Combination of two pharmacophoric systems: Synthesis and pharmacological evaluation of spirocyclic pyranopyrazoles with high σ1 receptor affinity

Article abstract of DOI:10.1021/jm200585k

The novel class of spirocyclic σ₁ ligands 3 (6′,7′-dihydro-1′H-spiro[piperidine-4,4′-pyrano[4,3-c] pyrazoles]) was designed by the combination of the potent σ₁ ligands 1 and 2 in one molecule. Thorough structure affinity relationship

Full text of DOI:10.1021/jm200585k

ARTICLE
pubs.acs.org/jmc
Combination of Two Pharmacophoric Systems: Synthesis and
Pharmacological Evaluation of Spirocyclic Pyranopyrazoles with High
σ1 Receptor Affinity
Torsten Schl€ager,† Dirk Schepmann,† Kirstin Lehmkuhl,† J€org Holenz,‡ Jose Miguel Vela,‡
Helmut Buschmann,‡ and Bernhard W€unsch*,†
†Institut f€ur Pharmazeutische und Medizinische Chemie der Universit€at M€unster, Hittorfstrasse 58-62, D-48149 M€unster, Germany
‡Esteve, Av. Mare de Deu de Montserrat 221, 08041 Barcelona, Spain
S Supporting Information
b
ABSTRACT: The novel class of spirocyclic σ1 ligands 3 (60,70-
dihydro-10H-spiro[piperidine-4,40-pyrano[4,3-c]pyrazoles])
was designed by the combination of the potent σ1 ligands 1 and
2 in one molecule. Thorough structure affinity relationships
were derived by the variation of the substituents in position 10,
1, and 60. Whereas the small electron rich methylpyrazole
heterocycle was less tolerated by the σ1 receptor protein, the introduction of a phenyl substituent instead of the methyl group
led to ligands with a high σ1 affinity. It is postulated that the additional phenyl substituent occupies a previously unrecognized
hydrophobic region of the σ1 receptor resulting in additional lipophilic interactions. The spirocyclic pyranopyrazoles are very
selective against the σ2 subtype, the PCP binding site of the NMDA receptor, and further targets. Despite high σ1 affinity, the
cyclohexylmethyl derivative 17i (Ki (σ1) = 0.55 nM) and the isopentenyl derivative 17p (Ki (σ1) = 1.6 nM) showed only low
antiallodynic activity in the capsaicin assay.
’ INTRODUCTION
the σ1 receptor pharmacology, in particular to find clear correla-
tions between ligand binding properties, signal transduction
pathways, and pharmaceutical properties.
Originally the class of σ receptors was identified as an opioid
receptor subtype due to the particular effects caused by benzo-
morphans (e.g., SKF-10,047, pentazocine).1 Then, σ receptors
were discussed to be identical with the phencyclidine (PCP)
binding site of the N-methyl-D-aspartate (NMDA) receptor,2
until they were recognized as specific, nonopioid, non-PCP but
haloperidol-sensitive binding structures. Two subtypes of σ
receptors have been identified, which are termed σ1 and σ2
receptors.3
The σ1 receptor was cloned from different species (guinea pig,
mouse, rat, and human) and different tissues (liver, human
placental choriocarcinoma cell line, and brain),4ꢀ8 Whereas the
gene (≈7 kbp) and the amino acid sequence (223 amino acids)
of the σ1 receptor is well-known, the σ2 receptor has not been
cloned yet.
The influence of σ1 receptors on several signal transduction
pathways has been investigated. It was shown that the σ1 receptor
modulates some ion channels like Kv 1.4 K+-channels in nerve
terminals,9,10 Ca2+-channels in cultured cardiac myocytes,11 and
voltage-gated Na+-channels in cardiac myocytes.12 Additionally,
some neurotransmitter systems like NMDA receptors,13 in-
ositoltriphosphate (IP3) receptors in the endoplasmatic
reticulum,14 and ankyrin, a cytoskeletal adaptor protein, which
regulates Ca2+-influx at IP3 receptors, are modulated.15 A cha-
perone activity of σ1 receptors was also postulated.16 Never-
theless, further investigations are necessary to learn more about
σ1 receptors are expressed in high density in the central
nervous system (CNS), in particular in brain regions involved
in memory, emotion, sensoric, and motor functions. Addition-
ally, they are found in some peripheral organs (e.g., liver, kidney,
heart, lung, intestine, and pancreas)5,17,18 and, moreover, in some
human tumor cell lines.19,20 Because of their involvement in
different neurological processes, σ1 receptors represent an
attractive target for the development of novel drugs for CNS
diseases, including depression, schizophrenia, anxiety, cocaine,
alcohol and methamphetamine addiction, amnesia, and neuro-
pathic pain as well as some neurodegenerative disorders (e.g.,
Alzheimer’s Disease and Parkinson’s Disease).21ꢀ24
Neuropathic pain is a special kind of pain, which is character-
ized by a spontaneous hypersensitive pain response and which
can typically persist long after the original nerve injury has
healed.25,26 The treatment of neuropathic pain is very difficult
due to its diffuse origin. It has been shown with σ1 receptor
knockout mice that σ1 receptor antagonists can be used for the
therapy of neuropathic pain situations.27 Very recently, it has
been reported that indazole derivatives of type 1 (Figure 1) are
potent and selective σ1 receptor antagonists, which are
Received: May 10, 2011
Published: August 22, 2011
r
2011 American Chemical Society
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analgesically active in the capsaicin model of neuropathic pain.
In order to combine the excellent σ1 affinity and σ1/σ2
selectivity of the spirocyclic compounds 2 with the high analgesic
activity of the indazole derivatives 1, the structures of 1 and 2
should be combined in the new spirocyclic pyranopyrazole
derivatives 3. (Figure 1) The incorporation of the flexible
aminoethyl side chain of 1 into the rather rigid piperidine moiety
of 3 should result in an increased σ1/σ2 selectivity of the mixed
spirocyclic ligands. It has already been shown that the re-
placement of the benzene ring of spirocyclic compounds 2 by
an electron rich thiophene ring leads to very potent and selective
σ1 ligands.36,37 Herein, we report on the synthesis and pharma-
cological evaluation of spirocyclic pyranopyrazole derivatives 3
bearing various substituents R1, R2, and R3.
2
Compound 1a (R1 = CH3, NR2 = 4-phenylpiperidin-1-yl)
represents a typical example of a highly potent σ1 ligand (Ki
(σ1) = 7.0 nM, Ki (σ2) = 39.7 nM, see Table 1) with analgesic
activity. At a dose of 0.5 mg/kg body weight, 1a led to more than
50% analgesia in the late phase of the capsaicin assay
(neuropathic pain model).28ꢀ30
We have shown that spirocyclic piperidines of type 2 and
analogues interact with high affinity and selectivity with σ1
receptors.31ꢀ35 In particular, the benzofuran derivative 2a
(n = 0) and the benzopyran derivative 2b (n = 1) represent σ1
receptor antagonists with low nanomolar σ1 affinity and
extraordinarily high σ1/σ2 selectivity31 (see Table 1). More-
over, the benzofuran derivative 2a was also active in the mouse
capsaicin assay indicating its potential as a drug for the
treatment of neuropathic pain.35 The high σ1/σ2 selectivity
of the compounds 2 is attributed to the reduced conforma-
tional flexibility of the rigid spirocyclic ring system.
