Stereochemical requirements for the mineralocorticoid receptor antagonist activity of dihydropyridines

Article abstract of DOI:10.1021/jm1002827

A number of known 1,4-dihydropyridine CCBs were identified as having comparable potency to the steroidal MR antagonist eplerenone. Chiral resolution of mebudipine revealed that MR and CCB activity reside in opposite enantiomers. Small molecule X-ray crystal structures showed that the C4 stereochemistry of optimized selective MR analogues, e.g. 5, is consistent with MR-active mebudipine. Molecular modeling supports a binding pose consistent with that previously proposed for DHP diesters.

Full text of DOI:10.1021/jm1002827

4300 J. Med. Chem. 2010, 53, 4300–4304
DOI: 10.1021/jm1002827
Stereochemical Requirements for the Mineralocorticoid Receptor Antagonist
Activity of Dihydropyridines
Graciela B. Arhancet,* Scott S. Woodard, Jessica D. Dietz, Danny J. Garland, Grace M. Wagner, Kaliappan Iyanar,
Joe T. Collins, James R. Blinn, Randal E. Numann, Xiao Hu, and Horng-Chih Huang
Pfizer Global Research & Development, St. Louis Laboratories, Pfizer Inc., St. Louis, Missouri 63017
Received March 3, 2010
A number of known 1,4-dihydropyridine CCBs were identified as having comparable potency to the
steroidal MR antagonist eplerenone. Chiral resolution of mebudipine revealed that MR and CCB
activity reside in opposite enantiomers. Small molecule X-ray crystal structures showed that the C4
stereochemistry of optimized selective MR analogues, e.g. 5, is consistent with MR-active mebudipine.
Molecular modeling supports a binding pose consistent with that previously proposed for DHP diesters.
Introduction
It is estimated that almost one in three adults in the U.S. has
high blood pressure (BPa), putting them at a markedly
increased risk of major cardiovascular and renal diseases
and shortened life expectancy.1 Several classes of antihyper-
tensive drugs have been developed; these include diuretics,
calcium channel-blockers (CCB), and drugs that target the
renin-angiotensin-aldosterone system, including rennin in-
Figure 1. DHP diesters with MR antagonist activity comparable to
hibitors, angiotensin converting enzyme inhibitors (ACEi),
eplerenone.
and angiotensin receptor blockers (ARBs).2 Aldosterone is a
reported that during screening for MR antagonists a number
of 1,4-dihydropyridine (DHP) CCBs were found to inhibit
aldosterone-induced activation of a luciferase reporter driven
by MR ligand binding domain (LBD).8 4-Aryl-1,4-dihydro-
pyridines have also been claimed as inhibiting [3H]aldosterone
binding to MR at 10 μM, however no data was disclosed
regarding the functional activity of this series.9
1,4-DHP CCBs act at the L type voltage dependent calcium
channel and are potent peripheral vasodilators which have been
extensively studied since their introduction almost 30 years
ago.10 Although the first DHP CCBs developed (e.g., nifedipine,
Figure 1) were achiral molecules, chiral DHPs with nonidentical
ester groups in the 3- and 5-positions were later developed in
search of more potent and longer acting vasodilators (e.g.,
felodipine, mebudipine 2) although they are marketed as race-
mic mixtures. The absolute configuration at the C4 position of
enantiomerically pure 1,4-DHP CCBs, such as nicardipine and
barnidipine, has been established to be a crucial determinant of
CCB activity.11 These findings prompted us to examine the
stereochemical requirements for MR activity in DHPs with the
goal of designing potent MR selective inhibitors.
