Synthesis and?receptor binding studies of?3-substituted piperazine derivatives

Article abstract of DOI:10.1016/j.ejmech.2005.09.004

In order to find novel receptor ligands various substituents were introduced into the side chain in position 3 of the piperazine 5. During nucleophilic substitution of the hydroxy group of 5 aziridinium ions were formed, resulting in rearranged 1,4-diazep

Full text of DOI:10.1016/j.ejmech.2005.09.004

European Journal of Medicinal Chemistry 41 (2006) 387–396
http://france.elsevier.com/direct/ejmech
Original article
Synthesis and receptor binding studies of
3-substituted piperazine derivatives
Stephan Bedürftig, Bernhard Wünsch *
Institut für Pharmazeutische und Medizinische Chemie, Hittorfstraße 58-62, 48149 Münster, Germany
Received 5 April 2004; received in revised form 1 September 2005; accepted 8 September 2005
Available online 30 January 2006
Abstract
In order to find novel receptor ligands various substituents were introduced into the side chain in position 3 of the piperazine 5. During
nucleophilic substitution of the hydroxy group of 5 aziridinium ions were formed, resulting in rearranged 1,4-diazepanes and piperazines as side
products. 1,2-anellated piperazines 15, 18 and 19 were prepared by hydrogenation of the α,β-unsaturated ester 13 and by condensation of the
primary amine 16b with formaldehyde, respectively. Receptor binding studies with radioligands revealed that the phenylacetamide 17b interacts
with moderate affinity (Ki = 181 nM) and considerable selectivity with σ1 receptors.
© 2006 Elsevier SAS. All rights reserved.
Keywords: 3-Substituted piperazines; 1,2-Anellated piperazines; Aziridinium ions; σ Receptor ligands
1. Introduction
In this article we report on the synthesis and receptor affi-
nities (NMDA, κ-opioid, μ-opioid, σ1, σ2,) of piperazine deri-
vatives with various substituents in position 3. Modification of
the hydroxymethyl substituent of the piperazine ring is inves-
tigated and special aspects of the transformation are discussed.
With respect to receptor interactions introduction of nitrogen
containing substituents is of particular interest. Pharmacophoric
elements, which are crucial for high σ and κ receptor affinity
(compare compounds 1, 3, and 4 of Scheme 1), are especially
considered.
The piperazine ring represents the core structure of several
pharmacologically active compounds. In particular piperazine
derivatives bearing substituents in position 3 can strongly inter-
act with various receptors within the central nervous system. In
Fig. 1 some examples of piperazine derivatives with high re-
ceptor affinity are given.
The (pyrrolidinylalkyl) substituted piperazines 1 are among
the most active and selective κ-opioid receptor agonists [1,2].
The competitive NMDA receptor antagonist 2 is derived from
the lead compound (R)-2-aminoadipic acid [3]. Several 1,4-di-
substituted piperazine derivatives display high affinity to σ re-
ceptors, e.g. the 1-butyl-4-(3,4-dichlorophenethyl)piperazine
(3) binds with high affinity at σ1 receptors (Ki = 0.55 nM)
[4]. Recently we have described the synthesis and σ receptor
affinity of 2-(hydroxymethyl) substituted piperazine deriva-
tives. We found that an additional aromatic element in the N-
4 residue is favorable for high σ1 receptor affinity. As an ex-
ample the phenethyl derivative 4 reveals a Ki–value of
36.8 nM [5].
2. Chemistry
Starting from the proteinogenic amino acid (S)-serine the
ester 5 was prepared in seven steps including chloroacetylation
of serine methyl ester, N/O-acetal formation with benzaldehyde
dimethyl acetal, piperazine ring formation via an azide, and
reductive cleavage of the oxazolidine ring with LiAlH4 [5]. In
order to introduce further substituents in the side chain the OH-
group of 5 was reacted with methanesulfonyl chloride and
triethylamine to obtain the sulfonate 6, an activated intermedi-
ate for following nucleophilic substitutions. After stirring the
reaction mixture for 15 min at 0 °C thin layer chromatography
(TLC) showed complete transformation. However, after work
up with water only the chloride 7 could be isolated. In TLC the
* Corresponding author. Tel.: +49 251 833 3311; fax: +49 251 833 2144.
E-mail address: wuensch@uni-muenster.de (B. Wünsch).
0223-5234/$ - see front matter © 2006 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2005.09.004

388
S. Bedürftig, B. Wünsch / European Journal of Medicinal Chemistry 41 (2006) 387–396
Fig. 1.
Rf values of the originally formed compound (6) and the iso-
lated product (7) were not identical. We assume that the origin-
ally formed mesylate 6 reacted further with chloride via inter-
mediate aziridinium ion 11a (see Scheme 2) to yield the
chloride 7. The existence of similar aziridinium ions has been
described in literature [6].
In order to avoid the formation of chloride 7 nucleophilic
substitutions were performed immediately after preparation of
the mesylate 6. Reaction of the alcohol 5 with methanesulfonyl
chloride and subsequently with KCN in DMSO at room tem-
perature provided the cyanomethyl derivative 8a in 54% yield.
However, isolation of the rearranged 1,4-diazepane derivative
9a (11% yield) demonstrated that the formation of the aziridi-
nium intermediate 11a had not been completely suppressed. In
addition to these main products careful chromatographic se-
paration provided the isomeric nitrile 10a (0.4%) and the chlor-
ide 7 (1.0%). The structures of the isomeric nitriles 8a, 9a, and
10a and the chloride 7 were unequivocally proven by NMR
spectroscopy.
Scheme 1.
The formation of the isomeric nitriles 8a–10a is explained
with intermediate aziridinium ions. Nucleophilic attack of cya-
nide at the aziridinium corners of 11a results in the nitriles 8a
and 9a. A preceding isomerization of the aziridinium ion 11a
into the regioisomeric aziridinium ion 11b and subsequent cy-
anide attack is responsible for the generation of the nitriles 9a
and 10a. We assume that the isomerization of 11a into 11b
could occur via nucleophilic attack of chloride to form the se-
ven-membered chloro derivative 9d. Subsequent intramolecu-
lar SN2 reaction should provide the isomeric aziridinium ions
11a and 11b. Ring opening of the aziridinium ion 11a to give a
Scheme 2.
seven-membered carbenium ion is unlikely since the isolated
products 8a, 9a, and 10a show optical activity.
The analogous reaction of the alcohol 5 with methanesulfo-
nyl chloride and subsequently with NaN3 also resulted in re-
gioisomeric products. Purification of the reaction mixture by
flash chromatography (FC) led to the expected (azidomethyl)
piperazine 8b (65%) and the 6-azidodiazepane 9b (11%). In
spite of intensive search the third regioisomeric azide 10b
was not detected.

S. Bedürftig, B. Wünsch / European Journal of Medicinal Chemistry 41 (2006) 387–396
389
In order to study the temperature effect on the nucleophilic
substitution reactions of the mesylate 6 with cyanide and azide
transformations were performed at 20 and 50 °C and the com-
hydrogenation of 13 with the catalyst Pd/C at elevated H2 pres-
sure (4 bar) led to saturation of the double bond and simulta-
neously hydrogenolysis of the benzyl group providing a γ-ami-
noester which spontaneously cyclized to yield the γ-lactam 15.
Analogous transformations have recently been described in [9,
10] (Scheme 3).
Depending on the reaction conditions hydrogenation of the
azide 8b yielded the benzylated primary amine 16a (H2, Pd/C,
1 bar, 0.5 h, 20 °C) or the debenzylated primary amine 16b
(H2, Pd(OH)2/C, 4 bar, 8 h, 20 °C) in 96% and 97% yield,
respectively (Scheme 4).
Great efforts were undertaken to synthesize 1,3-bridged pi-
perazine derivatives. However, all attempts failed to obtain bi-
cyclic lactones or lactams by cyclization of the hydroxyester 5
or the aminoesters 8c, 16a or 16b. Moreover, the projected
Dieckmann cyclization of the diesters 12 and 14 failed to give
bicyclic derivatives.
The secondary amine 8c and the benzylated primary amine
16a were used for the introduction of the κ- [11] and σ-phar-
macophoric [12] dichlorophenylacetyl residue. Acylation with
(dichlorophenyl)acetyl chloride (DCPA-Cl) provided the sec-
ondary and tertiary amides 17a and 17b, respectively. The de-
benzylated primary amine 16b reacted with formaldehyde to
afford the aminal 18, which was acylated with DCPA-Cl to
yield the amide 19, which is regarded as conformationally con-
strained analogue of the methylated amide 17b.
