Synthesis of (1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-sp iro[4.5]decan-4-one, a potent and selective orphanin FQ (OFQ) receptor agonist with anxiolytic-like properties

Article abstract of DOI:10.1016/S0223-5234(00)00171-9

The development of 8-(2,3,3a,4,5,6-hexahydro-1H-phenalenl-yl)-1-phenyl-1,3,8-triaza-spiro[4.5]de can-4-ones 3 starting from (RS)-8-acenaphten-1-yl-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one 1 is reported. The synthesis and the binding affinities at human OFQ and opioid (μ, χ, δ) receptors of the stereoisomers 3a-f are described. In vitro the most selective compound, (1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalenl-yl)-1-phenyl-1,3,8-triaza-spi ro[4.5]decan-4-one 3c, was found to act as a full agonist at the OFQ receptor in the GTPγ³⁵S binding test. It turned out to be selective versus a variety of other neurotransmitter systems. When tested in vivo following intraperitoneal injection, compound 3c was found to decrease neophobia in a novel environment and to exhibit dose-dependent anxiolytic-like effects in the elevated plus-maze procedure, thus confirming the effects observed following intracerebroventricular infusion of the OFQ peptide in rat. (C) 2000 Editions scientifiques et medicales Elsevier SAS.

Full text of DOI:10.1016/S0223-5234(00)00171-9

Eur. J. Med. Chem. 35 (2000) 839−851
© 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
S0223523400001719/FLA
Original article
Synthesis of (1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-
triaza-spiro[4.5]decan-4-one, a potent and selective orphanin FQ (OFQ) receptor
agonist with anxiolytic-like properties
Jürgen Wichmann*, Geo Adam, Stephan Röver, Michael Hennig, Michelangelo Scalone, Andrea M.
Cesura, Frank M. Dautzenberg, François Jenck
Pharma Division, Preclinical Research, F. Hoffmann-La Roche Ltd., Grenzacherstr. 124, CH-4070 Basel, Switzerland
Received 18 February 2000; revised 7 April 2000; accepted 13 April 2000
Abstract – The development of 8-(2,3,3a,4,5,6-hexahydro-1H-phenalen1-yl)-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-ones 3 starting from
(RS)-8-acenaphten-1-yl-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one 1 is reported. The synthesis and the binding affinities at human OFQ and
opioid (µ, κ, δ) receptors of the stereoisomers 3a–f are described. In vitro the most selective compound, (1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-
1H-phenalen1-yl)-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one 3c, was found to act as a full agonist at the OFQ receptor in the GTPγ35S
binding test. It turned out to be selective versus a variety of other neurotransmitter systems. When tested in vivo following intraperitoneal
injection, compound 3c was found to decrease neophobia in a novel environment and to exhibit dose-dependent anxiolytic-like effects in the
elevated plus-maze procedure, thus confirming the effects observed following intracerebroventricular infusion of the OFQ peptide in rat.
© 2000 Éditions scientifiques et médicales Elsevier SAS
OFQ receptor / agonists / 1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-ones / stereoselective synthesis / anxiolytic activity
1. Introduction
widespread sites of action in the brain suggest that it may
have more general functions [8–10]. It has recently been
shown that orphanin FQ plays an important role in higher
brain functions and can act as an anxiolytic by attenuating
the behavioural inhibition of animals being acutely ex-
posed to anxiogenic environmental conditions [11].
Recently we reported on a series of 1-phenyl-1,3,8-
triaza-spiro[4.5]decan-4-one derivatives [12–13] which
show high affinity for the OFQ receptor. One of these
Orphanin FQ (OFQ, nociceptin) is a recently discov-
ered 17-amino acid neuropeptide that is structurally
related to the opioid peptides but does not act on opioid
(µ, κ, δ) receptors [1, 2]. Orphanin FQ selectively binds
to its own receptor (OFQ receptor or ORL-1), which is a
member of the G protein-coupled receptor superfamily
[3]. The amino acid sequence of the OFQ receptor is 47%
identical to the opioid (µ, κ, δ) receptors overall, and is
64% identical in the transmembrane domains, however,
when compared with classical opioid receptors, the OFQ
receptor has low affinity for opioid ligands [4–5]. As for
the opioid receptors, OFQ receptor activation has been
linked to the inhibition of adenylyl cyclase activity and/or
modulation of neuronal K+ and Ca2+ conductance [6–7].
Orphanin FQ and its receptor are widely expressed
throughout the central nervous system. Orphanin FQ has
been proposed to act as an anti-opioid peptide, but its
compounds,
(RS)-8-acenaphthen-1-yl-1-phenyl-1,3,8-
triaza-spiro[4.5]decan-4-one 1, which represents a com-
bination of 1- and 2-indanyl-1-phenyl-1,3,8-triaza-
spiro[4.5]decan-4-one derivatives [12–13], turned out to
be an OFQ receptor full agonist with high affinity and
moderate selectivity to the opioid (µ, κ, δ) receptors.
In this paper we report on the synthesis and pharma-
cology
of
1H-phenalen-1-yl-1-phenyl-1,3,8-triaza-
spiro[4.5]decan-4-one derivatives 2 and 3a–f (figure 1).
Within this series of compounds we have identified full
agonists of the OFQ receptor with high affinity and
selectivity versus the opioid (µ, κ, δ) receptors. The most
* Correspondence and reprints: juergen.wichmann@roche.com

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J. Wichmann et al. / Eur. J. Med. Chem. 35 (2000) 839–851
Figure 1. Structures of 1, 2 and 3.
selective compound, (1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-
1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4.5]decan-
4-one 3c, was evaluated for its behavioural effects in rats
with specific attention for effects on emotional reactivity
and anxiolytic-like properties in the elevated plus-maze
test in comparison to the classical anxiolytic alprazolam.
Figure 2. Synthesis of compound 2.
2. Chemistry
(RS)-8-(2,3-Dihydro-1H-phenalen-1-yl)-1-phenyl-
1,3,8-triaza-spiro[4.5]decan-4-one 2 was prepared ac-
cording to figure 2. 2,3-Dihydro-1H-phenalen-1-one 4
[14] was converted into the corresponding amine 5 by a
two step procedure via the oxime, which was hydroge-
nated on Ra-Ni. For the introduction of the piperidin-4-
one moiety a Hofmann-elimination-Michael-addition se-
quence [15] was used to give 6. A Strecker-reaction with
aniline and trimethylsilyl cyanide in acetic acid led to the
anilino-nitrile 7, which on treatment with formic acid and
acetic acid anhydride and subsequent reaction of the
crude product in formamide at 200 °C gave 2.
The preparation of the 8-(2,3,3a,4,5,6-hexahydro-1H-
phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4.5]decan-
4-one diastereoisomers 3a–f is shown in figure 3. A
Stobbe-type condensation [16–17] of α-tetralone and
diethyl succinate with potassium tert-butylate in tert-
butanol and subsequent hydrolysis/decarboxylation in
acetic acid/HCl yielded the γ, δ-unsaturated acid 9, that
contains 5% of the corresponding 3-(naphthalen-1-yl)-
propionic acid as an impurity. The percentage of the
impurity formed in the Stobbe-type condensation de-
pends on the reaction time.
Hydrogenation of 9 with palladium on carbon as
catalyst gave the racemic acid 10a. The asymmetric
hydrogenation of 9 in the presence of (S)- or (R)-6,6′-
dimethoxybiphenyl-2,2′-diyl)bis(diphenylphosphine)
(MeOBIPHEP)/Ru(OAc)2 as catalyst [18] afforded 10b
(S) and 10c (R), respectively, with 93–94% ee [19].
The acid 10a can be transformed into the correspond-
ing (RS)-2,3,3a,4,5,6-hexahydro-1H-phenalen-1-one 11a
Figure 3. Synthesis of compounds 3a–f.

