Synthesis and biological evaluation of conformationally restricted σ1 receptor ligands with 7,9-diazabicyclo[4.2.2]decane scaffold

Article abstract of DOI:10.1039/c0ob00402b

The key step in the synthesis of the 7,9-diazabicyclo[4.2.2]decane system was a modified Dieckmann condensation of piperazinebutyrate 11, which makes use of trapping the first cyclized intermediate with TMS-Cl. Reduction of the bicyclic ketone 14 with LiB

Full text of DOI:10.1039/c0ob00402b

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and biological evaluation of conformationally restricted r1 receptor
ligands with 7,9-diazabicyclo[4.2.2]decane scaffold†
Sunil K. Sunnam,a Dirk Schepmann,a Elisabeth Rack,a Roland Fro¨hlich,b Katharina Korpis,c
Patrick J. Bednarskic and Bernhard Wu¨nsch*a
Received 8th July 2010, Accepted 7th September 2010
DOI: 10.1039/c0ob00402b
The key step in the synthesis of the 7,9-diazabicyclo[4.2.2]decane system was a modified Dieckmann
condensation of piperazinebutyrate 11, which makes use of trapping the first cyclized intermediate with
TMS-Cl. Reduction of the bicyclic ketone 14 with LiBH4 at -90 ◦C provided diastereoselectively
(>99 : 1) the syn-configured alcohol 15a, which was converted into the final alcohol and ethers 16a–g.
The configuration at the 2-position was established by X-ray structure analysis of methyl and ethyl
ethers 15b and 15c. In contrast to bicyclic systems with a three-carbon bridge, inversion of the
configuration at the 2-position of the alcohol 15a failed to give the inverted alcohol 19a. However, an
unselective reduction of the ketone 24 with L-Selectride led to the diastereomeric alcohols 16a and 25a
in the ratio 36 : 64. LiAlH4 reduction of the tosylate 20 and the alkene 18 yielded the
diazabicyclo-decane 26 and -decene 27 without further substituents at the four-carbon bridge. The s1
and s2 receptor affinities were investigated in receptor binding studies with radioligands. All test
compounds showed a lower s1 affinity than the corresponding bicyclic derivatives with a
three-membered bridge. The reduced s1 receptor affinity is attributed to the larger four-membered
bridge. This hypothesis is supported by the alkene 27, which represents the most potent s1 ligand of this
series (Ki = 7.5 nM). In the alkene 27 the size and flexibility of the bridge is considerably reduced by the
double bond. The methyl ether 25b and the unsubstituted derivatives 26 and 27 revealed moderate
inhibition of the growth of the human tumor cell lines A-427, 5637 and MCF-7. Again, these
compounds are less potent than the analogues with a three-membered bridge. The IC50-value of the
most potent s1 ligand 27 against the small cell lung cancer cell line A-427 (IC50 = 10 mM) should be
emphasized, since this cell line is particularly sensitive to homologues with a three-carbon bridge.
various systems including the glutamatergic,7 dopaminergic8 and
1. Introduction
cholinergic9 neurotransmission. Additionally, the influence on the
s Receptors, which were initially misclassified as a subtype of
opioid receptors1 are now generally accepted as a separate class
of receptors including two subtypes termed s1 and s2 receptors.2
Cloning of the s1 receptor subtype has proved that this receptor
does not have any structural similarity with any other known
mammalian protein. However 30% homology to the yeast enzyme
D8/D7-isomerase was found.3 Aydar et al. have postulated a s1
receptor model with two transmembrane domains and both the
amino and carboxy termini located intracellularly.4 Site directed
mutagenesis experiments have unraveled that the acidic amino
acids aspartate 126 and glutamate 172 in the carboxy terminus,5
as well as serine 99 and tyrosine 103, which are located in or close
to the second transmembrane helix (first steroid binding domain)
are crucial for (+)-pentazocine binding.6
activity of a variety of ion channels including K+-channels10,11 and
Ca2+-channels12 is an important feature of s1 receptors.
Both s receptor subtypes are involved in neuromodulatory
processes and, in particular the s1 receptor can be exploited as
target for the development of novel drugs for the treatment of
different neurological disorders, e.g. schizophrenia,13 depression,14
dementia15 and cocaine induced locomotor activity and toxicity.16
In addition to the relevance of s receptors in neurological
disorders, overexpression of s1 and s2 receptors in various human
tumor cell lines, including breast, lung and prostate cancer cell
lines, has been found.17,18 Since antiproliferative and cytotoxic
activity of s1 antagonists and s2 agonists has been shown, both s
receptor subtypes represent interesting targets for the development
of novel antitumor drugs.9
The class of s1 receptor ligands comprises potent but struc-
turally very diverse ligands, including aryl alkyl amines (e.g. 1),19
guanidine derivatives (e.g. di-o-tolylguanidine 2),20 dextrorotatory
benzomorphans (e.g. (+)-pentazocine 3),21 and spirocyclic com-
pounds (e.g. 4) (Fig. 1).22–27
Although the intracellular signal transduction pathway is not
yet elucidated, s1 receptors are involved in the modulation of
aInstitut fu¨r Pharmazeutische und Medizinische Chemie der Univer-
sita¨t Mu¨nster, Hittorfstraße 58-62, D-48149, Mu¨nster, Germany. E-mail:
wuensch@uni-muenster.de; Fax: +49-251-8332144; Tel: +49-251-8333311
bOrganisch-Chemisches Institut der Westfa¨lischen Wilhelms-Universita¨t
Mu¨nster, Corrensstraße 40, D-48149, Mu¨nster, Germany
Based on structure affinity relationships of a series of N,N-
dialkylamine derivatives Glennon has established a pharma-
cophore model for s1 receptor ligands. According to this model
a potent s1 receptor ligand should contain a basic amine, which
is substituted with two hydrophobic substituents at distances of
cInstitut fu¨r Pharmazie der Ernst-Moritz-Arndt-Universita¨t Greifswald,
Friedrich-Ludwig-Jahn-Straße 17, D-17489, Greifswald, Germany
† Electronic supplementary information (ESI) available: Experimental
details. See DOI: 10.1039/c0ob00402b
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Fig. 2 Comparison of the lead compounds 7 and 8 with the planned s1
ligands 9.
9 with various substituents and different stereochemistry at the 2-
position.
2. Conformational analysis
At first the conformational flexibility of the largest bridge of
6,8-diazabicyclo[3.2.2]nonanes (three-carbon bridge) and 7,9-
diazabicyclo[4.2.2]decanes (four-carbon bridge) as well as the
orientation of the substituent at the 2-position were considered
theoretically. For this purpose a stochastic conformational search
(molecular modeling program MOE31) was performed with model
compounds (1R,2R,5S)-A (model for 7), (1R,2S,5S)-B (model for
8), (1R,2R,6S)-C (model for 16a) and (1R,2S,6S)-D (model for
25a), which are substituted with small methyl groups at both N-
atoms (Fig. 3). Subsequent geometry optimization resulted in 4–10
conformations (cutoff energy 7 kcal mol-1). (see Tables 6–9 in the
Supporting Information†).
Fig. 1 Structurally diverse s1 receptor ligands.
19
˚
˚
2.5–3.9 A and 6–10 A. However, the two-dimensional model
does not reflect the three dimensional orientation of the partic-
ular pharmacophoric elements. For the development of a more
detailed three dimensional model, novel s1 receptor ligands with
restricted conformational flexibility and defined stereochemistry
are required.
Several s1 receptor ligands contain the ethylenediamine sub-
structure as a pivotal pharmacophoric element. The piperazines 5
(s1: Ki = 0.47 nM)28 and 6 (s1: Ki = 12.4 nM)29 are further examples
for potent s1 ligands with the ethylenediamine substructure.
Recently we have reported on the synthesis and pharmacological
evaluation of 6,8-diazabicyclo[3.2.2]nonanes 7 and 8 representing
conformationally restricted piperazine derivatives with a bridge
consisting of three carbon atoms.30 The enantiomeric pairs 7/ent-
7 and 8/ent-8 have almost the same s1 affinity, whereas the
s1 affinities of the diastereomers 7/8 and ent-7/ent-8 differ
considerably. Obviously the relative orientation of the OH moiety
in the bicyclic framework is responsible for high s1 receptor affinity
(Fig. 2). In order to learn more about the influence of the bridge
size, its type of substituents and the relative configuration on
the s1 affinity, bicyclic ligands 9 with a four-carbon bridge were
envisaged. In addition to the increased size of the bridge in 9 the
orientation of the substituents at the 2-position is changed when
compared with compounds 7 and 8 with a three-carbon bridge.
Herein, we report on the conformational analysis, synthesis
and pharmacological evaluation of 7,9-diazabicyclo[4.2.2]decanes
For all types of compounds two different conformations of the
bridge were found with considerably lower energy than the residual
conformations. The relative energies and the dihedral angles N8/9
–
C1–C2–OH of these conformations are summarized in Table 1. As
examples the two energetically most favored conformations of the
model compounds (1R,2R,5S)-A and (1R,2R,6S)-C are depicted
in Fig. 3. It can be seen clearly that the bridges of both bicyclic
systems can adopt two generally different conformations, which
result in considerably different dihedral angles of 157◦ and -158◦
for the three-membered bridge of (1R,2R,5S)-A and -165◦ and
164◦ for the four-membered bridge of (1R,2R,6S)-C.
Although being rather similar the dihedral angles of the bicyclic
systems (1R,2R,5S)-A and (1R,2S,5S)-B with a three-membered
bridge differ by 2–16◦ from the dihedral angles of (1R,2R,6S)-C
and (1R,2S,6S)-D with a four-membered bridge indicating a fine
tuning of the orientation of the OH-moiety attached at the 2-
position. Moreover the additional methylene moiety within the
bridge of the bicyclic systems C and D leads to an increased
occupied space by the larger bridge.
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Table 1 Relative energies and dihedral angles of the energetically most favored conformations of model compounds A–D
Model for compd.
Compd.
Conformation
DE/kcal mol-1
Dihedral angle N8/9–C1–C2–OH (◦)
7
(1R,2R,5S)-A
(1R,2S,5S)-B
(1R,2R,6S)-C
(1R,2S,6S)-D
1
2
1
2
1
2
1
2
0.00
0.69
0.00
0.57
0.00
2.90
0.00
3.44
157
-158
-83
8
-35
16a
25a
-165
164
-51
-81
3. Chemistry
Recently we have reported on the synthesis of racemic chloroac-
etamide 10, which started from racemic 2-aminoadipic acid.32
Treatment of the chloroacetamide 10 with p-methoxybenzylamine
(PMB-NH2) afforded in a one-pot procedure the dioxopiperazine
11 in 96% yield. In the first step a SN2 reaction of 10 with p-
methoxybenzylamine led to a secondary amine, which reacted
directly with the ester in an intramolecular aminolysis to give
the dioxopiperazine 11 (Scheme 1).
The classical Dieckmann condensation of the dioxopiperazine
11 (e.g. with KHMDS), did not provide the bicyclic ketone 14
due to reduced stabilization of the anion of the resulting b-
ketoamide 14. Usually, the driving force in ester condensations
under equilibrium conditions is the formation of the enolate of
the corresponding b-ketoester. Due to the negative charge on the
bridgehead position, the enolate of 14 is rather unstable (compare
Bredt’s rule33), which explains the failure of the direct Dieckmann
cyclization to obtain 14. (Scheme 1).
In order to establish the four-carbon bridge, a Dieckmann
analogous cyclization34 was followed, which involves trapping of
the first cyclized intermediate 12 with trimethylsilyl chloride to
afford diastereoselectively the mixed methyl trimethylsilyl ketal
Fig. 3 Energetically most favored conformations of model compound
(1R,2R,5S)-A (model for 7, top) compared with those of model compound
(1R,2R,6S)-C (model for 9, bottom).
Scheme 1 Synthesis of dioxopiperazine 11 and its Dieckmann analogous cyclization. Reagents and reaction conditions: (a) 4-methoxybenzylamine,
NEt3, CH3CN, rt, 18 h, 96%. (b) LHMDS, THF, 0.5 h, -78 ◦C then (c) Me3SiCl, 2 h, -78 ◦C, 0.5 h, rt, 89%. (d) p-toluenesulfonic acid, THF–H2O, rt,
16 h, 99%.
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13. Hydrolysis of the mixed ketal 13 produced the bicyclic ketone
14 in 99% yield (Scheme 1). In analogy to the corresponding mixed
ketal of the N-methyl analogue32 and the mixed ketals of bicyclic
systems with shorter bridges34–38 we assume that the TMSO group
is oriented towards the carbonyl moiety in position 8, since the
corresponding Li+-alcoholate 12 will be stabilized by forming a
chelate with this carbonyl moiety.
The ketone 14 was the central building block for the synthesis of
s1 receptor ligands with a 7,9-diazabicyclo[4.2.2]decane scaffold.
At first 14 was reduced chemo- and diastereoselectively with LiBH4
at -90 ◦C to provide the alcohol 15a in 87% yield. The unequivocal
assignment of the configuration of the newly formed center of
chirality at the 2-position was not possible by different NMR
spectroscopic techniques. Since the alcohol 15a appeared to be a
spongy solid, performing of an X-ray crystal structure analysis was
not possible. However, the methyl ether 15b, which was prepared
by a Williamson ether synthesis of the alcohol 15a with CH3I and
NaH (Scheme 2) gave nice crystals, which upon recrystallization
from ethyl acetate–n-hexane mixture were suitable for X-ray
crystal structure analysis. The X-ray crystal structure analysis
revealed the relative configuration as (1RS,2SR,6RS) for the
methyl ether 15b (Fig. 4).