1
position of the crude product mixtures was analyzed by H
NMR spectroscopy. These experiments demonstrated that the
amounts of rearranged products were enhanced with increasing
temperature: Performing the substitution reactions with cyanide
or azide at 20 °C resulted in product ratios of 8a/9a/
10a = 84.5:15.0:0.5 and 8b/9b = 77:23, respectively. At 50 °C
the product ratios amounted 8a/9a/10a = 67.6:22.7:9.7 and 8b/
9b = 70:30. These results lead to the conclusion that lower
temperatures might suppress rearrangement and increase the
yields of the desired substitution products 8a and 8b. However,
the high melting point of the solvent DMSO (18.5 °C) prevents
further reduction of the temperature. Therefore a solvent mix-
ture THF/DMSO 3:1 was used for the 0 °C substitution reac-
tions. After a reaction time of 44 h at 0 °C the 1H NMR spectra
revealed large amounts of starting mesylate 6 and chloride 7
which impede the exact interpretation of the spectra. Ob-
viously, a reaction temperature of 20 °C represents the best
compromise between selectivity and reactivity.
The nucleophilic substitution of the alcohol 5 with methyla-
mine was performed according to the same procedure affording
the methylamine 8c in 52% yield. TLC showed additional
spots which might represent rearranged products 9c and/or
10c. Because of strong tailing it was not possible to separate
and analyze the corresponding compounds (Scheme 1).
The nitrile 8a was transformed into the diester 12 with etha-
nol and H2SO4 [7]. The synthesis of the homologous diester 14
started with the alcohol 5. Combination of a Swern oxidation
[8] (oxalyl chloride, DMSO) with a subsequent Wittig reaction
using the stabilized phosphor ylide Ph3P=CH–CO2Et provided
the α,β-unsaturated ester 13 as a mixture of (E) and (Z) isomers
(90:10). The hydrogenation of the α,β-unsaturated ester 13 was
controlled by the catalyst: Hydrogenation using Raney Nickel
and 1 bar H2 predominantly furnished the propionic acid ester
14 (64%) and only small amounts of the γ-lactam 15; whereas
Scheme 3.
Scheme 4.

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S. Bedürftig, B. Wünsch / European Journal of Medicinal Chemistry 41 (2006) 387–396
3. Receptor binding studies
viously, the most σ1 active compound 17b displays consider-
able selectivity versus σ2 receptors.
The affinities of the 3-substituted and 1,2-anellated pipera-
zine derivatives 15, 17a, 17b, 18 and 19 towards NMDA, κ-
opioid, μ-opioid, σ1 and σ2 receptors were determined in recep-
tor binding studies using radioligands with high affinity and
selectivity to the corresponding receptors. The affinity to the
phencyclidine binding site of the NMDA receptor was
measured with tritium labeled (+)-MK-801. [3H]-U-69593 and
[3H]-DAMGO were employed for the determination of κ-
opioid and μ-opioid receptor affinity. The σ receptors were la-
beled with [3H]-(+)-pentazocine (σ1) and [3H]-ditolylguanidine
in the presence of non-tritiated (+)-pentazocine (σ2). Membrane
preparations from pig brain cortex were employed in the
NMDA assay. In the κ-opioid, μ-opioid and σ1 assay homoge-
nates of guinea pig brains were used as receptor material.
Homogenates of rat liver served as source for σ2 receptors in
the σ2 assay [5,13].
5. Conclusion
Within this series of compounds the phenylacetamide 17b
reveals considerable affinity towards σ1 receptors, which is in
the range of the σ1 receptor affinity of ditolylguanidine (Ki
= 164 nM). Despite the fact that 17b contains the σ1- and κ-
pharmacophoric (dichlorophenyl)acetamide substructure affi-
nity towards κ receptors was not found indicating high σ1 re-
ceptor selectivity with regard to the investigated receptor sys-
tems. 17b represents a monoacylated 1,2-diamine. It is known,
that the stereochemistry of analogously substituted 1,2-dia-
mines (compare compounds 1, 3, and 4 in Scheme 1) deter-
mines, whether a compound reacts with σ1 or κ receptors
[12]. Therefore, it might be possible that the (R)-configuration
contributes to the σ1 receptor selectivity of 17b. Further inves-
tigations will be directed to improve the σ1 receptor affinity of
17b and to keep its selectivity versus σ2, κ and other receptors.
At first the receptor interaction of the test compounds was
screened with 1 and 10 μM test compound concentrations.
Only when considerable inhibition of the radioligand binding
was observed at a concentration of 10 μM the exact Ki-values
were determined.
6. Experimental
6.1. Chemistry, general
4. Results and discussion
Unless otherwise noted, moisture sensitive reactions were
conducted under dry nitrogen. – THF was distilled from so-
dium/benzophenone ketyl prior to use. Petroleum ether used re-
fers to the fraction boiling at 40–60 °C. – TLC: silica gel 60 F254
plates (Merck). – FC [14]: silica gel 60, 0.040–0.063 mm
(Merck); parentheses include: diameter of the column (cm), elu-
ent, fraction size (ml), Rf. – Optical rotation: Polarimeter 241
(Perkin–Elmer); 1.0 dm tube; concentration c (g/100 ml); tem-
perature 20 °C. – Elemental analyses: Vario EL (Elementarana-
lysesysteme GmbH). – MS: MAT 312, MAT 8200, MAT 44, and
TSQ 7000 (Finnigan); EI = electron impact, CI = chemical ioni-
zation. – High resolution MS (HRMS): MAT 8200 (Finnigan). –
IR: IR spectrophotometer 1605 FT-IR (Perkin–Elmer).
In Table 1 the results of the receptor binding studies are
summarized.
It is shown that the test compounds do not interact signifi-
cantly with the phencyclidine binding site of the NMDA recep-
tor and with κ- and μ-opioid receptors. However, considerable
σ1 receptor affinity was found for the ligands 17a, 17b and 19
containing the σ1- and κ-pharmacophoric (dichlorophenyl)acet-
amide substructure. The Ki value of the secondary amide 17a
is in the low micromolar range (Ki = 1341 nM). Methylation of
the secondary amide led to a 10-fold increase of σ1 receptor
affinity (17b: Ki = 181 nM). However, cyclization of the N-
methyl derivative 17b to the imidazolidine derivative 19 re-
duced the interaction with σ1 receptors. The loss of the benzyl
moiety (second aromatic system) might be responsible for the
diminished σ1 receptor affinity of 19. At a concentration of
10 μM all test compounds were inactive at σ2 receptors. Ob-
1
(br = broad, m = medium, s = strong). – H NMR (300 MHz),
13C NMR (75 MHz): Unity 300 FT NMR spectrometer (Varian),
δ in ppm related to tetramethylsilane, coupling constants are gi-
1
ven with 0.5 Hz resolution; the assignments of 13C and of H
NMR signals were supported by 2D NMR techniques.
Table 1
Inhibition of radioligand binding at a test compound concentration of 10 μM
Compound
NMDA
κ
μ
σ1
σ2
[(+)-MK-801]
(U-69593)
0%
(DAMGO) [(+)-Pentazocine]
Ditolylguanidine
14%
15
4%
22%
38%
n.d.
24%
15%
15%
17a
17b
18
0%
27%
n.d.
2%
Ki = 1341 nM 241 nM (n = 3)
Ki = 181 nM 12 nM (n = 3)
14%
14%
n.d.
53%
21%
46%
19
8%
Ki = 907 nM 93 nM (n = 3)
19%
41%
Haloperidol
Ditolylguanidine
BMY 14802
–
–
–
–
–
–
–
–
–
Ki = 2.20 nM 0.31 nM (n = 3) [13] Ki = 34.2 nM 2.3 nM (n = 3) [13]
Ki = 164 nM 47 nM (n = 3) [13]
Ki = 265 nM 32 nM (n = 3) [13]
Ki = 63.9 nM 10.8 nM (n = 3) [13]
Ki = 391 nM 63 nM (n = 3) [13]

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391
6.2. (–)-Ethyl 2-[(3R)-4-benzyl-3-(chloromethyl)piperazin-1-yl]
acetate (7), (–)-Ethyl 2-[(3S)-4-benzyl-3-(cyanomethyl)
piperazin-1-yl]acetate (8a), (+)-Ethyl 2-[(6R)-4-benzyl-6-
cyano-1,4-diazepan-1-yl]acetate (9a) and (+)-Ethyl 2-[(2R)-4-
benzyl-2-(cyanomethyl)piperazin-1-yl]acetate (10a)
J = 17.1 Hz, 1 H, NCH2CO2Et), 3.46 (d, J = 13.4 Hz, 1 H, PhC
H2N), 3.80 (d, J = 13.2 Hz, 1 H, PhCH2N), 4.19 (q, J = 7.1 Hz,
2 H, OCH2CH3), 7.26–7.36 (m, 5 H, arom).- 13C NMR
(CDCl3): δ = 14.2 (1 C, OCH2CH3), 16.2 (1 C, CH2CN),
48.2 (1 C, C-5 or C-6), 52.4 (1 C, C-6 or C-5), 55.6 (1 C, C-
3), 56.3 (1 C, C-2), 58.3 (1 C, PhCH2N), 59.1 (1 C, N
CH2CO2Et), 60.7 (1 C, OCH2CH3), 118.5 (1 C, CN), 127.3
(1 C, C-4 arom), 128.4 (2 C, C-3 and C-5 arom), 128.7 (2 C,
C-2 and C-6 arom), 137.8 (1 C, C-1 arom), 170.1 (1 C,
CO2Et).
