Discovery of Novel pERK1/2- or β-Arrestin-Preferring 5-HT1AReceptor-Biased Agonists: Diversified Therapeutic-like versus Side Effect Profile

Article abstract of DOI:10.1021/acs.jmedchem.0c00814

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.

Full text of DOI:10.1021/acs.jmedchem.0c00814

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Article
Discovery of novel pERK1/2- or #-Arrestin-Preferring 5-HT1A Receptor
Biased Agonists: Diversified Therapeutic-Like vs. Side Effects Profile
Joanna Sniecikowska, Monika G#uch-Lutwin, Adam Bucki, Anna Wi#ckowska, Agata Siwek,
Magdalena Jastrzębska-Więsek, Anna Partyka, Daria Wilczynska, Karolina
Pytka, Gniewomir Latacz, Katarzyna Przejczowska-Pomierny, El#bieta Wyska, Anna
Weso#owska, Maciej H. Paw#owski, Adrian Newman-Tancredi, and Marcin Ko#aczkowski
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00814 • Publication Date (Web): 04 Sep 2020
Downloaded from pubs.acs.org on September 4, 2020
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Discovery of novel pERK1/2- or β-Arrestin-Preferring 5-HT Receptor
1A
Biased Agonists: Diversified Therapeutic-Like vs. Side Effects Profile
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Joanna Sniecikowska , Monika Gluch-Lutwin , Adam Bucki , Anna Więckowska , Agata Siwek ,
a
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Magdalena Jastrzebska-Wiesek , Anna Partyka , Daria Wilczyńska , Karolina Pytka , Gniewomir Latacz
a
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Katarzyna Przejczowska-Pomierny , Elżbieta Wyska , Anna Wesołowska , Maciej Pawłowski , Adrian
b
a
Newman-Tancredi , Marcin Kolaczkowski *
a
Jagiellonian University Medical College, Faculty of Pharmacy, 9 Medyczna St., 30-688 Kraków, Poland;
b
Neurolixis Inc. 251 Little Falls Drive Wilmington, 19808 Delaware, USA
ABSTRACT
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 antidepressant-like effect in the Porsolt forced
swimming test (FST) in rat. 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 (LLR) 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 CNS
pathologies and possessing accentuated therapeutic activity and reduced side effects.
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INTRODUCTION
Although serotonin 5-HT1A receptors exert major influence in CNS functions such as mood, pain
1
,2
and movement, and were identified several decades ago 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
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anxiolytic buspirone (Buspar ), the antidepressant vortioxetine (Brintellix ), the antipsychotic
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® 3–6
aripiprazole (Abilify ), and the antiparkinsonian bromocriptine (Parlodel ). 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 post-synaptic 5-HT1A heteroreceptors in frontal
cortex elicits procognitive and antidepressant effects, whereas activation of pre-synaptic 5-HT1A
autoreceptors is associated with pro-depressive effects, notably by inhibiting the release of serotonin
7
,8
in terminal regions. 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 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
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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
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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 vs. other signaling pathways (Figure 1).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.
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
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data allowing for rational design of other functionally selective 5-HT1A receptor agonists. In this
context, our previous work investigated the SAR and Structure Functional Activity Relationships
1
3
(SFARs) of novel analogues designed based on the structure of 1. 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 GPCR biased agonist studies.
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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 1). Since our previous work studied various unsubstituted derivatives,
we focused herein on determining the influence of 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
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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.
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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
1
3
CNS MPO = 4.89, LELP = 6.7 and Fsp3 = 0.38, were considered favorable. 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
PAMPA assay, 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 was collected in Table 7 and Chart 1 as well as supporting information Table S3).
Compound 3 showed acceptable metabolic stability, high permeability, very low potential for
hepatotoxicity and significant (at least 500x) selectivity versus the off-targets, including 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
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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.
Synthesis
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To prepare the target compounds 28–57 we have utilized a method that we have previously used and
described (Scheme 1).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 2.
Scheme 1. Synthesis of 1-(1-benzoyl-4-fluoropiperidin-4-yl)methanamine derivativesa
a
Reagents and conditions: (i) DABCO, NaCNBH
3
, FeSO
4
× 7H
2
O, molecular sieves, MeOH, r.t., 36–72 h, yield: 18–
8
6
2%; (ii) 1.0 M HCl in EtOAc, r.t., 24 h, yield: 48%; (iii) CH
3
COOH, NaCNBH
3
, 15 °C – 15 min, then r.t. – 1 h, yield:
7%. X = F or Cl,
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Amines 6–23 were synthesized according to a three-step procedure starting with Williamson
reaction20 using appropriate phenols and 1,2-dibromoethane. The obtained 2-bromoethoxy
derivatives (I 6–23) were used in Gabriel’s synthesis21–23 leading to the desired primary amines.
