Homologous piperazine-alcanols: Chiral pool synthesis and pharmacological evaluation

Article abstract of DOI:10.1039/c2md20070h

Starting with the proteinogenic amino acids (S)-aspartate and (S)-glutamate the homologous piperazine-alcanols 3 and 4 were prepared in a five step synthesis. The diversity was introduced by N-1 alkylation of the piperazinediones 5 and 6 with various alkyl halides. Subsequent LiAlH ₄ reduction of the dioxopiperazine-esters 7 and 8 provided the alcohols 3 and 4. The ethanol derivatives 3 show similar σ₁ affinity as the methanol derivatives 2, but increased selectivity over the σ₂ subtype. The corresponding propanol derivatives 4 are considerably less potent. A benzyl or dimethylallyl residue at N-1 appears to be optimal for high σ₁ affinity.

Full text of DOI:10.1039/c2md20070h

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CONCISE ARTICLE
Homologous piperazine-alcanols: chiral pool synthesis and pharmacological
evaluation
*
€
Ralph Holl, Dirk Schepmann and Bernhard Wunsch
Received 9th March 2012, Accepted 6th May 2012
DOI: 10.1039/c2md20070h
Starting with the proteinogenic amino acids (S)-aspartate and (S)-glutamate the homologous
piperazine-alcanols 3 and 4 were prepared in a five step synthesis. The diversity was introduced by N-1
alkylation of the piperazinediones 5 and 6 with various alkyl halides. Subsequent LiAlH4 reduction of
the dioxopiperazine-esters 7 and 8 provided the alcohols 3 and 4. The ethanol derivatives 3 show similar
s1 affinity as the methanol derivatives 2, but increased selectivity over the s2 subtype. The
corresponding propanol derivatives 4 are considerably less potent. A benzyl or dimethylallyl residue at
N-1 appears to be optimal for high s1 affinity.
1. Introduction
The s1 receptor represents a promising target for the development
of innovative therapies of various neurological and neuropsy-
chiatric diseases. It has been shown that s1 receptor agonists can
be used as antidepressants and antiamnesic drugs. The neuro-
protective activity of s1 agonists permits the treatment of memory
disorders (e.g. Alzheimer’s disease, Parkinson’s disease) with s1
agonists. In animal studies s1 receptor antagonists are able to
reduce the unpleasant and dangerous effects after withdrawal of
cocaine, amphetamine or ethanol from addicted animals. The
development of s1 antagonists as innovative analgesics with
reduced side effects is driven by their potentiation of opioid
induced antinociception.1–4 Recently the potential of s1 antago-
nists for the treatment of neuropathic pain has been demon-
Fig. 1 Design of novel s1 ligands based on monocyclic and bicyclic
strated.5,6 In addition to their CNS effects, s1 antagonists are
investigated as novel antitumor drugs, since some human tumor
cell lines express a high amount of s1 and s2 receptors.7,8
piperazine scaffolds.
derivatives.20 The bicyclic compound 1a (R1 ¼ benzyl) represents
a potent s1 ligand (Ki ¼ 6.5 nM) with restricted conformational
flexibility of the piperazine ring (Fig. 1). The stereochemistry and
substitution patterns (substituent R1) are responsible for the high
s1 affinity.21 Disconnection of the C-2/C-3-bond of 1 leads to
2-hydroxymethyl substituted piperazines 2. The p-methoxy-
benzyl (PMB) derivative 2a (R ¼ PMB) interacts with high
affinity with s1 receptors (Ki ¼ 12 nM).22
Since an X-ray crystal structure of the s1 receptor protein is
not yet available, lead compounds and pharmacophore
models9,10 of s1 ligands are used for the design of novel ligands.
The s1 pharmacophore models developed by Glennon,11–13
Langer14 and Pricl15 for different classes of compounds show
similar features including a basic amino moiety surrounded by 2–
3 hydrophobic regions. The number and distances of the
particular structural elements are defined, respectively.
Herein we report the synthesis and pharmacological evalua-
tion of piperazine-alcanols 3 and 4. On the one hand the ethanols
3 (n ¼ 1) and propanols 4 (n ¼ 2) represent homologues of the
methanols 2. On the other hand they result from disconnection of
the potent bicyclic compound 1 between C-1 and C-2 (Fig. 1).
The piperazine ring represents a privileged structural element
for the development of potent s1 ligands, since both N-atoms can
be used for the formation of ionic interactions depending on the
N-substituents.16–18 High s1 affinities were reported for 1-butyl-
4-(2-phenylethyl)piperazine19
and
1,4-dibenzylpiperazine
2. Synthesis
Institut fu€r Pharmazeutische und Medizinische Chemie der Universita€t
For the synthesis of the piperazine-alcanols 3 and 4 enantio-
€
Munster, Hittorfstraße 58-62, D-48149 Munster, Germany. E-mail:
wuensch@uni-muenster.de; Fax: +49-251-8332144; Tel: +49-251-8333311
€
merically pure amino acids of nature’s chiral pool were used.
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resulting piperazinediones 8a–c were reduced with LiAlH4 to
afford the piperazine-propanol derivatives 4a–c.
In both series of compounds the p-methoxybenzyl moiety was
attached to the N-atom in 4-position of the piperazine ring, since
this residue is the preferred substituent of the lead compounds 1
and 2. The benzyl moiety at 1-position of 3a and 4a corresponds
to the benzyl group at 6-position of 1a and 1-position of 2.
Additionally the dimethylallyl residue, which is often found in s1
ligands, was introduced (3b and 4b). According to our experience
with bicyclic s1 ligands 1, it was expected that the allyl group is
too small to produce potent s1 ligands. Nevertheless, the allyl
derivatives 3c and 4c were prepared in order to analyze the
minimal size required for substituents at 1-position.
Scheme 1 Reagents and conditions: (a) 1. H3COH, (H3C)3SiCl, rt, 16 h;
2. ClCH2COCl, CH2Cl2, NaHCO3, rt, 16 h; 3. p-Methoxybenzylamine
(PMB–NH2), H3CCN, NEt3, Bu4NI, reflux, 48 h. (b) NaHMDS, THF,
Bu4NI, ꢀ78 ꢁC, 40 min, R–Br, ꢀ78 ꢁC, 1 h. (c) LiAlH4, THF, reflux, 16 h.
PMB ¼ p-methoxybenzyl.
