Synthesis of bicyclic σ receptor ligands with cytotoxic activity

Article abstract of DOI:10.1021/jm070620b

All possible stereoisomeric alcohols (6-benzyl-8-(4-methoxybenzyl)-6,8- diazabicyclo[3.2.2]nonan-2-ol) and methyl ethers (6-benzyl-2-methoxy-8-(4- methoxybenzyl)-6,8-diazabicyclo[3.2.2]nonane) are prepared from (R)- and (S)-glutamate. A Dieckmann analogous cyclization, which makes use of trapping the primary cyclization product with Me₃SiCl, generates the bicyclic framework. Stereoselective LiBH₄ reduction and Mitsunobu inversion establish the configuration in position 2. Enantiomeric alcohols 15 (1S,2S,5R) and ent-15 (1R,2R,5S) as well as diastereomeric methyl ethers ent-17 (1R,2R,5S) and ent-22 (1R,2S,5S) display high σ₁ receptor affinity. Cell growth inhibition of the stereoisomeric alcohols and methyl ethers against five human tumor cell lines is investigated. In particular, at a concentration of 20 μthe four methyl ethers stop completely the cell growth of the small cell lung cancer cell line A-427, indicating a specific target in this cell line. The IC₅₀-values of methyl ethers ent-17 and ent-22 are in the range of the antitumor drugs cisplatin and oxaliplatin. Binding assays show that the investigated tumor cell lines express considerable amounts of σ₁ and σ₂ receptors.

Full text of DOI:10.1021/jm070620b

6144
J. Med. Chem. 2007, 50, 6144-6153
Synthesis of Bicyclic σ Receptor Ligands with Cytotoxic Activity
Christian Geiger,† Christel Zelenka,† Manuela Weigl,† Roland Fro¨hlich,‡ Birgit Wibbeling,‡ Kirstin Lehmkuhl,†
Dirk Schepmann,† Renate Gru¨nert,§ Patrick J. Bednarski,§ and Bernhard Wu¨nsch*,†
Institut fu¨r Pharmazeutische und Medizinische Chemie, UniVersity Mu¨nster, Hittorfstrasse 58-62, D-48149 Mu¨nster, Germany,
Organisch-chemisches Institut, UniVersity Mu¨nster, Correnstrasse 40, D-48149 Mu¨nster, Germany, and Institut fu¨r Pharmazie,
UniVersity Greifswald, F.-L.-Jahnstrasse 17, D-17489 Greifswald, Germany
ReceiVed May 31, 2007
All possible stereoisomeric alcohols (6-benzyl-8-(4-methoxybenzyl)-6,8-diazabicyclo[3.2.2]nonan-2-ol) and
methyl ethers (6-benzyl-2-methoxy-8-(4-methoxybenzyl)-6,8-diazabicyclo[3.2.2]nonane) are prepared from
(R)- and (S)-glutamate. A Dieckmann analogous cyclization, which makes use of trapping the primary
cyclization product with Me3SiCl, generates the bicyclic framework. Stereoselective LiBH4 reduction and
Mitsunobu inversion establish the configuration in position 2. Enantiomeric alcohols 15 (1S,2S,5R) and
ent-15 (1R,2R,5S) as well as diastereomeric methyl ethers ent-17 (1R,2R,5S) and ent-22 (1R,2S,5S) display
high σ1 receptor affinity. Cell growth inhibition of the stereoisomeric alcohols and methyl ethers against
five human tumor cell lines is investigated. In particular, at a concentration of 20 µM the four methyl ethers
stop completely the cell growth of the small cell lung cancer cell line A-427, indicating a specific target in
this cell line. The IC50-values of methyl ethers ent-17 and ent-22 are in the range of the antitumor drugs
cisplatin and oxaliplatin. Binding assays show that the investigated tumor cell lines express considerable
amounts of σ1 and σ2 receptors.
Introduction
Originally the sigma receptor was termed enigmatic because
of the lack of an endogenous ligand and the indistinct intrac-
ellular signal transduction pathway.1 Cloning of the σ1 receptor
about 10 years ago proved the existence of σ receptors as
Figure 1. 125I-labeled potent σ receptor ligands used for tumor
binding site for various exogenous and endogenous ligands.2-5
The rat brain gene encodes for a protein of 223 amino acids
with a single transmembrane domain.4 However, further inves-
tigations revealed two transmembrane domains with the amino
and carboxy termini on the intracellular side of the membrane.6
Crucial amino acids for ligand binding are aspartate 126 and
glutamate 172 located in the intracellular carboxy terminus.6
σ Receptors are considered to play a modulatory role in the
activity of a variety of ion channels and neurotransmitter systems
including Ca2+-channels,7 K+-channels,8 NMDA type glutamate
receptors, dopamine receptors, and acetylcholine receptors.9
Therefore, ligands interacting with σ receptors possess a
potential for the treatment of acute and chronic neurological
disorders, including schizophrenia, depression, Alzheimer’s
disease, pain as well as alcohol and cocaine abuse.10 In order
to disclose the in vivo relevance of σ1 receptors σ1 knockout
mice were generated, which are viable and fertile, but show a
significant decrease in motility.11
imagining.
Figure 2. σ1 receptor antagonists (3, 4) and σ2 receptor agonists (5)
with cytotoxic and antitumor activity.
Moreover, σ receptors of tumor cells can be addressed by
drugs with cytotoxic potential for the treatment of cancer. In
particular it was shown that the human SK-N-SH neuroblas-
toma and the C6 glioma cell lines, which produce large amounts
of σ1 and σ2 receptors, are sensitive against σ1 receptor
antagonists (e.g., 3 (NE 100), 4 (haloperidol), Figure 2) and σ2
receptor agonists (e.g., 5 (PB 28), Figure 2).15,16 Additionally,
4 (haloperidol) inhibited B16 melanoma cell growth in single
digit micromolar concentration and led to cell death at higher
concentrations.17
In addition to the CNS effects of σ receptor ligands it has
been shown that some human tumor cell lines (e.g., malignant
melanoma, breast carcinoma, prostate tumor, non-small cell lung
cancer among others) express a large number of σ1 and/or σ2
receptor copies on their cell surface. The high σ receptor density
can be exploited for tumor imaging (tumors and their metastasis)
in diagnosis using radiolabeled highly potent σ ligands (e.g., 1,
2, Figure 1).12-14
Recently, we reported on the synthesis of hydroxymethyl-
substituted piperazine derivatives 6, which represent a novel
class of potent σ1 receptor ligands (e.g., 6a with R ) p-
methoxybenzyl: Ki ) 12.4 nM).18 The piperazine ring of 6 can
adopt two chair conformations with an equatorially or axially
oriented, rather flexible hydroxymethyl side chain (Figure 3).
* Author to whom correspondence should be addressed.
Tel.: +49-251-8333311; fax: +49-251-832144; e-mail: wuensch@
uni-muenster.de.
† Institut fu¨r Pharmazeutische und Medizinische Chemie, University
Mu¨nster.
‡ Organisch-chemisches Institut, University Mu¨nster.
§ Institut fu¨r Pharmazie, University Greifswald.
10.1021/jm070620b CCC: $37.00 © 2007 American Chemical Society
Published on Web 10/30/2007

Cytotoxic Bicyclic σ Receptor Ligands
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6145
Scheme 1a
Figure 3. Structural development of novel σ receptor ligands.
Obviously, the pharmacophoric elements of 6, which are respon-
sible for the interactions with σ1 receptors, are not in a defined
orientation to each other. Therefore, we planned to synthesize
bridged piperazines 7 with a 2-(hydroxymethyl)piperazine sub-
structure. In the bicyclic compounds 7 the conformational flexi-
bility is considerably reduced, which leads to a defined three-
dimensional orientation of the pharmacophoric nitrogen atoms
(together with their residues) and the hydroxy moiety. The ster-
eoselective receptor binding of the bridged piperazines should
be investigated after synthesizing all possible stereoisomers of
7. Moreover, the cytotoxic activity of the synthesized bridged
piperazines should be determined in a panel of human tumor
cell lines and correlated with the σ1 and/or σ2 receptor affinity.
Chemistry. The synthesis was started by esterification of (R)-
glutamate with methanol and trimethylsilyl chloride (Me3SiCl)
to give the hydrochloride of dimethyl glutamate 8‚HCl. Con-
densation of 8 with benzaldehyde and subsequent reduction with
NaBH4 yielded the benzylamine 9, which was acylated with
chloroacetyl chloride to form the chloroacetamide 10. Since the
intermediate imine tends to racemize even during storage at
4 °C and the benzylamine 9 gives rise to form a γ-lactam, the
synthesis of the chloroacetamide 10 has to be performed without
storing the intermediates. Reaction of the chloroacetamide 10
with p-methoxybenzylamine led to the nucleophilic substitution
followed by intramolecular aminolysis, affording the pipera-
zinedione 11 in 82% yield (Scheme 1).19
a (a) Me3SiCl, MeOH. (b) Benzaldehyde, MgSO4, NEt3, CH2Cl2 and then
NaBH4, MeOH. (c) ClCH2COCl, NEt3, CH2Cl2. (d) p-Methoxybenzylamine
(PMB-NH2), NEt3, CH3CN. (e) LiHMDS, THF, -78 °C, 30 min and then
Me3SiCl. (f) p-Toluenesulfonic acid, THF, H2O.
Scheme 2a
Since according to Bredt’s rule20 deprotonation at the
bridgehead position of 13 (position 1) during Dieckmann
cyclization is not allowed, the Dieckmann cyclization protocol
recently developed by us was applied.21-23 Trapping of the first
reaction product with Me3SiCl led diastereoselectively to the
mixed methyl silyl acetal 12 in excellent yields (96%), which
was carefully hydrolyzed (room temperature, low concentration
of acid) with p-toluenesulfonic acid in THF/H2O to provide the
bicyclic ketone 13 (Scheme 1).
a (a) LiBlH4, THF, -90 °C. (b) LiAlH4, THF, 66 °C. (c) NaH, MeI,
THF. (d) LiAlH4, THF, 66 °C.
