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§
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
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∥
Department of Chemistry, Brooklyn College, 2900 Bedford Avenue, Brooklyn, New York 11210, United States
■ INTRODUCTION
The mineralocorticoid receptor (MR) is a member of the
include, but are not limited to, (a) morphological remodeling in
epithelia such as the kidney and distal colon; (b) direct
nuclear receptor superfamily of transcription factors.1 The main modulation of cardiac and vascular function and remodeling;
physiological role of the MR is to transduce the effects of (c) regulation of nervous system activity and its control of the
aldosterone, a steroid hormone produced in the adrenal gland cardiovascular and renal systems; (d) regulation of energy
in response to intravascular volume depletion or hyperkalemia. metabolism; and (e) immune responses. Underlying the wide
Aldosterone interaction with MR initiates homeostatic variety of MR physiological and pathological roles are two
responses that oppose the original imbalance. During volume factors. First, the MR is widely expressed in different tissues.
depletion, aldosterone/MR increases renal NaCl reabsorption Second, glucocorticoids such as cortisol can also act as agonists
by activation of the epithelial Na+ channel (ENaC) and the of the receptor,1 particularly in those tissues that lack the
Na+/Cl− co-transporter (NCC). In hyperkalemia, aldosterone- enzyme 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2),
mediated ENaC activation provides an electrical gradient in the which metabolizes cortisol to cortisone, an inactive metabolite.4
renal tubule that potentiates K+ and H+ excretion. Consistent As all other members of the steroid receptor family, the MR
with this fundamental physiological role, inherited gain- or loss- displays a modular architecture. The NH2-terminal domain
of-function mutations in MR, ENaC, and their regulatory (NTD) mediates the interaction with transcriptional co-
proteins cause genetic diseases characterized by disturbances in regulators and displays the highest level of sequence
blood pressure homeostasis and mineral metabolism.2 dissimilarity with other steroid receptors, an essential feature
In addition, during the past few years it has become apparent to determine selectivity in mineralocorticoid responses.
that MR medium- and long-term systemic actions are far wider Following the NTD in the MR primary sequence, there is a
than previously thought.3 Regulated MR function is essential central DNA binding domain (DBD) formed by two zinc
not only to blood pressure and mineral homeostasis but also to
organ and tissue differentiation and morphological and Received: July 17, 2016
functional tissue remodeling in health and disease. These Published: January 4, 2017
fingers. The DBD interacts with specific DNA sequences antagonists can be divided in two categories. The already
named hormone response elements (HREs). The DBD of the mentioned sexual side effects, due to the close similarity in the
MR is 90−94% identical to DBDs of other steroid receptors LBDs of MR and related steroid receptors, make it difficult to
such as glucocorticoid, progesterone, and androgen receptors, develop steroid-derived drugs with high specificity for the MR
implying that HREs recognized by them are highly similar. A over the progesterone or androgen receptors. The second
hinge domain separates the DBD from a COOH-terminal category of the MR adverse effects can be ascribed to the
portion that harbors the ligand-binding domain (LBD) and an undesired interference with aldosterone classical function in
additional region that serves a transcriptional activation regulating electrolyte homeostasis, particularly in promoting K+
function in a ligand-dependent manner. Steroid receptor excretion in the kidney tubule. Indeed, it has been observed
LBDs present moderate identity between them (50−60%), that inappropriate dosage of spironolactone can lead to
and while they allow for specific activation by different hyperkalemia, which in turn is a risk factor for cardiac
hormones, there is some degree of cross-reactivity between complications, particularly in patients with renal insufficiency.19
them. For instance, the MR shows the same affinity for On the other hand, hypokalemia also worsens outcomes in
aldosterone and for glucocorticoids such as cortisol or patients with severe heart failure, and therefore the tendency of
corticosterone (Kd = 0.5−2 nM), although the half-maximal MR antagonists to increase plasma K+ levels may be desirable
effective concentration (EC50) of aldosterone is 100-fold lower, under controlled circumstances.20 To overcome these two
which has been explained due to a much lower off-rate of types of adverse effects, the focus of MR pharmacology has
aldosterone from the MR.5 Other steroids such as progesterone shifted toward developing non-steroidal inhibitors that ideally
act as antagonists of the MR. High-resolution crystal structures would have tissue-specific properties. A third generation of MR
of the MR LBD in combination with agonists 6,7 or antagonists based on non-steroidal compounds is currently
antagonists6,8,9 are available. In addition, a naturally occurring being developed, and some of these products, like the
mutant that causes pregnancy-related hypertension10 and dihydropyridine-like compound finerenone (see below), are
converts progesterone into an agonist has also been crystal- currently under clinical trials.18,21,22 In this Perspective we
lized.6,11 Therefore, a good deal of information about the examine the described non-steroidal ligands collected either in
structural determinants involved in ligand binding to the MR papers or patents. The review aims to be a useful tool for
LBD is available.12 structure−activity relationship (SAR) studies, and therefore
For many years, the main focus of drug discovery targeting includes different families of non-steroid ligands able to bind to
the MR has been on the use of its antagonist spironolactone in the MR, and several compounds within each family.