J. Wichmann et al. / Eur. J. Med. Chem. 35 (2000) 839–851
841
Table I. Binding affinities (pKi ± SEM) for human OFQ and opioid (µ, κ, δ) receptors (number of experiments performed in triplicate)
calculated according to Cheng and Prusoff [25].
pKi ± SEM
Compound
OFQ
µ
κ
δ
1 (RS) [12]
2 (RS)
9.28
8.23
7.59
6.61
8.95 ± 0.14 (5)
9.29 ± 0.08 (5)
8.60 ± 0.02 (4)
9.41 ± 0.06 (7)
7.56 ± 0.18 (3)
7.29 ± 0.09 (4)
7.47 ± 0.07 (4)
7.33 ± 0.09 (7)
7.05 ± 0.12 (3)
6.98 ± 0.09 (3)
6.80 ± 0.08 (3)
7.05 ± 0.07 (3)
6.02 (2)
6.20 ± 0.14 (3)
6.00 (2)
3a (1RS,3aRS)
3b (1RS,3aSR)
3c (1S,3aS)
3d (1R,3aR)
3e (1S,3aR)
3f (1R,3aS)
5.86 ± 0.04 (3)
7.91 (2)
7.2 (1)
6.7 (1)
6.2 (1)
8.68 ± 0.11 (4)
8.76 ± 0.08 (4)
7.05 ± 0.06 (3)
7.60 ± 0.10 (4)
6.27 ± 0.06 (3)
6.84 ± 0.12 (3)
5.92 ± 0.23 (3)
6.21 ± 0.05 (3)
with polyphosphoric acid at 130 °C, whereas the enanti-
omers (S)- and (R)-2,3,3a,4,5,6-hexahydro-1H-phenalen-
1-ones 11b–c [20] were prepared under milder conditions
with trifluoroacetic acid anhydride in trifluoroacetic acid
at room temperature from the corresponding acids 10b–c
[21–22].
Formation of the amine via the oxime and introduction
of the piperidin-4-one moiety as described for the syn-
thesis of 6 led to 12a–f. No diastereoselectivity could be
observed in the hydrogenation step, however, at this step
it is possible to separate the diastereoisomers 12a/12b,
12c/12e and 12d/12f, respectively, by column chroma-
tography. The products of the Strecker-reaction are crys-
talline compounds, thus, at this step of the synthesis the
pure diastereoisomers 13a–b as well as the pure enantio-
mers 13c–f were formed by crystallization.
It is possible to prepare 3a–f from 13a–f as described
for the synthesis of 2, but the yields are rather low. To get
3a–f in good yields, it is necessary to prepare 14a–f by
reaction of 13a–f with formic acid/acetic acid anhydride
and subsequently with formic acid/acetic acid. Intramo-
lecular ring-closure in triethyl orthoformiate under reflux
conditions and reduction of the crude product with
NaBH4 in methanol led to 3a–f.
The absolute stereochemistry was determined by X-ray
crystallography of 10b [23], thus, the acid obtained by
asymmetric hydrogenation of 9 in the presence of (S)-
6,6′-dimethoxybiphenyl-2,2′-
ration of the compounds 10b–c, 11b–c, 12c–f, 13c–f,
14c–f and 3c–f, respectively, could be defined.
3. Pharmacology
The affinities (pKi) of the compounds for human OFQ
and opioid (µ, κ, δ) receptors were determined from
competition binding curves using [3H]-orphanin FQ and
membranes prepared from permanently transfected
HEK293 cells expressing hOFQ receptors as well as
[3H]-naloxone (µ, κ receptors) and [3H]-deltorphin II (δ
receptors) and membranes prepared from BHK cells
transiently expressing hµ, hκ and hδ receptors. The
results are shown in table I.
From our previous work it is known that a combination
of 1- and 2-indanyl-1-phenyl-1,3,8-triaza-spiro[4.5]decan-
4-one derivatives gave the 8-acenaphtenyl-derivative 1,
an OFQ receptor agonist with high affinity, but moderate
selectivity versus the opioid (µ, κ, δ) receptors [12, 13].
The 1H-phenalen-1-yl derivative 2, a combination of the
1- and 2-tetralinyl series [12, 13] shows a better selectiv-
ity for the QFQ receptor compared to 1. However, a real
increase in selectivity was observed by introducing more
stereochemical information into the molecule, which led
to the 1-(2,3,3a,4,5,6)-hexahydro-1H-phenalen-1-yl)-1-
phenyl-1,3,8-triaza-spiro[4.5]decan-4-one
derivatives
3a–f (figure 4).
The two diastereoisomers 3a (trans) and 3b (cis) show
a significant difference regarding their affinities for the
OFQ receptor, whereas their affinities for the opioid (µ, κ,
δ) receptors is comparable. Diastereoisomer 3a with the
higher affinity for the OFQ receptor is therefore the more
selective derivative. The enantiomers of 3a (3c and 3d),
again differ with respect to their affinities for the OFQ
receptor (DpKi = 1.5), whereas in the case of the enanti-
omers of 3b (3e and 3f) the affinities for the OFQ receptor
diyl)bis(diphenylphosphine) (MeOBIPHEP)/Ru(OAc)2 as
catalyst was crystallized as its (R)-1-phenyl-
ethylammonium salt from diethyl ether and turned out to
have the (S)-configuration.
The relative stereochemistry was determined by X-ray
crystallography of 13b [24], which was crystallized from
diethyl ether. It was found that 13b is the (1RS,3aSR)-
diastereoisomer, thus, the relative and absolute configu-

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J. Wichmann et al. / Eur. J. Med. Chem. 35 (2000) 839–851
binoid, histamin H1, muscarin, CCKA and central nicotine
receptors. The compound was inactive on NE uptake, DA
uptake, GABA uptake, 5HT uptake as well as calcium
and potassium channels at concentrations up to 1 µM,
however, it showed some effects on histamine H2, sigma,
dopamine D2 receptors and sodium-site 2 channels (IC50
1 µM).
4. Results and discussion
Figure 4. Structures of stereoisomers 3a, 3c and 3b, 3e.
When tested in vivo following intraperitoneal admin-
istration in rats (0.3, 1, 3.2 mg/kg versus vehicle), 3c was
found to increase exploration in a novel environment (in
a modified open field test, data not shown) and to elicit
increased open arm activity in a standardized elevated
plus-maze procedure (figure 6, number of transitions
from closed to open arms). Increases were specifically
observed in the open arm for transitions (F3,62 = 10.23,
P = 0.0001), time spent (F3,62 = 9.82, P = 0.0001) and
distance moved (F3,62 = 10.85, P = 0.0001) on those
arms.
are comparable. As in the case of the diastereoisomers,
within the four enantiomers there is no major difference
with respect to the affinities for the opioid (µ, κ, δ)
receptors. Among the enantiomers (1S,3aS)-1-
(2,3,3a,4,5,6)-hexahydro-1H-phenalen-1-yl)-1-phenyl-
1,3,8-triaza-spiro [4, 5]decan-4-one 3c shows the highest
affinity for the OFQ receptor and therefore the best
selectivity versus the opioid (µ, κ, δ) receptors.
The compound 3c was tested for its potency to stimu-
late GTPγ35S binding [26, 27] using membranes prepared
from 293s cells transfected with the human OFQ receptor
and turned out to be a full agonist (figure 5).
In addition, 3c was found to have no significant
(pKi < 6.5) affinity for a variety of other receptors like
serotonin 5HT1Dα, 5HT2A, 5HT2C, 5HT6, 5HT7, dopa-
mine D1, D3, D4, CRF1, CRF2α, benzodiazepine, adeno-
sine A1, A2a, A3, somatostatin, NPY, galanin, canna-
When compared to vehicle, the doses of 1 and 3.2 mg/kg
were significantly active on all parameters (P < 0.05 for
Bonferroni-Dunn post-hoc test for multiple compari-
sons). The dose of 0.32 mg/kg was significant on transi-
tions and distance moved on the open arms. No statisti-
cally significant effects were detected in closed arm
activity (F3,62 = 0.795, P > 0.05 on transitions from
closed to closed, and F3,62 = 1.106, P > 0.05 on distance
moved in the closed arms). Forced motor performance
Figure 5. Effects of OFQ and 3c on stimulation of GTPγ35S binding. Membranes prepared from 293s cells transfected with human
OFQ receptors were used.