Scheme 2 Synthesis of bicyclic piperazines 15a–g and 16a–g. Reagents
and reaction conditions: (a) LiBH4, THF, -90 ◦C, 2.5 h, 87%. (b) NaH,
RX, n-Bu4N+I-, THF, 1.5–24 h, 62–99%. (c) LiAlH4, THF, reflux, 12–24 h,
23–96%.
with the conformation of the bridge in the energetically favored
solid state of the dilactams 15b and 15c.
Finally, the two lactam groups of the bridged piperazinediones
15a–g were reduced with LiAlH4 in boiling THF39 to produce the
tertiary amines 16a–g (Scheme 2).
For the evaluation of the pharmacological activity of the 7,9-
diazabicyclo[4.2.2]decane compound class the diastereomers of 16
with opposite configuration in position 2 were to be synthesized.
At first different reducing agents for the reduction of the ketone
14 were investigated with the aim of finding a method for
the stereoselective synthesis of the diastereomeric alcohol 19a
(Table 2).
The results in Table 2 reveal that all reducing agents led
preferentially to the alcohol 15a. LiBH4 at -90 ◦C gave exclusively
the alcohol 15a. The lowest diastereoselectivity was observed for
the reduction of 14 with NaBH4 at rt. However, the alcohol 19a
was still formed as minor product (21%). Moreover, the separation
of the two diastereomeric alcohols 15a and 19a was not possible by
flash chromatography due to their similar interactions with silica
gel.
Fig. 4 X-Ray crystal structure of methyl ether 15b.
The corresponding ethers 15c–g were prepared using the same
conditions (NaH, R-X) (Scheme 2). A second X-ray crystal
structure analysis of the ethyl ether 15c was also recorded (Fig. 5).
A comparison of the dihedral angles and angles between the planes
of different atoms of the crystal structures of methyl ether 15b and
ethyl ether 15c showed that the orientation of the bridge in both
compounds is very similar. We assume that the orientation of the
bridge in the test compounds 16 with different 2-alkoxy groups
and with the same configuration is very similar to the orientation
of the bridge in the dilactam systems 15b and 15c. This assumption
is supported by comparing the orientation of the four-membered
bridge of the energetically most favored conformation of model
compound (1R,2R,6S)-C (see Fig. 3, conformation of the left side)
Next it was planned to invert the configuration of the alco-
hol 15a by a Mitsunobu reaction.40 Unfortunately, Mitsunobu
reaction of the alcohol 15a with p-nitrobenzoic acid, diisopropyl
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Table 2 Reduction of ketone 14 with various reducing agents
tionally, the activation of alcohol 15a as mesylate, trichloromesy-
late, nosylate and triflate followed by SN2 reaction with different
nucleophiles (CH3CO2Na, p-NO2C6H4CO2K, KNO2 and KNO3)
in different solvents (DMF, acetone, acetonitrile) also failed to
give the inverted alcohol 19a. Generally inseparable mixtures
of retention and inversion products together with elimination
product 18 were formed.
Reducing agent
Temp.
Time
Ratioa 15a:19a
LiBH4
NaBH4
-90 ◦
rt
C
3 h
5 h
>99 : 1
79 : 21
BH3·THF
KS-Selectride
BH3·THF
L-Selectride
DIBAL
rt
1 h
2 h
1 h
3 h
1.5 h
15 min
84 : 16
93 : 7
93 : 7
97.5 : 2.5
98.0 : 2.0
99.0 : 1.0
-80 ◦
-78 ◦
-80 ◦
C
C
C
C
C
Since all attempts to invert the configuration of the conforma-
tionally restricted alcohol 15a had failed, the Mitsunobu inversion
was tried with the more flexible reduced bicyclic alcohol 16a.
The transformation of 16a with DIAD–PPh3 and p-nitrobenzoic
acid proceeded smoothly providing the p-nitrobenzoate 21 in 69%
yield. Subsequently, the ester 21 was cleaved with CH3OH–K2CO3
68 ◦
LiAlH4
-78 ◦
a The ratio of diastereomeric alcohols 15a and 19a was determined by 1H
NMR spectroscopy.
1
(Scheme 3). Surprisingly, the H NMR spectra of the hydrolysis
product and the starting alcohol 16a were completely identical.
The X-ray crystal structure analysis of the p-nitrobenzoate 21
(Fig. 6) confirmed that the Mitsunobu reaction of 16a had taken
place with retention of configuration at the 2-position. We assume
that the retention of configuration is due to the anchimeric
assistance of the tertiary amine (N-9) of the piperazine alcohol
16a initiating a double inversion.
Fig. 5 X-Ray crystal structure of ethyl ether 15c.
Fig. 6 X-Ray crystal structure of p-nitrobenzoate 21.
azodicarboxylate (DIAD) and triphenylphosphine (PPh3) gave an
inseparable mixture of invertedp-nitrobenzoate17 and elimination
product 18. The mixture was treated with CH3OH and K2CO3
to transform the ester 17 into the inverted alcohol 19a in 6%
yield, whereas the elimination product 18 was isolated in 50%
yield (Scheme 3).
Finally, reduction of the ketone in position 2 of the bridged
piperazine 24 was envisaged instead of reduction of the ketone
in the bicyclic dilactam 14. For the preparation of 24, the ketone
14 was protected as dimethyl ketal 22. Subsequent reduction of
22 with LiAlH4 in boiling THF afforded the ketal 23, which was
hydrolyzed with diluted HCl to yield the ketone 24.
Reduction of 24 with L-Selectride at rt provided the diastere-
omeric alcohols 16a and 25a in the ratio of 36 : 64, which were
separated by flash chromatography. The desired diastereomer 25a
was isolated in 27% yield (Scheme 4). The alkylation of the
alcohol 25a was performed using the same reaction conditions,
i.e. NaH, THF, RX, as for the alkylation of the alcohol 15a
containing lactam moieties in the bicyclic system. Surprisingly
these conditions did not lead to the ethers 25b and 25c. However,
after changing the solvent from THF to DMF the ethers 25b, 25c,
and 25g were obtained in 55–72% yield (Scheme 4).
In the 1H NMR spectrum of 19a, the coupling constants of 1-H
(singlet at 4.20 ppm) differ considerably from those of the starting
3
alcohol 15a (doublet at 4.06 ppm, J = 4.9 Hz) confirming the
inversion of configuration at position 2.
The low yield (6%) of the inverted alcohol 19a encouraged
us to modify the Mitsunobu reaction of the alcohol 15a.
In particular more reactive azodicarboxylates (azodicarbonyldi-
piperidine (ADDP), diethyl azodicarboxylate (DEAD)), modified
phosphines (PBu3) and different acids (acetic acid, p-nitrobenzoic
acid, p-methoxybenzoic acid) were employed.40 However, all these
variations did not improve the yield of inverted alcohol 19a.
Moreover, the three step inversion of the configuration by
activation of the alcohol 15a as tosylate 20 followed by substitution
with CH3CO2Na and p-NO2C6H4CO2K failed (Scheme 3). Addi-
In order to include bicyclic compounds without substituents in
position 2 into the structure–affinity study the tosylate 20, which
had been synthesized for purpose of substitution reactions, was
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Scheme 3 Inversion of the alcohols 15a and 16a. Reagents and reaction conditions: (a) p-toluenesulfonyl chloride, powdered KOH, CH2Cl2, 0 ◦C, 2.5 h,
94%. (b) PPh3, DIAD, p-nitrobenzoic acid, THF, reflux, 18 h. (c) CH3OH, K2CO3, rt, 16 h, 6% (19a, calculated over 2 steps), 50% (18 calculated over 2
steps). (d) LiAlH4, THF, reflux, 15 h, 95%. (e) PPh3, DIAD, p-nitrobenzoic acid, THF, rt, 16 h, 69%. (f) CH3OH–CH2Cl2 (5 : 1), K2CO3, rt, 16 h, 97%.
Scheme 4 Synthesis of bicyclic piperazines 25a–c, g with inverted configuration at the 2-position. Reagents and reaction conditions: (a) HC(OCH3)3,
MeOH, p-toluenesulfonic acid, reflux, 15 h, 99.9%. (b) LiAlH4, THF, reflux, 12 h, 80%. (c) 1 M HCl, THF–H2O (1 : 1), rt, 10 h, 77%. (d) L-Selectride,
THF, rt, 12 h, 25a (28%), 16a (24%) (e) NaH, DMF, rt, 3 h, 25b (70%), 25c (72%), 25g (55%).
treated with LiAlH4 to afford the unsubstituted compound 26 in
65% yield. Analogously the unsaturated dilactam 18, which had
been obtained as side product in the Mitsunobu reactions, was
reduced with LiAlH4 in boiling THF to give the bicyclic alkene 27
in 71% yield (Scheme 5).
and 30 was not possible by flash chromatography, the mixture of
29 and 30 was reduced with LiAlH4 to obtain the piperazines 31
and 32. After flash chromatography, the methylene derivative 32
was isolated in 54% yield whereas the methylated alkene 31 was
isolated in very low amounts (<1 mg, 5%). (Scheme 6)
The high s1 affinity and selectivity of the unsubstituted alkene
27 (Ki (s1) = 7.5 nM) stimulated the synthesis of the 2-methyl
substituted alkene 31. For this purpose, the ketone 14 was reacted
with CH3MgBr in THF to obtain the tertiary alcohol 28 as a single
diastereomer in 48% yield. The tertiary alcohol was dehydrated
with P4O10 in toluene to provide two regioisomeric alkenes 29 and
The introduction of fluorine atoms in position 2 as bioisosteric
replacements of O-substituents was envisaged. Thus, the ketone
14 was treated with DAST41 at -78 ◦C in CH2Cl2, which resulted
in an inseparable 3 : 1 mixture of difluoro compound 33 and
fluoroalkene 34 (Scheme 7). This mixture was reduced with LiAlH4
in THF under reflux conditions.39 Surprisingly, these reaction
conditions converted both compounds 33 and 34 into fluoroalkene
35, which was isolated in 54% yield.
1
30 in the ratio 2 : 3, according to the H NMR spectrum of the
crude sample (Scheme 6). Since separation of the regioisomers 29
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Scheme 5 Synthesis of bicyclic compounds 26 and 27 without sub-
stituents in position 2. Reagents and reaction conditions: (a) LiAlH4, THF,
rt, 12 h then reflux, 12 h, 65%. (b) LiAlH4, THF, reflux, 12 h, 71%.
Scheme 7 Synthesis of fluoroalkene 35. Reagents and reaction conditions:
(a) DAST, CH2Cl2, -78 ◦C, 12 h, 81% (33 +,34). (b) LiAlH4, THF, reflux,
12 h, 54%.
Scheme 8 Synthesis of difluoride 36. Reagents and reaction conditions:
(a) DAST, CH2Cl2, 0 ◦C, 12 h, 95%.
presence of an excess of non-radiolabeled (+)-pentazocine for
selective masking of s1 receptors. An excess of non-tritiated di-
o-tolylguanidine was used for determination of the non-specific
binding.22,26,42,43
In the (1RS,2RS,6SR) series, the alcohol 16a has moderate s1
affinity (Ki = 298 nM) and transformation of the alcohol 16a into
the methyl ether 16b led to decreased s1 affinity (Ki > 1 mM)
(Table 3). Enlargement of the alkyl residue to an ethyl group 16c
(Ki = 552 nM) or propyl group 16d (Ki = 503 nM) retained the
s1 affinity, whereas further extension of the O-residue to a butyl
residue 16e led to almost complete loss of s1 affinity (Ki > 1 mM).
Interestingly, introduction of a branched residue, e.g. an isopentyl
moiety, resulted in the most potent s1 ligand 16f (Ki = 123 nM) of
the ether series. In analogy to the butyl ether 16e the benzyl ether
16g reveals only very low s1 affinity (Ki > 1 mM).
Scheme 6 Synthesis of methylene substituted bicyclic compound 32.
Reagents and reaction conditions: (a) CH3MgBr, THF, -78 ◦C, 10 h then
rt, 24 h, 48%. (b) P4O10, toluene, 90 ◦C, 12 h, 32% (29 + 30). (c) LiAlH4,
THF, reflux, 12 h, 32 (54%).
The diastereomeric alcohol 25a and ethers 25b, 25c and 25g
((1RS,2SR,6SR)-configuration) show only very low s1 affinity.
The s1 receptor affinity of the 2,2-dimethoxy derivative 23 is also
very low. Since the smaller homologous alcohols 7, ent-7, 8, ent-8
(compare Fig. 2) and the corresponding methyl ethers represent
potent s1 receptor ligands,30 it can be concluded that expansion
of the bridge from three to four methylene moieties is unfavorable
for high s1 receptor affinity. Either the changed orientation of
the 2-OR moiety on the four-membered bridge or the increased
spatial demand of the larger bridge itself may be the reason for the
reduced s1 affinity. However, in both bicyclic systems with a three-
and four-membered bridge the same effect of the stereochemistry
is observed, because 7/ent-7 and 16a show higher s1 receptor
affinities than 8/ent-8 and 25a, respectively.
The synthesis of difluoro substituted compound 36 was achieved
by treatment of piperazine ketone 24 with DAST at 0 ◦C in CH2Cl2
in 95% yield (Scheme 8).
4. Receptor affinity
The s1 and s2 receptor affinities of the bridged piperazines were
determined in competition experiments with radioligands. In the
s1 assay membrane preparations of guinea pig brains were used
as receptor material and [3H]-(+)-pentazocine as radioligand.