’ SYNTHESIS
The synthesis of the spirocyclic pyranopyrazoles 3 started with
1-phenylpyrazole (4). As described in the literature, pyrazole 4
was α-metalated with n-butyllithium at ꢀ78 °C to generate
regioselectively the pyrazol-5-yllithium intermediate, which was
trapped with N,N-dimethylformamide to afford the pyrazole-5-
carbaldehyde 637,38 (Scheme 1). In order to avoid the formation
of the thermodynamically more stable phenyllithium intermediate
by intramolecular deprotonation, the complete transformation
had to be performed at low temperature (< ꢀ65 °C) in the solvent
tetrahydrofuran (THF).39,40
The methylated pyrazole-5-carbaldehyde 7 was prepared
in the same way by metalation of 1-methylpyrazole (5) with
n-butyllithium and subsequent trapping of the anion with N,N
dimethylformamide.41 However, in this case, the methyl group of
the pyrazole moiety was deprotonated first. In order to obtain
high yields of the ring substitution product 7, the intermediate
anion was stirred for 30 min at 0 °C to induce a transmetalation
from the methylllithium intermediate to the thermodynamically
more stable pyrazol-5-yllithium derivative.42,43
The aldehyde 6 was homologated by a Wittig reaction using
the phosphonium salt Ph3PCH2OCH3+ Clꢀ, which was depro-
tonated by KOtBu,44 to obtain the enol ether 8 in 92% yield.
Figure 1. Development of novel σ1 receptor ligands with spirocyclic
pyranopyrazole substructure.
Scheme 1a
a Reagents and reaction conditions: (a) 4: n-BuLi, THF, ꢀ78 °C, 2 h then DMF, THF, ꢀ78 °C, 1 h, then rt, 18 h, 90%. (b) 5: n-BuLi, THF, ꢀ60 °C, then
30 min at 0 °C, then DMF, THF, ꢀ60 °C, 1 h, then 0 °C, 2 h. (c) Ph3PCH2OCH3+ Clꢀ, KOtBu, THF, ꢀ50 °C, then rt, overnight, 92% (8). (d) MeOH,
TosOH H2O, rt, 72 h, 90% (10). (e) Pyridinium bromide perbromide, MeOH, HC(OMe)3, 0 °C, 1 h, then rt, 4 h, 95% (12), 29% over 4 steps (13). (f)
3
n-BuLi, THF, ꢀ78 °C, 15 min then 14, THF, ꢀ78 °C, 4.5 h, then rt, 67% (15), 67% (16). (g) TosOH H2O, MeOH, rt, 73% (17a), 41% (18a) together
3
with 30% (22b).
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Scheme 2a
Scheme 3a
a Reagents and reaction conditions: (a) NH4 HCO2ꢀ, Pd/C, MeOH,
reflux, 10ꢀ25 min, 88% (17b), 96% (18b). (b) RꢀX, acetonitrile, K2CO3,
(Bu4N+ Iꢀ), reflux. (c) RCHdO, NaBH(OAc)3, 1,2-dichloroethane, rt.
+
the tertiary alcohol 20, elimination of water, and subsequent
trapping of the tertiary carbenium ion by methanol (f 22a) or
deprotonation (f 23). This result is in sharp contrast to the
formation of the corresponding benzopyran derivatives, which
does not lead to substitution or elimination side products.31
We assume that the high electron density of the electron rich
pyrazole moiety is responsible for the good stabilization of
the tertiary carbenium ion, initiating the formation of the side
products 22a and 23.
Next, the cyclization of the hydroxy acetal 15 was performed
using 2.5 equivalents of p-toluenesulfonic acid (Scheme 1). The
excess of acid should inhibit the formation of the tertiary carbe-
nium ion by protonation of both the pyrazole system (reducing
the electron donating properties) and the piperidine N-atom
(destabilization of the second cation in position 3 to the first
cation). Indeed, cyclization of 15 in methanol and THF led to
the spirocyclic pyranopyrazole 17a in 73% and 60% yields,
respectively.
Applying these optimized reaction conditions on the cycliza-
tion of the hydroxy acetal 16 using 2.5 equivalents of p-
toluenesulfonic acid provided the spirocyclic pyranopyrazole
18a in only 30% yield together with large amounts of the
corresponding methyl ether 22b (R1 = CH3, R2 = Bn, 56%).
Increasing the amount of p-toluenesulfonic acid to 10 equivalents
resulted in increased yields of 18a (41%) and reduced yields of
the methyl ether 22b (30%).
For the generation of broad structure affinity relationships, the
N-benzyl residue of the pyranopyrazoles 17a and 18a was cleaved
off byhydrogenolysis(Scheme 3). A phasetransferhydrogenolysis
using ammonium formate as an H2 source and Pd/C as a catalyst45
provided the secondary amines 17b and 18b in 88% and 96%
yields, respectively. Various residues were introduced at the
secondary amines 17b and 18b by alkylation with alkyl halides
or reductive alkylation with aldehydes and NaBH(OAc)3.46,47
In order to modify the substitution pattern of the pyran moiety,
the hydroxy acetals 15 and 16 were hydrolyzed with diluted HCl
to form the cyclic hemiacetals 24 and 25 (Scheme 4). Elimination
of water was achieved upon treatment of the lactols 24 and 25
with methanesulfonyl chloride and an excess of triethylamine.
Careful hydrogenation (H2, 1 bar, rt, 1 h) of the cyclic enol ethers
26 and 27 resulted in the spirocyclic pyranopyrazoles 28a and
29a. Spirocyclic pyranopyrazoles 28cꢀf and 29cꢀe were pre-
pared by hydrogenolytic removal of the N-benzyl protecting
group and subsequent alkylation of the secondary amines 28b
and 29b, respectively.
a Reagents and reaction conditions: (a) n-BuLi, THF, ꢀ78 °C, 15 min, then
19, ꢀ78 °C, 4.5 h, then rt, 85%. (b) TosOH H2O, MeOH, rt, 21 h, 9%
3
(21), 81% (22a). (c) TosOH H2O, THF, rt, 70 h, 28% (21), 39% (23).
3
Addition of methanol to the enol ether 8 provided the acetalde-
hyde dimethyl acetal 10, which was brominated with pyridinium
bromide perbromide (PyH+ Br3ꢀ). Because of the high electron
density at the pyrazole 4-position, the bromination took place
with high regioselectivity. The bromo acetal 12 was obtained in
95% yield.
In order to prove the position of the acetalic side chain
unequivocally, a nuclear Overhauser effect (NOE) difference
spectrum of compound 12 was recorded. After irradiation at 2.95
ppm (CH2), the signal at 7.45ꢀ7.58 ppm (C6H5) was increased
showing the neighborhood of the acetalic side chain and the
phenyl moiety. A metalation of pyrazole 4 in the 3-position
would lead to a regioisomeric product, which could not give a
positive NOE between side chain and phenylic protons.
The methylated derivatives were synthesized in the same
manner. However, because of the high volatility of the methyl-
pyrazole derivatives 7, 9, and 11, these intermediates were not
isolated but directly converted into the brominated derivative 13,
which was isolated in 29% yield over four steps starting from
methylpyrazole (5).
Treatment of bromopyrazoles 12 and 13 with n-BuLi led to
the pyrazolyllithium derivatives, which were trapped with 1-ben-
zylpiperidone 14 to afford the hydroxy acetals 15 and 16 in 67%
yield, respectively. In order to increase the yields, benzylpiper-
idone 14 was replaced with ethoxycarbonyl protected piperidone
19, which provided the addition product 20 in 85% yield.
