Conformationally constrained κ receptor agonists: Stereoselective synthesis and pharmacological evaluation of 6,8-diazabicyclo[3.2.2]nonane Derivatives

Article abstract of DOI:10.1021/jm100182p

Three sets of stereoisomeric bicyclic κ agonists with defined orientation of the pharmacophoric elements pyrrolidine and dichlorophenylacetamide were stereoselectively prepared and pharmacologically evaluated. Stereoselective reduction, reductive amination, and Mitsunobu inversions were the key steps for the establishment of the desired stereochemistry. The κ affinity decreased in the following order depending on the N-substituent: CO₂CH₃ > benzyl > COCH ₂CH₃. Bicyclic derivatives with (1S,2R,5R)-configuration showed the highest κ receptor affinity, which led to dihedral angles of 97° and 45° for the N(pyrrolidine)-C-C-N(phenylacetamide) structural element. The most potent κ agonist of this series was (+)-methyl (1S,2R,5R)-8-[2-(3,4-dichlorophenyl)acetyl]-2-(pyrrolidin-1-yl)-6, 8-diazabicyclo[3.2.2]nonane-6-carboxylate (ent-23, WMS-0121) with an K _i value of 1.0 nM. ent-23 revealed high selectivity against the other classical opioid receptors and related receptor systems. In the [ ³⁵S]GTPγS-binding assay at human κ-opioid receptors, ent-23 was proved to be a full agonist with the same EC₅₀ value (87 nM) as the prototypical full agonist U-69,593 (EC₅₀ = 80 nM).

Full text of DOI:10.1021/jm100182p

4212 J. Med. Chem. 2010, 53, 4212–4222
DOI: 10.1021/jm100182p
Conformationally Constrained K Receptor Agonists: Stereoselective Synthesis and Pharmacological
Evaluation of 6,8-Diazabicyclo[3.2.2]nonane Derivatives†
‡
Christian Geiger, Christel Zelenka, Kirstin Lehmkuhl, Dirk Schepmann, Werner Englberger, and Bernhard Wunsch*
‡
‡
‡
§
,‡
€
‡
€
€
€
Institut fu€r Pharmazeutische und Medizinische Chemie der Westfalischen, Wilhelms-Universitat Munster, Hittorfstraβe 58-62, D-48149
Mu€nster, Germany, and §Gru€nenthal GmbH, Zieglerstraβe 6, D-52078 Aachen, Germany
Received February 11, 2010
Three sets of stereoisomeric bicyclic κ agonists with defined orientation of the pharmacophoric elements
pyrrolidine and dichlorophenylacetamide were stereoselectively prepared and pharmacologically
evaluated. Stereoselective reduction, reductive amination, and Mitsunobu inversions were the key steps
for the establishment of the desired stereochemistry. The κ affinity decreased in the following order
depending on the N-substituent: CO2CH3 > benzyl > COCH2CH3. Bicyclic derivatives with (1S,2R,
5R)-configuration showed the highest κ receptor affinity, which led to dihedral angles of 97° and 45° for
the N(pyrrolidine)-C-C-N(phenylacetamide) structural element. The most potent κ agonist of this
series was (þ)-methyl (1S,2R,5R)-8-[2-(3,4-dichlorophenyl)acetyl]-2-(pyrrolidin-1-yl)-6,8-diazabicy-
clo[3.2.2]nonane-6-carboxylate (ent-23, WMS-0121) with an Ki value of 1.0 nM. ent-23 revealed high
selectivity against the other classical opioid receptors and related receptor systems. In the [35S]GTPγ-
S-binding assay at human κ-opioid receptors, ent-23 was proved to be a full agonist with the same EC50
value (87 nM) as the prototypical full agonist U-69,593 (EC50=80 nM).
Introduction
A1,7), benzomorphans (e.g., ethylketocyclazocine1,7), arylacet-
amides (e.g., the prototypical κ agonist 2-(3,4-dichlorophenyl)-
N-methyl-N-[(1S,2S)-2-pyrrolidinocyclohexyl]acetamide (U-
50,488)8), and the nonbasic natural product salvinorin A.9,10
The prototypical κ agonist U-50,488 from the arylacet-
amide class has served as lead compound for the development
of various potent κ agonists. In particular, annulated com-
pounds,11,12 simplified ligands,13,14 as well as heterocyclic
analogues like 2-(aminomethyl)piperidines15,16 and 2-(amino-
methyl)piperazines17-19 have been developed. The (2R)-con-
figured 2-(aminomethyl)piperazine 1 (GR-89,69618) belongs
to the most potentκ agonists withan IC50 valueof 0.018 nM in
the rabbit vas deferens model17-19 and a Ki value of 0.36 nM
in receptor binding studies.20 (Figure 1) Introduction of an
additional methyl moiety into the pyrrolidinylmethyl side
chain of 1 led to the pyrrolidinylethyl derivative 2 with slightly
increased κ receptor affinity (Ki = 0.31 nM).20,21 In both
compounds 1 and 2, the stereochemistry is crucial for high κ
receptor affinity. In Figure 1, the stereoisomers with the
highest κ affinity are shown, respectively.
Because of allylic strain, the pyrrolidinylalkyl side chain in
position 2 of both 1,4-diacylpiperazines 1 and 2 is forced to
adopt an axial orientation.21 In both compounds, an almost
free rotation around the indicated axial bond is possible.22 To
investigate the bioactive conformation of 1 and 2, the con-
formational flexibility of the side chain should be reduced by
connection of the terminal methyl moiety to the piperazine
scaffold.
In the first series of ligands, the methyl moiety of 2 was
connected with the N-atom in position 4 of the piperazine
system, which resulted in the bicyclic κ agonists 3. The dia-
stereomeric compounds exo-3 (Ki > 1000 nM) and endo-3
(Ki=73 nM) showed negligible and low κ receptor affinity,
Agonists of the three classical opioid receptors, μ, κ, and δ
receptors, can be used for the treatment of strong pain, caused
e.g. by surgery, accident, or cancer. The clinically used opioid
analgesics are more orlessselectiveμ receptor agonists. There-
fore, their application is accompanied by severe side effects
like euphoria, respiratory depression, constipation, tolerance,
and dependence. The side effect profile of κ receptor agonists
differs considerably from that of μ agonists. In contrast to μ
agonists, they cause only minimal physical dependence, respi-
ratory depression, and inhibition of gastrointestinal motility.
However, sedation, dysphoria, and strong diuresis are the most
severe side effects associated with the application of κ agonists.
In addition to the strong analgesic effect, κ agonists have been
shown to be potent neuroprotective and antihyperalgesic agents
in in vivo.1,2 Moreover, growing interest is observed in periphe-
rally acting κ agonists for the selective treatment of inflamma-
tory visceral and neuropathic pain as well as pruritus in the peri-
phery, which are devoid of centrally mediated side effects.3,4
Although all three opioid receptor subtypes have already
been cloned,5,6 X-ray crystal structures of these G-protein
coupled membrane bound receptors showing the exact three-
dimensional construction of the ligand binding sites are not
yet available. In this project, compounds with high κ receptor
affinity were selected as lead compounds for the development
of novel potent and selective κ agonists.
Most of the reported κ agonists belong to four compound
classes:1,2,7 peptides (e.g., the physiological agonist dynorphin
†Dedicated to Prof. Dr. h.c. F. Eiden, Munich, on occasion of his
85th birthday.
*To whom correspondence should be addressed. Phone: þ49-251-
8333311. Fax: þ49-251-8332144. E-mail: wuensch@uni-muenster.de.
r
pubs.acs.org/jmc
Published on Web 05/04/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4213
Figure 1. Structural development of bicyclic κ receptor agonists.
Scheme 1a
a Reagents and reaction conditions: (a) LiBH4, THF, -90 °C, 84%, ref 26; (b) (1) DIAD, PPh3, 4-nitrobenzoic acid, THF, ref 26, (2) CH3OH, K2CO3, rt,
74% from 6, ref 26; (c) HN3, PPh3, DIAD, benzene, THF, rt, 98%; (d) Zn(N3)2 (pyridine)2, PPh3, DIAD, toluene, rt, 72%; (e) H2, Pd/C, 1 bar, rt, 99%; (f) H2, Pd/C,
3
1 bar, rt, 98%; (g) I(CH2)4I, CH3CN, NaHCO3, 18 h, reflux, 82% (from 8); (h) I(CH2)4I, CH3CN, NaHCO3, 16 h, reflux, 60% (from 9); (i) pyrrolidine,
NaBH(OAc)3, 1,2-dichloroethane, 0-12 °C, 76%, de 94%.
respectively, indicating that a dihedral angle (N(pyrrolidine)-
C-C-N(phenylacetamide)) of 180° (exo-3) is not tolerated
and a dihedral angle of 58° (endo-3) is better accepted by the
κ receptor protein.22,23
Chemistry
Synthesis of the designed κ agonists of type 4 started with
bicyclic ketone 5, which was derived from the proteinogenic
amino acid (S)-glutamate (Scheme 1).24-26 At first the ketone
in position 2 should be stereoselectively converted into the
desired pyrrolidine moiety. Then the orthogonal protective
groups at the N-atoms will allow successive cleavage and
introduction of different acyl residues. Because the transfor-
mations have been carefully optimized with (S)-glutamate
derived bicyclic ketone 5, the synthesesinthe reactionschemes
are shown with the corresponding enantiomers.