The non-specific binding was determined in the presence of a
large excess of non-tritiated (+)-pentazocine. Homogenates of rat
liver served as source for s2 receptors in the s2 assay. Since a
s2 selective radioligand is not commercially available, the non-
selective radioligand [3H]-di-o-tolylguanidine was employed in the
Some derivatives without a substituent at the 2-position have
been synthesized in order to check the tolerance of the s1 receptor
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Table 3 s1 and s2 receptor affinities of 7,9-diazabicyclo[4.2.2]decanes
Ki SEMa/nM
Compd.
s1
s2
s1/s2 selectivity
16a
OH
OCH3
298 28
>1 mM
552
4.8 mM
>1 mM
>1 mM
>1 mM
>1 mM
>1 mM
>1 mM
640 nM
>1 mM
>1 mM
>1 mM
>1 mM
674
184
>1 mM
387
619
—
78 2.3
57 18
—
—
—
—
—
—
—
—
—
—
—
—
2.7
25
—
ª 1
0.62
—
16b
16c
OCH2CH3
OCH2CH2CH3
OCH2CH2CH2CH3
OCH2CH2CH(CH3)2
OCH2C6H5
OH
16d
503
16e
>1 mM
123 19
17 mM
>1 mM
600 nM
>1 mM
>1 mM
>1 mM
253 34
7.5 1.57
1.0 mM
361
16f
16g
25a
25b
OCH3
25c
OCH2CH3
OCH2C6H5
2-C: C(OCH3)2
H
2/3-CH CH-
2-C: C CH2
2/3-CF CH
2-C: CF2
25g
23
26
27
32
35
36
1.0 mM
5.6 2.2
6.3 1.6
89 29
(+)-Pentazocine
Haloperidol
Ditolylguanidine
12
0.64
a SEM values are determined only for high affinity (Ki < 300 nM) compounds.
with respect to 2-substitution. The unsubstituted derivative 26 has
almost the same s1 affinity as the alcohol 16a. This result indicates
that the changed orientation of the 2-OH or 2-OCH3 moiety
is not the reason for the reduced s1 affinity but the increased
size of the four-carbon bridge itself. Moreover, the analogous
bicyclic compound with an unsubstituted three-carbon bridge
represents a very potent s1 ligand ((S,S): Ki = 0.91 nM; (R,R): Ki =
4.4 nM).44 This hypothesis is further supported by introduction of
a double bond into the four-carbon bridge (27), which results
in reduced bridge size and high s1 affinity of the bicyclic alkene
27 (Ki = 7.5 nM). The corresponding bicyclic compounds with
an unsaturated three-membered bridge bearing only a small allyl
substituent at the 6-position are also very potent s1 ligands.44
An additional fluorine atom in position 2 of the alkene
substructure (35) again leads to reduced s1 affinity in the range
of the s1 affinity of the alcohol 16a and the alkane 26. An exo-
methylene moiety (32) and two fluorine atoms (36) are too big
to be tolerated by the s1 receptor protein. Altogether it can be
concluded that a reduced size of the bridge is favoring s1 affinity.
The most potent s1 ligands of this series 27, 16a, 16f and
26 interact with higher affinity with s1 receptors than with s2
receptors and the most potent compound 27 is the most selective
one (s1 : s2 = 25). On the contrary a slight preference for the s2
subtype is observed for the inverted alcohol 25a and the difluoride
36. However the corresponding Ki-values (ca. 650 nM) are rather
high, so that both compounds cannot be considered as potent s2
ligands.
is particularly sensitive to these bicyclic s1 ligands.30 Therefore,
the antiproliferative effects of some of the synthesized compounds
were investigated in a panel of six human tumor cell lines, including
the cell lines A-427 (small cell lung cancer), 5637 (bladder cancer),
RT-4 (bladder cancer), LCLC-103H (large cell lung cancer), MCF-
7 (breast cancer) and DAN-G (pancreas cancer).
In the primary screening the tumor cells were incubated with
◦
a 20 mM 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. The IC50 values
of all active compounds were determined by subjecting the cells
to 5 serial dilutions of test compounds for 96 h and measuring
the remaining cell density by crystal violet staining followed by
comparison with untreated controls.45
In the primary screening the compounds 26 and 27 without
further substituents at the bridge showed considerable inhibition
of the growth of tumor cells. (Table 4) The growth of the tumor cell
lines A-427, 5637 and MCF-7 was moderately inhibited while little
or no activity for any of the compounds was seen in the DAN-G,
LCLC-103H and RT-4 cell lines. For compounds that inhibited
cell growth by > 50% at 20 mM, IC50-values were determined.
Table 5 shows that the bladder cancer cell line 5637 is particu-
larly sensitive towards the methyl ether 25b, and the unsubstituted
compounds 26 and 27, which gave IC50 values of 6.8, 8.8 and
13 mM, respectively. In addition to the growth inhibition of the
tumor cell line 5637, the growth of the small cell lung cancer cell
line A-427 and the breast cancer cell line MCF-7 are also inhibited.
The bicyclic derivative 27, with an unsaturated bridge, rep-
resents the most potent s1 ligand (Ki = 7.5 nM) of this novel
compound class. In addition to its high s1 receptor affinity 27
inhibits the growth of the cell line A-427 with an IC50-value of
10 mM, which represents the highest activity within this compound
class against this cell line. Although a clear correlation between
5. Inhibition of growth of human tumor cell lines
6,8-Diazabicycyclo[3.2.2]nonane derivatives with a methoxy moi-
ety in position 2 are able to inhibit the growth of some human
tumor cell lines. The human small cell lung cancer cell line A-427
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Table 4 Cell growth inhibitory activity (% of untreated control) of 7,9-diazabicyclo[4.2.2]decane derivatives in six human tumor cell linesa
A-427b
5637c
MCF-7d
DAN-Ge
LCLC-103Hf
RT-4g
16a
16b
25a
25b
26
86 18
53 23
85 23
78 15
45 18
71 19
28 13
63 29
68 22
63 17
37 17
92 11
76 11
125 30
122 45
147 40
114 12
37 34
72
1
62 13
82 14
53 16
37 12
52 14
80
70
6
3
25
9
-10 17
-6 14
11
6
9
7
18 23
50
27
29 11
44 24
6
99
8
a Relative cell growth [%] in relation to untreated control of the tumor cell lines after 96 h exposure to test compound at a dose of 20 mM. Values are
averages of three independent experiments. b Small cell lung cancer. c Bladder cancer. d Breast cancer. e Pancreas cancer. f Large cell lung cancer. g Bladder
cancer.
Table 5 IC50 values (mM) of growth inhibition of tumor cell lines following a continuous 96 h exposure to the respective compounds. Unless otherwise
noted, all values are the averages of three independent determinations
A-427a
5637b
MCF-7c
DAN-Gd
LCLC-103He
RT-4f
16a
>20
>20
>20
17
15
10
10
>20
19
n.d.
6.8 5.3
8.8 7.2
13
27
>20
>20.
n.d.
>20
>20
>20
>20
17
18
29
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
16b
2
25a
25b
26
27
2
2
1
2
13
16h
18
3
4
5
7
2
9
3
Haloperidol
25 10
0.05 0.02
23
5
16
5
Methotrexateg
5.5 3.6
0.016 0.009
0.077 0.005
0.025 0.012
0.04 0.02
a Small cell lung cancer. b Bladder cancer. c Breast cancer. d Pancreas cancer. e Large cell lung cancer. f Bladder cancer; average of two determinations.
g IC50values are from ref. 45. h n.d. not determined.
the s1 (or s2) receptor affinity and the cell growth inhibition in
any of the cell lines cannot be derived from these data, the growth
inhibition of the A-427 cell line may to some extent be associated
with interaction of the compounds with s1 receptors. This cell
line is selectively sensitive to haloperidol, a known s1 receptor
antagonist, which indicates that 27 could also be a s1 receptor
antagonist. (Table 5) While 27 shows growth inhibitory activity in
the A-427 line, it also shows activity in the 5637 cell line, which
is relatively insensitive to haloperidol. Presently, studies are under
way to clarify whether receptor binding is a cause for cytotoxicity.
Generally, the 7,9-diazabicyclo[4.2.2]decane system showed
considerably different chemical and pharmacological behavior
compared with the corresponding 6,8-diazabicyclo[3.2.2]nonane
derivatives: (1) A nucleophilic substitution at the 2-position either
via a Mitsunobu inversion or after activation of the alcohol as
sulfonate did not take place. It is assumed that this failure is due
to higher shielding of this position by the unfavorable orientation
of the larger bridge. (2) The s1 and s2 receptor affinities of all
alcohols and ethers with a four-membered bridge are considerably
lower than the s affinities of the corresponding analogues with a
three-carbon bridge. This may be attributed to the larger bridge
itself. This hypothesis is supported by introduction of a double
bond into the bridge (compound 27), which reduced the size and
flexibility of the bridge, and led to the most potent s1 ligand of this
study (Ki = 7.5 nM). (3) The activity against human tumor cell lines
is also reduced considerably. Again the larger, more flexible four-
carbon bridge might be responsible for the reduced cell growth
inhibition compared to the three-carbon bridge homologues.
6. Conclusion
In this manuscript, the conformational analysis, synthesis, s1 and
s2 receptor affinities as well as growth inhibition of some human
tumor cell lines of 7,9-diazabicyclo[4.2.2]decanes with a benzyl
group at N-7 and a p-methoxybenzyl group at N-9 are reported.
Starting from the chloroacetamide 10, the bicyclic framework was
established by a Dieckmann analogous cyclization which involves
trapping of the first cyclized intermediate with trimethylsilyl
chloride. The yield of the resulting mixed methyl silyl ketal 13 was
even higher than the yields of the bicyclic analogues with a three-
carbon bridge. This observation is in good agreement with our
calculations showing that the diazabicyclo[4.2.2]decane system is
slightly more stable than the diazabicyclo[3.2.2]nonane system.36
The bicyclic ketone 14 was used as central building block for the
synthesis of diastereoisomerically pure bicyclic alcohols, ethers
and fluorine derivatives. Whereas the ketone 14 was reduced with
high diastereoselectivity (dr > 99 : 1) to produce the alcohol 15a,
the diastereomeric alcohol 25a was only available by an unselective
reduction of the ketone 24.
7. Experimental part
7.1. Conformational analysis
3D-Structures were generated with MOE (Molecular Operating
Environment), Version 2009.10, Chemical computing group AG
(CCG, Montreal, Canada). Stochastic conformational search was
carried out at standard conditions. Method: stochastic, rejection
limit: 100, iteration limit 10000, RMS gradient: 0.005, MM
iteration limit: 500, RMSD limit: 0.25, strain cutoff: 7 kcal mol-1,
conformation limit: 10000). Tables 6–9 in the Electronic Supple-
mentary Information show the relative calculated energies and
dihedral angles of all conformations.†
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7.2. Chemistry
(1C, C-8), 50.6 (1C, C6H4OCH3), 54.3 (1C, CO2CH3), 57.9 (1C,
C-5), 113.3 (2C, C-3, C-5methoxybenzyl), 126.1 (1C, C-4benzyl), 127.0
(1C, C-1methoxybenzyl), 127.2 (2C, C-3, C-5benzyl), 127.9 (2C, C-2,
C-6benzyl), 128.8 (2C, C-2, C-6methoxybenzyl), 134.4 (1C, C-1benzyl), 158.4
(1C, C-4methoxybenzyl), 163.1 (1C, amide carbonyl), 164.8 (1C, amide
carbonyl), 172.0 (1C, CO2CH3). MS (EI): m/z (%) = 424 [(M)+,
59], 303 [(M-methoxybenzyl)+, 37], 121 [methoxybenzyl+, 100],
91 [benzyl+, 46]. IR (neat): n/cm-1 = 1732 (C O), 1654 (C O),
1243 (C–O), 1171 (C–O).
7.2.1. General. Unless otherwise noted, moisture sensitive
reactions were conducted under dry nitrogen. THF was dried
with sodium/benzophenone and was freshly distilled before use.
Thin layer chromatography (tlc): Silica gel 60 F254 plates (Merck).
Flash chromatography (fc): Silica gel 60, 40–64 mm (Merck);
parentheses include: eluent, diameter of the column, height of the
column packed, fraction size, Rf value. Melting point: Melting
point apparatus SMP 3 (Stuart Scientific), uncorrected. MS:
MAT GCQ (Thermo–Finnigan), EI (electron impact); Thermo
7.2.3. (1RS,2SR,6RS)-7-Benzyl-2-methoxy-9-(4-methoxy-
benzyl)-2-(trimethylsilyloxy)-7,9-diazabicyclo[4.2.2]decane-8,10-
dione (13). Lithium hexamethyldisilazide (LHMDS), freshly
prepared from n-BuLi (1.6 M in n-hexane, 11.5 mL, 18.4 mmol)
and hexamethyldisilazane (3.8 mL, 18.4 mmol) in dry THF
(25 mL) at 0 ◦C, was added slowly to a solution of 11 (4.7 g,
11.2 mmol) in dry THF (70 mL) at -78 ◦C. The mixture was
stirred for 0.5 h at -78 ◦C, chlorotrimethylsilane (CH3)3SiCl
(4.2 mL, 33.5 mmol) was slowly added to this solution over a
R
Finnigan LCQꢀ ion trap mass spectrometer with an ESI (electro-
spray ionization) interface, exact mass (ESI): MicroTof (Bruker
Daltonics) Finnigan MAT 4200s. IR: IR spectrophotometer
480Plus FT-ATR-IR (Jasco). H NMR (400 MHz), 13C NMR
(100 MHz): Mercury-400BB spectrometer (Varian); d in ppm
related to tetramethylsilane; coupling constants are given with
0.5 Hz resolution. The assignments of H NMR and 13C NMR
1
1
were supported by COSY and GHSQC two dimensional NMR
techniques. HPLC: Merck Hitachi Equipment; UV detector: L-
7400; autosampler: L-7200; pump: L-7100; degasser: L-7614;
◦
period of 10 min and the mixture was stirred at -78 C for 2 h,
then warmed to rt and stirred for additional 0.5 h. The solvent
was evaporated to half of the original volume and ethyl acetate
(50 mL) was added. The mixture was washed with H2O (50 mL)
and the aqueous layer was extracted with CH2Cl2 (5 ¥ 50 mL).
