Relationships between the structure of 6-allyl-6,8-diazabicyclo[3.2.2]nonane derivatives and their σ receptor affinity and cytotoxic activity

Article abstract of DOI:10.1016/j.bmc.2008.11.043

A series of bridged piperazine derivatives was prepared and the affinity toward σ₁ and σ₂ receptors by means of radioligand binding assays as well as the inhibition of the growth of six human tumor cell lines was investigated. All possible stereoisomers of the 2-hydroxy, 2-methoxy, 2,2-dimethoxy, 2-oxo, and 2-unsubstituted 6,8-diazabicyclo[3.2.2]nonanes were prepared in a chiral pool synthesis starting with (S)- and (R)-glutamate. A Dieckmann analogous cyclization was the key step in the synthesis of the bicyclic framework. The configuration in position 2 was established by a diastereoselective LiBH₄ reduction and subsequent Mitsunobu inversion. Structure-affinity relationships demonstrate that substituents in position 2 decrease σ₁ receptor affinity which might be due to unfavorable interactions with the σ₁ receptor protein. Without a substituent in position 2 high σ₁ affinity was obtained (23a ((+)-(1S,5S)-6-allyl-8-(4-methoxybenzyl)-6,8-diazabicyclo[3.2.2]nonane): K_i = 11 nM). Experiments with six human tumor cell lines showed a weak but selective growth inhibition of the human small cell lung cancer cell line A-427 by the methyl ethers ent-16b (IC₅₀ = 18.9 μM), 21a (IC₅₀ = 16.4 μM), ent-21a (IC₅₀ = 20.4 μM), and 21b (IC₅₀ = 27.1 μM) and the unsubstituted compounds 23a and 23b (42% inhibition at 20 μM).

Full text of DOI:10.1016/j.bmc.2008.11.043

Bioorganic & Medicinal Chemistry 17 (2009) 777–793
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Relationships between the structure of 6-allyl-6,8-diazabicyclo[3.2.2]nonane
derivatives and their
r receptor affinity and cytotoxic activity
Ralph Holl a, Dirk Schepmann a, Renate Grünert b, Patrick J. Bednarski 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 bridged piperazine derivatives was prepared and the affinity toward r1 and r2 receptors by
means of radioligand binding assays as well as the inhibition of the growth of six human tumor cell
lines was investigated. All possible stereoisomers of the 2-hydroxy, 2-methoxy, 2,2-dimethoxy, 2-
oxo, and 2-unsubstituted 6,8-diazabicyclo[3.2.2]nonanes were prepared in a chiral pool synthesis start-
ing with (S)- and (R)-glutamate. A Dieckmann analogous cyclization was the key step in the synthesis
of the bicyclic framework. The configuration in position 2 was established by a diastereoselective LiBH4
reduction and subsequent Mitsunobu inversion. Structure–affinity relationships demonstrate that sub-
stituents in position 2 decrease r1 receptor affinity which might be due to unfavorable interactions
with the r1 receptor protein. Without a substituent in position 2 high r1 affinity was obtained (23a
((+)-(1S,5S)-6-allyl-8-(4-methoxybenzyl)-6,8-diazabicyclo[3.2.2]nonane): Ki = 11 nM). Experiments with
six human tumor cell lines showed a weak but selective growth inhibition of the human small cell lung
Received 20 August 2008
Revised 12 November 2008
Accepted 15 November 2008
Available online 24 November 2008
Keywords:
Sigma receptor ligands
6,8-Diazabicyclo[3.2.2]nonanes
Cytotoxicity
Structure–activity relationships
cancer cell line A-427 by the methyl ethers ent-16b (IC50 = 18.9 lM), 21a (IC50 = 16.4 lM), ent-21a
(IC50 = 20.4
lM), and 21b (IC50 = 27.1
lM) and the unsubstituted compounds 23a and 23b (42% inhibi-
tion at 20
lM).
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
Therefore, high affinity r1 and r2 receptor ligands could be
developed as atypical antipsychotics, antidepressants, and anti-
tumor agents.6
The ethylenediamine substructure substituted with different
residues at the nitrogen atoms represents a crucial pharmaco-
phoric element of several r1 receptor ligands.7 For example, the
piperazine derivative 1 (Fig. 1) which contains the ethylenedi-
amine substructure within the piperazine moiety represents a very
potent r1 receptor ligand (Ki = 0.47 nM).8
In order to reduce the conformational flexibility of the ethylene-
diamine substructure the piperazine ring of 1 was bridged to ob-
tain diazabicyclo[3.2.2]nonane derivatives like 2a (Ki = 6.5 nM)
and 2b (Ki = 26 nM) which also bind with high affinity and selectiv-
ity to the r1 receptor.9
In this paper, we wish to report on the synthesis, r1 and r2
receptor affinity and growth inhibition of human tumor cell lines
of a series of N-6-allyl substituted bridged piperazines 3 bearing
different substituents in position 2. The introduction of the allyl
Originally it was assumed that the
r receptor belongs to the
opioid receptor family.1 However, since most of the effects caused
by activation of
nist naloxone, this classification was discarded. Later it became
clear that receptors represent an own class of haloperidol-sensi-
r receptors are not sensitive to the opioid antago-
r
tive, non-opioid, non-phencyclidine receptors with a characteristic
binding profile. This family of receptors is subdivided into r1 and
r2 receptors.2
The human r1 receptor has been cloned,3 however cloning of
the r2 receptor has not yet been reported. The amino acid se-
quence of the r1 receptor displays no similarity to any other
known mammalian protein but shows 30% homology to the
yeast enzyme sterol D8
/D
7-isomerase. The cloned receptor is
postulated to possess two transmembrane domains4 and neuro-
steroids such as progesterone are discussed to be the endogenous
ligands for r1 receptors.5 Although the biochemical and physio-
logical role of
r
receptors as well as their mechanism of signal
group was stimulated by the prototypical r receptor ligand (+)-
transduction are not completely understood so far, they seem
to play a crucial role in pathophysiological processes such as
psychosis, depression, and uncontrolled cell proliferation.
SKF-10,047 which is also substituted by an allyl residue. Further-
more, the N-allyl group was envisaged in order to find out whether
this group is able to replace bioisosterically the benzyl group of 2a
and 2b. In addition, the electronic properties of the aromatic resi-
due in position 8 were changed by introduction of a second meth-
oxy group.
* Corresponding author. Fax: +49 251 8332144.
E-mail address: wusensch@uni-musenster.de (B. Wünsch).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.11.043

778
R. Holl et al. / Bioorg. Med. Chem. 17 (2009) 777–793
allylated with allyl bromide and NaHMDS to give 7a and 7b
(Scheme 1).
The desired 6,8-diazabicyclo[3.2.2]nonane scaffold was finally
obtained by a Dieckmann analogous cyclization using LiHMDS
and trapping of the resulting intermediates with chlorotrimethyl-
silane to form stereoselectively the mixed methyl/silyl acetals 8a
and 8b, which were carefully hydrolyzed to give the bicyclic ke-
tones 9a and 9b.
The bicyclic ketone 9a was reacted with methanol in the pres-
ence of trimethyl orthoformate and p-toluenesulfonic acid to give
the dimethyl acetal 10. Both lactam groups of 10 were subse-
quently reduced with LiAlH4 to yield the basic piperazine deriva-
tive 11. After hydrolysis of the dimethyl acetal 11 the bicyclic
ketone 12 was obtained (Scheme 2).
H3CO
H3CO
N
N
1
6
N
8
N
1
RO
2
2a: OR = OH
2b: OR = OCH3
A diastereoselective reduction of the bicyclic ketones 9a and 9b
with LiBH4 in THF at ꢁ78 °C led to the (2R)-configured alcohols 13a
and 13b (de > 90%).9 The alcohols 13a and 13b were transformed
into the corresponding methyl ethers 14a and 14b with methyl io-
dide and NaH. Reaction of the bicyclic alcohols 13 and methyl
ethers 14 with LiAlH4 led to reduction of both lactam carbonyl
moieties to give the bridged piperazines 15 and 16 with a hydroxy
and a methoxy group in position 2, respectively (Scheme 3).
The (2S)-configured diastereomers of 15 and 16 were obtained
after inversion of the (2R)-configured alcohols 13 by a Mitsunobu
reaction.11 For this purpose 13a and 13b were reacted with 4-
nitrobenzoic acid, diisopropyl azodicarboxylate (DIAD), and PPh3
to yield the (2S)-configured 4-nitrobenzoates 17a and 17b. Cleav-
age of the 4-nitrobenzoates 17a and 17b with K2CO3 in methanol
provided the inverted alcohols 18a and 18b (Scheme 4). Methyla-
tion of the alcohols 18 with methyl iodide and NaH gave the (2S)-
configured methyl ethers 19. Reduction of the alcohols 18 and
methyl ethers 19 with LiAlH4 resulted in the basic piperazine
derivatives with a hydroxy (20a and 20b) and a methoxy moiety
(21a and 21b) in position 2.
H3CO
6
N
5
8
N
1
X
R
2
3
3
Figure 1. Lead compounds 1 and 2 compared with the structure of the planned
ligands 3.
2. Chemistry
The synthesis of the bicyclic ketone 9a was performed accord-
ing to a literature procedure.10 Its 2,4-dimethoxybenzyl derivative
9b was synthesized in an analogous manner. In this six step syn-
thesis the proteinogenic amino acid (S)-glutamate was first esteri-
fied with methanol in the presence of trimethylsilyl chloride to
give the hydrochloride of dimethyl glutamate 4ꢀHCl. Acylation of
4 with chloroacetyl chloride yielded the chloroacetamide 5, which
was subsequently reacted with 4-methoxybenzylamine and 2,4-
dimethoxybenzylamine to provide the monocyclic piperazinedi-
ones 6a and 6b, respectively. The bislactams 6a and 6b were then
In order to obtain compounds 23a and 23b without a substitu-
ent in position 2 the (2R)-configured alcohols 13a and 13b were
transformed into the mesylates 22a and 22b with methanesulfonyl
chloride in the presence of 4-(dimethylamino)pyridine (DMAP)
R
N
OCH3
HO
O
H3CO
O
Cl
O
OCH3
(a)
(b)
(c)
OH
OCH3
OCH3
O
H2N
Cl H3N
O
N
H
H
H
H
O
O
O
OCH3
O
N
H
5
(S)-glutamate
4 HCl
H
O
6a: R = H
6b: R = OCH3
OCH3
OCH3
R
OCH3
O
O
R
R
N
N
O
(d)
(e)
(f)
N
N
N
H3CO
N
O
OCH3
O
H
O
O
O
OSi(CH3)3
7a: R = H
7b: R = OCH3
8a: R = H
9a: R = H
9b: R = OCH3
8b: R = OCH3
Scheme 1. Reagents and conditions: (a) Me3SiCl, MeOH, rt, 16 h, 98%; (b) ClCH2COCl, CH2Cl2, NaHCO3, rt, 16 h, 66%; (c) 4-methoxybenzylamine, NEt3, H3CCN, Bu4NI, reflux,
48 h, 83% (6a) or 2,4-dimethoxybenzylamine, NEt3, H3CCN, Bu4NI, reflux, 48 h, 79% (6b); (d) NaHMDS, THF, allyl bromide, Bu4NI, ꢁ78 °C, 1 h, rt, 2 h, 53% (7a), 59% (7b); (e)
LiHMDS, THF, ꢁ78 °C, 40 min and then M3SiCl, 82% (8a), 79% (8b); (f) 2 M HCl, THF, rt, 16 h, 80% (9a), 85% (9b). The compounds of the a series have been published in Ref. 10.

R. Holl et al. / Bioorg. Med. Chem. 17 (2009) 777–793
779
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.9,12,14
O
PMB
N
(a)
(b)
N
9a
O
H3CO
OCH3
10
N
N
(c)
N
N
PMB
PMB
H3CO
O
3.2. Results and discussion
OCH3
11
12
In Table 1, the r1 and r2 receptor affinity of the synthesized
compounds is summarized. Compared to their N-6-benzyl counter-
parts 2a and 2b, the bicyclic alcohol 15a and methyl ether 16a with
an allyl group in position 6 show a more than 100-fold reduced r1
receptor affinity. Obviously the allyl moiety is not able to replace
bioisosterically the benzyl group. ent-15a, which is the most po-
tent r1 ligand (Ki = 343 nM) within the series of bicyclic alcohols
and methyl ethers, displays a considerable selectivity toward the
(PMB: p-methoxybenzyl)
Scheme 2. Reagents and conditions: (a) HC(OCH3)3, MeOH, p-toluenesulfonic acid,
reflux, 16 h, 98%; (b) LiAlH4, THF, reflux, 16 h, 20%; (c) 1 M HCl, 3 h, 50% (over 2
steps).
and N,N-diisopropylethylamine (DIEA). The reaction of the mesy-
lates 22a and 22b with LiAlH4 led in addition to the reduction of
the lactam carbonyl moieties to the substitution of the mesylate
moieties by hydride to give the unsubstituted bridged piperazines
23a and 23b (Scheme 5).
The enantiomers of the dimethyl acetal 11, ketone 12, alcohols
15 and 20, methyl ethers 16 and 21, and 2-unsubstituted com-
pounds 23 were prepared in the same manner, starting from (R)-
glutamate. Therefore, all possible stereoisomers of the described
compounds were available for pharmacological evaluation,
respectively.
r2 receptor (r1:r2 ꢂ 4).
The dimethyl acetals 11 and ent-11 as well as the bicyclic ke-
tones 12 and ent-12 also interact with low affinity with r1 recep-
tors. However, removal of the substituent in position 2 (23) led to a
dramatic increase of the r1 affinity. In particular, the (S,S)-config-
ured 4-methoxybenzyl derivative 23a displays an affinity toward
the
r1 receptor in the low nanomolar range (Ki = 11 nM). The eudi-
smic ratio (23a:ent-23a) is 25 and the selectivity against the r2
receptor is about 20.
Introduction of a second methoxy moiety in position 2 of the
4-methoxybenzyl residue leads generally to
a decreased r1
3. Pharmacological evaluation
3.1. Receptor binding studies
affinity. Typical pairs are ent-15a (Ki = 343 nM) and ent-15b
(Ki = 9670 nM) as well as 23a (Ki = 11 nM) and 23b
(Ki = 267 nM). In both examples the r1 affinity is 20- to 30-fold
reduced by the second methoxy group. Obviously, increase of
electron density of the N-8 substituent is not tolerated by the
r1 receptor protein.
The
r receptor affinities of the synthesized compounds were
determined in competition experiments with radioligands.
9a,b
(a)
OCH3
OCH3
O
O
R
R
(b)
N
N
N
N
O
O
HO
H3CO
13a: R = H
14a: R = H
13b: R = OCH3
14b: R = OCH3
(c)
(c)
R
R
H3CO
H3CO
N
N
N
N
HO
H3CO
16a: R = H
16b: R = OCH3
15a: R = H
15b: R = OCH3
Scheme 3. Reagents and conditions: (a) LiBH4, THF, ꢁ78 °C, 92% (13a), 92% (13b); (b) NaH, MeI, THF, rt, 16 h, 89% (14a), 77% (14b); (c) LiAlH4, THF, reflux, 16 h, 58% (15a), 23%
(15b), 27% (16a), 34% (16b).

780
R. Holl et al. / Bioorg. Med. Chem. 17 (2009) 777–793
OCH3
O
R
(a)
(b)
N
13a,b
N
O
O
O
17a: R = H
O2N
17b: R = OCH3
OCH3
OCH3
O
O
R
R
(c)
N
N
N
N
O
O
HO
H3CO
18a: R = H
19a: R = H
18b: R = OCH3
19b: R = OCH3
(d)
(d)
R
R
H3CO
H3CO
N
N
N
N
HO
H3CO
21a: R = H
21b: R = OCH3
20a: R = H
20b: R = OCH3
Scheme 4. Reagents and conditions: (a) DIAD, PPh3, p-nitrobenzoic acid, THF, rt, 6 h, 87% (17a), 78% (17b); (b) MeOH, K2CO3, rt, 16 h, 90% (18a), 92% (18b); (c) NaH, MeI, THF,
rt, 16 h, 81% (19a), 97% (19b); (d) LiAlH4, THF, reflux, 16 h, 35% (20a), 20% (20b), 55% (21a), 25% (21b).
13a,b
(a)
OCH3
O
R
H3CO
R
N
N
(b)
N
N
O
H3C
O
O
O
2
S
22a: R = H
23a: R = H
23b: R = OCH3
22b: R = OCH3
Scheme 5. Reagents and conditions: (a) H3CSO2Cl, DMAP, DIEA, CH2Cl2, rt, 16 h, 96% (22a), 88% (22b); (b) LiAlH4, THF, reflux, 16 h, 46% (23a), 36% (23b).
A hypothetical explanation for the surprisingly high
r
1 receptor
pared to the lead compounds 2a and 2b with an N-6-benzyl moi-
ety. Obviously the allyl group in position 6 is too small to interact
fully with the secondary hydrophobic region (A) of the r1
receptor.
On the other hand, when N-8 binds to the proton donor site of
the receptor protein (Fig. 2a) unfavorable interactions between the
substituent in position 2 and the receptor protein prevent the
affinity of 23a can be deduced from the pharmacophore model,
which has been proposed by Glennon et al.13 According to this
model a basic nitrogen atom with two hydrophobic residues in dif-
ferent distances from the nitrogen atom are required for a high r1
receptor affinity (Fig. 2).
The synthesized bridged piperazine derivatives can bind either
with N-6 or N-8 to the postulated proton donor site of the recep-
tor protein. Fig. 2b shows the situation for the 6,8-diazabicy-
clo[3.2.2]nonanes derived from (S)-glutamate with N-6 binding
to the proton donor site. Most of the bicyclic compounds bearing
an allyl moiety in position 6 show rather low r1 affinity com-
compounds from binding to the
postulated unfavorable interaction between the substituent in po-
sition 2 and the 1 receptor is supported by the high affinity of the
r1 receptor with high affinity. This
r
bridged piperazine 23a (Ki = 11 nM) which does not bear a substi-
tuent in position 2.