(Scheme 2) Therefore, initial experiments for the establishment
of the spirocyclic ring system were performed starting with the
hydroxy acetal 20.
Reaction of hydroxy acetal 20 with p-toluenesulfonic acid in
the solvent methanol44 provided the desired spirocyclic pyrano-
pyrazole 21 (9%) and the methyl ether 22a (82%). In order to
avoid methyl ether formation, the cyclization of 20 was per-
formed in THF leading to the spirocyclic pyranopyrazole 21
(28%) and the elimination product 23 (39%). The formation
of the side products 22a and 23 is explained by protonation of
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ARTICLE
Scheme 4a
which even increases σ1 affinity. Therefore, smaller N-substitu-
ents were included into this study.
The secondary amine 17b was almost inactive at the σ1
receptor. However, the σ1 receptor affinity was enhanced with
increasing size of the N-substituent. In this series, compounds
containing N-residues with five carbon atoms (17nꢀp) repre-
sent the highest affinity σ1 ligands with Ki-values in the range of
1 nM. Even 17q with the very large octyl residue showed a σ1
receptor affinity of 2.9 nM.
Replacement of the methoxy moiety in position 60 with an OH
moiety (24) led to a 20-fold decrease of the σ1 receptor affinity.
However, the compound with a double bond between C-60 and
C-70 (26) and the unsubstituted system (28a) showed the same
σ1 receptor affinity as the parent methoxy derivative 17a.
Variation of the N-substituent (R2) of the unsubstituted com-
pound 28a resulted in the same trends as those observed for the
methoxy derivatives 17. In particular, the very high σ1 affinity
of the cyclohexylmethyl derivative 28d should be emphasized
(Ki = 0.43 nM).
Despite the high σ1 affinity and analgesic potency of the
methyl substituted indazole derivatives 1 (R1 = CH3), the
corresponding spirocyclic pyranopyrazoles with a methyl sub-
stituent showed considerably lower σ1 receptor affinities than
the phenyl derivatives. As a rule, the σ1 affinity of the methyl
substituted derivatives is about 15ꢀ20-fold lower than the σ1
affinity of the phenyl substituted derivatives (e.g., 17a, Ki =
1.5 nM; 18a, Ki = 21 nM; 17o, Ki = 0.97 nM; 18f, Ki = 18 nM;
28c, Ki = 0.81 nM; 29c, Ki = 17 nM). However, the structure
affinity relationships within the class of methyl derivatives
correlate nicely with those derived for the phenyl substituted
derivatives, although at a lower level of σ1 affinity.
a Reagents and reaction conditions: (a) HCl 2M, rt, 82% (24), 77% (25).
(b) MeSO2Cl, NEt3, CH2Cl2, rt, 2 h, then reflux, 1 h, 65% (26), 79%
+
(27). (c) H2, Pd/C, HOAc, rt, 1 h, 62% (28a), 74% (29a). (d) NH4
HCO2ꢀ, Pd/C, MeOH, reflux, 25ꢀ39 min, 93% (28b), 90% (29b).
(e) RꢀX, acetonitrile, K2CO3, reflux. (f) RCHdO, NaBH(OAc)3,
1,2-dichloroethane, rt.
’ σ1 RECEPTOR AFFINITY
The σ1 receptor affinity of the spirocyclic pyranopyrazoles of
type 3 was determined in competition experiments with the
potent and σ1 selective radioligand [3H]-(+)-pentazocine.
Guinea pig brain membrane preparations were used as receptor
material, and the nonspecific binding was determined in the pres-
ence of a large excess of nontritiated (+)-pentazocine.31ꢀ33,48
The σ1 receptor affinities of the spirocyclic pyranopyrazoles
are summarized in Table 1. Whereas the N-benzyl derivative 17a
with the phenylpyrazole framework shows a very similar σ1
receptor affinity (Ki = 1.5 nM) as the corresponding benzopyran
derivative 2b (Ki = 1.3 nM), the methylpyrazole derived analogue
18a has a considerably reduced σ1 affinity (Ki = 21 nM). It can be
concluded from these results that replacement of the benzene
ring of 2b with the polar and electron rich pyrazole moiety
leads to a decreased σ1 affinity, which can be compensated by
the enlargement of the aromatic system by an additional phenyl
moiety at the pyrazole moiety (17a).
Replacement of the N-benzyl moiety of 17a with an electron rich
arylmethyl residue (methoxybenzyl (17d), furan-2-ylmethyl
(17e)) resulted in the same or slightly reduced σ1 affinity. How-
ever, an electron poor benzyl moiety (4-fluorobenzyl (17c)) led to
an increased σ1 affinity. This observation is in good accordance
with analogous spirocyclic piperidine σ1 ligands.36
Systematic extension of the aryl-N distance from one methy-
lene moiety (17a) to two (17f), three (17g), and four (17h)
methylene moieties led to a slightly reduced σ1 affinity from
1.5 nM (17a) to 3.2 nM (17h). Replacement of the terminal
phenyl moiety of the potent benzyl derivative 17a with a
hydrogenated cyclohexyl group (17i) afforded a σ1 ligand with
subnanomolar affinity (Ki = 0.55 nM). This result indicates that
the aromatic system of 17a can be replaced by a saturated system,
’ σ1 PHARMACOPHORE MODELS
Various pharmacophore models have been developed as tools
for the explanation of the affinity of known σ1 ligands and for the
design of novel σ1 ligands.49ꢀ53 A basic N-atom together with
hydrophobic regions are important features according to these
models. The first 3D computer-based σ1 pharmacophore model
was developed by Langer et al. and consists of four hydrophobic
groups and one positive ionizable group (Figure 2, top). The
postulated distances between the basic amino moiety and the
hydrophobic groups are 4.1 Å, 6.3 Å, and 9.8 Å, respectively.51
These distances are in good accordance with the distances
defined in the Glennon model.49,50
In order to define the corresponding distances in the
spirocyclic pyranopyrazoles 28a and 29a (achiral without the
60-OCH3 group), a stochastic conformational analysis (Mole-
cular Operating Environment (MOE)) was performed with
subsequent AM1 minimization of the resulting conformations.
For both compounds, six energetically favored conformations
were found in the energy range <1.66 kcal/mol. In all conforma-
tions, the N-benzyl substituent is equatorially oriented at the
piperidine ring. For the spirocyclic framework, two types of
conformations were found with the pyrazole ring in an equatorial
or axial orientation related to the piperidine chair. The distance
between the basic N-atom and the pyrazole ring is greater (6.5 Å)
for an equatorially oriented pyrazole ring than for an axially
oriented one (5.9 Å).