The combined organic layers were washed with brine, dried over
Na2SO4, concentrated under vacuum and the residue was purified
by fc (cyclohexane/ethyl acetate = 7/3, 8 cm, 14 cm, 50 mL,
Rf 0.25) to obtain a colorless viscous oil, yield 4.93 g (89%).
R
Method A: column: LiChrospherꢀ 60 RP-select B (5 mm), 250-
4 mm; flow rate: 1.00 mL min-1; injection volume: 5.0 mL; detection
at l = 210 nm; solvents: A: water with 0.05% (v/v) trifluoroacetic
acid; B: acetonitrile with 0.05% (v/v) trifluoroacetic acid: gradient
elution: (A%): 0–4 min: 90%, 4–29 min: 90% to 0%, 29–31 min:
0%, 31–31.5 min: 0% to 90%, 31.5–40 min: 90%. Method B:
R
column: LiChrospherꢀ 60 RP-select B (5 mm), 250-4 mm; flow
rate: 1.00 mL min-1; injection volume: 5.0 mL; detection at l =
210 nm; solvents: A: water with 0.05% (v/v) trifluoroacetic acid, B:
methanol with 0.05% (v/v) trifluoroacetic acid: gradient elution:
(A%): 0–1 min: 80%, 1–22 min: 80% to 0%, 22–30 min: 0%, 30–
31.5 min: 0% to 80%, 31.5–40 min: 80%.
C27H36N2O5Si (496.6). Purity (HPLC, method A): 98.2%, tR
=
23.2 min. Elemental analysis: calcd. C 65.29 H 7.31 N 5.64 found
1
C 65.39 H 7.65 N 5.06. H NMR (CDCl3): d (ppm) = 0.13 (s,
9H, (CH3)3Si), 1.34–1.53 (m, 3H, 3-H, 4-H), 1.74-1.92 (m, 3H,
3-H, 5-H), 3.29 (s, 3H, OCH3 ketal), 3.73 (s, 3H, C6H4OCH3), 3.88
(d, J = 14.6 Hz, 1H, NCH2C6H4OCH3), 3.96 (d, J = 1.0 Hz, 1H,
1-H), 4.0 (dd, J = 6.4/1.8 Hz, 1H, 6-H), 4.18 (d, J = 14.7 Hz, 1H,
NCH2C6H5), 4.72 (d, J = 14.7 Hz, 1H, NCH2C6H5), 5.29 (d, J =
14.6 Hz, 1H, NCH2C6H4OCH3), 6.78 (d, J = 8.7 Hz, 2H, 3-H, 5-
Hmethoxybenzyl), 7.02 (d, J = 8.5 Hz, 2H, 2-H, 6-Hmethoxybenzyl), 7.14 (dd,
J = 7.5/1.9 Hz, 2H, 2-H, 6-Hbenzyl), 7.20– 7.26 (m, 3H, 3-H, 4-H, 5-
Hbenzyl). 13C NMR (CDCl3): d (ppm) = 0.0 (3C, (CH3)3Si), 15.6 (1C,
C-4), 32.0 (1C, C-5), 32.1 (1C, C-3), 45.9 (1C, CH2C6H4OCH3),
46.2 (1C, CH2C6H5), 47.3 (1C, OCH3 ketal), 53.5 (1C, C6H4OCH3),
57.9 (1C, C-6), 62.1 (1C, C-1), 103.8 (1C, C-2), 112.4 (2C, C-3,
C-5methoxybenzyl), 126.1 (1C, C-4benzyl), 126.2 (1C, C-1methoxybenzyl), 126.4
(2C, C-3, C-5benzyl), 127.0 (2C, C-2, C-6benzyl), 127.7 (2C, C-2, C-
6methoxybenzyl), 134.6 (1C, C-1benzyl), 157.4 (1C, C-4methoxybenzyl), 162.7
(1C, carbonyl), 167.6 (1C, carbonyl). MS (EI): m/z (%) = 496
(M+, 89), 375 [(M-methoxybenzyl)+, 42], 121 [(methoxybenzyl)+,
100], 91 [(benzyl)+, 85]. IR (neat): n/cm-1 = 1666 (C O), 1451
(C–N), 1243 (C–O), 839 (Si–O).
7.2.2. Methyl
4-[(RS)-1-benzyl-4-(4-methoxybenzyl)-3,6-
dioxopiperazin-2-yl]butanoate (11). Under ice-cooling NEt3
(2.4 mL, 17.5 mmol) and 4-methoxybenzylamine (2.4 mL,
18.6 mmol) were added slowly to a solution of 10 (4.1 g,
11.6 mmol) in CH3CN (60 mL) over a period of 30 min. The
mixture was warmed to rt and stirred for 18 h. Then the solvent
was evaporated under vacuum, Et2O (150 mL) was added
to the residue, the mixture was filtered and the filtrate was
concentrated under vacuum. The residue was purified by fc
(cyclohexane/ethyl acetate = 1/1, 5 cm, 15 cm, 30 mL, Rf 0.27)
to yield a colorless oil, yield 1.25 g (96%). C24H28N2O5 (424.5).
Purity (HPLC, method A): 94.6%, tR = 19.3 min. 1H NMR
(CDCl3): d (ppm) = 1.39–1.51 (m, 1H, CH2CH2CH2CO2CH3),
1.52–1.63 (m, 1H, CH2CH2CH2CO2CH3), 1.69–1.78 (m, 1H,
CH2CH2CH2CO2CH3), 1.80–1.88 (m, 1H, CH2CH2CH2-
CO2CH3), 2.07–2.26 (m, 2H, CH2CH2CH2CO2CH3), 3.58 (s, 3H,
CH2CH2CH2CO2CH3), 3.73 (s, 3H, C6H4OCH3), 3.77 (d, J =
17.6 Hz, 1H, NCH2CO), 3.84–3.89 (m, 2H, NCH2CO, NCHCO),
3.94 (d, J = 14.9 Hz, 1H, NCH2C6H4OCH3), 4.22 (d, J = 14.3 Hz,
1H, NCH2C6H5), 4.67 (d, J = 14.3 Hz, 1H, NCH2C6H5), 5.17
(d, J = 14.9 Hz, 1H, NCH2C6H4OCH3), 6.79 (d, J = 8.6 Hz, 2H,
3-H, 5-Hmethoxybenzyl), 7.11 (d, J = 8.6 Hz, 2H, 2-H, 6-Hmethoxybenzyl),
7.15–7.28 (m, 5H, 2-H, 3-H, 4-H, 5-H, 6-Hbenzyl). 13C NMR
(CDCl3): d (ppm) = 18.6 (1C, C-3), 30.0 (1C, C-4), 32.1 (1C,
C-2), 46.1 (1C, NCH2C6H4OCH3), 47.8 (1C, NCH2C6H5), 47.9
7.2.4. (1RS,6RS)-7-Benzyl-9-(4-methoxybenzyl)-7,9-diazabi-
cyclo[4.2.2]decane-2,8,10-trione (14). The mixed methyl silyl ke-
tal 13 (0.15 g, 0.3 mmol) was dissolved in a mixture of THF
(2.5 mL) and H2O (1 drop, ª 0.05 mL). p-Toluenesulfonic acid
(30 mg, 0.18 mmol) was added and the mixture was stirred for
16 h at rt. The mixture was concentrated to half of its original
volume, then it was washed with saturated NaHCO3 solution
(5 mL) and the aqueous layer was extracted with CH2Cl2 (5 ¥
10 mL). The combined organic layers were washed with brine,
5534 | Org. Biomol. Chem., 2010, 8, 5525–5540
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dried over Na2SO4 and the solvent was evaporated under vacuum
to obtain a colorless solid. Purification by recrystallization with
n-hexane–ethyl acetate (4/1) mixture yielded colorless crystals,
mp 190–195 ◦C, yield 0.12 g (99%). C23H24N2O4 (392.4). Purity
(HPLC, method A): 99.9%, tR = 18.9 min. Elemental analysis:
91.1 [(benzyl)+, 42]. IR (neat): n/cm-1 = 3398 (O–H), 1657 (C O),
1451 (C–N), 1244 (C–O), 1173 (C–O).
7.2.6. (1RS,2SR,6RS)-7-Benzyl-2-methoxy-9-(4-methoxyben-
zyl)-7,9-diazabicyclo[4.2.2] decane-8,10-dione (15b). Under N2
atmosphere, 15a (0.1 g, 0.25 mmol) and CH3I (0.05 mL, 0.76
mmol) were added to a solution of NaH (0.1 g, 2.5 mmol, 60% in
paraffin oil) in dry THF (12 mL) under ice-cooling. The mixture
was warmed to rt and stirred for 3 h. Excess NaH was destroyed
with H2O (3 mL) under ice-cooling. The reaction mixture was
washed with 2 M NaOH (15 mL) solution. The aqueous layer
was extracted with CH2Cl2 (5 ¥ 20 mL). The combined organic
layers were washed with brine, dried over Na2SO4 and the solvent
was evaporated under vacuum. The crude solid was purified by
fc (cyclohexane/ethyl acetate = 1/1, 2 cm, 13 cm, 7 mL, Rf
1
calcd. C 70.39 H 6.16 N 7.14 found C 69.77 H 6.24 N 6.91. H
NMR (CDCl3): d (ppm) = 1.11–1.22 (m, 1H, 4-H), 1.67–1.75
(m, 1H, 4-H), 1.95–1.99 (m, 2H, 5-H), 2.01–2.07 (m, 1H, 3-H),
2.19–2.25 (m, 1H, 3-H), 3.78 (s, 3H, C6H4OCH3), 4.26–4.28 (m,
1.5H, 6-H, NCH2C6H4OCH3), 4.31 (s, 0.5H, NCH2C6H4OCH3),
4.35 (d, J = 14.2 Hz, 1H, NCH2C6H5), 4.56 (d, J = 0.7 Hz,
1H, 1-H), 4.64 (d, J = 14.2 Hz, 1H, NCH2C6H5), 4.82 (d, J =
14.6 Hz, 1H, NCH2C6H4OCH3), 6.82 (d, J = 8.7 Hz, 2H, 3-H,
5-Hmethoxybenzyl), 7.18 (d, J = 8.7 Hz, 2H, 2-H, 6-Hmethoxybenzyl), 7.24–
7.27 (m, 2H, 2-H, 6-Hbenzyl), 7.30–7.33 (m, 3H, 3-H, 4-H, 5-Hbenzyl).
13C NMR (CDCl3): d (ppm) = 19.4 (1C, C-4), 31.3 (1C, C-5),
38.9(1C, C-3), 48.8 (1C, CH2C6H4OCH3), 49.8 (1C, CH2C6H5),
55.5 (1C, C6H4OCH3), 59.6 (1C, C-6), 71.6 (1C, C-1), 114.6 (2C,
C-3, C-5methoxybenzyl), 125.8, (1C, C-4benzyl), 128.6, (1C, C-1methoxybenzyl),
128.7, (2C, C-3, C-5benzyl), 129.2, (2C, C-2, C-6benzyl), 131.1, (2C,
C-2, C-6methoxybenzyl), 135.3, (1C, C-1benzyl), 160.0, (1C, C-4methoxybenzyl),
161.4 (1C, carbonyl), 166.8, 1C, carbonyl), 203.5 (1C, C-2carbonyl).
MS (EI): m/z (%) = 392.0 [M+, 22], 301.0 [(M-benzyl)+, 6], 121.1
◦
0.21). to obtain colorless crystals, mp 136–140 C, yield 0.105 g
(99%). C24H28N2O4 (408.2) Purity (HPLC, method A): 99.7%, tR =
17.0 min. 1H NMR (CDCl3): d (ppm) = 1.32–1.41 (m, 1H, 4-H),
1.58–1.78 (m, 3H, 4-H, 3-H), 1.81–1.90 (m, 1H, 5-H), 1.96-2.04
(m, 1H, 5-H), 3.36 (s, 4H, CHOCH3), 3.81 (s, 3H, C6H4OCH3),
4.05 (dd, J = 5.1/2.9 Hz, 1H, 6-H), 4.11 (d, J = 14.8 Hz, 1H,
NCH2C6H4OCH3), 4.26 (d, J = 5.0 Hz, 1H, 1-H), 4.31 (d, J =
14.6 Hz, 1H, NCH2C6H5), 4.76 (d, J = 14.6 Hz, 1H, NCH2C6H5),
5.04 (d, J = 14.8 Hz, 1H, NCH2C6H4OCH3), 6.87 (d, J = 8.7 Hz,
2H, 3-H, 5-Hmethoxybenzyl), 7.20 (d, J = 8.7 Hz, 2H, 2-H, 6-Hmethoxybenzyl),
7.22-7.24 (m, 2H, 3-H, 5-Hbenzyl), 7.28-7.34 (m, 3H, 2-H, 4-H,
6-Hbenzyl). 13C NMR (CDCl3): d (ppm) = 17.8 (1C, C-4), 28.1
(1C, C-3), 32.1 (1C, C-5), 47.4 (1C, CH2C6H4OCH3), 48.4 (1C,
CH2C6H5), 55.4 (1C, C6H4OCH3), 57.7 (1C, CHOCH3), 59.4
(1C, C-6), 60.7 (1C, C-1), 81.63 (1C, C-2), 114.4 (2C, C-3, C-
5methoxybenzyl), 127.6 (1C, C-4benzyl), 128.3 (1C, C-1methoxybenzyl), 128.5
(2C, C-3, C-5benzyl), 129.0 (2C, C-2, C-6benzyl), 130.0 (2C, C-2, C-
6methoxybenzyl), 135.3 (1C, C-1benzyl), 159.5 (1C, C-4methoxybenzyl), 167.1
(1C, carbonyl), 167.6 (1C, carbonyl). MS (EI): m/z (%) = 406.6
[(M)+, 2], 120.1[(methoxybenzyl)+, 100], 91.1 [(benzyl)+, 25]. IR
(neat): n/cm-1 = 1653 (C O), 1509 (C C), 1244 (C–O).
