Defining Structure-Functional Selectivity Relationships (SFSR) for a Class of Non-Catechol Dopamine D1 Receptor Agonists

Article abstract of DOI:10.1021/acs.jmedchem.9b00351

G protein-coupled receptors (GPCRs) are capable of downstream signaling through distinct noncanonical pathways such as β-arrestins in addition to the canonical G protein-dependent pathways. GPCR ligands that differentially activate the downstream signaling pathways are termed functionally selective or biased ligands. A class of novel non-catechol G protein-biased agonists of the dopamine D₁ receptor (D₁R) was recently disclosed. We conducted the first comprehensive structure-functional selectivity relationship study measuring G_S and β-arrestin2 recruitment activities focused on four regions of this scaffold, resulting in over 50 analogs with diverse functional selectivity profiles. Some compounds became potent full agonists of β-arrestin2 recruitment, while others displayed enhanced G_S bias compared to the starting compound. Pharmacokinetic testing of an analog with an altered functional selectivity profile demonstrated excellent blood-brain barrier penetration. This study provides novel tools for studying ligand bias at D₁R and paves the way for developing the next generation of biased D₁R ligands.

Full text of DOI:10.1021/acs.jmedchem.9b00351

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Article
Defining Structure-Functional Selectivity Relationships (SFSR)
for a Class of Non-Catechol Dopamine D1 Receptor Agonists
Michael L. Martini, Jing Liu, Caroline Ray, Xufen Yu, Xi-Ping Huang, Aarti Urs,
Nikhil Mahabir Urs, John McCorvy, Marc G. Caron, Bryan L Roth, and Jian Jin
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00351 • Publication Date (Web): 15 Mar 2019
Downloaded from http://pubs.acs.org on March 17, 2019
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Defining Structure-Functional Selectivity
Relationships (SFSR) for a Class of Non-Catechol
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Dopamine D Receptor Agonists
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Michael L. Martini , Jing Liu , Caroline Ray , Xufen Yu , Xi-Ping Huang , Aarti Urs , Nikhil
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Urs , John D. McCorvy *, Marc G. Caron *, Bryan L. Roth *, and Jian Jin *
1Mount Sinai Center for Therapeutics Discovery, Departments of Pharmacological Sciences and
Oncological Sciences, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New
York, NY 10029, USA
2Medical Scientist Training Program, Icahn School of Medicine at Mount Sinai, New York, NY
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0029, USA
3Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA
4Department of Pharmacology and Therapeutics, College of Medicine, University of Florida,
Gainesville, FL 32610, USA
5Department of Pharmacology and National Institute of Mental Health Psychoactive Drug
Screening Program, School of Medicine, University of North Carolina at Chapel Hill, Chapel
Hill, North Carolina 27599, USA
6Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin,
Milwaukee, WI 53226, USA
7Department of Medicine and Neurobiology, Duke University Medical Center, Durham, NC
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7710, USA
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ABSTRACT
G protein-coupled receptors (GPCRs) are capable of downstream signaling through distinct
non-canonical pathways such as -arrestins in addition to the canonical G protein-dependent
pathways. GPCR ligands that differentially activate the downstream signaling pathways are termed
functionally-selective, or biased ligands. A class of novel non-catechol G protein-biased agonists
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of the dopamine D
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Receptor (D R) was recently disclosed. We conducted the first comprehensive
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structure-functional selectivity relationship (SFSR) study measuring Gs and -arrestin2
recruitment activities focused on four regions of this scaffold, resulting in over 50 analogs with
diverse functional selectivity profiles. Some compounds became potent full agonists of -arrestin2
recruitment, while others displayed enhanced G bias compared to the starting compound.
S
Pharmacokinetic testing of an analog with an altered functional selectivity profile demonstrated
excellent blood-brain barrier penetration. This study provides novel tools for studying ligand bias
at D
1
R and paves the way for developing the next generation of biased D R ligands.
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INTRODUCTION
As the largest superfamily of proteins in the druggable genome with functions that have
been implicated in diverse biological processes, G protein-coupled receptors (GPCRs) represent
an incredibly important target class in drug discovery.1, 2 For decades, G proteins were believed to
be the sole signaling effector molecules downstream of membrane-bound GPCRs. This canonical
G protein-mediated signaling pathway is known to proceed upon ligand binding, which induces a
conformational change in the GPCR, causing the release and dissociation of a heterotrimeric G
protein that then drives downstream signaling processes within the cell.3-5
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More recently, exciting discoveries in GPCR pharmacology have revealed that this
receptor superfamily is capable of signaling through non-canonical G protein-independent