Synthesis of amine 17 (R= 3-NH–CH ) required additional Boc-protection to prevent reductive
3
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amination at a secondary amine group. Deprotection of the amine was performed at the last step of
the synthesis of compound 45 (Scheme 1). Amines 24–26 were obtained in Mitsunobu reaction24,25
of 2-(methylamino)phenol with tert-butyl-2-hydroxyethyl carbamate (II 24), and quinolin-8-ol or
1
H-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 (NaCNBH
3
) as reducing
agent and with addition of iron sulfate heptahydrate (FeSO × 7H O) that complexes cyanide ions
4
2
and therefore contributes to the improvement of the reaction yields. In additional step, indole
derivative 56 was reduced to indoline analog 57.
Scheme 2. Synthesis of the amine intermediates (6–26)a
v / vi
ii
a
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,
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K
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CO
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, 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, CH
3
I, 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% MeNH
2
(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.
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Structure Affinity Relationships (SAfiRs)
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
i
ranging from 8.18 to 12.80 (Table 1, 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
LLE 7.59), which was the lead structure for this series (Figure 5).
Firstly, we focused on checking the effect of the substitution site on the phenyl ring, using three
commonly used substituents (F, Cl or OCH ). The substitution in ortho position, increased binding
i
and ClogD7.4. All the relationships were compared to the unsubstituted phenoxy derivative 3
(
3
affinity, however, the effect was rather modest and did not exceed 0.5 LLE units. The effect of
substitution in meta position was diversified, ranging from slight increase of affinity for fluoro and
methoxy analogs to decrease in the case of the chloro derivative. On the other hand, substitution in
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 4-
chloro substituent (LLE 5.19 for 35).
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Table 1. Influence of the substituent position in the phenyl ring on 5-HT1A receptor affinity and selectivity for the most
important off-targets.
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a
receptor affinity (pK
i
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5-HT1Ab,e
c,f
d,g
ClogD7.4h
LLEi
Compd
3
X
Ar
α
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D
2
F
10.21 ± 0.20
6.29 ± 0.13
< 6.00
< 6.00
2.62
7.59
28
F
10.80 ± 0.15
< 6.00
2.81
7.99
29
30
31
F
10.78 ± 0.18
10.69 ± 0.25
10.10 ± 0.15
< 6.00
6.20 ± 0.06*
< 6.00
< 6.00
< 6.00
< 6.00
2.78
3.24
2.76
8.00
7.45
7.34
Cl
F
32
F
10.89 ± 0.22
< 6.00
< 6.00
3.25
7.64
33
F
10.32 ± 0.25
10.60 ± 0.17
8.41 ± 0.08
< 6.00
< 6.00
< 6.00
< 6.00
< 6.00
< 6.00
3.23
3.70
3.23
7.08
6.90
5.19
3
4
5
Cl
F
3
3
6
7
F
10.51 ± 0.09
7.18 ± 0.29* 6.17 ± 0.09
7.34 ± 0.11* 6.47 ± 0.10
2.49
8.02
3
Cl
F
9.42 ± 0.01
2.95
2.47
6.48
7.75
38
39
40
10.22 ± 0.21
< 6.00
< 6.00
Cl
F
10.50 ± 0.20
8.18 ± 0.09
8.91 ± 0.04
< 6.00
< 6.00
< 6.00
< 6.00
< 6.00
< 6.00
2.93
2.45
2.91
7.57
5.73
6.00
41
1
Cl
8.66 ± 0.07
9.09 ± 0.03
8.30 ± 0.05
< 6.00
NTj
< 6.00
NT
2.29
1.47
1.35
6.37
7.68
6.95
(
±)8-OH-DPAT
Buspirone
NT
NT
a
All binding affinity values are represented as pK
i i
(i.e. –logK ) and expressed as means ± SEM from at least 3
b
experiments performed in duplicate, unless otherwise indicated; Radioligand binding was performed using CHO-K1
cells transfected with 5-HT1A receptors, c rat cortex, CHO-K1 cells transfected with D
d
receptors; receptor affinity
2
e
3
f
3
g 3
values were determined by competition binding using [ H]8-OH-DPAT, [ H]-prazosin and [ H]-methylspiperone. In
these conditions, pK of phentolamine at α receptors was 7.95, and pK of haloperidol at D receptors was 8.85;
i
1
i
2
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2
2
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2
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2
2
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3
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5
5
5
5
5
6
h
i
j
calculated distribution coefficient at pH 7.4; Ligand-Lipophilicity Efficiency referring to 5-HT1A receptor; Not tested
*
i
pK value was expressed as mean ± range from 2 experiments performed in duplicates.
