Novel 1-(1-benzoylpiperidin-4-yl)methanamine derivatives with high affinity and selectivity for serotonin 5-HT1A receptors were obtained and tested in four functional assays: ERK1/2 phosphorylation, adenylyl cyclase inhibition, calcium mobilization, and β-arrestin recruitment. Compounds 44 and 56 (2-methylaminophenoxyethyl and 2-(1H-indol-4-yloxy)ethyl derivatives, respectively) were selected as biased agonists with highly differential "signaling fingerprints" that translated into distinct in vivo profiles. In vitro, 44 showed biased agonism for ERK1/2 phosphorylation and, in vivo, it preferentially exerted an antidepressant-like effect in the Porsolt forced swimming test in rats. In contrast, compound 56 exhibited a first-in-class profile: it preferentially and potently activated β-arrestin recruitment in vitro and potently elicited lower lip retraction in vivo, a component of "serotonergic syndrome". Both compounds showed promising developability properties. The presented 5-HT1A receptor-biased agonists, preferentially targeting various signaling pathways, have the potential to become drug candidates for distinct central nervous system pathologies and possessing accentuated therapeutic activity and reduced side effects.
Novel 1-(1-benzoylpiperidin-4-yl)methanamine derivatives with high affinity and selectivity for serotonin 5-HT1A receptors were obtained and tested in four functional assays: ERK1/2 phosphorylation, adenylyl cyclase inhibition, calcium mobilization, and β-arrestin recruitment. Compounds 44 and 56 (2-methylaminophenoxyethyl and 2-(1H-indol-4-yloxy)ethyl derivatives, respectively) were selected as biased agonists with highly differential "signaling fingerprints" that translated into distinct in vivo profiles. In vitro, 44 showed biased agonism for ERK1/2 phosphorylation and, in vivo, it preferentially exerted an antidepressant-like effect in the Porsolt forced swimming test in rats. In contrast, compound 56 exhibited a first-in-class profile: it preferentially and potently activated β-arrestin recruitment in vitro and potently elicited lower lip retraction in vivo, a component of "serotonergic syndrome". Both compounds showed promising developability properties. The presented 5-HT1A receptor-biased agonists, preferentially targeting various signaling pathways, have the potential to become drug candidates for distinct central nervous system pathologies and possessing accentuated therapeutic activity and reduced side effects.
Although serotonin 5-HT1A receptors exert a major influence
on central nervous system (CNS) functions such as mood, pain, and
movement and were identified several decades ago,[1,2] it
is notable that there are still no selective 5-HT1A receptor
agonists approved for therapeutic intervention. There are, of course,
commercialized drugs that exhibit some agonist properties at 5-HT1A receptors, including the anxiolytic buspirone (Buspar),
the antidepressant vortioxetine (Brintellix), the antipsychotic aripiprazole
(Abilify), and the antiparkinsonian bromocriptine (Parlodel).[3−6] However, all of these compounds also interact with other targets,
including other monoamine receptors or transporters, and they only
partially activate 5-HT1A receptors (i.e., they function as “partial agonists”). Moreover, such
compounds do not discriminate between subpopulations of 5-HT1A receptors which are expressed in different brain regions and that
mediate various, sometimes opposing, physiological and behavioral
responses. For example, activation of postsynaptic 5-HT1A heteroreceptors in the frontal cortex elicits procognitive and antidepressant
effects, whereas activation of presynaptic 5-HT1A autoreceptors is associated with prodepressive
effects, notably by inhibiting the release of serotonin in terminal
regions.[7,8] These contrasting effects have long been
the object of discussion in the search for more efficacious antidepressants
and suggest that indiscriminate activation of multiple 5-HT1A receptor subpopulations may limit the therapeutic efficacy of 5-HT1A receptor agonists or elicit unacceptable
side effects. In contrast, recent advances have shown that it is possible
to selectively target 5-HT1A receptors in desired brain
areas, such as the cortex or brain stem, leading to significantly
improved and promising therapeutic-like outcomes.The basis for such preferential brain region targeting is the emerging
concept of “biased agonism” at G-protein-coupled receptors.
Accumulated studies in recent years provide compelling evidence that
different agonists can preferentially activate intracellular signaling via specific effectors, such as different G-protein subtypes
or β-arrestins. Given that coupling to particular signaling
mechanisms can vary from one brain region to another, this provides
a basis for biased agonists to differentially activate particular
brain regions. Such differential signaling may be associated with
specific neurochemical, physiological, and behavioral responses and
has been proposed in the context of drug discovery at a variety of
receptor subtypes as a strategy to achieve superior therapeutic outcomes.[9−11]In the case of 5-HT1A receptors, an important advance
was the discovery of a first highly selective biased agonist, NLX-101
(aka F15599, 1), which shows a marked preference for
ERK1/2 phosphorylation versus other signaling pathways (Figure ).[12,13]1 displayed a strikingly superior activity profile
in a variety of electrophysiology, microdialysis, behavior, and brain
imaging studies, as compared to older, canonical 5-HT1A receptor agonists.[14−16] In particular, 1 exhibited highly promising
properties in models of antidepressant and procognitive activity as
well as in models of respiratory deficits in Rett syndrome, an orphan
disorder.[17,18] The discovery of 1 therefore
opened the way for drug discovery of novel, selective biased agonists
that target 5-HT1A receptors in specific brain areas that
control CNS functions and that constitute, potentially, more efficacious
and safer pharmacotherapeutics.
Figure 1
Selective 5-HT1A receptor-biased agonists.
Selective 5-HT1A receptor-biased agonists.However, despite its broad pharmacological characterization, 1 remained isolated as a single example of a biased agonist
with a superior pharmacological profile but with no medicinal chemistry
data allowing for rational design of other functionally selective
5-HT1A receptor agonists. In this context, our previous
work investigated the structure–activity relationship (SAR)
and structure functional activity relationships (SFARs) of novel analogues
designed based on the structure of 1.[13] In that study, we identified a new, patentable, and synthetically
versatile chemotype of selective 5-HT1A receptor-biased
agonists that preferentially activate ERK1/2 phosphorylation in vitro and show potent antidepressant-like properties in vivo. These findings met our objectives but did not identify
structures that may exhibit other biased agonist profiles, notably
for β-arrestin recruitment, which, as mentioned above, is a
major target for G protein coupled receptor (GPCR) -biased agonist
studies.The present study builds upon the conclusion that the pyridine-2-oxy-
or phenoxy-ethyl or derivatives of 1-(1-benzoylpiperidin-4-yl)methanamine,
represented by lead structures 2 (NLX-204) and 3 (NLX-219), are the most promising chemotypes for obtaining
new selective full agonists of the 5-HT1A receptor (Figure ). As our previous work studied various unsubstituted
derivatives, we focused herein on determining the influence of the
substitution pattern at the phenyl ring. Based on molecular modeling
studies, we observed that phenyl moiety binds in the part of the receptor
that is responsible for the stabilization of various bioactive conformations
(between transmembrane helices 3, 5, and 6), thus justifying diversification
of this fragment to obtain biased agonists with novel profiles of
functional selectivity.[13,19] Specifically, we aimed
to obtain, on the one hand, agonists with higher levels of bias for
specific signaling pathways (notably pERK1/2) and, on the other hand,
agonists exhibiting bias for signaling pathways other than pERK1/2
(notably β-arrestin recruitment). Such biased agonists with
diversified functional profiles could prove to be beneficial for different
CNS disorders involving serotonergic dysregulation. A series of variously
substituted phenoxyethyl derivatives of 1-(1-benzoylpiperidin-4-yl)methanamine
was therefore synthesized and extensively tested in a stepwise manner
to yield novel, selective, and functionally diversified 5-HT1A receptor agonists. As well as broadening our knowledge about the
pharmacology of 5-HT1A receptors, such compounds could
constitute promising candidates for treatment of different disorders
involving serotonergic neurotransmission, some of which (such as depression)
may be anticipated to respond to pERK1/2-biased agonists, whereas
others may be better treated with β-arrestin-biased agonists.
Overall, the availability of novel compounds differentially targeting
these key signaling mechanisms raises the prospect of achieving increased
therapeutic efficacy with reduced side effects in the treatment of
CNS disorders.
Results and Discussion
Design of a Novel Series of Variously Substituted Phenoxyethyl
Derivatives of 1-(1-Benzoylpiperidin-4-yl)methanamine
In
the present study, we decided to use compound 3, a previously
described unsubstituted phenoxyethyl derivative of 1-(1-benzoylpiperidin-4-yl)methanamine,
as a lead structure for modifications. The previously assessed in silico developability measures for compound 3, namely, CNS MPO = 4.89, LELP = 6.7 and Fsp3 = 0.38, were considered
favorable.[13] However, to further confirm
the properties of this compound as a good lead structure, some in vitro studies were applied. They included metabolic stability
using rat liver microsomes (RLMs), membrane permeability using parallel
artificial membrane permeability assay (PAMPA), hepatotoxicity on
HepG2 cell line, as well as extended selectivity study on a multitarget
panel, including 45 receptors (for the sake of comparison between
the lead structure and the most interesting derivatives developed
within the present study, the abovementioned data for these compounds
are collected in Table and Chart as well
as Supporting Information Table S3). Compound 3 showed acceptable metabolic stability, high permeability,
very low potential for hepatotoxicity, and significant (at least 500×)
selectivity versus the off-targets, including the hERG channel, thus
proving to be a good starting point for further modifications. The
structural diversification was focused on introducing various substituents
to the phenoxy moiety in order to modulate the functional profile
while maintaining favorable developability. The choice of substituents
was controlled primarily by molecular weight, number of hydrogen bond
donors and lipophilicity, as well as synthetic feasibility of the
final molecules. As a result, a set of 30 novel compounds was proposed
for chemical synthesis and pharmacological evaluation.
Table 7
Permeability, Hepatotoxicity, and
Intrinsic Clearance of Compounds 3, 44,
and 56
compound
PAMPA Pe [10–6 cm/s] ± SD
hepatotoxicity
50% viability of HepG2 cells
intrinsic
clearance CLint [mL/min/kg]
3
8.6 ± 1.4
>50 μM
41.7
44
6.7 ± 1.1
>50 μM
48.8
56
4.7 ± 0.4
>50 μM
9.6
references
Caffeine
Doxorubicin
diazepam
15.1 ± 0.40
<1 μM
31.0
norfloxacin
CCCP
aripiprazole
0.56 ± 0.13
<10 μM
7.02
Chart 1
Graphical Visualization of Selectivity Profiles of Compounds 3, 44, and 56a
For the sake of clarity, 34 most
important targets are shown out of 46 tested. The pK values shown were estimated based on
screening data and rounded to the nearest half-log value. For full
selectivity data, see Supporting Information Tables S4 and S5. hERG p-c—hERG blockade determined using
the patch-clamp method.
Synthesis
To prepare the target compounds 28–57, we have utilized a method that we have previously used and described
(Scheme ).[13] The method is based on a reaction of reductive
amination between cyanohydrins 4 or 5 and
the appropriate amines (6–26). Briefly, cyanohydrines 4 and 5 were prepared in Darzens reaction from
benzoylpiperidin-4-on derivatives and chloroacetonitrile, followed
by a regioselective ring opening with poly(hydrogen fluoride)pyridine.
