Synthesis of N-substituted 9-azabicyclo[3.3.1]nonan-3α-yl carbamate analogs as σ2 receptor ligands

Article abstract of DOI:10.1016/j.bmc.2006.06.028

A series of N-substituted 9-azabicyclo[3.3.1]nonan-3α-yl phenylcarbamate analogs was prepared and their affinities for sigma (σ₁ and σ₂) receptors were measured in vitro. The results of their structure-activity relationship study ide

Full text of DOI:10.1016/j.bmc.2006.06.028

Bioorganic & Medicinal Chemistry 14 (2006) 6988–6997
Synthesis of N-substituted 9-azabicyclo[3.3.1]nonan-3a-yl
carbamate analogs as r2 receptor ligands
Suwanna Vangveravong, Jinbin Xu, Chenbo Zeng and Robert H. Mach*
Division of Radiological Sciences, Washington University School of Medicine,
Mallinckrodt Institute of Radiology, 510 S. Kingshighway, St. Louis, MO 63110, USA
Received 12 April 2006; revised 9 June 2006; accepted 9 June 2006
Available online 11 July 2006
Abstract—A series of N-substituted 9-azabicyclo[3.3.1]nonan-3a-yl phenylcarbamate analogs was prepared and their affinities for
sigma (r1 and r2) receptors were measured in vitro. The results of their structure–activity relationship study identified two new com-
pounds, N-(9-(4-aminobutyl)-9-azabicyclo[3.3.1]nonan-3a-yl)-N0-(2-methoxy-5-methylphenyl)carbamate and N-(9-(6-aminohexyl)-
9-azabicyclo[3.3.1]nonan-3a-yl)-N0-(2-methoxy-5-methylphenyl)carbamate, having a high affinity and selectivity for r2 versus r1
receptors. These compounds were also used in the preparation of biotinylated and fluorescent probes of the r2 receptor.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
for r1 and r2 receptors, in vitro binding studies using
this radiotracer to measure r2 receptor density require
the use of the 100 nM (+)-pentazocine in the binding as-
say in order to mask r1 receptors.
Sigma receptors represent a class of proteins that were
initially thought to be a subtype of the opiate receptors.1
The development of sigma selective ligands, such as
(+)-pentazocine, DTG (1,3-di-o-tolylguanidine), and
(+)-3-PPP (3-(3-hydroxyphenyl)-N-(1-propyl)piperidine),
allowed sigma binding sites to be distinguished as a
separate receptor system.2 It is now widely accepted that
there are at least two classes of sigma binding sites,
denoted sigma-1 (r1) and sigma-2 (r2). These receptors
are distinguishable functionally, pharmacologically, and
by molecular size. The r1 receptor has a molecular
weight of ꢀ25 kDa, whereas the r2 receptor has a
molecular weight of ꢀ21.5 kDa. The r1 receptor gene
has been cloned from guinea pig liver, human placental
choriocarcinoma, rat brain, and mouse kidney, and dis-
plays a 30% sequence homology with the enzyme, yeast
sterol isomerase.3–5 The r2 receptor gene has not been
cloned, although a number of studies have presented
evidence linking the r2 receptor to potassium channels
and intracellular calcium release in NCB-20 cells.2,6
Radioligand binding studies using [3H](+)-pentazocine
and [3H]DTG have revealed that both r1 and r2 recep-
tors have a widespread distribution in the central ner-
vous system and in a variety of tissues and organs2,6
However, since [3H]DTG possesses a similar affinity
Previous studies have reported that r2 receptors overex-
pressed in a wide variety of human and murine tumor
cells grown in cell culture.7–9 Furthermore, it has shown
that the density of r2 receptors is 10-fold higher in pro-
liferative versus quiescent mouse mammary adenocarci-
noma cells in vitro9,10 and in vivo.11 Based on these
data, we have proposed that the r2 receptor may serve
as a receptor-based biomarker of the proliferative status
of solid tumors. Additional studies have shown that r2
receptor ligands can induce apoptosis in tumor cells,
and raise the possibility that r2 selective ligands may
be useful as anticancer or chemosensitizing agent.12,13
A potential role of the r2 receptor in regulating cellular
proliferation and apoptosis has led to a renewed interest
in identifying the biological function of this receptor.
A number of structurally diverse compounds have been
shown to possess a high affinity to sigma receptors.
Most of these compounds display either a high selectiv-
ity for the r1 receptor or bind with equal affinity to both
r1 and r2 receptors. Until recently, only a few r2 selec-
tive ligands have been identified. For example, the phen-
yl morphan CD-184,14 the trishomocubane analog
ANSTO-20,15 the potent 5-HT3 and 5-HT4 ligand,
BIMU-1,16 have been shown to possess a moderate
affinity and selectivity for r2 versus r1 receptors (Fig. 1).
Keywords: r2 Receptors; Fluorescent probe; Two-photon microscopy.
Corresponding author. Tel.: +1 314 362 8538; fax: +1 314 362
0039; e-mail: rhmach@mir.wustl.edu
*
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.06.028

S. Vangveravong et al. / Bioorg. Med. Chem. 14 (2006) 6988–6997
6989
CH3
O
OH
NH
H3C
O
H
n
+
Br
N
N
Cl
OH
N
O
H
O
n = 4, 6, 10
10
OCH3
Cl
N
O
O
CH3
ANSTO-19
1 = 152 nM
2 = 20 nM
CB-184
1 = 7,436 nM
2 = 13.4 nM
n
N
σ
N
σ
σ
K2CO3, KI
O
σ
H
F
CH3CN
O
N
H
O
H
O
OCH3
11a (n = 4)
11b (n = 6)
11c (n = 10)
N
CH3
N
N
H
CH3
N
n
N
NH2
O
O
H
NH2NH2, EtOH
reflux
BIMU-1
1 = 6,300 nM
2 = 32 nM
σ
N
O
σ
H
OCH3
1a (n = 4)
1b (n = 6)
1c (n = 10)
Figure 1. Structures of the r2 selective compounds.
Scheme 1.
We previously reported the synthesis and in vitro bind-
ing of a series of N-substituted-9-azabicyclo[3.3.1]no-
nan-3a-yl carbamate analogs having a modest affinity
and selectivity for r2 versus r1 receptors.17,18 The goal
of the current study was to prepare radiolabeled, fluo-
rescent, and biotinylated probes of the r2 receptor using
the N-substituted-9-azabicyclo[3.3.1]nonan-3a-yl carba-
mate analogs as the lead structures. This strategy in-
volved the incorporation of an aminoalkyl substituent
in which the length of the spacer group separating the
primary amino group and the bridgehead nitrogen atom
varied by four, six, and ten methylene units. The pri-
mary amino group served as the point of attachment
of the corresponding radiolabeled, fluorescent, and bio-
tinylated probe, whereas the methylene spacer group
separated the probe from the r2 receptor recognition
fragment, the 9-azabicyclo[3.3.1]nonan-3a-yl carbamate
moiety. This study developed two biotinylated probes
that will be useful in the identification and purification
of r2 receptors utilizing Biotin-Avidin coupling tech-
niques, and one fluorescent probe that has been useful
in sub-cellular visualization of r2 receptors using two-
photon microscopy.