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,
■ NON-STEROIDAL MINERALOCORTICOID
RECEPTOR LIGANDS
particularly producing sexual dysfunction and gynecomastia in 1. 1,4-Dihydropyridine and 1,4-Dihydro-1,6-naph-
men. In these cases, an alternative is to treat patients with thyridinamide Compounds as MR Ligands. 1,4-Dihydro-
chemically unrelated drugs such as amiloride or triamterene, pyridines (DHP) represent an interesting scaffold for the search
which target a downstream target of the MR, the epithelial of non-steroidal MR antagonis and have attracted the interest
sodium channel (ENaC).13 However, renewed interest in MR of several pharmaceutical companies.
antagonists has been triggered by a combination of new Pfizer, through a screening of its in-house collection,
findings in the last two decades. First, the realization that identified a series of DHPs, previously recognized as calcium
aldosteronism is more widespread in the human population channel blockers (CCBs), as MR antagonists.23 Among them,
than previously thought indicates that MR antagonism will nimodipine and felodipine competed for aldosterone binding
probably expand its scope. Second, it has been firmly and were only slightly less active than eplerenone. As
established that the MR is expressed outside classic aldosterone stereoselective issues affect calcium channel activity, it was
target epithelia and can be considered a ubiquitous receptor. investigated whether the CCB and the MR activities resided in
Pioneering work from Weber’s laboratory using animal models the same DHP stereoisomer. The study of the CCB-
suggested that the MR was an important mediator in the asymmetric DHP mebudipine (1a) indicated that the 4R
development of direct, blood pressure-independent heart enantiomer was responsible for the MR antagonist activity,
fibrosis and inflammation.14 This prompted a clinical trial while the 4S enantiomer had the CCB activity (Table 1.1).24 In
(RALES) where patients with severe heart failure received the agreement with this result, a cyano DHP, identified at Bayer,
aldosterone antagonist spironolactone at doses that did not showed a preference for the R configuration in its binding to
significantly affect blood pressure. The treatment quickly the MR. However, this was also the more active isomer for L-
demonstrated very beneficial effects, with significant decreases type Ca2+ channels.25 Moreover, it was also observed that the
in morbidity and mortality.15 On the other hand, the discovery cyano group at R3 position induced high selectivity against
of eplerenone, a steroid MR antagonist with improved CCB and good selectivity against other nuclear hormone
selectivity over other steroid receptors and therefore displaying receptors (NHRs).26 Based on these results, a library of DHP
less sexual side effects, triggered a second large clinical trial, was prepared to perform SAR studies. Substitution at the DHP
EPHESUS, which further demonstrated the utility of MR NH group decreased activity, suggesting a potential hydrogen
antagonism.16 Recent studies on non-epithelial aldosterone/ bond with the MR.26 Similar results were also reported by
MR effects has expanded the interest in this signaling system to Bayer.25 Regarding the aromatic phenyl ring at position 4,
related pathophysiological settings, such as the vasculature,3 small, nonpolar substituents like F, Cl, or CF3 were suitable at
oxidative stress, inflammation, and metabolic diseases related to the ortho or the para position, with a 2-chloro,4-fluorophenyl
obesity. Given the already important and expanding clinical moiety giving the best results (1b−1e, Table 1.1).26 A
applications, the obvious question is whether new MR voluminous R1 group like phenyl or benzyl decreased potency,
antagonists are needed.17,18 The main adverse effects of MR while smaller C2−C4 alkyl groups were adequate (1c).
