Synthesis and in vitro biological evaluation of carbonyl group-containing inhibitors of vesicular acetylcholine transporter

Article abstract of DOI:10.1021/jm9017916

To identify selective high-affinity inhibitors of the vesicular acetylcholine transporter (VAChT), we have interposed a carbonyl group between the phenyl and piperidyl groups of the prototypical VAChT ligand vesamicol and its more potent analogues benzovesamicol and 5-aminobenzovesamicol. Of 33 compounds synthesized and tested, 6 display very high affinity for VAChT (K _i, 0.25-0.66 nM) and greater than 500-fold selectivity for VAChT over σ₁ and σ₂ receptors. Twelve compounds have high affinity (K_i, 1.0-10 nM) and good selectivity for VAChT. Furthermore, 3 halogenated compounds, namely, trans-3-[4-(4-fluorobenzoyl) piperidinyl]-2-hydroxy-1,2,3,4-tetrahydronaphthalene (28b) (K_i = 2.7 nM, VAChT/sigma selectivity index = 70), trans-3-[4-(5-iodothienylcarbonyl) piperidinyl]-2-hydroxy-1,2,3,4-tetrahydronaphthalene (28h) (K_i = 0.66 nM, VAChT/sigma selectivity index = 294), and 5-amino-3-[4-(p-fluorobenzoyl) piperidinyl]-2-hydroxy-1,2,3,4,-tetrahydronaphthalene (30b) (K_i = 2.40 nM, VAChT/sigma selectivity index = 410) display moderate to high selectivity for VAChT. These three compounds can be synthesized with the corresponding radioisotopes so as to serve as PET/SPECT probes for imaging the VAChT in vivo.

Full text of DOI:10.1021/jm9017916

J. Med. Chem. 2010, 53, 2825–2835 2825
DOI: 10.1021/jm9017916
Synthesis and in Vitro Biological Evaluation of Carbonyl Group-Containing Inhibitors of Vesicular
Acetylcholine Transporter
Simon M. N. Efange,†,^ Anil B. Khare,† Krystyna von Hohenberg,† Robert H. Mach,‡, Stanley M. Parsons,§ and
Zhude Tu*,†,
†Departments of Radiology, Medicinal Chemistry and Neurosurgery, University of Minnesota, Minneapolis, Minnesota 55455,
‡Departments of Radiology and Physiology & Pharmacology, Wake Forest University School of Medicine, Winston-Salem,
North Carolina 27157, §Department of Chemistry and the Graduate Program in Biochemistry and Molecular Biology, University of California,
Santa Barbara, California 93106, Department of Radiology, Washington University School of Medicine, St. Louis, Missouri 63110, and
^Department of Chemistry, University of Buea, P.O. Box 63, Buea, Cameroon
Received December 2, 2009
To identify selective high-affinity inhibitors of the vesicular acetylcholine transporter (VAChT), we
have interposed a carbonyl group between the phenyl and piperidyl groups of the prototypical VAChT
ligand vesamicol and its more potent analogues benzovesamicol and 5-aminobenzovesamicol. Of 33
compounds synthesized and tested, 6 display very high affinity for VAChT (Ki, 0.25-0.66 nM) and
greater than 500-fold selectivity for VAChT over σ1 and σ2 receptors. Twelve compounds have high
affinity (Ki, 1.0-10 nM) and good selectivity for VAChT. Furthermore, 3 halogenated compounds,
namely, trans-3-[4-(4-fluorobenzoyl)piperidinyl]-2-hydroxy-1,2,3,4-tetrahydronaphthalene (28b) (Ki =
2.7 nM, VAChT/sigma selectivity index = 70), trans-3-[4-(5-iodothienylcarbonyl)piperidinyl]-2-hydro-
xy-1,2,3,4-tetrahydronaphthalene (28h) (Ki = 0.66 nM, VAChT/sigma selectivity index = 294), and 5-
amino-3-[4-(p-fluorobenzoyl)piperidinyl]-2-hydroxy-1,2,3,4,-tetrahydronaphthalene(30b) (Ki =2.40nM,
VAChT/sigma selectivity index = 410) display moderate to high selectivity for VAChT. These three
compounds can be synthesized with the corresponding radioisotopes so as to serve as PET/SPECT probes
for imaging the VAChT in vivo.
Introduction
though studies have revealed that the neurophathologies of
neurodegenerative diseases such as Alzheimer’s Disease and
Parkinson’s Disease are associated with alterations in the levels
of AChE.13 With the discovery of the anticholinergic proper-
ties of the lipophilic aminoalcohol (-)-trans-2-(4-phenylpiper-
idino)cyclohexanol (vesamicol (1), Figure 1), and the sub-
sequent demonstration of a unique binding site for this mole-
cule (the erstwhile vesamicol receptor) on cholinergic synaptic
vesicles,14-16 efforts were initiated to further characterize this
site and to evaluate its potential utility as a cholinergic marker.
In rat brain sections, the binding of [3H]vesamicol was found to
exhibit a regional heterogeneity which is correlated with ChAT
activity and [3H]hemicholinium-3 binding (an indicator of
SDHAChT).17,18 These results suggested that the vesamicol
receptor may be a reliable cholinergic marker. Although
problems were subsequently encountered with [3H]vesamicol,
these earlier predictions have been confirmed with the help of
molecular biology. The vesamicol binding protein hereinafter
referred to as the vesicular acetylcholine transporter (VAChT),
was cloned from the nematode Caenorhabditis elegans, three
species of Torpedo, rat, and human beings.19-24 ACh accumu-
lation and vesamicol binding are expressed by a single poly-
peptide, suggesting that all the essential components of ACh
storage reside in that polypeptide. Thus, the terms vesamicol
receptor and VAChT are synonymous.
Alzheimer’s disease, Down’s syndrome, Parkinson’s dis-
ease, and schizophrenia are generally characterized by pro-
gressive diminution in cognitive function associated with the
loss of cholinergic neurons and synapses in the brain.1-5 For
example, in Alzheimer’s disease, the severity of cognitive
dysfunction is correlated with loss of cholinergic synaptic
elements in the cortex and subcortical brain areas.6-12 Until
recently, cholinergic neurons were identified by the expression
of only three marker proteins: choline acetyltransferase
(ChATa), the sodium-dependent high-affinity choline trans-
porter (SDHAChT), and acetylcholinesterase (AChE). To
date, ChAT and SDHAChT are widely accepted as reliable
cholinergic markers. However, AChE is viewed as less reliable
because of its occurrence in cholinergic and noncholinergic
neurons and oligodendrocytes. This view has persisted even
*Address correspondence to: Zhude Tu, Ph.D., Division of Radi-
ological Sciences, Washington University School of Medicine, Campus
Box: #8225 510 S. Kingshighway Blvd., St. Louis, MO 63110. Tele-
phone: 314-362-8487, Fax: 314-262-8555, E-mail: tuz@mir.wustl.edu.
aAbbreviations: ABV, aminobenzovesamicol; ACh, acetylcholine;
AChE, acetylcholinesterase; Anal., Analysis; Calcd., Calculated; ChAT,
choline acetyltransferase; CIMS.,Chemical ionization mass spectro-
metry (CIMS) choline acetyltransferase; DCC, N,N0-dicyclohexylcar-
bodiimide; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfox-
ide; DTG, 1,3-ditolylguanidine; NBS, N-bromosuccinimide; ND, not
determined; PET, positron emission tomography; SPECT, single photon
emission computed tomography; SDHAChT, sodium-dependent high-
affinity choline transporter; THF, tetrahydrofuran; TLC, thin layer
chromatography; VAChT, vesicular acetylcholine transporter; Vesami-
col, (-)-trans-2-(4-phenylpiperidino)cyclohexanol.
