Discovery of 4-Aryl-5,6,7,8-tetrahydroisoquinolines as Potent, Selective, and Orally Active Aldosterone Synthase (CYP11B2) Inhibitors: In Vivo Evaluation in Rodents and Cynomolgus Monkeys

Article abstract of DOI:10.1021/acs.jmedchem.5b00851

Inappropriately high levels of aldosterone are associated with many serious medical conditions, including renal and cardiac failure. A focused screen hit has been optimized into a potent and selective aldosterone synthase (CYP11B2) inhibitor with in vitro activity against rat, mouse, human, and cynomolgus monkey enzymes, showing a selectivity factor of 160 against cytochrome CYP11B1 in the last species. The novel tetrahydroisoquinoline compound (+)-(R)-6 selectively reduced aldosterone plasma levels in vivo in a dose-dependent manner in db/db mice and cynomolgus monkeys. The selectivity against CYP11B1 as predicted by cellular inhibition data and free plasma fraction translated well to Synacthen challenged cynomolgus monkeys up to a dose of 0.1 mg kg^-1. This compound, displaying good in vivo potency and selectivity in mice and monkeys, is ideally suited to perform mechanistic studies in relevant rodent models and to provide the information necessary for translation to non-human primates and ultimately to man.

Full text of DOI:10.1021/acs.jmedchem.5b00851

Article
pubs.acs.org/jmc
Discovery of 4‑Aryl-5,6,7,8-tetrahydroisoquinolines as Potent,
Selective, and Orally Active Aldosterone Synthase (CYP11B2)
Inhibitors: In Vivo Evaluation in Rodents and Cynomolgus Monkeys
Rainer E. Martin,† Johannes D. Aebi,*,† Benoit Hornsperger,† Hans-Jakob Krebs,† Bernd Kuhn,†
Andreas Kuglstatter,† Andre M. Alker,† Hans Peter Marki,† Stephan Muller,‡ Dominique Burger,‡
́
̈
̈
Giorgio Ottaviani,§ William Riboulet,∥ Philippe Verry,⊥ Xuefei Tan,# Kurt Amrein,*,∇
and Alexander V. Mayweg*,†
†Medicinal Chemistry, Roche Pharma Research and Early Development (pRED), Roche Innovation Center Basel, F. Hoffmann-La
Roche Ltd., Grenzacherstrasse 124, CH-4070 Basel, Switzerland
‡Discovery Technologies, Roche Pharma Research and Early Development (pRED), Roche Innovation Center Basel, F. Hoffmann-La
Roche Ltd., Grenzacherstrasse 124, CH-4070 Basel, Switzerland
§Pharmaceutical Sciences, Roche Pharma Research and Early Development (pRED), Roche Innovation Center Shanghai, F.
Hoffmann-La Roche Ltd., 720 Cai Lun Road, Building 5, Pudong, Shanghai 201203, China
∥Neuroscience, Ophthalmology and Rare Diseases, Roche Pharma Research and Early Development (pRED), Roche Innovation
Center Basel, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, CH-4070 Basel, Switzerland
⊥Discovery Cardiovascular and Metabolic Disease, Roche Pharma Research and Early Development (pRED), Roche Innovation
Center Basel, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, CH-4070 Basel, Switzerland
#Medicinal Chemistry, Roche Pharma Research and Early Development (pRED), Roche Innovation Center Shanghai, F.
Hoffmann-La Roche Ltd., 720 Cai Lun Road, Building 5, Pudong, Shanghai 201203, China
∇Discovery Infectious Disease, Roche Pharma Research and Early Development (pRED), Roche Innovation Center Basel, F.
Hoffmann-La Roche Ltd., Grenzacherstrasse 124, CH-4070 Basel, Switzerland
S
* Supporting Information
ABSTRACT: Inappropriately high levels of aldosterone are
associated with many serious medical conditions, including
renal and cardiac failure. A focused screen hit has been
optimized into a potent and selective aldosterone synthase
(CYP11B2) inhibitor with in vitro activity against rat, mouse,
human, and cynomolgus monkey enzymes, showing a
selectivity factor of 160 against cytochrome CYP11B1 in the
last species. The novel tetrahydroisoquinoline compound (+)-(R)-6 selectively reduced aldosterone plasma levels in vivo in a
dose-dependent manner in db/db mice and cynomolgus monkeys. The selectivity against CYP11B1 as predicted by cellular
inhibition data and free plasma fraction translated well to Synacthen challenged cynomolgus monkeys up to a dose of 0.1 mg
kg−1. This compound, displaying good in vivo potency and selectivity in mice and monkeys, is ideally suited to perform
mechanistic studies in relevant rodent models and to provide the information necessary for translation to non-human primates
and ultimately to man.
provide protection against renal injury in patients with chronic
kidney disease (CKD).3
INTRODUCTION
■
The renin angiotensin aldosterone system (RAAS) is a pathway
that has been linked to hypertension, volume, and salt balance
and more recently to contribute directly to end organ damage
in advanced stages of heart failure or kidney disease.1 Beyond
blood pressure control the RAAS plays an important role in the
pathogenesis of a variety of clinical conditions such as
atherosclerosis, left ventricular hypertrophy, myocardial in-
farction, and heart failure.1 In particular it has been
demonstrated that activation of RAAS is associated with an
increased risk of ischemic cardiovascular events independent
from blood pressure.2 Moreover blocking the RAAS can
Angiotensin-converting enzyme (ACE) inhibitors and
angiotensin receptor blockers (ARBs) are successfully used to
improve duration and quality of life of patients. However,
although indispensable from today’s treatment regiment, these
drugs do not yield maximum protection. In a relatively large
number of patients these drugs lead to so-called aldosterone
breakthrough, a phenomenon where aldosterone levels, after a
Received: June 4, 2015
© XXXX American Chemical Society
A
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Figure 1. Aldosterone synthase inhibition was tested in renal leiomyoblastoma cells, ectopically expressing human (h), mouse (m), or rat (r)
CYP11B2 or CYP11B1. All values are the mean of at least three experiments with a standard deviation of usually <25%. The selectivity factor (SF) is
defined as the ratio between the IC50 values on human CYP11B1 and CYP11B2.
first initial decline, return to pathological levels.1,2 It has been
demonstrated that the deleterious consequences of inappropri-
ately increased aldosterone levels can be minimized by
aldosterone blockade with mineralocorticoid receptor antago-
nists.4−6 A direct inhibition of aldosterone synthesis is expected
to provide even better protection, as it will also reduce
nongenomic effects of aldosterone. Aldosterone, regulated by
the RAAS, plays a central role in fluid and electrolyte
homeostasis and is therefore one of the principal regulators
of blood pressure.7,8
salt-stimulated PAI-1 mRNA expression in both aorta and
heart. In other studies the CYP11B2 inhibitor FAD286 (1)
only slightly improved blood pressure but improved
cardiovascular function and ameliorated cardiac hypertrophy,
albuminuria, cell infiltration, and matrix deposition in the heart
and kidney in rats with experimental heart failure.13
Recently, Novartis reported clinical data on the first
administration of LCI699 (2, Figure 1), an orally active
CYP11B2 inhibitor structurally closely related to FAD286 (1),
to patients with primary aldosteronism (PA).14,15 After a 2-
week placebo run-in phase, patients were orally dosed with 0.5
mg b.i.d. of LCI699 (2) for 2 weeks, followed by 1.0 mg b.i.d. of
LCI699 (2) for 2 weeks and a placebo for 1 additional week.
The authors concluded that the administration of 2, up to a
dose of 1.0 mg b.i.d., inhibited CYP11B2 effectively and safely
in patients with PA, resulting in significantly reduced levels of
aldosterone in both plasma and urine. The treatment resulted
in a rapid correction of hypokalemia and a modest decrease in
blood pressure. As a consequence of the low selectivity against
CYP11B1, biological signs of partial inhibition of cortisol
synthesis such as dose-dependent increases in both plasma
adrenocorticotropic hormone (ACTH) and 11-deoxycortisol
(precursor of cortisol synthesis) concentrations were observed.
In summary, these data support the concept that a CYP11B2
inhibitor can lower inappropriately high aldosterone levels and
thus might be an interesting target for the treatment of
hypertension and renal disorders. However, good selectivity
against CYP11B1 is critical in order to prevent undesired side
effects related to the hypothalamic−pituitary−adrenal (HPA)
axis.
This clearly illustrates that there is a need for more potent,
selective, and orally active CYP11B2 inhibitors for both proof-
of-concept studies in translational animal models and treatment
in man. The imidazole-type inhibitors FAD286 (1) and LCI699
(2) are potent compounds in man but considerably less potent
in rodents. Furthermore, both also show a lack of selectivity
against human CYP11B1. The recently disclosed isoquinoline
compound 3 from Hartmann et al. is a non-imidazole-type
CYP11B2 inhibitor potent against rat aldosterone synthase but
unfortunately not suitable for oral application in rodents
(Figure 1).16
Aldosterone is a steroid hormone belonging to the
mineralocorticoid family that is synthesized from cholesterol
through a cascade of linear transformations mainly catalyzed by
enzymes of the cytochrome P450 family.9 The last three steps
in this sequence are mediated by aldosterone synthase
(CYP11B2), an enzyme that is mainly present in adrenal
gland zona glomerulosa cells and to a lower extent in other
tissues and is exclusively involved in the synthesis of
aldosterone.10 Thus, CYP11B2 qualifies as a primary target
for the development of selective aldosterone synthesis
inhibitors. Human CYP11B2 shows a high sequence identity
of approximately 93% to 11β-hydroxylase (CYP11B1), an
enzyme that is critical for the synthesis of cortisol and is mainly
present in the zona fasciculata/reticularis.11 A further
considerable obstacle to the development and pharmacody-
namic characterization of selective CYP11B2 inhibitors is
encountered in the rather high species difference between
human and rodent enzymes. For instance, rat CYP11B2 and
human CYP11B2 display an overall sequence homology of only
68%, making it challenging to develop aldosterone synthase
inhibitors with comparable potency on both species.11 The
nonselective CYP11B2 inhibitor FAD286 (1, Figure 1), which
is the (+)-enantiomer of the aromatase inhibitor Fadrozole
originally introduced in Japan for the treatment of breast
cancer, and the mineralocorticoid antagonist spironolactone
were evaluated in a rat model of chronic kidney disease.12
Specifically angiotensin II and high salt treatment caused
albuminuria, azotemia, renovascular hypertrophy, glomerular
injury, tubulointerstitial fibrosis, and increased plasminogen
activator inhibitor 1 (PAI-1) and expression of osteopontin
mRNA. Both experimental compounds prevented these renal
effects and attenuated cardiac and aortic medial hypertrophy.
