Development of indazole mineralocorticoid receptor antagonists and investigation into their selective late-stage functionalization

Article abstract of DOI:10.1016/j.bmcl.2019.04.024

The derivatization of pharmaceuticals is a core activity in the discovery and development of new medicines. Late-stage functionalization via modern C–H functionalization chemistry has emerged as a powerful technique with which to diversify advanced pharma

Full text of DOI:10.1016/j.bmcl.2019.04.024

Accepted Manuscript
Development of indazole mineralocorticoid receptor antagonists and investiga-
tion into their selective late-stage functionalization
Kun Liu, Ravi Kurukulasuriya, Kevin Dykstra, Lisa DiMichelle, Jinchu Liu,
Petr Vachal, Anthony Ogawa, Robert J. DeVita, Dong-Ming Shen, Qiang Tan,
Yili Chen, Don Gauthier, Andreas Verras, Alejandro Crespo, Beata Zamlynny,
Jeffrey Madwed, Maarten Hoek, Thomas Bateman, Yun-Fang Yang, K.N.
Houk, Shane Krska, Tim Cernak
PII:
S0960-894X(19)30244-6
https://doi.org/10.1016/j.bmcl.2019.04.024
BMCL 26391
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 January 2019
11 April 2019
13 April 2019
Please cite this article as: Liu, K., Kurukulasuriya, R., Dykstra, K., DiMichelle, L., Liu, J., Vachal, P., Ogawa, A.,
DeVita, R.J., Shen, D-M., Tan, Q., Chen, Y., Gauthier, D., Verras, A., Crespo, A., Zamlynny, B., Madwed, J., Hoek,
M., Bateman, T., Yang, Y-F., Houk, K.N., Krska, S., Cernak, T., Development of indazole mineralocorticoid
receptor antagonists and investigation into their selective late-stage functionalization, Bioorganic & Medicinal
Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.04.024
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Development of indazole mineralocorticoid
receptor antagonists and investigation into
their selective late-stage functionalization
Leave this area blank for abstract info.
Kun Liu, Ravi Kurukulasuriya, Kevin Dykstra, Lisa DiMichelle, Jinchu Liu, Petr Vachal, Anthony Ogawa,
Robert J. DeVita, Dong-Ming Shen, Qiang Tan, Yili Chen, Don Gauthier, Andreas Verras, Alejandro Crespo,
Beata Zamlynny, Jeffrey Madwed, Maarten Hoek, Thomas Bateman, Yun-Fang Yang, K. N. Houk, Shane
Krska, Tim Cernak*

Bioorganic & Medicinal Chemistry Letters
Development of indazole mineralocorticoid receptor antagonists and
investigation into their selective late-stage functionalization
Kun Liua, Ravi Kurukulasuriyaa, Kevin Dykstraa, Lisa DiMichellea, Jinchu Liua, Petr Vachala, Anthony
Ogawaa, Robert J. DeVitaa, Dong-Ming Shena, Qiang Tana, Yili Chena, Don Gauthiera, Andreas Verrasa,
Alejandro Crespoa, Beata Zamlynnya, Jeffrey Madweda, Maarten Hoeka, Thomas Batemana, Yun-Fang
Yangc, K. N. Houkc, Shane Krskaa, Tim Cernaka, b

