Development of Highly Selective Pyrimidine-Based Aldosterone Synthase (CYP11B2) Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.9b00152

Excess aldosterone production and signaling are primary contributors to numerous cardiovascular disorders including primary aldosteronism and resistant hypertension. Recently, inhibition of aldosterone synthesis via the enzyme aldosterone synthase (CYP11B

Full text of DOI:10.1021/acsmedchemlett.9b00152

Letter
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Cite This: ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
Development of Highly Selective Pyrimidine-Based Aldosterone
Synthase (CYP11B2) Inhibitors
Steven M. Sparks,*,† Dana P. Danger,‡ William J. Hoekstra,† Tony Leesnitzer,‡ Robert J. Schotzinger,†
Christopher M. Yates,† and J. David Becherer†
†Selenity Therapeutics, 4505 Emperor Boulevard, Durham, North Carolina 27703, United States
‡OpAns, 4134 South Alston Avenue, Durham, North Carolina 27713, United States
S
* Supporting Information
ABSTRACT: Excess aldosterone production and signaling are primary contributors to numerous cardiovascular disorders
including primary aldosteronism and resistant hypertension. Recently, inhibition of aldosterone synthesis via the enzyme
aldosterone synthase (CYP11B2) has been pursued to ameliorate the negative effects of elevated aldosterone. Herein, we report
the development of aldosterone synthase inhibitors using a pyrimidine-based metal binding group leading to the highly selective
CYP11B2 inhibitor 22. Superior selectivity combined with robust pharmacokinetics afforded highly selective in vivo aldosterone
suppression in a monkey model of adrenal steroidogenesis, demonstrating the potential for selective aldosterone lowering in
humans with pyrimidine 22.
KEYWORDS: Aldosterone, cytochrome P450, CYP11B2, CYP11B2 inhibitor
inappropriately activates MR contributing to high blood
pressure.
hronically elevated aldosterone levels negatively impact
Cthe cardiovascular and renal systems through multiple
mechanisms including increased blood pressure and promotion
of fibrosis and inflammation.1 Hyperaldosteronism is also
associated with insulin resistance and therefore contributes to
the progression of metabolic syndrome.2,3 Consequently,
patients with elevated aldosterone are at greater risk for
serious cardiovascular and renal disorders including heart
failure, ischemic heart disease, arrhythmias, stroke, and kidney
disease.4−7 Due to the negative consequences associated with
excess aldosterone levels, interest in inhibiting the production
of aldosterone has recently gained attention.
Aldosterone synthase (CYP11B2) is the enzyme located in
the zona glomerulosa of the adrenal glands, which is
responsible for the production of aldosterone.8 Typically
under the control of the primary regulators potassium,
angiotensin II, and renin, aldosterone elicits its fluid balance
effects in the kidney where it activates the mineralocorticoid
receptor (MR) leading to sodium and water reabsorption.
These effects increase plasma volume resulting in increased
blood pressure. Given this sequence, excess aldosterone
CYP11B2 produces aldosterone through a three-step
sequence involving sequential oxidations carried out by the
heme-iron cofactor of the enzyme.8 Starting with 11-
deoxycorticosterone (DOC), initial hydroxylation at C-11
affords corticosterone which is hydroxylated at C-18 to furnish
18-hydroxycorticosterone. 18-Hydroxycorticosterone then
undergoes a final C-18 oxidation (alcohol to aldehyde) to
provide aldosterone. Importantly, CYP11B2 is the only
enzyme which catalyzes the final oxidation leading to
aldosterone.