Following a 4-week treatment period with FAD286 (1), plasma
aldosterone concentration was reduced, whereas spironolactone
increased aldosterone levels after 4 and 8 weeks of treatment
due to a compensatory mechanism. Similarly, spironolactone
only, but not FAD286 (1), enhanced angiotensin II levels and
Herein we report on our aldosterone synthase inhibitor drug
discovery program to identify compounds with excellent
potency and an improved selectivity profile that are suitable
for oral application and would enable the study of biological
effects of aldosterone synthase inhibition in both rodents and
non-human primates.
B
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a
Scheme 1. Synthesis of Aldosterone Synthase Tetrahydroisoquinoline Inhibitors 5−11
a
Reagents and conditions: (a) EtOH, EDCI, DMAP, CH2Cl2, rt, 16 h, 84%; (b) (i) n-BuLi, N,N-diisopropylamine, −78 °C; (ii) methyl acrylate, −78
°C, 2.5 h, 95% (70% purity); (c) 6 N aqueous HCl, reflux, 2.5 h, 69%; (d) 16: Ti(Oi-Pr)4, 2 M NH3 in MeOH, NaBH4, rt, 7 h, 85%. (+)-(R)-16: (i)
Ti(Oi-Pr)4, 2 M NH3 in MeOH, NaBH4, rt, 7 h, 85%; (ii) Chiralpak AD column (40% isopropanol in n-heptane). (−)-(S)-16, 37% and (+)-(R)-16,
36%; (e) boronic acid, Pd(Ph3)4, Na2CO3, EtOH, H2O, 85 °C, 16 h. 4: with 4-cyanophenylboronic acid, 81%. (+)-(R)-4: with 4-cyanophenylboronic
acid, 84%. 8: with 3,4-difluorophenylboronic acid, 85%. 9: with 2,4,5-trifluorophenylboronic acid, 40%. 10: with 4-chloro-2-fluorophenylboronic acid,
70%. 11: with 4-chloro-3-fluorophenylboronic acid, 83%; (f) 5: acetyl chloride, triethylamine, CH2Cl2, −20 °C, 30 min, 44%. 6: propionic acid,
EDCI, CH2Cl2, rt, 16 h, 95%. (+)-(R)-6: propionic acid, EDCI, CH2Cl2, rt, 16 h, 81%. 7: isobutyryl chloride, triethylamine, CH2Cl2, −20 °C, 30 min,
63%.
were not successful. Therefore, the absolute configuration of
(+)-6 was derived from X-ray crystallographic analysis of the
undesired bromotetrahydroisoquinoline enantiomer (−)-16,
which was determined to be of (S)-configuration (Figure 2).17
In an analogous fashion the chiral separation of
aminotetrahydroisoquinoline intermediate ( )-16 enabled the
direct formation of (+)-(R)-6 from (+)-(R)-16 using the
synthetic route described above.
CHEMISTRY
■
The synthesis of aldosterone synthase inhibitors 5−11 is
outlined in Scheme 1. Esterification of commercially available 5-
bromo-4-methylnicotinic acid (12) with ethanol under EDCI/
DMAP activation provided ethyl ester 13. Deprotonation of the
methyl group with freshly prepared lithium diisopropylamide
(LDA) followed by addition of methyl acrylate gave access to
intermediate 14. The first step in this domino reaction is the
1,4-Michael addition of the tolyl anion to the acrylate, followed
by cyclization with the ethyl ester group to afford the cyclic
keto ester 14, which was isolated in a yield of 95% next to about
30% of unreacted starting material 13.
The crude keto ester 14 was then engaged in a Krapcho
decarboxylation using 6 M aqueous HCl to provide ketone 15,
which could easily be separated from hydrolyzed starting
material 13 by chemical separation. Reductive amination of
ketone 15 with ammonia in methanol in the presence of
titanium(IV) isopropoxide and treatment with sodium
borohydride furnished key building block 16, which was central
for preparation of both left- and right-hand-side variations of
target compounds 5−11. Suzuki reaction of aryl bromide
compound 16 with 4-cyanophenylboronic acid provided access
to tetrahydroisoquinoline amine 4, which was then further
reacted with different carboxyl acids to provide racemic
inhibitors 5−7. Similarly, amide formation of amine 16 with
propionic acid yielded aryl bromide compound 17, which was
then reacted under Suzuki conditions with various phenyl-
boronic acids to provide racemic target compounds 8−11 in
good to excellent yields. Racemic compound ( )-6 was
separated by preparative chiral chromatography on a Chiralpak
AD column into the two enantiomercially pure forms (+)-6 and
(−)-6. Attempts to assign the absolute configuration of the
more potent enantiomer (+)-6 by single crystal X-ray analysis
Figure 2. ORTEP drawing derived from the single crystal X-ray
analysis of bromotetrahydroisoquinoline intermediate (−)-16 reveals
the chiral center to be of (S)-configuration. Ellipsoids are drawn at the
50% probability level.
C
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Table 1. Structure−Activity Relationship (SAR) Data for Tetrahydroisoquinoline Derivatives 4−11 and 18
RESULTS AND DISCUSSION
■
Initial attempts to enhance selectivity against CYP11B1 in the
Fadrozole-imidazole series proved challenging and prompted us
to look out for suitable replacements of the imidazole moiety.
To make use of potential heme-iron binding in CYP11B2, we
compiled a library of in-house compounds containing
heteroaromatic substructures featuring a nitrogen lone pair
and without substitution α to the nitrogen atom. A focused
screen of this library yielded pyridine compound 18 as a
promising hit structure with high ligand efficiency, demonstrat-
ing that the imidazole unit can be replaced by a pyridine group
with comparable potency (Table 1). However, the selectivity
factor (SF), defined as the ratio between the IC50 values on
human CYP11B1 and B2, for this compound was measured to
be 8 and thus offered no improvement over imidazole-type
inhibitor 1 (SF = 12). In addition, the moderate metabolic
stability of arylpyridine compound 18 in mouse microsomes
limited the use of this compound for oral proof-of-concept
Figure 3. Homology models of CYP11B1 (white) and B2 (yellow) in
cartoon representation. The heme cofactor is shown in green and
modeled compound 18 in cyan. Amino acid differences within 16 Å of
the modeled ligand are highlighted (B1, salmon; B2, magenta), which
are at positions 68, V/M; 109, C/H; 112, I/S; 147, D/E; 302, E/D;
320, A/V; 404, Q/R; and 439, H/Y for CYP11B1/B2, respectively.18
experiments.
With the aim to identify potential selectivity pockets we
generated homology models of CYP11B1 and B2, respectively,
and modeled the binding mode of inhibitor 18, assuming a
critical iron−pyridine nitrogen interaction (Figure 3). This
structural hypothesis indicated additional space at the 4,5-
position of the pyridine moiety. As illustrated in Figure 3, there
are several amino acid differences between CYP11B1 and B2
enzymes within the extended binding site. Although they are
not exposed for direct ligand interaction, they might still affect
details and plasticity of the binding region resulting in decent
selectivity. Structure-guided design to fill the pocket on the
D
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right-hand side prompted the synthesis of tetrahydroisoquino-
line 4.
The tetrahydroisoquinoline 4 showed a potency of
hCYP11B2 IC50 = 42 nM together with a poor SF of 5 but
revealed high stability in both human and rodent microsomes.
Interestingly, amide bond formation at the free amino group in
4 was well tolerated providing acetylated and propionylated
compounds 5 and 6 with a potency of IC50 of 21 and 25 nM
and improved selectivity values of SF of 13 and 28, respectively.
Further enlargement of the alkyl substitution to an isopropyl
side chain gave compound 7 with significantly inferior potency
(IC50 = 123 nM), albeit with a slightly improved selectivity
factor of 33. In the isoquinoline series 5−7 log D values
increased from 1.8 to 2.1 and 2.6,19 concomitantly with an
expected decrease in aqueous solubility from 415 to 360 and 90
μg mL−1, respectively.20 All three compounds 5−7 displayed
high passive permeability in the Pampa assay (Pe > 4.4 × 10−6
cm s−1),21 low clearance in both human and rodent
microsomes, and no inhibition of cytochrome P450 subtype
enzymes 3A4, 2D6, and 2C9 (all IC50 > 50 μM).
Figure 4. In vitro data of tetrahydroisoquinoline enantiomers 6.
Aldosterone synthase inhibition was tested in renal leiomyoblastoma
cells, ectopically expressing human (h), cynomolgus monkey (c),
mouse (m), or rat (r) CYP11B2 or CYP11B1. All values are the mean
of at least three experiments with a standard deviation of usually <25%.
The selectivity factor (SF) is defined as the ratio between the IC50
values on human CYP11B1 and CYP11B2.
