In vivo active aldosterone synthase inhibitors with improved selectivity: Lead optimization providing a series of pyridine substituted 3,4-dihydro-1H-quinolin-2-one derivatives

Article abstract of DOI:10.1021/jm800888q

Pyridine substituted naphthalenes (e.g., I-III) constitute a class of potent inhibitors of aldosterone synthase (CYP11B2). To overcome the unwanted inhibition of the hepatic enzyme CYP1A2, we aimed at reducing the number of aromatic carbons of these molecules because aromaticity has previously been identified to correlate positively with CYP1A2 inhibition. As hypothesized, inhibitors with a tetrahydronaphthalene type molecular scaffold (1-11) exhibit a decreased CYP1A2 inhibition. However, tetralone 9 turned out to be cytotoxic to the human cell line U-937 at higher concentrations. Consequent structural optimization culminated in the discovery of heteroaryl substituted 3,4-dihydro-1H-quinolin-2-ones (12-26), with 12, a bioisostere of 9, being nontoxic up to 200 μM. The investigated molecules are highly selective toward both CYP1A2 and a wide range of other cytochrome P450 enzymes and show a good pharmacokinetic profile in vivo (e.g., 12 with a peroral bioavailability of 71%). Furthermore, isoquinoline derivative 21 proved to significantly reduce plasma aldosterone levels of ACTH stimulated rats.

Full text of DOI:10.1021/jm800888q

J. Med. Chem. 2008, 51, 8077–8087
8077
In Vivo Active Aldosterone Synthase Inhibitors with Improved Selectivity: Lead Optimization
Providing a Series of Pyridine Substituted 3,4-Dihydro-1H-quinolin-2-one Derivatives
Simon Lucas,† Ralf Heim,† Christina Ries,† Katarzyna E. Schewe,† Barbara Birk,‡ and Rolf W. Hartmann*,†
Pharmaceutical and Medicinal Chemistry, Saarland UniVersity, P.O. Box 151150, D-66041 Saarbru¨cken, Germany, Pharmacelsus CRO,
Science Park 2, D-66123 Saarbru¨cken, Germany
ReceiVed July 17, 2008
Pyridine substituted naphthalenes (e.g., I-III) constitute a class of potent inhibitors of aldosterone synthase
(CYP11B2). To overcome the unwanted inhibition of the hepatic enzyme CYP1A2, we aimed at reducing
the number of aromatic carbons of these molecules because aromaticity has previously been identified to
correlate positively with CYP1A2 inhibition. As hypothesized, inhibitors with a tetrahydronaphthalene type
molecular scaffold (1-11) exhibit a decreased CYP1A2 inhibition. However, tetralone 9 turned out to be
cytotoxic to the human cell line U-937 at higher concentrations. Consequent structural optimization culminated
in the discovery of heteroaryl substituted 3,4-dihydro-1H-quinolin-2-ones (12-26), with 12, a bioisostere
of 9, being nontoxic up to 200 µM. The investigated molecules are highly selective toward both CYP1A2
and a wide range of other cytochrome P450 enzymes and show a good pharmacokinetic profile in vivo
(e.g., 12 with a peroral bioavailability of 71%). Furthermore, isoquinoline derivative 21 proved to significantly
reduce plasma aldosterone levels of ACTH stimulated rats.
Introduction
aldosterone produced locally in the heart triggers myocardial
fibrosis, too.3 Recent clinical studies with the MR antagonists
spironolactone and eplerenone gave evidence for the pivotal role
of aldosterone in the progression of cardiovascular diseases.
Blocking the aldosterone action by functional antagonism of
its receptor reduced the mortality and significantly reduced the
symptoms of heart failure.4 Furthermore, follow-up studies
revealed that cardiac fibrosis can not only be prevented but also
reversed by use of spironolactone.5
The progressive nature of congestive heart failure (CHFa) is
a consequence of a neurohormonal imbalance that involves a
chronic activation of the renin-angiotensin-aldosterone system
(RAAS) in response to reduced cardiac output and reduced renal
perfusion. Aldosterone and angiotensin II (Ang II) are exces-
sively released, leading to increased blood volume and blood
pressure as a consequence of epithelial sodium retention as well
as Ang II mediated vasoconstriction and finally to a further
reduction of cardiac output.1 The RAAS is pathophysiologically
stimulated in a vicious cycle of neurohormonal activation that
counteracts the normal negative feedback loop regulation. The
most important circulating mineralocorticoid, aldosterone, acts
by binding to specific mineralocorticoid receptors (MR) located
in the cytosol of target epithelial cells. Thereby, renal sodium
reabsorption and potassium secretion are promoted in the distal
tubule and the collecting duct of the nephron. Elevated blood
volume and thus blood pressure result from water that follows
the sodium movement via osmosis. In addition to these indirect
effects on the heart function, aldosterone exerts direct effects
on the heart by activating nonepithelial MRs in cardiomyocytes,
fibroblasts, and endothelial cells. Synthesis and deposition of
fibrillar collagen in the fibroblasts result in myocardial fibrosis.2
Relatively inelastic collagen fibers stiffen the heart muscle,
which deteriorates the myocardial function and as a consequence
enhances the neurohormonal imbalance by further stimulation
of the RAAS. In addition to the effects of circulating aldosterone
deriving from adrenal secretion, Satoh et al. reported that
However, several issues are unsolved by this therapeutic
strategy. Spironolactone binds rather unselectively to the al-
dosterone receptor and also has some affinity to other steroid
receptors, provoking adverse side effects.4 Although eplerenone
is more selective, clinically relevant hyperkalemia remains a
principal therapeutic risk.6 Another crucial point is the high
concentration of circulating aldosterone, which is not lowered
by MR antagonistic therapy and raises several issues. First, the
elevated aldosterone plasma levels do not induce a homologous
down-regulation but an up-regulation of the aldosterone receptor,
which complicates a long-term therapy because MR antagonists
are likely to become ineffective.7 Furthermore, the nongenomic
actions of aldosterone are in general not blocked by receptor
antagonists and can occur despite MR antagonistic treatment.8
A novel therapeutic strategy for the treatment of hyperaldos-
teronism, congestive heart failure, and myocardial fibrosis with
potential to overcome the drawbacks of MR antagonists was
recently suggested by us:9,10 blockade of aldosterone production
by inhibiting the key enzyme of its biosynthesis, aldosterone
synthase (CYP11B2), a mitochondrial cytochrome P450 enzyme
that is localized mainly in the adrenal cortex and catalyzes the
terminal three oxidation steps in the biogenesis of aldoster-
one in humans.11 Consequent structural optimization of a hit
discovered by compound library screening led to a series of
nonsteroidal aldosterone synthase inhibitors with high selectivity
versus other cytochrome P450 enzymes.12,13 Pyridine-substituted
naphthalenes14,15 such as I-III (Chart 1) and dihydronaphtha-
lenes,16 the most potent and selective compounds that emerged
from our drug discovery program, however, revealed two major
* To whom correspondence should be addressed. Phone: +49 681 302
2424. Fax: +49 681 302 4386. E-mail: rwh@mx.uni-saarland.de.
†
Pharmaceutical and Medicinal Chemistry, Saarland University.
Pharmacelsus CRO.
‡
a Abbreviations: ACTH, adrenocorticotrophic hormone; Ang II, angio-
tensin II; AUC, area under the curve; CHF, congestive heart failure; CYP,
cytochrome P450; CYP11B1, 11ꢀ-hydroxylase; CYP11B2, aldosterone
synthase; CYP17, 17R-hydroxylase-17,20-lyase; CYP19, aromatase; Het,
heteroaryl; KHMDS, potassium hexamethyldisilazane; MR, mineralocor-
ticoid receptor; NBS, N-bromosuccinimide; NCS, N-chlorosuccinimide;
PPB, plasma protein binding; RAAS, renin-angiotensin-aldosterone
system; Tf2NPh, N-phenyltrifluoromethanesulphonimide.
10.1021/jm800888q CCC: $40.75
2008 American Chemical Society
Published on Web 12/02/2008

8078 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Lucas et al.
Chart 1. Pyridylnaphthalene Type CYP11B2 Inhibitors I-III
and Design Strategy for 3,4-Dihydro-1H-quinolin-2-one
Derivatives (e.g., 12 and 14)
Scheme 1a
Chart 2. Title Compounds: Pyridine Substituted
3,4-Dihydro-1H-quinolin-2-one Derivatives and Structurally
Related Compounds
a Reagents and conditions: (i) Tf2NPh, K2CO3, THF, µw, 120 °C; (ii)
3-pyridineboronic acid, Pd(PPh3)4, aq NaHCO3, DMF, µw, 150 °C.
