EST64454: A Highly Soluble σ1Receptor Antagonist Clinical Candidate for Pain Management

Article abstract of DOI:10.1021/acs.jmedchem.0c01575

The synthesis and pharmacological activity of a new series of pyrazoles that led to the identification of 1-(4-(2-((1-(3,4-difluorophenyl)-1H-pyrazol-3-yl)methoxy)ethyl)piperazin-1-yl)ethanone (9k, EST64454) as a σ1 receptor (σ1R) antagonist clinical candidate for the treatment of pain are reported. The compound 9k is easily obtained through a five-step synthesis suitable for the production scale and shows an outstanding aqueous solubility, which together with its high permeability in Caco-2 cells will allow its classification as a BCS class I compound. It also shows high metabolic stability in all species, linked to an adequate pharmacokinetic profile in rodents, and antinociceptive properties in the capsaicin and partial sciatic nerve ligation models in mice.

Full text of DOI:10.1021/acs.jmedchem.0c01575

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Article
EST64454: a Highly Soluble σ1 Receptor Antagonist Clinical
Candidate for Pain Management
́
́
̃
Jose Luis Díaz, Monica García, Antoni Torrens, Ana María Caamano, Juan Enjo, Cristina Sicre,
́
́
Adriana Lorente, Adriana Port, Ana Montero, Sandra Yeste, Ines Alvarez, Miquel Martín,
́
́
Rafael Maldonado, Beatriz de laPuente, Alba Vidal-Torres, Cruz Miguel Cendan, Jose Miguel Vela,
and Carmen Almansa*
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ABSTRACT: The synthesis and pharmacological activity of a new series of
pyrazoles that led to the identification of 1-(4-(2-((1-(3,4-difluorophenyl)-
1H-pyrazol-3-yl)methoxy)ethyl)piperazin-1-yl)ethanone (9k, EST64454) as a
σ1 receptor (σ1R) antagonist clinical candidate for the treatment of pain are
reported. The compound 9k is easily obtained through a five-step synthesis
suitable for the production scale and shows an outstanding aqueous solubility,
which together with its high permeability in Caco-2 cells will allow its
classification as a BCS class I compound. It also shows high metabolic stability
in all species, linked to an adequate pharmacokinetic profile in rodents, and
antinociceptive properties in the capsaicin and partial sciatic nerve ligation
models in mice.
INTRODUCTION
■
The adequate management of pain is still an unmet medical
need because currently available treatments provide in many
cases only modest improvements, leaving many patients
unrelieved1 and causing a heavy socioeconomical burden.2
Currently approved pain therapies such as opioid agonists,
Figure 1. Structure of the σ1R antagonist E-52862 (1).
nonsteroidal anti-inflammatory drugs, calcium channel modu-
lators, and antidepressants show an efficacy limited by a variety
of secondary effects that can preclude their prolonged use. In
order to identify analgesic agents with an alternative
mechanism of action, we have been working for many years
in developing sigma-1 receptor (σ1R) antagonists.3
where 1 showed a promising reduction in pain scores.9 An
enhancement of the benefit−risk ratio of the opioid morphine
overall clinical profile was also obtained in a clinical trial of
postoperative pain due to abdominal hysterectomy.10
As a backup program, we explored the variation in the
morpholine-containing chain of 1, by elongation of the
distance of the O-atom to the pyrazole ring or its replacement
by carbon atoms. We thought that these changes could still
comply with Glennon’s σ1R pharmacophore,11 the model used
for the development of 1, and lead to the improvement of
some of its properties while maintaining selectivity over the
sigma-2 receptor (σ2R). The two sigma receptors, σ1 and σ2,
The σ1R is a 24 kDa protein of 223 amino acids anchored to
the endoplasmic reticulum (ER) and plasma membranes.4 It is
structurally different from other target classes because it has no
direct downstream signaling, and instead, it acts as a molecular
chaperone modulating the functions of diverse proteins.5 A
wide range of evidence is now available to support the role of
the σ1R in the treatment of pain,3 including potentiation of
opioid receptor-mediated analgesia by σ1R antagonists6 and
the fact that σ1R knockout mice (σ1R-KO)7 did not develop
pain behaviors after the nerve sensitization developed under
neuropathic pain conditions or after capsaicin or formalin
injection. The use of σ1R antagonists reinforced these findings
and was the basis for the selection of S1RA (E-52862, 1, Figure
1)8 as a clinical candidate for the treatment of different pain
types. These included chemotherapy-induced neuropathy,
Received: September 9, 2020
https://dx.doi.org/10.1021/acs.jmedchem.0c01575
© XXXX American Chemical Society
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A

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a
Scheme 1
a
Reagents and conditions: (a) AcOH, 120 °C; (b) (i) NaOAc, EtOH, rt, (ii) bicyclo[2.2.1]hepta-2,5-diene, Et3N, toluene, 80 °C, and (iii) xylenes,
135 °C; (c) NaBH4, EtOH, 80 °C; (d) NaH, THF, 70 °C; (e) MnO2, CH2Cl2, 40 °C; (f) KOtBu, THF, rt; (g) PtO2, H2, MeOH, rt; (h) 1-bromo-
3-chloropropane, Bu4NSO4H, NaOH, toluene, rt; and (i) 1-acetylpiperazine, DIPEA, NaI, DMF, 90 °C.
have different molecular weights, tissue distributions, and
subcellular locations and were initially classified based on their
different behaviors in the presence of (+)-pentazocine in rat
liver and kidney membranes. Initial assays involved binding on
guinea pig membranes, applied to the σ1R12 or to the σ2R.13
The σ1R was cloned soon enough,14 but the identification of
the σ2R has been troublesome.15 Although it was later on
proposed to be a part of the progesterone receptor membrane
component 1 (PGRMC1),16 this was ruled out mainly based
on the fact that overexpression or knockdown of PGRMC1
failed to affect prototypic σ2R ligand binding. The receptor was
finally isolated using classical affinity purification approaches17
and it was identified as the ER-resident membrane protein
TMEM97, also known as MAC30. Both TMEM97 and the
classical σ2R binding site show similar characteristics and
behaviors for a range of prototypic σ2R ligands.
