Discovery of benzimidazole CYP11B2 inhibitors with in vivo activity in rhesus monkeys

Article abstract of DOI:10.1021/acsmedchemlett.5b00054

We report the discovery of a benzimidazole series of CYP11B2 inhibitors. Hit-to-lead and lead optimization studies identified compounds such as 32, which displays potent CYP11B2 inhibition, high selectivity versus related CYP targets, and good pharmacokin

Full text of DOI:10.1021/acsmedchemlett.5b00054

Letter
pubs.acs.org/acsmedchemlett
Discovery of Benzimidazole CYP11B2 Inhibitors with in Vivo Activity
in Rhesus Monkeys
Scott B. Hoyt,*,† Min K. Park,† Clare London,† Yusheng Xiong,† Jim Tata,† D. Jonathan Bennett,‡
Andrew Cooke,‡ Jiaqiang Cai,‡ Emma Carswell,‡ John Robinson,‡ John MacLean,‡ Lindsay Brown,‡
Simone Belshaw,‡ Thomas R. Clarkson,‡ Kun Liu,† Gui-Bai Liang,† Mary Struthers,† Doris Cully,†
Tom Wisniewski,† Ning Ren,† Charlene Bopp,† Andrea Sok,† Tian-Quan Cai,† Sloan Stribling,†
Lee-Yuh Pai,† Xiuying Ma,† Joe Metzger,† Andreas Verras,† Daniel McMasters,† Qing Chen,†
Elaine Tung,† Wei Tang,† Gino Salituro,† Nicole Buist,† Jeff Kuethe,† Nelo Rivera,† Joe Clemas,†
Gaochao Zhou,† Jack Gibson,† Carrie Ann Maxwell,† Mike Lassman,† Theresa McLaughlin,†
Jose Castro-Perez,† Daphne Szeto,† Gail Forrest,† Richard Hajdu,† Mark Rosenbach,† and Amjad Ali†
†Merck Research Laboratories, Rahway, New Jersey 07065, United States
‡Merck Research Laboratories, Newhouse, Lanarkshire ML1 5SH, United Kingdom
S
* Supporting Information
ABSTRACT: We report the discovery of a benzimidazole series of CYP11B2 inhibitors. Hit-to-lead
and lead optimization studies identified compounds such as 32, which displays potent CYP11B2
inhibition, high selectivity versus related CYP targets, and good pharmacokinetic properties in rat and
rhesus. In a rhesus pharmacodynamic model, 32 produces dose-dependent aldosterone lowering
efficacy, with no apparent effect on cortisol levels.
KEYWORDS: aldosterone synthase, CYP11B2, hypertension
Our goal is to discover potent and selective CYP11B2
inhibitors as treatments for hypertension. Toward that end, we
initiated screening of the Merck sample collection and
identified benzimidazole 1 (Table 1) as a starting point for
optimization studies. The modification of 1, described herein,
has led to the discovery of potent benzimidazole CYP11B2
inhibitors that display high selectivity vs related CYPs, drug-like
pharmacokinetic (PK) and physiochemical properties, and
robust aldosterone lowering in a rhesus pharmacodynamic
model.
Compounds were synthesized as described in the Supporting
Information section, and were tested in cell-based assays for
inhibition of human CYP11B2 and CYP11B1. The preparation
of these and related compounds has also been reported
previously.10
Taking screening hit 1 as our starting point, we synthesized a
series of compounds wherein the effects of benzimidazole N-
substitution were examined. Compounds in this series were
optimized with respect to CYP11B2 potency, selectivity vs
CYP11B1 and other CYPs, lipophilic ligand efficiency (LLE),
and PK properties.11 As shown in Table 1, screening hit 1 is a
potent CYP11B2 inhibitor that displays moderate, 56-fold
selectivity vs CYP11B1. In contrast, the unsubstituted parent
ldosterone is a steroid hormone that promotes increased
Ablood pressure, inflammation, fibrosis, and organ dam-
age.1−4 The final three rate-limiting steps of its biosynthesis are
catalyzed by the mitochondrial cytochrome P450 enzyme
aldosterone synthase (CYP11B2). Inhibition of CYP11B2
should lead to lower circulating plasma aldosterone levels,
and thus may be an effective strategy for the treatment of a
variety of ailments, including hypertension and heart failure.
Biological targets that are structurally related to CYP11B2
include CYP11B1 (>93% homology), and CYPs 17, 19, and 21.
