Propionamide Derivatives as Dual μ-Opioid Receptor Agonists and σ1Receptor Antagonists for the Treatment of Pain

Article abstract of DOI:10.1021/acs.jmedchem.1c00417

A new series of propionamide derivatives was developed as dual μ-opioid receptor agonists and σ₁receptor antagonists. Modification of a high-throughput screening hit originated a series of piperazinylcycloalkylmethyl propionamides, which were e

Full text of DOI:10.1021/acs.jmedchem.1c00417

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Propionamide Derivatives as Dual μ‑Opioid Receptor Agonists and
σ1 Receptor Antagonists for the Treatment of Pain
Mónica García,* Virginia Llorente, Lourdes Garriga, Ute Christmann, Sergi Rodríguez-Escrich,
̃
̃
Marina Virgili, Begona Fernández, Magda Bordas, Eva Ayet, Javier Burgueno, Marta Pujol, Albert Dordal,
Enrique Portillo-Salido, Georgia Gris, José Miguel Vela, and Carmen Almansa
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ABSTRACT: A new series of propionamide derivatives was developed as dual μ-opioid receptor agonists and σ1 receptor
antagonists. Modification of a high-throughput screening hit originated a series of piperazinylcycloalkylmethyl propionamides, which
were explored to overcome the challenge of achieving balanced dual activity and convenient drug-like properties. The lead
compound identified, 18g, showed good analgesic effects in several animal models of both acute (paw pressure) and chronic (partial
sciatic nerve ligation) pain, with reduced gastrointestinal effects in comparison with oxycodone.
showing synergistic efficacy are considered a good way to
provide better therapeutic indexes.9 Further advantages of this
approach are (i) potentially reduced side effects due to the
possibility of using a relatively lower affinity of multimodal
compounds showing a synergistic therapeutic effect, (ii) better
treatment compliance in comparison to that of a combination
of monomodal drugs, (iii) lower potential for drug−drug
interactions, and (iv) a more convenient pharmacokinetic
(PK) profile.10
With the aim of finding new analgesics with reduced adverse
effects, we undertook a research program to discover
multimodal compounds with dual σ1 receptor (σ1R)
antagonistic and MOR agonistic activities. The rationale
behind this strategy is linked both to the potentiation of
opioid analgesia with σ1R antagonists11−14 and to the
important effects of σ1R antagonism in neuropathic pain
where opioids are poorly effective.15,16 The results obtained
with σ1R knockout mice,17,18 and with several σ1R antagonists,
INTRODUCTION
■
Chronic pain is a condition affecting a significant part of the
population at some point in their lifetime, and it is associated
with significant alterations not only in health but also in quality
and life style1 since it induces affective disorders, such as
depression and anxiety, sleep disorders, and different disability
levels.2 As a consequence, chronic pain is one of the great
health problems worldwide at present due to both its high
prevalence and the economic, social, and psychological costs
that it entails.3 Current therapeutic strategies are only partially
effective or carry the risk of severe side effects, as in the case of
μ-opioid receptor (MOR) agonists, which are associated with
limiting adverse effects such as respiratory depression, nausea,
vomiting, and constipation.4 Moreover, in long-term treat-
ments, opioid drugs induce tolerance,5,6 and as a consequence,
increasing the dosage of opioids is often required throughout
the chronic pain treatment to maintain effective pain relief,
which finally results in unbearable side effects and/or
addiction. Abuse of opioids due to their reinforcement
properties7 has recently led to abuse epidemics affecting the
United States with dramatic consequences.8 Figure 1 shows the
structure of three of the most relevant MOR agonists,
morphine (1), oxycodone (2), and fentanyl (3).
Received: March 8, 2021
Published: July 8, 2021
In view of the multicomponent nature of pain, drugs
addressing simultaneous action on multiple targets and
https://doi.org/10.1021/acs.jmedchem.1c00417
© 2021 American Chemical Society
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Figure 1. Structures of MOR agonists (1−3), σ1R antagonist (4), dual clinical candidate (5), and dual HTS hits (6 and 7).
Table 1. Exploration of Substituents in R1, R1′, and R2
a
b
c
d
Comp
R1, R1′
R2
Ki MOR (h, nM)
12
EC50 MOR (h, nM)
17
Emax MOR (%)
Ki σ1R (h, nM)
clog P
2
3
9
100
>10000
0
4
6
7
>10000
>10000
17
7
3.9
4
297 89
1005 319
>1000
1167 105
4527 559
>10000
91
74
955 217
699 31
10
23
4.3
4.8
6.4
4.4
5.1
4.7
4.7
4.2
3.1
13-3
15a
15b
15c
15d
15e
15f
18a
Me, Me
cyclohexyl
H, H
Me, Me
Me, Me
cyclopropyl
cyclopropyl
cyclohexyl
H
1
1
COEt
COEt
COEt
COCH2OCH3
COEt
43
3
158
5
71
82
37
212 70
95 41
>1000
515 85
299 14
1468 107
92 27
14
48
42
54
3
8
4
3
2
1
34
1
62
COCH2OCH3
COEt
72 20
>1000
>10000
>10000
496 82
a
Binding affinity (Ki) in an MOR binding assay using [3H]-DAMGO as the radioligand. Each value is the mean SD of two determinations.
b
c
MOR functionality (EC50) measuring cAMP on CHO-K1 cells. Each value is the mean SD of two determinations. An efficacy of 100% is
defined as the maximum effect induced by stimulation with DAMGO. It is provided for compounds showing EC50 below 1000 nM in the functional
d
assay. Binding affinity (Ki) to the human σ1R in transfected HEK-293 membranes using [3H](+)-pentazocine as the radioligand. Each value is the
mean SD of two determinations.
such as E-52862 (4, Figure 1),19,20 have also proven the
involvement of the σ1R in pain. Compound 4, both
preclinically21 and clinically,22 has shown evidence that the
σ1R would be a good target for enhancing MOR analgesia
without affecting opioid-induced side effects.
As a first approach to the development of dual σ1R
antagonists and MOR agonists, we recently reported a series
that was conceived through a merging approach of the two
target pharmacophores, considering common features in their
structures.23,24 From this research program, compound
EST73502 (5, Figure 1) was selected as a clinical candidate25
for the treatment of pain.
As a complementary strategy in this promising research line,
a high-throughput screening (HTS) campaign was undertaken,
leading to the identification of two dual hits, 6 and 7 (Figure
1), with the common structure N-((1-(4-methylpiperazin-1-
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Figure 2. Modification sequence from 6 and 7 to 18g.
a
Scheme 1. Synthesis Route of Compounds 18 Starting from Substituted Piperazines 9 and Ketone 8
a
t
Reagents and conditions: (a) Na2S2O5, H2O, KCN, rt, 2 days; (b) LiAlH4, Et2O, 0 °C to rt, 1 h; (c) Pd2(dba)3, BINAP, BuOK, THF, 50 °C,
overnight; (d) TEA, DCM, rt, 0.5 h or DIPEA, DCE, 85 °C, 0.5 h; (e) NH4HCO2, 20% Pd/C, MeOH, 65 °C, 1 h; and (f) K2CO3, acetonitrile, 50
°C, overnight or DIPEA, KI, acetonitrile, MW, 80 °C, 1 h.
yl)cyclohexyl)methyl)amide and an incipient dual affinity (see
Table 1). We report here the synthesis and SAR studies
(structure−activity relationships) of a new series of piper-
azinylcycloalkylmethyl-N-arylpropionamides26 that were de-
signed based on these initial dual HTS hits. Figure 2 shows a
summary of the modifications performed from hits 6 and 7
that led to the identification of the lead compound 18g.
corresponding acid chlorides 14 provided the benzyl (15a-t)
and methyl (18a) derivatives. Alternative groups in R4 (Table
4) were introduced by cleaving the benzyl group of 15
followed by N-alkylation of 16 with the corresponding alkyl
halides 17 to give final compounds 18. Compound 19 (Table
3) was prepared following the synthetic route depicted in
Scheme 1, but using 2-benzyloctahydropyrrolo[3,4-c]pyrrole
instead of 1-benzylpiperazine 9-1.
For compounds 18 bearing a hydroxyl group in the N-alkyl
chain in R4, the final derivatization from compound 16 was
performed, as depicted in Scheme 2. Opening of epoxide 20a
with piperazine 16a provided a reaction mixture of
regioisomers that was purified by preparative high-performance
liquid chromatography (HPLC) to yield compounds 18d and
18d′ as racemates. A similar reaction of amine 16a with
epoxide 20b provided exclusive attack on the unsubstituted
epoxide position to give compound 18q.
CHEMISTRY
■
Schemes 1−4 depict the synthesis of the compounds described
herein. Starting from substituted piperazines 9, a convenient
route was designed to prepare final compounds 15 and 18
(Scheme 1). The Strecker reaction27,28 of 9 with ketone 8 led
to nitrile intermediates 10. Reduction of the nitrile group to
amines 11 and subsequent arylation under Buchwald−Hartwig
coupling conditions with the appropriate bromoaryls 12 led to
arylamines 13 in good yields. Finally, N-acylation with the
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Table 2. Exploration of Aryl Groups in R3
a
Binding affinity (Ki) in an MOR binding assay using [3H]-DAMGO as the radioligand. Each value is the mean SD of two determinations.
b
c
MOR functionality (EC50) measuring cAMP on CHO-K1 cells. Each value is the mean SD of two determinations. An efficacy of 100% is
defined as the maximum effect induced by stimulation with DAMGO. It is provided for compounds showing EC50 below 1000 nM in the functional
d
assay. Binding affinity (Ki) to the human σ1R in transfected HEK-293 membranes using [3H](+)-pentazocine as the radioligand. Each value is the
e
f
mean SD of two determinations. Whole-cell patch clamp hERG blockade. Intrinsic clearance in human liver microsomes as a measure of
g
metabolic stability, obtained as described in ref 33. Not tested.
