Synthesis and evaluation of heteroaryl-substituted dihydronaphthalenes and indenes: Potent and selective inhibitors of aldosterone synthase (CYP11B2) for the treatment of congestive heart failure and myocardial fibrosis

Article abstract of DOI:10.1021/jm060055x

In this study, the synthesis and biological evaluation of heteroaryl-substituted dihydronaphthalenes and indenes (1-16) is described. The compounds were tested for activity by use of human CYP11B2 expressed in fission yeast and V79 MZh cells and for selectivity by use of human CYP11B1, CYP17, and CYP19. The most active inhibitor was the 6-methoxydihydronaphthalene 4 (IC ₅₀ = 2 nM), showing a K_i value of 1.3 nM and a competitive type of inhibition. The 5-methoxyindene 3 was found to be the most selective CYP11B2 inhibitor (IC₅₀ = 4 nM; CYP11B1 IC₅₀ = 5684 nM), which also showed only marginal inhibition of human CYP3A4 and CYP2D6. Docking and molecular dynamics studies using our homology-modeled CYP11B2 structure were performed to understand some structure-activity relationships. Caco-2 cell experiments revealed highly cell-permeable compounds, and metabolic studies with 4 using rat liver microsomes showed sufficient stability.

Full text of DOI:10.1021/jm060055x

2222
J. Med. Chem. 2006, 49, 2222-2231
Synthesis and Evaluation of Heteroaryl-Substituted Dihydronaphthalenes and Indenes: Potent
and Selective Inhibitors of Aldosterone Synthase (CYP11B2) for the Treatment of Congestive
Heart Failure and Myocardial Fibrosis
Marieke Voets,† Iris Antes,‡ Christiane Scherer,† Ursula Mu¨ller-Vieira,† Klaus Biemel,§ Sandrine Marchais-Oberwinkler,† and
Rolf W. Hartmann*,†
8.2 Pharmaceutical and Medicinal Chemistry, Saarland UniVersity, P.O. Box 15 11 50, D-66041 Saarbruecken, Germany,
Max Planck Institute for Informatics, Stuhlsatzenhausweg 85, D-66123 Saarbruecken, Germany, and Pharmacelsus CRO,
Science Park 2, D-66123 Saarbruecken, Germany
ReceiVed January 17, 2006
In this study, the synthesis and biological evaluation of heteroaryl-substituted dihydronaphthalenes and indenes
(1-16) is described. The compounds were tested for activity by use of human CYP11B2 expressed in
fission yeast and V79 MZh cells and for selectivity by use of human CYP11B1, CYP17, and CYP19. The
most active inhibitor was the 6-methoxydihydronaphthalene 4 (IC50 ) 2 nM), showing a Ki value of 1.3 nM
and a competitive type of inhibition. The 5-methoxyindene 3 was found to be the most selective CYP11B2
inhibitor (IC50 ) 4 nM; CYP11B1 IC50 ) 5684 nM), which also showed only marginal inhibition of human
CYP3A4 and CYP2D6. Docking and molecular dynamics studies using our homology-modeled CYP11B2
structure were performed to understand some structure-activity relationships. Caco-2 cell experiments
revealed highly cell-permeable compounds, and metabolic studies with 4 using rat liver microsomes showed
sufficient stability.
Introduction
inhibitors.12,13 One of the main advantages of this approach is
that nonsteroidal inhibitors are expected to have less side effects
on the endocrine system than the steroidal antagonists. Further-
more, the inhibition of aldosterone formation should be more
advantageous than interfering at the receptor level while leaving
the elevated aldosterone levels unaffected. However, it is crucial
that the inhibitors do not affect 11â-hydroxylase (CYP11B1),
the key enzyme of the glucocorticoid biosynthesis, to avoid
unwanted effects. It is quite a challenge to develop inhibitors
with selectivity toward CYP11B2, since CYP11B1 and CYP11B2
have a sequence homology of more than 93%.14
Aldosterone synthase (CYP11B2) is the key enzyme of
mineralocorticoid biosynthesis. This cytochrome P450 enzyme
catalyzes the conversion of its substrate, 11-deoxycorticosterone,
to the mineralocorticoid aldosterone.1 It is responsible for the
regulation of the salt and water household and thus for the
regulation of blood pressure. Besides the classical adrenal
biosynthetic pathway, extra-adrenal sites of aldosterone produc-
tion have been identified in brain, blood vessels, and heart.2-4
Mineralocorticoid receptors have been detected in heart tissue,
and it is known that aldosterone produced locally in the
myocardium triggers myocardial fibrosis.5,6 Thus, in addition
to the indirect effects of aldosterone on the failing heart from
sodium retention, hypervolemia, and hypertension, aldosterone
exerts also direct effects on the heart. These findings show the
importance of the blockade of aldosterone production as possible
treatment for congestive heart failure and myocardial fibrosis.
The mineralocorticoid receptor antagonists spironolactone and
eplerenone (Rales and Ephesus study) were able to reduce
mortality in heart failure patients and in patients after myocardial
infarction.7,8 The treatment with spironolactone, however, was
accompanied by severe side effects because of its poor selectiv-
ity for the aldosterone receptor.7,9 Additionally, a correlation
between the use of spironolactone and hyperkalemia-associated
mortality was observed.10 Eplerenone turned out to be more
selective, but the hyperkalemia problem can be expected to occur
as well. In 1994, aldosterone synthase was already propagated
as a novel pharmacological target by our group.11 More recently,
a new therapeutic option for the treatment of congestive heart
failure and myocardial fibrosis was suggested by us: inhibition
of aldosterone production using nonsteroidal, selective CYP11B2
Previously, we evaluated amidinohydrazones as potential
CYP11B2 inhibitors using a CYP11B2-expressing yeast cell
line and the adrenocortical tumor cell line NCI-H295R.15
However, the compounds displayed almost no inhibitory activity
toward CYP11B2. Recently, (imidazolyl- and pyridylmethyl-
ene)tetrahydronaphthalenes and -indanes I (Chart 1) were
described as highly active and in some cases selective CYP11B2
inhibitors by our group.16,17 By keeping the pharmacophore and
rigidifying the molecules, a new class of 3-pyridine-substituted
naphthalenes II was discovered, which turned out to be potent
and selective inhibitors of CYP11B2.18 The most interesting of
these was the 6-methoxy-substituted naphthalene (IC50 CYP11B2
) 6 nM, IC50 CYP11B1 ) 1577 nM, selectivity factor ) 263).18
Another way to make the pharmacophore rigid (compounds of
type III) is examined in this paper. The synthesis of these
compounds (Chart 2) and their biological activity toward the
human CYP enzymes CYP11B2, CYP11B1, CYP17, and
CYP19 are described. Molecular modeling and docking studies
using our homology-modeled CYP11B2 structure16-18 were
performed to understand some interesting structure-activity
relationships. Cell permeation properties of selected compounds
using Caco-2 cells and metabolic stability of compound 4 using
rat hepatic microsomes were also investigated.
* To whom correspondence should be addressed: phone + 49 681 302
3424; fax + 49 681 302 4386; e-mail rwh@mx.uni-saarland.de. Home-
page: http://www.uni-saarland.de/fak8/hartmann/index.htm.
† Saarland University.
‡ Max Planck Institute for Informatics.
§ Pharmacelsus CRO.
10.1021/jm060055x CCC: $33.50 © 2006 American Chemical Society
Published on Web 03/08/2006

Potent SelectiVe Inhibitors of Aldosterone Synthase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7 2223
Chart 1. Title Compounds III Derived from the Lead Compounds I and II
Chart 2. Title Compounds
Scheme 2a
a Conditions: (a) 3-pyridylacetonitrile, NaNH2, DMF, 0-80 °C, 6.5 h;
(b) NaOH, EtOH, reflux, 24 h; (c) PPA, 110 °C, 20 min; (d) NaBH4, MeOH,
0 °C to RT, 1 h; (e) CH3COOH/H2SO4, 100 °C, 1 h; (f) R′MgX, toluene,
reflux, 3 h; (g) HCl, 100 °C, 2 h.
Scheme 1a
An alternative synthetic route, based on the method by Bencze
and Barsky,20 was used for the synthesis of the 3-pyridine-
substituted compounds 3 and 6-10 (Scheme 2). The alkylation
of 3-pyridylacetonitrile with the brominated alkyl derivatives
3v, 6v, and 8v-10v yielded the nitriles 3iv, 6iv, and 8iv-10iv.
Hydrolysis with NaOH resulted in the acids 3iii, 6iii, and 8iii-
10iii, which were cyclized to the corresponding indanones and
tetralones 3ii, 6ii, and 8ii-10ii by treatment with PPA. The
reduction of the ketones 3ii and 8ii-10ii with NaBH4 to the
alcohols 3i and 8i-10i was followed by dehydration with
sulfuric and acetic acids to give the desired indenes and
dihydronaphthalenes 3 and 8-10. The Grignard reaction of the
tetralone 6ii with an alkylmagnesium halogenide and subsequent
dehydration of the resulting alcohols 6i and 7i with HCl yielded
the 1-alkyl-substituted dihydronaphthalenes 6 and 7. The isomers
of the intermediates 3iv, 6iv, and 8iv-10iv to 3i, 6i, and 8i-
10i were not separated but used as mixtures for the next reaction
steps. The end products 8-10 were obtained as racemic
mixtures.
The synthesis of the 1-imidazole-substituted indenes and
dihydronaphthalenes 12-14 is shown in Scheme 3.21 The
R-bromoketones 2iii, 4iii, and 12iii were stirred with imidazole
for 24 h to give the imidazole-substituted ketones 12ii-14ii.
