Target-Based Identification and Optimization of 5-Indazol-5-yl Pyridones as Toll-like Receptor 7 and 8 Antagonists Using a Biochemical TLR8 Antagonist Competition Assay

Article abstract of DOI:10.1021/acs.jmedchem.0c00130

Inappropriate activation of endosomal TLR7 and TLR8 occurs in several autoimmune diseases, in particular systemic lupus erythematosus (SLE). Herein, the development of a TLR8 antagonist competition assay and its application for hit generation of dual TLR7

Full text of DOI:10.1021/acs.jmedchem.0c00130

pubs.acs.org/jmc
Article
Target-Based Identification and Optimization of 5‑Indazol-5-yl
Pyridones as Toll-like Receptor 7 and 8 Antagonists Using a
Biochemical TLR8 Antagonist Competition Assay
́
Thomas Knoepfel,* Pierre Nimsgern, Sebastien Jacquier, Marjorie Bourrel, Eric Vangrevelinghe,
Ralf Glatthar, Dirk Behnke, Phil B. Alper, Pierre-Yves Michellys, Jonathan Deane, Tobias Junt,
́
Geraldine Zipfel, Sarah Limonta, Stuart Hawtin, Cedric Andre, Thomas Boulay, Pius Loetscher,
Michael Faller, Jutta Blank, Roland Feifel, and Claudia Betschart
Cite This: https://dx.doi.org/10.1021/acs.jmedchem.0c00130
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: Inappropriate activation of endosomal TLR7 and
TLR8 occurs in several autoimmune diseases, in particular systemic
lupus erythematosus (SLE). Herein, the development of a TLR8
antagonist competition assay and its application for hit generation
of dual TLR7/8 antagonists are reported. The structure-guided
optimization of the pyridone hit 3 using this biochemical assay in
combination with cellular and TLR8 cocrystal structural data
resulted in the identification of a highly potent and selective
TLR7/8 antagonist (27) with in vivo efficacy. The two key steps for
optimization were (i) a core morph guided by a TLR7 sequence
alignment to achieve a dual TLR7/8 antagonism profile and (ii)
introduction of a fluorine in the piperidine ring to reduce its
basicity, resulting in attractive oral pharmacokinetic (PK) properties and improved TLR8 binding affinity.
target- or pathway-specificity, effective counterassays are
required, and rational optimization can be complicated by the
complexity of various assays and cell types used. With the ligand
binding domain (or ectodomain, ECD) of both TLR7 and 8
being localized inside the endosomal lumen, these assays favor
identification of compounds that are locally enriched in the
endolysosomal compartment. Consequently, the mode of action
of some antagonists of endosomal TLRs has been attributed to
rely on a combination of high local concentration within the
endosome and binding to TLR ligand oligonucleotides, rather
than direct interaction with the receptors.9 Although such
compounds show cellular efficacy, the presence of several basic
moieties needed for such a behavior10 negatively influences
physicochemical properties, off-target effects, and risk of
accumulation upon repeated dosing.11
INTRODUCTION
■
Toll-like receptors (TLRs) are a class of molecular pattern
recognition receptors that are a part of the innate immune
system. They recognize a variety of specific molecular motifs
indicative of infections and initiate effective immune responses.
Both TLR7 and TLR8 are located at the endosomal membrane
and recognize guanidine-/uridine-rich single-stranded RNA
(ssRNA) inside the endosomal lumen. Inappropriate activation
of these endosomal TLRs occurs in several autoimmune
diseases, in particular systemic lupus erythematosus (SLE), as
the body’s own RNA gains access to endosomes, e.g., upon
complexation with autoantibodies.1 TLR7 is predominantly
expressed in plasmacytoid dendritic cells (pDCs) and B cells,
whereas TLR8 is expressed in monocytes/macrophages and
̈
neutrophils. In autoimmune diseases such as SLE or Sjogren’s
Our strategy was to develop a robust biochemical assay that
would allow identification and subsequent optimization of small
molecules acting directly on the ECD of these receptors. In
syndrome, there is involvement of a broad range of immune
cells.1 Therefore, as an approach toward treatment of such
diseases, we aimed at identifying potent dual TLR7/8
antagonists.
Identification and optimization of TLR7 and TLR8
antagonists have previously relied on phenotypic cellular
assays.2−8 While this hit generation approach has been
successfully applied, some limitations exist. For example,
validation and optimization of hits from cellular screening
campaigns can be time and resource intensive. To demonstrate
Received: March 27, 2020
https://dx.doi.org/10.1021/acs.jmedchem.0c00130
© XXXX American Chemical Society
J. Med. Chem. XXXX, XXX, XXX−XXX
A

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 1. TLR8 binder 1 and fluorescent probe 2 used in the time-resolved fluorescence resonance energy transfer (TR-FRET) assay. IC50 reported as
geometric mean standard deviation (SD).
Table 1. Evolution of Hit Compound 3 to Lead Compound 8
a
IC50 [μM]
3
4
5
6
7
8
TLR8 FRET
2.4 2.0
1.4 0.48
1.5 1.8
0.091 0.036
2.2 0.96
0.22 0.22
5.9 2.2
>10
0.045 0.012
>10
7.6 1.8
1.6 1.5
0.14 0.013
0.32 0.021
2.0 0.55
1.7 0.7
19 5.4
>4.2
0.014 0.0078
0.076 0.021
7.2 1.8
3.7 1.0
>10
0.028 0.00071
0.039 0.013
>10
TLR4 PBMCs
TLR7 PBMCs
TLR8 PBMCs
TLR9 PBMCs
0.25 0.079
0.034 0.0021
0.033 0.0042
0.20 0.035
a
IC50 determined as geometric mean (n ≥ 2) SD. Cytokine readouts for TLR7 and TLR9 (IFNα) and TLR8 and TLR4 (TNF) are shown.
2013, the expression and X-ray structure of human TLR8 ECD
were reported by the Shimizu group in the apo form and bound
to a small-molecule agonist.12 Since TLR7 and TLR8 show high
sequence homology (see the Supporting Information for a
sequence alignment), we assumed that their structure and
function would also be closely related. No TLR7 crystal
structure was available at the time this work was initiated. In
the meantime, the structures of monkey TLR7 in the apo and
agonist bound forms have been reported.13,14 As such, we
decided to use the recombinant human TLR8 ECD as a
surrogate system for hit generation of dual TLR7/8 antagonists.
Herein, we report the development and use of a TLR8
antagonist competition assay for high-throughput screening of
TLR8 antagonists. Furthermore, we describe the optimization of
a pyridone biaryl scaffold identified in this screen. Using a
combination of structural, binding, and cellular approaches, we
have identified a highly potent, selective, and orally active dual
TLR7/8 antagonist.
labeled antagonist. At that time, no TLR8 antagonist had been
reported, which showed direct receptor binding. In an
alternative approach aimed at identifying TLR7/8 pathway
antagonists based on cellular screening and phenotypic
optimization, we had identified compound 1 (Figure 1). The
discovery of compound 1 and related structures is described in a
separate publication.15 Based on structure−activity relationship
(SAR) data, modifications of the primary amine were well
tolerated, making it an attractive attachment point for a
fluorescent tracer. Coupling of a fluorescent Cy5 dye to the
primary amine in 1 resulted in probe 2. Binding experiments
using fluorescence polarization (FP) as a readout with 2 nM
compound 2 and varying concentrations of TLR8 ECD protein
yielded an apparent Kd of 65 nM. Binding of 2 to TLR8 could be
competed with excess of unlabeled compounds such as 1 and its
analogues.
TR-FRET Assay and High-Throughput Screen. Fluo-
rescence polarization does not require any labeling of the target
protein. However, these assays are run with low nM
concentrations of the labeled ligand and target concentrations
above the apparent Kd. In this case, the amounts required (>80
nM TLR8), would limit the utility of the assay for a large screen.
Consequently, we converted this assay to a TR-FRET format
(time-resolved fluorescence resonance energy transfer),16 using
RESULTS AND DISCUSSION
■
TLR8 Antagonist Competition Assay. To devise an assay
system that would be amenable for high-throughput screening,
we established a ligand competition assay using a fluorescently
B
https://dx.doi.org/10.1021/acs.jmedchem.0c00130
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
biotinylated TLR8 protein and Eu-labeled streptavidin. Binding
of 2 to TLR8 brings the Cy5 acceptor dye on the ligand in close
proximity to the Eu fluorophore. The TR-FRET assay was
miniaturized to 1536-well plates. A set of about 50 000
compounds from the Novartis compound collection was
selected based on chemical diversity; TLR8 structural
information was not considered for the selection. The
compounds were evaluated for their ability to compete with
the fluorescent probe 2 in the TR-FRET assay at 20 μM. In total,
approximately 1500 compounds were identified with potency
determined in dose-response experiments. Representatives of
the most interesting hit classes were assessed in relevant human
cell-based assay systems based on peripheral blood mononuclear
cells (PBMCs), which express both TLR7 and TLR8.
pocket making face-to-edge contact with Phe495* and Phe494*
side chains. The model revealed a small hydrophobic cavity,
defined by the side chains of residues Phe346, Tyr348, and
Val378, which could be reached from the 8-position of the
quinazoline.
The 8-methylquinazoline 4 indeed showed an improved
binding affinity and cellular potency, in agreement with the
TLR8 model. However, when 4 was profiled against TLR7/8/9/
4 in PBMCs, the compound lacked antagonism of TLR7 (Table
1), which was in contrast with our initial hypothesis that TLR7
and TLR8 are structurally and functionally equivalent. To
rationalize this finding, we aligned the sequences of TLR7 and
TLR8 (see the Supporting Information) looking for non-
conserved amino acids in TLR7 vs TLR8, which are in direct
contact with the ligand.
Hit-to-Lead Optimization. Pyridone 3 was identified as a
hit compound with moderate binding affinity and attractive
TLR8 cellular potency as assessed by inhibition of TNFα release
from stimulated human PBMCs (Table 1). The presence of a
moderately basic piperazine (pKa: 6.1) was assumed to lead to
some degree of enrichment of the compound in the acidic
endosome, possibly contributing to the improved cellular
inhibition relative to its binding affinity. Compound 3 was
docked into the antagonist binding site of TLR8 ECD of a
previously internally obtained TLR8 antagonist structure by
applying a combination of manual and automated docking with
Glide17 (PDB file of the model in the Supporting Information).
According to this model, two antagonist molecules bind to two
symmetrical pockets at the interface of two TLR8 ECDs that
form a homodimer (see Figure 5 for the detailed description of a
TLR8 antagonist cocrystal structure). As shown in Figure 2, the
pyridone carbonyl of compound 3 was predicted to make a key
hydrogen bond with the backbone NH of Gly351 and the
piperazine NH+ a salt bridge with the carboxylate of Glu427.
The quinazoline core is located in a hydrophobic part of the
As highlighted in purple in Figure 3, we identified two amino
acids that are different in TLR7. In TLR8, alanine 518* is a
Figure 3. Binding model of compound 4 with TLR8; amino acid
differences in TLR7 are shown in purple. Amino acids belonging to one
TLR8 monomer are in green, and the second monomer amino acids are
in light gray. Amino acids belonging to the green monomer are labeled
with an asterisk. PDB file of the model is provided in the Supporting
Information.
serine (530*) and glutamic acid 427 is a valine (430) in TLR7.
Based on this analysis, we decided to morph the quinazoline core
of 4 to an indazole as is the case in compound 5. Our hypothesis
was to avoid a steric clash of the hetero-bicyclic ring of the ligand
with Ser530* in TLR7 and at the same time change the exit
vector to the piperazine avoiding direct interaction with Glu427
in TLR8.
While compound 5 lost significant binding affinity to TLR8,
the cellular TLR7 activity did increase to a similar potency level
as TLR8, in line with our desired inhibition profile. The loss in
binding affinity was attributed to the inability of 5 to directly
interact with Glu427, but to get to a balanced TLR7/8
antagonist profile, we considered this necessary. The observed
low micromolar antagonistic activity of 5 on TLR4 and TLR9 in
PBMCs was not believed to be a result of receptor binding.
Figure 2. Binding model of compound 3 with TLR8. Amino acids
belonging to one TLR8 monomer in green and the second monomer
amino acid in light gray. Amino acids belonging to the green monomer
were labeled with an asterisk. The PDB file of the model in the
Supporting Information.
C
https://dx.doi.org/10.1021/acs.jmedchem.0c00130
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Based on the structural dissimilarity of TLR4 and TLR9 to
TLR8, we considered it highly unlikely that an equivalent
antagonist binding pocket exists in these receptors (see the
Supporting Information for sequence alignment of human
TLR7/8/9 ECD; the similarity to TLR4 was too low for an
alignment with sufficient confidence). Hence, the cellular
response on TLR4 and 9 is likely caused by an off-target effect.
A methyl scan on the pyridone revealed the possibility of
separating TLR7/8 from TLR4/9 antagonism (compounds 6−
8 in Table 1). The 6-methyl derivative 8 displayed the desired
cellular profile, with equipotent TLR7/8 antagonism, while
eliminating measurable TLR4/9 effects. Based on this promising
cellular TLR antagonism profile, the favorable physicochemical
and in vitro absorption, distribution, metabolism, and excretion
(ADME) profile (albeit with a moderate permeability) of
compound 8, it was selected as a lead structure (Figure 4).
residue compared to a methyl group was expected to optimally
fill the hydrophobic subpocket. In line with this, chloro-
substituted 14 was equipotent, but trifluoromethyl 15 and
cyclopropyl 16 both led to a small but consistent positive effect
on TLR8 binding and cellular antagonism.
According to the binding model, the piperidine portion of the
molecule (R″) is located at the exit of the binding pocket facing
the solvent. As a consequence, we did not expect a significant
gain in binding affinity by modifications in this area. The basic
piperidine nitrogen could be N-methylated (17), acylated with
dimethylamino acetamide (18), or moved to the 3-position (19)
without loss of potency, in line with the above hypothesis. For
the 3-piperidine, the impact of basicity on the ratio of binding
affinity and cellular potency was studied by modulation of pKa
either through introduction of fluorines or by aromatization.
While the TLR8 binding affinity remained comparable for the
fluorinated piperidines 20, 21, and trifluoroethyl-substituted 23,
it was increased for pyridine 22. Basic compounds with a pKa of
6.2 or higher consistently gained in cellular potency relative to
the binding affinity, whereas compounds with a pKa of 4.7 and
below had comparable binding affinities and cellular potencies.
These data are consistent with a pKa-dependent permeability
leading to the colocalization of basic compounds with the
receptor in acidic endosomes.10 Based on the SAR generated in
the piperidine (R″) position, we kept the 4-piperidine
substituent for further optimization; the pyridine was
deprioritized based on the lack of sp3 carbons.18
Cocrystal Structure of 11 with TLR8 ECD. Cocrystals of
compound 11 with the TLR8 ECD suitable for X-ray diffraction
analysis were obtained. The structure was solved with a
resolution of 2.79 Å, and it confirmed the expected binding
mode of the antagonist at the interface of the dimeric TLR8
units in two symmetrical pockets (Figure 5a,b). The protein
adopts a conformation very similar to the apo form, which is in
line with stabilization of the inactive state, leading to antagonism
as the mode of action of these compounds. The structural basis
of inhibition is believed to be the same as has been published and
well described in detail in the literature.19
The binding mode in the two individual sites is very similar
and can be superimposed. In the binding sites, there are only
minor changes in the orientation of residues compared to the
apo structure of TLR8. The postulated key interaction of the
pyridone carbonyl with the backbone NH of Gly351was nicely
confirmed. In addition, two water-mediated hydrogen bonds
from the pyridone carbonyl to Gln529* and from the indazole
nitrogen to Tyr576* were observed (Figure 5c). The 7-methyl
indazole substituent fills the hydrophobic subpocket described
above, and the piperidine points into the solvent without directly
contacting Glu427 (Figure 5d). Taken together, this exper-
imental data was in good agreement with our model that had
been used to drive the optimization and the SAR generated. This
indicates that even though the binding pocket is at the interface
of two large proteins and made up of flexible loops, it is well
suited for structure-based compound optimization.
Figure 4. Lead profile of compound 8.
Lead Optimization. TLR8 receptor binding and perme-
ation were key parameters for further optimization. Systematic
modification of the indazole substituents (R, R′, and R″) was
aimed at identifying the substituent with the strongest
contribution to TLR8 binding (Table 2), which ultimately
could be combined in a single molecule with an overall
acceptable profile. For this phase of the project, TLR8 binding
and cellular data were used for decision-making in terms of
compound potency. TLR7 and TLR4 cellular data were
generated for selected compounds as tertiary assays to confirm
that the desired dual antagonism profile was maintained.
For the pyridone (R) position, the binding model suggested
only limited space for further growth from the 6-position.
Consequently, we focused on substituents maintaining or
slightly decreasing the size. The 6-fluoro-substituted pyridone
9 displayed a slightly improved binding affinity, whereas the 6-
chloro derivative 10 was similar to the methyl pyridone. We
hypothesized that annulation to a 6,5-bicylic system would have
a similar effect of slight size reduction at the 6-position due to the
ring constraint and that such a compound could nicely fit into
the binding pocket. Indeed, incorporation of the annulated 5-
membered rings in 11 and 12 led to improved affinities in TLR8
binding and improved the cellular TLR8 antagonism.
Based on the SAR information generated in all three parts (R,
R′, and R″) of the molecule, we then explored the synergy of
single effects by preparing two combinations with substituents
we considered optimal (Table 3). Both compounds 24 and 25
showed additive effects for enhanced TLR8 affinity and were
considered as candidates for testing in vivo. For the 6-fluoro
pyridone 24, we had concerns about potential chemical
reactivity with nucleophiles, even though during the synthesis
(vide infra) we had not observed any byproducts resulting from
The important contribution to binding of the indazole
substituent (R′) at the 7-position was confirmed by the loss in
affinity for the unsubstituted compound 13. A slightly larger R′
D
https://dx.doi.org/10.1021/acs.jmedchem.0c00130
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
c
Table 2. Structure−Activity Relationship of 5-Indazol-5-yl Pyridones
E
https://dx.doi.org/10.1021/acs.jmedchem.0c00130
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Table 2. continued
a
bS
c
IC50 determined as the geometric mean (n ≥ 2) SD. ingle measurement. Measured pKa of the corresponding R-enantiomer. n.d., not
determined.
