Synthesis and structure-activity relationship study of a new series of selective σ1 receptor ligands for the treatment of pain: 4-aminotriazoles

Article abstract of DOI:10.1021/jm501920g

The synthesis and pharmacological activity of a new series of 4-aminotriazoles as potent σ₁ receptor (σ₁R) ligands are reported. The compounds were prepared using a 4-5-step process, involving as a key step a click chemistry reaction between ynamides and azides. The most active compounds exhibited nanomolar potency for the σ₁R, and the selectivity over the σ₂R was improved on decreasing the central amine basicity. It was concluded that in order to achieve good σ₁R potency a minimum lipophilicity was required, while limiting to a defined range of cLogP avoided human ether-a-go-go-related gene channel inhibition. This made the most interesting derivatives to be concentrated in a narrow margin of lipophilicity. Among them, compound 13g exhibited the most potent in vivo antinociceptive properties, which are indicative of its antagonist character.

Full text of DOI:10.1021/jm501920g

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Article
Synthesis and Structure-Activity Relationship (SAR) Study of a New Series
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of Selective # Receptor Ligands for the Treatment of Pain: 4-Aminotriazoles.
José Luis Díaz, Ute Christmann, Ariadna Fernández, Antoni Torrens, Adriana
Port, Rosalia Pascual, Inés Álvarez, Javier Burgueño, Xavier Monroy,
Ana Montero, Ariadna Balada, José Miguel Vela, and Carmen Almansa
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 06 Feb 2015
Downloaded from http://pubs.acs.org on February 7, 2015
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Journal of Medicinal Chemistry
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Synthesis and Structure-Activity Relationship (SAR)
Study of a New Series of Selective σ1 Receptor Ligands
for the Treatment of Pain: 4-Aminotriazoles.
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José Luis Díaz,* Ute Christmann, Ariadna Fernández, Antoni Torrens, Adriana Port, Rosalia Pascual,
Inés Álvarez, Javier Burgueño, Xavier Monroy, Ana Montero, Ariadna Balada, José Miguel Vela, and
Carmen Almansa*
Drug Discovery and Preclinical Development, Esteve, Baldiri Reixach, 4-8, 08028 Barcelona, Spain.
RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required
according to the journal that you are submitting your paper to)
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ABSTRACT: The synthesis and pharmacological activity of a new series of 4-aminotriazoles as
potent σ1 receptor (σ1R) ligands are reported. The compounds were prepared using a 4-5-step process,
involving as a key step a click chemistry reaction between ynamides and azides. The most active
compounds exhibited nanomolar potency for the σ1R and the selectivity over the σ2R was improved on
decreasing the central amine basicity. It was concluded that in order to achieve good σ1R potency a
minimum lipophilicity was required, while limiting to a defined range of clogP avoided hERG channel
inhibition. This made the most interesting derivatives to be concentrated in a narrow margin of
lipophilicity. Among them, compound 13g exhibited the most potent in vivo antinociceptive properties,
which are indicative of its antagonist character.
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INTRODUCTION
The σ1 receptor (σ1R) is an intracellular chaperone protein expressed in central nervous system (CNS)
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regions important for pain control.1 Although the σ1R does not have homology to opioid receptors, it
modulates opioid analgesia2 through a direct physical interaction3 where σ1R antagonists enhance the
antinociceptive effect of opioids. Besides this chaperone modulatory activity on opioid analgesia, there
is increasing evidence that σ1 receptors play a role in pain control per se, especially in pain conditions
involving central sensitization.4 In this sense, the σ1R knockout (σ1R-KO) mouse shows attenuated pain
responses in the formalin test5 and does not develop mechanical hypersensitivity following capsaicin
sensitization,6 while σ1R antagonists reproduce the pain-protective phenotype of knockout mice when
administered to wild-type mice.7 Additionally, pain behaviours such as allodynia induced by sciatic
nerve injury is attenuated in σ1R-KO mice8 and in rodents treated with σ1R antagonists.9,10 More
recently, σ1R antagonists have been shown to revert paclitaxel-induced neuropathic pain, suggesting a
role for this type of compounds in chemotherapy-induced neuropathy.11
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Pharmacological studies have identified an additional σ receptor subtype, namely the σ2R, which has a
different molecular weight, tissue distribution and sub-cellular location than the σ1R. Ligands acting on
the σ2R have been proposed as biomarkers for tumour cell proliferation and show pro-apoptotic
properties, thus suggesting a potential role in cancer imaging and treatment.12 Progesterone receptor
membrane component 1 (PGRMC1), a cytochrome-related protein that binds directly to heme and
regulates lipid and drug metabolism and hormone signaling, has been recently identified as the putative
σ2R binding site.13
Several non-selective σR ligands have been assayed in clinical studies as antidepressants, drug abuse
treatments, antipsychotics and learning and memory enhancers,14 but none made it through clinical
evaluation. Based on the increasing evidence of σ1R antagonism involvement in pain control, our group
has been actively working in the search of σ1R antagonists9 for the treatment of pain, and selectivity
over the σ2R has been pursued due to the clear pharmacological differentiation of the two σ receptors.
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We recently reported the discovery of compound 1 (Figure 1), as a new highly selective σ1R
antagonist, which is currently undergoing Phase II clinical trials for the treatment of diverse pain
states.15-17 As a back-up program we undertook a scaffold hopping approach around 1 and we report
herein the synthesis and structure-activity relationships (SAR) studies of one of the most interesting
families thus identified, a new series of 4-aminotriazoles I.18
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CHEMISTRY
The synthesis of the compounds described herein was accomplished in a four to five step process,
using as a key step a click chemistry reaction, involving a Huisgen’s [1,3] dipolar cycloaddition between
ynamides and azides. As indicated in Scheme 1, the starting Boc protected amines 5 were prepared
either on protection of amines 419 or by reaction of amines 6 with haloamines 7. The N-alkyne
functionality was installed by Cu-catalyzed amidation20 of 5 with (bromoethynyl)triisopropylsilane 8,21
employing CuSO4⋅5H2O and 1,10-phenanthroline as catalytic system, leading to ynamides 9 in moderate
to good yields. TIPS-removal by TBAF, followed by direct Cu-mediated cycloaddition (CuI/DIPEA)
with organic azides 10 provided the 1,4-di-substituted triazoles 11 in good yields. The one-step TIPS
deprotection/cycloaddition sequence was developed in view of the observed degradation of the
unprotected ynamides over time. Finally, Boc deprotection of 11 under acidic conditions yielded the
target compounds 12-16, which pharmacological properties are described in Tables 1-3.