CYP11B1 catalyzes the biosynthesis of cortisol, an important
regulator of glucose metabolism, while CYPs 17, 19, and 21
catalyze the formation of hormones such as estrone and
testosterone. To be used safely in the clinic, CYP11B2
inhibitors need to display high selectivity versus these related
targets, as well as the major hepatic CYP enzymes.
A variety of small molecule CYP11B2 inhibitors have
recently been reported in the literature.5,6 One of these, LCI-
699, has been shown to lower aldosterone levels and blood
pressure in the clinic, thereby validating this mechanism as a
treatment for hypertension.7−9 In cell-based in vitro assays, LCI-
699 inhibits CYP11B2 with only modest, 4-fold selectivity vs
CYP11B1. In the clinic, LCI-699 produces an undesired, dose-
limiting impairment of cortisol response, presumably as a result
of its CYP11B1 inhibition. Clinical candidates that inhibit
CYP11B2 more selectively than LCI-699 are thus desired as
improved antihypertensive therapies.
Received: February 2, 2015
Accepted: April 7, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.5b00054
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Letter
significantly higher intrinsic clearance, suggesting that the
additional methyl group may be a primary site of metabolic
oxidation. From the initial set of compounds shown in Table 1,
cyclopropyl analog 5 appeared to provide the best combination
of PK profile, CYP11B2 inhibition, B2/B1 selectivity, and LLE,
and the cyclopropyl substituent was thus selected for
incorporation in subsequent analogs.
With an improved N-substituent in place, we next optimized
substitution at each position on the benzimidazole phenyl ring.
Because the initial screening hit 1 was fluorinated, we began by
substituting fluorine at each ring position (Table 3). Of the
Table 1. Effect of Benzimidazole N-Substitution on
CYP11B2 and B1 Inhibition and LLE
a
a
CYP11B2
CYP11B1
b
c
compd
R
(IC50, nM)
(IC50, nM)
B1/B2
LLE
1
2
Me
H
12
677
56
5.39
n.d.
>10000
35
1987
<1
3
Et
2101
60
4.57
3.60
5.38
4.57
n.d.
4
i-Pr
c-Pr
138
4
3923
28
Table 3. Effect of Benzimidazole Substitution on CYP11B2
and B1 Inhibition and LLE
5
401
100
179
n.d.
n.d.
>15
6
(Me)c-Pr
t-Bu
16
2857
7
>5000
>5000
561
607
>10000
>10000
>8333
>8333
8
c-Bu
n.d.
9
CH2CF3
Bn
2.71
2.10
10
>14
a
b
Assay details reported in Supporting Information. Ratio of
c
hCYP11B1 IC50/hCYP11B2 IC50. Ligand lipophilic efficiency; LLE
a
a
= pIC50 − a Log P98.
CYP11B2
CYP11B1
b
c
compd
R
(IC50, nM)
(IC50, nM)
B1/B2
LLE
compound in this series (compound 2, R = H) shows no
inhibition of CYP11B2. When compared to 1, compounds
bearing larger acyclic N-alkyl substituents such as ethyl
(compound 3) or isopropyl (compound 4) are less active,
with potency decreasing as the size of the N-substituent
increases. Interestingly, N-cyclopropyl analog 5 is considerably
more potent than the closely related isopropyl analog 4,
suggesting that the cyclopropyl group may participate in a
beneficial π-type interaction with the protein. Compounds 6
and 7 show an analogous relationship, with the (methyl)-
cyclopropyl analog 6 exhibiting much more potent CYP11B2
inhibition than the sterically similar tert-butyl analog 7. Finally,
larger analogs that incorporate a methylene spacer such as 9
and 10 are also less potent when compared with truncated
analogs such as 1 or 3. Taken together, these data suggest that
binding space at this position is quite limited and that a small,
cyclopropyl-containing group provides an optimal solution for
potency, B2/B1 selectivity, and LLE.
11
12
13
5
H
49
>8333
>8333
3984
>170
>38
142
105
>93
n.d.
n.d.
>6
4.49
3.64
4.53
5.38
3.95
n.d.
4-F
5-F
6-F
7-F
219
28
3.8
400
14
15
16
17
18
19
20
21
22
23
24
25
107
>10000
>1000
1768
>1000
2.2
>10000
>10000
>10000
>10000
>10000
477
5-CF3
5-OCH3
5-CN
n.d.
