pattern, marked by high and prolonged retention in the five brain regions,
most notably the cerebellum. This isomer was screened for binding in a series of 26 different
biological assays; 7-IBVM (14) exhibited affinity only for the σ-receptor with an IC50 of ∼30
nM. Drug-blocking studies suggested that brain retention of 7-IBVM (14) reflects high-affinity
binding to both vesamicol and σ-receptors. Competitive binding studies using rat cortical
homogenates gave IC50 values for binding to the vesamicol receptor of 2.5 nM for 5-IBVM (5),
4.8 nM for 6-IBVM (10), and 3.5 nM for 7-IBVM (14). Ex vivo autoradiography of rat brain
after injection of (-)-5-[125I]IBVM ((-)-[125I]5) clearly delineated small cholinergic-rich areas
such as basolateral amygdala, interpeduncular nucleus, and facial nuclei. Except for cortex,
regional brain levels of (-)-5-[123I]IBVM ((-)-[123I]5) at 4 h exhibited a linear correlation (r2 )
0.99) with endogenous levels of choline acetyltransferase. Conclusion: Vesamicol receptor
mapping of cholinergic nerve terminals in murine brain can be achieved with 5-IBVM (5) and
less robustly with 6-IBVM (10), whereas the brain localization of 7-IBVM (14) reflects high-
affinity binding to both vesamicol and σ-receptors.
In tr od u ction
the vesamicol receptor in vivo. However, certain de-
rivatives of vesamicol including 5-substituted benzove-
samicols not only bind with higher affinity to the
vesamicol receptor than vesamicol itself but show little
affinity for the vesamicol-binding protein.8
Benzovesamicols have been shown to bind in vitro
with high affinity to a site on the acetylcholine trans-
porter complex2 in synaptic vesicle preparations isolated
from the marine ray Torpedo3 and rat brain.4 The
binding is highly enantioselective and noncompetitive
with acetylcholine, indicating that the vesamicol recep-
tor allosterically modulates the transporter. The ace-
tylcholine transporter from Torpedo electric organ has
been recently cloned, expressed in fibroblasts, and
shown to harbor vesamicol receptors.5
Over the past decade Parsons and co-workers have
utilized vesamicol as a pharmacological tool to study
cholinergic nerve terminal function.6 This group has
shown that vesamicol also binds to a second site in rat
brain called the vesamicol-binding protein.7 Although
vesamicol has a lower affinity for this second site, the
higher density of this site, compared to the vesamicol
receptor, might complicate efforts to quantitatively map
A number of benzovesamicol derivatives have been
labeled with positron-emitting isotopes for use in tomo-
graphic imaging of cholinergic nerve density in brain.9
In a previous communication, we reported that (-)-5-
[
125I]IBVM is a highly specific in vivo marker for
cholinergic nerve fields.10 Selective localization of (-)-
5-[125I]IBVM in cholinergic-rich regions of the mouse
brain was avid, prolonged, highly stereoselective, and
partially blocked by vesamicol. On the basis of these
findings, Kuhl and co-workers have used (-)-5-[123I]-
IBVM to obtain tomographic measures of cholinergic
nerve terminal density in brain of normal volunteers11
and patients with Alzheimer’s disease and Parkinson’s
disease.12
Using an in vitro acetylcholine active-transport assay
to determine relative activity, Rogers et al. evaluated
over 80 structural variants of vesamicol and found that
bulky substituents were tolerated in the 5-position but
not the 8-position of benzovesamicol (see Figure 1); 6-
and 7-substituted benzovesamicols were not evaluated
* Corresponding author: Donald M. Wieland, Ph.D., PET Center,
Kresge Building III, Room 3480, Division of Nuclear Medicine,
University of Michigan Medical Center, Ann Arbor, MI 48109-0552.
Tel: 313-763-9095. Fax: 313-764-0288.
† Present address: Department of Radiology, Indiana University
Medical Center, Indianapolis, IN 46202.
X Abstract published in Advance ACS Abstracts, August 1, 1996.
S0022-2623(95)00748-5 CCC: $12.00 © 1996 American Chemical Society

3332 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17
J ung et al.
chiral resolution of racemic 5-IBVM (5) was achieved
by use of (-)-(S)-R-methoxy-R-(trifluoromethyl)pheny-
lacetyl chloride (MTPA) to form the diastereomeric
MTPA esters.14 The (+)- and (-)-diastereomers were
readily separated by preparative TLC on silica gel after
several developments with hexane/EtOAc (19/1).10 Base
hydrolysis of the MTPA esters provided the enantiomers
of 5-IBVM ((+)-5, (-)-5) with optical purities greater
than 98% as determined by chiral HPLC using a
Chiracel OD column.15
F igu r e 1. Structures of vesamicol and benzovesamicol show-
ing their trans-2R,3R-configuration. The numbering system
is the same as that used previously.10,13 A different numbering
system has been employed by others in a recent report.8
The 8-IBVM (6) isomer was prepared from 8-ABVM
(3) by the diazotization route described in the synthesis
of 5-IBVM (5). Synthesis of 4′-IBVM (17) was achieved
by reacting 6,7-epoxy-1-(trifluoroacetamido)-5,8-dihy-
dronaphthalene (1) with 4-(4′-iodophenyl)piperidine16
followed by removal of the amino group of the resulting
product through the diazonium salt as was done for
5-IBVM (5). Selective functionalization of the 6- and
7-positions of benzovesamicol, however, posed some
difficulty. The key step was the regioselective ortho-
nitration of 5- and 8-acetamidobenzovesamicols using
fuming nitric acid in acetic anhydride (Scheme 2). The
ortho-directing ability of the acetamido group has been
demonstrated in other aromatic systems.17 The desired
6-IBVM (10) was prepared from 5-amino-6-nitroben-
zovesamicol (7) by reductive deamination and subse-
quent reduction of the nitro group to give 6-ABVM (9)
followed by diazotization and reaction with potassium
iodide. The 7-IBVM (14) isomer was synthesized in
similar fashion starting from 8-ABVM (3).
in this study.13 We sought to expand our initial study10
with 5-IBVM (5) by investigating the effect of system-
atically altering the aromatic ring position of iodine on
the in vivo cholinergic nerve-mapping characteristics of
IBVM. We report here the synthesis of four new
radioiodinated isomers of IBVM and a comparison of
their binding and biodistribution behavior with that of
5-IBVM (5) in rodents. This study found that vesamicol
receptor mapping of cholinergic nerve terminals in
murine brain can be achieved with 5-IBVM (5) and less
robustly with 6-IBVM (10). The brain localization of
7-IBVM (14), however, reflects high-affinity binding to
both vesamicol and σ-receptors.
Resu lts
Ch em istr y. The synthesis of racemic 5-IBVM (5)10
was accomplished by two routes as shown in Scheme 1.
Method A is a one-step diazotization of the previously
described 5-aminobenzovesamicol (5-ABVM, 2)13 in the
presence of iodide. Method B is a two-step sequence in
which 8-aminobenzovesamicol (8-ABVM, 3) was regi-
oselectively iodinated in the 5-position using iodine
monochloride followed by reductive removal of the
8-amino group with hypophosphorus acid. Method B
was developed not because of difficulties with method
A but because of the availability of 8-ABVM (3) as a
major byproduct in the synthesis of 5-ABVM (5). The
Iodine-125-labeled isomers were synthesized by solid-
state exchange labeling of the respective racemic iodo
compounds with sodium [125I]iodide. The exchange
reaction, which is a refinement of a method first
reported by this laboratory in 1982, is conducted under
acidic conditions at 140-160 °C and likely involves an
electrophilic iodine species.18 Radiochemical yields
ranged from 65% to 89%. Purification was readily
Sch em e 1. Synthesis of Racemic 5-IBVM (5) and 8-IBVM (6)a
a
The alternate synthesis of 5 (method B) was also employed due to availability of 8-aminobenzovesamicol (3) as a major byproduct in
the synthesis of 5-aminobenzovesamicol (2).

Vesamicol Receptor Mapping
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17 3333
Sch em e 2. Synthesis of 6-IBVM (10)a
a
The 7-IBVM (14) isomer was synthesized from 8-ABVM (3) using the same strategy.
