Tricyclic Triazoles as σ1Receptor Antagonists for Treating Pain

Article abstract of DOI:10.1021/acs.jmedchem.1c00244

The synthesis and pharmacological activity of a new series of 5a,7,8,8a-tetrahydro-4H,6H-pyrrolo[3,4-b][1,2,3]triazolo[1,5-d][1,4]oxazine derivatives as potent sigma-1 receptor (σ1R) ligands are reported. A lead optimization program aimed at improving the aqueous solubility of parent racemic nonpolar derivatives led to the identification of several σ1R antagonists with a good absorption, distribution, metabolism, and excretion in vitro profile, no off-target affinities, and characterized by a low basic pKa (around 5) that correlates with high exposure levels in rodents. Two compounds displaying a differential brain-to-plasma ratio distribution profile, 12lR and 12qS, exhibited a good analgesic profile and were selected as preclinical candidates for the treatment of pain.

Full text of DOI:10.1021/acs.jmedchem.1c00244

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Article
Tricyclic Triazoles as σ1 Receptor Antagonists for Treating Pain
́
́
José Luis Díaz, Félix Cuevas, Ana I. Oliva, Daniel Font, M. Angeles Sarmentero, Paula Alvarez-Bercedo,
̀
José M. López-Valbuena, Miquel A. Pericas, Raquel Enrech, Ana Montero, Sandra Yeste,
Alba Vidal-Torres, Inés Alvarez, Pilar Pérez, Cruz Miguel Cendán, Enrique J. Cobos, José Miguel Vela,
́
and Carmen Almansa*
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ABSTRACT: The synthesis and pharmacological activity of a new
series of 5a,7,8,8a-tetrahydro-4H,6H-pyrrolo[3,4-b][1,2,3]triazolo[1,5-
d][1,4]oxazine derivatives as potent sigma-1 receptor (σ1R) ligands are
reported. A lead optimization program aimed at improving the aqueous
solubility of parent racemic nonpolar derivatives led to the identification
of several σ1R antagonists with a good absorption, distribution,
metabolism, and excretion in vitro profile, no off-target affinities, and
characterized by a low basic pKa (around 5) that correlates with high
exposure levels in rodents. Two compounds displaying a differential
brain-to-plasma ratio distribution profile, 12lR and 12qS, exhibited a
good analgesic profile and were selected as preclinical candidates for the
treatment of pain.
pharmacological and molecular differentiation of the two σ
receptors. The two proteins have a low homology with very
low sequence similarity, although many medicinal chemistry
programs have shown that achieving selectivity is far from
obvious. This is probably due to the presence of common acid
residues in their binding sites, with two key acidic groups in
both cases, Asp126 and Glu172 in the σ1R and Asp29 and
Asp56 in the σ2R.19 In fact, the majority of σR ligands present
a basic amine that effects key interactions with these acid
groups, and the σ1R pharmacophore models available,
represented by Glennon’s22 and Laggner’s,23 reflect the need
for both a positive ionizable (PI) moiety and the hydro-
phobicity of the receptor that often requires two hydrophobic
features at opposite sides of the PI (amino group).
The development of σ1R antagonists for the treatment of
pain was pioneered by E-52862 (1, Figure 1),24 which showed
efficacy in a phase II clinical trial on oxaliplatin-induced
neuropathy25 and a relevant reduction of opioid-associated
adverse events and the need for concomitant antiemetic
medication when coadministered with morphine.26 In a back-
up program aimed at the identification of an alternative series
of σ1R antagonists,27,28 we identified the parent racemic
INTRODUCTION
■
The sigma receptors (σR), discovered in the mid-1970s, and
initially considered as a subset of opioid receptors,1 are actually
a class of unique proteins comprising at least two distinct
subtypes, namely, σ1 and σ2.2,3 The σ1R subtype is a ligand-
regulated chaperone4 of 223 amino acids and 25 kDa cloned in
19965,6 and crystallized 20 years later.7 Residing primarily in
the interface between the endoplasmic reticulum (ER) and the
mitochondrion, it can translocate to the plasma membrane and
regulate the activity of other proteins including N-methyl-D-
aspartate receptors8 and several ion channels.9 Owing to the
role played by the σ1R in modulating pain-related hyper-
sensitivity and sensitization phenomena,10 σ1R antagonists
have been proposed for the treatment of neuropathic
pain.11−13 Additionally, the σ1R modulates opioid analge-
sia,14,15 with σ1R antagonists enhancing the antinociceptive
effect of opioids.16
Based on growing evidence of the involvement of σ1R in
pain control,17 our group has been actively working in the
search of σ1R antagonists for the treatment of pain.
Compounds with a good selectivity profile, especially versus
the σ2R, have been pursued. More than 40 years were needed
to identify the σ2R, which was in 2011 putatively assigned to
the progesterone receptor membrane component 118 but later
on cloned and identified as transmembrane protein-97
(TMEM97),19 an ER-resident transmembrane molecule
implicated in cholesterol homeostasis.20 The σ2R has potential
for multiple therapeutic applications,21 but we aimed to
discover selective σ1R compounds due to the clear
Received: February 7, 2021
Published: April 7, 2021
https://doi.org/10.1021/acs.jmedchem.1c00244
© 2021 American Chemical Society
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Figure 1. Structure of the σ1R antagonist E-52862 (1) and tricyclic derivatives 2, 3, and 4.
tricyclic derivatives 2 and 3,29 which in contrast to the majority
of existing σ1R ligands exhibited a low basic pKa (around 5), an
attribute potentially linked to the improved safety and
penetration into the central nervous system (CNS).30 In fact,
in the initial in vitro profiling of compound 2, the only
drawback was its low aqueous solubility that could hamper an
adequate in vivo behavior. Otherwise, compound 2 exhibited a
good absorption, distribution, metabolism, and excretion
(ADME) profile, with adequate in vitro metabolic stability in
human and rodent liver microsomes,31 no potential for drug−
drug interactions based on the low inhibition (<50% at 10
μM) of recombinant human cytochrome P450 (rhCYP)
isoforms (1A2, 2C9, 2C19, 2D6, 2E1, and 3A4),32 and IC50
> 10 μM in the human ether-a-go-go-related gene (hERG)33
patch clamp assay, a well-known predictor of cardiac toxicity.
This last result is of particular relevance because hERG
inhibition was a recurrent issue in our previous σ1R
programs.24,28
Compounds 2 and 3 were consistent with the σ1R
pharmacophore requirements and contained two aromatic
rings at the opposite sides of the amine group. In a previous
work, it was shown that changing the phenyl ring attached to
the triazole ring by an alkyl group proved to be detrimental,29
while replacing the opposite benzyl group by a pentyl chain
(4a) maintained affinity,12 but was worse in terms of metabolic
stability. As expected, eliminating the basic amine by attaching
a phenyl group to the pyrrolidine nitrogen atom completely
abolished activity (4b).29
shown in Tables 1−3. Throughout the paper, the letter R
following the compound number refers to the 5R,8R
enantiomer, while the letter S refers to the 5S,8S enantiomer.
The study began with the preparation of the individual
enantiomers of the previously reported29 compounds 2 and 3.
In both cases, the R,R enantiomers (2R and 3R) were slightly
more active toward the σ1R than their S,S counterparts (2S and
3S). Selectivity versus the σ2R was high, and the four isomers
exhibited a kinetic solubility below 10 μM, which is the usual
cut-off value for progressing compounds in our workflows. As
outlined in the Introduction, compounds 2 and 3 exhibited
otherwise a good in vitro ADME profile, which made them
good leads for a lead optimization program focused on
improving the solubility. At the beginning of this work, the cis
derivative (20, Figure 2) of parent compound 3 was also
prepared and although it maintained the σ1R affinity, its
metabolic stability was much lower, and for this reason the
efforts were directed to the trans isomers. It is also worth
mentioning that the requirement of a substituted basic amine
mentioned in the introduction was confirmed by the inactivity
of Boc-protected and NH intermediates, represented by 10lR
and 11lR, respectively (Figure 2).
With the aim of improving solubility, we undertook a lead
optimization program that involved the preparation of more
than 300 compounds and only selected compounds, illustrating
the most representative findings, are reported here. We began
with the exploration of R1 and some of the 4-fluorobenzyl
derivatives prepared are depicted in Table 1. Introduction of
polarity led to some decrease in affinity and more differ-
entiation between enantiomers, with the R,R isomer still being
the eutomer. This trend was general and is illustrated for the
pair 12aR/12aS. Introduction of a nitrogen atom was
somehow better in the ortho (12aR) than in the meta
(12bR) or para (12cR) positions, while a second nitrogen
atom was clearly detrimental (12fR). Introduction of
heterocycloalkyl moieties was also assayed and only the
tetrahydropyran ring 12gR maintained affinity, in a similar way
as had been observed in other σ1R ligand families.27 Other
groups were less tolerated, as illustrated by N-methylpiperidine
12h. Except in the case of 4-fluoro-pyridine 12eR, all these
modifications led to an increase in kinetic solubility above the
10 μM threshold value. Particularly outstanding was the
solubility of 12gR, associated with its high polarity.
The tricyclic scaffold represented by 2 was considered to be
a good starting point for a lead optimization program, aimed at
improving the solubility by means of increasing the polarity.
This was not considered to be a trivial task because polar
groups are often not tolerated in the highly hydrophobic σ1R.
We report herein the synthesis and structure−activity relation-
ship (SAR) studies that led to the identification of two
5a,7,8,8a-tetrahydro-4H,6H-pyrrolo[3,4-b][1,2,3]triazolo[1,5-
d][1,4]oxazine preclinical candidates for the treatment of
pain.34
RESULTS AND DISCUSSION
■
All the compounds synthesized were evaluated in a primary
human σ1R-binding assay using [3H]-(+)-pentazocine24 as the
radioligand in human transfected HEK-293 membranes and
against the human σ2/TMEM97 cloned receptor-enriched
membranes using [3H]-di-o-tolylguanidine35 as the radioligand.
The kinetic solubility of all the compounds was measured as
described in the Experimental Section. The compounds were
tested initially under screening conditions that allow specific
kinetic solubility determination up to 10 μM [the final
maximum compound concentration in dimethyl sulfoxide
(DMSO)]. For those active compounds with a solubility
value higher than 10 μM, more accurate values were obtained
using a more concentrated DMSO solution. The results are
The introduction of heteroatoms to the benzyl group in R2
was also assayed (Table 2). Five-membered heterocyclylmethyl
substituents, represented by 12iR, showed decreased affinity.