Figure 5. Cocrystal structure of compound 11 with TLR8 ECD: (PDB 6TY5) (a) full view, one TLR8 monomer in green, the second monomer in light
gray, compound 11 in light blue; (b) top view, turned by 90°; (c) close-up view on one binding site showing amino acids within 5 Å of 11, except for
Phe261, which is not shown for clarity. Amino acids belonging to the green monomer were labeled with an asterisk. (d) Side view of the binding site
only showing one monomer with surface representation and the 2Fo − Fc map contoured at 1.0 σ for the bound inhibitor.
fluoride substitution even under basic conditions and heating.
To assess the potential reactivity of the 6-fluoro pyridone in a
physiologically more relevant system, we exposed 24 to 5 mM
glutathione (GSH)20 in aqueous phosphate buffer (pH 7) at
room temperature (see the Supporting Information). We
observed the formation of a new product by liquid
chromatography−mass spectrometry (LC−MS), which by
mass was consistent with fluoride substitution with GSH (32%
F
https://dx.doi.org/10.1021/acs.jmedchem.0c00130
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Table 3. Combination of R Groups
Table 4. Fluorinated Analogs of 25
a
IC50 determined as geometric mean (n ≥ 2) SD.
a
IC50 determined as geometric mean (n ≥ 2) SD.
conversion after 15.5 h assuming the same absorption of 24 and
adduct at 254 nm). This qualitative observation of reactivity led
to the decision not to pursue the 6-fluoro pyridone structural
motif any further due to the risk of unspecific covalent adduct
formation in cells and later in vivo.
The combination of the bicyclic pyridone with the CF3-
indazole in 25 resulted in the lowest cellular IC50 of 2.7 nM we
had observed so far. However, to our disappointment, the in vitro
passive permeability measured in the low efflux (LE)-MDCK
assay was significantly lower than for 24 (Papp 24: 4.8 cm/s*10−6
→ 25: 1.8 cm/s*10−6). This was in line with a reduction in log D
(24: 0.63 → 25: 0.3), indicating that the annulation at the
pyridone resulted in increased polarity, which was limiting the
permeation.
Figure 6. Calculated lowest energy conformations of 28 and 27 in water
at neutral pH and small-molecule crystal structure of 27 (CCDC
1979262). Orange lines indicate dihedral angles about the indazole−
piperdine C−C bond.
Our strategy to improve the permeation properties of
compound 25 was to slightly lower the pKa to reduce the
fraction ionized at neutral pH, but only to a degree at which the
cellular potency was not negatively impacted as previously
observed. Both cis and trans isomers of the 3-fluoropiperidine
derivatives shown in Table 4 were prepared, the trans isomer in
both enantiopure forms. This desired effect on lowering the pKa
translated into a significantly higher permeability for all
stereoisomers. Interestingly, we observed an improvement in
the TLR8 binding affinity for the trans isomers 26 and 27
relative to the nonfluorinated 25 and the racemic cis isomer 28.
In an attempt to rationalize the effect of the relative
stereochemistry in the 3-F-piperidines on the binding affinities,
the lowest energy solution conformations of compounds 27 and
28 were calculated with a high-accuracy quantum-chemistry-
based calculation method.21 As shown in Figure 6, the lowest
energy conformations of the cis and trans isomers have a
different preference for the dihedral angle about the bond
connecting the indazole and piperidine rings (63° for 27; 94° for
28). The conformation of the piperidine is nearly inverted
between the two isomers. The lowest energy conformation of
the trans isomer is characterized by a fluorine atom in an
equatorial position and the basic nitrogen pointing toward the
same direction as the nitrogen acceptor of the indazole ring. In
the case of the cis isomer, the fluorine atom is in an axial position
and the basic nitrogen is pointing toward the opposite direction.
The orientation of the fluorine could explain the observed
difference in pKa for both isomers,22 thereby further increasing
our confidence in the relevance of the calculated conformations.
The calculated lowest energy conformation is in agreement with
a conformation observed in a crystal structure of 27 where the
measured dihedral angle is similar (53°, Figure 6). The unit cell
in the crystal structure of compound 27 contains two molecules.
One of this is displayed in Figure 6; the other molecule is
disordered and has a 60:40 ratio of two rotamers about the
piperidine indazole bond (see the Supporting Information).
This structure also unambiguously proved the chemical
structure and absolute stereochemistry of 27.
While the moderate resolution of the TLR8 cocrystal
structure obtained with compound 11 does not allow one to
experimentally determine the exact binding conformation in the
region of the piperidine, we propose that the introduction of the
fluorine in the trans isomer is responsible for the stabilization of
the bioactive conformation, leading to the increased binding
affinity.
Compound 27 combined high TLR8 binding affinity and
TLR8 cellular potency in human PBMCs with good permeation
and was therefore selected for broader characterization in vitro
and in vivo. As summarized in Table 5, compound 27 is a highly
potent, dual TLR7/8 antagonist in human PBMCs and blood.
Furthermore, 27 was very selective against a panel of other
human TLR isoforms and IL1R-driven cytokine pathways with
an overall selectivity profile >1000-fold. It displayed cross-
species activity albeit slightly weaker, with selective inhibition of
G
https://dx.doi.org/10.1021/acs.jmedchem.0c00130
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Table 5. TLR Pathway and Selectivity, Physicochemical,
ADME, and Pharmacokinetic (PK) Profile of 27
Figure 7. Compound 27 inhibits TLR7-dependent IFNα secretion in
mice in vivo. Exposure (blue squares) in blood and concentration of
IFNα in serum (gray bars) at the indicated doses are shown. Data
points for compound-treated groups represent means of n = 5 mice and
for the vehicle-treated group means of n = 10 mice SD. Pooled data
from two experiments. Horizontal dotted blue line represents in vitro
TLR7 IC50 of compound 27 in mouse whole blood (Table 5). **, p <
0.01; ***, p < 0.001 for IFNα, analysis of variance (ANOVA) with
Dunnett’s post test, compared to the vehicle group.
2-chloro quinazolines 31 and 32 with N-alkyl piperazines
followed by Suzuki coupling with pyridone-3-pinacol boronic
ester 35 as shown in Scheme 1. 6-Bromo-2-chloro-8-methyl-
quinazoline (32) was prepared in four steps from anthranilic
acid 29.
The first synthetic route to the indazol-5-pyridones is shown
in Scheme 2. Indazoles lacking an 8-substituent could be
conveniently prepared starting with a reductive Cadogan
cyclization of 5-bromo-2-nitrobenzaldehyde (36) with primary
amines.25 The resulting 5-bromo indazoles were coupled with 5-
halo pyridinones in a one-pot borylation/Suzuki coupling
sequence.26 We found that, generally, borylation of the indazole
followed by coupling with a 5-halo pyridone was preferred over
borylation of the pyridone followed by coupling. Due to the lack
of efficient synthetic access to the corresponding nitro
benzaldehydes, the 8-substituted indazoles were accessed by
N-alkylation of the indazoles at the 2-position. However, this
reaction resulted in mixtures of regioisomers of 1- and 2-
alkylated products that needed to be separated by chromatog-
raphy. To avoid this, we developed an alternative synthesis that
is shown in Scheme 3.
The second-generation synthesis relied on a reductive
cyclization of azido benzaldehydes with primary amines to
build the 2-alkyl indazole core. We found that this reaction
proceeded smoothly when preformed in 1,2-dichloroethane at
80 °C in the presence of catalytic copper(I) oxide.27,28 The
appropriately substituted azido benzaldehyde 45 could be
conveniently accessed in four robust steps from readily available
anthranilic acid 44. The synthesis of the bicyclic bromo-
pyridone 51 started with bromo-lithium exchange on 2-bromo-
6-methoxypyridine (49) followed by alkylation with THP-
protected 3-bromo propanol to give 50. The following two steps
have been reported previously29,30 and gave rise to 51. Two-step
one-pot borylation/Suzuki coupling as described above
followed by Boc cleavage furnished compounds 26−28.
To be able to introduce the basic amine in the molecule at the
very end of the synthesis, we developed an alternative route
relying on the steps described in Scheme 3, but in a different
order. As shown in Scheme 4, pyridone-coupled azido
benzaldehyde 55 was prepared in three steps from 52. The
copper-catalyzed cyclization proceeded smoothly to furnish the
indazoles 20−23.
a
IC50 determined as geometric mean (n ≥ 2)
SD. Cytokine
readouts for TLR7, TLR9 (IFNα), and TLR8 (TNF) are shown.
Similar TLR8 IC50 value of 0.0021 0.0034 μM using LMW agonist
R848 (1 μg/mL) to induce TNF in PBMCs. mean (n = 3) SD.
b
n.a., not applicable; dn, dose normalized to 1 mg/kg.
TLR7-driven cytokine responses in mouse blood. Compound
27 has a favorable physicochemical and in vitro ADME profile
with good solubility, passive permeability, and low clearance in
liver microsomes of mouse, rat, and human. This translated into
a good pharmacokinetic profile of 27 in mice and rats (see Table
5). In both species, the compound had a low to moderate
clearance and a good to excellent oral bioavailability. Overall, the
profile was attractive to assess oral efficacy in an acute TLR-
driven mechanistic in vivo mouse model.
In Vivo Evaluation. The in vivo efficacy of 27 was assessed in
a short-term mechanistic model in mice. It is controversial
whether murine TLR8 is functional or not;23,24 however, cells
that express TLR8 in humans express TLR7 in mice and respond
to ssRNA40. Therefore, TLR7-dependent responses were used
as surrogate readouts. Groups of animals were dosed orally with
different doses of compound 27, one hour prior to intravenous
injection of ssRNA to elicit a TLR7 response. Two hours later,
IFNα protein and compound levels were determined from
circulation, and dose-dependent inhibition of IFNα release was
demonstrated (Figure 7). These data are consistent with the in
vitro efficacy of compound 27 in mouse blood, where TLR7-
driven IFNα inhibition was observed at IC50 = 180 nM.
Collectively, these results demonstrate that compound 27 is an
efficacious TLR7 pathway inhibitor in vivo and has potential for
further development toward use in TLR7/8-driven autoimmune
diseases.
Compound Synthesis. The initial compounds containing a
quinazoline core (3 and 4) were prepared by SNAr of 6-bromo-
H
https://dx.doi.org/10.1021/acs.jmedchem.0c00130
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
Scheme 1. Synthesis of the Quinazoline Core Containing Compounds 3 and 4
a
Reagents and conditions: (a) LiAlH4, tetrahydrofuran (THF), 0 °C; 79%; (b) MnO2, dichloromethane (DCM), room temperature (RT); 91%;
(c) urea, 180 °C; quant.; (d) POCl3, 110 °C; 50%; (e) N-methyl-2-pyrrolidinone (NMP), 150 °C; (f) PdCl2(dppf), aq. Na2CO3, 1,2-
dimethoxyethane (DME), 80 °C.
a
Scheme 2. First-Generation Syntheses of Indazole-Core-Containing Compounds
a
Reagents and conditions: (a) Ag2SO4, I2, EtOH, RT; 67%; (b) NaNO2, AcOH, RT; 83%; (c) Cs2CO3, N,N-dimethylacetamide (DMA), 150 °C;
(d) NaH, DMF, 100 °C; (e) 2-PrOH, 80 °C; then, PBu3, 80 °C; (f) CuI, phenanthroline, TMSCF3, KF, B(OMe)3, dimethyl sulfoxide (DMSO),
60 °C; (g) XPhos Pd G3, XPhos, B2(OH)4, KOAc, EtOH, 80 °C; (h) K2CO3, 80 °C; (i) trifluoroacetic acid (TFA) or HCl, DCM, RT; (j)
formaldehyde, AcOH, NaBH(OAc)3, DCM; (k) 2-(dimethylamino)acetic acid, O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium
hexafluorophosphate (HATU), N,N-diisopropylethylamine (DIPEA), DMF, RT.
I
https://dx.doi.org/10.1021/acs.jmedchem.0c00130
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
Scheme 3. Second-Generation Synthesis of Indazole-Core-Containing Compounds
a
Reagents and conditions: (a) NBS, DCM, RT; 91%; (b) LiAlH4, THF, 0 °C; 72%; (c) MnO2, DCM, RT; 96%; (d) HCl, NaNO2, H2O, 0 °C to
RT; then, NaN3, 0°C to RT; quant.; (e) RNH2, Cu2O, DCE, 80 °C; (f) XPhos Pd G3, XPhos, B2(OH)4, KOAc, EtOH, 80 °C; then, K2CO3, 80
°C; (g) TFA, DCM, RT; (h) n-BuLi, THF, −78°C; 47%; (i) 48% HBr, reflux, then NaOH; 98%; (j) NBS, DMF, RT; 42%.
a
Scheme 4. Alternative Route with Late-Stage Introduction of Amine
a
Reagents and conditions: (a) XPhos Pd G3, XPhos, B2(OH)4, KOAc, EtOH, 80 °C; then, K2CO3, 80 °C; 80%; (b) MnO2, DCM, RT; 91%; (c)
HCl, NaNO2, H2O, 0 °C to RT; then, NaN3, 0 °C to RT; 87%; (d) Cu2O, DCE, 80 °C; (e) HCl, DCM, RT.
CONCLUSIONS
EXPERIMENTAL SECTION
■
■
Chemistry: General information. Unless otherwise stated, all
commercial reagents were used as supplied without further purification
and all reactions were performed under a nitrogen atmosphere using
dry solvents under anhydrous conditions. Compounds were purified by
flash column chromatography using Redisep Rf flash columns with a
Teledyne Isco CombiFlash Rf companion using gradient elution with
the solid phase and eluents given in parentheses or by preparative high-
performance liquid chromatography (HPLC) on a Gilson HPLC prep-
system using C-18 OBD, 100 mm × 30 mm, a 5 μm SunFire Prep
column using decreasingly polar mixtures of acetonitrile in water with a
TFA modifier or Agilent 1260 Infinity systems equipped with DAD and
mass detectors using a Waters X-bridge column, 100 Å, 5 μm, 19 mm ×
100 mm with a SunFire C18 Prep Guard Cartridge, 100 Å, 10 μm, 19
mm × 10 mm, using a MeOH/water optimized gradient elution or a
supercritical fluid chromatography (SFC) system using Waters SFC
100 prep-system Reprospher C18 WCX, 5 μm, 100 Å, 250 mm × 30
mm with a Waters 2998 photodiode array (PDA) detector and a Waters
3100 mass detector using supercritical CO2/MeOH optimized gradient
elution. 1H and 13C NMR spectra were obtained on a Bruker
UltrashieldTM 600 (600 MHz) or a 400 MHz DRX Bruker CryoProbe
(400 MHz) spectrometer. Chemical shifts (δ-values) are reported in
ppm downfield from tetramethylsilane; spectra splitting patterns are
designated as singlet (s), doublet (d), triplet (t), quartet (q), pentet (p)
multiplet, unresolved or more overlapping signals (m), and broad signal
Herein, we have reported a novel biochemical TLR8 antagonist
binding assay that is amenable to high-throughput screening.
Together with structural data on TLR8, this assay proved to be
highly effective in optimizing pyridone screening hit 3 into the
highly potent and selective dual TLR7/8 antagonist 27, which
demonstrated in vivo efficacy after oral dosing in mice.
It is noteworthy that others have reported TLR8 selective
antagonists that bind in the same pocket,19,31 similar to our early
compound 4. An ECD sequence alignment with TLR7 allowed
us to morph this hit into a dually active TLR7/8 antagonist. We
assumed that for TLR7 an equivalent pocket for antagonist
binding exists. However, this ultimately would need to be proven
by a TLR7 structure in complex with an antagonist.
To the best of our knowledge, this is the first report of a ligand
binding assay for an endosomal TLR and the use of such an assay
as a screening tool enabling optimization of TLR8 antagonists
biochemically. This target-based approach is an attractive
alternative to cellular phenotypic-based screens in which
optimization of hits proved to be more complex.
J
https://dx.doi.org/10.1021/acs.jmedchem.0c00130
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(br). 19F data were recorded at 300 K using a Bruker 500 MHz
AVANCE III spectrometer equipped with a 5 mm BBO probe with a z-
gradient system; shifts were referenced to CCl3F = 0.0 ppm. All tested
compounds have purity ≥95% as determined by the Waters ACQUITY
ultraperformance liquid chromatograph (UPLC) using the reverse
phase column (Acquity HSS T3 1.8 μm, 2.1 mm × 50 mm) and a
solvent gradient of A (H2O with 0.05% formic acid and 3.75 mM
ammonium acetate) and solvent B (CH3CN with 0.04% formic acid)
coupled to electrospray ionization (ESI) with positive/negative ion
switching on a single-stage quadrupole detector acquiring mass spectra
over a mass range from 100 to 1200 m/z or by the Agilent 1100 Series
LC/MSD system with DAD/ELSD. Analytical chiral HPLC was
performed on a Shimadzu LC-10 analytical system with a UV detector,
Chiralpak IC column, 5 μm, 4.6 mm × 250 mm, detection wavelength
of 240 nm, using heptane/DCM/isopropanol 50:20:30 with 0.05%
diethylamine.
5-(2-(4-Cyclobutylpiperazin-1-yl)quinazolin-6-yl)-1-methylpyri-
din-2(1H)-one (3). Compound 3 was prepared according to the
procedure described for compound 4 replacing 32 by 6-bromo-2-
chloroquinazoline (31) and 1-isopropylpiperazine by 1-cyclobutylpi-
perazine. 1H NMR (600 MHz, DMSO-d6) δ 9.19 (s, 1H), 8.24 (d, J =
2.7 Hz, 1H), 8.03 (d, J = 2.2 Hz, 1H), 8.00 (dd, J = 8.9, 2.3 Hz, 1H), 7.93
(dd, J = 9.5, 2.8 Hz, 1H), 7.55 (d, J = 8.7 Hz, 1H), 6.52 (d, J = 9.4 Hz,
1H), 3.86 (t, J = 5.0 Hz, 4H), 3.53 (s, 3H), 2.73 (p, J = 7.8 Hz, 1H), 2.34
(t, J = 5.1 Hz, 4H), 2.04−1.96 (m, 2H), 1.88−1.80 (m, 2H), 1.65 (tt, J =
10.3, 7.3 Hz, 2H). 13C NMR (151 MHz, DMSO-d6) δ 161.88, 161.11,
158.61, 150.60, 138.58, 137.30, 132.14, 130.07, 125.67, 123.26, 119.37,
119.35, 116.86, 59.66, 48.80, 43.48, 37.06, 26.57, 14.05. HRMS-ESI [M
+ H]+ m/z calcd for C22H25N5O 376.2132; found 376.2144.
