Bicyclic σ receptor ligands by stereoselective Dieckmann analogous cyclization of piperazinebutyrate

Article abstract of DOI:10.1039/c003878d

Starting from racemic 2-aminoadipic acid (6) the piperazinedione 10 with a butyrate side chain was synthesized in four reaction steps. The four-carbon bridge was established upon deprotonation of 10 with LHMDS and subsequent trapping of the lithium alcoholate with Me₃SiCl to give diastereoselectively the mixed methyl silyl ketal 12 in 94% yield. The relative configuration of the new center of chirality was determined by X-ray crystal structure analysis of 12. The high diastereoselectivity during the conversion of the butyrate 10 into the mixed methyl silyl ketal 12 supports the formation of the lithium alcoholate 11 as central intermediate. Stereoselective reduction of the ketone 13 with LiBH₄ led to the alcohol 14, which was benzylated and reduced to provide the final bicyclic products 16 and 17. Whereas the alcohol 16 shows only moderate affinity to both σ receptor subtypes, the benzyl ether 17 represents a potent and selective σ₁ receptor ligand (K_i = 47 nM). Comparison of the σ receptor affinities of 16 and 17 with the smaller homologues 5a and 5c clearly indicates that the size of the bridge and the O-substituent determines subtype selectivity.

Full text of DOI:10.1039/c003878d

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Bicyclic r receptor ligands by stereoselective Dieckmann analogous
cyclization of piperazinebutyrate†
Sunil Kumar Sunnam,a Dirk Schepmann,a Birgit Wibbelingb and Bernhard Wu¨nsch*a
Received 5th March 2010, Accepted 24th May 2010
First published as an Advance Article on the web 16th June 2010
DOI: 10.1039/c003878d
Starting from racemic 2-aminoadipic acid (6) the piperazinedione 10 with a butyrate side chain was
synthesized in four reaction steps. The four-carbon bridge was established upon deprotonation of 10
with LHMDS and subsequent trapping of the lithium alcoholate with Me3SiCl to give
diastereoselectively the mixed methyl silyl ketal 12 in 94% yield. The relative configuration of the new
center of chirality was determined by X-ray crystal structure analysis of 12. The high
diastereoselectivity during the conversion of the butyrate 10 into the mixed methyl silyl ketal 12
supports the formation of the lithium alcoholate 11 as central intermediate. Stereoselective reduction of
the ketone 13 with LiBH4 led to the alcohol 14, which was benzylated and reduced to provide the final
bicyclic products 16 and 17. Whereas the alcohol 16 shows only moderate affinity to both s receptor
subtypes, the benzyl ether 17 represents a potent and selective s1 receptor ligand (Ki = 47 nM).
Comparison of the s receptor affinities of 16 and 17 with the smaller homologues 5a and 5c clearly
indicates that the size of the bridge and the O-substituent determines subtype selectivity.
Introduction
The restriction of conformational flexibility of molecules is a
general strategy for the improvement of affinity and selectivity
during the development of novel drugs. The energy, which is
produced during the interaction of a ligand with its target protein,
is considerably increased with conformationally constrained lig-
ands. This phenomenon originates from the entropy, since flexible
ligands lose freedom during interaction with target proteins.
Therefore, a ligand, which is forced by conformational constraints
into the bioactive conformation of a related flexible ligand, shows
considerably higher affinity than the more flexible analogue.1
We are interested in conformationally constrained piperazine
derivatives, which will be developed as selective k-opioid receptor
agonists and s ligands, since the piperazine scaffold is a common
structural element of some very potent k agonists2,3 and s
ligands.4,5 In example the 1,4-disubstituted piperazine derivatives
14 and 25 represent very potent ligands for the s1 receptor. (Fig. 1)
Very recently we have reported on the synthesis and k receptor
affinity of piperazine derivatives with an additional three-carbon-
Fig. 1 Piperazines without and with conformational restriction showing
high s receptor affinity.
bridge. In the resulting bicyclic systems the pharmacophoric
elements are fixed in a definite orientation to each other.6,7 In
the field of s receptor ligands the flexible hydroxymethyl group
of the potent s1 receptor ligands 38 was also incorporated into a
conformationally constrained bicyclic system. Depending on the
stereochemistry and the substitution pattern 4 and 5 represent
potent s1 and/or s2 ligands.9,10
Whereas the alcohol 4a (R = H) and the methyl ether 4b (R =
CH3) with (1R,2R,5S)-configuration are selective s1 receptor lig-
ands with Ki values of 6.5 nM and 26 nM (s1 : s2 = 124 and 22)9 the
corresponding alcohol 5a (R = H) with (1R,2R,5S)-configuration
and a N-8 methyl moiety does not interact significantly with both
s receptor subtypes. The methyl ether 5b (R = CH3) prefers
the s2 subtype (Ki (s2) = 1350 nM; (Ki (s2) = 327 nM) and
after introduction of a benzyl group (5c, R = Bn) the affinity to
both subtypes was dramatically increased leading to a potent but
aInstitut fu¨r Pharmazeutische und Medizinische Chemie der Uni-
versita¨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
† Electronic supplementary information (ESI) available: HPLC chro-
matograms and NMR spectra of stable compounds. See DOI:
10.1039/c003878d
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Scheme 1 Synthesis of the dioxopiperazine 10 with butyrate side chain. Reagents and conditions: (a) Me3SiCl, MeOH, 18 h, rt, 99%. (b) 1. C6H5CHO,
CH2Cl2, NEt3, MgSO4, rt, 3.5 h; 2. NaBH4, MeOH, rt, 4 h. (c) ClCOCH2Cl, NEt3, CH2Cl2, rt, 4 h, 51% (over three steps). (d) H3CNH2, CH3CN, rt,
16 h, 55%.
unselective s ligand ((Ki (s1) = 16 nM; (Ki (s2) = 30 nM) (compare
Table 1).10
the Dieckmann cyclization,12-14 which makes use of trapping the
first intermediate hemiketal lithium salt 11 with chlorotrimethyl-
silane (TMSCl) was applied, because theoretical calculations
have shown that piperazinediones with a four-carbon bridge are
almost as stable as piperazinediones with a three-carbon bridge.14
(Scheme 2)
Herein we report on the expansion of the three-carbon bridge
of the 6,8-diazabicyclo[3.2.2]nonane framework of 4 and 5 to
a four-carbon-bridge and the s1 and s2 receptor affinities of
the resulting 7,9-diazabicyclo[4.2.2]decanes. The influence of this
bridge expansion on the s1 and s2 receptor affinity is initially
investigated with racemic mixtures. Racemic mixtures contain
both enantiomers and so all stereoisomers will be included in the
study. After detection of potent ligands in a new compound class
(e.g. 7,9-diazabicyclo[4.2.2]decanes), the particular enantiomers
will be synthesized separately in a second optimization step.
