Diphenylpyridylethanamine (DPPE) derivatives as cholesteryl ester transfer protein (CETP) inhibitors

Article abstract of DOI:10.1021/jm300611v

A series of diphenylpyridylethanamine (DPPE) derivatives was identified exhibiting potent CETP inhibition. Replacing the labile ester functionality in the initial lead 7 generated a series of amides and ureas. Further optimization of the DPPE series for potency resulted in the discovery of cyclopentylurea 15d, which demonstrated a reduction in cholesterol ester transfer activity (48% of predose level) in hCETP/apoB-100 dual transgenic mice. The PK profile of 15d was suboptimal, and further optimization of the N-terminus resulted in the discovery of amide 20 with an improved PK profile and robust efficacy in transgenic hCETP/apoB-100 mice and in hamsters. Compound 20 demonstrated no significant changes in either mean arterial blood pressure or heart rate in telemeterized rats despite sustained high exposures.

Full text of DOI:10.1021/jm300611v

Article
pubs.acs.org/jmc
Diphenylpyridylethanamine (DPPE) Derivatives as Cholesteryl Ester
Transfer Protein (CETP) Inhibitors
Lalgudi S. Harikrishnan,*,† Heather J. Finlay,† Jennifer X. Qiao,† Muthoni G. Kamau,† Ji Jiang,†
Tammy C. Wang,† James Li,† Christopher B. Cooper,† Michael A. Poss,† Leonard P. Adam,‡
David S. Taylor,‡ Alice Ye A. Chen,‡ Xiaohong Yin,‡ Paul G. Sleph,‡ Richard Z. Yang,‡ Doree F. Sitkoff,§
Michael A. Galella,∥ David S. Nirschl,⊥ Katy Van Kirk,⊥ Arthur V. Miller,⊥ Christine S. Huang,#
Ming Chang,# Xue-Qing Chen,∞ Mark E. Salvati,† Ruth R. Wexler,† and R. Michael Lawrence†
‡
§
∥
†Department of Chemistry, Department of Biology, Department of Computer Aided Drug Design, Department of Materials
Science, Synthesis and Analysis Technology Team, Department of Preclinical Candidate Optimization, and ∞Department of
⊥
#
Pharmaceutics, Bristol-Myers Squibb Company, P.O. Box 4000, Princeton, New Jersey 08543-4000, United States
S
* Supporting Information
ABSTRACT: A series of diphenylpyridylethanamine (DPPE)
derivatives was identified exhibiting potent CETP inhibition.
Replacing the labile ester functionality in the initial lead 7
generated a series of amides and ureas. Further optimization of
the DPPE series for potency resulted in the discovery of
cyclopentylurea 15d, which demonstrated a reduction in
cholesterol ester transfer activity (48% of predose level) in
hCETP/apoB-100 dual transgenic mice. The PK profile of 15d
was suboptimal, and further optimization of the N-terminus
resulted in the discovery of amide 20 with an improved PK
profile and robust efficacy in transgenic hCETP/apoB-100 mice and in hamsters. Compound 20 demonstrated no significant
changes in either mean arterial blood pressure or heart rate in telemeterized rats despite sustained high exposures.
diphenylpyridylethanamine-based CETP inhibitors are de-
scribed.
INTRODUCTION
■
Lipid modifying therapies for the treatment of coronary heart
disease (CHD) have focused on lowering circulating levels of
low-density lipoprotein cholesterol (LDL-C).1 With the
recognition that there exists an inverse relationship between
high-density lipoprotein cholesterol (HDL-C) and the risk of
CHD,2 drug discovery efforts now also focus on investigating
mechanisms that have the potential for raising HDL-C in
plasma. Current therapies such as fibrates or niacin exert only a
modest effect on increasing HDL-C levels while displaying
considerable side effects.3 Therefore, the need exists for safer
and more efficacious methods of increasing HDL-C levels.
Cholesteryl ester transfer protein (CETP) is a 74 kDa plasma
glycoprotein secreted predominantly by the liver. CETP
facilitates the transfer of cholesterol ester from HDL to LDL
and VLDL in exchange for triglycerides.4 Recent data have
shown that CETP inhibition leads to increased HDL-C in
humans.5 As HDL particles are capable of accepting cholesterol
from peripheral tissues including macrophages, CETP inhib-
itors could promote reverse cholesterol transport and therefore
be antiatherogenic.6 Interest in this target by the pharmaceut-
ical community has been widespread as evidenced by the
number of clinical and/or preclinical lead compounds in
various stages of development (Figure 1).7 In this manuscript
the identification and optimization of a novel series of
RESULTS AND DISCUSSION
■
In an effort to identify potential CETP inhibitor leads, a virtual
screen was undertaken based on the published structures of
three noncovalent modulators of CETP (compounds 1, 3, and
4 shown in Figure 1). Similarity searches and molecule shape-
matching techniques were used to identify compounds for
initial screening8 using a BODIPY fluorescence assay.9
Compounds that demonstrated activity in the BODIPY assay
were followed up in concentration−response mode using a
scintillation proximity assay (SPA).10 Validated leads were then
checked for activity in a human whole plasma assay (WPA).11
This led to identification of racemic DPPE ester 7 with a
measurable IC50 in the human whole plasma assay.
Developing a parallel high throughput synthetic method to
access various analogues was challenging because of the
quaternary carbon center. However, an efficient sequence was
identified and utilized to generate compounds with diversity in
the A, B, and C rings (Scheme 1).
Ketone 10 was obtained by the addition of substituted benzyl
Grignard reagent 8 to ethyl picolinate.12 Alternatively, ketone
Received: May 2, 2012
Published: May 31, 2012
© 2012 American Chemical Society
6162
dx.doi.org/10.1021/jm300611v | J. Med. Chem. 2012, 55, 6162−6175

Journal of Medicinal Chemistry
Article
Figure 1. Reported CETP inhibitors.
Scheme 1. Synthetic Route to Analogues of Ester 7
10 was obtained by the addition of the anion of substituted
benzylic nitrile 9 to ethyl picolinate, followed by hydrolysis of
Table 1. SAR of Ester Series
the cyanide and decarboxylation of the resultant carboxylic acid
(Scheme 1).13 Addition of aryllithium 11 to ketone 10 yielded
the lithium alkoxide, which was quenched with various acyl
chlorides to generate ester 12. Substituted A and B rings were
employed to synthesize the compounds shown in Table 1.
The enantiomers of ester 7 were resolved, and it was
determined that CETP activity resided primarily in one
enantiomer (compound 12a, Table 1). A racemic synthesis of
this complex core was initially employed to synthesize
analogues, and the SAR with the racemates demonstrated
that a 5-chloro substituent on the A-ring increased potency
(compound 12c) and an alternative substitution such as
tetrafluoroethoxy was tolerated in the B-ring (compound 12d).
Ester functionalities can be hydrolyzed rapidly in vivo,14 and
as a result, replacement of this ester with a potentially more
metabolically stable amide was targeted. A series of
intermediate amines was generated and converted to the
corresponding amides and ureas. An efficient one-pot parallel
synthesis of the corresponding primary amine has been
a
IC50 (μM)
quaternary chiral
CETP
SPA
CETP
WPA
compd R1
R2
center
b
b
12a
12b
12c
12d
H
H
Cl
H
CF3
CF3
CF3
enantiomer 1
enantiomer 2
racemic
6.7
44
26
>97
9.4
28
1.3
1.1
OCF2CF2H
racemic
a
SPA and WPA percent inhibitions were measured in duplicate at six
concentrations and the mean values used to calculate IC50 values.
