Discovery of 3-Pyridyl Isoindolin-1-one Derivatives as Potent, Selective, and Orally Active Aldosterone Synthase (CYP11B2) Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.0c00233

Aldosterone synthase (CYP11B2) inhibitors have been explored in recent years as an alternative therapeutic option to mineralocorticoid receptor (MR) antagonists to reduce elevated aldosterone levels, which are associated with deleterious effects on various organ systems including the heart, vasculature, kidney, and central nervous system (CNS). A benzamide pyridine hit derived from a focused screen was successfully developed into a series of potent and selective 3-pyridyl isoindolin-1-ones CYP11B2 inhibitors. Our systematic structure-activity relationship study enabled us to identify unique structural features that result in high selectivity against the closely homologous cortisol synthase (CYP11B1). We evaluated advanced lead molecules, exemplified by compound 52, in an in vivo cynomolgus monkey acute adrenocorticotropic hormone (ACTH) challenge model and demonstrated a superior 100-fold in vivo selectivity against CYP11B1.

Full text of DOI:10.1021/acs.jmedchem.0c00233

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Article
Discovery of 3-Pyridyl Isoindolin-1-one Derivatives as Potent, Selective
and Orally Active Aldosterone Synthase (CYP11B2) Inhibitors
Yongfu Liu, Jun Wu, Mingwei Zhou, Wenming Chen, Dongbo Li, Zhan-Guo Wang, Benoit Hornsperger,
Johannes D. Aebi, Hans-Peter Märki, Bernd Kuhn, Lisha Wang, Andreas Kuglstatter, Jörg Benz,
Stephan Müller, Remo Hochstrasser, Giorgio Ottaviani, Jian Xin, Stephan Kirchner, Susanne Mohr,
Philippe Verry, William Riboulet, Hong C Shen, Alexander V. Mayweg, Kurt Amrein, and Xuefei Tan
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00233 • Publication Date (Web): 12 Jun 2020
Downloaded from pubs.acs.org on June 13, 2020
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Riboulet, William; Roche Pharma Research & Early Development, Roche
Innovation Center Basel
Shen, Hong; Roche R&D Center China, Medicinal chemistry
Mayweg, Alexander; Roche Pharma Research & Early Development,
Roche Innovation Center Basel; Roche Pharma Research & Early
Development, Roche Innovation Center Basel
Amrein, Kurt; Roche Pharma Research & Early Development, Roche
Innovation Center Basel
Tan, Xuefei; Roche Pharma Research & Early Development, Roche
Innovation Center Shanghai, Medicinal Chemistry
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Discovery of 3-Pyridyl Isoindolin-1-one
Derivatives as Potent, Selective and Orally Active
Aldosterone Synthase (CYP11B2) Inhibitors
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†
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Yongfu Liu, Jun Wu, Mingwei Zhou, Wenming Chen, Dongbo Li, Zhanguo Wang, Benoit
‡
‡
‡
§
#
Hornsperger, Johannes D. Aebi, Hans-Peter Märki, Bernd Kuhn, Lisha Wang, Andreas
¶
¶
¶
¶
ǁ
ǂ
Kuglstatter, Jörg Benz, Stephan Müller, Remo Hochstrasser, Giorgio Ottaviani, Jian Xin, Stephan
ǁ
ǁ
Δ
Δ
†
Kirchner, Susanne Mohr, Philippe Verry, William Riboulet, Hong C. Shen, Alexander V.
Mayweg,†,‡ Kurt Amrein, and Xuefei Tan*
Δ
,†
†
#
ǂ
Department of Medicinal Chemistry, CADD, Pharmaceutical Sciences, Roche Pharma Research &
Early Development, Roche Innovation Center Shanghai, Roche R&D Center (China) Ltd, 720 Cai
Lun Road, Building 5, Pudong, Shanghai 201203 PR China
‡
§
¶
ǁ
Δ
Department of Medicinal Chemistry, CADD, Lead Discovery, Pharmaceutical Sciences, Discovery
Cardiovascular and Metabolic Diseases, Roche Pharma Research & Early Development, Roche
Innovation Center Basel, F. Hoffmann-La Roche Ltd, Grenzacherstrasse 124, CH-4070 Basel,
Switzerland
Abstract: Aldosterone synthase (CYP11B2) inhibitors were explored in recent years as an alternative
therapeutic option to MR antagonists to reduce elevated aldosterone levels, which are associated with
deleterious effects on various organ systems including heart, vasculature, kidney, and CNS. A
benzamide pyridine hit derived from a focused screen was successfully developed into a series of potent
and selective 3-pyridyl isoindolin-1-ones CYP11B2 inhibitors. Our systematic structure-activity
relationship study enabled us to identify unique structural features that result in high selectivity against
the closely homologous cortisol synthase (CYP11B1). We evaluated advanced lead molecules,
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exemplified by compound 52, in an in vivo cynomolgus monkey acute ACTH challenge model and
demonstrated a superior 100-fold in vivo selectivity against CYP11B1.
Keywords: Aldosterone synthase inhibitor, CYP11B2, CYP11B1, selectivity, 3-pyridyl isoindolin-1-
one
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INTRODUCTION
The renin angiotensin aldosterone system (RAAS) is an endocrine system that regulates body’s
blood pressure and fluid balance. Its dysregulation contributes to the pathogenesis of a number of
cardiovascular, renal, and metabolic diseases. Therefore, it is a pathway that has been successfully
targeted by many RAAS inhibitors, including angiotensin AT1 receptor blockers (ARBs), angiotensin-
converting-enzyme inhibitors (ACEIs), renin inhibitors, and mineralocorticoid receptor antagonists
1
(
MRAs). Aldosterone, as one component of RAAS, besides its well-known function in regulating fluid
and electrolyte homeostasis, has recently been recognized as one important contributor to end-organ
damage in various cardiovascular and renal diseases.2-5 An elevated aldosterone level, especially in
conjunction with high sodium/oxidative stress is associated with deleterious effects on various organ
6
systems including heart, vasculature, kidney, and central nervous system (CNS). In addition, in
hypertensive patients that are treated with either ACEIs or ARBs for a longer duration, an “aldosterone
breakthrough” phenomenon leading to rebound aldosterone levels after an initial decline is associated
with end-organ damage.7
Antagonizing the aldosterone effect by an MR antagonist has been shown to reduce the risk of
death in congestive heart failure (CHF) patients when used in conjunction with ACEI (RALES trial),
and to improve survival and reduce hospitalisation in patients with post-myocardial infarction heart
failure when added to the standard treatment regimen (EPHESUS trial).8,9 However, a suppression of
the plasma aldosterone level by an MR antagonist, after a prolonged usage, would induce an increased
aldosterone level by a compensatory mechanism and therefore limit its efficacy. Therefore, blocking
the biosynthesis of aldosterone by an aldosterone synthase inhibitor (ASI) has been explored as an
alternative approach in recent years.10-12 Additionally, an ASI is expected to block MR-independent
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genomic and non-genomic effectsand it may provide even better protection against the aforementioned
diseases.13
Aldosterone is a steroid hormone synthesized from cholesterol via a cascade of enzymatic
reactions by a number of cytochrome P450s. Located mainly in adrenal gland zona glomerulosa,
aldosterone synthase (encoded by the CYP11B2 gene) is the enzyme responsible for the last three steps
of reactions by converting 11-deoxycorticosterone to aldosterone via corticosterone and 18-hydroxy-
corticosterone intermediates (Figure 1).10 Since in human, the aldosterone synthase shows a high
sequence identity (~93%) to cortisol synthase (encoded by the CYP11B1 gene, mainly present in the
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zona fasciculate/reticularis), the development of a highly selective aldosterone synthase inhibitor has
been challenging over the past several years.10-12
The proof-of-concept efficacy study was first demonstrated by Novartis ASI LCI699 in patients
with primary hyperaldosteronism. The oral administration of LCI699 first at 0.5 mg b.i.d. for 2 weeks,
followed by 1.0 mg b.i.d. for another 2 weeks, significantly reduced levels of aldosterone in both plasma
and urine, leading to a rapid correction of hypokalemia, and a modest decrease in blood pressure.
