Novel pyridyl- or isoquinolinyl-substituted indolines and indoles as potent and selective aldosterone synthase inhibitors

Article abstract of DOI:10.1021/jm500140c

Pathologically, high levels of aldosterone are associated with severe cardiovascular diseases such as congestive heart failure, hypertension, and myocardial fibrosis. The inhibition of aldosterone synthase (CYP11B2) to reduce aldosterone levels has been proposed as a promising treatment for diseases related to CYP11B2 because it is the crucial enzyme in the biosynthesis of aldosterone. A series of novel pyridyl- or isoquinolinyl-substituted indolines and indoles was designed via a ligand-based approach. The synthesized compounds were tested and found to be strong CYP11B2 inhibitors. The most potent ones showed IC₅₀ values of less than 3 nM, being similarly potent as fadrozole and LCI699. Among them, compounds 14 and 23 showed good selectivity over the highly homologous CYP11B1, with selectivity factors (SF = IC _{50 CYP11B1}/IC_{50 CYP11B2}) around 170; thus, they are superior to fadrozole and LCI699 (SFs < 15). These potent CYP11B2 inhibitors exhibited no inhibition (IC₅₀ > 50 μM) of a panel of hepatic CYP enzymes including CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 and the crucial steroidogenic enzymes, CYP17 and CYP19. Because of these advantageous profiles, compounds 14 and 23 are considered to be candidates for further in vivo evaluation.

Full text of DOI:10.1021/jm500140c

Article
pubs.acs.org/jmc
Novel Pyridyl- or Isoquinolinyl-Substituted Indolines and Indoles as
Potent and Selective Aldosterone Synthase Inhibitors
Lina Yin,†,‡, ⊥ Qingzhong Hu,†, ⊥ Juliette Emmerich,† Michael Man-Chu Lo,∥ Edward Metzger,∥
Amjad Ali,∥ and Rolf W. Hartmann*,†,§
†Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C2.3, D-66123 Saarbrucken, Germany
̈
‡ElexoPharm GmbH, Campus A1, D-66123 Saarbrucken, Germany
̈
§Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Campus C2.3, D-66123 Saarbrucken, Germany
∥Discovery Chemistry, Merck Research Laboratories, 126 East Lincoln Avenue, Rahway, New Jersey 07065, United States
̈
S
* Supporting Information
ABSTRACT: Pathologically, high levels of aldosterone are
associated with severe cardiovascular diseases such as congestive
heart failure, hypertension, and myocardial fibrosis. The inhibition of
aldosterone synthase (CYP11B2) to reduce aldosterone levels has
been proposed as a promising treatment for diseases related to
CYP11B2 because it is the crucial enzyme in the biosynthesis of
aldosterone. A series of novel pyridyl- or isoquinolinyl-substituted
indolines and indoles was designed via a ligand-based approach. The
synthesized compounds were tested and found to be strong
CYP11B2 inhibitors. The most potent ones showed IC50 values of
less than 3 nM, being similarly potent as fadrozole and LCI699.
Among them, compounds 14 and 23 showed good selectivity over
the highly homologous CYP11B1, with selectivity factors (SF =
IC50 CYP11B1/IC50 CYP11B2) around 170; thus, they are superior to
fadrozole and LCI699 (SFs < 15). These potent CYP11B2 inhibitors exhibited no inhibition (IC50 > 50 μM) of a panel of
hepatic CYP enzymes including CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 and the crucial steroidogenic enzymes,
CYP17 and CYP19. Because of these advantageous profiles, compounds 14 and 23 are considered to be candidates for further in
vivo evaluation.
escape” phenomenon observed for ACE inhibitors4 and the
overcoming of the MR blockage as a result of aldosterone
INTRODUCTION
■
Aldosterone is a major mineralocorticoid that regulates
accumulation caused by long-term application of MR
electrolyte and volume homeostasis after binding to the
antagonists.5 Recently, it has been demonstrated that
mineralocorticoid receptor (MR). Normally, its secretion is
CYP11B2 inhibitors are effective in decreasing plasma
controlled by the cooperation of the renin−angiotensin−
aldosterone levels in rats and humans.6 Experiments in heart
aldosterone system, adrenocorticotropic hormone, and potas-
failure models in rats indicated that the administration of the
sium concentration. However, in some pathological conditions,
potent CYP11B2 inhibitor FAD286 (R-enantiomer of
aldosterone levels can be abnormally elevated. High aldoster-
fadrozole, a nonselective aromatase inhibitor, Chart 1)
one levels are associated with severe cardiovascular diseases
improved cardiac hemodynamics and cardiac function, and
such as congestive heart failure, myocardial fibrosis, and certain
forms of hypertension,1 which can lead to sudden death.
the effects were more significant than those achieved by
spironolactone.7 Application of the CYP11B2 inhibitor LCI699
Aldosterone is synthesized from 11-deoxycorticosterone via
(Chart 1, IC50 = 0.2 nM) in patients with primary
three consecutive steps catalyzed by aldosterone synthase
aldosteronism or hypertension reduced blood pressure and
(CYP11B2), which is a mitochondial cytochrome P450 (CYP)
increased potassium and renin plasma concentrations.8
enzyme located in the outer layer of the adrenal cortex.2
Reduction of high aldosterone levels through the inhibition of
CYP11B2 has been proposed, therefore, as a novel strategy for
the treatment of aldosterone-related heart diseases.3 Such a
therapy is believed to be superior to treatments with the
currently used angiotensin-converting enzyme (ACE) inhib-
itors (like enalapril, Chart 1) and MR antagonists (like
spironolactone, Chart 1). This is due to the “aldosterone
Over the past decade, several classes of CYP11B2 inhibitors
have been designed by our group9 and others.10 The inhibitory
mechanism exploited for the design of these compounds makes
use of an sp2 hybrid N, which reversibly coordinates to the
Received: January 27, 2014
© XXXX American Chemical Society
A
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Chart 1. Structures of ACE Inhibitor Enalapril, MR
Antagonist Spironolatone, Aromatase Inhibitor Fadrozole
(R-enantiomer: FAD 286), and CYP11B2 Inhibitor LCI699
Chart 2. Inhibitor Design Conception
heme iron. Such a strategy was successful in the past for
aromatase (CYP19) inhibitors,11 17α-hydroxylase-17,20-lyase
(CYP17) inhibitors,12 and 11β-hydroxylase (CYP11B1)
inhibitors.13 In the case of CYP11B2 inhibitors, extremely
potent compounds with IC50 values in a subnanomolar range
were obtained.14 To avoid severe side effects, the compounds
should not inhibit other steroidogenic CYP enzymes because
they are crucial for the biosynthesis of other steroid hormones,
for example, CYP17 for androgens, CYP19 for estrogens, and
CYP11B1 for glucocorticoids. However, because of the
identical catalytic mechanism and the homology throughout
the steroidogenic CYP enzyme family, it is a challenge to
develop selective steroidogenic CYP enzyme inhibitors,
especially between CYP11B1 and CYP11B2, which share
more than 93% homology. Nevertheless, highly selective
inhibition can be achieved, as demonstrated by some
CYP1715 and CYP11B216 inhibitors. These results showed
that the key to high selectivity often resides in subtle structural
differences caused by small substituents,15 as has been observed
in dual CYP enzyme inhibitors.17
In this study, a series of novel CYP11B2 inhibitors was
designed via a ligand-based approach. Reference compound
114a is a potent CYP11B2 inhibitor (IC50 = 28 nM) with a
dihydroquinolinone core (Chart 2). The fusion of an additional
five-membered ring between the lactam N atom and the
adjacent benzene moiety strongly elevated inhibitory potency
(reference compound 2, IC50 = 1.1 nM).16a To look for another
class of potent inhibitors and further explore the enzyme
binding pocket, the five-membered ring was sustained, whereas
the lactam ring was cleaved, leading to an indoline core (Chart
2). Because the carbonyl oxygen of the lactam was identified as
a hydrogen-bond acceptor interacting with the enzyme,14a the
amido moiety was preserved. Various R1 substituents with
different bulkiness and electrostatic potential were subsequently
introduced into the amido moiety to probe the binding pocket,
leading to compounds 3−12. Because the heme-complexing
heterocycle was a determinant factor of inhibitory potency,
modifications on this ring were also performed, resulting in
compounds 13−24. The replacement of the indoline core by an
indole moiety led to compounds 25−29. These compounds
were subsequently tested for CYP11B2 inhibition and also for
selectivity over CYP11B1, CYP17, and CYP19 for safety
evaluation.