The backup program reported here was directed at finding
an alternative derivative to the compound 1 that could improve
some of its properties. First, 1 showed an acceptable, but
suboptimal, aqueous solubility (0.2 mg/mL) that could be
probably improved by reducing its lipophilicity (elog P = 3.5),
taking into account the inverse relationship of solubility with
clog P.18 Reducing lipophilicity while maintaining good affinity
for the σ1R and selectivity versus other targets was not
considered a trivial task because polar groups are poorly
tolerated in the highly hydrophobic σ1R and need to be placed
at very specific positions.19 However, it was worth to be
pursued because such a reduction would be also beneficial for
the improvement of the overall safety profile. It is well known
that decreasing lipophilicity is linked to reduced attrition and
promiscuity20 and even a cutoff value of clog P = 3 has been
proposed to increase the likelihood of not finding toxic events
in so-called Pfizer’s 3/75 rule.21 On the other hand, the
exposure of the compound 1 in rodents (with AUC 958 and
428 ng/mL and bioavailability of 34 and 15% after oral
administration of 10 mg/kg to mice and rats, respectively)
could be improved in order to better establish efficacy−safety
ratios along the compound development. With these objectives
in mind, we initiated the synthesis and structure−activity
relationship (SAR) study that allowed the identification of 1-
(4-(2-((1-(3,4-difluorophenyl)-1H-pyrazol-3-yl)methoxy)-
ethyl)piperazin-1-yl)ethanone 9k as a selective σ1R antagonist
clinical candidate for the treatment of pain.22
CHEMISTRY
■
As outlined in Scheme 1, the synthesis of the new pyrazoles 9−
15 was carried out using three alternative procedures from 3-
hydroxymethylpyrazoles 7. The process started by constructing
B
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Table 1. SAR Data around the 1,3,5-Trisubstituted Pyrazoles
a
b
c
d
comp
X
R1
R2
R3
Ki σ1R (h, nM) Ki σ2R (h, nM) clog P hERG IC50 (nM)
e
f
1
17
209 57
14
2
240 38
6
2
3
15 0.1
278 12
58
22
5
7
>1000
>1000
3.9
3.6
3.8
3.6
2.0
4.5
5.1
4.8
3.3
3.4
2.5
2.0
4.2
4.7
3.6
>10,000
e
g
9a
9b
9c
9d
9e
9f
9g
9h
9i
O(CH2)2
O(CH2)2
O(CH2)2
O(CH2)2
O(CH2)2
O(CH2)2
O(CH2)2
O(CH2)2
O(CH2)2
O(CH2)2
O(CH2)2
(CH2)2
naphthalen-2-yl
Me
Me
H
morpholyn-4-yl
morpholyn-4-yl
morpholyn-4-yl
morpholyn-4-yl
pyrrolidin-1-yl
piperidin-1-yl
(2S,6R)-2,6-dimethylmorpholin-4-yl
4-acetylpiperazin-1-yl
piperazin-1-yl
4-acetylpiperazin-1-yl
4-acetylpiperazin-1-yl
morpholyn-4-yl
morpholyn-4-yl
4-acetylpiperazin-1-yl
NT
NT
NT
NT
g
3,4-dichlorophenyl
3,4-dichlorophenyl
cyclopentyl
1
0.2
399 76
137 47
>10,000
g
f
g
Me
Me
Me
Me
Me
Me
Me
H
3,4-dichlorophenyl
3,4-dichlorophenyl
3,4-dichlorophenyl
3,4-dichlorophenyl
3,4-dichlorophenyl
3,4-difluorophenyl
3,4-difluorophenyl
3,4-dichlorophenyl
3,4-dichlorophenyl
3,4-dichlorophenyl
2
0.3
1
27
1
5123
g
28 19
NT
NT
g
180 38
e
f
>1000
>10,000
g
g
NT
NT
f
f
f
9j
6
3
0.4
1
0.1
>10,000
>10,000
>10,000
>10,000
f
9k
13a
13b
15
g
Me
Me
Me
232 100
116 20
NT
NT
g
(CH2)3
O(CH2)3
9
9
e
>1000
4084
a
Binding affinity (Ki, nM) to human in transfected HEK-293 membranes using [3H](+)-pentazocine as a radioligand. Each value is the mean SD
of two determinations. The Ki values (nM) in this assay for some standard reference compounds are as follows: (+)-pentazocine, 12
1;
b
haloperidol, 2 1; and DTG, 59 7. Binding affinity (Ki, nM) to σ2R in guinea pig brain membranes using [3H]di-o-tolylguanidine as a
radioligand. Each value is the mean SD of two determinations. The Ki values (nM) in this assay for some standard reference compounds are as
c
d
follows: (+)-pentazocine, 719 63; haloperidol, 22 1; and DTG, 23 11. clog P was calculated using ChemDraw. Whole-cell patch clamp
e
f
g
hERG blockade. Less than 50% inhibition at 1 μM. Less than 50% inhibition at 10 μM. Not tested.
a
Scheme 2
a
Reagents and conditions: (a) CuI, Cs2CO3, TMEDA, MeCN, 85 °C; (b) LiBH4, THF, 70 °C; (c) TsCl, KOH, THF, rt; (d) NaH, THF, rt; and
(e) HCl, IPA, 0 °C.
the ester derivatives 4, which were prepared by two different
procedures depending on their substitution pattern. Using one
of the most prevalent methods for obtaining pyrazoles, that is,
cyclization of 1,3-diketones with hydrazine derivatives,23 the
reaction of ethyl acetopyruvate (3) with hydrazines 2 afforded
the 5-methyl-pyrazoles (4, R2 = Me) with excellent yields. An
alternative method was used for the synthesis of 5-H-pyrazoles
(4, R2 = H) involving the reaction of diazonium salts 5 with
ethyl 2-chloroacetoacetate (6). This reaction proceeded
through an intermediate arylhydrazonoyl chloride24 that
underwent a [3 + 2] cycloaddition with bicyclo[2.2.1]hepta-
2,5-diene, followed by a retro Diels−Alder reaction in refluxing
xylenes.25 Reduction of derivatives 4 with NaBH4 led to the
key 3-(hydroxymethyl)pyrazoles 7, which were alkylated with
C
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chloroethylamine derivatives 8 using NaH in refluxing
tetrahydrofuran (THF) to give pyrazoles 9a−h with moderate
to good yields. The derivative 9i (Table 1) was obtained by
deprotection of 9h with HBr in AcOH, as described in the
experimental part. Alternatively, alcohols 7 were oxidized with
MnO2 to afford aldehydes 10, which were reacted with
phosphonium salts 11 to render alkenes 12. Reduction of 12
using PtO2 under a H2 atmosphere afforded the alkylated
pyrazoles 13 with good yields. Furthermore, a derivative with
an extended alkyl chain was synthesized from 7 by alkylation
with 1-bromo-3-chloropropane and subsequent substitution
with 1-acetylpiperazine, affording the pyrazole 15 with
moderate yield.
pig membranes using [3H]-di-o-tolylguanidine13 as a radio-
ligand. The active and selective compounds were assayed for
the blockade of the human ether-a-go-go-related gene (hERG),
an off-target related to cardiac toxicity29 that has been a
repeated issue in our previous σ1R programs, particularly in
structurally related compounds.8,26 The results are shown in
Table 1.
The SAR was initiated with the preparation of the direct
elongated analogue of the reference compound 1, derivative
9a, which showed a decrease in σ1R binding. Changing the
naphthalenyl group by one of the best alternatives identified in
previous studies,8,30 the 3,4-dichlorophenyl ring, provided an
increased affinity (9b) but a reduction in selectivity for the
σ2R. A similar profile was obtained with the unsubstituted
derivative in position 2, 9c. Replacement of the aromatic ring
in position 1 with a saturated cyclopentyl ring (9d) provided a
decrease in affinity.