Substitution of the nitrogen atom attached to the 4-position of the pyrazole ring was accomplished
following the methods described in Scheme 2. Thus, alkylation under reductive amination conditions
afforded derivatives 17 in good yields. Acylation to 18 or sulfonylation to 19 were performed under
standard conditions and finally, reaction with propylisothiocianate provided derivative 20.
The regioisomeric 1,5-disubstituted triazole 24 was obtained by complementary Ru-catalyzed
cycloaddition reaction using Cp∗RuCl(PPh3)2 as described by Folkin22 and depicted in Scheme 3.
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RESULTS AND DISCUSSION
The new series of 4-aminotriazoles I was identified after a scaffold hopping approach around the
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arylpyrazoles represented by compound 1.15 The SAR study around this family, which had been
developed based on Glennon’s σ1R pharmacophore,23 indicated that a hydrophobic group was necessary
in position 1, while an alkylene chain of 2-3 atoms between the basic nitrogen atom and the pyrazole
oxygen was the best in order to achieve good potency and selectivity. Based on these previous findings,
the SAR around the new 4-aminotriazoles I was developed. All the compounds synthesized were
evaluated in a primary σ1R binding assay using [3H]-(+)-pentazocine24 as radioligand, while [3H]-di-o-
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tolylguanidine25 was used for σ2R binding assays (Tables 1-3).
As indicated in Table 1, maintaining a piperidinylethylamino group in position 4 of the triazole, the
substituents of the phenyl ring in position 1 were modified (12a-g). Halogen atoms were introduced,
since in the pyrazole series unsubstitution or electron donating groups provided a complete loss of
activity. The 3,4-dichorophenyl derivative, 12a exhibited a good activity, while other halogen
substitution patterns (12b-g) showed a decrease in affinity over the σ1R and no selectivity improvement.
Compound 12a showed similar activity and selectivity as compound 215 (Figure 2), its closest analogue
in the pyrazole series. However, the lower lipophilicity of the aminotriazoles with respect to their
aminopyrazole counterparts was considered to be a positive attribute of the new series, in view of the
direct relationship of increasing lipophilicity to undesired promiscuity and potential ADMET issues.26
Changing the piperidine for a morpholine group provided compound 12h, which was less potent than
12a, but more selective (67 vs > 218 ratio). Activity was impaired on replacing the 3,4-dichloro with 3-
fluoro or 3,4-difluoro substituents (12i,j).
Other amino containing groups were introduced, and while the homopiperidin-1-yl derivative 12k or
the cyclohexyl piperazine 12l provided poorly selective compounds, the homomorpholinyl (12m) or
difluoropiperidinyl (12n) derivatives provided active and selective compounds. The acetylpiperazine
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derivative 12o showed diminished activity, in contrast to the finding in the pyrazole series, where this
group was among the most active.
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We also explored the change of phenyl by benzyl in position 1, since a good fitting was obtained on
the superposition of 12h and 13g (Figure 3A). In addition, the two compounds fitted the positive
ionizable feature and three (HYD2-4) out of the four hydrophobic features of the Laggner27 σ1R
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pharmacophore. This model was the first published 3D computer-generated σ1R pharmacophore and, in
relation to Glennon’s one,23 it contained two additional hydrophobic features (see Figure 3B for a
comparison). In fact, the hydrophobic groups HYD3 and HYD4, with average distances of 9.8 Å and 6.3
Å to the positive ionizable group, cover Glennon’s primary hydrophobic region, while the HYD2
corresponds to the secondary hydrophobic region. As indicated below, the phenyl (12h) and benzyl
(13g) derivatives were similarly active, in accordance to the 3D superposition, and showed as well
similar σ1R binding to the pyrazole 1. As shown in Figure 3C, compound 1 fulfils the HYD1 feature,
which is not covered by 12h and 13g due to the presence of the triazole nitrogen atoms in this region.
This indicates that the HYD1 identified in Laggner σ1R pharmacophore is not so relevant as the other
features, that were already covered by Glennon’s pharmacophore and are indeed the only fulfilled by
many of the published σ1R ligands.28
As shown in Table 2, the benzylic derivatives 13 displayed comparable activities to their phenyl
analogues, and again the piperidinyl derivatives 13a-e were active, but not very selective. Among the
morpholino derivatives, only 13g, with the 3,4-dichlorophenyl substitution pattern showed a good
affinity, while 13f and 13h-i were poorly active. This paralleled the results obtained for the phenyl
analogues (12h-j) and suggested that when the good fitting provided by the piperidinyl groups in HYD2
is somehow decreased by the introduction of an oxygen atom in the morpholino derivatives, the HYD4
has to be occupied with a hydrophobic group in order to maintain affinity. The superior activity of the
3,4-dichloro substitution pattern can be explained by the increased hydrophobicity in the HYD4 area.
These results can also be interpreted in terms of the global polarity of the molecules, and indicate that a
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minimum global polarity is needed to achieve affinity for the σ1R. Hence, 13a (cLogP 2.5) was active,
while its morpholine counterpart 13f (cLogP 1.4) showed a diminished activity, this being regained by
the more lipophilic 13g (cLogP 2.7). The homopiperidinyl derivatives 13j and 13k retained activity but
lost selectivity, as did the homomorpholinyl 13l. Introduction of a gem-difluoro group in position 4 of
the piperidine ring provided compounds 13m and 13n, with good and intermediate affinity, respectively.
The dimethylated piperidine 13o did not improve any of the properties of 13c, while ring opening
provided a decrease in affinity (13p). Finally, the piperazines 13q-r were poorly active. Overall, the
results shown in Tables 1-3 seem to indicate that compounds with cLogP above 2 are generally
preferred by the σ1R, in accordance to its lipophilic nature.
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Additional changes were undertaken over the morpholine derivatives, since this group provided some
of the best selectivities (Table 3). Elongation of the linker chain from two to three carbon atoms was
detrimental (14a-b vs 13g and 12h, respectively), a result that matched the one obtained in the pyrazole
series.15 Introduction of a saturated ring in position 1 provided the poorly active cyclohexyl derivative
15, while the adamantyl 16 was active and selective, probably due to its superior lipophilicity.
Substitution of the nitrogen atom provided in all cases a reduction in affinity, as shown by the results of
the alkylated (17a-c), acetylated (18a-b) and mesylated (19) derivatives and the thiourea 20.
The isomeric triazole 24 (Scheme 3) was devoid of activity, a result that correlated with that obtained
in the pyrazole series,15 and can be explained by the poor alignment with its regioisomer 13c.
As it is well known, blockade of the human Ether-a-go-go-Related Gene (hERG) potassium channel
has been linked to cardiac toxicity,29 so it is usually evaluated at some point in drug discovery programs.
