Overcoming undesirable CYP1A2 inhibition of pyridylnaphthalene-type aldosterone synthase inhibitors: Influence of heteroaryl derivatization on potency and selectivity

Article abstract of DOI:10.1021/jm800377h

Recently, we reported on the development of potent and selective inhibitors of aldosterone synthase (CYP11B2) for the treatment of congestive heart failure and myocardial fibrosis. A major drawback of these nonsteroidal compounds was a strong inhibition of the hepatic drug-metabolizing enzyme CYP1A2. In the present study, we examined the influence of substituents in the heterocycle of lead structures with a naphthalene molecular scaffold to overcome this unwanted side effect. With respect to CYP11B2 inhibition, some substituents induced a dramatic increase in inhibitory potency. The methoxyalkyl derivatives 22 and 26 are the most potent CYP11B2 inhibitors up to now (IC₅₀ = 0.2 nM). Most compounds also clearly discriminated between CYP11B2 and CYP11B1, and the CYP1A2 potency significantly decreased in some cases (e.g., isoquinoline derivative 30 displayed only 6% CYP1A2 inhibition at 2 μM concentration). Furthermore, isoquinoline derivative 28 proved to be capable of passing the gastrointestinal tract and reached the general circulation after peroral administration to male Wistar rats.

Full text of DOI:10.1021/jm800377h

5064
J. Med. Chem. 2008, 51, 5064–5074
Overcoming Undesirable CYP1A2 Inhibition of Pyridylnaphthalene-Type Aldosterone Synthase
Inhibitors: Influence of Heteroaryl Derivatization on Potency and Selectivity
Ralf Heim,† Simon Lucas,† Cornelia M. Grombein,† Christina Ries,† Katarzyna E. Schewe,† Matthias Negri,†
Ursula Mu¨ller-Vieira,‡ 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 April 2, 2008
Recently, we reported on the development of potent and selective inhibitors of aldosterone synthase
(CYP11B2) for the treatment of congestive heart failure and myocardial fibrosis. A major drawback of
these nonsteroidal compounds was a strong inhibition of the hepatic drug-metabolizing enzyme CYP1A2.
In the present study, we examined the influence of substituents in the heterocycle of lead structures with a
naphthalene molecular scaffold to overcome this unwanted side effect. With respect to CYP11B2 inhibition,
some substituents induced a dramatic increase in inhibitory potency. The methoxyalkyl derivatives 22 and
26 are the most potent CYP11B2 inhibitors up to now (IC50 ) 0.2 nM). Most compounds also clearly
discriminated between CYP11B2 and CYP11B1, and the CYP1A2 potency significantly decreased in some
cases (e.g., isoquinoline derivative 30 displayed only 6% CYP1A2 inhibition at 2 µM concentration).
Furthermore, isoquinoline derivative 28 proved to be capable of passing the gastrointestinal tract and reached
the general circulation after peroral administration to male Wistar rats.
Chart 1. Nonsteroidal Inhibitors of CYP11B2
Introduction
The most important circulating mineralocorticoid aldosterone
is secreted by the zona glomerulosa of the adrenal gland and is
to a minor extent also synthesized in the cardiovascular system.1
The hormone plays a key role in the electrolyte and fluid
homeostasis and thus for the regulation of blood pressure. Its
biosynthesis is accomplished by the mitochondrial cytochrome
P450 enzyme aldosterone synthase (CYP11B2a) and proceeds
via catalytic oxidation of the substrate 11-deoxycorticosterone
to corticosterone and subsequently to aldosterone.2 The adrenal
aldosterone synthesis is regulated by several physiological
parameters such as the renin-angiotensin-aldosterone system
(RAAS) and the plasma potassium concentration. Chronically
elevated plasma aldosterone levels increase the blood pressure
and are closely associated with certain forms of myocardial
fibrosis and congestive heart failure.3 An insufficient renal flow
chronically activates the RAAS, and aldosterone is excessively
released. The therapeutic benefit of reducing aldosterone effects
by use of the mineralocorticoid receptor (MR) antagonists
spironolactone and eplerenone has been reported in two recent
clinical studies (RALES and EPHESUS).4,5 The studies showed
that treatment with these antagonists reduces mortality in patients
with chronic congestive heart failure and in patients after
myocardial infarction, respectively. Spironolactone, however,
showed severe side effects, presumably due to its steroidal
structure.4,6 Although the development of nonsteroidal aldos-
terone receptor antagonists has been reported recently,7 several
issues associated with the unaffected and pathophysiologically
elevated plasma aldosterone levels remain unsolved by this
therapeutic strategy such as the up-regulation of the mineralo-
corticoid receptor expression8 and nongenomic aldosterone
effects.9 A novel approach for the treatment of diseases affected
by elevated aldosterone levels is the blockade of aldosterone
biosynthesis by inhibition of CYP11B2.10,11 Aldosterone syn-
thase has previously been proposed as a potential pharmacologi-
cal target,12 and preliminary work focused on the development
of steroidal inhibitors, i.e., progesterone13 and deoxycorticos-
terone14 derivatives with unsaturated C18-substituents. These
compounds were found to be mechanism-based inhibitors
binding covalently to the active site of bovine CYP11B,
however, data on inhibitory action toward human enzyme are
essentially absent in these studies. The strategy of inhibiting
the aldosterone formation has two main advantages compared
to MR antagonism. First, there is no nonsteroidal inhibitor of a
steroidogenic CYP enzyme known to have affinity to a steroid
receptor. For this reason, fewer side effects on the endocrine
system should be expected. Furthermore, CYP11B2 inhibition
can reduce the pathologically elevated aldosterone levels,
whereas the latter remain unaffected by interfering one step later
at the receptor level. By this approach, however, it is a challenge
to reach selectivity versus other CYP enzymes. Taking into
consideration that the key enzyme of glucocorticoid biosynthesis,
11ꢀ-hydroxylase (CYP11B1) and CYP11B2 have a sequence
homology of more than 93%,15 the selectivity issue becomes
especially critical for the design of CYP11B2 inhibitors.
* 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: CYP11B2, aldosterone synthase; CYP11B1, 11ꢀ-
hydroxylase; CYP17, 17R-hydroxylase-17,20-lyase; CYP19, aromatase;
CYP, cytochrome P450; RAAS, renin-angiotensin-aldosterone system; MR,
mineralocorticoid receptor; AUC, area under the curve; MEP, molecular
electrostatic potential.
10.1021/jm800377h CCC: $40.75
2008 American Chemical Society
Published on Web 08/01/2008

CYP1A2 Inhibition of Aldosterone Synthase Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 5065
Table 1. Inhibition of Human Adrenal CYP11B2, CYP11B1, and Human CYP1A2 in Vitro
IC50 valuea (nM)
compd
1g
R1
6-OMe
6-OMe-3-Me
6-CN
6-OMe
6-OMe-3-Me
6-CN
6-OMe
6-CN
6-OMe
6-OMe
R2
V79 11B2b hCYP11B2
V79 11B1c hCYP11B1
selectivity factord
% inhibitione CYP1A2f
H
H
H
H
H
H
4′-Me
4′-Me
6.2
7.0
2.9
2.1
3.3
4.5
0.8
0.6
13
1577
1047
691
578
248
461
114
52
1521
8925
238
875
100
373
n.d.