3. Receptor affinity
Competition experiments with radioligands were used to deter-
mine the s1 and s2 receptor affinities of the piperazine-alcanol
derivatives 3 and 4. In the s1 assay the test compounds competed
with the radioligand [3H]-(+)-pentazocine for the s1 receptors of
a standardized preparation from guinea pig brains. Homoge-
nates of rat liver served as the source of s2 receptors in the s2
assay. Since the radioligand [3H]-1,3-di(o-tolyl)guanidine inter-
acts with s1 and s2 receptors, an excess of non-tritiated
(+)-pentazocine was added to mask the s1 receptors in the s2
assay.25–27
(S)-Aspartate served as a starting compound for the preparation
of ethanol derivatives 3 (Scheme 1). The piperazinedione 5 with
an acetic acid ester side chain was prepared in a three step
procedure comprising esterification of aspartate, chlor-
oacetylation and cyclization with p-methoxybenzylamine.23
Deprotonation of 5 with NaHMDS and subsequent alkylation
with different alkyl bromides provided the piperazinediones 7a–c
with various substituents at the N-atom. Finally LiAlH4 reduc-
tion of 7a–c led to the piperazine-ethanols 3a–c.
The s receptor affinities of the homologous piperazine-ethanol
and -propanol derivatives 3 and 4 are depicted in Table 1 and
compared with the affinity data of the smaller homologue 2a and
the conformationally restricted bicyclic compound 1a. Extension
of the side chain from methanol (2a) to ethanol (3a) led to a slight
reduction of s1 receptor affinity, but extension from ethanol (3a)
to propanol (4a) led to significant reduction of s1 receptor
The piperazine-propanol derivatives 4 were prepared in
a similar manner starting with (S)-glutamate (Scheme 1). Ester-
ification, chloroacetylation and reaction with p-methoxybenzyl-
amine yielded the piperazinedione 6.24 After N-alkylation the
Table 1 s1 and s2 receptor affinities of piperazine-alcanol derivatives
Ki ꢂ SEM [nM] (n ¼ 3)a
Compound
R1
n
s1
s2
s1/s2 selectivity
1a (ref. 21)
2a (ref. 22)
3a
3b
3c
4a
4b
4c
CH2C6H5
CH2C6H5
CH2C6H5
CH2CH]C(CH3)2
CH2CH]CH2
CH2C6H5
—
0
1
1
1
2
2
2
6.5 ꢂ 0.67
12 ꢂ 1.4
20 ꢂ 6.0
18 ꢂ 8.0
1080
188 ꢂ 61
300 ꢂ 21
1870
3.9 ꢂ 1.5
61 ꢂ 18
4.2 ꢂ 1.1
806
124
6
>50
>55
>5
>26
>16
ꢃ1
20
0.7
—
70 ꢂ 10
>1000
>1000
>5000
>5000
>5000
>2000
78 ꢂ 2.0
42 ꢂ 15
—
CH2CH]C(CH3)2
CH2CH]CH2
Haloperidol
Di-o-tolylguanidine
(+)-Pentazocine
a
Triplicates were recorded only for high affinity compounds. The affinity of compounds, which did not reduce significantly the radioligand binding in
the first assay, was recorded only once.
674 | Med. Chem. Commun., 2012, 3, 673–679
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affinity. Piperazines 3b and 4b with the dimethylallyl moiety at
N-1 show similar s1 receptor affinities as the corresponding
benzyl derivatives 3a and 4a, respectively. However, the allyl
moiety (3c and 4c) appears to be too small to produce strong
lipophilic interactions during binding at the s1 receptor protein.
Generally the propanol derivatives 4 show considerably lower s1
affinities than the ethanol derivatives 3.
Variable Wavelength Monitor (Knauer); data acquisition:
D-2500 Chromato-Integrator (Merck Hitachi); injection volume:
20.0 mL; stop time: 2 ꢄ tR; (a) column: LiChroCARTꢀ 250-4
with Superspherꢀ 100 RP-18; flow rate: 0.6 mL minꢀ1; detection:
wavelength: 235 nm; solvent: methanol–water ¼ 65 : 35 + 0.1%
triethylamine; (b) column: LiChroCARTꢀ 250-4 with Supers-
pherꢀ 100 RP-18; flow rate: 0.6 mL minꢀ1; detection: wave-
length: 235 nm; solvent: methanol–water ¼ 50 : 50 + 0.1%
triethylamine; (c) column: Hibarꢀ RT 250-4 with LiChrospherꢀ
100 RP-18 (5 mm); flow rate: 1.0 mL minꢀ1; detection: wave-
length: 254 nm; solvent: acetonitrile–water ¼ 40 : 60 + 0.1%
triethylamine; (d) column: Hibarꢀ RT 250-4 with LiChrospherꢀ
100 RP-18 (5 mm); flow rate: 1.0 mL minꢀ1; detection: wave-
length: 254 nm; solvent: acetonitrile–water ¼ 25 : 75 + 0.1%
triethylamine.
The s2 affinity of the piperazine-ethanol and -propanol
derivatives 3 and 4 is generally very low (Ki > 1 mM), which leads
to high selectivity for the s1 receptor over the s2 receptor. The
s1/s2 selectivity of the ethanol derivatives 3a (>50-fold) and 3b
(>55-fold) is about 10-fold higher than the moderate s1/s2
selectivity of the methanol derivative 2a.
4. Conclusion
The homologous piperazine-alcanols 3 and 4 were prepared by
a five step synthesis starting with enantiomerically pure amino
acids (S)-aspartate and (S)-glutamate. Homologization of the
methanol side chain (2a) to the ethanol side chain (3a) retains s1
affinity but increases s1/s2 selectivity. A further extension to
a propanol side chain (4) led to reduced s1 affinity indicating the
ethanol side chain to be optimal for interaction with the s1
receptor protein. The substituent at N-1 should have a consid-
erable size (e.g. benzyl, dimethylallyl), since the allyl derivative 3c
shows only very low s1 affinity. This observation is in good
accordance with the pharmacophore models mentioned in the
Introduction, which postulate at least two hydrophobic pockets
at the s1 receptor protein accepting sterically demanding
substituents.
5.2. General procedures
5.2.1. General procedure A – alkylation of piperazinediones 5
and 6. Under N2 atmosphere a solution of piperazinedione 5 or 6
(1 eq.) and tetrabutylammonium iodide (0.2 eq.) in THF was
cooled to ꢀ78 ꢁC. Then a 2.0 M solution of sodium hexame-
thyldisilazane in THF (1.1 eq.) was added dropwise. After stir-
ring the mixture at ꢀ78 ꢁC for 40 min, the alkyl bromide (5 eq.)
ꢁ
was added. The mixture was stirred at ꢀ78 C for 1 h and then
allowed to warm to room temperature. After stirring the mixture
at ambient temperature for 2 h, water was added and the mixture
extracted with CH2Cl2 (3ꢄ). The combined organic layers were
dried (Na2SO4), filtered, and the solvent was removed in vacuo.
The residue was purified by FC.
5. Experimental, chemistry
5.2.2. General procedure B – LiAlH4 reduction of piper-
azinediones 7 and 8. Under N2 atmosphere LiAlH4 (6 eq.) was
added to an ice-cooled solution of piperazinedione 7 or 8 (1 eq.)
in THF. The mixture was stirred at 0 ꢁC for 10 min and then
heated to reflux for 16 h. Then water was added under ice-cooling
until H2 formation was stopped. The mixture was stirred at 0 ꢁC
for 10 min and then heated to reflux for 30 min. After cooling to
rt, the mixture was filtered, the solvent removed in vacuo and the
residue purified by FC.