In support of the observed structure-activity relationships, 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 2A). On the other hand, the para substituted
analog (40) was unable to adopt such a position (Figure 2B), probably due to 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 3). 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
structure-activity relationships are consistent with those established for the long-chain
arylpiperazines, which suggests a similar binding mode.26
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
A
B
Figure 2. The predicted binding mode of the methoxy derivatives i.e. compound 36 (light teal) together with 38 (pink)
(A) and compound 40 (gray) (B) in the site of 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, e.g. 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).
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9
Gln2.65
Tyr7.43
Tyr2.64
Trp6.48
Phe6.51
Phe6.52
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Phe3.28
Asp3.32
ECL2
Ile189
Ser5.42
Figure 3. The predicted binding mode of the fluoro derivatives i.e. compound 28 (yellow) together with 29 (pink) and
31 (green) in the site of 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, e.g. 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 2). All
the ligands achieved very high 5-HT1A receptor affinity, with subnanomolar or even picomolar K
i
values and generally very high LLE values.
The most pronounced increase in 5-HT1A receptor affinity was noted in the case of derivatives
containing 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 value (pK
i
12.80, K 0.16 pM, LLE 10.06). The increase in binding affinity of the
i
derivatives with HBD in the meta position can be explained by their ability to create an additional
hydrogen bond with serine Ser5.42 in the binding site of the 5-HT1A receptor (Figure 4). 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).
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Tyr7.43
Tyr2.64
ECL2
Trp6.48
Phe6.51
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Phe6.52
Phe3.28
Asp3.32
Ile189
Ser5.42
Figure 4. The predicted binding mode of compound 56 (with HBD in meta position) in the site of serotonin 5-HT1A
receptor. Amino acid residues engaged in ligand binding (within 4Å from the ligand atoms) are displayed as sticks,
whereas crucial residues, e.g. 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 suggesting that they exert a negative steric contribution (this was,
however, less pronounced than in the case of the para analogs).
Regarding the substitution at the benzoyl moiety, the 4-chloro analogs had overall lower ligand-
lipophilicity efficiencies than their 4-fluoro counterparts, suggesting that this modification is not
generally favorable.
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
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.
a
receptor affinity (pK
i
)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Compd
3
X
Ar
5-HT1Ab,e
α
1
c,f
D
2
d,g
ClogD7.4h
LLEi
F
10.21 ± 0.20
6.29 ± 0.13
< 6.00
< 6.00
2.62
7.59
42
F
F
10.22 ± 0.21
10.53 ± 0.24
< 6.00
1.51
1.49
8.71
9.05
43
< 6.00
< 6.00
4
4
5
F
F
9.93 ± 0.22
6.04 ± 0.01
< 6.00
< 6.00
2.08
2.09
7.86
8.38
4
10.47 ± 0.19
< 6.00
46
F
9.52 ± 0.15
10.08 ± 0.13
10.27 ± 0.21
9.85 ± 0.23
< 6.00
< 6.00
< 6.00
< 6.00
< 6.00
< 6.00
< 6.00
< 6.00
2.72
1.86
2.33
3.13
6.79
8.21
7.95
6.72
47
48
49
F
Cl
F
5
0
F
F
9.56 ± 0.02
< 6.00
< 6.00
< 6.00
3.51
2.82
6.05
7.40
51
10.22 ± 0.25 6.76 ± 0.03*
5
2
3
Cl
F
9.37 ± 0.03
9.86 ± 0.11
9.71 ± 0.12
6.69 ± 0.07
6.72 ± 0.13*
6.71 ± 0.02*
6.36 ± 0.14
< 6.00
3.28
2.17
2.63
6.09
7.69
7.08
5
54
55
Cl
6.04 ± 0.12
F
11.07 ± 0.06 7.76 ± 0.32*
7.54 ± 0.12*
3.22
7.85
5
6
F
F
12.80 ± 0.16
10.35 ± 0.02
7.00 ± 0.05
6.36 ± 0.02
< 6.00
< 6.00
2.74
2.12
10.06
8.23
57
a
All binding affinity values are represented as pK
i i
(i.e. –logK ) and expressed as means ± SEM from at least 3
b
experiments performed in duplicate, unless otherwise indicated; Radioligand binding was performed using CHO-K1
cells transfected with 5-HT1A receptors, c rat cortex, CHO-K1 cells transfected with D
d
receptors; receptor affinity
2
e
3
f
3
g 3
values were determined by competition binding using [ H]8-OH-DPAT, [ H]-prazosin and [ H]-methylspiperone. In
these conditions, pK of phentolamine at α receptors was 7.95, and pK of haloperidol at D receptors was 8.85;
i
1
i
2
h
i
*
calculated distribution coefficient at pH 7.4; Ligand-Lipophilicity Efficiency referring to 5-HT1A receptor; pK
i
value
was expressed as mean ± range from 2 experiments performed in duplicates.