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pathways. This concept of “functional selectivity”, or biased signaling, now refers to the process
by which a ligand with a given GPCR binding mode is capable of differentially activating distinct
subsequent signaling cascades in the cell, including most notably those driven by G proteins or -
arrestins.10-16 In addition, recent studies have also shown that it is possible for a compound to
differentially activate various G protein subtypes, such as G
biased agonism.17
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/Golf, leading to subtype-selective
Importantly, recent and ongoing studies on diverse classes of GPCRs have demonstrated
the direct therapeutic relevance of functional selectivity at these receptors as distinct signaling
pathways can mediate divergent processes. In some cases, one downstream signaling pathway may
even be responsible for the therapeutic benefit of the ligand while the other pathway may underlie
the observed adverse effects.18-25 For example, at the -opioid receptor (MOR), cellular processes
related to analgesia, antinociception, and respiratory depression have been ascribed to differential
signaling downstream of the receptor.20, 22, 26-28 Similarly, at the D2 receptor, biased ligands with
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various downstream functional activities have been shown to modulate diverse effects on motor,
cognitive and antipsychotic processes.29-41 These findings provide a strong rationale for the
discovery and characterization of functionally selective GPCR ligands with the potential to serve
as valuable chemical tools with utility in dissecting the molecular pathways implicated in various
pathophysiological processes.
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Recently, Gray et al. reported a novel non-catechol-containing D
1
dopamine receptor (D R)
1
agonist scaffold with a putative orthosteric binding mode that differs in several important aspects
from the binding mechanisms of virtually all catechol-containing agonists at aminergic GPCRs,
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including dopamine. As a direct result of this unique binding mechanism, the few reported
compounds that were tested potently induced downstream cAMP production, indicating
42
stimulatory G protein (G
S
) pathway activation, while failing to recruit -arrestin2. A subsequent
study by Davoren et al. detailing the discovery of this scaffold via a high throughput screening
campaign confirmed its potent induction of cAMP production, in addition to providing a detailed
biophysical characterization of the atropisomerism that exists due to the locked biaryl ring
4
3
system. Results from that study suggested that this property relates to the scaffold’s D R high
1
binding affinity and potent ability to stimulate cAMP production. Importantly, functional
selectivity and -arrestin2 recruitment activity were not considered in the study and all of the
reported analogue ligands focused around altering the atropisomerism through modification of the
locked biaryl ring system. In further studies, this non-catechol scaffold was also shown to be highly
R across a panel of class A aminergic GPCRs and neurotransmitter transporters.42,
selective for D
1
4
3
In an acute rodent model of Parkinson’s Disease (PD), this agonist scaffold was shown to elicit
a more sustained dopaminergic response in the animals in comparison with dopamine by virtue of
their inability to recruit -arrestin2, thereby resulting in diminished desensitization and
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tachyphylaxis after repeated dosing. In separate studies of downstream signaling at D
1
R in PD
animal models, it has been suggested that the G -mediated pathway may be responsible for the
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eventual development of Levodopa-induced dyskinesias (LIDs), while -arrestin2-mediated
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signaling pathways may attenuate LIDs while maintaining locomotor improvements.18, 44, 45 We
believe that such findings concerning D
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R G - and -arrestin2-mediated signaling pathways make
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this a particularly interesting system for studying GPCR functional selectivity. As such, we saw a
ripe opportunity to more completely characterize the SFSR of this unique scaffold and assess
ligand structural determinants of D
Herein, we report our SFSR study results that demonstrate certain robust trends in this
distinctive scaffold that help confer its exquisite G protein functional selectivity at D R. We
1
R functional selectivity.
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describe the design, synthesis, and in vitro pharmacological assessment of novel derivatives that
explore four regions of the scaffold represented by compound 1 (PF-6142), which was reported as
a potent, highly selective, full agonist of the G
S
signaling pathway at D R with minimal -arrestin2
1
4
2
recruitment. These comprehensive SFSR studies provide critical information regarding the steric,
electronic, and spatial determinants of G protein bias on this scaffold as well as an important
framework for designing, characterizing, and optimizing compounds for ideal levels of functional
selectivity at various GPCR targets.
RESULTS AND DISCUSSION
Traditional catechol D R agonists are less CNS penetrant and may be liable for metabolism
1
to inactive metabolites, as observed in several well-known D1-selective catechol agonists.46-48
Non-catechol agonists, on the other hand, are more attractive for further development to avoid