3.06
n.d.
5-SO2CH3
6-Cl
n.d.
215
7
5.18
2.84
6.22
4.82
5.45
5.71
6.32
6-CF3
254
1.2
1831
6-CN
374
309
83
6-OCH3
5,6-di-F
5-F-6-Cl
5-F-6-CN
24
1980
2.1
370
176
835
0.4
334
0.6
117
195
a
b
Assay details reported in Supporting Information. Ratio of
hCYP11B1 IC50/hCYP11B2 IC50. Ligand lipophilic efficiency; LLE
c
Selected compounds from this set displayed promising
profiles in a rat pharmacokinetic assay.12 As shown in Table
2, screening hit 1 exhibited moderate oral bioavailability and
high plasma clearance. In comparison, cyclopropyl analog 5
displayed reduced plasma clearance and significantly higher oral
bioavailability and exposure. As the intrinsic clearance rates of 1
and 5 are essentially identical, the reduced plasma clearance of
5 may derive from its increased binding to plasma protein.
When compared to 5, methylcyclopropyl analog 6 showed
= pIC50 − a Log P98.
monofluorinated derivatives 12, 13, 5, and 14, the 6-fluoro
analog 5 displayed CYP11B2 inhibition and LLE that were
significantly improved relative to both the other monofluori-
nated compounds and to the parent, unsubstituted compound
11. A broader examination of substitution at the 5-position
revealed that larger lipophilic groups such as trifluoromethyl
(compound 15) as well as a variety of polar groups
(compounds 16−18) were all poorly tolerated. At the 6-
position, incorporation of larger lipophilic groups like
trifluoromethyl (compound 20) again afforded compounds
with reduced potency and LLE. However, small polar groups
such as cyano (compound 21) and methoxy (compound 22)
were better tolerated here than at the 5-position, with cyano in
particular providing a potent and selective compound. Of the
multiple difluorinated derivatives that were prepared, the 5,6-
difluoro analog 23 offered the best combination of CYP11B2
inhibition, B2/B1 selectivity, and LLE (other difluoro
compounds not shown). Finally, several other disubstituted
analogs were synthesized, and of those, the highly potent 5-
fluoro-6-chloro derivative 24 displayed optimal B2/B1
Table 2. Effect of Benzimidazole N-Substitution on Rat
a
Pharmacokinetic Profile
b
c
d
e
f
compd
PPB
Mic Clint
F (%)
AUCN (po)
Clp
1
5
6
25
15
19
30
28
82
37
77
32
0.56
1.82
0.20
59
28
131
a
b
Assay details reported in Supporting Information. Protein binding in
c
rat plasma, reported as % free compound. Intrinsic clearance in rat
liver microsomes (mL/min/kg). Bioavailability. Area under the
curve, normalized for dose (μM·h·kg/mg). Plasma clearance (mL/
d
e
f
min/kg).
B
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Table 4. Effect of Pyridine Substitution on CYP11B2 and B1 Inhibition and LLE
a
a
b
c
compd
R1
R2
R3
R4
CYP11B2 (IC50, nM)
CYP11B1 (IC50, nM)
B1/B2
LLE
23
26
27
28
29
30
31
32
33
34
35
36
37
F
F
F
F
F
F
F
H
F
F
F
F
F
F
H
H
H
H
H
H
H
H
H
2.1
4.7
22.3
0.4
0.8
1.0
0.3
2.3
10.2
8.1
0.5
0.2
0.6
370
435
1552
250
251
225
42
176
93
5.45
5.01
4.54
6.12
5.20
5.89
6.32
5.70
4.28
3.92
5.38
5.50
5.52
F
F
F
CN
70
F
OCH3
606
296
225
140
71
Cl
CN
F
F
F
C(CH3)2OH
F
C(CH3)2OH
142
723
795
258
17.7
435
F
CH3
CF3
H
71
F
H
98
F
CH3
CH3
OCH3
F
553
74
F
CH3
CH3
F
709
a
b
c
Assay details reported in Supporting Information. Ratio of hCYP11B1 IC50/hCYP11B2 IC50. Ligand lipophilic efficiency; LLE = pIC50 − a Log
P98.
selectivity, while the 5-fluoro-6-cyano compound 25 exhibited
optimal LLE.
that were only slightly less potent than parent 23. Additional
substitution with fluorine at R4 afforded compound 35, which
was 20-fold more potent and nearly 10-fold more selective than
33. Interestingly, incorporation of fluorine at R4 yielded far
greater benefit when R3 was also substituted with methyl than
when it was unsubstituted (compare 33 and 35 versus 23 and
26), suggesting that the R3 methyl might alter the dihedral
angle about the benzimidazole−pyridine bond, thus altering the
binding pocket environment into which the fluorine is
projected. Substitution of both R3 and R4 with methyl provided
a very potent CYP11B2 inhibitor (compound 36); subsequent
conversion of the R3 methyl to methoxy (compound 37) then
produced a compound nearly as potent and also 10-fold more
B2/B1 selective.