The reduction of the ketone 13 with NaBH4 in aqueous THF
provided a mixture of the diastereomeric alcohols 14 and 19 in
the ratio 75:25 (Scheme 2). In order to improve the diastereo-
selectivity of this reduction several reducing agents and condi-
tions were investigated. It was found that LiBH4 in THF at
-90 °C led to the diastereomeric alcohols 14 and 19 in the
ratio of 99:1. Recrystallization of the crude reaction product
from ethyl acetate/methanol (9:1) provided 82% of the alcohol
14 in diastereomerically and enantiomerically pure form (HPLC
analysis).
Because of the flexibility of the three-carbon bridge of the
bicyclic alcohol 14, it was not possible to unequivocally prove
the configuration of the new center of chirality in position 2 by
NMR experiments. Therefore an X-ray crystal structure analysis
of the enantiomer of 14 (ent-14) was recorded, which was
analogously prepared starting with the proteinogenic amino acid
(S)-glutamate. Recrystallization of ent-14 from ethyl acetate gave
colorless crystals, which were suitable for X-ray crystal structure
analysis. Solution of the structure revealed the relative config-
uration of the alcohol ent-14. Since the absolute (S)-configu-
ration of the center of chirality in position 5 is given from the
synthesis educt (S)-glutamate, the absolute configuration of the
alcohol ent-14 is (1S,2R,5S) (Figure 4).
Methylation of the alcohol 14 with CH3I and NaH gave the
methyl ether 16 in 96% yield. Heating of the alcohol 14 and
the methyl ether 16 with LiAlH4 in THF led to the bridged
piperazines 15 and 17 with a hydroxy and a methoxy group in
position 2, respectively. The configuration of the alcohol 15
and the methyl ether 17 is (1S,2S,5R) (Scheme 2).

6146 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Geiger et al.
Figure 5. Structural comparison of the enantiomeric alcohols ent-15
and 15 with the lead compound 6a.
amino acid (S)-glutamate. Thus, the four possible stereoisomeric
alcohols 15, ent-15, 20, and ent-20 as well as methyl ethers 17,
ent-17, 22, and ent-22 were available for pharmacological
evaluation.
Figure 4. X-ray crystal structure analysis of ent-14.
Scheme 3a
Receptor Binding Studies. The σ1 and σ2 receptor affinity
of the stereoisomeric bicyclic alcohols 15, ent-15, 20, and ent-
20 and methyl ethers 17, ent-17, 22, and ent-22 was determined
in competition experiments with radioligands. In the σ1 assay,
guinea pig brain membrane preparations were used as receptor
material, and [3H]-(+)-pentazocine was the radioligand. The
nonspecific binding was determined in the presence of a large
excess of nontritiated (+)-pentazocine. The σ2 assay was
performed with rat liver membrane preparations and the
nonselective radioligand [3H]-ditolylguanidine. In order to gain
σ2 selectivity, an excess of the σ1 selective ligand (+)-
pentazocine was added to occupy the σ1 receptors. A concentra-
tion of 10 µM of nontritiated ditolylguanidine was used for the
determination of nonspecific binding. Since structural relation-
ships between σ receptor ligands and NMDA receptor antago-
nists exist,25,26 the affinity of the bridged piperazines toward
the phencyclidine binding site of the NMDA receptor (pig brain
cortex membrane preparations, [3H]-MK801) was also included
in this study.27-29
In Table 1 the σ1 and σ2 receptor affinity of the stereoisomeric
bicyclic alcohols and methyl ethers is summarized. In the alcohol
series, the σ1 receptor affinity is strongly dependent on the
configuration in position 2. Whereas 15 with (1S,2S,5R)-
configuration interacts in the low nanomolar range (Ki ) 7.5
nM) with σ1 receptors, its (2R)-diastereomer 20 shows a
considerably lower affinity (Ki ) 118 nM). The same trend is
observed for the enantiomeric alcohols ent-15 and ent-20. It is
striking that in this series the enantiomer pairs 15/ent-15 and
20/ent-20 display the same σ1 receptor affinity, respectively.
In Figure 5, the structures of the flexible σ1 receptor ligand
6a (Ki ) 12.4 nM)18 and the conformationally constrained
bicyclic σ1 receptor ligands ent-15 and 15 are compared. We
assume that the hydrophobic pockets of the σ1 receptor protein
accept both the phenyl and the 4-methoxyphenyl moiety of the
ligands in the same manner. Therefore, the phenyl and the
4-methoxyphenyl moiety are considered to be equivalent for
the receptor.
a (a) DIAD, PPh3, p-nitrobenzoic acid (NBzOH), THF. (b) MeOH,
K2CO3. (c) LiAlH4, THF, 66 °C. (d) NaH, MeI, THF. (e) LiAlH4, THF,
66 °C.
In order to synthesize the diastereomeric alcohol 20 and
methyl ether 22 with (R)-configuration in position 2 the
configuration of the (2S)-configured alcohol 14 was inverted
by a Mitsunobu reaction.24 Thus, 14 reacted with 4-nitrobenzoic
acid, diisopropyl azodicarboxylate (DIAD), and PPh3 to provide
the 4-nitrobenzoate 18 with inverted C-2-configuration. The
inverted alcohol 19 was obtained by cleavage of the 4-nitroben-
zoate 18 with CH3OH and K2CO3 (Scheme 3).
The methyl ether 21 was prepared by reaction of the alcohol
19 with CH3I and NaH. LiAlH4 reduction of the alcohol 19
and the methyl ether 21 resulted in the basic bicyclic piperazines
bearing a hydroxy (20) and a methoxy group (22) in position
2, respectively (Scheme 3).
Formally, the σ1 ligands 6a and ent-15, which are derived
from the (S)-configured amino acids (S)-serine and (S)-
glutamate, respectively, are related to each other. Therefore, we
postulate that the bioactive conformation of the rather flexible
2-(hydroxymethyl)piperazine 6a, which is shown in Figure 5,
is similar to the structure of the bicyclic alcohol ent-15.
The enantiomeric alcohols ent-15 and ent-20 as well as the
enantiomeric methyl ethers ent-17 and ent-22 were prepared in
the same manner, starting the synthesis with the enantiomeric

Cytotoxic Bicyclic σ Receptor Ligands
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6147
Table 1. σ1 and σ2 Receptor Affinity of the Stereoisomeric Alcohols and Methyl Ethers
Ki ( SEM [nM] (n ) 3)
σ2 affinity
σ1 affinity
compound
configuration
C-2 substituent
([3H](+)-pentazocine)
([3H]-ditolylguanidine)
σ1/σ2 selectivity
15
ent-15
20
ent-20
17
ent-17
22
ent-22
(1S,2S,5R)
(1R,2R,5S)
(1S,2R,5R)
(1R,2S,5S)
(1S,2S,5R)
(1R,2R,5S)
(1S,2R,5R)
(1R,2S,5S)
(2S,6S,11S)
-
OH
OH
OH
OH
OCH3
OCH3
OCH3
OCH3
-
7.5 ( 2.1
6.5 ( 0.67
118 ( 5.0
125 ( 18
258 ( 11
26 ( 8.8
126 ( 26
25 ( 3.0
2.2 ( 1.2
177 ( 6.6
1.9 ( 0.4
1700
806
441
705
2430
573
1440
325 ( 20
-
20 ( 2.3
78 ( 2.4
227
124
4
6
9
22
11
13
-
0.1
41
(+)-pentazocine
ditolylguanidine
haloperidol
-
-
-
Table 2. σ1 Receptor Affinities (Ki Values) and Cell Growth Inhibitory Activity (% of Untreated Control) of the Stereoisomeric Alcohols and Methyl
Ethers in Five Human Cancer Cell Linesa)
compound
configuration
σ1 Ki [nM]
5637b
RT-4c
A-427d
LCLC-103He
MCF-7f
15
ent-15
20
ent-20
17
ent-17
22
ent-22
haloperidol
(+)-pentazocine
(1S,2S,5R)
(1R,2R,5S)
(1S,2R,5R)
(1R,2S,5S)
(1S)
(1R,2R,5S)
(1S,2R,5R)
(1R,2S,5S)
7.5
6.5
118
125
258
26
126
25
72 ( 32.6
88 ( 10.7
88 ( 4.5
103 ( 7.7
27 ( 13.6
68 ( 10.4
77 ( 8.5
79 ( 7.5
63 ( 16.2
96 ( 10.1
72 ( 1.2
93 ( 10.3
67 ( 4.2
95 ( 13.6
29 ( 21.4
61 ( 21.7
65 ( 18.5
75 ( 20.0
55 ( 5.9
98 ( 1.0
46 ( 8.6
46 ( 12.0
33 ( 7.8
46 ( 11.1
-10 ( 7.5
-8 ( 3.9
-7 ( 3.6
-5 ( 3.0
5 ( 1.5
80 ( 3.3
94 ( 6.9
77 ( 18.0
96 ( 11.4
19 ( 4.4
72 ( 7.1
71 ( 4.4
86 ( 4.1
63 ( 12.1
95 ( 6.2
76 ( 1.9
91 ( 9.4
73 ( 9.4
96 ( 14.1
-22 ( 20.2
21 ( 6.0
30 ( 7.0
56 ( 14.9
60 ( 6.6
94 ( 6.6
1.9 ( 0.4
2.2 ( 1.2
76 ( 7.0
a Relative cell growth (%) in relation to an untreated control of the tumor cell lines after a 96 h exposure to substance at 20 µM. Results are averages (
standard deviation of three or more independent determinations. b Bladder cancer. c Bladder cancer. d Small cell lung cancer. e Large cell lung cancer. f Breast
cancer.