15
41557
255
37796
199
614
31
1760
435
99
254
150
239
275
79
103
143
87
117
95
57
230
83
73
98
93
97
98
73
91
98
86
58
93
91
91
18
85
n.d.
n.d.
n.d.
80
n.d.
n.d.
93
83
92
97
78
n.d.
n.d.
57
45
6
2
3g
4g
5
6
7
8
9
4′-NH2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
fadrozole
5′-OH
94
6-OMe
5′-OMe
5′-OMe
5′-OMe
4.2
3.8
1.2
5.1
n.a.h
0.8
94
2.1
1216
6.9
9.1
0.2
22
6-OMe-3-Me
6-OMe-3-Me
6-OMe
6-OMe
6-OMe
6-OMe
6-OMe
6-OMe
6-OMe
6-OMe
6-OMe
6-OMe
6-OMe
6-OMe
6-OMe
6-OMe
6-OMe
5′-OEt
5′-COOH
5′-COOMe
5′-CONH2
5′-COMe
5′-CH2COOH
5′-CH2COOMe
5′-CH2OH
5′-CH2OMe
4′-CH2OH
4′-CH2OMe
5′-CH(OH)Me
5′-CH(OMe)Me
5′-Ph
n.d.
19
442
121
31
29
68
155
80
198
198
50
2.2
0.5
0.2
4.8
0.6
3.1
0.5
1.0
10
151
67
843
64
10
32
112
272
128
10
6-OMe-3-Me
8
a Mean value of four experiments, standard deviation usually less than 25%, n.d. ) not determined. b Hamster fibroblasts expressing human CYP11B2;
substrate deoxycorticosterone, 100 nM. c Hamster fibroblasts expressing human CYP11B1; substrate deoxycorticosterone, 100 nM. d IC50 CYP11B1/IC50
CYP11B2. e Mean value of two experiments, standard deviation less than 5%; n.d. ) not determined. f Recombinantly expressed enzyme from baculovirus-
infected insect microsomes (Supersomes); inhibitor concentration, 2.0 µM; furafylline, 55% inhibition. g These compounds were described previously.22,23
h n.a. ) no activity (7% inhibition at an inhibitor concentration of 500 nM).
Scheme 1a
These findings underline the potential therapeutic utility of
aldosterone synthase inhibition and, up to now, several structur-
ally modified fadrozole derivatives are investigated as CYP11B2
inhibitors.18,19 Recently, the development of imidazolyl- and
pyridylmethylenetetrahydronaphthalenes and -indanes as highly
active and in some cases selective CYP11B2 inhibitors has been
described by our group.20,21 By keeping the pharmacophore and
rigidization of the core structure, pyridine-substituted naphtha-
lenes22 II and dihydronaphthalenes23 III were shown to be
potent and selective CYP11B2 inhibitors (Chart 1). Combining
the structural features of these substance classes to a hybrid
core structure led to pyridine-substituted acenaphthenes as potent
CYP11B2 inhibitors with remarkable selectivity.24 Furthermore,
a Reagents and conditions: (i) Pd(PPh3)4, DMF, aq NaHCO3, µw, 150
most of the naphthalene and dihydronaphthalene type com-
°C; (ii) Tf2NPh, K2CO3, THF, µw, 120 °C; (iii) Pd(dppf)Cl2, toluene/acetone,
pounds exhibited a favorable selectivity profile versus selected
hepatic CYP enzymes. However, they turned out to be potent
inhibitors of the hepatic CYP1A2 enzyme (see examples 1, 3,
and 4 in Table 1). CYP1A2 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.25 This experimental result turned these naphthalene
type aldosterone synthase inhibitors into unsuitable drug
candidates because adverse drug-drug interactions are mainly
caused by inhibition of hepatic cytochrome P450 enzymes and
aq Na2CO3, µw, 150 °C.
The aromatase (CYP19) inhibitor fadrozole (I, Chart 1),
which is used in the therapy of breast cancer, was found to
significantly reduce the corticoid formation.16 This compound
is a potent inhibitor of CYP11B2 displaying an IC50 value of 1
nM (Table 1). The R(+)-enantiomer of fadrozole (FAD 286)
was recently shown to reduce mortality and to ameliorate
angiotensin II-induced organ damage in transgenic rats over-
expressing both the human renin and angiotensinogen genes.17

5066 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Heim et al.
Scheme 2a
Scheme 4a
a Reagents and conditions: (i) Zn(CN)2, Pd(PPh3)4, DMF, 100 °C; (ii)
Tf2NPh, KHMDS, THF/toluene, -78 °C; (iii) 3-pyridineboronic acid,
Pd(PPh3)4, DMF, aq NaHCO3, µw, 150 °C.
a Reagents and conditions: (i) conc HBr, reflux; (ii) EtBr, K2CO3, DMF,
rt; (iii) NaBH4, methanol, 0 °C.
methanol under acid catalysis afforded the corresponding methyl
esters 16 and 20. The synthesis of 6-cyanodihydronaphthalene
6 was accomplished by the sequence shown in Scheme 4. Using
6-bromo-2-tetralone, Pd-catalyzed cyanation30 led to intermedi-
ate 6b, which was transformed into the alkenyltriflate 6a by
deprotonation with KHMDS and subsequent treatment with
Tf2NPh.31 Compound 6a underwent Suzuki coupling with
3-pyridineboronic acid to afford 6. The naphthalenes 2, 12, and
29 and the dihydronaphthalenes 5, 13, and 30 were obtained as
shown in Scheme 5. The sequence for the synthesis of
intermediate 2e was described previously and was only slightly
modified by us (see Supporting Information).32 Regioselective
R-bromination was accomplished by treating 2e with CuBr2 in
refluxing ethyl acetate/CHCl3.33 After a subsequent reduction/
elimination step,23 the intermediate alkenylbromide 2b under-
went Suzuki coupling34 with the appropriate boronic acid to
afford the dihydronaphthalenes 5, 13, and 30. The corresponding
naphthalenes 2, 12, and 29 were obtained by aromatization of
2b with DDQ in refluxing toluene,35 followed by Suzuki
coupling.27 The synthesis of compounds 1, 3, and 4 has been
reported previously by our group.22,23
Scheme 3a
a Reagents and conditions: (i) NaBH4, methanol, 0 °C; (ii) MeI, NaH,
THF, rt; (iii) methanol, H2SO4, reflux.
have to be avoided in either case. In our preceding studies, the
attention was focused on the optimization of the naphthalene
skeleton; the substitution pattern of the heme complexing
3-pyridine moiety, however, was not investigated in detail.
Herein, we describe the synthesis of a series of naphthalenes
and dihydronaphthalenes with various substituents in the pyri-
dine heterocycle to examine their influence on potency and
selectivity (Table 1). The biological activity of the synthesized
compounds was determined in vitro on human CYP11B2 for
potency and human CYP11B1 and CYP1A2 for selectivity. In
addition, selected compounds were tested for inhibitory activity
at human CYP17 (17R-hydroxylase-C17,20-lyase), CYP19, and
selected hepatic CYP enzymes (CYP2B6, CYP2C9, CYP2C19,
CYP2D6, CYP3A4). The in vivo pharmacokinetic profile of
two promising compounds was determined in a cassette dosing
experiment using male Wistar rats.
