Novel pyridyl substituted 4,5-dihydro-[1,2,4]triazolo[4,3-a ]quinolines as potent and selective aldosterone synthase inhibitors with improved in vitro metabolic stability

Article abstract of DOI:10.1021/acs.jmedchem.5b00079

CYP11B2 inhibition is a promising treatment for diseases caused by excessive aldosterone. To improve the metabolic stability in human liver miscrosomes of previously reported CYP11B2 inhibitors, modifications were performed via a combination of ligand-and

Full text of DOI:10.1021/acs.jmedchem.5b00079

Article
pubs.acs.org/jmc
Novel Pyridyl Substituted 4,5-Dihydro-
[1,2,4]triazolo[4,3‑a]quinolines as Potent and Selective Aldosterone
Synthase Inhibitors with Improved in Vitro Metabolic Stability
Qingzhong Hu,†,# Lina Yin,†,‡,# Amjad Ali,§ Andrew J. Cooke,∥ Jonathan Bennett,∥ Paul Ratcliffe,∥
Michael Man-Chu Lo,§ Edward Metzger,§ Scott Hoyt,⊥ and Rolf W. Hartmann*,†
†Pharmaceutical and Medicinal Chemistry, Saarland University and Helmholtz Institute for Pharmaceutical Research Saarland
(HIPS), Campus C2-3, D-66123, Saarbrucken, Germany
̈
‡ElexoPharm GmbH, Campus A1, D-66123 Saarbrucken, Germany
̈
§Discovery Chemistry, Merck Research Laboratories, 126 East Lincoln Avenue, Rahway, New Jersey 07065, United States
∥Exploratory Chemistry, Merck Research Laboratories, Bo’Ness Road, Newhouse, Lanarkshire ML1 5SH, United Kingdom
⊥Medicinal Chemistry, Merck Research Laboratories, 2000 Galloping Hill Road, Kenilworth, New Jersey 07033, United States
S
* Supporting Information
ABSTRACT: CYP11B2 inhibition is a promising treatment for
diseases caused by excessive aldosterone. To improve the metabolic
stability in human liver miscrosomes of previously reported
CYP11B2 inhibitors, modifications were performed via a combina-
tion of ligand- and structure-based drug design approaches, leading
to pyridyl 4,5-dihydro-[1,2,4]triazolo[4,3-a]quinolones. Compound
26 not only exhibited a much longer half-life (t1/2 ≫ 120 min), but
also sustained inhibitory potency (IC50 = 4.2 nM) and selectivity
over CYP11B1 (SF = 422), CYP17, CYP19, and a panel of hepatic
CYP enzymes.
over 11β-hydroxylase (CYP11B1) with a selectivity factor (SF
= IC50 CYP11B1/IC50 CYP11B2) of 15, low doses (<1.3 mg daily)
had to be applied to avoid impairments of cortisol biosynthesis
and consequently only moderate beneficial effects were
observed.5 This fact revealed the importance of selectivity
over steroidogenic CYP enzymes, in particular CYP11B1. The
reported CYP11B2 inhibitors6 competitively bind to the
enzyme via coordination of their sp2 hybrid N to the heme
iron, which is an important constituent in the catalytic centers
of all CYP enzymes. This mechanism of reversible inhibition
was first identified for CYP19 inhibitors7 and was later also
applied in the design of CYP178 and CYP11B1 inhibitors.9
However, this strategy together with the conserved con-
formation among steroidogenic CYP enzymes leads to the
challenge to achieve selectivity, in particular between CYP11B1
and CYP11B2, which share more than 93% of homological
identity.
INTRODUCTION
■
CYP11B2 is a mitochondrial cytochrome P450 (CYP) enzyme,
which is crucial in aldosterone biosynthesis. As a major
mineralocorticoid, aldosterone not only regulates homeostasis
of electrolytes and fluids but also acts as a potent pro-
inflammation factor1a that is capable of inducing reactive
oxygen species1b and up-regulating many fibrosis related
genes.1c High aldosterone levels are thus involved in some
chronic diseases such as hypertension, primary aldosteronism,
congestive heart failure, cardiac fibrosis, ventricular remodel-
ling, and diabetic nephropathy. Blocking the deleterious effects
of aldosterone by antagonism of mineralocorticoid receptors or
interference with the renin−angiotensin−aldosterone system
(e.g., inhibitors of angiotensin converting enzyme or renin) has
demonstrated clinical benefits. However, associated side
effects2a and/or the phenomenon of aldosterone escape2b
limit further application. In contrast, CYP11B2 inhibition has
been proposed as a superior therapy for these diseases.3
CONCEPT OF INHIBITOR DESIGN
■
Nonselective adrenal suppressor FAD286 (R-Fadrozole, IC50
=
Via ligand-based drug design or screening of focused
compound libraries and subsequent optimizations, potent and
selective CYP11B2 inhibitors were discovered with compound
16a (Figure 1) as an example (IC50 = 28 nM, SF = 241).
0.8 nM, Table 1) significantly reduced the plasma aldosterone
levels in rats with heart failure and hence improved cardiac
hemodynamics and functions.4 Another CYP11B2 inhibitor,
LCI699 (IC50 = 0.2 nM, Table 1), has been clinically evaluated
in patients with primary hyperaldosteronism or hypertension
and demonstrated the expected pharmacodynamic effects.
However, because this compound showed poor selectivity
Received: January 16, 2015
Published: February 25, 2015
© 2015 American Chemical Society
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Table 1. Inhibition of CYP11B1and CYP11B2 by
Compounds 4−29, Fadrazole, and LCI699
a
structure
IC50 (nM)
b
compd
F
R1
R2
CYP11B2 CYP11B1
SF
4
8
9
8
9
8
9
8
9
8
9
8
9
8
9
9
9
9
9
9
9
9
9
9
9
9
9
H
H
H
H
H
H
H
H
H
H
H
H
H
244.0
172.2
131.4
62.5
7900
16100
12620
16990
≫10000
≫10000
≫10000
≫10000
≫10000
≫10000
≫10000
≫10000
≫10000
≫10000
1810
32
94
5
H
6
Me
Me
Et
96
7
272
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
89
8
979.3
541.7
>10000
>10000
>10000
>10000
4354.4
707.7
>10000
>10000
20.3
9
Et
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
LCI699
fadrazole
i-Pr
i-Pr
Ph
Ph
Bz
Bz
4-Py
4-Py
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
H
H
m-OMe
m-(1-OH)-Et
m-Ac
28.3
3910
138
78
87.6
6970
m-CF3
m-CN
57.2
3570
63
1177.9
89.7
≫10000
29540
428
nd
329
60
m-F
m-Ph
7.1
Figure 1. Design of title compounds from lead 1 using a combination
of ligand- and structure-based drug design approaches. The docking of
lead 1 (A) and compound 26 (B) in the CYP11B2 crystal structure
(PDB ID: 4DVQ) revealed an identical binding manner with two
distinct poses (orange or celadon green) for each compounds.