Among the naked pyridylmethyl derivatives, the meta-isomer
12jR was the most active (ortho- and para-isomers showed Kis
above 500 nM, results not shown), although it showed a
decreased affinity versus its benzyl counterpart 2R. Shielding
the pyridine nitrogen with a trifluoromethyl (12kR) or
methoxy (12lR) substituent increased activity. Compound
12lR showed improved solubility versus the initial benzyl
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derivatives, both due to its increased polarity and the ortho-
fluoro substituent in the phenyl group in R1, which was
relevant in view of the decreased solubility of its meta-fluoro
analogue, 12mR. The R,R isomer was confirmed again to be
the eutomer, as shown with the S,S derivative 12lS, one order
of magnitude less active than 12lR.
Table 1. Initial SAR Data Maintaining a 4-Fluorobenzyl
Group in R2
Changing the R2 substituent to a tetrahydropyranyl group
provided an interesting set of derivatives (Table 3). Again,27
the tetrahydropyranyl group was the only tolerated among
other similar groups tested, such as the 4-piperidinyl (Ki above
1000 nM, not shown). The data of 12nR and 12nS showed
that in this case, the S,S isomer was the eutomer. Several
halophenyl derivatives were prepared and in general they
maintained affinity, but regarding solubility the presence of an
ortho-substituent was beneficial. In fact, the introduction of
halogens in the ortho position of R1 was designed from the
beginning as a means of increasing solubility by decreasing
planarity,36 a fact exemplified by the 54.5° dihedral angle of
compound 12pS (see Figure 4).
The introduction of a second polar point was also assayed,
but it generally reduced affinity because it is not easy to find
the correct positioning of polar functionalities in a highly
lipophilic receptor such as the σ1R. In fact, only a nitrogen
atom in the more shielded ortho position was tolerated, to give
compound 12qS, which emerged as the most interesting
derivative in this group. Its enantiomer, 12qR, was poorly
active, confirming that when the tetrahydropyranyl is directly
linked to the nitrogen atom, S,S is the eutomer. On the
contrary, when the tetrahydropyranyl-containing chain was
elongated, both enantiomers showed a similar σ1R affinity (see
12rR and 12rS). The two enantiomers of 12r maintained the
desired selectivity for the σ2R, but increasing lipophilicity as in
12sR, 12tR, and 12uR provided poorly selective derivatives.
The soluble and selective compounds were then tested in an
additional battery of tests, and two compounds, 12lR and
12qS, emerged as the most interesting derivatives. Both of
them showed a good selectivity (inhibition at 1 μM < 50% for
another 180 molecular targets),37 high permeability in human
Caco-2-cells, high metabolic stability in human liver micro-
somes, were devoid of hERG inhibition, and showed an
acceptable physicochemical profile, as indicated by their low
basicity and adequate lipophilicity (Table 4). Additionally, the
compounds exhibited low CYP inhibition or induction,
suggesting low potential for drug−drug interactions.38
The functional activity of compounds 12lR and 12qS was
established by incubating in the presence of phenytoin.39 Both
of them were identified as σ1R antagonists on account of the
small shift to a lower affinity that was observed in relation to Ki
obtained without phenytoin (Ki without phenytoin/Ki with
phenytoin: 12lR = 0.4 0.07, 12qS = 0.9 0.47).
The pharmacokinetic profiles of 12lR and 12qS were
determined in mice and rats after oral and intravenous
administration of single doses of 10 and 1 mg/kg, respectively
(Table 5). Oral bioavailability (F) was higher than 50% in both
species, except for 12qS in mice (25%). Peak plasma
concentrations (Cmax) following oral administration were
high (for 12lR, 4657 ng/mL in rats and 5363 ng/mL in
mice and for 12qS, 4036 ng/mL in rats and 1864 ng/mL in
mice). Total plasma clearances were low (inferior to 10% of
liver blood flow in rats and mice) and the volumes of
distribution (Vd) were higher than the total body water (≥0.6
L/kg in rats and mice). No species differences were observed
in elimination half-lives, with mean values higher than 2 h in
a
Binding affinity (Ki) to human σ1R in transfected HEK-293
membranes using [3H](+)-pentazocine as the radioligand. Each
value is the mean standard deviation (SD) of two determinations.
b
Binding affinity (Ki, nM) to σ2R in human σ2/TMEM97-cloned
receptor-enriched membranes using [3H]di-o-tolylguanidine as the
c
radioligand. Each value is the mean SD of two determinations. clog
P calculated using ChemDraw. Kinetic solubility measured according
to the method described in the Experimental Section part. Synthesis
performed according to Scheme 1. Less than 50% inhibition at 1 μM
d
e
f
g
concentration. Less than 50% inhibition at 10 μM concentration.
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Table 2. SAR Data of Heteroarylmethyl Derivatives in R2
a
Binding affinity (Ki) to human σ1R in transfected HEK-293 membranes using [3H](+)-pentazocine as the radioligand. Each value is the mean
b
SD of two determinations. Binding affinity (Ki, nM) to σ2R in human σ2/TMEM97-cloned receptor-enriched membranes using [3H]di-o-
tolylguanidine as the radioligand. Each value is the mean SD of two determinations. clog P calculated using ChemDraw. Kinetic solubility
measured according to the method described in the Experimental Section part. Synthesis performed according to Scheme 1. Less than 50%
inhibition at 1 μM concentration. Less than 50% inhibition at 10 μM concentration. Not tested.
c
d
e
f
g
h
rats and mice. Overall, both 12lR and 12qS showed reduced
volumes of distribution and reached a much higher plasma
exposition in relation to compound 1. It is generally accepted
that higher values of Vd are obtained for compounds with
increased lipophilicity and basicity.40 Hence, the results
observed here can be explained by the reduced pKa (around
5 for 12lR/12qS vs 6.4 for 1) and lipophilicity (exp log P 2.7
for 12lR, 1.7 for 12qS, and 3.5 for 1) of 12lR and 12qS versus
1.
The kinetics of 12lR and 12qS in brain and plasma was also
assessed in mice after single intraperitoneal (ip) administration
of 5 and 20 mg/kg, respectively. Brain and plasma peak
concentrations were observed at 5 min after administration in
both compounds. Brain concentrations of 12qS were higher
than the corresponding plasma concentrations as shown by a
brain-to-plasma AUC0−t ratio of 1.6. Conversely, brain
concentrations of 12lR were lower than the corresponding
plasma concentrations, with a brain-to-plasma AUC0−t ratio of
0.5.
Compounds 12lR and 12qS were profiled in vivo in the
intraplantar capsaicin-induced mechanical hypersensitivity41
mice model. After oral administration, both 12lR and 12qS
showed higher potency in comparison to compound 1 (12lR
ED50 5.3, 12qS 14 vs 28 mg/kg, respectively, Figure 3, Table
5). Capsaicin, the pungent ingredient of hot peppers, binds to
transient receptor potential vanilloid 1, a nonselective cation
channel gated by noxious heat and extracellular protons (acidic
pH), and activates pain pathways. When applied on the skin,
intraepidermally or subcutaneously, capsaicin induces secon-
dary hyperalgesia (mechanical hypersensitivity) that results
from sensitization of dorsal horn neurons in the spinal cord
and extends to supraspinal pain-modulating CNS regions.
Accordingly, capsaicin-induced sensitization to mechanical
stimulation is underpinned by central mechanisms as it
involves changes in central processing of pain, including
enhanced responses to peripheral stimuli, which is a hallmark
in chronic pain disorders, particularly neuropathic pain. Of
particular interest is the observation that it has been shown to
evoke similar sensory changes in healthy human volunteers,
allowing for translation between animals and humans.42
Capsaicin sensitization is especially relevant for pain research
in the σ1R field as σ1R knockout does not develop pain
following capsaicin sensitization,41 and σ1R antagonists inhibit,
whereas σ1R agonists promote capsaicin-induced pain.43
Hence, it is a unique tool to establish the functional profile
of the σ1R ligands in vivo.
Chemistry. As outlined in Scheme 1, the synthesis of the
new tricyclic trans-fused chiral triazoles 12 involves the
derivatization of the chiral azidoalcohols 5 (R,R or S,S),29
which were obtained in the enantiopure form from the achiral
epoxide using the appropriate Jacobsen chiral catalyst.44
Reaction of 5 with the corresponding propargyl bromides 6
with NaH as the base and tetrabutylammonium iodide (TBAI)
as the catalyst in tetrahydrofuran (THF) (method A) provided
alkynes 9. The propargyl derivatives 6 were prepared by the
treatment of alcohols 7 with CBr4 and PPh3 in dichloro-
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Table 3. SAR Data of Tetrahydropyranyl Derivatives in R2
a
Binding affinity (Ki) to human σ1R in transfected HEK-293 membranes using [3H](+)-pentazocine as the radioligand. Each value is the mean
b
SD of two determinations. Binding affinity (Ki, nM) to σ2R in human σ2/TMEM97-cloned receptor-enriched membranes using [3H]di-o-
tolylguanidine as the radioligand. Each value is the mean SD of two determinations. clog P calculated using ChemDraw. Kinetic solubility
measured according to the method described in the Experimental Section part. Synthesis performed according to Scheme 1. Less than 50%
inhibition at 1 μM concentration. Less than 50% inhibition at 10 μM concentration.
c
d
e
f
g
methane (DCM), which in turn were obtained by Sonogashira
coupling of the corresponding aryl halides 8 and propargyl
alcohol in the presence of CuI and Pd(PPh3)4 in triethylamine.
The intramolecular thermal azide−alkyne cycloaddition in
refluxing toluene or xylenes afforded the tricyclic triazoles 10
with excellent yields. Finally, the diversity in R2 to provide the
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Figure 2. Structures of compounds 10lR, 11lR, and 20.
Table 4. Physicochemical and In Vitro ADME Data for
Compounds 12lR and 12qS
c
Caco-2
a
b
d
e
hERG
HLM Clint
Papp A−B
(nm/s)
exp
exp
comp IC50 (μM) (μL/min/mg prot)
log P
pKa
12lR
12qS
>10
>10
3.6
0.2
371
374
2.7
1.7
4.9
5.2
a
b
Whole-cell patch clamp hERG blockade. Intrinsic clearance in
c
human liver microsomes. Permeability in the Caco-2 human cell line.
d
Experimental log P obtained as described in the Experimental
e
Section part. Experimental pKa obtained as described in the
Figure 3. Dose−response effect of 12lR, 12qS, and 1 on the
capsaicin-induced mechanical hypersensitivity test in mice.