Although Scheme 1 could be of general application, in the
case where R1 and R1′ are di-H, di-methyl, or spirocyclopropyl
groups, some variations were used for the preparation of
intermediates 13 (Scheme 3), which were then acylated to
compounds 15 as described in Scheme 1. The unsubstituted
derivative 13-2 was prepared by the acylation of 9-1 with
chloroacetyl chloride, subsequent arylation of 21 with aniline
(22), and final amide reduction of 23. The synthesis of 13-3
involved alkylation of 9-1 with ethyl 2-bromo-2-methylpropa-
noate under microwave irradiation. Ester saponification to give
carboxylic acid 24, conventional amide coupling with aniline,
and final reduction with alane (aluminum hydride) afforded
intermediate 13-3. Cyclopropyl derivatives 13-4 were synthe-
sized following a strategy of piperazinyl cyclization. Treatment
of diol 25 with thionyl chloride provided the dichloroethyl
derivative 26, which was cyclized with ethyl 1-amino-
cyclopropanecarboxylate and hydrolyzed to afford the key
carboxylic intermediate 27. Compound 27 was transformed
into the primary carboxamide under amide coupling conditions
with ammonium bicarbonate. Amide reduction and final
Buchwald−Hartwig arylation with bromoaryls 12 provided
intermediates 13-4.
Piperidine analogue 36 was obtained as described in Scheme
4. The carbanion generated from tetrahydro-2H-pyran-4-
carbonitrile 28 in the presence of lithium diisopropylamide
(LDA) was added over Boc-protected 4-piperidone 29 to
render intermediate 30, which was then dehydrated with a
Burgess reagent. The resulting tetrahydropyridine 31 was
hydrogenated to the piperidine intermediate 32. Boc-
deprotection and introduction of the benzyl chain via reductive
amination afforded piperidine 33, which was reduced, arylated
to install the R3 substituent, and acylated to provide 36, using
similar conditions to those described in Scheme 1.
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Table 3. Heterocycloalkyl Groups in R1 and R1′ and Piperazine Replacement
a
Binding affinity (Ki) in an MOR binding assay using [3H]-DAMGO as the radioligand. Each value is the mean SD of two determinations.
b
c
MOR functionality (EC50) measuring cAMP on CHO-K1 cells. Each value is the mean SD of two determinations. An efficacy of 100% is
defined as the maximum effect induced by stimulation with DAMGO. It is provided for compounds showing EC50 below 1000 nM in the functional
d
assay. Binding affinity (Ki) to the human σ1R in transfected HEK-293 membranes using [3H](+)-pentazocine as the radioligand. Each value is the
e
f
mean SD of two determinations. Whole-cell patch clamp hERG blockade. Intrinsic clearance in human liver microsomes as a measure of
g
metabolic stability, obtained as described in ref 33. Not tested.
functional, scaffold-related agonistic bias of piperazinylcycloal-
kylmethyl propionamides. The functionality versus the σ1R was
not generally studied since there is not an efficient screening
functionality assay due to the unique ligand-regulated
molecular chaperone nature of the target.
The selectivity of the dual active compounds identified was
evaluated at 1 μM in a small selectivity panel as previously
described.23 The best lead compounds 18e, 18g, and 18k
described here were selective versus this panel (% inhibition
lower than 50% at 1 μM).
RESULTS AND DISCUSSION
■
All synthetized compounds were tested in human σ1R and
MOR binding assays using [3H]-(+)-pentazocine29 and [3H]-
DAMGO30 as radioligands, respectively. The MOR functional
assay involves cyclic AMP (cAMP) measurement on CHO-K1
cells.31 Potency (EC50) as well as efficacy (Emax) data are
provided in Tables 1−4. Compounds with Emax above 80% are
assigned as full agonists, while compounds with Emax below
80% are considered as partial agonists at the MOR.
Compounds binding to the MOR but with an antagonist
profile at the MOR (inactive in the functional assay or Emax
below 20%) were not identified in our series, suggesting a
A significant challenge in the design of dual compounds is
getting suitable drug-like properties as the space for
optimization is narrowed by the need of maintaining both
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Table 4. Optimization of Piperazine Substituents R4
a
Binding affinity (Ki) in an MOR binding assay using [3H]-DAMGO as the radioligand. Each value is the mean SD of two determinations.
b
c
MOR functionality (EC50) measuring cAMP on CHO-K1 cells. Each value is the mean SD of two determinations. An efficacy of 100% is
defined as the maximum effect induced by stimulation with DAMGO. It is provided for compounds showing EC50 below 1000 nM in the functional
d
assay. Binding affinity (Ki) to the human σ1R in transfected HEK-293 membranes using [3H](+)-pentazocine as the radioligand. Each value is the
e
f
mean SD of two determinations. Whole-cell patch clamp hERG blockade. Intrinsic clearance in human liver microsomes as a measure of
g
metabolic stability, obtained as described in ref 33. Not tested.
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a
Scheme 2. Derivatization Route of Compounds 18 Starting from Compound 16a
a
Reagents and conditions: (a) EtOH, 70 °C, overnight; (b) TEA, MeOH, 75 °C, 3 h.
a
Scheme 3. Synthetic Routes of Intermediates 13
a
Reagents and conditions: (a) DCM, rt, 0.5 h; (b) TEA, NaI, DMF, rt, overnight; (c) LiAlH4, THF, 0 °C to 65 °C, overnight; (d) TEA, EtOH,
MW, 150 °C, 3.5 h; (e) NaOH, MeOH, 65 °C, 6 h; (f) HOBt, EDC, DMF, TEA, rt, overnight; (g) alane solution in THF, 0 °C, 1.5 h; (h) thionyl
chloride, DCM, rt, overnight; (i) NaHCO3, EtOH, 80 °C for 4 h, then overnight at rt; (j) KOH, EtOH, 120 °C, 5 h; (k) ammonium bicarbonate,
t
HATU, TEA, DMF, 0 °C to rt, 2 days; and (l) R3Br (12), Pd2(dba)3, BINAP, BuOK, THF, 50 °C, overnight.
activities.32 Metabolic stability measured as the intrinsic
clearance in human liver microsomes was obtained for the
dual compounds,33 and we also assayed the blockade of the
human ether-a-go-go-related gene (hERG),34 whose inhibition
has been a constant concern in our previous σ1R programs.14
The results of these tests are shown in Tables 2−4.
As mentioned before, screening of our internal compound
library led to the identification of the dual hits 6 and 7 (Figure
1 and Table 1) with an incipient submicromolar dual affinity.
Starting from the common structure of N-((1-(4-methylpiper-
azin-1-yl)cyclohexyl)methyl) amide, we decided to preserve
the methylpiperazin-1-ylcycloalkyl moiety and modify the
nature of the amide substitution. The acylarylamine present
in fentanyl (3) was installed instead of the bulky amide group
present in the HTS hits. The first synthesized compound 18a
slightly improved the σ1R affinity but was devoid of MOR
activity. In parallel, we prepared compound 15a, in which the
methyl group was replaced by a benzyl group, which usually
gives good σ1R affinities as a hydrophobic group is required at
a suitable distance from the basic amine, as described in the
new σ1R model35 built using the Langer pharmacophore.36 We
were delighted to see that 15a showed an improved σ1R
affinity, regaining at the same time MOR agonist functionality.
However, it exhibited a very low metabolic stability (human
Clint 67.9 μl/min/mg protein), which we reasoned could be
attributed to its high clog P.
Exploration of smaller substituents in R1 and R1′ to decrease
lipophilicity was first studied (Table 1). The unsubstituted 15b
was 5-fold less potent than 15a, but the dimethyl (15c) and
spirocyclopropyl (15e) derivatives showed a significant
improvement. The hydrogen-bond-acceptor propionyl moiety
in R2 was proven necessary to interact with the MOR, as
shown by the lack of activity of compound 13-3. However, an
additional heteroatom was not tolerated (15d and 15f).
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a
Scheme 4. Synthetic Process of Piperidine Analogue 36
a
Reagents and conditions: (a) LDA, THF, −78 °C, 2 h; (b) Burgess reagent, toluene, 90 °C, overnight; (c) H2, Pd/C, EtOH, rt, overnight; (d)
TFA, DCM, rt, 1 h; (e) benzaldehyde, NaBH(OAc)3, THF, a few drops of AcOH, rt, overnight; (f) LiAlH4, Et2O; (g) Pd2(dba)3, BINAP, tBuOK,
THF, 55 °C, overnight; and (h) TEA, DCM, rt, 0.5 h.
Among the initial compounds, propionamide 15e with
spirocyclopropyl substitution in R1 and R1′ displayed the best
dual MOR and σ1R affinities with potent partial MOR
agonism. Despite its substantial decrease in lipophilicity versus
that of 15a, its metabolic stability was again quite low (human
Clint 90.5 μL/min/mg protein), so additional efforts were
focused on further decreasing clog P or avoiding potential
metabolic hotspots.
the poor human metabolic stability observed so far. The data
reported in Table 3 show that introduction of polarity in R1
and R1′ led to greatly improved metabolic stability for piperidyl
(15o) and tetrahydropyranyl (15q) analogues, although at the
expense of dual affinity in the case of 15o. Other analogues
were prepared, maintaining a 6-(trifluoromethyl)-2-pyridinyl
group in position R3. Dimethyl (15t) substitution in the 2-
position of the tetrahydropyran ring and changing the oxygen
atom with a sulfur atom to give tetrahydrothiopyranyl
derivative 15s were tolerated by both targets but were clearly
detrimental regarding hERG inhibition.