The reduction with NaBH4 to the corresponding alcohols 12i-
14i and the subsequent acid-catalyzed dehydration yielded the
1-imidazolyl compounds 12-14. The intermediates 12ii-14ii
and 12i-14i were used as racemates or diastereomeric mictures,
respectively. The (E)- and (Z)-3-styrylpyridines 15 and 16 were
synthesized by a modified Wittig reaction (Scheme 4). The
benzyl alcohol 15ii was transformed into the phosphonium salt
15i, which reacted with 3-pyridinecarbaldehyde in the presence
of K2CO3 as a base and 18-crown-6 as phase-transfer catalyst.
a Conditions: (a)TBA Br3, CH2Cl2/MeOH, RT, 0.5-1 h; (b) NaBH4,
MeOH, 0 °C to RT, 1 h; (c) pTSA, toluene, reflux, 2 h; (d) 3- or
4-pyridylboronic acid, Na2CO3, Pd(PPh3)4, DME, 80 °C, 16 h.
Chemistry
The pyridine-substituted indenes and dihydronaphthalenes 1,
2, 4, 5, and 11 were synthesized by the route shown in Scheme
1. The R-bromoketones 2iii, 4iii, and 5iii, synthesized by
reacting the corresponding ketone 2iv, 4iv, or 5iv with tetrabu-
tylammonium tribromide at room temperature, were reduced
with NaBH4. The resulting alcohols 2ii, 4ii, and 5ii and the
commercially available 2-bromo-1-indanol 1ii were refluxed in
toluene with a catalytic amount of pTSA to yield the dehydrated
compounds 1i, 2i, 4i, and 5i. The last step was the Suzuki cross-
coupling with a 3- or 4-pyridylboronic acid in the presence of
sodium carbonate and tetrakis(triphenylphosphine)palladium as
a catalyst.19 The intermediates 2iii, 4iii, and 5iii and 1ii, 2ii,
4ii, and 5ii have one or two chiral centers. The resulting isomers
were not separated and the racemic mixtures were used for the
next reaction steps.

2224 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7
Voets et al.
Scheme 3a
a Conditions: (a) imidazole, DMF, RT, 24 h; (b) NaBH4, MeOH, 0 °C to RT, 1 h; (c) CH3COOH, H2SO4, 100 °C, 1 h.
Scheme 4a
Selected compounds with less than 60% inhibition in the yeast
assay (10, 11, 13, 14, and 16) also showed only moderate or
no activity at all in the CYP11B2 expressing V79 cells (IC50
values in the range between 176 and 2834 nM). An interesting
finding is that the Z-isomer 16 exhibited some selectivity for
CYP11B1 as compared to CYP11B2 with a 2.5-fold lower IC50
for the 11â-hydroxylase.
By use of V79 cells, selected compounds (1-4) were tested
for their type of CYP11B2 inhibition. They turned out to be
competitive inhibitors (in Figure 1, compound 3 is shown as
an example) and revealed Ki values of 8.4, 4.6, 2.6, and 1.3
nM, respectively (KM value of deoxycorticosterone ) 185 nM).
Inhibition of Human CYP17, CYP19, and Hepatic CYPs
in Vitro (Table 1). The selectivity of the compounds toward
other steroidogenic CYP enzymes (CYP19, producing estrogens,
and CYP17, forming androgens) was investigated. The IC50
values of the compounds for CYP19 were determined in vitro
by use of human placental microsomes and [1-3H]androstene-
dione as substrate as described by Thompson and Siiteri25 with
our modification.26 All the compounds showed low or no
inhibitory activity with IC50 values in the range between 814
and >36 000 nM, thus being much less active than the reference
fadrozole (IC50 ) 30 nM).
The percent inhibition values of the compounds toward
CYP17 were determined in vitro with progesterone as substrate
and the 50000g sediment of Escherichia coli recombinantly
expressing human CYP17.27,28 The indenes 1 and 3 and the 3-
and 4-methyl-3,4-dihydronaphthalenes 8 and 9 displayed a
weaker inhibition at a concentration of 2.5 µM than the reference
ketoconazole (40%), which is an antimycotic and an unspecific
inhibitor of several CYPs. The 3,4-dihydronaphthalenes 2, 4,
and 7 showed similar or slightly higher inhibition (37-62%).
The most potent CYP17 inhibitor was the 1-methyl-substituted
compound 6, exhibiting an IC50 value of 1085 nM. Thus, almost
all of the tested compounds are very weak CYP17 inhibitors,
having moderate to very low percent inhibition values that
correspond to IC50 values of approximately 2500 nM or higher.
The most selective compound 3 with respect to CYP11B1
inhibition was further tested toward two crucial human hepatic
CYP enzymes: CYP3A4, being responsible for 75% of the drug
metabolism, and CYP2D6, for which a genetic polymorphism
is described. Only marginal inhibitory effects could be observed
(CYP3A4, ketoconazole IC50 ) 50 nM, 3, 7.0% ( 1.0%
inhibition at 50 nM; CYP2D6, quinidine IC50 ) 20 nM, 3, 8.5%
( 2.2% inhibition at 20 nM)
a Conditions: (a) PPh3‚HBr, benzene, reflux, 12 h; (b) 3-pyridinecar-
baldehyde, K2CO3, 18-crown-6, CH2Cl2, reflux, 12 h.
The resulting E- and Z-isomers could be easily separated by
column chromatography.
Biological Results
Inhibition of Human Adrenal Corticoids Producing CYP11B1
and CYP11B2 In Vitro (Table 1). For the determination of the
inhibitory activity of our compounds toward human CYP11B2,
our screening assay was used.12 Human CYP11B2 expressing
fission yeast was incubated with [14C]deoxycorticosterone as
substrate and the inhibitor at a concentration of 500 nM. The
product formation was monitored by HPTLC using a phospho-
imager.
Most of the 3-pyridine-substituted derivatives showed a higher
inhibitory activity than the reference fadrozole (68%), an
aromatase (CYP19) inhibitor that is in use for the treatment of
breast cancer and is known to reduce aldosterone and cortisol
levels in adrenal slices22 and in vivo.23 Only the 7-methoxy
compound 5 exhibited a moderate activity (51%), and the
racemic 4-ethyl-substituted 3,4-dihydronaphthalene 10 had little
activity (20%). In general, the 3-pyridine-substituted indenes
showed a slightly lower activity than the 3,4-dihydronaphtha-
lenes. The shift of the methoxy substituent from 6- into
7-position of the 3,4-dihydronaphthalene core reduced activity,
resulting in a moderate inhibitor 5. Introduction of a methyl
substituent in 1-, 3-, or 4-position of the 3,4-dihydronaphthalene
moiety did not change potency (6, 8, 9), but a larger ethyl
substituent in the 4-position resulted in a loss of activity (10).
The 1-imidazolylnaphthalenes 12-14 and the ring open com-
pounds 15 and 16 showed moderate activity (25-54%). The
4-pyridine-substituted dihydronaphthalene 11 had almost no
activity (15%).
The most potent compounds, showing more than 60%
inhibition in Schizosaccharomyces pombe, and a few less potent
compounds were tested for activity and selectivity in V79 MZh
cells expressing either CYP11B1 or CYP11B2.12,24 [14C]-
Deoxycorticosterone was used as substrate, and the products
were monitored as in the yeast assay. In Table 1 the IC50 values
are presented. All the compounds with more than 60% inhibition
in the yeast assay exhibited very high activity toward CYP11B2
with IC50 values in the range of 2-30 nM. They were also
highly selective by showing only moderate inhibition of
CYP11B1 (IC50 ) 503-5684 nM). Compounds 1, 2, and 4 were
over 180-fold more selective for CYP11B2 in contrast to the
reference fadrozole, which displayed a selectivity factor of 10.
The 5-methoxy-substituted indene 3 was the most selective
compound with an exceptional selectivity factor of 1421.
Computational Results
Docking and protein modeling studies were performed to
verify our homology-modeled CYP11B2 structure16-18 and to
explain interesting structure-activity relationships of two imi-
dazolyl and one pyridyl compound (Table 2). Previously,
compound 23 was found to be a highly active inhibitor of
CYP11B2.18 In this study, we observed that the corresponding
dihydronaphthalene compound 13 shows a much lower activity.

Potent SelectiVe Inhibitors of Aldosterone Synthase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7 2225
Table 1. Inhibition of Human Adrenal CYP11B1 and CYP11B2, Human CYP17 and Human CYP19 In Vitro
% inhibitiona
IC50 valueb (nM)
IC50 valuec (nM)
% inhibitiond
humane
CYP11B2
V79 11B1f
CYP11B1
V79 11B2g
CYP11B2
humanh
CYP19
humani
CYP17
selectivityj
factor
compd
n
R
R′
R′′
1
2
3
4
5
6
7
8
0
1
0
1
1
H
H
5-OMe
6-OMe
7-OMe
90
95
66
98
51
94
81
84
82
20
15
33
34
35
25
54
36
68
2391
1729
5684
578
nd
1268
2117
503
1291
1615
2529
nd
639
763
nd
288
224
10
13
7
4
2
45
7
>36 000
4073
>36 000
814
nd
2
37
7
184
247
1421
275
62
nd
69k
57
27
23
nd
nd
nd
nd
nd
nd
nd
nd
5
Me
Et
H
H
H
H
H
3-Me
4-Me
4-Et
6045
1507
1787
3551
nd
nd
nd
nd
nd
nd
nd
40
30
166
71
97
99
9
30
5
9
13
176
2834
448
334
411
nd
735
81
1
10
11
12
13
14
15
16
1
0
1
1
H
H
2
2
6-OMe
E-isomer
Z-isomer
0.4
10
ketoconazole
fadrozole
a Mean value of four determinations, standard deviation less than 10%. b Mean value of four determinations, standard deviation less than 20%. nd ) not
determined. c Mean value of four determinations, standard deviation less than 5%. d Mean value of four determinations, standard deviation less than 10%.
nd ) not determined. e S. pombe expressing human CYP11B2; substrate deoxycorticosterone, 100 nM; inhibitor, 500 nM. f Hamster fibroblasts expressing
human CYP11B1; substrate deoxycorticosterone, 100 nM. g Hamster fibroblasts expressing human CYP11B2; substrate deoxycorticosterone, 100 nM. h Human
placental CYP19; 1 mg/mL protein; substrate androstenedione, 500 nM. i E.coli expressing human CYP17; 5 mg/mL protein; substrate progesterone, 2.5
µM; inhibitor, 2.5 µM. j IC50 CYP11B1/IC50 CYP11B2. k IC50 CYP17 ) 1085 nM.