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1
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2
2
2
2
2
2
2
2
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3
3
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5
5
6
Δ of activity of the novel compounds in relation to the unsubstituted lead structure 3
Δ pKi
Δ LLE = Δ pKi - Δ logD7.4
-
3
-2
-1
0
1
2
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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6
7
8
9
0
1
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-
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-100×
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Figure 5. Changes in LLE in relation to unsubstituted lead structure 3, due to substitution at phenyl ring.
Selectivity vs. key antitargets, the adrenergic α
1
and dopaminergic D receptors
2
Previous studies of 1-(1-benzoylpiperidin-4-yl)methanamine derivatives indicated that the highest
receptors.13,27–29
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
risk of off-target interactions is with adrenergic α
1
and dopaminergic D
2
Accordingly, all the novel compounds were tested for binding to these receptors, to confirm their
selectivity (Tables 1 and 2).
Concerning α
HT1A over α and D
was observed for the ortho substituted analogs (36, 37, 44). The highest affinities (pK
observed for the derivatives with ortho-methoxy substituent (36, 37) and their bicyclic analogs, with
oxygen or nitrogen in ortho position (51, 52, 53, 54, 55). High α 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 α receptor (pK < 6) indicating that halogen in this
receptor binding. In comparison, literature data indicates that ortho-methoxy- or
receptor affinity. For example, in the
1
receptor affinity, the majority of the compounds showed very high selectivity for 5-
receptors (over 1000–10000 times). In general, increased affinity for α receptor
6.6–7.8) were
1
2
1
i
1
i
(
1
i
position impairs α
1
ortho-ethoxy- substitution in the aryl moiety increase α
1
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 vs. α
1
receptors was generally very high, and was less
than 1000× for only two compounds (37, 52).
In the case of the D
2
receptor, only one compound (55) showed substantial affinity (pK
i
D 7.54).
2
This observation is in line with the fact, that the 2,2‐dimethyl‐2,3‐dihydro‐1‐benzofuran moiety was
31
previously used in the structure of dual acting, 5-HT1AR agonist/D
the affinity of 55 for D
2
R antagonist ligands. However,
2
receptor did not significantly affect selectivity, because its affinity for 5-
HT1A receptor was still over 3.5 orders of magnitude higher (> 3000×, pK 5-HT1A = 11.07).
i
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Summing up, most of the presented compounds showed substantial selectivity versus key antitargets
ratio over 1000-fold), despite the fact that the phenoxyethanamine scaffold is common also for
(K
i
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.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Structure Functional Activity Relationships (SFARs)
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)
2
+
and calcium mobilization (Ca ). 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 human
5
-HT1A receptor.
In terms of potency, the general trends (Tables 3 and 4), were similar to those established in affinity
studies. Compared to 3, the derivatives substituted with the HBD in meta position (43, 45, 47, 48)
were characterized by a rise in potency at all the signaling pathways. The same trend was observed
for the bicyclic analogs (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 analogs, respectively, which were characterized by generally higher potency than 3, as well
as compound 42, an ortho-carboxamido analog, 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 profile for some of them.
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2
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 calcium mobilization assay. Thirteen compounds were classified as
partial agonists and two even as negligibly active, as they showed marginal level of stimulation
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(15%). Both of these compounds were para-substituted analogs (35 and 40).
