Identification of a Potent and Selective 5-HT1AReceptor Agonist with in Vitro and in Vivo Antinociceptive Activity

Article abstract of DOI:10.1021/acschemneuro.0c00289

Opioids are the gold standard drugs for the treatment of acute and chronic severe pain, although their serious side effects constitute a big limitation. In the search for new and safer drugs, 5-HT1AR agonists are emerging as potential candidates in pain relief therapy. In this work, we evaluated the affinity and activity of enantiomers of the two newly synthesized, potent 5-HT1AR agonists N-[(2,2-diphenyl-1,3-dioxolan-4-yl)methyl]-2-[2-(pyridin-4-yl)phenoxy]ethan-1-ammonium hydrogenoxalate (rac-1) and N-((2,2-diphenyl-1,3-dioxolan-4-yl)methyl)-2-(2-(1-methyl-1H-imidazol-5-yl)phenoxy)ethan-1-ammonium hydrogenoxalate (rac-2) in vitro and in vivo. The role of chirality in the interaction with 5-HT1AR was evaluated by molecular docking. The activity of the rac-1 was tested in mouse models of acute pain (hot plate) and severe tonic nociceptive stimulation (intraplantar formalin test). Rac-1 was active in the formalin test with a reduction in paw licking in both phases at 10 mg/kg, and its effect was abolished by the selective 5-HT1AR antagonist, WAY-100635. The eutomer (S)-1, but not the racemate, was active during the hot plate test at 10 and 20 mg/kg, and this effect was abolished by 30 min treatment with WAY-100635 at 30 min. Similarly to 8-OH-DPAT, (S)-1 evoked a slow outward current and depressed spontaneous glutamatergic transmission in superficial dorsal horn neurons, more effectively than rac-1. The eutomer (S)-1 showed promising developability properties, such as high selectivity over 5-HT subtypes, no interaction with the μ receptors, and low hepato- and cardiotoxicity. Therefore, (S)-1 may represent a potential candidate for the treatment of acute and chronic pain without having the adverse effects that are commonly associated with the classic opioid drugs.

Full text of DOI:10.1021/acschemneuro.0c00289

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Research Article
Identification of a Potent and Selective 5‑HT1A Receptor Agonist
with In Vitro and In Vivo Antinociceptive Activity
̇
Pasquale Linciano, Claudia Sorbi, Antonella Comitato, Anna Lesniak, Magdalena Bujalska-Zadrozny,
́
̇
Agata Pawłowska, Anna Bielenica, Jolanta Orzelska-Gorka, Ewa Kędzierska, Grazyna Biała,
Simone Ronsisvalle, Silvia Limoncella, Livio Casarini, Elena Cichero, Paola Fossa, Grzegorz Satała,
Andrzej J. Bojarski, Livio Brasili, Rita Bardoni,⬢ and Silvia Franchini*,⬢
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ABSTRACT: Opioids are the gold standard drugs for the
treatment of acute and chronic severe pain, although their serious
side effects constitute a big limitation. In the search for new and
safer drugs, 5-HT1AR agonists are emerging as potential candidates
in pain relief therapy. In this work, we evaluated the affinity and
activity of enantiomers of the two newly synthesized, potent 5-
HT1AR agonists N-[(2,2-diphenyl-1,3-dioxolan-4-yl)methyl]-2-[2-
(pyridin-4-yl)phenoxy]ethan-1-ammonium hydrogenoxalate (rac-
1) and N-((2,2-diphenyl-1,3-dioxolan-4-yl)methyl)-2-(2-(1-meth-
yl-1H-imidazol-5-yl)phenoxy)ethan-1-ammonium hydrogenoxalate
(rac-2) in vitro and in vivo. The role of chirality in the interaction
with 5-HT1AR was evaluated by molecular docking. The activity of
the rac-1 was tested in mouse models of acute pain (hot plate) and
severe tonic nociceptive stimulation (intraplantar formalin test). Rac-1 was active in the formalin test with a reduction in paw licking
in both phases at 10 mg/kg, and its effect was abolished by the selective 5-HT1AR antagonist, WAY-100635. The eutomer (S)-1, but
not the racemate, was active during the hot plate test at 10 and 20 mg/kg, and this effect was abolished by 30 min treatment with
WAY-100635 at 30 min. Similarly to 8-OH-DPAT, (S)-1 evoked a slow outward current and depressed spontaneous glutamatergic
transmission in superficial dorsal horn neurons, more effectively than rac-1. The eutomer (S)-1 showed promising developability
properties, such as high selectivity over 5-HT subtypes, no interaction with the μ receptors, and low hepato- and cardiotoxicity.
Therefore, (S)-1 may represent a potential candidate for the treatment of acute and chronic pain without having the adverse effects
that are commonly associated with the classic opioid drugs.
KEYWORDS: Serotonin receptors, 5-HT1AR agonists, pain, behavioral profiling, mice
have shown to be effective in the treatment of pain.4−7 In
addition, the use of 5-HT1A agonists in combination with
INTRODUCTION
■
Acute and chronic pain, both nociceptive and neuropathic, are
involved in many illnesses and conditions, posing a great
challenge to public health. Although opioids are widely used for
the treatment of acute and chronic severe pain, their serious
opioids could be particularly advantageous, since they are able to
reduce addiction, by decreasing the reward effects of opioids8
and to revert opioid-induced respiratory depression.9
5-HT2 receptors seem to exert opposite effects on pain, since
adverse effects, primarily respiratory depression, in addition to
tolerance and drug addiction, represent a big limitation.1 Thus,
the activation of the 5-HT2A receptors located on rat spinal cord
dorsal horn interneurons was shown to potentiate inflammatory
there is still an urgent medical need for better therapeutic
options. The serotoninergic system, originating in the brain stem
pain and facilitate excitatory synaptic transmission.10,11
Furthermore, 5-HT2 receptor agonists mediate major side
and exerting a prevalent antinociceptive effect in the spinal cord,
represents an innovative target for new analgesic compounds.2
Received: May 13, 2020
Accepted: November 11, 2020
All seven types of serotoninergic receptors (5-HTRs), including
various isoforms, are present in the spinal cord, where they play
complex roles in modulating pain.3 5-HT1ARs are highly
expressed throughout the pain neuroaxis where they exert a
clear antinociceptive action by inhibiting the transmission of
nociceptive signals.3 Indeed, full and partial 5-HT1AR agonists
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effects like anxiety, insomnia, sexual dysfunction, and
dysfunction in platelet aggregation, as well as side effects related
to the inhibition of dopamine signaling (e.g., Parkinsonian-like
tremor, galactorrhea, cognitive dysfunction, extrapyramidal
symptoms).12−16 5-HT2 receptors are also involved in fibrosis
in a variety of tissues like lung, kidney, heart, or liver, which is
particularly important with chronic use of 5-HT2 receptor
ligands or agents that inhibit serotonin reuptake.17,18 Thus,
designing ligands featuring low 5-HT2 receptor affinity and
higher selectivity at the 5-HT1A subtype could reduce the
occurrence of unwanted effects. 5-HT6 receptors are coupled to
Gαs protein and are expressed in regions regulating pain
processing, such as the cortex, thalamus, PAG, spinal cord, and
dorsal root ganglia (DRG). They seem to exert a prevalent
pronociceptive effect, in both formalin-evoked nociceptive
behavior and animal models of neuropathic pain.19−21 5-HT7
receptors are also located in several regions along the pain axis,
in nociceptive DRG neurons and in subpopulations of the spinal
cord dorsal horn. These receptors could exert pronociceptive
activity, since 5-HT7 agonists increase pain behavior during the
second phase of the formalin test, while antagonists reduce
tactile allodynia induced by nerve injury. However, antinoci-
ceptive effects of 5-HT7 receptors have also been reported in
several pain models.3,22 These data indicate that both 5-HT6 and
5-HT7 receptors are actively involved in nociception, with a
possible prevalent pronociceptive action. For this reason,
serotoninergic compounds with high selectivity for 5-HT1A
and low affinity for both 5-HT6 and 5-HT7 receptors could be
candidates as antinociceptive agents.
exhibit different biological activity. Indeed, the disconnection
between the high binding affinity and the low potency of 2, in the
GTPγS assay, might suggest that enantiomers exert opposite
effects.
In this study, the enantiomeric pairs (R)-1/(S)-1 and (R)-2/
(S)-2 were prepared using stereoselective synthesis and tested
for binding affinity and functional activity at the molecular target
5-HT1AR, as well as for binding affinity to other subtypes (5-
HT2AR, 5-HT2CR, 5-HT6R, and 5-HT7R) closer to the 5-HT1A.
Molecular docking studies were performed to investigate the
role of chirality in the interaction with the 5-HT1AR. The impact
on μ opioid receptors, as well as the hepato- and cardiotoxicity,
were also investigated in vitro. Considering the key role played
by 5-HT1AR agonists in pain, the racemate 1 and the most potent
enantiomer (S)-1 were evaluated in vivo in two pain models to
assess the antinociceptive potential. To confirm their efficacy in
modulating spinal nociceptive circuitries through 5-HT1AR, rac-
1 and (S)-1 were also tested in vitro by electrophysiological
recordings from spinal cord dorsal horn neurons.
RESULTS AND DISCUSSION
■
Chemistry. The tested compounds were synthesized as
previously reported with slight modifications.28 (S)- and (R)-1
and 2 were prepared as oxalate salts by reaction of the
corresponding enantiopure free amines (S)/(R)-3 and (S)/(R)-
4 with anhydrous oxalic acid, followed by crystallization from
dry diethyl ether. The free amines (S)/(R)-3 and (S)/(R)-4
were directly obtained by standard SN2 reaction between the
appropriate (S) or (R)-4-(chloromethyl)-2,2-diphenyl-1,3-
dioxolane ((S) or (R)-5) and 2-(2-(pyridin-4-yl)phenoxy)-
ethan-1-amine (6) or 2-(2-(1-methyl-1H-imidazol-5-yl)-
phenoxy)ethan-1-amine (7). The SN2 reaction was performed
in DMSO at 90 °C for 24−48 h, using potassium iodide as a
catalyst (Scheme 1). The enantiomeric pure aliphatic chlorides
(S) or (R)-5 were prepared by condensation of benzophenone
and the chiral (S)- or (R)-3-chloro-propan-1,2-diols, in refluxing
toluene, using p-toluensulfonic acid (pTSA) as a catalyst and a
Dean−Stark trap to remove the water formed. No racemization
was observed, and the two aliphatic chlorides (S) and (R)-5
were obtained.
Phenoxyethylamines are promising chemotypes for obtaining
potent and selective 5-HT1AR agonists.23−27 In our previous
work, a library of 2-heteroaryl-phenoxyethylamines was
prepared, and the role of an electron-poor/rich heteroaryl
moiety was investigated throughout the SAR studies.28 In that
study, the 4-pyridinyl (1) and imidazolyl (2) derivatives
emerged as the most promising compounds, showing excellent
5-HT1AR affinity (pKi = 9.2 and 8.98, respectively) and
selectivity (5-HT1A/α1 = 135 and 132, respectively) (Figure
1). The study of functional activity revealed that both
The two 2-heteroarylphenoxyethylamines 6 and 7 were
prepared by slightly improving the previously reported synthetic
approach.29 As shown in Scheme 2, the amino group of 2-
bromoethylamine hydrobromide was protected by reacting with
di-tert-butyl dicarbonate in THF/saturated NaHCO3 aqueous
solution, providing N-Boc-2-bromoethylamine (8) in quantita-
tive yield. 8 was then reacted with 2-iodophenol in standard SN2
conditions, obtaining N-Boc-(2-iodophenoxy)ethylamine (9).