Table 2. IC50 Values for CYP11B2 Inhibition of Selected Compounds
amino acid side-chain interactions, a distortion of the Fe-N
binding geometry can be observed. The corresponding highly
active naphthalene compound 23, shown in Figure 2c, obviously
forms phenyl-CH3 interactions with both aromatic rings of the
naphthalene and Leu382, Thr318, Leu227, and Phe231. How-
ever, the imidazole moiety is forming only the Fe-N interaction,
and no distortion of the Fe-N binding geometry by interactions
with binding pocket residues was found. In Figure 2a, the
docked complex of compound 2 is presented. Its binding pattern
is the same as for compound 13. However, due to the larger
size of the pyridine ring and the different geometry, the
dihydronaphthalene moiety of the inhibitor is placed differently
in the pocket, leading to interactions with other amino acids
(Val129 and Ala313 instead of Leu227, Phe231, and Leu382).
Its Fe-N interaction is not distorted as it is with compound
13. The main difference between the binding patterns of
compounds 2/23 and compound 13 is the distortion of the Fe-N
Figure 1. Lineweaver-Burk plot of 3 indicating competitive type of
inhibition: ([) no inhibitor; (2) 3, 3 nM; (9) 3, 15 nM.
However, its corresponding pyridyl compound 2 is again highly
active. To explain this interesting phenomenon, compounds 2,
13, and 23 were docked into the CYP11B2 model and the
structural and energetic aspects of the protein-inhibitor interac-
tions of the energetically most favorable docked complexes were
analyzed. Subsequently the dynamics of the complexes were
studied through molecular dynamics simulations of the docked
complexes. In Figure 2, the best scoring docking poses for the
three compounds are shown. The ligand-protein interactions
are indicated by green lines. Figure 2b shows the least active
compound 13. In addition to the Fe-N interaction with the heme
group, it forms aromatic-hydrophobic interactions between its
phenyl and imidazolyl ring and the binding pocket amino acids
Leu227/Phe231 and Leu382/Thr318. Due to the imidazolyl/

2226 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7
Voets et al.
Figure 2. Structure of the CYP11B2 binding pocket with the docked inhibitors 2 (a), 13 (b), and 23 (c). The structures shown correspond to the
docking poses with the lowest energy score, and all amino acids of the binding pocket that interact with the inhibitors are given. The inhibitors are
presented in yellow, nitrogen atoms are colored blue, and oxygen atoms are in red. The green lines correspond to the protein-inhibitor interactions
found by FlexX.
Table 3. Caco-2 Cell Permeation of Highly Active and Selective
CYP11B2 Inhibitors by a Multiple Dosing Approach
Papp (×10-6 cm/s)a,b
compd
multiple dosing ( SD
1
2
3
4
6
7
8
9
6.6 ( 0.5
14.6 ( 0.6
12.5 ( 0.2
13.5 ( 0.6
3.4 ( 0.1
21.6 ( 1.6
14.7 ( 0.6
16.3 ( 3.0
0.1 ( 0.03
25.7 ( 0.5
9.4 ( 0.2
<LOD
Figure 3. Superimposition of the pyridine-substituted compounds 2,
4, 6, 8, and 9 (gray) and the imidazole-substituted compounds 12-14
(yellow).
atenolol
ketoprofene
testosterone
erythromycin
interaction in the latter case, which indicates that this might be
the main reason for the lower activity of compound 13.
The same tendency as observed for the IC50 values (Table 2)
can be seen for the energy scores of the docking poses shown
in Figure 2, which are -50.137 for compound 2, -42.739 for
compound 13, and -48.116 for compound 23. However, these
energies cannot solely explain the large difference in the IC50
values of compounds 2/23 and compound 13. Obviously, not
static interactions alone but also the dynamics of the system
must be considered. Consequently, molecular dynamics simula-
tions were performed for the docked complexes of compounds
2 and 13. Identical simulations had been carried out for
compound 23 in a previous study,18 and a correlation between
the stability of the docked poses during molecular dynamics
simulations and the IC50 values of 23 and other inhibitors was
observed. These results were confirmed in the present work:
During the 1 ns simulations, compound 13 moved away (∼4
Å) from the heme cofactor, whereas compound 2 stayed close
to its docked position, as did compound 23 in the previous
work.18 This separates the three complexes in two classes:
compound 2 and 23 form a strong and dynamically stable Fe-
inhibitor interaction, whereas compound 13 does not. To further
explain the differences in the orientation of the pyridyl and
imidazolyl compounds in the binding pocket, superimposition
of these two classes of compounds was performed. From Figure
3 it is clear that when the lone pairs of the heterocyclic nitrogens
are pointing in the same direction, the corresponding dihy-
dronaphthalene cores are located with a different angle toward
the plane of the heme.
a Permeability of research compounds was classified according to
described reference compounds: testosterone29 (high), atenolol30 (low),
ketoprofene31 (high), and erythromycin32 (not detectable). b Mean value of
three determinations.
throughput of the Caco-2 assay, a multiple dosing approach was
used for the test compounds. Compound mixtures are admin-
istered, and by use of reference compounds,29-32 the inhibitors
can be classified as having low, medium, or high cell-
permeability. All the tested compounds had medium to high
cell permeability with Papp values in the range between 3.4 and
15.1 × 10-6 cm/s (Table 3).
The 6-methoxydihydronaphthalene 4, a highly active and
selective CYP11B2 inhibitor with favorable cell permeability
properties, was selected to be further tested for its metabolic
stability in rat liver microsomes. Samples were taken at various
time points to determine the remaining parent compound. The
assay showed that compound 4 has a half-life of 67 min, which
is an indication that the compound shows reasonable stability.
By use of the supernatant of the incubation, the major
metabolites of compound 4 were isolated and characterized by
liquid chromatography-tandem mass spectrometry (LC-MS/
MS). The fragmentation of the metabolite with [M + H]+ at
m/z 224 suggests that 4 has undergone O-demethylation,
resulting in the formation of the corresponding naphthol. Two
other major metabolites with [M + H]+ at m/z 254 bear an
additional oxygen atom in their dihydronaphthalene structure.
Caco-2 Cell Permeability Screening and Metabolic Stabil-
ity. Selected compounds (1-4 and 6-9) that showed high
CYP11B2 inhibitory activity and selectivity toward other CYP
enzymes were further screened for cell permeability properties
on Caco-2 monolayers. The Caco-2 cells are a human epithelial
colon carcinoma cell line and a suitable in vitro model for the
evaluation of intestinal absorption of drugs.29 To increase the
Discussion and Conclusion
Recently, we had reported that 3-pyridyl-substituted naph-
thalenes (II, Chart 1) are potent and selective CYP11B2
inhibitors.18 In this study, it is demonstrated that, in the class
of pyridyl- and 1-imidazolyldihydronaphthalenes and -indenes,
even more potent and selective compounds could be discovered.

Potent SelectiVe Inhibitors of Aldosterone Synthase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7 2227
The dihydronaphthalene compounds exhibited in general a
slightly higher activity than the corresponding indenes. The
3-pyridyl compounds exceeded the imidazolyl compounds and
especially the 4-pyridyl derivative. The most active inhibitor,
compound 4, exhibited a very low IC50 value of 2 nM. It is the
most potent compound described so far. The methoxy-
substituted indene 3 was very potent (IC50 value of 4 nM) and
the most selective compound toward CYP11B1. It showed an
extraordinary selectivity factor of 1421, which is very remark-
able since the amino acid homology between CYP11B2 and
B1 is more than 93%.14 Compound 3 affects the hepatic CYP
enzymes CYP3A and CYP2D6 only marginally and, just like
the other compounds of this paper, shows inhibition of the other
steroidogenic CYP enzymes, CYP17 and CYP19, only at very
high concentrations. Interestingly, compound 16 turned out to
be a slightly selective CYP11B1 inhibitor, being 2.5 times more
active toward CYP11B1 than CYP11B2. CYP11B1 inhibitors
could be used for the treatment of Cushing’s syndrome and
metabolic syndrome.
aldosterone levels in transgenic rats overexpressing human renin
and angiotensinogen genes.33
Experimental Section
Chemical Methods. Melting points were measured on a Mettler
FP1 melting point apparatus and are uncorrected. IR spectra were
measured neat on a Bruker Vector 33FT-infrared spectrometer. 1H
NMR spectra were recorded on a Bruker DRX-500 (500 MHz)
instrument. Chemical shifts are given in parts per million (ppm),
and tetramethylsilane (TMS) was used as internal standard for
spectra obtained in CDCl3. All coupling constants (J) are given in
hertz. Mass spectra (electrospray ionization (ESI)) were measured
on a TSQ quantum (Thermo Electron Corporation) instrument.
Elemental analyses were performed at the Department of Instru-
mental Analysis and Bioanalysis, Saarland University. Reagents
and solvents were used as obtained from commercial suppliers
without further purification. Column chromatography (CC) was
performed on silica gel (70-200 µm), and the reaction progress
was determined by thin-layer chromatography (TLC) analyses on
Alugram SIL G/UV254 (Macherey-Nagel).