In a new series of conformationally constrained κ agonists,
the methyl moiety of 2 was connected with the C-atom in
position 5 of the piperazine ring. This connection leads to
6,8-diazabicyclo[3.2.2]nonanes of type 4, which allows further
modifications by introduction of various residues R at the
N-atom in position 6. Herein, we report on the stereoselec-
tive synthesis and pharmacological evaluation of all possible
stereoisomers of the κ receptor agonists 4 with a 6,8-diaza-
bicyclo[3.2.2]nonane framework.
LiBH4 reduction of 5 at -90 °C provided diastereoselec-
tively alcohol 6 (6:7=99:1), which was transformed into the

4214 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Geiger et al.
diastereomeric alcohol 7 by a Mitsunobu inversion using
p-nitrobenzoic acid (Scheme 1).26 According to our first idea,
the alcohols 6 and 7 were activated as mesylates.27 But when
the mesylates were subsequently reacted with pyrrolidine,
starting material with few elimination products was obtained
instead of the desired substitution products 12 and 13.
Scheme 2a
Therefore, a stepwise setup of the pyrrolidine ring was
envisaged (Scheme 1). For this purpose, alcohol 6 should be
converted into azide 8, which should be reduced and alkylated
to form the pyrrolidine ring. In the literature, several methods
for the direct conversion of alcohols into azides by a
Mitsunobu reaction28 have been reported. Reaction of alco-
hol 6 with diphenyl phosphoryl azide ((PhO)2PON3), triphe-
nylphosphane (PPh3), and diisopropyl azodicarboxylate
(DIADa)29 led to bicyclic azide 8 in 78% yield. Using the
complex Zn(N3)2 (pyridine)230 increased the yield of azide 8
3
a Reagents and reaction conditions: (a) pyrrolidine, NaBH3CN,
Ti(OiPr)4, CH2Cl2, CH3OH, rt, 27% of 12/13 (34:66), 44% of 14;
(b) pyrrolidine, NaBH(OAc)3, 1,2-dichloroethane, 0-12 °C, 76% of
13, 12:13 = 3:97, traces of 14.
to91%. However, the bestyield ofazide8 (98%) was obtained
using a solution of HN3 in benzene31 as azide source. Nucleo-
philic substitution of mesylate of 6 with NaN3 also gave the
bicyclic azide 8 in 70% yield, but additionally considerable
amounts of the elimination product were produced. Because
occur by a pyrrolidine induced ester cleavage, which is similar
to the ring-opening observed after treatment of ketone 5 with
of the excellent yields, HN3 and Zn(N3)2 (pyridine)2 complex
3
were the standard reagents in this project. The diastereomeric
azide 9 was synthesized from diastereomeric alcohol 7 in
a Mitsunobu reaction with Zn(N3)2 (pyridine)2 complex.
35
methanolate.34 The reaction of ketone 5 with NaBH(OAc)3
in dichloroethane at 0-12 °C without Ti(OiPr)4 provided the
pyrrolidines 12 and 13 in 76% yield with an excellent diaster-
eoselectivity of 3:97. Only traces of the amide 14 were formed
under these reaction conditions. The reductive amination of
ketone 5 represents a considerable improvement of the six-
step synthesis of pyrrolidine 13 that includes two Mitsunobu
inversion reactions. Removal of small amounts (3%) of dia-
stereomer 12 succeeded only after reduction of 13 to piper-
azine derivative 16.
Next, bicyclic dilactams 12 and 13 were reduced with
LiAlH4 to give piperazine derivatives 15 and 16 with ortho-
gonal protective groups at the N-atoms (Scheme 3). The
p-methoxybenzyl protective group at N-8 was selectively
removed with trifluoroacetic acid, and the resulting secondary
amines 17 and 18 were acylated with (3,4-dichlorophenyl)-
acetyl chloride (DCPA-Cl) to afford acetamides 19 and 20,
respectively. The benzyl protective group at N-6 was removed
hydrogenolytically. However, dechlorinated product 27 was
formed exclusively when the hydrogenolytic cleavage was
performed with ammonium formate and Pd/C in boiling
methanol (Scheme 4). Selective N-debenzylation without
dechlorination was achieved using H2 (balloon) and Pd/C in
aqueous THF. The benzylamino moiety was activated by
addition of a few drops of concentrated HCl, the reaction time
was limited to 30 min, and the reaction mixture was stirred
at room temperature. These reaction conditions led to the
(dichlorophenyl)acetamides 21 and 22 in 69% and 83% yield,
respectively. Finally, the secondary amines 21 and 22 were
acylated with methyl chloroformate or propionyl chloride
to provide the methoxycarbonyl and propionyl derivatives
23-26 in more than 90% yields, respectively.
3
Under the Mitsunobu conditions, all reactions took place
with complete inversion of configuration at position 2 and
elimination products were not formed.
The diastereomeric azides 8 and 9 were reduced with H2 in
the presence of Pd/C to give the primary amines 10 and 11 in
99% and 98% yield, respectively. To develop an one-pot
consecutive Mitsunobu-Staudinger reaction azide, 8 was
treated with PPh3 in THF to provide the primary amine 10.
A quantitative transformation of azide 8 into primary amine
10 was observed by thin layer chromatography, however, the
isolated yield of the primary amine 10 was only 37% due to
problems during separation of Ph3PO.
Reaction of the primary amines 10 and 11 with 1,4-diiodo-
butane in boiling acetonitrile led to the pyrrolidine derivatives
12 and 13. High yields of 12 (82%) and 13 (60%) were only
achieved by use of freshly distilled 1,4-diiodobutane.
Altogether, pyrrolidine 12 was stereoselectively prepared in
four reaction steps starting from ketone 5. The overall yield of
67% over four steps was obtained only, when the primary
amine 10 was directly transformed into the pyrrolidine 12.
The stereoselective synthesis of diastereomeric pyrrolidine 13
required six reaction steps starting with ketone 5 because an
additional Mitsunobu inversion (6 f 7) was necessary. The
overall yield was 26% over six steps.
To shorten the synthesis of the diastereomeric pyrrolidines
12 and 13, the direct reductive amination of ketone 5 with
pyrrolidine was investigated (Scheme 2). Reaction of ketone 5
with pyrrolidine and NaBH3CN32 at pH 6 led to a mixture of
the diastereomeric pyrrolidines 12 and 13 (yield 49%, ratio 40:
60) and the diastereomeric alcohols 6 and 7 (yield 20%, ratio
The corresponding enantiomers of the κ agonists 19/20, 23/
24, and 25/26, which are termed using the prefix ent (e.g.,
ent-19, ent-26), were prepared in the same manner.
33
75: 25). Addition of the Lewis acid Ti(OiPr)4 did not
improve the yield of pyrrolidines 12/13 (27-52%) but led to
considerable amounts of amide 14 as an additional side pro-
duct (27-44%). The formation of amide 14 does obviously
K Receptor Affinity
The κ receptor affinities of the bicyclic amines were deter-
mined in competition experiments with the radioligand [3H]-
U-69,593.20,22 Membrane homogenates prepared from guinea
pig brains were used as receptor material. Nonspecific binding
a Abbreviations: DIAD, diisopropyl azodicarboxylate; DCPA-Cl,
(dichlorophenyl)acetyl chloride; MOE, molecular operating system;
ent, enantiomeric; PCP, phencyclidine [(1-phenylcyclohexyl)piperidine];
NMDA, N-methyl-D-aspartate.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4215
Scheme 3a
a Reagents and reaction conditions: (a) LiAlH4, THF, 66 °C, 19 h; (b) LiAlH4, THF, 66 °C, 16 h; (c) CF3CO2H, CH2Cl2, 40 °C, 24 h, 60% (from 12);
(d) CF3CO2H, CH2Cl2, 40 °C, 16 h; (e) (3,4-dichlorophenyl)acetyl chloride (DCPA-Cl), CH2Cl2, rt, 3 h, 95%; (f) DCPA-Cl, CH2Cl2, rt, 16 h, 49% (from
13); (g) H2,Pd/C, THF, H2O, HCl conc, rt, 30 min, 69%; (h) H2, Pd/C, THF, H2O, HCl conc, rt, 30 min, 83%; (i) H3COCOCl, NEt3, CH2Cl2, rt, 16 h,
92%; (k) H3COCOCl, NEt3, CH2Cl2, rt, 1 h, 91%; (l) H3CCH2COCl, NEt3, CH2Cl2, rt, 16 h, 92%; (m) H3CCH2COCl, NEt3, CH2Cl2, rt, 16 h, 92%.
