Discovery of novel cyanodihydropyridines as potent mineralocorticoid receptor antagonists

Article abstract of DOI:10.1021/jm100506y

A new 1,4-dihydropyridine 5a, containing a cyano group at the C3 position, was recently reported to possess excellent mineralocorticoid receptor (MR) antagonist in vitro potency and no calcium channel-blocker (CCB) activity. In the present study, we report the structure-activity relationships of this novel series of cyano ester dihydropyridines that resulted in R₆ substituted analogues with improved metabolic stability while maintaining excellent MR antagonist activity and selectivity against other nuclear receptors. Further structure optimization with the introduction of five-membered ring heterocycles at R₆ resulted in compounds with excellent MR antagonist potency and a suitable pharmacokinetic profile. In vivo studies of a promising tool compound in the Dahl salt-sensitive rat model of hypertension showed similar blood pressure (BP) reduction as the steroidal MR antagonist eplerenone, providing proof-of-concept (POC) for a nonsteroidal, orally efficacious MR antagonist.

Full text of DOI:10.1021/jm100506y

5970 J. Med. Chem. 2010, 53, 5970–5978
DOI: 10.1021/jm100506y
Discovery of Novel Cyanodihydropyridines as Potent Mineralocorticoid Receptor Antagonists
Graciela B. Arhancet,* Scott S. Woodard, Kaliappan Iyanar, Brenda L. Case, Rhonda Woerndle, Jessica D. Dietz,
Danny J. Garland, Joe T. Collins, Maria A. Payne, James R. Blinn, Silvia I. Pomposiello, Xiao Hu, Marcia I. Heron,
Horng-Chih Huang, and Len F. Lee
St. Louis Laboratories, Pfizer Global Research and Development, Pfizer, Inc., 700 Chesterfield Parkway West, St. Louis, Missouri 63017
Received April 26, 2010
A new 1,4-dihydropyridine 5a, containing a cyano group at the C3 position, was recently reported to possess
excellent mineralocorticoid receptor (MR) antagonist in vitro potency and no calcium channel-blocker
(CCB) activity. In the present study, we report the structure-activity relationships of this novel series of
cyano ester dihydropyridines that resulted in R6 substituted analogues with improved metabolic stability
while maintaining excellent MR antagonist activity and selectivity against other nuclear receptors. Further
structure optimization with the introduction of five-membered ring heterocycles at R6 resulted in com-
pounds with excellent MR antagonist potency and a suitable pharmacokinetic profile. In vivo studies of a
promising tool compound in the Dahl salt-sensitive rat model of hypertension showed similar blood pressure
(BP) reduction as the steroidal MR antagonist eplerenone, providing proof-of-concept (POC) for a
nonsteroidal, orally efficacious MR antagonist.
Introduction
treat this abnormal activation of the MR. One approach
involves lowering elevated aldosterone levels using aldosterone
synthase inhibitors.4 Alternatively, MR antagonists, such as
spironolactone 2 and more recently eplerenone 1 (Figure 1),
bind to the receptor, blocking the activation of target gene
transcription. This results in lowering of blood pressure in
hypertensive patients and improves survival in heart failure
patients.5 Human clinical trials have also shown that adminis-
tration of MR antagonists spironolactone and eplerenone
resulted in a greater reduction in albuminuria compared to
ACE inhibitors with similar hypotensive effects in hypertensive
patients, demonstrating the therapeutic potential of MR antago-
nists for diabetic nephropathy.6
In general, steroidal MR antagonists present issues of com-
plex chemical synthesis, undesirable physical properties, and
poor selectivity against other steroid hormone receptors. The
last results in unwanted side effects in the clinic. For example,
poor selectivity against the androgen receptor is thought to be
responsible for inducing gynocomastia in male patients trea-
ted with spironolactone.7 Thus, there has been a keen interest
in discovering novel classes of selective nonsteroidal MR
antagonists in recent years.8
The 1,4-dihydropyridine (DHP) scaffold has been used to
develop nonsteroidal MR antagonists. 4-Aryl-1,4-dihydro-
pyridines have been claimed as inhibiting [3H]aldosterone bind-
ing to MR at 10 μM. However, no data were disclosed regarding
the functional activity of this series.9 We previously reported that
a number of approved 1,4-dihydropyridine CCBs were found to
inhibit aldosterone-induced activation of a luciferase reporter
driven by the MR ligand binding domain (LBD).10 Further
study of asymmetric diester DHPs, such as mebudipine 3,
showed that only the R-isomer was MR active while the other
enantiomer was CCB active.11 Replacement of the methyl ester
in the mebudipine scaffold with a cyano group resulted in 5a
which possesses excellent MR in vitro potency and is devoid of
Hypertension or high blood pressure (BPa) is the world’s
most common killer, with an estimated one in three adults in
the U.S. being affected by this condition, putting them at a
markedly increased risk of major cardiovascular and renal
diseases and shortened life expectancy. Almost 70% of hyperten-
sive patients fail to reach their BP targets, with many needing
multiple agents to maintain their BP goal.1 Despite the anti-
hypertensive market being both mature and highly competi-
tive, current drugs are poorly differentiated in terms of clinical
efficacy. Major antihypertensive drug classes in the market
comprise diuretics, calcium channel blockers (CCB), and
drugs that target the renin-angiotensin-aldosterone system,
including rennin inhibitors, angiotensin converting enzyme
inhibitors (ACEi), and angiotensin receptor blockers (ARBs).2
Aldosterone is a steroid hormone that mediates sodium
reabsorption by binding to the mineralocorticoid receptor
(MR, NR3C2), a member of the nuclear receptor superfamily
of ligand-dependent transcription factors. Aldosterone bind-
ing initiates a series of events that lead to the interaction of
MR with the regulatory regions of target genes. Abnormal
activation of the MR by elevated levels of aldosterone and salt
imbalance causes hypertension and other detrimental effects
to the cardiovascular system such as heart failure and myo-
cardial fibrosis.3 Two approaches have been developed to
*To whom correspondence should be addressed. Phone: 636-926-7471.
Fax: 636-926-7449. E-mail: graciela.arhancet@novusint.com.
