Conformationally constrained ethylenediamines: Synthesis and receptor binding of 6,8-diazabicyclo[3.2.2]nonanes

Article abstract of DOI:10.1016/S0968-0896(02)00043-3

The synthesis and receptor affinity of 6,8-diazabicyclo[3.2.2]nonanes representing conformationally constrained ethylenediamines are described. The Dieckmann analogous cyclization of the (piperazin-2-yl)propionate 9 provided the bicyclononane 10 only, when the first cyclization product was trapped with chlorotrimethylsilane. 10 was stereoselectively transformed into the bicyclic amines 19a,b and amides 22a,b, which were investigated in competition experiments with radioligands for their σ₁-, σ₂-, κ-, and μ-receptor affinities. The (2R)-configured dimethylamine 19a showed promising σ₁-receptor affinity (K_i=23.8 nM) and selectivity, whereas the (2S)-configured (dichlorophenyl)acetamide 22b displayed a σ-receptor binding profile (σ₁: K_i=184 nM; σ₂: K_i=263 nM) very similar to the binding profile of the atypical antipsychotic BMY-14802 (26).

Full text of DOI:10.1016/S0968-0896(02)00043-3

Bioorganic & Medicinal Chemistry 10 (2002) 2245–2257
Conformationally Constrained Ethylenediamines: Synthesis and
Receptor Binding of 6,8-Diazabicyclo[3.2.2]nonanes
Manuela Weigl, Stephan Bedurftig, Christoph A. Maier and Bernhard Wunsch*
Pharmazeutisches Institut der Universitat Freiburg, Albertstraße 25, D-79104 Freiburg i. Br., Germany
¨
Received 23 November 2001; accepted 24 January 2002
Abstract—The synthesis and receptor affinity of 6,8-diazabicyclo[3.2.2]nonanes representing conformationally constrained ethyl-
enediamines are described. The Dieckmann analogous cyclization of the (piperazin-2-yl)propionate 9 provided the bicyclononane
10 only, when the first cyclization product was trapped with chlorotrimethylsilane. 10 was stereoselectively transformed into the
bicyclic amines 19a,b and amides 22a,b, which were investigated in competition experiments with radioligands for their s1-, s2-, k-,
and m-receptor affinities. The (2R)-configured dimethylamine 19a showed promising s1-receptor affinity (Ki=23.8 nM) and selec-
tivity, whereas the (2S)-configured (dichlorophenyl)acetamide 22b displayed a s-receptor binding profile (s1: Ki=184 nM; s2:
Ki=263 nM) very similar to the binding profile of the atypical antipsychotic BMY-14802 (26). # 2002 Elsevier Science Ltd. All
rights reserved.
Introduction
diamine 1 binds with high affinity to s-receptors
(Ki=0.34 nM).10 Insertion of the ethylenediamine sub-
structure into a piperazine ring systemleads to a high
affinity s-receptor ligand as well (compound 2, s1:
Ki=0.55 nM).11 In the cyclohexane derivative 3, which
also comprises the ethylenediamine substructure, the
stereochemistry is essential for high s-receptor binding.
Among the stereoisomers the cis-configured (1R,2S)-
cyclohexane 3 shown in Figure 1 is the most active
s-receptor ligand (Ki=118 nM).12
Ligands interacting with high affinity and selectivity
with central nervous system(CNS) receptors are able to
modulate (patho)physiological events and to influence
psychiatric disorders. Therefore, we are interested in the
development of novel ligands for s- and opioid-recep-
tors—particularly k-opioid-receptors.
s-Receptors, which have been originally classified into
the opioid receptor family,1 are now well accepted as
real receptors.2,3 Their ligands possess potential as
atypical antipsychotics,4,5 antidepressants,6 and anti-
tumor agents.7,8
However, changing the cis-(1R,2S)-configuration of the
cyclohexane derivative 3 into the trans-(1S,2S)-config-
uration provides the prototypical k-receptor agonist
U-50488 (4, Ki=0.89 nM).12 In the class of k-receptor
agonists the ethylenediamine substructure may also be
inserted into a piperazine heterocycle and its side
chain. Hence, the 2-(pyrrolidinylmethyl)piperazines 5
(IC50=0.018 nM)13 and 6 (Ki=0.34 nM)14 belong to the
most active k-receptor agonists. The k-receptor affinity
of 5 and 6 strongly depends on the stereochemistry.
Agonists at each of the opioid-receptor subtypes (m-, k-,
d-receptors) cause strong analgesic effects. Among these
analgesics k-receptor agonists display an advantageous
side effect profile with minimal physical dependence,
respiratory depression and inhibition of gastrointestinal
motility.9
The ethylenediamine substructure substituted with dif-
ferent residues at the nitrogen atoms represents a crucial
pharmacophoric element of several s- and k-receptor
ligands (see Fig. 1). For example, the simple ethylene-
Herein, we report on the synthesis of enantiopure 6,8-
diazabicyclo[3.2.2]nonanes 8 and their affinity for s1-,
s2-, k-, and m-receptors (see Fig. 2). The bicyclic deri-
vatives 8 comprise the ethylenediamine substructure
2-fold—in the bridged piperazine ring systemand,
moreover, in the side chain nitrogen atom combined with
C1, C2 and N8. In the bicyclic derivatives 8 the relative
orientation of the pharmacophoric elements—the nitro-
*Corresponding author. Tel.: +49-761-203-6320; fax: +49-761-203-
6321; e-mail: bernhard.wuensch@pharmazie.uni-freiburg.de
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00043-3

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M. Weigl et al. / Bioorg. Med. Chem. 10 (2002) 2245–2257
Figure 1.
Chemistry
gen atoms and their substituents—is fixed due to the
limited conformational flexibility of the ethylenediamine
substructures. The receptor affinity of conformationally
constrained ligands may give insight into the pharma-
cologically active conformation of analogous flexible
ligands.15
In our initial attempts the intramolecular ester con-
densation (Dieckmann analogous cyclization)21 was
investigated with the 1-benzyl-4-methyl derivative 9.20
However, the standard conditions usually applied for
Dieckmann condensations (NaOCH3/CH3OH; KOtBu/
toluene; LDA/THF; KHMDS/THF at roomtempera-
ture and at reflux temperature) did not lead to the
desired bicyclic ketone 11. We presume, that the equili-
briumof this cyclization is shifted towards the dioxo-
ester 9 since the formed b-dicarbonyl compound 11
cannot be stabilized by deprotonation. Abstraction of
a proton fromthe formed b-dicarbonyl compound in
the last step is the driving force of the otherwise
endergonic ester condensation (Dieckmann condensa-
tion). In the case of the bicyclic b-dicarbonyl compound
11 the proton has to be removed from a bridgehead
center, which is impeded according to Bredt’s rule
(Scheme 1).22
In the literature, two approaches for the synthesis of
6,8-diazabicyclo[3.2.2]nonanes are described. First, the
2-fold intramolecular aminolysis of like-configured
2,6-diaminopimelic acid derivatives provides racemic
6,8-diazabicyclo[3.2.2]nonanes without further sub-
stituents at the propano bridge.16,17 In the second
approach an intramolecular enolate epoxide cyclization
was used as key step. However, only poor yields of a
racemic 6,8-diazabicyclo[3.2.2]nonane (16%) were
obtained since the epoxide opening occurred with unfa-
vourable regioselectivity.18
According to our plan a Dieckmann analogous cycliza-
tion of 3-(dioxopiperazin-2-yl)propionic acid esters 7
should represent the key step in the synthesis of enan-
tiomerically pure 6,8-diazabicyclo[3.2.2]nonanes 8.19
This strategy enables the introduction of further sub-
stituents (e.g., nitrogen containing groups) at the pro-
pano bridge. Recently, we have described a general
method for the synthesis of enantiopure 3-(dioxopiper-
azin-2-yl)propionates 7 with various substituents at the
nitrogen atoms. The proteinogenic amino acid (S)-glu-
tamate has been used as enantiomerically pure educt.20
Therefore, the dioxoester 9 was deprotonated quantita-
tively with the strong base lithiumhexamethyldisilazane
(LiHMDS) and the resulting anion was trapped with
chlorotrimethylsilane (ClSiMe3). Indeed, the bicyclic,
mixed methyl silyl acetal 10 was obtained in 97% yield
according to this procedure. Careful hydrolysis of the
mixed methyl silyl acetal 10 with p-toluenesulfonic acid
in a mixture of THF and water provided the bicyclic
ketone 11 in 90% yield.
Figure 2.

M. Weigl et al. / Bioorg. Med. Chem. 10 (2002) 2245–2257
2247
In order to support the hypothesis of suppressed
deprotonation during attempted cyclization of 9, the
b-dicarbonyl compound 11 was treated with NaOCH3 in
methanol. Within a few minutes at room temperature,
the bicyclic ketone 11 was transformed into the dioxoester
9 which could be isolated. Obviously, nucleophilic
attack at the ketone carbonyl moiety followed by ring
opening occurred with NaOCH3 instead of deprotonation.
The same observation (opening of the bicyclic ring
system) was made during attempts of reductive amina-
tion of the bicyclic ketone 11. Therefore, the amino
moiety in position 2 was introduced on a different way
(Scheme 2).
Thus, the ketone 11 was reduced carefully with NaBH4
in methanol to yield the diastereomeric alcohols 12a and
12b in a ratio of 85:15. The main diastereomer 12a was
activated with methane sulfonyl chloride to provide the
methane sulfonate 13, which subsequently reacted in a
SN2 reaction with NaN3 to afford the inverted (S)-con-
figured azide 14. Reduction of the azide 14 with hydro-
gen in the presence of the catalyst Pd/C led to the
primary amine 15. Reductive alkylation of the primary
amine 15 with formaldehyde or acetaldehyde and
NaBH3CN23 yielded the tertiary amines 16 and 17,
respectively. The pyrrolidine derivative 18 was obtained
only in a low yield by 2-fold alkylation of the primary
amine 15 with 1-bromo-4-chlorobutane. In the last step
the piperazinedione substructure of 16 was reduced with
24,25
LiAlH4
(Scheme 2).
to provide the bridged piperazine 19b
Alternatively, the ketone 11 was condensed with hydro-
xylamine to yield the diastereomeric oximes (E)-20 and
(Z)-20 in a ratio of 1:1. The isomeric oximes (E)-20 and
(Z)-20 could be separated partly providing the pure (E)-
isomer (20%) and enriched (Z)-isomer. The diastereo-
meric mixture of (E)-20 and (Z)-20 was reduced with an
excess of LiAlH4. Thereby the oxime moiety as well as
both lactamcarbonyl groups were reduced to furnish
the primary amine 21, which was employed for further
transformations without purification. Reductive methyl-
ation23 of the primary amine 21 with formaldehyde and
NaBH3CN yielded the dimethylamines 19a and 19b,
which were isolated in a ratio of 2:3. In this sequence the
dimethylamine 19b with (S)-configuration in position 2
was obtained in 7.4% yield in three steps fromthe
Scheme 1. Reagents and reaction conditions: (a) (1) LiHMDS, THF,
30 min, ꢀ78 ꢁC; (2) ClSi(CH3)3, 30 min, ꢀ78 ꢁC, 60 min, rt, 97%;
(b) p-TosOH, THF/H2O, 16 h, rt, 90%.
Scheme 2. Reagents and reaction conditions: (a) NaBH4, THF/propan-2-ol/H2O, 16 h, rt, 77%; (b) CH3SO2Cl, NEt3, CH2Cl2, 30 min, 0 ꢁC,
2.5 h, rt, 92%; (c) NaN3, DMF, 2 h, 155 ꢁC, 79%; (d) H2, Pd/C, CH3OH, 4.5 h, rt, 99%; (e) CH2¼O, NaBH3CN, CH3CN, 3 h, rt, 66% (16);
(f) CH3CH¼O, NaBH3CN, CH3CN, 3 h, 0 ꢁC, 77% (17); (g) Br(CH2)4Cl, NEt3, DMF, 16 h, 155 ꢁC, 11% (18). (h) LiAlH4, THF, 88 h, 66 ꢁC, 97%.

