Novel hexahydrospiro[piperidine-4,1′-pyrrolo[3,4-c]pyrroles: Highly selective small-molecule nociceptin/orphanin FQ receptor agonists

Article abstract of DOI:10.1021/jm0209174

Novel hexahydrospiro[piperidine-4,1′-pyrrolo[3,4-c]pyrroles that act as potent and selective orphanin FQ/nociceptin (N/OFQ) receptor (NOP) agonists were identified. The best compound, (+)-5a, potently inhibited ³H-N/OFQ binding to the NOP recep

Full text of DOI:10.1021/jm0209174

J . Med. Chem. 2003, 46, 255-264
255
Novel Hexa h yd r osp ir o[p ip er id in e-4,1′-p yr r olo[3,4-c]p yr r oles]: High ly Selective
Sm a ll-Molecu le Nocicep tin /Or p h a n in F Q Recep tor Agon ists
Sabine Kolczewski,* Geo Adam, Andrea M. Cesura, Franc¸ois J enck,† Michael Hennig, Thomas Oberhauser,
Sonia M. Poli, Felix Ro¨ssler, Stephan Ro¨ver, J u¨rgen Wichmann, and Frank M. Dautzenberg*,‡
Pharma Division, Preclinical Research, F. Hoffmann-La Roche Ltd., Grenzacher Strasse 124, CH-4070 Basel, Switzerland
Received April 22, 2002
Novel hexahydrospiro[piperidine-4,1′-pyrrolo[3,4-c]pyrroles that act as potent and selective
orphanin FQ/nociceptin (N/OFQ) receptor (NOP) agonists were identified. The best compound,
3
(+)-5a , potently inhibited H-N/OFQ binding to the NOP receptor (Ki ) 0.49 nM) but was
>1000-fold less potent in binding to MOP, KOP, and DOP opiate receptors. Further, (+)-5a
potently stimulated GTPγS binding to NOP membranes (EC50 ) 65 nM) and inhibited forskolin-
mediated cAMP accumulation in NOP-expressing cells (EC50 ) 9.1 nM) with a potency
comparable to that of the natural peptide agonist N/OFQ. These results indicate that (+)-5a
is a highly selective and potent small-molecule full agonist of the NOP receptor.
Sch em e 1a
In tr od u ction
The 17 amino acid peptide orphanin FQ/nociceptin (N/
OFQ)1,2 is the endogenous ligand of the opiate-like
receptor NOP (previously named ORL1).3 NOP, like
DOP (δ), KOP (κ), and MOP (µ), is mainly coupled to
the inhibitory G protein (Gi), which results in the
inhibition of cAMP production upon receptor activation.
Furthermore, it has been shown that NOP may modu-
late neuronal Ca2+ and K+ channels in vivo.4
NOP plays important physiological roles in pain
transmission,5 cognition,6 anxiety,7 and anorexia ner-
vosa.8 We have recently published several studies on
potent small-molecule NOP receptor agonists;9-11 the
most potent compound, Ro 64-6198, was >100-fold
selective for the NOP receptor12 and displayed anxio-
lytic-like activity without causing tolerance in vivo.11-13
Although this compound may mediate its in vivo effects
through the NOP receptor, it also shows moderate
affinity toward KOP and MOP receptors as well as
toward other receptor sites.12,13
In this paper we report the synthesis and character-
ization of a series of novel hexahydrospiropiperidine-
4,1′-pyrrolo[3,4-c]pyrroles with improved selectivities
versus opioid receptors.
a
(a) Aniline, molecular sieves 4 Å, pentane; (b) (CH3)3SiCH2OTf,
CsF, dimethoxyethane; (c) N-methylmaleimide; (d) LiAlH4, CH2Cl2,
ether.
Ch em istr y
and ylide generation with cesium fluoride.15 Imine 2a
was obtained from the corresponding piperidone 1a and
aniline by stirring in pentane at room temperature in
the presence of molecular sieves.16
Racemic N-methylated hexahydropyrrolo[3,4-c]pyr-
role rac-5a was resolved by fractional crystallization
with O,O-dibenzoyltartaric acid, and both enantiomers
(+)-5a and (-)-5a were obtained with >98% ee. The
absolute configuration was determined by X-ray crystal-
lography of the tartrate salt of (+)-5a .
In cases where the N-substituted maleimides were not
commercially available or gave insufficient yields in the
[2 + 3] cycloaddition, the intermediate imides (rac-4a ,
rac-4b, rac-4d -f, rac-4m ) were synthesized via Mit-
sunobu reaction of rac-4g and rac-4l, which gave
exclusively N-alkylation as outlined in Scheme 2.17
Spirocyclic hexahydropyrrolo[3,4-c]pyrroles, for ex-
ample, rac-5a , were synthesized as outlined in Scheme
1. The key step is a [2 + 3] cycloaddition of N-
methylmaleimide and azomethine ylide 3a to give rac-
4a and subsequent reduction with lithium aluminum
hydride. The azomethine ylide 3a was generated in situ
from the imine precursor 2a by sequential alkylation
with (trimethylsilyl)methyl trifluoromethanesulfonate
* To whom correspondence should be addressed. For S.K.: phone,
+41-61-6880404; fax, +41-61-6888714, e-mail, sabine.kolczewski@
roche.com. For F.M.D.: phone, +41-61-4877678; fax, +41-61-4877600;
e-mail, frank.dautzenberg@axovan.com.
† Present address: Actelion Ltd., Innovation Center, Gewerbestrasse
14, 4123 Allschwil, Switzerland.
‡ Present address: Axovan Ltd., Innovation Center, Gewerbestrasse
16, 4123 Allschwil, Switzerland.
10.1021/jm0209174 CCC: $25.00 © 2003 American Chemical Society
Published on Web 12/11/2002

256 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2
Kolczewski et al.
Sch em e 2a
Sch em e 4a
a
(a) PPh3, EtO2C-NdN-CO2Et, R2OH, THF.
Sch em e 3a
a
(a) 5% Rh/Al2O3, 100 bar of H2, i-PrOH; (b) 1-ethyl-1-methyl-
4-oxopiperidinium iodide (7), K2CO3, i-PrOH, H2O.
Sch em e 5a
a
(a) LiAlH4, THF; (b) H2, Pd/C, EtOH; (c) 1-ethyl-1-methyl-4-
oxopiperinium iodide (7), K2CO3, EtOH, H2O.
a
(a) H2, Pd/C, HOAc, MeOH; (b) (1) ketone, Ti(i-PrO)4, THF,
(2) NaBH3CN, EtOH; (c) LiAlH4, CH2Cl2, ether.
11. With Rh/C (Degussa G 101 S/G) in hexane, 8 and 9
were formed in a 4:1 ratio along with ca. 50% 11.
Addition of NH3 to the hydrogenation decreased the
dimer formation to 11%. Unfortunately, 11% starting
material also remained. The best results were obtained
with Rh/Al2O3 (Degussa G 207 R/D) in i-PrOH: com-
plete conversion was achieved; 8 and 9 were obtained
in a 2:1 ratio; and only about 5% 11 formed. In a
Hofmann elimination and Michael addition sequence,
a mixture of 8 and 9 was reacted with 1-ethyl-1-methyl-
4-oxopiperinium iodide (7)19 to give the piperidones 1a
and 10. The desired cis-piperidone 1a was separated
from the trans compound 10 by careful chromatography
on silica gel.
Racemic (2RS,4aSR,8aRS)1-(decahydronaphthalen-
2yl)piperidin-4-one (rac-1k ) was synthesized starting
from the corresponding benzamide rac-1220 by reduction
and subsequent hydrogenation to give free amine rac-
14, which in turn was converted to the piperidone rac-
1k as shown in Scheme 5.
Cyclononyl (rac-4i) and cyclodecyl (rac-4j) substituted
compounds were synthesized by reductive amination of
the intermediate amine rac-4o and the corresponding
ketones in the presence of titanium(IV) isopropoxide18
as shown in Scheme 3. This methodology could be used
with a variety of cyclic ketones and resulted in hexa-
hydropyrrolo[3,4-c]pyrroles, which were less active as
OFQ agonists (data not shown). Unfortunately, this
reductive amination was not useful for 4-isopropyl-
cyclohexanone and 2-oxodecahydronaphthalen because
it produced diastereomeric mixtures of products.
The synthesis of cis-1-(4-isopropylcyclohexyl)piperi-
din-4-one (1a ) starting from 4-isopropylaniline 6 is
outlined in Scheme 4. A number of optimization experi-
ments were carried out at 80-120 °C and 100 bar of H2
to find suitable conditions for the hydrogenation of
4-isopropylaniline 6. Ru/Al2O3 (Degussa H 213 B/D) in
i-PrOH or hexane gave unfavorable cis/trans ratios of
1.1 to 1.4:1. Ru/C (J MC 19A) resulted in a 2:1 ratio of 8
and 9 and the formation of ca. 50% biscyclohexylamine
11. Hydrogenation with both Ru/C (Degussa H 101 B/W)
and Rh/C (Engelhard 8000) in HOAc gave substantial
amounts (30-50%) of the N-acetylated amines. A good
cis/trans ratio of 3:1 was obtained with Rh/Al2O3 (De-
gussa G 207 R/D) in hexane. However, the reaction was
incomplete and accompanied by the formation of 11%
Resu lts a n d Discu ssion
The binding characteristics of spirocyclic hexahydro-
pyrrolo[3,4-c]pyrroles 5a -k to membranes isolated from
cells expressing hDOP, hKOP, hMOP, or hNOP recep-
tors were assessed (see Tables 1 and 2). In control
experiments, high-affinity binding of the ligands N/OFQ

NOP Receptor Agonists
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2 257
Ta ble 1. Binding Affinities (pKi ( SEM) for Human NOP and Opioid (MOP, KOP, DOP) Receptors
pKi ( SEMa
compd
R1
R2
NOP
MOP
KOP
DOP
(+)-5a
(-)-5a
rac-5b
rac-5c
rac-5d
rac-5e
rac-5f
rac-5g
rac-5h
H
H
H
H
H
H
4-F
H
H
CH3 (3aS,6aR)
CH3 (3aR,6aS)
CH2Ph
Ph
n-Bu
(CH2)2morphol
CH2-cyPr
H
9.26 ( 0.11 (5)
8.92 ( 0.06 (3)
8.67 ( 0.29 (3)
8.78 ( 0.16 (3)
9.18 ( 0.15 (4)
8.68 ( 0.07 (4)
8.78 ( 0.09 (3)
8.66 ( 0.25 (3)
9.11 ( 0.23 (3)
6.27 ( 0.02 (5)
6.47 ( 0.13 (3)
6.81 ( 0.11 (3)
6.87 ( 0.23 (3)
6.56 ( 0.26 (3)
6.75 ( 0.10 (3)
6.18 ( 0.04 (3)
6.30 ( 0.13 (3)
6.76 ( 0.13 (3)
6.51 ( 0.02 (4)
6.55 ( 0.04 (3)
6.81 ( 0.04 (2)
6.29 ( 0.22 (2)
6.60 ( 0.01 (2)
6.83 ( 0.04 (2)
6.77 ( 0.19 (2)
6.59 ( 0.07 (2)
6.88 ( 0.20 (2)
5.67 ( 0.08 (3)
5.63 ( 0.22 (3)
6.04 ( 0.08 (2)
6.66 ( 0.15 (2)
6.41 ( 0.05 (2)
5.53 ( 0.14 (2)
5.97 ( 0.01 (2)
5.75 ( 0.06 (2)
6.03 ( 0.09 (2)
(CH2)2OH
a
In parentheses is the number of experiments in triplicate.