Scheme 4a
Table 1. Affinities of Bicyclic (3,4-Dichlorophenyl)acetamides towards
κ-Opioid Receptors
Ki ( SEM
(nM), n = 3 κ eudismic
compd
R
configuration ([3H]-U-69,593)
ratio
220
ent-220
C(dO)OCH3
(2S,10S)
(2R,10R)
0.31 ( 0.04
2.40 ( 0.81
8
19
CH2Ph
(1R,2S,5S)
(1S,2R,5R)
(1R,2R,5S)
(1S,2S,5R)
>1000 (25%a)
8.7 ( 1.3
152 ( 27
>110
2.8
ent-19
20
ent-20
a Reagents and reaction conditions: (a) NH4HCO2, Pd/C, CH3OH,
65 °C, 3.5 h, 74%.
421 ( 23
was determinedinthe presenceofa large excess of nontritiated
U-69,593 (10 μM).20,22
23
C(dO)OCH3
(1R,2S,5S)
(1S,2R,5R)
(1R,2R,5S)
(1S,2S,5R)
150 ( 4
150
6
ent-23
24
ent-24
1.0 ( 0.09
515 ( 80
80 ( 3.7
The κ receptor affinities of the bicyclic dichlorophenylace-
tamides are summarized in Table 1. A strong dependence of
the κ receptor affinity from the stereochemistry and the
N-substituent is demonstrated. Generally, the compounds
with (1S,2R,5R)-configuration ent-19, ent-23, and ent-25
show the highest κ receptor affinities with excellent eudismic
ratios. The corresponding diastereomers are considerably less
potent κ ligands.
The most potent κ agonist of this series is the (1S,2R,5R)-
configured methyl carbamate ent-23 (WMS-0121) with a Ki
value of 1.0 nM. The structural element of the methoxycar-
bonyl moiety of ent-23 has been derived from the lead
compounds 1 and 2. The bioisosteric replacement of the
oxygen atom of the methoxycarbonyl group by a methylene
moiety led to the propionyl derivative ent-25 (WMS-0122)
with a >10-fold reduced κ receptor affinity. Surprisingly, the
benzylamine ent-19 (WMS-0114) displayed a κ affinity of
8.7 nM. Although this κ affinity is considerably lower than
the κ affinity of ent-23, it demonstrates that the benzyl moiety
of ent-19 is better tolerated by the κ receptor than the propio-
nyl residue of ent-25. Moreover, ent-19 represents the first
potent κ agonist with an additional basic structural element.
25
C(dO)CH2CH3 (1R,2S,5S)
(1S,2R,5R)
(1R,2R,5S)
464 ( 98
35
ent-25
26
ent-26
13 ( 1.6
>1000 (6%a)
>1000 (35%a)
(1S,2S,5R)
U-50,488
U-69,593
naloxone
(1S,2S)
(1S,2S,4R)
0.31 ( 0.1
0.97 ( 0.4
7.3 ( 0.4
a Inhibition of the radioligand binding at a test compound concentra-
tion of 1 μM.
This additional N-atom allows further fine-tuning of the
pharmacological and pharmacokinetic properties of this com-
pound class.
Discussion of the Dihedral Angle N-C-C-N
To achieve high κ affinity, the relative orientation of the
pharmacophoric structural elements is of particular releva-
nce. Therefore, the dihedral angles N(pyrrolidine)-C-C-N-
(phenylacetamide) defining the relative orientation of the

4216 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Geiger et al.
Figure 2. Correlation of the dihedral angle N(pyrrolidine)-C-C-N(phenylacetamide) of the bicyclic compounds with their κ affinity.
Table 2. Correlation of the Dihedral Angle N(Pyrrolidine)-C-C-N-
(Phenylacetamide) of the Bicyclic Methyl Carbamates 23/24 with Their
κ Receptor Affinity
compd κ: Ki (nM) conformer dihedral anglea ΔH0 (kcal/mol)b
pharmacophoric pyrrolidine and dichlorophenylacetyl moi-
eties were calculated and correlated with the κ affinities of the
corresponding compounds. The detection of all favorable
orientations of the three-carbon bridge was of particular
interest.
23
150
I
-96
-45
-0.8
-1.2
þ2.4
þ2.3
II
The calculations were performed with the force field
MMFF94x of the Molecular Modeling Program MOE
(molecular operating environment). In Figure 2, the dihedral
angle of interest is illustrated for ent-23, the most potent κ
agonist of this series. In general, the three-carbon bridge of the
bicyclic compounds can adopt two energetically favored
conformations: in conformer I, the methylene moiety in the
middle of the bridge (3-CH2) is pointing to the “left side”
(to the dichlorophenylacetyl moiety) and in conformer II
3-CH2 is directed to the “right side” (to the methoxycarbonyl
moiety). According to AM1 calculations, the energy of the
two conformers is rather similar, with a little preference for
conformer IIinthecaseof23/ent-23and for conformer I in the
case of 24/ent-24 (Table 2). However, the small difference of
1-2 kcal/mol does not allow the unequivocal definition of the
bioactive conformation of the bicyclic κ agonists 23/ent-23
and 24/ent-24.
Recently, we have reported on a systematic conformational
analysis of the flexible κ agonist 2, showing an energy mini-
mum at a dihedral angle N(pyrrolidine)-C-C-N(phenyl-
acetamide) of 70°.22 The calculated dihedral angles of 97° and
45° for ent-23 are rather close to this energy minimum. The
difference of 25-27° might contribute tothe reducedκ affinity
of ent-23 compared with 2.
ent-23
24
1.0
I
97
45
II
515
80
I
-149
170
II
ent-24
I
150
II
-170
a N(Pyrrolidine)-C-C-N(Phenylacetamide). b ΔH0 = H0(confor-
mer II) - H0(conformer I); H0 = heat of formation calculated with
AM1.
CHO-K1 cell lines expressing the human μ- and δ-opioid
receptors served as receptor material. The following radioli-
gands were used: [3H]-Cl-977 in the κ assay, [3H]-naloxone in
the μ assay, and [3H]-deltorphine II in the δ assay.36,37
The results of the receptor binding studies are summarized
in Table 3. Generally, the κ assay using transfected HEK-293
cell lines and the radioligand [3H]-Cl-977 led to 6-10-fold
higher Ki values (lower κ affinities) compared with the assay
employing guinea pig brains and [3H]-U-69,593. However, the
relative order of κ affinities in both assays is the same. The
methyl carbamate ent-23 displays the highest κ affinity (Ki=
6.0 nM), followed by the benzylamine ent-19 (Ki=70 nM) and
the propionyl derivative ent-25 (Ki=120 nM).
The μ receptor affinity of almost all bicyclic compounds is
rather low. Thus, the most potent κ-agonists ent-19, ent-23,
and ent-25 show high selectivity against the μ-opioid receptor
with κ/μ selectivity factors of 6.7, 23, and 5.4, respectively. The
most potent κ-agonist ent-23 reveals the highest selectivity
(23fold) over the μ-opioid receptor.
The calculated dihedral angles of the stereoisomers of
ent-23 are summarized in Table 2. It is obvious that the
dihedral angles of 23, 24, and ent-24 differ considerably from
thecalculated optimal dihedral angle of70° for thevery potent
κ agonist 2. The modified orientation of the pharmacophoric
elements in the different stereoisomers might be responsible
for their various κ receptor affinities.
Selectivity against Opioid and Other Related Receptors
It should be noted that changing of the configuration in
position 2 of the potent κ agonist ent-19 led to ent-20, showing
a considerable preference for the μ-opioid receptor.
The bicyclic dichlorophenylacetamides showed only negli-
gible affinity toward the δ-opioid receptor, indicating high
selectivity of the κ agonists over the δ receptor.