Replacement of the morpholine ring by other basic groups
was investigated over the 1-(3,4-dichlorophenyl)-2-methylpyr-
azoles. Using more lipophilic cyclic amines, such as pyrrolidine
(9e), piperidine (9f), or dimethyl morpholine (9g) provided
low nanomolar affinities but was detrimental in relation to
selectivity versus the σ2R, in agreement with previous
findings.30 Introducing a 4-acetylpiperazine ring was favorable
because the compound 9h maintained a good activity for the
σ1R and a good selectivity versus the σ2R and was devoid of
hERG inhibition. Its naked piperazine counterpart 9i was
substantially less active. Elongation of the acetyl group to
bulkier groups was also studied, but either selectivity versus the
σ2R was impaired or hERG inhibition started to appear (results
not shown). In a similar way, elongation of one carbon atom of
the linker chain provided the compound 15, which was
interesting in terms of primary affinities, but showed a hERG
inhibition IC50 of around 4 μM.
Replacement of the oxygen-containing linker chain with an
alkylidene linker provided compounds 13a and 13b, with good
σ1R affinities but low selectivity. Overall, it seems that
selectivity versus the σ2R is more easily achieved with more
polar compounds, which is consistent with previous results.8,30
More analogues were prepared maintaining a 4-acetylpiper-
azin-1-ylethyloxymethyl linker and changing the nature of the
aromatic substituents of the phenyl ring in position 1 and
keeping methyl or hydrogen in position 2. Compounds 9h, 9j,
and 9k emerged as the most interesting derivatives because
they showed good affinity for the σ1R and good selectivity and
were devoid of hERG inhibition.
As an alternative synthesis for the large-scale production of
9k, the route described in Scheme 2 was developed.26 It is well
known that diazonium salts are explosive and their precursor
anilines are genotoxic.27 As a consequence, processes avoiding
their use are strongly preferred. Furthermore, the yield of the
final alkylation was difficult to reproduce when performing the
reactions in a bigger scale and needed further improvement.
An Ullmann-type reaction was envisaged for the synthesis of 3-
(ethoxycarbonyl)-pyrazole 18, based on the existing literature
describing the synthesis of pyrazoles using this methodology.28
Optimization of the reaction conditions included testing of
different copper salts as Cu2O and CuI; solvents such as
toluene, DMA, NMP, MeOH, DMF, THF, MeCN, and
dioxane; bases such as K3PO4, Cs2CO3, K2CO3, Na2CO3,
NaHCO3, and NaOH; and ligands such as bipyridine, 1,10-
phenanthroline, ethane-1,2-diamine, N1,N2-dimethylethane-
1,2-diamine, (1R,2R)-cyclohexane-1,2-diamine, (1R,2R)-
N1,N2-dimethylcyclohexane-1,2-diamine, TMEDA, and 2-
hydroxybenzaldehyde oxime. We observed no difference in
the reaction outcomes when using CuI or Cu2O and that
reactions performed in THF or MeCN showed full conversion
and cleaner crude products compared to the other tested
solvents. Finally, the best base/solvent combination was found
to be Cs2CO3/MeCN and the best ligand was TMEDA
because its use afforded good conversions and minimized
byproduct formation. The optimized conditions led to the
formation of 18 in an excellent yield and with high
regioselectivity (97:3), obtaining only traces of the 5-
substituted pyrazole 19. Reduction of the regioisomeric
mixture was performed with LiBH4 to give 3-(hydroxymethyl)-
pyrazole 20, which was obtained as a single regioisomer after
crystallization of the reaction mixture. An alternative alkylation
method in relation to that used in Scheme 1 was also envisaged
in order to avoid the variability previously observed.
Derivatization to the tosyl derivative 21 followed by
substitution with the alkoxide of alcohol 22 afforded the
pyrazole 9k in good yields. In this reaction, the hydrochloride
salt of the alkylating agent was directly used to afford the final
compound as hydrochloride salt, which was the form selected
for development. Overall, the process depicted in Scheme 2
allows the synthesis of 9k with improved efficiency, applying
reaction conditions suitable for large-scale production, without
the need of chromatographic purification throughout the
whole process and using easily available, nontoxic starting
materials.
The compound 9k was selected for further progression
because in addition to its good activity profile, it showed the
lowest clog P, almost 2 log units below that of the reference
compound 1, which was corroborated by the experimental
values (log P/log D7.4 1.7 for 9k and 3.5 for 1). This reduction
translated into an exceptionally high thermodynamic solubility
both at pH 2 and 7.4 (2.3 g/mL, 4 orders of magnitude higher
than the 0.23 mg/mL of 1). Since 9k shows as well a high
permeability in Caco-2 cells31 (515 nm/s), it will be
undoubtedly classified as a biopharmaceutics classification
system (BCS) class I compound. The BCS differentiates drugs
on the basis of their solubility and permeability, allowing the
prediction of drug absorption.32 The system divides drugs into
four classes: I, high solubility and permeability; II, low
solubility and high permeability; III, high solubility and low
permeability; and IV, low solubility and low permeability. In
the BCS, a drug is considered highly soluble when the highest
RESULTS AND DISCUSSION
■
The new compounds synthesized were tested in a primary σ1R
binding assay using [3H]-(+)-pentazocine8 as a radioligand in
human transfected HEK-293 membranes and in σ2R in guinea
D
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dose strength is soluble in 250 mL or less of aqueous media
(initial gastric volume) over the pH range of 1−7.5. The
compound 9k is predicted to be class I and therefore well
absorbed even at doses well above the gram.
The compound 9k also complies with Lipinski’s rules and
shows moderate basicity (pKa 6.4), an interesting attribute to
comply with the central nervous system multiparameter
optimization (CNS MPO) algorithm.33 An MPO value of
6.0 was calculated for 9k, which is the highest possible score,
predicting good ADME and safety properties for CNS-directed
drugs.
(Cmax) after po administration were relatively high: 1178 ng/
mL in rats and 771 ng/mL in mice. Total plasma clearances
were high, 70% of liver blood flow in rats and 89% in mice.
The volumes of distribution were higher than the total body
water, 1.2 L/kg in rat and 4.4 L/kg in mice. Marked species
differences were observed in elimination half-lives, with mean
values of <1 and 3.4 h in rats and mice, respectively. It can be
concluded that 9k shows lower metabolic clearance than 1,
which results in higher in vivo exposure (AUCs: 1.5-fold and
sixfold higher in mice and rats, respectively) and better
bioavailability (Fs: twofold and fourfold higher in mice and
rats, respectively).
The compound 9k was shown to be devoid of potential for
drug−drug interactions based on both direct and time-
dependent CYP inhibition for CYP1A2, 2C9, 2C19, 2D6,
and 3A4 in human liver microsomes, where it showed a IC50 of
between 100 and 1000 μM. Studies of 9k with HepaRG cells
revealed no CYP induction at concentrations 50 μM31 and
adequate in vitro metabolic stability in human, rat, and mouse
liver microsomes.31 It showed as well a good in vitro safety,
with lack of 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 cells34 and lack of genotoxic potential in the SOS/umu
and Ames bacterial mutation assays.35 Finally, 9k can be
considered as highly selective because it did not show any
significant affinity for another 180 molecular targets (inhibition
at 1 μM below 50% in a set of receptors, transporters, ion
channels, and enzymes), indicating that the relevant in vivo
activity shown below is only due to σ1R binding. The summary
in vitro profile of 9k is shown in Figure 2.