Ta ble 2. Binding Affinities (pKi ( SEM) for Human NOP and Opioid (MOP, KOP, DOP) Receptors
pKi ( SEMa
compd
R3
NOP
MOP
KOP
DOP
rac-5i
9.19 ( 0.13 (4)
6.14 ( 0.09 (4)
7.25 ( 0.06 (2)
6.05 ( 0.04 (2)
rac-5j
rac-5k
9.35 ( 0.09 (3)
9.28 ( 0.06 (3)
7.06 ( 0.11 (2)
6.38 ( 0.02 (2)
7.57 ( 0.18 (2)
6.88 ( 0.07 (2)
6.43 ( 0.06 (2)
5.99 ( 0.05 (2)
a
In parentheses is the number of experiments in triplicate.
(NOP), DAMGO (MOP), naloxone (KOP), and deltorphin
II (DOP) was observed.12
All compounds show a high affinity at the NOP
receptor and a good to excellent selectivity versus the
opioid receptors. As already shown before for the 8-
cycloalkyl-substituted 1-phenyl-1,3,8-triazaspiro[4.5]-
decan-4-one series,21 cyclodecyl (rac-5j), cyclononyl (rac-
5i), and decahydronaphtha-2-yl (rac-5k , mixture of
diastereomers) substituted spirocyclic hexahydro-
pyrrolo[3,4-c]pyrroles are less selective compared to the
cis-4-isopropylcyclohexyl substituted compounds (+)-5a
and (-)-5a , especially regarding activity at the KOP
receptor. We therefore focused our main efforts on the
cis-4-isopropylcyclohexyl substituted compounds.
rac-5a was exemplarily resolved into its enantiomers.
The 3aS,6aR enantiomer (+)-5a turned out to be
superior concerning affinity at the NOP receptor and
selectivity toward the other opiod receptors.
F igu r e 1. Binding properties of (+)-5a . Competitive binding
was performed as described in Experimental Section. Data
represent triplicates of one experiment repeated at least three
times.
opiate receptors to assess (+)-5a ’s agonist properties.
Like N/OFQ, (+)-5a was a full agonist at the NOP
receptors and produced a ∼3.2-fold stimulation over
basal levels of GTPγS binding to membranes of NOP-
expressing cells (Figure 2A). The EC50 values for N/OFQ
(EC50 ) 38.1 ( 3.6 nM, n ) 6) or (+)-5a mediated
The binding properties of (3aS,6aR)-1-(cis-4-isopro-
pylcyclohexyl)-5′-methyl-2′-phenylhexahydrospiro[pip-
eridine-4,1′-pyrrolo[3,4-c]pyrrole], (+)-5a , are shown in
Figure 1. We conducted GTPγS stimulation studies on
isolated membranes from cells expressing the NOP or

258 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2
Kolczewski et al.
Thus, (+)-5a displays a significantly higher degree of
selectivity than Ro 64-6198,7,11,12 which is less selective
toward the KOP and MOP receptors.11 We believe that
a >1000-fold selectivity especially toward the MOP
receptor is an interesting property for a small-molecule
N/OFQ mimetic to minimize undesired side effects due
to opioid activity. Indeed, since activation of the MOP
receptor may produce symptoms of drug addiction,14
a
non-peptide N/OFQ agonist with considerable affinity
for this receptor is highly undesirable. (+)-5a was
further assessed in a standard binding analysis against
more than 30 additional receptors and channels. The
compound interacted with histamine H3, muscarinic
and σ receptors, and sodium channels only at a concen-
tration of 10 µM and above.
In two functional assays, (+)-5a behaved as a full
agonist of the NOP receptor. In the GTPγS binding
assay, (+)-5a was almost as potent as the natural ligand
N/OFQ. The observed 2-fold lower potency of (+)-5a
compared to N/OFQ most likely reflects its 2-fold lower
binding affinity. In contrast to its high potency and full
efficacy at the NOP receptor, (+)-5a only weakly stimu-
lated GTPγS binding at membranes of cells expressing
KOP or MOP receptors and was inactive at the DOP
receptor. It appears unlikely that the compound acti-
vates opiate receptors in vivo.
(+)-5a was also a full agonist when tested for its
ability to inhibit forskolin-mediated cAMP accumulation
in HEK293 cells stably expressing the NOP receptor.
However, in contrast to the small differences between
N/OFQ and (+)-5a in the GTPγS binding assays, we
observed a ∼25-fold lower potency of (+)-5a in the cAMP
assays. This difference most likely reflects the hydro-
phobic nature of (+)-5a . We routinely observed a low
aqueous solubility of the compound and nonspecific
binding to plastic, which could not be improved by
addition of carrier molecules such as bovine serum
albumin. Also, the increase of organic solvents such as
DMSO interfered with the cellular response and did not
improve the potency of (+)-5a . Because the GTPγS
assay, like the binding but unlike the cAMP assay,
tolerates higher amounts of organic solvents, we believe
that the potency differences of (+)-5a between both
functional readouts are mostly reflected by the com-
pound’s low aqueous solubility. In agreement with these
findings, oral and intraperitoneal application of (+)-5a
resulted in low bioavailability, a high volume of distri-
bution, and unspecific tissue binding. In addition, no
anxiolytic-like properties could be observed in vivo, in
contrast to (1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-
phenalen-1-yl)-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-
one11 described previously. Thus, we believe that (+)-
5a represents a valuable and highly selective NOP
receptor agonist for in vitro but not in vivo use. Future
attempts in this project will address improved in vivo
activity of N/OFQ agonists.
F igu r e 2. Stimulation of GTPγS binding (A) and inhibiton
of forskolin-mediated cAMP production (B) by (+)-5a : (A)
GTPγS-binding was assessed as described in Experimental
Section; (B) HEK293 cells expressing the NOP receptor were
stimulated with 1 µM forskolin and increasing concentrations
(10-6-10-11 M) of agonists. Data are triplicates from one
experiment repeated three times.
GTPγS binding (EC50 ) 65.4 ( 6.3 nM, n ) 4) did not
differ more than 2-fold. In contrast to its high potency
at the NOP receptor, (+)-5a was a weak agonist at the
MOP receptor. While DAMGO (EC50 ) 220 ( 44 nM, n
) 8), a full MOP agonist,12 stimulated a ∼2.8-fold
increase over basal levels in GTPγS-binding, (+)-5a
(EC50 ) 7.3 ( 1.1 µM, n ) 3) elicited only a small (∼1.3-
fold) increase of GTPγS binding (Figure 2A). Similarly,
(+)-5a weakly activated the KOP receptor (EC50 ≈ 10
µM, n ) 2) and displayed no agonistic activity at the
DOP receptor (not shown).
We further assessed (+)-5a ’s agonist potency in cAMP
inhibition studies using intact NOP-expressing HEK293
cells. (+)-5a and N/OFQ were equally efficacious and
inhibited >80% of the forskolin-stimulated cAMP pro-
duction (Figure 2B). However, in contrast to its only
2-fold lower agonist potency in the GTPγS binding
assays, (+)-5a (EC50 ) 9.1 ( 4.8 nM, n ) 3) was
approximately 25-fold less potent than N/OFQ (EC50
0.36 ( 0.08 nM, n ) 6).
)
In conclusion, we have characterized (+)-5a as a high-
affinity and specific ligand for the NOP receptor. In two
functional in vitro assays, (+)-5a behaves as a full
agonist of the NOP receptor and only weakly activates
the closely related KOP and MOP receptors. (+)-5a
serves as an ideal tool for further improvement of non-
peptide N/OFQ agonists.
We have pharmacologically characterized (+)-5a as
a highly selective full agonist of the NOP receptor. In
binding experiments, (+)-5a showed subnanomolar af-
finity for NOP comparable to the affinity of the natural
peptide N/OFQ.1,2 In contrast, higher (+)-5a concentra-
tions (>1000-fold at KOP and MOP; >4000-fold at DOP)
were needed to compete for opiate receptor binding.

NOP Receptor Agonists
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2 259
with Na2SO4, and concentrated. The residue was filtered with
ethyl acetate through silica gel to yield the product (5.7 g, 79%)
as a yellow oil. An analytical sample was crystallized as the
HCl salt from ethyl acetate. (2RS,4aSR,8aRS)-1-(Deca-
hydronaphthalen-2-yl)piperidin-4-one hydrochloride (1:1): mp
174-175 °C; 1H NMR (250 MHz, CDCl3) δ 1.1-1.8 (m, 13 H),
1.8-2.0 (m, 3H), 2.1 (m, 1H), 2.5 (d, 2H), 3.2 (m, 2H), 3.7 (m,
4H), 13.2 (bs, 1H); MS m/z 235 (M+). Anal. (C15H26ClN) C, H,
N.