42
such extensive metabolism. Thus, we chose to study the SFSR of the scaffold represented by 1
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PF-6142), which features a neutral heterocyclic arylphenoxyaryl core with an interlocking biaryl
ring system.
To determine whether modifying various structural elements of 1 could result in biased
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compounds favoring either cAMP or -arrestin2 signaling, we divided the arylphenoxyaryl
scaffold into four primary regions: 1) a right-hand side (RHS) aromatic heterocycle with various
ring substitutions, 2) a substituted middle phenyl ring, 3) a linking heteroatom, and 4) a left-hand
side (LHS) pyridine-containing heterocycle (Figure 1). We then investigated each region by
making analogues with structural, spatial, and electronic changes isolated to that region and
comparing their resulting potencies, efficacies and functional selectivity profiles.
Figure 1. The four regions of the arylphenoxyaryl scaffold of compound 1 that were investigated
for SFSR.
SFSR of the RHS Heterocycle
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To determine the effects of various heteroaromatic rings on the RHS, we designed and
synthesized the compounds presented in Table 1. We sought to create a diverse array of analogues
of 1 with variously substituted bicyclic rings, including imidazopyridines, imidazopyrazines and
pyrimidines, and monocyclic rings, including pyridines, to evaluate the ligand determinants of
potency, efficacy and functional selectivity.
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All of these compounds were prepared according to the synthetic strategy depicted in
Scheme 1. Briefly, the designed compounds were prepared by Suzuki coupling of a known
intermediate 3 42 with known49 or commercially available brominated heterocycles.50 These
syntheses employed tetrakis(triphenylphosphine)palladium(0) as a catalyst and resulted in the
corresponding desired product in moderate to good yields.
Scheme 1. Synthesis of compounds for exploring the RHS heterocyclic moiety.a
The synthesized compounds were evaluated for potency and efficacy in stimulating D
1
R
mediated cAMP production as measured in a D R-Gs GloSensor assay, and in activating D R
1
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mediated -arrestin2 recruitment via a bioluminescence resonance energy transfer (BRET)
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platform-based assay, with both assays using dopamine (Figure 2A) and dihydrexidine (Figure 2B
as positive full agonist controls.51-53 Of note, the D
R-Gs GloSensor assay involves a signal
1
amplification mechanism, meaning that an EMAX may be achieved at less than full receptor
occupancy, while the -arrestin2 recruitment assay measures an unamplified, stoichiometric
protein-protein interaction, meaning that an EMAX is achieved at full receptor occupancy. We began
our SFSR study with compound 1, which was recently disclosed following an extensive HTS and
optimization campaign.42, 43 We identified 1 to be a potent partial agonist in the GloSensor assay
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(EC50 = 0.25 nM, EMAX = 61%) with some partial agonist activity in the -arrestin2 recruitment
BRET assay (EC50 = 110 nM, EMAX = 48%) (bias factor for cAMP accumulation: 7.7) (Figure 2C).
Through manipulation of compound 1’s RHS with diverse heterocyclic entities, we found
a wide variation among compounds in terms of their activities and functional selectivity profiles.
Compound 4 was designed to feature the same imidazopyrazinyl moiety as 1 but with a different
methyl substitution position and a different point of attachment to the middle phenyl ring of the
scaffold. Compound 4 possessed 23-fold reduced potency in the G
(EC50 = 5.8 nM; EMAX = 63%) and 22-fold reduction in potency for -arrestin2 recruitment (EC50
2,600 nM; EMAX = 89%) (bias factor for cAMP accumulation: 4.1) compared to 1. Compound 5
featured an unsubstituted imidazopyridinyl group for the RHS and was found to maintain a
similarly potent partial agonism towards -arrestin2 recruitment as 1 (EC50 = 180 nM; EMAX
S
-cAMP accumulation assay
=
=
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3%). While potency in the G
S
-cAMP accumulation assay was approximately 10-fold weaker than
pathway with an EC50 and Emax similar to
1
, 5 was determined to be a full agonist of the G
S
dopamine (EC50 = 2.7 nM; EMAX = 95%) (bias factor for cAMP accumulation: 1.3). The RHS of 6
featured a similar imidazopyridinyl moiety as 5, with alterations to the position of attachment and
a methyl ester substitution on the ring’s 8-position. These changes resulted in a dramatically
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reduced potency and efficacy towards G -cAMP accumulation (EC50 = 1,000 nM; EMAX = 44%)
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and no detectable -arrestin2 recruitment activity.
Switching the bicyclic imidazopyrazinyl moiety of 1 to a monocyclic aminopyrazinyl
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group (7) increased the efficacy of the ligand in G
potency (EC50 = 22 nM; EMAX = 91%). This modification also diminished the -arrestin2
recruitment activity, resulting in a compound that is almost completely biased compound for G
S
-cAMP accumulation, however, with sacrificed
S
compared to 1 (Figure 2D). Compared to 7, a disubstituted pyrimidinyl RHS (8) and a substituted
pyridazinyl RHS (9) showed similar and improved potency (8: EC50 = 12 nM; 9: EC50 = 1 nM),