Ta ble 1. Tissue Distribution of [125I]Iodobenzovesamicols in Micea,b
5-IBVM (5)
6-IBVM (10)
7-IBVM (14)
8-IBVM (6)
4′-IBVM (17)
0.5 h
cortex
2.29 ( 0.08
3.81 ( 0.73
2.09 ( 0.34
1.69 ( 0.22
1.28 ( 0.06
10.98 ( 1.90
7.57 ( 0.78
1.73 ( 0.41
1.27 ( 0.44
0.82 ( 0.42
2.99 ( 0.32
3.90 ( 0.52
2.73 ( 0.34
2.51 ( 0.27
2.05 ( 0.24
18.07 ( 1.47
3.93 ( 0.23
2.09 ( 0.22
1.34 ( 0.11
1.06 ( 0.07
2.32 ( 0.24
2.34 ( 0.33
1.99 ( 0.41
2.16 ( 0.33
2.06 ( 0.32
19.30 ( 5.72
13.11 ( 5.01
4.37 ( 0.44
3.29 ( 0.55
1.13 ( 0.12
1.53 ( 0.18
1.64 ( 0.22
3.01 ( 0.29
2.68 ( 0.17
2.91 ( 0.28
2.73 ( 0.35
2.33 ( 0.26
15.44 ( 1.53
8.86 ( 1.07
3.03 ( 0.32
2.90 ( 0.58
1.85 ( 0.15
striatum
hippocampus
hypothal
cerebellum
liver
lung
atria
ventricles
blood
1.22 ( 0.15
1.64 ( 0.73
1.25 ( 0.10
1.00 ( 0.10
4 h
cortex
1.17 ( 0.20
3.30 ( 0.52
1.06 ( 0.19
0.61 ( 0.08
0.21 ( 0.04
5.92 ( 2.04
1.11 ( 0.90
1.43 ( 0.50
0.31 ( 0.02
0.53 ( 0.08
1.28 ( 0.13
3.07 ( 0.24
1.03 ( 0.10
0.68 ( 0.06
0.29 ( 0.03
6.33 ( 0.50
0.89 ( 0.07
0.97 ( 0.18
0.53 ( 0.11
0.51 ( 0.08
1.62 ( 0.21
1.96 ( 0.36
1.27 ( 0.18
1.42 ( 0.19
1.23 ( 0.13
12.69 ( 1.20
5.86 ( 1.10
2.45 ( 0.66
1.77 ( 0.32
0.47 ( 0.04
0.10 ( 0.02
0.12 ( 0.02
0.32 ( 0.03
0.33 ( 0.03
0.31 ( 0.03
0.28 ( 0.03
0.28 ( 0.03
9.12 ( 0.23
2.95 ( 0.59
1.35 ( 0.21
1.88 ( 0.46
0.66 ( 0.07
striatum
hippocampus
hypothal
cerebellum
liver
lung
atria
ventricles
blood
0.08 ( 0.02
0.34 ( 0.10
0.23 ( 0.04
0.19 ( 0.03
24 h
cortex
0.88 ( 0.15
2.20 ( 0.26
0.71 ( 0.12
0.42 ( 0.04
0.05 ( 0.01
0.89 ( 0.06
0.09 ( 0.01
0.82 ( 0.38
0.06 ( 0.01
0.04 ( 0.01
0.22 ( 0.06
0.76 ( 0.15
0.16 ( 0.03
0.10 ( 0.01
0.05 ( 0.01
0.88 ( 0.08
0.10 ( 0.01
0.10 ( 0.02
0.10 ( 0.04
0.05 ( 0.004
1.74 ( 0.14
2.32 ( 0.17
1.48 ( 0.12
1.35 ( 0.18
1.06 ( 0.09
6.48 ( 0.57
1.74 ( 0.20
0.77 ( 0.13
0.27 ( 0.02
0.09 ( 0.01
striatum
hippocampus
hypothal
cerebellum
liver
lung
atria
ventricles
blood
a
b
Tissue radioactivity concentration expressed as % dose/g normalized to a standard 25-g weight ((SD). N ) 5/time interval for 5-,
6-, and 8-IBVM (5, 10, 6); N ) 4 or 5 for 7- and 4′-IBVM (14, 17).
accomplished by silica Sep-Pak chromatography using
a hexane/ethyl acetate gradient. We confirmed by
HPLC analysis15 that no ring scrambling of the iodine
label occurred during the radioiodide exchange reaction.
An alternative radiolabeling approach which provides
(-)-5-[123I]IBVM ((-)-[123I]5) with specific activities >
20 000 Ci/mmol has been developed for use in clinical
studies.19 However, the solid-state exchange method
utilized in this study gave specific activities sufficient
(50-200 Ci/mmol) for use in animal studies.10
Tissu e Distr ibu tion Stu d ies. Radioactivity con-
centrations obtained in mouse brain and peripheral
tissues 0.5, 4, and 24 h after intravenous injection of
five radioiodinated benzovesamicol isomers are pre-
sented in Table 1. At 0.5 h postinjection, all five tracers
showed relatively uniform localization in the brain
regions evaluated. However, at 4 h postinjection, as
further illustrated in Figure 2, three regional brain
distribution patterns became evident: The 5- and 6-
isomers 5 and 10 were retained in a pattern highly
consistent with cholinergic nerve density in mouse
brain, striatum . hippocampus g cortex > hypothala-
mus . cerebellum. In contrast, the 8- and 4′-isomers
6 and 17 effluxed so rapidly and uniformly from the
brain that further studies were not performed. A third,
unexpected pattern was observed at 4 h with the
7-isomer 14 which was strongly retained in all five brain
regions, most notably the cerebellum which is known
to have sparse cholinergic innervation.
At 24 h postinjection, the regional brain patterns

3334 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17
J ung et al.
F igu r e 2. Comparison of key distribution parameters in
mouse brain 4 h after iv tracer injection of IBVM isomers.
Position of iodine is shown on the x-axis. Panel A compares
the striatum concentrations; B and C compare the striatum/
cerebellum and cortex/cerebellum activity ratios, respectively;
D compares the cerebellum concentrations. Tissue concentra-
tions are given in % injected dose/g ((SD) of tissue normalized
to a 25-g mouse.
F igu r e 3. (A) Bar graph showing effect of haloperidol pre-
treatment (1.0 mg/kg ip) on 24-h regional brain distribution
of (()-7-[125I]IBVM ([125I]14) in CD-1 mice. Tissue concentra-
tions are given in % injected dose/g ((SD) of tissue normalized
to a 25-g mouse. (B) Effect of spiroperidol pretreatment (0.1
mg/kg ip) using the same protocol. In both studies, those
sample pairs for which the means are statistically different
(p < 0.01) are marked with an asterisk (*).
obtained with 6-IBVM (10) and especially with 5-IBVM
(5) were again highly consistent with cholinergic nerve
density. The absolute retention values were higher for
the 5-isomer 5 with tissue-to-cerebellum concentration
ratios of 44 for striatum, 18 for cortex, and 14 for
hippocampus; these ratios were 3-4-fold higher than
those obtained with 6-IBVM (10). The 24-h distribution
of 7-IBVM (14), like its 4-h distribution, was anomalous
in that all five brain regions retained large amounts of
radioactivity. Most notable was the 24-h concentration
of 7-IBVM (14) in cerebellum, a value 21-fold higher
than cerebellar levels of either 5-IBVM (5) or 6-IBVM
(10). The heart atria-to-ventricles concentration ratio,
an index of peripheral cholinergic neuronal selectiv-
ity,20,21 was highest for 5-IBVM (5), approaching 5 at 4
h postinjection and 14 at 24 h.
Recep tor Scr een in g of 7-IBVM (14). To determine
if binding to any other known receptor types might be
contributing to the anomalous brain retention pattern
of 7-IBVM (14), the compound was screened for binding
in a series of 26 different binding assays. Of the 26
binding assays performed with 7-IBVM (14) at a con-
centration of 1 × 10-5 M, >50% inhibition was observed
only in the σ- and R1-adrenergic receptor assays. Sub-
sequent evaluation of 7-IBVM binding to the σ-receptor
at five concentrations of the compound gave an esti-
mated IC50 of approximately 30 nM. A similar evalu-
ation of 7-IBVM in a 5-point R1-adrenergic assay gave
an estimated value of IC50 > 500 nM.
regional tissue distribution of this radiotracer were
evaluated in mice (see Figure 3). Haloperidol, a potent
antagonist of both dopamine D2 and σ-receptors,22
lowered radioactivity levels 55-58% in cortex, hippoc-
ampus, and hypothalamus and 76% in cerebellum (p <
0.01); no change was observed in striatal activity.
Spiroperidol, a potent D2 antagonist with only weak
σ-receptor blocking activity,23 did not have a significant
effect on radioactivity levels in the five brain areas
evaluated.
Vesa m icol Recep tor Bin d in g. The IC50 values for
vesamicol receptor binding were determined by means
of a competitive binding assay employing rat cortical
homogenates and [3H](methylamino)benzovesamicol,
[3H]MABV, a ligand that selectively binds to the vesa-
micol receptor with an apparent Kd ) 0.2 nM.24 Place-
ment of the iodine atom in position 5, 6, or 7 gave <5
nM IC50 values, with 5-IBVM (5) being the most potent
at 2.5 nM. A 22-fold decrease in binding affinity
occurred when iodine was moved from the 7- to the
8-position. Iodine placement in the 4′-position of the
phenylpiperidino group also lowered binding affinity
(20.4 nM) but less dramatically. Using this assay,
racemic vesamicol had an IC50 of 15.2 nM. The esti-
mated IC50 values are presented in Figure 4.
Au tor a d iogr a p h ic Distr ibu tion of (-)-5-[125I]-
IBVM. The distribution of (-)-5-[125I]IBVM ((-)-[125I]-
5) in brain was examined in two rats each at 5 and 120
min after iv injection. Initial tracer uptake delineated
gray and white matter structures, with greater levels
in the former due most likely to higher blood flow
Dr u g-Block in g Stu d ies. To further determine if
σ-receptor binding might be contributing to this anoma-
lous brain retention pattern of 7-IBVM (14), the effects
of haloperidol and spiroperidol pretreatment on the

Vesamicol Receptor Mapping
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17 3335
used in the determination of ChAT levels in brain.
ChAT activity in atria was approximately 2-fold higher
than the activity levels in the ventricles, whereas the
iodine-123 activity ratio was 3-4-fold higher in atria.
The levels of brain ChAT decreased in the order stria-
tum > hippocampus g cortex > cerebellum; the iodine-
123 concentrations decreased in the order striatum >
cortex > hippocampus . cerebellum. The linear cor-
relation coefficient for ChAT activities versus iodine-
123 concentrations in the four brain regions sampled
was only 0.79. If the results from cortex were neglected,
however, this relationship became linear (r2 ) 0.99). A
bar graph, Figure 7, summarizes the results obtained
in this correlation study.
