Synthesis and biological evaluation of a new series of hexahydro-2 H-pyrano[3,2-c ]quinolines as novel selective σ1 receptor ligands

Article abstract of DOI:10.1021/jm400181k

The synthesis and pharmacological activity of a new series of hexahydro-2H-pyrano[3,2-c]quinoline derivatives as potent σ₁ receptor (σ₁R) ligands are reported. This family, which does not contain the highly basic amino group usually present in other σ₁R ligands, showed high selectivity over the σ₂ receptor (σ₂R). The activity was shown to reside in only one of the four possible diastereoisomers, which exhibited a perfect match with known σ₁R pharmacophores. A hit to lead program based on a high-throughput screening hit (8a) led to the identification of compound 32c, with substantially improved activity and physicochemical properties. Compound 32c also exhibited a good ADMET (absorption, distribution, metabolism, excretion, toxicity) profile and was identified as a σ₁R antagonist on the basis of its analgesic activity in the mouse capsaicin and formalin models of neurogenic pain.

Full text of DOI:10.1021/jm400181k

Article
pubs.acs.org/jmc
Synthesis and Biological Evaluation of a New Series of
Hexahydro‑2H‑pyrano[3,2‑c]quinolines as Novel Selective σ1
Receptor Ligands
Jose Luis Díaz,*,† Ute Christmann,† Ariadna Fernandez,† Monica Luengo,† Magda Bordas,†
́
́
́
Raquel Enrech,† Monica Carro,† Rosalia Pascual,† Javier Burgueno,† Manuel Merlos,†
́
̃
Jordi Benet-Buchholz,‡ Jordi Ceron-Bertran,‡ Jesus Ramírez,‡ Raquel F. Reinoso,†
́
́
Antonio R. Fernandez de Henestrosa,† Jose Miguel Vela,† and Carmen Almansa*,†
́
́
†Drug Discovery and Preclinical Development, Esteve, Baldiri Reixach 4-8, 08028 Barcelona, Spain
‡Crysforma, Institute of Chemical Research of Catalonia (ICIQ), Avinguda Països Catalans 16, E-43007 Tarragona, Spain
S
* Supporting Information
ABSTRACT: The synthesis and pharmacological activity of a new series
of hexahydro-2H-pyrano[3,2-c]quinoline derivatives as potent σ1
receptor (σ1R) ligands are reported. This family, which does not contain
the highly basic amino group usually present in other σ1R ligands,
showed high selectivity over the σ2 receptor (σ2R). The activity was
shown to reside in only one of the four possible diastereoisomers, which
exhibited a perfect match with known σ1R pharmacophores. A hit to lead
program based on a high-throughput screening hit (8a) led to the
identification of compound 32c, with substantially improved activity and
physicochemical properties. Compound 32c also exhibited a good
ADMET (absorption, distribution, metabolism, excretion, toxicity)
profile and was identified as a σ1R antagonist on the basis of its
analgesic activity in the mouse capsaicin and formalin models of neurogenic pain.
nerve injury.11 The key role played by σ1R in pain-related
INTRODUCTION
■
central sensitization phenomena11,12 and the effect shown by
The σ receptors were discovered in the mid-1970s and
originally proposed as a new class of opioid receptors. At
present it is known that σ receptors are unique binding sites
with no homology to opioid receptors or other mammalian
several of our σ1R antagonists13 in different models of
nociceptive and neuropathic pain support the involvement of
σ1R in the control of nociception. Several compounds have
been evaluated for the treatment of pain, and compound 1,14
proteins. Pharmacological studies have identified at least two
distinct subtypes, namely, σ1 and σ2.1 The receptor (σ1R) has
which is currently in clinical trials for the treatment of diverse
pain states, is the most advanced one. Compound 1 was
been cloned and shown to encode a protein anchored to the
endoplasmic reticulum and plasma membranes.2 This protein
developed on the basis of the pharmacophoric model described
by Glennon et al.,15 which involves a basic amino group and at
least two hydrophobic regions at a certain distance from this
basic amino group, a structural feature shared by other
prototypical σ1R antagonists,16 such as BD-1063 (2) and
haloperidol (3) (Figure 1A,B).
acts as a unique ligand-regulated molecular chaperone3 that
modulates the activity of different proteins, such as N-methyl-D-
aspartic (NMDA) receptors4 and several ion channels.5
Additionally, σ1R has been known to modulate opioid
analgesia,6 and the relationship between the μ-opioid and σ1
receptors has been recently shown to involve direct physical
interaction,7 which explains why σ1R antagonists enhance the
antinociceptive effect of opioids.
On the other hand, σ2R still remains to be unambiguously
identified, although progesterone receptor membrane compo-
nent 1 (PGRMC1), a cytochrome-related protein that binds
directly to heme and regulates lipid and drug metabolism and
hormone signaling, has been recently proposed as the putative
σ2R binding site.17 Ligands acting on σ2R have been proposed
as biomarkers for tumor cell proliferation and show pro-
apoptotic properties, thus suggesting a potential role in cancer
No selective σ1R ligands have so far been marketed, but
several nonselective compounds have been assayed in clinical
studies as antidepressants, drug abuse treatments, antipsy-
chotics, and learning and memory enhancers.8 Our group
became interested in the potential role of σ1R in pain control,
and a σ1R knockout mouse was developed, showing attenuated
pain responses in the formalin test9 and no mechanical
hypersensitivity following capsaicin sensitization10 or sciatic
Received: February 4, 2013
Published: April 8, 2013
© 2013 American Chemical Society
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in terms of yields and purity, especially when aliphatic cyclic
aldehydes were employed. As expected, in this formal [4 + 2]
acid-catalyzed aza-Diels−Alder cycloaddition with cyclic
dienophiles,19 the fused furan or pyran rings maintained the
cis stereochemistry and the theoretically possible trans
orientation was never detected. Hence, only the two epimers
at the α-amino center, cis (a) and trans (b), were obtained as
racemic mixtures (Scheme 1). The SAR study shown in the
next section indicated that the cis (a) isomers always showed
much higher σ1R binding potencies than the corresponding
trans (b) derivatives. In this sense, the Mg(ClO4)2-mediated
protocol was also the best, since it provided cis diastereomers as
major isomers (Table 1 shows the proportion of representative
Table 1. cis:trans Ratios for Representative Pyranoquinolines
a
compd
R1
R2
a:b ratio
8
4-chlorophenoxy
3-chlorophenoxy
isopentyloxy
phenyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
78:22
83:17
87:13
81:19
90:10
b
9
17
20
32
Figure 1. Representative σ1R antagonists (A), pharmacophoric σ1R
model by Glennon (B),25 and structure of the initial hit 8a (C).
isopentyloxy
tetrahydro-2H-pyran-4-yl
a
1
Diastereomeric ratio determined by H NMR of the crude product.
imaging and treatment.18 Because the pharmacologies of the
two σ receptors are clearly differentiated, selectivity between
both of them and against other molecular targets is desired.