Experimental Section part.
three different synthetic methods. Although the absolute
configuration of the azidoalcohols and the subsequent cyclized
intermediates was clearly established in previous studies,29 the
X-ray single crystal analysis of compound 12pS (Figure 4)
confirmed its absolute configuration to be S,S (Parsons
absolute structure parameter, 0.04(5)). This structure also
showed that the chlorophenyl ring is out of the plane of the
triazole, with a dihedral angle of 54.5(5), indicating that, as
expected, the presence of a chlorine atom in ortho provides a
twist from planarity.
Racemic cis compound 20 was prepared using the same
general methodology (method A) from racemic cis azidoalco-
hol 19 (Scheme 2). Compound 19 was prepared from trans-
azido alcohol 18 by inversion of the configuration of the
hydroxyl group. This involved the formation of the mesylate
with methanesulfonyl chloride, substitution by acetate with
KOAc in DMF, and hydrolysis of the acetate with K2CO3 in
MeOH.45
desired tricyclic triazoles 12 was introduced by standard Boc
deprotection and subsequent reductive amination of 11 with
the corresponding aldehyde or ketone 13, in the presence of
NaBH(OAc)3 as the reductant and 1,2-dichloroethane (DCE)
as the solvent.
As an alternative process (method B) for the synthesis of
propargyl intermediates 9, azidoalcohols 5 were alkylated with
propargyl bromide and the Sonogashira reaction was then
carried out to afford the corresponding substituted alkynes
with a moderate to good yield.
Depending on the nature of substituent R1, a further
alternative process (method C) to prepare tricyclic triazoles 10
was developed. Reaction of 14 with N-iodomorpholine
hydriodide in the presence of CuI as the catalyst gives the
iodo intermediate 15, which is cyclized under the above
described thermal conditions to afford the two enantiopure
iodo triazoles 16. These in turn are derivatized using the
Suzuki reaction with the corresponding boronic acids 17 in the
presence of Pd(PPh3)4 as the catalyst and K2CO3 as the base.
The enantiomeric excess of the enantiopure compounds 12
was determined by chiral high-performance liquid chromatog-
raphy (HPLC) and found to be always above 95%, correlating
with that of the starting enantiopure azido alcohol 5 and
indicating that no epimerization took place in any step of the
CONCLUSIONS
■
The lead optimization program over a new series of 5a,7,8,8a-
tetrahydro-4H,6H-pyrrolo[3,4-b][1,2,3]triazolo[1,5-d][1,4]-
oxazine derivatives as σ1R ligands, mainly focusing on the
introduction of polarity to improve aqueous solubility, led to
Table 5. In Vivo Data for Compounds 12lR and 12qS in Comparison with 1
b
c
mouse pharmacokinetics 10 mg/kg, po
rat pharmacokinetics 10 mg/kg, po
a
capsaicin ED50
Cmax
t1/2
AUC0−∞
Cl
Vd
Cmax
t1/2 AUC0−∞ Cl Vd
comp
(95% CI) (mg/kg)
(ng/mL) (h)
(ng·h/mL) (% LBF) (L/kg) F (%) (ng/mL) (h) (ng·h/mL) (% LBF) (L/kg) F (%)
12lR
12qS
1
5.3 (4.4−6.1)
14 (13−15)
28 (20−41)
5363
1864
622
3.8
2.6
0.7
34,738
16,320
958
6
6
65
1.5
0.6
2.2
104
25
34
4657
4036
301
4.5
4.1
1.2
37,900
45,434
428
6
6
109
0.5
0.9
2.9
54
87
15
a
b
Efficacy dose 50 (ED50) and 95% CI for the reduction of mechanical hypersensitivity after po administration in the mouse capsaicin test. Mouse
c
pharmacokinetic parameters obtained as described in the Experimental Section part. Rat pharmacokinetic parameters obtained as described in the
Experimental Section part.
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a
Scheme 1
a
Reagents and conditions: (a) NaH, n-Bu4NI, THF, 0 °C to rt; (b) CBr4, PPh3, DCM, rt; (c) CuI, Pd(PPh3)4, Et3N, 40−60 °C; (d) toluene or
xylenes, reflux; (e) HCl, 1,4-dioxane, rt; (f) NaBH(OAc)3, DCE, rt; (g) CuI, N-iodomorpholine hydriodide, THF, rt; and (h) K2CO3, Pd(PPh3)4,
dimethyl ether/EtOH/H2O, 90 °C.
1
disposable columns. H spectra were recorded on a Bruker Fourier
300 MHz, Bruker Avance I 400 MHz, a Bruker Avance I 500 MHz or
Agilent Mercury 400 MHz. Chemical shifts (δ) are in parts per
million. Commercially available reagents and solvents (HPLC grade)
were used without further purification for all the analytical tests.
Analytical HPLC for all final compounds and intermediates was
performed on an Agilent HP1200 6120 system. UV spectra were
recorded at 254 nm using an Agilent HP1200 VWD detector. Data
were integrated and reported using Agilent ChemStation software.
Method 1: column Agilent Eclipse XDB-C18 (4.6 × 150 mm, 5 μm),
Figure 4. X-ray structure of compound 12pS (CCDC 2027373).
gradient 5−95% B [A = H2O (0.05% trifluoroacetyl) and B =
acetonitrile] over 7 min, then isocratic 95% B over 5 min, injection
volume: 1 μL, and flow: 1 mL/min. All final compounds displayed
purity equal to or higher than 95% as determined using the said
methods, except for 12gR, whose purity was 94.4%. Accurate mass
measurements were carried out using a Waters LCT-Premier system
and by electron spray ionization in a positive mode. Chiral analytical
HPLC was performed on an Agilent 1260 system. The coated or
immobilized polysaccharide columns were acquired from Chiral
Technologies Europe. Method 2: Chiralcel AS-H column (4.6 × 250
mm, 5 μm), isocratic n-heptane/ethanol + 0.33% diethanolamine
70:30, injection volume: 5 μL, and flow: 0.8 mL/min. Method 3:
ChiralPak IB column (4.6 × 250 mm, 5 μm), isocratic n-heptane/
ethanol + 0.33% DEA 70:30, injection volume: 5 μL, and flow: 0.8
mL/min. Method 4: ChiralPak IB column (4.6 × 250 mm, 5 μm),
isocratic n-heptane/isopropanol 90:10, injection volume: 5 μL, and
flow: 0.8 mL/min. All compounds active in biological assays were
electronically filtered for structural attributes common to pan assay
interference compounds and were found to be negative.46
the identification of several highly selective σ1R antagonists
that exhibited a good ADME in vitro profile. The compounds
were also characterized by a low basic pKa (around 5) that
correlates with high exposure levels in rodents and could be
linked to improved safety and penetration into the CNS. Two
compounds, 12lR and 12qS, exhibited a good analgesic profile
in vivo. Due to their differential brain-to-plasma ratio
distribution profile and thus their potential to treat different
indications in the neuropathic pain field, both compounds
were selected as preclinical development candidates. Their
further characterization will be reported in due course.
EXPERIMENTAL SECTION
■
Unless otherwise noted, all materials were obtained from commercial
suppliers and used without further purification. Flash chromatography
was performed on a Teledyne Isco CombiFlash RF system with
a
Scheme 2
a
Reagents and conditions: (a) MsCl, Et3N, DCM, rt; (b) KOAc, 18-crown-6, DMF, 100 °C; (c) K2CO3, MeOH, rt; (d) NaH, 1-(3-bromoprop-1-
yn-1-yl)-3-fluorobenzene, n-Bu4NI, THF, 0 °C to rt; and (e) toluene, reflux.
5163
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Determination of Physicochemical Properties. pKa was
calculated using ACD Labs 9.0.3 and clog P using ChemDraw Ultra
10.0.3. Kinetic solubility was measured by HPLC after centrifugation
of a sample obtained by adding phosphate buffer at pH = 7.4 (1 mL)
to 10 μL of a 10 mM solution of the test compound in DMSO and
stirring for 4 h. For determining more accurate values of kinetic
solubility in those compounds showing a solubility higher than 10
μM, the experiment was repeated with 15 μL of a 100 mM solution of
the test compound in DMSO. 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 values were determined
using a pH-metric technique47 on a GlpKa or a Sirius-T3 Analytical
instrument.
7.7, 1.3 Hz, 1H), 7.28 (ddd, J = 11.1, 8.2, 1 Hz, 1H), 5.43 (dd, J =
15.8, 1.3 Hz, 1H), 5.27 (m, 1H), 4.69 (m, 1H), 4.39 (m, 1H), 4.33
(dd, J = 11, 7.3 Hz, 1H), 3.85 (dd, J = 11, 7.3 Hz, 1H), 3.73 (t, J =
11.4 Hz, 1H), 3.44 (t, J = 11.4 Hz, 1H).
Step 5. (5aR,8aR)-3-(2-Fluorophenyl)-7-((6-methoxypyridin-3-
yl)methyl)-5a,7,8,8a-tetrahydro-4H,6H-pyrrolo[3,4-b][1,2,3]-
triazolo[1,5-d][1,4]oxazine Hydrochloride (12lR). To a suspension
of compound 11lR (12.0 g, 36 mmol) in dichloroethane (800 mL)
cooled at 0 °C, a solution of DIPEA (25 mL, 144 mmol) in
dichloroethane (100 mL) was added. After stirring at rt for 10 min, 6-
methoxynicotinaldehyde (5.9 g, 43.2 mmol) and NaBH(OAc)3 (15.2
g, 72 mmol) were added and the reaction mixture was stirred at rt for
48 h. DCM was added and the mixture was washed with NaHCO3
salt solution and brine, dried over Na2SO4, filtered, and concentrated.
Purification by flash chromatography, using silica gel and gradient
cyclohexane to ethyl acetate, afforded the title product as the free base
General Procedures for the Synthesis of Compounds 12
and 20. (5aR,8aR)-3-(2-Fluorophenyl)-7-((6-methoxypyridin-3-yl)-
methyl)-5a,7,8,8a-tetrahydro-4H,6H-pyrrolo[3,4-b][1,2,3]triazolo-
[1,5-d][1,4]oxazine Hydrochloride (12lR, Method A). Step 1. 1-(3-
Bromoprop-1-yn-1-yl)-2-fluorobenzene (6l). To a mixture of CuI
(2.23 g, 11.7 mmol) and Pd(PPh3)4 (2.71 g, 2.3 mmol) in Et3N (300
mL) under Ar at rt, 1-fluoro-2-iodobenzene (40 g, 180 mmol) was
added and the reaction mixture was stirred at rt for 30 min. Propargyl
alcohol (11.5 mL, 198 mmol) was added and the mixture was heated
at 60 °C for 20 h. Water was added, extracted with EtOAc, washed
with brine, dried with Na2SO4, filtered through a pad of Celite, and
the solvent was removed under vacuum. The crude was dissolved in
DCM (900 mL), CBr4 (47.7 g, 140 mmol) was added, and the
solution was cooled at 0 °C. PPh3 (42.4 g, 162 mmol) was slowly
added and the reaction mixture was allowed to reach rt and stirred for
16 h. EtOH (100 mL) was added and the mixture was stirred for 1 h.