steroid hormone that mediates sodium reabsorption by bind-
ing to the mineralocorticoid receptor (MR, NR3C2) a mem-
ber of the nuclear receptor superfamily of ligand-dependent
transcription factors. Abnormal activation of the MR by
elevated levels of aldosterone and salt imbalance cause hy-
pertension and other detrimental effects to the cardiovascular
system such as heart failure and myocardial fibrosis.3 Two
approaches have been developed to treat this abnormal
activation of the MR. One approach involves lowering ele-
vated aldosterone levels using aldosterone synthase inhibi-
tors.4 Alternatively MR antagonists, such as spironolactone
and more recently eplerenone 1, block MR activation, which
results in lowering of blood pressure in hypertensive patients
and improves survival in heart failure patients.5 However,
steroidal MR antagonists in general present issues of complex
chemical synthesis, undesirable physical properties, and poor
selectivity versus other steroid hormone receptors. The latter
results in unwanted clinical side effects, for example, gynoco-
mastia in male patients due to spironolactone’s poor selecti-
vity against the androgen receptor.6 Thus, there has been a
keen interest in discovering novel classes of nonsteroidal
selective MR antagonists in recent years.7 We previously
Results and Discussion
*To whom correspondence should be addressed. Phone: 314-991-
7932. E-mail: grace.arhancet@gmail.com.
Screening of the Pfizer compound collection identified a
number of widely used calcium channel antagonist 1,4-DHPs,
such as nifedipine and felodipine, as having MR antagonist
activity comparable to eplerenone 1 (Figure 1).
Since a number of groups have previously reported on the
stereoselectivity of calcium channel antagonism activity for
a Abbreviations: BP, blood pressure; CCB, calcium channel blocker;
ACEi, angiotensin converting enzyme inhibitor; ARBs, angiotensin
receptor blockers; MR, mineralocorticoid receptor; DHP, 1,4-dihydro-
pyridine; LBD, ligand binding domain; ER, estrogen receptor; PR,
progesterone receptor; GR, glucocorticoid receptor; AR, androgen
receptor; PK, pharmacokinetic; CL, clearance.
r
pubs.acs.org/jmc
Published on Web 04/21/2010
2010 American Chemical Society

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4301
Table 2. MR and CCB Activity of 3-Cyano-DHPsa
Scheme 1. Synthesis and Resolution of Mebudipine 2a
MR
IC50
(nM)
L-type Caþþ
channel
IC50 (nM)
compd
R1
R2
R3
Me
conf
5
A
A
A
B
B
B
B
B
B
B
A
A
t-Bu
t-Bu
t-Bu
Me
Me
Et
racemic
S
59
>1550
3840
6
Me
Me
3520
3.7
b
*
7
R
8
Me
Me
S
469
77
14570
7730
1100
1330
2200
11270
7930
570
9
R
racemic
a Reagents: (i) EtOH/piperidine (cat.), reflux, 12 h; (ii) chiral SFC.
10
11
12
13
14
15
16
Pr
Pr
31
Et
Et
S
R
S
R
S
R
3280
10
Table 1. MR and CCB Activity of Mebudipine and its Stereoisomersa
Pr
Me
Me
Me
Me
-CH2Cl
-CH2Cl
-CH2Cl
-CH2Cl
2800
340
2200
41
MRAT
IC50 (nM)
L-type Caþþ channel
IC50 (nM)
compd
stereoisomer
1
2
3
4
135
126
46
>5000
>20
607
2070
a IC50 values were obtained through curve-fitting of dose response
(n g 3/concentration,6-10 concentrations) using the 4-parameter logis-
tic model. Standard error of the IC50 was generally less than 30%. b * not
determined.
racemic
(þ)-isomer
(-)-isomer
536
>0.5
a IC50 values were obtained through curve-fitting of dose response
(n g 3/concentration,6-10 concentrations) using the 4-parameter logis-
tic model. Standard error of the IC50 was generally less than 30%.
Scheme 2. Synthesis of 3-Cyano DHPsa
DHPs, we were particularly interested in studying the MR
activity of asymmetric DHPs where different substitutions of
the ester groups render the molecule chiral. We envisioned
that the possibility of calcium channel and MR antagonist
activity residing in different stereoisomers of an asymmetric
DHP would allow for the development of a new class of
selective MR antagonists. To investigate the stereochemical
origin of the MR activity observed in traditional DHPs, we
chose 2, a newer asymmetric 1,4-DHP that has been reported
to have improved pharmacokinetic properties with respect to
nifedipine.12 Compound 2 was prepared by a variation of the
Hantzsch synthesis from tert-butyl oxobutanoate, (Z)-methyl
3-aminobut-2-enoate, and 3-nitrobenzaldehyde as shown in
Scheme 1.13
The resulting racemic product was then resolved into the
corresponding enantiomers, (þ)-mebudipine 3 and (-)-me-
budipine 4, using chiral supercritical fluid chromatography
(SFC) on an asymmetric resin with a mobile phase containing
a mixture of ethanol/CO2. The ability of racemic 2 and
enantiomers 3 and 4 to antagonize MR and L-type calcium
channel was investigated (Table 1).