With refinements to the benzimidazole core complete, we
conducted a final round of optimization on the pyridine ring,
focusing our efforts on reducing clearance and further
improving PK in this series. Metabolite identification studies
of partly optimized compounds such as 5 had implicated the
pyridine ring as the primary site of oxidative metabolism. In an
attempt to block any pyridyl N-oxidation, we substituted each
position adjacent to the pyridine nitrogen, but found that
incorporation of even small groups such as methyl at these
positions resulted in a complete loss of potency, presumably
due to interference with binding of the pyridine to the
CYP11B2 heme (results not shown). Given that, we focused
subsequent optimization efforts at positions nonadjacent to the
pyridine nitrogen (R3 and R4 in Table 4). In an effort to reduce
metabolic oxidation, we introduced electron-withdrawing
groups that would not only block those positions specifically
but also deactivate the ring generally, as well as sterically bulky
groups that could block access to the ring. As shown in Table 4,
incorporation of an electron-withdrawing fluorine at R4 was
tolerated (compound 26) and yielded a compound that was
nearly as potent as the unsubstituted parent 23 despite its
presumably diminished capacity to bind the CYP heme. Other
electron-withdrawing groups such as nitrile (compound 27)
were somewhat less well tolerated, while electron donating
groups such as methoxy (compound 28) imparted improved
potency and selectivity. Comparing compounds 26, 29, and 30,
one sees that conversion of the R2 substituent from fluorine
(26) to chlorine (29) or nitrile (30) again improved potency
and LLE in the series where R4 = fluorine. Introduction of a
bulky tertiary alcohol group at R4 as in 31 generated
improvements in potency, LLE, and clearance (see Table 6
for effects on PK). In the tertiary alcohol series, fluorination at
R1 provided a nearly 10-fold boost in potency as well as a gain
in LLE (compare compounds 31 and 32). Moving around the
ring, substitution of the R3 position with methyl (compound
33) or trifluoromethyl (compound 34) provided compounds
Compounds from this series displayed high selectivity versus
CYPs 17, 19 and 21, and hepatic CYPs 3A4, 2C9, and 2D6. As
shown in Table 5, compounds in this series consistently
Table 5. Activity of Selected Compounds at Related CYP
Enzyme Targets
a
a
a
a
CYP17 (IC50, CYP19 (IC50, CYP21 (IC50,
CYP3A4
compd
nM)
nM)
nM)
(IC50, nM)
26
31
32
35
>10000
>10000
>10000
>10000
4513
1268
1021
n.d.
>10000
>10000
>10000
>10000
>50000
>50000
>50000
8235
a
Assay details reported in Supporting Information.
displayed no inhibition of CYP17 or CYP21. Inhibition of
CYP19 was observed, though compounds generally exhibited at
least 500-fold selectivity or better for CYP11B2 vs CYP19.
Compounds with monosubstituted pyridine heme-binding
domains such as 26, 31, and 32 typically showed no inhibition
of CYPs 3A4, 2C9, and 2D6, and this held true whether the
pyridine was substituted with a lipophilic group as in 26, or a
more polar group as in 31 or 32. Representative data for
inhibition of CYP3A4 are shown in Table 5. Compounds with
C
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disubstituted heme binding domains such 35 did display
hepatic CYP inhibition, although even in these cases selectivity
vs CYP11B2 was >1000-fold. The results shown in Table 5 are
representative of the series as a whole, and indicate high
selectivity versus related CYP targets.
also displays an acceptable PK profile in rhesus, with low
plasma clearance, good oral exposure, and a half-life of 7.4 h.