The solvent was removed under vacuum, cyclohexane was added, and
the resulting solid was filtered. The filtrate was filtered through a pad
of silica with cyclohexane as the eluent to afford the title product
1
(11.6 g, 84% yield). H NMR (400 MHz, CD3OD): δ 8.12 (dd, J =
2.4, 0.8 Hz, 1H), 7.78 (td, J = 7.6, 1.8 Hz, 1H), 7.74 (dd, J = 8.5, 2.5
Hz, 1H), 7.45 (dddd, J = 8.2, 7.2, 5.3, 1.8 Hz, 1H), 7.31 (td, J = 7.6,
1.2 Hz, 1H), 7.24 (ddd, J = 11.2, 8.3, 1.2 Hz, 1H), 6.82 (dd, J = 8.5,
0.7 Hz, 1H), 5.29 (dd, J = 15.7, 1.4 Hz, 1H), 5.15 (ddd, J = 15.7, 1.9,
1.1 Hz, 1H), 4.47 (td, J = 9.6, 7.3 Hz, 1H), 4.18 (td, J = 9.7, 7.4 Hz,
1H), 3.93 (d, J = 12.9 Hz, 1H), 3.91 (s, 3H), 3.88 (d, J = 13.1 Hz,
1H), 3.68 (dd, J = 9.2, 7.1 Hz, 1H), 3.18 (dd, J = 9.1, 7.4 Hz, 1H),
3.21−3.11 (m, 1H), 2.96 (dd, J = 9.9, 9.1 Hz, 1H). 13C NMR (101
MHz, CD3OD): δ 165.26, 160.55 (d, J = 246.1 Hz), 147.71, 141.30,
137.83 (d, J = 2.6 Hz), 131.78 (d, J = 8.6 Hz), 131.10 (d, J = 3.5 Hz),
130.93, 128.48, 125.97 (d, J = 3.4 Hz), 119.27 (d, J = 14.3 Hz),
116.94 (d, J = 22.1 Hz), 111.63, 79.80, 65.72 (d, J = 15.7 Hz), 60.32,
58.39, 54.11, 52.77, 51.61. HPLC: RT 5.27 min, purity 99.2%;
HRMS: calcd for C20H21N5O2F [M + H]+, 382.1679; found,
382.1692 [M + H]+. The free base product (10.8 g, 28.3 mmol)
was dissolved in ethyl acetate (300 mL) and 1.25 M HCl solution in
EtOH (22.7 mL, 28.3 mmol) was added. The mixture was stirred at rt
for 15 min and the solvent was removed under reduced pressure until
1
(32.8 g, 86% yield). H NMR (500 MHz, CDCl3): δ 7.46 (t, J = 7.3
Hz, 1H), 7.35 (m, 1H), 7.12 (t, J = 7.7 Hz, 1H), 7.10 (t, J = 8.5 Hz,
1H), 4.21 (s, 2H).
Step 2. tert-Butyl (3R,4R)-3-Azido-4-((3-(2-fluorophenyl)prop-2-
yn-1-yl)oxy)pyrrolidine-1-carboxylate (9lR, R1 = 2-Fluorophenyl).
To a solution of (3R,4R)-tert-butyl 3-azido-4-hydroxypyrrolidine-1-
carboxylate44 (15.0 g, 65.7 mmol) in dry THF (400 mL) at 0 °C
under Ar, a suspension of NaH (6.05 g, 60% in mineral oil, 151
mmol) in THF (200 mL) was added. When the bubbling was
finished, the reaction mixture was allowed to warm to rt and stirred
for 1 h. The reaction mixture was cooled at 0 °C and TBAI (7.3 g,
19.7 mmol) and a solution of 6l (18.2 g, 85 mmol) in THF (200 mL)
were slowly added at 0 °C. The reaction mixture was stirred at rt for
16 h and after cooling to 0 °C, the NH4Cl sat solution (100 mL) and
water (100 mL) were added. The aqueous phase was extracted with
ethyl acetate and washed with water. Purification by flash
chromatography, using silica gel and the gradient from hexane to
1
dryness to afford 12lR (11.6 g, quant) as hydrochloride. H NMR
(400 MHz, CD3OD): δ 8.36 (d, J = 2.5 Hz, 1H), 7.89 (dd, J = 8.6, 2.5
Hz, 1H), 7.83 (td, J = 7.7, 1.8 Hz, 1H), 7.48 (dddd, J = 8.4, 7.3, 5.3,
1.8 Hz, 1H), 7.33 (td, J = 7.6, 1.2 Hz, 1H), 7.26 (ddd, J = 11.3, 8.4,
1.1 Hz, 1H), 6.94 (dd, J = 8.6, 0.7 Hz, 1H), 5.41 (dd, J = 15.9, 1.5 Hz,
1H), 5.30−5.20 (m, 1H), 4.81−4.74 (m, 1H), 4.53 (t, J = 15.0 Hz,
2H), 4.48−4.38 (m, 1H), 4.39−4.28 (m, 1H), 3.97 (s, 3H), 3.85 (t, J
= 9.2 Hz, 2H), 3.57 (t, J = 11.1 Hz, 1H); Chiral HPLC, method 2:
RT 21.74 min, 99.0% ee; [α]2D0 −2.8 (c 0.20, MeOH).
(5aS,8aS)-3-(5-Fluoropyridin-2-yl)-7-(tetrahydro-2H-pyran-4-yl)-
5a,7,8,8a-tetrahydro-4H,6H-pyrrolo[3,4-b][1,2,3]triazolo[1,5-d]-
[1,4]oxazine Hydrochloride (12qS, Method B). Step 1. (3S,4S)-tert-
Butyl-3-azido-4-(prop-2-ynyloxy)pyrrolidine-1-carboxylate (14S).
To a solution of 5S (12.5 g, 54.8 mmol) in dry THF (100 mL)
cooled at 0 °C, a suspension of NaH (4.38 g, 60% in mineral oil, 110
mmol) in THF (100 mL) was added. When the bubbling was
finished, the reaction mixture was allowed to warm to rt and stirred
for 45 min. Propargyl bromide (12.2 mL, 80% solution in toluene,
110 mmol) and TBAI (2.17 g, 5.48 mmol) were slowly added at 0 °C
and the reaction was stirred at rt for 16 h. The NH4Cl aqueous
saturated solution was added and the mixture was extracted with ethyl
acetate, washed with water, dried over Na2SO4, filtered, and
concentrated. Purification by flash chromatography using silica gel
and gradient hexane to hexane/ethyl acetate (1:1) afforded
1
ethyl acetate, afforded 9lR (17.7 g, 75% yield). H NMR (400 MHz,
CDCl3): δ 7.45 (m, 1H), 7.39−7.29 (m, 1H), 7.15−7.05 (m, 2H),
4.52−4.46 (m, 2H), 4.20 (m, 1H), 4.14 (m, 1H), 3.72−3.61 (m, 2H),
3.57−3.38 (m, 2H), 1.47 (s, 9H).
Step 3. tert-Butyl (5aR,8aR)-3-(2-Fluorophenyl)-5a,6,8,8a-tetra-
hydro-4H,7H-pyrrolo[3,4-b][1,2,3]triazolo[1,5-d][1,4]oxazine-7-car-
boxylate (10lR, R1 = 2-Fluorophenyl). Compound 9lR (9.0 g, 24.9
mmol) was dissolved in xylene (850 mL) and heated at reflux for 20
h. The solvent was removed under vacuum and the product was
purified by precipitation with MeOH to give compound 10lR (6.0 g,
1
83% yield). H NMR (300 MHz, CDCl3): δ 7.98 (m, 1H), 7.38 (m,
1
compound 14S (12.5 g, 86% yield) as a yellow oil. H NMR (400
1H), 7.27 (td, J = 8, 1 Hz, 1H), 7.14 (t, J = 9 Hz, 1H), 5.33 (dd, J =
16, 2 Hz, 1H), 5.15 (d, J = 16 Hz, 1H), 4.6−4.2 (m, 2H), 4.1−3.8 (m,
2H), 3.59 (m, 1H), 3.39 (m, 1H), 1.50 (s, 9H).
MHz, CDCl3): δ 4.27−4.17 (m, 2H), 4.13 (m, 1H), 4.07 (m, 1H),
3.70−3.56 (m, 2H), 3.52−3.37 (m, 2H), 2.51 (m, 1H), 1.48 (s, 9H).
Step 2. tert-Butyl (3S,4S)-3-Azido-4-((3-(5-fluoropyridin-2-yl)-
prop-2-yn-1-yl)oxy) pyrrolidine-1-carboxylate (9qS, R1 = 5-Fluo-
ropyridin-2-yl). To a mixture of compound 14S (3.3 g, 12.4 mmol),
CuI (94 mg, 0.49 mmol), Pd(PPh3)4 (286 mg, 0.24 mmol), 2-iodo-5-
fluoropyridine (4.42 g, 19.8 mmol) in DMF (60 mL) under Ar at rt,
Et3N (5.2 mL, 37.2 mmol) was added and the mixture was heated at
40 °C for 2.5 h. The reaction mixture was cooled to rt, and an
aqueous saturated solution of NH4Cl was added. The mixture was
Step 4. (5aR,8aR)-3-(2-Fluorophenyl)-4,5a,6,7,8,8a-
hexahydropyrrolo[3,4-b][1,2,3]triazolo[1,5-d][1,4]oxazine (11lR, R1
= 2-Fluorophenyl). To a solution of compound 101R (13.0 g, 36.1
mmol) in dioxane (86 mL), a solution of 4 M HCl in dioxane (117
mL, 469 mmol) was added and the mixture was stirred at rt for 16 h.
The solvent was concentrated to dryness to afford compound 11lR
1
(12.0 g, quantitative yield) as dihydrochloride. H NMR (500 MHz,
CD3OD): δ 7.84 (td, J = 7.7, 1.9 Hz, 1H), 7.50 (m, 1H), 7.35 (td, J =
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extracted with a mixture of ethyl acetate/cyclohexane several times.