2630 DOI: 10.1021/acs.jmedchem.6b01065
J. Med. Chem. 2017, 60, 2629−2650
Journal of Medicinal Chemistry Perspective
Table 1.1. Selected Pfizer DHP MR Antagonists and Their Table 1.2. MR Activity of DHP by Bayer Pharma25,29
IC50 Values24,26
a
Gal4/LBD reporter assay in CHO-K1 cells. bc-Bu = cyclobutyl.
IC50 (nM)a
compd R1 R2 R3 MR GR AR PR
1n CN H H 100
S-1n CN H H 47 6900 2800 5400
S-1o CN H Me 26 5800 2400 4200
S-1p CONH2 H Me 1825 ≥10000 ≥10000 ≥10000
(58)40
a
Functional cell-based transactivation assay. Luciferase assay.
which resides in the known A-ring binding pocket of the steroid introduced at positions 7 and 8 (e.g., 1o).25 Interestingly, the
MR antagonist binding mode, and lipophilic moieties at 3- and/ replacement of the 3-cyano group by a primary amide led to a
or 5-positions (e.g., alkyl esters). Specifically, the NH group of potent MR antagonist with remarkable selectivity toward GR,
the 1,4-DHP ring (e.g., compounds R-1b and R-1e26 and R- AR, and PR (finerenone, BAY 94-8862, 1p).25 This compound
1k27) forms a hydrogen bond with H3 helix Asn770.23 The has higher selectivity than spironolactone and improved affinity
hydrophobic ester group fills the α-face pocket formed by for the MR than eplerenone. Compound 1p was explored on
hydrophobic residues Leu814, Leu827, Phe829, Met845, 65 different enzyme and ion channel assays without significant
Cys849, Met852, and Leu938 (e.g., compounds R-1b, R-1e, effects at 10 μM.25 Clinical studies with 1p suggested that, in
and R-1k). On the other hand, substituents at the opposite site, comparison with spironolactone, a lower dose may be at least as
a methyl and either a CN or COMe group, protrude toward the effective in reducing ventricular remodeling, with lower
β-face pocket (e.g., Ala773, Trp806, Ser810, and Leu 960), incidence of hyperkalemia and renal adverse events.34 More
either directly impinging on Leu960 in H12 helix or indirectly recently it was shown that it was able to protect rats from
by perturbing Trp806 in H5 helix (e.g., compounds R-1b, R-1e, cardiorenal injury.35 These findings provided impetus for
and R-1k). The 4-aryl groups occupy the A-ring pocket, and further clinical evaluation of 1p, which entered in phase II
depending on the DHP derivatives, they interact with Phe829 studies for the treatment of congestive heart failure and diabetic
(e.g., compounds R-1b and R-1e), Arg817, or Gln776 (e.g., nephropathy. The latter phase II trial focused on the effect of
compound R-1k). The formation of hydrogen bonds between 1p on albuminuria in patients with diabetic nephropathy
the DHP derivatives and Arg817 and Gln776 indicates that the receiving an angiotensin-converting enzyme inhibitor or an
4-aryl groups are equivalent to the steroid C3-carbonyl in the angiotensin receptor blocker. The data showed that admin-
A-ring moiety. It has also been shown that DHPs partially
istration of 1p led to an improvement in the urinary albumin-
overlap with the steroidal skeleton of MR antagonists.