The synthesis of the two biaryl scaffolds was performed using
two different methods. The 4-phenylpyridine scaffold was
obtained by reaction of 9 via Suzuki cross-coupling with the
commercially available 4-pyridylboronic acid, in dioxane using
tetrakis(triphenylphosphine)palladium(0) as a catalyst and 2N
K2CO3 aqueous solution as a base, to give 10 in 72% yield.
Since the 1-methylimidazole-5-boronic acid was not
commercially available, the synthesis of the 1-methyl-5-
phenyl-1H-imidazole scaffold was performed by conversion of
9 into the corresponding phenylboronic acid pinacol ester (11)
by Miyaura borylation reaction. In order to avoid the loss of
product due to decomposition on silica gel, 11 was not purified
and the crude extract was used directly in the next step. Suzuki
cross-coupling of 11 with 5-bromo-1-methylimidazole afforded
12 in 71% yield over the two synthetic steps. Lastly, the N-Boc
Figure 1. (A) General structure of the heteroaryl-phenoxyethylamines
library. (B) Most promising compounds 1 and 2.
compounds behaved as agonists, with 1 having the highest
potency (pD2 = 8.83). Despite the quite low potency of 2 (pD2 =
5.46), this compound presented peculiar behavior: in its series, it
was the only one to show efficacy above 100%, with a relative
effectiveness (Emax) of 240%. Therefore, 1 and 2 were selected
for further investigation. Since both compounds present one
chiral center, we first evaluated the pharmacological profile of
each enantiomer, as a fundamental step in the drug development
process. It is well-known that enantiomers of chiral drugs may
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Scheme 1. Reagents and Conditions: (a) benzophenone (1 equiv), (R)- or (S)-3-chloro-propan-1,2-diol (1.5 equiv), pTSA (0.1
equiv), dry toluene, refl., 18 h, 90−05% yield; (b) 6 or 7 (1.2 equiv), KI (cat.), DMSO, 90 °C, 24−48 h, yield: 47% (for (S)-3), 18%
(for (R)-3), 21% (for (S)-4) and 36% (for (R)-4); (c) anhydrous oxalic acid (1.1 equiv), diethyl ether, r.t., 48 h, yield: 85% (for
(S)-1), 75% (for (R)-1), 49% (for (S)-2), 27% (for (R)-2)
Scheme 2. Reagents and Conditions: (a) Di-tert-butyl dicarbonate (1.1 equiv), THF/aq. NaHCO3 sat. sol. 1:1, r.t., 6 h, quant.
yield; (b) 2-iodo-phenol (0.9 equiv), K2CO3 (2.5 equiv), DMF, 60 °C, 2 h, 97% yield; (c) 4-pyridyl boronic acid (1.5 equiv), 2N
K2CO3 (2.5 equiv), tetrakis(triphenylphosphine)palladium(0) (0.03 equiv), dioxane, 80 °C, 18 h, 72% yield; (d) TFA/DCM 1:1,
0 °C to r.t., 3 h, yield: 95%
Scheme 3. Reagents and Conditions: (a) Bis(pinacolato)diboron (1.5 equiv), PdCl2(dppf) (0.05 equiv), AcOK (3 equiv), DMSO,
80 °C, overnight; (b) 5-bromo-1-methyl-imidazole (1 equiv), 2N K2CO3 (2.5 equiv), tetrakis(triphenylphosphine)palladium(0)
(0.1 equiv), dioxane, 80 °C, 24 h, 71% yield; (c) TFA/DCM 1:1, 0 °C to r.t., 2 h, yield: 89%
cleavage of 10 and 12 under acid conditions (TFA/DCM 1:1)
provided the final amines 6 and 7 (Scheme 3).
Table 1. Binding Affinity (pKi), Agonist Potency (pEC50),
and Relative Effectiveness (Emax) of the Enantiomeric Pairs
(S)-1/(R)-1 and (S)-2/(R)-2 and Racemates at the 5-HT1AR
Competition Binding and Functional Studies at 5-HT1AR.
The two enantiomeric pairs (R)-1/(S)-1 and (R)-2/(S)-2 and
the corresponding racemates 1 and 2 were tested for their
binding affinity and functional activity at the main target 5-
HT1AR. The results are shown in Table 1. As revealed by one-
way ANOVA, all the compounds showed high, nanomolar
affinities, pKi ranging from 7.92 to 8.39. Moreover, the
compound pairs did not differ in their binding affinity for
these receptor subtypes, thus highlighting lack of stereo-
selectivity, as previously reported by us for a 1,3-dioxolane-
based compound.24
Functional activity at 5-HT1AR was evaluated in rat
hippocampal membrane preparations. As shown in Table 1, all
optical isomers and racemates stimulated G-protein activation
with nanomolar affinity, pEC50 ranging from 7.0 to 7.8. One-way
ANOVA revealed differences in binding affinities within each
a
b,c
5-HT1A
5-HT1A
compound
pKi SEM
pEC50 SEM
Emax (%) SEM
rac-1
(S)-1
(R)-1
rac-2
(S)-2
(R)-2
buspirone
8-OH-DPAT
serotonin
8.04 0.05
8.03 0.10
8.35 0.9
8.13 0.10
8.39 0.11
7.92 0.07
7.52 0.04
-
7.0 0.2
7.8 0.12*
7.2 0.18
7.4 0.09
7.6 0.11*
7.2 0.09
-
151 4.2
138 2.9
158 5.9
184 2.7##
175 2.7##,***
143 3.6
-
7.5 0.11
6.8 0.08
152 2.4
192 4.9
-
a
HEK293 cells transfected with human 5-HT1AR. The results are
expressed as means SEM from at least two independent
experiments. Rat hippocampal membranes. The results are expressed
as the means SEM of three independent experiments. Basal 5-
b
compound pair and when compared with 8-OH-DPAT (F3,11
=
c
HT1AR activation was set to 100%. ***p < 0.001 and *p < 0.05 vs
7.76, p < 0.01 for pair 1 and F3,11 = 6.8, p < 0.05 for pair 2).
Unexpectedly, (R)-2 and (S)-2 enantiomers did not show an
opposite contribution to the functional readout, both acting as
agonists. Noteworthy, G-protein activation elicited by the four
optical isomers, as well as their racemates, involved 5-HT1AR
receptors. As seen in Table SI-1, WAY-100635 shifted the
agonist-stimulated [35S]GTPγS binding curve to the right
the (R) enantiomer; ##p < 0.01, and vs 8-OH-DPAT.
without affecting the maximal response. In addition, WAY-
100635 completely abolished agonist-induced stimulation,
implying a fully 5-HT1A-dependent effect (Table SI-2).
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Figure 2. Binding mode of (S)-1 (A, yellow carbon) and (R)-1 (B, magenta carbon) within the modeled 5-HT1AR (green diagram). H-bonds and π−π
stacking are represented as yellow and blue dashed lines, respectively. The derived ligplots are shown on the right; the polar and hydrophobic residues
are colored in magenta and green, respectively.
In detail, (S)-2 and rac-2 acted as full agonists and expressed
efficacy superior to 8-OH-DPAT (p < 0.01), while (S)-1, rac-1,
(R)-1, and (R)-2 were partial agonists as their stimulation of
[35S]GTPγS binding was half (or lower) as efficacious as for
serotonin. Interestingly, both S optical isomers of each couple
were more potent than the R pairs (p < 0.05), with a eudismic
ratio (EC50 S/R) of 4.0 and 2.6 for 1 and 2, respectively.
Promisingly, both S optical isomers showed an pEC50 value
comparable ((S)-2) or higher ((S)-1) than the reference
agonist, 8-OH-DPAT. Overall, the functional binding analysis
carried out on the new enantiomeric pairs highlighted a slight
degree of enantioselectivity for the S enantiomers in terms of 5-
HT1AR activation.
Molecular Modeling of 5-HT1A Receptor and Docking
Studies. To get a better understanding of the role of chirality on
the interaction with the biological target, molecular docking
studies were performed at the binding site of the human 5-
HT1AR protein. In the absence of the crystallographic structure
for 5-HT1AR, before proceeding with the docking calculation,
our previous ligand-based homology model was refined by using
the recently available X-ray crystallographic structure of the
human 5-HT1BR (PDBID = 5V54; resolution = 3.9 Å).30 As
shown by the alignment of the 5-HT1AR primary sequence with
that of the template, a consistent number of residues proved to
be conserved between these two receptor subtypes (Figure SI-
1). Accordingly, the modeled 5-HT1A backbone conformation
featured good correspondence with that of the GPCR template
(RMSD = 0.854 Å) (Figure SI-2 and SI-3). Superimposition of
the theoretical model of the 5-HT1A receptor onto the template
allowed us to identify the putative binding site of compounds
targeting 5-HT1AR, based on the corresponding binding site of
methiothepin. A number of studies describe a unique receptor
cavity involved in the binding with 5-HT1AR full agonists, partial
agonists, and antagonists.31,32 In particular, H-bond interactions
between agonists and Asp116 and Asn386 were suggested,
falling in a crevice delimited by Phe112, Ile113, Asp116, Lys191,
while partial agonists, as well as antagonists, were H-bonded at
least with Asp116. In agreement with the literature, the putative
binding mode of the antagonist methiothepin displayed one salt-
bridge with the key residue Asp116 and cation−π interactions
with Phe112. The tricyclic core detected hydrophobic contacts
with Ile189, Ala203, Trp358, Phe361, and Phe362 (Figure SI-4).
Then, (S)-1 and (R)-1 enantiomers were analyzed. According
to our calculations, they feature the same positioning within the
protein cavity. Both optical isomers displayed the key salt-bridge
with Asp116 and one H-bond with Asn386, through the
protonated nitrogen atom of the amine chain. The diphenyl-
dioxolane ring was located in proximity to Tyr96, Phe112, and
Tyr390, featuring π−π stacking and van der Waals contacts,
while one of the two oxygen atoms of the dioxolane was H-
bonded to Ile189 (Figure 2). The biaryl moiety moved toward a
hydrophobic pocket delimited by Val117, Thr121, Ile167,
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Figure 3. Binding mode of (S)-2 (A, yellow carbon) and (R)-2 (B, pink carbon) within the modeled 5-HT1AR (green diagram). The H-bonds and π−π
stacking are represented as yellow and blue dashed lines, respectively. The derived ligplots are shown on the right; the polar and hydrophobic residues
are colored in pink and green, respectively.