Absolute stereochemistry was not determined.14
b
6163
dx.doi.org/10.1021/jm300611v | J. Med. Chem. 2012, 55, 6162−6175

Journal of Medicinal Chemistry
Article
Table 2. Optimization of N-Substituents
a
SPA and WPA percent inhibitions were measured in duplicate at six concentrations and the mean values used to calculate IC50 values.
described previously and was used to generate extensive SAR in
this series.15
tested), certain monosubstituted phenylamides, for example,
13c and 13d, resulted in measurable activity in the SPA assay.
The combination of the two substituents (13f) resulted in
increased WPA potency. The urea analogue 13e showed
potency comparable to ester 7. Cyclopentyl urea 13g was
identified with improved SPA potency as well as good WPA
potency (comparable to 13f), and this moiety was used in the
further optimization of the A, B, and C rings.
An initial survey of N-substituents showed that a variety of
amides and ureas were tolerated (Table 2). The amide analogue
13b resulted in a decrease in CETP inhibition compared to its
ester counterpart 7. A survey of additional amides showed that
while unsubstituted benzamide 13a demonstrated no meas-
urable inhibition of CETP at 96 μM (highest concentration
6164
dx.doi.org/10.1021/jm300611v | J. Med. Chem. 2012, 55, 6162−6175

Journal of Medicinal Chemistry
Article
Table 3. Optimization of A-Ring
a
b
SPA and WPA percent inhibitions were measured in duplicate at six concentrations and the mean values used to calculate IC50 values. These
compounds were synthesized as racemates and resolved by chiral HPLC, and the stereochemistry was determined by X-ray analysis of Mosher’s
amide.16
6165
dx.doi.org/10.1021/jm300611v | J. Med. Chem. 2012, 55, 6162−6175

Journal of Medicinal Chemistry
Article
Examples of A- and B-ring analogues are shown in Tables 3
and 4, respectively. Small electron withdrawing groups at the 5-
Although the one-pot synthetic methodology allowed rapid
access to racemic analogues, a chiral route to the S amine
intermediates was developed to further drive the SAR in this
series and to enable subsequent scale up of compounds for in
vivo studies (Scheme 2).15 This route was then utilized to
explore the SAR of the C-ring.
Table 4. Optimization of B-Ring
Examples of C-ring analogues are shown in Table 5. Triazole
18a and oxadiazole 18e were prepared as shown in Scheme 2
from the corresponding nitrile obtained by the addition of the
anion of acetonitrile to the key (R)-sulfinamide followed by
direct conversion to the oxadiazole. Compounds 18b−d were
prepared from the corresponding intermediate primary amines
described previously.15 Addition of Grignard reagents to
sulfinamide 16 followed by deprotection and acylation afforded
compounds 18f−i.15 Heterocyclic replacement of the C-ring
was not tolerated (examples 18a, 18b, 18d, and 18e), and small
substitutions at the C-ring phenyl were less potent than the
corresponding unsubstituted phenyl compound 14e (examples
18f, 18h, and 18i). Therefore, an unsubstituted phenyl ring was
chosen as the C-ring for further SAR studies. Urea 15d proved
to be the most potent compound resulting from concomitant
optimization of the A, B, and C rings of the DPPE chemotype
and was advanced to in vivo studies.
a
B-ring
IC50 (μM)
quaternary center
chirality
CETP
SPA
CETP
WPA
compd
3-
OCF3
5-
15a
15b
15c
15d
H
H
F
rac
rac
R
0.031
0.025
0.43
2.7
OCF2CF2H
OCF2CF2H
OCF2CF2H
0.84
36
F
S
0.0069
0.63
a
SPA and WPA percent inhibitions were measured in duplicate at six
concentrations and the mean values used to calculate IC50 values.
Compound 15d inhibited cholesterol ester transfer from 3H-
CE/HDL to LDL to 48% of predose activity after 2 h in
hCETP/apoB-100 transgenic mice when dosed at 30 mg/kg
orally (po) (Table 6).17 Maximal inhibition of CETP in this
model reduces CE transfer to between ∼30% and 35% of
control. The area under the curve (AUC) of the plasma
concentration vs time profile, AUC (0−8 h), of 15d was 9.7
μM·h, demonstrating only moderate exposure in the mouse
pharmacodynamic (PD) model.
The mouse liver microsomal stability of urea 15d was poor
(10% remaining after 10 min; rate of 0.27 (nmol/min)/mg
protein),18 and biotransformation studies indicated that the
cyclopentyl urea was the principal site of metabolism.19 Liver
microsomal stability of earlier compounds revealed that the
profile of amide 13f (68% remaining after 10 min incubation)
was superior to that of the urea 13g (2% remaining after 10 min
position of the A-ring led to improvements in potency (14a−g
compared to 13g) as noted earlier in the ester series.
Regioisomeric 3-pyridyl compounds (14h and 14i) demon-
strated reduced potency. With the optimized 5-chloropyridine
A-ring, addition of fluorine at the 5-position of the B-ring led to
a further increase in potency (14c versus 14b). Consistent with
the ester series, the S enantiomer was the more potent
antipode. Extending the fluoroalkyl groups at the 5-position
farther from the B-ring led to a progressive improvement in
potency (compare 14b, 15a, and 15b). Incorporating the SAR
at the 3-position of the B-ring from the ester series (compound
12d) and 3,5-disubstitution SAR from Table 3 (compounds
14d−i) led to urea 15d and yielded one of the first analogues in
this series with submicromolar WPA potency.
a
Scheme 2. Synthetic Route to Heterocyclic C-Ring Analogues
a
Reagents and conditions: (a) 2 equiv of CH3CN, LDA, BF3Et2O, THF, −78 °C (28% S,R and 11% S,S yield); (b) NH2OH, EtOH 70 °C, 2 h; (c)
Ac2O TEA, DCM 0 °C to rt, 14 h; (d) Cs2CO3, THF, 105 °C to dryness 3 h (44% yield for three steps); (e) 4 M HCl/dioxane (100% and 100%
yield); (f) cyclopentyl isocyanate, DCE, catalytic citric acid, rt, 14 h (41% yield for 18e and 36% for 19); (g) DMF/DMA, rt (100% yield); (h)
hydrazine, AcOH, 60 °C, 2 h (21% yield).
6166
dx.doi.org/10.1021/jm300611v | J. Med. Chem. 2012, 55, 6162−6175

Journal of Medicinal Chemistry
Article
Table 5. Optimization of C-Ring
Figure 2. Optimized amide resulting in metabolically stable compound
20.
Although amide 20 inhibited CETP in h-CETP/apoB-100
transgenic mice, it remained to be evaluated in a species that
naturally expresses CETP. Since hamsters (unlike mice and
rats) express CETP, compound 20 was tested in a hamster PD
model. Compound 20 was dosed in moderately fat-fed
hamsters at 30 mg/kg, once-a-day (q.d.), and 21 μM plasma
exposures were achieved at 2 h after dosing on day 3.21 An
increase of 40% in HDL-C was also observed after 3 days
(Figure 3), demonstrating robust efficacy for the compound in
hamsters.
a
SPA and WPA percent inhibitions were measured in duplicate at six
concentrations and the mean values used to calculate IC50 values.