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M. Weigl et al. / Bioorg. Med. Chem. 10 (2002) 2245–2257
ꢁ
.
Scheme 3. Reagents and reaction conditions: (a) NH2OH HCl, CH3OH, NEt3, 16 h, rt, 83%; (b) LiAlH4, THF, 20 h, 66 C, 65%; (c) CH2¼O,
NaBH3CN, CH3CN, 21 h, rt, 6.3% (19a), 9.4% (19b); (d) (3,4-dichlorophenyl)acetic acid, CDI, CH2Cl2, 30 min, 0 ꢁC, 40 h, rt, 11% (22a), 15%.
ketone 11 (reaction with hydroxylamine, LiAlH4-reduc-
tion, CH2¼O/NaBH3CN reductive alkylation). Although
the synthesis of 19b outlined in Scheme 2 comprises six
steps the overall yield of 19b is significantly higher
(30%). However, the diastereomeric dimethylamine 19a
is only available via the oxime route (6.3%). Thus, the
two routes complement one another.
ꢀ71.8 ppm, respectively. The ratio of the signal inten-
sities was determined to be 98.8:1.2 (ee=97.6%) and
1.6:98.4 (ee=96.8%), respectively. The HPLC analysis
of the unpurified Mosher esters 24 and 25 succeeded
using a LiChrospher1 100 RP-18, endcapped stationary
phase. With the eluent acetonitrile/water=1:1 a baseline
separation of the diastereomeric Mosher esters 24 and
25 was achieved, providing an enantiomeric excess of
94.8% (peak ratio 97.4:2.6). In conclusion, in the worst
case (HPLC analysis) the ratio of diastereomeric
Mosher esters 24 and 25 and thus the ratio of enantio-
mers of the alcohol 12a amounts to at least 97.4:2.6.
Therefore, a significant racemization of the dioxoester 9
during LiHMDS cyclization can be excluded.
Additionally, the crude primary amine 21 was acylated
with (3,4-dichlorophenyl)acetic acid and the coupling
reagent 1,10-carbonyldiimidazole (CDI) to afford the
diastereomeric amides 22a and 22b in a ratio of 2:3. The
(3,4-dichlorophenyl)acetyl moiety was introduced,
because several s- and k-ligands include this acyl resi-
due as pharmacophoric element (cf., Fig. 1).
The Dieckmann analogous cyclization of the dioxoester
9 was performed using the strong base LiHMDS. In the
presence of LiHMDS, the asymmetric center in position
2 bearing the propionate side chain might be unstable.
Therefore, the enantiomeric purity of the bicyclic pro-
ducts has to be controlled. For this purpose, the alcohol
12a, the main diastereomer formed during NaBH4
reduction of the ketone 11, was acylated with (R)- and
(S)-3,3,3-trifluoro-2-methoxy-2-phenylpropionyl chlor-
ide [(R)-23 and (S)-23, Mosher’s acid chlorides]26,27 to
yield the diastereomeric Mosher esters 24 and 25,
respectively. The analytical methods were elaborated
with purified esters 24 and 25, the determination of the
enantiomeric purity, however, was performed with
unpurified esters 24 and 25 (Scheme 4).
Scheme 4. Reagents and reaction conditions: (a) (R)-23 or (S)-23,
NEt3, 4-dimethylaminopyridine, CH2Cl2, 9 h, 41 ꢁC.
Receptor binding studies
The s1-, s2-, k-, and m-receptor affinities of the diaster-
eomeric dimethylamines 19a and 19b and the phenyl-
acetamides 22a and 22b were determined in competition
experiments with radioligands.
1
In the H NMR spectra of 24 and 25 the signal for the
methoxy group is found at 3.52 and 3.64 ppm, respec-
tively. Integration of these signals results in a ratio of
99:1 (ee=98%). The 19F NMR spectra of 24 and 25
reveal the signals for the F3C-moiety at ꢀ72.2 and
In the s1-assay homogenates of guinea pig brains were
used as receptor material. The s1-selective ligand [3H]-
(+)-pentazocine was employed as radioligand, and the

M. Weigl et al. / Bioorg. Med. Chem. 10 (2002) 2245–2257
2249
Table 1. Affinities of the diazabicyclo[3.2.2]nonanes 19 and 22 for s1-, s2-, k- and m-receptors
Compd
s1 ([3H]-(+)-pentazocine)
s2 ([3H]-ditolylguanidine)
Ki (nM) SEM
k ([3H]-U69593)
m ([3H]-DAMGO)
19a
19b
22a
22b
23.8ꢂ5.3
240ꢂ39
131ꢂ27
184ꢂ22
265ꢂ32
164ꢂ47
3.58ꢂ0.20
—
353ꢂ9.0
1400ꢂ480
1110ꢂ560
263ꢂ52
391ꢂ62
63.9ꢂ10.8
—
13930ꢂ570
15900ꢂ1670
2650ꢂ360
2600ꢂ360
—
>10 mM
>10 mM
5220ꢂ2240
6300ꢂ520
—
BMY-14802 (26)
Ditolylguanidine
(+)-Pentazocine
U-50488 (4)
Naloxone
—
—
—
—
—
—
0.49ꢂ0.16
—
0.68ꢂ0.04
—
3.17
nonspecific binding was determined in the presence of a
large excess of haloperidol.28 Homogenates of rat liver
served as source for s2-receptors in the s2-assay. Since a
s2-selective radioligand is not available the nonselective
radioligand [3H]-ditolylguanidine was employed in the
presence of an excess of non-radiolabeled (+)-pentazo-
cine (100 nM) for selective labeling of s1-receptors.
Performing the s2-assay in the presence of an excess of
non-tritiated ditolylguanidine led to the non-specific
binding of the radioligand.28 The k-receptor binding of
the test compounds was determined with homogenates
of guinea pig brain (without cerebellum) membranes as
receptor material using the k-selective radioligand [3H]-
U-69593.14 The non-specific binding was determined
with an excess of U-50488 (4). In the m-receptor assay
the same membrane preparation as described for the
k-assay was used. The radioligand [3H]-DAMGO was
employed for labeling the m-receptors and the non-spe-
cific binding was determined in the presence of 1 mM
naloxone.14
dimethylamines 19, the phenylacetamides 22 bind with
lower affinity at s2-receptors than at s1-receptors.
However, in the phenylacetamide series the (2S)-config-
ured diastereomer 22b displays higher s2-receptor affi-
nity than its (2R)-diastereomer 22a (factor 4).
Altogether, the s-receptor binding profile of 22b is very
similar to the s-binding profile of the reference com-
pound BMY-14802 (26, cf., Table 1), which has been
evaluated as atypical antipsychotic in clinical trials.
In Figure 3, the structure of the phenylacetamide 22b is
compared with the structure of the reference compound
BMY-14802 (26). We presume, that the aryl moieties
and the nitrogen atoms of the piperazine ring systems
occupy similar positions at s1- and s2-receptors.
The dimethylamines 19a and 19b display very low affi-
nities in the k- and m-receptor assays (Ki>10 mM).
Considerable k-receptor affinities were found for the
compounds 22 containing the k-pharmacophoric phenyl-
acetamide substructure. Surprisingly, the k-receptor does
not discriminate between the diastereomers 22a and 22b.
Since the Ki-values of the phenylacetamides 22a and 22b
are m uch higher than theKi-values of the lead compounds
4–6 we suppose that the absolute configuration of the
bicycles 22 has to be changed for high k-receptor affinity.
Results and Discussion
The results of the receptor binding studies are summar-
ized in Table 1. It can be seen that the dimethylamine
19a with (R)-configuration in position 2 displays the
highest affinity for s1-receptors (Ki=23.8 nM), whereas
the s1-receptor affinity of the diastereomer 19b is 10-
fold lower. Although the (2R)-configured phenyl-
acetamide 22a contains the pharmacophoric dichloro-
phenylacetamide substructure of lead structure 3 it
reveals lower s1-receptor affinity (Ki=131 nM) than the
analogous dimethylamine 19a. Again the (2S)-diaster-
eomer 22b is less active at s1-receptors. However, the
difference between the Ki-values of the diastereomeric
phenylacetamides 22a and 22b is very small.
The s2-receptor affinities of both diastereomeric dime-
thylamines 19a and 19b are significantly lower than
their s1-receptor affinities (factors 15 and 6). The (2R)-
configured diastereomer 19a is more active than the
(2S)-diastereomer 19b by the factor 4. In analogy to the
Figure 3.