In several studies of the rat brain, staining for VAChT
protein and mRNA and ChAT protein and mRNA has
unequivocally revealed that VAChT is localized to synaptic
vesicles within cholinergic terminals.25-29 Consequently,
r
2010 American Chemical Society
Published on Web 03/10/2010
pubs.acs.org/jmc

2826 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Efange et al.
Figure 1. Representative compound structures for VAChT.
VAChT is now firmly established as a reliable cholinergic
marker.
(-)-trans-2-hydroxy-3-(4-(4-[18F]fluorobenzoyl)piperidino)-
tetralin, in this new class, and (b) in vivo evaluation of the
compound in rodents and monkeys. The initial evaluation
suggests that this radiotracer has specific binding to the
VAChT enriched striatum. The process of determining the
potential clinical utility of this tracer for PET imaging of
VAChT is still in progress.
In this paper, we will detail our exploration of this new class
of compounds to identify selective high-affinity analogues for
future investigation of VAChT in vivo. In order to discuss the
comprehensive structure-activity relationships, we include in
this article the molecular structures and in vitro data for
compounds that wereportedearlier.33 Theinvestigationswere
inspired by (1) the observation that VAChT expression in
brain is correlated with the severity of dementia associated
with cholinergic deficiency in patients with neurodegenerative
diseases;34-37 (2) the marginal selectivity of [3H]vesamicol14,38,39
and the inability of this ligand to adequately reflect the loss of
cholinergic neurons in experimental animals17,18,40-42 or in
Alzheimer’s disease;35 and (3) the need for a suitable radio-
ligand that could serve as PET probe for imaging VAChT
in vivo.
Recently, Prado and colleagues30 reported a strong rela-
tionship between the levels of VAChT expression and ACh
release in both the peripheral and central nervous systems. A
marked reduction of VAChT expression can affect neuro-
transmission at the neuromuscular junction. Even a modest
deficiency of VAChT is sufficient to interfere with release of
ACh in the brain and to alter cognitive behavior in object and
social recognition and memory function.30 Alterations in
VAChT expression levels have also been implicated in drug
addiction, including addiction tonicotine, ethanol, andneuro-
stimulants such as cocaine and amphetamine and opiates.31,32
The levels of VAChT are subject to both up- and down-
regulation as part of compensatory processes that attempt to
maintain homeostasis of neuronal cholinergic activity.32
Recently, we reported the synthesis and in vitro character-
ization of five analogues of a new class of VAChT ligands in
which a carbonyl group was interposed between the two rings
of the 4-phenylpiperidinyl fragment in the vesamicol struc-
ture.33 We also reported (a) the radiosynthesis of an 18F-labeled
positron emission tomography (PET) lead radiotracer, namely,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2827
Scheme 1a
a Reagents and conditions: (a) aryl Grignard reagents/toluene, reflux; (b) 70% of sulfuric acid, 160-170 ꢀC; (c) ethanol/Na2CO3, refluxed.
Results and Discussion
accommodate simultaneously both the phenyl group and a
second fragment. Because the existence of a large binding
pocket would allow for greater flexibility in structure modi-
fication, we synthesized and tested a number of analogues
designed to (a) probe the size of the pocket and (b) extend
structure modification beyond the 4-phenylpiperidine backbone.
Chemistry. The target compounds were synthesized as
depicted in Schemes 1-3. The previously reported52,53 4-
cyanopiperidine 16 reacted with Grignard reagents to pro-
duce 17a-c in moderate yields (Scheme 1). Hydrolytic
removal of the tosyl group in 17a-c was followed by reaction
of the resulting secondary amines 18a-c with epoxides 19 or
21 to yield the target compounds 20 or 22a-c in moderate
yield.
The synthesis of aryl 4-piperidyl ketones 26a-e has been
reported.33 Construction of the aryl 4-piperidyl ketones
26f-h was used similar procedure of making 26a-e by
starting with 23 (Scheme 2). Conversion of the latter into
the N-benzoyl protected isonipecotic acid 24 provided a
versatile intermediate for Friedel-Crafts acylation. Hydro-
lytic removal of the tosyl group from the Friedel-Cratfts
acylation products 25f-h provided 26f-h. Refluxing these
aminoketones 26a-c, f-h with the epoxides 19 or 21 pro-
vided the compounds of type 27a-c, f-h, or 28f-h in
moderate yields (Scheme 2). The procedure for making 28a-e
was reported in the literature.33 Similarly, 26b-c and 26f-g
were reacted with 29 and then hydrolyzed by sodium hydroxide
aqueous solution to produce the regioisomeric pairs 30b,c, 30f,g,
and 31b,c, 31f,g.
Previously, we and others have described several high-
affinity VAChT ligands.5,39,43-50 However, the majority of
these can be represented by only a few structural classes
(Figure 1, 1-15). A common attribute of these ligands is the
presence of a 4-phenylpiperidyl substructure. The recurrence
of this structural motif is based on the structure of the parent
compound and further justified by the observation that
removal of the phenyl group (to yield 10) or replacement of
the piperidyl fragment with the aminoethyl moiety results in
complete loss of potency.43 However, the continued use of the
4-phenylpiperidyl fragment in the design of new VAChT
ligands limits the scope of structure modification in this class
of ligands. Since the 4-phenylpiperidyl group is also recog-
nized by σ receptors,51 many vesamicol analogues bind to
both VAChT and σ receptors.5,38,39 The discovery of new
VAChT ligands that are devoid of this structural attribute
therefore presents an attractive prospect for the development
of novel selective VAChT ligands.
The present study was initiated by analyzing previously
reported structure-activity data. The structure of vesamicol
can be divided into distinct fragments: A, B, and C (Figure 1).
In reviewing the data, we observed that, while removal of the
phenyl group of vesamicol (to yield 10) causes a drastic
reduction in potency, addition of a propionyl group to the
C40 position of vesamicol yields a ligand, 11,43 that is compar-
able in affinity to the parent.35 On the basis of these observa-
tions, we proposed that, within the vesamicol binding site,
the binding pocket around fragment C is large enough to

2828 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Efange et al.
Scheme 2a
a Reagents and conditions:(a) (i) BzCl, Et3N; (ii) NaOH, aq. EtOH; (b) (i) Oxalyl chloride, CH2Cl2; (ii) arene, AlCl3, CS2, reflux; (c) 6 N HCl, heat;
(d) EtOH, Et3N, reflux; (e) NaOH, aq. EtOH.
To obtain the target amides 34a-d, ethyl isonipecotate
was converted in three steps to the activated ester 32
(Scheme 3). The latter was reacted with a number of sub-
stituted piperidines and the products were treated with HCl
(g) to yield the hydrochlorides 33a-d. Intermediates 33a-d
and 23 were refluxed with trans-2-hydroxy-3-bromo-1,2,3,4-
tetrahydronaphthalene in EtOH in the presence of Et3N to
provide 34a-e. Similarly, 33a was reacted with the epoxide
35 to produce 36 and 37. All compounds were tested as the
hydrochlorides.
In Vitro Binding Studies. Compounds were tested for
VAChT binding using highly purified Torpedo synaptic
vesicles. The σ1 and σ2 binding affinities were tested in rat
brain and in guinea pig membranes, respectively. Equilibr-
ium binding constants (Ki) are reported in Table 1. As in
previous studies, we define ligand selectivity in terms of a
selectivity index which is calculated as Ki (σ1 or σ2)/Ki
(VAChT). In the ensuing discussion, binding affinities are
characterized as very high (Ki<1.0 nM), high (Ki = 1-10
nM), moderate (Ki = 11-50 nM), or poor (Ki > 50 nM).