The following compounds were prepared according to previously
described procedures: 2-bromo-1H-indene (1i),34 [1-(bromomethyl)-
propyl]benzene (10v),35 3-pyridylboronic acid,36 and 4-pyridylbo-
ronic acid.36
Docking studies with compounds 2, 13, and 23 using our
CYP11B2 protein model led to interesting observations. The
imidazolyl-substituted dihydronaphthalene compound 13, which
is a less active inhibitor, shows a different binding behavior
than the corresponding naphthalene compound 23. The dihy-
dronaphthalene moiety forms less interactions with the protein
than the naphthalene ring. On the other hand, additional
aromatic-hydrophobic interactions are observed for the imi-
dazole ring of compound 13. To form these interactions, the
heterocycle is shifted with respect to the heme plane, weakening
the Fe-N interaction. For strong inhibitor binding, the direc-
tionality of the Fe-N interaction must be almost perpendicular
with respect to the plane of the heme. As a result of the distortion
of this interaction, 13 cannot bind as strongly as the others and
is less active. Due to the different geometry of the pyridyl
compounds, the dihydronaphthalene compound 2 is docked
differently, leading to a much smaller distortion of the Fe-N
interaction than is observed for 13. These findings are confirmed
by the energy scores of the docked poses.
General Procedure for Synthesis of Compounds 2iii, 4iii, 5iii,
and 12iii. To a solution of ketone (11.75 mmol) in dichloromethane/
methanol (140 mL/56 mL) was added TBABr3 (12.92 mmol) at
room temperature. The mixture was stirred until decoloration of
the orange solution took place. The solvent was removed in vacuo
and the obtained solid was extracted with diethyl ether. The ether
layer was dried (MgSO4), filtered, and evaporated in vacuo.
2-Bromo-3,4-dihydro-2H-naphthalen-1-one (2iii). Purification
by CC (CH2Cl2); yield 50%. 1H NMR (CDCl3) δ 2.45-2-57 (m,
2
3
2H, H-4), 2.92 (dt, 1H, J ) 17.0 Hz, J ) 4.4 Hz, H-3), 3.29-
3.36 (m, 1H, H-3′), 4.73 (t, 1H, 3J ) 4.4 Hz, H-2), 7.28 (d, 1H, 3J
) 7.6 Hz, Ar H), 7.36 (t, 1H, 3J ) 7.6 Hz, Ar H), 7.52 (td, 1H, 3J
) 7.6 Hz, 4J ) 1.3 Hz, Ar H), 8.10 (dd, 1H, 3J ) 7.9 Hz, 4J ) 1.3
Hz, Ar H). IR (cm-1): νmax 3069, 2933, 1665, 1595.
2-Bromo-6-methoxy-3,4-dihydro-2H-naphthalen-1-one (4iii).
No purification; yield 95%.
2-Bromo-7-methoxy-3,4-dihydro-2H-naphthalen-1-one (5iii).
Purification by CC (CH2Cl2); yield 60%.
Looking at the ligand-protein interactions in our homology
model, it is obvious that there exists a specific “network” of
hydrophobic groups in the binding pocket, which are important
for interaction and which have to be complemented by the
inhibitor in a way that the geometry of the Fe-N interaction is
retained. Strong binding is possible only in this case. This is
very interesting, because such specific interactions normally
consist of hydrogen bonds and the hydrophobic interactions are
supposed to play only a minor role for the exact positioning of
the ligand. In addition, our dynamics studies indicate that small
differences in this interaction network can lead to large
differences in the dynamics of the system. Thus, studying the
dynamics of the docked complexes, we could clearly distinguish
between strong (compounds 2 and 23) and weak (compound
13) inhibitors.
2-Bromoindan-1-one (12iii). Purification by CC (CH2Cl2); yield
78%.
General Procedure for Synthesis of Compounds 2ii, 4ii, and
5ii. A solution of the R-bromoketone (1.77 mmol) in anhydrous
methanol/tetrahydrofuran (1/1.6 mL) was stirred under nitrogen
atmosphere in an ice bath. NaBH4 (1.24 mmol) was added
portionwise. After being stirred for 15 min at 0 °C and 0.5 h at
room temperature, the mixture was poured into water and extracted
with diethyl ether. The organic layer was dried (MgSO4), filtered,
and evaporated in vacuo. This compound was used for further
reaction without purification.
2-Bromo-1, 2, 3, 4-tetrahydronaphthalen-1-ol (2ii). Yield 88%.
1H NMR (CDCl3) δ 2.28-2.34 (m, 1H, H-3), 2.50-2.57 (m, 1H,
H-3′), 2.86-2.92 (m, 1H, H-4), 3.01-3.15 (m, 1H, H-4′), 4.71-
4.74 (m, 1H, H-2), 4.81 (d, 1H, 3J ) 3.5 Hz, H-1), 7.12-7.14 (m,
1H, Ar H), 7.25-7.28 (m, 2H, Ar H), 7.49-7.50 (m, 1H, Ar H).
IR (cm-1): νmax 3413, 3022, 2955, 2923, 2849, 1603, 1490, 1455,
1225.
2-Bromo-6-methoxy-1,2,3,4-tetrahydro-naphthalen-1-ol (4ii).
Yield 86%.
2-Bromo-7-methoxy-1,2,3,4-tetrahydro-naphthalen-1-ol (5ii).
Yield 84%.
General Procedure for Synthesis of Compounds 1i, 2i, 4i, and
5i. A mixture of the alcohol (2.44 mmol) and p-toluenesulfonic
acid (0.24 mmol) in 10 mL of toluene was heated at reflux (with
Dean-Starktrap) for 2 h to remove any water. The mixture was
washed with saturated sodium bicarbonate and water. The organic
layer was dried (MgSO4), filtered, and evaporated in vacuo.
Cell permeation screening using Caco-2 cells showed that
the 3-pyridine-substituted dihydronaphthalenes and indenes are
highly cell-permeable. This is an indication that these com-
pounds might be perorally available. A metabolic stability test
using rat hepatic microsomes revealed that the highly active
compound 4 shows a reasonable metabolic stability with a half-
life time of 67 min. Presently this compound is being further
investigated for its in vivo activity.
We expect a reduction of the plasma aldosterone level by
these compounds since FAD286sone enantiomer of the racemic
fadrozoleswas recently shown to reduce circulating and cardiac

2228 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7
Voets et al.
2-Bromo-1H-indene (1i). Purification by CC (hexane); yield
47%. 1H NMR (CDCl3) δ 3.61 (s, 2H, H-3), 6.94 (t, 1H, 4J ) 1.9
General Procedure for Synthesis of Compounds 3iii, 6iii, and
8iii-10iii. To a solution of the nitrile compound (2.04 mmol) in 3
mL of ethanol was added sodium hydroxide (22.46 mmol) in 1
mL of water. After reflux for 24 h, some water was added and the
pH was adjusted to 5 with 2 N HCl solution and aqueous acetic
acid. The mixture was extracted with ethyl acetate and, after drying
(MgSO4) and filtration, evaporated in vacuo. This compound was
used for further reaction without purification.
3-(3-Methoxyphenyl)-2-pyridin-3-ylpropionic acid (3iii). Yield
99%. IR (cm-1): νmax 2939, 2836, 2526, 1721, 1585, 1491.
4-Phenyl-2-pyridin-3-ylbutyric acid (6iii). Yield 74%.
3-Methyl-4-phenyl-2-pyridin-3-ylbutyric acid (8iii). Yield
99%.
4-Phenyl-2-pyridin-3-ylpentanoic acid (9iii). Yield 68%.
4-Phenyl-2-pyridin-3-ylhexanoic acid (10iii). Yield 99%.
General Procedure for Synthesis of Compounds 3ii, 6ii, and
8ii-10ii. A mixture of polyphosphoric acid (2.2 g) and the acid
(0.53 g, 2.05 mmol) was stirred at 110 °C for 20 min. The reaction
was poured into ice water and neutralized with 6% NaOH solution.
The pH was adjusted to 8 with sodium bicarbonate, and the solution
was extracted with diethyl ether. The organic layer was dried
(MgSO4), filtered, and evaporated in vacuo.
3
4
Hz, H-1), 7,17 (td, 1H, J ) 7.6 Hz, J ) 1.3 Hz, Ar H), 7.24 (t,
3
3
1H, J ) 7.6 Hz, ArH), 7.31 (d, 1H, J ) 7.6 Hz, Ar H), 7.38 (d,
3
1H, J ) 7.6 Hz, Ar H). IR (cm-1): νmax 3072, 2897, 1556.
3-Bromo-1,2-dihydronaphthalene (2i). Purification by CC
(hexane); yield 81%.
3-Bromo-7-methoxy-1,2-dihydronaphthalene (4i). Purification
by CC (hexane/EtOAc, 80:20); yield 60%.
3-Bromo-6-methoxy-1,2-dihydronaphthalene (5i). Purification
by CC (hexane/EtOAc, 80:20); yield 58%.
General Procedure for Synthesis of Compounds 1, 2, 4, 5,
and 11. A mixture of the bromo compound (2.02 mmol), 3- or
4-pyridylboronic acid (2.63 mmol), sodium carbonate (4.24 mmol),
and tetrakis(triphenylphosphine)palladium (0.04 mmol) in 15 mL
of ethylene glycol dimethyl ether was heated at 80 °C overnight
under a nitrogen atmosphere. The mixture was cooled to room
temperature and water was added. The mixture was extracted with
dichloromethane and the organic layer was dried (MgSO4), filtered,
and evaporated in vacuo.
3-(1H-Inden-2-yl)pyridine (1). Purification by CC (CH2Cl2/
1
MeOH, 99:1); yield 51%, mp 102 °C. H NMR (CDCl3): δ )
5-Methoxy-2-pyridin-3-ylindan-1-one (3ii). Purification by CC
3
4
3.81 (s, 2H, H-3), 7.23 (td, 1H, J ) 7.2 Hz, J ) 0.9 Hz, Ar H),
7.29-7.32 (m, 3H, H-1, Ar H, pyr H-5), 7.44 (d, 1H, 3J ) 7.2 Hz,
Ar H), 7.50 (d, 1H, 3J ) 7.2 Hz, Ar H), 7.89 (dt, 1H, 3J ) 8.1 Hz,
4J ) 1.9 Hz, pyr H-4), 8.50 (s, 1H, pyr H-6), 8.90 (s, 1H, pyr
H-2). IR (cm-1): νmax 3054, 2916, 1533. MS m/z 194 (MH+). Anal.
