Perspective

pubs.acs.org/jmc

Modulating Mineralocorticoid Receptor with Non-steroidal

Antagonists. New Opportunities for the Development of Potent and
Selective Ligands without Off-Target Side Effects
Mercedes Martín-Martínez,*,† Felipe L. Pérez-Gordillo,† Diego Á lvarez de la Rosa,‡ Yoel Rodríguez,§,⊥
Guillermo Gerona-Navarro,∥ Rosario González-Muñiz,† and Ming-Ming Zhou§
†
Instituto de Química Médica (IQM-CSIC), Juan de la Cierva, 3, 28006 Madrid, Spain
‡
Institute of Biomedical Technologies and Department of Physiology, Campus de Ciencias de la Salud, Facultad de Medicina,
Universidad de La Laguna, 38204 Tenerife, Spain
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

§
Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York 10029, United States
⊥
Department of Natural Sciences, Hostos Community College of CUNY, 475 Grand Concourse, Bronx, New York 10451, United
Downloaded via UNIV DE BARCELONA on October 3, 2018 at 01:30:18 (UTC).

States
∥
Department of Chemistry, Brooklyn College, 2900 Bedford Avenue, Brooklyn, New York 11210, United States

ABSTRACT: Steroidal mineralocorticoid receptor (MR)

antagonists are used for treatment of a range of human
diseases, but they present challenging issues of complex
chemical synthesis, undesirable physical properties, and poor
selectivity along with unwanted side effects. Therefore, there is
a great interest in the discovery of non-steroidal ligands able to
bind to the ligand-binding domain of the MR and recruit
different co-regulators to produce tissue-specific therapeutic
effects. Several academic groups and pharmaceutical companies
have been developing a series of non-steroidal ligands that consist of different chemical scaffolds, yielding MR antagonists
currently evaluated in clinical studies for the treatment of congestive heart failure, hypertension, or diabetic nephropathy. The
main focus of this Perspective is to review the reported structure−activity relationships of the different series of compounds, as
well as the structural studies that contribute to a better understanding of the receptor active site and are also helpful for
optimization processes.

■ INTRODUCTION
The mineralocorticoid receptor (MR) is a member of the
nuclear receptor superfamily of transcription factors.1 The main
physiological role of the MR is to transduce the effects of
aldosterone, a steroid hormone produced in the adrenal gland
in response to intravascular volume depletion or hyperkalemia.
Aldosterone interaction with MR initiates homeostatic
responses that oppose the original imbalance. During volume
depletion, aldosterone/MR increases renal NaCl reabsorption
by activation of the epithelial Na+ channel (ENaC) and the
Na+/Cl− co-transporter (NCC). In hyperkalemia, aldosterone-
mediated ENaC activation provides an electrical gradient in the
renal tubule that potentiates K+ and H+ excretion. Consistent
with this fundamental physiological role, inherited gain- or loss-
of-function mutations in MR, ENaC, and their regulatory
proteins cause genetic diseases characterized by disturbances in
blood pressure homeostasis and mineral metabolism.2
In addition, during the past few years it has become apparent
that MR medium- and long-term systemic actions are far wider
than previously thought.3 Regulated MR function is essential
not only to blood pressure and mineral homeostasis but also to
organ and tissue differentiation and morphological and
functional tissue remodeling in health and disease. These
include, but are not limited to, (a) morphological remodeling in
epithelia such as the kidney and distal colon; (b) direct
modulation of cardiac and vascular function and remodeling;
(c) regulation of nervous system activity and its control of the
cardiovascular and renal systems; (d) regulation of energy
metabolism; and (e) immune responses. Underlying the wide
variety of MR physiological and pathological roles are two
factors. First, the MR is widely expressed in different tissues.
Second, glucocorticoids such as cortisol can also act as agonists
of the receptor,1 particularly in those tissues that lack the
enzyme 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2),
which metabolizes cortisol to cortisone, an inactive metabolite.4
As all other members of the steroid receptor family, the MR
displays a modular architecture. The NH2-terminal domain
(NTD) mediates the interaction with transcriptional co-
regulators and displays the highest level of sequence
dissimilarity with other steroid receptors, an essential feature
to determine selectivity in mineralocorticoid responses.
Following the NTD in the MR primary sequence, there is a
central DNA binding domain (DBD) formed by two zinc
Received: July 17, 2016
Published: January 4, 2017

