Relationships between the structure of 6-substituted 6,8-diazabicyclo[3.2.2]nonan-2-ones and their σ receptor affinity and cytotoxic activity

Article abstract of DOI:10.1016/j.bmc.2009.01.012

A series of 2-oxo-6,8-diazabicyclo[3.2.2]nonane derivatives was prepared and the affinity towards σ₁ and σ₂ receptors was investigated by means of radioligand binding assays as well as their inhibition of the growth of six human tumo

Full text of DOI:10.1016/j.bmc.2009.01.012

Bioorganic & Medicinal Chemistry 17 (2009) 1445–1455
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Relationships between the structure of 6-substituted 6,8-diazabicyclo
[3.2.2]nonan-2-ones and their
r
receptor affinity and cytotoxic activity
Ralph Holl a, Dirk Schepmann a, Patrick J. Bednarski b, Renate Grünert b, Bernhard Wünsch a,
*
a Institut für Pharmazeutische und Medizinische Chemie der Westfälischen Wilhelms-Universität Münster, Hittorfstraße 58-62, D-48149 Münster, Germany
b Institut für Pharmazie der Ernst-Moritz-Arndt-Universität Greifswald, Friedrich-Ludwig-Jahn-Straße 17, D-17489 Greifswald, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2-oxo-6,8-diazabicyclo[3.2.2]nonane derivatives was prepared and the affinity towards
r1 and
Received 17 November 2008
Revised 5 January 2009
Accepted 10 January 2009
Available online 15 January 2009
r2 receptors was investigated by means of radioligand binding assays as well as their inhibition of the
growth of six human tumor cell lines was studied. Starting from the enantiopure bicyclic ketones 3
and ent-3 bridged piperazines with different residues in position 6 were synthesized. The N-6 allyl pro-
tective group was removed by a RhCl3 catalyzed double bond isomerization and subsequent hydrolysis of
the resulting enamide 8. After acetalization the secondary amide 10 was alkylated and arylated. Structure
affinity relationships show that a relatively large substituent, which has not necessarily to be an aromatic
one, is required in position 6 for high r1 receptor affinity (e.g., 12 and ent-12 with a dimethylallyl resi-
due: Ki = 20 nM and 17 nM). Furthermore, it was shown that substituents that reduce the basicity of N-6
led to a severe decrease in r1 affinity. Growth inhibition experiments with six human tumor cell lines
revealed that the allyl and benzyl substituted 6,8-diazabicyclo[3.2.2]nonan-2-one derivatives 5, ent-5
and ent-14 are able to selectively inhibit the growth of the bladder cancer cell line 5637.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Diazabicyclo[3.2.2]nonanes
Sigma receptor ligands
Cytotoxicity
Structure–activity-relationships
1. Introduction
being important for high r
1 receptor binding.7 According to the pro-
posed two dimensional model, two hydrophobic substituents in dif-
ferent distances from a basic nitrogen atom, which is supposed to
bind to a proton donor site (Asp 126 and/or Glu 172) of the receptor,
The
type.1 However today it is accepted to be a distinct pharmacologi-
cal entity, with the class of receptors being subdivided into r1
and
2 receptors.2 Although their biochemical role as well as their
mechanism of signal transduction are not yet completely under-
stood so far, they have been implicated in a multitude of biological
and phathophysiological processes such as cancer biology.3
r receptor was first described as an opioid receptor sub-
r
are required for a high r1 receptor affinity (compare Fig. 2).
r
We recently reported on the synthesis and r receptor affinity of
a series of 6,8-diazabicyclo[3.2.2]nonane derivatives 1 (Fig. 1).8
These compounds possess two basic nitrogen atoms, which in prin-
cipal could both bind to the postulated proton donor site of the r1
The
other mammalian protein but shows about 30% homology to the
yeast enzyme sterol D8 7-isomerase.4 The cloned receptor is pos-
r1 receptor has been cloned and shows no homology to any
H3CO
6
5
8
N
/D
N
tulated to possess two transmembrane domains with the amino
and carboxy termini on the intracellular side of the membrane.
Mutation of the human r1 receptor revealed aspartate 126 and
glutamate 172 to be crucial amino acids for ligand binding. These
observations indicate that the binding domain of the receptor is
in its intracellular carboxy terminus.5,6 However, so far little is
known about the exact structure of the ligand binding site of the
membrane bound r1 receptor and the receptor features being
important for ligand binding.
R
1
X
2
3
1
H3CO
6
N
8
N
Glennon et al. reported on the structure affinity relationships of a
series of phenylalkylamine derivatives with respect to their binding
at r1 receptors and elaborated the features of these compounds
2
2
Figure 1. General structure of the 6,8-diazabicyclo[3.2.2]nonane derivatives (1)
and structure of the 2-unsubstituted compound 2: Ligands for the r1 receptor.
* Corresponding author. Fax: +49 251 8332144.
E-mail address: wuensch@uni-muenster.de (B. Wünsch).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.01.012

1446
R. Holl et al. / Bioorg. Med. Chem. 17 (2009) 1445–1455
receptor. However, the N-6 allyl derivatives 1 bearing a substituent
in position 2 showed a considerably reduced affinity towards the
8-phenylderivative 15 was subsequently reduced and hydrolyzedto
give the aniline derivative 16 (Scheme 3). Direct LiAlH4 reduction
and acetal cleavage of 10 yielded the secondary amine 17, which
was acylated with benzoyl chloride to form the benzamide 18.
The enantiomers of the bicyclic ketones 5, 7, 12, 14, 16 and 18
were prepared in the same manner, starting with (R)-glutamate.
Therefore, all possible stereoisomers of the described compounds
were available for pharmacological evaluation. The purity of all
compounds tested was evaluated by RP-HPLC analysis with UV
detection at wavelength of 210 nm (Method 1) and 235 nm (Meth-
od 2). All compounds were found to have at least a purity of 95%.
r1 receptor compared to the unsubstituted compound
2
(Ki = 11 nM). These findings led to the conclusion that unfavorable
interactions of the C-2 substituent arise with the r1 receptor pro-
tein when N-8 binds to the proton donor site (Fig. 2a). In case of N-
6 binding to the proton donor site, the allyl group seems to pro-
duce a low free energy during interaction with the secondary
hydrophobic region of the receptor (Fig. 2b). In order to further ex-
pand on these results a series of 6,8-diazabicyclo[3.2.2]nonan-2-
one derivatives was synthesized bearing various N-6-substituents,
which modify the hydrophobic interactions with the receptor pro-
tein and the basicity of the N-atom.
3. Pharmacological evaluation
3.1. Receptor binding studies
2. Chemistry
The synthesis of the central bicyclic building block 3 emanating
from (S)-glutamate has been described in the literature.8,9 Starting
from the enantiomerically pure bicyclic ketone 3 the dimethyl ace-
tal 4 was formed in methanol in the presence of trimethyl ortho-
formate and p-toluenesulfonic acid.8,9 Reaction of 4 with LiAlH4
led to the reduction of both lactam carbonyl moieties and a subse-
quent acetal cleavage yielded the bridged piperazine derivative 5
(Scheme 1). Hydrogenation of the allyl group of 4 gave the N-6 pro-
pyl derivative 6, which was also reduced with LiAlH4 and subse-
quently hydrolyzed with diluted HCl to yield the bicyclic ketone 7.8
In order to introduce further substituents in position 6 the allyl
group of 4 must be removed. The allyl group was cleaved by a
RhCl3 catalyzed isomerization and subsequent hydrolysis of the
The r receptor affinities of the synthesized compounds were
determined in competition experiments with radioligands.
Homogenates of guinea pig brains were used as receptor material
in the r1 assay and the r1 selective ligand [3H]-(+)-pentazocine
was employed as radioligand. The non-specific binding was deter-
mined in the presence of a large excess of cold (+)-pentazocine. In
the r2 assay homogenates of rat liver served as source for r2
receptors. The non-selective radioligand [3H]-1,3-di(o-tolyl)guani-
dine was employed in the presence of an excess of non-tritiated
(+)-pentazocine for selective occupation of r1 receptors. The
non-specific binding of the radioligand was determined by per-
forming the r2 assay in the presence of an excess of non-tritiated
1,3-di(o-tolyl)guanidine.11,12
resulting enamide 8, which yielded the secondary amide
9
(Scheme 2). After regeneration of the acetal moiety the secondary
amide 10 was alkylated. Deprotonation with NaHMDS and subse-
quent reaction with dimethylallyl bromide and benzyl bromide
yielded the dimethylallyl and benzyl derivatives 11 and 13, respec-
tively. Subsequent LiAlH4 reduction and acetal cleavage afforded
the basic piperazine derivatives 12 and 14.
3.2. Inhibition of cell growth of human tumor cell lines
In the literature the overexpression of r1 and r2 receptors in
human tumor cell lines has been reported.6 Furthermore, some
r
2 agonists and r1 antagonists showed antiproliferative and cyto-
toxic effects in some tumor cell lines.13 Therefore, the antiprolifer-
ative effects of the synthesized compounds were investigated in a
panel of six human tumor cell lines, including the cell lines 5637
The arylation of the secondary amide 10 was achieved with iodo-
benzene in DMF in the presence of CuI and K2CO3 at 150 °C.10 The N-
OCH3
OCH3
O
O
H3CO
(b)
(a)
N
N
N
N
O
N
N
O
O
H3CO
O
OCH3
3
4
5
(c)
OCH3
O
H3CO
(d)
CH3
N
N
N
N
CH3
O
H3CO
O
OCH3
6
7
Scheme 1. Reagents and conditions: (a) HC(OCH3)3, MeOH, p-toluenesulfonic acid, reflux, 16 h, 99% (according to Ref. 8); (b) LiAlH4, THF, reflux, 16 h, then 1 M HCl, 3 h, 50%
(according to Ref. 8); (c) H2, Pd/C, MeOH, rt, 1 h, 92%; (d) LiAlH4, THF, reflux, 16 h, then 1 M HCl, 3 h, 34%.