respectively, but decreased efficacy in the G
61%;). Similar to 7, 8 and 9 were not active in the -arrestin2 recruitment assay. Compound 10
is a close analogue of 7, featuring a pyridinyl ring rather than a pyrazinyl ring. Compound 10
possessed similar potency in the G pathway, but as a partial agonist compared to 7 (EC50 = 13
S
-cAMP accumulation assay (8: EMAX = 67%; 9: EMAX
=
S
nM; EMAX = 67%). Notably, 10 was more active than 7 in the -arrestin2 recruitment assay,
however, it was still only a weak recruiter of -arrestin2 overall (EC50 = 1,100 nM; EMAX = 64%)
(
bias factor for cAMP accumulation: 1.2). Compounds 11, 12, and 13 featured different substituted
pyridinyl RHS moieties, and displayed different degrees of potency (11: EC50 = 77 nM; 12: EC50
= 1,300 nM; 13: EC50 >10,000 nM) as partial agonists in the G -cAMP accumulation assay, while
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all being inactive in the -arrestin2 recruitment assay. This suggests that the positions of the
nitrogen in the pyridine and the ring substitutions can have a particularly important impact on both
cAMP production and -arrestin2 recruitment activity.
Table 1. SFSR of the RHS Moiety of Compound 1.
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G
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pEC50
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Bias for cAMP
SEM (%) accumulation
Cmpd
RHS
SEM
SEM (%)
9
0
.61 
.18
6.94 
1
0.25
5.8
61  3
110
2,600
180
48  3
89  4
63  3
N.A.
7.7
4.1
1.3
N.C
N.C
0.16
8
8
5
.24 
.11
5.59 
0.10
4
5
6
7
63  2
95  1
44  2
91  1
0
.57 
.03
6.75 
0.09
2.7
0
.99 
.07
1,000
22
N.A.
N.A.
0
7.66 
.04
>10,000
<5.00
N.D.
0
7.91 
.12
8
9
12
1.2
13
67  3
61  2
67  2
>10,000
N.A.
<5.00
N.A.
N.D.
N.A.
N.C
N.C
1.2
0
8.93 
.15
0
7.89 
.09
5.95 
0.08
10
1,100
64  3
0
1
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9
7
0
.12 
.06
1
1
77
80  2
59  3
N.D.
>10,000
N.A.
<5.00
N.A.
N.D.
N.A.
N.A.
N.C
N.C
N.C
5.88 
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
12
13
1,300
0
.08
>10,000
<5.00
N.A.
N.A.
EC50 and Emax values are the mean of at least three independent experiments performed in triplicate
with standard error of the mean (SEM) values. N.A. no activity at maximum concentration tested
-4
(
10 M), N.D. not determined, N.C. not calculable.
SFSR of the Middle Phenyl Ring
To determine the effects of the middle phenyl ring on functional selectivity, we designed
and synthesized a set of compounds with differentially substituted phenyl rings using the synthetic
route outlined in Scheme 2. Various commercially available bromo phenols were substituted onto
4
2
the same pyridofuran LHS group of 1, using previously described methods. The bromo group of
the resulting intermediates was subsequently converted to a boronic ester before Suzuki coupling
with the bromo imidazopyrazine RHS of 1 to afford the corresponding desired products in
moderate to good yields.
Scheme 2. Synthesis of compounds for exploring the middle phenyl ring moiety.a
1
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2
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
A few interesting SFSR trends emerged from this series of analogues. First, changing the
position of the methyl group from ortho to meta relative to the RHS, as in 14, dramatically reduced
potency in the G -cAMP accumulation assay (EC50 = 610 nM; EMAX = 62%) and completely
S
eliminated -arrestin2 recruitment activity. A dimethyl ring substitution at both the ortho and meta
positions were introduced in compound 15 and resulted in an intermediate activity profile. In the
G
S
-cAMP accumulation assay, 15 was found to be more potent than 14, but less potent than 1
EC50 = 55 nM; EMAX = 54%). Compound 15 was also inactive in -arrestin2 recruitment, similar
to 14. Replacing the methyl group of 1 with an amino group resulted in compound 16, which was
characterized as a potent, full agonist of the G signaling pathway (EC50 = 1.29 nM; EMAX = 104%),
while also displaying good potency and efficacy in recruiting -arrestin2 (EC50 = 209 nM; EMAX
81%) (bias factor for cAMP accumulation: 2.7). In compound 17, this substitution instead
(
S
=
featured a trifluoromethyl group. This single change resulted in potent, full agonism of both the G
protein (EC50 = 1.73 nM; EMAX = 94%) and -arrestin2 (EC50 = 230 nM; EMAX = 103%) assays
(
bias factor for cAMP accumulation: 1.6), comparable to dopamine (Figure 2E). The enhanced
efficacy of 16 and 17 in stimulating the G protein pathway may in part be due to the amino and
1
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trifluoromethyl groups being slightly bulkier and more electronegative, possibly leading to an
4
3
increased energy barrier preventing torsion in this biaryl ring system. When this trifluoromethyl
substitution was moved over one position to be meta to the RHS (compound 18), the resulting
ligand was completely devoid of activity in both the G protein and -arrestin2 recruitment assays.
Interestingly, however, difluoro substitutions at both of the positions meta to the RHS, as in
compound 19, eliminated -arrestin2 recruitment activity but preserved partial agonist activity in
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
promoting G -cAMP accumulation (EC50 = 143 nM; EMAX = 66%).
S
The above findings from our SFSR studies of the middle phenyl ring suggest that the
substituent on the phenyl ring positioned ortho, but not meta, to the RHS is important for the
observed potency and efficacy in the G signaling pathway.
S
Table 2. SFSR of the Middle Phenyl Ring Moiety of Compound 1.
G
S
-cAMP
Emax
SEM
%)
훃-arrestin2
pEC50
 SEM