F igu r e 4. IC50 values (nM), concentration of compound
required to inhibit 50% of [3H](methylamino)benzovesamicol
specific binding to homogenates of rat cerebral cortical syn-
aptosomes; values represent mean of quadruplicate determi-
nations ( SD. The value obtained for racemic vesamicol was
15.2 ( 1.3 nM.
Discu ssion
Radiotracers can often be tested for their potential
to map specific neuronal systems by simply determining
their gross regional distribution pattern in vivo. This
radiotracer screening method is especially applicable to
the cholinergic network which is heterogeneously dis-
tributed in murine brain.27,28 The striatum, cortex, and
hippocampus are densely innervated, in contrast to the
cerebellum which has very sparse cholinergic innerva-
tion. Thus, striatum-to-cerebellum and cortex-to-cer-
ebellum concentration ratios may serve as approximate
indices of neuronal specificity. Regional brain concen-
trations at 4 and 24 h strongly suggest that 5-IBVM
(5) and 6-IBVM (10) are specific markers for cholinergic
nerves. The 24-h tissue-to-cerebellum concentration
ratios for 5-IBVM (5) were 3-4-fold higher for striatum,
cortex, and hippocampus than the respective ratios for
6-IBVM (10). The more pronounced neuronal retention
of 5-IBVM (5) in brain was also consistent with the
observed regional heart distribution of this tracer. The
24-h atria-to-ventricles concentration ratio for 5-IBVM
(5) was 14-fold higher than the respective ratio for
6-IBVM (10). Atria are known to harbor a higher
density of cholinergic innervation than ventricles.20,21,27
In vivo autoradiography of (-)-5-[125I]IBVM ((-)-[125I]-
5) in rat brain provided additional support for the
cholinergic nerve specificity of 5-IBVM (5). The brain
distribution of 5-IBVM (5) at 5 min postinjection was
typical of patterns obtained with cerebral blood flow
tracers such as [14C]iodoantipyrene.29,30 In contrast, the
heterogeneous retention pattern of 5-IBVM (5) in brain
at 120 min postinjection was consistent with the re-
gional density of cholinergic nerve terminals as revealed
by in vitro autoradiography of [3H]hemicholinium-3
binding31-33 and by the regional brain distribution
patterns of acetylcholine and its synthesizing and
hydrolyzing enzymes ChAT and acetylcholinesterase.21,28
Small cholinergic-rich areas of the brain such as baso-
lateral amygdala, interpeduncular nucleus, and the
facial nuclei could be clearly delineated. On the basis
of known brain cholinergic neuroanatomy, the binding
of 5-IBVM (5) in the striatum likely reflects the presence
of cholinergic interneurons, whereas cortical and hip-
pocampal labeling is due predominantly to projection
terminals arising in the cholinergic basal forebrain
nuclei.34-37
F igu r e 5. Autoradiographic distribution of 5-[125I]IBVM ([125I]-
5) in coronal hemisections of rat brain at the level of the
hippocampus 5 min (left hemisection) and 120 min (right
hemisection) following tracer injection. Areas of highest tracer
concentration are represented by increased density in the
images, which are scaled independently to demonstrate rela-
tive distribution patterns. Note the initial homogeneity of
tracer activity in gray matter (left) and the distinct, laminar
cortical and hippocampal and selective subcortical retention
at longer postinjection times (right).
(Figure 5, left hemisection). However, by 120 min a
distinct pattern of tracer retention was observed, result-
ing in preferential labeling of a subset of gray matter
structures (Figure 5, right hemisection). In Figure 6, a
color-coded autoradiographic distribution at 120 min
posttracer injection indicated densest labeling in the
interpeduncular nucleus followed by the striatum (cau-
date-putamen and olfactory tubercle), anterior and
intralaminar thalamic nuclei, cranial nerve motor nu-
clei, cerebral cortex, and hippocampus. Lowest levels
of delayed tracer retention were observed in the cerebel-
lar hemispheres and white matter.
Cor r elation of Ch olin e Acetyltr an sfer ase (Ch AT)
a n d (-)-5-[123I]IBVM. Radioactivity concentrations
were determined in the atria and ventricles of heart and
in four brain regions 4 h after tail-vein injection of (-)-
5-[123I]IBVM ((-)-[123I]5) in CD-1 mice. After counting,
tissue samples were stored at -20 °C for 4 days to allow
the iodine-123 to decay before assaying for ChAT. The
radioenzymatic method of Fonnum,25 which utilizes
[14C]acetyl-CoA and choline to assay ChAT, was modi-
fied when applied to heart tissue to eliminate interfer-
ence from endogenous carnitine and carnitine-acetyl-
transferase.21,26 This modified ChAT assay was also
The brain distribution pattern of 7-IBVM (14) was
unique in that high radioactivity levels were observed
in all brain regions sampled, including the cerebellum,
even 24 h after tracer injection. The evaluation of
7-IBVM by NovaScreen in 26 different biological assays

3336 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17
J ung et al.
F igu r e 6. Autoradiographic distribution of 5-[125I]IBVM ([125I]5) retention in rat brain 120 min following bolus iv injection. Coronal
sections at various rostrocaudal levels are presented from the level of the head of the caudate (upper left) to the cervical spinal
cord (lower right). Tracer concentration is indicated by pseudocolor transformation, according to the scale at the lower right, with
red and violet representing highest (1920 dpm/µg of protein) and lowest (13 dpm/µg of protein) levels, respectively. Numbers
below each section refer to distance from the interaural line in mm, according to the stereotaxic atlas of Paxinos and Watson.53
Note distinct labeling of cholinergic terminals in the striatum, cerebral neocortex, hippocampus, select diencephalic nuclei, and
brainstem and spinal cord motor nuclei. Abbreviations: AM, anteromedial thalamus; AV, anteroventral thalamus; BL, basolateral
amygdala; CBL, cerebellum; CPU, caudate-putamen; CTX, cerebral neocortex; HPC, hippocampus; IPN, interpeduncular nucleus;
RT, reticular thalamus; TU, olfactory tubercle; 3, occulomotor nucleus; 6, abducens nucleus; 7, facial nucleus; 12, hypoglossal
nucleus.
was blocked by a haloperidol dose of 1 mg/kg ip; a dose
of 2.5 mg/kg ip (data not shown) decreased cerebellar
retention 86%. The lack of effect of spiroperidol pre-
treatment on 7-IBVM (14) brain retention demonstrated
that dopamine D2 receptor binding was not involved.
The high cerebellar retention of 7-IBVM (14) is also
inconsistent with D2 binding since the density of this
receptor is very low in mouse cerebellum.40,41 The
concentrations of radioactivity in liver, lung, and heart
24 h after injection of 7-IBVM (14) were also markedly
higher than the respective levels obtained with 5- and
6-IBVM (5, 10). Haloperidol (2.5 mg/kg ip) pretreat-
ment lowered radioactivity levels in liver (-74%), lung
(-88%), atria (-60%), and ventricles (-38%). This
finding is consistent with the high density of σ-receptors
in these peripheral tissues.42,43
F igu r e 7. Comparison of choline acetyltransferase (ChAT)
It is notable that, although the mouse striatum has
activity and (-)-5-[123I]IBVM ((-)-[123I]5) concentrations in four
a σ-receptor density only slightly lower than either
regions of CD-1 mouse brain (N ) 5). The concentrations have
hippocampus or hypothalamus,38 haloperidol pretreat-
been normalized to the levels obtained in striatum and are
expressed as a percentage of the striatal level. Mice were
ment did not block 7-IBVM (14) retention in this brain
region (Figure 3). It is possible that σ-receptor blockade
in the striatum may have been masked by an increase
in vesamicol receptor binding. Dopamine D2 receptors
located on striatal cholinergic neurons are generally
thought to be involved in the inhibitory control of
striatal cholinergic function.44 Enhanced retention of
sacrificed 4 h after iv injection of radiotracer. Abbreviations:
striatum (STR), hippocampus (HIPPO), cortex (CTX), cerebel-
lum (CEREB).
suggested that the σ-receptor might be involved in the
cerebellar retention of 7-IBVM. This hypothesis is
consistent with the high density of σ-receptors known
to be present in the mouse cerebellum.38 Also, while
this work was in progress, Efange et al. reported that
certain vesamicol derivatives bind to the σ-receptor with
affinities in the 10-25 nM range.39 Further evidence
that σ-receptor binding may be a major determinant of
the 24-h brain retention pattern of 7-IBVM (14) was
obtained from blocking studies with haloperidol, a
potent in vivo antagonist of both σ- and dopamine D2
receptors. Over 75% of 7-IBVM (14) cerebellar retention
a
18F-labeled 5-aminobenzovesamicol derivative in mon-
key striatum in response to pretreatment with halo-
peridol or the dopamine D2 antagonist raclopride has
been reported by Ingvar et al.45 They attributed this
phenomenon to augmentation of vesamicol receptor
binding due to enhanced cholinergic activity occurring
in response to dopamine D2 receptor blockade. How-
ever, our observation, that spiroperidol pretreatment at
a dose of 0.1 mg/kg ip did not significantly increase
striatal retention of racemic 7-IBVM (14), is not con-

Vesamicol Receptor Mapping
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17 3337
sistent with this hypothesis. It should be pointed out
that the study by Ingvar et al. was performed in
monkeys, and striatal radioactivity levels were deter-
mined by positron emission tomography up to 60 min
after radiotracer injection; the study described here was
performed in mice 24 h after tracer injection. A better
understanding of the striatal response of 7-IBVM (14)
to haloperidol pretreatment may be made possible by
drug-blocking studies of the enantiomers of 7-IBVM (14)
which have now become available.46
The relative in vitro binding affinities of 5- and
6-IBVM (5, 10) for the vesamicol receptor, as determined
in the competitive binding assay using rat cortical
homogenates, are consistent with the relative tenacity
with which these two isomers map cholinergic innerva-
tion in vivo. Although the 5-isomer 5 had only a two-
fold lower IC50 than the 6-isomer 10, the former showed
a 4-8-fold higher retention in cortical and hippocampal
brain regions 24 h after injection.