€
In collaboration with Grunenthal GmbH, the κ, μ, and δ
receptor affinities of the bridged piperazine derivatives 19, 20,
23-26, and their corresponding enantiomers with the prefix
ent were investigated. In these assays, transfected HEK-293
cell lines expressing the human κ-opioid receptor and

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4217
Table 3. Affinities of Bicyclic (3,4-Dichlorophenyl)acetamides towards
κ-, μ-, and δ-Opioid Receptors
Ki ( SEM (nM)
κ [3H]-Cl-
977
μ [3H]-
naloxone
δ [3H]-deltor-
phine II
κ/μ
selectivity
compd
19
ent-19
>1 μMa
70
>1 μMa
470
>1 μMa
>1 μM
(42%b)
6.7
20
ent-20
23
920
1510
1150
180
>1 μMa
>1 μMa
>1 μMa
>1 μMa
>1 μMa
>1 μMa
>1 μMa
>1 μMa
>1 μMa
>1 μMa
1.3
0.12
>1 μMa
6.0
>1 μM (57%b)
140
ent-23
24
23
>1 μMa
850
>1 μMa
6120
ent-24
25
7.2
5.4
>1 μMa
120
>1 μMa
650
Figure 3. Intrinsic activity of the methyl carbamate ent-23 com-
pared with the full agonist U-69,593 in the [35S]GTPγS-assay. 9
U-69,593; 2 ent-23.
ent-25
26
ent-26
>1 μMa
>1 μMa
>1 μMa
>1 μMa
norbinaltor 0.34 ( 0.23
phimine
Table 5. Intrinsic Activity and EC50 Values of the Most Potent κ
Agonists in the [35S]GTPγS Assay
U-50,488
naloxone
1.5 ( 0.9
14.7 ( 9.3
compound
EC50 (nM)
intrinsic activity (%)a
2.3 ( 1.1
103
a The inhibition of the radioligand binding at a test compound
concentration of 1 μM was lower than 38%. b Inhibition of the radi-
oligand binding at a test compound concentration of 1 μM.
ent-19
ent-23
820
87
90
99
ent-25
U-69,593
1800
80
100
100
a Intrinsic activity relative to the full κ agonist U-69,593 (= 100%).
Table 4. Affinities of Bicyclic (3,4-Dichlorophenyl)acetamides towards
σ1 and σ2 Receptors and the PCP Binding Site of the NMDA Receptor
agonist, whereas its enantiomer 19 interacts preferably with σ1
and σ2 receptors.
Ki ( SEM (nM), n = 3
σ
pentazocine
1 [3H]-(þ)-
σ2 [3H]-di-o-
tolylguanidine
NMDA (PCP)
[3H]-MK-801
In the class of benzomorphans, the stereochemistry and the
N-substituent determine whether a ligand interacts with κ, σ,
or NMDA receptors.42,43 Therefore, the affinity at NMDA
receptors was also included in this study. Even at a test
compound concentration of 10 μM in the assay, the prepared
dichlorophenylacetamides did not compete with the radio-
ligand [3H]-MK-801.41
In summary, the most potent κ agonists ent-19, ent-23, and
ent-25 showed high selectivity for the κ receptor related to
μ-opioid, δ-opioid, σ1, and σ2 receptors as well as the phency-
clidine binding site of the NMDA receptor.
compd
19
ent-19
20
433 ( 39
>1 μMb
198 ( 37
3700
880
3200
>1 μMa
>1 μMa
>1 μMa
>1 μMa
>1 μMa
>1 μMa
>1 μMa
>1 μMa
>1 μMa
>1 μMa
>1 μMa
>1 μMa
1700
ent-20
23
5100
>1 μMa
>1 μMa
>1 μMa
>1 μMa
>1 μMa
>1 μMa
>1 μMa
>1 μMa
4.2 ( 1.1
3.9 ( 1.5
61 ( 18
>1 μMa
>1 μMa
624
ent-23
24
ent-24
25
1500
>1 μMa
>1 μMa
1100
ent-25
26
ent-26
2100
Functional Activity
(þ)-pentazocine
haloperidol
78 ( 2.3
42 ( 17
To demonstrate the agonist activity of the most potent κ
ligands ent-19, ent-23, and ent-25, a [35S]GTPγS-binding assay
at human κ-opioid receptors was performed. In Figure 3, the
binding curve of ent-23 is compared with the binding curve of
the prototypical full agonist U-69,593.
In this assay, the bicyclic piperazines ent-19, ent-23, and
ent-25 behaved as full κ agonists with 90-100% relative
potency compared with U-69,593 (see Table 5). The EC50
value of the most potent κ agonist ent-23 (87 nM) is compar-
able with the EC50 value of U-69,593 (80 nM), indicating a
comparable activation of the κ receptor. The less potent κ
ligands ent-19 and ent-25 show reduced EC50 values, which
correlate well with their reduced κ receptor affinities. Alto-
gether, the 8-12-fold reduced κ receptor affinity of ent-19 and
ent-25 found in the receptor bindingstudies is confirmed in the
[35S]GTPγS-assay.
di-o-tolylguanidine
MK-801
2.9 ( 0.6
a The inhibition of the radioligand binding at a test compound
concentration of 10 μM was lower than 10%. b The inhibition of the
radioligand binding at a test compound concentration of 90 μM was
50%.
It has been reported that variation of the stereochemistry or
reduction of the amide moiety of prototypical κ agonists led to
potent σ receptor ligands.8,38 Therefore, the σ1 and σ2 receptor
affinities of the bicyclic κ agonists were also recorded in recep-
tor binding studies using the radioligands [3H]-(þ)-pentazo-
cine and [3H]-di-o-tolylguanidine, respectively.39-41 Table 4
summarizes the results.
The bicyclic piperazines 23-26 with two acyl moieties at the
N-atoms showed only negligible σ1 and σ2 receptor affinities,
indicating high selectivities of the most potent κ agonists
ent-23 and ent-25 against these receptors. Considerable σ1
and σ2 receptor affinities were found for the N-benzyl derivative
19, which even exceeded its κaffinity. This result reveals that the
stereochemistry strongly influences the receptor selectivity in
this compound class, as one enantiomer (ent-19) is a selective κ
Conclusion
In conclusion, a series of bicyclic κ receptor agonists with
restricted conformational flexibility was synthesized. Com-
pounds with (1S,2R,5R)-configuration showed the highest κ
receptor affinity. The methyl carbamate ent-23 was the most

4218 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Geiger et al.
potent κ agonist, followed by the benzyl derivative ent-19 and
the propionyl derivative ent-25. The reduction of the confor-
mational flexibility of the κ agonists by introduction of the
pharmacophoric elements into a bicyclic framework allowed
the determination of their relative orientation by definition of
the dihedral angle N(pyrrolidine)-C-C-N(phenylacetamide).
For the most potent stereoisomer ent-23, dihedral angles of
97° (conformer I) and 45° (conformer II) were determined,
which are close to the optimal dihedral angle of 70°. The novel
κ agonists were selective against μ-opioid, δ-opioid, σ1, and σ2
receptors and the PCP binding site of the NMDA receptor. In
the [35S]GTPγS-assay the κ ligands showed full agonism and
ent-23 (EC50=87 nM) was equipotent with the prototypical κ
agonist U-69,593 (EC50=80 nM).
7/3 to petroleum ether/ethyl acetate 60/40, Rf=0.31 (petroleum
ether/ethyl acetate 6/4)). Colorless solid, mp 123 °C, yield 0.485
g (91%). [R]=-17.7 (c=0.815, CH2Cl2). Anal. (C22H23N5O3) C,
H; N: calcd N 17.27; found N 16.75.
Spectroscopic Data of 8. 1H NMR (CDCl3): δ (ppm) =
1.33-1.43 (m, 1 H, 3-H or 4-H), 1.68-1.80 (m, 1 H, 3-H or
4-H), 1.95-2.07 (m, 2 H, 3-H and/or 4-H), 3.81 (s, 3 H,
PhOCH3), 3.89 (td, J = 4.5/2.0 Hz, 1 H, 2-H), 3.92 (dd, J =
5.1/2.7 Hz, 1 H, 5-H), 3.98 (d, J=2.0 Hz, 1 H, 1-H), 4.00 (d, J=
14.7 Hz, 1 H, NCH2Ar), 4.48 (d, J=14.7 Hz, 1 H, NCH2Ar),
4.58 (d, J=14.7 Hz, 1 H, NCH2Ar), 5.23 (d, J=14.7 Hz, 1 H,
NCH2Ar), 6.88 (d broad, J = 8.6 Hz, 2 H, aromat 3-H,
5-Hmethoxybenzyl), 7.16 (d, J = 8.6 Hz, 2 H, aromat 2-H,
6-Hmethoxybenzyl), 7.21 (dd, J = 7.6/1.8 Hz, 2 H, aromat 2-H,
6-Hbenzyl), 7.29-7.37 (m, 3 H, aromat H).