R. Holl et al. / Bioorg. Med. Chem. 17 (2009) 777–793
781
Table 1
1 and
reference compounds
which is able to interact with the secondary hydrophobic region of
the r1 receptor.
Generally, the r2 receptor affinity of the bicyclic compounds is
rather low. However, some compounds like the methyl ethers 16a
and 16b show a small preference for the r2 receptor over the r1
receptor.
r
r2 receptor affinity of the 6,8-diazabicyclo[3.2.2]nonane derivatives and some
Compound
Configuration
C-2 Substituents
Ki SEM [nM]
r1 affinity r2 affinity
6.5 0.7
26
2a
2b
11
(R,R,S)
(R,R,S)
(R,S)
H, OH
H, OCH3
OCH3, OCH3
OCH3, OCH3
@O
806
Since some
r receptor ligands show considerable affinity to-
9
573
ward the NMDA receptor,14 the affinity of the bridged pipera-
zines toward the phencyclidine binding site of the NMDA
receptor (pig brain cortex membrane preparations, radioligand
[3H]-MK801) was also determined. However, at a concentration
1250
588
1600
548
2240
343
9140
9670
0%a
ent-11
12
(S,R)
(R,S)
19%a
9100
1900
4780
1210
1460
5170
1690
1550
2100
13,500
ent-12
15a
ent-15a
15b
ent-15b
16a
ent-16a
16b
ent-16b
20a
ent-20a
20b
ent-20b
21a
ent-21a
21b
(S,R)
@O
(R,R,S)
(S,S,R)
(R,R,S)
(S,S,R)
(R,R,S)
(S,S,R)
(R,R,S)
(S,S,R)
(R,S,S)
(S,R,R)
(R,S,S)
(S,R,R)
(R,S,S)
(S,R,R)
(R,S,S)
(S,R,R)
(S,S)
H, OH
H, OH
H, OH
H, OH
H, OCH3
H, OCH3
H, OCH3
H, OCH3
H, OH
H, OH
H, OH
H, OH
H, OCH3
H, OCH3
H, OCH3
H, OCH3
H, H
of 1 lM the replacement of the radioligand by the synthesized
compounds was always lower than 25% indicating a very low
affinity toward the phencyclidine binding site of the NMDA
receptor.
4030
16,100
51%a
37%a
a
3.3. Inhibition of cell growth of human tumor cell lines
23,100 0
17,200
0%a
%
5%a
6%a
In the literature, it has been reported that some human tumor
22%a
3500
8280
5740
35%a
2100
22,400
7660
36%a
203
cell lines overexpress r1 and r2 receptors.15 Furthermore, some
r
2 agonists and r1 antagonists showed antiproliferative and cyto-
toxic effects in tumor cell lines.16 Therefore, the antiproliferative
effects of the synthesized compounds were investigated in a panel
of six human tumor cell lines, including the cell lines 5637 (bladder
cancer), RT-4 (bladder cancer), A-427 (small cell lung cancer),
LCLC-103H (large cell lung cancer), MCF-7 (breast cancer), and
DAN-G (pancreas cancer).
ent-21b
23a
10,600
11 4.7
27
ent-23a
23b
(R,R)
(S,S)
H, H
335
H, H
H, H
(S,S,S)
—
—
267 32
561 167
4.2 1.1
61 18
3.9 1.5
2340
1850
—
42 17
78 2.3
ent-23b
(+)-Pentazocine
Ditolylguanidine
Haloperidol
(R,R)
—
—
—
In the primary screening the tumor cells were incubated with
a 20 lM solution of the test compound 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.17 In
Table 2, the cell growth inhibiting activity of the test compounds
is given as part of living cells (in %) in relation to a control with-
out test compound.
a
Inhibition of radioligand binding at a test compound concentration of 1 lM.
Therefore, we conclude that potent
diazabicyclo[3.2.2]nonanes are unsubstituted in position 2 (23a) or
bear a relatively large lipophilic substituent in position 6 (2a, 2b),
r1 ligands result when 6,8-
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
2a)
2b)
A
N
B
A
N
B
OCH3
H3CO
N
N
N
N
R
R
receptor
protein
receptor
protein
Figure 2. Pharmacophore model of r1 receptor ligands (modified according to Ref. 13) and hypothetical binding of the bridged piperazines to the r1 receptor.

782
R. Holl et al. / Bioorg. Med. Chem. 17 (2009) 777–793
Table 2
4. Conclusion
Cell growth inhibitory activity (% of untreated control) of the 6,8-diazabicy-
clo[3.2.2]nonane derivatives in six human cancer cell linesa
6,8-Diazabicyclo[3.2.2]nonanes bearing a N-6-allyl moiety with-
out a substituent in position 2 interact with high affinity (23a:
Ki = 11 nM), high enantioselectivity (eudismic ratio 23a:ent-
Configuration 5637b RT-4c A-427d LCLC-103He MCF-7f DAN-Gg
2a9
2b9
11
ent-11
12
ent-12
15a
(1R,2R,5S)
(1R,2R,5S)
(R,S)
(S,R)
(R,S)
88
68
83
109
38
19
100
104
100
98
96
86
93
61
86
112
80
76
103
107
103
100
100
103
88
46
-8
94
72
101
104
81
91
21
62
84
72
65
92
99
86
100
85
91
61
79
93
91
84
102
80
84
84
87
65
74
61
67
—
—
23a = 25) and high selectivity over
r2 receptors (23a: Ki = 203 nM)
37
111
60
50
81
90
78
77
67
79
60
49
81
91
79
87
45
48
36
62
42
55
42
77
102
109
75
69
101
100
92
94
92
93
82
84
102
93
87
91
95
95
101
85
92
87
83
82
and NMDA receptors with r1 receptors. Substituents in position 2
such as a hydroxy, an oxo or one or two methoxy groups decrease
r1 receptor affinity, since these substituents lead to unfavorable
(S,R)
(R,R,S)
64
101
111
103
105
103
107
90
interactions with the
such a substituent is present in position 2 a large lipophilic moiety
like a benzyl group is required in position 6 to achieve high 1 recep-
r1 receptor protein (compare Fig. 2a). When
ent-15a (S,S,R)
15b (R,R,S)
ent-15b (S,S,R)
16a (R,R,S)
ent-16a (S,S,R)
16b (R,R,S)
ent-16b (S,S,R)
20a (R,S,S)
ent-20a (S,R,R)
20b (R,S,S)
ent-20b (S,R,R)
21a (R,S,S)
ent-21a (S,R,R)
21b (R,S,S)
ent-21b (S,R,R)
23a (S,S)
ent-23a (R,R)
23b (S,S)
r
tor affinity (compounds 2a, 2b). This observation is explained by a
reversed binding mode (compare Fig. 2b). Altogether, the allyl moi-
ety is too small to replace bioisosterically the N-benzyl group and to
interact efficiently with the secondary hydrophobic region.
Furthermore, in a screening some of the synthesized bridged
piperazine derivatives show a moderate cell growth inhibition of
the human small cell lung cancer cell line A-427. Since the r1
receptor affinity of the new N-allyl derivatives is significantly low-
er than the r1 affinity of the corresponding N-benzyl derivatives
2a and 2b, the growth inhibition of the human tumor cell lines,
in particular the A-427 cell line, is also considerably reduced.
61
63
87
98
103
101
100
105
95
81
102
84
87
90
73
75
109
108
103
109
89
94
68
79
65
107
106
103
103
99
107
78
86
91
82
74
59
71
66
65
ent-23b (R,R)
5. Experimental
a
Relative cell growth (%) in relation to untreated control of the tumor cell lines
after 96 h exposure to substance at 20
experiments.
lM. Results are averages of two independent
5.1. Chemistry: General
b
Bladder cancer.
Bladder cancer.
Small cell lung cancer.
Large cell lung cancer.
c
Unless otherwise noted, moisture sensitive reactions were con-
ducted under dry nitrogen. THF was dried with sodium/benzophe-
none and was freshly distilled before use. Thin layer
chromatography (TLC): Silica gel 60 F254 plates (Merck). Flash col-
d
e
f
Breast cancer.
g
Pancreas Cancer.
umn chromatography (FC): Silica gel 60, 40–64 lM (Merck);
parentheses include: diameter of the column, length of the silica
gel bed, eluent, fraction size, Rf value. Melting point: Melting point
apparatus SMP 3 (Stuart Scientific), uncorrected. 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 spectrometer (Varian); d in ppm related to tetramethylsil-
ane; coupling constants are given with 0.5 Hz resolution. HPLC:
method 1: Merck Hitachi Equipment; UV detector: L-7400; auto-
sampler:L-7200; pump: L-7100; degasser: L-7614; column:
3.4. Results and discussion
TheThe resulting data are summarized in Table 2. Compared
with their N-6-benzyl analogues,9 the alcohols 15, 20 and
methyl ethers 16, 21 led to a considerably decreased inhibition
of cell growth. The growth of the cell line 5637 was selectively
inhibited by the bicyclic ketones 12 and ent-12. On the other
hand the bicyclic acetal 11, the methyl ethers ent-16b, 21a,
ent-21a, and 21b as well as the 2-unsubstituted compounds
23a and 23b show cell growth inhibiting activity selectively
for the small cell lung cancer cell line A-427. The growth of
the other cell lines was reduced to a lower extent by these
compounds. Exemplarily the IC50-values for the methyl ethers
LiChrospherÒ 60 RP-select B (5
l
M); LiCroCARTÒ 250-4 mm car-
L; detec-
tridge; flow rate: 1.000 mL/min; injection volume: 5.0
l
tion at k = 210 nM; solvents: A: water with 0.05% (v/v)
trifluoroacetic acid; B: acetonitrile with 0.05% (v/v) trifluoroacetic
acid: gradient 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 Mon-
itor (Knauer); data acquisition: D-2500 Chromato-Integrator
ent-16b (IC50 = 18.9 7.0
lM), 21a (IC50 = 16.4 2.0
lM), ent-
M) were re-
21a (IC50 = 20.4 12.6 M), and 21b (IC50 = 27.1
l
l
corded. The low potency in this assay is correlating with low
r1 and r2 receptor affinity. However, the selective growth inhi-
bition of only few cell lines (5637, A-427) by few compounds
clearly indicates that the cell growth inhibiting activity is not
simply due to an unspecific toxicity, but is mediated by a spe-
cific mechanism.
The unsubstituted bridged piperazine 23a with the highest
affinity toward r1 and r2 receptors does not show signifi-
cantly higher cell growth inhibiting activity than its enantio-
mer ent-23a and its methoxy derivative 23b, which possess
(Merck Hitachi); injection volume: 20.0
l
L; stop time: 2ꢃ tR; (a)
column: LiChroCARTÒ 250-4 with SuperspherÒ 100 RP-18; flow
rate: 1.0 mL/min; detection: wavelength: 235 nM; solvent: metha-
nol/water = 60:40 + 0.1% triethylamine; (b) column: LiChroCARTÒ
250-4 with SuperspherÒ 100 RP-18; flow rate: 0.6 mL/min; detec-
tion: wavelength: 235 nM; solvent: methanol/water = 85:15 + 0.1%
triethylamine; (c) column LiChroCARTÒ 250-4 with SuperspherÒ
100 RP-18; flow rate: 0.6 mL/min; detection: wavelength:
235 nM; solvent: methanol/water = 75:25 + 0.1% triethylamine;
(d) column: LiChroCARTÒ 250-4 with SuperspherÒ 100 RP-18; flow
rate: 0.6 mL/min; detection: wavelength: 235 nM; solvent: metha-
nol/water = 65:35 + 0.1% triethylamine; (e) column: LiChroCARTÒ
considerably lower
r affinity. This might be due to the differ-
ent (partial) r1 agonistic and/or r2 antagonistic properties of
the compounds or the different penetration of the compounds
through the cell membrane to reach their site of action at the
r1 receptor.
250-4 with LiChrospherÒ RP-8e (5
lM); flow rate: 1.0 mL/min;

R. Holl et al. / Bioorg. Med. Chem. 17 (2009) 777–793
783
detection: wavelength: 254 nM; solvent: acetonitrile/water =
50:50 + 0.1% triethylamine; (f) column: LiChroCARTÒ 250-4 with
moved in vacuo. The residue was purified by FC (Ø = 3 cm,
h = 15 cm, cyclohexane/ethyl acetate = 1:2, 20 mL, Rf = 0.28) to give
7b as a yellow oil, yield 340 mg (59%). C20H26N2O6 (390.4). Purity
LiChrospherÒ RP-8e (5
lM); flow rate: 1.0 mL/min; detection:
wavelength: 254 nM; solvent: acetonitrile/water = 40:60 + 0.1%
by HPLC: method 1: tR = 17.6 min, purity 96.1%. ½a D20
ꢄ
+30.9 (c
triethylamine; (g) column: LiChroCARTÒ 250-4 with LiChrospherÒ
0.69; CH2Cl2). MS (EI): m/z [%] = 390 (M, 16), 151 (2,4-dimethoxy-
RP-8e (5
l
M); flow rate: 1.0 mL/min; detection: wavelength:
benzyl, 100). 1H NMR (CDCl3):
d [ppm] = 1.95–2.05 (m, 1H,
254 nM; solvent: acetonitrile/water = 35:65 + 0.1% triethylamine;
CH2CH2CO2CH3), 2.18–2.27 (m, 1H, CH2CH2CO2CH3), 2.32–2.49
(m, 2H, CH2CH2CO2CH3), 3.49 (dd, J = 15.7/7.8 Hz, 1H,
NCH2CH@CH2), 3.67 (s, 3H, CO2CH3), 3.80 (s, 6H, ArOCH3), 3.89 (s,
2H, O@CCH2N), 3.99 (dd, J = 8.6/3.9 Hz, 1H, CHCH2CH2CO2CH3),
4.37 (d, J = 14.1 Hz, 1H, NCH2Ar), 4.52–4.59 (m, 1H, NCH2CH@CH2),
4.71 (d, J = 14.1 Hz, 1H, NCH2Ar), 5.20–5.26 (m, 2H, NCH2CH@
CH2), 5.67–5.78 (m, 1H, NCH2CH@CH2), 6.43–6.47 (m, 2H, 30-
H2,4-dimethoxybenzyl, 50-H2,4-dimethoxybenzyl), 7.19 (d, J = 8.6 Hz, 1H, 60-
~
(h) column: HibarÒ RT 250-4 with LiChrospherÒ 100 RP-18
(5
solvent: acetonitrile/water = 60:40 + 0.1% triethylamine; (i) col-
umn: HibarÒ RT 250-4 with LiChrospherÒ 100 RP-18 (5
M); flow
lM); flow rate: 1.0 mL/min; detection: wavelength: 254 nM;
l
rate: 1.0 mL/min; detection: wavelength: 254 nM; solvent: aceto-
nitrile/water = 50:50 + 0.1% triethylamine.
5.2. (ꢁ)-Methyl (S)-3-[4-(2,4-dimethoxybenzyl)-3,6-
H2,4-dimethoxybenzyl). IR (neat):
m
[cmꢁ1] = 3080 (w,
m
CAH arom.), 2951
amide), 1612 (m)/
mC@O
dioxopiperazin-2-yl]propanoate (6b)
(w, mCAH aliph.), 1733 (m, mC@O
ester), 1658 (s,
1587 (m)/1508 (m, mC@ C arom.), 1464 (m, dCA H aliph.), 1208 (m)/
1157 (m)/1032 (m, CAO), 835 (w, Ctri-subst. arom.).
The chloroacetamide 5 (1.51 g, 6.0 mmol) was dissolved in ace-
tonitrile (50 mL) and 2,4-dimethoxybenzylamine hydrochloride
(1.83 g, 9.0 mmol), triethylamine (2.53 mL, 1.84 g, 18.2 mmol)
and tetrabutylammonium iodide (222 mg, 0.60 mmol) were added.
The mixture was heated to reflux for 48 h. Then 2/3 of the solvent
was removed in vacuo. The residue was filtered and poured into
0.5 M HCl. The mixture was extracted with CH2Cl2 (3ꢃ) and the
combined organic layers were dried (Na2SO4), filtered and concen-
trated in vacuo. The residue was purified by FC (Ø = 6 cm,
h = 15 cm, petroleum ether/acetone = 1:1, V = 30 mL, Rf = 0.27) to
give 6b as a colorless solid, mp 123 °C, yield 1.67 g (79%).
C17H22N2O6 (350.4). Purity by HPLC: method 1: tR = 14.6 min, purity
m
5.5. (-)-Methyl (R)-3-[1-allyl-4-(2,4-dimethoxybenzyl)-3,6-
dioxopiperazin-2-yl]propanoate (ent-7b)
As described for the preparation of 7b, the enantiomer ent-6
(600 mg, 1.71 mmol), tetrabutylammonium iodide (127 mg,
0.34 mmol), 2 M solution of sodium hexamethyldisilazane in THF
(0.94 mL, 1.88 mmol) and allyl bromide (0.75 mL, 1.04 g,
8.56 mmol) were reacted in THF (30 mL) to give ent-7b as a yellow
oil, yield 380 mg (57%). C20H26N2O6 (390.4). Purity by HPLC: meth-
od 1: tR = 17.5 min, purity 98.1%. ½a D20
ꢁ32.1 (c 0.68; CH2Cl2).
ꢄ
98.1%. ½a 2D0
ꢁ4.1 (c 0.64; CH2Cl2). MS (EI): m/z [%] = 350 (M, 25),
ꢄ
151 (2,4-dimethoxybenzyl, 100). 1H NMR (CDCl3): d [ppm] = 2.15–
2.21 (m, 2H, CH2CH2CO2CH3), 2.39–2.53 (m, 2H, CH2CH2CO2CH3),
3.67 (s, 3H, CO2CH3), 3.80 (s, 3H, ArOCH3), 3.81 (s, 3H, ArOCH3),
3.89 (s, 2H, O@CCH2N), 4.05–4.10 (m, 1H, CHCH2CH2CO2CH3), 4.47
(d, J = 14.1 Hz, 1H, NCH2Ar), 4.63 (d, J = 14.1 Hz, 1H, NCH2Ar),
6.43–6.47 (m, 2H, 30-H2,4-dimethoxybenzyl, 50-H2,4-dimethoxybenzyl), 6.70
(s, 1H, NH), 7.20 (d, J = 8.6 Hz, 1H, 60-H2,4-dimethoxybenzyl). IR (neat):
~
5.6. (+)-(1S,2R,5S)-6-Allyl-8-(2,4-dimethoxybenzyl)-2-methoxy-
2-(trimethylsiloxy)-6,8-diazabicyclo[3.2.2]nonane-7,9-dione
(8b)
Under N2 7b (5.76 g, 14.8 mmol) was dissolved in THF (150 mL)
and cooled to ꢁ78 °C. Then a 1 M solution of lithium hexamethyl-
disilazane in THF (22.1 mL, 22.1 mmol) was added dropwise. After
stirring at ꢁ78 °C for 40 min, chlorotrimethylsilane (6.79 mL,
5.77 g, 53.1 mmol) was slowly added. The mixture was stirred at
ꢁ78 °C for 1 h and at rt for 2 h. Then a saturated aqueous solution
of NaHCO3 was added and the mixture was extracted with CH2Cl2
(3ꢃ). The combined organic layers were dried (Na2SO4), filtered
and the solvent was removed in vacuo. The residue was purified
by FC (Ø = 8 cm, h = 15 cm, cyclohexane/ethyl acetate = 1:1,
V = 30 mL, Rf = 0.30) to give 8b as a yellow oil, yield 5.41 g (79%).
m
m
[cmꢁ1] = 3209 (m br,
CAH aliph.), 1729 (m,
C@C arom.), 1456 (m, dCAH aliph.), 1216 (m)/1161 (m)/
CAO), 818 (w, Ctri-subst. arom.).
mNAH), 3075 (w, mCA H arom.), 2951 (m,
C@O ester), 1673 (s, mC@O amide), 1615 (m)/1585
m
(m)/1510 (m,
1033 (m,
m
m
5.3. ( + )-Methyl (R)-3-[4-(2,4-dimethoxybenzyl)-3,6-
dioxopiperazin-2-yl]propanoate (ent-6b)
As described for the preparation of 6b, the enantiomer ent-5
(1.07 g, 4.25 mmol) was reacted with 2,4-dimethoxybenzylamine
hydrochloride (1.30 g, 6.38 mmol), triethylamine (1.95 mL, 1.42 g,
14.0 mmol) and tetrabutylammonium iodide (157 mg, 0.43 mmol)
in acetonitrile (40 mL) to give ent-6b as a colorless solid, mp
125 °C, yield 750 mg (50%). C17H22N2O6 (350.4). Purity by HPLC:
C23H34N2O6Si (462.6). ½a D20
ꢄ
+28.7 (c 0.71; CH2Cl2). MS (EI): m/z
[%] = 462 (M, 25), 311 (Mꢁ2,4-dimethoxybenzyl, 12), 151 (2,4-
dimethoxybenzyl, 100). 1H NMR (CDCl3): d [ppm] = 0.19 (s, 9H,
OSi(CH3)3), 1.74–1.83 (m, 1H, 4-H), 1.87–1.98 (m, 2H, 3-H), 2.00–
2.09 (m, 1H, 4-H), 3.22 (s, 3H, OCH3), 3.78 (s, 6H, 2ꢃ ArOCH3),
3.81 (dd, J = 5.5/2.3 Hz, 1H, 5-H), 3.93 (d, J = 6.3 Hz, 2H,
NCH2CH@CH2), 4.10 (d, J = 14.9 Hz, 1H, NCH2Ar), 4.13 (s, 1H, 1-
H), 4.96 (d, J = 14.9 Hz, 1H, NCH2Ar), 5.12–5.19 (m, 2H,
NCH2CH@CH2), 5.71 (ddt, J = 17.2/10.2/6.3 Hz, 1H, NCH2CH@CH2),
6.39–6.44 (m, 2H, 30-H2,4-dimethoxybenzyl, 50-H2,4-dimethoxybenzyl), 7.14
[cmꢁ1] = 2956
~
m
method 1: tR = 14.6 min, purity 99.5%. ½a D20
ꢄ
+4.6 (c 0.57; CH2Cl2).
5.4. (+)-Methyl (S)-3-[1-allyl-4-(2,4-dimethoxybenzyl)-3,6-
dioxopiperazin-2-yl]propanoate (7b)
(d, J = 8.6 Hz, 1H, 60-H2,4-dimethoxybenzyl). IR (neat):
Under N2 a solution of 6b (520 mg, 1.48 mmol) and tetrabutyl-
ammonium iodide (110 mg, 0.30 mmol) in THF (60 mL) was cooled
to ꢁ78 °C. Then a 2.0 M solution of sodium hexamethyldisilazane
in THF (0.82 mL, 1.63 mmol) was added dropwise. After stirring
the mixture at ꢁ78 °C for 40 min, allyl bromide (0.65 mL,
898 mg, 7.42 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 ambient temperature for 2 h, water was added
and the mixture was extracted with CH2Cl2 (3ꢃ). The combined or-
ganic layers were dried (Na2SO4), filtered and the solvent was re-
(m,
mC@C arom.), 1453 (m, dCA H aliph.), 1249 (m)/1207 (m,
(m, SiAO), 872 (m, Ctri-subst. arom.).
m
CAH aliph.), 1683 (s, mC@O amide), 1612 (m)/1588 (m)/1507 (m,
mCAO), 1106
m
5.7. (ꢁ)-(1R,2S,5R)-6-Allyl-8-(2,4-dimethoxybenzyl)-2-
methoxy-2-(trimethylsiloxy)-6,8-diazabicyclo[3.2.2]nonane-
7,9-dione (ent-8b)
As described for the preparation of 8b, the enantiomer ent-7b
(9.0 g, 23.1 mmol) was reacted with a 1 M solution of lithium hex-