Table 2. Binding Affinity (pKi) of the Enantiomeric Pairs (S)-1/(R)-1 and (S)-2/(R)-2 and Racemates at the 5-HT2AR, 5-HT2CR,
5-HT6R, and 5-HT7R
a
a
b
b
5-HT2A
5-HT2C
5-HT6
5-HT7
compound
pKi SEM
pKi SEM
pKi SEM
pKi SEM
rac-1
(S)-1
(R)-1
rac-2
(S)-2
(R)-2
ketanserin
RS-102221
olanzapine
clozapine
5.95 0.054
6.10 0.055*
5.80 0.056
6.38 0.12
6.00 0.044)
6.05 0.056
8.27 0.06
-
6.44 0.21
6.02 0.12
6.04 0.12
6.48 0.18
6.50 0.32*
5.90 0.14)
-
5.12 0.05
5.49 0.03
5.37 0.05
5.30 0.04
5.26 0.02
5.30 0.05
-
6.29 0.08
6.40 0.02
6.30 0.03
6.18 0.04
6.22 0.06
6.10 0.03
-
8.34 0.12
-
-
-
-
-
-
7.91 0.05 (
-
-
7.21 0.02
a
b
Rat frontal cortex membranes. The results are expressed as means SEM of three independent experiments. HEK293 cells transfected with
human 5-HT1AR. The results are expressed as means SEM from at least two independent experiments. *p < 0.05 vs the (R) enantiomer.
Ile189, Thr200, Trp358, Phe361, Phe362, and Ala365. This
allowed the agonist to be better stabilized at the protein crevice,
exhibiting additional π−π and hydrophobic contacts. While (S)-
1 featured a further H-bond with Asn386 thanks to the
dioxolane substituent, the (R)-1 was properly oriented within
the receptor crevice by means of additional polar contacts
involving the protonated basic group and the Tyr390 side-chain.
Remarkably, this kind of interaction was featured by the (S)-2
dioxolane ring, described as follows, exhibiting comparable
binding affinity.
The (S)-2 and (R)-2 enantiomers were also analyzed. As
described for (S)-1, the (S)-2 enantiomer experienced a similar
docking pose (Figure 3A), being endowed with numerous
hydrophobic interactions with the receptor, by means of the
imidazole ring with respect to the (R)-2 enantiomer (Figure
3B). Once again, (S)-2, but not (R)-2, displayed a further H-
bond with Asn386, via the amino group, while one of the two
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oxygen atoms of the dioxolane core was engaged in polar
contacts with Tyr390. These findings are in agreement with the
binding affinity data at 5-HT1AR, with (S)-2 being 3-fold more
potent than (R)-2.
Taken together, these results revealed the relevant role played
by the hydrogen bond between the amino group and the Asn386
residue and/or Tyr390, contributing to the stabilization of the
(R)-1 and (S)-2 optical isomers.
Cell Viability and Toxicity Studies. Hepatotoxicity and
cardiotoxicity are two of the major causes of failure during drug
development. In vitro cell-based viability assay using the Hep-G2
cell line of hepatic origin was used as a tool for safety evaluation
in the early stages of drug discovery. The impact of nanomolar−
micromolar concentrations of compounds (S)-1, (R)-1, (S)-2,
and (R)-2 on hepatic cell viability was evaluated by 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay after 72 h of treatment (Figure 4). Compounds (S)-1, (R)-
Developability Studies. Competition Binding Studies at
5-HT2A, 5-HT2C, 5-HT6, and 5-HT7 (Off-Targets). The two
enantiomeric pairs (R)-1/(S)-1 and (R)-2/(S)-2 and the
corresponding racemates 1 and 2 were tested for their binding
affinity at the 5-HT subtypes (5-HT2A, 5-HT2C, 5-HT6, and 5-
HT7) that are structurally and evolutionally closest to the 5-
HT1AR. Affinity at the 5-HT2AR and 5-HT2CR subtypes was
studied in rat frontal cortex membrane preparations, whereas
affinity at 5-HT6R and 5-HT7R was performed in stably
transfected HEK293 cells. The results are reported in Table 2.
All the compounds showed weaker binding affinity at both 5-
HT2R subtypes compared with 5-HT1AR. As revealed using one-
way ANOVA, all the compounds bound to 5-HT2AR with
relatively low affinity (F3,11 = 433.1, p < 0.001 for pair 1 and F3,11
= 201, p < 0.001 for pair 2). All the compounds also showed
considerably lower affinity to 5-HT2AR (pKi ranging from 5.80
to 6.38), when compared with the selective antagonist,
ketanserin (pKi = 8.27). (S)-1 showed almost 2-fold improved
affinity compared with (R)-1 (pKi S/R = 1.99, p < 0.05).
Conversely, no stereoselectivity was observed for the (R) and
(S) enantiomers of compound 2.
One-way ANOVA revealed that all the compounds also
showed low binding affinity at the 5-HT2CR (pKi ranging from
5.9 to 6.48), compared with the selective 5-HT2CR antagonist,
RS-102221 (pKi = 8.34) (F3,11 = 6.7, p < 0.05 for pair 1 and F3,11
= 4.5, p < 0.05 for pair 2). No stereoselectivity was observed for
compound 1 at the 5-HT2CR; however, the (S)-2 enantiomer
showed preference for the 5-HT2CR over the (R)-2 enantiomer
(pKi S/R = 3.98). On the other hand, all the compounds showed
low, micromolar or submicromolar affinity at the 5-HT6R and 5-
HT7R, respectively.
Figure 4. Impact on liver hepatocellular carcinoma Hep-G2 cell
viability of (S)-1, (R)-1, (S)-2, and (R)-2. Cells were maintained 72 h in
the presence of increasing concentrations of the compounds and
viability was assessed by MTT assay. 200 mM ethanol and 5% Triton X-
100 treated cells served as controls and data were shown by the box and
whiskers plot. Two-way ANOVA and Dunnett’s post-test, p < 0.001*, p
< 0.0001**, n = 4, versus untreated samples.
1, and (R)-2 showed very low toxicity at concentrations ≤10
μM, while (S)-2 decreased cell viability at 10 and 5 μM (two-
way ANOVA, p < 0.001, n = 4). At 50 μM, (R)-1, (S)-2, and (R)-
2 negatively impacted cell viability compared with untreated
(control cells), while (S)-1 had no effect (two-way ANOVA, p <
0.0001, n = 4). Therefore, compound (S)-1 had lowest
hepatotoxicity.
CiPA hERG QPatch Assay. Given the best functional potency
and the lower impact on hepatic cell viability of (S)-1, this
compound was profiled for activities on the human voltage gated
potassium ion channel hERG, using the QPatch electro-
physiological platform. The results showed that (S)-1 does not
inhibit hERG at the concentrations of 0.1 and 1 μM, indicating
that this compound is suitable for drug development (Table 4).
Interestingly, all the compounds showed favorable selectivity
profiles toward the 5-HT1AR (Table 3).
Notably, the affinity profile revealed a difference within the
same enantiomeric pair, (R)-1 and (S)-2 showing the highest
selectivity profile. These compounds might represent useful
pharmacological tools to study the role of 5-HT1AR subtype in
different biological systems.
Table 4. Mean % Inhibition of the Maximum Tail Current of
(S)-1 and the Reference Compound E-4031 in hERG-CHO
Automated Patch-Clamp
% inhibition of tail current in hERG-CHO
compound
10 μM
1 μM
0.1 μM
Table 3. Selectivity Profile of the Enantiomeric Pairs (S)-1/
(R)-1 and (S)-2/(R)-2 and the Racemates at the 5-HT1A vs 5-
HT2A, 5-HT2C, 5-HT6, and 5-HT7 Receptors
(S)-1
E-4031
66.65
-
5.61
-
−3.35
83.14
a
selectivity
To exclude any interaction with the opioid receptors, (S)-1
was tested in vitro on mouse spinal cord slices, in the presence of
naloxone, a nonselective and competitive opioid receptor
antagonist. These data are presented in the section regarding
the electrophysiological studies (Figure 11).
Based on the high potency, good safety profile, and favorable
ADME properties (including the ability of 1 to permeate, by
passive diffusion, MDCK-MDR1 monolayers, mimicking the
BBB, indicating high brain uptake, and low efflux ratio28), the
racemate 1 and its eutomer (S)-1 were selected for in vivo
studies.
5-HT2A/5-
HT1A
5-HT2/5-
HT1A
5-HT6/5-
HT1A
5-HT7/5-
HT1A
compound
rac-1
(S)-1
(R)-1
rac-2
(S)-2
(R)-2
123
85
355
56
245
74
40
102
204
45
78
105
832
347
955
676
1349
417
56
43
112
89
148
66
a
Selectivity ratio was calculated as an Antilog [pKi (5-HT1AR) − pKi
(5-HT2AR or 5-HT2CR or 5-HT6R or 5-HT7R)].
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In Vivo Studies. Assessment of the Antinociceptive
Activity in the Formalin Test. The formalin test was chosen
as a tonic pain model for the assessment of potential analgesic
activity of compound 1 in mice. Intraplantar administration of
formalin (5%, 10 μL) produces a biphasic nocifensive behavioral
response (i.e., biting or licking the injected hind paw). The acute
nociceptive phase, reflecting the chemical activation of sensory
C-fibers, lasts for the first 10 min, while the second inflammatory
phase takes place between 15 and 50 min and corresponds with
the development of nociceptive sensitization in the dorsal horn
of the spinal cord.33
As shown in Figure 5, compound 1 was administered 15 min
before formalin, at doses of 3, 5, and 10 mg/kg i.p. The 10 mg/kg
Figure 6. Effect of WAY-100635 (3 mg/kg, i.p.) on analgesia induced
by 1 (10 mg/kg, i.p.) during the first (0−10 min) and second (10−60
min) phases of the formalin test. The test compound or vehicle was
injected 15 min prior to the intraplantar injection of formalin. WAY-
100635 was injected 30 min prior to 1 or vehicle. The data are expressed
as the means
SEM of 8−10 mice per group. *p < 0.05 vs the
respective groups of mice injected with the vehicle. #p < 0.05 vs mice
treated with 1 (10 mg/kg, i.p.).
Figure 5. Effect of intraperitoneal (i.p.) injection of 1 (3, 5, or 10 mg/
kg) on the first (0−10 min) and second (10−60 min) phases of the
formalin test. The test compound or vehicle was injected 15 min before
the intraplantar injection of formalin. The data are expressed as the
means SEM of 8−10 mice per group. *p < 0.05 vs the respective
groups of mice injected with the vehicle.
dose was able to induce significant analgesic effects during the
first and second phases of the formalin test (*p < 0.05).
Morphine (10 mg/kg, i.p.) was used as a positive control and
essentially eliminated the response to formalin in both phases.
Pretreatment with the selective 5-HT1AR antagonist WAY-
100635 (3 mg/kg, i.p.), 30 min before the administration of 1
(10 mg/kg i.p.), prevented its analgesic effect during the second
phase (#p < 0.05), confirming the activation of the 5-HT1A
receptor.
Figure 7. Influence of rac-1 on nociceptive reactions assessed using the
hot plate test in mice. Nociceptive reactions were measured over the
total period of 180 min (30, 60, 90, 120, and 180 min after compound
administration). The data are expressed as the mean SEM values.