MR inhibition potency was evaluated on aldosterone-
induced activation of a luciferase reporter driven by MR
ligand binding domain fused to a heterologous DNA binding
domain from yeast transcription factor Gal4. A fluorescence-
based calcium indicator dye was used to quantify the influx of
extracellular calcium through the L-type calcium channel in
the A10 cell line of rat thoracic origin. The L-type calcium
channel was activated upon depolarization induced by the
addition of elevated extracellular potassium, producing an
increase in calcium sensitive fluorescence within the cells that
was measured on the kinetic plate reader. Enantiomer (-)-4
exhibited excellent CCB activity and more than 1000 times
greater activity than the corresponding (þ) isomer 3 in the
L-type Caþþ channel assay. Previous studies on the CCB
antagonist activity of asymmetric DHPs showed the S-con-
figuration at the C4 position to be required for activity.11 On
a Reagents and conditions: (i) EtOH/piperidine (cat.), reflux, 8-12 h
the basis of this precedent, we assigned the absolute config-
uration of CCB active isomer 4, (-)-isomer, as S (Scheme 1).
To our pleasant surprise, CCB inactive isomer (þ)-3
(R-isomer), showed excellent MR antagonist activity while
(-)-4 was roughly 10 times less potent. These findings in-
dicated that it was possible to design MR selective antagonists
with a 1,4-DHP core and prompted us to continue our
investigation. In an attempt to improve its metabolic stability,
we turned our attention to the replacement of the methyl ester
group with a cyano group, which led to very potent MR
antagonists (Table 2). Racemic compounds, 5 and 10, pre-
pared as shown in Scheme 2, exhibited good MR antagonist
activity and no CCB activity. Chiral resolution and testing of
the corresponding enantiomers confirmed our findings with 3
and 4 isomers, and only one enantiomer of 5 and 10 (7 and 12,
respectively) exhibited MR antagonist activity. The complete
SAR studies for the 5-cyano series will be reported elsewhere.
On the basis of MR potency, a group of enantiomerically
pure analogues was selected for evaluation of their selectivity
versus other steroidal nuclear hormone receptors, determined
in a similar Gal4 cellular assay format (Table 3).
The new cyano ester DHPs were highly selective against the
estrogen receptor (ER) and the progesterone receptor (PR)
while also exhibiting greater than 20-fold selectivity over the
glucocorticoid receptor (GR) and the androgen receptor (AR).
To get an early reading on any potential liabilities of the
new scaffold that could hinder further optimization, com-
pounds 7 and 12 were selected for evaluation of their phar-
macokinetic parameters in rats. For pharmacokinetic (PK)
studies, male Sprague-Dawley rats (n = 2) were dosed

4302 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Arhancet et al.
Table 3. Nuclear Receptor Selectivity for Select Enantiomerically
Pure DHPsa
MR
compd IC50 (nM) IC50 (nM) IC50 (nM) IC50 (nM) IC50 (nM)
GR
AR
PR
ER
b
3
46
3.7
10
3260
1370
270
1270
246
768
3520
1340
*
7
12
>10000
>10000
730
a IC50 values were obtained through curve-fitting of dose response
(n g 3/concentration, 6-10 concentrations) using the 4-parameter
logistic model. Standard error of the IC50 was generally less than 30%.
b * not determined.
Figure 3. Representative docking pose of MR active DHP 7.
(a) Top induced fit docking pose in wt 2A3I MR-LBD crystal structure
with H12 truncated. (b) Comparison with native steroid pose (red),
showing induced clash between Trp 806 and helix 12 Leu 960.