As a result of a high-throughput screening (HTS) program
devoted to the discovery of new σ1R antagonists, we identified a
new series of hexahydro-2H-pyrano[3,2-c]quinolines which
showed high selectivity over σ2R but contained an aniline-like
weak basic nitrogen, instead of the usual tertiary or secondary
alkylamino group. We report herein the structure−activity
relationship (SAR) studies that allowed the optimization of the
activity and physicochemical properties of the initial hit 8a
(Figure 1C).
b
Isolated yields: 71% (a) and 18% (b).
examples). In all cases cis/trans mixtures were obtained and
separated by column purification or by recrystallization. The
stereochemical assignment of the isolated diastereomers was
established by NMR with NOE studies: the cis adducts in
dihydropyran derivatives showed NOE effects between H5 and
H10b and H5 and H4a, while the trans adducts showed NOEs
between H4a and protons of the R2 group.
The synthesis of the piperidinyl-substituted pyranoquinoline
analogues 34a−36a, was accomplished from the Boc-protected
intermediate 33a (Scheme 2). Deprotection of 33a with TFA
provided 34a, and subsequent reductive amination gave the
alkylated derivatives 35a and 36a. Finally, the acetylated
pyranoquinoline 37a (Figure 2) was obtained from 17a with
Ac2O under microwave irradiation.
The enantiomeric resolution of the most active compounds
(Table 5) was undertaken by chiral HPLC. Several analytical
conditions were tried, and an n-heptane/ethanol (98:2) mixture
as the mobile phase with a Chiralpak AD-H column was found
to provide the best separation of the two enantiomers. These
conditions were used for enantiomeric excess (ee) determi-
CHEMISTRY
■
The synthesis of the new furano- and pyranoquinolines was
accomplished by the Povarov reaction,19 which provided a
“one-pot” ready access to the compounds. The multi-
component reaction of substituted anilines (4), aldehydes
(5), and dihydrofuran (6) or dihydropyran (7) mediated by the
Lewis catalyst Mg(ClO4)2 in acetonitrile provided the fused
quinolines 8−33 (Scheme 1). Other Lewis catalysts such as I2
20
21
or (TMS)Cl22 were tested, but Mg(ClO4)2 gave the best results
a
Scheme 1
a
Reagents and conditions: (a) Mg(ClO4)2 (5 mol %), MeCN, rt, 16 h.
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a
Scheme 2
a
Reagents and conditions: (a) DCM/TFA (5%) rt, 16 h; (b) (for 35a) MeCHO, NaBH3CN, MeOH, 90 °C, 24 h; (for 36a) BnCHO,
NaBH(OAc)3, DCE, microwave, 90 °C, 10 min.
which provided enantiomer 32c as the corresponding salt. After
final liberation, 32c was isolated with an ee > 99% and an
overall yield of 31%. The enantiomers of 32b (32e and 32f,
Figure 4) were not separated, and both of them were assumed
to be inactive in view of the inactivity of the racemic mixture.
RESULTS AND DISCUSSION
Figure 2. Structure of acetylated compound 37a.
■
The new series was identified from an HTS campaign through
an in vitro σ1R binding assay using [3H]-(+)-pentazocine23 as
nation and applied to a preparative method which allowed the
separation of the pairs 15c/d, 17c/d, and 32c/d to give the
individual enantiomers with good ee. As indicated below, only
one of the enantiomers (c) was active, and to elucidate the
absolute configuration of the eutomers, a single-crystal X-ray
diffraction (SCXRD) study of the most interesting compound,
32c, was undertaken. Since no suitable crystals were obtained
using the free base, a limited salt screening was carried out. The
hydrochloride provided crystals suitable for SCXRD analysis,
from which the absolute configuration was reliably determined
by anomalous dispersion effects (Flack −0.12(0.05)). As shown
in Figure 3, the absolute configuration 4aS,5R,10bS was
established for 32c, confirming the cis stereochemistry earlier
assigned.
the radioligand. Compound 8a, which was part of our internal
compound library and had not been previously described,
emerged as a hit with submicromolar affinity for σ1R and Ki
greater than 1000 nM for σ2R ([3H]di-o-tolylguanidine24 was
used as the radioligand for σ2R binding assays). This prompted
the selection of 8a for a hit to lead program, since we knew
from our previous work that it is quite challenging to find high
selectivity for the σ1 versus the σ2 receptor, while it is highly
desirable on the basis of the clear-cut distinctive pharmaco-
logical functions attributed to both receptors. Another
interesting feature of compound 8a was that it did not contain
a highly basic amino group, typically present in σ1R ligands. As
recently summarized by Wunsch et al.,25 several pharmaco-
̈
phores reported in the literature (Figure 1B depicts Glennon’s
σ1R pharmacophore) share an amine and at least two
hydrophobic regions at a certain distance from the basic
amino group, whose pKa is normally above 6.5. Compound 8a
contained two hydrophobic groups at an adequate distance, but
its calculated pKa of 4.6 (ACD), was well below the usual
values. The main drawback of compound 8a was its high
lipophilicity (cLogP of 7.0), which translated into a low kinetic
solubility (<0.4 μM) and anticipated promiscuity and other
ADMET (absorption, distribution, metabolism, excretion,
toxicity) problems.26 Thus, reduction of lipophilicity was one
of the main drivers in the optimization program.
Tables 2−5 contain the binding affinities of the most relevant
compounds in the series, and for the sake of comparison, the
results of reference compounds 1−3 are included (Table 2). All
the compounds showing a Ki below 1000 nM for σ1R were
tested against σ2R, showing in all cases binding affinities
superior to 1000 nM, which confirmed the good selectivity of
the series.
Figure 3. ORTEP plot (50%) showing the molecular structure of the
hydrochloride salt of 32c, which established the absolute configuration
as 4aS,5R,10bS.
As indicated in Table 2, there was a clear preference for the
cis orientation, since the trans isomer (8b) of the hit was
inactive, a result confirmed in the pair 9a/b as well as in other
examples of the series (not shown). Regarding phenoxy
substitution, the 3-chloro (9a) and the 4-chloro (8a)
derivatives were equipotent, while the 2-chloro (10a) and the
unsubstituted (12a) analogues were devoid of activity. The 3,4-
dichloro derivative 11a showed an improved potency,
The preparative chiral HPLC method was scaled up for the
separation of 4 g of racemic 32a to provide 32c with an ee >
99% in 35% yield. A diastereomeric salt resolution method was
also assayed, using several chiral resolving agents: [(1S)-endo]-
(+)-3-bromo-10-camphorsulfonic acid, (1S)-(+)-10-camphor-
sulfonic acid, and (S)-(+)-1,1′-binaphthalene-2,2′-diyl hydro-
gen phosphate. Enantiomerical enrichment was only achieved
by crystallization with the binaphthalene derivative in acetone,
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Figure 4. Minimized structures of the four accessible diastereoisomers of compound 32. The upper left in gray shows the 4aS,5R,10bS diastereomer
(32c) and its overlap with the σ1R pharmacophore described by Laggner,27 the upper right in pink the 4aR,5S,10bR diastereomer (32d), the lower
left in green the 4aS,5S,10bS diastereomer (32e), and the lower right in orange the 4aR,5R,10bR diastereomer (32f).