(C14H11N) C, H, N.
1
(CH2Cl2/MeOH, 97:3); yield 48%. H NMR (CDCl3) δ 3.20 (dd,
1H, 2J ) 17.3 Hz, 3J ) 4.1 Hz, H-3), 3.67 (dd, 1H, 2J ) 17.3 Hz,
3J ) 8.5 Hz, H-3′), 3.90-3.92 (m, 4H, OCH3, H-2), 6.96-6.98
(m, 2H, Ar H), 7.28-7.31 (m, 1H, pyr H-5), 7,57 (dt, 1H, 3J ) 8.2
Hz, 4J ) 1.9 Hz, pyr H-4), 7.74 (d, 1H, 3J ) 8.2 Hz, Ar H), 8.50-
8.51 (m, 2H, pyr H-6, pyr H-2). IR (cm-1): νmax 2945, 2841, 1702,
1599, 1261.
3-(3,4-Dihydronaphthalen-2-yl)pyridine (2). Purification by CC
(CH2Cl2/MeOH, 97:3); yield 62%.
3-(6-Methoxy-3,4-dihydronaphthalen-2-yl)pyridine (4). Puri-
fication by CC (CH2Cl2/MeOH, 99:1); yield 89%.
3-(7-Methoxy-3,4-dihydronaphthalen-2-yl)pyridine (5). The
reaction was stirred for 2 h. Purification by CC (CH2Cl2/MeOH,
99:1); yield 79%.
4-(6-Methoxy-3,4-dihydronaphthalen-2-yl)pyridine (11). The
reaction was stirred for 2 h. Purification by CC (CH2Cl2/MeOH,
97:3); yield 51%.
General Procedure for Synthesis of Compounds 3iv, 6iv, and
8iv-10iv. To a suspension of NaNH2 (9.40 mmol) in 20 mL of
anhydrous dimethylformamide (three-neck flask, reflux cooler, gas
inlet and dropping funnel with septum) under nitrogen was added
dropwise 3-pyridylacetonitrile (8.46 mmol) while the reaction
mixture was stirred and cooled with an ice bath. After being stirred
for 1 h at room temperature and 1 h at 80 °C, the mixture was
cooled with an ice bath and the corresponding brominated alkyl
derivative 3v, 6v, or 8v-10v (7.99 mmol) was added dropwise.
The reaction was stirred for 3 h at room temperature and then 1 h
at 80 °C. After cooling, an excess of water was added and the
mixture was extracted with diethyl ether. The organic layer was
washed with water, dried (MgSO4), filtered, and evaporated in
vacuo. The product was purified by column chromatography and
eluted with CH2Cl2/MeOH (99:1).
2-Pyridin-3-yl-3,4-dihydro-2H-naphthalen-1-one (6ii). Puri-
fication by CC (CH2Cl2/MeOH, 98:2); yield 90%.
3-Methyl-2-pyridin-3-yl-3,4-dihydro-2H-naphthalen-1-one (8ii).
Purification by CC (CH2Cl2/MeOH, 96:4); yield 38% (mixture of
stereoisomers).
4-Methyl-2-pyridin-3-yl-3,4-dihydro-2H-naphthalen-1-one (9ii).
Purification by CC (CH2Cl2/MeOH, 99:1); yield 98% (mixture of
stereoisomers).
4-Ethyl-2-pyridin-3-yl-3,4-dihydronaphthalen-1(2H)-one (10ii).
Purification by CC (CH2Cl2/MeOH, 97:3); yield 63% (mixture of
stereoisomers).
General Procedure for Synthesis of Compounds 3i and 8i-
10i. A solution of the ketone (1.77 mmol) in 5 mL of anhydrous
methanol was stirred under nitrogen in an ice bath. NaBH4 (3.54
mmol) was added portionwise. After being stirred for 1 h at room
temperature, the mixture was poured into water and extracted with
dichloromethane. The organic layer was dried (MgSO4), filtered,
and evaporated in vacuo. The resulting alcohol was used for further
reaction without purification.
5-Methoxy-2-pyridin-3-ylindan-1-ol (3i). Yield 53%.
3-Methyl-2-pyridin-3-yl-1,2,3,4-tetrahydronaphthalen-1-ol (8i).
Yield 72%.
4-Methyl-2-pyridin-3-yl-1,2,3,4-tetrahydronaphthalen-1-ol (9i).
Yield 45%.
3-(3-Methoxyphenyl)-2-pyridin-3-ylpropionitrile (3iv). Yield
4-Ethyl-2-pyridin-3-yl-1,2,3,4-tetrahydronaphthalen-1-ol (10i).
Yield 98%.
1
26%. H NMR (CDCl3) δ 3.13-3.25 (m, 2H, H-3), 3.76 (s, 3H,
3
4
OCH3), 4.20 (t, 1H, J ) 7.2 Hz, H-2), 6.65 (t, 1H, J ) 2.2 Hz,
Ar H), 6.67 (d, 1H, 3J ) 7.6 Hz, Ar H), 6.82 (dd, 1H, 3J ) 8.2 Hz,
General Procedure for Synthesis of Compounds 3 and 8-10.
A mixture of 1 mL of acetic acid, 0.14 mL of sulfuric acid, and
the alcohol (0.30 mmol) was stirred at 100 °C for 1 h. The reaction
was poured into ice water and basified with a 6% sodium hydroxide
solution. After extractions with dichloromethane, the organic layer
was dried (MgSO4), filtered, and evaporated in vacuo. The product
was purified by column chromatography, eluting with CH2Cl2/
MeOH (98:2).
4J ) 2.2 Hz, Ar H), 7.21 (t, 1H, J ) 8.2 Hz, Ar H), 7.43-7.46
3
3
4
(m, 1H, pyr H-5), 7,74 (dt, 1H, J ) 8.2 Hz, J ) 1.9 Hz, pyr
4
3
H-4), 8.61 (d, 1H, J ) 2.5 Hz, pyr H-2), 8.62 (dd, 1H, J ) 5.0
Hz, J ) 1.6 Hz, pyr H-6). IR (cm-1): νmax 3030, 2938, 2837,
4
2244, 1602, 1491, 1264.
4-Phenyl-2-pyridin-3-ylbutyronitrile (6iv). Yield 47% (mixture
of stereoisomers).
3-Methyl-4-phenyl-2-pyridin-3-ylbutyronitrile (8iv). Yield
29% (mixture of stereoisomers).
4-Phenyl-2-pyridin-3-ylpentanenitrile (9iv). Yield 25% (mix-
ture of stereoisomers).
4-Phenyl-2-pyridin-3-ylhexanenitrile (10iv). Purification by CC
(CH2Cl2/MeOH, 98:2); yield 59%.
3-(6-Methoxy-1H-inden-2-yl)pyridine (3). Yield 45%, mp 82
1
°C. H NMR (CDCl3) δ 3.77 (s, 2H, H-3), 3 85 (s, 3H, OCH3),
3
4
6.86 (dd, 1H, J ) 8.2 Hz, J ) 2.2 Hz, Ar H), 7.09 (d, 1H,4J )
1.5 Hz, Ar H), 7.30 (s, 1H, H-1), 7.34 (d, 1H, 3J ) 8.2 Hz, Ar H),
3
4
7.36-7.39 (m, 1H, pyr H-5), 7.94 (dt, 1H, J ) 7.9 Hz, J ) 1.5
3
4
Hz, pyr H-4), 8.46 (dd, 1H, J ) 4.9 Hz, J ) 1.5 Hz, pyr H-6),
8.85 (d, 1H, J ) 1.8 Hz, pyr H-2). IR (cm-1): νmax 3059, 2905,
4

Potent SelectiVe Inhibitors of Aldosterone Synthase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7 2229
2836, 1685, 1607, 1493, 1259. MS m/z 224 (MH+). Anal. (C15H13-
NO‚0.09H2O) C, H, N.
romethane. The organic layer was dried (MgSO4), filtered, and
evaporated in vacuo. The resulting alcohol was used for further
reaction without purification.
3-(3-Methyl-3,4-dihydronaphthalen-2-yl)pyridine (8). Yield
63%, mp 169 °C (HCl salt).
2-(1H-Imidazol-1-yl)indan-1-ol (12i). Yield 96%. IR (cm-1):
νmax 3541, 3032, 1660, 1504, 1276, 1084.
3-(4-Methyl-3,4-dihydronaphthalen-2-yl)pyridine (9). Yield
51%, mp 189 °C (HCl salt).
3-(4-Ethyl-3,4-dihydronaphthalen-2-yl)pyridine (10). Yield
55%, mp 174 °C (HCl salt).
2-(1H-Imidazol-1-yl)-1,2,3,4-tetrahydronaphthalen-1-ol (13i).
Yield 91%.
2-(1H-Imidazol-1-yl)-6-methoxy-1,2,3,4-tetrahydronaphtha-
len-1-ol (14i). Yield 83%.
General Procedure for Synthesis of Compounds 6i and 7i.
To a solution of 2-pyridin-3-yl-3,4-dihydronaphthalen-1-one 6ii
(0.15 g, 0.65 mmol) in 15 mL of dry toluene was added
alkylmagnesium chloride (3 M) in THF (0.44 mL, 1.31 mmol)
dropwise under nitrogen atmosphere. After the mixture was stirred
at reflux for 3 h and at room temperature overnight, an ammonium
chloride solution was added. The mixture was extracted with ethyl
acetate, dried (MgSO4), filtered, and evaporated in vacuo.