Since hERG blockade was a recurrent issue in the related pyrazole series,15 we decided to monitor it
from the beginning, and the results obtained in a patch-clamp assay are shown in Tables 1-3. The first
compounds prepared (12a-c), which contained a piperidine group, showed a high hERG inhibition.
Changing the piperidine by a morpholine group was clearly positive, a fact that could be attributed to the
reduction of either cLogP or pKa of 12h (cLogP: 3.4, ACD-pKa: 7.0) vs 12a (cLogP: 4.6, ACD-pKa:
9.1), since these are two factors known to affect hERG activity.30 In view of the absence of hERG
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inhibition of the piperidine derivatives 13a and 13c-e, with the same pKa as 12a-c and 13b, it was
concluded that pKa was not the key parameter. Instead, the difference could be rationalized in terms of
lipophilicity, since 13a and 13c-e showed more than one cLogP unit reduction over 12a-c and 13b. In
fact, the results shown in Tables 1-3, indicate that cLogP was the key factor affecting hERG blockade,
since all compounds having a cLogP above 3.5 show IC50 below 10 µM, while those with cLogP below
3.5, are above this limit. In summary, as frequently encountered in medicinal chemistry programs, two
key properties, in this case σ1R potency and hERG blockade, showed a clear opposite relationship with
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cLogP. Hence, a minimum lipophilicity was required to achieve good σ1R potency, with a superior
cLogP limit of 3.5 to not block the hERG channel. This concentrated the most interesting compounds in
a narrow margin of lipophilicity, with cLogP between 2.2 and 3.4.
The pKa of the nitrogen atom could, however, be related to selectivity (see Figure 4), since
compounds containing a piperidine ring (pKa around 9.1) were less selective than those with a
morpholino (pKa 7.0) or difluoropiperidino (pKa 5.7) group, a result consistent with that in the pyrazole
series.15 Therefore, the piperidines 12a and 13b showed selectivity ratios of 67 and 6.8, while their
morpholine counterparts, 12h and 13g, showed selectivity ratios of >218 and >165 times, respectively.
This may suggest that the σ2R preferentially binds compounds with a superior basicity than the σ1R and
could explain the high selectivity observed in the pyranoquinoline series recently reported by us,31 where
amine basicity (pKa 4.6) was among the lowest ever described in σ1R ligands. Another potential
explanation for the selectivity trend is that the σ2R does not tolerate the increase in polarity provided by
the replacement of the piperidine methylene by an oxygen atom or a difluoromethylene group.
The morpholino group revealed itself again as an interesting feature. Although the presence of a polar
atom near the hydrophobic region HYD-2 causes a decrease in σ1R affinity, it causes as well an increase
in selectivity and a decrease in hERG channel blockade versus the piperidine counterparts.
The compounds that showed high selectivity and IC50 for hERG blockade above 10 µM were
evaluated in vivo using the formalin-induced pain model in mice,32 at 40 mg/kg by intraperitoneal (i.p.)
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and at 80 mg/kg by oral (p.o.) routes. The best potencies were achieved by compounds 12h and 13g,
which contain a morpholino group, also present in the best compounds identified in the pyrazole series.
Table 4 shows the in vivo activity of 12h and 13g in several models in comparison to reference
compound 1. The intraplantar (i.pl.) injection of formalin elicits an early phase (phase I) and a delayed
phase (phase II) of pain, characterized by paw licking, biting and other behaviours. The phase I is
predominantly caused by direct activation of C-fibers, whereas the phase II appears to be dependent on
the combination of an inflammatory reaction in the peripheral tissue and functional changes in the spinal
cord, involving both peripheral and central sensitization.32 Compounds 12h and 13g were active both by
i.p. and p.o. routes, in phase I (data not shown) but mainly in the delayed phase II, in which central
sensitization mechanisms are involved. This antinociceptive activity was indicative of their antagonistic
nature, in accordance to the reported analgesic effect of σ1R antagonists, particularly in the formalin
test.33 In fact, both compounds showed a similar profile to 1, since although the potency by the ip route
was clearly superior for 12h and 13g, the oral potencies were very similar in the three cases.
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In addition, 12h and 13g showed good activity in the mouse model of mechanical hypersensitivity
induced by i.pl. capsaicin, where increased sensitivity in the area surrounding capsaicin injection (area
of secondary mechanical hypersensitivity) is known to result from central sensitization phenomena.6
Accordingly, the involvement of σ1R in the modulation of capsaicin-induced mechanical
hypersensitivity has been demonstrated by several studies.6,16,31 The compounds were also evaluated in
the partial sciatic nerve ligation (PSNL) model in mice,34 which is one of the most widely accepted
neuropathic pain models. This model of peripheral nerve injury leads to a pain syndrome that is
characterized in rodents by pain in response to normally innocuous stimuli (allodynia) and exaggerated
pain in response to noxious stimuli (hyperalgesia). Both compounds showed clear antiallodynic activity
when administered p.o. but 13g was the most potent in these last two models, in which central
sensitization mechanisms are involved.
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Compounds 12h and 13g were profiled in additional in vitro ADME tests (Table 4) and were shown
to be highly stable in human liver microsomes and highly permeable in Caco-2 cells, with values in a
similar range to those of compound 1.15 Compound 12h exhibited a similar solubility to 1, while 13g
was more soluble, possibly reflecting the reduced lipophilicity. This fact, together with the superior in
vivo potency of 13g, led to its selection for further preclinical profiling.
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In summary, the synthesis and pharmacological activity of a new series of potent and selective σ1R
ligands is reported herein. This family of 4-aminotriazoles, which originated from a scaffold hopping
approach around a previous series of pyrazoles represented by compounds 1-3, exhibited nanomolar
potency for the σ1R and high selectivity over the σ2R. The SAR study showed that less basic amines
provided superior σ1R selectivity and that σ1R potency and hERG blockage showed a clear opposite
relationship with cLogP. It was concluded that achievement of good σ1R potency requires a minimum
lipophilicity but that a superior cLogP limit of 3.5 should not be exceeded to avoid blocking the hERG
channel function, which made the most interesting compounds in this series to be concentrated in a
narrow margin of lipophilicity with cLogP between 2.2 and 3.4. Compound 13g exerted the most potent
in vivo antinociceptive effects, which points up its antagonist character.