Table 2. cis/trans Diastereomers of Pyranoquinolines
Table 3. rac-cis-Furano- and Pyranoquinolines
a
compd
R1
n
cLogP
Ki(hσ1) (nM)
a
compd
R
cLogP
Ki(hσ1) (nM)
13a
14a
15a
16a
17a
18a
19a
20a
21a
22a
23a
24a
25a
26a
H
2
2
2
2
2
2
2
2
2
2
1
1
1
1
4.2
6.3
6.8
5.9
6.3
6.2
6.8
6.1
4.4
4.7
6.6
6.4
5.5
5.9
>1000
>1000
1
3.9
2.6
3.8
7.0
7.0
7.0
7.0
6.8
7.6
6.3
17.0 7.0
6.3 1.7
2.1 0.3
194 75
>1000
butyl
pentyl
butoxy
2
43
178 18
52
4
3
8a
8b
9a
9b
10a
11a
12a
4-Cl
isopentyloxy
(4-fluorobenzyl)oxy
cyclohexyl
3
4-Cl
3-Cl
3-Cl
2-Cl
3,4-di-Cl
H
>1000
377 81
>1000
>1000
phenyl
>1000
>1000
(N-propylamino)sulfonyl
benzamido
>1000
79 11
>1000
>1000
(4-chlorophenyl)oxy
pentyl
302 17
245 159
>1000
a
Binding affinity to the human σ1 (hσ1) receptor in transfected HEK-
293 membranes using [3H]-(+)-pentazocine as the radioligand. Each
value is the mean SD of two determinations.
butoxy
isopentyloxy
108 33
a
Binding affinity to the human σ1 (hσ1) receptor in transfected HEK-
293 membranes using [3H]-(+)-pentazocine as the radioligand. Each
value is the mean SD of two determinations.
confirming the positive effect of chlorine atoms in the 3 and 4
positions.
The R1 group was subsequently explored over the
tetrahydropyran (n = 2) or -furan (n = 1) fused frameworks,
while maintaining the hit’s cyclohexyl ring in R2 (Table 3). The
unsubstituted 13a and other short-chain-substituted tetrahy-
dropyrans of diverse electronic nature were inactive (F, OH,
SMe, not shown). Interestingly enough, going from a butyl
(14a) to a pentyl (15a) group resulted in a dramatic
improvement in potency, suggesting that a minimum chain
length of five atoms was essential for activity. Alkyl groups
shorter than butyl were also prepared, but as expected, they
were completely inactive (results not shown). This trend was
confirmed in the alkoxy derivatives where the isopentyloxy 17a
was more potent than the butoxy 16a, and this in turn exhibited
a marked improved potency versus shorter alkoxides, which
were completely devoid of activity. The (4-fluorobenzyl)oxy
18a was inactive, in contrast with the (4-chlorophenyl)oxy
present in the initial hit. Direct attachment of a ring (19a and
20a) was clearly detrimental, as well as introduction of polar
functions such as sulfonamido (21a) or benzamido (22a). The
most active substituents found in the tetrahydropyran series
were introduced in the fused tetrahydrofuran analogues 23a−
26a, and while the trends observed were essentially the same, it
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became clear that the tetrahydropyrans were always superior to
the tetrahydrofurans.
Table 5. Enantiomers of Selected Pyranoquinolines
Keeping the best substituents in R1, different groups were
introduced in R2 over the tetrahydropyran scaffold (Table 4).
Table 4. rac-cis-Pyranoquinolines
a
Ki(hσ1)
(nM)
compd
R1
pentyl
R2
cLogP
15a
cyclohexyl
cyclohexyl
cyclohexyl
6.8
6.8
6.8
6.3
6.3
6.3
3.9
3.9
3.9
43
19
3
5
b
15c
pentyl
pentyl
a
b
Ki (hσ1)
15d
>1000
compd
R1
pentyl
R2
cLogP
(nM)
17a
isopentyloxy cyclohexyl
isopentyloxy cyclohexyl
isopentyloxy cyclohexyl
52
14
3
b
27a
28a
29a
30a
31a
32a
34a
35a
36a
pentan-3-yl
6.8
4.4
4.2
4.8
5.7
3.9
3.9
4.8
6.1
410 104
17c
1
b
pentyl
butoxy
butoxy
tetrahydro-2H-pyran-4-yl
cyclopropyl
67
9
17d
>1000
>1000
>1000
124
32a
isopentyloxy tetrahydro-2H-pyran-4-yl
isopentyloxy tetrahydro-2H-pyran-4-yl
isopentyloxy tetrahydro-2H-pyran-4-yl
84 23
c
phenyl
32c
30
8
c
isopentyloxy cyclopentyl
5
32d
>1000
isopentyloxy tetrahydro-2H-pyran-4-yl
isopentyloxy piperidin-4-yl
84 23
536 68
317 20
a
Binding affinity to the human σ1 (hσ1) receptor in transfected HEK-
293 membranes using [3H]-(+)-pentazocine as the radioligand. Each
value is the mean
configuration inferred from 32. Absolute configuration determined
b
isopentyloxy 1-ethylpiperidin-4-yl
isopentyloxy 1-benzylpiperidin-4-yl
SD of two determinations. Absolute
c
15
2
a
by X-ray diffraction (see Figure 3).
Binding affinity to human σ1 (hσ1) receptor in transfected HEK-293
membranes using [3H](+)-pentazocine as radioligand. Each value is
the mean SD of two determinations.
site of the pharmacophore, confirming thereby its function as
the traditional amine site of σ1R ligands. This explains why,
even if weak, certain basicity is needed, as shown by the lack of
affinity of the acetylated derivative 37a. This model also
explains that, to fit with HYD4, a minimum five-atom chain
length is required in position R1. Regarding stereospecificity,
the minimum energy conformations of the remaining three
diastereomers were obtained as well, after a systematic
conformational search with minimization at each step. The
minima thus obtained were mapped into Laggner’s pharmaco-
phore, the result being that none of them fit into the model
without missing any feature. As shown in Figure 4, either one
or both of HYD1 and HYD2 are not covered by the inactive
diastereomers, highlighting the importance of these two
hydrophobic features for the σ1R−ligand interaction.
