Discovery and Optimization of Novel SUCNR1 Inhibitors: Design of Zwitterionic Derivatives with a Salt Bridge for the Improvement of Oral Exposure

Article abstract of DOI:10.1021/acs.jmedchem.0c01020

G-protein-coupled receptor SUCNR1 (succinate receptor 1 or GPR91) senses the citric cycle intermediate succinate and is implicated in various pathological conditions such as rheumatoid arthritis, liver fibrosis, or obesity. Here, we describe a novel SUCNR1 antagonist scaffold discovered by high-throughput screening. The poor permeation and absorption properties of the most potent compounds, which were zwitterionic in nature, could be improved by the formation of an internal salt bridge, which helped in shielding the two opposite charges and thus also the high polarity of zwitterions with separated charges. The designed compounds containing such a salt bridge reached high oral bioavailability and oral exposure. We believe that this principle could find a broad interest in the medicinal chemistry field as it can be useful not only for the modulation of properties in zwitterionic compounds but also in acidic or basic compounds with poor permeation.

Full text of DOI:10.1021/acs.jmedchem.0c01020

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Article
Discovery and Optimization of Novel SUCNR1 Inhibitors: Design of
Zwitterionic Derivatives with a Salt Bridge for the Improvement of
Oral Exposure
Juraj Velcicky,* Rainer Wilcken, Simona Cotesta, Philipp Janser, Achim Schlapbach, Trixie Wagner,
Philippe Piechon, Frederic Villard, Rochdi Bouhelal, Fabian Piller, Stephanie Harlfinger, Rowan Stringer,
Dominique Fehlmann, Klemens Kaupmann, Amanda Littlewood-Evans, Matthias Haffke,
and Nina Gommermann
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ABSTRACT: G-protein-coupled receptor SUCNR1 (succinate receptor 1 or GPR91) senses the citric cycle intermediate succinate
and is implicated in various pathological conditions such as rheumatoid arthritis, liver fibrosis, or obesity. Here, we describe a novel
SUCNR1 antagonist scaffold discovered by high-throughput screening. The poor permeation and absorption properties of the most
potent compounds, which were zwitterionic in nature, could be improved by the formation of an internal salt bridge, which helped in
shielding the two opposite charges and thus also the high polarity of zwitterions with separated charges. The designed compounds
containing such a salt bridge reached high oral bioavailability and oral exposure. We believe that this principle could find a broad
interest in the medicinal chemistry field as it can be useful not only for the modulation of properties in zwitterionic compounds but
also in acidic or basic compounds with poor permeation.
succinate has been linked to several other pathological
conditions such as liver fibrosis,6 atherothrombosis,7 hyper-
tension,8,9 obesity,10 or retinal vascularization.11 Succinate also
plays an important role in inflammation as it serves as an
inflammatory signal inducing IL-1β through HIF1α stabiliza-
tion in macrophages.4 Furthermore, dendritic cells express high
levels of SUCNR1 and succinate proved to be required for
dendritic cell trafficking12 and Th17 cell expansion.13 In
arthritis patients, extracellular succinate accumulates in high
concentration in the synovial fluid.14 The importance of
INTRODUCTION
■
SUCNR1 (succinate receptor 1, initially named GPR91) is a
G-protein-coupled receptor (GPCR), which was deorphanized
in 2004 by He et al.,1 identifying succinate as its endogenous
ligand.2 Succinic acid is a small dicarboxylic acid deprotonated
under physiological conditions, thus acting in its anionic form,
succinate. While used under the code E363 as a dietary
supplement (flavor enhancer and acidifier), it is a crucial citric
acid cycle intermediate and therefore an important part of the
ATP formation and energy supply of the cell. As such,
succinate is normally located in the mitochondria. However,
under certain pathological conditions, succinate can be
released into the extracellular space and function as a signaling
molecule3 that is recognized as a danger signal4 by SUCNR1
located on the plasma membrane.
Received: June 15, 2020
Succinate and SUCNR1 signaling were implicated in local
stress conditions including ischemia, hypoxia, toxicity, and
hyperglycemia.2,5 Moreover, the activation of SUCNR1 by
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Figure 1. Known SUCNR1 antagonist 116 and HTS hit (2) to lead (5) optimization.
succinate in this disease could further be demonstrated using
SUCNR1-deficient mice that were protected not only from the
development of the disease but also from the activation of
macrophages and IL-1β production compared to the wild-type
animals.15 Moreover, SUCNR1-deficient mice demonstrated
reduced neutrophil infiltration, lower amounts of proinflam-
matory cytokines in the joints, and a decreased number of
antigen-specific Th17 cells.15 Based on these data, SUCNR1
represents an interesting pharmacological target and SUCNR1
antagonists could be useful for the treatment of various
diseases. Despite the importance of succinate and its receptor
in the pathology of various diseases, so far only one class of
SUCNR1 antagonists has been disclosed by the Advinus/
Merck group16 represented by compound 1 (Figure 1).
Interestingly, succinate analogs, which act as SUCNR1
agonists, were recently disclosed by two independent groups,
demonstrating that succinate seems to bind to SUCNR1 in its
cis-conformation.17−19 In this article, we describe the discovery
and optimization of a novel class of SUCNR1 antagonists for
which our group has recently disclosed an X-ray structure
resolving their binding into this GPCR.20 While in the previous
publication we focused on the structural biology aspects, here,
we describe hit-to-lead efforts as well as optimization of the
properties in our zwitterionic lead.
Table 1. Optimization of the Hit 2
RESULTS AND DISCUSSION
■
To identify novel SUCNR1 antagonists that are structurally
different from the known compound series, we performed a
high-throughput screening (HTS) campaign using CHEM-1
cells stably expressing human SUCNR1. Out of several
interesting hits identified, 2 (JC-59-GF68,20 Figure 1) stood
out due to its good drug-like properties,21 especially its rather
low molecular weight (381 Da), good water solubility
(>286 g/L), and low intrinsic clearance (25 μL/min/mg) in
rat liver microsomes. In addition, there was a clear preference
for the (S)-enantiomer (3, 12 μM, Table 1), while the
corresponding (R)-enantiomer (not shown) did not inhibit
SUCNR1 signaling even at 100 μM concentration. This
enantiodifferentiaton indicated real binding into SUCNR1,
making the scaffold rather appealing for further investigation.
During early hit optimization, the scaffold could be
simplified without losing potency by moving the carboxylate
onto the aryl ring while replacing the pyridine by a phenyl ring
(4). Further homologation of the carboxylate led to phenyl-
acetic acid 5 (PB-20-OV24,20 Figure 1) with almost 20-fold
improvement in SUCNR1 inhibition compared to 4 (Table 1).
Compared to the original hit 2, the lead compound 5 also
displayed an improved permeability by 1.5 log units as
determined by the parallel artificial membrane permeability
assay (PAMPA)22 while keeping the lipophilicity in a rather
a
IC50 determined as a mean (n ≥ 3) in the [35S]GTPγS assay run
with membranes prepared from stably transfected SUCNR1 in human
b
CHEM-1 cells. Permeability determined by high-throughput
PAMPA.22
polar range (log D7.423 of 1.9). The addition of a fluorine atom
into the ortho-position of the amide group on B-ring (for the
annotation of the three rings, see Figure 1) was tested for its
B
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possible introduction of a conformational lock24 that could
further help in improving the potency. Unfortunately, this
proved not to be the case since both the fluorinated analog 6
and nonfluorinated 5 showed the same potency (1.3 μM,
Table 1). However, the permeability was slightly enhanced,
thus following a known effect of a fluorine atom placed into
close proximity of a polar NH such as an amide24 or pyrrole
group.25 Counterintuitively, the fluorine atom in 6 also led to
decreased lipophilicity (by 0.5 log unit), potentially as a result
of an increased dipole moment in such a molecule.26,27 This
was in line with the calculated dipole moment from quantum
chemical calculations being higher for 6 (4.8) than for 5 (3.1).
A fruitful modification of the scaffold could be achieved by the
introduction of basic amines in the para-position of the C-ring
because such analogs, as exemplified by 7 (NF-58-EJ40,20
Table 1), inhibited SUCNR1 with high potency. Unfortu-
nately, these derivatives became highly polar and hence poorly
permeable.
an additional chlorine atom in the para-position in the C-ring
of 8 (10) could rescue the SUCNR1 potency, whereas it
completely prevented binding of such analogs to FD but not to
FXIa (Table 2).
The negative effect of the chlorine atom on binding to FD
can be explained when analyzing the published X-ray structure
of 8 bound to FD (PDB: 6QMT, Figure 2a).28 Whereas the
C-ring is nicely filling the S1 pocket of this serine protease and
its benzylamine is interacting with Asp189 and Ser190 as well
as two water molecules, the Ser190 side chain is rather close to
the para-position of the C-ring with a distance of 3.4 Å. This
indicates that an additional chlorine atom in this position
would not be favorable, which was confirmed by docking of a
chloro-analog 10 into FD. This study demonstrated that to
accommodate the chlorine atom in 10, the compound had to
shift away from Ser190 by roughly 1 Å and, as a consequence,
the amine was not able to interact with Asp189 and Ser190
anymore (Figure 2b). Gratifyingly, compound 10 also showed
improved potency against SUCNR1 compared to the simple
chloro-analog 5, thus indicating a synergistic effect of these two
substituents for inhibition of SUCNR1. The meta-benzylamine
in FD- and FXIa-inhibitors was shown to be crucial for these
serine proteases28,29 as it participates in an interaction with an
essential aspartate.30 Therefore, our strategy to avoid cross-
reactivity with these proteases was to keep the potency-
boosting chlorine atom in the para-position as well as to probe
larger substituents in the meta-position varying the position of
the basic amine.
Due to a pronounced similarity of this scaffold to the
complement factor D (FD) inhibitors recently described by
our colleagues,28 it became obvious to test some of the meta-
benzylamine-containing FD inhibitors (8 and 9, Table 2) for
Table 2. Selectivity Profile of Selected Analogs against
Selected Serine Proteases
Similar to analog 7, the presence of the amine in compound
10 led to much lower permeability (logPAMPA: −5.3 cm/s)
compared to nonamine analog 6 (logPAMPA: −3.6 cm/s,
Table 3). The reason for such behavior could be in the
zwitterionic nature of such compounds.31−33 The carboxylic
acid in this scaffold was shown to be essential for binding to
SUCNR1 as it makes several interactions with the receptor:
Tyr261.39, Tyr792.64, and Arg2767.39 residues (Figure 3).20
Arg2767.39 (Arg2817.39 in human SUCNR1) is one of the
four amino acids implicated in the binding of succinate by
SUCNR11, which illustrates the importance of the carboxylic
acid group in these antagonists. The amine could potentially be
omitted to avoid the amphiphilicity of these inhibitors;
however, its presence not only helped in increasing the
potency (10 vs 5, Table 2) but the zwitterionic nature of the
compounds also had a positive effect on plasma protein
binding (PPB). Whereas the single ionic compound 5 was
bound tightly (>99%) to the human plasma, its zwitterionic
analog 10 displayed a reduced human PPB of 97.9%. Since the
free drug concentration is crucial for the action of a bioactive
compound,34 we wished to keep the zwitterionic nature of this
class of SUCNR1 inhibitors. Despite options to improve the
permeability and bioavailability of zwitterions by formulations,
e.g., using salts or delivery systems,35 we preferred to explore
possibilities for the improvement of the intrinsic properties
rather than to rely on such efforts.
a
IC50 determined as a mean (n ≥ 3) in the [35S]GTPγS assay run
with membranes prepared from stably transfected SUCNR1 in human
b
CHEM-1 cells. IC50 (n ≥ 2) determined by the recombinant human
complement factor D time-resolved fluorescence resonance energy
transfer (TR-FRET) assay.28 IC50 (n ≥ 2) determined by the human
c
factor XIa TR-FRET assay.29
their inhibition of SUCNR1. Although beneficial in the para-
position, as demonstrated by the potency of 7 and its X-ray
structure20 showing a direct interaction of the adjacent
piperazine nitrogen with E181.31, the basic amine in the
meta-position of the C-ring (8) proved much less potent
(27 μM) compared to 7 (0.025 μM) or even compound 5
(1.3 μM) missing the basic amine (Table 1). A second FD
inhibitor, the nonamide analog 9 (Table 2), lost SUCNR1
potency completely (>100 μM), while it was highly potent
against FD28 and also factor XIa (FXIa).29 Rather surprisingly,
As previously observed,28 the two close analogs 8 and 9
differ significantly in their permeability and oral bioavailability
despite showing similar clearance in mice (Table 3). This is
really interesting, especially because also the pKa values36 for
both compounds were measured to be rather similar (4.4 and
9.4 for 9; 4.0 and 9.5 for 8), indicating that at neutral pH both
of them should be doubly charged (zwitterionic) and hence
poorly permeable.37 Nevertheless, 9 with the methyleneoxy
linker was fully absorbed in mice, whereas the bioavailability of
C
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Figure 2. (a) Crystal structure of 8 in complex with factor D (PDB: 6QMT).28 The protein is in surface representation, the ligand as well as amino
acids/water involved in polar interactions with the ligand are shown as sticks. Yellow: polar interactions between the compound and the protein.
Magenta: the distance between the phenyl carbon of 8 in the para-position to Ser190 side chain equals 3.4 Å. (b) 10 (yellow sticks) docked in
factor D cavity (6QMT)28 superimposed on the co-crystal structure of 8 (green).
Table 3. ADME Properties of Selected Compounds
Figure 3. Key interactions of amide carbonyl and carboxylate in 7
with SUCNR1 (PDB: 6RNK).20
as well as the Arg953.29 backbone carbonyl of SUCNR120
(Figure 3). In human SUCNR1, these amino acids (Arg993.29
and His1033.33) were shown to be required for the binding of
its endogenous ligand succinate.1 Therefore, the amide
replacement by methyleneoxy linker (9) resulted in a complete
loss of potency against SUCNR1 (Table 2).
a
b
Permeability determined by high-throughput PAMPA.22 Oral
bioavailability calculated as the dose-normalized ratio of extravascular
AUCextrap to IV AUCextrap; both parameters were determined as a
mean of 3 animals (male C57BL/6 mice). Exposure (AUC; dn =
dose-normalized to 1 mg/kg) measured as a mean of three animals
c
Being puzzled by such a significant difference in the
permeability and oral absorption of the zwitterions 9 and 10,
low molecular weight X-ray structures of both compounds (as
HCl salts) were generated (Figure 4), which revealed a
substantial difference in the conformations of the two closely
related analogs. While 10 showed an HCl-mediated interaction
of the amine with the acid moiety, 9 displayed a charge-assisted
hydrogen bond between these two functional groups, which
corresponds to the formation of a salt bridge in the aqueous
phase. Intramolecular salt bridges as electrostatic interactions
are known to contribute to peptide stabilization, protein
folding, flexibility, and function.39 Similar to the effect of
intramolecular hydrogen bonds,40 we speculated that the
presence of the intramolecular salt bridge in 9 could explain its
superior permeation and absorption properties because it
would allow for significant shielding of the polarity of the two
charged groups. Apparently, the formation of the intra-
molecular salt bridge was feasible in 9 thanks to a greater
flexibility of the methyleneoxy linker, whereas in 10, the salt
bridge seemed to be prevented by the rigid amide bond. In
addition, even the HCl-mediated interaction in 10 did not
seem optimal (Figure 4). To get engaged in such an interaction
(HCl-mediated), the A-ring got significantly twisted (60°)
(male C57BL/6 mice) after po dosing (3 mg/kg) using
MC:Water:Tween80 (0.5:99:0.5) formulation. Clearance measured
d
as a mean of three animals (male C57BL/6 mice) after iv dosing (1
e
mg/kg) using an NMP:plasma (10:90) formulation. F and AUC po
dn determined in three animals (male C57BL/6 mice) after po dosing
(10 mg/kg) using 30% PEG300, 50% of (20% Cremophor EL), and a
f
PBS formulation.28 F and AUC po dn determined in three animals
(male C57BL/6 mice) after po dosing (10 mg/kg) using an
MC:water:Tween80 (0.5:99.4:0.1) formulation.28
its amide analog 8 was only 10% (Table 3). This comparison
could suggest that the amide might be responsible for these
unfavorable properties, a well-known phenomenon in
peptides.38 However, the good oral absorption of non-
zwitterionic amide 6, again with a comparable clearance to 8
(Table 3), demonstrated that the poor permeability and
bioavailability of 8 was not necessarily caused by the amide
group alone but rather by a combination of amide and the
zwitterion. Whereas the amide was not required for binding of
this scaffold to FD and could therefore be replaced by the
methyleneoxy linker, in our case, the amide carbonyl was
needed for a water-mediated interaction with His993.33 residue
D
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Figure 4. Structure of the hydrochlorides of 10 (left) and 9 (right) in the crystal. Hydrogen bonds are shown as dotted lines.
toward the amide bond,41 whereas the biaryl group was
Table 4. Optimization toward the Formation of a Salt
Bridge for Improved Permeability
unusually bent (10°)42 and the two phenyl rings displayed a
somewhat lower torsion angle than usual (24°). Of course,
such a conformation could also be caused by crystal packing;
nevertheless, it clearly demonstrated that the rigidity of the
amide group made it difficult in 10 to bring the two charged
groups into close proximity of 4 Å as required for a productive
electrostatic interaction.39
Based on these observations, we decided to explore the
feasibility of salt bridge formation within the amide scaffold to
study whether such an intramolecular salt bridge could rescue
the poor permeability and absorption properties of the amide
zwitterions. We envisaged to investigate different types of
amines attached to a meta-alkoxy group. Gratifyingly, the
simplest analog 11 retained SUCNR1 potency, although
permeability was not changed (Table 4).