J. Wichmann et al. / Eur. J. Med. Chem. 35 (2000) 839–851
843
Figure 6. Effect of 3c in comparison to alprazolam in the elevated plus-maze test in rats (30 min pre-treatment for both drugs). This
test is based on the natural aversion of rodents for open spaces and uses a maze with two open and two closed arms; the number of
entries into open arms are indices of neophobic anxiety in animals. Anxiolytics increase and anxiogenics decrease these measures (see
methods section).
also remained intact. Animals of all dose/vehicle groups
performed to maximal scores on the traction test and their
grip strength non-significantly varied between 565 ±
7.6 g and 548 ± 7.9 g under vehicle and 3.2 mg/kg i.p.,
respectively.
was inactive when tested in the elevated plus-maze
procedure in a dose range of 1–10 mg/kg i.p. (data not
shown).
Agonists at OFQ receptors may offer interesting pos-
sibilities for the discovery of innovative anxiolytics and
the role of OFQ in fear responses to stress opens novel
avenues for exploring the pathophysiology of anxiety
disorders. Agonists at OFQ receptors may also prove
useful in the treatment of other stress-related psychiatric
disorders such as depression or eating disorders. This
innovative approach involving a novel peptidergic mecha-
nism of neuromodulatory action may propose interesting
possibilities for selective normalization of stress-related
neuronal dysfunctions with limited side effects.
5. Conclusions
Compound 3c was identified which exhibited high-
affinity binding to the human OFQ receptor with good
selectivity against the human opioid (µ, κ, δ) receptors
(> 100-fold). The compound was identified as an OFQ
receptor full agonist in the GTPγ35S assay. In a broad
binding evaluation the compound showed no significant
affinity for a variety of other receptors.
Following intraperitoneal injection, compound 3c was
found to decrease neophobia in a novel environment and
to exhibit dose-dependent anxiolytic-like effects in the
elevated plus-maze procedure, thus confirming the effects
observed following intracerebroventricular infusion of
the OFQ peptide in rat [11]. Increased exploration was
very similar to that elicited by benzodiazepines in these
novel environments and are interpreted as a decrease in
emotional reactivity of animals treated with the drugs.
Unlike psychostimulants however, 3c and alprazolam did
not non-specifically increase all parameters of locomo-
tion but rather specifically decreased thygmotaxis (wall-
seeking tendency). The opioid receptor agonist morphine
6. Experimental protocols
6.1. Chemistry
Reagent grade solvents were used without further
purification. All reactions were performed under Argon.
Evaporation means removal of the solvent by use of a
Büchi rotary evaporator at 30–40 °C in vacuo. Melting
points were determined in capillary tubes (Büchi B-540
apparatus) and are uncorrected. Column chromatography
was carried out by using silica gel (230–400 mesh;
Merck) and 0.3–1 bar pressure. Spectra were recorded
1
with the following instruments: H-NMR (δ values in

844
J. Wichmann et al. / Eur. J. Med. Chem. 35 (2000) 839–851
ppm relative to internal TMS): BrukerAS-250 (250 MHz)
and WM-400 (400 MHz). MS: MS 9 updated with a
Finnigan MAT data system SS 300. Microanalysis (C, H,
N) were performed on a Heraeus Vario EL. Starting
materials were high-grade commercial products unless
stated otherwise.
7.75 (d, J = 7.5 Hz, 1 H), 7.84 (d, J = 7.5 Hz, 1 H); MS
(EI) m/z 265 (M+), 165 (100), 152 (28), 100 (39).
6.1.1.3. (RS)-1-(2,3-Dihydro-1H-phenalen-
1-yl)-4-phenylamino-piperidine-4-carbonitrile 7
To a stirred solution of (RS)-1-(2,3-dihydro-1H-
phenalen-1-yl)-piperidin-4-one 6 (3.03 g, 11.4 mmol) in
acetic acid (11 mL) was added at 0 °C aniline (1.20 mL,
12.6 mmol) and afterwards trimethylsilyl cyanide
(1.43 mL, 11.4 mmol). The reaction mixture was stirred
at room temperature for 4 h, poured into ice-water
(150 mL) and ammonium hydroxide solution (80 mL)
and extracted with dichloromethane (2 × 100 mL). The
combined organic layers were washed with brine (80 mL),
dried (Na2SO4) and evaporated to give 7 (4.06 g, 96%) as
6.1.1. Preparation of (RS)-8-(2,3-
dihydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-
triaza-spiro[4.5]decan-4-one hydrochloride 2
6.1.1.1. (RS)-2,3-Dihydro-1H-phenalen-1-ylamine 5
A mixture of 2,3-dihydro-1H-phenalen-1-one 4 [14]
(2.83 g, 15.5 mmol), hydroxylamine hydrochloride
(1.73 g, 24.9 mmol) and water (14 mL) was stirred at
70 °C. Methanol (21 mL), THF (4.7 mL) and a solution
of sodium acetate (3.19 g, 38.9 mmol) in water (9.4 mL)
was added and stirring was continued for 85 min at
70 °C. Water (30 mL) was added, the mixture was stirred
for 2 h at room temperature, the crude product was
collected by filtration and subsequently hydrogenated on
Ra/Ni in MeOH–NH3 (200 mL, 3.5 N) over a period of
20 h at room temperature. The catalyst was filtered off,
and the solution was evaporated to give 5 (2.47 g, 87%)
1
a light brown oil: H-NMR (CDCl3) δ 1.88–2.17 (m, 3
H), 2.21–2.69 (m, 4 H), 2.80–3.19 (m, 4 H), 3.29 (mc, 1
H), 3.68 (s, 1 H), 4.12 (mc, 1 H), 6.88–6.96 (m, 3 H),
7.18–7.32 (m, 3 H), 7.33–7.50 (m, 2 H), 7.63–7.79 (m, 3
H); MS (FAB) m/z 368.2 (M + H+).
6.1.1.4. (RS)-8-(2,3-Dihydro-1H-phenalen-1-yl)-1-
phenyl-1,3,8-triaza-spiro[4.5]decan-4-one hydrochloride 2
1
as a light brown oil: H-NMR (CDCl3) δ 1.70 (s, 2 H),
To a stirred solution of (RS)-1-(2,3-dihydro-1H-
phenalen-1-yl)-4-phenylamino-piperidine-4-carbonitrile 6
(4.06 g, 11.0 mmol) in formic acid (24 mL) was added at
0 °C acetic acid anhydride (24 mL). The reaction mixture
was stirred at room temperature for 41 h, poured into
ice-sat. sodium hydrogen carbonate solution (400 mL)
and extracted with dichloromethane (4 × 200 mL). The
combined organic layers were washed with brine
(300 mL), dried (MgSO4) and evaporated to give a brown
oil, which was dissolved in formamide (50 mL) and
heated at 200 °C for 2 h. The reaction mixture was poured
into water (200 mL) and sat. sodium hydrogen carbonate
solution (200 mL) was added. The crude product was
collected by filtration, dissolved in dichloromethane
(100 mL), dried (Na2SO4) and evaporated to give a
brown foam (3.40 g), which was purified by column
chromatography (1. dichloromethane/MeOH/NH4OH,
99:1:0.1 and 2. ethyl acetate) to yield, after formation of
the hydrochloride from MeOH/HCl (15 mL) and diethyl
ether (100 mL), 2 (0.53 g, 11%) as a pale brown solid:
m.p. 251 °C; 1H-NMR (DMSO-d6) δ 1.76–1.98 (m, 2 H),
2.12–2.31 (m, 1 H), 2.72–3.09 (m, 3 H), 3.11–3.25 (m, 1
H), 3.27–3.48 (m, 2 H), 3.50–3.63 (m, 1 H), 3.75–3.99
(m, 2 H), 4.60 (s, 2 H), 4.94 (s, br, 1 H), 6.76 (t, J = 8 Hz,
1 H), 7.03 (d, J = 8 Hz, 2 H), 7.19 (t, J = 8 Hz, 2 H),
7.47–7.64 (m, 3 H), 7.89 (t, J = 7.5 Hz, 2 H), 8.05 (d, J =
7.5 Hz, 1 H), 9.05 (s, 1 H), 10.27 (s, br, 1 H); MS (FAB)
m/z 398.3 (M + H+). Anal. (C26H27N3O.HCl) C, H, N.
1.91–2.04 (m, 1 H), 2.17–2.28 (m, 1 H), 3.03–3.38 (m, 2
H), 4.28 (mc, 1 H), 7.26 (dd, J = 8.5, 1.5 Hz, 1 H),
7.35–7.54 (m, 3 H), 7.69 (dd, J = 8.5, 1.5 Hz, 2 H), 7.73
(dd, J = 8.5, 1.5 Hz, 1 H); MS (EI) m/z 183 (M+), 165
(100).
6.1.1.2. (RS)-1-(2,3-Dihydro-
1H-phenalen-1-yl)-piperidin-4-one 6
To a stirred and boiling mixture of (RS)-2,3-dihydro-
1H-phenalen-1-ylamine 5 (2.47 g, 13.5 mmol), potassium
carbonate (1.86 g, 13.5 mmol) and ethanol (30 mL) was
added dropwise over a period of 10 min a solution of
N-ethyl-N-methyl-4-oxo-piperidinium iodide (5.0 g,
18.6 mmol) in water (9 mL). The reaction mixture was
refluxed for 2 h, subsequently poured into water (200 mL)
and 3 N NaOH solution was added (60 mL). The mixture
was extracted with ethyl acetate (2 × 200 mL), the
combined organic layers were washed with brine
(150 mL), dried (Na2SO4) and evaporated to give 4.51 g
of a dark brown oil, which was further purified by column
chromatography (toluene/ethyl acetate, 5:1) to yield 6
1
(3.03 g, 84%) as a light orange oil: H-NMR (CDCl3) δ
1.93 (ddd, J = 18.5, 12, 5 Hz, 1 H), 2.22–2.34 (m, 1 H),
2.53 (t, J = 6.5 Hz, 4 H), 2.76–2.91 (m, 2 H), 3.01–3.19
(m, 3 H), 3.29 (dt, J = 18.5, 5 Hz, 1 H), 4.25 (dd, J = 12,
5 Hz, 1 H), 7.24 (d, J = 7.5 Hz, 1 H), 7.38 (t, J = 7.5 Hz,
1 H), 7.48 (t, J = 7.5 Hz, 1 H), 7.70 (d, J = 7.5 Hz, 1 H),