The synthesis of compounds 2-6 and 9-11 (Scheme 2)
started from either 6-bromo-2-tetralone (n ) 1) or 5-bromo-1-
indanone (n ) 0) as key building blocks. Suzuki coupling
afforded the heterocycle-substituted analogues 9-11. Dihy-
dronaphthalene derivative 6 was prepared by treatment of
tetralone 9 with KHMDS, in situ quenching the enolate with
Tf2NPh,19 and Pd-catalyzed cyanation20 of the intermediate
enoltriflate. The hydroxy-substituted tetrahydronaphthalene 2
was obtained by sodium borohydride reduction of the carbonyl
group21 to afford 2a and subsequent Suzuki coupling. O-
Alkylation of 2 afforded the corresponding methoxy- (3) and
ethoxy-substituted (4) derivatives. The 6-cyano-derivatized
analogue 5 was prepared in three consecutive steps starting with
a one-pot cyanohydrin/elimination step.22 The intermediate R,ꢀ-
unsaturated nitrile 5b was treated with NaBH4 in refluxing
ethanol to reduce the double bond.23 Suzuki coupling of 5a with
3-pyridineboronic acid afforded the tetrahydronaphthalene 5.
pharmacological drawbacks: a strong inhibition of the hepatic
drug metabolizing enzyme CYP1A2 and no inhibitory effect
on the aldosterone production in vivo by using a rat model.
In the present study, we describe the design and the synthesis
of pyridine-substituted 3,4-dihydro-1H-quinolin-2-ones and
structurally related compounds as highly potent and selective
CYP11B2 inhibitors (Chart 2). The design concept toward these
molecules is based on a systematic reduction of aromaticity by
saturation of the hydrocarbons C5 to C8 of the naphthalene
moiety and subsequent chemical modification of the fully
saturated ring (Chart 1). The inhibitory activity of the title
compounds was determined in V79 MZh cells expressing human
CYP11B2. The selectivity was investigated with respect to the
highly homologous 11ꢀ-hydroxylase (CYP11B1) as well as
other crucial steroid- or drug-metabolizing cytochrome P450
enzymes (CYP17, CYP19, CYP1A2, CYP2B6, CYP2C9,
CYP2C19, CYP2D6, and CYP3A4). The in vivo pharmacoki-
netic profile of some promising compounds was determined in
male Wistar rats in both cassette and single dosing experiments.
Furthermore, plasma protein binding and cytotoxicity studies
were performed. The isoquinoline derivative 21 was investigated
for aldosterone lowering effects in vivo using ACTH stimulated
rats.
The synthesis of the compounds with a dihydro-1H-quinolin-
2-one and structurally related scaffold was accomplished as
outlined in Scheme 3. The initial bromination procedures
yielding either 12a or 13a have been described previously.24,25
Subsequent N-alkylation was accomplished by treating the
quinolinones with alkyl halide and potassium tert-butylate in
dimethylformamide to afford the intermediates 14a-16a.26
A
nitro substituent was selectively introduced in the 8-position of
10a by treating with a mixture of concentrated sulfuric acid
and concentrated nitric acid to yield 18a.27 The obtained
bromoarenes were transformed into the heterocycle-substituted
analogues 12-16 and 18-23 by Suzuki coupling. Treating 12
with N-chlorosuccinimide in dimethylformamide afforded the
8-chloro derivative 17 as the only regioisomer. Conversion of
the dihydroquinolinones 12 and 17 into the thio analogues 24
and 25 was carried out using Lawessons reagent in refluxing
toluene.
Results
Chemistry. The key step for the synthesis of the title
compounds was a Suzuki coupling to introduce the heterocycle,
mostly 3-pyridine. In case of the unsubstituted tetrahydronaph-
thalene 1, the cross-coupling was accomplished by a microwave
enhanced method17 using 3-pyridineboronic acid and triflate 1a,
which was prepared from the corresponding tetrahydronaphthol
(Scheme 1).18 Compounds 7, 8, and 26 were synthesized via
Suzuki coupling from commercially available arylbromides and
3-pyridineboronic acid under microwave heating.
Biology. Inhibition of Human Adrenal Corticoid Produc-
ing CYP11B2 and CYP11B1 In Vitro (Table 1). The
inhibitory activities of the compounds were determined in V79
MZh cells expressing either human CYP11B2 or CYP11B1.10,28
The V79 MZh cells were incubated with [14C]-deoxycorticos-
terone as substrate and the inhibitor in different concentrations.
The product formation was monitored by HPTLC using a
phosphoimager. Fadrozole, an aromatase (CYP19) inhibitor with

Aldosterone Synthase Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8079
Scheme 2a
a Reagents and conditions: (i) heteroarylboronic acid, Pd(PPh3)4, aq NaHCO3, DMF, µw, 150 °C; (ii) NaBH4, methanol, 0 °C; (iii) Me3SiCN, ZnI2,
toluene, rt, then POCl3, pyridine, reflux; (iv) Tf2NPh, KHMDS, THF/toluene, -78 °C; (v) Zn(CN)2, Pd(PPh3)4, DMF, 100 °C; (vi) NaBH4, ethanol, reflux;
(vii) alkyl halide, NaH, THF, 50 °C.
Scheme 3a
a Reagents and conditions: (i) NBS, DMF, 0 °C (n ) 1) or Br2, KBr, water, reflux (n ) 0); (ii) alkyl halide, KOtBu, DMF, rt; (iii) heteroarylboronic acid,
Pd(PPh3)4, aq NaHCO3, DMF, µw, 150 °C or Pd(PPh3)4, aq Na2CO3, toluene/ethanol, reflux; (iv) HNO3/H2SO4, rt; (v) NCS, DMF, 65 °C; vi) Lawesson’s
reagent, toluene, reflux.
ability to reduce corticoid formation in vitro29 and in vivo,30
was used as a reference (CYP11B2, IC50 ) 1 nM; CYP11B1,
IC50 ) 10 nM).
(12-26) exhibit a range of both inhibitory potency at the target
enzyme (IC50 ) 0.1-64 nM) and selectivity with respect to
CYP11B1 inhibition (selectivity factor ) 44-440). A significant
decrease in inhibitory potency can be observed for derivatization
of the lactam nitrogen by an isopropyl residue (compound 16),
whereas methyl (compound 14) and ethyl (compound 15) are
tolerated in this position. The activity also decreases when the
3-pyridine is replaced by the 5-pyrimidine (compound 23) as
the heterocyclic moiety. Introduction of a nitro substituent in
the 8-position results in the rather moderate CYP11B2 inhibitor
18 (IC50 ) 64 nM) with lower CYP11B1 selectivity. Contrari-
wise, a chloro substituent in the same position (compound 17)
increases the inhibitory potency by a factor of 7 compared to
the hydrogen analogue 12. The most potent inhibitors are
obtained when the 3-pyridine moiety is modified by 5-meth-
oxylation or replaced by 4-isoquinoline, resulting in subnano-
molar IC50 values for compounds 20-22 (IC50 ) 0.1-0.2 nM).
Isoquinoline derivative 22 displays an IC50 value as low as 0.1
Most of the compounds presented in Table 1 show a strong
inhibition of the target enzyme. Within the tetrahydro- and
dihydronaphthalene series (compounds 1-6), a substituent in
6-position of the carbocyclic skeleton induces an increased
inhibitory potency in case of methoxy- (compound 3) and cyano-
substituents (compounds 5, 6) as well as a dramatic increase in
selectivity toward CYP11B1, most notably in methoxy deriva-
tive 3 (selectivity factor ) 347). Contrariwise, introduction of
heteroatoms into the saturated ring leads to a decrease of
CYP11B2 inhibition (e.g., 7 and 8). The carbonyl derivatives
9-11 are also highly potent (IC50 ) 1.8-7.8 nM) and selective
aldosterone synthase inhibitors. Tetralone 9 is the most selective
compound of the present series displaying a CYP11B1 IC50
value 496-fold higher than the CYP11B2 IC50 value. The
derivatives with dihydro-1H-quinolin-2-one molecular scaffold

8080 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Lucas et al.