(-)-(1S,2S,5S)-6-Benzyl-8-(4-methoxybenzyl)-2-(pyrrolidin-
1-yl)-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (12). A mixture of
Pd/C (10%, 520 mg), 8 (2.1 g, 5.17 mmol), and methanol (200
mL) was stirred under a H2 atmosphere (balloon) for 1.5 h at rt.
The catalyst was removed upon filtration over celite, and the
solvent was removed in vacuo. The residue (primary amine 10)
was dissolved in acetonitrile (200 mL). Then 1,4-diiodobutane
(6.37 g, 20.55 mmol) and NaHCO3 (2.93 g, 34.88 mmol) were
added. The mixture was refluxed for 18 h. After cooling down to
rt, the mixture was concentrated in vacuo (T < 40 °C) to a
volume of approximately 45 mL. Then 2 M HCl (400 mL) was
added, the mixture was washed with Et2O, and the aqueous
layer was made alkaline by addition of 2 M NaOH (pH 13-14)
and extracted with Et2O (5ꢀ). The combined Et2O layers were
dried (K2CO3), filtered, and evaporated in vacuo. The residue
was purified by fc (5 cm, h=15 cm, ethyl acetate, Rf=0.30).
Colorless oil, yield 1.83 g (82%). [R]=-12.9 (c=0.325, CHCl3).
Anal. (C26H31N3O3) C, H, N.
(þ)-(1R,2R,5R)-6-Benzyl-8-(4-methoxybenzyl)-2-(pyrrolidin-
1-yl)-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (ent-12). Method
A. A mixture of Pd/C (10%, 120 mg), ent-8 (489 mg, 1.21 mmol),
ammonium formate (400 mg, 6.35 mmol), and methanol (20 mL)
was heated to reflux for 2 h. It was filtered, and the solvent was
removed in vacuo. The residue was dissolved in CH2Cl2, the
solution was washed with a half-saturated solution of NaCl
(1ꢀ), dried (Na2SO4) and concentrated in vacuo. The residue
(primary amine ent-10) was dissolved in acetonitrile (40 mL),
and 1,4-diiodobutane (1.48 g, 4.79 mmol) and NaHCO3 (0.74 g,
8.81 mmol) were added and the mixture was heated to reflux for
16 h. Workup and purification were performed as described for
the enantiomer 12. Colorless oil, yield 320 mg (61%).
Method B. Under cooling with ice a solution of ent-6 (750 mg,
1.97 mmol) in dry CH2Cl2 (30 mL) was treated with NEt3
(0.81 mL, 5.84 mmol) and methanesulfonyl chloride (0.26 mL,
3.29 mmol). The mixture was stirred at rt for 1.5 h. The mixture
was washed with 0.5 M NaOH (1ꢀ) and 0.5 M HCl (1ꢀ), dried
(Na2SO4), filtered, and concentrated in vacuo. The pale-yellow
residue (mesylate of ent-6) was dissolved in DMF (20 mL),
NaN3 (950 mg, 14.6 mmol) was added, and the mixture was
stirred at 130 °C for 18 h. Then water (200 mL) was added, the
colorless precipitate (azide ent-8) was separated by filtration,
and the filtrate was extracted with Et2O (5ꢀ). The Et2O layer
was dried (Na2SO4), concentrated in vacuo, and the residue was
combined with the first precipitate. The azide ent-8 was dis-
solved in methanol (40 mL), Pd/C (10%, 200 mg), and ammo-
nium formate (660 mg, 10.5 mmol) were added, and the mixture
was heated to reflux for 1 h. After filtration, the solvent was
removed in vacuo and the residue was purified by fc (3 cm, h=
10 cm, petroleum ether/EtOH 50/10, Rf=0.16). The pale-yellow
product (primary amine ent-10, 499 mg) was dissolved in
acetonitrile (50 mL), 1,4-diiodobutane (0.70 mL, 5.31 mmol)
and NaHCO3 (750 mg, 8.93 mmol) were added, and the mixture
was heated to reflux for 16 h. Workup and purification were
performed as described for the enantiomer 12. Colorless oil,
yield 463 mg (54% calculated from the alcohol 6 over 4 reaction
Experimental Section
General. Unless otherwise noted, moisture sensitive reactions
were conducted under dry nitrogen. Flash chromatography (fc):
silica gel 60, 40-64 μm (Merck); parentheses include: diameter
of the column, height of silica gel, eluent, Rf value. Melting
point: melting point apparatus SMP 3 (Stuart Scientific), un-
corrected. Optical rotation: Polarimeter 341 (Perkin-Elmer); 1.0
dm tube; concentration c in g/100 mL; T=20 °C; wavelength 589
1
nm (D-line of Na light); unit of [R] is grad mL dm-1.g-1. H
3
3
NMR (400 MHz), 13C NMR (100 MHz): Mercury-400BB
spectrometer (Varian), δ in ppm related to tetramethylsilane,
coupling constants are given with 0.5 Hz resolution. Elemental
analyses: Vario EL (Elementaranalysesysteme GmbH). The
purity of all test compounds was proved by elemental analysis;
all values are within (0.4%. The syntheses of (3,4-dichlorophe-
nyl)acetyl chloride (DCPA-Cl), Zn(N3)2 (pyridine)2 complex,
3
HN3 solution in benzene, and 1,4-diiodobutane are detailed in
the Supporting Information.
(þ)-(1S,2S,5S)-2-Azido-6-benzyl-8-(4-methoxybenzyl)-6,8-
diazabicyclo[3.2.2]nonane-7,9-dione (8). Method A. Under N2,
triphenylphosphane (6.74 g, 25.7 mmol) and a solution of HN3
in benzene (16.0 mL, 1.4 M, 22.4 mmol) were added to a solution
of 626 (2.00 g, 5.26 mmol) in THF (100 mL). At 0 °C, diisopropyl
azodicarboxylate (DIAD, 5.0 mL, 25.7 mmol) was added and
the mixture was stirred for 10 h at rt. Then CH2Cl2 (100 mL)
was added and the mixture was extracted with 0.5 M NaOH
(100 mL). Subsequently, the organic layer was dried (K2CO3),
the solvent was removed in vacuo, and the residue was purified
by fc (8 cm, h=15 cm, elution with a gradient: petroleum ether/
ethyl acetate 70/30 to petroleum ether/ethyl acetate 60/40, Rf=
0.31 (petroleum ether/ethyl acetate 60/40)). Colorless solid, mp
124 °C, yield 2.09 g (98%).
Method B. A solution of 6 (250 mg, 0.66 mmol) and triphe-
nylphosphane (0.80 g, 3.05 mmol) in CH2Cl2 (4 mL) was diluted
with toluene (38 mL). Subsequently, Zn(N3)2 (pyridine)2
3
(0.45 g, 1.46 mmol) and diisopropyl azodicarboxylate (0.6 mL,
3.08 mmol) were added under ice cooling. The reaction mixture
was stirred for 72 h at rt. Then the solvent was removed in vacuo.
The residue was purified by fc (3 cm, h=15 cm, petroleum ether/
ethyl acetate 60/40, Rf=0.31). Colorless solid, mp 124 °C, yield
243 mg (91%). [R]=þ17.8 (c=0.965, CH2Cl2). Anal. (C22H23-
N5O3) C, H, N.
(-)-(1R,2R,5R)-2-Azido-6-benzyl-8-(4-methoxybenzyl)-6,8-
diazabicyclo[3.2.2]nonane-7,9-dione (ent-8). Under N2, triphe-
nylphosphane (1.68 g, 6.42 mmol) and a solution of HN3 in
benzene (4.0 mL, 1.4 M, 5.60 mmol) were added to a solution of
ent-626 (0.50 g, 1.31 mmol) in THF (25 mL). At 0 °C, diisopropyl
azodicarboxylate (1.25 mL, 6.43 mmol) was added and the
mixture was stirred for 2 h at rt. Then NaOH (25 mL, 2 M)
was added and the mixture was extracted with CH2Cl2 (3ꢀ). The
organic layer was washed with water, dried (K2CO3), the solvent
was removed in vacuo and the residue was purified by fc (6 cm,
h=15 cm, elution with a gradient: petroleum ether/ethyl acetate

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4219
steps). [R]=þ20.5 (c=0.17, CH2Cl2); [R]=þ13.0 (c=0.485,
CHCl3). Anal. (C26H31N3O3) H, N; C: calcd C 72.03; found C
71.59.