WAY-100635 (3 mg/kg, i.p.), per se, at least at the dose used,
did not alter the licking response after injection with formalin
(Figure 6). These data show that 1 was effective in decreasing
the acute activation of nociceptive fibers (first phase) and the
inflammatory response, leading to a facilitated state of the
nociceptive system (second phase). These results are consistent
with the previously described antinociceptive action of 5-HT1AR
agonists in the formalin pain model.34,35 A discrepancy between
the analgesic effect of 5-HT1AR agonists in both phases and the
blockade by the antagonist WAY-100635, only during the
second phase, has also been observed in previous studies.36 This
discordance could be due to the different involvement of 5-
HT1AR in acute pain and central pain sensitization.
receptors.37,38 The paws of mice are extremely sensitive to heat
at temperatures that do not cause damage to their skin. The
animals respond by jumping, withdrawing, or licking their paws.
The response time can be increased by the administration of
central acting analgesics. Drugs like acetylsalicylic acid or
metamizole type, with peripheral action, do not have an effect on
these responses. The hot plate test has been used by many
researchers and has been deemed suitable for the evaluation of
analgesics that act centrally but not peripherally.39
The hot plate test was performed before and after intra-
peritoneal administration of the compound 1 in mice at three
different doses (5, 10, and 20 mg/kg). Statistical analysis of the
results obtained did not reveal any significant change in the MPE
values at the chosen time intervals at all tested doses. At 30 min,
administration of the rac-1 at 5 and 10 mg/kg seemed to have a
pronociceptive action. However, this effect was not statistically
Assessment of Analgesic Activity in the Hot Plate Test. The
antinociceptive properties of 1 were also assessed using the hot
plate test, an acute, thermally induced pain model (Figure 7).
This procedure involves a supraspinal reflex mediated by opioid
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significant, nor did it seem to be attributable to 5-HT1AR
activation, being not inhibited by the 5-HT1A antagonist WAY-
100635.
Since in the functional experiments the (S)-1 enantiomer
showed 6-fold higher activity compared with the racemate 1, the
hot plate test was also performed on (S)-1. As shown in Figure 8,
one-way ANOVA showed significant changes in the MPE values
at 30 (F5,37 = 3.952; p < 0.01), 90 (F5,36 = 2.081; p < 0.05), 120
(F5,34 = 4.084; p < 0.01), and 180 min (F5,34 = 1.763; p = 1.1471)
after the injection for (S)-1. This compound, at the dose of 20
mg/kg, significantly increased the reaction time to the thermal
stimulus at 30 (p < 0.05), 90 (p < 0.05), 120 (p < 0.01), and 180
min (p < 0.05). At the dose of 10 mg/kg, the reaction time to the
thermal stimulus was significantly increased at 30, 90, 120, and
180 (p < 0.05) min, while at 5 mg/kg a significant effect was only
noted at 120 and 180 min (p < 0.05). The selective 5-HT1AR
antagonist WAY-100635 (3 mg/kg) significantly reversed the
antinociceptive effect of the (S)-1 enantiomer at the dose of 10
mg/kg at 30 min (p < 0.05). Morphine was employed as a
positive control at the dose of 3.2 mg/kg.
The behavioral data show that racemate 1 has an
antinociceptive action in the formalin test, while it is not
effective in the hot plate test, where only the (S)-1 enantiomer
has significant activity. This discrepancy could be due to a higher
agonist sensitivity of spinal versus supraspinal 5-HT1AR, as
previously reported.5
In Vitro Electrophysiological Studies. Effects of rac-1,
(S)-1, and 8-OH-DPAT on Activity of Spinal Cord Dorsal Horn
Neurons. Rac-1 and its more active enantiomer (S)-1 were also
tested in vitro by electrophysiological recordings from spinal
cord dorsal horn neurons. Recordings were obtained from a total
of 65 superficial dorsal horn neurons. At the beginning of the
experiment, the resting potential was measured: neurons
showing a value more positive than −50 mV were discarded.
Lamina I−II neurons were then recorded in voltage clamp at
−60 mV. Bath application of the 5-HT1A agonist 8-OH-DPAT
(10 μM) evoked a slow outward current in 6 out of the 11 tested
neurons (Figure 9A). This current, carried by potassium ions,
Figure 8. Influence of (S)-1 enantiomer on nociceptive reactions
assessed using the hot plate test in mice. Nociceptive reactions were
measured over the total period of 180 min (30, 60, 90, 120, and 180 min
after compound administration). The data are expressed as the mean
SEM values.
Figure 9. (A) Representative traces of the outward currents observed after 2 min of the compound application. Outward currents evoked by rac-1 and
(S)-1 had similar kinetics to those generated by 8-OH-DPAT. Application of WAY-100635 (10 μM) blocked the effect of (S)-1 in all 7 neurons tested.
(B) Top: (S)-1 (10 μM) was effective in evoking outward currents in a higher percentage of neurons (5 out of 12) than rac-1 50 μM (5 out of 23). 8-
OH-DPAT generated an outward current in 6 out of 11 cells. Neurons were considered responsive if they showed an outward current distinguishable
from the baseline noise (>3 pA). Bottom: Mean amplitudes of the outward currents evoked by the tested compounds in dorsal horn neurons, showing
no significant differences among the 3 groups (one-way ANOVA, P = 0.53).
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corresponds with neuron hyperpolarization and inhibition of
neuron excitability, as previously described in most lamina I−II
neurons.3,40,41 The rac-1 induced outward currents of similar
amplitudes in 5 out of 23 neurons when applied at 50 μM, while
the concentration of 10 μM was not effective. Its S enantiomer
exerted a more potent effect, evoking an outward current, at 10
μM, in 5 out of 12 tested neurons. The current amplitudes and
kinetics were similar to those obtained by applying 10 μM 8-
OH-DPAT (Figure 9B). In the presence of the 5-HT1AR
antagonist WAY-100635 (10 μM), (S)-1 failed to evoke the
outward currents in all 7 neurons tested, showing that the
compound activates 5-HT1AR on the recorded dorsal horn
neurons.
inhibiting nociceptive transmission, by decreasing neuronal
excitability and glutamate release.
A 5-fold concentration (50 vs 10 mM) was required for rac-1
to produce similar effects on dorsal horn neurons compared to
its S enantiomer. Interestingly, a similar ratio was observed
between the EC50 values determined for the two compounds
(92.1 and 14.6 nM, respectively).
Effects of (S)-1 on Dorsal Horn Neuron in the Presence of
Naloxone. To exclude any interaction with the opioid receptors,
the more active compound (S)-1 was tested in vitro on mouse
spinal cord slices, in the presence of naloxone, a nonselective and
competitive opioid receptor antagonist. As shown in Figure 11,
As shown in Figure 10A, fast inward currents, corresponding
with spontaneous excitatory postsynaptic currents (sEPSCs)
Figure 11. Effects of the enantiomer (S)-1 on spinal cord dorsal horn
neurons, in the presence of the opioid receptor antagonist naloxone. A:
Representative recording, showing the appearance of a slow outward
current, superimposed by a decrease of the spontaneous excitatory
postsynaptic currents (sEPSCs). B: Amplitudes of outward currents
(left) and percentage sEPSC frequency decreases (right), produced by
(S)-1, in the absence or presence of naloxone. No significant differences
were detected for both outward currents and sEPSC frequency (t test, P
= 0.86 and P = 0.88, respectively).
Figure 10. (A) Left: Representative traces of spontaneous excitatory
postsynaptic currents (sEPSCs) recorded from superficial dorsal horn
neurons at −60 mV. Right: Application of (S)-1 (10 μM) caused a
significant decrease of sEPSC frequency. In this particular neuron, (S)-
1 did not evoke any slow outward current. (B) Left: (S)-1 (10 μM)
caused a similar sEPSC frequency decrease as rac-1 (50 μM) and 8-OH-
DPAT (10 μM) (one-way ANOVA, P = 0.80). Right: sEPSC frequency
decrease was observed in a higher percentage of neurons for (S)-1 (5
out of 12) than for rac-1 (7 out of 23). 8-OH-DPAT was effective in 4
out of 11 tested neurons.
mediated by glutamate, were also visible in voltage-clamp
recordings. During the application of rac-1 (50 μM) or (S)-1 (10
μM), the frequency of the synaptic currents significantly
decreased in a subpopulation of neurons (Figure 10). A decrease
of EPSC frequency was also produced by 10 μM 8-OH-DPAT,
as previously reported in the superficial dorsal horn.11,42 The
enantiomer (S)-1 was more potent than the rac-1, producing
comparable effects at a lower concentration (10 vs 50 μM). This
indicates that, similarly to 8-OH-DPAT, rac-1 and (S)-1 can
inhibit glutamate release from presynaptic neurons and fiber
terminals, by acting on 5-HT1AR.
application of 10 μM (S)-1 still caused an outward current in 5
out of 12 neurons and a significant decrease in sEPSC frequency
in 6 out of 12 neurons. The recorded cells were located in
superficial dorsal horn (laminae I and II outer), where opioid
receptors (particularly μ receptors) are abundantly expressed at
both the pre- and post-synaptic sites.43−45 The amplitude of the
outward current and the percentage decrease in sEPSC
frequency were not significantly different from those observed
in the absence of naloxone, indicating that the effects of the
enantiomer (S)-1 on dorsal horn neurons are not due to the
activation of μ receptors. The lack of activity on μ opioid
receptors is fundamental for the development of (S)-1 as an
alternative non-opioid analgesic drug.
In summary, the data from the electrophysiological experi-
ments show that the more potent enantiomer (S)-1 activates 5-
HT1AR in the mouse superficial dorsal horn and is effective in
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(R)-1 (0.045 g, 0.082 mmol, 75% yield), as a white solid, mp 165−
CONCLUSIONS
■
1
166 °C. H NMR (400 MHz, DMSO-d6) and 13C NMR (101 MHz,
The enantiomeric pairs (S)/(R)-1 and (S)/(R)-2 were
synthesized and tested for their affinity and functional activity
at the main target 5-HT1AR. They were all selective for 5-HT1AR,
acting as full or partial agonists. The compound pairs did not
differ in their binding affinity for 5-HT1AR, thus highlighting a
lack of stereoselectivity, as confirmed by the docking
calculations. However, a slight degree of enantioselectivity was
seen for both S optical isomers with respect to 5-HT1AR
activation. In vivo experiments showed that the racemate 1 exerts
a potent analgesic action at both peripheral and spinal levels, as
highlighted by the effect of this compound in the formalin test.
At the supraspinal level, the enantiomer (S)-1 was able to
produce an analgesic effect, as shown in the hot plate test. In
both pain models, the effects were antagonized by WAY-100635,
confirming the involvement of the 5-HT1AR. In addition, the
results obtained in the in vitro spinal cord dorsal horn
preparation confirmed that (S)-1 is more effective than the
racemate at inhibiting neuron excitability. Moreover, (S)-1
exhibited a good developability profile, not interacting with the μ
opioid receptors and showing low hepato- and cardiotoxicity.
Taken together, our data suggest that (S)-1 is a valid alternative
to opioids in the treatment of acute and chronic pain.
DMSO-d6) identical to that of (S)-1. Elemental analysis (CHN)
calculated for C31H30N2O7: C: 68.62; H: 5.57; N: 5.16. Found: C:
68.48; H: 5.62; N: 5.06.