R. Holl et al. / Bioorg. Med. Chem. 17 (2009) 1445–1455
1447
OCH3
OCH3
O
O
(c)
(a)
(b)
CH3
NH
N
4
N
N
O
O
H3CO
O
OCH3
(E)-8, (Z)-8
9
OCH3
O
N
CH3
CH3
H3CO
(e)
N
N
CH3
CH3
OCH3
N
(d)
O
H3CO
O
O
OCH3
11
NH
N
12
OCH3
O
(f)
H3CO
OCH3
O
H3CO
(g)
10
N
N
N
N
O
H3CO
O
OCH3
13
14
Scheme 2. Reagents and conditions: (a) RhCl3, DABCO, MeOH, reflux, 16 h, 66% (E)-8, 9% (Z)-8; (b) H2O/HOAc, reflux 16 h, 24%; (c) HC(OCH3)3, MeOH, p-toluenesulfonic acid,
reflux, 16 h, 80%; (d) NaHMDS, 1-bromo-3-methylbut-2-ene, Bu4NI, THF, ꢀ78 °C, 82%; (e) LiAlH4, THF, reflux, 16 h, then 1 M HCl, 3 h, 75%; (f) NaHMDS, benzyl bromide, Bu4NI,
THF, ꢀ78 °C, 67%; (g) LiAlH4, THF, reflux, 16 h, then 1 M HCl, 3 h, 50%.
OCH3
O
H3CO
(b)
N
N
O
N
N
(a)
O
H3CO
O
OCH3
10
(c)
15
16
H3CO
H3CO
(d)
N
NH
N
N
O
O
17
18
Scheme 3. Reagents and conditions: (a) Iodobenzene, CuI, K2CO3, DMF, 150 °C, 72 h, 72%; (b) LiAlH4, THF, reflux, 16 h, then 1 M HCl, 3 h, 20%; (c) LiAlH4, THF, reflux, 16 h, then
1 M HCl, 3 h, 59%; (d) benzoyl chloride, NEt3, CH2Cl2, rt, 16 h, 61%.
(bladder cancer), RT-4 (bladder cancer), A-427 (small cell lung can-
cer), LCLC-103H (large cell lung cancer), MCF-7 (breast cancer) and
DAN-G (pancreas cancer).
In the primary screening the tumor cells were exposed to 20 lM
solutions of the test compounds at 37 °C. After a 96 h exposure the
medium was removed and the density of adherent cells (living

1448
R. Holl et al. / Bioorg. Med. Chem. 17 (2009) 1445–1455
cells) was measured by staining with crystal violet.14 In Table 2 the
cell growth inhibiting activity of the test compounds is expressed
as the percent of living cells after 96 h in relation to a control with-
out test compound.
potency, whilst the N-6 phenyl derivative ent-16 inhibits unselec-
tively the growth of most of the investigated cell lines including
5637, A-427, LCLC-103H and MCF-7. The growth of the other cell
lines was reduced to a lower extent by these compounds. The selec-
tive inhibition of the growth of only few cell lines by 5, ent-5 and ent-
14 indicates a specific mechanism of growth inhibition.
4. Biological results and discussion
The most potent
r1 ligands of this series, the dimethylallyl com-
pounds 12 and ent-12 as well as the benzyl derivative 14, possess Ki
values of 20 nM, 17 nM and 53 nM, respectively. The cell growth
inhibition of all six tumor cell lines together induced by 12, ent-12,
and 14 was rather low. However, looking selectively at the breast
Table 1 shows the
compounds. Whereas the introduction of a carbonyl group in posi-
tion 2 leads to a severe decrease in 1 affinity (5: Ki = 1600 nM) com-
r1 and r2 receptor affinity of the synthesized
r
pared to the unsubstituted compound (2: Ki = 11 nM), the
cancer cell line MCF-7 these three most potent
r1 ligands represent
introduction of different substituents in position 6 reverse this ef-
themostpotentgrowthinhibitorsof thistumorcell lineaswell. Nev-
fect. TheN-6propylderivative7interactsbetterwiththe
(Ki = 325 nM) thantheallylderivative5 (Ki = 1600 nM). Increasing of
the -system of the allyl derivative 5 to a benzyl group (14) leads to
r1 receptor
ertheless, the potency of around 55% growth inhibition at a test com-
pound concentration of 20 lM is still moderate. This might be due to
the different abilities of the compounds to penetrate through the tu-
p
an increased r1 receptor affinity (14: Ki = 53 nM). In this series of
6,8-diazabicyclo[3.2.2]nonan-2-ones the dimethylallyl derivative
12 shows the highest r1 affinity (Ki = 20 nM). Apparently, a rela-
tively large lipophilic substituent, which is able to interact with
mor cell membrane to attain the intracellularly located binding site
of the
r1 receptor. Furthermore, it should be considered that the r1
affinitiesof 12, ent-12, and 14 are considerablylower thanthat of the
cytotoxic r1 ligand haloperidol. A further explanation would be a
potential agonistic or partial agonistic instead of antagonistic activ-
ther1 receptor, is requiredinposition6forhighr1 receptorbinding.
The effect of the stereochemistry of the bicyclic system on r1
receptor binding is inconsistent. Whilst in case of the allyl and pro-
pyl derivatives ent-5 and ent-7 show higher r1 affinity than their
enantiomers derived from (S)-glutamate, the N-6 benzyl derivative
ity at
r1 receptors.
5. Conclusion
14 is the eutomer with respect to
meric dimethylallyl substituted compounds 12 and ent-12 display
comparable affinities towards the r1 receptor.
The phenyl and benzoyl derivatives 16 and 18, each with re-
duced basicity of N-6 because of the substituents, show consider-
r1 receptor binding. The enantio-
6,8-Diazabicyclo[3.2.2]nonan-2-one derivatives show consider-
able affinity towards the r1 receptor. This affinity is strongly
dependent on the substituent in position 6. Whereas an allyl moiety
leads to a rather low affinity (5: Ki = 1600 nM), propyl and benzyl
substituents increase the
r
1 affinity considerably. The dimethylal-
1 affinity (12: Ki = 20 nM, ent-12:
ably lower
r1 receptor affinities than the other compounds of this
lyl derivatives show the highest
r
series. This indicates that in this series of 6,8-diazabicy-
clo[3.2.2]nonan-2-ones the basicity of N-6 is crucial for r1 recep-
tor binding and therefore this nitrogen atom is likely to bind to
the proton donor site of the r1 receptor. After reduction of the
N-6-basicity the other basic N-atom in position 8 presumably
occupies the proton donor site of the r1 receptor protein. How-
ever, in this orientation (Fig. 2, part 2a) the unfavorable interac-
tion of the carbonyl oxygen with the r1 receptor protein lead
to low affinity of 16 and 18.
These results further underline the proposed binding mode of the
2-oxo-6,8-diazabicyclo[3.2.2]nonan-2-one derivatives (Fig. 2b).
When a basic N-6 is binding to the proton donor site, a relatively
large lipophilic substituent in position 6 (e.g., 12: dimethylallyl,
14: benzyl) is required for favorable interactions with the secondary
Ki = 17 nM) in this series of compounds.
Furthermore, substituents like a phenyl and a benzoyl group,
which decrease the basicity of N-6, lead to almost complete loss of
r1 receptor affinity in this series of 6,8-diazabicyclo[3.2.2]nonan-
2-one derivatives. Obviously, basic properties are required in posi-
tion 6 to attain high
r1 receptor affinity.
Some of the synthesized bridged piperazine derivatives possess
modest antiproliferative activity against some human tumor cell
lines. However, further structure–activity investigations are re-
quired to establish the relationships between this antiproliferative
activity and the r1 and/or r2 receptor affinity of these interesting
compounds.
6. Experimental
hydrophobic region (A) of the r1 receptor resulting in high r1 recep-
tor affinity. The lipophilic N-6 substituent must not necessarily be an
aromaticresidue. This hypothesis of interactionof thebridged piper-
6.1. Chemistry, general
azine derivatives with the
the observations made with
2 of the bicyclic framework.8
Generally, the r2 receptor affinity of the bicyclic compounds is
rather low. Therefore in particular the compounds with high 1 affinity
show a very good selectivity for the 1 receptor over the 2 receptor.
Since some receptor ligands show considerable affinity
r
r
1 receptor protein is in accordance with
1 receptor ligands modified in position
Unless otherwise noted, moisture sensitive reactions were
conducted under dry nitrogen. THF was dried with sodium/ben-
zophenone and was freshly distilled before use. Thin layer chro-
matography (tlc): Silica gel 60 F254 plates (Merck). Flash
r
chromatography (fc): Silica gel 60, 40–64 lm (Merck); parenthe-
r
r
ses include: diameter of the column, eluent, fraction size, Rf
value. Melting point: Melting point apparatus SMP 3 (Stuart Sci-
entific), uncorrected. Optical rotation: Polarimeter 341 (Perkin–
r
towards the NMDA receptor, the affinity of the bridged piperazines
towards the phencyclidine binding site of the NMDA receptor (pig
brain cortex membrane preparations, [3H]-MK801) was also deter-
Elmer); 1.0 dm tube; concentration c [g/100 mL], the unit of [a]
[grad mL dmꢀ1 gꢀ1]. MS: MAT GCQ (Thermo-Finnigan); IR: IR
spectrophotometer 480Plus FT-ATR-IR (Jasco). 1H NMR
(400 MHz), 13C NMR (100 MHz): Unity Mercury Plus 400 spec-
trometer (Varian); d in ppm related to tetramethylsilane; cou-
pling constants are given with 0.5 Hz resolution. HPLC: Method
1: Merck Hitachi Equipment; UV detector: L-7400; autosam-
pler:L-7200; pump: L-7100; degasser: L-7614; column: LiChro-
mined.12 However, at a concentration of 1
lM the replacement of
the radioligand by the synthesized compounds was always lower
than 25%. These results indicate high selectivity of the bicyclic
gands over the PCP binding site of the NMDA receptor.
r1 li-
Table 2 shows the cell growth inhibiting properties of the synthe-
sized compounds. At a concentration of 20 M the N-6 allyl deriva-
l
tives 5 and ent-5 as well as the benzyl derivative ent-14 selectively
inhibit the growth of the bladder cancer cell line 5637 with modest
spherÒ 60 RP-select B (5 m); LiCroCARTÒ 250-4 mm cartridge;
l

R. Holl et al. / Bioorg. Med. Chem. 17 (2009) 1445–1455
1449
Table 1
1 and
some reference compounds
Table 2
r
r
2 receptor affinity of the new 6,8-diazabicyclo[3.2.2]nonane derivatives and
Cell growth inhibitory activity (% of untreated control) of the 6,8-diazabicy-
clo[3.2.2]nonane derivatives in six human cancer cell linesa
Compound
N-6 substituent
r1 Ki SEM (nM)
r2 Ki SEM (nM)
5637b
RT-4c
A-427d
LCLC-103He
MCF-7f
DAN-Gg
5
Allyl
Allyl
1600
548
325
9100
1900
23%a
1800
841
1300
704
3600
3780
5%a
5
38
19
119
80
119
72
62
35
94
17
80
76
60
50
94
105
83
81
64
60
111
37
81
64
102
96
99
86
91
79
88
47
64
102
91
82
72
65
69
60
63
57
56
50
66
43
72
84
65
74
75
69
117
101
109
99
89
84
109
62
79
ent-5
7
ent-7
12
ent-12
14
ent-14
16
ent-16
18
ent-18
2
ent-5
7
ent-7
12
ent-12
14
ent-14
16
ent-16
18
ent-18
2
Propyl
Propyl
Dimethylallyl
Dimethylallyl
Benzyl
Benzyl
Phenyl
Phenyl
Benzoyl
Benzoyl
Allyl
101
97
102
100
89
67 8.2
20 5.5
17 4.6
53 2.1
133 23
6250
84
102
102
102
108
65
5%a
20%a
29%a
11%a
2038
3358
—
86
120
87
68
110
42
59%a
116
92
87
11 4.78
ent-2
Allyl
—
—
—
2708
ent-2
90
71
55
(+)-Pentazocine
Di-o-tolylguanidine
haloperidol
4.2 1.1
61 18
3.9 1.5
a
Relative cell growth [%] in relation to untreated control of the tumor cell lines
42 17
78 2.3
after 96 h exposure to compound at 20
lM.
b
Bladder cancer.