RESULTS AND DISCUSSION
■
Chemistry. The synthetic routes are depicted in Schemes
1−3. Two consecutive key steps were used for the synthesis of
compounds 3−16 and 24−29, which include a Suzuki coupling
reaction with pyridin-3-yl or isoquinolin-4-yl boronic acid to
introduce the N-containing heterocycle and an acylation in
order to furnish various substituents on the indoline or indole
core (Schemes 1 and 3). For indoline derivatives 3−16 and 24,
the amino group was first protected with Boc to ensure high
yields in the Suzuki coupling. Before acylation, the protecting
group was cleaved using trifluoroacetic (Scheme 1). With
regard to the compounds with different substituents on the
pyridyl moiety (17−23), boride 17a was employed as a
common building block to achieve last-stage diversification via
Suzuki coupling with various substituted bromo-3-pyridines
(Scheme 2). Intermediate 17a was prepared via acetylation of
5-bromoindoline and subsequent borylation using bis-
(pinacolato)diboron and Pd(dppf)2Cl2 as a catalyst. A simple
stoichiometric reduction of ketone 19 with sodium borohydride
was performed to obtain alcohol 21 as a racemate. Interestingly,
for compounds 8 and 9, in which the indoline core was
furnished with a substituted phenylcarbonyl moiety, the signal
of one aromatic proton was missing from the 1H NMR when it
was measured at room temperature. However, after heating to
373 K, the signal appeared at around 7.7 ppm (see the
Supporting Information for details). This observation might be
caused by the bulky phenylcarbonyl substituents, which block
the rotation of amid bond at room temperature and thus
conceal one signal of the aromatic protons. The structures of
compounds 8 and 9 were further confirmed by 13C NMR, MS,
and X-ray diffraction (see the Supporting Information).
Biological Results. Inhibition of CYP11B2 and CYP11B1.
The synthesized compounds were tested for their inhibitory
potencies using V79 MZh cells expressing either human
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a
Scheme 1
a
Reagents and conditions: (i) Boc2O, NaHCO3, THF, H2O, 0 °C to rt; (ii) method A: pyridin-3-ylboronic acid or isoquinolin-4-ylboronic acid,
Ph(PPh3)4, Na2CO3, DME, H2O, 90 °C; (iii) method B: CF3COOH, CH2Cl2, 0 °C to rt; (iv) method C: RCOCl, pyridine, THF, 0 °C to rt.
a
Scheme 2
a
Reagents and conditions: (i) method C: CH3COCl, pyridine, THF, 0 °C to rt; (ii) bis(pinacolato)diboron, Pd(dppf)2Cl2, KOAc, dioxane, 105 °C;
(iii) method A: R-substituted 3-bromopyridine, Ph(PPh3)4, Na2CO3, DME, H2O, 90 °C; (iv) NaBH4, MeOH, 0 °C.
a
Scheme 3
a
Reagents and conditions: (i) method A: pyridin-3-yl boronic acid or isoquinolin-4-yl boronic acid, Ph(PPh3)4, Na2CO3, DME, H2O, 90 °C; (ii)
method D: Ac2O, Et3N, DMAP, CH2Cl2, 60 °C.
CYP11B1 or CYP11B2.18 The results are presented with
fadrozole and LCI699 as references in Tables 1 and 2.
Various acyl moieties were introduced into the indoline core
(Table 1) to probe the enzyme binding pocket, and steric
hindrance was consequently identified as a crucial determinant
for CYP11B2 inhibition. Bearing a methyl group, compound 3
exhibited a strong inhibition of CYP11B2 with an IC50 value of
with isopropyl (6) and 2-chloroethyl (7) showed even weaker
inhibition, with IC50 values over 5000 nM. A steric clash for
bulky substituents at the indoline core was further demon-
strated by the finding that ethyl analogue 4 was around 200-fold
less potent than the corresponding ring-closed parent
compound 2 (IC50 = 1.1 nM, Table 1). Moreover, introduction
of aromatic rings (4-F Ph, 4-OMe Ph, and 3-OMe Ph in
compounds 8−10, respectively, as well as 2-thienyl in
compound 11) led to only moderate inhibitors (IC50 values
ranging from 600 to 3000 nM) with moderate to low selectivity
(SF < 50). A benzyl substitution (12) even resulted in a
preference for CYP11B1 inhibition.
60 nM and a selectivity factor (SF = IC50 CYP11B1/IC50 CYP11B2
)
of 47 over CYP11B1. In contrast, ethyl analogue 4 showed a
significant decrease in both potency and selectivity (IC50 = 228
nM and SF = 21). With a further increase in the bulkiness of
the substituent, the inhibitory potency of the corresponding
compounds dropped dramatically. Cyclopropyl substitution (3)
led to a 20-fold decrease in potency (IC50 = 1285 nM)
compared to that of methyl analogue 3. Similarly, compounds
To further validate the finding that the size of the
substituents at the amido moiety strongly influences
CYP11B2 inhibition and also to enhance inhibitory potency,
C
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wide range of substituents were introduced at the 4- or 5-
position, including electron-withdrawing (F, CN, and Ac),
electron-donating (Me, MeO, 1-OH-Et, and Ph), and bulky
(Ph) groups as well as hydrogen-bond donor (1-OH-Et) and
acceptors (F, MeO, and 1-OH-Et) (Table 2). Positions
adjacent to the N, however, were left unsubstituted to avoid
steric hindrance for the coordination. These modifications
sustained or improved both potency and selectivity in
comparison to that of nonsubstituted analogue 3 (IC50 = 60
nM), except for compound 17 (5-CN), which is 4-fold less
active, with an IC50 value of 426 nM. This is probably because
the CN group reduces the electron density at the sp2 hybrid N
and thus weakens the coordination to the heme iron.
Intriguingly, although having strong electron-withdrawing
groups similar to that of CN, 5-F (18) led to a similar
inhibition (IC50 = 61 nM) as that ofcompound 3, whereas 5-Ac
(19) resulted in an IC50 value of 36 nM, which is 2-fold
stronger. This interesting observation might be a compromise
between the weakened coordination and the hydrogen bonds
formed by F or Ac with Glu310. In contrast, all electron-
donating groups that were introduced promoted CYP11B2
inhibition. The OMe group (20) significantly increased
inhibitory potency to 16 nM, whereas the 1-OH-Et moiety
(21) rendered only a slight improvement (IC50 = 55 nM). It is
interesting that the bulky 5-phenyl group (22) elevated
CYP11B2 inhibition to 3 nM, which indicates the occupation
of an additional hydrophobic pocket near the heme that has
been exploited by fadrazole (PDB ID: 4FDH).19 The same
explanation could also be valid for compound 13 with an
isoquinoline moiety. Similarly, methyl substitution at the 4-
position of the pyridyl in compound 3 boosted the CYP11B2
inhibition from 60 to 2.2 nM (compound 23). This could be
explained by a conformation induced by this Me group being
located in ortho to the aryl−aryl bond, which is favorable for
the interaction with the active site of the enzyme. Such an ortho
effect might also contribute to the high potency of compound
13, in which the fused benzene nucleus twists the isoquinolinyl
moiety out of the indoline plane. More importantly, compound
23 exhibited excellent selectivity over CYP11B1 (SF = 166),
which is much better than that of the references, fadrozole and
LCI699 (SFs of 7.9 and 15, respectively). Because an Et group
at the amido moiety is tolerated and, in the case of compound
14, high selectivity is obtained (SF = 184), an Et analogue of
compound 23 was synthesized by combining the Et amido
moiety and the 4-Me substitution of the pyridyl into one
molecule, leading to compound 24. Although 24 is also very
potent (IC50 = 14 nM), the selectivity is much worse compared
to that of Me compound 23 (SF of 21 vs 166).
Table 1. Inhibition of CYP11B1 and CYP11B2 by
Compounds 3−16
percent
inhibition at
c
d
500 nM
IC50 (nM)
a
b
a
b
e
compd
R
11B2
11B1
11B2
11B1
SF
3
4
5
6
7
8
9
Me
Et
87
14
60
2848
4737
1423
4100
47
21
1
66
0.8
26
228
c-propyl
i-propyl
ClCH2CH2
4-F Ph
4-MeO Ph
3-MeO Ph
2-thienyl
Bn
28
1285
>5000
>5000
2833
1433
607
7.3
7.0
15
11
<1
6.0
2.1
1.6
>5000
>5000
>5000
2300
>5000
1523
52
n.d.
n.d.
n.d.
3.8
n.d.
<1
26
f
10
40
28
11
14
4.9
26
92
39
29
11
3070
>5000
0.7
12
2.2
100
100
98
13
Me
74
14
Et
2.8
516
184
124
40
15
c-propyl
i-propyl
30
3723
6093
715
16
90
152
2
1.1
650
7.9
15
Fadrozole
LCI699
0.8
6.3
0.2
2.9
a
Hamster fibroblasts expressing human CYP11B2; substrate: deoxy-
b
corticosterone, 100 nM. Hamster fibroblasts expressing human
CYP11B1; substrate: deoxycorticosterone, 100 nM. Mean value of
c
d
at least three experiments; standard deviation less than 10%. Mean
value of at least three experiments; relative standard deviation less than
e
25%. SF (selectivity factor) = IC50 CYP11B2/IC50 CYP11B1; n.d. = not
f
determined. Inhibitory percentage at a compound concentration of
1000 nM.
compounds 13−16 were synthesized. In these compounds, 4-
isoquinolinyl, which has been demonstrated to largely promote
CYP11B2 inhibition,14a was employed instead of 3-pyridyl,
whereas methyl, ethyl, cyclopropyl, and isopropyl were
sustained as substituents on the amid moiety. The exchange
of the nitrogen-containing heterocycle elevated the inhibitory
potency by approximately 80-fold for the methyl (13) and ethyl
(14) analogues to 0.7 and 2.8 nM, respectively. The inhibition
by the cyclopropyl (15) and isopropyl (16) compounds was
also increased to 30 and 152 nM, respectively. As expected, the
influence of the steric bulkiness was also observed in this series
of compounds. With the size increasing from methyl to ethyl,
cyclopropyl, and isopropyl, the IC50 values rose from 0.7 to 152
nM. It is notable that among these potent inhibitors,
compounds 13 and 14 exhibited similar inhibitory potency as
that of reference compound 2 (IC50 = 1.1 nM), fadrazole (IC50
= 0.8 nM and SF = 7.9), and LCI699 (IC50 = 0.2 nM and SF =
15). The selectivity (SF of 74 and 184 for compounds 13 and
14, respectively) is also largely improved, which, although is not
as good as that of reference compound 2 (SF = 600), is much
better than that of fadrazole and LCI699.