creatinine ratio, with a dose-dependent reduction at day 90
Interestingly, the proposed DHP derivatives binding mode
ranging from 21% to 38% when compared to placebo in the
fails to overlap the entire steroid D-ring region, impeding the
dosage group of 7.5 to 20 mg/d compared with placebo.36−38
stabilizing contacts with the H11 helix, including Thr945. Thus,
it has been suggested that the incomplete occupancy and In the second phase II trial, 1p was studied in patients with
stabilization of the receptor-binding pocket, as well as the steric worsening heart failure, who also had diabetes mellitus and/or
clashes caused by the branched DHPs, explain the passive MR chronic kidney disease.39 These patients are at higher risk of
antagonism of these ligands.27 These binding modes have been developing hyperkalemia, so they are less likely to receive
supported by site-directed mutagenesis studies.25,27 The high steroidal MR antagonists. The overall result of these studies
selectivity has been linked to the fact that the MR is the only indicated that 1p reduces the levels of pro-B-type natriuretic
oxosteroid receptor having an alanine residue (Ala773) in the peptide (NT-proBNP), as shown by the decrease of over 30%
H3 helix and a serine residue (Ser810) in the H5 helix, while from baseline found in 38.8% and 34.2% of the patients in the
the AR, GR, and PR have glycine and methionine at their 10 → 20 mg and 15 → 20 mg 1p groups, respectively (initial
corresponding positions, respectively.31,32 For example, the dose → up-titrated dose), a similar extent to that of eplerenone.
DHP derivative R-1k is able to form contacts with Ala773 and Moreover, and similar to eplerenone, 1p showed a good safety
Ser810, while neither glycine nor methionine is able to make profile, with hyperkalemia (serum potassium concentrations
contacts in AR, PR, and GR, thereby explaining their high ≥5.6 mmol/L) only observed in 4.3% of the patients. This
selectivity toward the MR. study also indicated that 10 → 20 mg of 1p is the most suitable
The SAR was further extended with a series of heterobicyclic dosage. The promising results of these phase II studies have
analogues. Among them, 1,4-dihydro-1,6-naphthyridine deriv- allowed the clinical development program for 1p to be
atives, as 1n, can be considered as conformationally frozen expanded with phase III studies for chronic heart failure and
bioisosteres of 1,4-DHP esters (Table 1.3).5,33 In an effort to diabetic kidney disease, as recently announced by Bayer in
improve selectivity vs other NHRs, a methyl group was September 2015.
2632 DOI: 10.1021/acs.jmedchem.6b01065
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Journal of Medicinal Chemistry Perspective
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.
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-
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
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
2635 DOI: 10.1021/acs.jmedchem.6b01065
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the R1 phenyl ring (2ai), as well as the introduction of a ring ion channels (IC50 > 6 μm), and CYPs (IC50 > 19 μm) as well
fused to the phenyl group (2aj) maintained the affinity.54 The as a reasonable rat PK profile.57,59 As previously observed, the
benzyl group at position 5 of the oxazolidinedione ring was also incorporation of a 3,5-dimethoxyphenyl substituent at position
an important element, as its replacement by H led to an R increased potency (2an vs 2am).
inactive compound. The optimization of the group attached to Authors also envisaged the benzimidazole as a replacement of
the amide bond revealed that certain substituents on the benzyl the amide moiety of compounds like 2ag. These benzimida-
moiety (R2, R3, R4) increased the affinity, as in 2ag and 2ah. In zole-based oxazolidine derivatives combined similar MR
contrast, the incorporation of monocyclic or fused bicyclic aryl potency with improved rat liver microsomal activity (Table
or heteroaryl moieties generally led to a decreased MR activity. 2.9).60 The SAR studies with respect to R1 and R2 showed that
Interestingly, an increase in activity was observed for the
cyclohexyl analogue 2ak, the most potent compound within Table 2.9. MR Activity of Benzimidazole-Based Oxazolidine
this series.54 Finally, attempts to change the oxazolidinedione Compounds
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
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.
a
Commercially available protein−protein interaction cell-based
assay.56 small substituents such as halides (2ar, 2ax, 2ay) or
trifluoromethyl (2az) led to modest MR activity, whereas the
hydrolysis, one of the primary metabolic pathways of these incorporation of heteroaryls or heterocycles at R2 led to several
compounds.57−59 The authors analyzed different rings, among compounds with improved IC50 values (2as−2aw).60 These
them imidazole, oxazole, triazole, oxadiazole, and benzimida- derivatives had acceptable human microsomal stability (>50%
zole. Imidazole and 1,3,4-oxadiazole derivatives, 2an and 2ap, of parent compound remaining after 30 min of incubation).
showed improved potency and lipophilic ligand efficiency Moreover, in a natriuresis rat model the effect of compound
(LLE) compared with the 1,2,4-triazole, 2ao. The inclusion of a 2au at 100 mg/kg was comparable to that of spironolactone at
cyclopropyl ring at the benzylic position, as in 2aq, led to an 30 or 100 mg/kg. Authors noted that previous monocyclic
enhancement in human metabolic stability. Both 2ap and 2aq heteroaryls (Table 2.8) exhibited much weaker efficacy in the
showed significant selectivity over other NRs (IC50 > 5 μm), same model.