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; POC, proof of concept; NHR,
nuclear hormone receptor; SS, salt sensitive; SAR, structure-activity
relantionship; ER, estrogen receptor; PR, progesterone receptor; GR,
glucocorticoid receptor; AR, androgen receptor; PK, pharmacokinetic;
CL, clearance, HLM, human liver microsome; RLM, rat liver micro-
some; CYP, cytochrome P450.
r
pubs.acs.org/jmc
Published on Web 07/30/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 5971
Figure 1
Scheme 1. Preparation of Diester DHPs 4a-ta
driven by MR ligand binding domain fused to a hetero-
logous DNA binding domain from yeast transcription factor
Gal4.16 Initial structure-activity relationship (SAR) studies
consisted of a library focused on 4-aryl substituents to identify
preferred groups for the replacement of the nitro group in 3.
Selected examples are shown in Table 1. Small nonpolar
substituents, e.g., F, Cl, and CF3, were well tolerated at the
R1 (4a-c) and R3 positions (4m, 4o, 4r). Disubstitution with
Cl at R1 and F at R3 was found to be optimal (4s).
a Reagents and conditions: piperidine (cat.), MeOH, 65 °C, overnight.
The same general SAR for 4-aryl substituents in the diester
series was observed in the cyano tert-butyl ester series where
disubstitution with halogens (e.g., 5a and 5d) produced the
most potent analogues, as shown in Table 2. Substitution at
the nitrogen in the DHP ring (5e, 5f, 5t) was poorly tolerated,
suggesting a potential H-bond interaction of the DHP NH
groupwiththe receptor. Replacementofthetert-butylesterby
methyl ester in 5g did not result in significant changes in
potency.
Substitution of the methyl group at the R6 position by phenyl
or benzyl groups was detrimental to potency (5n, 5o, 5p).
However, substitution with smaller C2-C4 alkyl groups yielded
more potent compounds (5i-l), with the gains in potency
reaching a plateau for propyl and butyl. The CF3 group was
also well-tolerated at this position (5s). An initial assessment on
the MR selectivity for a select group of potent analogues
against other steroidal nuclear hormone receptors was deter-
mined in a similar Gal4 cellular assay format (Table 3). In
general, cyano DHPs exhibit excellent selectivity against the
estrogen receptor (ER) (data not shown). They exhibit good
selectivity against the glucocorticoid receptor (GR), while
selectivity against the progesterone receptor (PR) and the
androgen receptor (AR) tends to be more compound depen-
dent. Compound 5s was found to be especially selective across
all other nuclear hormone receptors (NHRs) tested.
CCB activity. These findings increased our interest in the deve-
lopment of DHPs as selective MR antagonists. (R)-Mebudipine
was an attractive starting point in terms of its potent MR in vitro
activity, selectivity against the L-calcium channel, and being a
druggable scaffold.12 However, the presence of a nitro group and
poor physicochemical properties, such as high lipophilicity and
low water solubility, required significant modifications to trans-
form mebudipine into a viable MR antagonist lead. Herein, we
report the discovery and optimization of novel cyano DHPs that
led to identification of potent and selective MR antagonists.
Compound 6d was advanced to in vivo studies using the Dahl
salt-sensitive (SS) rat model of hypertension13 where it provided
proof of concept (POC) for a nonsteroidal, orally efficacious
MR antagonist.
Results and Discussion
Chemistry. A library of dihydropyridine diesters 4a-t was
prepared by a modified Hantzsch reaction as shown in
Scheme 1.14 Condensation of tert-butyl oxobutanoate with
(Z)-methyl 3-aminobut-2-enoate and the corresponding
benzaldehyde in the presence of piperidine afforded the
desired dihydropyridines in acceptable yields.
Cyano ester DHPs 5a-vwere prepared by a similar procedure
as described above from 3-aminobutenenitrile as shown in
Scheme 2. Chiral separation using supercritical fluid chromato-
graphy (SFC) on an asymmetric resin with a mobile phase
containing a mixture of ethanol/CO2 afforded the corresponding
enantiomers. Chloromethyl DHP 611 was used as an advanced
intermediate to obtain R6 substituted analogues 7a-d via
nucleophilic displacement of the chloro group with the desired
heterocycle in the presence of potassium carbonate in NMP, as
shown in Scheme 2.
4,4-Disubstituted DHP 5c was prepared in three steps from
pyridine 8 by intramolecular addition of sulfonyl carbanion to
generate intermediate 9, as shown in Scheme 3.15
DHPs 5d and 5s were further derivatized as shown in Scheme 4.
The sodium salt of 5d was alkylated with bromoethane and
1-bromo-2-methoxyethane to give 5e and 5f, respectively. The
sodium salt of 5s in turn was alkylated with iodomethane to give 5t.
Despite the significant gains in potency for this series with
respect to the corresponding diester DHPs, the analogues had
highlipophilicityand thus suffered from poor solubilityandin
vitro metabolic stability as shown in Table 3.
Replacement of alkyl groups at R6 by a methylene linker
tethering five-membered ring heterocycles resulted in ana-
logues that maintained excellent MR potency and showed
improved selectivity against the other NHRs, as shown in
Table 4. Although imidazole 7a and triazole 7d showed
improved rat microsomal stability (RLM), they were potent
CYP inhibitors. In contrast, tetrazole 7d showed improved in
vitro metabolic stability and solubility without CYP inhibi-
tion liability. On the basis of its MR potency and favorable in
vitro pharmacokinetic profile, compound 7d was advanced
for rat pharmacokinetic (PK) studies. Its moderate clearance
and good half-life, summarized in Table 5, made 7d a suitable
candidate for in vivo efficacy studies.
Results
The effect of 7d on blood pressure (BP) and kidney injury
was tested in male Dahl SS rats fed with 4% NaCl. Rats were
MR antagonist potency was evaluated in a cellular assay
based on aldosterone-induced activation of a luciferase reporter

5972 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Scheme 2. General Synthesis of Cyano Ester DHPs
Arhancet et al.
Scheme 3. Synthesis of DHP 5ca
a (a) 30% H2O2, hexfluoro-2-propanol, 2 h; (b) LDA, THF, -78 °C; (c) Raney Ni, EtOH, 90 C, 30 min.
Scheme 4. Synthesis of DHPs 5e-f and 5ta
Table 1. Initial Screening Results for Diester DHPsa
a (a) NaH, DMA, room temp, 4 h; (b) 10, room temp, overnight.
compd
R1
R2
R3
yield (%)
MR IC50 (nM)
1
122
126
3
H
NO2
H
H
dosed orally via gavage with 7d in vehicle at 10 and 60 (mg/kg)/d,
twice a day. For comparison, 1was dosed in chow at 100 mpk/d
because of its very short half-life in rats. Treatment was
initiated at the beginning of salt feeding, and BP was moni-
tored using telemetry units. The results are shown in Figure 3.