Biological Results
Inhibition of Human Adrenal Corticoid Producing
CYP11B2 and CYP11B1 In Vitro (Table 1). The inhibitory
activities of the compounds were determined in V79 MZh cells
expressing either human CYP11B2 or CYP11B1.10,36 The V79
MZh cells were incubated with [14C]-deoxycorticosterone as
substrate and the inhibitor at different concentrations. The
product formation was monitored by HPTLC using a phospho-
imager. Fadrozole, an aromatase (CYP19) inhibitor with proven
ability to reduce corticoid formation in vitro and in vivo,16 was
used as a reference compound (CYP11B2, IC50 ) 1 nM;
CYP11B1, IC50 ) 10 nM).
Most of the substituted pyridylnaphthalenes showed a high
inhibitory activity at the target enzyme CYP11B2 with IC50
values in the low nanomolar range (Table 1). Some of the
compounds displayed subnanomolar potency (IC50 < 1 nM) and
turned out to be even stronger aldosterone synthase inhibitors
than the reference substance fadrozole. The methoxyalkyl
substituted compounds 22 and 26 exhibited IC50 values of 0.2
nM each. Hence, they are 5-fold more active than fadrozole
(IC50 ) 1 nM) and 30-fold more active than the unsubstituted
parent compound 1 (IC50 ) 6.2 nM). However, derivatization
by polar and acidic residues in 5′-position resulted in a decrease
in potency. This particularly applies to the carboxylic acids 15
and 19, showing no or only low inhibitory activity and to a
minor extent also to the phenolic compound 10 and the
carboxamide 17, with IC50 values of 94 nM each. Beside
introduction of small residues in 4′- and 5′-position, an extension
of the heterocyclic moiety by a condensed phenyl ring afforded
the extraordinary potent isoquinoline compounds 28-30 with
IC50 values in the range of 0.5-3.1 nM. Even the sterically
demanding 5′-phenyl residue of compound 27 was still tolerated
Results
Chemistry. The key step for the synthesis of pyridine
substituted naphthalenes was a Suzuki cross-coupling (Scheme
1).26 A microwave enhanced method developed by van der
Eycken et al. was chosen for this purpose.27 By applying this
method, various substituted bromopyridines were coupled with
6-methoxy-2-naphthaleneboronic acid to afford compounds 7,
9-11, 14, 15, 17-19, 21, 23, 27, and 28. Compound 8 was
obtained by coupling of 4-methyl-3-pyridineboronic acid with
triflate 8a, which was accessible by treating 6-cyano-2-naphthol
with Tf2NPh and K2CO3 in THF under microwave irradiation.28
The bromopyridines could be derivatized prior to Suzuki
coupling according to Scheme 2 to provide heterocycles bearing
a hydroxy, ethoxy, or hydroxymethyl substituent (10a, 14a, 21a,
and 23a).29 For compounds 21-26, the substitution pattern was
modified after the cross-coupling reaction as shown in Scheme
3 by sodium borohydride reduction and optional methylation.
Esterification of the carboxylic acids 15 and 19 by refluxing in

CYP1A2 Inhibition of Aldosterone Synthase Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 5067
Scheme 5a
a Reagents and conditions: (i) 3-methoxyphenylmagnesium bromide, THF, -5 °C; (ii) KOH, NaOH/water, reflux; (iii) H2, Pd/C, AcOH, 60 °C; (iv)
(COCl)2, CH2Cl2, rt, then AlCl3, CH2Cl2, -10 °C; (v) CuBr2, ethyl acetate/CHCl3, reflux; (vi) NaBH4, methanol, 0 °C; (vii) p-toluenesulfonic acid, toluene,
reflux; (viii) boronic acid, Pd(OAc)2, TBAB, acetone, aq Na2CO3, µw, 150 °C; (ix) DDQ, toluene, reflux; (x) boronic acid, Pd(PPh3)4, DMF, aq NaHCO3,
µw, 150 °C.
Table 2. Inhibition of Selected Steroidogenic and Hepatic CYP Enzymes in Vitro
% inhibitiona
IC50 valueb (nM)
CYP2C9e,h CYP2C19e,i
compd
CYP17c
CYP19d
CYP1A2e,f
CYP2B6e,g
CYP2D6e,j
CYP3A4e,k
9
42
41
36
39
47
14
<5
7
1420
83
488
1619
>50000
>25000
>50000
16540
48970
1888
45800
>25000
>200000
3540
11100
>25000
>200000
33110
21070
1913
9070
3540
11
18
28
>200000
1270
a Mean value of four 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 2.0 µM; ketoconazole, IC50 ) 2780 nM. 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). f Furafylline, IC50 ) 2419 nM. g Tranylcypromine, IC50 ) 6240 nM. h Sulfaphenazole, IC50 ) 318 nM. i Tranylcypromine, IC50
) 5950 nM. j Quinidine, IC50 ) 14 nM. k Ketoconazole, IC50 ) 57 nM.
(IC50 ) 4.8 nM). In general, changing the carbocyclic core
(naphthalene, 3-methyl- or dihydro-derivative) while simulta-
neously keeping the substitution pattern of the heterocycle had
only little effect on the CYP11B2 inhibition as shown by the
series 11-13 (IC50 ) 1.2-4.2 nM) and 28-30 (IC50 ) 0.5-3.1
nM). With regard to the inhibitory activity at the highly
homologous CYP11B1, most of the tested compounds were less
active than at CYP11B2. However, a noticeable inhibition with
IC50 values in the range of 10-100 nM was observed in some
cases. In particular, the 5′-methoxyalkylpyridine derivatives 22
(IC50 ) 31 nM) and 26 (IC50 ) 10 nM) as well as the methyl
ester 16 (IC50 ) 15 nM) turned out to be potent CYP11B1
inhibitors. Although introduction of substituents in the hetero-
cyclic moiety mostly resulted in a moderate decrease in
selectivity compared to the unsubstituted derivatives, the
selectivity factors were still high for most of the tested
compounds (factor 100-200). In case of 6-methoxy-3-methyl-
naphthalene 2, the introduction of substituents in the heterocyclic
moiety led to an enhanced selectivity. A methoxy substituent
in 5′-position as accomplished in compound 12 increased the
selectivity factor from 150 to 230 and the isoquinoline derivative
29 proved to be one of the most selective CYP11B2 inhibitors
of the series with a selectivity factor of 272, thus being 27-fold
more selective than fadrozole (selectivity factor ) 10).
heme-coordinating heterocycle, e.g., 1-4 exhibited more than
90% inhibition at an inhibitor concentration of 2 µM (Table 1).
With regard to the potent CYP1A2 inhibitor 1, derivatization
of the heterocycle gave rise to compounds with a slightly
reduced inhibitory potency, e.g., compounds 14, 18, 22, and
25, displaying approximately 80% inhibition. A pronounced
decrease of CYP1A2 inhibition was observed in case of
compounds 9, 13, and 29-30 (6-57%). However, the dihy-
dronaphthalenes 6, 13, and 30 turned out to be chemically
unstable and decomposition in DMSO solution was observed
after storage at 2 °C (∼80% purity after three days), yielding
considerable amounts of the aromatized analogues and traces
of unidentified degradation products. Therefore, they were not
taken into account for further biological evaluations despite their
outstanding CYP1A2 selectivity. The CYP1A2 inhibition of
some compounds was not determined at all due to either a low
CYP11B2 potency (15, 17, and 19) or low CYP11B1 selectivity
(16, 20, 26, and 27).
