Novel spiropiperidines as highly potent and subtype selective σ-receptor ligands. Part 1

Article abstract of DOI:10.1021/jm010992z

A series of spiro[[2]benzopyran-1,4′-piperidines] and spiro[[2]benzofuran-1,4′-piperidines] of general structure 10 is prepared, and the affinity for σ₁- and σ₂-receptors is investigated by means of radioligand binding assays. The sy

Full text of DOI:10.1021/jm010992z

438
J . Med. Chem. 2002, 45, 438-448
Novel Sp ir op ip er id in es a s High ly P oten t a n d Su btyp e Selective σ-Recep tor
Liga n d s. P a r t 1
Christoph A. Maier and Bernhard Wu¨nsch*
Pharmazeutisches Institut der Universita¨t Freiburg, Albertstrasse 25, 79104 Freiburg i. Br., Germany
Received J uly 19, 2001
A series of spiro[[2]benzopyran-1,4′-piperidines] and spiro[[2]benzofuran-1,4′-piperidines] of
general structure 10 is prepared, and the affinity for σ1- and σ2-receptors is investigated by
means of radioligand binding assays. The synthesis of the spiropiperidines 14a and 23 proceeds
from bromine/lithium exchange of the bromoacetals 11 and 21, addition to piperidin-4-one 12a ,
and subsequent cyclization. Systematic variations of the substituent R at the nitrogen atom,
the group X in position 3, and the ring size of the oxygen heterocycle are performed. The σ1-
and σ2-receptor affinities are determined with guinea pig brain and rat liver membrane
preparations using [3H]-labeled (+)-pentazocine and ditolylguanidine, respectively. Test results
show that a benzyl residue at the piperidine nitrogen atom and a methoxy group in position 3
are advantageous for high σ1-receptor affinity. In this series the 1′-benzyl-3-methoxy-3,4-
dihydrospiro[[2]benzopyran-1,4′-piperidine] (14a ) and the 1′-benzyl-3-methoxy-3H-spiro[[2]-
benzofuran-1,4′-piperidine] (23) are among the most potent σ1-ligands interacting in the low
nanomolar range with σ1-receptors (14a , Ki ) 1.29 nM; 23, Ki ) 1.14 nM). Variation of the
nitrogen substituent R from benzyl to H, alkyl, phenyl, or ω-phenylalkyl and the group X from
methoxy to hydroxy, carbonyl, or alkyloxy led to reduced σ1-receptor affinity. In addition to
their high σ1-receptor affinity, the spiropiperidines 14a and 23 display excellent selectivity
toward σ2-receptors (σ1/σ2 ) 2708 and 1130) and several other receptor and reuptake systems.
Introduction of a polar hydroxy group in position 3 and elongation of the distance between the
piperidine nitrogen atom and the phenyl moiety result in ligands with considerable σ2-receptor
affinity and therefore diminished σ1/σ2-receptor selectivity. The hemiacetalic 1′-(3-phenylpropyl)-
3,4-dihydrospiro[[2]benzopyran-1,4′-piperidin]-3-ol (15e) represents the most active σ2-receptor
ligand in this series with a Ki value of 83.1 nM.
In tr od u ction
Up to now, at least two distinct σ-receptor subtypes
termed σ1- and σ2-receptors have been pharmacologi-
cally characterized. According to this classification,
haloperidol (1) and ditolylguanidine (2) bind with high
affinity at both σ1- and σ2-receptors (compare Table 1,
entries 9 and 11), whereas (+)-benzomorphans (e.g., (+)-
pentazocine (3)) display high affinity for σ1-receptors but
low affinity for σ2-receptors (compare Table 1, entry 12).
The stereochemistry of the benzomorphans is important
for σ-binding, with σ1-receptors binding enantioselec-
tively the (+)-enantiomers, whereas (-)-benzomorphans
are mixed σ1- and σ2-ligands with moderate affinity.14-16
The existence of a third σ3-subtype has been intensively
discussed.17 However, recent investigations revealed
that there are no significant differences of ligand
selectivity and CNS distribution between histamine
H1- and σ3-receptors.18
Meanwhile, the gene coding for the σ1-receptor of
different species and tissues has been cloned. Whereas
the σ1-receptors from human placental cells, guinea pig
liver, and rat brain are >92% identical and >95%
similar at the level of amino acid sequence, there is no
significant homology between the σ1-receptor and any
other known mammalian receptor or even protein.
However, a significant homology (∼30%) exists between
the cloned σ1-receptor and the yeast sterol-C8/C7-
isomerase. The role of this observation still remains to
be established. The protein, coded by the rat brain σ1-
receptor gene, comprises 223 amino acids (23 kDa) and
Originally Martin and co-workers suggested the σ-re-
ceptor to be a subtype of the opioid receptors.1 However,
since most of the σ-effects caused by typical σ-ligands
are not abolished by the opioid antagonist naloxone, this
classification was discarded.2 Nowadays, σ-receptors are
well recognized as a class of haloperidol-sensitive,
nonopioid, nonphencyclidine receptors with their own
binding profile and characteristic distribution within the
central nervous system (CNS) as well as in many tissues
outside the CNS (e.g., kidney, liver, lung).3,4
σ-Receptors are involved in several physiological and
pathophysiological processes. Therefore, ligands inter-
acting with σ-receptors are prone to modulate these
(patho)physiological events and influence psychiatric
disorders.3,4 A particular interest has been evoked by
the perspective of using σ-agents as atypical antipsy-
chotics, which are devoid of the typical extrapyramidal
motoric side effects5 associated with classical antipsy-
chotic D2 antagonists. Like haloperidol (1), several well-
established neuroleptic agents bind not only at dop-
amine D2 receptors but also at σ-receptors.6,7 Further-
more, σ-ligands could be used in the treatment of
cocaine abuse,8 depressions,9 and epileptic disorders.10
They also have potential as neuroprotective,11 antiam-
nesic,11 antineoplastic,12 and tumor imaging agents.13
* To whom correspondence should be addressed. Phone: +49-
761-203-6320. Fax: +49-761-203-6321. E-mail: wuensch@ruf.uni-
freiburg.de.
10.1021/jm010992z CCC: $22.00 © 2002 American Chemical Society
Published on Web 12/19/2001

Spiropiperidines
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 439
Ch a r t 1
3.8 nM). Thereby, the σ-receptor affinity was determined
in a receptor binding assay using [3H]-ditolylguanidine
(2) as the radioligand, which does not discriminate
between σ1- and σ2-receptors.24 In the meantime it was
shown that the tetraline derivative 7 possesses signifi-
cant preference for σ1-receptors.25 Some variations of the
piperidine nitrogen substituent have been performed,
but introduction of heteroatoms or substituents in the
aliphatic part of the tetraline substructure is not
described.24
The spiro[[2]benzopyran-1,4′-piperidine] 8 belongs to
the most potent and σ2-selective ligands described so
far (σ1, IC50 ) 53 nM; σ2, IC50 ) 0.90 nM). Whereas the
oxygen heterocycle of 8 has been extensively modified,
introduction of substituents into the benzopyran moiety
and modifications of the σ2-pharmacophoric 4-[1-(4-
fluorophenyl)indol-3-yl]butyl residue in the benzopyran
moiety are not reported.25 Furthermore, the 1-benzo-
thiopyran derivative 9 with the spiro connection shifted
to position 2 represents a very potent and selective
ligand for σ1-receptors (σ1, Ki ) 0.5 nM; σ2, Ki ) 416
nM).26
This prompted us to undertake the synthesis of novel
spirocyclic σ-ligands with the general structure 10. In
particular, we focused on the systematic variation of the
substituent R at the piperidine nitrogen atom, the
substituent X in position 3 of the 2-benzopyran ring
system, and the ring size of the oxygen heterocycle (n
) 0 or 1). The affinities for σ1- and σ2-receptors of the
synthesized compounds were determined in radioligand
binding assays. Relationships between ligand structure
and σ1- and σ2-receptor affinities and, moreover, σ1/σ2-
selectivity are discussed.
Ch a r t 2
Ch em istr y
The synthesis of spiro[[2]benzopyran-1,4′-piperidines]
of general formula 10 proceeds from the treatment of
2-(2-bromophenyl)acetaldehyde dimethyl acetal (11)27
with n-butyllithum at -78 °C to generate an aryllithium
intermediate that was trapped with 1-substituted pip-
eridin-4-ones 12a -c to afford the hydroxyacetals 13a -c,
respectively. The subsequent cyclization of the hydroxy-
acetals 13a -c was catalyzed by p-toluenesulfonic acid.
The use of methanol as a solvent is crucial in this step
to obtain the spiro[[2]benzopyran-1,4′-piperidines] 14a -c
in high yields.
a single putative transmembrane domain. In compari-
son, the molecular weight of the σ2-receptor, which has
not been cloned yet, is estimated to be about 16-18
kDa.19,20
The group of high-affinity σ-ligands contains several
structurally unrelated compounds6 including butyrophe-
none neuroleptics (e.g., haloperidol, 1), guanidines (e.g.,
2), dextrorotatory benzomorphans (e.g., (+)-pentazocine,
3), and cis-configured cyclohexane-1,2-diamines (e.g.,
4).21,22 The structurally unique cyclopropylmethylamine
igmesine (5) is in phase II trials for the treatment of
depressive disorder.9 The haloperidol related piperazine
derivative BMY-14802 (6), which interacts unselectively
with σ1- and σ2-receptors, has been investigated in
clinical trials (phase II) as atypical antipsychotic23 (see
Chart 1 for the structures of compounds 1-6).