5.1. General
Unless otherwise noted, moisture sensitive reactions were con-
ducted under dry nitrogen. THF was dried with sodium/benzo-
phenone 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); paren-
theses include: diameter of the column, eluent, fraction size, Rf
value. Melting point: Melting point apparatus SMP 3 (Stuart
Scientific), uncorrected. Optical rotation: Polarimeter 341 (Per-
kinElmer); 1.0 dm tube; concentration c [g per 100 mL], the unit
of [a]2D0 [grad mL dmꢀ1 gꢀ1]. MS: MAT GCQ (Thermo-Finnigan);
IR: IR spectrophotometer 480Plus FT-ATR-IR (Jasco). 1H
NMR (400 MHz), 13C NMR (100 MHz): Unity Mercury Plus
400 spectrometer (Varian); d in ppm related to tetramethylsilane;
coupling constants are given with 0.5 Hz resolution.
5.3. Synthesis of the compounds
5.3.1. (+)-(S)-2-[1-Benzyl-4-(4-methoxybenzyl)piperazin-2-yl]
ethanol (3a). According to general procedure B piperazinedione
7a (163 mg, 0.41 mmol) was reacted with LiAlH4 (94 mg, 2.47
mmol) in THF (50 mL) and the product was purified by FC (2
cm, h ¼ 15 cm, CH2Cl2–methanol ¼ 9.5/0.5, V ¼ 10 mL, Rf ¼
0.20). Yellow oil, yield 51 mg (36%). Purity by HPLC: method
2a: tR ¼ 15.1 min, purity 95.1%; method 2c: tR ¼ 11.9 min, purity
97.8%. [a]D20 ¼ +26.9 (c ¼ 1.76; CH2Cl2). C21H28N2O2 (340.5).
MS (EI): m/z [%] ¼ 340 (M, 9), 295 (M ꢀ CH2CH2OH, 13), 219
(M ꢀ CH2PhOCH3, 5), 121 (CH2PhOCH3, 100). 1H NMR
(CDCl3): d [ppm] ¼ 1.79–1.89 (m, 1H, CH2CH2OH), 1.98–2.08
(m, 1H, CH2CH2OH), 2.25–2.35 (m, 2H, piperazine-H), 2.41
(dd, J ¼ 11.7/7.0 Hz, 1H, piperazine-H), 2.45–2.52 (m, 1H,
piperazine-H), 2.62 (dd, J ¼ 11.7/3.1 Hz, 1H, piperazine-H),
2.79–2.85 (m, 1H, piperazine-H), 2.87–2.94 (m, 1H, piperazine-
H), 3.39 (d, J ¼ 13.3 Hz, 1H, NCH2Ph), 3.41 (d, J ¼ 12.5 Hz, 1H,
HPLC: method 1: Merck Hitachi Equipment; UV detector:
L-7400; autosampler: L-7200; pump: L-7100; degasser: L-7614;
column: LiChrospherꢀ 60 RP-select B (5 mm); LiCroCARTꢀ
250-4 mm cartridge; flow rate: 1.000 mL minꢀ1; injection volume:
5.0 mL; detection at l ¼ 210 nm; solvents: A: water with 0.05%
(v/v) trifluoroacetic acid; B: acetonitrile with 0.05% (v/v)
trifluoroacetic acid; gradient elution: 0.0 min: 90.0% of A, 10.0%
of B; 4.0 min: 90.0% of A, 10.0% of B; 29.0 min: 0.0% of A,
100.0% of B; 31.0 min: 0.0% of A, 100.0% of B; 31.5 min: 90.0%
of A, 10.0% of B; 40.0 min: 90.0% of A, 10.0% of B. Method
2: equipment: pump: HPLC pump 64 (Knauer); UV-Detector:
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NCH2Ar), 3.45 (d, J ¼ 12.5 Hz, 1H, NCH2Ar), 3.71–3.77 (m,
1H, CH2CH2OH), 3.79 (s, 3H, ArOCH3), 3.82–3.90 (m, 1H,
CH2CH2OH), 4.14 (d, J ¼ 13.3 Hz, 1H, NCH2Ph), 6.85 (d, J ¼
8.6 Hz, 2H, 30-H4-methoxybenzyl, 50-H4-methoxybenzyl), 7.20–7.27 (m,
3H, 20-H4-methoxybenzyl, 60-H4-methoxybenzyl, NCH2C6H5 (1H)),
7.28–7.34 (m, 4H, NCH2C6H5). The signal for the proton of the
5.3.4. (+)-(S)-3-[1-Benzyl-4-(4-methoxybenzyl)piperazin-2-yl]
propan-1-ol (4a). According to general procedure B piper-
azinedione 8a (98 mg, 0.24 mmol) was reacted with LiAlH4
(54 mg, 1.43 mmol) in THF (40 mL) and the product was purified
by FC (2 cm, h ¼ 15 cm, CH2Cl2–methanol ¼ 9.5/0.5, V ¼ 10 mL,
Rf ¼ 0.20). Yellow oil, yield 57 mg (67%). Purity by HPLC:
method 2a: tR ¼ 16.8 min, purity 95.9%; method 2c: tR ¼ 14.9
min, purity 97.6%. [a]2D0 ¼ +49.9 (c ¼ 1.95; CH2Cl2). C22H30N2O2
1
OH group is not seen in the H NMR spectrum. IR (neat): ~n
[cmꢀ1] ¼ 3379 (m br, nO–H), 2931 (m, nC–H aliph.), 1034 (m, nC–O),
812 (m, Gp-subst. arom.).
(354.5). MS (EI): m/z [%]
¼
354 (M, 3), 295 (M
ꢀ
1
CH2CH2CH2OH, 17), 121 (CH2PhOCH3). H NMR (CDCl3):
d [ppm] ¼ 1.52–1.63 (m, 1H, CH2CH2CH2OH), 1.67–1.94 (m,
3H, CH2CH2CH2OH), 2.12–2.33 (m, 3H, piperazine-H), 2.49–
2.61 (m, 2H, piperazine-H), 2.62–2.67 (m, 1H, piperazine-H),
2.71–2.77 (m, 1H, piperazine-H), 3.20 (d, J ¼ 12.5 Hz, 1H,
NCH2Ph), 3.39 (d, J ¼ 12.5 Hz, 1H, NCH2Ar), 3.44 (d, J ¼ 12.5
Hz, 1H, NCH2Ar), 3.61–3.71 (m, 2H, CH2CH2CH2OH), 3.79 (s,
3H, ArOCH3), 4.17 (d, J ¼ 12.5 Hz, 1H, NCH2Ph), 6.84 (d, J ¼
8.6 Hz, 2H, 30-H4-methoxybenzyl, 50-H4-methoxybenzyl), 7.19–7.27 (m,
3H, 20-H4-methoxybenzyl, 60-H4-methoxybenzyl, NCH2C6H5 (1H)),
7.28–7.36 (m, 4H, NCH2C6H5). The signal for the proton of the
5.3.2. (+)-(S)-2-[4-(4-Methoxybenzyl)-1-(3-methylbut-2-en-1-
yl)piperazin-2-yl]ethanol (3b). According to general procedure B
piperazinedione 7b (142 mg, 0.38 mmol) was reacted with
LiAlH4 (86 mg, 2.28 mmol) in THF (40 mL) and the product
was purified by FC (2 cm, h ¼ 15 cm, CH2Cl2–methanol ¼ 9.5/
0.5, V ¼ 10 mL, Rf ¼ 0.13). Yellow oil, yield 30 mg (25%).