pEC 
SEM
50
Middle Phenyl EC50
EC50
(nM)
E
max

Bias for cAMP
SEM (%) accumulation
Cmpd
Ring
(nM)
(
9
0
.61 
.18
6.94 
0.16
1
0.25
61  3
110
48  3
7.7
6.22 
.05
14
610
62  2 N.A.
N.A.
N.A.
N.C.
0
1
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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
7
0
.26 
.11
1
5
55
1.3
54  2 N.A.
104  1 210
N.A.
N.A.
81  3
103  2
N.A.
N.C.
2.7
8.89 
6.68 
0.10
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
16
17
0
.04
8.76 
6.64 
0.06
1.7
94  1
230
1.6
0
.05
1
8
9
N.A.
140
N.A.
N.A.
N.A.
N.A.
N.A.
N.C.
N.C.
6.85 
0
1
66  1 N.A.
N.A.
.04
EC50 and Emax values are the mean of at least three independent experiments performed in triplicate
with standard error of the mean (SEM) values. N.A. no activity at maximum concentration tested
-4
(
10 M), N.D. not determined, N.C. not calculable.
SFSR of the Linking Heteroatom
A series of ligands were generated to explore the role of the linking heteroatom in D R
1
activity and functional selectivity. This series was synthesized using the protocol outlined in
Scheme 3 and essentially involved replacing the linking oxygen atom with a variety of secondary
and tertiary amine analogues. The commercially available bromo aniline was substituted onto the
same pyridofuran group, using similar conditions above. The bromo group of the resulting
intermediate was then replaced with a boronic ester before coupling with the same bromo
1
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imidazopyrazine RHS to afford compound 20, which was then alkylated to yield compounds 21 –
2
5 in moderate to good yields.
Scheme 3. Synthesis of compounds for exploring the linking heteroatom moiety.a
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
Compound 20, which featured an unsubstituted secondary amino group linking the middle
phenyl ring and LHS moieties, was more G protein-biased than 1 as it became only minimally
active in the -arrestin2 recruitment assay but displayed moderately potent, full agonism in the
S
G -cAMP accumulation assay (EC50 = 16 nM; EMAX = 98%). We next sought to explore the
immediate space around this amino group. We prepared several analogues (21 – 25) featuring
tertiary amines alkylated with side groups of varying size as the linking heteroatom moiety. The
addition of a methyl group to the secondary amine to produce 21 reduced the compound’s potency
and efficacy in activating the G pathway (EC50 = 88 nM; EMAX = 79%). Methylating this amine
S
did not alter potency for -arrestin2 as both 20 and 21 were completely inactive in the -arrestin2
recruitment assay. Increasing the size of alkyl group revealed a size tolerance limit to retain
potency towards G
intermediate potency in the G
larger alkyl group at this position, such as isopropyl (23) (EC50 = 100 nM; EMAX = 67%),
S
-cAMP accumulation. An ethyl group at this position (22) possessed an
S
-cAMP accumulation assay (EC50 = 36 nM; EMAX = 80%). Any
1
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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
methylcyclopropyl (24) (EC50 = 320 nM; EMAX = 78%), or cyclopentyl (25) (EC50 = 1,100 nM;
E
MAX = 68%), significantly reduced potency and moderately reduced efficacy towards G activity.
S
Comparing to the ether linker in 1, the amino group linkers in 20 – 25 dramatically reduced potency
towards stimulating -arrestin2 recruitment, rendering the compounds inactive for -arrestin2
recruitment.
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
Our SFSR studies indicate that replacing the linking oxygen with unsubstituted and various
substituted amino groups is not well-tolerated, as these changes reduced this scaffold’s potency
towards G protein activation while eliminating ability to recruit -arrestin2. This suggests that
perhaps this region is spatially confined within the D R binding pocket, or that an electronic
1
interaction with the heteroatom at this position is important for the binding modality of this
scaffold, with an oxygen atom being the preferred entity over both a secondary and tertiary amine.
Table 3. SFSR of the Linking Heteroatom Moiety of Compound 1.
G
S
-cAMP
훃-arrestin2
pEC50
SEM

