Combination of cyclohexane and piperazine based κ-opioid receptor agonists: Synthesis and pharmacological evaluation of trans,trans-configured perhydroquinoxalines

Article abstract of DOI:10.1016/j.bmc.2014.04.054

Desymmetrization of the pseudochiral (2r)-configured cyclohexane-1,2,3- triamines 8 with dimethyl oxalate led to racemic aminoquinoxaline-2,3-diones 9. Selective introduction of the κ pharmacophoric structural elements pyrrolidine and 3,4-dichlorophenylacetamide with a two-carbon distance afforded conformationally restricted κ agonists 13-15 based on the quinoxaline ring system. In competitive radioligand receptor binding studies the benzylamine 13b, the secondary amine 14b, and the carbamate 15 displayed high κ receptor affinity. The K_i value of the lead compound derived methoxycarbonyl derivative 15 is 9.7 nM. However, the κ affinity of 15 is exceeded by 13b and 14b with a basic functional group instead of the methoxycarbonyl group in 1-position of the quinoxaline system. The chlorine atoms of the dichlorophenylacetyl residue are essential, since the corresponding phenylacetyl analogs show considerably reduced κ affinity. The potent κ ligands 13b, 14b and 15 are selective over the related μ- and δ-opioid receptors, σ₁, σ₂ and NMDA receptors. In the [³⁵S]GTPγS-binding assay 13b behaved as partial agonist with lower activity than U-69,593.

Full text of DOI:10.1016/j.bmc.2014.04.054

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Combination of cyclohexane and piperazine based
j
-opioid receptor
agonists: Synthesis and pharmacological evaluation
of trans,trans-configured perhydroquinoxalines
⇑
Christian Bourgeois, Elena Werfel, Dirk Schepmann, Bernhard Wünsch
Institut für Pharmazeutische und Medizinische Chemie der Universität Münster, Corrensstraße 48, D-48149 Münster, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Desymmetrization of the pseudochiral (2r)-configured cyclohexane-1,2,3-triamines 8 with dimethyl oxa-
Received 6 March 2014
Revised 23 April 2014
Accepted 26 April 2014
Available online xxxx
late led to racemic aminoquinoxaline-2,3-diones 9. Selective introduction of the
tural elements pyrrolidine and 3,4-dichlorophenylacetamide with a two-carbon distance afforded
conformationally restricted agonists 13–15 based on the quinoxaline ring system. In competitive radi-
oligand receptor binding studies the benzylamine 13b, the secondary amine 14b, and the carbamate 15
displayed high receptor affinity. The Ki value of the lead compound derived methoxycarbonyl derivative
15 is 9.7 nM. However, the affinity of 15 is exceeded by 13b and 14b with a basic functional group
j pharmacophoric struc-
j
j
Keywords:
j
j
Agonists
instead of the methoxycarbonyl group in 1-position of the quinoxaline system. The chlorine atoms of
the dichlorophenylacetyl residue are essential, since the corresponding phenylacetyl analogs show
Perhydroquinoxalines
Desymmetrization
Nitroaldol
Conformational restriction
Pseudochiral center
Structure–affinity relationships
Selectivity
considerably reduced
- and d-opioid receptors, r1
as partial agonist with lower activity than U-69,593.
j
affinity. The potent
j
ligands 13b, 14b and 15 are selective over the related
l
,
r2 and NMDA receptors. In the [35S]GTP
c
S-binding assay 13b behaved
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
(1)10 (Fig. 1). The pharmacophore of the fourth class of
j agonists
is represented by an ethylenediamine, whose terminal N-atoms
are incorporated into a pyrrolidine ring and an arylacetamide moi-
ety, respectively. In U-50,488 (1, Ki = 0.34 nM) this ethylenedia-
mine structural element is part of a trans-configured cyclohexane
j
receptors belonging to the class of opioid receptors are widely
distributed throughout the central nervous system and the periph-
ery. Activation of centrally localized opioid receptors leads to
strong analgesia.1 Compared to clinically used
receptor agonists
(e.g. morphine, fentanyl, pethidine, levomethadone), receptor
agonists show a quite different side effect profile. In particular dan-
gerous receptor mediated side effects like respiratory depression
and addiction are not caused by activation of opioid receptors.
However receptor agonists are not devoid of side effects: dys-
phoria, sedation and strong diuresis are the most common side
effects associated with activation of recep-
receptors.2,3 Since
tors are also found in the periphery, agonists which are restricted
j
l
ring. In the potent j agonist 2 (Ki = 0.31 nM) the ethylenediamine
j
pharmacophore is found as part of a piperazinylmethylamine
system.11
l
Since we are interested in conformationally restricted
nists with novel scaffolds, it was planned to combine the structural
features of the potent agonists 1 and 2 containing the ethylene-
j ago-
j
j
j
diamine substructure (Fig. 1). A superposition of the ethylenedia-
mine substructures of 1 and 2 is only possible by annulation of
the cyclohexane and piperazine rings to afford the perhydroquin-
oxalines 3, which also result from addition of an ethylene moiety
between the cyclohexane ring and the N-methyl moiety of 1 or
between the piperazine ring and the methyl group of 2 (see arrows
at compounds 1 and 2). Whereas the configuration of the centers of
chirality in 8- and 8a-position are defined by the lead compounds 1
and 2, the configuration of the third center of chirality (C-4a) is not
defined by the lead compounds 1 and 2.
j
j
j
to the periphery can be used for the treatment of visceral pain and
inflammatory and itching skin diseases.4–6
The known
j agonists can be classified into four compound
classes: peptides including the physiological agonist dynorphin
A,2,7 morphinoids with the prototypical ligand ketocyclazocine giv-
ing name to this opioid receptor subtype,2,7 the natural product
salvinorin A without a basic functional group8,9 and ethylenediam-
ines (arylacetamides) with the first synthetic
j
agonist U-50,488
In this communication we report on the synthesis of perhydro-
quinoxalines 3 with the relative trans,trans-configuration of the
three N-functionalities attached to the cyclohexane ring (Fig. 1).
The additional N-4-atom of the perhydroquinoxaline ring, which
⇑
Corresponding author. Tel.: +49 251 8333311; fax: +49 251 8332144.
E-mail address: wuensch@uni-muenster.de (B. Wünsch).
http://dx.doi.org/10.1016/j.bmc.2014.04.054
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Bourgeois, C.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.054

2
C. Bourgeois et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
CO2CH3
N
Although the reduction of the nitrodiamine 7a to form the tri-
amine 8a has been reported in literature using H2 and Raney
Ni,12,13 this reaction step required special attention and careful
selection of the reaction conditions. Reproducible yields of 8a
could only be achieved by a certain combination of reaction condi-
tions (H2 pressure, temperature) and amount and quality of Raney
Ni. Alternative reduction methods (Zn/HCl, Sn/HCl, SmI2, LiAlH4)
did not improve the reproducibility and yields. Finally, the triam-
ines 8a–c and 8e were prepared by reduction of the nitrodiamines
7a–c and 7e with H2 (1 bar) in the presence of Raney Ni as catalyst.
For the establishment of the quinoxaline ring two amino moie-
ties of the triamines 8 had to be connected by a C2-building block.
For this purpose the benzyl derivative 8a was reacted with oxalyl
chloride at ꢀ78 °C. According to tlc several products were formed,
but the desired quinoxalinedione 9a could not be isolated. There-
fore the triamines 8a–c were treated with the less reactive
dimethyl oxalate in boiling methanol for 48 h (Scheme 1). After
complete conversion the quinoxalinediones 9a–c were isolated
by recrystallization in yields of 33–64%. The corresponding 3,4-
dichlorobenzyl derivative 9d was not obtained due to side reac-
tions during the reduction of the nitro moiety of nitrodiamine 7d.
The nitrodiol 6, the nitrodiamines 7 and the triamines 8 repre-
sent achiral compounds with a pseudochiral center in 2-position
having (2r)-configuration. Treatment of the triamines 8 with
dimethyl oxalate led to desymmetrization since both enantiotopic
benzylamino moieties reacted with the same probability with
dimethyl oxalate. However the original trans,trans-configuration
of the triamines 8 is retained in the quinoxalinediones 9. Although
the quinoxalinediones 9 contain three centers of chirality, this syn-
thetic strategy led to only one out of four possible diastereomers.
In this manuscript only one enantiomer of the racemic mixtures
is shown in the Schemes.
The construction of the quinoxalinedione 9a led to a differentia-
tion of the benzylamino moieties; one benzylamine is part of an
amine, whereas the other one is part of an amide allowing their
chemoselective conversion. Treatment of 9a with ammonium for-
mate in the presence of Pd/C as catalyst15 cleaved chemoselectively
the benzyl moiety of the benzylamine affording the primary amine
10 in 97% yield (Scheme 2). Reaction of the primary amine 10 with
1,4-diiodobutane provided the pyrrolidine 11 in 76% yield. Several
reducing agents were tried for the reduction of the quinoxalinedione
11. A 3:1 mixture of LiAlH4 and AlCl3 forming AlH3 in situ16 turned
out to give the highest yield (96%) of the quinoxaline 12. The reduced
quinoxaline 12 contains a secondary (N-4) and a tertiary amine (N-
1) within the heterocycle. Acylation of the secondary amino moiety
(N-4) with phenylacetyl chloride and 3,4-dichlorophenylacetyl
chloride led to the phenylacetamides 13a and 13b, respectively.
In order to introduce the methoxycarbonyl moiety of the lead
compound 2 the remaining benzyl moiety should be removed by
conventional hydrogenolysis. However, treatment of the benzyl-
amines 13 with H2 and Pd/C in a THF/H2O mixture led to incom-
plete transformation and provided considerable amounts
(approx. 30%) of dechlorinated products from 13b. Therefore concd
HCl (10%) was added to the solvent, which should increase the
hydrogenolysis rate. After limitation of the reaction time to
30 min, the secondary amines 14a and 14b were isolated in 90%
and 94% yield, respectively. The amount of dechlorinated products
found after hydrogenolysis of 13b was less than 1%. Finally,
acylation of the secondary amine 14b with methyl chloroformate
afforded the carbamate 15 in 59% yield.
H
H3C
CH3
N
N
N
N
O
O
Cl
Cl
Cl
Cl
2
1
(U-50,488)
R
H
4
5
N
4a
Cl
Cl
H
O
8a
N
1
8
N
N
O
R
N
N
Cl
Cl
3
Figure 1. Development of the novel
j agonists 3 by combining the cyclohexane and
piperazine rings of the lead compounds 1 and 2 to give a perhydroquinoxaline ring
system.
is located outside of the
j pharmacophore, will allow fine tuning of
the pharmacodynamic and more importantly the pharmacokinetic
properties including the passage of the blood brain barrier of the
new compounds by introducing various substituents.