2.2
110
49
p-Me
4.2
1770
422
232
nd
134
15
m-F, p-Me
m-F
19.5
4520
335.0
42.5
>10000
5705
H
m-F, p-Me
0.2
2.9
dihydroquinolinone core because they could also be vulnerable
to metabolism. These aromatic protons were therefore replaced
by F at different positions to block potential metabolism
(Figure 1). Because the carbonyl of the amido moiety was
demonstrated to form hydrogen bonds,6a the CO oxygen was
exchanged by N as a bioisostere that can sustain these
interactions. An additional ring was also considered to connect
the resulting two N atoms and thus conceal the amido moiety.
At this stage, a structure-based approach was employed in
combination to decide how to build this extra ring. Lead 1 was
therefore docked into the CYP11B2 crystal structure (PDB ID:
4DVQ),11 in which this compound was anchored by the
coordination between pyridyl N and heme iron in a nearly
perpendicular manner (Figure 1A). An orientation similar to
that of the natural substrate deoxycorticosterone was adopted
(Figures 1A and 2A, the latter was depicted with compound 26,
which exhibited the same binding poses), namely the
hydrophobic core pointing to the β2-sheet rather than I-helix.
π−π interactions were observed between the core and Phe130
(perpendicular) and Phe487 (parallel). Two binding poses
were actually identified in which the amido O formed hydrogen
bonds with the OH of Glu383 or Tyr485. The bioisosteric
exchange of carbonyl O with an N sustained these interactions
(Figure 1B), while an adjacent second N could form additional
hydrogen bonds with the backbone NH of Glu383 or Phe381.
0.8
6.3
8.3
a
Standard deviations were within < 5%; All the data are the mean
b
values of at least 3 tests. SF, selective factor = IC50 CYP11B1
IC50 CYP11B2; nd, not determined.
/
Further modifications led to more potent inhibitors 26b and 3,6c
with IC50 values of less than 3 nM and excellent SFs of 626 and
166 (Figure 1), respectively. However, their metabolic
stabilities in human liver microsomes were not satisfying: the
half-life of compound 1 was 58 min (intrinsic clearance, CLint
=
25 μL/(mg·min)), whereas those of 2 and 3 were shorter than
20 min (CLint of 153 and 81 μL/(mg·min), respectively). This
shortcoming largely limits their potential as drug candidates
and urged us to perform scaffold hopping to increase stability
while sustaining potency and selectivity. A study on compound
1 revealed that the major metabolite shows a mass of 224.0899
and a loss of an OH radical in the fragmentation spectrum,
indicating the formation of an N-oxide of the pyridine or a
hydroxamic acid yielded from the hydroxylation of the lactam.10
Because the sp2 hybrid N is essential for CYP11B2 inhibition,
the pyridyl moiety has to be sustained and the o-positions to
the N have to be unsubstituted to avoid steric hindrance of
coordination. In contrast, the bare lactam is considered as a site
for further modifications. So are aromatic protons in the
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RESULTS AND DISCUSSION
■
Chemistry. The synthesis of compounds 4−29 started from
the corresponding m- or p-fluoroanilines (Scheme 1), which
underwent amidation with 3-chloropropanoyl chloride and
cyclization via Friedel−Crafts alkylation to form 3,4-dihydro-
quinolin-2(1H)-ones 4d and 5d. For p-fluoro 5d, the Friedel−
Crafts reaction was simply performed in neat, whereas N,N-
dimethylacetamide had to be employed for m-fluoro 4d to
prevent the ring from closing at the position next to the F.
Subsequently, site-specific bromination was achieved on these
intermediates using N-bromosuccinimide at low temperature,
which was followed by a Suzuki coupling with 3-pyridyl boronic
acid to insert a pyridyl as heme-coordinating group. The
resulting pyridyl dihydroquinolinones 4b and 5b were
subsequently sulphurated with Lawesson’s reagent to facilitate
the following annulation with various acylhydrazines in
cyclohexanol, which afforded the 4,5-dihydro-[1,2,4]triazolo-
[4,3-a]quinolinones with different substituents at the 1-
position, leading to compounds 4−17. Similarly, diversification
was reserved to the last step when modifications on the pyridyl
(18−29) were performed. The 9-fluoro-4,5-dihydro-[1,2,4]-
triazolo[4,3-a]quinoline core in 18b and 28b was first built as
described above. Their bromo groups were subsequently
converted to boronic acid (18a and 28a) using triisopropyl
borate and n-BuLi, which coupled with the corresponding
heterocyclic bromines to yield compounds 18−29.
CYP11B2 and CYP11B1 Inhibition. The inhibitory
activities of the synthesized compounds were determined in
V79MZh cells expressing hCYP11B2 or hCYP11B1. IC50 values
are presented in comparison to fadrozole and LCI699 in Table
1. Metabolic stability in human hepatic microsomes was also
determined for selected compounds.
Substituents on the dihydrotriazoloquinoline core demon-
strated significant influence on CYP11B2 inhibition with
bulkiness as a crucial determinant. Analogues without any
substitution at 1-position (4 and 5) exhibited potent inhibition
with IC50 values around 200 nM (Table 1). The introduction of
a methyl group (6 and 7) promoted the potency by 2−3-fold to
131 and 63 nM, respectively. In contrast, dramatically, further
augmentation of bulkiness to ethyl (8 and 9), i-propyl (10 and
11), phenyl (12 and 13), benzyl (14 and 15), and 4-pyridyl (16
and 17) resulted in large decrease or even a loss of inhibitory
potency, indicating steric clashes with the binding pocket.
Interestingly, benzyl analogues 14 and 15 exhibited weak to
moderate CYP11B2 inhibition (IC50 values of 4354 and 708
nM, respectively). This is probably because the benzyl moiety
could fit into a nearby hydrophobic pocket confined by Arg110-
Met111-Ile112-Leu113-Val117-Leu131-Phe130 (Figure 3) at
the cost of attenuating H-bonds formed by triazolo moiety.
Furthermore, the position of fluoro substitution also impacted
CYP11B2 inhibition. The 9-F analogue 7 (IC50 = 63 nM) is
Figure 2. Binding of 11-deoxycorticosterone ((A) green) and
fadrazole ((B) celadon green) as well as the docking poses of
compounds 24 ((B,C) magenta), 25 ((C) yellow), and 26 ((A,C)
orange) in the CYP11B2 crystal structure (PDB ID: 4DVQ).