Replacement of the piperazine by a piperidine ring (36)
maintained the duality, suggesting that N4 of the piperazine
nucleus was the positive ionizable feature required by both
targets. Surprisingly, changing the piperazine with bicycle
octahydropyrrolo[3,4-c]pyrrole (19) led to a complete loss of
MOR activity.
In the following optimization round, we set out to explore
different alternatives to the benzyl moiety in R4 while
maintaining the best substituents in the remaining positions
(Table 4). As expected, and in line with the data obtained with
compound 18a, the unsubstituted piperazine 16a was poorly
active, while the phenethyl derivative 18b lost MOR agonism.
One of the strategies for detuning hERG inhibition is the
generation of steric bulk near the basic amine or reducing the
planarity.39 With this aim, branched analogues were prepared.
The α-methylbenzyl derivative 18c showed a favorable hERG
inhibition result, but it had poor metabolic stability in humans.
The decrease in clog P by the introduction of a 2-hydroxy
group to 18c (18d) provided an impairment in the σ1R affinity,
a result not totally unexpected in view of the hydrophobicity of
the σ1R. Propyl (18e) and butyl (18f) derivatives showed a
We next explored the aryl substitution in R3 over the most
promising cyclopropyl derivative (Table 2). While the N-
benzyl substituent had low affinity for the MOR (15g), N-
phenyl significantly increased the affinity as well as agonist
potency, keeping also the affinity for the σ1R (compounds 15e
and 15h−j). Introduction of fluorine atoms (15i and 15j)
failed at improving the metabolic stability. A hydroxyl group in
the ortho position (15h) gave an improvement in hERG
inhibition but with diminished σ1R affinity and metabolic
stability. Introduction of the three possible pyridyl isomers in
R3 was also explored. Only the 2-pyridyl derivative (15k)
maintained the desired affinity for both targets, while MOR
affinity and functionality worsened for the 3- and 4-pyridyl
analogues (data not shown). Many substituents were tested at
different positions of the 2-pyridyl group, and the derivatives
containing a trifluoromethyl or fluorine group in the ortho
position (15l−n) were particularly interesting in terms of their
good dual activities. Unfortunately, the best compound (15n)
was not optimal regarding metabolic stability and hERG
inhibition.
With the key learnings achieved at this point of the SAR
study, we considered further optimization of R1 and R1′
because it is well known that cycloalkyl motifs are prone to
suffer oxidation by CYP3A4,37,38 which could be the cause of
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a
Table 5. PK Parameters of Compound 18g in Mice
route
dose(mg/kg)
t1/2 (h)
Cmax (ng/mL)
tmax (h)
AUC0‑t (ng·h/mL)
AUC (ng·h/mL)
Cl (l/h/kg)
10.8
Vss (l/kg)
F (%)
po
ip
iv
10
40
1
n.c.
0.6
0.4
130
11319
0.5
0.08
122
5953
92
n.c.
5955
93
13
>100
2.9
a
n.c.: not calculable (extrapolated AUC >20%).
Figure 3. Dose−response antinociception curve in the paw pressure test of 18g (A) and oxycodone (B) administered in male CD1 mice after ip
administration. Each point and vertical line represent the mean S.E.M of the maximum possible effect percentage of the effect (n = 6−8 per
group).
significant decrease in σ1R affinity and MOR agonism in
comparison with the parent benzyl (15r), although 18e
showed improved hERG inhibition and metabolic stability. A
slight increase in the hydrophobicity in compound 18g
provided a 2-fold improvement in σ1R affinity, with a moderate
metabolic stability and hERG IC50 above 10 μM. Introduction
of oxygen atoms in the alkyl chains was detrimental in the ester
(18h) and hydroxyl (18q) derivatives but was tolerated in the
case of ethers, with the exception of 18i. Some of them
displayed interesting dual affinities for the primary targets with
potent MOR agonism (18j, 18l, 18m, and 18o) but were still
suboptimal regarding metabolic stability.
Overall, compounds 18e, 18g, and 18k emerged as some of
the best derivatives, showing good affinity for primary targets,
balanced in the case of 18g to a higher σ1R affinity. Although
the optimal ratio for the σ1R and MOR in vitro affinities is
difficult to establish, a predominant affinity for the MOR
would not be desirable because it would increase the likelihood
for opioid-related adverse effects and, importantly, it would
mask the σ1R antagonism enhancement of opioid analgesia. In
fact, compound 4 showed the best results when combined with
low doses of morphine: E-52862, 40 mg/kg and morphine,
2.5−5 mg/kg.15
Compound 18g was selected for further in vitro and in vivo
characterizations. Regarding the selectivity, exhaustive in vitro
characterization of compound 18g revealed the lack of
significant affinity (inhibition % at 10 μM < 50%) for 180
molecular targets (receptors, transporters, ion channels, and
enzymes),40 except for the σ2R (Ki 413 nM). Phenytoin
binding experiments41 showed an antagonistic behavior for the
σ1R, while experiments with β-funaltrexamine31 confirmed the
partial agonism at the MOR (results not shown). Additionally,
compound 18g did not show in vitro cytotoxic potential at 100
μM in HepG2 cells, both in the neutral red uptake and in the
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide] assays.42 It was also devoid of genotoxic potential
in the SOS/umu and Ames bacterial mutation assays.43
Compound 18g exhibited an adequate physicochemical
profile, with moderate basicity (pKa 8.9) and lipophilicity
(experimental log P 3.7, log D7.4 2.2) and good solubility in the
whole physiologically meaningful pH range (thermodynamic
solubility > 0.5 mg/mL at pH 7.4 and pH 2). This, together
with its good permeability in Caco-2 cells (PappA‑to‑B 169 nm/
s), predicts a good oral (po) absorption in humans.44 It also
complied with Lipinski’s rules and presented a good value
(4.8) in the central nervous system (CNS) multi-parameter
optimization algorithm developed by Wager et al.45 as a holistic
approach for the prediction of good ADME (absorption,
distribution, metabolism, and excretion) and safety attributes
in the case of CNS-directed compounds.
The PK behavior of 18g was evaluated in mice after po,
intraperitoneal (ip), and intravenous (iv) administration of
single doses of 10, 40, and 1 mg/kg, respectively, and
comparative data are outlined in Table 5. The po plasma
exposure was not very high, but the exposure after ip
administration of 40 mg/kg was high, and it was selected as
a representative dose for establishing initial PK/pharmacody-
namics relationships as discussed below. Furthermore, the
CNS distribution of 18g was evaluated in rats as a
representative rodent species, where it showed a similar
exposure in the plasma and brain (AUC 2811 and 2434 ng·h/
mL in the plasma and brain, respectively, a brain-to-plasma
ratio of 0.9) after ip administration of 40 mg/kg.
Compound 18g was evaluated for its in vivo activity in the
mouse paw pressure test of acute pain in CD1 mice (n = 6−8
per group). After ip administration, 18g showed a dose−
response analgesic effect, reaching a maximum of 66 5.2%
and an ED50 of 24
0.78 mg/kg (Figure 3). The ip
administration of oxycodone also induced a dose−response
effect, reaching a maximum of 89 3.5% and an ED50 of 4
0.4 mg/kg.
The antinociceptive activity of 18g was also evaluated
following repeated/subchronic administration for 10 days in
the partial sciatic nerve ligation (PSNL) model of neuropathic
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pain in CD1 male mice in comparison to that of oxycodone
(2). Mechanical hypersensitivity (mechanical allodynia) was
evaluated as a neuropathic pain-related behavior, as described
in the Supporting Information. Repeated administration of
compound 18g (20 mg/kg, ip) and oxycodone (compound 2,
1.25 mg/kg, ip) showed that both 18g and 2 attenuated
mechanical allodynia induced by PSNL, reaching maximal
effects of 96 13.9 and 41 9.1%, respectively. Interestingly,
repeated administration of 18g twice daily over 10 days did not
induce tolerance but an increased antinociceptive effect, with
the analgesic effect being higher at day 22 than that at day 13
(p = 0.0052) (Figure 4).
distance traveled by the charcoal meal versus the total length of
the small intestine after drug administration and comparing to
that of the control group that received the vehicle. Figure 5
shows that, at the doses used, 18g and 2 induced similar
antinociceptive effects (18g = 59.4 8.3%, 2 = 77 5.7%, p =
0.10), whereas they produced a differentiated effect on the
intestinal transit. Compound 18g did not reduce the intestinal
transit (p = 0.35 vs vehicle), whereas oxycodone produced
significant constipation in comparison with the vehicle group
(p = 0.01 vs vehicle).
CONCLUSIONS
■
A new series of piperazinyl-cycloalkylmethyl-N-aryl-propiona-
mide derivatives as dual ligands for the MOR and σ1R is
reported. This dual approach aims at enhancing opioid
analgesia by additionally targeting the σ1R, which may provide
an increased effect in nociceptive and neuropathic analgesia
without affecting opioid-induced side effects. Starting from two
HTS dual hits, significant improvements in dual affinities were
achieved after the first round of modifications in the amide,
cyclohexyl, and methyl-piperazine groups of the initial hits
structure, as exemplified by derivatives 15a and 15e. Since
these compounds exhibited very low metabolic stability, a hit-
to-lead program was initiated to decrease clog P, maintaining
dual activities and also dealing with hERG inhibition, which
emerged as an additional challenge in the optimization of this
series. The best compounds identified (18e, 18g, and 18k)
showed good dual affinities, low hERG inhibition, and
acceptable metabolic stability. Compound 18g was selected
for further characterization and demonstrated analgesic activity
in acute and chronic pain mice models, with less opioid-
induced tolerance and constipation than oxycodone. These
results place the 18g lead compound and the chemical series
described herein as potential subjects for a lead optimization
program to develop a backup compound for candidate
EST73502 currently in clinical trials.