After 21 days of salt feeding, animals treated with vehicle
showed a dramatic increase (40-50 mmHg) in BP (mean 24 h
SBP), typical of Dahl SS rats. As has been demonstrated
previously, BP increase was much lower in the group fed with 1,
which represented a significant BP reduction compared to
the vehicle group. At 60 (mg/kg)/d b.i.d. 7d caused BP
reduction approaching the effect by 1, demonstrating POC for
this class of nonsteroidal MR antagonist. The PK of 7d, its
short half-life and low bioavailability, needs to be further
optimized to achieve the maximum BP lowering effect of
eplerenone. Dahl SS rats also develop kidney damage after
high salt feeding as evidenced by increased urinary albumin.
Both eplerenone and 60 (mg/kg)/d 7d achieved comparable
reduction of albumin levels, although statistical significance
was not achieved because of small sample size (data not
shown). Together, these results demonstrated that the DHP
class MR antagonist is capable of reducing BP and renal
injury in vivo.
4a
4b
4c
4d
4e
4f
4g
4h
4i
Cl
F
H
22
11
8
621
H
H
2020
224
CF3
Me
OMe
H
H
H
H
H
19
17
41
26
27
30
19
24
38
17
35
25
24
19
24
44
28
208
553
H
H
Me
CN
OMe
Cl
CF3
OH
F
H
2790
3200
888
H
H
H
H
H
H
518
702
4j
H
H
4k
4l
H
H
2830
1390
309
H
H
4m
4n
4o
4p
4q
4r
4s
4t
H
H
CF3
OH
F
H
H
2570
288
H
H
H
H
Me
OMe
Cl
F
12010
2620
319
H
H
H
H
Cl
H
H
78
574
F
Cl
a IC50 values were obtained through curve-fitting of dose response
(n g 3/concentration, 6-10 concentrations) using the four-parameter
logistic model. Standard error of the IC50 was generally less than 30%.
Chiral resolution of 7d by supercritical fluid chromatogra-
phy (SFC) afforded only one MR-active enantiomer, assigned
as (R)-7d based on our previous studies.11 Intermediate (R)-6
wasalsoobtainedbySFC,aspreviously described, and used in the
preparation of (R)-7d to confirm its configuration assignment.
(R)-7d showed a similar in vitro metabolic profile and similar
solubility to the racemic compound (see Table 4).
Induced-fit docking17 studies suggest that (R)-7d can adopt
the previously proposed DHP MR binding pose10,11 (Figure 2).
In this pose, the 4-aryl group occupies the A-ring pocket of the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 5973
Table 2. Cyano Ester DHP SARa
compd
R1
R2
R3
R4
R5
R6
MR IC50 (nM)
5a
5b
5c
5d
5e
5f
H
H
H
H
Et
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
2-chloro-4-fluorophenyl
4-fluorophenyl
4-fluorophenyl
tBu
tBu
tBu
tBu
tBu
tBu
Me
Me
Me
Me
Me
Me
Me
Et
Me
Me
Me
Me
Me
Me
Me
Me
Et
59
329
661
201
1620
3740
92
2,4-difluorophenyl
2,4-difluorophenyl
2,4-difluorophenyl
2-chloro-4-fluorophenyl
2-chlorophenyl
(CH2)2OMe
5g
5h
5i
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
594
232
64
2-chlorophenyl
2-chlorophenyl
5j
Pr
5k
5l
2-chlorophenyl
2-chlorophenyl
iPr
Bu
208
58
5m
5n
5o
5p
5q
5r
5s
5t
2-chlorophenyl
2-chlorophenyl
CH2OCH3
Ph
655
923
1090
852
7430
16
2-chlorophenyl
2-chlorophenyl
phenyl
Et
4-ClPh
Bn
Pr
Me
Me
Me
Me
Me
2-chloro-4-fluorophenyl
2-chloro-4-fluorophenyl
2-chloro-4-fluorophenyl
Pr
CF3
CF3
52
275
a IC50 values were obtained through curve-fitting of dose response (n g 3/concentration, 6-10 concentrations) using the four-parameter logistic
model. Standard error of the IC50 was generally less than 30%.
Table 3. NHR Selectivity and in Vitro PK Properties of Select 2,6-Dimethyl Cyano Ester DHPsa
HLM
compd MR IC50 (nM) GR IC50 (μM) AR IC50 (μM) PR IC50 (μM) % rem % rem
RLM CYP 3A4 CYP 2C9 CYP 2D
% inh
% inh
6% inh solubility (μM)
5g
5j
92
64
58
16
52
5.1
1.6
1.6
0.7
3.7
0.3
0.3
0.9
0.1
0.8
0.9
0.2
37
36
11
4
14
13
3
40
71
44
83
0
19
10
<5
<5
5
5l
0.3
>10.0
7.8
5r
5s
4
0
6
30
72
6.8
a IC50 values were obtained through curve-fitting of dose response (n g 3/concentration, 6-10 concentrations) using the four-parameter logistic
model. Standard error of the IC50 was generally less than 30%.
receptor, making a face-edge (tee) interaction with Phe 829,
while the DHP ring plane is approximately orthogonal to the
typical steroidal plane and the DHP NH donates a hydrogen
bond to Asn 770. In this orientation, the ester is accommodated
in the hydrophobic R-face pocket, and the 2-methyl and 3-cyano
groups protrude toward the β face, either directly impinging on
Leu 960 from helix 12 or doing so indirectly by perturbing Trp
806. Although no explicit hydrogen bonds are formed between
the cyano nitrogen and the hydroxyl of Ser 810 in this type of
nifedipine and mebudipine. Replacement of one ester group
by cyano, rendering the MR antagonists devoid of CCB
activity, followed by optimization of the dihydropyridine core
substituents resulted in the identification of lead compounds
5j, 5g, 5r, and 5s, which showed comparable in vitro MR
antagonist potency to steroid 1 and good selectivity against
other NHRs. However, in vitro PK data indicated these
compounds did not possess adequate metabolic stability to
be studied in vivo. Appending five-membered heterocycles at
the R6 position resulted in further improvements in the physico-
chemical properties of the analogues while maintaining ex-
cellent potency and selectivity for MR. Compound 7d was
advanced to in vivo studies using the Dahl SS rat model of
hypertension where it provided POC for a nonsteroidal, orally
active MR antagonist.
