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)

Article abstract of DOI:10.1021/jm3007323

The synthesis and pharmacological activity of a new series of 1-arylpyrazoles as potent σ₁ receptor (σ₁R) antagonists are reported. The new compounds were evaluated in vitro in human σ₁R and guinea pig σ₂ receptor (σ₂R) binding assays. The nature of the pyrazole substituents was crucial for activity, and a basic amine was shown to be necessary, in accordance with known receptor pharmacophores. A wide variety of amines and spacer lengths between the amino and pyrazole groups were tolerated, but only the ethylenoxy spacer and small cyclic amines provided compounds with sufficient selectivity for σ₁R vs σ₂R. The most selective compounds were further profiled, and compound 28, 4-{2-[5-methyl-1- (naphthalen-2-yl)-1H-pyrazol-3-yloxy]ethyl}morpholine (S1RA, E-52862), which showed high activity in the mouse capsaicin model of neurogenic pain, emerged as the most interesting candidate. In addition, compound 28 exerted dose-dependent antinociceptive effects in several neuropathic pain models. This, together with its good physicochemical, safety, and ADME properties, led compound 28 to be selected as clinical candidate.

Full text of DOI:10.1021/jm3007323

Featured Article
pubs.acs.org/jmc
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)
Jose Luis Díaz,*,† Rosa Cuberes,† Joana Berrocal,† Montserrat Contijoch,† Ute Christmann,†
́
Ariadna Fernandez,† Adriana Port,† Jorg Holenz,† Helmut Buschmann,† Christian Laggner,‡
́
̈
Maria Teresa Serafini,† Javier Burgueno,† Daniel Zamanillo,† Manuel Merlos,† Jose Miguel Vela,†
́
̃
and Carmen Almansa†
†Drug Discovery and Preclinical Development, Esteve, Av. Mare de Deu
́
de Montserrat, 221, 08041, Barcelona, Spain
‡Department of Drug and Natural Product Synthesis, Faculty of Life Sciences, University of Vienna, Althanstrasse 14, A-1090 Vienna,
Austria
S
* Supporting Information
ABSTRACT: The synthesis and pharmacological activity of a new series of
1-arylpyrazoles as potent σ1 receptor (σ1R) antagonists are reported. The
new compounds were evaluated in vitro in human σ1R and guinea pig σ2
receptor (σ2R) binding assays. The nature of the pyrazole substituents was
crucial for activity, and a basic amine was shown to be necessary, in
accordance with known receptor pharmacophores. A wide variety of amines
and spacer lengths between the amino and pyrazole groups were tolerated,
but only the ethylenoxy spacer and small cyclic amines provided compounds
with sufficient selectivity for σ1R vs σ2R. The most selective compounds were
further profiled, and compound 28, 4-{2-[5-methyl-1-(naphthalen-2-yl)-1H-
pyrazol-3-yloxy]ethyl}morpholine (S1RA, E-52862), which showed high activity in the mouse capsaicin model of neurogenic
pain, emerged as the most interesting candidate. In addition, compound 28 exerted dose-dependent antinociceptive effects in
several neuropathic pain models. This, together with its good physicochemical, safety, and ADME properties, led compound 28
to be selected as clinical candidate.
(Figure 1). On the other hand, σ2R has recently been shown to be
involved in cellular proliferation and cell death. Indeed, agonists
acting on this receptor show proapoptotic properties, thus
suggesting a potential role in cancer treatment.10 Because the two
INTRODUCTION
■
The σ receptors were discovered 35 years ago and later on
classified as σ1 and σ2, based on their size, tissue distribution, and
drug selectivity patterns.1 σ1R has been cloned and shown to
encode a protein of 223 amino acids anchored to the
endoplasmic reticulum and plasma membranes.2 This protein
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, since the early 1990s σ1R has been known to
modulate opioid analgesia,6 and the relationship between the μ-
opioid and σ1R has been recently shown to involve direct physical
interaction,7 which explains why σ1R antagonists enhance the
antinociceptive effect of opioids.
Research has been done on the therapeutic potential of σ1R
ligands. While several compounds have undergone clinical
studies as antidepressants, drug abuse treatments, antipsychotics,
and agents for improvement in learning and memory, no
selective σ1R ligands have so far been marketed.8 Several
compounds have been extensively used as pharmacological tools:
(+)-pentazocine (1) and PRE-084 (2) as σ1R agonists.6
Conversely, BD-1063 (3) and haloperidol (4), which also
antagonize dopamine receptors, are typical σ1R antagonists9
Figure 1. Representative σ1R agonists (1, 2) and antagonists (3, 4).
Received: May 25, 2012
Published: July 12, 2012
© 2012 American Chemical Society
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a
Scheme 1
a
Reagents and conditions: (a) Ac2O, toluene, rt; (b) PCl3, 50 °C; (c) t-BuOK, t-BuOH, rt; (d) K2CO3, NaI, DMF, 70 °C or NaH, DMF, 60 °C; (e)
K2CO3, NaI, DMF, rt; (f) NHR4R5, K2CO3, NaI, DMF, toluene, reflux.
Table 1. 3-Aminoethoxy-1-(3,4-dichlorophenyl)pyrazoles
a
Binding affinity to human σ1R in transfected HEK-293 membranes using [3H](+)-pentazocine as radioligand. Each value is the mean SD of two
b
determinations. Binding affinity to σ2R in guinea pig brain membranes using [3H]di-o-tolylguanidine as radioligand. Each value is the mean SD of
two determinations. Methods used for the synthesis of compounds according to Scheme 1.
c
receptors are clearly differentiated pharmacologically, selectivity
between the two and versus other targets is desired.
behaviors and molecular mechanisms underlying central
sensitization (i.e., phosphorylation of the NR1 subunit of the
NMDA receptor in the spinal cord) were inhibited by spinal
administration of a σ1R antagonist.15 These results, together with
the key role played by σ1R in pain-related central sensitization
phenomena,13,16 support the involvement of σ1R in the control
of nociception.
On the basis of these findings, an internal program aimed at
identifying σ1R antagonists for the treatment of pain was initiated
and led to several active series.17 In continuation of our efforts in
Our group became interested in the potential role of σ1R in
pain control, and a σ1R knockout mouse was developed. σ1R
knockout mice show attenuated pain responses in the formalin
test,11 and no mechanical hypersensitivity following capsaicin
sensitization12 or sciatic nerve injury was observed.13 Haloper-
idol was also shown to inhibit formalin-induced pain and
capsaicin-induced hypersensitivity.14 Other authors identified
the spinal cord as the main site of action, as both nociceptive
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a
Scheme 2
a
Reagents and conditions: (a) 2-bromoethanol, K2CO3, NaI, DMF, 60 °C, 18 h; (b) oxalyl chloride, CH2Cl2, 0 °C; (c) triethylamine, CH2Cl2, rt, 18
h; (d) K2CO3, NaI, DMF, rt, 18 h; (e) NHR4R5, K2CO3, NaI, DMF, 100 °C, 18 h; (f) (i) 2-(chloromethyl)oxirane, K2CO3, NaI, DMF, 100 °C, 4 h;
(ii) NHR4R5, K2CO3, NaI, DMF, 100 °C, 8 h; (g) (i) P2S5, xylene, reflux, 19 h; (ii) NaBH4, EtOH, reflux, 16 h; (h) K2CO3, NaI, DMF, 95 °C, 18 h.
this field, we report herein the structure−activity relationship
(SAR) studies of a series of 1-arylpyrazoles that led to the
discovery of 4-{2-[5-methyl-1-(naphthalen-2-yl)-1H-pyrazol-3-
yloxy]ethyl}morpholine (S1RA, E-52862, 28). Compound 28 is
a highly selective σ1R antagonist18 and has successfully
completed phase I safety and pharmacokinetic evaluation in
humans.19 It is currently undergoing phase II clinical trials for the
treatment of pain and is the first-in-class compound to be
developed for this indication.
yields.21 Method B afforded 8 in one step and around 50% yield
on treatment of 5 with ethyl 3-functionalized propinoates 9.22
The high regioselectivity of both methods was shown on account
of 5-hydroxypyrazole not being isolated in any case. Final
compounds 13−34 were obtained in good yields by treatment of
8 with commercially available chloroalkylamines 10 under basic
conditions (method C). Alternatively, a two-step procedure
(method D) was used. In this case, reaction of 8 with 1-bromo-ω-
chloroalkanes 11 occurred with good regioselectivity to provide
chloro derivatives 12, which upon reaction with the correspond-
ing amines under mild basic conditions afforded the final
compounds in moderate yields. In the case of amide 33f (Table
4), NaH was required for nucleophilic substitution. General
methods A−D are described in the experimental part, and the
specific procedures used for the preparation of each of the
derivatives 13−34 are indicated in Tables 1−4.
CHEMISTRY
■
The synthesis of the new compounds was accomplished using
different routes, as outlined in Schemes 1−4.20 As depicted in
Scheme 1, aminoalkoxypyrazoles 13−34 were obtained using
four alternative procedures. Depending on the substitution
pattern of the final pyrazole, the key intermediate 3-
hydroxypyrazole 8 was prepared through a one-step (method
B) or a two-step (method A) procedure from aryl- or
alkylhydrazines 5. Method A involved acetylation with acetic
anhydride to give hydrazides 6 in good yields and subsequent
cyclization with ethyl 3-oxoalkanoates 7 to give 8 in moderate
Elongated analogues 36, 40, and 41a,b were obtained starting
from 1-(3,4-dichlorophenyl)-1H-pyrazol-3-ol 8b (Scheme 2).