CH3
n
N
NH2
O
H
N
H
O
1a (n = 4)
1b (n = 6)
OCH3
X
, benzene, 90 0
C
OHC
1.
2. NaBH4 / EtOH
3. HCl / EtOH
CH3
n
N
N
H
X
O
H
O
N
H
OCH3
n = 4, 2a (X = 3-Br)
2b (X = 3-F)
2c (X = 3-I)
n = 6, 3a (X = 3-Br)
3b (X = 3-F)
3c (X = 3-I)
2d (X = 4-Br)
2e (X = 4-F)
2f (X = 4-I)
3d (X = 4-Br)
3e (X = 4-F)
3f (X = 4-I)
Scheme 2.
2. Chemistry
in moderate yield (75% and 78%). Similarly, 1b and 1c
were also condensed with (+)-biotinamidocaproate N-
hydroxysuccinimidyl ester to give 7 and 8 (Scheme 5)
in high yield (88% and 95%).
The synthesis of the target compounds is outlined in
Schemes 1–6. The reaction between the secondary amine
1017,18 and N-(x-bromoalkyl)phthalimides gave the
intermediates 11a–c. Treatment with anhydrous
hydrazine gave the primary amines 1a–c (Scheme 1).
Reductive amination of compounds 1a,b with halo-
benzaldehydes and sodium borohydride afforded the
N-halobenzyl derivatives (2a–f and 3a–f) (Scheme 2).
Only amine 1b was reacted with 4-halobenzoic acids to
give the 4-halobenzoyl derivatives (4a–d) (Scheme 3).
In an effort to gain some insight into the sub-cellular
localization of r2 receptors in tumor cells, the fluores-
cent analog, 9, was prepared by reacting 1b with dansyl
chloride as outlined in Scheme 6.19
3. Radioligand binding studies at sigma receptors
Compounds 1b and 1c were condensed with (+)-biotin
N-hydroxysuccinimide ester to give 5 and 6 (Scheme 4)
In vitro binding studies were conducted in order to deter-
mine the affinity of the target compounds at r1 and r2

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S. Vangveravong et al. / Bioorg. Med. Chem. 14 (2006) 6988–6997
CH3
receptors. The r1 binding studies were conducted using
the r1-selective radioligand, [3H](+)-pentazocine in guin-
ea pig brain membranes; r2 sites were assayed in rat liver
membranes with [3H]DTG in the presence of (+)-pentaz-
ocine (1 lM) to mask r1 sites or the r2 selective ligand
[3H]RHM-1 alone.6,21
HOOC
N
NH2
6
O
+
H
X
N
O
H
X = Cl, Br, F, I
OCH3
1b
The results of the in vitro binding studies are shown in
Table 1. For compounds 1a–c, the extension of the
methylene linker separating the amino group and the
bridgehead nitrogen atom (n = 4, 6, and 10) had some
affect on the binding affinity for sigma receptors. Com-
pounds 1a and 1b which have a linker group of 4 and
6 methylene units, respectively, between the amino
group and the bridgehead nitrogen atom, both had
moderate affinity and selectivity for r2 versus r1 recep-
tors. Compound 1c with the 10 methylene spacer group
had high affinity at r2 receptor but low selectivity for r2
versus r1 receptor relative to 1a and 1b.
N-hydroxysuccinimide, DCC, CH2Cl2
O
CH3
6
N
N
H
O
H
X
N
H
O
OCH3
4a (X = Cl)
4b (X = Br)
4c (X = F)
4d (X = I)
Scheme 3.
O
CH3
HN
NH
N
NH2
n
O
O
+
H
H
H
O
N
H
O
3
N
S
OCH3
O
1b (n = 6)
1c (n = 10)
O
Et3N, DMF
O
HN
NH
O
H
CH3
H
n
N
N
3
H
O
S
H
N
H
O
OCHa
5 (n = 6)
6 (n = 10)
Scheme 4.
O
CH3
HN
NH
O
N
N
NH2
n
O
O
H
O
+
H
H
H
N
N
H
O
S
3
5
OCH3
O
O
1b (n = 6)
1c (n = 10)
Et3N, DMF
O
NH
HN
H
O
H
CH3
H
N
N
N
S
n
3
5
H
O
H
O
N
H
O
OCH3
7 (n = 6)
8 (n = 10)
Scheme 5.

S. Vangveravong et al. / Bioorg. Med. Chem. 14 (2006) 6988–6997
6991
SO2Cl
CH3
N
NH2
6
O
H
+
N
H
O
N(CH3)2
OCH3
1b
K2CO3, CH3CN
CH3
O2
S
N
N
N(CH3)2
6
H
O
H
O
N
H
OCH3
9
Scheme 6.
Table 1. In vitro binding data
ity at r2 receptors (compounds 4a–d). An exception tothis
was compound 4a, which had a high affinity and selectiv-
ity for r2 receptors versus r1 receptors.
Compound
Kia (nM)
r1
r2
r1/r2 Ratiod
b
c
1a
1b
2490 271
1418 439
134.3 11.9
66.29 5.30
0.30 0.05
63.87 5.61
1.70 0.28
22.15 1.58
0.34 0.03
112.40 4.80
0.46 0.05
0.56 0.06
43.16 2.88
1.00 0.26
0.65 0.09
1397 64
12.94 0.46
5.19 0.80
7.07 1.27
3.06 0.41
4.13 0.33
1.21 0.14
4.07 0.61
29.07 3.48
4.00 0.54
8.94 0.61
4.85 0.49
32.68 5.93
9.93 1.61
70.88 4.38
35.93 1.50
3.56 0.08
719 84
193
273
Coupling of compounds 1b and 1c to two different bio-
tin activated esters showed that the biotinylated deriva-
tives having a short chain (compounds 5 and 6)
displayed a higher affinity and selectivity for r2 versus
r1 receptor than the biotin analogs having the addition-
al amidocaproate spacer group between the biotin and
bridgehead nitrogen atom (compounds 7 and 8).