Table 4. Rat Pharmacokinetic Data for 7 and 12
dose
(mpk)
CL
(mL/min/kg)
T1/2
(h)
Vdss
(L kg)
A-ring pocket, making a tee interaction with Phe 829 and
(in compound 12) a hydrogen bond to Gln (Figure 3a). The
DHP fails to make stabilizing contacts with the steroid D-
ring-binding area of helix 11, including Thr 945.15 Perturba-
tion of Trp 806 by the cyano and methyl groups may also
contribute an active antagonism component by inducing close
contact with helix 12 residue Leu 960 (Figure 3b). In contrast,
the S enantiomers 6 and 11 (as well as 4) typically placed the
4-aryl group in the R-face pocket and occupied the A-ring
pocket poorly if at all.
compd
3
7
12
2
2
84.8
9.7
1.14
6.54
4720
0.42
Conclusions
The present study demonstrates that the stereochemical
configuration of asymmetric 1,4-DHPs is critical for their
CCB or MR antagonist activity. Indeed, while it has been
previously reported that CCB active asymmetric DHPs pos-
sess the S configuration at C4, this study establishes that MR
activity requires the R configuration at C4. Replacement of
the ester group at the C3 position by a cyano group led to a
series of very active MR antagonists with no CCB activity.
These findings provide important pharmacophore informa-
tion for the exploration of an attractive target and a drug-like
scaffold for the design of novel MR antagonists.
Figure 2. X-ray crystal structure of MR active enantiomer 16.
intravenously (iv) at 2 mg/kg. Compounds were formulated
for iv dosing in 10% ethanol/40% PEG400/50% phosphate
buffered saline pH7.4. Plasma samples were analyzed by
LC/MS/MS. The PK profiles of compounds 7 and 12 are
shown in Table 4. While 7 exhibited a moderate to high
clearance (CL), the CL measured for 12 was low and the
half-life was adequate for future proof-of-concept study in rats.
To positively establish the configuration at C4 for the MR
active 5-cyano DHP series, compound 16 was prepared in
racemic form according to the procedure shown in Scheme 2.
The enantiomers were resolved by chiral chromatography
and tested for MR activity. An X-ray crystal structure of
MR-active enantiomer 16 was obtained (Figure 2), and the
configuration at C4 was determined to be R. Similarly, the
structure of a derivative of 14 was solved and the stereochem-
istry at C4 was again shown to be R (see Supporting Informa-
tion, SI). These results confirmed our initial C4 configuration
assignment based on the CCB activities of (þ) and (-)-
mebudipine.
Experimental Section
All materials were obtained from commercial sources and used
as purchased. Chromatography solvents were HPLC grade and
were used without further purification. Thin layer chromatogra-
phy (TLC) analysis was performed using Merck silica gel
60 F-254 thin layer plates. LC-MS analyses were performed on
Mariner TOF from Perseptive Biosystems. The scan range was
100-1000 d. The sample was introduced by flow inject from an
Agilent 1100 with 100 μL/min MeOH (10 mM ammonium
acetate) into the electrospray source. Preparative reverse phase
HPLC was performed on a Gilson 215 liquid handler equipped
with a Dynamax Microsorb C18; (300 A) column (41.4 mm ꢀ 25
cm, 8 μm) and Gilson 156 variable length UV detector. Analytical
chiral chromatography was performed on a OJ-H column
(Chiral Technologies, 4.6 mm ꢀ 250 mm) eluted with 5% etha-
nol/hexane. The purity of tested compounds was g95% as
determined by combustion analysis or by HPLC conducted on
an Agilent 1100 system using a reverse phase C8 column with
diode array detector. NMR spectra were recorded on a Bruker
400 spectrometer. The signal of the deuterated solvent was used
as internal reference. Chemical shifts (d) are given in ppm and
are referenced to residual not fully deuterated solvent signal.
Coupling constants (J) are given in Hz.
Induced-fit docking14 was used to examine potential bind-
ing mode differences between the R and S enantiomers, with
similar results being obtained for both the wild-type (2A3I)
and S810L (2AA6) MR crystal structures, in each case with
helix 12 truncated to allow for active antagonism. For the
R enantiomers 7 and 12, the top group of poses consistently
favored the binding mode previously hypothesized for DHP
diesters.8 In this pose, the NH of the DHP ring donates a
hydrogen to helix 3 Asn 770, the hydrophobic ester group
fills the R-face pocket, and the DHP 4-aryl ring resides in the
General Procedure for the Preparation of 3-Cyanodihydropyr-
idines. tert-Butyl 4-(2-Chloro-4-fluorophenyl)-5-cyano-2,6-di-
methyl-1,4-dihydropyridine-3-carboxylate (5). 4-Chloro-2-fluoro-
benzaldehyde (1 mmol), 3-aminocrotonitrile (1 mmol), and tert-
butyl acetoacetate (1 mmol) were dissolved in EtOH (20 mL).