Compound 35 displays a broadly similar rhesus PK profile,
though it suffers from a higher rate of plasma clearance, perhaps
as a result of oxidative metabolism at the pyridyl 4-methyl
group. The best overall rat PK profiles from this set belong to
compounds 31 and 32, which both contain a tertiary alcohol
substituent at the pyridine 3-position. These compounds
display excellent oral bioavailability and oral exposure in rat.
They also exhibit plasma clearance rates that are notably lower
than their unsubstituted or meta-fluoro substituted counterparts
(compare 31 to 23 or 26, and 32 to 5). Their improved
clearance rates may result from steric blockade, as the bulky
alcohol group may inhibit or prevent metabolizing enzymes
from accessing the pyridine. Compound 32 also displays a good
rhesus PK profile, with low plasma clearance and acceptable
oral exposure.
Compounds from this series exhibited potent inhibition of
human and rhesus CYP11B2, but only weak inhibition of rat
CYP11B2. Given the low identity/homology between human
and rat CYP11B2 (∼80%) and the higher homology between
human and rhesus CYP11B2 (∼95%), this is perhaps not
surprising. Consequently, these compounds could not be tested
in known rodent models of hypertension, and were instead
evaluated in a recently described rhesus pharmacodynamic
(PD) model.14
Several compounds from the current study exhibited profiles
promising enough to warrant testing in the PD model.
However, as the model was resource-intensive, throughput
was restricted. From the set of promising compounds,
compound 32 was identified earliest and was thus the
compound chosen for proof-of-concept PD testing.
Compound 32 exhibited dose-dependent aldosterone low-
ering efficacy in a rhesus PD model. In this model, monkeys are
anesthetized to maintain stable baseline levels of aldosterone.
Thus, although compound 32 displayed a PK profile suitable
for oral dosing, the use of anesthetized animals necessitated i.v.
dosing. Compound 32 was given at i.v. doses ranging from
0.001 to 0.3 mg/kg. Plasma levels of aldosterone and 11-
deoxycortisol (11-DOC), a biosynthetic precursor of cortisol,
were quantified by LC−MS at time points up to 180 min
postdose, allowing the determination of AUCs for aldosterone
and 11-DOC.15 Plasma concentrations of compound 32 were
measured at 90 min postdose, and free (unbound) concen-
trations were calculated based on 32’s 7% free fraction in rhesus
plasma.
A benchmark benzimidazole also displayed high selectivity
versus a broad panel of biological targets. Compound 26 was
assayed for activity against 114 receptors, enzymes, and ion
channels, and showed mid-micromolar binding activity against
just one target (adrenergic α2B receptor, IC50 = 2.49 μM). At a
concentration of 10 μM, 26 displayed <40% activity against all
other targets in the screen. Compound 26 was also screened
against a collection of >100 kinase enzymes, and at a
concentration of 10 μM, exhibited <40% activity against the
entire panel. Finally, compound 26 exhibited an IC50 > 30 μM
in functional assays that measure block of Nav1.5 and Cav1.2
ion channels, and in a binding assay that measures displacement
of the known hERG blocker MK-0499.13 The lack of ion
channel block observed with compound 26 is representative of
the series as a whole, and suggests reduced risk for
cardiovascular adverse effects.
Key compounds from this series exhibited good pharmaco-
kinetic profiles in both rat and rhesus (Table 6).12 When
Table 6. Rat and Rhesus Pharmacokinetic Profiles of
a
Selected Compounds
b
c
d
e
f
compd
species
PPB
Mic Clint
F (%)
AUCN (po)
Clp
23
26
26
35
31
32
32
rat
15
14
23
<20
<20
<20
<20
<20
<20
22
92
86
53
88
94
76
15
1.32
2.10
3.07
2.49
6.55
4.32
43
37
10
19
7
rat
rhesus
rhesus
rat
n.d.
14
rat
13
10
5
rhesus
15
1.53
a
b
Assay details reported in Supporting Information. Plasma protein
c
binding, reported as % free compound. Intrinsic clearance in rat or
rhesus liver microsomes (mL/min/kg). Bioavailability. Area under
the curve, normalized for dose (μM·h·kg/mg). Plasma clearance
(mL/min/kg).
d
e
f
compared to compound 5 (Table 2), compound 23, which
contains an additional fluorine at the benzimidazole 5-position,
displays a comparable rat PK profile, albeit with a slightly
higher rate of plasma clearance. Compound 26, which adds a
meta-fluorine to the pyridine, shows improvements in plasma
clearance, half-life, and oral exposure as a result. Compound 26
Figure 1. Effects of compound 32 on plasma AUCs of aldosterone and 11-DOC in rhesus monkeys.