1c
2a
19
21.66
0.07
52.77
0.42
0.76
0.08
12.57
0.09
0.02
4.35
0.01
0.02
392
2b
2c
2d
2e
2f
3a
3b
3c
4. Fluorescent r2 ligands
3d
3e
Fluorescent r2 ligands can be used to study the subcell-
uar localization of r2 receptors in cells growing under
cell culture conditions, or in brain tissue sections, using
two-photon microscopy. In vitro binding studies dem-
onstrated that the fluorescent analog, 9, prepared as out-
lined in Scheme 6, had a moderate affinity and selectivity
for r2 versus r1 receptors.
3f
4a
4b
4c
2041 98
2.84
2.50
1.40
29.87
147
1243 212
1412 318
3402 538
10,234 2895
10,365 2313
3819 518
12,644 3754
1.45 0.33
497 103
1,009 110
114 28
4d
5
6
69.70 7.67
1351 78
733 45
7
7.67
5.21
85.43
0.06
Excitation and emission spectra demonstrated that 9
exhibited the maximum excitation wavelength at
333 nm, and the maximum emission wavelength with a
range of 480–520 nm (Fig. 2). In order to use two-pho-
ton microscopy to study the sub-cellular localization of
8
9
Haloperidol
148 8.85
24.20 3.00
a Mean SEM, Ki values were determined by at least three
experiments.
b Ki for inhibiting the binding of [3H](+)-pentazocine to guinea pig
brain homogenates.
400
350
300
c Ki for inhibiting the binding of [3H]DTG or [3H]RHM-1 to rat liver
homogenates.
d Ki for r1/Ki for r2.
250
excitation
200
150
100
50
emission
Attachment of the substituted benzyl groups to the amino
side chain of compounds 1a and 1b resulted in compounds
(2a–f and 3a–f) having a dramatic increase in affinity at r1
receptors. The affinity at r2 receptors either slightly in-
creased (2a–d and 2f vs 1a) or decreased relative to the pri-
mary amine (3a–f vs 1b). On the contrary, attachment of
the substituted benzoyl groups to the amino side chain
of compound 1b resulted in a dramatic reduction in affin-
0
300
350
400
450
500
550
600
Wavelength (nm)
Figure 2. Excitation and emission spectra of 9.

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S. Vangveravong et al. / Bioorg. Med. Chem. 14 (2006) 6988–6997
A Excitation
B Emission
Figure 3. Excitation spectra (A) and emission spectra (B) of 9 in EMT6 cells by two-photon microscopy.
r2 receptors, EMT6 cells were incubated with 9 at a
concentration of 200 nM. The maximum excitation
wavelength and maximum emission wavelength were
examined by two-photon microscopy. Compound 9
was found to localize in the cytoplasm of EMT6 cells.
The maximum excitation wavelength for two-photon
microscopy was found to be 720 nm, whereas the maxi-
mum emission wavelength ranged from 480 to 540 nm.
The results of the two-photon sub-cellular localization
study are shown in Figure 3.
The results of these initial studies revealed that extension
of the length of the methylene spacer group between the
bridgehead nitrogen atom and the benzene ring had lit-
tle effect on binding to r receptors. The goal of the pres-
ent study was to further explore the structure–activity
relationships of the groups attached to the bridgehead
nitrogen atom of the granatane ring system in order to
prepare probes of the r2 receptor that could be used
in imaging studies and in the purification of the r2
receptor protein from tissue.
The first strategy involved extending the length of the
methylene spacer group between the bridgehead nitrogen
atom and the free amino group. The results of this study
(Table 1) revealed that compounds 1a and 1b, having
either a 4 or 6 methylene spacer group between the bridge-
head nitrogen and the free amino group, had a high affin-
ity for r2 versus r1 receptors. Extension of the spacer
group to 10 methylene units resulted in an increase in
affinity for r1 receptors and a reduction in the r1:r2 selec-
tivity ratio (Table 1). As a next step, we prepared a series
5. Discussion
The current study is a continuation of our effort to
develop ligands having a high affinity and selectivity
for r2 versus r1 receptors. In earlier studies, we
prepared a number of structural analogs of the mixed
serotonin 5-HT3/5-HT4 ligand, BIMU-1, having a high
affinity for r2 versus r1 receptors and a low (or negligi-
ble) affinity at serotonin 5-HT3 and 5-HT4 receptors.17,18

S. Vangveravong et al. / Bioorg. Med. Chem. 14 (2006) 6988–6997
6993
of analogs having substituted benzyl groups attached to
the primary amino group of 1a and 1b. In this study, the
aromatic ring of the benzyl group was substituted with
the halogen atoms F, Br, and I in the 3- or 4-position since
the corresponding radiolabeled versions (i.e., 18F-, 76Br-,
and 125I-labeled analogs) could be used in imaging studies
to assess the r2 receptor status of solid tumors. An unex-
pected finding was the dramatic increase in r1 receptor
affinity and reduction in r1:r2 selectivity ratio when mak-
ing this substitution on the amino group of 1a and 1b. This
result was in stark contrast to that obtained when the ami-
no group of 1b was substituted with a benzoyl group. This
substitution led to a large reduction in r2 receptor affinity
and no change in r1 receptor affinity (Table 1). An excep-
tion to this observation was the 5-chlorobenzoyl analog,
4a, which maintained a high affinity and selectivity for
r2 versus r1 receptors.
abbreviations are used for multiplicity of NMR signals:
br s = broad singlet, d = doublet, m = multiplet,
q = quintet, s = singlet, t = triplet. Melting points were
determined with an electrothermal melting point appa-
ratus and are uncorrected. Elemental analyses were per-
formed by Atlantic Microlab, Inc., Norcross, GA, and
were within 0.4% of the calculated values. Mass spec-
trometry was provided by the Washington University
Mass Spectrometry Resource, an NIH Research Re-
source (Grant No. P41RR0954). All reactions were car-
ried out under an inert atmosphere of nitrogen.
The general procedure for conversion to an HCl salt was
the addition of excess ethereal HCl solution to a solu-
tion of the compound in dry ethanol. The solvent was
evaporated and the resulting salt was triturated with
anhydrous ether and dried in vacuo.
A second goal of the current study was to prepare
molecular probes that could be used in the purification
of r2 receptors from tissue sources and in two-photon
microscopy studies aimed at visualizing the sub-cellular
localization of r2 receptor. In this regard, two potential-
ly useful compounds were identified, the biotinylated
analog, 6, and the dansyl analog, 9. Both compounds
have a moderate affinity for r2 receptors and moderate
r1:r2 selectivity ratio. We are currently using com-
pounds 6 and 9 in studies aimed at identifying the bio-
logical function of r2 receptors.
The general procedure for conversion to an oxalate salt
was the addition of a stoichiometric amount of a solu-
tion of oxalic acid in ethyl acetate to a solution of the
compound in ethyl acetate. The solvent was evaporated
and the resulting salt was triturated with anhydrous
ether and dried in vacuo.