9a (Rf = 0.37): Pale yellow oil, yield 43 mg (11 %), [α]589
= +5.5 (c = 0.145, CH2Cl2).- C17H23N3O2 (301.39).- MS (EI):
m/z (%) = 301 (M, 26), 228 (M –CO2Et, 45), 210 (M – benzyl,
13), 91 (benzyl, 100).- IR (film): n (cm−1) = 2924 (m, C–H),
2844 (m, C–H), 2268 (m, C≡N), 1732 (s, C=O), 740 and 700
(m, aryl-C–H).- 1H NMR (CDCl3): δ = 1.28 (t, J = 7.2 Hz, 3 H,
OCH2CH3), 2.69–2.79 (m, 2 H, 2-H and 3-H), 2.81–2.96 (m, 3
H, 2-H, 3-H, and 6-H), 2.98–3.09 (m, 2 H, 5-H or 7-H), 3.18
(dd, J = 14.0/6.6 Hz, 1 H, 5-H or 7-H), 3.27 (dd, J = 14.0/
5.2 Hz, 1 H, 5-H or 7-H), 3.48 (d, J = 17.5 Hz, 1 H,
Triethylamine (0.35 ml, 2.5 mmol) and methanesulfonyl
chloride (0.15 ml, 1.5 mmol) were added to a cooled (0 °C)
solution of 5 [5] (0.367 g, 1.25 mmol) in CH2Cl2 (30 ml).
The mixture was stirred at 0 °C for 15 min. The organic layer
was washed with water (2 × 10 ml), dried (Na2SO4) and con-
centrated in vacuo. The residue was dissolved in DMSO (5 ml)
and potassium cyanide (0.817 g, 12.5 mmol) was added. The
mixture was stirred for 24 h at room temperature. Then a satu-
rated solution of NaHCO3 (5 ml), water (10 ml) and diethyl
ether (20 ml) were added and the organic layer was separated.
The DMSO/water-layer was extracted with diethyl ether
(5 × 10 ml). The organic layers were combined, washed with
brine (30 ml), dried (Na2SO4) and evaporated in vacuo. Purifi-
cation of the residue by FC (3 cm, petroleum ether/ethyl acet-
ate 1:1, 5 ml) afforded 7, 8a, 9a and 10a, as well as a mixture
of 8a/10a (yield 41 mg, 11%).
NCH2CO2Et), 3.55 (d, J = 17.3 Hz, 1 H, NCH2CO2Et), 3.76 (s,
2 H, PhCH2N), 4.18 (q, J = 7.1 Hz, 2 H, OCH2CH3), 7.23–
7.36 (m, 5 H, arom).
7 (Rf = 0.41): Colorless oil, yield 4 mg (1.0%), [α]589 = –
12.9 (c = 0.605, CH2Cl2).- C16H23ClN2O2 (310.82) calcd. C
61.83 H 7.46 N 9.01 found C 61.14 H 7.86 N 9.37.- MS
(EI): m/z (%) = 312/310 (M, 1/4), 275 (M – Cl, 10), 239/237
(M –CO2Et, 4/12), 91 (benzyl, 100).- MS (CI): m/z (%) = 313/
311 (MH+, 37/100), 275 (M – Cl, 21).- IR (film): n (cm−1)
= 2939 (m, C–H), 2817 (m, C–H), 1748 (s, C=O), 834 (m,
C–Cl), 736 and 699 (m, aryl-C–H).- 1H NMR (CDCl3):
δ = 1.27 (t, J = 7.2 Hz, 3 H, OCH2CH3), 2.37–2.50 (m, 2 H,
5-H and 6-H), 2.61–2.76 (m, 3 H, 2-H, 5-H and 6-H), 2.78–
2.89 (m, 2 H, 2-H and 3-H), 3.17 (d, J = 16.4 Hz, 1 H, NC
H2CO2Et), 3.23 (d, J = 16.5 Hz, 1 H, NCH2CO2Et), 3.45 (d,
J = 13.4 Hz, 1 H, PhCH2N), 3.70 (dd, J = 11.0/2.8 Hz, 1 H, C
H2Cl), 3.87 (dd, J = 11.3/7.7 Hz, 1 H, CH2Cl), 3.93 (d,
J = 13.7 Hz, 1 H, PhCH2N), 4.18 (q, J = 7.1 Hz, 2 H, OC
H2CH3), 7.21–7.34 (m, 5 H, arom).- 13C NMR (CDCl3):
δ = 14.2 (1 C, OCH2CH3), 42.5 (1 C, CH2Cl), 49.2 (1 C, C-5
or C-6), 52.6 (1 C, C-6 or C-5), 55.1 (1 C, C-2), 58.0 (1 C, Ph
CH2N), 59.3 (1 C, NCH2CO2Et), 60.2 (1 C, C-3), 60.6 (1 C, O
CH2CH3), 127.1 (1 C, C-4 arom), 128.3 (2 C, C-3 and C-5
arom), 128.8 (2 C, C-2 and C-6 arom), 138.2 (1 C, C-1 arom),
170.2 (1 C, CO2Et).
10a (Rf = 0.26): Pale yellow oil, yield 2 mg (0.4%), [α]589
= +3.2 (c = 0.335, CH2Cl2).- C17H23N3O2 (301.39).- MS (EI):
m/z (%) = 301 (M, 36), 261 (M – CH2CN, 33), 228 (M
–CO2Et, 67), 210 (M – benzyl, 5), 170 (M – benzyl. –
CH2CN, 59), 91 (benzyl, 100).- IR (film): n (cm−1) = 2936
(m, C–H), 2819 (m, C–H), 2246 (m, C≡N), 1732 (s, C=O),
1
742 and 700 (m, aryl-C–H).- H NMR (CDCl3): δ = 1.28 (t,
J = 7.0 Hz, 3 H, OCH2CH3), 2.41–2.48 (m, 2 H, 3-H and 5-
H or 6-H), 2.51–2.56 (m, 2 H, CH2CN and 5-H or 6-H), 2.68
(dd, J = 11.3/2.5 Hz, 1 H, 3-H), 2.73–2.82 (m, 3 H, CH2CN, 5-
H and 6-H), 3.15–3.22 (m, 1 H, 2-H), 3.34 (d, J = 16.5 Hz, 1
H, NCH2CO2Et), 3.44 (d, J = 16.4 Hz, 1 H, NCH2CO2Et),
3.52 (s, 2 H, PhCH2N), 4.18 (q, J = 7.2 Hz, 2 H, OCH2CH3),
7.25–7.35 (m, 5 H, arom).- 13C NMR (CDCl3): δ = 14.2 (1 C,
OCH2CH3), 17.4 (1 C, CH2CN), 49.7 (1 C, C-5 or C-6), 52.5
(1 C, C-6 or C-5), 54.9 (1 C, C-2), 55.3 (1 C, NCH2CO2Et),
56.9 (1 C, C-3), 60.8 (1 C, OCH2CH3), 62.6 (1 C, PhCH2N),
118.1 (1 C, CN), 127.2 (1 C, C-4 arom), 128.3 (2 C, C-3 and
C-5 arom), 128.9 (2 C, C-2 and C-6 arom), 137.7 (1 C, C-1
arom), 170.0 (1 C, CO2Et).
8a (Rf = 0.24): Pale yellow oil, yield 0.206 g (54 %), [α]589
= –5.8 (c = 0.595, CH2Cl2).- C17H23N3O2 (301.39) calcd. C
67.75 H 7.69 N 13.94 found C 68.41 H 7.25 N 13.73.- HRMS:
Calcd. 301.1790, found 301.1798 (+ 0.8 ppm).- MS (EI): m/z
(%) = 301 (M, 15), 261 (M – CH2CN, 56), 228 (M –CO2Et,
54), 170 (M – benzyl. – CH2CN, 90), 91 (benzyl, 100).- IR
(film): n (cm−1) = 2938 (m, C–H), 2818 (m, C–H), 2245 (m,
6.3. (–)-Ethyl 2-[(3R)-3-(azidomethyl)-4-benzylpiperazin-1-yl]
acetate (8b) and (+)-ethyl 2-[(6R)-6-azido-4-benzyl-1,4-
diazepan-1-yl]acetate (9b)
Triethylamine (1.03 ml, 7.42 mmol) and methanesulfonyl
chloride (0.43 ml, 4.45 mmol) were added to a cooled (0 °C)
solution of 5 (1.085 g, 3.71 mmol) in CH2Cl2 (100 ml). The
mixture was stirred at 0 °C for 15 min. The organic layer was
washed with water (2 × 30 ml), dried (Na2SO4) and concen-
trated in vacuo. The residue was dissolved in DMSO (30 ml)
and sodium azide (1.207 g, 18.55 mmol) was added. The sus-
pension was stirred for 22 h at room temperature. Then water
(50 ml) and diethyl ether (70 ml) were added and the organic
1
C≡N), 1741 (s, C=O), 739 and 699 (m, aryl-C–H).- H NMR
(CDCl3): δ = 1.29 (t, J = 7.2 Hz, 3 H, OCH2CH3), 2.45 (ddd,
J = 11.0/5.3/3.2 Hz, 1 H, 5-H or 6-H), 2.54–2.71 (m, 5 H, 2-H,
5-H, 6-H and CH2CN), 2.80 (dd, J = 11.0/3.1 Hz, 1 H, 2-H),
2.91 (dd, J = 16.7/8.1 Hz, 1 H, CH2CN), 3.01–3.07 (m, 1 H, 3-
H), 3.19 (d, J = 17.1 Hz, 1 H, NCH2CO2Et), 3.24 (d,

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layer was separated. The DMSO/water-layer was extracted
with diethyl ether (5 × 30 ml). The organic layers were com-
bined, washed with brine (100 ml), dried (Na2SO4) and evapo-
rated in vacuo. Purification of the residue by FC (4 cm, petro-
leum ether/ethyl acetate 3:1, 20 ml) afforded 8b and 9b.