Some of the most active compounds (15c, 17c, and 36a)
showed up to a 1 unit reduction in lipophilicity versus 8a, but
still their kinetic solubility was below 1 μM. However,
compound 32c exhibited a much more reduced lipophilicity
(cLogP = 3.9) than that of 8a (cLogP = 7.0), which translated
into a solubility of 6.6 μM. The change of the 4-
chlorophenyloxy group to isopentyloxy reduced the cLogP by
almost 1 unit, but most relevant was the change of cyclohexyl to
pyranyl, which provided an additional 2.4 unit reduction. The
reduction of lipophilicity resulted in an improvement in ligand
efficiency (LE; 8a, 0.2; 32c, 0.3), lipophilic ligand efficiency
(LLE; 8a, −0.3; 32c, 3.6), and ligand-efficiency-dependent
lipophilicity (LELP; 8a, 35; 32c, 12.7) indices. However, the
topological polar surface area (tPSA) was maintained in the
same range for the two compounds (8a, 30.5 Å2; 32c, 39.7 Å2),
predicting a good blood−brain barrier (BBB) penetration.
Compound 32c was selected for further profiling. Addition-
ally to its high selectivity versus σ2R, it did not show significant
affinity (inhibition at 10 μM <50%) for another 163 molecular
targets (receptors, transporters, ion channels, and enzymes).28
The only exception was the human serotonin 5-HT2B receptor,
to which it showed a low binding affinity (Ki = 5.3 μM). This
result together with that observed in 1,14 which only hits the 5-
Ring opening (27a vs 15a), decreasing the ring size (29a vs 16a
and 31a vs 17a), and unsaturation to phenyl (30a vs 16a) were
detrimental for activity. On the contrary, polar atoms were
tolerated in position 4 of the cyclohexyl ring: the pyranyl group
(28a/32a) provided a good potency, while the unsubstituted
piperidine (34a) showed an intermediate activity, which was
improved on substitution with ethyl (35a) or benzyl (36a).
As mentioned before, in this series the pKa of the NH (the
experimental pKa of 32c is 3.8) is well below the usual values
(pKa above 6.5) found in σ1 receptor ligands. The negative
result of compound 37a (Figure 2) seems to indicate that some
basicity is needed, although it cannot be completely discarded
that the steric bulk of the acetamide group impairs activity.
The individual cis enantiomers of some of the most active
compounds were isolated by preparative chiral HPLC, and
activity was again shown to be clearly dependent on
stereochemistry, since only one enantiomer was active (Table
5). As indicated above, the absolute configuration 4aS,5R,10bS
was established for compound 32c on the basis of the X-ray
diffraction of its hydrochloride (Figure 3). This configuration
was also tentatively assigned to the eutomers 15c and 17c on
the basis of their activity.
The fact that only one of the four accessible diastereoisomers
was active indicated that the 3D distribution of the hydro-
phobic groups matched the spatial requirements of σ1R, thus
compensating for the lack of nitrogen basicity. The global
minimum conformation of compound 32c, which corresponds
with the experimental X-ray crystal structure, was modeled into
Laggner’s σ1R pharmacophore, reproduced on the basis of the
reported coordinates.27 As indicated in Figure 4, compound
32c matched quite well the pharmacophore: the cis-
tetrahydropyrano ring exquisitely maps the hydrophobic feature
HYD1, the tetrahydropyranyl group at R2 matches the second
one (HYD2), and substitution at R1 by the isopentyloxy group
covers the two remaining hydrophobic regions (HYD3 and
HYD4). The amino group matches the positive ionizable (PI)
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(HPLC grade) were used without further purification for all the
analytical tests. Analytical HPLC−MS was performed on a Waters
2795-MS ZQ system using reversed-phase XBridge C18 columns (4.6
× 50 mm, 2.5 μm), gradient 2−95% solvent B (solvent A, 10 mM
ammonium bicarbonate; solvent B, acetonitrile) over 5.5 min,
injection volume 10 μL, flow 2.0 mL/min. Photodiode array (PDA)
spectra were recorded at 220−310 nm using a Waters 2996 PDA
detector. Mass spectra were obtained over the range m/z 100−800 at a
sampling rate of 0.3 scan/s using a Waters ZQ instrument. Data were
integrated and reported using Water Masslynx software. All
compounds display purity higher than 95% as determined by this
method. Accurate mass measurements were carried out using an
Agilent 6540 ultra-high-definition (UHD) accurate-mass quadrupole
time-of-flight (QTOF) system and obtained by electron spray
ionization (ESI) in positive mode. Chiral analytical HPLC was
performed on Agilent 1100 series equipment using Daicel Chiralpak
AD-H columns (4.6 × 250 mm, 5 μm). UV spectra were recorded at
208, 210, and 250 nm using an Agilent 1100 diode array detector
(DAD). Chiral preparative HPLC was performed on semipreparative
Agilent 1100 series equipment using a Chiralpak AD-H column (20 ×
250 mm, 5 μm).
HT2B receptor in the same selectivity panel, suggests some
structural similarity between the σ1 and 5-HT2B receptor
binding sites.
In vivo evaluation of 32c was done using a mouse model of
neurogenic pain induced by intraplantar injection of
capsaicin,10 where immediate paw licking and delayed
mechanical hypersensitivity are indicators of ongoing pain
and pain sensitization, respectively. It inhibited mechanical
hypersensitivity by 63% after oral administration at 80 mg/kg.
It was also active in the formalin test, where it exerted a dose-
dependent analgesic effect on phase II of formalin-induced pain
(40% and 81% inhibition after intraperitoneal (ip) admin-
istration at 40 and 80 mg/kg, respectively). This antinoci-
ceptive activity was indicative of its antagonist nature, in
accordance with the reported analgesic effect of σ1R
antagonists, particularly on the formalin test.29
Compound 32c showed a good preliminary ADMET profile:
the inhibition of cytochrome P450 was assessed in recombinant
human cytochrome P450 isoforms (1A2, 2C9, 2C19, 2D6, and
3A4),30 and the IC50 values were higher than 5 μM, suggesting
a low potential of compound 32c for drug−drug interactions.
The intrinsic clearance found in liver microsomes31 (human,
5.0 μL/min/mg of protein; rat, 21.7 μL/min/mg of protein;
mouse, 15.1 μL/min/mg of protein) predicts a moderate to low
in vivo clearance. Additionally, compound 32c did not show in
vitro cytotoxic potential in the MTT (3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide) and neutral red uptake
assays when tested up to 100 μM in HepG2 cells32 and lacked
genotoxic potential in the SOS/umu and Ames bacterial
mutation assays.33 Finally, in the human ether-a-go-go-related
gene (hERG) potassium channel34 patch clamp assay, it
showed an IC50 > 10 μM, which predicts a low potential for
cardiac toxicity.
In summary, the synthesis and pharmacological activity of a
new series of hexahydro-2H-pyrano[3,2-c]quinolines as potent
and selective σ1R ligands are reported herein. This family,
which was identified in an HTS campaign, showed high
selectivity versus σ2R and did not contain the highly basic
amino group usually present in other σ1R ligands. The activity
was shown to reside in only one of the four accessible
diastereoisomers, which perfectly oriented the hydrophobic
groups to match with the receptor, as shown by the good
overlap of the eutomers with Laggner’s σ1R pharmacophore.