E
max

SEM
(%)
pEC50
SEM

Linking
Heteroatom
EC50
(nM)
EC50
(nM)
E
max

Bias for cAMP
SEM (%) accumulation
Cmpd
9
0
.61 
.18
6.94 
0.16
1
0.25
61  3
114
48  3
7.7
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7
8
9
7
0
.81 
.04
2
0
16
88
98  1 >10,000
<5.00
N.A.
N.D.
N.A.
N.A.
N.A.
N.A.
N.D.
N.C.
N.C.
N.C.
N.C.
N.C.
N.C.
7.05 
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
21
22
79  1
80  1
67  3
78  1
N.A.
N.A.
N.A.
N.A.
0
.03
7.45 
36
N.A.
0
.05
6.98 
23
24
25
100
320
1,100
N.A.
0
.09
6.50 
.03
N.A.
0
5.94 
68  3 >10,000
<5.00
0.07
EC50 and Emax values are the mean of at least three independent experiments performed in triplicate
with standard error of the mean (SEM) values. N.A. no activity at maximum concentration tested
-4
(
10 M), N.D. not determined, N.C. not calculable.
SFSR of the LHS Heterocycle
A fourth set of compounds was synthesized to explore the SFSR of the LHS portion of the
non-catechol scaffold. This compound set preserved the RHS, middle phenyl ring, and linking
heteroatom moieties from compound 1, while installing diverse heterocyclic entities on the LHS
of the scaffold. These compounds were all synthesized from commercially available starting
materials according to the synthetic routes outlined in Scheme 4. Various commercially available
1
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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
chlorinated heterocycles were reacted with 4-bromo-3-methylphenol, using previously described
4
2
methods. The bromo group of the resulting intermediates was converted to a boronic ester before
Suzuki coupling with the bromo imidazopyrazine RHS of 1 to afford the desired products in
moderate to good yields.
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
Scheme 4. Synthesis of compounds for exploring the LHS heterocyclic moiety.a
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Cyclopropyl group substitution at the 3-position on the furopyridinyl LHS (26) dramatically
reduced potency for G
arrestin2 recruitment activity as well. The roles of the LHS pyridine nitrogen was examined with
benzofuro compound 27. Omission of the nitrogen in 27 resulted in a reduction in potency for G
S
-cAMP production (EC50 = 62 nM; EMAX = 67%) and diminished -
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
S
-
cAMP accumulation (EC50 = 79 nM; EMAX = 69%) and inactivity for -arrestin2 recruitment.
Switching the furopyridinyl group of 1 to an isoquinolinyl moiety of 28 was also inactive for -
arrestin2 recruitment but retained more potency for G
MAX = 82%). Heteroatom placement within the bicyclic aromatic LHS ring was explored with the
next set of compounds. A thienopyridinyl moiety was introduced in 29, which was found to be a
potent full agonist of the G pathway (EC50 = 0.23 nM; EMAX = 100%) and a potent partial agonist
for -arrestin2 recruitment (EC50 = 22 nM; EMAX = 63%) (bias factor for cAMP accumulation:
.9). When this entity was replaced with a pyrrolopyrazinyl moiety (30), the activity in the G
S
-cAMP accumulation assay (EC50 = 14 nM;
E
S
1
S
-
cAMP accumulation assay was retained (EC50 = 0.87 nM; EMAX = 83%), while -arrestin2
recruitment activity dropped (EC50 = 690 nM; EMAX = 59%) (bias factor for cAMP accumulation:
1
5). However, the addition of a third nitrogen to create an imidazopyrazinyl moiety in this position
(
31) was not well-tolerated as potency for the G pathway was dramatically reduced (EC50 = 260
S
nM; EMAX = 85%) and -arrestin2 recruitment was undetectable. Rearrangement of the three
nitrogen atoms in this ring to generate a pyrazolopyridinyl on the LHS (32) almost fully restored
G
S
-cAMP accumulation activity (EC50 = 1.8 nM; EMAX = 104%). -arrestin2 recruitment activity,
however, was not recovered, making 32 a highly G -biased D R agonist. Our last set of compounds
in this series sought to briefly explore a few pyridinyl groups as the LHS. The unsubstituted
pyridinyl group led to a moderately potent ligand 33 with near full agonism in the G -cAMP
accumulation assay (EC50 = 120 nM; EMAX = 83%) and very weak activity in the -arrestin2
S
1
S
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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
recruitment assay. The addition of a bromine substituent at the 3-position (34) rendered the
scaffold completely inactive in both pathways. When a disubstituted 3-methyl-4-hydroxy analogue
(35) that mimicked an opened ring version of 1 was examined, only weak partial agonist activity
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
was observed in the G
S
-cAMP accumulation assay (EC50 = 160 nM; EMAX = 34%). Finally, a
similar effect on G -cAMP accumulation activity (EC50 = 640 nM; EMAX = 26%) was observed
S
with a piperidinyl substituted pyridinyl analogue (36). All of these pyridinyl analogues were also
inactive in -arrestin2 recruitment assays.
Table 4. SFSR of the LHS Moiety of Compound 1.
G
S
-cAMP
훃-arrestin2
pEC50
SEM