1-Methyl-2-pyridin-3-yl-1,2,3,4-tetrahydronaphthalen-1-ol (6i).
General Procedure for Synthesis of Compounds 12-14. A
mixture of 3 mL of acetic acid, 0.2 mL of sulfuric acid, and the
alcohol (0.50 mmol) was stirred at 100 °C for 4 h. The reaction
was poured into ice water and basified with a 6% sodium hydroxide
solution. After extractions with dichloromethane, the organic layer
was dried (MgSO4), filtered, and evaporated in vacuo.
1-(1H-Inden-2-yl)-1H-imidazole (12). Purification by CC
(CH2Cl2/MeOH, 97:3); yield 70%, mp 64 °C. 1H NMR (CDCl3) δ
3.87 (s, 2H, H-3), 6.78 (s, 1H, H-1), 7.22-7.24 (m, 2H, im H-4,
1
Purification by CC (CH2Cl2/MeOH, 98:2); yield 41%. H NMR
3
(CDCl3) δ 1.07 (s, 3H, CH3), 2.04-2.08 (m, 1H, H-3), 2.25-2.31
(m, 1H, H-3′), 2.96-2.30 (m, 2H, H-4), 3.28-3.31 (m, 2H, H-2,
OH), 7.03 (d, 1H, 3J ) 7.9 Hz, Ar H), 7.12 (t, 1H, 3J ) 7.9 Hz, Ar
Ar H), 7.30-7.33 (m, 2H, im H-5, Ar H), 7.38 (d, 1H, J ) 7.6
Hz, Ar H), 7.45 (d, 1H, 3J ) 7.6 Hz, Ar H), 8.09 (s, 1H, im H-2).
IR (cm-1): νmax 2940, 1707, 1616, 1498, 1264, 1238. MS m/z 183
(MH+).
3
3
H), 7.17 (t, 1H, J ) 7.9 Hz, Ar H), 7.56 (d, 1H, J ) 7.9 Hz, Ar
3
H), 7.69-7.71 (m, 1H, pyr H-5), 8.14 (d, 1H, J ) 7.9 Hz, pyr
1-(3,4-Dihydronaphthalen-2-yl)-1H-imidazole (13). Purifica-
tion by CC (CH2Cl2/MeOH, 98:2); yield 96%, mp 186 °C (HCl
salt).
1-(6-Methoxy-3,4-dihydronaphthalen-2-yl)-1H-imidazole (14).
Purification by CC (CH2Cl2/MeOH, 98:2); yield 54%, mp 106 °C
(HCl salt).
H-4), 8.54 (d, 1H, 3J ) 5.4 Hz, pyr H-6), 8.65 (s, 1H, pyr H-2). IR
(cm-1): νmax 3316, 3065, 2934, 1687, 1603, 1428.
1-Ethyl-2-pyridin-3-yl-1,2,3,4-tetrahydronaphthalen-1-ol (7i).
Purification by CC (CH2Cl2/MeOH, 99:1); yield 20%.
General Procedure for Synthesis of Compounds 6 and 7. A
mixture of 1 mL of HCl and the alcohol (0.30 mmol) was stirred
at 100 °C for 2 h. The reaction was poured into ice water and
basified with a 6% sodium hydroxide solution. After extractions
with dichloromethane, the organic layer was dried (MgSO4), filtered,
and evaporated in vacuo.
Synthesis of (E)- and (Z)-3-Styrylpyridine (15 and 16) A
mixture of benzyl alcohol 15ii (0.50 g, 4.62 mmol) and triph-
enylphosphonium bromide (1.59 g, 4.62 mmol) in 40 mL of toluene
was refluxed for 12 h under a nitrogen atmosphere. After cooling,
the phosphonium salt 15i was filtered off, washed with diethyl ether,
and dried in air. A suspension of the phosphonium salt 15i (0.76
g, 1.75 mmol), K2CO3 (2.42 g, 17.50 mmol), pyridine-3-carbalde-
hyde (0.19 g, 1.75 mmol) and a few milligrams of 18-crown-6 in
50 mL of anhydrous dichloromethane was refluxed for 12 h. After
being washed with water, the organic layer was dried (MgSO4),
filtered, and evaporated in vacuo. The mixture of isomers was
purified by column chromatography and eluted with hexane/ethyl
acetate (1:1).
3-(1-Methyl-3,4-dihydronaphthalen-2-yl)-pyridine (6). Puri-
fication by CC (CH2Cl2/MeOH, 97:3); yield 63%, mp 174 °C (HCl
salt). 1H NMR (CDCl3) δ 2.05 (s, 3H, CH3), 2.57 (td, 2H, 3J ) 8.2
Hz, 4J ) 1.6 Hz, H-3), 2.92 (t, 2H, 3J ) 8.2 Hz, H-4), 7.19-7.23
(m, 2H, Ar H), 7.27 (t, 1H, 3J ) 7.6 Hz, Ar H), 7.38 (d, 1H, 3J )
3
7.6 Hz, Ar H), 7.41-7.45 (m, 1H, pyr H-5), 7.72 (td, 1H, J )
4
3
4
7.92 Hz, J ) 1.6 Hz, pyr H-4), 8.54 (dd, 1H, J ) 5.0 Hz, J )
1.6 Hz, pyr H-6), 8.57 (s, 1H, pyr H-2). IR (cm-1): νmax 3024,
2937, 2831, 1602, 1487, 1408, 1250. MS m/z 222 (MH+). Anal.
(C16H15N‚HCl‚0.19H2O) C, H, N.
3-[(E)-2-Phenylvinyl]pyridine (15). Yield 38%, mp 81 °C. 1H
NMR (CDCl3) δ 7.08 (d, 1H, 3J ) 16.4 Hz, CH-Ph), 7.20 (d, 1H,
3J ) 16.4 Hz, CH-pyr), 7.31 (tt, 1H, 3J ) 7.6 Hz, 4J ) 1.3 Hz, Ar
3-(1-Ethyl-3,4-dihydronaphthalen-2-yl)-pyridine (7). Purifica-
tion by CC (CH2Cl2/MeOH, 99:1); yield 60%, mp 141 °C (HCl
salt).
3
H), 7.35-7.40 (m, 3H, pyr H-5, Ar H), 7.54 (d, 2H, J ) 8.8 Hz,
Ar H), 7.91 (dt, 1H,3J ) 7.9 Hz, 4J ) 1.6 Hz, pyr H-4), 8.50 (dd,
1H, 3J ) 4.7 Hz, 4J ) 1.6 Hz, pyr H-6), 8.75 (d, 1H, 4J ) 1.9 Hz,
pyr H-2). IR (cm-1): νmax 3022, 1566, 962. MS m/z 182 (MH+),
167, 115, 77, 51. Anal. (C13H11N) C, H, N.
General Procedure for Synthesis of Compounds 12ii-14ii.
A solution of R-bromoketone (4.79 mmol) and imidazole (23.94
mmol) in 20 mL of DMF was stirred overnight at room temperature.
The mixture was poured into ice water and extracted with
dichloromethane. After exhaustive washing with water, the organic
layer was dried (MgSO4), filtered, and evaporated in vacuo. The
product was purified by column chromatography and eluted with
CH2Cl2/MeOH (98:2).
3-[(Z)-2-Phenylvinyl]pyridine (16). Yield 52%, mp 145 °C (HCl
salt).
Biological Methods
1. Enzyme Preparations. CYP19 and CYP17 preparations were
obtained by described methods: microsomes from human placenta
for CYP1926 and the 50000g sediment of E. coli expressing human
CYP17.28
2. Enzyme Assays. The following enzyme assays were per-
formed as previously described: CYP1926 and CYP17.28
3. Fission Yeast Assay. Fission yeast expressing human
CYP11B2 (S. pombe PE1) was incubated with [4-14C]-11-deoxy-
corticosterone as substrate and inhibitor at a concentration of 500
nM.11 The enzyme reactions were stopped by addition of ethyl
acetate. The conversion of the substrate was analyzed by HPTLC
and a phosphoimaging system as described.12,18
2-(1H-Imidazol-1-yl)indan-1-one (12ii). Yield 39%, mp 64 °C.
2
3
1H NMR (CDCl3) δ 3.32 (dd, 1H, J ) 17.2 Hz, J ) 5.4 Hz,
H-3), 3.87 (dd, 1H, 2J ) 17.2 Hz, 3J ) 8.6 Hz, H-3), 5.06 (dd, 1H,
3J ) 8.6 Hz, 3J ) 5.4 Hz, H-2), 6.91 (s, 1H, im H-4), 7.12 (s, 1H,
im H-5), 7.49 (t, 1H, 3J ) 7.6 Hz, Ar H), 7.54 (d, 1H, 3J ) 7.6 Hz,
Ar H), 7.70 (s, 1H, im H-2), 7.73 (t, 1H, 3J ) 7.6 Hz, Ar H), 7.84
3
(d, 1H, J ) 7.6 Hz, Ar H). IR (cm-1): νmax 3009, 1722, 1603,
1262.
2-(1H-Imidazol-1-yl)-3,4-dihydronaphthalen-1(2H)-one (13ii).
Yield 65%, mp 71 °C.
2-(1H-Imidazol-1-yl)-6-methoxy-3,4-dihydronaphthalen-1(2H)-
one (14ii). Yield 50%, mp 106 °C.
General Procedure for Synthesis of Compounds 12i-14i. A
solution of the ketone (2.00 mmol) in 5 mL of anhydrous methanol
was stirred under nitrogen in an ice bath. NaBH4 (4.00 mmol) was
added portionwise. After being stirred for 1 h at room temperature,
the mixture was poured into water and extracted with dichlo-
4. Activity and Selectivity Assay with V79 Cells. V79 MZh
11B1 and V79 MZh 11B2 cells24 were incubated with [4-14C]-11-
deoxycorticosterone as substrate and inhibitor on at least three
different concentrations. The enzyme reactions were stopped by
addition of ethyl acetate. After vigorous shaking and a centrifugation

2230 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7
Voets et al.