Results and discussion
Chemistry
The synthesis of the novel 7,9-diazabicyclo[4.2.2]decane ring
system started with esterification of racemic 2-aminoadipic acid
(6) with methanol and chlorotrimethylsilane to give HCl salt
of diester 7.11 Reaction of primary amine 7 with benzaldehyde
afforded an imine which was subsequently reduced with NaBH4
to yield the monobenzylamine 8. Acylation of 8 with chloroacetyl
chloride led to chloroacetamide 9. Heating of chloroacetamide 9
with methylamine provided in a one-pot procedure the dioxopiper-
azinebutyrate 10 in 55% yield. In the first step an SN2 reaction
of 9 with methylamine led to a secondary amine, which upon
intramolecular aminolysis formed the dioxopiperazinebutyrate 10.
(Scheme 1)
Scheme 2 Dieckmann analogous cyclization of 10: Reagents and condi-
tions: (a) LHMDS, THF, 0.5 h, -78 ◦C. (b) Me3SiCl, 2 h, -78 ◦C, warmed
to rt, 1 h, 94%.
The classical Dieckmann cyclization of the corresponding
propionates12,13 and acetates14 of dioxopiperazines was not success-
ful, since the corresponding enolates of the resulting b-dicarbonyl
products contain a trans-configured double bond in a small cyclic
substructure, which is forbidden according to Bredt’s rule.15,16
Similarly butyrate 10 should not cyclize under normal Dieckmann
cyclization conditions, although the trans-configured enolate of
the corresponding bicyclic b-dicarbonyl compound 13 is located
within an eight-membered ring, which is allowed according to
Bredt’s rule. Nevertheless, the recently described variation of
For this purpose, the piperazinebutyrate 10 was treated with
LHMDS at -78 ◦C and after 30 min TMSCl was added. After
flash chromatography purification the mixed methyl silyl ketal 12
was isolated in 94% yield as single diastereomer. Since the relative
configuration of the newly formed center of chirality could not be
assigned unambiguously by NMR spectroscopy an X-ray crystal
structure analysis of the crystalline ketal 12 was performed after
recrystallization with iPr2O.
In Fig. 2 the X-ray crystal structure of 12 is displayed. It is clearly
shown that the siloxy group is directed to the carbonyl moiety in
3716 | Org. Biomol. Chem., 2010, 8, 3715–3722
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Table 1 Comparison of s affinities of piperazines with a three-carbon
bridge (5) and with a four-carbon bridge (16, 17)
Ki SEM/nM (n = 3)
s1
s2
(3[H]-(+)-
(3[H]-di-o-
s1/s2
Compound
R
H
pentazocine) tolylguanidine) selectivity
5a10
>2 000
CH2C6H5 16 5.6
2 000
>2 000
30 3.6
644 23 nM
—
2
0.33
16
—
0.7
5c10
16
17
H
CH2C6H5 47 3.2 nM 754 43 nM
(+)-pentazocine
di-o-tolylguanidine
42 1.1
61 18
—
42 17
Fig. 2 X-ray crystal structure analysis of the mixed methyl silyl ketal 12.
tumors19,20 the s1 and s2 affinities of 16 and 17 were evaluated in
receptor binding studies with radioligands.
position 8. This configuration supports our hypothesized reaction
path, which postulates the Li-chelate 11 as driving force for this
cyclization. (Scheme 2) A coordination of Li+ with the other
carbonyl O-atom in position 10 would result in a less favorable
seven-membered ring.
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
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.21,22
In Table 1 the s1 and s2 receptor affinities of alcohol 16 and
benzyl ether 17 are compared with the s affinities of the lead
compounds 5a and 5c. The alcohol 16 shows low affinity at both
s1 and s2 receptors with a slight preference for the s2 subtype.
However, the s affinities of the alcohol 16 are higher than the s
affinities of the smaller homologue 5a, which is almost inactive at
both s receptor subtypes. Introduction of an O-benzyl group in
17 increases the s1 receptor affinity enormously (Ki = 47 nM)
but does not change the s2 affinity. This result indicates that
the O-substituent influences the subtype selectivity. A similar
observation was made with the smaller homologue 5c. Here the
introduction of the O-benzyl group leads to a very potent s1 and
s2 receptor ligand. In contrast to the bicyclic benzyl ether 5c with
a three-carbon bridge, which shows a slight preference for the
s1 subtype, the homologous benzyl ether 17 with a four-carbon
bridge displays remarkable s1 selectivity (s1 : s2 = 16). Obviously
the size of the bridge controls the subtype selectivity together with
the O-substituent.
Since the novel 7,9-diazabicyclo[4.2.2]decane scaffold of 12 rep-
resents an interesting system for the synthesis of conformationally
constrained s receptor ligands the mixed methyl silyl ketal 12 was
further preceded. At first the ketal 12 was hydrolyzed with diluted
HCl to provide the ketone 13 in 97% yield. (Scheme 3) Reduction
of the ketone 13 with LiBH4 at -78 ◦C took place with high
diastereoselectivity giving exclusively the alcohol 14. Comparison
of NMR data, tlc behavior and reaction path with those of the
corresponding smaller homologues9,10 proved unequivocally the
relative configuration of the alcohol 14. Benzylation of 14 with
benzyl bromide led to the benzyl ether 15 and final LiAlH4
reduction of the alcohol 14 and the benzyl ether 15 provided the
basic bicyclic systems 16 and 17, respectively.