[(methoxybenzyl)+, 100], 91.2 [(benzyl)+, 17]. IR (neat): n/cm-1
=
1716 (C O), 1668 (C O), 1449 (C–N), 1240 (C–O).
7.2.5. (1RS,2SR,6RS)-7-Benzyl-2-hydroxy-9-(4-methoxy-
benzyl)-7,9-diazabicyclo[4.2.2] decane-8,10-dione (15a). Under
N2 LiBH4 (4.3 mL, 8.56 mmol, 2 M solution in THF) was slowly
added to a solution of 14 (2.23 g, 5.7 mmol) in dry THF (120 mL)
at -90 ◦C and the mixture was stirred for 2.5 h at -90 ◦C. The
excess LiBH4 was destroyed with 1 M HCl (50 mL), then 2 M
NaOH (20 mL) was added until the solution attained a pH of
9 and the mixture was stirred for 0.5 h at rt. The aqueous layer
was extracted with CH2Cl2 (5 ¥ 30 mL). The combined organic
layers were washed with brine, dried (Na2SO4) and the solvent
was evaporated under vacuum to obtain a colorless viscous oil.
The crude oil was purified by fc (cyclohexane/ethyl acetate = 3/7,
3.5 cm, 15 cm, 30 mL, Rf 0.1) to obtain a spongy colorless solid.
Yield 1.95 g (87%). C23H26N2O4 (394.4). Purity (HPLC, method
A): 97.2%, tR = 17.4 min. Diastereomeric purity: >99% (from the
7.2.7. (1RS,2RS,6SR)-7-Benzyl-9-(4-methoxybenzyl)-7,9-di-
azabicyclo[4.2.2]decan-2-ol (16a).
Method 1 from 15a. Under N2 LiAlH4 (13.6 mL, 13.6 mmol,
1 M solution in THF) was added dropwise under ice cooling to
a solution of 15a (0.89 g, 2.27 mmol) in dry THF (73 mL). The
mixture was warmed to rt and stirred under reflux for 15 h. The
excess LiAlH4 was destroyed with H2O (3 mL) under ice-cooling.
The mixture was again stirred under reflux for 1 h, cooled to rt
and filtered. The precipitate was washed with CH2Cl2 (50 mL) and
the solvent was evaporated under vacuum. The crude viscous oil
was purified by fc (petroleum ether/ethyl acetate = 95/5 + 0.2%
N,N-dimethylethylamine, 3 cm, 13 cm, 12 mL, Rf 0.27). Colorless
oil, yield 0.79 g (95%).
1
1H NMR spectrum of the crude sample). H NMR (CDCl3): d
(ppm) = 1.26–1.35 (m, 1H, 4-H), 1.35–1.58 (m, 2H, 3-H, 4-H),
1.63–1.69 (m, 1H, 3-H), 1.74–1.81 (m, 1H, 5-H), 1.91–1.98 (m,
1H, 5-H), 2.99 (d, J = 7.5 Hz, 0.8H, CHOH), 3.73 (s, 3H, OCH3),
3.81–3.87 (m, 1H, 2-H), 3.99 (dd, J = 4.7/3.2 Hz, 1H, 6-H), 4.05 (d,
J = 14.7 Hz, 1H, NCH2C6H4OCH3), 4.06 (d, J = 4.9 Hz, 1H, 1-H),
4.18 (d, J = 14.6 Hz, 1H, NCH2C6H5), 4.76 (d, J = 14.6 Hz, 1H,
NCH2C6H5), 4.96 (d, J = 14.7 Hz, 1H, NCH2C6H4OCH3), 6.79 (d,
J = 8.7 Hz, 2H, 3-H, 5-Hmethoxybenzyl), 7.13 (d, J = 8.7 Hz, 2H, 2-H, 6-
Hmethoxybenzyl), 7.16–6.18 (m, 2H, 2-H, 6-Hbenzyl), 7.23–7.29 18 (m, 3H,
3-H, 4-H, 5-Hbenzyl). 13C NMR (CDCl3): d (ppm) = 17.6 (1C, C-4),
32.2 (1C, C-3), 32.3 (1C, C-5), 47.7 (1C, CH2C6H4OCH3), 48.0 (1C,
CH2C6H5), 55.5 (1C, C6H4OCH3), 59.5 (1C, C-6), 63.1 (1C, C-1),
72.0 (1C, C-2), 114.6 (2C, C-3, C-5methoxybenzyl), 127.7 (1C, C-4benzyl),
128.5 (1C, C-1methoxybenzyl), 128.6 (2C, C-3, C-5benzyl), 129.2 (2C, C-2,
C-6benzyl), 130.1 (2C, C-2, C-6methoxybenzyl), 135.4 (1C, C-1benzyl), 159.7
(1C, C-4methoxybenzyl), 167.3 (1C, carbonyl), 167.7 (1C, carbonyl). MS
(EI): m/z (%) = 394.0 [(M)+, 32], 121.0 [(methoxybenzyl)+, 100],
Method 2 from 21. To a suspension of 21 (1.2 g, 2.3 mmol) in
CH3OH–CH2Cl2 (5/1, 50 mL), K2CO3 (0.96 g, 6.9 mmol) was
added and the mixture was stirred at rt for 16 h. The solvent
was evaporated under vacuum and H2O (20 mL) was added. The
aqueous layer was extracted with CH2Cl2 (5 ¥ 20 mL) and the
solvent was evaporated under vacuum to obtain 16a as colorless
viscous oil. Purification by fc (petroleum ether/ethyl acetate =
95/5 + 0.2% N,N-dimethylethylamine, 4 cm, 15 cm, 20 mL, Rf
0.27). Colorless oil, yield 0.82 g (97%).
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C23H30N2O2 (366.5). Purity (HPLC, method A): 98.0%, tR
=
0.38 mmol), p-nitrobenzoic acid (0.040 g, 0.25 mmol) were added
to a solution of 15a (50 mg, 0.13 mmol) in THF (15 mL).
Diisopropyl azodicarboxylate (0.07 mL, 0.38 mmol) was added
dropwise to this mixture under ice-cooling. The mixture was
heated to reflux and stirred for 18 h. The solvent was evaporated
under vacuum. Without purification, the residue (mixture of 17
and 18) was dissolved in CH3OH–CH2Cl2 (14 mL (1/1)), K2CO3
was added and the mixture was stirred at rt for 30 h. The solvent
was evaporated under vacuum. H2O (20 mL) was added to the
crude residue and extracted with CH2Cl2 (5 ¥ 20 mL). The
combined organic layers were dried (Na2SO4) and the solvent was
evaporated under vacuum. The crude residue was separated by
fc (ethyl acetate/cyclohexane = 1/4–ethyl acetate/cyclohexane =
7/3, 2.5 cm, 16 cm, 10 mL) to obtain alkene 18 as a colorless solid
and alcohol 19a as a colorless viscous oil.
18.7 min. 1H NMR (600 MHz, CDCl3): d (ppm) = 1.21-1.31 (m,
1H, 5-H), 1.48-1.53 (m, 1H, 5-H), 1.61-1.69 (m, 1H, 4-H), 1.95-
1.99 (m, 1H, 3-H), 2.22 (ddd, J = 17.5/13.8/4.9 Hz, 1H, 4-H),
2.37 (q, J = 10.7 Hz, 1H, 3-H), 2.56 (dd, J = 11.1/1.3 H, 1H,
10-H), 2.79-2.81 (m, 2H, 2-H, 8-H), 2.85 (s(b), 1H, 6-H), 2.95 (dd,
J = 12.5/3.1 Hz, 1H, 8-H), 3.01 (ddd, J = 10.9/4.1/1.5 Hz, 1H,
10-H), 3.52-3.65 (m, 5H, 1-H, NCH2C6H4OCH3, NCH2C6H5),
3.77 (s, 3H, OCH3), 6.81 (d, J = 8.6 Hz, 2H, 3-H, 5-Hmethoxybenzyl),
7.21-7.24 (m, 3H, 2-H, 6-Hmethoxybenzyl,4-Hbenzyl), 7.28-7.31 (m, 2H, 2-
H, 6-Hbenzyl), 7.34-7.36 (m, 2H, 3-H, 5-Hbenzyl). 13C NMR (CDCl3):
d (ppm) = 21.7 (1C, C-4), 33.9 (1C, C-3), 36.2 (1C, C-5), 44.6
(1C, C-10), 50.5 (1C, C-8), 55.4 (1C, C6H4OCH3), 57.9 (1C, C-6),
62.9 (1C, CH2C6H5), 63.4 (1C, C-1), 63.6 (1C, CH2C6H4OCH3),
74.2 (1C, C-2), 113.7 (2C, C-3, C-5methoxybenzyl), 127.1 (1C, C-4benzyl),
128.3 (1C, C-1methoxybenzyl), 129.1 (2C, C-3, C-5benzyl), 130.3 (2C, C-
2, C-6benzyl), 131.8 (2C, C-2, C-6methoxybenzyl), 140.0 (1C, C-1benzyl),
158.8 (1C, C-4methoxybenzyl). MS (EI): m/z (%) = 366.2 [(M)+, 64],
245.3 [(M-methoxybenzyl)+, 56], 121.0 [(methoxybenzyl)+, 100],
18. Rf 0.40 (ethyl acetate/cyclohexane = 1/1). Colorless solid,
mp 143–147 ◦C, yield 24 mg (52%). C23H24N2O3 (376.4). Purity
1
(HPLC, method A): 97.6%, tR = 18.8 min. H NMR (CDCl3):
d (ppm) = 1.73-1.82 (m, 1H, 5-H), 1.97-2.09 (m, 2H, 4-H, 5-H),
2.15-2.24 (m, 1H, 4-H), 3.73 (s, 3H, OCH3), 4.05 (t, J = 4.4 Hz,
1H, 6-H),4.11 (d, J = 14.8 Hz, 1H, NCH2C6H4OCH3), 4.12 (d,
J = 14.5 Hz, 1H, NCH2C6H5), 4.27 (d, J = 6.8 Hz, 1H, 1-H),
4.76 (d, J = 14.5 Hz, 1H, NCH2C6H5), 4.85 (d, J = 14.8 Hz,
1H, NCH2C6H4OCH3), 5.68-5.78 (m, 2H, 2-H, 3-H), 6. 78 (d,
J = 8.5 Hz, 2H, 3-H, 5-Hmethoxybenzyl), 7.11 (d, J = 8.6 Hz, 2H,
2-H, 6-Hmethoxybenzyl), 7.15-7.17 (m, 2H, 2-H, 6-Hbenzyl), 7.22-7.29
(m, 3H, 3-H, 4-H, 5-Hbenzyl). 13C NMR (CDCl3): d (ppm) = 22.1
(1C, C-5), 30.1 (1C, C-4), 46.6 (1C, NCH2C6H4OCH3), 47.2 (1C,
NCH2C6H5), 54.2 (1C, OCH3), 57.9 (1C, C-6), 58.2 (1C, C-1),
113.2 (2C, C-3, C-5methoxybenzyl), 126.5 (1C, C-3), 127.0 (1C, C-3),
127.3 (1C, C-4benzyl), 127.6 (1C, C-1methoxybenzyl), 127.9 (2C, C-3, C-
5benzyl), 128.9 (2C, C-2, C-6benzyl), 132.1 (2C, C-2, C-6methoxybenzyl),
134.5 (1C, C-1benzyl), 159.6 (1C, C-4methoxybenzyl), 165.0 (d, J = 14.8 Hz,
1C, carbonyl), 169.0 (1C, carbonyl). MS (EI): m/z (%) = 375 [(M-
H)+, 43], 255 [(M-methoxybenzyl)+, 32], 91 [(benzyl)+, 100]. IR
(neat): n/cm-1 = 1658 (C O), 1511 (C C aromatic), 1243 (C–
O), 699 (C–H).
91.1 [(benzyl)+, 38]. IR (neat): n/cm-1 = 3405 (O–H), 1509 (C
aromatic), 1442 (C–N), 1244 (C–O).
C
7.2.8. (1RS,2RS,6SR)-7-Benzyl-2-methoxy-9-(4-methoxy-
benzyl)-7,9-diazabicyclo[4.2.2] decane (16b). Under N2 LiAlH4
(0.76 mL, 0.76 mmol, 1 M solution in THF) was slowly added to
a solution of 15b (0.10 g, 0.25 mmol) in dry THF (25 mL) under
ice-cooling. The mixture was warmed to rt and stirred under reflux
for 14 h. Excess LiAlH4 was destroyed with H2O (1 mL) under ice-
cooling. The mixture was again stirred under reflux for 1 h. The
solution was cooled to rt and filtered through a sintering funnel.