Elemental analyses
Compound
% C
% H
% N
Calcd Found Calcd Found Calcd Found
1a
1b
1c
2a
2b
2c
2d
2e
2f
3a
3b
3c
3d
3e
3f
52.06 51.93 8.11 7.89 8.67 8.41
53.90 54.12 8.46 8.46 8.20 8.10
59.88 59.92 8.93 8.83 7.76 7.57
53.73 53.23 6.64 6.20 6.22 6.01
60.90 60.63 7.15 6.83 7.10 6.97
50.20 49.88 6.18 5.83 5.86 5.51
53.73 53.08 6.64 6.12 6.22 5.99
59.10 59.02 7.27 7.04 6.89 6.53
52.18 52.01 5.98 5.88 6.08 5.90
56.47 56.79 6.81 6.70 6.17 6.06
62.02 61.82 7.48 7.23 6.78 6.61
52.82 52.52 6.37 6.23 5.77 5.58
55.73 55.98 6.87 6.58 6.09 5.92
58.61 58.61 7.69 7.38 6.41 6.25
52.18 52.09 6.43 6.17 5.70 5.60
57.52 57.48 6.94 6.81 6.29 6.07
55.33 55.32 6.38 6.16 6.05 5.82
59.80 59.79 7.06 6.67 6.54 6.17
51.20 51.55 6.04 5.78 5.60 5.46
59.30 59.05 7.06 6.86 7.48 7.18
6. Conclusion
The results of the current study provided additional
information on the structure–activity relationships of
9-azabicyclo[3.3.1]nonan-3a-yl carbamate analogs with
respect to binding at r1 and r2 receptors. The length
of the methylene spacer group separating the primary
amino group and the bridgehead nitrogen atom had ef-
fect on the binding affinity for r2 receptors and r1:r2
selectivity ratio. From this study, the six methylene link-
er group gave a compound (1b) having the highest affin-
ity and selectivity for r2 versus r1 receptors. The
substituted benzyl or benzoyl groups attached to the
amino side chain (2a–f, 3a–f, and 4a–d) did not enhance
the selectivity for r2 versus r1 receptors. The attach-
ment of a shorter chain biotin moiety to either 1b or
1c gave compounds (5 and 6) with moderate affinity
and selectivity for r2 receptors. Finally, the dansyl
derivative, 9, had a moderate affinity and selectivity
for r2 receptors and may be a useful probe for two-pho-
ton microscopy studies of this receptor in cells growing
under cell culture conditions and in tissue slices.
4a
4b
4c
4d
9
7.2. General procedure for the synthesis of compounds
1a–c
7. Experimental
A mixture of secondary amine 10 (1.42 g, 4.68 mmol), N-
(x-bromoalkyl)phthalimides (1 equiv), KI (1 equiv), and
K2CO3 (5 equiv) in acetonitrile was stirred at reflux over-
night. After filtration, volatile components were evapo-
rated in vacuo. The resulting residue was purified by
silica gel column chromatography (2% CH3OH in
7.1. Chemical analysis
1H NMR spectra were recorded on a Varian 300 MHz
NMR spectrometer. Chemical shifts are reported in d
values (parts per million, ppm) relative to an internal
standard of tetramethylsilane (TMS). The following

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S. Vangveravong et al. / Bioorg. Med. Chem. 14 (2006) 6988–6997
CH2Cl2) to give the intermediate products (11a–c), which
were then refluxed with anhydrous hydrazine (1.2 equiv)
in ethanol (30 mL) for 2 h. The solvent was evaporated
and an aqueous solution of 10% NaOH (25 mL) was add-
ed. The mixture was extracted with CH2Cl2, dried over
Na2SO4, and evaporated to give the target compounds.
The products were converted to the corresponding hydro-
chloride salts for elemental analysis.
1a and 3-bromobenzaldehyde to give an off-white pow-
der, mp 189–190 ꢁC (dec); 1H NMR (free base, CDCl3) d
7.95 (br s, 1H), 7.13–7.50 (m, 5H), 6.72–6.80 (m, 2H),
5.12 (q, J = 6.8 Hz, 1H), 3.84 (s, 3H), 3.76 (s, 2H),
3.04–3.08 (m, 2H), 2.57–2.64 (m, 4H), 2.38–2.49 (m,
2H), 2.29 (s, 3H), 2.09–2.24 (m, 1H), 1.81–1.91 (m,
2H), 1.47–1.56 (m, 8H), 1.19–1.25 (m, 2H); Anal.
(C28H38BrN3O3ÆC2H2O4Æ2H2O) C, H, N.
7.2.1. N-(9-(4-Aminobutyl)-9-azabicyclo[3.3.1]nonan-3a-
yl)-N-(2-methoxy-5-methylphenyl)carbamate hydrochlo-
ride (1a). Obtained in 70% yield from N-(4-bromobu-
tyl)phthalimide to give an off-white powder, mp 130–
7.3.2.
N-(9-(4-(30-Fluorobenzylamino)butyl)-9-azabicy-
clo[3.3.1]nonan-3a-yl)-N-(2-methoxy-5-methylphenyl)car-
bamate oxalate (2b). Obtained in 55% yield from 1a and
3-fluorobenzaldehyde to give a white powder, mp 185–
186 ꢁC (dec); H NMR (free base, CDCl3) d 7.95 (br s,
1
1
131 ꢁC (dec); H NMR (free base, CDCl3) d 7.95 (br s,
1H), 7.14 (s, 1H), 6.72–6.79 (m, 2H), 5.13 (q, J = 6.8 Hz,
1H), 3.84 (s, 3H), 3.05–3.10 (m, 2H), 2.68–2.72 (m, 2H),
2.57–2.61 (m, 2H), 2.39–2.49 (m, 2H), 2.29 (s, 3H),
2.09–2.19 (m, 1H), 1.83–1.91 (m, 2H), 1.19–1.54 (m,
11H); Anal. (C21H33N2O3Æ2HClÆ2H2O) C, H, N.
1H), 6.72–7.31 (m, 7H), 5.12 (q, J = 6.7 Hz, 1H), 3.84
(s, 3H), 3.79 (s, 2H), 3.04–3.08 (m, 2H), 2.57–2.65 (m,
4H), 2.38–2.48 (m, 2H), 2.29 (s, 3H), 2.08–2.22 (m,
1H), 1.80–1.94 (m, 2H), 1.47–1.68 (m, 8H), 1.18–1.24
(m, 2H); Anal. (C28H38FN3O3ÆC2H2O4ÆH2O) C, H, N.