Table 6. Pharmacodynamic (PD) Data in hCETP/apoB-100
Tg Mice for Advanced Compounds
CETP activity
(% of predose)
a
IC50 (μM)
CETP
hWPA
CETP
mWPA
AUC (0−8 h)
(μM·h)
compd
2 h 4 h 8 h
Figure 3. In vivo efficacy data for amide 20 in hamsters. Male Golden
Syrian hamsters 12 weeks of age were used. Predose blood samples
were collected by retro-orbital bleed. Compounds were formulated in
ethanol/Cremophor/water at a 1:1:8 ratio and dosed at 30 mg/kg, po,
q.d., for 3 days. Blood was drawn 2 h postdose on day 3, and HDL-C
levels were measured in the predose and postdose samples using an
automated lipid analyzer (COBAS). Percentage increases in HDL-C
with treatment are reported as the mean SEM (N = 6).
torcetrapib
15d
0.11
0.63
3.6
0.16
0.94
9.0
65
9.7
93
31
48
55
31
58
48
25
65
40
20
a
SPA and WPA percent inhibitions were measured in duplicate at six
concentrations and the mean values used to calculate IC50 values.
incubation). Therefore, the cyclopentylurea moiety in com-
pound 15d was replaced with the metabolically stable amide to
obtain compound 20 (Figure 2).15 Compound 20 demon-
strated significantly improved mouse liver microsomal stability
(100% remaining after 10 min; rate of 0.0025 (nmol/min)/mg
protein), as well as a superior pharmacokinetic profile in mice
(Table 6). Compound 20 was advanced to PD evaluation and
Torcetrapib was reported to increase blood pressure in
humans,22 as well as in telemeterized rats.23 Since rats do not
express CETP, the elevation in blood pressure was due to an
off-target effect.24 To ensure that amide 20 did not cause effects
on blood pressure, it was also evaluated in rat telemetry studies.
Significantly, unlike torcetrapib, amide 20 did not produce
changes in mean arterial blood pressure (MABP) or heart rate
(HR) in telemeterized rats at doses up to 60 mg/kg with
sustained high exposure (Cmax = 25 μM; T1/2 = 14 h).
3
inhibited CETP mediated transfer of H-CE/HDL to LDL to
32% of predose activity after 8 h in h-CETP/apoB-100
transgenic mice when dosed at 30 mg/kg, po (Table 6).20
The AUC (0−8 h) of amide 20 was 93 μM·h.
In summary, ester 7 was identified from virtual screening as a
CETP inhibitor. Initial SAR in the ester series led to
6167
dx.doi.org/10.1021/jm300611v | J. Med. Chem. 2012, 55, 6162−6175

Journal of Medicinal Chemistry
Article
General Procedure A for Synthesis of DPPE Esters. To a
solution of aryl bromide (1 mmol) in ether (10 mL) under nitrogen at
−78 °C was added n-BuLi (1.6 M in hexane, 0.63 mL, 1 mmol), and
the mixture was stirred for 10 min. Then a solution of ketone 10 (1
mmol) in THF (0.5 mL) was added. The reaction mixture was stirred
at −78 °C for 30 min. An aliquot of the resultant alkoxide (1.1 mL,
0.10 mmol) was added into an array of vials containing freshly
prepared solutions of various acid chlorides (0.11 mmol) in ether (0.5
mL). The mixtures were warmed to room temperature. The crude
products were purified by reverse phase HPLC to obtain the desired
products in 18−60% yield.
General Procedure B for Synthesis of DPPE Ureas (40 μmol
Scale). To an array of vials containing various isocyanates (0.2 M in
dioxane, 400 μL, 80 μmol) was added primary amine 1415 (0.5 M in
dioxane, 80 μL, 40 μmol). The reaction mixture was agitated at room
temperature for 18 h. The solvents were evaporated, and the crude
product was purified by reverse phase HPLC.
optimization of the A-ring and B-ring. Replacement of the
metabolically labile ester group led to the discovery of N-
terminal ureas and amides which were further optimized in the
A-, B-, and C-ring positions. A one-pot route was developed to
access the precursor amines via parallel synthesis, and an
enantioselective route was subsequently developed to scale up
the desired S-enantiomers. The DPPE series SAR was further
extended to achieve over 1000-fold improvement in in vitro
potency and in vivo activity in h-CETP/apoB-100 transgenic
mice and hamsters. Improved metabolic stability was achieved
by replacing the cyclopentylurea group in compound 15d with
an optimized amide to yield 20 which had an improved
pharmacokinetic profile, demonstrated the ability to inhibit
CETP mediated cholesterol transfer in both h-CETP/apoB-100
transgenic mice and hamsters, and provided a 40% increase in
HDL levels in hamsters. No significant changes in MABP and
HR were observed in telemeterized rats when compound 20
was administered orally at sustained high exposures. The
studies described herein demonstrate that the DPPE chemo-
type shows promise as a novel scaffold for development of a
potent CETP inhibitor. Further efforts to optimize this series
will be reported in subsequent disclosures.
General Procedure C for Synthesis of DPPE Amides. To an
array of vials containing various acid chlorides (0.2 M in DCM, 400
μL, 80 μmol) and pyridine (8 μL, 98 μmol) was added primary amine
1415 (0.5 M in DCM, 80 μL, 40 μmol). The reaction mixture was
agitated at room temperature for 18 h. The solvents were evaporated,
and the crude product was purified by reverse phase HPLC.