˚
pose, they are typically about 3.5 A apart and an interaction
cannot be ruled out. Addition of the pendent tetrazole is readily
accommodated, occupying a position similar to that of the
steroid C20 side chain, with the potential for accepting hydrogen
bonds from Asn 770 or Thr 945 depending on the particular
pose. However, the DHP fails to overlap the entire steroidal
D-ring and much of the steroidal C-ring volume (Figure 2b).
Incomplete occupation/stabilization of the receptor binding
pocket18 and steric clashes induced by DHP binding are both
possible mechanisms for antagonism.
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 chromato-
graphy (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 m/z
Conclusions
We have designed novel nonsteroidal MR antagonists
by modifying marketed 1,4-dihydropyridine CCBs, such as

5974 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Arhancet et al.
Table 4. Cyano DHPs Containing Five-Membered Ring Heterocyclesa
a IC50 values were obtained through curve-fitting of dose response (n g 3/concentration, 6-10 concentrations) using the four-parameter logistic
model. Standard error of the IC50 was generally less than 30%. (/) Determined as CL intrinsic 8.71 mL/(kg min).
3
Table 5. Rat Pharmacokinetic Data for 7da
General Procedure for the Preparation of Cyano DHPs. To a
solution of the appropriate aldehyde (4 mmol) in THF (10 mL)
was added the acetoacetate (4 mmol) followed by 3 drops of
piperidine. The mixture was stirred at reflux under nitrogen
atmosphere for 4 h. The solvent was concentrated in vacuo, and
the residue was dissolved in ethanol (10 mL). 3-Aminocrotono-
nitrile (4 mmol) was added to the solution, and the reaction
mixture was stirred at reflux under nitrogen overnight. Solvent
was evaporated in vacuo and the resulting crude product was
purified by reverse phase HPLC (ACN/water with 0.1%TFA,
35-95% ACN) to give the desired cyano DHP.
tert-Butyl 5-Cyano-4-(4-fluorophenyl)-2,6-dimethyl-1,4-dihydro-
pyridine-3-carboxylate 5b. This compound was prepared by the
general method with 4-fluorobenzaldehyde and tert-butyl aceto-
acetate as starting materials. Yield 37%; 1H NMR (400 MHz,
DMSO-d6) δ ppm 9.06 (s, 1 H), 7.11-7.21 (m, 4 H), 4.41 (s, 1 H),
2.71 (s, 1 H), 2.56 (s, 1 H), 2.23 (s, 3 H), 1.99 (s, 3 H), 1.46 (s, 1 H),
1.22 (s, 11 H); ES-MS m/z 329 (M þ H).
compd
dose (mpk)
5
CL ((mL/min)/kg)
14.1
T1/2 (h)
Vdss (L kg)
3
7d
4.84
0.9
a Male Sprague-Dawley rats (n= 2) dosed intravenously (iv) at 5 mg/kg.
Compound 7d was formulated for iv dosing in 10% ethanol/40% PEG400/
50% phosphate buffered saline, pH 7.4. Plasma samples were analyzed by
LC/MS/MS.
100-1000. 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. The
purity of tested compounds was >95% as determined by com-
bustion 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
(δ) are given in ppm and are referenced to residual not fully
deuterated solvent signal. Coupling constants (J) are given in Hz.
General Procedure for the Preparation of Diester DHPs 4a-t.
To a solution of methyl 3-aminocrotonate (4.8 mmol) in MeOH
(5 mL) in a 4 dram vial was added tert-butyl acetoacetate
(1 equiv), the relevant aldehyde (1 equiv), and piperidine (1 drop).
The reaction mixture was stirred overnight at 65 °C. The solvent
was evaporated under a stream of nitrogen and the residue was
dissolved in DMSO and purified by reverse phase HPLC (ACN/
water with 0.1%TFA, 20-75% ACN) to yield the desired
diester DHP.
tert-Butyl 5-Cyano-4-(4-fluoro-2-(methylthio)phenyl)-2,6-dimethyl-
nicotinate 8. 4-Fluoro-2-methylthiobenzaldehyde and tert-butyl
acetoacetate were reacted according the general procedure for
the preparation of cyano DHPs to give the tert-butyl 5-cyano-
4-(4-fluoro-2-(methylthio)phenyl)-2,6-dimethyl-1,4-dihydro-
pyridine-3-carboxylate intermediate. Yield 72%. To a solution
of tert-butyl 5-cyano-4-(4-fluoro-2-(methylthio)phenyl)-2,6-di-
methyl-1,4-dihydropyridine-3-carboxylate (400 mg, 1.1 mmol)
in toluene (5 mL) in a capped vial was added chloranil
(330 mg, 1.3 mmol). The reaction mixture was heated at 120 °C
for 18 h and then cooled to room temperature. A precipitate deve-
loped and was filtered off. The solid was purified on silica gel

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 5975
Figure 2. (a) Representative induced-fit docking pose for tetrazole (R)-7d. (b) Overlay of induced-fit docking pose (gray) with the native
corticosterone-MR crystal structure 2A3I (red). Steroid hydrogen bonding in the native steroid structure is shown. Movement of W806 induced
by DHP is shown, as is close contact with L960 by the 2-methyl group.
Figure 3. Blood pressure lowering effect of 7d in Dahl SS rats. Radiotelemetrized arterial systolic blood pressure (SBP) was measured with the
DATAQUEST A.R.T., version 3.0, Gold software. The values represent the average of all data points collected from each animal every 15 min
for a 10 s interval over a 24 h period (6:00 a.m. to 6:00 a.m. the following day). SBP data were collected continuously over the course of the entire
study (days 1-21). Blood was drawn at day 5 and day 16 for PK analysis. N = 6 for the 100 mpk group, and N = 9 for all other groups.
(dichloromethane 100%) to give pure 8. Yield 75%; HRMS
M þ H calcd for C20H22FN2O2S, 373.1381; obsd, 373.1358.
tert-Butyl 5-Cyano-2,4-dimethyl-4-spiro-30-(50-fluoro-20,30-di-
hydro-10-benzothiophene 10-oxide)-1,4-dihydropyridine-3-carboxy-
late 9. Step 1. To a stirred solution of pyridine 8 (147 mg) in
hexafluoro-2-propanol (2 mL) was added 30% hydrogen peroxide
(0.6 mmol), and the mixture was stirred at room temperature. After
2 h the reaction was quenched with a saturated solution of potas-
sium metabisulfite and the mixture was concentrated under a stream
of nitrogen. The residue was filtered through a short column of silica
gel, eluting with methanol to give crude tert-butyl 5-cyano-4-(4-
fluoro-2-(methylsulfinyl)phenyl)-2,6-dimethylnicotinate (85%).
