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Modulating Mineralocorticoid Receptor with Non-steroid Antagonists. New Opportunities for the Development of Potent and Selective Ligands without Off-target Side-effects Mercedes Martín-Martínez, Felipe Luis Pérez-Gordillo, Diego Alvarez De la Rosa, Yoel Rodríguez, Guillermo Gerona-Navarro, Rosario González-Muñiz, and Ming-Ming Zhou J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01065 • Publication Date (Web): 04 Jan 2017 Downloaded from http://pubs.acs.org on January 5, 2017

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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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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,a* Felipe L. Pérez-Gordillo,a Diego Álvarez de la Rosa,b Yoel Rodríguez,c,e Guillermo Gerona-Navarro,d

Rosario González-Muñiz,a Ming-Ming,

Zhouc

a

Instituto de Química Médica (IQM-CSIC), Juan de la Cierva, 3, 28006 Madrid, Spain

b

Institute of Biomedical Technologies and Department of Physiology, Universidad de La Laguna, Campus

de Ciencias de la Salud, Facultad de Medicina, La Laguna, Tenerife, Spain. c

Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY

10029, USA. e

Department of Natural Sciences, Hostos Community College of CUNY, 475 Grand Concourse, Bronx, NY

10451, USA. d

Department of Chemistry, Brooklyn College, 2900 Bedford Avenue, Brooklyn, NY 11210.

Abstract Steroidal mineralocorticoid receptor (MR) antagonists are used for treatment of a range of human diseases, but 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 (LBD) of the MR, and recruit different coregulators 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 1 ACS Paragon Plus Environment

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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 last 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.

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Second, glucocorticoids such as cortisol can also act as agonists of the receptor,1 particularly in those tissues that lack the enzyme 11-β 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 fingers. The DBD interacts with specific DNA sequences named hormone response elements (HRE). 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 agonists6, 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 crystalized.6, 11 Therefore, a good deal of information about the structural determinants involved in ligand binding to the MR LBD is available.12

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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 potassiumsparing 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. Pioneer 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 nonepithelial aldosterone/MR effects has expanded the interest on 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 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, 4 ACS Paragon Plus Environment

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makes it difficult to develop steroid-derived drugs with high specificity for the MR over the progesterone or the 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 worsen 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 towards 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 studies, and therefore includes different families of non-steroid ligands able to bind to the MR, and several compounds within each family.

Non-steroidal MR ligands

1. 1,4-Dihydropyridine and 1,4-dihydro-1,6-naphthyridinamide compounds as MR ligands 1,4-Dihydropyridines (DHP) represent an interesting scaffold for the search of non-steroidal MR antagonist, 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

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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 or not. 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 Ltype 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 structure-activity relationship (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, non-polar substituents like F, Cl or CF3 were suitable at the ortho or the para position, with a 2-chloro,4fluorophenyl 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). 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 Table 1.1: Selected Pfizer DHP MR antagonists (IC50 values).24, 26

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2

R

R

R

t

Bu

CO2Me

H

NO2

H

MR IC50 a (nM) 126

Me

t

Bu

CO2Me

H

NO2

H

46

S-1a

Me

t

Bu

CO2Me

H

NO2

H

536

R-1b

Me

t

Bu

CN

Cl

H

F

3.7

S-1b

Me

t

Bu

CN

Cl

H

F

3520

1c

Pr

Me

CN

Cl

H

F

16

1d

Me

CN

Cl

H

F

9

1e

Me

CN

Cl

H

F

1

Compd

R

1a

Me

R-1a

R

3

4

5

6

R

64

b

a

Determined using a Gal4/MR LBD (Gal4/LBD) reporter assay in HUH7 human hepatocyte cells.

b

Chiral resolution of 1e afforded the R-enantiomer (IC50=52 nM)

Bayer Pharma, in an ultrahigh throughput screening of almost one million compounds, found a single

cluster

of

around

one

hundred

MR-modulating

compounds,

including

dihydropyridines.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 1,4dihydropyrimidines,

1,4-dihydropyridazines

and

3,4-dihydropyrimidin-2-ones,

or

the

attachment of highly hydrophilic groups to various positions of the DHP core.25 Several

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substituents had been analyzed at C4 position (Table 1.2), with the fluorenyl moiety B (compound 1g) leading to an increase in potency with respect to the quinoline analogue 1f.25, 28 Subsequent attempts to decrease 4-Ar size and liophilicity allowed the identification of chromenone derivatives (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 dosedependent manner, with effect for a dose as low as 1mg/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

Table 1.2. MR activity of DHP by Bayer Pharma25, 29

1

2

MR IC50

Compd

Ar

R

R

1f

A

CN

i

Pr

310

1g

B

CN

i

Pr

23

1h

C

CN

i

Pr

160

1i

C

COMe

i

Pr

39

(nM)a

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a

R-1j

C

COMe

c-Bu

R-1k

C

COMe

Et

1l

C

COCF3

i

1m

C

b

7 2825 (4329)

Pr

19

Me

18

Gal4/LBD reporter assay in CHO-K1 cells. b c-Bu: cycloButyl

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 deoxicorticosterone (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, 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 R1b 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 towards 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, R1e 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. compounds 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 Aring moiety. It has also been shown that DHPs partially overlap with the steroidal skeleton of 9 ACS Paragon Plus Environment

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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 sitedirected 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 are able to make contacts in AR, PR and GR, thereby explaining their high selectivity towards the MR. The SAR was further extended with a series of heterobicyclic analogues. Among them, 1,4dihydro-1,6-naphthyridine derivatives, 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 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 10 ACS Paragon Plus Environment

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inhibitor or an angiotensin receptor blocker. The data showed that administration 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→uptritrated dose), a similar extent to that of eplerenone. Moreover, and similar to eplerenone, 1p showed a good safety profile, with hyperlkalemia (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.