In Figure 2, energetically favored spirocyclic pyranopyrazoles
28a and 29a with an equatorially oriented N-benzyl moiety and
pyrazole ring are compared with the pharmacophore model
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Table 1. σ1 and σ2 Receptor Affinities of Spirocyclic Pyranopyrazoles Compared with Those of Lead Compounds and Reference
Compounds
compd
R1
R2
R3
σ1Ki ( SEM (nM)
σ2Ki ( SEM (nM)
selectivity σ1/σ2
1aa,28
2a31
2b31
17a
7.0
39.7
5.7
Ph-CH2
Ph-CH2
Ph-CH2
H
OCH3
1.1 ( 0.22
1.3 ( 0.18
1.5 ( 0.08
>1 μM
1280
1130
2708
>680
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OH
3500
Ph
>1 μM
>1 μM
687
17b
17c
Ph
Ph
p-F-C6H4-CH2
p-H3CO-C6H4-CH2
furan-2-yl-CH2
Ph-(CH2)2
Ph-(CH2)3
Ph-(CH2)4
C6H11-CH2
n-propyl
0.94 ( 0.21
1.5 ( 0.29
2.2 ( 0.37
2.7 ( 0.54
3.2 ( 0.70
3.2 ( 0.29
0.55 ( 0.17
33 ( 4.3
730
600
>450
209
260
134
200
>30
>5
17d
17e
Ph
925
Ph
>1 μM
570
17f
Ph
17g
Ph
833
17h
17i
Ph
428
Ph
109 ( 13
>1 μM
>1 μM
752
17j
Ph
17k
17l
Ph
isopropyl
210 ( 35
8.0 ( 2.2
6.3 ( 1.1
0.82 ( 0.06
0.97 ( 0.16
1.6 ( 0.33
2.9 ( 0.47
21 ( 2.3
Ph
n-butyl
94
17m
17n
17o
17p
17q
18a
Ph
isobutyl
933
150
415
326
610
73
Ph
n-pentyl
340 ( 32
316 ( 55
>1 μM
209 ( 22
>1 μM
>1 μM
>1 μM
>1 μM
>1 μM
>1 μM
>1 μM
557
Ph
isopentyl
Ph
isopentenyl
octyl
Ph
CH3
CH3
CH3
CH3
CH3
Ph
PhꢀCH2
p-F-C6H4-CH2
Ph-(CH2)3
n-propyl
>48
>48
>11
18c
21 ( 6.3
18d
18e
93 ( 19
>1 μM
18f
isopentyl
18 ( 6.3
>56
>37
>5
24
Ph-CH2
27 ( 6.7
25
CH3
Ph
Ph-CH2
OH
190 ( 9.5
1.48 ( 0.27
12 ( 3.7
26
Ph-CH2
HC6dC7H7
HC6dC7H
H
376
36
27
CH3
Ph
Ph-CH2
429
28a
Ph-CH2
1.71 ( 0.08
0.81 ( 0.15
0.43 ( 0.09
0.98 ( 0.17
0.97 ( 0.07
9.2 ( 2.8
17 ( 11
773
452
125
100
85
28c
Ph
Ph-(CH2)3
C6H11-CH2
isopentyl
H
102 ( 11
43 ( 4.8
83 ( 22
269
28d
28e
Ph
H
Ph
H
28f
Ph
isopentenyl
Ph-CH2
H
297
21
29a
CH3
CH3
CH3
CH3
H
191
29c
Ph-(CH2)3
isopentyl
H
>1 μM
>1 μM
>1 μM
>59
>33
>71
29d
29e
H
30 ( 11
isopentenyl
H
14 ( 2.9
(+)-pentazocine
haloperidol
4.2 ( 1.1
3.9 ( 1.5
61 ( 18
78 ( 2.3
42 ( 15
20
di-o-tolylguanidine
0.7
progesterone
660 ( 115
a Compound 1a: R1 = CH3, NR22 = 4-phenylpiperidin-1-yl.
developed by Langer. The corresponding distances were calcu-
lated using the end points of the hydrophobic substituents. The
distances determined for the spirocyclic systems fit nicely to the
distances of the model. The higher σ1 affinity of the phenyl
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The removal of the methoxy group in position 60 led in most
cases to an increased σ2 affinity (e.g., 17a, Ki > 1 μM; 28a, Ki =
773 nM; 17o, Ki = 316 nM; 28e, Ki = 83 nM). This result
indicates that high σ1 affinity and selectivity against the σ2
subtype can be achieved by introducing a substituent in position
60 of the pyranopyrazole system.
Since some potent σ ligands also interact with NMDA
receptors and vice versa,54,55 the affinity of all synthesized
pyranopyrazoles toward the NMDA receptor was also included
into this study. The affinity to the PCP binding site of the NMDA
receptor was determined in competition experiments using fresh
pig brain cortex membrane preparations as the receptor material
and the radioligand [3H]-(+)-MK-801.56 At a concentration of
1 μM, all synthesized pyranopyrazoles showed no interaction
with the PCP binding site of the NMDA receptor.
Additionally, the affinities of the highly affine σ1 ligands 17a,
17i, 17m, 17o, and 17q toward α1, α2, and 5-HT1A receptors as
well as the 5-HT-transporter were investigated. The correspond-
ing IC50-values were higher than 1 μM for all five test compounds
indicating a more than 1000-fold selectivity of these compounds
against these targets.
’ ANTIALLODYNIC ACTIVITY
Sensitization by subplantar capsaicin injection was used to
assess the effect of the cyclohexylmethyl derivative 17i (Ki (σ1) =
0.55 nM) and the isopentenyl derivative 17p (Ki (σ1) = 1.6 nM)
on mechanical allodynia of mice.27,28 In this assay, capsaicin
(8-methyl-N-vanillylnon-6-enamide) was injected subplantarly
to evoke a nocifensive behavior that is characterized by lifting and
guarding the injected paw and typically lasts up to 5 min
following injection. Afterward, hypersensitivity to both thermal
and mechanical stimuli is evidenced.27,28,57
Figure 2. Comparison of spirocyclic pyranopyrazole derivatives 28a
and 29a with the σ1 pharmacophore model of Langer et al.51 Top: σ1
pharmacophore model with indicated distances;51 middle and bottom:
distances between the basic N-atom and hydrophobic regions of the
energetically most favored conformations of 28a and 29a calculated
with AM1.
Mice were treated with the σ1 ligands 17i and 17p 30 min
before the capsaicin injection into the midplantar surface of the
right hind paw. Withdrawal latencies to mechanical stimuli by
a von Frey filament (1 g) were determined 15 min after the
capsaicin injection. Whereas the indazole derivative 1a (Ki (σ1) =
7.0 nM) was very potent in the capsaicin assay,28 the more potent
σ1 ligands 17i and 17p showed only low antiallodynic activity.
Even at the highest dose tested (32 mg/kg body weight), the
antiallodynic effect of 17i and 17p was only 70% and 25%,
respectively, indicating low in vivo activity. Although the anti-
allodynic activity is rather low, both compounds can be con-
sidered as partial σ1 receptor antagonists.
derivative 28a (Ki = 1.7 nM) compared with that of the methyl
derivative 29a (Ki = 9.2 nM) is attributed to the additional phenyl
residue. Whereas the methyl group of 29a is too small to produce
high lipophilic interactions with the σ1 receptor protein, the
phenyl moiety of 28a is able to occupy an additional hydrophobic
pocket resulting in increased lipophilic interactions (lower Ki
value).
’ RECEPTOR SELECTIVITY
The selectivity of the spirocyclic pyranopyrazoles against
related receptor systems was investigated. At first, the affinity
toward the σ2 subtype was determined using homogenates of rat
liver as the σ2 receptor source. Since a σ2 selective radioligand is
not commercially available, the nonselective radioligand [3H]-
ditolylguanidine was employed in the presence of an excess of
nonlabeled (+)-pentazocine (500 nM) for the selective masking
of σ1 receptors. An excess of nontritiated ditolylguanidine was
used for determining the nonspecific binding.31ꢀ33,48
Generally, all spirocyclic pyranopyrazoles reveal high selec-
tivity for the σ1 receptor subtype over the σ2 subtype. The σ1/σ2
selectivity is particularly high for very potent σ1 ligands (e.g., 17c,
Ki = 0.94 nM, selectivity 730; 28a, Ki = 1.71 nM, selectivity 452),
but compounds with reduced σ1 affinity display lower σ1/σ2
selectivity.