Spectroscopic Data of 19. 1H NMR (CDCl3): δ (ppm) =
1.41-1.92 (m, 8 H, 2ꢀ 3-H, 2ꢀ 4-H, N(CH2CH2)2), 2.47-2.65
(m, 3ꢀ 0.3 H and 4ꢀ 0.7 H, 2-HNR, N(CH2CH2)2NR, N(CH2-
CH2)2HR), 2.65-2.78 (m, 2ꢀ 0.3 H and 2ꢀ 0.7 H, N(CH2-
CH2)2NR, 2-HHR, 7-HHR), 2.82 (dd, J = 11.0/2.0 Hz, 0.3 H,
7-HNR), 2.96 (dd, J=10.9/2.7 Hz, 0.7 H, 7-HHR), 2.98-3.06 (m,
2ꢀ 0.3 H and 1ꢀ 0.7 H, 5-HHR, 5-HNR, 7-HNR), 3.44 (d, J=10.2
Hz, 0.3 H, 9-HNR), 3.56 (dd, J = 13.7/3.3 Hz, 0.7 H, 9-HHR),
3.64-3.72 (m, 5ꢀ 0.3 H, 9-HNR, NCH2ArNR, COCH2ArNR), 3.73
(s, 2ꢀ 0.7 H, NCH2ArHR), 3.75 (dd, J = 14.1/1.6 Hz, 0.7 H,
9-HHR), 3.83 (d, J=15.3 Hz, 0.7 H, COCH2ArHR), 4.01 (d, J=15.3
Hz, 0.7 H, COCH2ArHR), 4.11 (t, J=2.7 Hz, 0.7 H, 1-HHR), 5.08
(d, J=5.5 Hz, 0.3 H, 1-HNR), 7.10 (dd, J=8.2/1.9 Hz, 0.7 H,
aromat 6-HHRdichlorophenyl), 7.15 (dd, J=8.2/2.3 Hz, 0.3 H, aromat
6-HNRdichlorophenyl), 7.22-7.40 (m, 6.7 H, aromat H), 7.44 (d, J=
1.9 Hz, 0.3 H, aromat 2-HNRdichlorophenyl). Ratio of rotamers
70(HR):30(NR).
Spectroscopic Data of 12. 1H NMR (CDCl3): δ (ppm) =
1.22-1.36 (m, 1 H, 4-H), 1.56-1.69 (m, 1 H, 3-H), 1.70-1.83
(m, 4 H, N(CH2CH2)2), 1.90-2.05 (m, 2 H, 3-H, 4-H),
2.45-2.58 (m, 5 H, N(CH2CH2)2, 2-H), 3.80 (s, 3 H, PhOCH3),
3.85 (d, J = 5.5 Hz, 1 H, 5-H), 4.02 (d, J = 14.9 Hz, 1 H,
NCH2Ar), 4.09 (s broad, 1 H, 1-H), 4.38 (d, J=14.5 Hz, 1 H,
NCH2Ar), 4.64 (d, J=14.9 Hz, 1 H, NCH2Ar), 5.33 (d, J=14.5
Hz, 1 H, NCH2Ar), 6.86 (d broad, J=8.6 Hz, 2 H, aromat 3-H,
5-Hmethoxybenzyl), 7.13 (d, J = 8.6 Hz, 2 H, aromat 2-H,
6-Hmethoxybenzyl), 7.21 (d broad, J = 7.8 Hz, 2 H, aromat H),
7.25-7.33 (m, 3 H, aromat H).
(þ)-(1R,2S,5S)-6-Benzyl-2-(pyrrolidin-1-yl)-6,8-diazabicyclo-
[3.2.2]nonane (17). LiAlH4 (725 mg, 19.1 mmol) was carefully
added to an ice cold solution of 12 (1.66 g, 3.82 mmol) in THF
(220 mL). The reaction mixture was heated to reflux for 19 h.
A small amount of water was carefully added, it was filtered, and
the solvent was removed in vacuo. The oily residue (15) was
dissolved in CH2Cl2 (50 mL), trifluoroacetic acid (50 mL) was
added, and the mixture was heated to reflux for 24 h. After
cooling down to rt, the mixture was poured into cold water
(300 mL) and made alkaline by addition of solid NaOH (pH
13-14). The aqueous layer was extracted with CH2Cl2 (5ꢀ), and
the organic layer was dried (K2CO3) and concentrated in vacuo.
The residue was dissolved in 2 M HCl, washed with Et2O (2ꢀ),
made alkaline with solid NaOH, and extracted with CH2Cl2.
The combined organic layers were dried (K2CO3), concentrated
in vacuo, and the residue was purified by fc (4.5 cm, h=16 cm,
ethyl acetate/methanol/ethyldimethylamine 50/50/1, Rf=0.12).
Pale-yellow oil, yield 0.652 g (60%). [R]=þ46 (c=0.15, CHCl3).
C18H27N3 (285.4).
(-)-(1S,2R,5R)-6-Benzyl-2-(pyrrolidin-1-yl)-6,8-diazabicyclo-
[3.2.2]nonane (ent-17). As described for the synthesis of 17, the
enantiomeric dilactam ent-12 (720 mg, 1.66 mmol) was reduced
with LiAlH4 (315 mg, 8.31 mmol) in THF (95 mL) and the
residue (ent-15) was treated with trifluoroacetic acid (40 mL)
and CH2Cl2 (40 mL). Workup and fc purification gave a pale-
yellow oil, yield 309 mg (65%). [R]=-55.7 (c=0.37, CHCl3).
C23H26N2O4 (285.4).
(-)-1-[(1R,2S,5S)-2-(Pyrrolidin-1-yl)-6,8-diazabicyclo[3.2.2]-
nonan-8-yl]-2-(3,4-dichlorophenyl)ethanone (21). Pd/C (10%, 75 mg)
was added to a solution of 19 (202 mg, 0.427 mmol) in THF/
H2O (1:1, 6.2 mL) and conc HCl (0.62 mL), and the mixture was
stirred under a H2 atmosphere (balloon) for 30 min at rt. The
catalyst was filtered off, and the organic solvent was removed in
vacuo. The remaining aqueous solution was treated with solid
NaOH (pH 13-14) and subsequently extracted with CH2Cl2 (2ꢀ)
and CHCl3/ethanol (2:1, 3ꢀ). The combined organic layers were
dried (K2CO3), filtered, and concentrated in vacuo and the residue
was purified by fc (2 cm, h=19 cm, gradient CH2Cl2/MeOH/NH3
250/8/3 to CH2Cl2/MeOH/NH3 200/8/3, Rf = 0.20 (CH2Cl2/
MeOH/NH3 200/8/3)). Colorless resin, yield 113 mg (69%). [R]=
-56.5 (c = 0.115, CHCl3). C19H25Cl2N3O (382.3). 1H NMR
(CDCl3): δ (ppm) = 1.44-2.10 (m, 8 H, 2 ꢀ 3-H, 2ꢀ 4-H,
N(CH2CH2)2), 2.48-2.57 (m, 3 ꢀ 0.7 H, 2-HHR, N(CH2CH2)2HR),
2.58-2.70 (m, 3ꢀ 0.3 H and 2 ꢀ 0.7 H, N(CH2CH2)2HR, 2-HNR
,
N(CH2CH2)2NR), 2.76-2.84 (m, 2 ꢀ 0.3 H, N(CH2CH2)2NR), 3.01
(dd, J=12.3/2.1 Hz, 0.7 H, 7-HHR), 3.06 (d broad, J=12.1 Hz, 0.3
H, 7-HNR), 3.19 (dd, J=12.1/3.5 Hz, 0.7 H, 7-HHR), 3.28 (dd, 12.1/
5.7 Hz, 0.3 H, 7-HNR), 3.37 (s broad, 1 H, 5-HHR, 5-HNR), 3.47 (dd,
J=13.5/2.1 Hz, 0.7 H, 9-HHR), 3.55-3.74 (m, 4ꢀ 0.3 H, 2ꢀ 9-HNR
,
COCH2ArNR), 3.81 (d, J=14.9 Hz, 2ꢀ 0.7 H, 9-HHR, COCH2-
ArHR), 4.01 (d, J=15.3 Hz, 0.7 H, COCH2ArHR), 4.09 (s broad, 0.7
H, 1-HHR), 5.04 (d, J=5.5 Hz, 0.3 H, 1-HNR), 7.09 (dd, J=8.2/1.6
Hz, 0.7 H, aromat 6-HHdichlorophenyl), 7.17 (dd, J=8.2/2.0 Hz, 0.3 H,
aromat 6-HNRdichlorophenyl), 7.34-7.40 (m, 2ꢀ 0.7 H and 1ꢀ 0.3 H,
aromat 2-HHR, 5-HHR, 5-HNRdichlorophenyl), 7.42 (d, J = 1.6 Hz,
0.3 H, aromat 2-HNRdichlorophenyl). Ratio of rotamers 70(HR):
30(NR).
