Discovery of novel non-steroidal reverse indole mineralocorticoid receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2016.04.052

Reported herein are a series of reverse indoles that represent novel non-steroidal mineralocorticoid receptor (MR) antagonists. The key structure-activity relationships (SAR) are presented below. This reverse indole series is exemplified by a compound tha

Full text of DOI:10.1016/j.bmcl.2016.04.052

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel non-steroidal reverse indole mineralocorticoid
receptor antagonists
a
a
a
a
Anthony K. Ogawa a, , Ellen Vande Bunte , Rudrajit Mal , Ping Lan , Zhongxiang Sun ,
Alejandro Crespo b, Judyann Wiltsie a, Joseph Clemas a, Jack Gibson a, Lisa Contino a, JeanMarie Lisnock a,
Gaochao Zhou a, Margarita Garcia-Calvo a, Nina Jochnowitz a, Xiuying Ma a, Yi Pan a, Patricia Brown a,
Beata Zamlynny a, Thomas Bateman a, Dennis Leung b, Ling Xu b, Xinchun Tong b, Kun Liu a,
Martin Crook a, Peter Sinclair a
⇑
a Early Development and Discovery Sciences, Merck and Co., 2000 Galloping Hill Rd., Kenilworth, NJ 07033, USA
b Early Development and Discovery Sciences, Merck and Co., 126 E. Lincoln Ave., Rahway, NJ 07065, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Reported herein are a series of reverse indoles that represent novel non-steroidal mineralocorticoid
receptor (MR) antagonists. The key structure–activity relationships (SAR) are presented below. This
reverse indole series is exemplified by a compound that demonstrated efficacy in an acute natriuresis
rodent model comparable to marketed MR antagonists, spironolactone and eplerenone.
Ó 2016 Elsevier Ltd. All rights reserved.
Received 11 March 2016
Revised 18 April 2016
Accepted 19 April 2016
Available online xxxx
Keywords:
Mineralocorticoid receptor antagonist
Hypertension
Spironolactone
Eplerenone
Mineralocorticoid receptor antagonists (MRA) have tradition-
ally been prescribed as hypotensive drugs that were intended to
blunt the potential for aldosterone escape as a means of greater
control over of the RAAS signaling pathway.1 The first generation
MRA, spironolactone, demonstrated good efficacy, but was beset
with adverse effects (AE), such as gynocomastia and impotence
in male patients.2 The observed AE profile was attributed to a lack
of nuclear hormone receptor selectivity, which was overcome with
the approval of eplerenone in 2002.3
Recent clinical trials involving both spironolactone and eplere-
none have demonstrated a strong link between MR antagonism
and positive outcome benefits to heart failure patients, albeit with
an elevated risk of hyperkalemia that is believed to be mechanism-
related.4 The observed benefits to mortality, cardiovascular
and selective reverse indole MRA class that has demonstrated
acute PD efficacy (Fig. 1).
Initial efforts sought to expand on the SAR established around a
number of efficient central scaffolds.7 For example, qualitative
analysis of literature examples featuring 3,7-disubstituted indole
and 4,7-disubstituted benzoxazine scaffolds, e.g., suggested that
the analogous display of key functional groups from N1 and C4
of an indole central scaffold could be successful.8 Further,
alkylation at the indole N1-position facilitated the synthesis of
more structurally diverse pendant hydrophobic functionality,
which would, subsequently, enable SAR refinement.
To this end, the SAR derived from an initial basis set of
compounds 1–7,9 supported the aforementioned hypothesis that
the ‘reverse indole’ scaffold could yield MR antagonists of high
lipophilic ligand efficiency (LLE), as well as providing evidence that
a second hydrogen bond donor was not required for activity. The
initial array of MRAs also revealed subtle but potentially significant
determinants of activity, as seen from the improved activity for
adverse events and hospitalization were independent of
a
hypotensive effect long presumed to be the primary benefit of an
MRA.
Over the past decade, a number of reports have detailed efforts
to identify a next generation, non-steroidal MRA.5,6 Reported
herein are the background rationale and discovery of a potent
compounds 5–6, which contain
a conformational-directing
ortho-Cl atom. Lastly, the loss in activity observed for biaryl com-
pound, 7, suggests a steric limit to the binding pocket.