© 2017 American Chemical Society 2629 DOI: 10.1021/acs.jmedchem.6b01065

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Journal of Medicinal Chemistry
fingers. The DBD interacts with specific DNA sequences
named hormone response elements (HREs). The DBD of the
MR is 90−94% identical to DBDs of other steroid receptors
such as glucocorticoid, progesterone, and androgen receptors,
implying that HREs recognized by them are highly similar. A
hinge domain separates the DBD from a COOH-terminal
portion that harbors the ligand-binding domain (LBD) and an
additional region that serves a transcriptional activation
function in a ligand-dependent manner. Steroid receptor
LBDs present moderate identity between them (50−60%),
and while they allow for specific activation by different
hormones, there is some degree of cross-reactivity between
them. For instance, the MR shows the same affinity for
aldosterone and for glucocorticoids such as cortisol or
corticosterone (Kd = 0.5−2 nM), although the half-maximal
effective concentration (EC50) of aldosterone is 100-fold lower,
which has been explained due to a much lower off-rate of
aldosterone from the MR.5 Other steroids such as progesterone
act as antagonists of the MR. High-resolution crystal structures
of the MR LBD in combination with agonists 6,7 or
antagonists6,8,9 are available. In addition, a naturally occurring
mutant that causes pregnancy-related hypertension10 and
converts progesterone into an agonist has also been crystal-
lized.6,11 Therefore, a good deal of information about the
structural determinants involved in ligand binding to the MR
LBD is available.12
For many years, the main focus of drug discovery targeting
the MR has been on the use of its antagonist spironolactone in
the context of primary hyperaldosteronism or as potassium-
sparing diuretics to be used in combination with loop diuretics
and thiazides.13 Spironolactone displays undesired side effects,
particularly producing sexual dysfunction and gynecomastia in
men. In these cases, an alternative is to treat patients with
chemically unrelated drugs such as amiloride or triamterene,
which target a downstream target of the MR, the epithelial
sodium channel (ENaC).13 However, renewed interest in MR
antagonists has been triggered by a combination of new
findings in the last two decades. First, the realization that
aldosteronism is more widespread in the human population
than previously thought indicates that MR antagonism will
probably expand its scope. Second, it has been firmly
established that the MR is expressed outside classic aldosterone
target epithelia and can be considered a ubiquitous receptor.
Pioneering work from Weber’s laboratory using animal models
suggested that the MR was an important mediator in the
development of direct, blood pressure-independent heart
fibrosis and inflammation.14 This prompted a clinical trial
(RALES) where patients with severe heart failure received the
aldosterone antagonist spironolactone at doses that did not
significantly affect blood pressure. The treatment quickly
demonstrated very beneficial effects, with significant decreases
in morbidity and mortality.15 On the other hand, the discovery
of eplerenone, a steroid MR antagonist with improved
selectivity over other steroid receptors and therefore displaying
less sexual side effects, triggered a second large clinical trial,
EPHESUS, which further demonstrated the utility of MR
antagonism.16 Recent studies on non-epithelial aldosterone/
MR effects has expanded the interest in this signaling system to
related pathophysiological settings, such as the vasculature,3
oxidative stress, inflammation, and metabolic diseases related to
obesity. Given the already important and expanding clinical
applications, the obvious question is whether new MR
antagonists are needed.17,18 The main adverse effects of MR
2630
Perspective
antagonists can be divided in two categories. The already
mentioned sexual side effects, due to the close similarity in the
LBDs of MR and related steroid receptors, make it difficult to
develop steroid-derived drugs with high specificity for the MR
over the progesterone or androgen receptors. The second
category of the MR adverse effects can be ascribed to the
undesired interference with aldosterone classical function in
regulating electrolyte homeostasis, particularly in promoting K+
excretion in the kidney tubule. Indeed, it has been observed
that inappropriate dosage of spironolactone can lead to
hyperkalemia, which in turn is a risk factor for cardiac
complications, particularly in patients with renal insufficiency.19
On the other hand, hypokalemia also worsens outcomes in
patients with severe heart failure, and therefore the tendency of
MR antagonists to increase plasma K+ levels may be desirable
under controlled circumstances.20 To overcome these two
types of adverse effects, the focus of MR pharmacology has
shifted toward developing non-steroidal inhibitors that ideally
would have tissue-specific properties. A third generation of MR
antagonists based on non-steroidal compounds is currently
being developed, and some of these products, like the
dihydropyridine-like compound finerenone (see below), are
currently under clinical trials.18,21,22 In this Perspective we
examine the described non-steroidal ligands collected either in
papers or patents. The review aims to be a useful tool for
structure−activity relationship (SAR) studies, and therefore
includes different families of non-steroid ligands able to bind to
the MR, and several compounds within each family.
■ NON-STEROIDAL MINERALOCORTICOID
RECEPTOR LIGANDS
1. 1,4-Dihydropyridine and 1,4-Dihydro-1,6-naph-
thyridinamide Compounds as MR Ligands. 1,4-Dihydro-
pyridines (DHP) represent an interesting scaffold for the search
of non-steroidal MR antagonis and have attracted the interest
of several pharmaceutical companies.
Pfizer, through a screening of its in-house collection,
identified a series of DHPs, previously recognized as calcium
channel blockers (CCBs), as MR antagonists.23 Among them,
nimodipine and felodipine competed for aldosterone binding
and were only slightly less active than eplerenone. As
stereoselective issues affect calcium channel activity, it was
investigated whether the CCB and the MR activities resided in
the same DHP stereoisomer. The study of the CCB-
asymmetric DHP mebudipine (1a) indicated that the 4R
enantiomer was responsible for the MR antagonist activity,
while the 4S enantiomer had the CCB activity (Table 1.1).24 In
agreement with this result, a cyano DHP, identified at Bayer,
showed a preference for the R configuration in its binding to
the MR. However, this was also the more active isomer for L-
type Ca2+ channels.25 Moreover, it was also observed that the
cyano group at R3 position induced high selectivity against
CCB and good selectivity against other nuclear hormone
receptors (NHRs).26 Based on these results, a library of DHP
was prepared to perform SAR studies. Substitution at the DHP
NH group decreased activity, suggesting a potential hydrogen
bond with the MR.26 Similar results were also reported by
Bayer.25 Regarding the aromatic phenyl ring at position 4,
small, nonpolar substituents like F, Cl, or CF3 were suitable at
the ortho or the para position, with a 2-chloro,4-fluorophenyl
moiety giving the best results (1b−1e, Table 1.1).26 A
voluminous R1 group like phenyl or benzyl decreased potency,
while smaller C2−C4 alkyl groups were adequate (1c).
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Table 1.1. Selected Pfizer DHP MR Antagonists and Their
IC50 Values24,26
a increase in potency with respect to the quinoline analogue
Determined using a Gal4/MR LBD (Gal4/LBD) reporter assay in
HUH7 human hepatocyte cells. bChiral resolution of 1e afforded the
R-enantiomer (IC50 = 52 nM).
However, a methylene linker tethering an imidazole, 1d,
triazole or tetrazole, 1e, maintained potency while increasing
selectivity against other NHRs. The tetrazole derivative 1e
showed an in vitro pharmacokinetic (PK) profile adequate for in
vivo studies (CL = 14.1 mL/min/kg, T1/2 = 4.84 h). Compound
1e was able to reduce blood pressure and renal injury in rats in
vivo. Induced fit docking (IFD) studies of this compound
suggested partial overlapping with the native corticosterone MR
binding pocket.26
Bayer Pharma, in an ultra-high-throughput screening of
almost one million compounds, found a single cluster of around
one hundred MR-modulating compounds, including dihydro-
pyridines.27 Among them, 1f was identified as a selective sub-
micromolar MR antagonist (Table 1.2), but it was associated
with a number of liabilities, such as low metabolic stability in
microsomes and significant interaction with L-type Ca2+
channels.25 As previously observed, the R configuration at the
DHP core was preferred, and N-alkylation led to loss of
potency. Other modifications that also decreased potency were
the dehydrogenation of the DHP nucleus to pyridine, the
change of the DHP ring by similar scaffolds as 1,4-
dihydropyrimidines, 1,4-dihydropyridazines, and 3,4-dihydro-
pyrimidin-2-ones, or the attachment of highly hydrophilic
groups to various positions of the DHP core.25 Several
2631
Perspective
Table 1.2. MR Activity of DHP by Bayer Pharma25,29
a
Gal4/LBD reporter assay in CHO-K1 cells. bc-Bu = cyclobutyl.
substituents had been analyzed at C4 position (Table 1.2),
with the fluorenyl moiety B (compound 1g) leading to an
1f.25,28 Subsequent attempts to decrease 4-Ar size and
lipophilicity allowed the identification of chromenone deriva-
tives (1h, Ar = C), at the cost of lower activity.25 In this latter
subseries, the potency could be restored by modulating R1 and
R2 substituents (1i−1m).25,29 Because of the low bioavailability
of 1j, the less lipophilic ethyl ester BR-4628 (R-1k) was
preferred due to its favorable PK profile.25 R-1k, a potent MR
LBD antagonist, 160-fold more selective for the MR over the
AR and with low CCB activity, was considered a drug
candidate. Different from spironolactone and eplerenone, R-
1k retained its antagonist character at the MRS810L mutant. In
vivo assays in rats probed that it increased the urinary sodium/
potassium ratio in a dose-dependent manner, with effect for a
dose as low as 1 mg/kg.27 R-1k also prevented the aldosterone-
induced increased expression of connective tissue growth factor
and hydroxyproline in cardiac fibroblasts, thus suggesting that
the MR could be a biological target for the treatment of fibrosis
in the atrial myocardium.30
The binding mode of DHP antagonists has been studied by
means of induced-fit-molecular docking against available MR-
LBD X-ray structures, such as MR-LBD complexed with
corticosterone (PDB ID 2A3I),7 for derivatives 1b and 1e, or
with deoxycorticosterone (PDB ID 2ABI) to study R-1k.24,26,27
The helix 12 of the MR LBD was truncated, since the binding
pocket was too small to accommodate the branched ligands.
These studies have identified key phamachophore points within
the 1,4-DHP derivatives, namely a hydrogen bond acceptor
(e.g., donor DHP-NH), an aromatic moiety at the 4-position,
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Table 1.3. IC50 of Naphthyridinamide Derivatives on Several Steroid Hormone Receptors
compd R1 R2 R3
1n CN H H
S-1n CN H H
S-1o CN H Me
S-1p CONH2 H Me
a
Functional cell-based transactivation assay. Luciferase assay.
which resides in the known A-ring binding pocket of the steroid
MR antagonist binding mode, and lipophilic moieties at 3- and/
or 5-positions (e.g., alkyl esters). Specifically, the NH group of
the 1,4-DHP ring (e.g., compounds R-1b and R-1e26 and R-
1k27) forms a hydrogen bond with H3 helix Asn770.23 The
hydrophobic ester group fills the α-face pocket formed by
hydrophobic residues Leu814, Leu827, Phe829, Met845,
Cys849, Met852, and Leu938 (e.g., compounds R-1b, R-1e,
and R-1k). On the other hand, substituents at the opposite site,
a methyl and either a CN or COMe group, protrude toward the
β-face pocket (e.g., Ala773, Trp806, Ser810, and Leu 960),
either directly impinging on Leu960 in H12 helix or indirectly
by perturbing Trp806 in H5 helix (e.g., compounds R-1b, R-1e,
and R-1k). The 4-aryl groups occupy the A-ring pocket, and
depending on the DHP derivatives, they interact with Phe829
(e.g., compounds R-1b and R-1e), Arg817, or Gln776 (e.g.,
compound R-1k). The formation of hydrogen bonds between
the DHP derivatives and Arg817 and Gln776 indicates that the
4-aryl groups are equivalent to the steroid C3-carbonyl in the
A-ring moiety. It has also been shown that DHPs partially
overlap with the steroidal skeleton of MR antagonists.
Interestingly, the proposed DHP derivatives binding mode
fails to overlap the entire steroid D-ring region, impeding the
stabilizing contacts with the H11 helix, including Thr945. Thus,
it has been suggested that the incomplete occupancy and
stabilization of the receptor-binding pocket, as well as the steric
clashes caused by the branched DHPs, explain the passive MR
antagonism of these ligands.27 These binding modes have been
supported by site-directed mutagenesis studies.25,27 The high
selectivity has been linked to the fact that the MR is the only
oxosteroid receptor having an alanine residue (Ala773) in the
H3 helix and a serine residue (Ser810) in the H5 helix, while
the AR, GR, and PR have glycine and methionine at their
corresponding positions, respectively.31,32 For example, the
DHP derivative R-1k is able to form contacts with Ala773 and
Ser810, while neither glycine nor methionine is able to make
contacts in AR, PR, and GR, thereby explaining their high
selectivity toward the MR.
The SAR was further extended with a series of heterobicyclic
analogues. Among them, 1,4-dihydro-1,6-naphthyridine deriv-
atives, as 1n, can be considered as conformationally frozen
bioisosteres of 1,4-DHP esters (Table 1.3).5,33 In an effort to
improve selectivity vs other NHRs, a methyl group was
2632
Perspective
IC50 (nM)a
MR GR AR PR
100
47 6900 2800 5400
26 5800 2400 4200
1825 ≥10000 ≥10000 ≥10000
(58)40
introduced at positions 7 and 8 (e.g., 1o).25 Interestingly, the
replacement of the 3-cyano group by a primary amide led to a
potent MR antagonist with remarkable selectivity toward GR,
AR, and PR (finerenone, BAY 94-8862, 1p).25 This compound
has higher selectivity than spironolactone and improved affinity
for the MR than eplerenone. Compound 1p was explored on
65 different enzyme and ion channel assays without significant
effects at 10 μM.25 Clinical studies with 1p suggested that, in
comparison with spironolactone, a lower dose may be at least as
effective in reducing ventricular remodeling, with lower
incidence of hyperkalemia and renal adverse events.34 More
recently it was shown that it was able to protect rats from
cardiorenal injury.35 These findings provided impetus for
further clinical evaluation of 1p, which entered in phase II
studies for the treatment of congestive heart failure and diabetic
nephropathy. The latter phase II trial focused on the effect of
1p on albuminuria in patients with diabetic nephropathy
receiving an angiotensin-converting enzyme inhibitor or an
angiotensin receptor blocker. The data showed that admin-
istration of 1p led to an improvement in the urinary albumin-
creatinine ratio, with a dose-dependent reduction at day 90
ranging from 21% to 38% when compared to placebo in the
dosage group of 7.5 to 20 mg/d compared with placebo.36−38
In the second phase II trial, 1p was studied in patients with
worsening heart failure, who also had diabetes mellitus and/or
chronic kidney disease.39 These patients are at higher risk of
developing hyperkalemia, so they are less likely to receive
steroidal MR antagonists. The overall result of these studies
indicated that 1p reduces the levels of pro-B-type natriuretic
peptide (NT-proBNP), as shown by the decrease of over 30%
from baseline found in 38.8% and 34.2% of the patients in the
10 → 20 mg and 15 → 20 mg 1p groups, respectively (initial
dose → up-titrated dose), a similar extent to that of eplerenone.
Moreover, and similar to eplerenone, 1p showed a good safety
profile, with hyperkalemia (serum potassium concentrations
≥5.6 mmol/L) only observed in 4.3% of the patients. This
study also indicated that 10 → 20 mg of 1p is the most suitable
dosage. The promising results of these phase II studies have
allowed the clinical development program for 1p to be
expanded with phase III studies for chronic heart failure and
diabetic kidney disease, as recently announced by Bayer in
September 2015.
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Docking studies of S-1p at the MR LBD X-ray structure Table 2.1. Pyrrole Derivatives as MR Antagonist (IC50 < 500
(PDB ID 2ABI9 devoid of helix 12) indicated a similar binding nM)
mode as the above-mentioned for other DHP derivatives,
forming hydrogen bonds with Asn770, Gln776, and Arg817.40
Moreover, it confirmed the key roles of Ala773 and Ser810 for
the high MR selectivity over the AR, PR, and GR. These
structural studies also found that the binding pocket of the MR
LBD in its agonistic conformation seems to be too small to
accommodate non-steroidal ligands, thereby suggesting that the
DHP-type MR antagonists act as bulky antagonists. This latter
docking model of MR LBD complexed with S-1p was validated
by carrying out point mutations within the binding pocket.
Strikingly, both A773G and S810M mutations led to a dramatic
decrease in the S-1p antagonistic potency. IC50 value increased
respectively by ∼24- and 88-fold for MRWT.40 These results
strongly corroborate that Ala773 and Ser810 favor the selective
binding of S-1p to the MR, while glycine and methionine at
these respective positions, as in the AR, PR, and GR, impair S-
1p binding.
Merck also claimed a number of 1,4-DHPs as MR
modulators. After the analysis of different substituents at
every position of the DHP ring, they discovered several
compounds with sub-micromolar binding affinity (e.g., 1q−1t,
Table 1.4).41 a
Gal4/LBD cell-based reporter assay.