Table 1.3. IC50 of naphthyridinamides derivatives on several steroid hormone receptors.

IC50 (nM) Compd

1

R

2

R

a

3

R

MR

GR

AR

PR

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1n

CN

H

H

100

S-1n

CN

H

H

47

6900

2800

5400

S-1o

CN

H

Me

26

5800

2400

4200

≥ 10000

≥ 10000

≥ 10000

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1825 S-1p

a

CONH2

H

Me (58)40

Functional Cell-based transactivation assay. Luciferase assay.

Docking studies of S-1p at the MR LBD X-ray structure (PDB ID: 2ABI9 devoid of helix 12) indicated a similar binding mode as the above-mentioned for other DHP derivatives, forming hydrogen bonds with Asn770, Gln776 and Arg817.40 Moreover, it confirmed the key role 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 LDB 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 submicromolar binding affinity (i.e. 1q – 1t, Table 1.4).41 Table 1.4. Binding affinity of selected Merck DHPs for the MR

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Compd

1

Ar

R

2

R

3

MR Ki a

R

(nM) 1q

Naphthyl

Me

CO2Me

1r

2-Cl-Ph

SEt

CN

53.0

1s

2-Cl-Ph

SPr

CN

14.9

1t

3-OMe-4-OH-Ph

Me

CO2Ph

a

CO2Me

CO2Ph

61.0

21.5

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-2carboxamide structural core with different substituents attached. Table 2.1. Pyrrole derivatives as MR antagonist. IC50 lower than 500 nM.

Compd

X

Y

N

CH

CH2-CH2-Ph

2a 2b

R

3-Cl-4-Me-Ph

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naphthyl

2c

2d

2e a

C

NH

Gal4/LBD cell-based reporter assay

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 atroposiomers exhibited high 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 1.000-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-induce decrease in urinary Na+/K+ ratio in rats by 56% at 20 h, whereas spironolactone decline at 20 h of least than 20% and eplerenone at 8h of around 8%, 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, 3mg/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. Table 2.2. IC50 values of 1H-pyrrole 3-carboxamide MR inhibition

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Compd

X

Y

1

R

R

3

R

MR IC50 a (nM) b A+B Ac

CF3

Cl

11

3.7

CF3

Cl

6.2

3.1

2h

4-F-Ph

Cl

2.7

1.2

2i

OCHF2

F

14

6.5

CF3

F

6.2

3.8

CF3

H

-

9.4

2f 2g N

C

2j C

N

2k a

2

Gal4/LBD cell-based reporter assay.

atropoisomers A and B.

c

b

Mixture of

The corresponding 2f-2k

atropoisomers have an IC50 >1000 nM

2.2 Pyrazoline derivatives Pfizer identified pyrazoline 2l (Figure 2.1) as an acceptable MR antagonist during a highthroughput screening (HTS) campaign.47 Starting from this hit, and in an effort to improve activity and solubility, they analysed 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 improvement 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 group led to potent MR antagonists with reduced hERG channel inhibitory potency. At position 5, the R configuration was the most favored (R-m vs S-2m, Table 2.3), and the replacement of the 4-fluorophenyl group by a

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Page 16 of 68

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

Figure 2.1 Pfizer pyrazoline derivative 2l Table 2.3. MR pyrazoline antagonists

Compd

1

R

2

R

3

R

4

MR IC50

R

(nM) 2m

H

H

H

246

R-2m

H

H

H

101

S-2m

H

H

H

8360

2n

Cl

H

H

56

2o

CH3

H

H

56

2p

H

H

H

151

R-2q

Cl

H

H

16

R-2r

Cl

H

OMe

14

R-2s

Cl

OMe

OMe

2

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R-2t a

Cl

H

OEt

6

Gal4/LBD cell-based reporter assay.

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 stereochemistry 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 kidney 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 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 itself could explain their MR antagonism, because it has previously been suggested that MR steroidal activation, in addition to the C3ketone 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). Table 2.4. IC50 values of conformationally restricted pyrazoline derivatives.

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Compd

R1

R2

MR IC50

Page 18 of 68

a

(nM) cis-2u

Cl

223

3R,3aR-2u

Cl

41

cis-2v

Me

4

cis-2w

Cl

21

3S,3aR-2w

Cl

9

a

Gal4/LBD cell-based reporter assay .