The organic phase was dried over Na2SO4, filtered, and concentrated.
Purification by flash chromatography, using silica gel and gradient
cyclohexane to 30% ethyl acetate, afforded 9qS (3.2 g, 71% yield). 1H
NMR (300 MHz, CDCl3): δ 8.46 (d, J = 3 Hz, 1H), 7.49 (dd, J = 9, 5
Hz, 1H), 7.40 (dt, J = 8, 3 Hz, 1H), 4.47 (m, 2H), 4.18 (m, 1H), 4.10
(m, 1H), 3.73−3.58 (m, 2H), 3.57−3.36 (m, 2H), 1.47 (s, 9H).
Step 3. tert-Butyl (5aS,8aS)-3-(5-Fluoropyridin-2-yl)-5a,6,8,8a-
tetrahydro-4H,7H-pyrrolo[3,4-b][1,2,3]triazolo[1,5-d][1,4]oxazine-
7-carboxylate (10qS, R1 = 5-Fluoropyridin-2-yl). Compound 9qS
(8.0 g, 22.1 mmol) was dissolved in xylene (738 mL) and the mixture
was heated at reflux for 20 h. The reaction mixture was cooled at rt
and the crystallized solid was filtered to afford compound 10qS (5.85
g, 73% yield). 1H NMR (300 MHz, CDCl3): δ 8.45 (d, J = 3 Hz, 1H),
8.25 (dd, J = 9, 4 Hz, 1H), 7.51 (dt, J = 8, 3 Hz, 1H), 5.65 (d, J = 17
Hz, 1H), 5.29 (d, J = 17 Hz, 1H), 4.58−4.28 (m, 2H), 4.07−3.88 (m,
2H), 3.57 (m, 1H), 3.42 (m, 1H), 1.52 (s, 9H).
Step 2. (3R,4R)-tert-Butyl-3-azido-4-((3-iodoprop-2-yn-1-yl)oxy)-
pyrrolidine-1-carboxylate (15R). To a solution of 14R (0.69 g, 2.6
mol) in THF (15 mL), CuI (25 mg, 0.13 mmol) and N-
iodomorpholine hydriodide (1.0 g, 2.9 mmol) were added. The
reaction mixture was stirred at rt for 2 h. The suspension obtained was
poured onto a pad of neutral alumina and the filtrate was collected
under vacuum. The solid phase was washed with DCM and the
combined filtrate was concentrated under vacuum to afford 15R (0.99
g, 97% yield) as a yellow oil. 1H NMR (300 MHz, CDCl3): δ 4.38 (m,
2H), 4.07 (m, 1H), 4.03 (m, 1H), 3.61 (m, 2H), 3.44 (m, 2H), 1.46
(s, 9H).
Step 3. (5aR,8aR)-tert-Butyl-3-iodo-5a,6,8,8a-tetrahydropyrrolo-
[3,4-b][1,2,3]triazolo[1,5-d][1,4]oxazine-7(4H)-carboxylate (16R). A
solution of 15R (0.99 g, 2.5 mmol) in toluene (65 mL) was heated at
reflux for 20 h. The reaction mixture was cooled and the solvent
evaporated under vacuum. Purification by flash chromatography using
silica gel and gradient hexane to hexane/ethyl acetate (8:2) afforded
16R (0.76 g, 77% yield). 1H NMR (400 MHz, CDCl3): δ 5.11 (d, J =
16 Hz, 1H), 4.92 (d, J = 16 Hz, 1H), 4.5−4.2 (m, 2H), 4.0−3.8 (m,
2H), 3.51 (m, 1H), 3.37 (m, 1H), 1.49 (s, 9H).
Step 4. (5aS,8aS)-3-(5-Fluoropyridin-2-yl)-5a,7,8,8a-tetrahydro-
4H,6H-pyrrolo[3,4-b][1,2,3]triazolo[1,5-d][1,4]oxazine (11qS, R1
=
5-Fluoropyridin-2-yl). To a solution of 10qS (2.14 g, 5.9 mmol) in
dioxane (10 mL), a solution of 4 M HCl in dioxane (20 mL) was
added and stirred at rt for 20 h. The mixture was concentrated to
dryness to afford compound 11qS as dihydrochloride (1.95 g, 99%
yield). 1H NMR (400 MHz, CD3OD): δ 8.63 (m, 1H), 8.24 (dd, J =
7.9, 3.9 Hz, 1H), 7.84 (td, J = 8.5, 2.9 Hz, 1H), 5.76 (d, J = 16.8, Hz,
1H), 5.46 (dd, J = 16.6, 1.1 Hz, 1H), 4.75 (m, 1H), 4.43 (m, 1H),
4.39 (m, 1H), 3.91 (dd, J = 11.1, 7.7 Hz, 1H), 3.78 (t, J = 11.4 Hz,
1H), 3.51 (t, J = 11.2 Hz, 1H).
Step 4. (5aR,8aR)-tert-Butyl-3-(3,6-dihydro-2H-pyran-4-yl)-
5a,6,8,8a-tetrahydropyrrolo [3,4-b][1,2,3]triazolo[1,5-d][1,4]-
oxazine-7(4H)-carboxylate (9gR, R1 = 3,6-Dihydro-2H-pyran-4-yl).
To a mixture of 16R (250 mg, 0.64 mmol), 3,6-dihydro-2H-pyran-4-
boronic acid pinacol ester (161 mg, 0.76 mol), K2CO3 (352 mg, 2.55
mmol), and Pd(Ph3)4 (37 mg, 0.032 mmol) under nitrogen, a mixture
of dimethoxyethane/ethanol/water 4/1/0.2 (21 mL) was added. The
reaction mixture was heated at 90 °C for 20 h and after cooling it was
diluted with DCM. The mixture was washed with the NaHCO3
aqueous saturated solution, dried over Na2SO4, filtered, and
concentrated under vacuum. Purification by flash chromatography
using silica gel and gradient hexane to hexane/ethyl acetate (1:1)
Step 5. (5aS,8aS)-3-(5-Fluoropyridin-2-yl)-7-(tetrahydro-2H-
pyran-4-yl)-5a,7,8,8a-tetrahydro-4H,6H-pyrrolo[3,4-b][1,2,3]-
triazolo[1,5-d][1,4]oxazine Hydrochloride (12qS). To a suspension
of compound 11qS (1.95 g, 5.84 mmol) in dichloroethane (117 mL),
DIPEA (3.06 mL, 17.51 mmol) was added and the mixture was stirred
at rt for 10 min. Tetrahydro-4H-pyran-4-one (808 μL, 8.75 mmol)
and NaBH(OAc)3 (2.47 g, 11.67 mmol) were added and the reaction
mixture was stirred at rt for 48 h. DCM was added and the mixture
was washed with the NaHCO3 aqueous saturated solution and brine.
The organic phase was dried over Na2SO4, filtered, and concentrated.
Purification by flash chromatography using silica gel and gradient
hexane to ethyl acetate afforded the title compound as a free base
(1.88 g, 93% yield). 1H NMR (400 MHz, DMSO): δ 8.63 (dt, J = 3.0,
0.7 Hz, 1H), 8.16 (ddd, J = 8.9, 4.6, 0.7 Hz, 1H), 7.85 (td, J = 8.8, 2.9
Hz, 1H), 5.47 (d, J = 16.4 Hz, 1H), 5.23 (dd, J = 16.4, 1.1 Hz, 1H),
4.42 (td, J = 9.6, 7.8 Hz, 2H), 4.16−4.04 (m, 1H), 3.93−3.81 (m,
2H), 3.60 (dd, J = 8.7, 7.2 Hz, 1H), 3.28 (d, J = 9.6 Hz, 1H), 3.15
(dd, J = 8.5, 7.3 Hz, 1H), 3.01 (dd, J = 10.1, 8.7 Hz, 1H), 2.86 (dd, J
= 9.8, 8.6 Hz, 1H), 2.72 (tt, J = 10.6, 3.9 Hz, 1H), 1.86−1.70 (m,
2H), 1.51−1.30 (m, 2H). 13C NMR (101 MHz, DMSO): δ 158.04
(d, J = 253.5 Hz), 147.32 (d, J = 3.6 Hz), 140.39, 137.67 (d, J = 24.2
Hz), 130.44, 124.50 (d, J = 18.9 Hz), 120.94 (d, J = 4.6 Hz), 77.61,
65.81, 65.72, 64.99, 58.50, 58.26, 48.01, 46.91, 31.05, 30.74. HPLC:
RT 4.75 min, purity 99.4%. HRMS: calcd for C17H21N5O2F [M +
H]+, 346.1679; found, 346.1663. The free base product (279 mg, 0.81
mmol) was dissolved in a mixture of DCM/ethyl acetate 3:1 (8 mL)
and HCl 1.25 M solution in EtOH (0.68 mL, 0.81 mmol) was added.
The mixture was stirred at rt for 20 min and the solvent was removed
under reduced pressure until dryness to afford 12qS (308 mg, quant)
as hydrochloride. 1H NMR (400 MHz, CD3OD): δ 8.52 (dt, J = 3.0,
0.7 Hz, 1H), 8.18 (ddd, J = 8.9, 4.4, 0.7 Hz, 1H), 7.71 (td, J = 8.6, 2.9
Hz, 1H), 5.68 (d, J = 16.7 Hz, 1H), 5.38 (d, J = 16.4 Hz, 1H), 4.72−
4.57 (m, 1H), 4.53−4.38 (m, 1H), 4.38−4.20 (m, 1H), 4.09 (dd, J =
11.6, 4.4 Hz, 2H), 3.99−3.68 (m, 2H), 3.64−3.39 (m, 4H), 2.25−
2.02 (m, 2H), 1.86−1.60 (m, 2H). Chiral HPLC, method 2: RT 9.9
min, 98.4% ee; [α]2D0 +23.1 (c 0.17, MeOH).
1
afforded 9gR (160 mg, 72% yield). H NMR (400 MHz, CDCl3): δ
5.85 (s, 1H), 5.28 (d, J = 16 Hz, 1H), 5.08 (d, J = 16 Hz, 1H), 4.5−
4.2 (m, 4H), 4.0−3.8 (m, 4H), 3.53 (m, 1H), 3.37 (m, 1H), 2.68 (m,
2H), 1.49 (s, 9H).