6-position is only slightly less favorable. Evidence
regarding 7-IBVM (14) is not conclusive because of
tracer cobinding to the σ-receptor. Final assessment of
7-IBVM (14) will require evaluation of its enantiomers.
Exp er im en ta l Section
1H NMR spectra were obtained in CDCl3 on a Bruker 360
or 300 MHz NMR spectrometer and are reported in parts per
million downfield from tetramethylsilane. Mass spectra were
obtained on a Finnigan 4021 GC/MS spectrometer in the
electron impact (EI) ionization mode at 70 eV. Molecular
masses are given in atomic mass units (amu) followed by
percent intensity relative to the most abundant ion. Accurate
mass spectral determinations were also obtained in the EI
mode at 70 eV. Elemental analyses were carried out by Spang
Microanalytical Laboratory, Eagle Harbor, MI. Melting points
were determined on a Thomas-Hoover capillary melting point
apparatus in open capillary tubes and are uncorrected. Flash-
chromatography utilized Merck 230-400 mesh silica gel.
Thin-layer chromatography (TLC) used Analtech 0.25-mm
glass-backed plates with fluorescent background. Visualiza-
tion was achieved by phosphomolybdic acid (PMA), iodine, or
UV illumination. High-pressure liquid chromatography (HPLC)
was performed on a Beckman Instrument Model 344 gradient
liquid chromatograph with an ABS Model 757 UV/vis detector
and a Waters Model 740 data module. Neutral alumina and
silica Sep-Paks were purchased from Waters Associates,
Milford, MA. Borosilicate glass beads (3 mm) were obtained
from Macalaster-Bicknell, Millville, NJ . Reagents and sol-
vents were purchased from commercial sources and used
without further purification unless otherwise noted. The 5-
and 8-aminobenzovesamicols (2, 3) were prepared by reacting
6,7-epoxy-1-(trifluoroacetamido)-5,8-dihydronaphthalene (1)
with 4-phenylpiperidine by published procedures.13 Polysor-
bate-80, used to dissolve IBVM isomers for in vitro receptor
binding studies using rat brain cortices, was purchased from
American Research Products Co., Solon, OH.
The ability of 5-IBVM (5) to map cholinergic innerva-
tion is also supported by its selective retention in the
atrial wall of the myocardium. From slightly above
unity at 30 min, the atria-to-ventricles radioactivity
ratio of 5-IBVM (5) rose to almost 5 at 4 h and to over
13 at 24 h. The levels of ChAT in rat heart21 indicate
a density of cholinergic innervation in atria that are
approximately 5-fold higher than in ventricles.22 Our
assay of ChAT in CD-1 mouse heart indicated a greater
than 2-fold higher level in the atria; the actual ratio,
however, may be higher because ChAT activity in
ventricle is near the sensitivity level of the assay. Our
finding of higher ChAT levels in hippocampus than in
cortex of CD-1 mouse brain is consistent with other
reports in mice27 and rats.28 The low correlation (r2 )
0.79) obtained between ChAT activities and (-)-5-[123I]-
IBVM ((-)-[123I]5) levels in the four brain regions of
CD-1 mice was due mainly to higher tracer levels in
The iodine-125 was a no-carried-added solution of Na125I (ca.
700-800 mCi/mL) in reductant free 0.1 N NaOH obtained from
Nordion International Inc., Kanata, Ontario, Canada. Radio-
activity was quantified with a Capintec Model CRC-12 radio-
isotope calibrator which was calibrated against an NBS
standard solution of Na125I. Radio-TLC analyses were per-
formed on 2.5 × 20 cm silica gel-coated glass plates (Whatman
K6F). The plates were analyzed on a Berthold automatic TLC
linear analyzer LB2832 radiochromatogram scanner immedi-
ately after development and drying. Percent exchange was
determined by integration of the chromatogram peaks and
calculated as the ratio of activity of exchanged product to the
total activity chromatographed.
(()-tr a n s-2-Hyd r oxy-5-iod o-3-(4-p h en ylp ip er id in o)te-
tr a lin (5-IBVM) (5). Meth od A. To a cooled (5 °C) solution
of (()-5-aminobenzovesamicol (2) (120 mg, 37.0 µmol) in acetic
acid (2 mL) and concentrated HCl (1 mL) was added dropwise
a solution of NaNO2 (27 mg, 40.0 µmol) in H2O (2 mL) while
the temperature of the solution was maintained under 10 °C.
After the mixture was stirred for 30 min, a solution of KI (74
mg, 44.7 µmol) and I2 (57 mg, 22.3 µmol) in H2O (1 mL) was
added dropwise. The reaction mixture was stirred at 5 °C for
3 h, allowed to warm to room temperature, and stirred
overnight. The reaction mixture was poured into saturated
NaHCO3 solution (20 mL) and extracted with ethyl acetate (3
× 20 mL). The combined extracts were washed with 10%
NaHSO3 solution, dried over anhydrous Na2SO4, and concen-
trated under reduced pressure. The residue was flash-chro-
matographed on silica gel with 15% ethyl acetate in hexane
to afford 109 mg (68%) of (()-5-iodobenzovesamicol (5) as a
white solid: mp 138-140 °C (recrystallized from ether/
hexane); IR (KBr) 3610-3275 (br), 3059, 3025, 2929, 2907,
1645, 1602 cm-1; 1H NMR (CDCl3) δ 1.68-1.97 (m, 4H), 2.44-
3.03 (m, 9H), 3.25 (d, d, J ) 16.1, 5.7 Hz, 1H), 3.84 (t, d, J )
10.2, 5.7 Hz, 1H), 4.3 (br s, OH), 6.84 (t, J ) 7.7 Hz, 1H), 7.10
(d, J ) 7.7 Hz, 1H), 7.19-7.35 (m, 5H), 7.70 (d, J ) 7.7 Hz,
1H); MS (EI, 70 eV) m/ z (rel intensity) 433 (M+, 15.12), 306
(1.73), 231 (1.10), 216 (1.13), 202 (6.32), 186 (1.53), 174
cortex than in hippocampus; a linear correlation (r2
)
0.99) was obtained if cortical results were omitted.
Higher levels of radioactivity in cortex than in hippoc-
ampus have also been observed in mouse brain with
iodine-125-labeled 5-IBVM (5) and 6-IBVM (10) (see
Table 1) as well as with carbon-11- and fluorine-18-
labeled 5-substituted benzovesamicols.24,47,48 This mis-
match between ChAT and radioactivity levels, and thus
vesamicol receptor density, was not observed in normal
human brain using (-)-5-[123I]IBVM ((-)-[123I]5).11
The distribution data in Table 1 indicated that
5-IBVM (5) and 6-IBVM (10) cleared very slowly from
nonneuronal areas of the brain. At 4 h after tracer
injection, concentrations of radioactivity were still high
in cerebellum and blood. The synthesis of new analogs
of IBVM which are less lipophilic than 5-IBVM (5) may
provide tracers that show enhanced brain extraction as
well as faster clearance of nonspecifically bound radio-
activity. This would potentially enable the determina-
tion of brain cholinergic density within a few hours of
radiotracer injection as opposed to the 22-h time period
presently required to obtain a neuronal map of the
human brain.11,12
Con clu sion
The evaluation of five positional isomers of radioio-
dinated benzovesamicol as in vivo markers for brain
cholinergic nerve density indicates that radioiodine
substitution may be optimal at the 5-position. The

3338 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17
J ung et al.
(100.00), 160 (22.81), 146 (6.25), 128 (14.46), 115 (26.41), 91
(21.44), 56 (9.80), 42 (6.10); high-resolution MS (EI, 70 eV)
((-)-5) (mp 138-140 °C; [R]23D ) -45.3°, c ) 1.5, EtOH). The
(+)-5-IBVM enantiomer ((+)-5) was obtained in 89% yield from
the (+)-diastereomer by the above-described method (mp 138-
calcd for C21H24INO 433.0903, found 433.0910. Anal. (C21H24
INO) C, H, N.