With the annulation of a 5-membered aromatic ring, additional
π−π interactions with Phe381 were also observed. The F atoms
introduced could also increase binding affinity via forming
orthogonal multipolar interactions with the carbonyl of Phe487
and hydrogen bonds with the backbone NH of Ile488 or the
guanidyl of Arg110. Thus, the fluorinated 4,5-dihydro-[1,2,4]-
triazolo[4,3-a]quinoline core was designed (Figure 1). The
influence of F in different positions and various substituents at
the 1-position of the core was investigated, leading to
compounds 4−17. After ascertaining the most suitable
substituents pattern, the heme binding pyridyl was modified
by introducing various substituents yielding compounds 18−
29.
twice as potent as the corresponding 8-F compound 6 (IC50
=
131 nM). Similar observations were noted when comparing
further compound pairs (4 and 5, 8 and 9, as well as 14 and
15). This finding indicates that F at the 9-position might be
able to interact with the protein. In contrast, CYP11B1
inhibition was found to be unsusceptible to the change of
substituents. All compounds exhibited weak to no inhibition,
which thus endowed these potent CYP11B2 inhibitors with
high selectivity, in particular, compound 7, exhibiting an IC50
value of 63 nM and a SF of 272. This profile is similar to that of
lead 1 (IC50 = 28 nM, SF = 241). More importantly, compound
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a
Scheme 1
a
Reagents and conditions: (i) 3-chloropropanoyl chloride, pyridine, THF, rt; (ii) AlCl3, 140 °C, 4 h (8-F, DMA, or 9-F, neat); (iii) NBS, DMF, 0 °C,
9 h; (iv) method A, Pd(OAc)2, corresponding boronic acid, Na2CO3, TBAB, toluene, H2O, ethanol, reflux, 6 h; (v) Lawesson’s reagent, toluene,
reflux, 2 h; (vi) method B, corresponding acylhydrazine, cyclohexanol, reflux, 6 h. (vii) n-BuLi, triisopropyl borate, toluene, THF, HCl, −40 °C, 3 h.
did not improve selectivity (SF = 49) due to an elevated
CYP11B1 inhibition (IC50 = 110 nM), it resulted in the most
potent CYP11B2 inhibitor in this study (IC50 = 2.2 nM). Three
possible reasons were conjectured for the elevation of
CYP11B2 inhibition. First, the fused benzene moiety increased
the electron density at the N atom, thus promoting the
coordination with the heme iron. Second, the fused benzene
moiety reduced the rotation freedom between the core and the
pyridyl ring, resulting in a more favorable conformation. Lastly,
the fused benzene moiety, similar to the m-Ph substituent in
compound 24, occupied a hydrophobic pocket confined by
Met309-Glu310-Leu311-Ala313-Arg120-Leu131-Leu227-
Met230 (Figure 2B), which is exploited by fadrazole in the
cocrystal structure (PDB ID: 4FDH)11 and formed perpendic-
Figure 3. Docking of compound 15 in CYP11B2 crystal (PDB ID:
4DVQ). The benzyl moiety could fit into the hydrophobic pocket
confined by Arg110-Met111-Ile112-Leu113-Val117-Leu131-Phe130.
ular π−π interactions with Trp116, Phe231, and Trp260.
However, the occupation of an additional pocket was also
thought to be responsible for the elevation of CYP11B1
inhibition. Therefore, to improve selectivity, the fused phenyl
moiety was removed; whereas a methyl group was sustained at
the p-position as an electron donating residue rigidifying the
conformation (Figure 2C). The resulting compound 26
7 showed an elongated half-life in human hepatic microsomes
(t1/2 > 120 min), which is more than twice as long as that of
lead 1 (t1/2 = 58 min). Encouraged by this finding, further
optimizations on the heme-coordinating pyridyl moiety were
showed potent inhibition toward CYP11B2 with an IC50
performed to improve inhibitory potency and selectivity with
value of 4.2 nM, whereas its inhibition of CYP11B1 was largely
the 9-F, 1-Me substitution pattern sustained. Accordingly,
decreased as expected (IC50 = 1770 nM), leading to an
various substituents with different bulkiness and electrostatic
excellent selectivity of 422. The combination of m-F and p-Me
(27) resulted in potent CYP11B2 inhibition (IC50 = 20 nM) in
accordance with the SAR described above (compare with
compounds 23 and 26). Very good selectivity of 232 was also
exhibited, however, not as excellent as those of compounds
with only m-F or p-Me (329 and 422, respectively). Analogues
of compounds 23 and 27 without substituents at the 1-position
of the core (28 and 29) were also synthesized to reverify the
importance of the 1-Me group. Both compounds were less
potent (IC50 values of 335 and 43 nM, respectively) and less
selective than their corresponding analogues. Furthermore, the
most selective CYP11B2 inhibitors in this series, namely
compounds 23 and 26, were subsequently evaluated for
metabolic stability in human hepatic microsomes. Both
compounds exhibited low intrinsic clearance (CLint of 12 and
4 μL/(mg·min), respectively) and thus very long half-lives (t1/2
≫ 120 min; 80% of compound 26 remained unchanged after
120 min of incubation).
potential were introduced into the pyridyl ring leading to
compounds 18−27. When electron donating groups such as
methoxy (18) and 1-OH-ethyl (19) were furnished at the m-
position of the pyridyl ring, the analogues showed a 3-fold more
potent inhibition than their precursor 7 with IC50 values around
25 nM. These two compounds also exhibited good selectivity
over CYP11B1, with SFs of 89 and 138, respectively. However,
electron withdrawing groups such as acetyl (20), trifluor-
omethyl (21), and fluoro (23) led to similar or slightly reduced
CYP11B2 inhibition compared to the parent compound 7, with
IC50 values ranging from 57 to 90 nM. Interestingly, a cyano
substituent (22) strongly decreased the inhibitory potency to
1178 nM. Good selectivity over CYP11B1 (SFs around 70)
were shown by compounds 20 and 21, while the fluoro
analogue 23 achieved an excellent selectivity of 329. Moreover,
the introduction of a bulky electron donating phenyl group at
the m-position of the pyridyl ring (24) significantly elevated
both inhibition of CYP11B2 (IC50 = 7 nM) and CYP11B1
(IC50 = 428 nM). Subsequently, via a strategy of bioisosteric
transformation, this phenyl group was fused to the pyridyl,
forming a 4-isoquinolinyl moiety (25). Although this exchange
Selectivity over CYP17 and CYP19. The inhibition of
human CYP17 and CYP19 by the synthesized potent CYP11B2
inhibitors (IC50 < 100 nM) were determined to evaluate safety
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smephenytoin, 34 μM; bufuralol, 11 μM; midazolam, 3 μM;
testosterone, 40 μM; nifedipine, 8 μM). Test compounds were run
in duplicate at six concentrations up to 50 μM. At a single time point,
incubations were quenched with acetonitrile and centrifuged, and the
supernatant was transferred and diluted with water prior to LC−MS/
MS analysis of probe metabolites. Amounts of metabolites produced
were normalized to vehicle controls, and IC50 values were calculated
using XLFit.