Having established preliminary aryl ring SAR, the next goal
involved gaining a similar understanding with respect to indole
⇑
Corresponding author.
http://dx.doi.org/10.1016/j.bmcl.2016.04.052
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Ogawa, A. K.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.04.052

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A. K. Ogawa et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Table 2
O
O
In vitro SAR at the indole C4-position, compounds 8–14
O
O
F
MeO2C
O
N
Cl
O
SAc
Spironolactone
O
CO2Me
Eplerenone
R
F
Compda
R
hMR NH Pro IC50
(nM)
LLEc
Other NHR
counterscreens
b
F
3
8
9
10
11
H
F
Cl
CN
2700
2310
940
1.4
1.2
1.3
2.9
F
7
N
O
ARag = 58 nMd (76%)
N
4
O
O
7
H
200
GRantag = 0.3
l
Md
NH
S
ARag = 9 nMd (76%)
S
N
O
H
12
5
13
14
CO2Et
4800
530
7900
2120
1.1
2.9
1.4
2.1
O
O
GRantag = 3.2 l
Md
cpd. 14 from
J. Med. Chem. 2007, 50, 6443.
NHSO2Me
NHC(O)CF3
NHSO2CF3
WO2007/089034
Figure 1. Known mineralocorticoid receptor antagonists.
a
b
c
Compounds are racemic.
Values are the average of two 10-pt titrations.
LLE = pIC50 – clogP.
substitution (8–14). A brief SAR examination revealed two signifi-
cant findings. First, although replacement of the sulfonamide with
a nitrile (11) led to a significant improvement in activity, the off-
target activity at other NHR precluded its employment. The second
finding was that the sulfonamide SAR was also highly sensitive as
close homologs (13–14) resulted in erosion of activity (Tables 1
and 2).
d
Value is from a single 10-pt titration.
Table 3
In vitro SAR for the
a
-alkyl substituents, compounds 17–22
R
Cl
Despite the knowledge gained from probing the indole and aryl
ring SAR, the conclusions largely supported what was previously
MeO2C
N
Table 1
In vitro SAR for the pendant phenyl ring, compounds 1–7
O
NH
S
O
MeO2C
R
Compda
R
hMR NH Pro IC50 (nM)
LLEc
b
N
1
2
H
Me
Et
1860
200
60
275
30
1.8
3.2
3.4
2.3
—
4
15
16
17
18
19
20
O
NH
Bn
S
CH2cPr
CH2OMe
CH2CN
O
1900
70
—
4.3
Compda
R
hMR NH Pro IC50 (nM)
LLEc
—
b
a
1
Ph
4000
Compounds are racemic.
Values are the average of two 10-pt titrations.
b
c
Cl
F
LLE = pIC50 – clogP.
2
3
4
5
6
1700
1.8
2.1
—
established in the literature. Less well known, however is the
SAR governing substitution at the benzylic carbon linking the
aforementioned moieties. Further, the reverse indole scaffold read-
ily lent itself to rapid SAR development, and more significantly,
unsymmetrical substitution (see Table 3).
The introduction of a methyl substituent to form a quaternary
carbon (15) applied a common strategy to improve potency by
restricting rotation.10 The functional activity for 15 affirmed this
strategy, but expanding the SAR revealed a separate and distinct
preference for small compact lipophilic substituents, highlighted
by compounds 16, 18, and 20. Extending the hydrophobic surface
proved to be deleterious (17), and introducing polar functionality
in the form of a methoxymethyl group, also had an adverse effect
on activity.
10,000
9800
530
OMe
F
Cl
Cl
2.9
3.8
Cl
80
7
3920
1.4
Cl
Chromatographic separation of the enantiomers from racemic
16, indicated a clear preference for one stereoisomer (21), which
was tentatively assigned the R-stereochemistry based on correla-
tion studies using Vibrational Circular Dichroism (VCD).11 Also
a
b
c
Compounds are racemic.
Values are the average of two 10-pt titrations.
LLE = pIC50 À clogP.
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A. K. Ogawa et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
Table 4
Table 5
Comparison of in vitro data for
a
-ethyl compounds 21–22
Comparison of in vitro data for a-ethyl compounds 23–28
Cl
Cl
R
MeO2C
*
N
N
O
O
NH
NH
S
S
O
O
Compd Chiralitya hMR NH Pro IC50
(nM)
LLEc Other NHR
Compd
R
hMR NH Pro
IC50 (nM)
LLEb Other NHR
b
a
counterscreens
counterscreens
16
21
22
Racemic
R
S
90
15
330
3.4
3.3
1.8
—
23
24
25
AC(O)NHMe
115
97
3.8
2.7
1.6
GRantag = 3.2
(31%)c
GRantag 27%
l
M
PRantag = 3.6
PRantag = 0.4
l
l
Md
Me
AC(O)
NHCH2CF3
a
b
c
AC(O)NHtBu
960
—
Absolute stereochemistry inferred from VCD correlations.
Values are the average of two 10-pt titrations.
LLE = pIC50 – clogP.
Value is from one 10-pt titration.
Value is from three 10-pt titrations.
O
N
GRantag = 5.9
(48%)c
l
M
6
33
3.2
d
e
N
O
N
c
Ph
7
1900
21
3.9
4.2
PRantag = 1.5 lM
N
2812
ACH2OH
GRantag >10
lM;
PRantag = 6.7
lM
a
b
c
Values are the average of two 10-pt titrations.