Having optimized the right-hand side of the molecule with
respect to potency, selectivity, and rodent microsomal
clearance, we next turned our attention to the optimization
of the substitution pattern on the aryl ring. Exchange of the 4-
cyanophenyl side chain in 6 to a 3,4-difluorophenyl motif
provided compound 8 with reduced potency (IC50 = 47 nM)
and a slightly decreased selectivity factor of 18. Switching to a
2,4,5-trifluoro (9), 4-chloro-2-fluoro (10), and 4-chloro-3-
fluoro (11) substitution pattern on the aryl ring resulted in a
gain in potency (IC50 of 27, 28, and 14 nM) and also improved
selectivity to a SF of 42, 53, and 95, respectively. In the
sequence 8−11 lipophilicity increased from log D of 3.0, 3.1 to
3.6 and 3.6 with aqueous solubility decreasing in the same order
from 310, 239 to 129 and 99 μg mL−1, respectively. Again, all
compounds showed high passive permeability in the Pampa
assay (Pe > 4.0 × 10−6 cm s−1) and no inhibition of cytochrome
P450 subtype enzymes 3A4, 2D6, and 2C9. Although
tetrahydroisoquinoline 11 was found to be the most potent
and selective compound in the entire series, and thus seemed to
be an attractive candidate for further evaluation, the decreased
rodent microsomal stability precluded its further evaluation.
Similarly, compound 9 which showed an acceptable selectivity
profile was not further considered due to poor rat microsomal
stability. Finally, on the basis of the overall profile, tetrahydro-
isoquinoline 6 was selected for chiral separation owing to its
good potency in combination with medium selectivity, high
microsomal stability, and a balanced physicochemical profile
(lipophilicity, log D = 2.1; permeability, Pe = 5.2 × 10−6 cm s−1;
solubility, 360 μg mL−1). The separated enantiopure isomers of
6 showed significantly different affinities on hCYP11B2, with
the (+)-enantiomer being 105-fold more potent than the
(−)-enantiomer (Figure 4). The enantiomerically pure
compound (+)-(R)-6 displayed not only single digit nanomolar
potency on human aldosterone synthase (IC50 = 9 nM) but was
also very active on the cynomolgus monkey enzyme (IC50 = 3
nM), with an attractive selectivity factor of 160 against
CYB11B1 in the latter species. Similar to racemic 6 the
physicochemical profile of (+)-(R)-6 was very attractive (log D
= 2.2, Pe = 4.8 × 10−6 cm s−1, solubility of 315 μg mL−1),
making it an interesting candidate for in vivo proof-of-concept
studies.
Figure 5. X-ray structure of hCYP11B2 with compound 11 (PDB
code 4ZGX). The protein is displayed in green and the heme and the
tetrahydroisoquinoline 11 in cyan sticks. Overlaid onto this structure
are heme and Fadrozole from the complex structure with hCYP11B2
(magenta, PDB code 4FDH).22
Å, several features of the binding mode such as the close
contact of the heme iron with the nitrogen lone pair of the
tetrahydroisoquinoline core (d ≈ 2.0 Å) and the orientation of
the ligand in the binding site are unambiguous conclusions
from the electron density. The crystal structure confirms the
inhibitor binding mode as predicted by our homology model
and therefore further validates the original design strategy.
However, the absolute stereoconfiguration of the chiral center
in compound 11 could not be resolved. Figure 5 also displays
an overlay with the X-ray complex structure of Fadrozole with
hCYP11B2, which became recently available.22 The structure
reveals that the iron interaction with the imidazole lone pair of
Fadrozole is analogous to that with the tetrahydroisoquinoline
nitrogen in our series and that the phenyl substituent occupies
a similar position in both structures, engaging in aromatic
interactions with the side chains of Trp116 and Phe130.
The tetrahydroisoquinoline (+)-(R)-6 showed an IC50 of 530
nM on CYP11B2 in mouse and thus is of comparable potency
with 3,4-dihydro-1H-quinolin-2-one 3 and about half the
potency of reference compounds 1 or 2. However, the higher
Figure 5 shows the X-ray structure of human CYP11B2 in
complex with inhibitor 11. Despite the limited resolution of 3.2
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Figure 6. Lowering of aldosterone levels in db/db mice after food admix treatment with tetrahydroisoquinoline (+)-(R)-6 for 21 days at doses of 6
and 20 mg kg−1. Steroid levels are presented as % vs vehicle. Data are expressed as the mean SEM with n = 10 mice per group. Statistical analysis
was performed by ANOVA followed by post hoc test: (∗) p < 0.05, (∗∗) p < 0.01, or (∗∗∗) p < 0.001 between control and the indicated test group.
(a) Enzymatic step mediated by both CYP11B1 and CYP11B2. (b) Enzymatic step mediated by CYP11B2 only.
Figure 7. Steroid profile in cynomologus monkey 60 min after ACTH challenge and treatment with tetrahydroisoquinoline (+)-(R)-6. Due to the
natural variations of steroid levels of the individual animals, steroid baseline levels of each group at t = 0 were normalized to 0. The mean differences
at t = 60 min are reported as % change vs vehicle (mean SEM with n = 2 monkeys per group except for vehicle with n = 7 × 2 animals). Note that
this mathematical transformation might render mean values below 0. (a) Enzymatic step mediated by both CYP11B1 and CYP11B2. (b) Enzymatic
step mediated by CYP11B2 only. (c) Enzymatic step mediated by CYP11B1 only.
metabolic stability makes (+)-(R)-6 the first selective
compound suitable for oral dosing in mouse. Despite significant
effort, it was not possible to establish a reliable in vitro
expression and assay system for measuring mouse and rat
CYP11B1 inhibition. Therefore, we assessed efficacy and
selectivity of compound (+)-(R)-6 directly in obese diabetic
db/db mice, a well-known mouse model displaying con-
stitutively elevated steroid levels.23,24 Since both CYP11B1 and
CYP11B2 catalyze the oxidation of 11-deoxycorticosterone to
corticosterone, but only CYP11B2 can oxidize corticosterone to
aldosterone, the selectivities of inhibition can be inferred from
analysis of the levels of these steroids. A prerequisite for this
study was the accurate and concurrent measurement of a panel
of up to seven steroid concentrations in plasma samples, which
was accomplished by a highly optimized LC−MS−MS analysis
protocol (for detailed information, see Supporting Informa-
tion). At doses of 6 and 20 mpk (food admix, 21 days)
significant and dose-dependent reduction of aldosterone plasma
levels with no concurrent increase of 11-deoxycorticosterone,
demonstrating selective inhibition of CYP11B2, was observed
(Figure 6). In a similar experiment, where db/db mice were
treated with a single dose (po) of 3 and 30 mg kg−1 of
F
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unselective inhibitor LCI699 (2), precursor 11-deoxycorticos-
terone showed at t = 2 h a dramatic 14- and 59-fold increase,
respectively. In parallel, LCI699 (2) lowered both aldosterone
and corticosterone levels (aldosterone to 51% and 37% of
control, corticosterone to 29% and 9% of control; Figure S1 in
Supporting Information). Together these results underscore
the superiority of compound (+)-(R)-6 for future in vivo
assessment of aldosterone synthase blockage in rodent disease
models.
On the basis of the encouraging mouse data, compound
(+)-(R)-6 was next tested in an angiotension II ZSF1 rat
infusion model relevant for chronic kidney disease (20 mpk,
food admix, 6 weeks) showing similarly a selective decrease of
aldosterone production concomitant with an improvement in
renal parameters (including albuminuria, glomerular filtration
rate, and histology) to a better extent than the mineralocorti-
coid receptor antagonist eplerenone.25
The relatively high selectivity factor of 160 for (+)-(R)-6 in
cynomologus monkeys prompted us to test this compound also
in primates in order to elucidate the effects on steroid profiles
(Figure 7). In contrast to rodents, primates produce 11-
deoxycortisol in addition to 11-deoxycorticosterone.11 Since the
conversion of 11-deoxycortisol to cortisol is only catalyzed by
CYP11B1, this allowed an independent assessment of the
selectivity of inhibition. Cynomologus monekys were treated
with (+)-(R)-6 at doses of 0.01, 0.1, and 1.0 mg kg−1 and with
reference compound LCI699 (2) at 0.5 mg kg−1 at t = 0.
Adrenocorticotropic hormone (ACTH, Synacthen) was
administred 5 min later. Although the hormone levels were
measured over a time period of 6 h, percentage of changes were
calculated 60 min after Synacthen application where cmax is
observed and which is the clinically relevant time point (Table
S1). An additional argument to evaluate the efficacy of
compound (+)-(R)-6 at the single time point t = 60 min
rather than using steroid AUC0−6h profiles is the natural
variation of steroid levels observed during the day. The
maximum decrease of aldosterone levels for compound
(+)-(R)-6 was obtained for both doses of 0.1 and 1.0 mg
kg−1 which was comparable to the level observed with LCI699
(2) at a dose of 0.5 mg kg−1. However, at a dose of 0.5 mg kg−1,
LCI699 (2) resulted in a clear reduction of cortisol levels with a
concurrent build-up of 11-deoxycortisol precursor concen-
trations. In contrast, tetrahydroisoquinoline (+)-(R)-6 gave no
decrease of cortisol levels at all levels investigated and only
displayed an increase of plasma 11-deoxycortisol at the highest
applied dose of 1.0 mg kg−1; from this it was concluded that in
vivo selectivity against CYP11B1 is seen up to a dose of at least
0.1 mg kg−1. This experimental observation is in good
accordance with PK measurements that showed that the free
plasma levels measured for tetrahydroisoquinoline (+)-(R)-6 at
doses of ≤0.1 mg kg−1 are significantly below the IC50 against
cytochrome CYP11B1 corresponding to a calculated in vivo
CYP11B1 inhibition of <10% (Table S2).
cynomolgus monkeys selectivity against CYP11B1 was
demonstrated up to a dose of 0.1 mg kg−1 with complete
inhibition of aldosterone synthesis. Although compound
(+)-(R)-6 displays only moderate activity in rodents, the high
metabolic stability allows for oral application, making it a
suitable tool compound for in vivo assessment of aldosterone
synthase blockage in rodent disease models. The improved
properties of tetrahydroisoquinoline (+)-(R)-6 differentiate this
compound from the recently published benzimidazole-type
CYP11B2 inhibitors which were not suitable for evaluation in
classical rodent models of aldosteronism due to their rather
weak rat potency.26
EXPERIMENTAL SECTION
■
General Methods for Chemistry. Reactions were carried out
under an atmosphere of argon. Solvents and reagents were obtained
from commercial sources and were used without further purification.