(S)- or (R)-N-((2,2-Diphenyl-1,3-dioxolan-4-yl)methyl)-2-(2-(1-
methyl-1H-imidazol-5-yl)phenoxy)ethan-1-ammonium hydroge-
noxalate, (S)/(R)-2. (S)-2 (0.041 g, 0.075 mmol, 49% yield), as a
pale yellow solid, mp 102−103 °C. 1H NMR (400 MHz, DMSO-d6) δ
2.91 (dd, J = 8.76, 12.9 Hz, 1H), 3.00 (dd, J = 3.36, 12.9 Hz, 1H), 3.30−
3.37 (m, 2H), 3.47 (s, 3H), 3.68 (dd, J = 6.54, 8.46 Hz, 1H), 4.03 (dd, J
= 7.08, 8.46 Hz, 1H), 4.28 (t, J = 5.34 Hz, 2H), 4.28−4.33 (m, 1H), 6.98
(s, 1H), 7.10 (t, J = 6.0 Hz, 1H), 7.17 (d, J = 8.28 Hz, 1H), 7.29−7.35
(m, 5H), 7.38−7.48 (m, 7H), 7.85 (s, 1H). 13C NMR (101 MHz,
DMSO-d6) δ 32.06, 46.35, 49.77, 64.87, 67.25, 72.74, 109.83, 112.61,
118.11, 121.31, 125.78, 125.92, 126.69, 128.05, 128.22, 128.24, 128.31,
129.39, 130.41, 131.82, 138.26, 141.67, 141.77, 155.55, 163.04.
Elemental analysis (CHN) calculated for C30H31N3O7: C: 66.04; H:
5.73; N: 7.70. Found: C: 65.82; H: 5.98; N: 7.49.
(R)-2 (0.035 g, 0.064 mmol, 27% yield), as a yellow solid, 104−105
°C. 1H NMR (400 MHz, DMSO-d6) and 13C NMR (101 MHz,
DMSO-d6) identical to that of (S)-2. Elemental analysis (CHN)
calculated for C30H31N3O7: C: 66.04; H: 5.73; N: 7.70. Found: C:
65.92; H: 5.87; N: 7.69.
General Procedure for the Synthesis of Amines (S)/(R)-3 and (S)/
(R)-4. To a solution of (R)-5 or (S)-5 (1 equiv) in DMSO (3 mL) a
solution of the appropriate amine 6 or 7 (1.2 equiv) in DMSO (2 mL)
and KI (cat.) were added. The solution was stirred at 90 °C for 24−48 h
and quenched with water. The aqueous layer was alkalinized at pH 14
with 1 N NaOH and extracted with EtOAc. The organic layer was
washed with water, brine, dried over anhydrous Na2SO4, and
concentrated. The crude extract was chromatographed over silica gel
using EtOAc:MeOH 9:1 as the mobile phase.
(S)- or (R)-N-[(2,2-Diphenyl-1,3-dioxolan-4-yl)methyl]-2-[2-(pyr-
idin-3-yl)phenoxy]ethanamine, (R)/(S)-3. (S)-3 (240 mg, 0.53 mmol,
48% yield), as a yellow oil. 1H NMR (400 MHz, Chloroform-d) δ 1.79
(bs, 1H), 2.75 (dd, J = 4.4, 12.2 Hz, 1H), 2.84 (dd, J = 7.1, 12.2 Hz, 1H),
2.85−3.01 (m, 2H), 3.78 (dd, J = 6.4, 7.9 Hz, 1H), 3.91−4.19 (m, 3H),
4.27 (qd, J = 7.1, 4.2 Hz, 1H), 7.01 (dd, J = 1.0, 8.3 Hz, 1H), 7.08 (td, J =
1.0, 7.5 Hz, 1H), 7.17−7.39 (m, 8H), 7.39−7.51 (m, 6H), 8.56 (d, J =
3.9 Hz, 2H). 13C NMR (101 MHz, Chloroform-d): δ 48.69, 52.28,
68.06, 68.09, 76.19, 109.25, 112.75, 121.41, 124.30, 126.16, 126.28,
128.07, 128.16, 128.20, 130.15, 130.54, 142.20, 142.36, 146.38, 149.49,
155.75. [α]20D = −10.9° (c = 0.01, CHCl3). Elemental analysis (CHN)
calculated for C29H28N2O3: C: 76.97; H: 6.24; N: 6.19. Found: C:
76.92; H: 6.20; N: 6.26.
METHODS
■
Chemistry. All the reagents and solvents were commercially
available from Sigma-Aldrich. The moisture-sensitive reactions were
performed under an inert atmosphere of nitrogen. The following
solvents have been abbreviated: diethyl ether (Et2O), dichloromethane
(DCM), dimethylformamide (DMF), dimethyl sulfoxide (DMSO),
chloroform (CHCl3), cyclohexane (Cy), ethyl acetate (EtOAc),
methanol (MeOH). Each reaction was monitored by TLC on Merck
60G F254 plates and detected at 254 nm. All the compounds, unless
otherwise specified, were purified by flash column chromatography
using silica gel 60 (230−400 mesh, ASTM) supplied by Merck. The
melting points were determined with Stuart SMP3 apparatus and are
uncorrected. NMR spectra were recorded on a Bruker 400
spectrometer with 1H at 400.134 MHz and 13C at 100.62 MHz.
Chemical shifts were referenced to the solvent residual peaks. 1H NMR
peak patterns are as follows: s (singlet), d (doublet), t (triplet), m
(multiplet), br (broad singlet). The purity of the compounds was
determined by elemental analysis (C, H, N), which was performed on a
Carlo Erba 1106 Analyzer and the results shown here are within 0.4%
of the theoretical values. Optical rotation (λ) was measured with a
Polarimeter 240C (cell-length 100 mm, volume 1 mL) from
PerkinElmer (Milan, Italy).
(R)-3 (89 mg, 0.19 mmol, 18% yield) as a yellow oil. 1H NMR (400
MHz, Chloroform-d) and 13C NMR (101 MHz, Chloroform-d)
identical to that of (S)-3. [α]20D = +11.3° (c = 0.005, CHCl3).
(S)- or (R)-N-((2,2-diphenyl-1,3-dioxolan-4-yl)methyl)-2-(2-(1-
methyl-1H-imidazol-5-yl)phenoxy)ethan-1-amine, (S)/(R)-4. (S)-4
(222 mg, 0.48 mmol, 36% yield), as an amber oil. 1H NMR (400 MHz,
Chloroform-d) δ 2.13 (bs, 1H), 2.79 (m, 2H), 2.98 (t, J = 5.16 Hz, 2H),
3.46 (s, 3H), 3.83 (dd, J = 7.8, 6.4 Hz, 1H), 4.08 (m, 3H), 4.30 (qd, J =
6.7, 4.5 Hz, 1H), 7.02 (m, 2H), 7.07 (m, 1H), 7.21−7.60 (m, 13H). 13C
NMR (101 MHz, Chloroform-d): δ 32.04, 48.67, 52.24, 67.97, 68.35,
76.14, 109.93, 112.52, 119.30, 121.14, 126.20, 126.33, 128.06, 128.18,
General Procedure for the Synthesis of the Oxalate Salts (S)/(R)-1
and (S)/(R)-2. To a solution of the appropriate amine (S)-3, (R)-3, (S)-
4, or (R)-4 (1 equiv) in 5 mL of anhydrous diethyl ether at room
temperature and under nitrogen atmosphere, anhydrous oxalic acid
(1.2 equiv) was added. The suspension was stirred for 30 min and left to
settle for 48 h. The precipitate was collected by filtration, washed with
anhydrous diethyl ether, and dried to afford the title compound.
(S)- or (R)-N-[(2,2-Diphenyl-1,3-dioxolan-4-yl)methyl]-2-[2-(pyr-
idin-4-yl)phenoxy]ethan-1-ammonium hydrogenoxalate, (S)/(R)-1.
(S)-1 (0.050 g, 0.092 mmol, 85% yield) as a white solid, m.p 162−164
D
128.22, 128.45, 130.21, 132.29, 138.30, 142.18, 142.36, 156.60. [α]20
= −15.3° (c = 0.01, CHCl3). Elemental analysis (CHN) calculated for
C28H29N3O3: C: 73.82; H: 6.42; N: 9.22. Found: C: 73.75; H: 6.49; N:
9.30.
1
°C. H NMR (400 MHz, DMSO-d6) δ 2.95 (dd, J = 8.16, 12.68 Hz,
(R)-4 (169 mg, 0.37 mmol, 21% yield), as a yellow oil. 1H NMR (400
MHz, Chloroform-d) and 13C NMR (101 MHz, Chloroform-d)
identical to that of (S)-4. [α]20D = +16.5° (c = 0.01, CHCl3).
Synthesis of (S)- or (R)-4-(chloromethyl)-2,2-diphenyl-1,3-dioxo-
lane (5). The synthesis of the titled compounds was performed as
previously reported, using (S)-3-chloro-propan-1,2-diol or (R)-3-
1H), 3.02−3.06 (m, 1H), 3.27−3.29 (m, 2H), 3.64 (dd, J = 6.52, 8.32
Hz, 1H), 4.01 (dd, J = 7.00, 8.32 Hz, 1H), 4.24−4.27 (m, 2H), 4.29−
4.34 (m, 1H), 7.13 (t, J = 7.52 Hz, 1H), 7.19 (d, J = 7.96 Hz, 1H), 7.26−
7.45 (m, 12H), 7.54 (d, J = 6.12 Hz, 2H), 8.54 (d, J = 6.12 Hz, 2H). 13C
NMR (101 MHz, DMSO-d6) δ 46.77, 50.05, 65.23, 67.28, 73.13,
109.70, 113.14, 121.67, 124.08, 125.73, 125.81, 127.20, 128.02, 128.16,
128.26, 130.34, 130.44, 141.76, 141.87, 145.36, 149.33, 154.90, 163.61.
Elemental analysis (CHN) calculated for C31H30N2O7: C: 68.62; H:
5.57; N: 5.16. Found: C: 68.42; H: 5.70; N: 4.99.
1
chloropropan-1,2-diol to prepare (S)-5 and (R)-5, respectively. H
NMR (400 MHz, Chloroform-d) δ 3.40 (dd, J = 8.0, 10.9 Hz, 1H), 3.61
(dd, J = 4.7, 10.9 Hz, 1H), 3.95 (dd, J = 5.1, 8.6 Hz, 1H), 4.05 (dd, J =
J
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6.6, 8.6 Hz, 1H), 4.36 (ddt, J = 4.9, 6.6, 8.1 Hz, 1H), 7.18−7.34 (m,
6H), 7.34−7.52 (m, 4H). [α]20D = −43.6° (c = 0.03, CHCl3) for (S)-5
and [α]20D = +42.9° (c = 0.03, CHCl3) for (R)-5.