In general, the study identified several potent new ana-
logues. Moreover, a number of potentially useful trends are
discernible. In contrast to 40-propionylvesamicol,43 40-ben-
zoylvesamicol 20 displayed 35-fold lower affinity for VAChT
than vesamicol, while compound 22a was 30-fold less potent
than the corresponding tetralin benzovesamicol (2). No
change in affinity was observed following the introduction
of a fluorine atom or replacement of the benzoyl group with
the phenylacetyl fragment (compare 22a with 22b and 22c).
Given the nanomolar affinity of 22a (and its homologues), it
does appear that there is indeed a large binding pocket within
the receptor around fragment-C. However, the lower potency
of the 4-phenyl-4-aroylpiperidines 20 and 22a relative to the
corresponding 4-phenylpiperidines suggests that, in contrast
to the propionyl group, the more rigid benzoyl substituent is
not as well tolerated in this binding pocket.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2829
Scheme 3a
a Reagents and conditions: (a) di-tert-butyldicarbonate, CHCl3, NaCl, H2O, reflux; (b) NaOH (aq.), EtOH; (c) N-hydroxysuccinimide, DCC,
CH2Cl2; (d) 4-substituted piperidine, CH3CN, reflux; (e) HCl(g), EtOAc, 0 ꢀC; (f) trans-2-hydroxy-3-bromo-1,2,3,4-tetrahydronaphthalene, EtOH,
Et3N, reflux; (g) EtOH, Et3N, reflux; (h) NaOH, aq. EtOH.
To further probe the binding pocket around fragment-C,
we sought to examine vesamicol analogues that are devoid of
the 4-phenyl group. Since potent VAChT ligands can be
obtained from both the 4-phenylpiperidines and 4-phenyl-
4-acylpiperidines, we reasoned that the corresponding
4-acylpiperidines would also yield VAChT ligands of high
affinity. Surprisingly, the 4-aroylpiperidines 27a,b and 27f
were 200-380 times less potent than 1 and 6-11-fold less
potent than the corresponding 4-phenyl-4-acylpiperidine 20.
To explain the difference in affinity, molecular models of 4-
phenylpiperidine and 4-benzoylpiperidine were constructed
and inspected. When these models are rotated around the
long axis (which passes through N1 and C4 of the piperidyl
group), it is observed that the C4-phenyl and 4-benzoyl
groups sweep through different regions of conformational
space. Moreover, the C4-benzoyl group appears to sweep
through a larger spatial volume than the C4-phenyl group.
Therefore, the diminished affinity of the 4-aroylpiperidines
27a,b and 27f may be due in part to unfavorable steric
interaction of the C4-aroyl groups with the binding pocket
around fragment-C. Since compound 1 displays higher
affinity than compound 27a, it would appear that when
fragment-A is cyclohexyl, the 4-phenyl group interacts
more efficiently with the binding pocket than the angular
benzoyl group. We note, however, that the affinity of the
aroyl compounds can be enhanced by appropriate substitu-
tion on the aroyl group (compare 27a vs 27b or 27c, and 27f
vs 27g).
In the tetralin series, replacement of the 4-phenyl group in
2 with a benzoyl group also resulted in reduction of affinity
for VAChT (2 vs 28a). However, the 4-benzoylpiperidine 28a
was more closely matched with the 4-benzoyl-4-phenylpiper-
idine 22a, suggesting that the tetrahydronaphthyl fragment
partially compensates for the unfavorable interaction be-
tween the benzoyl group and the binding pocket around
fragment-C. As observed in the cyclohexane series, the para-
fluoro group was ineffective at altering affinity for VAChT
(28a vs 28b); however, a 10-20-fold increase in affinity was
observed when substitution was extended to include a
bromo or nitro group (compare 28a with 28c or 28e), thus
bringing the affinity of the benzoylpiperidines closer to that
of the potent 4-phenylpiperidines. The use of a 2-thienyl-
carbonyl fragment in place of the 4-benzoylpiperidyl group
provided trends similar to those observed above. Compound
28f displayed comparable affinity with 28a, while the
2-bromo and iodothienyl compounds, 28g and 28h, respec-
tively, showed 8-10-fold higher affinity. We therefore con-
clude that, with suitable substitution, the benzoyl and
related aroyl groups can replace the phenyl group in frag-
ment C.
To extend our structure modification studies, the benzoyl
group was replaced with the carbethoxy group (34e) and a
small selection of carbamoyl fragments (34a-d). While the 1-
piperidylcarbonyl group in 34a brought about 7- and 545-
fold reductions in affinity relative to 28a and 2, respectively;
analogues containing substituted piperidyl/piperazinyl groups

2830 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Efange et al.
Table 1. a: Affinities (Ki ( SD, nM) of Vesamicol and Selected Analogues for Torpedo VAChT and σ1 and σ2 Receptors
selectivity index
compounds
VAChTb (Ki [nM])
σ1c (Ki [nM])
σ2d (Ki[nM])
Ki-σ1/Ki-VAChT
Ki-σ2/Ki-VAChT
Vesamicol (1)e
2.0 ( 1.0
0.055 ( 0.010
50
25.8 ( 8.0
ND
ND
34.5 ( 2.0
ND
ND
12.9
ND
ND
17.25
ND
ND
Benzovesamicol (2) f
11g
20
68.5 ( 2.54
1.64 ( 0.26
1.83 ( 0.39
1.38 ( 0.36
650 ( 330
423 ( 2
51.4 ( 6.0
763 ( 94
46.8 ( 5.0
560 ( 50
4.30 ( 1.00
2.70 ( 0.40
0.25 ( 0.02
1.68 ( 0.14
0.48 ( 0.03
5.00 ( 1.20
0.54 ( 0.08
0.66 ( 0.06
2.40 ( 0.26
0.53 ( 0.09
4.90 ( 1.90
0.41 ( 0.02
7.60 ( 0.96
134 ( 17
29.4 ( 3.3
31.0 ( 4.7
2.07 ( 0.34
2.40 ( 0.25
2.08 ( 0.14
68.5 ( 9.0
89.5 ( 1.1
447 ( 55
ND
ND
ND
ND
ND
ND
ND
ND
22a
22b
22c
27a
27b
27c
27f
27g
27h
28a
28b
28c
28d
28e
28f
28g
28h
30b
30c
30f
30g
31b
31f
31g
34a
34b
34c
34d
34e
36
ND
ND
ND
ND
ND
ND
ND
ND
280.8 ( 18.8
302.79 ( 25.23
15.91 ( 2.82
ND
341.4 ( 28.7
112.74 ( 8.14
30.13 ( 3.80
ND
0.4
0.7
0.5
0.3
0.3
0.6
ND
ND
29.46 ( 0.94
9.39 ( 1.75
219.6 ( 17.3
191.1 ( 57.7
297.69 ( 18.73
ND
35.73 ( 1.13
48.86 ( 3.26
320.0 ( 6.6
251.3 ( 39.2
592.61 ( 95.19
ND
0.6
0.02
0.8
0.1
51.1
70.8
74.4
93.1
1190.8
ND
ND
2370.4
ND
ND
ND
ND
ND
ND
ND
238.7
294
ND
133.4
522
128.92 ( 1.65
194.03 ( 28.70
>1000
72.02 ( 9.40
344.50 ( 13.34
>1000
>416.7
745.5
>204.1
>2439.0
65.4
>416.7
1886.8
>204.1
>2439.0
46.4
395.11 ( 33.8
>1000
>1000
>1000
>1000
>1000
352.7 ( 13.2
>1000
497.1 ( 42.1
>1000
>7.5
16.4
>7.5
4.1
483.24 ( 33.71
>1000
120.66 ( 31.54
>1000
>1000
>32.3
24.3
8.3
>32.3
>483.1
>416.7
>480.8
>14.6
ND
50.24 ( 13.07
19.88 ( 5.71
118.12 ( 47.24
>1000
>1000
>1000
>1000
56.8
>14.6
ND
ND
ND
ND
ND
ND
37
ND
a Ki values (mean ( SEM) were determined in at least three experiments. b Ki for VAChT is measured using Torpedo synaptic vesicles. c σ1 binding is
measured using guinea pig brain membranes. d σ2 binding is measured using rat liver preparations. e Ref 59. f Ref 54. g Ref 43.