Piperidine (cat.) was added, and the reaction mixture was refluxed

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4303
gently overnight. The solvent was evaporated under vacuum, and
the resulting crude product was purified by preparative HPLC
(30-100% CH3CN/H2O/0.1% TFA over 40 min at a flow rate of
35 mL/min) to yield 236 mg (65%) of 5 as a white solid. 1H NMR
(400 MHz, DMSO-d6) δ ppm 1.18 (s, 9 H), 1.95 (s, 3 H), 2.25 (s, 3
H), 4.99 (s, 1 H), 7.20-7.30 (m, 2 H), 7.38 (dd, J = 8.99, 2.74 Hz,
1 H), 9.11 (s, 1 H). MS (ES) m/z 363 ([M þ H]þ).
Methyl (4S)-2-(Chloromethyl)-5-cyano-6-methyl-4-quinolin-
4-yl-1,4-dihydropyridine-3-carboxylate (13) and Methyl (4R)-
2-(Chloromethyl)-5-cyano-6-methyl-4-quinolin-4-yl-1,4-dihydro-
pyridine-3-carboxylate (14). To a solution of quinoline-4-
carbaldehyde (4 mmol) in 2-propanol was added methyl
4-chloroacetoacetate (4 mmol), acetic acid (20 mg), and piper-
idine (35 mg). The reaction was stirred at room temperature (rt)
under nitrogen for 20 h. 3-Aminocrotonitrile (4 mmol) was
added, and the resulting mixture was stirred at rt for another
20 h. Concentrated hydrochloric acid (0.25 mL) was then added
and the stirring continued for 2 h. The reaction mixture was
concentrated in vacuo, and the resulting solid (50% yield) was
then subjected to chiral separation of the enantiomers under the
same conditions described for 8 and 9. 13 and 14 were character-
ized as the HCl salt. 13: 1H NMR (400 MHz, DMSO-d6) δ ppm
2.00(s, 3 H), 3.28(s, 3 H), 4.74(d, J = 11.00 Hz, 1 H), 4.78(d, J =
11.00 Hz, 1 H), 5.75 (s, 1 H), 7.67 (d, J = 5.10 Hz, 1 H), 7.87
(t, J = 7.92 Hz, 1 H), 8.02 (t, J = 7.52 Hz, 1 H), 8.21 (d, J = 8.59
Hz, 1 H), 8.67 (d, J = 8.86 Hz, 1 H), 9.12 (d, J = 5.37 Hz,
1 H), 9.96 (s, 1 H); HRMS (C19H16ClN3O2 þ Hþ) calcd 354.1004,
found 354.0972; >99.5% ee by chiral HPLC. 14: 1H NMR (400
MHz, DMSO-d6) δ ppm 2.00 (s, 3 H), 3.28 (s, 3 H), 4.74 (d, J =
11.00 Hz, 1 H), 4.79 (d, J = 11.01 Hz, 1 H), 5.77 (s, 1 H), 7.71 (d,
J = 5.37 Hz, 1 H), 7.90 (ddd, J = 8.46, 7.11, 1.07 Hz, 1 H), 8.05
(ddd, J = 8.32, 7.11, 0.94 Hz, 1 H), 8.26 (d, J = 8.05 Hz, 1 H),
8.69 (d, J = 8.59 Hz, 1 H), 9.15 (d, J = 5.37 Hz, 1 H), 10.01 (s,
1 H); HRMS (C19H16ClN3O2 þ Hþ) calcd 354.1004, found
354.0974; >99.5% ee by chiral HPLC.
tert-Butyl (4S)-4-(2-Chloro-4-fluorophenyl)-5-cyano-2,6-dime-
thyl-1,4-dihydropyridine-3-carboxylate (6) and tert-Butyl (4R)-
4-(2-Chloro-4-fluorophenyl)-5-cyano-2,6-dimethyl-1,4-dihydropyr-
idine-3-carboxylate (7) were obtained by chiral chromatography
of 5, same conditions as described for 2. 6 (white solid): 1H
NMR (400 MHz, DMSO-d6) δ ppm 1.09 (s, 9 H), 1.87 (s, 3 H),
2.17 (s, 3 H), 4.91 (s, 1 H,) 7.08-7.33 (m, 3 H), 9.02 (s, 1 H); MS
(ES) m/z 363 ([M þ H]þ); >99.5% ee by chiral HPLC. 7 (white
solid): MS (ES) m/z 363 ([M þ H]þ); 99.5% ee by chiral HPLC.
