Selective dual inhibitors of CYP19 and CYP11B2: Targeting cardiovascular diseases hiding in the shadow of breast cancer

Article abstract of DOI:10.1021/jm3004637

Postmenopausal women are at high risk for cardiovascular diseases because of the estrogen deficiency. As for postmenopausal breast cancer patients, this risk is even higher due to inhibition of estrogens biosyntheses in peripheral tissue by the aromatase (CYP19) inhibitors applied. Because estrogen deficiency results in significantly elevated aldosterone levels, which are a major cause of cardiovascular diseases, dual inhibition of CYP19 and CYP11B2 (aldosterone synthase) is a promising treatment for breast cancer and the coinstantaneous cardiovascular diseases. By combination of important structural features of known CYP19 and CYP11B2 inhibitors, we succeeded in obtaining compounds 3 and 5 as selective dual inhibitors with IC₅₀ values around 50 and 20 nM toward CYP19 and CYP11B2, respectively. These compounds showed also good selectivity toward CYP11B1 (selectivity factors (IC_{50 CYP11B1}/IC _{50 CYP11B2}) around 50) and CYP17 (no inhibition).

Full text of DOI:10.1021/jm3004637

Article
pubs.acs.org/jmc
Selective Dual Inhibitors of CYP19 and CYP11B2: Targeting
Cardiovascular Diseases Hiding in the Shadow of Breast Cancer
Qingzhong Hu,*,† Lina Yin,†,‡ and Rolf W. Hartmann*,†
†Pharmaceutical and Medicinal Chemistry, Saarland University & Helmholtz Institute for Pharmaceutical Research Saarland (HIPS),
Campus C2-3, P.O. Box 151150, D-66123 Saarbrucken, Germany
̈
‡ElexoPharm GmbH, Campus A1, D-66123 Saarbrucken, Germany
̈
S
* Supporting Information
ABSTRACT: Postmenopausal women are at high risk for cardiovascular diseases because of the estrogen
deficiency. As for postmenopausal breast cancer patients, this risk is even higher due to inhibition of
estrogens biosyntheses in peripheral tissue by the aromatase (CYP19) inhibitors applied. Because estrogen
deficiency results in significantly elevated aldosterone levels, which are a major cause of cardiovascular
diseases, dual inhibition of CYP19 and CYP11B2 (aldosterone synthase) is a promising treatment for
breast cancer and the coinstantaneous cardiovascular diseases. By combination of important structural
features of known CYP19 and CYP11B2 inhibitors, we succeeded in obtaining compounds 3 and 5 as
selective dual inhibitors with IC50 values around 50 and 20 nM toward CYP19 and CYP11B2, respectively. These compounds
showed also good selectivity toward CYP11B1 (selectivity factors (IC50 CYP11B1/IC50 CYP11B2) around 50) and CYP17 (no
inhibition).
strated AIs as adjuvant therapeutics, significantly improving
disease-free and relapse-free survival with the overall survival
increased accordingly.6
However, it has been unveiled that only around 40% of BC
patients’ deaths are eventually caused by BC.7 A lot of people
survive BC but die of other medical conditions, especially
cardiovascular diseases (CVD).7 Therefore, there is an urgent
need to manage CVD in order to further improve the overall
survival.
Recently, estrogens have been proven to exhibit protective
effects on heart8 and kidney.9 The administration of estrogens
prevents the development of heart failure postmyocardial
infarction8b and attenuates ventricular hypertrophy and
remodelling.8c,d Moreover, the incidences of CVD in
postmenopausal women triple those of premenopausal
women at the same age,10 indicating that low estrogen levels
are closely correlated with CVD. Regarding postmenopausal
BC patients under endocrine therapy, AIs further decrease
estrogen formation, leading to an even higher risk of CVD.11
The ischemic side effects observed in clinical trials with AIs
were considered as results of lipid metabolism dysfunction
caused by estrogen deficiency11c and can be managed with
antihyperlipidemic agents. However, the up-regulation of the
renin−angiotensin−aldosterone system (RAAS) during this
pathological procedure, especially aldosterone, was still
neglected. It has been showed that the depletion of estrogens
not only directly increases circulating aldosterone levels, but
also augments the concentrations of other RAAS components12
(Chart 2). Elevated levels of renin, angiotensin II (Ang II),
angiotensin converting enzyme (ACE), and angiotensin type 1
INTRODUCTION
■
Breast cancer (BC) is the carcinoma with the highest morbidity
in females in the western countries. Although BC is still the
second leading cause of death, the mortality is significantly
reduced because of the cancer screening to identify cases in
early stages1 and, more importantly, the employment of
adjuvant endocrine therapy. Endocrine therapy is based on
the fact that estrogen stimulates the growth of “hormone
sensitive” breast cancer, in which estrogen receptor (ER) and/
or progesterone receptor (PgR) are expressed.2 Therefore,
deprivation of estrogens is a feasible treatment for the hormone
sensitive BC, which accounts for more than 60% of all cases.