Amines 6–26 were prepared from the corresponding
phenols according to two synthetic pathways depicted in Scheme .
Scheme 1
Synthesis of 1-(1-Benzoyl-4-fluoropiperidin-4-yl)methanamine Derivatives
Reagents and conditions: (i)
DABCO, NaCNBH3, FeSO4 × 7H2O,
molecular sieves, MeOH, r.t., 36–72 h, yield: 18–82%;
(ii) 1.0 M HCl in EtOAc, r.t., 24 h, yield: 48%; (iii) CH3COOH, NaCNBH3, 15 °C—15 min, then r.t—1
h, yield: 67%. X = F or Cl.
Scheme 2
Synthesis of the Amine Intermediates (6–26)
Reagents and conditions: (i)
for compounds II 24–26, tert-butyl-2-hydroxyethyl
carbamate or 2-(2-hydroxyethyl)isoindoline-1,3-dion, PPh3, DIAD, THF, 0 °C then r.t, 24 h, and 50 °C, 24 h; (ii)
1,2-dibromoethane, K2CO3, acetone, 40–80
°C, 24–72 h; (iii) for compounds II 6–16, 18–23, potassium phthalimide, 18-crown-6 ether,
DMF, 50 °C, 3 h; (iv) NaH, CH3I, THF, 0 °C, 30
min, then r.t, 1 h; for compound II 17 (iv) and then
(iii); (v) for compounds 6–23, 25, 26, 40% MeNH2(aq), 10% NaOH, 50 °C,
2 h, then r.t., 1 h. (vi) For compound 24, 1.0 M HCl
in EtOAc, r.t, 24 h; R1 = phthalimide or tert-butyl
carbamate.
Synthesis of 1-(1-Benzoyl-4-fluoropiperidin-4-yl)methanamine Derivatives
Reagents and conditions: (i)
DABCO, NaCNBH3, FeSO4 × 7H2O,
molecular sieves, MeOH, r.t., 36–72 h, yield: 18–82%;
(ii) 1.0 M HCl in EtOAc, r.t., 24 h, yield: 48%; (iii) CH3COOH, NaCNBH3, 15 °C—15 min, then r.t—1
h, yield: 67%. X = F or Cl.
Synthesis of the Amine Intermediates (6–26)
Reagents and conditions: (i)
for compounds II 24–26, tert-butyl-2-hydroxyethyl
carbamate or 2-(2-hydroxyethyl)isoindoline-1,3-dion, PPh3, DIAD, THF, 0 °C then r.t, 24 h, and 50 °C, 24 h; (ii)
1,2-dibromoethane, K2CO3, acetone, 40–80
°C, 24–72 h; (iii) for compounds II 6–16, 18–23, potassium phthalimide, 18-crown-6 ether,
DMF, 50 °C, 3 h; (iv) NaH, CH3I, THF, 0 °C, 30
min, then r.t, 1 h; for compound II 17 (iv) and then
(iii); (v) for compounds 6–23, 25, 26, 40% MeNH2(aq), 10% NaOH, 50 °C,
2 h, then r.t., 1 h. (vi) For compound 24, 1.0 M HCl
in EtOAc, r.t, 24 h; R1 = phthalimide or tert-butyl
carbamate.Amines 6–23 were synthesized according to a
three-step procedure starting with Williamson reaction[20] using appropriate phenols and 1,2-dibromoethane.
The obtained 2-bromoethoxy derivatives (I 6–23) were used in Gabriel’s synthesis,[21−23] leading to
the desired primary amines. Synthesis of amine 17 (R
= 3-NH–CH3) required additional Boc-protection to
prevent reductive amination at a secondary amine group. Deprotection
of the amine was performed at the last step of the synthesis of compound 45 (Scheme ). Amines 24–26 were obtained in Mitsunobu reaction[24,25] of 2-(methylamino)phenol with tert-butyl-2-hydroxyethyl
carbamate (II 24) and quinolin-8-ol or 1H-indol-4-ol with 2-(2-hydroxyethyl)isoindoline-1,3-dione (II
25 and II 26). The following Boc-deprotection
or methylaminolysis provided the desired amines. For a detailed description
of the synthetic procedures used in the synthesis on amines, see the Supporting Information.The final reaction of reductive amination was carried out in the
presence of 1,4-diazabicyclo[2.2.2]octane (DABCO) as a base, sodium
cyanoborohydride (NaCNBH3) as a reducing agent, and with
addition of iron sulfate heptahydrate (FeSO4 × 7H2O) that complexes cyanide ions and therefore contributes to
the improvement of the reaction yields. In an additional step, indole
derivative 56 was reduced to indoline analogue 57.
Structure-Affinity Relationships
5-HT1A Receptor Affinity
All the obtained
compounds were subjected to affinity determination using radioligand
binding studies. The affinity of the compounds at 5-HT1A receptors was generally high, with pK values ranging from 8.18 to 12.80 (Tables and 2). To determine the influence of various phenyl ring substitutions
on affinity and exclude the contribution of lipophilicity, we primarily
focused on analyzing changes of ligand-lipophilicity efficiency (LLE),
calculated as the difference between pK and ClogD7.4. All the
relationships were compared to the unsubstituted phenoxy derivative 3 (LLE 7.59), which was the lead structure for this series
(Figure ).
Table 1
Influence of the Substituent Position
in the Phenyl Ring on 5-HT1A Receptor Affinity and Selectivity
for the Most Important Off-Targets
All binding affinity values are
represented as pK (i.e., −log K) and expressed as means ± SEM from at least three experiments
performed in duplicate, unless otherwise indicated; radioligand binding
was performed using
CHO-K1 cells transfected with 5-HT1A receptors.
Rat cortex.
CHO-K1 cells transfected with D2 receptors; receptor affinity values were determined by competition
binding using
[3H]8-OH-DPAT.
[3H]-prazosin and
[3H]-methylspiperone.
In these conditions, pK of phentolamine at α1 receptors was 7.95 and pK of haloperidol at D2 receptors was 8.85.
Calculated distribution coefficient
at pH 7.4.
LLE referring to the 5-HT1A receptor.
Not tested *pK value was expressed as mean ±
range from two experiments performed in duplicate.
Table 2
Influence of the Type of Substituent
in the Phenyl Ring on 5-HT1A Receptor Affinity and Selectivity
for the Most Important Off-Targets
All binding affinity values are
represented as pK (i.e., −logKi) and expressed
as means ± SEM from at least three experiments performed in duplicate,
unless otherwise indicated; radioligand binding was performed using
CHO-K1 cells transfected with 5-HT1A receptors.
Rat cortex.
CHO-K1 cells transfected with D2 receptors; receptor affinity values were determined by competition
binding using
[3H]8-OH-DPAT.
[3H]-prazosin and
[3H]-methylspiperone.
In these conditions, pK of phentolamine at α1 receptors was 7.95, and pK of haloperidol at D2 receptors was 8.85.
Calculated distribution coefficient
at pH 7.4.
LLE referring to the 5-HT1A receptor. * pK value
was expressed as mean ± range from two experiments performed
in duplicate.
Figure 5
Changes in LLE in relation to unsubstituted lead structure 3 due to substitution at the phenyl ring.
Predicted binding mode of the methoxy derivatives, that is, compound 36 (light teal) together with 38 (pink) (A) and
compound 40 (gray) (B) in the site of the serotonin 5-HT1A receptor. Amino acid residues engaged in ligand binding
(within 4 Å from the ligand atoms) are displayed as sticks, whereas
crucial residues, for example, forming H-bonds (dotted yellow lines),
π–π/CH−π stacking (dotted cyan lines),
and cation−π interactions (dotted green line) are represented
as thick sticks. ECL2 residues were hidden for clarity; ECL—extracellular
loop. The homology model of the 5-HT1A receptor is based
on the crystal structure of the 5-HT1B receptor (PDB ID: 4IAR).Predicted binding mode of the fluoro derivatives, that is, compound 28 (yellow) together with 29 (pink) and 31 (green) in the site of the serotonin 5-HT1A receptor.
Amino acid residues engaged in ligand binding (within 4 Å from
the ligand atoms) are displayed as sticks, whereas crucial residues,
for example, forming H-bonds (dotted yellow lines), π–π/CH−π
stacking (dotted cyan lines), and cation−π interactions
(dotted green line) are represented as thick sticks. ECL2 residues
were hidden for clarity; ECL—extracellular loop. The homology
model of the 5-HT1A receptor is based on the crystal structure
of the 5-HT1B receptor (PDB ID: 4IAR).Predicted binding mode of compound 56 (with HBD in
meta position) in the site of the serotonin 5-HT1A receptor.
Amino acid residues engaged in ligand binding (within 4 Å from
the ligand atoms) are displayed as sticks, whereas crucial residues,
for example, forming H-bonds (dotted yellow lines), π–π/CH−π
stacking (dotted cyan lines), and cation−π interactions
(dotted green line) are represented as thick sticks. ECL2 residues
were hidden for clarity; ECL—extracellular loop. The homology
model of the 5-HT1A receptor is based on the crystal structure
of the 5-HT1B receptor (PDB ID: 4IAR).Changes in LLE in relation to unsubstituted lead structure 3 due to substitution at the phenyl ring.All binding affinity values are
represented as pK (i.e., −log K) and expressed as means ± SEM from at least three experiments
performed in duplicate, unless otherwise indicated; radioligand binding
was performed usingCHO-K1 cells transfected with 5-HT1A receptors.Rat cortex.CHO-K1 cells transfected with D2 receptors; receptor affinity values were determined by competition
binding using[3H]8-OH-DPAT.[3H]-prazosin and[3H]-methylspiperone.
In these conditions, pK of phentolamine at α1 receptors was 7.95 and pK of haloperidol at D2 receptors was 8.85.Calculated distribution coefficient
at pH 7.4.LLE referring to the 5-HT1A receptor.Not tested *pK value was expressed as mean ±
range from two experiments performed in duplicate.All binding affinity values are
represented as pK (i.e., −logKi) and expressed
as means ± SEM from at least three experiments performed in duplicate,
unless otherwise indicated; radioligand binding was performed usingCHO-K1 cells transfected with 5-HT1A receptors.Rat cortex.CHO-K1 cells transfected with D2 receptors; receptor affinity values were determined by competition
binding using[3H]8-OH-DPAT.[3H]-prazosin and[3H]-methylspiperone.