784
R. Holl et al. / Bioorg. Med. Chem. 17 (2009) 777–793
amethyldisilazane in THF (34.6 mL, 34.6 mmol) and chlorotrimeth-
ylsilane (10.6 mL, 9.02 g, 83.0 mmol) in THF (200 mL) to give ent-
3H, ArOCH3), 3.87 (ddt, J = 15.7/6.3/1.6 Hz, 1H, NCH2CH@CH2),
3.89–3.92 (m, 1H, 5-H), 3.96 (d, J = 14.9 Hz, 1H, NCH2Ar), 3.99 (s,
1H, 1-H), 4.11 (ddt, J = 15.7/6.3/1.6 Hz, 1H, NCH2CH@CH2), 5.18–
5.25 (m, 2H, NCH2CH@CH2), 5.20 (d, J = 14.9 Hz, 1H, NCH2Ar),
5.73 (ddt, J = 16.4/10.2/6.3 Hz, 1H, NCH2CH@CH2), 6.84 (d,
8b as a yellow oil, yield 8.09 g (76%). C23H34N2O6Si (462.6). ½a D20
ꢄ
ꢁ30.1 (c 0.73; CH2Cl2).
J = 8.6 Hz, 2H, 30-H4-methoxybenzyl
,
50-H4-methoxybenzyl), 7.12 (d,
5.8. (+)-(1S,5S)-6-Allyl-8-(2,4-dimethoxybenzyl)-6,8-
diazabicyclo[3.2.2]nonane-2,7,9-trione (9b)
J = 8.6 Hz, 2H, 20-H4-methoxybenzyl, 60-H4-methoxybenzyl). IR (neat):
m
~
[cmꢁ1] = 2938 (m,
(m, C@C arom.), 1453 (m, dCAH aliph.), 1243 (m)/1035 (m,
(w, Cp-subst. arom.).
m
CAH aliph.), 1677 (s,
m
C@O amide), 1612 (m)/1512
CAO), 808
Under N2 8b (518 mg, 1.12 mmol) was dissolved in a degassed
mixture of THF/2 M HCl (9:1, 70 mL) and the mixture was stirred
at rt for 2 h. Then water was added and the mixture was extracted
with CH2Cl2 (3ꢃ). The organic layer was dried (Na2SO4), filtered
and the solvent was removed in vacuo. The residue was purified
by FC (Ø = 3 cm, h = 15 cm, cyclohexane/ethyl acetate = 1:2,
V = 20 mL, Rf = 0.26) to give 9b as a colorless solid, mp 119 °C, yield
340 mg (85%). C19H22N2O5 (358.4). Calculated C, 63.68; H, 6.19; N,
m
m
5.11. (ꢁ)-(1R,5R)-6-Allyl-2,2-dimethoxy-8-(4-methoxybenzyl)-
6,8-diazabicyclo[3.2.2]nonane-7,9-dione (ent-10)
As described for the preparation of 10, the enantiomer ent-9a
(400 mg, 1.22 mmol) was reacted with trimethyl orthoformate
(1.7 mL, 1.42 g, 13.4 mmol) and p-toluenesulfonic acid (461 mg,
2.68 mmol) in methanol (60 mL) to give ent-10 as a colorless oil,
yield 450 mg (99%). C20H26N2O5 (374.4). Purity by HPLC: method
7.82; found: C, 63.58; H, 6.01; N, 7.69. ½a D20
ꢄ
+96.8 (c 0.26; CH2Cl2).
MS (EI): m/z [%] = 358 (M, 45), 151 (2,4-dimethoxybenzyl, 100). 1H
NMR (CDCl3): d [ppm] = 2.00–2.10 (m, 1H, 4-H), 2.22–2.30 (m, 1H,
3-H), 2.38–2.54 (m, 2H, 3-H (1H), 4-H (1H)), 3.70 (s, 3H, ArOCH3),
3.78 (s, 3H, ArOCH3), 3.83 (dd, J = 15.1/6.4 Hz, 1H, NCH2CH@CH2),
3.93–3.96 (m, 1H, 5-H), 4.15 (dd, J = 15.1/6.4 Hz, 1H,
NCH2CH@CH2), 4.29 (d, J = 13.5 Hz, 1H, NCH2Ar), 4.47 (s, 1H, 1-
H), 4.83 (d, J = 13.5 Hz, 1H, NCH2Ar), 5.18–5.26 (m, 2H,
NCH2CH@CH2), 5.66–5.77 (m, 1H, NCH2CH@CH2), 6.37 (d,
J = 2.4 Hz, 1H, 30-H2,4-dimethoxybenzyl), 6.43 (dd, J = 8.8/2.4 Hz, 1H,
50-H2,4-dimethoxybenzyl), 7.32 (d, J = 8.8 Hz, 1H, 60-H2,4-dimethoxybenzyl).
13C NMR (CDCl3): d [ppm] = 30.6 (1C, C-4), 36.8 (1C, C-3), 46.0
(1C, NCH2Ar), 48.0 (1C, NCH2CH@CH2), 55.2 (1C, ArOCH3),
55.6 (1C, ArOCH3), 59.2 (1C, C-5), 73.6 (1C, C-1), 98.5
(1C, C-302,4-dimethoxybenzyl), 104.7 (1C, C-502,4-dimethoxybenzyl), 115.8
(1C, C-102,4-dimethoxybenzyl), 120.0 (1C, NCH2CH@CH2), 131.7 (1C,
NCH2CH@CH2), 133.1 (1C, C-602,4-dimethoxybenzyl), 158.5 (1C,
C-402,4-dimethoxybenzyl), 161.5 (1C, C-202,4-dimethoxybenzyl), 163.7 (1C, C@O),
~
1: tR = 18.3 min, purity 97.2%. ½a D20
ꢁ11.8 (c 0.55; CH2Cl2).
ꢄ
5.12. (+)-(1R,5S)-6-Allyl-2,2-dimethoxy-8-(4-methoxybenzyl)-
6,8-diazabicyclo[3.2.2]nonan-2-one dimethyl acetal (11)
Under N2 LiAlH4 (66 mg, 1.74 mmol) was added to an ice-cooled
solution of 10 (130 mg, 0.35 mmol) in THF (30 mL). The mixture
was stirred at 0 °C for 10 min and then heated to reflux for 16 h.
Then water was added under ice-cooling until H2 formation was
finished. The mixture was stirred at 0 °C for 10 min and then
heated to reflux for 30 min. After cooling down the mixture was fil-
tered, the solvent was removed in vacuo and the residue was puri-
fied by FC (Ø = 2 cm, h = 15 cm, CH2Cl2/methanol = 50:1, V = 10 mL,
Rf = 0.04) to give 11 as
a colorless oil, yield 24 mg (20%).
C20H30N2O3 (346.5). Purity by HPLC: method 2c: tR = 24.0 min, pur-
ity 97.9%; method 1: tR = 16.7 min, purity 97.6%. ½a D20
ꢄ
+45.1 (c 0.24;
167.1 (1C, C@O), 201.4 (1C, C@Oketone). IR (neat):
(w, CAH arom.), 2934 (w, CAH aliph.), 1721 (m, mC@O ketone), 1678
(s, C@O amide), 1610 (m)/1509 (m, C@C arom.), 1456 (m, dCAH aliph.),
1210 (m)/1027 (m, CAO), 836 (m, Ctri-subst. arom.).
m
[cmꢁ1] = 3007
CH2Cl2). MS (EI): m/z [%] = 346 (M, 48), 315 (MꢁOCH3, 6), 225
(MꢁCH2PhOCH3, 6), 121 (CH2PhOCH3, 100). 1H NMR (CDCl3): d
[ppm] = 1.57–1.67 (m, 1H, 4-H), 1.76–1.91 (m, 2H, 3-H (1H), 4-H
(1H)), 1.95–2.04 (m, 1H, 3-H), 2.61–2.69 (m, 2H, 9-H), 2.74 (dd,
J = 11.7/3.9 Hz, 1H, 7-H), 2.84–2.87 (m, 1H, 1-H), 2.90–2.95 (m,
1H, 5-H), 3.07 (dd, J = 11.7/1.6 Hz, 1H, 7-H), 3.11 (s, 3H, OCH3),
3.12–3.19 (m, 2H, NCH2CH@CH2), 3.20 (s, 3H, OCH3), 3.76 (s, 2H,
NCH2Ar), 3.79 (s, 3H, ArOCH3), 5.08 (d, J = 10.2 Hz, 1H,
NCH2CH@CH2), 5.16 (dd, J = 17.2/1.6 Hz, 1H, NCH2CH@CH2), 5.85
(ddt, J = 17.2/10.2/6.3 Hz, 1H, NCH2CH@CH2), 6.84 (d, J = 8.6 Hz,
2H, 30-H4-methoxybenzyl, 50-H4-methoxybenzyl), 7.31 (d, J = 8.6 Hz, 2H,
~
m
m
m
m
m
5.9. (ꢁ)-(1R,5R)-6-Allyl-8-(2,4-dimethoxybenzyl)-6,8-
diazabicyclo[3.2.2]nonane-2,7,9-trione (ent-9b)
As described for the preparation of 9b, the enantiomer ent-8b
(9.0 g, 23.1 mmol) was reacted in a degassed mixture of THF/2 M
HCl (9:1, 200 mL) to give ent-9b as a colorless solid, mp 119 °C,
yield 4.62 g (75%). C19H22N2O5 (358.4). Calculated C, 63.68; H,
6.19; N, 7.82; found: C, 63.62; H, 6.08; N, 7.78. ½a D20
ꢄ
ꢁ96.1 (c
20-H4-methoxybenzyl
,
60-H4-methoxybenzyl). IR (neat): [cmꢁ1] =
m
3071 (w, mCAH arom.), 2932 (m, mCAH aliph.), 1611 (m)/1510
0.27; CH2Cl2).
(m, mC@C arom.), 1442 (m, dCAH aliph.), 1243 (m)/1038 (m,
(m, Cp-subst. arom.).
mCAO), 829
5.10. (+)-(1S,5S)-6-Allyl-2,2-dimethoxy-8-(4-methoxybenzyl)-
6,8-diazabicyclo[3.2.2]nonane-7,9-dione (10)
5.13. (ꢁ)-(1S,5R)-6-Allyl-2,2-dimethoxy-8-(4-methoxybenzyl)-
6,8-diazabicyclo[3.2.2]nonan-2-one dimethyl acetal (ent-11)
Under N2 trimethyl orthoformate (1.2 mL, 1.17 g, 11.1 mmol)
was added to a solution of 9a (330 mg, 1.00 mmol) and p-toluene-
sulfonic acid (381 mg, 2.21 mmol) in methanol (50 mL). The mix-
ture was heated to reflux for 16 h. Then a saturated aqueous
solution of NaHCO3 was added and the mixture was extracted with
CH2Cl2 (3ꢃ). The combined organic layers were dried (Na2SO4), fil-
tered and concentrated in vacuo. The residue was purified by FC
(Ø = 2 cm, h = 15 cm, cyclohexane/ethyl acetate = 2/1, V = 10 mL,
Rf = 0.10) to give 10 as a colorless oil, yield 368 mg (98%).
C20H26N2O5 (374.4). Purity by HPLC: method 1: tR = 18.3 min, pur-
As described for the preparation of 11, the enantiomer ent-10
(176 mg, 0.47 mmol) was reacted with LiAlH4 (89 mg, 2.35 mmol)
in THF (40 mL) to give ent-11 as a colorless oil, yield 30 mg (18%).
C20H30N2O3 (346.5). Purity by HPLC: method 2c: tR = 25.0 min,
purity 96.8%; method 1: tR = 16.4 min, purity 98.4%. ½a D20
ꢁ45.1
ꢄ
(c 0.49; CH2Cl2).
5.14. (ꢁ)-(1R,5S)-6-Allyl-8-(4-methoxybenzyl)-6,8-
ity 97.7%. ½a 2D0
ꢄ
+11.7 (c 0.66; CH2Cl2). MS (EI): m/z [%] = 374 (M, 2),
diazabicyclo[3.2.2]nonan-2-one (12)
343 (MꢁOCH3, 1), 121 (CH2PhOCH3, 31), 101 (H2CCHC(OCH3)2,
100). 1H NMR (CDCl3): d [ppm] = 1.73–1.88 (m, 2H, 3-H), 2.00–
2.11 (m, 2H, 4-H), 3.18 (s, 3H, OCH3), 3.27 (s, 3H, OCH3), 3.78 (s,
Under N2 a 1.0 M solution of LiAlH4 in THF (2.13 mL, 2.13 mmol)
was added to an ice-cooled solution of 10 (200 mg, 0.53 mmol) in