2. Synthesis
According to literature,12,13 the one-pot reaction of glutaralde-
hyde (4), nitromethane (5) and an excess of benzylamine led to
the nitrocyclohexanediamine 7a in 60% yield (Scheme 1). However,
due to the formation of several side products and subsequent puri-
fication problems the stepwise synthesis was preferred. Thus, a
double Henry reaction (nitroaldol reaction) of nitromethane (5)
with glutaraldehyde (4) in the presence of NaOH13,14 afforded the
nitrocyclohexanediol 6 in 80% yield. Subsequent treatment of 6
with benzylamine in an aqueous solution provided the nitrodi-
amine 7a.13 Reduction of the amount of benzylamine and increas-
ing the amount of H2O resulted in an optimized yield of 92%,
exceeding the previously reported yield of 75%.13 The nitrodiam-
ines 7b, 7d, and 7e were obtained under the same conditions in
86–93% yields. Due to purification problems the p-chlorobenzyl-
amine 7c could only be isolated in 49% yield.
The exchange of the OH moieties of the nitrodiol 6 with amino
groups is explained either by a base induced b-elimination of water
followed by conjugated addition of amine or by a retro-nitroaldol
reaction followed by imine formation and subsequent addition of
the nitroalkane to the intermediate imine. The formation of the
nitrodiol 6 and its transformation into nitrodiamines 7 occurred
with high diastereoselectivity giving only the thermodynamically
most stable trans,trans-configured diastereomers with all substitu-
ents in the favorable equatorial orientation. The trans,trans-config-
uration of 6 and 7 is confirmed by the triplet for the proton
adjacent to the nitro moiety (CH-NO2) with large coupling con-
stants of 10.3–10.6 Hz indicating the axial orientation of three
adjacent protons.
3. Pharmacological evaluation
The
j receptor affinity of the quinoxalines 9–15 was deter-
mined in competition receptor binding studies. Guinea pig brain
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C. Bourgeois et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
7 9
N
R2
-
CH
O
O
O2N
(b)
+
CH3-NO2
HO
OH
HN
a
CH
4
5
6
HN
HN
OCH3
Cl
b
c
(c)
(a)
HNR2
HNR2
O2N
R2N
NR2
HN
N
d
e
Cl
7a-e
(d)
Cl
O
O
N
H2N
HN
R2N
NR2
(e)
R
RNH
8a-e
9a-c*
Scheme 1. Synthesis of racemic quinoxalinediones 9. Reagents and reaction conditions: (a) BnNH2, 60%;12,13 (b) CH3OH, NaOH, rt, 4 h, 80%;13,14 (c) HNR2, H2O, rt, 16–96 h; (d)
H2, 1 bar, Raney Ni, CH3OH, rt, 3–20 h; (e) dimethyl oxalate (MeO2C–CO2Me), CH3OH, 65 °C, 24–48 h. ⁄Residues R in 9 refer to benzyl (9a), p-methoxybenzyl (9b) and p-
chlorobenzyl (9c). Only one enantiomer of the racemic mixture 9 is shown.
O
O
O
O
N
O
N
O
N
HN
HN
HN
(c)
(b)
(a)
Bn
N
Bn
Bn
BnHN
H2N
11
9a
10
Cl
Cl
X
O
O
HN
X
Bn (d)
(f)
N
N
N
N
CO2CH3
R
N
N
N
N
13a,b
: R = Bn
14a,b
: R = H
15
12
(e)
a
: X = H
b: X = Cl
Scheme 2. Synthesis of the quinoxaline based
j receptor agonist 15. Reagents and reaction conditions: (a) NH4ꢁHCO2, Pd/C, CH3OH, 65 °C, 2 h, 97%; (b) 1,4-diiodobutane,
CH3CN, NaHCO3, 80 °C, 18 h, 76%; (c) AlCl3/LiAlH4 1:3, THF, 0 °C, 45 min, then rt, 20 min, 96%; (d) phenylacetyl chloride or (3,4-dichlorophenyl)acetyl chloride, CH2Cl2, NaOH,
rt, 2–18 h, 91% (13a), 86% (13b); (e) H2, 1 bar, Pd/C, THF, H2O, HCl, rt, 30 min, 90% (14a), 94% (14b); (f) ClCO2CH3, CH2Cl2, rt, 2 h, 59%. Only one enantiomer of the racemic
mixtures is shown.
membrane preparations were used as receptor material and tri-
tium labeled [3H]-U-69,593 served as radioligand. The non-specific
binding of the radioligand was determined after addition of a large
N-atom for modifications. The j receptor tolerates various substit-
uents at that position including a proton (14b), a benzyl moiety
(13b) and a methoxycarbonyl group (15). The tolerance of a second
excess of non-tritiated U-69,593 (10
l
M).17,18
basic structural element within the
particular interest. This observation correlates nicely with the
results obtained with agonists based on the 6,8-diazabicy-
clo[3.2.2]nonane scaffold.18
j agonists 13b and 14b is of
In Table 1 the receptor affinities of the quinoxaline deriva-
j
tives are summarized. It is obvious that the quinoxalinediones
9–11 with lactam moieties within the piperazine ring do not
j
interact significantly with the
oxaline 12 without a substituent at N-4 shows very low
ity. However, introduction of the dichlorophenylacetyl moiety as
second pharmacophoric element resulted in the potent
receptor agonist 13b with a Ki value of 9.4 nM. Removal of the
benzyl moiety led to a fourfold increase of the affinity (14b:
Ki = 2.1 nM), whereas the introduction of the methoxycarbonyl
j
receptor. Additionally the quin-
The relevance of the two chlorine atoms of the dichloropheny-
lacetyl moiety is documented by the data in Table 1. The ligands
13a and 14a without chlorine atoms at the phenyl moiety show
considerably reduced j affinity when compared with their chlori-
nated analogs 13b and 14b.
j
affin-
j
j
j
In order to test the opioid receptor selectivity, the l- and d-opi-
oid receptor affinities of the
radioligand receptor binding studies.18 At a concentration of
M the compounds did not interact significantly with - and d-
opioid receptors (IC50 >1 M) indicating high selectivity for the
receptor over these related opioid receptors.
j agonists 13–15 were recorded in
moiety of the lead compound 2 did not change the
(15: Ki = 9.7 nM).
j affinity
1
l
l
In the
j
agonists 13–15 the ethylenediamine
j
pharmacophore
l
j
is embedded in the quinoxaline ring, which contains an additional
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4
C. Bourgeois et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Table 1
4. Conclusion
j
-Opioid receptor affinity of quinoxaline derivatives 9–15 and reference compounds
Compd
Ki SEM# (nM)
([3H]-U-69,593)
The conformational flexibility of
introduction of the ethylenediamine
j
agonists was restricted by
j
pharmacophore into a quin-
j
oxaline ring system. The quinoxalines 13b, 14b, and 15 represent
potent agonists with low nanomolar affinity. Unexpectedly,
the affinity of the quinoxalines 14b with a basic functional group
in 4-position is higher than the affinity of the methoxycarbonyl
derivative 15. The impact of the aromatic chlorine atoms on the
affinity is shown by comparison of the affinity of the dichlor-
ophenylacetyl derivatives 13b and 14b with those of unsubstituted
phenylacetyl analogs 13a and 14a. The high affinity and receptor
9a
9b
9c
10
11
12
13a
0%
5%
10%
0%
3%
2%
129 46
9.4 1.6
435 91
2.1 0.4
9.7 1.8
0.34 0.07
0.31 0.04
0.88 0.10
6.9 0.50
j
j
j
j
j
j
13b (WMS-0610)
14a
14b (WMS-0611)
15 (WMS-0622)
U-50, 488 (1)
2
j
selectivity of 13b, 14b, and 15 together with the agonistic activity
of 13b render these compounds a new compound class with prom-
ising
j agonistic properties. The additional N-atom outside the j
U-69,593
Naloxone
pharmacophore allows further fine tuning of the pharmacokinetic
and pharmacodynamics properties.
#
Generally the Ki values were determined in triplicates (n = 3). For compounds
showing very low affinity in the first experiment repetitions were not performed
(n = 1). For low affinity compounds the residual binding of the radioligand (in %) at a
test compound concentration of 1 lM is given.
5. Experimental
5.1. Experimental, chemistry
19,20
Additionally the affinity towards r1
binding site)21,22 receptors was recorded. The
did not compete with the radioligands even at the high concentra-
tion of 1 M. It can be concluded that among the new agonists
the potent dichlorophenylacetamides 13b, 14b, and 15 are very
selective over r1 2, and NMDA receptors.
The synthetic intermediates 9–12 were also included into the
d, r1 2, and NMDA (PCP binding site) assays. With exception of
,
r2
,
and NMDA (PCP
5.1.1. General
j
agonists 13–15
Unless otherwise noted, moisture sensitive reactions were
conducted under dry nitrogen. THF was dried with sodium/
benzophenone and was distilled freshly before use. Thin layer
chromatography (tlc): Silica gel 60 F254 plates (Merck). Flash chro-
l
j
,
r
matography (fc): Silica gel 60, 40–64 lm (Merck); parentheses
l,
include: diameter of the column, length of the column, eluent, frac-
tion size, Rf value. Melting point: Melting point apparatus SMP 3
(Stuart Scientific), uncorrected. Elemental analysis: CHN-Rapid
Analysator (Foss-Heraeus). MS: MAT GCQ (Thermo-Finnigan);
EI = electron impact; Thermo Finnigan LCQÒ ion trap mass spec-
trometer with an ESI = electrospray ionization interface. IR: IR spec-
trophotometer 480Plus FT-ATR-IR (Jasco). 1H NMR (400 MHz), 13C
NMR (100 MHz): Mercury-400BB spectrometer (Varian); d in ppm
related to tetramethylsilane; coupling constants are given with
0.5 Hz resolution. HPLC: Merck Hitachi Equipment; UV detector:
L-7400; autosampler: L-7200; pump: L-7100; degasser: L-7614;
,
r
9b and 9c, these test compounds did not show affinity towards
these receptor systems. Unexpectedly, submicromolar r1 affinity
was found for the quinoxalinediones 9b (Ki(
r
1) = 368 nM) and 9c
receptor.
(Ki( 1) = 543 nM), which did not interact with the
r
j
Although the r1 affinity is rather low, 9b and 9c can be regarded
as lead compounds for optimizations.