5-(2-(4-Isopropylpiperazin-1-yl)-8-methylquinazolin-6-yl)-1-
methylpyridin-2(1H)-one (4). Step a: (2-Amino-5-bromo-3-
methylphenyl)methanol (52). A solution of 2-amino-5-bromo-3-
methylbenzoic acid (29, 500 mg, 2.17 mmol) in THF (9.00 mL) at 0
°C was treated dropwise with LiAlH4 in THF (2.3 M, 1.89 mL, 4.35
mmol). The reaction mixture was allowed to warm to RT for 1 h; then,
more LiAlH4 in THF (2.3 M, 1.89 mL, 4.35 mmol) was added and the
reaction mixture was stirred at RT for 1.5 h. The reaction mixture was
quenched by slow addition of water (0.352 mL, 19.6 mmol) (warning:
gas evolution) followed by addition of 1 M aq. NaOH (1.52 mL, 1.52
mmol). The resulting suspension was filtered, and the solid was rinsed
with EtOAc. The filtrate was concentrated, and the residue was purified
by flash chromatography (silica gel, DCM/MeOH) to give 52 as a light-
brown powder (371 mg, 79%). 1H NMR (400 MHz, DMSO-d6): δ 7.07
(d, J = 2.1 Hz, 1H), 7.02 (d, J = 2.1 Hz, 1H), 5.12 (t, J = 5.5 Hz, 1H),
4.77 (s, 2H), 4.35 (d, J = 5.5 Hz, 2H), 2.05 (s, 3H). ESI-MS m/z 216.1
[M + H]+. Step b: 2-Amino-5-bromo-3-methylbenzaldehyde (30). To a
solution of 52 (370 mg, 1.71 mmol) in DCM (8.00 mL) was added
MnO2 (1.16 g, 12.00 mmol), and the suspension was stirred at RT for 5
h. Subsequently, more MnO2 (447 mg, 5.14 mmol) was added and the
reaction mixture was stirred at RT for 16 h. The suspension was filtered
over a pad of hyflo, and the solid was rinsed with DCM. The filtrate was
concentrated to give 30 as an orange powder (334 mg, 91%). 1H NMR
(400 MHz, DMSO-d6): δ 9.78 (s, 1H), 7.62 (d, J = 2.3 Hz, 1H), 7.38 (d,
J = 1.9 Hz, 1H), 7.08 (s, 2H), 2.10 (s, 3H). ESI-MS m/z 214.1 [M +
H]+. Step c: 6-Bromo-8-methylquinazolin-2(1H)-one. A mixture of 30
(333 mg, 1.56 mmol) and urea (1.40 g, 23.3 mmol) was stirred at 180
°C for 1 h. The obtained yellow solid was cooled to RT, triturated in
water, filtered, rinsed with water, and dried under high vacuum to give
the title compound (444 mg, 100%). ESI-MS m/z 239.1 [M + H]+. Step
d: 6-Bromo-2-chloro-8-methylquinazoline (32). A suspension of 6-
bromo-8-methylquinazolin-2(1H)-one (443 mg, 1.56 mmol) and
POCl3 (2.03 mL, 21.8 mmol) was stirred at 110 °C for 30 min. The
reaction mixture was cooled to RT and poured into ice water. It was
then neutralized with saturated aq. NaHCO3 and extracted with DCM
(3 times). The combined organic phases were dried over Na2SO4,
filtered, and concentrated. The residue was purified by flash
chromatography (silica gel, heptanes/EtOAc) to give 32 as a light-
yellow powder (215 mg, 50%). 1H NMR: δ 9.52 (s, 1H), 8.33 (d, J = 2.1
Hz, 1H), 8.11 (s, 1H), 2.62 (s, 3H). ESI-MS m/z 257.1 [M + H]+. Step
e: 6-Bromo-2-(4-isopropylpiperazin-1-yl)-8-methylquinazoline (34).
In a sealed tube, a mixture of 32 (100 mg, 0.388 mmol) and 1-
isopropylpiperazine (149 mg, 1.16 mmol) in NMP (1.00 mL) was
stirred at 150 °C for 1 h. The reaction mixture was cooled to RT and
poured into water; the product was then extracted twice with EtOAc.
The combined organic phases were dried over Na2SO4, filtered, and
concentrated. The residue was purified by flash chromatography (silica
gel, DCM/MeOH) to give 34 as an orange oil (107 mg, 79%). 1H NMR
(400 MHz, DMSO-d6): δ 9.11 (s, 1H), 7.89 (d, J = 2.1 Hz, 1H), 7.72−
7.68 (m, 1H), 3.88−3.79 (m, 4H), 2.72−2.63 (m, 4H), 2.53−2.50 (m,
4H), 0.98 (d, J = 6.5 Hz, 6H). ESI-MS m/z 349.2 [M + H]+. Step f: 5-
(2-(4-Isopropylpiperazin-1-yl)-8-methylquinazolin-6-yl)-1-methylpyr-
idin-2(1H)-one (4). A mixture of 34 (30 mg, 0.086 mmol), 1-methyl-5-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one (40.4
mg, 0.172 mmol), PdCl2(dppf) (3.14 mg, 4.29 μmol), and aq. Na2CO3
(2 M, 0.129 mL, 0.258 mmol) in DME (500 μL) was stirred at 80 °C for
2 h. The reaction mixture was cooled to RT, diluted with EtOAc, and
washed with water. The organic phase was dried over MgSO4, filtered,
and concentrated. The residue was purified by flash chromatography
(C18, (H2O + 0.1% TFA)/MeCN), and then a basic workup was
performed to remove the TFA salt (aq. Na2CO3 and extraction with
1
DCM) to give 4 (10.4 mg, 32%) as a yellow powder. H NMR (400
MHz, DMSO-d6): δ 9.13 (s, 1H), 8.20 (d, J = 2.6 Hz, 1H), 7.90 (dd, J =
9.5, 2.6 Hz, 1H), 7.85 (d, J = 13.7 Hz, 2H), 6.49 (d, J = 9.5 Hz, 1H),
3.90−3.81 (m, 4H), 3.51 (s, 3H), 2.73−2.67 (m, 1H), 2.55−2.51 (m, 7
H), 0.99 (d, J = 6.5 Hz, 6H). ESI-MS m/z 378.4 [M + H]+.
5-Bromo-7-iodo-1H-indazole (37d). 4-Bromo-2-methylaniline (2.0
g, 10.8 mmol) was dissolved in EtOH (40.0 mL). Silver sulfate (3.35 g,
10.8 mmol) and iodine (2.75 g, 10.8 mmol) were added, and the
mixture was stirred at RT for 4 h. The solvent was evaporated, and the
residue was dissolved in DCM (50.0 mL), washed with 1 M aq. NaOH
(3 times 40.0 mL) and water (30.0 mL), dried with sodium sulfate, and
evaporated to dryness to give 4-bromo-2-iodo-6-methylaniline (2.30 g,
67%) as a solid. 1H NMR (400 MHz, DMSO-d6) δ 7.55 (d, J = 2.3 Hz,
1H), 7.16 (d, J = 2.3 Hz, 1H), 5.03 (s, 2H), 2.14 (s, 3H). ESI-MS m/z
311.9, 313.9 [M + H]+. A solution of 4-bromo-2-iodo-6-methylaniline
(2.3 g, 7.37 mmol) in acetic acid (30.0 mL) was treated with sodium
nitrite (0.560 g, 8.11 mmol) in water (2.00 mL) and stirred at RT for 0.5
h. The solvent was evaporated to dryness. The residue was triturated
with water (20.0 mL) for 15 min. The suspension was filtered and dried
in vacuo to give 37d (2.20 g, 83%). 1H NMR (400 MHz, DMSO-d6) δ
13.55−13.32 (m, 1H), 8.23 (s, 1H), 8.03 (d, J = 1.6 Hz, 1H), 7.87 (d, J
= 1.9 Hz, 1H). ESI-MS m/z 322.8, 324.8 [M + H]+.
tert-Butyl 4-(5-Bromo-2H-indazol-2-yl)piperidine-1-carboxylate
(38). A mixture of 5-bromo-2-nitrobenzaldehyde 36 (100 mg, 0.435
mmol) and tert-butyl 4-aminopiperidine-1-carboxylate (96.0 mg, 0.478
mmol) in 2-PrOH (1.50 mL) was stirred at 80 °C for 2 h, cooled to RT,
and treated with PBu3 (0.322 mL, 1.30 mmol). The reaction mixture
was then stirred at 80 °C overnight. The reaction mixture was cooled to
RT, diluted with EtOAc, and washed with saturated aq. NH4Cl and
brine. The organic phase was then dried over Na2SO4, filtered, and
evaporated. The crude material was purified by flash chromatography
(silica gel, heptanes/EtOAc) to give 38 (121 mg, 69%) as a yellow
powder. 1H NMR (400 MHz, DMSO-d6): δ 8.43 (s, 1H), 7.93 (d, J =
1.3 Hz, 1H), 7.58 (d, J = 9.1 Hz, 1H), 7.29 (dd, J = 9.1, 1.9 Hz, 1H),
4.76−4.65 (m, 1H), 4.07 (d, J = 12.6 Hz, 2H), 3.02−2.82 (m, 2H), 2.09
(d, J = 10.1 Hz, 2H), 1.99−1.85 (m, 2H), 1.41 (s, 9H). ESI-MS m/z
380.2 [M + H]+.
tert-Butyl 4-(5-Bromo-7-methyl-2H-indazol-2-yl)piperidine-1-
carboxylate (39). In a sealed tube, a mixture of 5-bromo-7-methyl-
1H-indazole (37a) (500 mg, 2.37 mmol), tert-butyl 4-
((methylsulfonyl)oxy)piperidine-1-carboxylate (794 mg, 2.84 mmol),
and Cs2CO3 (1.54 g, 4.74 mmol) in DMA (7.00 mL) was stirred at 150
°C overnight. The reaction mixture was cooled to room temperature,
diluted with EtOAc, and washed with water. The organic phase was
then dried over Na2SO4, filtered, and evaporated. The crude material
was purified twice by flash chromatography ((silica gel, heptanes/
EtOAc) and (C18, (H2O + 0.1%TFA)/MeCN)). The obtained
material was treated with saturated aq. Na2CO3 and extracted with
DCM. The organic phase was dried over Na2SO4, filtered, and
concentrated to give 39 (243 mg, 26%) as an oil. 1H NMR (400 MHz,
K
https://dx.doi.org/10.1021/acs.jmedchem.0c00130
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
DMSO-d6): δ 8.39 (s, 1H), 7.67−7.78 (m, 1H), 7.15−7.03 (m, 1H),
4.80−4.60 (m, 1H), 4.08 (d, J = 12.4 Hz, 2H), 3.03−2.82 (m, 2H), 2.48
(s, 3H), 2.13−2.06 (m, 2H), 1.98−1.85 (m, 2H), 1.41 (s, 9H). ESI-MS
m/z 394.2 [M + H]+ and its regioisomer tert-butyl 4-(5-bromo-7-
(0.038 mL, 0.344 mmol), and TMSCF3 in THF (2 M, 0.172 mL, 0.344
mmol) was added via a syringe, and the reaction mixture was stirred at
60 °C for 16 h. The reaction mixture was diluted with EtOAc and
washed with saturated aq. NaHCO3. The organic phase was then dried
over Na2SO4, filtered, and evaporated. The crude material was purified
by flash chromatography (silica gel, heptanes/EtOAc) to give 43 (35.5
mg, 69%) as a brown resin. 1H NMR (400 MHz, DMSO-d6) δ 8.69 (s,
1H), 8.31 (s, 1H), 7.72 (s, 1H), 4.81 (tt, J = 11.4, 3.9 Hz, 1H), 4.09 (d, J
= 12.4 Hz, 2H), 2.95 (s, 2H), 2.12 (d, J = 10.1 Hz, 2H), 1.94 (qd, J =
12.3, 4.3 Hz, 2H), 1.42 (s, 9H): ESI-MS m/z 448.2, 450.2 [M + H]+.
1-Methyl-5-(7-methyl-2-(piperidin-4-yl)-2H-indazol-5-yl)pyridin-
2(1H)-one Hydrochloride (5). A glass microwave vial was charged with
tert-butyl 4-(5-bromo-7-methyl-2H-indazol-2-yl)piperidine-1-carboxy-
late 39 (44.0 mg, 0.112 mmol), tetrahydroxydiboron (28.6 mg, 0.319
mmol), XPhos Pd G3 (9.00 mg, 10.6 μmol), XPhos (10.1 mg, 0.021
mmol), and potassium acetate (31.3 mg, 0.319 mmol) and flushed with
argon; then, degassed EtOH (500 μL) was added, and the reaction
mixture was stirred at 80 °C for 30 min; then, aq. K2CO3 (2 M, 0.160
mL, 0.319 mmol) (degassed) was added followed by a solution of 5-
bromo-1-methylpyridin-2(1H)-one (20 mg, 0.106 mmol) in degassed
EtOH (200 μL). The reaction mixture was stirred at 80 °C for 1 h. The
reaction mixture was poured into water and extracted twice with DCM.
The organic phase was then dried over MgSO4, filtered, and evaporated.
The residue was purified by flash chromatography (C18, (H2O + 0.1%
TFA)/MeCN) to give tert-butyl 4-(7-methyl-5-(1-methyl-6-oxo-1,6-
dihydropyridin-3-yl)-2H-indazol-2-yl)piperidine-1-carboxylate as a
1
methyl-1H-indazol-1-yl)piperidine-1-carboxylate (214 mg, 23%). H
NMR (400 MHz, DMSO-d6): δ 8.03 (s, 1H), 7.80 (d, J = 1.3 Hz, 1H),
7.32−7.22 (m, 1H), 5.04−4.89 (m, 1H), 4.10−4.01 (m, 2H), 3.06−
2.87 (m, 2H), 2.72 (s, 3H), 1.97−1.88 (m, 4H), 1.41 (s, 9H). ESI-MS
m/z 394.2, 396.2 [M + H]+.
tert-Butyl 3-(5-Bromo-7-methyl-2H-indazol-2-yl)piperidine-1-
carboxylate (40). A solution of 5-bromo-7-methyl-1H-indazole 37a
(200 mg, 0.948 mmol) in DMF (4.00 mL, ratio: 2.00) was treated with
NaH (60% dispersion in mineral oil) (45.5 mg, 1.14 mmol) and stirred
for 10 min. Subsequently, tert-butyl 3-((methylsulfonyl)oxy)-
piperidine-1-carboxylate (318 mg, 1.14 mmol) was added, and the
reaction mixture was heated to 100 °C and stirred for 18 h. The reaction
mixture was cooled to room temperature and treated with more NaH
(60% dispersion in mineral oil) (45.5 mg, 1.14 mmol) and tert-butyl 3-
((methylsulfonyl)oxy)piperidine-1-carboxylate (318 mg, 1.14 mmol),
heated to 100 °C, and stirred for 4 h. The reaction mixture was cooled
to RT, treated with water, and extracted with EtOAc. The organic layer
was washed with water and brine, dried over anhydrous Na2SO4,
filtered, and concentrated under reduced pressure. To facilitate the
separation of the product from the remaining indazole starting material,
the crude mixture was dissolved in DCM (2.00 mL, ratio: 1.00), treated
with Ac2O (0.089 mL, 0.948 mmol) and NEt3 (0.132 mL, 0.948 mmol),
and stirred for 14 h. More Ac2O (0.089 mL, 0.948 mmol) and NEt3
(0.132 mL, 0.948 mmol) were added, and the reaction was stirred at RT
for 1 h. The reaction mixture was treated with water and extracted with
EtOAc. The organic layer was dried over anhydrous Na2SO4, filtered,
and concentrated under reduced pressure. The crude material was
purified by flash chromatography (silica gel, heptanes/EtOAc) to give
40 (72.8 mg, 0.185 mmol, 19.5 % yield) as an orange oil. 1H NMR (400
MHz, DMSO-d6): δ 8.42 (s, 1H), 7.79−7.73 (m, 1H), 7.13 (s, 1H),
4.62−4.49 (m, 1H), 4.30−4.07 (m, 1H), 3.82 (s, 1H), 3.05−2.87 (m,
1H), 2.50−2.49 (m, 3H), 2.26−2.09 (m, 2H), 1.88−1.75 (m, 1H),
1.68−1.51 (m, 2H), 1.42−1.35 (m, 9H). ESI-MS m/z 394.1, 396.1 [M
+ H]+.
tert-Butyl 4-(5-Bromo-7-chloro-2H-indazol-2-yl)piperidine-1-car-
boxylate (41). To a solution of 37c (300 mg, 1.30 mmol) in DMF (6.00
mL) was added tBuOK (175 mg, 1.55 mmol) followed by tert-butyl 4-
((methylsulfonyl)oxy)piperidine-1-carboxylate (434 mg, 1.55 mmol).
The reaction mixture was stirred at 60 °C for 24 h. Then, the reaction
mixture was cooled to RT and Ac2O (0.122 mL, 1.30 mmol) was added,
and the reaction mixture was stirred at RT for 15 min. The reaction
mixture was poured into water, and the product was extracted with
EtOAc. The organic phase was then dried over Na2SO4, filtered, and
evaporated. The crude material was purified by flash chromatography
(silica gel, Heptane/EtOAc) to give 41 (162.5 mg, 30%) as a brown
solid. 1H NMR (400 MHz, DMSO-d6): δ 8.58 (s, 1H), 7.95 (d, J = 1.6
Hz, 1H), 7.50 (d, J = 1.6 Hz, 1H), 4.82−4.68 (m, 1H), 4.15−4.02 (m,
2H), 3.07−2.82 (m, 2H), 2.15−2.06 (m, 2H), 2.00−1.85 (m, 2H), 1.42
(s, 9H). ESI-MS m/z 414.1 [M + H]+.
