Identification of spirooxindole and dibenzoxazepine motifs as potent mineralocorticoid receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2016.02.014

Mineralocorticoid receptor (MR) antagonists continue to be a prevalent area of research in the pharmaceutical industry. Herein we report the discovery of various spirooxindole and dibenzoxazepine constructs as potent MR antagonists. SAR analysis of our spirooxindole hit led to highly potent compounds containing polar solubilizing groups, which interact with the helix-11 region of the MR ligand binding domain (LBD). Various dibenzoxazepine moieties were also prepared in an effort to replace a known dibenzoxepane system which interacts with the hydrophobic region of the MR LBD. In addition, an X-ray crystal structure was obtained from a highly potent compound which was shown to exhibit both partial agonist and antagonist modes of action against MR.

Full text of DOI:10.1016/j.bmc.2016.02.014

Accepted Manuscript
Identification of Spirooxindole and Dibenzoxazepine Motifs as Potent Minera-
locorticoid Receptor Antagonists
Stephen D. Lotesta, Andrew P. Marcus, Yajun Zheng, Katerina Leftheris, Paul
B. Noto, Shi Meng, Geeta Kandpal, Guozhou Chen, Jing Zhou, Brian
McKeever, Yuri Bukhtiyarov, Yi Zhao, Deepak S. Lala, Suresh B. Singh, Gerard
M. McGeehan
PII:
S0968-0896(16)30096-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.02.014
BMC 12816
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
18 December 2015
1 February 2016
8 February 2016
Please cite this article as: Lotesta, S.D., Marcus, A.P., Zheng, Y., Leftheris, K., Noto, P.B., Meng, S., Kandpal, G.,
Chen, G., Zhou, J., McKeever, B., Bukhtiyarov, Y., Zhao, Y., Lala, D.S., Singh, S.B., McGeehan, G.M.,
Identification of Spirooxindole and Dibenzoxazepine Motifs as Potent Mineralocorticoid Receptor Antagonists,
Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.02.014
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
Identification of Spirooxindole and Dibenzoxazepine Motifs as Potent
Mineralocorticoid Receptor Antagonists
*
Stephen D. Lotesta, Andrew P. Marcus, Yajun Zheng, Katerina Leftheris, Paul B. Noto, Shi Meng, Geeta
Kandpal, Guozhou Chen, Jing Zhou, Brian McKeever, Yuri Bukhtiyarov, Yi Zhao, Deepak S. Lala, Suresh
B. Singh, Gerard M. McGeehan
Vitae Pharmaceuticals, 502 West Office Center Drive, Fort Washington, Pennsylvania 19034, United States
*
Corresponding author. Tel.: +1 215-461-2014. E-mail address: slotesta@vitaerx.com (S.D. Lotesta). Fax: +1 215-461-2006
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Available online
Mineralocorticoid receptor (MR) antagonists continue to be a prevalent area of
research in the pharmaceutical industry. Herein we report the discovery of various
spirooxindole and dibenzoxazepine constructs as potent MR antagonists. SAR
analysis of our spirooxindole hit led to highly potent compounds containing polar
solubilizing groups, which interact with the helix-11 region of the MR ligand binding
domain (LBD). Various dibenzoxazepine moieties were also prepared in an effort to
replace a known dibenzoxepane system which interacts with the hydrophobic region
of the MR LBD. In addition, an X-ray crystal structure was obtained from a highly
potent compound which was shown to exhibit both partial agonist and antagonist
modes of action against MR.
Keywords:
Mineralocorticoid Receptor
MR
MR Antagonist
Spirooxindole
Dibenzoxazepine
©
2015 Elsevier Ltd. All rights reserved.
medicine, spironolactone suffers from a lack of selective binding
to MR leading to a variety of unwanted side effects such as
gynecomastia/feminization in men and menstrual irregularities in
women. The more current MR antagonist, eplerenone, also
known by its trade name Inspra®, was brought to market by
Pfizer in 2002. Although this newer steroidal medicine does
possess good selectivity to the MR LBD, it suffers from a lack of
potency and is therefore taken orally twice daily. There is a clear
unmet medical need for new MR antagonists without harmful
side effects due to poor selectivity while maintaining high levels
1
. Introduction
The mineralocorticoid receptor (MR), one of the 48
members of the nuclear hormone receptor (NHR) superfamily, is
responsible for regulating gene expression. Aberrant activation of
MR leads to a variety of medical conditions such as
cardiovascular disease, chronic kidney disease and hypertension.
Discovered over 50 years ago, aldosterone is considered the
major endogenous ligand for MR. Thus, elevated levels of
aldosterone ultimately cause an increase in blood pressure
through the inhibition of natriuresis leading to elevated levels of
3
of potency for better dosing regimens and patient compliance.
1-2
sodium in the blood.
MR is part of an NHR subfamily commonly referred to
as the estrogen receptor-like family which consists of estrogen
(ER), glucocorticoid (GR), progesterone (PR) and androgen (AR)
receptors. The ligand binding domains (LBDs) of GR and AR are
structurally homologous to the LBD of MR. These high structural
homologies present a challenge in developing motifs that
selectively bind to MR in the LBD in order to circumvent any
potential side effects resulting from other estrogen receptor-like
interactions.
To date, only two steroidal MR antagonists,
spironolactone (1) and eplerenone (2), are marketed as drugs and
used for the treatment of heart failure and hypertension (Figure
Figure 1. Marketed MR antagonist drugs
1). Discovered over 50 years ago and still a widely used