In these conditions, pK of phentolamine at α1 receptors was 7.95, and pK of haloperidol at D2 receptors was 8.85.Calculated distribution coefficient
at pH 7.4.LLE referring to the 5-HT1A receptor. * pK value
was expressed as mean ± range from two experiments performed
in duplicate.First, we focused on checking the effect of the substitution site
on the phenyl ring using three commonly used substituents (F, Cl,
or OCH3). The substitution in the ortho position increased the binding affinity; however, the effect was
rather modest and did not exceed 0.5 LLE units. The effect of substitution
in the meta position was diversified, ranging from slight increase
of affinity for fluoro and methoxy analogues to decrease in the case
of the chloro derivative. On the other hand, substitution in the para
position generally decreased the affinity by up to 2 LLE units. The
negative effect of a para substituent depended on its size, being
the least pronounced for the 4-fluoro derivative (LLE 7.34 for 31) through 4-methoxy derivative (LLE 5.73 for 40), to reach the lowest value in the case of the 4-chloro substituent
(LLE 5.19 for 35).In support of the observed SARs, docking studies showed that, in
the group of the derivatives with methoxy substituent (36, 38, 40), both ortho-
and meta-substituted compounds took almost the same position in the
binding site (Figure A). On the other hand, the para-substituted analogue (40) was unable to adopt such a position (Figure B) probably because of being too sterically
hindered in the area adjacent to the helix 6. In contrast to the above,
all the compounds with a smaller fluorine substituent (28, 29, 31) showed a common binding mode
(Figure ). These results
support the conclusion from the binding studies that the affinity
of para-substituted derivatives depends on the size of substituent
(the smaller the substituent, the higher the affinity). These SARs
are consistent with those established for the long-chain arylpiperazines,
which suggests a similar binding mode.[26]
Figure 2
Predicted binding mode of the methoxy derivatives, that is, compound 36 (light teal) together with 38 (pink) (A) and
compound 40 (gray) (B) in the site of the serotonin 5-HT1A receptor. Amino acid residues engaged in ligand binding
(within 4 Å from the ligand atoms) are displayed as sticks, whereas
crucial residues, for example, forming H-bonds (dotted yellow lines),
π–π/CH−π stacking (dotted cyan lines),
and cation−π interactions (dotted green line) are represented
as thick sticks. ECL2 residues were hidden for clarity; ECL—extracellular
loop. The homology model of the 5-HT1A receptor is based
on the crystal structure of the 5-HT1B receptor (PDB ID: 4IAR).
Figure 3
Predicted binding mode of the fluoro derivatives, that is, compound 28 (yellow) together with 29 (pink) and 31 (green) in the site of the serotonin 5-HT1A receptor.
Amino acid residues engaged in ligand binding (within 4 Å from
the ligand atoms) are displayed as sticks, whereas crucial residues,
for example, forming H-bonds (dotted yellow lines), π–π/CH−π
stacking (dotted cyan lines), and cation−π interactions
(dotted green line) are represented as thick sticks. ECL2 residues
were hidden for clarity; ECL—extracellular loop. The homology
model of the 5-HT1A receptor is based on the crystal structure
of the 5-HT1B receptor (PDB ID: 4IAR).
Further studies focused on exploration of different types of substituents
in meta and ortho positions (well tolerated by the
receptor) as well as various benzo-fused heteroaromatic moieties,
which can be also considered as a kind of ortho–meta-substituted
derivatives at the phenyl ring (Table ). All the ligands achieved very high 5-HT1A receptor affinity, with subnanomolar or even picomolar Ki values and generally very high LLE values.The most pronounced increase in 5-HT1A receptor affinity
was noted in the case of derivatives containing H-bond donor (HBD)
moiety at the meta position (45, 47, 56, and 57). Worth mentioning is the fact that
one of the meta-HBD derivatives, compound 56, had an
exceptionally high affinity, reaching subpicomolar values (pK 12.80, Ki 0.16 pM, LLE 10.06). The increase in binding affinity
of the derivatives with HBD in the meta position can be explained
by their ability to create an additional hydrogen bond with serineSer5.42 in the binding site of the 5-HT1A receptor (Figure ). This stabilizes
the ligand–receptor complex and lowers the binding energy (IFD
score for compound 56 = −465.26 compared to −463.69
for compound 3).
Figure 4
Predicted binding mode of compound 56 (with HBD in
meta position) in the site of the serotonin 5-HT1A receptor.
Amino acid residues engaged in ligand binding (within 4 Å from
the ligand atoms) are displayed as sticks, whereas crucial residues,
for example, forming H-bonds (dotted yellow lines), π–π/CH−π
stacking (dotted cyan lines), and cation−π interactions
(dotted green line) are represented as thick sticks. ECL2 residues
were hidden for clarity; ECL—extracellular loop. The homology
model of the 5-HT1A receptor is based on the crystal structure
of the 5-HT1B receptor (PDB ID: 4IAR).
The hypothesis that creating a hydrogen bond in this region improves
binding affinity was further supported by the marked increase in LLE
for the benzamide derivatives (42 and 43), which are also capable of forming this interaction. Moreover,
the meta derivatives not capable of forming H-bonds (46, 49, 50) displayed lower affinity, which
is consistent with the observation made for compound 33 (a meta-chloro derivative) and suggests that they exert a negative
steric contribution (this was, however, less pronounced than in the
case of the para analogues).Regarding the substitution at the benzoyl moiety, the 4-chloro
analogues had overall lower LLE than their 4-fluoro counterparts,
suggesting that this modification is not generally favorable.
Selectivity versus Key Antitargets, the Adrenergic α1 and Dopaminergic D2 Receptors
Previous
studies of 1-(1-benzoylpiperidin-4-yl)methanamine derivatives indicated
that the highest risk of off-target interactions is with adrenergic
α1 and dopaminergic D2 receptors.[13,27−29] Accordingly, all the novel compounds were tested
for binding to these receptors to confirm their selectivity (Tables and 2).Concerning α1 receptor affinity,
the majority of the compounds showed very high selectivity for 5-HT1A over α1 and D2 receptors (over
1000–10,000 times). In general, increased affinity for the
α1 receptor was observed for the ortho-substituted analogues (36, 37, 44). The highest affinities (pK 6.6–7.8) were observed for the derivatives with the ortho-methoxy substituent (36, 37) and their bicyclic analogues, with oxygen or nitrogen in the ortho position (51, 52, 53, 54, 55). High α1 receptor affinity was also observed for the indole derivative 56 (pK 7.00).
Interestingly, the ortho-fluoro- and ortho-chloro-derivatives (28, 32) did not show
significant affinity for the α1 receptor (pK < 6), indicating that
halogen in this position impairs α1 receptor binding.
In comparison, literature data indicate that ortho-methoxy- or ortho-ethoxy-substitution in the aryl
moiety increase α1 receptor affinity. For example,
in the structure of tamsulosin, a selective α1A receptor
antagonist used for the treatment of benign prostatic hyperplasia,
a 2-(2-ethoxyphenoxy)ethanamine fragment, can be highlighted.[30] Nevertheless, it should be noted that selectivity
for 5-HT1A versus α1 receptors was generally
very high and was less than 1000× for only two compounds (37, 52).In the case of the D2 receptor, only one compound (55) showed substantial affinity (pK D2 7.54). This observation is in line
with the fact that the 2,2-dimethyl-2,3-dihydro-1-benzofuran moiety
was previously used in the structure of dual acting, 5-HT1AR agonist/D2R antagonist ligands.[31] However, the affinity of 55 for the D2 receptor
did not significantly affect selectivity because its affinity for
the 5-HT1A receptor was still over 3.5 orders of magnitude
higher (>3000×, pK 5-HT1A = 11.07).Summing up, most of the presented compounds showed substantial
selectivity versus key antitargets (Ki ratio over 1000-fold) although the phenoxyethanamine scaffold is
common also for ligands of other monoaminergic receptors. This supports
the finding that the 1-[4-(aminomethyl)-4-fluoropiperidin-1-yl]ethan-1-one
core is the essential scaffold for providing both high affinity and
high selectivity for the 5-HT1A receptor.
Structure-Functional Activity Relationships
Based on
the results of the studies described above, 25 compounds were selected
for functional studies. The functional profiles of the novel compounds
were measured at several pathways engaged in 5-HT1A receptor
signal transduction. Compounds were tested in four functional assays:
ERK1/2 phosphorylation (pERK1/2), adenylyl cyclase inhibition (cAMP),
β-arrestin recruitment (β-arrestin), and calcium mobilization
(Ca2+). To classify the agonist efficacy of the compounds,
we assumed that Emax values higher than
80% relative to the maximal effect of serotonin are characteristic
of a full agonist, between 79 and 21% of a partial agonist, and 20%
or less, indicating negligible agonist activity. The experiments were
carried out using cell lines expressing the recombinant human5-HT1A receptor.In terms of potency, the general trends
(Tables and 4) were similar to those established in affinity
studies. Compared to 3, the derivatives substituted with
the HBD in the meta position (43, 45, 47, 48) were characterized by a rise in potency
in all the signaling pathways. The same trend was observed for the
bicyclic analogues (51, 55, 56, 57), but the other derivatives showed mostly decreased
potency in functional assays. The notable exceptions were compound 36 and 38, the ortho- and meta-methoxy analogues, respectively, which were characterized
by generally higher potency than 3 as well as compound 42, an ortho-carboxamido analogue, which
displayed potency similar to 3, with modest variations
in both sides. The extent of potency change varied between individual
analogues in terms of signaling pathways, resulting in diversified
functional selectivity profiles for some of them.
Table 3
Functional Activity of Compounds 28–33, 35, 36, 38–41 at 5-HT1A Receptors
All the functional activity values
were expressed as means from at least three experiments performed
in duplicate, unless otherwise indicated. For the sake of clarity,
the SEM values were omitted in this table and are presented in the Supporting Information—Table S1; the functional
assay was performed using
CHO-K1 cells.
U2OS cells (Tango LiveBLAzer assay
kit).
NT—not tested; * value was
expressed as mean from two experiments performed in duplicate.
Data for Serotonin on ERK, cAMP,
and β-arrestin are reproduced from the previous paper.[13]
Table 4
Functional Activity of Compounds 42–51 and 55–57 at 5-HT1A Receptors
All the functional activity values
were expressed as means from at least three experiments performed
in duplicate, unless otherwise indicated. For the sake of clarity,
the SEM values were omitted in this table and are presented in the Supporting Information—Table S1; the functional
assay was performed using
CHO-K1 cells.
U2OS cells (Tango LiveBLAzer assay
kit); * value was expressed as mean from two experiments performed
in duplicate.
All the functional activity values
were expressed as means from at least three experiments performed
in duplicate, unless otherwise indicated. For the sake of clarity,
the SEM values were omitted in this table and are presented in the Supporting Information—Table S1; the functional
assay was performed usingCHO-K1 cells.U2OS cells (Tango LiveBLAzer assay
kit).NT—not tested; * value was
expressed as mean from two experiments performed in duplicate.Data for Serotonin on ERK, cAMP,
and β-arrestin are reproduced from the previous paper.[13]All the functional activity values
were expressed as means from at least three experiments performed
in duplicate, unless otherwise indicated. For the sake of clarity,
the SEM values were omitted in this table and are presented in the Supporting Information—Table S1; the functional
assay was performed usingCHO-K1 cells.U2OS cells (Tango LiveBLAzer assay
kit); * value was expressed as mean from two experiments performed
in duplicate.The efficacy of the ligands for the ERK1/2, cAMP, and β-arrestin
pathways was generally high, falling slightly below 80% in only a
few cases. The vast majority of the compounds can therefore be considered
as full agonists in those signaling pathways. On the other hand, most
of the compounds showed lower efficacies in the calcium mobilization
assay. Thirteen compounds were classified as partial agonists and
two even as negligibly active, as they showed marginal level of stimulation
(15%). Both these compounds were para-substituted analogues (35 and 40).
Bias Factors
The functional selectivity of the ligands
was analyzed by calculating bias factors. These compare the efficacy
and potency of compounds for pairs of signaling pathways using the
following equation[32−35]The calculations in the present study
follow the same approach as in our previous study.[13] Briefly, the bias factor provides a measure that integrates Emax and EC50 values of both a test
ligand and a reference compound (i.e. serotonin).