R. Holl et al. / Bioorg. Med. Chem. 17 (2009) 777–793
785
THF (60 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
heated to reflux 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 puri-
fied by FC (Ø = 2 cm, h = 15 cm, CH2Cl2/methanol = 100:1,
V = 10 mL, Rf = 0.15) to give 12 as a colorless oil, yield 80 mg
(50%). C18H24N2O2 (300.4). Purity by HPLC: method 2d:
tR = 24.5 min, purity 98.0%; method 1: tR = 15.6 min, purity 98.6%.
(2C, C-204-methoxybenzyl, C-604-methoxybenzyl), 132.0 (1C, NCH2CH@CH2),
159.7 (1C, C-404-methoxybenzyl), 167.6 (1C, C@O), 169.1 (1C, C@O). IR
(neat):
[cmꢁ1] = 3567 (m br,
mOAH), 2932 (w,
mCAH aliph.), 1652 (s,
~
m
mC@O amide), 1612 (m)/1510 (m,
mC@C arom.), 1453 (m, dCAH aliph.), 1241
(m)/1079 (m,
mCAO), 839 (w, Cp-subst. arom.).
5.17. (ꢁ)-(1R,2S,5R)-6-Allyl-2-hydroxy-8-(4-methoxybenzyl)-
6,8-diazabicyclo[3.2.2]nonane-7,9-dione (ent-13a)
As described for the preparation of 13a, the enantiomer ent-9a
(510 mg, 1.6 mmol) was reacted with a 2 M solution of LiBH4 in
THF (3.1 mL, 6.4 mmol) in THF (50 mL) to give ent-13a as a color-
less solid, mp 109 °C, yield 451 mg (88%). C18H22N2O4 (330.4). Cal-
culated C, 65.44; H, 6.71; N, 8.48; found: C, 65.12; H, 6.58; N, 8.31.
½
a 2D0
ꢄ
ꢁ73.7 (c 0.59; CH2Cl2). MS (EI): m/z [%] = 301 (MH, 2), 272
(MꢁCO, 16), 121 (CH2PhOCH3, 100). 1H NMR (CDCl3):
d
[ppm] = 1.66–1.76 (m, 1H, 4-H), 1.83–1.92 (m, 1H, 4-H), 2.28
(ddd, J = 13.3/8.6/1.6 Hz, 1H, 3-H), 2.78–2.87 (m, 3H, piperazine-
H), 2.92 (dd, J = 11.0/2.3 Hz, 1H, piperazine-H), 3.05–3.21 (m, 4H,
NCH2CH@CH2, piperazine-H), 3.27 (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), 3.79 (s, 3H, ArOCH3), 5.09 (dd, J = 10.2/1.6 Hz, 1H,
NCH2CH@CH2), 5.15 (ddd, J = 17.2/3.1/1.6 Hz, 1H, NCH2CH@CH2),
5.75–5.86 (m, 1H, NCH2CH@CH2), 6.83 (d, J = 8.6 Hz, 2H,
½
a 2D0
ꢄ
ꢁ122 (c 0.28; CH2Cl2).
5.18. (+)-(1S,2R,5S)-6-Allyl-8-(2,4-dimethoxybenzyl)-2-
hydroxy-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (13b)
Under N2 9b (1.8 g, 5.0 mmol) was dissolved in THF (100 mL)
and the solution was cooled to ꢁ78 °C. Then a 2 M solution of LiBH4
in THF (10.0 mL, 20.0 mmol) was slowly added. The reaction mix-
ture was stirred for 3 h at ꢁ78 °C. Then the mixture was warmed to
rt and carefully hydrolyzed with 1 M aq HCl (80 mL). The resulting
mixture was extracted with CH2Cl2 (3ꢃ). The combined organic
layers were dried (Na2SO4), filtered and the solvent was removed
in vacuo. The residue was purified by FC (Ø = 5 cm, h = 15 cm, ethyl
acetate, V = 30 mL, Rf = 0.22) to give 13b as a colorless oil, yield
30-H4-methoxybenzyl
,
50-H4-methoxybenzyl), 7.18 (d, J = 8.6 Hz, 2H,
20-H4-methoxybenzyl, 60-H4-methoxybenzyl). IR (neat):
[cmꢁ1] = 3067
~
m
(w, mCA H arom.), 2911 (m, mCA H aliph.), 1708 (s, mC@ O ketone), 1611
(m)/1510 (s, mC@C arom.), 1441 (m, dCAH aliph.), 1243 (m)/1033 (m,
m
CAO), 820 (w, Cp-subst. arom.).
5.15. (+)-(1S,5R)-6-Allyl-8-(4-methoxybenzyl)-6,8-
diazabicyclo[3.2.2]nonan-2-one (ent-12)
1.7 g (92%). C19H24N2O5 (360.4). ½a D20
ꢄ
+119 (c 0.67; CH2Cl2). MS
(EI): m/z [%] = 360 (M, 65), 151 (2,4-dimethoxybenzyl, 100). 1H
NMR (CDCl3): d [ppm] = 1.50–1.60 (m, 1H, 3-H), 1.78–1.87 (m,
1H, 4-H), 1.89–2.02 (m, 2H, 3-H (1H), 4-H (1H)), 2.26 (s br, 1H,
OH), 3.48–3.56 (m, 1H, 2-H), 3.79 (s, 3H, ArOCH3), 3.81 (s, 3H, Ar-
OCH3), 3.87 (dd, J = 5.5/2.3 Hz, 1H, 5-H), 3.95 (dd, J = 14.9/6.3 Hz,
1H, NCH2CH@CH2), 4.05–4.12 (m, 2H, 1-H, NCH2CH@CH2), 4.46
(d, J = 14.1 Hz, 1H, NCH2Ar), 4.60 (d, J = 14.1 Hz, 1H, NCH2Ar),
5.20–5.28 (m, 2H, NCH2CH@CH2), 5.71–5.82 (m, 1H, NCH2CH@
CH2), 6.42–6.46 (m, 2H, 30-H2,4-dimethoxybenzyl, 50-H2,4-dimethoxybenzyl),
7.22 (d, J = 8.6 Hz, 1H, 60-H2,4-dimethoxybenzyl). 13C NMR (CDCl3): d
[ppm] = 23.5 (1C, C-4), 29.6 (1C, C-3), 44.0 (1C, NCH2Ar),
47.8 (1C, NCH2CH@CH2), 55.6 (1C, ArOCH3), 55.7 (1C, ArOCH3),
59.2 (1C, C-5), 65.8 (1C, C-1), 66.6 (1C, C-2), 98.8 (1C,
C-302,4-dimethoxybenzyl), 104.7 (1C, C-502,4-dimethoxybenzyl), 116.5
(1C, C-102,4-dimethoxybenzyl), 119.6 (1C, NCH2CH@CH2), 132.1 (1C,
NCH2CH@CH2), 132.2 (1C, C-602,4-dimethoxybenzyl), 158.9 (1C,
C-402,4-dimethoxybenzyl), 161.2 (1C, C-202,4-dimethoxybenzyl), 167.7 (1C,
~
As described for the preparation of 12, the enantiomer ent-10
(357 mg, 0.95 mmol) was reacted with LiAlH4 (3.81 mL of a 1.0 M
solution in THF, 3.81 mmol) in THF (80 mL) and afterward hydro-
lyzed with 1.0 M HCl (20 mL) to give ent-12 as a colorless oil, yield
95 mg (33%). C18H24N2O2 (300.4). Purity by HPLC: method 2d:
tR = 24.5 min, purity 98.0%; method 1: tR = 15.6 min, purity 99.5%.
½
a 2D0
+76.1 (c 0.73; CH2Cl2).
ꢄ
5.16. (+)-(1S,2R,5S)-6-Allyl-2-hydroxy-8-(4-methoxybenzyl)-
6,8-diazabicyclo[3.2.2]nonane-7,9-dione (13a)
Under N2 9a (1.3 g, 4.0 mmol) was dissolved in THF (70 mL) and
the solution was cooled to ꢁ78 °C. Then a 2 M solution of LiBH4 in
THF (8.0 mL, 16.0 mmol) was slowly added. The reaction mixture
was stirred for 3 h at ꢁ78 °C. Then the mixture was warmed to rt
and carefully hydrolyzed with 1 M HCl (70 mL). The resulting mix-
ture was extracted with CH2Cl2 (3ꢃ). The combined organic layers
were dried (Na2SO4), filtered and the solvent was removed in va-
cuo. The residue was purified by FC (Ø = 4 cm, h = 15 cm, ethyl ace-
tate, V = 30 mL, Rf = 0.30) to give 13a as a colorless solid, mp 109 °C,
yield 1.2 g (92%). C18H22N2O4 (330.4). Calculated C, 65.44; H, 6.71;
C@O), 169.1 (1C, C@O). IR (neat):
2938 (m, mCAH aliph.), 1660 (s, mC@O amide), 1611 (m)/1507
(m, C@C arom.), 1455 (m, dCAH aliph.), 1208 (m)/1030 (m, CAO), 833
(w, Ctri-subst. arom.).
m mOAH),
[cmꢁ1] = 3381 (m br,
m
m
N, 8.48; found: C, 65.30; H, 6.66; N, 8.29. ½a D20
ꢄ
+120 (c 1.41; CH2Cl2).
5.19. (ꢁ)-(1R,2S,5R)-6-Allyl-8-(2,4-dimethoxybenzyl)-2-
MS (EI): m/z [%] = 330 (M, 100), 209 (MꢁCH2PhOCH3, 7), 121
(CH2PhOCH3, 72). 1H NMR (CDCl3): d [ppm] = 1.50–1.64 (m, 1H,
3-H), 1.78–2.06 (m, 3H, 3-H (1H), 4-H (2H)), 2.54 (d br, J = 3.9 Hz,
1H, OH), 3.43–3.49 (m, 1H, 2-H), 3.78 (s, 3H, ArOCH3), 3.90–4.00
(m, 3H, 1-H, 5-H, NCH2CH@CH2), 4.08 (dd, J = 15.7/6.3 Hz,
1H, NCH2CH@CH2), 4.40 (d, J = 14.1 Hz, 1H, NCH2Ar), 4.60 (d,
J = 14.1 Hz, 1H, NCH2Ar), 5.20–5.28 (m, 2H, NCH2CH@CH2),
5.70–5.82 (m, 1H, NCH2CH@CH2), 6.84 (d, J = 8.6 Hz, 2H,
hydroxy-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (ent-13b)
As described for the preparation of 13b, the enantiomer ent-9b
(3.0 g, 8.4 mmol) was reacted with a 2 M solution of LiBH4 in THF
(16.8 mL, 33.6 mmol) in THF (150 mL) to give ent-13b as a color-
less oil, yield 2.3 g (76%). C19H24N2O5 (360.4). Purity by HPLC:
method 1: tR = 15.6 min, purity 99.5%. ½a D20
ꢁ119 (c 0.49; CH2Cl2).
ꢄ
30-H4-methoxybenzyl
,
,
50-H4-methoxybenzyl), 7.19 (d, J = 8.6 Hz, 2H,
5.20. (+)-(1S,2R,5S)-6-Allyl-2-methoxy-8-(4-methoxybenzyl)-
6,8-diazabicyclo[3.2.2]nonane-7,9-dione (14a)
20-H4-methoxybenzyl
60-H4-methoxybenzyl). 13C NMR (CDCl3):
d
[ppm] = 23.5 (1C, C-4), 29.5 (1C, C-3), 47.9 (1C, NCH2CH@CH2),
48.7 (1C, NCH2Ar), 55.5 (1C, ArOCH3), 59.1 (1C, C-5), 65.4 (1C, C-1),
66.3 (1C, C-2), 114.6 (2C, C-304-methoxybenzyl, C-504-methoxybenzyl),
119.8 (1C, NCH2CH@CH2), 128.0 (1C, C-104-methoxybenzyl), 130.1
Under N2 and ice-cooling 60% NaH suspension in paraffin oil
(75 mg, ca. 45 mg NaH, 1.87 mmol) was added to a solution of
13a (103 mg, 0.31 mmol) in THF (30 mL). After 20 min methyl io-

786
R. Holl et al. / Bioorg. Med. Chem. 17 (2009) 777–793
dide (0.06 mL, 132 mg, 0.94 mmol) was added dropwise and the
mixture was stirred at rt for 16 h. Then the solvent was removed
in vacuo, the residue was dissolved in CH2Cl2 and washed with
water (2ꢃ), 0.5 M HCl (1ꢃ) and 0.5 M NaOH (1ꢃ). The organic layer
was dried (Na2SO4), filtered and the solvent was removed in vacuo.
The residue was purified by FC (Ø = 1 cm, h = 15 cm, cyclohexane/
ethyl acetate = 1:2, V = 5 mL, Rf = 0.18) to give 14a as a colorless so-
lid, mp 127 °C, yield 96 mg (89%). C19H24N2O4 (344.4). Calculated C,
5.23. (ꢁ)-(1R,2S,5R)-6-Allyl-8-(2,4-dimethoxybenzyl)-2-
methoxy-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (ent-14b)
As described for the preparation of 14b, the enantiomer ent-13b
(148 mg, 0.41 mmol), 60% NaH suspension in paraffin oil (99 mg,
ca. 59 mg NaH, 2.46 mmol) and methyl iodide (0.08 mL, 175 mg,
1.23 mmol) were reacted in THF (30 mL) to give ent-14b as a col-
orless oil, yield 136 mg (88%). C20H26N2O5 (374.4). ½a D20
ꢁ144.7 (c
ꢄ
66.26; H, 7.02; N, 8.13; found: C, 66.36; H, 6.97; N, 8.00. ½a D20
ꢄ
0.18; CH2Cl2).
+149.0 (c 0.22; CH2Cl2). MS (EI): m/z [%] = 344 (M, 30), 303
(MꢁCH2CH@CH2, 4), 223 (MꢁCH2PhOCH3, 7), 121 (CH2PhOCH3,
100). 1H NMR (CDCl3): d [ppm] = 1.55–1.65 (m, 1H, 3-H), 1.80–
1.91 (m, 2H, 3-H (1H), 4-H (1H)), 1.92–2.01 (m, 1H, 4-H), 2.86–
2.92 (m, 1H, 2-H), 3.16 (s, 3H, OCH3), 3.79 (s, 3H, ArOCH3), 3.92
(dd, J = 5.5/2.3 Hz, 1H, 5-H), 3.94–4.01 (m, 2H, 1-H, NCH2CH@CH2
(1H)), 4.08 (dd, J = 14.9/6.3 Hz, 1H, NCH2CH@CH2), 4.29 (d,
J = 14.1 Hz, 1H, NCH2Ar), 4.75 (d, J = 14.1 Hz, 1H, NCH2Ar), 5.21–
5.27 (m, 2H, NCH2CH@CH2), 5.76 (ddt, J = 17.2/10.2/6.3 Hz, 1H,
5.24. (+)-(1R,2R,5S)-6-Allyl-8-(4-methoxybenzyl)-6,8-
diazabicyclo[3.2.2]nonan-2-ol (15a)
Under N2 LiAlH4 (40 mg, 1.1 mmol) was added to an ice-cooled
solution of the 13a (70 mg, 0.21 mmol) in THF (20 mL). The mix-
ture was stirred at 0 °C for 10 min and then heated to reflux for
16 h. Then water was added under ice-cooling until H2 formation
was finished. The mixture was stirred at 0 °C for 10 min and then
heated to reflux for 30 min. After cooling down the mixture was fil-
tered, the solvent was removed in vacuo and the residue was puri-
fied by FC (Ø = 1 cm, h = 15 cm, CH2Cl2/methanol = 9.5:0.5,
V = 5 mL, Rf = 0.12) to give 15a as a yellow oil, yield 37 mg (58%).
C18H26N2O2 (302.4). Purity by HPLC: method 2a: tR = 19.9 min, pur-
NCH2CH@CH2), 6.86 (d, J = 8.6 Hz, 2H, 30-H4-methoxybenzyl
,
,
50-H4-methoxybenzyl), 7.21 (d, J = 8.6 Hz, 2H, 20-H4-methoxybenzyl
60-H4-methoxybenzyl). IR (neat):
m
[cmꢁ1] = 3080 (w,
m
CAH arom.), 2951
~
(w, mCAH aliph.), 1663 (s, mC@O amide), 1609 (m)/1508 (m,
mC@C arom.), 1459 (m, dCAH aliph.), 1242 (m)/1096 (m,
mCAO), 835
(w, Cp-subst. arom.).
ity 99.9%; method 2g: tR = 17.4 min, purity 97.2%. ½a D20
ꢄ
+14.3 (c
0.30; CH2Cl2). MS (EI): m/z [%] = 302 (M, 14), 181 (MꢁCH2PhOCH3,
24), 121 (CH2PhOCH3, 100). 1H NMR (CDCl3): d [ppm] = 1.65–1.78
(m, 2H, 3-H (1H), 4-H (1H)), 1.88–1.98 (m, 1H, 4-H), 2.14–2.24
(m, 1H, 3-H), 2.68–2.80 (m, 3H, 7-H (1H), 9-H), 2.86–2.91 (m, 1H,
1-H), 2.92–2.98 (m, 1H, 5-H), 3.12–3.27 (m, 3H, 7-H (1H),
NCH2CH@CH2), 3.60 (d, J = 17.2 Hz, 1H, NCH2Ar), 3.63 (d,
J = 17.2 Hz, 1H, NCH2Ar), 3.79 (s, 3H, ArOCH3), 4.00 (q, J = 5.7 Hz,
1H, 2-H), 5.12–5.24 (m, 2H, NCH2CH@CH2), 5.83–5.95 (m,
1H, NCH2CH@CH2), 6.84 (d, J = 8.5 Hz, 2H, 30-H4-methoxybenzyl, 50-
5.21. (ꢁ)-(1R,2S,5R)-6-Allyl-2-methoxy-8-(4-methoxybenzyl)-
6,8-diazabicyclo[3.2.2]nonane-7,9-dione (ent-14a)
As described for the preparation of 14a, the enantiomer ent-13a
(81 mg, 0.25 mmol), 60% NaH suspension in paraffin oil (59 mg, ca.
35 mg NaH, 1.47 mmol) and methyl iodide (0.05 mL, 104 mg,
0.74 mmol) were reacted in THF (30 mL) to give ent-14a as a color-
less solid, mp 128 °C, yield 70 mg (83%). C19H24N2O4 (344.4). Calcu-
lated C, 66.26; H, 7.02; N, 8.13; found: C, 66.18; H, 7.07; N, 8.04.
H4-methoxybenzyl), 7.23 (d, J = 8.5 Hz, 2H, 20-H4-methoxybenzyl
,
60-H4-methoxybenzyl). The signal for the proton of the OH group could
not be detected. 13C NMR (CDCl3): d [ppm] = 29.3 (1C, C-4), 30.6
(1C, C-3), 46.6 (1C, C-7), 51.2 (1C, C-9), 54.7 (1C, C-5), 55.5 (1C, Ar-
OCH3), 59.7 (1C, NCH2CH@CH2), 59.8 (1C, C-1), 60.5 (1C, NCH2Ar),
74.5 (1C, C-2), 113.9 (2C, C-304-methoxybenzyl, C-50H4-methoxybenzyl),
118.1 (1C, NCH2CH@CH2), 129.0 (1C, C-104-methoxybenzyl), 129.9
(2C, C-204-methoxybenzyl, C-604-methoxybenzyl), 135.7 (1C, NCH2CH@CH2),
~
½
a 2D0
ꢄ
ꢁ149.4 (c 0.33; CH2Cl2).
5.22. (+)-(1S,2R,5S)-6-Allyl-8-(2,4-dimethoxybenzyl)-2-
methoxy-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (14b)
Under N2 and ice-cooling 60% NaH suspension in paraffin oil
(54 mg, ca. 32 mg NaH, 1.35 mmol) was added to a solution of
13b (81 mg, 0.31 mmol) in THF (20 mL). After 20 min methyl io-
dide (0.04 mL, 96 mg, 0.67 mmol) was added dropwise and the
mixture was stirred at rt for 16 h. Then the solvent was removed
in vacuo, the residue was dissolved in CH2Cl2 and washed with
water (2ꢃ), 0.5 M HCl (1ꢃ) and 0.5 M NaOH (1ꢃ). The organic layer
was dried (Na2SO4), filtered and the solvent was removed in vacuo.
The residue was purified by FC (Ø = 1 cm, h = 15 cm, cyclohexane/
ethyl acetate = 1:2, V = 5 mL, Rf = 0.18) to give 14b as a colorless
158.9 (1C, C-404-methoxybenzyl). IR (neat):
m
[cmꢁ1] = 3362 (m br,
mOAH), 2919 (m, mCAH aliph.), 1610 (w)/1510 (m, mC@C arom.), 1242
(m)/1036 (m, CAO), 830 (w, Cp-subst. arom.).
m
5.25. (ꢁ)-(1S,2S,5R)-6-Allyl-8-(4-methoxybenzyl)-6,8-
diazabicyclo[3.2.2]nonan-2-ol (ent-15a)
As described for the preparation of 15a, the enantiomer ent-13a
(110 mg, 0.33 mmol) was reacted with LiAlH4 (63 mg, 1.7 mmol) in
THF (30 mL) to give ent-15a as a yellow oil, yield 41 mg (41%).
C18H26N2O2 (302.4). Purity by HPLC: method 2a: tR = 20.0 min,
oil, yield 65 mg (77%). C20H26N2O5 (374.4). ½a D20
ꢄ
+141.1 (c 0.40;
CH2Cl2). MS (EI): m/z [%] = 374 (M, 24), 343 (MꢁOCH3, 8), 223
(Mꢁ2,4-dimethoxybenzyl, 2), 151 (2,4-dimethoxybenzyl, 100). 1H
NMR (CDCl3): d [ppm] = 1.54–1.65 (m, 1H, 3-H), 1.77–1.98 (m,
3H, 3-H (1H), 4-H (2H)), 2.95–3.00 (m, 1H, 2-H), 3.20 (s, 3H,
OCH3), 3.79 (s, 3H, ArOCH3), 3.81 (s, 3H, ArOCH3), 3.87 (dd,
J = 5.5/3.1 Hz, 1H, 5-H), 3.96 (ddt, J = 15.7/6.3/1.6 Hz, 1H,
NCH2CH@CH2), 4.06 (ddt, J = 14.9/6.3/1.6 Hz, 1H, NCH2CH@CH2),
4.15 (d, J = 3.9 Hz, 1H, 1-H), 4.42 (d, J = 14.1 Hz, 1H, NCH2Ar),
4.66 (d, J = 14.1 Hz, 1H, NCH2Ar), 5.18–5.25 (m, 2H, NCH2CH@CH2),
5.75 (ddt, J = 16.4/10.2/6.3 Hz, 1H, NCH2CH@CH2), 6.41–6.47 (m,
2H, 30-H2,4-dimethoxybenzyl, 50-H2,4-dimethoxybenzyl), 7.22 (d, J = 8.6 Hz,
~
purity 97.6%; method 2g: tR = 17.4 min, purity 96.7%. ½a D20
ꢁ14.4
ꢄ
(c 0.28; CH2Cl2).
5.26. (+)-(1R,2R,5S)-6-Allyl-8-(2,4-dimethoxybenzyl)-6,8-
diazabicyclo[3.2.2]nonan-2-ol (15b)
Under N2 LiAlH4 (58 mg, 1.53 mmol) was added to an ice-cooled
solution of 13b (110 mg, 0.31 mmol) in THF (30 mL). The mixture
was stirred at 0 °C for 10 min and then heated to reflux for 16 h.
Then water was added under ice-cooling until H2 formation was
finished. The mixture was stirred at 0 °C for 10 min and then heated
to reflux for 30 min. After cooling down the mixture was filtered,
the solvent was removed in vacuo and the residue was purified
by FC (Ø = 2 cm, h = 15 cm, CH2Cl2/methanol = 50:1, V = 10 mL,
1H, 60-H2,4-dimethoxybenzyl). IR (neat):
2937 (w, CAH aliph.), 1673 (s,
(m, C@C arom.), 1452 (m, dCAH aliph.), 1208 (m)/1091 (m,
(w, Ctri-subst. arom.).
m
[cmꢁ1] = 3081 (w,
C@O amide), 1611 (m)/1587 (m)/1507
CAO), 834
mCAH arom.),
m
m
m
m