In spite of the good properties of 3S,3aR-2w, there was still room for improvement. To this end, Pfizer focused again on non-conformationally restricted pyrazoline analogues, in particular R-2r, because of its increased selectivity over PR and its improved solubility.50 The MR potency and selectivity rose by the replacement of the 1-(3-chlorophenyl) ring by the corresponding 3-methyl-substituted analogue, and even more by incorporation of a nitrogen in the benzoic acid moiety, resulting in compound 2x, with a selectivity over PR greater than 500fold (Table 2.5).50 The incorporation of a nitrogen within 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 On the contrary, acylsulfonamide analogues (2aa, 2ab) increased the MR potency while maintaining low PR activity.50 Thus, compound 2aa displayed greater than 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 18 ACS Paragon Plus Environment

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2x was dose-dependent. This compound behaved as a MR antagonist, with good selectivity over others NHRs and improved solubility in comparison with 2w, and showed in vivo efficacy. Therefore, 2x was selected for further preclinical profiling.50

Table 2.5. Activity of pyrazoline derivatives in the MR and the PR

a

Solubility

MR

PR

(µM)

IC50 (nM) Compd

a

Y

b

R

2x

CH

OH

4.5

2530

312

2y

N

OH

7.6

1590

324

2z

CH

NH2

2.7

494

40 µM), as well as the positive contribution of a methyl group as R1 substituent (2ag-2ai).54 According to molecular modelling studies, this methyl plays a key role in favouring the conformation which is preferred for receptor binding. The incorporation of fluoro- or chloro- substituents on 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. On the contrary, the incorporation of monocyclic or fused bicyclic

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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 greater than 20 µM and IC50 antagonist mode assay greater than 8 µM).54

Table 2.7. MR ligands with an oxazolidinedione central scaffold.

Compd

a

R

1

R

2

R

R

MR IC50a

3

4

2af

H

H

H

(nM) 6000

2ag

OMe

H

OMe

54

2ah

H

CN

H

37

2ai

OMe

H

OMe

100

2aj

OMe

H

OMe

33

2ak

OMe

H

OMe

14

Values obtained using a commercially available protein-protein interaction cell-based

assay.56

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Next, aiming at improving physicochemical, 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 hydrolysis, one of the primary metabolic pathways of these compounds.57-59 The authors analyzed different rings, among them imidazole, oxazole, triazole, oxadiazole and benzimidazole. Imidazole and 1,3,4-oxadiazole derivatives, 2an and 2ap, showed improved potency and lipophilic ligand efficiency (LLE) compared with 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), ion channels (IC50 > 6 µm) and CYP’s (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).

Table 2.8 IC50 of oxazolidinedione derived

MR

heteroaromatic

ligands

with

substituents

at

position 5.

MR IC50a Compd

R

X

Y (nM)

2al

Bn

O

CH

240

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NH

CH

270

2an

NH

CH

68

2ao

NH

N

670

2ap

O

N

39

2aq

O

N

82

2am

a

Bn

Commercial available protein-protein interaction cell-based assay.56

Authors also envisaged the benzimidazole as a replacement of the amide moiety of compounds like 2ag. These benzimidazole-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 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 100mg/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.

Table 2.9 MR activity of benzimidazole-based oxazolidine compounds

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Compd

1

R

R

2

MR IC50

a

hLM

(nM)

(rLM)b

160

55 (14)

2as

42

97 (63)

2at

33

72 (26)

2au

40

81 (92)

2av

34

94 (45)

2aw

36

68 (23)

72

57 (0)

130

66 (6)

150

77 (51)

Cl

2ar

Page 24 of 68

H

a

2ax

F

2ay

Cl

2az

CF3

H

Commercially available protein-protein interaction

cell-based assay.56 b Percentage of compound (1µM) remaining at 0.5 h and incubation in human hLM and rat (rLM) liver microsomes.

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 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 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

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which the replacement of the isoxazole ring with substituted phenyls afforded derivatives with improved properties, like 3d,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 permeability (RRCK, Papp, 10-6 cm/s, 15,4) and metabolic stability (CLint,app=9.6 µM/min/million cells).

Figure 3.1. Sulfonamide based MR antagonist identify through HTS Table 3.1. MR binding (IC50) and physicochemical properties of aryl sulfonamide derivatives

MR Compd

R

1

2

R

IC50

a

LipE

b

elogD

62

(nM)

3b

356

2.3

4.1

3c

4698

3.8

1.5

3d

342

4.5

2

3e

191

4.4

2.3

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a

Page 26 of 68

Competitive binding assays using [3H]aldosterone. bLipE=Pic50-elogD

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 comprised among those with the higher activity (Table 3.2). Additionally, different R1 and R2 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

Table 3.2. MR IC50 values for representative sulfonamide substituted biaryl amide derivatives.

MR Compd

L

R1

R2

IC50a (nM)

3f

CH2OCOCH3

CF3

31

3g

CH2OH

OCF3

60

3h

CH2OH

84

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3i

OCF3

22

3j

OCF3

61

CF3

100

3k a

CH2OCH2CH3

Competitive binding binding assays using [3H]aldosterone in a rat renal soluble fraction in the

presence of RU-486 to avoid binding to the glucocorticoid receptor.

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 incorporation of F at R3, as in 4e, improved binding affinity, and led to derivatives with binding IC50 values below 30 nM, whereas the substitution by CH3, CF3 or OCH3 led to values over 100 nM, as in compound 4f.