The functional activity of 9k was evaluated using the
phenytoin assay,36 where σ1R agonists are shifted by the
allosteric ligand to significantly higher affinities (Ki ratios
without phenytoin vs with phenytoin > 1), while σ1R
antagonists show small or no shift to lower affinity values (Ki
ratios without phenytoin vs with phenytoin ≤1). The behavior
of the compound 9k may be considered indicative of σ1R
antagonism (Ki without phenytoin/Ki with phenytoin = 0.9
0.2). Although this value is not completely conclusive, the
antagonist nature of 9k is confirmed by its in vivo analgesia, as
shown below.
The compound 9k showed in vivo efficacy after oral
administration in two different pain models in mice:
intraplantar capsaicin-induced mechanical hypersensitivity37
and partial sciatic nerve ligation (PSNL)-induced mechanical
hypersensitivity,38 a model representative of neuropathic pain.
In the capsaicin test, the antiallodynic potency of 9k was
similar to that of the compound 1 (ED50 33 vs 28 mg/kg,
respectively, Figure 3, Table 2). In the PSNL model, oral
administration of 9k at 80 mg/kg, revealed an increased
antiallodynic efficacy when comparing with the same dose of
the compound 1 (95% vs 54%, respectively).
CONCLUSIONS
■
In summary, we have described the synthesis and pharmaco-
logical activity of a new series of potent and selective σ1R
ligands. Several compounds in this family of arylpyrazoles
exhibited high in vitro affinity and selectivity versus σ2R/
TMEM97 and low potential for cardiac toxicity. The most
promising compound 9k (EST64454) behaves as a σ1R
antagonist and shows a remarkable physicochemical profile,
with a very high aqueous solubility at any physiologically
meaningful pH, which in view of its high permeability in Caco-
2 cells, will allow its classification as a BCS class I compound at
any dose range. It also shows high metabolic stability in all
species and an adequate pharmacokinetic profile in rodents.
This adequate drug metabolism and pharmacokinetic (DMPK)
profile, together with its in vivo antinociceptive properties in
Figure 2. Structure and relevant in vitro data of the clinical candidate
9k.
The pharmacokinetic profile of 9k was determined in mice
and rats after oral and intravenous administration of single
doses of 10 and 1 mg/kg, respectively, and comparative data
versus 1 are outlined in Table 2. Bioavailability (F) was higher
than 60% in both species and peak plasma concentrations
Table 2. In Vivo Comparative Data for Compounds 9k and 1
c
d
mouse pharmacokinetics 10 mg/kg, po
rat pharmacokinetics 10 mg/kg, po
b
PSNL
a
capsaicin ED50
(95% CI)
efficacy SEM
(% at 80 mg/kg,
po)
Cmax
t1/2
AUC0−∞
Cl
Vss
Cmax
t1/2
AUC0−∞
Cl
Vss
comp
(mg/kg, po)
(ng/mL) (h) (ng·h/mL) (% LBF) (l/kg) F (%) (ng/mL) (h) (ng·h/mL) (% LBF) (l/kg) F (%)
9k
1
33 (32−35)
28 (20−41)
95 21
54 13
771
622
3.4
0.7
1431
958
89
65
4.4
2.2
69
34
1178
301
<1
2645
428
70
1.2
2.9
60
15
1.2
109
a
b
Reduction in mechanical hypersensitivity after po administration of test compounds in the mouse capsaicin test. Reduction in mechanical
c
hypersensitivity after po administration of test compounds in the mouse PSNL model. Mouse pharmacokinetic parameters calculated as described
in the Experimental Section. Rat pharmacokinetic parameters calculated as described in the Experimental Section.
d
E
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(74 mg, 0.47 mmol) was added and the mixture was stirred at 120 °C
for 1 h. After this time, dichloromethane (DCM) was added and the
organic layer was washed with H2O, 10% aqueous NaOH, and brine.
The organic phase was dried over Na2SO4, filtered, and concentrated
to dryness. The residue was purified by flash chromatography (SiO2,
hexane/EtOAc 80:20) to give the compound 4a as an orange solid
(139 mg, 99%). 1H NMR (250 MHz, CDCl3): δ 7.62 (d, J = 2.4 Hz,
1H), 7.54 (d, J = 8.3 Hz, 1H), 7.32 (dd, J = 2.4, 8.3 Hz, 1H), 6.72 (s,
1H), 4.39 (c, J = 6.8 Hz, 2H), 2.34 (s, 3H), 1.39 (t, J = 6.8 Hz, 3H).
Ethyl 1-(3,4-Dichlorophenyl)-1H-pyrazole-3-carboxylate (4b, R1
= 3,4-Dichlorophenyl, R2 = H). To a solution of ethyl 2-
chloroacetoacetate (6) (1.52 g, 9.26 mmol) and NaOAc (2.41 g,
29.4 mmol) in EtOH (5 mL) at 0 °C, 3,4-dichlorobenzenediazonium
chloride (5) (1.88 g, 9.26 mmol) was added and the mixture was
stirred at 0 °C for 2 h. After this time, the suspension was filtered and
the solid was washed with EtOH. The obtained solid was dried and
dissolved in toluene (20 mL). Then, bicyclo[2.2.1]hepta-2,5-diene
(2.06 mL, 20.31 mmol) and Et3N (2.64 mL, 18.96 mmol) were added
and the mixture was stirred at 80 °C for 2 h. The suspension was
filtered again; the solid was dissolved in xylenes (20 mL) and heated
to 140 °C for 16 h more. The reaction mixture was concentrated to
dryness and the residue was purified by flash chromatography (SiO2,
hexane/EtOAc 100:0 to 85:15) to give the compound 4b as a white
Figure 3. Dose−response effect of 9k and 1 on the capsaicin-induced
mechanical hypersensitivity test in mice. Each point and vertical line
represent the mean SEM of the values obtained in 8 (compound
9k) or 8−31 (compound 1) animals.
different animal models, prompted us to select the compound
9k as a clinical candidate for the treatment of pain.
1
solid (1.28 g, 66%). H NMR (250 MHz, CDCl3): δ 7.92 (m, 2H),
7.63−7.52 (m, 2H), 7.01 (d, J = 2.5 Hz, 1H), 4.45 (q, J = 7.1 Hz,
2H), 1.43 (t, J = 7.1 Hz, 3H).
EXPERIMENTAL SECTION
■
General Procedure for the Synthesis of 3-(Hydroxymethyl)-
pyrazole Derivatives 7. (1-(3,4-Dichlorophenyl)-5-methyl-1H-
pyrazol-3-yl)methanol (7a). To a suspension of 4a (551 mg, 1.84
mmol) in EtOH (12 mL), NaBH4 (208 mg, 5.5 mmol) was added in
portions and the mixture was stirred at 80 °C for 5 h. After this time,
H2O was added, and the mixture was extracted with DCM. The
combined organic layers were washed with aqueous sat NH4Cl, dried
over Na2SO4, filtered, and concentrated to dryness. The residue was
purified by flash chromatography (SiO2, hexane/EtOAc 60:40) to
Unless otherwise noted, all materials were obtained from commercial
suppliers and used without further purification. The reference
compound E-52862 (1) was obtained as previously described8 and
(+)-pentazocine, haloperidol, and [3H]-Di-o-tolylguanidine (DTG)
were commercially available. Flash chromatography was performed on
1
a Teledyne Isco CombiFlash RF system with disposable columns. H
NMR and 13C NMR spectra were recorded on a Agilent Mercury 300
or 400 MHz (spectrometer fitted with a 5 mm ATB 1H/19F/X
probe) with a 2 H lock in deuterated solvents. Chemical shifts (δ) are
in parts per million. Commercially available reagents and solvents
[high-performance liquid chromatography (HPLC) grade] were used
without further purification for all the analytical tests. Analytical
HPLC-MS for all final compounds and intermediates was performed
on an Agilent HP1200-MS 6110 system. A reverse-phase XBridge
C18 XP column was used (4.6 × 30 mm, 2.5 μm), gradient 5−100%
B (A = 10 mM ammonium bicarbonate, B = acetonitrile) over 8 min,
injection volume: 1 μL, flow: 2.0 mL/min, and temperature: 40 °C.