Furthermore, replacement of the indoline core by an indole
moiety elevated the CYP11B2 inhibition of the compound with
a nonsubstituted pyridyl by nearly 3-fold to 23 nM (25, Table
2) compared to that of its parent compound, 3. In contrast, for
analogues comprising isoquinolinyl and 4-methyl-pyridin-3-yl,
this exchange only led to similar potency as compared to that of
the indoline compounds (for comparison, see compound pairs
13 and 27, 23 and 28, and 24 and 29). Similar to that observed
in the indoline series, the employment of Et instead of a Me
group at the amido moiety decreased CYP11B2 inhibition by 3-
to 8-fold (for comparison, see compound pairs 25 and 26 as
well as 28 and 29). Although these indole analogues were
demonstrated to be very potent CYP11B2 inhibitors with IC50
values less than 25 nM, with the exception of compound 26
Because a methyl group on the amido moiety led to more
potent inhibitors, it was preserved when further modifications
were performed on the heme-coordinating 3-pyridyl ring. A
D
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Table 2. Inhibition of CYP11B1 and CYP11B2 by Compounds 17−29
c
d
percent inhibition at 500 nM
IC50 (nM)
a
b
a
b
e
compd
17
core
R1
R2
5-CN
11B2
11B1
11B2
11B1
SF
A
A
A
A
A
A
A
A
B
B
B
B
B
Me
Me
Me
Me
Me
Me
Me
Et
54
7.3
11
24
48
8.7
74
70
66
25
42
97
80
57
426
61
6642
6812
3125
803
3756
160
366
302
1379
736
34
15
18
5-F
93
112
87
19
5-Ac
97
36
20
5-MeO
5-(1-OH-Et)
5-Ph
100
99
16
50
21
55
68
22
100
100
100
98
3.0
2.2
14
53
23
4-Me
4-Me
H
166
21
24
25
Me
Et
23
60
26
H
85
77
10
27
Me
Me
Et
100
100
100
0.6
1.7
13
57
28
4-Me
4-Me
61
36
29
435
715
6.3
33
2
1.1
0.8
0.2
650
7.9
15
Fadrozole
LCI699
2.9
a
b
Hamster fibroblasts expressing human CYP11B2; substrate: deoxycorticosterone, 100 nM. Hamster fibroblasts expressing human CYP11B1;
c
d
substrate: deoxycorticosterone, 100 nM. Mean value of at least three experiments; standard deviation less than 10%. Mean value of at least three
experiments; relative standard deviation less than 25%. SF (selectivity factor) = IC50 CYP11B2/IC50 CYP11B1; n.d. = not determined.
e
(IC50 = 77 nM), no significant improvement of selectivity was
observed (SF < 60) for the exchange of indoline by indole.
Docking Studies. Because the crystal structure of
CYP11B2 (PDB ID: 4DVQ) was reported recently,19
compounds 19, 22, and 23 were docked into CYP11B2 to
further explore the binding of this series of inhibitors to the
enzyme. In the predominant binding mode, there is
coordination to the heme iron by the pyridyl sp2 hybrid N in
a nearly perpendicular way (Figures 1 and 2). The orientation
of these inhibitors is similar to that of the natural substrate
deoxycorticosterone, in which the indoline core points to a β2-
sheet (Figure 1, illustrated with compound 23). The carbonyl
O of the amido moiety forms hydrogen bonds with the OH of
Glu383 and/or the NH of Phe381. π−π interactions between
Figure 2. (A, B) Steric clash around the amido moiety. (C) Binding of
compounds 19 and 22 in CYP11B2 as well as the interactions formed
by substituents on the pyriyl group. Images are illustrated with
compounds 19 (celadon green), 22 (yellow), and 23 (magenta) in
CYP11B2 (PDB ID: 4DVQ).
the indoline core and Phe130 (perpendicular) and Phe487
(parallel) are also observed. These interactions seem to be the
Figure 1. Docking of compound 23 (depicted in magenta) in
CYP11B2 (PDB ID: 4DVQ).
E
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most important ones to anchor binding and to grant affinity. As
expected from the SAR, steric clashes were observed for bulky
substituents at the amido moiety. In the binding pocket, the
amido substituents can either orient to the right (Figure 2A) or
to the left (Figure 2B), depending on the carbonyl O
interacting with Glu383 or Phe381, respectively. However, in
either situation, there is not much space left before clashing
with Glu383−Cys76−Arg110 (Figure 2A) and Phe487−
Ile449−Leu380 (Figure 2B). Furthermore, the 5-Ph group at
the pyridyl leans against the I-helix and occupies the
hydrophobic pocket that is exploited by fadrazole in the co-
crystal structure (PDB ID: 4FDH).19 It also forms
perpendicular π−π interactions with Phe231, Trp260, and
Trp116 (Figure 2C). Hydrogen-bond acceptors like OMe, Ac,
and F on the pyridyl are found to interact with Glu310 (Figure
2C). These interactions enhance the affinity toward the enzyme
and thus promote inhibitory potency.
Inhibition of CYP17 and CYP19. To investigate the
selectivity profiles of the synthesized compounds, potent
CYP11B2 inhibitors (IC50 < 100 nM) were selected for further
evaluation against CYP17 and CYP19. Inhibition of CYP17 was
tested using human CYP17 co-expressed with cytochrome
P450 reductase in Escherichia coli at a compound concentration
of 2 μM.20a As for CYP19 inhibition, microsomal preparations
of human placenta were employed, and 500 nM of tested
compound was applied.21 No compound exhibited significant
inhibition of CYP17 and CYP19 at the tested concentrations,
with inhibition values of less than 7% (see the Supporting
Information for details). This indicates that there should be no
side effects with regard to sex hormone production.
Inhibition of Hepatic CYP Enzymes. Because of the
important roles of hepatic CYP enzymes in the metabolism of
endogenous compounds and xenobiotic-like drugs, the
inhibition of these enzymes is considered to be an important
criterion for safety evaluation of drug candidates. Compounds
14 and 23 were therefore tested for their inhibition against a
panel of hepatic CYP enzymes including CYP1A2, CYP2C9,
CYP2C19, CYP2D6, and CYP3A4. With IC50 values higher
than 50 μM, these two compounds showed almost no
inhibition, indicating good safety profiles in this regard.
In Vitro Metabolic Stability. Compounds 14 and 23 were
subsequently evaluated for metabolic stability using human
hepatic microsomes in comparison to that from ref compound
2. Compound 2 showed a short half-life of 9 min. In contrast,
moderate intrinsic clearance of 91 and 81 μL·mg−1·min−1 as
well as half-lives of 15 and 17 min were observed for
compounds 14 and 23, respectively. Although the metabolic
stability is not so exciting, it is viable for further development.
this ortho substituent. Finally, compounds 14 and 23 were
identified as the most potent and selective CYP11B2 inhibitors
in this series, with IC50 values around 2 nM (similar potency as
that of parent compound 2 as well as reference compounds
fadrozole and LCI699) and SFs around 170. According to our
experience, a SF over CYP11B1 of more than 100 would
significantly reduce the possibility of impairing cortisol
biosynthesis. Although compounds 14 and 23 were not as
selective as reference compound 2, they were much more
selective than fadrozole and LCI699. These two compounds
exhibited no inhibition of CYP17, CYP19, and a panel of
hepatic CYP enzymes, and they also demonstrated acceptable
metabolic stability in human hepatic microsomes, which is
better than reference compound 2. Because of their superior
profiles, compounds 14 and 23 are considered to be candidates
for further evaluation in vivo. The SAR and information on the
enzyme binding pocket from this study could guide future
design of CYP11B2 inhibitors. More drug candidates in novel
classes also provide the opportunity to choose the best one for
further development.