Figure 4.1. An example of a ([pyrazol-3-yl]pyridine-2-yl)-urea MR antagonist

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Table 4.1. IC50 values of MR binding and functional assays of pyridyl ureas.

Compd

Y

R1

R2

MR Binding

MR Functional

IC50 (nM)a

IC50 (nM)b

7.1

19

R3

4b

H

Me

H

4c

Ph

H

H

13

44

Me

Me

H

11

43

4e

H

Me

F

4f

H

H

Me

480

2800

H

OMe

H

25

97

4d

4g a

X

N

CH

CH

N

4.7

31

MR competitive molecular binding assay based on the binding and displacement of a TAMRA-

labeled Dexamethasone probe with fluorescence polarization (FP) detection.

b

Commercially

available Gal4/LBD cell-based reporter assay (GeneBLAzer® UAS-bla HEK 293 cell line).

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 supporting an indole ring and at least another aromatic group.

5.1. 3,3-Bisaryloxoindole derivatives

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SAR studies on the oxoindole derivative 5a, a human MR (hMR) ligand identified through screening (Table 5.1),65 showed the importance of the 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 other steroid receptors (GR, PR, AR and ER).65 The authors suggested that the presence of a hydroxy group at R4 forces a conformation that allowed the discrimination between the binding pockets of MR and GR. The isolation of the 5g enantiomers showed that the (-)-isomer was more potent and selective, than the (+)-isomer.

Table 5.1. hMR binding of bisaryloxoindol derivatives

Ki (nM) Compd

n

1

R

2

R

3

R

4

R

a

hGR

hPR

hAR

hERα

hERβ

-

-

-

-

b

733

>1100

hMR H

Me

Me

H

65

-

H

Me

Me

H

27

147

Me

Me

Me

H

7

84

138

306

183

844

5d

OMe

Me

Me

H

0.6

51

715

402

242

930

5e

OMe

H

H

OH

3

793c

678

19%b

>1100

>1100

5a

1

5b 5c

44%

b

41%

0

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5f

OMe

H

Me

OH

1

693

c

395

1105

>1100

>1100

5g

Me

H

Me

OH

2

334

245

37%

579

>1100

5g(-)

Me

H

Me

OH

1

582

33%

b

359

>1100

5g(+)

Me

H

Me

OH

40

686c

49%

20%

519

>1100

a

16%

Competitive binding assay using [3H]aldosterone b Percentage of inhibition at 1 µM antagonist

concentration. c One determination.

5.2. Indol-7-yl-methanesulfonamide derivatives Bell et al. explored the use of indole-derived compounds as modulators of steroid hormone nuclear receptors.66 In particular, they focused on the MR and the GR. It is worth noting that the great majority of the compounds reported interact both with the MR and the GR with Ki values lower than 500 nM (Table 5.2). In general, these compounds have two substituents on the indole 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 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,

benzothiophene,

benzodioxin,

benzodioxol,

benzoimidazol, benzothiazole, benzooxazol, indazol or 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 lower than 500 nM, the SAR studies showed the favorable effect of a proton donor at position 7 of the indole ring, particularly a methane 30 ACS Paragon Plus Environment

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sulfonamide moiety, and the best combination for the dialkyl substituents, was methyl/cyclopropyl (5n-5q Table 5.2).

Table 5.2. Indol-7-yl-methanesulfonamide derivatives with Ki equal or lower than 500 nM for the MR. Ki values were calculated with a competitive binding assay using radioligands.66, 67

MR Ki Compd

R (nM)

5h

≤ 500

5i

≤ 500

5j

≤ 500

5k

Ki (nM) Compd

R1

R2 MR

GR

5n

H

H

≤ 500

≤ 500

S-5o

H

F

1.56

167

R-5o

H

F

7.5

387

≤ 500

S-5p

F

F

0.49

8.9

5l

≤ 500

5q

H

Me

≤ 500

≤ 500

5m

≤ 500

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 better in vitro selectivity profile than spironolactone.

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Page 32 of 68

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, 5r5y, 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).

Table 5.3. MR antagonist activity of indazole derivatives

MR IP Compd

R

1

2

R

3

4

R

R

R

a,b

5

(nM) 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

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a

IP: Inflection point of the non-linear regression curve. The IP

value is equal to the IC50 value when the slope is 1, and the minimum is 0 and the maximum is 100.

b

Commercially

available protein-protein interaction cell-based assay.56

Table 5.4. PK profile in rats and metabolic stability for indazole derivatives

3.6

Human liver microsome stability t1/2 (min) 48

Human hepatocyte stability t1/2 (min) 69

24

5.3

72

>90

3.8

9.9

62

>90

Compd

Cl (mL/min/kg)

t1/2 (h)

5r

18

5t 5w

6.