A significant obstacle to the development of CYP11B2
inhibitors has been the identification of compounds which
selectivity inhibit CYP11B2 over CYP11B1, the enzyme
responsible for cortisol production.9 The serious complications
associated with dysregulated cortisol production, including
adrenal insufficiency and adrenal crisis, make inhibition of
Received: April 3, 2019
Accepted: June 6, 2019
Published: June 7, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.9b00152
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
CYP11B1 a serious liability that needs to be avoided. Indeed,
the development of the first CYP11B2 inhibitor to reach the
clinic, LCI699, suffered from poor selectivity against CYP11B1
and was eventually discontinued for the treatment of
hypertension for this reason.10,11 Given the high homology
between the two enzymes (93%) it is not surprising this
challenge exists.8 However, several recent reports detail
significant progress in the development of selective inhib-
itors,12−15 including the identification of highly selective
clinical candidates LFF269 and RO-6836191.16,17
Our approach to the design of highly selective metal-
loenzyme inhibitors focuses on the optimization of the
inhibitor metal binding group (MBG)−enzyme metal ion
interaction and has been successfully employed for the
development of highly selective inhibitors of related
cytochrome P450 enzymes, fungal CYP51 and CYP17.18,19
Recognition that overly potent MBGs (pyridines and
imidazoles for heme-iron) exert potent off-target activities
causing side-effects, led to consideration of less metal-avid
MBGs to improve selectivity, and provide an opportunity for
an improved therapeutic window.20 As this strategy typically
identifies less potent inhibitors with improved selectivity,
modifications to the rest of the scaffold are utilized to enhance
potency while retaining the high selectivity imparted by the
optimized MBG. Given our success employing this strategy for
related cytochrome P450s, application to CYP11B2 was
undertaken.
Table 1. MBG Evaluation of Benzimidazole Scaffold 2
CYP11B2 IC50
CYP11B1 IC50
a
cmpd
MBG
(μM)
(μM)
selectivity
8.5
3
1−1,2,3-
1.064
9.072
triazole
4
5
6
7
8
9
10
5-oxazole
4-pyrazole
5-thiazole
5-pyrimidine
2-pyrazine
3-pyridazine
4-pyridazine
>10.0
0.782
0.062
0.040
0.541
0.412
0.330
0.004
>10.0
1.668
4.523
5.380
>10.0
>10.0
>10.0
0.401
N.A.
2
73
136
>18
>24
>30
100
1112 3-pyridine
a
Selectivity: CYP11B1 IC50/CYP11B2 IC50.
Examination of simple substitution of the pyrimidine is
shown in Table 2 utilizing the 5,6-difluorobenzimidazole
scaffold. As expected, simple 2-methyl substitution on the
pyrimidine ring provided compounds devoid of CYP11B2
activity (data not shown), likely due to disruption of the
pyrimidine nitrogen−heme iron interaction. In addition, 4,6-
dimethyl substitution was also inactive (data not shown),
potentially suggesting some degree of coplanarity between the
MBG and benzimidazole core is required for binding in the
CYP11B2 pocket. Increasing the size of simple alkyl
substituents at C4 (12 → 13 → 14 → 15) led to increased
potency at CYP11B2 with compounds 14 and 15 providing
extremely high selectivity (>300-fold) over CYP11B1.
Interestingly, cyclopropyl derivative 16 had a falloff in
CYP11B2 potency. Polarity at C4 was well tolerated with
nitrile 17, achieving high selectivity and amine 18 providing
excellent CYP11B2 inhibition, albeit with more potent
CYP11B1 inhibition than seen with previous pyrimidine-
based inhibitors (18 vs 12−17). N,N-Disubstitution (19) was
also well tolerated as was elongation of the alkylamine (20).
The stability of select compounds was evaluated in
cynomolgus monkey liver microsomes, which revealed the
alkyl substituted compounds (13, 14, and 15) had moderate
stability while the aminoalkyl compounds (19 and 20) were
rapidly metabolized.
Given the overall profile of the simple alkyl-substituted
pyrimidines, exploration of fluoroalkyl derivatives was under-
taken (Table 3). Di- and trifluoromethyl analogues 21 and 22
potently inhibited CYP11B2 with excellent selectivity over
CYP11B1. 1,1-Difluoroethyl derivative 23 had a modest
decrease in CYP11B2 potency leading to slightly diminished
selectivity. Analogues 21 and 22 both showed excellent in vitro
stability. Examination of the thermodynamic aqueous solubility
of trifluoromethyl derivative 22 revealed moderate solubility (9
μM) in a pH 7.4 buffer.
A survey of the CYP11B2 literature revealed several
screening hits utilizing pyridine and imidazole MBGs affording
modest selectivity which were subsequently developed into
selective inhibitors. Given the favorable developability proper-
ties, the simple 2-(3-pyridyl) indole and benzimidazole
screening hits 1 and 2 were selected for further evaluation
(Figure 1).12,16
Figure 1. Indole and benzimidazole based CYP11B2 scaffolds.