-
140 °C; [R]23 ) +44.0°, c ) 1.5, EtOH). The optical purity of
D
the (-)- and (+)-enantiomers was >98% as determined by
chiral HPLC using a Chiracel OD column (4.6 × 250 mm)
eluting with 10% 2-propanol in hexane at a flow rate of 1 mL/
min with UV detection at 254 nm. Retention times of (-)-5
and (+)-5 were 6.8 and 10.0 min, respectively.15
(()-tr a n s-2-Hyd r oxy-5-iod o-3-(4-p h en ylp ip er id in o)te-
tr a lin (5-IBVM) (5). Meth od B: (()-tr a n s-2-Hyd r oxy-8-
a m in o-5-iod o-3-(4-p h en ylp ip er id in o)t et r a lin (8-Am in o-
5-IBVM) (4). To a solution of (()-8-aminobenzovesamicol (3)
(4.28 g, 13.27 mmol) in acetic acid (125 mL) was added
dropwise a solution of iodine monochloride (2.16 g, 13.27 mmol)
in acetic acid (5 mL) at room temperature. The resulting
solution was stirred overnight, and the solvent was evaporated
under reduced pressure. The residue was dissolved in CHCl3
(30 mL), and the solution was washed with saturated NaHCO3
solution (30 mL) and extracted with CHCl3 (2 × 50 mL). The
combined extracts were dried over anhydrous Na2SO4 and
concentrated under reduced pressure. The residue was flash-
chromatographed on silica gel with 70% ethyl acetate in
hexane to afford 4.97 g (84%) of (()-8-amino-5-iodobenzove-
samicol (4) as a white solid: mp 165-169 °C (recrystallized
from ether/hexane); 1H NMR (CDCl3) δ 1.68-1.99 (m, 4H),
2.33-2.98 (m, 9H), 3.03 (d, d, J ) 16.5, 5.7 Hz, 1H), 3.67 (br
s, NH2), 3.76 (t, d, J ) 10.2, 5.7 Hz, 1H), 4.38 (br s, OH), 6.32
(d, J ) 8.4 Hz, 1H), 7.15-7.36 (m, 5H), 7.49 (d, J ) 8.4 Hz,
1H); MS (EI, 70 eV) m/ z (rel intensity) 448 (M+, 10.76), 321
(6.60), 289 (16.75), 287 (17.38), 269 (4.13), 256 (6.25), 203
(7.61), 174 (33.89), 160 (100.00), 144 (21.87), 143 (23.70), 130
(39.66), 56 (50.77), 42 (36.78); high-resolution MS (EI, 70eV)
(()-tr a n s-2-Hyd r oxy-8-iod o-3-(4-p h en ylp ip er id in o)te-
tr a lin (8-IBVM) (6). The 8-iodobenzovesamicol (6) was
obtained as a white solid in 57% yield from 8-aminobenzove-
samicol (3) by the same method (method A) described for the
synthesis of 5-iodobenzovesamicol (5): mp 194-197 °C (re-
crystallized from ether/hexane);
1H NMR (CDCl3) δ 1.72-2.05
(m, 4H), 2.36 (d, d, J ) 11.4, 2.2 Hz, 1H), 2.52-2.99 (m, 8H),
3.41 (d, d, J ) 16.8, 6.2 Hz, 1H), 3.87 (t, d, J ) 10.1, 6.2 Hz,
1H), 4.34 (br s, OH), 6.83 (t, J ) 7.8 Hz, 1H), 7.09 (d, J ) 7.8
Hz, 1H), 7.19-7.35 (m, 5H), 7.71 (d, J ) 7.8 Hz, 1H); MS (EI,
70 eV) m/ z (rel intensity) 433 (17.51), 306 (4.29), 231 (1.04),
216 (1.63), 203 (8.46), 1.86 (1.11), 174 (100.00), 160 (23.03),
145 (9.61), 56 (44.40), 42 (42.08); high-resolution MS (EI, 70
eV) calcd for C21
H24INO 433.0903, found 433.0894. Anal.
(C21H24INO) C, H, N.
(()-tr a n s-5-Am in o-2-h yd r oxy-6-n it r o-3-(4-p h en ylp ip -
er id in o)tetr a lin (5-Am in o-6-n itr o-BVM) (7). (()-5-Ami-
nobenzovesamicol (2) (3.04 g, 9.42 mmol) was added to acetic
anhydride (50 mL). After the resulting solution was stirred
for 30 min, p-toluenesulfonic acid monohydrate (2.15 g, 11.30
mmol) was added. The solution was cooled at 5 °C in an ice
bath, and 90% fuming nitric acid (1.12 mL, 22.60 mmol) was
added dropwise at a rate which maintained the reaction
temperature under 7 °C. After stirring at 5 °C for 3.5 h, the
solution was poured with stirring into ice water, and the
aqueous layer was extracted with CH2Cl2 (3 × 30 mL). The
combined organic layers were washed with saturated NaHCO3
solution and evaporated to dryness under reduced pressure.
The residue was refluxed in 6 N HCl (40 mL) solution with
stirring for 2 h to hydrolyze the acetamide. After adjusting
to pH 10 by the addition of 6 N NaOH, the solution was
extracted with CH2Cl2 (3 × 30 mL), and the combined extracts
were dried over anhydrous Na2SO4 and concentrated under
reduced pressure. The residue was flash-chromatographed on
silica gel, eluting with 40% ethyl acetate in hexane to afford
2.95 g (85%) of (()-5-amino-6-nitrobenzovesamicol (7) as a
calcd for C21H25IN2O 448.1012, found 448.1021. Anal. (C21H25
IN2O) C, H, N.
-
(()-tr a n s-2-Hyd r oxy-5-iod o-3-(4-p h en ylp ip er id in o)te-
t r a lin (5-IBVM) (5). A solution of NaNO2 (811 mg, 11.75
mmol) in H2O (5 mL) was added dropwise to a cooled (5 °C)
solution of 8-amino-5-iodobenzovesamicol (4) (4.97 g, 11.09
mmol) in 50% hypophosphorous acid (40 mL) and concentrated
HCl (10 mL) while maintaining the reaction temperature
under 10 °C. The mixture was stirred under 5 °C for 1 h,
allowed to warm to room temperature, stirred overnight, and
poured into saturated aqueous NaCl solution. The aqueous
layer was extracted with CH2Cl2 (3 × 40 mL). The combined
extracts were washed with saturated NaHCO3 solution, dried
over anhydrous sodium sulfate, and concentrated under
reduced pressure. The residue was flash-chromatographed on
silica gel, eluting with 15% ethyl acetate in hexane to afford
4.18 g (87%) of (()-5-iodobenzovesamicol (5). The 1H NMR
and IR spectra of this product were identical with those of 5
prepared from 2 by the diazotization route described above.
R esolu t ion of (()-tr a n s-2-H yd r oxy-5-iod o-3-(4-p h e-
n ylp ip er id in o)tetr a lin ((()-5-IBVM) ((+)-5 a n d (-)-5). To
a solution of (()-5-iodobenzovesamicol (5) (286 mg, 0.66 mmol),
4-(dimethylamino)pyridine (24 mg, 0.20 mmol), and triethy-
lamine (2.76 mL, 1.98 mmol) in dry chloroform (5 mL) was
added dropwise (S)-(-)-R-methoxy-R-(trifluoromethyl)pheny-
lacetyl chloride (184 mg, 0.73 mmol) by syringe at room
temperature.10,14 The resulting solution was stirred for 6 h
and then poured into ethyl acetate (20 mL). The solution was
washed with saturated NaHCO3 solution, and the aqueous
layer was extracted with ether (2 × 20 mL). The combined
extracts were dried over anhydrous Na2SO4 and concentrated
under reduced pressure. The two diastereomeric MTPA esters
were separated by preparative thin layer chromatography on
silica gel (20 cm × 20 cm, 1.0 mm) after several developments
with 5% ethyl acetate in hexane. The less polar compound
(Rf ) 0.41, 10% ethyl acetate in hexane) was the (+)-
1
yellow solid: mp 176-180 °C; H NMR (CDCl3) δ 1.70-1.99
(m, 4H), 2.42-3.02 (m, 9H), 3.26 (d, d, J ) 17.0, 5.5 Hz, 1H),
3.86 (t, d, J ) 10.4, 5.5 Hz, 1H), 4.21 (br s, OH), 6.28 (br s,
NH2
), 6.47 (d, J ) 8.8 Hz, 1H), 7.19-7.35 (m, 5H), 7.97 (d, J
) 8.8 Hz, 1H); MS (EI, 70 eV) m/ z (rel intensity) 367 (M+,
10.88), 350 (1.81), 322 (2.10), 206 (2.69), 203 (5.30), 190 (16.62),
174 (27.82), 160 (51.22), 144 (11.38), 130 (16.65), 43 (100.00);
high-resolution MS (EI, 70eV) calcd for C21H25N3O3 367.1896,
found 367.1896.
(()-tr a n s-2-Hyd r oxy-6-n itr o-3-(4-p h en ylp ip er id in o)te-
tr a lin (6-Nitr o-BVM) (8). To a cooled (5 °C) solution of
5-amino-6-nitrobenzovesamicol (7) (2.93 g, 7.97 mmol) in acetic
acid (20 mL) was added dropwise a solution of NaNO2 (605
mg, 8.77 mmol) in H2O (5 mL) while maintaining the reaction
temperature under 10 °C. The mixture was stirred at 5 °C
for 1 h, and then hypophosphorous acid (30 mL) was added.
The mixture was allowed to warm to room temperature, stirred
overnight, and then poured into saturated saline solution. The
aqueous layer was extracted with CH2Cl2 (3 × 40 mL). The
combined extracts were washed with a saturated NaHCO3
solution, dried over anhydrous Na2SO4, and concentrated
under reduced pressure. The residue was flash-chromato-
graphed on silica gel, eluting with 40% ethyl acetate in hexane
to afford 2.61 g (93%) of (()-6-nitrobenzovesamicol (8) as a
diastereomer ([R]23 ) +36.7°, c ) 1.5, EtOH), of which 195
D
mg (46%) was obtained. The more polar compound (Rf ) 0.35,
10% ethyl acetate in hexane) was the (-)-diastereomer ([R]23
D
) -56.7°, c ) 1.5, EtOH), of which 182 mg (43%) was obtained.