D
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When dosed i.v. at 0.01 and 0.3 mg/kg, compound 32
produced reductions in aldosterone AUC of 62% and 95%,
respectively, compared to baseline (Figure 1). Compound 32
achieved these effects at free plasma concentrations 90 min
postdose of 12 and 27 nM, respectively. The reduction in
aldosterone AUC produced by a 0.3 mg/kg dose of compound
32 is comparable to that of a 1.0 mg/kg i.v. dose of the positive
control Fadrozole, a pan-CYP19/CYP11B2/CYP11B1 inhib-
itor that has been shown to reduce blood pressure in rodent
models of hypertension.16−18 At doses up to 0.3 mg/kg,
compound 32 does not produce an increase in 11-DOC AUC,
suggesting that CYP11B1, the enzyme that converts 11-DOC
to cortisol, has not been significantly inhibited. In contrast, a
1.0 mg/kg dose of Fadrozole, which displays potent inhibition
of both CYP11B2 and B1, elicits a significant increase in 11-
DOC AUC. These data demonstrate that, in an acute setting,
compound 32 is capable of producing significant reductions in
rhesus plasma aldosterone levels with no apparent effect on
cortisol levels.
At a free plasma concentration of 12 nM, compound 32
produces a 62% reduction in aldosterone AUC, which is
consistent with its potent inhibition of rhesus CYP11B2 (IC50 =
0.3 nM). Additionally, at free plasma concentrations up to 27
nM, no reduction in 11-DOC AUC and no apparent inhibition
of CYP11B1 are observed. Although that plasma concentration
approaches compound 32’s IC50 for inhibition of rhesus
CYP11B1 (IC50 = 41 nM), higher concentrations are
apparently required to affect reductions in 11-DOC AUC in
this acute setting.
Reductions in plasma aldosterone levels have been shown to
produce reductions in blood pressure in the clinic. In a Phase II
clinical trial, the aldosterone synthase inhibitor LCI-699
produced a mean 34% reduction in plasma aldosterone at 8
weeks, with concomitant reductions in systolic (−9.7 mmHg)
and diastolic (−4.7 mmHg) blood pressure.5 In light of those
results, the fact that compound 32 can reduce plasma
aldosterone levels by >90% in rhesus suggests that a compound
from this series could produce significant blood pressure
reduction in the clinic.
In summary, we have reported the discovery of a structurally
novel series of benzimidazole CYP11B2 inhibitors. Benchmark
compound 32 from this series displays potent and selective
inhibition of CYP11B2, and a good pharmacokinetic profile in
rat and rhesus. In a rhesus pharmacodynamic model,
compound 32 exhibits dose-dependent aldosterone lowering
efficacy, with maximal reductions in plasma aldosterone AUC
of >90%. Additional optimization in this series has been
completed and will be reported in due course.
ABBREVIATIONS
■
11-DOC, 11-deoxycortisol; AUC, area under curve; BP, blood
pressure; CNS, central nervous system; LLE, lipophilic ligand
efficiency; PK, pharmacokinetics
REFERENCES
■
(1) Carey, R. M. Aldosterone and cardiovascular disease. Curr. Opin.
Endocrinol. Diabetes Obes. 2010, 17, 194−198.
(2) Shavit, L.; Lifschitz, M. D.; Epstein, M. Aldosterone blockade and
the mineralocorticoid receptor in the management of chronic kidney
disease: current concepts and emerging treatment paradigms. Kidney
Int. 2012, 81, 955−968.
(3) Tomaschitz, A.; Pilz, S.; Ritz, E.; Obermayer-Pietsch, B.; Pieber,
T. R. Aldosterone and arterial hypertension. Nat. Rev. Endocrinol.
2010, 6, 83−93.
(4) Gilbert, K. C.; Brown, N. J. Aldosterone and inflammation. Curr.
Opin. Endocrinol. Diabetes Obes. 2010, 17, 199−204.
(5) Hu, Q.; Yin, L.; Hartmann, R. W. Aldosterone synthase inhibitors
as promising treatments for mineralocorticoid dependent cardiovas-
cular and renal diseases. J. Med. Chem. 2014, 57, 5011−5022.
(6) Cerny, M. A. Progress toward clinically useful aldosterone
synthase inhbitors. Curr. Top. Med. Chem. 2013, 13, 1385−1401.