Alkylation of 8b with 1-bromo-2-(bromoethoxy)ethane under
basic conditions provided exclusively the monosubstituted 35,
which on treatment with piperidine afforded compound 36 in
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Table 2. 1-(3,4-Dichlorophenyl)pyrazoles
a
Binding affinity to human σ1R in transfected HEK-293 membranes using [3H](+)-pentazocine as radioligand. Each value is the mean SD of two
b
determinations. Binding affinity to σ2R in guinea pig brain membranes using [3H]di-o-tolylguanidine as radioligand. Each value is the mean SD of
two determinations. Methods used for the synthesis of compounds according to Scheme 1.
c
a
Scheme 3
a
Reagents and conditions: (a) NaOEt, EtOH, acrylonitrile, reflux, 24 h; (b) MnO2, CH2Cl2, rt; (c) (i) triethylamine, THF, 2-chloroacetyl chloride,
rt, 18 h; (ii) amine, K2CO3, NaI, DMF, 100 °C, 18 h; (d) 2 M BH3·Me2S in THF, THF, reflux, 4 h.
70% overall yield. Sulfonate ester 40 was obtained by alkylation
of 8b with 2-bromoethanol and subsequent sulfonation of the
resulting 37 with sulfonyl chloride 39, which was prepared by
treatment of 38 with oxalyl chloride. Hydroxyl derivatives 41
were prepared by treatment of 8b with 2-(chloromethyl)oxirane
under basic conditions, followed by ring-opening with morpho-
line or piperidine, which took place over the less substituted
oxirane position to afford 41a and 41b, respectively (Table 2).
Thiopyrazole 43 was prepared by treatment of 8c with
phosphorus pentasulfide to provide 42, which was subsequently
alkylated with chloroethylmorpholine using conditions similar to
those used for hydroxy derivatives.
The route depicted in Scheme 3 was used for the preparation
of aminopyrazoles 47−49. 3,4-Dichlorophenylhydrazine, 5a, was
reacted with acrylonitrile23 to provide the red pyrazoline 44,
which upon oxidation with manganese dioxide gave amino-
pyrazole 45. Treatment of 45 with 2-chloroacetyl chloride and
subsequent substitution with morpholine or piperidine afforded
amides 46 and 47 in excellent yields. Final reduction with borane
dimethylsulfide provided amines 48 and 49 in moderate yields.
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For the sake of comparison, the preparation of the 5-oxy
regioisomer of the most interesting compound, 28, was
undertaken (Scheme 4). N-Boc protection of hydroxypyrazole
are reported in Tables 1−5. For the sake of comparison, the
results of reference compounds 3 and 4 are included in all tables.
The SAR study was initiated with the introduction, at position
1 of the pyrazole ring, of a 3,4-dichlorophenyl group which was
present in known σ1R ligands, such as 3,30 and using a
morpholino group also common in σ1R ligands, such as 2. The
ethylene linker was selected because the resulting seven bond
length between the aromatic and the amine group could fit
pharmacophoric requirements. The first compound prepared,
13a, showed good σ1R binding affinity and selectivity toward the
σ2R, which encouraged further exploration of the different
positions. The 3,4-dichlorophenyl group at position 1 and the
ethylene linker were maintained to explore the nature of the
amino group (Table 1). The substitution of morpholino by
carbonated cyclic analogues provided good σ1R potency, but
selectivity vs the σ2R receptor decreased in the following order:
morpholinyl (13a), pyrrolidinyl (13b), piperidinyl (13c),
homopiperidinyl (13d), and 4-methylpiperidinyl (13e). Bulkier
substituents, such as the pyridoimidazolyl group of 13f or the
phenyl group of 13i, which probably occupy the second
hydrophobic region of the receptor, were tolerated but led to
decreased selectivity. The piperazine derivatives substituted with
smaller groups, 13g,h, were potent and selective, thus suggesting
that small polar groups are preferred in terms of selectivity. Open
chain amines, such as diethylamino 13j and cyclohexylamino
13k, showed good potency but low selectivity, and the
introduction of an imidazole group resulted in significantly
reduced potency for both receptors (13l), unsurprisingly so
given the low basicity of the imidazole nitrogen.
a
Scheme 4
a
Reagents and conditions: (a) (i) (Boc)2O, CH2Cl2, triethylamine, rt,
18 h; (ii) 4-(2-chloroethyl)morpholine hydrochloride, K2CO3, KI,
DMF, 95 °C; (iii) EtOH, 3 N HCl; (iv) NaOH; (b) 2-
naphthaleneboronic acid, Cu(OAc)2, pyridine, 4 Å molecular sieves,
CH2Cl2, air, 16 h, rt.
50, followed by treatment with 4-(2-chloroethyl)morpholine and
deprotection, provided compound 51. Arylation of 51 with 2-
naphthylboronic acid under Chan−Lam conditions24 provided a
2:1 mixture of the 5- and 3-oxy regioisomers, which were
separated by chromatography to give 52 in 30% yield along with
15% yield of 28.
RESULTS AND DISCUSSION
The design of the new series was performed taking into account
■
the known pharmacophoric features of σ1R. As recently
summarized by Wunsch et al.,25 several pharmacophores
̈
reported in the literature share a basic amino group and at
least two hydrophobic regions at a certain distance from the basic
amino group. According to the model by Glennon et al.,26 the
distances are defined as 2.5−3.9 and 6−10 Å (Figure 2 A), while
Some 5-unsubstituted analogues were prepared that were
shown to have similar σ1R binding affinity but diminished
selectivity compared to their methylated counterparts (see 14a−
e vs 13a−e, respectively). Conversely, the 4-acetylpiperazinyl
derivative 14f showed excellent selectivity, again suggesting that
this more polar group was not tolerated by the σ2R binding site.
Position 5 was further explored by introducing bulkier groups,
such as i-Pr (15) and phenyl (16), which resulted in great loss of
potency.
The effect of increasing the distance between the pyrazole ring
and the basic nitrogen atom was then explored. As shown in
Table 2, propylene derivatives 17a−c maintained σ1R potency,
but affinity for σ2R increased vs their ethylene counterparts
(13a,b,h, respectively). In this case, the 5-unsubstituted
derivatives 18a−c were quite similar to their methylated
analogues in terms of potency and selectivity. Further elongation
of the spacer provided derivatives 19 and 20, which showed
similar or increased σ2R affinity vs their propylene counterparts.
The introduction of an additional aromatic group to reach the
second hydrophobic region provided potent compounds 19d
and 20e, which again were equipotent for σ2R. Selectivity was not
improved by morpholino replacement with thiomorpholino
(20b) or by methylation of the morpholine ring (20c) or by
introduction of oxygen atoms in the alkyl chains (20f). As shown
with the ethylene spacer, the reduced basicity of the nitrogen
atoms was clearly detrimental for activity (19g,h). A five-atom
spacer with an intercalated oxygen atom (36) maintained
potency for both receptors, while the six-atom sulfonate ester
spacer, 40, was not tolerated. The introduction of a hydroxyl
group in the linker provided a decrease in the potency of both
receptors (41a,b vs 18a,c), thus suggesting that polar groups are
not preferred in this region.
Figure 2. Pharmacophoric σ1R model by Glennon26 (A) and general
structure of the new 1-arylpyrazoles I (B).
the model by Laggner et al.27 suggests two additional
hydrophobic groups. Compounds of general structure I (Figure
2 B) were designed in which the pyrazole ring was foreseen as a
good scaffold to be decorated with different groups in order to
comply with said pharmacophoric requirements as well as to
explore other possibilities.
The new compounds were first evaluated in vitro by
determining their binding affinities for σ1 and σ2 receptors
using [3H](+)-pentazocine28 and [3H]di-o-tolylguanidine,29
respectively, as radioligands. The results of these primary assays
The morpholinoethoxy group emerged as one of the best
moieties in terms of selectivity and was thus selected for further
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Table 3. Morpholinoethyloxypyrazoles
a
b
c
compd
R1
R2
R3
Ki σ1 (h, nM)
Ki σ2 (gp, nM)
M
3
6.3 1.7
2.1 0.3
9.4 1.8
741 134
>1000
318.4 62.3
22.0 1.4
351 400
>1000
4
21
22
23
24
25
26
27
28
29
30
31
32
3,4-dichlorophenyl
3,4-dichlorophenyl
phenyl
Me
Me
Me
Me
Me
H
Me
A, D
A, C
A, C
A, C
A, C
B, C
A, C
A, C
B, C
A, C
B, C
B, C
COMe
H
>1000
4-methoxyphenyl
3-chlorophenyl
4-chlorophenyl
2,4-dichlorophenyl
2-naphthyl
H
>1000
>1000
H
180 28
17.8 1.5
44.0 11.0
>1000
H
357 357
>1000
Me
Me
H
H
d
H
17.0 7.0
>1000
2-naphthyl
H
33.3 4.0
>1000
2-naphthyl
Me
H
Me
H
139
9
>1000
tert-butyl
>1000
>1000
cyclohexyl
H
H
23.1 3.9
>1000
a
Binding affinity to human σ1R in transfected HEK-293 membranes using [3H](+)-pentazocine as radioligand. Each value is the mean SD of two
b
determinations. Binding affinity to σ2R in guinea pig brain membranes using [3H]di-o-tolylguanidine as radioligand. Each value is the mean SD of
two determinations. Methods used for the synthesis of compounds according to Scheme 1. See ref 18.
c
d
Table 4. 1-Naphthalen-2-ylpyrazoles
a
b
c
compd
X
R1
Ki σ1 (h, nM)
Ki σ2 (gp, nM)
M
3
6.3 1.7
2.1 0.3
10.0 0.7
9.9 0.4
47.0 1.4
33.8 4.8
83.4 3.7
>1000
318.4 62.3
22.0 1.4
84.7 1.1
395 96
589 65
>1000
4
33a
33b
33c
33d
33e
33f
34a
34b
34c
(CH2)2
(CH2)2
(CH2)2
(CH2)2
(CH2)2
(CH2)2
(CH2)4
(CH2)4
(CH2)4
piperidin-1-yl
pyrrolidin-1-yl
diethylamino
A, C
A, C
A, C
A, D
A, D
other
A, D
A, D
A, D
(2S,6R)-2,6-dimethylmorpholin-4-yl
4-acetylpiperazin-1-yl
>1000
d
3-oxomorpholin-4-yl
>1000
morpholin-4-yl
99.1 19.5
65.0 21.2
296 15
428 122
226 26
324 69
(2S,6R)-2,6-dimethylmorpholin-4-yl
4-acetylpiperazin-1-yl
a
Binding affinity to human σ1R in transfected HEK-293 membranes using [3H](+)-pentazocine as radioligand. Each value is the mean SD of two
b
determinations. Binding affinity to σ2R in guinea pig brain membranes using [3H]di-o-tolylguanidine as radioligand. Each value is the mean SD of
two determinations. Methods used for the synthesis of compounds according to Scheme 1. See experimental part for the synthesis of 33f.