Table 3. IC50 Values of Growth Inhibition of the Cancer Cell Lines
However, the structure of the enantiomeric bicyclic alcohol
A-427 and MCF-7 Following a Continuous 96 h Exposure to the
Various Compounds
15 is also very similar to the postulated bioactive conformation
of 6a: The three-dimensional orientation of both the p-
methoxybenzyl and benzyl residues of 15 and 6a are quite
similar. Moreover, the hydroxy moiety of 15 points in a similar
direction as the hydroxy moiety of 6a (and also ent-15) does.
Therefore, a properly positioned H-bond acceptor group in the
σ1 receptor protein would be able to form H-bonds with the
OH moieties of all three potent σ1 ligands 6a, 15, and ent-15.
The methyl ethers 17, ent-17, 22, and ent-22 display lower
σ1 receptor affinities than the more active alcohols 15 and ent-
15. This result supports the idea that an H-bond contributes to
the overall interaction of the ligands with σ1 receptors, since
the methoxy moiety cannot function as H-bond donor group.
In the methyl ether series the diastereomers ent-17 and ent-22,
which are derived from (S)-glutamate, display the highest σ1
receptor affinity with Ki-values of 26 and 25 nM, respectively.
Generally, the σ2 receptor affinity of the investigated stere-
oisomeric alcohols and methyl ethers is very low, indicating
high selectivity for σ1 receptors over σ2 receptors. The best σ1/
σ2 selectivity was observed for the most potent σ1 receptor
ligands 15, ent-15, ent-17, and ent-22.
In the NMDA assay the stereoisomeric alcohols and ethers
did not interact with the phencyclidine binding site of the
NMDA receptor. At a concentration of 10 µM the test
compounds were not able to compete with the radioligand
(100% radioligand binding) for the binding sites. Thus the
bicyclic alcohols and ethers display high preference for σ1
receptors compared with NMDA receptors.
IC50 [µM]a
compound
configuration
A-427
MCF-7
17
ent-17
22
ent-22
haloperidol
cisplatinb
oxaliplatinb
methotrexateb
(1S,2S,5R)
(1R,2R,5S)
(1S,2R,5R)
(1R,2S,5S)
9.5 ( 2.32
1.23 ( 0.46
8.00 ( 1.61
0.51 ( 0.21
10.0 ( 1.71
1.96 ( 0.54
0.76 ( 0.09
5.52 ( 3.55
13.4 ( 0.75
12.2 ( 2.05
15.3 ( 3.34
13.5 ( 2.34
24.9 ( 9.77
1.38 ( 0.29
0.32 ( 0.04
0.05 ( 0.02
a Results are averages ( standard deviation of three or more independent
determinations. b IC50 values are from ref 30.
five human tumor cell lines. This panel includes the cell lines
5637 (bladder cancer), RT-4 (bladder cancer), A-427 (small cell
lung cancer), LCLC 103H (large cell lung cancer), and MCF-7
(breast cancer).
In the primary screening the tumor cells were incubated with
a 20 µM solution of the test compounds at 37 °C. After 96 h
the medium was removed and the density of adherent cells
(living cells) was measured by staining with crystal violet.30 In
Table 2 the results are given as part of living cells (in %) in
relation to a control without test compound.
The data in Table 2 clearly indicate the tendency of the methyl
ethers 17, ent-17, 22, and ent-22 to reduce the cell growth of
the tumor cells to a greater extent than the corresponding
alcohols 15, ent-15, 20, and ent-20. It is possible that the higher
lipophilicity of the methyl ethers is responsible for their stronger
activity.
The cell growth inhibition of the small cell lung cancer cell
line A-427 is striking. Whereas the stereoisomeric alcohols
caused about 40% cell growth inhibition, the values of the
stereoisomeric methyl ethers are negative, indicating not only
Cytotoxicity Assay. The overexpression of σ1 and σ2
receptors in human tumor cell lines and the cytotoxic activity
of some σ ligands have been documented in the literature (see
Introduction). Therefore, we investigated the cytotoxic effects
of the stereoisomeric alcohols and methyl ethers in a panel of

6148 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Geiger et al.
Table 4. Expression of σ1 and σ2 Receptors in the Five Human Tumor Cell Lines
c
c
cell line
SBa (%)
σ1 Nb (109)
Np (107) (N/µg)
cpd (pmol/mg)
SBa (%)
σ2 Nb (109)
Np (107) (N/µg)
cpd (pmol/mg)
5637
RT-4
A-427
LCLC-103H
MCF-7
23
92
73
87
74
0.99
18.5
5.97
8.36
10.4
1.1
21
6.6
9.3
11.5
0.018
0.35
0.11
0.15
0.19
52
97
43
88
89
3.61
110
2.33
14.9
12.6
4.0
122
2.6
16.6
14.1
0.066
2.03
0.043
0.28
0.23
a SB: specific binding in relation to the total binding in %. b N: Number of binding sites in the assay. c Np: Number of bindings sites per µg protein.
d cp: molar amount of binding sites (pmol) per mg protein.
inhibition of cell growth but also death of existing cells. It is
very likely that the growth inhibition of the A-427 cell line is
not simply unspecific toxicity of the test compounds because
the growth inhibition of the other four tumor cell lines is
considerably lower. Obviously a definite target, which is
selectively produced by A-427 cells and to a lesser extent in
the MCF-7 cells, is interacting with the test compounds, possibly
the σ1 receptor. It is notable that at a concentration of 20 µM
haloperidol, a known σ1 receptor antagonist, also strongly and
selectively inhibits the growth of the A-427 cell line while (+)-
pentazocine, a known σ1 receptor agonist, does not. Similar
cytotoxic behavior of haloperidol and a lack of cytotoxic
behavior of (+)-pentazocine in other cell lines expressing σ
receptors have been documented.42
The IC50-values of the diastereomeric methyl ethers ent-17
and ent-22, which combine high σ1 receptor affinity and good
growth inhibition against both A-427 and MCF-7 cells, and the
IC50-values of their enantiomers 17 and 22 were determined.
In Table 3 the IC50-values of the test compounds are compared
with the IC50-values of three standard anticancer drugs. The
cytotoxic effect of the (R,R,S)-configured methyl ether ent-17
(IC50 ) 1.23 µM) is comparable with the activity of cisplatin
(IC50 ) 1.27 µM). Moreover, the diastereomeric methyl ether
ent-22 (IC50 ) 0.51 µM) is equipotent with the more active
drug oxaliplatin (IC50 ) 0.68 µM). The A-427 cell line is at
least 10 times more sensitive to the antiproliferative effects of
ent-17 and ent-22 than the MCF-7 cell lines. Comparing the
IC50 values in the A-427 and MCF-7 cell lines, there is much
less selectivity for haloperidol and the enantiomeric ethers 17
and 22 (Table 3) than for ent-17 and ent-22.
σ1 and σ2 Receptor Expression in Tumor Cell Lines. In
order to investigate the σ1 and σ2 receptor expression of the
tumor cell lines under consideration cell membrane preparations
of these tumor cells were produced by homogenization and
centrifugation. For a better comparison of the data, the protein
amount was standardized with the Bradford method.31 The
specific σ1 receptor binding (SB) was determined by incubation
of the membrane preparations (1.8 mg protein/mL) with the σ1
selective radioligand [3H]-(+)-pentazocine in the absence (total
binding, TB) and the presence (nonspecific binding, NSB) of
the non-radiolabeled competitor (+)-pentazocine. The σ2 recep-
tor expression of the tumor cell lines was determined in the
same manner using the radioligand [3H]ditolylguanidine in the
absence (TB) and the presence (NSB) of the non-radiolabeled
competitor ditolylguanidine. In order to selectively label σ2
receptors (ditolylguanidine is an unselective σ ligand) these
experiments were performed in the presence of an excess of
(+)-pentazocine, which selectively blocks σ1 receptors.
With only few exceptions the five tumor cell preparations
show a specific σ1 and σ2 receptor binding greater than 50%
(Table 4). A particular high σ1 (92% SB) and σ2 (97% SB)
receptor expression was found for the bladder cancer cell line
RT-4. The high σ1 and σ2 receptor density of this cell line will
be exploited to establish new σ1 and σ2 receptor assays on the
basis of RT-4 cell membrane preparations. We were able to
confirm the relatively high density of both σ1 and σ2 receptors
in the MCF-7 cell line, which was reported previously by John
and co-workers.43 The specific σ1 receptor binding in the small
cell lung cancer cell line A-427 is also relatively high (73%). It
is possible that the strong cytotoxic effects of the methyl ethers
ent-17 and ent-22, which interact in the nanomolar range with
σ1 receptors, are mediated by these receptors.
It should be noted that strong correlations between σ receptor
affinities, tumor cell growth inhibition, and σ receptor expression
are not apparent in our data. However, relationships between
these three parameters are complicated and direct correlations
may not be apparent. For example, the cell growth inhibition
assay uses whole cells while the receptor binding assays use
cell membranes isolated from cell homogenates. Thus, additional
parameters influencing cell membrane penetration (e.g., lipo-
philicity) may be important for the compounds to access the
receptors and act on the cells. The intrinsic function of the
receptors may also be different in each cell line. In confirmation
of this, Spruce and co-workers found no correlation between
the expression levels of the σ1 receptor and the susceptibility
of tumor cells to the antiproliferative effects of σ antagonists.42
Finally, the intrinsic activities of the new σ ligands (agonist,
partial agonist, antagonist) have not yet been established, and
this property will be a deciding factor as to whether a compound
has antiproliferative activity (e.g., compare the σ1 antagoinst
haloperidol with the σ1 agonist (+)-pentazocine in Table 2).