For a set of four structurally diverse compounds (9, 11, 18,
and 28), an extended selectivity profile including inhibition of
the steroidogenic enzymes CYP17 and CYP19 as well as
inhibition of some crucial hepatic CYP enzymes (CYP2B6,
CYP2C9, CYP2C19, CYP2D6, CYP3A4) was determined
(Table 2). The inhibition of CYP17 was determined with the
50000g sediment of the E. coli homogenate recombinantly
expressing human CYP17, progesterone (25 µM) as substrate,
and the inhibitors at a concentration of 2 µM.37 The tested
compounds turned out to be moderately potent inhibitors of
CYP17. The inhibition values ranked around 40%, correspond-
ing with IC50 values of approximately 2000 nM or higher. The
Inhibition of Hepatic and Steroidogenic CYP Enzymes
(Tables 1 and 2). To further examine the influence of heteroaryl
substitution on selectivity, the compounds were tested for
inhibition of the hepatic CYP1A2 enzyme. CYP1A2 was
strongly inhibited by all previous CYP11B2 inhibitors of the
naphthalene and dihydronaphthalene type with unsubstituted

5068 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Table 3. Pharmacokinetic Profile of Compounds 1, 9, and 28
Heim et al.
and CYP11B1 was observed: an increased or decreased inhibi-
tory activity at the one enzyme was accompanied by an
increased or decreased inhibitory activity at the other enzyme.
For instance, based on the unsubstituted parent compound 1,
introduction of the methoxyalkyl substituent in compound 26
compda
t1/2 z (h)b tmax (h)c Cmax (ng/mL)d AUC0-∞ (ng·h/mL)e
1
9
28
5.4
n.d. f
3.2
1.0
n.d. f
6.0
222
<1.5g
81
1544
n.d. f
762
fadrozole
2.2
1.0
454
3575
resulted in an enhanced inhibition of both CYP11B2 (IC50
)
a All compounds were applied perorally at a dose of 5 mg per kg body
weight in four different cassette dosing experiments using male Wistar rats.
b Terminalhalf-life.c Timeofmaximalconcentration.d Maximalconcentration.
e Area under the curve. f n.d. ) not detectable. g Below the limit of
quantification.
0.2 nM) and CYP11B1 (IC50 ) 10 nM), whereas introduction
of the hydroxy group in compound 10 resulted in a decreased
inhibitory potency at both CYP11B isoforms in a comparable
order of magnitude (CYP11B2, IC50 ) 94 nM; CYP11B1, IC50
) 8925 nM). This trend becomes particularly evident when
plotting the CYP11B2 versus the CYP11B1 pIC50 values of the
compounds presented in Table 1, revealing a reasonable linear
correlation (r2 ) 0.86, n ) 29). The finding that it is to some
extent possible to change the inhibitory potency by the heteroaryl
derivatization without significantly changing the selectivity
versus either CYP11B2 or CYP11B1 is an indication that the
inhibitor binding proceeds via similar protein-inhibitor interac-
tions of the heterocyclic moiety with both CYP11B isoforms.
Contrariwise, it has been shown earlier by us that variation of
the carbocyclic skeleton instead of the heterocycle can signifi-
cantly influence the selectivity. Therefore, no correlation is
observed for a plot of the CYP11B2 and CYP11B1 pIC50 values
of the naphthalenes22 and dihydronaphthalenes23 described
previously by us that are functionalized with an unsubstituted
3-pyridine as heme complexing heterocycle (r2 ) 0.30, n )
20). Consistent with these findings, it can be assumed that both
enzymes, CYP11B2 and CYP11B1, are structurally more
diverse in the naphthalene binding site than in the heterocyclic
binding site.
inhibition of CYP19 at an inhibitor concentration of 500 nM
was determined in vitro by use of human placental microsomes
and [1ꢀ-3H]androstenedione as substrate as described by
Thompson and Siiteri38 using our modification.39 In this assay,
no inhibition of CYP19 was observed for compounds 11, 18,
and 28. Only the amino-substituted compound 9 displayed a
moderate inhibition of 47%. The IC50 values of the compounds
for the inhibition of the hepatic CYP enzymes CYP1A2,
CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 were
determined using recombinantly expressed enzymes from bacu-
lovirus-infected insect microsomes (Supersomes). The values
of the CYP1A2 inhibition matched well the previously deter-
mined percental inhibition (Table 1). Methoxy compound 11
with 91% inhibition at 500 nM turned out to be a potent
CYP1A2 inhibitor (IC50 ) 83 nM), whereas the inhibitory
potency decreased to 488 nM in the case of the ketone derivative
18. A pronounced selectivity regarding the CYP1A2 inhibition
was observed in case of compounds 9 and 28, with IC50 values
of approximately 1.5 µM. In most cases, the other investigated
CYP enzymes were unaffected, e.g., IC50 values of 9 were
greater than 10 µM versus CYP2B6, CYP2C9, CYP2C19,
CYP2D6, and CYP3A4. (Table 2)
Pharmacokinetic Profile of Compounds 1, 9, and 28
(Table 3). The pharmacokinetic profile of compounds 9 and
28 was determined after peroral application to male Wistar rats
and compared to the unsubstituted parent compound 1. After
administration of a 5 mg/kg dose in a cassette (N ) 5), plasma
samples were collected over 24 h and plasma concentrations
were determined by HPLC-MS/MS. Fadrozole, which was used
as a reference compound, displayed the highest plasma levels
(AUC0-∞ ) 3575 ng ·h/mL), followed by 1 (1544 ng ·h/mL) and
28 (762 ng ·h/mL). At all sampling points, the amounts of 9
detected were found below the limit of quantification (1.5 ng
per mL plasma). This experimental result may be either due to
a fast metabolism of the aromatic amine or due to a lacking
ability of this compound to permeate the cell membrane under
physiological conditions. The half-lives were between 2.2-5.4
h, in which the elimination of fadrozole occurs faster than the
elimination of the naphthalenes 1 and 28. Compound 28 is
slowly absorbed as indicated by the tmax of 6 h, whereas 1 is
absorbed as fast as fadrozole (tmax ) 1 h). Furthermore, no
obvious sign of toxicity was noted in any animal over the
duration of the experiment (24 h).
Interesting structure-activity relationships could also be
observed with respect to electronic properties. Compounds
bearing protic substituents in 5′-position rather poorly inhibited
CYP11B2, whereas bioisosteric exchange by aprotic residues
gave rise to highly potent aldosterone synthase inhibitors, e.g.,
the inhibitory potency increased by a factor of 40 from
carboxamide 17 (IC50 ) 94 nM) to the ethanone 18 (IC50
)
2.1 nM). A comparable increase of potency was observed when
the protic hydroxy group was replaced by the aprotic methoxy
group, e.g., the IC50 value decreased by a factor of 20 in case
of compound 11 compared to the phenol 10 or by a factor of
40 in case of compound 22 compared to the primary alcohol
21. Similarly, the methyl esters 16 and 20 were more active
than the corresponding carboxylic acids 15 and 19. However, a
lack of membrane permeability must be taken into consideration
as an alternative explanation. Figure 1 shows the molecular
electrostatic potentials (MEP) mapped on the electron density
surface of compounds 17, 10, and 21 and their bioisosteric
analogues 18, 11, and 22. Both the shape and the geometry of
the compounds as well as the electrostatic potential distribution
in the naphthalene moiety are very similar. In addition, all
compounds contain a region in which the nitrogen of the
pyridine ring presents a negative potential. However, areas with
a distinct positive potential in the pyridine moiety are present
in compounds 17, 10, and 21, showing low inhibitory potency.