1
colorless solid (28.9 mg, 64%). H NMR (400 MHz, DMSO-d6): δ
8.40 (s, 1H), 8.07 (d, J = 2.6 Hz, 1H), 7.82 (dd, J = 9.4, 2.7 Hz, 1H),
7.25 (s, 1H), 7.62 (s, 1H), 6.46 (d, J = 9.4 Hz, 1H), 4.75−4.62 (m, 1H),
4.10 (d, J = 11.4 Hz, 2H), 3.50 (s, 3H), 3.08−2.84 (m, 2H), 2.52 (s,
3H), 2.06−2.14 (m, 2H), 2.01−1.89 (m, 2H), 1.42 (s, 9H). ESI-MS m/
z 423.4 [M + H]+. To a solution of tert-butyl 4-(7-methyl-5-(1-methyl-
6-oxo-1,6-dihydropyridin-3-yl)-2H-indazol-2-yl)piperidine-1-carboxy-
late (28.0 mg, 0.066 mmol) in DCM (500 μL) was added HCl in Et2O
(2 M, 0.663 mL, 1.33 mmol); then, the suspension was stirred at RT for
30 min. The reaction mixture was concentrated. The residue was
dissolved in water and lyophilized to give 5 (21.5 mg, 90%) as a
colorless powder. 1H NMR (400 MHz, DMSO-d6): δ 8.92−9.03 (m,
1H), 8.55−8.71 (m, 1H), 8.38 (s, 1H), 8.07 (d, J = 2.6 Hz, 1H), 7.82
(dd, J = 9.4, 2.7 Hz, 1H), 7.65 (s, 1H), 7.28 (s, 1H), 6.47 (d, J = 9.4 Hz,
1H), 4.76−4.90 (m, 1H), 3.50 (s, 3H), 3.41−3.48 (m, 2H), 3.03−3.17
(m, 2H), 2.53 (s, 3H), 2.25−2.34 (m, 4H). 13C NMR (151 MHz,
DMSO-d6) δ 161.10, 147.41, 139.16, 136.74, 129.32, 127.27, 123.48,
122.88, 121.20, 119.15, 118.62, 113.78, 56.65, 42.25, 37.01, 29.02,
17.02. HRMS-ESI [M + H]+ m/z calcd for C19H23ON4 323.18664;
found 323.18683.
1,3-Dimethyl-5-(7-methyl-2-(piperidin-4-yl)-2H-indazol-5-yl)-
pyridin-2(1H)-one Hydrochloride (6). Compound 6 was prepared
starting from 39 and 5-bromo-1,3-dimethylpyridin-2(1H)-one accord-
ing to the procedure used to make compound 5. 1H NMR (400 MHz,
DMSO-d6) δ 9.12−8.88 (m, 1H), 8.82−8.52 (m, 1H), 8.36 (s, 1H),
7.93 (d, J = 2.6 Hz, 1H), 7.76−7.72 (m, 1H), 7.65 (s, 1H), 7.28 (s, 1H),
4.89−4.78 (m, 1H), 3.51 (s, 3H), 3.48−3.41 (m, 2H), 3.18−3.02 (m,
2H), 2.53 (s, 3H), 2.35−2.25 (m, 4H), 2.08 (s, 3H). ESI-MS m/z 337.3
[M + H]+.
1,4-Dimethyl-5-(7-methyl-2-(piperidin-4-yl)-2H-indazol-5-yl)-
pyridin-2(1H)-one Hydrochloride (7). Compound 7 was prepared
starting from 39 and 5-bromo-1,4-dimethylpyridin-2(1H)-one accord-
ing to the procedure used to make compound 5. 1H NMR (400 MHz,
DMSO-d6): δ 9.23−9.05 (m, 1H), 8.90−8.71 (m, 1H), 8.38 (s, 1H),
7.56 (s, 1H), 7.41 (s, 1H), 6.97 (s, 1H), 6.33 (s, 1H), 4.91−4.78 (m,
1H), 3.49−3.39 (m, 5H), 3.18−3.04 (m, 2H), 2.51 (s, 3H), 2.37−2.26
(m, 4H), 2.05 (s, 3H). ESI-MS m/z 337.3 [M + H]+. 5-bromo-1,4-
dimethylpyridin-2(1H)-one. Step 1: A mixture of 5-bromo-2-fluoro-4-
methylpyridine (523 mg, 2.75 mmol) and NaOH (385 mg, 9.63 mmol)
in water (2.50 mL) was stirred at 100 °C for 16 h and then at 150 °C
under microwave irradiation for 30 min. The reaction mixture was
cooled to 0 °C, and conc. HCl (0.904 mL, 11.0 mmol) was slowly added
dropwise. The solid was collected by filtration, rinsed with water, and
dried under high vacuum to give 5-bromo-4-methylpyridin-2(1H)-one
tert-Butyl 4-(5-Bromo-7-iodo-2H-indazol-2-yl)piperidine-1-car-
boxylate (42). Compound 42 was prepared starting from 37d
1
according to the procedure used to make compound 41. H NMR
(400 MHz, DMSO-d6): δ 8.64 (s, 1H), 7.97 (d, J = 1.6 Hz, 1H), 7.79 (d,
J = 1.6 Hz, 1H), 4.65−4.83 (m, 1H), 3.99−4.18 (m, 2H), 2.95 (s, 2H),
2.04−2.17 (m, 2H), 1.82−2.00 (m, 2H), 1.42 (s, 9H). ESI-MS m/z
506.0 [M + H]+.
tert-Butyl 4-(5-Bromo-7-(trifluoromethyl)-2H-indazol-2-yl)-
piperidine-1-carboxylate (43). Compound 43 was prepared by three
different methods. Method 1 included starting from 37b according to
the procedure used to make compound 40; method 2 included starting
from 45 according to the procedure used to make compound 47; and in
method 3 (step f, shown in Scheme 2), KF (20.0 mg, 0.344 mmol), CuI
(4.36 mg, 0.023 mmol), and 1,10-phenanthroline (4.13 mg, 0.023
mmol) were combined in a vial and dried under high vacuum at 40 °C
for 1 h. Under argon, a solution of 42 (58.0 mg, 0.115 mmol)
(previously dried under high vacuum) in DMSO (1.00 mL), B(OMe)3
L
https://dx.doi.org/10.1021/acs.jmedchem.0c00130
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(539 mg, 100%) as a colorless powder. 1H NMR (400 MHz, DMSO-
d6): δ 11.56 (s, 1H), 7.66 (s, 1H), 6.38 (s, 1H), 2.16 (s, 3H). ESI-MS
m/z 188.0 [M + H]+. Step 2: To a suspension of 5-bromo-4-
methylpyridin-2(1H)-one (539 mg, 2.75 mmol) and K2CO3 (761 mg,
5.50 mmol) in DMF (6.00 mL) was added MeI (0.258 mL, 4.13 mmol),
and the reaction mixture was stirred at RT for 2 h. The reaction mixture
was diluted with water and acidified with conc. HCl (0.376 mL, 12.4
mmol). The product was then extracted with DCM (3 times). The
organic phase was then dried over Na2SO4, filtered, and evaporated.
The crude material was purified by flash chromatography (silica gel,
heptanes/EtOAc) to give 5-bromo-1,4-dimethylpyridin-2(1H)-one
1H), 8.43 (s, 1H), 7.50 (s, 1H), 7.47 (d, J = 9.3 Hz, 1H), 7.01−6.97 (m,
1H), 6.50 (d, J = 9.3 Hz, 1H), 4.91−4.79 (m, 1H), 3.64 (s, 3H), 3.51−
3.41 (m, 2H), 3.19−3.04 (m, 2H), 2.51 (s, 3H), 2.34−2.27 (m, 4H).
13C NMR (151 MHz, DMSO-d6) δ 161.38, 147.33, 141.64, 135.75,
130.16, 126.70, 126.36, 123.20, 120.67, 119.26, 118.73, 117.20, 56.76,
42.18, 33.41, 29.02, 16.89. HRMS-ESI [M + H]+ m/z calcd for
C19H22ON4Cl 357.14767; found 357.14767. 5-bromo-6-chloro-1-
methylpyridin-2(1H)-one. Step 1: 6-chloro-1-methylpyridin-2(1H)-one.
A solution of 6-chloropyridin-2(1H)-one (10.0 g, 77.0 mmol) in DMF
(100 mL) was treated with Li2CO3 (11.4 g, 154 mmol) and then cooled
to 0 °C. The resulting suspension was treated with methyl iodide (7.24
mL, 116 mmol), stirred for 1 h at 0 °C, allowed to warm to room
temperature, and then stirred for 24 h. The reaction was treated with
water and extracted with DCM (3 times). The combined organic layers
were dried over anhydrous Na2SO4, filtered, and concentrated under
reduced pressure. The crude material was purified by flash
chromatography (silica gel, DCM/MeOH) to give 6-chloro-1-
1
(359 mg, 64%) as a colorless powder. H NMR (400 MHz, DMSO-
d6): δ 8.01 (s, 1H), 6.39 (s, 1H), 3.36 (s, 3H), 2.14 (s, 3H). ESI-MS m/z
202.1 [M + H]+.
5-Bromo-1,6-dimethylpyridin-2(1H)-one (53). A suspension of 5-
bromo-6-methylpyridin-2(1H)-one (10.0 g, 53.2 mmol) and K2CO3
(14.7 g, 106 mmol) in DMF (100 mL) was treated with iodomethane
(5.00 mL, 80.0 mmol) and stirred at RT for 2 h. The reaction mixture
was diluted with water and extracted with DCM (3 times) and EtOAc
(twice). The combined organic phases were dried over Na2SO4,
filtered, and concentrated. The crude material was purified by flash
chromatography (silica gel, heptanes/EtOAc) to give 5-bromo-1,6-
dimethylpyridin-2(1H)-one (53) (6.92 g, 61%) as a light-yellow
powder. 1H NMR (400 MHz, DMSO-d6) δ 7.51 (d, J = 9.7 Hz, 1H),
6.25 (d, J = 9.7 Hz, 1H), 3.48 (s, 3H), 2.49 (s, 3H). ESI-MS m/z 202.0,
204.0 [M + H]+.
1,6-Dimethyl-5-(7-methyl-2-(piperidin-4-yl)-2H-indazol-5-yl)-
pyridin-2(1H)-one (8). Compound 8 was prepared starting from 39 and
5-bromo-1,6-dimethylpyridin-2(1H)-one (53) according to the
procedure used to make compound 5. The HCl salt of 8 was converted
into the free base by basic extraction using saturated aq. Na2CO3 and
EtOAc. 1H NMR (600 MHz, DMSO-d6) δ 8.38 (s, 1H), 7.34 (d, J = 1.6
Hz, 1H), 7.32 (d, J = 9.2 Hz, 1H), 6.88 (t, J = 1.4 Hz, 1H), 6.34 (d, J =
9.2 Hz, 1H), 4.54 (tt, J = 11.7, 4.2 Hz, 1H), 3.51 (s, 3H), 3.08 (dt, J =
12.8, 3.3 Hz, 2H), 2.64 (t, J = 11.7 Hz, 2H), 2.51 (s, 3H), 2.30 (s, 3H),
2.03 (dd, J = 12.6, 3.6 Hz, 2H), 1.96 (qd, J = 12.0, 4.1 Hz, 2H). 13C
NMR (151 MHz, DMSO-d6) δ 161.85, 146.90, 144.65, 141.42, 131.82,
126.73, 126.69, 122.00, 120.90, 119.57, 118.30, 115.52, 60.81, 45.20,
33.96, 31.15, 18.24, 16.98. HRMS-ESI [M + H]+ m/z calcd for
C20H25ON4 337.20229; found 337.20218.
6-Fluoro-1-methyl-5-(7-methyl-2-(piperidin-4-yl)-2H-indazol-5-
yl)pyridin-2(1H)-one Hydrochloride (9). Compound 9 was prepared
starting from 39 and 5-bromo-6-fluoro-1-methylpyridin-2(1H)-one
according to the procedure used to make compound 5. 1H NMR (400
MHz, DMSO-d6) δ 8.89−8.78 (m, 1H), 8.61−8.45 (m, 1H), 8.42 (s,
1H), 7.71 (dd, J = 11.3, 9.4 Hz, 1H), 7.58 (s, 1H), 7.11 (d, J = 1.5 Hz,
1H), 6.40 (d, J = 9.4 Hz, 1H), 4.90−4.78 (m, 1H), 3.51−3.41 (m, 5H),
3.18−3.05 (m, 2H), 2.52 (s, 3H), 2.36−2.26 (m, 4H). ESI-MS m/z
341.2 [M + H]+. 5-bromo-6-fluoro-1-methylpyridin-2(1H)-one. A
solution of 5-bromo-6-fluoropyridin-2(1H)-one (7.64 g, 39.8 mmol)
in DMF (100 mL) was treated with Li2CO3 (5.90 g, 80.0 mmol) and
then cooled to 0 °C. The resulting suspension was treated with
iodomethane (3.70 mL, 60.0 mmol), allowed to warm to room
temperature, and stirred for 3 h. The reaction mixture was heated to 50
°C and stirred for 1 h and then treated with iodomethane (2.0 mL, 32
mmol) and stirred at 50 °C for 16 h. The reaction mixture was cooled to
room temperature, diluted with water, and extracted with DCM (3
times). The combined organic layers were dried over Na2SO4, filtered,
and concentrated. The crude material containing 3-bromo-2-fluoro-6-
methoxypyridine as the major component and the title compound as
the minor component was purified by flash chromatography (silica gel,
heptanes/EtOAc) to give 5-bromo-6-fluoro-1-methylpyridin-2(1H)-
one (1.33 g, 15%) as a brown solid. 1H NMR (400 MHz, DMSO-d6) δ
7.67 (t, J = 10.0 Hz, 1H), 6.29 (d, J = 9.8 Hz, 1H), 3.41 (d, J = 3.6 Hz,
3H). ESI-MS m/z 206.1, 208.0 [M + H]+.
1
methylpyridin-2(1H)-one (9.98 g, 90%) as a white solid. H NMR
(400 MHz, DMSO-d6) δ 7.38 (dd, J = 9.2, 7.3 Hz, 1H), 6.47 (dd, J = 7.3,
1.2 Hz, 1H), 6.39 (dd, J = 9.2, 1.1 Hz, 1H), 3.54 (s, 3H). ESI-MS m/z
144.0 [M + H]+. Step 2: 5-bromo-6-chloro-1-methylpyridin-2(1H)-one. A
solution of 6-chloro-1-methylpyridin-2(1H)-one (9.69 g, 67.5 mmol)
in acetic acid (50.0 mL) at 0 °C was treated with N-bromosuccinimide
(10.0 g, 56.2 mmol), allowed to warm to room temperature, and stirred
for 1.5 h. The reaction mixture was concentrated; the residue was
treated with saturated aq. NaHCO3 and extracted twice with DCM.
The combined organic layers were dried over Na2SO4, filtered, and
concentrated. The crude material was purified by flash chromatography
(silica gel, heptanes/EtOAc) to give 5-bromo-6-chloro-1-methylpyr-
idin-2(1H)-one (4.97 g, 32%) as a brown solid. 1H NMR (400 MHz,
DMSO-d6) δ 7.67 (d, J = 9.7 Hz, 1H), 6.42 (d, J = 9.7 Hz, 1H), 3.61 (s,
3H). ESI-MS m/z 222.0, 224.0 [M + H]+.
5-Methyl-7-(7-methyl-2-(piperidin-4-yl)-2H-indazol-5-yl)furo-
[3,2-c]pyridin-4(5H)-one Hydrochloride (11). Compound 11 was
prepared starting from 39 and 7-bromo-5-methylfuro[3,2-c]pyridin-
4(5H)-one according to the procedure used to make compound 5. 1H
NMR (400 MHz, DMSO-d6) δ 9.17−8.97 (m, 1H), 8.83−8.62 (m,
1H), 8.46 (s, 1H), 7.99 (d, J = 2.1 Hz, 1H), 7.96 (s, 1H), 7.93 (s, 1H),
7.39 (s, 1H), 7.05 (d, J = 2.1 Hz, 1H), 4.91−4.80 (m, 1H), 3.60 (s, 3H),
3.51−3.39 (m, 2H), 3.20−3.05 (m, 2H), 2.56 (s, 3H), 2.36−2.29 (m,
4H). ESI-MS m/z 363.2 [M + H]+. 7-Bromo-5-methylfuro[3,2-c]pyridin-
4(5H)-one. A suspension of 7-bromofuro[3,2-c]pyridin-4(5H)-one
(0.20 g, 0.94 mmol) and K2CO3 (0.26 g, 1.90 mmol) in DMF (2.00
mL) was treated with iodomethane (0.088 mL, 1.40 mmol) and stirred
at room temperature for 1.5 h. The reaction mixture was diluted with
water, and the precipitate was collected by centrifugation and dried to
give 7-bromo-5-methylfuro[3,2-c]pyridin-4(5H)-one (173 mg, 80%)
as a yellow powder. 1H NMR (400 MHz, DMSO-d6): δ 8.03 (s, 1H),
7.99 (d, J = 2.0 Hz, 1H), 7.07 (d, J = 2.1 Hz, 1H), 3.49 (s, 3H). ESI-MS
m/z 228.0, 230.0 [M + H]+.
8-(7-Methyl-2-(piperidin-4-yl)-2H-indazol-5-yl)-2,3-dihydroindo-
lizin-5(1H)-one (12). Compound 12 was prepared starting from 39 and
8-bromo-2,3-dihydroindolizin-5(1H)-one (51) according to the proce-
1
dure used to make compound 5. H NMR (400 MHz, DMSO-d6) δ
8.35 (s, 1H), 7.51 (d, J = 9.2 Hz, 1H), 7.44 (s, 1H), 7.02 (s, 1H), 6.30
(d, J = 9.2 Hz, 1H), 4.59−4.48 (m, 1H), 4.06−3.98 (m, 2H), 3.16 (t, J =
7.6 Hz, 2H), 3.12−3.04 (m, 2H), 2.68−2.59 (m, 2H), 2.51 (s, 3H),
2.11−1.89 (m, 6H). 13C NMR (151 MHz, DMSO-d6) δ 160.19,
148.20, 146.88, 141.77, 130.24, 126.79, 125.31, 121.90, 120.98, 116.67,
116.45, 115.34, 60.66, 48.77, 45.12, 33.82, 31.71, 21.10, 16.94. HRMS-
ESI [M + H]+ m/z calcd for C21H25ON4 349.20229; found 349.20224.
1-Methyl-5-(2-(piperidin-4-yl)-2H-indazol-5-yl)pyridin-2(1H)-one
Hydrochloride (13). Compound 13 was prepared starting from 38
according to the procedure used to make compound 5. 1H NMR (400
MHz, DMSO-d6): δ 8.96−8.83 (m, 1H), 8.73−8.59 (m, 1H), 8.42 (s,
1H), 8.09 (d, J = 2.3 Hz, 1H), 7.89−7.79 (m, 2H), 7.66 (d, J = 9.0 Hz,
1H), 7.49 (d, J = 9.1 Hz, 1H), 6.48 (d, J = 9.4 Hz, 1H), 4.87−4.78 (m,
1H), 3.50−3.34 (m, 5H), 3.20−3.04 (m, 2H), 2.34−2.24 (m, 4H). ESI-
MS m/z 309.2 [M + H]+.