Benzoxazinones-derived MR ligands

6.1. 1,4-Benzoxazin-3-ones The 1,4-benzoxazin-3-one bicycle is an 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 bezoxazin-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 with smaller substituents, such as methyl or fluorine. Several of these derivatives were able to bind to the MR (Table 6.1, compound 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, 6e.70

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Table 6.1. Binding data of a series of 1,4benzoxazin-3-ones derivatives

Compd

R1

R2

R3

MR IC50 (nM)

6a

H

H

10.000

6o

Me

H

H

O

120

740

3.800

6p

Me

H

H

N-Et

33

770

1.600

6q

Me

H

H

N-c-Prb

68

400

2.200

6r

Me

H

H

NH

94

5.600

9.100

6s

Me

H

Cl

NH

23

1.600

1.100

6t

Me

H

Me

NH

43

>10.000

4.900

Competitive binding assay using [3H]-Aldosterone, [3H]-Progesterone or [3H]-Dexamethasone

to test binding to MR, PR and GR, respectively. IC50 binding to AR was greater than 10.000 nM in all cases. b c-Pr: cyclopropyl

A

B

C

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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 color). (B) 6o (S-enantiomer, pink color). (C) 6z (blue color) (PDB IDs: 3WFF, 3WFG and 4PF3, respectively). The hydrogen bonds and water molecules are depicted as yellow dashed lines and red spheres, respectively.

In functional assays, compound 6t and most of the dihydropyrrol-2-one derivatives showed also moderate partial agonistic activity at high concentrations (20-30 % at 10 µM), whereas the values obtained for pyrazole analogues, as 6k, were low. The X-ray structure of the MR LBD bound to 6o (PDB ID: 3WFG),73 close analogue of 6t, suggested a role of the central ring carbonyl group in a water-mediated hydrogen bonding network. Since this network was considered not to be present in 6k, it was assumed that it was important for the partial agonist effect. As clinical efficacy and safety of partial agonists remains unclear, other azole rings, with no possibility to form these water-mediated hydrogen bonds, were selected, with the pyrazole scaffold providing the best MR activities (6u-6z, Table 6.5).74,

75

However, derivatives 6v-6x

also showed high 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 partial agonistic activities and high selectivity over other steroid receptors, like 6y, 6z (binding IC50 AR and PR>10.000 nM, GR>1.700 nM).75 Compound 6z showed an acceptable metabolic clearance (42

µL/mg/min) and it was able to lower the blood pressure in DOCA-salt hypertensive rats, without antiandrogenic effect. The crystal structure of the complex between 6z and the MR LBD (1.10 Å PDB ID: 4PF3)75 showed that it binds to the steroid binding pocket of the MR. As expected, the binding mode of compound 6z is similar to that of compounds 6n (Figure 6.3 C, right). The NH group and the carbonyl oxygen of the benzoxazin-3-one moiety form hydrogen bonds to Asn770 and Thr945, and the 4-fluorobenzene ring occupies the α-face hydrophobic 41 ACS Paragon Plus Environment

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pocket. The ligand side chain at the 1-position of the pyrazole ring, the 2,2-difluoropropyl-3hydroxy group, points out towards Arg817 and Gln776. Its hydroxyl group directly forms a hydrogen bond to Gln776. The two fluorine atoms do not form any specific hydrogen bonding interactions suggesting that the major contribution of these fluorine atoms to the binding is hydrophobic.

Table 6.5. MR activity of 1,4-benzoxazin-3-one-containing pyrazole derivatives

Compd

a

MR bindinga

MR activity b

IC50 (nM)

IC50 (nM)

R

6u

n-Pr

22

45 (0 %)

6v

CH2CH2CF3

8.4

33 (6 %)

6w

CH2CF2CH3

10

18 (4 %)

6x

CF2CH2CH3

5.8

27 (11 %)

6y

3-Py

36

36 (-4 %)

6z

CF2CH2OH

51

71 (1 %)

Competitive binding assay using [3H]-aldosterone

b

Cell-based reporter gene assay. Antagonist activity and, in brackets, agonist activity at 10 µM concentration.

Pfizer has also explored compounds with a morpholine central ring (Table 6.6). They observed that the incorporation at morpholine N4 of benzoxazin-3-one, 6aa, or pyridoxazinone moieties,

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6ab-6ae, led to derivatives with IC50 values below 100 nM, nevertheless the former provide the most active derivative.76 There was a decrease in affinity caused either by the removal of the aromatic ring at position 5 (IC50=3.050 nM) or the methyl at position 2 (IC50=1.970 nM) in derivative 6ab. It was also observed that the incorporation of a fluorine atom in the ortho or meta-position of the phenyl ring keeps the affinity (6ac, 6ad), whereas it is detrimental in the para-position.76

Table 6.6. MR IC50 of 1,4-benzoxazin-3-one-containing morpholine derivatives

Compd

1

R

2

R

MR IC50a X

Y (nM)

a

6aa

H

H

6ab

H

H

6ac

F

H

6ad

H

F

6ae

F

H

CH

CH

24 44.4

N

CH

33.4 55

CH

N

53.9

Gal4/LBD cell-based reporter gene assay.