2-Phenyl-1-(pyridin-2-yl)-1-(3-(trifluoromethyl)phenyl)ethyl
3,3-Dimethylbutanoate, 7. Ester 7 was obtained using general
procedure A, in 49% yield. The racemic 2-phenyl-1-(pyridin-2-yl)-1-
(3-(trifluoromethyl)phenyl)ethyl 3,3-dimethylbutanoate 7 (100 mg,
22.6 μmol) was separated by chiral HPLC using Chiralpak AD
column, eluting with 2% EtOH/MeOH (1:1) in heptane to give (R)-
2-phenyl-1-(pyridin-2-yl)-1-(3-(trifluoromethyl)phenyl)ethyl 3,3-di-
methylbutanoate 12b (45.3 mg, white solids) as the faster eluting
enantiomer (analytical Chiralpak AD 10 μm, 4.6 mm × 250 mm, SN
AD00CE-DD007 column, 2% EtOH/MeOH (1:1) in heptane, 0.5
mL/min, retention time of 9.29 min) and (S)-2-phenyl-1-(pyridin-2-
yl)-1-(3-(trifluoromethyl)phenyl)ethyl 3,3-dimethylbutanoate 12a (43
mg, white solids) as the slower eluting enantiomer (analytical
Chiralpak AD 10 μm, 4.6 mm × 250 mm; SN AD00CE-DD007
column, 2% EtOH/MeOH (1:1) in heptane, 0.5 mL/min, retention
time of 11.36 min). Data for ester 6: LC−MS ESI 464.5 (M + Na),
retention time of 2.1 min (10−90% MeOH in H2O with 0.1% TFA in
EXPERIMENTAL SECTION
■
General Information. All reactions were carried out under a static
atmosphere of argon or nitrogen and stirred magnetically unless
otherwise stated. All reagents used were of commercial quality and
were obtained from Aldrich Chemical Co., Sigma Chemical Co.,
Lancaster Chemical Co., Oakwood Chemical Co., and Matrix
Chemical Co. 1H (400 MHz) and 13C (100 MHz) NMR spectra
were recorded on a JEOL GSX400 spectrometer using tetramethylsi-
1
lane as an internal standard unless otherwise stated. H (500 MHz)
and 13C (125 MHz) NMR spectra were recorded on a JEOL JNM-
ECP500 spectrometer. Chemical shifts are given in parts per million
(ppm) downfield from internal reference trimethylsilane in δ units,
and coupling constants (J) are given in hertz (Hz). Selected data are
reported in the following manner: chemical shift, multiplicity, coupling
constants. All reactions were carried out using commercially available
anhydrous solvents from Aldrich Chemical Co. or EM Science
Chemical Co. unless otherwise stated. All flash chromatographic
separations were performed using E. Merck silica gel (particle size,
0.040−0.063 mm). Reactions were monitored by TLC using 0.25 mm
E. Merck silica gel plates (60 F254) and were visualized with UV light,
with 5% phosphomolybdic acid in 95% EtOH, or by sequential
treatment with 1 N HCl/MeOH followed by ninhydrin staining. LC−
MS data were recorded on a Shimadzu LC-10AT equipped with a SIL-
10A injector, a SPD-10AV detector, normally operating at 220 nm, and
interfaced with a Micromass ZMD mass spectrometer. LC−MS or
HPLC retention times, unless otherwise noted, are reported using a
Phenomenex Luna C-18 4.6 mm × 50 mm column, and elution was
with a 4 min gradient from 0% to 100% B, where A is 10% MeOH−
90% H2O−0.1% TFA and B is 90% MeOH−10% H2O−0.1% TFA. All
solvents were removed by rotary evaporation under vacuum using a
standard rotovap equipped with a dry ice condenser. All filtrations
were performed with a vacuum unless otherwise stated. Unless
otherwise specified the purity of all intermediates and final compounds
was determined to be >95% by Sunfire C18 3.5 μm, 3.0 mm × 150
mm, 1.0 mL/min, gradient 10−100% 95:5 AcCN in H2O (0.05%
TFA) in 5:95 AcCN in H2O (0.05% TFA) (method A), Xbridge
Phenyl 3.5 μm, 4.6 mm × 150 mm, 1.0 mL/min, gradient 10−100%
95:5 AcCN in H2O (0.05% TFA) in 5:95 AcCN in H2O (0.05% TFA)
(method B), Phenomenex Luna 5 μm, 4.6 mm × 50 mm, 4.0 mL/min,
gradient 10−100% 95:5 MeOH in H2O (0.1% HP3O4) in 5:95 MeOH
in H2O (0.1% HP3O4) (method C), Sunfire 5 μm, 4.6 mm × 50 mm,
4.0 mL/min, gradient 10−100% 90:10 MeOH in H2O (0.1% TFA) in
10:90 MeOH in H2O (0.1% TFA) (method D) and/or elemental
analyses.
1
a 2 min gradient). H NMR (500 MHz, chloroform-d) δ 8.60−8.69
(m, 1H), 7.63 (dt, J = 1.76, 7.68 Hz, 1H), 7.34−7.57 (m, 5H), 7.06−
7.22 (m, 4H), 6.62 (d, J = 7.05 Hz, 2H), 4.42 (d, J = 13.60 Hz, 1H),
4.10 (d, J = 13.60 Hz, 1H), 2.27 (s, 2H), 1.02 (s, 9H). 13C NMR (126
MHz, chloroform-d) δ 170.5, 162.0, 148.9, 144.6, 136.5, 135.7, 130.2,
130.3 (q, J = 32.9 Hz, 1C), 129.5, 128.5, 127.7, 126.5, 124.17−123.70
(m, 1C), 123.1, 122.3, 120.9, 125.3 (q, J = 271.3 Hz, 1C), 86.5, 48.4,
41.9, 30.7, 29.5. Orthogonal HPLC purity: 95%, retention time of
11.84 min (HPLC method A); 98%, retention time of 13.12 min
(HPLC method B).
1-(5-Chloropyridin-2-yl)-2-phenyl-1-(3-(trifluoromethyl)-
phenyl)ethyl 3,3-Dimethylbutanoate, 12c. Ester 12c was
obtained using general procedure A, in 60% yield. LC−MS ESI
476.13 (M + H), retention time of 4.23 min (10−90% MeOH in H2O
with 0.1% TFA in a 4 min gradient). 1H NMR (500 MHz, chloroform-
d) δ 8.58 (d, J = 2.22 Hz, 1H), 7.60 (dd, J = 2.50, 8.60 Hz, 1H), 7.47−
7.53 (m, 3H), 7.39−7.45 (m, 1H), 7.30 (d, J = 8.60 Hz, 1H), 7.08−
7.19 (m, 3H), 6.61 (d, J = 7.21 Hz, 2H), 4.35 (d, J = 13.87 Hz, 1H),
4.06 (d, J = 13.59 Hz, 1H), 2.26 (s, 2H), 1.02 (s, 9H). 13C NMR (126
MHz, chloroform-d) δ 170.5, 160.3, 147.8, 144.2, 136.3, 135.4, 130.6,
130.2 (s, 2C), 130.85−129.96 (m, J = 33.6, 33.6, 33.6 Hz, 1C), 129.4,
128.6, 127.8 (s, 2C), 126.7, 124.29−124.04 (m, 1C), 123.08−122.83
(m, 1C), 121.8, 86.2, 48.4, 41.9, 30.7, 29.5 (s, 3C). Orthogonal HPLC
purity: 92%, retention time of 12.70 min (HPLC method A)25
2-Phenyl-1-(pyridin-2-yl)-1-(3-(1,1,2,2-tetrafluoroethoxy)-
phenyl)ethyl 3,3-Dimethylbutanoate, 12d. Ester 12d was
obtained using general procedure A, in 27% yield. LC−MS ESI
490.17 (M + H), retention time of 3.38 min (10−90% MeOH in H2O
with 0.1% TFA in a 4 min gradient). 1H NMR (500 MHz, chloroform-
d) δ 8.59−8.67 (m, 1H), 7.56−7.66 (m, 1H), 7.34 (d, J = 8.05 Hz,
6168
dx.doi.org/10.1021/jm300611v | J. Med. Chem. 2012, 55, 6162−6175

Journal of Medicinal Chemistry
Article
1H), 7.21−7.32 (m, 2H), 7.17 (ddd, J = 0.97, 4.86, 7.49 Hz, 1H),
7.03−7.15 (m, 5H), 6.60−6.68 (m, 2H), 4.39 (d, J = 13.59 Hz, 1H),
4.06 (d, J = 13.87 Hz, 1H), 2.24 (d, J = 1.39 Hz, 2H), 1.00 (s, 9H). 13C
NMR (126 MHz, chloroform-d) δ 170.4, 162.2, 148.6, 145.7, 136.6,
135.8, 130.3 (s, 2C), 129.2, 127.7 (s, 2C), 126.5, 124.3, 122.3, 121.0,
120.1, 119.7, 116.4 (tt, J = 271.8, 28.6 Hz, 1C), 107.6 (tt, J = 251.8,
41.0 Hz, 1C), 86.4, 48.4, 42.0, 30.6, 29.5 (s, 3C). Orthogonal HPLC
purity: 93%, retention time of 11.7 min (HPLC method A); 95%,
retention time of 13.44 min (HPLC method B).