A variety of non-steroidal MR antagonists have been
mode of tanaproget with the progesterone receptor (PR) as
revealed in the crystal structure (PDB code, 1ZUC). If the
proposed binding modes for compounds 3a and 4a are correct,
6
disclosed in the patent and journal literature over the past ten
4
years (Figure 2). The first crystal structure of a nonsteroidal MR
antagonist (4b) in the MR LBD was elucidated just three years
one
4b
ago by a team from Takeda. Other chemotypes have also been
4c
disclosed including compound 3b from Eli Lilly as well as
compounds 5 and 6 which were discovered by Dainippon
4d
4e
Sumitomo and Novartis, respectively. In addition, Bayer’s
BAY-94-8862, also known as Finerenone (6b), is currently in
clinical trials as an MR antagonist for the treatment of heart
4
i
failure. Of the several structural motifs in the literature
possessing MR antagonist activity, we were particularly drawn to
compounds 3-6 as a starting point for our efforts. Utilizing
4h
Contour®, our structure-based design platform , as a molecular
modeling tool, our goal for this project was to develop novel,
potent and selective nonsteroidal MR antagonists.
When Vitae initiated the MR project, there were ten
liganded MR structures in the PDB (Protein Data Bank), all of5
which were steroid ligands that bound very similarly to MR.
However, in the structure of the S810L mutant of MR with bound
Figure 3. The proposed binding mode for compound 3a.
5d
spironolactone (PDB code, 2OAX), the Met-852 sidechain
swings away, creating a new pocket to accommodate the
thioester sidechain of spironolactone. We hypothesized that this
new pocket could be useful for optimizing off-target selectivity.
Therefore, 2OAX was used to model various molecular designs.
First, we docked two known non-steroidal MR antagonists from
4f
4a
the literature: compound 3a and 4a (Figure 2).
Figure 4. The proposed binding mode for compound 4a.
Figure 2. MR antagonists from literature and patents
Figure 5. Hybrid structures 3c and 4c
The proposed binding modes based on the molecular
modeling for these two compounds are shown in Figures 3 and 4.
These binding modes appeared to be consistent with the binding
can imagine hybrid structures 3c and 4c (Figure 5) as potential
ligands for MR. To test this hypothesis, compound 4c was
synthesized and was found to bind to MR with a K of 390 nM
i