Bladder cancer.
Small cell lung cancer.
Large cell lung cancer.
Breast cancer.
Pancreas cancer.
a
c
Inhibition of radioligand binding at a concentration of the test compound 1
l
m.
d
e
f
flow rate: 1.000 mL/min; injection volume: 5.0 lL; detection at
g
k = 210 nm; solvents: A: water with 0.05% (v/v) trifluoroacetic
acid; B: acetonitrile with 0.05% (v/v) trifluoroacetic acid: gradi-
ent elution: 0.0 min: 90.0% of A, 10.0% of B; 4.0 min: 90.0% of
A, 10.0% of B; 29.0 min: 0.0% of A, 100.0% of B; 31.0 min: 0.0%
of A, 100.0% of B; 31.5 min: 90.0% of A, 10.0% of B; 40.0 min:
90.0% of A, 10.0% of B. Method 2: Equipment: pump: HPLC pump
64 (Knauer); UV-Detector: Variable Wavelength Monitor
(Knauer); data acquisition: D-2500 Chromato-Integrator (Merck
Hitachi); column: LiChroCARTÒ 250-4 with SuperspherÒ 100
6.2. (+)-(1S,5S)-2,2-Dimethoxy-8-(4-methoxybenzyl)-6-propyl-
6,8-diazabicyclo[3.2.2]nonane-7,9-dione (6)
The dimethyl acetal 48 (140 mg, 0.37 mmol) was dissolved in
methanol (30 mL) and 10% Pd/C (14 mg) was added. The mixture
was stirred under hydrogen (balloon) for 1 h at rt. Then the suspen-
sion was filtered through Celite and the filtrate was concentrated in
vacuo. The residue was purified by fc (Ø = 2 cm, h = 15 cm, cyclohex-
ane/ethyl acetate = 2/1, 10 mL, Rf = 0.08) to give 6 as a colorless solid,
RP-18; flow rate: 0.6 mL/min; injection volume: 20.0 lL; detec-
tion: wavelength: 235 nm; stop time: 2 ꢁ tR; method (2a) sol-
vent: methanol/water = 75:25 + 0.1% triethylamine; method (2b)
solvent: methanol/water = 65:35 + 0.1% triethylamine.
Proton Donor Site
x
y
2.5 –3.9 Å
6 –10 Å
A
N
B
Region tolerates
only small groups
Primary
Hydrophobic
Region
Secondary
Hydrophobic
Region
a
b
A
N
B
A
N
B
OCH3
H3CO
6
N
8
N
8
N
6
N
R
R
O
O
σ1 receptor
protein
σ1 receptor
protein
Figure 2. Pharmacophore model of
r1 receptor ligands (modified according to Ref. 7) and hypothetical binding modes of the bridged piperazine derivatives at the r1 receptor
protein. Binding mode (a) N-8 interacts with the proton donor site. Binding mode (b) N-6 interacts with the proton donor site.

1450
R. Holl et al. / Bioorg. Med. Chem. 17 (2009) 1445–1455
mp 98 °C, yield 130 mg (92%). C20H28N2O5 (376.5). Purity by HPLC:
method 1: tR = 18.4 min, purity 99.1%. = +35.7 (c = 0.15;
CH2Cl2). MS (EI): m/z [%] = 376 (M, 1), 121 (CH2PhOCH3, 40), 101
(H2CCHC(OCH3)2, 100). 1H NMR (CDCl3):
[ppm] = 1.25 (t,
6.6. (ꢀ)-(1S,5S,E)-2,2-Dimethoxy-8-(4-methoxybenzyl)-6-(prop-
1-en-1-yl)-6,8-diazabicyclo[3.2.2]nonan-7,9-dione ((E)-8) and
(ꢀ)-(1S,5S,Z)-2,2-dimethoxy-8-(4-methoxybenzyl)-6-(prop-1-
en-1-yl)-6,8-diazabicyclo[3.2.2]nonan-7,9-dione ((Z)-8)
½ ꢂ
a 2D0
d
J = 7.0 Hz, 3H, NCH2CH2CH3), 1.46–1.62 (m, 2H, NCH2CH2CH3),
1.74–1.86 (m, 2H, 3-H), 2.01–2.15 (m, 2H, 4-H), 3.15–3.23 (m, 1H,
NCH2CH2CH3), 3.17 (s, 3H, OCH3), 3.26 (s, 3H, OCH3), 3.41–3.50 (m,
1H, NCH2CH2CH3), 3.77 (s, 3H, ArOCH3), 3.88 (dd, J = 5.5/1.6 Hz, 1H,
5-H), 3.96 (d, J = 14.9 Hz, 1H, NCH2Ar), 3.97 (s, 1H, 1-H), 5.18 (d,
Under N2 the dimethyl acetal 48 (229 mg, 0.61 mmol) was dis-
solved in methanol (30 mL). Then 1,4-diazabicyclo[2.2.2]octane
(69 mg, 0.61 mmol) and rhodium(III) chloride trihydrate (16 mg,
0.06 mmol) were added and the mixture was heated to reflux
for 16 h. Then a saturated aqueous solution of NaHCO3 was
added and the mixture was extracted with CH2Cl2 (3ꢁ). The
combined organic layers were dried (Na2SO4), filtered and con-
centrated in vacuo. The residue was purified by fc (Ø = 2 cm,
h = 15 cm, cyclohexane/ethyl acetate = 2/1, V = 10 mL) to give
(E)-8 and (Z)-8.
J = 14.9 Hz, 1H, NCH2Ar), 6.83 (d, J = 8.6 Hz, 2H, 30-H4-methoxybenzyl
50-H4-methoxybenzyl), 7.11 (d, J = 8.6 Hz, 2H, 20-H4-methoxybenzyl
,
,
60-H4-methoxybenzyl). IR (neat):
1675 (s, mC
m
[cmꢀ1] = 2940
***
(m, m C–H aliph.),
~
@
O
amide), 1613 (m)/1512 (m, mC
@
C
arom.), 1457
(m, dC–H aliph.), 1238 (m)/1038 (m, mC–O), 832 (w, Cp-subst. arom.).
6.3. (ꢀ)-(1R,5R)-2,2-Dimethoxy-8-(4-methoxybenzyl)-6-propyl-
(E)-8 (Rf = 0.22): Colorless oil, yield 150 mg (66%). C20H26N2O5
(374.4). Purity by HPLC: method 1: tR = 20.0 min, purity 97.9%.
6,8-diazabicyclo[3.2.2]nonane-7,9-dione (ent-6)
½
a 2D0
ꢂ
= ꢀ23.3 (c = 0.39; CH2Cl2). MS (EI): m/z [%] = 374 (M, 23), 342
(M–HOCH3, 28), 121 (CH2PhOCH3, 100), 101 (H2CCHC(OCH3)2, 99).
1H NMR (CDCl3):
[ppm] = 1.73 (dd, J = 7.0/1.6 Hz, 3H,
As described for the preparation of 6, the enantiomer ent-48
(100 mg, 0.27 mmol) was reacted with hydrogen (balloon) and
10% Pd/C (10 mg) in methanol (30 mL) to give ent-6 as a colorless
solid, mp 98 °C, yield 76 mg (76%). C20H28N2O5 (376.5). Purity by
d
NCH@CHCH3), 1.74–1.90 (m, 2H, 3-H), 2.02–2.08 (m, 1H, 4-H),
2.11–2.18 (m, 1H, 4-H), 3.18 (s, 3H, OCH3), 3.24 (s, 3H, OCH3), 3.78
(s, 3H, ArOCH3), 3.96 (d, J = 14.9 Hz, 1H, NCH2Ar), 4.00 (s, 1H, 1-H),
4.39–4.42 (m, 1H, 5-H), 5.12 (dq, J = 14.1/7.0 Hz, 1H, NCH@CHCH3),
5.22 (d, J = 14.9 Hz, 1H, NCH2Ar), 6.83–6.88 (m, 1H, NCH@CHCH3),
6.84 (d, J = 8.6 Hz, 2H, 30-H4-methoxybenzyl, 50-H4-methoxybenzyl), 7.12
~
HPLC: method 1: tR = 18.5 min, purity 98.0%.
(c = 0.30; CH2Cl2).