8b (Rf = 0.14): Colorless oil, yield 0.767 g (65%), [α]589 = –
3.2 (c = 0.445, CH2Cl2).- C16H23N5O2 (317.39) calcd. C 60.55
H 7.30 N 22.07 found C 60.63 H 7.33 N 21.76.- MS (EI): m/z
(%) = 275 (M – N3, 3), 261 (M – CH2N3, 81), 170 (M – benzyl
– CH2N3, 100).- MS (CI): m/z (%) = 318 (MH+, 100).- IR
(film): n (cm−1) = 2940 (m, C–H), 2814 (m, C–H), 2097 (s,
and a solution of methylamine in ethanol (8 M, 0.41 ml,
3.3 mmol) was added dropwise. The reaction mixture was stir-
red for 48 h at room temperature. Then water (10 ml) and
diethyl ether (8 ml) were added and the organic layer was se-
parated. The DMSO/water layer was extracted with diethyl
ether (5 × 5 ml). The organic layers were combined, washed
with brine (10 ml), dried (Na2SO4) and evaporated in vacuo.
Purification of the residue by FC (2 cm, at first ethanol [130
ml], then ethyl acetate/acetone 8:2 + 2% ethyldimethylamine, 2
ml, Rf = 0.10 (EtOH), Rf = 0.21 (ethyl acetate/acetone 8:2
+ 2% ethyldimethylamine)) afforded 8c as a colorless, viscous
oil. Yield 53 mg (52%), [α]589 = –29.2 (c = 0.55, CH2Cl2).-
C17H27N3O2 (305.42) calcd. C 66.85 H 8.91 N 13.76 found
C 66.04 H 8.92 N 13.46.- MS (EI): m/z (%) = 261 (M –
CH2NHCH3, 74), 232 (M – CO2Et, 5), 170 (261 – benzyl,
100).- MS (CI): m/z (%) = 306 (MH+, 100).- IR (film): n
(cm−1) = 2935 (m, C–H), 2807 (s, C–H), 1747 (s, C=O), 736
and 699 (m, aryl-C–H).- 1H NMR (CDCl3): δ = 1.24 (t,
J = 7.2 Hz, 3 H, OCH2CH3), 1.46 (s broad, 1 H, NH), 2.25–
2.39 (m, 2 H, 5-H and 6-H), 2.35 (s, 3 H, NHCH3), 2.48 (dd,
J = 10.7/8.5 Hz, 1 H, 2-H), 2.61–2.80 (m, 4 H, 2-H, 3-H, 5-H
and 6-H), 2.84 (dd broad, J = 11.9/5.4 Hz, 2 H, CH2NHCH3),
3.11 (d, J = 16.5 Hz, 1 H, NCH2CO2Et), 3.19 (d, J = 16.5 Hz,
1 H, NCH2CO2Et), 3.27 (d, J = 13.6 Hz, 1 H, PhCH2N), 3.99
(d, J = 13.8 Hz, 1 H, PhCH2N), 4.15 (q, J = 7.2 Hz, 2 H, OC
H2CH3), 7.19–7.30 (m, 5 H, arom).- 13C NMR (CDCl3):
δ = 14.2 (1 C, OCH2CH3), 36.9 (1 C, NHCH3), 50.7 (1 C, C-
5 or C-6), 51.9 (1 C, CH2NHCH3), 52.8 (1 C, C-6 or C-5),
56.6 (1 C, C-2), 57.9 (1 C, PhCH2N), 59.4 (1 C, C-3), 59.5
(1 C, NCH2CO2Et), 60.6 (1 C, OCH2CH3), 126.9 (1 C, C-4
arom), 128.3 (2 C, C-3 and C-5 arom), 128.7 (2 C, C-2 and
C-6 arom), 139.0 (1 C, C-1 arom), 170.4 (1 C, CO2Et).
1
N3), 1745 (s, C=O), 738 and 699 (m, aryl-C–H).- H NMR
(CDCl3): δ = 1.27 (t, J = 7.0 Hz, 3 H, OCH2CH3), 2.35–2.52
(m, 3 H, 2-H, 5-H and 6-H), 2.60–2.68 (m, 1 H, 5-H or 6-H),
2.72–2.83 (m, 3 H, 2-H, 3-H and 6-H or 5-H), 3.16 (d,
J = 16.4 Hz, 1 H, NCH2CO2Et), 3.22 (d, J = 16.5 Hz, 1 H, NC
H2CO2Et), 3.42 (d, J = 13.5 Hz, 1 H, PhCH2N), 3.52 (dd,
J = 12.8/4.1 Hz, 1 H, CH2N3), 3.62 (dd, J = 12.8/6.5 Hz, 1 H,
CH2N3), 3.96 (d, J = 13.4 Hz, 1 H, PhCH2N), 4.18 (q,
J = 7.1 Hz, 2 H, OCH2CH3), 7.21–7.34 (m, 5 H, arom).- 13C
NMR (CDCl3): δ = 14.2 (1 C, OCH2CH3), 49.6 (1 C, C-5 or
C-6), 50.3 (1 C, CH2N3), 52.4 (1 C, C-6 or C-5), 55.6 (1 C, C-
2), 58.2 (1 C, PhCH2N), 58.8 (1 C, C-3), 59.3 (1 C, N
CH2CO2Et), 60.6 (1 C, OCH2CH3), 127.1 (1 C, C-4 arom),
128.3 (2 C, C-3 and C-5 arom), 128.7 (2 C, C-2 and C-6
arom), 138.3 (1 C, C-1 arom), 170.2 (1 C, CO2Et).
9b (Rf = 0.29): Colorless oil, yield 0.127 g (11 %), [α]589
=
+4.5 (c = 1.025, CH2Cl2).- C16H23N5O2 (317.39) calcd. C
60.55 H 7.30 N 22.07 found C 60.58 H 7.35 N 21.96.- MS
(EI): m/z (%) = 275 (M – N3, 100), 244 (M – CO2Et, 13),
189 (M– N3 – CH2CO2Et, 36), 91 (benzyl, 100).- MS (CI):
m/z (%) = 318 (MH+, 100).- IR (film): n (cm−1) = 2938 (m,
C–H), 2095 (s, N3), 1735 (s, C=O), 738 and 698 (m, aryl-C–
H).- 1H NMR (CDCl3): δ = 1.27 (t, J = 7.1 Hz, 3 H, OCH2CH3
), 2.64–2.70 (m, 2 H, 2-H and 3-H), 2.79–2.90 (m, 3 H, 5-H or
7-H, 2-H and 3-H), 2.96–3.03 (m, 2 H, 5-H and 7-H), 3.23 (dd,
J = 14.1/4.6 Hz, 1 H, 5-H or 7-H), 3.41–3.49 (m, 1 H, 6-H),
3.43 (d, J = 17.0 Hz, 1 H, NCH2CO2Et), 3.50 (d, J = 17.3 Hz,
1 H, NCH2CO2Et), 3.67 (d, J = 13.4 Hz, 1 H, PhCH2N), 3.74
(d, J = 13.4 Hz, 1 H, PhCH2N), 4.17 (q, J = 7.2 Hz, 2 H, OC
H2CH3), 7.21–7.36 (m, 5 H, arom).- 13C NMR (CDCl3):
δ = 14.2 (1 C, OCH2CH3), 56.3 (1 C, C-2 or C-3), 56.6 (1 C,
C-3 or C-2), 58.2 (1 C, C-5 or C-7), 59.2 (1 C, C-7 or C-5),
59.6 (1 C, NCH2CO2Et), 60.0 (1 C, C-6), 60.4 (1 C, O
CH2CH3), 62.8 (1 C, PhCH2N), 127.1 (1 C, C-4 arom),
128.3 (2 C, C-3 and C-5 arom), 128.7 (2 C, C-2 and C-6
arom), 139.1 (1 C, C-1 arom), 171.3 (1 C, CO2Et).