Spectroscopic Data of 17. 1H NMR (CDCl3): δ (ppm) =
1.55-1.65 (m, 1 H, 4-H), 1.70-1.88 (m, 6 H, 3-H, 4-H, N-
(CH2CH2)2), 2.06-2.15 (m, 1 H, 3-H), 2.21 (s breit, 1 H, N-H),
2.46-2.49 (m, 1 H, 2-H), 2.50-2.58 (m, 2 H, N(CH2CH2)2),
2.58-2.67 (m, 2 H, N(CH2CH2)2), 2.75 (d breit, J=12.1 Hz, 1 H,
9-H), 2.82-2.88 (m, 1 H, 5-H), 2.94 (d, J=3.1 Hz, 2 H, 7-H),
3.26-3.35 (m, 2 H, 1-H, 9-H), 3.68 (d, J = 13.3 Hz, 1 H,
NCH2Ar), 3.73 (d, J=13.7 Hz, 1 H, NCH2Ar), 7.20-7.40 (m,
5 H, aromat H).
(-)-1-[(1R,2S,5S)-6-Benzyl-2-(pyrrolidin-1-yl)-6,8-diazabicyclo-
[3.2.2]nonan-8-yl]-2-(3,4-dichlorophenyl)ethanone (19). (3,4-Di-
chlorophenyl)acetyl chloride (DCPA-Cl, 648 mg, 2.90 mmol)
was added to a solution of 17 (552 mg, 1.93 mmol) in CH2Cl2
(20 mL), and the mixture was stirred for 3 h at rt. Then
2 M NaOH (20 mL) was added, the mixture was stirred for an
additional hour at rt, and the organic layer was separated
and washed with saturated NaCl. The CH2Cl2 layer was
dried (K2CO3), filtered, concentrated in vacuo, and the residue
was purified by fc (3.5 cm, h= 17 cm, CH2Cl2/ethyl acetate/
ethyldimethylamine 90/10/1, Rf = 0.31). Pale-yellow oil, yield
869 mg (95%). [R]=-63.5 (c=0.23, CHCl3). Anal. (C26H31-
Cl2N3O) C, H, N.
(-)-Methyl (1R,2S,5S)-8-[2-(3,4-Dichlorophenyl)acetyl]-2-
(pyrrolidin-1-yl)-6,8-diazabicyclo[3.2.2]nonane-6-carboxylate
(23). Under cooling with ice, NEt3 (18 μL, 0.13 mmol) and
methyl chloroformate (15 μL, 0.195 mmol) were added to a
solution of 21 (50 mg, 0.13 mmol) in CH2Cl2 (1 mL). The
reaction mixture was stirred for 16 h at rt. Then CH2Cl2 was
added, the organic layer was washed with 0.5 M NaOH, dried
(K2CO3), and concentrated in vacuo, and the residue was puri-
fied by fc (2 cm, h=10 cm, CH2Cl2/ethyl acetate/ethyldimethyl-
amine 90/10/1, Rf = 0.13). Pale-yellow resin, yield 53 mg
(92%). [R]=-59.7 (c=0.37, CHCl3). Anal. (C21H27Cl2N3O3)
C, H, N.
(þ)-Methyl (1S,2R,5R)-8-[2-(3,4-Dichlorophenyl)acetyl]-2-
(pyrrolidin-1-yl)-6,8-diazabicyclo[3.2.2]nonane-6-carboxylate
(ent-23). As described for the synthesis of 21, the benzyl deriva-
tive ent-19 (150 mg, 0.32 mmol) was dissolved in THF (2.35 mL).
After addition of water (2.35 mL), conc HCl (0.47 mL), and Pd/
C (10%, 56 mg), the mixture was hydrogenated (balloon) for 28
min at rt. After workup and fc purification the residue (ent-21,
32 mg) was acylated with methyl chloroformate (10 μL, 0.13
mmol), NEt3 (12 μL, 0.09 mmol), and CH2Cl2 (1 mL) for 16 h at
rt as described for the synthesis of 23. Pale-yellow resin, yield
36 mg (26% from ent-19). [R]=þ57.1 (c=0.34, CHCl3). Anal.
(þ)-1-[(1S,2R,5R)-6-Benzyl-2-(pyrrolidin-1-yl)-6,8-diazabi-
cyclo[3.2.2]nonan-8-yl]-2-(3,4-dichlorophenyl)ethanone (ent-19).
As described for the synthesis of 19 the enantiomeric secondary
amine ent-17 (309 mg, 1.08 mmol) was acylated with DCPA-Cl
(360 mg, 1.61 mmol) in CH2Cl2 (11 mL). Pale-yellow oil, yield
475 mg (93%). [R]=þ60.0 (c=0.25, CHCl3). Anal. (C26H31-
Cl2N3O) C, H, N.

4220 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Geiger et al.
(C21H27Cl2N3O3) H; C, N: calcd C 57.28, N 9.54; found C 56.84,
N 9.11.
5-HHR(2)þNR(2)), 4.22-4.27 (broad signal, 0.35 H, 1-HHR(1)),
4.29-4.33 (broad signal, 0.35 H, 1-HHR(2)), 4.73-4.79 (broad
signal, 0.35 H, 5-HHR(1)), 4.83-4.89 (broad signal, 0.2 H, 5-HNR(1)),
5.18-5.25 (broad signal, 0.2 and 0.1 H, 1-HNR(1)þNR(2)), 7.03-7.12
(m, 1 H, aromat 6-H), 7.31-7.39 (m, 2 H, aromat 2-H, 5-H). Ratio
of rotamers 35(HR1):35(HR2):20(NR1):10(NR2).
Receptor Binding Studies. K Receptor Affinity Using Guinea
Pig Brain Membrane Preparations. Membrane Preparation for
the K Assay (Modified According to refs 20,22). Five guinea pig
brains were homogenized with the potter (500-800 rpm, 10 up-
and-down strokes) in 6 volumes of cold 0.32 M sucrose. The
suspension was centrifuged at 1200g for 10 min at 4 °C. The
supernatant was separated and centrifuged at 23500g 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 23500g (20 min,
4 °C). This procedure was repeated twice. The final pellet was
resuspended in 5-6 volumes of buffer, the protein concentra-
tion was determined according to the method of Bradford44
using bovine serum albumin as standard, and subsequently the
preparation was frozen (-80 °C) in 1.5 mL portions containing
about 1.5 mg protein/mL.
Performing of the K Assay (Modified According to refs 20,22).
The test was performed with the radioligand [3H]-U-69,593 (55
Ci/mmol, Amersham, Little Chalfont, UK). The thawed mem-
brane preparation (about 75 μg of the protein) was incubated
with various concentrations of test compounds, 1 nM [3H]-U-
69,593, and TRIS-MgCl2 buffer (50 mM, 8 mM MgCl2, pH 7.4)
in a total volume of 200 μL for 150 min at 37 °C. The incubation
was terminated by rapid filtration through the presoaked filter-
mats using a cell harvester. After washing each well five times
with 300 μL of water, the filtermats were dried at 95 °C.
Subsequently, the solid scintillator was placed on the filtermat
and melted at 95 °C. After 5 min, the scintillator was allowed to
solidify at rt. The bound radioactivity trapped on the filters was
counted in the scintillation analyzer. The nonspecific binding
was determined with 10 μM unlabeled U-69,593. The Kd value of
U-69,593 is 0.69 nM.
Data Analysis. All experiments were carried out in triplicates
using standard 96-well-multiplates (Diagonal). The IC50 values
were determined in competition experiments with six concen-
trations of the test compounds and were calculated with the
program GraphPad Prism 3.0 (GraphPad Software) by non-
linear regression analysis. The Ki values were calculated accord-
ing to Cheng and Prusoff.45 The Ki values are given as mean
values ( SEM from three independent experiments.