Conclusion
The stereoisomeric alcohols 15, ent-15, 20, and ent-20 and
methyl ethers 17, ent-17, 22, and ent-22 with the bridged
piperazine framework represent a novel class of potent σ1
receptor ligands. The σ1 receptor affinity appears to be correlated
with cytotoxic effects against the A-427 cell line; in particular
the diastereoisomeric methyl ethers ent-17 and ent-22 show
promising σ1 receptor affinity (Ki ) 26 nM and 25 nM) and
growth inhibition of the A-427 cell line (IC50 ) 1.23 µM and
0.51 µM) while enantiomeric compounds 17 and 22 show
weaker σ1 receptor binding and also weaker antiproliferative
activity. In the A-427 cell line, both σ1 receptors with 73%
specific binding and σ2 receptors with only 43% specific binding
were found. This lends support to the hypothesis that ent-17
and ent-22 act as antiproliferative agents through their interac-
tions with the σ1 receptor, possibly as antagonists. Ongoing
competition experiments with known agonists and antagonists
of the σ receptors are aiming to elucidate the mechanism of
cell growth inhibition by these interesting compounds.
Experimental
Chemistry. General. Flash chromatography (fc):32 silica gel 60,
40-64 µm (Merck; parentheses include: diameter of the column,
eluent, Rf-value). Optical rotation: Polarimeter 341 (Perkin-Elmer);
1
1.0 dm tube; concentration c [g/100 mL]. H NMR (400 MHz),
13C NMR (100 MHz): Mercury-400BB spectrometer (Varian), δ
in ppm related to tetramethylsilane, coupling constants are given
with 0.5 Hz resolution; the assignments of 13C and 1H NMR signals
were supported by 2D NMR techniques.

Cytotoxic Bicyclic σ Receptor Ligands
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6149
(-)-Dimethyl (R)-Glutamate Hydrochloride (8‚HCl). As de-
scribed for the preparation of ent-8‚HCl,19 (R)-glutamate (25.1 g,
171 mmol) was reacted with dry MeOH (350 mL) and Me3SiCl
(75 mL, 594 mmol). After workup, the residual viscous oil was
dried in vacuo at 40 °C to obtain 8‚HCl as colorless crystals, yield
was stirred for 16 h at rt. Then, the mixture was concentrated in
vacuo to half of the original volume, and after dilution with CH2-
Cl2 (150 mL), the organic layer was washed with a half-saturated
solution of NaHCO3 (50 mL), H2O (50 mL), and brine (50 mL),
dried (Na2SO4), and then concentrated in vacuo. The residue was
washed with Et2O to afford 13 as colorless solid, yield 3.6 g (85%),
mp 160 °C. [R]20589 ) -72.6 (c 0.113, CH2Cl2). Anal. (C22H22N2O4)
33 g (91%), mp 91 °C. [R]20 ) -25.4 (c 5.0, H2O).
589
(+)-Dimethyl (S)-Glutamate Hydrochloride (ent-8‚HCl).19 ent-
8‚HCl was prepared as described in ref 19 from (S)-glutamate (15
g, 102 mmol). Colorless solid, yield 21.3 g (99%), mp 83 °C.
1
C, H, N. H NMR (CDCl3): δ 1.73-1.84 (m, 1H, 4-H), 2.22-
2.37 (m, 2H, 4-H, 3-H), 2.40-2.50 (m, 1H, 3-H), 3.79 (s, 3H,
PhOCH3), 4.05 (dd, J ) 4.7/2.3 Hz, 1H, 5-H), 4.30 (s, 1H, 1-H),
4.39 (d, J ) 14.3 Hz, 1H, NCH2Ph), 4.56 (d, J ) 14.7 Hz, 1H,
NCH2Ph), 4.62 (d, J ) 14.7 Hz, 1H, NCH2Ph), 4.76 (d, J ) 14.5
[R]20 ) +25.4 (c 5.05, H2O).
589
(+)-Dimethyl (R)-2-[N-Benzyl-N-(2-chloroacetyl)amino]pen-
tanedioate (10). As described for the preparation of ent-10,19 the
hydrochloride 8‚HCl (10.0 g, 47.2 mmol) was treated with
benzaldehyde (4.72 mL, 46.7 mmol), MgSO4 (8 g), and triethy-
lamine (6.5 mL, 46.9 mmol) in dry CH2Cl2 (75 mL). The formed
imine was reduced with NaBH4 (3.05 g, 80.9 mmol) in dry MeOH
(120 mL), and the resulting secondary amine 9 (ca. 12 g) was
acylated with chloroacetyl chloride (6.1 mL, 76.5 mmol) and
triethylamine (5.5 mL, 39.6 mmol) in dry CH2Cl2 (65 mL). After
workup and fc, the chloroacetamide 10 was isolated as colorless
Hz, 1H, NCH2Ph), 6.84 (d, J ) 8.6 Hz, 2H, arom 3-H, 5-Hmethoxy
-
benzyl), 7.16-7.38 (m, 7H, arom H).
(+)-(1S,5S)-6-Benzyl-8-(4-methoxybenzyl)-6,8-diazabicyclo-
[3.2.2]nonane-2,7,9-trione (ent-13). As described for the prepara-
tion of 13, the enantiomer ent-11 (8.5 g, 20.7 mmol) was treated
with LiHMDS (1 M in THF, 37.5 mL, 37.5 mmol) and Me3SiCl
(9.85 mL, 77.6 mmol) in dry THF (210 mL), and the resulting
methyl silyl acetal ent-12 was hydrolyzed in a mixture of THF (150
mL), H2O (15 mL) and p-TosOH (2.76 g, 14.4 mmol). After
purification by fc (8 cm, cyclohexane/EtOAc 1:1, Rf ) 0.25), ent-
13 was obtained as colorless solid, yield 6.6 g (84%), mp 163 °C.
[R]20589 ) +72.4 (c 0.132, CH2Cl2). Anal. (C22H22N2O4) C, H, N.
Purity by HPLC analysis.
oil, Rf ) 0.27 (clohexane/EtOAc 7:3), yield 11.33 g (70%). [R]20
589
) +63.0 (c 1.925, CHCl3). Anal. (C16H20ClNO5) C, H, N.
(-)-Dimethyl (S)-2-[N-Benzyl-N-(2-chloroacetyl)amino]pen-
tanedioate (ent-10).19 ent-10 was prepared as described in ref 19
from ent-8‚HCl (10 g, 47.2 mmol). Colorless oil, yield 9.15 g (56%).
[R]20 ) -63.0 (c 1.455, CHCl3). Purity by HPLC analysis.
(-)-(1R,2S,5R)-6-Benzyl-2-hydroxy-8-(4-methoxybenzyl)-6,8-
diazabicyclo[3. 2.2]nonane-7,9-dione (14). Under N2 at -90 °C
a solution of LiBH4 (2 M in THF, 25.3 mL, 50.6 mmol) was slowly
added to a solution of 13 (4.8 g, 12.68 mmol) in dry THF (250
mL), and the reaction mixture was stirred for 3.5 h at low
temperature. The reaction mixture was cautiously hydrolyzed at
-90 °C with 2 M aq HCl (250 mL). The mixture was warmed to
rt and was stirred for an additional 0.5 h. The resulting solution
was neutralized under ice cooling with 2 M NaOH, and the pH
was brought to 8-9. The mixture was extracted three times with
CH2Cl2, and the combined organic layers were dried (Na2SO4) and
concentrated in vacuo. The residual solid was recrystallized (EtOAc/
MeOH 9:1) to afford 14 as colorless crystals, yield 3.98 g (82%),
589
(+)-Methyl (R)-3-[1-Benzyl-4-(4-methoxybenzyl)-3,6-diox-
opiperazin-2-yl]propanoate (11). As described for the preparation
of ent-11,19 a solution of the chloroacetamide 10 (11.3 g, 33.1
mmol) in acetonitrile (160 mL) was reacted with 4-methoxyben-
zylamine (6.2 mL, 47.9 mmol) and triethylamine (6.5 mL, 46.9
mmol). After workup and fc, the piperazinedione 11 was isolated
as colorless solid, Rf ) 0.33 (cyclohexane/EtOAc 1:1), yield 11.2
g (82%), mp 63 °C (Et2O). [R]20589 ) +8.2 (c 1.10, CH2Cl2). Anal.
(C23H26N2O5) C, H, N.
(-)-Methyl (S)-3-[1-Benzyl-4-(4-methoxybenzyl)-3,6-diox-
opiperazin-2-yl]propanoate (ent-11).19 ent-11 was prepared as
described in ref 19 from ent-10 (9.15 g, 26.8 mmol) and 4-meth-
oxybenzylamine (4.97 mL, 38.4mmol). Pale yellow oil, yield 8.5
mp 183 °C (EtOAc). [R]20 ) -91.0 (c 1.25, CH2Cl2). Anal.
589
1
g (77%). [R]20 ) -7.8 (c 0.712, CH2Cl2). Purity by HPLC
(C22H24N2O4) C, H, N. H NMR (CDCl3): δ 1.39-1.51 (m, 1H,
589
3-H or 4-H), 1.54-1.66 (m, 1H, 3-H or 4-H), 1.67-1.77 (m, 1H,
3-H or 4-H), 1.78-1.89 (m, 1H, 3-H or 4-H), 3.41-3.51 (m, 1H,
2-H), 3.79 (s, 3H, PhOCH3), 3.93 (dd, J ) 5.6/2.6 Hz, 1H, 5-H),
4.00 (d, J ) 3.7 Hz, 1H, 1-H), 4.43 (d, J ) 14.5 Hz, 1H, NCH2-
aryl), 4.51 (d, J ) 14.5 Hz, 1H, NCH2aryl), 4.59 (d, J ) 14.5 Hz,
1H, NCH2aryl), 4.70 (d, J ) 14.5 Hz, 1H, NCH2aryl), 6.86 (d, J )
8.6 Hz, 2H, arom 3-H, 5-Hmethoxybenzyl), 7.20 (d, J ) 8.6 Hz, 2H,
arom 2-H, 6-Hmethoxybenzyl), 7.25-7.37 (m, 5H, arom H). HPLC:
Column: Merck Superspher 100 (4 µm) LiChroCART 250-4 mm;
eluent CH3OH/H2O 40:60; 0.5 mL/min; retention times: 14: 90
min; 19: 102 min.
analysis.