6.5. (–)-Ethyl 2-[(2S)-1-benzyl-4-(ethoxycarbonylmethyl)
piperazin-2-yl]acetate (12)
H2SO4 conc. (4.5 ml) was added slowly to a solution of 8a
(0.110 g, 0.367 mmol) in ethanol (13.5 ml). The mixture was
heated to reflux for 3 h and, subsequently, concentrated in va-
cuo. The residue was dissolved in CH2Cl2 (15 ml), extracted
with water (2 × 10 ml), the pH of the combined aqueous layers
was adjusted to 10 with NaOH and then the aqueous layer was
extracted with CH2Cl2 (5 × 15 ml). The organic layer was
washed with a saturated solution of NaHCO3 (2 × 40 ml), dried
(Na2SO4), concentrated in vacuo and the residue was purified
by FC (2 cm, petroleum ether/ethyl acetate 1:1, 5 ml, Rf
= 0.36). Pale yellow oil, yield 60 mg, 47%, [α]589 = –5.9
(c = 0.535, CH2Cl2).- C19H28N2O4 (348.44) calcd. C 65.49 H
8.10 N 8.04 found C 64.74 H 8.09 N 7.85.- MS (EI): m/z (%)
= 348 (M, 46), 275 (M – CO2Et, 67), 261 (M – CH2CO2Et,
100), 170 (261 – Benzyl, 97).- IR: ˜ν ˜ ˜[cm−1] = 2938 (m, C–
H), 2811 (m, C–H), 1732 (s, C=O), 1167 (s, C-O), 738 and 699
(m, aryl-C–H).- 1H NMR (CDCl3): δ = 1.22 (t, J = 7.1 Hz, 3 H,
OCH2CH3), 1.25 (t, J = 7.2 Hz, 3 H, OCH2CH3), 1.92–2.02
(m, 2 H, CH2CH2CO2Et), 2.24–2.37 (m, 4 H, 3-H, 5-H, 6-H
and CH2CH2CO2Et), 2.45 (“dt”, J = 15.8/8.1 Hz, 1 H,
CH2CH2CO2Et), 2.51–2.60 (m, 1 H, 2-H), 2.62–2.76 (m, 3
6.4. (–)-Ethyl 2-[(3S)-4-benzyl-3-(methylaminomethyl)
piperazin-1-yl]acetate (8c)
Triethylamine (0.09 ml, 0.66 mmol) and methanesulfonyl
chloride (39 μl, 0.396 mmol) were added to a cooled (0 °C)
solution of 5 (98 mg, 0.33 mmol) in CH2Cl2 (10 ml). The
mixture was stirred at 0 °C for 15 min. The organic layer
was washed with water (8 ml), dried (Na2SO4) and concen-
trated in vacuo. The residue was dissolved in DMSO (3 ml)

S. Bedürftig, B. Wünsch / European Journal of Medicinal Chemistry 41 (2006) 387–396
393
H, 3-H, 5-H and 6-H), 3.11 (d, J = 16.4 Hz, 1 H, NCH2CO2Et),
3.18 (d, J = 16.4 Hz, 1 H, NCH2CO2Et), 3.21 (d, J = 13.4 Hz,
1 H, PhCH2N), 4.01 (d, J = 13.7 Hz, 1 H, PhCH2N), 4.09 (q,
J = 7.1 Hz, 2 H, OCH2CH3), 4.16 (q, J = 7.1 Hz, 2 H, OC
H2CH3), 7.21–7.34 (m, 5 H, arom).- 13C NMR (CDCl3):
δ = 14.2 (2 C, 2 × OCH2CH3), 24.6 (1 C, CH2CH2CO2Et),
30.2 (1 C, CH2CH2CO2Et), 50.2 (1 C, C-5 or C-6), 52.6 (1
C, C-6 or C-5), 56.8 (1 C, C-3), 57.4 (1 C, PhCH2N), 58.5
(1 C, C-2), 59.6 (1 C, NCH2CO2Et), 60.4 (1 C, OCH2CH3),
60.6 (1 C, OCH2CH3), 126.9 (1 C, C-4′), 128.2 (2 C, C-3′ and
C-5′), 128.8 (2 C, C-2′ and C-6′), 138.9 (1 C, C-1′), 170.2 (1 C,
C=O), 173.6 (1 C, C=O).
and 698 (m, aryl-C–H).- 1H NMR (CDCl3): δ = 1.26 (t,
J = 7.2 Hz, 3 H, OCH2CH3), 2.29–2.43 (m, 2 H, 5-H and 6-
H), 2.47–2.60 (m, 2 H, 2-H and 3-H), 2.65–2.73 (m, 1 H, 5-H
or 6-H), 2.76–2.85 (m, 3 H, 2-H and 6-H or 5-H and C
H2NH2), 3.11 (dd, J = 13.2/5.7 Hz, 1 H, CH2NH2), 3.14 (d,
J = 16.5 Hz, 1 H, NCH2CO2Et), 3.21 (d, J = 16.5 Hz, 1 H, NC
H2CO2Et), 3.29 (d, J = 13.5 Hz, 1 H, PhCH2N), 4.02 (d,
J = 13.4 Hz, 1 H, PhCH2N), 4.17 (q, J = 7.0 Hz, 2 H, OC
H2CH3), 7.21–7.33 (m, 5 H, arom). The signals for the protons
of the NH2 group could not be detected.- 13C NMR (CDCl3):
δ = 14.2 (1 C, OCH2CH3), 41.1 (1 C, CH2NH2), 50.3 (1 C, C-5
or C-6), 52.7 (1 C, C-6 or C-5), 55.5 (1 C, C-2), 57.7 (1 C, Ph
CH2N), 59.5 (1 C, NCH2CO2Et), 60.6 (2 C, C-3 and O
CH2CH3), 126.9 (1 C, C-4 arom), 128.3 (2 C, C-3 and C-5
arom), 128.8 (2 C, C-2 and C-6 arom), 138.8 (1 C, C-1 arom),
170.3 (1 C, CO2Et).
6.6. (+)-Ethyl 2-[(6S)-9-oxo-1,4-diazabicyclo[4.3.0]nonan-4-
yl]acetate (15)
Pd/C (10%, 47 mg) was added to a solution of 13 (50 mg,
0.14 mmol) in ethanol (10 ml). The suspension was stirred un-
der an H2 atmosphere (4 bar) at room temperature for 8 h. The
mixture was filtered through a pad of Celite®AFA and the fil-
trate was stirred at room temperature for additional 40 h. Re-
moval of the solvent in vacuo and purification by FC (1 cm,
ethanol, 1 ml, Rf = 0.26) gave 15 as a pale yellow oil. Yield
10 mg (30%), [α]589 = +17.7 (c = 0.81, CH2Cl2).- C11H18N2O3
(226.27).- HRMS: Calcd. 226.1317, found 226.1317
( 0 ppm).- MS (EI): m/z (%) = 226 (M, 9), 153 (M – CO2Et,
100), 138 (M – CH2CO2Et, 11).- IR (film): n (cm−1) = 2934
(m, C–H), 1736 (s, C=O ester), 1682 (s, C=O amide), 1034
6.8. (–)-Ethyl 2-[(3S)-3-(aminomethyl)piperazin-1-yl]acetate
(16b)
A suspension of 8b (0.423 g, 1.33 mmol), Pd(OH)2/C
(Pearlman catalyst, 0.237 g) and ethanol (10 ml) was stirred
under an H2 atmosphere (4 bar) at room temperature for 8 h.
The mixture was filtered through a pad of Celite®AFA and the
filtrate was concentrated in vacuo. The residue was character-
ized without further purification and then reacted with formal-
dehyde to give 18. Pale yellow, viscous oil, yield 0.260 g
(97%), [α]589 = −0.6 (c = 0.86, CH2Cl2).- C9H19N3O2
(201.27).- MS (EI): m/z (%) = 201 (M, 3), 171 (M –
CH2NH2, 100), 128 (M – CO2Et, 17).- MS (CI): m/z (%) = 202
(MH+, 100).- IR (film): n (cm−1) = 3262 (br, N–H), 2934 (m,
C–H), 2814 (s, C–H), 1739 (s, C=O).- 1H NMR (CDCl3):
δ = 1.25 (t, J = 7.1 Hz, 3 H, OCH2CH3), 1.66 (s broad, 3 H,
NH and NH2), 1.94 (t, J = 9.6 Hz, 1 H, 2-H), 2.23 (ddd,
J = 15.3/9.8/4.4 Hz, 1 H, 5-H or 6-H), 2.58 (dd, J = 12.4/
7.2 Hz, 1 H, 6-H or 5-H), 2.70–2.86 (m, 4 H, 2-H, 3-H, 5-H
and 6-H), 2.94–2.98 (m, 2 H, CH2NH2), 3.18 (s, 2 H, NC
H2CO2Et), 4.16 (q, J = 7.2 Hz, 2 H, OCH2CH3).- 13C NMR
(CDCl3): δ = 14.2 (1 C, OCH2CH3), 45.3, (2 C, C-5 or C-6 and
CH2NH2), 53.7 (1 C, C-6 or C-5), 57.0 (1 C, C-3), 57.2 (1 C,
C-2), 59.8 (1 C, NCH2CO2Et), 60.5 (1 C, OCH2CH3), 170.2 (1
C, C=O).