aMerck & Co., Inc., Rahway, NJ, USA, 07065
bPresent address: University of Michigan, Department of Medicinal Chemistry, Ann Arbor, MI, 48109
cUniversity of California at Los Angeles, Department of Chemistry and Biochemistry, Los Angeles, CA, 90095
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
The derivatization of pharmaceuticals is a core activity in the discovery and development of new
medicines. Late-stage functionalization via modern C−H functionalization chemistry has emerged
as a powerful technique with which to diversify advanced pharmaceutical intermediates. We
report herein a case study in late-stage functionalization towards the development of a new class
of indazole-based mineralocorticoid receptor antagonists (MRA). An effort to modify the
electronics of the core indazole heterocycle inspired the use of modern C−H borylation chemistry.
New reactivity patterns were revealed and studied computationally. Ultimately, a de novo
synthesis delivered a key 6-fluoroindazole compound 26, a potent MRA with excellent metabolic
stability.
Keywords:
Late-stage functionalization
Mineralocorticoid receptor antagonists
C−H functionalization
2009 Elsevier Ltd. All rights reserved.
C−H borylation
High-throughput experimentation
Mineralocorticoid receptor antagonists (MRAs) have a long
history as anti-hypertensive agents dating back to the marketing of
spironolactone in 1959 and eplerenone in 2002. More recently, the
RALES clinical trial demonstrated that chronic heart failure
patients taking spironolactone had prolonged lives with 35% fewer
hospital admissions owing to worsening heart failure compared to
placebo and substantial lower incidence of death.1 These exciting
validation results inspired a renewed interest in MRAs.2 The poor
selectivity of spironolactone against other steroid receptors leads
to several off-target effects such as male gynecomastia, which is a
counter-effect in a typical hypertension treatment regime but may
be a desirable effect in cross-sex hormone therapy of transgender
patients.3 We were interested in developing non-steroidal MRAs
with improved selectivity against related hormone receptors. We
have previously reported on a reverse indole lead class of MRAs
exemplified by 1, which demonstrated robust efficacy in a cellular
assay and in in vivo models.4 This letter details further
development of this series and documents an early case study from
the Late Stage Functionalization group at Merck & Co., Inc.
Reverse indole 1 was previously reported to be a potent MRA
with in vivo efficacy comparable to the marketed MRA
spironolactone and 54 nM half-maximal inhibition (IC50) of
mineralocorticoid receptor activity in vitro. We sought to further
develop this lead for several reasons. First, incubation with rat and
human microsomes5 led to significant oxidative metabolism of the
electron-rich indole core as determined by HRMS/MS2
fragmentation analysis (Figure 1). We were concerned with the
potential of these oxidized metabolites (2) to engage in
unfavorable off-target interactions and reasoned that the
pharmacokinetic profile of 1 might be improved if this oxidative
metabolism could be blocked. Additionally, compounds in this
series exhibited poor to modest selectivity in gel filtration binding
studies over related nuclear hormone receptors such as the
glucocorticoid receptor (GR).6 Moreover, chemical instability
upon irradiation by UV light, a property which could complicate
the development of the series, presented an additional concern. We
reasoned that the electron-rich nature of the indole ring was a
likely contributor to these deleterious properties and, therefore,
explored opportunities to temper the electronics of the heterocyclic
core to develop structure activity relationships (SAR) around this
promising lead class.
———
 Corresponding author. Tel.: +1-734-615-2178; e-mail: tcernak@med.umich.edu

Figure 1. Metabolism in the reverse-indole series.
Our initial forays into modifying the electronics of the
electron-rich indole core focused on the installation of electron-
withdrawing substituents. Inspection of the predicted binding
mode of indole compound 3, docked using a reported MR x-ray
structure (PDB code: 2A3I),7 suggested that there would be room
in the active site to accommodate small groups (Figure 2) with
which we could attenuate electronics on the heterocycle. Indeed, a
small fluoro group could be installed at the 6-position with only a
modest loss in MRA potency (Figure 2) and with comparable
pharmacokinetic properties (4). Larger groups such as 6-Br (5) and
6-CN (6) were less tolerable and led to a marked reduction in
potency. While a 6-fluoro substituent led to only a slight loss in
MR potency (IC50 = 116 nM versus 54 nM for 3), the selectivity
against GR and the pharmacokinetic profile were effectively
unchanged so other core replacements were investigated.
Figure 3. Modification of the heterocyclic core.
Compounds in the indazole class were prepared by a route
involving conversion of an arylacetic acid (11) into the
corresponding -brominated ester (12) followed by SN2
displacement with 4-nitroindazole (13) (Figure 4). This reaction
typically gave 14 along with a significant amount of an undersired
byproduct, wherein alkylation occurred at N2 instead of N1.
Separation of this undesired byproduct was achieved by column
chromatography on silica gel, and the enolate of the desired
regioisomer (14) was then alkylated with ethyl iodide to give
racemic 15. Reduction of the nitro group and trityl protection of
the resultant amine (16) was followed by a chiral separation using
supercritical fluid chromatography (SFC) to give 17.8 Eventually,
this route was used to produce >50 grams of intermediate 17 for
SAR studies.
Figure 2. Predicted binding pose of indole lead 3 in a homology model (based
on structure 2A3I) of the mineralocorticoid receptor. Profiles of analogs of
reverse-indole lead with 6-substituents are shown.
Another opportunity to modulate the electronics of the ring
presented itself in the conversion of the indole to related
heterocycles (Figure 3), which we explored using the simplified
series exemplified by 7, which has gem-diethyl substituents.
Replacement of the indole ring with an imidazopyridine or a
triazolopyridine led to a marked reduction of in vitro activity
against the mineralocorticoid receptor (8 & 9); however, simple
introduction of an extra nitrogen atom, as in indazole 10, produced
a compound with modest MRA activity (IC50 114 nM). This
indazole series was prioritized for follow up.
Figure 4. Synthesis of key intermediate 17.
Intermediate 17 was used to access compounds such as 18 and
19 (Figure 5). The profile of these indazole compounds was better
than that of 2, with comparable MR potency, enhanced binding
selectivity over GR, and reduced clearance. For 19, the half-life
(t1/2) in rats was 5.7 hours, which marked a significant step towards
our desired profile. While availability of 17 enabled the
preparation of diverse analogs, such as 18 and 19, we sought more
synthetic flexibility in our chemical space explorations. Our