6.2. 1,3-Benzoxazin-2-ones 1,3-Benzoxazin-2-one and -2-thione derivatives also provided compounds able to interact with the MR, which are collected in two Knobbe Martens Olson & Bear LLP patents77, 78 and one from Dainippon Sumitomo Pharma Co (6af, SM-368229, Figure 6.2).79 In particular, a study on

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the pharmacological profile of 6af indicated that this compound was able to increase urinary Na+/K+ ratio in adrenalectomized rats treated with deoxycorticosterone acetate. Even at doses of 300 mg/kg, only very weak antiandrogenic effects were observed in methyltestosteronetreated male rats.79

Figure

1,3-Benzoxazin-2-ona

6.2.

derivative from Dainippon Sumitomo Pharma Co

Mitsubishi Tanabe has investigated related 2H-1,3-benzoxazine (Y=N, 6ag, 6ah) and 2Hchromene (Y=C, 6ai, 6aj) derived compounds, reporting several compounds that showed submicromolar MR dissociation constants (Table 6.7).80 Most of them have a phenyl ring at position 4 of the scaffold, although there is an example with a benzothiophene ring. The most frequent substituents at position 2 are methyl groups, whereas either methyl or hydrogen are suitable at position 5. In the chromene (Y=C) scaffold, H, CN, or Br are adequate as R4 substituents for interacting with the MR (Ki < 500 nM).

Table 6.7. Examples of 2H-1,3benzoxazine

and

2H-chromene

derivatives with MR Ki values below 500 nM. 44 ACS Paragon Plus Environment

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Compd

Y

R1

R2

R3

R4

6ag

N

Cl

H

H

-

6ah

N

F

H

Me

-

6ai

C

F

H

H

Me

6aj

C

Cl

Me

H

H

Competitive binding assay using [3H]-aldosterone.

7.

Macrolide MR antagonists

In an screening program to search for non-steroidal MR antagonist, compound 7a, methyllasiodiplodin, was found and reported as the first macrolide MR antagonist (Table 7.1).81 The antagonist effect 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

Table 7.1. Inhibitory activity of Omethyllasiodiplodin analogues

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Compd

R

7a

a

1

2

R

R

H

H

7b

Ac

7c

Ac

3

n

IC50 (nM)

Me

5

8930

Ac

Me

5

2780

Ac

H

4

580

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a

Values determined using a yeast two-hybrid assay to asses inhibition of MR interaction with a

co-regulator.

8.

Fused heterotricyclic derivatives as MR ligands

Eli Lilly and Company described a series of tricyclic steroid hormone receptor modulators with the capacity to bind to the MR and the GR with Ki≤500 nM. The general structures are shown in Figure 8.1. For derivatives of general formula III, the tricyclic structure of the reported compounds with Ki lower than 500 nM corresponds mainly to a thioxanthene scaffold (X=S), but there are also examples in which X is a methylene group or an oxygen atom. R1 is usually hydrogen or methyl, and Ar is an aromatic group as phenyl, benzimidazol-2-one, benzoxazol-2one or benzothiazol-2-one.82 A tricyclic structure with a seven membered ring has also been explored, structure IV (Figure 8.1), and the authors reported almost 200 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 46 ACS Paragon Plus Environment

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phenyl, benzimidazol-2-one, benzimidazol, indolyl-2-one, benzoxazol-2-one or benzothiazol-2one. Subsequent patents focused on a tricyclic scaffold with a central seven membered ring of general formula V (Figure 8.1 and Table 8.1). A 2005 patent described a series of derivatives with either a tricyclic dihydrodibenz[b,e]oxepine (X=O) or dihydrodibenz[a,d][7]annulene (X=CH2) showing good Ki values.84 For the active 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 Within this family, compound 8a showed good affinity for the MR and selectivity over the AR, BR and PR, and behaved as a potent and selective antagonist of the hMR (IC50= 21 nM in functional assays).86 In in vivo models, 8a displayed potent reno-protective activity, antihypertensive effects and a reduced probability of producing hyperkalemia.

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.

In 2009 Eli Lilly and Company focused on a series of dihydrodibenz[b,e]oxepine based on the previously reported tricyclic steroid hormone receptor modulators (Figure 8.1, IV and V).83, 84 Representative compounds with Ki ≤10 nM are collected in Table 8.1. At R3 position several heterocycles led to compounds with significant activity, where R4 was O, N-CN or 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 R2. In general

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for each R3 and R4 substituent the corresponding 3-fluoro (R1=H, R2=H), 3,7- (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 The authors also carried out an exhaustive modification of the configuration of the chiral centers of R3 substituents, and for the reported examples, Ki values were below 10 nM regardless of their stereochemistry (8a-8c vs 8d-8f, or 8h-8k).85,

86

Several of the prepared

compounds were tested in a functional assay and behaved as antagonist of the hMR. Moreover, three of them, namely 8b (0.79 nM), 8g (0.08 nM) 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 7.500 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 phosphodiesterase type 5 (PDE5) inhibitor and behaves as 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 Table 8.1. Tricyclic compounds with Ki ≤ 10 nM for hMR

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Kia Compd

1

R

2

R

3

R

R

4

(nM) 8a

H

H

0.40

8b

H

F

0.79

8c

F

H

≤ 10

8d

H

H

≤ 10

8e

H

F

≤ 10

8f

F

H

≤ 10

8g

H

H

0.08 N-CN

8h

0.23

8i

≤ 10 F

H

8j

≤ 10

8k

≤ 10

8l

H

H

≤ 10

8m

H

H

0.33 N-CONH2

8n

H

H

≤ 10

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a

8o

H

H

8p

H

H

H

O

12

O

9

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3

8a-8n: Competitive binding assay using [ H]-aldosterone. 8o,

8p: MR Competitive binding assay using [3H]-cortisol.