6-Chloro-1-methyl-5-(7-methyl-2-(piperidin-4-yl)-2H-indazol-5-
yl)pyridin-2(1H)-one Hydrochloride (10). Compound 10 was
prepared starting from 39 and 5-bromo-6-chloro-1-methylpyridin-
2(1H)-one according to the procedure used to make compound 5. 1H
NMR (400 MHz, DMSO-d6): δ 8.86−8.75 (m, 1H), 8.55−8.45 (m,
M
https://dx.doi.org/10.1021/acs.jmedchem.0c00130
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
5-(7-Chloro-2-(piperidin-4-yl)-2H-indazol-5-yl)-1,6-dimethylpyri-
din-2(1H)-one Hydrochloride (14). Compound 14 was prepared
starting from 42 and 5-bromo-1,6-dimethylpyridin-2(1H)-one (53)
according to the procedure used to make compound 5. 1H NMR (400
MHz, DMSO-d6) δ 8.94−8.77 (m, 1H), 8.62 (s, 1H), 8.60−8.47 (m,
1H), 7.60 (d, J = 1.3 Hz, 1H), 7.37 (d, J = 9.3 Hz, 1H), 7.31 (d, J = 1.3
Hz, 1H), 6.38 (d, J = 9.3 Hz, 1H), 5.01−4.83 (m, 1H), 3.54 (s, 3H),
3.53−3.47 (m, 2H), 3.22−3.09 (m, 2H), 2.38−2.30 (m, 7 H). ESI-MS
m/z 357.2, 359.3 [M + H]+.
DMSO-d6) δ = 8.40 (s, 1H), 7.51 (s, 1H), 6.75 (s, 1H), 4.75−4.66 (m,
1H), 4.09 (br s, 2H), 2.98 (br s, 2H), 2.41−2.32 (m, 1H), 2.10 (br d, J =
11.9 Hz, 2H), 1.93 (dq, J = 4.2, 12.1 Hz, 2H), 1.43 (s, 9H), 1.07−0.99
(m, 4H). ESI-MS m/z 376.3 [M + H]+.
1,6-Dimethyl-5-(7-methyl-2-(1-methylpiperidin-4-yl)-2H-inda-
zol-5-yl)pyridin-2(1H)-one (17). A mixture of 8 (30 mg, 89 μmol),
acetic acid (6.10 μL, 0.110 mmol), formaldehyde in water (37%, 9.96
μL, 0.134 mmol), and DCM (500 μL) was treated with sodium
triacetoxyborohydride (28.3 mg, 0.134 mmol) and stirred at room
temperature for 1.5 h. The reaction mixture was slowly poured into
saturated aq. Na2CO3 and extracted with DCM (4 times). The
combined organic phases were dried over Na2SO4, filtered, and
concentrated. The crude material was purified by flash chromatography
(silica gel, DCM/(7 M NH3·in MeOH)) to give the title compound 17
(26.7 mg, 85%) as a colorless powder. 1H NMR (400 MHz, DMSO-d6)
δ 8.41 (s, 1H), 7.37−7.26 (m, 2H), 6.88 (s, 1H), 6.34 (d, J = 9.3 Hz,
1H), 4.52−4.40 (m, 1H), 3.52 (s, 3H), 2.96−2.85 (m, 2H), 2.53−2.51
(m, 3H), 2.31 (s, 3H), 2.23 (s, 3H), 2.16−2.05 (m, 6H). ESI-MS m/z
351.3 [M + H]+.
1,6-Dimethyl-5-(2-(piperidin-4-yl)-7-(trifluoromethyl)-2H-inda-
zol-5-yl)pyridin-2(1H)-one Hydrochloride (15). Compound 15 was
prepared starting from 41 and 5-bromo-1,6-dimethylpyridin-2(1H)-
1
one (53) according to the procedure used to make compound 5. H
NMR (400 MHz, DMSO-d6) δ 9.01−8.83 (m, 1H), 8.69 (s, 1H), 8.64−
8.48 (m, 1H), 7.93 (s, 1H), 7.55 (s, 1H), 7.39 (d, J = 9.3 Hz, 1H), 6.38
(d, J = 9.3 Hz, 1H), 5.04−4.89 (m, 1H), 3.54−3.47 (m, 5H), 3.22−3.06
(m, 2H), 2.37−2.29 (m, 7 H). ESI-MS m/z 391.3 [M + H]+.
5-(7-Cyclopropyl-2-(piperidin-4-yl)-2H-indazol-5-yl)-1,6-dime-
thylpyridin-2(1H)-one (16). Compound 16 was prepared starting from
tert-butyl 4-(5-chloro-7-cyclopropyl-2H-indazol-2-yl)piperidine-1-car-
boxylate and 5-bromo-1,6-dimethylpyridin-2(1H)-one (53) according
5-(2-(1-(2-(Dimethylamino)acetyl)piperidin-4-yl)-7-methyl-2H-
indazol-5-yl)-1,6-dimethylpyridin-2(1H)-one (18). A mixture of 8, 2-
(dimethylamino)acetic acid (12.7 mg, 0.120 mmol), HATU (46.7 mg,
0.120 mmol), and N-ethyl-N-isopropylpropan-2-amine (0.030 mL,
0.170 mmol) was stirred at room temperature for 1 h. The reaction
mixture was diluted with DCM and washed with saturated aq. Na2CO3.
The organic phase was dried over Na2SO4, filtered, and concentrated.
The crude material was purified by flash chromatography (silica gel,
DCM/(7 M NH3·in MeOH)) to give 18 (39.0 mg, 82%) as a colorless
1
to the procedure used to make compound 5. H NMR (600 MHz,
DMSO-d6) δ = 8.37 (s, 1H), 7.31 (d, J = 9.2 Hz, 1H), 7.28 (s, 1H), 6.64
(s, 1H), 6.33 (d, J = 9.2 Hz, 1H), 4.57−4.50 (m, 1H), 3.51 (s, 3H), 3.08
(br d, J = 12.7 Hz, 2H), 2.64 (br t, J = 11.6 Hz, 2H), 2.42−2.36 (m, 1H),
2.29 (s, 3H), 2.08−2.00 (m, 2H), 1.95 (dq, J = 3.9, 11.8 Hz, 2H), 1.06−
0.96 (m, 4H). ESI-MS m/z 363.4 [M + H]+. tert-Butyl 4-(5-chloro-7-
cyclopropyl-2H-indazol-2-yl)piperidine-1-carboxylate. Step 1: (2-
Amino-5-chloro-3-iodophenyl)methanol. A solution of methyl-2-
amino-5-chloro-3-iodobenzoate (10.0 g, 31.1 mmol) in THF (160
mL) was treated portionwise with LiBH4 (2.00 g, 93.0 mmol). The
reaction mixture was stirred at RT for 1.75 h, cautiously quenched with
saturated aq. NH4Cl, and extracted twice with EtOAc. The combined
organic phases were washed with brine, dried over Na2SO4, filtered, and
concentrated. The crude material was recrystallized from hot EtOAc.
The mother liquor was concentrated, and the residue was triturated
with hot MeOH. Both solids were combined and dried to give the title
1
powder. H NMR (400 MHz, DMSO-d6) δ 8.42 (s, 1H), 7.36−7.29
(m, 2H), 6.89 (s, 1H), 6.34 (d, J = 9.2 Hz, 1H), 4.86−4.73 (m, 1H),
4.52 (d, J = 12.9 Hz, 1H), 4.23 (d, J = 13.0 Hz, 1H), 3.52 (s, 3H), 3.27−
3.10 (m, 3H), 2.87−2.75 (m, 1H), 2.51−2.51 (m, 3H), 2.31 (s, 3H),
2.25−2.12 (m, 8 H), 2.10−2.01 (m, 1H), 1.98−1.86 (m, 1H). 13C
NMR (151 MHz, DMSO-d6) δ 167.85, 161.78, 146.93, 144.61, 141.32,
131.93, 126.79, 126.70, 122.32, 120.91, 119.43, 118.24, 115.48, 62.08,
59.71, 45.18, 45.17, 44.02, 33.08, 32.30, 31.10, 18.19, 16.89. HRMS-ESI
[M + H]+ m/z calcd for C24H32O2N5 422.25505; found 422.25500.
1,6-Dimethyl-5-(7-methyl-2-(piperidin-3-yl)-2H-indazol-5-yl)-
pyridin-2(1H)-one (19). Compound 19 was prepared starting from 40
and 5-bromo-1,6-dimethylpyridin-2(1H)-one (53) according to the
procedure used to make compound 5. 1H NMR (400 MHz, DMSO-d6)
δ 8.42 (s, 1H), 7.37−7.28 (m, 2H), 6.89 (s, 1H), 6.34 (d, J = 9.2 Hz,
1H), 4.59−4.46 (m, 1H), 3.51 (d, J = 4.1 Hz, 4H), 3.29−3.29 (m, 1H),
3.04−2.92 (m, 2H), 2.64−2.56 (m, 1H), 2.31 (s, 3H), 2.24−2.16 (m,
1H), 2.14−2.02 (m, 1H), 1.83−1.75 (m, 1H), 1.65−1.55 (m, 1H). ESI-
MS m/z 337.2 [M + H]+.
1
compound as a light-pink solid (7.07 g, 79%). H NMR (400 MHz,
DMSO-d6) δ 7.52 (d, J = 2.5 Hz, 1H), 7.17 (d, J = 2.5 Hz, 1H), 5.32 (t, J
= 5.5 Hz, 1H), 5.07 (s, br, 2H), 4.38 (d, J = 5.5 Hz, 2H). ESI-MS m/z
284.0, 286.0 [M + H]+. Step 2: 2-Amino-5-chloro-3-iodobenzaldehyde.
2-Amino-5-chloro-3-iodobenzaldehyde was prepared starting from (2-
amino-5-chloro-3-iodophenyl)methanol according to the procedure
used to make compound 30. 1H NMR (400 MHz, DMSO-d6) δ 9.70 (s,
1H), 7.94 (d, J = 2.4 Hz, 1H), 7.78 (d, J = 2.4 Hz, 1H), 7.12 (s, br, 2H).
Step 3: 2-Azido-5-chloro-3-iodobenzaldehyde. 2-Azido-5-chloro-3-
iodobenzaldehyde was prepared starting from 2-amino-5-chloro-3-
iodobenzaldehyde according to the procedure used to make compound
45. 1H NMR (400 MHz, DMSO-d6) δ 10.02 (s, 1H), 8.28 (d, J = 2.5
Hz, 1H), 8.00 (d, J = 2.5 Hz, 1H). Step 4: tert-Butyl 4-(5-chloro-7-iodo-
2H-indazol-2-yl)piperidine-1-carboxylate. tert-Butyl 4-(5-chloro-7-
iodo-2H-indazol-2-yl)piperidine-1-carboxylate was prepared starting
from 2-azido-5-chloro-3-iodobenzaldehyde according to the procedure
used to make compound 46. 1H NMR (400 MHz, DMSO-d6): δ 8.65
(s, 1H), 7.82 (s, 1H), 7.70 (s, 1H), 4.86−4.62 (m, 1H), 4.17−4.00 (m,
2H), 3.04−2.81 (m, 2H), 2.17−2.04 (m, 2H), 2.01−1.83 (m, 2H), 1.42
(s, 9H). ESI-MS m/z 462.1, 464.1 [M + H]+. Step 5: tert-Butyl 4-(5-
chloro-7-cyclopropyl-2H-indazol-2-yl)piperidine-1-carboxylate. A sus-
pension of tert-butyl 4-(5-chloro-7-iodo-2H-indazol-2-yl)piperidine-1-
carboxylate (810 mg, 1.75 mmol), cyclopropylboronic acid (301 mg,
3.51 mmol), and potassium phosphate (1.30 g, 6.14 mmol) in toluene
(16 mL) and water (1.6 mL) was degassed with nitrogen for 5 min.
Then, Pd(OAc)2 (19.69 mg, 0.088 mmol) and tricyclohexylphospho-
nium tetrafluoroborate (64.6 mg, 0.175 mmol) were added and the
reaction mixture was stirred at 100 °C for 18 h. The reaction mixture
was cooled to RT, quenched with water, and extracted with DCM (3
times). The combined organic phases were dried over Na2SO4, filtered,
and concentrated. The crude material was purified by flash
chromatography (silica gel, heptanes/EtOAc) to give the title
compound (673 mg, 92%) as a light-yellow oil. 1H NMR (600 MHz,
5-(4-Amino-3-(hydroxymethyl)-5-methylphenyl)-1,6-dimethyl-
pyridin-2(1H)-one (54). A mixture of 52 (5.43 g, 25.1 mmol),
tetrahydroxydiboron (6.76 g, 75.0 mmol), XPhos Pd G3 (0.850 g, 1.01
mmol), XPhos (0.960 g, 2.01 mmol), and potassium acetate (7.40 g,
75.0 mmol) in ethanol (100 mL) was stirred under reflux for 20 min
(reaction mixture color turned from yellow to orange). The reaction
mixture was treated with 2 M aq. K2CO3 (37.7 mL, 75.0 mmol)
followed by a solution of 53 (5.08 g, 25.1 mmol) in ethanol (20.0 mL),
stirred at reflux for 1 h, and concentrated, and the residue was diluted
with water. The mixture was extracted with EtOAc (5 times) and DCM
(twice); the combined organic phases were dried over Na2SO4, filtered,
and concentrated. The crude material was purified by flash
chromatography (silica gel, DCM/MeOH) to give the title compound
54 (5.19 g, 80%) as a brown powder. 1H NMR (400 MHz, DMSO-d6):
δ 7.21 (d, J = 9.2 Hz, 1H), 6.83−6.75 (m, 2H), 6.28 (d, J = 9.2 Hz, 1H),
5.04 (t, J = 5.4 Hz, 1H), 4.76 (s, 2H), 4.42 (d, J = 5.4 Hz, 2H), 3.48 (s,
3H), 2.28 (s, 3H), 2.10 (s, 3H). ESI-MS m/z 259.1 [M + H]+.
2-Azido-5-(1,2-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-3-meth-
ylbenzaldehyde (55). Step b: 2-Amino-5-(1,2-dimethyl-6-oxo-1,6-
dihydropyridin-3-yl)-3-methylbenzaldehyde was prepared starting
from 54 according to the procedure used to make compound 30. 1H
NMR (400 MHz, DMSO-d6): δ 9.84 (s, 1H), 7.32 (d, J = 1.9 Hz, 1H),
7.29 (d, J = 9.3 Hz, 1H), 7.15 (s, 1H), 7.04 (s, 2H), 6.32 (d, J = 9.2 Hz,
1H), 3.49 (s, 3H), 2.29 (s, 3H), 2.14 (s, 3H). ESI-MS m/z 257.2 [M +
N
https://dx.doi.org/10.1021/acs.jmedchem.0c00130
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
H]+. Step c: To a suspension of amino-5-(1,2-dimethyl-6-oxo-1,6-
dihydropyridin-3-yl)-3-methylbenzaldehyde (4.68 g, 18.3 mmol) in
water (45 mL) and conc. HCl (15.0 mL, 183 mmol) at 0 °C was added
NaNO2 (1.58 g, 22.8 mmol) in 2.5 mL of water. The reaction mixture
was stirred at 0 °C for 30 min and allowed to warm to RT for 30 min.
The reaction mixture was then cooled to 0 °C, and a solution of NaN3
(1.19 g, 18.3 mmol) in 2.5 mL of water was added slowly (warning: gas
evolution). The reaction mixture was stirred at RT for 1 h and diluted
with 100 mL of water. Because the reaction was not complete, NaN3
(0.594 g, 9.13 mmol) was added, and the resulting red suspension was
stirred at RT for 1 h; then, NaN3 (0.594 g, 9.13 mmol) was added and
the reaction mixture was stirred at RT for 3 h. The precipitate was
collected by filtration and rinsed with water. The solid was then dried
under high vacuum to give 55 (4.48 g, 87%) as a light-brown powder.
1H NMR (400 MHz, DMSO-d6): δ 10.19 (s, 1H), 7.64 (d, J = 2.2 Hz,
55 and 1-(2,2,2-trifluoroethyl)piperidin-3-amine. 1H NMR (400 MHz,
chloroform-d) δ 8.14 (s, 1H), 7.35−7.27 (m, 2H), 6.88 (s, 1H), 6.53 (d,
J = 9.2 Hz, 1H), 4.78−4.66 (m, 1H), 3.63 (s, 3H), 3.33 (dd, J = 11.2, 3.9
Hz, 1H), 3.17−2.99 (m, 3H), 2.98−2.85 (m, 1H), 2.73−2.65 (m, 1H),
2.63 (s, 3H), 2.32 (s, 3H), 2.26−2.15 (m, 1H), 2.12−2.00 (m, 1H),
1.91−1.71 (m, 2H). ESI-MS 419.2 [M + H]+.
6-Fluoro-1-methyl-5-(2-(piperidin-4-yl)-7-(trifluoromethyl)-2H-
indazol-5-yl)pyridin-2(1H)-one (24). Compound 24 was prepared
starting from 43 and 5-bromo-6-fluoro-1-methylpyridin-2(1H)-one
according to the procedure used to make compound 5. 1H NMR (400
MHz, DMSO-d6) δ 8.69 (s, 1H), 8.08 (s, 1H), 7.79 (dd, J = 11.3, 9.4
Hz, 1H), 7.71 (s, 1H), 6.42 (d, J = 9.4 Hz, 1H), 4.69−4.60 (m, 1H),
3.47 (d, J = 3.7 Hz, 3H), 3.13−3.05 (m, 2H), 2.71−2.61 (m, 2H),
2.11−2.03 (m, 2H), 2.02−1.90 (m, 2H). 13C NMR (151 MHz, DMSO-
d6) δ 160.00 (d, J = 5.6 Hz), 152.91 (d, J = 269.0 Hz), 141.48 (d, J = 8.0
Hz), 141.47, 124.82 (d, J = 7.5 Hz), 124.68−124.40 (m), 124.28 (d, J =
2.7 Hz), 123.59 (q, J = 272.7 Hz), 123.58, 122.70, 117.12 (q, J = 32.2
Hz), 114.48 (d, J = 4.4 Hz), 100.54 (d, J = 14.0 Hz), 61.23, 45.01, 33.77,
28.02 (d, J = 7.2 Hz). HRMS-ESI [M + H]+ m/z calcd for
C19H19ON4F4 395.14895; found 395.14883.