½
a 2D0
ꢂ
= ꢀ36.3
6.4. (ꢀ)-(1R,5S)-8-(4-Methoxybenzyl)-6-propyl-6,8-diazabicyclo
[3.2.2]nonan-2-one (7)
(d, J = 8.6 Hz, 2H, 20-H4-methoxybenzyl, 60-H4-methoxybenzyl). IR (neat):
m
[cmꢀ1] = 2939 (m,
(m, mC C arom.), 1422 (m, dC–H aliph.), 1242 (m)/1102 (m)/1036 (m,
C–O), 809 (w, Cp-subst. arom.).
mC–H aliph.), 1680 (s, mC@O amide), 1612 (m)/1512
Under N2 a 1.0 M solution of LiAlH4 in THF (1.17 mL, 1.17 mmol)
was added to an ice-cooled solution of 6 (110 mg, 0.29 mmol) in
THF (40 mL). The mixture was stirred at 0 °C for 10 min and then
heated to reflux for 16 h. Then 1.0 M HCl (10 mL) was added under
ice-cooling and the mixture was stirred at 0 °C for 10 min and then
refluxed for 3 h. After cooling down the mixture was alkalized with
a saturated aqueous solution of NaHCO3 and extracted three times
with CH2Cl2. The combined organic layers were dried (Na2SO4), fil-
tered and concentrated in vacuo. The residue was purified by fc
@
m
(Z)-8 (Rf = 0.12): Colorless oil, yield 20 mg (9%). C20H26N2O5
(374.4). ½a 2D0
= ꢀ25.0 (c = 0.25; CH2Cl2). MS (EI): m/z [%] = 374 (M,
ꢂ
4), 342 (M–HOCH3, 8), 121 (CH2PhOCH3, 64), 101 (H2CCHC(OCH3)2,
100). 1H NMR (CDCl3): d [ppm] = 1.65 (dd, J = 7.0/2.3 Hz, 3H,
NCH@CHCH3), 1.81–1.93 (m, 2H, 3-H), 2.09–2.21 (m, 2H, 4-H),
3.19 (s, 3H, OCH3), 3.29 (s, 3H, OCH3), 3.79 (s, 3H, ArOCH3), 4.00
(d, J = 14.9 Hz, 1H, NCH2Ar), 4.03 (s, 1H, 1-H), 4.08 (dd, J = 6.3/
1.6 Hz, 1H, 5-H), 5.23 (d, J = 14.9 Hz, 1H, NCH2Ar), 5.43 (dq,
J = 8.6/7.0 Hz, 1H, NCH@CHCH3), 5.96–6.00 (m, 1H, NCH@CHCH3),
6.85 (d, J = 8.6 Hz, 2H, 30-H4-methoxybenzyl, 50-H4-methoxybenzyl), 7.15
(d, J = 8.6 Hz, 2H, 200-H4-methoxybenzyl, 600-H4-methoxybenzyl). IR (neat):
~
amide), 1612 (m)/
O
(Ø = 2 cm,
h = 15 cm,
CH2Cl2/methanol = 100/1,
V = 10 mL,
Rf = 0.11) to give 7 as a yellow oil, yield 30 mg (34%). C18H26N2O2
(302.4). Purity by HPLC: method 2a: tR = 16.2 min, purity 99.3%;
method 1: tR = 16.0 min, purity 97.6%.
CH2Cl2). MS (EI): m/z [%] = 302 (M, 2), 181 (M–CH2PhOCH3, 3),
121 (CH2PhOCH3, 100). 1H NMR (CDCl3):
[ppm] = 0.84 (t,
½
a 2D0
ꢂ
= ꢀ97.7 (c = 0.43;
m
[cmꢀ1] = 2944 (m, mC–H aliph.), 1681 (s, mC
@
d
1512 (m, mC@ arom.), 1419 (m, dC–H aliph.), 1244 (m)/1101 (m)/
C
J = 7.0 Hz, 3H, NCH2CH2CH3), 1.36–1.46 (m, 2H, NCH2CH2CH3),
1.68–1.78 (m, 1H, 4-H), 1.84–1.92 (m, 1H, 4-H), 2.27 (ddd,
J = 13.3/8.6/1.6 Hz, 1H, 3-H), 2.46 (t, J = 7.0 Hz, 2H, NCH2CH2CH3),
2.77 (dd, J = 10.2/2.3 Hz, 1H, piperazine-H), 2.81–2.88 (m, 2H,
piperazine-H), 2.91 (dd, J = 11.0/2.3 Hz, 1H, piperazine-H), 2.97–
3.01 (m, 1H, piperazine-H), 3.17 (t, J = 3.1 Hz, 1H, piperazine-H),
3.26 (ddd, J = 13.3/10.2/8.6 Hz, 1H, 3-H), 3.60 (d, J = 12.5 Hz, 1H,
NCH2Ar), 3.64 (d, J = 12.5 Hz, 1H, NCH2Ar), 3.78 (s, 3H, ArOCH3),
6.83 (d, J = 8.6 Hz, 2H, 30-H4-methoxybenzyl, 50-H4-methoxybenzyl), 7.18
(d, J = 8.6 Hz, 2H, 20-H4-methoxybenzyl, 60-H4-methoxybenzyl). IR (neat):
~
1035 (m, mC–O), 808 (w, Cp-subst. arom.).
6.7. (+)-(1R,5R,E)-2,2-Dimethoxy-8-(4-methoxybenzyl)-6-prop-
1-en-1-yl-6,8-diazabicyclo[3.2.2]nonan-7,9-dione (ent-(E)-8)
and (+)-(1R,5R,Z)-2,2-Dimethoxy-8-(4-methoxybenzyl)-6-prop-
1-en-1-yl-6,8-diazabicyclo[3.2.2]nonan-7,9-dione (ent-(Z)-8)
As described for the preparation of (E)-8 and (Z)-8, the enantio-
mer ent-48 (130 mg, 0.35 mmol) was reacted with 1,4-diazabicy-
clo[2.2.2]octane (39 mg, 0.35 mmol) and rhodium(III) chloride
trihydrate (9 mg, 0.03 mmol) in methanol (20 mL) to give ent-
(E)-8 and ent-(Z)-8.
m
[cmꢀ1] = 2955 (m, mC–H aliph.), 1708 (s, mC
@
O
ketone), 1611 (m)/
1510 (s, mC@C arom.), 1244 (m)/1033 (m, mC–O), 820 (w, Cp-subst. arom.).
ent-(E)-8: Colorless oil, yield 79 mg (61%). C20H26N2O5 (374.4).
Purity by HPLC: method 1: tR = 19.9 min, purity 97.9%.
6.5. (+)-(1S,5R)-8-(4-Methoxybenzyl)-6-propyl-6,8-
diazabicyclo[3.2.2]nonan-2-one (ent-7)
½
a 2D0
a 2D0
ꢂ
= +22.7 (c = 0.63; CH2Cl2).
ent-(Z)-8: Colorless oil, yield 20 mg (15%). C20H26N2O5 (374.4).
= +24.1 (c = 0.22; CH2Cl2).
As described for the preparation of 7, the enantiomer ent-6
(70 mg, 0.19 mmol) was reacted with LiAlH4 (0.74 mL of a 1.0 M
solution in THF, 0.74 mmol) in THF (30 mL) and afterwards hydro-
lyzed with 1.0 M HCl (5 mL) to give ent-7 as a yellow oil, yield
39 mg (69%). C18H26N2O2 (302.4). Purity by HPLC: method 2a:
tR = 15.8 min, purity 99.1%; method 1: tR = 15.9 min, purity 98.9%.
½
ꢂ
6.8. (+)-(1S,5S)-8-(4-Methoxybenzyl)-6,8-diazabicyclo[3.2.2]-
nonane-2,7,9-trione (9)
(E)-8 (170 mg, 0.45 mmol) was dissolved in a mixture of
water/glacial acetic acid (1/1, 20 mL) and the mixture was heated
½
a 2D0
ꢂ
= +94.1 (c = 1.40; CH2Cl2).

R. Holl et al. / Bioorg. Med. Chem. 17 (2009) 1445–1455
1451
to reflux for 16 h. Then the mixture was neutralized with a satu-
6.12. (ꢀ)-(1S,5S)-2,2-Dimethoxy-8-(4-methoxybenzyl)-6-(3-me
rated aqueous solution of NaHCO3 and the mixture was extracted
with CH2Cl2 (3ꢁ). The combined organic layers were dried
(Na2SO4), filtered and the solvent was removed in vacuo. The res-
idue was purified by fc (Ø = 2 cm, h = 15 cm, ethyl acetate, 10 mL,
Rf = 0.25) to give 9 as a colorless solid, mp 264 °C (decomposi-
tion), yield 31 mg (24%). C15H16N2O4 (288.3). Purity by HPLC:
thylbut-2-en-1-yl)-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (11)
Under N2 a solution of 10 (61 mg, 0.18 mmol) and tetrabutylam-
monium iodide (13 mg, 0.04 mmol) in THF (30 mL) was cooled to
ꢀ78 °C. Then a 2.0 M solution of sodium hexamethyldisilazane in
THF (0.10 mL, 0.20 mmol) was added dropwise. After stirring the
mixture at ꢀ78 °C for 40 min, the 1-bromo-3-methylbut-2-ene
(0.11 mL, 136 mg, 0.91 mmol) was added. The mixture was stirred
at ꢀ78 °C for 1 h and was then allowed to warm to rt. After stirring
the mixture at rt for 2 h, water was added and the mixture was ex-
tracted with CH2Cl2 (3ꢁ). The combined organic layers were dried
(Na2SO4), filtered and the solvent was removed in vacuo. The resi-
due was purified by fc (Ø = 1 cm, h = 15 cm, cyclohexane/ethyl ace-
tate = 2/1, 5 mL, Rf = 0.14) to give 11 as a colorless oil, yield 60 mg
(82%). C22H30N2O5 (402.5). Purity by HPLC: method 1: tR = 20.3 min,
method 1: tR = 13.4 min, purity 96.5%.
½
a 2D0
= +48.1 (c = 0.08;
ꢂ
H3CCN). MS (EI): m/z [%] = 288 (M, 4), 121 (CH2PhOCH3, 100).