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M. Weigl et al. / Bioorg. Med. Chem. 10 (2002) 2245–2257
Conclusion
next reaction without further purification. Colorless
solid, yield 1.05 g (97%); tlc: ethyl acetate, Rf 0.50.
C19H28N2O4Si (376.4). MS (EI): m/z (%)=376 (M, 48),
361 (MꢀCH3, 15), 345 (MꢀOCH3, 12), 285
(MꢀCH2Ph, 4). [a]25859=+60.4 (c 0.56, CH2Cl2). IR
(film): n (cmꢀ1)=3032 (w, nCH arom.), 2959 (m, nCH
aliph.), 1682 (s, nC¼O, tert. amides), 1452 (s, dCH aliph.), 1254,
1017 (each m, nCOC), 1105, 875 (each m, O–Si), 843 (m,
Si(CH3)3), 733, 701 (each m, gmonosubst. aromate). 1H
NMR (CDCl3): d=0.21 (s, 9H, Si(CH3)3), 1.40–1.51 (m,
1H, 4-H), 1.65–1.89 (m, 3H, 4-H, 3-H), 3.00 (s, 3H,
NCH3), 3.25 (s, 3H, OCH3), 3.82 (dd, J=5.5/2.4 Hz,
1H, 5-H), 3.93 (s, 1H, 1-H), 4.30 (d, J=14.6 Hz, 1H,
NCH2Ph), 4.72 (d, J=14.6 Hz, 1H, NCH2Ph), 7.20–
7.36 (m, 5H, aromat. H). 13C NMR (CDCl3): d=1.3 (3
C, Si(CH3)3), 24.2 (1 C, C-4), 32.7 (1 C, C-3), 33.1 (1 C,
NCH3), 48.6 (1 C, NCH2Ph), 48.8 (1 C, OCH3), 59.0 (1
C, C-5), 69.5 (1 C, C-1), 98.3 (1 C, C-2), 127.7 (1 C,
aromat CH), 128.3 (2 C, aromat. CH), 128.6 (2 C, aro-
mat. CH), 135.8 (1 C, aromat. C), 166.0 (1 C, C¼O),
168.4 (1 C, C¼O).
Within the novel class of 6,8-diazabicyclo[3.2.2]nonane
derivatives we have found the (2R)-configured dimethyl-
amine 19a with high affinity for s1-receptors and good
selectivity towards s2- (factor 15), k- (factor 580) and
m-receptors (factor>400). The (2S)-configured phenyl-
acetamide 22b has almost the same affinity for s1- and
s2-receptors resulting in a receptor binding profile
similar to that of the atypical antipsychotic BMY-14802
(26). The k-receptor affinity of the phenylacetamides 22
is low presumably by reason of the stereochemistry.
Experimental
Chemistry, general
Unless otherwise noted, moisture sensitive reactions
were conducted under dry nitrogen. THF was distilled
fromsodium/benzophenone ketyl prior to use. Petro-
leumether used refers to the fraction boiling at 40–
60 ꢁC. Thin layer chromatography (tlc): Silica gel 60
F254 plates (Merck). Flash chromatography (fc):29 Silica
gel 60, 0.040–0.063 mm (Merck); parentheses include:
Diameter of the column (cm), eluent, fraction size (mL),
Rf. Melting points: Melting point apparatus SMP 2
(Stuart Scientific), uncorrected. Optical rotation: Polari-
meter 241 (Perkin-Elmer); 1.0 dm tube; concentration c
(g/100 mL). Elemental analyses: CHN-Elementar-
analysator Rapid (Heraeus), Elemental Analyzer 240
(Perkin-Elmer) and Vario EL (Elementaranalysesysteme
GmbH). MS: MAT 312, MAT 8200, MAT 44, and TSQ
7000 (Finnigan); EI=electron impact, CI=chemical
ionization. High resolution MS (HRMS): MAT 8200
(Finnigan). IR: IR spectrophotometer 1600 FT-IR and
2000 FT-IR (Perkin-Elmer); s=strong, m=medium,
(1S,5S) - 6 - Benzyl - 8 - methyl - 6,8 - diazabicyclo[3.2.2]no-
nane-2,7,9-trione (11). As described for 10 the piper-
azinedione 9 (8.28 g, 27.2 mmol) was reacted with
lithium bis(trimethylsilyl)amide (30.0 mL, 30.0 mmol)
and chlorotrimethylsilane (11.2 mL, 87.4 mmol, in 8 mL
THF) to yield the mixed methyl silyl acetal 10. Without
purification 10 was dissolved in a mixture of THF
(100 mL) and water (10 mL), p-toluenesulfonic acid
(1.00 g, 5.25 mmol) was added and the mixture was stir-
red for 16 h at roomtemperature. The solvent was
removed under reduced pressure and the residue was
purified by fc (8 cm, ethyl acetate, 100 mL, Rf 0.41).
Colorless solid, mp 183–184 ꢁC, yield 6.70 g (90%, with
regard to 9). C15H16N2O3 (272.3) calcd C 66.2H 5.92 N
10.3, found C 65.9H 6.41 N 10.4. MS (EI): m/z
(%)=272 (M, 49), 215 (MꢀCO–NCH3, 8), 181
(MꢀCH2Ph, 97). MS (CI): m/z (%)=273 (MH+, 100),
181 (MH+ꢀPhCH3, 2). [a]52859=+5.2 (c 0.55, CH2Cl2).
IR (film): n (cmꢀ1)=3030 (w, nCH arom.), 2975, 2935
(each m, nCH aliph.), 1728 (s, nC¼O, ketone), 1682 (s, nC¼O,
1
w=weak. H NMR (300 MHz), 13C NMR (75 MHz):
Unity 300 FT NMR spectrometer (Varian), d in ppm
related to tetramethylsilane, coupling constants are
given with 0.5 Hz resolution; the assignments of 13C and
1
of H NMR signals were supported by 2D NMR tech-
niques. HPLC: Gradient pump 2249 (Pharmacia); UV-
detector VWM 2141 (Pharmacia); integrator Chroma-
topac C-r6A (Shimadzu); column LiChroCart1 250–4
(Merck); stationary phase LiChroSpher1 100 RP-18
endcapped; injection volume 20 mL.
amides), 1452 (s, dCH aliph.), 1253, 1177 (each m,
COC), 732 (m, gmonosubst. aromate). H NMR (CDCl3):
tert.
1
n
d=1.84 (dddd, J=14.5, 8.8, 7.2, 3.3 Hz, 1H, 4-H), 2.28
(ddt, J=14.5/8.4/4.1 Hz, 1H, 4-H), 2.46 (ddd, J=15.5,
7.3, 4.3 Hz, 1H, 3-H), 2.67 (dt, J=15.5, 8.4 Hz, 1H, 3-
H), 3.03 (s, 3H, NCH3), 4.03 (t, J=3.7 Hz, 1H, 5-H),
4.18 (s, 1H, 1-H), 4.53 (d, J=14.6 Hz, 1H, NCH2Ph),
4.66 (d, J=14.6 Hz, 1H, NCH2Ph), 7.21–7.36 (m, 5H,
aromat. H). 13C NMR (CDCl3): d=29.2 (1 C, C-4), 32.9
(1 C, NCH3), 36.9 (1 C, C-3), 49.0 (1 C, NCH2Ph), 59.1
(1 C, C-5), 73.5 (1 C, C-1), 128.3 (2 C, aromat. CH),
128.4 (1 C, aromat. CH), 129.1 (2 C, aromat. CH),
135.3 (1 C, aromat. C), 163.3 (1 C, C¼O), 167.4 (1 C,
C¼O), 199.2 (1 C, C¼Oketone).
(1S,5S)-6-Benzyl-2-methoxy-8-methyl-2-(trimethylsiloxy)-
6,8-diazabicyclo[3.2.2]nonane-7,9-dione (10). Under nitro-
gen a solution of lithium bis(trimethylsilyl)amide (1 M
in THF; 3.30 mL, 3.30 mmol) was added dropwise to a
solution of 920 (877 mg, 2.88 mmol) in THF (50 mL) at
ꢀ78 ꢁC. After a reaction time of 30 min at ꢀ78 ꢁC a
solution of chlorotrimethylsilane (1.15 mL, 8.97 mmol)
in THF (6.0 mL) was added and the reaction mixture
was stirred for 30 min at ꢀ78 ꢁC, then for 60 min at
room temperature. The solvent was removed in vacuo
and the residue was dissolved in ethyl acetate (80 mL).
The solution was washed with NaOH (0.5 N, 2ꢃ50 mL),
with HCl (0.5 N, 2ꢃ50 mL) and with brine (50 mL),
dried (Na2SO4) and concentrated in vacuo. The result-
ing product 10 was characterized and applied for the
(1S,2R,5S)-6-Benzyl-2-hydroxy-8-methyl-6,8-diazabicy-
clo[3.2.2]nonane-7,9-dione (12a) and (1S,2S,5S)-6-benzyl-
2-hydroxy-8-methyl-6,8-diazabicyclo[3.2.2]nonane-7,9-
dione (12b). Sodium borohydride (0.83 g, 21.9 mmol)
was added to a cooled (ice bath) solution of 11 (1.95 g,
7.16 mmol) in a mixture of THF/propan-2-ol/water