interest in modulating the electronic nature of the core heterocycle
by installing substituents at the 6-position, as in 4, led us to target
C–H functionalization at C6. However, functionality at this
position would need to be installed in the first step of the current
synthetic route. While such a de novo route was expected to be
successful, given the large batch of 17 we had in hand, the potential
for modifying the C−H bonds of 17 via emerging late stage
functionalization techniques was indeed tempting.9 Importantly,
this work on MRA’s was contemporaneous with the formation of
a Late Stage Funcationalization (LSF) Group at Merck & Co., Inc.
that aimed to explore the reach of C−H functionalization in drug
discovery. Presented herein is the first reported LSF case study
from our drug discovery pipeline. While we ultimately achieved
our goals through de novo synthesis (vida infra), some peculiar
reactivity patterns were observed and studied. These early studies
in C−H functionalization on high complexity intermediates such
as 17 launched additional LSF efforts at Merck & Co., Inc.
Figure 6. C–H borylation of 20 occurs at C3, then C6. Reaction conditions:
a. [Ir(OMe)(cod)]2, dtbpy, B2pin2, CyH, 70 °C, 79% yield.
We initially sought to explore ligand effects in this
transformation and to learn if bis-borylation could be mitigated.
Using miniaturized high-throughput experimentation (HTE),17 we
executed a screen of 4 solvents × 6 ligands (Figure 7)18 which
demonstrated that clean monoborylation was only achieved at low
conversion under the conditions tested, again exclusively at the 3-
position. The 3,6-bis-borylated product was observed as a major
component in reactions where conversion went to completion or
near completion. We therefore elected to explore if we could
completely bis-borylate 20 to give 22 and selectively proto-
deborylate at the 3-position.
Figure 5. Profile of 18 and 19.
To explore the possibility of achieving
a late-stage
functionalization on 20, the trifluoromethyl analog of 17, we chose
to investigate the possibility of performing a sterically-guided
borylation.10 The pinacol-boronate esters that would result from
this transformation could then be converted into other functional
groups such as cyano,11 chloro/bromo,12 fluoro,13 or small alkyl
groups14 for example. Our labs, in collaboration with the Smith
and Malezcka groups,15 and others16 have demonstrated the utility
of iridium-catalyzed borylation methods in heterocycle
functionalization. Therefore, 20 was subjected to iridium-
catalyzed borylation using 4,4-di-tert-butyl-2,2-dipyridyl (dtbpy,
L1) as ligand and cyclohexane as solvent. A mono-borylated
product and bis-borylated product were isolated from the reaction
screen and their structures determined by NMR. We anticipated
that borylation would occur cleanly at the desired 6-position of the
indazole since it bears the most sterically accessible C−H bond, but
were surprised to observe that the mono-borylated product had the
Bpin in the 3-position (21), proximal to the nitrogen of the
indazole. The bis-borylated product had Bpin groups installed
again at the indazole 3-position and at the desired 6-position (22).
Figure 7. High-throughput experimentation enabled by a C–H borylation
“kit”.
In collaboration with the Smith and Malezcka groups, we had
developed a protocol for achieving selective deborylation of poly-
borylated products using catalytic bismuth(III) acetate and
methanol,19 Movassaghi and coworkers reported similar findings
using acetic acid,20 and Steel and colleagues demonstrated
deborylation of indazoles using KOH.21 From this precedent, we
anticipated 22 could be selectively deborylated to arrive at the
desired 6-borylated product. Starting from 500 mg of 20, we were
able to isolate 80% of the bis-borylated material starting when