Chemistry Section. General. Melting points were determined on
a Mettler FP1 melting point apparatus and are uncorrected. IR spectra
were recorded neat on a Bruker Vector 33FT-infrared spectrometer.
1H 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
determined 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).
Method A: 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.
because these enzymes are crucial in the biosynthesis of sex
hormones. All tested compounds exhibited IC50 values of more
than 10 μM, indicating no inhibition of both enzymes at
relevant concentrations.
Inhibition of Hepatic CYP Enzymes. Because of the
important roles of hepatic CYP enzymes in the metabolism of
xenobiotics and drugs, the inhibition of these enzymes is
another important criterion in evaluating the safety of drug
candidates. Compounds 23 and 26 showed no inhibition (IC50
> 50 μM) against a panel of hepatic CYP enzymes including
CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4,
indicating good safety profiles regarding this aspect.
CONCLUSION
■
CYP11B2 inhibition is a promising treatment for diseases that
are related to excessive aldosterone. Although several classes of
potent and selective CYP11B2 inhibitors were reported by our
group, the compounds suffered from low metabolic stability in
human liver microsomes. Scaffold hopping via a combination of
ligand- and structure-based drug design approaches was
performed to solve this problem, leading to pyridyl 4,5-
dihydro-[1,2,4]triazolo[4,3-a]quinolones. This strategy was
demonstrated to be successful because the resulting compounds
not only sustained CYP11B2 inhibition and selectivity but also
significantly prolonged half-lives. Compound 26 is more potent
and selective over CYP11B1 (IC50 = 4.2 nM, SF = 422) than
lead compound 1 (IC50 = 28 nM, SF = 241). It is similarly
potent as LCI699 and fadrazole but far more selective. Inhibitor
26 exhibited largely improved in vitro metabolic stability in
human liver microsomes (t1/2 ≫ 120 min), which is much
longer than that of lead 1 (t1/2 = 58 min). Satisfying selectivity
over steroidogenic CYP17, CYP19, and a panel of hepatic CYP
enzymes was also achieved. Because of these superior activities,
compound 26 is considered as a promising candidate for
further evaluation in vivo.
Method B: Cyclization with acylhydrazine. The suspension of
dihydroquinoline-2-thione (1.0 equiv) and corresponding acylhydra-
zine (1.2 equiv) in cyclohexanol (1 mL/mmol) was refluxed for 6 h
under inert atmosphere. After cooling down to ambient temperature,
cyclohexanol was distilled off and the residue was taken up with ethyl
acetate (10 mL) and water (10 mL). Then the organic phase was
separated, and the water phase was extracted with ethyl acetate (2 ×
10 mL). The combined organic phases were washed with brine, dried
over Na2SO4, and evaporated under reduced pressure to afford the
crude products. The compounds were purified by flash chromatog-
raphy on silica gel.
EXPERIMENTAL SECTION
■
Inhibition of CYP11B1 and CYP11B2. V79 MZh cells expressing
human CYP11B2 or CYP11B112a were incubated with [14C]-
deoxycorticosterone as substrate and inhibitors in various concen-
trations. After 6 h, the reaction was stopped and the steroids were
extracted with ethyl acetate, separated by HPTLC, and quantified
using a phosphoimager. Then the inhibitory potency was calculated
from the reduced substrate conversion.12b
8-Fluoro-7-pyridin-3-yl-4,5-dihydro-[1,2,4]triazolo[4,3-a]-
quinoline (4). Synthesized according to method B using 4a (0.50 g,
1.94 mmol) and formohydrazide (0.14 g, 2.32 mmol); yield 0.39 g
(75%); white solid; mp 246−247 °C; Rf = 0.21 (DCM/MeOH, 20:1).
δH (CDCl3, 500 MHz): 3.11 (t, J = 7.5 Hz, 2H, CH2), 3.27 (t, J = 7.5
Hz, 2H, CH2), 7.27 (d, 3JHF = 10.0 Hz, 1H), 7.41 (ddd, J = 0.7, 4.8, 8.0
Inhibition of CYP19. The inhibition of CYP19 was determined in
vitro using human placental microsomes with [1β-3H]androstenedione
as substrate.12c
Inhibition of CYP17. Human CYP17 was expressed in Escherichia
coli12d (coexpressing human CYP17 and NADPH-P450 reductase),
and the assay was performed as previously described.12e
In Vitro Metabolic Stability. Human (pooled, mixed gender)
liver microsomes were diluted with potassium phosphate−MgCl2
buffer (100 mM, pH 7.4) to a final protein concentration of 0.5
mg/mL. After the addition of test compounds (final concentration 1
μM) and NADPH system, it was incubated at 37 °C. Samples were
taken at time points of 2, 5, 15, 30, and 45 min and quenched with
acetonitrile. They were further vortexed and centrifuged at 4000 rpm
for 15 min. Supernatant was subsequently aliquoted for LC−MS/MS
analysis.
Inhibition of Hepatic CYP Enzymes. Inhibition of drug
metabolizing CYP enzymes was determined following a single
incubation approach using recombinant human CYP enzymes
(CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4). Incubations
included a cocktail of probe substrates at final concentrations
approximating their Km (phenacetin, 50 μM; diclofenac, 6 μM;
4
Hz, 1H), 7.45 (d, JHF = 7.7 Hz, 1H), 7.88 (dq, J = 1.8, 7.9 Hz, 1H),
8.61 (s, 1H), 8.66 (dd, J = 1.6, 4.8 Hz, 1H), 8.79 (s, 1H). MS (ESI):
m/z = 267 [M+ + H]. δC (CDCl3, 125 MHz) 20.6 (CH2), 25.4 (CH2),
105.1 (d, 2JCF = 27.8 Hz), 123.4 (2C), 123.6 (d, 4JCF = 4.2 Hz), 130.3,
131.3 (d, 4JCF = 4.1 Hz), 132.6 (d, 3JCF = 9.9 Hz), 136.1 (d, 4JCF = 3.5
4
1
Hz), 137.5, 149.4, 149.5 (d, JCF = 4.4 Hz), 150.3, 158.9 (d, JCF
=
250.3 Hz).
9-Fluoro-7-pyridin-3-yl-4,5-dihydro-[1,2,4]triazolo[4,3-a]-
quinoline (5). Synthesized according to method B using 5a (0.50 g,
1.94 mmol) and formohydrazide (0.14 g, 2.32 mmol); yield 0.41 g
(79%); white solid; mp 210−211 °C; Rf = 0.19 (DCM/MeOH, 20:1).