Our work dedicated to the development of novel
σ-ligands was inspired by the already existing structure-
activity relationship studies on the spirocyclic piperidine
derivatives 7-9 (Chart 2). The tetraline derivative 7
developed by Merck, Sharp, and Dohme revealed an
Attempts to cleave the N-benzyl substituent of 14a
with hydrogen in the presence of the catalyst Pd/C led
to a mixture of the desired secondary amine 14d and,
surprisingly, the methyl derivative 14c. The identity of
the methyl derivative 14c was unequivocally proven by
comparison of the spectroscopic data with an authentic
sample prepared by addition of the bromo acetal 11 to
1-methylpiperidin-4-one 12c and subsequent cycliza-
tion.28 However, using ammonium formate29 instead of
hydrogen led to clean cleavage of the N-benzyl substitu-
ent of 14a to provide the secondary amine 14d in
70% yield (Scheme 1). The secondary amine 14d reacted
with 1-bromo-3-phenylpropane, 1-chloro-4-phenylbu-
tane, and 3-chloro-1-phenylprop-1-ene (cinnamyl chlo-
ride) to afford spiro[[2]-benzopyran-1,4′-piperidine]
derivatives with N-(3-phenylpropyl) 14e, N-(4-phenyl-
butyl) 14f, and N-cinnamyl 14g residues, respectively
(Scheme 1).
affinity for σ-receptors in the nanomolar range (IC50
)

440 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2
Maier and Wu¨nsch
Sch em e 1a
Sch em e 2a
a
Reagents and reaction conditions: (a) n-BuLi, THF, -78 °C;
13a , 39%; 13b, 44%; 13c, 51%. (b) TsOH, CH3OH, room temp; 14a ,
86%; 14b, 92%; 14c, 75%. (c) Ammonium formate, Pd/C, CH3OH,
65 °C, 70%. (d) Ph(CH2)3Br, K2CO3, THF, 66 °C; 14e, 64%. (e)
Ph(CH2)4Cl or PhCHdCHCH2Cl, K2CO3, CH3CN, 82 °C; 14f, 38%;
14g, 24%. (f) PhBr, Pd(OAc)2, PtBu3, NaOtBu, o-xylene, 130 °C;
14h , 21%.
The results of the pharmacological tests indicated
that a phenyl moiety in the nitrogen substituent is
essential for high σ1- and σ2-receptor affinities. There-
fore, we reasoned that the spiro[[2]benzopyran-1,4′-
piperidine] 14h with the phenyl moiety attached directly
to the nitrogen atom should also be included in the
study. The synthesis of the N-phenyl derivative 14h
succeeded by Pd-catalyzed phenylation of the secondary
amine 14d .30,31 Thus, the secondary amine 14d was
heated with bromobenzene, PtBu3, and NaOtBu in the
presence of 0.25 mol % of the catalyst Pd(OAc)2 to yield
the N-phenyl derivative 14h . Thus, the synthesis of the
first series of test compounds 14a -h bearing different
substituents R at the piperidine nitrogen atom was
completed.
a
Reagents and reaction conditions: (a) HCl, THF, room temp;
15a , 69%; 15b, 64%. (b) HCl, THF, 66 °C; 15e, 37%. (c) C2H5OH
or C6H5CH2OH, H2SO4, room temp; 16, 81%; 17, 77%. (d) (NPr4)-
RuO4, NMMO, molecular sieves 4 Å, CH2Cl2, room temp, 43%.
Sch em e 3a
Next, we investigated the analogues with modifica-
tions on the 2-benzopyran ring system. Treatment of the
hydroxy acetals 13a and 13b with diluted HCl yielded
the lactols ()hemiacetals) 15a and 15b, respectively
(Scheme 2). The corresponding N-(3-phenylpropyl)-
substituted 2-benzopyran-3-ol 15e was obtained by
hydrolysis of the methoxy derivative 14e. Since the
N-benzyl derivatives showed the most promising σ-re-
ceptor affinities (compare Table 1), further modifications
were carried out using the N-benzyl-substituted lactol
15a . The homologous ethyl acetal 16 and the benzyl
acetal 17 were prepared by acetalization of the lactol
15a with ethanol and benzyl alcohol, respectively, in the
presence of sulfuric acid. Oxidation of the lactol 15a
with a catalytic amount of tetrapropylammonium per-
ruthenate [(NPr4)RuO4] and an excess of the reoxidant
N-methylmorpholine N-oxide (NMMO)32 provided the
lactone 18.33
a
t
Reagents and reaction conditions: (a) BuBr, CHCl3, 61 °C;
19‚HBr, 24%. (b) H2, Pd/C, HOAc, room temp, 39%.
Since the five membered indane analogue of 7 (L-
693403) and the isobenzofuran analogue of 8 (Lu 28-
179) are superior to the parent six-membered spiro
compounds 7 and 8 in σ-receptor affinity,24,25 the five-
membered spiro compounds of general structure 10 (n
) 0) were also included into the study. Thus, the ring
size represents a third structural parameter influencing
σ-receptor affinity. Starting with 2-bromobenzaldehyde
dimethyl acetal (21) halogen/metal exchange with n-
butyllithium at -95 °C and subsequent reaction with
1-benzylpiperidin-4-one (12a ) afforded the hydroxy
acetal 22 (Scheme 4). Cyclization of the hydroxy acetal
22 with p-toluenesulfonic acid in methanol led to the
cyclic methyl acetal 23, whereas hydrolysis with diluted
HCl provided the lactol 24. The lactone 2536 was
Elimination of methanol succeeded upon heating of
the methyl acetal 14a with tert-butyl bromide34 to yield
the dehydro derivative 19‚HBr (Scheme 3). Careful
hydrogenation (low H2 pressure, short reaction time,
solvent HOAc) of 19 afforded the N-benzyl-substituted
spiropiperidine 2035 without further substituents in the
benzopyran ring system.

Spiropiperidines
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 441
Sch em e 4a
14a , displayed the highest σ1/σ2-selectivity (2708-fold,
entry 1). However, the σ2-receptor affinity was increased
with elongation of the nitrogen-phenyl distance. In this
series the highest σ2-receptor affinity (311 nM) and,
therefore, the lowest σ1/σ2-selectivity (30-fold) was found
for the 4-phenylbutyl derivative 14f (entry 6).
The results of the systematic variation of the sub-
stituent X in position 3 of the benzopyran scaffold
compared with the most active σ1-ligand 14a with the
benzyl residue at the nitrogen atom (Ki ) 1.29 nM) are
shown in Table 2. The lactol 15a with a polar hydroxy
group in position 3 (entry 2) displayed a somewhat lower
affinity for σ1-receptors than the methyl acetal 14a .
Enlargement of the acetalic methoxy group led to the
ethoxy and benzyloxy derivatives 16 and 17 with
reduced σ1-receptor affinity (entries 5, 6). Oxidation to
the lactone 18 was detrimental for the σ1-receptor
affinity (entry 7). A low Ki value (Ki ) 1.87 nM) indi-
cating high σ1-receptor affinity was determined for the
corresponding elimination product 19 (entry 8). The
most active σ1-receptor ligand, however, was the unsub-
stituted benzopyran derivative 20 with a Ki value in the
subnanomolar range (Ki ) 0.69 nM). Nevertheless, since
20 exhibits a high σ2-affinity (Ki ) 99.7 nM), the σ1/σ2-
selectivity of 20 was reduced (146-fold, entry 9).
a
Reagents and reaction conditions: (a) n-BuLi, THF, -95 °C.
(b) TsOH, CH3OH, room temp, 36%. (c) HCl, THF, 66 °C, 11%. (d)
(NPr4)RuO4, NMMO, molecular sieves 4 Å, CH2Cl2, room temp,
58%.
prepared by oxidation of the lactol 24 with (NPr4)RuO4
and NMMO.32
In the hemiacetalic series of ligands 15, the σ1-
receptor affinity was influenced by the nitrogen sub-
stituent in the same way as in the acetalic series 14.
Hence, the benzyl derivative 15a displayed the highest
σ1-receptor affinity (entry 2), whereas the 2-phenylethyl
derivative 15b was considerably less active (entry 3).
Once more, the 3-phenylpropyl derivative 15e (entry 4)
showed higher σ1-receptor affinity than the 2-phenyl-
ethyl derivative 15b but lower affinity than the benzyl
derivative 15a .
Recep tor Bin d in g Stu d ies
The σ-receptor affinities of the test compounds were
determined in competition experiments with radioli-
gands. In the σ1-assay, homogenates of guinea pig brains
were used as the receptor material. The σ1-selective
ligand [3H]-(+)-pentazocine (3) was employed as the
radioligand, and the nonspecific binding was determined
in the presence of a large excess of haloperidol (1).37
Homogenates of rat liver served as source for σ2-re-
ceptors in the σ2-assay. Since a σ2-selective radioligand
is not available, the nonselective radioligand [3H]-di-
tolylguanidine (2) was employed in the presence of an
excess of nonradiolabeled (+)-pentazocine (100 nM) for
selective labeling of σ1-receptors. Performing the σ2-
assay in the presence of an excess of non-tritiated 1,3-
di(o-tolyl)guanidine led to the nonspecific binding of the
radioligand.38,39
A polar hydroxy group (15b, entry 3; 15e, entry 4) or
two protons in position 3 of the benzopyran moiety (20,
entry 9) were beneficial for σ2-receptor binding. Among
these ligands, the 3-phenylpropyl-substituted lactol 15e
with a trimethylene spacer between the piperidine
nitrogen atom and the phenyl residue was the most
active σ2-ligand (Ki ) 83.1 nM, entry 4). However, the
σ1-receptor affinity still predominated with a σ1/σ2-
selectivity factor of 7. The lowest σ1/σ2-selectivity factor
was found for the 2-phenylethyl derivative 15b (entry
3) because of its reduced σ1-receptor affinity.
The σ-receptor affinities of the five-membered 2-ben-
zofuran derivatives 23-25 are shown in Table 3. As in
the six-membered 2-benzopyran series, the methoxy
derivative 23 revealed best interaction with σ1-receptors
(Ki ) 1.14 nM, entry 1). Although statistically not
significant, the σ1-receptor affinity of 23 even exceeds
the σ1-receptor affinity of the analogous six-membered
acetal 14a . For comparison, the five-membered ana-
logues of the spirocyclic lead structures 7 and 8 also
display higher σ-receptor affinity than their six-mem-
bered counterparts.24,25 In analogy to the six-membered
benzopyran derivatives, the lactol 24 (entry 2) and the
lactone 25 (entry 3) were less active than the methoxy
derivative 23 at σ1-receptors.