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 3. Functional activity of compounds 28–33, 35, 36, 38-41 at 5-HT1A receptors.
-HT1A functional activitya
5
ERK1/2b
cAMPb
β-arrestinc
Ca2+ b
Compd
3
X
Ar
E
MAX
pEC50
E
MAX
pEC50
E
MAX
pEC50
E
MAX
pEC50
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
F
92%
95%
9.10
90%
89%
8.09
96%
7.98
91%
90%
7.20
7.61*
6.82*
28
29
30
31
32
33
F
F
Cl
F
F
F
F
F
8.24
8.64
7.70
8.24
NTd
7.74
7.80
7.40
7.37
7.59
7.71
6.32
9.86*
97%
91%
96%
93%
98%
NTd
99%
89%
96%
87%
88%
63%
83%
7.46
73%
90%
73%
36%
80%
15%
54%
7.16*
6.19*
7.00
104% 6.21*
99% 7.06*
94%
7.76
6.26*
6.60*
5.28
97%
88%
NTd
7.99
6.93
NTd
99% 6.65*
109% 6.01*
35
36
90%
9.43
9.52*
8.64*
3
3
4
8
9
0
1
F
97%
95%
86%
78%
9.40
8.14
7.68
7.32
89%
96%
88%
59%
8.98
7.69
6.25
6.48
90%
95%
82%
64%
74%
15%
24%
7.15
6.50
6.21
Cl
F
6.91
5.79
4
Cl
65%
98%
5.60
6.71
5.88*
6.52
1
100% 8.33
92%
63%
7.22
7.50
7.14
7.51
66%
35%
(
±) 8-OH-DPAT
93%
44%
8.09
7.82
101% 7.84
100% 6.73
100% 6.89
7.66
7.42*
Buspirone
Serotonine
49%
8.3%
100%
100% 7.48
100%
7.23
a
All the functional activity values were expressed as means from at least 3 experiments performed in duplicates, unless
otherwise indicated. For the sake of clarity, the SEM values were omitted in this table and presented in the supporting
b
c
information – Table S1; The functional assay was performed using CHO-K1 cells, U2OS cells (Tango LiveBLAzer
d
*
e
assay kit) ; NT – not tested; value was expressed as mean from 2 experiments performed in duplicates. Data for
Serotonin on ERK, cAMP and β-arrestin are reproduced from the previous paper.13
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Table 4. Functional activity of compounds 42–51 and 55–57 at 5-HT1A receptors.
0
1
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5-HT1A functional activitya
ERK1/2b
cAMPb
β-arrestinc
Ca2+ b
Compd
3
X
Ar
E
MAX
pEC50
E
MAX
pEC50
E
MAX
pEC50
E
MAX
pEC50
F
92%
92%
9.10
90%
95%
8.09
96%
7.98
91%
84%
7.20
*
42
F
F
8.68
9.74
8.22
101% 8.08
7.53
*
4
3
4
87%
92%
77%
9.18
7.82
101%
96%
8.55
105% 7.42
*
4
F
F
89%
83%
8.86
6.79
43%
6.99
45
46
47
10.13
87%
91%
9.95
7.77
100% 10.05
94%
82%
7.49
*
F
87%
102%
100%
93%
8.22
9.75
9.23
8.72
101%
93%
7.22
6.86
*
*
F
89% 10.86
12.68 100% 7.66
*
*
48
Cl
F
94%
82%
10.18
7.83
96% 10.60
100% 7.36
*
4
9
0
98%
7.27
59%
73%
6.83
*
5
F
F
100%
96%
7.25
9.30
99%
6.56
9.47
104% 6.14
6.14
7.87
*
5
1
5
104%
92% 11.83
67%
89%
*
5
F
81%
10.99
88%
8.18
99%
9.49
7.27
5
6
7
F
F
102% 10.53
94% 11.44
96%
89%
10.06
10.88
94%
92%
13.67
11.29
79%
56%
7.84
*
5
8.45
a
All the functional activity values were expressed as means from at least 3 experiments performed in duplicates, unless
otherwise indicated. For the sake of clarity, the SEM values were omitted in this table and presented in the supporting
b
c
information – Table S1; The functional assay was performed using CHO-K1 cells, U2OS cells (Tango LiveBLAzer
assay kit) ; value was expressed as mean from 2 experiments performed in duplicates.