Since the late 1970s, selective estrogen receptor modulators
(SERMs),3 such as Tamoxifen and Raloxifen (Chart 1), were
introduced into the clinic. These SERMs competitively bind to
ER inside breast cancer tissue, inhibiting transcription and the
subsequent mitogenic effects. However, the unsatisfying risk/
benefit profiles prevented Tamoxifen from being applied for
more than five years, and severe toxicities such as endometrial
cancer and thrombosis were observed.4 On the contrary, the
third-generation aromatase inhibitors (AIs), for example,
Anastrozole, Letrozole, and Exemestane (Chart 1), exhibited
better efficacy and tolerability compared to Tamoxifen, which
rendered AIs to become the first choice as adjuvant
therapeutics for postmenopausal women, the majority of breast
cancer patients. Aromatase (CYP19) is the crucial enzyme
catalyzing the final aromatization of the steroidal A-ring in the
biosynthesis of estrogens from corresponding androgen
precursors: testosterone and androstenedione. Inhibition of
CYP19 can totally block estrogen production and consequently
prevent BC cells from proliferation. After administration of the
third-generation AIs, plasma estrogen concentration can be
reduced to undetectable level.5 Several clinical trials demon-
Received: April 3, 2012
© XXXX American Chemical Society
A
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Chart 1. Structures of Selective Estrogen Receptor Modulators (Tamoxifen and Raloxifen) and Aromatase Inhibitors
(Anastrozole, Letrozole, Fadrozole, and Exemestane)
Chart 2. Deleterious Effects of High Aldosterone Levels Resulting from Estrogen Deficiency
receptor (AT1R) further stimulate aldosterone biosynthesis.13
Moreover, estrogen deficiency also increases the potassium
plasma concentration, resulting in an enhancement of
aldosterone secretion.14
The resulting high aldosterone levels exhibit deleterious
effects on kidney, vessels, brain, and most severe on heart15
(Chart 2, for detailed description and the corresponding
references, see Supporting Information). Therefore, reducing
B
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Chart 3. Design of Heterocyclic Dihydroquinolinones As Dual CYP19/CYP11B2 Inhibitors
the high plasma aldosterone concentration rendered by
estrogen deficiency should be an effective treatment for
cardiovascular conditions in BC patients. Although ACE
inhibitors and/or MR antagonists, which have been imple-
mented in the clinic for a long time, could be options for
combinatory application with AIs to meet this need, they are
not the best choice due to the side effects observed and the
phenomenon of “aldosterone escape”. Because aldosterone
synthase (CYP11B2) catalyzing the conversion of 11-
deoxycorticosterone to aldosterone is the key enzyme in
aldosterone biosynthesis, the inhibition of CYP11B2 is the
method of choice to reduce aldosterone levels. Because AIs as
adjuvant therapeutics have to be applied for at least five years
and there are no selective CYP11B2 inhibitors in clinical use
until now, it is our aim to design dual inhibitors of CYP19 and
CYP11B2, which are selective to other steroidogenic CYP
enzymes like CYP17 and CYP11B1. It is not possible to adjust
individually the suppression degree of estrogens and aldoster-
one productions with dual inhibitors, which projects the
demands of careful balancing on the inhibition toward both
enzymes for the drug candidates. When doing so, the exposure
of the inhibitors to both enzymes should also be taken into
consideration because CYP19 is expressed in endoplasmic
reticulum whereas CYP11B2 is located in mitochondria.
Despite the potential issues to be addressed, the benefits of
dual CYP19 and CYP11B2 inhibitors are apparent. This kind of
multitarget-directed agents16,17 should not only improve
patients’ compliance but should also avoid possible drug−
drug interactions caused by combinative application. Further-
more, accurate designation of the suitable patient cohort at the
drug design stage by narrowing down the patient scope from
whole population to the most promising ones to response can
probably improve the performances and outcomes of the drug
candidates in clinical trials. In this study, important structural
features of CYP19 inhibitors were incorporated into CYP11B2
inhibitors. Thus, a series of pyridyl dihydroquinolinone
derivatives were designed and synthesized, leading to
compounds 1−13. The inhibition of these compounds toward
CYP11B2 and CYP19 are presented with the unselective
CYP19 inhibitor Fadrozole (Chart 1) as a reference.
Furthermore, the selectivity of these compounds against 11β-
hydroxylase (CYP11B1) and 17α-hydroxylase-17,20-lyase
(CYP17), which are the crucial enzymes in the biosynthesis
of glucocorticoids and androgens, respectively, were also
determined as safety criteria.
Design Conception for Dual Inhibitors. Besides
Anastrozole and Letrozole, the CYP19 inhibitors in clinical
use, a lot of potent nonsteroidal CYP19 inhibitors have been
designed and synthesized.18 Almost all of these inhibitors
coordinate with their sp2 hybrid nitrogen to the heme iron,
leading to tight binding and reversible inhibition of this
enzyme.18a Because all CYP enzymes contain a heme iron as
the catalytic center, inhibitors toward other steroidogenic
enzymes, such as CYP17,19 CYP11B2,20 and CYP11B1,21 have
also been designed based on this mechanism.
Two potent CYP19 inhibitors have been employed in the
design of the compounds presented in this investigation (Chart
3), Ref. 1 (IC50 CYP19 = 71 nM)18b with a phenyl group
furnishing the flavone scaffold, and Ref. 2 (IC50 CYP19 = 12
nM),18c with a phenyl core substituted by a phenoxy group.
Both compounds possess an imidazolylmethyl moiety adjacent
to the bulky phenyl or phenoxy substituents. Three important
structural features can be noticed in these two potent CYP19
inhibitors: a heterocycle providing a sp2 hybrid N (imidazolyl),
an aromatic core (chromone or phenyl), and a bulky
hydrophobic substituent nearby. Besides, the groups furnishing
the core are also important for CYP19 inhibition. This was
demonstrated by Ref. 3 (Chart 3),20b which is the only
compound exhibiting potent CYP19 inhibition (IC50 CYP19 = 38
nM) in a series of CYP11B2 inhibitors, despite all compounds
in that series containing the structural features described above.
This inhibition is considered to be rendered by the methoxy
substitution at the 6 position of the naphthalene core.20b
Accordingly, we selected 6-(pyridin-3-yl)-3,4-dihydroquinolin-
2(1H)-one (Ref. 4, Chart 3)20a as the template for CYP11B2
inhibition to design dual inhibitors of CYP19 and CYP11B2,
because Ref. 4 is already composed of two structural features
important for CYP19 inhibition: the 3-pyridyl as N containing
C
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a
Scheme 1
a
Reagents and conditions: (i) method A, 3-chloropropanoyl chloride, pyridine, THF, rt; (ii) method B, AlCl3, DMA, 140 °C, 4 h; (iii) method C,:
NBS, DMF, 0 °C, 9 h; (iv) method D, Pd(OAc)2, corresponding boronic acid, Na2CO3, TBAB, toluene, H2O, ethanol, reflux, 6 h; (v) method E,
corresponding halide, K2CO3, KI, EtOH, 70 °C, overnight; (vi) method F, MeI, KOt-Bu, DMF, 50 °C, overnight.
a
Scheme 2
a
Reagents and conditions: (i) method G, NBS, DMF, 65 °C, 6 h; (ii) method D, Pd(OAc)2, corresponding boronic acid, Na2CO3, TBAB, toluene,
H2O, ethanol, reflux, 6 h.
heterocycle and the dihydroquinolinone as aromatic core.