In case of the highly potent bioisosters, these areas display less
positive potential values with a more uniformly distributed
electron charge. Hence, the difference in the electrostatic
potential distribution is a reasonable explanation for the varying
binding behavior within this set of compounds.
Discussion and Conclusion
The results obtained in the present study revealed that a
variety of substituents in 4′- and 5′-position is tolerated with
regard to the CYP11B2 potency. Most of the tested compounds
were more potent than the unsubstituted parent compounds, and
IC50 values less than 1 nM were observed in 7 cases (e.g., 22
and 26, IC50 ) 0.2 nM). Some of the compounds were also
potent CYP11B1 inhibitors (e.g., 26, IC50 ) 10 nM). Interest-
ingly, a precise relationship between the inhibition of CYP11B2
The heteroaryl derivatization had also a noticeable influence on
the CYP1A2 potency of the compounds. Most of the substituted
derivatives were still inhibiting CYP1A2 for more than 90% at a
concentration of 2 µM. With respect to the compounds with a

CYP1A2 Inhibition of Aldosterone Synthase Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 5069
Figure 1. MEP of compounds 17, 18, 10, 11, 21, and 22 (front and back view). The electrostatic potential surfaces were plotted with GaussView
3.0 in a range of -18.83 kcal/mol (red) to +21.96 kcal/mol (blue).
6-methoxynaphthalene core, a slight decrease to approximately
80% inhibition was observed in some cases. This effect was
due to the introduction of substituents in 5′-position of the
heterocycle. While no decrease of CYP1A2 inhibition was
observed in the case of the rather small substituents in
compounds 10, 11, and 21 (hydroxy, methoxy, and hydroxy-
methyl), a slight increase of the sterical bulk in compounds 14,
18, 22, and 25 (ethoxy, acetyl, methoxymethyl, and hydroxy-
methyl) resulted in a decrease in CYP1A2 inhibition to
78-85%. On the other hand, some derivatives proved to be
significantly less active with approximately 50% inhibition of
CYP1A2, including the 4′-amino-substituted compound 9 and
the isoquinoline 28 with IC50 values of 1420 and 1619 nM,
respectively. The effect of changing 3-pyridine by 4-isoquinoline
as heme-complexing heterocycle is particularly noteworthy. The
three isoquinoline derivatives 28, 29, and 30 are considerably
less active at CYP1A2 (6-57% inhibition) than their unsub-
stituted analogues 1, 2, and 5 (73-98% inhibition). An
explanation might be found in the geometry of these molecules.
The isoquinoline constrains the rotation around the carbon-carbon
bond between the heterocycle and the naphthalene, especially
in presence of the additional ortho-methyl groups in 29 and
30. Thus, a coplanar conformation becomes energetically
disfavored compared to the pyridine analogues and the sterically
demanding heterocycle rotates out of the naphthalene plane. This
loss of planarity is a reasonable explanation for the reduced
inhibitory potency because both CYP1A2 substrates and inhibi-
tors are usually small-volume molecules with a planar shape
(e.g., caffeine40 and furafylline41). An even more drastic effect
on the CYP1A2 potency was observed in the case of the
dihydronaphthalene type compounds. While the unsubstituted
parent compound 5 exhibited 74% inhibition, introduction of
the methoxy substituent in compound 13 led to a reduction to
18% and the isoquinoline derivative 30 displayed only 6%
inhibition. The partly saturated core structure becomes flexible
and disturbs the planarity. Factors other than steric might play
an additional role for the decreased CYP1A2 inhibition, e.g.,
disturbed π-π stacking contacts with aromatic amino acids in
the CYP1A2 binding pocket due to the reduced number of
aromatic carbons. Aromaticity has been identified to correlate
positively with CYP1A2 inhibition in recent QSAR studies.42
As dihydronaphthalenes 13 and 30 were found to be unstable
in DMSO solution, the low potencies might be due to substance
degradation. However, the decomposition (∼20% after three
days) afforded mainly the aromatized naphthalene analogues,
i.e., 12 and 29, both displaying higher CYP1A2 inhibition than
13 and 30.
selectivity profile. Some substituents induced a significant
increase in inhibitory potency versus CYP11B2. Compounds
22 and 26 with subnanomolar IC50 values are the most potent
aldosterone synthase inhibitors so far. The undesirable high
CYP1A2 inhibition that is present in the previously investigated
derivatives could be overcome by certain residues, giving rise
to compounds with an advantageous overall selectivity profile.
It was also demonstrated that the naphthalene type aldosterone
synthase inhibitors 1 and 28 were able to cross the gastrointes-
tinal tract and reached the general circulation. Presently, the
elucidated concepts are used to systematically modify other lead
structures whereof some are under investigation for their ability
to reduce aldosterone levels in vivo.
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 (Hz). 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. 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 chromatography was performed on
silica gel 40 (35/40-63/70 µM) with hexane/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.
The following compounds were prepared according to previously
described procedures: 6-methoxy-3-methyl-3,4-dihydronaphthalen-
1(2H)-one (2e),32 (2E)-4-hydroxy-4-(3-methoxyphenyl)-3-methyl-
2-butenoic acid (2g),32 5-bromopyridin-3-ol (10a).29
Synthesis of the Target Compounds. Procedure A.27 Pyridine
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 a 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
In conclusion, we have shown that modifying the lead
compounds I and II by introduction of substituents in the
heterocyclic moiety has a clear effect on the activity and

5070 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Heim et al.
products were obtained after flash chromatography on silica gel
(petroleum ether/ethyl acetate mixtures) and/or crystallization. If
an oil was obtained, it was transferred into the hydrochloride salt
by 1N HCl solution in diethyl ether.
matography on silica gel (petroleum ether/ethyl acetate, 7/3, Rf )
0.10) pure 7 was obtained as a white solid (65 mg, 0.26 mmol,
52%), mp (HCl salt) 172-174 °C. MS m/z 250.30 (MH+). Anal.
(C17H15NO·HCl·0.1H2O) C, H, N.
Procedure B.34 In a microwave tube, alkenyl bromide 7 (1
equiv), pyridine boronic acid (1.3 equiv), tetrabutylammonium
bromide (1 equiv), sodium carbonate (3.5 equiv), and palladium
acetate (1.5 mol %) were suspended in water/acetone 3.5/3 to give
a 0.15 M solution of bromide 7 under an atmosphere of nitrogen.
The septum-sealed vessel was irradiated under stirring and simul-
taneous cooling for 15 min at 150 °C with an initial irradiation
power of 150 W. The reaction mixture was cooled to room
temperature, diluted with a saturated ammonium chloride solution,
and extracted several times with diethyl ether. The combined
extracts were washed with brine, dried over MgSO4, concentrated,
and purified by flash chromatography on silica gel. The resulting
oil was transferred into the hydrochloride salt by a 5-6 N HCl
solution in 2-propanol and crystallized from ethanol.
6-(4-Methylpyridin-3-yl)-2-naphthonitrile (8). Triflate 8a (151
mg, 0.50 mmol), 4-methyl-3-pyridineboronic acid (89 mg, 0.65
mmol), K2CO3 (138 mg, 1.0 mmol), and Pd(dppf)Cl2 (37 mg, 0.05
mmol) were suspended in 4.0 mL of a 4:4:1 mixture of toluene/
acetone/water. This mixture was heated to 125 °C by microwave
irradiation for 15 min (initial irradiation power 150 W). After
cooling to room temperature, 15 mL of distilled water were added
and the reaction mixture was extracted four times with 10 mL of
Et2O. After washing the combined organic fractions with water
(twice) and brine, drying over MgSO4, and evaporation of the
solvent crude product, 8 was obtained as a yellow solid (127 mg).