1H), 7.51 (d, J = 1.6 Hz, 1H), 7.33 (d, J = 9.3 Hz, 1H), 6.37 (d, J = 9.3
Hz, 1H), 3.51 (s, 3H), 2.41 (s, 3H), 2.30 (s, 3H). ESI-MS m/z 283.1 [M
+ H]+.
5-(2-(4,4-Difluoropiperidin-3-yl)-7-methyl-2H-indazol-5-yl)-1,6-
dimethylpyridin-2(1H)-one (20). In a capped vial, a suspension of 55
(0.125 mmol), tert-butyl 3-amino-4,4-difluoropiperidine-1-carboxylate
(0.138 mmol), and Cu2O (12.6 μmol) in DCE (500 μL) was stirred at
80 °C for 2 h. The reaction mixture was filtered. HCl in dioxane (10%)
was added to the filtrate, and the mixture was left stirring overnight. The
solvent was evaporated under reduced pressure, and the residue was
purified by flash chromatography (C18, (H2O + 0.3% NH3)/MeOH +
0.3%NH3) to give the title compound 20. 1H NMR (400 MHz, DMSO-
d6) δ 9.35 (s, 1H), 8.59 (s, 1H), 7.43 (s, 1H), 7.33 (d, J = 9.3 Hz, 1H),
6.99 (s, 1H), 6.36 (d, J = 9.2 Hz, 1H), 5.65−5.37 (m, 1H), 4.10−3.84
(m, 2H), 3.64−3.46 (m, 5H), 2.68−2.58 (m, 1H), 2.55 (s, 3H), 2.47−
2.36 (m, 1H), 2.31 (s, 3H). ESI-MS m/z 373.4 [M + H]+.
2-Azido-5-bromo-3-(trifluoromethyl)benzaldehyde (45). Step a:
2-Amino-5-bromo-3-(trifluoromethyl)benzoic acid. A suspension of 2-
amino-3-(trifluoromethyl)benzoic acid (44) (5.00 g, 24.4 mmol) in
DCM (150 mL) at room temperature was treated with N-
bromosuccinimide (4.60 g, 25.6 mmol) and stirred at room
temperature for 1 h. The resulting suspension was filtered; the solid
was washed with heptanes, leading to precipitation in the filtrate. The
mother liquor was filtered through the same filter funnel. The solid was
washed with heptanes and dried to give 2-amino-5-bromo-3-
1
(trifluoromethyl)benzoic acid (6.30 g, 91%) as a colorless solid. H
NMR (400 MHz, DMSO-d6) δ 13.53 (s, br, 1H), 8.07 (d, J = 2.4 Hz,
1H), 7.75 (d, J = 2.4 Hz, 1H), 7.16 (s, br, 2H). ESI-MS m/z 282.0, 284.0
[M − H]−. Step b: (2-amino-5-bromo-3-(trifluoromethyl)phenyl)-
methanol. A solution of 2-amino-5-bromo-3-(trifluoromethyl)benzoic
acid (9.50 g, 33.4 mmol) in THF (120 mL) at 0 °C was treated
dropwise with LiAlH4 in THF (2 M, 33.4 mL, 66.9 mmol) and stirred at
room temperature for 1 h. The reaction mixture was cooled to 0 °C and
quenched dropwise with water (3.60 mL, 200 mmol). The resulting
mixture was treated with aq. NaOH (2 M, 8.40 mL, 16.8 mmol) and
stirred until a well-stirrable suspension was obtained and then filtered
through celite (rinse with EtOAc); the filtrates were then concentrated.
The crude material was purified by flash chromatography (silica gel,
DCM/MeOH) to give (2-amino-5-bromo-3-(trifluoromethyl)-
phenyl)methanol (6.53 g, 72%) as a light-yellow solid. 1H NMR (400
MHz, DMSO-d6) δ 7.51 (d, J = 2.3 Hz, 1H), 7.41 (d, J = 2.4 Hz, 1H),
5.46 (s, 2H), 5.40 (t, J = 5.5 Hz, 1H), 4.43 (d, J = 5.4 Hz, 2H). ESI-MS
m/z 270.0, 272.0 [M + H]+. Step c: 2-Amino-5-bromo-3-
(trifluoromethyl)benzaldehyde. A solution of (2-amino-5-bromo-3-
(trifluoromethyl)phenyl)methanol (6.53 g, 24.2 mmol) in DCM
(150 mL) was treated with manganese(IV) oxide (23.4 g, 242
mmol), and the resulting suspension was stirred at room temperature
for 1 h. The reaction mixture was filtered through celite (rinse with
DCM), and the filtrates were concentrated to give 2-amino-5-bromo-3-
5-(2-(5,5-Difluoropiperidin-3-yl)-7-methyl-2H-indazol-5-yl)-1,6-
dimethylpyridin-2(1H)-one Hydrochloride (21). In a sealed tube, a
suspension of 56 (30 mg, 0.106 mmol), tert-butyl 5-amino-3,3-
difluoropiperidine-1-carboxylate (27.6 mg, 0.117 mmol), and Cu2O
(1.52 mg, 10.6 μmol) in DCE (500 μL) was stirred at 80 °C for 5 h. The
reaction mixture was filtered. The crude material was purified by SFC to
give tert-butyl 5-(5-(1,2-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-7-
methyl-2H-indazol-2-yl)-3,3-difluoropiperidine-1-carboxylate (15.6
1
mg, 31%) as a colorless resin. H NMR (400 MHz, DMSO-d6): δ
8.57 (s, 1H), 7.41 (s, 1H), 7.35 (d, J = 9.3 Hz, 1H), 6.96 (s, 1H), 6.37
(d, J = 9.3 Hz, 1H), 4.97−4.79 (m, 1H), 4.40−4.17 (m, 2H), 3.71−3.46
(m, 5H), 2.99−2.72 (m, 2H), 2.55 (s, 3H), 2.33 (s, 3H), 1.45 (s, 9H).
ESI-MS m/z 473.3 [M + H]+. To a solution of tert-butyl 5-(5-(1,2-
dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-7-methyl-2H-indazol-2-yl)-
3,3-difluoropiperidine-1-carboxylate (15.6 mg, 0.033 mmol) in DCM
(400 μL) was added HCl in Et2O (2 M, 0.330 mL, 0.660 mmol), and
the suspension was stirred at room temperature for 1 h. Then, HCl in
diethylether (2 M, 0.330 mL, 0.660 mmol) was added and the reaction
mixture was stirred at RT for 2 h. The reaction mixture was evaporated.
The product was then dissolved in water, frozen, and lyophilized to give
the title compound 21 (14.2 mg, quant.) as a light-brown powder. 1H
NMR (400 MHz, DMSO-d6): δ 8.57 (s, 1H), 7.40 (s, 1H), 7.32 (d, J =
9.3 Hz, 1H), 6.95 (s, 1H), 6.35 (d, J = 9.2 Hz, 1H), 5.19−5.06 (m, 1H),
3.89−3.72 (m, 2H), 3.71−3.64 (m, 2H), 3.51 (s, 3H), 3.00−2.83 (m,
2H), 2.53 (s, 3H), 2.30 (s, 3H). ESI-MS m/z 373.3 [M + H]+.
1,6-Dimethyl-5-(7-methyl-2-(pyridin-3-yl)-2H-indazol-5-yl)-
pyridin-2(1H)-one (22). The title compound 22 was prepared
according to the procedure used to make 20 starting from 55 and 3-
aminopyridine. 1H NMR (400 MHz, DMSO-d6): δ 9.33 (d, J = 2.5 Hz,
1H), 9.17 (s, 1H), 8.65 (dd, J = 4.7, 1.3 Hz, 1H), 8.54−8.45 (m, 1H),
7.65 (dd, J = 8.3, 4.7 Hz, 1H), 7.44 (s, 1H), 7.37 (d, J = 9.3 Hz, 1H),
7.01 (s, 1H), 6.36 (d, J = 9.2 Hz, 1H), 3.52 (s, 3H), 2.59 (s, 3H), 2.34 (s,
3H). 13C NMR (151 MHz, DMSO-d6) δ 161.80, 148.86, 148.74,
144.84, 141.52, 141.16, 136.48, 133.42, 128.59, 127.86, 127.32, 124.46,
122.74, 122.40, 119.01, 118.42, 115.58, 31.13, 18.26, 16.81. HRMS-ESI
[M + H]+ m/z calcd for C20H19ON4 331.15534; found 331.15527.
1,6-Dimethyl-5-(7-methyl-2-(1-(2,2,2-trifluoroethyl)piperidin-3-
yl)-2H-indazol-5-yl)pyridin-2(1H)-one (23). The title compound 23
was prepared according to the procedure used to make 20 starting from
1
(trifluoromethyl)benzaldehyde (6.20 g, 96%) as a yellow solid. H
NMR (400 MHz, DMSO-d6) δ 9.88 (s, 1H), 8.14 (d, J = 2.3 Hz, 1H),
7.82 (d, J = 2.3 Hz, 1H), 7.49 (s, br, 2H). Step d: 2-Azido-5-bromo-3-
(trifluoromethyl)benzaldehyde (45). A solution of 2-amino-5-bromo-3-
(trifluoromethyl)benzaldehyde (5.87 g, 21.9 mmol) in TFA (30.0 mL)
at 0 °C was treated with sodium nitrite (2.27 g, 32.9 mmol), stirred for 1
h at 0 °C, and then treated portionwise with sodium azide (2.14 g, 32.9
mmol). The reaction mixture was stirred for 0.5 h at 0 °C; the resulting
suspension was diluted with DCM and cautiously quenched at 0 °C
with 30% solution of NaOH until the pH was above 7. The resulting
mixture was diluted with saturated aq. NaHCO3 and extracted with
DCM (3 times). The combined organic phases were dried over
Na2SO4, filtered, and concentrated to give 45 (6.66 g, quant.) as an
orange solid. 1H NMR (400 MHz, DMSO-d6) δ 10.11 (s, 1H), 8.49 (d,
J = 2.3 Hz, 1H), 8.24 (d, J = 2.3 Hz, 1H). ESI-MS m/z mass not
observed.
O
https://dx.doi.org/10.1021/acs.jmedchem.0c00130
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(3R,4R)-tert-Butyl-4-(5-bromo-7-(trifluoromethyl)-2H-indazol-2-
yl)-3-fluoropiperidine-1-carboxylate (46). Compound 46 was pre-
pared starting from 45 and (3R,4R)-tert-butyl 4-amino-3-fluoropiper-
idine-1-carboxylate according to the procedure used to make
compound 47. ESI-MS m/z 466.3, 468.3 [M + H]+.
2-yl)piperidine-1-carboxylate. A mixture of bis(pinacolato)diboron
(1.21 g, 4.68 mmol), XPhos Pd G3 (0.20 g, 0.23 mmol), XPhos (0.15 g,
0.31 mmol), and potassium acetate (0.92 g, 9.37 mmol) was treated
with a solution of 43 (1.40 g, 3.12 mmol) in dioxane (20.0 mL). The
reaction mixture was purged with nitrogen, sealed, and stirred at 80 °C
for 18 h. The reaction mixture was diluted with water and extracted with
DCM (3 times). The combined organic phases were dried over
Na2SO4, filtered, and concentrated. The crude material was purified by
flash chromatography (silica gel, heptanes/EtOAc) to give tert-butyl 4-
(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-7-(trifluoromethyl)-
2H-indazol-2-yl)piperidine-1-carboxylate (1.38 g, 80%) as a colorless
foam. 1H NMR (400 MHz, methanol-d4) δ 8.53 (s, 1H), 8.41 (s, 1H),
7.86 (s, 1H), 4.79−4.69 (m, 1H), 4.33−4.20 (m, 2H), 3.11−2.95 (m,
2H), 2.28−2.18 (m, 2H), 2.16−2.03 (m, 2H), 1.49 (s, 9H), 1.37 (s,
12H). ESI-MS m/z 496.4 [M + H]+. Step 2: tert-Butyl 4-(5-(5-oxo-
1,2,3,5-tetrahydroindolizin-8-yl)-7-(trifluoromethyl)-2H-indazol-2-
yl)piperidine-1-carboxylate. A mixture of tert-butyl 4-(5-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-7-(trifluoromethyl)-2H-indazol-
2-yl)piperidine-1-carboxylate (80 mg, 0.162 mmol), 51 (34.6 mg, 0.162
mmol), aq. Na2CO3 (2 M, 0.162 mL, 0.323 mmol), and DME (1.50
mL) was purged with argon; then, PdCl2(dppf) (5.91 mg, 8.08 μmol)
was added, and the reaction mixture was stirred at 80 °C for 3 days. The
reaction mixture was poured into water and extracted twice with
EtOAc. The combined organic phases were dried over Na2SO4, filtered,
and concentrated. The residue was purified by flash chromatography
(silica gel, DCM/MeOH) to give tert-butyl 4-(5-(5-oxo-1,2,3,5-
tetrahydroindolizin-8-yl)-7-(trifluoromethyl)-2H-indazol-2-yl)-
piperidine-1-carboxylate (44.0 mg, 54%) as a colorless resin. ESI-MS
m/z 504.3 [M + H]+. Step 3: 8-(2-(Piperidin-4-yl)-7-(trifluoromethyl)-
2H-indazol-5-yl)-2,3-dihydroindolizin-5(1H)-one (25). A solution of
tert-butyl 4-(5-(5-oxo-1,2,3,5-tetrahydroindolizin-8-yl)-7-(trifluoro-
methyl)-2H-indazol-2-yl)piperidine-1-carboxylate (44.0 mg, 0.088
mmol) in DCM (1 mL) was treated with TFA (500 μL) and allowed
to stand for 1 h. The reaction mixture was diluted with DCM, quenched
with saturated aq. NaHCO3 (caution: gas evolution), and extracted
with DCM (3 times). The combined organic layers were dried over
anhydrous Na2SO4, filtered, and concentrated under reduced pressure.
The residue was lyophilized from MeCN/water to give 25 (24.0 mg,
64%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 8.64 (s, 1H),
7.99 (s, 1H), 7.63 (s, 1H), 7.59 (d, J = 9.2 Hz, 1H), 6.34 (d, J = 9.2 Hz,
1H), 4.71−4.57 (m, 1H), 4.05 (t, J = 7.2 Hz, 2H), 3.18 (t, J = 7.5 Hz,
2H), 3.13−3.04 (m, 2H), 2.70−2.61 (m, 2H), 2.14−1.89 (m, 6H). 13C
NMR (151 MHz, DMSO-d6) δ 160.23, 148.99, 141.58, 141.46, 129.00,
124.73 (q, J = 5.2 Hz), 124.44, 123.34, 123.98 (q, J = 272.4 Hz), 122.73,
117.05 (q, J = 32.2 Hz), 116.64, 113.80, 61.20, 48.86, 45.04, 33.79,
31.59, 21.04. HRMS-ESI [M + H]+ m/z calcd for C21H22ON4F3
403.17402; found 403.17410.
8-(2-((3R,4R)-3-Fluoropiperidin-4-yl)-7-(trifluoromethyl)-2H-in-
dazol-5-yl)-2,3-dihydroindolizin-5(1H)-one (26). Compound 26 was
prepared according to the method of 27 by replacing 47 with 46. 1H
NMR (400 MHz, DMSO-d6): δ 8.71 (s, 1H), 8.02 (s, 1H), 7.65 (s, 1H),
7.59 (d, J = 9.2 Hz, 1H), 6.33 (d, J = 9.2 Hz, 1H), 5.08−4.78 (m, 2H),
4.04 (t, J = 7.2 Hz, 2H), 3.38−3.32 (m, 1H), 3.18 (t, J = 7.5 Hz, 2H),
3.03−2.93 (m, 1H), 2.64−2.53 (m, 2H), 2.18−2.02 (m, 4H). ESI-MS
m/z 421.3 [M + H]+. Chiral HPLC Rt = 28.25 min, >99% ee.
8-(2-((3S,4S)-3-Fluoropiperidin-4-yl)-7-(trifluoromethyl)-2H-in-
dazol-5-yl)-2,3-dihydroindolizin-5(1H)-one (27). Step f: (3S,4S)-tert-
Butyl-3-fluoro-4-(5-(5-oxo-1,2,3,5-tetrahydroindolizin-8-yl)-7-(tri-
fluoromethyl)-2H-indazol-2-yl)piperidine-1-carboxylate. A vial was
charged with 47 (1.36 g, 2.92 mmol), tetrahydroxydiboron (748 mg,
8.34 mmol), XPhos Pd G3 (235 mg, 0.278 mmol), XPhos (265 mg,
0.556 mmol), and potassium acetate (818 mg, 8.34 mmol) and flushed
with argon and capped. Then, degassed EtOH (10.0 mL) was added
and the reaction mixture was stirred at 80 °C for 30 min. The reaction
mixture was cooled to room temperature, and aq. K2CO3 (2 M, 4.20
mL, 8.40 mmol) was added dropwise followed by a solution of 51 (700
mg, 2.78 mmol) in degassed EtOH (2.00 mL). The reaction mixture
was stirred at 80 °C for 1 h, poured into water, and extracted twice with
EtOAc. The combined organic phases were dried over Na2SO4, filtered,
and concentrated. The crude material was purified twice by flash
(3S,4S)-tert-Butyl-4-(5-bromo-7-(trifluoromethyl)-2H-indazol-2-
yl)-3-fluoropiperidine-1-carboxylate (47). A suspension of 2-azido-5-
bromo-3-(trifluoromethyl)benzaldehyde (45) (400 mg, 1.36 mmol),
(3S,4S)-tert-butyl 4-amino-3-fluoropiperidine-1-carboxylate (312 mg,
1.43 mmol), and Cu2O (19.5 mg, 0.14 mmol) in DCE (7.0 mL) was
stirred at 80 °C for 15 h. The reaction mixture was cooled to RT,
directly absorbed on isolute, and dried under vacuum. The absorbed
crude material was purified by flash chromatography (silica gel,
heptanes/EtOAc) to give the title compound 47 (561 mg, 88%) as a
light-yellow foam. 1H NMR (400 MHz, DMSO-d6) δ 8.79 (s, 1H), 8.36
(s, 1H), 7.76 (s, 1H), 5.11−4.87 (m, 2H), 4.44−4.28 (m, 1H), 4.07−
3.96 (m, 1H), 3.15−2.87 (m, 2H), 2.21−2.03 (m, 2H), 1.44 (s, 9H).