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.12,18
5. Inhibition of Human Hepatic CYPs. Compound 3 was tested
for inhibition of human hepatic CYP3A4 and CYP2D6 at concen-
trations corresponding to the IC50 values of the well-known
inhibitors ketoconazole and quinidine, respectively. The recombi-
nantly expressed enzymes from baculovirus-infected insect mi-
crosomes (Supersomes) were used and the manufacturer’s instruc-
tions were followed (www.gentest.com).
permeability after each assay. All samples were analyzed by LC-
MS/MS after 1:1 dilution with buffer of the opposite transwell
chamber containing 2% acetic acid. The apparent cell permeability
coefficients (Papp) were calculated from the equation Papp ) (dQ/
dtAc0, where dQ/dt is the mass appearance rate in the acceptor
compartment, A is the surface area of the transwell membrane, and
c0 is the initial concentration in the donor compartment.
8. Metabolic Stability Assay. The assay was performed with
rat microsomes (male pool, Gentest, Woburn, MA). The incubation
solution contained a microsomal suspension of 0.15 mg of protein/
mL in 0.1 M phosphate buffer, pH 7.4, and a NADPH-regenerating
system (NADP 1 mM, glucose-6-phosphate 5 mM, glucose-6-
phosphate dehydrogenase 5 units/mL, MgCl2 5 mM). After prein-
cubation at 37 °C, the reaction was initiated by the addition of the
test compound (a stock solution of 10 mM in 100% DMSO, diluted
in 0.1 mM phosphate buffer, pH 7.4, to reach the final concentration
of 5 µM with 2% final DMSO concentration). After 0, 30, 60, 120,
and 180 min, 200 µL from the incubation was removed and added
to ethyl acetate, containing the internal standard methoxyverapamil
(5 µM), to stop the reaction. Subsequently, the samples were
vortexed for 5 min and the organic layer was evaporated in a
vacuum centrifuge, reconstituted in mobile phase, and analyzed by
LC-MS/MS. The percentage of the remaining test compound was
plotted against the corresponding time points, and the half-life time
was calculated by using a standard fit of the data.
9. Metabolite Detection. The metabolites were identified by
comparison of the total ion chromatograms between the incubation
of time point zero and after 180 min. Once quasi-molecular ions
were detected, they were subjected to further MS/MS analysis. The
product ion MS/MS spectra of the parent compound were subse-
quently compared with the corresponding fragmentation pattern of
putative metabolite structures. The specific fragment ion that
showed a shift in its m/z was used for metabolite identification.
6. Modeling and Docking. By use of the recently resolved
human cytochrome CYP2C9 structure (PDB code1OG5)37 as
template, a homology model was build and refined for CYP11B2
as described in refs 16-18. In this study selected compounds were
docked into the refined homology model by use of FlexX-Pharm.38
Pharmacophore constraints were applied to ensure the right binding
mode of the inhibitors with the heme cofactor. For this purpose
the standard Fe-N interaction parameters of FlexX39,40 were
modified and a directed heme-Fe-N interaction was defined
perpendicular to the heme plane. The constraint requires the
existence of an inhibitor-nitrogen-atom on the surface of an
interaction cone with a 20° radius, which has its origin at the Fe
atom and points perpendicular to the heme plane (with a length of
2.2 Å). Only docking solutions were accepted, which fulfill this
constraint. For all other ligand-protein interactions, the standard
FlexX interaction parameters and geometries were used.39,40 The
protein-ligand interactions were analyzed with the FlexX soft-
ware.39,40 The docked protein-inhibitor complex structures were
used as a starting point for the subsequent molecular dynamics
simulations, which were performed by applying the GROMOS96
force field41 and the GROMACS program.42 For the heme group
the GROMOS96 force field parameters were used as provided with
the GROMACS program package. A cutoff of 14 Å was used for
the nonbonded interactions, and a time step of 1 fs was applied.
The temperature was maintained by weak coupling to an external
bath with a temperature coupling relaxation time of 0.1 ps.43
Throughout the simulations, the bond lengths were constrained to
ideal values by use of the LINCS procedure. No explicit solvent
was included, because of the mainly hydrophobic character of the
binding pocket. Potential solvent molecules were approximated
through a dielectric constant of 4.0. Harmonic restraints were
applied to all backbone atoms outside the binding pocket (all
residues except residues 106-133, 212-221, 244-262, 305-332,
372-384, and 483-494). The systems were heated from 0 to 300
K over 200 ps and afterward 800 ps of molecular dynamics were
performed at 300 K.
Acknowledgment. We thank the Deutsche Forschungsge-
meinschaft (Ha 1513/6), the Saarland Ministry of Education,
Culture and Science (ETTT Project), and the Fonds der
Chemischen Industrie for financial support. C.S. is grateful to
the European Postgraduate School 532 (DFG) for a scholarship.
We thank Ms. Martina Palzer and Ms. Anja Palusczak for their
help in performing the in vitro tests. Thanks are due to Professor
Rita Bernhardt, Saarland University, and Organon NV, Oss, The
Netherlands, for supplying us with the V79 cells.
Supporting Information Available: Elemental analysis results
of compounds 1-17 and spectroscopic data of synthesized com-
pounds 2, 4, 5, 7-11, 13, 14, 16, 2i, 4i, 5i, 7i-10i, 13i, 14i, 3ii-
6ii, 8ii-10ii, 14ii, 4iii-6iii, 8iii, 10iii, 12iii, 6iv, and 8iv-10iv.
This material is available free of charge via the Internet at http://
pubs.acs.org.
Regarding the superposition of Figure 3, the compounds were
first generated in Hyperchem and the geometry was optimized in
the steepest descent mode. Subsequently, all molecules were
transferred to DS Viewer Pro. These compounds were aligned by
use of three fixed atoms: the nitrogen of the heterocycle and the
two adjacent carbons.
References
(1) Kawamoto, T.; Mitsuuchi, Y.; Toda, K., Yokoyama, Y.; Miyakara,
K.; Miura, S.; Onishi, T.; Ichihawa, Y.; Nakao, K.; Imura, H.; Ulick,
S.; Shizuta, Y. Role of steroid 11â-hydroxylase and steroid 18-
hydroxylase in the biosynthesis of glucocorticoids and mineralocor-
ticoids in humans. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 1458-
1462.
(2) Gomez-Sanchez, C. E.; Zhou, M. Y.; Cozza, E. N.; Morita, H.;
Foecking, M. F.; Gomez-Sanchez, E. P. Aldosterone biosynthesis in
the rat brain. Endocrinology 1997, 138, 3369-3373.
(3) Hatakeyama, H.; Miyamori, I.; Fujita, T.; Takeda, Y.; Takeda, R.;
Yamamoto, H. Vascular aldosterone. J. Biol. Chem. 1994, 269,
24316-24320.
(4) White, P. C. Aldosterone: Direct effects on and production by the
heart. J. Clin. Endocrinol. Metab. 2003, 88, 2376-2383.
(5) Satoh, M.; Nakamura, M.; Saitoh, H.; Satoh, H.; Akatsu, T.; Iwasaka,
J.; Masuda, T.; Hiramori, K. Aldosterone synthase (CYP11B2)
expression and myocardial fibrosis in the failing human heart. Clin.
Sci. 2002, 102, 381-386.
(6) Sylvestre, J.-S.; Robert, V.; Heymes, C.; Aupetit-Faisant, B.; Mouas,
C.; Moalic, J.-M.; Swynghedauw, B.; Delcayre, C. Myocardial
production of aldosterone and corticosterone in the rat. J. Biol. Chem.
1998, 273, 4883-4891.
7. Caco-2 Transport Experiments. Caco-2 cell culture and
transport experiments were performed according to Yee29 with small
modifications. Cell culture time was reduced to 10 days by
increasing seeding density from 6.3 × 104 to 1.65 × 105 cells/cm2.
Four reference compounds, atenolol, ketoprofene, testosterone, and
erythromycin, were used in each assay for validation of the transport
properties of the Caco-2 cells. The compounds were applied to the
cells as mixtures (cassette dosing) to increase the throughput of
the cell permeability tests. The starting concentration of the
compounds in the donor compartment was 50 µM in buffer
containing either 1% ethanol or dimethyl sulfoxide (DMSO). After
a preincubation step of 20 min at 37 °C, the reaction was started.
The 12-well Transwellplates (Corning Costar) were stirred (20 rpm)
at 37 °C. Samples were taken from the acceptor side after 60, 120,
and 180 min and from the donor side after 0 and 180 min. Each
experiment was run in triplicate. Monolayer integrity was checked
by measuring the transepithelial electrical resistance (TEER) before
the transport experiments and by measuring lucifer yellow cell

Potent SelectiVe Inhibitors of Aldosterone Synthase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7 2231
(7) Pitt, B.; Zannad, F.; Remme, W. J.; Cody, R.; Castaigne, A.; Perez,
A., Palensky, J.; Wittes, J. The effect of spironolactone on morbidity
and mortality in patients with severe heart failure. N. Engl. J. Med.
1999, 341, 709-717.
(8) Pitt, B.; Remme, W. J.; Zannad, F.; Neaton, J.; Martinez, F.; Roniker,
B.; Bittman, R.; Hurley, S.; Kleiman, J.; Gatlin, M. Eplerenone, a
selective aldosterone blocker, in patients with left ventricular
dysfunction after myocardial infarction. N. Engl. J. Med. 2003, 348,
1309-21.
(9) Khan, N. U. A.; Movahed, A. The role of aldosterone and aldosterone-
receptor antagonists in heart failure. ReV. CardioVasc. Med. 2004,
5, 71-81.
(10) Juurlink, D. N.; Mamdani, M. M.; Lee, D. S.; Kopp, A.; Austin, P.