Conclusion
The stepwise Dieckmann cyclization making use of trapping of
the first intermediate lithium alcoholate 11 with Me3SiCl was
successfully applied on the synthesis of piperazine derivatives
with a four-membered bridge. The increased conformational
freedom of the butyrate side chain compared with propionate
and acetate side chains does not inhibit this bridging reaction of
the piperazinedione scaffold. The configuration of the resulting
mixed methyl silyl ketal 12, which was unambiguously proved by
an X-ray crystal structure analysis, supports the hypothesis of
the lithium chelate 11 being the driving force of this Dieckmann
analogous cyclization. Whereas introduction of an O-benzyl group
Scheme 3 Reagents and conditions: (a) HCl, THF–H2O, rt, 17.5 h, 97%.
(b) LiBH4, THF, -78 ◦C, 3 h, 99%. (c) NaH, BnBr, DMF, rt, 1 h, 82%. (d)
LiAlH4, THF, reflux, 15–16 h, 67% (16), 59% (17).
r Receptor binding studies
Since s receptor ligands have a great potential in the treatment of
neuropsychiatric disorders17,18 and in the diagnosis and therapy of
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into piperazine derivatives with a three-carbon bridge (5c) resulted
in a very potent but unselective s ligand, the corresponding
benzyl ether 17 with a four-carbon bridge represents a potent
and selective s1 receptor ligand. Obviously the size of the bridge
has a considerable influence on the s receptor subtype selectivity.
(1C, CH2CH2CH2CO2CH3), 177.7 (1C, CHCO2CH3). MS (EI):
m/z (%) = 190 [MH+ - Cl, 100]. IR (neat): n/cm-1 = 3392 (N–H),
=
1730 (C O), 1225 (C–O).
Dimethyl
(RS)-2-[N-benzyl-N-(2-chloroacetyl)amino]hexa-
nedioate (9). Benzaldehyde (10.3 mL, 102 mmol), triethylamine
(14.1 mL, 120 mmol) and anhydrous MgSO4 (5.6 g, 46.5 mmol)
were added to a solution of 7.HCl (21 g, 93 mmol) in dry CH2Cl2
(165 mL) under ice-cooling. The mixture was stirred at rt for
3.5 h. The solvent was evaporated in vacuum. Et2O (600 mL) was
added to the residue, the mixture was filtered and concentrated
in vacuum to obtain the corresponding imine as pale yellow oil.
The oily residue (21.5 g) was dissolved in dry CH3OH (210 mL)
and NaBH4 (6.2 g, 163 mmol) was added slowly over a period of
1 h under ice-cooling. The mixture was then stirred at rt for 4 h.
The solvent was evaporated slowly in vacuum, H2O (100 mL) was
added to the residue and the aqueous layer was extracted with
CH2Cl2 (6 ¥ 100 mL). The combined organic layers were washed
with brine, dried over Na2SO4 and concentrated in vacuum to
obtain amine 8 as a pale yellow oil. The crude oil (8, 15.8 g) was
dissolved in CH2Cl2 (130 mL), chloroacetyl chloride (5.7 mL,
71.5 mmol) and triethylamine (5.0 mL, 35.7 mmol) were added
slowly under ice-cooling and the mixture was stirred at rt for
4 h. The solvent was evaporated in vacuum, Et2O (100 mL) was
added to the residue and the mixture was filtered. The filtrate
was concentrated in vacuum to obtain a pale yellow viscous
oil. This process was repeated three times with Et2O. The crude
oil was purified by fc (cyclohexane/ethyl acetate = 7 : 3, 8 cm,
25 cm, 100 mL, Rf 0.27) to obtain a colorless viscous oil. Yield
10.5 g (51%) (over three steps). C17H22ClNO5 (355.8). Purity
(HPLC, method A): 94.6%, tR = 19.3 min. 1H NMR (CDCl3): d
(ppm) = 1.50 (tt, J = 6.8/5.8 Hz, 0.3H, CH2CH2CH2CO2CH3min),
1.61 (tt, J = 7.7/7.4 Hz, 2 ¥ 0.7H, CH2CH2CH2CO2CH3maj),
1.69 (s broad, 0.3H, CH2CH2CH2CO2CH3min), 1.76–1.85
(m, 1H, CH2CH2CH2CO2CH3maj+min), 1.98–2.08 (m, 1H,
Experimental
General Chemistry
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 Finnigan
R
LCQꢀ ion trap mass spectrometer with an ESI (electrospray ion-
ization) interface, Exact mass (ESI): MicroTof (Bruker Daltonics)
Finnigan MAT 4200s. IR: IR spectrophotometer 480Plus FT-
1
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 reso-
lution. The assignment of 1H and 13CNMR signals was supported
by various 2D techniques including H/H-COSY and GHSQC
two dimensional NMR techniques. HPLC: Merck Hitachi Equip-
ment; UV detector: L-7400; autosampler: L-7200; pump: L-7100;
R
degasser: L-7614; 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%,
R
31.5–40 min: 90%. Method B: LiChrospherꢀ 60 RP-select B (5
CH2CH2CH2CO2CH3maj+min), 2.14–2.18 (m,
2
¥
0.3H,
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%.
CH2CH2CH2CO2CH3min), 2.25 (2 ¥ t, J = 7.3 Hz, 2 ¥ 0.7H,
CH2CH2CH2CO2CH3maj), 3.46 (s, 3 ¥ 0.3H, CHCO2CH3min),
3.60 (s, 3 ¥ 0.7H, CO2CH3maj), 3.63 (s, 3H, CH2CO2CH3maj+min),
4.02 (d, J = 12.7 Hz, 0.7H, NCH2C6H4OCH3maj), 4.07 (d, J =
12.6 Hz, 0.7H, NCH2C6H5maj), 4.18 (d, J = 12.4 Hz, 0.3H,
NCH2C6H5min), 4.32 (d, J = 12.4 Hz, 0.3H, NCH2C6H4OCH3min),
4.42 (t, J = 7.3 Hz, 0.3H, CHCO2CH3min), 4.54 (t, J =
7.5 Hz, 0.7H, CHCO2CH3maj), 4.58–4.62 (m, 0.7H + 2 ¥
0.3H, COCH2Clmaj, COCH2Clmin), 4.69 (d, J = 17.2 Hz, 0.7H,
COCH2Clmaj), 7.21–7.38 (m, 5H, 2-H, 3-H, 4-H, 5-H, 6-Hbenzyl).