2636 DOI: 10.1021/acs.jmedchem.6b01065
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Journal of Medicinal Chemistry Perspective
3. Aryl Sulfonamide Derivatives as MR Antagonists. permeability (RRCK, Papp = 10−6 cm/s, 15.4) and metabolic
Pfizer identified an aryl sulfonamide based MR antagonist, 3a, stability (CLint,app = 9.6 μM/min/million cells).
through a HTS of its in-house collection (Figure 3.1).61 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
Figure 3.1. Sulfonamide-based MR antagonist identified through HTS. Table 3.2. MR IC50 Values for Representative Sulfonamide-
Substituted Biaryl Amide Derivatives
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
a
Competitive binding assays using [3H]aldosterone. bLipE = Pic50 −
a
elogD. Competitive binding assays using [3H]aldosterone in a rat renal
soluble fraction in the presence of RU-486 to avoid binding to the
However, both compounds had a low lipophilic efficiency glucocorticoid receptor.
(LipE). Consequently, an optimization process was carried out
to improve this parameter and the metabolic stability. groups could be incorporated while keeping high affinity (e.g.,
Modifications of the R1 substituent led to compound 3c that 3f−3k), whereas substituents at other positions rendered less
showed the best balance of LipE and metabolic stability.61 To active compounds (Table 3.2).63
improve affinity over 3c a sulfonamide library was prepared, 4. Pyridyl Ureas as MR Antagonists. Boehringer
Ingelheim International GMBH studied a series of compounds
from which the replacement of the isoxazole ring with
involving a pyridyl urea linked to a phenyl moiety by a pyrazole,
substituted phenyls afforded derivatives with improved proper- 4a (Figure 4.1), or an ethylene spacer, 4b−4g (Table 4.1).64 In
ties, like 3d and 3e.61 Compound 3e was considered a general, the best results were obtained with the ethylene linker,
promising lead for optimization because it was selective against particularly in the functional assay (Table 4.1). A variety of
PR (>48-fold selectivity), AR (no antagonist activity up to 10 substituents are permitted on the ethylene moiety, at R1, such
μM), and GR (>11-fold selectivity) and exhibited good passive as aliphatic chains or aromatic moieties (4c, 4d, Table 4.1). The
2637 DOI: 10.1021/acs.jmedchem.6b01065
J. Med. Chem. 2017, 60, 2629−2650
Journal of Medicinal Chemistry Perspective
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.
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 6.1. Binding Data of a Series of 1,4-Benzoxazin-3-ones Table 6.2. MR IC50 Values of 1,4-Benzoxazin-3-one-
Derivatives Containing Triazolothidiazine Derivatives
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
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
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
2642 DOI: 10.1021/acs.jmedchem.6b01065
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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 from SRC1, PGC1α, PGC1β, and ASC2 co-activators, which
vivo renal protective activity in a rat model of aldosterone- have an extra leucine before the LXXLL motif in the NR-box.
mediated renal disease.85 However, not all LLXXLL-containing peptides were capable of
Compound 8m showed a good selectivity for binding the strong interactions. In the presence of eplerenone the
MR, from 800- to 7500-fold over GR, AR, and PR, and was recruitment of these peptides is blocked, indicating that
selected for a combination therapy with tadalafil for the eplerenone changes the conformation of the MR LBD.