(34b-d) displayed affinities comparable to 28a. The angular
carbamoyl group thus permits deep penetration of substitu-
ents into the binding pocket around fragment-C without a
substantial loss of binding affinity.
agents. Among the compounds tested, there was wide varia-
bility in affinity for σ1 or σ2 receptors; only one compound
(27h) displayed high affinity for σ1 receptors, while five
compounds displayed moderate affinity for either σ1 or σ2
receptors. All other compounds exhibited poor affinity for σ
receptors. As in previous studies of vesamicol analogues,5,39,45
we have derived estimates of ligand selectivity, expressed as a
selectivity index, by comparing Ki values for VAChT ob-
tained from highly purified Torpedo synaptic vesicles, with
corresponding values for σ1 and σ2 receptors determined
from rat or guinea pig brain preparations. In the present
study, all cyclohexyl-containing analogues examined (27a-h)
displayed poor VAChT/σ receptors (Table 1). Among the
benzovesamicol analogues, two compounds (28a,b) showed
moderate selectivity while three others (28c,g,h) displayed
high VAChT/σ selectivity. In the latter group, the bromo
analogue 28c emerged as the most selective compound.
Among the aminobenzovesamicol analogues 30b-g, all com-
pounds except 31b displayed high VAChT/σ selectivity. In
contrast, among the amide-containing analogues 34a-d, only
34d exhibited moderate selectivity while the rest showed poor
selectivity for VAChT. Collectively, these observations are
consistent with previous studies, which suggest that benzo-
vesamicol (2) and aminobenzovesamicol (3) are more selective
VAChT ligands than vesamicol (1).
The introduction of an amino group into 2 (benzovesam-
icol) increases affinity for VAChT by an order of magnitude,
making 3 (Ki = 0.0065 ( 0.0005nM) the most potent
VAChT inhibitor reported to date.54 In the present study,
we therefore sought to investigate the effect of the 5-amino
group on the affinity of 4-benzoylpiperidines. Surprisingly,
no improvement was observed for the 4-benzoylpiperidines
(28b vs 30b and 28c vs 30c) or the 4-(thien-2-yl)carbonyl-
piperidines (28f vs 30f and 28g vs 30g). There was, never-
theless, a separation in affinity between the regioisomeric
pairs (30b vs 31b, 30f vs 31f, 30g vs 31g, and 36 vs 37) similar
to that observed with 2 and its 8-amino isomer.5 Thus, it
would appear that the structure-activity relationships of the
4-aroylpiperidines largely parallel, but are not identical to,
those of the 4-phenylpiperidines.
The moderate to high affinity of vesamicol and many of its
analogues for σ1 and σ2 receptors reduces the selectivity of
these compounds for the VAChT and potentially compro-
mises their utility as VAChT ligands. Consequently, we
screened the target compounds for sigma binding to identify
selective VAChT ligands from this new structural class of

Article
We have therefore shown that the phenyl group of vesa-
micol can be successfully replaced with aroyl groups. The
current demonstration serves as a useful point of departure
for expanding the search for new structural classes of
VAChT inhibitors.
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2831
prepared from bromobenzene (4.74 g, 30.2 mmol) and Mg
(0.73 g, 30.0 mmol) in 10 mL of dry THF. A solution of 16
(5.1 g, mmol) in dry toluene (20 mL) was added under nitrogen
to the stirring solution of phenylmagnesium bromide in THF.
The latter solvent was removed by distillation (at 70 ꢀC oil bath
temp), and the resulting mixture was refluxed overnight, cooled,
and acidified with 10% aqueous HCl. The precipitate was
collected by filtration, washed with diethyl ether, and recrystal-
lized from ethanol to give 17a (5.5 g, 88%). 1H NMR (CDCl3) δ
2.12-2.63 (m, 9H), 3.60-3.71 (m, 2H), 7.10-7.70 (m, 14H).
4-Fluorobenzoyl-4-phenyl-1-(p-toluenesulfonyl)piperidine (17b).
Conclusion
In the present study, we have synthesized a new class of
compounds targeting VAChT. In an effort to identify com-
pounds that have high affinity and high selectivity for
VAChT, we modified the vesamicol structure by interposing
a carbonyl group between the B region and the C region of the
vesamicol structure, and exploited the observation that ben-
zovesamicol analogs have increased VAChT binding affinity
compared with vesamicol. From this study, we have identified
six compounds, 28c (0.25 nM), 28e (0.48 nM), 28g (0.54 nM),
28h (0.66 nM), 30c (0.53 nM), and 30g (0.41 nM), that have
very high affinity for VAChT. These compounds also display
very high selectivity for VAChT versus σ1 and σ2 receptors,
and could serve as useful pharmacological probes. For 28c
and 30g, the VAChT/σ selectivity ratios are greater than 1000,
while for 30c, the selectivity ratio is greater than 500-fold. We
also have identified 12 compounds that have high affinity for
VAChT (Ki, 1.0-10 nM) some of these also display very high
selectivity for VAChT vs σ receptors. Furthermore, promising
analogues which contain fluorine, iodine, or bromine provide
the possibility to make corresponding radio halogenated
ligands to serve as PET/SPECT probes. Further evaluation
of these radioligands could provide useful tools for studying
the relationship between alteration of VAChT expression in
the brain and the cholinergic deficiency associated with the
severity of dementia associated with cholinergic deficiency in
patients with neurodegenerative diseases.
1
Procedure A was used to prepare 17b (1.2 g, 90%). H NMR
(CDCl3) δ 2.11-2.62 (m, 9H), 3.62-3.74 (m, 2H), 6.91-7.71 (m,
13H).
4-Phenylacetyl-4-phenyl-1-(p-toluenesulfonyl)piperidine (17c).
1
Procedure A was used to prepare 17c (0.9 g, 61%). H NMR
(CDCl3) δ 2.10-2.62 (m, 9H), 3.39 (s, 2H), 3.55-3.59 (m, 2H),
6.74-7.59 (m, 14H).
Procedure B: General Method of Preparing 4-Acyl-4-phenyl-
piperidines (18a-c). 4-Benzoyl-4-phenylpiperidine (18a). Com-
pound 17a (2.0 g, 4.8 mmol) was dissolved in 7 mL of 70%
sulfuric acid and the solution was heated at 160-170 ꢀC for 2 h.
The reaction mixture was cooled to room temperature, and then
carefully neutralized with dilute aqueous NaOH and extracted
with methylene chloride. The organic extract was dried over
anhydrous Na2SO4 and concentrated under reduced pressure to
give 18a (1.4 g, 99%) of the desired compound. 1H NMR
(CDCl3) 2.05-3.41 (m, 8H), 7.16-7.44 (m, 10H). 18a was used
without further purification.
Procedure B was used to prepare 18b and 18c in 95% and 45%
yield, respectively, starting with the corresponding sulfon-
amides. For 18b, 1H NMR (CDCl3) is 2.0-3.45 (m, 8H), 7.15-
1
7.50 (m, 7H), 7.75-8.15 (2H); for 18c, H NMR (CDCl3) is
2.09-2.70 (m, 8H), 3.56-3.61 (m, 2H), 7.08-7.50 (m, 10H).