UV spectra were recorded at 210 nm using an Agilent HP1200 VWD
detector. Mass spectra were obtained over the range m/z 50−1000 by
electron spray ionization (ESI) in the positive mode using an Agilent
MS6110 simple quadrupole. Data were integrated and reported using
Agilent ChemStation software. All final compounds displayed purity
equal or higher than 95%, as determined by said methods. Accurate
mass measurements were carried out using an Agilent 6540 UHD
Accurate-Mass QTOF system and obtained by ESI in the positive
mode. Chiral analytical and preparative HPLC was performed in
Agilent 1100 analytical and preparative HPLC systems, respectively.
The coated or immobilized polysaccharide columns were acquired
from Chiral Technologies Europe; heptane and ethanol or IPA with
or without DEA at variable proportions was used as a mobile phase.
All compounds active in biological assays were electronically filtered
for structural attributes common to pan-assay interference com-
pounds and were found to be negative.39
1
give the compound 7a as a yellow solid (354 mg, 75%). H NMR
(250 MHz, CDCl3): δ 7.82 (d, J = 2.5 Hz, 1H), 7.75 (d, J = 8.5 Hz,
1H), 7.52 (m, 1H), 6.43 (s, 1H), 4.91 (s, 2H), 2.57 (s, 3H).
General Procedure for the Synthesis of 3-Methoxyalkyl-
amine-pyrazole Derivatives 9. 4-(2-((1-(3,4-Dichlorophenyl)-5-
methyl-1H-pyrazol-3-yl)methoxy)ethyl)morpholine (9b). To a
solution of 7a (80 mg, 0.31 mmol) in THF (3 mL), NaH (60% in
mineral oil, 25 mg, 0.62 mmol) was added in portions and the mixture
was stirred at rt for 5 min. Then, a solution of 4-(2-iodoethyl)-
morpholine (225 mg, 0.93 mmol) in THF (2 mL) was added, and the
mixture was stirred at 70 °C for 2 h. After this time, aqueous sat
NaHCO3 was added and the mixture was extracted with DCM. The
combined organic layers were washed with aqueous sat NaHCO3 and
H2O, dried over Na2SO4, filtered, and concentrated to dryness. The
residue was purified by flash chromatography (SiO2, DCM/MeOH
1
96:4) to give the compound 9b as a white solid (114 mg, 99%). H
NMR (250 MHz, CDCl3): δ 7.59 (d, J = 2.5 Hz, 1H), 7.52 (d, J = 8.6
Hz, 1H), 7.32−7.27 (dd, J = 2.5,8.6 Hz, 1H), 6.24 (s, 1H), 4.54 (s,
2H), 3.73 (t, J = 4.6 Hz, 4H), 3.65 (t, J = 5.8 Hz, 2H), 2.62 (t, J = 5.8
Hz, 2H), 2.51 (t, J = 4.6 Hz, 4H), 2.35 (s, 3H). 13C NMR (101 MHz,
CDCl3): δ 150.94, 140.18, 139.08, 133.25, 131.72, 130.79, 126.61,
123.72, 107.37, 67.59, 67.00, 66.86, 58.41, 54.15, 12.77. HPLC-MS:
purity 98%. HRMS [M + H]+ (diff ppm) 369.1015 (1.04).
1-(2-((1-(3,4-Dichlorophenyl)-5-methyl-1H-pyrazol-3-yl)-
methoxy)ethyl)piperazine (9i). A solution of 9h (115 mg, 0.28
mmol) in 48% HBr in AcOH (7 mL) was heated at 110 °C during 2
h. The solution was cooled to 0 °C, and a 6 M aqueous NaOH
solution was added dropwise until pH = 8−9. The mixture was
extracted with DCM. The combined organic layers were dried over
Na2SO4, filtered, and concentrated to dryness. The residue was
purified by flash chromatography (SiO2, DCM/MeOH/NH4OH
Determination of Physicochemical Properties. pKa was
calculated using ACDLABS 9.0.3 and clog P was calculated using
ChemDraw Ultra 10.0.3. Thermodynamic solubility was measured by
HPLC after stirring the solid compound for 24 h in phosphate buffer
at pH = 7.4. Log P and pKa were determined using a pHmetric
technique40 in a GlpKa or Sirius-T3 analytical instrument.
General Procedures for the Synthesis of 3-(Ethoxycarbon-
yl)-pyrazole Derivatives 4. Ethyl 1-(3,4-Dichlorophenyl)-5-meth-
yl-1H-pyrazole-3-carboxylate (4a, R1 = 3,4-Dichlorophenyl, R2 =
Me). To a suspension of 3,4-dichlorophenyl hydrazine hydrochloride
(2) (100 mg, 0.47 mmol) in AcOH (5 mL), ethyl acetopyruvate (3)
1
95:4:1) to give the compound 9i as a yellow oil (88 mg, 85%). H
NMR (400 MHz, CDCl3): δ 7.60 (dd, J = 0.3, 2.5 Hz, 1H), 7.52 (dd,
J = 0.3, 8.6 Hz, 1H), 7.30 (dd, J = 2.5, 8.6 Hz, 1H), 6.24 (q, J = 0.8
Hz, 1H), 4.54 (s, 2H), 3.64 (t, J = 5.9 Hz, 2H), 2.89 (t, J = 4.9 Hz,
F
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4H), 2.60 (t, J = 5.9 Hz, 2H), 2.54−2.39 (m, 4H), 2.35 (d, J = 0.8 Hz,
3H). 13C NMR (101 MHz, CD3OD): δ 151.03, 140.14, 139.10,
133.23, 131.69, 130.78, 126.61, 123.73, 107.39, 67.79, 66.84, 58.61,
55.07, 46.04, 12.77. HPLC-MS: purity 97%. HRMS [M + H]+ (diff
ppm) 368.1167 (−0.96).
and the mixture was stirred at 90 °C for 3 h. After this time, the
reaction mixture was concentrated to dryness and the residue was
purified by flash chromatography (SiO2, DCM/MeOH 100:0 to 93:7)
to give the compound 15 as a yellow oil (45 mg, 47%). 1H NMR (400
MHz, CDCl3): δ 7.60 (dd, J = 0.3, 2.5 Hz, 1H), 7.52 (dd, J = 0.3, 8.6
Hz, 1H), 7.30 (dd, J = 2.5, 8.6 Hz, 1H), 6.24 (q, J = 0.8 Hz, 1H), 4.50
(s, 2H), 3.62−3.59 (m, 2H), 3.57 (t, J = 6.5 Hz, 2H), 3.47−3.42 (m,
2H), 2.50−2.37 (m, 6H), 2.35 (d, J = 0.8 Hz, 3H), 2.08 (s, 3H), 1.81
(ddt, J = 6.4, 7.3, 8.7 Hz, 2H). 13C NMR (101 MHz, CD3OD): δ
169.04, 151.06, 140.14, 139.09, 133.24, 131.72, 130.78, 126.62,
123.72, 107.34, 68.95, 66.73, 55.43, 53.44, 52.92, 46.43, 41.54, 27.18,
21.49, 12.77. HPLC-MS: purity 97%. HRMS [M + H]+ (diff ppm)
424.1438 (1.3).