treatment of resistant hypertension.87 Taladafil is a phospho- Li et al. also focused on peptides derived from known co-
diesterase type 5 (PDE5) inhibitor and behaves as a mild activators containing LXXLL motifs (Table 9.1). They also
vasodilator. Authors also indicated that this therapeutic found a preferential binding of the fourth LXXLL motif of
combination would alleviate PDE5-monotherapy resistant steroid receptor co-activator-1 (SRC1-4).7 In contrast, a weaker
erectile dysfuntion. interaction was established for the first motif of SRC1-1 and
An Eli Lilly and Company derivative, LY2623091 (structure SRC3-1 (over 16 μM).7 Interestingly, the X-ray structure of the
not disclosed), entered phase II clinical studies for the MR LBD bound to corticosterone and SRC1-4 (1.95 Å, PDB
treatment of hypertension and chronic kidney disease. ID 2A3I7) suggested two molecular structural features that
Although to the best of our knowledge there is no publication could account for the high affinity of SRC1-4. First, the
that revealed its structure, on the Internet this compound has glutamic acid at position +7 (relative to the first leucine in the
been associated with derivative 8m.88,89 LXXLL motif) that is hydrogen bonded with K782 of MR.
In 2016, Vitae pharmaceuticals explored several linkers Second, the high stability of the SRC1-4 helix due to the
between the tricyclic scaffold and a series of bicyclic moieties existence of several intramolecular hydrogen bonds between Q
(Table 8.1, 8o, 8p, Figure 8.2).70 The selection of the bicyclic +3 and K−3 as well as Q+2 and S−2.
system was based on previous SAR studies, which suggested the In order to identify antagonist peptides able to interact with
1,4 benzoxazin-3-one (8q), 1,3-benzoxazin-2-thione (8r), and the MR in a ligand-selective manner, Yang et al. recently used
several spirooxindoles (8s−8u) as suitable sub-groups.70 two phage libraries, expressing 19mer peptides, to screen 108
Interestingly, an X-ray structure was described for the MR peptides for MR binding in the presence of three different
LBD complex with compound 8q (2.5 Å, PDB ID 5HCV).70 In agonists: aldosterone, cortisol, and deoxycorticosterone (Table
a similar way to previous X-ray structures, there are two 9.2).91 One of the libraries was designed to target the AF-2
hydrogen bonds involving Asn770 and the NH group and region of the MR, and consequently included a central LXXLL
carbonyl oxygen of the benzoxazinone moiety, besides one motif flanked by seven random amino acids on either side. The
more between the carbonyl group and the Thr945 side chain other was a random library created to identify peptides that
(Figure 8.3). In contrast, no hydrogen bond is observed could bind to any site on the MR. The analysis of the 165
between the receptor and the tricyclic structure. peptides using the mammalian two-hybrid assay showed that 18
9. Peptide MR Ligands. The MR LBD, the key regulatory peptides had an enhanced interaction by the addition of the
domain of MR, contains an activation function-2 domain (AF- ligand, one from the random library and 17 from the LXXLL-
2) in addition to the binding pocket for the endogenous constrained phage library (9a−9d).91 Within these latter
ligands. The AF-2 domain has been implicated in the peptides, approximately 50% of them contained the unique
interaction with co-activators that contain multiple LXXLL motif MPXLXXLL. Six of these 18 peptides were selective for
motifs, organized as α-helices. These co-regulators are critical the MR, five of them from the constrained phage library, and
for MR-mediated gene expression and may confer specificity to interacted significantly better in the presence of aldosterone
MR-mediated responses. Thus, there is a great interest in (9e−9g), while the one from the random library (9h) showed
knowing whether different MR ligands can induce distinct MR increased interaction in the presence of cortisol.91 Thus, these
conformations that might lead to different co-regulator studies have demonstrated that different peptides interact
recruitment and ligand-specific gene regulation. differentially with the MR in the presence of different ligands,
Hultman et al. focused on co-activator and co-repressor thus suggesting that agonists can induce different conforma-
peptides and their interaction with the LBD of the MR in the tional changes in the MR that might lead to the recruitment of
presence of various MR ligands.90 They analyzed 50 co- specific co-regulatory proteins.