13C NMR (CDCl3): d (ppm) = 21.6 (CH2CH2CH2CO2CH3min),
22.0 (CH2CH2CH2CO2CH3 maj), 28.8 (CH2CH2CH2CO2CH3maj),
29.9 (CH2CH2CH2CO2CH3min), 33.4 (CH2CH2CH2CO2CH3min),
33.6 (CH2CH2CH2CO2CH3maj), 41.8 (NCH2C6H5maj), 46.8
(COCH2Clmin), 51.2 (COCH2Clmaj), 51.8 (CH2CO2CH3maj),
52.4 (CHCO2CH3maj), 52.7 (2C, CH2CO2CH3maj+min), 58.8
(CHCO2CH3maj), 60.1 (CHCO2CH3min), 127.1 (2C, C-2, C-
6benzylmaj), 127.6 (2C, C-2, C-6benzylmin), 128.3 (1C, C-1benzylmaj),
128.6 (1C, C-1benzylmin), 129.2 (4C, C-3, C-5benzylmaj+min), 135.9
(1C, C-4benzylmaj), 137.2 (1C, C-4benzylmin), 167.9 (1C, N-C=Omaj),
168.3 (1C, N+=C-O- min), 170.6 (1C, CHCO2CH3min), 171.0
(1C, CHCO2CH3maj), 173.5 (1C, CH2CO2CH3min), 173.7 (1C,
CH2CO2CH3maj). MS (EI): m/z (%) = 278 [(M-COCH2Cl)+,
Dimethyl (RS)-2-aminohexanedioate hydrochloride (7·HCl)11.
Chlorotrimethylsilane (51 mL, 652 mmol) was slowly added over
a period of 30 min using a dropping funnel to a solution of
2-aminoadipic acid (6, 30.0 g, 186 mmol) in methanol (370
mL) under ice cooling. The mixture was stirred at rt for 18 h.
The solvent was evaporated in vacuum. Et2O (300 mL) was
added to the crude viscous residue, the mixture was stirred
intensively and concentrated in vacuum. This procedure was
repeated four times to get yellow viscous oil. Yield 41.6 g (99%).
C8H16ClNO4 (225.7). 1H NMR (D2O): d (ppm) = 1.47–1.67
(m, 2H, CH2CH2CH2CO2CH3), 1.73–1.91 (m, 2H, CH2CH2CH2
CO2CH3), 2.31 (t, J = 7.2 Hz, 2H, CH2CH2CH2CO2CH3), 3.54 (s,
3H, CH2CH2CH2CO2CH3), 3.69 (s, 3H, CHCO2CH3), 4.01 (t, J =
6.4 Hz, 1H, +NH3CHCO2CH3). 13C NMR (D2O): d (ppm) = 19.9
(1C, CH2CH2CH2CO2CH3), 29.1 (1C, CH2CH2CH2CO2CH3),
33.0 (1C, CH2CH2CH2CO2CH3), 52.8 (1C, +NH3CHCO2CH3),
52.9 (1C, CH2CH2CH2CO2CH3), 53.8 (1C, CHCO2CH3), 170.6
3718 | Org. Biomol. Chem., 2010, 8, 3715–3722
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100], 106 [(C6H5CH2NH)+, 21], 91 [(benzyl)+, 50]. IR (neat):
15.5 (1C, C-4), 27.9 (1C, C-5), 31.9 (1C, C-3), 32.9 (1C, NCH3),
46.4 (1C, NCH2C6H5), 47.1 (1C, OCH3), 57.7 (1C, C-6), 66.3
(1C, C-1), 103.5 (1C, C-2), 126.2 (1C, C-4benzyl), 126.7 (2C, C-2,
C-6benzyl), 127.0 (2C, C-3, C-5benzyl), 134.3 (1C, C-1benzyl), 162.6
n/cm-1 = 1731 (C O, ester), 1656 (C O, amide), 1200 (C–O,
=
=
ester), 1171 (C–O, ester).
Methyl
(RS)-4-[1-benzyl-4-methyl-3,6-dioxopiperazin-2-yl]-
=
=
(1C, N–C O), 189.2 (1C, N–C O). Exact mass (ESI): m/z =
butyrate (10). Under ice-cooling NEt3 (1.0 mL, 7.5 mmol)
and methylamine (2.0 M in THF, 3.8 mL, 7.5 mmol) were
added slowly to a solution of 9 (2.3 g, 6.6 mmol) in CH3CN
(12 mL) over a period of 10 min. The mixture was warmed
to rt and stirred for 16 h. Then the solvent was evaporated in
vacuum, Et2O (150 mL) was added to the residue in portions, the
mixture was filtered and concentrated in vacuum. The residue
was purified by fc (acetone/dichloromethane = 1 : 9 + 1%
N,N-dimethylethylamine, 4 cm, 15 cm, 20 mL, Rf 0.26) to yield
a colorless viscous oil. Yield 1.15 g (55%). C17H22N2O4 (318.1).
Purity (HPLC, method B): 98.5%, tR = 14.3 min. 1H NMR
(CDCl3): d (ppm) = 1.47–1.59 (m, 1H, CH2CH2CH2CO2CH3),
calculated for C20H30N2O4SiNa+ 413.1873, found 413.1867. IR
(neat): n/cm-1 = 1654 (C O amide), 1472 (C–N amide), 1255
=
(C–O ketal), 836 (Si–O ketal).
X-ray crystal structure analysis of 12. A sample of mixed
methyl silyl ketal 12 was recrystallized from iPr2O to get crystals,
which were suitable for X-ray crystal structure analysis.