and showed inhibition of MR activity in a cellular Gal4 assay
IC50 = 889 nM), suggesting the validity of our proposed binding
model. Moreover, the binding mode of 4a was subsequently
(
4b, g
confirmed crystallographically by Takeda.
To generate ideas, we computationally grew various
structures to fill the ligand binding pocket starting from the
benzoxazinone fragment (Figure 6). One of the best scoring
structures generated had an aromatic ring attached to the
benzoxazinone moiety through a two-atom linker as shown in
Figure 7. Interestingly, 2,4-difluorophenyl compound 7 (Figure
7
6
) which came out of this analysis was quite similar to compound
(Figure 2) claimed in an earlier Novartis patent.
4e
Figure 6. Growth from benzoxazinone moiety.
Figure 8. Modifications of the Hydrophobic Region. Efficacy
levels for compounds 7, 12-18 were 100%.
Therefore, various bifurcated constructs were identified
and synthesized in order to fill more space in the hydrophobic
pocket in an attempt to improve the binding affinity. From this
analysis, compound 16 exhibited a binding potency of 55 nM
which was roughly a 3- to 5-fold improvement from 13 and 14
but was still 3-fold less potent than 7 and suffered a significant
shift in the cellular assay. Initially, more polar aniline motifs,
such as 17, showed promising data with virtually no shift when
comparing the binding and cellular potencies. However, larger
substituents on the amine (18) as well as sulfonamide
incorporation (19) led to diminished potencies.
Figure 7. Development of phenethyl scaffold from Contour®
2. Chemistry and SAR Analysis
Compound was prepared in two steps from
7
commercially available bromobenzoxazinone 8 via Sonogashira
coupling followed by hydrogenation to give this target which
exhibited good binding and cellular potency in our MR assays
By comparing compound 3b (Figure 2) with the
phenethyl scaffold, it became clear that one of the fused benzene
rings of the tricyclic dibenzoxepane moiety was filling the Met-
(Figure 8). SAR analysis of compound 7 was performed in an
attempt to gain insight into the effects of changing the phenethyl
sidechain shape in the hydrophobic pocket of the MR LBD where
the Met-845, Met-852 and Cys-849 reside. Interestingly, more
rigid templates housing either an olefin or alkyne resulted in a 7-
to 13-fold loss in binding potency (12-14). Apparently, the
conformational flexibility of the saturated phenethyl sidechain
was more compatible with the ligand binding pocket giving rise
to its tighter binding affinity. It is also interesting to note that
incorporation of an oxygen into the phenethyl sidechain (15)
resulted in a ~50-fold loss in potency compared to 7, presumably
due to the conformational preference of phenethyl versus
phenoxymethyl.
852 pocket. We decided to combine the tricyclic system
contained in compound 3b with the Asn-770 interacting
benzoxazinone scaffold seen in 4a. Hence, coupling of
4c
compound 8 with known boronate ester 20 gave compound 21
which exhibited excellent binding and cellular potency (Scheme
1). Although this compound had exquisite potency, it was shown
to behave as both a partial agonist as well as a partial antagonist.

benzoxazinone cis-amide moiety and the amide side chain of
Asn-770, in addition to the hydroxyl group of Thr-945.
Moreover, MR bound with 21 existed as a trimer in the crystal
form. The overall conformations observed were very similar
among the three monomers except for minor differences in the
side chain conformations as well as small differences in the
intermolecular hydrogen bonding distances.
A 6-nanosecond molecular dynamics simulation was
performed on MR with bound 21 to probe the conformational
flexibility of the complex. The overall structure was quite stable
during the course of the simulation. The complex appeared to
oscillate between two main ensembles of conformations: one
with and one without the hydrogen bonding between the carbonyl
oxygen of 21 and the OH of the Thr-945, while both had the Asn-
770 interaction.
Scheme 1. Discovery of potent partial MR antagonist 21. PXPd2
dichloro[di-tert-butyl(chloro)phosphine] palladium(II) dimer
-
Figure 10. Modifications of the hydrophilic region
Although compound 21 suffered from a lack of
7
selectivity, in addition to CYP (cytochrome P450) and off-target
liabilities, we decided to explore other Asn-770 interacting
scaffolds to try and improve upon these areas while maintaining
high levels of potency. Thus, other bicyclic systems were also
examined with tricyclic scaffold 20 which exhibited reasonable
levels of binding and cellular potencies (Figure 10, 22-25).
However, all of these compounds suffered from a lack of
selectivity to AR, PR, GR and ER. Based on these results, we
speculated that the pyrrolo-oxazine substituent in compound 3b
was necessary for achieving high levels of selectivity as well as
improving the physical properties of the molecule.
Therefore, we explored the feasibility of preparing
compounds with substitution directed towards the helix-11 region
of the MR LBD, similar to that of the pyrrolo-oxazine substituent
in compound 3b. Ideally, a polar substituent with functionality
amenable to rapid SAR was preferred. This led us to consider an
oxindole construct bearing a spirocyclic piperidine due to its
intrinsic polarity in addition for the opportunity to perform
various derivatizations of the amine functionality. Thus, Suzuki
coupling of commercially available spirooxindole 26 with 20
gave compound 27 which possessed not only excellent binding
and cellular potency but better selectivity against AR, PR, GR
and ER (Scheme 2). Although this compound still suffered from
Figures 9. The binding pose of 21 in the MR ligand binding
pocket. The electron density map for 21 is contoured at 1.0 σ
(PDB code 5HCV). The hydrogen bonds between 21 and the
Asn-770 and Thr-945 residues are indicated along with a
ChemDraw rendering of the crystal structure.
We subsequently obtained an X-ray crystal structure of
MR bound to 21 at 2.5 Å (Figure 9, PDB Code 5HCV). X-ray
structural analysis of compound 21 in the MR LBD clearly
indicated hydrogen bonding interactions between the
7
off-target activities as well as CYP and RLM (rat liver
microsome) issues, the improved selectivity against the NHRs