Resu lts a n d Discu ssion
The σ-receptor affinities of 3-methoxy-3,4-dihydro-
spiro[[2]benzopyran-1,4′-piperidines] 14 with varying
piperidine nitrogen substituents R are summarized in
Table 1. Ligands with a proton (14d , entry 4), a methyl
substituent (14c, entry 3), or a phenyl residue directly
attached to the piperidine nitrogen (14h , entry 8) did
not interact significantly with σ1-receptors. However,
introduction of a methylene spacer between the nitrogen
atom and the phenyl residue led to the very potent σ1-
receptor ligand 14a (entry 1) showing the highest σ1-
affinity (Ki ) 1.29 nM) in this series. Whereas the
3-phenylpropyl derivative 14e (entry 5) was almost as
active as the benzyl derivative 14a , the 2-phenylethyl
(14b, entry 2), the 4-phenylbutyl (14f, entry 6), and the
cinnamyl derivatives (14g, entry 7) revealed lower σ1-
receptor affinity.
J ust like the six-membered benzopyrans, the five-
membered benzofurans 23-25 revealed selectivity for
σ1-receptors. Again, a polar hydroxy group in position
Generally, the σ2-receptor affinities of the spiropi-
peridines 14 were lower than their σ1-receptor affinities.
Thus, the most active σ1-ligand, the benzyl derivative

442 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2
Maier and Wu¨nsch
Ta ble 1. σ-Receptor Affinities of Spiro[[2]benzopyran-1,4′-piperidines] 14a -h with Various N-Substituents
Ki ( SEM (3) (nM)
σ1
σ2
σ1/σ2
selectivity
entry
compd
R
([3H]-(+)-pentazocine)
([3H]-ditolylguanidine)
1
2
3
4
5
6
7
8
9
14a
14b
14c
14d
14e
14f
14g
14h
CH2C6H5
(CH2)2C6H5
CH3
1.29 ( 0.18
6.64 ( 1.14
4400 ( 1440
19800 ( 1760
2.07 ( 0.43
10.2 ( 0.94
13.3 ( 1.55
10100 ( 2280
2.20 ( 0.31
265 ( 32
3500 ( 352
410 ( 58
>10000
2708
62
H
>10000
(CH2)3C6H5
(CH2)4C6H5
CH2CHdCHC6H5
C6H5
307 ( 99
311 ( 84
4000 ( 499
>10000
148
30
301
haloperidol (1)
BMY-14802 (6)
ditolylguanidine (2)
(+)-pentazocine (3)
34.2 ( 2.3
391 ( 62
63.9 ( 10.8
16
1.5
0.4
10
11
12
164 ( 47
3.58 ( 0.20
Ta ble 2. σ-Receptor Affinities of Spiro[[2]benzopyran-1,4′-piperidines] 15-20 with Various Substituents X in Position 3 of the
2-Benzopyran Moiety
Ki ( SEM (3) (nM)
σ1
σ2
σ1/σ2
selectivity
entry
compd
X
R
([3H]-(+)-pentazocine)
([3H]-ditolylguanidine)
1
2
3
4
5
6
7
8
9
14a
15a
15b
15e
16
17
18
19
20
OCH3
OH
OH
OH
OC2H5
OCH2C6H5
dO
3,4-d
H
CH2C6H5
CH2C6H5
(CH2)2C6H5
(CH2)3C6H5
CH2C6H5
CH2C6H5
CH2C6H5
CH2C6H5
CH2C6H5
1.29 ( 0.18
2.17 ( 0.40
43.7 ( 8.32
12.4 ( 3.2
5.83 ( 1.48
95.3 ( 22.3
29.3 ( 8.96
1.87 ( 0.27
0.69 ( 0.17
3500 ( 352
513 ( 72
2708
236
2
107 ( 24
83.1 ( 16.0
2960 ( 729
9890 ( 2200
1157 ( 148
302 ( 97
7
508
104
39
162
146
99.7 ( 19.8
Ta ble 3. σ-Receptor Affinities of
assays. The results in Table 4 point out that interactions
of the spiropiperidines 14a , 14e, and 23 with the inves-
tigated receptor and reuptake systems are very low. Ob-
viously, the selectivity of the spiropiperidines 14a , 14e,
and 23 for the σ1-receptor is very high, usually greater
than 1000-fold. However, it should be emphasized that
the 3-phenylpropyl derivative 14e was bound in the
submicromolar range at serotonine 5-HT2A receptors
(IC50 ) 0.46 µM, entry 6), at the serotonine reuptake
system (IC50 ) 0.70 µM, entry 13), and the noradrena-
line reuptake system (IC50 ) 0.51 µM, entry 14).
Nevertheless, the selectivity of 14e for σ1-receptors is
still greater than 100-fold with regard to these systems.
Spiro[[2]benzofuran-1,4′-piperidines] 23-25
Ki ( SEM (3) (nM)
σ1 ([3H]-
σ2 ([3H]-
σ1/σ2
entry compd
X
(+)-pentazocine) ditolylguanidine) selectivity
1
2
3
23
24
25
OCH3
OH
dO
1.14 ( 0.22
7.33 ( 0.60
21.3 ( 3.45
1280 ( 137
761 ( 13
1460 ( 108
1130
104
68
Additionally, the µ-opioid and κ-opioid receptor affini-
ties40 of the spiropiperidines 14a and 20 were investi-
gated. In the µ-receptor screening with test concentra-
tions of 1 and 10 µM, the test compounds 14a and 20
displayed low competition with the radioligand [3H]-
DAMGO. Obviously, the IC50 values are greater than 1
µM. However, the screenings with the same test con-
centrations pointed to promising κ-receptor affinity.
3 (24, entry 2) is advantageous for σ2-binding, but the
σ1-receptor affinity of the lactol 24 still predominates.
The receptor binding profiles of the σ1-selective spiropi-
peridines 14a , 14e, and 23 were determined by screen-
ing these compounds in several receptor and reuptake

Spiropiperidines
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 443
Ta ble 4. Affinities of the Spiropiperidines 14a , 14e, and 23 for
Various Receptor and Reuptake Systems
FT NMR spectrometer (Varian), and chemical shifts (δ) are
reported in ppm downfield from tetramethylsilane. Coupling
constants are given with 0.5 Hz resolution, and the assign-
ments of 13C and of 1H NMR signals were supported by 2D
NMR techniques (correlation spectroscopy (COSY)). Com-
pounds 13c and 14c have been prepared according to ref 28.
IC50 (µM)
entry
receptor system (radioligand)
14a
14e
23
1
2
3
4
5
6
7
8
9
histamine H1 (mepyramine)
dopamine D1 (SCH 23390)
NMDA, PCP (MK 801)
NMDA, glycine (MDL 105519)
serotonine 5-HT1A (5-OH DPAT)
serotonine 5-HT2A (ketanserine)
serotonine 5-HT3 (GR 65630)
noradrenaline R1 (prazosine)
noradrenaline R2 (idazoxane)
neurokinin NK1 (h)
>10
>10
>10
>10
>1
6.6
>10
5.9
3.0
>10
8.0
>10
>10
>10
>1
>10
>10
>10
>10
>1
2-[2-(1-Ben zyl-4-h yd r oxyp ip er id in -4-yl)p h en yl]a ceta l-
d eh yd e Dim eth yl Aceta l (13a ). Under an N2 atmosphere, a
1.6 M solution of n-butyllithium in hexane (5.4 mL, 8.4 mmol)
was slowly added to a cooled (-78 °C) solution of 11 (1.72 g,
7.0 mmol) in THF (20 mL). The mixture was stirred at -78
°C for 5 min, and then a solution of 12a (1.4 g, 7.4 mmol) in
THF (6 mL) was slowly added. The solution was stirred at -78
°C for 3 h. Then it was allowed to warm to room temperature
and stirred for another 2 h. Then water (40 mL) was added,
the mixture was extracted with CH2Cl2, the organic layer was
dried (Na2SO4), and the solvent was removed in vacuo to give
1.76 g of crude product. The crude product was purified by fc
(5.5 cm, petroleum ether/ethyl acetate 1:2, 50 mL, Rf ) 0.05)
to afford a colorless oil, yield 0.963 g (39%). Anal. (C22H29NO3)
H, N; C: calcd, 74.3; found, 73.8. MS (EI): m/z 355 [M+], 340
[M+ - CH3], 264 [M+ - CH2Ph], 91 [CH2Ph+]. IR (film), ν
(cm-1): 3436 (O-H); 2939, 2826 (C-H); 1119, 1067 (C-O);
744, 699 (C-H). 1H NMR (CDCl3): δ (ppm) 1.85 (dd, J ) 13.7/
2.4 Hz, 2 H, N(CH2CH2)2), 2.15 (td, J ) 12.4/4.2 Hz, 2 H,
N(CH2CH2)2), 2.61 (td, J ) 11.7/2.2 Hz, 2 H, N(CH2CH2)2), 2.78
(br d, J ) 10.7 Hz, 2 H, N(CH2CH2)2), 3.34 (s, 6 H, CH(OCH3)2),
3.45 (d, J ) 5.9 Hz, 2 H, ArCH2CH), 3.60 (s, 2 H, NCH2Ph),
4.01 (s, 1 H, OH), 4.54 (t, J ) 5.9 Hz, 1 H, CH2CH(OCH3)2),
7.17-7.44 (m, 9 H, arom).
0.46 >10
>10
>10
>10
1.4
3.1
6.2
10
>10
>10
>10
>10
([Sar9,Met(O2)11]-SP)
11
12
neurokinin NK2 (h)
>10
>10
>10
>10
([125I]-NKA)
neurokinin NK3 (h)
([125I]-[MePhe7]-NKB)
13
14
serotonine reuptake (serotonine)
noradrenaline reuptake
(noradrenaline)
6.0
3.0
0.70
0.51 >10
8.0
15
dopamine reuptake (dopamine)
5.9
1.6
>10
Therefore, the complete competition curves were re-
corded using the radioligand [3H]-U-69593. Both test
compounds 14a and 20 revealed submicromolar κ-recep-
tor affinity with Ki values of 418 nM ((37 nM, n ) 2)
and 586 nM ((11 nM, n ) 2), respectively. We reasoned
that the 4-phenylpiperidine substructure of the spiropi-
peridines 14a and 20, which is also present in opioid
analgesics (e.g., morphine, pethidine), is responsible for
the interaction with κ-opioid receptors.