Note: optical purity of 6 and 7 was checked from stock solutions
at 1, 2, and 4 weeks.
Methyl (4S)-5-Cyano-2,6-dimethyl-4-quinolin-4-yl-1,4-dihy-
dropyridine-3-carboxylate (8) and Methyl (4R)-5-Cyano-2,6-di-
methyl-4-quinolin-4-yl-1,4-dihydropyridine-3-carboxylate (9).
Following the general procedure, quinoline-4-carbaldehyde was
reacted with 3-aminocrotonitrile and methyl acetoacetate to gen-
erate the corresponding cyanodihydropyridine as a solid (60%
yield), which was then subjected to chiral separation of the
enantiomers on a OD-H column (Chiral Technologies, 21 mm ꢀ
250 mm), eluted with 10% ethanol/CO2, 70 mL/min to yield
1
each enantiomer. 8 (solid): H NMR (400 MHz, DMSO-d6) δ
ppm 1.97 (s, 3 H), 2.33 (s, 3 H), 3.25 (s, 3 H), 5.45 (s, 1 H), 7.30
(d, J = 4.56 Hz, 1 H), 7.63 (ddd, J = 8.46, 6.85, 1.34 Hz, 1 H), 7.74
(ddd, J = 8.32, 6.98, 1.34 Hz, 1 H), 7.99 (d, J = 8.32 Hz, 1 H), 8.40
(d, J = 8.32 Hz, 1 H), 8.84 (d, J = 4.57 Hz, 1 H), 9.34 (s, 1 H);
HRMS (C19H17N3O2 þ Hþ) calcd 320.1934, found 320.1389;
>99.5% ee by chiral HPLC. 9 (solid): 1H NMR (400 MHz,
DMSO-d6) δ ppm 1.97 (s, 3 H), 2.33 (s, 3 H), 3.25 (s, 3 H), 5.45
(s, 1 H), 7.29 (d, J = 4.56 Hz, 1 H), 7.62 (ddd, J = 8.39, 6.91, 1.34
Hz, 1 H), 7.74 (ddd, J = 8.39, 6.91, 1.34 Hz, 1 H), 7.99 (dd, J =
8.46, 0.94 Hz, 1 H), 8.40 (d, J = 8.32 Hz, 1 H), 8.83 (d, J = 4.56
Hz, 1 H), 9.33 (s, 1 H); HRMS (C19H17N3O2 þ Hþ) calcd
320.1394, found 320.1393; >99.5% ee by chiral HPLC.
Methyl (4S)-(2-Chloro-4-fluorophenyl)-2-(chloromethyl)-5-cy-
ano-6-methyl-1,4-dihydropyridine-3-carboxylate (15) and Methyl
(4R)-(2-Chloro-4-fluorophenyl)-2-(chloromethyl)-5-cyano-6-meth-
yl-1,4-dihydropyridine-3-carboxylate (16). Racemic product was
prepared following the same procedure for 14 using 4-chloro-2-
fluorobenzaldehyde in place of quinoline-4-carbaldehyde to gen-
erate methyl 4-(2-chloro-4-fluorophenyl)-2-(chloromethyl)-5-cy-
ano-6-methyl-1,4-dihydropyridine-3-carboxylate as a solid (62%
yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 2.01 (s, 3 H), 3.47
(s, 3 H), 4.71 (q, J = 10.83 Hz, 2 H), 5.07 (s, 1 H), 7.22 (m, 1 H),
7.31 (m, 1 H), 7.37 (dd, J = 9.00, 2.55 Hz, 1 H), 9.68 (s, 1 H).
HRMS (C16H13Cl2FN2O2 þ NH4þ) calcd 372.0676, found
372.0719. Product was then subjected to chiral separation of the
enantiomers on an OJ-H column (Chiral Technologies, 30 mm ꢀ
250 mm) eluted with 10%n-butanol/CO2, 70 mL/min flow. 15:
MS(ES) m/z 355 ([Mþ H]þ); >99.5% ee by chiral HPLC. 16:MS
(ES) m/z 355 ([M þ H]þ); >99.5% ee by chiral HPLC.