Exp er im en ta l Section
1. Ch em istr y. Gen er a l P r oced u r es. Melting points were
taken with a Bu¨chi 510 melting point apparatus and are
uncorrected. The 1H spectra were recorded on a Bruker AC250
instrument in DMSO (unless noted otherwise). Low-resolution
EI-MS spectra (EI: 70 eV) were recorded on a MS9 updated
with a VG ZAB console, Finnigan data system SS300, with
direct sample introduction. Microanalysis (C, H, N) were
performed on a Heraeus Vario EL. Halogens were determined
by standard titrimetric procedures. NMR data are reported
in parts per million (δ) relative to internal tetramethylsilane
and are referenced to the deuterium lock signal from the
sample solvent. Coupling constants (J ) are in hertz. All
reactions were performed under argon. Drying of organic
solutions was with Na2SO4, and evaporation was done in a
rotary evaporator at 45 °C in vacuo as appropriate. For
chromatography, Merck silica gel 60 (size 70-230 mesh) was
used. Analytical TLC plates employed were Merck silica gel
60 F-254 plates. Starting materials were high-grade com-
mercial products unless stated otherwise.
cis-[1-(4-Isop r op ylcycloh e xyl)p ip e r id in -4-ylid e n e ]-
p h en yla m in e (2a ). St a n d a r d P r oced u r e A. cis-1-(4-Iso-
propylcyclohexyl)piperidine-4-on (1a) (5.0 g, 23.4 mmol), aniline
(3.3 g, 35.3 mmol), and molecular sieves (20 g, 4 Å) were stirred
in 100 mL of pentane at room temperature for 6 days. The
molecular sieves was filtered off, and the solvent was evapo-
rated. The crude product, which crystallized on storage at 4°C,
was used without any further purification for the following
1
step. H NMR (250 MHz, CDCl3) δ 0.88 (d, J ) 6.6 Hz, 6H),
1.05-1.19 (m, 1H), 1.31-1.78 (m, 9H), 2.29-2.34 (m, 3H), 2.59
(dd, J ) 8.9, 5.9 Hz, 4H), 2.82 (dd, J ) 6.0 Hz, 2H), 6.73 (d, J
) 7.3 Hz, 2H), 7.05 (d, 7.4 Hz, 1H), 7.29 (dd, 7.4, 7.3, 2H).
cis-1-(4-Isop r op ylcycloh exyl)p ip er id in -4-on e (1a ). To
the crude reaction solution obtained in the hydrogenation step
containing 4-isopropylcyclohexylamine (8 + 9) (5.26 L contain-
ing 2.690 mol of 8 + 9) was added 2.1 L of deionized water
and K2CO3 (744 g, 5.38 mol, 2 equiv). The clear, brown solution
was heated to boiling temperature (81°C). Within 2 h, a
solution of ammonium salt 7 (869 g, 3.229 mol, 1.2 equiv) in
2.75 L of water was added, and stirring at boiling temperature
was continued for 2 h. GC analysis of the reaction mixture
indicated that only 1.2% of the starting material 8 + 9 were
left. The reaction mixture was allowed to cool to 50 °C, and
i-PrOH was removed by distillation at reduced pressure. The
residue, 5 L of a biphasic mixture of water and precipitated
product, was extracted with 5 L of EtOAc. The organic layer
was washed with 5 L of 20% aqueous NaCl. To remove an
apolar byproduct, the organic layer was extracted with 8 L of
5% aqueous HOAc. The organic layer was discarded. The
aqueous layer was adjusted to pH 14 with 1 L of 28% NaOH
and extracted with 5 L of EtOAc. The organic layer was dried
over 300 g of Na2SO4, filtered, and concentrated at reduced
pressure. A total of 544 g of a brown oil was obtained, a
mixture of 56% cis product 1a and 34% trans product 10
according to GC. The crude product was combined with
another batch (509 g obtained from 2.562 mol 8 + 9) and
chromatographed on silica gel (12 kg, hexane/acetone/Et3N 95:
5:0.4) to afford 419 g (1.876 mol, 35.7%) of cis compound 1a
and 193 g (0.864 mol, 16.4%) of trans compound 9 as yellow
oils. For GC analyses, an RTx-200 (trifluoromethylpolysilox-
ane) fused silica column (30 m × 0.32 mm; 0.25 µm) was used
with helium as carrier gas (150 kPa). A 1% solution in EtOAc
was injected. The program was 50°C at 8°C/min to 300°C. Data
for 1a : 1H NMR (250 MHz, CDCl3) δ 0.88 (d, J ) 6.6, 6H),
1.15 (m, 1H), 1.34-1.76 (mm, 9H), 2.43 (t, J ) 6.1, 4H), 2.45
(m, 1H), 2.83 (t, J ) 6.1, 4H); IR (neat) 2953 (s), 2935 (s), 2868
(s), 2801 (s), 2761 (m), 1719 (s), 1445 (m), 1225 (s) cm-1; MS
(EI), m/z 223 (M+, 13), 180 (5), 152 (9), 138 (100), 96 (32); GC
tR ) 17.0 min; TLC Rf ) 0.32 (hexane/acetone/Et3N 90:10:0.4).
Data for 10: 1H NMR (250 MHz, CDCl3) δ 0.87 (d, J ) 6.8,
6H), 0.94-1.13 (mm, 3H), 1.18-1.35 (mm, 2H), 1.43 (m, 1H),
1.75-1.95 (mm, 4H), 2.43 (m, 1H), 2.44 (t, J ) 6.2, 5H), 2.86
(t, J ) 6.2, 4H); IR (neat) 2955 (s), 2930 (s), 2857 (s), 2804 (s),
2757 (m), 1720 (s), 1205 (s) cm-1; MS (EI), m/z 223 (M+, 16),
205 (4), 180 (4), 152 (6), 138 (100), 96 (15); GC tR ) 18.0 min;
TLC Rf ) 0.20 (hexane/acetone/Et3N 90:10:0.4).
(4-F lu or op h en yl)-[1-(cis-4-isop r op ylcycloh exyl)p ip er i-
d in -4-ylid en e]a m in e (2f). The title compound was obtained
according to the standard procedure A as a yellow oil from
cis-1-(4-isopropylcyclohexyl)piperidine-4-on (1a ) and 4-fluoro-
aniline and was used without any further purification for the
1
following step. H NMR (250 MHz, CDCl3) δ 0.88 (d, J ) 6.6
Hz, 6H), 1.07-1.21 (m, 1H), 1.31-1.75 (m, 9H), 2.27-2.42 (m,
3H), 2.58 (dt, J ) 5.8, 10.0 Hz, 4H), 2.81 (dd, J ) 5.8 Hz, 2H),
6.67 (dd, J ) 4.9, 8.7 Hz, 2H), 6.97 (t, J ) 8.7 Hz, 2H).
(3′a RS,6′a SR)-1-(cis-4-Isop r op ylcycloh exyl)-5′-m eth yl-
2′-p h en ylh exa h yd r osp ir o[p ip er id in e-4,1′-p yr r olo[3,4-c]-
p yr r ole]-4′,6′-d ion e (r a c-4a ). cis-[1-(4-Isopropylcyclohexyl)-
piperidin-4-ylidene]phenylamine (2a ) (2.0 g, 6.7 mmol) in 70
mL of 1,2-dimethoxyethane was treated at 0 °C with (tri-
methylsilyl)methyltrifluoromethane sulfonate (1.58 g, 6.7
mmol) and stirred at room temperature for 1 h. N-Methyl-
maleimide (2.0 g, 18.0 mmol) and then cesium fluoride (1.02
g, 6.7 mmol) were added, and the reaction mixture was stirred
at room temperature for 40 h. The title compound precipitated
from the reaction mixture and was obtained after filtration
and washing with 1,2-dimethoxyethane as a white solid (1.08
g, 38%): mp 184 °C; 1H NMR (250 MHz, DMSO-d6) δ 0.82 (d,
J ) 6.5, 6H), 1.01-1.93 (m, 15H), 2.88 (s, 3H), 3.10-3.42 (m,
6H), 3.38 (m, 1H), 3.89 (m, 1H), 7.04 (d, J ) 7.5, 2H), 7.13 (t,
J ) 7.8, 1H), 7.30 (dd, J ) 7.8, 7.5, 2H); MS m/z 424.5 (MH)+.
(3a RS,6a SR)-1-(cis-4-Isop r op ylcycloh exyl)-5′-ben zyl-2′-
p h en ylh exa h yd r osp ir o[p ip er id in e-4,1′-p yr r olo[3,4-c]p yr -
r ole]-4′,6′-d ion e (r a c-4b). Sta n d a r d P r oced u r e B. cis-[1-
(4-Isopropylcyclohexyl)piperidin-4-ylidene]phenylamine (2a )
(1.0 g, 3.35 mmol) in 35 mL of 1,2-dimethoxyethane was
treated at 0 °C with (trimethylsilyl)methyltrifluoromethane
sulfonate (950 mg, 4.02 mmol) and stirred at room temperature
for 1 h. N-Benzylmaleimide (3.14 g, 16.8 mmol) and then
cesium fluoride (610 mg, 4.02 mmol) were added and the
reaction mixture was stirred at room temperature for 20 h.
The solvent was evaporated, and the residue was taken up in
150 mL of diethyl ether and extracted with sodium carbonate
solution (2 N, 150 mL). The phases were separated, and the
aqueous phase was extracted twice with diethyl ether (100 mL
each). The combined organic phases were dried (magnesium
sulfate) and concentrated. The residue was purified by column
chromatography (ethyl acetate/hexane/triethylamine 30:10:1)
to yield the title compound (400 mg, 24%) as an off-white foam.