Results are presented in Tables and 6 and compounds which displayed
that a significant bias (over 1 log) was highlighted in green (for
positive values) or in blue (for negative values). Those compounds
which showed significant bias, but with low pEC50 values,
were marked in gray.
Table 5
Bias Factors of Compounds (28–33, 35, 36, 38–41) and
References at 5-HT1A Receptors
No data for pERK assay. Compounds
that displayed a significant bias (over 1 log) are highlighted in
green (for positive values) or in blue (for negative values). Those
compounds that showed significant bias but with low pEC50 values are marked in gray.
Table 6
Bias Factors of Compounds 42–51 and 55–57 at 5-HT1A Receptors
Compounds that displayed a significant bias (over
1 log) are highlighted in green (for positive values) or in blue (for
negative values).
No data for pERK assay. Compounds
that displayed a significant bias (over 1 log) are highlighted in
green (for positive values) or in blue (for negative values). Those
compounds that showed significant bias but with low pEC50 values are marked in gray.Compounds that displayed a significant bias (over
1 log) are highlighted in green (for positive values) or in blue (for
negative values).
ERK1/2 versus cAMP
Most of the compounds showed a preference
for ERK1/2 phosphorylation, with the highest bias factors (>1 log)
being found for compounds 40, 44, and 55. The highest ERK1/2 phosphorylation preference was found
for compound 55 with a bias factor of 2.8 log. Three
compounds (47, 48, and 51),
preferred the cAMP pathway and significant bias was observed for compound 47 (bias factor −1.03).When comparing 3-chloro-4-fluorobenzoyl
derivatives (29, 38, 40, 47) with their 3,4-dichlorobenzoyl derivative counterparts
(30, 39, 41, 48), it is noticeable that the former always show a more pronounced
biased profile than the latter compounds.
ERK1/2 versus β-Arrestin
Three compounds preferred
ERK1/2 phosphorylation versus β-arrestin, and four compounds
preferred β-arrestin recruitment versus ERK1/2 phosphorylation.
This is the first time, to our knowledge, that β-arrestin-biased
agonists have been reported for the selective 5-HT1A receptor
ligands. Bias factors for the ERK1/2-biased agonists ranged from 1.21
for compound 41 to 1.43 for compound 44,
whereas in the case of β-arrestin-biased agonists, their bias
factors were much more pronounced (from −1.95 for 48 to −3.71 log for 56).The ERK1/2-preferring
compounds were the para-methoxy derivatives (40 and 41) and the ortho-methylamine
derivative 44. On the other hand, the compounds that
showed bias for β-arrestin recruitment were either the bicyclic
aromatic derivatives (51 and 56) or meta-acetamido derivatives (47 and 48).It is noticeable that the ortho-methylamine-substituted
derivative (44) showed substantial ERK1/2 bias (1.43),
while the meta-methylamine-substituted derivative
(45) showed an opposite preference (bias factor −0.60).
ERK1/2 versus Ca2+
In general, most of the
compounds showed substantial bias for ERK1/2 phosphorylation versus
calcium mobilization (Ca2+) and none of the tested compounds
showed preference for Ca2+. The highest ERK1/2 preference
was found for the benzo-fused, five-membered ring derivatives (55–57), reaching a bias factor of 3.42 for compound 55.
cAMP versus β-Arrestin
Only five compounds (30, 33, 39, 41, 44) exhibited some preference toward cAMP inhibition, however
not exceeding half a log, whereas the rest of the compounds preferred
β-arrestin recruitment. As seen for ERK1/2 versus cAMP bias,
a favorable influence of the 3,4-dichlorobenzoyl moiety on cAMP potency
was observed here, as compared to the corresponding 3-chloro-4-fluorobenzoyl
analogues.Among the derivatives with marked β-arrestin
recruitment bias, compounds 47, 48, 51, and 56 were identified again, as in the case
of preference for β-arrestin versus ERK1/2. Interestingly, compound 55 also preferred β-arrestin pathway versus cAMP, although
previously it exhibited the highest preference for ERK1/2 versus β-arrestin.
This is due to its very high potency in the ERK1/2 assay (pEC50 10.99) and also relatively high β-arrestin potency
(pEC50 9.49), as compared to other assays, where its potency
was noticeably weaker.Overall, it should be noted that preference toward β-arrestin
recruitment was much higher than for the most biased reference compound,
(±) 8-OH-DPAT (−1.18), and reached an extremely high value
(−4.24) for compound 56.Previous studies by Stroth and co-workers are worth mentioning,
where the authors identified 5-HT1A-biased ligands with
a strong preference for cAMP over β-arrestin signaling.[36] However, it should be noted that those arylpiperazine
derivatives had only partial agonist properties in the cAMP assay
(53–73%). They also showed very low Emax values in the β-arrestin assay (6–36%). Noteworthily,
Stroth et al. reported that the reference agonist
(±) 8-OH-DPAT achieved only 44% efficacy in the β-arrestin
assay, while herein it reached 101%, so the observed differences in
signaling bias may be at least partially due to the methodological
differences (β-arrestin assay in that study was performed using
PathHunter eXpress HTR1ACHO-K1 β-Arrestin GPCR Assay DiscoveRx
and in the current study using Tango HTR1A-bla U2OS LiveBLAzer assay
kit, Life Technologies).
cAMP versus Ca2+
Eleven compounds markedly
preferred the cAMP pathway versus Ca2+. Noteworthily, the
most biased compounds (with bias factors over 2) had HBD in the meta
position (45, 47, 48, 56, and 57), further indicating the positive
influence of this substituent on cAMP inhibition potency. Because
of relatively low potency of all the compounds in the calcium mobilization
assay, none of them exhibited bias toward this signaling pathway.
β-Arrestin versus Ca2+
Thirteen compounds
were markedly biased for β-arrestin. Four of the compounds (47, 48, 51, and 56)
showed extremely high bias for β-arrestin (over 3.5 log), reaching
a 6.25 log value (over 1,000,000 times) for compound 56. The relatively lower ability of the compounds to stimulate Ca2+ mobilization resulted in a lack of noticeable Ca2+-preferring biases.
“Signaling Fingerprint” Analysis
The
functional studies enabled selection of biased agonists that exhibit
preference for specific pathways. To describe the pattern of behavior
of the compounds in the different pathways, we calculated “signaling
fingerprints” based on measures of potency and efficacy and
represented them as bars of particular height and color intensity
(heat map), respectively. The potency of each ligand in a particular
assay was normalized according to the performance of the native neurotransmitter
(i.e., serotonin) in this assay. It was calculated
using the following equation:A “signaling fingerprint”
therefore allows for the simultaneous comparison of a ligand’s
functional profile in all pathways. “Signaling fingerprints”
were calculated for both the reference and the novel compounds, including
all four tested pathways, with serotonin as the native ligand and
cAMP as the reference pathway (due to a relatively higher potency
of serotonin in this assay). Significant preference of a given pathway
was defined in this study as a difference in normalized ligand potency
of at least 1 order of magnitude (1 log).Among the reference compounds (Figure ), the most biased was compound 1, showing significant preference for ERK1/2 phosphorylation over
all other assays, which is in line with previous studies.[12,13] On the other hand, (±) 8-OH-DPAT displayed a 1 log preference
for the β-arrestin versus cAMP pathway but was unbiased with
respect to other pathways. Buspirone, consistent with its partial
agonist properties, showed low efficacy in all assays but β-arrestin,
which was particularly evident for calcium mobilization (Emax 8.3%); the potencies, however, did not differ significantly.
Figure 6
“Signaling fingerprints” for reference compounds
(bar height—normalized ligand potency in log scale, bar color—ligand
efficacy, as percent Emax).
“Signaling fingerprints” for reference compounds
(bar height—normalized ligand potency in log scale, bar color—ligand
efficacy, as percent Emax).The “signaling fingerprints” for the most interesting
novel compounds, in comparison with the lead structures 2 and 3, are shown in Figure . In the rows, the analogues with structurally
closest substituents were collected to show the gradual impact of
their modification on changes in the functional profile. The pERK1/2-preferring
analogues are shown in the left column, the more balanced in the middle,
and the β-arrestin-biased agonists in the right column.
Figure 7
“Signaling fingerprints” for the novel compounds
(bar height—normalized ligand potency in log scale, bar color—ligand
efficacy, as percent Emax). The signaling
fingerprints for 2 and 3 are shown for comparison
with our previous work.[13]
“Signaling fingerprints” for the novel compounds
(bar height—normalized ligand potency in log scale, bar color—ligand
efficacy, as percent Emax). The signaling
fingerprints for 2 and 3 are shown for comparison
with our previous work.[13]Based on the more detailed analysis of the “signaling fingerprints”,
the novel 5-HT1A receptor agonists can be categorized into
three types, divided into five subtypes, each with a different functional
selectivity profile. Type I includes ligands with a significant preference
for ERK1/2 phosphorylation and a diverse profile of activity in other
assays, which is mainly differentiated by the level of activation
of β-arrestin recruitment. Type IA, including compounds 44 and 2, consists of ligands which showed significant
preference for ERK1/2 phosphorylation over all other pathways, similar
to the reference biased agonist, 1. Compounds 3 and 55, which were classified into type IB, were characterized
by a significant preference for ERK1/2 phosphorylation over cAMP and
Ca2+, but not β-arrestin. Type II includes compounds 45 and 57, which show a similar level of activity
in ERK1/2, cAMP, and β-arrestin assays, with a slight preference
for the latter.In contrast to types I and II, and of particular interest in the
present study, are type III compounds: these include first-in-class
ligands that strongly prefer β-arrestin recruitment over all
other signaling pathways. It is noteworthy that such a profile was
not observed for any of the reference compounds, and, to our knowledge,
has not been previously described in the literature, which could imply
that these compounds may exhibit novel pharmacological and, potentially,
therapeutic properties. Type IIIA (compounds 51 and 56) includes ligands characterized by the strongest preference
of the β-arrestin pathway, similar levels of activity in ERK1/2
and cAMP assays, and much lower stimulation of Ca2+. Type
IIIB, represented by compound 47, is characterized by
the high levels of activity in both β-arrestin and cAMP assays
(with especially marked activity of β-arrestin pathway) and
lower ability to activate ERK1/2 phosphorylation and calcium mobilization.