The precipitate was washed with CH2Cl2 (50 mL). The solvent
was evaporated under vacuum. The crude oil was purified by fc
(ethyl acetate/petroleum ether = 5/95, 2.5 cm, 13 cm, 10 mL, Rf
0.45 (ethyl acetate/cyclohexane = 3/7)). Colorless oil, yield 0.092 g
(91%). C24H32N2O2 (380.2). Purity (HPLC, method A): 98.6%, tR =
1
19.1 min. H NMR (CDCl3): d (ppm) = 1.22-1.29 (m, 1H, 5-H),
1.35-1.45 (m, 1H, 5-H), 1.56-1.64 (m, 1H, 4-H), 1.87-1.94 (m, 1H,
3-H), 2.12-2.18 (m, 1H, 4-H), 2.25-2.34 (m, 1H, 3-H),2.55 (dd, J =
10.9/1.8 Hz, 1H, 10-H), 2.75-2.80 (m, 2H, 6-H, 8-H), 2.84-2.91
(m, 3H, 1-H, 2-H, 8-H), 2.98 (s, 4H, CHOCH3, 10-H), 3.45 (d,
J = 12.4 Hz, 2H, CH2Ph, CH2C6H4OCH3), 3.52 (d, J = 12.5 Hz,
1H, CH2C6H5), 3.59 (d, J = 13.5 Hz, 1H, CH2C6H4OCH3), 3.72
(s, 3H, C6H4OCH3), 6.79 (d, J = 8.7 Hz, 2H, 3-H, 5-Hmethoxybenzyl),
7.13-7.24 (m, 5H, 2-H, 6-Hmethoxybenzyl, 2-H, 4-H, 6-Hbenzyl), 7.26-7.29
(m, 2H, 3-H, 5-Hbenzyl). 13C NMR (CDCl3): d (ppm) = 22.0 (1C,
C-4), 30.1 (1C, C-3), 36.3 (1C, C-5), 45.4 (1C, C-8), 50.7 (1C, C-
10), 55.5 (1C, C6H4OCH3), 56.9 (1C, CHOCH3), 57.6 (1C, C-6),
59.5 (1C, C-1), 62.8 (1C, CH2C6H5), 63.3 (1C, CH2C6H4OCH3),
83.9 (1C, C-2), 113.6 (2C, C-3, C-5methoxybenzyl), 126.9 (1C, C-4benzyl),
128.2 (1C, C-1methoxybenzyl), 129.3 (2C, C-3, C-5benzyl), 130.6 (2C,
C-2, C-6benzyl), 131.8 (2C, C-2,C-6methoxybenzyl), 140.0 (1C, C-1benzyl),
158.8 (1C, C-4methoxybenzyl). MS (EI): m/z (%) = 380.1 [(M)+, 66],
289.0 [(M-benzyl)+, 12], 259.0 [(M-methoxybenzyl)+, 55], 121.0
19a. Rf 0.10 (cyclohexane/ethyl acetate = 3/7). Colorless oil,
yield (3 mg, 6%, calculated over two steps from 15a). C23H26N2O4
1
(394.4). H NMR (CDCl3): d (ppm) = 1.26-1.35 (m, 1H, 4-H),
1.35-1.58 (m, 2H, 3-H, 4-H), 1.63-1.69 (m, 1H, 3-H), 1.74-1.81
(m, 1H, 5-H), 1.91-1.98 (m, 1H, 5-H), 3.73 (s, 3H, OCH3), 3.99
(d, J = 14.5 Hz, 1H, NCH2C6H4OCH3), 4.05 (d, J = 3.7 Hz, 1H,
6-H), 4.07 (s, 1H, 2-H), 4.11 (d, J = 14.6 Hz, 1H, NCH2C6H5), 4.20
(s, 1H, 1-H), 4.86 (d, J = 14.7 Hz, 1H, NCH2C6H5), 5.27 (d, J =
14.5 Hz, 1H, NCH2C6H4OCH3), 6.79 (d, J = 8.7 Hz, 2H, 3-H, 5-
Hmethoxybenzyl), 7.13 (d, J = 8.7 Hz, 2H, 2-H, 6-Hmethoxybenzyl), 7.16-7.18
(m, 2H, 2-H, 6-Hbenzyl), 7.23-7.29 18 (m, 3H, 3-H, 4-H, 5-Hbenzyl).
MS (EI): m/z (%) = 394.0 [(M)+, 32], 121.0 [(methoxybenzyl)+,
100], 91.1 [(benzyl)+, 42]. IR (neat): n/cm-1 = 3398 (O–H), 1657
(C O), 1451 (C–N), 1244 (C–O), 1173 (C–O).
7.2.10. [(1RS,2SR,6RS)-7-Benzyl-9-(4-methoxybenzyl)-8,10-
dioxo-7,9-diazabicyclo[4.2.2]dec-2-yl] p-toluenesulfonate (20).
The alcohol 15a (0.10 g, 0.25 mmol) was dissolved in CH2Cl2
(5 mL). p-Toluenesulfonyl chloride (0.070 g, 0.38 mmol) and
powdered KOH ◦(15 mg, 0.28 mmol) were added and the mixture
was stirred at 0 C for 2.5 h. The reaction mixture was acidified
with 1 M HCl and sat. NaHCO3 solution was added (pH 7.5).
[(methoxybenzyl)+, 100], 91.1 [(benzyl)+, 33]. IR (neat): n/cm-1
1510 (C C aromatic), 1244 (C–O), 1088 (C–O).
=
7.2.9. (1RS,6RS)-7-Benzyl-9-(4-methoxybenzyl)-7,9-diaza-
bicyclo[4.2.2]dec-2-ene-8,10-dione (18) and (1RS,2RS,6RS)-7-
benzyl-2-hydroxy-9-(4-methoxybenzyl)-7,9-diazabicyclo[4.2.2]dec-
ane-8,10-dione (19a). Under N2 triphenylphosphine (0.10 g,
5536 | Org. Biomol. Chem., 2010, 8, 5525–5540
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The aqueous layer was extracted with CH2Cl2 (5 ¥ 10 mL). The
combined organic layers were washed with brine, dried (Na2SO4)
and evaporated under vacuum to obtain a colorless viscous oil.
Purification by fc (ethyl acetate/petroleum ether = 1/1, 3 cm,
13 cm, 10 mL, Rf 0.26) gave a colorless oil, yield 0.13 g (94%).
1.11 (dd, J = 13.5/3.2 Hz, 1H, 5-H), 1.32–1.40 (m, 1H, 5-H), 1.54
(s(b), 1H, 4-H), 2.02 (t, J = 11.1 Hz, 1H, 3-H), 2.31 (d, J = 10.6 Hz,
2H, 4-H, 10-H), 2.42 (s(b), 1H, 3-H), 2.71 (dd, J = 11.2/4.0 Hz,
2H, 6-H, 8-H), 2.77 (ddd, J = 10.7/3.7/1.3 Hz, 1H, 10-H), 2.88 (d,
J = 11.1 Hz, 1H, 8-H), 2.97 (s(b), 1H, 1-H), 3.09 (s, 3H, OCH3 ketal),
3.16 (s, 3H, OCH3 ketal),3.37 (d, J = 12.7 Hz, 1H, CH2C6H4OCH3),
3.47 (d, J = 13.2 Hz, 1H, CH2C6H5), 3.58 (d, J = 13.2 Hz, 1H,
CH2C6H5),3.71 (s, 3H, C6H4OCH3), 3.85 (d, J = 12.7 Hz, 1H,
CH2C6H4OCH3), 6.76 (d, J = 8.6 Hz, 2H, 3-H, 5-Hmethoxybenzyl), 7.11-
1
C30H32N2O6S (548.6). H NMR (CDCl3): d (ppm) = 1.24-1.37
(m, 1H, 4-H), 1.56-1.67 (m, 2H, 4-H, 5-H) 1.69-1.78 (m, 2H,
3-H, 5-H), 1.90-1.96 (m, 1H, 3-H), 2.39 (s, 3H, CH3 tosyl), 3.76
(s, 3H, OCH3), 3.98 (dd, J = 4.6/3.2 Hz, 1H, 6-H), 4.06 (d, J =
14.7 Hz, 1H, NCH2C6H4OCH3), 4.15 (d, J = 4. 8 Hz, 1H, 1-H),
4.18 (d, J = 14.5 Hz, 1H, NCH2C6H5), 4.45-4.48 (m, 1H, 2-H),
4.62 (d, J = 14.4 Hz, 1H, NCH2C6H5), 4.92 (d, J = 14.7 Hz, 1H,
NCH2C6H4OCH3), 6. 76 (d, J = 8.5 Hz, 2H, 3-H, 5-Hmethoxybenzyl),
7.15-7.25 (m, 5H, 2-H, 6-Hmethoxybenzyl,2-H, 4-H, 6-Hbenzyl), 7.28 (d,
J = 7.3 Hz, 2H, 3-H, 5-Hbenzyl), 7.30 (d, J = 8.0 Hz, 2H, 3-H,
5-Htosyl), 7.76 (d, J = 8.2 Hz, 2H, 4-H, 6-Htosyl). Exact mass (ESI):
m/z = calculated for C30H32N2O6SNa+ 571.1878, found 571.1874.
7.27 (m, 7H, 2-H, 6-Hmethoxybenzyl, 2-H, 3-H, 4-H, 5-H, 6-Hbenzyl). 13
C
NMR (CDCl3): d (ppm) = 19.8 (1C, C-4), 31.9 (1C, C-3), 36.0 (1C,
C-5), 46.9 (1C, C-10), 48.1 (1C, OCH3 ketal), 48.2 (1C, OCH3 ketal),
49.1 (1C, C-8), 55.4 (1C, C6H4OCH3), 57.6 (1C, C-6), 61.9 (1C, 1C,
C-1), 63.1 (1C, CH2C6H5), 63.6 (1C, CH2C6H4OCH3), 103.5 (1C,
C-2), 113.6 (2C, C-3, C-5methoxybenzyl), 126.9 (1C, C-4benzyl), 128.2 (1C,
C-1methoxybenzyl), 129.1 (2C, C-3, C-5benzyl), 130.1 (2C, C-2, C-6benzyl),
132.8 (2C, C-2, C-6methoxybenzyl), 140.1 (1C, C-1benzyl), 158.6 (1C, C-
4methoxybenzyl). Exact mass (ESI): m/z = calculated for C25H35N2O3H+
411.2648, found 411.2642. I.R. (neat): n/cm-1 = 2909 (C–H), 1509
(C C), 1245 (C–O), 1108 (C–O).
7.2.11. (1RS,6RS)-7-Benzyl-2,2-dimethoxy-9-(4-methoxyben-
zyl)-7,9-diazabicyclo[4.2.2]
decane-8,10-dione
(22). p-
Toluenesulfonic acid (1.21 g, 6.37 mmol) and trimethyl
orthoformate (2.0 mL, 17.85 mmol) were slowly added to a
solution of ketone 14 (1.0 g, 2.55 mmol) in CH3OH (70 mL)
under ice-cooling. The solution was warmed to rt and stirred
under reflux for 15 h. The mixture was cooled to rt, the solvent
was evaporated under vacuum and CH2Cl2 (10 mL) was added
to residue. The mixture was washed with sat. NaHCO3 and
the aqueous layer was extracted with CH2Cl2 (5 ¥ 25 mL). The
combined organic layers were washed with brine, dried over
Na2SO4 and the solvent was evaporated under vacuum. The crude
viscous oil was purified by fc (cyclohexane/ethyl acetate = 2/3,
3.5 cm, 15 cm, 30 mL, Rf 0.26) to obtain a colorless viscous oil,
7.2.13. (1RS,6SR)-7-Benzyl-9-(4-methoxybenzyl)-7,9-diaza-
bicyclo[4.2.2]decan-2-one (24). 1M HCl (1.6 mL, 1.6 mmol) was
added to a solution of 23 (0.66 g, 1.6 mmol) in THF–H2O (1/1,
30 mL) and the mixture was stirred for 10 h at rt. Saturated
NaHCO3 solution was added until the mixture attained a pH
of 7.5. The aqueous layer was extracted with Et2O (5 ¥ 20 mL).
The combined organic layers were washed with brine, dried over
Na2SO4 and the solvent was evaporated under vacuum. The crude
viscous oil was purified by fc (ethyl acetate/petroleum ether =
5/95+0.05% N,N-dimethylethylamine, 4 cm, 10 cm, 30 mL, Rf 0.4
(ethyl acetate/cyclohexane = 1/4)) to obtain a colorless viscous oil,
yield 0.45 g (77%). C23H28N2O2 (364.2). Purity (HPLC, method A):
96.0%, tR = 17.5 min. 1H NMR (CDCl3): d (ppm) = 1.36 (dt, J =
11.4/4.3 Hz, 1H, 5-H), 1.54-1.61 (m, 1H, 5-H), 1.70–1.85 (m, 2H,
4-H), 2.10(dt, J = 11.4/3.2 Hz, 1H, 3-H), 2.54 (d, J = 10.6 Hz,
1H, 10-H), 2.92 (dd, J = 10.6/2.4 Hz, 1H, 10-H), 2.96-3-00 (m,
2H, 6-H, 8-H), 3.02 (t, J = 3.4 Hz, 1H, 8-H), 3.08 (s(b), 1H, 1-H),
3.31 (dt, J = 11.4/3.2 Hz, 1H, 3-H), 3.47 (d, J = 13.0 Hz, 1H,
CH2C6H4OCH3), 3.56 (t, J = 12.5 Hz, 2H, CH2C6H5), 3.71 (s, 3H,
OCH3), 6.75 (d, J = 8.6 Hz, 2H, 3-H, 5-Hmethoxybenzyl), 7.11-7.22 (m,
7H, 2-H, 6-Hmethoxybenzyl, 2-H, 3-H, 4-H, 5-H, 6-Hbenzyl). 13C NMR
(CDCl3): d (ppm) = 22.4 (1C, C-4), 35.3 (1C, C-5), 40.7 (1C, C-
3), 50.9 (1C, C-10), 52.33 (1C, C-8), 55.4 (1C, OCH3), 55.8 (1C,
C-6), 61.8 (1C, CH2C6H5), 62.5 (1C, CH2C6H4OCH3), 69.0 (1C,
C-1), 114.0 (2C, C-3, C-5methoxybenzyl), 127.2 (1C, C-4benzyl), 128.4 (1C,
C-1methoxybenzyl), 129.1 (2C, C-2, C-6benzyl), 130.1 (2C, C-3, C-5benzyl),
130.7 (2C, C-2, C-6methoxybenzyl), 139.1 (1C, C-1benzyl), 158.9 (1C, C-
4methoxybenzyl), 222.6 (1C, C-2). MS (EI): m/z (%) = 365.1 [M+H)+,
3] 121.1 [(methoxybenzyl)+, 100], 91.1 [(benzyl)+, 48]. IR (neat)
n/cm-1 = 1698 (C O), 1510 (C C aromatic), 1244 (C–O).