EXPERIMENTAL SECTION
■
Biology. Inhibition of CYP11B1 and CYP11B2. V79 MZh cells
expressing human CYP11B1 or CYP11B2 were incubated with
[1,2-3H]-11-deoxycorticosterone as substrate and the inhibitor at
different concentrations. The assay was performed as previously
described.18
CYP17 Preparation and Assay. Human CYP17 co-expressed
with NADPH-P450 reductase in E. coli20a was used, and the assay was
performed as previously described with progesterone as substrate and
NADPH as cofactor.20b
CYP19 Preparation and Assay. Human CYP19 was obtained
from microsomal preparations of human placenta, and the assay was
performed as previously described with [1β-3H]androstenedione as
the substrate.21
In Vitro Metabolic Stability. Human (pooled, mixed gender)
liver microsomes were diluted with potassium phosphate−MgCl2
buffer (100 mM, pH 7.4) to a final protein concentration of 0.5
mg/mL. After the addition of test compounds (final concentration 1
μM) and NADPH system, it was incubated at 37 °C. Samples were
taken at time points of 2, 5, 15, 30, and 45 min and quenched with
acetonitrile. They were further vortexed and centrifuged at 4000 rpm
for 15 min. Supernatant was subsequently aliquoted for LC−MS/MS
analysis.
Inhibition of Hepatic CYP Enzymes. Inhibition of drug
metabolizing CYP enzymes was determined following a single
incubation approach using recombinant human CYP enzymes
(CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4). Incubations
included a cocktail of probe substrates at final concentrations
approximating their Km (phenacetin, 50 μM; diclofenac, 6 μM; S-
mephenytoin, 34 μM; bufuralol, 11 μM; midazolam, 3 μM;
testosterone, 40 μM; and nifedipine, 8 μM). Test compounds were
run in duplicate at six concentrations up to 50 μM. At a single time
point, incubations were quenched with acetonitrile and centrifuged,
and the supernatant was transferred and diluted with water prior to
LC−MS/MS analysis of probe metabolites. Amounts of metabolites
produced were normalized to vehicle controls, and IC50 values were
calculated using XLFit.
CONCLUSIONS
■
A series of novel 3-pyridyl- or 4-isoquinolinyl-substituted
indolines and indoles was synthesized and identified as potent
and selective CYP11B2 inhibitors. These compounds were
designed via a ligand-based approach: cleaving the lactam ring
of reference compound 2 to form an indoline core and
introducing various substituents onto the amido moiety to
probe the binding pocket. Bulky groups were found to reduce
inhibitory potency, indicating steric hindrance around that
region, which was subsequently demonstrated by modeling
studies. Moreover, 4-Me substitution at the heme-coordinating
3-pyridyl moiety significantly enhanced CYP11B2 inhibition
and selectivity over CYP11B1 (compound 23). This effect is
probably due to the induction of a favorable conformation by
Chemistry. General Methods. Melting points were determined
on a Mettler FP1 melting point apparatus and are uncorrected. 1H and
13C NMR spectra were measured on a Bruker DRX-500 (500 MHz).
Chemical shifts are given in parts per million (ppm), and TMS was
used as an internal standard for all spectra obtained. All coupling
constants (J) are given in hertz. ESI (electrospray ionization) mass
spectra were determined on a TSQ quantum (Thermo Electron
Corporation) instrument. The purities of the final compounds were
controlled by a Surveyor LC system. Purities were greater than 95%.
F
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Column chromatography was performed using silica-gel 60 (50−200
μm), and reaction progress was determined by TLC analysis on
Alugram SIL G/UV254 (Macherey−Nagel). Reagents and solvents
were used as obtained from commercial suppliers without further
purification.
Method A: Suzuki Coupling. A suspension of bromide (1.0
equiv), boride (0.8−1.2 equiv), sodium carbonate (4.1−5.0 mmol),
and tetrakis(triphenylphosphine)palladium (0) (0.05 equiv) in
dimethoxyethane/water (3:1) was stirred at 90 °C under nitrogen
for 2 h. The reaction mixture was cooled to room temperature slowly
and then diluted with water. The aqueous layer was extracted with
EtOAc, and the combined organic layers were washed with brine twice
and dried over MgSO4. After evaporation in vacuo, the resulting
residue was purified by flash chromatography to afford the
corresponding product.
Method B: N-Boc Deprotection. Trifluoroacetic acid (9.0 equiv)
was added to a solution of Boc-protected indoline (1.0 equiv) in
anhydrous dichloromethane (25 mL) at 0 °C. The reaction was
warmed to room temperature and stirred overnight. Subsequently, the
reaction mixture was poured into a solution of saturated aqueous
NaHCO3 (25 mL). The resulting mixture was separated, and the
aqueous layer was extracted with dichloromethane (2 × 15 mL). The
combined organic layers were washed with brine, dried over MgSO4
and concentrated in vacuo. The residue was purified by flash
chromatography on silica gel (EtOAc/n-hexane, 1:50 to 1:1) to
yield the deprotected product.
Method C: Acylation of Indoline. Acyl chloride (1.2 equiv) was
added dropwise to a solution of substituted indoline (1.0 equiv) and
pyridine (1.5 equiv) in anhydrous THF at 0 °C. The reaction was
warmed to room temperature and stirred overnight. After water (5
mL) was added to quench, the resulting mixture was separated. The
aqueous layer was extracted with EtOAc (3 × 5 mL). The combined
organic layers were washed with brine, dried over MgSO4, and
concentrated in vacuo. The resulting crude product was purified by
flash chromatography on silica gel (EtOAc/n-hexane, 1:50 to 1:2).
Method D: Acetylation of Indole. To a stirred solution of 3-
pyridinyl or isoquinolinyl substituted indole (1.0 equiv) in 1,2-
dichloroethane (6 mL) were added triethylamine (1.5 equiv), acetic
anhydride (3.9 equiv), and DMAP (0.2 equiv) in sequence under N2.
The solution was heated at 60 °C for 8 h. Upon cooling to ambient
temperature, the reaction was diluted with EtOAc (6 mL) and washed
with a saturated solution of ammonium chloride, and the aqueous
layer was extracted with EtOAc (3 × 10 mL). The combined organic
layers were dried over MgSO4 and concentrated in vacuo. The crude
product was purified by flash column chromatography on silica gel
(EtOAc/n-hexane, 1:50 to 1:2).
column chromatography, recrystallization from THF gave offwhite
needles (220 mg, 86%). mp 155−157 °C. Rf = 0.06 (EtOAc/n-hexane,
1:1). 1H NMR (500 MHz, CDCl3): δ 1.26 (t, J = 7.3 Hz, 3H), 2.48 (q,
J = 7.3 Hz, 2H), 3.28 (t, J = 8.3 Hz, 2H), 4.11 (t, J = 8.3 Hz, 2H), 7.33
(dd, J = 4.9, 7.8 Hz, 1H), 7.41 (d, J = 3.4 Hz, 1H), 7.42 (s, 1H), 7.84
(dt, J = 2.0, 7.8 Hz, 1H), 8.34 (d, J = 8.0 Hz, 1H), 8.55 (d, J = 4.0
Hz,1H), 8.82 (d, J = 2.2 Hz, 1H). 13C NMR (125 MHz, CDCl3): δ
8.7, 28.0, 29.1, 48.0, 117.3, 123.1, 123.5, 126.7, 132.0, 133.0, 133.9,
136.3, 143.3,143.4, 148.0, 172.1. MS (ESI) m/z = 253 [M + H]+.
4-(2,3-Dihydro-1H-indol-5-yl)isoquinoline (13a). The title
compound was synthesized according to method B using crude 13b
(1.97 g) and trifluoroacetic acid (2.43 mL, 31.8 mmol) and was
1
obtained as yellow oil (0.58 g, 67% for three steps). H NMR (500
MHz, CDCl3): δ 3.14 (t, J = 8.4 Hz, 2H), 3.67 (t, J = 8.4 Hz, 2H), 6.78
(d, J = 7.9 Hz, 1H), 7.17 (dd, J = 1.7, 7.9 Hz, 1H), 7.27 (m, 1H), 7.61
(m, 1H), 7.66 (m, 1H), 8.01 (dd, J = 1.0, 8.4 Hz, 2H), 8.46 (s, 1H),
9.19 (s, 1H).
4-(1-Acetyl-2,3-dihydro-1H-indol-5-yl)isoquinoline (13). The
title compound was synthesized according to method C using 13a
(100 mg, 0.41 mmol), acetyl chloride (28 μL, 0.49 mmol), and
pyridine (49 μL, 0.61 mmol) in anhydrous THF (5 mL). Purification
by flash column chromatography twice gave a pale yellow solid (78
1
mg, 66%). mp 170−172 °C. Rf = 0.05 (EtOAc/n-hexane, 1:1). H
NMR (500 MHz, CDCl3): δ 2.28 (s, 3H), 3.30 (t, J = 8.4 Hz, 2H),
4.16 (t, J = 8.4 Hz, 2H), 7.32 (s, 1H), 7.35 (d, J = 8.2 Hz, 1H), 7.62 (t,
J = 7.4 Hz, 1H), 7.67 (t, J = 7.4 Hz, 1H), 7.93 (d,3J = 8.4 Hz, 1H), 8.03
(d, J = 7.9 Hz, 1H), 8.35 (d, J = 8.2 Hz, 1H), 8.46 (s, 1H), 9.24 (s,
1H). 13C NMR (125 MHz, CDCl3): δ 24.2, 28.0, 49.0, 116.9, 124.8,
126.1, 127.2, 127.9, 128.4, 129.6, 130.6, 131.7, 132.2, 133.2, 134.4,
142.6, 142.9, 151.6, 168.8. MS (ESI) m/z = 289 [M + H]+.