Figure 4. Effect of 18g (blue bars) or oxycodone (2, gray) in
comparison to the vehicle (white) on PSNL-induced mechanical
allodynia in male CD1 mice. Compounds 18g (20 mg/kg) and 2
(1.25 mg/kg) were systemically (ip) administered twice daily for 10
EXPERIMENTAL SECTION
■
Unless otherwise noted, all materials were obtained from commercial
suppliers and used without further purification. Reference compounds
used in biological assays were obtained from the following vendors:
oxycodone hydrochloride (2) was obtained from Macfarlan Smith Ltd
(UK); E-52862 (4) was obtained as previously described;14 and HTS
compounds 6 and 7 were acquired from Cerep.
days. Each bar and vertical line represent the mean
S.E.M. paw
withdrawal threshold (n = 6−12 per group). ***p < 0.001 versus basal
mean; ##p < 0.01; and ###p < 0.001 versus each vehicle group (one-
way ANOVA, followed by Tukey’s test).
Flash chromatography was performed on a Teledyne Isco
CombiFlash RF system with disposable columns. H spectra were
recorded on an Agilent Mercury 400 MHz spectrometer fitted with a
1
Intestinal transit reduction (as a surrogate of constipation)
was evaluated in CD1 mice by measuring the percentage of the
Figure 5. Effect of 18g (40 mg/kg, ip, blue bars) or oxycodone (2) (10 mg/kg, ip, gray bars) on the analgesic response (paw pressure test) (A) and
on the intestinal transit measured in the charcoal test (B) in male CD1 mice after drug administration. Each bar and vertical line represent the
mean S.E.M. percentage of effect (n = 4−14 per group). *p < 0.05 comparing with the vehicle (HPMC 0.5%, white bar; one-way ANOVA,
followed by Tukey’s test).
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5 mm ATB 1H/19F/X probe with a 2 H lock in deuterated solvents.
Chemical shifts (δ) are in parts per million. Commercially available
reagents and solvents (HPLC grade) were used without further
purification for all the analytical tests. Analytical HPLC−mass
spectrometry (HPLC-MS) for all final compounds and intermediates
were performed on a Waters H-Class-ZQ MS system. A reverse-phase
Acquity C18 BHE column was used (2.1 × 5 mm, 1.7 μm) under the
following conditions: gradient 0.3 min in 98% A, 98% A to 5% A in
2.52 min, and isocratic 1.02 min in 5% A (A = 10 mM ammonium
bicarbonate and B = acetonitrile); injection volume: 2 μL; flow: 0.61
mL/min; and temperature: 35 °C. UV spectra were recorded from
195 to 315 nm using a Waters Acquity photodiode array detector.
Mass spectra were obtained over the range m/z 100−800 by
electrospray ionization (ESI) in positive and negative modes using a
Waters ZQ Single Quadrupole. Data were integrated and reported
using Waters MassLynx software. All final compounds displayed a
purity equal or higher than 95%, as determined by said methods,
except for 15a, whose purity was 85%. Accurate mass measurements
were carried out using an Agilent 6540 UHD high-resolution mass
spectrometer quadrupole time-of-flight system and obtained by ESI in
the positive mode. Data were acquired and processed using
MassHunter software. 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
Physicochemical properties of compounds were determined as
previously described.24,47 All compounds have been named using
ChemBioDraw Ultra 12.0.2.
General Procedures for the Synthesis of Piperazinyl-
cycloalkylmethyl-N-aryl-propionamide Derivatives 15 and
18. N-((4-(4-Benzylpiperazin-1-yl)tetrahydro-2H-pyran-4-yl)-
methyl)-N-(6-(trifluoromethyl)pyridin-2-yl)propionamide (15r).
Step 1. In a 2 L round-bottomed flask, dihydro-2H-pyran-4(3H)-
one (8-1, 10.8 g, 107.8 mmol) was dissolved in water (500 mL) along
with sodium metabisulfite (10.2 g, 54 mmol).48 The mixture was
allowed to stir at room temperature (rt) for 1.5 h, and then,
benzylpiperazine (9-1, 19 g, 108 mmol) was added. The mixture was
stirred for 2 h, and potassium cyanide (11.2 g, 173 mmol) was added
to the reaction mixture. After stirring at rt for 2 days, the solid formed
was filtered and dried to give 4-(4-benzylpiperazin-1-yl)tetrahydro-
2H-pyran-4-carbonitrile (10-1) as a white solid (27.8 g, 90% yield).
1H NMR (400 MHz, CDCl3): δ 7.35−7.28 (m, 4H), 7.31−7.22 (m,
thus obtained was purified by flash chromatography on silica gel,
gradient CH/ethyl acetate from (100:0) to (0:100), to give N-((4-(4-
benzylpiperazin-1-yl)tetrahydro-2H-pyran-4-yl)methyl)-6-
(trifluoromethyl)pyridin-2-amine (13-1) as a solid (3.53 g, 61%
1
yield). H NMR (400 MHz, CD3OD): δ 7.50 (t, J = 7.9 Hz, 1H),
7.38−7.20 (m, 5H), 6.84 (d, J = 7.2 Hz, 1H), 6.71 (d, J = 8.5 Hz,
1H), 3.81 (ddd, J = 3.3, 7.6, 11.1 Hz, 2H), 3.64 (ddd, J = 3.4, 6.7, 11.0
Hz, 2H), 3.61 (s, 2H), 3.54 (s, 2H), 2.80 (t, J = 4.5 Hz, 4H), 2.58−
2.45 (m, 4H), 1.90−1.76 (m, 2H), 1.62 (ddd, J = 3.3, 7.5, 12.4 Hz,
2H). HPLC-MS: purity 99%, m/z = 435 [M + H]+.
Step 4. Propionyl chloride (14-1, 1.38 mL, 15.88 mmol) was added
to a solution of compound 13-1 (2.3 g, 5.29 mmol) and N-ethyl-N-
isopropylpropan-2-amine (DIPEA) (3.68 mL, 21.17 mmol) in
dichloroethane under a nitrogen atmosphere. The reaction mixture
was heated for 30 min at 85 °C, after which it was allowed to reach rt.
The reaction mixture was diluted with dichloromethane (DCM) (25
mL), and water (25 mL) was added. The aqueous phase was acidified
with 10% HCl, and the phases were separated. The organic phase was
extracted with 10% HCl, and the aqueous phases were made alkaline
with 20% NaOH while cooling. Ethyl acetate (10 mL) was added, the
phases were separated, and the aqueous phase was extracted with
ethyl acetate. The combined organic phases were dried over Na2SO4,
filtered, and concentrated to give N-((4-(4-benzylpiperazin-1-yl)-
tetrahydro-2H-pyran-4-yl)methyl)-N-(6-(trifluoromethyl)pyridin-2-
1
yl)propionamide (15r) (2.45 g, 94% yield). H NMR (400 MHz,
CD3OD): δ 8.13 (t, J = 6.9 Hz, 1H), 7.91−7.70 (m, 2H), 7.60−7.48
(m, 5H), 4.49−4.18 (m, 4H), 3.73 (ddt, J = 3.7, 6.3, 10.5 Hz, 2H),
3.68−2.67 (m, 10H), 2.25 (q, J = 7.3 Hz, 2H), 1.95−1.50 (m, 4H),
1.05 (t, J = 7.4 Hz, 3H). HPLC-MS: purity 99%. HRMS [M + H]+
(diff ppm) 491.2645 (−3.38).
N-((4-(4-Isobutylpiperazin-1-yl)tetrahydro-2H-pyran-4-yl)-
methyl)-N-(6-(trifluoromethyl)pyridin-2-yl)propionamide (18g).
Step 1. Compound 15r (427 mg, 0.87 mmol) was dissolved in
MeOH (10 mL), and ammonium formate (246 mg, 3.91 mmol) and
Pd/C (85 mg, 10% Wt) were added. The suspension was stirred
under a N2 atmosphere for 1 h at 65 °C. The reaction mixture was
filtered through Celite, washed with MeOH, and concentrated to give
N-((4-(piperazin-1-yl)tetrahydro-2H-pyran-4-yl)methyl)-N-(6-
(trifluoromethyl)pyridin-2-yl)propionamide (16a) (347 mg, 99%
1
yield). H NMR (400 MHz, CDCl3): δ 8.42 (s, 1H), 7.97 (t, J =
8.0 Hz, 1H), 7.63 (d, J = 7.6 Hz, 1H), 7.42 (d, J = 8.1 Hz, 1H), 4.21
(s, 2H), 3.77−3.56 (m, 4H), 3.28−2.37 (m, 8H), 2.23 (q, J = 7.3 Hz,
2H), 1.74−1.59 (m, 2H), 1.52−1.39 (m, 2H), 1.09 (t, J = 7.3 Hz,
3H). HPLC-MS: purity 95%, m/z = 401 [M + H]+.
1H), 4.09−3.91 (m, 2H), 3.65 (td, J = 2.1, 12.3 Hz, 2H), 3.52 (s,
2H), 2.75−2.58 (m, 4H), 2.61−2.41 (m, 4H), 2.08 (dq, J = 2.5, 13.5
Hz, 2H), 1.72 (ddd, J = 4.5, 11.7, 13.2 Hz, 2H). HPLC-MS: m/z =
286 [M + H]+.