Interestingly, conformationally restricted amines such as
azetidine (12), pyrrolidine (13), and the two possible
piperidine isomers (14 and 15) showed similar potency as
the flexible amine 11, indicating that the amino-substituent was
devoid of any crucial interaction with the receptor. With
respect to permeability, these derivatives also did not show any
improvement, except for 15 (Table 4). With only a marginal
change in the structure compared to the 4-piperidyl analog 14,
but also to pyrrolidine 13, the 3-piperidyl compound 15
demonstrated quite a remarkable increase (1 log unit) in
permeability as well as lipophilicity (Table 4). Based on
compound 9, this improvement was interpreted by a successful
formation of an intramolecular salt bridge being mediated by
the 3-piperidyloxy substituent but not by any other substituent
tried. Apparently, this particular substituent allowed for the
right geometry and distance to bring the two charged groups in
this scaffold into close proximity (<4 Å).39 As a consequence,
the intramolecular salt bridge may be responsible for shielding
of the charges, similar to an intramolecular hydrogen bond,43
and the compound may appear as much less polar and hence
more permeable than analogs with separated charges. Another
hint for the existence of the salt bridge in 15 arose from the
ionic profile for the two piperidine isomers. The pKa values for
the acid and base of 4-piperidine analog 14 were determined to
be 4.1 and 9.9, respectively, being virtually the same as for
3-piperidine 15 (4.1 and 9.4). For this reason, both
compounds were expected to be in the zwitterionic form at
neutral pH and, as such, to display similar permeation
properties. This was clearly not the case, as evidenced by the
observed log D and permeability data for these two analogs.
We concluded that to explain the difference in permeability for
these two analogs, 15 must have displayed a different
conformational behavior compared to 14, allowing for
a
IC50 determined as a mean (n ≥ 3) in the [35S]GTPγS assay run
with membranes prepared from stably transfected SUCNR1 in human
b
CHEM-1 cells. Permeability determined by high-throughput
PAMPA.22
shielding of the two opposite charges. Applying an in-house-
developed quantum chemical workflow (ReSCoSS, described
elsewhere)44 for the elucidation of conformational behavior
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and associated changes in polarity revealed that 14 and 15
populated remarkably different shapes in aqueous solution
(Figure 5). While 14 preferred an elongated conformation, 15
keeping good permeability and oral bioavailability. As a
consequence, 20 reached a very high oral exposure of 37 000
nmol·h/L (Table 5). Interestingly, the SUCNR1 potency was
not much influenced by these modifications.
To confirm the presence of the intramolecular salt bridge, a
low molecular weight X-ray structure of compound 20 was
generated (Figure 6). This structure confirmed our hypothesis,
i.e., 3-piperidyl is capable of participating in an internal salt
bridge with the carboxylate since the piperidine nitrogen was
within a favorable distance of 3.1 Å to the carboxylate oxygen.
In addition, compared to the X-ray structure of the analog 10,
the structure of 20 seemed closer to the expected minimum
conformation with ring A twisted by 42° toward the amide
bond, the biaryl group not being bent anymore, and the two
phenyl rings displaying a rather normal torsion angle of 54°.
Unfortunately, a poor species cross-reactivity was observed
for this scaffold, as the advanced inhibitors displayed virtually
no inhibition of rat and mouse SUCNR1. For example,
compound 20 showed only 23% inhibition of mouse and 56%
of rat SUCNR1 activity at 100 μM concentration, whereas IC50
on human SUCNR1 was 88 nM. Due to this drawback, these
compounds could not be tested in any of the rodent models of
arthritis or any other diseases. To understand the species
difference, an X-ray structure of 20 bound to SUCNR1 was
solved (Figure 7a), using a humanized rat construct recently
disclosed by our group.20 As already described,20 the rat
SUCNR1 proved to be more suitable for crystallization due to
the lack of glycosylation sites as well as higher biochemical
stability than human SUCNR1. Mutagenesis studies identified
a rat SUCNR1 double mutant (K18E, K269N) that allowed
for binding of human selective SUCNR1 inhibitor 7, thus
indicating the key residues responsible for the species
selectivity.20 Strikingly, the residues of the required two
mutations are of a very different polarity, especially the
mutation of K18 in rats into an oppositely charged glutamate
found in human SUCNR1. While the inhibitor 20 does not
make any direct interaction with E18 or N269 in the X-ray
structure (Figure 7a, in gray), in the case of E18, this may
actually happen in solution since the highest-scoring predicted
pose from docking (Figure 7a, in green) forecasts a salt bridge
interaction between the piperidine moiety of the ligand and
E18 by virtue of a simple rotation around the biaryl axis. Even
more strikingly, the two amino acid mutations (K18E and
K269N), i.e. away from lysines, decreased the positive formal
charge present in the binding site of the rat receptor. This is
clearly reflected in a marked change in electrostatics in the
binding site (Figure 7b,c), allowing the positively charged
piperidine moiety to be accommodated in the humanized
receptor.
Figure 5. Minimum-energy conformation in water at the TZVPD-
FINE19 level for (a) 14 and (b) 15 (first row) and associated
σ-surfaces (second row). The (R)-enantiomer was modeled for 15.
The folded conformation observed for 15 with the intramolecular salt
bridge leads to a drastic reduction in the overall polar surface area as
quantified by COSMO-3D-PSA.44
was predominantly folded with an intramolecular salt bridge
formed between the piperidyl moiety and the carboxylic acid
moieties. Comparing the polar surface areas derived from the
electrostatic surfaces of these two different structures
(COSMO-3D-PSA)44 as well as visual inspection of these
surfaces revealed that the salt bridge in 15 dramatically reduces
the polarity of both charged moieties. Even taking into account
that the permeating species may or may not correspond to the
minimum-energy conformer in water, the reduction of the
polar surface area observed here seems causative in allowing
for better permeation.
After the successful identification of a suitable substituent
forming the internal salt bridge in the zwitterionic amide
scaffold, the stereochemistry of chiral 3-oxopiperidine was
investigated for potency and physicochemical properties.
Surprisingly, both enantiomers were found to be equally
potent, even with similar properties, except for PPB. For
unknown reasons, PPB was measured to be lower for the
R-enantiomer as exemplified by 16 (Table 5) showing human
PPB of 96.0% and mouse PPB of 97.4%, while the binding of
its (S)-enantiomer (not shown) to human plasma was 98.6%
and mouse plasma 99.7%. Therefore, the (R)-enantiomer was
preferred for further exploration (Table 5). Adding a fluorine
atom into the B-ring (16) or C-ring (17) led to slightly
improved potency compared to 15. Both compounds displayed
good bioavailability and oral exposure in mice, which
supported our internal salt bridge hypothesis. The addition
of a second fluorine atom into the A-ring (para to the anilide-
NH) of 17 resulted in a metabolically more stable derivative
18, which led to a further significant (10-fold) improvement in
oral exposure compared to 17 (Table 5). Moving the fluoro-
substituent in the C-ring from the ortho- to the para-position
to the alkoxy substituent (19) caused an increase in clearance,
leading to diminished oral exposure despite reaching full
bioavailability. The addition of another fluorine atom into the
B-ring resulted in compound 20 with very low clearance while
Remarkably, this class of inhibitors embodied by 20 displays
a chameleonic nature,45 with the extended conformation
binding to the receptor (Figure 7), while the closed
conformation with an intramolecular salt bridge seems to be
favorable in aqueous solution (quantum chemical studies,
Figure 5) as well as in the solid state for the isolated compound
(low-molecular-weight crystal structure, Figure 6). The
coexistence of the two classes of conformations that are
decidedly different in polarity46 in structures like 20 likely
allows transport across membranes during absorption in the
closed conformation, while these groups may be released when
binding to the target. We believe that this intriguing
characteristic of the zwitterions described in this publication
will find wider application in medicinal chemistry, especially in
F
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Table 5. Optimization of Piperidine-Based Intramolecular Zwitterion
a
IC50 determined as a mean (n ≥ 3) in the [35S]GTPγS assay run with membranes prepared from stably transfected SUCNR1 in human CHEM-1
cells. Permeability determined by high-throughput PAMPA.22 Oral bioavailability SD calculated as the dose-normalized ratio of extravascular
AUCextrap to IV AUCextrap; both parameters were determined as a mean of three animals (male C57BL/6 mice). Exposure (AUC; dn = dose-
b
c
d
normalized to 1 mg/kg) SD reported in nM·h and measured as a mean of three animals (male C57BL/6 mice) after po dosing (3 mg/kg) using
e
an MC:water:Tween80 (0.5:99:0.5) formulation. Clearance measured as a mean SD of three animals (male C57BL/6 mice) after iv dosing (1
mg/kg) using an NMP:plasma (10:90) formulation.
CHEMISTRY
■
The majority of compounds described in this publication were
prepared by a convergent route using a coupling of amide
building blocks with simple or complex C-rings (Scheme 3).
Whereas the amide building blocks 21 and 22 were made by a
direct acylation of the commercially available methyl 2-(2-
aminophenyl)acetate with corresponding benzoic acid chlor-
ides, their analogs 25 and 26 required an access to 2-(2-amino-
5-fluorophenyl)acetic acid (Scheme 1). This intermediate
could be reached from 5-fluorooxindole by opening the lactam
upon its heating with 10% aqueous NaOH47 and was
subsequently acylated with benzoic acid chlorides to provide
the corresponding amide acids 23 and 24 in good yields
(Scheme 1). These acids were then converted into
Figure 6. Structure of the hydrochloride of compound 20 in the
crystal. Hydrogen bonds are shown as dotted lines.
corresponding methyl esters 25 and 26, and all bromine
intermediates were then transformed into boronates (27−30)
(Scheme 1).
The C-ring building blocks were rather diverse (Scheme 2).
Whereas intermediate 31 could be made by Boc-introduction
into corresponding commercial benzylamine, the alkyloxy
derivatives (32−rac-36) were prepared by alkylation of
the design of new bioactive molecules containing basic or
acidic groups and lacking good permeability and/or oral
absorption properties.
G
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Figure 7. (a) X-ray crystal structure of 20 (grey carbons) bound to humanized rat SUCNR1 (PDB: 6Z10) overlaid onto the top-scoring binding
pose from docking (green carbons) forming salt bridge interaction with Glu18. (b) Electrostatic potential mapped onto the molecular surface for
humanized rat SUCNR1 and (c) for wild-type rat SUCNR1 (model based on PDB: 6Z10) showing a large concentration of positively charged
amino acid residues flanking the binding site. Calculations were done using the APBS plugin in PyMOL.
a
Scheme 1. Preparation of Amide Building Blocks
a
Reagents and conditions: (a) Et3N or N,N-diisopropylethylamine (DIPEA), CH2Cl2, 23 °C, 1 h (89−97%); (b) 10% NaOH (aq.), 100 °C, 16 h;
(c) THF/H2O, 23 °C, 16 h (76−92% over 2 steps); (d) MeOH, HCl, 23 °C, 16 h (86−98%); (e) (PinB)2, PdCl2dppf, KOAc, dioxane, 110 °C, 2 h
(62−94%).
5-bromo-2-chlorophenol. For acyclic alkoxy intermediate 32,
Boc-2-bromoethylamine was used, while for the cyclic
substituents, the appropriate mesylates were employed
(Scheme 2). In contrast, the enantiomerically pure
(R)-3-piperidyloxy building blocks (36−38) were accessed
by the Mitsunobu reaction48 of the (S)-N-Boc-3-piperidinol
and various phenols (Scheme 2).
hydrolysis and Boc-cleavage in 40 (Scheme 3). Derivatives
possessing diverse alkoxy-substituents (11−15) were made by
Suzuki coupling of boronate 27 and alkoxy-phenyl bromides
32−rac-36 followed by Boc-cleavage of the resulting acids 41−
45 (Scheme 3). In analogy, the enantiomerically pure
piperidyloxy analogs 16−20 were prepared via the correspond-
ing acid intermediates 46−50.
The assembly of different final products is summarized in
Scheme 3. Compound 4 was made by saponification of
compound 39, which was prepared from 4′-chloro-[1,1′-
biphenyl]-3-carboxylic acid by its activation into acyl chloride
and reaction with methyl 2-aminobenzoate. Derivative 6 was
synthesized by Suzuki coupling of 22 with p-chlorophenyl
boronate, whereas compound 10 was accessed from the boron
ester 27 and bromo-building block 31 followed by ester
CONCLUSIONS
■
In this paper, we described the discovery and optimization of a
new SUCNR1 antagonist scaffold. Starting from an HTS hit 2
(32 μM), the scaffold could be optimized to SUCNR1
antagonists with high potency such as, e.g. 7 (25 nM).
However, the new inhibitors displayed poor permeability and
oral absorption, possibly as a result of their zwitterionic nature
H
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a
Scheme 2. Preparation of C-Ring Building Blocks
a
Reagents and conditions: (a) Boc2O, DIPEA, CH2Cl2, 23 °C, 16 h (83%); (b) Cs2CO3, DMF, 120 °C, 6 h (91%); (c) Cs2CO3, DMF, 80 °C, 16 h
(16−72%); (d) Ph3P, diisopropyl azodicarboxylate (DIAD), THF, 60 °C, 16 h (42−77%).
due to the presence of the essential acid group and amine
required for potency and favorable properties. As demon-
strated by the pair of two closely related zwitterions 8 and 9
with very different absorption properties, the issue for poor
permeability and absorption could be caused by the separation
of the two adjacent, inversely charged groups as hypothesized
from the X-ray structures of 8 and 9. Whereas the flexible
methyleneoxy linker in the well-absorbed compound 9 allowed
for a salt bridge formation, its poorly permeable and
bioavailable analog 8 contained a rigid amide linker that
prevented the salt bridge formation. Since the amide makes key
interactions with SUCNR1, a search for suitable amines was
performed that would allow for the formation of the salt bridge
also in the amide subseries. This effort led to the identification
of the 3-piperidyloxy substituent in the C-ring that favored the
salt bridge formation as confirmed by the X-ray structure of 20.
According to our hypothesis, the formation of the internal salt
bridge provided derivatives with much improved oral
absorption. For example, the optimized analog 20 displayed
nearly full bioavailability and a very high oral exposure in mice
(37 μmol·h/L), also achieved thanks to its very low clearance
(0.6 mL/min/kg). Finally, the X-ray structure of 20 bound to
SUCNR1 demonstrated an excellent property of such
zwitterions, namely, their coexistence in two different
conformations. While the closed conformation is forced by
the salt bridge and may play an important role during
permeation, it allows for the breaking of the salt bridge, leading
to the stretched conformation required by the receptor. We
believe that this work allows for the further development of
SUCNR1 inhibitors, thanks to the presented SAR and
disclosure of the second X-ray structure of an antagonist
bound to SUCNR1. In addition, this work might also find a
wide interest in the medicinal chemistry community as it
describes the design of zwitterions with an internal salt bridge
that can be highly useful for the improvement of permeability
and/or oral absorption of new bioactive compounds
containing basic or acidic groups.
EXPERIMENTAL SECTION
■
Chemistry. All reagents and solvents were purchased from
commercial suppliers and used without further purification. All
reactions were performed under inert conditions (argon) unless
otherwise stated. NMR spectra were recorded on a Bruker 400 MHz
NMR spectrometer. Chemical shifts are reported in parts per million
(ppm) relative to an internal solvent reference. Significant peaks are
tabulated in the order of multiplicity (s, singlet; d, doublet; t, triplet;
q, quartet; quintet; m, multiplet; br, broad), coupling constants, and
the number of protons. Final compounds were purified to ≥95%
purity as assessed by analytical liquid chromatography:
LCMS method a. Waters UPLC Acquity; column: CORTECS C18
+ 2.7 μm, 2.1 mm × 50 mm at 80 °C, eluent A: water + 4.76%
isopropanol + 0.05% HCOOH + 3.75 mM NH4OAc, B: isopropanol
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a
Scheme 3. Preparation of the Final Products
a
Reagents and conditions: (a) (COCl)2, DMF (cat.), CH2Cl2, 23 °C, 2 h, then methyl 2-aminobenzoate, Et3N, CH2Cl2, 23 °C, 20 h (86%); (b)
LiOH, H2O, MeOH, 50 °C, 3−18 h (45−89%); (c) Pd(PPh3)4, NaHCO3, DME/H2O, 80 °C, 16 h (50−92%); (d) PdCl2dppf, Na2CO3, DMF/
H2O, 80 °C, 0.5 h (87%); (e) TFA, CH2Cl2, 23 °C, 4 h (57% over 2 steps); (f) HCl, dioxane/CH2Cl2, 23 °C, 16 h (53−95%).