Further purification by flash chromatography on silica gel (petro-
leum ether/ethyl acetate, 2/5, Rf ) 0.20) and subsequent crystal-
lization of the free base as hydrochloride salt gave 52 mg (0.19
mmol, 37%) of pure 8 (HCl salt) as an yellowish solid, mp (HCl
salt) decomposition above 210 °C. MS m/z 245.30 (MH+). Anal.
(C17H12N2 ·HCl·0.5H2O) C, H, N.
3-(6-Methoxynaphthalen-2-yl)pyridin-4-amine (9). The com-
pound was prepared according to procedure A starting from
6-methoxy-2-naphthaleneboronic acid (131 mg, 0.65 mmol) and
3-bromopyridin-4-amine (86 mg, 0.50 mmol). After crystallization
from acetone, pure 9 was obtained as a white solid (39 mg, 0.16
mmol, 31%), mp 155-156 °C. MS m/z 251.28 (MH+). Anal.
(C16H14N2O) C, H, N.
Procedure C. To a suspension of NaH (1.15 equiv, 60%
dispersion in oil) in 5 mL dry THF was added dropwise a solution
of alcohol (1 equiv) in 5 mL THF at room temperature under an
atmosphere of nitrogen. After hydrogen evolution ceased, a solution
of methyliodide (3.3 equiv) in 5 mL THF was added dropwise,
and the resulting mixture was stirred for 5 h at room temperature.
The mixture was then treated with saturated aqueous NH4Cl solution
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. The crude product was flash
chromatographed on silica gel (petroleum ether/ethyl acetate
mixtures) to afford the pure methylether. If an oil was obtained, it
was transferred into the hydrochloride salt by 1N HCl solution in
diethyl ether.
Procedure D. To a 0.05 M solution of carbonyl compound in
dry methanol was added sodium borohydride (2 equiv). The reaction
mixture was stirred for 1 h, diluted with diehtylether, and treated
with saturated aqueous NaHCO3 solution. The mixture was then
extracted three times with ethyl acetate, washed twice with saturated
aqueous NaHCO3 solution and once with brine, and dried over
MgSO4. The filtrate was concentrated in vacuo, and the residue
was filtered through a short pad of silica gel or flash chromato-
graphed on silica gel (petroleum ether/ethyl acetate mixtures) to
afford the corresponding alcohols.
3-(6-Methoxy-3-methylnaphthalen-2-yl)pyridine (2). The com-
pound was obtained according to procedure A starting from 2a (377
mg, 1.50 mmol) and 3-pyridineboronic acid (240 mg, 1.95 mmol)
after flash chromatography on silica gel (petroleum ether/ethyl
acetate, 4/1, Rf ) 0.16) as a white solid (304 mg, 1.22 mmol, 81%),
mp 106-107 °C. MS m/z 250.06 (MH+). Anal. (C17H15NO) C, H,
N.
3-(6-Methoxy-3-methyl-3,4-dihydronaphthalen-2-yl)pyridine (5).
The compound was obtained according to procedure B starting from
2b (127 mg, 0.50 mmol) and 3-pyridineboronic acid (80 mg, 0.65
mmol) after flash chromatography on silica gel (petroleum ether/
ethyl acetate, 4/1, Rf ) 0.20), precipitation as HCl salt, and
crystallization from ethanol as a white solid (50 mg, 0.17 mmol,
35%), mp (HCl salt) 186-187 °C. MS m/z 252.02 (MH+). Anal.
(C17H17NO·HCl·0.2H2O) C, H, N.
5-(6-Methoxynaphthalen-2-yl)pyridin-3-ol (10). The compound
was prepared according to procedure A starting from 6-methoxy-
2-naphthaleneboronic acid (131 mg, 0.65 mmol) and 10a (87 mg,
0.50 mmol). After crystallization from acetone/diethyl ether, pure
10 was obtained as an off-white solid (86 mg, 0.34 mmol, 68%),
mp 172-175 °C. MS m/z 252.02 (MH+). Anal. (C16H13NO2 ·
0.7H2O) C, H, N: calcd, 5.31; found, 5.79.
3-Methoxy-5-(6-methoxynaphthalen-2-yl)pyridine (11). The com-
pound was prepared according to procedure A starting from
6-methoxy-2-naphthaleneboronic acid (131 mg, 0.65 mmol) and
3-bromo-5-methoxypyridine (94 mg, 0.50 mmol). After flash
chromatography on silica gel (petroleum ether/ethyl acetate, 7/3,
Rf ) 0.10), pure 11 was obtained as a white solid (80 mg, 0.30
mmol, 60%), mp (HCl salt) 211-214 °C. 1H NMR (500 MHz,
CD3OD): δ 3.96 (s, 3H), 4.15 (s, 3H), 7.23 (dd, 3J ) 9.1 Hz, 4J )
4
3
4
2.5 Hz, 1H), 7.32 (d, J ) 2.2 Hz, 1H), 7.85 (dd, J ) 8.5 Hz, J
3
3
) 1.9 Hz, 1H), 7.92 (d, J ) 8.8 Hz, 1H), 7.97 (d, J ) 8.5 Hz,
1H), 8.29 (d, 4J ) 1.5 Hz, 1H), 8.52 (s, 1H), 8.54 (s, 1H), 8.86 (s,
1H). 13C NMR (125 MHz, CD3OD): δ 57.9, 58.3, 108.5, 120.9,
121.9, 128.1, 128.5, 130.2, 131.4, 132.5, 134.5, 136.7, 138.9, 139.1,
142.6, 158.4, 160.4. MS m/z 266.26 (MH+). Anal. (C17H15NO2 ·
HCl·0.3H2O) C, H, N.
3-Methoxy-5-(6-methoxy-3-methylnaphthalen-2-yl)pyridine (12).
The compound was obtained according to procedure A starting from
2a (377 mg, 1.50 mmol) and 5-methoxy-3-pyridineboronic acid
(298 mg, 1.95 mmol) after flash chromatography on silica gel
(petroleum ether/ethyl acetate, 3/1, Rf ) 0.16) as a white solid (328
mg, 1.17 mmol, 78%), mp 106-107 °C. 1H NMR (500 MHz,
CDCl3): δ 2.40 (s, 3H), 3.91 (s, 3H), 3.94 (s, 3H), 7.12 (m, 2H),
4
4
7.23 (dd, J ) 2.8 Hz, J ) 1.9 Hz, 1H), 7.62 (s, 1H), 7.64 (s,
1H), 7.71 (d, 3J ) 8.8 Hz, 1H), 8.28 (d, 4J ) 1.9 Hz, 1H), 8.33 (d,
4J ) 2.8 Hz, 1H). 13C NMR (125 MHz, CDCl3): δ 21.0, 55.3,
55.6, 104.9, 118.6, 121.5, 127.4, 127.5, 128.6, 129.2, 134.1, 134.4,
134.5, 135.8, 138.0, 142.6, 155.2, 158.1. MS m/z 280.08 (MH+).
Anal. (C18H17NO2) C, H, N.