Facile synthetic access to the benzimidazole scaffold allowed
for efficient preparation of a series of inhibitors with varied
MBGs (Table 1).21 Following precedent, the benzimidazole
core was adorned with N1 cyclopropyl and 6-fluoro
substituents for the MBG scan with published data for
pyridine-containing inhibitor 11 included for comparison.12
Compounds were tested for inhibition of human recombinant
CYP11B2 or CYP11B1 utilizing an HPLC−MS readout to
monitor both substrate and product steroids.22
A variety of five- and six-membered ring nitrogen-containing
heterocyclic MBGs were evaluated with positions adjacent to
the nitrogen atom unencumbered to allow for effective
interaction with the heme-iron. In general, six-membered
ring heterocycles were preferred over five-membered hetero-
cycles (7−10 vs 3−5) with a single five-membered hetero-
cycle, thiazole 6, affording good potency and encouraging
selectivity. Intrigued by the weak CYP11B1 activity and high
selectivity afforded by the pyrimidine analogue 7, further
exploration was undertaken.23
To improve upon the solubility of the pyrimidine 22,
modifications to the benzimidazole core were explored.16 6-
Cyanobenzimidazole and 6-cyano-7-azabenzimidazole cores
with the 4-di- and trifluoromethyl pyrimidine MBGs were
prepared (Table 4). All compounds were potent CYP11B2
inhibitors with analogues 24, 25, and 27 affording over 200-
fold selectivity and moderate (25) to excellent in vitro stability
(24, 26, and 27). Gratifyingly, 6-cyano-7-azabenzimidazole 27
B
DOI: 10.1021/acsmedchemlett.9b00152
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ACS Medicinal Chemistry Letters
Letter
Table 2. Evaluation of C4-Pyrimidine SAR
a
b
cmpd
R
CYP11B2 IC50 (μM)
CYP11B1 IC50 (μM)
S
microsome stability
12
13
14
15
16
17
18
19
20
−H
0.022
0.017
0.008
0.005
0.172
0.013
<0.002
0.029
2.267
4.157
3.330
1.907
>10.0
4.987
0.390
4.629
0.378
103
244
416
381
>58
383
>195
159
126
98
72
78
80
−Me
−Et
−iPr
−cyclopropyl
−CN
−NHMe
−N(CH3)2
−NH(CH2)2CF3
85
6
55
0.003
a
b
S, Selectivity (CYP11B1 IC50/CYP11B2 IC50). Percentage of parent compound remaining after a 65 min incubation in cynomolgus monkey liver
microsomes.
Table 3. In Vitro Evaluation of 4-Fluoroalkylpyrimidines
Table 5. Pharmacokinetic Parameters of Pyrimidine-Based
CYP11B2 Inhibitors
a
cmpd
Cmax (μM)
Tmax (h)
AUC0−24 (μM h)
t1/2 (h)
13
21
22
24
26
27
1.26
0.62
0.41
1.00
2.88
1.68
0.5
1.0
2.0
2.0
2.0
0.5
3.0
8.8
7.8
8.3
32.2
10.1
4.2
15.3
30.0
5.4
7.5
5.3
CYP11B2
CYP11B1
microsome
b
a
cmpd
R
IC50 (μM)
IC50 (μM)
S
stability
21
22
23
−CF2H
−CF3
−CF2CH3
0.018
0.025
0.050
4.325
4.771
5.723
240
190
114
106
99
a
Dosing vehicle: 0.5% CMC, 0.1% Tween-80, 1% DMSO.
a
b
S, selectivity (CYP11B1 IC50/CYP11B2 IC50). Percentage of
compound remaining after a 65 min incubation in cynomolgus
monkey liver microsomes.
moderate Cmax values, which are well below the concentrations
required to effectively inhibit CYP11B1 but well above the
levels expected for robust CYP11B2 inhibition based on the
compounds’ in vitro profiles. Additionally, both compounds
had excellent half-lives which would be expected to provide
once a day (qd) dosing in humans, thus affording an ideal PK
profile for durable inhibition of aldosterone synthesis. While
the pharmacokinetic profiles of compounds 24, 26, and 27
were acceptable in terms of drug exposures and half-lives,
compound 22 was ultimately chosen for further evaluation
given its superior pharmacokinetic profile. Evaluation of
protein binding for compound 22 in cynomolgus monkey
plasma revealed an unbound fraction of 16%, thus affording
robust free drug concentrations for efficacy studies.