1
(m, C-O).- H NMR (CDCl3): δ = 1.26 (t, J = 7.3 Hz, 3 H,
OCH2CH3), 1.50–1.63 (m, 1 H, 7-H), 2.00 (t, J = 10.7 Hz, 1
H, 5-H), 2.09–2.20 (m, 1 H, 7-H), 2.22 (td, J = 11.5/3.7 Hz, 1
H, 3-H), 2.34–2.41 (m, 2 H, 8-H), 2.83–2.88 (m, 1 H, 3-H),
2.94 (td, J = 12.4/3.4 Hz, 1 H, 2-H), 3.02 (ddd, J = 10.9/3.7/
1.6 Hz, 1 H, 5-H), 3.22 (d, J = 16.6 Hz, 1 H, NCH2CO2Et),
3.28 (d, J = 16.7 Hz, 1 H, NCH2CO2Et), 3.69 (dtd, J = 10.6/
7.1/3.6 Hz, 1 H, 6-H), 3.99 (ddd, J = v12.9/3.6/1.4 Hz, 1 H,
2-H), 4.17 (q, J = 7.2 Hz, 2 H, OCH2CH3).- 13C NMR
(CDCl3): δ = 14.2 (1 C, OCH2CH3), 22.0 (1 C, C-7), 30.1 (1
C, C-8), 39.4 (1 C, C-2), 51.5 (1 C, C-3), 55.4 (1 C, C-6), 59. 0
(1 C, NCH2CO2Et), 59.5 (1 C, C-5), 60.7 (1 C, OCH2CH3),
170.0 (1 C, C=O), 173.3 (1 C, C-9).
6.7. (–)-Ethyl 2-[(3S)-3-(aminomethyl)-4-benzylpiperazin-1-yl]
acetate (16a)
6.9. (–)-Ethyl 2-[(3S)-4-benzyl-3-{N-[2-(3,4-dichlorophenyl)
acetyl]aminomethyl}-piperazin-1-yl]acetate (17a)
Pd/C (10%, 45 mg) was added to a solution of 8b (100 mg,
0.315 mmol) in ethanol (10 ml). The suspension was stirred
under an H2 atmosphere (balloon) at room temperature for
30 min. The mixture was filtered through a pad of
Celite®AFA and the filtrate was concentrated in vacuo. The
residue was characterized without further purification and then
acylated to afford 17a. Pale yellow, viscous oil, yield 88 mg
(96%), [α]589 = –8.2 (c = 1.15, CH2Cl2).- C16H25N3O2
(291.39).- MS (EI): m/z (%) = 261 (M – CH2NH2, 72), 179
(M – CH2NH2 – benzyl, 100), 91 (benzyl, 46).- MS (ESI):
m/z (%) = 292 (MH+, 100).- IR (film): n (cm−1) = 3397 (broad,
N–H), 2935 (m, C–H), 2812 (m, C–H), 1733 (s, C=O), 731
To a stirred and cooled (0 °C) solution of 3,4-dichlorophe-
nylacetic acid (0.137 g, 0.67 mmol) in CH2Cl2 (10 ml), 1,1′-
carbonyldiimidazole (CDI) (0.127 g, 0.78 mmol) was added
under N2 atmosphere. It was allowed to warm to room tem-
perature and the reaction mixture was stirred for 2 h. Under
N2 atmosphere and cooling (ice bath) a cooled solution of
16a (0.163 g, 0.56 mmol) in CH2Cl2 (10 ml) was added drop-
wise. The reaction mixture was stirred for 24 h at room tem-
perature. The organic layer was washed with a saturated solu-
tion of Na2CO3 (2 × 10 ml) and brine (10 ml), dried with
Na2SO4 and concentrated in vacuo. FC purification of the re-

394
S. Bedürftig, B. Wünsch / European Journal of Medicinal Chemistry 41 (2006) 387–396
sidue (3 cm, ethyl acetate, 7 ml, Rf = 0.24) afforded 17a as a
pale yellow, viscous oil; yield 0.233 g (87%), [α]589 = –6.7
(c = 0.385, CH2Cl2).- C24H29Cl2N3O3 (478.42) calcd. C
60.25 H 6.11 N 8.78 found C 59.96 H 6.40 N 8.05.- MS
(EI): m/z (%) = 478 (M, 1), 261 (M – CH2NHCOCH2PhCl2,
100), 170 (261 – benzyl, 76).- MS (CI): m/z (%) = 482/480/478
(MH+, 14/67/100).- IR (film): n (cm−1) = 3299 (broad, N–H),
2938 (m, C–H), 2815 (m, C–H), 1737 (s, C=O ester), 1648 (s,
C=O amide), 1470 (s, N–H amide II), 1031 (s, C–Cl), 826, 738
and 699 (m, aryl-C–H).- 1H NMR (CDCl3): δ = 1.28 (t,
J = 7.2 Hz, 3 H, OCH2CH3), 2.32–2.42 (m, 2 H, 5-H and 6-
H), 2.47–2.62 (m, 3 H, 2-H and 5-H or 6-H), 2.67–2.73 (m, 1
H, 6-H or 5-H), 2.75–2.80 (m, 1 H, 3-H), 3.06 (d, J = 17.1 Hz,
1 H, NCH2CO2Et), 3.17 (d, J = 16.7 Hz, 1 H, NCH2CO2Et),
3.42 (d, J = 13.4 Hz, 1 H, PhCH2N), 3.43 (dt, J = 14.0/4.6 Hz,
1 H, CH2NHCO), 3.51 (s, 2 H, COCH2PhCl2), 3.62 (dt,
J = 14.0/4.7 Hz, 1 H, CH2NHCO), 3.78 (d, J = 13.5 Hz, 1 H,
PhCH2N), 4.13–4.22 (m, 2 H, OCH2CH3), 6.92 (s broad, 1 H,
NH), 7.13–7.17 (m, 3 H, arom), 7.23–7.32 (m, 3 H, arom),
7.39 (d, J = 6.5 Hz, 1 H, 6-H PhCl2), 7.41 (s, 1 H, 2-H
PhCl2).- 13C NMR (CDCl3): δ = 14.2 (1 C, OCH2CH3), 38.4
(1 C, CH2NHCO), 42.9 (1 C, COCH2PhCl2), 49.0 (1 C, C-5 or
C-6), 52.8 (1 C, C-6 or C-5), 55.6 (1 C, C-2), 56.1 (1 C, C-3),
57.8 (1 C, PhCH2N), 58.9 (1 C, NCH2CO2Et), 60.8 (1 C, O
CH2CH3), 127.1 (1 C, C-4 benzyl), 128.4 (2 C, C-3 and C-5
benzyl), 128.5 (2 C, C-2 and C-6 benzyl), 128.9, 130.6, 131.2,
131.4, 132.6 and 135.5 (6 C, PhCl2), 138.3 (1 C, C-1 benzyl),
170.5 (1 C, CO2Et).
rotamer 2), 3.07 (s, 3 × 0.5 H, NCH3 rotamer 2), 3.14–3.24
(m, 2 H, NCH2CO2Et), 3.31 (dd broad, J = 14.4/4.3 Hz, 0. 5
H, COCH2PhCl2 rotamer 1), 3.50–3.69 (m,
2 H, C
H2N(CH3)CO and 3 × 0.5 H, COCH2PhCl2 rotamer 2 and PhC
H2N rotamer 1 and 2), 3.79–3.97 (m, 4 × 0.5 H, COCH2PhCl2
rotamer 1 and 2 and PhCH2N rotamer 1 and 2), 4.16 (q,
J = 7.1 Hz, 2 H, OCH2CH3), 7.06–7.12 (m, 1 H, arom),
7.25–7.35 (m, 7 H, arom).- 1H NMR (DMSO, 27 °C):
δ = 1.12 (t, J = 7.0 Hz, 3 × 0.5 H, OCH2CH3 rotamer 1), 1.16
(t, J = 7.2 Hz, 3 × 0.5 H, OCH2CH3 rotamer 2), 2.20–2.59 (m,
6 H, 2-H, 5-H and 6-H), 2.72 (s, 3 × 0.5 H, NCH3 rotamer 1),
3.01 (s, 3 × 0.5 H, NCH3 rotamer 2), 3.07–3.14 (m, 2 H, 3-H
and CH2N(CH3)CO or NCH2CO2Et), 3.19–3.45 (m, 3 H, C
H2N(CH3)CO and/or NCH2CO2Et), 3.64–3.88 (m, 4 H, PhC
H2N and COCH2PhCl2), 4.03 (q, J = 6.8 Hz, 2 × 0.5 H, OC
H2CH3 rotamer 1), 4.05 (q, J = 6.9 Hz, 2 × 0.5 H, OCH2CH3
rotamer 2), 7.31–7.41 (m, 6 H, arom), 7.41 (s broad, 0.5 H, 2-
H PhCl2 rotamer 1), 7.44 (s broad, 0.5 H, 2-H PhCl2 rotamer
1
2), 7.51 (d, J = 8.3 Hz, 1 H, 5-H PhCl2).- H NMR (DMSO,
120 °C): δ = 1.19 (t, J = 7.1 Hz, 3 H, OCH2CH3), 2.40–2.52
(m, 5 H, 2-H, 5-H and 6-H), 2.62 (dd, J = 11.3/3.4 Hz, 1 H, 2-
H), 2.76–2.96 (m, 4 H, 3-H and NCH3), 3.15 (s, 2 H, NC
H2CO2Et), 3.59 (d, J = 14.7 Hz, 1 H, PhCH2N), 3.70 (s broad,
4 H, CH2N(CH3)CO and COCH2PhCl2), 3.85 (d, J = 14.2 Hz,
1 H, PhCH2N), 4.09 (q, J = 7.1 Hz, 2 H, OCH2CH3), 7.15–
7.21 (m, 2 H, arom), 7.28 (m, 4 H, arom), 7.42 (s broad, 1
H, 2-H PhCl2), 7.46 (d, J = 8.2 Hz, 1 H, 5-H PhCl2).- 13C
NMR (CDCl3): δ = 14.5 (1 C, OCH2CH3), 34.2 (0.5 C, N
CH3 rotamer 1), 37.7 (0.5 C, NCH3 rotamer 2), 40.2 1 C,
CH2N(CH3)CO), 46.5 (0.5 C, COCH2PhCl2 rotamer 1), 47.7
(0.5 C, COCH2PhCl2 rotamer 2), 51.9 (1 C, C-5 or C-6), 52.9
(1 C, C-6 or C-5), 55.4 (1 C, C-2 and 0.5 C, C-3 rotamer 1),
58.1 (2 × 0.5 C, C-3 rotamer 2 and PhCH2N rotamer 1), 58.4
(0.5 C, PhCH2N rotamer 2), 59.7 (1 C, NCH2CO2Et), 60.8 (0.5
C, OCH2CH3 rotamer 1), 60.9 (0.5 C, OCH2CH3 rotamer 2),
127.2 and 127.6 (both 0.5 C, C-4 benzyl rotamer 1 and 2),
128.69 and 128.72 (both 2 × 0.5 C, C-3 and C-5 benzyl rota-
mer 1 and 2), 128.8 and 128.9 (both 2 × 0.5 C, C-2 and C-6
benzyl rotamer 1 and 2), 130.6, 130.7, 131.2, 131.3, 132.6 and
135.3 (6 C, PhCl2), 136.2 (1 C, C-1 benzyl), 170.2, 170.4,
170.5 and 171.0 (all 0.5 C, 2 × CO2Et rotamer 1 and 2).