6-(Pyridin-3-yl)-7,8-dihydronaphthalene-2-carbonitrile (6). The
copound was prepared according to procedure A starting from
3-pyridineboronic acid (107 mg, 0.87 mmol) and 6a (189 mg, 0.62
mmol). After flash chromatography on silica gel (petroleum ether/
ethyl acetate, 2/1, Rf ) 0.10) pure 6 was obtained as a white,
crystalline solid (100 mg, 0.43 mmol, 69%). Treatment with
hydrochloride acid (0.1 N in Et2O) yielded the hydrochloride salt
of 6 (110 mg, 0.41 mmol, 66%) as a white solid, mp (HCl salt)
264-268 °C. MS m/z 223.23 (MH+). Anal. (C16H12N2 · HCl ·
0.4H2O) C, H, N.
3-(6-Methoxynaphthalen-2-yl)-4-methylpyridine (7). The com-
pound was prepared according to procedure A starting from
6-methoxy-2-naphthaleneboronic acid (131 mg, 0.65 mmol) and
3-bromo-4-methylpyridine (86 mg, 0.50 mmol). After flash chro-
3-Methoxy-5-(6-methoxy-3-methyl-3,4-dihydronaphthalen-2-
yl)pyridine (13). The compound was obtained according to proce-
dure B starting from 2b (253 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, 4/1, Rf ) 0.14),
precipitation as HCl salt, and crystallization from ethanol as a
yellow solid (84 mg, 0.26 mmol, 26%), mp (HCl salt) 181-182
1
3
°C. H NMR (500 MHz, CD3OD): δ 0.94 (d, J ) 7.0 Hz, 3H),

CYP1A2 Inhibition of Aldosterone Synthase Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 5071
2.71 (dd, 2J ) 15.3 Hz, 3J ) 1.2 Hz, 1H), 3.02 (m, 1H), 3.13 (dd,
3-(Methoxymethyl)-5-(6-methoxynaphthalen-2-yl)pyridine (22).
The compound was prepared according to procedure C starting from
21 (150 mg, 0.57 mmol) using methyl iodide (82 µL, 1.32 mmol).
After flash chromatography on silica gel (petroleum ether/ethyl
acetate, 1/1, Rf ) 0.22), pure 22 was obtained as an off-white solid
(80 mg, 0.29 mmol, 50%), mp 121-122 °C. MS m/z 279.91 (MH+).
Anal. (C18H17NO2) C, H, N.
[4-(6-Methoxynaphthalen-2-yl)pyridin-3-yl]methanol (23). The
compound was prepared according to procedure A starting from
6-methoxy-2-naphthaleneboronic acid (131 mg, 0.65 mmol) and
23a (94 mg, 0.50 mmol). After crystallization from acetone/diethyl
ether, pure 23 was obtained as a white solid (90 mg, 0.34 mmol,
68%), mp decomposition above 240 °C. MS m/z 266.05 (MH+).
Anal. (C17H15NO2 ·0.2H2O) C, H, N.
4-(Methoxymethyl)-3-(6-methoxynaphthalen-2-yl)pyridine (24).
The compound was prepared according to procedure C starting from
23 (150 mg, 0.57 mmol) using methyl iodide (82 µL, 1.32 mmol).
After flash chromatography on silica gel (petroleum ether/ethyl
acetate, 1/1, Rf ) 0.23), pure 24 was obtained as an off-white solid
(74 mg, 0.26 mmol, 46%), mp (HCl salt) 174-177 °C. MS m/z
279.91 (MH+). Anal. (C18H17NO2 ·0.2H2O) C, H, N.
1-[5-(6-Methoxynaphthalen-2-yl)-pyridin-3-yl]ethanol (25). The
compound was prepared according to procedure D starting from
18 (50 mg, 0.18 mmol) using NaBH4 (8.0 mg, 0.21 mmol). After
flash chromatography on silica gel (petroleum ether/ethyl acetate,
1/1, Rf ) 0.24), pure 25 was obtained as a white solid (28 mg,
0.10 mmol, 56%), mp 154-155 °C. MS m/z 280.05 (MH+). Anal.
(C18H17NO2) C, H, N.
3
2J ) 15.5 Hz, J ) 6.4 Hz, 1H), 3.74 (s, 3H), 4.01 (s, 3H), 6.72
3
(m, 2H), 7.15 (d, J ) 8.2 Hz, 1H), 7.19 (s, 1H), 8.22 (m, 1H),
4
4
8.34 (d, J ) 2.4 Hz, 1H), 8.59 (d, J ) 1.5 Hz, 1H). 13C NMR
(125 MHz, CD3OD): δ 17.8, 30.6, 36.7, 55.8, 57.9, 112.9, 115.6,
116.6, 127.0, 127.2, 127.8, 129.8, 130.3, 132.3, 137.1, 137.2, 150.9,
160.1. MS m/z 281.96 (MH+). Anal. (C18H19NO2 ·HCl·0.2H2O)
C, H, N.
3-Ethoxy-5-(6-methoxynaphthalen-2-yl)pyridine (14). The com-
pound was prepared according to procedure A starting from
6-methoxy-2-naphthaleneboronic acid (131 mg, 0.65 mmol) and
14a (101 mg, 0.50 mmol). After crystallization from ethyl acetate/
petroleum ether, pure 14 was obtained as a white solid (33 mg,
0.17 mmol, 23%), mp decomposition above 130 °C. MS m/z 280.05
(MH+). Anal. (C18H17NO2 ·0.2H2O) C, H, N.
5-(6-Methoxynaphthalen-2-yl)pyridine-3-carboxylic acid (15).
The compound was prepared according to procedure A starting from
6-methoxy-2-naphthaleneboronic acid (131 mg, 0.65 mmol) and
5-bromonicotinic acid (101 mg, 0.50 mmol). After crystallization
from methanol/water, pure 15 was obtained as an off-white solid
(74 mg, 0.26 mmol, 53%), mp decomposition above 300 °C. MS
m/z 279.98 (MH+). Anal. (C17H13NO3 ·HCl·0.3H2O) C, H, N.
Methyl 5-(6-methoxynaphthalen-2-yl)pyridine-3-carboxylate (16).
Carboxylic acid 15 (45 mg, 0.16 mmol) was dissolved in 20 mL
dry methanol and 0.05 mL concentrated H2SO4 (98%) was added.
The whole mixture was refluxed for 10 h, and thereafter the excess
methanol was distilled off. The residue was taken up in 50 mL of
ethyl acetate, and the organic layer was washed several times with
5% aqueous Na2CO3 solution, water, and brine. After drying over
MgSO4, the solvent was evaporated in vacuo. After flash chroma-
tography on silica gel (petroleum ether/ethyl acetate, 7/3, Rf ) 0.34),
pure 16 was obtained as an off-white solid (28 mg, 0.10 mmol,
60%), mp 150-151 °C. MS m/z 293.97 (MH+). Anal. (C18H14NO3)
C, H, N.
3-(1-Methoxyethyl)-5-(6-methoxynaphthalen-2-yl)pyridine (26).
The compound was prepared according to procedure C starting from
25 (70 mg, 0.25 mmol) using methyl iodide (41 µL, 0.66 mmol).
After flash chromatography on silica gel (petroleum ether/ethyl
acetate, 1/1, Rf ) 0.23), pure 26 was obtained as a yellowish solid
(26 mg, 0.08 mmol, 35%), mp 124-125 °C. MS m/z 294.11 (MH+).