Noteworthily, compound 47 was the only one that showed
significant preference for activity (above 1 log) in the cAMP assay
over ERK1/2 phosphorylation.Summing up, the following structure functional selectivity relationships
could be inferred:the presence of an H-bond-forming
substituent in the ortho position of the phenoxyethyl
moiety (44, 55) or a nitrogen atom built
in the aryl ring in the same position (2) decreases the
ability to activate β-arrestin recruitment in the tested group
of 5-HT1A receptor agonists, thus relatively enhancing
a preference for ERK1/2 phosphorylation.substitution of the HBD moiety in
the meta position of the phenoxyethyl moiety (45, 57) increases agonist potency in all signaling pathways, with
the effect being especially pronounced for cAMP inhibition and β-arrestin
recruitment. Except for calcium mobilization being substantially weaker,
those potent agonists do not distinguish significantly between other
pathways.in contrast, the derivatives with
a bicyclic aromatic moiety (56 and 51) or
a flat, π-electron-containing substituent (e.g.47) exhibited particular preference for β-arrestin
recruitment, yielding very strong activity in this assay. Noteworthily,
replacement of an aromatic indole moiety of 56 with a
partially saturated indoline (57) markedly decreased
β-arrestin recruitment, resulting in no particular preference
over ERK1/2 phosphorylation or cAMP inhibition, thus confirming this
finding. It should be noted that, to our knowledge, these compounds
are the first 5-HT1A ligands to show such a strong biased
agonism for β-arrestin recruitment and this, in itself, constitutes
an intriguing novel finding in drug discovery at this receptor.More broadly, these in vitro data strongly indicate
that it is possible to identify specific structural motifs that are
responsible for directing 5-HT1A receptor signaling to
distinct intracellular responses.
Developability Studies
The developability of the novel
compounds was initially assessed in silico using
LELP, Fsp3, and CNS-MPO measures (Table S2). Majority of the compounds showed favorable score values, thus
testifying to the overall promising developability potential of the
explored series. In order to support the choice of proper candidates
for in vivo tests, selected in vitro developability studies were performed. As a first step, the novel
compounds displaying the most interesting functional profiles were
tested for preliminary metabolic stability using RLMs (Table S3). The stability was assessed referring
to the marketed drugs of different stabilities, aripiprazole and verapamil,
showing high or low stability in the given conditions, respectively.
Various levels of stability were found for the novel compounds, ranging
from high stability for compounds 47, 48, 51, and 56 (73–87%), through medium
stability for compounds 2, 3, and 44 (54–59%), to low stability for compounds 55 and 57 (21 and 37%). Based on the functional studies
and the above results, compound 56, a β-arrestin
recruitment-biased agonist with high metabolic stability, and compound 44, an ERK1/2 phosphorylation-preferring ligand with medium
metabolic stability, were selected for further studies. To confirm
preliminary metabolic stability data, for the lead structure 3 as well as compounds 44 and 56, intrinsic clearance was determined in comparison with the reference
CNS drugs aripiprazole and diazepam (Table ). As expected, compound 44 and
the lead structure 3 showed the same level of medium
metabolic stability (CLint 48.8 and 41.7 mL/min/kg, respectively),
similar to diazepam, a reference CNS drug with medium but acceptable
stability, whereas compound 56 was more stable, with
intrinsic clearance close to aripiprazole, a reference CNS drug showing
a very high stability in this experimental setting (CLint 9.6 and 7.2 mL/min/kg, respectively).As a next step, compounds 44 and 56 were tested for membrane permeability
using PAMPA and for potential hepatotoxicity using HepG2 cell viability
(Table ). Both compounds, similar to the lead structure 3, showed satisfying permeability (>1 × 10–6 cm/s), suggesting good absorption and brain penetration as well
as very low hepatotoxicity, not reaching statistically significant
reduction of viability even in a concentration as high as 50 μM.Finally, compounds 44 and 56 were tested
for selectivity against a broad group of 45 off-targets, including
those structurally and evolutionally closest to the 5-HT1A receptor as well as the most troublesome for drug development (e.g., hERG channel, Chart , Supporting Information Tables S4 and S5). In most cases, the affinity for the off-targets
proved to be in the micromolar range (<50% binding in 1 ×
10–6). For some of the targets, binding was stronger,
but considering the very high affinity of the tested compounds for
the 5-HT1A receptor, the estimated selectivity was still
over 3 orders of magnitude (>1000×), even relatively higher than
for the lead structure 3. Interestingly, compound 56, which displayed relatively highest affinity for some of
the off-targets (reaching pK ∼ 8), proved to be also relatively the most selective
(>10,000×) because of its extremely high affinity for the 5-HT1A receptor (pK = 12.80). Based on all the data mentioned above, compounds 44 and 56 were ultimately selected for in vivo studies.
In Vivo Studies
So far, there is only
sparse information connecting particular signaling transduction pathways
with physiological effects. Evidence indicates that increased cortical
ERK1/2 phosphorylation is associated with antidepressant activity,[37,38] whereas inhibition of cAMP production by hippocampal 5-HT1A receptors may interfere with memory process.[39,40] On the other hand, it is currently not known what physiological
effects are associated with activation of β-arrestin recruitment
mediated by 5-HT1A receptors. Nevertheless, it is very
important for drug discovery to establish a link between particular
functional profiles and the desired pharmacological effects.[41,42] Therefore, two compounds with significantly differing in
vitro functional pERK1/2 versus β-arrestin selectivity
profiles were compared in various in vivo measures
relevant to 5-HT1A receptor agonism. Compound 44 has a pERK1/2 versus β-arrestin bias factor of 1.43 (i.e., its EC50 is almost 30-fold lower for ERK1/2
phosphorylation than for β-arrestin recruitment), while compound 56 has a bias factor of −3.49, translating to over
3000-fold greater potency for β-arrestin recruitment than for
pERK1/2. Interestingly, while both compounds displayed similar effectiveness
in the Porsolt forced swimming test (FST) for antidepressant activity,
with 44 being slightly more potent (minimal effective
dose MED = 0.16 mg/kg p.o. for 44vs 0.63 mg/kg p.o. for 56), they differed significantly
in their ability to induce lower lip retraction (LLR). LLR is an autonomic
response, a component of the rat “serotonergic syndrome”,
attributed to 5-HT1A receptor activation.[43] Compound 44 did not induce any significant
LLR, even up to a dose 4× higher than the MED for antidepressant
activity, while compound 56 elicited a full LLR in a
dose 2× lower than the MED in Porsolt test (Chart , Supporting Information Tables S6 and S8).
Chart 2
Differential Profiles in the FST (in Blue) and LLR (in Red) of Compounds 44 (A) and 56 (B), the 5-HT1AR-Biased
Agonists with Contrasting Functional Selectivity Signaling Fingerprints
(Preferential for pERK1/2 and β-Arrestin, Respectively)a
****p < 0.0001,
*****p < 0.00001.Noteworthily, at the time point that the in vivo effects were observed, we verified that there was a detectable exposure
of the tested compounds in both serum and brain (Supporting Information Chart S1, Table S10). Moreover, the
abovementioned pharmacological effects were reversed by the selective
5-HT1A receptor antagonist WAY100635, thus testifying for
their full 5-HT1A receptor dependence (Supporting Information Tables S7 and S9).The significance of the LLR effect for human condition is so far
unknown. However, it is undoubtedly an autonomic side effect, not
connected with the antidepressant-like response in rat, and has previously
been considered to be inseparable from the desired therapeutic-like
effects resulting from the 5-HT1A receptor activation.[44] Interestingly, antidepressant-like activity
in the FST is mediated by activation of the cortical postsynaptic
subpopulation of 5-HT1A receptors, while induction of LLR
is thought to be mediated by presynaptic 5-HT1A autoreceptors
localized in the Raphe nuclei.[45,46] In the case of 5-HT1A receptors, the contrasting roles of pre- and postsynaptic
receptors in different brain regions have been extensively investigated,
also in the context of therapeutic effectiveness.[16] The diverse pharmacological profiles of 44 and 56 are therefore of considerable interest because
they suggest that different preferences for β-arrestin recruitment
relative to ERK1/2 phosphorylation (functional selectivity at the
cellular level) may be associated with preferential activation of
particular subpopulations of 5-HT1A receptors (brain region
selectivity) and thus lead to separate therapeutic and side effects.
It should also be considered that such a high level of β-arrestin-biased
agonism, reported for the first time in the present study, could open
the way to novel opportunities for targeting 5-HT1A receptors,
with previously unexplored physiological or behavioral outcomes. However,
these are preliminary suggestions that need to be carefully and thoroughly
evaluated using more numerous biased agonists and diversified technical
approaches. Although these observations are promising and warrant
further investigation, formal demonstration of superior therapeutic
activity by biased agonists ultimately requires appropriately designed
clinical trials and a clear understanding linking in vitro biased agonism to disease mechanisms. Nevertheless, the present
work provides compelling evidence that chemical modifications of 5-HT1A receptor-biased agonists allow for their functional diversification,
which in turn translates to distinct pharmacological effects in vivo.
Conclusions
The present work describes the SARs and SFARs of 5-HT1A receptor agonists and proves that novel and highly selective biased
agonists can be designed to exhibit distinct and innovative signaling
profiles. Thus, a series of 30 novel derivatives of 1-(1-benzoylpiperidin-4-yl)methanamine
was synthesized and found to exhibit high affinity for the 5-HT1A receptor (pK > 8.0, LLE > 5.0). Twenty-seven of these had subnanomolar affinities
(pK > 9.0, LLE > 6)
and 15 compounds possessed higher lipophilic-ligand efficiencies than
the lead compound 3. Noteworthily, compound 56 was found to be extremely potent, one of the highest affinity 5-HT1A receptor ligands discovered to date (based on the ChEMBL
database). Moreover, most of the presented compounds showed substantial
selectivity versus key antitargets—the adrenergic α1 and dopaminergic D2 receptors (Ki ratio over 1000-fold). Twenty-five compounds were selected
and tested in four functional assays connected with the 5-HT1A receptor activation, that is, ERK1/2 phosphorylation (pERK1/2),
adenylyl cyclase inhibition (cAMP), calcium mobilization (Ca2+), and β-arrestin recruitment. Based on analysis of SFARs and
of bias factors, nine novel 5-HT1A receptor-biased agonists
were identified that exhibit diversified functional activity profiles
(i.e., “signaling fingerprints”). The
selected, most interesting biased agonists 44 and 56 displayed high selectivity versus a panel of 45 off-target
sites as well as promising metabolic stability, high permeability,
and low hepatotoxicity, thus testifying for their favorable developability
profiles. Strikingly, whereas 44 exhibited marked biased
agonism for activation of pERK1/2, 56 exhibited the opposite
profile, with very potent biased agonism for β-arrestin recruitment.
The profile of 56 is, to our knowledge, unprecedented
and could constitute a novel class of 5-HT1A receptor-biased
agonists, an interpretation reinforced by the differential in vivo activity of the two compounds in tests of antidepressant-like
activity (FST) and behavioral syndrome (LLR). 44 preferentially
elicited antidepressant-like effects, whereas 56 more
potently elicited LLR, thus suggesting that the balance of β-arrestin
recruitment relative to ERK1/2 phosphorylation (functional selectivity)
may be associated with accentuated activity in specific physiological
and/or behavioral models. As discussed previously, such differences
likely reflect activation of particular subpopulations of the 5-HT1A receptors (regional selectivity) and may account for differential
separation of therapeutic and side effects.[16,47,48] The novel 5-HT1A agonists described
herein, displaying diversified functional profiles, may constitute
promising tool drugs to investigate the activity of 5-HT1A receptor subpopulations and, potentially, could be developed as
pharmacotherapeutics to treat CNS disorders involving dysfunctional
serotonergic neurotransmission.