In order to demonstrate the agonistic activity at
benzyl derivative 13b was tested exemplarily in the [35S]GTP
binding assay employing human
-opioid receptors.18,23 In this
assay 13b demonstrated the properties of a partial agonist with
j
receptors, the
c
S-
j
method 1: column: LiChrospherÒ 60 RP-select B (5
4 mm; flow rate: 1.00 mL/min; injection volume: 5.0 L; detection
at k = 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: gradient from 90% to 0%,
29–31 min: 0%, 31–31.5 min: gradient from 0% to 90%, 31.5–
40 min: 90%; method 2: column: phenomenexÒ Gemini C6-Phenyl
lm), 250–
j
70% relative efficacy compared with the full agonist U-69,593
(Fig. 2). The recorded EC50 value of 490 nM for 13b indicates
l
reduced
69,593 (EC50 = 80 nM). This result documents that an additional
basic moiety in -opioid receptor ligands does not inhibit activa-
tion of receptors.
j agonistic activity compared to the full agonist U-
j
j
110A (5
lm), 250–4.6 mm; flow rate: 1.00 mL/min; injection vol-
ume: 5.0
lL; detection at k = 220 nm; solvents: A: water with
0.05% (v/v) trifluoroacetic acid; B: acetonitrile with 0.05% (v/v) tri-
fluoroacetic acid: gradient elution: (A%): 0–3 min: 90%, 3–28 min:
gradient from 90% to 0%, 28–31 min: 0%, 31–31.5 min: gradient
from 0% to 90%, 31.5–40 min: 90%. According to two different HPLC
methods the purity of all test compounds was greater than 95%.
5.1.2. Synthetic procedures
5.1.2.1. (2r)-2-Nitrocyclohexane-1,3-diol (6)13,14
.
An aque-
ous solution of glutaraldehyde (4, 25 %, 182 mL, 460 mmol), nitro-
methane (5, 38 mL, 0.71 mol) and CH3OH (600 mL) was cooled to
0–5 °C. Then 2 M NaOH (12 mL) was added dropwise and the mix-
ture was stirred at rt for 4 h. An acid cation exchange resin (16.8 g)
was added and the mixture was stirred for 20 min. Then it was fil-
tered and the filtrate was concentrated in vacuo. The residue was
treated with EtOH (100 mL) and toluene (250 mL) and the solvents
were removed in vacuo to obtain a solid, which was dissolved in
EtOH (a00 mL). Crystallization was induced by addition of toluene
Figure 2. [35S]GTP
employing human
c
S-binding assay of 13b (N) in comparison with U-69,593 (j)
j-opioid receptors.
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C. Bourgeois et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
(250 mL). Colorless solid, mp 154 °C, yield 58.7 g (80%). C6H11NO4
(161.2). Rf = 0.69 (CH3OH). 1H NMR (DMSO-d6): d (ppm) = 1.13–
1.32 (m, 3H, 4-H, 5-H, 6-H), 1.48–1.66 (m, 1H, 5-H), 1.74–1.86
(m, 2H, 4-H, 6-H), 3.28–3.39 (m, 2H, 1-H, 3-H), 4.17 (t,
~
5.1.2.5. (2r)-N1,N3-Bis(3,4-dichlorobenzyl)-2-nitrocyclohexane-
1,3-diamine (7d). 3,4-Dichlorobenzylamine (221 mg, 1.3 mmol)
was added to a solution of nitrodiol 6 (101 mg, 0.6 mmol) in H2O
(5 mL) and the suspension was stirred overnight at rt. A few drops
of CH3OH were added and the mixture was stirred at rt for another
24 h. The mixture was cooled to ꢀ6 °C for a few days. The resulting
precipitate was collected by filtration and dried. Colorless solid, mp
92 °C, yield 258 mg (86%). C20H21CI4N3O2 (477.2). Rf = 0.78 (CH3OH).
3J = 9.9 Hz, 1H, 2-H), 5.45 (d, 3J = 6.1 Hz, 2H, OH). IR:
m
m
(cmꢀ1) = 3.500–3.100 (s,
m (OH)), 1550 (s, m (NO2)), 1339 (m,
(NO2)).
5.1.2.2. (2r)-N1,N3-Dibenzyl-2-nitrocyclohexane-1,3-diamine
(7a)13
The nitrodiol 6 (19.3 g, 0.12 mol) was added to a solution of
1H NMR (CDCl3): (ppm) = 1.08 (qd broad, 2J = 3J = 12.6 Hz,
d
.
3J = 3.6 Hz, 2H, 4-Ha, 6-Ha), 1.29 (qt, 2J = 3J = 13.2 Hz, 3J = 3.2 Hz,
1H, 5-Ha), 1.81 (dquint, 2J = 13.8 Hz, 3J = 3.3 Hz, 1H, 5-He), 2.16
(dq, 2J = 13.1 Hz, 3J = 3.4 Hz, 2H, 4-He, 6-He), 3.00 (td, 3J = 11.1 Hz,
3J = 4.1 Hz, 2H, 1-H, 3-H), 3.65 (d, 2J = 13.7 Hz, 2H, Ph-CH2),
3.81 (d, 2J = 13.7 Hz, 2H, Ph-CH2), 4.17 (t, 3J = 10.4 Hz, 1H, 2-H),
7.08–7.12 (m, 2H, Ph-H), 7.33–7.38 (m, 4H, Ph-H). Signals for the
~
benzylamine (26.4 mL, 0.24 mol) in H2O (60 mL) and the mixture
was stirred at rt for 16 h. The precipitate was removed by filtration
and recrystallized from CH3OH. Colorless solid, mp 91 °C, yield
37.6 g (92 %). C20H25N3O2 (339.4). Rf = 0.65 (CH3OH). 1H NMR
(CDCl3): d (ppm) = 1.10 (qd broad, 2J = 3J = 12.5 Hz, 3J = 3.6 Hz, 2H,
4-Hax, 6-Hax), 1.29 (qt, 2J = 3J = 13.4 Hz, 3J = 3.6 Hz, 1H, 5-Hax), 1.77
(dquint, 2J = 13.8 Hz, 3J = 3.5 Hz, 1H, 5-Heq), 2.16 (dq, 2J = 13.7 Hz,
3J = 3.7 Hz, 2H, 4-Heq, 6-Heq), 3.06 (td, 3J = 11.0 Hz, 3J = 4.0 Hz, 2H,
1-H, 3-H), 3.70 (d, 2J = 13.1 Hz, 2H, 2ꢂ Ph-CH2), 3.85 (d,
2J = 13.1 Hz, 2H, 2ꢂ Ph-CH2), 4.20 (t, 3J = 10.3 Hz, 1H, 2-H), 7.22–
7.32 (m, 10H, Ph-H). Signals for the NH-protons are not visible in
NH-protons are not visible in the 1H NMR spectrum. IR:
m
(cmꢀ1) = 3329 (m,
m (N-H)), 1557 (s, m (NO2)), 1359 (m, m (NO2)),
820 (m, out-of-plane (Ar-H)). MS (EI): m/z (%) = 477 (M+, <1), 174
(C7H5Cl2NH+, 56), 159 (C7H5Cl+2, 100).
5.1.2.6. (2r)-1,3-Di(pyrrolidin-1-yl)-2-nitrocyclohexane (7e).
Pyrrolidine (2.02 g, 29 mmol) was added dropwise to a solution
of nitrodiol 6 (2.09 g, 13 mmol) in H2O (21 mL) and the mixture
was stirred at rt overnight. The resulting precipitate was collected
by filtration and dried. Colorless solid, mp 98 °C, yield 3.23 g (93%),
1
the H NMR spectrum. IR:
(cmꢀ1) = 3336 (s,
m
(N-H)), 1545 (s,
m
~
m
(NO2)), 1338 (s,
m (CNO2)), 743 (s, out-of-plane (Ar-H)), 701 (s,
out-of-plane (Ar-H)). MS (EI): m/z (%) = 304 ((MꢀH2OꢀOH)+, 6),
199 ((MꢀH2OꢀOHꢀC7H7)+, 25), 91 (C7H7+, 100).
C
14H25N3O2 (267.4). Rf = 0.68 (CH3OH). 1H NMR (CDCl3):
d
5.1.2.3. (2r)-N1,N3-Bis(4-methoxybenzyl)-2-nitrocyclohexane-
1,3-diamine (7b). The nitrodiol 6 (102 mg, 0.64 mmol) was added
to a solution of 4-methoxybenzylamine (0.18 g, 1.3 mmol) in H2O
(5 mL) and the mixture was stirred at rt for 3 d. the resulting
precipitate was removed by filtration and dried. Colorless
solid, mp 94 °C, yield 226 mg (87%). C22H29N3O4 (399.5). Rf = 0.71
(CH3OH). 1H NMR (CDCl3): d (ppm) = 1.09 (qd broad, 2J = 3J =
12.3 Hz, 3J = 3.6 Hz, 2H, 4-Ha, 6-Ha), 1.28 (qt, 2J = 3J = 13.4 Hz,
3J = 3.5 Hz, 1H, 5-Ha), 1.77 (dquint, 2J = 13.7 Hz, 3J = 3.3 Hz, 1H, 5-
He), 2.14 (dq, 2J = 13.5 Hz, 3J = 3.4 Hz, 2H, 4-He, 6-He), 3.03 (td,
3J = 11.0 Hz, 3J = 4.3 Hz, 2H, 1-H, 3-H), 3.64 (d, 2J = 12.8 Hz, 2H,
Ph-CH2), 3.78 (d, 2J = 12.9 Hz, 2H, Ph-CH2), 3.79 (s, 6H, OCH3),
4.18 (t, 3J = 10.3 Hz, 1H, 2-H), 6.80–6.89 (m, 4H, ortho-Ph-H),
7.13–7.27 (m, 4H, meta-Ph-H). Signals for the NH-protons are not
(ppm) = 1.19–1.28 (m, 3H, 4-Ha, 5-Ha, 6-Ha), 1.63–1.71 (m, 8H,
N(CH2CH2)2), 1.85–1.94 (m, 3H, 4-He, 5-He, 6-He), 2.53–2.60 (m,
4H, N(CH2CH2)2), 2.67–2.73 (m, 4H, N(CH2CH2)2), 3.26 (td,
3J = 10.9 Hz, 3J = 3.2 Hz, 2H, 1-H, 3-H), 4.49 (t, 3J = 10.8 Hz, 1H, 2-
~
H). IR:
m m (NO2)), 1373 (m, m (NO2)). MS (EI):
(cmꢀ1) = 1555 (s,
m/z (%) = 268 (MH+, 4), 197 ((MꢀC4H8N)+, 27), 150 ((MꢀC4H8
NꢀHNO2)+, 100).
5.1.2.7. (2r)-N1,N3-Dibenzylcyclohexane-1,2,3-triamine (8a)12,13
.