However, due to the low selectivity of LCI699 (CYP11B1 IC /CYP11B2 IC = 3.6-fold), a blunted
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cortisol response to adrenocorticotropic hormone (ACTH) was observed at dose 1 mg/d or greater in
the safety evaluation.15,16 Good selectivity against CYP11B1 is critical in order to prevent undesired
side-effects related to the hypothalamic–pituitary–adrenal (HPA) axis. We recently reported our own
clinical study of a highly selective ASI RO6836191 (CYP11B1 IC /CYP11B2 IC = 100-fold). In a
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phase I SAD study (0-360 mg/d), RO6836191 achieved high CYP11B1/CYP11B2 selectivity with
maximum suppression of aldosterone production at 10 mg/d, and no change of cortisol level up to 360
mg/d. Such good selectivity was also reflected by increase of precursors 11-deoxycorticosterone and
1-deoxycortisol only at ≥ 90 mg.17
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Figure 1. Terminal steroidogenesis pathways leading to aldosterone and cortisol.
Herein, we report a new type of ASIs with even superior CYP11B1/CYP11B2 selectivity both
in vitro and in vivo.
RESULT AND DISCUSSION
SAR and Lead Optimization Strategy
To identify novel ASIs we performed a focused screen of a subset of the Roche library. First
selection criterion for this library was the presence of a nitrogen lone pair in a heteroaromatic ring
without substitution alpha to the nitrogen atom to enable potential heme-iron binding in CYP11B2.
This initial list was further reduced to a final set of 2’000 compounds by 2D fingerprint and 3D shape
similarity to several selected reference inhibitors (SI, Figure S1),18-21 molecular property filtering, and
structure clustering. Screening of this library against human CYP11B2 yielded a significant number of
hits, several of them with very high ligand efficiencies. However, only few compounds of the hit list
showed initial selectivity against human CYP11B1. This is not surprising as the active site residues are
22
fully conserved between the two isoforms. Interestingly, despite the presence of unshielded nitrogen
lone pairs in our hit structures, which are privileged motifs for iron-heme binding, we did not find strong
cross-reactivities against the drug-metabolizing CYP isoforms 3A4, 2C9, 2D6, 2C19, and 1A2.
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Among the promising hits, a class of N-(3-pyridyl)benzamides (1-6)23 was selected as our
chemistry starting point. These initial hits showed sub-micromolar CYP11B2 IC values and some
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selectivity against human CYP11B1 in in-house developed cellular assays (Table 1). Initial structure-
activity relationship (SAR) indicated that a chlorine (1) or nitrile (2) substitution at the para-position of
the benzamide was favored for better potency. In addition, in the ortho-position small substituents like
fluorine (5) or methyl (6) slightly improved the potency. Methylation at the benzamide nitrogen led to
almost inactive compounds (7-9), presumably because of the drastic conformational change compared
to the unsubstituted analogs.
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Table 1. Preliminary SAR for initial N-(3-pyridyl)benzamide hits, 1-9.
3
R
O
2
R
N
1
R
N
a
Structure
IC (M)
5
0
SFb
Cmpd
R1
H
R2
H
R3
Cl
CYP11B2
0.42
CYP11B1
1
2
3
4
5
6
7
8
9
> 3
> 3
> 3
> 3
> 3
> 3
> 3
> 3
> 3
> 7
> 7
na
H
H
CN
Me
H
0.454
> 3
H
H
H
Cl
F
0.967
0.297
0.277
> 3
> 3
> 10
> 11
na
H
Cl
H
Me
F
Cl
Me
Me
Me
Cl
Me
H
Cl
> 3
na
CN
> 3
na
aIC50: the concentration of compound that inhibited 50% enzyme activity; SF: the selective factor defined as the
b
ratio between the IC50 values of human CYP11B1 and CYP11B2.
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Subsequent conformational restriction of 4-chloro-N-(3-pyridyl)benzamide 1 via cyclization
gave either 6-membered lactam, 3,4-dihydroisoquinolin-1-one derivative 10 (IC50 = 61 nM) or 5-
membered lactam, isoindolin-1-one derivative 13 (IC50 = 179 nM), which showed 7- and 2-fold
CYP11B2 potency improvements, respectively. Additional substitution on the phenyl ring, such as 11,
did not gain further potency. The CN substituted isoindolin-1-one analog 14 (IC = 232 nM), despite a
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two-fold potency improvement (vs. 2), was still inferior to a chlorine substitution. Apart from their
better potencies, the cyclized lactams also showed encouraging double-digit selectivity factors against
CYP11B1 (Table 2).
Table 2. Potency, selectivity, and microsomal stability of 3,4-dihydroisoquinolin-1-one and isoindolin-
1-one derivatives, 10-14.
2
R
O
1
R
N
n
(
)
N
a
SFb
LMc
mL/min/kg)
Cmpd
Structure
IC (M)
5
0
(
n
R1
Cl
Cl
H
R2
H
F
CYP11B2
0.061
CYP11B1
5.002
human
< 3.5
< 3.5
nd
mouse
< 20.2
30.9
nd
1
1
1
0
1
2
2
2
1
1
1
82
34
> 3
60
67
0.104
3.541
H
H
H
1.12
> 3
13
Cl
CN
0.179
10.74
12.8
8.7
76.5
45.6
1
4
0.232
15.544
aIC50: the concentration of compound that inhibited 50% enzyme activity; SF: the selective factor defined as the
b
c
ratio between the IC50 values of human CYP11B1 and CYP11B2; LM: scaled intrinsic clearance of compound in
liver microsomes.