N-(2-Phenyl-1-(pyridin-2-yl)-1-(3-(trifluoromethyl)phenyl)-
ethyl)benzamide, 13a. Amide 13a was obtained using general
procedure C, in 31% yield. LC−MS ESI 446.99 (M + H), retention
time of 3.89 min (10−90% MeOH in H2O with 0.1% TFA in a 4 min
gradient). 1H NMR (500 MHz, chloroform-d) δ 9.43 (br s, 1H),
8.36−8.40 (m, 1H), 7.76−7.82 (m, 3H), 7.69−7.75 (m, 2H), 7.42−
7.56 (m, 5H), 7.18−7.23 (m, 2H), 7.06−7.11 (m, 1H), 6.96−7.02 (m,
2H), 6.57−6.62 (m, 2H), 4.70 (d, J = 12.76 Hz, 1H), 3.71 (d, J = 12.76
Hz, 1H). 13C NMR (126 MHz, chloroform-d) δ 166.4, 160.3, 146.5,
146.0, 137.6, 135.5, 135.1, 131.5, 130.7 (q, J = 31.5 Hz, 1C), 130.2 (m,
3C, two overlapping peaks), 128.9, 128.5, 127.7, 127.0, 126.69−126.46
(m, 1C), 124.17−124.00 (m, 1C), 123.3 (q, J = 3.8 Hz, 1C), 122.9,
122.5, 124.1 (q, J = 270.8 Hz, 1C), 64.0, 41.7. Orthogonal HPLC
purity: 94%, retention time of 11.39 min (HPLC method A); 99%,
retention time of 12.75 min (HPLC method B).
3,3-Dimethyl-N-(2-phenyl-1-(pyridin-2-yl)-1-(3-
(trifluoromethyl)phenyl)ethyl)butanamide, 13b. Amide 13b was
obtained using general procedure C, in 55% yield. LC−MS ESI 441.07
(M + H), retention time of 4.00 min (10−90% MeOH in H2O with
0.1% TFA in a 4 min gradient). 1H NMR (500 MHz, chloroform-d) δ
8.58 (s, 1H), 8.35−8.39 (m, 1H), 7.71 (dt, J = 1.94, 7.77 Hz, 1H), 7.68
(d, J = 7.49 Hz, 1H), 7.59 (s, 1H), 7.49−7.53 (m, 1H), 7.43−7.49 (m,
1H), 7.17−7.24 (m, 2H), 7.10−7.15 (m, 1H), 7.03−7.08 (m, 2H),
6.53−6.58 (m, 2H), 4.46 (d, J = 12.76 Hz, 1H), 3.67 (d, J = 12.76 Hz,
1H), 2.69 (br s, 1H), 2.10−2.21 (m, 2H), 0.97 (s, 9H). 13C NMR (126
MHz, chloroform-d) δ 171.4, 160.4, 146.5, 146.2, 137.6, 135.7, 130.7
(q, J = 31.5 Hz, 1C), 130.2, 130.0, 128.7, 127.7, 126.6, 124.08−123.63
(m, 2C), 122.4, 124.1 (q, J = 271.8 Hz, 1C), 64.0, 50.7, 41.7, 34.9,
31.1, 29.8. Orthogonal HPLC purity: 94%, retention time of 11.49 min
(HPLC method A); 95%, retention time of 13.24 min (HPLC method
B).
N-(2-Phenyl-1-(pyridin-2-yl)-1-(3-(trifluoromethyl)phenyl)-
ethyl)-3-(trifluoromethyl)benzamide, 13c. Amide 13c was
obtained using general procedure C, in 65% yield. LC−MS ESI
515.02 (M + H), retention time of 4.08 min (10−90% MeOH in H2O
with 0.1% TFA in a 4 min gradient). 1H NMR (500 MHz, chloroform-
d) δ 9.53 (s, 1H), 8.39−8.44 (m, 1H), 8.06 (s, 1H), 7.92 (d, J = 7.77
Hz, 1H), 7.72−7.84 (m, 4H), 7.54−7.62 (m, 2H), 7.47−7.53 (m, 1H),
7.21−7.27 (m, 2H), 7.10−7.15 (m, 1H), 6.99−7.05 (m, 2H), 6.55−
6.61 (m, 2H), 4.70 (d, J = 12.76 Hz, 1H), 3.74 (d, J = 12.76 Hz, 1H).
13C NMR (126 MHz, chloroform-d) δ 165.1, 160.0, 146.4, 145.2,
138.2, 135.7, 135.1, 130.9 (q, J = 32.4 Hz, 1C), 131.2 (q, J = 32.7 Hz,
1C), 130.2, 130.1 (s, 2C, 129.2, 129.1, 128.2 (q, J = 3.8 Hz, 1C), 127.8
(s, 2C), 126.9, 124.58−124.22 (m, J = 3.8, 3.8, 3.8, 3.8 Hz, 1C), 123.4
(q, J = 3.8 Hz, 1C), 123.2, 123.99−122.99 (m, 1C), 122.8, 124.0 (q, J
= 272.8 Hz, 1C), 127.09−120.28 (m, 1C), 64.2, 41.9. Orthogonal
HPLC purity: 95%, retention time of 11.87 min (HPLC method A);
100%, retention time of 13.47 min (HPLC method B).
4-Fluoro-N-(2-phenyl-1-(pyridin-2-yl)-1-(3-(trifluoromethyl)-
phenyl)ethyl)benzamide, 13d. Amide 13d was obtained using
general procedure C, in 50% yield. LC−MS ESI 465.01 (M + H),
retention time of 3.93 min (10−90% MeOH in H2O with 0.1% TFA in
a 4 min gradient). 1H NMR (500 MHz, chloroform-d) δ 9.40 (s, 1H),
8.34−8.42 (m, 1H), 7.76−7.83 (m, 3H), 7.68−7.75 (m, 2H), 7.51−
7.57 (m, 1H), 7.43−7.50 (m, 1H), 7.18−7.24 (m, 2H), 7.06−7.15 (m,
3H), 6.95−7.03 (m, 2H), 6.52−6.61 (m, 2H), 4.68 (d, J = 12.76 Hz,
1H), 3.70 (d, J = 12.76 Hz, 1H). 13C NMR (126 MHz, chloroform-d)
δ 165.6, 164.8 (d, J = 252.7 Hz, 1C), 160.1, 146.3, 145.3, 138.1, 135.2,
131.0 (d, J = 2.9 Hz, 1C), 130.8 (q, J = 32.2 Hz, 1C), 130.1, 129.5 (d, J
= 8.6 Hz), 129.0, 127.8, 126.7, 124.37−124.16 (m, 1C), 123.4 (q, J =
3.8 Hz), 123.2, 122.7, 124.0 (q, J = 271.8 Hz, 1C), 115.6 (d, J = 21.9
Hz, 2C), 64.1, 41.9. Orthogonal HPLC purity: 97%, retention time of
11.53 min (HPLC method A); 98%, retention time of 12.91 min
(HPLC method B).