Moreover, the presence of a ketone group mimicking the 6-
methoxy substituent of Ref. 3 as hydrogen bond acceptor
fulfilled another precondition of potent CYP19 inhibition.
Because Ref. 4 is a highly potent and selective CYP11B2
inhibitor (IC50 CYP11B2 = 28 nM) showing no inhibition of
CYP19, the desired CYP19 inhibition could probably be
acquired after introduction of the third structural feature into
this template while its CYP11B2 activity and selectivity over
CYP11B1 and CYP17 should be maintained. Therefore,
hydrophobic groups, such as alkoxyl, halogen, and aryl in
various sizes, were introduced into the 7- or 8-position of the
core. For substituents at the 7-position, an oxygen was
exploited as a linker, similar to that of Ref. 2, because of our
previous observation that bulky groups adjacent to the pyridyl
substituent significantly reduce CYP11B2 inhibition.20a Fur-
thermore, methylation of the amide was also performed to
explore the role of the NH proton.
group was cleaved simultaneously to afford the hydroxy
substituent. Second, 6-position specific bromination was
achieved with N-bromosuccinimide (NBS), which was followed
by Suzuki coupling with 3-pyridyl boronic acids, leading to
compound 1. Further derivatives 2−9 were obtained after
alkylation of the phenol or the amide, which were performed
before the final step of Suzuki coupling. As for 8-substituted
analogues, 6-(pyridin-3-yl)-3,4-dihydroquinolin-2(1H)-one was
prepared similarly as described above as a common
intermediate, which after 8-position specific bromination gave
compound 10. Further aryl groups were subsequently
introduced via another Suzuki coupling reaction resulting in
the desired compounds 11−13.
Inhibition of CYP19, CYP11B2, and CYP11B1. Inhib-
itory activity of the synthesized compounds toward CYP19 was
determined using human placental microsomes with [1β-3H]-
androstenedione as substrate,22d whereas V79MZh cells
expressing human CYP11B2 were employed for the
CYP11B2 assay with [14C]-11-deoxycorticosterone as sub-
strate.22c Because CYP11B1 and CYP11B2 exhibit more than
93% of homology and the inhibition of CYP11B1 results in
cortisol deficiency, the selectivity toward CYP11B1 was also
examined for safety evaluation using a similar procedure as
described for CYP11B2, except for V79MZh cells expressing
human CYP11B1 as source of enzyme.22c The IC50 values of
the synthesized compounds toward these three enzymes are
presented in Table 1 with the reference Fadrozole.
RESULTS AND DISCUSSION
■
Chemistry. The synthesis of compounds 1−13 is shown in
Schemes 1−2. First, the substituted 3,4-dihydroquinolin-
2(1H)-one core 1b was built from the corresponding methoxy
aniline after amidation with 3-chloropropanoyl chloride,
followed by cyclization via Friedel−Craft alkylation in the
presence of N,N-dimethylacetamide (DMA) to suppress side
reactions. During the Friedel−Craft alkylation, the methoxy
D
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Table 1. Inhibition of CYP11B1, CYP11B2, and CYP19 by Compounds 1−13
CYP19
CYP11B2
CYP11B1
bc
,
bd
,
bc
,
e
compd
R1
R2
R3
IC50 nM
IC50 nM
IC50 nM
SF
Ref. 4
H
H
H
H
H
H
H
H
H
H
H
H
H
Br
Ph
H
>5000
426
3073
447
49
28
2.6
6747
742
241
289
38
Ref. 520a
Me
H
1
OH
312
268
19
11940
2867
1098
3737
790
2
OMe
OMe
OEt
OEt
O-c-Pent
O-c-Pent
OBz
OBz
H
H
9
3
Me
H
59
4
488
48
674
19
5.5
41
5
Me
H
6
162
22
1703
759
22
1077
1167
44
0.6
1.5
2
7
Me
H
8
35
9
Me
H
11
178
12
139
0.8
120
13.9
3.7
22
10
11
12
13
Ref. 3
2901
394
954
275
38
1422
16170
4087
4589
4329
H
H
1167
1097
208
11
H
3-Py
H
H
3-Thienyl
H
394
8.3
a
FDZ
52
0.8
6.3
a
b
FDZ: Fadrozole. Concentration of inhibitors required to give 50% inhibition. Standard deviations were within < 5%; all the data are the mean
c
values of at least three tests. Hamster fibroblasts expressing human CYP11B1 or CYP11B2 are used with deoxycorticosterone as the substrate at a
concentration of 100 nM. Human placental CYP19 is used with androstenedione as the substrate at a concentration of 500 nM. SF: selective factor
d
e
= IC50 CYP11B1/IC50 CYP11B2
.