dtbpy was used as the ligand and CPME was used as the solvent
with 2.0 equivalents of B2pin2. Indeed, 22 could be cleanly 3-
deborylated to give the desired 6-borylated product 23 in 80%
yield simply by adding a large excess of methanol and heating in
CPME (Figure 8). While this procedure proved fruitful as a route
to secure 23, we were unable to achieve successful transformation
of boron to fluorine.22 As described below, we ultimately secured
24, and subsequent analogs, by de novo synthesis. Nonetheless,
this example served as an important first foray in applying late-
stage functionalization to the Merck & Co., Inc. pipeline, and the
HTE array of C−H borylation conditions developed for Figure 7
proved applicable to many subsequent programs.
explanation is that the NHBoc group serves as a directing group to
guide borylation to C3 in 20. We have also found an alternative
Figure 9. Calculated transition states in the C-H borylation of an indazole.
pathway for C3-borylation, which does not rely on this hydrogen
bonding interaction. The N−H bond is weaker than the C−H bond,
and it can be cleaved via TS3 to form an N-coordinated iridium
complex. The subsequent oxidative addition of C−H bond at the
C3-position via TS4 has a barrier close to TS1. There is only 0.4
kcal/mol difference between these two pathways for C3-
borylation. Therefore, both mechanisms may operate in this
system and are consistent with the experimentally observed C3-
regioselectivity.
Parallel to our efforts to fluorinate the 6-position through late-
stage functionalization, we had completed the installation of the 6-
fluoro substituent via de novo synthesis using a route similar to
that shown in Figure 4.25 Compounds 25 and 26 were prepared by
this route (Figure 10). As expected based on precedent from the
indole series, the 6-fluoro substitution was tolerated on the
indazole core with MR potency effectively unchanged between 19
and 25 and only a two-fold loss in potency for diastereomeric
compound 26. For diastereomer A, the pharmacokinetic profile
was somewhat less favorable than 5 but for diastereomer B, the
pharmacokinetic profile was very similar with slightly reduced
clearance in the 6-fluoro compound. As we had set out to reduce
the oxidative metabolism of the lead by tempering the electronics
of the core heterocycle, and investigated the power of late-stage
functionalization to achieve this, it was most encouraging to note
that the installation of a 6-fluoro substituent indeed reduced the
amount of observed decomposition in 30-minute incubations with
human liver microsomes (52% remaining for 19, X = H; versus
84% remaining for 26, X = F).
Figure 8. Selective borylation of 22. Fluorination of 23 was unsuccessful.
Reaction conditions: a. MeOH, CPME, 50 °C, 18 h, 80% yield; b. ref 21.
The observation of borylation at the 3-position in preference to
the desired 6-position of 20 was an unexpected result. Several
studies have shown that while the regioselectivity of iridum-
catalyzed borylation of the C−H bonds of substituted benzene rings
is typically governed by steric effects, the regioselectivity of
borylation on heterocycles can be more complicated and an
interplay of directing, electronic and steric factors is involved. In
2015, Steel and colleagues showed that alkylated indazoles can
undergo C3 borylation, but the work presented herein predated
publication of those results. Houk and Merlic have applied the
distortion-interaction model to describe the observed
regioselectivity patterns for a variety of substrates.23 We applied
DFT calculations24 to the current system in an effort to understand
the unexpected borylation at the 3-position. Figure 9 shows the
oxidative addition transition structures for the reaction. TS1 and
TS2 are the oxidative addition transition structures for C3-
borylation and C6-borylation, respectively. TS1 is more favorable
than TS2 by 3.7 kcal/mol. In TS1, the hydrogen bond interaction
between the NHBoc at the 4-position and oxygen of Bpin is
beneficial for the oxidative addition of C−H bond at C3-position,
while TS2 lacks this favorable interaction. Therefore, our current
We report herein the development of a new indazole series of
potent antagonists of MR with good to excellent pharmacokinetic
profiles in rat. A major focus of our explorations was the
development of selective iridium catalyzed C−H borylation of a
key intermediate, 17, which we had prepared in bulk. Notably, an
unanticipated regioselectivity was observed in the borylation step,
however, through
a sequence of bis-borylation / proto-
deborylation we arrived at the selective functionalization of the
desired C−H bond. Two energetically similar transition states were
located in DFT studies to explain the unexpected borylation at the
3-position. One transition state invokes an internal H-bond
between a free N−H on the heterocycle and an oxygen on the active