δH (CDCl3, 500 MHz): 3.19 (t, J = 7.5 Hz, 2H, CH2), 3.30 (t, J = 7.5
Hz, 2H, CH2), 7.39−7.43 (m, 3H), 7.87 (dt, J = 2.1, 7.9 Hz, 1H), 8.66
(dd, J = 1.3, 4.8 Hz, 1H), 8.83 (d, JHF = 3.5 Hz, 1H), 8.84 (d, J = 2.0
Hz, 1H). MS (ESI): m/z = 267 [M+ + H]. δC (CDCl3, 125 MHz) 26.7
2
3
(CH2), 30.9 (CH2), 114.2 (d, JCF = 20.7 Hz), 120.6 (d, JCF = 11.2
Hz), 123.4 (d, 4JCF = 2.8 Hz), 123.8, 131.1, 134.2 (2C), 137.8 (d, 3JCF
2534
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Article
= 7.6 Hz), 140.1 (d, 3JCF = 13.7 Hz), 148.0, 149.6, 150.1, 152.5 (d, 1JCF
= 249.4 Hz).
9-Fluoro-1-methyl-7-(5-trifluoromethyl-pyridin-3-yl)-4,5-dihydro-
[1,2,4]triazolo[4,3-a] quinoline (21). Synthesized according to
method A using 18a (0.35 g, 1.42 mmol) and 3-bromo-5-
(trifluoromethyl)pyridine (0.38 g, 1.70 mmol); yield 0.43 g (88%);
white solid; mp 243−244 °C; Rf = 0.21 (DCM/MeOH, 20:1). δH
(CDCl3, 500 MHz) 2.66 (d, JHF = 8.3 Hz, 3H, CH3), 3.07 (t, J = 7.5
Hz, 2H, CH2), 3.14 (t, J = 7.5 Hz, 2H, CH2), 7.43 (dd, J = 1.8 Hz, 3JHF
= 10.2 Hz, 1H), 7.45 (d, J = 1.7 Hz, 1H), 8.10 (dd, J = 2.0, 2.1 Hz,
1H), 8.94 (d, J = 2.2 Hz, 1H), 9.03 (d, J = 2.0 Hz, 1H). MS (ESI): m/z
8-Fluoro-1-methyl-7-pyridin-3-yl-4,5-dihydro-[1,2,4]triazolo[4,3-
a]quinoline (6). Synthesized according to method B using 4a (0.50 g,
1.94 mmol) and acetohydrazide (0.17 g, 2.32 mmol); yield 0.42 g
(78%); white solid; mp 227−228 °C; Rf = 0.19 (DCM/MeOH, 20:1).
δH (CDCl3, 500 MHz): 2.78 (s, 3H, CH3), 3.04 (t, J = 7.5 Hz, 2H,
CH2), 3.15 (t, J = 7.5 Hz, 2H, CH2), 7.33 (d, 3JHF = 10.9 Hz, 1H), 7.39
(ddd, J = 0.7, 4.8, 8.0 Hz, 1H), 7.44 (d, 4JHF = 8.0 Hz, 1H), 7.86 (dq, J
= 1.8, 8.0 Hz, 1H), 8.62 (dd, J = 1.6, 4.8 Hz, 1H), 8.77 (s, 1H). MS
(ESI): m/z = 281 [M+ + H]. δC (CDCl3, 125 MHz) 13.8 (CH3), 21.4
= 349 [M++H]. δC (CDCl3, 125 MHz) 13.3 (d, JCF = 17.2 Hz, CH3),
2
22.2 (CH2), 28.1 (CH2), 115.1 (d, JCF = 22.8 Hz), 121.3 (q, JCF3
=
287.3 Hz), 122.2 (d, 4JCF = 6.3 Hz), 123.7 (d, 4JCF = 2.5 Hz), 131.4 (d,
4JCF = 3.7 Hz), 134.5, 135.0, 135.2, 136.5 (d, 3JCF = 8.1 Hz), 146.5 (d,
(CH2), 26.5 (CH2), 106.2 (d, 2JCF = 28.3 Hz), 123.4, 123.6 (d, 3JCF
=
4
4
13.8 Hz), 125.5 (d, JCF = 3.7 Hz), 130.3, 131.0 (d, JCF = 4.4 Hz),
4JCF = 4.0 Hz), 150.0, 151.3, 152.9, 153.1 (d, JCF = 251.9 Hz).
1
133.9 (d, 3JCF = 10.8 Hz), 136.0 (d, 4JCF = 3.3 Hz), 148.1, 149.2, 149.4
4
1
5-(9-Fluoro-1-methyl-4,5-dihydro-[1,2,4]triazolo[4,3-a]quinolin-
7-yl)-nicotinonitrile (22). Synthesized according to method A using
18a (0.35 g, 1.42 mmol) and 5-bromonicotinonitrile (0.31 g, 1.70
mmol); yield 0.38 g (89%); white solid; mp 267−268 °C; Rf = 0.20
(DCM/MeOH, 20:1). δH (CDCl3, 500 MHz): 2.66 (d, JHF = 8.3 Hz,
3H, CH3), 3.07 (t, J = 7.5 Hz, 2H, CH2), 3.14 (t, J = 7.5 Hz, 2H,
(d, JCF = 3.3 Hz), 151.7, 158.6 (d, JCF = 249.1 Hz).
9-Fluoro-1-methyl-7-pyridin-3-yl-4,5-dihydro-[1,2,4]triazolo[4,3-
a]quinoline (7). Synthesized according to method B using 5a (0.50 g,
1.94 mmol) and acetohydrazide (0.17 g, 2.32 mmol); yield 0.44 g
(81%); white solid; mp 195−196 °C; Rf = 0.20. (DCM/MeOH, 20:1).
δH (CDCl3, 500 MHz): 2.51 (d, JHF = 8.2 Hz, 3H, CH3), 2.89 (t, J =
7.5 Hz, 2H, CH2), 2.99 (t, J = 7.5 Hz, 2H, CH2), 7.11 (s, 1H), 7.23−
7.28 (m, 2H), 7.73 (dt, J = 2.1, 8.0 Hz, 1H), 8.51 (dd, J = 1.3, 4.8 Hz,
1H), 8.71 (d, J = 2.0 Hz, 1H). MS (ESI): m/z = 281 [M+ + H]. δC
(CDCl3, 125 MHz) 13.1 (d, JCF = 17.9 Hz, CH3), 22.0 (CH2), 27.9
(CH2), 114.6 (d, 2JCF = 21.9 Hz), 121.5 (d, 3JCF = 11.7 Hz), 123.2 (d,
3
CH2), 7.41 (dd, J = 1.8 Hz, JHF = 11.9 Hz, 1H), 7.42 (d, J = 1.7 Hz,
1H), 8.15 (dd, J = 2.0, 2.1 Hz, 1H), 8.92 (d, J = 1.9 Hz, 1H), 9.03 (d, J
= 2.3 Hz, 1H). MS (ESI): m/z = 306 [M+ + H]. δC (CDCl3, 125
MHz) 13.0 (d, JCF2 = 17.8 Hz, CH3), 21.9 (CH2), 27.8 (CH2), 110.5
4
(CN), 114.9 (d, JCF = 23.1 Hz), 116.0, 123.3 (d, JCF = 2.6 Hz),
3
4JCF = 2.2 Hz), 123.8, 134.1, 134.2, 134.5, 138.1 (d, JCF = 8.2 Hz),
125.7, 126.2, 134.4, 135.1, 137.1, 149.8, 151.1, 151.7, 152.6, 152.9 (d,
1JCF = 256.0 Hz).