Experimental Section
Molecular Modeling
Computer-aided ligand design and
further studies on SARs were based on ligand–receptor interaction
analysis. The previously built template of the 5-HT1B crystal
structure (PDB ID 4IAR)[49] and preoptimized serotonin 5-HT1A receptor homology model served as a structural target for
docking studies.[50] To capture distinctive
binding mode of a variety of functionally biased ligands, the general
procedure for developing ligand-optimized models using induced-fit
technique[51] served as both a ligand-steered
binding site optimization method (in terms of amino acid side chains)
and a routine docking approach, predicting bioactive conformation.[13] Glide SP flexible docking procedure using an
OPLS3 force field was set for the induced-fit docking (IFD). H-bond
constraint and centroid of a grid box for docking to the 5-HT1A receptor were located on Asp3.32. Ligand structures were
sketched in Maestro 2D Sketcher and optimized using a LigPrep tool.
The aforementioned tools were implemented in Small-Molecule Drug Discovery
Suite (Schrödinger, Inc. New York, USA), which was licensed
for Jagiellonian University Medical College. Instant JChem was used
for structure database management and property prediction, Instant
JChem 20.8.0, 2020, ChemAxon (http://www.chemaxon.com).
Chemistry
General Chemistry Information
All the reagents were
purchased from commercial suppliers (Sigma-Aldrich, Merck, Chempur,
Fluorochem, Enamine, Acros Organics, Manchester Organics, POCh, Activate
Scientific, Chem-Impex International, Apollo Scientific) and were
used without further purification. Analytical thin-layer chromatography
was performed on Merck Kieselgel 60 F254 (0.25 mm) precoated
aluminum sheets (Merck, Darmstadt, Germany). Compounds were visualized
with UV light and by suitable visualization reagents 2.9% solution
of ninhydrin in a mixture of 1-propanol and acetic acid (100/3, v/v)
and Pancaldi reagent [solution of 12.0 g (NH4)6Mo7O24, 0.5 g Ce(SO4)2 and 6.8 mL of 98% H2SO4 in 240 mL of water].
Flash chromatography was performed on CombiFlash RF (Teledyne Isco)
using disposable silica gel flash columns RediSep Rf (silica gel 60,
particle size 40–63 μm) and RediSep Gold (silica gel
60, particle size 20–40 μm) purchased from Teledyne Isco.
The ultraperformance liquid chromatography (UPLC)–mass spectrometry
(MS) or UPLC–tandem mass spectrometry (MS/MS) analysis was
done on a UPLC–MS/MS system comprising a Waters ACQUITY UPLC
(Waters Corporation, Milford, MA, USA) coupled with a Waters tandem
quadrupole (TQD) mass spectrometer (electrospray ionization (ESI)
mode with TQD). Chromatographic separations were carried out using
the ACQUITY UPLC BEH (bridged ethyl hybrid) C18 column: 2.1 ×
100 mm and 1.7 μm particle size. The column was maintained at
40 °C and eluted under gradient conditions using 95–0%
of eluent A over 10 min at a flow rate of 0.3 mL/min. Eluent A: 0.1%
solution of formic acid in water (v/v); eluent B: 0.1% solution of
formic acid in acetonitrile (v/v). A total of 10 μL of each
sample was injected and chromatograms were recorded using a Waters
eλ photodiode array detector. The spectra were analyzed in the
range of 200–700 nm with 1.2 nm resolution and at a sampling
rate of 20 points/s. The UPLC–MS purity of all the test compounds
and key intermediates were determined to be >95%. 1H NMR, 13C NMR, and 19F NMR spectra were obtained in a
Varian Mercury spectrometer (Varian Inc., Palo Alto, CA, USA) and
JEOL spectrometer (JEOL SAS., Tokyo, Japan), in CDCl3,
CD3OD, or DMSO-d6 operating
at 300 MHz (1H NMR), 75 MHz or 126 MHz (13C
NMR), and 282 MHz (19F NMR). Chemical shifts are reported
as δ values (ppm) relative to TMS δ = 0 (1H)
as internal standard (IS). The J values are expressed
in Hertz (Hz). Signal multiplicities are represented by the following
abbreviations: s (singlet), br s (broad singlet), bd (broad doublet),
d (doublet), dd (doublet of doublets), dt (doublet of triplets), t
(triplet), td (triplet of doublets), tdd (triplet of doublet of doublets),
q (quartet), dq (doublet of quartets), and m (multiplet). Melting
points were determined on a Büchi Melting Point B-540 apparatus
using open glass capillaries and are uncorrected.
Synthetic Procedures
Previously reported or commercially
available compounds:2-(1-(3-Chloro-4-fluorobenzoyl)-4-fluoropiperidin-4-yl)-2-hydroxyacetonitrile
(4),[13]2-(1-(3,4-Dichlorobenzoyl)-4-fluoropiperidin-4-yl)-2-hydroxyacetonitrile
(5),[13]2-(2-Chlorophenoxy)ethanamine (9).
Detailed Procedures for the Preparation of the Amine Intermediates 6–26 Are Described in the Supporting Information.
General Procedures for the Preparation of 1-(1-Benzoylpiperidin-4-yl)methanamine
Derivatives (27–57)
To appropriate cyanohydrin
(4 or 5)[13] (1.0
equiv) dissolved in methanol, DABCO (2.0–12.5 equiv) was added
in one portion, followed by the appropriate amine (6–26) (1.0–1.6 equiv), 4 c5 molecular sieves, sodium cyanoborohydride
(1.6–7.8 equiv), and iron sulfate heptahydrate (FeSO4 × 7 H2O) (1.1 equiv). The mixture was stirred at
room temperature until the cyanohydrin was consumed (24–72
h); then, the reaction mixture was filtered, concentrated in vacuo,
and next brine was added. The resulting mixture was extracted with
EtOAc (3×), organics were combined and dried over magnesium sulfate,
filtered, and concentrated. The crude product was purified by flash
chromatography.
The title compound was prepared by the
reduction of ((4-(((2-((1H-indol-4-yl)oxy)ethyl)amino)methyl)-4-fluoropiperidin-1-yl)(3-chloro-4-fluorophenyl)methanone)
(56).To a solution of compound 56 (1.0 equiv, 0.177 g, 0.40 mmol) in acetic acid (1.0 equiv, 0.23
mL, 0.40 mmol) sodium cyanoborohydride (2.0 equiv 0.051 g, 0.80 mmol)
was added in portions at 15 °C. Then, the mixture was warmed
up to room temperature and stirred for an hour. The reaction mixture
was then cooled to 0 °C, quenched, and adjusted to pH 8 with
a saturated aqueous solution of sodium bicarbonate and extracted with
EtOAc (3×). The organic layers were combined and dried over magnesium
sulfate, filtered, and concentrated to yield the crude product that
was purified by flash chromatography in n-hexane/EtOAc/methanol/NH3(aq) (6/3/1/0.02, v/v/v/v). Yield: 67%; pale pink transparent
oil. 1H NMR (300 MHz, CDCl3, δ): 7.48
(dd, J = 2.3, 7.0 Hz, 1H), 7.35–7.26 (m, 1H),
7.23–7.11 (m, 1H), 6.97 (t, J = 7.9 Hz, 1H),
6.29 (dd, J = 7.9, 14.4 Hz, 2H), 4.51 (br s, 1H),
4.08 (t, J = 5.0 Hz, 2H), 3.56 (t, J = 8.5 Hz, 3H), 3.47–3.08 (m, 2H), 3.06–2.93 (m, 4H),
2.91–2.76 (m, 2H), 2.02 (br s, 2H), 1.85–1.44 (m, 2H),
NH protons not detected. 19F NMR (282
MHz, CDCl3, δ): −112.6 (s, 1F), −166.7
(s, 1F). 13C NMR (75 MHz, CDCl3, δ): 168.0,
158.8 (d, J = 254 Hz), 155.6, 153.5, 132.9 (d, J = 3.5 Hz), 129.7, 128.6, 127.1 (d, J =
6.9 Hz), 121.5 (d, J = 18 Hz), 116.8 (d, J = 22 Hz), 116.0, 103.3, 102.6, 94.4 (d, J = 172 Hz), 67.2, 57.2 (d, J = 22 Hz), 49.3, 47.4,
43.5, 38.2, 33.5, 32.7, 26.9. Formula: C23H26ClF2N3O2; MS (ESI+) m/z: 450 [M + H+].
In Vitro Studies
The tested compounds
were examined for known classes of assay interference compounds. None
of the compounds contain substructural features recognized as pan
assay interference compounds (PAINS), according to the SwissADME tool.[52]
Radioligand Binding Assays for 5-HT1AR, α1R, D2R
Preparation of Solutions of Test and Reference Compounds
Stock solutions (1 mM) of tested compounds were prepared in DMSO.
Serial dilutions of compounds were prepared in a 96-well microplate
in assay buffers using automated pipetting system epMotion 5070 (Eppendorf).
The final concentration of DMSO in the test solutions was 0.1%. Each
compound was tested in 10 concentrations from 1.0 × 10–6 to 1.0 × 10–12 M (final concentration).
Serotonin 5-HT1A Receptor Binding Assay
Radioligand binding was performed using membranes from CHO-K1 cells
stably transfected with the human5-HT1A receptor (PerkinElmer).
All assays were carried out in duplicate. A working solution (50 μL)
of the tested compounds, 50 μL of [3H]-8-OH-DPAT
(final concentration 1 nM), and 150 μL of diluted membranes
(10 μg protein per well) prepared in assay buffer (50 mM Tris,
pH 7.4, 10 mM MgSO4, 0, 5 mM EDTA, 0.1% ascorbic acid)
were transferred to a polypropylene 96-well microplate using a 96-well
pipetting station Rainin Liquidator (MettlerToledo). Serotonin (10
μM) was used to define nonspecific binding. The microplate was
covered with a sealing tape, mixed, and incubated for 60 min at 27
°C. The reaction was terminated by rapid filtration through a
GF/C filter mate presoaked with 0.3% polyethyleneimine for 30 min.
Ten rapid washes with 200 μL 50 mM Tris buffer (4 °C, pH
7.4) were performed using an automated harvester system Harvester-96
MACH III FM (Tomtec). The filter mates were dried at 37 °C in
forced air fan incubator and then solid scintillator MeltiLex was
melted on filter mates at 90 °C for 4 min. Radioactivity was
counted in MicroBeta2 scintillation counter (PerkinElmer). Data were
fitted to a one-site curve-fitting equation with Prism 6 (GraphPad
Software) and Ki values were estimated
from the Cheng–Prusoff equation.
Adrenergic α1 Receptor Binding Assay
Radioligand binding was performed using rat cortex. Tissue was homogenized
in 20 volumes of ice-cold 50 mM Tris-HCl buffer, pH 7.6, using an
Ultra Turrax T25B (IKA) homogenizer. The homogenate was centrifuged
at 20,000g for 20 min. The resulting supernatant
was decanted and the pellet was resuspended in the same buffer and
centrifuged again in the same conditions. The final pellet was resuspended
in an appropriate volume of buffer (10 mg/1 mL). All assays were carried
out in duplicate. The working solution (50 μL) of the tested
compounds, 50 μL of [3H]-prazosin (final concentration
0.2 nM), and 150 μL of tissue suspension were transferred to
a polypropylene 96-well microplate using a 96-well pipetting station
Rainin Liquidator (MettlerToledo). Phentolamine (10 μM) was
used to define nonspecific binding. The microplate was covered with
a sealing tape and the content mixed and incubated for 30 min at 30
°C. The incubation was terminated by rapid filtration over glass
fiber filters FilterMate B (PerkinElmer, USA) using a 96-well FilterMate
harvester (PerkinElmer, USA). Five rapid washes were performed with
ice-cold 50 mM Tris-HCl buffer, pH 7.6. The filter mates were dried
at 37 °C in a forced air fan incubator and then solid scintillator
MeltiLex was melted on filter mates at 90 °C for 5 min. Radioactivity
was counted in a MicroBeta2 scintillation counter (PerkinElmer). Data
were fitted to a one-site curve-fitting equation with Prism 6 (GraphPad
Software) and Ki values were estimated from the Cheng–Prusoff
equation.