1-(4-(2-((1-(3,4-Difluorophenyl)-1H-pyrazol-3-yl)methoxy)ethyl)-
piperazin-1-yl)ethanone Hydrochloride (9k, Scheme 2 Method).
Step 1: To a solution of ethyl 1H-pyrazole-3-carboxylate (16) (31.0 g,
221.2 mmol), CuI (8.42 g, 44.2 mmol) and Cs2CO3 (144 g, 441.9
mmol) in MeCN (310 mL), 1,2-difluoro-4-iodobenzene (17) (71.6 g,
298.3 mmol), and TMEDA (13.35 mL, 88.5 mmol) were added and
the mixture was stirred at 85 °C for 6 h. After this time, the mixture
was cooled to rt and the formed salts were filtered and washed with
MeCN. The filtered salts were dissolved in H2O and the remaining
MeCN was removed under vacuum. The suspension was stirred at rt
during 1 h and was filtered again washing with H2O. The obtained
solid was dried under vacuum to give a 97:3 mixture of ethyl 1-(3,4-
difluorophenyl)-1H-pyrazole-3-carboxylate (18) and ethyl 1-(3,4-
difluorophenyl)-1H-pyrazole-5-carboxylate (19) as a white solid
(44.8 g, 80%). 1H NMR (250 MHz, CDCl3): δ (major isomer)
7.87 (d, J = 2.5 Hz, 1H), 7.66 (ddd, J = 10.8, 6.9, 2.7 Hz, 1H), 7.50−
7.41 (m, 1H), 7.32−7.20 (m, 1H), 6.99 (d, J = 2.5 Hz, 1H), 4.43 (q, J
= 7.1 Hz, 2H), 1.42 (t, J = 7.1 Hz, 3H).
Step 2: To a solution of a 97:3 mixture of 18 and 19 (33.5 g, 133.0
mmol) in THF (160 mL) under a nitrogen atmosphere, a 2 M
solution of LiBH4 in THF (66.5 mL, 133.0 mmol) was added and the
reaction mixture was stirred at 70 °C for 4 h. After cooling to rt, H2O
(200 mL) and 10% aqueous HCl were added until pH = 4−5. Then,
20% aqueous NaOH was added until pH = 6−7 and half of the initial
volume was removed under vacuum. The suspension was stirred at rt
for 1 h and was filtered washing with H2O. The obtained solid was
dried under vacuum to give (1-(3,4-difluorophenyl)-1H-pyrazol-3-
yl)methanol (20) as a white solid (27.4 g, 98%). 1H NMR (250 MHz,
CDCl3): δ 7.80 (d, J = 2.2 Hz, 1H), 7.54 (ddd, J = 11.1, 7.0, 2.6 Hz,
1H), 7.40−7.30 (m, 1H), 7.29−7.16 (m, 1H), 6.46 (d, J = 2.2 Hz,
1H), 4.76 (d, J = 5.1 Hz, 2H), 2.38 (t, J = 5.1 Hz, 1H).
Step 3: To a solution of 20 (27.4 g, 130.2 mmol) in THF (130
mL), KOH (36.53 g, 651.1 mmol) was added. Then, a solution of
TsCl (32.3 g, 169.3 mmol) in THF (130 mL) was slowly added and
the reaction was stirred for 2 h. H2O was added and it was extracted
with EtOAc. The combined organic phases were washed with H2O,
dried over Na2SO4, filtered, and concentrated to dryness obtaining a
brown solid that was triturated with hexane and filtered to give (1-
(3,4-difluorophenyl)-1H-pyrazol-3-yl)methyl 4-methylbenzenesulfo-
nate (21) as a white solid (42.4 g, 89%). 1H NMR (250 MHz,
CDCl3): δ 7.80 (d, J = 8.2 Hz, 2H), 7.76 (d, J = 2.4 Hz, 1H), 7.56
(ddd, J = 11.2, 6.6, 2.2 Hz, 1H), 7.34−7.17 (m, 4H), 6.46 (d, J = 2.7
Hz, 1H), 5.14 (s, 2H), 2.43 (s, 3H).
4-(3-(1-(3,4-Dichlorophenyl)-5-methyl-1H-pyrazol-3-yl)propyl)-
morpholine (13a). Step 1: To a solution of 7a (542 mg, 2.11 mmol)
in DCM (30 mL), MnO2 (2.16 g, 21.1 mmol) was added and the
mixture was stirred at 40 °C for 16 h. The mixture was cooled to rt,
filtered through Celite and concentrated to dryness. The residue was
purified by flash chromatography (SiO2, hexane/EtOAc 80:20) to
give 1-(3,4-dichlorophenyl)-5-methyl-1H-pyrazole-3-carbaldehyde
(10a) as a yellow solid (403 mg, 75%). 1H NMR (250 MHz,
CDCl3): δ 9.98 (s, 1H), 7.65 (d, J = 2.5 Hz, 1H), 7.61 (d, J = 8.5 Hz,
1H), 7.36 (dd, J = 2.5, 8.5 Hz, 1H), 6.73 (s, 1H), 2.39 (s, 3H).
Step 2: To a solution of 4-(2-iodoethyl)morpholine (2.0 g, 8.29
mmol) in xylenes (15 mL), PPh3 (3.26 g, 12.44 mmol) was added and
the mixture was stirred at 135 °C for 16 h. The mixture was cooled to
rt; toluene (8 mL) was added and it was stirred for 2 h more. The
obtained solid was filtered, washed with Et2O, and dried under
vacuum to give 2-(morpholin-4-yl)ethyl]triphenylphosphonium io-
1
dide (11a) as a white solid (3.96 g, 95%). H NMR (250 MHz,
CDCl3): δ 7.93−7.83 (m, 15H), 3.84 (m, 2H), 3.29 (m, 4H), 2.59
(m, 2H), 2.30 (m, 4H).
Step 3: To a solution of 11a (177 mg, 0.353 mmol) in THF (3
mL), KOtBu (79 mg, 0.7 mmol) was added and the mixture was
stirred at rt for 1 min. Then, a solution of 10a (108 mg, 0.42 mmol) in
THF (3 mL) was added and the mixture was stirred at rt for 1 h more.