1
148.0, 149.6, 150.6, 152.1 (d, JCF = 255.6 Hz), 153.8.
9-Fluoro-7-(5-fluoro-pyridin-3-yl)-1-methyl-4,5-dihydro-[1,2,4]-
triazolo[4,3-a]quinoline (23). Synthesized according to method A
using 18a (0.35 g, 1.42 mmol) and 3-bromo-5-fluoropyridine (0.30 g,
1.70 mmol); yield 0.39 g (92%); white solid; mp 215−216 °C; Rf =
0.23 (DCM/MeOH, 20:1). δH (CDCl3, 500 MHz): 2.65 (d, JHF = 8.3
Hz, 3H, CH3), 3.04 (t, J = 7.5 Hz, 2H, CH2), 3.13 (t, J = 7.5 Hz, 2H,
9-Fluoro-7-(5-methoxy-pyridin-3-yl)-1-methyl-4,5-dihydro-
[1,2,4]triazolo[4,3-a]quinoline (18). Synthesized according to method
A using 18a (0.35 g, 1.42 mmol) and 3-bromo-5-methoxypyridine
(0.32 g, 1.70 mmol); yield 0.39 g (89%); white solid; mp 196−197 °C;
Rf = 0.22 (DCM/MeOH, 20:1). δH (CDCl3, 500 MHz): 2.65 (d, JHF
=
8.2 Hz, 3H, CH3), 3.04 (t, J = 7.5 Hz, 2H, CH2), 3.12 (t, J = 7.5 Hz,
2H, CH2), 3.95 (s, 3H, OCH3), 7.34 (dd, J = 1.9, 2.6 Hz, 1H), 7.38
(dd, J = 1.8 Hz, 3JHF = 11.4 Hz, 1H), 7.40 (d, J = 1.7 Hz, 1H), 8.35 (d,
J = 2.7 Hz, 1H), 8.45 (d, J = 1.8 Hz, 1H). MS (ESI): m/z = 311 [M+ +
H]. δC (CDCl3, 125 MHz) 13.0 (d, JCF = 17.7 Hz, CH3), 22.0 (CH2),
27.8 (CH2), 55.8 (OCH3), 114.7 (d, 2JCF = 22.5 Hz), 118.9, 121.2 (d,
3JCF = 11.4 Hz), 123.3 (d, 4JCF = 2.6 Hz), 134.4, 134.7, 137.2, 138.0 (d,
3
CH2), 7.39 (dd, J = 1.9 Hz, JHF = 14.6 Hz, 1H), 7.41 (d, J = 1.7 Hz,
1H), 7.59 (ddd, J = 2.0, 2.6 Hz, 3JHF = 9.2 Hz, 1H), 8.52 (d, J = 2.7 Hz,
1H), 8.67 (t, J = 1.5 Hz, 1H). MS (ESI): m/z = 299 [M+ + H]. δC
(CDCl3, 125 MHz) 13.1 (d, JCF = 17.6 Hz, CH3), 22.0 (CH2), 17.8
(CH2), 114.8 (d, 2JCF = 22.5 Hz), 120.9 (d, 2JCF = 19.0 Hz), 121.7 (d,
3JCF = 11.3 Hz), 123.4 (d, 4JCF = 2.7 Hz), 134.8, 135.6, 136.5 (d, 3JCF
=
2
4
8.6 Hz), 137.9 (d, JCF = 23.0 Hz), 143.8 (d, JCF = 3.9 Hz), 149.8,
1
3JCF = 7.8 Hz), 140.3, 149.8, 152.7, 152.8 (d, JCF = 251.2 Hz), 155.9.
1
1
152.7, 152.8 (d, JCF = 251.9 Hz), 159.6 (d, JCF = 258.8 Hz).
9-Fluoro-1-methyl-7-(5-phenyl-pyridin-3-yl)-4,5-dihydro-[1,2,4]-
triazolo[4,3-a]quinoline (24). Synthesized according to method A
using 18a (0.35 g, 1.42 mmol) and 3-bromo-5-phenylpyridine (0.40 g,
1.70 mmol); yield 0.43 g (85%); white solid; mp 209−210 °C; Rf =
0.20 (DCM/MeOH, 20:1). δH (CDCl3, 500 MHz) 2.65 (d, JHF = 8.3
Hz, 3H, CH3), 3.05 (t, J = 7.5 Hz, 2H, CH2), 3.14 (t, J = 7.5 Hz, 2H,
CH2), 7.44−7.47 (m, 3H), 7.51 (dd, J = 7.6, 7.8 Hz, 2H), 7.64 (d, J =
7.7 Hz, 1H), 8.03 (t, J = 2.1 Hz, 1H), 8.81 (d, J = 1.8 Hz, 1H), 8.88 (d,
J = 2.3 Hz, 1H). MS (ESI): m/z = 357 [M+ + H]. δC (CDCl3, 125
MHz) 13.1 (d, JCF = 17.6 Hz, CH3), 22.0 (CH2), 27.8 (CH2), 114.7
(d, 2JCF = 22.4 Hz), 121.2 (d, 3JCF = 11.7 Hz), 123.3 (d, 4JCF = 2.1 Hz),
127.3 (2C, Ph), 128.6, 129.3 (2C, Ph), 132.7, 134.1, 134.6, 137.0,
1-[5-(9-Fluoro-1-methyl-4,5-dihydro-[1,2,4]triazolo[4,3-a]-
quinolin-7-yl)-pyridin-3-yl]-ethanol (19). Synthesized according to
method A using 18a (0.35 g, 1.42 mmol) and 1-(5-bromopyridin-3-
yl)ethanol (0.34 g, 1.70 mmol); yield 0.33 g (72%); white solid; mp
228−229 °C; Rf = 0.12 (DCM/MeOH, 20:1). δH (CDCl3, 500 MHz):
1.59 (d, J = 6.5 Hz, 3H, CH3), 2.01 (s, br, 1H, OH), 2.65 (d, JHF = 8.2
Hz, 3H, CH3), 3.04 (t, J = 7.5 Hz, 2H, CH2), 3.09 (t, J = 7.5 Hz, 2H,
CH2), 5.08 (q, J = 6.5 Hz, 1H, CH), 7.42 (dd, J = 1.8 Hz, 3JHF = 13.2
Hz, 1H), 7.43 (d, J = 1.7 Hz, 1H), 7.99 (dd, J = 1.9, 2.6 Hz, 1H), 8.61
(d, J = 2.7 Hz, 1H), 8.71 (d, J = 1.8 Hz, 1H). MS (ESI): m/z = 325
[M+ + H]. δC (CDCl3, 125 MHz) 12.9 (d, JCF = 17.8 Hz, CH3), 21.9
(CH2), 25.5 (CH3), 27.7 (CH2), 67.6 (CH), 114.6 (d, 2JCF = 22.2 Hz),
121.0 (d, 3JCF = 11.8 Hz), 123.2 (d, 4JCF = 2.6 Hz), 131.4, 133.8, 134.4,
138.2 (d, 3JCF = 6.8 Hz), 141.7 (d, 3JCF = 8.9 Hz), 146.6 (d, 4JCF = 5.9
3
137.2, 138.0 (d, JCF = 8.0 Hz), 146.6, 148.1, 149.8, 152.8, 152.9 (d,
1JCF = 251.2 Hz).