4-(1-Propanoyl-2,3-dihydro-1H-indol-5-yl)isoquinoline (14).
The title compound was synthesized according to method C using 13a
(80 mg, 0.32 mmol), propanoyl chloride (34 μL, 0.39 mmol), and
pyridine (39 μL, 0.49 mmol) in anhydrous THF (3 mL). Purification
by flash column chromatography twice gave a pale yellow solid (65
1
mg, 67%). mp 153−155 °C. Rf = 0.12 (EtOAc/n-hexane, 1:1). H
NMR (500 MHz, CDCl3): δ 1.28 (t, J = 7.3 Hz, 3H), 2.51 (q, J = 7.3
Hz, 2H), 3.30 (t, J = 8.45 Hz, 2H), 4.14 (t, J = 8.45 Hz, 2H), 7.32 (s,
1H), 7.35 (d, J = 8.3 Hz, 1H), 7.62 (t, J = 7.3 Hz, 1H), 7.67 (t, J = 7.3
Hz, 1H), 7.93 (d, J = 8.4 Hz, 1H), 8.03 (d, J = 8.0 Hz, 1H), 8.39 (t, J =
8.1 Hz, 1H), 8.46 (s, 1H), 9.23 (s, 1H). 13C NMR (125 MHz,
CDCl3): δ 8.7, 28.1, 29.2, 48.1, 116.8, 124.8, 126.1, 127.1, 127.9, 128.4,
129.6, 130.5, 131.5, 132.1, 133.1, 134.3, 136.3, 143.1, 151.7, 172.2. MS
(ESI) m/z = 303 [M + H]+.
4-[1-(2-Methylpropanoyl)-2,3-dihydro-1H-indol-5-yl]-
isoquinoline (15). The title compound was synthesized according to
method C using 13a (80 mg, 0.32 mmol), isobutyryl chloride (36 μL,
0.40 mmol), and pyridine (39 μL, 0.49 mmol) in anhydrous THF (3
mL). Purification by flash column chromatography twice gave a pale
yellow solid (70 mg, 69%). mp 149−151 °C. Rf = 0.12 (EtOAc/n-
hexane, 1:1). 1H NMR (500 MHz, CDCl3): δ 1.28 (d, J = 6.7 Hz, 6H),
2.84 (m, 1H), 3.31 (t, J = 8.5 Hz, 2H), 4.24 (t, J = 8.5 Hz, 2H), 7.35
(m, 2H), 7.66 (m, 1H), 7.72 (m, 1H), 7.97 (d, J = 8.1 Hz, 1H), 8.07
(d, J = 7.8 Hz, 1H), 8.42 (s, J = 7.8 Hz, 1H), 8.47 (s, 1H), 9.26 (s,
1H). 13C NMR (125 MHz, CDCl3): δ 19.2, 28.1, 33.5. 48.1, 117.2,
124.8, 126.0, 127.1, 127.9, 128.4, 129.6, 130.5, 131.8, 132.2, 133.1,
134.3, 142.8, 143.2, 151.7, 175.8. MS (ESI) m/z = 317 [M + H]+.
4-[1-(Cyclopropylcarbonyl)-2,3-dihydro-1H-indol-5-yl]-
isoquinoline (16). The title compound was synthesized according to
method C using 13a (80 mg, 0.32 mmol), cyclopropanecarbonyl
chloride (36 μL, 0.40 mmol), and pyridine (39 μL, 0.49 mmol) in
anhydrous THF (3 mL). Purification by flash column chromatography
twice gave a pale yellow solid (77 mg, 76%). mp 159−162 °C. Rf =
0.11 (EtOAc/n-hexane, 1:1). 1H NMR (500 MHz, CDCl3): δ 0.93 (m,
2H), 1.17 (m, 2H), 1.81 (s, br, 1H), 3.33 (s, br, 2H), 4.37 (s, br, 2H),
7.34 (m, 2H), 7.62−7.70 (m, 2H), 7.94 (d, J = 8.1 Hz, 1H), 8.04 (d, J
= 7.7 Hz, 1H), 8.32 (s, br, 1H), 8.47 (s, 1H), 9.24 (s, 1H). 13C NMR
(125 MHz, CDCl3): δ 8.3, 13.7, 27.9, 48.3, 116.8, 124.8, 126.1, 127.2,
127.9, 128.4, 129.6, 130.6, 131.7, 131.9, 133.3, 134.4, 142.4, 143.2,
151.5, 172. MS (ESI) m/z = 315 [M + H]+.
5-Pyridin-3-yl-2,3-dihydro-1H-indole (3a). The title compound
was synthesized according to method B using crude 3b (1.40 g, 4.73
mmol) and trifluoroacetic acid (3.26 mL, 42.5 mmol) as a brown solid
1
(0.93 g, 100%). H NMR (500 MHz, CDCl3): δ 3.11 (t, J = 8.4 Hz,
2H), 3.64 (t, J = 8.4 Hz, 2H), 6.71 (d, J = 8.1 Hz, 1H), 7.25 (m, 1H),
7.29 (ddd, J = 0.8, 4.9, 7.9 Hz, 1H), 7.35 (d, J = 1.2 Hz, 1H), 7.80 (dt,
J = 2.1, 7.9 Hz, 1H), 8.49 (dd, J = 1.6, 4.8 Hz, 1H), 8.79 (dd, J = 0.6,
2.3 Hz, 1H).
1-Acetyl-5-pyridin-3-yl-2,3-dihydro-1H-indole (3). The title
compound was synthesized according to method C using 3a (80 mg,
0.41 mmol), acyl chloride (28 μL, 0.49 mmol), and pyridine (50 μL,
0.62 mmol) in anhydrous THF (3 mL). After flash column
chromatography, recrystallization from THF gave yellow crystals (93
1
mg, 95%). mp 125−127 °C. Rf = 0.05 (EtOAc/n-hexane, 1:1). H
NMR (500 MHz, CDCl3): δ 2.25 (s, 3H), 3.27 (t, J = 8.5 Hz, 2H),
4.12 (t, J = 8.5 Hz, 2H), 7.33 (dd, J = 4.8, 7.8 Hz, 1H), 7.42 (m, 2H),
7.83 (dt, J = 1.9, 7.8 Hz, 1H), 8.29 (d, J = 8.3 Hz, 1H), 8.55 (d, J = 4.5
Hz, 1H), 8.81 (d, J = 1.9 Hz, 1H). 13C NMR (125 MHz, CDCl3): δ
24.2, 28.0, 49.0, 117.3, 123.1, 123.5, 126.6, 132.1, 133.2, 134.0, 136.3,
143.0, 148.0, 148.1, 168.8. MS (ESI) m/z = 239 [M + H]+.
1-Propanoyl-5-pyridin-3-yl-2,3-dihydro-1H-indole (4). The
title compound was synthesized according to method C using 3a
(200 mg, 1.02 mmol), propanoyl chloride (0.11 mL, 1.22 mmol), and
pyridine (0.12 mL, 1.53 mmol) in anhydrous THF (3 mL). After flash
G
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1-[5-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-2,3-dihy-
dro-indol-1-yl]-ethanone (17a). To a solution of 17b (1.86 g, 7.75
mmol) in dioxane (31 mL) were added bis(pinacolato)diboron (3.93
g, 15.5 mmol), Pd(dPPf)2Cl2 (0.57 g, 0.78 mmol), and anhydrous
potassium acetate (3.80 g, 38.7 mmol) under N2. The reaction was
heated at 105 °C for 2 h. Upon cooling to room temperature, water
(20 mL) was added to dilute it, and the resulting mixture was extracted
with EtOAc (3 × 15 mL). The combined organic layers were washed
with brine, dried over MgSO4, and concentrated in vacuo. The crude
product was purified by flash column chromatography on silica gel
(EtOAc/n-hexane, 1:50 to 1:2) to yield a pale yellow solid (2.08 g,
Hz, 2H), 3.92 (s, 3H), 4.12 (t, J = 8.5 Hz, 2H), 7.35 (t, J = 2.1 Hz,1H),
7.40 (m, 2H), 8.26 (d, J = 2.6 Hz,1H), 8.29 (d, J = 8.3 Hz,1H), 8.43
(d, J = 1.8 Hz,1H). 13C NMR (125 MHz, CDCl3): δ 24.2, 28.0, 49.0,
55.7, 117.3, 119.1, 123.2, 126.8, 132.1, 132.8, 135.2, 137.3, 140.0,
143.2, 155.9, 168.8. MS (ESI) m/z = 269 [M + H]+.
1-[5-(1-Acetyl-2,3-dihydro-1H-indol-5-yl)pyridin-3-yl]-
ethanol (21). To a solution of 19 (120 mg, 0.43 mmol) in methanol
(5 mL) was added sodium borohydride (32 mg, 0.86 mmol) at 0 °C.