c
d
exploration of the SAR around I (Table 3). Substitution at
position 4 of the pyrazole ring provided dimethyl derivative 21,
which maintained σ1R potency but lost selectivity as compared to
13a, and 4-acetyl derivative 22, which in turn lost potency for
both receptors. The nature of the group at position 1 was key for
activity. Indeed, phenyl (23) and methoxyphenyl (24) were
devoid of activity, while 3-chlorophenyl (25) led to substantially
decreased potency. In contrast, the 4-chlorophenyl (26), 2,4-
dichlorophenyl (27), and naphthyl (28, 29) derivatives showed
good affinity for σ1R and selectivity against σ2R. In the naphthyl
series, methylation at position 4 was again shown to be
detrimental for activity (30). Replacing the aromatic group at
position 1 with tert-butyl (31) provided a complete loss of
potency, while the cyclohexyl group (32) maintained potency
and selectivity, thus suggesting the need for a ring in this position.
Naphthyl 29 and cyclohexyl 32 were the only 5-unsusbtituted
derivatives that showed comparatively good selectivities for σ1R
vs their methylated counterparts.
After identification of the 2-naphthyl group as an interesting
substituent at position 1, both in terms of potency and selectivity,
several analogues were prepared where the methyl group at
position 5 was also fixed. As described in Table 4, the 1-
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pyrrolidinyl (33b), 2,6-dimethyl-4-morpholinyl (33d), and 4-
acetylpiperazinyl (33e) derivatives were rather selective, but this
good selectivity was lost on elongation to the butylene spacer
(34a−c). Several unsubstituted analogues at position 5 were also
prepared in the naphthyl series but failed to show any substantial
improvement vs their methylated counterparts (results not
shown).
increased selectivity vs unsubstituted derivatives. However, the
spacer length and the nature of the amine were most crucial. As
indicated in Figure 3A, where the general structure of
Finally, the replacement of the oxygen atom adjacent to the
pyrazole ring by NH or S (Table 5) showed that the thio (43)
Table 5. Amino and Thiopyrazoles
Figure 3. (A) General structure of compounds with IC50 < 50 nM for
the σ1R and IC50 > 1000 nM for the σ2R (Table 6). (B) Structure and
physicochemical properties (obtained as described in the Experimental
Section) of compound 28.
a
b
compd
R1
X
Y
Z
Ki σ1 (h, nM) Ki σ2 (gp, nM)
3
6.3 1.7
2.1 0.3
14.2 4.4
80.8 30.2
39.4 6.4
9.5 4.7
318.4 62.3
22.0 1.4
>1000
4
43
47
48
49
Me
H
S
H2
O
O
NH
NH
NH
CH2
O
>1000
compounds with σ1R IC50 < 50 nM and σ2R IC50 > 1000 nM
is shown, only the ethyleneoxy spacer and cyclic amines with
small polar groups (morpholine and piperazines) fulfilled these
criteria (see Figure 3A). σ2R thus requires more hydrophobicity
in this region, and binding affinity is favored by spacer elongation,
as suggested by the increased potency of the compounds shown
in Table 2.
H
H2
H2
562 60
90.5 42.8
H
CH2
a
Binding affinity to human σ1R in transfected HEK-293 membranes
using [3H](+)-pentazocine as radioligand. Each value is the mean
b
SD of two determinations. Binding affinity to σ2R in guinea pig brain
membranes using [3H]di-o-tolylguanidine as radioligand. Each value is
the mean SD of two determinations.
The compounds that fulfilled the above-mentioned selectivity
criteria were further profiled (Table 6). The in vitro metabolic
stability in human liver microsomes31 was evaluated and a cutoff
value of intrinsic clearance (Clint) < 7 (μL/min)/mg protein was
established to meet the desired profile of once-daily oral
administration. Compounds were also tested in the human
ether-a-go-go-related gene (hERG)32 patch clamp assay, a well-
known predictor of cardiac toxicity. In this case, a cutoff value of
and amino derivatives (48, 49) bound both σ1R and σ2R in a way
similar to their oxygenated analogues 13a, 14a, and 14c,
respectively. The amide 47 showed better selectivity but
somewhat diminished potency.
The SAR summarized in Tables 1−5 shows that the nature of
the pyrazole substituents is crucial for the potency of both σ1R
and σ2R. Only certain substituted aromatics and cyclohexyl
groups were tolerated at position 1. Substitution at position 4 was
clearly detrimental, and only small substituents (H and methyl)
were tolerated at position 5. There was also a clear need for a
basic amine, as substantiated by the poor potency of derivatives
13l and 19g,h, which was further confirmed by the inactivity of
compound 33f (Table 4), the amide analogue of 28. Compound
52, a regioisomer of 28, was also devoid of activity (Ki of σ1, σ2,
>1000 nM), thus suggesting that requirements for achieving σ1R
activity are not as straightforward as they seem to be, based on
the relatively simple pharmacophore. Regarding the amine
nature, a wide variety of amines was tolerated and, interestingly,
potency was not improved by adding aromatic groups (13f, 13i,
19d, 20e) likely to reach the second hydrophobic pocket
described in known σ1R pharmacophores. Finally, spacer lengths
of three to six atoms between the amine and the pyrazole groups
were tolerated, although their substitution with polar groups
such as hydroxyl was detrimental for activity.
Table 6. Additional Data for Compounds Complying with
Ki(σ1) < 50 nM and Ki(σ2) > 1000 nM
a
b
c
hERG IC50
HLM Clint
capsaicin (% inhibtion at
compd
(μM)
((μL/min)/mg protein)
16 mg/kg)
13a
13g
13h
14f
27
3.5
12
3
25
3
8
7.4
27
d
d
4.1
26
7
NT
73
4.5
6
>10
>10
25
3
NT
43
e
28
7
f
62
7
f
29
>10
>10
7.4
6
55
7
32
0
20
6
d
d
33d
43
25
37
NT
NT
>10
a
b
Whole-cell patch clamp hERG blockade. Intrinsic clearance in
Much more exquisite were the requirements for achieving
good selectivity for σ1R vs σ2R. As mentioned above, good
selectivity is desirable based on the clear-cut distinctive
pharmacological functions attributed to both receptors. Gen-
erally speaking, the presence of a methyl group at position 5
c
human liver microsomes as a measure of metabolic stability.
%
reduction of mechanical hypersensitivity after ip administration of test
d
compounds at 16 mg/kg in the mouse capsaicin test. NT: not tested.
e
f
Capsaicin and hERG data were previously reported in ref 18. Dose:
32 mg/kg.
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Figure 4. Dose−response effect of 28 and pregabalin on mechanical allodynia (von Frey test) and thermal (heat) hyperalgesia (plantar test) in the
partial sciatic nerve ligation model of neuropathic pain. Treatments were administered by the ip route 30 (von Frey test) and 45 (plantar test) min before
behavioral testing. Data were obtained from 10−12 mice per group and expressed as the mean SEM percentage of antiallodynic or antihyperalgesic
effect: (∗∗) p < 0.01, (∗∗∗) p < 0.001 vs vehicle (dose 0) (Bonferroni’s multiple comparison test after one-way ANOVA).
IC50 > 10 μM was established. Finally, the compounds were
evaluated in vivo in a mouse model of neurogenic pain induced
by intraplantar injection of capsaicin,12 where immediate paw
licking and delayed mechanical hypersensitivity are indicators of
ongoing pain and pain sensitization, respectively. The inhibition
of mechanical hypersensitivity was evaluated after ip admin-
istration of test compounds at 16 mg/kg.
pain. Its pharmacological activity was shown to correlate closely
with drug levels in the brain and with actual occupancy of brain
σ1Rs.18 Additionally, 28 potentiated morphine analgesia but not
its rewarding effects34 and showed moderate to high activity in a
battery of different pain models, including carrageenan- and
CFA-induced inflammatory pain and TNBS-induced chronic
visceral pain models.35
As shown in Table 6, half of the 10 most selective compounds
showed sufficient metabolic stability. Regarding hERG, five
compounds showed IC50 inhibition higher than 10 μM and,
interestingly, all contained a morpholinoethoxy group. Com-
pounds displaying a dimethylmorpholino or a piperazino group,
irrespective of the substituent type, were below the cutoff values.
Regarding in vivo analgesia, several amines showed good
potencies and 2-naphthyl proved to be the best group at position
1, given that 28 was superior to the 3,4-dichlorophenyl (13a) and
cyclohexyl (32) derivatives. Interestingly, the two best
compounds, 28 and 29, only differed in the nature of the
substituent at position 5 (Me and H, respectively). Compound
28 was somewhat superior to 29 in all tests and was thus selected
for further evaluation.
Compound 28 was evaluated in a representative model of
neuropathic pain, the partial sciatic nerve ligation model in
mice,36 and was shown to dose-dependently inhibit both
mechanical allodynia and thermal hypersensitivity, with ED50
of 23.4 0.9 and 18.8 1.2 mg/kg, respectively. Interestingly,
28 showed increased potency compared to pregabalin, the gold
standard for neuropathic pain treatment in humans (Figure 4). In
order to discard that the observed efficacy could be due to
interference with motor coordination, compound 28 was tested
in the rotarod test (Figure 5), and no effect at analgesic doses was
found. In contrast, analgesic doses of pregabalin induced marked
motor impairment.