EXPERIMENTAL SECTION
Unless otherwise noted, all materials were obtained from commercial suppliers and used without
further purification. Microwave assisted reactions were conducted with an Explorer Synthesizer from
CEM. Flash chromatography was performed with a forced flow of the indicated solvent system on SDS
silica gel Chromagel 60 ACC-(230-400 mesh) or on a CombiFlash Companion system with Redisep Rf
disposable columns. 1H spectra were recorded on an Agilent UNITY 300 MHz (spectrometer fitted with
a 5 mm H/F/X ATB probe) or an Agilent Mercury 400 MHz (spectrometer fitted with a 5 mm ID/PFG
probe) with 2 H lock in deuterated solvents. Chemical shifts (δ) are in parts per million. Commercially
available reagents and solvents (HPLC grade) were used without further purification for all the
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analytical tests. Analytical HPLC−MS were performed on a Waters 2795-MS ZQ or an Acquity H-
Class-MS ZQ systems. For the Waters 2795 system a reverse phase XBridge C18 column was used (4.6
x 50 mm, 2.5 µm), gradient 2−95% B (A = 10 mM Ammonium bicarbonate, B = acetonitrile) over 5.5
min, injection volume: 10 μL, flow: 2.0 mL/min. PDA spectra were recorded at 220-310 nm using a
Waters 2996 PDA detector. For the Acquity H-Class system a reverse phase Acquity BEH C18 column
was used (2.1 x 50 mm, 1.7 µm), gradient 2-95% B (A= 10 mM Ammonium bicarbonate, B =
acetonitrile) over 4.75 min, injection volume: 2 μL, flow: 0.61 mL/min. In both systems, mass spectra
were obtained over the range m/z 100−800 at a sampling rate of 0.3 scans per second using Waters ZQ.
Data were integrated and reported using Water Masslynx software. All compounds display purity higher
than 95% as determined by those methods. Accurate mass measurements were carried out using an
Agilent 6540 UHD Accurate-Mass QTOF system and obtained by electron spray ionization (ESI) in
positive mode.
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Determination of physicochemical properties. pKa was calculated using ACDLABS 9.0.3 and
clogP using ChemDraw Ultra 10.0.3. Solubility was measured as thermodynamic solubility from solid
compound in phosphate buffer at pH = 7.4 by HPLC. LogP and pKa were determined by using a
pHmetric technique35 in a GlpKa or a Sirius-T3 Analytical instrument.
tert-Butyl 2-(4-acetylpiperazin-1-yl)ethylcarbamate (5, NR2R3 = 4-acetylpiperazinyl). To a stirred
solution of tert-butyl 2-bromoethylcarbamate (4.37 g, 19.5 mmol) in dry CH2Cl2 (40 mL) 1-(piperazin-
1-yl)ethanone (5 g, 39.0 mmol) was added, followed by TEA (4.07 mL, 29.2 mmol). The reaction
mixture was heated under reflux for 16 h. After cooling back to room temperature, the reaction was
quenched with H2O and extracted with CH2Cl2. The combined organic fractions were dried over Na2SO4
and the solvent was removed under reduced pressure to give tert-butyl 2-(4-acetylpiperazin-1-
yl)ethylcarbamate as a red oil (5.39 g, quant.). The product was used without further purification in the
next step. 1H NMR (300 MHz, CD3OD) δ 3.68 – 3.48 (m, 4H), 3.20 (t, J = 6.6 Hz, 2H), 2.56 – 2.37 (m,
6H), 2.09 (s, 3H), 1.44 (s, 9H).
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tert-Butyl 2-morpholinoethylcarbamate (5, NR2R3 = morpholinyl).19 To a solution of 2-
morpholinoethanamine (10.0 g, 76.8 mmol) in H2O (80 mL) di-tert-butyl dicarbonate (18.9 g, 86.6
mmol) was added and the mixture stirred at room temperature overnight. The reaction mixture was
partitioned between H2O and EtOAc and the aqueous phase was extracted with EtOAc. The combined
organic fractions were dried over Na2SO4 and filtered. The solvent was removed under reduced pressure
to give tert-butyl 2-morpholinoethylcarbamate (16.8 g, 94%) as yellow oil. 1H NMR (300 MHz, CDCl3)
δ 4.98 (bs, 1H), 3.82 – 3.50 (m, 4H), 3.17 (q, J = 5.8 Hz, 2H), 2.49 – 2.32 (m, 6H), 1.40 (s, 9H).
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tert-Butyl [2-(morpholin-4-yl)ethyl][(tripropan-2-ylsilyl)ethynyl]carbamate (9, NR2R3
=
morpholinyl). A schlenk tube was charged with tert-butyl 2-morpholinoethylcarbamate (16.8 g, 72.9
mmol), CuSO4·5H2O (1.80 g, 7.29 mmol), K3PO4 (30.9 g, 145 mmol) and 1,10-phenanthroline (2.63 g,
14.6 mmol), evacuated and backfilled with argon. Then, a solution of 8 (20.9 g, 80.2 mmol) dissolved in
dry toluene (300 mL) was added and the reaction solution was heated at 110 ºC for 2 days. The solvent
was removed under reduced pressure and the crude product was taken up in EtOAc and partitioned
between H2O and EtOAc. The aqueous phase was extracted with EtOAc and the combined organic
fractions were dried over Na2SO4. The solvent was removed under reduced pressure and the residue was
purified by combiflash chromatography (SiO2, c-Hexane/EtOAc up to 10%) to give the title compound
1
as a yellow oil (21.8 g, 73%). H NMR (300 MHz, CDCl3) δ 3.77 – 3.64 (m, 4H), 3.55 (t, J = 6.7 Hz,
2H), 2.64 (t, J = 6.7 Hz, 2H), 2.56 – 2.43 (m, 4H), 1.49 (s, 9H), 1.14 – 1.00 (m, 21H).
tert-Butyl 1-(3,4-dichlorobenzyl)-1H-1,2,3-triazol-4-yl(2-morpholinoethyl)carbamate (11, R1
=3,4-dichlorobenzyl, NR2R3
=
morpholine).
A
stirred solution of tert-butyl 2-
morpholinoethyl((triisopropylsilyl)ethynyl)carbamate (9, 10.0 g, 24.4 mmol) in dry THF (300 mL) was
cooled to 0 ºC under an argon atmosphere, and TBAF (1 M in THF, 24.4 mL, 24.4 mmol) was added
dropwise. The reaction mixture was stirred at 0 ºC for 30 min. and additionally at room temperature for
10 min, after which the TIPS deprotection had been completed as judged by TLC. At this point CuI
(2.32 g, 12.2 mmol) and 4-(azidomethyl)-1,2-dichlorobenzene (10, 5.41 g, 26.8 mmol) were added,
followed by DIPEA (4.17 mL, 24.4 mmol) and the reaction mixture stirred at room temperature
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Journal of Medicinal Chemistry
overnight. The solvent was removed under reduced pressure and the crude product taken up in EtOAc.