The initial hit (8a) potency as well as its physicochemical
properties were substantially improved in a hit to lead program
that led to the identification of compound 32c, which showed a
good selectivity and ADMET profile as well as antinociceptive
properties in several in vivo tests, reinforcing the notion that
σ1R antagonism could be a useful strategy for treating pain.
Further profiling of compound 32c and evolution of the series
will be reported in due curse.
Determination of Physicochemical Properties. The topolog-
ical polar surface area (tPSA) was calculated using ChemDraw Ultra
10.0.3. pKa was calculated using ACDLABS 12.0.2 and cLogP using
ChemDraw Ultra 10.0.3. Solubility was measured as kinetic solubility
from the compound solution at 1% DMSO in phosphate buffer at pH
7.4 by HPLC.35 Experimental pKa was determined by using a
pHmetric technique36 in a Sirius Analytical GlpKa instrument.
(4aS*,5R*,10bS*)-9-(3-Chlorophenoxy)-5-cyclohexyl-
3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinoline Hydro-
chloride (9a) and (4aS*,5S*,10bS*)-9-(3-Chlorophenoxy)-5-
cyclohexyl-3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]-
quinoline Hydrochloride (9b). To a solution of 4-(3-
chlorophenoxy)aniline (220 mg, 1 mmol) in dry acetonitrile (3 mL)
under an argon atmosphere were sequentially added cyclohexane-
carbaldehyde (121 μL, 1 mmol) and 3,4-dihydro-2H-pyran (90 μL, 1
mmol), followed by Mg(ClO4)2 (12.3 mg, 0.05 mmol). The reaction
mixture was stirred at room temperature for 18 h. The solvent was
removed under reduced pressure and the residue taken up in CH2Cl2
and passed through Dicalite. The solvent was removed under reduced
pressure and the crude crystallized from EtOAc to afford pure cis
diastereomer (4aS*,5R*,10bS*)-9-(3-chlorophenoxy)-5-cyclohexyl-
3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinoline (138 mg, 34%)
as a white solid. The solvent from the mother liquid was evaporated
and the remaining residue purified by CombiFlash chromatography
(SiO2, cyclohexane/EtOAc up to 10%) to give additional cis
diastereomer (144 mg, overall yield 71%) along with trans
diastereomer (4aS*,5S*,10bS*)-9-(3-chlorophenoxy)-5-cyclohexyl-
3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinoline as a yellow oil
(78 mg, 18%). To an ice-cooled solution of the cis diastereomer (50
mg, 0.13 mmol) in acetone (500 μL) was added dropwise a 2 N HCl
solution in Et2O (69 μL, 1.4 mmol). After 30 min of stirring at room
temperature, a solid precipitated. The solid was filtered off and dried in
vacuum to give 9a (42 mg, 76%) as a white solid: 1H NMR (400 MHz,
CDCl3) δ 8.12−7.93 (m, 1H), 7.26−7.22 (m, 1H), 7.21 (s, 1H), 7.10
(d, J = 7.8 Hz, 1H), 7.00 (s, 1H), 6.93 (d, J = 6.8 Hz, 1H), 6.88 (d, J =
8.3 Hz, 1H), 4.89 (d, J = 4.4 Hz, 1H), 3.62 (d, J = 10.9 Hz, 1H), 3.36−
3.18 (m, 2H), 2.69−2.46 (m, 2H), 2.30−2.11 (m, 1H), 1.95−1.75 (m,
4H), 1.75−1.45 (m, 3H), 1.42−1.12 (m, 4H), 1.12−0.95 (m, 1H).
The same procedure was repeated for the trans diastereomer to give
9b (52 mg, 79%) as a white solid: 1H NMR (400 MHz, CDCl3) δ 8.11
(d, J = 7.9 Hz, 1H), 7.25 (t, J = 8.1 Hz, 1H), 7.10 (d, J = 7.9 Hz, 1H),
7.06 (d, J = 2.0 Hz, 1H), 7.02 (s, 1H), 6.92 (dd, J = 8.8, 2.2 Hz, 1H),
6.88 (dd, J = 8.4, 1.7 Hz, 1H), 4.57 (d, J = 2.5 Hz, 1H), 3.84 (dt, J =
10.3, 3.9 Hz, 1H), 3.72−3.58 (m, 2H), 2.55−2.45 (m, 1H), 2.05−1.94
(m, 2H), 1.93−1.74 (m, 6H), 1.74−1.60 (m, 2H), 1.59−1.43 (m, 2H),
1.39−1.16 (m, 3H).
EXPERIMENTAL SECTION
■
Unless otherwise noted, all materials were obtained from commercial
suppliers and used without further purification. Microwave-assisted
reactions were conducted with an Explorer synthesizer from CEM.
Flash chromatography was performed with a forced flow of the
indicated solvent system on SDS silica gel Chromagel 60 ACC (230−
400 mesh) or on a CombiFlash Companion system with Redisep Rf
1
disposable columns. H spectra were recorded on an Agilent UNITY
300 MHz spectrometer (fitted with a 5 mm H/F/X ATB probe) or an
Agilent Mercury 400 MHz spectrometer (fitted with a 5 mm ID/PFG
probe) with 2 H lock in deuterated solvents. Chemical shifts (δ) are in
parts per million. Commercially available reagents and solvents
(4aR*,5S*,10bR*)-9-(Isopentyloxy)-5-(tetrahydro-2H-pyran-
4-yl)-3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinoline Hy-
drochloride (32a). To a solution of 4-(isopentyloxy)aniline (1.08 g,
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6.0 mmol) in dry acetonitrile (6 mL) were sequentially added
tetrahydro-2H-pyran-4-carbaldehyde (822 mg, 7.0 mmol), 3,4-
dihydro-2H-pyran (668 μL, 7.0 mmol), and Mg(ClO4)2 (74.2 mg,
0.3 mmol). The reaction mixture was stirred under an argon
atmosphere at room temperature for 18 h. The solvent was removed
under reduced pressure and the residue taken up in CH2Cl2 and
filtered through Dicalite. The solvent was removed under reduced
pressure and the crude crystallized from EtOAc to afford pure cis
diastereomer (4aR*,5S*,10bR*)-9-(isopentyloxy)-5-(tetrahydro-2H-
pyran-4-yl)-3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinoline as a
white solid (865 mg, 40%). To an ice-cooled solution of the previous
compound (50 mg, 0.14 mmol) in acetone (5 mL) was added
dropwise a 2 N HCl solution in Et2O (76 μL, 0.15 mmol). After 30
min of stirring at room temperature, the solid formed was filtered off
CDCl3) δ 9.80−9.58 (m, 1H), 9.58 − 9.30 (m, 1H), 6.97 (s, 1H),
6.82−6.58 (m, 2H), 4.97 (d, J = 5.36 Hz, 1H), 3.92 (t, J = 6.68 Hz,
2H), 3.81−3.66 (m, 1H), 3.66−3.51 (m, 2H), 3.39 (t, J = 11.65 Hz,
1H), 3.27−3.05 (m, 1H), 3.05−2.74 (m, 2H), 2.32−2.07 (m, 2H),
2.07−1.33 (m, 11H), 0.95 (d, J = 6.57 Hz, 6H).