J. Wichmann et al. / Eur. J. Med. Chem. 35 (2000) 839–851
845
6.1.2. Preparation of (1RS,3aRS)-8-
(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-
1,3,8-triaza-spiro[4.5]decan-4-one hydrochloride 3a
and polyphosphoric acid (220 g) was kept at 130 °C over
a period of 1 h, subsequently poured into ice-water (1 L)
and extracted with ethyl acetate (3 × 1 L). The combined
organic layers were washed with water (1 L), sat. sodium
hydrogen carbonate solution (1 L), water (1 L) and brine
(1 L), dried (MgSO4) and evaporated to give the crude
product as a brown solid. Purification by column chro-
matography (ethyl acetate/hexane, 1:4) yielded 11a
(17.6 g, 94%) as brown oil, which crystallized upon
standing: 1H-NMR (CDCl3) δ 1.01–1.22 (m, 1 H),
1.38–1.49 (m, 2 H), 2.20–2.39 (m, 1 H), 2.41–2.64 (m, 4
H), 6.94 (t, J = 7.5 Hz, 1 H), 7.01 (dd, J = 7.5, 1.5 Hz, 1
H), 7.59 (dd, J = 7.5, 1.5 Hz, 1 H); MS (EI) m/z 186 (M+),
158 (37), 144 (100), 129 (48), 115 (28).
6.1.2.1. 3-(3,4-Dihydro-naphthalen-1-yl)-propionic acid
9
Astirred mixture of α-tetralone 8 (36.4 mL, 274 mmol),
diethyl succinate (38.4 mL, 229 mmol), potassium tert
butylate (46.9 g, 418 mmol) and tert butanol (200 mL)
was heated under reflux conditions for 45 min, evapo-
rated, poured into hydrochloric acid solution (8 N, 80 mL)
and extracted with diethyl ether (2 × 200 mL). The
combined organic layers were washed with brine
(150 mL), dried (MgSO4) and evaporated to give a brown
oil (86.3 g), which was subsequently heated in a mixture
of concentrated hydrochloric acid (200 mL) and acetic
acid (200 mL) under reflux conditions for 3 h. The
reaction mixture was evaporated, poured into water
(500 mL) and extracted with diethyl ether (2 × 400 mL).
The combined organic layers were washed with sodium
hydroxide solution (3 N, 120 mL) and water (2 × 200 mL).
The combined water phases were acidified with sulfuric
acid (3 N, 220 mL), extracted with diethyl ether (2 ×
150 mL), the combined organic layers washed with water
(2 × 150 mL) and brine (150 mL), dried (MgSO4) and
evaporated to give the crude product as a brown solid
(39.2 g). Crystallization from ethyl acetate/hexane yielded
9 (10.5 g, 23%) as an off-white solid, that contains 5% of
3-(naphthalen-1-yl)-propionic acid as an impurity, and
28.4 g (61%) of crude product as a brown oil: m.p.
107 °C; 1H-NMR (CDCl3) δ 2.19–2.34 (m, 2 H),
2.58–2.67 (m, 2 H), 2.68–2.91 (m, 4 H), 5.91 (t, J =
4.5 Hz, 1 H), 7.08–7.29 (m, 4 H); MS (EI) m/z 202 (M+),
142 (70), 129 (100). Anal. (C13H14O2) C, H.
6.1.2.4. (1RS,3aRS)-1-(2,3,3a,4,5,6-
Hexahydro-1H-phenalen-1-yl)-piperidin-4-one 12a
and (1RS,3aSR)-1-(2,3,3a,4,5,6-
hexahydro-1H-phenalen-1-yl)-piperidin-4-one 12b
The mixture of (1RS,3aRS)- and (1RS,3aSR)-
2,3,3a,4,5,6-hexahydro-1H-phenalen-1-ylamine (10.5 g,
84% of a light yellow oil) was prepared by reaction of
(RS)-2,3,3a,4,5,6-hexahydro-phenalen-1-one 11a (12.5 g,
67.1 mmol) with hydroxylamine hydrochloride (7.90 g,
115 mmol) and subsequent hydrogenation of the oxime
according to the procedure described for 5 (6.1.1.1.):
1H-NMR (CDCl3) δ 1.19–2.35 (m, 10 H), 2.44–2.93 (m,
3 H), 3.91–4.07 (m, 1 H), 6.91–7.38 (m, 3 H); MS (EI)
m/z 187 (M+), 170 (100), 158 (27), 142 (40), 12 (17), 115
(16).
Reaction of a mixture of (1RS,3aRS)- and (1RS,3aSR)-
2,3,3a,4,5,6-hexahydro-1H-phenalen-1-ylamine (10.5 g,
54.2 mmol) and N-ethyl-N-methyl-4-oxo-piperidinium
iodide (20.0 g, 74.7 mmol) as described for 6 (6.1.1.2.)
gave, after purification and separation of the diastereo-
isomers by column chromatography (ethyl acetate/toluene,
1:4), a mixture of 12a–b (2.47 g, 17%) as a pale yellow
oil, 12a (3.65 g, 25%) as a colourless oil and 12b (5.35 g,
36%) as a colourless oil: 1H-NMR (CDCl3, 12a) δ
1.19–1.51 (m, 2 H), 1.62–2.05 (m, 6 H), 2.34–2.64 (m, 5
H), 2.71–2.98 (m, 6 H), 4.09 (dd, J = 9.5, 5.5 Hz, 1 H),
6.98 (d, J = 7.5 Hz, 1 H), 7.12 (t, J = 7.5 Hz, 1 H), 7.60
(d, J = 7.5 Hz, 1 H); 1H-NMR (CDCl3, 12b) δ 1.19–1.42
(m, 2 H), 1.62–2.08 (m, 6 H), 2.33–2.56 (m, 5 H),
2.71–2.99 (m, 6 H), 3.92 (dd, J = 6.5, 4.5 Hz, 1 H), 6.99
(d, J = 7.5 Hz, 1 H), 7.14 (t, J = 7.5 Hz, 1 H), 7.53 (d, J =
7.5 Hz, 1 H); MS (FAB) m/z 270.3 (M + H+).
6.1.2.2. (RS)-3-(1,2,3,4-Tetrahydro-
naphthalen-1-yl)-propionic acid 10a
3-(3,4-Dihydro-naphthalen-1-yl)-propionic acid
9
(28.4 g, 0.14 mol, crude product from B1) was hydroge-
nated on palladium (2.84 g, 5% on carbon) in methanol
(250 mL) for 40 h at room temperature. The catalyst was
filtered off and the solution was evaporated to give the
crude product as a brown oil (25.7 g), which was purified
by column chromatography (toluene/ethyl acetate, 3:1) to
yield 10a (21.4 g, 74%) as a white solid: m.p. 78 °C;
1H-NMR (CDCl3) δ 1.55–1.99 (m, 5 H), 2.01–2.19 (m, 1
H), 2.37–2.51 (m, 2 H), 2.64–2.92 (m, 3 H), 7.01–7.23
(m, 4 H); MS (EI) m/z 204 (M+), 144 (58), 131 (100).
Anal. (C13H16O2) C, H.
6.1.2.3. (RS)-2,3,3a,4,5,6-Hexahydro-phenalen-1-one 11a
A
stirred mixture of (RS)-3-(1,2,3,4-tetrahydro-
naphthalen-1-yl)-propionic acid 10a (21.4 g, 0.10 mol)