Synthesis of tert-Butyl (2-(2-(1-methyl-1H-imidazol-5-yl)-
phenoxy)ethyl)carbamate (12). To a solution of 9 (500 mg, 1.38
mmol, 1 equiv) in DMSO (5 mL), bis(pinacolato)diboron (530 mg,
2.07 mmol, 1.5 equiv), PdCl2(dppf) (51 mg, 0.07 mmol, 0.05 equiv),
and potassium acetate (405 mg, 4.14 mmol, 3 equiv) were added and
stirred in a closed vessel at 80 °C, overnight. The mixture was diluted
with water and extracted with Et2O. The organic layer was dried over
anhydrous Na2SO4 and concentrated to give a sticky white liquid (11)
which was used directly in the next Suzuki cross-coupling reaction. 5-
Bromo-1-methylimidazole (221 mg, 1.38 mmol, 1 equiv) was
solubilized in dry dioxane (20 mL) at room temperature and under
argon atmosphere. The crude of the previous Miyaura reaction (11)
and the 2N K2CO3 aqueous solution (0.7 mL, 1.38 mmol, 1 equiv) were
added. The mixture was degassed with argon-vacuum cycles before the
addition of tetrakis(triphenylphosphine)palladium(0) (159 mg, 0.138
mmol, 0.1 equiv). The reaction was heated to 80 °C under vigorous
stirring for 24 h and concentrated. The residue was partitioned between
EtOAc and Na2CO3 saturated solution. The organic phase was
collected, washed with brine, dried over anhydrous Na2SO4, and
concentrated. The crude was chromatographed over silica gel (mobile
phase EtOAc:MeOH 95:5) to give 311 mg (71% yield) of a yellow
liquid.
Synthesis of 2-(2-(Pyridin-4-yl)phenoxy)ethan-1-amine (6). 8
(440 mg, 1.40 mmol, 1 equiv) was solubilized in a 1:1 mixture of
TFA:DCM at 0 °C. The solution was stirred at room temperature for 3
h and concentrated. The residue was solubilized in water and the pH
adjusted to 14 with pellets of NaOH. The aqueous phase was extracted
with EtOAc and the organic layer washed with brine, dried over
anhydrous Na2SO4, and concentrated. The titled compound was
obtained as a pale yellow liquid (290 mg, 97% yield), which was pure
enough to be used in the next step without further purification.
1H NMR (400 MHz,Chloroform-d) δ 1.71 (br s, 2H), 2.99 (t, J = 5.0
Hz, 2H), 3.99 (t, J = 5.0 Hz, 2H), 6.99 (d, J = 8.3 Hz, 1H), 6.91−6.99
(m, 1H), 7.27−7.42 (m, 2H), 7.39−7.47(m, 2H), 8.51−8.60 (m, 2H);
13C NMR (101 MHz, Chloroform-d): δ 52.48, 68.62, 112.57, 121.23,
124.14, 127.79, 129.90, 130.62, 146.18, 149.41, 153.67.
Synthesis of 2-(2-(1-Methyl-1H-imidazol-5-yl)phenoxy)ethan-1-
amine (7). The N-Boc cleavage of 9 was performed as reported above
for the synthesis of 6. Dark yellow liquid (89% yield). 1H NMR (400
MHz, Chloroform-d) δ 1.37 (bs, 2H), 2.98 (t, J = 5.3 Hz, 2H), 3.55 (s,
3H), 4.00 (t, J = 5.3 Hz, 2H), 6.95−7.18 (m, 3H), 7.21−7.32 (m, 1H),
7.37−7.47 (m, 1H), 7.53 (s, 1H). 13C NMR (101 MHz, Chloroform-d)
δ 32.18, 41.42, 70.93, 112.67, 130.20, 132.18, 138.34, 156.68.
Synthesis of tert-Butyl (2-bromoethyl)carbamate (8). To a stirred
suspension of 2-bromoethylamine hydrobromide (1.0 g, 4.88 mmol, 1
equiv) in THF/aq. NaHCO3 saturated solution 1:1 (20 mL), di-tert-
butyl decarbonate (1.17 g, 5.40 mmol, 1.1 equiv) was added. The
mixture was stirred at room temperature for 6 h. The organic phase was
evaporated, and the aqueous residue was extracted with Et2O. The
organic layer was washed with brine, dried over anhydrous Na2SO4, and
concentrated to give the titled compound as colorless liquid (1.09 g,
quant. yield) that was pure enough to be used in the next step without
further purification.
1H NMR (400 MHz, Chloroform-d) δ 1.42 (s, 9H), 3.40 (m, 2H),
3.51 (s, 3H), 4.03 (t, J = 5.2 Hz), 4.84 (bs, 1H), 6.89−7.07 (m, 3H),
7.20−7. 32 (m, 1H), 7.33−7.42 (m, 1H), 7.50 (s, 1H). 13C NMR (101
MHz, Chloroform-d) δ 28.51, 32.43, 40.38, 67.82, 79.67, 112.30,
121.51, 121.66, 127.34, 127.88, 128.59, 129.93, 138.47, 154.71, 155.92.
Binding Studies. Radioligand Binding Assays for 5-HT1A, 5-HT6,
and 5-HT7 Receptors in Transfected HEK293 Cells. Cell Culture.
HEK293 cells with stable expression of human 5-HT1A, 5-HT6, and 5-
HT7b (prepared with the use of Lipofectamine 2000) were grown in
Dulbecco’s Modified Eagle Medium (DMEM) containing 10%
dialyzed fetal bovine serum (FBS) and 500 μg/mL G418 sulfate.
Liver hepatocellular carcinoma Hep-G2 cells were cultured in DMEM
enriched by 10% FBS, 2 mM L-glutamine, 100 IU/mL penicillin, and 50
μg/mL streptomycin (all from Sigma-Aldrich Corporation, a division of
Merck KGaA, Darmstadt, Germany). Cell lines were maintained at 37
°C in a humidified atmosphere with 5% CO2. For membrane
preparation, the cells were subcultured in 150 cm2 flasks, grown to
90% confluence, washed twice with prewarmed to 37 °C phosphate
buffered saline (PBS), and pelleted by centrifugation (200g) in PBS
containing 0.1 mM EDTA and 1 mM dithiothreitol. Prior to membrane
preparation, pellets were stored at −80 °C.
The cell pellets were thawed and homogenized in 10 volumes of
assay buffer using an Ultra Turrax tissue homogenizer and centrifuged
twice at 35 000g for 15 min at 4 °C, with incubation for 15 min at 37 °C
in between. The composition of the assay buffers was as follows: for 5-
HT1AR: 50 mM Tris HCl, 0.1 mM EDTA, 4 mM MgCl2, 10 μM
pargyline, and 0.1% ascorbate; for 5-HT6R: 50 mM Tris HCl, 0.5 mM
EDTA, and 4 mM MgCl2; for 5-HT7bR: 50 mM Tris HCl, 4 mM
MgCl2, 10 μM pargyline, and 0.1% ascorbate. All the assays were
incubated in a total volume of 200 μL in 96-well microtiter plates for 1 h
at 37 °C, except 5-HT1AR which was incubated at room temperature.
The process of equilibration was terminated by rapid filtration through
Unifilter plates with a 96-well cell harvester and radioactivity retained
on the filters was quantified on a Microbeta plate reader (PerkinElmer,
USA). For the displacement studies, the assay samples contained the
following radioligands (PerkinElmer, USA): 2.5 nM [3H]-8-OH-DPAT
(135.2 Ci/mmol) for 5-HT1AR; 2 nM [3H]-LSD (83.6 Ci/mmol) for 5-
HT6R; 0.8 nM [3H]-5-CT (39.2 Ci/mmol) for 5-HT7R. Nonspecific
binding was defined with 10 μM of 5-HT in 5-HT1AR and 5-HT7R
binding experiments, where 10 μM of methiothepine was used in 5-
HT6R assays. Each compound was tested in triplicate at 7
concentrations (10−10−10−4 M). The results were analyzed with one-
way ANOVA followed by the Bonferroni’s posthoc test. The inhibition
constants (pKi) were calculated from the Cheng-Prusoff equation.46
The results were expressed as means of at least two separate
experiments.
1H NMR (400 MHz, Chloroform-d) δ 1.44 (s, 9H), 3.44 (t, J = 5.9
Hz, 2H), 3.51 (t, J = 5.9 Hz, 2H), 4.94 (s, 1H). 13C NMR (101 MHz,
Chloroform-d) δ 146.6, 85.0, 42.3, 28.2, 27.2.
Synthesis of tert-Butyl (2-(2-iodophenoxy)ethyl)carbamate (9).
To a solution of 2-iodophenol (1.18 g, 5.35 mmol, 1.2 equiv) in DMF
(5 mL), K2CO3 (1.53 g, 11.15 mmol, 2.5 equiv) was added. The
mixture was stirred at room temperature for 30 min and 8 (1.0 g, 4.46
mmol, 1 equiv) was added in one portion. The suspension was heated
to 60 °C for 2 h and concentrated. The residue was resuspended in
water and extracted with EtOAc. The organic layer was washed with
Na2CO3 saturated solution, brine, dried over anhydrous Na2SO4, and
concentrated. The crude was chromatographed over silica gel (mobile
phase Cy:EtOAc 8:2) to give 1.57 g (97% yield) of a pale yellow liquid.
1H NMR (400 MHz, Chloroform-d) δ 1.47 (s, 9H), 3.60−3.61 (m,
2H), 4.08 (t, J = 4.8 Hz, 2H), 5.14 (bs, 1H), 6.75 (t, J = 8.0 Hz, 1H),
6.82 (d, J = 8.0 Hz, 1H), 7.30 (t, J = 8.0 Hz, 1H), 7.78 (d, J = 8.0 Hz,
2H). 13C NMR (101 MHz, Chloroform-d) δ 28.4, 40.0, 68.6, 79.5, 86.8,
112.5, 123.0, 129.6, 139.4, 155.9, 157.0.
Synthesis of tert-Butyl (2-(2-(pyridin-4-yl)phenoxy)ethyl)-
carbamate (10). To a solution of 9 (726 mg, 2 mmol, 1 equiv) in
dry dioxane (15 mL) at room temperature and under argon
atmosphere, 4-pyridylboronic acid (369 mg, 3 mmol, 1.5 equiv) and
the 2 N K2CO3 aqueous solution (3 mL, 6 mmol, 2 equiv) were added.
The mixture was degassed with argon-vacuum cycles before the
addition of tetrakis(triphenylphosphine)palladium(0) (70 mg, 0.06
mmol, 0.03 equiv). The reaction was heated to 80 °C under vigorous
stirring for 18 h and concentrated. The residue was partitioned between
EtOAc and Na2CO3 saturated solution. The organic phase was
collected, washed with brine, dried over anhydrous Na2SO4, and
concentrated. The crude was chromatographed over silica gel (mobile
phase EtOAc 100%) to give 480 mg (72% yield) of a yellow liquid.
1H NMR (400 MHz, Chloroform-d) δ 1.41 (s, 9H), 3.44 (q, J = 5.2
Hz, 2H), 4.03 (t, J = 5.2 Hz, 2H), 4.72 (bs, 1H), 6.98 (d, J = 8.2 Hz,
1H), 7.06 (t, J = 8.2 Hz, 1H), 7.31−7.34 (m, 2H), 7.44 (d, J = 4.8 Hz,
1H), 8.62 (d, J = 6.0 Hz, 2H).