LLE = pIC50 – clogP.
Value is from one 10-pt titration.
Table 6
Rat PK for compounds 23 and 28
Compd Cl (mL/min/kg) Vdss (L/kg)
t
(h) AUCNpo
(
lM h kg/mg) %F
½
23
28
67
41
2.8
4.1
0.6
1.5
0.13
0.67
21
65
Cl
CO2Me
MeO2C
Figure 2. Predicted binding mode of compound 21 within MR pocket.
c , d
a , b
N
gratifying was that the off-target PR antagonism exhibited the
opposite stereochemical preference (see Table 4).
Cl
16a
Cl
NO2
Cl
Figure 2 shows the predicted binding mode of compound 21
obtained by molecular modeling using MR X ray structure 2A3I.8
The indole core binds in the hydrophobic middle region of the
pocket and it is placed in the same plane as aldosterone’s sterol
ring.12 The C4-sulfonamide makes hydrogen bonds with canonical
MR resides Q766 and R817 (resembling the 3-keto group of
aldosterone). The methyl ester moiety binds in the similar region
where eplerenone ester does, interacting with residues located in
helix 7 (M845, L848, C849 and M852). The ethyl group points
down towards residues located in helix 10 (L938 and L939). Finally,
the chlorophenyl ring stacks between residues located in helix 3
(N770 and L766) and helix 10 (F941). This orientation is predicted
to prevent the formation of the canonical hydrogen bond network
(between the ligand, N770 in helix 3 and T945 in helix 10) required
for receptor activation,12 thus conveying the antagonism behavior
to the series.
MeO2C
MeO2C
N
16b
N
e
16c
NHBoc
NHBoc
Cl
MeO2C
f , g
N
16
NHSO2Me
Scheme 1. Synthesis of compound 16. Reagents and conditions: (a) NBS, AIBN,
80 °C; (b) 4-nitroindole, NaH, DMF, 0 0C; (c) 5% Pt–C, H2 (1 atm), EtOAc; (d) Boc2O;
(e) addition of 16b in DMF to NaH, DMF, 0 °C, then iodoethane; (f) TFA; (g) MsCl.
The synthesis of compound 16 follows a general route to
reverse indole MRA, in which the appropriate phenylacetate is
treated with NBS to afford the corresponding
a-bromophenylac-
etate that is alkylated with 4-nitroindole. Reduction of the nitro
group of 16a, followed by Boc-protection yields intermediate
16b. It should be noted the early stage reduction and protection
was necessary to facilitate alkylation to generate the a-quaternary
ester functionality, as attempts to directly convert 16a resulted in
fragmentation. Conversion of 16b to 16c proved to also require
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A. K. Ogawa et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
demonstrated a comparable natriuretic effect. The acute pharma-
codynamic data demonstrating comparable efficacy for compound
28 versus well-established benchmark like spironolactone
a
affirms the promise for the reverse indole series of MR antagonists.
References and notes
1. Reviewed in Jaisser, F. et al Pharm. Rev. 2015, 68, 49.
2. (a) Menard, J. Mol. Cell. Endocrinol. 2004, 217, 45; (b) Kolkhof, P.; Borden, S. A.
Mol. Cell. Endocrinol. 2012, 350, 310.
3. Craft, J. BUMC Proc. 2004, 17, 217.
4. (a) Pitt, B. et al N. Eng. J. Med. 1999, 341, 709; (b) Pitt, B. et al N. Eng. J. Med. 2003,
348, 1309; (c) Juurlink, D. N. et al N. Eng. J. Med. 2004, 351, 543; (d) Preiss, D.
et al Eur. J. Heart Fail. 2012, 14, 909; (e) Eschalier, R. et al J. Am. Coll. Cardiol.
2013, 62, 1585.
5. Reviewed in Jasser, F. et al Expert Opin. Ther. Patents 2013, 24, 1; Kolkhof, P. et al
Curr. Opin. Nephrol. Hypertens. 2015, 24, 1.
6. (a) Yang, C. et al Bioorg. Med. Chem. Lett. 2013, 23, 4388; (b) Cox, J. M. et al
Bioorg. Med. Chem. Lett. 2014, 24, 1681; (c) Liu, L. C. Y. et al Expert Opin. Invest.
Drugs 2015, 24, 1.
7. (a) Kawaguchi, T. et al. PCT Int. Appl., WO 2007/089034.; (b) Bell, M. G. et al J.
Med. Chem. 2007, 50, 6443; (c) Casimiro-Garcia, A. et al J. Med. Chem. 2014, 57,
4273.
Figure 3. Acute pharmacodynamic evaluation of spironolactone and compound 28
in oral dosed WKY rats.