The (-)-diastereomer (163 mg, 0.25 mmol) was dissolved
in THF (10 mL) and MeOH (5 mL) to which was added 2 N
NaOH solution (10 mL). The mixture was stirred for 10 h at
room temperature and then extracted with ethyl acetate (3 ×
30 mL). The combined extracts were dried over anhydrous
Na2SO4 and concentrated under reduced pressure. The resi-
due was flash-chromatographed on silica gel, eluting with 15%
ethyl acetate in hexane to afford 94 mg (86%) of (-)-5-IBVM
1
yellow solid: mp 174-177 °C; H NMR (CDCl3) δ 1.68-1.98
(m, 4H), 2.40 (t, d, J ) 11.5, 2.2 Hz, 1H), 2.58 (m, 1H), 2.77-
3.09 (m, 7H), 3.42 (d, d, J ) 17.0, 5.9 Hz, 1H), 3.91 (t, d, J )
10.1, 5.9 Hz, 1H), 4.30 (br s, OH), 7.19-7.36 (m, 6H), 7.97 (d,
J
) 9.3 Hz, 1H), 7.99 (s, 1H); MS (EI, 70 EV) m/ z (rel
intensity) 352 (M+, 29.26), 335 (3.38), 321 (1.46), 203 (7.86),
191 (2.04), 174 (100.00), 160 (20.10), 144 (11.54), 128 (19.13),

Vesamicol Receptor Mapping
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17 3339
115 (45.31); high-resolution MS (EI, 70 eV) calcd for C21H24N2O3
352.1787, found 352.1791.
132 (13.8), 120 (16.3), 91 (12.4); high-resolution MS (EI, 70
eV) calcd for C21H26N2O 322.2045, found 322.2028. Anal.
(C21H26N2O) C, H, N.
(()-tr a n s-2-Hyd r oxy-6-a m in o-3-(4-p h en ylp ip er id in o)-
tetr a lin (6-Am in o-BVM) (9). The 6-nitrobenzovesamicol (8)
(1.24 g, 3.51 mmol) was dissolved in ethyl acetate (20 mL) and
ethanol (30 mL), and 10% palladium on activated carbon (400
mg) was added. The mixture was hydrogenated at 40 psi
overnight. After filtration, the solution was concentrated
under reduced pressure, and the residue was flash-chromato-
graphed on silica gel, eluting with 50% ethyl acetate in hexane
to afford 1.05 g (93%) of (()-6-aminobenzovesamicol (9) as a
white solid: mp 172-173 °C; 1H NMR (CDCl3) δ 1.68-1.94
(m, 4H), 2.36 (t, d, J ) 11.4, 2.1 Hz, 1H), 2.55 (m, 1H), 2.65-
2.86 (m, 6H), 2.96 (d, J ) 11.2 Hz, 1H), 3.21 (d, d, J ) 15.5,
5.8 Hz, 1H), 3.54 (br s, NH2) 3.83 (t, d, J ) 9.7, 5.8 Hz, 1H),
4.38 (br s, OH), 6.44 (d, J ) 2.4 Hz, 1H), 6.51 (d, d, J ) 8.1,
2.4 Hz, 1H), 6.90 (d, J ) 8.1 Hz, 1H), 7.19-7.34 (m, 5H); MS
(EI, 70 eV) m/ z (rel intensity) 322 (M+, 5.63), 304 (20.46), 291
(4.36), 279 (1.99), 215 (2.67), 202 (4.48), 174 (42.65), 161 (9.34),
160 (9.67), 144 (100.00), 131 (23.37), 57 (39.74), 43 (85.29);
high-resolution MS (EI, 70 eV) calcd for C21H26N2O 322.2045,
found 322.2036. Anal. (C21H26N2O) C, H, N.
(()-tr a n s-2-Hyd r oxy-6-iod o-3-(4-p h en ylp ip er id in o)te-
tr a lin (6-IBVM) (10). 6-Iodobenzovesamicol (10) was ob-
tained as a white solid in 66% yield from 6-aminobenzovesa-
micol (9) by using method A described for the synthesis of
5-iodobenzovesamicol (5): mp 142-143 °C (recrystallized from
ether/hexane); 1H NMR (CDCl3) δ 1.71-1.95 (m, 4H), 2.36 (t,
d, J ) 11.4, 2.2 Hz, 1H), 2.52-2.57 (m, 1H), 2.69-2.97 (m, 7H),
3.26 (d, d, J ) 16.3, 5.8 Hz, 1H), 3.84 (t, d, J ) 10.0, 5.8 Hz,
1H), 4.35 (br s, OH), 6.85 (d, J ) 8.1 Hz, 1H), 7.19-7.35 (m,
5H), 7.44 (d, J ) 8.1 Hz, 1H), 7.46 (s, 1H); MS (EI, 70 eV) m/ z
(rel intensity) 433 (M+, 19.27), 415 (2.15), 314 (1.10), 307 (4.17),
245 (2.01), 231 (2.26), 203 (8.37), 202 (10.41), 174 (100.00), 160
(8.58), 146 (7.62), 128 (24.39), 115 (41.54), 56 (49.43), 42
(56.26); high-resolution MS (EI, 70 eV) calcd for C21H24INO
433.0903, found 433.0889. Anal. (C21H24INO) C, H, N.
(()-tr a n s-8-Am in o-2-h yd r oxy-7-n it r o-3-(4-p h en ylp ip -
er id in o)tetr a lin (8-Am in o-7-n itr o-BVM) (11). 8-Amino-7-
nitrobenzovesamicol (11) was obtained in 78% yield from
8-aminobenzovesamicol (3) by using the method described for
the synthesis of 5-amino-6-nitrobenzovesamicol (7): mp 153-
154 °C (recrystallized from ether/hexane); 1H NMR (CDCl3) δ
1.69-1.98 (m, 4H), 2.36-2.48 (m, 2H), 2.53-2.62 (m, 1H),
2.80-3.03 (m, 6H), 3.12 (d, d, J ) 15.7, 6.3 Hz, 1H), 3.94 (t, d,
J ) 9.4, 6.3 Hz, 1H), 6.30 (br s, NH2), 6.48 (d, J ) 8.9 Hz, 1H),
7.19-7.36 (m, 5H), 7.98 (d, J ) 8.9 Hz, 1H); MS (EI, 70 eV)
m/ z (rel intensity) 367 (M+, 9.39), 350 (2.12), 349 (3.14), 332
(1.87), 229 (2.03), 216 (3.15), 206 (2.63), 204 (2.38), 203 (11.49),
190 (6.79), 189 (15.46), 174 (100.00), 172 (18.37), 162 (52.40),
161 (22.50), 160 (50.92), 144 (11.54), 143 (17.76), 130 (30.96),
117 (28.74), 115 (29.11), 103 (30.36), 91 (48.06); high-resolution
MS (EI, 70 eV) calcd for C21H25IN3O3 367.1896, found 367.1887.
(()-tr a n s-2-Hyd r oxy-7-n itr o-3-(4-p h en ylp ip er id in o)te-
tr a lin (7-Nitr o-BVM) (12). 7-Nitrobenzovesamicol (12) was
obtained in 86% yield from 8-amino-7-nitrobenzovesamicol (11)
by using the method described for the synthesis of 6-nitroben-
zovesamicol (8): mp 166-170 °C; 1H NMR (CDCl3) δ 1.73-
1.96 (m, 4H), 2.23-3.09 (m, 9H), 3.42 (d, d, J ) 16.3, 5.8 Hz,
1H), 3.90 (t, d, J ) 10.0, 5.8 Hz, 1H), 4.32 (br s, OH), 7.20-
7.36 (m, 6H), 7.97 (d, J ) 8.7 Hz, 1H), 7.99 (s, 1H); MS (EI, 70
eV) m/ z (rel intensity) 352 (M+, 100.00), 335 (26.3), 322 (11.6),
307 (11.1), 219 (8.0), 203 (20.2), 174 (91.2), 160 (20.8), 145
(14.5), 115 (26.0), 91 (23.7); high-resolution MS (EI, 70 eV)
calcd for C21H24N2O3 352.1787, found 352.1791.
(()-tr a n s-2-Hyd r oxy-7-iod o-3-(4-p h en ylp ip er id in o)te-
tr a lin (7-IBVM) (14). 7-Iodobenzovesamicol (14) was ob-
tained as a white solid in 45% yield from 7-aminobenzovesa-
micol (13) by using method A described for the synthesis of
5-iodobenzovesamicol (5): mp 185-187 °C (recrystallized from
ether/hexane); 1H NMR (CDCl3) δ 1.65-1.95 (m, 4H), 2.37 (t,
d, J ) 11.4, 2.2 Hz, 1H), 2.51-2.58 (m, 1H), 2.70-2.99 (m, 7H),
3.26 (d, d, J ) 16.3, 5.8 Hz, 1H), 3.84 (t, d, J ) 10.0, 5.8 Hz,
1H), 4.37 (br s, OH), 6.84 (d, J ) 8.1 Hz, 1H), 7.19-7.35 (m,
5H), 7.43 (d, J ) 8.1 Hz, 1H), 7.47 (s, 1H); MS (EI, 70 eV) m/ z
(rel intensity) 433 (M+, 25.00), 203 (11.70), 174 (100.00), 160
(14.38), 146 (7.30), 128 (20.58), 115 (37.65), 91 (31.77), 56
(47.96), 42 (46.73); high-resolution MS (EI, 70 eV) calcd for
C21H24INO 433.0903, found 433.0881. Anal. (C21H24INO) C,
H, N.
(()-tr a n s-5-Am in o-2-h yd r oxy-3-[4-(4′-iod op h en yl)p ip -
er id in o]tetr a lin (5-Am in o-4′-iod o-BVM) (15) a n d (()-
tr a n s-8-Am in o-2-h ydr oxy-3-[4-(4′-iodoph en yl)piper idin o]-
tetr a lin (8-Am in o-4′-iod o-BVM) (16). To a solution of 6,7-
epoxy-1-(trifluoroacetamido)-5,8-dihydronaphthalene (1) (420
mg, 1.63 mmol) in EtOH (20 mL) was added 4-(4′-iodophenyl)-
piperidine16 (920 mg, 3.32 mmol). The resulting solution was
refluxed for 32 h and concentrated under reduced pressure.