The mixture was stirred at the same temperature for 1 h before
removal of the solvent. The residue was diluted with water (5 mL) and
then extracted with EtOAc (2 × 5 mL). The combined organic layers
were washed with brine, dried over MgSO4, and concentrated in
vacuo.The residue was purified by flash column chromatography on
silica gel (MeOH/CH2Cl2, 0 to 5%) and crystallization from acetone
to yield a pale yellow solid (120 mg, 98%). mp 179−181 °C. Rf = 0.06
(MeOH/CH2Cl2, 1:20). 1H NMR (500 MHz, MeOD-d4): δ 1.53 (s, J
= 6.5 Hz, 3H), 2.67 (s, 3H), 3.29 (d, J = 5.3 Hz, 2H), 4.19 (d, J = 3.8
Hz, 2H), 4.98 (q, J = 6.5 Hz, 1H), 7.49 (s, 1H), 7.55 (s, 1H), 8.05 (s,
1H), 8.20 (s, 1H), 8.49 (s, 1H), 8.66 (s, 1H). 13C NMR (125 MHz,
MeOD-d4): δ 24.0, 25.5, 28.8, 50.3, 68.5, 118.4, 124.5, 127.3, 133.2,
134.4, 134.8, 138.0, 143.9, 144.3, 146.1, 146.8, 171.7. MS (ESI) m/z =
283 [M + H]+.
1
94%). H NMR (500 MHz, CDCl3): δ 1.34 (s, 12H), 2.32 (s, 3H),
3.18 (t, J = 8.45 Hz, 2H), 4.05 (t, J = 8.45 Hz, 2H), 7.62 (s, 1H), 7.67
(d, J = 8.0 Hz, 1H), 8.19 (d, J = 8.1 Hz, 1H).
5-(1-Acetyl-2,3-dihydro-1H-indol-5-yl)pyridine-3-carboni-
trile (17). The title compound was synthesized according to method
A using 17a (150 mg, 0.52 mmol), 5-bromonicotinonitrile (118 mg,
0.63 mmol), sodium carbonate (277 mg, 2.61 mmol), and tetrakis-
(triphenylphosphine)palladium(0) (30 mg, 0. 03 mmol) in dimethoxy-
ethane (9 mL) and water (3 mL). The crude product was purified by
flash column chromatography on silica gel (MeOH/CH2Cl2, 0 to 2%)
and crystallization from acetone to yield a white solid (123 mg, 90%).
1
mp 206−207 °C. Rf = 0.31 (MeOH/CH2Cl2, 1:20). H NMR (500
1-Acetyl-5-(5-phenylpyridin-3-yl)-2,3-dihydro-1H-indole
(22). The title compound was synthesized according to method A
using 17a (150 mg, 0.52 mmol), 3-bromo-5-phenylpyridine (153 mg,
0.63 mmol), sodium carbonate (277 mg, 2.61 mmol), and tetrakis-
(triphenylphosphine)palladium(0) (30 mg, 0. 03 mmol) in dimethoxy-
ethane (9 mL) and water (3 mL). The crude product was purified by
flash column chromatography on silica gel (MeOH/CH2Cl2, 0 to 2%)
and crystallization from acetone to yield pale yellow crystals (137 mg,
MHz, CDCl3): δ 2.27 (s, 3H), 3.30 (t, J = 8.45 Hz, 2H), 4.15 (t, J =
8.45 Hz, 2H), 7.40 (m, 2H), 8.10 (t, J = 2.1 Hz, 1H), 8.34 (d, J = 8.3
Hz, 1H), 8.80 (d, J = 1.5 Hz, 1H), 9.00 (d, J = 2.2 Hz, 1H). 13C NMR
(125 MHz, CDCl3): δ 24.2, 27.9, 49.0, 110.1, 116.5, 117.6, 123.1,
126.9, 130.4, 132.7, 136.8, 136.9, 144.1, 150.0, 151.0, 169.0. MS (ESI)
m/z = 264 [M + H]+.
1-Acetyl-5-(5-fluoropyridin-3-yl)-2,3-dihydro-1H-indole (18).
The title compound was synthesized according to method A using 17a
(150 mg, 0.52 mmol), 3-bromo-5-fluoropyridine (111 mg, 0.63
mmol), sodium carbonate (277 mg, 2.61 mmol), and tetrakis-
(triphenylphosphine)palladium(0) (30 mg, 0.03 mmol) in dimethoxy-
ethane (9 mL) and water (3 mL). The crude product was purified by
flash column chromatography on silica gel (MeOH/CH2Cl2, 0 to 2%)
and crystallization from acetone to yield colorless crystals (108 mg,
1
84%). mp 168−170 °C. Rf = 0.24 (MeOH/CH2Cl2, 1:20). H NMR
(500 MHz, CDCl3): δ 2.26 (s, 3H), 3.29 (t, J = 8.4 Hz, 2H), 4.13 (t, J
= 8.4 Hz, 2H), 7.42−7.52 (m, 5H), 7.64 (m, 2H), 8.03 (d, J = 2.0
Hz,1H), 8.32 (d, J = 8.3 Hz,1H), 8.79 (m, 2H). 13C NMR (125 MHz,
CDCl3): δ 24.2, 28.0, 49.0, 117.4, 123.2, 126.8, 127.2, 128.3, 129.1,
132.2, 132.8, 132.9, 136.5, 136.8, 137.6, 143.3, 146.2, 146.3, 168.8. MS
(ESI) m/z = 315 [M + H]+.
1
81%). mp 178−180 °C. Rf = 0.26 (MeOH/CH2Cl2, 1:20). H NMR
1-Acetyl-5-(4-methylpyridin-3-yl)-2,3-dihydro-1H-indole
(23). The title compound was synthesized according to method A
using 17a (150 mg, 0.52 mmol), 3-bromo-4-methylpyridine (70 μL,
0.63 mmol), sodium carbonate (277 mg, 2.61 mmol), and tetrakis-
(triphenylphosphine)palladium(0) (30 mg, 0. 03 mmol) in dimethoxy-
ethane (9 mL) and water (3 mL). The crude product was purified by
flash column chromatography on silica gel (MeOH/CH2Cl2, 0 to 2%)
and crystallization from acetone to yield pale yellow crystals (85 mg,
(500 MHz, CDCl3): δ 2.26 (s, 3H), 3.28 (t, J = 8.4 Hz, 2H), 4.13 (t, J
= 8.4 Hz, 2H), 7.40 (m, 2H), 7.56 (dt, J = 2.0, 9.6 Hz, 1H), 8.30 (d, J
= 8.2 Hz,1H), 8.41 (d, J = 1.8 Hz,1H), 8.64 (s, 1H). 13C NMR (125
2
MHz, CDCl3): δ 24.2, 27.9, 49.0, 117.4, 118.1, 120.7 (d, JC,F = 18.6
2
Hz), 123.2, 126.8, 131.5, 132.3, 136.0 (d, JC,F = 23.3 Hz), 138.1 (d,
4
1
4JC,F = 3.6 Hz), 143.6 (d, JC,F = 3.2 Hz), 159.7 (d, JC,F = 257 Hz),
168.9. MS (ESI) m/z = 257 [M + H]+.
1
65%). mp 180−182 °C. Rf = 0.50 (MeOH/CH2Cl2 1:20). H NMR
1-[5-(1-Acetyl-2,3-dihydro-1H-indol-5-yl)pyridin-3-yl]-
ethanone (19). The title compound was synthesized according to
method A using 17a (300 mg, 1.04 mmol), 3-acetyl-5-bromopyridine
(259 mg, 1.26 mmol), sodium carbonate (551 mg, 5.20 mmol), and
tetrakis(triphenylphosphine)palladium(0) (60 mg, 0. 05 mmol) in
dimethoxyethane (15 mL) and water (5 mL). The crude product was
purified by flash column chromatography on silica gel (MeOH/
CH2Cl2, 0 to 1:50) and crystallization from acetone to yield colorless
crystals (250 mg, 86%). mp 152−154 °C. Rf = 0.23 (MeOH/CH2Cl2,
1:20). 1H NMR (500 MHz, CDCl3): δ 2.26 (s, 3H), 2.69 (s, 3H), 3.29
(t, J = 8.5 Hz, 2H), 4.13 (t, J = 8.5 Hz, 2H), 7.45 (m, 2H), 8.32 (t, J =
8.3 Hz,1H), 8.38 (t, J = 2.1 Hz,1H), 8.98 (d, J = 2.0 Hz,1H), 9.01 (s,
1H). 13C NMR (125 MHz, CDCl3): δ 24.2, 26.9, 27.9, 49.0, 117.4,
123.2, 126.7, 131.8, 132.3, 132.4, 133.2, 136.6, 143.6, 147.8, 151.4,
168.9, 196.7. MS (ESI) m/z = 281 [M + H]+.
(500 MHz, CDCl3): δ 2.25 (s, 3H), 2.29 (s, 3H), 3.26 (t, J = 8.5 Hz,
2H), 4.12 (t, J = 8.5 Hz, 2H), 7.12−7.18 (m, 3H), 8.27 (d, J = 8.2
Hz,1H), 8.41 (m, 2H). 13C NMR (125 MHz, CDCl3): δ 19.9, 24.1,
28.0, 48.9, 116.7, 125.2, 125.3, 128.8, 131.5, 133.1, 137.6, 142.5, 144.8,
147.8, 149.7, 168.8. MS (ESI) m/z = 253 [M + H]+.