All reactions were monitored by thin-layer chromatography (TLC) on
Merck silica gel 60 F254 TLC glass plates (visualized by UV
fluorescence at λ = 254 nm). 1H nuclear magnetic resonance
(NMR) spectra were recorded on a Bruker 300 or 600 MHz
instrument. The respective 13C NMR spectra were measured on a 150
MHz instrument. The chemical shifts (δ in ppm) are reported relative
1
to tetramethylsilane as internal standard. Resonances in the H NMR
spectra are reported to the nearest 0.01 ppm. NMR abbreviations are
as follows: s, singlet; d, doublet; t, triplet; q, quadruplet; m, multiplet;
br, broad. The purity of final compounds as measured by LC−MS was
1
at least >95%. Purity of compounds was analyzed by H NMR and
LC−MS. LC−MS spectra were recorded on a Waters UPLC−MS
system equipped with Waters Acquity CTC PAL autosampler and a
Waters SQD single quadrupole mass spectrometer. Analytical
separation was conducted on a Zorbax Eclipse Plus C18 2.1 mm ×
30 mm, 1.7 μm column at 50 °C; eluent A = 0.01% formic acid in
water; eluent B = acetonitrile at a flow of 1 mL min−1. Gradient: 0 min
3% B, 0.2 min 3% B, 2 min 97% B, 1.7 min 97% B, and 2.0 min 97% B.
The injection volume was 2 μL. LC−MS high resolution spectra were
recorded on an Agilent LC system consisting of an Agilent 1290 high
pressure gradient system, a CTC PAL autosampler, and an Agilent
6520 QTOF. Analytical separation was achieved on a Zorbax Eclipse
Plus C18 column (2.1 mm × 50 mm, 1.7 μm) at 55 °C; eluent A =
0.01% formic acid in water; eluent B = 0.01% formic acid in
acetonitrile at a flow of 1 mL min−1. Gradient: 0 min 5% B, 0.3 min 5%
B, 4.5 min 99% B, and 5 min 99% B. The injection volume was 2 μL.
Ionization was performed in multimode source. The mass
spectrometer was run in 2 GHz extended dynamic range mode,
resulting in a resolution of about 10 000 at m/z = 922. Mass accuracy
was ensured by internal drift correction. Service measurements were
performed by the NMR and MS service teams at F. Hoffmann-La
Roche, Basel.
Representative Synthetic Procedures. Preparation of Ethyl
5-Bromo-4-methylnicotinate (13). To a stirred light brown
suspension of 5-bromo-4-methylnicotinic acid (12, 10.00 g, 46.3
mmol) and ethanol (2.35 g, 2.97 mL, 50.9 mmol) in CH2Cl2 (231
mL) at 0 °C under argon were added EDCI (10.9 g, 55.5 mmol) and
DMAP (566 mg, 4.63 mmol). Stirring was continued overnight and
the reaction mixture allowed to warm to rt. The reaction mixture was
poured on aqueous 10% KH2PO4 solution followed by extraction with
EtOAc (3×). The organic phases were washed once with aqueous 10%
KH2PO4, aqueous saturated NaHCO3, and with aqueous saturated
NaCl solution. The combined organic phases were dried over Na2SO4,
filtered, and evaporated to afford the title compound (9.49 g, 84%) as
brown solid. LC−MS: tR = 1.19 min, 97% (265 nm); tR = 1.19 min,
CONCLUSION
■
The novel tetrahydroisoquinoline compound (+)-(R)-6 shows
excellent inhibitory potency against human and cynomolgus
monkey aldosterone synthase, with a remarkable selectivity
factor of 160 against CYB11B1 in the latter species. The
compound significantly lowered aldosterone plasma levels in a
dose-dependent manner in mice and cynomolgus monkeys and
protected kidney structure and function in a ZSF1 rat model of
chronic kidney disease. In Synacthen challenged conscious
1
97% (225 nm). H NMR (600 MHz, DMSO-d6): δ = 8.86 (s, 1H),
8.82 (s, 1H), 4.35 (q, J = 7.1 Hz, 2H), 2.58 (s, 3H), 1.33 (t, J = 7.1 Hz,
3H). 13C NMR (150 MHz, DMSO-d6): δ = 165.1, 153.6, 149.0, 146.9,
128.4, 124.7, 61.6, 19.7, 14.0. HRMS (C9H10BrNO2): calcd 242.9895;
found 242.9898.
G
DOI: 10.1021/acs.jmedchem.5b00851
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Preparation of Methyl 4-Bromo-8-oxo-5,6,7,8-tetrahydro-
isoquinoline-7-carboxylate (14). Ethyl 5-bromo-4-methylnicoti-
nate (13, 7.04 g, 28.8 mmol) in THF (28.8 mL) was added over a
period of 20 min to a solution of LDA (31.7 mmol), which was freshly
prepared from N,N-diisopropylamine (4.52 mL, 31.7 mmol) and n-
BuLi (19.8 mL, 31.7 mmol; 1.6 M in hexane) in THF (144 mL) at
−78 °C. The resulting dark red solution was stirred for 20 min, then
methyl acrylate (6.5 mL, 72.1 mmol) in THF (28.8 mL) was added
within 15 min. The reaction was stirred an additional 1.5 h, then
aqueous 10% AcOH (57.8 mL, 101 mmol) was added (pH 4−5) and
the reaction was allowed to warm to rt. After evaporation, the residue
was partitioned between aqueous saturated NaHCO3 and EtOAc, and
extracted with EtOAc (3×). The combined organic phases were dried
over Na2SO4 and concentrated to afford the title compound (7.80 g,
95% in 70% purity as keto−enol mixture and 30% of starting material
13) as brown solid. The material was used crude in the consecutive
reaction step without further purification. LC−MS: 280.0 ([M + H]+,
1 Br).
(C9H11BrN2): calcd 226.0106; found 226.0104. [α]20 +8.00 (c 1.0,
MeOH).
D
Enantiomer (−)-(S)-16. LC−MS: tR = 0.92 min, 89% (265 nm); tR
= 0.92 min, 94% (220 nm). 1H NMR (600 MHz, DMSO-d6): δ = 8.59
(s, 1H), 8.48 (s, 1H), 3.92 (t, J = 5.7 Hz, 1H), 2.70−2.63 (m, 1H),
2.62−2.56 (m, 1H), 1.98−1.90 (m, 1H), 1.87−1.81 (m, 1H), 1.75−
1.66 (m, 1H), 1.63−1.55 (m, 1H). 13C NMR (150 MHz, DMSO-d6):
δ = 149.0, 148.1, 144.6, 140.1, 122.7, 46.9, 31.6, 29.3, 18.0. HRMS
(C9H11BrN2): calcd 226.0106; found 226.0104. [α]20 −8.72 (c 0.41,
D
MeOH). Crystallization from n-pentane followed by recrystallization
from n-heptane provided single crystals suitable for X-ray crystallo-
graphic analysis.
Preparation of (rac)-N-(4-Bromo-5,6,7,8-tetrahydro-
isoquinolin-8-yl)propionamide (17). To a stirred black solution
of (rac)-4-bromo-5,6,7,8-tetrahydroisoquinolin-8-amine (16, 317 mg,
1.4 mmol) and propionic acid (115 μL, 1.54 mmol) in CH2Cl2 (7.0
mL) at 0 °C was added EDCI (295 mg, 1.54 mmol). Stirring was
continued overnight and the reaction mixture allowed to warm to rt.
The reaction mixture was poured on aqueous 10% KH2PO4 solution
followed by extraction with EtOAc (3×). The organic phases were
washed once with aqueous 10% KH2PO4, aqueous saturated NaHCO3,
and aqueous saturated NaCl solution. The organic phase was dried
over Na2SO4, filtered, evaporated, and purified by precipitation from
CH2Cl2 with n-pentane to afford the title compound (365 mg, 92%) as
light brown solid. LC−MS: tR = 1.42 min, 92% (265 nm); tR = 1.42
Preparation of 4-Bromo-6,7-dihydroisoquinolin-8(5H)-one
(15). The crude methyl 4-bromo-8-oxo-5,6,7,8-tetrahydro-
isoquinoline-7-carboxylate (14, 7.79 g, 27.4 mmol) was dissolved
(small amount of insoluble material remained) in aqueous 6 M HCl
(84.1 mL, 505 mmol) and heated at reflux for 2.5 h (dark brown
solution, no more SM visible on TLC). The acidic solution was
concentrated in vacuo, suspended in water (∼25 mL), cooled in an ice
bath, and basified with 6.0 M KOH. The aqueous solution was washed
with diethyl ether (2×) and CH2Cl2 (3×), and the combined organic
layers were dried over Na2SO4, filtered, and concentrated to afford the
title compound (4.30 g, 69%) as brown solid. LC−MS: tR = 1.08 min,
1
min, 91% (225 nm). H NMR (600 MHz, DMSO-d6): δ = 8.54 (s,
1H), 8.29 (s, 1H), 8.23 (d, J = 8.5 Hz, 1H), 5.05−5.01 (m, 1H), 2.69−
2.64 (m, 2H), 2.13 (quin, J = 7.4 Hz, 2H), 1.92−1.87 (m, 1H), 1.84
(br dd, J = 5.0, 2.5 Hz, 1H), 1.83−1.76 (m, 1H), 1.72−1.67 (m, 1H),
1.03 (t, J = 7.6 Hz, 3H). 13C NMR (150 MHz, DMSO-d6): δ = 172.5,
148.8, 148.6, 145.5, 136.4, 122.8, 44.2, 29.0, 28.5, 28.4, 18.6, 10.0.