Step 5. (5aR,8aR)-tert-Butyl-3-(tetrahydro-2H-pyran-4-yl)-
5a,6,8,8a-tetrahydropyrrolo[3,4-b][1,2,3]triazolo[1,5-d][1,4]-
oxazine-7(4H)-carboxylate (10gR, R1 = Tetrahydro-2H-pyran-4-yl).
A suspension of 9gR (325 mg, 0.93 mmol), ammonium formate (882
mg, 14.0 mmol), and Pd/C (20% w/w, 65 mg) in MeOH/THF (1:1)
(30 mL) under a nitrogen atmosphere was heated at 75 °C for 20 h.
The suspension was filtered through Celite and washed with MeOH.
The filtrate was evaporated under vacuum and the residue obtained
was portioned with DCM and water. The organic layer was washed
with the NaHCO3 aqueous saturated solution, dried over Na2SO4,
filtered, and the solvent was removed under vacuum to afford 10gR as
1
a white solid (319 mg, 98% yield). H NMR (400 MHz, CDCl3): δ
5.24 (d, J = 15.3 Hz, 1H), 5.03 (d, J = 14.6 Hz, 1H), 4.5−4.2 (m,
2H), 4.06 (m, 2H), 4.0−3.8 (m, 2H), 3.53 (m, 3H), 3.36 (m, 1H),
3.00 (m, 1H), 1.85 (m, 4H), 1.49 (s, 9H).
Step 6. (5aR,8aR)-3-(Tetrahydro-2H-pyran-4-yl)-5a,7,8,8a-tetra-
hydro-4H,6H-pyrrolo[3,4-b][1,2,3]triazolo[1,5-d][1,4]oxazine
(11gR, R1 = Tetrahydro-2H-pyran-4-yl). To a solution of 10gR (318
mg, 0.90 mmol) in dioxane (1.6 mL), a solution of 4 M HCl in
dioxane (3.4 mL) was added. The mixture was stirred at rt for 4 h and
concentrated to dryness to afford 11gR as dihydrochloride (290 mg,
1
99% yield). H NMR (500 MHz, CD3OD): δ 5.48 (d, J = 15 Hz,
1H), 5.25 (d, J = 15 Hz, 1H), 4.6 (m, 1H), 4.36 (m, 1H), 4.33 (m,
1H), 4.11 (m, 1H), 4.09 (m, 1H), 3.88 (dd, J = 10.7, 7.1 Hz, 1H),
3.71 (t, J = 10.4 Hz, 1H), 3.63 (m, 2H), 3.46 (t, J = 10.7 Hz, 1H),
3.10 (m, 1H), 1.92 (m, 4H).
Step 7. (5aR,8aR)-7-(4-Fluorobenzyl)-3-(tetrahydro-2H-pyran-4-
yl)-5a,7,8,8a-tetrahydro-4H,6H-pyrrolo[3,4-b][1,2,3]triazolo[1,5-d]-
[1,4]oxazine Hydrochloride (12gR). To a suspension of 11gR (290
mg, 0.89 mmol) in dichloroethane (25 mL), DIPEA (500 μL, 2.71
mmol) was added and the mixture was stirred at rt for 5 min. 4-
Fluorobenzaldehyde (135 μL, 1.26 mmol) and NaBH(OAc)3 (380
mg, 1.79 mmol) were added and the reaction mixture was stirred at rt
for 20 h. DCM was added and the mixture was washed with the
NaHCO3 aqueous saturated solution and brine, dried over Na2SO4,
(5aR,8aR)-7-(4-Fluorobenzyl)-3-(tetrahydro-2H-pyran-4-yl)-
5a,7,8,8a-tetrahydro-4H,6H-pyrrolo[3,4-b][1,2,3]triazolo[1,5-d]-
[1,4]oxazine Hydrochloride (12gR, Method C). Step 1. (3R,4R)-tert-
Butyl-3-azido-4-(prop-2-ynyloxy)pyrrolidine-1-carboxylate (14R).
The compound was prepared as described above for 14S but starting
from 5R.
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filtered, and concentrated. Purification by flash chromatography using
silica gel and gradient hexane to ethyl acetate afforded the title
product as a free base (222 mg, 69% yield). H NMR (300 MHz,
1H), 7.14 (m, 1H), 7.06 (m, 1H), 4.51 (m, 2H), 4.39 (m, 1H), 3.86
(m, 1H), 3.69 (s, 2H), 3.06 (dd, J = 10.2, 7 Hz, 1H), 3.00 (dd, J =
10.2, 6.4 Hz, 1H), 2.73 (m, 2H).
1
CDCl3): δ 7.29 (m 2H), 7.01 (m, 2H), 5.19 (d, J = 15 Hz, 1H), 4.98
(d, J = 15.4 Hz, 1H), 4.28 (m, 1H), 4.05 (m, 2H), 3.95 (m, 1H), 3.87
(m, 2H), 3.65 (dd, J = 9.5, 7.2 Hz, 1H), 3.51 (m, 2H), 3.12 (m, 2H),
2.97 (m, 2H), 1.83 (m, 4H). HPLC: RT 4.70 min, purity 94.4%.
HRMS: calcd for C19H24N4O2F [M + H]+, 359.1883; found,
359.1888 [M + H]+. The free base product (222 mg, 0.62 mmol)
was dissolved in ethyl acetate (2 mL) and HCl 1.25 M solution in
EtOH (0.50 mL, 0.62 mmol) was added. The mixture was stirred at rt
for 30 min and the solvent was removed under reduced pressure until
Step 5. (5a,8a-cis)-7-Benzyl-3-(3-fluorophenyl)-5a,7,8,8a-tetra-
hydro-4H,6H-pyrrolo[3,4-b][1,2,3]triazolo[1,5-d][1,4]oxazine (20).
(3,4-cis)-3-Azido-1-benzyl-4-((3-(3-fluorophenyl) prop-2-yn-1-yl)-
oxy)pyrrolidine (68 mg, 0.19 mmol) was dissolved in toluene (8
mL) and heated at 120 °C for 20 h. The solvent was removed under
vacuum and the product was purified by flash chromatography, using
silica gel and gradient from hexane to ethyl acetate, to afford 20 (50
mg, 73% yield). 1H NMR (400 MHz, CDCl3): δ 7.45−7.25 (m, 8H),
7.05 (m, 1H), 5.14 (d, J = 15 Hz, 1H), 4.91−4.85 (m, 2H), 4.60 (td, J
= 6.5, 2.4 Hz, 1H), 3.71 (m, 2H), 3.38 (dd, J = 9.3, 6.8 Hz, 1H), 3.25
(dd, J = 11.4, 6.0 Hz, 1H), 2.94 (dd, J = 9.0, 6.8 Hz, 1H), 2.85 (dd, J
= 11.4, 2.4 Hz, 1H). HPLC: RT 5.50 min, purity 96.6%; HRMS: calcd
for C20H20N4OF [M + H]+, 351.1621; found, 351.1631 [M + H]+.
Single-Crystal X-ray Structure Determination of 12pS.
Deposition number was CCDC 2027373. Crystals of 12pS were
obtained by slow evaporation in diethylether. The analyzed crystal
was prepared for measurement under inert conditions and immersed
in perfluoropolyether as a protecting oil for manipulation. Data
collection: intensity measurements were carried out in a Bruker DUO
microsource anode (Mo Kα radiation, λ = 0.71073 Å) equipped with
a APEX II CCD detector. The data collections were performed at 100
K using an Oxford Cryostream 700+ low temperature device.
Programs used: data collection and data processing were carried out
using a Bruker APEX II v2009.1-02.48 Absorption correction was
performed using TWINABS-Version 2012/1.49 Structure Solution
and Refinement: the structures were solved through the dual-space
algorithm and refined on F2 by the full-matrix least-squares method
using the programs SHELXT and SHELXL 2018/350 under the
SHELXle51 interface. All the H atoms were introduced in calculated
positions and refined as riding on their parent atoms with idealized
geometries and with restrained isotropic displacement parameters.
ORTEP figures were performed using the XP program included in the
SHELX50 interface. Crystal Data: 12pS C19H20Cl1F3N4O2, 428.84 g/
mol, orthorhombic, P21, a = 6.2165(8) Å, b = 8.4282(10) Å, c =
18.327(2) Å, α = 90°, β = 92.987(4), γ = 90°, V = 958.9(2) Å3, Z = 2,
ρcalcd = 1.485 g/cm3, 24,322 measured reflections, 5078 unique
reflections, Rint = 0.0626, R1 = 0.0509, wR2 = 0.11198, Parsons
parameter = 0.04(5), for 4161 reflections with I > 2σ(I), goodness-of-
fit = 1.034, and highest peak/deepest hole = 0.499/−0.294 e/Å3.
Human σ1R Radioligand Assay.24 The binding properties of the
test compounds to human σ1R were studied in transfected HEK-293
membranes using [3H](+)-pentazocine (PerkinElmer, NET-1056) as
the radioligand. The assay was carried out with 7 μg of membrane
suspension, [3H]-(+)-pentazocine (5 nM, 100 μL), 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, 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 (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 (PerkinElmer) using the EcoScint liquid scintillation cocktail.
Human σ2R Radioligand Assay. Transfected HEK-293 σ1R
knockout membranes (15 μg) were incubated with 10 nM [3H]-1,3-
di-o-tolylguanidine in the assay buffer containing 50 mM Tris-HCl at
pH 7.4. Nonspecific binding was measured by adding 10 μM
haloperidol. The binding of the test compound was measured at
either one concentration (% inhibition at 1 or 10 μM concentration)
or five different concentrations to determine affinity values (Ki).
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 washed three times with ice-cold 10
mM Tris-HCL (pH 8.0). Filters were dried and counted at
approximately 40% efficiency on a MicroBeta scintillation counter
(PerkinElmer) using the EcoScint liquid scintillation cocktail.
1
dryness to afford 12gR (232 mg, 95% yield) as hydrochloride. H
NMR (400 MHz, CD3OD): δ 7.62 (dd, J = 8.6, 5.3 Hz, 2H), 7.26 (t, J
= 8.7 Hz, 2H), 5.39 (d, J = 15.2 Hz, 1H), 5.15 (d, J = 15.2 Hz, 1H),
4.72−4.56 (m, 2H), 4.52 (d, J = 12.7 Hz, 1H), 4.42−4.16 (m, 2H),
4.11−3.95 (m, 2H), 3.89−3.68 (m, 2H), 3.67−3.47 (m, 3H), 3.06−
2.94 (m, 1H), 1.95−1.70 (m, 4H). Chiral HPLC, method 2: RT 10.8
min, 97.0% ee; [α]2D0 −20.1 (c 0.19, MeOH).