(+)-(1R,2S,5R)-6-Benzyl-2-methoxy-8-(4-methoxybenzyl)-2-
(trimethylsiloxy)-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (12).23
As described in ref 23, a solution of 11 (3.20 g, 7.79 mmol) in
THF (70 mL) was treated with lithium hexamethyldisilazane
(LiHMDS, 1 M in THF, 8.9 mL, 8.9 mmol) and Me3SiCl (3.6 mL,
28.36 mmol) to yield 12. Colorless solid (methanol/water (1:1)),
mp 95 °C. [R]589 ) +8.7, (c 1.72, CH2Cl2).
(-)-(1S,2R,5S)-6-Benzyl-2-methoxy-8-(4-methoxybenzyl)-2-
(trimethylsiloxy)-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (ent-
12). As described for the synthesis of 12, the enantiomer ent-11
(233 mg, 0.57 mmol) was reacted with LiHMDS (1 M in THF,
0.95 mL, 0.95 mmol) and Me3SiCl (0.25 mL, 1.97 mmol) in THF
(7.5 mL). After workup and purification, ent-12 was obtained as
colorless solid, yield 214 mg (78%), mp 96 °C (petroleum ether/
ethyl acetate). [R]589 ) -8.7 (c 1.72, CH2Cl2). Anal. (C26H34N2O5-
Si) C, H, N.
(-)-(1R,5R)-6-Benzyl-8-(4-methoxybenzyl)-6,8-diazabicyclo-
[3.2.2]nonane-2,7,9-trione (13). Under N2 at -78 °C a solution
of 11 (4.6 g, 11.2 mmol) in dry THF (40 mL) was added dropwise
during 10 min to a solution of LiHMDS (1 M in THF, 18.5 mL,
18.5 mmol) in dry THF (100 mL). After the mixture was stirred
for 30 min at -78 °C, Me3SiCl (4.9 mL, 38.6 mmol) was added
slowly and the reaction mixture was stirred for 30 min at -78 °C
and for 3 h at rt. Then, the solvent was removed in vacuo, the
residue was dissolved in EtOAc (75 mL), and the organic layer
was washed with 0.5 M HCl (30 mL), 0.5 M NaOH (30 mL), and
brine (30 mL), dried (Na2SO4), and concentrated in vacuo. Since
further purification was not necessary, the resulting residue (methyl
silyl acetal 12) was dissolved in THF (70 mL), H2O (7 mL) and
p-TosOH (1.38 g, 7.25 mmol) were added, and the reaction mixture
(+)-(1S,2R,5S)-6-Benzyl-2-hydroxy-8-(4-methoxybenzyl)-6,8-
diazabicyclo[3.2.2]nonane-7,9-dione (ent-14). As described for the
preparation of 14, ketone ent-13 (5.0 g, 13.21 mmol) was reduced
with LiBH4 (2 M in THF, 26.4 mL, 52.8 mmol) in dry THF (250
mL). The crude product was recrystallized (EtOAc) to afford ent-
14 as colorless solid, yield 4.22 g (84%), mp 182-183 °C (EtOAc).
[R]20 ) +91.5 (c 1.40, CH2Cl2). Anal. (C22H24N2O4) C, H, N.
589
Further recrystallization from ethyl acetate gave colorless crystals,
which were suitable for X-ray crystal structure analysis.
X-rayCrystalStructureAnalysisofent-14.FormulaC22H24N2O4,
M ) 380.43, T ) 223 K, colorless crystal, crystal size 0.35 × 0.10
× 0.03 mm, a ) 9.973(1), b ) 7.210(1), c ) 13.836(1) Å, â )
105.35(1)°, V ) 959.4(2) Å,3 Fcalcd ) 1.317 g cm-3, µ ) 0.741
mm-1, empirical absorption correction (0.781 e T e 0.978), Z )
2, monoclinic, space group P21 (no. 4), λ ) 1.54178 Å, ω and æ
scans, 4392 reflections collected ((h, (k, (l), [(sinθ)/λmax] ) 0.59
Å-1, 2457 independent (Rint ) 0.034) and 2024 observed reflections
[I g 2 σI)], 255 refined parameters, R ) 0.038, wR2 ) 0.099, max.
residual electron density 0.14 (-0.13) e Å-3, Flack parameter 0.2-
(3), hydrogens calculated and refined as riding atoms. The data set

6150 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Geiger et al.
was collected with a Nonius KappaCCD diffractometer. Programs
used: data collection COLLECT,33 data reduction Denzo-SMN,34
absorption correction Denzo,35 structure solution SHELXS-97,36
structure refinement SHELXL-97,37 graphics SCHAKAL.38
CCDC-635641 contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge
at www.ccdc.cam.ac.uk/conts/retrieving.html [or from the Cam-
bridge Crystallographic Data Centre, 12 Union Road, Cambridge
CB2 1EZ, UK; fax: (internat.) +44(1223)336-033, E-mail:
deposit@ccdc.cam.ac.uk].
(-)-(1S,2S,5R)-6-Benzyl-8-(4-methoxybenzyl)-6,8-diazabicyclo-
[3.2.2]nonan-2-ol (15). Under N2, LiAlH4 (75 mg, 1.98 mmol) was
added in small portions to an ice-cooled solution of 14 (151 mg,
0.40 mmol) in dry THF (13 mL). The reaction mixture was heated
to reflux for 16 h. Then, H2O was added under ice cooling until
H2 formation was finished. After filtration the filtrate was evapo-
rated in vacuo. The resulting crude product was purified by fc (2
cm, petroleum ether/EtOAc 8:2 + 1% N,N-dimethylethanamine,
Rf ) 0.28) to afford 15 as colorless viscous oil, yield 118 mg (84%).
[R]20589 ) -12.7 (c 0.845, CH2Cl2). Anal. (C22H28N2O2) H. C, N:
calcd, C 74.97, N 7.95; found, C 74.52, N 7.53. 1H NMR
(CDCl3): δ ) 1.59-1.69 (m, 1H, 4-H), 1.74-1.87 (m, 2H, 3-H,
4-H), 2.13-2.24 (m, 1H, 3-H), 2.67-2.74 (m, 2H, 7-H, 9-H), 2.76-
2.87 (m, 3H, 1-H, 5-H, 9-H), 3.10 (dd, J ) 11.2/2.4 Hz, 1H, 7-H),
3.57-3.74 (m, 4H, 2 × NCH2aryl), 3.79 (s, 3H, PhOCH3), 3.97
(q, J ) 5.7 Hz, 1H, 2-H), 6.84 (d, J ) 8.6 Hz, 2H, arom 3-H,
5-Hmethoxybenzyl), 7.12-7.26 (m, 3H, arom H), 7.27-7.35 (m, 4H,
arom H). 13C NMR (CDCl3): δ ) 29.3 (1C, C-3), 30.5 (1C, C-4),
46.7 (1C, C-7), 51.0 (1C, C-9), 54.5 (1C, C-5), 55.2 (1C, PhOCH3),
60.2 (1C, C-1), 60.4 (1C, NCH2atyl), 60.9 (1C, NCH2aryl), 75.1
(1C, C-2), 113.6 (2C, arom CHC-3,C-5 methoxybenzyl), 126.9 (1C, arom
CH), 128.2 (2C, arom CH), 128.5 (2C, arom CH), 129.6 (2C, arom
(-)-(1S,2S,5R)-6-Benzyl-2-methoxy-8-(4-methoxybenzyl)-6,8-
diazabicyclo[3.2.2]nonane (17). As described for the reduction of
the alcohol 14, the methyl ether 16 (139 mg, 0.35 mmol) dissolved
in dry THF (15 mL) was reduced with LiAlH4 (66 mg, 1.74 mmol).
After purification by fc (3 cm, petroleum ether/EtOAc 9:1, Rf )
0.11), 17 was obtained as colorless viscous oil, yield 70 mg (54%).
[R]20 ) -12.0 (c 0.70, CH2Cl2). Anal. (C23H30N2O2) C, H. N:
589
1
calcd, N 7.64; found, N 7.11. H NMR (CDCl3): δ ) 1.50-1.60
(m, 1H, 4-H), 1.78-1.87 (m, 1H, 4-H), 1.88-1.96 (m, 2H, 3-H),
2.63 (dd, J ) 10.7/3.7 Hz, 1H, 9-H), 2.68-2.76 (m, 2H,1-H, 7-H),
2.82-2.88 (m, 2H, 5-H, 9-H), 3.05 (s, 3H, OCH3), 3.05-3.10 (m,
1H, 7-H), 3.24 (t, J ) 8.2 Hz, 1H, 2-H), 3.64 (d, J ) 12.9 Hz, 1H,
NCH2aryl), 3.67 (s, 2H, NCH2aryl), 3.73 (d, J ) 12.9 Hz, 1H,
NCH2aryl), 3.78 (s, 3H, PhOCH3), 6.84 (d, J ) 8.6 Hz, 2H, arom
3-H, 5-Hmethoxybenzyl), 7.17-7.35 (m, 7H, arom H). 13C NMR
(CDCl3): δ ) 26.7 (1C, C-3), 28.4 (1C, C-4), 47.8 (1C, C-7), 51.8
(1C, C-9), 53.4 (1C, C-5), 55.2 (1C, PhOCH3), 56.2 (2C, C-1,
OCH3), 60.1 (1C, NCH2aryl), 60.6 (1C, NCH2aryl), 85.3 (1C, C-2),
113.4 (2C, arom CHC-3,C-5 methoxybenzyl), 126.6 (1C, arom CH), 128.1
(2C, arom CH), 128.3 (2C, arom CH), 130.0 (2C, arom CH), 131.6
(1C, arom CC-1 methoxybenzyl), 140.1 (1C, arom CC-1 benzyl), 158.6 (1C,
arom CC-4 methoxybenzyl).