Compared to the 3,4-dihydroisoquinolin-1-one analog 10 (logD = 2.25), the slightly more
lipophilic isoindolin-1-one analog 13 (logD = 2.52) was much less stable in liver microsomes, likely
due to oxidative metabolism at the benzylic C-H position. Although metabolic stability could be
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improved by lowering the lipophilicity, as demonstrated by analog 14 (logD = 1.35), we also envisioned
that increasing the steric hindrance at the benzylic carbon could reduce metabolic oxidation. As shown
in Table 3, a number of such analogs were prepared as either a racemic mixture or an achiral compound.
Indeed, benzylic substitutions (Me, Et, and cyclopropyl) did improve the metabolic stability (15, 16,
and 19), and gem-dimethyl (17) as well as spirocyclopropyl (18) analogs showed the most significant
improvement in liver microsomal stability. Except for the benzyl substituted 20, all analogs (15-19)
substituted at the benzylic position retained comparable CYP11B2 potency and selectivity against
CYP11B1. Encouraging, but difficult to rationalize, a surprising 202-fold of CYP11B1 selectivity was
observed for analog 19 (IC = 161 nM). On the other hand, analogs derived from derivatization at the
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3
- or 4- position of 3,4-dihydroisoquinolin-1-one core such as 21-22 (Me) and 23 (gem-dimethyl),
either decreased potency, selectivity or had reduced microsomal stability (vs. 10). These new types of
compounds also showed excellent selectivity against a common cytochrome P450 screening panel
(
3A4, 2D6, and 2C9) with IC values generally greater than 50 M.
5
0
Table 3. Potency, selectivity, and microsomal stability of 3,4-dihydroisoquinolin-1-one and
isoindolin-1-one derivatives, 15-23.
Cl
O
O
Cl
N
N
4
R
3
3
R
R
N
N
1
5-20
21-23
a
SFb
LMc
(mL/min/kg)
Cmpd
Structure
IC (M)
5
0
3
4
R /R
CYP11B2
CYP11B1
human
mouse
1
1
1
1
1
2
5
6
7
8
9
0
Me
Et
0.123
0.121
0.108
0.120
0.161
1.248
3.341
4.276
4.450
32.522
10
28
12.2
6.6
66.5
65.9
25
gem-dimethyl
40
< 3.5
3.5
spirocyclopropyl
cyclopropyl
37
nd
202
9.4
nd
Bn
1.07
0.214
>10
0.2
nd
nd
nd
nd
2
1
Me / -
0.761
>13
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- / Me
0.164
1.538
2.588
> 10
16
6.6
nd
45
nd
23
- / gem-dimethyl
> 7
aIC50: the concentration of compound that inhibited 50% enzyme activity; SF: the selective factor defined as the
b
c
ratio between the IC50 values of human CYP11B1 and CYP11B2; LM: scaled intrinsic clearance of compound in
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5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
liver microsomes.
Having optimized the pyridine left-hand side moiety, in particular the isoindolin-1-one core for
potency, selectivity, and liver microsomal stability, the lead optimization effort then focused onto the
central pyridine. An overlay between the modeled binding mode of compound 13 in the X-ray complex
structure of deoxycorticosterone with CYP11B2 indicated room for extension at the steroid D ring and
its polar substituent (Figure 2). We first tried to mimic this motif with a bicyclic tetrahydroisoquinoline
core. Thus, a number of 5,6,7,8-tetrahydroisoquinolin-8-amine derived analogs were prepared with a
small set of amide or sulfonamide modifications to both mimic the deoxycorticosterone hydroxymethyl
ketone and as a handle for physico-chemical property modulation (Table 4). Similar to previously
2
4
reported examples, the chirality of C-8 was found to be critical for CYP11B2 potency, with the (R)-
configuration enantiomer being the preferred one (25, IC = 44 nM vs. 24, IC = 5.14 M). In general,
5
0
50
amide substitutions were more potent than sulfonamide (25 vs. 27, and 26 vs. 28), and bigger
substitutions were better tolerated than small ones (e.g., propyl amide 26 vs. acetyl amide 25, and ethyl
sulfonamide 28 vs. methyl sulfonamide 27). Interestingly, for this new series of compounds, even
without benzylic substitution at the isoindolin-1-one core, all analogs were quite stable in liver
microsomes. The most potent propyl amide analog 26 showed a CYP11B2 IC of 28 nM and a 186-
50
fold selectivity vs. CYP11B1, together with a very good physico-chemical profile (lipophilicity, logD
-6
= 2.14, permeability, Pampa Pe = 3.63 x 10 cm/s, and solubility, LYSA > 460 g/mL). Subsequent
hybrids with isoindolin-1-one derivatives having small alkyl substitutions at the benzylic position did
result in further potency improvements and single digit nanomolar IC values. Unfortunately, several
5
0
of the most potent analogs (30, IC = 4 nM, 31, IC = 7 nM, and 34, IC = 6 nM) did not pass the
5
0
50
50
1
00-fold CYP11B1 selectivity threshold that we had set to achieve. The combination with 3,4-
dihydroisoquinolin-1-one core (39-41) also did not yield better candidates for further development.
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The relative configuration of compounds 29-38 were tentatively assigned based on optical
rotation and logD values correlation between each pair of diasteromers, in which (3R,8R) vs. (3S,8R)
has consistent trend (SI, Table S6). Corresponding gem-dimethyl and spirocyclopropyl hybrid analogs
could not be synthesized via the Ullman coupling reaction with the 5,6,7,8-tetrahydroisoquinolin-8-
amine building block due to significant steric hindrance at the alpha position of the lactam nitrogen.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H
H
O
H
O
OH
deoxycorticosterone
Figure 2. Overlay of modeled binding mode of compound 13 (magenta) with the X-ray co-crystal
structure of human CYP11B2 with deoxycorticosterone (PDB code: 4DVQ, protein in green, heme and
steroid ligand in cyan). In the model, the pyridine nitrogen atom engages in a short contact with the
heme iron atom (dashed line).
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8
9
Table 4. Potency, selectivity, and microsomal stability of 5,6,7,8-tetrahydroisoquinolin-8-amine
derived analogs, 24-41.