C.; Laupacis, A.; Redelmeier, D. A. Rates of hyperkalemia after
publication of the Randomized Aldactone Evaluation Study. N. Engl.
J. Med. 2004, 351, 543-551.
(11) Hartmann, R. W. Selective inhibition of steroidogenic P450 en-
zymes: current status and future perspectives. Eur. J. Pharm. Sci.
1994, 2, 15-16.
(12) Ehmer, P. B.; Bureik, M.; Bernhardt, R.; Mu¨ller, U.; Hartmann, R.
W. Development of a test system for inhibitors of human aldosterone
synthase (CYP11B2): Screening in fission yeast and evaluation of
selectivity in V79 cells. J. Steroid Biochem. Mol. Biol. 2002, 81,
173-179.
(13) Hartmann, R. W.; Mu¨ller, U.; Ehmer, P. B. Discovery of selective
CYP11B2 (aldosterone synthase) inhibitors for the therapy of
congestive heart failure and myocardial fibrosis. Eur. J. Med. Chem.
2003, 38, 363-366.
(26) Hartmann, R. W.; Batzl, C. Aromatase inhibitors. Synthesis and
evaluation of mammary tumor inhibiting activity of 3-alkylated 3-(4-
aminophenyl)piperidine-2,6-diones. J. Med. Chem. 1986, 29, 1362-
1369.
(27) Ehmer, P. B.; Jose, J.; Hartmann, R. W. Development of a simple
and rapid assay for the evaluation of inhibitors of human 17R-
hydroxylase-C17,20-lyase (P450c17) by coexpression of P450c17 with
NADPH-cytochrome-P450-reductase in Escherichia coli. J. Steroid
Biochem. Mol. Biol. 2000, 75, 57-63.
(28) Hutschenreuter, T. U.; Ehmer, P. B.; Hartmann, R. W. Synthesis of
hydroxy derivatives of highly potent nonsteroidal CYP17 inhibitors
as potential metabolites and evaluation of their activity by a non
cellular assay using recombinant enzyme. J. Enzyme Inhib. Med.
Chem. 2004, 19, 17-32.
(29) Yee, S. In vitro permeability across Caco-2 cells (colonic) can predict
in vivo (small intestinal) absorption in mensfact or myth. Pharm.
Res. 1997, 14, 763-766.
(30) Tannergren, C.; Langguth, P.; Hoffmann, K. J. Compound mixtures
in Caco-2 cell permeability screens as a means to increase screening
capacity. Pharmazie 2001, 56, 337-342.
(31) Laitinen, L.; Kangas, H.; Kaukonen, A. M.; Hakala, K.; Kotiaho,
R.; Kostiainen, R.; Hirvonen, J. N-in-one permeability studies of
heterogeneous sets of compounds across Caco-2 cell monolayers.
Pharm. Res. 2003, 20, 187-197.
(32) Saito, H.; Fukasawa, Y.; Otsubo, Y.; Yamada, K.; Sezaki, H.;
Yamashita, S. Carrier-mediated transport of macrolide antimicrobial
agents across Caco-2 cell monolayers. Pharm. Res. 2000, 17, 761-
765.
(33) Fiebeler, A.; Nussberger, J.; Shagdarsuren, E.; Rong, S.; Hilfenhaus,
G.; Al-Saadi, N.; Dechend, R.; Wellner, M.; Meiners, S.; Maser-
Gluth, C.; Jeng, A. Y.; Webb, R. L.; Luft, F. C.; Muller, D. N.
Aldosterone synthase inhibitor ameliorates angiotensin II-induced
organ damage. Circulation 2005, 111, 3087-3094.
(14) Taymans, S. E.; Pack, S.; Pak, E.; Torpy, D. J.; Zhuang, Z.; Stratakis,
C. A. Human CYP11B2 (aldosterone synthase) maps to chromosome
8q24.3. J. Clin. Endocrinol. Metab. 1998, 83, 1033-1036.
(15) Voets, M.; Mu¨ller-Vieira, U.; Marchais-Oberwinkler, S.; Hartmann,
R. W. Synthesis of amidinohydrazones and evaluation of their
inhibitory effect towards aldosterone synthase (CYP11B2) and the
formation of selected steroids. Arch. Pharm. Pharm. Med. Chem.
2004, 337, 411-416.
(16) Ulmschneider, S.; Mu¨ller-Vieira, U.; Mitrenga, M.; Hartmann, R.
W.; Oberwinkler-Marchais, S.; Klein, C. D.; Bureik, M.; Bernhardt,
R.; Antes, I.; Lengauer, T. Synthesis and evaluation of imidazolyl-
methylene-tetrahydronaphthalenes and imidazolylmethylene-in-
danes: Potent inhibitors of aldosterone synthase. J. Med. Chem. 2005,
48, 1796-1805.
(17) Ulmschneider, S.; Mu¨ller-Vieira, U.; Klein, C. D.; Antes, I.; Lengauer,
T.; Hartmann, R. W. Synthesis and evaluation of (pyridylmethylene)-
tetrahydronaphthalenes/-indanes and structurally modified deriva-
tives: Potent and selective inhibitors of aldosterone synthase. J. Med.
Chem. 2005, 48, 1563-1575.
(18) Voets, M.; Antes, I.; Scherer, C.; Mu¨ller-Vieira, U.; Biemel, K.;
Barassin, C.; Oberwinkler-Marchais, S.; Hartmann, R. W. Heteroaryl
substituted naphthalenes and structurally modified derivatives: Selec-
tive inhibitors of CYP11B2 for the treatment of congestive heart
failure and myocardial fibrosis. J. Med. Chem. 2005, 48. 6632-6642.
(19) Miyaura, N.; Suzuki, N. Palladium-catalyzed cross-coupling reactions
of organoboron compounds. Chem. ReV. 1995, 95, 2457-2483.
(20) Bencze, W. L.; Barsky, L. I. Selective adrenal cortical and gonadal
inhibitors. J. Med. Pharm. Chem. 1962, 5, 1298-1306.
(21) Cozzi, P.; Branzoli, U.; Ferti, C.; Pillan, A.; Severino, D.; Tonani,
R. N-Imidazolyl derivatives of the naphthalene and chroman rings
as thromboxane A2 synthase inhibitors. Eur. J. Med. Chem. 1991,
26, 423-433.
(22) Ha¨usler, A.; Monnet, G.; Borer, C.; Bhatnagar, A. S. Evidence that
corticosterone is not an obligatory intermediate in aldosterone
biosynthesis in the rat adrenal. J. Steroid Biochem. 1989, 34, 567-
570.
(23) Demers, L. M.; Melby, J. C.; Wilson, T. E.; Lipton, A.; Harvey, H.
A.; Santen, R. J. The effects of CGS 16949A, an aromatase inhibitor,
on adrenal mineralocorticoid biosynthesis. J. Clin. Endocrinol. Metab.
1990, 70, 1162-1166.
(24) Denner, K.; Doehmer, J.; Bernhardt, R. Cloning of CYP11B1 and
CYP11B2 from normal human adrenal and their functional expression
in COS-7 and V79 chinese hamster cells. Endocr. Res. 1995, 21,
443-448.
(25) Thompson, E. A.; Siiteri, P. K. Utilization of oxygen and reduced
nicotinamide adenine dinucleotide phosphate by human placental
microsomes during aromatization of androstenedione. J. Biol. Chem.
1974, 249, 5364-5372.
(34) Lindley, W. A.; MacDowell, D. W. H. Keto-enol tautomerism in
the thiophene analogues of naphthacen-5-one. J. Org. Chem. 1982,
47, 705-709.
(35) Hauser, C. R.; Skell, P. S.; Bright, R. D.; Renfrow, W. B. Removal
of hydrogen bromide from certain â-phenylalkyl bromides by means
of potassium amide in liquid ammonia. J. Am. Chem. Soc. 1947, 69,
589-593.
(36) Li, W.; Nelson, D. P.; Jensen, M. S.; Hoerrner, R. S.; Cai, D.; Larsen,
R. D.; Reider, P. J. An improved protocol for the preparation of
3-pyridyl- and some arylboronic acids. J. Org. Chem. 2002, 67,
5394-5397.
(37) Williams, P. A.; Cosme, J.; Ward, A.; Angove, H. C.; Matak
Vinkovic, D.; Jhoti, H. Crystal structure of human cytochrome
P4502C9 with bound warfarin. Nature 2003, 424, 464-468.
(38) Hindle, S. A.; Rarey, M.; Buning, C.; Lengauer, T. Flexible docking
under pharmacophore type constraints. J. Comput.-Aided Mol. Des.
2002, 16, 129-149.
(39) Rarey, M.; Kramer, B.; Lengauer, T.; Klebe G. A fast flexible docking
method using an incremental construction algorithm. J. Mol. Biol.
1996, 261, 470-489.
(40) Rarey, M.; Kramer, B.; Lengauer, T. Multiple automatic base
selection: Protein-ligand docking based on incremental construction
without manual intervention. J. Comput.-Aided Mol. Des. 1997, 11,
369-384.
(41) Scott, W. R. P.; Hu¨nenberger, P. H.; Tironi, I. G.; Mark, A. E.;
Billeter, S. R.; Fennen, J.; Torda, A. E.; Huber, T.; Kru¨ger, P.; van
Gunsteren, W. F. The GROMOS Biomolecular Simulation Program
Package. J. Phys. Chem. A 1999, 103, 3596-3607.
(42) Lindahl, E.; Hess, B.; van der Spoel, D. GROMACS 3.0: a package
for molecular simulation and trajectory analysis. J. Mol. Mod. 2001,
7, 306-317.
(43) Berendsen, H. J. C.; Postma, J. P. M.; van Gunsteren, W. F.; Hermans,
J. Interaction models for water in relation to protein hydration. In
Intermolecular Forces; Pullman, B., Ed.; Reidel Publishing Co.:
Dordrecht, The Netherlands, 1981; pp 331-342.
JM060055X