5-(4-Methylpyridin-3-yl)indoline (24a). The title compound
was synthesized using 5-bromoindoline (540 mg, 2.73 mmol) and 4-
methylpyridine-3-boronic acid (560 mg, 4.09 mmol) according to
method A. The crude product was purified by flash chromatography
on silica gel using a mixture of hexane/ethyl acetate (2:1) as eluent to
give a yellow solid (390 mg, 68%). mp 110−112 °C. 1H NMR
(CDCl3, 500 MHz): δ 2.32 (s, 3 H), 3.10 (t, J = 8.4 Hz, 2 H), 3.63 (t, J
= 8.4 Hz, 2 H), 6.70 (d, J = 7.9 Hz, 1 H), 6.95−6.98 (m, 1 H), 7.07 (d,
J = 1.3 Hz, 1 H), 7.15 (d, J = 5.0 Hz, 1 H), 8.39 (d, J = 5.0 Hz, 1 H),
8.42 (s, 1 H). 13C NMR (CDCl3, 125 MHz): δ 20.2, 30.0, 47.7, 109.2,
125.3, 125.9, 128.3, 128.7, 129.8, 138.5, 144.8, 147.7, 150.4, 151.4.
1-(5-(4-Methylpyridin-3-yl)indolin-1-yl)propan-1-one (24).
The title compound was synthesized using compound 24a (323 mg,
1.56 mmol), propionyl chloride (0.41 mL, 4.68 mmol), and NaOH
(156 mg, 3.90 mmol) according to method C. The crude product was
purified by flash chromatography on silica gel using a mixture of
hexane/ethyl acetate (2:1) as eluent to give a white solid. (45 mg,
1-Acetyl-5-(5-methoxypyridin-3-yl)-2,3-dihydro-1H-indole
(20). The title compound was synthesized according to method A
using 17a (150 mg, 0.52 mmol), 3-bromo-5-methoxylpyridine (122
mg, 0.63 mmol), sodium carbonate (277 mg, 2.61 mmol), and
tetrakis(triphenylphosphine) palladium(0) (30 mg, 0. 03 mmol) in
dimethoxyethane (9 mL) and water (3 mL). The crude product was
purified by flash column chromatography on silica gel (MeOH/
CH2Cl2, 0 to 2%) and crystallization from acetone to yield colorless
crystals (99 mg, 71%). mp 166−168 °C. Rf = 0.22 (MeOH/CH2Cl2,
1
11%). mp 144−146 °C. H NMR (CDCl3, 500 MHz): δ 1.22−1.31
1
1:20). H NMR (500 MHz, CDCl3): δ 2.25 (s, 3H), 3.27 (t, J = 8.5
(m, 3H), 2.28 (s, 3H), 2.47 (q, J = 7.3 Hz, 2H), 3.25 (t, J = 8.4 Hz,
H
dx.doi.org/10.1021/jm500140c | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
2H), 4.10 (t, J = 8.4 Hz, 2H), 7.10−7.20 (m, 3H), 8.30 (d, J = 8.2 Hz,
1H), 8.38−8.44 (m, 2H). 13C NMR (CDCl3, 125 MHz): δ 8.7, 19.8,
28.0, 29.1, 48.0, 116.6, 125.1, 125.3, 128.7, 131.3, 133.0, 137.5, 142.7,
144.5, 148.0, 149.9, 172.1. MS (ESI): m/z = 267 [M + H]+.
1H), 8.99 (br s, 1H). 13C NMR (CDCl3, 125 MHz): δ 20.0, 102.6,
111.0, 110.9, 121.3, 123.4, 125.1, 128.0, 129.3, 135.3, 139.0, 145.0,
147.5, 150.3. MS (ESI): m/z = 209 [M + H]+.
1-(5-(4-Methylpyridin-3-yl)-1H-indol-1-yl)ethanone (28). The
title compound was synthesized using compound 28a (60.0 mg, 0.29
mmol), triethylamine (0.06 mL, 0.43 mmol), acetic anhydride (0.11
mL, 1.13 mmol), and DMAP (7.1 mg, 0.06 mmol) according to
method D. The crude product was purified by flash chromatography
on silica gel using a mixture of hexane/ethyl acetate (2:1) as eluent to
5-Pyridin-3-yl-1H-indole (25a). The title compound was
synthesized according to method A using crude 5-bromoindole (250
mg, 1.26 mmol), pyridin-3-ylboronic acid (186 mg, 1.51 mmol),
sodium carbonate (667 mg, 6.30 mmol), and tetrakis-
(triphenylphosphine)palladium(0) (73 mg, 0.06 mmol) in dimethoxy-
ethane (30 mL) and water (10 mL). The crude product was purified
by flash column chromatography on silica gel (EtOAc/n-hexane, 1:100
1
give a light yellow solid (28 mg, 38%). mp 112−114 °C. H NMR
(CDCl3, 500 MHz): δ 2.31 (s, 3H), 2.68 (s, 3H), 6.69 (dd, J = 0.6, 3.8
Hz, 1H), 7.20 (dd, J = 0.6, 5.0 Hz, 1H), 7.31 (dd, J = 1.9, 8.5 Hz, 1H),
7.50 (d, J = 3.8 Hz, 1H), 7.51 (dd, J = 0.6, 1.9 Hz, 1H), 8.46 (d, J = 5.0
Hz, 1H), 8.49 (s, 1H), 8.51 (d, J = 8.2 Hz, 1H). 13C NMR (CDCl3,
125 MHz): δ 19.9, 23.9, 109.1, 116.4, 121.4, 125.1, 125.9, 126.4, 130.5,
133.4, 134.9, 137.9, 144.6, 148.2, 150.2, 168.6. MS (ESI): m/z = 251
[M + H]+.
1
to 1:2) to yield a pale yellow solid (202 mg, 82%). H NMR (500
MHz, CDCl3): δ 7.37 (ddd, J = 0.8, 4.8, 7.9 Hz, 1H), 7.41 (m, 1H),
7.49 (m, 2H), 7.59 (m, 2H), 7.88 (dt, J = 2.0, 7.9 Hz, 1H), 8.60 (dd, J
= 1.6, 4.8 Hz, 1H), 8.86 (dd, J = 0.6, 2.4 Hz, 1H).
1-Acetyl-5-pyridin-3-yl-1H-indole (25). The title compound
was synthesized according to method D using 25a (163 mg, 0.84
mmo), triethylamine (0.17 mL, 1.26 mmol), acetic anhydride (0.30
mL, 3.28 mmol), and DMAP (20 mg, 0.16 mmol) and was obtained as
pale yellow crystals (100 mg, 50%). mp 138−140 °C. Rf = 0.16
(EtOAc/n-hexane, 1:1). 1H NMR (500 MHz, CDCl3): δ 2.67 (s, 3H),
6.71 (d, J = 3.8 Hz, 1H), 7.38 (dd, J = 4.8, 7.9 Hz, 1H), 7.48 (d, J = 3.7
Hz, 1H), 7.57 (dd, J = 1.8, 8.5 Hz, 1H), 7.77 (d, J = 1.7 Hz, 1H), 7.93
(dt, J = 1.8, 7.9 Hz, 1H), 8.54 (d, J = 8.5 Hz, 1H), 8.59 (dd, J = 1.5, 4.8
Hz, 1H), 8.90 (d, J = 1.8 Hz, 1H). 13C NMR (125 MHz, CDCl3): δ
23.9, 109.2, 117.1, 119.4, 123.6, 124.4, 126.1, 131.1, 133.4, 134.7,
135.4, 137.0, 148.0, 148.3, 168.5. MS (ESI) m/z = 237 [M + H]+.
1-(5-(Pyridin-3-yl)-1H-indol-1-yl)propan-1-one (26). Synthe-
sized using compound 25a (659 mg, 3.39 mmol), propionyl chloride
(0.89 mL, 10.2 mmol), and NaOH (678 mg, 17.0 mmol) according to
method D. Crude product was purified by flash chromatography on
silica gel using a mixture of hexane/ethyl acetate (2:1) as eluent to give
a white solid (583 mg, 69%). mp 116−118 °C. 1H NMR (CDCl3, 500
MHz): δ 1.35−1.40 (m, 3H), 2.98 (q, J = 7.3 Hz, 2H), 6.70 (dd, J =
0.6, 3.8 Hz, 1H), 7.37 (ddd, J = 0.9, 4.7, 7.9 Hz, 1H), 7.53 (d, J = 3.8
Hz, 1H), 7.57 (dd, J = 1.9, 8.5 Hz, 1H), 7.77 (dd, J = 0.6, 1.9 Hz, 1H),
7.90−7.95 (m, 1H), 8.54−8.61 (m, 2H), 8.91 (dd, J = 0.8, 2.4 Hz,
1H). 13C NMR (CDCl3, 125 MHz): δ 8.6, 29.1, 109.0, 117.1, 119.3,
123.5, 124.3, 125.4, 131.0, 133.3, 134.4, 135.4, 136.9, 148.1, 148.5,
172.1. MS (ESI): m/z = 251 [M + H]+.
4-(1H-indol-5-yl)isoquinoline (27a). The title compound was
synthesized according to method A using crude 5-bromoindole (250
mg, 1.26 mmol), isoquinoline-4-ylboronic acid (240 mg, 1.39 mmol),
sodium carbonate (667 mg, 6.30 mmol), and tetrakis-
(triphenylphosphine)palladium(0) (73 mg, 0.06 mmol) in dimethoxy-
ethane (12 mL) and water (4 mL). The crude product was purified by
flash column chromatography on silica gel (EtOAc/n-hexane, 1:100 to
1:2) to yield a yellow solid (190 mg, 62%).