Table 1.4. Binding Affinity of Selected Merck DHPs for the Table 2.2. IC50 Values of 1H-Pyrrole 3-Carboxamide MR
MR Inhibition

a
Binding affinity was determined using a competitive binding assay
with 3H-aldosterone and recombinant rhesus MR.

2. Five-Membered Nitrogen Heterocyclic Rings as

Central Scaffolds in MR Ligands. 2.1. Pyrrole Derivatives.
Exelixis developed a series of compounds with a common
structural pyrrole motif. One of them, CS-3150 (2k), entered in
phase III clinical trials in 2016.42 This work started from a series
of derivatives able to modulate 50% of the MR activity at
concentrations below 500 nM (2a−2e, Table 2.1).43 These
compounds presented a 1H-pyrrole-3-carboxamide or a 1H-
pyrrole-2-carboxamide structural core with different substitu-
ents attached.
In March 2006, Exelixis and Daiichi Sankyo entered into a
joint research agreement to develop modulators of the MR,
which led to two patents in 2010. These new 1H-pyrrole-3-
carboxamide compounds showed atropisomerism (2f−2k,
Table 2.2), and only one of the atropisomers exhibited high
2633
a
Gal4/LBD cell-based reporter assay. bMixture of atropisomers A and
B. cThe corresponding 2f−2k atropisomers have an IC50 > 1000 nM.
MR antagonist action.44,45 The pharmacological profile of
compound 2k was further investigated.42 In these studies 2k
showed a good selectivity profile over other human steroid
hormone receptors (at least 1000-fold more selective for MR
over GR, AR, and PR), which could result in reduced sex-
hormone-related side effects. Compound 2k also showed more
potent MR antagonism and longer-lasting activity than the two
currently marketed drugs, since it was able to inhibit the
aldosterone-induced decrease in urinary Na+/K+ ratio in rats by
56% at 20 h, whereas spironolactone inhibited the decrease at
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20 h by less than 20% and eplerenone at 8 h by around 8%, Table 2.3. MR Pyrazoline Antagonists
following oral administration to rats. Moreover, compound 2k
exhibits more potent antihypertensive and cardiorenal
protective effects in a Dahl salt-sensitive hypertensive rat
model than the two marketed drugs.46 Indeed, 3 mg/kg of 2k
inhibited the elevation in systolic blood pressure in DOCA/
salt-loading rats, whereas spironolactone and eplerenone did
not show significant effects at 30 mg/kg. The overall results
prompted the initiation of phase II clinical trials to treat
patients with hypertension and diabetic nephropathy. In March
2016 this compound started phase III clinical trials in essential
hypertension.
2.2. Pyrazoline Derivatives. Pfizer identified pyrazoline 2l
(Figure 2.1) as an acceptable MR antagonist during a high-

Figure 2.1. Pfizer pyrazoline derivative 2l.

throughput screening (HTS) campaign.47 Starting from this hit,

and in an effort to improve activity and solubility, they analyzed
substituents at the pyrazoline 1, 3, and 5 positions. Regarding
the aromatic moiety at N1, the cyano substituent was the most
appropriate replacement for the nitro group (Table 2.3), while
a small nonpolar substituent at R2 brought a 2-fold improve- a
Gal4/LBD cell-based reporter assay.
ment in potency (2m vs 2n and 2o, Table 2.3).47,48 As for the
aromatic ring at position 3, the introduction of a 4-carboxylate
Table 2.4. IC50 Values of Conformationally Restricted
group led to potent MR antagonists with reduced hERG
Pyrazoline Derivatives
channel inhibitory potency. At position 5, the R configuration
was the most favored (R-2m vs S-2m, Table 2.3), and the
replacement of the 4-fluorophenyl group by a cyclopentyl
moiety led to a slight improvement in potency (2m vs 2p, 2n vs
2q).47,48 Further modifications by addition of alkoxy groups
resulted also in potency enhancement for 2r−2t.47,48
A related series resulted from the conformational restriction
through cycling the phenyl ring at position 3 to the pyrazoline
scaffold. A six-membered ring (2u−2w, Table 2.4) was
preferred over the corresponding lower five-membered
homologue.47,49 As in the non-restricted series, the stereo-
chemistry at C3 was essential for potency, whereas that of C3a
was only of marginal importance. Compound PF-3882845
(3S,3aR-2w) displayed high affinity for the MR, selectivity over
other steroid receptors (PR IC50 = 416 nM, AR IC50 = 8960
nM, GR and ER IC50 > 10000 nM) as well as a good
pharmacokinetic profile.47 Moreover, 3S,3aR-2w reduced blood
pressure and protected the kidneys in a preclinical model of
hypertension induced in Dahl salt-sensitive rats. 3S,3aR-2w
entered clinical trials for diabetic nephropathy,47 but its
development was discontinued in 2012.
Molecular docking studies has been carried out to get a
Gal4/LBD cell-based reporter assay.
insights into the binding pocket of 2w.47 Unlike the DHP
compounds, pyrazoline derivatives do not make the stabilizing
contact to Asn770 residue (H3 helix). This binding feature by
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itself could explain their MR antagonism, because it has Table 2.6. MR Binding Affinity of Imidazole Derivatives (Ki
previously been suggested that MR steroidal activation, in Values)
addition to the C3-ketone group, requires ligands able to
engage in hydrogen bonding to Asn770 (H3 helix) and Thr945
(H11 helix).23,47 Similarly to the binding mode of some DHP
derivatives (e.g., S-1p), the cyano phenyl group of 2w sits in the
A-ring pocket forming hydrogen bonds, with Gln776 (H3
helix) and Arg817 (H5 helix), thus mimicking the A-ring C3-
carbonyl group of steroidal ligands (e.g., corticosterone).
In spite of the good properties of 3S,3aR-2w, there was still
room for improvement. To this end, Pfizer focused again on compd R1 R2 R3 MR Ki (nM)a
non-conformationally restricted pyrazoline analogues, in 2ac H Me H 134.4
particular R-2r, because of its increased selectivity over PR 2ad H 3,5-di-Cl-Ph-CONH H 161.7
and its improved solubility.50 The MR potency and selectivity 2ae Me H H 285
rose upon the replacement of the 1-(3-chlorophenyl) ring by a
Binding affinity was determined using a competitive binding assay
the corresponding 3-methyl-substituted analogue, and even with 3H-aldosterone and recombinant rhesus MR.
more upon incorporation of a nitrogen in the benzoic acid
moiety, resulting in compound 2x, with a selectivity over PR subcutaneous dose but showed lower activity than spirono-
>500-fold (Table 2.5).50 The incorporation of a nitrogen within lactone at 10 mg/kg.53
2.4. Oxazolidinedione Derivatives. Merck Research Labo-
Table 2.5. Activity of Pyrazoline Derivatives in the MR and ratories recently described the discovery of a new class of MR
the PR antagonists based on the oxazolidine-2,4-dione scaffold. Starting
from a HTS of an in-house collection, compound 2af was
selected as a modest MR ligand for a thorough optimization
strategy (Table 2.7).54,55 First, the importance of the absolute

Table 2.7. MR Ligands with an Oxazolidinedione Central

Scaffold

IC50 (nM)a
compd Y R MR PR solubilityb (μM)
2x CH OH 4.5 2530 312
2y N OH 7.6 1590 324
2z CH NH2 2.7 494 <25
2aa CH NHSO2Me 2.8 2480 239
2ab N NHSO2Me 4.1 831 39
a
Gal4/LBD cell-based reporter assay. bKinetic solubility determined as
previously described.51