2-{2-[4-H y d r o x y -1-(2-p h e n y le t h y l)p ip e r id in -4-y l]-
p h en yl}a ceta ld eh yd e Dim eth yl Aceta l (13b). Under an N2
atmosphere a 1.6 M solution of n-butyllithium in hexane (0.8
mL, 1.28 mmol) was slowly added to a cooled (-78 °C) solution
of 11 (0.273 g, 1.11 mmol) in THF (6 mL). The mixture was
stirred at -78 °C for 15 min, then a solution of 12b (0.266 g,
1.31 mmol) in THF (2 mL) was slowly added and the mixture
was stirred at -78 °C for 3.25 h. The mixture was warmed to
room temperature and stirred for another 1 h. Then water (9
mL) was added and the mixture was extracted with CH2Cl2.
The organic layer was dried (Na2SO4), and the solvent was
removed in vacuo to give 0.424 g of crude product, which was
purified by fc (3 cm, ethyl acetate, 20 mL, Rf ) 0.08) to afford
a colorless solid (petroleum ether), mp 117 °C, yield 0.182 g
Con clu sion
The presented data indicate that high σ1-receptor
affinity and good σ1/σ2-selectivity can be obtained with
spiropiperidines 10 bearing a benzyl residue at the
piperidine nitrogen atom and a methoxy group in
position 3. This result is found in both the six-membered
2-benzopyran series and the five-membered 2-benzofu-
ran series of ligands, the methoxy derivatives 14a and
23 representing the most active σ1-ligands, respectively.
The σ1-receptor affinities of 14a and 23 are only
exceeded by that of the unsubstituted spirobenzopyran
20 (Ki ) 0.69 nM). However, the unsubstituted spiroben-
zopyran 20 revealed a significantly lower σ1/σ2-receptor
selectivity (146-fold) in contrast to the σ1-selective
methoxy derivatives 14a (2708-fold) and 23 (1130-fold).
(44%). Anal. (C23H31NO3) C, H, N. MS (EI), m/z: 278 [M+
CH2Ph]. MS (CI, isobutane), m/z: 370 [MH+], 338 [M+
-
-
OCH3], 278 [M+ - CH2Ph]. IR (film), ν (cm-1): 3441 (O-H);
2938, 2828 (C-H); 1114, 1061 (C-O); 756, 697 (C-H). 1H NMR
(CDCl3): δ (ppm) 1.90 (dd, J ) 13.7/2.4 Hz, 2 H, N(CH2CH2)2),
2.22 (td, J ) 13.1/3.7 Hz, 2 H, N(CH2CH2)2), 2.71 (m, 4 H,
N(CH2CH2)2 (2 H), NCH2CH2Ph (2 H)), 2.90 (m, 4 H, N(CH2-
CH2)2 (2 H), NCH2CH2Ph (2 H)), 3.35 (s, 6 H, CH(OCH3)2),
3.46 (d, J ) 5.8 Hz, 2 H, ArCH2CH), 4.06 (s, 1 H, OH), 4.55 (t,
J ) 5.8 Hz, 1 H, ArCH2CH), 7.19-7.33 (m, 8 H, arom), 7.38-
7.44 (m, 1 H, arom).
Exp er im en ta l Section
1′-Ben zyl-3-m eth oxy-3,4-d ih yd r osp ir o[[2]ben zop yr a n -
1,4′-p ip er id in e] (14a ). A solution of 13a (0.75 g, 2.1 mmol)
and p-toluenesulfonic acid monohydrate (0.48 g, 2.5 mmol) in
methanol (60 mL) was stirred at room temperature for 4 d.
Then a solution of Na2CO3 (5% in water, 100 mL) was added.
The mixture was extracted with CH2Cl2, the organic layer was
dried (Na2SO4), and the solvent was removed in vacuo. The
crude product was purified by fc (3 cm, ethyl acetate, 20 mL,
Rf ) 0.38) to afford a colorless oil 14a , which slowly solidified
on standing, giving white needles (iPr2O), mp 41 °C, yield 0.587
g (86%). 14a was converted into the hydrochloride in the usual
manner to obtain 14a ‚HCl as colorless crystals, mp 182-183
°C. Anal. (C21H26NO2Cl) C, H, N. MS (base, EI), m/z: 323 [M+],
308 [M+ - CH3], 292 [M+ - OCH3], 232 [M+ - CH2Ph], 91
[CH2Ph+]. IR (base, film), ν (cm-1): 2938, 2812 (C-H); 1077,
1044 (C-O); 735, 698 (C-H). 1H NMR (base, CDCl3): δ (ppm)
1.86 (d, J ) 13.7 Hz, 1 H, N(CH2CH2)2), 1.96-2.00 (m, 2 H,
N(CH2CH2)2), 2.22 (td, J ) 12.7/4.4 Hz, 1 H, N(CH2CH2)2),
2.46-2.56 (m, 1 H, N(CH2CH2)2), 2.60 (td, J ) 11.7/2.4 Hz, 1
1. Gen er a l. Unless otherwise noted, moisture-sensitive
reactions were conducted under dry nitrogen. Tetrahydrofuran
(THF) was distilled from sodium/benzophenone ketyl prior to
use. Thin layer chromatography (TLC) was conducted with
silica gel 60 F254 plates (Merck). Flash chromatography (fc)41
was conducted with silica gel 60, 0.040-0.063 mm (Merck),
and the terms in parentheses for fc include the following:
diameter of the column [cm], eluent, fraction size [mL], Rf.
Melting points were determined with an SMP 2 (Stuart
Scientific) apparatus and were uncorrected. Elemental analy-
ses were conducted with an Vario EL (Elementaranalysesys-
teme GmbH) instrument. The mass spectrometer used was
model 5989A (Hewlett-Packard) and models MAT 312, MAT
8200, MAT 44, and TSQ 7000 (Finnigan). In the paragraphs
below, EI is defined as electron impact and CI is defined as
chemical ionization. IR data were collected from a 1605 FT-
IR (Perkin-Elmer) spectrophotometer. The 1H NMR (300 MHz)
and 13C NMR (75 MHz) data were collected from a Unity 300

444 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2
Maier and Wu¨nsch
H, N(CH2CH2)2), 2.77-2.85 (m, 2 H, N(CH2CH2)2, 2.89-2.95
(m, 2 H, ArCH2CH), 3.56 (s, 3 H, OCH3), 3.62 (s, 2 H, NCH2-
Ph), 4.87 (dd, J ) 6.5/4.4 Hz, 1 H, ArCH2CH, 7.09 (d, J ) 6.3
Hz, 1 H, 5-H), 7.14-7.43 (m, 8 H, arom). 13C NMR (base,
CDCl3): δ (ppm) 35.3 (1 C, ArCH2CH), 36.5 (1 C, N(CH2CH2)2),
39.0 (1 C, N(CH2CH2)2), 49.3 (2 C, N(CH2CH2)2), 56.1 (1 C,
OCH3), 63.4 (1 C, NCH2Ph), 74.9 (1 C, ArCO), 96.2 (1 C,
ArCH2CH), 124.8 (1 C, C-8), 126.4 (1 C, C-7), 126.6 (1 C, C-6),
126.9 (1 C, C-4′′), 128.1 (2 C, C-3′′ + C-5′′), 129.1 (1 C, C-5),
129.2 (2 C, C-2′′ + C-6′′), 131.5 (1 C, C-4a), 138.5 (1 C, C-1′′),
141.2 (1 C, C-8a).
acetonitrile (5 mL). This mixture was refluxed for 40 h. Then
it was filtered and concentrated in vacuo. The crude product
(72 mg) was purified by fc (2 cm, ethyl acetate, 10 mL, Rf )
0.18) to give a colorless oil, yield 26 mg (38%), which solidified
on standing, giving a colorless solid, mp 42-44 °C. HRMS for
C
24H31NO2: calcd 365.235 479, found 365.235 630 (+0.4
ppm). MS (EI), m/z: 365 [M+], 334 [M+ - OCH3], 246 [M+
-
CH2CH2CH2Ph]. IR (film), ν (cm-1): 2935, 2811 (C-H); 1076,
1044 (C-O); 753, 699 (C-H). 1H NMR (CDCl3): δ (ppm) 1.54-
1.75 (m, 4 H, NCH2CH2CH2CH2Ph), 1.85-2.03 (m, 3 H,
N(CH2CH2)2), 2.21 (td, J ) 13.2/4.4 Hz, 1 H, N(CH2CH2)2),
2.39-2.59 (m, 4 H, NCH2CH2CH2CH2Ph (2 H), N(CH2CH2)2
(2 H)), 2.68 (t, J ) 7.3 Hz, 2 H, NCH2CH2CH2CH2Ph), 2.78-
2.88 (m, 2 H, N(CH2CH2)2), 2.91-2.96 (m, 2 H, ArCH2CH), 3.58
(s, 3 H, OCH3), 4.88 (dd, J ) 6.3/3.9 Hz, 1 H, ArCH2CH), 7.10
(d, J ) 6.8 Hz, 1 H, arom), 7.15-7.34 (m, 8 H, arom).
3-Me t h oxy-1′-(2-p h e n yle t h yl)-3,4-d ih yd r osp ir o[[2]-
ben zop yr a n -1,4′-p ip er id in e] (14b). A solution of 13b (55
mg, 0.15 mmol) and p-toluenesulfonic acid monohydrate (34
mg, 0.18 mmol) in methanol (10 mL) was stirred at room
temperature for 20 h. Then NaOH (15 mg) was added and the
solvent was removed under reduced pressure. The residue was
purified by fc (2 cm, petroleum ether/ethyl acetate 1:1, 20 mL,
Rf ) 0.04) to give a colorless oil, yield 46 mg (92%). The base
14b was converted into the hydrochloride 14b‚HCl in the usual
manner to obtain colorless crystals, mp 263-264 °C (dec).