Raney Ni (1.6 mL suspension, ca. 0.96 g Raney Ni, Sigma–Aldrich)
was added to a solution of 7a (0.34 g, 1.0 mmol) in CH3OH
(2.5 mL) and the suspension, was stirred at rt under H2 (1 bar)
for 3 h. The suspension was filtered and the filtrate was concen-
trated in vacuo. Pale yellow oil, yield 0.25 g (81%). C20H27N3
(309.5). Rf = 0.21 (CH3OH). 1H NMR (CD3OD): d (ppm) = 1.12
(qd broad, 2J = 3J = 11.6 Hz, 3J = 3.2 Hz, 2H, 4-Ha, 6-Ha), 1.22 (qt,
2J = 3J = 12.9 Hz, 3J = 2.9 Hz, 1H, 5-Ha), 1.77 (dquint, 2J = 12.8 Hz,
3J = 3.2 Hz, 1H, 5-He), 2.11 (dq, 2J = 12.5 Hz, 3J = 3.1 Hz, 2H, 4-He,
6-He), 2.21 (td, 3J = 10.1 Hz, 3J = 2.1 Hz, 2H, 1-H, 3-H), 2.25 (t,
3J = 9.0 Hz, 1H, 2-H), 3.64 (d, 2J = 12.9 Hz, 2H, Ph-CH2), 3.88 (d,
2J = 12.9 Hz, 2H, Ph-CH2), 7.20–7.36 (m, 10H, Ph-H). Signals for
~
1
visible in the H NMR spectrum. IR:
(cmꢀ1) = 3313 (s,
(C-O)), 1011 (s, m
m (N-H)),
~
m
1553 (s,
m
(NO2)), 1353 (s,
m
(NO2)), 1031 (s,
m
(C-O)), 819 (s, out-of-plane (Ar-H)), 807 (s, out-of-plane (Ar-H)).
MS (EI): m/z (%) = 399 (M+, 1), 354 ((MꢀNO2)+, 2), 136 (CH3OC7H6
NH+, 100), 121 (CH3OC7H+6, 78).
5.1.2.4. (2r)-N1,N3-Bis(4-chlorobenzyl)-2-nitrocyclohexane-1,3-
diamine (7c). 4-Chlorobenzylamine (0.90 g, 6.3 mmol) was added
to a solution of nitrodiol 6 (509 mg, 3.2 mmol) in H2O (15 mL)
and the suspension was stirred at rt for 4 d. The resulting precipi-
tate was collected by filtration and dried under high vacuum.
Colorless solid, mp 101 °C, yield 636 mg (49%). C20H23CI2N3O2
(408.3). Rf = 0.72 (CH3OH).1H NMR (CDCl3): d (ppm) = 1.08 (qd
broad, 2J = 3J = 12.5 Hz, 3J = 3.7 Hz, 2H, 4-Ha, 6-Ha), 1.27 (qt,
2J = 3J = 13.3 Hz, 3J = 3.5 Hz, 1H, 5-Ha), 1.79 (dquint, 2J = 13.9 Hz,
3J = 3.6 Hz, 1H, 5-He), 2.15 (dq, 2J = 13.1 Hz, 3J = 3.8 Hz, 2H, 4-He,
6-He), 3.00 (td, 3J = 11.0 Hz, 3J = 4.1 Hz, 2H, 1-H, 3-H), 3.65 (d,
2J = 13.4 Hz, 2H, Ph-CH2), 3.81 (d, 2J = 13.5 Hz, 2H, Ph-CH2), 4.14
(t, 3J = 10.5 Hz, 1H, 2-H), 7.16–7.20 (m, 4H, meta-Ph-H), 7.25–7.28
(m, 4 H, ortho-Ph-H). Signals for the NH-protons are not visible in
~
the NH-protons are not visible in the 1H NMR spectrum. IR:
m
(cmꢀ1) = 3303 (s,
m (N-H)) 730 (s, out-of-plane (Ar-H)), 696
(s, out-of-plane (Ar-H)).
5.1.2.8. (2r)-1,3-Di(pyrrolidin-1-yl)cyclohexan-2-amine (8e).
Raney Ni (ca. 9.0 g. Sigma–Aldrich) was added to a solution of 7e
(3.23 g, 12 mmol) in CH3OH (24 mL) and the mixture was stirred
at rt under H2 (1 bar) for 3 h. The suspension was filtered and
the solvent was removed in vacuo. Colorless oil, yield 2.33 g
(81%). C14H27N3 (237.4 g/mol). Rf = 0.16 (CH3OH). 1H NMR (CD3OD):
d (ppm) = 1.21–1.38 (m, 3H, 4-Ha, 5-Ha, 6-Ha), 1.74–1.82 (m,
8H, N(CH2CH2)2), 1.86–1.95 (m, 1H, 5-He), 2.10–2.14 (m, 4H, 1-H,
3-H, 4-He, 6-He), 2.63–2.70 (m, 4H, N(CH2CH2)2), 2.70–2.78 (m,
4H, N(CH2CH2)2), 2.89 (t, 3J = 10.8 Hz, 1H, 2-H). A signal for the
protons of the NH2 group is not visible in the 1H NMR spectrum.
~
the 1H NMR spectrum. IR:
(NO2)), 1363 (s,
m/z (%) = 408 (M+, <1), 127 (C7H6Cl+, 33), 125 (C7H6Cl+, 100).
m
(cmꢀ1) = 3322 (m,
m (N-H)), 1548 (s,
m
m (NO2)), 810 (m, out-of-plane (Ar-H)). MS (EI):
IR:
m m (N-H)), 3263 (w, m (N-H)). MS (EI): m/z
(cmꢀ1) = 3356 (w,
Please cite this article in press as: Bourgeois, C.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.054

6
C. Bourgeois et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
(%) = 238 (MH+, 2), 167 (C4H8NC6H9NH2+, 16), 150 (C4H8NC6H8, 79),
98 (C6H9NH+3, 30).
5.1.2.11. (4aRS,5SR,8aRS)-1-(4-Chlorobenzyl)-5-(4-chlorobenz-
ylamino)perhydroquinoxaline-2,3-dione (9c). Raney Ni (180
mg, Sigma–Aldrich) was added to a solution of 7c (636 mg,
1.6 mmol) in CH3OH (2 mL) and ethyl acetate (20 mL) and the
mixture was stirred at rt under H2 (1 bar) for 20 h. The suspension
was filtered and the solvent was removed in vacuo. Pale yellow
5.1.2.9.
(4aRS,5SR,8aRS)-1-Benzyl-5-(benzylamino)perhydro-
quinoxaline-2,3-dione (9a). A solution of triamine 8 (100 mg,
0.32 mmol) and dimethyl oxalate (38 mg, 0.32 mmol) in CH3OH
(2.0 mL) was heated to reflux for 24 h. The solvent was evaporated
in vacuo and the residue was recrystallized from ethyl acetate. Col-
orless solid, mp 194 °C, yield 75 mg (64%). C22H25N3O2 (363.4).
oil (8c), yield 505 mg (83%).
C
22H23CI2N3O2 (432.3 g/mol).
(N-H)), 799 (m,
(cmꢀ1) = 3350–3100 (w,
m
~
Rf = 0.27 (CH3OH). IR:
m
out-of-plane (Ar-H)).
Rf = 0.61 (CH3OH). 1H NMR (CDCl3):
d
(ppm) = 1.05 (qd,
The crude product 8c (493 mg, 1.30 mmol) and dimethyl oxa-
late (154 mg, 1.30 mmol) were dissolved in CH3OH (20 mL) and
the mixture was heated to reflux for 18 h. The solvent was evapo-
rated in vacuo and the residue was recrystallized from ethyl ace-
tate. Colorless solid, mp 194 °C, yield 262 mg (47%).
2J = 3J = 12.9 Hz, 3J = 3.3 Hz, 1H, 6-Ha), 1.21 (qt, 2J = 3J = 13.4 Hz,
3J = 3.4 Hz, 1H, 7-Ha), 1.34 (qd, 2J = 3J = 12.4 Hz, 3J = 3.4 Hz, 1H, 8-
Ha), 1.85 (dquint, 2J = 13.7 Hz, 3J = 3.1 Hz, 1H, 7-He), 2.11 (dq,
2J = 12.1 Hz, 3J = 3.2 Hz, 1H, 8-He), 2.26 (dq, 2J = 12.8 Hz,
3J = 3.0 Hz, 1H, 6-He), 2.44 (td, 3J = 10.8 Hz, 3J = 3.6 Hz, 1H, 5-H),
3.13 (t, 3J = 10.5 Hz, 1H, 4a-H), 3.42 (td, 3J = 11.6 Hz, 3J = 3.9 Hz,
1H, 8a-H), 3.67 (d, 2J = 12.7 Hz, 1H, Ph-CH2-NH), 3.94 (d,
2J = 12.7 Hz, 1H, Ph-CH2-NH), 4.39 (d, 2J = 15.6 Hz, 1H, N1-CH2-Ph),
5.21 (d, 2J = 15.6 Hz, 1H, N1-CH2-Ph), 7.16–7.36 (m, 10H, Ph-H).
Signals for the NH-protons are not visible in the 1H NMR spectrum.
13C NMR (CDCl3): d (ppm) = 22.5 (C-7), 28.2 (C-8), 30.2 (C-6), 46.0
(N1-C), 50.7 (Ph-CH2), 57.7 (C-5), 58.2 (C-8a), 58.4 (C-4a), 127.3 (2
C, Ph-C), 127.7 (Ph-C), 127.8 (Ph-C), 128.5 (2 C, Ph-C), 128.9 (2 C,
Ph-C), 129.1 (2 C, Ph-C), 136.7 (2 C, quart. Ph-C), 157.5 (C-3),
~
C
22H23CI2N3O2 (432.3). Rf = 0.66 (CH3OH). 1H NMR (CDCl3): d
(ppm) = 1.07(q broad, 2J = 3J = 12.1 Hz, 3J = Hz, 1H, 6-Ha), 1.17–
1.39 (m, 2H, 7-Ha, 8-Ha), 1.88 (dquint, 2J = 13.5 Hz, 3J = 2.9 Hz, 1H,
7-He), 2.03–2.10 (m, 1H, 8-He), 2.21–2.28 (m, 1H, 6-He), 2.44 (t,
3J = 10.2 Hz, 1H, 5-H), 3.16 (t, 3J = 10.3 Hz, 1H, 4a-H), 3.42 (td,
3J = 11.4 Hz, 3J = 3.8 Hz, 1H, 8a-H), 3.65 (d, 2J = 13.0 Hz, 1H, Ph-
CH2-NH), 3.92 (d, 2J = 13.0 Hz, 1H, Ph-CH2-NH), 4.41 (d,
2J = 15.7 Hz, 1H, N1-CH2-Ph), 5.10 (d, 2J = 15.7 Hz, 1H, N1-CH2-
Ph), 7.14 (d, 3J = 8.4 Hz, 2H, meta-Ph-H), 7.23–7.31 (m, 6H, Ph-H).