(2R S ,4a S R ,8a R S )-1-(De ca h yd r on a p h t h a le n -2-yl)p i-
p er id in -4-on e (r a c-1k ). To a boiling suspension of (2RS,
4aSR,8aRS)-decahydronaphthalen-2-ylamine (rac-14) (31 mmol)
and potassium carbonate (18 mmol) in ethanol (70 mL) was
added 1-ethyl-1-methyl-4-oxopiperidinium iodide (7) (46 mmol)
dissolved in water (25 mL). The reaction mixture was boiled
for another hour, cooled, and diluted with water (60 mL).
Ethanol was removed in vacuo, and the residue was parti-
tioned between sodium hydroxide solution (1 N, 100 mL) and
ethyl acetate (3 × 150 mL). Organic phases were pooled, dried
Sta n d a r d P r oced u r e C. A total of 7.15 g (17.5 mmol) of
(3aRS,6aSR)-1-(cis-4-isopropylcyclohexyl)-2′-phenylhexahy-
drospiro[piperidine-4,1′-pyrrolo[3,4-c]pyrrole]-4′,6′-dione (rac-
4g) were dissolved in 150 mL of dry tetrahydrofuran. Tri-
phenylphosphine (5.51 g, 21.0 mmol), benzyl alcohol (2.27 g,
21.0 mmol), and diethyl azodicarboxylate (3.66 g, 21.0 mmol)
were added subsequently, and the mixture was stirred at room
temperature for 24 h. The tetrahydrofuran was evaporated and
the residue was purified by column chromatography (hexane/
ethyl acetate/triethylamine 40:10:1) to yield 6.33 g (73%) of

260 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2
Kolczewski et al.
1
the title compound. H NMR (250 MHz, CDCl3) δ 0.82 (d, J )
on e (r a c-4k ). Aniline (6.3 mmol) and molecular sieves (4 Å,
4 g) were added to a solution of (2RS,4aSR,8aRS)-1-(deca-
hydro-naphthalen-2-yl)piperidin-4-one (rac-1k ) (4.2 mmol) in
pentane (60 mL). The mixture was stirred at room temperature
for a week, molecular sieves were filtered off, and the solvent
was removed to yield inter alia the corresponding imine as a
yellow oil (raw 1.5 g). The material was, without further puri-
fication, dissolved in dimethoxyethane (30 mL). (Trimethyl-
silyl)methyltrifluoromethanesulfonate (2.4 mmol) was added
at 0 °C within 15 min. The mixture was stirred at room
temperature for 15 min. N-Methylmaleimide (8.4 mmol) and
cesium fluoride (2.4 mmol) were consecutively added, and the
mixture was stirred at room temperature for 24 h. The solvent
was removed in vacuo, and sodium bicarbonate solution (10%,
80 mL) was added to the residue. The mixture was extracted
with CH2Cl2 (3 × 40 mL), the organic phases were pooled, dried
with Na2SO4, and evaporated. The residue (2.6 g), a yellow
oil, was purified by chromatography on silica gel with CH2Cl2/
MeOH (2%) to yield the product (0.21 g, 10%) as a yellow oil.
An analytical sample was crystallized as the HCl salt from
ethyl acetate. (3′aRS,6′aSR) and (3′aSR,6′aRS)-1-[(2RS,4aSR,
8aRS)-Decahydronaphthalen-2-yl]-5′-methyl-2′-phenylhexa-
hydrospiro[piperidine-4,1′-pyrrolo[3.4-c]pyrrole]-4′,6′-dione hy-
drochloride (1:1): mp 168-171 °C; 1H NMR (250 MHz, DMSO-
d6) δ 1.1-2.1 (m, 21 H), 1.8-2.0 (m, 3H), 2.90 (s, 3H), 3.1-3.7
(m, 8H), 3.9 (t, 1H), 7.07 (d, 2H), 7.15 (t, 1H), 7.31 (t, 2H), 9.1
(bs, 1H); MS m/z 436.5 (M + H)+. Anal. (C27H38ClN3O2) C, H,
N.
6.6, 6H), 1.03-1.12 (m, 1H), 1.21-1.88 (m, 13H), 2.10-2.28
(m, 2H), 2.55-2.70 (m, 1H), 2.93-3.08 (m, 2H), 3.28 (t, J )
7.7, 1H), 3.43 (d, J ) 7.7, 1H), 3.49 (d, J ) 9.7, 1H), 3.88 (t, J
) 9.7, 1H), 4.72 (s, 2H), 6.97 (d, J ) 7.4, 2H), 7.09 (t, J ) 7.2,
1H), 7.23-7.35 (m, 7H); MS m/z 500.3 (MH)+.
(3′a R S ,6′a SR )-1-(cis-4-Isop r op ylcycloh e xyl)-2′,5′-d i-
p h en ylh exa h yd r osp ir o[p ip er id in e-4,1′-p yr r olo[3,4-c]-
p yr r ole]-4′,6′-d ion e (r a c-4c). The title compound was ob-
tained according to standard procedure B from cis-[1-(4-
isopropylcyclohexyl)piperidin-4-ylidene]phenylamine (2a ) and
N-phenylmaleimide as a yellow foam (58%). 1H NMR (250
MHz, CDCl3) δ 0.83 (d, J ) 6.6, 6H), 1.01-1.15 (m, 1H), 1.23-
2.03 (m, 14H), 2.13-2.31 (m, 2H), 2.62-2.74 (m, 1H), 2.96-
3.11 (m, 2H), 3.40 (t, J ) 7.4 Hz, 1H), 3.59 (d, J ) 8.2 Hz,
2H), 3.96 (dd, J ) 7.4, 8.2, 1H), 7.02-7.07 (m, 3H), 7.24-7.33
(m, 4H), 7.40-7.48 (m, 3H); MS m/z 486.4 (MH)+.
(3′a RS,6′a SR)-1-(cis-4-Isop r op ylcycloh exyl)-5′-bu tyl-2′-
p h en ylh exa h yd r osp ir o[p ip er id in e-4,1′-p yr r olo[3,4-c]p yr -
r ole]-4′,6′-d ion e (r a c-4d ). The title compound was obtained
from (3aRS,6aSR)-1-(cis-4-isopropylcyclohexyl)-2′-phenylhexa-
hydrospiro[piperidine-4,1′-pyrrolo[3,4-c]pyrrole]-4′,6′-dione (rac-
4g) and n-butanol according to standard procedure C as a
yellow waxy solid (83%). 1H NMR (250 MHz, CDCl3) δ 0.83
(d, J ) 6.6 Hz, 6H), 0.92 (t, J ) 7.2 Hz, 3H), 1.02-1.13 (m,
1H), 1.20-1.94 (m, 17H), 2.13-2.30 (m, 2H), 2.61-2.75 (m,
1H), 2.97-3.08 (m, 2H), 3.20 (t, J ) 7.2 Hz, 1H), 3.42 (t, J )
9.9 Hz, 2H), 3.54 (t, J ) 7.1 Hz, 2H), 3.86 (dd, J ) 7.2, 9.6 Hz,
1H), 6.98 (d, J ) 7.4 Hz, 2H), 7.08 (t, J ) 7.9 Hz, 1H), 7.25
(dd, 7.3, 7.9 Hz, 2H); MS m/z 466.4 (MH)+.
(3′a RS,6′a SR)-1-(cis-4-Isop r op ylcycloh exyl)-5′-(2-m or -
p h olin -4-yl-eth yl)-2′-p h en ylh exa h yd r osp ir o[p ip er id in e-
4,1′-p yr r olo[3,4-c]p yr ole]-4′,6′-d ion e (r a c-4e). The title
compound was obtained from (3aRS,6aSR)-1-(cis-4-isopropyl-
cyclohexyl)-2′-phenylhexahydrospiro[piperidine-4,1′-pyrrolo-
[3,4-c]pyrrole]-4′,6′-dione (rac-4g) and N-(2-hydroxyethyl)mor-
pholine according to standard procedure C as a crystalline solid
(3′a RS,6′a SR)-1-(cis-4-Isop r op ylcycloh exyl)-2′-(4-flu o-
r op h en yl)tetr a h yd r osp ir o[p ip er id in e-4,1′-p yr r olo[3,4-c]-
p yr r ole]-4′,6′-d ion e (r a c-4l). The title compound was ob-
tained according to standard procedure B from (4-fluorophenyl)-
[1-(cis-4-isopropylcyclohexyl)piperidin-4-ylidene]amine (2f) and
1
maleimide as a white solid (20%): mp 217°C; H NMR (250
MHz, CDCl3) δ 0.83 (d, J ) 6.6 Hz, 6H), 1.02-1.16 (m, 1H),
1.24-1.76 (m, 12H), 1.84-1.96 (m, 1H), 2.12-2.30 (m, 2H),
2.44-2.70 (m, 1H), 2.94-3.12 (m, 2H), 3.22 (t, J ) 7.8 Hz, 1H),
3.36-3.45 (m, 2H), 3.77 (t, J ) 9.0 Hz, 1H), 6.93-7.00 (m, 4H);
MS m/z 428.6 (MH)+.
1
(95%). H NMR (250 MHz, CDCl3) δ 0.83 (d, J ) 6.6 Hz, 6H),
1.02-1.13 (m, 1H), 1.24-2.05 (m, 15H), 2.12-2.30 (m, 2H),
2.40-2.71 (m, 6H), 2.96-3.10 (m, 2H), 3.23 (t, J ) 6.6 Hz, 1H),
3.41 (d, J ) 6.6 Hz, 1H), 3.46 (d, J ) 11.9 Hz, 1H), 3.55-3.71
(m, 5H), 3.86 (dd, J ) 6.5, 10.1 Hz, 1H), 6.97 (d, J ) 7.0 Hz,
2H), 7.07 (t, 7.5 Hz, 1H), 7.25 (dd, J ) 7.5, 7.0 Hz, 2H); MS
m/z 523.3 (MH)+.