was encouraging and therefore we sought to overcome these
liabilities through SAR exploration on the nitrogen of the
piperidine ring system.
dimethylamine were tolerated in these molecules without any
9b
significant loss in potency. However, further analysis still
showed some level of off-target activity for the 4 panel PanLabs
7
screen. Subsequently, we found that Eli-Lilly’s compound 3b
(bottom of Figure 11) had a similar profile when tested in our in-
house assays. In an effort to circumvent these off-target issues, a
number of other spirocyclic systems were prepared (35-41,
Figure 12). Although these compounds still maintained high
levels of potency they still suffered from the same off-target
activities.
At this stage, we decided to investigate potential
modifications of the tricyclic system in the hydrophobic region.
Based on earlier efforts, it was clear to us that the tricyclic
dibenzoxepine system was adequately filling the desired space in
the hydrophobic region leading to high levels of potency.
Therefore, we investigated various tricyclic heterocyclic motifs
which would mimic the space filling ability of the dibenzoxepane
system but provide more polarity. We reasoned that an increase
in polarity could lead to a better off-target profile.
Scheme 2. Discovery of potent spirooxindole compound 27
Consequently, benzyl protected spirooxindole 29 was
prepared in one step from commercially available bromo-8
oxindole 28 in excellent yield on a multi-gram scale (Scheme 3).
Suzuki coupling of 29 with 20 gave compound 30 followed by
9
benzyl deprotection using 1-chloroethyl chloroformate afforded
unprotected piperidine 31 as the HCl salt. Various modifications
of the piperidine were performed introducing a variety of polar
substituents. This SAR analysis led to the identification of three
compounds (32-34, Figure 11) which showed good binding and
cellular potencies as well as excellent selectivities (400 to >1000
fold) against GR, PR, and ER. In addition, these compounds had
reasonable CYP profiles and excellent HLM stabilities. It is
interesting to note that polar groups such as a carboxylic acid and
Figure 11. Discovery of lead compounds in spirooxindole series
Scheme 3. Gram scale synthesis of spirooxindole amine 31

2
bond formation. For benzylic amine substrate 48, Pd(OAc) -
catalyzed conditions employing XPhos with NaOt-Bu as the base
gave a smooth conversion to tricyclic dibenoxazepane system 49
in 50% yield. Alternatively, sulfonamide substrate 50 underwent
2 3
clean and rapid cyclization to 51 using CuI with K CO as the
base. Although these compounds suffered from a loss in potency,
we were encouraged by the off-target profile of sulfonamide 51.
Thus, incorporation of a sulfonamide in the hydrophobic region
seemed to alleviate the previous off-target liabilities seen in
compound 21.
Figure 12. Analogs and modifications of the spirooxindole
hydrophilic region
We
were
immediately
drawn
to
various
dibenzoxazepane systems of type 43 (Figure 13). Although there
1
0
is literature precedence for such motifs, installation of such
constructs into our spirocyclic scaffolds did not seem like a
trivial task. Indeed, this hypothesis was confirmed when
attempted couplings of either sulfonyl or acid chlorides 44
(bottom of Figure 13) with various tricyclic systems (45) suffered
from very low conversions (<5%) to the desired sulfonamides
and amides (46). Alternatively, we investigated the feasibility of
a late-stage 7-membered ring cyclization event to form the
dibenzoxazepane system. Since intramolecular C-N bond
11
formations are known for medium-size rings , we thought that
this was a reasonable strategy to pursue.
Scheme 4. Synthesis of dibenzoxazepanes via 7-membered
cyclization
Figure 13. Modification of the dibenzoxepane system
Thus, coupling of known aniline 47 with commercially
available benzoxazinones 10 and 9 gave benzylic amine 48 and
sulfonamide 50 respectively (Scheme 4). Various conditions
were investigated for the 7-membered ring cyclizations via C-N
We decided to prepare our previously explored
spirooxindole templates with the dibenzoxazepane scaffolds in
the hydrophobic region (Scheme 5). Thus, bromide 29 was