Signals for the NH-protons are not visible in the 1H NMR spectrum.
~
159.5 (C-2). IR:
m
(cmꢀ1) = 3338 (m,
m
(N-H)), 3283 (m,
m
(N-H)),
IR:
(s,
m
(cmꢀ1) = 3350 (m,
m
(N-H)), 1700 (s,
m
(C@O), tert amide), 1675
1704 (s, (C=O), tert. amide), 1663 (s,
m
m
(C=O), sec. amide), 748
m (C@O), sec. amide), 796 (m, out-of-plane (Ar-H)). MS (EI): m/z
(s, out-of-plane (Ar-H)), 701 (s, out-of-plane (Ar-H)).MS (EI): m/z
(%) = 363 (M+, 46), 335 ((MꢀCO)+, 11), 272 ((MꢀC7H7)+, 81), 258
((MꢀC7H7NH)+, 95), 106 (C7H7NH+, 71), 91 (C7H+7, 100). Anal. Calcd
C, 72.70 H 6.93; N 11.56. Found C, 72.55; H, 6.79; N, 11.77. HPLC
(method 1): purity 99.6%, tR = 15.5 min. HPLC (method 2): purity
98.6%, tR = 13.5 min.
(%) = 431 (M+, 7), 308 ((MꢀC7H6Cl)+, 15), 306 ((MꢀC7H6Cl)+, 50),
142 (C7H6ClNH+, 15), 140 (C7H6ClNH+, 46), 127 (C7H6Cl+, 31), 125
(C7H6Cl+, 100). HPLC (method 1): purity 97.9%, tR = 18.1 min. HPLC
(method 2): purity 95.8%, tR = 15.7 min.
5.1.2.12. (4aRS,5SR,8aRS)-5-Amino-1-benzylperhydroquinoxa-
line-2,3-dione (10).
A mixture of benzylamine 9a (1.19 g,
5.1.2.10. (4aRS,5SR,8aRS)-1-(4-Methoxybenzyl)-5-(4-methoxy-
benzylamino)-perhydro-quinoxaline-2,3-dione (9b). Raney Ni
(360 mg, Sigma–Aldrich) was added to a solution of 7b (1.16 g,
2.91 mmol) in CH3OH (2 mL) and ethyl acetate (20 mL) and the
mixture was stirred at rt under H2 (1 bar) for 20 h. The suspension
was filtered and the solvent was removed in vacuo. Pale yellow oil
(8b), yield 928 mg (86%). C24H29N3O4 (423.5 g / mol). Rf = 0.26
~
3.28 mmol), NH4HCO2 (2.07 g, 32.8 mmol) and Pd/C (120 mg) in
CH3OH (40 mL) was heated to reflux for 2 h. The suspension was
filtered, the filtrate was concentrated in vacuo, the residue was dis-
solved in CH2Cl2 and the solution was washed with 0.1 M NaOH
(3ꢂ). The organic layer was dried (Na2SO4), filtered and concen-
trated in vacuo. Colorless solid, mp 69 °C, yield 0.89 g (97%).
C15H19N3O2 (273.3). Rf = 0.20 (CH3OH); 0.16 (CH2Cl2/MeOH/
(CH3OH). IR:
m
(cmꢀ1) = 3350–3100 (w,
m
(N–H)), 1242 (s,
m
NH3 = 9:1:0.1). 1H NMR (CD3OD): d (ppm) = 1.15–1.40 (m, 3H, 6-
Ha, 7-Ha, 8-Ha), 1.72–1.79 (m, 1H, 7-He), 1.84–1.91 (m, 1H, 6-He),
2.08–2.15 (m, 1H, 8-He), 2.61 (td, 3J = 10.0 Hz, 3J = 3.3 Hz, 1H, 5-
H), 3.20 (t, 3J = 10.1 Hz, 1H, 4a-H), 3.56 (td, 3J = 11.4 Hz,
3J = 3.9 Hz, 1H, 8a-H), 4.61 (d, 2J = 15.8 Hz, 1H, Ph-CH2), 5.05 (d,
2J = 15.8 Hz, 1H, Ph-CH2), 7.21–7.28 (m, 3H, ortho-Ph-H, para-Ph-
H), 7.30–7.36 (m, 2H, meta-Ph-H). Signals for the NH-protons are
(C–O–C)), 1031 (s,
m
(C–O–C)), 813 (s, out-of-plane (Ar-H)).
The crude product 8b (0.49 g, 1.33 mmol) and dimethyl oxalate
(0.31 g, 2.66 mmol) were dissolved in CH3OH (20 mL) and the mix-
ture was heated to reflux for 48 h. The solvent was evaporated in
vacuo and the residue was recrystallized from ethyl acetate. Color-
less solid, mp 155 °C, yield 0.19 g (33%). C24H29N3O4 (423.5).
Rf = 0.62 (CH3OH). 1H NMR (CDCl3): d (ppm) = 1.02–1.15 (m, 1H,
6-Ha), 1.15–1.28 (m, 1H, 7-Ha), 1.35 (qd, 2J = 3J = 12.3 Hz,
3J = 2.8 Hz, 1H, 8-Ha), 1.86 (dquint, 2J = 13.4 Hz, 3J = 3.0 Hz, 1H, 7-
He), 2.11–2.19 (m, 1H, 8-He), 2.21–2.29 (m, 1H, 6-He), 2.39–2.50
(m, 1H, 5-H), 3.11–3.20 (m, 1H, 4a-H), 3.39 (td, 3J = 10.7 Hz,
3J = 2.8 Hz, 1H, 8a-H), 3.62 (d, 2J = 12.6 Hz, 1H, Ph-CH2-NH), 3.78
(s, 3H, OCH3), 3.79 (s, 3H, OCH3), 3.89 (d, 2J = 12.5 Hz, 1H, Ph-
CH2-NH), 4.29 (d, 2J = 15.3 Hz, 1H, N1-CH2-Ph), 5.19 (d,
2J = 15.3 Hz, 1H, N1-CH2-Ph), 6.82 (d, 3J = 8.7 Hz, 2H, ortho-Ph-H),
6.85 (d, 3J = 8.7 Hz, 2H, ortho-Ph-H), 7.13 (d, 3J = 8.6 Hz, 2H, meta-
Ph-H), 7.23 (d, 3J = 8.2 Hz, 2H, meta-Ph-H). Signals for the NH-pro-
not visible in the 1H NMR spectrum. 13C NMR (CDCl3):
d
(ppm) = 22.7 (C-7), 28.3 (C-8), 36.0 (C-6), 46.1 (N1-CH2-Ph), 52.6
(C-5), 58.1 (C-8a), 60.0 (C-4a), 127.3 (2 C, ortho-Ph-C), 127.7
(para-Ph-C), 129.1 (2 C, meta-Ph-C), 136.8 (quart. Ph-C), 158.0 (C-
~
3), 159.6 (C-2). IR:
m m (N–H)), 1704 (s, m
(cmꢀ1) = 3300–3100 (m,
(C@O), tert amide), 1663 (s,
m (C@O), sec. amide). MS (EI): m/z
(%) = 273 (M+, 55), 255 ((MꢀNH4)+, 24), 106 (C7H7NH+, 71), 91
(C7H+7, 100). HPLC (method 1): purity 98.4%, tR = 12.4 min. HPLC
(method 2): purity 99.1%, tR = 10.9 min.
5.1.2.13. (4aRS,5SR,8aRS)-1-Benzyl-5-(pyrrolidin-1-yl)perhydro-
1
(cmꢀ1) = 3345
quinoxaline-2,3-dione (11).
A mixture of primary amine 10
~
m
tons are not visible in the H NMR spectrum. IR:
(m,
1674 (s,
m
(N–H)), 3282 (m,
m
(N–H)), 1694 (s,
m
(C@O), tert amide),
(3.06 g, 11.2 mmol), 1,4-diiodobutane (13.9 g, 44.8 mmol, 5.9 mL),
NaHCO3 (6.4 g, 76.2 mmol) and CH3CN (300 mL) was heated to
reflux for 18 h. NaHCO3 was filtered off and the solvent was
removed in vacuo. The residue was dissolved in CH2Cl2 and the
solution was extracted with 1 M HCl (3ꢂ). After addition of 2 M
NaOH until pH 8, the aqueous layer was extracted with CH2Cl2
m
(C@O), sec. amide), 811 (s, out-of-plane (Ar-H)). MS
(EI): m/z (%) = 423 (M+, 18), 302 ((MꢀH3COꢀC7H6)+, 100), 136
(H3COꢀC7H6NH+, 61), 121 (CH3OꢀC7H6+, 77), 91 (C7H+7, 10). HPLC
(method 1): purity 97.8%, tR = 16.2 min. HPLC (method 2): purity
97.1%, tR = 14.0 min.
Please cite this article in press as: Bourgeois, C.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.054

C. Bourgeois et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
7
(3ꢂ). The organic layer was dried (Na2SO4), filtered and concen-
trated in vacuo. Colorless solid, mp 104 °C, yield 2.80 g (76%).