regulator peptides, derived from 23 co-activators and co- Rogerson et al., with the aim of identifying co-regulatory
repressors, containing the NR-box or CoRNR-box motifs molecules that could exhibit a ligand-specific interaction with
(small peptide motifs that are necessary and sufficient to the MR LBD, screened a yeast-2-hybrid kidney cDNA library in
interact with NRs). Only a few of these peptides showed strong the presence of aldosterone and cortisol.92 A clone encoding
interactions with the MR LBD in the presence of aldosterone the region of the tesmin (metallothionein-like 5) gene that has
or the glucocorticoid cortisol. These include peptides derived two LXXLL motifs provided a 7-fold greater response in the
2644 DOI: 10.1021/acs.jmedchem.6b01065
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Journal of Medicinal Chemistry Perspective
■ 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-
a
8a−8n: competitive binding assay using [3H]-aldosterone. 8o, 8p: cular injuries. Moreover, some compounds have proved to
MR competitive binding assay using [3H]-cortisol. reduce sex-hormone-related adverse effects and to lower the
incidence of hyperkalemia compared with the marketed steroid-
presence of aldosterone over cortisol. Therefore, tesmin was a like antagonists. Several non-steroidal compounds are currently
ligand-selective co-activator of the hMR, providing further in clinical evaluation for congestive heart failure, hypertension,
evidence of the adoption of ligand-dependent conformations by or diabetic nephropathy. These derivatives belong to different
the MR-LBD. The understanding of these interactions may chemical families and have been designated as 1p, 2k,
2645 DOI: 10.1021/acs.jmedchem.6b01065
J. Med. Chem. 2017, 60, 2629−2650
Journal of Medicinal Chemistry Perspective
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.
LY2623091 (structure not disclosed), and MT-3995 (a coactivators, together with allosteric modulators of the MR
Mitsubishi Tanabe Pharma Corporation non-steroidal deriva- action targeting protein−protein interaction, may be interesting
tive in phase II studies for diabetic nephropathy, structure not areas to explore.
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.
■ AUTHOR INFORMATION
Corresponding Author
Over the past few years, a variety of tissue-specific MR targets *E-mail: mercedes@iqm.csic.es. Telephone: +34 912587478.
with prominent roles in several physiological and pathophysio-
ORCID
logical processes played by MR have been described. Thus, it is
now clear that many of the most important MR target genes are Mercedes Martín-Martínez: 0000-0002-6449-0905
highly tissue-specific. This, together with the similarities of the Notes
MR with other steroid receptors, highlights the importance of The authors declare no competing financial interest.
finding tissue-selective non-steroidal MR antagonists with the Biographies
desired therapeutic potential and diminished adverse effects. ́
Mercedes Martin-Marti ́
nez received her B.Sc. and Ph.D. (1996) in
The determinants of the MR tissue-specific regulation of genes chemistry from Complutense University in Madrid. After her Ph.D.
are unclear, but presumably the process is at least partially she joined the group of Prof. Tom Blundell at Cambridge University
based on differential interaction with co-regulators of tran- (U.K.), where she got expertise in structure-based computer-aided
scription. Therefore, the search of compounds able to bind to drug design. In 1999, she moved back to the Medicinal Chemistry
the MR LBD and modulate the recruitment of different Institute (IQM-CSIC), where she is currently a staff scientific
researcher and a member of the peptidomimetics group. Her research School of Medicine at Mount Sinai. His research interest lies in basic
interests focus on the application of molecular modeling tools to the molecular mechanisms of epigenetic control of gene transcription.
design of novel molecular entities able to modulate protein Among his contributions are the discovery of the bromodomain as the
interactions. One of her currently interests is the search of non- acetyl-lysine binding domain (Nature, 1999) and validation of
steroidal modulators of MR. bromodomain proteins as drug targets for cancer and inflammation
Felipe L. Pérez-Gordillo received his B.Sc. in chemistry from the (Cancer Cell 2014). Dr. Zhou received a Ph.D. degree in chemistry at
University of Granada, Spain, in 1997, and became a specialist in Purdue University, did postdoctoral studies at Abbott Laboratories,
clinical biochemistry at Hospital Universitario La Paz, Madrid, in 2004. and has published over 150 research papers. Dr. Zhou is a Director at
He joined the Spanish National Council for Scientific Research New York Structural Biology Center and a fellow of the American
(CSIC) as a research collaborator in 2009. He is currently pursuing a Association for the Advancement of Science (2012).
́
Ph.D. under the supervision of Dr. Mercedes Martin-Marti ́
nez.
a strong interest in drug discovery, especially in small-molecule
He has
■
Yoel Rodriguez received his B.S. in chemistry from Havana University
in 1995, and his Ph.D. in chemistry/theoretical biophysics at
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