6.10. (–)-Ethyl 2-[(3R)-4-benzyl-3-{N-[2-(3,4-dichlorophenyl)
acetyl]-N-methylamino-methyl}-piperazin-1-yl]acetate (17b)
To a stirred and cooled (0 °C) solution of 3,4-dichlorophe-
nylacetic acid (43 mg, 0.21 mmol) in CH2Cl2 (5 ml), 1,1′-CDI
(40 mg, 0.245 mmol) was added under N2 atmosphere. It was
allowed to warm to room temperature and the reaction mixture
was stirred for 2 h. Under N2 atmosphere and cooling (ice
bath) a cooled solution of 8c (53 mg, 0.175 mmol) in CH2Cl2
(10 ml) was added dropwise. The reaction mixture was stirred
for 24 h at room temperature. The organic layer was washed
with a saturated solution of Na2CO3 (2 × 7 ml) and brine (7
ml), dried (Na2SO4) and concentrated in vacuo. FC of the re-
sidue (2 cm, ethyl acetate, 3 ml, Rf = 0.32) yielded 17b as a
pale yellow, viscous oil. Yield 62 mg (72%), [α]589 = –5.6
(c = 0.51, CH2Cl2).- C25H31Cl2N3O3 (492.44) calcd. C 60.98
H 6.34 N 8.53 found C 60.42 H 6.28 N 8.22.- MS (EI):
6.11. (+)-Ethyl 2-[(6S)-1,4,8-triazabicyclo[4.3.0]nonan-4-yl]
acetate (18)
An aqueous solution of formaldehyde (37%, 24 μl,
0.3 mmol) was added to a solution of 16b (50 mg, 0.25 mmol)
in ethanol (5 ml). The mixture was heated to reflux for 15 min,
then stirred at 35 °C for 30 h. After removal of the solvent the
residue was characterized without further purification. Pale yel-
low, viscous oil, yield 53 mg (99%), [α]589 = +11.9 (c = 0.12,
CH2Cl2).- C10H19N3O2 (213.28).- MS (EI): m/z (%) = 213 (M,
29), 170 (piperazinylethylacetate, 26), 140 (M – CO2Et, 64),
97 (170 – CH2CO2Et, 100).- MS (CI): m/z (%) = 214 (MH+,
100).- IR (film): n (cm−1) = 3387 (br, N–H), 2934 (m, C–H),
m/z (%) = 418 (M
– H – CO2Et, 3), 261 (M –
CH2N(CH3)COCH2PhCl2, 100), 170 (261 – benzyl, 75).- MS
(CI): m/z (%) = 496/494/492 (MH+, 12/69/100).- IR (film): n
(cm−1) = 2937 (m, C–H), 2816 (m, C–H), 1743 (s, C=O ester),
1643 (s, C=O amide), 1031 (s, C–Cl), 832, 738 and 699 (m,
1
aryl-C–H).- H NMR (CDCl3): δ = 1.25 (t, J = 7.1 Hz, 3 × 0.5
H, OCH2CH3 rotamer 1), 1.26 (t, J = 7.2 Hz, 3 × 0.5 H,
OCH2CH3 rotamer 2), 2.43–2.51 (m, 2 H, 2-H and 5-H or 6-
H), 2.52–2.60 (m, 2 H, 5-H and 6-H), 2.67–2.71 (m, 2 H, 2-H
and 5-H or 6-H), 2.81 (s, 3 × 0.5 H, NCH3 rotamer 1), 2.89–
2.93 (m, 0.5 H, 3-H rotamer 1), 2.93- 2.99 (m, 0.5 H, 3-H
1
2814 (s, C–H), 1736 (s, C=O).- H NMR (CDCl3): δ = 1.26 (t,
J = 7.2 Hz, 3 H, OCH2CH3), 2.18–2.25 (m, 1 H, 2-H, 3-H, 5-H

S. Bedürftig, B. Wünsch / European Journal of Medicinal Chemistry 41 (2006) 387–396
395
or 7-H), 2.41–2.59 (m, 4 H, 2-H, 3-H, 5-H, 6-H and/or 7-H),
2.82–2.97 (m, 4 H, 2-H, 3-H, 5-H, 6-H and/or 7-H), 3.22 (d,
J = 5.2 Hz, 1 H, 9-H), 3.25 (s, 2 H, NCH2CO2Et), 3.93 (d,
J = 5.4 Hz, 1 H, 9-H), 4.17 (q, J = 7.1 Hz, 2 H, OCH2CH3).
The signal for the proton of the NH group could not be de-
tected.
J = 6.6 Hz, 2 H, OCH2CH3), 4.38 (d, J = 7.3 Hz, 0.6 H, 9-H
rotamer 2), 4.53 (d, J = 4.7 Hz, 0.4 H, 9-H rotamer 1), 7.21 (d,
J = 7.6 Hz, 1 H, 6-H arom), 7.49 (s, 1 H, 2-H arom), 7.53 (d,
J = 7.9 Hz, 1 H, 5-H arom).- 1H NMR (DMSO, 160 °C):
δ = 1.22 (t, J = 7.0 Hz, 3 H, OCH2CH3), 2.42–2.52 (m, 2 H,
2-H, 3-H and/or 5-H), 2.56–2.71 (m, 2 H, 2-H, 3-H, 5-H and/or
6-H), 2.80–2.90 (m, 3 H, 2-H, 3-H, 5-H and/or 6-H), 3.19 (d
broad, J = 11.6 Hz, 1 H, 7-H), 3.25 (s, 2 H, NCH2CO2Et), 3.46
(d broad, J = 11.5 Hz, 1 H, 7-H), 3.62 (s, 2 H, COCH2PhCl2),
3.78–3.82 (m, 1 H, 9-H), 4.13 (q, J = 7.1 Hz, 2 H, OCH2CH3),
4.47 (d, J = 6.7 Hz, 1 H, 9-H), 7.23 (d broad, J = 8.2 Hz, 1 H,
6-H arom), 7.45 (s, 1 H, 2-H arom), 7.47 (d broad, J = 7.6 Hz,
1 H, 5-H arom).- 13C NMR (CDCl3): δ = 14.2 (1 C,
OCH2CH3), 40.6 (0.55 C, COCH2PhCl2 rotamer 2), 40.7
(0.45 C, COCH2PhCl2 rotamer 1), 47.4 (1 C, C-7), 47.9 (1
C, C-3), 51.3 (0.45 C, C-2 rotamer 1), 51.6 (0.55 C, C-2 rota-
mer 2), 53.2 (0.55 C, C-5 rotamer 2), 54.0 (0.45 C, C-5 rota-
mer 1), 58.9 (1 C, NCH2CO2Et), 59.3 (0.45 C, C-6 rotamer 1),
60.2 (0.55 C, C-6 rotamer 2), 60.7 (1 C, OCH2CH3), 68.1
(0.55 C, C-9 rotamer 2), 68.3 (0.45 C, C-9 rotamer 1), 128.5
(0.45 C, C PhCl2 rotamer 1), 128.6 (0.55 C, C PhCl2 rotamer
2), 130.4 (1 C, C PhCl2), 131.0 (0.45 C, C PhCl2 rotamer 1),
131.1 (0.55 C, C PhCl2 rotamer 2), 132.5, 134.4 and 134.5 (3
C, C PhCl2), 167.1 (0.45 C, C=O amide rotamer 1), 167.9
(0.55 C, C=O amide rotamer 2), 170.1 (1 C, C=O ester).