It can be seen that compared to Ref. 4 (IC50 CYP11B2 = 28 nM
and IC50 CYP19 > 5000 nM), the hydroxy substitution at the 7-
position (compound 1) slightly improved CYP19 inhibition but
decreased CYP11B2 inhibitory potency to about 300 nM and
strongly increased to IC50 values below 20 nM. It is important
that the selectivity over CYP11B1 of these compounds was
enhanced to 59 and 41, respectively. Accordingly, by a
combination of introducing small alkoxy groups into the 7-
position and amide methylation, the desired dual inhibitors of
CYP19/11B2 were successfully obtained with compounds 3
and 5, showing good selectivity over CYP11B1. As for more
bulky substituents like cyclopentyloxy (compound 7), the
promotion of CYP11B2 inhibition rendered by amide
methylation could not neutralize the deterioration from the
increase of bulkiness. As the inhibition of CYP19 was increased
to 22 nM, this compound turned out to be a selective CYP19
selectivity factor over CYP11B1 (SF, IC50 CYP11B1/IC50 CYP11B2
)
to 38. After alkylation of the hydroxy group with subnstituents
of different sizes, strong effects were observed. With increasing
bulkiness of the substituents, the inhibition of the correspond-
ing compounds toward CYP19 rose from 450−500 nM
(compounds 2 and 4 with methoxy and ethoxy, respectively)
to 162 nM (compound 6 with cyclopentyloxy), and finally to
35 nM (compound 8 with benzyloxy). In parallel, the inhibitory
activities of these compounds toward CYP11B2 decreased, the
methoxy compound 2 exhibiting an IC50 value of 268 nM,
while 673 nM for the ethoxy compound 4 and 1700 nM for the
cyclopentyloxy compound 6. Surprisingly, the benzyloxy
analogue 8 turned out to be a potent CYP11B2 inhibitor
with an IC50 value of 22 nM, probably due to a different
binding mode. The inhibition of CYP11B1 followed the same
trend. However, it seemed that CYP11B1 is less sensitive to an
increase in bulkiness compared to CYP11B2. Accordingly,
significant reduction of selectivity was observed with SFs of less
than 10 for these compounds. However, methylation of the
amide N dramatically improved the inhibition of CYP19 and
CYP11B2 as well as slightly increased CYP11B1 inhibition. It is
apparent that after amide methylation, the methoxy (3) and
ethoxy (5) analogues exhibited 10-fold more potent inhibition
of CYP19 than their parent compounds (2 and 4), with IC50
values around 50 nM. The inhibition of CYP11B2 was also
inhibitor with selectivity of 35 and 53 (IC50 CYP19
/
IC50 CYP11B2 or CYP11B1) over CYP11B2 and CYP11B1, respec-
tively. As for the benzyloxy analogues 8 and 9, which were
believed to bind in a different mode to the two CYP11Bs, the
amide methylation significantly decreased CYP11B inhibition
to around 150 nM. Because compound 9 showed potent
inhibition of CYP19 (IC50 = 11 nM), it has to be considered as
a selective CYP19 inhibitor, showing selectivities around 15-
fold toward CYP11B2 and CYP11B1.
Furthermore, the influence of substituents at the 8-position
was also investigated. The bromo analogue 10 turned out to be
a potent and selective CYP11B2 inhibitor, exhibiting an IC50
value of 11 nM and a SF of 120 over CYP11B1. However, this
compound showed only weak inhibition of CYP19 (IC50
=
2900 nM). The introduction of aryl groups (17 with phenyl
and 18 with 3-pyridyl) increased CYP19 inhibition, with the
potency toward CYP11B2 reduced accordingly. It is notable
E
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that unlike its bioisostere phenyl, the 3-thienyl group with its
smaller size rendered compound 19 as a modest dual inhibitor
of CYP19/11B2 (IC50 values of 275 and 208 nM, respectively),
with a fair SF of 22 over CYP11B1.
Selectivity over CYP17. The inhibition of CYP17 by the
synthesized compounds was also determined as a criterion to
evaluate safety because this enzyme is crucial in the biosynthesis
of androgens. It turned out that all compounds exhibited IC50
values of more than 10000 nM (data not shown), indicating no
inhibition of CYP17.
Selectivity over Hepatic CYP3A4. Because CYP3A4 is
responsible for the metabolism of a large percentage of drugs
and xenobiotics and therefore is a key factor in drug−drug
interactions, compounds 3 and 5 were tested for the inhibition
against CYP3A4. Both compounds showed IC50 values around
90 μM, which grants the compounds a wide safety window.
Solubility in Water. Solubility is always an important issue
in drug discovery and development because low solubility not
only limits oral absorption and reduces bioavailability but also is
an obstacle to formulation. Therefore, the solubility of selected
compounds 3, 5, 7, and 10 in water with 2% of DMSO were
determined at 25 °C. Compounds 3, 5, and 10 showed
solubility of 145.5, 136.7, and 177.3 μM, respectively, whereas
the cyclopentyloxy analogue 7 is less soluble (76.8 μM). The
solubility can be further improved simply by forming salts due
to the presence of pyridine moiety.
selective CYP19 inhibitor with an IC50 value of 22 nM and
selectivity factors of more than 35 for other enzymes, as well as
compound 10 as a selective CYP11B2 inhibitor (IC50 = 12
nM), with selectivity factors of more than 120 over other
enzymes. The dual inhibitors 3 and 5 are promising lead
compounds to be further developed for treating BC patients
with risk of CVD.
EXPERIMENTAL SECTION
■
Inhibition of CYP11B1 and CYP11B2. V79MZh cells expressing
human CYP11B1 or CYP11B222e were incubated with [14C]-11-
deoxycorticosterone as substrate. The assay was performed as
previously described.22c
Inhibition of CYP19. The inhibition of CYP19 was determined in
vitro using human placental microsomes with [1β-3H]androstenedione
as substrate.22d
Inhibition of CYP17. Human CYP17 was expressed in Escherichia
coli22b (coexpressing human CYP17 and NADPH-P450 reductase),
and the assay was performed with progesterone as substrate at high
concentrations of 25 μM as previously described.22a
Chemistry. General. Melting points were determined on a Mettler
FP1 melting point apparatus and are uncorrected. IR spectra were
1
recorded neat on a Bruker Vector 33FT-infrared spectrometer. H
NMR and 13C NMR spectra were measured on a Bruker DRX-500
(500 MHz). Chemical shifts are given in parts per million (ppm), and
TMS was used as an internal standard for spectra obtained. All
coupling constants (J) are given in Hz. ESI (electrospray ionization)
mass spectra were determined on a TSQ quantum (Thermo Electron
Corporation) instrument. The purities of the final compounds were
controlled by a Surveyor-LC system. Purities were greater than 95%.
Column chromatography was performed using Silica Gel 60 (50−200
μm), and reaction progress was determined by TLC analysis on
Alugram SIL G/UV254 (Macherey-Nagel).