Step 2. 1-Bromo-2-methylpropane (17, 39 μL, 0.36 mmol) was
added to a solution of 16a (80 mg, 0.2 mmol) and K2CO3 (83 mg, 0.6
mmol) in ACN (5 mL). The reaction mixture was stirred at 50 °C
overnight and then was cooled down to rt. Ethyl acetate (10 mL) and
saturated aqueous NaHCO3 solution (10 mL) were added, and the
phases were separated. The organic layer was dried over Na2SO4,
filtered, and concentrated to give N-((4-(4-isobutylpiperazin-1-
yl)tetrahydro-2H-pyran-4-yl)methyl)-N-(6-(trifluoromethyl)pyridin-
2-yl)propionamide (18g free base) as an oil (67 mg, 73% yield). To a
slurry of the previous free base (12 mg, 0.026 mmol) in ethyl acetate
(1 mL), HCl (2 M solution in diethyl ether, 26 μL, 0.052 mmol) was
added, and the mixture was stirred at rt for 0.5 h. The solids were
dried in vacuo to give the 18g HCl salt as a white solid (12.5 mg, 95%
yield). 1H NMR (400 MHz, D2O): δ 8.25 (t, J = 7.9 Hz, 1H), 7.95 (d,
J = 7.9 Hz, 1H), 7.86 (d, J = 8.1 Hz, 1H), 4.42 (s, 2H), 3.80 (dt, J =
4.2, 12.3 Hz, 2H), 3.75−3.30 (m, 8H), 3.25 (t, J = 11.1 Hz, 3H), 3.04
(d, J = 7.4 Hz, 2H), 2.25 (q, J = 7.3 Hz, 2H), 2.21−2.04 (m, 1H),
1.99−1.87 (m, 2H), 1.78 (d, J = 14.7 Hz, 2H), 0.99 (d, J = 6.6 Hz,
8H), 0.98 (t, J = 7.4 Hz, 6H). HPLC-MS: purity 99%. HRMS [M +
H]+ (diff ppm) 457.2783 (0.41).
Step 2. Compound 10-1 (2 g, 7 mmol) in tetrahydrofuran (THF)/
Et2O (40/20 mL) was added at 0 °C to a stirred solution of fresh
lithium aluminum hydride in Et2O (1 M, 14 mL, 14 mmol). The
reaction was stirred at rt for 1 h, a few drops of an aqueous saturated
solution of potassium sodium tartrate were added, and the mixture
was stirred overnight at rt. Then, water and ethyl acetate were added,
and the aqueous layer was separated and extracted several times with
ethyl acetate. The organic layers were combined, dried over Na2SO4,
filtered, and concentrated to give (4-(4-benzylpiperazin-1-yl)-
tetrahydro-2H-pyran-4-yl)methanamine (11-1) as a solid (1.8 g,
1
88% yield). H NMR (300 MHz, CD3OD): δ 7.64−7.39 (m, 5H),
4.38 (s, 2H), 3.96−3.78 (m, 2H), 3.59 (t, J = 11.3 Hz, 2H), 3.47 (d, J
= 11.9 Hz, 2H), 3.25 (s, 2H), 3.34−3.10 (m, 4H), 2.73 (t, J = 12.3
Hz, 2H), 2.02−1.84 (m, 2H), 1.53 (d, J = 13.7 Hz, 2H). HPLC-MS:
purity 100%, m/z = 290 [M + H]+.
Step 3. Compound 11-1 (3.6 g, 13.13 mmol), Pd2(dba)3 (1.14 g,
1.25 mmol), 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (BINAP,
0.93 g, 1.50 mmol), and tBuOK (2.8 g, 25.01 mmol) were dissolved in
anhydrous THF (430 mL). 2-Bromo-6-(trifluoromethyl)pyridine (12-
1, 2.96 g, 13.13 mmol) was added, and the reaction mixture was
stirred under an inert atmosphere at 50 °C overnight. The solvents
were evaporated, and the residue was dissolved in ethyl acetate and an
aqueous saturated NaHCO3 solution. The aqueous layer was
extracted with ethyl acetate, and the combined organic layers were
dried over Na2SO4, filtered, and concentrated. The crude product
N-((4-(4-(2-Hydroxy-1-phenylethyl)piperazin-1-yl)tetrahydro-2H-
pyran-4-yl)methyl)-N-(6-(trifluoromethyl)pyridin-2-yl)-
propionamide (18d) and N-((4-(4-(2-Hydroxy-2-phenylethyl)-
piperazin-1-yl)tetrahydro-2H-pyran-4-yl)methyl)-N-(6-
(trifluoromethyl)pyridin-2-yl)propionamide (18d′). A solution of
16a (80 mg, 0.2 mmol) and 2-phenyloxirane (20a, 211 mg, 1.75
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mmol) in EtOH (5 mL) was heated at 70 °C in a sealed tube
overnight. The reaction mixture was cooled to rt, and the solvent was
evaporated. The residue was purified by preparative HPLC (Column
X-Bridge C18, ACN: NH4HCO3 10 mM from 2:98 to 95:5, flow 20
ml/min, rt) to give N-((4-(4-(2-hydroxy-1-phenylethyl)piperazin-1-
yl)tetrahydro-2H-pyran-4-yl)methyl)-N-(6-(trifluoromethyl)pyridin-
2-yl)propionamide (18d) (13 mg, 13% yield) and N-((4-(4-(2-
hydroxy-2-phenylethyl)piperazin-1-yl)tetrahydro-2H-pyran-4-yl)-
methyl)-N-(6-(trifluoromethyl)pyridin-2-yl)propionamide (18d′) (6
Step 3. Compound 23 (0.5 g, 1.61 mmol) in THF (18 mL) was
added at 0 °C to a stirred solution of fresh lithium aluminum hydride
in THF (1 M, 3.23 mL, 3 mmol). The reaction was stirred at 65 °C
overnight, and then, it was cooled down to 0 °C. A few drops of an
aqueous solution of 5% NaOH were added, and the mixture was
allowed to reach rt and stirred overnight. The resulting suspension
was filtered and washed with ethyl acetate. The combined filtrates
were washed with water, and the combined organic layers were dried
over Na2SO4, filtered, and concentrated to dryness to give 7-(2-(4-
benzylpiperazin-1-yl)ethyl)aniline (13-2) as a dark oil (0.381 g, 79%
1
mg, 6% yield). H NMR (18d) (400 MHz, CD3OD): δ 7.70 (t, J =
1
8.1 Hz, 1H), 7.57 (d, J = 8.1 Hz, 1H), 7.44−7.29 (m, 4H), 7.17 (d, J
= 7.7 Hz, 2H), 4.24 (s, 2H), 3.89 (dd, J = 7.1, 11.1 Hz, 1H), 3.81−
3.64 (m, 5H), 3.40−3.34 (m, 1H), 2.48 (d, J = 8.9 Hz, 4H), 2.13 (q, J
= 7.4 Hz, 2H), 2.06−1.80 (m, 4H), 1.73 (tt, J = 4.6, 8.7 Hz, 2H),
1.55−1.38 (m, 2H), 0.99 (t, J = 7.4 Hz, 3H). HPLC-MS (18d): purity
yield). H NMR (400 MHz, CD3OD): δ 7.47−7.19 (m, 5H), 7.18−
7.03 (m, 2H), 6.79−6.54 (m, 3H), 3.55 (s, 2H), 3.22 (dd, J = 6.2, 7.2
Hz, 2H), 2.61 (dd, J = 6.3, 7.1 Hz, 2H), 2.68−2.39 (m, 8H). HPLC-
MS: m/z = 296 [M + H]+.
N-(2-(4-Benzylpiperazin-1-yl)-2-methylpropyl)aniline (13-3).
Step 1. TEA (1.4 mL, 10 mmol) and ethyl 2-bromo-2-
methylpropanoate (1.5 mL, 10 mmol) were added to a solution of
1-benzylpiperazine (9-1, 0.6 g, 3.4 mmol) in EtOH (15 mL) in a
microwave vial under a nitrogen atmosphere. The reaction mixture
was heated under microwave irradiation for 3.5 h at 150 °C, after
which it was allowed to reach rt. The solvent was concentrated in
vacuo, and the residue was dissolved in DCM and washed twice with
water. The aqueous phase was acidified to pH 2 and extracted twice
with DCM. The organic layers were combined, dried over Na2SO4,
filtered, and concentrated to give ethyl 2-(4-benzylpiperazin-1-yl)-2-
methylpropanoate (656 mg, 66% yield) as a crude ester that was
submitted to hydrolysis without further purification. HPLC-MS: m/z
= 291 [M + H]+. Aqueous NaOH (1 M, 4.2 mL, 40 mmol) was added
to a solution of the previous compound (648 mg, 2.23 mmol) in
MeOH (20 mL). The mixture was refluxed for 6 h and then
concentrated under vacuum to afford 2-(4-benzylpiperazin-1-yl)-2-
methylpropanoic acid (24), which was used in the following step
without further purification. HPLC-MS: m/z = 263 [M + H]+.