Table 1. Inhibition of Human Adrenal CYP11B2 and CYP11B1 In Vitro
% inhibitiona
IC50 valueb (nM)
compd
R
X
Y
Het
V79 11B2c hCYP11B2 V79 11B2c hCYP11B2 V79 11B1d hCYP11B1 selectivity factore
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
H
OH
CH2 CH2
CH2 CH2
82
86
97
92
94
97
71
70
97
90
94
88
85
92
93
39
97
78
91
94
94
99
57
97
97
92
29
44
1977
4921
1145
4371
745
68
112
347
146
146
181
59
OMe CH2 CH2
OEt
CN
3.3
30
5.1
1.6
101
154
7.8
4.4
1.8
28
14
2.6
22
nd
3.8
64
2.7
0.2
0.2
0.1
CH2 CH2
CH2 CH2
290
H
H
H
H
H
H
H
H
H
H
Cl
NMe
O
O
O
5970
13378
3964
819
87
CH2 CH2 3-pyridine
CH2 3-pyridine
CH2 CH2 5-methoxy-3-pyridine
496
186
106
241
425
289
235
nd
440
84
126
435
187
69
191
NH
NH
CH2 3-pyridine
3-pyridine
6746
5952
742
5177
nd
1671
5402
339
87
33
6.9
nd
580
769
525
10
NMe CH2 3-pyridine
NEt CH2 3-pyridine
NiPr CH2 3-pyridine
NH
CH2 3-pyridine
NO2 NH
CH2 3-pyridine
H
H
H
H
H
H
Cl
H
NH
CH2 5-methoxy-3-pyridine
NMe CH2 5-methoxy-3-pyridine
NH CH2 4-isoquinoline
NMe CH2 4-isoquinoline
22
23
24
25
26
fadrozole
NH
CH2 5-pyrimidine
nd
3.1
4.2
12
1.0
nd
187
183
44
NH
S
3-pyridine
10
a Mean value of at least four experiments, standard deviation less than 10%; inhibitor concentration, 500 nM. b Mean value of at least four experiments,
standard deviation usually less than 25%, nd ) not determined. c Hamster fibroblasts expressing human CYP11B2; substrate deoxycorticosterone, 100 nM.
d Hamster fibroblasts expressing human CYP11B1; substrate deoxycorticosterone, 100 nM. e IC50 CYP11B1/IC50 CYP11B2, nd ) not determined.
nM and is the most potent aldosterone synthase inhibitor known
so far and also shows a pronounced inhibitory potency at
CYP11B1 (IC50 ) 6.9 nM). The same trend was observed
previously for the binding properties of a series of heteroaryl-
substituted naphthalenes.31 Changing the lactam moiety into the
corresponding thiolactam results in a slightly reduced CYP11B1
selectivity as seen in compounds 24 and 25 compared to the
oxygen analogues 12 and 17. Moreover, incorporation of a sulfur
atom into the lactam moiety in compound 26 induces a dramatic
loss of CYP11B1 selectivity (selectivity factor ) 44).
Inhibition of Steroidogenic and Hepatic CYP Enzymes
(Tables 2 and 3). The inhibition of CYP17 was investigated
using the 50000g sediment of the E. coli homogenate recom-
binantly expressing human CYP17 and progesterone (25 µM)
as substrate.32 The percent inhibition values were measured at
an inhibitor concentration of 2.5 µM. Most of the investigated
compounds display rather low inhibitory action on CYP17
(Table 2). However, a distinct inhibition in the range of 31-74%
at a concentration of 2.5 µM is observed in case of the
tetrahydro- and dihydronaphthalenes 2-6, which is comparable
to the naphthalene parent compounds I-III (40-73%). All other
derivatives, including the keto analogues 9-11 and the inves-
tigated dihydro-1H-quinolin-2-ones, are considerably less active
at CYP17 (<22%). Exceptions from this are lactam 12 (41%
inhibition) and the thiolactam analogue 24 (72%). The inhibition
of CYP19 at a concentration of 500 nM was determined in vitro
with human placental microsomes and [1ꢀ-3H]androstenedione
as substrate as described by Thompson and Siiteri33 using our
Table 2. Inhibition of Human CYP17, CYP19, and CYP1A2 In Vitro
% inhibitiona [IC50 valueb (µM)]
compd
CYP17c
CYP19d
CYP1A2e
I
40
72
73
49
44
31
38
74
20
<5
21
41
<5
8
<5
7
12
<5
22
72
[5.7]
[0.6]
[>36]
23
22
10
<5
<5
31
53
58
21
62
54
63
17
<5
5
99 [nd]
98 [nd]
97 [nd]
II
III
2
3
4
5
6
9
10
11
12
13
14
15
17
19
20
21
24
74 [0.60]
80 [0.44]
73[0.62]
72 [0.66]
90 [0.18]
60 [1.55]
57 [1.55]
57 [1.56]
50 [1.95]
25 [6.58]
53 [1.79]
36 [3.48]
14 [30.6]
25 [5.24]
20 [16.5]
<5 [>150]
77 [0.64]
<5
<5
a Mean value of three experiments, standard deviation less than 10%.
b Mean value of two experiments, standard deviation less than 5%. c E.
coli expressing human CYP17; substrate progesterone, 25 µM; inhibitor
concentration, 2.5 µM; ketoconazole, IC50 ) 2.78 µM. d Human placental
CYP19; substrate androstenedione, 500 nM; inhibitor concentration, 500
nM; fadrozole, IC50 ) 30 nM. e Recombinantly expressed enzymes from
baculovirus-infected insect microsomes (Supersomes); inhibitor con-
centration, 2.0 µM; furafylline, IC50 ) 2.42 µM.

Aldosterone Synthase Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8081
Table 3. Inhibition of Selected Hepatic CYP Enzymes In Vitro
IC50 valuea (µM)
Table 4. Plasma Protein Binding of Compounds 9, 10, and 12
PPBa (% bound)
CYP
CYP
CYP
CYP
CYP
compd
rat
human
compd
2B6b,c
2C9b,d
2C19b,e
2D6b,f
3A4b,g
9
10
12
25
24
60
24
22
61
3
9
10
11
12
14
21
47.4
<50
16.3
28.3
123
12.9
58.9
125
<200
41.5
147
45.4
<200
122
<200
171
<200
<200
171
<200
<50
12.2
6.21
<50
<200
a Determined by analysis of the ultrafiltrates via LC-MS/MS; the degree
of binding to the plasma proteins (PPB) is calculated by the following
equation: % PPB ) (1 - [ligandultrafiltrate]/[ligandtotal])·100.
<50
3.8
<50
127
<200
<50
<50
<100
2.9
9.2
a Mean value of two experiments, standard deviation less than 5%.
b Recombinantly expressed enzymes from baculovirus-infected insect mi-
crosomes (Supersomes). c Tranylcypromine, IC50 ) 6.24 µM. d Sulfaphenazole,
Table 5. Pharmacokinetic Profile of Compounds 9-12, 14, and 21
dose
t1/2 z
tmax
Cmax
AUC0-∞
compd
(mg/kg)a
(h)b
(h)c
(ng/mL)d
(ng·h/mL)e
IC50 ) 318 nM. e Tranylcypromine, IC50 ) 5.95 µM. f Quinidine, IC50
14 nM. g Ketoconazole, IC50 ) 57 nM.
)
9f
5
5
5
3.5
2.4
3.9
3.8
1.2
1.7
2.3
2.9
3.2
4
4
4
4
1
104
300
35
261
1537
727
3178
212
1753
4762
270
659
1658
3207
10f
11f
modification.34 In most cases, the inhibitory action on CYP19
is low (<30%) at the chosen concentration (Table 2). Exceptions
are observed in case of the keto derivatives 10 and 11 as well
as the lactam derivatives 13-15, displaying aromatase inhibition
in the range of 53-63%.
12f,g
5
12
25
1h
5
25
5
12
14f
6
2
1
86
134
471
21
fadrozolef,g
A selectivity profile relating to inhibition of crucial hepatic
CYP enzymes (CYP1A2, CYP2B6, CYP2C9, CYP2C19,
CYP2D6, and CYP3A4) was determined by use of recombi-
nantly expressed enzymes from baculovirus-infected insect
microsomes. As previous studies have shown that aldosterone
synthase inhibitors of the naphthalene type are potent inhibitors
of CYP1A2 but otherwise rather selective versus important
hepatic CYP enzymes,14,15 most of the newly prepared com-
pounds were initially tested for their inhibitory action on
CYP1A2 (Table 2). The parent compounds I-III are highly
potent inhibitors of CYP1A2 (>95% inhibition at concentration
of 2 µM). With regard to this high CYP1A2 activity of these
naphthalene type compounds, the inhibitory potency slightly
decreases in case of the dihydronaphthalene derivative 6 (90%)
and the tetrahydronaphthalene derivatives 2-5 (72-80%). The
keto analogues 9-11 exhibit 57-60% inhibition corresponding
with IC50 values of approximately 1.6 µM. A further decrease
of CYP1A2 inhibition to less than 55% is observed for the
investigated lactam bioisosteres 12-15, 17, and 19-21. This
is especially true for chloro-substituted 17 as well as compounds
20 and 21 with modified heterocycles displaying IC50 values
greater than 15 µM but also for indanone 13 and methoxypy-
ridine derivative 19 with IC50 values greater than 5 µM.