(4aS*,5R*,10bS*)-5-(1-Ethylpiperidin-4-yl)-9-(isopentyloxy)-
3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinoline Hydro-
chloride (35a). To a solution of 34a (80 mg, 0.22 mmol) in
MeOH (2.5 mL) was added acetaldehyde (31 μL, 0.55 mmol)
followed by NaBH3CN (42 mg, 0.66 mmol). The reaction solution
was heated at 90 °C for 24 h. After the solution was cooled to room
temperature, the solvent was removed under reduced pressure. The
residue was taken up in EtOAc and washed with saturated aqueous
NaHCO3 solution. The organic phase was dried over MgSO4 and the
solvent removed under reduced pressure. The residue was purified by
CombiFlash chromatography (SiO2, cyclohexane/EtOAc) to afford
(4aS*,5R*,10bS*)-5-(1-ethylpiperidin-4-yl)-9-(isopentyloxy)-
3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinoline (21 mg, 26%).
Formation of the hydrochloride in the same conditions used for the
1
and dried in vacuum to give 32a as a white solid (41 mg, 74%): H
NMR (300 MHz, DMSO) δ 7.21−6.75 (m, 3H), 4.95 (d, J = 5.35 Hz,
1H), 4.05−3.81 (m, 4H), 3.56 (d, J = 10.79 Hz, 1H), 3.40−3.04 (m,
4H), 2.25 (d, J = 12.23 Hz, 1H), 2.07−1.84 (m, 2H), 1.85−1.38 (m,
6H), 1.38−1.07 (m, 4H), 0.92 (d, J = 6.65 Hz, 6H).
Separation of the Racemic Mixtures of 15a, 17a, and 32a by
Chiral HPLC. Analytical Setting. Analytical HPLC was performed
using a Chiralpak AD-H (Daicel) column (4.6 × 250 mm, 5 μm) with
n-heptane/ethanol (98:2, v/v) as the mobile phase at a 0.8 mL/min
flow rate (diode array detector; wavelength, 210 nm for 15a, 208 nm
for 17a, and 250 nm for 32a; retention times, 6.7 min for 15c, 9.4 min
for 15d, 13.6 min for 17c, 34.9 min for 17d, 46.2 min for 32d
(4aR,5S,10bR), and 66.7 min for 32c (4aS,5R,10bS). These conditions
were used for enantiomeric excess determination after separation, and
in all cases ee was higher than 99%.
Preparative Setting. Preparative HPLC was performed using a
Chiralpak AD-H (Daicel) column (20 × 250 mm, 5 μm) with n-
heptane/ethanol (98:2, v/v) as the mobile phase at a 10 mL/min flow
rate. In this way the pairs 15c/d, 17c/d, and 32c/d were separated at
the 100 mg scale. In the case of 32a the method was scaled up to
separate 4 g of the racemic mixture to provide 1.58 g of 32d and 1.46 g
of 32c.
(4aS,5R,10bS)-9-(Isopentyloxy)-5-(tetrahydro-2H-pyran-4-
yl)-3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinoline (32c)
by Diastereomeric Salt Formation. Compound 32a (415 mg,
1.15 mmol) was suspended in acetone (6 mL), and (S)-(+)-1,1′-
binaphthalene-2,2′-diyl hydrogen phosphate (0.20 g, 0.57 mmol) was
added. The mixture was heated to reflux and stirred overnight. The
suspension thus obtained was cooled to room temperature and
filtered. The solid was washed with cold acetone and dried in vacuum
(2−5 mmHg) at 45 °C for 4 h to give the (S)-(+)-1,1′-binaphthalene-
2,2′-diyl hydrogen phosphate salt of 32c (0.27 g, 33%). The previous
compound was dissolved in CH2Cl2 (17 mL), and 1 M aqueous
NaOH (17 mL) and water (17 mL) were added. The mixture was
stirred for 10 min at room temperature. The organic layer was
separated, and the aqueous layer was washed with CH2Cl2 (2 × 10
mL). The organic layers were joined and washed with 1 M aqueous
NaOH (20 mL), brine (20 mL), and water, followed by filtration
through cotton. The solution thus obtained was evaporated under
vacuum to give the free base of 32c as a white solid (127 mg, 93%,
>99% ee).
1
preparation of 32a provided 35a as a white solid (7.5 mg, 50%): H
NMR (300 MHz, CDCl3) δ 6.95 (d, J = 2.5 Hz, 1H), 6.68 (dd, J = 8.6,
2.6 Hz, 1H), 6.50 (d, J = 8.7 Hz, 1H), 5.00 (d, J = 5.4 Hz, 1H), 3.92 (t,
J = 6.7 Hz, 2H), 3.75−3.51 (m, 3H), 3.42 (t, J = 11.2 Hz, 1H), 3.13 (d,
J = 9.6 Hz, 1H), 3.05 (q, J = 7.2 Hz, 2H), 2.70−2.46 (m, 2H), 2.27−
2.02 (m, 4H), 2.01−1.89 (m, 1H), 1.89−1.59 (m, 5H), 1.49 (t, J = 7.4
Hz, 3H), 1.56−1.40 (m, 3H), 0.95 (d, J = 6.6 Hz, 6H).
(4aR*,5S*,10bR*)-5-(1-Benzylpiperidin-4-yl)-9-(isopenty-
loxy)-3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinoline Hy-
drochloride (36a). A microwave vial was charged, under an argon
atmosphere, with 39a (80 mg, 0.22 mmol), benzaldehyde (24 mg, 0.22
mmol), and NaBH(OAc)3 (95 mg, 0.44 mmol) followed by DCE (2.5
mL). The reaction mixture was heated using a microwave reactor at 90
°C for 10 min. After the mixture was cooled back to room
temperature, the reaction mixture was quenched with saturated
NaHCO3 solution and diluted with CH2Cl2. The phases were
separated, and the aqueous phase was extracted with CH2Cl2. The
combined organic phases were dried over MgSO4, and the solvent was
removed under reduced pressure. The residue was purified by
CombiFlash chromatography (SiO2, cyclohexane/EtOAc) to afford
(4aR*,5S*,10bR*)-5-(1-benzylpiperidin-4-yl)-9-(isopentyloxy)-
3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinoline (38 mg, 38%).