E
max

pEC50
SEM

E
SEM
max

EC50
EC50
(nM)
Bias for cAMP
accumulation
Cmpd
LHS
SEM
(
(nM)
%)
(%)
9
0
.61 
.18
6.94 
0.16
1
0.25
61  3
67  2
69  1
110
N.A.
N.A.
48  3
N.A.
7.7
7
7
7
.21 
0
26
27
62
79
15
N.A.
N.A.
N.C.
N.C.
N.C.
.08
.10 
0
N.A.
.05
.84 
28
82  2 >10,000 <5.00
N.D.
0
.06
2
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.64 
100 
1
7.67 
0.10
2
9
0.23
0.87
260
1.8
22
63  2
59  4
N.A.
1.9
15
0.05
.06 
6.16 
0.19
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
30
31
83  2
85  1
690
N.A.
0.09
.59 
N.A.
N.C.
N.C.
N.C.
N.C.
N.C.
0.03
.76 
.04
104 
1
32
33
34
35
>10,000 <5.00
N.D.
0
6.91 
.06
120
83  2 >10,000 <5.00
N.D.
0
>10,000 <5.00
N.D.
N.A.
N.A.
N.A.
N.A.
N.A.
6.79 
160
640
34  1
N.A.
0
.11
6.19 
36
26  1
N.A.
N.A.
N.A.
N.C.
0.07
EC50 and Emax values are the mean of at least three independent experiments performed in triplicate
with standard error of the mean (SEM) values. N.A. no activity at maximum concentration tested
-4
(
10 M), N.D. not determined, N.C. not calculable.
Hybrid SFSR Exploration
2
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1
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3
3
3
4
4
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4
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4
4
4
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5
5
5
5
5
5
5
5
5
5
6
During our SFSR campaign of the RHS portion of this non-catechol scaffold, we
discovered compound 5, a close analogue of 1 that featured an unsubstituted imidazopyridine
moiety and displayed enhanced efficacy and comparable potency in both downstream pathways
relative to its parent compound. Given these robust activities, we wanted to explore whether our
initial SFSR trends with 1 were consistent with 5 and whether this scaffold could also be altered
to display considerably different functional selectivity profiles.
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
First, a set of analogues of 5 were generated by modifying the methylation pattern on the
middle phenyl ring of the scaffold using the same synthetic route outlined in Scheme 2. Various
commercially available bromo phenols were substituted onto the same pyridofuran LHS group of
4
2
5, using previously described methods. The bromo group of the resulting intermediates was
subsequently converted to a boronic ester before Suzuki coupling with the bromo imidazopyridine
RHS of 5 to afford the desired products in moderate to good yields.
Similar to before, migration of the methyl substituent on the middle phenyl ring to the meta
position relative to the RHS (37) severely dampened G activity (EC50 = 530 nM; EMAX = 29%)
S
and eliminated -arrestin2 recruitment activity. A more mild reduction in G protein activity (EC50
= 110 nM; EMAX = 30%) was observed for the substituted dimethyl analogue, 38, while -arrestin2
recruitment was eliminated. Interestingly, this scaffold was completely inactive when both methyl
groups were moved ortho to the RHS, as in 39.
When the methyl substituent on 1 was converted to an amino group, the resulting
compound 16 possessed high potency and efficacy in both signaling pathways, however, when this
same conversion was implemented starting with 5, the resulting analogue (40) was a G
compound with full agonistic activity towards G -cAMP accumulation (EC50 = 10 nM; EMAX
1%) and relatively weak -arrestin2 recruitment activity (EC50 = 790 nM; EMAX = 54%) (bias
S
-biased
S
=
9
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factor for cAMP accumulation: 1.7). Conversely, converting the methyl substituent on the middle
phenyl ring of 5 into a trifluoromethyl group (compound 41) closely recapitulated the functional
selectivity profile of the analogue 17 in both pathways (bias factor for cAMP accumulation: 1.5).
When this trifluoromethyl substitution was moved meta to the RHS (compound 42), the resulting
ligand was inactive in both G protein and -arrestin2 recruitment assays. Difluoro substitutions at
both of the positions meta to the RHS (compound 43) showed moderate partial agonist activity in
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
the G -cAMP accumulation assay with an EC50 = 190 nM and EMAX = 57%, and minimal -
S
arrestin2 recruitment.
Table 5. SFSR of the Middle Phenyl Ring Moiety of Compound 5.
G
S
-cAMP
훃-arrestin2
pEC50
SEM

E
max

SEM
%)
pEC50
SEM

E
SEM
max

Middle Phenyl
Ring
EC50
(nM)
EC50
(nM)
Bias for cAMP
accumulation
Cmpd
(
(%)
8
0
.57 
.03
6.75 
0.09
5
2.7
530
110
95  1
29  1
30  1
180
N.A.
N.A.
63  3
1.3
N.C.
N.C.
6.27 
.09
3
7
8
N.A.
N.A.
N.A.
0
6.96 
0.13
3
N.A.
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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
3
9
N.A.
10
N.A.
N.A.
91  1
94  1
N.A.
N.A.
790
270
N.A.
N.A.
N.A.
54  3
93  5
N.A.
N.C.
1.7
8
0
.00 
.05
6.10 
0.11
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
40
41
8.64 
6.57 
0.12
2.3
1.5
0.03
4
2
3
N.A.
190
N.A.
N.A.
N.C.
N.C.
6.71 
4
57  1 >10,000
<5.00
N.D.
0.04
EC50 and Emax values are the mean of at least three independent experiments performed in triplicate
with standard error of the mean (SEM) values. N.A. no activity at maximum concentration tested
-4
(
10 M), N.D. not determined, N.C. not calculable.
Because the goal of this SFSR study was to explore the structural determinants of the non-
catechol scaffold’s potency and efficacy in both the G
S
and -arrestin2 pathways at D R, we
1
wanted to use one of the more balanced ligands we developed with potent full agonism in both
pathways to further explore this topic. We decided to use 41 for this purpose because it featured
slight structural modifications on the RHS and middle phenyl moieties that led to potent full
agonism of both the G and -arrestin2 pathways. The compound set derived from 41 preserved
S
the RHS, middle phenyl ring, and linking heteroatom moieties from compound 41, while making
diverse modifications to the heterocyclic entities on the LHS portion of the scaffold. Compounds
2
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in this set were all synthesized from commercially available starting materials using the synthetic
routes similar to the ones outlined in Scheme 4. Various commercially available chlorinated
heterocycles were reacted with 4-bromo-3-trifluoromethylphenol, using previously described
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
42
methods. The bromo group of the resulting intermediate was converted to a boronic ester before
Suzuki coupling with the bromo imidazopyridine RHS of 41 to afford the desired products in
moderate to good yields.
Substitution on the 3-position of the LHS pyridofuran moiety with a cyclopropyl group
(
44) abolished -arrestin2 recruitment and dramatically hindered G pathway activation (EC50 =
S
770 nM; EMAX = 83%), suggesting spatial constraints in this binding region. G protein activity was
only mildly reduced (EC50 = 67 nM; EMAX = 61%) and -arrestin2 recruitment still eliminated for
compound 45 in which the furan moiety was replaced with a phenyl group on the LHS bicycle,
suggesting that spatial or electronic properties on this portion of the LHS may be important for
binding.
When heteroatom substitutions and rearrangements were made within the LHS core of 41,
the scaffold tended to become more G
the LHS (46), G -cAMP accumulation activity showed mild attenuation (EC50 = 2.2 nM; EMAX
3%) while -arrestin2 recruitment activity was greatly weakened (Figure 2F). A similar profile
was also observed for the pyrrolopyrazine-containing analogue 47. Compound 47 retained full
agonism towards G -cAMP accumulation with a slight reduction in potency (EC50 = 7.8 nM; EMAX
78%), while -arrestin2 recruitment activity was completely abrogated. Only moderate partial
agonist activity was observed in the G -cAMP accumulation assay for 48, which featured an
imidazopyrazine moiety on the LHS (EC50 = 180 nM; EMAX = 54%). The pyridine-containing
analogue 49 displayed modest partial agonist activity in the G pathway (EC50 = 270 nM; EMAX
S
-biased. When a thienopyridine analogue was installed on
S
=
9
S
=
S
S
=
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1
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1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
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5
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5
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5
5
5
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5%) and no activity in the -arrestin2 pathway. Finally, the substituted pyridine analogue 50 was
determined to be inactive in both the G
that the electronic and spatial properties of 41’s LHS moiety are critical for maintaining its potent
-arrestin2 recruitment activity, as many rearrangements and substitutions disproportionately
impacted -arrestin2 recruitment over G -cAMP accumulation activity.
S
and -arrestin2 pathways. Together, these findings suggest
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