the aromatic ring displaying the nitrile (2y) did not change
significantly the MR activity or solubility.50 The replacement of
the carboxylic acid by amide derivatives did not improve the
MR potency or the selectivity over the PR and decreased
kinetic solubility (2x vs 2z).50 In contrast, acylsulfonamide
analogues (2aa, 2ab) increased the MR potency while
maintaining low PR activity.50 Thus, compound 2aa displayed
>800-fold selectivity for the MR over the PR. However, the
high in vitro liver microsomal intrinsic clearance of 2aa (Clint =
175 μL/min/mg) motivated the authors to focus additional
efforts on 2x (Clint < 14 μL/min/mg). Oral administration of
2x in rats significantly increased the urinary Na+/K+ ratio at the
doses of 10 and 30 mg/kg. The effect of 2x was dose-
dependent. This compound behaved as a MR antagonist, with a
good selectivity over other NHRs and improved solubility in Values obtained using a commercially available protein−protein
comparison with 2w, and showed in vivo efficacy. Therefore, 2x interaction cell-based assay.56
was selected for further preclinical profiling.50
2.3. Imidazole Derivatives. In 2008, Merck patented several configuration of 2af was recognized, as its enantiomer was
imidazole derivatives with antagonist MR activity (some inactive (IC50 > 40 μM), as well as the positive contribution of
representative examples, 2ac−2ae, are collected in Table a methyl group as R1 substituent (2ag−2ai).54 According to
2.6).52,53 Compound 2ae was selected to test a protocol for molecular modeling studies, this methyl plays a key role in
determining the anti-mineralocorticoid activity of compounds favoring the conformation which is preferred for receptor
dosed in rats. This compound was active at a 30 mg/kg binding. The incorporation of fluoro or chloro substituents on
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the R1 phenyl ring (2ai), as well as the introduction of a ring
fused to the phenyl group (2aj) maintained the affinity.54 The
benzyl group at position 5 of the oxazolidinedione ring was also
an important element, as its replacement by H led to an
inactive compound. The optimization of the group attached to
the amide bond revealed that certain substituents on the benzyl
moiety (R2, R3, R4) increased the affinity, as in 2ag and 2ah. In
contrast, the incorporation of monocyclic or fused bicyclic aryl
or heteroaryl moieties generally led to a decreased MR activity.
Interestingly, an increase in activity was observed for the
cyclohexyl analogue 2ak, the most potent compound within
this series.54 Finally, attempts to change the oxazolidinedione
core led to inactive analogues. Theoretical studies suggested
that this scaffold was essential to place the pharmacophore
groups in the right pocket regions. Compound 2ai showed an
acceptable selectivity profile against several nuclear receptors,
including GR, AR, ERα, ERβ, and PRβ (EC50 agonist mode
assay values >20 μM and IC50 antagonist mode assay >8 μM).54
Next, aiming at improving physicochemical and ADME
(absorption, distribution, metabolism, and excretion) properties
and safety profiles, the cyclization of the amide moiety was
explored (2al−2aq, Table 2.8) as a way of avoiding amide
Table 2.8. IC50 of Oxazolidinedione-Derived MR Ligands
with Heteroaromatic Substituents at Position 5
a
Commercially available protein−protein interaction cell-based
assay.56
hydrolysis, one of the primary metabolic pathways of these
compounds.57−59 The authors analyzed different rings, among
them imidazole, oxazole, triazole, oxadiazole, and benzimida-
zole. Imidazole and 1,3,4-oxadiazole derivatives, 2an and 2ap,
showed improved potency and lipophilic ligand efficiency
(LLE) compared with the 1,2,4-triazole, 2ao. The inclusion of a
cyclopropyl ring at the benzylic position, as in 2aq, led to an
enhancement in human metabolic stability. Both 2ap and 2aq
showed significant selectivity over other NRs (IC50 > 5 μm),
2636
Perspective
ion channels (IC50 > 6 μm), and CYPs (IC50 > 19 μm) as well
as a reasonable rat PK profile.57,59 As previously observed, the
incorporation of a 3,5-dimethoxyphenyl substituent at position
R increased potency (2an vs 2am).
Authors also envisaged the benzimidazole as a replacement of
the amide moiety of compounds like 2ag. These benzimida-
zole-based oxazolidine derivatives combined similar MR
potency with improved rat liver microsomal activity (Table
2.9).60 The SAR studies with respect to R1 and R2 showed that
Table 2.9. MR Activity of Benzimidazole-Based Oxazolidine
Compounds
a
Commercially available protein−protein interaction cell-based
assay.56 bPercentage of compound (1 μM) remaining at 0.5 h and
incubation in human (hLM) and rat (rLM) liver microsomes.
small substituents such as halides (2ar, 2ax, 2ay) or
trifluoromethyl (2az) led to modest MR activity, whereas the
incorporation of heteroaryls or heterocycles at R2 led to several
compounds with improved IC50 values (2as−2aw).60 These
derivatives had acceptable human microsomal stability (>50%
of parent compound remaining after 30 min of incubation).
Moreover, in a natriuresis rat model the effect of compound
2au at 100 mg/kg was comparable to that of spironolactone at
30 or 100 mg/kg. Authors noted that previous monocyclic
heteroaryls (Table 2.8) exhibited much weaker efficacy in the
same model.
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3. Aryl Sulfonamide Derivatives as MR Antagonists.
Pfizer identified an aryl sulfonamide based MR antagonist, 3a,
through a HTS of its in-house collection (Figure 3.1).61
Figure 3.1. Sulfonamide-based MR antagonist identified through HTS.
Subsequent studies revealed that the replacement of the
thiophene ring by a phenyl ring led to a modest improvement
in MR binding affinity, as in derivative 3b (Table 3.1).61
Table 3.1. MR Binding (IC50) and Physicochemical
Properties of Aryl Sulfonamide Derivatives
a
Competitive binding assays using [3H]aldosterone. bLipE = Pic50 −
elogD.
However, both compounds had a low lipophilic efficiency
(LipE). Consequently, an optimization process was carried out
to improve this parameter and the metabolic stability.
Modifications of the R1 substituent led to compound 3c that
showed the best balance of LipE and metabolic stability.61 To
improve affinity over 3c a sulfonamide library was prepared,
from which the replacement of the isoxazole ring with
substituted phenyls afforded derivatives with improved proper-
ties, like 3d and 3e.61 Compound 3e was considered a
promising lead for optimization because it was selective against
PR (>48-fold selectivity), AR (no antagonist activity up to 10
μM), and GR (>11-fold selectivity) and exhibited good passive
2637
Perspective
permeability (RRCK, Papp = 10−6 cm/s, 15.4) and metabolic
stability (CLint,app = 9.6 μM/min/million cells).
The sulfonamide moiety is also within a series of biaryl amide
derivatives, developed by Dainippon Sumitomo Pharma, which
also provided molecules able to bind to the MR.63 They
explored a wide variety of substituents at the aromatic rings by
preparing more than five hundred compounds. The methoxy
and sulfamide substituents were always among those with the
higher activity (Table 3.2). Additionally, different R1 and R2
Table 3.2. MR IC50 Values for Representative Sulfonamide-
Substituted Biaryl Amide Derivatives
a
Competitive binding assays using [3H]aldosterone in a rat renal
soluble fraction in the presence of RU-486 to avoid binding to the
glucocorticoid receptor.
groups could be incorporated while keeping high affinity (e.g.,
3f−3k), whereas substituents at other positions rendered less
active compounds (Table 3.2).63
4. Pyridyl Ureas as MR Antagonists. Boehringer
Ingelheim International GMBH studied a series of compounds
involving a pyridyl urea linked to a phenyl moiety by a pyrazole,
4a (Figure 4.1), or an ethylene spacer, 4b−4g (Table 4.1).64 In
general, the best results were obtained with the ethylene linker,
particularly in the functional assay (Table 4.1). A variety of
substituents are permitted on the ethylene moiety, at R1, such
as aliphatic chains or aromatic moieties (4c, 4d, Table 4.1). The
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incorporation of F at R3, as in 4e, improved binding affinity and

led to derivatives with binding IC50 values below 30 nM,
whereas substitution by CH3, CF3, or OCH3 led to values over
100 nM, as in compound 4f.
5. Indole- and Indazole-Derived MR Ligands. Eli Lilly
and Company, in a search for non-steroidal human MR
antagonists, and on the basis of screening, identified a series of
indole derivatives with interesting properties. They reported
two series, one with a 3,3-bisaryloxoindol skeleton, and the
other with a quaternary carbon serving as the scaffold
Figure 4.1. Example of a [(pyrazol-3-yl)pyridin-2-yl]urea MR
supporting an indole ring and at least another aromatic group.
antagonist. 5.1. 3,3-Bisaryloxoindole Derivatives. SAR studies on the
oxoindole derivative 5a, a human MR (hMR) ligand identified
Table 4.1. IC50 Values of MR Binding and Functional Assays through screening (Table 5.1),65 showed the importance of the
of Pyridyl Ureas phenol substituents, in particular of hydroxy groups, as their
replacement with H or OMe, led to inactive derivatives. An
increase in potency and selectivity was observed when the
phenyl ring was directly attached to the nitrogen atom of the
oxoindole core (5b), and particularly when a meta-substituent
was present, 5c and 5d.65 Unsymmetrical phenol derivatives,
like 5e−5g, showed good selectivity for the MR compared to
IC50 (nM) other steroid receptors (GR, PR, AR, and ER).65 The authors
MR MR suggested that the presence of a hydroxy group at R4 forces a
compd X Y R1 R2 R3 bindinga functionalb conformation that allowed the discrimination between the
4b N CH H Me H 7.1 19 binding pockets of MR and GR. The isolation of the 5g
4c N CH Ph H H 13 44 enantiomers showed that the (−)-isomer was more potent and
4d N CH Me Me H 11 43 selective than the (+)-isomer.
4e N CH H Me F 4.7 31 5.2. Indol-7-yl-methanesulfonamide Derivatives. Bell et al.
4f N CH H H Me 480 2800 explored the use of indole-derived compounds as modulators of
4g CH N H OMe H 25 97 steroid hormone nuclear receptors.66 In particular, they focused
a
MR competitive molecular binding assay based on the binding and on the MR and the GR. It is worth noting that the great
displacement of a TAMRA-labeled dexamethasone probe with majority of the compounds reported interact with both the MR
fluorescence polarization (FP) detection. bCommercially available and the GR with Ki values lower than 500 nM (Table 5.2). In
Gal4/LBD cell-based reporter assay (GeneBLAzer UAS-bla HEK 293 general, these compounds have two substituents on the indole
cell line).
ring at positions 3 and 7, the latter quite frequently a methane
sulfonamide moiety. A thorough exploration was carried out at
the indole position 3: through this position the indole ring was

Table 5.1. hMR Binding of Bisaryloxoindole Derivatives

Ki (nM)
compd n R1 R2 R3 R4 hMRa hGR hPR hAR hERα hERβ
5a 1 H Me Me H 65 − − − − −
5b 0 H Me Me H 27 147 44%b 41%b 733 >1100
5c 0 Me Me Me H 7 84 138 306 183 844
5d 0 OMe Me Me H 0.6 51 715 402 242 930
5e 0 OMe H H OH 3 793c 678 19%b >1100 >1100
5f 0 OMe H Me OH 1 693c 395 1105 >1100 >1100
5g 0 Me H Me OH 2 334 245 37% 579 >1100
5g(−) 0 Me H Me OH 1 582 33% 16%b 359 >1100
5g(+) 0 Me H Me OH 40 686c 49% 20% 519 >1100

a
Competitive binding assay using [3H]aldosterone bPercentage of inhibition at 1 μM antagonist concentration. cOne determination.