(3′a RS,6′a SR)-5′(2-Ben zyloxyeth yl)-1-(cis-4-isop r op yl-
cycloh exyl)-2′-p h en ylh exa h yd r osp ir o[p ip er id in e-4,1′-
p yr r olo[3,4-c]p yr ole]-4′,6′-d ion e (r a c-4m ). The title com-
pound was obtained from (3aRS,6aSR)-1-(cis-4-isopropylcyclo-
hexyl)-2′-phenylhexahydrospiro[piperidine-4,1′-pyrrolo[3,4-c]-
pyrrole]-4′,6′-dione (rac-4g) and 2-benzyloxyethanol according
to standard procedure C as a yellow waxy solid that was
further purified by salt formation. A total of 200 mg (0.37
mmol) of the basic compound was dissolved in 10 mL of diethyl
ether and treated with 41 mg (0.35 mmol) of fumaric acid in
1 mL of methanol. After being stirred for 16 h, the white
precipitate was filtered off and washed with diethyl ether. The
product (150 mg, 61%) was further characterized as its 1:1
fumarate salt: mp 155°C; 1H NMR (250 MHz, DMSO-d6) δ
0.82 (d, J ) 6.4, 6H), 1.00-1.86 (m, 14H), 2.42-2.62 (m, 3H),
2.69-2.91 (m, 1H), 3.03-3.18 (m, 3H), 3.38 (t, J ) 7.8 Hz, 1H),
3.51-3.72 (m, 5H), 3.86 (t, J ) 9.0 Hz, 1H), 4.46 (s, 2H), 6.49
(s, 2H), 6.95 (d, J ) 7.8 Hz, 2H), 7.07 (t, J ) 6.6 Hz, 1H), 7.19-
7.29 (m, 7H); MS m/z 544.3 (MH)+.
(3′aRS,6′aSR)-5′-Cyclopr opylm eth yl-2′-(4-flu or oph en yl)-
1-(cis-4-isopr opylcycloh exyl)h exa h yd r osp ir o[p iper idin e-
4,1′-p yr r olo[3,4-c]p yr r ole]-4′,6′-d ion e (r a c-4f). Reaction of
(3′aRS,6′aSR)-1-(cis-4-Isopropylcyclohexyl)-2′-(4-fluorophenyl)-
tetrahydrospiro[piperidine-4,1′-pyrrolo[3,4-c]pyrrole]-4′,6′-di-
one (rac-4l) (1.10 g, 2.57 mmol) and hydroxymethylcyclo-
propane (0.25 mL, 3.16 mmol) according to standard procedure
C yielded rac-4f (0.84 g, 68%) as a light-yellow solid: mp
1
139°C; H NMR (250 MHz, DMSO-d6) δ 0.27-0.36 (m, 2 H),
0.41-0.55 (m, 2 H), 0.83 (d, J ) 6.5 Hz, 6 H), 0.92-1.74 (m,
14 H), 1.84 (mc, 1 H), 2.04-2.31 (m, 2 H), 2.64 (mc, 1 H), 2.92-
3.09 (m, 2 H), 3.22 (t, J ) 7.0 Hz, 1 H), 3.31-3.49 (m, 4 H),
3.79 (dd, J ) 8.5 Hz, 1 H), 6.94 (d, J ) 6.5 Hz, 4 H). MS (FAB)
m/z 482.5 (M + H+).
(3′a RS,6′a SR)-1-(cis-4-Isop r op ylcycloh exyl)-2′-p h en yl-
h exa h yd r osp ir o[p ip er id in e-4,1′-p yr r olo[3,4-c]p yr r ole]-
4′,6′-d ion e (r a c-4g). The title compound was obtained ac-
cording to standard procedure B from cis-[1-(4-isopropyl-
cyclohexyl)piperidin-4-ylidene]phenylamine (2a ) and male-
imide as a yellow solid (57%). 1H NMR (250 MHz, CDCl3) δ
0.82 (d, J ) 6.6 Hz, 6H), 1.01-1.13 (m, 1H), 1.24-1.69 (m,
13H), 1.79 (dt, J ) 4.0, 12.3 Hz, 1H), 1.90-2.00 (m, 1H), 2.12-
2.27 (m, 2H), 2.61 (dt, J ) 2.1 Hz, 12.3, 1H), 2.97-3.09 (m,
2H), 3.24 (t, J ) 8.2 Hz, 1H), 3.42 (d, J ) 6.2 Hz, 1H), 3.45 (d,
J ) 7.6 Hz, 1H), 3.84 (dd, J ) 8.2, 9.2 Hz, 1H), 7.01 (d, J )
7.3 Hz, 2H), 7.08 (t, J ) 8.2 Hz, 1H), 7.25 (dd, J ) 7.3, 8.2 Hz,
2H); MS m/z 410.5 (MH)+.
(3′a R S ,6′a S R )-1-Be n zyl-5′-m e t h yl-1′-p h e n ylh e xa h y-
d r osp ir o[p ip er id in e-4,1′(2′H)-p yr r olo[3,4-c]p yr r ole]-4′,6′-
d ion e (r a c-4n ). A solution of 1-benzyl-4-piperidone (9.44 mL,
52.8 mmol), aniline (4.82 mL, 52.8 mmol), and p-toluenesulfonc
acid (120 mg) in toluene (300 mL) was heated on a Dean-
Stark trap over a period of 17 h. The solvent was evaporated
to give the crude imine, which was subsequently dissolved in
DME (300 mL) and treated with N-methylmaleimide (21.0 g,
189 mmol), trimethylsilylmethyl trifluoromethansulfonate
(10.5 mL, 52.8 mmol), and cesium fluoride (8.02 g, 52.8 mmol)
according to standard method B to give rac-4n (10.4 g, 51%)
as a white solid: mp 196 °C; 1H NMR (250 MHz, CDCl3) δ
1.43 (dt, J ) 12, 4.5 Hz, 1 H), 1.63 (mc, 2 H), 1.81-1.91 (m, 1
H), 2.14 (dt, J ) 12, 3 Hz, 1 H), 2.65 (dt, J ) 10.5, 4.5 Hz, 1
H), 2.85-2.98 (m, 2 H), 3.02 (s, 3 H), 3.22 (t, J ) 7.5 Hz, 1 H),
3.43 (d, J ) 8.5 Hz, 2 H), 3.48 (s, 2 H), 3.83 (t, J ) 8.5 Hz, 1
(3′a RS,6′a SR)- a n d (3′a SR,6′a RS)-1-[(2RS,4a SR,8a RS)-
Deca h yd r on a p h th a len -2-yl]-5′-m eth yl-2′-p h en ylh exa h y-
d r osp ir o[p ip er id in e-4,1′-p yr r olo[3.4-c]p yr r ole]-4′,6′-d i-

NOP Receptor Agonists
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2 261
20
20
H), 6.98 (d, J ) 7.5 Hz, 2 H), 7.09 (t, J ) 7.5 Hz, 1 H), 7.18-
7.33 (m, 7 H); MS (FAB) m/z 390.3 (M + H+).
as described for rac-4m : [R]589 -112.49°, [R]546 -137.91°
(fumarate salt, c 0.7947, methanol). Anal. (C26H41N3‚1.8C4H4O4)
C, H, N.
(3′a RS,6′a SR)-5′-Meth yl-1′-p h en ylh exa h yd r osp ir o[p i-
p er id in e-4,1′(2′H)-p yr r olo[3,4-c]p yr r ole]-4′,6′-d ion e (r a c-
4o). (3′aRS,6′aSR)-1-Benzyl-5′-methyl-1′-phenylhexahydro-
spiro[piperidine-4,1′(2′H)-pyrrolo[3,4-c]pyrrole]-4′,6′-dione (rac-
4n ) (10.4 g, 26.6 mmol) was dissolved in methanol (500 mL)
and acetic acid (50 mL) and hydrogenated on Pd/C (1.5 g, 10%)
at room temperature over a period of 16 h. The catalyst was
filtered off, the filtrate was evaporated, and the residue was
dissolved in ammonium hydroxide (25%)/ice (100 mL) and
extracted with dichloromethane (2 × 200 mL). The combined
organic layers were washed with brine (100 mL), dried
(MgSO4), and evaporated. Crystallization of the crude product
from diethyl ether yielded rac-4o (7.32 g, 92%) as a light-yellow
(3′a RS,6′a SR)-4-(cis-4-Isop r op ylcycloh exyl)-5′-ben zyl-
2′-p h en ylh exa h yd r osp ir o[p ip er id in e-4,1′(2′H )-p yr r olo-
[3,4-c]p yr r ole (r a c-5b). The title compound was obtained
from (3aRS,6aSR)-1-(cis-4-isopropylcyclohexyl)-5′-benzyl-2′-
phenylhexahydrospiro[piperidine-4,1′-pyrrolo[3,4-c]pyrrole]-
4′,6′-dione (rac-4b) according to standard procedure D as a
colorless oil (95%), which was transformed into its HCl salt
by the following procedure. An amount of 800 mg (1.70 mmol)
of the basic compound was dissolved in 20 mL of diethyl ether,
and 1.5 mL of HCl in dioxane (approximately 2.26 N) were
added dropwise. The precipitate was filtered off, washed with
diethyl ether, and recrystallized from a mixture of ethyl acetate
and ethanol to give 514 mg (54%) of a 1:2.3 HCl salt: mp (salt)
1
solid: mp 181 °C; H NMR (250 MHz, CDCl3) δ 1.29 (dt, J )
1
>190 °C (dec); H NMR (250 MHz, CDCl3, free base) δ 0.83
12, 4.5 Hz, 1 H), 1.59-1.73 (m, 2 H), 1.83-1.97 (m, 2 H), 2.79
(dt, J ) 12, 3 Hz, 1 H), 3.03 (s, 3 H), 3.06-3.19 (m, 2 H), 3.20-
3.36 (m, 2 H), 3.43 (m, 2 H), 3.88 (t, J ) 8.5 Hz, 1 H), 7.01 (d,
J ) 7.5 Hz, 2 H), 7.10 (t, J ) 7.5 Hz, 1 H), 7.26 (t, J ) 7.5 Hz,
2 H); MS (FAB) m/z 300.3 (M + H+).
(d, J ) 6.6 Hz, 6H), 1.00-1.13 (m, 1H), 1.17-2.29 (m, 18H),
2.68-2.99 (m, 5H), 3.05 (t, J ) 7.2 Hz, 1H), 3.31 (t, J ) 9.0
Hz, 1H), 3.56-3.74 (m, 3H), 6.95 (t, J ) 7.8 Hz, 1H), 7.03 (d,
J ) 8.4 Hz, 2H), 7.20-7.35 (m, 7H); MS m/z 472.4 (MH)+. Anal.