Purity by HPLC: method 2a: tR ¼ 14.7 min, purity 99.3%;
method 2c: tR ¼ 8.4 min, purity 99.5%. [a]2D0 ¼ +16.4 (c ¼ 0.46;
CH2Cl2). C19H30N2O2 (318.5). MS (EI): m/z [%] ¼ 318 (M, 10),
197 (M ꢀ CH2PhOCH3, 38), 121 (CH2PhOCH3, 100). 1H
NMR (CDCl3): d [ppm] ¼ 1.64 (s, 3H, NCH2CH]C(CH3)2),
1.67–1.78 (m, 1H, CH2CH2OH), 1.72 (s, 3H, NCH2CH]
C(CH3)2), 1.91–2.01 (m, 1H, CH2CH2OH), 2.26–2.46 (m, 3H,
piperazine-H), 2.51–2.63 (m, 2H, piperazine-H), 2.79–2.86 (m,
1H, piperazine-H), 2.97–3.13 (m, 2H, NCH2CH]
C(CH3)2(1H), piperazine-H), 3.39–3.52 (m, 3H, NCH2Ar,
NCH2CH]C(CH3)2(1H)), 3.67–3.74 (m, 1H, CH2CH2OH),
3.79 (s, 3H, ArOCH3), 3.82–3.90 (m, 1H, CH2CH2OH), 5.18–
5.24 (m, 1H, NCH2CH]C(CH3)2), 6.85 (d, J ¼ 8.6 Hz, 2H, 30-
1
OH group is not seen in the H NMR spectrum. IR (neat): ~n
[cmꢀ1] ¼ 3362 (m br, nO–H), 3028 (w, nC–H arom.), 2938
(m, nC–H aliph.), 1035 (m, nC–O), 815 (m, Gp-subst. arom.), 734 (m)/698
(m, Gmono-subst. arom.).
5.3.5. (+)-(S)-3-[4-(4-Methoxybenzyl)-1-(3-methylbut-2-en-1-
yl)piperazin-2-yl]propan-1-ol (4b). According to general proce-
dure B piperazinedione 8b (169 mg, 0.44 mmol) was reacted with
LiAlH4 (99 mg, 2.61 mmol) in THF (50 mL) and the product was
purified by FC (2 cm, h ¼ 15 cm, CH2Cl2–methanol ¼ 9.5/0.5, V
¼ 10 mL, Rf ¼ 0.16). Yellow oil, yield 46 mg (32%). Purity by
H
H
4-methoxybenzyl, 50-H4-methoxybenzyl), 7.22 (d, J ¼ 8.6 Hz, 2H, 20-
4-methoxybenzyl, 60-H4-methoxybenzyl). A signal for the proton of
1
the OH group is not seen in the H NMR spectrum. IR (neat):
~n [cmꢀ1] ¼ 3379 (m br, nO–H), 2931 (m, nC–H aliph.), 1034 (m,
nC–O), 812 (m, Gp-subst. arom.).
HPLC: method 2a: tR ¼ 16.5 min, purity 96.8%; method 2c: tR ¼
10.4 min, purity 96.9%. [a]2D0 ¼ +24.0 (c ¼ 1.42; CH2Cl2).
C20H32N2O2 (332.5). MS (EI): m/z [%] ¼ 332 (M, 6), 211 (M ꢀ
1
CH2PhOCH3, 37), 121 (CH2PhOCH3, 100). H NMR (CDCl3):
5.3.3. (+)-(S)-2-[1-Allyl-4-(4-methoxybenzyl)piperazin-2-yl]-
ethanol (3c). According to general procedure B piperazinedione
7c (123 mg, 0.36 mmol) was reacted with LiAlH4 (81 mg, 2.13
mmol) in THF (40 mL) and the product was purified by FC (2
cm, h ¼ 15 cm, CH2Cl2–methanol ¼ 9.5/0.5, V ¼ 10 mL, Rf ¼
0.13). Yellow oil, yield 67 mg (65%). Purity by HPLC: method
2b: tR ¼ 22.9 min, purity 96.9%; method 2d: tR ¼ 13.1 min, purity
97.7%. [a]2D0 ¼ +6.7 (c ¼ 0.19; CH2Cl2). C17H26N2O2 (290.4). MS
(EI): m/z [%] ¼ 290 (M, 6), 169 (M ꢀ CH2PhOCH3, 22), 121
(CH2PhOCH3, 100). 1H NMR (CDCl3): d [ppm] ¼ 1.67–1.78 (m,
1H, CH2CH2OH), 1.90–2.02 (m, 1H, CH2CH2OH), 2.26–2.43
(m, 3H, piperazine-H), 2.48–2.61 (m, 2H, piperazine-H), 2.75–
2.82 (m, 1H, piperazine-H), 2.95–3.06 (m, 2H, NCH2CH]CH2
(1H), piperazine-H (1H)), 3.40 (d, J ¼ 12.5 Hz, 1H, NCH2Ar),
3.44 (d, J ¼ 12.5 Hz, 1H, NCH2Ar), 3.52 (dd, J ¼ 14.1/5.5 Hz,
1H, NCH2CH]CH2), 3.69 (ddd, J ¼ 11.7/6.3/4.7 Hz, 1H,
CH2CH2OH), 3.79 (s, 3H, ArOCH3), 3.84 (ddd, J ¼ 11.7/7.8/4.7
Hz, 1H, CH2CH2OH), 5.13–5.22 (m, 2H, NCH2CH]CH2),
5.77–5.88 (m, 1H, NCH2CH]CH2), 6.85 (d, J ¼ 8.6 Hz, 2H,
30-H4-methoxybenzyl, 50-H4-methoxybenzyl), 7.22 (d, J ¼ 8.6 Hz, 2H,
20-H4-methoxybenzyl, 60-H4-methoxybenzyl). The signal for the proton
of the OH group is not seen in the 1H NMR spectrum. IR (neat):
~n [cmꢀ1] ¼ 3377 (m br, nO–H), 2936 (m, nC–H aliph.), 1033 (m, nC–O),
813 (w, Gp-subst. arom.).