Ethyl 5-Cyano-6-methyl-2-propyl-4-quinolin-4-yl-1,4-dihydro-
pyridine-3-carboxylate (10). Following the general procedure,
quinoline-4-carbaldehyde was reacted with 3-aminocrotonitrile
1
and ethyl acetoacetate to yield 10 as a yellow solid (72%). H
NMR (400 MHz, DMSO-d6) δ ppm 0.67 (t, J = 7.03 Hz, 3 H),
0.98 (t, J = 7.42 Hz, 3 H), 1.59-1.69 (m, 2 H), 2.00 (s, 3 H),
2.63-2.74 (m, 2 H), 3.67-3.75 (m, J = 10.57, 7.02, 3.71 Hz, 2 H),
5.49 (s, 1 H), 7.32 (d, J = 4.69 Hz, 1 H), 7.64 (t, J = 7.81 Hz, 1 H),
7.76 (t, J = 7.03 Hz, 1 H), 8.02 (d, J = 7.42 Hz, 1 H), 8.43 (d, J =
8.20 Hz, 1 H), 8.87 (d, J = 4.69 Hz, 1 H), 9.31 (s, 1 H). Anal.
(C22H23N3O2) C, H, N.
Single Crystal X-ray Analysis. A representative crystal of
˚
compound 16 was surveyed and a 1 A data set (maximum sin
Θ/λ = 0.5) was collected on a Bruker P4/R diffractometer.
Friedel pairs were collected in order to facilitate the determina-
tion of the absolute configuration. Atomic scattering factors
were taken from the International Tables for X-ray Crystal-
lography.16 All crystallographic calculations were facilitated
by the SHELXTL system.17 C16H13N2O2FCl2; FW = 355.18;
monoclinic; space group P2(1); unit cell dimensions: a =
Ethyl 5-Cyano-6-methyl-2-propyl-4-quinolin-4-yl-1,4-dihydro-
pyridine-3-carboxylate (11) and Ethyl 5-Cyano-6-methyl-2-pro-
pyl-4-quinolin-4-yl-1,4-dihydropyridine-3-carboxylate (12) were
obtained by chiral separation of 10 under same conditions
for the preparation of 8 and 9. 11 (yellow solid): 1H NMR
(400 MHz, DMSO-d6) δ ppm 0.62 (t, J = 7.03 Hz, 3 H), 0.94
(t, J = 7.33 Hz, 3 H), 1.44-1.62 (m, 2 H), 1.96 (s, 3 H), 2.55-
2.77 (m, 2 H), 3.66 (m, 2 H), 5.44 (s, 1 H), 7.27 (d, J = 4.49 Hz,
1 H), 7.60 (dd, J = 15.24, 1.17 Hz, 1 H), 7.72 (t, J = 7.62 Hz,
1 H), 7.97 (d, J = 8.40 Hz, 1 H), 8.38 (d, J = 8.40 Hz, 1 H), 8.82
(d, J = 4.69 Hz, 1 H), 9.26 (s, 1 H); (ES) m/z 362 ([M þ H]þ);
˚
˚
˚
12.1090(10) A, b = 9.0150(10) A, c = 15.5900(10) A; volume =
3
3
˚
1628.0(2) A ; Z = 4; D calcd = 1.449 mg/m ; absorption co-
efficient = 3.779 mm-1; F(000) = 728; GOF on F2 = 0.988;
final R indices [I > 2σ(I)]; R1 = 0.0374, wR2 = 0.0992. The re-
fined structure was plotted using the SHELXTL plotting pack-
age(Figure 2). Theabsolute configurationwasdetermined bythe
method of Flack.18 Coordinates, anisotropic temperature fac-
tors, distances, and angles are available as SI (Tables S1-S5).
Cell-Based Gal4 Response Element-Controlled Luciferase Re-
porter Assays. MR Luciferase Reporter Antagonist Assay.