Anal. (C22H28NO2Cl) C, H, N. MS (EI), m/z: 246 [M+ - CH2-
Ph]. MS (CI, NH3), m/z: 338 [MH+], 246 [M+ - CH2Ph]. IR
(film), ν (cm-1): 2941, 2813 (C-H); 1076, 1043 (C-O); 756,
1′-C in n a m y l-3-m e t h o x y -3,4-d ih y d r o s p ir o [[2]b e n -
zop yr a n -1,4′-p ip er id in e] (14g). 3-Chloro-1-phenylprop-1-ene
(cinnamyl chloride, 27 mg, 0.18 mmol) and potassium carbon-
ate (190 mg, 1.4 mmol) were added to a solution of 14d (40
mg, 0.17 mmol) in acetonitrile (5 mL). This mixture was re-
fluxed for 9 h. Then it was filtered and concentrated in vacuo.
The crude product (36 mg) was purified by fc (2 cm, ethyl
acetate, 10 mL, Rf ) 0.19) to give a colorless oil, yield 14 mg
(24%). HRMS for C23H27NO2: calcd 349.204 179, found
1
699 (C-H). H NMR (CDCl3): δ (ppm) 1.91 (dd, J ) 13.7/2.2
349.204 127 (-0.1 ppm). MS (EI), m/z: 349 [M+], 334 [M+
-
Hz, 1 H, N(CH2CH2)2), 1.98-2.05 (m, 2 H, N(CH2CH2)2), 2.23
(td, J ) 12.8/4.3 Hz, 1 H, N(CH2CH2)2), 2.56 (td, J ) 11.0/4.9
Hz, 1 H, N(CH2CH2)2), 2.56-2.74 (m, 3 H, N(CH2CH2)2 (1 H),
NCH2CH2Ph (2 H)), 2.85-2.98 (m, 6 H, N(CH2CH2)2 (2 H),
NCH2CH2Ph (2 H), ArCH2CH (2 H)), 3.58 (s, 3 H, OCH3), 4.89
(dd, J ) 6.7/4.3 Hz, 1 H, ArCH2CH), 7.10 (d, J ) 6.4 Hz, 1 H,
arom), 7.15-7.34 (m, 8 H, arom).
CH3], 117 [CH2CHCHPh+]. IR (film), ν (cm-1): 2932, 2813
(C-H); 1671 (CdC (E)); 1071, 1044 (C-O); 750, 699 (C-H).
1H NMR (CDCl3): δ (ppm) 1.90 (d, J ) 13.7 Hz, 1 H,
N(CH2CH2)2), 1.98-2.06 (m, 2 H, N(CH2CH2)2), 2.25 (td, J )
12.7/4.4 Hz, 1 H, N(CH2CH2)2), 2.47-2.68 (m, 2 H, N(CH2-
CH2)2), 2.88-2.98 (m, 4 H, ArCH2CH, N(CH2CH2)2), 3.27 (d, J
) 6.8 Hz, 2 H, NCH2CHdCHPh), 3.57 (s, 3 H, OCH3), 4.88
(dd, J ) 6.5/4.2 Hz, 1 H, ArCH2CH), 6.36 (dt, J ) 15.6/6.8 Hz,
1 H, (E) NCH2CHdCHPh), 6.58 (d, J ) 16.1 Hz, 1 H, (E) NCH2-
CHdCHPh), 7.09 (d, J ) 6.8 Hz, 1 H, arom), 7.12-7.43 (m, 8
H, arom).
3-Meth oxy-3,4-d ih yd r osp ir o[[2]ben zop yr a n -1,4′-p ip e-
r id in e] (14d ). Under an N2 atmosphere, ammonium formate
(231 mg, 3.65 mmol) was added to a stirred mixture of 14a
(237 mg, 0.73 mmol) and 10% Pd/C (40 mg) in dry methanol
(30 mL). This mixture was refluxed for 2.5 h. Then it was
filtered through Celite and concentrated in vacuo. The crude
product was purified by fc (2 cm, methanol/ammonia 98:2, 12
mL, Rf ) 0.09) to yield 14d as colorless needles (petroleum
ether), mp 70-71 °C, yield 120 mg (70%). Anal. (C14H19NO2)
C, H, N. MS (EI), m/z: 233 [M+], 218 [M+ - CH3]. IR (KBr), ν
(cm-1): 3561 (N-H); 2941, 2870 (C-H); 1141, 1073 (C-O);
764 (C-H). 1H NMR (CDCl3): δ (ppm) 1.80-1.92 (m, 2 H,
N(CH2CH2)2), 1.99 (dd, J ) 14.2/2.4 Hz, 1 H, N(CH2CH2)2),
2.09 (td, J ) 13.2/4.6 Hz, 1 H, N(CH2CH2)2), 2.30 (br s, 1 H,
NH), 2.85-2.95 (m, 2 H, ArCH2CH), 3.01 (br d, J ) 12.7 Hz,
2 H, N(CH2CH2)2), 3.09-3.33 (m, 2 H, N(CH2CH2)2), 3.57 (s, 3
H, OCH3), 4.88 (dd, J ) 6.3/3.9 Hz, 1 H, ArCH2CH), 7.09 (d, J
) 6.3 Hz, 1 H, arom), 7.14-7.25 (m, 3 H, arom).
3-Met h oxy-1′-(3-p h en ylp r op yl)-3,4-d ih yd r osp ir o[[2]-
ben zop yr a n -1,4′-p ip er id in e] (14e). 1-Chloro-3-phenylpro-
pane (328 mg, 1.65 mmol) and potassium carbonate (1.5 g, 10.9
mmol) were added to a solution of 14d (320 mg, 1.37 mmol) in
THF (40 mL). This mixture was refluxed for 19 h. Then it was
filtered and concentrated in vacuo. The crude product was
purified by fc (3 cm, petroleum ether/ethyl acetate 1:1, 20 mL,
Rf ) 0.04) to yield a colorless oil (14e, 310 mg, 64%). The base
14e was converted into the hydrochloride in the usual manner
to obtain 14e‚HCl as colorless crystals, mp 196-198 °C. Anal.
(C23H30NO2Cl) C, H, N. MS (base, EI), m/z: 351 [M+], 246 [M+
- CH2CH2Ph]. IR (base, film), ν (cm-1): 2940, 2815 (C-H);
1128, 1076, 1044 (C-O); 754, 700 (C-H). 1H NMR (CDCl3): δ
(ppm) 1.84-2.03 (m, 5 H, CH2CH2CH2Ph (2 H), N(CH2CH2)2
(3 H)), 2.21 (td, J ) 13.1/4.2 Hz, 1 H, N(CH2CH2)2), 2.40-2.59
(m, 4 H, NCH2CH2CH2Ph (2 H), N(CH2CH2)2 (2 H)), 2.69 (t, J
) 7.6 Hz, 2 H, NCH2CH2CH2Ph), 2.80-2.89 (m, 2 H, N(CH2-
CH2)2), 2.91-2.95 (m, 2 H, ArCH2CH), 3.57 (s, 3 H, OCH3),
4.88 (dd, J ) 6.4/4.0 Hz, 1 H, ArCH2CH), 7.09 (d, J ) 6.4 Hz,
1 H, arom), 7.14-7.34 (m, 8 H, arom).
3-Meth oxy-1′-p h en yl-3,4-d ih yd r osp ir o[[2]ben zop yr a n -
1,4′-p ip er id in e] (14h ). Under an N2 atmosphere, Pd(OAc)2
(0.1 mg, 0.000 437 mmol, 0.25 mol %) and PtBu3 (0.35 mg,
0.001 75 mmol, 1 mol %) were added to a suspension of
bromobenzene (27.5 mg, 0.175 mmol), 14d (49.0 mg, 0.21
mmol), and NaOtBu (28.3 mg, 0.29 mmol) in dry o-xylene (3
mL). This mixture was heated at 130 °C for 4 h. After the
mixture cooled to room temperature, petroleum ether (3 mL)
was added and the mixture was washed with a saturated
solution of NaCl (6 mL). The organic layer was dried (MgSO4)
and concentrated in vacuo. The residue was purified by fc (1
cm, petroleum ether/ethyl acetate 95:5, 5 mL, Rf ) 0.10) to
give a colorless oil, yield 11.3 mg (21%), which solidified on
standing. HRMS for C20H23NO2: calcd 309.172 879, found
309.172 914 (+0.1 ppm). MS (EI), m/z: 309 [M+], 294 [M+
-
CH3], 77 [Ph+]. IR (film), ν (cm-1): 2940, 2823 (C-H); 1078,
1
1035 (C-O); 757, 694 (C-H). H NMR (CDCl3): δ (ppm) 1.98
(d, J ) 13.2 Hz, 1 H, N(CH2CH2)2), 2.10 (br d, J ) 4.9 Hz, 2 H,
N(CH2CH2)2), 2.31 (td, J ) 12.7/3.9 Hz, 1 H, N(CH2CH2)2),
2.89-3.03 (m, 2 H, ArCH2CH), 3.19-3.40 (m, 2 H, N(CH2-
CH2)2), 3.57 (s, 3 H, OCH3), 3.62 (br d, J ) 11.7 Hz, 2 H, N(CH2-
CH2)2), 4.93 (dd, J ) 6.3/3.9 Hz, 1 H, ArCH2CH), 6.88 (t, J )
7.8 Hz, 1 H, arom), 7.04 (d, J ) 8.3 Hz, 2 H, arom), 7.09-7.35
(m, 6 H, arom).