(C32H45N3‚2.3HCl) C, H, N.
(3a RS,6a SR)-1-(cis-4-Isop r op ylcycloh exyl)-5′-m et h yl-
2′-p h en ylh exa h yd r osp ir o[p ip er id in e-4,1′-p yr r olo[3,4-c]-
p yr r ole] (r a c-5a ). Sta n d a r d P r oced u r e D. (3aRS,
6aSR)1-(cis-4-Isopropylcyclohexyl)-5′-methyl-2′-phenylhexa-
hydrospiro[piperidine-4,1′-pyrrolo[3,4-c]pyrrol]-4′,6′-dione (rac-
4a ) (212 mg, 0.5 mmol) was dissolved in methylene chloride
(4 mL) and diethyl ether (5 mL), and lithium aluminum
hydride (55 mg, 1.45 mmol) was added at 0°C. The reaction
mixture was stirred at room temperature for 1.5 h and then
subsequently treated with 0.05 mL of water, 0.05 mL of 15%
sodium hydroxide solution, and 0.15 mL of water. The solid
material was filtered off, and the filtrate was evaporated.
Column chromatography (hexane/ethyl acetate/triethylamine
10:10:1) of the residue gave the desired product (140 mg, 70%),
which was precipitated as its 1:1.5 fumarate salt from a
mixture of methanol and diethyl ether as described for rac-
4m : mp (salt) > 165 °C (dec); 1H NMR (250 MHz, CDCl3, free
base) δ 0.84 (d, J ) 6.6 Hz, 6H), 1.02-1.15 (m, 1H), 1.24-
1.75 (m, 13H), 1.88-2.05 (m, 3H), 2.06-2.27 (m, 3H), 2.38 (s,
3H), 2.72-3.09 (m, 5H), 3.32 (t, J ) 7.2 Hz, 1H), 3.65 (dd, J )
7.2, 9.00 Hz, 1H), 6.96 (t, J ) 7.2 Hz, 1H), 7.02 (d, J ) 7.2 Hz,
2H), 7.22 (t, J ) 7.2 Hz, 2H); MS m/z 396.4 (MH)+.
(3′a R S ,6′a SR )-4-(cis-4-Isop r op ylcycloh e xyl)-2′,5′-d i-
p h e n ylh e xa h yd r osp ir o[p ip e r id in e -4,1′(2′H )-p yr r olo-
[3,4-c]p yr r ole (r a c-5c). The title compound was obtained
from (3′aRS,6′aSR)-1-(cis-4-isopropylcyclohexyl)-2′,5′-diphenyl-
hexahydrospiro[piperidine-4,1′-pyrrolo[3,4-c]pyrrole]-4′,6′-di-
one (rac-4c) according to standard procedure D as a colorless
oil (88%) and was precipitated as its 1:1 fumarate salt from a
mixture of methanol and diethyl ether as described for rac-
1
4m : mp (salt) > 255 °C (dec); H NMR (250 MHz, DMSO-d6,
salt) δ 0.87 (d, J ) 6.5 Hz, 6H), 1.07-1.18 (m, 1H), 1.27-1.42
(m, 2H), 1.48-1-78 (m, 9H), 2.29-2.82 (m, 5H), 3.03-3.31
(m, 7H), 3.50-3.64 (m, 3H), 6.56 (s, 2H), 7.08-7.20 (m, 4H);
MS m/z 458.5 (MH)+. Anal. (C31H43N3‚1C4H4O4) C, H, N
(3′a RS,6′a SR)-4-(cis-4-Isop r op ylcycloh exyl)-5′-bu tyl-2′-
p h en ylh exa h yd r osp ir o[p ip er id in e-4,1′(2′H)-p yr r olo[3,4-
c]p yr r ole (r a c-5d ). The title compound was obtained from
(3′aRS,6′aSR)-1-(cis-4-isopropylcyclohexyl)-5′-butyl-2′-phenyl-
hexahydrospiro[piperidine-4,1′-pyrrolo[3,4-c]pyrrole]-4′,6′-di-
one (rac-4d ) according to standard procedure D as a colorless
oil (89%) and was precipitated as its 1:2.2 fumarate salt from
a mixture of methanol and diethyl ether as described for rac-
1
4m : mp (salt) >220°C (dec); H NMR (250 MHz, CDCl3, free
base) δ 0.84 (d, J ) 6.6 Hz, 6H), 0.93 (t, J ) 7.2 Hz, 3H), 1.00-
1.13 (m, 1H), 1.23-1.74 (m, 16H), 1.93-2.26 (m, 6H), 2.37-
2.51 (m, 2H), 2.69-3.00 (m, 5H), 3.10 (t, J ) 7.9 Hz, 1H), 3.39
(t, J ) 8.5 Hz, 1H), 3.65 (dd, J ) 7.9, 9.3 Hz, 1H), 6.97 (t, J )
7.2 Hz, 1H), 7.01 (d, J ) 7.6 Hz, 2H), 7.22 (dd, J ) 7.2, 7.4 Hz,
2H); MS m/z 438.5 (MH)+. Anal. (C29H47N3‚2.2C4H4O4) C, H,
N.
(3a S,6a R)-1-(cis-4-Isop r op ylcycloh exyl)-5′-m et h yl-2′-
p h en ylh exa h yd r osp ir o[p ip er id in e-4,1′-p yr r olo[3,4-c]p yr -
r ole] ((+)-5a ). Sta n d a r d P r oced u r e E. An amount of 3.04
g (7.68 mmol) of the racemic mixture (3aSR,6aRS)-1-(cis-4-
isopropylcyclohexyl)-5′-methyl-2′-phenylhexahydrospiro[piperi-
dine-4,1′-pyrrolo[3,4-c]pyrrole] (rac-5a ) was dissolved in 70 mL
of methanol and water (95:5), and an amount of 1.65 g (4.61
mmol) of (-)-O,O-dibenzoyl-L-tartaric acid was added. The
mixture was refluxed until a complete solution was attained,
and the solvent was partly distilled off until the first precipi-
tate occurred. Slow cooling resulted in the precipitation of 1.3
g (22%) of crystals. The enantiomeric excess was determined
by chiral HPLC to be 98.6%. The tartrate salt was reconverted
to the free base by extraction with dichloromethane/sodium
bicarbonate solution and precipitated as its 1:2.1 fumarate salt
from a mixture of methanol and diethyl ether as described for
(3′a RS,6′a RS)-1-(cis-4-Isop r op ylcycloh exyl)-5′-(2-m or -
p h olin -4-yl-eth yl)-2′-p h en ylh exa h yd r osp ir o[p ip er id in e-
4,1′-p yr r olo[3,4-c]p yr r ole] (r a c-5e). The title compound was
obtained from (3′aRS,6′aSR)-1-(cis-4-isopropylcyclohexyl)-5′-
(2-morpholin-4-yl-ethyl)-2′-phenylhexahydrospiro[piperidine-
4,1′-pyrrolo[3,4-c]pyrrole]-4′,6′-dione (rac-4e) according to stand-
ard procedure D as a colorless oil (62%) and was precipitated
as its 1:3.2 fumarate salt from a mixture of methanol and
diethyl ether as described for rac-4m : mp (salt) 145 °C; 1H
NMR (250 MHz, CDCl3, free base) δ 0.84 (d, J ) 6.6 Hz, 6H),
1.02-1.15 8m, 1H), 1.24-1.38 (m, 3H), 1.42-1.72 (m, 10H),
1.89-2.26 (m, 7H), 2.46-3.00 (m, 12H), 3.12 (t, J ) 7.8 Hz,
1H), 3.42 (t, J ) 8.7 Hz, 1H), 3.62-3.74 (m, 4H), 6.95 (t, J )
6.6 Hz, 1H), 7.01 (d, J ) 7.6 Hz, 2H), 7.22 (dd, J ) 6.6, 7.6 Hz,
2H); MS m/z 495.5 (MH)+. Anal. (C31H50N4O‚3.2C4H4O4) C, H,
N.
(3′aRS,6′aSR)-5′-Cyclopr opylm eth yl-2′-(4-flu or oph en yl)-
1-(cis-4-isop r op ylcycloh exyl)h exa h yd r osp ir o[p ip er in e-
4,1′-p yr r olo[3,4-c]p yr r ole] (r a c-5f). Reduction of (3′aRS,
6′aSR)-5′-cyclopropylmethyl-2′-(4-fluorophenyl)-1-(cis-4-iso-
propylcyclohexyl)hexahydrospiro[piperidine-4,1′-pyrrolo[3,4-c]-
pyrrole]-4′,6′-dione (rac-4f) (0.84 g, 1.74 mmol) with lithium
aluminum hydride (199 mg, 5.24 mmol) according to standard
20
20
rac-4m : [R]589 +101.08°, [R]546 +124.22° (fumarate salt, c
0.9982, methanol). Anal. (C26H41N3‚2.1C4H4O4) C, H, N.