Saturated aqueous NaHCO3 solution was added and the phases were separated. The aqueous phase was
additionally extracted with EtOAc. The combined organic fractions were dried over MgSO4 and the
solvent removed under reduced pressure after filtration. The residue was purified by combiflash column
chromatography (SiO2, c-Hexane/EtOAc up to 40%) to give the title compound as a yellow solid (8.03
g, 72%). 1H NMR (300 MHz, CDCl3) δ 7.84 (s, 1H), 7.44 (d, J = 8.2 Hz, 1H), 7.38 (d, J = 2.1 Hz, 1H),
7.12 (dd, J = 8.3, 2.1 Hz, 1H), 5.39 (s, 2H), 4.12 (t, J = 7.2 Hz, 2H), 3.79 – 3.60 (m, 4H), 2.66 (t, J = 7.2
Hz, 2H), 2.61 – 2.47 (m, 4H), 1.51 (s, 9H).
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1-(3,4-Dichlorobenzyl)-N-(2-morpholinoethyl)-1H-1,2,3-triazol-4-amine hydrochloride (13g).
tert-butyl 1-(3,4-dichlorobenzyl)-1H-1,2,3-triazol-4-yl(2-morpholinoethyl)carbamate (11, 8.03 g, 17.6
mmol) was dissolved in CH2Cl2 (110 mL) and TFA (11 mL, 148 mmol) was added dropwise. The
reaction mixture was stirred at room temperature until the deprotection process had been completed as
judged by TLC. The reaction mixture was quenched with saturated aqueous NaHCO3 and the product
extracted with CH2Cl2 twice. The combined organic fractions were dried over Na2SO4 and the solvent
was removed under reduced pressure to give 1-(3,4-dichlorobenzyl)-N-(2-morpholinoethyl)-1H-1,2,3-
triazol-4-amine as yellow oil (6.01 g, 95%). This compound (4.50 g, 12.6 mmol) was dissolved in
acetone (40 mL) and HCl solution (2 M in Et2O, 6.95 mL, 13.9 mmol) was added dropwise. After
stirring for 30 min. at room temperature, the white solid formed was filtered off and dried in vacuo to
give 13g as white solid (4.56 g, 92%). 1H NMR (300 MHz, CD3OD) δ 7.55 (d, J = 8.4 Hz, 1H), 7.53 (d,
J = 1.9 Hz, 1H), 7.48 (s, 1H), 7.30 (dd, J = 8.3, 2.2 Hz, 1H), 5.52 (s, 2H), 4.10 – 3.98 (m, 2H), 3.91 –
3.77 (m, 2H), 3.59 (t, J = 5.8 Hz, 2H), 3.58 – 3.50 (m, 2H), 3.41 (t, J = 5.9 Hz, 2H), 3.28 – 3.15 (m,
2H).
1-(3,4-Dichlorophenyl)-N-(2-morpholinoethyl)-N-propyl-1H-1,2,3-triazol-4-amine
hydrochloride, (17b). To a solution of 1-(3,4-dichlorophenyl)-N-(2-morpholinoethyl)-1H-1,2,3-triazol-
4-amine (12h, 85 mg, 0.25 mmol) in DCE (2.5 mL) in a microwave vial under argon atmosphere,
propionaldehyde (49 µL, 0.67 mmol) was added followed by NaBH(OAc)3 (105.2 mg, 0.49 mmol). The
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reaction mixture was heated under microwave radiation at 120 °C for 10 min. After cooling back to
room temperature, the reaction mixture was quenched with saturated aqueous NaHCO3 and extracted
with CH2Cl2 twice. The combined organic fractions were washed with brine and dried over MgSO4. The
solvent was removed under reduced pressure after filtration. The residue was purified by Combiflash
chromatography (SiO2, c-Hexane/EtOAc up to 100%) to give 1-(3,4-dichlorophenyl)-N-(2-
morpholinoethyl)-N-propyl-1H-1,2,3-triazol-4-amine as a red oil (60.8 mg, 63%). The compound was
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prepared as a hydrochloride salt following the method described in the preparation of 13g. H NMR
(300 MHz, CD3OD) δ 8.11 (d, J = 2.5 Hz, 1H), 8.04 (s, 1H), 7.84 (dd, J = 8.8, 2.5 Hz, 1H), 7.73 (d, J =
8.8 Hz, 1H), 4.09 (d, J = 12.9 Hz, 2H), 3.90 – 3.83 (m, 2H), 3.81 (t, J = 6.3 Hz, 2H), 3.67 (d, J = 12.5
Hz, 2H), 3.47 (t, J = 6.3 Hz, 2H), 3.30 – 3.19 (m, 4H), 1.68 (h, J = 7.4 Hz, 2H), 0.99 (t, J = 7.4 Hz, 3H).
N-(1-(3,4-Dichlorobenzyl)-1H-1,2,3-triazol-4-yl)-N-(2-morpholinoethyl)acetamide hydrochloride
(18b). A mixture of 13g (70 mg, 0.20 mmol) and acetic anhydride (0.5 mL, excess) was heated using
microwave heating at 120 ºC for 10 min. After cooling back to room temperature, the reaction mixture
was quenched with H2O and extracted with EtOAc twice. The combined organic fractions were dried
over Na2SO4 and the solvent was removed under reduced pressure to give N-(1-(3,4-dichlorobenzyl)-
1H-1,2,3-triazol-4-yl)-N-(2-morpholinoethyl)acetamide as a colourless oil (60 mg, 76%). The compound
was prepared as a hydrochloride salt following the method described in the preparation of 13g. 1H NMR
(300 MHz, CD3OD) δ 8.26 (s, 1H), 7.60 (d, J = 2.0 Hz, 1H), 7.57 (d, J = 8.3 Hz, 1H), 7.34 (dd, J = 8.4,
2.1 Hz, 1H), 5.64 (s, 2H), 4.14 – 4.08 (m, 2H), 4.07 (t, J = 5.7 Hz, 2H), 3.82 (t, J = 12.5 Hz, 2H), 3.68
(d, J = 12.4 Hz, 2H), 3.43 (t, J = 5.8 Hz, 2H), 3.20 (t, J = 12.1 Hz, 2H), 1.98 (s, 3H).