R. Holl et al. / Bioorg. Med. Chem. 17 (2009) 777–793
787
Rf = 0.03) to give 15b as a yellow oil, yield 23 mg (23%). C19H28N2O3
(332.5). Purity by HPLC: method 2d: tR = 19.6 min, purity 97.4%;
THF (30 mL) to give ent-16a as a yellow oil, yield 13 mg (13%).
C
19H28N2O2 (316.5). Purity by HPLC: method 2c: tR = 20.1 min,
method 2g: tR = 16.6 min, purity 95.6%. ½a D20
ꢄ
+12.1 (c 0.28; CH2Cl2).
MS (EI): m/z [%] = 332 (M, 7), 181 (Mꢁ2,4-dimethoxybenzyl, 21),
purity 95.7%; method 2e: tR = 15.8 min, purity 97.6%. ½a D20
ꢁ6.4
ꢄ
(c 0.57; CH2Cl2).
151 (2,4-dimethoxybenzyl, 100). 1H NMR (CDCl3):
d
[ppm] = 1.66–1.75 (m, 2H, 3-H (1H), 4-H (1H)), 1.85–1.94 (m, 1H,
4-H), 2.14–2.22 (m, 1H, 3-H), 2.72 (dd, J = 11.7/3.1 Hz, 1H, 7-H),
2.78 (dd, J = 11.7/3.1 Hz, 1H, 9-H), 2.84 (dd, J = 11.7/3.1 Hz, 1H, 9-
H), 2.87–2.93 (m, 2H, 1-H, 5-H), 3.11 (dd, J = 11.7/2.3 Hz, 1H, 7-H),
3.13–3.23 (m, 2H, NCH2CH@CH2), 3.62 (d, J = 14.1 Hz, 1H,
NCH2Ar), 3.68 (d, J = 14.1 Hz, 1H, NCH2Ar), 3.78 (s, 3H, ArOCH3),
3.79 (s, 3H, ArOCH3), 4.03 (q, J = 5.5 Hz, 1H, 2-H), 5.11 (dd,
J = 10.2/1.6 Hz, 1H, NCH2CH@CH2), 5.18 (dd, J = 17.2/1.6 Hz, 1H,
NCH2CH@CH2), 5.86 (ddt, J = 17.2/10.2/6.3 Hz, 1H, NCH2CH@CH2),
6.43 (d, J = 2.3 Hz, 1H, 30-H2,4-dimethoxybenzyl), 6.46 (dd, J = 8.6/
2.3 Hz, 1H, 50-H2,4-dimethoxybenzyl), 7.27 (d, J = 8.6 Hz, 1H,
60-H2,4-dimethoxybenzyl). The signal for the proton of the OH group
~
5.30. (+)-(1R,2R,5S)-6-Allyl-8-(2,4-dimethoxybenzyl)-2-
methoxy-6,8-iazabicyclo[3.2.2]nonane (16b)
Under N2 LiAlH4 (67 mg, 1.76 mmol) was added to an ice-
cooled solution of 14b (132 mg, 0.35 mmol) in THF (30 mL). The
mixture was stirred at 0 °C for 10 min and then heated to reflux
for 16 h. Then water was added under ice-cooling until H2 forma-
tion was finished. The mixture was stirred at 0 °C for 10 min and
then heated to reflux for 30 min. After cooling down the mixture
was filtered, the solvent was removed in vacuo and the residue
was purified by FC (Ø = 2 cm, h = 15 cm, CH2Cl2/methanol = 50:1,
V = 10 mL, Rf = 0.05) to give 16b as a yellow oil, yield 41 mg
(34%). C20H30N2O3 (346.5). Purity by HPLC: method 2c:
tR = 19.3 min, purity 96.4%; method 2e: tR = 15.1 min, purity
could not be detected. IR (neat):
m mOAH),
[cmꢁ1] = 3367 (m br,
2919 (m, mCA H aliph.), 1610 (m)/1587 (m)/1505 (m, mC@ C arom.),
1205 (m)/1037 (m, CAO), 831 (w, Ctri-subst. arom.).
m
99.1%. ½a 2D0
ꢄ
+12.6 (c 0.43; CH2Cl2). MS (EI): m/z [%] = 346 (M,
80), 195 (Mꢁ2,4-dimethoxybenzyl, 81), 151 (2,4-dimethoxyben-
zyl, 100). 1H NMR (CDCl3): d [ppm] = 1.53–1.63 (m, 1H, CH2CH2),
1.70–1.81 (m, 1H, CH2CH2), 1.86–1.94 (m, 2H, CH2CH2), 2.65–2.77
(m, 2H, piperazine-H), 2.82 (s br, 1H, piperazine-H), 2.88–2.97 (m,
2H, piperazine-H), 3.06 (d, J = 11.0 Hz, 1H, piperazine-H), 3.13–
3.18 (m, 2H, NCH2CH@CH2), 3.14 (s, 3H, OCH3), 3.30–3.36 (m,
1H, 2-H), 3.70 (d, J = 13.3 Hz, 1H, NCH2Ar), 3.73–3.83 (m, 7H,
NCH2Ar (1H), Ar(OCH3)2 (6H)), 5.07 (d, J = 10.2 Hz, 1H,
NCH2CH@CH2), 5.17 (d, J = 17.2 Hz, 1H, NCH2CH@CH2),
5.80–5.91 (m, 1H, NCH2CH@CH2), 6.43 (d, J = 2.3 Hz, 1H,
5.27. (ꢁ)-(1S,2S,5R)-6-Allyl-8-(2,4-dimethoxybenzyl)-6,8-
diazabicyclo[3.2.2]nonan-2-ol (ent-15b)
As described for the preparation of 15b, the enantiomer ent-13b
(95 mg, 0.26 mmol) was reacted with LiAlH4 (50 mg, 1.32 mmol) in
THF (30 mL) to give ent-15b as a yellow oil, yield 27 mg (26%).
C19H28N2O3 (332.5). Purity by HPLC: method 2d: tR = 19.4 min, pur-
ity 99.1%; method 2g: tR = 16.4 min, purity 98.1%. ½a D20
ꢁ12.3 (c
ꢄ
0.26; CH2Cl2).
30-H2,4-dimethoxybenzyl),
6.46
(dd,
J = 8.6/2.3 Hz,
1H,
50-
H2,4-dimethoxybenzyl), 7.31 (d, J = 8.6 Hz, 1H, 60-H2,4-dimethoxybenzyl). IR
5.28. (+)-(1R,2R,5S)-6-Allyl-2-methoxy-8-(4-methoxybenzyl)-
6,8-diazabicyclo[3.2.2]nonane (16a)
(neat):
[cmꢁ1] = 3075 (w,
m
CAH arom.), 2925 (m,
mCAH aliph.), 1611
~
m
(m)/1587 (m)/1505 (m, mC@ C arom.), 1455 (m, d CAH aliph.), 1206
(m)/1097 (m, CAO), 832 (w, Ctri-subst. arom.).
Under N2 LiAlH4 (34 mg, 0.89 mmol) was added to an ice-cooled
solution of 14a (61 mg, 0.17 mmol) in THF (20 mL). The mixture was
stirred at 0 °C for 10 min and then heated to reflux for 16 h. Then
water was added under ice-cooling until H2 formation was finished.
The mixture was stirred at 0 °C for 10 min and then heated to reflux
for 30 min. After cooling down the mixture was filtered, the solvent
was removed in vacuo and the residue was purified by FC (Ø = 1 cm,
h = 15 cm, CH2Cl2/methanol = 50:1, V = 5 mL, Rf = 0.05) to give 16a
as a yellow oil, yield 15 mg (27%). C19H28N2O2 (316.5). Purity by
HPLC: method 2c: tR = 21.8 min, purity 96.3%; method 2e:
m
5.31. (ꢁ)-(1S,2S,5R)-6-Allyl-8-(2,4-dimethoxybenzyl)-2-
methoxy-6,8-diazabicyclo[3.2.2]nonane (ent-16b)
As described for the preparation of 16b, the enantiomer ent-14b
(87 mg, 0.23 mmol) was reacted with LiAlH4 (44 mg, 1.16 mmol) in
THF (30 mL) to give ent-16b as a yellow oil, yield 39 mg (48%).
C20H30N2O3 (346.5). Purity by HPLC: method 2c: tR = 19.3 min, pur-
ity 96.6%; method 2e: tR = 14.7 min, purity 98.4%. ½a D20
ꢁ12.0 (c
ꢄ
tR = 14.4 min, purity 96.6%. ½a D20
ꢄ
+6.7 (c 0.95; CH2Cl2). MS (EI): m/z
0.09; CH2Cl2).
[%] = 316 (M, 100), 275 (M–CH2CH@CH2, 11), 195 (MꢁCH2PhOCH3,
47), 121 (CH2PhOCH3, 100). 1H NMR (CDCl3): d [ppm] = 1.53–1.63
(m, 1H, CH2CH2), 1.70–1.82 (m, 1H, CH2CH2), 1.86–1.94 (m, 2H,
CH2CH2), 2.61–2.67 (m, 1H, piperazine-H), 2.68–2.78 (m, 2H, piper-
azine-H), 2.79–2.86 (m, 1H, piperazine-H), 2.90–2.96 (m, 1H, piper-
azine-H), 2.99–3.10 (m, 1H, piperazine-H), 3.08 (s, 3H, OCH3), 3.13–
3.18 (m, 2H, NCH2CH@CH2), 3.25–3.31 (m, 1H, 2-H), 3.64 (d,
J = 13.3 Hz, 1H, NCH2Ar), 3.72 (d, J = 13.3 Hz, 1H, NCH2Ar), 3.79 (s,
3H, ArOCH3), 5.06 (d, J = 10.2 Hz, 1H, NCH2CH@CH2), 5.17 (dd,
J = 17.2/1.6 Hz, 1H, NCH2CH@CH2), 5.85 (ddt, J = 17.2/10.2/5.5 Hz,
5.32. (+)-[(1S,2S,5S)-6-Allyl-8-(4-methoxybenzyl)-7,9-dioxo-6,8-
diazabicyclo[3.2.2]non-2-yl] 4-nitrobenzoate (17a)
Under N2 atmosphere 13a (503 mg, 1.52 mmol), triphenylphos-
phine (2.00 g, 7.61 mmol) and 4-nitrobenzoic acid (1.27 g,
7.61 mmol) were dissolved in THF (70 mL). Under ice-cooling
diisopropyl azodicarboxylate (1.51 mL, 1.54 g, 7.61 mmol) was
added dropwise. The mixture was stirred at 0 °C for 10 min and
then at rt for 6 h. Thereafter the solvent was removed in vacuo
and the residue was purified by FC (Ø = 6 cm, h = 15 cm, cyclohex-
ane/ethyl acetate = 2:1, V = 30 mL, Rf = 0.11) to give 17a as a color-
1H, NCH2CH@CH2), 6.84 (d, J = 8.6 Hz, 2H, 30-H4-methoxybenzyl
,
,
50-H4-methoxybenzyl), 7.26 (d, J = 8.6 Hz, 2H, 20-H4-methoxybenzyl
[cmꢁ1] = 3073 (w,
m
CAH arom.), 2924
C@C arom.), 1462 (w, dCAH aliph.),
CAO), 830 (w, Cp-subst. arom.).
less solid, mp 173 °C, yield 634 mg (87%). C25H25N3O7 (479.5). ½a D20
ꢄ
60-H4-methoxybenzyl). IR (neat):
~
m
(m,
mCAH aliph.), 1611 (w)/1510 (m, m
+101 (c 0.53; CH2Cl2). MS (EI): m/z [%] = 479 (M, 92), 313
(MꢁO2CPhNO2, 73), 121 (CH2PhOCH3, 100). 1H NMR (CDCl3): d
[ppm] = 1.76–1.86 (m, 1H, CH2CH2), 1.89–2.00 (m, 1H, CH2CH2),
2.18–2.30 (m, 2H, CH2CH2), 3.74 (s, 3H, ArOCH3), 3.88 (dd,
J = 14.9/6.3 Hz, 1H, NCH2CH@CH2), 4.01 (dd, J = 4.7/2.3 Hz, 1H, 5-
H), 4.14–4.22 (m, 2H, 1-H, NCH2CH@CH2 (1H)), 4.44 (d,
J = 14.9 Hz, 1H, NCH2Ar), 4.82 (d, J = 14.9 Hz, 1H, NCH2Ar), 5.22–
5.35 (m, 3H, 2-H, NCH2CH@CH2), 5.59–5.70 (ddt, J = 16.4/10.2/
1243 (m)/1098 (m,
m
5.29. (ꢁ)-(1S,2S,5R)-6-Allyl-2-methoxy-8-(4-methoxybenzyl)-
6,8-diazabicyclo[3.2.2]nonane (ent-16a)
As described for the preparation of 16a, the enantiomer ent-14a
(109 mg, 0.32 mmol) was reacted with LiAlH4 (60 mg, 1.6 mmol) in