C
(toluene-d8, 100 °C): d (ppm) = 0.87–0.96 (m, 1H, 7-Ha), 1.02 (qd,
2J = 3J = 14.4 Hz, 3J = 3.3 Hz, 1H, 5-Ha), 1.11 (qt, 2J = 3J = 13.1 Hz,
3J = 3.1 Hz, 1H, 6-Ha), 1.49–1.56 (m, 5H, 6-He, N(CH2CH2)2), 1.64–
1.71 (m, 1H, 5-He), 1.76–1.83 (m, 1H, 7-He), 1.83–1.91 (m, 1H, 3-
He), 2.34 (td, 3J = 11.0 Hz, 3J = 4.0 Hz, 1H, 8-H), 2.41–2.48 (m, 1H,
3-H), 2.50–2.59 (m, 4H, N(CH2CH2)2), 2.87 (d, 2J = 13.5 Hz, 1H, Ph-
CH2-N), 3.02–3.11 (m, 3H, 2-Ha, 2-He, 8a-H), 3.48 (d, 2J = 15.2 Hz,
1H, Ph-CH2-C@O), 3.54 (d, 2J = 15.2 Hz, 1H, Ph-CH2-C@O), 3.60–
3.66 (m, 1H, 4a-H), 3.68 (d, 2J = 13.9 Hz, 1H, Ph-CH2-N), 6.89–7.11
~
19H25N3O2 (327.4). Rf = 0.55 (CH3OH). 1H NMR (CDCl3):
d
(ppm) = 1.14–1.36 (m, 3H, 6-Ha, 7-Ha, 8-Ha), 1.68–1.82 (m, 5H,
N(CH2CH2)2 (4H), 6-He), 1.84–1.92 (m, 1H, 7-He), 2.06–2.14 (m,
1H, 8-He), 2.49–2.57 (m, 2H, N(CH2CH2)2), 2.57–2.64 (m, 2H,
N(CH2CH2)2), 2.68 (td, 3J = 11.2 Hz, 3J = 3.1 Hz, 1H, 5-H), 3.27 (t,
3J = 10.6 Hz, 1H, 4a-H), 3.46 (td, 3J = 11.3 Hz, 3J = 3.7 Hz, 1H, 8a-
H), 4.42 (d, 2J = 15.6 Hz, 1H, Ph-CH2), 5.18 (d, 2J = 15.6 Hz, 1H, Ph-
CH2), 7.17–7.33 (m, 5H, Ph-H). A signal for the NH-proton is not
visible in the 1H NMR spectrum. 13C NMR (CDCl3): d (ppm) = 20.5
(C-6), 22.4 (C-7), 23.8 (2C, N(CH2CH2)2), 28.3 (C-8), 46.0 (Ph-CH2),
47.2 (2C, N(CH2CH2)2), 56.2 (C-4a), 58.7 (C-8a), 59.6 (C-5), 127.3
(2 C, Ph-C), 127.7 (Ph-C), 129.0 (2 C, Ph-C), 136.9 (quart. Ph-C),
~
(m, 8H, Ph-H), 7.19–7.23 (m, 2H, Ph-H).IR:
m m
(cmꢀ1) = 1645 (s,
(C@O)), 729 (s, out-of-plane (Ar-H)), 696 (s, out-of-plane (Ar-H)).
MS (ESI): m/z (%) = 418 (MH+, 100). HPLC (method 1): purity
98.3 %, tR = 15.5 min. HPLC (method 2): purity 96.0 %, tR = 11.8 min.
157.6 (C-3), 159.7 (C-2). IR:
m
(cmꢀ1) = 3186 (m,
(C@O), sec. amide). MS (EI):
m
(N–H)), 1695
5.1.2.16. 1-[(4aRS,8SR,8aRS)-4-Benzyl-8-(pyrrolidin-1-yl)perhy-
droquinoxalin-1-yl]-2-(3,4-dichlorophenyl)ethan-1-one (13b).
2-(3,4-Dichlorophenyl)acetyl chloride (291 mg, 1.3 mmol) was
(s, (C@O), tert amide), 1667 (s,
m
m
m/z (%) = 327 (M+, 100), 258 ((MꢀC4H7N)+, 29), 236 ((MꢀC7H7)+,
98), 167 ((MꢀC7H7ꢀC4H7N)+, 16), 91 (C7H+7, 23). Anal. Calcd C,
69.70; H, 7.70; N, 12.83. Found C, 69.5; H, 7.66; N, 12.78. HPLC
(method 1): purity 99.6%, tR = 13.5 min. HPLC (method 2): purity
98.8%, tR = 12.0 min.
added dropwise to
a solution of quinoxaline 12 (325 mg,
1.09 mmol) in CH2Cl2 (35 mL) and the mixture was stirred at rt
for 30 min. Then 2 M NaOH (35 mL) was added and the mixture
was stirred for 2 h at rt. The aqueous layer was separated and
the organic layer was extracted with 1 M HCl (3x). Then the pH
value of the aqueous layer was adjusted to pH 8 by addition
of 2 M NaOH. The aqueous layer was extracted with CH2Cl2
(3ꢂ), the organic layer was dried (Na2SO4), filtered and concen-
trated in vacuo. Pale yellow solid, mp 73 °C, yield 458 mg (86%).
5.1.2.14.
droquinoxaline (12).
(4aRS,5SR,8aRS)-1-Benzyl-5-(pyrrolidin-1-yl)perhy-
Under N2, dry AlCl3 (45 mg, 0.33 mmol)
was dissolved in absolute THF (2.5 mL) and the solution was cooled
to 0 °C. A solution of LiAlH4 (1.0 M in THF, 1.0 mL, 1.00 mmol) was
added dropwise. The suspension was warmed to rt and stirred for
20 min. A solution of diamide 11 (59 mg, 0.18 mmol) in THF (3 mL)
was added to the freshly prepared solution of AlH3 (1.33.M AlH3) at
0 °C. The mixture was stirred at 0 °C for 45 min and at rt for 20 min.
Then 2 M NaOH (2 mL) was added dropwise under cooling and the
mixture was extracted with CH2Cl2 (5 ꢂ 15 mL). The organic layer
was dried (Na2SO4), filtered and concentrated in vacuo. Pale yellow
solid, mp 54 °C, yield 52 mg (96%). C19H29N3 (299.4). Rf = 0.24 (CH3-
C27H33CI2N3O (486.5). Rf = 0.11 (CH3OH); 0.77 (CH2Cl2/MeOH/
NH3 = 9:1:0.1). 1H NMR (toluene-d8, 100 °C): d (ppm) = 0.91 (qd,
2J = 3J = 12.1 Hz, 3J = 4.1 Hz, 1H, 7-Ha), 0.99 (qd, 2J = 3J = 12.5 Hz,
3J = 3.3 Hz, 1H, 5-Ha), 1.09 (qt, 2J = 3J = 13.1 Hz, 3J = 3.4 Hz, 1H, 6-
Ha), 1.48–1.54 (m, 5H, 6-He, N(CH2CH2)2 (4H)), 1.62–1.68 (m, 1H,
5-He), 1.77–1.83 (m, 1H, 7-He), 1.83–1.89 (m, 1H, 3-H), 2.31
(td, 3J = 10.7 Hz, 3J = 3.6 Hz, 1H, 8-H), 2.44–2.54 (m, 5H, 3-H,
N(CH2CH2)2 (4H)), 2.89 (d, 2J = 13.7 Hz, 1H, Ph-CH2-N), 2.91–2.97
(m, 1H, 2-H), 2.97–3.05 (m, 2H, 2-H, 8a-H), 3.30 (s, 2H,
Ph-CH2-C@O), 3.47–3.57 (m, 1H, 4a-H), 3.69 (d, 2J = 13.5 Hz, 1H,
Ph-CH2-N), 6.86–6.92 (m, 2H, para-Ph-H, Ph-6-H), 6.98–7.04 (m,
3H, ortho-Ph-H, Ph-5-H), 7.08–7.12 (m, 2H, meta-Ph-H), 7.24 (d,
OH); 0.71 (CH2Cl2/MeOH/NH3 = 9:1:0.1). 1H NMR (CDCl3):
d
(ppm) = 1.16 (qd, 1H, 8-Ha), 1.23–1.37 (m, 2H, 6-Ha, 7-Ha), 1.65–
1.72 (m, 4H, N(CH2CH2)2), 1.72–1.78 (m, 1H, 6-He), 1.82–1.89 (m,
1H, 7-He), 2.04 (td, 3J = 8.7 Hz, 3J = 3.7 Hz, 1H, 8a-H), 2.15–2.24
(m, 2H, 2-Ha, 8-He), 2.36 (t, 3J = 10.2 Hz, 1H, 4a-H), 2.53–2.62 (m,
4H, N(CH2CH2)2), 2.62–2.66 (m, 1H, 5-H), 2.69 (dt, 2J = 11.2 Hz,
3J = 2.5 Hz, 1H, 2-He), 2.81 (td, 2J = 3J = 11.3 Hz, 3J = 2.8 Hz, 1H, 3-
Ha), 2.92 (dt, 2J = 10.9 Hz, 3J = 2.4 Hz, 1H, 3-He), 3.13 (d,
2J = 13.2 Hz, 1H, Ph-CH2), 4.13 (d, 2J = 13.3 Hz, 1H, Ph-CH2), 7.18–
7.33 (m, 5H, Ph-H). A signal for the NH-proton is not visible in
the 1H NMR spectrum. 13C NMR (CDCl3): d (ppm) = 20.9 (C-6),
22.9 (C-7), 23.9 (2 C, N(CH2CH2)2), 28.9 (C-8), 46.1 (C-3), 47.3 (2
C, N(CH2CH2)2), 53.1 (C-2), 57.9 (Ph-CH2), 60.7 (C-5), 62.8 (C-4a),
65.9 (C-8a), 127.0 (Ph-C), 128.4 (2 C, Ph-C), 129.4 (2 C, Ph-C)
~
4J = 1.9 Hz, 1H, Ph-2-H). 13C NMR (toluene-d8, 100 °C):
d
(ppm) = 22.8 (C-6), 23.8 (2 C, N(CH2CH2)2), 24.9 (C-5), 30.5 (C-7),
40.9 (Ph-CH2-C@O), 44.8 (C-2), 47.9 (2 C, N(CH2CH2)2), 52.7 (C-3),
56.7 (Ph-CH2-N), 58.0 (C-4a), 61.5 (C-8), 66.4 (C-8a), 126.5 (2 C,
Ph-C), 128.3 (2 C, Ph-C), 129.7 (2 C, Ph-C), 130.4 (Ph-C), 130.8 (2
C, Ph-C), 132.1 (Ph-C), 136.8 (quart. Ph-C), 139.7 (quart. Ph-C),
~
169.5 (C@O). IR:
m m (C@O)), 875 (w, out-of-plane
(cmꢀ1) = 1646 (s,
(Ar-H)), 814 (w, out-of-plane (Ar-H)). MS (ESI): m/z (%) = 486 (MH+,
2ꢁ35Cl, 100), 488 (MH+, 35Cl/37Cl, 61), 490 (MH+, 2 37Cl, 9). Anal.
Calcd C, 66.66; H, 6.84; N, 8.64. Found C, 66.15; H, 6.81; N, 8.61.
HPLC (method 1): purity 99.8 %, tR = 17.5 min. HPLC (method 2):
purity 99.1%, tR = 14.2 min.
139.4 (quart. Ph-C). IR:
m m (N-H)). MS (EI): m/z
(cmꢀ1) = 3329 (w,
(%) = 299 (M+, 78), 229 ((MꢀC4H8N)+, 26), 208 ((MꢀC7H7)+, 12),
91 (C7H+7, 29). Anal. Calcd C, 76.21; H, 9.76; N, 14.03. Found C,
75.20; H, 9.89; N, 13.71. HPLC (method 1): purity 96.9%, tR = 9.5
min. HPLC (method 2): purity 97.4%, tR = 3.2 min.