Spectroscopic Data of 23. 1H NMR (CDCl3): δ (ppm) =
1.46-2.01 (m,
8
H, N(CH2CH2)2, 2ꢀ 3-H, 2ꢀ 4-H),
2.41-2.49 (m, 2ꢀ 0.4 H, 2-HHR(1)þHR(2)), 2.49-2.66 (m, 8ꢀ
0.4 H, N(CH2CH2)2HR(1)þHR(2)), 2.66-2.86 (m, 10ꢀ 0.1 H,
2-HNR(1)þNR(2), N(CH2CH2)2NR(1)þNR(2)), 3.32-4.40 (m, 2ꢀ
0.4 H, 7-H HR(1)þHR(2)), 3.42-3.52 (m, 2ꢀ 0.4 H and 4ꢀ 0.1
H, 9-HHR(1)þHR(2), 2ꢀ 7-HNR(1)þNR(2)), 3.54-3.62 (m, 2ꢀ 0.4
H, 7-HHR(1)þHR(2)), 3.62-3.76 (m, 3 H and 8ꢀ 0.1 H, OCH3,
COCH2ArNR(1)þNR(2), 2ꢀ 9-HNR(1)þNR(2)), 3.78-3.94 (m, 4ꢀ
0.4 H, COCH2ArHR(1)þHR(2), 9-H HR(1)þHR(2)), 3.99-4.10 (m,
2ꢀ 0.4 H, COCH2ArHR(1)þHR(2)), 4.20-4.24 (broad signal, 0.4
H, 1-HHR(1)), 4.27-4.32 (broad signal, 2ꢀ 0.4 H, 5-HHR(1)
,
1-HHR(2)), 4.33-4.37 (broad signal, 0.1 H, 5-HNR(1)), 4.40-4.45
(broad signal, 0.4 H, 5-HHR(2)), 4.47-4.53 (broad signal, 0.1 H,
5-HNR(2)), 5.16-5.20 (broad signal, 0.1 H, 1-HNR(1)), 5.20-5.24
(broad signal, 0.1 H, 1-HNR(2)), 7.09 (d broad, J=7.8 Hz, 0.8 H,
aromat 6-H HR(1)þHR(2)), 7.13 (d broad, J = 8.2 Hz, 0.2 H,
aromat 6-HNR(1)þNR(2)), 7.34-7.41 (m, 2 H, aromat 2-H, 5-H).
Ratio of rotamers: 40(HR1):40(HR2):10(NR1):10(NR2).
(-)-1-{(1R,2S,5S)-8-[2-(3,4-Dichlorophenyl)acetyl]-2-(pyrro-
lidin-1-yl)-6,8-diazabicyclo[3.2.2]nonan-8-yl}propan-1-one (25).
Propanoyl chloride (25 μL, 0.286 mmol) was added under ice
cooling to a solution of 21 (55 mg, 0.144 mmol) in CH2Cl2
(1 mL) and NEt3 (20 μL, 0.144 mmol), and the mixture was
stirred for 16 h at rt. Then CH2Cl2 was added, the mixture was
extracted with 2 M NaOH, and the organic layer was dried
(K2CO3), concentrated in vacuo, and the residue was purified by
fc (2 cm, h=15 cm, CH2Cl2/ethyl acetate/ethyldimethylamine
90/10/1, Rf = 0.36). Colorless resin, yield 58 mg (92%). [R]=
-60.1 (c=0.506, CHCl3). Anal. (C22H29Cl2N3O2) C, H, N.
(þ)-1-{(1S,2R,5R)-8-[2-(3,4-Dichlorophenyl)acetyl]-2-(pyrro-
lidin-1-yl)-6,8-diazabicyclo[3.2.2]nonan-8-yl}propan-1-one (ent-25).
The benzyl derivative ent-19 (50 mg, 0.106 mmol) was dissolved
in THF (0.8 mL). Water (0.8 mL), conc HCl (0.16 mL), and
Pd/C (10%, 18.7 mg) were added and the mixture was stirred
under H2 (balloon) for 30 min at rt. This procedure was repeated
with ent-19 (47 mg, 0.10 mmol). After combining the reaction
mixtures, it was filtered and concentrated in vacuo and the
residue was purified by fc (2 cm, h=15 cm, gradient CH2Cl2/
MeOH/NH3 300/8/3 to CH2Cl2/MeOH/NH3 175/8/3, Rf=0.20
(CH2Cl2/MeOH/NH3 200/8/3)]. The resulting colorless oil
(ent-21, 38 mg, 50%) was dissolved in CH2Cl2 (1 mL), NEt3
(16 μL, 0.115 mmol), and propanoyl chloride (19 μL, 0.217
mmol) were added, and the mixture was stirred for 16 h at rt.
Then CH2Cl2 was added, the mixture was extracted with 2 M
NaOH, and the organic layer was dried (K2CO3), concentrated
in vacuo, and the residue was purified by fc (2 cm, h=15 cm,
CH2Cl2/ethyl acetate/ethyldimethylamine 90/10/1, Rf = 0.36).
Colorless resin, yield 38 mg (42% from ent-19). [R]=þ58.1 (c=
0.26, CHCl3). Anal. (C22H29Cl2N3O2) C, H; N: calcd N 9.59;
found N 10.10.
K, μ, and δ Receptor Affinity Using Cell Lines Expressing
Human Receptors. The affinity toward κ, μ, and δ receptors was
determined as described in refs 36,37
σ1 and σ2 Receptor Affinity. The affinity toward σ1 and σ2
receptors was determined as described in refs 39-41
Affinity toward the Phencyclidine Binding Site of The NMDA
Receptor. The affinity toward the phencyclidine binding site of
the NMDA was determined as described in ref 41.
[35S]GTPγS Binding Assay. The [35S]-guanosine-50-3-O-(thio)-
triphosphate (GTPγS) assay 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). To test the agonist activity of
test compounds on human recombinant κ-opioid receptors, cell
membranes from HEK-293-cells expressing human κ-opioid
receptors (PerkinElmer Life Sciences), 10 μg membrane proteins
per assay, were incubated with 0.4 nmol/L [35S]GTPγS and
series concentrations of agonists in buffer 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 10 μmol/L GDP for
45 min at rt. The microtiter plates were thereafter centrifuged for
10 min at 2100 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,
Spectroscopic Data of 25. 1H NMR (CDCl3): δ (ppm) =
1.07-1.18 (m, 3 H, CH2CH3), 1.46-2.01 (m, 7 and 0.35 H
and 0.2 H, N(CH2CH2)2, 2ꢀ 3-H, 1ꢀ 4-H, 4-HHR(1)þNR(1)),
2.03-2.12 (m, 0.35 and 0.1 H, 4-HHR(2)þNR(2)), 2.16-2.44 (m, 2
H and 2ꢀ 0.35 H, CH2CH3, 2-HHR(1)þHR(2)), 2.46-2.68 (m,
4 H, N(CH2CH2)2, 2.69-2.75 (m, 1ꢀ 0.2 H and 1ꢀ 0.1 H,
2-HNR(1)þNR(2)), 3.29 (dd, J=13.0/3.7 Hz, 0.35 H, 7-HHR(2)),
3.36-3.74 [In this m, a broad d (J=13.7 Hz) at 3.40 ppm for
9-HHR(1) is seen and at 3.47 ppm appears the broad d (J=13.7
Hz) for 9-HHR(2).] (m, 4ꢀ 0.35 H and 6ꢀ 0.2 H and 6ꢀ 0.1 H, 2ꢀ
7-HHR(1)þNR(1)þNR(2), 9-HHR(1)þHR(2), 2ꢀ 9-HNR(1)þNR(2), 2ꢀ
COCH2ArNR(1)þNR(2)), 3.79 (d, J = 15.3 Hz, 0.35 H, CO-
CH2ArHR(2)), 3.82-3.94** [In this m, a d (J = 15.3 Hz)
at 3.85 ppm for COCH2ArHR(1) is seen.] (m, 4ꢀ 0.35 H,
9-HHR(1)þHR(2), 7-HHR(2), COCH2ArHR(1)), 4.00 (d, J = 15.3
Hz, 0.35 H, COCH2ArHR(1)), 4.05 (d, J = 15.3 Hz, 0.35 H,
COCH2ArHR(2)), 4.11-4.18 (broad signal, 0.35 and 0.1 H,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4221
Germany). The enhancement of [35S]GTPγS 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, CA).
Molecular Modeling. The conformational analysis was per-
formed with force field MMFF94x of the Molecular Modeling
Program MOE (molecular operating environment), version
2008.10 (Chemical Computing Group AG).
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