O
O
Cl
Cl
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
3
N
n
R
N
R
(
)
N
H
N
H
8
3
(8R)
2
R
R
N
N
n = 1, 24-28
n = 2, 39-41
29-38
a
SFb
LMc
mL/min/kg)
human mouse
Cmpd
Structure
IC (M)
5
0
(
C8
R1
R2
R3
CYP11B2 CYP11B1
O
24
(S)
(R)
---
---
---
---
5.141
0.044
39.623
2.332
8
nd
nd
O
2
5
53
< 3.5
< 20.2
< 20.2
< 20.2
27
O
2
2
2
6
7
8
(R)
(R)
(R)
(R)
---
---
---
---
---
H
0.028
0.998
0.040
0.130
5.208
9.352
1.788
5.137
186
9
< 3.5
< 3.5
< 3.5
< 3.5
O
O
S
O
O
S
---
45
40
O
O
O
O
29
Me
43.5
3
3
3
0
1
2
(R)
(R)
(R)
(R)
(R)
(R)
(R)
(R)
(R)
(R)
H
Et
H
Me
H
0.004
0.007
0.097
0.066
0.006
0.090
0.012
0.1
0.081
0.334
3.634
9.414
0.414
10.246
0.345
22.8
20
48
< 3.5
< 3.5
< 3.5
< 3.5
< 3.5
< 3.5
< 3.5
nd
< 20.2
< 20.2
< 20.2
45.6
Et
H
37
O
O
O
O
33
Me
H
143
69
3
3
3
4
5
6
Me
H
< 20.2
Et
H
114
29
44.9
< 20.2
nd
Et
H
O
O
O
cyclo-
propyl
37
228
>85
73
cyclo-
propyl
3
3
8
9
H
0.354
0.054
>30
nd
nd
< 20.2d
---
---
3.921
4.3
1
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
O
40
(R)
(R)
---
---
---
---
0.119
0.076
3.326
78
68
< 3.5
< 3.5
< 20.2d
< 20.2d
O
4
1
S
5.148
aIC50: the concentration of compound that inhibited 50% enzyme activity; SF: the selective factor defined as the
b
c
ratio between the IC50 values of human CYP11B1 and CYP11B2; LM: scaled intrinsic clearance of compound in
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
d
liver microsomes using well-stirred liver model: LM measured in rat.
The structural model in Figure 2 suggests that extension in the meta-position at the right-hand
side of the central pyridyl ring could be a good direction to further grow into the CYP11B2 binding
site. Consequently, a number of 5-pyridyl substituted analogs with different size and polarity were
synthesized while keeping the 5-chloro-3,3-dimethyl-2-(3-pyridyl)isoindolin-1-one constant at the left-
hand side (Table 5). Based on the assumption that electron-donating substitution might help to improve
potency via enhancing pyridine nitrogen binding affinity to the heme-iron, we decided to limit
substitutions to carbon, oxygen, and nitrogen linked attachments. Various types of linkers with variable
length were explored and a terminal amine group was specifically chosen for further derivatization with
either amides or sulfonamides. As shown in Table 5, the majority of analogs showed improved
CYP11B2 potencies compared to the unsubstituted analog 17 (IC = 108 nM). For a few methylene
5
0
amine-linked compounds (42-47), either the installation of an (R)-methyl at the methylene carbon (45,
IC50 = 8 nM) or a 3-methylpyridyl amide substitution (47, IC50 = 9 nM) resulted in single digit
nanomolar potencies. While the binding affinity towards CYP11B2 could be improved >10-fold in this
way, it is worth mentioning that the ligand efficiency (LE) is significantly reduced (17: LE = 0.50, 45:
LE = 0.43, 47: LE = 0.37). This indicates that protein interactions made at the right-hand side of the
scaffold contribute less binding energy per atom compared to the rest of the molecule.
To further guide our inhibitor design efforts, we determined the X-ray structure of human
CYP11B2 in complex with compound 45 (Figure 3). Despite the limited resolution of 3.0 Å, the
structure clearly confirmed our proposed binding mode with the central pyridine nitrogen in close
contact with the heme iron atom (d = 2.2 Å) and the isoindolin-1-one core positioned at the left-hand
side of the binding pocket. Pyridine and isoindolin-1-one ring systems are rotated against each other by
~
60° and the gem-dimethyl group is engaged in a number of van der Waals interactions with Phe 130
1
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and Trp 116, respectively. Several additional protein interactions are made around the isoindolin-1-one
moiety. In contrast, only few short protein contacts are visible around the ethylmethanesulfonamide
substituent, which fills the right-hand side of the binding pocket only partly. This is in line with the
generally reduced ligand efficiency when substituting at the 5-pyridyl position.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Several factors likely contribute to the enhanced potency of the substitutions displayed in Table
5
. First, electron-donating substituents might increase the interaction strength of the central pyridine
nitrogen atom with the heme iron. Second, additional favorable protein interactions can be formed in
some cases, as in the matched pair of compound 44 (IC = 33 nM) with the methylated analogue 45
5
0
(IC = 8 nM). In this example, the extra methyl group in 45 engages in a short dispersion interaction
5
0
with the side chain of Ile 488 (Figure 3), which likely contributes to its 4-fold potency improvement.
On the other hand, suboptimal interactions for some substitutions, as exemplified by the amide and
sulfonamide pairs 42 vs. 43 and 44 vs. 46, respectively, can lead to reduced CYP11B2 binding. In
Figure 3, the terminal methyl group of compound 46 would point to a rather polar region of the binding
site, and the resulting desolvation penalty might explain the loss of binding affinity.
Unfortunately, none of the methylene amine-linked analogs showed a selectivity against
CYP11B1 greater than 100-fold. Therefore, linkers with different shape and length including piperidine
(48-49), piperazine (50-51), 2,6-diazaspiro[3.3]heptane (59-62), oxygen-linked azetidine (52, and 56-
5
8), pyrrolidine (53-54), and piperidine (55) were prepared. Notably, now we found analogs for each
of these types of linkers (48-62) which had superior CYP11B2 activities (< 10 nM), revealing that quite
diverse substituents are well tolerated in this region of the binding site. It suggests that this part of the
binding site is quite flexible, to be able to accommodate such a variety of substitutions. One would
expect an entropic penalty for binding to such a region, which is supported by the generally reduced
ligand efficiency for 5-pyridyl substituents. For some substitutions at the terminal nitrogen, we finally
achieved very good CYP11B1 selectivities (> 100-fold). In particular, analogs containing the unique
heteroaryl amide substitution patterns in 57, 58, and 62 gave superior CYP11B1 selectivity even over
5
00-fold while maintaining excellent CYP11B2 potency.
1
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2
3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 5. Potency and selectivity of 5-chloro-3,3-dimethyl-2(3-pyridyl)isoindolin-1-one derivatives, 42-
6
2.