M. Weigl et al. / Bioorg. Med. Chem. 10 (2002) 2245–2257
2251
(100 mL, 20 mL, 5 mL). After stirring for 16 h at room
temperature the solvent was removed in vacuo, the
residue was dissolved in ethyl acetate (80 mL) and
washed with water (2ꢃ50 mL) and HCl (0.5 N,
1ꢃ50 mL). The organic layer was dried (Na2SO4) and
concentrated in vacuo to yield a mixture of the diaster-
eomeric alcohols 12a and 12b as a colorless solid. The
diastereomers 12a/12b (ratio 85:15 according to the H
NMR spectrum) were separated by fc (4 cm, ethyl ace-
tate/acetone=1:1, 20 mL).
methanesulfonyl chloride (0.10 mL, 1.28 mmol), dis-
solved in CH2Cl2 (0.9 mL), were added successively.
After stirring for 30 min at 0 ꢁC and 2.5 h at roomtem-
perature NaOH (0.5 N, 20 mL) was added, followed by
stirring for another 30 min. The aqueous layer was
separated and extracted with CH2Cl2 (10 mL) once. The
combined organic layers were washed with 0.5 N NaOH
(1ꢃ20 mL), 0.5 N HCl (1ꢃ20 mL) and brine (1ꢃ20 mL),
dried (Na2SO4) and concentrated in vacuo. The residue
was purified by fc (2 cm, ethyl acetate/acetone=8:2,
10 mL, Rf 0.56). Colorless solid, mp 159–161 ꢁC, yield
246 mg (92%). C16H20N2O5S (352.4) calcd C 54.5H
5.76 N 7.85 S 9.10, found C 54.6H 5.72 N 7.95 S 8.87.
MS (EI): m/z (%)=352 (M, 44), 261 (MꢀCH2Ph, 2),
256 (MꢀCH3SO2OH, 15), 228 (MꢀCH3CH2OSO2CH3,
58), 137 (228ꢀCH2Ph, 10). [a]25849=+147 (c 1.16,
CH2Cl2). IR (film): n (cmꢀ1)=3024 (w, nCH arom.), 2935
(w, nCH aliph.), 1682 (s, nC¼O, tert. amides), 1451 (m, dCH
1
12a (Rf 0.38): Colorless solid, mp 165–167 ꢁC yield
0.53 g (27%). C15H18N2O3 (274.3) calcd C 65.7H 6.61 N
10.2, found C 65.7H 6.55 N 10.1. MS (EI): m/z
(%)=274 (M, 35), 217 (MꢀCH3NCO, 9), 183
(MꢀCH2Ph, 39). [a25859]=+166 (c 1.06, CH2Cl2). IR
(film): n (cmꢀ1)=3419 (m, nOH), 3055 (w, nCH arom.),
2976 (w, nCH aliph.), 1678 (s, nC¼O, tert. amides), 1516, 1456
(each w, dCH aliph.), 1265, 1073 (each m, nCOC), 737, 700
(each m, gmonosubst. aromate). 1H NMR (CDCl3):
d=1.48–1.75 (m, 3H, 4-H, 3-H), 1.90–2.02 (m, 1H, 3-
H), 2.99 (s, 3H, NCH3), 3.35–3.45 (s broad, 1H,
CHOH), 3.82 (dt, J=4.6, 3.3 Hz, 1H, 2-H), 3.86 (dd,
J=5.4, 2.4 Hz, 1H, 5-H), 4.00 (d, J=3.1 Hz, 1H, 1-H),
4.50 (d, J=14.3 Hz, 1H, NCH2Ph), 4.68 (d, J=14.6 Hz,
1H, NCH2Ph), 7.22–7.36 (m, 5H, aromat. H); 13C
NMR (CDCl3): d=23.4 (1 C, C-4), 29.1 (1 C, C-3), 32.7
(1 C, NCH3), 48.7 (1 C, NCH2Ph), 59.0 (1 C, C-5), 65.8
(1 C, C-2), 67.7 (1 C, C-1), 128.1 (1 C, aromat. CH),
128.4 (2 C, aromat. CH), 128.9 (2 C, aromat. CH),
135.6 (1 C, aromat. C), 167.7 (1 C, C¼O), 169.5 (1 C,
C¼O).
aliph.), 1354, 1173 (each m, CH3–SO2O–R), 1253 (w,
1
n
COC), 733, 701 (each m, gmonosubst. aromate). H NMR
(CDCl3): d=1.52–1.64 (m, 1H, 4-H), 1.70–1.87 (m, 2H,
4-H, 3-H), 2.02 (ddt, J=13.7, 9.4, 4.8 Hz, 1H, 3-H), 3.03
(s, 3H, NCH3), 3.15 (s, 3H, OSO2CH3), 3.91 (dd,
J=6.1, 2.4 Hz, 1H, 5-H), 4.20 (d, J=3.4 Hz, 1H, 1-H),
4.39 (d, J=14.3 Hz, 1H, NCH2Ph), 4.73 (ddd, J=8.6,
5.2, 3.4 Hz, 1H, 2-H), 4.83 (d, J=14.3 Hz, 1H,
NCH2Ph), 7.25–7.39 (m, 5H, aromat. H). 13C NMR
(CDCl3): d=23.4 (1 C, C-4), 27.1 (1 C, C-3), 32.7 (1 C,
NCH3), 39.0 (1 C, OSO2CH3), 49.0 (1 C, NCH2Ph),
58.7 (1 C, C-5), 64.8 (1 C, C-1), 72.0 (1 C, C-2), 128.3 (1
C, aromat. CH), 128.5 (2 C, aromat. CH), 128.9 (2 C,
aromat. CH), 135.6 (1 C, aromat. C), 165.1 (1 C, C¼O),
169.0 (1 C, C¼O).
12b (Rf 0.45): Colorless solid, mp 171–173 ꢁC, yield
0.20 g (10%).C15H18N2O3 (274.3) calcd C 65.7H 6.61 N
10.2, found C 65.4H 6.61 N 10.2. MS (EI): m/z
(%)=274 (M, 79), 246 (MꢀCO, 6), 217 (MꢀCH3NCO,
23), 183 (MꢀCH2Ph, 52). [a]25839=+88.1 (c 0.50,
CH2Cl2). IR (film): n (cmꢀ1)=3394 (m, nOH), 3063,
3030 (w, nCH arom.), 2934 (m, nCH aliph.), 1668 (s, nC¼O,
(1S,2S,5S)-2-Azido-6-benzyl-8-methyl-6,8-diazabicy-
clo[3.2.2]nonane-7,9-dione (14).
A
solution of 13
(982 mg, 2.79 mmol) and sodium azide (920 mg,
14.1 mmol) in DMF (60 mL) was heated to reflux for
2 h. Then it was concentrated in vacuo, the oily residue
was dissolved in ethyl acetate (120 mL) and washed with
water (3ꢃ80 mL). The organic layer was dried
(Na2SO4), concentrated in vacuo and the residue was
purified by fc (3 cm, ethyl acetate, 10 mL, Rf 0.55). Col-
orless solid, mp 142–144 ꢁC, yield 656 mg (79%).
C15H17N5O2 (299.3) calcd C 60.2H 5.73 N 23.39, found
C 60.3H 5.82 N 23.21. MS (EI): m/z (%)=299 (M, 8),
271 (MꢀN2, 4), 256 (MꢀN3H, 3), 208 (MꢀCH2Ph, 2),
amides), 1455 (m, dCH aliph.), 1254, 1063 (each m,
COC), 737, 701 (each m, gmonosubst. aromate). H NMR
tert.
1
n
(CDCl3): d=1.23–1.38 (m, 1H, 4-H), 1.49–1.71 (m, 1H,
3-H), 1.85–1.98 (m, 2H, 3-H, 4-H), 3.11 (s, 3H, NCH3),
3.36–3.54 (s broad, 1H, CHOH), 3.82 (dd, J=4.9,
2.4 Hz, 1H, 5-H), 4.05 (d, J=1.8 Hz, 1H, 1-H), 4.14
(ddd, J=8.5, 4.8, 1.9 Hz, 1H, 2-H), 4.43 (d, J=14.6 Hz,
1H, NCH2Ph), 4.58 (d, J=14.6 Hz, 1H, NCH2Ph),
7.11–7.35 (m, 5H, aromat. H). 13C NMR (CDCl3):
d=24.5 (1 C, C-4), 29.1 (1 C, C-3), 34.5 (1 C, NCH3),
48.5 (1 C, NCH2Ph), 59.0 (1 C, C-5), 67,2 (1 C, C-2),
69.6 (1 C, C-1), 128.1 (1 C, aromat. CH), 128.2 (2 C,
aromat. CH), 128.9 (2 C, aromat. CH), 135.4 (1 C,
aromat. C), 167.9 (1 C, C¼O), 168.3 (1 C, C¼O).
22
589
180 (MꢀN2–CH2Ph, 51). [a] =+92.0 (c 0.80,
CH2Cl2). IR (film): n (cmꢀ1)=3031 (w, nCH arom.), 2936
(w, nCH aliph.), 2101 (s, nN3), 1682 (s, nC¼O, tert. amides),
1453 (m, dCH aliph.), 1251 (m, nCOC), 733, 702 (each m,
1
g
monosubst. aromate). H NMR (CDCl3): d=1.26–1.39 (m,
1H, 4–H), 1.59–1.74 (m, 1H, 3-H), 1.87–2.02 (m, 2H, 4-
H, 3-H), 3.07 (s, 3H, NCH3), 3.85 (dd, J=4.6, 3.0 Hz,
1H, 5-H), 3.90 (ddd, J=8.6, 4.9, 2.1 Hz, 1H, 2-H), 3.97
(d, J=2.1 Hz, 1H, 1-H), 4.48 (d, J=14.6 Hz, 1H,
NCH2Ph), 4.56 (d, J=14.3 Hz, 1H, CH2Ph), 7.19–7.36
(m, 5H, aromat. H). 13C NMR (CDCl3): d=24.2 (1 C,
C-4), 26.0 (1 C, C-3), 34.1 (1 C, NCH3), 48.8 (1 C,
NCH2Ph), 57.3 (1 C, C-2), 58.8 (1 C, C-5), 66.4 (1 C, C-
1), 128.3 (1 C, aromat. CH), 128.4 (2 C, aromat. CH),
129.0 (2 C, aromat. CH), 135.3 (1 C, aromat. C), 166.7
(1 C, C¼O), 168.0 (1 C, C¼O).
Additionally, a mixture of 12a and 12b was isolated.
Colorless solid (Rf 0.45/0.38) yield 0.78 g (40%). Total
yield 1.51 g (77%).
[(1S,2R,5S)-(6-Benzyl-8-methyl-7,9-dioxo-6,8-diazabicy-
clo[3.2.2]nonan-2-yl)]methane sulfonate (13). To
cooled (ice bath) solution of 12a (209 mg, 0.76 mmol) in
a
CH2Cl2 (20 mL) triethylamine (0.32 mL, 2.30 mmol) and