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), 1,3benzoxazin-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). On the contrary, no hydrogen bond is observed between the receptor and the tricyclic structure.

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.

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A

B

Figure 8.3. (A) Close-up view of the X-ray structure of the MRC808S/S810L-LBD double mutant bound to 8q (cyan color) (PDB IDs: 5HCV). (B) Superimposition of the X-ray structures of compound 8q (cyan color) and spironolactone (orange color) bound to the MRC808S/S810L-LBD (PDB ID: 5HCV and 3VHU, respectively). The hydrogen bonds and water molecules are depicted as yellow dashed lines and red spheres, respectively.

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 coactivators that contain multiple LXXLL motifs, organized as α-helices. These coregulators 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 coregulator recruitment and ligand-specific gene regulation. Hultman et al. focused on coactivator and corepressor peptides, and their interaction with the LBD of the MR in the presence of various MR ligands.90 They analyzed 50 coregulator peptides, 51 ACS Paragon Plus Environment

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derived from 23 coactivators and corepressors, 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 from SRC1, PGC1α, PGC1β and ASC2 coactivators, 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 coactivators containing LXXLL motifs. They also found a preferential binding of the fourth LXXLL motif of steroid receptor coactivator-1 (SRC1-4).7 On the contrary, 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.

Table 9.1. Peptides derived from coactivators with LXXLL motifs. IC50

a

Peptide

µM SRC2-1

S KGQT KL L QLLT C S S

3.9

SRC1-2

T E R H K I LH R LLQE S S

1.4

SRC2_2

K E K H K I LH R LLQD S S

2.2

SRC3-2

QE K H R I LH K LLQNGN

4.6

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SRC1-3

S KDHQLLR Y LLDKDE

1.6

SRC2-3

K ENALLR Y LLDKDD

4.8

SRC3-3

K ENNALLR Y LLDRDD

1.4

SRC1-4

AQQK S LLQQLL 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 (Perkin Elmer).

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.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 conformational changes in the MR that might lead to the recruitment of specific coregulatory proteins.

Figure 9.2. Peptides identify as MR antagonist using phage display. 53 ACS Paragon Plus Environment

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Peptide 9a

VPE P MSML R AL L SNDDF S G

9b

PP P E Q S I L H RL L TADVS D L

9c

L S E T H P L L WT L L S S EGD SM

9d

L E E R MP L L S GL L TG T Y L T G

9e

DF G PMP L L R S L L E E N I G T F

9f

LDH Q F P L L T QL L RS YDAG L

9g

GEMR MP I L T GL L T S HP YQ E

9h

S C D N S Y C N I RSWF S DRV I S

Rogerson et al., with the aim of identifying coregulatory molecules that could exhibit a ligandspecific 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 presence of aldosterone over cortisol. Therefore, tesmin was a ligand-selective coactivator of the hMR, providing further evidence of the adoption of ligand-dependent conformations by the MR-LBD. The understanding of these interactions may 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

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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 last years this search has been intensified and has mainly been undertaken by pharmaceutical companies. In general, the researches have 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, selectivity as well as pharmacokinetic properties. These medicinal chemistry approaches 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, selectivity against other nuclear receptors and displayed in vivo activity. Recently X-ray structures, describing not only the MR LBD 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 improve 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 cardiovascular 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

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different chemical families and have been designated as 1p, 2k, LY2623091 (structure not disclosed), MT-3995 (a Mitsubishi Tanabe Pharma Corporation non-steroidal derivative 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 pathophysiological 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 coregulators of transcription. 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.

Acknowledgements Supported by grants from Ministerio de Economía y Competitividad (MINECO, Spain; grant BFU2012-39092-C02-02, BFU2013-47089-R and SAF2015-66275-C2-R), European Cooperation in Science and Technology (COST) action ADMIRE (BM-1301), the European Union Seventh Framework Program “Capacities” (FP7-REGPOT-2012-CT2012-31637-IMBRAIN) and CSIC (201280E096). YR is grateful to CUNY for being a recipient of the CUNY Chancellor’s Research Fellowship Award for the 2015 - 2016 Academic Year.

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Mercedes Martín-Martínez 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), she is currently a staff scientific research at this institute, and a member of the peptidomimetics group. Her research interests focused on the application of molecular modelling tools to the design of novel molecular entities able to modulate protein interactions. One of her currently interest 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. 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 Martín Martínez. He has a strong interest in drug discovery especially in small molecule inhibitors for MR. His expertise is in the development and synthesis of druglike compounds and computational modeling. Diego Alvarez de la Rosa is a Professor of Physiology at Universidad de La Laguna (Spain) since 2009. He established his research group in this 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. Yoel Rodríguez 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 with Dr. Roman Osman at Mount Sinai School of Medicine (MSSM) in NY, U.S. in Computational Biophysics from 2003 to 2007. In 2008, Rodríguez was appointed Assistant