ES-MS m/z 389 (M þ H).
and the mixture was heated at 90 °C for 30 min. The mixture was
cooled and filtered. The solid was rinsed with hot ethanol, and
the combined ethanol solution was concentrated under a N2
stream. The residue was chromatographed on silica gel (hexanes/
EtOAc) to yield 5c (60%). 1H NMR (400 MHz, chloroform-d)
δ ppm 7.36 (dd, J = 8.86, 5.37 Hz, 2 H), 7.25 (s, 1 H), 6.99 (t, J =
8.73 Hz, 2 H), 2.15 (s, 3 H), 2.04 (s, 3 H), 1.83 (s, 4 H), 1.54 (s,
2 H), 1.09 (s, 10 H), 1.05 (s, 1 H); ES-MS m/z 343 (M þ H).
tert-Butyl 5-Cyano-4-(2,4-difluorophenyl)-2,6-dimethyl-1,4-
dihydropyridine-3-carboxylate 5d. This compound was prepared
by the general method with 2,4-difluorobenzaldehyde and tert-
1
butyl acetoacetate as starting materials. Yield 72%; H NMR
(400 MHz, DMSO-d6) δ ppm 9.08 (s, 1 H), 7.20 (td, J = 8.59,
6.71 Hz, 1 H), 7.15 (td, J = 9.87, 2.55 Hz, 1 H), 7.03 (td, J =
8.53, 2.55 Hz, 1 H), 4.73 (s, 1 H), 2.21 (s, 3 H), 1.94 (s, 3 H), 1.16
(s, 9 H); ES-MS m/z 347 (M þ H).
Step 2. A solution of LDA (0.5 mmol) prepared from
diisopropylamine and n-BuLi in THF was added to a solution
of tert-butyl 5-cyano-4-(4-fluoro-2-(methylsulfinyl)phenyl)-2,6-
dimethylnicotinate (130 mg) in THF (5 mL) cooled at -78 °C.
Reaction was quenched with saturated NH4Cl and the resulting
solid was washed with water to give 9 (50%). ES-MS m/z 387
(M þ H).
tert-Butyl 5-Cyano-4-(2,4-difluorophenyl)-1-ethyl-2,6-dimethyl-
1,4-dihydropyridine-3-carboxylate 5e. To a solution of 5d (16.6 g)
in DMA (46 mL) was added sodium hydride (60%, 1.2 equiv),
and the solution was stirred at room temperature for 4 h. An aliquot
of the solution (0.58 mL) was placed in a vial under nitrogen,
and bromoethane (2.5 equiv) was added. The reaction mixture
was stirred at room temperature overnight. The solution was
tert-Butyl 5-Cyano-4-(4-fluorophenyl)-2,4,6-trimethyl-1,4-di-
hydropyridine-3-carboxylate 5c. To a solution of intermediate 9
(25 mg) in ethanol (3 mL) was added Raney Ni (slurry, 200 μL),

5976 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Arhancet et al.
purified by reverse phase HPLC (ACN/water with 0.1%TFA,
25-95% ACN) to give pure 5e (84%). H NMR (400 MHz,
Ethyl 4-(2-Chlorophenyl)-5-cyano-2-methyl-6-phenyl-1,4-di-
hydropyridine-3-carboxylate 5n. This compound was prepared
by the general procedure with 2-chlorobenzaldehyde and ethyl
benzoylacetate. Yield 82%; 1H NMR (400 MHz, DMSO-d6) δ
ppm 0.66 (t, J = 7.03 Hz, 3 H), 2.01 (s, 3 H), 3.61 (qd, J = 7.03,
3.12 Hz, 2 H), 5.18 (s, 1H), 7.27 (dt, J = 7.52, 3.86 Hz, 2 H), 7.36
(m, 2 H), 7.44 (m, 4 H), 7.50 (dd, J = 7.81, 1.56 Hz, 1 H), 9.39
(s 1 H); ES-MS m/z 379 (M þ H).
1
DMSO-d6) δ ppm 7.19 (dt, J = 8.59, 4.30 Hz, 2 H), 4.76 (s, 1 H),
3.72 (s, 1 H), 3.66 (s, 1 H), 2.42 (s, 3 H), 2.19 (s, 3 H), 1.23 (s, 9 H),
1.15 (t, J = 7.12 Hz, 3 H); ES-MS m/z 375 (M þ H).
tert-Butyl 5-Cyano-4-(2,4-difluorophenyl)-1-(2-methoxyethyl)-
2,6-dimethyl-1,4-dihydropyridine-3-carboxylate 5f. 5f was pre-
pared as example 5e using 1-bromo-2-methoxyethane (2.5 equiv)
in place of bromoethane. Yield 31%; 1H NMR (400 MHz,
DMSO-d6) δ ppm 7.26 (td, J = 8.59, 6.71 Hz, 1 H), 7.19 (td, J =
9.87, 2.55 Hz, 1 H), 7.00-7.12 (m, 1 H), 4.76 (s, 1 H), 3.87-4.09
(m, 1 H), 3.72-3.87 (m, 1 H), 2.40 (s, 3 H), 2.15 (s, 3 H), 1.24 (s,
9 H), signal at 3.45 blocked for water peak. HRMS M þ H calcd
for C22H27F2N2O2, 406.2016; obsd 406.2049.
Ethyl 4-(2-Chlorophenyl)-6-(4-chlorophenyl)-5-cyano-2-ethyl-
1,4-dihydropyridine-3-carboxylate 5o. This compound was pre-
pared by the general procedure with 2-chlorobenzaldehyde and
ethyl 4-chlorobenzoylacetate. Yield 80%; 1H NMR (400 MHz,
DMSO-d6) δ ppm 0.70 (t, J = 7.03 Hz, 3 H), 2.00 (s, 3 H), 3.63
(dd, J = 7.23, 2.15 Hz, 2 H), 5.18 (s, 1 H), 7.27 (dd, J = 7.82,
1.95 Hz, 1 H), 7.41 (d, J = 7.82 Hz, 1 H), 7.37-7.42 (m, 3 H),
7.50 (d, J = 8.60 Hz, 2 H), 7.48-7.51 (m, 1 H); ES-MS m/z 413
(M þ H).