1H NMR (CDCl3): d [ppm] = 2.10–2.22 (m, 1H, 4-H), 2.40–2.46
(m, 1H, 3-H), 2.50–2.60 (m, 2H, 3-H (1H), 4-H (1H)), 3.79 (s,
3H, ArOCH3), 4.10–4.14 (m, 1H, 5-H), 4.19 (d, J = 2.3 Hz, 1H, 1-
H), 4.38 (d, J = 14.1 Hz, 1H, NCH2Ar), 4.79 (d, J = 14.1 Hz, 1H,
NCH2Ar), 6.37 (s br, 1H, NH), 6.85 (d, J = 8.6 Hz, 2H, 30-H4-methoxybenzyl
,
50-H
), 7.21 (d, J = 8.6 Hz, 2H, 20-H4-methoxybenzyl 60-
,
4-methoxybenzyl
~
H4-methoxybenzyl). IR (neat):
mC–H aliph.), 1724 (m, mC
m
[cmꢀ1] = 3241 (m br,
m
N–H), 2973 (m,
amide), 1613 (m)/
O
@
O
ketone), 1663 (s, mC
@
purity 99.4%. ½a D20
= ꢀ10.9 (c = 0.59; CH2Cl2). MS (EI): m/z [%] = 402
ꢂ
1513 (m, mC
@C arom.), 1450 (m, dC–H aliph.), 1248 (m)/1025 (m,
mC–O),
(M, 5), 121 (CH2PhOCH3, 54), 101 (H2CCHC(OCH3)2, 100). 1H NMR
(CDCl3): d [ppm] = 1.67 (s, 3H, NCH2CH@C(CH3)2), 1.73 (s, 3H,
NCH2CH@C(CH3)2), 1.75–1.83 (m, 2H, 3-H), 1.97–2.07 (m, 2H, 4-
H), 3.16 (s, 3H, OCH3), 3.26 (s, 3H, OCH3), 3.77 (s, 3H, ArOCH3),
3.87 (dd, J = 5.5/2.3 Hz, 1H, 5-H), 3.91 (dd, J = 14.9/7.0 Hz, 1H,
NCH2CH@C(CH3)2), 3.95 (d, J = 14.9 Hz, 1H, NCH2Ar), 3.96 (s, 1H,
1-H), 4.05 (dd, J = 14.9/7.0 Hz, 1H, NCH2CH@C(CH3)2), 5.08–5.13
(m, 1H, NCH2CH@C(CH3)2), 5.17 (d, J = 14.9 Hz, 1H, NCH2Ar), 6.83
(d, J = 8.6 Hz, 2H, 30-H4-methoxybenzyl, 50-H4-methoxybenzyl), 7.11 (d,
~
833 (m, Cp-subst. arom.).
6.9. (ꢀ)-(1R,5R)-8-(4-Methoxybenzyl)-6,8-diazabicyclo[3.2.2]
no nane-2,7,9-trione (ent-9)
As described for the preparation of 9, the enantiomer ent-(E)-8
(236 mg, 0.63 mmol) was reacted in a mixture of water/glacial ace-
tic acid (1/1, 30 mL) to give ent-9 as a colorless solid, mp 260 °C
(decomposition), yield 77 mg (42%). C15H16N2O4 (288.3). Purity
J = 8.6 Hz, 2H, 20-H4-methoxybenzyl, 60-H4-methoxybenzyl). IR (neat):
m
by HPLC: method 1: tR = 13.4 min, purity 100%. ½a D20
ꢂ
= ꢀ48.6
[cmꢀ1] = 2933 (m,
(m, mC C arom.), 1450 (m, dC–H aliph.), 1244 (m)/1035 (m,
(w, Cp-subst. arom.).
m
C–H aliph.), 1678 (s, mC
@
O amide), 1612 (m)/1513
(c = 0.08; H3CCN).
@
m
C–O), 808
6.10. (+)-(1S,5S)-2,2-Dimethoxy-8-(4-methoxybenzyl)-6,8-
diazabicyclo[3.2.2]nonane-7,9-dione (10)
6.13. (+)-(1R,5R)-2,2-Dimethoxy-8-(4-methoxybenzyl)-6-(3-
methylbut-2-en-1-yl)-6,8-diazabicyclo[3.2.2]nonane-7,9-dione
(ent-11)
5Under N2 trimethyl orthoformate (0.50 mL, 486 mg,
4.58 mmol) was added to a solution of 9 (120 mg, 0.42 mmol)
and p-toluenesulfonic acid (158 mg, 0.92 mmol) in methanol
(30 mL). The mixture was heated to reflux for 16 h. Then a sat-
urated aqueous solution of NaHCO3 was added and the mixture
was extracted with CH2Cl2 (3ꢁ). The combined organic layers
were dried (Na2SO4), filtered and concentrated in vacuo. The
residue was purified by fc (Ø = 2 cm, h = 15 cm, cyclohexane/
ethyl acetate = 1/2, V = 10 mL, Rf = 0.06) to give 10 as a colorless
oil, yield 112 mg (80%). C17H22N2O5 (334.4). Purity by HPLC:
As described for the preparation of 11, the enantiomer ent-10
(66 mg, 0.20 mmol), tetrabutylammonium iodide (15 mg, 0.04
mmol), 2 M solution of sodium hexamethyldisilazane in THF
(0.11 mL, 0.22 mmol) and 1-bromo-3-methylbut-2-ene (0.12 mL,
147 mg, 0.99 mmol) were reacted in THF (30 mL) to give ent-11 as
a colorless oil, yield63 mg(79%). C22H30N2O5 (402.5). PuritybyHPLC:
method 1: tR = 20.1 min, purity 96.8%. ½a D20
ꢂ
= +10.9 (c = 0.26; CH2Cl2).
method 1: tR = 15.9 min, purity 95.9%.
½
a 2D0
ꢂ
= +60.7 (c = 0.22;
6.14. (ꢀ)-(1R,5S)-8-(4-Methoxybenzyl)-6-(3-methylbut-2-en-1-
CH2Cl2). MS (EI): m/z [%] = 334 (M, 6), 213 (M–CH2PhOCH3, 1),
121 (CH2PhOCH3, 40), 101 (H2CCHC(OCH3)2, 100). 1H NMR
(CDCl3): d [ppm] = 1.77–1.90 (m, 2H, 3-H), 2.05–2.20 (m, 2H,
4-H), 3.18 (s, 3H, OCH3), 3.27 (s, 3H, OCH3), 3.78 (s, 3H, Ar-
OCH3), 3.91 (d, J = 2.3 Hz, 1H, 1-H), 3.93–3.97 (m, 1H, 5-H),
3.95 (d, J = 14.9 Hz, 1H, NCH2Ar), 5.22 (d, J = 14.9 Hz, 1H,
NCH2Ar), 6.67 (d br, J = 5.5 Hz, 1H, NH), 6.85 (d, J = 8.6 Hz, 2H,
30-H4-methoxybenzyl, 50-H4-methoxybenzyl), 7.14 (d, J = 8.6 Hz, 2H, 20-
~
yl)-6,8-diazabicyclo[3.2.2]nonan-2-one (12)
Under N2 a 1.0 M solution of LiAlH4 (0.74 mL, 0.74 mmol) was
added to an ice-cooled solution of 11 (74 mg, 0.18 mmol) in THF
(30 mL). The mixture was stirred at 0 °C for 10 min and then heated
to reflux for 16 h. Then 1.0 M HCl (5 mL) was added under ice-cool-
ing and the mixture was stirred at 0 °C for 10 min and then refluxed
for 3 h. After cooling down the mixture was alkalized with a satu-
rated aqueous solution of NaHCO3 and extracted three times with
CH2Cl2. The combined organic layers were dried (Na2SO4), filtered
and concentrated in vacuo. The residue was purified by fc
(Ø = 1 cm, h = 15 cm, CH2Cl2/methanol = 100/1, V = 5 mL, Rf = 0.09)
to give 12 as a yellow oil, yield 45 mg (75%). C20H28N2O2 (328.5). Pur-
ity by HPLC: method 2a: tR = 19.5 min, purity 100%; method 1:
H4-methoxybenzyl, 60-H4-methoxybenzyl). IR (neat):
br,
m
[cmꢀ1] = 3239 (m
mN–H), 2943 (m, mC–H aliph.), 1679 (s, mC@ amide), 1612 (m)/
O
1512 (m, mC
@
arom.), 1436 (m, dC–H aliph.), 1244 (m)/1035 (m,
C
m
C–O), 807 (w, Cp-subst. arom.).
6.11. (ꢀ)-(1R,5R)-2,2-Dimethoxy-8-(4-methoxybenzyl)-6,8-
diazabicyclo[3.2.2]nonane-7,9-dione (ent-10)
tR = 18.0 min, purity 99.8%. ½a D20
= ꢀ77.8 (c = 0.34; CH2Cl2). MS (EI):
ꢂ
m/z [%] = 328 (M, 4), 207 (M–CH2PhOCH3, 17), 121 (CH2PhOCH3,
100). 1H NMR (CDCl3): d [ppm] = 1.62 (s, 3H, NCH2CH@C(CH3)2),
1.67–1.75 (m, 1H, 4-H), 1.70 (s, 3H, NCH2CH@C(CH3)2), 1.83–1.91
(m, 1H, 4-H), 2.27 (ddd, J = 13.3/8.6/1.6 Hz, 1H, 3-H), 2.77 (dd,
J = 10.2/2.3 Hz, 1H, piperazine-H), 2.83–2.93 (m, 3H, piperazine-H),
3.00–3.04 (m, 1H, piperazine-H), 3.05–3.17 (m, 2H,
NCH2CH@C(CH3)2), 3.17–3.19 (m, 1H, piperazine-H), 3.25 (ddd,
As described for the preparation of 10, the enantiomer ent-9
(77 mg, 0.27 mmol) was reacted with trimethyl orthoformate
(0.32 mL, 312 mg, 2.94 mmol) and p-toluenesulfonic acid
(101 mg, 0.59 mmol) in methanol (30 mL) to give ent-10 as color-
less oil, yield 70 mg (78%). C17H22N2O5 (334.4). ½a D20
= ꢀ61.8
ꢂ
(c = 0.13; CH2Cl2).

1452
R. Holl et al. / Bioorg. Med. Chem. 17 (2009) 1445–1455
J = 13.3/10.2/8.6 Hz, 1H, 3-H), 3.60 (d, J = 12.5 Hz, 1H, NCH2Ar), 3.64
(d, J = 12.5 Hz, 1H, NCH2Ar), 3.79 (s, 3H, ArOCH3), 5.15–5.21 (m, 1H,
NCH2CH@C(CH3)2), 6.83 (d, J = 8.6 Hz, 2H, 30-H4-methoxybenzyl, 50-
(30 mL). The mixture was stirred at 0 °C for 10 min and then
heated to reflux for 16 h. Then 1.0 M HCl (5 mL) was added under
ice-cooling and the mixture was stirred at 0 °C for 10 min and
then refluxed for 3 h. After cooling down the mixture was alkal-
ized with a saturated aqueous solution of NaHCO3 and extracted
three times with CH2Cl2. The combined organic layers were dried
(Na2SO4), filtered and concentrated in vacuo. The residue was
purified by fc (Ø = 1 cm, h = 15 cm, CH2Cl2/methanol = 50/1,
V = 5 mL, Rf = 0.39) to give 14 as a colorless oil, yield 33 mg
(R,S)-107 (50%). C22H26N2O2 (350.5). Purity by HPLC: method
2a: tR = 23.2 min, purity 99.6%; method 1: tR = 18.3 min, purity
H4-methoxybenzyl), 7.19 (d, J = 8.6 Hz, 2H, 20-H4-methoxybenzyl
,
60-H4-methoxybenzyl). IR (neat):
(s, mC
m
[cmꢀ1] = 2912 (m,
m
C–H aliph.), 1708
C arom.), 1441 (m, dC-H aliph.),
C–O), 820 (m, Cp-subst. arom.).