5.1.2.17. 2-Phenyl-1-[(4aRS,8SR,8aSR)-8-(pyrrolidin-1-yl)perhy-
droquinoxalin-1-yl]ethan-1-one (14a).
Pd/C (10%, 52 mg)
was added to a solution of 13a (111 mg, 0.27 mmol) in THF/H2O
(1:1, 30 mL) and conc. HCl (3 mL) and the reaction mixture was
stirred at rt under H2 (1 bar) for 30 min. The suspension was fil-
tered, the organic solvent was removed in vacuo, the aqueous layer
was adjusted to pH 8 by addition of 2 M NaOH and extracted with
CH2Cl2 (5ꢂ). The organic layer was dried (Na2SO4), filtered, concen-
trated in vacuo and the residue was purified by fc (2 cm, CH2Cl2/
MeOH/NH3 = 9:1:0.1, 16 cm, 5 mL). Pale yellow resin, yield 78 mg
(90%). C20H29N3O (327.5). Rf = 0.02 (CH3OH); 0.27 (CH2Cl2/MeOH/
NH3 9:1:0.1). 1H NMR (toluene-d8, 100 °C): d (ppm) = 0.71–0.81
(m, 1H, 7-Ha), 0.94–1.03 (m, 1H, 5-Ha), 1.03–1.16 (m, 1H, 6-Ha),
1.41–1.48 (m, 2H, 6-He, 7-He), 1.48–1.55 (m, 4H, N(CH2CH2)2),
1.63–1.70 (m, 1H, 5-He), 2.35–2.45 (m, 3H, 3-Ha, 3-He, 8-H),
5.1.2.15. 1-[(4aRS,8SR,8aRS)-4-Benzyl-8-(pyrrolidin-1-yl)perhy-
droquinoxalin-1-yl]-2-phenylethan-1-one
(13a).
Phenyl-
acetyl chloride (160 mg, 1.0 mmol) was added dropwise to a
solution of 12 (257 mg, 0.86 mmol) in CH2Cl2 (30 mL) and the mix-
ture was stirred overnight at rt. Then 2 M NaOH (30 mL) was added
and the mixture was stirred for 2 h at rt. The aqueous layer was
separated, and the organic layer was extracted with 1 M HCl
(3ꢂ). Then 2 M NaOH was added to the combined aqueous layers
until pH 8. The aqueous layer was extracted with CH2Cl2 (3ꢂ)
the organic layer was dried (Na2SO4), filtered and concentrated in
vacuo. Pale yellow oil, yield 325 mg (91%). C27H25N3O (417.6).
Rf = 0.14 (CH3OH); 0.76 (CH2Cl2/MeOH/NH3 = 9:1:0.1). 1H NMR
Please cite this article in press as: Bourgeois, C.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.054

8
C. Bourgeois et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
2.48–2.58 (m, 4H, N(CH2CH2)2), 2.77 (t, 3J = 9.8 Hz, 1H, 8a-H), 2.97–
3.05 (m, 1H, 2-H), 3.07–3.15 (m, 1H, 2-H), 3.51–3.55 (m, 3H, Ph-
CH2-C@O (2H), 4a-H), 6.89–7.08 (m, 3H, ortho-Ph-H, para-Ph-H),
7.19–7.23 (m, 2H, meta-Ph-H). A signal for the NH-proton is not
~
5.2. Receptor binding studies
5.2.1. Materials and general procedures
The guinea pig brains, rat liver and rat brains for the r1,r2, and
j-opioid receptor binding assays were commercially available
visible in the 1H NMR spectrum. IR:
m
(cmꢀ1) = 3288 (w,
m
(N–
H)), 1637 (s,
m
(C@O)), 729 (s, out-of-plane (Ar-H)), 697 (s, out-
(Harlan-Winkelmann, Borchen, Germany). Homogenizer: Elvehjem
Potter (B. Braun Biotech International, Melsungen, Germany) and
Soniprep 150, MSE, London, UK). Centrifuges: Cooling centrifuge
model Rotina 35R (Hettich, Tuttlingen, Germany) and High-speed
cooling centrifuge model Sorvall RC-5C plus (Thermo Fisher
Scientific, Langenselbold, Germany). Multiplates: standard 96-well
multiplates (Diagonal, Muenster, Germany). Shaker: self-made
device with adjustable temperature and tumbling speed (scientific
workshop of the institute). Vortexer: Vortex Genie 2 (Thermo
Fisher Scientific, Langenselbold, Germany). Harvester: MicroBeta
FilterMate-96 Harvester. Filter: Printed Filtermat Typ A and B.
Scintillator: Meltilex (Typ A or B) solid state scintillator. Scintillation
analyzer: MicroBeta Trilux (all Perkin Elmer LAS, Rodgau-Jügesheim,
Germany). Chemicals and reagents were purchased from different
commercial sources and of analytical grade.
of-plane (Ar-H)). MS (ESI): m/z (%) = 328 (MH+, 100). HPLC (method
1): purity 99.9%, tR = 12.7 min.
5.1.2.18. 2-(3,4-Dichlorophenyl)-1-[(4aRS,8SR,8aRS)-8-(pyrroli-
din-1-yl)-perhydroquinoxalin-1-yl]ethan-1-one
(14b).
A
mixture of 13b (244 mg, 0.50 mmol), concd HCl (5 mL), Pd/C
(98.4 mg) and THF/H2O (1:1, 50 mL) was stirred at rt under H2
(1 bar) for 30 min. The suspension was filtered and the solvent
was removed in vacuo. The pH value of the aqueous layer was
adjusted to pH 8 by addition of 2 M NaOH. The aqueous layer
was extracted with CH2Cl2 (5ꢂ), the combined organic layers were
dried (Na2SO4), filtered, concentrated in vacuo and the residue was
purified by fc (3 cm, 17 cm, CH2Cl2/MeOH/NH3 = 9:1:0.1, 10 mL,
Rf = 0.28). Pale yellow resin, yield 186 mg (94%). C20H27CI2N3O
(396.4). Rf = 0.02 (CH3OH); 0.28 (CH2Cl2/MeOH/NH3 = 9:1:0.1). 1H
The test compound solutions were prepared by dissolving
approximately 10 lmol (usually 2–4 mg) of test compound in
NMR (toluene-d8, 100 °C):
d
(ppm) = 0.80 (qd, 2J = 11.2 Hz,
3J = 4.2 Hz, 1H, 7-Ha), 0.96 (qd, 2J = 11.8 Hz, 3J = 3.4 Hz, 1H, 5-Ha),
1.10 (qt, 2J = 3J = 13.1 Hz, 3J = 3.6 Hz, 1H, 6-Ha), 1.43–1.49 (m, 2H,
6-He, 7-He), 1.49–1.55 (m, 4H, N(CH2CH2)2), 1.65 (dq,
2J = 12.6 Hz, 3J = 1.8 Hz, 1H, 5-He), 2.37–2.45 (m, 2H, 3-Ha, 8-H),
2.46–2.53 (m, 5H, 3-He, N(CH2CH2)2 (4H)), 2.71 (t, 3J = 10.1 Hz,
1H, 8a-H), 2.92–3.04 (m, 2H, 2-Ha, 2-He), 3.29 (d, 2J = 15.4 Hz,
1H, Ph-CH2-C@O), 3.37 (d, 2J = 15.4 Hz, 1H, Ph-CH2-C@O), 3.45–
3.55 (m, 1H, 4a-H), 6.87 (dd, 3J = 7.9 Hz, 4J = 2.1 Hz, 1H, Ph-6-H),
7.03 (d, 3J = 8.2 Hz, 1H, Ph-5-H), 7.24 (d, 4J = 1.9 Hz, 1H, Ph-2-H).
A signal for the NH-proton is not visible in the 1H NMR spectrum.
13C NMR (toluene-d8, 100 °C): d (ppm) = 23.5 (C-6), 24.6 (2C,
N(CH2CH2)2), 26.2 (C-5), 33.6 (C-7), 41.7 (Ph-CH2-C@O), 46.7 (2C,
C-2, C-3), 48.7 (2C, N(CH2CH2)2), 56.8 (C-8), 58.9 (C-4a), 69.7 (C-
8a), 128.4 (Ph-C), 129.1 (Ph-C), 129.3 (Ph-C), 130.5 (Ph-C), 131.6
~
DMSO so that a 10 mM stock solution was obtained. To obtain
the required test solutions for the assay, the DMSO stock solution
was diluted with the respective assay buffer. The filtermats were
presoaked in 0.5% aqueous polyethylenimine solution for 2 h at
room temperature before use. All binding experiments were car-
ried out in duplicates in 96-well multiplates. The concentrations
given are the final concentrations in the assay. Generally, the
assays were performed by addition of 50
buffer, 50 L test compound solution in various concentrations
(10ꢀ5, 10ꢀ6, 10ꢀ7, 10ꢀ8, 10ꢀ9 and 10ꢀ10 mol/L), 50
L of corre-
sponding radioligand solution and 50 L of the respective receptor
preparation into each well of the multiplate (total volume 200 L).
lL of the respective assay
l
l
l
l
The receptor preparation was always added last. During the incu-
bation, the multiplates were shaken at a speed of 500–600 rpm
at the specified temperature. Unless otherwise noted, the assays
were terminated after 120 min by rapid filtration using the har-
vester. During the filtration each well was washed five times with
(Ph-C), 137.7 (quart. Ph-C), 170.4 (C=O). IR:
(N–H)), 1636 (s,
m
(cmꢀ1) = 3301 (w,
m (C@O)), 873 (w, out-of-plane (Ar-H)), 814
m
(w, out-of-plane (Ar-H)). MS (ESI): m/z (%) = 396 (MH+, 2ꢁ35Cl,
100), 398 (MH+, 35Cl/37Cl, 63), 400 (MH+, 2ꢁ37Cl, 10). HPLC (method
1): purity 99.8%, tR = 14.7 min. HPLC (method 2): purity 96.0%,
tR = 11.9 min.
300 lL of water. Subsequently, the filtermats were dried at 95 °C.
The solid scintillator was melted on the dried filtermats at a tem-
perature of 95 °C for 5 min. After solidifying of the scintillator at
room temperature, the trapped radioactivity in the filtermats was
measured with the scintillation analyzer. Each position on the fil-
termat corresponding to one well of the multiplate was measured
for 5 min with the [3H]-counting protocol. The overall counting
efficiency was 20%. The IC50-values were calculated with the pro-
gram GraphPad PrismÒ 3.0 (GraphPad Software, San Diego, CA,
USA) by non-linear regression analysis. Subsequently, the IC50 val-
ues were transformed into Ki-values using the equation of Cheng
and Prusoff.24 The Ki-values are given as mean value SEM from
three independent experiments.