J
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+ 0.05% HCOOH, gradient: 1−50% B in 1.4 min, 50−98% in 0.3
min. Flow: 1.0 mL/min.
LCMS method b. Waters UPLC Acquity; column: BEH C18 1.7
μm, 2.1 mm × 100 mm at 80 °C, eluent A: water + 4.76% isopropanol
+ 0.05% HCOOH + 3.75 mM NH4OAc, B: isopropanol + 0.05%
HCOOH, gradient: 1−60% B in 8.4 min, 60−98% in 1.0 min. Flow:
0.4 mL/min.
The preparation of compounds 2, 5, 7, 8, and 9 has been previously
described.20,28 Compound 3 was prepared according to the procedure
described for compound 2,20 using the commercially available (S)-3-
amino-3-(pyridin-3-yl)propanoic acid (Chem-Impex).
Preparation of Boronic Esters. A mixture of bromo-compound
(1 equiv), bis(pinacolato)diboron (1.1 equiv), and KOAc (2 equiv) in
dioxane (0.2 M concentration) was degassed with argon. After the
addition of PdCl2dppf·CH2Cl2 (0.1 equiv), the reaction mixture was
heated at 110 °C for 2 h. After being cooled to 23 °C, the reaction
mixture was filtered through Celite and washed with EtOAc. The
reaction mixture was then treated with water, the aqueous layer was
extracted with EtOAc, and the combined organic layers were dried
(Na2SO4) and concentrated. The residue was purified by column
chromatography to provide the boronates.
Phenol Alkylation by Mesylates. A mixture of 5-bromo-2-
chlorophenol (1 equiv), mesylate (1 equiv), and Cs2CO3 (1.1 equiv)
in DMF (0.05 M concentration) was stirred at 80 °C for 16 h. After
being cooled to 23 °C, the reaction mixture was diluted with ether
and washed with water and brine, dried (Na2SO4) and concentrated.
The crude product was purified by column chromatography to
provide the phenylalkyl ethers.
Phenol Alkylation by the Mitsunobu Reaction. (S)-1-N-Boc-
3-hydroxypiperidine (1 equiv) was added at 23 °C to a mixture of
phenol (1 equiv), DIAD (1.5 equiv) and PPh3 (1.5 equiv), in THF
(0.1 M concentration), and the resulting solution was stirred at 23 °C
for 16 h. The reaction mixture was concentrated and purified by
column chromatography to provide phenylalkyl ethers.
Suzuki Coupling. A mixture of the bromo intermediate (1 equiv),
boronate (1 equiv), and saturated NaHCO3 (5 equiv) in DME/water
(4:1, 0.04 M concentration) was degassed with argon. Pd(PPh3)4
(0.03 equiv) was then added, and the mixture was heated at 80 °C for
16 h. After being cooled to 23 °C, the beige suspension was diluted
with 0.2 M HCl and extracted with ethyl acetate. The organic layers
were washed with water and brine, dried (Na2SO4), and concentrated.
The crude material was purified by column chromatography to
provide the coupling products.
Boc-Cleavage with HCl. HCl (4 M) in dioxane (10 equiv) was
added at 0 °C to a solution of Boc-intermediate (1 equiv) in CH2Cl2
(0.05 M concentration). After being stirred at 23 °C for 16 h, the
formed suspension was diluted with ether and the solid was filtered
off, washed with ether, and dried in high vacuum to provide the amine
products as HCl salts.
methyl 2-(2-aminophenyl)acetate (1.9 g, 11.5 mmol) and DIPEA (4.1
mL, 23 mmol) in CH2Cl2 (30 mL). After being stirred at 23 °C for 1
h, the reaction mixture was treated with CH2Cl2 and 10% NaHCO3
(aq.). The water phase was extracted with CH2Cl2, and the organic
layers were washed with water and brine, dried (Na2SO4), and
concentrated. The crude product was purified by column chromatog-
raphy (0−25% ethyl acetate in cyclohexane) to provide the title
product as a yellowish solid in 4.1 g (97%) yield. 1H NMR (400 MHz,
DMSO-d6, δ) 10.06 (s, 1H), 7.88 (ddd, J = 8.2, 6.7, 1.8 Hz, 1H), 7.66
(ddd, J = 7.9, 6.3, 1.7 Hz, 1H), 7.45 (d, J = 8.0 Hz, 1H), 7.38−7.19
(m, 4H), 3.77 (s, 2H), 3.59 (s, 3H). 13C NMR (101 MHz, DMSO-d6,
δ) 171.2, 161.9, 155.2 (d, J = 249.3 Hz), 135.9, 135.5, 131.1, 123.0,
129.4 (d, J = 2.0 Hz), 127.5, 126.3, 126.2, 125.99, 125.97 (d, J = 13.1
Hz), 108.9 (d, J = 21.5 Hz), 51.6, 36.8. 19F NMR (376 MHz, DMSO-
d6, δ) −108.9. LCMS (method a) m/z: 366.0 [M + H]+, tR = 0.99
min.
2-(2-(3-Bromobenzamido)-5-fluorophenyl)acetic Acid (23). 5-
Fluoroindolin-2-one (5.0 g, 33.1 mmol) was dissolved in 10%
NaOH (aq.) (45 mL) and the mixture was stirred at 100 °C for 16 h.
After being cooled to 23 °C, concentrated HCl (8.5 mL) was added
and a half of the volume of this mixture was treated at 23 °C with the
solution of 3-bromobenzoyl chloride (2.92 g, 13.3 mmol) in THF (10
mL) and the reaction mixture was stirred at 23 °C for 16 h. The solid
was filtered off, washed with water, and dried in high vacuum to
1
provide the title product as a brown solid in 4.25 g (76%) yield. H
NMR (400 MHz, DMSO-d6, δ) 12.33 (s, 1H), 10.14 (s, 1H), 8.10 (t,
J = 1.9 Hz, 1H), 7.92 (dt, J = 7.8, 1.4 Hz, 1H), 7.80 (ddd, J = 8.0, 2.1,
1.0 Hz, 1H), 7.50 (t, J = 7.9 Hz, 1H), 7.40 (dd, J = 8.8, 5.6 Hz, 1H),
7.21 (dd, J = 9.6, 3.0 Hz, 1H), 7.15 (td, J = 8.6, 3.1 Hz, 1H), 3.65 (s,
2H). 13C NMR (101 MHz, DMSO-d6, δ) 172.0, 164.1, 159.8 (d, J =
242.3 Hz), 136.6, 134.3, 133.9 (d, J = 8.6 Hz), 132.7 (d, J = 2.7 Hz),
130.7, 130.3, 128.5 (d, J = 8.6 Hz), 126.8, 121.7, 117.5 (d, J = 22.8
Hz), 113.8 (d, J = 21.8 Hz), 37.3. 19F NMR (376 MHz, DMSO-d6, δ)
−116.8. LCMS (method a) m/z: 352.0 [M + H]+, tR = 0.84 min.
Methyl 2-(2-(3-Bromobenzamido)-5-fluorophenyl)acetate (25).
HCl (4 M) in dioxane (9.5 mL, 38 mmol) was added at 23 °C to a
solution of 2-(2-(3-bromobenzamido)-5-fluorophenyl)acetic acid (23,
3.4 g, 9.54 mmol) in MeOH (100 mL). The formed suspension was
stirred at 23 °C for 16 h. The reaction mixture was concentrated and
the crude product was purified by column chromatography (100%
CH2Cl2) to provide the title product as a yellowish solid in 3.0 g
1
(86%) yield. H NMR (400 MHz, DMSO-d6, δ) 10.09 (s, 1H), 8.08
(t, J = 1.8 Hz, 1H), 7.91 (dt, J = 7.9, 1.3 Hz, 1H), 7.80 (ddd, J = 8.0,
2.1, 1.0 Hz, 1H), 7.50 (t, J = 7.9 Hz, 1H), 7.37 (dd, J = 8.7, 5.5 Hz,
1H), 7.24 (dd, J = 9.6, 3.0 Hz, 1H), 7.17 (td, J = 8.5, 3.0 Hz, 1H),
3.74 (s, 2H), 3.52 (s, 3H). 13C NMR (101 MHz, DMSO-d6, δ) 170.8,
164.2, 159.9 (d, J = 242.5 Hz), 136.4, 134.3, 133.6 (d, J = 8.6 Hz),
132.6 (d, J = 2.9 Hz), 130.7, 130.3, 128.9 (d, J = 8.7 Hz), 126.7,
121.6, 117.5 (d, J = 22.9 Hz), 114.2 (d, J = 22.1 Hz), 51.7, 36.8. 19F
NMR (376 MHz, DMSO-d6, δ) −116.4. LCMS (method a) m/z:
366.0 [M + H]+, tR = 0.99 min.
Methyl 2-(2-(3-Bromobenzamido)phenyl)acetate (21). A solution
of 3-brombenzoylchlorid (18.0 g, 82 mmol) in CH2Cl2 (20 mL) was
added dropwise at 23 °C to a solution of methyl 2-(2-aminophenyl)-
acetate (15.2 g, 82 mmol) and Et3N (13.7 mL, 98 mmol) in CH2Cl2
(400 mL). After being stirred at 23 °C for 1 h, the reaction mixture
was concentrated and the residue was treated with t-BuOMe and
stirred at 23 °C for 16 h. The mixture was then cooled to 0 °C, and
the solid was filtered off and washed with n-hexane. The filtrate was
concentrated to 25% of the volume, and the suspension was filtered
off again and washed with n-hexane. Collected solids were dried in
high vacuum to provide the title product as a colorless solid in 25.6 g
2-(2-(3-Bromo-2-fluorobenzamido)-5-fluorophenyl)acetic Acid
(24). 5-Fluoroindolin-2-one (5.0 g, 33.1 mmol) was dissolved in a
10% solution of NaOH (aq.) (45.3 mL, 125 mmol) and the mixture
was stirred at 100 °C for 16 h. After being cooled to 23 °C,
concentrated HCl (8.5 mL) was added and a half of the volume of
this mixture was treated at 23 °C with the solution of 3-bromo-2-
fluorobenzoyl chloride (3.16 g, 13.3 mmol) in THF (10 mL) and the
reaction mixture was stirred at 23 °C for 16 h. The solid was filtered
off, washed with water, and dried in high vacuum to provide the title
1
1
(89%) yield. H NMR (400 MHz, DMSO-d6, δ) 10.09 (s, 1H), 8.09
product as a brown solid in 4.7 g (92%) yield. H NMR (400 MHz,
DMSO-d6, δ) 12.43 (s, 1H), 10.04 (s, 1H), 7.88 (t, J = 7.3 Hz, 1H),
7.67 (t, J = 7.0 Hz, 1H), 7.51−7.42 (m, 1H), 7.29 (t, J = 7.7 Hz, 1H),
7.25−7.10 (m, 2H), 3.68 (s, 2H). 13C NMR (101 MHz, DMSO-d6, δ)
171.8, 162.0, 159.7 (d, J = 242.4 Hz), 155.2 (d, J = 250.0 Hz), 135.5,
133.1 (d, J = 8.5 Hz), 132.2 (d, J = 2.8 Hz), 129.5 (d, J = 2.4 Hz),
128.1 (d, J = 8.4 Hz), 126.0 (d, J = 4.3 Hz), 125.8 (d, J = 3.1 Hz),
117.5 (d, J = 22.9 Hz), 113.9 (d, J = 22.0 Hz), 109.0, 36.9. 19F NMR
(376 MHz, DMSO-d6, δ) −108.7, −116.7. LCMS (method a) m/z:
372.1 [M + H]+, tR = 0.82 min.
(t, J = 1.8 Hz, 1H), 7.92 (dt, J = 7.8, 1.2 Hz, 1H), 7.80 (ddd, J = 8.0,
2.1, 1.0 Hz, 1H), 7.51 (t, J = 7.9 Hz, 1H), 7.40−7.29 (m, 3H), 7.25
(td, J = 7.3, 1.7 Hz, 1H), 3.74 (s, 2H), 3.52 (s, 3H). 13C NMR (101
MHz, DMSO-d6, δ) 171.4, 164.0, 136.6, 136.3, 134.3, 131.0, 130.9,
130.7, 130.3, 127.5, 126.9, 126.7, 126.3, 121.6, 51.6, 37.2. LCMS
(method a) m/z: 349.9 [M + H]+, tR = 0.98 min.
Methyl 2-(2-(3-Bromo-2-fluorobenzamido)phenyl)acetate (22).
A solution of 3-bromo-2-fluorobenzoyl chloride (3.0 g, 12.65 mmol)
in CH2Cl2 (10 mL) was added dropwise at 23 °C to a solution of
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Methyl 2-(2-(3-Bromo-2-fluorobenzamido)-5-fluorophenyl)-
acetate (26). HCl (4 M) in dioxane (11.7 mL, 46.7 mmol) was
added at 23 °C to a solution of 2-(2-(3-bromo-2-fluorobenzamido)-5-
fluorophenyl)acetic acid (24, 4.5 g, 11.7 mmol) in MeOH (100 mL),
and the formed suspension was stirred at 23 °C for 16 h. After
concentration, the crude product was purified by column chromatog-
raphy (100% CH2Cl2) to provide the title product as a pale yellow
yield. 1H NMR (400 MHz, DMSO-d6, δ) 9.91 (s, 1H), 7.83−7.74 (m,
2H), 7.43 (dd, J = 8.8, 5.6 Hz, 1H), 7.34 (t, J = 7.4 Hz, 1H), 7.22 (dd,
J = 9.6, 3.0 Hz, 1H), 7.16 (td, J = 8.5, 3.1 Hz, 1H), 3.77 (s, 2H), 3.61
(s, 3H), 1.32 (s, 12H). 13C NMR (101 MHz, DMSO-d6, δ) 170.7,
163.0, 162.9 (d, J = 254.6 Hz), 159.8 (d, J = 242.3 Hz), 138.7 (d, J =
8.4 Hz), 133.6 (d, J = 3.7 Hz), 132.7 (d, J = 8.5 Hz), 132.5 (d, J = 2.9
Hz), 128.3 (d, J = 8.7 Hz), 124.30, 124.19 (d, J = 3.7 Hz), 124.12,
117.5 (d, J = 22.9 Hz), 114.1 (d, J = 22.1 Hz), 83.9, 51.6, 36.5, 24.6.
19F NMR (376 MHz, DMSO-d6, δ) −103.8, −116.7. LCMS (method
a) m/z: 432.2 [M + H]+, tR = 1.16 min.
tert-Butyl 5-Bromo-2-chlorobenzylcarbamate (31). DIPEA (1.25
mL, 7.14 mmol) and Boc2O (1.26 mL, 5.40 mmol) were added at 23
°C to a solution of 5-bromo-2-chlorobenzyl amine (750 mg, 3.40
mmol) in CH2Cl2 (34 mL). After being stirred at 23 °C for 16 h, the
mixture was treated with saturated NaHCO3 (aq.) and extracted with
CH2Cl2. The organic layers were washed with water and brine, dried
(Na2SO4), and concentrated. The crude product was purified by
column chromatography (0−20% ethyl acetate in cyclohexane) to
provide the title product as a pale yellow solid in 905 mg (83%) yield.
1H NMR (400 MHz, DMSO-d6, δ) 7.50−7.44 (m, 2H), 7.44−7.42
(m, 1H), 7.40 (d, J = 8.5 Hz, 1H), 4.18 (d, J = 6.2 Hz, 2H), 1.41 (s,
9H). 13C NMR (101 MHz, DMSO-d6, δ) 155.7, 139.5, 131.2, 131.1,
131.0, 130.8, 120.0, 78.3, 41.0, 28.1. LCMS (method a) m/z: 220.1
[M + H-Boc]+, tR = 1.29 min.