Step 2. A mixture of compound 24 (0.6 g, 2.28 mmol) and HOBt
(0.7 g, 4.57 mmol) was added to a solution of 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDC) (0.87 g, 4.57 mmol) in
DMF (3 mL). Then, aniline (22, 311 μL, 3.43 mmol) and TEA (495
μL, 3.55 mmol) were added, and the reaction mixture was stirred at rt
overnight. The mixture was partitioned between water and ethyl
acetate/ethyl ether (1:1), and the organic layer was washed twice with
an aqueous basic solution, twice with an aqueous acid solution, and
twice with water. Finally, the organic layer was dried over Na2SO4,
filtered, and concentrated to give 2-(4-benzylpiperazin-1-yl)-2-methyl-
N-phenylpropanamide (174.5 mg, 22% yield). HPLC-MS: m/z = 338
[M + H]+. The alane solution was then prepared for reduction of this
carboxamide compound: to a vigorously stirred solution of LiAlH4 (1
M in THF, 15 mmol) at −70 °C, sulfuric acid (0.3 mL) was added
dropwise. The reaction was stirred with the temperature increasing
from −70 °C to −50 °C for 2 h and left at rt for 2 h without stirring
(a white solid appeared). In a round-bottomed flask, the previously
prepared fresh alane solution (1.07 mL, 3 eq) decanted via a syringe
was cooled to 0 °C under an argon atmosphere. 2-(4-Benzylpiperazin-
1-yl)-2-methyl-N-phenylpropanamide (120 mg, 0.36 mmol) dissolved
in THF (5 mL) was added dropwise, and the mixture was stirred at 0
°C for 1.5 h. The reaction was quenched with ethyl acetate and ice
water. The aqueous layer was extracted several times with ethyl
acetate. The combined organic layers were washed with water, dried
over Na2SO4, filtered, and concentrated to dryness. The residue was
purified by flash chromatography on silica gel, gradient DCM to
MeOH/DCM (90:10), to give N-(2-(4-benzylpiperazin-1-yl)-2-
1
100%. HRMS [M + H]+ (diff ppm) 521.2754 (−3.65). H NMR
(18d′) (400 MHz, CD3OD): δ 8.10 (ddq, J = 0.7, 7.7, 8.4 Hz, 1H),
7.76 (dd, J = 0.7, 7.7 Hz, 2H), 7.73 (dq, J = 0.7, 8.0 Hz, 2H), 4.74
(dd, J = 3.5, 9.2 Hz, 1H), 4.23 (s, 2H), 3.85−3.72 (m, 2H), 3.71−
3.60 (m, 2H), 2.70−2.54 (m, 4H), 2.49 (dd, J = 9.2, 13.0 Hz, 1H),
2.31 (dd, J = 3.5, 13.0 Hz, 1H), 2.41−2.10 (m, 4H), 2.23 (q, J = 7.4
Hz, 2H), 1.81−1.65 (m, 2H), 1.59−1.43 (m, 2H), 1.05 (t, J = 7.4 Hz,
3H). HPLC-MS (18d′): purity 98%. HRMS [M + H]+ (diff ppm)
521.2753 (−3.31).
N-((4-(4-(2-Hydroxy-2-methylpropyl)piperazin-1-yl)tetrahydro-
2H-pyran-4-yl)methyl)-N-(6-(trifluoromethyl)pyridin-2-yl)-
propionamide (18q). A solution of compound 16a (500 mg, 1.25
mmol), 2,2-dimethyloxirane (243 mg, 3.37 mmol), and triethylamine
(TEA) (379 mg, 3.74 mmol) in MeOH (20 mL) was stirred at 75 °C
for 3 h. The solvent was concentrated in vacuum, and the residue was
partitioned between DCM and water. The mixture was made alkaline
with 20% NaOH, and the organic layer was separated, dried over
Na2SO4, filtered, and concentrated. The crude residue was purified by
flash chromatography on neutral alumine, gradient CH/ethyl acetate
from (100:0) to (50:50), to give N-((4-(4-(2-hydroxy-2-
methylpropyl)piperazin-1-yl)tetrahydro-2H-pyran-4-yl)methyl)-N-(6-
(trifluoromethyl)pyridin-2-yl)propionamide (18q) as an oil (200 mg,
34% yield). 1H NMR (400 MHz, CD3OD): δ 8.11 (ddq, J = 0.7, 7.6,
8.2 Hz, 1H), 7.76 (dd, J = 0.7, 7.7 Hz, 1H), 7.73 (dq, J = 0.7, 8.1 Hz,
1H), 4.22 (s, 2H), 3.79 (ddd, J = 3.1, 7.9, 11.2 Hz, 2H), 3.64 (ddd, J
= 3.3, 6.2, 10.9 Hz, 2H), 2.54 (t, J = 4.8 Hz, 4H), 2.35−2.21 (m, 4H),
2.23 (q, J = 7.4 Hz, 2H), 2.16 (s, 2H), 1.78−1.65 (m, 2H), 1.49 (ddd,
J = 3.5, 7.9, 14.0 Hz, 2H), 1.13 (s, 9H), 1.05 (t, J = 7.4 Hz, 3H).
HPLC-MS: purity 99%. HRMS [M + H]+ (diff ppm) 473.2749
(−3.15).
N-(2-(4-Benzylpiperazin-1-yl)ethyl)aniline (13-2). Step 1. To a
solution of chloroacetyl chloride (0.53 mL, 6.60 mmol) in dry DCM
(25 mL) at 0 °C, 1-benzylpiperazine (9-1, 0.98 mL, 5.50 mmol)
dissolved in dry DCM (25 mL) was added. The reaction mixture was
stirred at rt for 0.5 h, the mixture was poured into cold H2O (150
mL) and made alkaline with an aqueous saturated NaHCO3 solution,
and the phases were separated. The organic layer was dried over
Na2SO4 and evaporated under reduced pressure to give 1-(4-
benzylpiperazin-1-yl)-2-chloroethanone (21) as a yellow oil (1.46 g,
1
quantitative yield). H NMR (400 MHz, CD3OD): δ 7.38−7.30 (m,
4H), 7.29−7.22 (m, 1H), 4.24 (s, 2H), 3.63−3.56 (m, 4H), 3.56 (s,
2H), 2.49 (dt, J = 5.2, 20.2 Hz, 4H). HPLC-MS: purity 94%, m/z =
253 [M + H]+.
Step 2. Compound 21 (1.39 g, 5.5 mmol), TEA (2.3 mL, 16
mmol), and NaI (50 mg, 0.33 mmol) were dissolved in anhydrous
dimethylformamide (DMF) (35 mL). Aniline (22, 0.51 mL, 6 mmol)
was added, and the reaction was stirred at rt overnight. The mixture
was partitioned between water and ethyl acetate/ethyl ether (1:1).
The organic layer was washed with water, dried over Na2SO4, filtered,
and concentrated to give 1-(4-benzylpiperazin-1-yl)-2-
(phenylamino)ethanone (23) as a crude oil that was used in the
1
methylpropyl)aniline (13-3) (32.5 mg, 28% yield). H NMR (400
MHz, CD3OD): δ 7.38−7.30 (m, 4H), 7.30−7.22 (m, 1H), 7.14−
7.06 (m, 2H), 6.64−6.57 (m, 3H), 3.53 (s, 2H), 2.98 (s, 2H), 2.74−
2.57 (m, 4H), 2.57−2.43 (m, 4H), 1.13 (s, 6H). HPLC-MS: purity
94%, m/z = 324 [M + H]+.
N-((1-(4-Benzylpiperazin-1-yl)cyclopropyl)methyl)-6-
(trifluoromethyl)pyridin-2-amine (13-4a). Step 1. Thionyl chloride
(19.75 mL, 266 mmol) in anhydrous DCM (40 mL) was added to a 0
°C cooled solution of 2,2′-(benzylazanediyl)diethanol (25, 17.29 g,
89 mmol) in DCM (15 mL), and the reaction mixture was stirred at rt
1
following step without further purification (1.029 g, 60% yield). H
NMR (400 MHz, CD3OD): δ 7.48−7.32 (m, 5H), 7.18−7.06 (m,
2H), 6.80−6.63 (m, 3H), 3.98 (s, 2H), 3.86 (s, 2H), 3.78−3.59 (m,
4H), 2.85−2.65 (m, 4H). HPLC-MS: m/z = 310 [M + H]+.
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overnight. The solvent was removed to dryness to give N-benzyl-2-
chloro-N-(2-chloroethyl)ethanamine hydrochloride (26, 23.78 g,
100% crude yield), which was used in the next step without further
solution of tetrahydro-2H-pyran-4-carbonitrile (28, 4.85 g, 43.6
mmol) in dry THF (41 mL), cooled at −78 °C, LDA solution
(30.5 mL, 1.5 M in a mixture of THF/ethylbenzene/heptane, 45.8
mmol) was added dropwise under a nitrogen atmosphere. The
mixture was stirred at −50 °C for 45 min, and then, it was cooled at
−78 °C. A solution of tert-butyl 4-oxopiperidine-1-carboxylate (29,
8.69 g, 43.6 mmol) in dry THF (5.2 mL) was added, and the reaction
mixture was stirred at −78 °C for 2 h. Then, NH4Cl saturated
aqueous solution was added, and the mixture was extracted with ethyl
acetate. The organic phases were combined, dried over MgSO4,
filtered, and concentrated to dryness. The residue was purified by
flash chromatography on silica gel, gradient DCM to MeOH/DCM
(10:90), to give tert-butyl 4-(4-cyanotetrahydro-2H-pyran-4-yl)-4-
1
purification. H NMR (400 MHz, CDCl3): δ 7.76−7.63 (m, 2H),
7.57−7.42 (m, 3H), 4.38 (s, 2H), 4.06 (t, J = 6.8 Hz, 4H), 3.47 (t, J =
6.8 Hz, 4H). HPLC-MS: m/z = 232 [M + H]+.
Step 2. Compound 26 (10.37 g, 38.61 mmol) was added to a
solution of ethyl 1-aminocyclopropanecarboxylate hydrochloride (6.4
g, 38.6 mmol) and NaHCO3 (17.5 g, 208.5 mmol) in EtOH (200
mL). The reaction mixture was stirred at 80 °C for 4 h and overnight
at rt. The solvent was concentrated, and the crude product was
diluted with ethyl acetate and water. The layers were separated, and
the organic layer was dried over Na2SO4, filtered, and concentrated.