HRMS (C12H15BrN2O): calcd 282.0368; found 282.0372.
1
97% (265 nm); tR = 1.08 min, 97% (225 nm). H NMR (300 MHz,
DMSO-d6): δ = 8.89 (s, 1H), 8.88 (s, 1H), 2.95 (dd, J = 6.1 Hz, 2H),
2.66 (dd, J = 7.5, 5.9 Hz, 2H), 2.21−2.02 (m, 2H). 13C NMR (150
MHz, DMSO-d6): δ = 196.7, 154.3, 151.9, 146.7, 129.2, 122.6, 37.6,
28.6, 20.9. HRMS (C9H8BrNO): calcd 224.9789; found 224.9789.
Preparation of (rac)-4-Bromo-5,6,7,8-tetrahydroisoquinolin-
8-amine (16). 4-Bromo-6,7-dihydroisoquinolin-8(5H)-one (15,
4.81g, 21.3 mmol), titanium(IV) isopropoxide (12.5 mL, 42.6
mmol), and ammonia (53.2 mL, 106 mmol; 2.0 M solution in
MeOH) were stirred at rt for 5 h. The reaction was cooled to 0 °C,
NaBH4 (1.21 g, 31.9 mmol) added portionwise over 10 min, and the
resulting mixture stirred at rt for an additional 2 h. The reaction was
quenched by pouring it into aqueous ammonium hydroxide (25%) and
the formed precipitate filtered and washed with EtOAc (3×, each time
suspended in EtOAc and stirred for 5 min). The organic layer was
separated, and the remaining aqueous layer was extracted with EtOAc.
The combined organic phases were extracted with 1 M HCl. The
acidic aqueous layer was washed with EtOAc (1×), then treated with
aqueous sodium hydroxide (2 M) to give pH 10−12 and extracted
with EtOAc (3×). The combined second organic extracts were washed
with brine, dried over Na2SO4, and concentrated in vacuo to give the
title compound (4.11 g, 85%) as brown solid. LC−MS: tR = 0.26 min,
Preparation of (rac)-4-(8-Amino-5,6,7,8-tetrahydro-
isoquinolin-4-yl)benzonitrile (4). (rac)-4-Bromo-5,6,7,8-
tetrahydroisoquinolin-8-amine (16, 681 mg, 3 mmol) and 4-
cyanophenylboronic acid (540 mg, 3.6 mmol) were dissolved in
ethanol (54 mL) to give a light brown solution. Na2CO3 (350 mg, 3.3
mmol) dissolved in water (8.9 mL) was added followed by
tetrakis(triphenylphosphine)palladium (0) (104 mg, 90 μmol) after
evacuation and replacing with argon (cycle repeated 5×). The solution
was then heated at 85 °C overnight. The reaction was treated with an
aqueous 10% NaCl solution and extracted with EtOAc (3×). The
organic phases were washed again with an aqueous 10% NaCl solution,
dried over Na2SO4, filtered, and evaporated under reduced pressure to
give a brown foam which was purified by flash chromatography [50 g
SiO2, Telos cartridge, CH2Cl2/MeOH (5% → 7.5%)] and
precipitation from CH2Cl2 with n-pentane. The title compound
(605 mg, 81%) was isolated as a light brown foam. LC−MS: tR = 0.98
1
min, 99% (265 nm); tR = 0.98 min, 98% (225 nm). H NMR (300
MHz, DMSO-d6): δ = 8.69 (s, 1H), 8.18 (s, 1H), 7.94 (d, J = 8.1 Hz,
2H), 7.58 (d, J = 8.1 Hz, 2H), 3.98 (dd, J = 5.4 Hz, 1H), 2.47−2.29
(m, 2H), 2.00−1.72 (m, 2H), 1.70−1.49 (m, 2H). 13C NMR (150
MHz, DMSO-d6): δ = 150.3, 146.5, 142.7, 142.6, 137.1, 134.6, 132.3,
130.4, 118.7, 110.5, 46.9, 31.9, 27.0, 18.5. HRMS (C16H15N3): calcd
249.1266; found 249.1268.
Preparation of (+)-4-[(8R)-8-Amino-5,6,7,8-tetrahydro-
isoquinolin-4-yl]benzonitrile ((+)-(R)-4). (+)-(R)-4-Bromo-
5,6,7,8-tetrahydroisoquinolin-8-amine ((+)-(R)-16, 90.8 mg, 400
μmol) and 4-cyanophenylboronic acid (72 mg, 480 μmol) were
dissolved in ethanol (7.1 mL) to give a light brown solution. Na2CO3
(46.6 mg, 440 μmol) dissolved in water (0.8 mL) was added followed
by tetrakis(triphenylphosphine)palladium (0) (13.9 mg, 12 μmol)
after evacuation and replacing with argon (cycle repeated 5×). The
solution was then heated at 85 °C overnight. The reaction was treated
with an aqueous 10% NaCl solution and extracted with EtOAc (3×).
The organic phases were washed again with an aqueous 10% NaCl
solution, dried over Na2SO4, filtered, and evaporated under reduced
pressure to give a brown foam which was purified by flash
chromatography [20 g SiO2, Telos cartridge, CH2Cl2/MeOH (5%
→ 10%)] and precipitation from CH2Cl2 with n-pentane. The title
1
89% (265 nm); tR = 0.26 min, 91% (225 nm). H NMR (600 MHz,
DMSO-d6): δ = 8.59 (s, 1H), 8.48 (s, 1H), 3.92 (t, J = 5.7 Hz, 1H),
2.70−2.63 (m, 1H), 2.62−2.56 (m, 1H), 1.98−1.90 (m, 1H), 1.87−
1.81 (m, 1H), 1.75−1.66 (m, 1H), 1.63−1.55 (m, 1H). 13C NMR
(150 MHz, DMSO-d6): δ = 149.1, 148.1, 144.6, 140.1, 122.7, 46.9,
31.7, 29.3, 18.1. HRMS (C9H11BrN2): calcd 226.0106; found
226.0107.
Preparation of (+)-(R)-4-Bromo-5,6,7,8-tetrahydro-
isoquinolin-8-amine ((+)-(R)-16) and (−)-(S)-4-Bromo-5,6,7,8-
tetrahydroisoquinolin-8-amine ((−)-(S)-16). The two enantiomers
of 16 (300 mg) were separated on a Chiralpak AD column (40%
isopropanol in n-heptane) to give (+)-(R)-16 (109 mg, 36%) and
(−)-(S)-16 (112 mg, 37%) as light brown crystals.
Enantiomer (+)-(R)-16. LC−MS: tR = 0.92 min, 94% (265 nm); tR
= 0.92 min, 96% (220 nm). 1H NMR (600 MHz, DMSO-d6): δ = 8.59
(s, 1H), 8.48 (s, 1H), 3.92 (t, J = 5.7 Hz, 1H), 2.70−2.63 (m, 1H),
2.62−2.56 (m, 1H), 1.98−1.90 (m, 1H), 1.87−1.81 (m, 1H), 1.75−
1.66 (m, 1H), 1.63−1.55 (m, 1H). 13C NMR (150 MHz, DMSO-d6):
δ = 149.0, 148.1, 144.6, 140.1, 122.7, 46.9, 31.6, 29.3, 18.0. HRMS
H
DOI: 10.1021/acs.jmedchem.5b00851
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
compound (84 mg, 84%) was isolated as a light yellow foam. LC−MS:
tR = 0.98 min, 99% (265 nm); tR = 0.98 min, 98% (225 nm). 1H NMR
(300 MHz, DMSO-d6): δ = 8.69 (s, 1H), 8.18 (s, 1H), 7.94 (d, J = 8.1
Hz, 2H), 7.58 (d, J = 8.1 Hz, 2H), 3.98 (dd, J = 5.4 Hz, 1H), 2.47−
2.29 (m, 2H), 2.00−1.72 (m, 2H), 1.70−1.49 (m, 2H). 13C NMR
(150 MHz, DMSO-d6): δ = 150.3, 146.5, 142.7, 142.6, 137.1, 134.6,
132.3, 130.4, 118.7, 110.5, 46.9, 31.9, 27.0, 18.5. HRMS (C16H15N3):
calcd 249.1266; found 249.1268.
2.22−2.08 (m, 2H), 1.92−1.84 (m, 1H), 1.82−1.63 (m, 3H), 1.05 (t, J
= 7.6 Hz, 3H). 13C NMR (150 MHz, DMSO-d6): δ = 172.9, 150.3,
147.4, 144.0, 142.7, 135.2, 134.3, 132.9, 130.8, 119.2, 111.1, 44.7, 29.4,
29.0, 27.2, 19.5, 10.5. HRMS (C19H19N3O): calcd 305.1528; found
305.1527.