Cl
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
N
R
O
N
a
a
Structure
IC (M)
hSFb
IC (M)
cySFc
5
0
50
Cmpd
R1
hCYP11B2
cyCYP11B2
O
4
4
4
2
3
4
0.059
47
28
0.014
333
na
N
H
O
0.282
0.033
0.008
0.092
0.009
0.005
0.003
0.009
0.002
0.004
0.011
na
N
H
O
O
S
49
0.01
179
93
N
H
O
O
45
S
71
0.004
na
N
H
O
O
O
S
4
4
4
6
7
8
31
na
N
H
N
89
0.009
0.002
0.0004
0.003
0.0002
0.002
0.007
191
466
430
236
170
664
643
N
O
N
77
O
S
49
N
106
80
O
O
N
5
5
5
0
1
2
N
O
O
S
N
146
162
113
N
O
O
N
S O
O
O
53
N
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5
6
7
8
9
O
O
5
5
4
5
N
0.007
0.004
0.005
0.004
0.0005
0.023
0.016
0.014
0.002
81
0.005
0.002
0.0005
0.0009
na
193
225
1120
1772
na
O
N
O
61
Cl
O
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
N
56
174
N
O
O
N
O
O
5
5
5
6
7
8
9
0
553
N
N
N
4164
640
O
N
O
na
na
N
O
O
S
N
855
0.001
na
19699
na
N
O
N
61
1870
5683
N
N
O
N
N
6
2
na
na
N
N
a
b
IC : the concentration of compound that inhibited 50% enzyme activity; hSF: the selective factor
5
0
c
defined as the ratio between the IC values of human CYP11B1 and CYP11B2; cySF: the selective
5
0
factor defined as the ratio between the IC values of cynomolgus monkey CYP11B1 and CYP11B2.
5
0
1
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Figure 3. X-ray structure of human CYP11B2 in complex with compound 45 (PDB code: 6XZ8). The
protein is displayed in green, and the heme cofactor and compound 45 are shown as cyan sticks. Protein
residues with short contacts to the ligand (d < 4.0 Å) are highlighted as green sticks.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
We were able to determine the X-ray co-crystal structure of the highly selective compound 58,
revealing particular protein interactions of the terminal heterocycle (Figure 4). Apart from a hydrogen
bond of the pyrazole nitrogen atom with the sidechain of Tyr 485, we observe significant protein
induced fit around Phe 381 and Phe 406. Interestingly, there is one amino acid difference between
CYP11B2 and B1 in a 5 Å environment around the 5-pyridyl substituent, which is at position 404 (B2:
Gln vs. B1: Arg). We hypothesize that the increased selectivity for 58 and related compounds might be
a consequence of a) different free energy costs for CYP11B2 and B1 to accommodate the terminal
heterocycle in the induced fit pocket, and b) potentially different positions of the side chain of Tyr 485
leading to different hydrogen bond interactions. Co-crystal structures of these compounds with
CYP11B1 would be highly valuable to shed further light on the reasons for selectivity.
1
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Tyr 485
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Phe 381
Phe 406
Gln 404
Figure 4. Overlay of X-ray structures of human CYP11B2 with compounds 45 (PDB code: 6XZ8;
protein: green, heme and ligand: cyan) and 58 (PDB code: 6XZ9; protein: orange, heme and ligand:
magenta) illustrating protein induced fit around the pyrazole tail. The heme iron…pyridine nitrogen
contact and the hydrogen bond between the ligand pyrazole and Tyr 485 are shown as dashed lines. At
position 404, the Gln residue CYP11B2 is mutated to Arg in CYP11B1.
One of the challenges to identify selective ASIs and to validate them in in vivo studies is the
fact that the primary structures between human and rodent CYP11B2 and CYP11B1 enzymes is
relatively poorly conserved (68% for human and rodent CYP11B2, and 63% for human and rodent
CYP11B1). Moreover, rodents produce corticosterone as their major glucocorticoid rather than cortisol
1
0
observed in man and monkeys. As such, pharmacodynamic characterization of a selective ASI in
rodent animal model can not be easily translated into a human situation. Therefore, in recent years, due
to higher species homology between human and monkey (93% for both CYP11B2 and CYP11B1),
functional animal models in non-human primates were developed to support the discovery and
1
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
development of selective ASIs.24-28 To validate candidate molecule in cynomolgus monkey, good in
vitro potency and selectivity towards the cynomolgus monkey derived enzymes was assessed. Table 5
summarizes the potency and selectivity results of compounds 42-62 using cell line G401 expressing the
corresponding monkey enzymes. In general, this series of analogs showed more potent cyCYP11B2
activities (1~16-fold of increase) and a higher cyCYP11B1 selectivity. For analogs that already
achieved > 100-fold of CYP11B1/CYP11B2 selectivity in human enzymes, their selectivity values for
the monkey enzymes were even higher (49, 51, 52, 56, and 57).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In Vivo Rat and Cynomolgus Monkey Pharmacokinetics Studies
Compounds with sub-nanomolar human CYP11B2 potency (< 10 nM) and > 100-fold human
CYP11B1 selectivity were evaluated in rat SDPK study first (Table 6). A good in vitro and in vivo
clearance correlation was observed for this series of compounds, as the in vivo clearance could be well
predicted from rat liver microsomal clearance. Meanwhile, due to acceptable solubility and permeability,
these analogs also showed good to excellent oral bioavailability. Among three sulfonamides (49, 51,
and 52), the oxygen-linked azetidine analog 52 showed a better metabolic stability than those of
piperidine (49) and piperazine (51) analogs. Therefore, it achieved a higher oral exposure with a
bioavailability of 45%. A similar or better PK profile was also maintained for the two heteroaryl amides
that were derived from the same linker (56 and 58). A spiro analog 62, which was designed to address
the metabolic liability of the piperazine linker, also gave good oral exposure due to the decreased
metabolism of 2,6-diazaspiro[3.3]heptane ring (e.g., rat LM for compounds 50, 51, 59, and 60 are 17.7
and 16.8 vs. 3.71, and 0.87 mL/min/kg, respectively).
Compounds with a good oral exposure in rats were subsequently evaluated in cynomolgus
monkey SDPK study. As shown in Table 7, although low in vivo clearances were maintained, lower
oral bioavailabilities were observed for both compounds 52 and 62. Portal vein cannulated cynomolgus
monkey PK study later suggested that for these analogs high intestinal CYP3A4 metabolism could be
the cause for the lower bioavailability observed in cynomolgus monkey PK studies.
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Table 6. Physico-chemical property and single-dose pharmacokinetic evaluation of representative
compounds in rat.