Methyl 4-(2-Chloro-4-fluorophenyl)-5-cyano-1,4-dihydro-2,6-
dimethylpyridine-3-carboxylate 5g. This compound was pre-
pared by the general method with 2-chloro-4-fluorobenzaldehyde
1
and methyl acetoacetate as starting materials. Yield 56%; H
Methyl 2-Benzyl-4-(2-chlorophenyl)-5-cyano-1,4-dihydro-6-
methylpyridine-3-carboxylate 5p. This compound was prepared
by the general method with 2-chlorobenzaldehyde and methyl
3-oxo-4-phenylbutanoate. Yield 50%; 1H NMR (300 MHz,
DMSO-d6) δ ppm 1.99 (s, 3 H), 3.40 (s, 3 H), 4.01 (d, J =
14.10 Hz, 1 H), 4.16 (m, 1 H), 5.13 (s, 1 H), 7.27 (m, 9 H), 9.37
(s, 1 H); ES-MS m/z 379 (M þ H).
NMR (300 MHz, DMSO-d6) δ ppm 1.98 (s, 3 H), 2.28 (s, 3 H),
3.42 (s, 3 H), 5.02 (s, 1 H), 7.19 (m, 1 H), 7.31 (m, 2 H), 9.26 (s,
1 H); ES-MS m/z 321 (M þ H).
Methyl 4-(2-Chlorophenyl)-5-cyano-1,4-dihydro-2,6-dimethyl-
pyridine-3-carboxylate 5h. This compound was prepared by
the general method with 2-chlorobenzaldehyde and methyl aceto-
acetate as starting materials. Yield 60%; 1H NMR (400 MHz,
DMSO-d6) δ ppm 1.98 (s, 3 H), 2.28 (s, 3 H), 3.41 (s, 3 H), 5.05
(s, 1 H), 7.20 (m, 1 H), 7.26 (dd, J = 7.79, 2.15 Hz, 1 H), 7.31 (m,
1H), 7.36(dd, J= 7.79, 1.34 Hz, 1 H), 9.22 (s, 1 H). HRMS M þ H
calcd for C16H16ClN2O2, 303.0895; obsd 303.0902.
Methyl 5-Cyano-1,4-dihydro-6-methyl-4-phenyl-2-propyl-
pyridine-3-carboxylate 5q. This compound was prepared by the
general method with benzaldehyde and methyl butyrylacetate.
1
Yield 54%; H NMR (400 MHz, DMSO-d6) δ ppm 0.91 (m,
3 H), 1.70 (m, 2 H), 2.73 (m, 2 H), 2.73 (s, 3 H), 3.52 (s, 3 H), 4.44
(s, 1 H), 7.37 (m, 2 H), 7.52 (m, 3 H), 9.16 (m, 1 H); ES-MS m/z
297 (M þ H).
Methyl 4-(2-Chlorophenyl)-5-cyano-2-ethyl-1,4-dihydro-6-methyl-
pyridine-3-carboxylate 5i. This compound was prepared by the
general method with 2-chlorobenzaldehyde and methyl pro-
Methyl 4-(2-Chloro-4-fluorophenyl)-5-cyano-1,4-dihydro-
6-methyl-2-propylpyridine-3-carboxylate 5r. This compound
was prepared by the general method with 2-chloro-4-fluoroben-
zaldehyde and methyl butyrylacetate. Yield 70%; 1H NMR (400
MHz, DMSO-d6) δ ppm 0.94 (t, J = 7.38 Hz, 3 H), 1.56 (m, 2 H),
1.98 (s, 3 H), 2.56 (m, 1 H), 2.71 (m, 1 H), 3.42 (s, 3 H), 5.03 (s,
1 H), 7.20 (m, 1 H), 7.27 (m, 1 H), 7.34 (dd, J = 9.00, 2.55 Hz,
1 H), 9.22 (s, 1 H); ES-MS m/z 349 (M þ H).
1
pionylacetate. Yield 57%; H NMR (400 MHz, DMSO-d6) δ
ppm 1.14 (t, J = 7.38 Hz, 3 H), 1.98 (s, 3 H), 2.62 (m, 1 H), 2.72
(m, 1 H), 3.41 (s, 3 H), 5.05 (s, 1 H), 7.21 (m, J = 7.79, 1.88 Hz,
1 H), 7.24 (dd, J = 7.79, 1.88 Hz, 1 H), 7.30 (m, 1 H), 7.36 (dd,
J = 8.06, 1.34 Hz, 1 H), 9.21 (s, 1 H). HRMS M þ H calcd for
C17H18ClN2O2, 317.1051; obsd 317.1010. Anal. (C17H18ClN2O2)
C, H, N.
Methyl 4-(2-Chlorophenyl)-5-cyano-1,4-dihydro-6-methyl-2-
propylpyridine-3-carboxylate 5j. This compound was prepared
by the general method with 2-chlorobenzaldehyde and methyl
butyrylacetate. Yield 50%; 1H NMR (300 MHz, DMSO-d6) δ
ppm 0.94 (t, J = 7.35 Hz, 3 H), 1.57 (m, 2 H), 1.98 (s, 3 H), 2.57
(m, 1 H), 2.72 (m, 1 H), 3.41 (s, 3 H), 5.05 (s, 1 H), 7.27 (m, 4 H),
9.19 (s, 1 H); ES-MS m/z 331 (M þ H).
Methyl 4-(2-Chloro-4-fluorophenyl)-5-cyano-6-methyl-2-(trifluoro-
methyl)-1,4-dihydropyridine-3-carboxylate 5s. To a solution of
2-chlorobenzaldehyde (0.215 g, 1.53 mmol) in THF (10 mL) was
added methyl trifluoroacetoacetate (0.236 g, 1.39 mmol) fol-
lowed by piperidine (3 drops), and the reaction mixture was
stirred at reflux under nitrogen. After 4 h the solution was
concentrated in vacuo to yield a solid. This crude product was
dissolved in EtOH (10 mL), and 3-aminocrotononitrile (0.114 g,
1.39 mmol) was added. The reaction mixture was stirred at
reflux under nitrogen overnight. The solvent was evaporated
and the resulting product was purified by reverse phase HPLC
(ACN/water with 0.1%TFA, 35-95% ACN) to give a solid.
Solid was dissolved in acetonitrile (1 mL) and 4 N HCl in
dioxane (1 mL), and the mixture was stirred at 55 °C for 18 h.