Formula C20H30N2O4Si, M = 390.55, colorless crystal 0.40 ¥
0.05 ¥ 0.03 mm, a = 8.9326(1), b = 10.1193(1), c = 11.8959(1)
◦
˚
˚
A, a = 82.230(1), b = 80.314(1), g = 86.226(1) V = 1049.32(2)
3
-3
-1
A , rc = 1.236 g cm , m = 1.210 mm , empirical absorption
correction (0.643 SYMBOL 163 \ f "Symbol" T SYMBOL
¯
1.62–1.73
(m,
1H,
CH2CH2CH2CO2CH3),
1.78–1.94
163 \ f "Symbol" 0.965), Z = 2, triclinic, space group P1 (No.
˚
(m, 2H, CH2CH2CH2CO2CH3), 2.18–2.34 (m, 2H,
CH2CH2CH2CO2CH3), 2.97 (s, 3H, NCH3), 3.66 (s, 3H,
OCH3), 3.88 (t, J = 3.6 Hz, 1H, NCHCO), 3.93 (d, J = 17.5 Hz,
1H, NCH2CO), 3.98 (d, J = 14.8 Hz, 1H, NCH2C6H5), 4.17
(d, J = 17.5 Hz, 1H, NCH2CO), 5.31 (d, J = 14.8 Hz, 1H,
NCH2C6H5), 7.23–7.26 (m, 2H, 3-H, 5-Hbenzyl), 7.27–7.32 (m,
3H, 2-H, 4-H, 6-Hbenzyl). 13C NMR (CDCl3): d (ppm) = 19.8
(1C, CH2CH2CH2CO2CH3), 31.1 (1C, CH2CH2CH2CO2CH3),
33.3 (1C, CH2CH2CH2CO2CH3), 33.7 (1C, NCH3), 47.1 (1C,
NCH2C6H5), 51.8 (1C, NCH2CO), 51.9 (1C, OCH3), 58.8 (1C,
NCHCO), 128.3 (1C, C-4benzyl), 128.6 (2C, C-2, C-6benzyl), 129.1
2), l = 1.54178 A, T = 223(2) K, w and j scans, 12 315
reflections collected (SYMBOL 177 \ f "Symbol"h, SYMBOL
177 \ f "Symbol"k, SYMBOL 177 \ f "Symbol"l), [(sinq)/l] =
-1
˚
0.60 A , 3630 independent (Rint = 0.057) and 3025 observed
reflections [I SYMBOL 179 \ f "Symbol" 2 SYMBOL 115 \ f
"Symbol"(I)], 249 refined parameters, R = 0.048, wR2 = 0.127,
-3
˚
max. (min.) residual electron density 0.26 (-0.23) e A , hydrogen
atoms calculated and refined as riding atoms.
Data set was collected with a Nonius KappaCCD diffrac-
tometer. Programs used: data collection COLLECT (Nonius
B.V., 1998), data reduction Denzo-SMN,28 absorption correction
Denzo,29 structure solution SHELXS-97,30 structure refinement
SHELXL-97,31 graphics SCHAKAL.32
CCDC 766603 contains the supplementary crystallographic
data for this paper.† These data can be obtained free of
charge at www.ccdc.cam.ac.uk/conts/retrieving.html (or from
the Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; Fax: +44 1223 336033, E-mail:
deposit@ccdc.cam.ac.uk).
=
(2C, C-3, C-5benzyl), 135.5 (1C, C-1benzyl), 163.9 (1C, N–C O),
=
166.1 (1C, N–C O), 173.3 (1C, C-1ester carbonyl). Exact mass (ESI):
m/z = calculated for C17H22N2O4Na+ 341.1477, found 341.1472.
IR (neat): n/cm-1 = 1732 (C O ester), 1656 (C O amide), 1173
=
=
(C–O ester).
(1RS,2SR,6RS)-7-Benzyl-2-methoxy-9-methyl-2-(trimethylsi-
lyloxy)-7,9-diazabicyclo[4.2.2]decane-8,10-dione (12). Lithium
hexamethyldisilazide (LHMDS), freshly prepared from n-BuLi
(1.3 mL, 1.5 mmol) and hexamethyldisilazane (0.34 mL, 1.5
mmol) in dry THF (5 mL) at 0 ◦C, was slowly added to a solution
of 10 (0.37 g, 1.2 mmol) in dry THF (10 mL) at -78 ◦C. The
mixture was stirred for 0.5 h at -78 ◦C, chlorotrimethylsilane
(CH3)3SiCl (0.54 mL, 4.1 mmol) was slowly added to the solution,
the mixture was stirred at -78 ◦C for 2 h and then warmed to
rt and stirred for 1 h. The solvent was evaporated to half of
the original volume and ethyl acetate (10 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 and dried over Na2SO4. The solvent
was evaporated in vacuum to obtain a colorless solid which was
purified by fc (cyclohexane/ethyl acetate = 3 : 7, 3 cm, 16 cm,
10 mL, Rf 0.46) to obtain colorless crystals, mp 118–121 ◦C. Yield
0.43 g (94%). C20H30N2O4Si (390.3). Purity (HPLC, method A):
100%, tR = 19.7 min. 1H NMR (CDCl3): d (ppm) = 0.00 (s, 9H,
(CH3)3Si), 1.11–1.26 (m, 3H, 3-H, 4-H), 1.51–1.55 (m, 1H, 5-H),
1.56–1.67 (m, 2H, 3-H, 5-H), 2.72 (s, 3H, NCH3), 3.00 (s, 3H,
OCH3), 3.74 (dd, J = 6.4/1.8 Hz, 1H, 6-H), 3.79 (d, J = 0.9 Hz,
1H, 1-H), 4.09 (d, J = 14.6 Hz, 1H, NCH2C6H5), 4.44 (d, J =
14.6 Hz, 1H, NCH2C6H5), 6.99–7.05 (m, 5H, 2-H, 3-H, 4-H,
5-H, 6-Hbenzyl). 13C NMR (CDCl3): d (ppm) = 0.0 (3C, (CH3)3Si),
(1RS,6RS)-7-Benzyl-9-methyl-7,9-diazabicyclo[4.2.2]decane-2,
8,10-trione (13). The ketal 12 (0.78 g, 2.0 mmol) was dissolved in
a mixture of THF (9 mL) and H2O (9 mL). 1 M HCl (3.5 mL) was
added to the mixture and the mixture was stirred for 17.5 h at rt.