2252
M. Weigl et al. / Bioorg. Med. Chem. 10 (2002) 2245–2257
(1S,2S,5S)-2-Amino-6-benzyl-8-methyl-6,8-diazabicy-
clo[3.2.2]nonane-7,9-dione (15). Pd/C catalyst (10%,
30 mg) was added to a solution of 14 (180 mg,
0.60 mmol) in methanol (12 mL). The suspension was
stirred under a H2 atmosphere (1 bar, balloon) at room
temperature for 4.5 h. The mixture was filtered through
a pad of Celite1 AFA and the filtrate was concentrated
in vacuo. Colorless solid, mp 171–174 ꢁC, yield 163 mg
(99%). C15H19N3O2 (273.3) calcd C 65.9H 7.01 N 15.37,
found C 65.5H 7.36 N 14.92. MS (EI): m/z (%)=273
Removal of the solvent in vacuo furnished a yellow oil,
which was dissolved in ethyl acetate (30 mL) and
washed with NaOH (0.5 N, 2ꢃ20 mL). The dried
organic layer (Na2SO4) was evaporated in vacuo and
purified by fc (2 cm, ethyl acetate/acetone=8:2, 5 mL,
Rf 0.32). Pale yellow oil, yield 51 mg (77%).
C19H27N3O2 (329.2). HRMS: calcd 329.2103, found
329.2104 (+0.3 ppm). MS (EI): m/z (%)=329 (M, 4),
167 (MꢀCH2Ph–NC4H9, 1), 139 (167ꢀNCH3, 3), 112
(CH2CHCHN(C2H5)2, 100). MS (CI): m/z (%)=330
(MH+, 100), 302 (MH+ꢀNCH3, 5), 271 (Mꢀ2ꢃC2H5,
2), 243 (MꢀCH2N(C2H5)2, 4). [a]25819=+37.3 (c 0.36,
CH2Cl2). IR (film): n (cmꢀ1)=3029 (w, nCH arom.), 2970,
2934 (each m, nCH aliph.), 2815 (w, nNCH3), 1676 (s, nC¼O,
(M, 7), 230 (MꢀCH2CHNH2, 28), 182 (MꢀCH2Ph, 51).
23
589
[a] =+71.7 (c 0.79, CH2Cl2). IR (film):
n
(cmꢀ1)=3445 (m, nNH2), 3058 (w, nCH arom.), 2933 (w,
n
CH aliph.), 1671 (s, nC¼O, tert. amides), 1455 (m, dCH aliph.),
1261, 1181 (each w, nCOC), 735, 702 (each m, gmonosubst.
aromate). H NMR (CDCl3): d=1.24–1.48 (m, 4H, 3-H,
amides), 1456 (s, dCH aliph.), 1232, 1060 (each m,
tert.
1
1
n
COC), 733, 702 (each m, gmonosubst. aromate). H NMR
4-H, NH2), 1.75–1.92 (m, 2H, 3-H, 4-H), 3.13 (s, 3H,
NCH3), 3.34 (ddd, J=8.5, 4.9, 1.6 Hz, 1H, 2-H), 3.77–
3.84 (m, 2H, 1-H, 5-H), 4.44 (d, J=14.6 Hz, 1H,
NCH2Ph), 4.55 (d, J=14.6 Hz, 1H, NCH2Ph), 7.17–
7.33 (m, 5H, aromat. H). 13C NMR (CDCl3): d=24.3 (1
C, C-4), 29.7 (1 C, C-3), 35.0 (1 C, NCH3), 48.4 (1 C,
NCH2Ph), 48.6 (1 C, C-2), 59.0 (1 C, C-5), 69.7 (1 C, C-
1), 128.0 (1 C, aromat. CH), 128.2 (2 C, aromat. CH),
128.9 (2 C, aromat. CH), 135.6 (1 C, aromat. C), 168.3
(1 C, C¼O), 168.5 (1 C, C¼O).
(CDCl3): d=1.03 (t, J=7.0 Hz, 6H, N(CH2CH3)2),
1.23–1.38 (m, 1H, 4-H), 1.62–1.78 (m, 2H, 3-H, 4-H),
1.96 (dtd, J=14.6, 5.6, 3.5 Hz, 1H, 3-H), 2.45 (dq,
J=13.7, 6.8 Hz, 2H, N(CH2CH3)2), 2.63 (dq, J=14.3,
7.0 Hz, 2H, N(CH2CH3)2), 2.97–3.07 (m, 1H, 2-H), 3.05
(s, 3H, NCH3), 3.79 (dd, J=6.1, 1.5 Hz, 1H, 5-H), 3.92
(s, 1H, 1-H), 4.43 (d, J=14.6 Hz, 1H, NCH2Ph), 4.58
(d, J=14.6 Hz, 1H, NCH2Ph), 7.17–7.35 (m, 5H, aro-
mat. H).
(1S,2S,5S)-6-Benzyl-8-methyl-2-(pyrrolidin-1-yl)-6,8-di-
azabicyclo[3.2.2]nonane-7,9-dione (18). Triethylamine
(0.4 mL, 2.9 mmol) and a solution of 1-bromo-4-chloro-
butane (0.3 mL, 2.6 mmol) in DMF (0.7 mL) were added
to a solution of 15 (63 mg, 0.23 mmol) in DMF (20 mL).
The reaction mixture was heated to reflux for 16 h.
Then, the mixture was concentrated in vacuo, the
resulting brown oil was dissolved in ethyl acetate
(50 mL), washed with NaOH (2ꢃ30 mL) and water
(1ꢃ30 mL) and dried (Na2SO4). The solvent was eva-
porated in vacuo and the residue was purified by fc
(2 cm, acetone, 4 mL, Rf 0.38). Pale yellow oil, yield 8 mg
(11%). C19H25N3O2 (327.2). HRMS: calcd 327.1947,
found 327.1946 (ꢀ0.2 ppm). MS (EI): m/z (%)=327 (M,
2), 236 (MꢀCH2Ph, 1), 217 (MꢀCH2CHCHN(CH2)4,
(1S,2S,5S)-6-Benzyl-2-(dimethylamino)-8-methyl-6,8-di-
azabicyclo[3.2.2]nonane-7,9-dione (16). A solution of
formaldehyde (37% in water; 1.2 mL, 15 mmol) and
subsequently NaBH3CN (62 mg, 1.0 mmol) were added
to a solution of 15 (104 mg, 0.38 mmol) in acetonitrile
(10 mL). After stirring for 3 h at room temperature the
solvent was removed and the residue was dissolved in
ethyl acetate (50 mL). The organic solution was washed
with NaOH (0.5 N, 2ꢃ30 mL), dried (Na2SO4), con-
centrated in vacuo and the residue was purified by fc
(2 cm, acetone/C2H5OH=9:1, 8 mL, Rf 0.33). Colorless
oil, yield 76 mg (66%). C17H23N3O2 (301.2). HRMS:
calcd 301.1790, found 301.1792 (+0.5 ppm). MS (EI):
m/z (%)=301 (M, 9), 256 (MꢀHN(CH3)2, 2), 230
(MꢀCH2CHN(CH3)2, 1), 165 (256ꢀCH2Ph, 1). MS
(CI): m/z (%)=302 (MH+, 78), 256 (MꢀHN(CH3)2, 3),
167 (MH+ꢀCH2Ph–N(CH3)2, 14). [a]25809=+80.4 (c
0.53, CH2Cl2). IR (film): n (cmꢀ1)=3030 (w, nCH arom.),
2946 (m, nCH aliph.), 2782 (w, nNCH3), 1677 (s, nC¼O, tert.
amides), 1455 (m, dCH aliph.), 1256, 1038 (each m, nCOC),
732, 700 (each m, gmonosubst. aromate). 1H NMR (CDCl3):
d=1.23 (dddd, J=14.0, 12.5, 5.2, 1.5 Hz, 1H, 4-H), 1.53
(dtd, J=13.6, 11.9, 5.5 Hz, 1H, 3-H), 1.73 (dtd, J=13.6,
4.9, 3.1 Hz, 1H, 3-H), 1.89 (dtd, J=14.3, 5.8, 3.0 Hz,
1H, 4-H), 2.22 (s, 6H, N(CH3)2), 2.55 (dd, J=11.3,
4.6 Hz, 1H, 2-H), 3.00 (s, 3H, NCH3), 3.74 (dd, J=6.1,
1.5 Hz, 1H, 5-H), 3.95 (s, 1H, 1-H), 4.40 (d, J=14.6 Hz,
1H, NCH2Ph), 4.50 (d, J=14.6 Hz, 1H, NCH2Ph),
7.14–7.29 (m, 5H, aromat. H).
21
589
2). [a] =+73.2 (c 0.38, CH2Cl2). IR (film): n
(cmꢀ1)=2965 (w, nCH aliph.), 1669 (s, nC¼O, tert. amides),
1457 (m, dCH aliph.), 1261 (w, nCOC), 733, 699 (each m,
1
g
monosubst. aromate). H NMR (CDCl3): d=1.22–1.34 (m,
1H, 4-H), 1.48–1.66 (m, 1H, 3-H), 1.68–1.82 (m, 4H,
CH2CH2NCH2CH2), 1.86–2.00 (m, 2H, 3-H, 4-H),
2.50–2.68 (m, 5H, CH2CH2NCH2CH2, 2-H), 3.10 (s,
3H, NCH3), 3.80 (dd, J=6.4, 1.4 Hz, 1H, 5-H), 4.15 (s,
1H, 1-H), 4.46 (d, J=14.6 Hz, 1H, NCH2Ph), 4.59 (d,
J=14.6 Hz, 1H, NCH2Ph), 7.20–7.38 (m, 5H, aromat.
H).
(E)- and (Z)-(1S,5S)-6-Benzyl-2-(hydroxyimino)-8-methyl-
6,8-diazabicyclo[3.2.2]nonane-7,9-dione ((E)-20 and (Z)-
20). The bicyclic ketone 11 (2.05 g, 7.51 mmol) was
added to a solution of hydroxylamine hydrochloride
(2.64 g, 38 mmol) and triethylamine (5.3 mL, 38 mmol)
in methanol (25 mL). After stirring for 16 h at room
temperature the solvent was evaporated in vacuo. The
residue was dissolved in ethyl acetate (120 mL), washed
with water (2ꢃ100 mL) and dried (Na2SO4). After
removal of the solvent under reduced pressure the resi-
(1S,2S,5S)-6-Benzyl-2-(diethylamino)-8-methyl-6,8-di-
azabicyclo[3.2.2]nonane-7,9-dione (17). Acetaldehyde
(0.45 mL, 8.0 mmol) was added to a cooled (ice bath)
solution of 15 (55 mg, 0.20 mmol) in acetonitrile (6 mL).
After 15 min NaBH3CN (35 mg, 0.56 mmol) was added
and the reaction mixture was stirred for 3 h at 0 ꢁC.