However, compound 24, the thio analogue of 12, displays a
pronounced inhibitory potency (IC50 ) 0.64 µM). Some
compounds were also scrutinized for inhibition of other crucial
hepatic CYP enzymes (Table 3). The data presented in Table 3
reveal that the investigated CYP enzymes are rather unaffected
by the compounds with tetrahydronaphthalene (3), tetralone
(9-11), as well as dihydro-1H-quinolin-2-one (12, 14, and 21)
type molecular scaffold, and with few exceptions (i.e., 9, 11 at
CYP3A4 and 21 at CYP2C9) the IC50 values measured are
significantly greater than 10 µM.
a Compounds were applied perorally to male Wistar rats. b Terminal
half-life. c Time of maximal concentration. d Maximal concentration. e Area
under the curve. f These compounds were investigated in a cassette dosing
approach. g Mean value of two experiments. h Intravenous application.
the amount of freely available compound is lower (approxi-
mately 60% bound).
In Vivo Pharmacokinetics (Table 5). The pharmacokinetic
profile of selected compounds was determined after peroral
application to male Wistar rats. Plasma samples were collected
over 24 h and plasma concentrations were determined by HPLC-
MS/MS. Compounds 9-12 and 14 were investigated in cassette
dosing experiments (peroral dose ) 5 mg/kg) and compared to
fadrozole. All five compounds show comparable absorption rates
(tmax ) 4-6 h) and terminal half-lifes (t1/2z ) 2.3-3.9 h). The
slowest elimination is observed in case of tetralone 11 (t1/2z
)
3.9 h) and dihydro-1H-quinolin-2-one 12 (t1/2z ) 3.8 h). Within
this series, compound 10 shows the highest maximal concentra-
tion (Cmax) in plasma (300 ng/mL), followed in the same range
by compound 12 (261 ng/mL). Using the area under the curve
(AUC0-∞) as a ranking criterion, the bioavailability after peroral
cassette dosing increases in the order 11 (212 ng·h/mL) < 14
(659 ng·h/mL) < 9 (727 ng·h/mL) < 12 (1753 ng·h/mL) <
10 (3178 ng ·h/mL). Compounds 12 and 21 were investigated
in single dosing experiments (peroral dose ) 25 mg/kg). The
amounts of the test compounds found in the plasma after peroral
application are rather high in case of 21 (AUC0-∞ ) 1658 ng ·h/
mL) and high in case of 12 (AUC0-∞ ) 4762 ng·h/mL).
Comparing the AUC of peroral with intravenous (dose ) 1 mg/
kg) application of dihydro-1H-quinolin-2-one 12 reveals an
absolute bioavailability of 71%. Although the maximal con-
centration of 21 found in the plasma is significantly lower than
in the case of compound 12 (factor 11), the AUC0-∞ values
differ only by a factor of 3, which is due to the greater terminal
half-life of isoquinoline derivative 21. This becomes particularly
apparent from Figure 1, where the mean profile of plasma levels
(ng/mL) in rat versus time after oral application (25 mg/kg) of
compounds 12 (Figure 1a) and 21 (Figure 1b) are shown. The
concentrations of 21 are rather constant and measure between
90-130 ng/mL in a time frame of 0.5-8 h after application,
Plasma Protein Binding (Table 4). The plasma protein
binding of compounds 9, 10, and 12 was determined by
ultrafiltration. Test solutions of an aliquot of concentrated test
compound and rat or human plasma were incubated at 37 °C
for 1 h and then centrifuged at 8000g for 20 min. Ultrafiltrates
were analyzed for drug concentrations by LC-MS/MS. The
plasma protein binding of the investigated CYP11B2 inhibitors
was found to be low. The bound form of keto compounds 9
and 10 range between 22-25% in both human and rat plasma.
In the case of the bioisosteric dihydro-1H-quinolin-2-one 12,

8082 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Lucas et al.
Figure 2. Mean profile (() SEM of fractional survival (%) of human
U-937 cells in presence of compounds 9 or 12.
compounds are potent aldosterone synthase inhibitors with
pronounced selectivity toward CYP11B1. As hypothesized, a
decrease of CYP1A2 inhibition can be observed with decreased
aromaticity (i.e., number of aromatic carbons) and planarity
within this series. While the fully aromatized naphthalenes I-III
exhibit 97-99% inhibition at a concentration of 2 µM, dihy-
dronaphthalene 6 is slightly less potent (90%) and the tetrahydro
derivatives 1-5 are significantly less potent (72-80%) inhibitors
of CYP1A2. However, IC50 values are still below 1 µM and
thus the molecules are rather strongly inhibiting CYP1A2.
Further increase in CYP1A2 selectivity is achieved by introduc-
tion of a keto group into the saturated ring as accomplished in
compounds 9-11. The inhibitory potencies toward CYP1A2
clearly decrease to IC50 values of approximately 1.5 µM.
Presumably, the decrease in CYP1A2 inhibition is due to a
reduced lipophilicity of the cyclic ketone scaffold compared to
the tetrahydronaphthalene scaffold. Lipophilicity has been
hypothesized to be one of the most important variables
determining CYP1A2 inhibition.36 Furthermore, the highly
potent aldosterone synthase inhibitors 9 (IC50 ) 7.8 nM) and
10 (IC50 ) 4.4 nM) show reasonable plasma levels after peroral
application to male Wistar rats (Table 5). However, due to the
risk of the dialkyl ketone moiety to trigger unwanted cytotoxic
effects, tetralone 9 was tested against the human cell line U-937
prior to further optimization. The experiment revealed that this
compound significantly reduces the fractional survival of U-937
cells at concentrations greater than 100 µM (Figure 2). Although
the cytotoxic effect is seen only at rather high concentrations,
which might not play a critical role under physiological
conditions, a structural modification of the cyclic ketone moiety
was performed with the aim to prevent this potential pharma-
cological drawback at an early stage by choosing a nontoxic
scaffold for further structural modifications. Subsequent bioi-
sosteric exchange by a lactam moiety gave rise to the dihydro-
1H-quinolin-2-one derivatives 12-26. Contrary to tetralone 9,
dihydro-1H-quinolin-2-one 12 exhibits no distinct cytotoxic
effect on U-937 cells up to the highest concentration tested
(Figure 2). In addition, compound 12 is an even slightly less
potent inhibitor of CYP1A2 (IC50 ) 1.95 µM) than the
analogous tetralone (IC50 ) 1.55 µM), which is again in
correspondence with the reduced lipophilicity compared to the
bioisosteric 9. Contrariwise, lipophilicity does not influence the
CYP1A2 potency within the series of dihydro-1H-quinolin-2-
ones (12-21) as does the substitution pattern of the molecules,
especially in the heterocyclic moiety (e.g., 20 and 21). It is also
striking that even minor structural variations such as introduction
of a chloro substituent into the dihydro-1H-quinolin-2-one core
as accomplished in 17 can induce an almost complete loss of
CYP1A2 activity.
Figure 1. Mean profile (() SEM of plasma levels (ng/mL) in rat versus
time after oral application (25 mg/kg) of compounds 12 (a) and 21 (b)
determined in single dosing experiments.
albeit the plasma levels are considerably lower than the plasma
levels of 12.
Discussion and Conclusion
Selectivity is a prerequisite of any drug candidate to avoid
adverse side effects. In the development process of aldosterone
synthase inhibitors, it is a crucial point to investigate the
selectivity profile toward other cytochrome P450 enzymes at
an early stage. It is known that the concept of heme-iron
complexation (e.g., by nitrogen-containing heterocycles) is an
appropriate strategy to discover highly potent and selective
inhibitors. Because of this binding mechanism, however, a
putative CYP11B2 inhibitor is potentially capable of interacting
with other CYP enzymes by similarly binding to the heme
cofactor with its metal binding moiety. Taking into consideration
that the key enzyme of glucocorticoid biosynthesis, 11ꢀ-
hydroxylase (CYP11B1), and CYP11B2 have a sequence
homology of approximately 93%,35 the selectivity issue is
especially critical for the design of CYP11B2 inhibitors.