’ CONCLUSIONS
The combination of potent indazole-based (1) and spirocyclic
(2) σ1 ligands resulted in the spirocyclic pyranopyrazoles 3,
which interact with high affinity and selectivity with σ1 receptors.
The core structure was extensively modified in positions 1, 10,
and 60. In contrast to the indazole class of σ1 ligands 1, spirocyclic
pyranopyrazoles 18 with a methyl group (R1) showed lower
σ1 affinities than their phenyl (R1) substituted analogues 17.
The lower σ1 affinities of compounds containing the methylpyr-
azole substructure (e.g., 18) compared with those of analogous
benzene derivatives 2 were attributed to the small, electron rich
pyrazole heterocycle. However, this effect was compensated by
an additional phenyl ring at the pyrazole heterocycle, which is
able to occupy an additional hydrophobic region of the σ1
receptor protein. This observation is in good accordance with
σ1 pharmacophore models. Despite the high σ1 affinity and
Relatively high σ2 affinities were observed for the cyclohex-
ylmethyl derivatives 17i (Ki = 109 nM) and 28d (Ki = 43 nM).
Because of the very high σ1 affinities of these compounds, the
σ1/σ2 selectivities are still greater than 100.
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Journal of Medicinal Chemistry
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selectivity, the in vivo activity of two promising representatives
(17i, 17p) in the capsaicin neuropathic pain model was rather
low. Nevertheless, the low antiallodynic activity of 17i and 17p
indicates at least a partial σ1 receptor antagonistic activity.
and stirred for 4 h. Then, water and a saturated solution of NaHCO3
were added, and the aqueous solution was extracted with CH2Cl2. The
organic layer was dried (K2CO3), the solvent was removed in vacuo, and
the residue was purified by fc (Ø = 8 cm, n-hexane:EtOAc = 8:2, 40 mL,
Rf = 0.30). Colorless oil, yield 12.7 g (95%). Anal. (C13H15BrN2O2,
311.2) C, H, N. 1H NMR (DMSO-D6): δ (ppm) = 2.95 (d, J = 5.9 Hz,
2H, CH2CH(OCH3)2), 3.10 (s, 6H, CH2CH(OCH3)2), 4.40 (t, J =
5.7 Hz, 1H, CH2CH(OCH3)2), 7.45ꢀ7.58 (m, 5H, phenyl-CH), 7.76
(s, 1H, pyrazole-3-CH). NOE difference spectrum (CDCl3): irrad. at
2.95 ppm (CH2CH(OCH3)2), increase of signals at 4.40 ppm (CH2CH-
(OCH3)2), 7.45ꢀ7.58 ppm (C6H5). 13C NMR (DMSO-D6): δ (ppm) =
29.6 (1C, CH2CH(OCH3)2), 54.2 (2C, CH2CH(OCH3)2), 95.8 (1C,
pyrazole-4C), 102.7 (1C, CH2CH(OCH3)2), 126.3 (2C, phenyl-CH,
ortho), 129.3 (1C, phenyl-CH, para), 130.0(2C, phenyl-CH, meta), 137.4
(1C, phenyl-C), 140.2 (1C, pyrazole-5C), 140.5 (1C, pyrazole-3-CH).
2-[4-(1-Benzyl-4-hydroxypiperidin-4-yl)-1-phenylpyra-
zol-5-yl]acetaldehyde dimethyl acetal (15). Under N2, a 1.53
M* solution of n-butyllithium in hexane (10.5 mL, 16.1 mmol) was
added slowly to a cooled (ꢀ78 °C) solution of 12 (5.0 g, 16.1 mmol) in
THF (80 mL). The mixture was stirred at ꢀ78 °C for 15 min, then a
solution of piperidone 14 (3.3 g, 17.7 mmol) in THF (10 mL) was added
slowly, and the mixture was stirred at ꢀ78 °C for 4.5 h and for 1 h at rt.
Then, water (∼40 mL) was added until no more precipitate was formed.
The mixture was extracted with CH2Cl2, the organic layer was dried
(K2CO3) and concentrated in vacuo, and the residue was purified by fc
(Ø = 8 cm, n-hexane:EtOAc = 2:8 + 2% N,N-dimethylethanamine,
80 mL, Rf = 0.23). Pale yellow oil, which solidified slowly upon standing.
Pale yellow solid, mp 107 °C, yield 4.6 g (67%). C25H31N3O3 (421.5).
1H NMR (DMSO-D6): δ (ppm) = 1.77 (d broad, J = 12.5 Hz, 2H,
N(CH2CH2)2), 1.92 (td, J = 12.5/3.1 Hz, 2H, N(CH2CH2)2), 2.42 (t,
J = 10.6 Hz, 2H, N(CH2CH2)2), 2.58 (d broad, J = 11.0 Hz, 2H,
N(CH2CH2)2), 3.04 (s, 6H, CH2CH(OCH3)2), 3.12 (d, J = 5.5 Hz,
2H, CH2CH(OCH3)2), 3.49 (s, 2H, NCH2Ph), 4.50 (t, J = 5.8 Hz, 1H,
CH2CH(OCH3)2), 4.66 (s, 1H, OH), 7.21ꢀ7.27 (m, 1H, aromat.
CH, para), 7.32 (m, 4H, aromat. CH, meta), 7.40ꢀ7.46 (m, 3H, aromat.
CH, para, ortho), 7.49 (d, J = 7.0 Hz, 2H, aromat. CH, ortho), 7.50
(s, 1H, pyrazole-3-CH).
’ EXPERIMENTAL SECTION
General. Unless otherwise noted, moisture sensitive reactions were
conducted under dry nitrogen. THF was dried with sodium/benzophe-
none and was freshly distilled before use. The concentration of n-BuLi
marked with an asterisk (*) was determined by titration with 1,3-
diphenylpropan-2-one p-toluenesulfonylhydrazone. Flash chromatogra-
phy (fc), Silica gel 60, 40ꢀ64 μm (Merck); parentheses include
diameter of the column, eluent, fraction size, and Rf value. Melting
point: melting point apparatus SMP 3 (Stuart Scientific), uncorrected.
1H NMR (400 MHz) and 13C NMR (100 MHz): Mercury-400BB
spectrometer (Varian); δ in ppm related to tetramethylsilane; coupling
constants are given with 0.5 Hz resolution; the assignments of 13C and
1H NMR signals were supported by 2D NMR techniques. Elemental
analysis: CHN-Rapid Analysator (Fons-Heraeus). The purity of all test
compounds was proved by elemental analysis; all values are within
(0.4%. The purity of four test compounds was proved by HPLC
analysis (purity >95%).