7.2.2. N-(9-(6-Aminohexyl)-9-azabicyclo[3.3.1]nonan-3a-
yl)-N-(2-methoxy-5-methylphenyl)carbamate hydrochloride
(1b). Obtained in 74% yield from N-(6-bro-
mohexyl)phthalimide to give a white powder, mp 119–
7.3.3. N-(9-(4-(30-Iodobenzylamino)butyl)-9-azabicyclo-
[3.3.1]nonan-3a-yl)-N-(2-methoxy-5-methylphenyl)carba-
mate oxalate (2c). Obtained in 12% yield from 1a and
3-iodobenzaldehyde to give a white powder, mp
1
1
120 ꢁC (dec); H NMR (free base, CDCl3) d 7.95 (br s,
200–201 ꢁC (dec); H NMR (free base, CDCl3) d 7.94
1H), 7.14 (s, 1H), 6.73–6.80 (m, 2H), 5.14 (q, J =
6.8 Hz, 1H), 3.85 (s, 3H), 3.05–3.10 (m, 2H), 2.66–2.71
(m, 2H), 2.55–2.60 (m, 2H), 2.40–2.50 (m, 2H), 2.30 (s,
3H), 2.08–2.24 (m, 1H), 1.82–1.94 (m, 2H), 1.20–1.55
(m, 14H); Anal. (C23H37N2O3Æ2HClÆ2H2O) C, H, N.
(br s, 1H), 7.70 (s, 1H), 7.55–7.60 (m, 1H), 7.30–7.34
(m, 1H), 7.03–7.13 (m, 2H), 6.72–6.80 (m, 2H), 5.12
(q, J = 6.7 Hz, 1H), 3.84 (s, 3H), 3.74 (s, 2H),
3.06–3.10 (m, 2H), 2.59–2.65 (m, 4H), 2.39–2.49 (m,
2H), 2.29 (s, 3H), 2.14–2.24 (m, 1H), 1.80–1.90 (m,
2H), 1.46–1.56 (m, 8H), 1.20–1.26 (m, 2H); Anal.
(C28H38IN3O3ÆC2H2O4Æ2H2O) C, H, N.
7.2.3. N-(9-(10-Aminodecyl)-9-azabicyclo[3.3.1]nonan-3a-
yl)-N-(2-methoxy-5-methylphenyl)carbamate hydrochloride
(1c). Obtained in 70% yield from N-(10-bromode-
cyl)phthalimide to give a white powder, mp 114–
7.3.4.
N-(9-(4-(40-Bromobenzylamino)butyl)-9-azabicy-
clo[3.3.1]nonan-3a-yl)-N-(2-methoxy-5-methylphenyl)car-
bamate oxalate (2d). Obtained in quantitative yield from
1a and 4-bromobenzaldehyde to give an off-white pow-
der, mp 197–198 ꢁC (dec); 1H NMR (free base, CDCl3) d
7.95 (br s, 1H), 7.44 (d, J = 8.3 Hz, 2H), 7.21 (d,
J = 8.3 Hz, 2H), 7.13 (s, 1H), 6.73–6.80 (m, 2H), 5.12 (q,
J = 6.8 Hz, 1H), 3.84 (s, 3H), 3.74 (s, 2H), 3.02–3.08 (m,
2H), 2.54–2.64 (m, 2H), 2.37–2.47 (m, 2H), 2.30 (s, 3H),
2.07–2.22 (m, 1H), 1.78–1.92 (m, 2H), 1.16–1.54 (m,
10H); Anal. (C28H38BrN3O3ÆC2H2O4Æ2H2O) C, H, N.
1
115 ꢁC (dec); H NMR (free base, CDCl3) d 7.96 (br s,
1H), 7.14 (s, 1H), 6.72–6.78 (m, 2H), 5.14 (q,
J = 6.6 Hz, 1H), 3.85 (s, 3H), 3.04–3.09 (m, 2H), 2.65–
2.70 (m, 2H), 2.53–2.58 (m, 2H), 2.39–2.49 (m, 2H),
2.30 (s, 3H), 2.09–2.20 (m, 1H), 1.81–1.93 (m, 2H),
1.19–1.53 (m, 23H); Anal. (C27H45N2O3Æ2HClÆ0.5H2O)
C, H, N.
7.3. General procedure for the synthesis of compounds
2a–f and 3a–f
7.3.5.
N-(9-(4-(40-Fluorobenzylamino)butyl)-9-azabicy-
Primary amines 1a or 1b (200 mg) and 3-halo- or 4-halo-
benzaldehydes (1.3 equiv) in benzene (6 mL) were heat-
ed at 90 ꢁC for 2 h. After evaporation, the resulting
residue was treated with sodium borohydride (4 equiv)
in ethanol (10 mL) at ambient temperature overnight.
The reaction mixture was quenched with 10% HCl solu-
tion and concentrated in vacuo. The residue was dis-
solved in water (8 mL), the pH was adjusted to 10 by
dropwise addition of an aqueous solution of 10%
NaOH, and the product was extracted with CH2Cl2
(2· 25 mL). The organic layer was dried over Na2SO4
and evaporated to give the products. The oxalate salts
were made for elemental analysis.
clo[3.3.1]nonan-3a-yl)-N-(2-methoxy-5-methylphenyl)car-
bamate oxalate (2e). Obtained in quantitative yield from
1a and 4-fluorobenzaldehyde to give a white powder, mp
195–196 ꢁC (dec); H NMR (free, base, CDCl3) d 7.95
(br s, 1H), 7.29–7.34 (m, 2H), 7.13 (s, 1H), 6.97–7.07
(m, 2H), 6.73–6.80 (m, 2H), 5.12 (q, J = 6.8 Hz, 1H),
3.84 (s, 3H), 3.76 (s, 2H), 3.04–3.08 (m, 2H), 2.56–2.65
(m, 2H), 2.38–2.48 (m, 2H), 2.29 (s, 3H), 2.09–2.19 (m,
1H), 1.79–1.90 (m, 2H), 1.19–1.56 (m, 9H); Anal.
(C28H38FN3O3ÆC2H2O4Æ2H2O) C, H, N.
1
7.3.6. N-(9-(4-(40-Iodobenzylamino)butyl)-9-azabicyclo-
[3.3.1]nonan-3a-yl)-N-(2-methoxy-5-methylphenyl)carba-
mate oxalate (2f). Obtained in 10% yield from 1a and
4-iodobenzaldehyde to give a white powder, mp 159–
7.3.1.