4-(1-Acetyl-1H-indol-5-yl)isoquinoline (27). The title com-
pound was synthesized according to method D using 22a (190 mg,
0.78 mmol), triethylamine (0.16 mL, 1.17 mmol), acetic anhydride
(0.28 mL, 3.03 mmol), and DMAP (27 mg, 0.22 mmol) and was
obtained as a pale yellow solid (130 mg, 58%). mp 120−122 °C. Rf =
0.18 (EtOAc/n-hexane, 1:1). 1H NMR (500 MHz, CDCl3): δ 2.70 (s,
3H), 6.73 (d, J = 3.7 Hz, 1H), 7.51 (dd, J = 1.7, 8.5 Hz, 1H), 7.53 (d, J
= 3.7 Hz, 1H), 7.66 (m, 2H), 7.70 (d, J = 1.4 Hz, 1H), 7.94 (d, J = 8.4
Hz, 1H), 8.06 (dd, J = 1.3, 7.4 Hz, 1H), 8.54 (s, 1H), 8.59 (d, J = 8.4
Hz, 1H), 9.28 (s, 1H). 13C NMR (125 MHz, CDCl3): δ 24.0, 109.2,
116.2, 122.3, 125.0, 126.0, 127.2, 127.3, 127.9, 128.4, 130.7, 130.8,
132.4, 133.6, 134.6, 135.2, 142.7, 151.6, 168.6. MS (ESI) m/z = 287
[M + H]+.
1-(5-(4-Methylpyridin-3-yl)-1H-indol-1-yl)propan-1-one (29).
The title compound was synthesized using compound 28a (500 mg,
2.40 mmol), propionyl chloride (0.63 mL, 7.20 mmol), and NaOH
(480 mg, 12.0 mmol) according to method D. The crude product was
purified by flash chromatography on silica gel using a mixture of
hexane/ethyl acetate (2:1) as eluent to give a white solid (200 mg,
32%). mp 91−93 °C. 1H NMR (CDCl3, 500 MHz): δ 1.37 (t, J = 7.4
Hz, 3H), 2.30 (s, 3H), 2.99 (q, J = 7.5 Hz, 2H), 6.69 (d, J = 3.8 Hz,
1H), 7.20 (d, J = 5.0 Hz, 1H), 7.31 (dd, J = 1.9, 8.5 Hz, 1H), 7.51 (d, J
= 1.3 Hz, 1H), 7.54 (d, J = 3.8 Hz, 1H), 8.45 (d, J = 5.0 Hz, 1H), 8.48
(s, 1H), 8.54 (d, J = 8.5 Hz, 1H). 13C NMR (CDCl3, 125 MHz): δ 8.7,
19.9, 29.1, 109.0, 116.4, 121.4, 125.1, 125.2, 126.4, 130.4, 133.3, 135.0,
138.0, 144.6, 148.1, 150.2, 172.2. MS (ESI): m/z = 265 [M + H]+.
Docking Studies. The redundant protein copies in the CYP11B2
crystal (PDB ID: 4DVQ) as well as the substrate (Doc) and water
were removed. Hydrogens and partial charges were added via the
Protonate3D application in MOE. Ligands were built and energy-
minimized in the MMFF94s force field with MOE. The docking
software GOLD was employed in the docking studies with the
following settings: the automatic active-site detection was switched on,
heme iron was selected as the active-site origin, and the radius of the
active site was set to 19 Å. A distance constraint of 1.9−2.5 Å between
the sp2 hybrid N and the iron was set as well. Ligand was docked in 50
independent genetic algorithm (GA) iterations for each of the three
GOLD-docking runs. Moreover, the goldscore.p450_pdb parameters
were exploited, and the genetic algorithm default parameters were set.
The resulting poses were subsequently ranked according to fitness and
further evaluated with the LigX module in MOE.
ASSOCIATED CONTENT
* Supporting Information
■
S
Inhibition data regarding CYP17 and CYP19; synthetic
procedures and characterization of intermediates 3c, 3b, 13b,
17b, and final compounds 9−12; and X-ray crystal structure
data of compounds 8 and 9. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +(49) 681 302 70300. Fax: +(49) 681 302 70308. E-
mail: rolf.hartmann@helmholtz-hzi.de. Home Page: http://
www.helmholtz-hzi.de/?id=3897.
■
Author Contributions
5-(4-Methylpyridin-3-yl)-1H-indole (28a). The title compound
was synthesized using 5-bromindole (954 mg, 4.87 mmol) and 4-
methylpyridine-3-boronic acid (1.00 g, 7.30 mmol) according to
method A. The crude product was purified by flash chromatography
on silica gel using a mixture of hexane/ethyl acetate (2:1) as eluent to
give a white solid (900 mg, 89%). mp 131−133 °C. 1H NMR (CDCl3,
500 MHz): δ 2.35 (s, 3H), 6.61−6.62 (m, 1H), 7.15 (dd, J = 1.6, 8.2
Hz, 1H), 7.23 (dd, J = 0.6, 5.0 Hz, 1H), 7.29−7.31 (m, 1H), 7.45−
7.50 (m, 1H), 7.59−7.62 (m, 1H), 8.47 (d, J = 5.0 Hz, 1H), 8.55 (s,
⊥L.Y. and Q.H. contributed to this article equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We appreciate the help of Dr. Jorg Haupenthal, Dr. Christina
Zimmer, Jeannine Jung, and Jannine Ludwig for performing the
■
̈
I
dx.doi.org/10.1021/jm500140c | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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(10) Roumen, L.; Peeters, J. W.; Emmen, J. M. A.; Beugels, I. P. E.;
Custers, E. M. G.; de Gooyer, M.; Plate, R.; Pieterse, K.; Hilbers, P. A.
J.; Smits, J. F. M.; Vekemans, J. A. J.; Leysen, D.; Ottenheijm, H. C. J.;
Janssen, H. M.; Hermans, J. J. R. Synthesis, biological evaluation, and
molecular modeling of 1-benzyl-1H-imidazoles as selective inhibitors
of aldosterone synthase (CYP11B2). J. Med. Chem. 2010, 53, 1712−
1725.
in vitro tests, Dr. Josef Zapp for the NMR spectra at elevated
temperature, and Dr. Volker Huch for determination of the X-
ray crystal structure. We also thank Professor Hermans
(University of Maastricht, The Netherlands) for providing us
with V79MZh11B1 cells expressing human CYP11B1.
ABBREVIATIONS USED
(11) (a) Gobbi, S.; Hu, Q.; Negri, M.; Zimmer, C.; Belluti, F.;
Rampa, A.; Hartmann, R. W.; Bisi, A. Modulation of cytochromes
P450 with xanthone-based molecules: from aromatase to aldosterone
synthase and steroid 11β-hydroxylase inhibition. J. Med. Chem. 2013,
56, 1723−1729. (b) Leze, M. P.; Le Borgne, M.; Pinson, P.; Palusczak,
A.; Duflos, M.; Le Baut, G.; Hartmann, R. W. Synthesis and biological
evaluation of 5-[(aryl)(1H-imidazol-1-yl)methyl]-1H-indoles: potent
and selective aromatase inhibitors. Bioorg. Med. Chem. Lett. 2006, 16,
1134−1137. (c) Hartmann, R. W.; Frotscher, M.; Ledergerber, D.;
■
CYP, cytochrome P450; CYP11B2, aldosterone synthase; MR,
mineralocorticoid receptor; ACE, angiotensin converting
enzyme; CYP17, 17α-hydroxylase-17,20-lyase; CYP11B1, 11β-
hydroxylase; CPY19, aromatase, estrogen synthase; SF,
selectivity factor = IC50 CYP11B1/IC50 CYP11B2; Pd(dppf)2Cl2,
[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II);
DME, dimethoxyethane; Ph(PPh3 )4 , tetrakis-
(triphenylphosphine)palladium (0)
Wachter, G. A.; Grun, G. L.; Sergejew, T. F. Synthesis and evaluation
̈
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of azole-substituted tetrahydronaphthalenes as inhibitors of P450
arom, P450 17, and P450 TxA2. Arch. Pharm. (Weinheim, Ger.) 1996,
329, 251−261. (d) Hille, U. E.; Hu, Q.; Pinto-Bazurco Mendieta, M.
A. E.; Bartels, M.; Vock, C. A.; Lauterbach, T.; Hartmann, R. W.
Steroidogenic cytochrome P450 (CYP) enzymes as drug targets:
combining substructures of known CYP inhibitors leads to compounds
with different inhibitory profile. C. R. Chim. 2009, 12, 1117−1126.
(e) Gobbi, S.; Cavalli, A.; Negri, M.; Schewe, K. E.; Belluti, F.; Piazzi,
L.; Hartmann, R. W.; Recanatini, M.; Bisi, A. Imidazolylmethylbenzo-
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