846
J. Wichmann et al. / Eur. J. Med. Chem. 35 (2000) 839–851
6.1.2.5. (1RS,3aRS)-1-
(2,3,3a,4,5,6-Hexahydro-1H-phenalen-1-yl)-
4-phenylamino-piperidine-4-carbonitrile 13a
stirred for 3 h. The reaction mixture was evaporated,
poured into ice-sodium hydroxide solution (3 N, 40 mL)
and extracted with dichloromethane (2 × 80 mL). The
combined organic layers were washed with brine (70 mL),
dried (MgSO4) and evaporated to give a light brown
foam. Further purification by column chromatography (1.
Reaction of (1RS,3aRS)-1-(2,3,3a,4,5,6-hexahydro-1H-
phenalen-1-yl)-piperidin-4-one 12a (1.50 g, 5.57 mmol),
aniline (0.56 mL) and trimethylsilyl cyanide (0.69 mL,
5.57 mmol) as described for 7 (6.1.1.3.) yielded 13a
(2.03 g, 98%) as a colourless oil, which crystallized upon
standing: 1H-NMR (CDCl3) δ 1.21–1.47 (m, 2 H),
1.69–2.11 (m, 8 H), 2.27 (mc, 1 H), 2.38 (mc, 1 H),
2.48–2.99 (m, 7 H), 3.64 (s, 1 H), 3.91 (dd, J = 9.5,
5.5 Hz, 1 H), 6.89–6.98 (m, 4 H), 7.09 (t, J = 7.5 Hz, 1
H), 7.21–7.29 (m, 2 H), 7.47 (d, J = 7.5 Hz, 1 H); MS
(FAB) m/z 372.2 (M + H+).
dichloromethane/methanol/ammonium
hydroxide,
150:10:1 and 2. ethyl acetate) and subsequent formation
of the hydrochloride with methanol/hydrochloric acid
(3 N, 5 mL) and diethyl ether (100 mL) yielded 3a
(1.08 g, 48%) as a light brown solid: m.p. 250 °C;
1H-NMR (DMSO-d6) δ 1.05–1.34 (m, 2 H), 1.59–2.04
(m, 7 H), 2.25–2.46 (m, 1 H), 2.62 (mc, 1 H), 2.71–2.93
(m, 4 H), 3.19–3.49 (m, 2 H), 3.61–3.97 (m, 2 H), 4.60
(mc, 2 H), 4.99 (mc, 1 H), 6.79 (t, J = 7.0 Hz, 1 H),
7.04–7.29 (m, 6 H), 7.90 (d, J = 7.0 Hz, 1 H), 9.01 (s, 1
H), 10.48 (s, br, 1 H); MS (FAB) m/z 402.3 (M + H+).
Anal. (C26H27N3O.HCl) C, H, N.
6.1.2.6. (1RS,3aRS)-4-(Formyl-
phenylamino)-1-(2,3,3a,4,5,6-hexahydro-1H-
phenalen-1-yl)-piperidine-4-carboxylic acid amide 14a
To a stirred solution of (1RS,3aRS)-1-(2,3,3a,4,5,6-
hexahydro-1H-phenalen-1-yl)-4-phenylamino-piperidine-
4-carbonitrile 13a (2.03 g, 5.46 mmol) in formic acid
(20 mL) was added, dropwise at 0 °C over a period of
10 min, acetic acid anhydride (20 mL). The reaction
mixture was stirred at room temperature for 3 h, evapo-
rated and the crude product dissolved in formic acid
(20 mL). Subsequently acetic acid (2 mL) was added, the
solution was stirred at room temperature for 16 h, evapo-
rated, poured into ice-sat. sodium hydrogen carbonate
solution (70 mL) and extracted with dichlormethane (2 ×
100 mL). The combined organic layers were washed with
brine (70 mL) dried (MgSO4) and evaporated to give the
crude product as a light brown oil, which was further
purified by column chromatography (dichloromethane/
methanol, 19:1) to give 14a (2.15 g, 94%) as a pale
6.1.3. Preparation of (1RS,3aSR)-8-
(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-
1,3,8-triaza-spiro[4.5]decan-4-one hydrochloride 3b
6.1.3.1. (1RS,3aSR)-1-
(2,3,3a,4,5,6-Hexahydro-1H-phenalen-1-yl)-
4-phenylamino-piperidine-4-carbonitrile 13b
Reaction of (1RS,3aSR)-1-(2,3,3a,4,5,6-hexahydro-1H-
phenalen-1-yl)-piperidin-4-one 12b (1.50 g, 5.57 mmol),
aniline (0.56 mL) and trimethylsilyl cyanide (0.69 mL,
5.57 mmol) as described for 7 (6.1.1.3.) yielded 13b
(1.92 g, 93%) as a light yellow oil, which crystallized
1
upon standing: H-NMR (CDCl3) δ 1.17–1.41 (m, 2 H),
1.64–2.15 (m, 8 H), 2.29 (mc, 1 H), 2.31–2.53 (m, 2 H),
2.55 (mc, 1 H), 2.63–2.98 (m, 5 H), 3.62 (s, 1 H), 3.79
(dd, J = 6.5, 4.5 Hz, 1 H), 6.85–7.02 (m, 4 H), 7.09 (t, J =
7.5 Hz, 1 H), 7.21–7.29 (m, 2 H), 7.41 (d, J = 7.5 Hz, 1
H); MS (FAB) m/z 372.2 (M + H+).
1
yellow foam: H-NMR (CDCl3) δ 1.15–1.39 (m, 2 H),
1.59–2.11 (m, 8 H), 2.27–2.65 (m, 7 H), 2.78 (dd, J = 7.5,
5.5 Hz, 2 H), 3.80 (dd, J = 9.5, 5.5 Hz, 1 H), 5.51 (br, 1
H), 6.92 (d, J = 7.5 Hz, 1 H), 7.04 (t, J = 7.5 Hz, 1 H),
7.15 (br, 1 H), 7.21–7.46 (m, 6 H), 8.24 (s, 1 H); MS
(FAB) m/z 418.3 (M + H+).
6.1.3.2. (1RS,3aSR)-4-
(Formyl-phenylamino)-1-(2,3,3a,4,5,6-hexahydro-
1H-phenalen-1-yl)-piperidine-4-carboxylic acid amide
14b
6.1.2.7. (1RS,3aRS)-8-(2,3,3a,4,5,6-
Hexahydro-1H-phenalen-1-yl)-1-phenyl-
1,3,8-triaza-spiro[4.5]decan-4-one hydrochloride 3a
Reaction of (1RS,3aSR)-1-(2,3,3a,4,5,6-hexahydro-1H-
phenalen-1-yl)-4-phenylamino-piperidine-4-carbonitrile
13b (1.92 g, 5.17 mmol) as described for 14a (6.1.2.6.)
gave 14b (2.03 g, 94%) as a white foam: 1H-NMR
(CDCl3) δ 1.13–1.38 (m, 2 H), 1.59–2.14 (m, 8 H),
2.32–2.59 (m, 6 H), 2.64 (mc, 1 H), 2.88 (dd, J = 7.5,
5.5 Hz, 2 H), 3.65 (dd, J = 6.5, 4.5 Hz, 1 H), 5.51 (br, 1
H), 6.94 (d, J = 7.5 Hz, 1 H), 7.08 (t, J = 7.5 Hz, 1 H),
7.15 (br, 1 H), 7.24–7.46 (m, 6 H), 8.24 (s, 1 H); MS
(FAB) m/z 418.3 (M + H+).
A
stirred solution of (1RS,3aRS)-4-(formyl-
phenylamino)-1-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-
yl)-piperidine-4-carboxylic acid amide 14a (2.15 g,
5.15 mmol) in triethyl orthoformiate (70 mL) was heated
under reflux conditions for 4 days, evaporated and dis-
solved in methanol (60 mL) and THF (30 mL). To the
stirred solution was added, at room temperature, sodium
boronhydride (0.39 g, 10.3 mmol) and the solution was