d [ppm] ¼ 1.47–1.56 (m, 1H, CH2CH2CH2OH), 1.64 (s, 3H,
NCH2CH]C(CH3)2), 1.66–1.84 (m, 3H, CH2CH2CH2OH),
1.73 (s, 3H, NCH2CH]C(CH3)2), 2.19–2.30 (m, 2H, piperazine-
H), 2.35–2.43 (m, 1H, piperazine-H), 2.51–2.58 (m, 1H, pipera-
zine-H), 2.60–2.69 (m, 2H, piperazine-H), 2.93–3.04 (m, 2H,
NCH2CH]C(CH3)2(1H), piperazine-H), 3.37–3.46 (m, 3H,
NCH2Ar, NCH2CH]C(CH3)2(1H)), 3.52–3.58 (m, 1H,
CH2CH2CH2OH), 3.60–3.67 (m, 1H, CH2CH2CH2OH), 3.79
(s, 3H, ArOCH3), 5.23–5.29 (m, 1H, NCH2CH]C(CH3)2), 6.84
(d, J ¼ 8.6 Hz, 2H, 30-H4-methoxybenzyl, 50-H4-methoxybenzyl), 7.21 (d,
J ¼ 8.6 Hz, 2H, 20-H4-methoxybenzyl, 60-H4-methoxybenzyl). The signal
1
for the proton of the OH group is not seen in the H NMR
spectrum. IR (neat): ~n [cmꢀ1] ¼ 3342 (m br, nO–H), 2933
(m, nC–H aliph.), 1035 (m, nC–O), 814 (m, Gp-subst. arom.).
5.3.6. (+)-(S)-3-[1-Allyl-4-(4-methoxybenzyl)piperazin-2-yl]
propan-1-ol (4c). According to general procedure B piper-
azinedione 8c (146 mg, 0.41 mmol) was reacted with LiAlH4
(92 mg, 2.43 mmol) in THF (40 mL) and the product was purified
by FC (2 cm, h ¼ 15 cm, CH2Cl2–methanol ¼ 9.5/0.5, V ¼ 10 mL,
Rf ¼ 0.10). Yellow oil, yield 42 mg (34%). Purity by HPLC:
method 2b: tR ¼ 29.1 min, purity 96.8%; method 2d: tR ¼ 16.7
min, purity 97.2%. [a]2D0 ¼ +8.0 (c ¼ 0.38; CH2Cl2). C18H28N2O2
(304.4). MS (EI): m/z [%] ¼ 304 (M, 6), 183 (M ꢀ CH2PhOCH3,
676 | Med. Chem. Commun., 2012, 3, 673–679
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28), 121 (CH2PhOCH3, 100). 1H NMR (CDCl3): d [ppm] ¼ 1.48–
1.58 (m, 1H, CH2CH2CH2OH), 1.64–1.79 (m, 3H,
CH2CH2CH2OH), 2.16–2.26 (m, 2H, piperazine-H), 2.33–2.41
(m, 1H, piperazine-H), 2.46–2.52 (m, 1H, piperazine-H), 2.58–
2.67 (m, 2H, piperazine-H), 2.87–2.96 (m, 2H, NCH2CH]CH2
(1H), piperazine-H (1H)), 3.39 (d, J ¼ 13.3 Hz, 1H, NCH2Ar),
3.43 (d, J ¼ 13.3 Hz, 1H, NCH2Ar), 3.49–3.59 (m, 2H,
CH2CH2CH2OH (1H), NCH2CH]CH2 (1H)), 3.60–3.66 (m,
1H, CH2CH2CH2OH), 3.79 (s, 3H, ArOCH3), 5.14–5.22 (m, 2H,
NCH2CH]CH2), 5.82–5.94 (m, 1H, NCH2CH]CH2), 6.84 (d,
J ¼ 8.6 Hz, 2H, 30-H4-methoxybenzyl, 50-H4-methoxybenzyl), 7.21 (d, J
¼ 8.6 Hz, 2H, 20-H4-methoxybenzyl, 60-H4-methoxybenzyl). A signal for
the proton of the OH group is not seen in the 1H NMR spectrum.
IR (neat): ~n [cmꢀ1] ¼ 3393 (m br, nO–H), 2935 (m, nC–H aliph.), 1034
(m, nC–O), 815 (w, Gp-subst. arom.).
1H, NCH2Ar), 4.61 (d, J ¼ 14.1 Hz, 1H, NCH2Ar), 5.04–5.10
(m, 1H, NCH2CH]C(CH3)2), 6.86 (d, J ¼ 8.6 Hz, 2H,
30-H4-methoxybenzyl, 50-H4-methoxybenzyl), 7.21 (d, J ¼ 8.6 Hz, 2H,
20-H4-methoxybenzyl, 60-H4-methoxybenzyl). IR (neat): ~n [cmꢀ1] ¼ 2932
(m, nC–H aliph.), 1735 (m, nC]O ester), 1654 (s, nC]O amide), 1031
(m, nC–O), 811 (m, Gp-subst. arom.).