Procedure C: General Method of Preparing Cyclohexanols
(20,27a-c and 27f-h). 2-(4-Benzoyl-4-phenylpiperidinyl)cyclo-
hexanol Hydrochloride (20). A mixture of 18a, anhydrous so-
dium carbonate, and cyclohexene oxide in absolute ethanol
(25 mL) was refluxed for 48 h, and then it was cooled and
filtered to remove insoluble material. The precipitate was
washed with a small amount of ethanol (5 mL) and set aside.
Concentration of the filtrate provided a residue that was puri-
fied by radial flow chromatography using acetone/hexane (20/
80) as mobile phase to give 20 (1.0 g, 45%). 1H NMR (CDCl3). δ
1.10-2.79 (m, 17H), 3.30-3.40 (m, 1H), 3.80-4.20 (s, 1H),
7.20-7.50 (m, 10H). The hydrochloride was obtained from
methanolic HCl and recrystallized from i-PrOH; mp 240-
Experimental Section
General. Synthetic intermediates were purchased from
Aldrich Chemical Co. (Milwaukee, WI) and Lancaster Synth-
esis (Windham, MA) and used as received. Tetrahydrofuran
(THF) was distilled from sodium hydride immediately prior to
use. All other reagents and solvents were purchased as reagent
grade and used without further purification.
All air-sensitive reactions were carried out under nitrogen.
Standard handling techniques for air-sensitive materials were
employed throughout this study. Yields were not optimized.
Melting points were determined on a Haake-Buchler or Mel-
241 ꢀC. Anal. (C24H29NO2 HCl) C, H, N.
3
Procedure D: General Method of Preparing Tetralins (22a-c
and 28f-h). trans-3-(4-Benzoyl-4-phenylpiperidinyl)-2-hydro-
xy-1,2,3,4-tetrahydronaphthalene Hydrochloride (22a). A mix-
ture of dihydronaphthalene bromohydrin (2.0 g, 8.8 mmol) in
CHCl3 (25 mL) and 2 N NaOH (25 mL) was refluxed for 2 h and
allowed to cool. The organic layer was separated and the
aqueous phase was extracted with methylene chloride (2 ꢀ
25 mL). The combined organic extracts were dried over anhy-
drous Na2SO4 and then concentrated to yield crude dihydro-
naphthalene oxide (21). The latter was dissolved in ethanol, and
to this solution was added 18a (2.2 g, 8.3 mmol) and sodium
carbonate (4.0 g, 37.7 mmol). The resulting mixture was refluxed
for 42 h, and then cooled and filtered. The precipitate was
washed with a small amount of ethanol and set aside. The
filtrate was concentrated to a residue that was redissolved in
methylene chloride (60 mL). The solution was dried over
anhydrous Na2SO4 and concentrated to a residue. The crude
product was purified on radial flow chromatography to give 22a
1
Temp melting point apparatus and are uncorrected. H NMR
spectra were recorded on a 200 MHz IBM-Bruker spectrometer
or a 300 MHz GE spectrometer. NMR spectra are referenced to
the deuterium lock frequency of the spectrometer. With this
condition, the chemical shifts (in ppm) of residual solvents are
observed at 7.26 (CHCl3) or 4.78 (CD3OH). The following
abbreviations were used to describe peak patterns wherever
appropriate: b = broad, d = doublet, t = triplet, q = quartet,
m = multiplet. Preparative chromatography was performed on
Harrison Research Chromatotron using Merck 60 PF254 silica
gel or a preparative HPLC system (Rainin Instrument Co.)
using a 41.1 mm i.d. Dynamax silica gel column (delivering
solvent at 80 mL/min). Analytical TLC was carried out on
Analtech GHLF silica gel glass plates, and visualization was
aided by UV. Elemental analysis and HPLC method were used
to determine the purity of the target compounds that were
assessed biological data. All the compounds reported in the
manuscript have a purity g95%.
1
(1.0 g, 46%). H NMR (CDCl3) δ 2.72-3.70 (m, 13H), 4.19-
Procedure A: General Method of Preparing 4-Acyl-4-phenyl-
N-tosylpiperidines (17a-c). 4-Benzoyl-4-phenyl-1-(p-toluenesu-
lfonyl)piperidine (17a). Phenylmagnesium bromide was freshly
4.22 (m, 1H), 5.00-5.50 (s, 1H), 7.07-7.56 (m, 14H). The free
base was converted to the hydrochloride by dissolving in metha-
nolic HCl and recrystallizing from isopropyl alcohol-diethyl

2832 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
ether; mp 248-249 ꢀC. Anal. (C28H29NO2 HCl 0.25H2O) C,
H, N.
Efange et al.
prepared by suing procedure D to give (0.6 g, 20%). mp
253-255 ꢀC. 1H NMR [free base] (CDCl3) δ 1.70-3.40 (m,
14H), 3.80-4.00 (m, 1H), 4.00-4.60 (s, 1H), 7.00-7.85 (m, 7H).
CIMS: Calcd 342.1485 (Mþ); Found 342.1485 (Mþ). The
purity of 28f was greater than 95% (determined by HPLC).
trans-3-[4-(5-Bromothienylcarbonyl)piperidinyl]-2-hydroxy-1,2,
3,4-tetrahydronaphthalene Hydrochloride (28g). Compound 28g
was prepared by using procedure D to give (1.3 g, 30%). mp
273-274 ꢀC. 1H NMR [free base] (CDCl3) δ 1.80-3.14 (m,
13H), 3.28-3.36 (m, 1H), 3.83-3.92 (m, 1H), 4.17 (s, 1H), 7.00-
7.60 (m, 6H). Anal. (C20H22BrNO2S HCl 0.5H2O) C, H, N.
trans-3-[4-(5-Iodothienylcarbonyl)piperidinyl]-2-hydroxy-1,2,-
3,4-tetrahydronaphthalene Hydrochloride (28h). Compound 28h
was prepared by using procedure D to give (0.65 g, 42%). mp
275-277 ꢀC. 1H NMR [free base] (CDCl3) δ 1.60-2.80 (m,
14H), 4.10-4.40 (m, 2H), 7.00-7.90 (m, 6H). CIMS: Calcd,
468.0449 (Mþ); Found, 468.0449 (Mþ). The purity of 28h was
greater than 95% (determined by HPLC).
Procedure E: General Method of Preparing Amino Tetralins
30b-c and 30f-g, 31b-c and 30f-g, as well as 36 and 37. 5-Amino-
3-[4-(p-fluorobenzoyl)piperidinyl]-2-hydroxy-1,2,3,4,-tetrahydrona-
phthalene Hydrochloride (30b) and 8-Amino-3-[4-(p-fluorobenzoyl)-
piperidinyl]-2-hydroxy-1,2,3,4,-tetrahydronaphthalene Hydrochlor-
ide (31b). A mixture of 26b (2.2 g, 9.1 mmol), 29 (0.94 g, 3.7 mmol),
and sodium carbonate (3.86 g, 36 mmol) in absolute ethanol
(50 mL) was refluxed for 48 h. The reaction mixture was cooled
to room temperature and filtered to remove insoluble material. The
filtrate was concentrated to a residue that was redissolved into
acetone (150 mL). More insoluble material was removed by
filtration. The solvent was removed by rotary evaporator. The
residue was dissolved into 70% aqueous ethanol (50 mL). NaOH
pellets (0.3 g, 7.5 mmol) were added into the solution. The reaction
mixture was stirred overnight and then concentrated to a residue in
vacuum. The residue was treated with water (20 mL) and extracted
with methylene chloride (3 ꢀ 20 mL). After drying over anhydrous
Na2SO4, the organic extract was concentrated to a residue. The
residue was purified on silica gel column using isopropyl alcohol/
hexane (20/80) as mobile phase to give 30b (1.24 g, 37%) and 31b
(1.24 g, 37%) as 1:1 ratio. In this system, compound 30b displayed
greater chromatographic mobility. The free bases were converted
to the corresponding hydrochlorides by using methanolic HCl. The
hydrochloride salt was recrystallized from i-PrOH. For compound
3
3
trans-3-(4-Fluorobenzoyl-4-phenylpiperidinyl)-2-hydroxy-1,2,
3,4-tetrahydronaphthalene Hydrochloride (22b). Procedure D
1
was used to prepare 22b (0.72 g, 40%). mp 242-243 ꢀC. H
NMR [free base] (CDCl3) δ 2.10-3.34 (m, 13H), 3.79-3.88 (m,
1H), 4.10-4.50(s, 1H), 6.91-7.54 (m, 13H). Anal. (C28H28FNO2
3
HCl 0.25H2O) C, H, N.