(+)-(1R,2R,5S)-6-Benzyl-2-methoxy-8-(4-methoxybenzyl)-6,8-
diazabicyclo[3.2.2]nonane (ent-17). As described for the prepara-
tion of 17, the enantiomer ent-16 (630 mg, 1.60 mmol) was reduced
with LiAlH4 (312 mg, 8.22 mmol) in dry THF (50 mL) to give
ent-17 as a colorless viscous oil, yield 413 mg (70%). [R]20
)
589
+10.5 (c 1.005, CH2Cl2). Anal. (C23H30N2O2) H, N. C: calcd, C
75.37; found, C 74.86.
(+)-[(1S,2S,5S)-6-Benzyl-8-(4-methoxybenzyl)-7,9-dioxo-6,8-
diazabicyclo[3.2.2]nonan-2-yl] 4-Nitrobenzoate (ent-18). Under
N2, diisopropyl azodicarboxylate (DIAD, 1.0 mL, 5.14 mmol) was
added dropwise to an ice-cooled solution of ent-14 (500 mg, 1.31
mmol), PPh3 (1.69 g, 6.44 mmol), and p-nitrobenzoic acid (960
mg, 5.74 mmol) in dry THF (25 mL). The reaction temperature
was allowed to rise to rt. After 6 h, the solvent was removed in
vacuo and the residue was purified by fc (6 cm, petroleum ether/
EtOAc 1:1, Rf ) 0.42) to obtain ent-18 as pale yellow solid, yield
CH), 131.5 (1C, arom CC-1 methoxybenzyl), 139.4 (1C, arom C
C-1
benzyl), 158.5 (1C, arom CC-4 methoxybenzyl).
(+)-(1R,2R,5S)-6-Benzyl-8-(4-methoxybenzyl)-6,8-diazabicyclo-
[3.2.2]nonan-2-ol (ent-15). As described for the preparation of 15,
the enantiomer ent-14 (600 mg, 1.58 mmol) was reduced with
LiAlH4 (300 mg, 7.91 mmol) in dry THF (50 mL) to give ent-15
555 mg (80%), mp 143 °C. [R]20 ) +51.1 (c 0.19, CH2Cl2).
589
as a colorless viscous oil, yield 480 mg (86%). [R]20 ) +11.4
589
1
Anal. (C29H27N3O7) C, H, N: calcd. C 65.78; found C 65.36. H
(c 0.775, CH2Cl2). Anal. (C22H28N2O2) H, N. C: calcd, C 74.97;
NMR (CDCl3): δ ) 1.42-1.54 (m, 1H, 3-H or 4-H), 1.82-1.94
(m, 1H, 3-H or 4-H), 2.05-2.16 (m, 2H, 3-H or 4-H), 3.75 (s, 3H,
PhOCH3), 4.00 (dd, J ) 4.9/2.5 Hz, 1H, 5-H), 4.27 (d, J ) 2.0 Hz,
1H, 1-H), 4.37 (d, J ) 14.9 Hz, 1H, NCH2aryl), 4.59 (s, 2H, NCH2-
aryl), 4.87 (d, J ) 14.9 Hz, 1H, NCH2aryl), 5.34 (ddd, J ) 9.3/
5.0/1.9 Hz, 1H, 2-H), 6.79 (d, J ) 9.0 Hz, 2H, arom 3-H,
5-Hmethoxybenzyl), 7.12 (d, J ) 8.6 Hz, 2H, arom 2-H, 6-Hmethoxybenzyl),
7.22-7.39 (m, 5H, arombenzyl), 7.94 (d, J ) 9.0 Hz, 2H, arom 2-H,
6-Hnitrophenyl), 8.28 (d, J ) 9.0 Hz, 2H, arom 3-H, 5-Hnitrophenyl).
(+)-(1R,2R,5R)-6-Benzyl-2-hydroxy-8-(4-methoxybenzyl)-6,8-
diazabicyclo[3.2.2]nonane-7,9-dione (19). Under N2, DIAD (1.0
mL, 5.14 mmol) was added dropwise to an ice-cooled solution of
14 (500 mg, 1.31 mmol), PPh3 (1.69 g, 6.44 mmol), and p-
nitrobenzoic acid (960 mg, 5.74 mmol) in dry THF (25 mL). The
reaction temperature was allowed to rise to rt. After 16 h, the solvent
was removed in vacuo and the residue was purified by fc (3 cm,
petroleum ether/EtOAc 1:1, Rf ) 0.42). The resulting pale yellow
solid (p-nitrobenzoate 18) was immediately dissolved in MeOH
(30 mL), K2CO3 (300 mg, 2.17 mmol) was added, and the reaction
mixture was stirred for 16 h at rt. Then, H2O (120 mL) was added,
the aqueous solution was extracted with CH2Cl2, and the combined
organic layers were dried (Na2SO4) and concentrated in vacuo. The
residual solid was purified by fc (3 cm, EtOAc, Rf ) 0.40) to obtain
19 as a colorless solid, yield 301 mg (60%), mp 173-174 °C.
[R]20589 ) +23.0 (c 1.045, CH2Cl2). Anal. (C22H24N2O4) C, H, N.
1H NMR (CDCl3): δ 1.29-1.41 (m, 1H, 4-H), 1.50-1.64 (m, 1H,
3-H), 1.86-1.94 (m, 1H, 3-H), 1.94-2.01 (m, 1H, 4-H), 3.80 (s,
3H, PhOCH3), 3.89 (dd, J ) 5.4/2.3 Hz, 1H, 5-H), 3.99-4.06 (m,
2H, 1-H, 2-H), 4.42 (d, J ) 14.5 Hz, 1H, NCH2Ph), 4.44 (d, J )
14.7 Hz, 1H, NCH2Ph), 4.60 (d, J ) 14.7 Hz, 1H, NCH2Ph), 4.88
(d, J ) 14.5 Hz, 1H, NCH2Ph), 6.87 (d, J ) 8.6 Hz, 2H, arom
found, C 74.47.
(-)-(1R,2S,5R)-6-Benzyl-2-methoxy-8-(4-methoxybenzyl)-6,8-
diazabicyclo[3.2.2]nonane-7,9-dione (16). The alcohol 14 (150 mg,
0.39 mmol) and MeI (0.12 mL, 1.88 mmol) were consecutively
added to a suspension of NaH (135 mg obtained from 225 mg 60%
dispersion in oil, 5.63 mmol) in dry THF (20 mL). The reaction
mixture was stirred at rt for 3 h. After hydrolysis with H2O (2 mL)
under ice cooling, CH2Cl2 (50 mL) was added and the mixture was
extracted twice with 0.5 M NaOH. The organic layer was dried
(Na2SO4) and concentrated in vacuo. The residue was purified by
fc (2 cm, petroleum ether/EtOAc 3:7, Rf ) 0.38) to give 16 as
colorless viscous oil, which solidified slowly. Colorless solid, yield
150 mg (96%), mp 102 °C. [R]20 ) -119.4 (c 0.535, CH2Cl2).
Anal. (C23H26N2O4) C, H, N. H NMR (CDCl3): δ ) 1.42-1.60
589
1
(m, 2H, 3-H and/or 4-H), 1.63-1.80 (m, 2H, 3-H and/or 4-H),
2.86-2.92 (m, 1H, 2-H), 3.18 (s, 3H, OCH3), 3.81 (s, 3H, PhOCH3),
3.91 (dd, J ) 5.7/2.4 Hz, 1H, 5-H), 4.05 (d, J ) 3.3 Hz, 1H, 1-H),
4.33 (d, J ) 14.5 Hz, 1H, NCH2aryl), 4.45 (d, J ) 14.6 Hz, 1H,
NCH2aryl), 4.72 (d, J ) 14.5 Hz, 1H, NCH2aryl), 4.75 (d, J )
14.5 Hz, 1H, NCH2aryl), 6.88 (d, J ) 8.6 Hz, 2H, arom 3-H,
5-Hmethoxybenzyl), 7.22 (d, J ) 8.6 Hz, 2H, arom 2-H, 6-Hmethoxybenzyl),
7.26-7.35 (m, 5H, arom H).
(+)-(1S,2R,5S)-6-Benzyl-2-methoxy-8-(4-methoxybenzyl)-6,8-
diazabicyclo[3.2.2]nonane-7,9-dione (ent-16). As described for the
preparation of 16, the enantiomer ent-14 (530 mg, 1.39 mmol) was
allowed to react with NaH (474 mg obtained from 790 mg 60%
dispersion in oil, 19.8 mmol) and MeI (0.44 mL, 6.9 mmol) in dry
THF (30 mL) to yield ent-16 as a colorless solid, yield 491 mg
(89%), mp 98 °C. [R]20 ) +102.4 (c 0.505, CH2Cl2). Anal.
(C23H26N2O4) C, H, N.
589

Cytotoxic Bicyclic σ Receptor Ligands
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6151
3-H, 5-Hmethoxybenzyl), 7.20 (d, J ) 8.6 Hz, 4H, arom H), 7.27-7.36
(m, 3H, arom H).