Preparation of (+)-(R)-6 from Enantiopure Benzonitrile
(+)-(R)-4. (+)-(R)-N-(4-(4-Cyanophenyl)-5,6,7,8-tetrahydro-
isoquinolin-8-yl)propionamide ((+)-(R)-6). To a stirred solution
of (+)-(R)-4-[(8R)-8-amino-5,6,7,8-tetrahydroisoquinolin-4-yl]-
benzonitrile ((+)-(R)-4, 71 mg, 285 μmol) and propionic acid (23.4
μL, 313 μmol) in CH2Cl2 (1.5 mL) at 0 °C under argon was added
EDCI (60.1 mg, 313 μmol). Stirring was continued overnight and the
reaction mixture was allowed to warm to rt. The reaction mixture was
poured into aqueous 10% KH2PO4 solution followed by extraction
with EtOAc (3×). The organic phases were washed once with aqueous
10% KH2PO4, aqueous saturated NaHCO3, and aqueous saturated
NaCl solution. The organic phase was dried over Na2SO4, filtered, and
evaporated to afford after precipitation (CH2Cl2/n-pentane) the title
compound (70 mg, 81%) as light brown solid. The analytical
characterization data obtained are identical to the data for (+)-(R)-6
derived from chiral separation of (rac)-6.
Preparation of (rac)-N-(4-(4-Cyanophenyl)-5,6,7,8-
tetrahydroisoquinolin-8-yl)acetamide (5). A solution of (rac)-4-
(8-amino-5,6,7,8-tetrahydroisoquinolin-4-yl)benzonitrile (4, 62.3 mg,
250 μmol) in CH2Cl2 (3.6 mL) cooled to −20 °C was treated with
acetyl chloride (21.3 μL, 275 μmol) and triethylamine (41.8 μL, 300
μmol). After 30 min, an additional drop of acetyl chloride was added,
and after 5 min the mixture was treated with MeOH (0.2 mL) and
extracted with water/EtOAc (3×). The organic phases were dried over
Na2SO4, filtered, and evaporated under reduced pressure. Purification
by flash chromatoraphy [50 g SiO2, Telos cartridge, CH2Cl2/MeOH
(2% → 3%)] and precipitation from CH2Cl2 with n-pentane gave the
title compound (32 mg, 44%) as an off-white powder. LC−MS: tR =
1
1.18 min, 96% (265 nm); tR = 1.18 min, 96% (225 nm). H NMR
Preparation of (rac)-N-(4-(4-Cyanophenyl)-5,6,7,8-
tetrahydroisoquinolin-8-yl)isobutyramide (7). A solution of
(rac)-4-(8-amino-5,6,7,8-tetrahydroisoquinolin-4-yl)benzonitrile (4,
62.3 mg, 250 μmol) in CH2Cl2 (3.6 mL) cooled to −20 °C was
treated with isobutyryl chloride (32.1 μL, 300 μmol) and triethylamine
(69.7 μL, 500 μmol). After 30 min, an additional drop of isobutyryl
chloride was added, and after 5 min the mixture was treated with
MeOH (0.2 mL) and extracted with water/EtOAc (3×). The organic
phases were dried over Na2SO4, filtered, and evaporated under
reduced pressure. Purification by flash chromatography [20 g SiO2,
Telos cartridge, CH2Cl2/MeOH (1% → 2%)] and precipitation from
CH2Cl2 with n-pentane gave the title compound (50 mg, 63%) as an
off-white powder. LC−MS: tR = 1.60 min, 100% (265 nm); tR = 1.60
(300 MHz, DMSO-d6): δ = 8.41 (s, 1H), 8.36 (d, J = 8.5 Hz, 1H),
8.25 (s, 1H), 7.95 (d, J = 8.5 Hz, 2H), 7.59 (d, J = 8.5 Hz, 2H), 5.09
(ddd, J = 7.3 Hz, 1H), 2.60−2.53 (m, 2H), 1.95−1.85 (m, 1H), 1.89
(s, 3H), 1.81−1.62 (m, 3H). 13C NMR (150 MHz, DMSO-d6): δ =
168.7, 149.9, 147.0, 143.7, 142.2, 134.8, 133.8, 132.4, 130.4, 118.7,
110.7, 44.3, 28.9, 26.8, 22.7, 19.0. HRMS (C18H17N3O): calcd
291.1372; found 291.1376.
Preparation of (rac)-N-(4-(4-Cyanophenyl)-5,6,7,8-
tetrahydroisoquinolin-8-yl)propionamide (6). To a stirred
solution of (rac)-4-(8-amino-5,6,7,8-tetrahydroisoquinolin-4-yl)-
benzonitrile (4, 480 mg, 1.93 mmol) and propionic acid (158 μL,
2.12 mmol) in CH2Cl2 (10 mL) at 0 °C under argon was added EDCI
(406 mg, 2.12 mmol). Stirring was continued overnight, and the
reaction mixture was allowed to warm to rt. The reaction mixture was
poured into aqueous 10% KH2PO4 solution followed by extraction
with EtOAc (3×). The organic phases were washed once with aqueous
10% KH2PO4, aqueous saturated NaHCO3, and aqueous saturated
NaCl solution. The organic phase was dried over Na2SO4, filtered, and
evaporated to afford the title compound (558 mg, 95%) as light brown
solid. LC−MS: tR = 1.56 min, 97% (265 nm); tR = 1.18 min, 98% (225
1
min, 99% (225 nm). H NMR (300 MHz, DMSO-d6): δ = 8.36 (s,
1H), 8.25 (d, J = 8.3 Hz, 1H), 8.24 (s, 1H), 7.95 (d, J = 8.3 Hz, 2H),
7.59 (d, J = 8.5 Hz, 2H), 5.08 (ddd, J = 6.1 Hz, 1H), 2.60−2.53 (m,
2H), 2.42 (dq, J = 6.9 Hz, 1H), 1.96−1.83 (m, 1H), 1.84−1.60 (m,
3H), 1.07 (d, J = 6.9 Hz, 3H), 1.04 (d, J = 6.9 Hz, 3H). 13C NMR (150
MHz, DMSO-d6): δ = 175.7, 149.7, 147.0, 143.6, 142.2, 134.8, 133.9,
132.4, 130.3, 118.7, 110.6, 44.2, 33.9, 28.9, 26.8, 19.7, 19.5, 19.1.
HRMS (C20H21N3O): calcd 319.1685; found 319.1685.
1
nm). H NMR (300 MHz, DMSO-d6): δ = 8.39 (s, 1H), 8.28 (d, J =
Preparation of (rac)-N-(4-(3,4-Difluorophenyl)-5,6,7,8-
tetrahydroisoquinolin-8-yl)propionamide (8). (rac)-N-(4-
Bromo-5,6,7,8-tetrahydroisoquinolin-8-yl)propionamide (17, 60 mg,
212 μmol) and 3,4-difluorophenylboronic acid (40.2 mg, 254 μmol)
were dissolved in ethanol (3.8 mL) to give a light brown solution.
Na2CO3 (24.7 mg, 233 μmol) dissolved in water (0.6 mL) was added
followed by tetrakis(triphenylphosphine)palladium (0) (7.4 mg, 6.4
μmol) after evacuation and replacing with argon (cycle repeated 5×).
The solution was then heated at 85 °C overnight. The reaction was
treated with an aqueous 10% NaCl solution and extracted with EtOAc
(3×). The organic phases were washed again with an aqueous 10%
NaCl solution, dried over Na2SO4, filtered, and evaporated under
reduced pressure to give a brown foam which was purified by flash
chromatography [20 g SiO2, Telos cartridge, CH2Cl2/isopropanol (3%
→ 5%)]. Precipitation from CH2Cl2 with n-pentane gave the final
compound (57 mg, 85%) as a light brown foam. LC−MS: tR = 1.50
8.5 Hz, 1H), 8.24 (s, 1H), 7.95 (d, J = 8.5 Hz, 2H), 7.59 (d, J = 8.3 Hz,
2H), 5.10 (ddd, J = 6.5 Hz, 1H), 2.59−2.54 (m, 2H), 2.15 (qd, J = 7.6,
2.9 Hz, 2H), 1.96−1.82 (m, 1H), 1.81−1.62 (m, 3H), 1.05 (t, J = 7.6
Hz, 3H). 13C NMR (150 MHz, DMSO-d6): δ = 172.5, 149.8, 147.0,
143.6, 142.2, 134.8, 133.9, 132.4, 130.3, 118.7, 110.7, 44.2, 28.9, 28.5,
26.8, 19.0, 10.0. HRMS (C19H19N3O): calcd 305.1528; found
305.1537.
Preparation of (+)-(R)-N-(4-(4-Cyanophenyl)-5,6,7,8-
tetrahydroisoquinolin-8-yl)propionamide ((+)-(R)-6) and
(−)-(S)-N-(4-(4-Cyanophenyl)-5,6,7,8-tetrahydroisoquinolin-8-
yl)propionamide ((−)-(S)-6). The two enantiomers of 6 (548 mg)
were separated on a Chiralpak AD column (30% isopropanol in n-
heptane) to give after precipitation from CH2Cl2 with n-pentane
(+)-(R)-6 (202 mg, 37%) and (−)-(S)-6 (125 mg, 36%) as off-white
solids.
Enantiomer (+)-(R)-6. LC−MS: tR = 1.59 min, 100% (265 nm); tR
= 1.59 min, 99% (220 nm). 1H NMR (600 MHz, DMSO-d6): δ = 8.39
(s, 1H), 8.28 (d, J = 8.5 Hz, 1H), 8.24 (s, 1H), 7.99−7.92 (m, 2H),
7.61−7.57 (m, 2H), 5.15−5.04 (m, 1H), 2.56 (t, J = 6.0 Hz, 2H),
2.22−2.08 (m, 2H), 1.92−1.84 (m, 1H), 1.82−1.63 (m, 3H), 1.05 (t, J
= 7.6 Hz, 3H). 13C NMR (150 MHz, DMSO-d6): δ = 172.9, 150.3,
147.4, 144.0, 142.7, 135.2, 134.3, 132.9, 130.8, 119.2, 111.1, 44.7, 29.4,
29.0, 27.2, 19.5, 10.5. HRMS (C19H19N3O): calcd 305.1528; found
305.1534.