Recently, we have demonstrated that 3-pyridine substituted
naphthalenes such as I-III provide an ideal molecular scaffold
for high inhibitory potency at the target enzyme CYP11B2 as
well as high selectivity toward several other CYP enzymes (e.g.,
CYP11B1, CYP17, CYP19).14 However, these compounds
strongly inhibit the hepatic enzyme CYP1A2 (e.g., compounds
I-III in Table 2) that makes up about 10% of the overall
cytochrome P450 content in the liver and metabolizes aromatic
and heterocyclic amines as well as polycyclic aromatic hydro-
carbons. In recent QSAR studies, CYP1A2 inhibition has been
identified to correlate positively with aromaticity and lipophi-
licity.36 Furthermore, both CYP1A2 substrates and inhibitors
are usually small-volume molecules with a planar shape (e.g.,
caffeine37 and furafylline38). Rationalizing these findings, our
design strategy aimed at reducing the aromaticity and disturbing
the planarity of the molecules while keeping the pharmacophoric
points39 of the naphthalene molecular scaffold (see Chart 1).
These considerations led to the development of pyridine
substituted dihydro- and tetrahydronaphthalenes 1-6. The
Pharmacokinetic investigations performed with compound 12
reveal a peroral absolute bioavailability of 71%. Compounds

Aldosterone Synthase Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8083
14 and 21 are also capable of crossing the gastrointestinal tract
and reach the general circulation after peroral application.
However, their total range of absorption is below that of 12.
The plasma protein binding of inhibitor 12 was found to be
low in both rat and human plasma (approximately 60%),
indicating that a sizable free fraction of circulating compound
is present in the plasma. Within the series of dihydro-1H-
quinolin-2-one type inhibitors 12-26, most compounds are
highly active at the target enzyme. This particularly applies to
the derivatives with a functionalized pyridine heterocycle, for
example, methoxy derivative 20 (IC50 ) 0.2 nM) and isoquino-
line derivatives 21 (IC50 ) 0.2 nM) and 22 (IC50 ) 0.1 nM).
By introduction of a chloro substituent in the 8-position or
methoxy in the 5-position of the pyridine heterocycle, the
selectivity increases to a factor of greater than 400 (17, 20).
Hence, these compounds are approximately 40-fold more
selective than fadrozole (selectivity factor ) 10). With respect
to the high homology of the two CYP11B isoforms, this
experimental result is particularly noteworthy.
To measure aldosterone-lowering effects in rats, we inves-
tigated the most potent and selective inhibitors of the present
series for their ability to block aldosterone biosynthesis in V79
MZh cells expressing rat CYP11B2 prior to in vivo experiments.
The results revealed that only compound 21 (and to a minor
degree also the N-methyl analogue 22) shows a moderate
inhibitory action toward rat CYP11B2 in vitro, which cor-
roborates our expectations concerning potential problems due
to species cross-over, presumably as a consequence of a rather
low sequence identity of the human and the rat CYP11B2
enzyme (approximately 69%).40 The most potent compound in
the rat assay, isoquinoline derivative 21, is also a highly potent
inhibitor of human CYP11B2 in vitro (IC50 ) 0.2 nM) and
exhibits a pronounced selectivity toward other CYP enzymes.
Examination of availability in plasma following peroral admin-
istration of this compound to rats revealed a good half-life (2.9
h) and reasonable plasma levels (AUC0-∞ ) 1658 ng·h/mL
following a 25 mg/kg dose). For the in vivo study, we used the
animal model of Ha¨usler et al., where the animals receive a
subcutaneous injection of ACTH (1 mg/kg) 16 h before test
item application to stimulate the gluco- and mineralocorticoid
biosynthesis and the aldosterone levels are determined using
RIA.40,41 The plasma aldosterone levels of the individual animals
of this study are shown in Figure 3. It becomes apparent that
ACTH treatment induces a significant increase of the aldosterone
levels. Within the vehicle-treated group (animals 7-10, Figure
3b), the concentrations found in the plasma are rather constant
over the duration of the experiment (6 h). To six animals
(compound 21 group) isoquinoline derivative 21 was intrave-
nously applied in a 20 mg/kg dose after 16 h upon ACTH
treatment (animals 1-6, Figure 3a). A significant aldosterone-
lowering effect can already be observed after 15 min. Within a
time frame of 0.5-3 h, the aldosterone concentrations are
maximally reduced to 36-63% of the prior ACTH level for
the individual animals of the compound 21 group.
Figure 3. Lowering of aldosterone plasma levels in vivo of (a) the
compound 21 treated group (animals 1-6) and (b) the vehicle treated
group (animals 7-10). Shown are the aldosterone plasma levels of the
individual animals predose before sc application of 1 mg/kg ACTH,
and before iv application of 20 mg/kg 21 (animals 1-6) or vehicle
(animals 7-10), followed by sampling 0.25, 0.5, 1, 2, 3, and 6 h
postdose.
is significantly lower in the case of the dihydro-1H-quinolin-
2-ones with IC50 values in the micromolar range. The investi-
gated molecules reach the circulation after peroral administration
to rats (e.g., 12 with a peroral bioavailability of 71%). Moreover,
it has been found that isoquinoline derivative 21 significantly
reduces the plasma aldosterone levels of ACTH stimulated rats.
Our current research focuses on further in vivo investigations
of compound 21 and structurally related compounds in disease
oriented models to determine their capability to prevent or
reverse myocardial fibrosis and reduce CHF induced mortality.
Experimental Section
Chemical and Analytical Methods. Melting points were
measured on a Mettler FP1 melting point apparatus and are
uncorrected. 1H NMR and 13C spectra were recorded on a Bruker
DRX-500 instrument. Chemical shifts are given in parts per million
(ppm), and tetramethylsilane (TMS) was used as internal standard
for spectra obtained in DMSO-d6 and CDCl3. All coupling constants
(J) are given in hertz. Mass spectra (LC/MS) were measured on a
TSQ Quantum (Thermo Electron Corporation) instrument with a
RP18 100-3 column (Macherey Nagel) and with water/acetonitrile
mixtures as eluents. GC/MS spectra were measured on a GCD
Series G1800A (Hewlett-Packard) instrument with an Optima-5-
MS (0.25 µM, 30 m) column (Macherey Nagel). Elemental analyses
were carried out at the Department of Chemistry, University of
Saarbru¨cken. Reagents were used as obtained from commercial
suppliers without further purification. Solvents were distilled before
use. Dry solvents were obtained by distillation from appropriate
drying reagents and stored over molecular sieves. Flash chroma-
tography was performed on silica gel 40 (35/40-63/70 µM) with
petroleum ether/ethyl acetate mixtures as eluents, and the reaction
progress was determined by thin-layer chromatography analyses
on Alugram SIL G/UV254 (Macherey Nagel). Visualization was
accomplished with UV light and KMnO4 solution. All microwave
irradiation experiments were carried out in a CEM-Discover
monomode microwave apparatus.
In summary, nonsteroidal aldosterone synthase inhibitors with
a dihydro-1H-quinolin-2-one molecular scaffold are superior to
the previously investigated pyridylnaphthalenes such as I-III.
Most compounds exhibit a potent inhibitory activity at the target
enzyme and isoquinoline derivative 22 is the most potent
CYP11B2 inhibitor described so far (IC50 ) 0.1 nM). The
selectivity versus other steroidogenic as well as hepatic cyto-
chrome P450 enzymes is generally high. Most notably, the
strong inhibition of the hepatic CYP1A2 enzyme (>95% at a
concentration of 2 µM) present in the naphthalene type inhibitors
The following compounds were prepared according to previously
described procedures: 6-bromo-1,2,3,4-tetrahydronaphthalen-2-ol

8084 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Lucas et al.
2
(2a),21 6-bromo-3,4-dihydroquinolin-2(1H)-one (12a),24 5-bromo-
1,3-dihydro-2H-indol-2-one (13a),25 6-bromo-8-nitro-3,4-dihydro-
quinolin-2(1H)-one (18a).27
2.23 (m, 1H), 2.92 (m, 1H), 3.02-3.13 (m, 3H), 3.18 (dd, J )
3
3
16.4 Hz, J ) 5.7 Hz, 1H), 7.19 (d, J ) 7.9 Hz, 1H), 7.31 (s,
3
4
1H), 7.33-7.37 (m, 2H), 7.83 (m, 1H), 8.57 (dd, J ) 5.0 Hz, J
4
Synthesis of the Target Compounds. Procedure A.17 Boronic
acid (0.75 mmol, 1 equiv), aryl bromide or -triflate (0.9-1.3 equiv),
and tetrakis(triphenylphosphane)palladium(0) (43 mg, 37.5 µmol,
5 mol %) were suspended in 1.5 mL DMF in a 10 mL septum-
capped tube containing a stirring magnet. To this was added a
solution of NaHCO3 (189 mg, 2.25 mmol, 3 equiv) in 1.5 mL water
and the vial was sealed with an Teflon cap. The mixture was
irradiated with microwaves for 15 min at a temperature of 150 °C
with an initial irradiation power of 100 W. After the reaction, the
vial was cooled to 40 °C, the crude mixture was partitioned between
ethyl acetate and water and the aqueous layer was extracted three
times with ethyl acetate. The combined organic layers were dried
over MgSO4 and the solvents were removed in vacuo. The coupling
products were obtained after flash chromatography on silica gel
(petroleum ether/ethyl acetate mixtures) and/or crystallization.