788
R. Holl et al. / Bioorg. Med. Chem. 17 (2009) 777–793
6.3 Hz, 1H, NCH2CH@CH2), 6.78 (m, 2H, 30-H4-methoxybenzyl, 50-
H4-methoxybenzyl), 7.12 (m, 2H, 20-H4-methoxybenzyl, 60-H4-methoxybenzyl),
7.92 (m, 2H, 20-H4-nitrobenzoate, 60-H4-nitrobenzoate), 8.27 (m, 2H, 30-
~
in vacuo. The residue was purified by FC (Ø = 3 cm, h = 15 cm, ethyl
acetate, V = 20 mL, Rf = 0.30) to give 18a as a colorless solid, mp
111 °C, yield 393 mg (90%). C18H22N2O4 (330.4). Calculated C,
H4-nitrobenzoate
,
50-H4-nitrobenzoate). IR (neat):
m
[cmꢁ1] = 3076 (m,
65.44; H, 6.71; N, 8.48; found: C, 65.17; H, 6.62; N, 8.30. ½a D20
ꢄ
m
m
CAH arom.), 2964 (w,
C@O amide), 1614 (m,
N@O), 1233 (s)/1110 (m, m
m
m
CAH aliph.), 1715 (s,
C@C arom.), 1514 (s,
CAO), 824 (m, Cp-subst. arom.).
m
C@O ester), 1692 (s)/1679 (s,
+31.5 (c 0.26; CH2Cl2). MS (EI): m/z [%] = 330 (M, 100), 209
(MꢁCH2PhOCH3, 7), 121 (CH2PhOCH3, 83). 1H NMR (CDCl3): d
[ppm] = 1.56–1.69 (m, 2H, 3-H (1H), 4-H (1H)), 1.93–2.03 (m, 1H,
3-H), 2.05–2.17 (m, 1H, 4-H), 2.31 (d br, J = 5.5 Hz, 1H, OH), 3.71–
3.80 (m, 4H, ArOCH3 (3H), NCH2CH@CH2 (1H)), 3.88 (dd, J = 5.5/
1.8 Hz, 1H, 5-H), 3.96–4.03 (m, 2H, 1-H, 2-H), 4.14 (ddt, J = 15.7/
6.4/1.8 Hz, 1H, NCH2CH@CH2), 4.38 (d, J = 14.6 Hz, 1H, NCH2Ar),
4.91 (d, J = 14.6 Hz, 1H, NCH2Ar), 5.18–5.26 (m, 2H, NCH2CH@CH2),
5.65–5.76 (m, 1H, NCH2CH@CH2), 6.82–6.87 (m, 2H, 30-
mN@O), 1452 (s, dCAH aliph.),
1344 (m,
m
5.33. (ꢁ)-[(1R,2R,5R)-6-Allyl-8-(4-methoxybenzyl)-7,9-dioxo-
6,8-diazabicyclo[3.2.2]non-2-yl] 4-nitrobenzoate (ent-17a)
As described for the preparation of 17a, the enantiomer ent-13a
(250 mg, 0.76 mmol) was reacted with triphenylphosphine
(992 mg, 3.78 mmol), 4-nitrobenzoic acid (632 mg, 3.78 mmol)
and diisopropyl azodicarboxylate (0.75 mL, 765 mg, 3.78 mmol)
in THF (50 mL) to give ent-17a as a colorless solid, mp 172 °C, yield
H4-methoxybenzyl
,
50-H4-methoxybenzyl), 7.16–7.21 (m, 2H, 20-
H4-methoxybenzyl, 60-H4-methoxybenzyl). IR (neat):
[cmꢁ1] = 3337 (m
~
m
br,
(m)/1515 (m,
CAO), 829 (w, Cp-subst. arom.).
mOH), 2931 (m, mCAH aliph.), 1672 (s)/1652 (s, mC@O amide), 1613
275 mg (76%). C25H25N3O7 (479.5). ½a D20
ꢄ
ꢁ98 (c 0.13; CH2Cl2).
m
C@C arom.), 1455 (m, dCAH aliph.), 1241 (m)/1066 (m,
m
5.34. (+)-[(1S,2S,5S)-6-Allyl-8-(2,4-dimethoxybenzyl)-7,9-dioxo-
6,8-diazabicyclo[3.2.2]non-2-yl] 4-nitrobenzoate (17b)
5.37. (ꢁ)-(1R,2R,5R)-6-Allyl-2-hydroxy-8-(4-methoxybenzyl)-
6,8-diazabicyclo[3.2.2]nonane-7,9-dione (ent-18a)
Under N2 13b (1.70 g, 4.72 mmol), triphenylphosphine (4.95 g,
18.9 mmol) and 4-nitrobenzoic acid (3.15 g, 18.9 mmol) were dis-
solved in THF (300 mL). Under ice-cooling diisopropyl azodicar-
boxylate (3.74 mL, 3.82 g, 18.9 mmol) was added dropwise. The
mixture was stirred at 0 °C for 10 min and then at rt for 6 h.
Thereafter the solvent was removed in vacuo and the residue
was purified by FC (Ø = 8 cm, h = 15 cm, cyclohexane/ethyl ace-
tate = 1:1, V = 30 mL, Rf = 0.16) to give 17b as a colorless solid,
As described for the preparation of 18a, the enantiomer ent-17a
(275 mg, 0.57 mmol) was reacted with potassium carbonate
(198 mg, 1.43 mmol) in methanol (50 mL) to give ent-18a as a col-
orless solid, mp 111 °C, yield 158 mg (83%). C18H22N2O4 (330.4).
Calculated C, 65.44; H, 6.71; N, 8.48; found: C, 65.21; H, 6.67; N,
8.33. ½a 2D0
ꢁ29.3 (c 0.18; CH2Cl2).
ꢄ
mp 169 °C, yield 1.87 g (78%). C26H27N3O8 (509.5). ½a 20
ꢄ
+79.5 (c
0.51; CH2Cl2). MS (EI): m/z [%] = 509 (M, 44), 343 (MꢁDO2CPhNO2,
5.38. (+)-(1S,2S,5S)-6-Allyl-8-(2,4-dimethoxybenzyl)-2-hydroxy-
6,8-diazabicyclo[3.2.2]nonane-7,9-dione (18b)
26), 151 (2,4-dimethoxybenzyl, 100). 1H NMR (CDCl3):
d
[ppm] = 1.73–1.83 (m, 1H, CH2CH2), 1.90–2.01 (m, 1H, CH2CH2),
2.18–2.28 (m, 2H, CH2CH2), 3.67 (s, 3H, ArOCH3), 3.74 (s, 3H, Ar-
OCH3), 3.84 (dd, J = 14.9/6.3 Hz, 1H, NCH2CH@CH2), 3.94 (m, 1H,
5-H), 4.16 (dd, J = 14.9/6.3 Hz, 1H, NCH2CH@CH2), 4.34 (d,
J = 14.1 Hz, 1H, NCH2Ar), 4.38 (d, J = 1.6 Hz, 1H, 1-H), 4.94 (d,
J = 14.9 Hz, 1H, NCH2Ar), 5.18–5.24 (m, 2H, NCH2CH@CH2), 5.35
(ddd, J = 9.4/4.7/1.6 Hz, 1H, 2-H), 5.74 (ddt, J = 17.2/10.2/6.3 Hz, 1H,
NCH2CH@CH2), 6.33 (d, J = 2.3 Hz, 1H, 30-H2,4-dimethoxybenzyl), 6.38
(dd, J = 8.6/2.3 Hz, 1H, 50-H2,4-dimethoxybenzyl), 7.13 (d, J = 8.6 Hz,
The ester 17b (1.87 g, 3.67 mmol) and potassium carbonate
(1.27 g, 9.18 mmol) were suspended in methanol (200 mL) and
the mixture was stirred at rt for 16 h. Then water was added and
the mixture was extracted with CH2Cl2 (3ꢃ). The combined organic
layers were dried (Na2SO4), filtered and the solvent was removed
in vacuo. The residue was purified by FC (Ø = 5 cm, h = 15 cm, ethyl
acetate, V = 30 mL, Rf = 0.22) to give 18b as a colorless solid, mp
159 °C, yield 1.22 g (92%). C19H24N2O5 (360.4). Calculated C,
63.32; H, 6.71; N, 7.77; found: C, 63.44; H, 6.76; N, 7.42. ½a D20
ꢄ
1H, 60-H2,4-dimethoxybenzyl), 8.08 (d, J = 8.6 Hz, 2H, 20-H4-nitrobenzoate
,
+41.6 (c 0.20; CH2Cl2). MS (EI): m/z [%] = 360 (M, 38), 151 (2,4-
dimethoxybenzyl, 100). 1H NMR (CDCl3): d [ppm] = 1.56–1.71 (m,
2H, 3-H (1H), 4-H (1H)), 1.92–2.02 (m, 1H, 3-H), 2.04–2.14 (m,
1H, 4-H), 3.70–3.80 (m, 7H, ArOCH3 (6H), NCH2CH@CH2 (1H)),
3.84 (dd, J = 5.5/2.3 Hz, 1H, 5-H), 3.98–4.03 (m, 1H, 2-H), 4.06–
4.16 (m, 2H, 1-H, NCH2CH@CH2 (1H)), 4.40 (d, J = 14.1 Hz, 1H,
NCH2Ar), 4.87 (d, J = 14.1 Hz, 1H, NCH2Ar), 5.13–5.23 (m, 2H,
NCH2CH@CH2), 5.69 (ddt, J = 17.2/10.2/6.3 Hz, 1H, NCH2CH@CH2),
6.42–6.47 (m, 2H, 30-H2,4-dimethoxybenzyl, 50-H2,4-dimethoxybenzyl), 7.19
(d, J = 7.8 Hz, 1H, 60-H2,4-dimethoxybenzyl). The signal for the proton
~
60-H4-nitrobenzoate), 8.30 (d, J = 8.6 Hz, 2H, 30-H4-nitrobenzoate
,
50-
[cmꢁ1] = 3076 (w,
m
CAH arom.), 2934 (m,
~
H4-nitrobenzoate). IR (neat):
m
mC AH aliph.), 1720 (s, mC@ O ester), 1685 (s, mC@ O amide), 1608 (m,
mC@C arom.), 1523 (s, N@O), 1455 (m, dCAH aliph.), 1347 (m, N@O),
1269 (s)/1103 (m, CAO), 824 (m, Ctri-subst. arom.).
m
m
m
5.35. (ꢁ)-[(1R,2R,5R)-6-Allyl-8-(2,4-dimethoxybenzyl)-7,9-
dioxo-6,8-diazabicyclo[3.2.2]non-2-yl] 4-nitrobenzoate (ent-17b)
As described for the preparation of 17b, the enantiomer ent-13b
(513 mg, 1.42 mmol) was reacted with triphenylphosphine (1.87 g,
7.12 mmol), 4-nitrobenzoic acid (1.19 g, 7.12 mmol) and diisopro-
pyl azodicarboxylate (1.41 mL, 1.44 g, 7.12 mmol) in THF (70 mL)
to give ent-17b as a colorless solid, mp 168 °C, yield 574 mg
of the OH group could not be detected. IR (neat):
(m br,
1615 (m)/1585 (m)/1510 (m, m C@C arom.), 1454 (m, d CAH aliph.),
1211 (m)/1129 (m, CAO), 819 (w, Ctri-subst. arom.).
m
[cmꢁ1] = 3419
OAH), 2945 (m, mCAH aliph.), 1675 (s)/1652 (s, mC@O amide),
m
m
(79%). C26H27N3O8 (509.5). ½a D20
ꢁ79.1 (c 0.27; CH2Cl2).
ꢄ
5.39. (ꢁ)-(1R,2R,5R)-6-Allyl-8-(2,4-dimethoxybenzyl)-2-
hydroxy-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (ent-18b)
5.36. (+)-(1S,2S,5S)-6-Allyl-2-hydroxy-8-(4-methoxybenzyl)-6,8-
diazabicyclo[3.2.2]nonane-7,9-dione (18a)
As described for the preparation of 18b, the enantiomer ent-17b
(512 mg, 1.00 mmol) was reacted with potassium carbonate
(347 mg, 2.51 mmol) in methanol (70 mL) to give ent-18b as a col-
orless solid, mp 159 °C, yield 343 mg (95%). C19H24N2O5 (360.4).
Calculated C, 63.32; H, 6.71; N, 7.77; found: C, 63.47; H, 6.67; N,
The ester 17a (634 mg, 1.32 mmol) and potassium carbonate
(457 mg, 3.31 mmol) were suspended in methanol (70 mL) and
the mixture was stirred at rt for 16 h. Then water was added and
the mixture was extracted with CH2Cl2 (3ꢃ). The combined organic
layers were dried (Na2SO4), filtered and the solvent was removed
7.65. ½a 2D0
ꢁ40.4 (c 0.23; CH2Cl2).
ꢄ