5.3.9. (+)-Methyl
(S)-2-[1-allyl-4-(4-methoxybenzyl)-3,6-
dioxopiperazin-2-yl]acetate (7c).23 According to general proce-
dure A piperazinedione 5 (6.24 g, 20.4 mmol), tetrabutylammo-
nium iodide (1.50 g, 4.07 mmol), 2 M solution of sodium
hexamethyldisilazane in THF (11.2 mL, 22.4 mmol) and allyl
bromide (8.9 mL, 12.3 g, 102 mmol) were reacted in THF
(150 mL). The product was purified by FC (8 cm, cyclohexane–
ethyl acetate ¼ 1/2, 30 mL, Rf ¼ 0.20). Colorless solid, m.p. 89 ꢁC,
yield 3.64 g (52%). Purity by HPLC: method 1: tR ¼ 17.3 min,
purity 98.2%. [a]2D0 ¼ +57.1 (c ¼ 0.33; CH2Cl2). C18H22N2O5
(346.4). MS (EI): m/z [%] ¼ 346 (M, 39), 225 (M ꢀ CH2PhOCH3,
10), 121 (CH2PhOCH3, 100). 1H NMR (CDCl3): d [ppm] ¼ 2.88
(dd, J ¼ 17.2/4.7 Hz, 1H, CH2CO2CH3), 3.13 (dd, J ¼ 17.2/3.1 Hz,
1H, CH2CO2CH3), 3.60 (s, 3H, CO2CH3), 3.63 (dd, J ¼ 15.7/7.0
5.3.7. (+)-Methyl (S)-2-[1-benzyl-4-(4-methoxybenzyl)-3,6-
dioxopiperazin-2-yl]acetate (7a). According to general procedure
A piperazinedione 5 (306 mg, 1.00 mmol), tetrabutylammonium
iodide (74 mg, 0.20 mmol), 2 M solution of sodium hexame-
thyldisilazane in THF (0.55 mL, 1.10 mmol) and benzyl bromide
(0.6 mL, 854 mg, 4.99 mmol) were reacted in THF (50 mL). The
product was purified by FC (3 cm, h ¼ 15 cm, cyclohexane–ethyl
acetate 1/1, 20 mL, Rf ¼ 0.30). Colorless solid, m.p. 95 ꢁC, yield
130 mg (33%). [a]2D0 ¼ +71.9 (c ¼ 0.25; CH2Cl2). C22H24N2O5
(396.5). MS (EI): m/z [%] ¼ 396 (M, 44), 305 (M ꢀ CH2Ph, 7),
Hz, 1H, NCH2CH]CH2), 3.78 (d,
J
¼
17.2 Hz, 1H,
O]CCH2N), 3.80 (s, 3H, ArOCH3), 4.03 (d, J ¼ 17.2 Hz, 1H,
O]CCH2N), 4.23–4.27 (m, 1H, CHCH2CO2CH3), 4.43 (ddt, J ¼
15.7/5.5/1.6 Hz, 1H, NCH2CH]CH2), 4.50 (d, J ¼ 14.9 Hz, 1H,
NCH2Ar), 4.59 (d, J ¼ 14.9 Hz, 1H, NCH2Ar), 5.20–5.27 (m, 2H,
NCH2CH]CH2), 5.67–5.78 (m, 1H, NCH2CH]CH2), 6.86 (d,
J ¼ 8.6 Hz, 2H, 30-H4-methoxybenzyl, 50-H4-methoxybenzyl), 7.21 (d, J ¼
8.6 Hz, 2H, 20-H4-methoxybenzyl, 60-H4-methoxybenzyl). IR (neat): ~n
[cmꢀ1] ¼ 3080 (w, nC–H arom.), 2924 (m, nC–H aliph.), 1735
(m, nC]O ester), 1660 (s, nC]O amide).
1
275 (M ꢀ CH2PhOCH3, 5), 121 (CH2PhOCH3, 100). H NMR
(CDCl3): d [ppm] ¼ 2.81 (dd, J ¼ 17.2/4.7 Hz, 1H, CH2CO2CH3),
3.07 (dd, J ¼ 17.2/3.1 Hz, 1H, CH2CO2CH3), 3.55 (s, 3H,
CO2CH3), 3.80 (s, 3H, ArOCH3), 3.85 (d, J ¼ 17.2 Hz, 1H, O]
CCH2N),
4.09–4.15
(m,
2H,
O]CCH2N
(1H),
CHCH2CO2CH3), 4.20 (d, J ¼ 14.9 Hz, 1H, NCH2Ph), 4.43 (d,
J ¼ 14.9 Hz, 1H, NCH2Ar), 4.65 (d, J ¼ 14.9 Hz, 1H, NCH2Ar),
5.07 (d, J ¼ 14.9 Hz, 1H, NCH2Ph), 6.87 (d, J ¼ 8.6 Hz, 2H,
30-H4-methoxybenzyl, 50-H4-methoxybenzyl), 7.19–7.24 (m, 4H, 20-
5.3.10. (ꢀ)-Methyl (S)-3-[1-benzyl-4-(4-methoxybenzyl)-3,6-
dioxopiperazin-2-yl]propanoate (8a). According to general
procedure A piperazinedione 6 (680 mg, 2.12 mmol), tetrabuty-
lammonium iodide (157 mg, 0.42 mmol), 2 M solution of sodium
hexamethyldisilazane in THF (1.2 mL, 2.33 mmol) and benzyl
bromide (1.3 mL, 1.82 g, 10.6 mmol) were reacted in THF (70
mL). The product was purified by FC (4 cm, h ¼ 15 cm, cyclo-
hexane–ethyl acetate ¼ 1/1, 30 mL, Rf ¼ 0.35). Colorless oil, yield
98 mg (11%). [a]2D0 ¼ ꢀ3.1 (c ¼ 1.09; CH2Cl2). C23H26N2O5
(410.5). MS (EI): m/z [%] ¼ 410 (M, 46), 289 (M ꢀ CH2PhOCH3,
11), 121 (CH2PhOCH3, 100). 1H NMR (CDCl3): d [ppm] ¼ 1.99–
2.08 (m, 1H, CH2CH2CO2CH3), 2.17–2.27 (m, 1H,
CH2CH2CO2CH3), 2.32–2.48 (m, 2H, CH2CH2CO2CH3), 3.67
(s, 3H, CO2CH3), 3.80 (s, 3H, ArOCH3), 3.83 (d, J ¼ 17.2 Hz,
1H, O]CCH2N), 3.92–4.00 (m, 3H, O]CCH2N (1H),
CHCH2CH2CO2CH3, NCH2Ph (1H)), 4.34 (d, J ¼ 14.1 Hz, 1H,
NCH2Ar), 4.65 (d, J ¼ 14.1 Hz, 1H, NCH2Ar), 5.26 (d, J ¼ 14.9
H
4-methoxybenzyl, 60-H4-methoxybenzyl, NCH2C6H5 (2H)), 7.29–7.35
(m, 3H, NCH2C6H5). IR (neat): ~n [cmꢀ1] ¼ 3003 (w, nC–H arom.),
2952 (w, nC–H aliph.), 1734 (m, nC]O ester), 1649 (s, nC]O amide),
1030 (m, nC–O), 817 (m, Gp-subst. arom.), 731 (m, Gmono-subst. arom.).
5.3.8. (+)-Methyl (S)-2-[4-(4-methoxybenzyl)-1-(3-methylbut-
2-en-1-yl)-3,6-dioxopiperazin-2-yl]acetate (7b). According to
general procedure A piperazinedione 5 (605 mg, 1.98 mmol),
tetrabutylammonium iodide (146 mg, 0.40 mmol), 2 M solution
of sodium hexamethyldisilazane in THF (1.1 mL, 2.17 mmol)
and 1-bromo-3-methylbut-2-ene (1.16 mL, 1.47 mg, 9.88 mmol)
were reacted in THF (70 mL). The product was purified by FC
(4 cm, h ¼ 15 cm, cyclohexane–ethyl acetate ¼ 1/1, 30 mL,
Rf ¼ 0.30). Yellow oil, yield 225 mg (30%). [a]2D0 ¼ +52.0 (c ¼
0.57; CH2Cl2). C20H26N2O5 (374.4). MS (EI): m/z [%] ¼ 374
(M, 18), 253 (M ꢀ CH2PhOCH3, 87), 121 (CH2PhOCH3, 100).