Radioligand Binding Assays for 5-HT2A and 5-HT2C Receptors on
Rat Brain Membranes. Male Sprague−Dawley rats were decapitated
K
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and their brains removed and placed on ice. The hippocampi (for the
functional 5-HT1A receptor assay) or frontal cortexes (for the
competitive 5-HT2A and 5-HT2C receptor assays) were dissected and
homogenized with a glass homogenizer in 30 vol. ice-cold TED buffer
(50 mMTris-HCl, 1 mM EDTA, 1 mM dithiotheritol, pH 7.4). Next,
the homogenate was centrifuged at 21 000g for 30 min at 4 °C. The
pellet was suspended in 30 vol TED buffer (pH 7.4) and incubated in a
water bath for 10 min at 37 °C to remove endogenous ligands. The
suspension was centrifuged again at 21 000g for 30 min at 4 °C. The
pellet was resuspended in 30 vol TED buffer (pH 7.4) and the
centrifugation step was repeated. The final pellet was suspended in 10
vol 50 mM Tris-HCl (pH 7.4) and stored at −80 °C until use.47
The 5-HT2A assay was performed according to a previously described
protocol with some modifications.48 Briefly, the frontal cortex
homogenates (160 μg protein/mL) were incubated in triplicate with
1 nM [3H]ketanserin for 60 min at 36 °C in a 50 mM Tris-HCl (pH
7.4) buffer containing 0.1% ascorbate, 3 mM CaCl2, and 10 μM
pargyline) and increasing concentrations (10−11−10−5 M) of the
compound of interest. Nonspecific binding was determined in the
presence of 10 μM mianserin. For the 5-HT2C assay, the frontal cortex
homogenates (250 μg protein/mL) were incubated in triplicate with 1
nM [3H]mesulergine for 60 min at 36 °C in a 50 mM Tris-HCl (pH
7.4) buffer containing 0.1% ascorbate, 10 mM MgCl2, 10 μM pargyline,
100 nM spiperone, and increasing concentrations (10−10−10−5 M) of
the compound tested. After incubation, the reaction mixture was
deposited onto UniFilter-96 GF/B plates, with the aid of a FilterMate-
96 Harvester. The filter plates were presoaked with 0.4% PEI for 1 h.
Next, each filter well was washed with 1.75 mL of 50 mM Tris-HCl (pH
7.4) and left to dry on a heating block set to 50 °C for 2 h. Then, 45 μL
of Microscint-20 scintillation fluid was added to each filter well and left
to equilibrate overnight. The filter-bound radioactivity was counted in a
MicroBeta2 Microplate Counter. Curves were fitted with a one-site
sigmoidal dose−response equation and pKi for each compound was
calculated with the Cheng-Pursoff equation. Results were analyzed with
one-way ANOVA followed by the Bonferroni’s posthoc test. The results
are expressed as means SEM of three independent experiments.
Functional 5-HT1A Binding Assay. The [35S]GTPγS assay was
performed according to the method described previously.49 In the
agonist mode, 25 μg/mL of hippocampus homogenate was incubated in
triplicate with 0.8 nM [35S]GTPγS in an assay buffer (50 mM Tris-HCl,
pH = 7.4, 1 mM EGTA, 3 mM MgCl2, 100 mM NaCl, 30 μM GDP) in
the presence of increasing concentrations of the tested compounds
(10−10−10−5 M). Nonspecific binding was determined with 100 μM of
unlabeled GTPγS. The reaction mixture was incubated for 90 min at 36
°C in a volume of 250 μL. To test whether G-protein stimulation was 5-
HT1A receptor dependent, in a separate experiment, the EC80
concentration of each compound was incubated with increasing
concentrations (10−10−10−5 M) of WAY-100635. Next, 96-well
Unifilter Plates (PerkinElmer, USA) were presoaked for 1 h with 50
mM Tris-HCl (pH = 7.4) before harvesting. The reaction was
terminated by vacuum filtration onto filter plates with the Filter Mate
Harvester (PerkinElmer, USA). The samples were then rapidly washed
with 2 mL of 50 mM Tris-HCl (pH = 7.4) buffer. The filter plates were
dried for 2 h at 50 °C. After drying, 45 μL of EcoScint-20 scintillant
(PerkinElmer, USA) was added to every well. The radioactivity was
counted in a Trilux MicroBeta2 counter (PerkinElmer, USA). The data
were analyzed with GraphPad Prism 5.0 software (GraphPad Software,
San Diego California USA, www.graphpad.com). To determine
whether compound-stimulated [35S]GTPγS binding involved 5-HT1A
receptors, two protocols were used. In the first experiment, a single
WAY-100635 concentration (10−7 M) was used against increasing
concentrations of the agonist (10−10−10−5 M) to determine the
rightward shift in EC50 (see Table SI-1). In the second protocol,
increasing concentrations of WAY-100635 (10−10−10−5 M) were used
against a single EC80 concentration of the compound studied to check if
[35S]GTPγS binding was 5-HT1A-dependent (see Table SI-1).
on their Emax values. Compounds that produced half of the stimulation
for the endogenous full agonist serotonin were considered as partial
agonists.50 The differences in compound potency and efficacy were
evaluated with one-way ANOVA followed by the Bonferroni’s post-hoc
test. The baseline G-protein stimulation was set at 100%. One, two, or
three symbols represent statistical significance of 0.05, 0.01, and 0.001,
respectively.
Molecular Modeling. Ligand Preparation. All the herein
investigated couples of enantiomers were built, parametrized
(Gasteiger-Huckel method), and energy minimized, within MOE
̈
using an MMFF94 force field (the root-mean-square gradient has been
set to 0.00001) [MOE: Chemical Computing Group Inc., Montreal,
H3A 2R7 Canada. http://www.chemcomp.com].51
Homology Modeling and Docking Studies. As most of the key
residues characteristic of GPCRs are conserved within the serotoni-
nergic system, a novel 5-HT1A receptor homology model was generated,
taking into account the X-ray structure of human 5-HT1B (PDB code:
5V54; resolution = 3.9 Å), in complex with methiotepin.30 This model
was built by applying the ligand-based homology modeling strategy, as
proposed by Moro, in order to take into account the role played by the
ligand placed within the receptor crevice.52 This method was previously
performed and discussed by us, for building other protein theoretical
models.53,54 In this case, during the homology modeling calculations,
we maintained the methiothepin structure, being the cocrystallized
compound at the template cavity. Notably, methiothepin is a
nonselective serotoninergic ligand, opening the possibility of building
a more reliable model of the biological target, rather than taking into
account only the coordinates of the template. The amino acid sequence
of 5-HT1AR (P08908) was retrieved using the swissprot databank,55
while the three-dimensional structure coordinates file of the GPCR
template was downloaded from the Protein Data Bank.56 The amino
acid sequence of the biological target 5-HT1AR was aligned with the
corresponding residues of 5V54, on the basis of the Blosum62 matrix
(MOE software), obtaining a high value of the pairwise percentage
residue identity (PPRI = 53%). The connecting loops were constructed
by the loop search method implemented in MOE. The MOE output file
included a series of ten models for the 5-HT1A protein, independently
built on the basis of a Boltzmann-weighted randomized procedure57
combined with specialized logic for the handling of sequence insertions
and deletions.58 Among the derived models, there were no significant
main chain deviations. The best scored model as packing quality
functions was selected for a further full energy minimization, using the
AMBER94 force field.59 Then, the putative binding site of 5-HT1A
targeting ligands was identified based on the alignment onto the known
binding site of the serotoninergic template cocrystallized with
methiothepin. This approach allowed us to identify the protein crevice
involved in the ligand binding, as we previously successfully discussed
for other GPCRs.60−63 Docking calculations of each enantiomer were
then performed by means of LeadIT 2.1.8 software suite (www.
biosolveit.com). The final docking poses were prioritized using the
score values of the lowest energy pose of the compounds docked to the
protein structure. All the ligands were refined and rescored by
assessment with the algorithm HYDE, included in the LeadIT 2.1.8
software. The HYDE module considers dehydration enthalpy and
hydrogen bonding.64,65
Analysis of Cell Viability. Liver hepatocellular carcinoma Hep-G2
cells were seeded in 96-well plates (1 × 104 cells/well) and maintained
under continuous stimulation with increasing concentrations (nM−μM
range) of the tested compounds. 200 mM ethanol and 5% Triton X-100
(Sigma-Aldrich Corporation) served as controls. The viability of cells
was assessed by MTT assay as previously described.66,67 The
absorbance was detected by a Victor3 plate reader (PerkinElmer Inc.,
Waltham, MA, USA) and represented by box and whiskers plot, as a
measure of cell viability. Differences between values from treated and
untreated cells were evaluated by two-ANOVA followed by Dunnett’s
multiple comparison test (p < 0.05).
The curves were fitted with a one-site nonlinear regression model.
Efficacy (Emax) and potency (pEC50) were calculated from the Cheng-
Prusoff equation from three separate experiments and expressed as the
means SEM. The drugs were classified as full or partial agonists based
CYL8038QP2 hERG Human Potassium Ion Channel Cell
Based QPatch CiPA Assay. After whole cell configuration is achieved,
the cell is held at −80 mV. A 500 ms pulse to −40 mV is delivered to
measure the leaking current, which is subtracted from the tail current
L
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online. Then, the cell is depolarized to +40 mV for 500 ms and then to
−80 mV over a 100 ms ramp to elicit the hERG tail current. This
paradigm is delivered once every 8 s to monitor the current amplitude.
(tylose). All the compounds were given in a manner generally
accepted in experimental pharmacology, in a volume of 10 mL/
kg body weight. The animals were weighed immediately before
injection. Each group consisted of 8 members. The control
animals received an equivalent volume of the solvent at the
respective time before the tests. Morphine was used as positive
control. Between injections, the mice were provided with stable
living conditions and unrestricted access to food and water. All
the experiments were conducted in the light phase between
09.00 a.m. and 05.00 p.m.
For the hot plate test, the mice were placed on a hot plate
(Ugo Basile Srl, Gemonio, Italy) maintained at a constant
temperature of 55 °C with a cutoff time of 20 s. The baseline
latency response (in seconds), induced by the thermal stimulus,
with the mouse lifting either of the hind paws or jumping with all
four feet off the hot plate, before administration of a compound
was measured first. The animals were then administered with
compounds and the post-treatment latency responses were
determined at 30 min time intervals up to 180 min after drug
injection. The antinociceptive effects of the compounds were
expressed as a percentage of maximum possible effect (%MPE),
which was calculated according to the following equation: [(T1
− T0)/(20 − T0)] × 100, where T0 and T1 are the predrug and
post-drug latencies for hot plate response, respectively.37,38 The
results were calculated using the one-way analysis of variance
(ANOVA) followed by Bonferroni’s post hoc test. The results are
presented as the means standard errors of mean (S.E.M.). The
level of p < 0.05 was considered statistically significant. All the
figures were prepared using the GraphPad Prism ver. 5.00 for
Windows (GraphPad Software, San Diego, CA, USA), www.
graphpad.com.