*
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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:
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9
0
relative activityퟏ2,ꢀꢁꢂ
퐸mꢇxpꢈꢉh1 × EC50−ꢊꢇꢋꢌ2
EC50−ꢊꢇꢋꢌꢍ × 퐸mꢇxpꢈꢉhꢎ
퐸mꢇxpꢈꢉh1 × EC50−ꢊꢇꢋꢌ2
EC50−ꢊꢇꢋꢌꢍ × 퐸mꢇxpꢈꢉhꢎ
Bias factor = log (
) = log ꢆ(
)
÷ (
)
ꢏ
relative activityퟏ2,ꢃꢄꢅ
ꢀꢁꢂ
ꢃꢄꢅ
1
3
The calculations in the present study follow the same approach as in our previous study. 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 5 and 6 and compounds which
displayed significant bias (over 1 log) were 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.
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Table 5. Bias factors of compounds (28–33, 35, 36, 38-41) and references at 5-HT1A receptors.
5-HT1A receptor bias factor (logarithmic value)
ERK1/2 vs.
cAMP
ERK1/2 vs.
β-arrestin
ERK1/2
vs. Ca2+
cAMP vs.
β-arrestin
cAMP
β-arrestin
Compd
3
X
R
vs. Ca2+
vs. Ca2+
0
1
2
3
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8
9
0
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9
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8
9
0
1
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3
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9
0
1
2
3
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7
8
9
0
F
F
F
1.05
0.51
1.65
-0.54
0.60
1.15
28
0.56
0.03
1.19
-0.53
0.63
1.16
2
9
0
0.85
0.35
0.90
0.60
0.85
0.57
1.35
1.27
1.11
-0.25
0.50
0.50
0.92
0.20
0.75
0.42
0.54
3
Cl
F
31
-0.33
– a
– a
– a
32
F
-0.83
1.42
2.25
3
3
5
F
F
0.35
0.78
0.73
0.23
1.22
2.15
0.38
0.86
1.38
0.48
1.92
3
-0.55
– a
– a
– a
3
6
8
F
F
-0.23
-0.30
0.24
1.68
0.47
1.98
3
0.49
0.19
2.17
39
Cl
F
0.48
1.44
1.00
0.63
1.31
1.21
1.49
1.98
1.70
0.15
-0.13
0.21
1.02
0.54
0.70
0.87
0.67
0.49
40
4
1
1
Cl
1.17
0.79
0.66
0.00
1.02
-0.39
0.14
0.00
1.76
0.59
0.86
0.00
-0.15
-1.18
-0.52
0.00
0.59
-0.20
0.21
0.00
0.74
0.98
0.73
0.00
(
±) 8-OH-DPAT
Buspirone
5-HT
a- no data for pERK assay
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Table 6. Bias factors of compounds 42–51 and 55–57 at 5-HT1A receptors.
5-HT1A receptor bias factor (logarithmic value)
ERK1/2
vs. cAMP β-arrestin
ERK1/2 vs. ERK1/2
cAMP vs.
cAMP
β-arrestin
Compd
3
X
Ar
vs. Ca2+ β-arrestin vs. Ca2+
vs. Ca2+
0
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F
1.05
0.51
1.65
-0.54
0.60
1.15
4
4
4
2
3
4
F
F
0.47
-0.04
0.93
-0.51
0.46
0.97
0.57
0.53
1.98
-0.04
1.42
1.45
F
F
1.13
0.20
1.43
1.93
2.33
0.31
0.81
2.14
0.50
2.93
4
5
6
-0.60
-0.80
4
F
0.45
-1.03
-0.90
0.97
0.33
-3.49
-1.95
0.83
1.12
1.85
1.61
1.84
-0.12
-2.47
-1.06
-0.14
0.67
2.87
2.50
0.87
0.79
5.34
3.56
1.01
47
F
4
8
9
Cl
F
4
5
0
1
F
F
0.73
0.49
1.00
1.33
-0.23
-2.94
0.27
1.50
0.50
4.44
5
-0.17
-3.11
5
5
6
F
2.80
0.81
3.42
-1.99
0.62
2.61
5
F
F
0.53
0.61
-3.71
-0.44
2.54
2.95
-4.24
-0,99
2.02
2.34
6.25
3.39
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ERK1/2 vs. cAMP
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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
0
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1), preferred cAMP pathway and the significant bias was observed for compound 47 (bias factor -
1
.03).