1
min, 98% (265 nm); tR = 1.50 min, 97% (225 nm). H NMR (600
MHz, DMSO-d6): δ = 8.36 (s, 1H), 8.27 (d, J = 8.5 Hz, 1H), 8.23 (s,
1H), 7.56−7.52 (m, 1H), 7.52−7.49 (m, 1H), 7.23−7.20 (m, 1H),
5.11−5.07 (m, 1H), 2.58 (t, J = 6.1 Hz, 2H), 2.18−2.12 (m, 2H),
1.90−1.85 (m, 1H), 1.81−1.75 (m, 1H), 1.74−1.68 (m, 1H), 1.72−
1.66 (m, 1H), 1.05 (t, J = 7.6 Hz, 3H). 13C NMR (150 MHz, DMSO-
d6): δ = 172.5, 149.5, 149.3 (m), 149.2 (m), 147.2, 143.8, 134.6 (m),
134,3, 133.8, 126.4 (dd, JCF = 6.3, 3.2 Hz), 118.5 (d, JCF = 17.4 Hz),
117.6 (d, JCF = 17.0 Hz), 44.2, 28.9, 28.5, 26.7, 19.1, 10.0. HRMS
(C18H18F2N2O): calcd 316.1387; found 316.1387.
Enantiomer (−)-(S)-6. LC−MS: tR = 1.21 min, 99% (265 nm); tR =
1
1.21 min, 99% (225 nm). H NMR (600 MHz, DMSO-d6): δ = 8.39
(s, 1H), 8.28 (d, J = 8.5 Hz, 1H), 8.24 (s, 1H), 7.99−7.92 (m, 2H),
7.61−7.57 (m, 2H), 5.15−5.04 (m, 1H), 2.56 (t, J = 6.0 Hz, 2H),
Preparation of (rac)-N-(4-(2,4,5-Trifluorophenyl)-5,6,7,8-
tetrahydroisoquinolin-8-yl)propionamide (9). (rac)-N-(4-
I
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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Bromo-5,6,7,8-tetrahydroisoquinolin-8-yl)propionamide (17, 105 mg,
370 μmol) and 2,4,5-trifluorophenylboronic acid (78.1 mg, 444 μmol)
were dissolved in ethanol (6.7 mL) to give a light brown solution.
Na2CO3 (43.1 mg, 407 μmol) dissolved in water (1.1 mL) was added
followed by tetrakis(triphenylphosphine)palladium (0) (12.8 mg, 11
μmol) after evacuation and replacing with argon (cycle repeated 5×).
The solution was then heated at 85 °C overnight. The reaction was
treated with an aqueous 10% NaCl solution and extracted with EtOAc
(3×). The organic phases were washed again with an aqueous 10%
NaCl solution, dried over Na2SO4, filtered, and evaporated under
reduced pressure to give a brown foam which was purified by flash
chromatography [20 g SiO2, Telos cartridge, CH2Cl2/isopropanol (3%
→ 5%)]. Precipitation from CH2Cl2 with n-pentane gave the final
compound (49 mg, 40%) as a white foam. LC−MS: tR = 1.73 min,
2.64−2.55 (m, 2H), 2.15 (qd, J = 7.6, 2.6 Hz, 2H), 1.95−1.63 (m,
4H), 1.05 (t, J = 7.6 Hz, 3H). 13C NMR (150 MHz, DMSO-d6): δ =
172.9, 157.5 (d, JCF = 247.3 Hz), 150.0, 147.5, 144.3, 138.8 (d, JCF
7.4 Hz), 134.6, 134.3, 131.1, 127.2 (d, JCF = 3.3 Hz), 119.5 (d, JCF
=
=
17.3 Hz), 118.3 (d, JCF = 3.3 Hz), 44.7, 29.4, 29.0, 27.2, 19.5, 10.5.
HRMS (C18H18ClFN2O): calcd 332.1092; found 332.1101.
Experimental Details for Protein Crystallization and
Structure Determination of CYP11B2 with Compound 11.
CYP11B2 protein was overexpressed in E. coli and purified with nickel
affinity and ion exchange chromatography. The ligand complex was
cocrystallized by vapor diffusion in sitting drops with 10% PEG3350 in
90 mM ammonium citrate (pH 7.0). Thin plates grew after several
days and were flash-frozen after addition of 20% ethylene glycol. X-ray
diffraction images were collected at 100 K at the beamline X10SA
(PXII) of the Swiss Light Source (SLS) using a Pilatus pixel detector.
In Vitro Assay for the Determination of CYP11B2 and
CYP11B1 Inhibition. CYP11B1 and CYP11B2 proteins were
expressed in a human renal leiomyoblastoma cell line (ATCC number
CRL-1440). qPCR revealed that these cells express FDXR and FDX,
two proteins that are crucial for the enzymatic activity of CYP11B
enzymes and importantly do not express detectable levels of CYP11B1
or CYP11B2 activity. The cells are grown in ATCC-formulated
McCoy’s 5a medium modified (catalog no. 30-2007) containing 10%
fetal bovine serum. Stable cells expressing ectopically CYP11B1 or
CYP11B2 from human, cynomolgus monkey, or mouse were
developed by transfecting appropriate expression plasmids. The
plasmids contain the cDNA (GeneCopoeia) for either CYP11B1 or
CYP11B2 from the appropriate species (mouse, cynomolgus monkey,
or man) under the control of a CMV promoter and the neomycin
resistance marker. Using electroporation expression plasmid were
transfected into cells, and these cells were then selected for expressing
the given resistance markers. Individual cell clones are then selected
(400 μg mL−1 G-418; Geneticin) and assessed for displaying the
desired enzymatic activity using 11-deoxycorticosterone (CYP11B2)
or 11-deoxycortisol (CYP11B1) as a substrate. Cellular enzyme assays
were performed in DMEM/F12 medium containing 2.5% charcoal
treated FCS and appropriate concentration of substrate (1 μM 11-
deoxycorticosterone or 11-deoxycortisol). For assaying enzymatic
activity, cells were plated onto 96-well plates and incubated for 16 h.
An aliquot of the supernatant was then transferred and analyzed for
the concentration of the expected product (aldosterone for CYP11B2;
cortisol for CYP11B1). The concentrations of these steroids can be
determined using HTRF (homogeneous time-resolved fluorescence)
assays from CisBio analyzing either aldosterone or cortisol. Inhibition
of the release of produced steroids can be used as a measure of the
respective enzyme inhibition by test compounds added during the
cellular enzyme assay. The dose dependent inhibition of enzymatic
activity by a compound is calculated by means of plotting added
inhibitor concentrations (x-axes) vs measured steroid/product level (y-
axes). The inhibition is then calculated by fitting the following four-
parameter sigmoidal function [Morgan−Mercer−Flodin (MMF)
model] to the raw data points using the least-squares method:
1
98% (265 nm); tR = 1.73 min, 96% (225 nm). H NMR (600 MHz,
DMSO-d6): δ = 8.40 (s, 1H), 8.36−8.26 (m, 1H), 8.24 (s, 1H), 7.75−
7.70 (m, 1H), 7.58 (br s, 1H), 5.09 (q, J = 6.9 Hz, 1H), 2.46 (br d, J =
6.2 Hz, 2H), 2.19−2.13 (m, 2H), 1.91−1.86 (m, 1H), 1.82−1.77 (m,
1H), 1.72−1.67 (m, 2H), 1.06 (t, J = 7.6 Hz, 3H). 13C NMR (150
MHz, DMSO-d6): δ = 172.5, 154.6 (dd, JCF = 243.0, 9.8 Hz), 150.0,
147.7, 147.7 (m), 146.0 (m), 144.7, 133.9, 128.6, 121.3 (m), 119.6
(m), 106.4 (dd, JCF = 29.2, 21.1 Hz), 44.2, 29.0, 28.5, 26.1, 19.0, 10.0.
HRMS (C18H17F3N2O): calcd 334.1293; found 334.1294.
Preparation of (rac)-N-(4-(4-Chloro-2-fluorophenyl)-5,6,7,8-
tetrahydroisoquinolin-8-yl)propionamide (10). (rac)-N-(4-
Bromo-5,6,7,8-tetrahydroisoquinolin-8-yl)propionamide (17, 116 mg,
565 μmol) and 4-chloro-2-fluorophenylboronic acid (122 mg, 678
μmol) were dissolved in ethanol (10.2 mL) to give a light brown
solution. Na2CO3 (65.9 mg, 622 μmol) dissolved in water (1.7 mL)
was added followed by tetrakis(triphenylphosphine)palladium(0)
(19.6 mg, 17 μmol) after evacuation and replacing with argon (cycle
repeated 5×). The solution was then heated at 85 °C overnight. The
reaction was treated with an aqueous 10% NaCl solution and extracted
with EtOAc (3×). The organic phases were washed again with an
aqueous 10% NaCl solution, dried over Na2SO4, filtered, and
evaporated under reduced pressure to give a brown foam which was
purified by flash chromatography [50 g SiO2, Telos cartridge, CH2Cl2/
isopropanol (3% → 6%)]. Precipitation from CH2Cl2 with n-pentane
gave the final compound (132 mg, 70%) as an off-white foam. LC−
1
MS: tR = 2.19 min, 93% (265 nm); tR = 2.11 min, 99% (220 nm). H
NMR (600 MHz, DMSO-d6): δ = 8.39 (s, 1H), 8.35−8.25 (m, 1H),
8.23 (s, 1H), 7.60 (dd, J = 9.8, 1.9 Hz, 1H), 7.43−7.40 (m, 1H), 7.40
(br d, J = 7.9 Hz, 1H), 5.12−5.08 (m, 1H), 2.46−2.41 (m, 2H), 2.19−
2.13 (m, 2H), 1.91−1.85 (m, 1H), 1.82−1.76 (m, 1H), 1.69 (br t, J =
7.0 Hz, 1H), 1.71−1.66 (m, 1H), 1.05 (t, J = 7.6 Hz, 3H). 13C NMR
(150 MHz, DMSO-d6): δ = 172.5, 159.2 (d, JCF = 247.9 Hz), 149.9,
147.5, 144.7, 133.9 (d, JCF = 10.5 Hz), 133.8, 132.9, 129.5, 125.1 (d,
JCF = 3.3 Hz), 123.6 (d, JCF = 16.8 Hz), 116.4 (d, JCF = 26.1 Hz), 44.2,
29.0, 28.5, 26.2, 18.9, 10.0. HRMS (C18H18ClFN2O): calcd 332.1091;
found 332.10949.