J. Wichmann et al. / Eur. J. Med. Chem. 35 (2000) 839–851
847
6.1.3.3. (1RS,3aSR)-8-(2,3,3a,4,5,6-
Hexahydro-1H-phenalen-1-yl)-1-phenyl-
(hexane/ethyl acetate, 5:1) to yield 11b (2.36 g, 84%) as
a white solid: m.p. 59 °C; [α]D20 = –34.5° (c = 0.21,
CHCl3).
1,3,8-triaza-spiro[4.5]decan-4-one hydrochloride 3b
Transformation of (1RS,3aSR)-4-(formyl-phenyl-
amino)-1-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-
piperidine-4-carboxylic acid amide 14b (2.03 g,
4.86 mmol) as described for 3a (6.1.2.7.) yielded 3b
6.1.4.3. (1S,3aS)-1-(2,3,3a,4,5,6-
Hexahydro-1H-phenalen-1-yl)-4-phenylamino-
piperidine-4-carbonitrile 13c
1
(0.98 g, 46%) as a white solid: m.p. 233 °C; H-NMR
Transformation
phenalen-1-one
of
11b
(S)-2,3,3a,4,5,6-hexahydro-
described for 13a
(DMSO-d6) δ 1.33–1.64 (m, 2 H), 1.72–2.19 (m, 7 H),
2.37–2.64 (m, 2 H), 2.80 (t, J = 6.0 Hz, 2 H), 2.92 (mc, 1
H), 3.01–3.43 (m, 3 H), 3.73–4.02 (m, 2 H), 4.62–4.73
(m, 3 H), 6.78 (t, J = 7.0 Hz, 1 H), 7.07–7.29 (m, 6 H),
7.80 (d, J = 7.0 Hz, 1 H), 9.04 (s, 1 H), 10.41 (s, br, 1 H);
MS (FAB) m/z 402.3 (M + H+). Anal. (C26H27N3O.HCl)
C, H, N.
as
(6.1.2.4.–6.1.2.5.) gave, after crystallization from diethyl
ether/hexane, 13c as a light yellow solid (ee > 99%,
HPLC): m.p. 152 °C; [α]D20 = +28.4° (c = 0.26, CHCl3).
6.1.4.4. (1S,3aS)-8-(2,3,3a,4,5,6-
Hexahydro-1H-phenalen-1-yl)-1-phenyl-
1,3,8-triaza-spiro[4.5]decan-4-one hydrochloride 3c
Transformation of (1S,3aS)-1-(2,3,3a,4,5,6-hexahydro-
1H-phenalen-1-yl)-4-phenylamino-piperidine-4-
carbonitrile 13c as described for 3a (6.1.2.6.–6.1.2.7.)
yielded 3c as an off-white solid (ee > 98%, HPLC):
m.p. = 262 °C, [α]D20 = –98.5° (c = 0.11, MeOH). Anal.
(C26H27N3O.HCl) C, H, N.
6.1.4. Preparation of (1S,3aS)-8-(2,3,3a,4,5,6-
hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-
triaza-spiro[4.5]decan-4-one hydrochloride 3c
6.1.4.1. (S)-3-(1,2,3,4-Tetrahydro-
naphthalen-1-yl)-propionic acid 10b
In a glove box (argon, < 1 ppm oxygen) a 185 mL
stainless steel autoclave was charged with 9 (18.0 g,
89.0 mmol, containing 5% of the 3-(naphthalen-1-yl)-
propionic acid as an impurity), a solution of (S)-
MeOBIPHEP/Ru(OAc)2 (713 mg, 0.89 mmol) in metha-
nol (128 mL) and triethylamine (1.80 g, 17.8 mmol).
After having been connected to a hydrogen source
(99.9999% purity) the argon was replaced with hydrogen
and then the hydrogenation was run at 20 °C under a
constant pressure of 100 bar. After 114 h the conversion
was complete. The dark hydrogenation mixture was
evaporated, the residue was dissolved in sodium hydrox-
ide solution (0.5 N, 200 mL) and extracted with dichlo-
romethane (3 × 100 mL). Addition of hydrochloric acid
37% (ca. 250 mmol) to the aqueous phase until the pH
was ca. 1 led to separation of 10b as a dark oil, which was
extracted into dichloromethane (3 × 100 mL). This or-
ganic phase was washed with water (2 × 100 mL), dried
(Na2SO4) and evaporated to afford 10b (17.8 g, 99%) as
a light grey oil (94% ee) [19], which crystallized upon
standing: [a]D20 = +6.75° (c = 0.32, CHCl3). Anal.
(C13H16O2) C, H.
6.1.5. Preparation of (1R,3aR)-8-(2,3,3a,4,5,6-
hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-
triaza-spiro[4.5]decan-4-one hydrochloride 3d
6.1.5.1. (R)-3-(1,2,3,4-Tetrahydro-
naphthalen-1-yl)-propionic acid 10c
The asymmetric hydrogenation of 9 (4.04 g, 20.0 mmol)
in methanol (46 mL) in the presence of (R)-MeOBIPHEP/
Ru(OAc)2 (160 mg, 0.20 mmol) as catalyst under the
same conditions as described for 10b afforded 10c (3.7 g,
92%) as a light grey oil (93% ee) [19], which crystallized
upon standing: [α]D20 = –6.43° (c = 0.26, CHCl3). Anal.
(C13H16O2) C, H.
6.1.5.2. (R)-2,3,3a,4,5,6-Hexahydro-phenalen-1-one 11c
Reaction of (R)-3-(1,2,3,4-tetrahydro-naphthalen-1-yl)-
propionic acid 10c (3.64 g, 17.8 mmol) as described for
11b (6.1.4.2.) gave 11c (3.13 g, 94%) as a light orange
solid: m.p. 57 °C; [α]D20 = +35.1° (c = 0.30, CHCl3).
6.1.5.3. (1R,3aR)-1-(2,3,3a,4,5,6-
Hexahydro-1H-phenalen-1-yl)-4-
phenylamino-piperidine-4-carbonitrile 13d
6.1.4.2. (S)-2,3,3a,4,5,6-Hexahydro-phenalen-1-one 11b
To a stirred solution of (S)-3-(1,2,3,4-tetrahydro-
naphthalen-1-yl)-propionic acid 10b (3.10 g, 15.2 mmol)
in trifluoroacetic acid (30 mL) was added at 0 °C trifluo-
roacetic acid anhydride (3 mL). The reaction mixture was
stirred at room temperature for 75 min, evaporated and
the crude product purified by column chromatography
Transformation
of
(R)-2,3,3a,4,5,6-hexahydro-
phenalen-1-one 11c as described for 13a (6.1.2.4.–6.1.2.5.)
gave, after crystallization, from diethyl ether/hexane 13d
as a light yellow solid: m.p. 151 °C; [α]D20 = +29.2°
(c = 0.31, CHCl3).