converted to the corresponding benzylic thioether via Pd-
conversion to the corresponding aldehyde followed by reductive
amination and Pd-catalyzed ring closure to afford tricylic amine
system 56 after benzyl deprotection. However, this compound
also suffered from a significant loss in potency when compared
to that of 31.
catalyzed coupling to give compound 52 in 64% yield. An
1
2
oxidation/chlorination sequence was employed using chlorine
gas in the presence of aq. AcOH to give sulfonyl chloride 53
which was taken without purification and coupled with known
aniline 47 to give sulfonamide substrate 54 in 21% yield from
thioether 52. Cyclization of 54 proceeded without incident to
give compound 55 after benzyl deprotection.
Although we had not explored the amide construct
(Figure 13, 46, R = C=O) for the benoxazinone system
containing the new tricyclic motif, we reasoned that this
cyclization could also proceed as smoothly as the transformation
from 50 to 51. Thus, carboxylic acid 58 was prepared in excellent
yield from bromide 57 using a Pd-catalyzed carbonylation
method employing Mo(CO)
(
6
as the carbon monoxide source
Scheme 6) . HATU-promoted coupling of 58 with known
aniline 47 gave 59 which was subjected to the previously used
CuI/K CO conditions to give 60 after benzyl deprotection.
1
3
2
3
However, this compound also suffered from a significant loss in
potency compared to that of 31. Based on this SAR analysis, it
appeared to us that these types of compounds housing the more
polar dibenzoxazepane ring system in the hydrophobic pocket
were adopting a different conformation compared to that of 42
(Figure 13). However, these conformational differences are not
definitive based on our models and we can only hypothesize that
this is the reason for the substantial loss in potency.
Scheme 6. Synthesis of dibenzoxazepine 60
4d
A group from Sumitomo reported in a patent
compound 5 (Figure 2), for which we modeled its sulfonamide in
a similar region of the MR LBD. It also exhibited a para
relationship between the sulfonamide and the Asn-770
interacting NH group. The cyclic thiocarbamate motif of 5 was
then incorporated with our tricyclic sulfonamide scaffold.
Conversion of commercially available bromide 61 proceeded in
high yield to the corresponding thioether (62, Scheme 7).
Oxidation/chlorination sequence followed by sulfonamide
formation with 47 gave compound 63 in 81% yield from 62.
Similar to our earlier conditions, CuI/K CO -promoted
Scheme 5. Synthesis of dibenzoxazepines 55 and 56
Unfortunately, comparison of 55 with earlier
spirooxindole compound 31 (Scheme 3), showed close to a 1000-
fold loss in potency. The benzyl amine analog of 55, analogous
to compound 49, was also prepared from bromide 29 via
2
3

cyclization afforded the desired tricyclic motif which was
subjected to Lawesson’s reagent to give final target 64. Although
the cyclic carbamate analog of 63 (not shown) exhibited a
moderate level of potency, thiocarbamate 64 showed a very high
level of binding and cellular potency. Even though this
compound shows moderate a level of selectivity, in addition to
some off-target liabilities and CYP issues, we feel that further
optimizations to 64 (molecular weight of 470 g/mol) could lead
to a more promising series. Based on our results from compounds
Acknowledgements
We would like to thank Angel Morales-Ramos and
Colin M. Tice for reviewing and editing this manuscript. We also
thank the members and staff of BNL’s Protein Crystallography
Research Resource (PXRR) for help using beam lines X25 and
X29. Use of the National Synchrotron Light Source, Brookhaven
National Laboratory, was supported by the U.S. Department of
Energy, Office of Science, Office of Basic Energy Sciences,
under Contract No. DE-AC02-98CH10886.
32-34 (Figure 11), introduction of polar functionality at the gem-
dimethyl region of 64 could potentially circumvent some of these
issues.
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6
.
7
8
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.
For the initial off-target screening assays, the compounds were sent to
PanLabs for a “mini-Pan Labs” screening of 4 targets: Serotonin
5
HT28, Histamine H1, hERG and Adrenergic 2A.
(a) Kyle, D. J.; Mavunkel, B. J.; Chakravarty, S.; Lu, Z. “Pseudo- and
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1
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2
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6
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Supplementary Material
Experimental details for the synthesis of compounds,
biological assays and molecular modeling studies are available
free of charge via the Internet at http://www.sciencedirect.com.

Identification of Spirooxindole and
Dibenzoxazepine Motifs as Potent
Mineralocorticoid Receptor Antagonists
Stephen D. Lotesta, Andrew P. Marcus, Yajun Zheng, Katerina Leftheris, Paul B. Noto, Shi Meng, Geeta
Kandpal, Guozhou Chen, Brian McKeever, Yuri Bukhtiyarov, Yi Zhao, Deepak S. Lala, Suresh B. Singh,
Gerard M. McGeehan
Vitae Pharmaceuticals, 502 West Office Center Drive, Fort Washington, Pennsylvania 19034, United States