Cmpd
49
LYSAa
PAMPA
b
RLMc
Clivd
AUC(0-)e
po
hr*ng/mL)
Ff
(%)
-6
(g/mL)
(10 cm/s)
(mL/min/kg) (mL/min/kg)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
39
91
4.01
6.03
4.17
3.32
5.4
35.1
16.8
5.0
0
29.7
16.6
3.8
1465
1640
6370
10818
1020
7880
5660
87
54
45
58
40
60
69
5
1
52
78
5
6
20
3.1
57
290
310
44
10.4
6.4
6.4
15.8
3.8
5
8
3.45
3.63
62
6.4
a
c
b
LYSA: Lyophilisation solubility assay; PAMPA: parallel artificial membrane permeability assay;
d
e
RLM: scaled intrinsic clearance of compound in rat liver microsomes; Cl : plasma clearance; AUC:
iv
f
area under the curve; F: oral bioavailability; intravenous dosing (iv) at 1 mg/kg, oral dosing (po) at 3
mg/kg.
Table 7. Single-dose pharmacokinetic parameters comparison between rat and cynomolgus monkey for
compounds 52 and 62.
Cmpd
Species
fua
Hepa Clb
Clivc
AUC_Dosed
Fe
(%)
(mL/min/kg) (mL/min/kg) (hr*kg*ng/mL/mg)
(%)
5
6
2
2
Rat
5.2
26.7
7.2
3.8
4.6
2123
1085
45
19
Cynomolgus
monkey
Rat
13.8
6.6
3.6
< 4.4
< 3.0
6.4
2.1
1889
3111
69
37
Cynomolgus
monkey
a
b
fu: percentage of compound free fraction in a plasma protein binding assay; Hepa Cl: scaled intrinsic
c
d
clearance of compound in liver hepatocytes; Cliv: plasma clearance; AUC_Dose: dose normalized area
e
under the curve; F: oral bioavailability.
In Vivo Evaluation of ASI in Cynomolgus Monkey ACTH Model
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2
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2
2
2
2
3
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3
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3
4
4
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5
6
In clinical studies, adrenocorticotropic hormone (ACTH, Synacthen®) stimulation test is used
as a diagnostic tool to assess adrenocortical function. Plasma aldosterone and cortisol both increase in
response to an ACTH stimulation. This test can be used to measure quantitatively the aldosterone
2
9
(
potency) / cortisol (selectivity) production change as a consequence of CYP11B2/B1 inhibition. As
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
previously described, we chose to adapt this test to cynomolgus monkeys and developed an acute
2
4
Synacthen® challenge animal model for PKPD profiling of ASI in-house. To measure steroid levels
reliably in monkey requires a lot of skill and the right cooperative animals together with well trained
animal keepers. Indeed, in the cohort that we were using only selected animals that displayed robust
ACTH induced steroid responses; and that were cooperative and voluntarily donated blood samples
were selected. For animal welfare reasons we had to limit the number of primates used in this study to
an n=2 and the number of compounds tested to an absolute minimum.
Using compound 52 as an example, both plasma exposure and several steroids profile changes
in cynomologus monkeys (n =2) were recorded over a period of 24 hours following the oral doses of
isoindolin-one compound 52 at 0.01, 0.1, 1, and 10 mg/kg (t = 0 min) and an ACTH challenge (t = 1 hr,
time point close to the Cmax of 52). Highly sensitive LC/MS-MS analysis was applied to guarantee
each hormone level’s accurate measurement. 60 min post ACTH stimulation was selected in agreement
with the human protocol for the Synacthen test and our experience of achieving stable and maximal
steroid induction. Figure 5 shows the levels of 5 steroids 1 hour after the ACTH challenge. Compound
52 reduced the stimulated aldosterone level below basal levels with 3 out of the 4 tested doses (0.1, 1,
and 10 mg/kg). The corresponding CYP11B2-dependent steroid precursors, corticosterone and 11-
deoxycorticosterone remained comparable to vehicle treated animals displaying a natural variability.
Concomitantly, selectivity against CYP11B1 was investigated independently by assessing both the
precursor level (11-deoxycortisol) and the product level (cortisol) of the CYP11B1 enzyme. There were
no or minimum cortisol changes at doses 0.01, 0.1, and 1 mg/kg. At the top dose of 10 mg/kg, cortisol
levels were observed below average pre-treatment levels (vehicle t = 0 min); importantly the precursor
1
1-deoxycortisol changed minimally or not at all. This in marked contrast to LCI699 that led to a
dramatic increase in the precursors of deoxycorticosterone and deoxycortiosl. This phenomenon has
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been reported in clinical studies as well when another type of ASI was tested at high, non-selective
17
doses. Selectivity in enzyme inhibition at the 10 mg/kg dose is also suggested based on simple PK/PD
modeling (Figure 6). Four different doses were applied to cynomolgus monkey. For each dose, the
average free exposure was calculated from AUC0-7hr and corrected for plasma protein binding. There is
a good linear correlation between applied doses and average free drug concentrations. When comparing
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
exposure data with measured cellular IC values, we predict that the compound 52 is efficacious and
50
selective at doses between ~0.1-72 mg/kg, this modeling data is in agreement with data shown in Figure
5. For a definitive answer, doses up to 100 mg/kg would have to be tested, which are expected to result
in free exposures clearly above the IC of CYP11B1.
5
0
Figure 5. Steroid levels in cynomologus monkey 60 min post ACTH challenge and treatment with
compound 52 are compared to the changes of steroid levels induced by LCI699, a less selective ASI.
Note the 3 highest doses of compound 52 lowered the aldosterone clearly below basal levels (vehicle t
=
0 min). LCI699 treatment led to a dramatic increase in deoxycorticosterone and deoxycortisol levels.
Enzymatic step mediated by CYP11B1 or
CYP11B2 are indicated.
2
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. PK/PD prediction in cynomolgus monkeys. Different doses of compound 52 were applied
and the PK profile was determined (see also SI, Table S8). For each dose, the average free exposure
was calculated from AUC0-7hr and corrected for plasma protein binding. Overall, a good linear
correlation between applied doses and achieved free plasma exposures was observed. Horizontal
crosslines mark the IC values for CYP11B2 and CYP11B1. This predicts that the doses of 0.1, 1 and
5
0
1
0 mg/kg display significant and selective inhibition of aldosterone.