~
@O ketone), 1611 (m)/1510 (s, mC@
1244 (s)/1033 (m,
m
6.15. (+)-(1S,5R)-8-(4-Methoxybenzyl)-6-(3-methylbut-2-en-1-
yl)-6,8-diazabicyclo[3.2.2]nonan-2-one (ent-12)
As described for the preparation of 12, the enantiomer ent-11
(78 mg, 0.19 mmol) was reacted with LiAlH4 (0.78 mL of a 1.0 M
solution in THF, 0.78 mmol) in THF (30 mL) and afterwards hydro-
lyzed with 1.0 M HCl (5 mL) to give ent-12 as a yellow oil, yield
42 mg (66%). C20H28N2O2 (328.5). Purity by HPLC: method 2a:
tR = 19.8 min, purity 97.2%; method 1: tR = 18.2 min, purity 99.5%.
99.5%. ½a 2D0
= ꢀ61.5 (c = 0.85; CH2Cl2). MS (EI): m/z [%] = 350 (M,
ꢂ
3), 229 (M–CH2PhOCH3, 3), 121 (CH2PhOCH3, 100), 91 (CH2Ph,
36). 1H NMR (CDCl3): d [ppm] = 1.63–1.73 (m, 1H, 4-H), 1.76–
1.85 (m, 1H, 4-H), 2.31 (ddd, J = 13.3/8.6/1.6 Hz, 1H, 3-H), 2.82
(dd, J = 10.2/2.3 Hz, 1H, piperazine-H), 2.86–2.92 (m, 3H, pipera-
zine-H), 3.03–3.07 (m, 1H, piperazine-H), 3.20 (t, J = 3.1 Hz, 1H,
piperazine-H), 3.28 (ddd, J = 13.3/10.2/8.6 Hz, 1H, 3-H), 3.61 (d,
J = 12.5 Hz, 1H, NCH2Ar), 3.66 (d, J = 12.5 Hz, 2H, NCH2Ar), 3.74 (d,
J = 12.5 Hz, 1H, NCH2Ar), 3.79 (s, 3H, ArOCH3), 6.83 (d, J = 8.6 Hz,
2H, 30-H4-methoxybenzyl, 50-H4-methoxybenzyl), 7.18 (d, J = 8.6 Hz, 2H,
20-H4-methoxybenzyl, 60-H4-methoxybenzyl), 7.20–7.25 (m, 1H, NCH2C6H5),
~
½
a 2D0
= +76.1 (c = 0.16; CH2Cl2).
ꢂ
6.16. (ꢀ)-(1S,5S)-6-Benzyl-2,2-dimethoxy-8-(4-methoxybenzyl)-
6,8-diazabicyclo[3.2.2]nonane-7,9-dione (13)
Under N2 a solution of 10 (26 mg, 0.08 mmol) and tetrabutylam-
monium iodide (6 mg, 0.02 mmol) in THF (20 mL) was cooled to
ꢀ78 °C. Then a 2.0 M solution of sodium hexamethyldisilazane in
THF (0.05 mL, 0.09 mmol) was added dropwise. After stirring the
mixture at ꢀ78 °C for 40 min, benzyl bromide (0.05 mL, 66 mg,
0.39 mmol) was added. The mixture was stirred at ꢀ78 °C for 1 h
and was then allowed to warm to rt. After stirring the mixture at
rt for 2 h, water was added and the mixture was extracted with
CH2Cl2 (3ꢁ). The combined organic layers were dried (Na2SO4), fil-
tered and the solvent was removed in vacuo. The residue was puri-
fied by fc (Ø = 1 cm, h = 15 cm, cyclohexane/ethyl acetate = 2/1,
5 mL, Rf = 0.12) to give 13 as a colorless oil, yield 22 mg (67%).
C24H28N2O5 (424.5). Purity by HPLC: method 1: tR = 20.2 min, purity
7.26–7.30 (m, 4H, NCH2C6H5). IR (neat):
C–H arom.), 2913 (m, C–H aliph.), 1708 (s,
(s, C@C arom.), 1453 (m, dC-H aliph.), 1244 (m)/1030 (m,
Cp-subst. arom.), 698 (m, Cmono-subst. arom.).
m
[cmꢀ1] = 3027 (w,
C@O ketone), 1610 (m)/1510
C–O), 819 (w,
m
m
m
m
m
6.19. (+)-(1S,5R)-6-Benzyl-8-(4-methoxybenzyl)-6,8-
diazabicyclo[3.2.2]nonan-2-one (ent-14)
As described for the preparation of 14, the enantiomer ent-13
(118 mg, 0.28 mmol) was reacted with LiAlH4 (1.11 mL of a 1.0 M
solution in THF, 1.11 mmol) in THF (40 mL) and afterwards hydro-
lyzed with 1.0 M HCl (10 mL) to give ent-14 as a colorless oil, yield
20 mg (21%). C22H26N2O2 (350.5). Purity by HPLC: method 2a:
tR = 23.8 min, purity 99.6%; method 1: tR = 18.5 min, purity 99.0%.
97.5%. ½a 2D0
= ꢀ21.1 (c = 0.42; CH2Cl2). MS (EI): m/z [%] = 424 (M, 3),
ꢂ
121 (CH2PhOCH3, 29), 101 (H2CCHC(OCH3)2, 100), 91 (CH2Ph, 13).
1H NMR (CDCl3): d [ppm] = 1.44–1.54 (m, 1H, 3-H), 1.70–1.80 (m,
1H, 3-H), 1.87–1.99 (m, 2H, 4-H), 3.17 (s, 3H, OCH3), 3.28 (s, 3H,
OCH3), 3.79 (s, 3H, ArOCH3), 3.88 (dd, J = 5.5/1.6 Hz, 1H, 5-H), 3.94
(d, J = 14.9 Hz, 1H, NCH2Ar), 4.05 (s, 1H, 1-H), 4.51 (d, J = 14.9 Hz,
1H, NCH2Ph), 4.59 (d, J = 14.9 Hz, 1H, NCH2Ph), 5.18 (d,
½
a 2D0
= +64.1 (c = 0.44; CH2Cl2).
ꢂ
6.20. (ꢀ)-(1S,5S)-2,2-Dimethoxy-8-(4-methoxybenzyl)-6-phenyl-
6,8-diazabicyclo[3.2.2]nonane-7,9-dione (15)
J = 14.9 Hz, 1H, NCH2Ar), 6.86 (d, J = 8.6 Hz, 2H, 30-H4-methoxybenzyl
,
Under N2 a mixture of 10 (50 mg, 0.15 mmol), iodobenzene
(0.34 mL, 610 mg, 2.99 mmol), K2CO3 (21 mg, 0.15 mmol) and
CuI (3 mg, 0.015 mmol) in DMF (20 mL) was heated at 150 °C
for 72 h. After the mixture was cooled to rt, water was added
and the mixture was extracted with diethyl ether (6ꢁ). The
combined organic layers were dried (Na2SO4), filtered and con-
centrated in vacuo. The residue was purified by fc (Ø = 1 cm,
h = 15 cm, cyclohexane/ethyl acetate = 2/1, V = 5 mL, Rf = 0.16)
50-H4-methoxybenzyl), 7.12 (d, J = 8.6 Hz, 2H, 20-H4-methoxybenzyl, 60-
H4-methoxybenzyl), 7.20–7.24 (m, 2H, NCH2C6H5), 7.27–7.35 (m, 3H,
~
NCH2C6H5). IR (neat):
mC–H aliph.), 1676 (s, mC
m
[cmꢀ1] = 3029 (w, mC–H arom.), 2942 (m,
amide), 1612 (m)/1512 (m, m C@C arom.),
@
O
1451 (m, dC-H aliph.), 1245 (m)/1036 (m,
mC-O), 808 (w, Cp-subst. arom.),
732 (m)/700 (m, ).
C
mono-subst. arom.
to give 15 as
a yellow oil, yield 44 mg (72%). C23H26N2O5
6.17. (+)-(1R,5R)-6-Benzyl-2,2-dimethoxy-8-(4-methoxybenzyl)-
6,8-diazabicyclo[3.2.2]nonane-7,9-dione (ent-13)
(410.5). Purity by HPLC: method 1: tR = 20.2 min, purity 100%.
½
a 2D0
ꢂ
= ꢀ46.2 (c = 0.18; CH2Cl2). MS (EI): m/z [%] = 410 (M, 5),
121 (CH2PhOCH3, 32), 101 (H2CCHC(OCH3)2, 100). 1H NMR
(CDCl3): d [ppm] = 1.86–1.96 (m, 1H, 3-H), 1.99–2.09 (m, 1H,
3-H), 2.13–2.23 (m, 2H, 4-H), 3.21 (s, 3H, OCH3), 3.32 (s, 3H,
OCH3), 3.78 (s, 3H, ArOCH3), 4.06 (d, J = 14.9 Hz, 1H, NCH2Ar),
4.11 (s, 1H, 1-H), 4.40 (dd, J = 5.5/1.6 Hz, 1H, 5-H), 5.24 (d, J =
As described for the preparation of 13, the enantiomer ent-10
(30 mg, 0.09 mmol), tetrabutylammonium iodide (7 mg,
0.02 mmol), 2 M solution of sodium hexamethyldisilazane in THF
(0.05 mL, 0.10 mmol) and benzyl bromide (0.05 mL, 77 mg,
0.45 mmol) were reacted in THF (20 mL) to give ent-13 as a colorless
oil, yield 18 mg (47%). C24H28N2O5 (424.5). Purity by HPLC: method
14.9 Hz, 1H, NCH2Ar), 6.86 (d, J = 8.6 Hz, 2H, 3‘-H4-methoxybenzyl
,
,
50-H4-methoxybenzyl), 7.18 (d, J = 8.6 Hz, 2H, 20-H4-methoxybenzyl
1: tR = 20.4 min, purity 96.3%. ½a D20
ꢂ
= +20.4 (c = 0.33; CH2Cl2).