M. Weigl et al. / Bioorg. Med. Chem. 10 (2002) 2245–2257
2253
due was purified by fc (4 cm, ethyl acetate, 20 mL, Rf
0.38/0.30) providing a mixture of (E)-20 and (Z)-20
(ratio 1:1 according to the 1H NMR spectrum), col-
orless solid, yield 1.79 g (83%). The diastereomers
were separated by a further fc (4 cm, ethyl acetate,
10 mL).
(1R,2R,5S) - 6 - Benzyl - N,N,8 - trimethyl - 6,8 - diazabicy-
clo[3.2.2]nonan-2-amine (19a) and (1R,2S,5S)-6-benzyl-
N,N,8-trimethyl-6,8- diazabicyclo[3.2.2]nonan-2-amine
(19b). Method A. Synthesis of both diastereomers 19a
and 19b via reduction of the oxime 20 and reductive
methylation. A cooled (ice bath) solution of the primary
amine 21 (338 mg, 1.38 mmol) in acetonitrile (25 mL)
was reacted with an aqueous solution of formaldehyde
(37%, 4.2 mL, 56 mmol) and after 15 min NaBH3CN
(202 mg, 3.2 mmol) was added. After stirring for 1 h at
roomtemperature, the pH of the solution was brought
to pH 7 by dropwise addition of glacial acetic acid. The
reaction mixture was stirred for 20 h at room tempera-
ture, then the solvent was evaporated in vacuo. The
residue was dissolved in ethyl acetate (100 mL), the
solution was washed with NaOH (0.5 N, 2ꢃ70 mL) and
water (1ꢃ70 mL), dried (Na2SO4) and concentrated
under reduced pressure. The residue was purified by fc
(2 cm, 24 cm, acetone/C2H5OH=8:2+2% ethyldi-
methylamine, 2 mL).
(E)-20 (Rf 0.38): Colorless solid, mp 155–158 ꢁC, yield
0.43 g (20%). C15H17N3O3 (287.1). HRMS: calcd
287.1270, found 287.1279 (+3.2 ppm). MS (EI): m/z
(%)=287 (M, 11), 270 (MꢀOH, 48), 196 (MꢀCH2Ph,
20
589
48). [a] =+128 (c 0.23, CH2Cl2). IR (film): n
(cmꢀ1)=3300 (m, nNꢀOH), 3085 (w, nCH arom.), 2932 (w,
n
d
CH aliph.), 1682 (s, nC¼O, tert. amides+C=N, oxime), 448 (m,
CH aliph.), 1254 (m, nCOC), 732, 699 (each m, gmonosubst.
aromate). 1H NMR (CDCl3): d=1.61 (dddd, J=14.3, 7.6,
6.6, 4.7 Hz, 1H, 4-H), 1.95 (dddd, J=14.6, 7.7, 6.7,
3.1 Hz, 1H, 4-H), 2.60 (ddd, J=17.1, 7.6, 6.7 Hz, 1H, 3-
H), 2.70 (ddd, J=17.1, 7.4, 6.6 Hz, 1H, 3-H), 2.98 (s,
3H, NCH3), 3.99 (dd, J=4.6, 3.0 Hz, 1H, 5-H), 4.38 (s,
1H, 1-H), 4.53 (d, J=14.6 Hz, 1H, NCH2Ph), 4.70 (d,
J=14.6 Hz, 1H, NCH2Ph), 7.24–7.38 (m, 5H, aromat.
H), 8.59 (s broad, 1H, N–OH).
19a (Rf 0.16): Pale yellow oil, yield 36 mg (6.3% with
regard to the oxime 20). C17H27N3 (273.2). HRMS:
calcd 273.2205, found 273.2205 (+0.2 ppm). MS (EI):
m/z (%)=273 (M, 20), 228 (MꢀHN(CH3)2, 4), 203
(MꢀH2CCHN(CH3)2, 47), 187 (MꢀCH2CH2CH2
N(CH3)2, 12), 137 (228ꢀCH2Ph, 45). [a]15869=+13.1 (c
0.38, CH2Cl2). IR (film): n (cmꢀ1)=3028 (w, nCH arom.),
2934 (s, nCH aliph.), 2782 (s, nNCH3), 1452 (m, dCH aliph.),
730, 698 (each m, gmonosubst. aromate). 1H NMR (CDCl3):
d=1.41–1.54 (m, 1H, 4-H), 1.65–1.77 (m, 1H, 3-H),
1.86–2.14 (m, 2H, 3-H, 4-H), 2.28 (s, 6H, N(CH3)2),
2.43 (s, 3H, NCH3), 2.48–2.57 (m, 1H, 2-H), 2.52 (dd,
J=10.7, 2.1 Hz, 1H, 9-H), 2.72–2.76 (m, 1H, 1-H), 2.84
(ddt, J=5.8, 4.2, 2.0 Hz, 1H, 5-H), 2.91 (dd, J=14.6,
3.9 Hz, 1H, 7-H), 2.96 (dd, J=14.5, 3.8 Hz, 1H, 7-H),
2.99 (dd, J=11.3, 1.8 Hz, 1H, 9-H), 3.68 (s, 2H,
NCH2Ph), 7.15–7.37 (m, 5H, aromat. H). 13C NMR
(CDCl3): d=23.8 (1 C, C-3), 30.4 (1 C, C-4), 42.6 (2 C,
N(CH3)2), 43.6 (1 C, NCH3), 50.2 (1 C, C-7), 52.4 (1 C,
C-5), 55.0 (1 C, C-9), 58.8 (1 C, C-1), 60.3 (1 C,
NCH2Ph), 67.6 (1 C, C-2), 126.7 (1 C, aromat. CH),
128.1 (2 C, aromat. CH), 128.4 (2 C, aromat. CH),
139.9 (1 C, aromat. C).
(Z)-20: In a second fraction (Rf 0.34–0.28), the (Z)-iso-
mer (Z)-20 predominated [(E)-20/(Z)-20=33:67
1
according to the H NMR spectrum]. Colorless solid,
mp 158–164 ꢁC, yield 1.28 g (59%). C15H17N3O3 (287.1).
HRMS: calcd 287.1270, found 287.1276 (+2.2 ppm).
MS (EI): m/z (%)=287 (M, 12), 270 (MꢀOH, 30), 196
(MꢀCH2Ph, 42). [a]25809=+125 (c 0.59, CH2Cl2). IR
(film): n (cmꢀ1)=3301 (m, nNꢀOH), 3087 (w, nCH arom.),
2930 (w, nCH aliph.), 1680 (s, nC¼O, tert. amides+C¼N, oxime),
1449 (m, dCH aliph.), 1254 (m, nCOC), 732, 700 (each m,
1
g
monosubst. aromate). H NMR (CDCl3): d=1.55–1.68 (m,
0.33H, 4-HE), 1.59 (ddd, J=13.7, 11.3, 6.9 Hz, 0.67H, 3-
HZ), 1.88–2.03 (m, 1.67H, 4-HZ+E), 2.33–2.42 (m,
0.67H, 3-HZ), 2.54–2.73 (m, 2ꢃ0.33H, 3-HE), 2.98 (s,
3ꢃ0.33H, NCHE3 ), 3.04 (s, 3ꢃ0.67H, NCHZ3 ), 3.99 (dd,
J=8.1, 4.5 Hz, 1H, 5-HZ+E), 4.37 (s, 0.33H, 1-HE), 4.49
(d, J=14.5 Hz, 0.67H, NCH2PhZ), 4.52 (d, J=14.5 Hz,
0.33H, NCH2PhE), 4.67 (d, J=14.5 Hz, 0.33H,
NCH2PhE), 4.69 (d, J=14.5 Hz, 0.67H, NCH2PhZ),
5.39 (s, 0.67H, 1-HZ), 7.21–7.37 (m, 5H, aromat. H),
9.57 (s broad, 0.67H, N–OHZ). A signal for the OH-
proton of (E)-20 was not found.
19b (Rf 0.30): Pale yellow oil, yield 54 mg (9.4% with
regard to the oxime 20). C17H27N3 (273.2). HRMS:
calcd 273.2205, found 273.2205 (+0.2 ppm). MS (EI):
m/z (%)=273 (M, 20), 228 (MꢀHN(CH3)2, 5), 203
(MꢀH2CCHN(CH3)2, 48), 187 (MꢀCH2CH2CH2
N(CH3)2, 13), 137 (228ꢀCH2Ph, 48). [a]15869=+32.4 (c
0.49, CH2Cl2). IR (film): n (cmꢀ1)=3028 (w, nCH arom.),
2927 (s, nCH aliph.), 2788 (s, nNCH3), 1451 (m, dCH aliph.),
727, 697 (each m, gmonosubst. aromate). 1H NMR (CDCl3):
d=1.48–1.57 (m, 1H, 3-H), 1.62 (dddd, J=13.7, 8.8,
4.3, 2.1 Hz, 1H, 4-H), 1.72 (dddd, J=13.6, 8.8, 4.6,
1.6 Hz, 1H, 4-H), 2.01–2.16 (m, 1H, 3-H), 2.24 (s, 6H,
N(CH3)2), 2.29 (s, 3H, NCH3), 2.60–2.69 (m, 4H, 1-H,
5-H, 7-H, 9-H), 2.76 (dd, J=9.2, 1.5 Hz, 1H, 7-H), 2.90
(dd, J=10.4, 2.0 Hz, 1H, 9-H), 2.94 (dd, J=12.2,
3.4 Hz, 1H, 2-H), 3.64 (d, J=13.4 Hz, 1H, NCH2Ph),
3.71 (d, J=13.4 Hz, 1H, NCH2Ph), 7.18–7.38 (m, 5H,
aromat. H). 13C NMR (CDCl3): d=21.1 (1 C, C-3), 33.5
(1R,2R,5S)- and (1R,2S,5S)-6-Benzyl-8-methyl-6,8-di-
azabicyclo[3.2.2]nonan-2-amine (21).
A solution of
LiAlH4 (1 M in Et2O; 10.0 mL, 10.0 mmol) was added to
a cooled (ice bath) suspension of 20 (mixture of dia-
stereomers, 606 mg, 2.11 mmol) in THF (70 mL) and the
reaction mixture was heated to reflux for 20 h. The
excess of LiAlH4 was carefully destroyed by successive
addition of Na2SO4ꢃ10H2O (2.5 g). Then the suspen-
sion was heated to reflux for 60 min. After cooling to
roomtemperature, the precipitate was filtered off and
thoroughly extracted with ethyl acetate. The organic
layer was concentrated in vacuo. With regard to the
instability of the primary amine 21, the resulting color-
less, transparent oil (yield: 338 mg, 65%) was neither
purified nor characterized, but directly reacted further
on.