N-(1-(3,4-Dichlorophenyl)-1H-1,2,3-triazol-4-yl)-N-(2-morpholinoethyl)methanesulfonamide
hydrochloride (19). To a stirred solution of 12h (100 mg, 0.29 mmol) in dry CH2Cl2 (10 mL), DIPEA
(110 µL, 0.64 mmol) was added under an argon atmosphere and the reaction mixture stirred for 10 min
at room temperature. The mixture was cooled down to 0 ºC and methanesulfonyl chloride (24.9 µL,
0.321 mmol) was added dropwise. The reaction mixture was stirred overnight at room temperature, after
which one additional equivalent of reactants was added. The reaction was stirred at room temperature
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Journal of Medicinal Chemistry
until full conversion was reached and the resulting mixture was diluted with CH2Cl2, washed with 3%
aqueous HCl solution and then with saturated aqueous NaHCO3 followed by brine. The combined
organic fractions were dried over MgSO4 and the solvent removed under reduced pressure to give 19 as
a yellow solid (102 mg, 83%). The compound was prepared as a hydrochloride salt following the
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method described in the preparation of 13g. H NMR (300 MHz, CD3OD) δ 8.51 (s, 1H), 8.07 (d, J =
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2.1 Hz, 1H), 7.78 (dd, J = 8.5, 2.1 Hz, 1H), 7.67 (d, J = 8.5 Hz, 1H), 3.91 (t, J = 6.2 Hz, 2H), 3.65 –
3.50 (m, 4H), 3.08 (s, 3H), 2.67 – 2.54 (m, 2H), 2.54 – 2.39 (m, 4H).
1-(1-(3,4-Dichlorophenyl)-1H-1,2,3-triazol-4-yl)-1-(2-morpholinoethyl)-3-propylthiourea
hydrochloride (20). To a stirred solution of 12h (100 mg, 0.29 mmol, 1 equiv.) in CH2Cl2 (8 mL), TEA
(130 µL, 0.94 mmol, 3.2 equiv.) was added and the resulting reaction mixture stirred for 10 min at room
temperature. The mixture was cooled down to 0 ºC and 1-isothiocyanatopropane (79.8 µL, 0.760 mmol,
2.6 equiv.) was added, after which it was allowed to reach room temperature and slowly heated to 50 ºC
for 16 h. After cooling back to room temperature, the resulting mixture was diluted with CH2Cl2,
washed with 3% aqueous HCl (three times) and with saturated aqueous NaHCO3 (three times) followed
by brine. The combined organic fractions were dried over MgSO4 and the solvent removed under
reduced pressure to give 20 as an orange-red oil (83 mg, 64%). The compound was prepared as a
1
hydrochloride salt following the method described in the preparation of 13g. H NMR (400 MHz,
CD3OD) δ 8.81 (s, 1H), 8.19 (d, J = 2.6 Hz, 1H), 7.91 (dd, J = 8.7, 2.4 Hz, 1H), 7.81 (d, J = 8.7 Hz, 1H),
4.72 (t, J = 6.2 Hz, 2H), 4.14 – 4.05 (m, 2H), 3.88 – 3.76 (m, 2H), 3.73 (d, J = 12.6 Hz, 2H), 3.59 – 3.49
(m, 4H), 3.32 – 3.23 (m, 2H), 1.62 (h, J = 7.5 Hz, 2H), 0.90 (t, J = 7.5 Hz, 3H).
tert-Butyl 1-(4-fluorobenzyl)-1H-1,2,3-triazol-5-yl(2-(piperidin-1-yl)ethyl)carbamate (23). A
stirred solution of 9a (prepared as described above for 9, 356 mg, 0.87 mmol) in dry THF (30 mL) was
cooled to 0 ºC under an argon atmosphere, and TBAF (1 M in THF, 871 µL, 0.87 mmol) was added
dropwise. The reaction mixture was stirred at 0 ºC for 30 min and additionally at room temperature for
10 min, after which the TIPS deprotection had been completed as judged by TLC. The reaction was
quenched with saturated aqueous NaHCO3 solution and the deprotected alkyne extracted with EtOAc.
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The combined organic fractions were washed with brine and dried over MgSO4. The solvent was
removed under reduced pressure after filtration to give crude 21. This crude was transferred without
further purification to a schlenk and 1-(azidomethyl)-4-fluorobenzene 22 (144 mg, 0.96 mmol) and
Cp*RuCl(PPh3)2 (33.5 mg, 0.44 mmol) were added, evacuated and backfilled with argon (three cycles).
Toluene (5 mL) was added and the reaction mixture heated at 80 ºC for 25 h. After cooling back to room
temperature, the solvent was removed under reduced pressure. The crude tert-butyl 2-(piperidin-1-
yl)ethyl((triisopropylsilyl)ethynyl)carbamate was partitioned between H2O and EtOAc. The aqueous
phase was additional extracted with EtOAc twice. The combined organic fractions were dried over
Na2SO4. The solvent was removed under reduced pressure after filtration. The residue was purified by
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Combiflash chromatography (SiO2, c-Hexane/ EtOAc up to 50%) to give 23 (45 mg, 13%). %). H
NMR (300 MHz, CDCl3) δ 7.54 (s, 1H), 7.33 – 7.19 (m, 2H), 7.04 (t, J = 8.5 Hz, 2H), 5.43 (s, 2H), 3.72
– 3.25 (m, 2H), 2.59 – 2.18 (m, 6H), 1.90 – 1.12 (m, 6H), 1.33 (s, 9H).
1-(4-Fluorobenzyl)-N-(2-(piperidin-1-yl)ethyl)-1H-1,2,3-triazol-5-amine hydrochloride (24). To a
stirred solution of 23 (45 mg, 0.11 mmol) in CH2Cl2 (10 mL), TFA (500 µL, 6.73 mmol) was added
dropwise. The reaction mixture was stirred at room temperature until the deprotection process had been
completed as judged by TLC. The reaction mixture was quenched with saturated aqueous NaHCO3 and
the product extracted with CH2Cl2 twice. The combined organic fractions were dried over Na2SO4 and
the solvent was removed under reduced pressure to give 1-(4-fluorobenzyl)-N-(2-(piperidin-1-yl)ethyl)-
1H-1,2,3-triazol-5-amine as colourless oil (32 mg, 94%). This compound (32 mg, 0.11 mmol) was
dissolved in acetone (2 mL) and HCl solution (2 M in Et2O, 58 µL, 0.12 mmol) was added dropwise.
After stirring for 30 min. at room temperature, the white solid formed was filtered off and dried in vacuo
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to give 24 as white solid (16 mg, 45%). H NMR (300 MHz, CD3OD) δ 7.79 (s, 1H), 7.45 – 7.32 (m,
2H), 7.14 (t, J = 8.8 Hz, 2H), 5.62 (s, 2H), 3.64 (t, J = 6.2 Hz, 2H), 3.59 – 3.47 (m, 2H), 3.33 (t, J = 6.2
Hz, 2H), 3.02 – 2.86 (m, 2H), 2.05 – 1.69 (m, 5H), 1.63 – 1.37 (m, 1H).