848
J. Wichmann et al. / Eur. J. Med. Chem. 35 (2000) 839–851
6.1.5.4. (1R,3aR)-8-(2,3,3a,4,5,6-
Hexahydro-1H-phenalen-1-yl)-1-phenyl-
[α]D20 = +58.8°
(c = 0.10,
MeOH).
Anal.
(C26H27N3O.HCl) C, H, N.
1,3,8-triaza-spiro[4.5]decan-4-one hydrochloride 3d
Transformation of (1R,3aR)-1-(2,3,3a,4,5,6-hexahydro-
1H-phenalen-1-yl)-4-phenylamino-piperidine-4-
carbonitrile 13d as described for 3a (6.1.2.6.–6.1.2.7.)
yielded 3d as a light brown solid: m.p. = 253 °C,
6.1.8. X-ray crystallography
6.1.8.1. Crystal data for 10b (figure 7)
(S)-3-(1,2,3,4-Tetrahydro-naphthalen-1-yl)-propionic
acid as (R)-(+)-1-phenylethylammonium salt: C21H27NO2
[C13H16O2 (1:1) C8H11N], M = 325.44, monoclinic, space
group P2(1), a = 11.992 (3) Å, b = 6.198 (2) Å, c =
12.709 (3) Å, ꢀ = 101.50 (5)°, T = 180 K, Z = 2; 1 612
unique reflections measured on a Siemens P3 diffracto-
meter with graphite-monochromated Mo-Kα radiation,
which were used in all calculations, structure solved by
direct methods and expanded using Fourier techniques
and the program SHELX-97 [28]. All non-hydrogen
atoms were refined anisotropically using full matrix
least-squares refinement without restraints, final R-factor
0.0803 and wR-factor 0.1490 (all data). The structure has
been deposited at the Cambridge Crystallographic Data
Centre, deposition number CCDC 133359.
[α]D20 = +94.6°
(C26H27N3O.HCl) C, H, N.
(c = 0.10,
MeOH).
Anal.
6.1.6. Preparation of (1S,3aR)-8-(2,3,3a,4,5,6-
hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-
triaza-spiro[4.5]decan-4-one hydrochloride 3e
6.1.6.1. (1S,3aR)-1-(2,3,3a,4,5,6-
Hexahydro-1H-phenalen-1-yl)-4-
phenylamino-piperidine-4-carbonitrile 13e
Transformation
of
(R)-2,3,3a,4,5,6-hexahydro-
phenalen-1-one 11c as described for 13a (6.1.2.4.–6.1.2.5.)
gave, after crystallization from diethyl ether/hexane, 13c
as a white solid: m.p. 142 °C; [α]D20 = +50.4° (c = 0.28,
CHCl3).
6.1.8.2. Crystal data for 13b (figure 8)
(1RS,3aSR)-1-(2,3,3a,4,5,6-Hexahydro-1H-phenalen-
1-yl)-4-phenylamino-piperidine-4-carbonitrile: C25H29N3,
M = 371.51, monoclinic, space group P2(1)/c, a =
18.419 (4) Å, b = 8.3060 (17) Å, c = 14.076 (3) Å, ꢀ =
111.18 (3)°, T = 180 K, Z = 4; 3 246 reflections measured
6.1.6.2. (1S,3aR)-8-(2,3,3a,4,5,6-
Hexahydro-1H-phenalen-1-yl)-1-phenyl-
1,3,8-triaza-spiro[4.5]decan-4-one hydrochloride 3e
Transformation of (1S,3aR)-1-(2,3,3a,4,5,6-hexahydro-
1H-phenalen-1-yl)-4-phenylamino-piperidine-4-
carbonitrile 13e as described for 3a (6.1.2.6.–6.1.2.7.)
yielded 3e as an off-white solid: m.p. = 239 °C,
on
a
Siemens P3 diffractometer with graphite-
monochromated Mo-Kα radiation, 2 610 unique
(Rint = 0.0284) which were used in all calculations,
structure solved by direct methods and expanded using
Fourier techniques and the program SHELX-97 [28]. All
non-hydrogen atoms were refined anisotropically using
full matrix least-squares refinement without restraints,
final R-factor 0.1087 and wR-factor 0.1387 (all data). The
structure has been deposited at the Cambridge Crystallo-
graphic Data Centre, deposition number CCDC 133360.
[a]D20 = –60.7°
(C26H27N3O.HCl) C, H, N.
(c = 0.10,
MeOH).
Anal.
6.1.7. Preparation of (1R,3aS)-8-(2,3,3a,4,5,6-
hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-
triaza-spiro[4.5]decan-4-one hydrochloride 3f
6.1.7.1. (1R,3aS)-1-(2,3,3a,4,5,6-
Hexahydro-1H-phenalen-1-yl)-4-
phenylamino-piperidine-4-carbonitrile 13f
6.2. Pharmacology
Transformation
phenalen-1-one
(6.1.2.4.–6.1.2.5.) gave, after crystallization from diethyl
ether/hexane, 13c as a white solid (ee > 99%, HPLC):
m.p. 141 °C; [α]D20 = –50.4° (c = 0.27, CHCl3).
of
11b
(S)-2,3,3a,4,5,6-hexahydro-
described for 13a
6.2.1. Cell culture and membrane preparation
as
The cDNAs encoding human µ opioid receptor (hµR),
human κ opioid receptor (hκR) and human δ opioid
receptor (hδR) were subcloned into the pSFV2gen vector
and recombinant Semliki Forest Virus stocks were gen-
erated as described previously [29]. Baby hamster kidney
(BHK) cells were infected and harvested for membrane
preparation 24 h after infection. The cDNA encoding
human OFQ receptor (hOFQR) was inserted into the
pcDNA3 vector and stably transfected into human em-
bryonic kidney 293 (HEK293) cells. One cell clone was
selected for pharmacological characterization.
6.1.7.2. (1R,3aS)-8-(2,3,3a,4,5,6-
Hexahydro-1H-phenalen-1-yl)-1-phenyl-
1,3,8-triaza-spiro[4.5]decan-4-one hydrochloride 3f
Transformation of (1R,3aS)-1-(2,3,3a,4,5,6-hexahydro-
1H-phenalen-1-yl)-4-phenylamino-piperidine-4-
carbonitrile 13f as described for 3a (6.1.2.6.–6.1.2.7.)
yielded 3f as an off-white solid: m.p. = 240 °C,