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Professor of Physics and Chemistry in the Natural Sciences Department of Hostos Community College of CUNY; currently Associate Professor. He is 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 Prof Ming-Ming Zhou at Mount Sinai School of Medicine (MSSM) from 20072012, both in Chemical Biology, in NY, U.S. 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 proteinprotein 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). She is currently the head of the peptidomimetics group at the IQM-CSIC. Specialist in peptides, peptidomimetics and bioactive small-molecules, 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. Zhou Ming-Ming

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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 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 PhD degree in Chemistry at Purdue University and did postdoctoral study 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 American Association for the Advancement of Science (2012). Corresponding Author information: +34 912587478, [email protected] REFERENCES

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33. Bärfacker, L.; Kolkhof, P.; Schlemmer, K. H.; Grosser, R.; Nitsche, A.; Klein, M.; Münter, K.; Albrecht-Küpper, B.; Hartmann, E. Substituted-4-aryl-1,4-dihydro-1,6-naphthyridinamides and use thereof. US20100136142A1, 2010. 34. Pitt, B.; Kober, L.; Ponikowski, P.; Gheorghiade, M.; Filippatos, G.; Krum, H.; Nowack, C.; Kolkhof, P.; Kim, S.-Y.; Zannad, F. Safety and tolerability of the novel non-steroidal mineralocorticoid receptor antagonist BAY 94-8862 in patients with chronic heart failure and mild or moderate chronic kidney disease: a randomized, double-blind trial. Eur. Heart J. 2013, 34, 2453-2463. 35. Kolkhof, P.; Delbeck, M.; Kretschmer, A.; Steinke, W.; Hartmann, E.; Bärfacker, L.; Eitner, F.; Albrecht-Küpper, B.; Schäfer, S. Finerenone, a novel selective nonsteroidal mineralocorticoid receptor antagonist protects from rat cardiorenal injury. J. Cardiovascular Pharm. 2014, 64, 69-78. 36. Bakris, G. L.; Agarwal, R.; Chan, J. C.; Cooper, M. E.; Gansevoort, R. T.; Haller, H.; Remuzzi, G.; Rossing, P.; Schmieder, R. E.; Nowack, C.; Kolkhof, P.; Joseph, A.; Pieper, A.; Kimmeskamp-Kirschbaum, N.; Ruilope, L. M. Effect of finerenone on albuminuria in patients with diabetic nephropathy: A randomized clinical trial. JAMA 2015, 314, 884-894. 37. Haller, H.; Bertram, A.; Stahl, K.; Menne, J. Finerenone: a new mineralocorticoid receptor antagonist without hyperkalemia: an opportunity in patients with CKD? Curr. Hypertension Rep. 2016, 18, 1-9. 38. Feng, Y.; Zeng, X.; Fu, P. FInerenone for albuminuria in patients with diabetic nephropathy. JAMA 2016, 315, 305-306. 39. Filippatos, G.; Anker, S. D.; Böhm, M.; Gheorghiade, M.; Køber, L.; Krum, H.; Maggioni, A. P.; Ponikowski, P.; Voors, A. A.; Zannad, F.; Kim, S.-Y.; Nowack, C.; Palombo, G.; Kolkhof, P.; Kimmeskamp-Kirschbaum, N.; Pieper, A.; Pitt, B. A randomized controlled study of finerenone vs. eplerenone in patients with worsening chronic heart failure and diabetes mellitus and/or chronic kidney disease. Eur. Heart J. 2016, in press (DOI: http://dx.doi.org/10.1093/eurheartj/ehw132). 40. Amazit, L. L. B., F.; Kolkhof, P.; Lamriber, K.; Viengchareum, S.; Fay, M. R.; Khan, J. A.; Hillisch, A.; Lombès, M.; Rafestin-Oblin, M.-E.; Fagart, J. Finerenone impedes aldosteronedependent nuclear import of the mineralocorticoid receptor and prevents genomic recruitment of ateroid recpetor coactivator-1. J. Biol. Chem. 2015, 290, 21876-21889 41. Brandish, P., E. Fraley, Mark, E. Hershey, James, C. Steem, Justin, T. Mineralocorticoid receptor modulators. WO2009/078934A1, 2009. 42. Arai, K.; Homma, T.; Morikawa, Y.; Ubukata, N.; Tsuruoka, H.; Aoki, K.; Ishikawa, H.; Mizuno, M.; Sada, T. Pharmacological profile of CS-3150, a novel, highly potent and selective non-steroidal mineralocorticoid receptor antagonist. Eur. J. Pharmacol. 2015, 761, 226-234. 43. Canne, B. L.; Chen, J.; Dalrymple, L. E.; Flatt, B. T.; Forsyth, T. P.; Gu, X. H.; Mac, M. B.; Mann, G.; Mann, L. W.; Martin, R. Pyrrole derivatives as pharmaceutical agents. WO2006/012642, 2006.

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difluoro-phenyl)-ethyl]-1H-indol-7-yl}-methanesulfonamide: A potent, nonsteroidal, functional antagonist of the mineralocorticoid receptor. J. Med. Chem. 2007, 50, 6443-6445. 68. Cernak, T. A.; Dykstra, WO2014/014794A2, 2014.

K.

D.

Mineralocorticoid

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