Preparation of (rac)-N-(4-(4-Chloro-3-fluorophenyl)-5,6,7,8-
tetrahydroisoquinolin-8-yl)propionamide (11). (rac)-N-(4-
Bromo-5,6,7,8-tetrahydroisoquinolin-8-yl)propionamide (17, 116 mg,
565 μmol) and 4-chloro-3-fluorophenylboronic acid (84.9 mg, 300
μmol) were dissolved in ethanol (5.4 mL) to give a light brown
solution. Na2CO3 (35.0 mg, 330 μmol) dissolved in water (1.0 mL)
was added followed by tetrakis(triphenylphosphine)palladium (0)
(10.4 mg, 9 μmol) after evacuation and replacing with argon (cycle
repeated 5×). The solution was then heated at 85 °C overnight. The
reaction was treated with an aqueous 10% NaCl solution and extracted
with EtOAc (3×). The organic phases were washed again with an
aqueous 10% NaCl solution, dried over Na2SO4, filtered, and
evaporated under reduced pressure to give a brown foam which was
purified by flash chromatography [20 g SiO2, Telos cartridge, CH2Cl2/
isopropanol (3% → 5%)]. Precipitation from CH2Cl2 with n-pentane
gave the final compound (83 mg, 83%) as a light brown foam. LC−
AB + CxD
y =
B + xD
where A is the maximum y value, B is the EC50 value determined using
XLfit, C is the minimum y value, and D is the slope value. The
maximum value A corresponds to the amount of steroid produced in
the absence of an inhibitor, and the value C corresponds to the
amount of steroid detected when the enzyme is fully inhibited.
Experimental Details for in Vivo db/db Mouse Experiments.
Male db/db mice (5 weeks old) were ordered from Taconic
Biosciences (Hudson, NY, USA) and were housed in groups to
acclimate to the Roche facility. The animals were kept on a 12 h light/
dark cycle and were fed with NIH no. 31M rodent diet (Harlan
Laboratories, Indianapolis, IN, USA) with access to tap water ad
libitum. Right before the age of 12 weeks, mice were randomized
according to body weight and blood glucose (fed conditions),
measured with Alphatrak glucometer (Abbott Laboratories, Chicago,
IL, USA). Treatments were prepared as food admix by SSniff (Soest,
1
MS: tR = 2.11 min, 98% (265 nm); tR = 2.11 min, 99% (220 nm). H
NMR (300 MHz, DMSO-d6): δ = 8.37 (s, 1H), 8.27 (d, J = 8.3 Hz,
1H), 8.24 (s, 1H), 7.69 (dd, J = 8.1 Hz, 1H), 7.49 (dd, J = 10.3, 1.8
Hz, 1H), 7.24 (dd, J = 8.3, 1.4 Hz, 1H), 5.09 (ddd, J = 7.3 Hz, 1H),
J
DOI: 10.1021/acs.jmedchem.5b00851
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Germany) in NIH no. 31M rodent diet and were given for 3 weeks. At
the end of the treatment, animals were sacrificed by decapitation after
Accession Codes
Atomic coordinates of compound 11 have been deposited in
the Protein Data Bank with accession number 4ZGX.
isoflurane (Foren
̀
e, Abbott Laboratories, Chicago, IL, USA) anesthesia,
and blood was sampled on EDTA-coated tubes (Microvette 500 K3E,
Sarstedt, Numbrecht, Germany) for steroid level determinations.
̈
AUTHOR INFORMATION
Corresponding Authors
*J.D.A.: phone, 0041 61 6887738; fax, 0041 61 6886459; e-
mail:, johannes.aebi@roche.com.
■
Blood was centrifuged at 10 000 rpm for 15 min at 4 °C, and plasma
was sampled and steroid levels were measured by LC−MS−MS
methodology (see Supporting Information).
Experimental Details for in Vivo Cynomolgus Monkey
Experiments. Non-naive grouped male cynomolgus monkeys
(Macaca fascicularis) were kept in cages on a 12 h light−dark cycle
with natural light in 50−60% humidity and 22.5−22.8 °C. Animals
received the diet 3448 EM S12 (80 g 7 kg−1 day−1 animal−1; Provimi-
Kliba, Kaiseraugst, Switzerland) 2−3 times daily, on top of daily fruits,
vegetables, and nuts. Water access was ad libitum. Animals identified as
good Synacthen (Novartis, Basel, Switzerland) responders participated
in the study under the following conditions: at least a 2-fold increase of
plasma aldosterone, cortisol, and corticosterone production should be
measured 1 h after intramuscular acute application of Synacthen. On
the day of the experiment, the monkeys were moved from their group
cages and individually housed in stainless-steel mesh cages (H 1.5 m ×
L 1.2 m × W 0.8 m) according to the Swiss regulation on animal
welfare. For oral gavage (po) all samples were analyzed using routine
protein precipitation technique followed by LC−MS−MS analysis. PK
parameters were calculated using noncompartmental analysis (Table
S1). Oral treatment and Synacthen were given as follows: T = 0, oral
dosing of 2 monkeys per group (vehicle or compound (+)-(R)-6) with
compound administered as suspension in hydroxyethylcellulose,
polysorbate 80, methyparaben, and propylparaben vehicle using 2
mL kg−1 administration volume; T = +5 min, intramuscular application
of 14.5 μg kg−1 Synacthen. Blood samples were collected 15 min, 30
min, 1 h, 2 h, 4 h, and 6 h after compound or vehicle application. At
each time point, 1.2 mL blood was collected in EDTA-coated tubes
(BD Vacutainer K2 EDTA, Becton Dickinson, Franklin Lakes, NJ,
USA) for plasma mineral corticoids and compound concentration
measurements. Blood was centrifuged at 10 000 rpm for 15 min at 4
°C. Subsequently, plasma was separated and stored at −20 °C for
further analysis. Plasma steroids, % inhibition of CYP11B1 and
CYP11B2, and exposure data for (+)-(R)-6 were analyzed from all
time points by LC−MS−MS methodology (see Supporting
Information).
*K.A.: phone, 0041 61 6889587; e-mail, kurt.amrein@roche.
com.
*A.V.M.: phone, 0041 61 6871384; e-mail, alexander_v.
mayweg@roche.com.
Author Contributions
All authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We acknowledge Liliane Forzy for excellent technical
■
assistance; Markus Burkler, Christian Bartelmus, and Dr.
̈
Inken Plitzko for spectroscopical analysis of compounds;
Daniel Zimmerli for chiral separation of compound ( )-6;
Dr. Jean-Michel Adam, Christophe Pfleger, and Tanja Gasser
for upscaling the synthesis of enantiomerically pure (+)-(R)-6;
Isabelle Parrilla, Bjorn Wagner, and Severin Wendelspiess for
̈
physicochemical characterization of compounds; Sandrine
Simon for microsomal stability data, Dr. Stephen Fowler for
CYP inhibition measurements; Thomas Thelly for formulation
work, Dr. Doris Roth, Dr. Remo Hochstrasser, Henry Cabrier
and Angelika Schuler for developing and performing the
CYP11B1 and CYP11B2 in vitro assays; Dr. Agnes Benardeau,
̀
e,
̀
́
Emmanuelle Hainaut, Anthony Vandjour, Anthony Retournard,
and Peter Schrag for in vivo work; Monique Wittig for running
the LC−MS−MS steroid analytics; Dr. Daniel Schlatter for
protein production; Dr. Jorg Benz and Catherine Joseph for
̈
protein crystallization; Dr. Manfred Schneider for helpful
comments; and Dr. Fionn O’Hara for careful reading of the
manuscript. This work was supported exclusively by F.
Hoffmann-La Roche Ltd.
Animals in Research. Animal work was performed according to
the Swiss federal law for animal protection and was approved by the
Veterinary Office Basel. Animals were provided an acclimation period
of >5 days before use, conventional hygienic conditions for housing
(temperature of 20−24 °C, minimum relative humidity of 40%, light/
dark cycle of 12 h), and standard diet; access to food and drinking
water was ad libitum. Specific details on permissions can be provided
upon request.
ABBREVIATIONS USED
■
AcOH, acetic acid; n-BuLi, n-butyllithium; CYP, cytochrome
P450 enzyme; DMAP, 4-dimethylaminopyridine; EDCI, N-(3-
dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride;
EtOAc, ethyl acetate; EtOH, ethanol; MeOH, methanol; PA,
primary aldosteronism; rt, room temperature; SF, selectivity
factor
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b00851.
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Operating Environment (MOE), version 2011; Chemical Computing
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3MZS were kept during model building, and the best scoring
minimized homology model was selected. This was further refined by
subsequent relaxation of the binding site using Moloc. See the
following: Gerber, P. R.; Muller, K. MAB, a generally applicable
̈
molecular force field for structure modelling in medicinal chemistry. J.
Comput.-Aided Mol. Des. 1995, 9, 251−268.
(19) The log D values were measured spectrophotometrically at pH
7.4 in an 1-octanol/50 mM TAPSO buffer system containing 5% (v/v)
DMSO.
L
DOI: 10.1021/acs.jmedchem.5b00851
J. Med. Chem. XXXX, XXX, XXX−XXX