1
solid in 4.4 g (98%) yield. H NMR (400 MHz, DMSO-d6, δ) 10.06
(s, 1H), 7.88 (ddd, J = 8.2, 6.7, 1.8 Hz, 1H), 7.65 (ddd, J = 7.8, 6.3,
1.7 Hz, 1H), 7.44 (dd, J = 8.8, 5.6 Hz, 1H), 7.30 (t, J = 7.9 Hz, 1H),
7.23 (dd, J = 9.6, 3.0 Hz, 1H), 7.18 (td, J = 8.5, 3.0 Hz, 1H), 3.78 (s,
2H), 3.59 (s, 3H). 13C NMR (101 MHz, DMSO-d6, δ) 170.7, 162.1,
159.8 (d, J = 242.6 Hz), 155.2 (d, J = 249.4 Hz), 135.5, 132.7 (d, J =
8.5 Hz), 132.2 (d, J = 2.5 Hz), 129.4 (d, J = 2.1 Hz), 128.3 (d, J = 8.7
Hz), 126.0 (d, J = 4.3 Hz), 125.8 (d, J = 16.5 Hz), 117.6 (d, J = 22.9
Hz), 114.2 (d, J = 22.2 Hz), 108.9 (d, J = 21.4 Hz), 51.7, 36.5. 19F
NMR (376 MHz, DMSO-d6, δ) −108.9, −116.4. LCMS (method a)
m/z: 384.1 [M + H]+, tR = 0.99 min.
Methyl 2-(2-(3-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-
benzamido)phenyl)acetate (27). According to the general procedure
for the preparation of boronesters, the reaction of methyl 2-(2-(3-
bromobenzamido)phenyl)acetate (21, 7.5 g, 21.5 mmol) and
bis(pinacolato)diboron (5.5 g, 21.5 mmol) provided the title product
after column chromatography (0−30% ethyl acetate in cyclohexane)
1
as a yellow sticky oil in 8.3 g (94%) yield. H NMR (400 MHz,
tert-Butyl (2-(5-Bromo-2-chlorophenoxy)ethyl)carbamate (32).
A mixture of 5-bromo-2-chlorophenol (0.50 g, 2.34 mmol), tert-butyl
(2-bromoethyl)carbamate (1.35 g, 5.84 mmol), and Cs2CO3 (1.92 g,
5.84 mmol) in DMF (4.7 mL) was stirred at 120 °C for 6 h. After
being cooled to 23 °C, the reaction mixture was diluted with ether
and washed with water. The organic phases were washed with 2 M
NaOH, water, and brine; dried (Na2SO4); and concentrated. The
crude product was purified by column chromatography (0−77% ethyl
acetate in n-heptane) to provide the title product as a colorless oil in
DMSO-d6, δ) 10.07 (s, 1H), 8.22 (s, 1H), 8.06−7.99 (m, 1H), 7.86
(dt, J = 7.4, 1.2 Hz, 1H), 7.54 (t, J = 7.6 Hz, 1H), 7.38−7.29 (m, 3H),
7.24 (td, J = 7.3, 1.7 Hz, 1H), 3.73 (s, 2H), 3.53 (s, 3H), 1.33 (s,
12H). 13C NMR (101 MHz, DMSO-d6, δ) 171.4, 165.5, 137.3, 136.7,
134.1, 133.6, 131.0, 130.9, 130.5, 128.0, 127.4, 126.9, 126.1, 83.9,
51.5, 37.2, 24.7 (the signal for boron α carbon not resolved). LCMS
(method a) m/z: 396.2 [M + H]+, tR = 1.11 min.
Methyl 2-(2-(2-Fluoro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-
lan-2-yl)benzamido)phenyl)acetate (28). According to the general
procedure for the preparation of boronesters, the reaction of methyl
2-(2-(3-bromo-2-fluorobenzamido)phenyl)acetate (22, 1.0 g, 2.7
mmol) and bis(pinacolato)diboron (0.77 g, 3 mmol) provided the
title product after column chromatography (0−30% ethyl acetate in
1
768 mg (91%) yield. H NMR (400 MHz, DMSO-d6, δ) 7.40−7.33
(m, 2H), 7.15 (dd, J = 8.4, 2.2 Hz, 1H), 6.94 (t, J = 5.8 Hz, 1H), 4.08
(t, J = 5.8 Hz, 2H), 3.34−3.30 (m, 2H), 1.38 (s, 9H). LCMS (method
a) m/z: 249.9 [M + H-Boc]+, tR = 1.36 min.
tert-Butyl 3-(5-Bromo-2-chlorophenoxy)azetidine-1-carboxylate
(33). According to the general procedure for phenol alkylation by
mesylates, the reaction of 5-bromo-2-chlorophenol (300 mg, 1.45
mmol) and 1-(tert-butoxycarbonyl)-3-(methansulfonyloxy)azetidine
(363 mg, 1.45 mmol) provided the title product after column
chromatography (0−20% ethyl acetate in cyclohexane) as a colorless
solid in 259 mg (45%) yield. 1H NMR (400 MHz, DMSO-d6, δ) 7.43
(d, J = 8.4 Hz, 1H), 7.21 (dd, J = 8.4, 2.2 Hz, 1H), 7.09 (d, J = 2.2 Hz,
1H), 5.19−5.04 (m, 1H), 4.33 (dd, J = 9.2, 7.2 Hz, 2H), 3.81 (dd, J =
9.7, 3.6 Hz, 2H), 1.39 (s, 9H). 13C NMR (101 MHz, DMSO-d6, δ)
155.5, 152.6, 131.7, 125.2, 121.0, 120.6, 116.8, 79.0, 67.0, 55.3, 28.0.
LCMS (method a) m/z: 262.1 [M + H-Boc]+, tR = 1.44 min.
tert-Butyl 3-(5-Bromo-2-chlorophenoxy)pyrrolidine-1-carboxy-
late (34). According to the general procedure for phenol alkylation
by mesylates, the reaction of 5-bromo-2-chlorophenol (100 mg, 0.48
mmol) and tert-butyl 3-((methylsulfonyl)oxy)pyrrolidine-1-carboxy-
late (128 mg, 0.48 mmol) provided the title product after column
chromatography (0−10% ethyl acetate in cyclohexane) as a colorless
resin in 136 mg (72%) yield. 1H NMR (400 MHz, DMSO-d6, δ) 7.45
(d, J = 2.2 Hz, 1H), 7.40 (d, J = 8.4 Hz, 1H), 7.19 (dd, J = 8.5, 2.2 Hz,
1H), 5.29−5.06 (m, 1H), 3.60−3.47 (m, 1H), 3.46−3.31 (m, 3H),
2.21−1.96 (m, 2H), 1.40 + 1.39 (s, 9H). 13C NMR (101 MHz,
DMSO-d6, δ) 153.7, 153.1, 131.5, 124.9, 122.1, 120.4, 118.6, 78.5,
(78.3 + 77.4), (51.2 + 51.0), (43.9 + 43.7), (30.8 + 30.0), 28.1.
LCMS (method a) m/z: 276.1 [M + H-Boc]+, tR = 1.44 min.
tert-Butyl 4-(5-Bromo-2-chlorophenoxy)piperidine-1-carboxy-
late (35). According to the general procedure for phenol alkylation
by mesylates, the reaction of 5-bromo-2-chlorophenol (300 mg, 1.45
mmol) and tert-butyl 4-((methylsulfonyl)oxy)piperidine-1-carboxy-
late (404 mg, 1.45 mmol) provided the title product after column
chromatography (0−10% ethyl acetate in cyclohexane) as a colorless
resin in 321 mg (57%) yield. 1H NMR (400 MHz, DMSO-d6, δ) 7.48
(d, J = 2.2 Hz, 1H), 7.39 (d, J = 8.5 Hz, 1H), 7.16 (dd, J = 8.5, 2.2 Hz,
1
cyclohexane) as a yellow sticky oil in 0.80 g (62%) yield. H NMR
(400 MHz, DMSO-d6, δ) 9.90 (s, 1H), 7.84−7.74 (m, 2H), 7.45 (d, J
= 7.7 Hz, 1H), 7.38−7.27 (m, 3H), 7.26−7.20 (m, 1H), 3.77 (s, 2H),
3.61 (s, 3H), 1.33 (s, 12H). 13C NMR (101 MHz, DMSO-d6, δ)
171.2, 163.0 (d, J = 254.5 Hz), 162.8, 138.7 (d, J = 8.4 Hz), 136.2,
133.6 (d, J = 3.6 Hz), 131.0, 130.0, 127.4, 126.3, 126.0, 124.4 (d, J =
17.2 Hz), 124.2 (d, J = 3.7 Hz), 83.9, 51.5, 36.8, 24.6 (the signal for
boron α carbon not resolved). 19F NMR (376 MHz, DMSO-d6, δ)
−103.8. LCMS (method a) m/z: 414.4 [M + H]+, tR = 1.09 min.
Methyl 2-(5-Fluoro-2-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-
lan-2-yl)benzamido)phenyl)acetate (29). According to the general
procedure for the preparation of boron esters, the reaction of methyl
2-(2-(3-bromobenzamido)-5-fluorophenyl)acetate (25, 700 mg, 1.9
mmol) and bis(pinacolato)diboron (534 mg, 2.1 mmol) provided the
title product after column chromatography (0−60% ethyl acetate in
1
cyclohexane) as a beige solid in 612 mg (77%) yield. H NMR (400
MHz, DMSO-d6, δ) 10.07 (s, 1H), 8.22 (s, 1H), 8.02 (d, J = 7.9 Hz,
1H), 7.87 (d, J = 7.3 Hz, 1H), 7.54 (t, J = 7.6 Hz, 1H), 7.36 (dd, J =
8.8, 5.6 Hz, 1H), 7.23 (dd, J = 9.6, 3.0 Hz, 1H), 7.16 (td, J = 8.5, 3.0
Hz, 1H), 3.74 (s, 2H), 3.54 (s, 3H), 1.33 (s, 12H). 13C NMR (101
MHz, DMSO-d6, δ) 170.9, 165.7, 159.9 (d, J = 242.2 Hz), 137.4,
133.9, 133.6, 133.5, 133.0 (d, J = 2.6 Hz), 130.6, 128.9, 128.8, 128.0,
117.5 (d, J = 22.9 Hz), 114.1 (d, J = 22.1 Hz), 83.9, 51.6, 36.9, 24.7.
19F NMR (376 MHz, DMSO-d6, δ) −116.8. LCMS (method a) m/z:
414.2 [M + H]+, tR = 1.15 min.
Methyl 2-(5-Fluoro-2-(2-fluoro-3-(4,4,5,5-tetramethyl-1,3,2-diox-
aborolan-2-yl)benzamido)phenyl)acetate (30). According to the
general procedure for the preparation of boron esters, the reaction of
methyl 2-(2-(3-bromo-2-fluorobenzamido)-5-fluorophenyl)acetate
(26, 1.76 g, 4.6 mmol) and bis(pinacolato)diboron (1.28 g, 5.0
mmol) provided the title product after column chromatography (0−
40% ethyl acetate in cyclohexane) as a beige solid in 2.1 g (86%)
L
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1H), 4.83−4.69 (m, 1H), 3.69−3.49 (m, 2H), 3.30−3.22 (m, 2H),
1.92−1.80 (m, 2H), 1.63−1.51 (m, 2H), 1.41 (s, 9H). 13C NMR (101
MHz, DMSO-d6, δ) 153.9, 153.2, 131.5, 124.7, 122.1, 120.5, 118.8,
78.8, 73.5, 40.2, 30.0, 28.0. LCMS (method a) m/z: 290.0 [M + H-
Boc]+, tR = 1.56 min.
tert-Butyl 3-(5-Bromo-2-chlorophenoxy)piperidine-1-carboxy-
late (rac-36). According to the general procedure for phenol
alkylation by mesylates, the reaction of 5-bromo-2-chlorophenol
(300 mg, 1.45 mmol) and tert-butyl 3-((methylsulfonyl)oxy)-
piperidine-1-carboxylate (404 mg, 1.45 mmol) provided the title
product after column chromatography (0−20% ethyl acetate in
cyclohexane) as a colorless solid in 91 mg (16%) yield. For analytical
data, see 36.
7.99−7.94 (m, 2H), 7.84−7.79 (m, 2H), 7.76−7.67 (m, 2H), 7.62−
7.57 (m, 2H), 7.28 (ddd, J = 8.1, 7.4, 1.2 Hz, 1H), 3.89 (s, 3H).
LCMS (method a) m/z 366.1 [M + H]+, tR = 1.41 min.
2-(4′-Chloro-[1,1′-biphenyl]-3-carboxamido)benzoic Acid (4).
LiOH·H2O (85 mg, 2.0 mmol) was added at 23 °C to a mixture of
methyl 2-(4′-chloro-[1,1′-biphenyl]-3-carboxamido)benzoate (39,
0.40 g, 1.0 mmol) in MeOH (8 mL) and water (2 mL). After
being stirred at 50 °C for 3 h, the mixture was concentrated and the
crude product was treated with concentrated HCl (11 mL). After
being stirred at 60 °C for 2 h and cooled to 23 °C, the suspension was
filtered off, washed with water, and dried in vacuum to provide the
1
product 4 as a white solid in 161 mg (45%) yield. H NMR (400
MHz, DMSO-d6, δ) 13.83 (s, 1H), 12.24 (s, 1H), 8.71 (d, J = 7.9 Hz,
1H), 8.21 (s, 1H), 8.07 (dd, J = 7.9, 1.5 Hz, 1H), 8.00−7.89 (m, 2H),
7.78 (d, J = 8.6 Hz, 2H), 7.74−7.63 (m, 2H), 7.58 (d, J = 8.5 Hz,
2H), 7.23 (td, J = 7.6, 1.2 Hz, 1H). 13C NMR (101 MHz, DMSO-d6,
δ) 170.0, 164.5, 141.0, 139.5, 138.2, 135.4, 134.3, 133.0, 131.3, 130.4,
129.8, 129.1, 128.6, 126.5, 125.1, 123.1, 120.0, 116.9. LCMS (method
a) m/z 352.3 [M + H]+, tR = 1.37 min. LCMS (method b) m/z: 352.2
[M + H]+, tR = 6.55 min. HRMS m/z: [M + H]+ calcd for
C20H15ClNO3 352.0735; found 352.0736.
2-(2-(4′-Chloro-2-fluoro-[1,1′-biphenyl]-3-carboxamido)phenyl)-
acetic Acid (6). According to the general procedure for Suzuki
coupling, the reaction of methyl 2-(2-(3-bromo-2-fluorobenzamido)-
phenyl)acetate (22, 150 mg, 0.41 mmol) and 2-(4-chlorophenyl)-
4,4,5,5-tetramethyl-1,3,2-dioxaborolane (117 mg, 0.49 mmol) pro-
vided the title compound after column chromatography (0−50% ethyl
acetate in cyclohexane) as a white powder in 130 mg (79%) yield.
1H NMR (400 MHz, DMSO-d6, δ) 12.35 (s, 1H), 9.98 (d, J = 1.6 Hz,
1H), 7.75−7.56 (m, 6H), 7.52 (d, J = 7.9 Hz, 1H), 7.42 (t, J = 7.6 Hz,
1H), 7.31 (t, J = 7.4 Hz, 2H), 7.22 (td, J = 7.7, 1.5 Hz, 1H), 3.69 (s,
2H). 13C NMR (101 MHz, DMSO-d6, δ) 172.4, 162.7, 155.9 (d, J =
251.2 Hz), 136.1, 133.3 (d, J = 41.2 Hz), 132.8 (d, J = 3.2 Hz), 131.0,
130.8 (d, J = 3.0 Hz), 129.9 (d, J = 47.3 Hz), 129.6, 128.7, 127.8 (d, J
= 14.2 Hz), 127.2, 125.9, 125.9, 125.3 (d, J = 16.5 Hz), 124.8 (d, J =
4.1 Hz), 37.2. 19F NMR (376 MHz, DMSO-d6, δ) −119.8. LCMS
(method a) m/z: 383.8 [M + H]+, tR = 1.12 min. LCMS (method b)
m/z: 384.2 [M + H]+, tR = 5.41 min. HRMS m/z: [M + H]+ calcd for
C21H16ClFNO3 384.0797; found 384.0797.
Methyl 2-(2-(3′-(((tert-Butoxycarbonyl)amino)methyl)-4′-chloro-
[1,1′-biphenyl]-3-carboxamido)phenyl)acetate (40). A mixture of
methyl 2-(2-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
benzamido)phenyl)acetate (27, 0.28 g, 0.67 mmol), tert-butyl
5-bromo-2-chlorobenzylcarbamate (31, 0.24 g, 0.67 mmol) and 2
M Na2CO3 (aq.) (1.0 mL, 2.0 mmol) in DMF (6.7 mL) was degassed
with argon. PdCl2dppf·CH2Cl2 (27 mg, 0.034 mmol) was added, and
the reaction mixture was heated in a microwave oven at 80 °C for 30
min. After being cooled to 23 °C, the mixture was filtered over Celite
and washed with ethyl acetate and the filtrate was concentrated. The
crude product was purified by column chromatography (0−80% ethyl
acetate in n-heptane) to provide the title product as a pale yellow
resin in 0.30 g (87%) yield. 1H NMR (400 MHz, DMSO-d6, δ) 10.09
(s, 1H), 8.18 (t, J = 1.9 Hz, 1H), 7.93 (dt, J = 7.8, 1.4 Hz, 1H), 7.84
(d, J = 7.8 Hz, 1H), 7.72−7.66 (m, 2H), 7.64 (d, J = 7.8 Hz, 1H),
7.57 (d, J = 8.7 Hz, 1H), 7.50 (t, J = 5.6 Hz, 1H), 7.43−7.39 (m, 1H),
7.38−7.30 (m, 2H), 7.25 (td, J = 7.4, 1.5 Hz, 1H), 4.28 (d, J = 6.1 Hz,
2H), 3.76 (s, 2H), 3.51 (s, 3H), 1.40 (s, 9H). 13C NMR (101 MHz,
DMSO-d6, δ) 171.4, 165.3, 156.0, 139.2, 138.3, 137.4, 136.5, 135.3,
133.4, 132.9, 131.0, 130.9, 129.7, 129.5, 129.2, 127.5, 126.92, 126.84,
126.80, 126.2, 125.8, 78.2, 51.6, 41.4, 37.2, 28.2. LCMS (method a)
m/z: 507.2 [M − H]−, tR = 1.26 min.