2254
M. Weigl et al. / Bioorg. Med. Chem. 10 (2002) 2245–2257
(1 C, C-4), 40.8 (2 C, N(CH3)2), 43.4 (1 C, NCH3), 49.8
(1 C, C-7), 52.9 (1 C, C-9), 54.8 (1 C, C-5), 60.1 (1 C, C-
1), 61.0 (1 C, NCH2Ph), 67.6 (1 C, C-2), 126.7 (1 C,
aromat. CH), 128.1 (2 C, aromat. CH), 128.6 (2 C,
aromat. CH), 139.9 (1 C, aromat. C).
2.6 Hz, 1H, 1-H), 2.95 (dt, J=7.9, 2.7 Hz, 1H, 5-H),
3.48 (d, J=15.6 Hz, 1H, COCH2Aryl), 3.55 (d,
J=15.6 Hz, 1H, COCH2Aryl), 3.76 (s, 2H, NCH2Ph),
4.46 (dtd, J=8.9, 6.7, 5.8 Hz, 1H, 2-H), 6.20 (d broad,
J=8.9 Hz, 1H, NH), 7.17 (dd, J=8.2, 2.1 Hz, 1H, aro-
mat. 6-Hdichlorophenyl), 7.34–7.50 (m, 7H, aromat. H).
13C NMR (CDCl3): d=26.5 (1 C, C-3), 31.6 (1 C, C-4),
42.8 (1 C, COCH2Aryl), 44.2 (1 C, NCH3), 46.6 (1 C, C-
7), 52.6 (1 C, C-2), 52.7 (1 C, C-9), 54.6 (1 C, C-5), 60.4
(1 C, C-1), 61.0 (1 C, NCH2Ph), 127.2 (1 C, aromat.
CH), 128.4 (2 C, aromat. CH), 128.5 (2 C, aromat. CH),
128.6 (1 C, aromat. CH dichlorophenyl, C-6), 130.6 (1 C,
Method B. Diastereoselective synthesis of 19b via
reduction of the dimethylamine 16. A solution of
LiAlH4 (1 M in Et2O; 3.0 mL, 3.0 mmol) was slowly
added to an ice-cold solution of 16 (190 mg, 0.63 mmol)
in THF (45 mL). The reaction mixture was heated to
reflux for 88 h, then the excess of LiAlH4 was carefully
hydrolyzed with Na2SO4ꢃ10H2O (5 g). The suspension
was stirred for 30 min under reflux, the precipitate was
filtered off and extracted with ethyl acetate. Removal of
the solvent in vacuo directly provided the dimethyl-
amine 19b without further purification. Colorless oil,
yield 166 mg (97%).
aromat. CHdichlorophenyl,
mat. CH
C-5), 131.1 (1 C, aro-
C-5 or C-2), 131.3 (1 C, aromat.
C-2 or
dichlorophenyl,
C-Cl), 132.6 (1 C, aromat. C-Cl), 135.4 (1 C, aromat.
Cdichlorophenyl), 139.2 (1 C, aromat. C), 168.7 (1 C, C¼O).
22b (Rf 0.34): Pale yellow oil, yield 133 mg (15% with
regard to 20). C23H27Cl2N3O (431.2) calcd C 63.9H
6.29 N 9.7, found C 63.4H 6.27 N 9.1. HRMS: calcd
431.1531, found 431.1530 (ꢀ0.2 ppm). MS (EI): m/z
(%)=435/433/431 (M, 1/4/7), 342/340 (MꢀCH2Ph, 3/
5), 285 (MꢀC6H3Cl2, 77). [a]15869=+53.6 (c 0.55,
CH2Cl2). IR (film): n (cmꢀ1)=3324 (m, nNH, s. amide),
3061, 3028 (each w, nCH arom.), 2933 (m, nCH aliph.), 2801
(m, nNCH3), 1649 (s, nC¼O, s. amide), 1550 (w, amide II),
1497, 1471 (each m, dCH aliph.), 1257, 1135 (each m,
N-[(1R,2R,5S)-6-Benzyl-8-methyl-6,8-diazabicyclo[3.2.2]-
nonan-2-yl]-2-(3,4-dichlorophenyl)acetamide (22a) and
N-[(1R,2S,5S)-6-benzyl-8-methyl-6,8-diazabicyclo[3.2.2]-
nonan-2-yl]-2-(3,4-dichlorophenyl)acetamide (22b). As
described for the synthesis of 21 a solution of LiAlH4 in
Et2O (1 M, 10.0 mL, 10.0 mmol) was cautiously added
to a cooled (ice-bath) suspension of the oxime 20 (mix-
ture of diastereomers; 585 mg, 2.04 mmol) in THF
(75 mL). After stirring for 15 min at 0 ꢁC, the reaction
mixture was heated to reflux for 36 h. The mixture was
cooled and the excess of LiAlH4 was carefully destroyed
by addition of Na2SO4ꢃ10H2O (4.5 g). The suspension
was refluxed for another 30 min, then the precipitate
was filtered off and washed with ethyl acetate. The fil-
trate was concentrated in vacuo to yield the primary
amine 21 (colorless oil, yield 384 mg, 77%), which was
directly reacted further on. The residue (384 mg) was
dissolved in CH2Cl2 (25 mL) and treated with (3,4-
dichlorophenyl)acetic acid (656 mg, 3.2 mmol) and car-
bonyldiimidazole (CDI, 520 mg, 3.2 mmol). After stir-
ring for 30 min at 0 ꢁC and for 40 h at room
temperature, the reaction mixture was washed with a
saturated solution of NaHCO3 (20 mL) and with brine
(20 mL). The organic layers were dried (MgSO4), con-
centrated in vacuo and the residue was purified by fc
(4 cm, petroleum ether/ethyl acetate 2:8+2% ethyldi-
methylamine, 5 mL).
n
COC), 910 (m, C-Cl), 819 (w, gdichlorophenyl), 732, 698
1
(each m, gmonosubst. aromate). H NMR (CDCl3): d=1.57
(td broad, J=12.8, 4.3 Hz, 1H, 4-H), 1.69 (dq, J=13.6,
3.2 Hz, 1H, 3-H), 1.87 (ddt, J=13.3, 8.6, 4.3 Hz, 1H, 4-
H), 2.25–2.39 (m, 1H, 3-H), 2.35 (s, 3H, NCH3), 2.57
(dt, J=4.0, 3.0 Hz, 1H, 1-H), 2.76 (d, J=2.7 Hz, 2H, 9-
H), 2.90 (dd, J=11.6, 3.6 Hz, 1H, 7-H), 2.93–2.97 (m,
1H, 5-H), 3.01 (dd, J=12.2, 3.2 Hz, 1H, 7-H), 3.62 (s,
2H, NCH2Ph), 3.78 (s, 2H, COCH2Aryl), 4.18 (tt,
J=7.9, 2.7 Hz, 1H, 2-H), 7.05 (d broad, J=8.2 Hz,
1H, NH), 7.25 (dd, J=8.2, 2.1 Hz, 1H, aromat.
6-Hdichlorophenyl), 7.30–7.45 (m, 5H, aromat. H), 7.51 (d,
J=2.1 Hz, 1H, aromat. 2-Hdichlorophenyl), 7.52 (d,
J=7.9 Hz, 1H, aromat. 5-Hdichlorophenyl). 13C NMR
(CDCl3): d=27.2 (1 C, C-3), 28.8 (1 C, C-4), 43.0 (1 C,
COCH2Aryl), 44.7 (1 C, NCH3), 48.7 (1 C, C-7), 51.6 (1
C, C-2), 53.5 (1 C, C-5), 53.6 (1 C, C-9), 60.5 (1 C, C-1),
60.6 (1 C, NCH2Ph), 126.8 (1 C, aromat. CH), 128.1 (2
C, aromat. CH), 128.2 (2 C, aromat. CH), 128.7 (1 C,
aromat. CHdichlorophenyl, C-6), 130.5 (1 C, aromat. CH
dichlorophenyl, C-2 or C-5), 131.0 (1 C, aromat. C–Cl), 131.1
22a (Rf 0.23): Pale yellow oil, yield: 94 mg (11% with
regard to 20). C23H27Cl2N3O (431.2). HRMS: calcd
431.1531, found 431.1530 (ꢀ0.2 ppm). MS (EI): m/z
(%)=435/433/431 (M, 8/41/59), 344/342/340 (MꢀCH2Ph,
4/21/35), 284 (MꢀC6H3Cl2, 5). [a]15869=+24.8 (c 0.99,
(1 C, aromat. CHdichlorophenyl,
aromat. C–Cl), 135.8 (1 C, aromat. Cdichlorophenyl), 139.6
(1 C, aromat. C), 168.1 (1 C, C¼O).
C-2), 132.5 (1 C,
C-5 or
[(1S,2R,5S)-6-Benzyl-8-methyl-7,9-dioxo-6,8-diazabicy-
clo[3.2.2]nonan-2-yl]
CH2Cl2). IR (film): n (cmꢀ1)=3303 (m, nNH, amide),
(2S)-3,3,3-trifluoro-2-methoxy-2-
s.
3061, 3029 (each w, nCH arom.), 2931 (m, nCH aliph.), 2801
(m, nNCH3), 1647 (s, nC¼O, s. amide), 1545 (w, amide II),
1469 (m, dCH aliph.), 1257, 1136 (each m, nCOC), 910 (m,
C–Cl), 818 (w, gdichlorophenyl), 732, 698 (each m, gmonosubst.
aromate). 1H NMR (CDCl3): d=1.68 (dq, J=13.1,
6.6 Hz, 1H, 3-H), 1.78–1.93 (m, 2H, 4-H), 2.21 (dq,
J=13.4, 6.6 Hz, 1H, 3-H), 2.49 (s, 3H, NCH3), 2.78 (d,
J=2.7 Hz, 2H, 7-H), 2.82 (dd, J=11.0, 2.8 Hz, 1H, 9-
H), 2.88 (dd, J=11.0, 2.4 Hz, 1H, 9-H), 2.90 (dt, J=5.8,
phenylpropanoate (24). A solution of 12a (22 mg,
0.080 mmol) in CH2Cl2 (4 mL) was treated with (R)-(ꢀ)-
3,3,3-trifluoro-2-methoxy-2-phenylacetyl chloride [(R)-
Mosher’s acid chloride; (R)-23, 13 mL, 0.070 mmol],
triethylamine (0.3 mL, 2.1 mmol) and dimethylamino-
pyridine (17 mg, 0.14 mmol). The reaction mixture was
heated to reflux for 9 h, then it was cooled to room
temperature, diluted with CH2Cl2 (6 mL) and washed
with saturated solutions of NaHCO3 (5 mL) and NH4Cl