The functional activity of compound 28 was evaluated using
phenytoin,33 a low potency allosteric modulator of σ1R that shifts
known σ1R agonists to significantly higher affinities (Ki ratios
without phenytoin vs with phenytoin of >1), while σ1R
antagonists show small or no shift to lower affinity values (Ki
ratios without phenytoin vs with phenytoin of ≤1). Compound
28 was shown to be a σ1R antagonist18 on account of a small shift
to lower affinity being observed when incubated in the presence
of phenytoin (Ki(without phenytoin)/Ki(with phenytoin) =
0.8). Additionally, 28 was confirmed to be a highly selective
compound: in addition to its low affinity for the σ2R (Ki > 1000
nM for guinea pig and 9300 nM for rat σ2R), it failed to show
significant affinity (inhibition % at 1 μM of <50%) for another
170 molecular targets (receptors, transporters, ion channels, and
enzymes).18 The only exception to this was the human serotonin
5-HT2B receptor, to which it showed moderate binding affinity
(Ki = 328 nM) but very weak antagonistic activity (IC50 = 4700
nM).
Figure 5. Dose−response effect of 28 and pregabalin on motor
coordination (rotarod test). Data were obtained from 7−10 mice per
group and expressed as the mean SEM latency (seconds) to fall-down
from the rod: (∗∗) p < 0.01, (∗∗∗) p < 0.001 vs. vehicle (Bonferroni’s
multiple comparison test after one-way ANOVA).
Further in vivo evaluation of 28 was done by completing its
profile in the mouse capsaicin and formalin tests. It exerted a clear
dose-dependent analgesic effect on capsaicin-induced mechan-
ical hypersensitivity and on both phases of formalin-induced
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higher than 95% as determined by this method. Accurate mass
measurements were carried out using an Agilent 6540 UHD accurate-
mass QTOF system and obtained by electron spray ionization (ESI) in
positive mode. Elemental analyses were carried out in Institut
As shown in Figure 3, compound 28 complied with Lipinski’s
rules and was not highly basic, this being a desirable parameter as
recently pointed out by Wager et al.37 These authors have
developed the central nervous system multiparameter optimiza-
tion (CNS MPO) algorithm as a holistic approach for the
prediction of good ADME and safety attributes in the case of
CNS-directed compounds. An MPO value of 5.0 was calculated
for 28, which is considered to be in the high desirability range.
Additionally, it also showed good permeability in Caco-2 cells38
(370 nm/s) and an efflux ratio of 0.9, thus suggesting that it is not
a substrate of efflux transporters such as glycoprotein P. All these
results, together with the compound’s acceptable solubility and
high metabolic stability in liver microsomes, suggest good oral
bioavailability in humans. Additionally, its pharmacokinetic
profile was characterized in the mouse, the species used in the
efficacy models. After oral administration of 10 mg/kg,
compound 28 achieved a peak concentration of 622 ng/mL at
0.5 h postdosing and an AUC (area under the plasma
concentration−time curve) of 958 ng·h/mL.
Finally, its potential for drug−drug interactions based on the
inhibition of cytochrome P450 was evaluated in recombinant
human cytochrome P450 (rhCYP) isoforms (1A2, 2B6, 2C8,
2C9, 2C19, 2D6, 2E1, and 3A4) and further studied in human
liver microsomes.39 IC50 values were above 10 μM in all cases,
thus suggesting a low potential of compound 28 for these
interactions.
In view of its interesting pharmacological, physicochemical,
and ADME profile, compound 28 was selected as a preclinical
candidate. Then, upon successful completion of the preclinical
regulatory toxicological and pharmacodynamic package, it was
selected as a clinical candidate. The ongoing clinical trials will
hopefully help establish the usefulness of σ1R antagonism as a
new approach for the treatment of pain in humans, a field with
strong need for new therapies.
In summary, the synthesis and pharmacological activity of a
new series of 1-arylpyrazoles as potent and selective σ1R ligands
are reported here. Several compounds showed high in vitro
potency as well as good antinociceptive properties, thus
suggesting that σ1R antagonization may be a useful strategy for
treating pain. The most promising compound in the preclinical
tests, 28, has recently completed single and multiple dose phase I
clinical studies. Upon treatment of over 250 male and female
human subjects, compound 28 has shown good safety and
tolerability, as well as a pharmacokinetic profile compatible with a
good absorption and once-daily oral administration.19 Proof of
concept phase II studies for the treatment of neuropathic pain of
different etiologies and potentiation of opioid analgesia are
currently ongoing, and results will be reported in due course.
́
d’Investigacions Quimiques i Ambientals de Barcelona (CSIC), and
results are within 0.4% of the theoretical values. Commercially
available reagents and solvents (HPLC grade) were used without further
purification for all the analytical tests. Flash chromatography was
performed with a forced flow of the indicated solvent system on SDS
silica gel Chromagel 60 ACC (230−400 mesh).
Determination of Physicochemical Properties. Polar surface
area (PSA) was calculated using ChemDraw Ultra 10.0.3. The log D,
log P, and pKa were determined by using a pH-metric technique40 in a
T3 Sirius Analytical instrument. Solubility was measured as thermody-
namic solubility from solid compound in phosphate buffer at pH 7.4 by
HPLC.
General Procedures for the Synthesis of 2-(1H-Pyrazol-3-
yloxy)alkylamine Derivatives (13−34). Method A. 5-Methyl-1-
(naphthalen-2-yl)-1H-pyrazol-3-ol (8a, R1 = 2-naphthyl, R2 = Me,
R3 = H). To a suspension of naphthalen-2-ylhydrazine hydrochloride
(25 g, 0.128 mol) in H2O (250 mL) was added K2CO3 (25 g, 0.18 mol)
portionwise. After 15 min at room temperature, EtOAc (250 mL) was
added and the organic layer was extracted, washed with H2O, dried over
Na2SO4, and concentrated to dryness. The residue was diluted in
toluene (300 mL) and EtOAc (200 mL), and Ac2O (13.1 g, 0.13 mol)
was added dropwise. The solution was stirred at room temperature for 1
h, after which petroleum ether (200 mL) was added. The solution was
cooled to 7 °C, and the precipitate thus formed was filtered and washed
with petroleum ether to give N′-(naphthalen-2-yl)acetohydrazide, 6a, as
a white solid (20 g, 79%). 1H NMR (DMSO-d6) δ 9.7 (d, J = 2.6 Hz, 1
H), 7.95 (d, J = 2.6 Hz, 1 H), 7.7−7.6 (m, 3 H), 7.3 (m, 1 H), 7.15 (m, 1
H), 7.05 (dd, J = 2.3, 8.9 Hz, 1 H), 6.9 (d, J = 2.2 Hz, 1 H), 1.95 (s, 3 H).
A mixture of 6a (20 g, 0.1 mol), ethyl 3-oxobutanoate (7, R2 = Me, R3
= H; 13.1 g, 0.1 mol), and PCl3 (13.85 g, 0.1 mol) was heated at 50 °C
for 2 h. After this time, ice was added and the solution stirred for 1 h. The
suspension was poured into EtOAc and the precipitate filtered. The
organic layer of the filtrate was washed, dried over Na2SO4, and
concentrated to dryness. The residue was suspended in EtOH, filtered,
and washed with cool EtOH to obtain an additional amount of 5-methyl-
1-(naphthalen-2-yl)-1H-pyrazol-3-ol (6.47 g, 29%). 1H NMR (DMSO-
d6) δ 7.95 (m, 4 H), 7.65 (dd, J = 2.3, 8.7 Hz, 1 H), 7.5 (m, 2 H), 5.65 (s,
1 H), 2.35 (s, 3 H).
Method B. 1-(3,4-Dichlorophenyl)-1H-pyrazol-3-ol (8b, R1 =
3,4-dichlorophenyl, R2 = R3 = H). A solution of 1-(3,4-
dichlorophenyl)hydrazine (7.95 g, 44.9 mmol) in t-BuOH (65 mL)
was warmed with stirring at 30 °C, and ethyl propiolate (9, R2 = H)
(5.05 g, 51.5 mmol) was added dropwise. The resulting solution was
cooled to 0 °C, and t-BuOK (11.45 g, 93.7 mmol) was added
portionwise. The mixture was left to reach room temperature and stirred
for 2 days. Ice was added, t-BuOH evaporated under vacuum, and the
aqueous solution extracted with CH2Cl2. The remaining aqueous
solution was cooled with ice, neutralized with AcOH and the resulting
precipitate filtered off to afford 1-(3,4-dichlorophenyl)-1H-pyrazol-3-ol
(8b; 5.3 g, 50%). 1H NMR (CDCl3) δ 7.65 (d, J = 2.6 Hz, 1 H), 7.6 (d, J
= 2.5 Hz, 1 H), 7.5 (d, J = 8.6 Hz, 1 H), 7.35 (dd, J = 8.6 and 2.5 Hz, 1 H),
5.95 (d, J = 2.6 Hz, 1 H).