5-(2-Methoxyvinyl)-1-phenylpyrazole (8). Under N2, dry
+
Ph3PCH2OCH3 Clꢀ (6.22 g, 18.1 mmol) was suspended in THF
(60 mL) for 30 min. The suspension was cooled down to ꢀ50 °C, and
then a solution of KOtBu in THF (1 M, 16.5 mL, 16.5 mmol) was added
dropwise. After stirring for 15 min, a solution of 6 (1.42 g, 8.3 mmol) in
abs. THF (40 mL) was added dropwise. The mixture was allowed to
warm to rt overnight. After the addition of water (∼25 mL), the mixture
was extracted with EtOAc. The organic layer was dried (K2CO3) and
filtered, the solvent was removed in vacuo, and the residue was purified
by fc (Ø = 8 cm, n-hexane:EtOAc = 9:1, 40 mL, Rf = 0.10). Colorless oil,
1
yield 1.53 g (92%). C12H12N2O (200.2). H NMR (DMSO-D6): δ
(ppm) = 3.56 (s, 3 ꢁ 0.75H, OCH3, trans), 3.77 (s, 3 ꢁ 0.25H, OCH3,
cis), 5.18 (d, J = 6.8 Hz, 0.25H, CHdCHOCH3, cis), 5.59 (d, J = 12.8
Hz, 0.75H, CHdCHOCH3, trans), 6.37 (d, J = 6.8 Hz, 0.25H,
CHdCHOCH3, cis), 6.47 (d, J = 1,6 Hz, 0.75H, pyrazole-4-CH, trans),
6.63 (d, J = 1.6 Hz, 0.25H, pyrazole-4-CH, cis), 7.26 (d, J = 12.8 Hz,
0.75H, CHdCHOCH3, trans), 7.39ꢀ7.54 (m, 5H, phenyl-CH), 7.55
(d, J = 1.6 Hz, 0.75H, pyrazole-3-CH, trans), 7.58 (d, J = 1.2 Hz, 0.25H,
pyrazole-3-CH, cis). The ratio of cis-8:trans-8 is 25:75.
2-(1-Phenylpyrazol-5-yl)acetaldehyde dimethyl acetal
(10). A solution of the enol ether 8 (1.0 g, 5.0 mmol) and p-tol-
uenesulfonic acid monohydrate (475 mg, 2.5 mmol) in MeOH (80 mL)
was stirred at rt for 72 h. Then, a solution of saturated NaHCO3 was
added. The mixture was extracted with CH2Cl2, the organic layer was
dried (K2CO3), the solvent was removed in vacuo, and the residue was
purified by fc (Ø = 6 cm, n-hexane:EtOAc = 8:2, 40 mL, Rf = 0.12).
Colorless oil, yield 1.04 g (90%). C13H16N2O2 (232.3). 1H NMR
(DMSO-D6): δ (ppm) = 2.92 (d, J = 5.9 Hz, 2H, CH2CH(OCH3)2),
3.39 (s, 6H, CH2CH(OCH3)2), 4.55 (t, J = 5.5 Hz, 1H, CH2CH-
(OCH3)2), 6.39 (d, J = 1.6 Hz, 1H, pyrazole-4-CH), 7.40ꢀ7.55 (m, 5H,
phenyl-CH), 7.59 (d, J = 1.6 Hz, 1H, pyrazole-3-CH). 13C NMR
(DMSO-D6): δ (ppm) = 30.5 (1C, CH2CH(OCH3)2), 53.8 (2C,
CH2CH(OCH3)2), 103.6 (1C, CH2CH(OCH3)2), 107.3 (1C, pyra-
zole-4-CH), 126.0 (2C, phenyl-CH, ortho), 128.6 (1C, phenyl-CH,
para), 129.9 (2C, phenyl-CH, meta), 139.1 (1C, phenyl-C), 140.2 (1C,
pyrazole-5-CH), 140.3 (1C, pyrazole-3-CH).
1-Benzyl-60-methoxy-10-phenyl-60,70-dihydro-10H-spiro-
[piperidine-4,40-pyrano[4,3-c]pyrazole] (17a). A solution of 15
(4.5 g, 10.7 mmol) and p-toluenesulfonic acid monohydrate (4.5 g, 23.5
mmol) in MeOH (150 mL) was stirred at rt for 21 h. After the addition
of NaOH (0.5 M, 5 mL), the mixture was extracted with CH2Cl2. The
organic layer was dried (K2CO3) and concentrated in vacuo, and the
residue was purified by fc (Ø = 8 cm, EtOAc, 80 mL, Rf = 0.27).
Colorless solid, mp 151 °C, yield 3.0 g (73%). Anal. (C24H27N3O2,
389.5) C, H, N. 1H NMR (CDCl3): δ (ppm) = 1.92 (dd, J = 14.1/3.1
Hz, 2H, N(CH2CH2)2), 1.98ꢀ2.05 (m, 1H, N(CH2CH2)2), 2.09 (td,
J = 12.5/3.7 Hz, 1H, N(CH2CH2)2), 2.45 (t broad, J = 11.7 Hz, 1H,
N(CH2CH2)2), 2.55 (t broad, J = 11.7 Hz, 1H, N(CH2CH2)2), 2.77 (m,
2H, N(CH2CH2)2), 2.88 (dd, J = 15.6/7.0 Hz, 1H, CH2CHOCH3),
2.96 (dd, J = 15.7/3.9 Hz, 1H, CH2CHOCH3), 3.53 (s, 3H, OCH3),
3.58 (s, 2H, NCH2Ph), 4.84 (dd, J = 7.0/3.9 Hz, 1H, CH2CHOCH3),
7.23ꢀ7.28 (m, 1H, phenyl-CH), 7.29ꢀ7.39 (m, 5H, phenyl-CH),
7.40ꢀ7.47 (m, 4H, phenyl-CH), 7.49 (s, 1H, pyrazole-3-CH). 13C
NMR (CDCl3): δ (ppm) = 31.2 (1C, CH2CHOCH3), 36.7 (1C,
N(CH2CH2)2), 39.5 (1C, N(CH2CH2)2), 49.5 (1C, N(CH2CH2)2),
49.6 (1C, N(CH2CH2)2), 56.9 (1C, OCH3), 63.7 (1C, NCH2Ph), 71.9
(1C, spiro-C), 77.5 (1C, pyrazole-4-C), 96.8 (1C, CH2CHOCH3),
122.8 (2C, phenyl-CH), 124.4 (1C, phenyl-C), 127.3, 128.5, 129.5
(8C, phenyl-CH), 133.8 (1C, phenyl-C), 135.9 (1C, pyrazole-3-CH),
139.5 (1C, pyrazole-5-C).
2-(4-Bromo-1-phenylpyrazol-5-yl)acetaldehyde dimethyl
acetal (12). Trimethyl orthoformate (7.0 mL, 64.3 mmol) and
pyridinium bromide perbromide (PBB) (13.7 g, 42.8 mmol) were added
in portions to a solution of 10 (9.9 g, 42.8 mmol) in MeOH (500 mL) at
0 °C. After stirring for 1 h at 0 °C, the mixture was allowed to warm to rt
60-Methoxy-10-phenyl-60,70-dihydro-10H-spiro[piperidine-
4,40-pyrano[4,3-c]pyrazole] (17b). Dry ammonium formate
(64.8 mg, 1.28 mmol) was added to a stirred suspension of 17a
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ARTICLE
(80 mg, 0.21 mmol) and 10% Pd/C (16 mg) in MeOH (5 mL). This
mixture was heated to reflux for 10 min. Then, it was filtered and
concentrated in vacuo, and the residue was purified by fc (Ø = 2 cm,
methanol + 2% NH3 (conc.), 10 mL, Rf = 0.10). After removing the
solvent of the respective fractions, the residue was dissolved in
CH2Cl2, the solution was filtered, and the solvent was evaporated in
vacuo. Pale yellow oil, yield 55 mg (88%). Anal. (C17H21N3O2,
299.4) H, N; C calcd. 68.2; found, 67.2. Purity (HPLC): 100% (tR =
(454 μL, 3.26 mmol) and MeSO2Cl (127 μL, 1.63 mmol) were added.