1
yield 1.12 g (99.9%). C25H30N2O5 (438.2). H NMR (CDCl3): d
(ppm) = 1.43-1.64 (m, 3H, 4-H, 5-H), 1.88-2.44 (m, 3H, 3-H,
5-H), 3.21 (s, 3H, OCH3 ketal), 3.33 (s, 3H, OCH3 ketal), 3.79 (s, 3H,
C6H4OCH3), 3.93 (d, J = 14.8 Hz, 1H, CH2C6H4OCH3), 4.03
(d, J = 14.8 Hz, 1H, CH2C6H5), 4.07 (dd, J = 5.7/2.5 Hz, 1H,
6-H), 4.17 (d, J = 0.6 Hz, 1H, 1-H), 5.04 (d, J = 14.8 Hz, 1H,
CH2C6H5), 5.38 (d, J = 14.8 Hz, 1H, CH2C6H4OCH3), 6.84 (d,
J = 8.7 Hz, 2H, 3-H, 5-Hmethoxybenzyl), 7.08 (d, J = 8.7 Hz, 2H, 2-H,
6-Hmethoxybenzyl), 7.19 (dd, J = 7.6 Hz, 1.6 Hz, 2H, 3-H, 5-Hbenzyl),
7.26-7.33 (m, 3H, 2-H, 4-H, 6-Hbenzyl). Exact mass (ESI): m/z =
calculated for C25H30N2O5Na+ 461.2052, found 461.2051. I.R.
(neat): n/cm-1 = 1663 (C O), 1511 (C C), 1244 (C–O), 1091
(C–O), 730 (C C).
7.2.12. (1RS,6SR)-7-Benzyl-2,2-dimethoxy-9-(4-methoxyben-
zyl)-7,9-diazabicyclo[4.2.2] decane (23). Under N2 LiAlH4
(10.8 mL, 0.10.8 mmol, 1 M solution in THF) was slowly added to
a solution of 22 (0.95 g, 2.15 mmol) in dry THF (43 mL) under ice-
cooling. The mixture was warmed to rt and stirred under reflux
for 12 h. Excess LiAlH4 was destroyed with H2O (3 mL) under
ice-cooling and the mixture was again stirred under reflux for 1 h.
The solution was cooled to rt and filtered through a sintering
funnel. The precipitate was washed with CH2Cl2 (100 mL) and the
solvent was evaporated under vacuum. The crude oil was purified
by fc (petroleum ether/ethyl acetate = 95/5, 4.5 cm, 16 cm, 30 mL,
Rf 0.42 (cyclohexane/ethyl acetate = 4/1)) to obtain a colorless
viscous oil, yield 0.71 g (80%). C25H34N2O3 (410.2). Purity (HPLC,
method B): 97.2%, tR = 16.33 min. 1H NMR (CDCl3): d (ppm) =
7.2.14. (1RS,2SR,6SR)-7-Benzyl-9-(4-methoxybenzyl)-7,9-
diazabicyclo[4.2.2]decan-2-ol (25a) and (1RS,2RS,6SR)-7-benzyl-
9-(4-methoxybenzyl)-7,9-diazabicyclo[4.2.2]decan-2-ol
(16a).
Under N2 L-Selectride (Lithium tri-sec-butylborohydride, 1M in
THF, 4.15 mL, 4.15 mmol) was slowly added to a solution of 24
(0.76 g, 2.07 mmol) in dry THF (40 mL) under ice-cooling and the
mixture was stirred for 10 min at low temperature and for 12 h at
rt. The excess L-Selectride was destroyed by addition of 1 M HCl
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Org. Biomol. Chem., 2010, 8, 5525–5540 | 5537
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(5 mL) under ice-cooling. Saturated NaHCO3 solution was added
until the solution attained a pH of 7.5, then the aqueous layer was
extracted with ethyl acetate (5 ¥ 30 mL). The combined organic
layers were washed with brine, dried over K2CO3 and the solvent
was evaporated under vacuum to obtain the diastereomeric
alcohols 16a and 25a. The 1H NMR spectrum of the crude sample
showed a ratio of 16a : 25a = 36 : 64. The diastereomers were
separated by fc (started from ethyl acetate/petroleum ether =
5/95+0.05% N,N-dimethylethylamine to ethyl acetate/petroleum
ether = 7/93+0.05% N,N-dimethylethylamine, 1.5 cm, 13 cm,
10 mL).
H
methoxybenzyl, 2-H, 4-H, 6-Hbenzyl), 7.26-7.29 (m, 2H, 3-H, 5-Hbenzyl).
13C NMR (CDCl3): d (ppm) = 19.2 (1C, C-4), 29.2 (1C, C-3), 34.4
(1C, C-5), 48.8 (1C, C-10), 48.9 (1C, C-8), 54.2 (1C, C6H4OCH3),
55.2 (1C, CHOCH3), 56.3 (1C, C-6), 59.9 (1C, C-1), 62.0 (1C,
CH2C6H5), 62.5 (1C, CH2C6H4OCH3), 85.7 (1C, C-2), 112.3 (2C,
C-3, C-5methoxybenzyl), 125.7 (1C, C-4benzyl), 127.0 (1C, C-1methoxybenzyl),
127.8 (2C, C-2, C-6benzyl), 129.0 (2C, C-3, C-5benzyl), 131.3 (2C, C-2,
C-6methoxybenzyl), 138.9 (1C, C-1benzyl), 157.3 (1C, C-4methoxybenzyl). Exact
mass (ESI): m/z = calculated for C24H33N2O2H+ 381.2542, found
381.2537. I.R. (neat): n/cm-1 = 2905 (C–H), 1509 (C–O), 1244
(C–O), 698 (C C).
25a (Rf 0.27, ethyl acetate/cyclohexane = 3/7): Colorless viscous
oil, yield 0.21 g (28%). C23H30N2O2 (366.2). Purity (HPLC, method
B): 96.0%, tR = 14.9 min. 1H NMR (CDCl3): d (ppm) = 1.18–1.27
(m, 1H, 5-H), 1.39-1.51 (m, 2H, 4-H, 5-H), 1.76-1.83 (m, 1H,
3-H), 1.95-2.07 (m, 1H, 4-H), 2.36 (ddd, J = 14.0/11.1/2.9 Hz,
1H, 3-H), 2.51 (s, 1H, 8-H), 2.54-2.55 (m, 1H, 10-H), 2.77 (d (b),
J = 4.5 Hz, 0.5H, 8-H), 2.80 (s(b), 1.5H, 8-H, 10-H), 2.86 (dd, J =
11.6/4.2 Hz, 1H, 6-H), 2.98 (s(b), 1H, 2-H), 3.15 (s(b), 0.6H, OH),
3.46 (d, J = 12.7 Hz, 2H, 1-H, CH2C6H5), 3.53 (d, J = 12.9 Hz,
1H, CH2C6H4OCH3), 3.58 (d, J = 12.9 Hz, 1H, CH2C6H4OCH3),
3.66 (d, J = 12.7 Hz, 1H, CH2C6H5), 3.72 (s, 3H, OCH3), 6.79 (d,
J = 8.4 Hz, 2H, 3-H, 5-Hmethoxybenzyl), 7.16 (d, J = 8.5 Hz, 2H, 2-H,
6-Hmethoxybenzyl), 7.22-7.24 (m, 3H, 3-H, 4-H, 5-Hbenzyl). 13C NMR
(CDCl3): d (ppm) = 18.9 (1C, C-4), 33.6 (1C, C-3), 35.9 (1C, C-5),
47.5 (1C, C-10), 51.0 (1C, C-8), 55.4 (1C, C6H4OCH3), 57.9 (1C, C-
6), 63.2 (1C, CH2Ph), 63.7 (1C, C-1), 64.9 (1C, CH2C6H4OCH3),
71.4 (1C, C-2), 114.1 (2C, C-3, C-5methoxybenzyl), 127.1 (1C, C-4benzyl),
128.3 (1C, C-1methoxybenzyl), 129.1 (2C, C-2, C-6benzyl), 130.0 (2C, C-
3, C-5benzyl), 131.3 (2C, C-2, C-6methoxybenzyl), 139.8 (1C, C-1benzyl),
7.2.16. (1RS,6RS)-7-Benzyl-9-(4-methoxybenzyl)-7,9-diaza-
bicyclo[4.2.2]decane (26). Under N2 LiAlH4 (0.03 g, 0.87 mmol)
was added to a solution of 20 (0.12 g, 0.22 mmol) in THF (10 mL)
under ice-cooling. The mixture was stirred at rt for 12 h and
under reflux for 3 h. The excess LiAlH4 was destroyed with H2O
(1 mL) under ice-cooling and the mixture was refluxed for an
additional hour. The mixture was cooled to rt and filtered. The
filtrate was concentrated under vacuum to obtain a colorless vis-
cous oil. Purification by fc (t-butyl methyl ether/petroleum ether =
7.5/92.5, 1.5 cm, 18 cm, 2 mL, Rf 0.26 ethyl acetate/cyclohexane =
1/9)) gave a colorless oil, yield 0.05 g (65%). C23H30N2O (350.5).
Purity (HPLC, method A): 96.5%, tR = 19.4 min. 1H NMR
(CDCl3): d (ppm) = 1.26 (d, J = 12.8 Hz, 2H, 2-H, 5-H), 1.47
(s (broad), 2H, 2-H, 5-H), 1.69 (d, J = 9.9 Hz, 2H, 3-H, 4-H), 2.09
(t, J = 10.2 Hz, 2H, 3-H, 4-H), 2.47 (d, J = 10.2 Hz, 2H, 8-H,
10-H), 2.80 (s, 2H, 1-H, 6-H), 2.88 (dd, J = 10.8/2.7 Hz, 2H, 8-H,
10-H), 3.48 (s, 2H, NCH2C6H5), 3.55 (s, 2H, NCH2C6H4OCH3),
3.72 (s, 3H, OCH3), 6. 76 (d, J = 8.5 Hz, 2H, 3-H, 5-Hmethoxybenzyl),
7.13-7.24 (m, 5H, 2-H, 6-Hmethoxybenzyl,2-H, 4-H, 6-Hbenzyl), 7.28 (d,
J = 7.3 Hz, 2H, 3-H, 5-Hbenzyl). 13C NMR (CDCl3): d (ppm) = 25.5
(2C, C-2, C-5), 36.3 (2C, C-3, C-4), 50.9 (2C, C-8, C-10), 55.4 (1C,
OCH3), 58.1 (1C, C-6), 58.3 (1C, C-1), 62.8 (1C, NCH2C6H5),
63.6 (1C, NCH2C6H4OCH3), 113.6 (2C, C-3, C-5methoxybenzyl), 126.9
(1C, C-4benzyl), 128.3 (1C, C-1methoxybenzyl), 129.2 (2C, C-3, C-5benzyl),
130.2 (2C, C-2, C-6benzyl), 132.5 (2C, C-2, C-6methoxybenzyl), 140.5
(1C, C-1benzyl), 160.2 (1C, C-4methoxybenzyl). Exact mass (ESI): m/z =
calculated for C23H30N2OH+ 351.2436, found 351.2435. IR (neat):
n/cm-1 = 1611, 1511 (C C), 1242 (C–O), 699 (C C).
159.0 (1C, C-4methoxybenzyl). Exact mass (ESI): m/z = calculated for
+
C23H31N2O2H+ 367.2386, found 367.2380 . IR (neat): n/cm-1
=
3401 (O–H), 1510 (C C), 1442 (C–N), 1247 (C–O).
16a (Rf 0.20, ethyl acetate/cyclohexane = 3/7): Colorless viscous
oil, yield 0.18 g (24%).
7.2.15. (1RS,2SR,6SR)-7-Benzyl-2-methoxy-9-(4-methoxy-
benzyl)-7,9-diazabicyclo[4.2.2] decane (25b). Under N2 NaH
(10 mg, 0.22 mmol, 60% in paraffin oil) was slowly added to a
solution of 25a (55 mg, 0.15 mmol) and CH3I (0.02 mL, 0.3 mmol)
in dry DMF (2 mL) under ice-cooling. The reaction mixture was
stirred for 3 h at rt. The excess NaH was destroyed with H2O (5 mL)
under ice-cooling. The aqueous layer was extracted with ethyl
acetate (7 ¥ 10 mL). The combined organic layers were washed
with brine, dried over K2CO3 and the solvent was evaporated
under vacuum. The crude viscous oil was purified by fc (ethyl
acetate/petroleum ether = 5/95+0.05% N,N-dimethylethylamine,
1.5 cm, 13 cm, 10 mL, Rf 0.36 (ethyl acetate/cyclohexane = 3/7))
to obtain a colorless viscous oil, yield 40 mg (70%). C24H32N2O2
(380.2). Purity (HPLC, method B): 97.2%, tR = 16.3 min. 1H
NMR (CDCl3): d (ppm) = 1.09–1.14 (m, 1H, 5-H), 1.46 (dt,
J = 12.9/5.6 Hz, 1H, 5-H), 1.56-1.64 (m, 1H, 4-H), 1.92-2.13
(m, 3H, 3-H, 4-H), 2.28 (d, J = 10.3 Hz, 1H, 10-H), 2.50 (d,
J = 11.1 Hz, 1H, 8-H), 2.78 (dd, J = 10.3/6.4 Hz, 2H, 6-H,
10-H), 2.86 (dd, J = 11.1/4.4 Hz, 1H, 8-H), 2.94-2.97 (m, 1H,
2-H), 3.11 (s(b), 1H, 1-H), 3.21 (s, 3H, CHOCH3), 3.46 (d, J =
12.7 Hz, 1H, CH2C6H4OCH3), 3.53 (s, 2H, CH2C6H5), 3.72 (s,
3H, C6H4OCH3), 3.76 (d, J = 12.7 Hz, 1H, CH2C6H4OCH3), 6.79
(d, J = 8.7 Hz, 2H, 3-H, 5-Hmethoxybenzyl), 7.13-7.24 (m, 5H, 2-H, 6-
7.2.17. (1RS,6RS)-7-Benzyl-9-(4-methoxybenzyl)-7,9-di-
azabicyclo[4.2.2]dec-2-ene (27). Under N2 LiAlH4 (0.01 g,
0.26 mmol) was added to a solution of 18 (50 mg, 0.13 mmol)
under ice-cooling. The mixture was heated to reflux for a period
of 12 h. H2O (1 mL) was added to the mixture under ice-cooling
and the mixture was heated to reflux for an additional hour. The
solution was cooled to rt and filtered through a sintering funnel.