EXPERIMENTAL SECTION
■
Method C. 4-{2-[5-Methyl-1-(naphthalen-2-yl)-1H-pyrazol-3-
yloxy]ethyl}morpholine Hydrochloride (28). To a suspension of
NaH (60% dispersion in mineral oil, 1.07 g, 26.8 mmol) in dry DMF (75
mL) at 0 °C was added a solution of 8a (5.0 g, 22.3 mmol) in DMF (45
mL) dropwise. The reaction mixture was stirred at room temperature
under a nitrogen atmosphere for 45 min, and then a solution of 4-(2-
chloroethyl)morpholine (10, n = 1, NR4R5 = morpholine; 3.34 g, 22.3
mmol) in dry DMF (5 mL) was added. The reaction mixture was heated
to 60 °C for 4 h, quenched with some drops of H2O and the solvent
removed under vacuum. The crude residue thus obtained was
partitioned between diethyl ether and 2 N HCl solution. The acidic
aqueous phase was washed with diethyl ether, basified by addition of
10% NaOH solution, and extracted with diethyl ether. The combined
organic phases were dried over Na2SO4 and filtered, and the solvent was
Melting points were determined in open capillary tubes on a Buchi B-
540 melting point apparatus and are uncorrected. H spectra were
̈
1
recorded on a Varian UNITY 300 MHz (spectrometer fitted with a 5
mm H/F/X ATB probe) or a Varian 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. Analytical HPLC−MS was
performed on a Waters 2795-MS ZQ system using reverse phase
XBridge C18 columns (4.6 mm × 50 mm, 2.5 μm), gradient 2−95% B
(A = 10 mM ammonium bicarbonate, B = acetonitrile) over 5.5 min,
injection volume of 10 μL, and flow rate of 2.0 mL/min. 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 scans per second using Waters ZQ. Data were integrated and
reported using Water Masslynx software. All compounds display purity
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eliminated to dryness to afford 4-{2-[5-methyl-1-(naphthalen-2-yl)-
pyrazol-3-yloxy]ethyl}morpholine (6.39 g, 85%) as an oil. The oil was
dissolved in HCl-saturated EtOH, and the solvent was evaporated to
dryness. The crude residue thus obtained was crystallized from i-PrOH
to give the monohydrochloride as a white solid (5.24 g, 63%): mp 197−
mmol) in DMF (5 mL) was stirred at 100 °C under a nitrogen
atmosphere for 18 h. The solvent was evaporated in vacuo, and H2O and
diethyl ether were added. The aqueous phase was extracted with diethyl
ether, and the combined organic phases were washed with H2O, dried
over Na2SO4, filtered, and evaporated to dryness in vacuo. The crude
dark oil thus obtained was purified by column chromatography using
mixtures of petroleum ether/EtOAc/MeOH of increasing polarity to
give 1-{2-[2-(1-(3,4-dichlorophenyl)-1H-pyrazol-3-yloxy)ethoxy]-
ethyl}piperidine as an oil (0.12 g, 69%): 1H NMR (300 MHz,
CDCl3) δ 7.74 (d, J = 2.3 Hz, 1 H), 7.68 (d, J = 2.7 Hz, 1 H), 7.46 (d, J =
8.7 Hz, 1 H), 7.41 (dd, J = 8.8, 2.4 Hz, 1 H), 5.94 (d, J = 2.7 Hz, 1 H),
4.47−4.36 (m, 2 H), 3.91−3.78 (m, 4 H), 3.00−2.44 (m, 6 H), 1.89−
1.60 (m, 4 H), 1.59−1.40 (m, 2 H).
2-[1-(3,4-Dichlorophenyl)-1H-pyrazol-3-yloxy]ethanol (37). A
mixture of 8b (0.24 g, 1.04 mmol), 2-bromoethanol (0.27 g, 2.08 mmol),
K2CO3 (0.43 g, 3.12 mmol), and NaI (0.16 g, 1.04 mmol) in DMF (5
mL) was stirred at 60 °C under a nitrogen atmosphere for 18 h. The
solvent was evaporated to dryness and the residue partitioned between
H2O and diethyl ether. The combined organic phases were washed with
H2O and dried over Na2SO4, filtered, and evaporated in vacuo. The
residue was stirred with petroleum ether at 45 °C and decanted 3 times.
The solid thus obtained was dried to give 37 (0.26 g, 91%) which was
used without further purification in the next step: 1H NMR (CDCl3) δ
7.7 (d, J = 2.3 Hz, 1 H), 7.65 (d, J = 2.7 Hz, 1 H), 7.45 (d, J = 8.7 Hz, 1
H), 7.4 (dd, J = 2.3 and 8.7 Hz, 1 H), 5.95 (d, J = 2.7 Hz, 1 H), 4.4 (m, 2
H), 4.0 (m, 2 H).
2-[1-(3,4-Dichlorophenyl)-1H-pyrazol-3-yloxy]ethyl 2-Mor-
pholinoethanesulfonate (40). To a solution of 37 (0.15 g, 0.53
mmol) and triethylamine (0.22 mL, 1.58 mmol) in CH2Cl2 (9 mL) was
added a solution of 39 (prepared from 0.69 mmol of 38 and 0.14 mL,
1.57 mmol of oxalyl chloride in CH2Cl2 at 0 °C), and the final mixture
was stirred at room temperature for 18 h. Water was added, and the
organic phase was separated. The aqueous phase was extracted with
more CH2Cl2, and the combined organic phases were washed with a
saturated aqueous NaHCO3 solution and dried over Na2SO4. The
solvent was evaporated to give a residue, which was purified by column
chromatography using petroleum ether/EtOAc, 8:2, as eluent. The solid
thus obtained was crystallized from acetone−petroleum ether to give 40
as a white solid (0.094 g, 40%): mp 120−122 °C; 1H NMR (DMSO-d6)
δ 8.49 (d, J = 2.7 Hz, 1 H), 8.03 (d, J = 2.3 Hz, 1 H), 7.76 (dd, J = 8.9, 2.3
Hz, 1 H), 7.72 (d, J = 8.9 Hz, 1 H), 6.15 (d, J = 2.7 Hz, 1 H), 4.62−4.52
(m, 2 H), 4.52−4.43 (m, 2 H), 3.59 (t, J = 7.3 Hz, 2 H), 3.53 (t, J = 4.7
Hz, 4 H), 2.69 (t, J = 7.3 Hz, 2 H), 2.39 (t, J = 4.6 Hz, 4 H).
1-[1-(3,4-Dichlorophenyl)-1H-pyrazol-3-yloxy]-3-morpholi-
nopropan-2-ol (41a). A mixture of 8b (0.20 g, 0.87 mmol), 2-
(chloromethyl)oxirane (0.10 g, 1.05 mmol), K2CO3 (0.36 g, 2.62
mmol), and NaI (0.13 g, 0.87 mmol) in DMF (6 mL) was stirred at 100
°C for 4 h. The solvent was evaporated to dryness and the residue
partitioned between H2O and CH2Cl2. The combined organic phases
were dried over Na2SO4, filtered, and evaporated in vacuo to yield 1-
(3,4-dichlorophenyl)-3-(oxiran-2-ylmethoxy)-1H-pyrazole as a dark oil
(0.24 g, 95%), which was used without further purification in next step.
1H NMR (CDCl3) δ 7.75 (d, J = 2.4 Hz, 1 H), 7.7 (d, J = 2.6 Hz, 1 H),
1
199 °C; H NMR (DMSO-d6) δ 10.95 (sb, 1H), 8.07−7.96 (m, 4H),
7.69 (dd, J = 8.8, 2.2 Hz, 1H), 7.63−7.52 (m, 2H), 5.92 (s, 1H), 4.57 (t, J
= 4.9 Hz, 2H), 4.05−3.90 (m, 2H), 3.86−3.72 (m, 2H), 3.62−3.53 (m,
2H), 3.53−3.43 (m, 2H), 3.27−3.11 (m, 2H), 2.40 (s, 3H). Anal.
(C20H24ClN3O2) C, H, N.
Method D. 3-(4-Chlorobutoxy)-1-(3,4-dichlorophenyl)-4,5-
dimethyl-1H-pyrazole (12a, R1 = 3,4-Dichlorophenyl, R2 = R3 =
Me, n = 3). . A mixture of 1-(3,4-dichlorophenyl)-4,5-dimethyl-1H-
pyrazol-3-ol (3 g, 11.7 mmol), 1-bromo-4-chlorobutane (11, n = 3; 6.06
g, 35 mmol), K2CO3 (4.83 g, 35.0 mmol), and a catalytic amount of NaI
in dry DMF (25 mL) was stirred at room temperature for 18 h. The
solvent was evaporated in vacuo, and the crude residue thus obtained
was partitioned between H2O and CH2Cl2. The organic extracts were
washed with H2O, dried over Na2SO4, and concentrated in vacuo to give
12a as an oil (3.22 g, 80%): 1H NMR (DMSO-d6) δ 7.75 (d, J = 2.5 Hz, 1
H), 7.7 (d, J = 8.8 Hz, 1 H), 7.45 (dd, J = 2.5 and 8.8 Hz, 1 H), 4.2 (m, 2
H), 3.7 (m, 2 H), 2.25 (s, 3 H), 1.8 (m, 7 H).
1-(3,4-Dichlorophenyl)-4,5-dimethyl-3-[4-(pyrrolidin-1-yl)-
butoxy]-1H-pyrazole (21). A mixture of 12a (0.80 g, 2.3 mmol),
pyrrolidine (0.19 g, 2.6 mmol), K2CO3 (0.95 g, 6.9 mmol), and a
catalytic amount of NaI in dry DMF (20 mL) was stirred at 90 °C, under
a nitrogen atmosphere, for 18 h. The solvent was evaporated in vacuo
and H2O/diethyl ether added to the residue. The organic extracts were
washed with H2O, dried over Na2SO4, and concentrated in vacuo to give
0.77 g of a crude product, which was purified by column
chromatography (eluent, petroleum ether/EtOAc, 8/2) to afford 21
as an oil (0.43 g, 48%): 1H NMR (DMSO-d6) δ 10.85 (sb, 1 H), 7.77 (d,
J = 2.4 Hz, 1 H), 7.74 (d, J = 8.8 Hz, 1 H), 7.51 (dd, J = 8.7, 2.5 Hz, 1 H),
4.59 (t, J = 4.8 Hz, 2 H), 4.04−3.91 (m, 2 H), 3.78 (t, J = 12.1 Hz, 2 H),
3.65−3.53 (m, 2 H), 3.54−3.42 (m, 2 H), 3.29−3.08 (m, 2 H), 2.28 (s, 3
H), 1.88 (s, 3 H).