The solvent was evaporated to half of the original volume, washed
with saturated NaHCO3 solution (30 mL) and the aqueous layer
was extracted with CH2Cl2 (6 ¥ 20 mL). The combined organic
layers were washed with brine, dried over Na2SO4 and the solvent
was evaporated in vacuum to obtain a colorless solid. Purification
by fc (acetone/dichloromethane = 1 : 9, 4 cm, 14 cm, 30 mL,
Rf 0.22) yielded colorless crystals, mp 193–198 ◦C. Yield 0.56 g
(97%). C16H18N2O3 (286.1). Purity (HPLC, method B): 99.4%,
1
tR = 11.9 min. H NMR (CDCl3): d (ppm) = 1.24–1.35 (m, 1H,
4-H), 1.73–1.81 (m, 1H, 4-H), 1.95–1.99 (m, 2H, 5-H), 2.34 (ddd,
J = 14.2/9.1/0.9 Hz, 1H, 3-H), 2.51 (dd, J = 14.2/9.2 Hz, 1H,
3-H), 2.88 (s, 3H, NCH3), 4.16 (dd, J = 5.8/2.8 Hz, 1H, 6-H), 4.18
(d, J = 14.6 Hz, 1H, NCH2C6H5), 4.41 (s, 1H, 1-H), 4.86 (d, J =
14.6 Hz, 1H, NCH2C6H5), 7.19–7.29 (m, 5H, 2-H, 3-H, 4-H, 5-H,
6-Hbenzyl). 13C NMR (CDCl3): d (ppm) = 19.3 (1C, C-4), 31.3 (1C,
C-5), 33.5 (1C, C-3), 39.0 (1C, NCH3), 48.6 (1C, NCH2C6H5),
59.1 (1C, C-6), 73.6 (1C, C-1), 128.7 (1C, C-4benzyl), 128.8 (2C,
C-2, C-6benzyl), 129.3 (2C, C-3, C-5benzyl), 135.1 (1C, C-1benzyl), 161.2
This journal is
The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 3715–3722 | 3719
©

View Article Online
=
=
=
(1C, N–C O), 167.4 (1C, N–C O), 203.1 (1C, C-2). Exact mass
C-1N-benzyl), 138.0 (1C, C-1O-benzyl), 165.3 (1C, N–C O, 169.0 (1C,
+
(ESI): m/z = calculated for C16H18N2O3Na+ 309.1215, found
N–C O). Exact mass (ESI): m/z = calculated for C23H26N2O3Na
=
309.1210. IR (neat): n/cm-1 = 1715 (C O ketone), 1666 (C O
401.1841, found 401.1836. IR (neat): n/cm-1 = 1654 (C O amide),
=
=
=
amide), 1448 (C–N amide).
723, 696 (aromatic out-of-plane bend).
(1RS,2SR,6RS)-7-Benzyl-2-hydroxy-9-methyl-7,9-diazabicy-
clo[4.2.2]decane-8,10-dione (14). Under N2 atmosphere LiBH4
(0.09 g) was slowly added to a solution of 13 (0.42 g, 1.4 mmol)
in dry THF (30 mL) at -78 ◦C and the mixture was stirred for
3 h. The excess LiBH4 was destroyed with 1 M HCl (pH 4) then
1 M NaOH was added until the solution attained pH 9 and the
mixture was stirred for 0.5 h at rt. The aqueous layer was extracted
with ethyl acetate (5 ¥ 15 mL). The combined organic layers were
washed with brine, dried (Na2SO4) and the solvent was evaporated
in vacuum to obtain a colorless sponge. The residue was purified
by fc (cyclohexane/ethyl acetate = 1 : 9, 3 cm, 15 cm, 20 mL,
Rf 0.05) to obtain a colorless solid, mp 115–120 ◦C. Yield 0.42 g
(99%). C16H20N2O3 (288.1). Purity (HPLC, method A): 97.7%, tR =
11.7 min. 1H NMR (CDCl3): d (ppm) = 1.27–1.36 (m, 1H, 3-H),
1.55–1.72 (m, 3H, 3-H, 4-H), 1.76–1.83 (m, 1H, 5-H), 1.88–1.96
(m, 1H, 5-H), 2.94 (s, 3H, NCH3), 3.07 (s, 1H, OH), 3.95 (t, J =
3.6 Hz, 1H, 6-H), 4.01 (d, J = 14.7 Hz, 1H, NCH2C6H5), 4.10 (s
broad, 2H, 1-H, 2-H), 5.04 (d, J = 14.7 Hz, 1H, NCH2C6H5), 7.19–
7.29 (m, 5H, 2-H, 3-H, 4-H, 5-H, 6-Hbenzyl). 13C NMR (CDCl3): d
(ppm) = 17.6 (1C, C-3), 32.1 (1C, C-4), 32.3 (1C, C-5), 32.7 (1C,
NCH3), 47.6 (1C, NCH2C6H5), 59.1 (1C, C-6), 66.0 (1C, C-1), 71.7
(1C, C-2), 128.5 (1C, C-4benzyl), 128.7 (2C, C-2, C-6benzyl), 129.2 (2C,
(1RS,2RS,6SR) - 7 - Benzyl - 9 - methyl - 7,9 - diazabicyclo[4.2.2]-
decan-2-ol (16). Under N2 atmosphere LiAlH4 (0.9 mL,
0.86 mmol, 1 M solution in THF) was added dropwise under
ice cooling to a solution of 14 (0.05 g, 0.17 mmol) in dry THF (10
mL). The mixture was warmed to rt and stirred under reflux for
16 h. The excess LiAlH4 was destroyed with H2O (1 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 (20 mL)
and the solvent was evaporated in vacuum. The crude viscous oil
was purified by fc (ethyl acetate/cyclohexane = 8.5 : 1.5 + 0.5%
N,N-dimethylethylamine, 2 cm, 15 cm, 10 mL, Rf 0.19) to obtain a
colorless viscous oil. Yield 0.79 g (67%). C16H24N2O (260.2). Purity
(HPLC, method A): 97.3%, tR = 11.0 min. 1H NMR (CDCl3): d
(ppm) = 1.30–1.36 (m, 1H, 5-H), 1.43–1.50 (m, 1H, 5-H), 1.54–
1.62 (m, 1H, 4-H), 1.92 (ddd, J = 12.6/9.9/5.7 Hz, 1H, 3-H),
2.06 (ddd, J = 15.0/11.7/4.9 Hz, 1H, 4-H), 2.28 (s, 3H, NCH3),
2.34 (t, J = 10.8 Hz, 1H, 3-H), 2.54 (s broad, 1H, 1-H), 2.57
(s broad, 1H, 10-H), 2.73 (dd, J = 11.6/3.9 Hz, 1H, 8-H), 2.80
(s broad, 1H, 6-H), 2.86 (d broad, J = 11.0 Hz, 1H, 8-H), 2.95
(ddd, J = 11.0/4.1/1.7 Hz, 1H, 10-H), 3.50 (d, J = 12.8 Hz, 1H,
NCH2C6H5), 3.57 (d, J = 12.8 Hz, 1H, NCH2C6H5), 3.67 (dt, J =
10.2/5.0 Hz, 1H, 2-H), 7.16 (t, J = 7.2 Hz, 1H, 4-HN-benzyl), 7.23
(t, J = 7.3 Hz, 2H, 3-H, 5-HN-benzyl), 7.28 (d, J = 7.1 Hz, 2H, 2-H,
6-HN-benzyl). A signal for the OH proton is not seen in the spectrum.