S
In light of the fact that our SFSR campaign resulted in numerous compounds with varying
functional selectivity profiles, an open question remains as to what degree of bias measured in
vitro is sufficient to observe significant change in the biological activity of this ligand class at D R
1
in vivo. An excellent example of this is G protein-biased agonism at the -opioid receptor (MOR),
which has shown the potential for improving therapeutic on-target effects for anti-nociception with
reduced constipation and respiratory depression effects.20, 22, 26-28 Among the most clinically-
advanced MOR G protein-biased agonists includes TRV130, a potent G protein-biased ligand that
recently completed phase 3 clinical studies for the treatment of acute pain. In in vitro studies,
TRV130 demonstrated nanomolar activation of G protein signaling (pEC50 = 8.1; Emax = 84%) and
20
diminished activity in recruiting -arrestin (pEC50 = 7.3; Emax = 15%). Several other examples
of MOR G protein-biased agonists show a correlation between their in vitro profile and reduced
2
3
side effects. While the novel biological effects of TRV130 and other MOR G protein-biased
agonists are attributed to their G protein bias at MOR, it is unclear about the degree of bias
threshold necessary to produce these significant effects in vivo, especially for other GPCRs. Future
studies using sets of tool compounds with a variety of bias profiles, both toward either G protein
or -arrestin, could help provide further insight into the degree of in vitro bias necessary for
therapeutic effects.
Table 6. SFSR of the LHS Moiety of Compound 41.
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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
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8
9
0
1
2
3
4
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8
9
0
1
2
3
4
5
6
7
8
9
0
G
S
-cAMP
훃-arrestin2
EC50 pEC50

E
max

EC50
(nM)
pEC50
SEM

E
max

Bias for cAMP
SEM (%) accumulation
Cmpd
LHS
(
nM)
SEM
SEM (%)
8.64 
0.03
6.57 
4
4
4
1
4
5
2.3
94  1
270
N.A.
N.A.
93  5
N.A.
N.A.
N.D.
N.A.
N.A.
N.A.
1.5
0.12
6
.11 
0.02
770
67
83  1
61  2
N.A.
N.A.
<5.00
N.A.
N.A.
N.A.
N.C.
N.C.
N.C.
N.C.
N.C.
N.C.
7
.18 
0.07
8
.66 
46
2.2
7.8
180
270
93  2 >10,000
0.06
8
.11 
47
48
49
78  2
54  1
65  1
N.A.
N.A.
N.A.
0.07
6
.74 
0.06
6.57 
0.05
50
N.A.
N.A.
N.A.
>10,000
<5.00
N.D.
N.C.
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2
3
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3
4
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5
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5
5
5
5
5
5
6
EC50 and Emax values are the mean of at least three independent experiments performed in triplicate
with standard error of the mean (SEM) values. N.A. no activity at maximum concentration tested
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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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
-4
(
10 M), N.D. not determined, N.C. not calculable.
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0
1
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3
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9
0
1
2
3
4
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6
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8
9
0
Figure 2. Full concentration-response curves for representative compounds and control
compounds measured using the Glosensor assay for Gs pathway activation (Gs-cAMP) and the
2
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