(5a,8a-cis)-7-Benzyl-3-(3-fluorophenyl)-5a,7,8,8a-tetrahydro-
4H,6H-pyrrolo[3,4-b][1,2,3]triazolo[1,5-d][1,4]oxazine (20). Step 1.
(3,4-trans)-4-Azido-1-benzylpyrrolidin-3-yl Methanesulfonate. To a
solution of (3,4-trans)-4-azido-1-benzylpyrrolidin-3-ol (18, 815 mg,
3.73 mmol) in DCM (15 mL) cooled at 0 °C, Et3N (630 μL, 4.48
mmol) was added and the solution was stirred for 10 min.
Methanesulfonyl chloride (442 μL, 5.60 mmol) was added at 0 °C
and the mixture was stirred at rt for 6 h. Water was added and the
aqueous phase was extracted with DCM, washed with brine, dried
over Na2SO4, filtered, and concentrated under vacuum. Purification
by flash chromatography using silica gel and gradient from hexane to
1
ethyl acetate afforded the title compound (650 mg, 59% yield). H
NMR (400 MHz, CDCl3): δ 7.37−7.28 (m, 5H), 4.97 (m, 1H), 4.10
(m, 1H), 3.66 (m, 2H), 3.10 (dd, J = 10.5, 6.2 Hz, 1H), 3.07 (s, 3H),
3.04 (dd, J = 10.5, 6.9 Hz, 1H), 2.79 (dd, J = 11.1, 3.6 Hz, 1H), 2.62
(dd, J = 9.8, 4.6 Hz, 1H).
Step 2. (3,4-cis)-4-Azido-1-benzylpyrrolidin-3-yl Acetate. A
mixture of the previous compound (200 mg, 0.67 mmol), KOAc
(146 mg, 1.48 mmol), and 18-crown-6 (39 mg, 0.14 mmol) in DMF
(3 mL) was heated at 100 °C for 6 h. Additional amounts of KOAc
(146 mg, 1.48 mmol) and 18-crown-6 (50 mg, 0.19 mmol) were
added and the mixture was heated at 120 °C for 16 h. Water was
added, the aqueous phase was extracted with diethylether, dried over
Na2SO4, filtered, and concentrated to afford the title compound,
which was used in the next step without further purification (176 mg).
1H NMR (400 MHz, CDCl3): δ 7.37−7.27 (m, 5H), 5.28 (m, 1H),
3.96 (m, 1H), 3.67 (s, 2H), 3.01 (dd, J = 10.6, 7.2 Hz, 1H), 2.92 (dd,
J = 9.6, 6.2 Hz, 1H), 2.72 (m, 2H), 2.16 (s, 3H).
Step 3. (3,4-cis)-4-Azido-1-benzylpyrrolidin-3-ol (19). A solution
of (3,4-cis)-4-azido-1-benzylpyrrolidin-3-yl acetate (176 mg, 0.67
mmol) and K2CO3 (206 mg, 1.48 mmol) in MeOH (6 mL) was
stirred at rt for 6 h. The solvent was removed under vacuum.
Purification by flash chromatography using silica gel and gradient
1
from hexane to ethyl acetate afforded 19 (86 mg, 58% yield). H
NMR (400 MHz, CDCl3): δ 7.40−7.25 (m, 5H), 4.34 (m, 1H), 3.92
(m, 1H), 3.67 (s, 2H), 2.97−2.88 (m, 3H), 2.71 (dd, J = 10.1, 5.7 Hz,
1H), 2.62 (dd, J = 10.2, 4.2 Hz, 1H).
Step 4. (3,4-cis)-3-Azido-1-benzyl-4-((3-(3-fluorophenyl)prop-2-
yn-1-yl)oxy)pyrrolidine. To a solution of 19 (86 mg, 0.39 mmol) in
dry THF (4 mL) at 0 °C under Ar, NaH (24 mg, 60% in mineral oil,
0.59 mmol) was added and the mixture was stirred at 0 °C for 10 min
and then at rt for 1 h. The reaction mixture was cooled again at 0 °C,
TBAI (102 mg, 0.27 mmol) and a solution of 1-(3-bromoprop-1-yn-
1-yl)-3-fluorobenzene (102 mg, 0.27 mmol) in THF (1 mL) were
slowly added and the reaction mixture was stirred at rt for 16 h. The
reaction mixture was cooled at 0 °C and the NH4Cl aqueous saturated
solution and water were added. The aqueous phase was extracted with
ethyl acetate and washed with water. Purification by flash
chromatography using silica gel and gradient from hexane to ethyl
1
acetate afforded the title compound (68 mg, 49% yield). H NMR
(400 MHz, CDCl3): δ 7.36−7.30 (m, 5H), 7.28 (m, 1H), 7.23 (m,
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hERG Assay.33 CHO cells stably expressing hERG channels
(Millipore) were cultured in the F12 HAM medium supplemented
with 10% fetal bovine serum and 400 μg/L Geneticin. The
extracellular Ringer’s solution consisted of (in mM): 2 CaCl2, 1
MgCl2, 10 HEPES, 4 KCl, 145 NaCl, 10 glucose, pH 7.4, and 305
mOsm. The intracellular Ringer’s solution composition (in mM):
5.37 CaCl2, 1.75 MgCl2, 31.25/10 KOH/EGTA, 10 HEPES, 210 KCl,
pH 7.2, and 295 mOsm. 4 mM Na2-ATP 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 manufacturer’s
recommendations. Upon 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 a Vh for 3100 ms. Thus, voltage protocols were
repeated every 15 s. For each cell, the extracellular solution was
applied before increasing the concentrations of the tested compound.
In Vitro Metabolic Stability in Human Liver Microsomes.
Compounds 12lR and 12qS were incubated in 96-well plates at 37 °C
for 1 h under standard incubation conditions: sodium−potassium
phosphate buffer (50 mM, pH 7.4), MgCl2 (3 mM), the NADPH-
generating system, and the 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 spiked with an
internal standard (testosterone). Upon centrifugation of the resultant
mixture, supernatants were analyzed by a generic ultraperformance
liquid chromatography−triple quadrupole mass spectrometry
(UPLC−MS/MS) method. Metabolic stability was determined by
the disappearance of compound over time. 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 using the
equation 0.693/k, where k is the biotransformation rate constant and
corresponds to the slope of the ln−linear curve. The microsomal
96-well plates in Tris-HCl 50 mM (pH 7.4), each well containing
[3H](+)-pentazocine (100 mg of 3 nM of the tissue suspension).
Nonspecific binding was determined using 1 μM haloperidol.
Diphenylhydantoin sodium salt (DPH, 1 mM) was used to evaluate
its effect on the σ1R binding of the tested drugs. Plates were incubated
at 37 °C for 150 min and the reaction mixture was transferred to a
pretreated 0.1% PEI, Millipore filter 96-well plate (GFC), filtered, and
washed four times with buffer solution Tris-HCl 10 mM (pH 7.4).
Filters were allowed to dry, the scintillation liquid (30 μL) was
dispensed into the wells, and the mixture was allowed to equilibrate
overnight. The amount of bound radioactivity was determined by
liquid scintillation spectrometry. Nonspecific binding was subtracted
for each well and the modulation of DPH on the σ1R binding of the
tested drugs was expressed as the ratio of the IC50 value for the
inhibitor drugs in the absence of DPH (control) to the value obtained
in the presence of DPH.
Pharmacokinetic Studies. Treatment of both rodent species in
the pharmacokinetic experiments was essentially similar. Groups of 2
male Wistar rats (250−300 g; Harlan) or 16 male CD1 mice (25−30
g; Charles River) were given oral or intravenous doses of 1, 12lR, or
12qS 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 HPLC−MS/MS after plasma protein
precipitation.
Plasma and Brain Pharmacokinetic Studies. 10 male CD1
mice (25−30 g; Charles River) were given ip doses of test compound
in 0.5% hydroxypropyl methylcellulose. Dose levels were 5 and 20
mg/kg for 12lR and 12qS, respectively. Blood samples were collected
at different times 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. Brain was extracted and
homogenized in phosphate-buffered saline. Test compound concen-
trations in plasma and brain homogenate were determined by
HPLC−MS/MS after protein precipitation with acetonitrile.
Pharmacokinetic Parameters. Standard pharmacokinetic pa-
rameters, such as the area under the curve (AUC), peak plasma
concentration (Cmax), time to peak concentration (tmax), volume of
distribution (Vd), and terminal half-life (t1/2), were determined by
noncompartmental analysis of the plasma and brain concentration−
time curves (Phoenix v.6.2.1.51, Pharsight, CA).
In Vivo Studies. Animals: 6−8-week-old CD1 female mice
(Charles River, France) were used. Animals had access to food and
water ad libitum and were kept under controlled laboratory
conditions with a temperature of 21 1 °C and a light−dark cycle
of 12 h. Experimental behavioral testing was carried out in a
soundproof and air-regulated experimental rooms and was done blind
with respect to treatment. 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.52 Animal
studies are reported in compliance with the ARRIVE guidelines.53,54
Drug Administration. Compounds 12lR, 12qS, and 1 were
dissolved in 0.5% hydroxypropylmethylcellulose (HPMC) to their
final concentrations immediately before oral administration. Drugs
were administered in a volume of 10 mL/kg; an equal volume of
vehicle was used in control animals. Drug doses refer to their base
forms.
intrinsic clearance (Clint) was calculated using the equation: Clint
=
0.693/[t1/2 × (mg of microsomal protein/volume of incubation)].
Caco-2 Permeability. Compounds 12lR and 12qS were
evaluated in human colon adenocarcinoma (Caco-2) cells, which
were grown and seeded in Readycell (Barcelona, Spain). Caco-2 cells
were seeded in 24-transwell HTS plates, and the assay was performed
with 21-day confluent monolayer cells. The incubation media were
Hank’s balanced salt solution, pH 7.4, supplemented with 10 mM
glucose. Incubations were carried out in an atmosphere of 5% CO2
with a relative humidity of 95% at 37 °C. Experiment was carried out
in duplicate. 10 mM stock solutions in DMSO were diluted with
incubation media, and the final concentration being 10 μM (1%
DMSO). Compounds 121R and 12qS were placed in the apical side
to assess the permeability in the apical to basolateral direction (A →
B) or in the basolateral side to assess the permeability in the reverse
direction (B → A). After 2 h incubation, both sides were sampled for
analysis using a generic UPLC−MS/MS method. The concentrations
of 121R and 12qS were determined using a seven-point calibration
curve. Apparent permeability was calculated using the equation: Papp
=
(dQ/dt)/(A × C0), where dQ/dt is the amount of compound
transported across the cells during the incubation time, A is the
surface area of the monolayer, and C0 is the concentration in the
donor side at time 0. The efflux ratio was estimated as the Papp A→B-to-
P
app B→A ratio. The integrity of the monolayer was checked before the
experiment by measuring the transepithelial electrical resistance (cut-
off value ≥ 1000 Ω cm2) and after the experiment by determining the
permeability of Lucifer yellow (cut-off value of Papp A→B ≤ 10 nm/s).