The crude residue was purified by flash chromatography on silica gel
and eluents CH/ethyl acetate (100:0 to 0:100) to give ethyl 1-(4-
benzylpiperazin-1-yl)cyclopropanecarboxylate (2.86 g, 24% yield).
HPLC-MS: m/z = 289 [M + H]+. The previous compound (2.36 g,
8.21 mmol) was dissolved in EtOH (100 mL), and KOH (5.4 g, 82.07
mmol) in EtOH (50 mL) was added. The solution was heated at 120
°C for 5 h. The reaction mixture was cooled at 0 °C, and acetic acid
(4.7 mL, 82.07 mmol) was added. After stirring for 10 min, the
mixture was concentrated under vacuum. The residue was treated
with ethyl acetate, and the solid thus obtained was filtered and washed
several times with water. Then, it was dried in vacuo to afford 1-(4-
benzylpiperazin-1-yl)cyclopropanecarboxylic acid (27) as a beige
1
hydroxypiperidine-1-carboxylate (30, 7.11 g, 53% yield). H NMR
(400 MHz, CDCl3): δ 4.21−3.95 (m, 4H), 3.72 (td, J = 2.5, 12.0 Hz,
2H), 3.03 (t, J = 12.3 Hz, 2H), 1.91−1.71 (m, 6H), 1.71−1.60 (m,
2H), 1.46 (s, 9H). HPLC-MS: m/z = 255 [M + H − 56]+ (−tBu, Boc
fragmentation).
Step 2. To a solution of compound 30 (6.10 g, 19.7 mmol) in
toluene (71 mL), (methoxycarbonylsulfamoyl)triethylammonium
hydroxide inner salt (“Burgess reagent”, 7.03 g, 29.5 mmol) was
added, and the mixture was heated at 90 °C overnight under a
nitrogen atmosphere. It was then cooled to rt, and water and DCM
were added. The aqueous phase was back-extracted with DCM. The
organic phases were combined, washed with saturated NaHCO3
solution, dried over Na2SO4, filtered, and concentrated to dryness
to give tert-butyl 4-(4-cyanotetrahydro-2H-pyran-4-yl)-5,6-dihydro-
pyridine-1(2H)-carboxylate (31, 6.14 g crude product, 5.75 g
theoretical weight; quantitative yield). 1H NMR (400 MHz,
CDCl3): δ 5.78 (br s, 1H), 4.06−3.94 (m, 4H), 3.84−3.73 (m,
2H), 3.54 (t, J = 5.6 Hz, 2H), 2.27−2.18 (m, 2H), 1.90−1.77 (m,
4H), 1.48 (s, 9H). HPLC-MS: m/z = 237 [M + H − 56]+ (−tBu, Boc
fragmentation).
1
solid (1.76 g, 82% yield). H NMR (400 MHz, CD3OD): δ 7.60−
7.37 (m, 5H), 4.19 (s, 2H), 3.37−3.17 (m, 4H), 3.13−2.94 (m, 4H),
1.27 (q, J = 3.8 Hz, 2H), 0.95 (q, J = 3.7 Hz, 2H). HPLC-MS: purity
100%, m/z = 261 [M + H]+.
Step 3. N-[(Dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-
ylmethylene]-N-methylmethanaminium hexafluorophosphate N-
oxide (HATU, 6.3 g, 17 mmol), TEA (4.2 mL, 30 mmol), and
ammonium bicarbonate (1.19 g, 15 mmol) were added to a solution
of 27 (12.7 g, 15.07 mmol) in DMF (100 mL) at 0 °C, and the
mixture was stirred at rt for 2 days. The reaction mixture was
partitioned between water and ethyl acetate/Et2O (1/1), and the
combined organic layers were washed twice with water, dried over
MgSO4, filtered, and concentrated to afford 1-(4-benzylpiperazin-1-
yl)cyclopropanecarboxamide (1.75 g, 45% yield). HPLC-MS: m/z =
260 [M + H]+. A solution of LiAlH4 (1 M in THF, 13.5 mL, 13.5
mmol) was added to a −10 °C cooled solution of the previous
compound (1.75 g, 6.76 mmol) in dry THF (20 mL). The reaction
mixture was stirred at this temperature for 5 h, allowed to reach rt,
and stirred overnight. The suspension thus obtained was cooled down
to 0 °C, and diluted aqueous NaOH was added. The mixture was
filtered and rinsed with ethyl acetate. The filtrate was washed with
H2O, and the combined organic layers were dried over Na2SO4,
filtered, and concentrated to give (1-(4-benzylpiperazin-1-yl)-
cyclopropyl)methanamine as an oil (0.9 g, 54% yield). HPLC-MS:
m/z = 246 [M + H]+. A portion of this compound (665 mg, 2.7
mmol), 2-bromo-6-(trifluoromethyl)pyridine (12-1, 735 mg, 3.26
mmol), Pd2(dba)3 (101.2 mg, 0.163 mmol), BINAP (124 mg, 0.136
Step 3. A mixture of compound 31 (6.14 g crude, 19.7 mmol) and
palladium (1.23 g, 5% wt on charcoal, wet) in EtOH (115 mL) was
stirred at rt under 1 bar of H2 overnight. Then, the solids were filtered
off over a pad of Celite, and the solvent was evaporated to dryness.
The residue was purified by flash chromatography, silica gel, gradient
DCM to MeOH/DCM (10:90), to give tert-butyl 4-(4-cyanotetrahy-
dro-2H-pyran-4-yl)piperidine-1-carboxylate (32, 4.04 g, 70% yield).
1H NMR (400 MHz, CDCl3): δ 4.38−4.12 (m, 2H), 4.05−3.93 (m,
2H), 3.71 (td, J = 1.9, 12.4 Hz, 2H), 2.65 (t, J = 11.4 Hz, 2H), 1.88
(dd, J = 2.0, 13.5 Hz, 2H), 1.80 (d, J = 10.9 Hz, 2H), 1.60 (ddd, J =
4.5, 10.0, 17.0 Hz, 2H), 1.46 (s, 9H), 1.50−1.34 (m, 3H). HPLC-MS:
purity 91%, m/z = 239.1 [M + H − 56]+ (−tBu, Boc fragmentation).
Step 4. To a solution of compound 32 (4.0 g, 13.6 mmol) in DCM
(40 mL), TFA (10.4 mL, 136 mmol) was added, and the reaction
mixture was stirred at rt for 1 h. The solvent was evaporated to
dryness to give 4-(piperidin-4-yl)tetrahydro-2H-pyran-4-carbonitrile
trifluoroacetate (7.18 g, 4.19 g theoretical weight, quantitative yield).
This crude compound and benzaldehyde (1.3 mL, 17.7 mmol) were
dissolved in dry THF (92 mL), and AcOH (1.73 mL, 30.2 mmol) was
added. The mixture was stirred at rt for 15 min, and then, sodium
triacetoxyborohydride (7.99 g, 40.8 mmol) was added in portions.
The resulting mixture was stirred at rt overnight. Then, concentrated
NH4OH (50 mL) was carefully added, and it was extracted with ethyl
acetate. The organic phases were combined, washed with brine, dried
over MgSO4, filtered, and concentrated to dryness. The residue was
purified by flash chromatography, silica gel, gradient DCM to MeOH/
DCM (25:75), to give 4-(1-benzylpiperidin-4-yl)tetrahydro-2H-
t
mmol), and BuOK (395 mg, 3.52 mmol) were added to a Schlenk
tube, that was evacuated and backfilled with argon (three cycles).
Then, anydrous THF (15 mL) was added, and the reaction mixture
was stirred at 50 °C overnight. The solvents were evaporated, and the
residue was dissolved in ethyl acetate and aqueous saturated NaHCO3
solution. The aqueous layer was extracted with ethyl acetate, and the
combined organic layers were dried over Na2SO4, filtered, and
concentrated. The crude product was purified by flash chromatog-
raphy on silica gel, gradient CH/ethyl acetate from (100:0) to
(0:100), to give N-((1-(4-benzylpiperazin-1-yl)cyclopropyl)methyl)-
6-(trifluoromethyl)pyridin-2-amine (13-4a) as a solid (628 mg, 53%
1
pyran-4-carbonitrile (33, 1.75 g, 45% yield). H NMR (400 MHz,
CDCl3): δ 7.34−7.29 (m, 4H), 7.28−7.22 (m, 1H), 3.97 (ddt, J = 1.0,
4.6, 12.3 Hz, 2H), 3.71 (td, J = 1.9, 12.3 Hz, 2H), 3.50 (s, 2H), 3.06−
2.93 (m, 2H), 1.93 (td, J = 2.4, 11.9 Hz, 2H), 1.86 (dq, J = 2.5, 13.5
Hz, 2H), 1.81−1.72 (m, 2H), 1.65−1.50 (m, 4H), 1.31 (tt, J = 3.7,
12.1 Hz, 1H). HPLC-MS purity 98%; m/z 285 [M + H]+.
1
yield). H NMR (400 MHz, CD3OD): δ 7.48 (ddq, J = 0.7, 7.3, 8.7
Hz, 1H), 7.33−7.20 (m, 5H), 6.82 (dd, J = 0.7, 7.2 Hz, 1H), 6.64 (dq,
J = 0.7, 8.6 Hz, 1H), 3.54 (s, 2H), 3.48 (s, 2H), 2.84 (t, J = 4.9 Hz,
4H), 2.50−2.32 (m, 4H), 0.68−0.62 (m, 2H), 0.62−0.54 (m, 2H).
HPLC-MS: purity 95%, m/z = 391 [M + H]+.