13C NMR (CDCl3): d (ppm) = 21.6 (1C, C-4), 33.9 (1C, C-3), 36.2
(1C, C-5), 43.9 (1C, C-8), 46.3 (1C, NCH3), 52.7 (1C, C-10), 57.9
(1C, C-6), 63.4 (1C, NCH2C6H5), 65.5 (1C, C-1), 74.1 (1C, C-2),
127.1 (1C, C-4N-benzyl), 128.3 (2C, C-2, C-6N-benzyl), 129.2 (2C, C-3, C-
5N-benzyl), 139.9 (1C, C-1N-benzyl). Exact mass (ESI): m/z = calculated
=
C-3, C-5benzyl), 135.3 (1C, C-1benzyl), 167.1 (1C, N–C O), 167.8 (1C,
+
=
N–C O). Exact mass (ESI): m/z = calculated for C16H20N2O3Na
311.1372, found 311.1366. IR (neat): n/cm-1 = 3293 (O–H), 1661,
=
1634 (C O), 1474, 1456 (C–N), 1132 (C–O).
(1RS,2SR,6RS)-7-Benzyl-2-benzyloxy-9-methyl-7,9-diazabicy-
clo[4.2.2]decane-8,10-dione (15). Under N2 atmosphere NaH
(0.05 g, 0.8 mmol, 60% dispersion in paraffin oil) was slowly added
to a solution of 14 (0.05 g, 0.18 mmol) and C6H5CH2Br (0.04 mL,
0.3 mmol) in dry DMF (4 mL) under ice-cooling. The reaction
mixture was warmed to rt and stirred for 1 h. The excess NaH
was destroyed with H2O (5 mL) under ice-cooling. The aqueous
layer was extracted with ethyl acetate (5 ¥ 10 mL). The combined
organic layers were washed with brine, dried (Na2SO4) and the
solvent was evaporated in vacuum. The crude solid was purified
by fc (ethyl acetate/cyclohexane = 7 : 3, 2 cm, 15 cm, 10 mL, Rf
for C16H24N2OH+ 261.1967, found 261.1961. IR (neat): n/cm-1
=
3361 (O–H), 2776 (C–H), 717, 696 (aromatic out-of-plane bend).
(1RS,2RS,6SR)-7-Benzyl-2-benzyloxy-9-methyl-7,9-diazabi-
cyclo[4.2.2]decane (17). Under N2 atmosphere LiAlH4 (0.03, 0.7
mmol) was added slowly to a solution of 15 (0.05 g, 0.14 mmol)
in dry THF (10 mL) under ice-cooling. The mixture was warmed
to rt and then stirred under reflux for 15 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 (20 mL). The solvent was evaporated in vacuum. The
crude oil was purified by fc (petroleum ether/ethyl acetate = 9 : 1,
2 cm, 13 cm, 10 mL, Rf 0.42 (ethyl acetate/cyclohexane = 3 : 7))
to obtain a colorless viscous oil. Yield 0.03 g (59%). C23H30N2O
(350.2). Purity (HPLC, method A): 94.9%, tR = 19.2 min. 1H NMR
(CDCl3): d (ppm) = 1.26–1.31 (m, 1H, 5-H), 1.40–1.48 (m, 1H,
5-H), 1.54–1.62 (m, 1H, 4-H), 1.96–2.06 (m, 2H, 3-H, 4-H), 2.23
(s, 3H, NCH3), 2.39 (q, J = 11.0 Hz, 1H, 3-H), 2.54 (s broad,
1H, 1-H), 2.67 (s broad, 1H, 6-H), 2.77 (dd, J = 11.1/4.1 Hz,
2H, 8-H, 10-H), 2.93–3.00 (m, 2H, 8-H, 10-H), 3.35 (s broad, 1H,
2-H), 3.48 (d, J = 13.0 Hz, 1H, NCH2C6H5), 3.60 (d, J = 13.0 Hz,
1H, NCH2C6H5), 4.42 (d, J = 12.2 Hz, 1H, OCH2C6H5), 4.49
(d, J = 12.2, 1H, OCH2C6H5), 7.13–7.24 (m, 8H, 2-H, 3-H, 4-H,
5-H, 6-HN-benzyl, 3-H, 4-H, 5-HO-benzyl), 7.30 (d, J = 7.3 Hz, 2H, 2-H,
6-HO-benzyl). 13C NMR (CDCl3): d (ppm) = 21.7 (1C, C-4), 32.1 (1C,
◦
0.18). to obtain colorless crystals, mp 126–130 C. Yield 0.057 g
(82%). C23H26N2O3 (378.2). Purity (HPLC, method A): 97.2%,
tR = 18.6 min. 1H NMR (CDCl3): d (ppm) = 1.23–1.32 (m,
1H, 4-H), 1.55 (dt, J = 14.4/2.9 Hz, 1H, 3-H), 1.67 (dquint,
J = 14.6/4.6 Hz, 1H, 4-H), 1.74–1.82 (m, 2H, 3-H, 5-H), 1.84–
1.92 (m, 1H, 5-H), 2.78 (s, 3H, NCH3), 3.74–3.78 (m, 1H, 2-H),
3.92 (dd, J = 4.9/2.9 Hz, 1H, 6-H), 4.06 (d, J = 14.7 Hz, 1H,
NCH2C6H5), 4.19 (d, J = 5.0 Hz, 1H, 1-H), 4.60 (d, J = 12.2 Hz,
1H, OCH2C6H5), 4.74 (d, J = 12.2 Hz, 1H, OCH2C6H5), 5.00 (d,
J = 14.7 Hz, 1H, NCH2C6H5), 7.19–7.25 (m, 6H, 2-H, 3-H, 4-H,
5-H, 6-HN-benzyl, 4-HO-benzyl), 7.29 (t, J = 7.3 Hz, 2H, 3-H, 5-HO-benzyl),
7.36 (d, J = 6.9 Hz, 2H, 2-H, 6-HO-benzyl). 13C NMR (CDCl3): d
(ppm) = 17.7 (1C, C-4), 29.1 (1C, C-3), 32.1 (1C, C-5), 32.9 (1C,
NCH3), 47.7 (1C, NCH2C6H5), 59.2 (1C, C-6), 63.9 (1C, C-1), 71.5
(1C, OCH2C6H5), 77.9 (1C, C-2), 128.0 (2C, C-2, C-6O-benzyl), 128.1
(1C, C-4N-benzyl), 128.2 (1C, C-4O-benzyl), 128.6 (2C, C-2, C-6N-benzyl),
128.7 (2C, C-3, C-5N-benzyl), 129.1 (2C, C-3, C-5O-benzyl), 136.0 (1C,
3720 | Org. Biomol. Chem., 2010, 8, 3715–3722
This journal is
The Royal Society of Chemistry 2010
©

View Article Online
C-3), 36.1 (1C, C-5), 44.9 (1C, C-8), 46.2 (1C, NCH3), 52.7 (1C, C-