4-{2-[5-Methyl-1-(naphthalen-2-yl)-1H-pyrazol-3-yloxy]-
ethyl}morpholin-3-one (33f). To a stirred suspension of NaH (0.065
g, 60% dispersion in mineral oil, 1.63 mmol) in DMF (3 mL), cooled to
0−5 °C, was added a solution of morpholin-3-one (0.091 g, 0.91 mmol)
in DMF (3 mL) dropwise. The mixture was stirred at room temperature
for 3 h. After this time, a solution of 3-(2-chloroethoxy)-5-methyl-1-
(naphthalen-2-yl)-1H-pyrazole (12, R1 = 2-naphthyl, R2 = Me, R3 = H, n
= 1; 0.20 g, 0.7 mmol) in DMF (4 mL) was added and the mixture was
heated to 50 °C for 14 h. The reaction mixture was cooled, quenched
with water (2 mL), and evaporated to dryness. The residue was
partitioned between CH2Cl2 and H2O. The organic layer was washed
with water, dried over Na2SO4, filtered, and evaporated to give a crude
residue, which was purified by column chromatography using EtOAc as
eluent to afford 33f as a white solid (0.19 g, 78%): 1H NMR (CDCl3) δ
7.97−7.79 (m, 4 H), 7.59 (dd, J = 8.8, 2.1 Hz, 1 H), 7.57−7.45 (m, 2 H),
5.71 (s, 1 H), 4.43 (t, J = 5.1 Hz, 2 H), 4.19 (s, 2 H), 3.87 (dd, J = 6.0, 4.2
Hz, 2 H), 3.82 (t, J = 5.1 Hz, 2 H), 3.60 (dd, J = 6.0, 4.2 Hz, 2 H), 2.37 (s,
3 H).
3-[2-(2-Bromoethoxy)ethoxy]-1-(3,4-dichlorophenyl)-1H-
pyrazole (35). A mixture of 8b (0.23 g, 1 mmol), 1-bromo-2-(2-
bromoethoxy)ethane (0.51 g, 2 mmol), K2CO3 (0.42 g, 3 mmol), and
NaI (0.15 g, 1 mmol) in DMF (12 mL) was stirred at room temperature
under a nitrogen atmosphere for 18 h. The inorganic solid was filtered
off, and the solvent was evaporated to dryness. The remaining residue
was partitioned between H2O and diethyl ether. The combined organic
phases were washed with H2O and dried over Na2SO4, filtered, and
evaporated in vacuo. The residue was stirred with petroleum ether at 45
°C and filtered to yield, after drying, 35 which was used in the next step
without further purification (466 mg): 1H NMR (CDCl3) δ 7.75 (d, J =
2.2 Hz, 1 H), 7.7 (d, J = 2.7 Hz, 1 H), 7.45 (m, 2 H), 5.95 (d, J = 2.7 Hz, 1
H), 4.4 (t, J = 4.4 Hz, 2 H), 4.0−3.8 (m, 4 H), 3.5 (t, J = 6.4 Hz, 2 H).
1-{2-[2-(1-(3,4-Dichlorophenyl)-1H-pyrazol-3-yloxy)ethoxy]-
ethyl}piperidine (36). A mixture of 35 (0.17 g, 0.45 mmol), piperidine
(0.062 g, 0.72 mmol), K2CO3 (0.25 g, 1.8 mmol), and NaI (0.067 g, 0.45
7.45 (d, J = 8.8 Hz, 1 H), 7.4 (dd, J = 2.5 and 8.8 Hz, 1 H), 5.95 (d, J = 2.6
Hz, 1 H), 4.55 (dd, J = 3.1 and 11.7 Hz, 1 H), 4.2 (dd, J = 6.0 and 11.7
Hz, 1 H), 3.4 (m, 1 H), 2.9 (dd, J = 5.0 and 9.1 Hz, 1 H), 2.75 (dd, J = 2.7
and 5.0 Hz, 1 H).
A mixture of the previous compound (0.23 g, 0.82 mmol),
morpholine (0.072 g, 0.82 mmol), K2CO3 (0.34 g, 2.47 mmol), and
NaI (0.123 g, 0.82 mmol) in DMF (6 mL) was heated to reflux for 8 h,
after which the solvent was evaporated in vacuo. To the crude residue
thus obtained were added H2O and CH2Cl2. The aqueous solution was
extracted several times with CH2Cl2, and the combined organic phases
were washed with H2O and dried over Na2SO4. The suspension was
filtered and the solvent evaporated to dryness to give a crude compound,
which was purified by column chromatography using mixtures of
petroleum ether/EtOAc/MeOH of increasing polarity to give 41a as an
oil (0.114 g, 37%): 1H NMR (300 MHz, CDCl3) δ 7.72 (d, J = 2.5 Hz, 1
H), 7.70 (d, J = 2.7 Hz, 1 H), 7.47 (d, J = 8.8 Hz, 1 H), 7.41 (dd, J = 8.9,
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2.5 Hz, 1 H), 5.95 (d, J = 2.7 Hz, 1 H), 4.49−4.25 (m, 3 H), 4.04−3.83
(m, 4 H), 3.11−2.74 (m, 6 H).
NaI (0.11 g, 0.74 mmol) in dry DMF (5 mL) was heated to 100 °C
under a nitrogen atmosphere for 18 h. The solvent was evaporated in
vacuo, and H2O was added to the crude residue. The dark solid thus
precipitated was filtered and dried to give a crude product, which was
purified by crystallization from EtOAc. Compound 46 was obtained as a
beige solid (0.23 g, 87%): mp 125−127 °C; 1H NMR (CDCl3) δ 9.5 (br
s, 1 H), 7.8 (2d, J = 2.8 and 2.4 Hz, 2 H), 7.5 (d, J = 8.5 Hz, 1 H), 7.45
(dd, J = 2.4 and 8.5 Hz, 1 H), 7.0 (d, J = 2.8 Hz, 1 H), 3.8 (m, 4 H), 3.2
(m, 2 H), 2.6 (m, 4 H).
1-(3,4-Dichlorophenyl)-N-(2-morpholinoethyl)-1H-pyrazol-
3-amine (48). To an ice cooled solution of 46 (0.10 g, 0.28 mmol) in
dry THF (4 mL), under a nitrogen atmosphere, a 2 M solution of
BH3·Me2S in THF (0.7 mL, 1.4 mmol) was added dropwise. The
solution was slowly warmed to reflux, kept at reflux temperature for 4 h,
and stirred at room temperature for 16 h. After the mixture was cooled to
0 °C, H2O (2 mL) and 6 M HCl (0.5 mL) were cautiously added and the
mixture was heated to reflux for 2 h. Solvents were evaporated to
dryness, and the residue was partitioned between 10% NaOH aqueous
solution and EtOAc. The combined organic phases were washed with
H2O, dried over Na2SO4, filtered, and evaporated to dryness. The crude
residue was purified by column chromatography using mixtures of
petroleum ether/EtOAc of increasing polarity as eluent to give 48 as an
oil (0.053 g, 56%): 1H NMR (300 MHz, CDCl3) δ 7.72 (d, J = 2.4 Hz, 1
H), 7.66 (d, J = 2.7 Hz, 1 H), 7.43 (d, J = 8.7 Hz, 1 H), 7.38 (dd, J = 8.8,
2.4 Hz, 1 H), 5.87 (d, J = 2.7 Hz, 1 H), 3.97−3.68 (m, 4 H), 3.54−3.29
(m, 2 H), 3.02−2.39 (m, 6 H).
1-(3,4-Dichlorophenyl)-5-methyl-1H-pyrazole-3-thiol (42). A
suspension of 8c (R1 = 3,4-dichlorophenyl, R2 = Me, R3 = H; obtained
using method A; 8.00 g, 33 mmol) and P2S5 (7.33 g, 33 mmol) in xylenes
(250 mL) was heated at reflux under a nitrogen atmosphere for 20 h.
The mixture was cooled to room temperature. The precipitated solid
was filtered off, and the filtrate was evaporated to dryness. The residue
thus obtained was partitioned between toluene and 10% aqueous KOH
solution, and the phases were separated. The combined organic phases
were washed with H2O, dried over Na2SO4, and concentrated to dryness
to afford a yellow solid (2.20 g), which was identified as a mixture of 42
and 1,2-bis[1-(3,4-dichlorophenyl)-5-methyl-1H-pyrazol-3-yl]-
disulfane. The aqueous phase was acidified with 2 N HCl solution and
extracted again with EtOAc to obtain a mixture of the same two
compounds (5.80 g). The two fractions (total 8.0 g) were suspended in
EtOH (300 mL). NaBH4 (11.13 g, 0.294 mol) was slowly added, and the
reaction mixture was heated to reflux for 18 h. The solvent was
evaporated under vacuum, and the residue was partitioned between
CH2Cl2 and 2 N HCl aqueous solution. The combined organic phases
were washed with H2O, dried over Na2SO4, filtered, and evaporated to
dryness to afford 42 as a brown oil (4.65 g, 55%): 1H NMR (CD3OD-d4)
δ 7.7 (m, 2 H), 7.45 (dd, J = 2.5 Hz, J′ = 8.6 Hz, 1 H), 6.25 (s, 1 H), 2.3
(s, 3 H).
4-{2-[1-(3,4-Dichlorophenyl)-5-methyl-1H-pyrazol-3-ylthio]-
ethyl}morpholine (43). A mixture of 42 (0.16 g, 0.63 mmol), 4-(2-
chloroethyl)morpholine (0.13 g, 0.7 mmol), K2CO3 (0.26 g, 1.9 mmol),
and a catalytic amount of NaI in dry DMF (6 mL) was stirred at 95 °C
under a nitrogen atmosphere for 18 h. The solvent was evaporated and a
mixture of H2O/diethyl ether added to the crude residue. The organic
extracts were washed with H2O, dried over Na2SO4, and concentrated in
vacuo to obtain 43 as an oil (0.17 g, 72%): 1H NMR (300 MHz, CDCl3)
δ 7.59−7.54 (m, 2 H), 7.31 (dd, J = 8.8, 2.1 Hz, 1 H), 6.18 (s, 1 H),
4.04−3.92 (m, 4 H), 3.69−3.28 (m, 2 H), 3.67−3.54 (m, 2 H), 3.46−
3.36 (m, 2 H), 3.36−3.25 (m, 2 H), 2.99−2.80 (m, 2 H), 2.33 (s, 3 H).