Anal. (C19H19NO2) C, H, N.
3-(6-Methoxynaphthalen-2-yl)-5-phenylpyridine (27). The com-
pound was prepared according to procedure A starting from
6-methoxy-2-naphthaleneboronic acid (394 mg, 1.95 mmol) and
3-bromo-5-phenylpyridine (351 mg, 1.50 mmol). After flash
chromatography on silica gel (petroleum ether/ethyl acetate, 2/1,
Rf ) 0.23), pure 27 was obtained as a white, crystalline solid (455
mg, 1.46 mmol, 97%), mp 216-217 °C. MS m/z 312.09 (MH+).
Anal. (C22H17NO·0.4H2O) C, H, N.
4-(6-Methoxynaphthalen-2-yl)isoquinoline (28). The compound
was prepared according to procedure A starting from 6-methoxy-
2-naphthaleneboronic acid (131 mg, 0.65 mmol) and 4-bromoiso-
quinoline (104 mg, 0.50 mmol). After flash chromatography on
5-(6-Methoxynaphthalen-2-yl)pyridine-3-carboxamide (17). The
compound was prepared according to procedure A starting from
6-methoxy-2-naphthaleneboronic acid (131 mg, 0.65 mmol) and
5-bromonicotinamide (92 mg, 0.50 mmol). After crystallization
from acetone/diethyl ether, pure 17 was obtained as a white solid
(55 mg, 0.20 mmol, 40%), mp 245-247 °C. MS m/z 279.07 (MH+).
Anal. (C17H14N2O2) C, H, N.
1-[5-(6-Methoxynaphthalen-2-yl)pyridin-3-yl]ethanone (18). The
compound was prepared according to procedure A starting from
6-methoxy-2-naphthaleneboronic acid (131 mg, 0.65 mmol) and
3-acetyl-5-bromopyridine (100 mg, 0.50 mmol). After crystallization
from acetone/diethyl ether, pure 18 was obtained as a white solid
(75 mg, 0.27 mmol, 54%), mp 159-160 °C. MS m/z 278.09 (MH+).
Anal. (C18H15NO2 ·0.1H2O) C, H, N.
[5-(6-Methoxynaphthalen-2-yl)pyridin-3-yl]acetic acid (19). The
compound was prepared according to procedure A starting from
6-methoxy-2-naphthaleneboronic acid (131 mg, 0.65 mmol) and
5-bromo-3-pyridineacetic acid (108 mg, 0.50 mmol). After crystal-
lization from methanol/water, pure 19 was obtained as a white solid
(70 mg, 0.24 mmol, 48%), mp decomposition above 210 °C. MS
m/z 293.97 (MH+). Anal. (C18H15NO3 ·0.5H2O) C, H, N.
Methyl [5-(6-methoxynaphthalen-2-yl)pyridin-3-yl]acetate (20).
The compound was prepared as described for 16 starting from 19
(145 mg, 0.49 mmol). After flash chromatography on silica gel
(petroleum ether/ethyl acetate, 1/1, Rf ) 0.18), pure 20 was obtained
as a white solid (81 mg, 0.26 mmol, 53%), mp 145-146 °C. MS
m/z 308.04 (MH+). Anal. (C19H17NO3) C: calcd, 74.25, found,
74.72, H, N.
silica gel (petroleum ether/ethyl acetate, 7/3, Rf
) 0.21), pure 28
was obtained as a white solid (44 mg, 0.16 mmol, 31%), mp
185-186 °C. MS m/z 286.07 (MH+). Anal. (C20H15NO·0.1H2O)
C, H, N.
4-(6-Methoxy-3-methylnaphthalen-2-yl)isoquinoline (29). The
compound was obtained according to procedure A starting from
2a (377 mg, 1.50 mmol) and 4-isoquinolineboronic acid (337 mg,
1.95 mmol) after flash chromatography on silica gel (dichlo-
romethane/methanol 99/1, Rf ) 0.26) as yellow oil, which solidified
with diethyl ether as a pale-yellow solid (178 mg, 0.59 mmol, 40%),
mp 156-157 °C. MS m/z 300.10 (MH+). Anal. (C21H11NO) C, H,
N.
4-(6-Methoxy-3-methyl-3,4-dihydronaphthalen-2-yl)isoquino-
line (30). The compound was obtained according to procedure B
starting from 2b (253 mg, 1.00 mmol) and 4-isoquinolineboronic
acid (225 mg, 1.30 mmol) after two flash chromatographical
separations on silica gel (petroleum ether/ethyl acetate, 5/1, Rf )
0.20, and dichloromethane/methanol 99/1, Rf ) 0.27) and precipita-
tion as HCl salt as a yellow solid (112 mg, 0.33 mmol, 17%), mp
149-150 °C. MS m/z 302.18 (MH+). Anal. (C21H19NO · HCl ·
0.2H2O) C, H, N.
[5-(6-Methoxynaphthalen-2-yl)pyridin-3-yl]methanol (21). The
compound was prepared according to procedure A starting from
6-methoxy-2-naphthaleneboronic acid (131 mg, 0.65 mmol) and
21a (94 mg, 0.50 mmol). After crystallization from acetone/diethyl
ether, pure 21 was obtained as a white solid (86 mg, 0.32 mmol,
65%), mp 193-194 °C. MS m/z 266.05 (MH+). Anal.
(C17H15NO2 ·0.1H2O) 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 CYP1737 and

5072 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Heim et al.
microsomes from human placenta for CYP19.39 (2) Enzyme assays.
The following enzyme assays were performed as previously
described: CY1737 and CYP19.39 (3) ActiVity and selectiVity assay
using V79 cells. V79 MZh 11B1 and V79 MZh 11B2 cells36 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.10,22 (4) Inhibition of human
hepatic CYP enzymes. The recombinantly expressed enzymes from
baculovirus-infected insect microsomes (Supersomes) were used
and the manufacturer’s instructions (www.gentest.com) were fol-
lowed. (5) In ViVo pharmacokinetics. Animal trials were conducted
in accordance with institutional and international ethical guidelines
for the use of laboratory animals. Male Wistar rats weighing
317-322 g (Janvier, France) were housed in a temperature-
controlled room (20-22 °C) and maintained in a 12 h light/12 h
dark cycle. Food and water were available ad libitum. The animals
were anaesthetised 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 five 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). Then 400 µL
of blood were taken via jugularis catheter 1 h before application
and then 1 and 2 h after application. Immediately, equal volume
(400 µL) of 0.9% NaCl (37 °C) was reinjected intravenously to
keep the blood volume stable. Then 4, 6, 8, 10 and 24 h after
application, 250 µL of blood were sampled without balancing the
blood volume. Blood samples were centrifuged at 3000g for 10
min at 4 °C. Plasma was harvested and kept at -20 °C until
analysis. 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). 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).
Computational Methods. MEP. For each docked compound,
geometry optimization was performed at the B3LYP/6-31G* density
functional levels by means of the Gaussian03 software, and the
molecular electrostatic potential (MEP) maps were plotted using
GaussView3, the 3-D molecular graphics package of Gaussian.43
These electrostatic potential surfaces were generated by mapping
6-31G* electrostatic potentials onto surfaces of molecular electron
density (isovalue ) 0.002 electron/Å).44
8a, 14a, 21a, 23a, elemental analysis results of compounds 2, 5-30.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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regression of pIC50 values, NMR-spectroscopic data of compounds
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