1
Hz), 147.3, 149.8, 152.7 (d, JCF = 251.4 Hz), 152.7.
9-Fluoro-7-isoquinolin-4-yl-1-methyl-4,5-dihydro-[1,2,4]triazolo-
[4,3-a]quinoline (25). Synthesized according to method A using 18a
(0.35 g, 1.42 mmol) and 4-bromoisoquinoline (0.35 g, 1.70 mmol);
yield 0.25 g (86%); white solid; mp 209−210 °C; Rf = 0.19 (DCM/
MeOH, 20:1). δH (CDCl3, 500 MHz) 2.69 (d, JHF = 8.3 Hz, 3H,
CH3), 3.05 (t, J = 7.5 Hz, 2H, CH2), 3.17 (t, J = 7.5 Hz, 2H, CH2),
7.34−7.37 (m, 2H), 7.67−7.70 (m, 1H), 7.74−7.77 (m, 1H), 7.89 (d, J
= 8.5 Hz, 1H), 8.09 (d, J = 8.0 Hz, 1H), 8.49 (s, 2H), 9.30 (s, 1H). MS
(ESI): m/z = 331 [M+ + H]. δC (CDCl3, 125 MHz) 13.1 (d, JCF = 17.4
1-[5-(9-Fluoro-1-methyl-4,5-dihydro-[1,2,4]triazolo[4,3-a]-
quinolin-7-yl)-pyridin-3-yl]-ethanone (20). Synthesized according to
method A using 18a (0.35 g, 1.42 mmol) and 1-(5-bromopyridin-3-
yl)ethanone (0.34 g, 1.70 mmol); yield 0.39 g (86%); white solid; mp
216−217 °C; Rf = 0.19 (DCM/MeOH, 20:1). δH (CDCl3, 500 MHz):
2.66 (d, JHF = 8.3 Hz, 3H, CH3), 2.72 (s, 3H, CH3), 3.07 (t, J = 7.5 Hz,
2H, CH2), 3.14 (t, J = 7.5 Hz, 2H, CH2), 7.45 (dd, J = 1.8 Hz, 3JHF
=
13.3 Hz, 1H), 7.46 (d, J = 1.7 Hz, 1H), 8.43 (dd, J = 2.0, 2.1 Hz, 1H),
9.02 (d, J = 2.2 Hz, 1H), 9.19 (d, J = 1.9 Hz, 1H); MS (ESI): m/z =
323 [M+ + H]. δC (CDCl3, 125 MHz) 13.1 (d, JCF = 17.2 Hz, CH3),
22.0 (CH2), 26.9 (CH3), 27.8 (CH2), 114.8 (d, 2JCF = 22.8 Hz), 123.4
(d, 4JCF = 5.3 Hz), 132.4 (d, 4JCF = 3.4 Hz), 133.4, 133.8 (d, 4JCF = 3.8
Hz), 134.3 (d, 4JCF = 3.7 Hz), 134.8, 149.5, 149.8, 151.5, 152.7, 152.9
2
Hz, CH3), 22.1 (CH2), 27.8 (CH2), 117.6 (d, JCF = 21.8 Hz), 121.1
3
4
(d, JCF = 11.8 Hz), 124.0, 126.2 (d, JCF = 2.8 Hz), 127.7, 128.3,
3
128.4, 130.7, 131.2, 133.7, 134.0, 137.4 (d, JCF = 8.4 Hz), 142.8,
149.9, 151.4 (d, JCF = 252.1 Hz), 152.8, 153.0.
1
9-Fluoro-1-methyl-7-(4-methyl-pyridin-3-yl)-4,5-dihydro-[1,2,4]-
triazolo[4,3-a]quinoline (26). Synthesized according to method A
1
(d, JCF = 251.4 Hz), 161.6, 196.2 (CO).
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Journal of Medicinal Chemistry
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using 18a (0.35 g, 1.42 mmol) and 3-bromo-4-methylpyridine (0.29 g,
1.70 mmol); yield 0.38 g (91%); white solid; mp 157−158 °C; Rf =
0.22 (DCM/MeOH, 20:1). δH (CDCl3, 500 MHz) 2.62 (d, JHF = 8.2
Hz, 3H, CH3), 2.99 (t, J = 7.5 Hz, 2H, CH2), 3.09 (t, J = 7.5 Hz, 2H,
essential hypertensive patients with left ventricular hypertrophy. J. Int.
Med. Res. 2001, 29, 13−21.
(3) Hu, Q.; Yin, L.; Hartmann, R. W. Aldosterone synthase inhibitors
as promising treatments for mineralocorticoid dependent cardiovas-
cular and renal diseases. J. Med. Chem. 2014, 57, 5011−5022.
(4) Mulder, P.; Mellin, V.; Favre1, J.; Vercauteren, M.; Remy-Jouet,
I.; Monteil, C.; Richard, V.; Renet, S.; Henry, J. P.; Jeng, A. Y.; Webb,
R. L.; Thuillez, C. Aldosterone synthase inhibition improves
cardiovascular function in rats with heart failure: a comparison with
spironolactone. Eur. Heart J. 2008, 29, 2171−2179.
3
CH2), 7.12 (dd, J = 1.8 Hz, JHF = 11.7 Hz, 1H), 7.13 (d, J = 1.9 Hz,
1H), 7.20 (d, J = 5.0 Hz, 1H), 8.39 (s, 1H), 8.45 (d, J = 5.0 Hz, 1H).