Dopamine D2 Receptor Binding Assay
Radioligand
binding was performed using membranes from CHO-K1 cells stably transfected
with the human D2 receptor (PerkinElmer). All assays were
carried out in duplicate. A working solution (50 μL) of the
tested compounds, 50 μL of [3H]-methylspiperone (final
concentration 0.4 nM), and 150 μL of diluted membranes (3 μg
protein per well) prepared in assay buffer (50 mM Tris, pH 7.4, 50
mM HEPES, 50 mM NaCl, 5 mM MgCl2, 0.5 mM EDTA) were transferred
to a polypropylene 96-well microplate using a 96-well pipetting station
Rainin Liquidator (MettlerToledo). Haloperidol (10 μM) was used
to define nonspecific binding. The microplate was covered with a sealing
tape, and the mixture was mixed and incubated for 60 min at 37 °C.
The reaction was terminated by rapid filtration through a GF/B filter
mate presoaked with 0.5% polyethyleneimine for 30 min. Ten rapid washes
with 200 μL of 50 mM Tris buffer (4 °C, pH 7.4) were performed
using an automated harvester system Harvester-96 MACH III FM (Tomtec).
The filter mates were dried at 37 °C in a forced air fan incubator
and then solid scintillator MeltiLex was melted on filter mates at
90 °C for 5 min. Radioactivity was counted in a MicroBeta2 scintillation
counter (PerkinElmer). Data were fitted to a one-site curve-fitting
equation with Prism 6 (GraphPad Software) and Ki values were estimated
from the Cheng–Prusoff equation.
Functional Assays for the 5-HT1A Receptor
ERK1/2 Phosphorylation
Test and reference compounds
were dissolved in dimethyl sulfoxide (DMSO) at a concentration of
10 mM. Serial dilutions were prepared in a 96-well microplate in HBSS
with 0.1% BSA added and eight concentrations were tested. The final
concentration of DMSO in the test solutions was 0.1%.The CHO-5HT1A receptor cells were tested for agonist-induced ERK1/2-phosphorylation
using the SureFire ERK1/2-phosphorylation α LISA assay kit according
to the manufacturer’s instruction (Perkin Elmer). After thawing,
cells were cultured in medium [advanced DMEM/F12 with 1% fetal bovine
serum (FBS) dialyzed, 400 μg/mL G-418, 4 mM l-glutamine].
At the experiment, cells were plated at 50,000 cells/well of a 96-well
tissue culture plate and grown 7 h in an incubator (5% CO2, 37 °C); after this time, the cells were starving (DMEM/F12
with 0, 1% BSA (immunoglobulin- and protease-free) for 12 h. The serial
dilutions of compounds were added and incubated for 15 min in 37 °C.
The medium was removed, “lysis buffer” (70 μL)
was added and the plate gently agitated on a plate shaker (10 min).
The plates were frozen at −80 °C. The next day, the plate
was thawed on plate shaker for 10 min and 10 μL was transferred
to assay plates (384-OptiPlate, Perkin Elmer) in duplicate and 10 μL
of the reaction mix α LISA SureFire Ultra assay (Perkin Elmer)
was added. The plate was incubated for 2 h at 22 °C. After incubation,
the assay plate was measured in an EnVision multifunction plate reader
(PerkinElmer Life Science). Emax values
were defined as the response of the ligand expressed as a percentage
of the maximal response elicited by serotonin, determined by nonlinear
regression using GraphPad Prism 6.0 software. pEC50 values
correspond to the ligand concentration at which 50% of its own maximal
response was measured.
cAMP Inhibition
Tested and reference compounds were
dissolved in DMSO to the concentration of 10 mM. Dilutions were prepared
in a 96-well microplate in assay buffers. For 5-HT1A receptors,
adenylyl cyclase activity was determined using cryopreserved CHO-K1
cells with expression of the humanserotonin 5-HT1A receptor.
The final concentration of DMSO in the test solutions was 0.1%.The functional assay was performed with the CHO-K1 cells with expression
of the 5-HT1Ahumanserotonin receptor in which plasmid
containing the coding sequence was transfected. The cells were cultured
under selective conditions (400 μg/mL GeneticinG418) (PerkinElmer).
Thawed cells were resuspended in stimulation buffer (HBSS, 5 mM HEPES,
0.5 IBMX, and 0.1% BSA at pH 7.4) at 2 × 105 cells/mL.
The same volume (10 μL) of cell suspension was added to tested
compounds with 10 μM forskolin. Samples were loaded onto a white
opaque half-area 96-well microplate. Cell stimulation was performed
for 40 min at room temperature. After incubation, cAMP measurements
were performed with homogeneous time-resolved fluorescence resonance
energy transfer (TR-FRET) immunoassay using the LANCE Ultra cAMP kit
(PerkinElmer, USA). Ten microliters of EucAMP Tracer Working Solution
and 10 μL of ULight-anti-cAMP Tracer Working Solution were added,
mixed, and incubated for 1 h. The TR-FRET signal was read on an EnVision
microplate reader (PerkinElmer, USA). Emax values were defined as the response of the ligand expressed as a
percentage of the maximal response elicited by serotonin, determined
by nonlinear regression using GraphPad Prism 6.0 software. pEC50 values correspond to the ligand concentration at which 50%
of its own maximal response was measured.
β-Arrestin Recruitment
Test and reference compounds
were dissolved in DMSO at a concentration of 10 mM. Serial dilutions
were prepared in 96-well microplate in Dulbecco’s modified
Eagle’s medium (DMEM) with 10% FBS added and eight concentrations
were tested. The final concentration of DMSO in the test solutions
was 0.1%.The HTR1A-bla U2OS receptor cells containing the humanSerotonin Type 1A receptor linked to a TEV protease site and a Gal4-VP16
transcription factor were tested for agonist induced using the Tango
LiveBLAzer assay kit according to the manufacturer’s instruction
(Life Technologies). After thawing, cells were cultured in medium
(McCoy’s 5A with 10% FBS dialyzed, 0.1 mM NEAA, 25 mM HEPES,
1 mM sodium pyruvate, 100 μg/mL G-418, 100 U/mL penicillin/streptomycin
antibiotic, 200 μg/mL zeocin, 50 μg/mL hygromycin). At
the experiment, cells were plated at 10,000 cells/well of a 384-well
black, clear bottom, tissue culture plate and grown for 12 h in an
incubator (5% CO2, 37 °C) in DMEM with 10% FBS added.
The serial dilutions of compounds were added and incubated for 5 h
(5% CO2, 37 °C). After this time, 8 μL of reaction
mix was added. The plate was incubated for 2 h at 22 °C. After
incubation, the assay plate was measured in a FLUOstar Optima multifunction
plate reader (PerkinElmer Life Science). Emax values were defined as the response of the ligand expressed as a
percentage of the maximal response elicited by serotonin, determined
by nonlinear regression using GraphPad Prism 6.0 software. pEC50 values correspond to the ligand concentration at which 50%
of its own maximal response was measured.
Calcium Mobilization Assay
Test and reference compounds
were dissolved in DMSO at a concentration of 10 mM. Serial dilutions
were prepared in a 96-well microplate in assay buffer and 8–10
concentrations were tested. The final concentration of DMSO in the
test solutions was 0.1%.A cellular aequorin-based functional
assay was performed with recombinant CHO-K1 cells expressing mitochondrially
targeted aequorin, humanGPCR and the promiscuous G protein α16 for 5-HT1A receptor. Assay was executed according
to a previously described protocol. After thawing, cells were transferred
to assay buffer (DMEM/HAM’s F12 with 0.1% protease free BSA)
and centrifuged. The cell pellet was resuspended in assay buffer and
coelenterazine h was added at a final concentration of 5 μM.
The cell suspension was incubated at 16 °C, protected from light
with constant agitation for 16 h, and then diluted with assay buffer
to the concentration of 100,000 cells/mL. After 1 h of incubation,
50 μL of the cell suspension was dispensed using automatic injectors
built into the radiometric and luminescence plate counter MicroBeta2
LumiJET (PerkinElmer, USA) into white opaque 96-well microplates preloaded
with test compounds. Immediate light emission generated following
calcium mobilization was recorded for 30 s. In antagonist mode, after
25 min of incubation, the reference agonist was added to the above
assay mix and light emission was recorded again. The final concentration
of the reference agonist was equal to EC80 (300 nM serotonin).
Preliminary Metabolic Stability Assessment
The in vitro evaluation of metabolic stability of phenoxyethyl
derivatives of 1-(1-benzoyl-4-fluoropiperidin-4-yl)methanamine was
performed by using RLMs (Sigma-Aldrich, St. Louis, MO, USA) according
to previously described methods and protocols.[53−55] The reaction
mixtures were prepared first, consisting of 50 μM of the tested
compound, microsomes (1 mg/mL), and 10 mM Tris-HCl buffer, pH = 7.4.
Reaction mixtures were preliminarily incubated for 5 min in a temperature
of 37 °C. After preincubation, 50 μL of an NADPH Regeneration
System (Promega, Madison, WI, USA) was added to initiate the reaction.
The reaction mixtures were incubated for 2 h at a temperature of 37
°C. In order to terminate the reaction, 200 μL of cold
extrapure methanol was added. Then, the mixtures were centrifuged
(14,000 rpm, 15 min) and the supernatants were analyzed using an LC/MS
Waters ACQUITY TQD system with the TQ Detector (Waters, Milford, USA).In silico prediction of metabolic biotransformations
was performed by MetaSite 6.0.1 software (Molecular Discovery Ltd,
Hertfordshire, UK).[56] The computational
liver model of metabolism was used for determination of the most probable
sites of metabolism and identification of structures of obtained in vitro metabolites.
Intrinsic Clearance
For determination of the intrinsic
clearance (CLint) parameter, five independent reactions
were terminated by the addition of cold methanol containing IS at
different time points: 0, 5, 15, 30, 45 min. The reaction mixtures
were next centrifuged at 14,500 rpm for 10 min. The course of reaction
was followed by using the analyte/IS peak height ratio values. In
the determination of the in vitro t1/2 value, the slope of linear regression from log concentration remaining
versus time relationships (−k) was used according
to Obach (1999). The supernatants with 3, 44, and 56 were analyzed by an HPLC system (LaChrom Elite,
Merck-Hitachi, Germany) consisting of an L-2130 pump, an L-2200 autosampler,
and an L-2420 UV–VIS detector. EZChrome Elite v. 3.2 (Merck-Hitachi,
Germany) computer program was used for data acquisition and integration.