1-(3,4-Dichlorophenyl)-4,5-dihydro-1H-pyrazol-3-amine
(44). A solution of NaOEt in EtOH was prepared by dissolving Na (0.40
g, 17.6 mmol) in EtOH (23 mL) under a nitrogen atmosphere. To this
solution 3,4-dichlorophenylhydrazine hydrochloride, 5a (1.85 g, 8
mmol), was added, and the mixture was heated to reflux for 45 min
under vigorous stirring. The mixture was cooled to room temperature,
and acrylonitrile (0.53 mL, 8 mmol) was added and the resulting
solution heated again to reflux for 4 h. The red suspension was cooled
and filtered, and the solid thus obtained was washed with EtOH, H2O,
and diethyl ether and dried under vacuum. Compound 44 was obtained
as an off beige solid (1.10 g, 61% yield), which was used without further
purification in the next step: 1H NMR (DMSO-d6) δ 7.25 (d, J = 8.9 Hz,
1 H), 6.85 (d, J = 2.6 Hz, 1 H), 6.65 (dd, J = 2.6 and 8.9 Hz, 1 H), 5.9 (s, 2
H), 3.5 (t, J = 9.2 Hz, 2 H), 2.8 (t, J = 9.4 Hz, 2 H).
4-[2-(5-Methyl-1H-pyrazol-3-yloxy)ethyl]morpholine (51).
To a suspension of 50 (21.5 g, 0.22 mol) in CH2Cl2 (150 mL), a
solution of (Boc)2O (47.80 g, 0.22 mol) and triethylamine (32 mL, 0.23
mol) in CH2Cl2 (50 mL) was added. The mixture was stirred at room
temperature for 18 h, after which the organic solvent was evaporated in
vacuo. The resulting crude product was recrystallized from toluene to
give tert-butyl 3-hydroxy-5-methyl-1H-pyrazole-1-carboxylate as a white
solid (32.60 g, 75%): 1H NMR (400 MHz, DMSO-d6) δ 10.69 (sb, 1 H),
5.70 (d, J = 1.1 Hz, 1 H), 2.37 (d, J = 1.0 Hz, 3 H), 1.52 (s, 9 H).
To a solution of the previous compound (18.30 g, 0.092 mol) in DMF
(200 mL) were added KI (0.69 g, 0.005 mol), K2CO3 (38.28 g, 0.277
mol), and 4-(2-chloroethyl)morpholine hydrochloride (16.32 g, 0.088
mol). The reaction mixture was heated at 95 °C for 20 h and then
allowed to reach room temperature. The solvent was removed in vacuo,
and the crude residue was taken up in a mixture of H2O and diethyl
ether. The organic layers were dried over Na2SO4 and evaporated to
dryness to give tert-butyl 5-methyl-3-(2-morpholinoethoxy)-1H-pyr-
azole-1-carboxylate as a red solid (19.97 g, 72%), which was used
1
without further purification in the next step: H NMR (300 MHz,
DMSO-d6) δ 5.90 (s, 1 H), 4.21 (t, J = 5.7 Hz, 2 H), 3.63−3.49 (m, 4 H),
2.64 (t, J = 5.6 Hz, 2 H), 2.47−2.40 (m, 4 H), 2.38 (s, 3 H), 1.54 (s, 9 H).
To an ice-cooled solution of the previous compound (19.97 g, 0.064
mol) in EtOH (380 mL) was added 150 mL of aqueous 3 N HCl
solution, and the mixture was allowed to reach room temperature and
stirred for 1 h. The solvent was removed under reduced pressure to give
4-[2-(5-methyl-1H-pyrazol-3-yloxy)ethyl]morpholine hydrochloride as
a yellow solid (19.70 g).
A solution of the previous compound (3.00 g, 12.11 mmol) in H2O
was basified with 10% NaOH solution. EtOAc was added, and the
phases were separated. The combined organic fractions were dried over
Na2SO4, and the solvent was removed under reduced pressure. The
residue was dried under vacuum to give 51 as an orange oil (1.30 g,
50%), which was used without further purification in the next step: 1H
NMR (300 MHz, CD3OD) δ 6.02 (s, 1 H), 4.70−4.60 (m, 2 H), 4.16−
3.77 (m, 6 H), 3.72−3.62 (m, 2 H), 3.65−3.37 (m, 2 H), 2.36 (s, 3 H).
4-{2-[3-Methyl-1-(naphthalen-2-yl)-1H-pyrazol-5-yloxy]-
ethyl}morpholine (52). To a solution of 51 (0.185 g, 0.88 mmol) in
CH2Cl2 (10 mL) were added 2-naphthaleneboronic acid (0.165 g, 0.96
mmol), pyridine (141 μL, 1.75 mmol), Cu(OAc)2·H2O (0.26 g, 1.31
mmol), and 4 Å molecular sieves (1.0 g). The mixture was stirred at
room temperature for 18 h in air and then evaporated. The residue thus
obtained was purified by flash chromatography (cyclohexane/EtOAc)
to provide 52 (0.088 g, 30%): 1H NMR (400 MHz, CDCl3) δ 8.14 (d, J
= 2.0 Hz, 1 H), 7.93 (dd, J = 8.9, 2.1 Hz, 1 H), 7.90−7.79 (m, 3 H),
1-(3,4-Dichlorophenyl)-1H-pyrazol-3-amine (45). To a solution
of 44 (0.62 g, 2.7 mmol) in CH2Cl2 (45 mL) was added MnO2 (1.04 g,
10.8 mmol), and the mixture was stirred at room temperature for 4 h.
The resulting suspension was filtered through dicalite and the solvent
evaporated to dryness in vacuo. A red crude solid was obtained, which
was crystallized in ethyl ether/petroleum ether to give 45 (0.46 g, 74%).
1H NMR (DMSO-d6) δ 8.2 (d, J = 2.5 Hz, 1 H), 7.9 (bs, 1 H), 7.6 (m, 2
H), 5.75 (d, J = 2.5 Hz, 1 H), 5.2 (bs, 2 H).
N-[1-(3,4-Dichlorophenyl)-1H-pyrazol-3-yl]-2-morpholinoa-
cetamide (46). To a solution of 45 (0.36 g, 1.59 mmol) and
triethylamine (0.24 g, 2.38 mmol) in dry THF (5 mL) was added 2-
chloroacetyl chloride (0.20 g, 1.79 mmol) at 0 °C. The mixture was
stirred for 18 h at room temperature. H2O was added and the organic
solvent evaporated in vacuo. The resulting suspension was filtered and
dried to give 2-chloro-N-(1-(3,4-dichlorophenyl)-1H-pyrazol-3-yl)-
1
acetamide as a beige solid (0.45 g, 93%): H NMR (CDCl3) δ 8.75
(br s, 1 H), 7.75 (d, J = 2.7 Hz, 1 H), 7.7 (d, J = 2.4 Hz, 1 H), 7.45 (d, J =
8.6 Hz, 1 H), 7.4 (dd, J = 2.4 and 8.6 Hz, 1 H), 6.9 (d, J = 2.7 Hz, 1 H),
4.15 (s, 2 H).
A mixture of the previous compound (0.22 g, 0.74 mmol),
morpholine (0.071 g, 0.81 mmol), K2CO3 (0.22 g, 1.62 mmol), and
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7.53−7.41 (m, 2 H), 5.55 (s, 1 H), 4.25 (t, J = 5.6 Hz, 2 H), 3.78−3.64
(m, 4 H), 2.83 (t, J = 5.6 Hz, 2 H), 2.62−2.48 (m, 4 H), 2.32 (s, 3 H).
The regioisomer 28 was also isolated (0.044 g, 15%).
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 injection of
capsaicin (1 μg in 20 μL of 1% DMSO) into the mid-plantar surface of
the right hind paw. Fifteen minutes after ipl capsaicin, 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 withdraw 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. Mice (n = 8−
12 per group) received vehicle or drug through the ip route 15 min
before capsaicin injection, and withdrawal latencies to mechanical
stimulation were determined 15 min after capsaicin injection (30 min
after treatments). The effect of treatments on mechanical hyper-
sensitivity 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 time
in drug- and vehicle-treated animals, respectively, and CT is the cutoff
time (60 s).