ESI-MS m/z 466.2, 468.2 [M + H]+.
Racemic-(3S*,4R*)-tert-butyl-4-(5-bromo-7-(trifluoromethyl)-
2H-indazol-2-yl)-3-fluoropiperidine-1-carboxylate (48). Compound
48 was prepared starting from 45 and racemic (3S*,4R*)-tert-butyl 4-
amino-3-fluoropiperidine-1-carboxylate according to the procedure
used to make compound 47. 1H NMR (400 MHz, DMSO-d6) δ 8.67 (s,
1H), 8.32 (s, 1H), 7.76 (s, 1H), 5.26−4.98 (m, 2H), 4.44−4.09 (m,
2H), 3.00 (s, 2H), 2.42−2.29 (m, 1H), 2.12 (d, J = 10.6 Hz, 1H), 1.41
(s, 9H). ESI-MS m/z 466.2, 468.2 [M + H]+.
8-Bromo-2,3-dihydroindolizin-5(1H)-one (51). Step h: 2-Methoxy-
6-(3-((tetrahydro-2H-pyran-2-yl)oxy)propyl)pyridine (50). A solution of
2-bromo-6-methoxypyridine (49, 5.52 mL, 43.6 mmol) in THF (75.0
mL) at −78 °C was treated dropwise with n-butyllithium in hexanes
(2.5 M, 17.4 mL, 43.6 mmol). The reaction mixture was stirred at −78
°C for 30 min, treated dropwise with 2-(3-bromopropoxy)tetrahydro-
2H-pyran (4.91 mL, 29.0 mmol), stirred at −78 °C for 1 h, and allowed
to warm to room temperature over 1 h. The reaction mixture was
poured into ice water and extracted twice with EtOAc. The combined
organic phases were dried over Na2SO4, filtered, and concentrated. The
crude material was purified by flash chromatography (silica gel,
1
heptanes/EtOAc) to give 50 (3.46 g, 47%) as a yellow oil. H NMR
(400 MHz, DMSO-d6) δ 7.57 (dd, J = 8.1, 7.4 Hz, 1H), 6.81 (d, J = 7.2
Hz, 1H), 6.59 (d, J = 8.2 Hz, 1H), 4.55−4.49 (m, 1H), 3.81 (s, 3H),
3.76−3.68 (m, 1H), 3.68−3.62 (m, 1H), 3.43−3.33 (m, 2H), 2.73−
2.65 (m, 2H), 1.95−1.87 (m, 2H), 1.76−1.65 (m, 1H), 1.65−1.55 (m,
1H), 1.51−1.39 (m, 4H). ESI-MS m/z 252.3 [M + H]+. Step i: 2,3-
dihydroindolizin-5(1H)-one. A solution of 50 (3.46 g, 13.8 mmol) in
48% aq. HBr (78.0 mL, 0.690 mol) was stirred under reflux for 4 h. The
reaction mixture was cooled to RT, filtered, and slowly basified with
sodium hydroxide (27.5 g, 0.690 mol). The crude mixture was then
diluted with water and extracted twice with DCM. The organic phases
were dried over Na2SO4, filtered, and concentrated to give 2,3-
dihydroindolizin-5(1H)-one (1.83 g, 98%) as a brown solid. 1H NMR
(400 MHz, DMSO-d6) δ 7.35 (dd, J = 8.8, 7.0 Hz, 1H), 6.18−6.10 (m,
2H), 3.97−3.90 (m, 2H), 3.04 (t, J = 7.7 Hz, 2H), 2.06 (p, J = 7.6 Hz,
2H). ESI-MS m/z 136.1 [M + H]+. Step j: 8-Bromo-2,3-
dihydroindolizin-5(1H)-one (51). A solution of 2,3-dihydroindolizin-
5(1H)-one (1.82 g, 13.5 mmol) in DMF (35.0 mL) at 0 °C was treated
with N-bromosuccinimide (2.40 g, 13.5 mmol). The reaction mixture
was stirred at room temperature for 30 min, poured into water, and
extracted twice with DCM. The combined organic phases were dried
over Na2SO4, filtered, and concentrated. The residue was dissolved in a
mixture of DCM and heptanes. The solution was then concentrated
until precipitation occurred. The suspension was filtered, and the
filtrates were concentrated. The residue was purified by flash
chromatography (silica gel, DCM/MeOH) to give 51 (1.43 g, 42%)
as a light-yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 7.49 (d, J =
9.5 Hz, 1H), 6.18 (d, J = 9.5 Hz, 1H), 4.04 (t, J = 7.5 Hz, 2H), 3.05 (t, J
= 7.8 Hz, 2H), 2.12 (p, J = 7.7 Hz, 2H). ESI-MS m/z 214.1, 216.1 [M +
H]+.
8-(2-(Piperidin-4-yl)-7-(trifluoromethyl)-2H-indazol-5-yl)-2,3-di-
hydroindolizin-5(1H)-one (25). Step 1: tert-Butyl 4-(5-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-7-(trifluoromethyl)-2H-indazol-
P
https://dx.doi.org/10.1021/acs.jmedchem.0c00130
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
chromatography ((silica gel, DCM/MeOH) and (C18, (H2O + 0.1%
TFA)/MeCN)). The obtained material was treated with a saturated
solution of Na2CO3 and extracted with DCM. The organic phase was
dried over Na2SO4, filtered, and concentrated. The residue was
dissolved in DCM (15.0 mL), and metal scavenger resin (MP-TMT)
(2.67 g, 1.39 mmol) was added. The resulting suspension was shaken
overnight and filtered (one rinse with DCM), and the filtrate was
concentrated to afford (3S,4S)-tert-butyl-3-fluoro-4-(5-(5-oxo-1,2,3,5-
tetrahydroindolizin-8-yl)-7-(trifluoromethyl)-2H-indazol-2-yl)-
(PerkinElmer; excitation 340 nm, emission 615 and 665 nm). Control
wells without TLR8 protein and with DMSO were used to calculate the
% activity. IC50 values were estimated from a four-parameter logistic fit
in a data analysis and visualization package developed at Novartis.
TLR-Driven Cytokine Inhibition in Human PBMCs. Fresh
human blood, collected in S-Monovette Heparin tubes (Starstedt), was
obtained from healthy individuals with patient informed consent
́
(Santemed Gesundheitszentrum AG Basel Switzerland). PBMCs were
prepared by diluting blood 1:1 with phosphate-buffered saline (PBS)
and transferred to pre-prepared Leucosep tubes (Greiner Bio-one)
containing 15 mL of LSM1077 (EuroBio). The PBMC layer was
carefully removed from the plasma/separation medium interface
following centrifugation (800g, 20 min at 22 °C without brake).
PBMCs were washed with PBS, centrifuged (400g, 10 min at 22 °C),
and resuspended in growth media (RPMI1640+GlutaMAX-I supple-
mented with 0.05 mM 2-mercaptoethanol, 10 mM 4-(2-hydroxyethyl)-
1-piperazineethanesulfonic acid (HEPES), and 5% v/v FBS, GE
Healthcare). PBMCs were seeded in 384-well plates and incubated for
30 min in the presence of increased amounts of compound or vehicle
control (0.25% DMSO final per well). Cells were stimulated with fixed
amounts of agonists for TLR1/2 (Pam3CSK4, 0.1 μg/mL, Invivogen),
TLR4 (LPS, 5 ng/mL, Sigma), TLR5 (flagellin, 1 μg/mL), TLR9
(ODN2216, 0.3 μM), and IL-1β (3 μg/mL, produced internally),
TLR7 (ssRNA40, 10 μg/mL, synthesized by Microsynth, Switzerland),
or TLR8 (ssRNA40, 1 μg/mL). For both TLR7 and TLR8, ssRNA40
was precomplexed with N-[1-(2,3 dioleoyloxy) propyl]-N,N,N tri-
methyl ammonium methylsulfate (DOTAP) (10 or 25 μg/mL,
respectively; Roche Life Sciences). For each agonist, EC90 values
were experimentally determined to assess IC50 compounds across
cellular pathways. After 20 h at 37 °C in a humidified incubator,
supernatants were transferred to 384-well Optiplates (PerkinElmer)
and IFNα levels (TLR7 and TLR9) were quantified by AlphaLisa
technology using the human interferon AlphaLISA kit (PerkinElmer)
and the EnVision multiplate reader according to the manufacturer’s
protocols. TNFα was measured routinely for TLR4 and TLR8, and all
other TLRs and IL1R levels in the supernatant were quantified by
homogeneous time-resolved fluorescence (HTRF) technology using
the human kits (CisBio) and a RUBYstar (BMG Labtech) fluorescent
plate reader. Data were analyzed using Excel XL fit 5.0 (Microsoft) with
XLfit add-in (IDBS; version 5.2.0). Cytokine concentrations were
determined following extrapolation to standard curves using the
appropriate reference cytokine. Individual IC50 (median inhibition
concentration) values were determined by nonlinear regression after
fitting of curves to the experimental data with each point performed in
biological triplicate.
1
piperidine-1-carboxylate (980 mg, 68%) as a light-yellow solid. H
NMR (400 MHz, DMSO-d6): δ 8.79 (s, 1H), 8.04−7.97 (m, 1H), 7.66
(s, 1H), 7.59 (d, J = 9.2 Hz, 1H), 6.33 (d, J = 9.2 Hz, 1H), 5.15−4.91
(m, 2H), 4.44−4.31 (m, 1H), 4.10−3.98 (m, 3H), 3.18 (t, J = 7.5 Hz,
2H), 3.12−2.94 (m, 2H), 2.23−2.02 (m, 4H), 1.44 (s, 9H). ESI-MS m/
z 521.4 [M + H]+. Step g: 8-(2-((3S,4S)-3-Fluoropiperidin-4-yl)-7-
(trifluoromethyl)-2H-indazol-5-yl)-2,3-dihydroindolizin-5(1H)-one
(27). A solution of (3S,4S)-tert-butyl 3-fluoro-4-(5-(5-oxo-1,2,3,5-
tetrahydroindolizin-8-yl)-7-(trifluoromethyl)-2H-indazol-2-yl)-
piperidine-1-carboxylate (980 mg, 1.88 mmol) in DCM (6.00 mL) was
treated with TFA (2.90 mL, 37.7 mmol) and stirred at room
temperature for 30 min. The reaction mixture was concentrated, and
the residue was purified by flash chromatography (C18, (H2O + 0.1%
TFA)/MeCN). The pure product-containing fractions were concen-
trated until the bulk of the MeCN had been evaporated, treated with
saturated aq. Na2CO3, and extracted with DCM. The organic phase was
dried over Na2SO4, filtered, and concentrated. The residue was then
dissolved in MeOH and treated with diethylether. The resulting
solution was concentrated, and the residue was dried under vacuum to
1
give 27 (682 mg, 85%) as a colorless powder. H NMR (400 MHz,
DMSO-d6): δ 8.71 (s, 1H), 8.02 (s, 1H), 7.65 (s, 1H), 7.59 (d, J = 9.2
Hz, 1H), 6.33 (d, J = 9.2 Hz, 1H), 5.09−4.77 (m, 2H), 4.05 (t, J = 7.3
Hz, 2H), 3.39−3.32 (m, 1H), 3.18 (t, J = 7.6 Hz, 2H), 3.03−2.93 (m,
1H), 2.64−2.53 (m, 2H), 2.19−2.02 (m, 4H). 13C NMR (151 MHz,
DMSO-d6) δ 160.27, 149.03, 141.97, 141.56, 129.28, 125.55, 125.13 (d,
J = 5.6 Hz), 124.54, 123.9 (q, J = 272.8 Hz), 122.58, 117.19 (q, J = 32.5
Hz), 116.65, 113.78, 90.49 (d, J = 181.6 Hz), 65.23 (d, J = 16.6 Hz),
49.18 (d, J = 21.8 Hz), 48.87, 44.05, 33.32 (d, J = 4.7 Hz), 31.57, 21.02.
19F NMR (500 MHz, DMSO-d6) δ −184.83 (1F), -60.78 (3F). IR
(KBr) 3430, 3305, 3090, 2956, 2830, 1654, 1594, 1537, 1463, 1434,
1349, 1293, 1277, 1155, 1122, 1064, 830 cm−1. HRMS-ESI [M + H]+
m/z calcd for C21H20F4N4O 421.16460; found 421.16458. Chiral
HPLC Rt = 24.29 min, >99% ee.
Racemic-8-(2-((3S*,4R*)-3-fluoropiperidin-4-yl)-7-(trifluoro-
methyl)-2H-indazol-5-yl)-2,3-dihydroindolizin-5(1H)-one (28).
Compound 28 was prepared according to the method of 27 by
replacing 47 with 48. 1H NMR (400 MHz, DMSO-d6) δ 8.60 (d, J = 1.1
Hz, 1H), 8.01 (s, 1H), 7.65 (s, 1H), 7.59 (d, J = 9.2 Hz, 1H), 6.33 (d, J =
9.2 Hz, 1H), 5.07−4.89 (m, 2H), 4.04 (t, J = 7.2 Hz, 2H), 3.25−3.07
(m, 4H), 2.91 (dd, J = 39.8, 14.5 Hz, 1H), 2.76−2.67 (m, 1H), 2.36−
2.24 (m, 1H), 2.13−1.99 (m, 3H). ESI-MS m/z 421.3 [M + H]+.
Biology: TLR8 Protein Production and Purification. The
extracellular domain of TLR8 (residues 27−827) was expressed by
Express2ion and purified in-house according to the published
literature.12 In brief, TLR8 was expressed in a Drosophila S2 expression
system. The protein was purified by IgG Sepharose affinity
chromatography, subsequent saccharide trimming using EndoHF,
and Superdex 200 gel filtration chromatography followed by HiTrap Q
anion-exchange chromatography.
TLR8 Binding Assay. The TR-FRET binding assay was performed
in white 1536-well plates (Greiner) in a final volume of 5 μL. An Echo
acoustic dispenser was used to transfer 50 nL of 2 mM compound
solution in DMSO into the assay plate. Assay components were
prepared in 20 mM Tris/HCl pH 7.0, 0.2% Pluronic F127, and 0.1 mM
ethylenediamine tetraacetic acid (EDTA). The final concentrations
were 10 nM biotinylated TLR8, 50 nM Cy5-ligand 2, and 0.5 nM Eu-
Streptavidin (PerkinElmer). Then, 2.5 μL of TLR8 protein was added
to the compounds using a Certus dispenser, followed by 30 min of
incubation at room temperature. After addition of a 2.5 μL detection
mix (Eu-Streptavidin and 2), plates were incubated for 30 min before
measuring TR-FRET in an Envision multimode plate reader
Ex vivo TLR stimulated human and murine whole-blood cytokine
assays. For blood assays, fresh human blood or blood from 129/SV
mice was collected into citrate S-Monovette 9NC tubes (Sarstedt).
Blood was diluted 1:1 with RPMI1640 into sterile 96-well U-bottom
plates. Blood cells were incubated with increasing concentrations of 27
in RPMI1640 or vehicle control (0.25 % v/v DMSO final per well) for
30 min at 37 °C. Human blood was stimulated with ssRNA40 (1 μg/
mL) for TLR7-mediated IFNα and TLR8-mediated TNFα and
ODN2216 (0.2 μM) for TLR9-driven IFNα. Both agonists were
precomplexed with DOTAP (15 μg/mL) in a final assay volume of 100
μL/well. For murine, blood was stimulated with ssRNA40 (1 μg/mL
precomplexed with 15 μg/mL DOTAP) or ODN1585 (0.2 μM
precomplexed with 25 μg/mL DOTAP for IFNα) in a final assay
volume of 100 μL/well. After 20 h at 37 °C in a humidified incubator,
supernatants were collected for quantification of human and murine
IFNα (Platinum ELISA kit, Thermo Fisher Scientific) according to the
manufacturer’s instructions and a multiplate SpectraMax M5 reader
(Molecular Devices, Sunnyvale, CA). Human TNF release was
quantified using HTRF technology (CisBio, Bedford, MA) and a
RUBYstar (BMG Labtech, Ortenberg, Germany) fluorescence plate
reader. The remaining blood cell pellet was washed twice with PBS, and
cell viability was assessed using CellTiter Glo (Promega, Madison, WI)
or ATPLite assays (PerkinElmer) according to the manufacturer’s
protocols. Luminescence was measured using an EnVision multiplate
reader (PerkinElmer), SpectraMax M5 reader, or CLIPR (Molecular
Q
https://dx.doi.org/10.1021/acs.jmedchem.0c00130
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Devices, Sunnyvale, CA). Individual IC50 values were determined as
indicated above.
Animals. All animal studies described were performed according to
Swiss animal welfare guidelines.
X-ray crystal data and structure refinement details,
sequence alignment for TLRs, glutathione stability data
for compound 24, synthesis procedure and spectral data
for probe compound 2 (PDF)
In Vivo PK Experiments. Three male Sprague−Dawley rats or
C57BL/6 mice, respectively, were used to assess the principal PK
parameters. Compounds were prepared in suitable vehicles for
intravenous (i.v.) administration (as solution) and peroral (p.o.)
administration (as suspension) at the given doses (mg/kg). For rats,
both dosing arms were done as crossover, i.e., with respective washout
times in between. Each dosing arm was applied once (single dose), and
blood samples were taken from a suitable vein at regular time points
thereafter (0.083, 0.25, 0.5, 1, 2, 4, 7, and 24 h after i.v.; 0.25, 0.5, 1, 2, 4,
7, and 24 h after p.o., respectively). Compound blood concentrations
were determined by suitable LC/MS/MS methods, using internal
standards and calibration curves to integrate the peak areas. From these
concentrations, PK parameters were determined by standard methods,
i.e., area-under-curve (AUC) via trapezoidal calculation, clearance
(CL), and volume of distribution at steady state (Vss) using AUC and
dose information. Finally, oral bioavailability was determined by
relating AUCs derived from IV and PO arms including dose correction.
For comparison reasons, some of the PK parameters are presented as
“dose-normalized, dn”, i.e., related to a standard dose of 1 mg/kg.