Procedure B. Boronic acid (1 equivalent), aryl bromide or
(1.3-1.5 equiv), and tetrakis(triphenylphosphane)palladium(0) (5
mol%) were suspended in toluene/ethanol 4/1 to give a 0.07-0.1
M solution of boronic acid under an atmosphere of nitrogen. To
this was added a 1 N aqueous solution of Na2CO3 (6 equivalents).
The mixture was then refluxed for 12-18 h, cooled to room
temperature, diluted with water, and extracted several times with
ethyl acetate. The combined extracts were dried over MgSO4,
concentrated, and purified by flash chromatography on silica gel
(petroleum ether/ethyl acetate mixtures) and/or crystallization.
3-(5,6,7,8-Tetrahydronaphthalen-2-yl)pyridine (1). Compound
1 was obtained according to procedure A from 1a (280 mg, 1.0
mmol) and 3-pyridineboronic acid (160 mg, 1.3 mmol) after flash
chromatography on silica gel (petroleum ether/ethyl acetate, 2/1,
Rf ) 0.20) as a pale yellow oil (142 mg, 0.68 mmol, 68%), mp
(HCl salt) 200-202 °C. LC/MS m/z 210.27 (MH+). Anal.
(C15H15N·HCl) C, H, N.
6-Pyridin-3-yl-1,2,3,4-tetrahydronaphthalen-2-ol (2). Com-
pound 2 was obtained according to procedure A from 2a (114 mg,
0.50 mmol) and 3-pyridineboronic acid (80 mg, 0.65 mmol) after
flash chromatography on silica gel (ethyl acetate, Rf ) 0.27) as a
colorless solid (96 mg, 0.43 mmol, 86%), mp 118-120 °C. LC/
MS m/z 226.23 (MH+). Anal. (C15H15NO·0.1H2O) C, H, N.
3-(6-Methoxy-5,6,7,8-tetrahydronaphthalen-2-yl)pyridine (3). To a
suspension of NaH (73 mg, 1.84 mmol, 60% dispersion in oil) in
10 mL dry THF was added dropwise a solution of 2 (345 mg, 1.53
mmol) in 5 mL THF at room temperature. The mixture was heated
to 50 °C until evolution of hydrogen ceased and then cooled to
room temperature again. Thereupon, a solution of methyl iodide
(326 mg, 2.30 mmol) in 5 mL THF was added via canula and
stirring was continued at 50 °C for 3 h. The mixture was treated
with saturated aqueous NH4Cl solution and extracted three times
with ethyl acetate. The combined organic extracts were washed
with water and brine, dried over MgSO4, and evaporated to dryness.
The crude product was purified by flash chromatography on silica
gel (petroleum ether/ethyl acetate, 7/3, Rf ) 0.09) to afford 3 as a
colorless oil (289 mg, 1.21 mmol, 79%), mp (HCl salt) 188-190
°C. LC/MS m/z 240.29 (MH+). Anal. (C16H17NO·HCl·0.6H2O)
C, H, N.
) 1.6 Hz, 1H), 8.80 (d, J ) 1.6 Hz, 1H). 13C NMR (125 MHz,
CDCl3): δ 25.5, 26.1, 27.1, 32.1, 121.8, 123.5, 125.1, 127.8, 129.8,
132.3, 134.2, 135.5, 136.2, 136.4, 148.2, 148.5. LC/MS m/z 235.26
(MH+). Anal. (C16H14N2 ·0.1H2O) C, H, N.
6-Pyridin-3-yl-3,4-dihydronaphthalene-2-carbonitrile (6). To a so-
lution of 6a (562 mg, 1.58 mmol) in 10 mL degassed DMF were
added zinc cyanide (117 mg, 1.00 mmol) and tetrakis(triph-
enylphosphane)palladium(0) (173 mg, 0.15 mmol) and the mixture
was heated at 100 °C for 2 h. After cooling to room temperature,
the mixture was diluted with 200 mL of water and extracted three
times with ethyl acetate. The combined organic extracts were
washed with water and brine, dried over MgSO4, and evaporated
to dryness. The crude product was crystallized from petroleum ether/
ethyl acetate to afford 6 as colorless needles (286 mg, 1.23 mol,
78%), mp 142-143 °C. LC/MS m/z 233.23 (MH+). Anal.
(C16H12N2) C, H, N.
4-Methyl-7-pyridin-3-yl-3,4-dihydro-2H-1,4-benzoxazine (7). Com-
pound 7 was obtained according to procedure A from 7-bromo-4-
methyl-3,4-dihydro-2H-1,4-benzoxazine (228 mg, 1.00 mmol) and
3-pyridineboronic acid (160 mg, 1.30 mmol) after flash chroma-
tography on silica gel (petroleum ether/ethyl acetate, 1/1, Rf ) 0.26)
as an off-white solid (102 mg, 0.45 mmol, 45%), mp 70-72 °C.
LC/MS m/z 227.21 (MH+). Anal. (C14H14N2O) C, H, N.
3-(2,3-Dihydro-1,4-benzodioxin-6-yl)pyridine (8). Compound 8 was
obtained according to procedure A from 6-bromo-2,3-dihydro-1,4-
benzodioxine (215 mg, 1.00 mmol) and 3-pyridineboronic acid (160
mg, 1.30 mmol) after flash chromatography on silica gel (petroleum
ether/ethyl acetate, 7/3, Rf ) 0.14) as a colorless solid (184 mg,
0.86 mmol, 86%), mp 59-61 °C. LC/MS m/z 214.19 (MH+). Anal.
(C16H12N2) C, H, N.
6-Pyridin-3-yl-3,4-dihydronaphthalen-2(1H)-one (9). Compound 9
was obtained according to procedure A from 6-bromo-2-tetralone
(113 mg, 0.50 mmol) and 3-pyridineboronic acid (80 mg, 0.65
mmol) after flash chromatography on silica gel (petroleum ether/
ethyl acetate, 1/1, Rf ) 0.15) as a colorless oil (97 mg, 0.43 mmol,
86%), mp (HCl salt) 180-182 °C. LC/MS m/z 224.20 (MH+). Anal.
(C15H13NO·HCl·0.4H2O) C, H, N.
5-Pyridin-3-yl-2,3-dihydro-1H-inden-1-one (10). Compound 10 was
obtained according to procedure A from 5-bromo-1-indanone (211
mg, 1.00 mmol) and 3-pyridineboronic acid (160 mg, 1.30 mmol)
after flash chromatography on silica gel (petroleum ether/ethyl
acetate, 1/1, Rf ) 0.14) as a colorless solid (146 mg, 0.69 mmol,
69%), mp 122-123 °C. LC/MS m/z 210.69 (MH+). Anal. (C14H11-
NO·0.1H2O) C, H, N.
6-(5-Methoxypyridin-3-yl)-3,4-dihydronaphthalen-2(1H)-one (11).
11 was obtained according to procedure A from 6-bromo-2-tetralone
(225 mg, 1.00 mmol) and 5-methoxy-3-pyridineboronic acid (199
mg, 1.30 mmol) after flash chromatography on silica gel (petroleum
ether/ethyl acetate, 1/1, Rf ) 0.15) as an off-white solid (133 mg,
0.53 mmol, 53%), mp 109-110 °C. LC/MS m/z 254.01 (MH+).
Anal. (C16H15NO2) C, H, N.
6-Pyridin-3-yl-3,4-dihydroquinolin-2(1H)-one (12). Compound 12
was obtained according to procedure B from 12a (2.71 g, 12.0
mmol) and 3-pyridineboronic acid (1.23 g, 10.0 mmol) after
crystallization from acetone/diethyl ether as colorless needles (2.15
g, 9.59 mmol, 96%), mp 181-183 °C. 1H NMR (500 MHz, DMSO-
d6): δ 2.49 (t, 3J ) 7.3 Hz, 2H), 2.95 (t, 3J ) 7.3 Hz, 2H), 6.95 (d,
3-(6-Ethoxy-5,6,7,8-tetrahydronaphthalen-2-yl)pyridine (4). Com-
pound 4 was obtained as described for 3 starting from 2 (270 mg,
1.20 mmol), NaH (58 mg, 1.44 mmol, 60% dispersion in oil), and
ethyl bromide (196 mg, 1.80 mmol) after flash chromatography on
silica gel (petroleum ether/ethyl acetate, 1/1, Rf ) 0.31) as a
colorless oil (198 mg, 0.78 mmol, 65%), mp (HCl salt) 186-188
°C. LC/MS m/z 254.29 (MH+). Anal. (C17H19NO·HCl·0.5H2O)
C, H, N.