60-H4-methoxybenzyl), 7.23–7.27 (m, 1H, 40-Hphenyl), 7.28 (d,
J = 7.8 Hz, 2H, 20-Hphenyl
,
60-Hphenyl), 7.39 (t, J = 7.8 Hz, 2H,
6.18. (ꢀ)-(1R,5S)-6-Benzyl-8-(4-methoxybenzyl)-6,8-
30-Hphenyl, 50-Hphenyl). IR (neat):
[cm-1] = 2946 (m, mC–H aliph.),
~
m
diazabicyclo[3.2.2]nonan-2-one (14)
1682 (s, mC@O amide), 1612 (m)/1512 (m, mC@C arom.), 1456 (m,
dC-H aliph.), 1246 (m)/1032 (m,
(m, Cmono-subst. arom.).
mC–O), 808 (w, Cp-subst. arom.), 729
Under N2 a 1.0 M solution of LiAlH4 (0.76 mL, 0.76 mmol) was
added to an ice-cooled solution of 13 (80 mg, 0.19 mmol) in THF

R. Holl et al. / Bioorg. Med. Chem. 17 (2009) 1445–1455
1453
6.21. (+)-(1R,5R)-2,2-Dimethoxy-8-(4-methoxybenzyl)-6-
phenyl-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (ent-15)
for 3 h. After cooling down the mixture was alkalized with a satu-
rated aqueous solution of NaHCO3 and extracted three times with
CH2Cl2. The combined organic layers were dried (Na2SO4), filtered
and concentrated in vacuo. The residue was purified by fc
(Ø = 2 cm, h = 15 cm, CH2Cl2/methanol = 9.5/0.5, V = 10 mL, Rf =
0.08) to give 17 as a pale yellow oil, yield 50 mg (59%). C15H20N2O2
As described for the preparation of 15, the enantiomer ent-10
(70 mg, 0.21 mmol) was reacted with iodobenzene (0.47 mL,
854 mg, 4.19 mmol), K2CO3 (29 mg, 0.21 mmol) and CuI (4 mg,
0.021 mmol) in DMF (20 mL) to give ent-15 as a yellow oil, yield
73 mg (85%). C23H26N2O5 (410.5). Purity by HPLC: method 1:
(260.3). ½a 2D0
= ꢀ28.2 (c = 0.41; CH2Cl2). MS (EI): m/z [%] = 260 (M,
ꢂ
3), 232 (M–CO, 9), 121 (CH2PhOCH3, 100). 1H NMR (CDCl3): d
[ppm] = 1.57–1.67 (m, 1H, 4-H), 1.96–2.04 (m, 1H, 4-H), 2.32 (ddd,
J = 13.3/7.8/1.6 Hz, 1H, 3-H), 2.90–2.96 (m, 2H, 7-H, 9-H), 3.02 (dd,
J = 11.0/2.3 Hz, 1H, 9-H), 3.19–3.21 (m, 1H, 1-H), 3.25–3.33 (m, 2H,
3-H, 7-H), 3.47–3.51 (m, 1H, 5-H), 3.62 (d, J = 13.3 Hz, 1H, NCH2Ar),
3.69 (d, J = 13.3 Hz, 1H, NCH2Ar), 3.79 (s, 3H, ArOCH3), 6.83 (d,
tR = 20.0 min, purity 98.6%. ½a D20
ꢂ
= +48.4 (c = 0.32; CH2Cl2).
6.22. (+)-(1R,5S)-8-(4-Methoxybenzyl)-6-phenyl-6,8-
diazabicyclo[3.2.2]nonan-2-one (16)
Under N2 a 1.0 M solution of LiAlH4 (0.58 mL, 0.58 mmol) was
added to an ice-cooled solution of 15 (60 mg, 0.15 mmol) in THF
(20 mL). The mixture was stirred at 0 °C for 10 min and then heated
to reflux for 16 h. Then 1.0 M HCl (5 mL) was added under ice-cool-
ing and the mixture was stirred at 0 °C for 10 min and then refluxed
for 3 h. After cooling down the mixture was alkalized with a satu-
rated aqueous solution of NaHCO3 and extracted three times with
CH2Cl2. The combined organic layers were dried (Na2SO4), filtered
and concentrated in vacuo. The residue was purified by fc
(Ø = 1 cm, h = 15 cm, CH2Cl2, V = 5 mL, Rf = 0.26) to give 16 as a yel-
low oil, yield 10 mg (20%). C21H24N2O2 (336.4). Purity by HPLC:
method 2a: tR = 21.6 min, purity 98.7%; method 1: tR = 17.8 min,
J = 8.6 Hz, 2H, 30-H4-methoxybenzyl 50-H4-methoxybenzyl), 7.19 (d,
,
J = 8.6 Hz, 2H, 20-H4-methoxybenzyl, 60-H4-methoxybenzyl). The signal for
the proton of the NH group could not be detected. 13C NMR (CDCl3):
d [ppm] = 31.0 (1C, C-4), 37.6 (1C, C-3), 40.8 (1C, C-7), 49.8 (1C, C-5),
53.5 (1C, C-9), 55.5 (1C, ArOCH3), 60.1 (1C, NCH2Ar), 68.2 (1C, C-1),
114.0 (2C, C-304-methoxybenzyl C-504-methoxybenzyl), 129.9 (2C, C-
,
204-methoxybenzyl, C-604-methoxybenzyl), 130.6 (1C, C-104-methoxybenzyl),
0
159.0 (1C, C-4 4-methoxybenzyl), 216.9 (1C, 2-C). IR (neat):
3341 (m br, N–H), 2922 (s, C–H aliph.), 1705 (s, C@O ketone), 1610 (s)/
[cm-1] =
~
m
m
m
m
1510 (s, mC@C arom.), 1442 (m, dC-H aliph.), 1242 (s)/1031 (m,
mC–O),
815 (m, Cp-subst. arom.).
purity 98.3%. ½a D20
ꢂ
= +34.7 (c = 0.41; CH2Cl2). MS (EI): m/z [%] = 336
6.25. (+)-(1S,5R)-8-(4-Methoxybenzyl)-6,8-diazabicyclo[3.2.2]
nonan-2-one (ent-17)
(M, 12), 308 (M–CO, 16), 215 (M–CH2PhOCH3, 6), 121 (CH2PhOCH3,
100), 77 (Ph, 27). 1H NMR (CDCl3): d [ppm] = 1.99–2.13 (m, 2H, 4-H),
2.29 (ddd, J = 13.3/7.0/3.1 Hz, 1H, 3-H), 2.95–3.00 (m, 1H, 9-H),
3.19–3.23 (m, 1H, 9-H), 3.27–3.31 (m, 1H, 7-H), 3.40 (dt, J = 13.3/
9.4 Hz, 1H, 3-H), 3.46–3.48 (m, 1H, 1-H), 3.56 (dd, J = 11.0/2.3 Hz,
1H, 7-H), 3.73 (s, 2H, NCH2Ar), 3.80 (s, 3H, ArOCH3), 4.10–4.14 (m,
1H, 5-H), 6.63 (d, J = 7.8 Hz, 2H, 20-Hphenyl, 60-Hphenyl), 6.72 (t,
As described for the preparation of 17, the enantiomer ent-10
(70 mg, 0.21 mmol) was reacted with LiAlH4 (0.84 mL of a 1.0 M
solution in THF, 0.84 mmol) in THF (30 mL) and afterwards hydro-
lyzed with 1.0 M HCl (5 mL) to give ent-17 as a pale yellow oil, yield
21 mg (39%). C15H20N2O2 (260.3). ½a D20
ꢂ
= +26.9 (c = 0.11; CH2Cl2).
J = 7.8 Hz, 1H, 40-Hphenyl), 6.86 (d, J = 8.6 Hz, 2H, 30-H4-methoxybenzyl
50-H4-methoxybenzyl), 7.20–7.26 (m, 4H, 20-H4-methoxybenzyl 60-
H4-methoxybenzyl
30-Hphenyl 50-Hphenyl). 13C NMR (CDCl3):
,
,
6.26. (ꢀ)-(1R,5S)-6-Benzoyl-8-(4-methoxybenzyl)-6,8-
,
,
d
diazabicyclo[3.2.2]nonan-2-one (18)
[ppm] = 28.4 (1C, C-4), 37.3 (1C, C-3), 42.2 (1C, C-7), 53.6 (1C, C-
5), 54.6 (1C, C-9), 55.5 (1C, ArOCH3), 60.1 (1C, NCH2Ar), 67.9 (1C,
Under N2 17 (35 mg, 0.13 mmol) was dissolved in CH2Cl2
(20 mL). Under ice-cooling triethylamine (0.02 mL, 14 mg,
0.13 mmol) and benzoyl chloride (0.03 mL, 38 mg, 0.27 mmol)
were added and the mixture was stirred at rt for 16 h. Then a sat-
urated aqueous solution of NaHCO3 was added and the mixture
was extracted with CH2Cl2 (3ꢁ). The combined organic layers were
dried (Na2SO4), filtered and the solvent was removed in vacuo. The
residue was purified by fc (Ø = 1 cm, h = 15 cm, CH2Cl2/metha-
nol = 100/1, V = 5 mL, Rf = 0.13) to give 18 as a pale yellow oil, yield
30 mg (61%). C22H24N2O3 (364.5). Purity by HPLC: method 2b:
tR = 14.0 min, purity 95.5%; method 1: tR = 14.8 min, purity 96.2%.
C-1), 111.2 (2C, C-20phenyl, C-60phenyl), 114.1 (2C, C-304-methoxybenzyl
,
C-504-methoxybenzyl), 117.0 (1C, C-40phenyl), 129.6 (2C, C-30phenyl, C-
50phenyl), 129.9 (2C, C-204-methoxybenzyl, C-604-methoxybenzyl), 130.3 (1C,
C-104-methoxybenzyl), 148.2 (1C, C-10phenyl), 159.2 (1C, C-404-methoxybenzyl),
~
215.2 (1C, 2-C). IR (neat):
m
[cm-1] = 3037 (w,
m
C–H arom.), 2932 (m,
C@C arom.), 1463
mC–O), 836 (w, Cp-subst. arom.), 753
m
C–H aliph.), 1703 (s, mC@O ketone), 1598 (s)/1511 (s, m
(m, dC-H aliph.), 1240 (m)/1029 (m,
(m, Cmono-subst. arom.).