N-(9-(4-(30-Bromobenzylamino)butyl)-9-azabicy-
1
clo[3.3.1]nonan-3a-yl)-N-(2-methoxy-5-methylphenyl)car-
bamate oxalate (2a). Obtained in quantitative yield from
160 ꢁC (dec); H NMR (free base, CDCl3) d 7.95 (br s,
1H), 7.64 (d, J = 8.1 Hz, 2H), 7.14 (s, 1H), 7.10 (d,

S. Vangveravong et al. / Bioorg. Med. Chem. 14 (2006) 6988–6997
6995
J = 8.1 Hz, 2H), 6.72–6.80 (m, 2H), 5.12 (q, J = 6.7 Hz,
1H), 3.84 (s, 3H), 3.74 (s, 2H), 3.04–3.10 (m, 2H), 2.57–
2.64 (m, 4H), 2.38–2.49 (m, 2H), 2.30 (s, 3H), 2.10–2.20
(m, 1H), 1.80–1.92 (m, 2H), 1.18–1.58 (m, 10H); Anal.
(C28H38IN3O3ÆC2H2O4Æ0.5 H2O) C, H, N.
2H), 2.38–2.48 (m, 2H), 2.29 (s, 3H), 2.05–2.20 (m,
1H), 1.80–1.92 (m, 2H), 1.18–1.53 (m, 13H); Anal.
(C30H42FN3O3ÆC2H2O4Æ3H2O) C, H, N.
7.3.12.
N-(9-(6-(40-Iodobenzylamino)hexyl)-9-azabicy-
clo[3.3.1]nonan-3a-yl)-N-(2-methoxy-5-methylphenyl)car-
bamate oxalate (3f). Obtained in 85% yield from 1b and
4-iodobenzaldehyde to give a white powder, mp 144–
7.3.7. N-(9-(6-(30-Bromobenzylamino)hexyl)-9-azabicy-
clo[3.3.1]nonan-3a-yl)-N-(2-methoxy-5-methylphenyl)car-
bamate oxalate (3a). Obtained in 88% yield from 1b and
3-bromobenzaldehyde to give a white powder, mp 193–
1
145 ꢁC (dec); H NMR (CDCl3) d 7.95 (br s, 1H), 7.44
(d, J = 8.1 Hz, 2H), 7.20 (d, J = 8.1 Hz, 2H), 7.14 (s,
1H), 6.73–6.80 (m, 2H), 5.13 (q, J = 6.8 Hz, 1H), 3.85
(s, 3H), 3.74 (s, 2H), 3.04–3.08 (m, 2H), 2.54–2.62 (m,
4H), 2.40–2.50 (m, 2H), 2.29 (s, 3H), 2.10–2.23 (m,
1H), 1.83–1.92 (m, 2H), 1.19–1.51 (m, 14H); Anal.
(C30H42IN3O3ÆC2H2O4Æ1.5 H2O) C, H, N.
1
194 ꢁC (dec); H NMR (free base, CDCl3) d 7.95 (br s,
1H), 7.49 (s, 1H), 7.37 (d, J = 7.9 Hz, 1H), 7.22 (d,
J = 7.9 Hz, 1H), 7.18 (t, J = 7.9 Hz, 1H), 7.14 (s, 1H),
6.73–6.80 (m, 2H), 5.13 (q, J = 6.7 Hz, 1H), 3.84 (s,
3H), 3.76 (s, 2H), 3.04–3.09 (m, 2H), 2.55–2.64 (m,
4H), 2.40–2.50 (m, 2H), 2.29 (s, 3H), 2.10–2.22 (m,
1H), 1.84–1.93 (m, 2H), 1.20–1.52 (m, 14H); Anal.
(C30H42BrN3O3ÆC2H2O4ÆH2O) C, H, N.
7.4. General procedure for the synthesis of compounds
4a–d
7.3.8.
N-(9-(6-(30-Fluorobenzylamino)hexyl)-9-azabicy-
A solution of 1,3-dicyclohexylcarbodiimide (154 mg,
0.74 mmol) in CH2Cl2 (0.5 mL) was added dropwise to
a solution of 4-halobenzoic acids (1.2 equiv) and N-hy-
droxysuccinimide (64 mg, 0.74 mmol) in CH2Cl2
(2 mL) at 0 ꢁC (ice bath). After removal of the ice bath,
the mixture was stirred at ambient temperature for 1 h.
A solution of amine 1b (250 mg, 0.62 mmol) in CH2Cl2
(5 mL) was slowly added, and the reaction mixture was
stirred at ambient temperature for 3 h. The formed pre-
cipitate was filtered, the organic layer was washed with
water (1· 50 mL) and then saturated aqueous K2CO3
(1· 50 mL). The organic layer was dried over Na2SO4
and concentrated in vacuo. The resulting residue was
purified by silica gel column chromatography (5%
CH3OH in CH2Cl2) to give the desired compounds.
The oxalate salts were made for elemental analysis.
clo[3.3.1]nonan-3a-yl)-N-(2-methoxy-5-methylphenyl)car-
bamate oxalate (3b). Obtained in 86% yield from 1b and
3-fluorobenzaldehyde to give a white powder, mp 149–
150 ꢁC (dec); 1H NMR (CDCl3) d 7.95 (br s, 1H),
7.24–7.29 (m, 1H), 7.13 (s, 1H), 7.04–7.10 (m, 2H),
6.90–6.96 (m, 1H), 6.73–6.80 (m, 2H), 5.13 (q,
J = 6.5 Hz, 1H), 3.85 (s, 3H), 3.79 (s, 2H), 3.00–3.05
(m, 2H), 2.54–2.64 (m, 4H), 2.39–2.49 (m, 2H), 2.29 (s,
3H), 2.09–2.18 (m, 1H), 1.83–1.93 (m, 2H), 1.19–1.55
(m, 14H); Anal. (C30H42FN3O3ÆC2H2O4ÆH2O) C, H, N.
7.3.9. N-(9-(6-(30-Iodobenzylamino)hexyl)-9-azabicyclo-
[3.3.1]nonan-3a-yl)-N-(2-methoxy-5-methylphenyl)carbamate
oxalate (3c). Obtained in 86% yield from 1b and 3-iodo-
benzaldehyde to give a white powder, mp 192–193 ꢁC
1
(dec); H NMR (CDCl3) d 7.95 (br s, 1H), 7.69 (s, 1H),
7.57 (d, J = 7.8 Hz, 1H), 7.28 (d, J = 7.8 Hz, 1H), 7.13 (s,
1H), 7.05 (t, J = 7.8 Hz, 1H), 6.72–6.79 (m, 2H), 5.13 (q,
J = 6.6 Hz, 1H), 3.84 (s, 3H), 3.73 (s, 2H), 3.04–3.09 (m,
2H), 2.54–2.63 (m, 4H), 2.39–2.49 (m, 2H), 2.29 (s, 3H),
2.08–2.18 (m, 1H), 1.82–1.94 (m, 2H), 1.19–1.54 (m, 14H);
Anal. (C30H42IN3O3ÆC2H2O4ÆH2O) C, H, N.