6-Pyridin-3-yl-1,2,3,4-tetrahydronaphthalene-2-carbonitrile (5). Com-
pound 5 was obtained according to procedure A from 5a (130 mg,
0.55 mmol) and 3-pyridineboronic acid (88 mg, 0.72 mmol) after
flash chromatography on silica gel (petroleum ether/ethyl acetate,
1/1, Rf ) 0.17) as a colorless solid (91 mg, 0.39 mmol, 71%), mp
110-111 °C. 1H NMR (500 MHz, CDCl3): δ ) 2.12 (m, 1H),
3
3
5
3J ) 8.2 Hz, 1H), 7.43 (ddd, J ) 7.9 Hz, J ) 4.7 Hz, J ) 0.6
3
4
4
Hz, 1H), 7.51 (dd, J ) 8.2 Hz, J ) 2.2 Hz, 1H), 7.56 (d, J )
3
4
4
2.1 Hz, 1H), 8.00 (ddd, J ) 7.9 Hz, J ) 2.2 Hz, J ) 1.6 Hz,
3
4
4
1H), 8.50 (dd, J ) 4.7 Hz, J ) 1.5 Hz, 1H), 8.84 (d, J ) 2.2
Hz, 1H), 10.19 (s, 1H). 13C NMR (125 MHz, DMSO-d6): δ )
24.8, 30.3, 115.6, 123.8, 124.3, 125.6, 126.2, 130.6, 133.4, 135.2,
138.4, 147.2, 147.8, 170.2. LC/MS m/z 225.25 (MH+). Anal.
(C14H12N2O·0.1H2O) C, H, N.
5-Pyridin-3-yl-1,3-dihydro-2H-indol-2-one (13). Compound 13 was
obtained according to procedure A from 13a (159 mg, 0.75 mmol)
and 3-pyridineboronic acid (123 mg, 1.00 mmol) after crystallization

Aldosterone Synthase Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8085
from acetone/diethyl ether as colorless needles (129 mg, 0.61 mmol,
81%), mp 218-220 °C. LC/MS m/z 211.01 (MH+). Anal. (C13H10-
N2O·0.3H2O) C, H, N.
1-Methyl-6-pyridin-3-yl-3,4-dihydroquinolin-2(1H)-one (14). Com-
pound 14 was obtained according to procedure A from 14a (110
mg, 0.46 mmol) and 3-pyridineboronic acid (74 mg, 0.60 mmol)
after flash chromatography on silica gel (petroleum ether/ethyl
acetate, 2/3, Rf ) 0.07) as colorless needles (83 mg, 0.35 mmol,
76%), mp 100-101 °C. LC/MS m/z 239.80. Anal. (C15H14N2O·
0.1H2O) C, H, N.
1-Ethyl-6-pyridin-3-yl-3,4-dihydroquinolin-2(1H)-one (15). Com-
pound 15 was obtained according to procedure A from 15a (229
mg, 0.90 mmol) and 3-pyridineboronic acid (92 mg, 0.75 mmol)
after flash chromatography on silica gel (petroleum ether/ethyl
acetate, 1/1, Rf ) 0.09) and crystallization from acetone/diethyl
ether as colorless plates (125 mg, 0.50 mmol, 55%), mp 91-92
°C. LC/MS m/z 253.00 (MH+). Anal. (C16H16N2O·0.1H2O) C, H,
N.
1-(1-Methylethyl)-6-pyridin-3-yl-3,4-dihydroquinolin-2(1H)-
one (16). Compound 16 was obtained according to procedure A
from 16a (174 mg, 0.65 mmol) and 3-pyridineboronic acid (74 mg,
0.60 mmol) after flash chromatography on silica gel (petroleum
ether/ethyl acetate, 1/1, Rf ) 0.14) as a colorless solid (47 mg,
0.18 mmol, 29%), mp 100-101 °C. LC/MS m/z 267.10 (MH+).
Anal. (C17H18N2O) C, H, N.
8-Chloro-6-pyridin-3-yl-3,4-dihydroquinolin-2(1H)-one (17). To a
solution of 12 (560 mg, 2.50 mmol) in 5 mL DMF was added
N-chlorosuccinimide (368 mg, 2.75 mmol) in 5 mL DMF over a
period of 2 h at 65 °C. After additional 3 h at 65 °C, the mixture
was poured into ice water and extracted three times with ethyl
acetate. The combined organic layers were washed with water and
brine, dried over MgSO4, and the solvent was evaporated in vacuo.
17 was obtained after flash chromatography on silica gel (petroleum
ether/ethyl acetate, 3/7, Rf ) 0.15) and crystallization from acetone/
diethyl ether as colorless needles (225 mg, 0.87 mmol, 35%), mp
177-178 °C. GC/MS m/z 258.95 (M+). Anal. (C14H11ClN2O·0.1H2-
O) C, H, N.
8-Nitro-6-pyridin-3-yl-3,4-dihydroquinolin-2(1H)-one (18). Com-
pound 18 was obtained according to procedure B from 18a (1.0 g,
3.70 mmol) and 3-pyridineboronic acid (546 mg, 4.44 mmol) after
flash chromatography on silica gel (ethyl acetate, Rf ) 0.15) as
yellow needles (311 mg, 1.16 mmol, 31%), mp 187-189 °C. LC/
MS m/z 269.94 (MH+). Anal. (C14H11N3O3) C, H, N.
6-(5-Methoxypyridin-3-yl)-3,4-dihydroquinolin-2(1H)-one (19). Com-
pound 19 was obtained according to procedure A from 12a (170
mg, 0.75 mmol) and 5-methoxy-3-pyridineboronic acid (150 mg,
0.98 mmol) after crystallization from acetone/diethyl ether as
colorless needles (77 mg, 0.30 mmol, 40%), mp 213-215 °C. LC/
MS m/z 255.02 (MH+). Anal. (C15H14N2O2) C, H, N.
6-(5-methoxypyridin-3-yl)-1-methyl-3,4-dihydroquinolin-2(1H)-
one (20). Compound 20 was obtained according to procedure A
from 14a (200 mg, 0.83 mmol) and 5-methoxy-3-pyridineboronic
acid (115 g, 0.75 mmol) after crystallization from acetone/diethyl
ether as colorless needles (132 mg, 0.49 mmol, 66%), mp 158-159
°C. LC/MS m/z 268.95 (MH+). Anal. (C16H16N2O2 ·0.1H2O) C, H,
N.
crystallization from ethanol as colorless needles (75 mg, 0.33 mmol,
40%),mp232-233°C.LC/MSm/z225.74(MH+
).Anal.(C13H11N3O·
0.2H2O) C, H, N.
6-Pyridin-3-yl-3,4-dihydroquinoline-2(1H)-thione (24). Compound
24 A suspension of 12 (395 mg, 1.76 mmol) and Lawesson’s
reagent (356 mg, 0.88 mmol) in dry toluene was refluxed for 30
min under an atmosphere of nitrogen. After cooling to room
temperature, the solvent was removed in vacuo and the residue was
purified by flash chromatography on silica gel (petroleum ether/
ethyl acetate, 3/7, Rf ) 0.31) to afford 24 as yellow plates (63 mg,
0.26 mmol, 15%), mp 265-267 °C. LC/MS m/z 241.05 (MH+).
Anal. (C14H12N2S) C, H, N.
8-Chloro-6-pyridin-3-yl-3,4-dihydroquinoline-2(1H)-thione (25). Com-
pound 25 was obtained as described for 24 starting from 17 (900
mg, 3.48 mmol) and Lawesson’s reagent (985 mg, 2.44 mmol) after
flash chromatography on silica gel (ethyl acetate, Rf ) 0.26) and
crystallization from acetone/diethyl ether as yellow needles (212
mg, 0.77 mmol, 22%), mp 174-175 °C. GC/MS m/z 273.95
(M35Cl+), 275.95 (M37Cl+). Anal. (C14H11ClN2S) C, H, N.