1′-Ben zyl-3,4-d ih yd r osp ir o[[2]ben zop yr a n -1,4′-p ip er i-
d in ]-3-ol (15a ). A solution of 13a (557 mg, 1.57 mmol) in THF
(6 mL), HCl (2 M, 8 mL), and water (10 mL) was stirred at
room temperature for 11 days. Then a solution of NaOH (2
M) was added to give pH 10 and the mixture was extracted
with CH2Cl2. The organic layer was dried (Na2SO4), and the
solvent was removed under reduced pressure. The crude
product was purified by fc (3 cm, petroleum ether/ethyl acetate
2:1, 20 mL, Rf ) 0.02) to yield a colorless solid (petroleum
ether/ethyl acetate), mp 163 °C, yield 363 mg (69%). Anal.
(C20H23NO2) C, H, N. MS (EI), m/z: 309 [M+], 232 [M+ - Ph],
218 [M+ - CH2Ph], 91 [CH2Ph+]. IR (KBr), ν (cm-1): 3422
(O-H); 3064, 2942, 2836 (C-H); 1134, 1060, 1020 (C-O); 754,
3-Me t h oxy-1′-(4-p h e n ylb u t yl)-3,4-d ih yd r osp ir o[[2]-
ben zop yr a n -1,4′-p ip er id in e] (14f). 1-Bromo-4-phenylbutane
(32 mg, 0.19 mmol) and potassium carbonate (209 mg, 1.5
mmol) were added to a solution of 14d (44 mg, 0.19 mmol) in

Spiropiperidines
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 445
1
694 (C-H). H NMR (CDCl3): δ (ppm) 1.86 (dd, J ) 13.4/2.4
1′-Ben zyl-3-ben zyloxy-3,4-dih ydr ospir o[[2]ben zopyr an -
1,4′-p ip er id in e] (17). One drop of concentrated H2SO4 was
added to a solution of 15a (40 mg, 0.129 mmol) in benzyl
alcohol (0.5 g), and the mixture was stirred at room temper-
ature for 6 h. Then solid sodium hydroxide (28 mg) was added.
This mixture was directly purified by fc (3 cm, petroleum ether/
ethyl acetate initially 9:1, then 2:1, 20 mL, Rf ) 0.09) to yield
a colorless oil, yield 38 mg (77%). HRMS for C27H29NO2: calcd
399.219 829, found 399.219 726 (-0.3 ppm). MS (EI), m/z: 399
[M+], 308 [M+ - CH2Ph], 91 [CH2Ph+]. MS (CI, NH3), m/z: 400
[MH+]. IR (film), ν (cm-1): 2935, 2812 (C-H); 1093, 1040 (C-
Hz, 1 H, N(CH2CH2)2), 1.92-2.02 (m, 2 H, N(CH2CH2)2), 2.25
(td, J ) 13.0/4.9 Hz, 1 H, N(CH2CH2)2), 2.49 (td, J ) 10.7/4.9
Hz, 1 H, N(CH2CH2)2), 2.58 (td, J ) 12.2/2.4 Hz, 1 H, N(CH2-
CH2)2), 2.71-2.84 (m, 2 H, N(CH2CH2)2), 2.88 (dd, J ) 15.6/
7.3 Hz, 1 H, ArCH2CH), 3.03 (dd, J ) 15.6/2.9 Hz, 1 H,
ArCH2CH), 3.59 (s, 2 H, NCH2Ph), 5.31 (dd, J ) 7.8/2.9 Hz, 1
H, ArCH2CH), 7.10 (d, J ) 6.8 Hz, 1 H, arom), 7.16-7.44 (m,
8 H, arom).
1′-(2-P h en yleth yl)-3,4-dih ydr ospir o[[2]ben zopyr an -1,4′-
p ip er id in ]-3-ol (15b). A solution of 13b (80 mg, 0.22 mmol)
in THF (1 mL), HCl (2 M, 2 mL), and water (2 mL) was stirred
for 2.5 days at room temperature. Then diluted NaOH (2 M)
was added (pH 10) and the mixture was extracted with
CH2Cl2. The organic layer was dried (Na2SO4), and the solvent
was removed under reduced pressure. The crude product was
purified by fc (2 cm, petroleum ether/ethyl acetate 1:1, 20 mL,
Rf ) 0.03) to yield a colorless solid (petroleum ether/ethyl
acetate), mp 127 °C, yield 45 mg (64%). Anal. (C21H25NO2) C,
H, N. MS (EI), m/z: 232 [M+ - CH2Ph]. MS (CI, isobutane),
m/z: 324 [MH+], 232 [M+ - CH2Ph]. IR (film), ν (cm-1): 3401
(O-H); 2929, 2827 (C-H); 1117, 1065 (C-O); 757, 700
1
O); 738, 698 (C-H). H NMR (CDCl3): δ (ppm) 1.85-2.01 (m,
3 H, N(CH2CH2)2), 2.21 (td, J ) 13.1/4.4 Hz, 1 H, N(CH2CH2)2),
2.48-2.56 (m, 1 H, N(CH2CH2)2), 2.62 (td, J ) 12.8/2.7 Hz, 1
H, N(CH2CH2)2), 2.77-2.86 (m, 2 H, N(CH2CH2)2), 2.93 (dd, J
) 16.2/3.7 Hz, 1 H, ArCH2CH), 3.01 (ddd, J ) 15.6/7.3/0.9 Hz,
1 H, ArCH2CH), 3.63 (s, 2 H, NCH2Ph), 4.68 (d, J ) 12.2 Hz,
1 H, OCH2Ph), 4.97 (d, J ) 12.2 Hz, 1 H, OCH2Ph), 5.02 (dd,
J ) 7.3/3.7 Hz, 1 H, ArCH2CH), 7.08 (d, J ) 6.4 Hz, 1 H, arom),
7.13-7.43 (m, 13 H, arom).
1′-Ben zylsp ir o[[2]b en zop yr a n -1,4′-p ip er id in e]-3(4H )-
on e (18). Under an N2 atmosphere, solid tetrapropylammo-
nium perruthenate ((NPr4)RuO4, 3.3 mg, 0.0093 mmol, 5 mol
%) was added in one portion to a stirred mixture of 15a (57
mg, 0.185 mmol), N-methylmorpholine N-oxide (NMMO, 43
mg, 0.37 mmol), and powdered 4 Å molecular sieves (93 mg)
in dry CH2Cl2 (3 mL). This mixture was stirred for 1 h at room
temperature. Then it was filtered. The filtrate was evaporated
under reduced pressure, and the residue was purified by fc (2
cm, petroleum ether/ethyl acetate 2:1, 20 mL, Rf ) 0.21) to
afford a colorless oil, yield 24 mg (43%), which solidified on
standing, giving a colorless solid (iPr2O), mp 138 °C. Anal.
(C20H21NO2) C, H, N. MS (EI), m/z: 307 [M+], 230 [M+ - Ph],
216 [M+ - CH2Ph], 91 [CH2Ph+]. IR (film), ν (cm-1): 2939,
2820 (C-H); 1740 (CdO); 1237, 1105 (C-O); 745, 700 (C-H).
1H NMR (CDCl3): δ (ppm) 1.95 (dd, J ) 14.2/2.6 Hz, 2 H,
N(CH2CH2)2), 2.22 (td, J ) 13.0/4.6 Hz, 2 H, N(CH2CH2)2), 2.61
(td, J ) 12.0/2.1 Hz, 2 H, N(CH2CH2)2), 2.82 (br d, J ) 11.0
Hz, 2 H, N(CH2CH2)2), 3.58 (s, 2 H, NCH2Ph), 3.75 (d, J ) 0.8
Hz, 2 H, ArCH2CO), 7.11-7.16 (m, 1 H, arom), 7.21-7.38 (m,
8 H, arom).
1
(C-H). H NMR (CDCl3): δ (ppm) 1.91 (br d, J ) 13.1 Hz, 1
H, N(CH2CH2)2), 2.00-2.07 (m, 2 H, N(CH2CH2)2), 2.28 (td, J
) 12.9/4.4 Hz, 1 H, N(CH2CH2)2), 2.54-2.73 (m, 4 H, N(CH2-
CH2)2 (2 H), NCH2CH2Ph (2 H)), 2.84-2.96 (m, 5 H, N(CH2-
CH2)2 (2 H), NCH2CH2Ph (2 H), ArCH2CH (1 H)), 3.04 (dd, J
) 15.6/3.1 Hz, 1 H, ArCH2CH), 5.32 (dd, J ) 7.6/3.1 Hz, 1 H,
ArCH2CH), 7.11 (d, J ) 6.7 Hz, 1 H, arom), 7.16-7.34 (m, 8
H, arom).
1′-(3-P h en ylp r op yl)-3,4-d ih yd r osp ir o[[2]ben zop yr a n -
1,4′-p ip er id in ]-3-ol (15e). A solution of 14e (212 mg, 0.60
mmol) in THF (10 mL) and HCl (2 M, 3 mL) was stirred for
24 days at room temperature and for 4 h at reflux temperature.
Then the solution was treated with diluted NaOH (2 M) to
give pH 10 and extracted with CH2Cl2. The organic layer was
dried (Na2SO4), and the solvent was removed in vacuo. The
crude product was purified by fc (2 cm, petroleum ether/ethyl
acetate 1:1, 10 mL, Rf ) 0.03) to yield a colorless solid
(cyclohexane/ethyl acetate), mp 114-115 °C, yield 75 mg (37%).
HRMS for C22H27NO2: calcd 337.204 179, found 337.204 304
(+0.4 ppm). MS (EI), m/z: 337 [M+], 232 [M+ - CH2CH2Ph].
IR (film), ν (cm-1): 3269 (O-H); 2932 (C-H); 1064, 1017 (C-
1′-Ben zylsp ir o[[2]ben zop yr a n -1,4′-p ip er id in e] Hyd r o-
br om id e (19‚HBr ). A solution of 14a (37 mg, 0.11 mmol) and
2-bromo-2-methylpropane (78 mg, 0.55 mmol) in CHCl3 (10
mL) was refluxed for 24 h. Then it was concentrated in vacuo
and the residue was recrystallized from ethyl acetate to afford
colorless crystals, mp 251-252 °C, yield 10 mg (24%). Anal.