Prior to evaluation of in vivo efficacy, compound 22 was
profiled against the rodent and cynomolgus monkey CYP11B1
and CYP11B2 enzymes.24 As seen with previous inhibitors, the
pyrimidine-based CYP11B2 compounds suffered a significant
loss of potency against the rodent ortholog enzymes (data not
shown).12,16 Compound 22 potently inhibited monkey
CYP11B2 with an IC50 value of 13 nM and afforded excellent
selectivity against monkey CYP11B1, with an IC50 of 8.850 μM
leading to 702-fold selectivity. Against a panel of steroid and
drug-metabolizing cytochrome enzymes, compound 22 was
over 1000-fold selective against the majority of the panel
(CYP17, CYP2D6, CYP2C9, CYP2C19, CYP3A4) with a
CYP19 IC50 of 6.445 μM, providing a 250-fold window relative
to inhibition of human CYP11B2. With the desired potency
and selectivity profiles achieved along with excellent
pharmacokinetics, compound 22 was selected for evaluation
in in vivo efficacy studies.
Table 4. In Vitro Evaluation of 6-Cyanobenzimidazole and
6-Cyano-7-azabenzimidazole Scaffolds
CYP11B2
IC50 (μM)
CYP11B1
IC50 (μM)
microsome
b
a
cmpd
R, X
S
stability
24
25
26
27
−CF2H, CH
−CF3, CH
−CF2H, N
−CF3, N
0.009
0.016
0.029
0.024
2.035
4.171
3.926
4.809
226
261
135
200
84
62
101
87
a
b
S, selectivity (CYP11B1 IC50/CYP11B2 IC50). Percentage of
compound remaining after a 65 min incubation in cynomolgus
monkey liver microsomes.
showed a 17-fold improvement (153 μM) in aqueous solubility
relative to pyrimidine 22.
With multiple compounds meeting the desired profile for
selectivity and stability, the pharmacokinetic properties of
select compounds in cynomolgus monkeys were examined.
Utilizing male monkeys, systemic drug concentrations from the
plasma were measured following oral dosing at 1 mg/kg out to
24 or 72 h with data summarized in Table 5. 3-Methyl
derivative 13 was rapidly absorbed (Tmax = 0.5 h) with a robust
Cmax (1.26 μM) but shorter than desired half-life. In contrast,
di- and trifluoromethyl derivatives 21 and 22 provided more
C
DOI: 10.1021/acsmedchemlett.9b00152
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In vivo aldosterone lowering was assessed in an ACTH-
induced steroidogenesis model in conscious male cynomolgus
monkeys with concurrent measurement of cortisol and steroid
precursors (DOC and 11-deoxycortisol).25 In the study,
control steroid responses were measured on day 1 with vehicle
treatment followed by ACTH challenge to elicit adrenal
steroidogenesis. This protocol was repeated after a 7-day
recovery period with compound 22 administered orally at 0.2,
1, and 3 mg/kg 10 min prior to ACTH administration. Area
under the curve (AUC) calculations were made from the
plasma steroid concentrations and, using each animal’s steroid
AUC levels on day 1 as its own control, changes in day 8
steroid levels were determined. For comparative purposes, the
nonselective inhibitor LCI699 (CYP11B2 IC50 = 0.7 nM;
CYP11B1 IC50 = 2.5 nM; selectivity = 3.5) was also evaluated
in the model.10
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.9b00152.
■
S
Assay protocols, experimental procedures, and analytical
data for compounds 13, 22, and 27 (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: sparkss56@yahoo.com.
ORCID
Steven M. Sparks: 0000-0002-3869-3389
Author Contributions
All authors have given approval to the final version of the
manuscript.
■
Plasma aldosterone AUCs were dose-dependently reduced
by 68, 88, and 93% at the 0.2, 1.0, and 3.0 mg/kg doses of
compound 22, respectively, with no significant impact on
plasma cortisol levels (Figure 2). In addition, no change in the
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
ACTH, adrenocorticotropic hormone; AUC, area under the
curve; DOC, 11-deoxycorticosterone; MBG, metal binding
group; mpk, mg per kg; MR, mineralocorticoid receptor.
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