(R)-tert-Butyl 3-(5-Bromo-2-chlorophenoxy)piperidine-1-carbox-
ylate (36). According to the general procedure for phenol alkylation
by the Mitsunobu reaction, the reaction of (S)-1-N-Boc-3-
hydroxypiperidine (485 mg, 2.4 mmol) and 5-bromo-2-chlorophenol
(500 mg, 2.4 mmol) provided the title product after column
chromatography (0−60% ethyl acetate in cyclohexane) as a colorless
1
oil in 458 mg (43%) yield. H NMR (400 MHz, DMSO-d6, δ) 7.43
(d, J = 2.1 Hz, 1H), 7.38 (d, J = 8.5 Hz, 1H), 7.15 (dd, J = 8.4, 2.2 Hz,
1H), 4.71−4.53 (m, 1H), 3.97−3.65 (m, 1H), 3.61−3.33 (m, 1H),
3.30−3.17 (m, 1H), 3.08−2.90 (m, 1H), 1.94−1.69 (m, 3H), 1.53−
1.43 (m, 1H), 1.16 (s, 9H). 13C NMR (101 MHz, DMSO-d6, δ)
153.7, 153.3, 131.4, 124.6, 124.2, 120.3, 117.8, 78.3, 70.9, 45.3, 42.6,
28.7, 27.6, 20.4. LCMS (method a) m/z: 290.1 [M + H-Boc]+, tR =
1.48 min. [α]2D3 + 39.4 (c 1.0, MeOH).
(R)-tert-Butyl 3-(5-Bromo-2-chloro-4-fluorophenoxy)piperidine-
1-carboxylate (37). According to the general procedure for phenol
alkylation by the Mitsunobu reaction, the reaction of (S)-N-Boc-3-
hydroxy piperidine (1.8 g, 8.9 mmol) and 5-bromo-2-chloro-4-
fluorophenol (2.0 g, 8.9 mmol) provided the title product as a
colorless oil in 1.53 g (42%) yield. 1H NMR (400 MHz, DMSO-d6, δ)
7.73−7.50 (m, 2H), 4.69−4.45 (m, 1H), 3.96−3.39 (m, 2H), 3.27−
3.15 (m, 1H), 3.11−2.88 (m, 1H), 1.97−1.63 (m, 3H), 1.52−1.42
(m, 1H), 1.16 (s, 9H). 13C NMR (101 MHz, DMSO-d6, δ) 151.0 (d,
J = 256 Hz), 149.7, 122.0, 120.1, 118.8 (d, J = 2.3 Hz), 118.0 (d, J =
26.8 Hz), 106.6 (d, J = 22.1 Hz), 78.3, 71.5, 45.3, 42.7, 28.7, 27.7,
20.4.19F NMR (376 MHz, DMSO-d6, δ) −116.8. LCMS (method a)
m/z: 408.4 [M + H]+, tR = 1.51 min. [α]2D3 + 30.7 (c 1.0, MeOH).
(R)-tert-Butyl 3-(3-Bromo-6-chloro-2-fluorophenoxy)piperidine-
1-carboxylate (38). According to the general procedure for phenol
alkylation by the Mitsunobu reaction, the reaction of (S)-1-N-Boc-3-
hydroxypiperidine (0.75 g, 3.7 mmol) and 3-bromo-6-chloro-2-
fluorophenol (0.60 g, 2.7 mmol) provided the title product after
column chromatography (0−5% ethyl acetate in cyclohexane) in 877
mg (77%) yield. 1H NMR (400 MHz, DMSO-d6, δ) 7.48 (dd, J = 8.8,
6.9 Hz, 1H), 7.33 (dd, J = 8.8, 1.9 Hz, 1H), 4.45−4.19 (m, 1H),
3.74−3.33 (m, 3H), 3.28−3.08 (m, 1H), 1.99−1.86 (m, 1H), 1.85−
1.66 (m, 2H), 1.52−1.41 (m, 1H), 1.32 (s, 9H). 13C NMR (101
MHz, DMSO-d6, δ) 153.8, 152.7 (d, J = 242 Hz), 142.1 (d, J = 15
Hz), 127.8 (d, J = 9.1 Hz), 127.6 (d, J = 18 Hz), 126.5 (d, J = 3.4 Hz),
107.9 (d, J = 20 Hz), 78.7, 77.4, 47.1, 42.5, 29.3, 27.9, 20.9. 19F NMR
(376 MHz, DMSO-d6, δ) −119.6. LCMS (method a) m/z: 408.1 [M
+ H]+, tR = 1.53 min. [α]2D3 + 10.8 (c 1.0, MeOH).
Methyl 2-(4′-Chloro-[1,1′-biphenyl]-3-carboxamido)benzoate
(39). Oxalyl chloride (1.1 mL, 12 mmol) and DMF (1 drop) were
added rapidly at 23 °C to 4′-chloro-[1,1′-biphenyl]-3-carboxylic acid
(0.28 g, 1.2 mmol) in CH2Cl2 (12 mL) and the reaction mixture was
stirred at 23 °C for 2 h. After concentration in high vacuum, the
residue was dissolved in CH2Cl2 (12 mL) and treated at 23 °C with
methyl 2-aminobenzoate (0.17 mL, 1.26 mmol) and Et3N (0.42 mL,
3.0 mmol). After being stirred at 23 °C for 20 h, the reaction mixture
was quenched with saturated NaHCO3 (aq.) and extracted with
CH2Cl2. The combined organic layers were washed with water and
brine, dried (Na2SO4), and concentrated. The crude product was
purified by column chromatography (0−60% EtOAc in n-heptane) to
provide the title compound as a white solid in 0.41 g (86%) yield.
1H NMR (400 MHz, DMSO-d6, δ) 11.57 (s, 1H), 8.51 (dd, J = 8.4,
2-(2-(3′-(Aminomethyl)-4′-chloro-[1,1′-biphenyl]-3-
carboxamido)phenyl)acetic Acid (10). LiOH·H2O (48 mg, 1.1
mmol) was added rapidly at 23 °C to a mixture of methyl 2-(2-(3′-
(((tert-butoxycarbonyl)amino)methyl)-4′-chloro-[1,1′-biphenyl]-3-
carboxamido)phenyl)acetate (40, 0.29 g, 0.56 mmol) in MeOH (5
mL) and water (1.25 mL) and the mixture was stirred at 50 °C for 18
h. After being cooled to 23 °C, the mixture was concentrated and the
residue was treated with CH2Cl2 (5 mL) followed by TFA (0.88 mL,
11.3 mmol). The mixture was stirred at 23 °C for 4 h. After
1.2 Hz, 1H), 8.24 (t, J = 1.8 Hz, 1H), 8.02 (dd, J = 8.0, 1.7 Hz, 1H),
M
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concentration, the crude product was purified by HPLC (Gilson
SunFire 30 mm × 100 mm, 23 °C, eluent A: water + 0.1% TFA, B:
acetonitrile + 0.1% TFA, gradient: 5−80% B in 10 min, 80−100% B
in 0.5 min. Flow: 40 mL/min) to provide the title product (TFA salt)
J = 8.2, 2.0 Hz, 1H), 7.39−7.28 (m, 2H), 7.23 (td, J = 7.4, 1.4 Hz,
1H), 4.41 (t, J = 5.2 Hz, 2H), 3.70 (s, 2H), 3.36−3.27 (m, 2H). 13C
NMR (101 MHz, DMSO-d6, δ) 172.8, 165.1, 153.7, 140.0, 139.1,
136.6, 135.3, 131.1, 130.8, 130.6, 129.9, 129.2, 127.25, 127.18, 126.3,
125.99, 125.89, 121.6, 120.9, 113.1, 66.0, 48.6, 37.7. LCMS (method
a) m/z: 425.1 [M + H]+, tR = 0.68 min. LCMS (method b) m/z:
425.1 [M + H]+, tR = 3.13 min. HRMS m/z: [M + H]+ calcd for
C23H22ClN2O4 425.1263; found 425.1263.
1
as a white powder in 165 mg (57%) yield. H NMR (400 MHz,
DMSO-d6, δ) 12.38 (s, 1H), 10.14 (s, 1H), 8.41 (s, 3H), 8.29 (s, 1H),
8.04−7.97 (m, 2H), 7.93 (d, J = 7.8 Hz, 1H), 7.85 (dd, J = 8.4, 2.3
Hz, 1H), 7.73−7.63 (m, 2H), 7.47 (d, J = 7.6 Hz, 1H), 7.36 (d, J =
7.4 Hz, 1H), 7.32 (d, J = 7.7 Hz, 1H), 7.24 (t, J = 7.3 Hz, 1H), 4.26
(s, 2H), 3.69 (s, 2H). 13C NMR (101 MHz, DMSO-d6, δ) 172.7,
165.1, 138.6, 136.6, 135.5, 132.5, 132.2, 131.0, 130.9, 130.1, 129.6,
129.2, 129.0, 128.5, 127.3, 127.2, 126.3, 126.0, 37.6 (three carbons
missing). LCMS (method a) m/z: 395.2 [M + H]+, tR = 0.56 min.
LCMS (method b) m/z: 395.3 [M + H]+, tR = 2.59 min. HRMS m/z:
[M + H]+ calcd for C22H20ClN2O3 395.1157; found 395.1156.
2-(2-(3′-(2-((tert-Butoxycarbonyl)amino)ethoxy)-4′-chloro-
[1,1′-biphenyl]-3-carboxamido)phenyl)acetic Acid (41). A
mixture of tert-butyl (2-(5-bromo-2-chlorophenoxy)ethyl)carbamate
(32, 747 mg, 2.1 mmol), methyl 2-(2-(3-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)benzamido)phenyl)acetate (27, 0.86 g, 2.1 mmol),
and Na2CO3 (3.10 mL, 6.20 mmol) in DMF (21 mL) was degassed
with argon. After the addition of PdCl2(dppf)·CH2Cl2 (84 mg, 0.10
mmol), the mixture was stirred at 80 °C for 1 h. The mixture was
cooled to 23 °C, filtered over Celite, and washed with EtOAc. The
filtrate was concentrated and the crude product was purified by
column chromatography (0−75% ethyl acetate in n-heptane) to
provide the intermediate methyl 2-(2-(3′-(2-((tert-butoxycarbonyl)-
amino)ethoxy)-4′-chloro-[1,1′-biphenyl]-3-carboxamido)phenyl)-
2-(2-(3′-((1-(tert-Butoxycarbonyl)azetidin-3-yl)oxy)-4′-chloro-
[1,1′-biphenyl]-3-carboxamido)phenyl)acetic Acid (42). According
to the general procedure for Suzuki coupling, the reaction of tert-butyl
3-(5-bromo-2-chlorophenoxy)azetidine-1-carboxylate (33, 80 mg,
0.22 mmol) and methyl 2-(2-(3-(4,4,5,5-tetramethyl-1,3,2-dioxabor-
olan-2-yl)benzamido)phenyl)acetate (27, 87 mg, 0.22 mmol)
provided the title product after column chromatography (0−10%
1
MeOH in CH2Cl2) as a colorless solid in 114 mg (92%) yield. H
NMR (400 MHz, DMSO-d6, δ) 12.37 (s, 1H), 10.11 (s, 1H), 8.22 (s,
1H), 8.01−7.85 (m, 2H), 7.68−7.56 (m, 2H), 7.53 (d, J = 7.9 Hz,
1H), 7.42 (d, J = 8.3 Hz, 1H), 7.38−7.28 (m, 2H), 7.23 (t, J = 7.5 Hz,
1H), 7.18 (s, 1H), 5.35−5.20 (m, 1H), 4.50−4.30 (m, 2H), 3.97−
3.83 (m, 2H), 3.72 (s, 2H), 1.40 (s, 9H). 13C NMR (101 MHz,
DMSO-d6, δ) 172.8, 165.2, 155.5, 152.2, 140.1, 139.0, 136.6, 135.3,
131.1, 130.74, 130.68, 130.0, 129.2, 127.31, 127.25, 126.2, 126.0,
125.8, 121.3, 120.9, 112.3, 79.0, 66.8, 56.0, 37.8, 28.0. LCMS
(method a) m/z: 535.3 [M − H]−, tR = 1.33 min.
2-(2-(3′-(Azetidin-3-yloxy)-4′-chloro-[1,1′-biphenyl]-3-
carboxamido)phenyl)acetic Acid (12). Following the general
procedure for Boc-cleavage with HCl, 2-(2-(3′-((1-(tert-
butoxycarbonyl)azetidin-3-yl)oxy)-4′-chloro-[1,1′-biphenyl]-3-
carboxamido)phenyl)acetic acid (42, 100 mg, 0.19 mmol) provided
the title product (HCl salt) as a colorless solid in 60 mg (92%) yield.
1H NMR (400 MHz, DMSO-d6, δ) 13.44 (s, 1H), 8.72 (s, 1H), 8.10
(d, J = 8.0 Hz, 1H), 8.01 (d, J = 1.6 Hz, 1H), 7.99 (d, J = 1.6 Hz, 1H),
7.78 (s, 1H), 7.64 (t, J = 7.7 Hz, 1H), 7.58 (d, J = 8.3 Hz, 1H), 7.54
(dd, J = 8.3, 1.6 Hz, 1H), 7.27−7.19 (m, 3H), 7.04 (td, J = 7.4, 1.3
Hz, 1H), 6.65 (s, 2H), 5.78 (p, J = 6.9 Hz, 1H), 4.44 (t, J = 8.1 Hz,
2H), 4.12 (t, J = 8.6 Hz, 2H), 3.56 (s, 2H). 13C NMR (101 MHz,
DMSO-d6, δ) 172.9, 165.3, 151.9, 140.3, 138.8, 136.7, 135.3, 131.1,
130.9, 130.8, 130.2, 129.2, 127.5, 127.3, 126.5, 126.3, 125.9, 121.4,
121.3, 112.9, 68.4, 52.0, 37.4. LCMS (method a) m/z: 437.2 [M +
H]+, tR = 0.72 min. LCMS (method b) m/z: 437.4 [M + H]+, tR =
3.29 min. HRMS m/z: [M + H]+ calcd for C24H22ClN2O4 437.1263;
found 437.1263.
1
acetate as a yellow foam in 0.78 g (63%) yield. H NMR (400 MHz,
DMSO-d6, δ) 10.08 (s, 1H), 8.21 (s, 1H), 8.00−7.86 (m, 2H), 7.62
(t, J = 7.7 Hz, 1H), 7.55 (d, J = 8.3 Hz, 1H), 7.50 (s, 1H), 7.44 (d, J =
6.5 Hz, 1H), 7.39−7.31 (m, 3H), 7.25 (td, J = 7.4, 1.5 Hz, 1H), 6.99
(t, J = 5.8 Hz, 1H), 4.20 (t, J = 6.0 Hz, 2H), 3.78 (s, 2H), 3.50 (s,
3H), 3.37 (dt, J = 6.2 Hz, 2H), 1.37 (s, 9H). 13C NMR (101 MHz,
DMSO-d6, δ) 171.5, 165.3, 155.7, 154.2, 139.8, 139.2, 136.5, 135.1,
131.0, 130.8, 130.4, 129.9, 129.1, 127.5, 127.2, 126.8, 126.2, 125.9,
121.3, 120.1, 112.5, 77.8, 67.7, 51.6, 37.3, 28.2, 24.9, 24.5. LCMS
(method a) m/z: 537.3 [M − H]−, tR = 1.33 min. LiOH·H2O (35 mg,
0.84 mmol) was added at 23 °C to a mixture of products from the
previous step (methyl 2-(2-(3′-(2-((tert-butoxycarbonyl)amino)-
ethoxy)-4′-chloro-[1,1′-biphenyl]-3-carboxamido)phenyl)acetate,
0.25 g, 0.42 mmol) in MeOH (3.3 mL) and water (0.84 mL), and the
mixture was stirred at 23 °C for 4 h. The reaction mixture was then
concentrated, and the residue was treated with 0.2 M HCl. The
resulting suspension was filtered, and the solid was washed with water
and dried in high vacuum to provide the title product as a white solid
in 158 mg (70%). 1H NMR (400 MHz, DMSO-d6, δ) 12.35 (s, 1H),
10.17 (s, 1H), 8.24 (s, 1H), 7.95 (d, J = 1.7 Hz, 1H), 7.93 (d, J = 1.7
Hz, 1H), 7.62 (t, J = 7.7 Hz, 1H), 7.54 (d, J = 8.2 Hz, 1H), 7.53−7.47
(m, 2H), 7.40−7.29 (m, 3H), 7.22 (td, J = 7.4, 1.4 Hz, 1H), 7.00 (t, J
= 5.6 Hz, 1H), 4.20 (t, J = 6.0 Hz, 2H), 3.69 (s, 2H), 3.37 (td, J = 6.2,
5.6 Hz, 2H), 1.37 (s, 9H). 13C NMR (101 MHz, DMSO-d6, δ) 174.0,
164.2, 155.8, 154.3, 139.5, 138.8, 138.1, 135.9, 130.45, 130.30, 130.23,
129.27, 129.13, 127.4, 125.9, 125.6, 123.6, 122.6, 121.0, 119.3, 112.4,
77.3, 67.51, 67.45, 44.9, 28.2. LCMS (method a) m/z: 525.2 [M +
H]+, tR = 1.25 min.