6.12. (+)-Ethyl 2-{(6R)-8-[2-(3,4-dichlorophenyl)acetyl]-1,4,8-
triazabicyclo[4.3.0]nonan-4-yl}-acetate (19)
To a stirred and cooled (0 °C) solution of 3,4-dichlorophe-
nylacetic acid (0.111 g, 0.54 mmol) in CH2Cl2 (5 ml), 1,1′-CDI
(0.103 g, 0.63 mmol) was added under N2 atmosphere. It was
allowed to warm to room temperature and the reaction mixture
was stirred for 2 h. Under N2 atmosphere and cooling (ice
bath) a cooled solution of 18 (0.096 g, 0.45 mmol) in
CH2Cl2 (5 ml) was added slowly. The reaction mixture was
stirred for 24 h at room temperature. The organic layer was
washed with a saturated solution of NaHCO3 (2 × 7 ml) and
brine (7 ml), dried (Na2SO4) and concentrated in vacuo. FC
purification of the residue (2 cm, ethyl acetate/ethanol, 1:1,
3 ml, Rf = 0.43) afforded 19 as a viscous oil. Yield 77 mg
(43%), [α]589 = +12.1 (c = 0.195, CH2Cl2).- C18H23Cl2N3O3
(400.30) calcd. C 54.01 H 5.79 N 10.50 found C 53.81 H
5.99 N 11.10.- MS (EI): m/z (%) = 403/401/399 (M, 4/24/36),
240 (M – dichlorobenzyl, 15), 212 (M – COCH2PhCl2, 27),
163/161/159 (dichlorobenzyl, 11/53/82), 140 (212 – CO2Et,
100).- IR (film): n (cm−1) = 2937 (m, C–H), 2817 (m, C–H),
1739 (s, C=O ester), 1643 (s, C=O amide), 1030 (s, C–Cl), 821
(m, aryl-C–H).- 1H NMR (CDCl3): δ = 1.26 (t, J = 7.1 Hz,
3 × 0.45 H, OCH2CH3 rotamer 1), 1.27 (t, J = 7.2 Hz,
3 × 0.55 H, OCH2CH3 rotamer 2), 2.33 (dd, J = 10.8/8.9 Hz,
0.45 H, 5-H rotamer 1), 2,47 (dd, J = 11.0/8.0 Hz, 0.55 H, 5-H
rotamer 2), 2.54–2.64 (m, 2 H, 2-H and 3-H both rotamers),
2.71–2.78 (m, 0.45 H, 6-H rotamer 1, and 0.55 H, 2-H rotamer
2), 2.84–3.03 (m, 0.55 H, 6-H rotamer 2, and 0.45 H, 2-H
rotamer 1, and 2 H, 3-H and 5-H both rotamers), 3.17 (t,
J = 10.7 Hz, 0.45 H, 7-H rotamer 1), 3.26 (s, 2 × 0.55 H, NC
H2CO2Et rotamer 2), 3.27 (s, 2 × 0.45 H, NCH2CO2Et rotamer
1), 3.36 (t, J = 9.2 Hz, 0.55 H, 7-H rotamer 2), 3.49 (dd,
J = 8.6/6.4 Hz, 0.55 H, 7-H rotamer 2), 3.52 (s, 2 × 0.45 H,
COCH2PhCl2 rotamer 1), 3.56 (s, 2 × 0.55 H, COCH2PhCl2
rotamer 2), 3.67 (dd, J = 10.7/6.4 Hz, 0.45 H, 7-H rotamer 1),
3.82 (d, J = 7.1 Hz, 0.55 H, 7-H rotamer 2), 3.83 (d,
J = 4.7 Hz, 0.45 H, 9-H rotamer 1), 4.18 (q, J = 7.1 Hz, 2 H,
OCH2CH3), 4.46 (d, J = 4.9 Hz, 0.45 H, 9-H rotamer 1), 4.62
(d, J = 7.7 Hz, 0.55 H, 9-H rotamer 2), 7.10 (dd, J = 8.2/
2.1 Hz, 1 H, 6-H arom), 7.37 (d, J = 8.3 Hz, 1 H, 5-H arom),
7. Receptor binding studies
7.1. General information
Homogenizer: Potter®S (B. Braun Biotech International). –
Ultraturrax: Euroturrax® T20 (Ika Labortechnik). – Centrifuge:
High speed cooling centrifuge model J2-HS (Beckman). - Fil-
ter: Whatman glass fiber filters GF/B, presoaked in 0.5% poly-
ethylenimine in water for 2 h at 4 °C before use. - Filtration
was performed with a Brandel 24-well cell harvester. - Scintil-
lation cocktail: Rotiscint Eco Plus (Roth). - Liquid scintillation
analyzer: TriCarb 2100 TR (Canberra Packard), counting effi-
ciency 66%. – All experiments were carried out in triplicates. -
IC50-values were determined from competition experiments
with at least six concentrations of test compounds and were
calculated with the program GraphPad Prism® 3.0 (GraphPad
Software) by nonlinear regression analysis. – Ki-values were
calculated according to Cheng and Prusoff [15]. – The Ki-va-
lues are given as mean value S.E.M. from three independent
experiments.
7.2. Performance of the σ1-assay
1
7.36 (s broad, 1 H, 2-H arom).- H NMR (DMSO, 27 °C):
δ = 1.17 (t broad, J = 6.3 Hz, 3 H, OCH2CH3), 2.30–2.56 (m,
3 H, 2-H, 3-H and 5-H), 2.61–2.68 (m, 1 H, 2-H and 6-H
rotamers), 2.72–2.84 (m, 1 H, 2-H and 6-H rotamers, and 2
H, 3-H and 5-H), 2.96 (t, J = 10.6 Hz, 0.4 H, 7-H rotamer 1),
3.27 (s, 2 H, NCH2CO2Et), 3.40–3.70 (m, 0.6 H, 7-H rotamer
2, and 1 H, 7-H), 3.60 (s, 2 × 0.4 H, COCH2PhCl2 rotamer 1),
3.65 (s, 2 × 0.6 H, COCH2PhCl2 rotamer 2), 3.83 (d broad,
J = 5.2 Hz, 1 H, 9-H both rotamers), 4.07 (q, broad,
For the σ1-assay guinea pig brain membranes were prepared
as described in Ref. [13]. The test was performed with the radi-
oligand [3H]-pentazocine (1036 GBq mmol–1; NENTM Life
Science Products). The thawed membrane preparation (about
150 μg of protein) was incubated with various concentrations
of the test compound, 3 nM [3H]-pentazocine, and buffer
(50 mM Tris HCl, pH 7.4) in a total volume of 500 μl for
120 min at 37 °C. The incubation was terminated by rapid

396
S. Bedürftig, B. Wünsch / European Journal of Medicinal Chemistry 41 (2006) 387–396
[2] S. Soukara, C.A. Maier, U. Predoiu, A. Ehret, R. Jackisch, B. Wünsch, J.
filtration through presoaked Whatman GF/B filters (0.5% poly-
ethylenimine in water 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 radioactivity trapped on the filters was counted in
a liquid scintillation analyzer. Nonspecific binding was deter-
mined with 10 μM haloperidol.
Med. Chem. 44 (2001) 2814–2826.
[3] H. Bräuner-Osborne, J. Egebjerg, E. Nielsen, U. Madsen, P. Krogsgaard-
Larsen, J. Med. Chem. 43 (2000) 2609–2645.
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7.3. NMDA, κ-opioid, μ-opioid, and σ2 receptor assays
[7] G.L. Bihan, F. Rondu, A. Pelé-Tounian, X. Wang, S. Lidy, E. Touboul,
A. Lomouri, G. Dive, J. Huet, B. Pfeiffer, P. Renard, B. Guardiole-Le-
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NMDA, κ-opioid, μ-opioid, and σ2 receptor assays were
performed as described in reference [5].
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Acknowledgements
[9] S. Bedürftig, M. Weigl, B. Wünsch, Tetrahedron Asymmetry 12 (2001)
1293–1302.
Financial support of this work by the Deutsche Forschungs-
gemeinschaft and the Fonds der Chemischen Industrie is grate-
fully acknowledged. Thanks are also due to the Degussa AG,
Janssen-Cilag GmbH, and Bristol-Myers Squibb for donation
of chemicals and reference compounds.
[10] D. Manetti, C. Ghelardini, A. Bartolini, C. Bellucci, S. Dei, N. Galeotti,
F. Gualtieri, M.N. Romanelli, S. Scapecchi, E. Teodori, J. Med. Chem.
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