Formation of the hydrochloride in the same conditions used for the
1
preparation of 32a provided 36a as a white solid (20 mg, 54%): H
NMR (300 MHz, CDCl3) δ 11.21 (br s, 2H), 7.73−7.54 (m, 2H),
7.50−7.33 (m, 4H), 7.02 (s, 1H), 6.78 (d, J = 8.61 Hz, 1H), 5.02−4.86
(m, 1H), 4.56−4.36 (m, 1H), 4.37−4.16 (m, 1H), 3.94 (t, J = 6.65 Hz,
2H), 3.89−3.71 (m, 2H), 3.62 (d, J = 10.83 Hz, 1H), 3.50−3.24 (m,
3H), 3.24−3.06 (m, 1H), 3.06−2.52 (m, 3H), 2.50−2.23 (m, 2H),
2.11−1.31 (m, 8H), 0.95 (d, J = 6.56 Hz, 6H).
1-((4aS*,5R*,10bS*)-5-Cyclohexyl-9-(isopentyloxy)-3,4,4a,5-
tetrahydro-2H-pyrano[3,2-c]quinolin-6(10bH)-yl)ethanone
(37a). A mixture of 17a (100 mg, 0.28 mmol) and acetic anhydride
(480 μL, 5.08 mmol) was heated using a microwave reactor at 120 °C
for 20 min. After the mixture was cooled back to room temperature,
the reaction mixture was quenched with H2O and extracted with
EtOAc. The combined organic fractions were dried over Na2SO4, and
the solvent was removed under reduced pressure to give 37a (56 mg,
(4aR*,5S*,10bR*)-9-(Isopentyloxy)-5-(piperidin-4-yl)-
3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinoline Hydro-
chloride (34a). To a stirred solution of 33a, obtained by
multicomponent reaction (MCR) without further purification (400
mg, 0.87 mmol), in dry CH2Cl2 (5 mL), was added at 0 °C TFA (1
mL). The solution was allowed to reach room temperature and stirred
under an argon atmosphere for 18 h. The reaction was then
neutralized with NaOH solution and extracted with CH2Cl2. The
combined organic fractions were washed with saturated aqueous NaCl
solution, dried over Na2SO4, and concentrated under reduced
pressure. After filtration (4aR*,5S*,10bR*)-9-(isopentyloxy)-5-(piper-
idin-4-yl)-3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinoline was
obtained as a viscous yellow solid (270 mg, 86%). Formation of the
hydrochloride in the same conditions used for the preparation of 32a
1
51%) as an oil: H NMR (300 MHz, CDCl3) δ 7.13−6.92 (m, 2H),
6.80 (dd, J = 2.91, 8.77 Hz, 1H), 4.51 (d, J = 5.29 Hz, 1H), 4.12−3.90
(m, 3H), 3.71 (ddd, J = 3.26, 7.74, 11.00 Hz, 1H), 2.38 (p, J = 5.78 Hz,
1H), 2.07 (s, 3H), 1.95−1.44 (m, 14H), 1.31 − 0.79 (m, 5H), 0.97 (d,
J = 6.56 Hz, 6H).
Single-Crystal X-ray Structure Determination of 32c.
Crystallization and Sample Preparation. Crystals of the hydro-
chloride salt of 32c were obtained by slow evaporation of an ethanol
solution of the compound. The measured crystal was prepared under
inert conditions immersed in perfluoropolyether as a protecting oil for
manipulation.
Data Collection. Crystal structure determination for the hydro-
chloride salt of 32c was carried out using an Apex DUO Kappa four-
1
provided 34a as a white solid (57 mg, 68%): H NMR (300 MHz,
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axis goniometer equipped with an APPEX 2 4K charge-coupled device
(CCD) area detector, a Microfocus Source E025 IuS using Mo Kα
radiation, Quazar MX multilayer optics as the monochromator, and an
Oxford Cryosystems low-temperature device, Cryostream 700 plus (T
= −173 °C). Full-sphere data collection was used with ω and φ scans.
Programs used: data collection, APEX-2;37 data reduction, Bruker
Saint V/.60A;38 absorption correction, SADABS.39
and female mice for the capsaicin test. Animals had access to food and
water ad libitum and were kept in controlled laboratory conditions
with the temperature at 21 1 °C and a light−dark cycle of 12 h
(lights on at 7:00 a.m.). Experimental behavioral testing was carried
out in a soundproof and air-regulated experimental room and was
done blind with respect to treatment and surgical procedure.
Experimental procedures and animal husbandry were conducted
according to European guidelines regarding protection of animals used
for experimental and other scientific purposes (Council Directive of
Nov 24, 1986, 86/609/ECC) and received approval by the local
Ethical Committee.
Formalin Test. A diluted formalin solution (20 μL of a 2.5%
formalin solution, 0.92% formaldehyde) was injected into the
midplantar surface of the right hind paw of the mouse. Formalin-
induced nociceptive behavior was quantified as the time spent licking
or biting the injected paw during two different periods individually
recorded: the first period was recorded 0−5 min after the injection of
formalin and was considered indicative of phase I formalin-evoked
nociception; the second period was recorded 15−30 min after
formalin injection and was considered indicative of phase II formalin-
evoked nociception. The mice (n = 8−12 per group) received ip
administration of a 10 mL/kg volume of vehicle 0.5% hydroxypropyl
methyl cellulose (HPMC) (Sigma-Aldrich) or test compound 15 min
before intraplantar (ipl) formalin injection.
Capsaicin Test. Capsaicin (8-methyl-N-vanillyl-6-nonamide) was
purchased from Sigma-Aldrich and dissolved in 1% DMSO (vehicle)
in physiological saline. Mice were habituated for 2 h in individual test
compartments placed on an elevated mesh-bottomed platform with a
0.5 cm2 grid to provide access to the ventral side of the paws and were
then given an ipl capsaicin injection (1 μg in 20 μL of 1% DMSO) into
the midplantar surface of the right hind paw. Fifteen minutes after ipl
capsaicin injection, mechanical stimulation was applied onto the
plantar surface of the hind paw using an automated testing device
(dynamic plantar aesthesiometer, Ugo Basile, Italy). The device lifts a
straight monofilament (0.5 mm in diameter) exerting a constant
upward pressure of 0.5 g (4.90 mN) onto the plantar surface, and
when the animal withdraws its hind paw, the mechanical stimulus
automatically stops and the latency time is recorded. Latency was
defined as the time from the onset of exposure to the filament to the
cessation of the pressure when the sensor detected the paw
withdrawal. Paw withdrawal latencies were measured in triplicate for
each animal at 30 s intervals. A cutoff latency of 60 s was used in each
trial. The mice (n = 8−12 per group) received vehicle 0.5% HPMC
(Sigma-Aldrich) or test compound through the ip route in a volume of
10 mL/kg 15 min before capsaicin injection, and withdrawal latencies
to mechanical stimulation were determined 15 min after capsaicin
injection (30 min after the treatments). The effect of the treatments
on mechanical hypersensitivity induced by capsaicin was calculated
with the following equation: reduction of mechanical hypersensitivity
(%) = ((LTD − LTV)/(CT − LTV)) × 100, where LTD and LTV
are the latency times in drug- and vehicle-treated animals, respectively,
and CT is the cutoff time (60 s).