7.4.1. N-(9-(4-(40-Chlorobenzoylamino)hexyl)-9-azabicy-
clo[3.3.1]nonan-3a-yl)-N-(2-methoxy-5-methylphenyl)car-
bamate oxalate (4a). Obtained in 58% yield from
4-chlorobenzoic acid to give a white powder, mp 127–
1
128 ꢁC (dec); H NMR (free base, CDCl3) d 7.92 (br s,
1H), 7.72 (d, J = 8.8 Hz, 2H), 7.37 (d, J = 8.8 Hz, 2H),
7.14 (s, 1H), 6.73–6.80 (m, 2H), 6.29 (br s, 1H), 5.14 (q,
J = 6.8 Hz, 1H), 3.85 (s, 3H), 3.42–3.48 (m, 2H), 3.05–
3.08 (m, 2H), 2.38–2.60 (m, 4H), 2.26 (s, 3H), 1.16–2.11
(m, 16H); Anal. (C30H40ClN3O4ÆC2H2O4Æ2H2O) C, H, N.
7.3.10. N-(9-(6-(40-Bromobenzylamino)hexyl)-9-azabicy-
clo[3.3.1]nonan-3a-yl)-N-(2-methoxy-5-methylphenyl)car-
bamate oxalate (3d). Obtained in 85% yield from 1b and
4-bromobenzaldehyde to give a white powder, mp 164–
1
165 ꢁC (dec); H NMR (CDCl3) d 7.95 (br s, 1H), 7.44
7.4.2. N-(9-(4-(40-Bromobenzoylamino)hexyl)-9-azabicy-
clo[3.3.1]nonan-3a-yl)-N-(2-methoxy-5-methylphenyl)car-
bamate oxalate (4b). Obtained in 71% yield from
4-bromobenzoic acid to give a white powder, mp 152–
(d, J = 8.1 Hz, 2H), 7.20 (d, J = 8.1 Hz, 2H), 7.14 (s,
1H), 6.73–6.80 (m, 2H), 5.13 (q, J = 6.8 Hz, 1H), 3.85
(s, 3H), 3.74 (s, 2H), 3.04–3.08 (m, 2H), 2.54–2.62 (m,
4H), 2.40–2.50 (m, 2H), 2.29 (s, 3H), 2.10–2.23 (m,
1H), 1.83–1.92 (m, 2H), 1.19–1.51 (m, 14H); Anal.
(C30H42BrN3O3ÆC2H2O4Æ1.5H2O) C, H, N.
1
153 ꢁC (dec); H NMR (free base, CDCl3) d 7.91 (br s,
1H), 7.67 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H),
7.14 (s, 1H), 6.73–6.80 (m, 2 H), 6.39 (br s, 1H), 5.14 (q,
J = 6.8 Hz, 1H), 3.85 (s, 3H), 3.42–3.48 (m, 2H), 3.10–
3.15 (m, 2H), 2.49–2.66 (m, 4H), 2.27 (s, 3H), 1.22–2.22
(m, 16H); Anal. (C30H40BrN3O4ÆC2H2O4ÆH2O) C, H, N.
7.3.11. N-(9-(6-(40-Fluorobenzylamino)hexyl)-9-azabicy-
clo[3.3.1]nonan-3a-yl)-N-(2-methoxy-5-methylphenyl)car-
bamate oxalate (3e). Obtained in 92% yield from 1b and
4-fluorobenzaldehyde to give a white powder, mp 178–
179 ꢁC (dec); 1H NMR (CDCl3) d 7.95 (br s, 1H),
7.28–7.36 (m, 2H), 7.14 (s, 1H), 6.97–7.07 (m, 2H),
6.73–6.79 (m, 2H), 5.13 (q, J = 6.8 Hz, 1H), 3.84 (s,
3H), 3.75 (s, 2H), 3.03–3.08 (m, 2H), 2.58–2.63 (m,
7.4.3. N-(9-(4-(40-Fluorobenzoylamino)hexyl)-9-azabicy-
clo[3.3.1]nonan-3a-yl)-N-(2-methoxy-5-methylphenyl)car-
bamate oxalate (4c). Obtained in 81% yield from
4-fluorobenzoic acid to give a white powder, mp 119–
1
120 ꢁC (dec); H NMR (free base, CDCl3) d 7.91 (br s,

6996
S. Vangveravong et al. / Bioorg. Med. Chem. 14 (2006) 6988–6997
1H), 7.79–7.84 (m, 2H), 6.97–7.05 (m, 3H), 6.73–6.78
(m, 2H), 6.40 (br s, 1H), 5.14 (q, J = 6.5 Hz, 1H), 3.85
(s, 3H), 3.41–3.47 (m, 2H), 3.18–3.22 (m, 2H), 2.53–
2.74 (m, 4H), 2.27 (s, 3H), 1.28–2.17 (m, 16H); Anal.
(C30H40FN3O4ÆC2H2O4Æ1.5H2O) C, H, N.
2.30–3.10 (m, 15H), 2.22 (s, 3H), 1.10–2.10 (m, 30H);
MS (FAB+) exact mass calcd for C39H62N6O6S
[M+Li]+: 749.4612, found: 749.4628.
7.6.2. Compound 8. Obtained in 95% yield from 1c to
1
give an off-white solid; H NMR (DMSO-d6) d 8.02 (s,
7.4.4. N-(9-(4-(40-Iodobenzoylamino)hexyl)-9-azabicyclo-
[3.3.1]nonan-3a-yl)-N-(2-methoxy-5-methylphenyl)carba-
mate oxalate (4d). Obtained in 57% yield from 4-iodo-
benzoic acid to give a white powder, mp 150–151 ꢁC
2H), 7.94 (br s, 1H), 7.14 (s, 1H), 6.73–6.80 (m, 2H),
6.26 (s, 1H), 6.14 (s, 1H), 5.09–5.18 (m, 1H), 4.49–4.54
(m, 1H), 4.30–4.34 (m, 1H), 3.85 (s, 3H), 2.40–3.30 (m,
11H), 2.29 (s, 3H), 1.21–2.23 (m, 42H); MS (FAB+) ex-
act mass calcd for C43H70N6O6S [M+Li]+: 805.5238,
found: 805.5243.
1
(dec); H NMR (free base, CDCl3) d 7.91 (br s, 1H),
7.74 (d, J = 8.6 Hz, 2H), 7.52 (d, J = 8.6 Hz, 2H), 7.13
(s, 1H), 6.73–6.77 (m, 2H), 6.38 (br s, 1H), 5.13 (q, J =
6.8 Hz, 1H), 3.84 (s, 3H), 3.40–3.48 (m, 2H), 3.08–3.14
(m, 2H), 2.44–2.64 (m, 4H), 2.26 (s, 3H), 1.20–2.20 (m,
16H); Anal. (C30H40IN3O4ÆC2H2O4Æ1.5H2O) C, H, N.