MS (ESI): m/z = 295 [M+ + H]. δC (CDCl3, 125 MHz) 12.9 (d, JCF
17.8 Hz, CH3), 19.7 (CH3), 21.8 (CH2), 27.6 (CH2), 116.8 (d, 2JCF
=
=
3
4
21.8 Hz), 120.8 (d, JCF = 11.6 Hz), 125.3, 125.4 (d, JCF = 2.6 Hz),
133.8, 135.2, 138.2 (d, 3JCF = 8.1 Hz), 144.5, 149.1, 149.4, 149.7, 152.1
1
(d, JCF = 251.5 Hz), 152.7.
(5) Menard, J.; Watson, C.; Rebello, S.; Zhang, Y. M.; Dole, W. P.
Hormonal and electrolyte responses to the aldosterone synthase
inhibitor LCI699 in sodium depleted healthy subjects. J. Am. Coll.
Cardiol. 2010, 55, A61.E583.
9-Fluoro-7-(5-fluoro-4-methylpyridin-3-yl)-1-methyl-4,5-dihydro-
[1,2,4]triazolo[4,3-a]quinoline (27). Synthesized according to method
A using 18a (0.35 g, 1.42 mmol) and 3-bromo-5-fluoro-4-
1
methylpyridine (0.32 g, 1.70 mmol); yield 0.38 g (85%). H NMR
(6) (a) Lucas, S.; Heim, R.; Ries, C.; Schewe, K. E.; Birk, B.;
Hartmann, R. W. In vivo active aldosterone synthase inhibitors with
improved selectivity: lead optimization providing a series of pyridine
substituted 3,4-dihydro-1H-quinolin-2-one derivatives. J. Med. Chem.
2008, 51, 8077−8087. (b) Lucas, S.; Negri, M.; Heim, R.; Zimmer, C.;
Hartmann, R. W. Fine-tuning the selectivity of aldosterone synthase
inhibitors: insights from studies from studies of heteroaryl substituted
1,2,5,6-tetrahydropyrrolo[3,2,1-ij]quinoline-4-one derivatives. J. Med.
Chem. 2011, 54, 2307−2319. (c) Yin, L.; Hu, Q.; Emmerich, J.; Lo, M.
M.; Metzger, E.; Ali, A.; Hartmann, R. W. Novel pyridyl- or
isoquinolinyl-substituted indolines and indoles as potent and selective
aldosterone synthase inhibitors. J. Med. Chem. 2014, 57, 5179−5189.
(d) Yin, L.; Hu, Q.; Hartmann, R. W. Tetrahydropyrroloquinolinone
type dual inhibitors of aromatase/aldosterone synthase as a novel
strategy for breast cancer patients with elevated cardiovascular risks. J.
Med. Chem. 2013, 56, 460−470. (e) Roumen, L.; Peeters, J. W.;
Emmen, J. M. A.; Beugels, I. P. E.; Custers, E. M. G.; de Gooyer, M.;
Plate, R.; Pieterse, K.; Hilbers, P. A. J.; Smits, J. F. M.; Vekemans, J. A.
J.; Leysen, D.; Ottenheijm, H. C. J.; Janssen, H. M.; Hermans, J. J. R.
Synthesis, biological evaluation, and molecular modeling of 1-benzyl-
1H-imidazoles as selective inhibitors of aldosterone synthase
(CYP11B2). J. Med. Chem. 2010, 53, 1712−1725. (f) Yin, L.; Hu,
Q.; Hartmann, R. W. 3-Pyridinyl substituted aliphatic cycles as
CYP11B2 inhibitors: aromaticity abolishment of the core significantly
increased selectivity over CYP1A2. PLoS One 2012, 7 (11), e48048
DOI: 10.1371/journal.pone.0048048. (g) Grombein, C. M.; Hu, Q.;
Heim, R.; Rau, S.; Zimmer, C.; Hartmann, R. W. 1-Phenylsulfinyl-3-
(pyridin-3-yl)naphthalen-2-ols: a new class of potent and selective
aldosterone synthase inhibitors. Eur. J. Med. Chem. 2015, 89, 597−605.
(h) Grombein, C. M.; Hu, Q.; Rau, S.; Zimmer, C.; Hartmann, R. W.
Heteroatom insertion into 3,4-dihydro-1H-quinolin-2-ones leads to
potent and selective inhibitors of human and rat aldosterone synthase.
Eur. J. Med. Chem. 2015, 90, 788−796.
(500 MHz, CD3OD, δ): 2.36 (s, 3H), 2.82 (d, J = 8.0 Hz, 3H), 3.21−
3.25 (m, 4H), 7.49 (s, 1H), 7.53 (d, J = 12.2 Hz, 1H), 8.38 (s, 1H),
8.52 (s, 1H). MS (ESI): m/z = 313 [M+ + H].
ASSOCIATED CONTENT
* Supporting Information
■
S
The metabolite identification of compound 1, synthetic
methods and characterization of all intermediates and final
compounds 8−17, HPLC purity control of all final compounds,
and description of docking study. This material is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*Phone: +(49) 681 302 70300. Fax: +(49) 681 302 70308. E-
mail: rolf.hartmann@helmholtz-hzi.de. Home Page: http://
www.helmholtz-hzi.de/?id=3897.
Author Contributions
#Q.H. and L.Y. contributed to this article equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Jeannine Jung and Jannine Ludwig for bioactivity
determination, Professor J. Hermans (University of Maastricht,
The Netherlands) for V79MZh11B1 cells, and Dr. James Baker
and Thomas Bateman (Preclinical Pharmacokinetics, Merck
Research Laboratories) for determining in vitro microsomal
CLint, MetID, and CYP inhibition.
(7) (a) Hartmann, R. W.; Bayer, H.; Grun, G. Aromatase inhibitors.
̈
Syntheses and structure−activity studies of novel pyridyl-substituted
indanones, indans, and tetralins. J. Med. Chem. 1994, 37, 1275−1281.
(b) Hu, Q.; Yin, L.; Hartmann, R. W. Selective dual inhibitors of
CYP19 and CYP11B2: targeting cardiovascular diseases hiding in the
shadow of breast cancer. J. Med. Chem. 2012, 55, 7080−7089.
(c) Gobbi, S.; Hu, Q.; Negri, M.; Zimmer, C.; Belluti, F.; Rampa, A.;
Hartmann, R. W.; Bisi, A. Modulation of cytochromes P450 with
xanthone-based molecules: from aromatase to aldosterone synthase
and steroid 11β-hydroxylase inhibition. J. Med. Chem. 2013, 56, 1723−
1729. (d) Leze, M. P.; Palusczak, A.; Hartmann, R. W.; Le Borgne, M.
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ABBREVIATIONS USED
■
CYP, cytochrome P450; CYP11B2, aldosterone synthase;
CYP17, 17α-hydroxylase-17,20-lyase; CYP11B1, 11β-hydrox-
ylase; CPY19, aromatase; SF, selectivity factor = IC50 CYP11B1
IC50 CYP11B2; CLint, intrinsic clearance; t1/2, half-life
/
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