R. Holl et al. / Bioorg. Med. Chem. 17 (2009) 777–793
789
[cmꢁ1] = 3102 (w,
m
CAH arom.), 2952 (w,
m
CAH aliph.), 1672 (s,
~
5.40. (ꢁ)-(1S,2S,5S)-6-Allyl-2-methoxy-8-(4-methoxybenzyl)-
(neat):
m
6,8-diazabicyclo[3.2.2]nonane-7,9-dione (19a)
mC@ O amide), 1617 (m)/1591 (m)/1506 (m, m C@C arom.), 1456 (m,
dCAH aliph.), 1210 (m)/1096 (m, CAO), 831 (w, Ctri-subst. arom.).
m
Under N2 and ice-cooling 60% NaH suspension in paraffin oil
(52 mg, ca. 31 mg NaH, 1.31 mmol) was added to a solution of 18a
(72 mg, 0.22 mmol) THF (20 mL). After 20 min methyl iodide
(0.04 mL, 93 mg, 0.65 mmol) was added dropwise and the mixture
was stirred at rt for 16 h. Then the solvent was removed in vacuo,
the residue was dissolved in CH2Cl2 and washed with water (2ꢃ),
0.5 M HCl (1ꢃ) and 0.5 M NaOH (1ꢃ). The organic layer was dried
(Na2SO4), filtered and the solvent was removed in vacuo. The residue
was purified by FC (Ø = 1 cm, h = 15 cm, cyclohexane/ethyl ace-
tate = 2:1, V = 5 mL, Rf = 0.04) to give 19a as a colorless solid, mp
118 °C, yield 61 mg (81%). C19H24N2O4 (344.4). Calculated C, 66.26;
5.43. (ꢁ)-(1R,2R,5R)-6-Allyl-8-(2,4-dimethoxybenzyl)-2-
methoxy-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (ent-19b)
As described for the preparation of 19b, the enantiomer ent-18b
(102 mg, 0.28 mmol), 60% NaH suspension in paraffin oil (68 mg,
ca. 41 mg NaH, 1.70 mmol) and methyl iodide (0.05 mL, 121 mg,
0.85 mmol) were reacted in THF (20 mL) to give ent-19b as a col-
orless solid, mp 116 °C, yield 85 mg (80%). C20H26N2O5 (374.4). Cal-
culated C, 64.16; H, 7.00; N, 7.48; found: C, 64.21; H, 6.99; N, 7.32.
½
a 2D0
ꢄ
ꢁ5.9 (c 0.48; CH2Cl2).
H, 7.02; N, 8.13; found: C, 66.27; H, 6.89; N, 8.00. ½a D20
ꢁ9.9 (c 0.18;
ꢄ
5.44. (+)-(1R,2S,5S)-6-Allyl-8-(4-methoxybenzyl)-6,8-
diazabicyclo[3.2.2]nonan-2-ol (20a)
CH2Cl2). MS (EI): m/z [%] = 344 (M, 32), 303 (MꢁCH2CH@CH2, 4),
223 (MꢁCH2PhOCH3, 10), 121 (CH2PhOCH3, 100). 1H NMR (CDCl3):
d [ppm] = 1.55–1.75 (m, 2H, 3-H (1H), 4-H (1H)), 2.03–2.18 (m, 2H,
3-H (1H), 4-H (1H)), 3.31 (s, 3H, OCH3), 3.48 (ddd, J = 8.6/5.5/1.6 Hz,
1H, 2-H), 3.72–3.79 (m, 4H, ArOCH3, NCH2CH@CH2 (1H)), 3.88 (dd,
J = 5.5/1.6 Hz, 1H, 5-H), 3.97 (d, J = 14.1 Hz, 1H, NCH2Ar), 4.02 (d,
J = 1.6 Hz, 1H, 1-H), 4.15 (ddt, J = 14.9/6.3/1.6 Hz, 1H, NCH2CH@CH2),
5.18–5.28 (m, 3H, NCH2Ar (1H), NCH2CH@CH2), 5.71 (ddt,
J = 16.4/10.2/6.3 Hz, 1H, NCH2CH@CH2), 6.85 (d, J = 8.6 Hz, 2H, 30-
Under N2 LiAlH4 (68 mg, 1.78 mmol) was added to an ice-cooled
solution of 18a (118 mg, 0.36 mmol) in THF (20 mL). The mixture
was stirred at 0 °C for 10 min and then heated to reflux for 16 h. Then
water was added under ice-cooling until H2 formation was finished.
The mixture was stirred at 0 °C for 10 min and then heated to reflux
for 30 min. After cooling down the mixture was filtered, the solvent
was removed in vacuo and the residue was purified by FC (Ø = 2 cm,
h = 15 cm, CH2Cl2/methanol = 50:1, V = 10 mL, Rf = 0.11) to give 20a
as a yellow oil, yield 38 mg (35%). C18H26N2O2 (302.4). Purity by
HPLC: method 2d: tR = 23.3 min, purity 97.0%; method 2f:
H4-methoxybenzyl
,
50-H4-methoxybenzyl), 7.14 (d, J = 8.6 Hz, 2H,
20-H4-methoxybenzyl, 60-H4-methoxybenzyl). IR (neat):
[cmꢁ1] = 3086 (w,
~
m
m
CAH arom.), 2969 (w,
(m, C@C arom.), 1456 (m, dCAH aliph.), 1250 (m)/1093 (m,
(w, Cp-subst. arom.).
m
CAH aliph.), 1676 (s,
m
C@O amide), 1609 (m)/1510
m
mCAO), 837
tR = 16.6 min, purity 98.8%. ½a D20
ꢄ
+27.4 (c 0.39; CH2Cl2). MS (EI): m/z
[%] = 302 (M, 37), 181 (MꢁCH2PhOCH3, 31), 121 (CH2PhOCH3,
100). 1H NMR (CDCl3): d [ppm] = 1.52–1.64 (m, 2H, 3-H (1H), 4-H
(1H)), 1.71–1.80 (m, 1H, 4-H), 1.93–2.03 (m, 1H, 3-H), 2.57 (dd,
J = 11.0/2.3 Hz, 1H, 7-H or 9-H), 2.62 (dd, J = 11.0/2.3 Hz, 1H, 7-H or
9-H), 2.79 (dd, J = 11.0/3.9 Hz, 1H, 7-H or 9-H), 2.84–2.94 (m, 3H,
1-H, 5-H, 7-H or 9-H), 3.13 (d, J = 6.3 Hz, 2H, NCH2CH@CH2), 3.67
(d, J = 12.5 Hz, 1H, NCH2Ar), 3.72 (d, J = 12.5 Hz, 1H, NCH2Ar), 3.75–
3.80 (m, 1H, 2-H), 3.79 (s, 3H, ArOCH3), 5.07 (dd, J = 10.2/1.6 Hz,
1H, NCH2CH@CH2), 5.16 (dd, J = 17.2/2.3 Hz, 1H, NCH2CH@CH2),
5.80 (ddt, J = 17.2/10.2/6.3 Hz, 1H, NCH2CH@CH2), 6.85 (d,
5.41. (+)-(1R,2R,5R)-6-Allyl-2-methoxy-8-(4-methoxybenzyl)-
6,8-diazabicyclo[3.2.2]nonane-7,9-dione (ent-19a)
As described for the preparation of 19a, the enantiomer ent-18a
(112 mg, 0.34 mmol), 60% NaH suspension in paraffin oil (81 mg,
ca. 49 mg NaH, 2.03 mmol) and methyl iodide (0.06 mL, 144 mg,
1.02 mmol) were reacted in THF (30 mL) to give ent-19a as a color-
less solid, mp 118 °C, yield 97 mg (83%). C19H24N2O4 (344.4). Calcu-
lated C, 66.26; H, 7.02; N, 8.13; found: C, 66.23; H, 6.96; N, 8.03.
,
J = 8.6 Hz, 2H, 30-H4-methoxybenzyl 50-H4-methoxybenzyl), 7.23 (d,
½
a 2D0
+9.7 (c 0.33; CH2Cl2).
ꢄ
J = 8.6 Hz, 2H, 20-H4-methoxybenzyl, 60-H4-methoxybenzyl). The signal for
the proton of the OH group could not be detected. 13C NMR (CDCl3):
d (ppm) = 28.1 (1C, C-4), 30.1 (1C, C-3), 49.1 (1C, C-7 or C-9), 51.9 (1C,
C-9 or C-7), 53.7 (1C, C-5), 55.5 (1C, ArOCH3), 59.5 (1C,
NCH2CH@CH2), 60.2 (1C, C-1), 61.6 (1C, NCH2Ar), 70.6 (1C, C-2),
5.42. (+)-(1S,2S,5S)-6-Allyl-8-(2,4-dimethoxybenzyl)-2-
methoxy-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (19b)
Under N2 and ice-cooling 60% NaH suspension in paraffin oil
(55 mg, ca. 33 mg NaH, 1.37 mmol) was added to a solution of 18b
(82 mg, 0.23 mmol) in THF (20 mL). After 20 min methyl iodide
(0.04 mL, 97 mg, 0.68 mmol) was added dropwise and the mixture
was stirred at rt for 16 h. Then the solvent was removed in vacuo,
the residue was dissolved in CH2Cl2 and washed with water (2ꢃ),
0.5 M HCl (1ꢃ) and 0.5 M NaOH (1ꢃ). The organic layer was dried
(Na2SO4), filtered and the solvent was removed in vacuo. The residue
was purified by FC (Ø = 1 cm, h = 15 cm, cyclohexane/ethyl ace-
tate = 2:1, V = 5 mL, Rf = 0.03) to give 19b as a colorless solid, mp
116 °C, yield 83 mg (97%). C20H26N2O5 (374.4). Calculated C, 64.16;
114.1 (2C, C-304-methoxybenzyl
,
C-504-methoxybenzyl), 117.0 (1C,
NCH2CH@CH2), 130.4 (2C, C-204-methoxybenzyl C-604-methoxybenzyl),
,
130.6 (1C, C-104-methoxybenzyl), 136.7 (1C, NCH2CH@CH2), 159.2 (1C,
C-404-methoxybenzyl). IR (neat):
[cmꢁ1] = 3379 (m br,
m
OAH), 2922
~
m
(m,
m
CA H aliph.), 1611 (w)/1510 (m,
mC @C arom.), 1244 (m)/1035 (w,
mCAO), 829 (w, Cp-subst. arom.).
5.45. (ꢁ)-(1S,2R,5R)-6-Allyl-8-(4-methoxybenzyl)-6,8-
diazabicyclo[3.2.2]nonan-2-ol (ent-20a)
As described for the preparation of 20a, the enantiomer ent-18a
(98 mg, 0.30 mmol) was reacted with LiAlH4 (56 mg, 1.48 mmol) in
THF (20 mL) to give ent-20a as a yellow oil, yield 27 mg (30%).
C18H26-N2O2 (302.4). Purity by HPLC: method 2d: tR = 22.6 min,
H, 7.00; N, 7.48; found: C, 63.77; H, 6.81; N, 7.32. ½a 20
ꢄ
+5.8 (c 0.37;
CH2Cl2). MS (EI): m/z [%] = 374 (M, 42), 343 (MꢁDOCH3, 9), 223
(Mꢁ2,4-dimethoxybenzyl, 2), 151 (2,4-dimethoxybenzyl, 100). 1H
NMR (CDCl3): d [ppm] = 1.52–1.76 (m, 2H, 3-H (1H), 4-H (1H)),
2.00–2.16 (m, 2H, 3-H (1H), 4-H (1H)), 3.31 (s, 3H, OCH3), 3.43–3.48
(m, 1H, 2-H), 3.71 (ddt, J = 15.7/6.3/1.6 Hz, 1H, NCH2CH@CH2), 3.77–
3.83 (m, 7H, 5-H, ArOCH3 (6H)), 4.10–4.19 (m, 2H, NCH2CH@CH2
(1H), NCH2Ar (1H)), 4.20 (s, 1H, 1-H), 5.05 (d, J = 14.9 Hz, 1H, NCH2Ar),
5.12–5.22 (m, 2H, NCH2CH@CH2), 5.69 (ddt, J = 17.2/10.2/6.3 Hz, 1H,
purity 99.6%; method 2f: tR = 16.6 min, purity 99.0%. ½a D20
ꢁ26.7
ꢄ
(c 0.13; CH2Cl2).
5.46. (+)-(1R,2S,5S)-6-Allyl-8-(2,4-dimethoxybenzyl)-6,8-
diazabicyclo[3.2.2]nonan-2-ol (20b)
NCH2CH@CH2), 6.41–6.45 (m, 2H, 30-H2,4-dimethoxybenzyl
,
50-
Under N2 LiAlH4 (63 mg, 1.65 mmol) was added to an ice-cooled
solution of 18b (119 mg, 0.33 mmol) in THF (30 mL). The mixture
H2,4-dimethoxybenzyl), 7.14 (d, J = 8.6 Hz, 1H, 60-H2,4-dimethoxybenzyl). IR

790
R. Holl et al. / Bioorg. Med. Chem. 17 (2009) 777–793
was stirred at 0 °C for 10 min and then heated to reflux for 16 h.
Then water was added under ice-cooling until H2 formation was
finished. The mixture was stirred at 0 °C for 10 min and then
heated to reflux for 30 min. After cooling down the mixture was fil-
tered, the solvent was removed in vacuo and the residue was puri-
fied by FC (Ø = 2 cm, h = 15 cm, CH2Cl2/methanol = 50:1, V = 10 mL,
Rf = 0.11) to give 20b as a yellow oil, yield 22 mg (20%). C19H28N2O3
(332.5). Purity by HPLC: method 2d: tR = 38.3 min, purity 98.8%;
5.49. (+)-(1S,2R,5R)-6-Allyl-2-methoxy-8-(4-methoxybenzyl)-
6,8-diazabicyclo[3.2.2]nonane (ent-21a)
As described for the preparation of 21a, the enantiomer ent-19a
(97 mg, 0.28 mmol) was reacted with LiAlH4 (54 mg, 1.4 mmol) in
THF (20 mL) to give ent-21a as a yellow oil, yield 19 mg (21%).
C19H28N2O2 (316.5). Purity by HPLC: method 2c: tR = 18.1 min,
purity 98.8%; method 2f: tR = 24.0 min, purity 100%. ½a D20
ꢄ
+6.8
method 2g: tR = 31.2 min, purity 97.3%. ½a D20
ꢄ
+25.5 (c 0.54; CH2Cl2).
(c 0.58; CH2Cl2).
MS (EI): m/z [%] = 332 (M, 27), 181 (Mꢁ2,4-dimethoxybenzyl, 48),
151 (2,4-dimethoxybenzyl, 100). 1H NMR (CDCl3): d [ppm] = 1.47–
1.60 (m, 2H, 3-H (1H), 4-H (1H)), 1.67–1.77 (m, 1H, 4-H), 1.93–2.02
(m, 1H, 3-H), 2.43 (d, J = 9.4 Hz, 1H, 7-H or 9-H), 2.60 (d, J = 10.2 Hz,
1H, 7-H or 9-H), 2.77–2.98 (m, 4H, 1-H, 5-H, 7-H (1H), 9-H (1H)),
3.14 (d, J = 6.3 Hz, 2H, NCH2CH@CH2), 3.65 (d, J = 11.7 Hz, 1H,
NCH2Ar), 3.75–3.85 (m, 2H, 2-H, NCH2Ar, (1H)), 3.80 (s, 3H, Ar-
OCH3), 3.83 (s, 3H, ArOCH3), 5.02 (d, J = 10.2 Hz, 1H, NCH2CH@CH2),
5.17 (dd, J = 17.2/1.6 Hz, 1H, NCH2CH@CH2), 5.81 (ddt, J = 17.2/
10.2/6.3 Hz, 1H, NCH2CH@CH2), 6.40 (dd, J = 8.6/2.3 Hz, 1H, 50-
H2,4-dimethoxybenzyl), 6.46 (d, J = 2.3 Hz, 1H, 30-H2,4-dimethoxybenzyl),
7.08 (d, J = 8.6 Hz, 1H, 60-H2,4-dimethoxybenzyl). The signal for the pro-
~
5.50. (ꢁ)-(1R,2S,5S)-6-Allyl-8-(2,4-dimethoxybenzyl)-2-
methoxy-6,8-diazabicyclo[3.2.2]nonane (21b)
Under N2 LiAlH4 (41 mg, 1.08 mmol) was added to an ice-cooled
solution of 19b (81 mg, 0.22 mmol) in THF (20 mL). The mixture
was stirred at 0 °C for 10 min and then heated to reflux for 16 h.
Then water was added under ice-cooling until H2 formation was
finished. The mixture was stirred at 0 °C for 10 min and then
heated to reflux for 30 min. After cooling down the mixture was fil-
tered, the solvent was removed in vacuo and the residue was puri-
fied by FC (Ø = 1 cm, h = 15 cm, CH2Cl2/methanol = 50:1, V = 5 mL,
ton of the OH group could not be detected. IR (neat):
3373 (m br,
m
[cmꢁ1] =
Rf = 0.03) to give 21b as a yellow oil, yield 19 mg (25%).
m
OAH), 2921 (m, mCAH aliph.), 1612 (m)/1587 (m)/
C20H30N2O3 (346.5). Purity by HPLC: method 2c: tR = 19.2 min, pur-
1506 (m, mC@C arom.), 1207 (m)/1033 (m,
Ctri-subst. arom.).
mCAO), 833 (w,
ity 100%; method 2i: tR = 16.3 min, purity 97.2%. ½a D20
ꢁ12.7 (c 0.26;
ꢄ
CH2Cl2). MS (EI): m/z [%] = 346 (M, 100), 195 (Mꢁ2,4-dimethoxy-
benzyl, 68), 151 (2,4-dimethoxybenzyl, 80). 1H NMR (CDCl3): d
[ppm] = 1.60–1.92 (m, 3H, CH2CH2), 2.03–2.15 (m, 1H, CH2CH2),
2.76–2.90 (m, 4H, piperazine-H), 2.92–2.99 (m, 1H, piperazine-
H), 3.02–3.09 (m, 1H, piperazine-H), 3.14–3.24 (m, 2H,
NCH2CH@CH2), 3.18 (s, 3H, OCH3), 3.56 (dd, J = 10.2/4.7 Hz, 1H, 2-
H), 3.69–3.74 (m, 2H, NCH2Ar), 3.79 (s, 3H, ArOCH3), 3.80 (s, 3H, Ar-
OCH3), 5.11–5.22 (m, 2H, NCH2CH@CH2), 5.83–5.96 (m, 1H,
NCH2CH@CH2), 6.43 (d, J = 2.3 Hz, 1H, 30-H2,4-dimethoxybenzyl), 6.49
(dd, J = 7.8/2.3 Hz, 1H, 50-H2,4-dimethoxybenzyl), 7.43–7.49 (m, 1H, 60-
~
5.47. (ꢁ)-(1S,2R,5R)-6-Allyl-8-(2,4-dimethoxybenzyl)-6,8-
diazabicyclo[3.2.2]nonan-2-ol (ent-20b)
As described for the preparation of 20b, the enantiomer ent-18b
(110 mg, 0.31 mmol) was reacted with LiAlH4 (58 mg, 1.53 mmol)
in THF (30 mL) to give ent-20b as a yellow oil, yield 30 mg (30%).
C19H28N2O3 (332.5). Purity by HPLC: method 2d: tR = 34.6 min, pur-
ity 99.7%; method 2g: tR = 34.1 min, purity 97.3%. ½a D20
ꢁ26.1 (c
ꢄ
0.69; CH2Cl2).
H2,4-dimethoxybenzyl). IR (neat):
(m, CAH aliph.), 1611 (m)/1588 (m)/1506 (m,
dCAH aliph.), 1206 (m)/1097 (m, CAO), 831 (w, Ctri-subst. arom.).
m
[cmꢁ1] = 3075 (w,
m
CAH arom.), 2926
m
mC@C arom.), 1455 (m,
m
5.48. (ꢁ)-(1R,2S,5S)-6-Allyl-2-methoxy-8-(4-methoxybenzyl)-
6,8-diazabicyclo[3.2.2]nonane (21a)
5.51. (+)-(1S,2R,5R)-6-Allyl-8-(2,4-dimethoxybenzyl)-2-
methoxy-6,8-diazabicyclo[3.2.2]nonane (ent-21b)
Under N2 LiAlH4 (72 mg, 1.9 mmol) was added to an ice-cooled
solution of 19a (130 mg, 0.38 mmol) in THF (20 mL). The mixture
was stirred at 0 °C for 10 min and then heated to reflux for 16 h.
Then water was added under ice-cooling until H2 formation was
finished. The mixture was stirred at 0 °C for 10 min and then
heated to reflux for 30 min. After cooling down the mixture was fil-
tered, the solvent was removed in vacuo and the residue was puri-
fied by FC (Ø = 2 cm, h = 15 cm, CH2Cl2/methanol = 50:1, V = 10 mL,
Rf = 0.07) to give 21a as a yellow oil, yield 66 mg (55%). C19H28N2O2
(316.5). Purity by HPLC: method 2c: tR = 18.8 min, purity 97.8%;
As described for the preparation of 21b, the enantiomer ent-19b
(90 mmg, 0.24 mmol) was reacted with LiAlH4 (46 mg, 1.21 mmol)
in THF (20 mL) to give ent-21b as a yellow oil, yield 22 mg (26%).
C20H30N2O3 (346.5). Purity by HPLC: method 2c: tR = 18.9 min, pur-
ity 98.2%; method 2i: tR = 18.0 min, purity 97.4%. ½a D20
ꢄ
+13.2 (c
0.79; CH2Cl2).
5.52. (+)-[(1S,2R,5S)-6-Allyl-8-(4-methoxybenzyl)-7,9-dioxo-
6,8-diazabicyclo[3.2.2]nonan-2-yl] methanesulfonate (22a)
method 2f: tR = 23.9 min, purity 98.0%. ½a D20
ꢁ6.5 (c 0.38; CH2Cl2).
ꢄ
MS (EI): m/z [%] = 316 (M, 96), 275 (MꢁCH2CH@CH2, 6), 195
(MꢁCH2PhOCH3, 44), 121 (CH2PhOCH3, 100). 1H NMR (CDCl3): d
[ppm] = 1.59–1.69 (m, 1H, CH2CH2), 1.73–1.81 (m, 1H, CH2CH2),
1.82–1.90 (m, 1H, CH2CH2), 1.99–2.11 (m, 1H, CH2CH2), 2.65–2.71
(m, 1H, piperazine-H), 2.74–2.86 (m, 4H, piperazine-H), 2.99–
3.02 (m, 1H, piperazine-H), 3.11–3.22 (m, 2H, NCH2CH@CH2),
3.15 (s, 3H, OCH3), 3.52 (dd, J = 11.0/4.7 Hz, 1H, 2-H), 3.65 (d,
J = 13.3 Hz, 1H, NCH2Ar), 3.70 (d, J = 13.3 Hz, 1H, NCH2Ar), 3.79 (s,
3H, ArOCH3), 5.10 (d, J = 10.2 Hz, 1H, NCH2CH@CH2), 5.17 (dd,
J = 17.2/1.6 Hz, 1H, NCH2CH@CH2), 5.86 (ddt, J = 17.2/10.2/6.3 Hz,
The alcohol 13a (247 mg, 0.75 mmol), 4-(dimethylamino)pyri-
dine (183 mg, 1.50 mmol) and N,N-diisopropylethylamine
(0.62 mL, 483 mg, 3.74 mmol) were dissolved in CH2Cl2 (20 mL).
Under ice-cooling methanesulfonyl chloride (0.12 mL, 171 mg,
1.50 mmol) was slowly added and the mixture was stirred at 0 °C
for 2 h and at rt for 16 h. Then water was added and the mixture
was extracted with CH2Cl2 (3ꢃ). The combined organic layers were
dried (Na2SO4), filtered and the solvent was removed in vacuo. The
residue was purified by FC (Ø = 3 cm, h = 15 cm, cyclohexane/ethyl
acetate = 1:2, V = 20 mL, Rf = 0.28) to give 22a as a colorless solid,
1H, NCH2CH@CH2), 6.83 (d, J = 8.6 Hz, 2H, 30-H4-methoxybenzyl
,
,
mp 110 °C, yield 292 mg (96%). C19H24N2O6S (408.5). ½a D20
ꢄ
+97.8
50-H4-methoxybenzyl), 7.30 (d, J = 8.6 Hz, 2H, 20-H4-methoxybenzyl
(c 0.72; CH2Cl2). MS (EI): m/z [%] = 408 (M, 100), 121 (CH2PhOCH3,
46). 1H NMR (CDCl3): d [ppm] = 1.80–2.08 (m, 4H, 3-H, 4-H), 3.02
(s, 3H, CH3), 3.80 (s, 3H, ArOCH3), 3.95 (dd, J = 4.7/3.1 Hz, 1H, 5-
H), 3.97–4.08 (m, 2H, NCH2CH@CH2), 4.17 (d, J = 3.9 Hz, 1H, 1-H),
60-H4-methoxybenzyl). IR (neat):
m
[cmꢁ1] = 3072 (w,
m
CAH arom.), 2925
C@C arom.), 1463 (w, dCAH aliph.),
CAO), 828 (w, Cp-subst. arom.).
~
(m,
mCAH aliph.), 1611 (w)/1510 (m, m
1242 (m)/1097 (m,
m