1-tert-Butyl-3-(2-phenyl-1-(pyridin-2-yl)-1-(3-
(trifluoromethyl)phenyl)ethyl)urea, 13e. Urea 13e was obtained
using general procedure B, in 81% yield. LC−MS ESI 442.2 (M + H),
retention time of 2.3 min (10−90% MeOH in H2O with 0.1% TFA in
1
a 4 min gradient). H NMR (500 MHz, chloroform-d) δ 8.52 (d, J =
4.44 Hz, 1H), 8.11 (t, J = 7.21 Hz, 1H), 7.95 (br s, 1H), 7.81 (br s,
1H), 7.47−7.58 (m, 2H), 7.41 (t, J = 7.91 Hz, 1H), 7.27−7.37 (m,
2H), 7.14−7.21 (m, 1H), 7.06−7.14 (m, 2H), 6.56−6.70 (m, 2H),
4.12 (d, J = 12.21 Hz, 1H), 3.83 (d, J = 11.37 Hz, 1H), 1.27 (s, 9H).
13C NMR (126 MHz, chloroform-d) δ 161.6, 157.0, 144.3, 143.3,
141.5, 134.5, 130.6, 130.8 (q, J = 32.4 Hz, 1C), 130.1, 129.1, 127.9,
127.1, 124.77−124.53 (m, J = 3.8 Hz, 1C), 124.4, 123.6, 123.61−
123.50 (m, J = 3.8 Hz, 1C), 123.9 (q, J = 270.8 Hz, 1C), 64.6, 50.6,
43.7, 29.1. Orthogonal HPLC purity: 95%, retention time of 9.79 min
(HPLC method A); 97%, retention time of 10.56 min (HPLC method
B).
4-Fluoro-N-(2-phenyl-1-(pyridin-2-yl)-1-(3-(trifluoromethyl)-
phenyl)ethyl)-3-(trifluoromethyl)benzamide, 13f. Amide 13f
was obtained using general procedure C, in 48% yield. LC−MS ESI
533.03 (M + H), retention time of 4.12 min (10−90% MeOH in H2O
with 0.1% TFA in a 4 min gradient). 1H NMR (500 MHz, chloroform-
d) δ 9.49 (s, 1H), 8.37−8.42 (m, 1H), 8.06 (dd, J = 1.94, 6.66 Hz,
1H), 7.91 (ddd, J = 2.22, 4.58, 8.46 Hz, 1H), 7.69−7.80 (m, 3H),
7.54−7.58 (m, 1H), 7.46−7.52 (m, 1H), 7.20−7.27 (m, 3H), 7.08−
7.13 (m, 1H), 6.97−7.04 (m, 2H), 6.50−6.58 (m, 2H), 4.66 (d, J =
13.04 Hz, 1H), 3.72 (d, J = 12.76 Hz, 1H). 13C NMR (126 MHz,
chloroform-d) δ 163.8, 162.83−160.35 (m, J = 261.3, 1.9 Hz, 1C),
160.0, 146.5, 145.6, 137.9, 135.3, 132.6 (d, J = 9.5 Hz, 1C), 131.6 (d, J
= 3.8 Hz, 1C), 130.9 (q, J = 32.1 Hz, 1C), 130.1, 130.0 (s, 2C), 129.1,
127.8 (s, 2C), 127.10−126.87 (m, J = 1.9 Hz, 1C), 126.8, 124.48−
124.23 (m, 1C), 123.44−123.24 (m, 1C), 123.0, 122.7, 124.0 (q, J =
272.8 Hz, 1C), 122.2 (q, J = 273.2 Hz, 1C), 118.8 (qd, J = 33.4, 13.4
Hz, 1C), 117.1 (d, J = 21.0 Hz, 1C), 64.1, 41.7. Orthogonal HPLC
purity: 98%, retention time of 12.00 min (HPLC method A); 100%,
retention time of 13.60 min (HPLC method B).
1-Cyclopentyl-3-(2-phenyl-1-(pyridin-2-yl)-1-(3-
(trifluoromethyl)phenyl)ethyl)urea, 13g. Urea 13g was obtained
using general procedure B, in 45% yield. LC−MS ESI 454.05 (M + H),
retention time of 1.9 min (10−90% MeOH in H2O with 0.1% TFA in
1
a 4 min gradient). H NMR (500 MHz, chloroform-d) δ 8.27−8.30
(m, J = 0.96, 0.96, 0.96, 4.89 Hz, 1H), 7.73 (s, 1H), 7.70 (d, J = 7.77
Hz, 1H), 7.64 (dt, J = 1.66, 7.77 Hz, 1H), 7.47−7.51 (m, 1H), 7.42−
7.47 (m, 1H), 7.38 (br s, 1H), 7.09−7.16 (m, 3H), 7.02−7.07 (m,
2H), 6.58−6.62 (m, 2H), 4.53 (d, J = 12.48 Hz, 1H), 3.94 (br s, 1H),
3.60 (d, J = 12.76 Hz, 1H), 1.98−2.06 (m, 1H), 1.82−1.91 (m, 1H),
1.51−1.70 (m, 4H), 1.43 (dd, J = 6.38, 12.76 Hz, 1H), 1.25−1.31 (m,
1H). 13C NMR (126 MHz, chloroform-d) δ 160.7, 157.6, 145.5, 145.2,
139.1, 135.0, 130.7 (q, J = 32.0 Hz, 1C), 130.3, 127.7, 126.8, 124.3 (q,
J = 3.6 Hz, 1C), 123.4 (q, J = 3.8 Hz, 1C), 123.9 (q, J = 273.0 Hz, 1C),
122.9, 64.0, 52.6, 43.0, 33.1, 32.9, 23.6, 23.5. Orthogonal HPLC purity:
97%, retention time of 9.95 min (HPLC method A); 98%, retention
time of 10.77 min (HPLC method B).