The separation of studied compounds was performed using a 250 ×
4.6 mm Supelcosil LC-CN column with a particle size of 5 ìm
(Sigma-Aldrich, Germany) protected with a guard column (20 ×
4 mm) with the same packing material. The mobile phase consisted of
50 mM potassium dihydrogen phosphate (pH = 4.6), methanol, and acetonitrile
mixed at a ratio of 51:40:9 v/v/v and run at 1 mL/min. Chromatographic
analysis was carried out at 25 °C and the analytical wavelength
was 205 nm.
PAMPA Assay
The ability to passive transport across
a cell membrane was determined by precoated PAMPA Plate System Gentest
(Corning, Tewksbury, MA, USA) according to described previously protocols.[54,55] The compounds’ concentrations in acceptor and donor wells
were estimated by the UPLC–MS analyses, which were performed
by LC/MS Waters ACQUITY TQD system with the TQ Detector (Waters, Milford,
USA). The permeability coefficients (Pe, cm/s) were calculated according
described previously formulas.[57]The substrates of Pgp were determined by the luminescent Pgp-Glo
Assay System (Promega, Madison, WI, USA). The test measures luminescently
the ATP consuming by Pgp in the presence of substrates. The assay
was performed in triplicate as described previously.[55] Tested compounds (100 μM) were incubated with Pgp
membranes for 40 min at 37 °C. The positive (VL) and negative
(CFN) controls were incubated at 200 and 100 μM, respectively.
Basal activity of Pgp was considered as the difference in the luminescent
signal between samples treated with 100 μM of the potent and
selective Pgp inhibitor (Na3VO4) and untreated
samples. The luminescence signal was measured by microplate reader
EnSpire PerkinElmer (Waltham, MA, USA). Caffeine (CFN) and norfloxacin
were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Hepatotoxicity
The hepatotoxicity was investigated
with use of a hepatomaHepG2 (ATCC HB-8065) cell line. Cells were
grown under described previously conditions.[54] The cells viability was determined by CellTiter 96 AQueous Non-Radioactive
Cell Proliferation Assay (Promega, Madison, WI, USA) after 72 h of
incubation with tested compounds at a final concentration range (0.1–100
μM). The reference toxins CCCP and doxorubicin (DX) were used
at 10 and 1 μM, respectively. The absorbance was measured using
a microplate reader EnSpire (PerkinElmer, Waltham, MA USA) at 490
nm. The compounds and references were tested in quadruplicate. DX
was purchased from Sigma-Aldrich (St. Louis, MO, USA).
Extended Selectivity Studies
The off-target receptor
screen and cardiac toxicity (hERG automated patch-clamp method) assays
were performed by Eurofins Pharma Discovery Services according to
well-known methods. Further methodological details are available on
the company web site (www.eurofinsdiscoveryservices.com) and the appropriate publications.[58−65]
In Vivo Pharmacodynamics Studies
Animals
The experiments were performed on male Wistar
rats (170–200 g) obtained from an accredited animal facility
at the Jagiellonian University Medical College, Krakow, Poland. The
animals were housed in groups of four in a controlled environment
(ambient temperature 21 ± 2 °C; relative humidity 50–60%;
12 h light/dark cycles (lights on at 8:00). Standard laboratory food
(LSM-B) and filtered water were freely available. Animals were housed
for a period of 6 days in polycarbonate Makrolon type 3 cages (dimensions
26.5 × 15 × 42 cm, “open top”) without enrichment
environment (only wooden shavings litter). Each animal was assigned
randomly to treatment groups and only used once (no repeated use of
animals). All the experiments were performed by two observers unaware
of the treatment applied between 9:00 and 14:00 on separate groups
of animals. All experimental procedures involving animals were conducted
in accordance with European Union (Directive 2010/63/EU) and Polish
legislation acts concerning animal experimentation and approved by
the II Local Ethics Committee for Experiments on Animals in Cracow,
Poland (approval number: 108/2016). All efforts were made to minimize
suffering and to reduce the number of animals used in the experiments
and to use only the number of animals necessary to produce reliable
scientific data. Each experimental group consisted of 6–8 animals.
Drugs
All drugs were dissolved in distilled water immediately
before administration in a volume of 2 mL/kg. The examined compounds
were administered orally 60 min before tests. In antagonism experiments,
WAY100635 (Tocris, UK) was administered subcutaneously 75 min before
testing. Control animals received vehicle (distilled water) according
to the same schedule.
Porsolt FST
The experiment was carried out according
to the method of Porsolt et al.(66) On the first day of an experiment, the animals were individually
gently placed in plexiglas cylinders (40 cm high, 18 cm in diameter)
containing 17 cm of water maintained at 23–25 °C for 15
min. On removal from water, the rats were placed for 30 min in a plexiglas
box under a 60 W bulb to dry. On the following day (24 h later), the
rats were re-placed in the cylinder after administration of test compounds
and the total duration of immobility was recorded during the 5-min
test period. Immobility was considered to occur when no additional
activity was observed other than that necessary to keep the rat’s
head above the water.[67] Fresh water was
used for each animal.
Lower Lip Retraction
Observations were made according
to the method described by Kleven et al.(68) Animals were observed individually during a
10 min period for 10 s of observation per animal. During each of these
observation periods, the uninterrupted presence for at least 3 s (1)
or absence (0) of LLR was recorded. This cycle was repeated 10 times
over a 10 min period; thus, the incidence of a particular behavior
could vary from 0 to 10.
Statistical Analysis
The data of behavioral studies
were evaluated by an analysis of variance: one-way ANOVA (when one
drug was given) or two-way ANOVA (when two drugs were used) followed
by Bonferroni’s post hoc test (statistical significance set
at p < 0.05).
In Vivo Pharmacokinetics Studies
Male Wistar rats weighing 200–250 g
were used in this study. The investigated compounds were dissolved
in water and administered orally at three different doses, that is,
0.31, 0.63, and 1.25 mg/kg (56) and 0.04, 0.16, 0.63
mg/kg (44). One hour following compound administration,
the animals were sacrificed by decapitation and blood and brains were
harvested. The blood was allowed to clot at room temperature and subsequently
centrifuged at 3000 rpm for 10 min (Universal 32 centrifuge, Hettich,
Germany) to obtain serum. All collected samples were frozen at −80
°C until assayed.
Determination of 56 and 44 in Serum
and Brain Tissue
Serum and brain concentrations of the studied
compounds were measured by HPLC with UV detection. The brains were
homogenized in distilled water (1:4, w/v) with a tissue homogenizer
TH220 (Omni International, Inc., Warrenton, VA, USA). The extraction
of both compounds from serum and brain homogenates was performed using
a mixture of ethyl acetate and hexane (30:70, v/v)|. The IS for 56 was 6-[({[1-(3-chloro-4-fluorobenzoyl)-4-fluoropiperidin-4-yl]methyl}amino)methyl]-N,3-dimethylpyridin-2-amine (0.5 μg/mL for serum samples
and 2 μg/mL for brain homogenates) and for 44 it
was (3-chloro-4-fluorophenyl)(4-fluoro-4-((((5-methylpyridin-2-yl)methyl)amino)methyl)piperidin-1-yl)methanone
(2 μg/mL for both serum and brain samples) as methanolic solutions.To isolate 56 and 44 from serum (1.5
mL) or brain homogenate (2 mL) containing these compounds, an appropriate
IS (10 μL) was added and the samples were alkalized with 100
μL of 4 M NaOH. Then, the samples were extracted with 6 mL of
the extraction reagent by shaking for 20 min (IKA Vibrax VXR, Germany).
After centrifugation at 3000 rpm for 20 min (Universal 32, Hettich,
Germany), the organic layers were transferred to new tubes containing
150 μL solution of 0.1 M H2SO4 and methanol
(90:10 v/v). The mixtures were shaken for 20 min and centrifuged for
20 min (3000 rpm). The organic layer was discarded and 60–80
μL aliquots of the acidic solutions were injected into the HPLC
system.The analytical procedure for ultrafiltrate was similar to that
described above, with the exception that 300 μL of this matrix
was used for analysis, the volumes of 4 M NaOH and the extraction
solvent were 20 μL and 1 mL, respectively, and the organic layers
were transferred to 100 μL of the acidic solution.The HPLC system (LaChrom Elite, Merck-Hitachi, Germany) consisted
of an L-2130 pump, an L-2200 autosampler, and an L-2420 UV–VIS
detector. EZChrome Elite v. 3.2 (Merck-Hitachi, Germany) computer
program was used for data acquisition and integration. The separation
of studied compounds was performed using a 250 × 4.6 mm Supelcosil
LC-CN column with a particle size of 5 μm (Sigma-Aldrich, Germany)
protected with a guard column (20 × 4 mm) with the same packing
material. The mobile phase consisted of 50 mM potassium dihydrogen
phosphate (pH = 4.6), methanol, and acetonitrile mixed at a ratio
of 51:40:9, v/v/v and run at 1 mL/min. Chromatographic analysis was
carried out at 25 °C and the analytical wavelength was 205 nm.The calibration curves were constructed by plotting the ratio of
peak areas of the studied compound to that of an appropriate IS versus
the compound concentration. They were linear in the concentration
range of 0.5–5 ng/mL for 56 and 0.25–5
ng/mL for 44 in serum and 1–50 ng/g for 56 and 5–100 ng/g for 44 in brain homogenate.
In the case of ultrafiltrate, the calibration curves were linear in
the range of 5–700 ng/mL for both compounds. The lower limit
of quantification for all biological matrices studied was the lowest
calibration standard on the calibration curve, which after extraction
procedure was analyzed with a coefficient of variation (CV) of ≤20%
and a relative error of ≤20%. No interfering peaks were observed
in the chromatograms. The assays were reproducible with low intra-
and inter-day variation (CV < 10%). The concentrations were expressed
in ng/mL of serum or ultrafiltrate and ng/g of wet brain tissue.
Determination of in Vitro Rat Plasma Protein
Binding
Fresh blood was harvested from male adult Wistar
rats that were sacrificed by exsanguination. The blood was allowed
to clot for 20 min at room temperature and then centrifuged at 3000
rpm for 10 min (Universal 32, Hettich, Germany). 56 and 44 dissolved in water were added in volume of 10 μL
to separate glass tubes containing 1 mL aliquots of rat serum to achieve
final concentrations of 3 and 30 μg/mL each. All tests were
run in triplicate. After vortexing, the serum samples were incubated
in a water bath at 37 °C for 30 min with gentle shaking. Following
this incubation period, 100 μL of serum samples from each tube
were transferred to Eppendorf tubes and frozen at −80 °C
for analysis. The remaining serum was transferred into Centrifree
ultrafiltration devices with Ultracel regenerated cellulose membrane
(Merck, Darmstadt, Germany) and centrifuged at 1000g for 15 min (EBA III centrifuge, Hettich, Germany). The collected
ultrafiltrates were frozen (−80 °C) for further analysis.
Authors: Tarsis F Brust; Michael P Hayes; David L Roman; Kevin D Burris; Val J Watts Journal: J Pharmacol Exp Ther Date: 2014-12-24 Impact factor: 4.030
Authors: Ronan Depoortère; Agnès L Auclair; Laurent Bardin; Francis C Colpaert; Bernard Vacher; Adrian Newman-Tancredi Journal: Eur Neuropsychopharmacol Date: 2010-05-21 Impact factor: 4.600
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