Preparation of Spinal Cord Slices and Patch-Clamp
Recording. The Italian Ministry of Health approved all the
experiments that were conducted on postnatal CD1 mice (P18−
P25) in accordance with the Guide for the Care and Use of
Laboratory Animals and the EU and Italian regulations on
animal welfare. The spinal cord slices were obtained following
the procedure described previously.69,70 Briefly, the animals
were anesthetized with isoflurane and decapitated, the spinal
cord and vertebrae were rapidly removed and placed in ice-cold
dissecting Krebs’ solution (composition in mM:125 NaCl, 2.5
KCl, 1.25 NaH2PO4, 26 NaHCO3, 25 glucose, 6 MgCl2, 1.5
CaCl2, and 1 kynurenic acid, pH 7.4, 320 mOsm), bubbled with
95% O2, 5% CO2. The lumbar spinal cord was isolated,
embedded in an agarose block (low melting point agarose 3%,
Thermo Fisher Scientific, Waltham, USA), and transverse slices
(500 μm thick) were obtained using a vibrating microtome
(WPI, Sarasota, USA). The slices were incubated in oxygenated
incubation Krebs’ solution (the same as dissecting but without
kynurenic acid) at 35 °C for 30 min and used for recording. The
Patch-clamp recording in whole-cell configuration was per-
formed on lamina I−II neurons at room temperature. The slices
were perfused at 2 mL/min with recording Krebs’solution (in
mM: 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 25
glucose, 1 MgCl2, and 2 CaCl2, pH 7.4, 320 mOsm). Recordings
of post-synaptic currents (EPSCs) were performed in voltage
clamp, by using an intracellular solution with the following
composition (in mM): 120 potassium methanesulfonate, 10
NaCl, 10 EGTA, 1 CaCl2, 10 HEPES, 5 ATP-Mg, pH adjusted
to 7.2 with KOH, osmolarity 300 mOsm. 8-OH-DPAT was
obtained from Sigma-Aldrich (Saint Louis, USA). The data were
recorded and acquired using a Multiclamp 700A amplifier and
the pClamp 10 software (Molecular Devices, Sunnyvale, USA).
DATA CALCULATION AND ANALYSIS
■
The parameters measured were the maximum tail current
evoked on stepping to 40 mV and ramping back to −80 mV from
the test pulse. All data were filtered for seal quality, seal drop, and
current amplitude. The peak current amplitude was calculated
before and after compound addition, and the amount of block
was assessed by dividing the Test compound current amplitude
by the Control current amplitude. Control is the mean hERG
current amplitude collected 15 s at the end of the control; Test
Compound is the mean hERG current amplitude collected in
the presence of test compound at each concentration.
E-4031, a blocker of hERG-type potassium channels, was used
as reference compound.
Behavioral Tests. Formalin Test. Male Swiss CB1 mice
(Envigo, S. Pietro al Natisone (UD)) weighing 25−30 g were
used. The animals were kept at a constant room temperature (25
1 °C) under a 12:12 h light and dark cycle, with free access to
food and water. Each mouse was used for only one experiment.
The experimental procedures were conducted in accordance
with international guidelines, as well as the European
Communities Council Directive and National Regulations
(CEE Council 86/609 and DL 116/92). All the tests were
performed blind to treatment.
The formalin (5%, 10 μL; Sigma-Aldrich) was injected
subcutaneously into the plantar side of the right hind paw.68
After injection, the mice were immediately placed in a plexiglas
box: the total time (in seconds) spent on licking or biting the
injected hind paw was recorded every 5 min at selected intervals,
0−10 (phase I) and 10−60 (phase II) min, in the different
experimental groups, as an indicator of nociceptive behavior.
The formalin scores were separated into two phases: phase I (0−
10 min) and phase II (10−60 min). A mean response was then
calculated for each phase. The test compound and WAY-100635
(Sigma-Aldrich) were dissolved in normal saline solution,
containing 10% dimethyl sulfoxide (DMSO, Sigma-Aldrich).
A vehicle solution containing 10% DMSO was given as a control.
Morphine was used as positive control. The test compound and
vehicle were intraplantar (i.p.) administered (5 mL/kg) 15 min
before the formalin. The WAY-100635 (3 mg/kg i.p.) was
injected 30 min before the test compound or vehicle. The data
are expressed as mean values (SEM). Analyses of variance (two-
way repeated measures ANOVA followed by post hoc
Bonferroni test) were performed to assess significance using
the Instat 3.0 software (GraphPad Software, San Diego, CA). p <
0.05 was considered significant.
Hot Plate Test. The experiments were performed on male
Albino Swiss mice (20−25 g), where 4 animals were kept in a
cage, in an environmentally controlled room (ambient temper-
ature 22 1 °C; relative humidity 50−60%; 12 h light/dark
cycle, lights on at 8:00). Standard laboratory food (LSM,
Agropol-Motycz, Poland) and filtered water were available ad
libitum. All the behavioral experiments were carried out in
accordance with the European Community Council Directive
for Care and Use of Laboratory Animals (2010/63/EU) (2016),
and approved by the Local Ethics Committee for Animal
Experimentation. The tested compounds were administered
intraperitoneally (i.p.), dissolved in DMSO (its final concen-
tration of 0.1%), suspended in 0.5% Tween-80 (1−2 drops) and
then diluted using an aqueous solution of 0.5% methylcellulose
M
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ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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Research Article
The sampling rate was 10 kHz, and the data were filtered at 2
kHz. The data were analyzed off-line using pClamp10 software
and Minianalysis (Synaptosoft, USA). The data are expressed as
the mean SEM and differences were considered significant for
p < 0.05. The statistical significance for the effect on EPSC
frequency was determined using the Kolmogorov−Smirnov test
on individual neurons.
Medical Analytics, Medical University of Lublin, 20-093
Lublin, Poland
Grazyna Biała − Department of Pharmacology and
̇
Pharmacodynamics, Faculty of Pharmacy with Division of
Medical Analytics, Medical University of Lublin, 20-093
Lublin, Poland
Simone Ronsisvalle − Department of Drug Sciences, Medicinal
Chemistry Section, University of Catania, I-95125 Catania,
Italy; orcid.org/0000-0003-3488-7343
Silvia Limoncella − Unit of Endocrinology, Department
Biomedical, Metabolic, and Neural Sciences, University of
Modena and Reggio Emilia, 41125 Modena, Italy
Livio Casarini − Unit of Endocrinology, Department
Biomedical, Metabolic, and Neural Sciences and Center for
Genomic Research, University of Modena and Reggio Emilia,
41125 Modena, Italy
Elena Cichero − Department of Pharmacy, Medicinal
Chemistry Section, School of Medical and Pharmaceutical
Sciences, University of Genova, 16132 Genova, Italy
Paola Fossa − Department of Pharmacy, Medicinal Chemistry
Section, School of Medical and Pharmaceutical Sciences,
University of Genova, 16132 Genova, Italy
Grzegorz Satała − Department of Medicinal Chemistry, Maj
Institute of Pharmacology, Polish Academy of Sciences, 31-343
Kraków, Poland
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acschemneuro.0c00289.
Experimental Section: (i) Effect of a single/increasing
concentrations of WAY-100635 on compound-stimulated
[35S]GTPγS binding (Table SI-1/SI-2); (ii) Molecular
modeling: Alignment of the 5HT1A primary sequence
with that of the template 5HT1B (Figure SI-1); Super-
imposition of the modeled 5-HT1A receptor onto the X-
ray crystallographic structure of the 5-HT1B template
(Figure SI-2); Match of the conserved regions among the
modeled 5-HT1A receptor and the X-ray crystallographic
structure of the 5-HT1B template (Figure SI-3); Position-
ing of the antagonist methiothepin at the modeled 5-
HT1AR (Figure SI-4) (PDF)
Andrzej J. Bojarski − Department of Medicinal Chemistry, Maj
Institute of Pharmacology, Polish Academy of Sciences, 31-343
Kraków, Poland; orcid.org/0000-0003-1417-6333
Livio Brasili − Department of Life Sciences, University of
Modena and Reggio Emilia, 41125 Modena, Italy;
orcid.org/0000-0002-3280-3196
AUTHOR INFORMATION
■
Corresponding Author
Silvia Franchini − Department of Life Sciences, University of
Modena and Reggio Emilia, 41125 Modena, Italy;
orcid.org/0000-0002-6320-9712;
Phone: +390592058582; Email: silvia.franchini@
unimore.it
Complete contact information is available at:
https://pubs.acs.org/10.1021/acschemneuro.0c00289
Author Contributions
Authors
⬢S.F. and R.B. contributed equally. P.L., C.S., S.F., and L.B.
designed the novel compounds and planned the procedures for
their synthesis. P.L. and C.S. developed the chemical synthesis
and characterized the novel compounds. P.L. wrote the chemical
experimental parts of the manuscript. A.L., M.B.Z., A.P., and
A.B. performed the functional studies at 5-HT1A and the
competition binding studies at 5-HT2A, 5-HT2C receptors. A.J.B.
and G.S. performed the binding experiments at 5-HT1A, 5-HT6,
and 5-HT7. S.R. provided the in vivo experiments (formalin test)
and CiPA hERG QPatch Assay through the EUROFINS
contract and discussed the biological data. J.O., E.K., and G.B.
performed the hot plate test. E.C. and P.F. performed the
molecular modeling studies. L.C. and S.L. performed the
hepatotoxicity studies. A.C. and R.B. performed the electro-
physiological studies. S.F. and R.B. drafted the main text of the
manuscript. All authors critically discussed and approved the
final version of the manuscript.
Pasquale Linciano − Department of Life Sciences, University of
Modena and Reggio Emilia, 41125 Modena, Italy;
orcid.org/0000-0003-0382-7479
Claudia Sorbi − Department of Life Sciences, University of
Modena and Reggio Emilia, 41125 Modena, Italy;
orcid.org/0000-0001-6916-4933
Antonella Comitato − Department of Biomedical, Metabolic
and Neural Sciences, University of Modena and Reggio Emilia,
41125 Modena, Italy
Anna Lesniak − Department of Pharmacodynamics, Faculty of
Pharmacy, Centre for Preclinical Research and Technology,
Medical University of Warsaw, 02-097 Warsaw, Poland
Magdalena Bujalska-Zadrozny − Department of
̇
Pharmacodynamics, Faculty of Pharmacy, Centre for
Preclinical Research and Technology, Medical University of
Warsaw, 02-097 Warsaw, Poland
Agata Pawłowska − Department of Pharmacodynamics, Faculty
of Pharmacy, Centre for Preclinical Research and Technology,
Medical University of Warsaw, 02-097 Warsaw, Poland
Anna Bielenica − Department of Biochemistry, Medical
University of Warsaw, 02-097 Warsaw, Poland
Notes
The authors declare no competing financial interest.
́
Jolanta Orzelska-Gorka − Department of Pharmacology and
ACKNOWLEDGMENTS
■
Pharmacodynamics, Faculty of Pharmacy with Division of
Medical Analytics, Medical University of Lublin, 20-093
Lublin, Poland
This work was supported by FFABR grant (Finanziamento
Annuale Individuale delle Attivita Base Di Ricerca) to S.F. and
donation from BPER (Banca Popolare dell’Emilia Romagna) to
̀
Ewa Kędzierska − Department of Pharmacology and
Pharmacodynamics, Faculty of Pharmacy with Division of
R.B.
N
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ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
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ABBREVIATIONS USED
■
5-HT, serotonin; SAR, structure−activity relationship; GTPγS,
guanosine 5′-O-[gamma-thio]triphosphate; TMS, tetramethyl-
silane
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Signaling in Tissue Fibrosis. Biochim. Biophys. Acta, Mol. Basis Dis. 1832
(7), 905−910.
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