M. Weigl et al. / Bioorg. Med. Chem. 10 (2002) 2245–2257
2255
(5 mL). The organic layer was dried (MgSO4) and con-
centrated in vacuo. The diastereomeric ratio of the non-
Investigation of ꢀ1-receptor-affinity
purified residue was investigated by 19F and H NMR
[3H]-(+)-Pentazocine binding to guinea pig brain mem-
brane preparations was performed according to the
procedure described in ref 28.
1
spectroscopy and by HPLC. A sample of the residue
was purified by fc (2 cm, petroleum ether/ethyl ace-
tate=3:7, 2 mL, Rf 0.38). Colorless, viscous oil.
C25H25F3N2O5 (490.5). 1H NMR (CDCl3): d=1.55–
1.67 (m, 1H, 4-H), 1.70–1.86 (m, 2H, 4-H, 3-H), 1.91–
2.05 (m, 1H, 3-H), 3.06 (s, 3H, NCH3), 3.52 (s, 3H,
OCH3), 3.88 (dd, J=5.9, 1.9 Hz, 1H, 5-H), 4.06 (d,
J=3.2 Hz, 1H, 1-H), 4.42 (d, J=14.8 Hz, 1H,
NCH2Ph), 4.67 (d, J=14.4 Hz, 1H, NCH2Ph), 4.96
(ddd, J=8.5, 5.2, 3.3 Hz, 1H, 2-H), 7.20–7.60 (m, 10H,
aromat. H). 19F NMR (CDCl3): d=ꢀ72.2 (s, 3 F, CF3,
98.8% intensity), ꢀ71.8 (s, 3 F, CF3, 1.2% intensity).
HPLC (acetonitrile/water=1:1; flow rate: 0.80 mL/min):
retention time: 30.6 min (97.6% intensity).
Membrane preparation. Thawed guinea pig brains
(Dunkin Hartley, Harlan-Sera-Lab) were homogenized
with an Ultraturrax (8000 rpm) in 10 volumes of cold
0.32 M sucrose. The homogenate was centrifuged at
1000g for 10 min at 4 ꢁC. The supernatant was separated
and centrifuged at 22,000g for 20 min at 4 ꢁC. The pellet
was resuspended in 10 volumes of buffer (50 mM Tris–
HCl, pH 7.4) with an ultraturrax (8000 rpm), incu-
bated for 30 min at 25 ꢁC and centrifuged at 22,000g
(20 min, 4 ꢁC). The pellet was resuspended in buffer,
the protein concentration was determined according
to the method of Bradford31 using bovine serumalbu-
min as standard, and subsequently the preparation was
frozen (ꢀ83 ꢁC) in 5 mL portions of about 2 mg protein/
mL.
[(1S,2R,5S)-6-Benzyl-8-methyl-7,9-dioxo-6,8-diazabicy-
clo[3.2.2]nonan-2-yl)] (2R)-3,3,3-trifluoro-2-methoxy-2-
phenylpropanoate (25). As described for 24 the alcohol
12a (23 mg, 0.084 mmol) was acylated with (S)-(+)-
Mosher’s acid chloride [(S)-23, 15 mL, 0.080 mmol],
triethylamine (0.3 mL, 2.1 mmol) and dimethylamino-
pyridine (19 mg, 0.15 mmol) in CH2Cl2 (4 mL). The
diastereomeric ratio of the non-purified residue was
Performance of the ꢀ1-receptor binding assay. The test
was performed with the radioligand [ring-1,3-3H]-(+)-
pentazocine (1036 GBq/mmol; NEN Life Science Pro-
ducts). The thawed membrane preparation (about
150 mg of the protein) was incubated with various con-
centrations of test compounds, 3 nM [3H]-(+)-pentazo-
cine and buffer (50 mM Tris–HCl, pH 7.4) in a total
volume of 500 mL for 150 min at 37 ꢁC. The incubation
was terminated by rapid filtration through presoaked
Whatman GF/B filters using a cell harvester. After
washing four times with 2 mL of cold buffer 3 mL of
scintillation cocktail were added to the filters. After
at least 8 h, bound radioactivity trapped on the filters
was counted in a liquid scintillation analyzer. Non-
specific binding was determined with 10 mM haloper-
idol.
1
investigated by 19F and H NMR spectroscopy and by
HPLC. A sample of the residue was purified by fc (2 cm,
petroleumether/ethyl acetate=3:7, 2 mL, Rf 0.41). Col-
orless, viscous oil. C25H25F3N2O5 (490.5). 1H NMR
(CDCl3): d 1.47–1.60 (m, 1H, 4-H), 1.61–1.78 (m, 2H, 4-
H, 3-H), 1.81–1.91 (m, 1H, 3-H), 3.08 (s, 3H, NCH3),
3.64 (s, 3H, OCH3), 3.87 (dd, J=4.9, 1.5 Hz, 1H, 5-H),
4.10 (d, J=2.7 Hz, 1H, 1-H), 4.43 (d, J=14.6 Hz, 1H,
NCH2Ph), 4.69 (d, J=14.6 Hz, 1H, NCH2Ph), 4.89
(ddd, J=8.7, 5.2, 2.7 Hz, 1H, 2-H), 7.20–7.63 (m, 10H,
aromat. H). 19F NMR (CDCl3): d=ꢀ72.2 (s, 3 F, CF3,
1.6% intensity), ꢀ71.8 (s, 3 F, CF3, 98.4% intensity).
HPLC (acetonitrile/water=1:1; flow rate: 0.80 mL/min):
retention time: 33.0 min (97.4% intensity).
Investigation of ꢀ2-receptor-affinity
s2-Receptor-affinity was determined using rat liver
membranes with [3H]-ditolylguanidine in the presence of
100 nM (+)-pentazocine to mask s1-binding sites. The
assay was performed according to the procedure descri-
bed in ref 28.
Receptor binding studies, general
Teflon-glass-homogenizer: Potter1S (B. Braun Biotech
International). Rotor/stator homogenizer: Ultraturrax1
T25 basic (Ika Labortechnik). Centrifuge: High speed
refrigerating centrifuge model J2-HS (Beckman). Filter:
Whatman glass fibre filters GF/B, presoaked in 0.5%
polyethylenimine (in water) for 2 h at 4 ꢁC before use.
Filtration was performed with a Brandel 24-well cell
harvester. Scintillation cocktail: Rotiszint eco plus
(Roth). Liquid scintillation analyzer: Tri-Carb 2100 TR
(Canberra Packard), counting efficiency 66%. All
experiments were carried out in triplicate. IC50-values
were determined from competition experiments with at
least six concentrations of test compounds and were
calculated with the curve-fitting programGraphPad
Prism1 3.0 (GraphPad Software) by nonlinear regres-
sion analysis. Ki-values were calculated according to
Cheng and Prusoff.30 KD values for the radioligands
were taken fromthe literature. For compounds with
high affinity (low Ki-values) mean valuesꢂSEM fromat
least three independent experiments are given.
Membrane preparation. One frozen rat liver (Sprague
Dawley, Harlan-Sera-Lab) was allowed to thaw slowly
on ice. Then it was homogenized with a potter (800 rpm)
in 10 volumes of cold buffer (10 mM Tris–HCl/0.32 M
sucrose, pH 7.4). The homogenate was centrifuged at
1000g for 10 min at 4 ꢁC. The supernatant was separated
and saved on ice. The pellet was resuspended in 30 mL
of cold buffer and centrifuged again. Both supernatants
were then centrifuged at 31,000g for 20 min at 4 ꢁC. The
pellet was resuspended in 30 mL of buffer (10 mM Tris–
HCl, pH 7.4) by vortexing and gentle potter homo-
genization. Then it was incubated for 15 min at 25 ꢁC
and centrifuged at 31,000g (20 min, 4 ꢁC). The pellet was
resuspended in buffer, the protein concentration was
determined according to the method of Bradford31
using bovine serumalbumin as standard, and subse-

2256
M. Weigl et al. / Bioorg. Med. Chem. 10 (2002) 2245–2257
quently the preparation was frozen (ꢀ83 ꢁC) in 5 mL
Investigation of ꢂ-receptor-affinity
portions of about 2.5 mg protein/mL.
[3H]-DAMGO binding to guinea pig brain membrane
preparations was performed according to standard pro-
cedure described in ref 14.
Performance of the ꢀ2-receptor binding assay. The
membrane preparation (about 60 mg protein) was incu-
bated with 3 nM [3H]-ditolylguanidine (di-[p-ring-3H]-
1,3-di-o-tolylguanidine, 2220 GBq/mmol; American
Radiolabeled Chemicals Inc.) and different concentra-
tions of test compounds in buffer (50 mM Tris–HCl, pH
8.0) in the presence of 100 nM (+)-pentazocine. The
total volume was 250 mL. The incubation (120 min,
25 ꢁC) was stopped by addition of 2 mL of ice cold buf-
fer (10 mM Tris–HCl, pH 8.0) followed by rapid filtra-
tion through presoaked Whatman GF/B filters using a
cell harvester. After washing three times with 2 mL of
cold buffer, 3 mL of scintillation cocktail were added to
the filters. After at least 8 h, bound radioactivity trapped
on the filters was counted in a liquid scintillation ana-
lyzer. Non-specific binding was determined with 10 mM
nonradiolabeled ditolylguanidine.
Membrane preparation. Described under Investigation
of k-receptor-affinity.
Performance of the ꢂ-receptor binding assay. The test
was performed with the radioligand [3H]-DAMGO
(2016.5 GBq/mmol; NEN Life Science Products). The
TM
thawed membrane preparation (about 400 mg of the
protein) was incubated with various concentrations of
test compounds, 1 nM [3H]-DAMGO, 5 mM MgCl2,
100 mM PMSF (phenylmethanesulfonyl fluoride) and
buffer (50 mM Tris–HCl, pH 7.4) in a total volume of
500 mL at 25 ꢁC for 90 min. The incubation was termi-
nated by rapid filtration through presoaked Whatman
GF/B filters (50 mM Tris–HCl, pH 7.4 for 2 h at 4 ꢁC)
using a cell harvester. After washing four times with
2 mL of cold buffer, 3 mL of scintillation cocktail were
added to the filters. After at least 8 h, bound radio-
activity trapped on the filters was counted in a liquid
scintillation analyser. Non-specific binding was deter-
mined with 1 mM naloxone.
Investigation of ꢁ-receptor-affinity
[3H]-U-69593 binding to guinea pig brain membrane
preparations was performed according to standard pro-
cedure described in ref 14.
Membrane preparation. The cerebellumwas removed
fromguinea pig brains (Dunkin Hartley, Harlan) and
the brains were bisected. Three brain halves were
homogenised in 50 mL of buffer (50 mM Tris–HCl pH
7.4) with a potter (800 rpm, 10 up-and-down strokes).
The suspension was centrifuged at 49,000g for 10 min at
4 ꢁC. The supernatant was removed and the pellet was
resuspended in buffer (30 mL) with an Ultraturrax
(8000 rpm). Subsequently, it was centrifuged at 49,000g
for 10 min at 4 ꢁC. The pellet was resuspended in buffer,
incubated for 45 min at 37 ꢁC and centrifuged (49,000g,
10 min, 4 ꢁC). Again the pellet was resuspended and
centrifuged. Then, the pellet was resuspended in buffer
(30 mL), the protein concentration was determined
according to the method of Bradford31 using bovine
serumalbumin as standard, and subsequently the pre-
paration was frozen (ꢀ83 ꢁC) in 5 mL portions of about
3 mg protein/mL.
Acknowledgements
Financial support of this work by the Deutsche For-
schungsgemeinschaft, the Fonds der Chemischen
Industrie and the Wissenschaftliche Gesellschaft Frei-
burg is gratefully acknowledged. Thanks are also due to
the Degussa AG, Janssen-Cilag GmbH, and Bristol-
Myers Squibb for donation of chemicals and reference
compounds.
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