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Table 5.2. Indol-7-ylmethanesulfonamide Derivatives with Ki Table 5.3. MR Antagonist Activity of Indazole Derivatives
Equal to or Lower than 500 nM for the MRa

compd R1 R2 R3 R4 R5 MR IP (nM)a,b
5r H Et Me Br Me 7
5s H Et Et Br Me 3
5t H Et Et CF3 Me 4
5u F Et Et CF3 Me 9
5v H CH2CHF2 Et CF3 Me 15
5w H Et CHF2 CF3 Me 11
5x H Et CHF2 CF3 Et 4
5y F Et CHF2 CF3 Et 11
a
Inflection point of the nonlinear regression curve. The IP value is
equal to the IC50 value when the slope is 1, the minimum is 0, and the
maximum is 100. bCommercially available protein−protein interaction
cell-based assay.56

Table 5.4. PK Profile in Rats and Metabolic Stability for

a
Ki values were calculated with a competitive binding assay using
radioligands.66,67.
attached to a tetrasubstituted carbon, which, in general, bear
another aromatic ring and two aliphatic chains. As for the
aromatic substituent, a wide variety of mono- or bicyclic
aromatic and heterocyclic moieties have been explored. Among
them, phenyl, thiophene, furan, benzofuran, indole, benzothio-
phene, benzodioxin, benzodioxole, benzimidazole, benzothia-
zole, benzooxazole, indazole, and biphenyl led to affinity below
500 nM (e.g., compounds 5h, 5k−5m, Table 5.2).66 These
aromatic moieties supported in some cases an extra substituent,
such as methyl, chlorine or fluorine (5i, 5j, 5o−5q).66
Regarding the other two substituents in derivatives with
significant Ki, both groups could be equal or different, and
generally they were aliphatic groups, such as methyl (5n−5q),
ethyl (5h−5m), propyl, cyclopropyl (5n−5q), or cyclobutyl, or
both substituents were within a five- or six-membered ring.66,67
From the series of compounds with a Ki < 500 nM, the SAR
studies showed the favorable effect of a proton donor at
position 7 of the indole ring, particularly a methane
sulfonamide moiety, and the best combination for the dialkyl
substituents was methyl/cyclopropyl (5n−5q Table 5.2).
Derivative 5p was further studied, showing that the S-
enantiomer behaved as a potent, selective, and orally efficacious
antagonist of the MR.67 Furthermore, in a rat model of
hypertension, S-5p was more potent than the marketed drug
eplerenone and showed a better in vitro selectivity profile than
spironolactone.
5.3. Indazole Derivatives. Merck Sharp & Dohme Corp.
described a series of indazole derivatives which display MR
antagonist activity. Although the diastereoisomers and
enantiomers were isolated, their stereochemistry was not
disclosed within the patent. In Table 5.3, representative
examples 5r−5y are collected.68 Compounds 5r, 5t, and 5w
have good metabolic stability in liver microsomes and
hepatocytes as well as good PK profiles in rats (Table 5.4).
6. Benzoxazinones-Derived MR Ligands. 6.1. 1,4-
Benzoxazin-3-ones. The 1,4-benzoxazin-3-one bicycle is an
2639
Indazole Derivatives
stability, t1/2 (min)
CL t1/2 human liver human
compd (mL/min/kg) (h) microsome hepatocyte
5r 18 3.6 48 69
5t 24 5.3 72 >90
5w 3.8 9.9 62 >90
interesting moiety in the search of non-steroidal MR
antagonists whose use has been explored by several
pharmaceutical companies. In 2006, Novartis patented a series
of 333 derivatives with a benzoxazin-3-one central core, bearing
a great variety of large substituents attached either directly or
through a linker to position 6 (R2), including aromatic and
heteroaromatic rings (Table 6.1). Positions 5, 7, and 8 were less
explored and had smaller substituents, such as methyl or
fluorine. Several of these derivatives were able to bind to the
MR (Table 6.1, compounds 6a−6c).69 In 2016, Vitae
pharmaceuticals performed a SAR study with derivatives having
variable substituents at position 6, finding several compounds
with an acceptable MR affinity, derivatives 6d and 6e.70
Toru Iijima et al., from Tanabe Seiyaku Co., focused on
substituents at positions 2, 4, and 7 of the 1,4-benzoxazin-3-one
scaffold (Figure 6.1, I). Several derivatives were reported as
having MR Ki values lower or equal to 500 nM, and in general,
they had two methyl groups at position 2 and either NHSO2Me
or NH2 at position 7.71 More variety could be found at position
4, with aromatic or heteroaromatic rings, as 4-fluorophenyl, 4-
fluoro-3-methylphenyl, 3,5-dichloropyridin-2-yl, benzyl, and so
forth.
The benzoxazin-3-one ring is also within more complex
structures, having monocyclic aromatic rings or fused-hetero-
cycles as central scaffold (Figure 6.1, II). These compounds,
described in 2009, showed inhibition rates up to 70% at 10
μM.72 It was observed that the central scaffold A was tolerant to
modification, while keeping significant MR binding activity.
Thus, Takeda pharmaceuticals studied different skeletons as
replacement of this central scaffold. Starting from a HTS hit
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Table 6.1. Binding Data of a Series of 1,4-Benzoxazin-3-ones
Derivatives
a
Gal4/LBD cell-based functional assay in HEK293 cells. bCompetitive
binding assay using 3H-cortisol.
Figure 6.1. General formulas of 1,4-benzoxazine-containing deriva-
tives.
and guided by docking studies, they identified compound 6f,
with a triazolothiadiazine skeleton as the central scaffold (Table
6.2).72 Even more, they solved the X-ray structure of the
complex between 6f and the MRC808S/S810L-LBD (1.35 Å, PDB
ID 3VHV)the first X-ray structure of a non-steroidal
compound bound to the MR (Figure 6.2).8 This structure
revealed the binding mode of the benzoxazin-3-one ring, in
which the NH group and the carbonyl oxygen of this moiety
form hydrogen bonds to Asn770. Also, the nitrogen atoms of
the triazole scaffold establish hydrogen bonds to Gln776 and
through a water molecule to Arg817. The aryl group at the 2-
position of the triazolothiadiazine ring partially filled the α-face
pocket providing the bases for further chemical optimization.73
After superimposing the crystal structures of compound 6d and
spironolactone bound to the MR-LBD binding-site cavity, it
2640
Perspective
Table 6.2. MR IC50 Values of 1,4-Benzoxazin-3-one-
Containing Triazolothidiazine Derivatives
compd R1 R2 MR IC50 (nM)a
6f H H 510
6g F H 310
6h H F 270
a
Competitive binding assay using [3H]-aldosterone.
was found that both compounds have similar binding modes.
The 3D structure showed a small space around the phenyl
group; thereby, derivatives 6g and 6h were prepared.8
Compound 6h showed improved affinity and better selectivity
over other steroid hormone receptors than 6f.
The poor PK profile of 6h (metabolic clearance in rat
microsomes 60 μL/min/mg) led to the investigation of
alternative scaffolds without metabolically labile groups.8 The
replacement of the triazolothiadiazine ring by thiazole or
imidazole led to a decrease in the activity. In contrast,
derivatives 6i−6m with a pyrazole scaffold showed interesting
results (Table 6.3).8 The X-ray structure of the triazolothiadia-
zine derivative 6f was useful in the lead optimization of this
pyrazole series, in which a trifluoromethyl group was attached
to the 3-position of the pyrazole ring (as in 6i−6m) to fill the
space of the triazolothiadiazine ring (as in 6f).8 Subsequent
optimization of the substituents on the benzene and
benzoxazine rings showed that the introduction of a methyl
group as R1 substituent (compound 6j) considerably enhanced
binding affinity.8 Further increases in affinity were achieved by
adding fluorine at R2, and chlorine or methyl at R3 (compounds
6k−6m). Compound 6k showed a good PK profile in SD rats
[CL = 1328 mL/h/kg, Vdss = 4626 mL/kg], as well as the best
balance of potency and selectivity (binding IC50 for AR, PR,
and GR was 2500 nM or higher), and was selected for in vivo
evaluation.8 These studies showed that 6k behaved as an MR
antagonist with blood-pressure-lowering effect similar to that of
spironolactone.8
Taken together, the SAR studies on benzoxazin-3-one
derivatives identified three main key pharmacophores, namely
a benzoxazin-3-one moiety, a 4-fluorobenzene ring, and a
central scaffold linking these two aromatic rings at adjacent
positions, which can be exploited to develop more potent and
selective MR antagonists.8,73 Accordingly, a novel benzoxazin-3-
one derivative, having a dihydrofuran-2-one moiety as the
central scaffold, was selected as lead compound (Table 6.4).73
An X-ray co-crystal structure of the MR LBD/compound 6n
complex (2.05 Å, PDB ID 3WFF73) revealed the binding mode
of dihydrofuran-2-one derivatives and guided the lead
optimization of the new benzoxazin-3-one-based compounds
(Figure 6.3A). As expected, compound 6n binds to the MR-
LBD in a similar way as compound 6f. The NH group and the
carbonyl oxygen of the benzoxazin-3-one moiety forms
hydrogen bonds to Asn770, and its carbonyl oxygen established
a new hydrogen bond to Thr945. The carbonyl oxygen in the
dihydrofuran-2-one scaffold establishes hydrogen bonds to
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Figure 6.2. (A) Close-up view of the X-ray structure of the MRC808S/S810L-LBD double mutant bound to compound 6f showing its binding mode.
(B) Superimposition of the X-ray structures of compound 6f (yellow) and spironolactone (orange) bound to the MRC808S/S810L-LBD (PDB IDs
3VHV and 3VHU, respectively). The hydrogen bonds and water molecules are depicted as yellow dashed lines and red spheres, respectively.