1-Cyclopentyl-3-(1-(5-fluoropyridin-2-yl)-2-phenyl-1-(3-
(trifluoromethyl)phenyl)ethyl)urea, 14a. Urea 14a was obtained
using general procedure B, in 25% yield. LC−MS ESI 472.20 (M + H),
retention time of 3.38 min (10−90% MeOH in H2O with 0.1% TFA in
1
a 4 min gradient). H NMR (500 MHz, chloroform-d) δ 8.15 (d, J =
2.77 Hz, 1H), 7.66−7.74 (m, 2H), 7.49−7.54 (m, 1H), 7.43−7.49 (m,
1H), 7.35−7.42 (m, 1H), 7.05−7.17 (m, 5H), 6.58−6.65 (m, 2H),
4.53 (d, J = 12.48 Hz, 1H), 4.37 (d, J = 6.66 Hz, 1H), 3.88−3.99 (m,
1H), 3.58 (d, J = 12.48 Hz, 1H), 1.96−2.07 (m, 1H), 1.82−1.92 (m,
1H), 1.51−1.73 (m, 4H), 1.37−1.48 (m, 1H), 1.23−1.33 (m, 1H). 13C
NMR (126 MHz, chloroform-d) δ 157.33−157.25 (m, 1C), 158.3 (d, J
= 261.3 Hz, 1C), 155.9, 147.4, 136.1, 134.4 (d, J = 23.8 Hz, 1C), 130.7
(q, J = 31.5 Hz, 1C), 130.3 (s, 2C), 130.1, 128.8, 127.6 (s, 2C), 126.4,
124.2 (d, J = 19.1 Hz, 1C), 123.9 (q, J = 3.8 Hz, 1C), 123.8 (d, J = 4.8
Hz, 1C), 123.4 (q, J = 3.8 Hz, 1C), 124.2 (q, J = 271.8 Hz, 1C), 63.4,
6169
dx.doi.org/10.1021/jm300611v | J. Med. Chem. 2012, 55, 6162−6175

Journal of Medicinal Chemistry
Article
52.2, 42.8, 33.8, 33.3, 23.6 (s, 2C). Orthogonal HPLC purity: 84%,
retention time of 11.11 min (HPLC method A); 97%, retention time
of 12.45 min (HPLC method B).
iPA/heptane/DEA, isocratic), retention time of 5.30 min) and (S)-1-
(1-(5-cyanopyridin-2-yl)-1-(3-fluoro-5-(trifluoromethyl)phenyl)-2-
phenylethyl)-3-cyclopentylurea 14f as the slow eluting enantiomer:
analytical chiral HPLC AD (25% iPA/heptane/DEA, isocratic),
1-(1-(5-Chloropyridin-2-yl)-2-phenyl-1-(3-(trifluoromethyl)-
phenyl)ethyl)-3-cyclopentylurea, 14b. Urea 14b was obtained
using general procedure B, in 34% yield. LC−MS ESI 488.30 (M + H),
retention time of 3.32 min (10−90% MeOH in H2O with 0.1% TFA in
a 4 min gradient). 1H NMR (500 MHz, chloroform-d) δ 8.26 (dd, J =
2.5, 0.6 Hz, 1H), 7.76−7.66 (m, 2H), 7.63 (dd, J = 8.6, 2.5 Hz, 1H),
7.55−7.50 (m, 1H), 7.49−7.43 (m, 1H), 7.19−7.02 (m, 5H), 6.67−
6.60 (m, 2H), 4.54 (d, J = 12.8 Hz, 1H), 4.27 (d, J = 6.9 Hz, 1H),
3.99−3.89 (m, J = 6.7, 6.7, 6.7, 6.7, 6.7 Hz, 1H), 3.58 (d, J = 12.5 Hz,
1H), 2.08−1.97 (m, 1H), 1.93−1.83 (m, 1H), 1.74−1.56 (m, 4H),
1.48−1.38 (m, 1H), 1.34−1.23 (m, 1H). 13C NMR (126 MHz,
chloroform-d) δ 159.6, 155.9, 147.3, 145.4, 136.8, 136.0, 130.6, 130.8
(q, J = 32.4 Hz, 1C), 130.4 (s, 2C), 130.1, 128.9, 127.7 (s, 2C), 126.5,
124.09−123.88 (m, 1C), 123.7, 123.50−123.29 (m, J = 3.8 Hz, 1C),
124.2 (q, J = 271.8 Hz, 1C), 63.5, 52.2, 42.7, 33.8, 33.3, 23.6 (s, 2C).
Orthogonal HPLC purity: 97%, retention time of 11.55 min (HPLC
method A); 97%, retention time of 13.00 min (HPLC method B).
1-(1-(5-Chloropyridin-2-yl)-1-(3-fluoro-5-(trifluoromethyl)-
phenyl)-2-phenylethyl)-3-cyclopentylureas 14c, 14d, and 14e.
Urea 14c was obtained using general procedure B, in 40% yield. The
precursor amine was resolved by chiral SFC and acylated according to
procedure B to obtain (R)-1-(1-(5-chloropyridin-2-yl)-1-(3-fluoro-5-
(trifluoromethyl)phenyl)-2-phenylethyl)-3-cyclopentylurea 14d (SFC
conditions, chiral OJ column, 5% methanol, 2 mL/min, 35 °C, 100
bar, tR = 5.42 min) as the faster eluting enantiomer and (S)-1-(1-(5-
chloropyridin-2-yl)-1-(3-fluoro-5-(trifluoromethyl)phenyl)-2-phenyl-
ethyl)-3-cyclopentylurea 14e (SFC conditions, chiral OJ column, 5%
methanol, 2 mL/min, 35 °C, 100 bar, tR = 7.60 min) as the slower
eluting enantiomer. Data for urea 14e: LC−MS ESI 506.06 (M + H),
retention time of 3.59 min (10−90% MeOH in H2O with 0.1% TFA in
1
retention time of 9.72 min. Data for the S-isomer: H NMR (600
MHz, CDCl3) δ 8.62 (d, J = 1.4 Hz, 1H), 7.96 (dd, J = 8.4, 2.1 Hz,
1H), 7.55 (s, 1H), 7.40 (d, J = 9.6 Hz, 1H), 7.31 (d, J = 8.3 Hz, 1H),
7.29 (s, 1H), 7.26 (d, J = 8.0 Hz, 1H), 7.19 (t, J = 7.4 Hz, 1H), 7.12 (t,
J = 7.5 Hz, 2H), 6.99 (br s, 1H), 6.60 (d, J = 7.2 Hz, 2H), 4.55 (d, J =
12.8 Hz, 2H), 3.95 (br s, 1H), 3.59 (d, J = 12.8 Hz, 1H), 2.19−2.00
(m, 1H), 1.91 (td, J = 12.8, 6.8 Hz, 1H), 1.82−1.65 (m, 2H), 1.65−
1.55 (m, 2H), 1.55−1.39 (m, 1H), 1.33 (dt, J = 20.3, 6.5 Hz, 1H). 13C
NMR (151 MHz, CDCl3) δ 165.01, 162.87 (d, J = 249.3 Hz, 1C),
155.99, 150.18, 149.62 (d, J = 6.7 Hz), 140.10, 135.24, 130.37, 128.16,
127.14, 123.43 (d, J = 275.5 Hz, 1C), 123.17, 119.32 (m, 1C), 117.84
(d, J = 22.6 Hz, 1C), 116.27, 112.21 (dd, J = 24.6, 3.7 Hz, 1C), 108.94,
64.36, 52.51, 42.92, 34.00, 33.52, 23.78. 19F NMR (471 MHz, CDCl3)
δ −62.54, −109.77. Orthogonal HPLC purity: 97.7%, retention time
of 13.386 min (method A); 100.0%, retention time of 12.376 min
(method B). HRMS (M + H)+ calcd for C27H25F4N4O 497.1959,
found 497.1946.