1H NMR (CDCl3): d [ppm] ¼ 1.70 (s, 3H, NCH2CH]
C(CH3)2), 1.73 (s, 3H, NCH2CH]C(CH3)2), 2.83 (dd, J ¼
17.2/5.5 Hz, 1H, CH2CO2CH3), 3.13 (dd, J ¼ 17.2/3.1 Hz, 1H,
CH2CO2CH3), 3.59 (s, 3H, CO2CH3), 3.63 (dd, J ¼ 14.9/7.8
Hz, 1H, NCH2CH]C(CH3)2), 3.75 (d, J ¼ 17.2 Hz, 1H, O]
CCH2N), 3.80 (s, 3H, ArOCH3), 4.00 (d, J ¼ 17.2 Hz, 1H, O]
CCH2N), 4.22–4.26 (m, 1H, CHCH2CO2CH3), 4.40 (dd, J ¼
14.9/6.3 Hz, 1H, NCH2CH]C(CH3)2), 4.47 (d, J ¼ 14.1 Hz,
Hz, 1H, NCH2Ph), 6.87 (d, J ¼ 8.6 Hz, 2H, 30-H4-methoxybenzyl
50-H4-methoxybenzyl), 7.17 (d, J ¼ 8.6 Hz, 2H, 20-H4-methoxybenzyl
,
,
60-H4-methoxybenzyl), 7.23–7.35 (m, 5H, NCH2C6H5). IR (neat): ~n
[cmꢀ1] ¼ 2952 (m, nC–H aliph.), 1733 (m, nC]O ester), 1658 (s,
nC]O amide), 1029 (m, nC–O), 821 (w, Gp-subst. arom.), 730 (m)/700
(m, Gmono-subst. arom.).
5.3.11. (+)-Methyl (S)-3-[4-(4-methoxybenzyl)-1-(3-methyl-
but-2-en-1-yl)-3,6-dioxopiperazin-2-yl]propanoate (8b). Accord-
ing to general procedure
A piperazinedione 6 (650 mg,
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2.03 mmol), tetrabutylammonium iodide (150 mg, 0.41 mmol), 2
M solution of sodium hexamethyldisilazane in THF (1.1 mL,
2.23 mmol) and 1-bromo-3-methylbut-2-ene (1.19 mL, 1.51 mg,
10.1 mmol) were reacted in THF (70 mL). The product was
purified by FC (4 cm, h ¼ 15 cm, cyclohexane–ethyl acetate ¼ 1/
1, 30 mL, Rf ¼ 0.33). Yellow oil, yield 221 mg (28%). [a]2D0 ¼ +2.1
(c ¼ 0.70; CH2Cl2). C21H28N2O5 (388.5). MS (EI): m/z [%] ¼ 388
(M, 23), 267 (M ꢀ CH2PhOCH3, 34), 121 (CH2PhOCH3, 100).
1H NMR (CDCl3): d [ppm] ¼ 1.70 (s, 3H, NCH2CH]C(CH3)2),
1.73 (s, 3H, NCH2CH]C(CH3)2), 1.98–2.07 (m, 1H,
CH2CH2CO2CH3), 2.22–2.31 (m, 1H, CH2CH2CO2CH3), 2.32–
2.45 (m, 2H, CH2CH2CO2CH3), 3.56 (dd, J ¼ 14.9/8.6 Hz, 1H,
NCH2CH]C(CH3)2), 3.67 (s, 3H, CO2CH3), 3.76 (d, J ¼ 17.2
Hz, 1H, O]CCH2N), 3.80 (s, 3H, ArOCH3), 3.87 (d, J ¼ 17.2
The filtration was carried out with a MicroBeta FilterMate-96
Harvester (PerkinElmer). The scintillation analysis was per-
formed using Meltilex (Type A) solid scintillator (PerkinElmer).
The radioactivity bound to the filter was measured using
a MicroBeta Trilux scintillation analyzer (PerkinElmer). The
overall counting efficiency was 20%.
6.2. Membrane preparation for the s1 assay25–27
Five guinea pig brains were homogenized with the potter (500–
800 rpm, 10 up-and-down strokes) in 6 volumes of cold 0.32 M
sucrose. The suspension was centrifuged at 1200 ꢄ g for 10 min
at 4 ꢁC. The supernatant was separated and centrifuged at
23 500 ꢄ 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
Hz, 1H, O]CCH2N), 4.02 (dd,
J
¼
8.6/3.9 Hz, 1H,
ꢁ
23 500 ꢄ g (20 min, 4 C). This procedure was repeated twice.
CHCH2CH2CO2CH3), 4.38 (d, J ¼ 14.1 Hz, 1H, NCH2Ar), 4.47
(dd, J ¼ 14.9/6.3 Hz, 1H, NCH2CH]C(CH3)2), 4.61 (d, J ¼ 14.1
Hz, 1H, NCH2Ar), 5.07–5.13 (m, 1H, NCH2CH]C(CH3)2),
6.86 (d, J ¼ 8.6 Hz, 2H, 30-H4-methoxybenzyl, 50-H4-methoxybenzyl),
7.18 (d, J ¼ 8.6 Hz, 2H, 20-H4-methoxybenzyl, 60-H4-methoxybenzyl). IR
(neat): ~n [cmꢀ1] ¼ 2933 (m, nC–H aliph.), 1734 (m, nC]O ester), 1654
(s, nC]O amide), 1031 (m, nC–O), 821 (w, Gp-subst. arom.).
The final pellet was resuspended in 5–6 volumes of buffer, the
protein concentration was determined according to the method
of Bradford28 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 per mL.