Phenytoin Assay. Compounds 12lR and 12qS were evaluated in
the phenytoin-modulated σ1R-binding assay. Guinea pig brains were
homogenized in Tris-HCl 50 mM and 0.32 M sucrose (pH 7.4) and
centrifuged at 1000g for 10 min at 4 °C. The obtained pellet was
resuspended in 50 mM Tris-HCl (pH 7.4) incubated at 37 °C for 30
min and centrifuged at 48,000g for 20 min at 4 °C. Pellet aliquots
were frozen at −70 °C until use. The binding test was performed in
Capsaicin Test. Capsaicin (8-methyl-N-vanillyl-6-nonamide) was
purchased from Sigma-Aldrich and dissolved in 1% DMSO in
physiological saline to a concentration of 0.05 μg/μL. Mice were
habituated for 2 h in individual test compartments placed on an
elevated mesh-bottomed platform with a 0.5 cm2 grid to provide
access to the ventral surface of the hind paws. After this period, the
animals were then given an intraplantar capsaicin (or its solvent)
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injection (1 μg in 20 μL of 1% DMSO) into the midplantar surface of
the right hind paw. Fifteen minutes after the administration of
capsaicin or its solvent, mechanical stimulation was applied onto the
plantar surface of the hind paw. A nonflexible filament (0.5 mm
diameter) was electronically (dynamic plantar esthesiometer, Ugo
Basile, Italy) driven into the ventral side of the paw previously
injected, at least 5 mm away from the site of the injection toward the
fingers. The automated device exerts a constant upward pressure of
0.5 g (4.90 mN) onto the plantar surface. When a paw withdrawal
response occurred, the stimulus was automatically terminated and the
response latency time was automatically recorded. A cut-off time of 50
s was used. In all experiments, the filament was applied to the right
hind paw of each mouse three times, separated by an interval of 0.5
min, and the mean value of the three trials was considered the
withdrawal latency time of the animal. The mice (n = 8−31 per
treatment group) received vehicle 0.5% HPMC (Sigma-Aldrich) or
test compound through po in a volume of 10 mL/kg 30 min before
capsaicin injection, and withdrawal latencies to mechanical
stimulation were determined 15 min after capsaicin injection (45
min after the treatment). The effect of the treatments on mechanical
hypersensitivity induced by capsaicin was calculated with the
following equation: reduction of mechanical hypersensitivity (%) =
[(LTD − LTV)/(CT − LTV)] × 100, where LTD and LTV are the
latency times in drug- and vehicle-treated animals, respectively, and
CT is the cut-off time (50 s).
Data and Statistical Analysis. Data were expressed as means
standard error of the mean. ED50 values were determined using a four-
parameter logistic equation (sigmoidal dose−response curve, variable
slope). The effect of systemic treatments was expressed in the
capsaicin test as the percent of antinociception: % antinociception =
((individual test latency − vehicle mean latency)/(cut-off latency −
vehicle mean latency)) × 100. ED50 was defined as the dose that
produced 50% of the maximum possible effect. ED50 values with 95%
confidence intervals (CI) were calculated with GraphPad Prism
version 8.0.2 software (San Diego, CA, USA) using nonlinear
regression methods.
Daniel Font − The Barcelona Institute of Science and
Technology, Institute of Chemical Research of Catalonia
(ICIQ), 43007 Tarragona, Spain
́
M. Angeles Sarmentero − The Barcelona Institute of Science
and Technology, Institute of Chemical Research of Catalonia
(ICIQ), 43007 Tarragona, Spain
́
Paula Alvarez-Bercedo − The Barcelona Institute of Science
and Technology, Institute of Chemical Research of Catalonia
(ICIQ), 43007 Tarragona, Spain
José M. López-Valbuena − The Barcelona Institute of Science
and Technology, Institute of Chemical Research of Catalonia
(ICIQ), 43007 Tarragona, Spain
̀
Miquel A. Pericas − The Barcelona Institute of Science and
Technology, Institute of Chemical Research of Catalonia
(ICIQ), 43007 Tarragona, Spain; Departament de Química
̀
̀
Inorganica i Organica, Universitat de Barcelona, 08028
Barcelona, Spain; orcid.org/0000-0003-0195-8846
Raquel Enrech − Drug Discovery and Preclinical
Development, ESTEVE Pharmaceuticals S.A., 08028
Barcelona, Spain; WELAB, Parc Científic Barcelona, 08028
Barcelona, Spain
Ana Montero − Drug Discovery and Preclinical Development,
ESTEVE Pharmaceuticals S.A., 08028 Barcelona, Spain;
WELAB, Parc Científic Barcelona, 08028 Barcelona, Spain
Sandra Yeste − Drug Discovery and Preclinical Development,
ESTEVE Pharmaceuticals S.A., 08028 Barcelona, Spain;
WELAB, Parc Científic Barcelona, 08028 Barcelona, Spain
Alba Vidal-Torres − Drug Discovery and Preclinical
Development, ESTEVE Pharmaceuticals S.A., 08028
Barcelona, Spain; WELAB, Parc Científic Barcelona, 08028
Barcelona, Spain
́
Inés Alvarez − Drug Discovery and Preclinical Development,
ESTEVE Pharmaceuticals S.A., 08028 Barcelona, Spain;
WELAB, Parc Científic Barcelona, 08028 Barcelona, Spain
Pilar Pérez − Drug Discovery and Preclinical Development,
ESTEVE Pharmaceuticals S.A., 08028 Barcelona, Spain;
WELAB, Parc Científic Barcelona, 08028 Barcelona, Spain
Cruz Miguel Cendán − Department of Pharmacology, Faculty
of Medicine, University of Granada, 18016 Granada, Spain;
Biosanitary Research Institute ibs.GRANADA, 18012
Granada, Spain
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00244.
■
sı
Analytical data for all the final compounds and
intermediates and HPLC traces of final compounds
(PDF)
Enrique J. Cobos − Department of Pharmacology, Faculty of
Medicine, University of Granada, 18016 Granada, Spain;
Biosanitary Research Institute ibs.GRANADA, 18012
Granada, Spain
José Miguel Vela − Drug Discovery and Preclinical
Development, ESTEVE Pharmaceuticals S.A., 08028
Barcelona, Spain; WELAB, Parc Científic Barcelona, 08028
Barcelona, Spain
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Author
■
Carmen Almansa − Drug Discovery and Preclinical
Development, ESTEVE Pharmaceuticals S.A., 08028
Barcelona, Spain; WELAB, Parc Científic Barcelona, 08028
Barcelona, Spain; orcid.org/0000-0001-5665-4685;
Phone: 0034650697372; Email: calmansa@
welab.barcelona
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00244
Authors
Author Contributions
José Luis Díaz − Drug Discovery and Preclinical Development,
ESTEVE Pharmaceuticals S.A., 08028 Barcelona, Spain;
WELAB, Parc Científic Barcelona, 08028 Barcelona, Spain;
orcid.org/0000-0001-8812-4689
This manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Félix Cuevas − The Barcelona Institute of Science and
Technology, Institute of Chemical Research of Catalonia
(ICIQ), 43007 Tarragona, Spain
Ana I. Oliva − The Barcelona Institute of Science and
Technology, Institute of Chemical Research of Catalonia
(ICIQ), 43007 Tarragona, Spain
Notes
The authors declare no competing financial interest.
12pS: CCDC 2027373 “Authors will release the atomic
coordinates and experimental data upon article publication.”
PDB ID is provided in Figure 2 legend.
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Article
(11) de la Puente, B.; Nadal, X.; Portillo-Salido, E.; Sánchez-Arroyos,
R.; Ovalle, S.; Palacios, G.; Muro, A.; Romero, L.; Entrena, J. M.;
Baeyens, J. M.; López-García, J. A.; Maldonado, R.; Zamanillo, D.;
Vela, J. M. Sigma-1 receptors regulate activity-induced spinal
sensitization and neuropathic pain after peripheral nerve injury.
Pain 2009, 145, 294−303.
ACKNOWLEDGMENTS
■
We thank Magda Bordas, Adriana Port, Joan Andreu Morató,
Mónica Carro, Edmundo Ortega, Rosalía Pascual, Manel
̃
Merlos, Javier Burgueno, Marta Pujol, Enrique Hernández,
Javier Farré, Eva Ayet, Raquel Fernández-Reinoso, Maria
Teresa Serafini, Carmen Segalés, Daniel Zamanillo, Ma
Carmen Ruiz, and Lucía Romero for their expert contribution
to analytical, in vitro, and in vivo studies; Carlos Pérez and
Eduardo Villarroel for their contribution to compound
management; and Eduardo C. Escudero Adán for the single-
crystal X-ray structure determination of 12pS. This work was a
part of activities in R&D Projects IDI-20110577, IDI-
20130942, and IDI-20150914 supported by the Spanish
Ministerio de Economía y Competitividad (MINECO),
through the Centro para el Desarrollo Tecnológico Industrial
(CDTI), cofinanced by the European Union through the
European Regional Development Fund (ERDF; Fondo
Europeo de Desarrollo Regional, FEDER).
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Cent. Nerv. Syst. Agents Med. Chem. 2009, 9, 172−183.
̃
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R.; Vela, J. M. Pharmacological Modulation of the Sigma 1 Receptor
and the Treatment of Pain. Adv. Exp. Med. Biol. 2017, 964, 85−107.
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ABBREVIATIONS
■
ADME, absorption, distribution, metabolism, and excretion;
CNS, central nervous system; DMSO, dimethylsulphoxide;
DPH, diphenylhydantoin sodium salt; ER, endoplasmic
reticulum; hERG, human ether-a-go-go-related gene; NMDA,
N-methyl-D-aspartate; PGRMC1, progesterone receptor mem-
brane component 1; PI, positive ionizable; rhCYP, recombi-
nant human cytochrome P450; SAR, structure−activity
relationships; σR, sigma receptor; σ1R, sigma-1 receptor; σ2R,
sigma-2 receptor; TMEM97, transmembrane protein-97
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