2-(2-(3′-(2-Aminoethoxy)-4′-chloro-[1,1′-biphenyl]-3-
carboxamido)phenyl)acetic Acid (11). TFA (0.85 mL, 10.9 mmol)
was added at 23 °C to a solution of 2-(2-(3′-(2-((tert-
butoxycarbonyl)amino)ethoxy)-4′-chloro-[1,1′-biphenyl]-3-
carboxamido)phenyl)acetic acid (41, 148 mg, 0.27 mmol) in CH2Cl2
(2.7 mL). After being stirred at 23 °C for 3 h, the reaction mixture
was concentrated and the crude product was purified by HPLC
(Gilson SunFire 30 mm × 100 mm; 23 °C, eluent A: water + 0.1%
TFA, B: acetonitrile + 0.1% TFA, gradient: 5−80% B in 10 min, 80−
100% B in 0.5 min. Flow: 40 mL/min) to provide the title product
(TFA-salt) as a white powder in 120 mg (81%) yield. 1H NMR (400
MHz, DMSO-d6, δ) 12.45 (s, 1H), 10.11 (s, 1H), 8.23 (s, 1H), 8.02
(s, 3H), 7.98−7.89 (m, 2H), 7.64 (t, J = 7.7 Hz, 1H), 7.60 (d, J = 8.3
Hz, 1H), 7.53 (d, J = 2.0 Hz, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.43 (dd,
2-(2-(3′-((1-(tert-Butoxycarbonyl)pyrrolidin-3-yl)oxy)-4′-chloro-
[1,1′-biphenyl]-3-carboxamido)phenyl)acetic Acid (43). According
to the general procedure for Suzuki coupling, the reaction of tert-butyl
3-(5-bromo-2-chlorophenoxy)pyrrolidine-1-carboxylate (34, 90 mg,
0.23 mmol) and methyl 2-(2-(3-(4,4,5,5-tetramethyl-1,3,2-dioxabor-
olan-2-yl)benzamido)phenyl)acetate (27, 91 mg, 0.23 mmol)
provided the title product after column chromatography (0−30%
ethyl acetate in cyclohexane) as a colorless solid in 71 mg (56%)
1
yield. H NMR (400 MHz, DMSO-d6, δ) 12.35 (s, 1H), 10.15 (s,
1H), 8.24 (s, 1H), 7.98−7.89 (m, 2H), 7.63 (t, J = 7.7 Hz, 1H),
7.59−7.49 (m, 3H), 7.40 (dd, J = 8.3, 2.1 Hz, 1H), 7.37−7.29 (m,
2H), 7.22 (td, J = 7.4, 1.5 Hz, 1H), 5.32 (s, 1H), 3.71 (s, 2H), 3.63−
3.51 (m, 1H), 3.51−3.36 (m, 3H), 2.26−2.03 (m, 2H), 1.41 + 1.40 +
1.39 (s, 9H). 13C NMR (101 MHz, DMSO-d6, δ) 172.8, 165.1, 153.7,
152.7, 139.9, 139.1, 136.6, 135.3, 131.0, 130.66, 130.60, 129.9, 129.1,
127.21, 126.2, 125.94, 125.78, 122.47, 122.40, 120.7, 114.4, 78.5,
(78.1 + 77.2), (51.4 + 51.1), (44.0 + 43.7), 37.8, (31.0 + 30.1), 28.1.
LCMS (method a) m/z: 551.1 [M + H]+, tR = 1.31 min.
2-(2-(4′-Chloro-3′-(pyrrolidin-3-yloxy)-[1,1′-biphenyl]-3-
carboxamido)phenyl)acetic Acid (13). Following the general
procedure for Boc-cleavage with HCl, 2-(2-(3′-((1-(tert-
butoxycarbonyl)pyrrolidin-3-yl)oxy)-4′-chloro-[1,1′-biphenyl]-3-
carboxamido)phenyl)acetic acid (43, 60 mg, 0.11 mmol) provided the
title product (HCl salt) as a colorless solid in 47 mg (85%) yield. 1H
NMR (400 MHz, DMSO-d6, δ) 12.35 (s, 1H), 10.19 (s, 1H), 9.45 (s,
1H), 9.34 (s, 1H), 8.27 (s, 1H), 7.92−7.98 (m, 2H), 7.64 (t, J = 7.8
Hz, 1H), 7.61−7.57 (m, 2H), 7.52 (d, J = 7.7 Hz, 1H), 7.44 (dd, J =
N
https://dx.doi.org/10.1021/acs.jmedchem.0c01020
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
8.3, 1.9 Hz, 1H), 7.37−7.29 (m, 2H), 7.23 (td, J = 7.5, 1.5 Hz, 1H),
5.46−5.39 (m, 1H), 3.72 (s, 2H), 3.64−3.58 (m, 1H), 3.48−3.34 (m,
3H), 2.29−2.19 (m, 2H). 13C NMR (101 MHz, DMSO-d6, δ) 172.8,
165.1, 152.3, 140.0, 138.9, 136.6, 135.3, 131.01, 130.72, 130.67, 130.0,
129.1, 127.3, 127.2, 126.2, 126.0, 125.8, 122.3, 121.2, 114.5, 77.3,
49.8, 43.7, 37.8, 30.8. LCMS (method a) m/z: 451.1 [M + H]+, tR =
0.72 min. LCMS (method b) m/z: 451.1 [M + H]+, tR = 3.26 min.
HRMS m/z: [M + H]+ calcd for C25H24ClN2O4 451.1419; found
451.1419.
1.7 Hz, 1H), 7.31 (dd, J = 7.7, 1.8 Hz, 1H), 7.22 (td, J = 7.5, 1.5 Hz,
1H), 5.03−4.92 (m, 1H), 3.72 (s, 2H), 3.44 (d, J = 11.9 Hz, 1H),
3.24−3.10 (m, 2H), 3.09−2.97 (m, 1H), 2.11−1.92 (m, 2H), 1.92−
1.70 (m, 2H). 13C NMR (101 MHz, DMSO-d6, δ) 172.8, 165.1,
152.6, 140.1, 138.9, 136.6, 135.2, 131.0, 130.79, 130.72, 130.0, 129.1,
127.4, 127.1, 126.4, 126.1, 125.8, 122.6, 121.3, 115.2, 70.9, 45.3, 42.7,
37.8, 27.2, 19.0. LCMS (method a) m/z: 465.2 [M + H]+, tR = 0.86
min. LCMS (method b) m/z: 465.0 [M + H]+, tR = 4.02 min. HRMS
m/z: [M + H]+ calcd for C26H26ClN2O4 465.1576; found 465.1578.
(R)-2-(2-(3′-((1-(tert-Butoxycarbonyl)piperidin-3-yl)oxy)-4′-
chloro-2-fluoro-[1,1′-biphenyl]-3-carboxamido)phenyl)acetic Acid
(46). According to the general procedure for Suzuki coupling, the
reaction of (R)-tert-butyl 3-(5-bromo-2-chlorophenoxy)piperidine-1-
carboxylate (36, 95 mg, 0.24 mmol) and methyl 2-(2-(2-fluoro-3-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamido)phenyl)-
acetate (28, 100 mg, 0.24 mmol) provided the title product after
column chromatography (0−60% ethyl acetate in cyclohexane) as a
2-(2-(3′-((1-(tert-Butoxycarbonyl)piperidin-4-yl)oxy)-4′-chloro-
[1,1′-biphenyl]-3-carboxamido)phenyl)acetic Acid (44). According
to the general procedure for Suzuki coupling, the reaction of methyl
2-(2-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamido)-
phenyl)acetate (27, 81 mg, 0.21 mmol) and tert-butyl 4-(5-bromo-2-
chlorophenoxy)piperidine-1-carboxylate (35, 80 mg, 0.21 mmol)
provided the title product after column chromatography (0−50%
ethyl acetate in cyclohexane) as a colorless solid in 80 mg (69%)
1
1
colorless solid in 129 mg (86%) yield. H NMR (400 MHz, DMSO-
yield. H NMR (400 MHz, DMSO-d6, δ) 12.36 (s, 1H), 10.27 (s,
d6, δ) 12.15 (s, 1H), 10.05 (s, 1H), 7.77−7.67 (m, 2H), 7.65 (d, J =
9.1 Hz, 1H), 7.58−7.51 (m, 2H), 7.42 (t, J = 7.6 Hz, 1H), 7.32 (d, J =
7.6 Hz, 1H), 7.28−7.13 (m, 3H), 4.77−4.52 (m, 1H), 3.81−3.71 (m,
1H), 3.68 (s, 2H), 3.46−3.38 (m, 3H), 1.95−1.77 (m, 3H), 1.51−
1.45 (m, 1H), 1.12 (s, 9H). 13C NMR (101 MHz, DMSO-d6, δ)
172.5, 162.7, 155.9 (d, J = 251.5 Hz), 153.7, 152.4, 151.7 (d, J = 4.6
Hz), 136.1, 134.7, 132.9, 131.0, 130.8 (d, J = 7.4 Hz), 130.2, 130.0,
129.6, 127.2, 125.8 (d, J = 4.8 Hz), 125.3 (d, J = 16.3 Hz), 124.7 (d, J
= 4.1 Hz), 124.4, 122.0, 115.5 (d, J = 4.3 Hz), 78.2, 70.6, 45.4, 42.7,
37.4, 28.8, 27.6, 20.5. 19H NMR (376 MHz, DMSO-d6, δ) −119.5.
LCMS (method a) m/z: 581.3 [M − H]−, tR = 1.30 min. [α]D23 + 26.2
(c 1.0, MeOH).
(R)-2-(2-(4′-Chloro-2-fluoro-3′-(piperidin-3-yloxy)-[1,1′-biphen-
yl]-3-carboxamido)phenyl)acetic Acid (16). Following the general
procedure for Boc-cleavage with HCl, (R)-2-(2-(3′-((1-(tert-
butoxycarbonyl)piperidin-3-yl)oxy)-4′-chloro-2-fluoro-[1,1′-biphen-
yl]-3-carboxamido)phenyl)acetic acid (46, 120 mg, 0.2 mmol)
provided the title product (HCl-salt) as a colorless solid in 70 mg
(65%) yield. 1H NMR (400 MHz, DMSO-d6, δ) 12.36 (s, 1H), 10.03
(s, 1H), 9.37 (s, 1H), 8.87 (s, 1H), 7.80−7.66 (m, 2H), 7.61 (d, J =
8.2 Hz, 1H), 7.57−7.47 (m, 2H), 7.43 (t, J = 7.6 Hz, 1H), 7.36−7.15
(m, 4H), 4.90−4.77 (m, 1H), 3.70 (s, 2H), 3.50−3.25 (m, 3H),
3.13−2.96 (m, 2H), 2.10−1.91 (m, 2H), 1.90−1.68 (m, 2H). 13C
NMR (101 MHz, DMSO-d6, δ) 172.5, 162.7, 155.9 (d, J = 251.7 Hz),
152.2, 136.1, 135.0, 133.0 (d, J = 2.9 Hz), 131.1, 130.5, 130.1, 129.8
(d, J = 2.6 Hz), 127.9 (d, J = 13.8 Hz), 127.2, 125.9, 125.3 (d, J = 16.1
Hz), 124.73, 124.70, 123.5 (d, J = 2.2 Hz), 123.0, 117.4 (d, J = 2.6
Hz), 71.0, 45.3, 42.6, 37.3, 27.0, 18.8. 19F NMR (376 MHz, DMSO-
d6, δ) −119.0. LCMS (method a) m/z: 483.2 [M + H]+, tR = 0.72
min. LCMS (method b) m/z: 483.2 [M + H]+, tR = 3.28 min. HRMS
m/z: [M + H]+ calcd for C26H25ClFN2O4 483.1481; found 483.1482.
[α]2D3 + 1.4 (c 1.0, MeOH).
1H), 8.25 (s, 1H), 7.97−7.89 (m, 2H), 7.62 (t, J = 7.8 Hz, 1H),
7.59−7.51 (m, 3H), 7.37 (dd, J = 8.3, 2.1 Hz, 1H), 7.35−7.29 (m,
2H), 7.21 (td, J = 7.4, 1.4 Hz, 1H), 4.96−4.82 (m, 1H), 3.70 (s, 2H),
3.61 (ddd, J = 10.8, 8.9, 5.1 Hz, 2H), 3.29−3.25 (m, 2H), 2.01−1.85
(m, 2H), 1.72−1.58 (m, 2H), 1.41 (s, 9H). 13C NMR (101 MHz,
DMSO-d6, δ) 172.8, 165.1, 153.9, 152.7, 139.9, 139.1, 136.7, 135.3,
131.0, 130.62, 130.55, 129.9, 129.1, 127.22, 127.15, 126.03, 125.91,
125.67, 122.6, 120.5, 114.8, 78.7, 73.2, 69.2, 30.2, 28.1, 24.9. LCMS
(method a) m/z: 565.2 [M + H]+, tR = 1.38 min.
2-(2-(4′-Chloro-3′-(piperidin-4-yloxy)-[1,1′-biphenyl]-3-
carboxamido)phenyl)acetic Acid (14). Following the general
procedure for Boc-cleavage with HCl, 2-(2-(3′-((1-(tert-
butoxycarbonyl)piperidin-4-yl)oxy)-4′-chloro-[1,1′-biphenyl]-3-
carboxamido)phenyl)acetic acid (44, 70 mg, 0.12 mmol) provided the
1
title product (HCl-salt) as a colorless solid in 52 mg (84%) yield. H
NMR (400 MHz, DMSO-d6, δ) 12.36 (s, 1H), 10.18 (s, 1H), 8.80 (s,
2H), 8.25 (s, 1H), 7.95 (d, J = 7.8 Hz, 1H), 7.92 (d, J = 8.3 Hz, 1H),
7.67−7.61 (m, 2H), 7.58 (d, J = 8.3 Hz, 1H), 7.52 (d, J = 6.7 Hz,
1H), 7.40 (dd, J = 8.3, 2.1 Hz, 1H), 7.37−7.29 (m, 2H), 7.23 (td, J =
7.5, 1.5 Hz, 1H), 5.06−4.91 (m, 1H), 3.72 (s, 2H), 3.28−3.19 (m,
2H), 3.18−3.06 (m, 2H), 2.22−2.07 (m, 2H), 2.02−1.87 (m, 2H).
13C NMR (101 MHz, DMSO-d6, δ) 172.8, 165.1, 152.3, 140.1, 139.1,
138.6, 136.6, 135.3, 131.0, 130.7, 130.0, 129.1, 127.3, 127.2, 126.3,
126.0, 125.8, 122.5, 120.9, 114.8, 70.6, 40.4, 37.7, 27.0. LCMS
(method a) m/z: 465.3 [M + H]+, tR = 0.65 min. LCMS (method a)
m/z: 465.3 [M + H]+, tR = 0.65 min. LCMS (method b) m/z: 465.3
[M + H]+, tR = 3.17 min. HRMS m/z: [M − H]− calcd for
C26H24ClN2O4 463.1430; found 463.1431.