6.3. Protocol of the s1 assay25–27
5.3.12. (+)-Methyl
(S)-3-[1-allyl-4-(4-methoxybenzyl)-3,6-
The test was performed with the radioligand [3H]-(+)-pentazo-
cine (42.5 Ci mmolꢀ1; PerkinElmer). The thawed membrane
preparation (about 75 mg of the protein) was incubated with
dioxopiperazin-2-yl]propanoate (8c).24 According to general
procedure A piperazinedione 6 (2.80 g, 8.74 mmol), tetrabuty-
lammonium iodide (0.65 g, 1.75 mmol), 2 M solution of sodium
hexamethyldisilazane in THF (4.81 mL, 9.61 mmol) and allyl
bromide (3.8 mL, 5.29 g, 43.7 mmol) were reacted in THF
(80 mL). The product was purified by FC (8 cm, h ¼ 15 cm,
cyclohexane–ethyl acetate ¼ 1/2, 30 mL, Rf ¼ 0.29). Yellow oil,
yield 1.67 g (53%). Purity by HPLC: method 1: tR ¼ 17.9 min,
purity 98.1%. [a]2D0 ¼ +20.2 (c ¼ 11.3; CH2Cl2). C19H24N2O5
(360.4). MS (EI): m/z [%] ¼ 360 (M, 65), 239 (M ꢀ CH2PhOCH3,
54), 121 (CH2PhOCH3, 100). 1H NMR (CDCl3): d [ppm] ¼ 1.97–
2.07 (m, 1H, CH2CH2CO2CH3), 2.21–2.30 (m, 1H,
CH2CH2CO2CH3), 2.34–2.50 (m, 2H, CH2CH2CO2CH3), 3.51
(dd, J ¼ 15.7/7.8 Hz, 1H, NCH2CH]CH2), 3.68 (s, 3H,
CO2CH3), 3.78 (d, J ¼ 17.2 Hz, 1H, O]CCH2N), 3.80 (s, 3H,
ArOCH3), 3.90 (d, J ¼ 17.2 Hz, 1H, O]CCH2N), 4.03 (dd, J ¼
8.6/3.9 Hz, 1H, CHCH2CH2CO2CH3), 4.42 (d, J ¼ 14.9 Hz, 1H,
NCH2Ar), 4.52–4.61 (m, 2H, NCH2Ar (1H), NCH2CH]CH2
(1H)), 5.21–5.27 (m, 2H, NCH2CH]CH2), 5.68–5.79 (m, 1H,
NCH2CH]CH2), 6.86 (d, J ¼ 8.6 Hz, 2H, 30-H4-methoxybenzyl, 50-
various concentrations of test compounds,
2
nM [3H]-
(+)-pentazocine, and buffer (50 mM Tris, pH 7.4) in a total
volume of 200 mL for 180 min at 37 ꢁC. The incubation was
terminated by rapid filtration through the presoaked filtermats
by using the cell harvester. After washing each wellꢁfive times
with 300 mL of water, the filtermats were dried at 95 C. Subse-
quently, the solid scintillator was put on the filtermat and melted
at 95 ꢁC. After 5 min, the solid scintillator was allowed to solidify
at rt. The bound radioactivity trapped on the filters was counted
in the scintillation analyzer. The non-specific binding was
determined with 10 mM unlabeled (+)-pentazocine. The Kd-value
of the radioligand [3H]-(+)-pentazocine is 2.9 nM.29
6.4. Membrane preparation for the s2 assay25–27
Two rat livers were cut into small pieces and homogenized with
a potter (500–800 rpm, 10 up-and-down strokes) in 6 volumes of
cold 0.32 M sucrose. The suspension was centrifuged at 1200 ꢄ g
ꢁ
H
4-methoxybenzyl), 7.18 (d, J ¼ 8.6 Hz, 2H, 20-H4-methoxybenzyl
,
for 10 min at 4 C. The supernatant was separated and centri-
60-H4-methoxybenzyl). IR (neat): ~n [cmꢀ1] ¼ 2952 (w, nC–H aliph.),
1733 (m, nC]O ester), 1658 (s, nC]O amide), 1032 (m, nC–O), 820
(w, Gp-subst. arom.).
fuged at 31 000 ꢄ g for 20 min at 4 ꢁC. The pellet was resus-
pended in buffer (50 mM Tris, pH 8.0) and incubated at rt for 30
min. After the incubation, the suspension was centrifuged again
at 31 000 ꢄ g for 20 min at 4 ꢁC. The final pellet was resuspended
in buffer, the protein concentration was determined according to
the method of Bradford28 using bovine serum albumin as stan-
6. Experimental, receptor binding studies
ꢁ
dard, and subsequently the preparation was frozen (ꢀ80 C) in
6.1. Materials and general procedures
1.5 mL portions containing about 2 mg protein per mL.
Guinea pig brains and rat livers were commercially available
(Harlan-Winkelmann, Germany). Homogenizer: Elvehjem
Potter (B. Braun Biotech International). Centrifuge: High-speed
cooling centrifuge model Sorvall RC-5C plus (Thermo Fin-
nigan). Filter: Printed Filtermat Type A (PerkinElmer), pre-
soaked in 0.5% aqueous polyethylenimine for 2 h at rt before use.
6.5. Protocol of the s2 assay25–27
The test was performed with the radioligand [3H]-di-o-tolylgua-
nidine (50 Ci mmolꢀ1; ARC). The thawed membrane preparation
(about 100 mg of the protein) was incubated with various
678 | Med. Chem. Commun., 2012, 3, 673–679
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concentrations of test compounds, 3 nM [3H]-di-o-tolylguani-
dine, 500 nM (+)-pentazocine and buffer (50 mM Tris, pH 8.0) in
a total volume of 200 mL for 180 min at rt. 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 put on the filtermat and melted at 95 C.
After 5 min, the solid scintillator was allowed to solidify at rt.
The bound radioactivity trapped on the filters was counted in the
scintillation analyzer. The non-specific binding was determined
with 10 mM unlabeled ditolylguanidine. The Kd-value of the
radioligand [3H]-ditolylguanidine is 17.9 nM.30
7 K. Hashimoto and K. Ishiwata, Curr. Pharm. Des., 2006, 12, 3857–
3876.
8 J. Simony-Lafontaine, M. Esslomani, E. Bribes, S. Gougou,
N. Lequeux, R. Lavail, J. Grenier, A. Kramar and P. Casellas, Br.
J. Cancer, 2000, 82, 1958–1966.
€
€
9 E. Rack, R. Frohlich, D. Schepmann and B. Wunsch, Bioorg. Med.
Chem., 2011, 19, 3141–3151.
€
10 B. Wunsch, Curr. Pharm. Des., 2012, 18, 930–937.
11 R. A. Glennon, Mini-Rev. Med. Chem., 2005, 5, 927–940.
12 R. A. Glennon, S. Y. Ablordeppey, A. M. Ismaiel, M. B. El-
Ashmawy, J. B. Fischer and K. B. Howie, J. Med. Chem., 1994, 37,
1214–1219.
13 S. Y. Ablordeppey, J. B. Fischer, H. Law and R. A. Glennon, Bioorg.
Med. Chem., 2002, 10, 2759–2765.
14 C. Laggner, C. Schieferer, B. Fiechtner, G. Poles, R. D. Hoffmann,
H. Glossmann, T. Langer and E. F. Moebius, J. Med. Chem., 2005,
48, 4754–4764.
6.6. Data analysis
15 D. Zampieri, M. G. Mamolo, E. Laurini, C. Florio, C. Zanette,
M. Fermeglia, P. Posocco, M. S. Paneni, S. Pricl and L. Vio, J.
Med. Chem., 2009, 52, 5380–5393.
Usually, all experiments were carried out in triplicates using
standard 96-well-multiplates (Diagonal). The IC50-values were
determined in competition experiments with six concentrations
of the test compounds and were calculated with the program
GraphPad Prismꢀ 3.0 (GraphPad Software) by non-linear
regression analysis. The Ki-values were calculated according to
Cheng and Prusoff.31 The Ki-values of potent compounds are
€
€
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given as mean values
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ꢂ
SEM from three independent
€
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Acknowledgements
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This work was performed within the International Research
Training Group ‘‘Complex Functional Systems in Chemistry:
Design, Synthesis and Applications’’ in collaboration with the
University of Nagoya. Financial support of this IRTG by the
Deutsche Forschungsgemeinschaft is gratefully acknowledged.
€
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