Table 6.3. MR Binding IC50 of 1,4-Benzoxazin-3-one- Table 6.4. IC50 Binding to MR, PR, and GR of
Containing Pyrazole Derivatives Dihydrofuran-2-one and Dihydropyrrol-2-one Derivatives
Containing a 1,4-Benzoxazin-3-one Ring

compd R1 R2 R3 MR IC50 (nM)a

IC50 (nM)a
6i H F H 260
6j Me H H 80 compd R1 R2 R3 X MR PR GR
6k Me F H 41 6n H F H O 660 4600 >10000
6l Me F Cl 12 6o Me H H O 120 740 3800
6m Me F Me 21 6p Me H H N-Et 33 770 1600
a
Competitive binding assay using [3H]-aldosterone. 6q Me H H N-c-Prb 68 400 2200
6r Me H H NH 94 5600 9100
6s Me H Cl NH 23 1600 1100
Arg817 and Gln776 through a water molecule. It was also
6t Me H Me NH 43 >10000 4900
observed the existence of three lipophilic spaces that could be a
filled with adequate groups at X, R1, and R2.73 At dihydrofuran Competitive binding assay using [3H]-aldosterone, [3H]-progester-
position 5, a methyl substituent (6o) was optimal, whereas a one, or [3H]-dexamethasone to test binding to MR, PR, and GR,
bigger ethyl group led to a decrease in binding activity.73 The respectively. IC50 binding to AR was >10 000 nM in all cases. bc-Pr =
cyclopropyl.
binding mode of compound 6o is similar to that of compound
6n, as observed in the solution of the crystal structure of the
MR-LBD/compound 6o S-enantiomer complex (1.40 Å, PDB study the R3 substituent. Compounds 6s and 6t showed higher
ID 3WFG73) (Figure 6.3B). The X-ray co-structure of MR activity and were equipotent to spironolactone.73 More-
compound 6o in the MR LBD suggested that the S-enantiomer over, the methyl derivative 6t has decreased PR activity
should be more potent than its R counterpart, and that a compared with 6r (Table 6.4).
lipophilic group should be adequate at position 1 (Table 6.4).73 In functional assays, compound 6t and most of the
Therefore, the central scaffold was replaced by a dihydropyrrol- dihydropyrrol-2-one derivatives showed also moderate partial
2-one ring. As indicated in Table 6.4, ethyl and cyclopropyl agonistic activity at high concentrations (20−30% at 10 μM),
derivatives 6p and 6q showed increased binding activity.73 whereas the values obtained for pyrazole analogues, such as 6k,
Unexpectedly, the unsubstituted NH derivative 6r exhibited were low. The X-ray structure of the MR LBD bound to 6o
high binding activity, which could be explained by the increased (PDB ID 3WFG),73 close analogue of 6t, suggested a role of
electron density of the carbonyl group in the dihydropyrrol-2- the central ring carbonyl group in a water-mediated hydrogen
one scaffold compared to the dihydrofuran-2-one framework, bonding network. Since this network was considered not to be
reinforcing the water-mediated hydrogen bonds with Arg817 present in 6k, it was assumed that it was important for the
and/or Gln776.73 Regarding the high selectivity of 6r over the partial agonist effect. As the clinical efficacy and safety of partial
AR, PR, and GR compared to 6p and 6q (Table 6.4), its N- agonists remain unclear, other azole rings, with no possibility to
unsubstituted dihydropyrrol-2-one ring was shown to be a key form these water-mediated hydrogen bonds, were selected, with
scaffold for such a property compared to the dihydrofuran-2- the pyrazole scaffold providing the best MR activities (6u−6z,
one moiety. Further efforts were focused on compound 6r to Table 6.5).74,75 However, derivatives 6v−6x also showed high
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Figure 6.3. Close-up views of the MRC808S/S810L-LBD double mutant bound to benzoxazin-3-one-based compounds: (A) 6n (magenta), (B) 6o (S-
enantiomer, pink), and (C) 6z (blue) (PDB IDs 3WFF, 3WFG, and 4PF3, respectively). The hydrogen bonds and water molecules are depicted as
yellow dashed lines and red spheres, respectively.

Table 6.5. MR Activity of 1,4-Benzoxazin-3-one-Containing contribution of these fluorine atoms to the binding is
Pyrazole Derivatives hydrophobic.
Pfizer has also explored compounds with a morpholine
central ring (Table 6.6). They observed that the incorporation

Table 6.6. MR IC50 of 1,4-Benzoxazin-3-one-Containing

Morpholine Derivatives

IC50 (nM)
compd R MR bindinga MR activityb
6u n-Pr 22 45 (0%)
6v CH2CH2CF3 8.4 33 (6%)
6w CH2CF2CH3 10 18 (4%)
6x CF2CH2CH3 5.8 27 (11%)
compd R1 R2 X Y MR IC50 (nM)a
6y 3-Py 36 36 (−4%)
6z CF2CH2OH 51 71 (1%) 6aa H H CH CH 24
a 3 b 6ab H H N CH 44.4
Competitive binding assay using [ H]-aldosterone Cell-based
6ac F H 33.4
reporter gene assay. Antagonist activity and, in parentheses, agonist
activity at 10 μM concentration. 6ad H F 55
6ae F H CH N 53.9
a
Gal4/LBD cell-based reporter gene assay.
metabolic clearance in rat microsomes (>100 μL/mg/min),
which was attributed to their high lipophilicity (logD > 3).
Design of less lipophilic derivatives allowed the identification of
several potent compounds that have significantly reduced at morpholine N4 of benzoxazin-3-one, 6aa, or pyridoxazinone
partial agonistic activities and high selectivity over other steroid moieties, 6ab−6ae, led to derivatives with IC50 values below
receptors, like 6y and 6z (binding IC50 AR and PR > 10 000 100 nM, nevertheless the former provide the most active
nM, GR > 1700 nM).75 Compound 6z showed an acceptable derivative.76 There was a decrease in affinity caused either by
metabolic clearance (42 μL/mg/min), and it was able to lower the removal of the aromatic ring at position 5 (IC50 = 3050
the blood pressure in DOCA-salt hypertensive rats, without nM) or the methyl at position 2 (IC50 = 1970 nM) in derivative
antiandrogenic effect. The crystal structure of the complex 6ab. It was also observed that the incorporation of a fluorine
between 6z and the MR LBD (1.10 Å, PDB ID 4PF3)75 atom in the ortho or meta-position of the phenyl ring keeps the
showed that it binds to the steroid binding pocket of the MR. affinity (6ac, 6ad), whereas it is detrimental in the para-
As expected, the binding mode of compound 6z is similar to position.76
that of compounds 6n (Figure 6.3C). The NH group and the 6.2. 1,3-Benzoxazin-2-ones. 1,3-Benzoxazin-2-one and -2-
carbonyl oxygen of the benzoxazin-3-one moiety form thione derivatives also provided compounds able to interact
hydrogen bonds to Asn770 and Thr945, and the 4- with the MR, which are collected in two Knobbe Martens
fluorobenzene ring occupies the α-face hydrophobic pocket. Olson & Bear LLP patents77,78 and one from Dainippon
The ligand side chain at the 1-position of the pyrazole ring, the Sumitomo Pharma Co (6af, SM-368229, Figure 6.4).79 In
2,2-difluoropropyl-3-hydroxy group, points out toward Arg817 particular, a study on the pharmacological profile of 6af
and Gln776. Its hydroxyl group directly forms a hydrogen bond indicated that this compound was able to increase urinary Na+/
to Gln776. The two fluorine atoms do not form any specific K+ ratio in adrenalectomized rats treated with deoxycortico-
hydrogen bonding interactions suggesting that the major sterone acetate. Even at doses of 300 mg/kg, only very weak
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Journal of Medicinal Chemistry Perspective