CONCLUSIONS
■
Although AIs as BC therapeutics are successful by reducing
estrogen concentrations to prevent BC cell proliferation, the
estrogen deficiency they result in together with the
postmenopausal status of the patients results in elevations of
aldosterone levels, which sometimes are not detected in the
plasma but are intracellularly in the myocardia and other target
cells. The abnormally high aldosterone levels exert deleterious
effects on kidney, vessels, brain, and most severe on the
myocardia. These damages result in asymptomatic left
ventricular dysfunction and may exist long before the clinical
syndrome appears and sudden death occurs. Therefore, it is an
urgent need to find an appropriate way to cope with BC and
the coinstantaneous cardiovascular diseases. Because AIs as
adjuvant therapeutics have to be administered for a long time,
the compliance of the patients is another advantage of dual
inhibitors of CYP19 and CYP11B2, besides the reduction of
drug−drug interactions risks, which possibly happen when
administrating in combination.
Method A: Amidation. To the corresponding aniline (1.0 equiv)
solution in dry THF (10 mL/mmmol) was added pyridine (1.5 equiv)
before the mixture was cooled to 0 °C in an ice bath. Then 3-
chloropropanoyl chloride (1.1 equiv) was dropped in carefully. After
the addition, the reaction mixture was warmed to ambient temperature
and stirred overnight. THF was then removed by reduced pressure,
and the residues were taken up with ethyl acetate (10 mL) and water
(10 mL). After extraction of the water phase three times with ethyl
acetate (10 mL), the organic phases were combined, washed with
brine, dried over Na2SO4, and evaporated under reduced pressure to
give the crude product. The compounds were then purified by flash
chromatography on silica gel.
3-Chloro-N-(3-methoxy-phenyl)-propionamide (1c). Synthesized
according to method A using 3-methoxyaniline (0.50 g, 4.06 mmol)
and 3-chloropropanoyl chloride (0.62 g, 4.87 mmol). This
intermediate was used directly in the next step without further
purification and characterization
Method B: Friedel−Craft alkylation in DMA. To the cold mixture
of corresponding 3-chloro-N-phenylpropanamides (1.0 equiv) and
AlCl3 (5.0 equiv) was dropped in DMA (1.0 equiv). Then the reaction
mixture was heated to 140 °C for 4 h. After cooling down to ambient
temperature, the black mixture was worked up with ethyl acetate (20
mL) and ice water (20 mL). After extraction of the water phase three
times with ethyl acetate (10 mL), the organic phases were combined,
washed with brine, dried over Na2SO4, and evaporated under reduced
pressure to give the crude product. The compounds were then purified
by flash chromatography on silica gel.
7-Hydroxy-3,4-dihydro-1H-quinolin-2-one (1b). Synthesized ac-
cording to method B using 1c (0.55 g, 2.57 mmol). This intermediate
was used directly in the next step without further purification and
characterization.
The approach of combining important structural features of
CYP19 and CYP11B2 inhibitors to design dual inhibitors has
been demonstrated to be successful in this study. Structural
modifications exhibit different effects on the SARs toward the
different target enzymes. It has been found that the increase of
bulkiness of the alkoxy groups at the 7-position strongly
elevated CYP19 inhibition. However, it also reduced CYP11B2
inhibition and selectivity over CYP11B1. On the contrary,
methylation of the amide N led to an increase of inhibition
toward all of these three enzymes. After balancing the different
effects of the substituents on the SARs, compounds 3 and 5
were obtained as selective dual inhibitors with IC50 values
around 50 and 20 nM toward CYP19 and CYP11B2,
respectively, similar to that of Fadrozole. These compounds
also showed a better selectivity compared to Fadrozole against
CYP11B1, with selectivity factors around 50 and no inhibition
of CYP17. Also, in this study, compound 7 was identified as a
Method C: Selective Bromination at 6-Position. To the solution of
corresponding dihydroquinolinone (1.0 equiv) in DMF (5 mL/mmol)
cooling in an ice bath was dropped in NBS (1.1 equiv) solution in
DMF (2 mL/mmol). The addition lasted for 3 h, and then the
reaction mixture was hold at 0 °C for another 6 h before extraction
F
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with ethyl acetate (20 mL) and water (20 mL). After extraction of the
water phase three times with ethyl acetate (10 mL), the organic phases
were combined, washed with brine, dried over Na2SO4, and
evaporated under reduced pressure to give the crude product. The
compounds were then purified by flash chromatography on silica gel.
6-Bromo-7-hydroxy-3,4-dihydro-1H-quinolin-2-one (1a). Synthe-
sized according to method C using 7b (0.47 g, 2.88 mmol) and NBS
(0.51 g, 2.88 mmol); yield 0.61 g (87%); white solid; Rf = 0.25
(DCM/MeOH, 20:1); δH (CDCl3, 500 MHz) 2.38 (t, J = 7.5 Hz, 2H,
CH2), 2.75 (t, J = 7.7 Hz, 2H, CH2), 6.53 (s, 1H), 7.24 (s, 1H), 10.05
(s, br, 1H, NH); MS (ESI): m/z = 242 [M+ + H].
Method D: Suzuki Coupling. The corresponding brominated
aromatic compound (1.0 equiv) was dissolved in toluene (7 mL/
mmol), and an aqueous 2.0 M Na2CO3 solution (3.2 mL/mmol), an
ethanolic solution (3.2 mL/mmol) of the corresponding boronic acid
(1.5−2.0 equiv), and tetrabutylammonium bromide (1.0 equiv) were
added. The mixture was deoxygenated under reduced pressure and
flushed with nitrogen. After having repeated this cycle several times,
Pd(OAc)2 (5 mol %) was added and the resulting suspension was
heated under reflux for 2−6 h. After cooling, ethyl acetate (10 mL)
and water (10 mL) were added and the organic phase was separated.
The water phase was extracted with ethyl acetate (2 × 10 mL). The
combined organic phases were washed with brine, dried over Na2SO4,
filtered over a short plug of Celite, and evaporated under reduced
pressure. The compounds were purified by flash chromatography on
silica gel.