After this time, H2O was added and the mixture was extracted with
DCM. The combined organic layers were dried over Na2SO4, filtered,
and concentrated to dryness. The residue was purified by two
consecutive flash chromatography purifications (SiO2, hexane/EtOAc
50:50 and DCM/MeOH/NH4OH 98:2:1) to give 4-(3-(1-(3,4-
dichlorophenyl)-5-methyl-1H-pyrazol-3-yl)allyl)morpholine (12a) as
1
a yellow oil (45 mg, 36%). H NMR (250 MHz, CDCl3): δ (major
isomer) 7.62 (d, J = 2.5 Hz, 1H), 7.54 (d, J = 8.5 Hz, 1H), 7.33 (dd, J
= 2.5, 8.5 Hz, 1H), 6.45 (d, J = 11.8 Hz, 1H), 6.22 (s, 1H), 5.84 (m,
1H), 3.74 (m, 4H), 3.46 (dd, J = 1.9, 6.3 Hz, 2H), 2.53 (m, 4H), 2.37
(s, 3H).
Step 4: To a solution of 12a (123 mg, 0.35 mmol) in MeOH (4
mL), PtO2 (4 mg, 0.017 mmol) was added and the solution was
stirred under a H2 atmosphere during 2 h. After this time, the mixture
was filtered through Celite and concentrated to dryness. The residue
was purified by flash chromatography (SiO2, hexane/acetone 60:40)
1
to give 13a as a yellow solid (79 mg, 64%). H NMR (250 MHz,
CDCl3): δ 7.59 (d, J = 2.5 Hz, 1H), 7.50 (d, J = 8.8 Hz, 1H), 7.29
(dd, J = 2.5, 8.8 Hz, 1H), 6.02 (s, 1H), 3.72 (m, 4H), 2.64 (t, J = 7.7
Hz, 2H), 2.44 (m, 6H), 2.32 (s, 3H), 1.87 (m, 2H). 13C NMR (101
MHz, CDCl3): δ 154.04, 139.60, 139.36, 133.15, 131.20, 130.71,
126.39, 123.51, 107.00, 67.16, 58.72, 53.89, 26.55, 26.20, 12.80.
HPLC-MS: purity 96%. HRMS [M + H]+ (diff ppm) 353.1071
(2.62).
1-(4-(3-((1-(3,4-Dichlorophenyl)-5-methyl-1H-pyrazol-3-yl)-
methoxy)propyl)piperazin-1-yl)ethanone (15). Step 1: A mixture of
7a (100 mg, 0.39 mmol), Bu4NSO4H (6 mg, 0.02 mmol), and 1-
bromo-3-chloropropane (0.09 mL, 0.97 mmol) in 40% aqueous
NaOH (0.5 mL) and toluene (0.5 mL) was vigorously stirred at rt for
20 h. After this time, H2O was added and the mixture was extracted
with DCM. The combined organic layers were dried over Na2SO4,
filtered, and concentrated to dryness. The residue was purified by
flash chromatography (SiO2, hexane/EtOAc 90:10) to give 3-((3-
chloropropoxy)methyl)-1-(3,4-dichlorophenyl)-5-methyl-1H-pyrazole
Steps 4 and 5: To a suspension of NaH (60%, 2.64 g, 66.0 mmol)
in THF (200 mL), 1-(4-(2-hydroxyethyl)piperazin-1-yl)ethanone
hydrochloride (22) (11.3 g, 65.8 mmol) was added in portions and
the suspension was stirred for 30 min. Then, 21 (20.0 g, 54.9 mmol)
was added in portions and the reaction mixture was stirred at rt for 3
h. After this time, H2O (200 mL) and 37% aqueous HCl were added
until pH = 7−7.5; excess THF was removed under vacuum and 37%
aqueous HCl was again added until pH = 2−3. The aqueous phase
was washed with EtOAc and 20% aqueous NaOH was added until pH
= 8.5−10. The aqueous phase was then extracted with EtOAc. The
combined organic phases were concentrated to dryness to afford the
free base of 9k. Methyl ethyl ketone was added and the solution was
heated to 50 °C. A 5 M solution of HCl in IPA (11 mL, 55 mmol)
was added and the solution was left to crystallize at 0 °C; the obtained
1
(14) as a yellow oil (78 mg, 56%). H NMR (250 MHz, CDCl3): δ
7.61 (d, J = 2.5 Hz, 1H), 7.57−7.50 (d, J = 8.7 Hz, 1H), 7.31 (dd, J =
8.7, 2.5 Hz, 1H), 6.25 (s, 1H), 4.52 (s, 2H), 3.66 (m, 4H), 2.35 (s,
3H), 2.06 (m, 2H).
Step 2. To a solution of 1-acetylpiperazine (29 mg, 0.22 mmol) in
DMF (5 mL), DIPEA was added (0.11 mL, 0.67 mmol) and the
mixture was stirred at rt for 15 min. Then, a solution of 14 (75 mg,
0.22 mmol) and NaI (50 mg, 0.33 mmol) in DMF (2 mL) was added
G
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Article
solid was filtered and dried under vacuum to give 9k as a white solid
(16.7 g, 76%). mp 155−157 °C; H NMR (250 MHz, CDCl3): δ
Pharmacokinetic Studies. Groups of two male Wistar rats
(250−300 g; Harlan) or 16 male CD1 mice (25−30 g; Charles River)
were given oral or intravenous doses of 9k and 1 in 0.5%
hydroxypropyl methylcellulose or saline solution, respectively. Dose
levels were 10 mg/kg for oral studies and 1 mg/kg for intravenous
studies. Blood samples were collected at different times from the
saphenous vein or by intracardiac puncture into heparinized tubes.
Plasma was obtained by blood centrifugation at 4 °C and 2280g for 10
min and kept at −80 °C until analysis. Plasma samples were assayed
by high-performance liquid chromatography−triple quadrupole mass
spectrometry (HPLC-MS/MS) after plasma protein precipitation.
Pharmacokinetic Parameters. Standard pharmacokinetic pa-
rameters, such as the area under the curve (AUC), peak plasma
concentration (Cmax), time to peak concentration (tmax), oral
bioavailability (F), total plasma clearance (Cl), volume of distribution
at the steady state (Vss), and terminal half-life (t1/2), were determined
by noncompartmental analysis of the plasma concentration−time
curves (Phoenix v.6.2.1.51, Pharsight, CA).
1
13.14 (bs, 1H), 7.82 (d, J = 2.5 Hz, 1H), 7.55 (ddd, J = 11.1, 6.9, 2.5
Hz, 1H), 7.42−7.32 (m, 1H), 7.31−7.17 (m, 1H), 6.46 (d, J = 2.5 Hz,
1H), 4.68 (s, 1H), 4.62 (s, 2H), 4.17−4.04 (m, 3H), 3.91−3.39 (m, 4
H), 3.27 (s, 2H), 2.87 (s, 2H), 2.09 (s, 3H). 13C NMR (101 MHz,
CD3OD): δ 171.76, 152.43, 151.83 (dd, J = 13.7, 247.6 Hz), 150.06
(dd, J = 12.7, 246.5 Hz), 138.05 (dd, J = 3.0, 5.3 Hz), 130.42, 119.23
(d, J = 18.8 Hz), 116.21 (dd, J = 3.7, 6.4 Hz), 109.90 (d, J = 22.0 Hz),
108.98, 67.43, 64.23, 57.42, 52.98, 52.78, 44.04, 39.28, 20.88. HPLC-
MS: purity 97%. HRMS [M + H]+ (diff ppm) 364.1717 (1.67).