5.1.2.19. Methyl (4aRS,8SR,8aRS)-1-[2-(3,4-dichlorophenyl)
acetyl]-8-(pyrrolidin-1-yl)-perhydroquinoxaline-4-carboxylate
(15).
Under N2, methyl chloroformate (28.9 mg, 0.31 mmol)
was added to a solution of 14b (100.9 mg, 0.25 mmol) in CH2Cl2
(13 mL) and the mixture was stirred at rt for 2 h. After evaporation
of the solvent in vacuo, the residue was purified by fc (2 cm,
16 cm, CH2Cl2/MeOH/NH3 = 9.5:0.5:0.05, 5 mL, Rf = 0.76). Pale
yellow resin, yield 67 mg (59%). C22H29CI2N3O3 (454.4). Rf = 0.17
(CH3OH); 0.76 (CH2Cl2/MeOH/NH3 9:1:0.1). 1H NMR (CD2Cl2,
ꢀ40 °C): d (ppm) = 0.74–0.85 (m, 1H, 5-Ha), 1.10–1.40 (m, 4H,
5-He, 6-Ha, 7-Ha, 7-He), 1.55–1.62 (m, 1H, 6-He), 1.63–1.75 (m, 4H,
N(CH2CH2)2), 1.82–1.92 (m, 2H, 2-H, 3-H), 2.00–2.07 (m, 1H, 8a-H),
2.56–2.71 (m, 4H, N(CH2CH2)2), 3.00–3.07 (m, 1H, 2-H), 3.44–3.54
(m, 3H, COOCH3), 3.55–3.61 (m, 2H, Ph-CH2-C@O), 3.76–3.82
(m, 1H, 8-H), 3.89–3.97 (m, 1H, 4a-H), 4.00–4.12 (m, 1H, 3-H),
6.98–7.03 (m, 1H, Ph-6-H), 7.28–7.32 (m, 1H, Ph-2-H), 7.33–7.38
~
5.2.2. Protein determination
The protein concentration was determined by the method of
Bradford,25 modified by Stoscheck.26 The Bradford solution was
prepared by dissolving 5 mg of Coomassie Brilliant Blue G 250 in
2.5 mL of EtOH (95%, v/v). 10 mL deionized H2O and 5 mL phospho-
ric acid (85%, m/v) were added to this solution, the mixture was
stirred and filled to a total volume of 50.0 mL with deionized
water. The calibration was carried out using bovine serum albumin
as a standard in 9 concentrations (0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.5, 2.0
(m, 1H, Ph-5-H). IR:
m m (NAC@OAO)), 1646 (s, m
(cmꢀ1) = 1700 (s,
(NAC@O)), 873 (w, out-of-plane (Ar-H)). MS (ESI): m/z (%) = 454
(MH+, 2ꢁ35Cl, 100), 456 (MH+, 35Cl/37Cl, 66), 458 (MH+, 2ꢁ37Cl, 10).
HPLC (method 1): purity 98.4%, tR = 18.6 min. HPLC (method 2):
purity 97.3%, tR = 15.4 min.
and 4.0 mg/mL). In a 96-well standard multiplate, 10
lL of the cal-
ibration solution or 10 L of the membrane receptor preparation
l
Please cite this article in press as: Bourgeois, C.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.054

C. Bourgeois et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
9
were mixed with 190
lL of the Bradford solution, respectively.
10 lmol/L GDP for 45 min at rt. The microtiter plates were thereaf-
After 5 min, the UV absorption of the protein-dye complex at
k = 595 nm was measured with a platereader (Tecan Genios, Tecan,
Crailsheim, Germany).
ter centrifuged for 10 min at 2,100 rpm in a GS6 microtiter plate
centrifuge (Beckman Coulter, Krefeld, Germany) to sediment the
SPA beads. The bound radioactivity was determined after a delay
of 15 min by means of a 1450 Microbeta Trilux (Wallac, Freiburg,
5.2.3. Preparation of membrane homogenates from guinea pig
brain
Germany). The enhancement of [35S]GTP
cS binding above the basal
activity was used to determine the potency (EC50) and the relative
efficacy (in % of maximal efficacy) of test compounds versus the
reference compound U-69,593, which was set as 100%. EC50 values
were calculated by means of nonlinear regression analysis with the
software ‘GraphPad Prism 4 for Windows’ (Version 4.03, GrapPad
Software, San Diego, USA).
5 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 1200ꢂg for 10 min at
4 °C. The supernatant was separated and centrifuged at 23,500ꢂg
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 23,500ꢂg
(20 min, 4 °C). This procedure was repeated twice. The final pellet
was resuspended in 5–6 volumes of buffer and frozen (ꢀ80 °C) in
1.5 mL portions containing about 1.5 mg protein/mL.
Acknowledgments
Financial support of this project by the Deutsche Forschungs-
gemeinschaft is gratefully acknowledged. We also thank Dr. Werner
Englberger, Grünenthal GmbH, Aachen, for performing the
5.2.4.
j Receptor affinity using guinea pig brain (modified
according to Refs. 17,18)
[ cS-binding assay of 13b.
35S]GTP
The assay was performed with the radioligand [3H]-U-69,593
(55 Ci/mmol, Amersham, Little Chalfont, UK). The thawed guinea
References and notes
pig brain membrane preparation (about 100 lg of the protein)
1. Vanderah, T. W. Clin. J. Pain 2010, 26, S10.
2. Scopes, D. I. C. Drugs Future 1993, 18, 933.
3. Holzgrabe, U.; Nachtsheim, C.; Siener, T.; Drohsin, S.; Brandt, W. Pharmazie
1997, 52, 4.
4. Riviére, P. J.-M. Br. J. Pharmcol. 2004, 141, 1331.
was incubated with various concentrations of test compounds,
1 nM [3H]-U-69,593, and TRIS-MgCl2-Puffer (50 mM, 8 mM MgCl2,
pH 7.4) at 37 °C. The non-specific binding was determined with
10
lM unlabeled U-69,593. The Kd-value of U-69,593 is 0.69 nM.
5. Vanderath, T. W. Clin. J. Pain 2010, 26, S10.
6. Togashi, Y.; Umeuchi, H.; Okano, K.; Ando, N.; Yoshizawa, Y.; Honda, T.;
Kawamura, K.; Endoh, T.; Utsumi, J.; Kamei, J.; Tanaka, T.; Nagase, H. Eur. J.
Pharmacol. 2002, 435, 259.
5.2.5.
j, l
and d Receptor affinity using cell lines expressing
human receptors
The affinity towards
described in Ref. 18.
7. Horwell, D. C. Drugs Future 1988, 13, 1061.
j, l and d receptors was determined as
8. Roth, B. L.; Baner, K.; Westkaemper, R.; Siebert, D.; Rice, K. C.; Steinberg, S.;
Ernsberger, P.; Rothman, R. B. PNAS 2002, 99, 11934.
9. Siebert, D. J. Ethnopharmacol. 1994, 43, 53.
10. de Costa, B. R.; Bowen, W. D.; Hellewell, S. B.; George, C.; Rothman, R. B.; Reid,
A. A.; Walker, J. M.; Jacobson, A. E.; Rice, K. C. J. Med. Chem. 1989, 32, 1996.
11. Soukara, S.; Maier, C. A.; Predoiu, U.; Ehret, A.; Jackisch, R.; Wünsch, B. J. Med.
Chem. 2001, 44, 2814.
12. Lichtenthaler, F. W.; Nakagawa, T.; El-Scherbiney, A. Angew. Chem. 1967, 79,
530.
5.2.6. r1 and r2 receptor affinity
The affinity towards r1 and r2 receptors was determined as
described in Refs. 19,20.
5.2.7. Affinity towards the phencyclidine binding site of the
NMDA receptor
13. Lichtenthaler, F. W.; Nakagawa, T.; El-Scherbiney, A. Chem. Ber. 1968, 101,
1837.
14. Eberle, M.; Missbach, M.; Seebach, D. Org. Synth. Coll. 2003, 8, 332.
15. Ram, S.; Spicer, L. D. Tetrahedron Lett. 1987, 28, 515.
16. Enders, D.; Schaumann, E. Science of Synthesis In Reduction of Carbonic and
Carboxylic Acid Derivatives; Wünsch, B., Geiger, C., Eds., 2009; 40a, pp 23–64.
17. Kracht, D.; Rack, E.; Schepmann, D.; Fröhlich, R.; Wünsch, B. Org. Biomol. Chem.
2010, 8, 212.
The affinity towards the phencyclidine binding site of the
NMDA was determined as described in Refs. 21,22.
5.3. [35S]GTP
cS binding assay
18. Geiger, C.; Zelenka, C.; Lehmkuhl, K.; Schepmann, D.; Englberger, W.; Wünsch,
B. J. Med. Chem. 2010, 53, 4212.
The [35S]-guanosine-50-3-O-(thio)triphosphate (GTP
cS) assay
19. Maier, C. A.; Wünsch, B. J. Med. Chem. 2002, 45, 4923.
20. Meyer, C.; Neue, B.; Schepmann, D.; Yanagisawa, S.; Yamaguchi, J.; Würthwein,
E.-U.; Itami, K.; Wünsch, B. Bioorg. Med. Chem. 2013, 21, 1844.
21. Köhler, J.; Bergander, K.; Fabian, J.; Schepmann, D.; Wünsch, B. J. Med. Chem.
2012, 55, 8953.
22. Banerjee, A.; Schepmann, D.; Köhler, J.; Würthwein, E.-U.; Wünsch, B. Bioorg.
Med. Chem. 2010, 18, 7855.
23. Strange, P. G. Br. J. Pharmacol. 2010, 16, 1238.
was carried out as an homogeneous scintillation proximity assay
using 1.5 mg of WGA-coated SPA-beads (Amersham, Cardiff, UK)
in microtiter luminescence plates (Costar, Cambridge MA, USA).
In order to test the agonist activity of test compounds on human
recombinant
cells expressing human
ences), 10 g membrane proteins per assay, were incubated with
0.4 nmol/L [35S]GTP
S and series concentrations of agonists in buf-
j-opioid receptors cell membranes from HEK-293-
j-opioid receptors (PerkinElmer Life Sci-
24. Cheng, Y.; Prusoff, H. W. Biochem. Pharmacol. 1973, 22, 3099.
25. Bradford, M. M. Anal. Biochem. 1976, 72, 248.
26. Stoscheck, C. Methods Enzymol. 1990, 182, 50.
l
c
fer containing 20 mmol/L HEPES pH 7.4, 100 mmol/L NaCl,
10 mmol/L MgCl2, 1 mmol/L EDTA, 1 mmol/L dithiothreitol, and
Please cite this article in press as: Bourgeois, C.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.054