7-Hydroxy-6-pyridin-3-yl-3,4-dihydro-1H-quinolin-2-one (1). Syn-
thesized according to method D using 1a (0.35 g, 1.45 mmol) and
pyridin-3-ylboronic acid (0.21 g, 1.74 mmol); yield 0.11 g (32%);
white solid; mp 297−298 °C; Rf = 0.19 (DCM/MeOH, 20:1); δH
(DMSO, 500 MHz) 2.44 (t, J = 7.9 Hz, 2H, CH2), 2.81 (t, J = 7.9 Hz,
2H, CH2), 6.55 (s, 1H), 7.13 (s, 1H), 7.37 (ddd, J = 0.7, 4.9, 8.0 Hz,
1H), 7.89 (dt, J = 2.2, 7.9 Hz, 1H), 8.43 (dd, J = 1.5, 4.7 Hz, 1H), 8.69
(d, J = 1.8 Hz, 1H), 9.68 (s, br, 1H, NH), 10.10 (s, br, 1H, OH); MS
(ESI): m/z = 241 [M+ + H].
7-Methoxy-6-pyridin-3-yl-3,4-dihydro-1H-quinolin-2-one (2).
Synthesized according to method D using 2a (0.35 g, 1.37 mmol)
and pyridin-3-ylboronic acid (0.16 g, 1.64 mmol); yield 0.30 g (85%);
white solid; mp 221−222 °C; Rf = 0.13 (DCM/MeOH, 50:1); δH
(CDCl3, 500 MHz) 2.68 (t, J = 7.9 Hz, 2H, CH2), 2.97 (t, J = 7.9 Hz,
2H, CH2), 3.81 (s, 3H, OCH3), 6.45 (s, 1H), 7.11 (s, 1H), 7.31 (ddd, J
= 0.7, 5.4, 7.9 Hz, 1H), 7.81 (dt, J = 2.0, 7.9 Hz, 1H), 8.52 (dd, J = 1.6,
4.7 Hz, 1H), 8.63 (s, br, 1H, NH), 8.72 (d, J = 1.7 Hz, 1H); MS (ESI):
m/z = 255 [M+ + H].
7-Methoxy-1-methyl-6-pyridin-3-yl-3,4-dihydro-1H-quinolin-2-
one (3). Synthesized according to method D using 3a (0.50 g, 1.85
mmol) and pyridin-3-ylboronic acid (0.22 g, 2.22 mmol); yield 0.46 g
(93%); white solid; mp 207−208 °C; Rf = 0.19 (DCM/MeOH, 50:1);
δH (CDCl3, 500 MHz) 2.62 (t, J = 7.9 Hz, 2H, CH2), 2.83 (t, J = 7.9
Hz, 2H, CH2), 3.35 (s, 3H, NCH3), 3.79 (s, 3H, OCH3), 6.56 (s, 1H),
7.01 (s, 1H), 7.25 (ddd, J = 0.7, 5.3, 8.3 Hz, 1H), 7.76 (dt, J = 2.0, 8.0
Hz, 1H), 8.46 (dd, J = 1.6, 4.8 Hz, 1H), 8.68 (d, J = 1.7 Hz, 1H); MS
(ESI): m/z = 269 [M+ + H].
7-Ethoxy-6-pyridin-3-yl-3,4-dihydro-1H-quinolin-2-one (4). Syn-
thesized according to method D using 4a (0.35 g, 1.30 mmol) and
pyridin-3-ylboronic acid (0.19 g, 1.55 mmol); yield 0.31 g (89%);
white solid; mp 206−207 °C; Rf = 0.13 (DCM/MeOH, 50:1); δH
(CDCl3, 500 MHz) 1.34 (t, J = 6.9 Hz, 3H, CH3), 2.67 (t, J = 7.9 Hz,
2H, CH2), 2.96 (t, J = 7.9 Hz, 2H, CH2), 4.01 (q, J = 6.9 Hz, 2H,
OCH2), 3.35 (s, 3H, CH3), 3.79 (s, 3H, OCH3), 6.49 (s, 1H), 7.10 (s,
1H), 7.31 (ddd, J = 0.7, 4.8, 7.8 Hz, 1H), 7.84 (dt, J = 1.8, 7.9 Hz, 1H),
8.51 (d, J = 3.8 Hz, 1H), 8.75 (s, 1H), 9.00 (s, br, 1H, NH); MS (ESI):
m/z = 269 [M+ + H].
4.9, 7.9 Hz, 1H), 7.84 (dt, J = 2.0, 7.9 Hz, 1H), 8.50 (dd, J = 1.6, 4.8
Hz, 1H), 8.76 (d, J = 1.7 Hz, 1H); MS (ESI): m/z = 283 [M+ + H].
7-Cyclopentyloxy-6-pyridin-3-yl-3,4-dihydro-1H-quinolin-2-one
(6). Synthesized according to method D using 6a (0.35 g, 1.13 mmol)
and pyridin-3-ylboronic acid (0.17 g, 1.35 mmol); yield 0.29 g (83%);
white solid; mp 181−182 °C; Rf = 0.15 (DCM/MeOH, 50:1); δH
(CDCl3, 500 MHz) 1.52−1.89 (m, 8H, c-Pent), 2.66 (t, J = 7.9 Hz,
2H, CH2), 2.94 (t, J = 7.9 Hz, 2H, CH2), 4.73 (sept, J = 2.8 Hz, 1H,
OCH), 6.50 (s, 1H), 7.10 (s, 1H), 7.28 (ddd, J = 0.7, 4.7, 7.8 Hz, 1H),
7.90 (dt, J = 1.9, 7.9 Hz, 1H), 8.50 (d, J = 4.5 Hz, 1H), 8.71 (d, J = 1.4
Hz, 1H),8.98 (s, br, 1H, NH); MS (ESI): m/z = 309 [M+ + H].