3
trans-2-Hydroxy-3-(4-phenylacetyl-4-phenylpiperidinyl)-1,2,
3,4-tetrahydronaphthalene Hydrochloride (22c). Compound 22c
was prepared using procedure D to give (0.14 g, 13%). mp 243-
244 ꢀC. 1H NMR [free base] (CDCl3) δ 2.09-3.58 (m, 13H), 3.53
(s, 2H), 3.83-3.87 (m, 1H), 4.27 (s, 1H), 6.87-7.47 (m, 14H).
Anal. (C29H32NO2 HCl 0.25H2O) C, H, N.
3
3
3
3
Compounds 26a-e were obtained as reported in literature.33
Compounds 26f-h were made by using similar procedure of
making compounds 26a-e.33
1-Benzoyl-4-(thien-2-yl)carbonylpiperidine (25f). Yield, 80%.
1H NMR (CDCl3) δ 1.60-5.00 (m, 9H), 7.10-7.90 (m, 8H).
1-Benzoyl-4-(5-bromothien-2-yl)carbonylpiperidine (25g). Yield,
80%. 1H NMR (CDCl3) δ 1.70-4.85 (m, 9H), 7.26-7.52 (m, 5H),
7.11-7.50 (d, 2H).
1-Benzoyl-4-(5-iodothien-2-yl)carbonylpiperidine (25h). Yield,
1
76%. H NMR (CDCl3) δ 1.60-5.00 (m, 9H), 7.10-7.80 (m,
8H).
4-(Thien-2-yl)carbonyliperidine Hydrochloride (26f). Yield,
1
72%. H NMR (CDCl3) δ 1.60-3.60 (m, 9H), 7.00-7.90 (m,
3H), 6.00-6.80 (s, 1H).
4-(5-Bromothien-2-yl)carbonylpiperidine Hydrochloride (26g).
Yield, 68%. 1H NMR not provided.
4-(5-Iodothien-2-yl)carbonylpiperidine Hydrochloride (26h).
Yield, 67%. 1H NMR (DMSO-d6) δ 1.50-3.80 (m, 9H), 7.10-
8.30 (m, 2H), 8.70-8.60 (s, 2H).
Compounds 27a-c, 27f-h were made by using procedure C
described above.
2-(4-Benzoylpiperidinyl)cycohexanol Hydrochloride (27a).
Compound 27a was prepared by using procedure C to give
(0.45 g, 51%). mp 232-233 ꢀC. 1H NMR [free base] (CDCl3) δ
1.00-3.20 (m, 18H), 3.30-3.60 (s, 1H), 3.80-4.20 (s, 1H), 7.40-
8.20 (m, 5H). Anal. (C18H25NO2 HCl) C, H, N.
3
2-[4-(p-Fluorobenzoyl)piperidinyl]cycohexanol Hydrochloride
(27b). Compound 27b was prepared by using procedure C to
1
1
give (0.35 g, 63%). mp 263-264 ꢀC. H NMR (DMSO-d6) δ
30b, mp 212 ꢀC (decomposed) H NMR [free base] (CDCl3) δ
1.20-1.36 (m, 4H), 1.60-1.70 (m, 2H), 1.98 (m, 4H), 2.86-3.66
(m, 8H), 4.62 (t, 1H), 7.36 (m, 2H), 8.08 (m, 2H). Anal. (C18H24-
FNO2 HCl) C, H, N.
1.82-1.94 (m, 4H), 2.44-3.22 (m, 8H), 3.26-3.33 (m, 2H), 3.62 (s,
2H), 3.87 (m, 1H), 4,20 (br s, 1H), 6.57 (t, 2H, J = 9 Hz), 6.99 (t,
1H, J = 9 Hz), 7.16 (t, 2H, J = 9 Hz), 7.97 (m, 2H). Anal.
(C22H25FN2O2 2HCl 1.25H20) C, H, N. For compound 31b, mp
3
2-[4-(p-Bromobenzoyl)piperidinyl]cycohexanol Hydrochloride
(27c). Compound 27c was prepared by using procedure C to give
(1.4 g, 80%). mp 294-296 ꢀC. 1H NMR [free base] (CDCl3) δ
1.00-3.30 (m, 18H), 3.30-3.60 (s, 1H), 3.80-4.25 (s, 1H), 7.50-
3
3
217 ꢀC (decomposed), 1H NMR [free base] (CDCl3) δ 1.71-1.96
(m, 4H), 2.36-2.44 (m, 2H), 2.75-3.30 (m, 8H), 3.63 (s, 2H), 3.93
(m, 1H), 4.30 (br s, 1H), 6.55 (d, 2H, J = 7.5 Hz), 6.99 (t, 1H, J =
8.20 (d, 4H). Anal. (C18H24BrNO2 HCl) C, H, N.
7.5Hz), 7.16(t,2H,J=9Hz),7.97(m,2H).Anal. (C22H25FN2O2
2HCl 2H2O) C, H, N.
3
3
2-[4-(Thien-2-yl)carbonylpiperidinyl]cyclohexanol Hydrochlo-
ride (27f). Compound 27f was prepared by using procedure C to
give (0.80 g, 42%). mp 247-248 ꢀC. 1H NMR [free base]
(CDCl3) δ 0.80-3.25 (m, 18H), 3.25-3.50 (m, 1H), 3.80-4.25
(s, 1H), 7.00-7.85 (m, 3H). CIMS: Calcd, 294.1483 (Mþ);
3
5-Amino-3-[4-(p-bromobenzoyl)piperidinyl]-2-hydroxy-1,2,3,4,-
tetrahydronaphthalene Hydrochloride (30c) and 8-Amino-3-[4-(p-
bromobenzoyl)-piperidinyl]-2-hydroxy-1,2,3,4,-tetrahydronapht-
halene (31c). Procedure E was used to prepare 30c (1.0 g, 36%)
and 31c (0.69 g, 25%). For compound 30c, 1H NMR [free base]
(CDCl3) δ 1.70-3.40 (m, 14H), 3.40-4.75 (m, 4H), 6.45-7.20
(m, 3H), 7.50-8.20 (d, 4H). Anal. (C22H25N2O2Br 2HCl) C, H,
N. For compound 31c, H NMR [free base] (CDCl3) δ 1.50-
3.40 (m, 14H), 3.40-4.70 (m, 4H), 6.40-7.20 (m, 3H), 7.50-
8.20 (d, 4H).
Found, 293.1483 (Mþ). Anal. (C16H23NO2S HCl) C, H, N.
3
2-[4-(5-Bromothien-2-yl)carbonylpiperidinyl]cyclohexanol Hydro-
chloride (27g). Compound 27g was prepared by using procedure
C to give (0.43 g, 30%). mp 283-285 ꢀC. 1H NMR [free base]
(CDCl3) δ 1.00-3.20 (m, 18H), 3.20-3.60 (s, 1H), 3.70-4.20 (s,
3
1
1H), 6.90-7.80 (m, 2H). Anal. (C16H22BrNO2S HCl) C, H, N.