Human Sigma-1 receptor radioligand assay.24 The binding properties of the test compounds to
human σ1R were studied in transfected HEK-293 membranes using [3H](+)-pentazocine (Perkin Elmer,
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Journal of Medicinal Chemistry
NET-1056) as the radioligand. The assay was carried out with 7 μg of membrane suspension, [3H]-(+)-
pentazocine (5 nM) in either absence or presence of either buffer or 10 μM haloperidol for total and non-
specific binding, respectively. Binding buffer contained Tris-HCl (50 mM, at pH 8). Plates were
incubated at 37 °C for 120 minutes. After the incubation period, the reaction mix was transferred to
MultiScreen HTS, FC plates (Millipore), filtered and plates were washed (3 times) with ice-cold Tris–
HCl (10 mM, pH 7.4). Filters were dried and counted at approximately 40% efficiency in a MicroBeta
scintillation counter (Perkin-Elmer) using EcoScint liquid scintillation cocktail.
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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 described25 with some modifications. [3H]-Di-o-
tolylguanidine (DTG) (Perkin Elmer, Code NET-986) was used as the radioligand. The assay was
carried out with 200 μg of membrane suspension, [3H]-DTG (10 nM) in either absence or presence of
either buffer or 10 μM haloperidol for total and non-specific 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 minutes. After the incubation period, the reaction mixture was transferred to
MultiScreen HTS, FC plates (Millipore), filtered and plates were washed 3 times with ice-cold 50 mM
Tris–HCl (pH 7.4). Filters were dried and counted at approximately 40% efficiency in a MicroBeta
scintillation counter (Perkin-Elmer) using EcoScint liquid scintillation cocktail.
Automated patch clamp electrophysiology studies using QPatch. CHO cells stably expressing
hERG channels (Millipore) were cultured in F12 HAM medium supplemented with 10% FBS and
400µg/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, 305 mOsm. The intracellular Ringer’s solution consisted
of (in mM): 5.37 CaCl2, 1.75 MgCl2, 31.25/10 KOH/EGTA, 10 HEPES, 210 KCl, pH 7.2, 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
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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 each 15 seconds. For each cell, extracellular solution was applied previous to increasing
concentrations of the tested compound.
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In vivo tests. Animals. CD1 mice (Charles River, France) from 6 to 8 weeks old were used. Male mice
were used for the formalin test and partial sciatic nerve ligation model and female mice for the capsaicin
test. Animals had access to food and water ad libitum and were kept in controlled laboratory conditions
with the temperature at 21 ± 1 °C and a light−dark cycle of 12 h. Experimental behavioural testing was
carried out in a soundproof and air-regulated experimental rooms and was done blind with respect to
treatment. Experimental procedures and animal husbandry were conducted according to European
guidelines regarding protection of animals used for experimental and other scientific purposes (Council
Directive of Nov 24, 1986, 86/609/ECC) and received approval by the local Ethical Committee.
Formalin test. A diluted formalin solution (20 μL of a 2.5% formalin solution, 0.92% formaldehyde)
was injected into the midplantar surface of the right hind paw of the mouse. Formalin induced
nociceptive behaviour was quantified as the time spent licking or biting the injected paw during two
different periods individually recorded: the first period was recorded 0−5 min after the injection of
formalin and was considered indicative of phase I formalin-evoked nociception (data not shown); the
second period was recorded 15−30 min after formalin injection and was considered indicative of phase
II formalin evoked nociception. The mice (n = 7-8 per group) received i.p. or p.o. administration of a 10
mL/kg volume of vehicle 0.5% hydroxypropyl methyl cellulose (HPMC) (Sigma-Aldrich) or test
compound 30 min before i.pl. formalin injection. The antinociception induced by the treatments in the
formalin test was calculated with the following equation: antinociception (%) = [(LTV – LTD) – LTV]
× 100, where LTV and LTD represent the licking-biting time in vehicle- and drug-treated animals,
respectively.
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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
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Journal of Medicinal Chemistry
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 i.pl. capsaicin (or its solvent) 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. Briefly, a nonflexible
filament (0.5 mm diameter) was electronically (dynamic plantar aesthesiometer, Ugo Basile, Italy)
driven into the ventral side of the paw previously injected, at least 5 mm away from the site of the
injection towards 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 intervals of 0.5 min, and the mean value of the three trials was considered the withdrawal
latency time of the animal. The mice (n = 8−26 per group) received vehicle 0.5% HPMC (Sigma-
Aldrich) or test compound through p.o. 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).
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Partial sciatic nerve ligation model. In this model of neuropathic pain the sciatic nerve is injured
(ligated) at mid-thigh level. Briefly, mice were anaesthetized with isoflurane (induction: 3%; surgery:
1%) and the common sciatic nerve was exposed at the level of the mid-thigh of the right paw. At about 1
cm, proximally to the nerve trifurcation, tight ligation was created around 33-50% of the sciatic nerve,
leaving the rest of the nerve “uninjured”. Care was taken to ensure that the ligation was not too tight so
as to occlude the perineural blood flow. The muscle was then stitched, and the incision was closed with
wound clips. Allodynia to mechanical stimuli was used as outcome measure of neuropathic pain by
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using the von Frey test (as described below). Animals were first habituated for 1 h to experimental test
once daily for 4 days. After the habituation period, baseline responses were established during 2
consecutive days. One day after baseline measurements, sciatic nerve injury was induced and mice were
tested on days 5 and 8 after the surgical procedure to monitor the development of the neuropathic pain-
related behaviour (mechanical allodynia). On day 8, mechanical allodynia was already apparent and
mice received a vehicle injection. The protocol used in these experiments was adapted to a screening
phase in which various σ1R ligands were consecutively evaluated in the same study. Therefore,
compounds 12h and 13g were administered on day 15, in two independent experiments. The mice (n =
7−8 per group) received test compound by p.o. route in a volume of 10 mL/kg 30 min before testing. As
an internal control to know if mechanical sensitivity was influenced by previous treatment, a baseline
measurement was recorded before drug administration, and also on day 18 after surgery in the absence
of drug treatments.