7-Cyclopentyloxy-1-methyl-6-pyridin-3-yl-3,4-dihydro-1H-quino-
lin-2-one (7). Synthesized according to method D using 7a (0.50 g,
1.54 mmol) and pyridin-3-ylboronic acid (0.23 g, 1.85 mmol); yield
0.42 g (85%); colorless oil; Rf = 0.23 (DCM/MeOH, 50:1); δH
(CDCl3, 500 MHz) 1.52−1.89 (m, 8H, c-Pent), 2.67 (t, J = 7.9 Hz,
2H, CH2), 2.88 (t, J = 7.9 Hz, 2H, CH2), 3.38 (s, 3H, NCH3), 4.75
(sept, J = 2.8 Hz, 1H, OCH), 6.62 (s, 1H), 7.11 (s, 1H), 7.29 (ddd, J =
0.7, 5.3, 7.9 Hz, 1H), 7.81 (dt, J = 1.9, 7.9 Hz, 1H), 8.50 (dd, J = 1.5,
4.8 Hz, 1H), 8.72 (d, J = 1.8 Hz, 1H); MS (ESI): m/z = 322 [M+ +
H].
7-Benzyloxy-6-pyridin-3-yl-3,4-dihydro-1H-quinolin-2-one (8).
Synthesized according to method D using 8a (0.35 g, 1.05 mmol)
and pyridin-3-ylboronic acid (0.16 g, 1.26 mmol); yield 0.30 g (86%);
white solid; mp 189−190 °C; Rf = 0.17 (DCM/MeOH, 50:1); δH
(CDCl3, 500 MHz) 2.67 (t, J = 7.9 Hz, 2H, CH2), 2.97 (t, J = 7.9 Hz,
2H, CH2), 5.06 (s, 2H, bz CH2), 6.50 (s, 1H), 7.14 (s, 1H), 7.26−7.35
(m, 6H, bz), 7.86 (dt, J = 1.8, 8.0 Hz, 1H), 8.47 (s, br, 1H, NH), 8.50
(dd, J = 1.5, 4.8 Hz, 1H), 8.76 (d, J = 1.7 Hz, 1H); MS (ESI): m/z =
331 [M+ + H].
7-Benzyloxy-1-methyl-6-pyridin-3-yl-3,4-dihydro-1H-quinolin-2-
one (9). Synthesized according to method D using 9a (0.50 g, 1.44
mmol) and pyridin-3-ylboronic acid (0.21 g, 1.73 mmol); yield 0.45 g
(90%); white solid; mp 209−210 °C; Rf = 0.23 (DCM/MeOH, 50:1);
δH (CDCl3, 500 MHz) 2.67 (t, J = 7.9 Hz, 2H, CH2), 2.89 (t, J = 7.9
Hz, 2H, CH2), 3.33 (s, 3H, NCH3), 5.09 (s, 2H, bz CH2), 6.67 (s,
1H), 7.14 (s, 1H), 7.29−7.38 (m, 6H, bz), 7.87 (dt, J = 1.9, 8.0 Hz,
1H), 8.53 (dd, J = 1.6, 4.9 Hz, 1H), 8.78 (d, J = 1.8 Hz, 1H); MS
(ESI): m/z = 345 [M+ + H].
8-Phenyl-6-pyridin-3-yl-3,4-dihydro-1H-quinolin-2-one (11). Syn-
thesized according to method D using 10 (0.35 g, 1.15 mmol) and
phenylboronic acid (0.17 g, 1.38 mmol); yield 0.30 g (86%); white
solid; mp 171−172 °C; Rf = 0.25 (DCM/MeOH, 50:1); δH (CDCl3,
500 MHz) 2.71 (t, J = 7.9 Hz, 2H, CH2), 3.12 (t, J = 7.9 Hz, 2H,
CH2), 7.34−7.36 (m, 2H), 7.38−7.46 (m, 4H), 7.49−7.53 (m, 3H),
7.86 (dt, J = 1.9, 7.9 Hz, 1H), 8.57 (dd, J = 1.5, 4.7 Hz, 1H), 8.84 (d, J
= 2.1 Hz, 1H); MS (ESI): m/z = 301 [M+ + H].
6,8-Dipyridin-3-yl-3,4-dihydro-1H-quinolin-2-one (12). Synthe-
sized according to method D using 10 (0.35 g, 1.15 mmol) and
pyridin-3-ylboronic acid (0.17 g, 1.38 mmol); yield 0.29 g (83%);
white solid; mp 177−178 °C; Rf = 0.19 (DCM/MeOH, 50:1); δH
(CDCl3, 500 MHz) 2.73 (t, J = 7.9 Hz, 2H, CH2), 3.14 (t, J = 7.9 Hz,
2H, CH2), 7.33−7.38 (m, 2H), 7.44−7.47 (m, 2H), 7.74 (dt, J = 1.8,
7.9 Hz, 1H), 7.85 (dt, J = 2.2, 8.4 Hz, 1H), 8.59 (dd, J = 1.5, 4.8 Hz,
1H), 8.68 (d, J = 2.3 Hz, 1H), 8.71 (dd, J = 1.6, 4.8 Hz, 1H), 8.84 (d, J
= 1.8 Hz, 1H); MS (ESI): m/z = 302 [M+ + H].
6-Pyridin-3-yl-8-thiophen-3-yl-3,4-dihydro-1H-quinolin-2-one
(13). Synthesized according to method D using 10 (0.47 g, 1.55
mmol) and thiophen-3-ylboronic acid (0.24 g, 1.86 mmol); yield 0.39
g (83%); white solid; mp 235−236 °C; Rf = 0.35 (DCM/MeOH,
50:1); δH (CDCl3, 500 MHz) 2.71 (t, J = 7.9 Hz, 2H, CH2), 3.11 (t, J
= 7.9 Hz, 2H, CH2), 7.18 (dd, J = 1.2, 4.9 Hz, 1H), 7.34−7.38 (m,
2H), 7.40 (s, 2H), 7.52 (dd, J = 4.8, 4.9 Hz, 1H), 7.66 (s, br, 1H, NH),
7.85 (dt, J = 1.8, 8.0 Hz, 1H), 8.58 (dd, J = 1.5, 4.9 Hz, 1H), 8.83 (d, J
= 2.1 Hz, 1H); MS (ESI): m/z = 307 [M+ + H].