Thesolution was stirred atrt for 2 h and then heated toreflux for 1 h. After
the addition of a saturated solution of NaHCO3 (5 mL), the mixture was
extracted with CH2Cl2 (3ꢁ). The organic layer was dried (K2CO3),
filtered, and concentrated in vacuo, and the residue was purified by fc
(L = 5 cm, n-hexane:EtOAc 6:4 + 2% N,N-dimethylethanamine, 65 mL,
Rf = 0.15). Colorless solid, mp 135 °C, yield 315 mg (65%). Anal.
(C23H23N3O, 357.5) C, H, N. 1H NMR (CDCl3): δ (ppm) = 1.92ꢀ2.02
(m, 2H, N(CH2CH2)2), 2.19ꢀ2.26 (m, 2H, N(CH2CH2)2), 2.47 (td, J =
11.5/1.7 Hz, 2H, N(CH2CH2)2), 2.70ꢀ2.76 (m, 2H, N(CH2CH2)2),
3.58 (s, 2H, NCH2Ph), 5.82 (dd, J = 6.1/0.8 Hz, 1H, ArCHdCHO), 6.50
(d, J = 5.9 Hz, 1H, ArCHdCHO), 7.26ꢀ7.29 (m, 1H, phenyl-CH),
7.30ꢀ7.39 (m, 6H, phenyl-CH), 7.43ꢀ7.52 (m, 4H, phenyl-CH (3H),
pyrazole-3-CH (1H)).
1
14.8 min). H NMR (DMSO-D6): δ (ppm) = 1.55ꢀ1.68 (m, 2H,
N(CH2CH2)2), 1.79ꢀ1.92 (m, 2H, N(CH2CH2)2), 2.75 (t broad,
J = 11.0 Hz, 2H, N(CH2CH2)2), 2.80ꢀ2.95 (m, 2H, N(CH2CH2)2),
2.82 (dd, J = 15.7/7.0 Hz, 1H, CH2CHOCH3), 2.91 (dd, J = 15.7/3.9
Hz, 1H, CH2CHOCH3), 3.31 (s broad, 1H, NH), 3.39 (s, 3H,
OCH3), 4.88 (dd, J = 6.7/3.5 Hz, 1H, CH2CHOCH3), 7.35 (t, J = 7.4
Hz, 1H, phenyl-CH, para), 7.44ꢀ7.55 (m, 4H, phenyl-CH, meta,
ortho), 7.59 (s, 1H, pyrazole-3-CH).
1-Benzyl-10-phenyl-60,70-dihydro-10H-spiro[piperidine-
4,40-pyrano[4,3-c]pyrazole] (28a). Ten percent Pd/C (35 mg)
was added to a solution of 26 (50 mg, 0.14 mmol) in HOAc (5 mL). The
mixture was stirred under H2 (balloon) for 1 h. Then, the catalyst was
filtered off, and the filtrate was alkalized with NaOH (2 M) and extracted
with CH2Cl2 (3ꢁ). The organic layer was dried (K2CO3) and filtered,
the solvent was removed in vacuo, and the residue was purified by fc
(L = 2.5 cm, n-hexane:EtOAc 6:4 + 2% N,N-dimethylethanamine,
10 mL, Rf = 0.23). Colorless solid, mp 141 °C, yield 31 mg (62%). Anal.
(C23H25N3O, 359.5) C, H, N. 1H NMR (CDCl3): δ (ppm) =
1.88ꢀ1.95 (m, 2H, N(CH2CH2)2), 1.98 (td, J = 13.5/4.3 Hz, 2H,
N(CH2CH2)2), 2.42 (td, J = 11.4/3.2 Hz, 2H, N(CH2CH2)2), 2.73
(d broad, J = 11.3 Hz, 2H, N(CH2CH2)2), 2.82 (t, J = 5.5 Hz, 2H,
ArCH2CH2O), 3.57 (s, 2H, NCH2Ph), 3.88 (t, J = 5.4 Hz, 2H,
ArCH2CH2O), 7.23ꢀ7.38 (m, 6H, phenyl-CH), 7.42ꢀ7.52 (m, 5H,
phenyl-CH (4H), pyrazole-3-CH (1H)).
Receptor Binding Studies. Materials and General Procedures.
Guinea pig brains and rat livers were commercially available (Harlan-
Winkelmann, Germany). Homogenizer: Elvehjem Potter (B. Braun
Biotech International). Centrifuge: High-speed cooling centrifuge mod-
el Sorvall RC-5C plus (Thermo Finnigan). Filter: Printed Filtermat
Type A (Perkin-Elmer), presoaked in 0.5% aqueous polyethylenimine
for 2 h at rt before use. The filtration was carried out with a MicroBeta
FilterMate-96 Harvester (Perkin-Elmer). The scintillation analysis was
performed using a Meltilex (Type A) solid scintillator (Perkin-Elmer).
The radioactivity bound to the filter was measured using a MicroBeta
Trilux scintillation analyzer (Perkin-Elmer). The overall counting
efficiency was 20%.
1-(4-Fluorobenzyl)-60-methoxy-10-phenyl-60,70-dihydro-
10H-spiro[piperidine-4,40-pyrano[4,3-c]pyrazole] (17c). p-
Fluorobenzaldehyde (36.0 μL, 0.33 mmol) and NaBH(OAc)3 (106 mg,
0.50 mmol) were added to a stirred solution of 17b (100 mg, 0.33 mmol)
in dichloroethane (2 mL). The mixture was stirred at rt for 19 h. After the
addition of a saturated solution of NaHCO3 (10 mL), the mixture was
extracted with CH2Cl2. The organic layer was dried (K2CO3) and
concentrated in vacuo, and the residue was purified by fc (L = 3 cm,
n-hexane:EtOAc 5:5 + 2% N,N-dimethylethanamine, 20 mL, Rf = 0.20).