(C20H22NOBr) C, H, N. MS (base, EI), m/z: 291 [M+], 214 [M+
- Ph], 200 [M+ - CH2Ph], 91 [CH2Ph+]. IR (base, film), ν
(cm-1): 2941, 2813 (C-H); 1630 (CdC); 1054 (C-O); 743, 698
1
O); 756, 701 (C-H). H NMR (CDCl3): δ (ppm) 1.84-2.00 (m,
5 H, CH2CH2CH2Ph (2 H), N(CH2CH2)2 (3 H)), 2.23 (td, J )
13.1/4.5 Hz, 1 H, N(CH2CH2)2), 2.43-2.59 (m, 4 H, NCH2CH2-
CH2Ph (2 H), N(CH2CH2)2 (2 H)), 2.67, (t, J ) 7.9 Hz, 2 H,
NCH2CH2CH2Ph), 2.77-2.91 (m, 3 H, N(CH2CH2)2 (2 H),
ArCH2CH (1 H)), 3.02 (dd, J ) 15.9/3.1 Hz, 1 H, ArCH2CH),
5.30 (dd, J ) 7.6/3.1 Hz, 1 H, ArCH2CH), 7.10 (d, J ) 6.4 Hz,
1 H, arom), 7.16-7.33 (m, 8 H, arom).
1
(C-H). H NMR (base, CDCl3): δ (ppm) 1.99 (td, J ) 13.4/4.4
Hz, 2 H, N(CH2CH2)2), 2.25 (dt, J ) 14.1/2.7 Hz, 2 H,
N(CH2CH2)2), 2.49 (td, J ) 12.0/2.4 Hz, 2 H, N(CH2CH2)2), 2.79
(br d, J ) 11.0 Hz, 2 H, N(CH2CH2)2), 3.60 (s, 2 H, NCH2Ph),
5.75 (d, J ) 5.7 Hz, 1 H, ArCHdCH), 6.50 (d, J ) 5.7 Hz, 1 H,
ArCHdCH), 6.93-6.96 (m, 1 H, arom), 7.08-7.42 (m, 8 H,
arom).
1′-Ben zyl-3-et h oxy-3,4-d ih yd r osp ir o[[2]b en zop yr a n -
1,4′-p ip er id in e] (16). A solution of 15a (50 mg, 0.16 mmol)
and one drop of concentrated H2SO4 in ethanol (2.3 g) was
stirred at room temperature for 3 days. Then solid sodium
hydroxide (66 mg) was added. The solvent was removed under
reduced pressure, and the residue was purified by fc (1 cm,
petroleum ether/ethyl acetate 1:1, 5 mL, Rf ) 0.10) to yield a
colorless oil, yield 44 mg (81%). The base 16 was converted
into the hydrochloride 16‚HCl in the usual manner to obtain
colorless crystals, mp 164 °C. Anal. (C22H28NO2Cl) H, N, C:
calcd, 70.67; found, 70.13. HRMS (base): calcd 337.204 179,
found 337.204 304 (+0.4 ppm). MS (base, EI), m/z: 337 [M+],
246 [M+ - CH2Ph], 91 [CH2Ph+]. IR (base, film), ν (cm-1):
2934, 2811 (C-H); 1125, 1041 (C-O); 753, 699 (C-H). 1H NMR
(CDCl3): δ (ppm) 1.30 (t, J ) 7.0 Hz, 3 H, OCH2CH3), 1.89
(dd, J ) 13.4/1.6 Hz, 1 H, N(CH2CH2)2), 1.97-2.02 (m, 2 H,
N(CH2CH2)2), 2.21 (td, J ) 12.8/4.4 Hz, 1 H, N(CH2CH2)2), 2.49
(td, J ) 10.6/5.0 Hz, 1 H, N(CH2CH2)2), 2.60 (td, J ) 11.7/2.6
Hz, 1 H, N(CH2CH2)2), 2.78-2.82 (m, 2 H, N(CH2CH2)2), 2.93
(d, J ) 5.5 Hz, 2 H, ArCH2CH), 3.52-3.63 (m, 1 H, OCH2-
CH3), 3.63 (s, 2 H, NCH2Ph), 3.98-4.10 (m, 1 H, OCH2CH3),
4.96 (t, J ) 5.5 Hz, 1 H, ArCH2CH), 7.10 (d, J ) 6.6 Hz, 1 H,
arom), 7.13-7.42 (m, 8 H, arom).
1′-Ben zyl-3,4-d ih yd r osp ir o[[2]ben zop yr a n -1,4′-p ip er i-
d in e] (20). A mixture of base 19 (27 mg, 0.092 mmol), 10%
Pd/C (19 mg), and acetic acid (4 mL) was stirred under a
hydrogen atmosphere (0.8 bar) for 1.25 h. Then it was filtered
through Celite, and diluted NaOH (2 M, 25 mL) was added.
This solution was extracted with CH2Cl2, and the organic layer
was dried (Na2SO4) and evaporated under reduced pressure.
The residue was purified by fc (1 cm, petroleum ether/ethyl
acetate 3:1, 5 mL Rf ) 0.06) to give a colorless oil, yield 11 mg
(39%), which solidified on standing, mp 50 °C. HRMS for
C20H23NO: calcd 293.177 964, found 293.177 874 (-0.3 ppm).
MS (EI), m/z: 293 [M+], 216 [M+ - Ph], 202 [M+ - CH2Ph],
91 [CH2Ph+]. IR (film), ν (cm-1): 2929, 2813 (C-H); 1092
(C-O); 737, 697 (C-H). 1H NMR (CDCl3): δ (ppm) 1.88 (dd, J
) 14.3/2.4 Hz, 2 H, N(CH2CH2)2), 2.05 (td, J ) 13.0/4.5 Hz, 2
H, N(CH2CH2)2), 2.43 (td, J ) 11.9/2.7 Hz, 2 H, N(CH2CH2)2),
2.73 (br d, J ) 10.7 Hz, 2 H, N(CH2CH2)2), 2.82 (t, J ) 5.6 Hz,

446 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2
Maier and Wu¨nsch
2 H, ArCH2CH2), 3.58 (s, 2 H, NCH2Ph), 3.90 (t, J ) 5.6 Hz, 2
H, ArCH2CH2), 7.05-7.41 (m, 9 H, arom).
7.51 (td, J ) 7.6/0.9 Hz, 1 H, 5-H), 7.66 (td, J ) 7.3/1.2 Hz, 1
H, 6-H), 7.87 (dt, J ) 7.3/1.2 Hz, 1 H, 4-H).
1′-Ben zyl-3-m et h oxy-3H -sp ir o[[2]b en zofu r a n -1,4′-p i-
p er id in e] (23). Under an N2 atmosphere, a 1.6 M solution of
n-butyllithium in hexane (8.13 mL, 10 mmol) was slowly added
to a cooled (-95 °C) solution of 21 (3.0 g, 13 mmol) in THF
(30 mL). The mixture was stirred at -95 °C for 5 min, and
then a solution of 12a (1.89 g, 10 mmol) in THF (10 mL) was
slowly added. The solution was stirred at -95 °C for 2 h. Then
it was allowed to warm to room temperature and was stirred
for another 4 h. Then water (50 mL) was added, the mixture
was extracted with CH2Cl2, the organic layer was dried
(Na2SO4), and the solvent was removed in vacuo to give 3.9 g
of crude product 22. The crude product was dissolved in
methanol (20 mL), and p-toluenesulfonic acid monohydrate (2.8
g, 14.7 mmol) was added. This mixture was stirred for 7 days
at room temperature. Then the solution was treated with
diluted NaOH (2 M) to give pH 10 and was extracted with
CH2Cl2. The crude product was purified by fc (8 cm, petroleum
ether/ethyl acetate 3:1, 50 mL, Rf ) 0.20) to afford a colorless
oil (23), yield 1.1 g (36%). 23 was converted into the hydro-
chloride in the usual manner to obtain 23‚HCl as colorless
crystals, mp 171-173 °C. Anal. (C20H24NO2Cl) C, H, N. MS
(EI), m/z: 309 [M+], 294 [M+ - CH3], 278 [M+ - OCH3], 218
[M+ - CH2Ph], 91 [CH2Ph+]. IR (base, film), ν (cm-1): 2944,
2813 (C-H); 1085, 1003 (C-O); 747, 699 (C-H). 1H NMR
(base, CDCl3): δ (ppm) 1.65 (ddd, J ) 13.4/5.5/2.4 Hz, 1 H,
N(CH2CH2)2), 1.82 (ddd, J ) 13.4/5.5/2.8 Hz, 1 H, N(CH2CH2)2),
2.05 (td, J ) 12.8/4.0 Hz, 1 H, N(CH2CH2)2), 2.13 (td, J ) 13.0/
4.4 Hz, 1 H, N(CH2CH2)2), 2.52 (br t, J ) 11.8 Hz, 2 H, N(CH2-
CH2)2), 2.81-2.95 (m, 2 H, N(CH2CH2)2), 3.48 (s, 3 H, OCH3),
3.62 (s, 2 H, NCH2Ph), 6.09 (s, 1 H, ArCH), 7.18-7.22 (m, 1
H, arom), 7.24-7.43 (m, 8 H, arom).