The solvent was removed and the crude product was purified by
reverse phase HPLC to give the title compound. Yield 72%; 1H
NMR (400 MHz, DMSO-d6) δ ppm 2.08 (s, 3 H), 3.50 (s, 3 H),
5.12 (s, 1 H), 7.28 (m, 2 H), 7.41 (m, 2 H), 9.68 (s, 1 H); HRMS
Mþ HcalcdforC16H11F4ClN2O2 NH4, 392.0783; obsd, 392.0820.
Methyl 4-(2-Chloro-4-fluorophenyl)-5-cyano-1,6-dimethyl-
2-(trifluoromethyl)-1,4-dihydropyridine-3-carboxylate 5t. This
compound was prepared by the same procedure described for
5e from 5s and iodomethane. Yield 40%; 1H NMR (400 MHz,
DMSO-d6) δ ppm 7.48 (dd, J = 8.86, 2.69 Hz, 1 H), 7.42 (dd,
J = 8.59, 6.18 Hz, 1 H), 7.29 (td, J = 8.46, 2.69 Hz, 1 H), 4.98
(s, 1 H), 3.61 (s, 3 H), 3.22 (m, 3 H), 2.28 (s, 3 H); ES-MS m/z 389
(M þ H).
Methyl 4-(2-Chlorophenyl)-5-cyano-1,4-dihydro-2-isopropyl-
6-methylpyridine-3-carboxylate 5k. This coumpound was pre-
pared by the general method with 2-chloro-4-fluorobenzalde-
hyde and methyl acetoacetate. Yield 52%; 1H NMR (400 MHz,
DMSO-d6) δ ppm 1.13 (d, J = 6.98 Hz, 3 H), 1.20 (d, J = 6.98
Hz, 3 H), 2.04 (s, 3 H), 3.41 (s, 3 H), 4.08 (m, 1 H), 5.04 (s, 1 H),
7.21 (m, 2 H), 7.32 (m, 1 H), 7.36 (m, 1 H), 8.60 (s, 1 H). HRMS
M þ H calcd for C18H20ClN2O2, 331.1208; obsd, 331.1192.
Methyl 2-Butyl-4-(2-chlorophenyl)-5-cyano-1,4-dihydro-6-methyl-
pyridine-3-carboxylate 5l. This compound was prepared by the
general procedure with 2-chlorobenzaldehyde and 3-oxohepta-
noic acid methyl ester. Yield 40%; 1H NMR (400 MHz, DMSO-
d6) δ ppm 0.91 (t, J = 7.25 Hz, 3 H), 1.36 (m, 2 H), 1.53 (m, 2 H),
1.98 (s, 3 H), 2.58 (m, 1 H), 2.75 (m, 1 H), 3.41 (s, 3 H), 5.05 (s,
1 H), 7.22 (m, 2 H), 7.30 (m, 1 H), 7.36 (m, 1 H), 9.19 (s, 1 H).
HRMS M þ H calcd for C19H22ClN2O2, 345.1364; obsd,
345.1339. Anal. (C19H22ClN2O2) C, H, N.
Methyl 4-(2-Chlorophenyl)-5-cyano-1,4-dihydro-2-(2-methoxy-
methyl)-6-methylpyridine-3-carboxylate 5m. This compound
was prepared by the general procedure with 2-chlorobenzalde-
hyde and methyl 4-methoxyacetoacetate. Yield 30%; 1H NMR
(400 MHz, DMSO-d6) δ ppm 2.03 (s, 3 H), 3.34 (s, 3 H), 3.43 (s,
3 H), 4.55 (s, 2 H), 5.08 (s, 1 H), 7.29 (m, 4 H), 9.07 (s, 1 H). HRMS
M þ H calcd for C17H18ClN2O3, 333.1000; obsd, 333.1005.
Methyl 2-((1H-Imidazol-1-yl)methyl)-4-(2-chloro-4-fluorophenyl)-
5-cyano-6-methyl-1,4-dihydropyridine-3-carboxylate 7a. To a
solution of 6 (2.0 mmol) in NMP (6 mL) were added imidazole
(2.0 mmol) and K2CO3 (4.4 mmol). The mixture was stirred at

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 5977
room temperature overnight. The mixture was poured into
water and extracted with EtOAc. The combined organic extract
was dried over sodium sulfate and the solvent evaporated in
vacuo to give the crude product which was purified by reverse
treatment groups and compounds were continued for 21 days.
The vehicle group received 0.5% methylcellulose/0.1% Tween 80.
All compounds given to the treatment groups were dissolved
in 0.5% methylcellulose/0.1% Tween 80. For compound treated
groups, animals were dosed with the compounds daily, via gavage.
For eplerenone treated groups, eplerenone was incorporated into
the 4% NaCl rodent chow at various concentrations (Research
Diets, Inc., New Brunswick, NJ). Radiotelemetrized arterial SBP
was measured with the DATAQUEST A.R.T., version 3.0, Gold
software (Data Sciences International, St. Paul, MN).
1
phase HPLC. Yield 64%; H NMR (400 MHz, DMSO-d6) δ
ppm 2.01 (s, 3 H), 3.49 (s, 3 H), 5.08 (s, 1 H), 5.14 (d, J = 2.95 Hz,
2 H), 6.91 (s, 1 H), 7.20 (m, 3 H), 7.37 (dd, J = 9.00, 2.55 Hz,
1 H), 7.69 (s, 1 H), 9.64 (s, 1 H). HRMS M þ H calcd for C19H17-
ClFN4O2, 387.1019; obsd, 387.1058. Anal. (C19H17ClFN4O2
0.3H2O) C, H, N.
3
Methyl 2-((1H-Pyrazol-1-yl)methyl)-4-(2-chloro-4-fluorophenyl)-
5-cyano-6-methyl-1,4-dihydropyridine-3-carboxylate 7b. This
compound was prepared by the same procedure described
Twenty-four hours prior to the termination of the study,
animals were placed in metabolism caging and urine was
collected at 24 h. Animals were not fasted for the 24 h period.
After 21 days of treatment, animals were exsanguinated using a
20 gauge needle inserted into the abdominal aorta. Blood
samples were immediately transferred into Vacutainer collec-
tion tubes (Becton-Dickinson and Co., Franklin Lakes, NJ) and
placed on wet ice. Blood was centrifuged for 15 min at 3000 rpm,
4 °C, and plasma was collected and frozen at -80 °C until
further analysis. Plasma and urine chemistries (e.g., albumin,
creatine, and electrolytes) were analyzed with the Hitachi 912
automated diagnostic clinical chemistry analyzer (Roche Diag-
nostics Corp., Indianapolis, IN) according to standard proce-
dures. The St. Louis Pfizer Institutional Animal Care and Use
Committee reviewed and approved the animal use in these
studies. The animal care and use program is fully accredited
by the Association for Assessment and Accreditation of La-
boratory Animal Care, International.