increased for the diacetylated analogue 7b, however further

increase of the steric hindrance at R1 and R2 substituents (Et,
iPr, or n-Pr) tended to decrease activity.81 The influence of the
ring size was also investigated from the 11- to 14-membered
lactone ring; the 11-membered ring derivative 7c showed the
best inhibitory activity.81
8. Fused Heterotricyclic Derivatives as MR Ligands. Eli
Figure 6.4. 1,3-Benzoxazin-2-ona derivative from Dainippon Sumito- Lilly and Company described a series of tricyclic steroid
mo Pharma Co. hormone receptor modulators with the capacity to bind to the
MR and the GR with Ki ≤ 500 nM. The general structures are
antiandrogenic effects were observed in methyltestosterone- shown in Figure 8.1. For derivatives of general formula III, the
treated male rats.79 tricyclic structure of the reported compounds with Ki lower
Mitsubishi Tanabe has investigated related compounds than 500 nM corresponds mainly to a thioxanthene scaffold (X
derived from 2H-1,3-benzoxazine (Y = N, 6ag, 6ah) and 2H- = S), but there are also examples in which X is a methylene
chromene (Y = C, 6ai, 6aj), reporting several compounds that group or an oxygen atom. R1 is usually hydrogen or methyl, and
showed sub-micromolar MR dissociation constants (Table Ar is an aromatic group such as phenyl, benzimidazol-2-one,
6.7).80 Most of them have a phenyl ring at position 4 of the benzoxazol-2-one, or benzothiazol-2-one.82 A tricyclic structure
with a seven-membered ring has also been explored, structure
Table 6.7. Examples of 2H-1,3-Benzoxazine and 2H- IV (Figure 8.1), and the authors reported almost 200
Chromene Derivatives with MR Ki Values below 500 nMa compounds with significant Ki values. In particular, within
these compounds the most recurrent were dihydrobenzo[a,d]-
[7]annulene derivatives (X = Y= C and two phenyl moieties
flanking the seven membered ring). However, there are also
several examples where X = S, SO, SO2, or O (Y = C), or
alternatively Y could be an oxygen atom, and X could be a
carbon. As for R8, it is mainly a hydrogen atom.83 More variety
of substituents is observed in the C ring, which might be a
phenyl, benzimidazol-2-one, benzimidazole, indolyl-2-one,
benzoxazol-2-one, or benzothiazol-2-one. Subsequent patents
compd Y R1 R2 R3 R4 focused on a tricyclic scaffold with a central seven-membered
6ag N Cl H H − ring of general formula V (Figure 8.1 and Table 8.1). A 2005
6ah N F H Me − patent described a series of derivatives with either a tricyclic
6ai C F H H Me dihydrodibenz[b,e]oxepine (X = O) or dihydrodibenz[a,d][7]-
6aj C Cl Me H H annulene (X = CH2) showing good Ki values.84 For the active
a
Competitive binding assay using [3H]-aldosterone. compounds reported, R1 and R2 are independently selected
from hydrogen or fluorine, and at the R3 position a variety of
heterocycles are suitable (Figure 8.1, formula V, Table 8.1).85,86
scaffold, although there is an example with a benzothiophene Within this family, compound 8a showed good affinity for the
ring. The most frequent substituents at position 2 are methyl MR and selectivity over the AR, BR, and PR, and behaved as a
groups, whereas either methyl or hydrogen are suitable at potent and selective antagonist of the hMR (IC50= 21 nM in
position 5. In the chromene (Y = C) scaffold, H, CN, and Br functional assays).86 In in vivo models, 8a displayed potent
are adequate as R4 substituents for interacting with the MR (Ki reno-protective activity, antihypertensive effects, and a reduced
< 500 nM). probability of producing hyperkalemia.
7. Macrolide MR Antagonists. In an screening program to In 2009, Eli Lilly and Company focused on a series of
search for non-steroidal MR antagonist, compound 7a, dihydrodibenz[b,e]oxepines based on the previously reported
methyllasiodiplodin, was found and reported as the first tricyclic steroid hormone receptor modulators (Figure 8.1, IV
macrolide MR antagonist (Table 7.1).81 The antagonist effect and V).83,84 Representative compounds with Ki ≤ 10 nM are
collected in Table 8.1. At R3 position several heterocycles led to
Table 7.1. Inhibitory Activity of O-methyllasiodiplodin compounds with significant activity, where R4 was O, N-CN, or
Analogues N-CONH2. Interestingly, these derivatives have at least one
fluorine atom at the C3 position, and the authors analyzed
systematically the presence of another fluorine either at R1 or
R 2 . In general, for each R 3 and R 4 substituent, the
corresponding 3-fluoro (R1 = H, R2 = H), 3,7-difluoro (R1 =
H, R2 = F), and 3,8-difluoro (R1 = F, R2 = H) derivatives have
been prepared and evaluated (i.e., 8a−c or 8d−f).85,86
compd R1 R2 R3 n IC50 (nM)a The authors also carried out an exhaustive modification of
7a H H Me 5 8930 the configuration of the chiral centers of R3 substituents, and
7b Ac Ac Me 5 2780 for the reported examples, Ki values were below 10 nM
7c Ac Ac H 4 580 regardless of their stereochemistry (8a−8c vs 8d−8f, or 8h−
8k).85,86 Several of the prepared compounds were tested in a
a functional assay and behaved as antagonists of the hMR.
Values determined using a yeast two-hybrid assay to asses inhibition
of MR interaction with a co-regulator. Moreover, three of them, namely 8b (0.79 nM), 8g (0.08 nM),
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Figure 8.1. General structures of tricyclic scaffolds developed at Eli Lilly that led to compounds with inhibitory constants lower that 500 nM for MR.
and 8h (0.23 nM), with Ki values below 1 nM, demonstrated in
vivo renal protective activity in a rat model of aldosterone-
mediated renal disease.85
Compound 8m showed a good selectivity for binding the
MR, from 800- to 7500-fold over GR, AR, and PR, and was
selected for a combination therapy with tadalafil for the
treatment of resistant hypertension.87 Taladafil is a phospho-
diesterase type 5 (PDE5) inhibitor and behaves as a mild
vasodilator. Authors also indicated that this therapeutic
combination would alleviate PDE5-monotherapy resistant
erectile dysfuntion.
An Eli Lilly and Company derivative, LY2623091 (structure
not disclosed), entered phase II clinical studies for the
treatment of hypertension and chronic kidney disease.
Although to the best of our knowledge there is no publication
that revealed its structure, on the Internet this compound has
been associated with derivative 8m.88,89
In 2016, Vitae pharmaceuticals explored several linkers
between the tricyclic scaffold and a series of bicyclic moieties
(Table 8.1, 8o, 8p, Figure 8.2).70 The selection of the bicyclic
system was based on previous SAR studies, which suggested the
1,4 benzoxazin-3-one (8q), 1,3-benzoxazin-2-thione (8r), and
several spirooxindoles (8s−8u) as suitable sub-groups.70
Interestingly, an X-ray structure was described for the MR
LBD complex with compound 8q (2.5 Å, PDB ID 5HCV).70 In
a similar way to previous X-ray structures, there are two
hydrogen bonds involving Asn770 and the NH group and
carbonyl oxygen of the benzoxazinone moiety, besides one
more between the carbonyl group and the Thr945 side chain
(Figure 8.3). In contrast, no hydrogen bond is observed
between the receptor and the tricyclic structure.
9. Peptide MR Ligands. The MR LBD, the key regulatory
domain of MR, contains an activation function-2 domain (AF-
2) in addition to the binding pocket for the endogenous
ligands. The AF-2 domain has been implicated in the
interaction with co-activators that contain multiple LXXLL
motifs, organized as α-helices. These co-regulators are critical
for MR-mediated gene expression and may confer specificity to
MR-mediated responses. Thus, there is a great interest in
knowing whether different MR ligands can induce distinct MR
conformations that might lead to different co-regulator
recruitment and ligand-specific gene regulation.
Hultman et al. focused on co-activator and co-repressor
peptides and their interaction with the LBD of the MR in the
presence of various MR ligands.90 They analyzed 50 co-
regulator peptides, derived from 23 co-activators and co-
repressors, containing the NR-box or CoRNR-box motifs
(small peptide motifs that are necessary and sufficient to
interact with NRs). Only a few of these peptides showed strong
interactions with the MR LBD in the presence of aldosterone
or the glucocorticoid cortisol. These include peptides derived
2644
Perspective
from SRC1, PGC1α, PGC1β, and ASC2 co-activators, which
have an extra leucine before the LXXLL motif in the NR-box.
However, not all LLXXLL-containing peptides were capable of
strong interactions. In the presence of eplerenone the
recruitment of these peptides is blocked, indicating that
eplerenone changes the conformation of the MR LBD.
Li et al. also focused on peptides derived from known co-
activators containing LXXLL motifs (Table 9.1). They also
found a preferential binding of the fourth LXXLL motif of
steroid receptor co-activator-1 (SRC1-4).7 In contrast, a weaker
interaction was established for the first motif of SRC1-1 and
SRC3-1 (over 16 μM).7 Interestingly, the X-ray structure of the
MR LBD bound to corticosterone and SRC1-4 (1.95 Å, PDB
ID 2A3I7) suggested two molecular structural features that
could account for the high affinity of SRC1-4. First, the
glutamic acid at position +7 (relative to the first leucine in the
LXXLL motif) that is hydrogen bonded with K782 of MR.
Second, the high stability of the SRC1-4 helix due to the
existence of several intramolecular hydrogen bonds between Q
+3 and K−3 as well as Q+2 and S−2.
In order to identify antagonist peptides able to interact with
the MR in a ligand-selective manner, Yang et al. recently used
two phage libraries, expressing 19mer peptides, to screen 108
peptides for MR binding in the presence of three different
agonists: aldosterone, cortisol, and deoxycorticosterone (Table
9.2).91 One of the libraries was designed to target the AF-2
region of the MR, and consequently included a central LXXLL
motif flanked by seven random amino acids on either side. The
other was a random library created to identify peptides that
could bind to any site on the MR. The analysis of the 165
peptides using the mammalian two-hybrid assay showed that 18
peptides had an enhanced interaction by the addition of the
ligand, one from the random library and 17 from the LXXLL-
constrained phage library (9a−9d).91 Within these latter
peptides, approximately 50% of them contained the unique
motif MPXLXXLL. Six of these 18 peptides were selective for
the MR, five of them from the constrained phage library, and
interacted significantly better in the presence of aldosterone
(9e−9g), while the one from the random library (9h) showed
increased interaction in the presence of cortisol.91 Thus, these
studies have demonstrated that different peptides interact
differentially with the MR in the presence of different ligands,
thus suggesting that agonists can induce different conforma-
tional changes in the MR that might lead to the recruitment of
specific co-regulatory proteins.
Rogerson et al., with the aim of identifying co-regulatory
molecules that could exhibit a ligand-specific interaction with
the MR LBD, screened a yeast-2-hybrid kidney cDNA library in
the presence of aldosterone and cortisol.92 A clone encoding
the region of the tesmin (metallothionein-like 5) gene that has
two LXXLL motifs provided a 7-fold greater response in the
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Table 8.1. Tricyclic Compounds with Ki ≤ 10 nM for hMR
a
8a−8n: competitive binding assay using [3H]-aldosterone. 8o, 8p:
MR competitive binding assay using [3H]-cortisol.
presence of aldosterone over cortisol. Therefore, tesmin was a
ligand-selective co-activator of the hMR, providing further
evidence of the adoption of ligand-dependent conformations by
the MR-LBD. The understanding of these interactions may
2645
Perspective
Figure 8.2. Structures of tricyclic scaffolds developed at Vitae
Pharmaceuticals that led to compounds able to bind to MR LBD.
Competitive binding assay using [3H]-cortisol.
open the venue for the development of selective MR
modulators.
■ CONCLUSIONS
The MR is a member of the nuclear receptor family that plays
an important role in regulating genes involved in cardiovascular
diseases and electrolyte homeostasis. The two currently
marketed MR antagonists, eplerenone and spironolactone,
both with a structure similar to that of steroid hormones, have
proven to be effective in the treatment of hypertension or
congestive heart failure. However, their therapeutic use is
limited due to unwanted side effects. These limitations have
propelled the search of new non-steroidal MR antagonists able
to circumvent these drawbacks. Over the past years this search
has been intensified and has mainly been undertaken by
pharmaceutical companies. In general, the research has started
with a HTS, which allowed the identification of a hit able to
interact with the MR. Next, hit-to-lead optimization processes
were carried out to improve potency and selectivity as well as
pharmacokinetic properties. These medicinal chemistry ap-
proaches have provided a series of scaffolds, such as five- and
six-membered heterocyclic rings or bicyclic and tricyclic
skeletons, able to support key pharmacophore groups in the
right 3D disposition to interact with the MR, thus leading to
novel MR antagonists. Interestingly, several of the non-steroid
derivatives showed excellent potency toward the MR and
selectivity against other nuclear receptors and displayed in vivo
activity.
Recently, X-ray structures describing the MR LBD not only
bound to steroid ligands but also in complex with non-steroidal
compounds have become available. These structures are
valuable tools for the computer-assisted design of new MR
ligands, or to guide the hit to lead optimization process. In fact,
as above indicated, docking studies have proved to be useful to
optimize initial HTS hits, leading to compounds with improved
PK and PD properties. The resolution of complexes of the MR
LBD bound to other families of antagonists could provide
further insight into the binding mode of these compounds.
In vivo studies with the most promising compounds have
shown that MR antagonists are able to reduce blood pressure
and protect against aldosterone-mediated renal and cardiovas-
cular injuries. Moreover, some compounds have proved to
reduce sex-hormone-related adverse effects and to lower the
incidence of hyperkalemia compared with the marketed steroid-
like antagonists. Several non-steroidal compounds are currently
in clinical evaluation for congestive heart failure, hypertension,
or diabetic nephropathy. These derivatives belong to different
chemical families and have been designated as 1p, 2k,
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Figure 8.3. (A) Close-up view of the X-ray structure of the MRC808S/S810L-LBD double mutant bound to 8q (cyan) (PDB ID 5HCV). (B)
Superimposition of the X-ray structures of compound 8q (cyan) and spironolactone (orange) bound to the MRC808S/S810L-LBD (PDB IDs 5HCV
and 3VHU, respectively). The hydrogen bonds and water molecules are depicted as yellow dashed lines and red spheres, respectively.