6.23. (ꢀ)-(1S,5R)-8-(4-Methoxybenzyl)-6-phenyl-6,8-diazabi
cyclo[3.2.2]nonan-2-one (ent-16)
½
a 2D0
ꢂ
= ꢀ77.8 (c = 0.22; CH2Cl2). MS (EI): m/z [%] = 364 (M, 59),
336 (M–CO, 21), 121 (CH2PhOCH3, 100), 105 (PhCO, 35). 1H NMR
(CDCl3): d [ppm] = 1.85–1.92 (m, 2ꢁ 0.3H, 4-Hb), 1.99––2.08 (m,
0.7H, 4-Ha), 2.13–2.23 (m, 0.7H, 4-Ha), 2.25–2.32 (m, 0.7H, 3-Ha),
2.36–2.44 (m, 0.3H, 3-Hb), 2.94–3.09 (m, 1ꢁ 0.7H + 3ꢁ 0.3H,
9-Ha, 3-Hb (1ꢁ 0.3H), 9-Hb (2ꢁ 0.3H)), 3.24–3.29 (m, 2ꢁ 0.7H, 1-
Ha, 9-Ha (1ꢁ 0.7H)), 3.34–3.49 (m, 2ꢁ 0.7H + 1ꢁ 0.3H, 3-Ha (1ꢁ
0.7H), 7-Ha (1ꢁ 0.7H), 1-Hb), 3.62–3.69 (m, 1H + 1ꢁ 0.7H, NCH2Ar,
7-Ha), 3.70–3.81 (m, 1H + 1ꢁ 0.3H, NCH2Ar, 7-Hb), 3.79 (s, 3H, Ar-
OCH3), 3.84–3.89 (m, 0.3H, 7-Hb), 4.07–4.12 (m, 0.3H, 5-Hb),
As described for the preparation of 16, the enantiomer ent-15
(70 mg, 0.17 mmol) was reacted with LiAlH4 (0.68 mL of a 1.0 M
solution in THF, 0.68 mmol) in THF (20 mL) and afterwards hydro-
lyzed with 1.0 M HCl (5 mL) to give ent-16 as a yellow oil, yield
43 mg (75%). C21H24N2O2 (336.4). Purity by HPLC: method 2a:
tR = 21.4 min, purity 98.4%; method 1: tR = 17.7 min, purity 99.1%.
½
a 2D0
ꢂ
= ꢀ36.2 (c = 0.35; CH2Cl2).
6.24. (ꢀ)-(1R,5S)-8-(4-Methoxybenzyl)-6,8-diazabicyclo[3.2.2]
4.84–4.87 (m, 0.7H, 5-Ha), 6.84 (d, J = 8.6 Hz, 2H, 30-H4-methoxybenzyl
50-H ), 7.18 (d, J = 8.6 Hz, 2H, 20-H4-methoxybenzyl 60-
,
nonan-2-one (17)
,
4-methoxybenzyl
H4-methoxybenzyl), 7.32–7.42 (m, 5H, NCOC6H5). Two rotamers exist
in the ratio 70: 30 (a: major rotamer; b: minor rotamer). 13C
NMR (CDCl3): d [ppm] = 24.3 (1C, C-4a), 27.3 (1C, C-4b), 34.6 (1C,
C-3b), 35.3 (1C, C-3a), 39.9 (1C, C-7b), 41.7 (1C, C-7a), 47.9 (1C,
C-5a), 50.7 (1C, C-5b), 51.4 (1C, C-9a), 52.0 (1C, C-9b), 53.4 (1C,
ArOCH3a,b), 57.8 (1C, NCH2Ara,b), 63.5 (1C, C-1b), 65.1 (1C, C-1a),
Under N2 a 1.0 M solution of LiAlH4 (1.30 mL, 1.30 mmol) was
added to an ice-cooled solution of 10 (109 mg, 0.33 mmol) in THF
(30 mL). The mixture was stirred at 0 °C for 10 min and then heated
to reflux for 16 h. Then 1.0 M HCl (10 mL) was added under ice-cool-
ing and the mixture was stirred at 0 °C for 10 min and then refluxed

1454
R. Holl et al. / Bioorg. Med. Chem. 17 (2009) 1445–1455
112.1 (2C, C-3a,b
,
C-5a,b 4-methoxybenzyl), 124.4 (2C,
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 lL 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 scintil-
lator was allowed to solidify at rt. The bound radioactivity trapped
on the filters was counted in the scintillation analyzer. The non-
0
0
4-methoxybenzyl
C-2b benzoyl, C-6b benzoyl), 124.9 (2C, C-2a benzoyl, C-6a benzoyl), 126.6
0
0
0
0
(2C, C-3a benzoyl, C-5a benzoyl), 126.8 (2C, C-3b benzoyl, C-5b benzoyl),
0
0
0
0
127.6(1C, C-1a,b 4-methoxybenzyl), 127.8 (2C, C-2a,b 4-methoxybenzyl, C-
0
0
6a,b 4-methoxybenzyl), 127.9 (1C, C-4b benzoyl), 128.0 (1C, C-4a benzoyl),
0
0
0
133.6 (1C, C-1a,b benzoyl), 157.1 (1C, C-4a,b 4-methoxybenzyl), 168.5
0
0
(1C, NCOC6H5a,b), 212.2 (1C, C-2a,b). Two rotamers exist in the ratio
~
m
70: 30 (a: major rotamer; b: minor rotamer). IR (neat):
2933 (m, mC–H aliph.), 1710 (s, mC@O ketone), 1628 (s, mC@O amide),
1511 (s, mC@C arom.), 1446 (m, dC-H aliph.), 1244 (s)/1030 (m,
[cm-1] =
specific binding was determined with 10 lM unlabeled (+) pentaz-
ocine. The Kd-value of the radioligand [3H]-(+)-pentazocine is
m
C–O),
2.9 nM.16
818 (m, Cp-subst. arom.), 702 (m, Cmono-subst. arom.).
7.4. Membrane preparation for the r2 assay (modified
6.27. (+)-(1S,5R)-6-Benzoyl-8-(4-methoxybenzyl)-6,8-diazabi
cyclo[3.2.2]nonan-2-one
according to Refs. 11,12)
Two rat livers were cut into smaller pieces and homogenized
with a potter (500–800 rpm, 10 up-and-down strokes) in 6 vol-
umes of cold 0.32 M sucrose. The suspension was centrifuged at
1200g for 10 min at 4 °C. The supernatant was separated and cen-
trifuged at 31,000g for 20 min at 4 °C. The pellet was resuspended
in buffer (50 mM Tris, pH 8.0) and incubated at rt for 30 min. After
the incubation, the suspension was centrifuged again at 31,000g
for 20 min at 4 °C. The final pellet was resuspended in buffer, the
protein concentration was determined according to the method
of Bradford15 by using bovine serum albumin as standard, and sub-
sequently the preparation was frozen (ꢀ80 °C) in 1.5 mL portions
containing about 2 mg protein/mL.
(ent-18)
As described for the preparation of 18, the enantiomer ent-17
(23 mg, 0.09 mmol) was reacted with benzoyl chloride (0.01 mL,
12 mg, 0.18 mmol) and triethylamine (0.01 mL, 9 mg, 0.09 mmol)
in CH2Cl2 (20 mL) to give ent-18 as a pale yellow oil, yield 15 mg
(47%). C22H24N2O3 (364.5). Purity by HPLC: method 2b:
tR = 14.6 min, purity 96.4%; method 1: tR = 14.8 min, purity 96.9%.
½
a 2D0
= +74.5 (c = 0.22; CH2Cl2).
****
ꢂ
7. Receptor binding studies
7.1. Materials and general procedures
7.5. Performing of the r2 assay (modified according to Refs.
11,12)
The guinea pig brains and rat livers were commercially avail-
able (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 rt before use. The filtration
was carried out with a MicroBeta FilterMate-96 Harvester (Per-
kin–Elmer). The scintillation analysis was performed using Melti-
lex (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 rt, the scintillation was
measured using a MicroBeta Trilux scintillation analyzer (Perkin–
Elmer). The counting efficiency was 20%.
The test was performed with the radioligand [3H]di-o-tolylgua-
nidine (50 Ci/mmol; ARC). The thawed membrane preparation
(about 100 lg of the protein) was incubated with various concen-
trations of test compounds, 3 nM [3H]-di-o-tolylguanidine, 500 nM
(+)-pentazocine, and buffer (50 mM Tris, pH 8.0) in a total volume
of 200
filtration through the presoaked filtermats using a cell harvester.
After each well was washed five times with 300 L of water, the fil-
lL for 180 min at rt. The incubation was terminated by rapid
l
termats 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 rt. The bound radioac-
tivity trapped on the filters was counted in the scintillation ana-
7.2. Membrane preparation for the r1 assay (modified
according to Refs. 11,12)
lyzer. The non-specific binding was determined with 10 lM
unlabeled di-o-tolylguanidine. The Kd-value of the radioligand
[3H]-di-o-tolylguanidine is 17.9 nM.17
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 su-
crose. The suspension was centrifuged at 1200 g for 10 min at 4 °C.
The supernatant was separated and centrifuged at 23,500g for
20 min at 4 °C. The pellet was resuspended in 5–6 volumes of buf-
fer (50 mM Tris, pH 7.4) and centrifuged again at 23,500 g (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 Bradford15 by using
bovine serum albumin as standard, and subsequently the prepara-
tion was frozen (ꢀ80 °C) in 1.5 mL portions containing about
1.5 mg protein/mL.
7.6. NMDA assay
The preparation of the receptor material and the assay were
performed according to a literature procedure.12
7.7. 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 com-
pounds 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.18 The Ki-values are
given as the mean value SEM from three independent experiments.
7.3. Performing of the r1 assay (modified according to Refs.
11,12)
The assay was performed with the radioligand [3H]-(+) pentaz-
ocine (42.5 Ci/mmol; Perkin–Elmer). The thawed membrane prep-
8. Cytotoxicity assay
aration (about 75
concentrations of test compounds, 2 nM [3H]-(+)-pentazocine,
and buffer (50 mM Tris, pH 7.4) in a total volume of 200 L for
lg of the protein) was incubated with various
All cell lines were obtained from the German Collection of Micro-
biology and Cell Culture (DSZK, Braunschweig, FRG). Cytotoxicity
l

R. Holl et al. / Bioorg. Med. Chem. 17 (2009) 1445–1455
1455
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with crystal violet as described in detail in Ref. 14 To determine the
IC50 values, five serially diluted stock solutions of test compound in
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between 10% and 90% were used to estimate the IC50 values, which
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Acknowledgements
This work was performed within the International Research
Training Group ‘Complex Functional Systems in Chemistry: Design,
Synthesis and Applications’ in collaboration with the University of
Nagoya. Financial support of this IRTG by the Deutsche Forschungs-
gemeinschaft is gratefully acknowledged.
References and notes
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17. Mach, R. H.; Smith, C. R.; Childers, S. R. Life Sci. 1995, 57, PL57.
K. C. Pharmacol. Rev. 1990, 42, 355.
18. Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099.