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Mechanical allodynia was quantified by measuring the hind paw withdrawal response to von Frey
filament stimulation. Briefly, animals were placed in a Plexiglas® box (20 cm high, 9 cm diameter) with
a wire grid bottom through which the von Frey filaments (bending force range from 0.008 to 2 g) (North
Coast Medical, Inc., San Jose CA, USA) was applied by using the up-down paradigm as previously
described.36 The filament of 0.4 g was first applied. Then, the strength of the next filament was
decreased when animal responded or increased when animal did not respond. This up-down procedure
was stopped 4 measures after the first change in animal responding (i.e. from response to no response or
from no response to response). The threshold of response was then calculated by using the up-down
Excel program generously provided by Basbaum’s laboratory (UCSF, San Francisco, CA, USA). Clear
paw withdrawal, shaking or licking was considered as a nociceptive-like response. Both ipsilateral and
contralateral hind paws were tested.
The effect of the treatments on mechanical hypersensitivity induced by partial sciatic nerve ligation
was calculated with the following equation: reduction of mechanical hypersensitivity (%) = [(PD −
Pd8)/(Baseline − Pd8)] × 100, where PD is the pressure (grams) required (threshold) to elicit the
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Journal of Medicinal Chemistry
response (paw withdrawal) to von Frey filament stimulation in drug-treated animals, Pd8 is the pressure
threshold measured on day 8 after surgery following vehicle administration, and Baseline is the pressure
threshold measure before surgery.
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Supporting Information Available: Analytical data for all the compounds. This material is available
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free of charge via the Internet at http://pubs.acs.org.
Corresponding author information: José Luis Díaz and Carmen Almansa. Drug Discovery and
Preclinical Development, Esteve, Baldiri Reixach, 4-8, 08028 Barcelona, Spain. Tel: 0034934466000,
email: jldiaz@esteve.es; calmansa@esteve.es
Acknowledgment. We thank Albert Dordal, Raquel Enrech, Magda Bordas, Enrique Portillo, Beatriz
de la Puente and Daniel Zamanillo for their expert contribution to analytical, in vitro and in vivo studies,
Joan Andreu Morató, Monica Carro, and Edmundo Ortega for their excellent technical assistance and
Carlos Pérez and Eduardo Villarroel for their contribution to compound management.
We thank also the support from CDTI (PROJECT IDI-20110577).
Abbreviations Used. ADMET, absorption, distribution, metabolism, excretion and toxicity; DIPEA,
N,N-diisopropylethylamine; TIPS, triisopropylsilyl ether; TBAF, tetra-N-butylammonium fluoride.
References and Notes
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Immunocytochemical localization of the sigma1 receptor in the adult rat central nervous system.
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phosphorylation of NMDA receptor subunit 1 and the second phase of formalin test in mice. Br. J.
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Journal of Medicinal Chemistry
Table 1. Aryltriazoles 12
1
2
3
4
5
6
7
H
N
R2
N
N
N
8
9
R1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
hERGc
a
b
Ki σ1
Ki σ2
comp
R1
R2
cLogP
IC50
(µM)
(h, nM)
(gp, nM)
---
---
---
17 ± 7
10 ± 5
>1000
>10
NTe
1
---
91 ± 43
562 ± 60
323 ± 57
62 ± 1 %d
65 ± 6 %d
59 ± 1 %d
54 ± 1 %d
65 ± 2 %d
53 ± 0 %d
>10000
2
---
---
39 ± 6
NTe
3
3,4-diCl
2,4-diCl
4-Cl
Piperidin-1-yl
Piperidin-1-yl
Piperidin-1-yl
Piperidin-1-yl
Piperidin-1-yl
Piperidin-1-yl
Piperidin-1-yl
Morpholin-4-yl
5 ± 1
4.6
4.8
4.0
3.4
3.0
3.5
3.6
3.4
1.7 ± 0.3
4.9 ± 0.2
3.1 ± 0.2
5.2 ± 0.7
NTe
12a
12b
12c
12d
12e
12f
12g
12h
31 ± 1
62 ± 11
58 ± 9
3-F
4-F
144 ± 15
31 ± 2
3,4-diF
2,4-diF
3,4-diCl
2.2 ± 0.3
NTe
273 ± 78
46 ± 18
>10
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1
2
3
4
5
6
7
8
3-F
3,4-diF
3-F
Morpholin-4-yl
781 ± 133
>1000
NTe
2.2
2.2
4.0
3.2
2.2
2.9
2.9
NTe
NTe
NTe
NTe
>10
>10
NTe
12i
12j
Morpholin-4-yl
Homopiperidin-1-yl
NTe
9
12 ± 2
86 ± 2 %d
74 ± 2 %d
47 ± 2 %d
29 ± 2 %d
NTe
12k
12l
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3-F
4-Cyclohexylpiperazin-1-yl
Homomorpholin-4-yl
4,4-Difluoropiperidin-1-yl
4-Acetylpiperazin-1-yl
56 ± 7
3-F
36 ± 8
12m
12n
12o
3,4-diF
3,4-diCl
70 ± 12
275 ± 90
aBinding affinity (Ki) to human σ1R in transfected HEK-293 membranes using [3H](+)-pentazocine as
radioligand. Each val ue i s the mean ± SD of two determinations. bBinding affinity (Ki, nM) to σ2R in
guinea pig brain membranes using [3H]di-o-tolylguanidine as radioligand. Each value is the mean ± SD
of two determinations. cWhole-cell patch clamp hERG blockade. Binding affinity to σ2R (% inhibition
d
at 1 µM). eNot tested.
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Journal of Medicinal Chemistry
Table 2. Arylmethyltriazoles 13
1
2
3
4
5
6
7
H
N
R2
N
N
N
8
9
R1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
hERGc
a
b
Ki σ1
Ki σ2
comp
R1
R2
cLogP
(h, nM)
(gp, nM)
IC50 (µM)
H
3,4-diCl
4-F
Piperidin-1-yl
Piperidin-1-yl
Piperidin-1-yl
Piperidin-1-yl
Piperidin-1-yl
49 ± 16
624 ± 134
2.5
4.0
2.8
2.8
2.9
1.4
2.7
1.6
1.6
4.5
3.1
>10
2.2 ± 0.7
>10
13a
13b
13c
13d
13e
13f
13g
13h
13i
10 ± 1
52 ± 7
35 ± 6
33 ± 7
>1000
61 ± 20
69 ± 30
66 ± 12 %d
43 ± 4 %d
946 ± 4
NTe
3-F
>10
3,4-diF
H
>10
Morpholin-4-yl
Morpholin-4-yl
NTe
3,4-diCl
4-F
>10000
NTe
>10
Morpholin-4-yl
662 ± 352
878 ± 229
9 ± 1
NTe
3,4-diF
3,4-diCl
H
Morpholin-4-yl
NTe
NTe
Homopiperidin-1-yl
Homopiperidin-1-yl
87 ± 2 %d
75 ± 1 %d
3.9 ± 0.5
>10
13j
13k
23 ± 0
ACS Paragon Plus Environment