N-((4-(1-Benzylpiperidin-4-yl)tetrahydro-2H-pyran-4-yl)methyl)-
N-(6-(trifluoromethyl)pyridin-2-yl)propionamide (36). Step 1. To a
Step 5. Following a similar procedure to the one described in step 2
of the synthesis of compound 15r, (4-(1-benzylpiperidin-4-yl)-
tetrahydro-2H-pyran-4-yl)methanamine (34) was obtained (879 mg,
1
50% yield). H NMR (400 MHz, CDCl3): δ 7.33−7.28 (m, 4H),
10151
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́
7.27−7.21 (m, 1H), 3.72 (dt, J = 4.3, 11.8 Hz, 2H), 3.59 (ddd, J = 2.8,
10.3, 11.8 Hz, 2H), 3.49 (s, 2H), 2.98 (ddd, J = 1.7, 3.4, 12.4 Hz,
2H), 2.82 (s, 2H), 1.96−1.84 (m, 2H), 1.66−1.56 (m, 4H), 1.46−
1.31 (m, 5H). HPLC-MS: purity 100%; m/z = 289 [M + H]+.
Step 6. Following a similar procedure to the one described in step 3
of the synthesis of compound 15r, N-((4-(1-benzylpiperidin-4-
yl)tetrahydro-2H-pyran-4-yl)methyl)-6-(trifluoromethyl)pyridin-2-
amine (35) was obtained (712 mg, 54% yield). 1H NMR (400 MHz,
CDCl3): δ 7.48 (t, J = 7.9 Hz, 1H), 7.36−7.29 (m, 5H), 6.89 (d, J =
7.3 Hz, 1H), 6.53 (d, J = 8.5 Hz, 1H), 4.63 (br s, 1H), 3.75−3.69 (m,
4H), 3.65−3.52 (m, 2H), 3.50 (s, 2H), 3.06−2.93 (m, 2H), 1.93 (t, J
=11.9 Hz, 2H), 1.80−1.58 (m, 4H), 1.55−1.45 (m, 1H), 1.44−1.30
(m, 2H). HPLC-MS: purity 96%, m/z = 434 [M + H]+.
Cientific de Barcelona, C/ Baldiri Reixac 4-8, 08028
Barcelona, Spain
Ute Christmann − ESTEVE Pharmaceuticals, Barcelona
08038, Spain; Present Address: WELAB Barcelona, Parc
́
Cientific de Barcelona, C/ Baldiri Reixac 4-8, 08028
Barcelona, Spain
Sergi Rodríguez-Escrich − ESTEVE Pharmaceuticals,
Barcelona 08038, Spain; Present Address: WELAB
Barcelona, Parc Cientific de Barcelona, C/ Baldiri Reixac
4-8, 08028 Barcelona, Spain
́
Marina Virgili − Enantia, S.L., Barcelona 08028, Spain
Begona Fernández − ESTEVE Pharmaceuticals, Barcelona
̃
Step 7. Following a similar procedure to the one described in step 4
of the synthesis of compound 15r, N-((4-(1-benzylpiperidin-4-
yl)tetrahydro-2H-pyran-4-yl)methyl)-6-(trifluoromethyl)pyridin-2-
08038, Spain; Present Address: WELAB Barcelona, Parc
́
Cientific de Barcelona, C/ Baldiri Reixac 4-8, 08028
1
Barcelona, Spain
Magda Bordas − ESTEVE Pharmaceuticals, Barcelona 08038,
Spain; Present Address: WELAB Barcelona, Parc Cientific
de Barcelona, C/ Baldiri Reixac 4-8, 08028 Barcelona,
Spain
Eva Ayet − ESTEVE Pharmaceuticals, Barcelona 08038,
Spain; Present Address: WELAB Barcelona, Parc Cientific
amine (36) was obtained (49 mg, 33% yield). H NMR (400 MHz,
CD3OD): δ 8.17 (t, J = 7.9 Hz, 1H), 7.82 (d, J = 8.0 Hz, 2H), 7.81 (d,
J = 7.7 Hz, 1H), 7.59−7.46 (m, 5H), 4.28 (s, 2H), 4.21 (s, 2H),
3.63−3.47 (m, 4H), 3.46−3.36 (m, 2H), 3.06−2.77 (m, 2H), 2.22 (q,
J = 7.3 Hz, 2H), 2.03 (d, J = 13.9 Hz, 2H), 1.83 (t, J = 12.3 Hz, 1H),
1.64 (t, J = 13.0 Hz, 2H), 1.50 (ddd, J = 4.2, 9.1, 13.6 Hz, 2H), 1.26
(d, J = 14.4 Hz, 2H), 1.04 (t, J = 7.4 Hz, 3H). HPLC-MS: purity 94%,
m/z = 490 [M + H]+.
In Vitro Studies. Human radioligand assays of the σ1R and MOR
and human MOR functionality and hERG assays were carried out
under the experimental conditions previously described.24
In Vitro Metabolic Stability and PK Studies. Experimental details
of in vitro metabolic stability in human and mice liver microsomes and
PKs in rodents are provided in the Supporting Information.
In Vivo Efficacy Studies. The experimental details of the paw
pressure test, PSNL, and intestinal transit inhibition models are given
in the Supporting Information. All animal husbandry and
experimental procedures complied with the European guidelines
regarding the protection of animals used for experimental and other
scientific purposes international standards (European Communities
Council directive 2010/63) and were approved by the local ethics
committee.
́
́
de Barcelona, C/ Baldiri Reixac 4-8, 08028 Barcelona,
Spain
Javier Burgueno − ESTEVE Pharmaceuticals, Barcelona
̃
08038, Spain; Present Address: WELAB Barcelona, Parc
́
Cientific de Barcelona, C/ Baldiri Reixac 4-8, 08028
Barcelona, Spain
Marta Pujol − ESTEVE Pharmaceuticals, Barcelona 08038,
Spain; Present Address: WELAB Barcelona, Parc Cientific
de Barcelona, C/ Baldiri Reixac 4-8, 08028 Barcelona,
Spain
Albert Dordal − ESTEVE Pharmaceuticals, Barcelona 08038,
Spain; Present Address: WELAB Barcelona, Parc Cientific
́
́
de Barcelona, C/ Baldiri Reixac 4-8, 08028 Barcelona,
Spain
Enrique Portillo-Salido − ESTEVE Pharmaceuticals,
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00417.
■
Barcelona 08038, Spain; Present Address: WELAB
sı
́
Barcelona, Parc Cientific de Barcelona, C/ Baldiri Reixac
4-8, 08028 Barcelona, Spain
Georgia Gris − ESTEVE Pharmaceuticals, Barcelona 08038,
Spain; Present Address: WELAB Barcelona, Parc Cientific
́
Molecular formula strings (CSV)
Analytical data for all the final compounds and
intermediates, HPLC traces for final compounds,
calculated log D7.4 values of final compounds, exper-
imental details of in vitro metabolic stability and PK
studies, and experimental details of in vivo efficacy
studies (PDF)
de Barcelona, C/ Baldiri Reixac 4-8, 08028 Barcelona,
Spain
José Miguel Vela − ESTEVE Pharmaceuticals, Barcelona
08038, Spain; Present Address: WELAB Barcelona, Parc
́
Cientific de Barcelona, C/ Baldiri Reixac 4-8, 08028
Barcelona, Spain
Carmen Almansa − ESTEVE Pharmaceuticals, Barcelona
08038, Spain; Present Address: WELAB Barcelona, Parc
Cientific de Barcelona, C/ Baldiri Reixac 4-8, 08028
Barcelona, Spain; orcid.org/0000-0001-5665-4685
AUTHOR INFORMATION
Corresponding Author
■
́
Mónica García − ESTEVE Pharmaceuticals, Barcelona
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00417
08038, Spain; Present Address: WELAB Barcelona, Parc
́
Cientific de Barcelona, C/ Baldiri Reixac 4-8, 08028
Barcelona, Spain; orcid.org/0000-0003-2338-8338;
Email: mgarcial@welab.barcelona
Notes
The authors declare no competing financial interest.
Authors
Virginia Llorente − ESTEVE Pharmaceuticals, Barcelona
ACKNOWLEDGMENTS
■
08038, Spain; Present Address: WELAB Barcelona, Parc
We thank Sandra Silvestre, Ma Paz Diez, and Joana Berrocal for
their expert technical contribution to the compound syntheses;
Adriana Port, Raquel Enrech, Joan Andreu Morató, Mónica
́
Cientific de Barcelona, C/ Baldiri Reixac 4-8, 08028
Barcelona, Spain
Lourdes Garriga − ESTEVE Pharmaceuticals, Barcelona
08038, Spain; Present Address: WELAB Barcelona, Parc
́
Carro, Edmundo Ortega, Manel Merlos, Inés Alvarez, Pilar
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Article
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Pérez, Xavier Monroy, Xavier Farré, Enric Hernández, Raquel
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Pardo, Ana Paz Marín, Antonio Rodriguez Fernández de
Henestrosa, and Daniel Zamanillo for their expert contribution
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management. This work was a part of activities in R&D
Projects IDI20130943 and IDI20150915 supported by the
Spanish Ministerio de Economía y Competitividad (MINE-
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ABBREVIATIONS
■
H.; Laggner, C.; Serafini, M. T.; Burgueno, J.; Zamanillo, D.; Merlos,
̃
ADME, absorption, distribution, metabolism, and excretion;
BCS, biopharmaceutics classification system; CNS, central
nervous system; ESI, electrospray ionization; hERG, human
ether-a-go-go-related gene; HPLC, high-performance liquid
chromatography; HTS, high-throughput screening; MOR, μ-
opioid receptor; MS, mass spectrometry; MW, microwave;
PDA, photodiode array; QTOF, quadrupole time-of-flight;
SAR, structure−activity relationships; σ1R, sigma-1 receptor;
TEA, triethylamine
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