When comparing 3-chloro-4-fluorobenzoyl (29, 38, 40, 47) with their 3,4-dichlorobenzoyl
derivatives counterparts (30, 39, 41, 48), it is noticeable that the former always show a more
pronounced biased profile than the latter compounds.
ERK1/2 vs. β-arrestin
Three compounds preferred ERK1/2 phosphorylation vs β-arrestin, and four compounds preferred
β-arrestin recruitment vs 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 vs. Ca2+
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In general, most of the compounds showed substantial bias for ERK1/2 phosphorylation vs. calcium
2
+
2+
mobilization (Ca ) and none of the tested compounds showed preference for Ca . The highest
ERK1/2 preference was found for the benzo-fused, five-membered ring derivatives (55–57),
reaching bias factor of 3.42 for compound 55.
0
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0
cAMP vs. β-arrestin
Only five compounds (30, 33, 39, 41, 44) exhibited some preference towards cAMP inhibition,
however not exceeding half a log, whereas the rest of the compounds preferred β-arrestin
recruitment. As seen for ERK1/2 vs. 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 analogs.
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, despite the fact that previously it exhibited the
highest preference for ERK1/2 vs. 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 towards 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.
Worth mentioning are the previous studies by Stroth and co-workers, where the authors identified
3
6
5-HT1A biased ligands with a strong preference for cAMP over β-arrestin signaling . 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%).
Noteworthy, Stroth et al. reported that the reference agonist (±) 8-OH-DPAT achieved only 44%
efficacy in β-arrestin assay, while herein it reached 101%, so the observed differences in signaling
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bias may be at least partially due to the methodological differences (β-arrestin assay in that study
was performed using PathHunter eXpress HTR1A CHO-K1 β-Arrestin GPCR Assay DiscoveRx,
while in the current study Tango™ HTR1A-bla U2OS LiveBLAzer assay kit, Life Technologies).
3
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cAMP vs. Ca2+
0
1
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2
+
Eleven compounds markedly preferred cAMP pathway versus Ca . Noteworthy, 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. Due to relatively
low potency of all the compounds in the calcium mobilization assay, none of them exhibited bias
towards this signaling pathway.
β-arrestin vs. Ca2+
Thirteen compounds were markedly biased for β-arrestin. Four of the compounds (47, 48, 51 and
5
6) showed extremely high bias for β-arrestin (over 3.5 log), reaching 6.25 log value (over 1 000 000
times) for compound 56. The relatively lower ability of the compounds to stimulate Ca2+
2+
mobilization, resulted in a lack of noticeable Ca -preferring biases.
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“
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:
0
1
2
3
4
5
6
7
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9
0
1
2
3
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9
0
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0
1
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9
0
1
2
3
4
5
6
7
8
9
0
EC50 ꢊꢇꢋꢌ
Norꢐalized ligand ꢑotency = ꢒ log ꢆEC50 ꢊꢇꢋꢌꢀꢁꢂ ÷ (
)
ꢏ
EC50 ꢃꢄꢅ. ꢊꢇꢋꢌ ꢓꢇꢋꢁꢔꢄ
ꢁꢂꢇꢓꢕ
ꢀ
A ‘signaling fingerprint’ therefore allows for the simultaneous comparison of 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 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 6) 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 1 log preference for β-arrestin vs 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, didn’t differ significantly.
2
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Journal of Medicinal Chemistry
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Serotonin
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Buspirone
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ERK cAMP β-arrestin Ca2+
ERK cAMP β-arrestin Ca2+
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ERK cAMP β-arrestin Ca2+
Figure 6. “Signaling fingerprints” for reference compounds (bar height – normalized ligand potency in log scale, bar
color – ligand efficacy, as percent Emax).
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Journal of Medicinal Chemistry
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The “signaling fingerprints” for the most interesting novel compounds, in comparison with the lead
structures 2 and 3, were shown in Figure 7. 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 analogs were shown in the left column, the more
balanced in the middle and the β-arrestin biased agonists in the right column.
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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
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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, similarly 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
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and Ca , 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 the Type I and II, and of particular interest in the present study, are type III compound:
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 β-arrestin
2+
pathway, similar levels of activity in ERK1/2 and cAMP assays and much lower stimulation of Ca .
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. Noteworthy, 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:
(1) the presence of an H-bond forming substituent in ortho position of the phenoxyethyl moiety (44,
5
5) or a nitrogen atom built in the aryl ring in the same position (2), decreases the ability to activate
2
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