HUH7 human hepatocyte cells were maintained in RPMI
1640 plus 10% FBS and transfected with Gal4-MRLBD con-
struct and a luciferase reporter under Gal4 control. After
transfection, compounds were added in RPMI 1640 media plus
10% heat-inactivated and charcoal dextran stripped FBS
(Hyclone) with and without 1 nM aldosterone. Cells were
1
>99.5% ee by chiral HPLC. 12 (yellow solid): H NMR (400
MHz, DMSO-d6) δ ppm 0.62 (t, J = 7.13 Hz, 3 H), 0.94 (t, J =
7.33 Hz, 3 H), 1.49-1.73 (m, 2 H), 1.96 (s, 3 H), 2.51-2.74 (m,
2 H), 3.27 (s, 3 H) 3.66 (m, 2 H), 5.44 (s, 1 H), 7.27 (d, J = 4.69
Hz, 1 H), 7.60 (ddd, J = 8.50, 6.93, 1.37 Hz, 1 H), 7.72 (ddd, J =
8.35, 6.89, 1.37 Hz, 1 H), 7.97 (dt, J = 8.45, 0.76 Hz, 1 H), 8.38
(dt, J = 8.64, 0.66 Hz, 1 H), 8.82 (d, J = 4.49 Hz, 1 H), 9.26 (s,
1 H); MS (ES) m/z 362 ([M þ H]þ); >99.5% ee by chiral HPLC.

4304 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Arhancet et al.
harvested 20 h later for reporter activity as described in ref 7.
Selectivity against other steroid receptors were assayed in the
same manner using Gal4-driven luciferase as reporter.
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Vieira, U.; Biemel, K.; Marchais-Oberwinkler, S.; Hartmann, R. W.
Synthesis and Evaluation of Heteroaryl-Substituted Dihydronaphtha-
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acic rat cell line A10 and a calcium sensing fluorescence dye run
on a kinetic plate reader. The A10 cells were plated into a
Biocoat 384-well black-walled, clear bottom plate at 1500 cells
per well. Cells were cultured for 48 h prior to running the
experiment. One hour before running, the media was removed
from the cells, and they were loaded with 50 μL of no wash
calcium dye Calcium 4 (Molecular Devices R-8141) following
the manufacturers instructions. The plate was then loaded onto
the FLIPRTETRA plate reader (Molecular Devices) along with
the compound plate and a stimulus plate (384 well Greiner)
containing 140 mM KCl to depolarize the cells and activate the
L type calcium channel. After establishing a baseline read, 25 μL
of compound from the compound plate (source 1) was dispensed
into each well of the cell plate. Ten minutes later, 25 μL of the
depolarizing KCl solution (source 2) was added to each well
(final [Kþ] = 45 mM). The peak of the depolarizing response
after the second addition (stimulus) was used to produce a
concentration response curve.
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Novel procedure for modeling ligand/receptor induced fit effects.
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Docking Studies. Molecular modeling was conducted using
the Schrodinger Suite 2009 (Schrodinger Suite 2009 Induced Fit
Docking protocol, Glide version 5.5, Prime version 2.1, Jaguar
version 7.6; Schrodinger LLC, New York, NY). The wild type
MR and S810L docking structures were prepared from the
˚
1.95 A MR structure with bound corticosterone (PDB:
2A3I)19 and the 1.95 A S810L crystal structure with bound
˚
progesterone (PDB: 2AA6),15 respectively by removal of waters,
truncation of helix 12 (AF-2) to the C-terminus (residues
958-984), and capping of Pro 957 as the N-methylacetamide.
Dihydropyridine ligands 7 and 12 were geometry optimized
using Jaguar (B3LYP/6-31G**//B3LYP/6-31G**) to ensure
appropriate dihydropyridine ring geometries; the correspond-
ing enantiomers 6 and 11 were obtained by chiral inversion. The
induced fit docking protocol14 was used to dock the dihydro-
pyridine ligands (3, 4, 6, 7, 11, 12, and R- and S-mebudipine) into
the truncated receptor models, using the default induced fit
docking protocol parameters with the exception of truncating
the side chain of Met 845 during initial Glide docking and using
XP scoring for the redock step. No hydrogen bonding or other
constraints were used. Pictures were generated using Maestro
9.0.111 (Schrodinger LLC).
Acknowledgment. We thank Jon Bordner and Ivan
Samardjiev for solving the X-ray crystal structure of compounds
16 and the methyl thio ether derivative of compound 14.
Supporting Information Available: X-ray data for compound
16 and methyl thio ether derivative of compound 14. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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