(CDCl3): δ ) 1.59-1.75 (m, 2H, 4-H), 1.82-1.90 (m, 1H, 3-H),
2.00-2.12 (m, 1H, 3-H), 2.67-2.77 (m, 3H, 5-H, 7-H, 9-H), 2.78-
2.87 (m, 2H, 7-H, 9-H), 3.00 (t, J ) 2.7 Hz, 1H, 1-H), 3.14 (s, 3H,
OCH3), 3.53 (dd, J ) 10.2/4.7 Hz, 1H, 2-H), 3.63-3.74 (m, 4H, 2
× NCH2aryl), 3.79 (s, 3H, PhOCH3), 6.84 (d, J ) 8.6 Hz, 2H,
arom 3-H, 5-Hmethoxybenzyl), 7.20-7.36 (m, 7H, arom H). 13C NMR
(CDCl3): δ ) 26.4 (1C, C-3), 31.7 (1C, C-4), 50.0 (1C, C-7), 51.2
(1C, C-9), 54.6 (1C, C-5), 55.2 (1C, PhOCH3), 55.7 (1C, OCH3),
59.0 (1C, C-1), 61.0 (2C, NCH2aryl), 86.0 (1C, C-2), 113.3 (2C,
arom CHC-3,C-5 methoxybenzyl), 126.8 (1C, arom CH), 128.1 (2C, arom
CH), 128.6 (2C, arom CH), 129.8 (2C, arom CH), 131.8 (1C, arom
CC-1 methoxybenzyl), 139.7 (1C, arom CC-1 benzyl), 158.4 (1C, arom
(-)-(1S,2S,5S)-6-Benzyl-2-hydroxy-8-(4-methoxybenzyl)-6,8-
diazabicyclo[3.2.2]nonane-7,9-dione (ent-19). K2CO3 (300 mg,
2.17 mmol) was added to a solution of ent-18 (514 mg, 0.97 mmol)
in MeOH (35 mL), and the reaction mixture was stirred for 16 h at
rt. Then, H2O (140 mL) was added, the aqueous solution was
extracted with CH2Cl2, and the combined organic layers were dried
(Na2SO4) and concentrated in vacuo. The residual solid was purified
by fc (2 cm, EtOAc, Rf ) 0.40) to obtain ent-19 as a colorless
solid, yield 340 mg (92%), mp 174 °C (EtOAc). [R]20589 ) -22.6
(c 1.485, CH2Cl2). Anal. (C22H24N2O4) C, H, N.
C
C-4 methoxybenzyl).
(-)-(1S,2R,5R)-6-Benzyl-8-(4-methoxybenzyl)-6,8-diazabicyclo-
[3.2.2]nonan-2-ol (20). As described for the reduction of 14, the
diastereomeric alcohol 19 (124 mg, 0.33 mmol) in dry THF (11
mL) was reduced with LiAlH4 (62 mg, 1.64 mmol). After workup
and fc purification (2 cm, petroleum ether/EtOAc 8:2 + 1% N,N-
dimethylethanamine, Rf ) 0.33), 20 was obtained as colorless
(-)-(1R,2S,5S)-6-Benzyl-2-methoxy-8-(4-methoxybenzyl)-6,8-
diazabicyclo[3.2.2]nonane (ent-22). As described for the prepara-
tion of 22, the enantiomer ent-21 (545 mg, 1.38 mmol) was reduced
with LiAlH4 (260 mg, 6.86 mmol) in dry THF (50 mL) to give
ent-22 as a colorless viscous oil, yield 415 mg (82%). [R]20
-7.8 (c 0.80, CH2Cl2). Anal. (C23H30N2O2) C, H, N.
)
589
viscous oil, yield 76 mg (66%). [R]20 ) -24.0 (c 0.325, CH2-
589
Cl2). Anal. (C22H28N2O2) C, H, N. 1H NMR (CDCl3): δ ) 1.53-
1.63 (m, 2H, 3-H, 4-H), 1.70-1.79 (m, 1H, 4-H), 2.07-2.18 (m,
1H, 3-H), 2.57 (dd, J ) 10.8/2.9 Hz, 1H, 9-H), 2.70 (dd, J ) 10.6/
2.4 Hz, 1H, 9-H), 2.79-2.86 (m, 2H, 5-H, 7-H), 2.88-2.96 (m,
2H, 1-H, 7-H), 3.67-3.73 (m, 4H, 2 × NCH2aryl), 3.80 (s, 4H,
PhOCH3, 2-H), 6.87 (d, J ) 8.6 Hz, 2H, arom 3-H, 5-Hmethoxybenzyl),
7.20-7.34 (m, 7H, arom H). 13C NMR (CDCl3): δ ) 27.8 (1C,
C-4), 30.0 (1C, C-3), 48.9 (1C, C-7), 51.6 (1C, C-9), 53.7 (1C,
C-5), 55.2 (1C, PhOCH3), 60.0 (1C, C-1), 60.5 (1C, NCH2aryl),
61.3 (1C, NCH2aryl), 70.3 (1C, C-2), 113.8 (2C, arom CHC-3,C-5
methoxybenzyl), 126.8 (1C, arom CH), 128.2 (4C, arom CH), 130.2
(2C, arom CH), 130.4 (1C, arom CC-1 methoxybenzyl), 139.8 (1C, arom
Receptor Binding Studies. Materials and General Procedures.
The guinea pig brains and rat livers were commercially available
(Harlan-Winkelmann, Germany). Homogenizer: Elvehjem Potter
(B. Braun Biotech International). Centrifuge: High-speed cooling
centrifuge model Sorvall RC-5C plus (Thermo Finnigan). Filter:
Printed Filtermat Typ B (Perkin-Elmer), presoaked in 0.5% aqueous
polyethylenimine for 2 h at room temperature before use. The
filtration was carried out with a MicroBeta FilterMate-96 Harvester
(Perkin-Elmer). The scintillation analysis was performed using
Meltilex (Typ A) solid scintillator (Perkin-Elmer). The solid
scintillator was melted on the filtermat at a temperature of 95 °C
for 5 min. After solidification of the scintillator at room temperature,
the scintillation was measured using a MicroBeta Trilux scintillation
analyzer (Perkin-Elmer). The counting efficiency was 20%.
Membrane Preparation for the σ1 Assay (modified according
to refs 27, 29). Five guinea pig brains were homogenized with the
potter (500-800 rpm, 10 up-and-down strokes) in 6 volumes of
cold 0.32 M sucrose. The suspension was centrifuged at 1200g for
10 min at 4 °C. The supernatant was separated and centrifuged at
23500g for 20 min at 4 °C. The pellet was resuspended in 5-6
volumes of buffer (50 mM Tris, pH 7.4) and centrifuged again at
23500g (20 min, 4 °C). This procedure was repeated twice. The
final pellet was resuspended in 5-6 volumes of buffer, the protein
concentration was determined according to the method of Bradford31
using bovine serum albumin as standard, and subsequently the
preparation was frozen (-80 °C) in 1.5 mL portions containing
about 1.5 mg protein/mL.
CC-1,benzyl), 158.9 (1C, arom CC-4,methoxybenzyl).
(+)-(1R,2S,5S)-6-Benzyl-8-(4-methoxybenzyl)-6,8-diazabicyclo-
[3.2.2]nonan-2-ol (ent-20). As described for the preparation of 20,
the enantiomer ent-19 (600 mg, 1.58 mmol) was reduced with
LiAlH4 (300 mg, 7.91 mmol) in dry THF (50 mL) to give ent-20
as a pale yellow viscous oil, yield 432 mg (77%). [R]20589 ) +20.4
(c 0.790, CH2Cl2). Anal. (C22H28N2O2) C, H, N.
(+)-(1R,2R,5R)-6-Benzyl-2-methoxy-8-(4-methoxybenzyl)-6,8-
diazabicyclo[3.2.2]nonane-7,9-dione (21). As described for the
synthesis of the methyl ether 16, the alcohol 19 (300 mg, 0.79
mmol) was reacted with MeI (0.12 mL, 1.88 mmol) and NaH (270
mg obtained from 450 mg 60% dispersion in oil, 11.25 mmol) in
dry THF (40 mL). Workup and fc purification (3 cm, petroleum
ether/EtOAc 3:7, Rf ) 0.38) gave 21 as colorless viscous oil, yield
300 mg (96%). [R]20
) +70.7 (c 0.42, CH2Cl2). Anal.
589
(C23H26N2O4) C, H, N. H NMR (CDCl3): δ ) 1.28-1.38 (m, 1H,
4-H), 1.58-1.71 (m, 1H, 3-H or 4-H), 1.94-2.06 (m, 2H, 3-H and/
or 4-H), 3.33 (s, 3H, OCH3), 3.51 (ddd, J ) 9.2/4.5/1.3 Hz, 1H,
2-H), 3.81 (s, 3H, PhOCH3), 3.88 (dd, J ) 5.5/2.0 Hz, 1H, 5-H),
3.95 (d, J ) 14.5 Hz, 1H, NCH2aryl), 4.07 (d, J ) 1.0 Hz,1H,
1-H), 4.42 (d, J ) 14.8 Hz, 1H, NCH2aryl), 4.63 (d, J ) 14.5 Hz,
1H, NCH2aryl), 5.27 (d, J ) 14.8 Hz, 1H, NCH2aryl), 6.88 (d, J )
8.6 Hz, 2H, arom 3-H, 5-Hmethoxybenzyl), 7.15 (d, J ) 8.6 Hz, 2H,
arom 2-H, 6-Hmethoxybenzyl), 7.21 (dd, J ) 7.4/1.6 Hz, 2H, arom H),
7.27-7.36 (m, 3H, arom H).
Performing of the σ1 Assay (modified according to refs 27,
29). The test was performed with the radioligand [3H]-(+) penta-
zocine (42.5 Ci/mmol; Perkin-Elmer). The thawed membrane
preparation (about 75 µg of the protein) was incubated with various
concentrations of test compounds, 2 nM [3H]-(+)-pentazocine, and
buffer (50 mM Tris, pH 7.4) in a total volume of 200 µL for 180
min at 37 °C. The incubation was terminated by rapid filtration
through the presoaked filtermats by using the cell harvester. After
washing each well five times with 300 µL of water, the filtermats
were dried at 95 °C. Subsequently, the solid scintillator was placed
on the filtermat and melted at 95 °C. After 5 min, the solid
scintillator was allowed to solidify at room temperature. The bound
radioactivity trapped on the filters was counted in the scintillation
analyzer. The nonspecific binding was determined with 10 µM
unlabeled (+) pentazocine. The Kd-value of the radioligand [3H]-
(+)-pentazocine is 2.9 nM.39
Membrane Preparation for the σ2 Assay (modified according
to refs 27, 29). Two rat livers were cut into smaller pieces and
homogenized with a potter (500-800 rpm, 10 up-and-down strokes)
in 6 volumes of cold 0.32 M sucrose. The suspension was
centrifuged at 1200g for 10 min at 4 °C. The supernatant was
separated and centrifuged at 31000g for 20 min at 4 °C. The pellet
was resuspended in buffer (50 mM Tris, pH 8.0) and incubated at
room temperature for 30 min. After the incubation, the suspension
was centrifuged again at 31000g for 20 min at 4 °C. The final pellet
(-)-(1S,2S,5S)-6-Benzyl-2-methoxy-8-(4-methoxybenzyl)-6,8-
diazabicyclo[3.2.2]nonane-7,9-dione (ent-21). As described for the
preparation of 21, the enantiomeric alcohol ent-19 (433 mg, 1.14
mmol) was allowed to react with NaH (390 mg obtained from 650
mg 60% dispersion in oil, 16.3 mmol) and MeI (0.36 mL, 5.66
mmol) in dry THF (25 mL) to give ent-21 as a colorless solid,
yield 367 mg (82%), mp 153 °C (petroleum ether/EtOAc). [R]20
589
) -67.1 (c 0.505, CH2Cl2). Anal. (C23H26N2O4) C, H, N.