(7) Calhoun, D. C.; White, W. B.; Krum, H.; Guo, W.; Bermann, G.;
Trapani, A.; Lefkowitz, M. P.; Menard, J. Effects of a novel aldosterone
synthase inhibitor for treatment of primary hypertension. Circulation
2011, 124, 1945−1955.
(8) Azizi, M.; Amar, L.; Menard, J. Aldosterone synthase inhibition in
humans. Neprhol. Dial. Transplant. 2013, 28, 36−43.
(9) Meredith, E. L.; Ksander, G.; Monovich, L. G.; Papillon, J. P. N.;
Liu, Q.; Miranda, K.; Morris, P.; Rao, C.; Burgis, R.; Capparelli, M.;
Hu, Q.-Y.; Singh, A.; Rigel, D. F.; Jeng, A. Y.; Beil, M.; Fu, F.; Hu, C.-
W.; LaSala, D. Discovery and in vivo evaluation of potent dual
CYP11B2 (Aldosterone Synthase) and CYP11B1 inhibitors. ACS Med.
Chem. Lett. 2013, 4, 1203−1207.
(10) Hoyt, S. B.; Park, M. K.; London, C.; Xiong, Y.; Bennett, D. J.;
Cai, J.; Ratcliffe, P.; Cooke, A.; Carswell, E.; MacLean, J.; Saxena, R.;
Kulkarni, B. A.; Gupta, A. Aldosterone Synthase Inhibitors. WO
2012012478, Jan 26, 2012.
(11) Lipophilic ligand efficiency (LLE) was calculated using the
equation LLE = pIC50 − a Log P98, as first proposed by: Leeson, P. D.;
Springthorpe, B. The influence of drug-like concepts on decision-
making in medicinal chemistry. Nat. Rev. Drug Discovery 2007, 6, 881−
890.
(12) For detailed experimental protocols, see Supporting Information
section.
(13) Wang, J.; Della Penna, K.; Wang, H.; Karczewski, J.; Connolly,
T. M.; Koblan, K. S.; Bennett, P. B.; Salata, J. J. Am. J. Physiol. Heart
Circ. Physiol. 2002, 284, H256.
(14) Cai, T.-Q.; Stribling, S.; Tong, X.; Xu, L.; Wisniewski, T.;
Fontenot, J. A.; Struthers, M.; Akinsanya, K. O. Rhesus monkey model
for concurrent analyses of pharmacodynamics, pharmacokinetics and
in vivo selectivity of aldosterone synthase inhibitors. J. Pharmacol.
Toxicol. Methods 2015, 71, 137−146.
(15) We had initially planned to monitor plasma levels of cortisol,
but development of an LC−MS analytical method that cleanly
resolved the cortisol signal proved challenging. Thus, rather than
monitoring decreases in cortisol levels, we monitor increases in levels
of 11-DOC, the direct biosynthetic precursor that is converted to
cortisol by CYP11B1.
(16) Rigel, D. F.; Fu, F.; Beil, M.; Hu, C.-W.; Liang, G.; Jeng, A. Y.
Pharmacodynamic and pharmacokinetic characterization of the
aldosterone synthase inhibitor FAD286 in two rodent models of
hyperaldosteronism: comparison with the 11beta-hydroxylase inhibitor
metapyrone. J. Pharmacol. Exp. Ther. 2010, 334, 232−243.
(17) LaSala, D.; Shibanaka, Y.; Jeng, A. Y. Coexpression of CYP11B2
or CYP11B1 with adrenodoxin and adrenodoxin reductase for
assessing the potency and selectivity of aldosterone synthase inhibitors.
Anal. Biochem. 2009, 394, 56−61.
ASSOCIATED CONTENT
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S
* Supporting Information
Assay protocols, synthesis schemes, representative procedures
for the synthesis of compounds 31, 32, 35, and related
intermediates. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: 1-908-740-3753. E-mail: scott_hoyt@merck.com.
Notes
The authors declare no competing financial interest.
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Letter
(18) Huang, B. S.; White, R. A.; Jeng, A. Y.; Leenen, F. H. H. Role of
central nervous system aldosterone synthase and mineralocorticoid
receptors in salt-induced hypertension in Dahl salt-sensitive rats. Am. J.
Physiol. Reg. Integr. Comp. Physiol. 2009, 296, R994−R1000.
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