Colorless solid, mp 158 °C, yield 107 mg (79%). Anal. (C24H26FN3O2,
1
407.5) C, H, N. H NMR (CDCl3): δ (ppm) = 1.86ꢀ1.98 (m, 2H,
N(CH2CH2)2), 2.00ꢀ2.06 (m, 1H, N(CH2CH2)2), 2.09 (td, J = 12.9/4.4
Hz, 1H, N(CH2CH2)2), 2.45 (t broad, J = 11.0 Hz, 1H, N(CH2CH2)2),
2.56 (t broad, J = 11.0 Hz, 1H, N(CH2CH2)2), 2.71ꢀ2.81 (m, 2H,
N(CH2CH2)2), 2.90 (dd, J = 15.3/6.7 Hz, 1H, CH2CHOCH3), 2.98 (dd,
J = 15.3/3.5 Hz, 1H, CH2CHOCH3), 3.55 (s, 5H, OCH3 (3H), NCH2Ph
(2H)), 4.86 (dd, J = 6.7/3.5 Hz, 1H, CH2CHOCH3), 7.03 (t, J = 8.6 Hz,
2H, phenyl-CH), 7.30ꢀ7.38 (m, 3H, phenyl-CH), 7.41ꢀ7.48 (m, 4H,
phenyl-CH), 7.50 (s, 1H, pyrazole-3-CH). 13CNMR(CDCl3): δ(ppm) =
31.2 (1C, CH2CHOCH3), 36.7 (1C, N(CH2CH2)2), 39.4 (1C, N-
(CH2CH2)2), 49.3 (1C, N(CH2CH2)2), 49.4 (1C, N(CH2CH2)2), 56.9
(1C, OCH3), 62.7 (1C, NCH2Ph), 71.8 (1C, spiro-C), 77.4 (1C, pyrazole-
4-C), 96.8 (1C, CH2CHOCH3), 115.1, 115.3 (2C, benzyl-20,60-CH),
122.8 (2C, phenyl-CH, ortho), 124.3 (1C, benzyl-40-C), 127.3 (1C,
phenyl-CH, para), 129.4 (2C, phenyl-CH, meta), 130.9 (2C, benzyl-
3050-CH), 133.7 (1C, phenyl-C), 134.3 (1C, benzyl-10-C), 135.8 (1C,
pyrazole-3-CH), 139.4 (1C, pyrazole-5-C).
Membrane Preparation for the σ1 Assay31ꢀ33,48. Five guinea pig
brains were homogenized with the potter (500ꢀ800 rpm, 10 up-and-
down strokes) in 6 volumes of cold 0.32 M sucrose. The suspension was
centrifuged at 1200g for 10 min at 4 °C. The supernatant was separated
and centrifuged at 23500g for 20 min at 4 °C. The pellet was
resuspended in 5ꢀ6 volumes of buffer (50 mM TRIS, pH 7.4) and
centrifuged again at 23500g (20 min, 4 °C). This procedure was repeated
twice. The final pellet was resuspended in 5ꢀ6 volumes of buffer, the
protein concentration was determined according to the method of
Bradford58 using bovine serum albumin as a standard, and, subsequently,
the preparation was frozen (ꢀ80 °C) in 1.5 mL portions containing
about 1.5 mg protein/mL.
1-(Cyclohexylmethyl)-60-methoxy-10-phenyl-60,70-dihy-
dro-10H-spiro[piperidine-4,40-pyrano[4,3-c]pyrazole] (17i).
(Bromomethyl)cyclohexane (48.5 μL, 0.35 mmol) and K2CO3 (295
mg, 2.14 mmol) were added to a solution of 17b (80 mg, 0.27 mmol) in
acetonitrile (5 mL). This mixture was heated to reflux for 26 h. Then, it
was filtered and concentrated in vacuo, and the residue was purified by fc
(Ø = 2.5 cm, n-hexane:EtOAc 7:3 + 1% N,N-dimethylethanamine,
10 mL, Rf = 0.18). Colorless solid, mp 151 °C, yield 82 mg (77%). Anal.
(C24H33N3O2, 395.6) C, H, N. 1H NMR (CDCl3): δ (ppm) = 0.84ꢀ0.97
(m, 2H, NCH2C6H11), 1.18ꢀ1.29 (m, 4H, NCH2C6H11), 1.47ꢀ1.59 (m,
1H, NCH2C6H11), 1.62ꢀ1.87 (m, 4H, NCH2C6H11), 1.90ꢀ1.98 (m, 2H,
N(CH2CH2)2), 2.02ꢀ2.15 (m, 2H, N(CH2CH2)2), 2.21 (d, J = 7.0 Hz,
2H, NCH2C6H11), 2.37 (td, J = 11.4/2.4 Hz, 1H, N(CH2CH2)2), 2.45 (td,
J = 11.7/2.4 Hz, 1H, N(CH2CH2)2), 2.70ꢀ2.79 (m, 2H, N(CH2CH2)2),
2.90 (dd, J = 15.7/7.0 Hz, 1H, CH2CHOCH3), 2.97 (dd, J = 15.7/3.5 Hz,
1H, CH2CHOCH3), 3.56 (s, 3H, OCH3), 4.84 (dd, J = 6.7/3.5 Hz, 1H,
CH2CHOCH3), 7.30ꢀ7.35 (m, 1H, phenyl-CH, para), 7.41ꢀ7.48 (m, 4H,
phenyl-CH), 7.51 (s, 1H, pyrazole-3-CH).
Performing of the σ1 Assay31ꢀ33,48. The test was performed with the
radioligand [3H]-(+)-pentazocine (22 Ci/mmol; Perkin-Elmer). The
thawed membrane preparation (about 75 μg of the protein) was
incubated with various concentrations of test compounds, 2 nM
[3H]-(+)-pentazocine, and buffer (50 mM TRIS, pH 7.4) in a total
volume of 200 μL for 180 min at 37 °C. The incubation was terminated
by rapid filtration through the presoaked filtermats by using the cell
harvester. After washing each well five times with 300 μL of water, the
filtermats were dried at 95 °C. Subsequently, the solid scintillator was
placed on the filtermat and melted at 95 °C. After 5 min, the solid
1-Benzyl-10-phenyl-10H-spiro[piperidine-4,40-pyrano-[4,3-
c]pyrazole] (26). Under N2, lactol 24 (510 mg, 1.36 mmol) was
dissolved in CH2Cl2 (12 mL). The mixture was cooled to 0 °C, and NEt3
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scintillator was allowed to solidify at rt. The bound radioactivity trapped
on the filters was counted in the scintillation analyzer. The nonspecific
binding was determined with 10 μM unlabeled (+)-pentazocine. The
Kd-value of the radioligand [3H]-(+)-pentazocine is 2.9 nM.59
Data Analysis. Usually, all experiments were carried out in triplicate
using standard 96-well-multiplates (Diagonal). The IC50-values were
determined in competition experiments with six concentrations of the
test compounds and were calculated with the program GraphPad Prism
3.0 (GraphPad Software) by nonlinear regression analysis. The Ki-values
were calculated according to Cheng and Prusoff.60 The Ki-values of
highly affine compounds are given as mean values ( SEM from three
independent experiments.
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’ ASSOCIATED CONTENT
S
Supporting Information. Physical and spectroscopic
b
data of all new compounds, purity data of all test compounds,
general chemistry methods, and details of the pharmacological
assays. This material is available free of charge via the Internet at
http://pubs.acs.org.
’ AUTHOR INFORMATION
(14) Wu, Z.; Bowen, W. D. Role of sigma-1 receptor C-terminal
segment in inositol 1,4,5-trisphosphate receptor activation: Constitutive
enhancement of calcium signaling in MCF-7 tumor cells. J. Biol. Chem.
2008, 283, 28198–28215.
Corresponding Author
*Tel: +49-251-8333311. Fax: +49-251-8332144. E-mail: wuensch@
uni-muenster.de.
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sigma-1 receptors. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 491–496.
(16) Hayashi, T.; Su, T.-P. Sigma-1 receptor chaperones at the er-
mitochondrion interface regulate Ca2+ signaling and cell survival. Cell
2007, 131, 596–610.
’ ACKNOWLEDGMENT
This work was supported by the Deutsche Forschungsgemeinschaft,
which is gratefully acknowledged.
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’ ABBREVIATIONS USED
NMDA, N-methyl-D-aspartate; IP3, inositoltriphosphate; CNS,
central nervous system; THF, tetrahydrofuran; MOE, molecular
operating system; NOE, nuclear Overhauser effect
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