In Vivo Efficacy Analysis in Mice. Compound 27 in methyl
cellulose/Tween-80 (0.5% methyl cellulose (BioConcept, Allschwil,
Switzerland)/0.5% Tween-80 (Sigma-Aldrich)) or vehicle alone was
administered p.o. to 129Sv mice (n = 5 per dose group). One hour later,
mice were injected i.v. with 20 μg of ssRNA R0006, complexed to 140
μg of N-[1-(2,3 dioleoyloxy) propyl]-N,N,N tri-methyl ammonium
methylsulfate (DOTAP, Roche) according to the manufacturer’s
instructions, and diluted in HEPES-buffered saline. Mice were
terminally bled from the vena cava into EDTA microvettes (Sarstedt,
Molecular formula strings including assay data (CSV)
Binding model of compound 3 to TLR8 (PDB)
Accession Codes
PDB code for the X-ray cocrystal structure of TLR8 ECD in
complex with compound 11 is 6TY5. CCDC deposition number
for the small-molecule crystal structure for compound 27 is
1979262. The authors will release the atomic coordinates and
experimental data upon publication of the article.
AUTHOR INFORMATION
Corresponding Author
■
Thomas Knoepfel − Global Discovery Chemistry, Novartis
Institutes for BioMedical Research, CH-4002 Basel, Switzerland;
orcid.org/0000-0002-1675-6297;
Email: thomas.knoepfel@novartis.com
Authors
Pierre Nimsgern − Global Discovery Chemistry, Novartis
Institutes for BioMedical Research, CH-4002 Basel, Switzerland
Sebastien Jacquier − Global Discovery Chemistry, Novartis
́
Institutes for BioMedical Research, CH-4002 Basel, Switzerland
Marjorie Bourrel − Global Discovery Chemistry, Novartis
Institutes for BioMedical Research, CH-4002 Basel, Switzerland
Eric Vangrevelinghe − Global Discovery Chemistry, Novartis
Institutes for BioMedical Research, CH-4002 Basel, Switzerland
Ralf Glatthar − Global Discovery Chemistry, Novartis Institutes
for BioMedical Research, CH-4002 Basel, Switzerland
Dirk Behnke − Global Discovery Chemistry, Novartis Institutes
for BioMedical Research, CH-4002 Basel, Switzerland
Phil B. Alper − Genomics Institute of the Novartis Research
Foundation, San Diego, California 92121, United States;
orcid.org/0000-0002-4115-0633
Numbrecht, Germany) 2 h after R0006/DOTAP injection. Serum was
̈
isolated by centrifugation (2000g, 10 min, 4 °C), and IFNα levels were
determined using the mouse IFNα Platinum ELISA (eBioscience, San
Diego, CA). Concentrations of compound 27 in whole blood were
determined by liquid chromatography, coupled to mass spectrometry
(LC−MS/MS). Data and statistics were analyzed using GraphPad
Prism version 8.
TLR8 Cocrystallization and Structure Determination. Purified
TLR8 ECD protein buffered in 50 mM Tris pH 8.0 and 175 mM NaCl
at a concentration of 5.9 mg*mL−1 and containing 2 mM compound 11
was used to set up a sparse-matrix screen. Then, 0.2 μL of the protein
solution was mixed with 0.2 μL of the well solution and equilibrated
against 80 μL of the reservoir using SWISSCI MRC 2 well
crystallization plates designed in the 96-well plate format and sealed
with Hampton Research ClearSeal Film. Crystallization plates were
incubated at 20 °C. Crystals were found under several conditions after
2−10 days, while crystals suitable for X-ray diffraction experiments were
obtained in condition E4: 20% PEG 3350, 200 mM magnesium
chloride, reaching 450 μm length on day 28. Crystals were vitrified by
plunging them directly into liquid nitrogen.
Data sets were collected at 1.0 Å wavelength with a PILATUS 6M
detector at the Swiss Light Source beamline X10SA (Villigen,
Switzerland). Data were collected by Expose GmbH. Diffraction
images were processed and scaled using XDS and XSCALE,
respectively. Structures were solved by molecular replacement (Phaser)
using 3W3G as the starting model. The initial model was subjected to
iterative cycles of manual rebuilding and subsequent structure
refinement in Coot and autoBuster, respectively. The ligand structure
was built into unbiased Fo − Fc difference electron density calculated by
autoBuster. Final structure refinement statistics are summarized in
Supporting Table S1. Refined coordinates were deposited to the PDB
with entry number 6TY5.
Pierre-Yves Michellys − Genomics Institute of the Novartis
Research Foundation, San Diego, California 92121, United
States
Jonathan Deane − Genomics Institute of the Novartis Research
Foundation, San Diego, California 92121, United States
Tobias Junt − Autoimmunity, Transplantation and
Inflammation, Novartis Institutes for BioMedical Research, CH-
4002 Basel, Switzerland
Geraldine Zipfel − Autoimmunity, Transplantation and
Inflammation, Novartis Institutes for BioMedical Research, CH-
4002 Basel, Switzerland
Sarah Limonta − Autoimmunity, Transplantation and
Inflammation, Novartis Institutes for BioMedical Research, CH-
4002 Basel, Switzerland
Stuart Hawtin − Autoimmunity, Transplantation and
Inflammation, Novartis Institutes for BioMedical Research, CH-
4002 Basel, Switzerland
Cedric Andre − Autoimmunity, Transplantation and
Inflammation, Novartis Institutes for BioMedical Research, CH-
4002 Basel, Switzerland
Thomas Boulay − Autoimmunity, Transplantation and
Inflammation, Novartis Institutes for BioMedical Research, CH-
4002 Basel, Switzerland
Pius Loetscher − Autoimmunity, Transplantation and
Inflammation, Novartis Institutes for BioMedical Research, CH-
4002 Basel, Switzerland
́
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c00130.
R
https://dx.doi.org/10.1021/acs.jmedchem.0c00130
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(6) Hawkins, L. D. E6887: A Novel and Selective Inhibitor of Toll-like
Receptors 7 and 8, Presented at the 252nd National Meeting, American
Chemical Society: Philadelphia, PA, MEDI 203, August 20−25, 2016.
(7) Padilla-Salinas, R.; Anderson, R.; Sakaniwa, K.; Zhang, S.;
Nordeen, P.; Lu, C.; Shimizu, T.; Yin, H. H. Discovery of Novel
Inhibitors Targeting Toll-like Receptor 7 and 8. J. Med. Chem. 2019, 62,
10221−10244.
Michael Faller − Chemical Biology & Therapeutics, Novartis
Institutes for BioMedical Research, CH-4002 Basel, Switzerland
Jutta Blank − Chemical Biology & Therapeutics, Novartis
Institutes for BioMedical Research, CH-4002 Basel, Switzerland
Roland Feifel − Pharmacokinetic Sciences, Novartis Institutes for
BioMedical Research, CH-4002 Basel, Switzerland
Claudia Betschart − Global Discovery Chemistry, Novartis
Institutes for BioMedical Research, CH-4002 Basel, Switzerland
(8) Bou Karroum, N.; Moarbess, G.; Guichou, J.-F.; Bonnet, P.-A.;
Patinote, C.; Bourharoun-Tayoun, H.; Chamat, S.; Cuq, P.; Diab-Assaf,
M.; Kassab, I.; Deleuze-Masquefa, C. Novel and Selective TLR7
Antagonists among the Imidazo[1,2-a]pyrazines, Imidazo[1,5-a]-
quinoxalines, and Pyrazolo[1,5-a]quinaxoline Series. J. Med. Chem.
2019, 62, 7015−7031.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c00130
Author Contributions
̌
(9) Kuznik, A.; Bencina, M.; Svajger, U.; Jeras, M.; Rozman, B.; Jerla,
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
R. Mechanism of Endosomal TLR Inhibition by Antimalarial Drugs and
Imidazoquinolines. J. Immunol. 2011, 186, 4794−4804.
(10) Lamphier, M.; Zheng, W.; Latz, E.; Spyvee, M.; Hansen, H.;
Rose, J.; Genest, M.; Yang, H.; Shaffer, C.; Zhao, Y.; Shen, Y.; Liu, C.;
Liu, D.; Mempel, T. R.; Rowbottom, C.; Chow, J.; Twine, N. C.; Yu, M.;
Gusovsky, F.; Ishizaka, S. Novel Small Molecule Inhibitors of TLR7 and
TLR9: Mechanism of Action and Efficacy in Vivo. Mol. Pharmacol.
2014, 85, 429−440.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
́
We thank T. Wager, P. Piechon, and I. Dix for crystallography
(11) Kaufmann, A. M.; Krise, J. P. Lysosomal Sequestration of Amine-
Containing Drugs: Analysis and Therapeutic Implications. J. Pharm. Sci.
2007, 96, 729−746.
(12) Tanji, H.; Ohto, U.; Shibata, T.; Miyake, K.; Shimizu, T.
Structural Reorganization of the Toll-Like Receptor 8 Dimer Induced
by Agonistic Ligands. Science 2013, 339, 1426−1429.
(13) Zhang, Z.; Ohto, U.; Shibata, T.; Krayukhina, E.; Taoka, M.;
Yamauchi, Y.; Tanji, H.; Isobe, T.; Uchiyama, S.; Miyake, K.; Shimizu,
T. Structural Analysis Reveals that Toll-like Receptor 7 is a Dual
Receptor for Guanosine and Single-Stranded RNA. Immunity 2016, 45,
737−748.
(14) Zhang, Z.; Ohto, U.; Shibata, T.; Taoka, M.; Yamauchi, Y.; Sato,
R.; Shukla, N. M.; David, S. A.; Isobe, K.; Shimizu, T. Structural
Analyses of Toll-like Receptor 7 Reveal Detailed RNA Sequence
Specificity and Recognition Mechanism of Agonistic Ligands. Cell Rep.
2018, 25, 3371−3381.e5.
(15) Alper, P. B.; Deane, J.; Betschart, C.; Buffet, D.; Collignon Zipfel,
G.; Gordon, P.; Hampton, J.; Hawtin, S.; Ibanez, M.; Jiang, T.; Junt, T.;
Knoepfel, T.; Liu, B.; Maginnis, J.; McKeever, U.; Michellys, P.-Y.;
Mutnick, D.; Nayak, B.; Niwa, S.; Richmond, W.; Rush, J. S.; Syka, P.;
Zhang, Y.; Zhu, X. Discovery of Potent, Orally Bioavailable in Vivo
Efficacious Antagonists of the TLR7/8 Pathway. Bioorg. Med. Chem.
Lett. [Online early access]. DOI: DOI: 10.1016/j.bmcl.2020.127366.
Published Online: June 24, 2020. https://www.sciencedirect.com/
science/article/pii/S0960894X20304777 (accessed June 24, 2020).
(16) Arkin, M. R.; Glicksman, M. A.; Fu, H.; Havel, J. J.; Du, Y.
Inhibition of Protein-Protein Interactions: Non-Cellular Assay
Formats. In Assay Guidance Manual [Online]; Sittampalam, G. S.;
Grossman, A.; Brimacombe, K., Eds.; Eli Lilly & Company and the
National Center for Advancing Translational Sciences: Bethesda
(MD), 2004; https://www.ncbi.nlm.nih.gov/books/NBK92000/ (ac-
cessed June 3, 2020).
support for compound 27; R. Denay for analytical support; D.
Langenegger for support setting up and running the biochemical
assay; Y. Zhang for the preparation of compound 1; R. Hemmig
and F. Zink for protein purification and crystallization; and G.
Ruzzante for support with compound profiling.
ABBREVIATIONS
■
aq., aqueous solution; DCM, dichloromethane; DCE, 1,2-
dichloroethane; DMA, N,N-dimethylacetamide; DME, 1,2-
dimethoxyethane; DMF, N,N-dimethylformamide; DMSO,
dimethylsulfoxide; dppf, 1,1′-bis(diphenylphosphino)-
ferrocene; ECD, ectodomain; ESI-MS, electrospray ionization
mass spectroscopy; FP, fluorescence polarization; HATU, O-(7-
azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexa-
fluorophosphate; HRMS-ESI, high-resolution mass spectrosco-
py electrospray ionization; IFN, interferon; MDCK, Madin−
Darby Canine Kidney cells; n.a., not applicable; n.d., not
determined; NMP, 1-methyl-2-pyrrolidinone; PBMCs, periph-
eral blood mononuclear cells; PEG, poly(ethylene glycol);
R848, 1-[4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]-
chinolin-1-yl]-2-methylpropan-2-ol; RT, room temperature;
SD, standard deviation; SFC, supercritical fluid chromatog-
raphy; ssRNA, single-stranded RNA; TFA, trifluoroacetic acid;
THF, tetrahydrofuran; TNF, tumor necrosis factor; TR-FRET,
time-resolved fluorescence resonance energy transfer; XPhos, 2-
dicyclohexylphosphino-2′,4′,6′-tri-isopropyl-1,1′-biphenyl;
XPhos, Pd G3 (XPhos)[2-(2′-amino-1,1′-biphenyl)]palladium-
(II) methanesulfonate
̈
(17) Glide, version 2015.3; Schrodinger, LLC: New York, NY, 2015.
REFERENCES
(18) Lovering, F.; Bikker, J.; Humblet, C. Escape from Flatland:
Increasing Saturation as an Approach to Improving Clinical Success. J.
Med. Chem. 2009, 52, 6752−6756.
■
(1) Junt, T.; Barchet, W. Translating Nucleic Acid-Sensing Pathways
into Therapies. Nat. Rev. Immunol. 2015, 15, 529−544.
(2) Lipard, G. B.; Zepp, C. M. Quinazoline Derivative Useful as Toll-
Like Receptor Antagonist. WO2008152471, 2008.
(3) Boivin, R.; Carlson, E.; Endo, A.; Hansen, H.; Hawkins, L. D.;
Ishizaka, S.; Mackey, M.; Narayan, S.; Satoh, T.; Schiller, S.
Tetrahydropyrazolopyrimidine compounds. US20130324547, 2013.
(4) Carlson, E.; Hansen, H.; Mackey, M.; Schiller, S.; Ogawa, C.;
Davis, H. Selectively Substituted Quinoline Compounds.
WO2015057659 A1, 2015.
(5) Boivin, R.; Hansen, H.; Ishizaka, S.; Mackey, M.; Schiller, S.;
Ogawa, C.; Narayan, S.; Bertinato, P.; Burger, G. Selectively Substituted
Quinoline Compounds. WO2015057655, 2015.
(19) Zhang, S.; Hu, Z.; Tanji, H.; Jiang, S.; Das, N.; Li, J.; Sakaniwa, K.;
Jin, J.; Bian, Y.; Ohto, U.; Shimizu, T.; Yin, H. Small-Molecule
Inhibition of TLR8 through Stabilization of its Resting State. Nat.
Chem. Biol. 2018, 14, 58−64.
(20) Forman, J. H.; Zhang, H.; Rinna, A. Glutathione: Overview of its
Roles, Measurement, and Biosynthesis. Mol. Aspects Med. 2009, 30, 1−
12.
(21) Pracht, P.; Wilcken, R.; Udvarhelyi, A.; Rodde, S.; Grimme, S.
High Accuracy Quantum-Chemistry-based Calculation and Blind
Prediction of Macroscopic pKa Values in the Context of the SAMPL6
Challenge. J. Comput.-Aided Mol. Des. 2018, 32, 1139−1149.
S
https://dx.doi.org/10.1021/acs.jmedchem.0c00130
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
̈
(22) Morgenthaler, M.; Schweizer, E.; Hoffmann-Roder, A.; Benini,
F.; Martin, R. E.; Jaeschke, G.; Wagner, B.; Fischer, H.; Bendels, S.;
Zimmerli, D.; Schneider, J.; Diederich, F.; Kansy, M.; Muller, K.
̈
Predicting and Tuning Physicochemical Properties in Lead Opti-
mization: Amine Basicities. ChemMedChem 2007, 2, 1100−1115.
(23) Gorden, K. K. B.; Qiu, X. X.; Binsfeld, C. C.; Vasilakos, J. P.;
Alkan, S. S. Cutting Edge: Activation of Murine TLR8 by a
Combination of Imidazoquinoline Immune Response Modifiers and
PolyT Oligodeoxynucleotides. J. Immunol. 2006, 177, 6584−6587.
(24) Guiducci, C.; Gong, M.; Cepika, A.-M.; Xu, Z.; Tripodo, C.;
Bennett, L.; Crain, C.; Quartier, P.; Cush, J. J.; Pascual, V.; Coffman, R.
L.; Barrat, F. J. RNA Recognition by Human TLR8 Can Lead to
Autoimmune Inflammation. J. Exp. Med. 2013, 210, 2903−2919.
(25) Genung, N. E.; Wei, L.; Aspnes, G. E. Regioselective Synthesis of
2H-Indazoles Using a Mild, One-Pot Condensation−Cadogan
Reductive Cyclization. Org. Lett. 2014, 16, 3114−3117.
(26) Molander, G. A.; Trice, S. L. J.; Kennedy, S. M. Scope of the Two-
Step, One-Pot Palladium-Catalyzed Borylation/Suzuki Cross-Coupling
Reaction Utilizing Bis-Boronic Acid. J. Org. Chem. 2012, 77, 8678−
8688.
(27) Hu, J.; Cheng, Y.; Yang, Y.; Rao, Y. A General and Efficient
Approach to 2H-Indazoles and 1H-Pyrazoles through Copper-
Catalyzed Intramolecular N−N Bond Formation under Mild
Conditions. Chem. Commun. 2011, 47, 10133−10135.
(28) Sharghi, H.; Aberi, M. Ligand-Free Copper(I) Oxide Nano-
particle Catalyzed Three-Component Synthesis of 2H-Indazole
Derivatives from 2-Halobenzaldehydes, Amines and Sodium Azide in
Polyethylene Glycol as a Green Solvent. Synlett 2014, 25, 1111−1115.
(29) Thomas, E. W. Synthesis of Indolizinones and a Pyridoazepi-
none: A New Method for the Annulation of Pyridinones. J. Org. Chem.
1986, 51, 2184−2191.
(30) McComas, C. C.; Kuduk, S. D.; Reger, T. S. Substituted Pyridone
Derivatives as PDE10 Inhibitors. WO201481617, 2014.
(31) Hu, Z.; Tanji, H.; Jiang, S.; Zhang, S.; Koo, K.; Chan, J.;
Sakaniwa, K.; Ohto, U.; Candia, A.; Shimizu, T.; Yin, H. Small-
Molecule TLR8 Antagonists via Structure-Based Rational Design. Cell
Chem. Biol. 2018, 25, 1286−1291.
T
https://dx.doi.org/10.1021/acs.jmedchem.0c00130
J. Med. Chem. XXXX, XXX, XXX−XXX