Figure 10. Profile of fluorinated compounds 25 and 26.
catalyst structure while the other transition state suggests a
surprising formation of an N-iridium bond. This novel N-Ir
binding arrangement could be useful in future modes of catalysis.
Finally, a series of indazole and 6-substituted indazole and indole
leads were developed as potent MRAs with potential to treat
hypertension and chronic heart failure.
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18. HTE reaction screening equipment was purchased from Analytical Sales
& Services. A 24-well aluminum reactor plate (catalog # 24253) was
equipped with 1 mL clear glass shell vials (catalog # 84001) then moved
to the glove box. A 10 mM stock solution of [(cod)Ir(OMe)]2 in THF (50
L / vial), and 20 mM stock solutions of the ligands (a 40 mM stock
solution was used for monodentate ligand L2) in THF (50 L / vial) were
added to the desired vials. Solvents were removed in vacuo with a
Genevac in the glovebox. A parylene-coated stir dowel (catalog # 13258)
was then added to each vial, and 100 L of a solution of 20 and B2pin2 in
the solvent noted was added to respective vials. The system was sealed
in the glovebox, then stirred at 80 ˚C for 14 hours. Each reaction was
quenched with 1 mL of 9:1 acetonitrile–methanol (1 mL), stirred 20
minutes, then diluted 10-fold into 8:1:1 acetonitrile–methanol–water
containing biphenyl as an internal standard, and analyzed by UPLC-MS
to obtain the UV (220 nm) peak area percentages reported in Figure 7.
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6.
7.
The functional activity of most compounds in this series in a cellular
assay of GR activity was >10 uM despite high measured affinity to a
truncated GR receptor. This disconnect between a binding affinity assay
and a cellular function assay was never fully resolved so we sought to
reduce GR binding to mitigate any risk of off-target activity.
Putative design structures were modeled based on docking experiments
performed starting from the 1.95
Å MR structure with bound
corticosterone (PDB code 2A3I: Li, Y., Suino, K., Daugherty, J., Xu, H.
E. Mol. Cell, 2005, 19, 367). Structures were prepared by removal of
water molecules, addition of hydrogens, and restrained energy
minimization using Macromodel (Macromodel 9.8.107, Schrödinger
LLC, New York, NY). Molecular docking was performed by employing
our in-house docking routine FLOG (Miller, M. D., Kearsley, S. K.,
Underwood, D. J., Sheridan, R. P. J. Comput. Aided Mol. Des. 1994, 8,
153), which ranked precalculated conformations of the target molecules
in a 6 Å grid centered on the crystallographic ligand. Conformations were
generated using our in-house metric matrix distance geometry algorithm
JG (Kearsley, S. K. Merck & Co., Inc., unpublished). The conformations
were subjected to energy minimization with Macromodel using the
MMFFs force field (Halgren, T. A. J. Comput. Chem. 1999, 20, 720). A
representative docking pose (data not shown) was selected by visual
inspection and was subjected to restrained energy minimization (using
Macromodel) to produce a putative model.
8.
Synthetic procedures for most compounds presented herein appear in: T.
A. Cernak, K. D. Dykstra, D.-M. Shen, K. Liu, A. Stamford, J. Qiang
Tan. “Preparation of indazole derivatives as mineralocorticoid receptor
antagonists for the treatment of aldosterone-mediated diseases”, PCT
International Application, 2014, WO2014014794.

was used for Ir, and the 6-31G (d) basis set was used for other atoms.
Frequency analysis was conducted at the same level of theory to verify
that the stationary points are minima or saddle points. The single-point
energies and solvent effects were computed with the more accurate M06
/SDD -6-311+G(d,p) method (density functional/basis sets for Ir and
other atoms). The SMD solvation model was for cyclohexane solvent.
The relative Gibbs free energies (at 298.15 K) are given in kcal/mol.
Computed structures are illustrated using CYLView.
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Movassaghi, J. Org. Chem., 2014, 79(22), 11254–11263.
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25. The de novo synthesis procedure appears in ref. 8.
24. All the calculations were carried out with Gaussian 09. Geometry
optimizations were performed with B3LYP. The LANL2DZ basis set