7-Ethoxy-1-methyl-6-pyridin-3-yl-3,4-dihydro-1H-quinolin-2-one
(5). Synthesized according to method D using 5a (0.50 g, 1.76 mmol)
and pyridin-3-ylboronic acid (0.26 g, 2.11 mmol); yield 0.43 g (86%);
white solid; mp 217−218 °C; Rf = 0.20 (DCM/MeOH, 50:1); δH
(CDCl3, 500 MHz) 1.35 (t, J = 6.9 Hz, 3H, CH3), 2.66 (t, J = 7.9 Hz,
2H, CH2), 2.88 (t, J = 7.9 Hz, 2H, CH2), 3.38 (s, 3H, NCH3), 4.04 (q,
J = 6.9 Hz, 2H, OCH2), 6.61 (s, 1H), 7.11 (s, 1H), 7.30 (ddd, J = 0.7,
Method E: Alkylation of Phenol. The suspension of K2CO3 (2.0
equiv) and 6-bromo-7-hydroxy-3,4-dihydroquinolin-2(1H)-one (1.0
equiv) in dry ethanol (5 mL/mmol) was refluxed for 2 h before KI
(0.05 equiv) and the corresponding halide (2.0 equiv) were added.
Then the white suspension was boiled overnight. After cooling down
G
dx.doi.org/10.1021/jm3004637 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
to ambient temperature, ethanol was removed with reduced pressure
and the residue was taken up with ethyl acetate (20 mL) and water (20
mL). After extraction of the water phase three times with ethyl acetate
(10 mL), the organic phases were combined, washed with brine, dried
over Na2SO4, and evaporated under reduced pressure to give the crude
product. The compounds were then purified by flash chromatography
on silica gel.
Method F: Alkylation of Amide. The suspension of potassium t-
butanolate (2.0 equiv) and corresponding dihydroquinolinone (1.0
equiv) in dry DMF (5 mL/mmol) was heated to 50 °C for 2 h before
KI (0.05 equiv) and the iodomethane (2.0 equiv) were added. Then
the white suspension was boiling overnight. After cooling down to
ambient temperature, the reaction mixture was diluted with ethyl
acetate (20 mL) and water (20 mL). After extraction of the water
phase three times with ethyl acetate (10 mL), the organic phases were
combined, washed with brine, dried over Na2SO4, and evaporated
under reduced pressure to give the crude product. The compounds
were then purified by flash chromatography on silica gel.
Method G: Selective Bromination at 8-Position. To the solution of
corresponding dihydroquinolinone (1.0 equiv) in DMF (5 mL/mmol)
was dropped in NBS (1.1 equiv) solution in DMF (2 mL/mmol) at 65
°C. The addition lasted for 3 h, and then the reaction mixture was hold
at 65 °C for another 3 h before extraction with ethyl acetate (20 mL)
and water (20 mL). After extraction of the water phase three times
with ethyl acetate (10 mL), the organic phases were combined, washed
with brine, dried over Na2SO4, and evaporated under reduced pressure
to give the crude product. The compounds were then purified by flash
chromatography on silica gel.
receptor modulator; AI, aromatase inhibitor; CPY19, aroma-
tase, estrogen synthase; CVD, cardiovascular diseases; RAAS,
renin−angiotensin−aldosterone system; Ang II, angiotensin II;
ACE, angiotensin converting enzyme; AT1R, angiotensin type
1 receptor; CYP11B2, aldosterone synthase; MR, mineralo-
corticoid receptor; CYP17, steroid 17α-hydroxylase-17,20-
lyase; CYP11B1, steroid 11β-hydroxylase; DMA, N, N-
dimethylacetamide; NBS, N-bromosuccinimide; SF, selectivity
factor
REFERENCES
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8-Bromo-6-pyridin-3-yl-3,4-dihydro-1H-quinolin-2-one (10). Syn-
thesized according to method G using 6-(pyridin-3-yl)-3,4-dihydro-
quinolin-2(1H)-one (0.50 g, 2.23 mmol) and NBS (0.40 g, 2.23
mmol); yield 0.61 g (90%); white solid; mp 175−176 °C; Rf = 0.25
(DCM/MeOH, 50:1); δH (CDCl3, 500 MHz) 2.70 (t, J = 7.9 Hz, 2H,
CH2), 3.09 (t, J = 7.9 Hz, 2H, CH2), 7.35−7.37 (m, 2H), 7.64 (d, J =
1.8 Hz, 1H), 7.81 (dt, J = 1.8, 7.9 Hz, 1H), 7.84 (s, br, 1H, NH), 8.61
(dd, J = 1.5, 5.4 Hz, 1H), 8.79 (d, J = 1.8 Hz, 1H); MS (ESI): m/z =
303 [M+ + H].
ASSOCIATED CONTENT
* Supporting Information
■
S
The detailed description of deleterious effects of excessive
aldosterone; the synthetic procedures and characterization of
rest intermediates as well as the 13C NMR spectra and HPLC
purities of all final compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*For R.W.H.: phone, +(49) 681 302 70300; fax, +(49) 681 302
70308; E-mail, rolf.hartmann@helmholtz-hzi.de; web site,
http://www.helmholtz-hzi.de/?id=3897. For Q.H.: E-mail, q.
hu@mx.uni-saarland.de.
Notes
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N.; Muss, H. B.; Palmer, M.; Yu, C.; Goss, P. E. Competing causes of
death from a randomized trial of extended adjuvant endocrine therapy
for breast cancer. J. Natl. Cancer Inst. 2008, 100, 252−260.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to Dr. Christina Zimmer, Jeannine Jung, and
Jannine Ludwig for the help in determination of bioactivity, and
we thank Professor J. Hermans (Cardiovascular Research
Institute, University of Maastricht, The Netherlands) for
providing us with V79MZh11B cells expressing human
CYP11B1.
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ABBREVIATIONS USED
BC, breast cancer; CYP, cytochrome P450; ER, estrogen
receptor; PgR, progesterone receptor; SERM, selective estrogen
■
H
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