(3a R,6a S)-1-(cis-4-Isop r op ylcycloh exyl)-5′-m et h yl-2′-
p h en ylh exa h yd r osp ir o[p ip er id in e-4,1′-p yr r olo[3,4-c]p yr -
r ole] ((-)-5a ). The title compound was prepared in accordance
with standard procedure E from the racemic mixture (3aSR,
6aRS)-1-(cis-4-isopropylcyclohexyl)-5′-methyl-2′-phenylhexa-
hydrospiro[piperidine-4,1′-pyrrolo[3,4-c]pyrrole] (rac-5a ) and
(+)-O,O-dibenzoyl-D-tartaric acid. The enantiomeric excess was
determined by chiral HPLC to be 98.4%. The tartrate salt was
reconverted to the free base by extraction with dichloromethane/
sodium bicarbonate solution and precipitated as its 1:1.8
fumarate salt from a mixture of methanol and diethyl ether

262 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2
Kolczewski et al.
procedure D and subsequent formation of the fumarate yielded
rac-5f (0.86 g, 74%) as a white solid: mp 209°C; 1H NMR
(DMSO-d6) δ 0.21-0.32 (m, 2 H), 0.44-0.61 (m, 2 H), 0.82 (d,
J ) 6.0 Hz, 6 H), 0.94-1.14 (m, 3 H), 1.21-1.43 (m, 3 H), 1.44-
1.87 (m, 10 H), 2.33-2.65 (m, 6 H), 2.66-2.93 (m, 2 H), 2.94-
3.07 (m, 1 H), 3.09-3.23 (m, 2 H), 3.42-3.55 (m, 1 H), 3.56-
3.74 (m, 2 H), 6.51 (s, 4 H), 7.06 (d, J ) 6.5 Hz, 4 H); MS (FAB)
m/z 454.6 (M + H+). Anal. (C29H44N3 F‚2 C4H4O4) C, H, N.
H), 3.44 (mc, 1 H), 3.61 (mc, 1 H), 6.52 (s, 4.5 H), 6.96 (t, J )
7.5 Hz, 1 H), 7.02 (d, J ) 7.5 Hz, 2 H), 7.22 (t, J ) 7.5 Hz, 2
H); MS (FAB) m/z 396.4 (M + H+). Anal. (C26H41N3‚2.25C4H4O4)
C, H, N.
(3′a RS,6′a SR)-1-Cyclod ecyl-5′-m et h yl-2′-p h en ylh exa -
h yd r osp ir o[p ip er id in e-4,1′(2′H )-p yr r olo[3,4-c]p yr r ole]
(r a c-5j). Reaction of (3′aRS,6′aSR)-5′-methyl-1′-phenylhexa-
hydrospiro[piperidine-4,1′(2′H)-pyrrolo[3,4-c]pyrrole]-4′,6′-dione
(rac-4o) (1.0 g, 3.34 mmol) with cyclodecanone (516 mg, 3.34
mmol) and subsequent reduction with lithium aluminum
hydride according to standard procedure E gave rac-5j (0.34
(3′a RS,6′a SR)-4-(cis-4-Isop r op ylcycloh exyl)-2′-p h en yl-
h exa h yd r osp ir o[p ip er id in e-4,1′(2′H )-p yr r olo[3,4-c]p yr -
r ole] (r a c-5g). (3′aRS,6′aSR)-4-(cis-4-Isopropylcyclohexyl)-5′-
benzyl-2′-phenylhexahydrospiro[piperidine-4,1′(2′H)-pyrrolo[3,4-
c]pyrrole] (rac-5b) (5.5 g, 11.6 mmol) and 10% Pd/C (0.67 g)
in 67 mL of methanol and 6.7 mL of glacial acetic acid were
hydrogenated with 1 atm of hydrogen for 20 h. Pd/C was
filtered off, and the methanol and acetic acid coevaporated with
toluene. Chromatography (dichloromethane/methanol/ammo-
nium hydroxide 140:10:1) of the residue gave 3.77 g (85%) of
the desired product as a white powder, mp 127°C, which was
precipitated as its 1:1.5 fumarate salt from a mixture of
1
g, 15%) as a pale-pink solid: mp 240°C; H NMR (250 MHz,
DMSO-d6) δ 1.31-1.79 (m, 20 H), 2.02 (mc, 1 H), 2.40 (mc, 1
H), 2.50 (s, 3 H), 2.51-3.13 (m, 16 H), 3.29 (mc, 1 H), 3.44 (mc,
1 H), 3.61 (mc, 1 H), 6.52 (s, 4.4 H), 6.94 (t, J ) 7.5 Hz, 1 H),
7.02 (d, J ) 7.5 Hz, 2 H), 7.22 (t, J ) 7.5 Hz, 2 H); MS (FAB)
m/z 410.5 (M + H+). Anal. (C27H43N3 ‚2.2C4H4O4) C, H, N.
(3′a RS,6′a SR)- a n d (3′a SR,6′a RS)-1-[(2RS,4a SR,8a RS)-
Deca h yd r on a p h th a len -2-yl]-5′-m eth yl-2′-p h en ylh exa h y-
d r osp ir o[p ip er id in e-4,1′-p yr r olo[3.4-c]p yr r ole] (r a c-5k ).
To a solution of (3′aRS,6′aSR)- and (3′aSR,6′aRS)-1-[(2RS,
4aSR,8aRS)-decahydronaphthalen-2-yl]-5′-methyl-2′-phenyl-
hexahydrospiro[piperidine-4,1′-pyrrolo[3.4-c]pyrrole]-4′,6′-di-
one (rac-4k ) (0.3 mmol) in THF (10 mL) was added lithium
aluminum hydride (1.5 mmol). The mixture was stirred for
30 min at room temperature and for 2 h at reflux temperature
and hydrolyzed by addition of water (50 µL), sodium hydroxide
solution (15%, 100 µL), and again water (150 µL). Ethyl acetate
(20 mL) and Na2SO4 were added, and the solvent was removed
to yield the product (120 mg, 99%) as a yellow oil. Crystal-
lization with fumaric acid in ethanol yielded (3′aRS,6′aSR)-
and (3′aSR,6′aRS)-1-[(2RS,4aSR,8aRS)-decahydronaphthalen-
2-yl]-5′-methyl-2′-phenylhexahydrospiro[piperidine-4,1′-pyrrolo-
[3.4-c]pyrrole] fumarate (1:2.4) as colorless solid: mp >152 °C
(dec); 1H NMR (250 MHz, DMSO-d6) δ 1.1-1.8 (m, 20 H), 1.95
(t, 1H), 2.2 (t, 1H), 2.43 (s, 3H), 2.4-3.2 (m, 9H), 3.34 (t, 1H),
3.62 (t, 1H), 6.90 (t, 1H), 6.52 (s, 4H), 7.01 (d, 2H), 7.22 (t,
2H), 12.9 (bs, 4H); MS m/z 408.5 (M + H)+. Anal. (C27H41N3‚
2.4C4H4O4) C,H,N.
(3′a RS,6′a SR)-5′(2-Ben zyloxyeth yl)-1-(cis-4-isop r op yl-
cycloh exyl)-2′-p h en ylh exa h yd r osp ir o[p ip er id in e-4,1′-
p yr r olo[3,4-c]p yr r ole] (r a c-5m ). The title compound was
obtained from (3′aRS,6′aSR)-5′(2-benzyloxyethyl)-1-(cis-4-iso-
propylcyclohexyl)-2′-phenylhexahydrospiro[piperidine-4,1′-pyr-
rolo[3,4-c]pyrole]-4′,6′-dione (rac-4m ) according to standard
procedure D as a colorless oil (62%) and was precipitated as
its 1:2 fumarate salt from a mixture of methanol and diethyl
ether as described for rac-4m , mp (salt) 169 °C; 1H NMR (250
MHz, CDCl3, free base) δ 0.84 (d, J ) 6.6 Hz, 6H), 1.01-1.15
(m, 1H), 1.24-1.38 (m, 1H), 1.41-1.76 (m, 10H), 1.82-2.25
(m, 7H), 2.68-3.00 (m, 17H), 3.15 (t, J ) 7.8 Hz, 1H), 3.42 (t,
J ) 8.7 Hz, 1H), 3.57-3.80 (m, 3H), 4.56 (s, 2H), 6.93 (t, J )
6.6 Hz, 1H), 7.01 (d, J ) 7.6 Hz, 2H), 7.19-7.36 (dd, m, 7H);
MS m/z 516.4 (MH)+.
1-Eth yl-1-m eth yl-4-oxop ip er id in iu m Iod id e (7). To a
stirred solution of 1-ethyl-4-piperidone (481 g, 3.782 mol) in 3
L of acetone was added CH3I (644 g, 1.2 equiv) over 30 min.
Occasionally the reaction mixture was cooled with a water bath
to maintain the temperature between 20 and 25 °C. The
reaction mixture changed color from brown to greenish, and a
yellow precipitate formed. Stirring at room temperature was
continued for 23 h. The reaction mixture was cooled to 3 °C
and stirred for 1 h at this temperature. The product was
collected by filtration, washed with 3 L of acetone at 0-5 °C,
and dried (50 °C, 1 mbar, 17 h). A total of 967 g (3.593 mol,
95%) of 1-ethyl-1-methyl-4-oxopiperidinium iodide was ob-
tained as a yellow solid. 1H NMR (250 MHz, DMSO-d6) δ 1.31
(t, J ) 7.2, 3H), 2.72 (m, 4H), 3.17 (s, 3H), 3.57 (q, J ) 7.2,
2H), 3.72 (t, J ) 7.2, 4H).
1
methanol and diethyl ether as described for rac-4m . H NMR
(250 MHz, DMSO-d6, free base) δ 0.82 (d, J ) 6.6 Hz, 6H),
0.98-1.12 (m, 1H), 1.22-1.72 (m, 17H), 1.72-2.25 (m, 5H),
2.50-2.79 (m, 7H), 2.81-3.07 (m, 5H), 3.17-3.27 (m, 1H),
3.53-3.63 (m, 1H), 3.86 (t, J ) 7.3 Hz, 1H), 6.93 (d, J ) 7.9
Hz, 2H), 7.20 (dd, J ) 7.3, 7.9 Hz, 2H); MS m/z 382.4 (MH)+.
Anal. (C25H39N3‚1.5C4H4O4) C, H, N.