1′-Ben zyl-3H -sp ir o[[2]b en zofu r a n -1,4′-p ip er id in ]-3-ol
(24). As described for 23 the bromobenzaldehyde acetal 21 (578
mg, 2.5 mmol) was reacted with n-butyllithum (1.6 M, 1.72
mL, 2.75 mmol) and 12a (402 mg, 2.1 mmol) to yield the
addition product 22 (510 mg). The crude product 22 was
dissolved in THF (2 mL), HCl (2 M, 10 mL), and water (10
mL). This solution was refluxed for 3.5 h. Then the solution
was treated with diluted NaOH (2 M) to give pH 10 and was
extracted with CH2Cl2. The crude product was purified by fc
(2 cm, petroleum ether/ethyl acetate 3:2, 10 mL, Rf ) 0.08) to
afford 24 as a colorless solid (petroleum ether/ethyl acetate),
mp 154 °C, yield 68 mg (11%). Anal. (C19H21NO2) C, H, N. MS
(EI), m/z: 295 [M+], 204 [M+ - CH2Ph], 91 [CH2Ph+]. IR (KBr),
ν (cm-1): 3420 (O-H); 2919, 2829 (C-H); 1038, 997 (C-O);
753, 697 (C-H). 1H NMR (CDCl3): δ (ppm) 1.63 (ddd, J ) 13.7/
5.3/2.9 Hz, 1 H, N(CH2CH2)2), 1.80 (ddd, J ) 13.7/5.4/2.4 Hz,
1 H, N(CH2CH2)2), 2.02-2.21 (m, 2 H, N(CH2CH2)2), 2.45-
2.60 (m, 2 H, N(CH2CH2)2), 2.83-2.93 (m, 2 H, N(CH2CH2)2),
3.62 (s, 2 H, NCH2Ph), 6.45 (s, 1 H, ArCH), 7.22 (dd, J ) 6.3/
2.0 Hz, 1 H, arom), 7.24-7.45 (m, 8 H, arom).
2. Recep tor Bin d in g Stu d ies. Gen er a l. Teflon-glass-
homogenizer was Potter S (B. Braun Biotech International).
The rotor/stator homogenizer was a Ultraturrax T25 basic (Ika
Labortechnik). The centrifuge was a high-speed refrigerating
centrifuge model J 2-HS (Beckman). Whatman glass fiber
filters GF/B were presoaked in 0.5% polyethylenimine in water
for 2 h at 4 °C before use. Filtration was performed with a
Brandel 24-well cell harvester. The scintillation cocktail was
Rotiszint eco plus (Roth). The liquid scintillation analyzer was
a Tri-Carb 2100 TR (Canberra Packard) with a counting
efficiency of 66%. All experiments were carried out in triplicate.
IC50 values were determined from competition experiments
with at least six concentrations of test compounds and were
calculated with the curve-fitting program GraphPad Prism 3.0
(GraphPad Software) by nonlinear regression analysis. Ki
values were calculated according to Cheng and Prusoff.42 Kd
values for the radioligands were taken from the literature. For
compounds with high affinity (low Ki values) mean values (
SEM from at least three independent experiments are given.
3. In vestiga tion of th e σ1-Recep tor Affin ity.37 [3H]-(+)-
Pentazocine binding to guinea pig brain membrane prepara-
tions was performed according to standard radioligand binding
assays,37 which were slightly modified as described below.
Mem b r a n e P r ep a r a t ion . Thawed guinea pig brains
(Dunkin Hartley, Harlan-Sera-Lab) were homogenized with
an ultraturrax (8000 rpm) in 10 volumes of cold 0.32 M
sucrose. The homogenate was centrifuged at 1000g for 10 min
at 4 °C. The supernatant was separated and centrifuged at
22 000g for 20 min at 4 °C. The pellet was resuspended in 10
volumes of buffer (50 mM Tris-HCl, pH 7.4) with an ultratur-
rax (8000 rpm), incubated for 30 min at 25 °C, and centrifuged
at 22 000g (20 min, 4 °C). The pellet was resuspended in buffer,
the protein concentration was determined according to the
method of Bradford43 using bovine serum albumin as standard,
and subsequently the preparation was frozen (-83 °C) in 5
mL portions of about 2 mg of protein/mL.
σ1-Recep tor Bin d in g Assa y. The test was performed with
the radioligand [ring-1,3-3H]-(+)-pentazocine (1036 GBq/mmol;
NEN Life Science Products). The thawed membrane prepara-
tion (about 150 µg of the protein) was incubated with various
concentrations of test compounds, 3 nM [3H]-(+)-pentazocine,
and buffer (50 mM Tris-HCl, pH 7.4) in a total volume of 500
µL for 150 min at 37 °C. The incubation was terminated by
rapid filtration through presoaked Whatman GF/B filters using
a cell harvester. After the sample was washed four times with
2 mL of cold buffer, a total of 3 mL of scintillation cocktail
was added to the filters. After at least 8 h, bound radioactivity
trapped on the filters was counted in a liquid scintillation
analyzer. Nonspecific binding was determined with 10 µM
haloperidol.
1′-Ben zylspir o[[2]ben zofu r an -1,4′-piper idin ]-3-on e (25).
Under an N2 atmosphere, solid tetrapropylammonium perru-
thenate ((NPr4)RuO4, 3.5 mg, 0.0098 mmol, 5 mol %) was
added in one portion to a stirred mixture of 24 (58 mg, 0.196
mmol), N-methylmorpholine N-oxide (NMMO, 46 mg, 0.39
mmol), and powdered 4 Å molecular sieves (100 mg) in dry
CH2Cl2 (3 mL). This mixture was stirred for 1.5 h at room
temperature. Then it was filtered. The filtrate was evaporated
under reduced pressure, and the residue was purified by fc (1
cm, petroleum ether/ethyl acetate 2:1, 5 mL, Rf ) 0.06) to
afford a colorless oil, yield 33 mg (58%), which solidified on
standing. Compound 25 was converted into the hydrochloride
in the usual manner to obtain 25‚HCl as colorless crystals,
mp 274-275 °C (ref 36, 280-283 °C). MS (EI) for C19H19NO2,
m/z: 293 [M+], 202 [M+ - CH2Ph], 91 [CH2Ph+]. IR (film), ν
(cm-1): 2944, 2813 (C-H); 1762 (CdO); 1072 (C-O); 741, 695
(C-H). 1H NMR (base, CDCl3): δ (ppm) 1.71 (dd, J ) 14.3/2.5
Hz, 2 H, N(CH2CH2)2), 2.23 (td, J ) 13.4/4.7 Hz, 2 H,
N(CH2CH2)2), 2.56 (td, J ) 12.2/2.7 Hz, 2 H, N(CH2CH2)2), 2.92
(br d, J ) 11.0 Hz, 2 H, N(CH2CH2)2), 3.62 (s, 2 H, NCH2Ph),
7.23-7.39 (m, 5 H, arom), 7.42 (dt, J ) 7.6/0.9 Hz, 1 H, 7-H),
4. In vestiga tion of th e σ2-Recep tor Affin ity.38,39 σ2-
Receptor affinity was determined using rat liver membranes
with [3H]-ditolylguanidine in the presence of 100 nM (+)-
pentazocine to mask σ1-binding sites. The assay was performed
according to the standard procedure,38,39 which was slightly
modified as described below.
Mem br a n e P r ep a r a tion . One frozen rat liver (Sprague
Dawley, Harlan-Sera-Lab) was allowed to thaw slowly on ice.
Then it was homogenized with a potter (800 rpm) in 10
volumes of cold buffer (10 mM Tris-HCl/0.32 M sucrose, pH
7.4). The homogenate was centrifuged at 1000g for 10 min at
4 °C. The supernatant was separated and saved on ice. The
pellet was resuspended in 30 mL of cold buffer and centrifuged
again. Both supernatants were then centrifuged at 31 000g
for 20 min at 4 °C. The pellet was resuspended in 30 mL of
buffer (10 mM Tris-HCl, pH 7.4) by vortexing and gentle potter
homogenization. Then it was incubated for 15 min at 25 °C
and centrifuged at 31 000g (20 min, 4 °C). The pellet was
resuspended in buffer, the protein concentration was deter-
mined according to the method of Bradford43 using bovine

Spiropiperidines
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 447
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Williams, W.; Sine, B.; Bowen, W. D.; Carroll, F. I. Synthesis
and σ Binding Properties of 2′-Substituted 5,9R-Dimethyl-6,7-
benzomorphans. J . Med. Chem. 1995, 38, 2978-2985.
(17) Wyrick, S. D.; Booth, R. G. Progress in sigma receptor research.
Drugs Future 1995, 20, 1033-1044.
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S. D. Synthesis, Evaluation, and Comparative Molecular Field
Analysis of 1-Phenyl-3-amino-1,2,3,4-tetrahydronaphthalenes as
Ligands for Histamine H1 Receptors. J . Med. Chem. 1999, 42,
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T.; Plumley, L. H.; Yang-Feng, T. L.; Leibach, F. H.; Ganapathy,
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σ
serum albumin as standard, and subsequently the preparation
was frozen (-83 °C) in 5 mL portions of about 2.5 mg of
protein/mL.
σ2-Recep tor Bin d in g Assa y. The membrane preparation
(about 60 µg of protein) was incubated with 3 nM [3H]-
ditolylguanidine (di-[p-ring-3H]-1,3-di-o-tolylguanidine, 2220
GBq/mmol; American Radiolabeled Chemicals, Inc.) and dif-
ferent concentrations of test compounds in buffer (50 mM Tris-
HCl, pH 8.0) in the presence of 100 nM (+)-pentazocine. The
total volume was 250 µL. The incubation (120 min, 25 °C)
was stopped by addition of 2 mL of ice-cold buffer (10 mM
Tris-HCl, pH 8.0) followed by rapid filtration through pre-
soaked Whatman GF/B filters using a cell harvester. After the
sample was washed three times with 2 mL of cold buffer, a
total of 3 mL of scintillation cocktail was added to the filters.
After at least 8 h, bound radioactivity trapped on the filters
was counted in a liquid scintillation analyzer. Nonspecific
binding was determined with 10 µM nonradiolabeled di-
tolylguanidine.
Ack n ow led gm en t . We thank the Deutsche
Forschungsgemeinschaft, the Fonds der Chemischen
Industrie,and the Wissenschaftliche Gesellschaft Freiburg
for financial support. Thanks are also due to Degussa
AG, J anssen Cilag GmbH, and Bristol-Myers Squibb for
donation of chemicals and reference compounds. Per-
formance of the receptor screening (Table 4) by Dr. C.
A. Seyfried, Department of CNS Research, Merck
KGaA, Darmstadt, is gratefully acknowledged.
(23) Monograph BMY-14802. Drugs Future 1995, 20, 821-822.
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Middlemiss, D. N.; Wong, E. H. F. Spiropiperidines as High-
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