2-(2-(3′-((1-(tert-Butoxycarbonyl)piperidin-3-yl)oxy)-4′-chloro-
[1,1′-biphenyl]-3-carboxamido)phenyl)acetic Acid (45). According
to the general procedure for Suzuki coupling, the reaction of methyl
2-(2-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamido)-
phenyl)acetate (27, 86 mg, 0.22 mmol) and tert-butyl 3-(5-bromo-2-
chlorophenoxy)piperidine-1-carboxylate (rac-36, 85 mg, 0.22 mmol)
provided the title product after column chromatography (0−50%
ethyl acetate in cyclohexane) as a colorless solid in 117 mg (90%)
(R)-2-(2-(3′-((1-(tert-Butoxycarbonyl)piperidin-3-yl)oxy)-4′-
chloro-2′-fluoro-[1,1′-biphenyl]-3-carboxamido)phenyl)acetic Acid
(47). According to the general procedure for Suzuki coupling, the
reaction of (R)-tert-butyl 3-(3-bromo-6-chloro-2-fluorophenoxy)-
piperidine-1-carboxylate (38, 103 mg, 0.25 mmol) and methyl 2-(2-
(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamido)phenyl)-
acetate (27, 100 mg, 0.25 mmol) provided the title product after
column chromatography (0−5% MeOH in CH2Cl2) as a colorless
1
yield. H NMR (400 MHz, DMSO-d6, δ) 12.42 (s, 1H), 10.22 (s,
1H), 8.23 (s, 1H), 7.94 (d, J = 7.8 Hz, 1H), 7.92−7.84 (m, 1H), 7.62
(t, J = 7.7 Hz, 1H), 7.57−7.47 (m, 3H), 7.39−7.28 (m, 3H), 7.22 (td,
J = 7.4, 1.4 Hz, 1H), 4.77 (s, 1H), 4.00−3.87 (m, 1H), 3.69 (s, 2H),
3.61−3.40 (m, 3H), 1.85 (s, 3H), 1.54−1.45 (m, 1H), 1.40 (s, 9H).
LCMS (method a) m/z: 563.4 [M − H]−, tR = 1.32 min.
2-(2-(4′-Chloro-3′-(piperidin-3-yloxy)-[1,1′-biphenyl]-3-
carboxamido)phenyl)acetic Acid (15). Following the general
procedure for Boc-cleavage with HCl, 2-(2-(3′-((1-(tert-
butoxycarbonyl)piperidin-3-yl)oxy)-4′-chloro-[1,1′-biphenyl]-3-
carboxamido)phenyl)acetic acid (45, 105 mg, 0.19 mmol) provided
the title product (HCl salt) as a colorless solid in 50 mg (53%) yield.
1H NMR (400 MHz, DMSO-d6, δ) 12.32 (s, 1H), 10.26 (s, 1H), 9.34
1
resin in 108 mg (70%) yield. H NMR (400 MHz, DMSO-d6, δ)
12.34 (s, 1H), 10.11 (s, 1H), 8.10 (s, 1H), 8.00 (d, J = 7.7 Hz, 1H),
7.78 (d, J = 7.7 Hz, 1H), 7.65 (t, J = 7.7 Hz, 1H), 7.50−7.43 (m, 2H),
7.42−7.28 (m, 3H), 7.23 (td, J = 7.5, 1.4 Hz, 1H), 4.44−4.18 (m,
1H), 3.85−3.68 (m, 1H), 3.67 (s, 2H), 3.64−3.52 (m, 1H), 3.49−
3.35 (m, 1H), 3.29−3.10 (m, 1H), 2.12−1.70 (m, 3H), 1.52−1.41
(m, 1H), 1.30 (s, 9H). 13C NMR (101 MHz, DMSO-d6, δ) 172.8,
164.9, 153.9, 153.0 (d, J = 252.1 Hz), 141.8 (d, J = 14.5 Hz), 136.6,
135.1, 134.2, 131.9 (d, J = 3.4 Hz), 131.0, 130.8, 128.8, 128.2 (d, J =
12.2 Hz), 127.9, 127.5, 127.2, 126.2, 125.78, 125.70 (d, J = 3.8 Hz),
125.30, 125.22, 78.7, (77.55 + 77.46), (47.4 + 46.6), (43.5 + 42.7),
38.0, 29.5, 27.9, (22.1 + 21.2). 19F NMR (376 MHz, DMSO-d6, δ)
(s, 1H), 8.84 (s, 1H), 8.31 (s, 1H), 7.98−7.91 (m, 2H), 7.75 (d, J =
2.2 Hz, 1H), 7.63 (t, J = 7.7 Hz, 1H), 7.59 (d, J = 8.2 Hz, 1H), 7.51
(d, J = 7.8 Hz, 1H), 7.44 (dd, J = 8.3, 2.0 Hz, 1H), 7.34 (dd, J = 7.3,
O
https://dx.doi.org/10.1021/acs.jmedchem.0c01020
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
−131.2. LCMS (method a) m/z: 583.3 [M + H]+, tR = 1.33 min.
(R)-2-(2-(5′-((1-(tert-Butoxycarbonyl)piperidin-3-yl)oxy)-4′-
chloro-2′-fluoro-[1,1′-biphenyl]-3-carboxamido)-5-fluorophenyl)-
acetic Acid (49). According to the general procedure for Suzuki
coupling, the reaction of (R)-tert-butyl 3-(5-bromo-2-chloro-4-
fluorophenoxy)piperidine-1-carboxylate (37, 99 mg, 0.24 mmol)
and methyl 2-(5-fluoro-2-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)benzamido)phenyl)acetate (29, 100 mg, 0.24 mmol) provided
the title product after column chromatography (0−5% MeOH in
CH2Cl2) as a colorless resin in 81 mg (50%) yield. 1H NMR
(400 MHz, DMSO-d6, δ) 12.38 (s, 1H), 10.06 (s, 1H), 8.09 (s, 1H),
7.99 (d, J = 7.7 Hz, 1H), 7.85−7.69 (m, 1H), 7.65 (t, J = 7.7 Hz, 1H),
7.60 (d, J = 10.0 Hz, 1H), 7.45 (dd, J = 8.8, 5.5 Hz, 1H), 7.43−7.29
(m, 1H), 7.22 (dd, J = 9.7, 3.0 Hz, 1H), 7.16 (td, J = 8.5, 3.0 Hz, 1H),
4.78−4.44 (m, 1H), 3.96−3.70 (m, 1H), 3.69 (s, 2H), 3.53−3.32 (m,
3H), 2.01−1.70 (m, 3H), 1.57−1.43 (m, 1H), 1.15 (s, 9H). 13C NMR
(101 MHz, DMSO-d6, δ) 172.1, 165.2, 159.7 (d, J = 242.0 Hz), 153.8,
152.6 (d, J = 252.2 Hz), 149.3 (d, J = 2.6 Hz), 134.8, 134.5, 133.6 (d,
J = 8.4 Hz), 132.8 (d, J = 2.7 Hz), 132.0 (d, J = 3.2 Hz), 128.8, 128.4
(d, J = 8.6 Hz), 128.1 (d, J = 2.5 Hz), 127.4, 127.1 (d, J = 14.5 Hz),
122.8, 117.9 (d, J = 27.5 Hz), 117.5 (d, J = 22.8 Hz), 116.4, 113.8 (d,
J = 22.0 Hz), 78.3, 71.3, 45.6, 42.7, 37.2, 28.8, 27.6, 20.6. 19F NMR
(376 MHz, DMSO-d6, δ) −116.9, −126.3. LCMS (method a) m/z:
599.4 [M − H]−, tR = 1.36 min. [α]2D3 + 34.9 (c 1.0, MeOH).
(R)-2-(2-(4′-Chloro-2′-fluoro-5′-(piperidin-3-yloxy)-[1,1′-biphen-
yl]-3-carboxamido)-5-fluorophenyl)acetic Acid (19). Following the
general procedure for Boc-cleavage with HCl, (R)-2-(2-(5′-((1-(tert-
butoxycarbonyl)piperidin-3-yl)oxy)-4′-chloro-2′-fluoro-[1,1′-biphen-
yl]-3-carboxamido)-5-fluorophenyl)acetic acid (49, 81 mg, 0.12
mmol) provided the title product (HCl-salt) as a colorless solid in
52 mg (81%) yield. 1H NMR (400 MHz, DMSO-d6, δ) 12.37 (s, 1H),
10.18 (s, 1H), 9.00 (s, 1H), 8.67 (s, 1H), 8.14 (s, 1H), 8.00 (d, J = 7.8
Hz, 1H), 7.80 (d, J = 7.5 Hz, 1H), 7.70−7.62 (m, 2H), 7.59 (d, J =
7.1 Hz, 1H), 7.52−7.41 (m, 1H), 7.22 (dd, J = 9.6, 3.0 Hz, 1H), 7.16
(td, J = 8.5, 3.0 Hz, 1H), 4.89−4.72 (m, 1H), 3.70 (s, 2H), 3.44−3.38
(m, 1H), 3.29−3.19 (m, 1H), 3.16−2.99 (m, 2H), 2.08−1.94 (m,
2H), 1.92−1.80 (m, 1H), 1.79−1.65 (m, 1H). 13C NMR (101 MHz,
DMSO-d6, δ) 171.1, 165.1, 159.7 (d, J = 240.8 Hz), 153.4 (d, J =
244.5 Hz), 149.2 (d, J = 2.8 Hz), 134.9, 134.1, 133.6, 132.8 (d, J = 7.8
Hz), 132.0 (d, J = 3.5 Hz), 130.8 (d, J = 31.8 Hz), 128.8, 128.1,
127.52, 127.35 (d, J = 14.5 Hz), 123.3, 118.8 (d, J = 3.5 Hz), 118.1
(d, J = 27.5 Hz), 117.4 (d, J = 22.6 Hz), 113.8 (d, J = 21.6 Hz), 71.9,
45.4, 42.7, 37.4, 27.0, 18.7. 19F NMR (376 MHz, DMSO-d6, δ)
−117.0, −124.1. LCMS (method a) m/z: 501.2 [M + H]+, tR = 0.97
min. LCMS (method b) m/z: 501.2 [M + H]+, tR = 4.53 min. HRMS
m/z: [M + H]+ calcd for C26H24ClF2N2O4 501.1387; found 501.1389.
[α]2D3 − 2.1 (c 1.0, MeOH).
[α]2D3 + 13.3 (c 1.0, MeOH).
(R)-2-(2-(3′-((1-(tert-butoxycarbonyl)piperidin-3-yl)oxy)-4′-
chloro-2′-fluoro-[1,1′-biphenyl]-3-carboxamido)phenyl)acetic Acid
(17). Following the general procedure for Boc-cleavage with HCl,
(R)-2-(2-(3′-((1-(tert-butoxycarbonyl)piperidin-3-yl)oxy)-4′-chloro-
2′-fluoro-[1,1′-biphenyl]-3-carboxamido)phenyl)acetic acid (47, 97
mg, 0.17 mmol) provided the title product (HCl salt) as a colorless
solid in 76 mg (88%) yield. 1H NMR (400 MHz, DMSO-d6, δ) 12.33
(s, 1H), 10.12 (s, 1H), 9.10 (s, 1H), 8.84 (s, 1H), 8.12 (s, 1H), 8.02
(d, J = 7.8 Hz, 1H), 7.79 (d, J = 7.0 Hz, 1H), 7.66 (t, J = 7.8 Hz, 1H),
7.53 (d, J = 9.5 Hz, 1H), 7.49−7.42 (m, 2H), 7.37−7.28 (m, 2H),
7.23 (td, J = 7.4, 1.5 Hz, 1H), 4.48 (tt, J = 7.0, 3.3 Hz, 1H), 3.68 (s,
2H), 3.48−3.37 (m, 2H), 3.16−2.97 (m, 2H), 2.11−1.96 (m, 2H),
1.96−1.82 (m, 1H), 1.79−1.65 (m, 1H). 13C NMR (101 MHz,
DMSO-d6, δ) 172.7, 164.9, 153.1 (d, J = 249.6 Hz), 141.9 (d, J = 13.4
Hz), 136.5, 135.1, 134.0, 131.9, 131.0, 130.8, 128.9, 128.3, 128.0,
127.5 (d, J = 9.0 Hz), 127.4, 127.2, 126.4, 126.2 (d, J = 2.7 Hz),
125.93, 125.79 (d, J = 3.1 Hz), 76.3, 46.2, 42.8, 37.6, 27.4, 18.7. 19F
NMR (376 MHz, DMSO-d6, δ) −130.8. LCMS (method a) m/z:
483.2 [M + H]+, tR = 0.73 min. LCMS (method b) m/z: 483.1 [M +
H]+, tR = 3.18 min. HRMS m/z: [M + H]+ calcd for C26H25ClFN2O4
483.1481; found 483.1484. [α]2D3 + 2.2 (c 0.5, MeOH).
(R)-2-(2-(3′-((1-(tert-Butoxycarbonyl)piperidin-3-yl)oxy)-4′-
chloro-2′-fluoro-[1,1′-biphenyl]-3-carboxamido)-5-fluorophenyl)-
acetic Acid (48). According to the general procedure for Suzuki
coupling, the reaction of (R)-tert-butyl 3-(3-bromo-6-chloro-2-
fluorophenoxy)piperidine-1-carboxylate (38, 99 mg, 0.24 mmol)
and methyl 2-(5-fluoro-2-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)benzamido)phenyl)acetate (29, 100 mg, 0.24 mmol) provided
the title product after column chromatography (0−5% MeOH in
1
CH2Cl2) as a colorless resin in 113 mg (70%) yield. H NMR (400
MHz, DMSO-d6, δ) 12.39 (s, 1H), 10.16 (s, 1H), 8.09 (s, 1H), 7.99
(d, J = 7.7 Hz, 1H), 7.77 (d, J = 7.7 Hz, 1H), 7.64 (t, J = 7.7 Hz, 1H),
7.50−7.35 (m, 3H), 7.22 (dd, J = 9.7, 3.0 Hz, 1H), 7.16 (td, J = 8.5,
3.0 Hz, 1H), 4.47−4.17 (m, 1H), 3.67 (s, 2H), 3.63−3.49 (m, 1H),
3.43−3.19 (m, 3H), 2.07−1.66 (m, 3H), 1.47−1.41 (m, 1H), 1.33 (s,
9H). 13C NMR (101 MHz, DMSO-d6, δ) 172.0, 165.1, 159.7 (d, J =
242.0 Hz), 153.9, 152.9 (d, J = 251.3 Hz), 141.8 (d, J = 14.5 Hz),
134.9, 134.2, 133.8 (d, J = 8.5 Hz), 132.9 (d, J = 2.8 Hz), 131.9,
128.8, 128.4 (d, J = 8.7 Hz), 128.2 (d, J = 12.3 Hz), 127.9 (d, J = 2.2
Hz), 127.50, 127.45, 125.7 (d, J = 3.8 Hz), 125.3, 117.4 (d, J = 22.8
Hz), 113.8 (d, J = 22.1 Hz), 78.7, (77.6 + 77.4), (47.4 + 46.6), (43.4
+ 42.6), 37.3, 29.5, 27.9, (22.0 + 21.2). 19F NMR (376 MHz, DMSO-
d6, δ) −117.0, −131.2. LCMS (method a) m/z: 601.5 [M + H]+, tR =
1.33 min. [α]2D3 + 12.6 (c 1.0, MeOH).
(R)-2-(2-(4′-Chloro-2′-fluoro-3′-(piperidin-3-yloxy)-[1,1′-biphen-
yl]-3-carboxamido)-5-fluorophenyl)acetic Acid (18). Following the
general procedure for Boc-cleavage with HCl, (R)-2-(2-(3′-((1-(tert-
butoxycarbonyl)piperidin-3-yl)oxy)-4′-chloro-2′-fluoro-[1,1′-biphen-
yl]-3-carboxamido)-5-fluorophenyl)acetic acid (48, 113 mg, 0.17
mmol) provided the title product (HCl salt) as a colorless solid in 73
(R)-2-(2-(5′-((1-(tert-Butoxycarbonyl)piperidin-3-yl)oxy)-4′-
chloro-2,2′-difluoro-[1,1′-biphenyl]-3-carboxamido)-5-
fluorophenyl)acetic Acid (50). According to the general procedure
for Suzuki coupling, the reaction of (R)-tert-butyl 3-(5-bromo-2-
chloro-4-fluorophenoxy)piperidine-1-carboxylate (37, 489 mg, 1.2
mmol) and methyl 2-(5-fluoro-2-(2-fluoro-3-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)benzamido)phenyl)acetate (30, 600 mg, 1.2
mmol) provided the title product as a white foam in 486 mg (62%)
1
mg (80%) yield. H NMR (400 MHz, DMSO-d6, δ) 12.39 (s, 1H),
10.16 (s, 1H), 9.45 (s, 1H), 9.01 (s, 1H), 8.12 (s, 1H), 8.01 (d, J = 7.8
Hz, 1H), 7.79 (d, J = 7.6 Hz, 1H), 7.66 (t, J = 7.8 Hz, 1H), 7.52 (d, J
= 9.3 Hz, 1H), 7.49−7.38 (m, 2H), 7.23 (dd, J = 9.7, 3.0 Hz, 1H),
7.16 (td, J = 8.5, 3.1 Hz, 1H), 4.57−4.39 (m, 1H), 3.69 (s, 2H),
3.50−3.30 (m, 1H), 3.23 (dd, J = 12.4, 7.2 Hz, 1H), 3.16−2.93 (m,
2H), 2.11−1.97 (m, 2H), 1.94−1.82 (m, 1H), 1.80−1.65 (m, 1H).