8. Putative design structures were modeled based on docking experiments
performed starting from the 1.95 Å MR structure with bound corticosterone
(PDB code 2A3I: Li, Y.; Suino, K.; Daugherty, J.; Xu, H. E. Mol. Cell 2005, 19, 367;
). Structures were prepared by removal of waters, addition of hydrogens, and
restrained energy minimization using Macromodel (Macromodel 9.8.107,
Schrödinger LLC, New York, NY). Molecular docking was performed by
employing our in-house docking routine FLOG ( Miller, M. D.; Kearsley, S. K.;
Underwood, D. J.; Sheridan, R. P. J. Comput. Aided Mol. Des. 1994, 8, 153. ), which
ranked precalculated conformations of the target molecules in a 6 Å grid
centered on the crystallographic ligand. Conformations were generated using
our in-house metric matrix distance geometry algorithm JG (Kearsley, S. K.
Merck & Co., unpublished).; The conformations were subjected to energy
minimization with Macromodel using the MMFFs force field ( Halgren, T. A. J.
Comput. Chem. 1999, 20, 720. ). A representative docking pose (data not shown)
was selected by visual inspection and was subjected to restrained energy
minimization (using Macromodel) to produce a putative model.
9. MR antagonist IC50 data were determined using a commercially available cell-
based PathHunterTM protein–protein interaction assay that measured the ability
of compounds to antagonize full-length human MR binding to a coactivator
peptide. Similar PathHunterTM assays were applied to determine other nuclear
hormone antagonist activities. Average potency in PathHunterTMassay:
spironolactone 11 nM and eplerenone 244 nM.
care, as slow reverse addition of starting material to base was
required to further suppress fragmentation. With 16c in hand,
the synthesis was readily completed by exchange of the Boc-pro-
tecting group for the desired methylsulfonamide moiety to afford
compound 16 (Tables 5 and 6).
Elaboration of the ester moiety in 21 remained as a clear avenue
to positively impact activity. The SAR for both simple amides, as
well as 1,2,4-oxadiazole bioisosteres, revealed a similar trend as
previously observed for other substituents.13 There was a clear
preference for compact aliphatic groups (23–24, 26) over sterically
larger groups (25, 27). Reduction of the ester to the primary alcohol
yielded compound 28,14 which exhibited a better off-target profile
along with excellent lipophilic ligand efficiency (Scheme 1).
Confirmation of in vivo efficacy followed from initial pharma-
cokinetic evaluation of key compounds.15 Intravenous and oral
dosing of compounds 23 and 28 in rats affirmed the potential for
the series to achieve oral efficacy. Despite modest clearance for
both representative compounds, both compounds 23 and 28 exhib-
ited suitable half-life and oral exposures to support investigation of
their effect in an acute rat natriuresis model (Fig. 3).
http://www.discoverx.com/nhrs/prod-nhrs.php.
10. Schonherr, H.; Cernak, T. Angew. Chem., Int. Ed. 2013, 52, 12256.
11. Nafie, L. A. Nat. Prod. Commun. 2008, 3, 451.
12. Bledsoe, R. K. et al J. Biol. Chem. 2005, 280, 31283.
13. Amides and heterocycles were prepared by derivatization of compound 17c.
WO2012/097744.
14. Additional off-target data: other NHR selectivity IC50 >10
lM; Ion Channel IC50
(Ikr, Na) >30 M; CYP (3A4, 2D6, 2C8, 2C9) IC50 >13 M.
l
l
An important experiment to validate the lead series involved
confirmation of in vivo efficacy in an animal model. A well charac-
terized pharmacodynamic model for assessing target engagement
in vivo involves quantifying the natriuretic effect in rats treated
with an MR antagonist.16 WKY rats on an established low-salt diet
were treated with compound 28 in a dose dependent manner, and
the urine collected over 6 h was analyzed for cumulative elec-
trolyte output. The effect of compound 28 was benchmarked
against a comparable dose titration of spironolactone (SPL) and
15. Formulation: 1 mg/mL PEG 200/water (70:30). Iv dose: 1 mg/kg (n = 2). PO
dose: 2 mg/kg (n = 3). Blood concentrations were determined by LC/MS/MS
following protein precipitation with acetonitrile.
16. Procedures were modified based on semilal references: Kagawa, C. M. et al
Science 1957, 126, 1015; Bicking, J. B. et al J. Med. Chem. 1965, 8, 638. Age-
matched male WKY rats (N = 6 per group) were fed a low salt diet and
maintained under a reverse dark-light cycle environment. Animals were dosed
PO using Imwitor:tween as a vehicle (2 mL/kg) at the doses indicated, and
urinary electrolytes were analyzed. Jugular vein bleeds were performed at 6 h
to determine terminal drug levels (data not shown).
Please cite this article in press as: Ogawa, A. K.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.04.052