The residue was flash-chromatographed on silica gel, eluting
with 50% ethyl acetate in hexane to afford 5-amino-4′-
iodobenzovesamicol (15) (301 mg, 41%) and 8-amino-4′-iodo-
benzovesamicol (16) (289 mg, 39%). 15 (Rf 0.27, 50% ethyl
acetate in hexane): mp 198-201 °C; 1H NMR (CDCl3) δ 1.68-
1.94 (m, 4H), 2.41-3.01 (m, 9H), 3.25 (d, d, J ) 16.0, 5.5 Hz,
1H), 3.58 (br s, NH2), 3.86 (t, d, J ) 10.4, 5.5 Hz, 1H), 6.55 (d,
J ) 7.7 Hz, 1H), 6.59 (d, J ) 7.7 Hz, 1H), 6.99 (t, J ) 7.7 Hz,
1H), 7.00 (d, J ) 8.4 Hz, 2H), 7.63 (d, J ) 8.4 Hz, 2H); MS
(EI, 70 eV) m/ z (rel intensity) 448 (M+, 8.03), 300 (20.77), 286
(21.32), 229 (2.23), 217 (2.43), 174 (4.62), 163 (59.72), 162
(34.30), 160 (17.54), 144 (100.00), 130 (43.38), 115 (23.86), 56
(43.60); high-resolution MS (EI, 70 eV) calcd for C21H25IN2O
448.1012, found 448.1006. Anal. (C21H25IN2O) C, H, N.
16 (Rf 0.20, 50% ethyl acetate in hexane): 1H NMR (CDCl3)
δ 1.62-1.92 (m, 4H), 2.30-3.01 (m, 9H), 3.13 (d, d, J ) 15.7,
6.3 Hz, 1H), 3.62 (br s, NH2), 3.92 (m, 1H), 6.56 (d, J ) 7.7
Hz, 2H), 6.98 (t, J ) 7.7 Hz, 1H), 7.00 (d, J ) 8.4 Hz, 1H),
7.63 (d, J ) 8.4 Hz, 2H).
(()-tr a n s-2-Hyd r oxy-3-[4-(4′-iod op h en yl)p ip er id in o]te-
tr a lin (4′-IBVM) (17). 4′-Iodobenzovesamicol (17) was ob-
tained in 81% yield from 8-amino-4′-iodobenzovesamicol (16)
by using the second step of method B (Scheme 1) described
for the synthesis of 5-iodobenzovesamicol (5) from 8-amino-5-
iodobenzovesamicol (4): mp 148-149 °C (recrystallized from
ether/hexane); 1H NMR (CDCl3) δ 1.66-1.92 (m, 4H), 2.39 (t,
d, J ) 11.3, 1.9 Hz, 1H), 2.51 (t, t, J ) 11.9, 3.9 Hz, 1H), 2.78-
3.01 (m, 7H), 3.32 (d, d, J ) 16.1, 5.8 Hz, 1H), 3.89 (t, d, J )
10.1, 5.8 Hz, 1H), 7.00 (d, J ) 8.4 Hz, 2H), 7.10-7.19 (m, 4H),
7.63 (d, J ) 8.4 Hz, 2H); MS (EI, 70 eV) m/ z (rel intensity)
443 (M+, 38.49), 328 (6.04), 300 (100.00), 288 (12.61), 229
(5.14), 217 (6.15), 174 (18.29), 156 (9.06), 144 (13.10), 129
(58.68), 115 (66.64), 56 (94.65), 42 (98.26); high-resolution MS
(EI, 70 eV) calcd for C21H24INO 433.0903, found 433.0904.
Anal. (C21H24INO) C, H, N.
Ra d ioch em ica l Syn th eses. In a general procedure, solid-
phase radioiodide exchange reactions were performed in a
septum-closed 3-mL multidose vial by modification of our
previously described method.18,19,49 Heating was accomplished
with an oil bath, and the temperature was that of the
equilibrated oil bath.
A solution of (NH4)2SO4 (5.0 mg in 15 µL of deionized H2O),
(()-5-iodobenzovesamicol (5) (20 µg in 20 µL of ethanol), and
three layers of 3-mm glass beads were successively placed in
a 3-mL multidose vial; 8 mCi of Na125I was added via syringe.
The syringe was rinsed with acetone (2 × 50 µL), which was
added to the reaction vial, and the sides of the reaction vial
were washed down with EtOH (2 × 50 µL). The reaction vial
was crimped and fitted with a distillate condenser, and the
reaction mixture was heated to dryness at 145 °C in an oil
(()-tr a n s-2-Hyd r oxy-7-a m in o-3-(4-p h en ylp ip er id in o)-
tetr a lin (7-Am in o-BVM) (13). 7-Aminobenzovesamicol (13)
was obtained in 73% yield from 7-nitrobenzovesamicol (12) by
using the method described for the synthesis of 6-amino-BVM
1
(9): mp 178-180 °C; H NMR (CDCl3) δ 1.69-1.94 (m, 4H),
2.36 (t, d, J ) 11.4, 2.2 Hz, 1H), 2.54 (m, 1H), 2.70-2.98 (m,
7H), 3.21 (d, d, J ) 16.0, 5.8 Hz, 1H), 3.54 (br s, NH2), 3.84
(m, 1H), 4.38 (br s, OH), 6.46 (d, J ) 2.3 Hz, 1H), 6.50 (d, d, J
) 8.1, 2.3 Hz, 1H), 6.89 (d, J ) 8.1 Hz, 1H), 7.19-7.34 (m,
5H); MS (EI, 70 eV) m/ z (rel intensity) 322 (M+, 100.00), 291
(3.1), 203 (14.2), 189 (6.9), 174 (36.5), 161 (10.2), 144 (16.6),

3340 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17
J ung et al.
bath. Air (20 mL) was then slowly introduced via syringe
through the septum of the reaction vial for 1 min to completely
dry the reaction mixture. The reaction mixture was main-
tained at 155 °C for an additional 30 min, cooled to room
temperature, dissolved in acetone (1 mL), and subjected to
radio-TLC analysis (silica gel, CHCl3/EtOH, 97/3). The radio-
chemical yield was 89%.
received bolus tail-vein injections of 9.5-10.5 µCi of racemic
7-[125I]IBVM ([125I]14) in 150-200 µL of isotonic acetate buffer.
The mice were sacrificed 24 h after radiotracer injection;
regional brain and peripheral tissue samples were processed
as described above. The same experiment was repeated at
higher drug dose levels: haloperidol (2.5 mg/kg), spiroperidol
(0.5 mg/kg); N ) 5/drug group and N ) 2 for the control group.
IC50 Deter m in a tion s. The competitive binding assay
employed here is a modification of that reported by Ruberg51
which was derived from the in vitro autoradiographic method
of Marien and co-workers.52 In place of [3H]vesamicol, we
utilized a tracer developed recently in our laboratory, [3H]-
MABV, to avoid possible complications that might arise from
the reported binding of [3H]vesamicol to a second site called
the vesamicol-binding protein.7 [3H]MABV (82 Ci/mmol) was
labeled using a procedure analogous to that described for
the preparation of [11C]MABV24 except that [3H]methyl iodide
was used in place of [11C]methyl iodide. There was no
significant difference in results obtained from incubations
conducted at 25 or 37 °C. Therefore, the latter temperature
was utilized.
Following removal of the acetone by a gentle stream of
argon, the dry reaction mixture was dissolved in CH2Cl2 (3 ×
1 mL) and transferred to a silica Sep-Pak cartridge. Elution
with hexane (6 mL) followed by hexane/ethyl acetate (9/1, 14
mL) removed the less polar impurities. Further elution with
hexane/ethyl acetate (1/1, 8 mL) afforded 6.36 mCi of (()-5-
[
125I]iodobenzovesamicol ([125I]5) in >96% radiochemical purity
as determined by radio-TLC. The specific activity of the
exchanged product was then estimated based on starting
quantities of substrate and final radiochemical yield. Prior
to biological evaluation, the hexane/ethyl acetate solution was
evaporated to dryness under argon flow. The radioligand was
formulated in ethyl alcohol/0.005 M acetate-buffered saline,
pH 4.5 (5/95).
1. Tissu e P r ep a r a tion . Five female Sprague-Dawley
rats weighing 200-250 g were decapitated while under ether
anesthesia, and whole cerebral cortices were quickly removed,
pooled, and homogenized in 9 vol of pH 7.2 Tris buffer (120
mM NaCl, 50 mM Tris, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2,
HCl to adjust pH) with a polytron at a setting of 7 for 10-15
s. The crude homogenate was centrifuged at 50000g for 1 h,
the supernatant discarded, and the pellet resuspended in 5
vol of buffer and recentrifuged at 50000g for 1 h. The resulting
pellet was reserved and reconstituted in buffer to 10 times the
original volume of tissue to furnish the final homogenate
preparation. Protein concentration, measured by the Bradford
assay, was 3-4 µg/µL. Preparations were stored at -70 °C
until used.
2. Bin d in g Assa y Con d ition s for IC50 Deter m in a tion s.
Incubations were performed at 37 °C for 1 h in a total volume
of 500 µL of Tris-salt buffer, containing test drug dissolved in
90/10 Tris buffer/ethanol:Polysorbate-80 (4:1), 0.6 nM [3H]-
MABV (∼400 fmol of MABV/sample), and 150 µg of cortex
homogenate protein (∼30-40 fmol of specific binding sites).