Additionally, selectivity against CYP11B1 is not the only concern. For example, an early ASI
FAD286 was derived from a known aromatase (CYP19A1) inhibitor Fadrozole. To exclude such un-
selectivity, we studied steroid production in the human adrenocarcinoma cell line H295R. A similar
assay was recently published as part of the ToxCast program that includes measurement of steroid
hormones, including progestagens, corticosteroids, androgens, and estrogens.30 Specifically we
induced steroidogenesis with 16 mM potassium chloride for 48 hours in the presence of various amounts
of inhibitors (0.001-20 µM) and analyzed 9 different steroids (deoxycorticosterone, corticosterone, 18-
hyroxycorticosterone, aldosterone, deoxycortisol, cortisol, 17-hydroxyprogesterone, testosterone and
estrone). Using non-linear 4 parameter fitting (GraphPad Prism 7.04), the concentration that led to a
5
0% reduction of steroid levels was calculated. In this analysis, aldosterone and 18-
hydroxycorticosterone were the only two steroids inhibited by compound 52 at less than 1 nM drug
concentrations (SI, Table S8).
CHEMISTRY
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The syntheses of compounds 10-23 are depicted in Scheme 1. These analogs were generally
prepared through copper(I)-catalyzed Ullman coupling reactions of substituted 3,4-dihydroisoquinolin-
1
-ones or isoindolin-ones intermediates with 3-bromo or 3-iodopyridine.31 For commercially not
available intermediates, their syntheses are described in Schemes 2-5. For example, the 3,4-
dihydroisoquinolin-1-one 63 was obtained via PPA catalyzed ring cyclization of methyl
phenethylcarbamate 63b (Scheme 2). Similar syntheses were also applied for the preparation of 3,4-
dihydroisoquinolin-1-ones 74-76. In another route, the 3,4-dihydroisoquinolin-1-one 64 was prepared
by first cyclization of N-trifluoroacylated phenethylamine 64e in the presence of paraformaldehyde and
acetic-sulfuric acid to provide tetrahydroisoquinoline 64f, followed by protecting group removal and
iodosylbenzene oxidation (Scheme 2). For the isoindolin-one intermediate 67, it started from the ester
formation of 4-bromo-2-methyl-benzoic acid to yield 67b, followed by nucleophilic aromatic
substitution with CuCN to give 67c. Bromination of 67c with NBS/BPO furnished 67d, which further
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
reacted with PMBNH to give an in situ cyclized product, isoindolin-one 67e. After removal of PMB
2
protecting group by the treatment with CAN then afforded intermediate 67 (Scheme 3).
Scheme 1. General syntheses of aldosterone synthase inhibitors, 10-23.
2
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
R
O
NH
O
O
1
O
Cl
2
R
R
a
a
N
N
n
NH
(
)
1
( )
Cl
3
R
n
3
R
R
N
N
6
3-67
10-14
68-73
15-20
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3
6
3
n=2 R =Cl R =H
68 R =Me
69 R =Et
2
3
1
64 n=2 R =Cl R =F
2
3
1
70 R =gem-dimethyl
65 n=1 R =H
R =H
2
3
7
7
1
2
R =spiro cyclopropyl
R =cyclopropyl
1
66 n=1 R =Cl R =H
3
2
1
67 n=1 R =CN R =H
3
2
73 R =Bn
Cl
Cl
O
O
a
4
N
4
NH
R
R
3
3
R
R
N
74-76
21-23
3
4
7
7
4
5
R =Me R =H
R =H R =Me
76 R =H R =gem-dimethyl
3
4
3
4
Reagents and conditions: a) 3-bromopyridine, CuI, K
2 3 2 3
CO (or Cs CO ), N,N’-dimethylethylenediamine (or (+)-
(
S,S)-1,2-diaminocyclohexane), 1,4-dioxane, 110~150 °C.
Scheme 2. Syntheses of 3,4-dihydroisoquinolin-1-one intermediates, 63 and 64.
Cl
H
Cl
NH2
Cl
N
O
b
a
NH
O
O
6
3a
63b
63
Cl
F
Cl
F
Cl
F
Cl
F
NH2
OH
c
Br
d
e
N
6
4a
64b
64c
64d
Cl
H
F
Cl
F
Cl
N
F
F
O
F
f
g
F
N
h, i
F
F
NH
F
O
O
6
4e
64f
64
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o
o
Reagents and conditions: a) methyl chloroformate, Et
trimethylsilyl cyanide, TBAF, CH CN, reflux; e) BH
g) paraformaldehyde, AcOH, H SO , rt; h) K CO , MeOH-H
3
N, DCM, rt; b) neat PPA, 120 C; c) PBr
-THF, reflux; f) trifluoroacetic anhydride, Et
O, rt; i) KBr, iodosylbenzene, H O, rt.
3
, DCM, 0 C; d)
3
3
3
N, DCM, rt;
2
4
2
3
2
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 3. Synthesis of isoindolin-one intermediate, 67.
Br
N
Br
N
Br
a
b
c
OH
O
O
O
O
O
N
O
O
6
7a
67b
67c
67d
O
N
d
e
NH
N
O
O
67e
67
Reagents and conditions: a) SOCl
2
, MeOH, 70 °C; b) CuCN, 1-methyl-pyrrolidin-2-one, 180 °C; c) NBS, BPO,
CN, 0 °C.
CCl , reflux; d) PMBNH , THF, rt; e) CAN, CH
4
2
3
For the several 3-substituted 5-chloro-isoindolin-1-one intermediates 68-72, an efficient two-
step synthesis was developed as outlined in Scheme 4. The first step involved the reaction of 2-bromo-5-
chloro-benzonitrile with corresponding Grignard reagents under the conditions of a) sodium
borohydride reduction; b) Ti(Oi-Pr) -mediated addition; and c) Lewis acid BF .OEt catalyzed addition
4
3
2
to give benzylic substituted phenylmethanamine intermediates 68a-72a, respectively. These
intermediates then underwent palladium catalyzed carbonylation and in situ cyclization to give 3-
substituted 5-chloro-isoindolin-1-ones 68-72. Alternatively, a 3-substituted 5-chloro-isoindolin-one
intermediate, such as 73 could also be synthesized via reacting 5-chloro-isoindole-1,3-diones with
benzylmagnesium chloride followed by Et SiH reduction; although under this route, a mixture of two
3
regioisomeric products were produced with only the desired one shown in Scheme 5. Additionally,
when there is a chiral center involved, chiral column separation was applied to obtain each individual
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
enantiomeric intermediate. Based on the optical rotation measurement, the relative configuration of an
enantiomer was assigned as either R (negative sign) or S (positive sign) accordingly.