Partial Sciatic Nerve Ligation Model. The partial sciatic nerve
ligation model was used to study the efficacy of treatments in
neuropathic pain. Briefly, mice were anesthetized with isoflurane
(Abbott-Esteve) (induction, 3%; surgery, 1%) and the common sciatic
nerve was exposed at the level of the mid-thigh of the right hind paw. At
about 1 cm proximal to the nerve trifurcation, a tight ligation using 9−0
nonabsorbable virgin silk suture (Alcon Surgical, U.S.) was created
enclosing the outer 33−50% of the diameter of the sciatic nerve. The
muscle was then stitched with 6−0 silk suture and the skin incision
closed with wound clips. Two tests were performed: von Frey test first
(30 min after treatment) and plantar test second (45 min after
treatment). At 1 day after baseline measurements, sciatic nerve injury
was induced and mice were tested after the surgical procedure to
monitor the development of neuropathic-pain-related behaviors. On day
10 these behaviors were already apparent and mice received a vehicle
injection. On days 11, 12, and 13, mice received ip administration of the
drugs following a Latin square design (n = 10−12 per group). Allodynia
to mechanical stimuli and hyperalgesia to noxious thermal stimulus were
used as outcome measures of neuropathic pain by using the von Frey
and plantar test, respectively. Briefly, in the von Frey test, animals were
placed into compartment enclosures in a test chamber with a framed
metal mesh floor through which the von Frey monofilaments (bending
force range from 0.008 to 2.0 g) (North Coast Medical, Inc., U.S.) were
applied and thresholds were measured in the ipsilateral, nerve-injured
paw using the up−down paradigm. Clear paw withdrawal, shaking, or
licking was considered as a nociceptive-like response. The effect of
treatments on mechanical hypersensitivity induced by the operation was
calculated with the following equation: % reduction of mechanical
hypersensitivity = [(TRD − TRV)/(B − TRV)] × 100, where TRD and
TRV are the threshold pressure responses in drug- and vehicle-treated
animals, respectively, and B is the basal pressure response (before
operation). Thermal (heat) hyperalgesia was assessed with a plantar test
apparatus (Ugo Basile), by measuring hind paw withdrawal latency in
response to radiant heat. Briefly, mice were placed into compartment
enclosures on a glass surface. The heat source was then positioned under
the plantar surface of the hind paw and activated with a light beam
intensity chosen based on preliminary studies to give baseline latencies
from 8 to 9 s in control mice. The digital timer connected to the heat
source automatically recorded the response latency for paw withdrawal
to the nearest 0.1 s. A cutoff time of 20 s was imposed to prevent tissue
damage in the absence of response. The mean withdrawal latencies for
the ipsilateral hind paw were determined from the average of three
separate trials done at 5 min intervals. The effect of treatments on
thermal (heat) hypersensitivity induced by the operation was calculated
with the following equation: % reduction of thermal hypersensitivity =
[(LTD− LTV)/(B − LTV)] × 100, where LTD and LTV are the
Human σ1 Receptor Radioligand Assay.28 The binding proper-
ties 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 presence of either buffer or 10 μM haloperidol for total and
nonspecific binding, respectively. Binding buffer contained Tris-HCl
(50 mM, at pH 8). Plates were incubated at 37 °C for 120 min. After the
incubation period, the reaction mix was transferred to MultiScreen HTS
FC plates (Millipore), filtered, and plates were washed (3 times) with
ice-cold Tris-HCl (10 mM, pH 7.4). Filters were dried and counted at
approximately 40% efficiency in a MicroBeta scintillation counter
(Perkin-Elmer) using EcoScint liquid scintillation cocktail.
Guinea Pig σ2 Receptor Radioligand Assay. The binding
properties of test compounds to guinea pig σ2R were studied in guinea
pig brain membranes as described29 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 absence or presence of either
buffer or 10 μM haloperidol for total and nonspecific binding,
respectively. Binding buffer contained Tris-HCl (50 mM, pH 8), and
σ1 receptor was blocked with (+)-SKF10047 at 400 nM. Plates were
incubated at 25 °C for 120 min. After the incubation period, the reaction
mix was transferred to MultiScreen HTS, FC plates (Millipore), filtered
and plates were washed 3 times with ice-cold 50 mM Tris-HCl (pH 7.4).
Filters were dried and counted at approximately 40% efficiency in a
MicroBeta scintillation counter (Perkin-Elmer) using EcoScint liquid
scintillation cocktail.
In Vitro Metabolic Stability in Human Liver Microsomes. The
assay was carried out in a robotic liquid handling system (Packard
Multiprobe II, Perkin-Elmer). All incubations were performed
individually for each test compound. Compounds (10 μM) were
incubated in 96-well plates at 37 °C during 1 h under standard
incubation conditions: sodium−potassium phosphate buffer (50 mM,
pH 7.4), MgCl2 (3 mM), the NADPH-regenerating system, and CYP
content (0.3 nmol/mL). At preset times (0, 10, 20, 40, and 60 min)
aliquots of the reaction mixture were stopped with an equal volume of
cold acetonitrile. Upon centrifugation of the resultant mixture,
supernatants were analyzed by a generic UPLC−MS/MS method.
Metabolic stability was determined by the disappearance of compound
over time.31,41The ln-linear plots of the % of compound remaining based
on chromatographic peak area versus time were plotted, and the slope
was calculated by linear fitting of the curve. The in vitro metabolic half-
life (t1/2) was estimated by using 0.693/k where k is the
biotransformation rate constant and corresponds to the slope of the
ln-linear curve. The microsomal intrinsic clearance (Clint) was calculated
using the equation Clint = 0.693/[(t1/2)(mg of microsomal protein/
volume of incubation)].
In Vivo Tests. Animals. CD1 mice (Charles River, France) aged
from 6 to 8 weeks old were used. Male mice were used for neuropathic
pain studies and female mice for the capsaicin test. Animals had access to
food and water ad libitum and were kept in controlled laboratory
conditions with 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
November 24, 1986, 86/609/ECC) and received approval by the
local ethical committee.
Capsaicin Test. Capsaicin (8-methyl-N-vanillyl-6-nonamide) was
purchased from Sigma-Aldrich and dissolved in 1% DMSO (vehicle) in
physiological saline. Test compounds were dissolved in 0.5%
hydroxypropylmethylcellulose (HPMC) (Sigma-Aldrich) and were
administered in a volume of 10 mL/kg through the ip route. Mice
were habituated for 2 h in individual test compartments placed on an
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latency times in drug- and vehicle-treated animals, respectively, and B is
the basal latency time.
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Motor Coordination (Rotarod Test). The motor performance of
mice was assessed by means of an automated rotarod (Panlab SL,
Spain). Before drug treatments, mice were trained and those that were
unable to stay moving on the rod for 240 s at 10 rpm were discarded for
the study. Mice were required to walk against the motion of an elevated
rotating drum at 10 rpm, and the latency to fall-down was recorded
automatically. With the selected animals rotarod latencies were
measured 30, 60, 120, and 180 min after ip administration of drugs.
(7) Kim, F. J.; Kovalyshyn, I.; Burgman, M.; Neilan, C.; Chien, C. C.;
Pasternak, G. W. Sigma-1 receptor modulation of G-protein-coupled
receptor signaling: potentiation of opioid transduction independent
from receptor binding. Mol. Pharmacol. 2010, 77, 695−703.
(8) (a) Zamanillo, D.; Portillo-Salido, E; Vela, J. M.; Romero, L. Sigma
1 Receptor Chaperone: Pharmacology and Therapeutic Perspectives. In
Therapeutic Targets; Botana, J. M., Loza, M.; Eds.; John Wiley & Sons:
Hoboken, NJ, 2012; Chapter 6, pp 225−278. (b) Maurice, T.; Su, T. P.
The pharmacology of sigma-1 receptors. Pharmacol. Ther. 2009, 124,
195−206.
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Ronsisvalle, S.; Pasquinucci, L.; Prezzavento, O.; Colabufo, N. A.;
Contino, M. A.; Gonzalez, L. G.; Scoto, M. G.; Ronsisvalle, G. Novel
potent and selective sigma ligands: evaluations of their agonist and
antagonist properties. J. Med. Chem. 2011, 54, 3669−3673.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Analytical and characterization data for all the compounds and
description of methods used for drug transport assay using Caco-
2 cells, CYP inhibition, and pharmacokinetic studies of
compound 28. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
́
(11) Cendan, C. M.; Pujalte, J. M.; Portillo-Salido, E.; Montoliu, L.;
Baeyens, J. M. Formalin-induced pain is reduced in sigma(1) receptor
knockout mice. Eur. J. Pharmacol. 2005, 511, 73−74.
́
(12) Entrena, J. M.; Cobos, E. J.; Nieto, F. R.; Cendan, C. M.; Gris, G.;
Corresponding Author
*Phone: 0034934466000. E-mail: jldiaz@esteve.es.
Notes
Del Pozo, E.; Zamanillo, D.; Baeyens, J. M. Sigma-1 receptors are
essential for capsaicin-induced mechanical hypersensitivity: studies with
selective sigma-1 ligands and sigma-1 knockout mice. Pain 2009, 143,
252−261.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(13) De la Puente, B.; Nadal, X.; Portillo-Salido, E.; San
R.; Ovalle, S.; Palacios, G.; Muro, A.; Romero, L.; Entrena, J. M.;
Baeyens, J. M; Lopez-García, J. A.; Maldonado, R.; Zamanillo, D.; Vela, J.
́
chez-Arroyos,
We thank Magda Bordas, Carme Calvet, and Raquel Enrech for
their expert contribution to the analysis of the compounds;
́
́
́
Alvarez, Eva
Albert Dordal, Xavier Monroy, Xavier Codony, Ines
Ayet, and Ariadna Balada for their expert contribution to ADME
and in vivo studies; Miguel Angel Blazquez, Monica Carro, Joan
Andreu Morato, and Edmundo Ortega for their excellent
technical assistance; and Carlos Perez, Oscar Ferre, and Eduardo
M. Sigma-1 receptors regulate activity-induced spinal sensitization and
neuropathic pain after peripheral nerve injury. Pain 2009, 145, 294−303.
(14) Cendan, C. M.; Pujalte, J. M.; Portillo-Salido, E.; Baeyens, J. M.
́
́
́
Antinociceptive effects of haloperidol and its metabolites in the formalin
test in mice. Psychopharmacology (Berlin) 2005, 182, 485−493.
(15) Roh, D. H.; Kim, H. W.; Yoon, S. Y.; Seo, H. S.; Kwon, Y. B.; Kim,
K. W.; Han, H. J.; Beitz, A. J.; Na, H. S.; Lee, J. H. Intrathecal injection of
the sigma 1 receptor antagonist BD1047 blocks both mechanical
allodynia and increases in spinal NR1 expression during the induction
phase of rodent neuropathic pain. Anesthesiology 2008, 109, 879−889.
(16) Drews, E.; Zimmer, A. Central sensitization needs sigma
receptors. Pain 2009, 145, 269−270.
́
́
́
Villarroel for their contributions to compound management.
ABBREVIATIONS USED
■
S1RA, E-52862, 4-{2-[5-methyl-1-(naphthalen-2-yl)-1H-pyra-
zol-3-yloxy]ethyl}morpholine; ADME, absorption, distribution,
metabolism, and excretion; CFA, Freund’s complete adjuvant;
TNBS, 2,4,6-trinitrobenzenesulfonic acid
(17) 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.
REFERENCES
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