R. Holl et al. / Bioorg. Med. Chem. 17 (2009) 777–793
791
4.43–4.48 (m, 1H, 2-H), 4.53 (s, 2H, NCH2Ar), 5.23–5.29
(m, 2H, NCH2CHH@CH2), 5.75 (ddt, J = 17.2/9.4/6.3 Hz, 1H,
5.56. (+)-(1S,5S)-6-Allyl-8-(4-methoxybenzyl)-6,8-
diazabicyclo[3.2.2]nonane (23a)
NCH2CHH@CH2), 6.87 (d, J = 8.6 Hz, 2H, 30-H4-methoxybenzyl
,
50-H4-methoxybenzyl), 7.22 (d, J = 8.6 Hz, 2H, 20-H4-methoxybenzyl, 60-
Under N2 LiAlH4 (172 mg, 4.53 mmol) was added to an ice-
cooled solution of 22a (370 mg, 0.91 mmol) in THF (50 mL). The
mixture was stirred at 0 °C for 10 min and then heated to reflux
for 16 h. Then water was added under ice-cooling until H2 forma-
tion was finished, the mixture was stirred at 0 °C for 10 min and
then heated to reflux for 30 min. After cooling down the mixture
was filtered, the solvent was removed in vacuo and the residue
was purified by FC (Ø = 2 cm, h = 15 cm, CH2Cl2/methanol = 9.5:0.5,
V = 10 mL, Rf = 0.24) to give 23a as a yellow oil, yield 120 mg (46%).
C18H26N2O (286.4). Purity by HPLC: method 2b: tR = 18.4 min, pur-
H4-methoxybenzyl). IR (neat):
(m, CAH aliph.), 1675 (s,
m
[cmꢁ1] = 3012 (w, mCA H arom.), 2935
~
m
m
C@O amide), 1612 (m)/1512 (m,
m
C@C arom.),
1455 (m, dCA H aliph.), 1244 (m,
mC-O), 1170 (m, mS@O), 829 (m,
Cp-subst. arom.).
5.53. (ꢁ)-[(1R,2S,5R)-6-Allyl-8-(4-methoxybenzyl)-7,9-dioxo-
6,8-diazabicyclo[3.2.2]nonan-2-yl] methanesulfonate (ent-22a)
As described for the preparation of 22a, the enantiomer ent-13a
(296 mg, 0.90 mmol) was reacted with 4-(dimethylamino)pyridine
(219 mg, 1.79 mmol), N,N-diisopropylethylamine (0.74 mL,
580 mg, 4.49 mmol) and methanesulfonyl chloride (0.14 mL,
214 mg, 1.89 mmol) in CH2Cl2 (20 mL) to give ent-22a as a color-
less solid, mp 111 °C, yield 256 mg (70%). C19H24N2O6S (408.5).
ity 95.8%; method 2h: tR = 15.6 min, purity 96.1%. ½a D20
ꢄ
+10.8 (c
0.18; CH2Cl2). MS (EI): m/z [%] = 286 (M, 35), 245 (MꢁCH2CH@CH2,
11), 165 (MꢁCH2PhOCH3, 81), 121 (CH2PhOCH3, 100). 1H NMR
(CDCl3): d [ppm] = 1.58–1.94 (m, 6H, 2-H, 3-H, 4-H), 2.74–2.79
(m, 2H, piperazine-H), 2.88–2.97 (m, 4H, piperazine-H), 3.17–
3.26 (m, 2H, NCH2CH@CH2), 3.65 (s, 2H, NCH2Ar), 3.79 (s, 3H, Ar-
OCH3), 5.10 (m, 1H, NCH2CH@CH2), 5.19 (m, 1H, NCH2CH@CH2),
5.92 (ddt, J = 16.4/10.2/6.3 Hz, 1H, NCH2CH@CH2), 6.84 (d,
½
a 2D0
ꢄ
ꢁ98.5 (c 0.33; CH2Cl2).
J = 8.6 Hz, 2H, 30-H4-methoxybenzyl
,
50-H4-methoxybenzyl), 7.28 (d,
5.54. (+)-[(1S,2R,5S)-6-Allyl-8-(2,4-dimethoxybenzyl)-7,9-
dioxo-6,8-diazabicyclo[3.2.2]nonan-2-yl] methanesulfonate
(22b)
J = 8.6 Hz, 2H, 20-H4-methoxybenzyl, 60-H4-methoxybenzyl). IR (neat):
~
m
[cmꢁ1] = 3073 (w,
(m, C@C arom.), 1452 (m, dCAH aliph.), 1241 (m)/1037 (m,
m
CAH arom.), 2913 (m,
mCAH aliph.), 1611 (m)/1510
m
m
CAO), 826
(m, Cp-subst. arom.).
The alcohol 13b (192 mg, 0.53 mmol), 4-(dimethylamino)pyri-
dine (130 mg, 1.07 mmol) and N,N-diisopropylethylamine
(0.44 mL, 344 mg, 2.66 mmol) were dissolved in CH2Cl2 (20 mL).
Under ice-cooling methanesulfonyl chloride (0.08 mL, 122 mg,
1.07 mmol) was slowly added and the mixture was stirred at 0 °C
for 2 h and at rt for 16 h. Then water was added and the mixture
was extracted with CH2Cl2 (3ꢃ). The combined organic layers were
dried (Na2SO4), filtered and the solvent was removed in vacuo. The
residue was purified by FC (Ø = 3 cm, h = 15 cm, cyclohexane/ethyl
acetate = 1:2, V = 20 mL, Rf = 0.24) to give 22b as a yellow oil, yield
205 g (88%). C20H26N2O7S (438.5). Purity by HPLC: method 1:
5.57. (ꢁ)-(1R,5R)-6-Allyl-8-(4-methoxybenzyl)-6,8-
diazabicyclo[3.2.2]nonane (ent-23a)
As described for the preparation of 23a, the enantiomer ent-22a
(240 mg, 0.59 mmol) was reacted with LiAlH4 (112 mg, 2.94 mmol)
in THF (40 mL) to give ent-23a as a yellow oil, yield 60 mg (36%).
C18H26N2O (286.4). Purity by HPLC: method 2b: tR = 18.9 min, pur-
ity 95.8%; method 2h: tR = 15.6 min, purity 96.8%. ½a D20
ꢁ10.9 (c
ꢄ
0.29; CH2Cl2).
tR = 18.4 min, purity 99.1%. ½a D20
ꢄ
+99.8 (c 0.64; CH2Cl2). MS (EI):
m/z [%] = 438 (M, 30), 343 (MꢁO3SCH3, 8), 287 (Mꢁ2,4-dimethoxy-
benzyl, 7), 151 (2,4-dimethoxybenzyl, 100). 1H NMR (CDCl3): d
[ppm] = 1.80–1.96 (m, 4H, 3-H, 4-H), 3.02 (s, 3H, CH3), 3.80 (s,
3H, ArOCH3), 3.83 (s, 3H, ArOCH3), 3.87–3.90 (m, 1H, 5-H), 3.96
(ddt, J = 14.9/6.3/1.6 Hz, 1H, NCH2CH@CH2), 4.05 (ddt, J = 14.9/
6.3/1.6 Hz, 1H, NCH2CH@CH2), 4.36 (d, J = 4.7 Hz, 1H, 1-H), 4.44
(d, J = 14.1 Hz, 1H, NCH2Ar), 4.55–4.59 (m, 1H, 2-H), 4.66 (d,
J = 14.1 Hz, 1H, NCH2Ar), 5.20–5.25 (m, 2H, NCH2CH@CH2), 5.74
(ddt, J = 18.0/9.4/6.3 Hz, 1H, NCH2CH@CH2), 6.43–6.46 (m, 2H, 30-
H2,4-dimethoxybenzyl, 50-H2,4-dimethoxybenzyl), 7.25 (d, J = 8.6 Hz, 1H, 60-
~
5.58. (+)-(1S,5S)-6-Allyl-8-(2,4-dimethoxybenzyl)-6,8-
diazabicyclo[3.2.2]nonane (23b)
Under N2 LiAlH4 (287 mg, 7.56 mmol) was added to an ice-
cooled solution of 22b (663 mg, 1.51 mmol) in THF (80 mL). The
mixture was stirred at 0 °C for 10 min and then heated to reflux
for 16 h. Then water was added under ice-cooling until H2 forma-
tion was finished and the mixture was stirred at 0 °C for 10 min
and then heated to reflux for 30 min. After cooling down the mix-
ture was filtered, the solvent was removed in vacuo and the resi-
due was purified by FC (Ø = 3 cm, h = 15 cm, CH2Cl2/
methanol = 9.5/0.5, V = 20 mL, Rf = 0.12) to give 23b as a yellow
oil, yield 170 mg (36%). C19H28N2O2 (316.5). Purity by HPLC: meth-
od 2b: tR = 18.2 min, purity 99.2%; method 2h: tR = 15.4 min, purity
H2,4-dimethoxybenzyl). IR (neat):
m
[cmꢁ1] = 2937 (m,
m
CAH aliph.), 1679
arom.), 1454 (m,
C@C
(s, mC@ amide), 1612 (m)/1508 (m,
m
O
dCAH aliph.), 1171 (m, mS@O), 1030 (m, mCAO), 830 (m, Ctri-subst. arom.).
5.55. (ꢁ)-[(1R,2S,5R)-6-Allyl-8-(2,4-dimethoxybenzyl)-7,9-dioxo-
95.3%. ½a 2D0
ꢄ
+6.5 (c 0.17; CH2Cl2). MS (EI): m/z [%] = 316 (M, 26), 275
6,8-diazabicyclo[3.2.2]nonan-2-yl] methanesulfonate (ent-22b)
(MꢁCH2CH@CH2, 3), 165 (Mꢁ2,4-dimethoxybenzyl, 74), 151 (2,4-
dimethoxybenzyl, 100). 1H NMR (CDCl3): d [ppm] = 1.59–1.99 (m,
6H, 2-H, 3-H, 4-H), 2.74 (d, J = 9.4 Hz, 1H, piperazine-H), 2.80 (d,
J = 10.1 Hz, 1H, piperazine-H), 2.88–3.00 (m, 4H, piperazine-H),
3.15–3.25 (m, 2H, NCH2CH@CH2), 3.63 (d, J = 14.9 Hz, 1H, NCH2Ar),
3.71 (d, J = 14.9 Hz, 1H, NCH2Ar), 3.79 (s, 3H, ArOCH3), 3.80 (s, 3H,
ArOCH3), 5.08 (d, J = 10.2 Hz, 1H, NCH2CH@CH2), 5.18 (dd, J = 17.2/
1.6 Hz, 1H, NCH2CH@CH2), 5.90 (ddt, J = 17.2/10.2/6.3 Hz, 1H,
NCH2CH@CH2), 6.43 (d, J = 2.3 Hz, 1H, 30-H2,4-dimethoxybenzyl), 6.47
(dd, J = 8.6/2.3 Hz, 1H, 50-H2,4-dimethoxybenzyl), 7.36 (d, J = 8.6 Hz,
~
The alcohol ent-13b (725 mg, 2.01 mmol) and triethylamine
(1.1 mL, 814 mg, 8.05 mmol) were dissolved in CH2Cl2 (30 mL). Un-
der ice-cooling methanesulfonyl chloride (0.31 mL, 461 mg,
4.02 mmol) was slowly added and the mixture was stirred at 0 °C
for 2 h and at rt for 16 h. Then water was added and the mixture
was extracted with CH2Cl2 (3ꢃ). The combined organic layers were
dried (Na2SO4), filtered and the solvent was removed in vacuo. The
residue was purified by FC (Ø = 4 cm, h = 15 cm, cyclohexane/ethyl
acetate = 1:2, V = 30 mL, Rf = 0.24) to give ent-22b as a yellow oil,
1H, 60-H2,4-dimethoxybenzyl). IR (neat):
2917 (m,
(m, dCAH aliph.), 1206 (m)/1037 (m,
m
[cmꢁ1] = 3073 (w,
C@C arom.), 1453
mCAO), 831 (m, Ctri-subst. arom.).
mCAH arom.),
yield 572 mg (65%). C20H26N2O7S (438.5). ½a D20
ꢄ
ꢁ96.8 (c 0.66;
mCAH aliph.), 1611 (m)/1588 (m)/1505 (m, m
CH2Cl2).

792
R. Holl et al. / Bioorg. Med. Chem. 17 (2009) 777–793
5.59. (ꢁ)-(1R,5R)-6-Allyl-8-(2,4-dimethoxybenzyl)-6,8-
umes 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 31,000g for 20 min at 4 °C. The pellet was resus-
pended 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 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 Bradford18 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.
diazabicyclo[3.2.2]nonane (ent-23b)
As described for the preparation of 23b, the enantiomer ent-22b
(572 mg, 1.30 mmol) was reacted with LiAlH4 (248 mg, 6.52 mmol)
in THF (70 mL) to give ent-23b as a yellow oil, yield 65 mg (16%).
C19H28N2O2 (316.5). Purity by HPLC: method 2b: tR = 17.1 min, pur-
ity 98.8%; method 2h: tR = 14.9 min, purity 96.6%. ½a D20
ꢁ6.3 (c 0.29;
ꢄ
CH2Cl2).
6. Receptor binding studies
6.1. Materials and general procedures
6.5. Performing of the r2 assay (modified according to Ref. 9)
The test was performed with the radioligand [3H]-ditolylguani-
dine (50 Ci/mmol; ARC). The thawed membrane preparation
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 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 rt, the scintillation was measured
using a MicroBeta Trilux scintillation analyzer (Perkin-Elmer). The
counting efficiency was 20%.
(about 100 lg of the protein) was incubated with various concen-
trations of test compounds, 3 nM [3H]-ditolylguanidine, 500 nM
(+)-pentazocine, and buffer (50 mM Tris, pH 8.0) in a total volume
of 200 lL 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 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 scintillator was allowed to solidify
at room temperature. The bound radioactivity trapped on the fil-
ters was counted in the scintillation analyzer. The non-specific
binding was determined with 10 lM unlabeled ditolylguanidine.
The Kd-value of the radioligand [3H]-ditolylguanidine is
6.2. Membrane preparation for the r1 assay (modified
according to Ref. 9)
17.9 nM.20
Five guinea pig brains were homogenized with the potter (500–
800 rpm, 10 up-and-down strokes) in six volumes of cold 0.32 M su-
crose. The suspension was centrifuged at 1200g 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 buffer
(50 mM Tris, pH 7.4) and centrifuged again at 23,500g (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 Bradford18 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.
6.6. NMDA assay
The preparation of the receptor material and the assay were
performed according to a literature procedure.14
6.7. 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
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.21 The Ki-
values are given as mean value SEM from three independent
experiments.
6.3. Performing of the r1 assay (modified according to Ref. 9)
The test was performed with the radioligand [3H]-(+) pentazo-
cine (42.5 Ci/mmol; Perkin-Elmer). The thawed membrane prepa-
ration (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
180 min at 37 °C. The incubation was terminated by rapid filtration
through the presoaked filtermats by using the cell harvester. After
lg of the protein) was incubated with various
6.8. Cytotoxicity assay
l
All cell lines were obtained from the German Collection of
Microbiology and Cell Culture (DSZK, Braunschweig, FRG). Cyto-
toxicity testing was done by using a microtiter assay based on
staining cells with crystal violet as described in detail elsewhere.17
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.
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 room temperature. The bound
radioactivity trapped on the filters was counted in the scintillation
analyzer. The non-specific binding was determined with 10 lM
unlabeled (+) pentazocine. The Kd-value of the radioligand [3H]-
(+)-pentazocine is 2.9 nM.19
Acknowledgments
6.4. Membrane preparation for the r2 assay (modified
according to Ref. 9)
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.
Two rat livers were cut into smaller pieces and homogenized
with a potter (500–800 rpm, 10 up-and-down strokes) in six vol-

R. Holl et al. / Bioorg. Med. Chem. 17 (2009) 777–793
793
9. Geiger, C.; Zelenka, C.; Weigl, M.; Fröhlich, R.; Wibbeling, B.; Lehmkuhl, K.;
Schepmann,D.;Grünert, R.;Bednarski, P.J.;Wünsch,B.J.Med.Chem.2007, 50, 6144.
10. Jung, B.; Englberger, W.; Wünsch, B. Arch. Pharm. Chem. Life Sci. 2005, 338, 281.
11. Mitsunobu, O. Synthesis 1981, 1.
12. Maier, C. A.; Wünsch, B. J. Med. Chem. 2002, 45, 438.
13. Glennon, R. A.; Ablordeppey, S. Y.; Ismaiel, A. M.; El-Ashmawy, M. B.; Fischer, J.
B.; Howie, K. B. J. Med. Chem. 1994, 37, 1214.
14. Wirt, U.; Schepmann, D.; Wünsch, B. Eur. J. Org. Chem. 2007, 462.
15. Aydar, E.; Palmer, C. P.; Djamgoz, M. B. A. Cancer Res. 2004, 64, 5029.
16. Colabufo, N. A.; Berardi, F.; Contino, M.; Niso, M.; Abate, C.; Perrone, R.;
Tortorella, V. Naunyn Schmiedebergs Arch. Pharmacol. 2004, 370, 106.
17. Bracht, K.; Boubakari; Grunert, R.; Bednarski, P. J. Anticancer Drugs 2006, 17, 41.
18. Bradford, M. M. Anal. Biochem. 1976, 72, 248.
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