Nonspecific binding of [3H]MABV was determined in the
presence of either 4 µM unlabeled (()-MABV or 10 µM (()-
vesamicol. Incubations were terminated by filtration through
0.1% poly(ethylenimine)-treated Whatman GF/B filters in a
Brandel cell harvester followed by washing of the filters with
fill-suction cycles with room temperature buffer (∼10 mL total
volume) and a final rinse with deionized water. Filters were
removed and counted by liquid scintillation using 10 mL of
scintillation cocktail. The mean ( SD of each data set was
calculated and corrected versus negative controls (no homo-
genate). The percent of inhibition of control [3H]MABV
binding versus the log of the test drug concentration was
plotted to determine the IC50 value for each drug. Inhibition
data were fit to a single binding site model using a nonlinear
least-squares algorithm. The equation used to relate specific
binding relative to control specific binding (% of control
binding) to the concentration of inhibitor added (I) is
Tissu e Distr ibu tion Stu d ies in Mice. These studies were
performed in 6-8 week old female CD-1 mice (18-32 g)
obtained from Charles River, Inc. For each time interval
evaluated, 4-5 mice under light ether anesthesia received
bolus tail-vein injections of 7.0-13.3 µCi of racemic 125I-labeled
compound in 0.05-0.10 mL of isotonic acetate buffer, pH 4.5.
The mice were sacrificed by decapitation at designated time
intervals. The whole brain was rapidly separated from the
skull, and the entire cerebral cortex, cerebellum, striatum,
hippocampus, and approximately 75% of the hypothalamus
were excised, weighed, and counted on
a Packard 5260
Autogamma counter. The remainder of the brain was also
weighed and counted, as were samples of four peripheral
tissues and blood (0.10 mL) obtained by cardiac puncture.
Tissue concentrations are expressed as % injected dose/g of
tissue (% dose/g ( SD) normalized to a standard 25-g mouse
weight.
Recep tor Scr een in g Assa ys. Receptor screening assays
were performed by NovaScreen, a commercial research and
development support service, located in Hanover, MD. The
study was funded by the National Institute of Mental Health
as part of their Psychoactive Drug Discovery Program.
1. Bin d in g Assa ys. The hydrochloride salt of 7-IBVM (14)
dissolved in 4% aqueous dimethyl sulfoxide was screened in a
panel of 26 assays (N ) 2) at a concentration of 1 × 10-5 M.
Binding of 7-IBVM (14) was determined in 14 neurotransmit-
ter receptor assays (adenosine, R1, R2, â, dopamine 1, dopamine
2, GABAA, GABAB, serotonin 1, serotonin 2, NMDA, kainate,
quisqualate, and strychnine-sensitive glycine), five regulatory
site assays (central benzodiazine, strychnine-insensitive gly-
cine, PCP, MK-801, and σ), four ion channel binding assays
(calcium type N, calcium types T and L, chloride, and low-
conductance potassium), and three second messenger site
assays (forskolin, phorbol ester, and inositol triphosphate).
2. σ-Recep tor Bin d in g Assa y. Solutions of [3H]di(2-tolyl)-
guanidine (DTG)50 were added to preparations of guinea pig
brain membranes containing varying concentrations of 7-IBVM
(14) in 50 mM Tris-HCl buffer, pH 7.4. The final concentration
of DTG was 2.0 nM. The preparations were incubated at 25
°C for 60 min. The reaction was terminated by rapid vacuum
filtration onto glass fiber filters. The amount of radioactivity
retained on the filters was determined by liquid scintillation
counting. The degree of nonspecific binding was determined
in the presence of 10 µM haloperidol. Reference compounds
using this assay had the following Ki values: haloperidol (2.8
nM), DTG (31.0 nM), (+)-3-PPP (124.6 nM), (+)SKF-10047
(1263.3 nM).
IC50
% of control binding ) (100.0 - BB)
IC50 + I
where BB is a constant background binding term that repre-
sents the nondisplaceable binding seen at the highest concen-
trations of added inhibitor. This nondisplaceable binding may
represent binding of [3H]MABV to a second, low-affinity
binding site.
Dr u g-Block in g Stu d ies. Haloperidol lactate (im haldol
brand) was purchased from McNeil Pharmaceutical, Spring
House, PA, and spiroperidol hydrochloride from RBI, Natick,
MA. Female CD-1 mice (20-27 g), prepared as described
above, received ip injections of either haloperidol (1.0 mg/kg
in 10-15 µL of vehicle) or spiroperidol (0.1 mg/kg in 150-200
µL of physiological saline); control animals received equal
volumes of vehicle; N ) 5/group. One hour later, all mice
In Vivo Au tor a d iogr a p h y. Female Sprague-Dawley rats
underwent cannulation of a femoral vein under light ether
anesthesia. Following recovery for a minimum of 1 h, 0.75-
1.0 mCi of (-)-5-[125I]IBVM ((-)-[125I]5) was injected as an
intravenous bolus followed immediately by flushing of the
catheter with fresh saline solution. Animals were killed by
injection of pentobarbital and KCl for cardioplegia followed
by decapitation. Brains were dissected, cut coronally, caudal

Vesamicol Receptor Mapping
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17 3341
to the inferior colliculus, and frozen in crushed dry ice. Brains
were mounted on microtome chucks and coated with frozen
section embedding medium (Lipshaw, Inc., Detroit, MI) to
prevent desiccation. Triplicate, 20 µm-thick, coronal sections
were cut throughout the brain at intervals of 0.5 mm on a
cryostat microtome at -18 °C. Frozen sections were mounted
on chilled glass microscope slides and rapidly desiccated on a
hotplate at 60-70 °C. Autoradiograms of radioactivity dis-
tribution were obtained by apposition of the tissue to tritium-
sensitive X-ray film (Hyperfilm-3H; Amersham) for 3-7 days.
Films were scanned and analyzed with the use of a micro-
computer-assisted video densitometry system (MCID system,
Imaging Research, St. Catherines, Ontario).
Cor r ela t ion Bet w een Ch AT a n d (-)-5-[123I]IBVM
((-)-[123I]5) in Mou se Br a in . 1. Tissu e Distr ibu tion
An a lysis of (-)-[123I]5. The tracer (-)-[123I]5 was synthesized
by the method of Van Dort and co-workers.19 Five female CD-1
mice under light ether anesthesia were injected via the femoral
vein with 35-46 µCi of (-)-[123I]5. Four hours later, the
animals were decapitated and selected tissues were quickly
excised, weighed, and counted in an autogamma counter. The
tissues were stored in their counting vials at -70 °C for 6 or
more days (>10 iodine-123 half-lives) and thawed as needed
for subsequent ChAT assay.
Refer en ces
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(2) Rogers, G. A.; Parsons, S. M. Photoaffinity Labeling of the
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2. Ch AT Assa y. Brain samples from five of the mice were
homogenized in a Kontes glass tissue homogenizer; heart
tissue samples were homogenized with a polytron; 50 µL of a
buffered choline solution,25 prepared from 1.49 g (4.0 mmol)
of EDTA (disodium salt), 6.5 mg (0.02 mmol) of eserine
(physostigmine), and 368 mg (2.0 mmol) of choline hydrobro-
mide dissolved in 100 mL of 0.05 M Tris-HCl buffer, was mixed
with 10 µL of [14C]acetyl-S-coenzyme A (DuPont NEN; CAT
assay grade, 0.01 µCi/µL, 2.5 × 10-3 M). The above mixture
was combined with 40 µL of brain homogenate supernatant
and incubated at ambient temperature for 15 min. The final
concentrations of the reagents in the incubation mixture were
2.5 × 10-4 M acetyl-S-coenzyme A (2.2 × 103 dpm/µL), 8.0 ×
10-3 M choline hydrobromide, 2 × 10-2 M EDTA, and 1 × 10-4
M eserine.
The incubation mixture was carefully transferred by mi-
cropipet to a preequilibrated neutral alumina Sep-Pak and
eluted with acetone-water (9:1, v/v). Fifteen fractions of
approximately 0.5 mL each were collected directly into scintil-
lation vials, mixed with 10 mL of biodegradable scintilla-
tion fluid, and counted. Control studies were conducted
demonstrating that under typical assay conditions, elution of
[14C]acetylcholine was complete after 15 fractions (>80%
recovery), while [14C]acetyl-S-coenzyme A was completely
retained.
Sta tistica l An a lysis. Data are expressed as the mean (
SD. To compare means, the two-tailed t-test for unpaired
samples was used. Linear regression analysis was performed
with the linear curve-fit option in CA-Cricket Graph III,
version 1.5.2, for the Macintosh computer, Computer Associ-
ates International, Inc., Islandia, NY.
Hu m a n e Tr ea tm en t of An im a ls. In all experiments with
living animals, studies were performed in accordance with
the United States Department of Agriculture Animal Welfare
Act.
Ack n ow led gm en t. This research was supported by
National Institutes of Health Grants NS 25656, NS
24896, and AG 08671. We appreciate National Institute
of Mental Health support of the Psychoactive Drug
Discovery Program which paid for the receptor screen-
ing of 7-IBVM (14). We thank the Phoenix Memorial
Laboratory of the University of Michigan for use of their
facilities, Clarke Hagen for preparation of (-)-5-[123I]-
IBVM, and Teresa Pisani, Anne Chen, and Mary Burton
for technical assistance in the biological studies. We
also thank Linder Markham for help in preparing this
manuscript.
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