3
2-[4-(5-Iodothien-2-yl)carbonylpiperidinyl]cyclohexanol Hydro-
chloride (27h). Compound 27h was prepared by using procedure
5-Amino-3-[4-(thien-2-yl)carbonylpiperidyl]-2-hydroxy-1,2,3,4-
tetrahydronaphthalene Hydrochloride (30f) and 8-Amino-3-[4-
(thien-2-yl)carbonylpiperidyl]-2-hydroxy-1,2,3,4-tetrahydronaph-
thalene Hydrochloride (31f). Procedure E was used to prepare
30f (0.8 g, 41%) and 31f (0.78 g, 40%). For compound 30f, mp
225-227 ꢀC. 1H NMR [free base] (CDCl3) δ 1.60-3.40 (m,
1
C to give (1.0 g, 71%); mp 236-238 ꢀC. H NMR (CDCl3) δ
0.80-3.80 (m, 18H), 3.80-4.80 (m, 2H), 7.00-7.90 (m, 2H).
CIMS: Calcd 420.0449 (Mþ); Found 420.0449 (Mþ).
Compounds 28a-e were made as reported.33
trans-2-Hydroxy-3-[4-(thien-2-yl)carbonylpiperidinyl]-1,2,3,4-
tetrahydronaphthalene Hydrochloride (28f). Compound 28f was
14H), 3.50-4.20 (m, 4H), 6.40-8.10 (m, 6H). Anal. (C20H24N2O2
3
HCl) C, H, N. For compound 31f, mp 243-247 ꢀC. 1H NMR

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2833
[free base] (CDCl3) δ 1.80-3.5 (m, 14H), 3.50-4.20 (m, 4H),
to give 34a (0.50 g, 16%) of white powder. mp 252-254 ꢀC. 1H
NMR (DMSO-d6) δ 1.39-1.57 (m, 6H), 1.74-2.11 (m, 4H),
2.73-2.82 (m, 1H), 2.92-3.45 (m, 13H), 4.11 (m, 1H), 7.13 (m,
6.40-8.10 (m, 6H). Anal. (C20H24N2O2 HCl) C, H, N.
3
5-Amino-3-[4-(5-bromothien-2-yl)carbonylpiperidinyl]-2-hydro-
xy-1,2,3,4-tetrahydronaphthalene Hydrochloride (30g) and 8-
Amino-3-[4-(5-bromothien-2-yl)carbonylpiperidinyl]-2-hydroxy-
1,2,3,4-tetrahydronaphthalene Hydrochloride (31g). Procedure E
was used to prepare 30g (1.0 g, 50%) and 31g (1.0 g, 50%) as 1:1
ratio. For compound 30g, mp 195 ꢀC (decomposed), 1H NMR
[free base] (CDCl3) δ 1.70-3.40 (m, 14H), 3.40-4.75 (m, 4H),
4H). Anal. (C21H30N2O2 HCl) C, H, N.
3
trans-3-[4-(4-Phenylpiperidyl)carbonylpiperidyl]-2-hydroxy-
1,2,3,4-tetrahydronaphthalene Hydrochloride (34b). Compound
34b was prepared by using procedure F to give (0.3 g, 8%). mp
238-240 ꢀC. 1H NMR (DMSO-d6) δ 1.34-1.56 (m, 2H),
1.74-2.18 (m, 5H), 2.54-2.62 (t, 1H), 2.74-2.79 (m, 2H), 2.99-
3.45 (m, 8H), 4.11 (m, 2H), 4.52 (d, 1H), 7.09-7.30 (m, 9H).
EIMS: Calcd, 454.2387; Found, 418.2625 (M-HCl)þ. Anal.
(C27H34N2O2 HCl 0.5H2O) C, H, N.
6.40-8.10 (m, 5H). Anal. (C20H23BrN2O2 2HCl) H, N, C. For
3
compound 31g, mp 215 ꢀC (decomposed), 1H NMR [free base]
(CDCl3) δ 1.70-3.40 (m, 14H), 3.40-4.75 (m, 4H), 6.40-8.10
3
3
(m, 5H). Anal. (C20H23BrN2O2 2HCl) C, H, N.
trans-3-[4-(4-Benzylpiperidyl)carbonylpiperidyl]-2-hydroxy-
1,2,3,4-tetrahydronaphthalene Hydrochloride (34c). Compound
34c was prepepared by using procedure F to give (0.6 g, 32%),
3
1-(t-Butoxycarbonyl)piperidine 4-Carboxylic Acid Succini-
mide Ester (32). A mixture of ethyl isonipecotate (24.8 g,
158 mmol), di-tert-butyldicarbonate (36.3 g, 166 mmol), sodium
chloride (42 g, 1.05 mmol) in chloroform (100 mL), and water
(125 mL) was refluxed for 2.5 h and cooled to room temperature.
The layers were separated, and the aqueous layer was extracted
with CHCl3 (2 ꢀ 35 mL). The organic extracts were combined,
dried over anhydrous Na2SO4, and then concentrated to pale
yellow syrup. The latter was dissolved into EtOH (60 mL), and
the resulting solution treated with 6.25 M NaOH solution
(40 mL). After stirring at room temperature for 14 h, the
reaction mixture was concentrated to half its original volume,
and treated with a saturated aqueous solution of NaCl (80 mL).
The resulting mixture was cooled in ice bath, stirred vigorously,
and treated carefully with glacial acetic acid (21.5 mL). Extrac-
tion of the resulting mixture followed by drying over anhydrous
Na2SO4. After concentrating the organic extracts, it gave the N-
protected isonipecotic acid as a white solid (33.1 g, 91%). The
latter was used without further purification. Accordingly, a cold
(ice bath) solution of this acid (11.46 g, 50.0 mmol) in methylene
chloride (90 mL) was treated consecutively with N-hydroxysuc-
cinimide (6.33 g, 55.0 mmol) and DCC (11.35 g, 55.0 mmol)
dissolved in 30 mL of methylene chloride). Cooling was stopped
after 20 min and stirring was continued at room temperature.
After 16 h, the reaction mixture was cooled in an ice bath and
filtered. The suspension was washed with a minimum volume of
methylene chloride. The filtrate was washed with saturated
aqueous sodium bicarbonate (2 ꢀ 35 mL), dried over anhydrous
Na2SO4, and then concentrated to a white solid. The latter was
triturated with hot hexane, and the mixture cooled and filtered
to give 32 (16 g, 98%) of the activated ester. 1H NMR (CDCl3) δ
1.56 (s, 9H), 1.7-1.85 (m 1H), 1.95-2.05 (m, 1H), 2.75-3.05 (m,
2H), 4.00 (m, 1H).
1
mp 246-248 ꢀC. H NMR (DMSO-d6) δ 0.89-1.11 (m, 2H),
1.57 (m, 2H), 2.47 (m, 3H), 2.73-3.41 (m, 12H), 4.00 (d, 1H),
4.11 (m, 1H), 4.33 (d, 1H), 6.08 (s, 1H), 7.08-7.29 (m, 9H).
EIMS: Calcd, 433.2810 (Mþ); Found, 433.2812 (Mþ). Anal.
(C28H36N2O2 HCl) C, H, N.
3
trans-3-[4-(4-Benzylpiperazinyl)carbonylpiperidyl]-2-hydroxy-
1,2,3,4-tetrahydronaphthalene Hydrochloride (34d). Procedure F
1
was used to prepare 34d (0.82 g, 77%). mp 250-252 ꢀC. H
NMR (DMSO-d6) δ 1.80-2.15 (m, 5H), 2.73-3.72 (m, 14H),
4.02-4.48 (m, 6H), 6.08 (s, 1H), 7.12 (m, 5H), 7.38 (m, 2H), 7.66
(m, 2H). EIMS: Calcd, 434.2780 (Mþ); Found, 434.2780 (Mþ).
Anal. (C27H35N3O2 2HCl 0.75H2O) C, H, N.