7-Pyridin-3-yl-2H-1,4-benzothiazin-3(4H)-one (26). Compound
26 was obtained according to general procedure B from 7-bromo-
2H-1,4-benzothiazin-3(4H)-one (1.15 g, 4.71 mmol) and 3-py-
ridineboronic acid (695 mg, 5.56 mmol) after flash chromatography
on silica gel (petroleum ether/ethyl acetate, 1/1, Rf ) 0.20) and
crystallization from ethanol as colorless needles (486 mg, 2.01
mmol, 43%), mp 238-240 °C. LC/MS m/z 242.99 (MH+). Anal.
(C13H10N2OS·0.2H2O) C, H, N.
Biological Methods. 1. Enzyme Preparations. CYP17 and
CYP19 preparations were obtained by described methods: the
50000g sediment of E. coli expressing human CYP1732 and
microsomes from human placenta for CYP19.34
2. Enzyme Assays. The following enzyme assays were per-
formed as previously described: CY1732 and CYP19.34
3. Activity and Selectivity Assay Using V79 Cells. V79 MZh
11B1 and V79 MZh 11B2 cells10,28 were incubated with [4-14C]-
11-deoxycorticosterone as substrate and inhibitor in at least three
different concentrations. The enzyme reactions were stopped by
addition of ethyl acetate. After vigorous shaking and a centrifugation
step (10000g, 2 min), the steroids were extracted into the organic
phase, which was then separated. The conversion of the substrate
was analyzed by HPTLC and a phosphoimaging system as
described.
4. Inhibition of Human Hepatic CYP Enzymes. The recom-
binantly expressed enzymes from baculovirus-infected insect mi-
crosomes (Supersomes) were used and the manufacturer’s instruc-
tions (www.gentest.com) were followed.
5. In Vivo Pharmacokinetics. Animal trials were conducted
in accordance with institutional and international ethical guidelines
for the use of laboratory animals. Cassette dosing: Male Wistar
rats weighing 297-322 g (Janvier, France) were housed in a
temperature-controlled room (20-24 °C) and maintained in a 12 h
light/12 h dark cycle. Food and water were available ad libitum.
The animals were anaesthetized with a ketamine (135 mg/kg)/
xylazine (10 mg/kg) mixture and cannulated with silicone tubing
via the right jugular vein. Prior to the first blood sampling, animals
were connected to a counterbalanced system and tubing to perform
blood sampling in the freely moving rat. Separate stock solutions
(5 mg/mL) were prepared for the tested compounds in labrasol/
water (1:1; v/v), leading to a clear solution. Immediately before
application, the cassette dosing mixture was prepared by adding
equal volumes of the stock solutions to end up with a final
concentration of 1 mg/mL for each compound. The mixture was
applied perorally to 3 rats with an injection volume of 5 mL/kg
(Time 0). Blood samples (250 µL) were collected 1 h before
application and 1, 2, 4, 6, 8, and 24 h thereafter. They were
centrifuged at 650g for 10 min at 4 °C and then the plasma was
harvested and kept at -20 °C until LC/MS analysis. To 50 µL of
rat plasma sample and calibration standard 100 µL of acetonitrile
containing the internal standard was added. Samples and standards
were vigorously shaken and centrifuged for 10 min at 6000g and
6-Isoquinolin-4-yl-3,4-dihydroquinolin-2(1H)-one (21). Compound
21 was obtained according to procedure B from 12a (1.55 g, 6.85
mmol) and 4-isoquinolineboronic acid (950 mg, 5.50 mmol) after
crystallization from acetone/diethyl ether as colorless needles (800
mg, 2.92 mmol, 53%), mp 221-222 °C. LC/MS m/z 275.04 (MH+).
Anal. (C18H14N2O·0.1H2O) C, H: calcd, 5.18, found, 5.60, N.
6-Isoquinolin-4-yl-1-methyl-3,4-dihydroquinolin-2(1H)-one (22). Com-
pound 22 was obtained according to procedure A from 14a (264
mg, 1.10 mmol) and 4-isoquinolineboronic acid (172 mg, 1.00
mmol) after crystallization from acetone/diethyl ether as colorless
needles (163 mg, 0.57 mmol, 57%), mp 175-176 °C. LC/MS m/z
289.91 (MH+). Anal. (C19H16N2O) C, H, N.
6-Pyrimidin-5-yl-3,4-dihydroquinolin-2(1H)-one (23). Compound
23 was obtained according to procedure A from 12a (226 mg, 1.00
mmol) and 5-pyrimidineboronic acid (103 mg, 0.83 mmol) after

8086 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Lucas et al.
20 °C. For the test items, an additional dilution was performed by
mixing 50 µL of the particle free supernatant with 50 µL water.
An aliquot was transferred to 200 µL sampler vials and subsequently
subjected to LC-MS/MS. HPLC-MS/MS analysis and quantification
of the samples was carried out on a Surveyor-HPLC-system coupled
with a TSQ Quantum (ThermoFinnigan) triple quadrupole mass
spectrometer equipped with an electrospray interface (ESI). The
mean of absolute plasma concentrations ((SEM) was calculated
for the three rats, and the regression was performed on group mean
values. The pharmacokinetic analysis was performed using a
noncompartment model (PK Solutions 2.0, Summit Research
Services). Single dosing: the single dose experiments were per-
formed as described for the cassette dosing procedure with male
Wistar rats weighing 234-276 g (Janvier, France). Separate stock
solutions (5 mg/mL) were prepared for compound 12 in PEG400/
water/ethanol (50:40:10; v/v/v) and for compound 21 in labrasol/
water (1:1; v/v), leading to clear solutions. Compound 12 was
applied at 25 mg/kg perorally and 1 mg/kg intravenously and
compound 21 at 25 mg/kg perorally to 4 rats each. Additional blood
samples were taken 10 and 12 h after application in case of peroral
application and 0.08, 0.25, 0.50, and 0.75 h in case of intravenous
application of compound 12, respectively.
6. Plasma Protein Binding. A 10 mM test compound solution
and ketoprofen solution is prepared in acetonitrile. The test
compound solution is diluted with solvent to the 50 fold concentra-
tion (150 µM, working solution) used in the assay (3 µM). In a 1.5
mL Eppendorf vial, 3 µL of working solution are given to 147 µL
of serum (rat or human) and mixed. For recovery, the same dilutions
are done in ultrafiltrated serum. The solutions are incubated for
1 h at 37 °C. The whole samples (6 test solutions, 6 recovery
samples) are centrifuged at 8000g for 20 min using Centrifree
micropartition devices (Millipore). The 75 µL of ultrafiltrate (UF)
sample is removed for sample preparation. To 75 µL of sample or
150 µL calibration standard, 75 or 150 µL acetonitrile containing
the internal standard (ketoprofen, 1 µM) is added to precipitate
plasma proteins. Samples are then vigorously shaken (10 s) and
centrifuged for 10 min at 6000g and 20 °C. An aliquot (70 µL) of
the particle-free supernatant is subsequently subjected to LC-MS/
MS. The degree of binding to the plasma proteins (PPB) is
calculated by the following equation: % Protein binding ) (1 -
[ligandultrafiltrate]/[ligandtotal])·100.
7. Cytotoxicity. Cell viability upon drug exposure was deter-
mined using a fluorimetric alamar blue conversion assay using a
96-well plate format. Briefly, U-937 cells (human monocytic
leukemia) were seeded in growth medium into 96-well plates at a
final density of 5 × 104 cells/mL and exposed to the respective
compounds for the indicated time intervals (six replicates per
concentration). At the end of the exposure time, alamar blue
(Biosource International, Camarillo, CA) was added at 10% (v/v)
and incubated for 4 h. Fluorescence intensity was quantitated using
a Wallac Victor fluorescence plate reader (Perkin-Elmer, Wellesley,
MA) at 530 nm excitation and 590 nm emission. Relative viability
of cells was determined in relation to the untreated control. Control
wells containing compound only were included to detect potential
interference of the compound with the indicator system. Also, media
only controls were included to account for background fluorescence.
Viability of cells prior to cytotoxicity experiments was determine
by Trypan Blue staining. Cells were diluted 1:3 in 0.4% (w/v)
Trypan Blue (Sigma), and counted in a hemacytomer.
mination of the cyctotoxicity by Dr. Reiner Class, Pharmacelsus
CRO, Saarbru¨cken, as well as the investigation of the hepatic
CYP profile by Dr. Ursula Mu¨ller-Vieira, Pharmacelsus CRO,
is gratefully acknowledged.
Supporting Information Available: Aldosterone concentrations
in the individual animals at all sampling points, NMR spectroscopic
data of the target compounds 1-4, 6-11, 13-26, full experimental
details, and spectroscopic characterization of the reaction intermedi-
ates 1a, 5a, 5b, 6a, 14a-16a, elemental analysis results of
compounds 1-26. This material is available free of charge via the
Internet at http://pubs.acs.org.
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