Human Sigma-1 Receptor Radioligand Assay.8 The binding
properties of the test compounds to human σ1R were studied in
transfected HEK-293 membranes using [3H](+)-pentazocine (Perki-
nElmer, NET-1056) as the radioligand. The assay was carried out
with 7 μg of membrane suspension, [3H]-(+)-pentazocine (5 nM, 100
μL), 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) presoaked
in 100 μL of 0.1% polyethyleneimine and filtered. Then, plates were
washed (three 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 (PerkinElmer) using an EcoScint
liquid scintillation cocktail.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01575.
Analytical data for all the final compounds and
intermediates. HPLC traces for final compounds of
formula 9, 13, and 15. Experimental description of in
vivo studies (PDF)
Guinea Pig Sigma-2 Receptor Radioligand Assay. The
binding properties of test compounds to guinea pig σ2R were studied
in guinea pig brain membranes as described13 with some
modifications. DTG (PerkinElmer, 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 presence of
either buffer or 10 μM haloperidol for total and nonspecific binding,
respectively. Binding buffer contained Tris-HCl (50 mM, pH 8) and
σ1R was blocked with (+)-SKF10047 at 400 nM. 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 plates were washed three 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
(PerkinElmer) using an EcoScint liquid scintillation cocktail.
Molecular Formula Strings (CSV)
AUTHOR INFORMATION
■
Corresponding Author
Carmen Almansa − ESTEVE Pharmaceuticals, Torre Esteve,
08038 Barcelona, Spain; orcid.org/0000-0001-5665-
4685; Phone: 0034650697372; Email: calmansa@
welab.barcelona
hERG Assay.29 CHO cells stably expressing hERG channels
(Millipore) were cultured in F12 HAM medium supplemented with
10% FBS and 400 μg/L Geneticin. Extracellular Ringer’s solution
consisted of (in mM) 2 CaCl2, 1 MgCl2, 10 HEPES, 4 KCl, 145 NaCl,
and 10 glucose with pH 7.4 and 305 mOsm. Intracellular Ringer’s
solution consisted of (in mM) 5.37 CaCl2, 1.75 MgCl2, 31.25/10
KOH/EGTA, 10 HEPES, and 210 KCl with pH 7.2 and 295 mOsm.
Na2-ATP (4 mM) was added to intracellular Ringer’s solution shortly
before use. Whole-cell currents were measured with a QPatch system
(Sophion) in response to continuously executed voltage protocols as
per the manufacturer’s recommendations. Upon the onset of the
voltage protocol, cells were maintained at a holding potential (Vh) of
−80 mV, then clamped briefly to −50 mV (20 ms), subsequently
depolarized to 20 mV for 4800 ms, and finally repolarized to −50 mV
for 5000 ms, at which potential the peak outward tail current was
measured. Finally, the voltage returned to Vh for 3100 ms. Thus,
voltage protocols were repeated for each 15 s. For each cell, the
extracellular solution was applied previously to increasing concen-
trations of the tested compound.
In Vivo Studies. The experimental detail of capsaicin and PSNL
models is given in the Supporting Information. All animal research
was conducted in accordance with protocols approved by the local
Committee of Animal Use and Care, with the Care and Use of
Laboratory Animals Guidelines of the European Community
(European Directive 2010/63/EU), and with the International
Association for the Study of Pain Guidelines on ethical standards
for investigation in animals.41 Animal studies are reported in
compliance with the ARRIVE guidelines.42,43
Authors
́
Jose Luis Díaz − ESTEVE Pharmaceuticals, Torre Esteve,
08038 Barcelona, Spain; orcid.org/0000-0001-8812-
4689
́
Monica García − ESTEVE Pharmaceuticals, Torre Esteve,
08038 Barcelona, Spain
Antoni Torrens − ESTEVE Pharmaceuticals, Torre Esteve,
08038 Barcelona, Spain
Ana María Caaman
Coruña, Spain
̃
o − Galchimia, S.A., 15823 O Pino, A
Juan Enjo − Galchimia, S.A., 15823 O Pino, A Coruña, Spain
Cristina Sicre − Galchimia, S.A., 15823 O Pino, A Coruña,
Spain
Adriana Lorente − ESTEVE Pharmaceuticals, Torre Esteve,
08038 Barcelona, Spain
Adriana Port − ESTEVE Pharmaceuticals, Torre Esteve,
08038 Barcelona, Spain
Ana Montero − ESTEVE Pharmaceuticals, Torre Esteve,
08038 Barcelona, Spain
Sandra Yeste − ESTEVE Pharmaceuticals, Torre Esteve,
08038 Barcelona, Spain
́
́
Ines Alvarez − ESTEVE Pharmaceuticals, Torre Esteve,
08038 Barcelona, Spain
Miquel Martín − Laboratory of Neuropharmacology, Facultat
de Ciències de la Salut i de la Vida, Universitat Pompeu
Fabra, 08003 Barcelona, Spain
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Rafael Maldonado − Laboratory of Neuropharmacology,
Facultat de Ciències de la Salut i de la Vida, Universitat
Pompeu Fabra, 08003 Barcelona, Spain
Beatriz de laPuente − ESTEVE Pharmaceuticals, Torre
Esteve, 08038 Barcelona, Spain
Alba Vidal-Torres − ESTEVE Pharmaceuticals, Torre Esteve,
08038 Barcelona, Spain
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Cruz Miguel Cendan − Department of Pharmacology, Faculty
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of Medicine, University of Granada, 18016 Granada, Spain
́
Jose Miguel Vela − ESTEVE Pharmaceuticals, Torre Esteve,
08038 Barcelona, Spain
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https://pubs.acs.org/10.1021/acs.jmedchem.0c01575
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Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Ma Magdalena Bordas, Raquel Enrech, Joan Andreu
Morato, Monica Carro, Edmundo Ortega, Manel Merlos, Pilar
■
́
́
́
́
Perez, Javier Burgueno, Marta Pujol, Enrique Hernandez,
̃
́
́
Javier Farre, Eva Ayet, Raquel Fernandez-Reinoso, Maria
́
Teresa Serafini, Carmen Segales, Alicia Pardo, Daniel
Zamanillo, and Lucia Romero for their expert contribution
to analytical, in vitro, and in vivo studies and Carlos Perez and
Eduardo Villarroel for their contribution to compound
management. This work was a part of activities in R&D
Projects IDI-20110577 and IDI-20130942 supported by the
Spanish Ministerio de Economia y Competitividad (MINE-
CO), through the Centro para el Desarrollo Tecnologico
́
́
́
́
Industrial (CDTI), cofinanced by the European Union
through the European Regional Development Fund (ERDF;
Fondo Europeo de Desarrollo Regional, FEDER).
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ABBREVIATIONS
■
ADME, absorption, distribution, metabolism, and excretion;
ER, endoplasmic reticulum; BCS, biopharmaceutics classifica-
tion system; CNS MPO, central nervous system multi-
parameter optimization; DMPK, drug metabolism and
pharmacokinetics; hERG, human ether-a-go-go-related gene;
PGRMC1, progesterone receptor membrane component 1;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide; PSNL, partial sciatic nerve ligation; SAR, struc-
ture−activity relationships; σ1R, sigma-1 receptor; σ1R-KO,
sigma-1 receptor knockout mice
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