The precipitate was washed with CH2Cl2 (20 mL) and the solvent
was evaporated under vacuum. The crude oil was purified by fc
(petroleum ether/ethyl acetate = 95/5, 1.5 cm, 22 cm, 5 mL, Rf
0.23 (ethyl acetate/cyclohexane = 2/8)) to obtain 27 as a colorless
viscous oil, yield 32 mg (71%). C23H28N2O (348.4). Purity (HPLC,
1
method A): 99.1%, tR = 18.2 min. H NMR (CDCl3): d (ppm) =
1.43 (dq, J = 13.9/4.7 Hz, 1H, 5-H), 1.64 (s (broad), 1H, 5-H), 2.02
(s, 1H, 4-H), 2.58 (d, J = 8.6 Hz, 1H, 8-H), 2.65 (d, J = 10.4 Hz,
1H, 10-H), 2.87 (d, J = 9.2 Hz, 1H, 8-H), 2.93-3.03 (m, 3H; 4-H,
6-H, 10-H), 3.24 (s, 1H, 1-H), 3.58 (s, 2H, NCH2C6H5), 3.66 (d,
J = 13.8 Hz, 1H, NCH2C6H4OCH3), 3.72 (s, 3H, OCH3), 3.76 (d,
J = 13.1 Hz, 1H, NCH2C6H4OCH3), 5.37 (dd, J = 10.6/5.5 Hz,
5538 | Org. Biomol. Chem., 2010, 8, 5525–5540
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1H, 2-H),5.90 (dt, J = 11.3/7.6 Hz, 1H, 3-H), 6. 76 (d, J = 8.5 Hz,
2H, 3-H, 5-Hmethoxybenzyl), 7.15-7.25 (m, 5H, 2-H, 6-Hmethoxybenzyl,2-H,
4-H, 6-Hbenzyl), 7.28 (d, J = 7.3 Hz, 2H, 3-H, 5-Hbenzyl). 13C NMR
(CDCl3): d (ppm) = 24.2 (1C, C-4), 35.9 (1C, C-5), 49.6 (1C, C-8),
50.7 (1C, C-10), 55.4 (1C, OCH3), 56.2 (1C, C-6), 58.8 (1C, C-1),
61.3 (1C, NCH2C6H4OCH3), 61.9 (1C, NCH2C6H5), 113.7 (2C, C-
3, C-5methoxybenzyl), 127.0 (1C, C-4benzyl), 128.3 (3C, C-1methoxybenzyl, C-3,
C-5benzyl), 129.2 (2C, C-2, C-6benzyl), 129.8 (3C, C-2, C-6methoxybenzyl, C-
1benzyl), 130.3 (1C, C-3), 130.9 (1C, C-2), 158.7 (1C, C-4methoxybenzyl).
MS (EI): m/z (%) = 347 [M+, 3], 121 [methoxybenzyl+, 100], 91
[(benzyl)+, 56]. IR (neat): n/cm-1 = 1611 (C C), 1511 (C C),
1244 (C–O), 699 (C–H).
The scintillation analysis was performed using Meltilex (Typ
A or B) solid scintillator (Perkin Elmer). The solid scintillator
was melted on the filter mat at a temperature of 95 ◦C for
5 min. After solidifying of the scintillator at rt, the scintillation
was measured using a MicroBeta Trilux scintillation analyzer
(Perkin Elmer). The counting efficiency was 40%. All experiments
were carried out in triplicates using standard 96-well-multiplates
(Diagonal, Muenster, Germany). The IC50-values were determined
in competition experiments with at least six concentrations of the
test compounds and were calculated with the program GraphPad
R
Prismꢀ 3.0 (GraphPad Software, San Diego, CA, USA) by non-
linear regression analysis. The Ki-values were calculated according
to the formula of Cheng and Prusoff.46 The Ki-values are given as
mean value SEM from three independent experiments.
7.2.18. (1RS,6SR)-7-Benzyl-2,2-difluoro-9-(4-methoxybenzyl)-
7,9-diazabicyclo[4.2.2] decane (36). DAST (0.016 mL,
0.12 mmol) was added to an ice-cold solution of ketone 24
(22 mg, 0.06 mmol) in CH2Cl2 (2 mL) and the mixture was
stirred at 0 ◦C for 12 h. The reaction mixture was washed
with sat. NaHCO3 solution (10 mL) and the aqueous layer
was extracted with CH2Cl2 (5 ¥ 10 mL). The combined organic
layers were dried (K2CO3) and the solvent was evaporated under
vacuum to obtain a colorless viscous oil. Purification by fc
(ethyl acetate/petroleum ether = 3/97, 1.5 cm, 15 cm, 5 mL, Rf
0.52 (ethyl acetate/cyclohexane = 1/4)) gave a colorless viscous
oil, yield 0.22 g (95%). C23H28F2N2O (386.2). Purity (HPLC,
7.3.2. Membrane preparation for the r1 assay. Five guinea
pig brains were homogenized with the Potter (500–800 rpm, 10
up-and-down strokes) in 6 volumes of cold 0.32 M sucrose. The
◦
suspension was centrifuged at 1200 ¥ g for 10 min at 4 C. The
supernatant was separated and centrifuged at 23500 ¥ g 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
23500 ¥ g (20 min, 4 ◦C). This procedure was repeated twice.
The final pellet was resuspended in 5–6 volumes of buffer, the
protein concentration was determined according to the method
of Bradford47 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-1.
1
method A): 96.4%, tR = 19.7 min. H NMR (CDCl3): d (ppm) =
1.28-1.32 (m, 1H, 5-H), 1.43-1.52 (m, 1H, 5-H), 1.67 (ddd, J =
14.2/8.7/4.1 Hz, 1H, 4-H), 2.20 (ddd, J = 25.1/12.5/10.6 Hz,
1H, 3-H), 2.39 (ddd, J = 15.4/13.1/4.1 Hz, 1H, 4-H), 2.45 (d, J =
9.2 Hz, 1H, 10-H), 2.64-2.81 (m, 1H, 3-H), 2.84-2.92 (m, 4H, 6-H,
8-H, 10-H), 3.16 (t, J = 9.1 Hz, 1H, 1-H), 3.53 (d, J = 12.7 Hz,
1H, NCH2C6H4OCH3), 3.61 (d, J = 12.9 Hz, 1H, NCH2C6H5),
3.65 (d, J = 12.9 Hz, 1H, NCH2C6H5), 3.80 (s, 3H, OCH3), 3.88
(d, J = 12.9 Hz, 1H, NCH2C6H5), 6.84 (d, J = 8.8 Hz, 2H, 3-H,
5-Hmethoxybenzyl), 7.24 (d, J = 8.6 Hz, 3H, 2-H, 6-Hmethoxybenzyl, 4-Hbenzyl),
7.30-7.36 (m, 4H, 2-H, 3-H, 5-H, 6-Hbenzyl). 13C NMR (CDCl3): d
(ppm) = 19.8 (t, J = 5.6 Hz, 1C, C-4), 35.1 (t, J = 25.6 Hz, 1C,
C-3), 35.4 (1C, C-5), 45.5 (t, J = 4.9 Hz, 1C, C-8), 48.6 (1C, 10-C),
55.4 (1C, OCH3), 57.7 (1C, C-6), 62.8 (1C, NCH2C6H4OCH3),
63.2 (1C, NCH2C6H5), 64.7 (t, J = 29.2 Hz, 1C, C-1), 113.7 (2C,
C-3, C-5methoxybenzyl), 127.2 (1C, C-4benzyl), 124.8 (t, J = 246.2 Hz, 1C,
C-2), 128.4 (1C, C-1methoxybenzyl), 129.2 (2C, C-3, C-5benzyl), 130.2 (2C,
C-2, C-6benzyl), 131.5 (2C, C-2, C-6methoxybenzyl), 139.4 (1C, C-1benzyl),
158.8 (1C, C-4methoxybenzyl). Exact mass (ESI): m/z = calculated for
7.3.3. Performing the r1 assay. The test was performed
with the radioligand [3H]-(+)-pentazocine (32,2 Ci/mmol; Perkin
Elmer LAS). The thawed membrane preparation (about 75 mg
of the protein) was incubated with various concentrations of
test compounds, 2 nM [3H]-(+)-pentazocine, and buffer (50 mM
TRIS, pH 7.4) in a total volume of 200 mL for 150 min at
37 ◦C. The incubation was terminated by rapid filtration through
the presoaked filter mats using a cell harvester. After washing each
well five times with 300 ml of water, the filter mats were dried at
95 ◦C. Subsequently, the solid scintillator was placed on the filter
◦
mat and melted at 95 C. After 5 min, the solid scintillator was
allowed to solidify at rt. The bound radioactivity trapped on the
filters was counted in the scintillation analyzer. The non-specific
binding was determined with 10 mM unlabeled (+)-pentazocine.
The Kd-value of (+)-pentazocine is 2.9 nM.42
C23H28F2N2OH+ 387.2247, found 387.2242. IR (neat): n/cm-1
=
7.3.4. Membrane preparation for the r2 assay. Two rat livers
were cut into smaller pieces and homogenized with the potter (500–
800 rpm, 10 up-and-down strokes) in 6 volumes of cold 0.32 M
sucrose. The suspension was centrifuged at 1200 ¥ g for 10 min at
4 ◦C. The supernatant was separated and centrifuged at 31000 ¥ g
for 20 min at 4 ◦C. The pellet was resuspended in 5–6 volumes of
buffer (50 mM TRIS, pH 8.0) and incubated at rt for 30 min. After
the incubation, the suspension was centrifuged again at 31000 ¥ g
for 20 min at 4 ◦C. The final pellet was resuspended in 5–6 volumes
of buffer, the protein concentration was determined according to
the method of Bradford47 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-1.
1511 (C C), 1245 (C–F), 698 (C C).
7.3. Receptor binding studies
7.3.1. Materials and general procedures. The guinea pig
brains and rat livers were commercially available (Harlan–
Winkelmann, Borchen, Germany). The pig brains were a donation
of the local slaughterhouse (Coesfeld, Germany). Homogenizer:
Elvehjem Potter (B. Braun Biotech International, Melsungen,
Germany). Centrifuge: High-speed cooling centrifuge model
Sorvall RC-5C plus (Thermo Fisher Scientific, Langenselbold,
Germany). Filter: Printed Filtermat Typ A and B (Perkin Elmer
LAS, Rodgau-Ju¨gesheim, Germany), presoaked in 0.5% aqueous
polyethylenimine for 2 h at rt before use. The filtration was carried
out with a MicroBeta FilterMate-96 Harvester (Perkin Elmer).
7.3.5. Performing the r2 assay. The test was performed with
the radioligand [3H]-ditolylguanidine (50 Ci/mmol; ARC, St.
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Louis, MO, USA). The thawed membrane preparation (about
100 mg of the protein) was incubated with various concentra-
tions of test compounds, 3 nM [3H]-ditolylguanidine, and buffer
containing (+)-pentazocine (2 mM (+)-pentazocine in 50 mM
TRIS, pH 8.0) in a total volume of 200 mL for 150 min at rt.
The incubation was terminated by rapid filtration through the
presoaked filter mats using a cell harvester. After washing each
well five times with 300 mL of water, the filter mats were dried at
95 ◦C. Subsequently, the solid scintillator was placed on the filter
14 A. Urani, P. Romieu, F. J. Roman, K. Yamada, Y. Noda, H. Kamei,
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◦
mat and melted at 95 C. After 5 min, the solid scintillator was
allowed to solidify at rt. The bound radioactivity trapped on the
filters was counted in the scintillation analyzer. The non-specific
binding was determined with 10 mM unlabeled ditolylguanidine.
The Kd-value of ditolylguanidine is 17.9 nM.43
24 C. Oberdorf, D. Schepmann, J. M. Vela, J. L. Diaz, J. Holenz and B.
Wu¨nsch, J. Med. Chem., 2008, 51, 6531–6537.
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26 E. Große Maestrup, S. Fischer, C. Wiese, D. Schepmann, A. Hiller, W.
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A well established microtiter assay based on the staining of cell
components with crystal violet was used to measure the inhibition
of cell growth, as described in detail in ref. 45
27 A. Jasper, D. Schepmann, K. Lehmkuhl, J. M. Vela, H. Buschmann, J.
Holenz and B. Wu¨nsch, Eur. J. Med. Chem., 2009, 44, 4306–4314.
28 A. Foster, H. Wu, W. Chen, W. Williams, W. D. Bowen, R. R.
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29 S. Bedu¨rftig and B. Wu¨nsch, Bioorg. Med. Chem., 2004, 12, 3299–3311.
30 C. Geiger, C. Zelenka, M. Weigl, R. Fro¨hlich, B. Wibbeling, K.
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Acknowledgements
We wish to thank the NRW Graduate School of Chemistry (GSC-
MS) for a stipend, which was funded by the Government of the
state Northrhine-Westfalia and the DAAD. Financial support of
this project by the DFG is gratefully acknowledged.
31 Calculations were preformed with the MMFF94x of MOE (molecular
operating environment).
32 S. K. Sunnam, D. Schepmann, B. Wibbeling and B. Wu¨nsch, Org.
Biomol. Chem., 2010, 8, 3715–3722.
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