10), 57.3 (1C, C-6), 63.1 (1C, NCH2C6H5), 71.0 (2C, C-1, C-2), 77.4
(1C, OCH2C6H5), 127.0 (2C, C-2, C-6O-benzyl), 127.5(1C, C-4N-benzyl),
127.6 (1C, C-4O-benzyl), 128.2(2C, C-2, C-6N-benzyl), 128.4 (2C, C-3, C-
5N-benzyl), 129.3 (2C, C-3, C-5O-benzyl), 131.1 (1C, C-1N-benzyl), 139.5 (1C,
C-1O-benzyl). Exact mass (ESI): m/z = calculated for C23H30N2OH+
351.2436, found 351.2431. I.R. (neat): n/cm-1 = 2922 (C–H), 1509
the presoaked filtermats using a cell harvester. After washing each
well five times with 300 ml of water, the filtermats were dried
at 95 ◦C. Subsequently, the solid scintillator was placed on the
filtermat and melted at 95 ◦C. After 5 min, the solid scintillator was
allowed to solidify at room temperature. The bound radioactivity
trapped on the filters was counted in the scintillation analyzer.
The non-specific binding was determined with 10 mM unlabeled
(+)-pentazocine. The Kd-value of (+)-pentazocine is 2.9 nM.26
=
=
(C C aromatic), 1246 (C–O), 731 (C C aromatic out-of-plane
=
bend), 698 (C C aromatic out-of-plane bend).
Membrane preparation for the r2 assay22–24
. 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 31 000 ¥ 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 room temperature for
30 min. After the incubation, the suspension was centrifuged again
at 31 000 ¥ 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 Bradford25 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.
Receptor binding studies
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 polyethylen-
imine for 2 h at room temperature before use. The filtration was
carried out with a MicroBeta FilterMate-96 Harvester (Perkin
Elmer). The scintillation analysis was performed using Meltilex
(Typ A or B) solid scintillator (Perkin Elmer). The solid scintillator
Performing of the r2 assay22–24
. The test was performed with the
radioligand [3H]-ditolylguanidine (50 Ci/mmol; ARC, St. Louis,
MO, USA). The thawed membrane preparation (about 100 mg
of the protein) was incubated with various concentrations 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 room temperature.
The incubation was terminated by rapid filtration through the
presoaked filtermats using a cell harvester. After washing each
well five times with 300 mL of water, the filtermats were dried
at 95 ◦C. Subsequently, the solid scintillator was placed on the
filtermat and melted at 95 ◦C. After 5 min, the solid scintillator was
allowed to solidify at room temperature. The bound radioactivity
trapped on the filters was counted in the scintillation analyzer.
The non-specific binding was determined with 10 mM unlabeled
ditolylguanidine. The Kd-value of ditolylguanidine is 17.9 nM.27
◦
was melted on the filtermat at a temperature of 95 C for 5 min.
After solidifying of the scintillator at room temperature, 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
R
program GraphPad 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.21 The
Ki-values are given as mean value SEM from three independent
experiments.
Membrane preparation for the r1 assay22–24
brains were homogenized with the potter (500–800 rpm, 10 up-
and-down strokes) in 6 volumes of cold 0.32 M sucrose. The
. Five guinea pig
Acknowledgements
We wish to thank the NRW Graduate School of Chemistry for
a stipend, which was funded by the Government of the State
Nordrhein-Westfalen and the DAAD.
◦
suspension was centrifuged at 1200 ¥ g for 10 min at 4 C. The
supernatant was separated and centrifuged at 23 500 ¥ 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
23 500 ¥ g (20 min, 4 ◦C). This procedure was repeated twice.
The final pellet was resuspended in 5–6 volumes of buffer, the
protein concentration was determined according to the method
of Bradford25 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.
References
1 G. Klebe, Wirkstoffdesign, Entwurf und Wirkung von Arzneistoffen,
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. The test was performed with the
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