J. Wichmann et al. / Eur. J. Med. Chem. 35 (2000) 839–851
849
Figure 7. Crystal structure of 10b.
Membrane fractions were prepared by homogenization
of cells followed by a 30 min centrifugation at 39 000 g
using a Beckman JA-20 rotor. The resulting membrane
pellet was suspended in 50 mM Tris, pH 7.8, 1 mM
EDTA, 6 mM MgCl2 buffer at a concentration of 2 ×
107 cells/mL and frozen at –80 °C.
(OFQR), naloxone 1.3 × 10–9 M (µR), naloxone 2.8 × 10–9
(κR) and deltorphin II 0.36 × 10–9 M (δR).
Reactions were performed in 1 mL binding buffer
(50 mM Tris-HCl, pH 7.8; 1 mM EGTA; 5 mM MgCl2;
0.1% BSA) for 60 min at 22 °C. At the end of the
incubation, the reaction mix was filtered through GF/C-
filters (Amersham), which had been pre-coated (0.3%
polyethyleneimine plus 0.1% BSA). Filters were washed
three times with 0.5 mL cold wash buffer (50 mM Tris-
HCl, pH 7.5). Finally, 60 µL scintillation fluid (Micro
Scint, Canberra Packard) was added and samples were
counted in a Top Counter (Packard). Non-specific binding
was defined in the presence of 1 µM unlabelled OFQ
(hOFQR), 10 µM naloxone (hµR and hκR) or 10 µM
deltorphin II (hδR).
6.2.2. Radioligands
The radioligands [leucyl-3H]-OFQ (specific activity
150 Ci/mmol), [N-allyl-2-3-3H]-naloxone (specific activ-
ity 54.5 Ci/mmol) and [Ile5,6-3H]-deltorphin II {[3H]Tyr-
D-Ala-[3H]Phe-Glu-Ile-Ile-Gly-NH2} (specific activity
72 Ci/mmol) were purchased from Amersham (Little
Chalfont, UK).
6.2.3. Radioligand binding assays
Competitive binding displacement analysis was per-
formed with membranes prepared from permanently
transfected HEK293 cells expressing hOFQR (20 µg
membrane protein) and 0.1 nM [leucyl-3H]-OFQ. Com-
petitive binding displacement analyses for opioid recep-
tors were performed with membranes prepared from
BHK cells transiently expressing hµR, hκR or hδR (10 µg
membrane protein each) and 1.5 nM (hµR) and 3 nM
(hκR) [N-allyl-2-3-3H]-naloxone or 0.3 nM [Ile5,6-3H]-
6.2.4. GTPγ35S binding assay
Agonist-mediated binding of GTPγ35S was investi-
gated in 96-well plates using a scintillation proximity
assay (SPA) and membranes prepared from cells express-
ing hOFQR, hµR, hκR or hδR. Binding was performed in
200 µL 20 mM HEPES-buffer (pH 7.4, plus 6 mM MgCl2
and 100 mM NaCl), supplemented with 20 µM GDP,
10 µM cold GTPγS and 0.3 nM GTPγ35S (1 130 Ci/
mmol). Twenty micrograms of membrane, 1 mg wheat-
germ agglutinin SPA beads (Amersham, Little Chalfont,
deltorphin II (hδR). The KD values are: OFQ 7 × 10–11
M

850
J. Wichmann et al. / Eur. J. Med. Chem. 35 (2000) 839–851
Figure 8. Crystal structure of 13b.
UK) and either OFQ (10–5–10–10 M) or synthetic com-
pounds (10–4–10–10 M) were added.
The reaction mixture was incubated on a shaker for
60 min at 22 °C and then centrifuged for 5 min at
1 500 rpm in an Eppendorf 5403 centrifuge. Finally the
plates were read in a Top Counter (Packard).
white environment in a sound-proof observation room
and the light intensity on the central platform was
225 lux. All parts of the apparatus were made of grey
PVC plastic. The floor of the maze was thoroughly
cleaned after each test trial. Thirty minutes after i.p.
injection, rats were placed in the centre of the plus-maze
facing one of the closed arms and observed via a
closed-circuit TV camera by an observer located in an
adjacent room. Locomotor behaviour in the maze was
recorded for 5 min using an automated image analysis
system (Ethovision, Noldus Information Technology).
The number of entries, the distance and the total time
spent in the open arms were measured. Total distance
moved, distance and number of closed arm entries were
also recorded as measures of spontaneous general activ-
ity.
6.2.5. Elevated plus-maze test
This test is based on the natural aversion of rodents for
open spaces and heights and uses an elevated maze with
two open and two closed arms; exposure to an open maze
alley leads to an approach-avoidance conflict that is
considerably stronger than that evoked by exposure to a
closed maze alley. The time spent and the number of
entries into open arms are indices of neophobic anxiety in
animals [30]. Anxiolytics increase and anxiogenics de-
crease these two measures. The elevated plus-maze is
based on spontaneous behaviour and thus does not
necessitate training or use of appetitive behaviours in-
duced by food or water deprivation [31].
Male Sprague-Dawley rats weighing 110–200 g and
housed with ad libitum access to food and water on a 12 h
light/12 h dark cycle (6 am–6 pm) light were used. The
apparatus was 50 cm above the floor and consisted of two
open arms (50 × 10 cm) perpendicular to two closed arms
(50 × 10 × 50 cm high) extending from an open central
area (10 × 10 cm). The maze was positioned in a closed,
Forced motor performance was subsequently evaluated
in a traction test that consisted of forcing rats to grasp a
horizontally strung wire (20 cm above the bench level,
2 mm in diameter, 20 cm long) with their fore paws.
Various items were scored: the grasp reflex (score 1),
body weight support (score 2), climb reflex (score 3) and
escape (score 4); group scores were calculated by aver-
aging individual scores. The maximum of two permis-
sible readings was recorded. Fore limb grip strength was
then quantitatively assessed using a digital strain gauge.

J. Wichmann et al. / Eur. J. Med. Chem. 35 (2000) 839–851
851
[4] Chen Y., Fan Y., Liu J., Mestek A., Tian M., Kozak C.A., Yu L.,
Animals held by the tail grasped a triangular bar (2 mm
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triangle. The strain gauge remains fixed at its maximum
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the maximum of three permissible readings was recorded
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The experimental procedures used received approval
based on international guidelines and adherence to Swiss
federal regulations and guidelines on animal experimen-
tation provided by the Swiss Academy of Sciences
(1995).
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Compound 3c was freshly prepared in 0.3% v/v Tween
80 in physiological saline (NaCl 0.9%) and ultrasonified
under normal laboratory conditions. It was injected intra-
peritoneally in a volume of 5 mL/kg body weight. Three
doses (0.32, 1 and 3.2 mg/kg i.p.) administered versus
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[19] The ee of 10b–c was determined by HPLC using a Hewlett Packard
instrument equipped with a Chiracel OD-H column. The mobile
phase was A: hexane, B: 10% ethanol in hexane, C: 0.4% trifluo-
roacetic acid in hexane. The retention times were 12.0 min (10c),
14.0 min (10b), 16.7 min (9).
Acknowledgements
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We would like to express our thanks to Arthur Meili,
Philipp Oberli, Sandra Müller, Daniel Wehrli, Patrick
Biry and Jacqueline Higelin for their skilful technical
assistance, Drs Wolf Arnold, Walter Huber, Stephan
Müller, Mr Walter Meister, Roger Rueher, Vladimir
Meduna and Gottfried Oesterhelt for spectroscopic deter-
minations and analysis as well as Drs Kenneth Lundstrom
and Parichehr Malherbe for membranes expressing hu-
man OFQ and opioid receptors.
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