(3aRS,6aRS)-1-(cis-4-Isopr opylcycloh exyl)-5′-(2-h ydr oxy-
eth yl)-2′-p h en ylh exa h yd r osp ir o[p ip er id in e-4,1′-p yr r olo-
[3,4-c]p yr r ole] (r a c-5h ). (3aRS,6aRS)-5′-(2-Benzyloxyethyl)-
1-(cis-4-isopropylcyclohexyl)-2′-phenylhexahydrospiro[piperi-
dine-4,1′-pyrrolo[3,4-c]pyrrole] (rac-5m ) (430 mg, 0.833 mmol)
and 10% Pd/C (50 mg) in 10 mL of methanol and 5 mL of 2.7
N HCl/MeOH were hydrogenated with 1 atm of hydrogen for
20 h. Pd/C was filtered off, and the methanol was evaporated.
The residue was taken up in ethyl acetate and extracted with
2 N NaOH solution. The organic phase was dried with MgSO4
and concentrated. Column chromatography (dichloro methane/
methanol/ ammonium hydroxide 140:10:1) gave 170 mg (48%)
of the desired product, which was precipitated as its fumarate
salt from a mixture of methanol and diethyl ether as described
for rac-4m : mp 194°C; 1H NMR (250 MHz, CDCl3, free base)
δ 0.85 (d, 6.6 Hz, 6H), 1.02-1.14 (m, 1H), 1.24-1.38 (m, 3H),
1.42-1.73 (m, 10H), 1.89-2.45 (m, 6H), 2.66-3.02 (m, 7H),
3.12 (t, J ) 8.4 Hz, 1H), 3.40 (t, J ) 7.2 Hz, 1H), 3.62-3.72
(m, 4H), 6.97 (t, J ) 7.2 Hz, 1H), 7.02 (d, J ) 7.5 Hz, 2H),
7.23 (dd, J ) 7.2, 7.5 Hz, 2H); MS m/ z 426.6 (MH)+. Anal.
(C27H43N3O‚1.7C4H4O4) C, H, N.
(3′a RS,6′a SR)-1-Cyclon on yl-5′-m et h yl-2′-p h en ylh exa -
h yd r osp ir o[p ip er id in e-4,1′(2′H )-p yr r olo[3,4-c]p yr r ole]
(r a c-5i). Standar d P r ocedu r e E. A mixture of (3′aRS,6′aSR)-
5′-methyl-1′-phenylhexahydrospiro[piperidine-4,1′(2′H)-pyrrolo-
[3,4-c]pyrrole]-4′,6′-dione (rac-4o) (0.76 g, 2.54 mmol), cyclo-
nonanone (0.36 g, 2.57 mmol), tetraisopropyl orthotitanate
(0.92 mL, 3.12 mmol), and THF (3 mL) was stirred at room
temperature for 19 h. The reaction mixture was evaporated
and dissolved in ethanol (5 mL), and sodium cyanoboron-
hydride (0.12 g, 1.72 mmol) was added at room temperature.
The mixture was stirred for 22 h, water (12 mL) was added,
the precipitate was filtered off, and the filtrate was evaporated.
The crude product was dissolved in 1.5 N sodium hydroxide
solution (90 mL) and extracted with dichloromethane (3 × 70
mL). The combined organic layers were washed with brine (100
mL), dried (MgSO4), and evaporated. Further purification by
column chromatography (MeOH/dichloromethane, 1:9) yielded
(3′aRS,6′aSR)-1-cyclononyl-5′-methyl-2′-phenylhexahydrospiro-
[piperidine-4,1′(2′H)-pyrrolo[3,4-c]pyrrole]-4′,6′-dione (0.24 g,
0.57 mmol) as a white foam, which was subsequently reduced
with lithium aluminum hydride (41 mg, 1.71 mmol) according
to standard procedure D. Formation of the fumarate with
fumaric acid (122 mg, 1.05 mmol) in diethyl ether (10 mL) gave
4-Isop r op ylcycloh exyla m in e (8 + 9). To a solution of
4-isopropylaniline 6 (693 g, 5.125 mol) in 6.93 L of i-PrOH was
added 5% Rh/Al2O3 (70 g, Degussa G 207 R/D). The mixture
was hydrogenated at 80 °C and 100 bar of H2 in an oil-heated
12 L stainless steel autoclave equipped with a hollow shaft
1
rac-5i (0.24 g, 14%) as an orange solid: mp 242 °C; H NMR
(250 MHz, DMSO-d6) δ 1.35-1.86 (m, 18 H), 2.06 (mc, 1 H),
2.39 (mc, 1 H), 2.50 (s, 3 H), 2.51-3.17 (m, 16 H), 3.29 (mc, 1

NOP Receptor Agonists
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2 263
gassing agitator and a reaction temperature control via PC.
Hydrogen uptake was monitored by a temperature-controlled
hydrogen reservoir connected to the reactor via a reducing
valve to keep the autoclave pressure constant. After 10 h of
reaction time, the reaction mixture was allowed to cool to room
temperature and a sample was taken and analyzed by TLC.
Because that not all of the starting material was consumed,
an additional 20 g of catalyst was added and the hydrogenation
(80 °C, 100 bar of H2) was continued for 5 h. A third portion of
20 g of catalyst and another 3 h were needed to bring the
reaction to completion. The reaction mixture was allowed to
cool to room temperature, and the catalyst was removed by
filtration over a suction filter equipped with a cellulose filter
plate (Filtrox AF40) and a Speedex pad (20 g). The solid
residue was rinsed with 1 L of i-PrOH. Both filtrates were
combined and used in the next step.
For GC analyses a DB17+ (50% phenyl/50% methylpoly-
siloxane) fused silica column (15 m × 0.32 mm; 0.25 µm) was
used with helium as the carrier gas (50 kPa). A sample (0.5%
amine) was derivatized in a 5:1 mixture of pyridine and
MBTFA at room temperature. The program was 50 °C, 8°C/
min to 140°C, 16°C/min to 300°C, and 3.75 min at 300 °C. tR
of cis compound 8 was 7.73 min (59%), tR of trans compound 9
was 8.39 min (32%), tR of 4-isopropylaniline 6 was 9.36 min
(1.5%), tR of bis(4-isopropylcyclohexyl)amine 11 was 14.59 min
(2.8%) and 14.99 min (2.7%).
(2RS,4a SR,8a RS)-Ben zyl(d eca h yd r on a p h th a len -2-yl)-
a m in e (r a c-13). A solution of (2RS,4aSR,8aRS)-N-(deca-
hydronaphthalen-2-yl)benzamide (rac-12)20 (64 mmol) in THF
(150 mL) was added to a suspension of lithium aluminum
hydride (128 mmol) in THF (300 mL). This mixture was heated
to reflux for 20 h. After cooling, water (9 mL), sodium
hydroxide solution (15%, 25 mL), and again water (40 mL)
were added. THF was removed in vacuo, the residue was
suspended in CH2Cl2 and washed with sodium hydroxide
solution (2 N). The water phase was extracted with CH2Cl2,
the organic phases were pooled, dried with Na2SO4, and
concentrated to yield the product as a colorless oil (15.6 g,
99%). An analytical sample was crystallized as HCl salt from
ethyl acetate. (2RS,4aSR,8aRS)-Benzyl(decahydronaphthalen-
2-yl)amine hydrochloride (1:1): mp 196-200 °C; 1H NMR (250
MHz, CDCl3) δ 1.1-1.8 (m, 14 H), 1.8-2.0 (m, 1 H), 2.1 (q, J
) 12 Hz, 1 H), 2.8 (bs, 1 H), 4.0 (s, 2 H), 7.37 (m, 3 H), 7.65 (d,
J ) 6.3 Hz, 2 H), 9.8 (bs, 2 H); MS m/z 243 (M+). Anal. Calcd
(C17H26ClN) C,H,N.
presence of 1 µM unlabeled N/OFQ (hNOP), 1 µM DAMGO
(hMOP), 10 µM naloxone (hKOP), or 10 µM deltorphin (hDOP).
γ[35S]Th io-GTP (GTP γ35S) Bin d in g Assa y. Agonist-
stimulated binding of GTPγ35S was investigated in the SPA
format using membranes from hNOP, hDOP, hKOP, or hMOP
receptors as described previously.10,13 Binding was accom-
plished in the presence of 1 mg of wheat germ agglutinin SPA
beads (Amersham) and N/OFQ (0.1 nM to 10 µM), DAMGO,
or 5b (1 nM to 100 µM). Reactions were performed on a shaker
at 22 °C for 120 min and read in a TopCount scintillation
counter (Packard).
cAMP In h ibition Assa y. The inhibition of forskolin-
mediated cAMP accumulation was determined in 96-well
plates as described recently.12 Reactions were performed in
the presence of 0.1 mM rolipram and 1 µM forskolin (both from
Sigma) with increasing concentrations of agonists (10 pM to
100 nM) for 15 min at 37 °C. The cAMP content was
determined from the supernatant using the Biotrak non-
radioactive cAMP kit (Amersham) according to the manufac-
turer’s instructions.
Ack n ow led gm en t. We thank J aqueline Higelin,
Re`ne Mossie`re, Markus Vogler, Philipp Oberli, Daniel
Wehrli, Axel Maier, Mathias Burn, Reto Augsburger,
and Beat Frei for their skillful technical assistance, Wolf
Arnold, W. Meister, Roger Rueher, and Gottfried Oester-
helt for spectroscopic determinations and analysis, and
Kenneth Lundstrom and Paricher Malherbe for mem-
branes expressing human N/OFQ and opioid receptors.
Refer en ces
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Henningsen, R. A.; Bunzow, J . R.; Grady, D. K.; Langen, H.;
Monsma, F. J ., J r.; Civelli, O. Orphanin FQ: a neuropeptide that
activates an opioidlike G protein-coupled receptor. Science 1995,
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(2) Meunier, J .-C.; Mollereau, C.; Toll, L.; Suaudeau, C.; Moisand,
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Costentin, J . Isolation and structure of the endogeous agonist
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Mem br a n e Isola tion a n d Ra d ioliga n d Bin d in g Assa y.
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