13C NMR (101 MHz, DMSO-d6, δ) 172.0, 165.1, 159.8 (d, J = 242.1
Hz), 153.1 (d, J = 249.8 Hz), 141.8 (d, J = 14.8 Hz), 134.9, 133.9,
133.8 (d, J = 8.5 Hz), 132.8 (d, J = 2.9 Hz), 131.9 (d, J = 3.2 Hz),
128.8, 128.5 (d, J = 8.6 Hz), 128.3 (d, J = 12.2 Hz), 128.0, 127.6,
127.5 (d, J = 2.4 Hz), 126.2 (d, J = 3.4 Hz), 125.8 (d, J = 4.1 Hz),
117.4 (d, J = 22.8 Hz), 113.8 (d, J = 22.1 Hz), 76.3, 46.1, 42.7, 37.2,
27.6, 18.8. 19F NMR (376 MHz, DMSO-d6, δ) −116.9, −130.7.
LCMS (method a) m/z: 501.2 [M + H]+, tR = 0.73 min. LCMS
(method b) m/z: 501.2 [M + H]+, tR = 3.35 min. HRMS m/z: [M +
H]+ calcd for C26H24ClF2N2O4 501.1387; found 501.1392. [α]2D3 + 1.5
(c 1.0, MeOH).
1
yield. H NMR (400 MHz, DMSO-d6, δ) 12.45 (s, 1H), 10.05 (s,
1H), 7.77 (t, J = 6.4 Hz, 1H), 7.69−7.56 (m, 2H), 7.50 (dd, J = 8.8,
5.6 Hz, 1H), 7.45 (t, J = 7.7 Hz, 1H), 7.40−7.26 (m, 1H), 7.21 (dd, J
= 9.7, 3.0 Hz, 1H), 7.16 (td, J = 8.5, 2.9 Hz, 1H), 4.70−4.49 (m, 1H),
4.17−4.05 (m, 1H), 3.96−3.72 (m, 1H), 3.68 (s, 2H), 3.27−3.20 (m,
1H), 3.08−2.86 (m, 1H), 1.97−1.69 (m, 3H), 1.52−1.42 (m, 1H),
1.21 (s, 9H). 13C NMR (101 MHz, DMSO-d6, δ) 171.9, 162.7, 159.3
(d, J = 242.5 Hz), 156.1 (d, J = 252.5 Hz), 153.8, 150.3 (d, J = 234
Hz), 149.1, 133.8, 133.1 (d, J = 8.4 Hz), 132.4 (d, J = 2.6 Hz), 130.3,
128.0 (d, J = 8.8 Hz), 124.8 (d, J = 16.0 Hz), 124.5 (d, J = 3.9 Hz),
122.8 (d, J = 16.9 Hz), 121.6 (d, J = 17.2 Hz), 117.7 (d, J = 12.2 Hz),
117.4 (d, J = 7.8 Hz), 117.04, 114.0, 113.8, 78.3, 71.1, 45.4, 42.7, 37.0,
28.8, 27.7, 20.5.19F NMR (376 MHz, DMSO-d6, δ) −116.84,
−116.90, −123.1. LCMS (method a) m/z: 617.2 [M − H]−, tR
1.33 min. [α]2D3 + 29.0 (c 1.0, MeOH).
=
P
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(R)-2-(2-(5′-((1-(tert-Butoxycarbonyl)piperidin-3-yl)oxy)-4′-
chloro-2,2′-difluoro-[1,1′-biphenyl]-3-carboxamido)-5-
fluorophenyl)acetic Acid (20). Following the general procedure for
Boc-cleavage with HCl, (R)-2-(2-(5′-((1-(tert-Butoxycarbonyl)-
piperidin-3-yl)oxy)-4′-chloro-2,2′-difluoro-[1,1′-biphenyl]-3-carboxa-
mido)-5-fluorophenyl)acetic acid (50, 423 mg, 0.65 mmol) provided
the title product (HCl salt) as a colorless solid in 341 mg (95%) yield.
1H NMR (400 MHz, DMSO-d6, δ) 12.42 (s, 1H), 10.05 (s, 1H), 9.17
rat SUCNR1−Nanobody6 complex were performed as
described previously with the following modifications for
complex formation and crystallization:20
Compound 5 was added to purified humanized rat SUCNR1
to a final concentration of 1 mM from a 100 mM stock
solution in DMSO, giving a final DMSO concentration of 1%
(v/v). The receptor was mixed with a 1.2 M excess of purified
Nanobody6, incubated on ice for 30 min. The complex was
further purified without any antagonist by size-exclusion
chromatography on a Superose6 Increase 10/300 GL column
(GE Healthcare) equilibrated in 25 mM HEPES pH 7.5, 800
mM NaCl, 10% (w/v) glycerol, 0.002% (w/v) LMNG, and
0.0004% (w/v) CHS. Peak fractions were pooled and
concentrated using a 100 kDa MWCO concentrator
(Millipore) to a final concentration of 30 mg/mL. The
complex was flash-frozen in liquid nitrogen in small aliquots
and stored at −80 °C until crystallization.
The rat SUCNR1−Nanbody6 complex was reconstituted in
the lipidic cubic phase (LCP) by mixing protein at 30 mg/mL
with monoolein:cholesterol (9:1) (w:w) at a 2:3 ratio (v:w) in
50 μL Hamilton syringes using the two-syringe method.
Crystallization trials were performed using Innovadyne SD-2
vapor diffusion plates and dispensed using an Oryx8
crystallization robot (Douglas Instruments). Protein-laden
LCP (50 nL) was covered with 2.25 μL of precipitant,
containing 200 μM 20, and incubated at 20 °C in an RI-1000
imager (Formulatrix). First crystals appeared within 24 h and
grew to a maximum size of 40 μm × 40 μm × 40 μm in 10
days in 50 mM 2-[(2-amino-2-oxoethyl)-(carboxymethyl)-
amino]acetic acid (ADA) pH 7.0, 28% (w/v) poly(ethylene
glycol) monomethyl ether (PEG MME) 550, 0.55 M
(NH4)2SO4, 200 μM 20, and 2% (v/v) DMSO. Crystals
were directly collected from the LCP bolus with MiTeGen
micromount loops and flash-frozen in liquid nitrogen.
Data Collection, Structure Solution and Refinement.
Data for the humanized rat SUCNR1−Nanobody6-20
complex were collected at PXII at the Swiss Light Source,
Villigen, Switzerland, using a 10 μm diameter beam at a
wavelength of 0.999 Å on a Dectris Eiger-16M detector.
Crystals were exposed for 0.05 s per 0.1° oscillation per frame
using an attenuated beam to reduce radiation damage. Datasets
were integrated, scaled, and merged using XDS and XSCALE
in autoPROC and aP_Scale (Global Phasing). Data collection
and refinement statistics are reported in Extended Data Table
S1 (Supporting Information). The structure was solved by
molecular replacement in Phaser using the structure of the
humanized rat SUCNR1−Nanobody6−NF-56-EJ40 (7) com-
plex (PDB 6RNK) as a search model. The structural model
was adjusted by repetitive rounds of manual model building in
Coot and refinement against the anisotropic-scaled data in
Buster (Global Phasing). Structure factors and coordinates of
the humanized rat GRP91−Nanobody6−20 complex were
deposited in the Protein Data Bank (PDB) under accession
codes 6Z10.
(s, 1H), 8.75 (s, 1H), 7.78 (t, J = 6.3 Hz, 1H), 7.72−7.62 (m, 2H),
7.54−7.41 (m, 3H), 7.21 (dd, J = 9.7, 3.0 Hz, 1H), 7.16 (td, J = 8.5,
3.1 Hz, 1H), 4.78−4.64 (m, 1H), 3.69 (s, 2H), 3.42−3.28 (m, 1H),
3.20 (dd, J = 12.3, 6.2 Hz, 1H), 3.14−2.99 (m, 2H), 2.05−1.90 (m,
2H), 1.89−1.79 (m, 1H), 1.77−1.67 (m, 1H). 13C NMR (101 MHz,
DMSO-d6, δ) 171.9, 162.7, 159.7 (d, J = 242.1 Hz), 156.0 (d, J =
253.3 Hz), 152.3 (d, J = 245.3 Hz), 148.92 (d, J = 2.5 Hz) 133.9,
133.1 (d, J = 8.4 Hz), 130.4 (d, J = 3.0 Hz), 128.0 (d, J = 8.5 Hz),
124.8 (d, J = 15.7 Hz), 124.5 (d, J = 3.7 Hz), 124.2 (d, J = 10.5 Hz),
122.5 (d, J = 16.3 Hz), 121.9 (d, J = 17.1 Hz), 120.0, 117.9, 117.6 (d,
J = 6.1 Hz), 117.4, 113.9 (d, J = 22.3 Hz), 71.9, 45.2, 42.7, 37.0, 26.9,
18.6. 19F NMR (376 MHz, DMSO-d6, δ) −116.6, −116.8, −120.8.
LCMS (method a) m/z: 519.1 [M + H]+, tR = 0.81 min. LCMS
(method b) m/z: 519.1 [M + H]+, tR = 3.66 min. HRMS m/z: [M +
H]+ calcd for C26H23ClF3N2O4 519.1293; found 519.1293. [α]D23
1.3 (c 1.0, MeOH).
−
BIOLOGY
■
SUCNR1 GTPγS Assay. Assays were done as described
(Haffke et al. 2019)20 using membranes from stably trans-
fected Chem1 or CHO-K1 cells expressing human or rat
SUCNR1, respectively. The final assay mixture in 96-well
Optiplates (Perkin Elmer) contained 15 μg of rat or human
SUCNR1 membranes, wheat germ-agglutinin-coated scintilla-
tion proximity assay beads (Perkin Elmer), 200 pM
[35S]GTPγS (Perkin Elmer), 20 mM HEPES pH 7.4, 100
mM NaCl, 10 mM MgCl2, 25 μg/mL saponin, 10 μM GDP,
0.1% fat-free bovine serum albumin, 50 μM succinate, and the
test compound. After incubation for 60 min at room
temperature sealed plates were centrifuged for 10 min at
1200 rpm and the radioactivity was counted (TopCount NXT;
Perkin Elmer). IC50 values were determined from eight-point
concentration−response curves using GraphPad Prism.
In Vivo Experiments. All animals used were 5−12 weeks
of age and were maintained in the specific pathogen-free
facilities. All animal studies were performed in accordance with
the animal experimentation guidelines and laws laid down by
the Swiss Federal and Cantonal Authorities.
In Vivo Pharmacokinetic Studies in Mice. Pharmacoki-
netic studies were conducted using male C57/BL6 mice. For 1
mg/kg intravenous dosing, compounds were formulated as a
solution (NMP:plasma (10:90)) and mice (n = 3 per group)
were administered compounds by bolus injection; the dose
volume was 5 mL/kg. For oral dosing, compounds were
formulated as a suspension (MC:water:Tween80 (0.5:99:0.5)).
Mice (n = 3 per group) were administered by oral gavage; the
dose volume was 10 mL/kg. Serial intravenous blood samples
were taken at 0.083, 0.25, 0.5, 1, 2, 4, 7, and 24 h. For the oral
arm blood samples were taken at 0.25, 0.5, 1, 2, 4, 7, and 24 h;
precipitated with acetonitrile; and stored at −20 °C prior to
liquid chromatography−mass spectrometry (LC−MS)/MS
analysis. Pharmacokinetic parameters were calculated by
noncompartmental analysis. Pharmacokinetic studies with
compounds 8 and 9 were described previously.28
Following high-throughput assays were performed according
to the described procedures: PAMPA,22 LogD7.4,23 and UV-
metric pKa.36
Plasma Protein Binding (PPB). Compounds, dissolved in
DMSO, were added to the plasma to achieve a concentration
of 5 μM and 0.5% DMSO. Plasma samples were placed in one
chamber of the rapid equilibrium dialysis (RED) device.
Phosphate-buffered saline (100 mM) containing 0.5% DMSO
was placed in the other chamber. The RED device was shaken
Protein Expression, Purification, and Crystallization
of Humanized Rat SUCNR1 in Complex with 20. The
expression, purification, and crystallization of the humanized
Q
https://dx.doi.org/10.1021/acs.jmedchem.0c01020
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Journal of Medicinal Chemistry
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Article
at 250 rpm at 37 °C with 5% CO2 for 4 h to reach equilibrium.
After the incubation, 0.45 μL of the sample was removed from
each side of the membrane. An equal volume of 100 mM
phosphate buffer containing 0.5% DMSO was added to the
plasma sample. An equal volume of plasma containing 0.5%
DMSO was also added to the buffer sample. Samples were
protein-precipitated with 180 μL of chilled methanol
(containing the internal standard 0.55 μM metoprolol). After
being shaken, the samples were centrifuged at 2000g for 30
min, and 20 μL of the supernatant was removed and diluted
with 20 μL of water prior to LC−MS/MS analysis. The
fraction unbound in plasma (fu) was calculated by dividing the
concentration in the buffer chamber by the concentration in
the plasma chamber.
Fabian Piller − Novartis Institutes for BioMedical Research, CH-
4002 Basel, Switzerland
Stephanie Harlfinger − Novartis Institutes for BioMedical
Research, CH-4002 Basel, Switzerland
Rowan Stringer − Novartis Institutes for BioMedical Research,
CH-4002 Basel, Switzerland
Dominique Fehlmann − Novartis Institutes for BioMedical
Research, CH-4002 Basel, Switzerland
Klemens Kaupmann − Novartis Institutes for BioMedical
Research, CH-4002 Basel, Switzerland
Amanda Littlewood-Evans − Novartis Institutes for BioMedical
Research, CH-4002 Basel, Switzerland
Matthias Haffke − Novartis Institutes for BioMedical Research,
CH-4002 Basel, Switzerland
Nina Gommermann − Novartis Institutes for BioMedical
Research, CH-4002 Basel, Switzerland
Docking Studies. Docking studies were carried out using
̈
Glide SP at standard settings (cite: Schrodinger Release 2019-
̈
2: Glide, Schrodinger, LLC, New York, NY, 2019). Figures
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01020
were prepared using PyMOL (cite: The PyMOL Molecular
̈
Graphics System, Version 2.0 Schrodinger, LLC).
Author Contributions
ASSOCIATED CONTENT
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01020.
Funding
The authors declare no competing financial interest, and all
work described was funded by Novartis Institutes for
BioMedical Research Inc.
HPLC and NMR charts and X-ray crystallographic data
for compounds 9, 10, and 20 as well as SUCNR1-bound
20 (PDF)
Notes
Molecular formula strings (CSV)
The authors declare no competing financial interest.
Accession Codes
ACKNOWLEDGMENTS
We thank Daniel Pflieger, Gu
Gaelle Chenal, and Harald Schroeder for their assistance
during the preparation of this manuscript. We would also like
to thank Stephane Rodde and Damien Hubert for the
physicochemical measurements and Corinne Marx for
HRMS measurements.
Atomic coordinates and structure factors for the crystal
structures of compounds 9, 10, and 20 can be accessed
using the following codes: CCDC 2009710 (9), CCDC
2009711 (10), and CCDC 2009712 (20). For compound 20
bound to humanized rat SUCNR1, the PDB code is 6Z10. The
authors will release the atomic coordinates upon article
publication.
■
̈
nter Schwalm, Bernard Faller,
ABBREVIATIONS USED
AUTHOR INFORMATION
■
■
CHO-K1, Chinese hamster ovary cells; CHS, cholesteryl
hemisuccinate; DIAD, diisopropyl azodicarboxylate; DIPEA,
N,N-diisopropylethylamine; dn, dose-normalized; dppf, 1,1′-
ferrocenediyl-bis(diphenylphosphine); HEPES, 4-(2-hydrox-
yethyl)-1-piperazineethanesulfonic acid; HIF1α, hypoxia-indu-
cible factor 1α; extrap, extrapolated; LMNG, lauryl maltose
neopentyl glycol; MC, methyl cellulose; MWCO, molecular
weight cut-off; Pin, pinacol
Corresponding Author
Juraj Velcicky − Novartis Institutes for BioMedical Research,
CH-4002 Basel, Switzerland; orcid.org/0000-0001-6564-
1448; Email: juraj.velcicky@novartis.com
Authors
Rainer Wilcken − Novartis Institutes for BioMedical Research,
CH-4002 Basel, Switzerland; orcid.org/0000-0002-6159-
1460
REFERENCES
Simona Cotesta − Novartis Institutes for BioMedical Research,
■
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