Structure Solution and Refinement. Crystal structure solution was
achieved using direct methods as implemented in SHELXTL40 and
visualized using the program XP. Missing atoms were subsequently
located from difference Fourier synthesis and added to the atom list.
Least-squares refinement on F2 using all measured intensities was
carried out using the program SHELXTL. All non-hydrogen atoms
were refined, including anisotropic displacement parameters. The
chlorine anion corresponding to one cationic molecule is distributed in
two half-positions located on 2-fold rotation axes and shared with the
neighboring cationic molecules.
Crystal data at 100 K: C22H34Cl1N1O3, 395.95 g mol−1
monoclinic, C2, a = 24.7260(15) Å, b = 8.9820(5) Å, c = 9.8850(6)
Å, α = 90°, β = 104.524(6)°, γ = 90°, V = 2125.2(2) Å3, Z = 4, ρcalcd
,
=
1.238 Mg/m3, R1 = 0.0495 (0.0640), wR2 = 0.1186 (0.1273), Flack41
−0.12(5), for 7265 reflections with I > 2σ(I) (for 8603 reflections [Rint
= 0.0677] with a total of 33765 measured reflections), goodness-of-fit
on F2 1.053, largest difference peak (hole) 0.842 (−0.527) e Å−3.
Molecular Modeling Studies. Global mimimun energy con-
formations were found using the generate conformations protocol
implemented in Discovery Studio 3.542 with a systematic search
method, setting the sp3−sp3, sp3−sp2, and sp2−sp2 torsion increments
to 60, 60, and 180, respectively, and minimizing the conformation
candidates in the gas phase using the MMFF force field. As the starting
conformation the crystal structure of 32c was used, confirming it as a
global minima, whereas the diastereomers were obtained by
stereochemical inversion of the whole neutralized compound in the
case of the enantiomer (32d) or by inversion of the individual
stereogenic centers in the case of the trans disastereomers. Laggner’s
σ1 pharmacophore27 was reproduced as well in Discovery Studio 3.0
from the details on tolerance, coordinates, and distances given by the
authors, and the ligand−pharmacophore mappings were done by the
Catalyst program embedded in this system with a rigid fitting method.
Human σ1 Receptor Radioligand Assay.23 The binding
properties of the test compounds to human σ1R were studied in
transfected HEK-293 membranes using [3H]-(+)-pentazocine (Perkin-
Elmer, NET-1056) as the radioligand. The assay was carried out with 7
μg of membrane suspension, [3H]-(+)-pentazocine (5 nM) in either
the absence or the presence of either buffer or 10 μM haloperidol for
total and nonspecific binding, respectively. The binding buffer
contained Tris−HCl (50 mM, at pH 8). The plates were incubated
at 37 °C for 120 min. After the incubation period, the reaction mixture
was transferred to MultiScreen HTS, FC plates (Millipore) and
filtered, and the plates were washed (three times) with ice-cold Tris−
HCl (10 mM, pH 7.4). The filters were dried and counted at
approximately 40% efficiency in a MicroBeta scintillation counter
(Perkin-Elmer) using EcoScint liquid scintillation cocktail.
ASSOCIATED CONTENT
* Supporting Information
Guinea Pig σ2 Receptor Radioligand Assay. The binding
properties of test compounds to guinea pig σ2R were studied in guinea
pig brain membranes as described24 with some modifications. [3H]Di-
o-tolylguanidine (DTG) (Perkin-Elmer, code NET-986) was used as
the radioligand. The assay was carried out with 200 μg of membrane
suspension, [3H]DTG (10 nM) in either the absence or the presence
of either buffer or 10 μM haloperidol for total and nonspecific binding,
respectively. The binding buffer contained Tris−HCl (50 mM, pH 8),
and the σ1 receptor was blocked with (+)-SKF10047 at 400 nM. The
plates were incubated at 25 °C for 120 min. After the incubation
period, the reaction mixture was transferred to MultiScreen HTS, FC
plates (Millipore) and filtered, and the plates were washed three times
with ice-cold 50 mM Tris−HCl (pH 7.4). The filters were dried and
counted at approximately 40% efficiency in a MicroBeta scintillation
counter (Perkin-Elmer) using EcoScint liquid scintillation cocktail.
In Vivo Tests. Animals. CD1 mice (Charles River, France) from 6
to 8 weeks old were used. Male mice were used for the formalin test
■
S
Analytical and characterization data for all the compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org. CCDC 922502 contains the supplementary
crystallographic data for this paper. These data can be obtained
free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html
(or from the Cambridge Crystallographic Data Centre, 12
Union Rd., Cambridge CB21EZ, U.K. (fax (+44) 1223-336-
033, e-mail deposit@ccdc.cam.ac.uk).
AUTHOR INFORMATION
Corresponding Author
*Phone: 0034934466000. E-mail: jldiaz@esteve.es (J.L.D.);
calmansa@esteve.es (C.A.).
■
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Article
(13) Díaz, J. L.; Zamanillo, D.; Corbera, J.; Baeyens, J. M.;
Maldonado, R.; Pericas, M. A.; Vela, J. M.; Torrens, A. Selective
̀
sigma-1 receptor antagonists: emerging target for the treatment of
neuropathic pain. Cent. Nerv. Syst. Agents Med. Chem. 2009, 9, 172−
183.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(14) Díaz, J. L.; Cuberes, R.; Berrocal, J.; Contijoch, M.; Christmann,
We thank Albert Dordal, Xavier Monroy, Daniel Zamanillo,
a
U.; Fernan
́
dez, A.; Port, A.; Holenz, J.; Buschmann, H.; Laggner, C.;
́
Ines
́
Alvarez, Eva Ayet, M Jose
Balada, Ana Paz Martin, and Maite Ruiz for their expert
contribution to in vitro, ADME/TOX, and in vivo studies,
́
Pretel, Sandra Yeste, Ariadna
Serafini, M. T.; Burgueno, J.; Zamanillo, D.; Merlos, M.; Vela, J. M.;
̃
́
́
Almansa, C. Synthesis and biological evaluation of the 1-arylpyrazole
class of σ1 receptor antagonists: identification of 4-{2-[5-methyl-1-
(naphthalen-2-yl)-1H-pyrazol-3-yloxy]ethyl}morpholine (S1RA, E-
52862). J. Med. Chem. 2012, 55, 8211−8224.
Miguel Angel Blaz
Ortega for their excellent technical assistance, and Carlos Per
Oscar Ferre, and Eduardo Villarroel for their contribution to
́
quez, Joan Andreu Morato,
́
and Edmundo
́
ez,
́
(15) Glennon, R. A. Pharmacophore identification for sigma-1 (σ1)
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potent and selective σ ligands: evaluations of their agonist and
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(17) (a) Xu, J.; Zeng, C.; Chu, W.; Pan, F.; Rothfuss, J. M.; Zhang, F.;
Tu, Z.; Zhou, D.; Zeng, D.; Vangveravong, S.; Johnston, F.; Spitzer,
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