Scheme 4. Syntheses of isoindolin-one intermediates, 68-72.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
N
R
O
Cl
NH2
a
d
NH
Cl
Br
Cl
Br
3
R
3
3
7
7
68a R = Me
6
7
68 R = Me
3
3
9a R = Et
69 R = Et
3
3
2a R = cyclopropyl
72 R = cyclopropyl
O
NH2
Cl
b
d
d
NH
Cl
Br
7
0a
70
O
NH2
Br
Cl
c
NH
Cl
7
1a
71
o
Reagents and conditions: a) MeMgBr (or EtMgBr, or cyclopropyl MgBr), THF, 0 C then NaBH
4
, rt; b) MeMgBr,
o
o
THF, 0 C then Ti(Oi-Pr)
4
, rt; c) Ti(Oi-Pr)
4
, EtMgBr, -78 C to rt then BF
3
-Et
2
2
O; d) Pd(dppf)Cl , DIPEA, CO,
o
130 C.
Scheme 5. Synthesis of isoindolin-one intermediate, 73.
O
Bn
NH
O
Bn
OH
Cl
Cl
Cl
a
b
NH
NH
O
O
7
3
7
8
73a
o
3
Reagents and conditions: a) benzylmagnesium chloride, DCM, 0 C; b) Et SiH, TFA, rt.
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The syntheses of bicyclic tetrahydroisoquinoline series of isoindolin-one compounds 24-41 are
depicted in Schemes 6 and 7. The key intermediate 83 was prepared as previously disclosed.24
Esterification of commercially available 5-bromo-4-methyl nicotinic acid with ethanol under EDCI /
DMAP activation provided ethyl ester 80. Deprotonation of the methyl group with freshly prepared
lithium diisopropylamide (LDA) followed by addition of methyl acrylate gave access to intermediate
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
0 via a two-step cascade reaction. The crude keto ester 81 was then engaged in a Krapcho
decarboxylation using 6M aq. HCl to provide ketone 82. A reductive amination of ketone 82 with
ammonia in methanol in the presence of titanium(IV) isopropoxide and treatment with sodium
borohydride furnished key building block 83. After chiral column separation, the obtained individual
enantiomer (-)-(S)-83 and (+)-(R)-83 were applied in the subsequent Ullman coupling reactions with 5-
chloroisoindolin-1-one to give intermediates (-)-(S)-84 and (+)-(R)-85, which were further reacted with
either acid chlorides or sulfonyl chlorides to give compounds 24-28 (Scheme 6). Similarly, compounds
2
9-41 were prepared by first reacting (+)-(R)-83 with acetyl chloride, propanoyl propanoate, and
methane sulfonyl chloride to give intermediates (+)-(R)-86, (+)-(R)-87, and (+)-(R)-88, respectively.
Subsequent Ullman coupling reaction with appropriate 3-substituted isoindolin-ones or 3,4-
dihydroisoquinolin-1-one then afforded compounds 29-41 (Scheme 7).
Scheme 6. Syntheses of bicyclic tetrahydroisoquinoline series of isoindolin-one aldosterone synthase
inhibitors, 24-28.
2
6
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
O
O
O
O
Br
a
Br
b
c
Br
OH
O
Br
O
N
N
N
N
7
9
80
81
f
82
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Cl
Cl
d
e
Br
1
NH2
N
N
R
NH2
N
H
O
O
N
N
N
24-28
8
3 or (-)-(S)-83
or (+)-(R)-83
(-)-(S)-84
(+)-(R)-85
1
2
2
2
4
5
6
R = Ac (S-enantiomer)
1
R = Ac (R-enantiomer)
1
R = propyl amide (R-enantiomer)
1
2
2
7
8
R = Methyl sulfone (R-enantiomer)
1
R = Ethyl sulfone (R-enantiomer)
Reagents and conditions: a) EtOH, EDCI, DMAP, DCM, rt; b) LDA, methyl acrylate, THF then 10% AcOH; c)
o
6
M HCl, reflux; d) Ti(Oi-Pr)
4
, NH
3
-MeOH, 0 C then NaBH
4
; e) CuI, (1S,2S)-cyclohexane-1,2-diamine, Cs
2
CO
3
,
o
(
-)-(S)-83 (or (+)-(R)-83), 1,4-dioxane, microwave 150 C; f) acetyl chloride (or propanoyl propanoate,
o
methanesulfonyl chloride, ethanesulfonyl chloride), Et
3
N, DCM, 0 C.
Scheme 7. Syntheses of bicyclic tetrahydroisoquinoline series of isoindolin-one aldosterone synthase
inhibitors, 29-41.
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1
2
3
4
5
6
7
8
9
O
O
O
2
b,c
2
Br
a
Br
R
R
N
NH2
N
Cl
N
H
H
1
0
11
R
R
N
N
N
2
(
(
+)-(R)-86 R = H
29-38
(
+)-(R)-83
2
+)-(R)-87 R = Me
2
10
11
11
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
29 R = H, R = H,
R
= Me
1
1
1
1
0
0
0
0
30
R
R
R
= Me, R = H
= H,
= Et, R = H
a
d
11
3
3
1
2
R
= Et
11
11
Cl
O
O
37
38
R = H,
R = cyclopropyl,
R
= cyclopropyl
10
11
Br
S
O
R
= H
N
H
O
2
2
10
11
N
R
33 R = Me, R = H, R = Me
N
N
H
1
1
1
0
0
0
11
34
R
R
R
= Me, R = H
= Et
= Et, R = H
11
3
3
5
6
= H,
R
(+)-(R)-88
d
N
11
2
39 R = H
0 R = Me
2
4
Cl
O
N
O
O
S
N
H
N
4
1
Reagents and conditions: a) acetyl chloride (or propanoyl propanoate, methanesulfonyl chloride), Et
3
N, DCM, 0
oC; b) 68 (or 69, 72), CuI, (1S,2S)-cyclohexane-1,2-diamine, Cs
CO
, 1,4-dioxane, microwave 150 C; c) chiral
o
2
3
o
column separation; d) 63, CuI, (1S,2S)-cyclohexane-1,2-diamine, Cs
2
CO
3
, 1,4-dioxane, microwave 150 C.
The syntheses of 5-chloro-3,3-dimethyl-isoindolin-1-one derivatized compounds 42-47 are
described in Schemes 8. Starting from (5-bromo-3-pyridyl)methanol, treatment with thionyl chloride
followed by quenching with ammonia in methanol, afforded 3-pyridylmethanamine 90. Subsequent
Ullman coupling reaction with 5-chloro-3,3-dimethyl-isoindolin-1-one then provided compound 91,
which further reacted with various acid chlorides and sulfonyl chlorides to give compounds 42-47,
respectively. Compound 45 was obtained in a similar manner when 1-(5-bromo-3-pyridyl)ethanamine
92 was used as the corresponding starting material.
Scheme 8. Syntheses of 5-chloro-3,3-dimethyl-isoindolin-1-one derived aldosterone synthase
inhibitors, 42-47.
2
8
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