1
for 6a with 6 and pyrazole. Yield 48%; H NMR (400 MHz,
DMSO-d6) δ ppm 2.00 (s, 3 H), 3.45 (s, 3 H), 5.07 (s, 1 H), 5.21 (d,
J = 13.96 Hz, 1 H), 5.46 (d, J = 13.96 Hz, 1 H), 6.25 (m, 1 H), 7.14
(m, 1 H), 7.33 (m, 2 H), 7.51 (d, J = 1.88 Hz, 1 H), 7.72 (d, J =
2.42 Hz, 1 H), 9.67 (s, 1 H). HRMS M þ H calcd for C19H17-
ClFN4O2, 387.1019; obsd, 387.1052. Anal. (C19H15ClFN4O2
0.8HCl) C, H, N.
3
Methyl 2-((1H-1,2,4-Triazol-1-yl)methyl)-4-(2-chloro-4-fluoro-
phenyl)-5-cyano-6-methyl-1,4-dihydropyridine-3-carboxylate 7c.
This compound was prepared by the same procedure described
for 7a with 6 and 1,2,4-triazole. Yield 58%; 1H NMR (400 MHz,
DMSO-d6) δ ppm 2.01 (s, 3 H), 3.44 (s, 3 H), 5.07 (s, 1 H), 5.23
(d, J = 14.23 Hz, 1 H), 5.52 (d, J = 14.23 Hz, 1 H), 7.17 (m, 1 H),
7.35 (m, 2 H), 8.01 (s, 1 H), 8.50 (s, 1 H), 9.77 (s, 1 H). Anal.
(C18H15ClFN5O2 0.55HCl) C, H, N.
3
Modeling 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
Methyl 2-((1H-Tetrazol-1-yl)methyl)-4-(2-chloro-4-fluoro-
phenyl)-5-cyano-6-methyl-1,4-dihydropyridine-3-carboxylate 7d.
To a solution of 6 (1.8 mmol) in NMP (3 mL) was added a solu-
tion of tetrazole in ACN (3% w/w, 6.5 mL) and K2CO3 (4.35 mmol).
The mixture was stirred at room temperature overnight. The
mixture was poured into water and extracted with EtOAc. The
combined organic extract was dried over sodium sulfate and the
solvent evaporated in vacuo to give crude product which was puri-
fied by reverse phase HPLC. Yield 60%; 1H NMR (400 MHz,
DMSO-d6) δ ppm 2.02 (s, 3 H), 3.44 (s, 3 H), 5.10 (s, 1 H), 5.52
(d, J = 14.23 Hz, 1 H), 5.65 (m, 1 H), 7.26 (m, 3 H), 9.40 (s, 1 H),
9.88 (s, 1 H). HRMS M þ H calcd for C17H15ClFN6O2,
389.0924; obsd, 389.0924. Anal. (C17H15ClFN6O2) C, H, N.
(R)-Methyl 2-((1H-Tetrazol-1-yl)methyl)-4-(2-chloro-4-fluoro-
phenyl)-5-cyano-6-methyl-1,4-dihydropyridine-3-carboxylate (R)-7d.
Racemic methyl 2-((1H-tetrazol-1-yl)methyl)-4-(2-chloro-4-fluoro-
phenyl)-5-cyano-6-methyl-1,4-dihydropyridine-3-carboxylate 7d
was loaded on a Berger SFC MultiGram II SFC system equipped
with a Chiralpak AS-H (Chiral Technologies, 30 mm ꢀ 250 mm)
and eluted with 20% MeOH/CO2, 70 mL/min. The two peaks were
collected to yield the two enantiomers. Peak 1: (R)-methyl 2-((1H-
tetrazol-1-yl)methyl)-4-(2-chloro-4-fluorophenyl)-5-cyano-6-methyl-
1,4-dihydropyridine-3-carboxylate (R)-7d; ES-MS m/z 389 (M þ H);
>99% ee determined by analytical chiral chromatography per-
formed on the equivalent Chiralpak AS-H column (Chiral Techno-
logies, 4.6 mm ꢀ 100 mm, 3 mL/min).
˚
MR docking structure was prepared from the 1.95 A MR struc-
ture with bound corticosterone (PDB code 2A3I)19 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-methyl-
acetamide. Dihydropyridine (R)-7d was geometry-optimized
using Jaguar (HF/6-31G**//HF/6-31G**) to ensure appropri-
ate dihydropyridine ring geometry. The induced fit docking
protocol17 was used to dock (R)-7d into the truncated receptor
model, using the default induced fit docking protocol parameters
with the exception of truncating the side chain of Met 845 and Trp
806 during initial Glide docking, requirement of a hydrogen bond
˚
to Asn 770, refinement of side chains within 6.0 A of ligand poses,
and the use of XP scoring for the redock step. Pictures were
generated using Maestro 9.0.111 (Schrodinger LLC).
Acknowledgment. The authors thank Jon Bordner and
Ivan Samardjiev of Pfizer Global Research and Development
(PGRD) for determination of the X-ray crystal structures;
Silvia Portolan, Jay M. Wendling, Robert C. Chott, Mark E.
Smith, and Lesley Albin of PGRD for PK analysis; Kimberly
A. Fosterof PGRD Pharmaceutical Sciences for formulations
support; Monica L. Hultman of PGRD for steroid selecti-
vity assay support; Shengtian Yang for 2D and NOE NMR
structural determinations;and Donna L. Romero, Charles W.
Bolten, and Marvin J. Meyers of PGRD for helpful discus-
sions and support.
Biological Assays. Cell-Based Gal4 Response Element-Con-
trolled Luciferase Reporter Assays. MR Luciferase Reporter
Antagonist Assay. HUH7 human hepatocyte cells were main-
tained in RPMI 1640 plus 10% FBS and transfected with Gal4-
MRLBD construct and a luciferase reporter under Gal4 con-
trol. 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
harvested 20 h later for reporter activity as described.16 Selec-
tivity against other steroid receptors was assayed in the same
manner using Gal4-driven luciferase as reporter.
Supporting Information Available: Elemental analysis data
for compounds 5i, 5l, and 7a-d. This material is available free of
charge via the Internet at http://pubs.acs.org.
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was measured and all animals were then randomized to various
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