3
3
trans-3-[4-Ethoxycarbonylpiperidyl]-2-hydroxy-1,2,3,4-tetra-
hydronaphthalene Hydrochloride (34e). The ethyl isonipecotate
(3.63 g, 23 mmol) was dissolved in EtOH (60 mL) and the
solution treated with sodium bicarbonate (8.0 g, 75 mmol) and
dihydronaphthalene bromohydrin (5.22 g, 23 mmol). The resul-
ting mixture was refluxed overnight and concentrated to a
residue which was partitioned between water (60 mL) and
CH2Cl2 (2 ꢀ 60 mL). The organic extracts were combined, dried,
and concentrated to a dark brown residue. The product was
purified by silica gel column chromatography using ethyl
acetate/hexane (30/70) as mobile phase to give 34e free base,
which was converted to the hydrochloride in methanolic HCl
and recrystallized from ethanol to give 34e HCl (5.0 g, 71%) of
3
white powder. mp 191-192 ꢀC 1H NMR (CDCl3): δ 1.25-1.31
(t, 3H), 1.70-2.94 (m, 9H), 3.27-3.35 (m, 1H), 3.80-3.90 (m,
1H), 4.12-4.19 (q, 2H), 4.24 (s, 1H), 7.00-7.27 (m, 4H). EIMS:
Calcd, 303.1832 (Mþ); Found, 304.1832 (Mþ).
trans-5-Amino-3-[4-(piperidyl)carbonylpiperidyl]-2-hydroxy-
1,2,3,4,-tetrahydronaphthalene Hydrochloride (36) and trans-8-
Amino-3-(4-(piperidyl)carbonylpiperidyl)-2-hydroxy-1,2,3,4,-tetra-
hydronaphthalene Hydrochloride (37). Using procedure E gave
compound 36 (1.2 g, 43%) and compound 37 (1.2 g, 43%). For
compound 36, mp 256-258 ꢀC. 1H NMR [free base] (CDCl3) δ
1.25-3.80 (m, 26H), 3.80-4.00 (m, 1H), 6.50-7.10 (m, 3H).
EIMS: Calcd, 357.2416; Found, 357.2415. Anal. (C21H31N3O2).
For compound 37, mp 264-266 ꢀC. 1H NMR [free base]
(CDCl3) δ 1.25-3.80 (m, 26H), 3.80-4.00 (m, 1H), 6.50-7.10
(m, 3H). EIMS: Calcd, 357.2416; Found, 357.2416. Anal. (C21-
H31N3O2) H, N, C.
Procedure F: General Method of Preparing Isonipecotamide
Derivatives (34a-d). Trans-3-[4-(piperidyl)carbonylpiperidyl]-
2-hydroxy-1,2,3,4-tetrahydronaphthalene Hydrochloride (34a).
A mixture of piperidine (0.90 g, 10.6 mmol) and 32 (3.26 g,
10.1 mmol) in dry acetonitrile (50 mL) was refluxed for 26 h and
concentrated to a residue. The latter was redissolved into
methylene chloride (80 mL), and the solution was washed with
saturated aqueous sodium bicarbonate (2 ꢀ 20 mL), dried over
anhydrous Na2SO4, and concentrated to an off-white solid. The
solid was redissolved in ethyl acetate (50 mL) and the solution
cooled in an ice bath. HCl(g) was bubbled vigorously through
the solution for 20 min, and stirring was continued at room
temperature. After 3 h, the reaction mixture was concentrated to
give crude 33a. The latter was redissolved in EtOH (60 mL), and
the solution treated with sodium bicarbonate (2.15 g, 20 mmol)
and dihydronaphthalene bromohydrin (1.82 g, 8.0 mmol). The
resulting mixture was refluxed for 17 h, and concentrated to a
residue that was partitioned between water (60 mL) and methy-
lene chloride (2 ꢀ 60 mL). The organic extracts were combined,
dried, and concentrated to a dark brown residue. The product
was purified by silica gel radial flow chromatography using
acetone/hexane (20/80) as mobile phase. The free base was
converted to the hydrochloride in methanolic HCl, and then,
the hydrochloride salt was recrystallized from isopropyl alcohol
In Vitro Biological Evaluation. Vesicular Acetylcholine
Transporter Binding Assays. In vitro assays of the binding of
compounds to the VAChT were conducted with cholinergic
synaptic vesicles isolated from the electric organ of Torpedo
californica. The radioligand used was [3H]vesamicol, and the
assay conditions were conducted as described.43 (-)-Vesamicol
was used as an external standard in this assay. Averaged data
were fit by regression with a rectangular hyperbola to estimate
Ki value. All compounds were independently assayed at least
two times.
Sigma Receptor Binding Assays. The compounds were dis-
solved in DMF, DMSO, or ethanol, and then diluted in 50 mM
Tris-HCl buffer containing 150 mM NaCl and 100 mM EDTA
at pH 7.4 prior to performing the σ1 and σ2 receptor binding

2834 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Efange et al.
assays. The procedures for isolating the membrane homo-
genates and performing the σ1 and σ2 receptor binding assays
have been described in detail previously.55,56
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Briefly, the σ1 receptor binding assays were conducted in 96-
well plates using guinea pig brain membrane homogenates
(∼300 μg protein) and ∼5 nM [3H](þ)-pentazocine (34.9 Ci/
mmol, Perkin-Elmer, Boston, MA). The total incubation time
was 90 min at room temperature. Nonspecific binding was
determined from samples that contained 10 μM of cold haloper-
idol. After 90 min, the reaction was terminated by the addition
of 150 μL of ice-cold wash buffer (10 mM Tris-HCl, 150 mM
NaCl, pH 7.4) using a 96 channel transfer pipet (Fisher Scien-
tific, Pittsburgh, PA). The samples were harvested and filtered
rapidly through a 96-well fiberglass filter plate (Millipore,
Billerica, MA) that had been presoaked with 100 μL of 50 mM
Tris-HCl buffer at pH 8.0 for 1 h. Each filter was washed 3 times
with 200 μL of ice-cold wash buffer, and the filter counted in a
Wallac 1450 MicroBeta liquid scintillation counter (Perkin-
Elmer, Boston, MA).
The σ2 receptor binding assays were conducted using rat liver
membrane homogenates (∼300 μg protein) and ∼5 nM
[3H]DTG (58.1 Ci/mmol, Perkin-Elmer, Boston, MA) in the
presence of 1 μM (þ)-pentazocine to block σ1 sites. The incuba-
tion time was 2 h at room temperature. Nonspecific binding was
determined from samples that contained 10 μM of cold haloper-
idol. All other procedures were identical to those described for
the σ1 receptor binding assay above.
Data from the competitive inhibition experiments were mod-
eled using nonlinear regression analysis to determine the con-
centration that inhibits 50% of the specific binding of the
radioligand (IC50 value). Competitive curves were best fit to a
one-site fit and gave pseudo-Hill coefficients of 0.6-1.0. Ki
values were calculated using the method of Cheng and Prusoff57
and are presented as the mean ( 1 SEM. For these calculations,
we used a Kd value of 7.89 nM for [3H](þ)-pentazocine and
guinea pig brain and a Kd value of 30.73 nM for [3H]DTG and
rat liver.58
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Acknowledgment. Financial support for these studies was
provided by the National Institutes of Health under grants no.
AG13621 and NS33742, Washington University Alzheimer
Disease Research pilot grant (Grant NIA P50 AG05681-21)
and NIH grants 1R21NS061025-01A2 and NS15047.
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tion of the mouse vesicular acetylcholine transporter gene. Neuro-
report 1997, 8, 3467–3473.
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Supporting Information Available: Analytical data of new
analogues. This material is available free of charge via the
Internet at http://pubs.acs.org.
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