7.7. N-(9-(6-(5-Dimethylamino-1-naphthalenesulfonami-
do)hexyl)-9-azabicyclo[3.3.1]-nonan-3a-yl)-N-(2-meth-
oxy-5-methylphenyl)carbamate oxalate (9)
7.5. General procedure for the synthesis of compounds 5
and 6
A solution of dansyl chloride (135 mg, 0.50 mmol) in
CH3CN (6 mL) was added dropwise to a mixture of 1b
(200 mg, 0.50 mmol) and K2CO3 (104 mg, 0.75 mmol)
in CH3CN (2 mL). The reaction mixture was stirred at
ambient temperature for 24 h. The mixture was filtered,
and volatiles were removed in vacuo. The product was
purified by column chromatography (CH3OH–CH2Cl2–
NH4OH 10:90:0.1) to give 9 (93%) as a yellow oil. The
Primary amines 1b or 1c (150 mg), (+)-biotin N-hy-
droxysuccinimide ester (1.1 equiv), and triethylamine
(0.1 mL) in DMF (5 mL) were stirred at 65 ꢁC for
48 h. The mixture was allowed to cool to ambient tem-
perature, and volatiles were removed under reduced
pressure. The resulting residue was purified by silica
gel column chromatography (15% CH3OH, 1% NH4OH
in CH2Cl2) to give 5 or 6.
1
oxalate salt was made for analysis, mp 162–163 ꢁC; H
NMR (free base, CDCl3) d 8.54 (d, J = 8.6 Hz, 1H),
8.30 (d, J = 8.6 Hz, 1H), 8.23–8.26 (m, 1H), 7.94 (br s,
1H), 7.50–7.58 (m, 2H), 7.18 (d, J = 7.1 Hz, 1H), 7.13 (s,
1H), 6.73–6.80 (m, 2H), 5.10 (q, J = 6.7 Hz, 1H), 4.77
(br s, 1H), 2.99–3.11 (m, 2H), 2.89 (s, 6H), 2.42–2.56 (m,
4H), 2.29 (s, 3H), 1.17–2.15 (m, 18H); MS (FAB+) exact
mass calcd for C35H48N4O5S [M+Li]+: 643.3505, found:
643.3490; Anal. (C35H48N4O5SÆC2H2O4Æ1.25H2O) C, H, N.
7.5.1. Compound 5. Obtained in 76% yield from 1b to give a
whitepowder; 1H NMR (DMSO-d6) d8.12(brs,1H), 7.70–
7.74 (m, 1H), 7.47 (s, 1H), 6.82–6.89 (m, 2H), 6.41 (s, 1H),
6.35 (s, 1H), 4.90–4.99 (m, 1H), 4.27–4.31 (m, 1H), 4.09–
4.13 (m, 1H), 3.75 (s, 3H), 2.30–3.11 (m, 13H), 2.21 (s,
3H), 1.13–2.06 (m, 22H); MS (FAB+) exact mass calcd
for C33H51N5O5S [M+Li]+: 636.3771, found: 636.3762.
8. Sigma receptor binding assays
7.5.2. Compound 6. Obtained in 78% yield from 1c to give
a white powder; 1H NMR (DMSO-d6) d 8.07 (br s, 1H),
7.70–7.74 (m, 1H), 7.49 (s, 1H), 6.82–6.90 (m, 2H), 6.42
(s, 1H), 6.35 (s, 1H), 4.90–4.98 (m, 1H), 4.28–4.31 (m,
1H), 4.10–4.15 (m, 1H), 3.75 (s, 3H), 2.30–3.09 (m,
13H), 2.21 (s, 3H), 1.11–2.05 (m, 30H); MS (FAB+) exact
mass calcd for C37H59N5O5S [M+Li]+: 692.4397, found:
692.4411.
Test compounds were dissolved in N,N-dimethylform-
amide (DMF), dimethylsulfoxide (DMSO) or ethanol
and then diluted in 50 mM Tris–HCl, pH 7.4, buffer
containing 150 mM NaCl and 100 mM EDTA. Mem-
brane homogenates were made from guinea pig brain
for r1 binding assay and rat liver for r2 binding assay.
Membrane homogenates were diluted with 50 mM
Tris–HCl buffer, pH 8.0, and incubated at 25 ꢁC in a to-
tal volume of 150 lL in 96-well plates with the radioli-
gand and test compounds with concentrations ranging
from 0.1 nM to 10 lM. After incubation was completed,
the reactions were terminated by the addition of 150 lL
of ice-cold wash buffer (10 mM Tris–HCl, 150 mM
NaCl, pH 7.4) using a 96-channel transfer pipette (Fish-
er Scientific, Pittsburgh, PA), and the samples harvested
and filtered rapidly through 96-well fiber glass filter
plate (Millipore, Billerica, MA) that had been presoaked
with 100 lL of 50 mM Tris–HCl buffer, pH 8.0, for 1 h.
Each filter was washed three times with 200 lL of
ice-cold wash buffer. A Wallac 1450 MicroBeta liquid
scintillation counter (Perkin-Elmer, Boston, MA) was
used to quantitate the bound radioactivity.
7.6. General procedure for the synthesis of compounds 7
and 8
Primary amines 1b or 1c (150 mg), (+)-biotinamidocap-
roate N-hydroxysuccinimidyl ester (1.1 equiv), and tri-
ethylamine (0.1 mL) in DMF (5 mL) were stirred at
50 ꢁC for 48 h. The mixture was allowed to cool to
ambient temperature, and volatiles were removed under
reduced pressure. The resulting residue was purified by
silica gel column chromatography (15% CH3OH, 1%
NH4OH in CH2Cl2) to give 7 or 8.
7.6.1. Compound 7. Obtained in 88% yield from 1b to
1
give a white solid; H NMR (DMSO-d6) d 8.11 (br s,
1H), 7.70–7.75 (m, 2H), 7.48 (s, 1H), 6.82–6.90 (m,
2H), 6.42 (s, 1H), 6.36 (s, 1H), 4.92–4.99 (m, 1H),
4.25–4.32 (m, 1H), 4.08–4.15 (m, 1H), 3.76 (s, 3H),
The r1 receptor binding assay was conducted using
guinea pig brain membrane homogenates (ꢀ300 lg

S. Vangveravong et al. / Bioorg. Med. Chem. 14 (2006) 6988–6997
6997
protein) and ꢀ5 nM [3H](+)-pentazocine (34.9 Ci/mmol,
References and notes
Perkin-Elmer, Boston, MA), incubation time was
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Acknowledgments
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This research was funded by NIH Grant CA 102869
and the U.S. Army Medical Research and Material
Command under DAMD 17-01-1-0446.