(+)-(1S,2R,5R)-6-Benzyl-2-methoxy-8-(4-methoxybenzyl)-6,8-
diazabicyclo[3.2.2]nonane (22). As described for the reduction of
14 the methyl ether 21 (197 mg, 0.50 mmol) in dry THF (20 mL)
was reduced with LiAlH4 (94 mg, 2.48 mmol). Workup and fc
purification (3 cm, petroleum ether/EtOAc 8:2, Rf ) 0.24) led to
22 as colorless viscous oil, yield 105 mg (57%). [R]20 ) +8.9
589
(c 0.765, CH2Cl2). Anal. (C23H30N2O2) C, H, N. 1H NMR

6152 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Geiger et al.
(2) Hanner, M.; Moebius, F. F.; Flandorfer, A.; Knaus, H.-G.; Striessnig,
J.; Kempner, E.; Glossmann, H. Purification, molecular cloning, and
expression of the mammalian sigma1-binding site. Proc. Natl. Acad.
Sci. U.S.A. 1996, 93, 8072-8077.
(3) Kekuda, R.; Prasad, P. D.; Fei, Y.-J.; Leibach, F. H.; Ganapathy, V.
Cloning and Functional Expression of the Human Type 1 Sigma
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(4) Seth, P.; Fei, Y.-J.; Li, H. W.; Huang, W.; Leibach, F. H.; Ganapathy,
V. Cloning and Functional Characterization of a σ Receptor from
Rat Brain. J. Neurochem. 1998, 70, 922-931.
(5) Prasad, P. D.; Li, H. W.; Fei, Y.-J.; Ganapathy, M. E.; Fujita, T.;
Plumley, L. H.; Yang-Feng, T. L.; Leibach, F. H.; Ganapathy, V.
Exon-Intron structure, Analysis of Promoter Region, and Chromo-
somal Localization of the Human Type 1 σ Receptor Gene. J.
Neurochem. 1998, 70, 443-451.
(6) Aydar, E.; Palmer, C. P.; Djamgoz, M. B. A. Sigma receptors and
Cancer: Possible Involvement of Ion Channels. Cancer Res. 2004,
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(7) Monnet, F. P.; Morin-Surun, P.; Leger, J.; Comettes, L. Protein Kinase
C-Dependent Potentiation of Intracellular Calcium Influx by σ1
Receptor Agonists in Rat Hippocampal Neurons. J. Pharmacol. Exp.
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(8) Aydar, E.; Palmer, C. P.; Klyachko, V. A.; Meyer, B. J. The Sigma
Receptor as a Ligand-Regulated Auxiliary Potassium Channel
Subunit. Neuron 2002, 34, 399-410.
(9) Bowen, W. D. Sigma receptors: recent advances and new clinical
potentials. Pharm. Acta HelV. 2000, 74, 211-218.
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of Neuropsychiatric Disorders. CNS Drugs 2004, 18, 269 - 284.
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Scheffel, U. A. Synthesis and in vivo evaluation of a new PET
radioligand for Studying sigma-2 receptors. Biorg. Med. Chem. 2005,
13, 3623-3626.
(12) John, C. S.; Lim, B. B.; Viler, B. J.; Geyer, B.; C.; Bowen, W. D.
Substituted Halogenated Arylsulfonamides: A New Class of σ
Receptor Binding Tumor Imaging Agents. J. Med. Chem. 1998, 41,
2445-2450.
(13) John, C. S.; Bowen, W. D.; Varma, V. M.; McAfee, J. G.; Moody,
T. W. Sigma Receptors are Expressed in Human Non-Small Cell
Lung Carcinoma. Life Sci.1995, 56, 2385-2392.
(14) John, C. S.; Vilner, B. J.; Geyer, B. C.; Moody, T.; Bowen W. D.
Targeting Sigma Receptor-binding Benzamides as in ViVo Diagnostic
and Therapeutic Agents for Human Prostate Tumors. Cancer Res.
1999, 59, 4578-4583.
(15) Vilner, B. J.; de Costa, B. R.; Bowen, W. D. Cytotoxic Effects of
Sigma Ligands: Sigma Receptor-mediated Alterations in Cellular
Morphology and Viability. J. Neurosci. 1995, 15, 117-134.
(16) Colabufo, N. A.; Berardi, F.; Contino, M.; Niso, M.; Abate, C.;
Perrone, R.; Tortorella, V. Antiproliferative and cytotoxic effects of
some σ2 agonists and σ1 antagonists in tumour cell lines. Arch.
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was resuspended in buffer, the protein concentration was determined
according to the method of Bradford31 using bovine serum albumin
as standard, and subsequently the preparation was frozen (-80 °C)
in 1.5 mL portions containing about 2 mg protein/mL.
Performing of the σ2-Assay (modified according to refs 27,
29). The test was performed with the radioligand [3H]ditolylguani-
dine (50 Ci/mmol; ARC). The thawed membrane preparation (about
100 µg of the protein) was incubated with various concentrations
of test compounds, 3 nM [3H]-ditolylguanidine, 500 nM (+)-
pentazocine, and buffer (50 mM Tris, pH 8.0) in a total volume of
200 µL for 180 min at room temperature. The incubation was
terminated by rapid filtration through the presoaked filtermats using
a cell harvester. After each well was washed five times with 300
µL of water, the filtermats were dried at 95 °C. Subsequently, the
solid scintillator was placed on the filtermat and melted at 95 °C.
After 5 min, the solid scintillator was allowed to solidify at room
temperature. The bound radioactivity trapped on the filters was
counted in the scintillation analyzer. The nonspecific binding was
determined with 10 µM unlabeled ditolylguanidine. The Kd-value
of the radioligand [3H]-ditolylguanidine is 17.9 nM.40
NMDA Assay. The preparation of the receptor material and the
assay were performed according to ref 29.
Data Analysis. All experiments were carried out in triplicates
using standard 96-well multiplates (Diagonal). The IC50-values were
determined in competition experiments with six concentrations of
the test compounds and were calculated with the program GraphPad
Prism 3.0 (GraphPad Software) by nonlinear regression analysis.
The Ki-values were calculated according to Cheng and Prusoff.41
The Ki-values are given as the mean value + SEM from three
independent experiments.
Cytotoxicity Assay.30 All cell lines were obtained from the
German Collection of Microbiology and Cell Culture (DSZK,
Braunschweig, FRG). Cytotoxicity testing was done by using a
microtiter assay based on staining cells with crystal violet as
described in detail elsewhere.30 To determine the IC50 values, five
serially diluted stock solutions of test substance in DMF were used
in the studies; concentrations giving T/C values between 10 and
90% were used to estimate the IC50 values, which were calculated
by least-squares analysis of the dose-response curves.
σ1 and σ2 Expression in Tumor Cell Lines. A homogenized
and standardized membrane preparation (1.8 mg protein/mL) of
the corresponding tumor cell line was prepared as previously
described15 and incubated with the corresponding radioligand in
the presence or absence of an excess of an inhibitor ((+)-
pentazocine in the σ1 assay, ditolylguanidine in the σ2 assay). The
experimental details are given above for the σ1 and σ2 assays. In
the assay 90 µg protein/well were employed. For each tumor cell
line preparation, the specific binding of the radioligand was
calculated. Additionally the number of binding sites (receptors) in
the assay was calculated according to the following equation:
(18) Bedu¨rftig, S.; Wu¨nsch, B. Chiral, nonracemic (piperazin-2-yl)-
methanol derivatives with σ-receptor affinity. Biorg. Med. Chem.
2004, 12, 3299-3311.
(19) Weigl, M.; Wu¨nsch, B. The synthesis of the enantiomer ent-11 was
described in literature: Synthesis of chiral non-racemic 3-(dioxopip-
erazin-2-yl)propionic acid derivatives. Tetrahedron 2002, 58, 1173-
1183.
N ) Cpm × L/60 × EA
where N is the number of binding sites (receptors) in the assay,
Cpm is the counts per minute, L is Avogadro’s number (6.022 ×
1023 molecules/mol), E is the efficiency of the counter (20%), and
A is the specific activity of the radioligand in Bq/mol. Furthermore,
Np, the number of binding sites (receptors) per µg protein as well
as the molar amount of binding sites (pmol) per mg protein were
calculated.
(20) Warner, P. M. Strained Bridgehead Double Bonds. Chem. ReV. 1989,
89, 1067 1093.
(21) Weigl, M.; Wu¨nsch, B. Synthesis of 6,8-Diazabicyclo[3.2.2]nonanes:
Conformationally Restricted Piperazine Derivatives. Org. Lett. 2000,
2, 1177-1179.
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Acknowledgment. Thanks are due to the Degussa AG,
Janssen-Cilag GmbH, and Bristol-Myers Squibb for donation
of chemicals and reference compounds.
Supporting Information Available: Purity data of the prepared
compounds (elemental analysis, HPLC analysis), some experimental
details, and spectroscopic data. This material is available free of
charge via the Internet at http://pubs.acs.org.
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