Table 9.1. Peptides Derived from Co-activators with LXXLL Motifs

peptide IC50 (μM)a
SRC2-1 S K G Q T K L L Q L L T C S S 3.9
SRC1-2 T E R H K I L H R L L Q E S S 1.4
SRC2-2 K E K H K I L H R L L Q D S S 2.2
SRC3-2 Q E K H R I L H K L L Q N G N 4.6
SRC1-3 S K D H Q L L R Y L L D K D E 1.6
SRC2-3 K E N A L L R Y L L D K D D 4.8
SRC3-3 K E N N A L L R Y L L D R D D 1.4
SRC1-4 A Q Q K S L L Q Q L L T E 0.9
PGC1α-1 A E E P S L L K K L L L A P A 0.9
PGC1α-2 R R P C S E L L K Y L T T N D 1.1
a
In vitro peptide competition experiments using AlphaScreen Assays (PerkinElmer).

Table 9.2. Peptides Identified as MR Antagonists Using Phage Display

9a V P E P M S M L R A L L S N D D G S G
9b P P P E Q S I L H R L L T A D V S D L
9c L S E T H P L L W T L L S S E G D S M
9d L E E R M P L L S G L L T G T Y L T G
9e D F G P M P L L R S L L E E N I G T G
9f L D H Q F P L L T Q L L R S Y D A G L
9g G E M R M P I L T G L L T S H P Y Q E
9h S C D N S Y C N I R S W F S D R V I S

LY2623091 (structure not disclosed), and MT-3995 (a
Mitsubishi Tanabe Pharma Corporation non-steroidal deriva-
tive in phase II studies for diabetic nephropathy, structure not
disclosed to the best of our knowledge).93 These promising
results have encouraged the search of novel non-steroidal MR
antagonists that overcome the limitations of steroid molecules.
Over the past few years, a variety of tissue-specific MR targets
with prominent roles in several physiological and pathophysio-
logical processes played by MR have been described. Thus, it is
now clear that many of the most important MR target genes are
highly tissue-specific. This, together with the similarities of the
MR with other steroid receptors, highlights the importance of
finding tissue-selective non-steroidal MR antagonists with the
desired therapeutic potential and diminished adverse effects.
The determinants of the MR tissue-specific regulation of genes
are unclear, but presumably the process is at least partially
based on differential interaction with co-regulators of tran-
scription. Therefore, the search of compounds able to bind to
the MR LBD and modulate the recruitment of different
coactivators, together with allosteric modulators of the MR
action targeting protein−protein interaction, may be interesting
areas to explore.
■ AUTHOR INFORMATION
Corresponding Author
*E-mail: mercedes@iqm.csic.es. Telephone: +34 912587478.
ORCID
Mercedes Martín-Martínez: 0000-0002-6449-0905
Notes
The authors declare no competing financial interest.
Biographies
́
Mercedes Martin-Marti ́
nez received her B.Sc. and Ph.D. (1996) in
chemistry from Complutense University in Madrid. After her Ph.D.
she joined the group of Prof. Tom Blundell at Cambridge University
(U.K.), where she got expertise in structure-based computer-aided
drug design. In 1999, she moved back to the Medicinal Chemistry
Institute (IQM-CSIC), where she is currently a staff scientific

2646 DOI: 10.1021/acs.jmedchem.6b01065

J. Med. Chem. 2017, 60, 2629−2650
Journal of Medicinal Chemistry
researcher and a member of the peptidomimetics group. Her research
interests focus on the application of molecular modeling tools to the
design of novel molecular entities able to modulate protein
interactions. One of her currently interests is the search of non-
steroidal modulators of MR.
Felipe L. Pérez-Gordillo received his B.Sc. in chemistry from the
University of Granada, Spain, in 1997, and became a specialist in
clinical biochemistry at Hospital Universitario La Paz, Madrid, in 2004.
He joined the Spanish National Council for Scientific Research
(CSIC) as a research collaborator in 2009. He is currently pursuing a
́
Ph.D. under the supervision of Dr. Mercedes Martin-Marti ́
nez.
a strong interest in drug discovery, especially in small-molecule
He has
inhibitors for MR. His expertise is in the development and synthesis of
druglike compounds and computational modeling.
Diego Á lvarez de la Rosa has been a professor of physiology at
Universidad de La Laguna (Spain) since 2009. He established his
research group at the university as a junior group leader in 2005, after
completing postdoctoral training at Yale University School of
Medicine (1999−2004). His research focuses on mineralocorticoid
hormone actions, with special focus on molecular mechanisms of
receptor modulation and characterization of target genes.
́ Academic Year.
Yoel Rodriguez received his B.S. in chemistry from Havana University
in 1995, and his Ph.D. in chemistry/theoretical biophysics at
Complutense University of Madrid, Spain, in 2002 with Dr. Francisco
Montero Carnerero, both summa cum laude. He conducted
postdoctoral research in computational biophysics with Dr. Roman
Osman at Mount Sinai School of Medicine (MSSM), New York, from
́
2003 to 2007. In 2008, Rodriguez was appointed Assistant Professor of
Physics and Chemistry in the Natural Sciences Department of Hostos
Community College of CUNY, where he is currently Associate
Professor. He is also a visiting professor at MSSM in the Department
of Pharmacological Science, where he collaborates with Dr. Ming-
Ming Zhou. His research focuses on applying computational
theoretical biophysics methods to investigate molecular mechanisms
in biological processes.
Guillermo Gerona-Navarro received his B.S. in chemistry from
Havana University in 1996, and his Ph.D. in chemistry/medicinal
chemistry at Complutense University of Madrid, Spain, in 2002 with
Dr. Rosario Gonzalez-Muñiz, both summa cum laude. He conducted
postdoctoral research with Prof. Samie Jaffrey at Weill Medical College
of Cornell University from 2003 to 2006, and with Dr. Ming-Ming
Zhou at Mount Sinai School of Medicine (MSSM) from 2007 to 2012,
both in chemical biology. In 2013, Dr. Gerona-Navarro was appointed
assistant professor in the Chemistry Department of Brooklyn College
of CUNY. His research laboratory at Brooklyn College focuses on
developing chemical probes targeting protein−protein interactions
inside of the cell to investigate the role of these biologically relevant
proteins in human biology and disease.
Rosario González-Muñiz completed a Ph.D. in chemistry from
Autónoma University in Madrid, Spain (1987), and a post-doctoral
stay at René Descartes University (Paris V, 1988-1990). She currently
is a staff senior scientific researcher at the Medicinal Chemistry
Institute (IQM-CSIC), where she was the Deputy Director (2005−
2011) and is currently the head of the peptidomimetics group. She is a
specialist in peptides, peptidomimetics, and bioactive small-molecules,
and her recent research interests include the design and synthesis of
new modulators of protein−protein interactions and of different types
of ion channels and associated proteins.
Ming-Ming Zhou, Ph.D., is the Dr. Harold and Golden Lamport
Professor and Chairman of Department of Pharmacological Sciences,
and Co-Director of Experimental Therapeutics Institute at Icahn
2647
Perspective
School of Medicine at Mount Sinai. His research interest lies in basic
molecular mechanisms of epigenetic control of gene transcription.
Among his contributions are the discovery of the bromodomain as the
acetyl-lysine binding domain (Nature, 1999) and validation of
bromodomain proteins as drug targets for cancer and inflammation
(Cancer Cell 2014). Dr. Zhou received a Ph.D. degree in chemistry at
Purdue University, did postdoctoral studies at Abbott Laboratories,
and has published over 150 research papers. Dr. Zhou is a Director at
New York Structural Biology Center and a fellow of the American
Association for the Advancement of Science (2012).
■ ACKNOWLEDGMENTS
Supported by grants from Ministerio de Economiá y
Competitividad (MINECO, Spain; grants BFU2012-39092-
C02-02, BFU2013-47089-R, and SAF2015-66275-C2-R), Euro-
pean Cooperation in Science and Technology (COST) action
ADMIRE (BM-1301), the European Union Seventh Frame-
work Program “Capacities” (FP7-REGPOT-2012-CT2012-
31637-IMBRAIN), and CSIC (201280E096). Y.R. is grateful
to CUNY for being a recipient of the CUNY Chancellor’s
Research Fellowship Award for the 2015 - 20162015−2016
■
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2650 DOI: 10.1021/acs.jmedchem.6b01065

J. Med. Chem. 2017, 60, 2629−2650