(R)-1-Cyclopentyl-3-(1-(3-fluoro-5-(trifluoromethyl)phenyl)-
1-(5-fluoropyridin-2-yl)-2-phenylethyl)urea, 14g. Urea 14g was
obtained using general procedure B, in 55% yield. LC−MS ESI 490.55
(M + H), retention time of 3.97 min (10−90% MeOH in H2O with
0.1% TFA in a 4 min gradient). 1H NMR (500 MHz, chloroform-d) δ
8.16 (d, J = 2.77 Hz, 1H), 7.55 (s, 1H), 7.36−7.46 (m, 2H), 7.22 (d, J
= 8.32 Hz, 1H), 7.06−7.18 (m, 5H), 6.56−6.65 (m, 2H), 4.51 (d, J =
12.48 Hz, 1H), 4.32 (d, J = 6.94 Hz, 1H), 3.90−4.00 (m, J = 6.50, 6.50,
6.50, 6.50, 6.50 Hz, 1H), 3.54 (d, J = 12.48 Hz, 1H), 1.98−2.10 (m,
1H), 1.84−1.95 (m, 1H), 1.53−1.74 (m, 4H), 1.40−1.50 (m, 1H),
1.27−1.34 (m, 1H). 13C NMR (126 MHz, chloroform-d) δ 162.6 (d, J
= 248.9 Hz, 1C), 158.5 (d, J = 256.5 Hz, 1C), 156.6 (d, J = 2.9 Hz,
1C), 155.8, 150.7 (d, J = 7.6 Hz, 1C), 135.7, 134.6 (d, J = 23.8 Hz,
1C), 132.4 (qd, J = 32.4, 7.6 Hz, 1C), 130.3 (s, 2C), 127.8 (s, 2C),
126.6, 124.5 (d, J = 19.1 Hz, 1C), 123.7 (d, J = 4.8 Hz, 1C), 126.76−
119.76 (m, J = 275.6, 275.6, 275.6, 3.8 Hz, 1C), 119.26−119.04 (m, J
= 2.9 Hz, 1C), 117.6 (d, J = 21.9 Hz, 1C), 111.76−111.25 (m, 1C),
63.3, 52.3, 42.8, 33.9, 33.4, 23.6 (s, 2C). Orthogonal HPLC purity:
98%, retention time of 11.22 min (HPLC method A); 96%, retention
time of 12.63 min (HPLC method B).
1-(1-(6-Chloropyridin-3-yl)-1-(3-fluoro-5-(trifluoromethyl)-
phenyl)-2-phenylethyl)-3-cyclopentylurea, 14h. Urea 14h was
obtained using general procedure B, in 91% yield. LC−MS ESI
506.1(M + H), retention time of 2.25 min (MeOH/H2O/TFA,
Phenomenex Luna C18, 50 mm × 4.6 mm, 2 min gradient). 1H NMR
(500 MHz, chloroform-d) δ 8.33−8.22 (d, J = 2.7 Hz, 1H), 7.78−7.65
(dd, J = 8.5, 2.7 Hz, 1H), 7.61−7.50 (d, J = 8.3 Hz, 1H), 7.46−7.45
(m, 1H), 7.45−7.42 (dd, J = 8.4, 0.6 Hz, 2H), 7.19−7.07 (qd, J = 5.2,
1.8 Hz, 3H), 6.76−6.66 (dd, J = 7.4, 2.0 Hz, 2H), 6.47−6.39 (d, J = 7.1
Hz, 1H), 3.96−3.86 (m, 2H), 3.84−3.69 (dq, J = 12.9, 6.7 Hz, 1H),
1.83−1.66 (m, 2H), 1.66−1.56 (td, J = 7.3, 3.1 Hz, 2H), 1.55−1.41
(m, 2H), 1.39−1.21 (dt, J = 11.6, 5.3 Hz, 2H). 13C NMR (126 MHz,
DMSO) δ 161.73 (d, J = 245.8 Hz), 156.84, 148.37, 148.26, 140.37,
138.28, 136.11, 130.63, 131.10−130.04 (m), 127.59, 126.44, 123.48,
123.30 (d, J = 272.5 Hz), 119.39, 118.46, 118.29, 111.07 (d, J = 25.6
Hz), 62.56, 59.78, 50.81, 42.39, 32.95, 32.80, 23.07, 20.78, 14.10.
Orthogonal HPLC purity: 94%, retention time of 12.09 min (HPLC
method A); 94%, retention time of 10.62 min (HPLC method B).
(S)-1-Cyclopentyl-3-(1-(3-fluoro-5-(trifluoromethyl)phenyl)-
1-(6-methoxypyridin-3-yl)-2-phenylethyl)urea, 14i. By use of
general procedure B, 1-cyclopentyl-3-(1-(3-fluoro-5-(trifluoromethyl)-
phenyl)-1-(6-methoxypyridin-3-yl)-2-phenylethyl)urea (62 mg, 62%)
was obtained as white solids from 1-(3-fluoro-5-(trifluoromethyl)-
phenyl)-1-(6-methoxypyridin-3-yl)-2-phenylethanamine15 (75 mg)
and cyclopentyl isocyanate (0.075 mL). The racemic compound (50
mg) was separated by chiral HPLC using chiral AD column, eluting
with 20% IPA/heptane/0.1%DEA to give (R)-1-cyclopentyl-3-(1-(3-
fluoro-5-(trifluoromethyl)phenyl)-1-(6-methoxypyridin-3-yl)-2-
phenylethyl)urea (26 mg, white solids) as the faster eluting
1
a 4 min gradient). H NMR (500 MHz, chloroform-d) δ 8.27 (d, J =
2.2 Hz, 1H), 7.66 (dd, J = 8.6, 2.5 Hz, 1H), 7.55 (s, 1H), 7.39 (d, J =
9.7 Hz, 1H), 7.22 (d, J = 8.0 Hz, 1H), 7.19−7.13 (m, 1H), 7.12−7.02
(m, 4H), 6.63 (d, J = 7.2 Hz, 2H), 4.51 (d, J = 12.5 Hz, 1H), 4.36 (d, J
= 6.9 Hz, 1H), 3.99−3.88 (m, J = 6.5, 6.5, 6.5, 6.5, 6.5 Hz, 1H), 3.55
(d, J = 12.8 Hz, 1H), 2.09−1.97 (m, 1H), 1.94−1.83 (m, 1H), 1.72−
1.54 (m, 4H), 1.50−1.39 (m, 1H), 1.35−1.24 (m, J = 13.6, 6.6, 6.6, 6.6
Hz, 1H). 13C NMR (126 MHz, chloroform-d) δ 162.6 (d, J = 248.0
Hz, 1C), 158.9, 155.8, 150.5, 150.4, 145.6, 137.0, 135.6, 132.4 (qd, J =
32.4, 7.6 Hz, 1C), 130.9, 130.3 (s, 2C), 127.8 (s, 2C), 123.57−123.33
(m, 1C), 127.13−119.80 (m, J = 273.7, 273.7, 273.7, 2.9 Hz, 1C),
119.15−118.99 (m, 1C), 117.5 (d, J = 21.9 Hz, 1C), 111.5 (dq, J =
24.8, 3.8 Hz, 1C), 63.4, 52.2, 42.6, 33.8, 33.3, 23.6 (s, 2C). Orthogonal
HPLC purity: 97%, retention time of 11.72 min (HPLC method A);
98%, retention time of 13.30 min (HPLC method B).
( S ) - 1 - ( 1 - ( 5 - C y a n o p y r i d i n - 2 - y l ) - 1 - ( 3 - fl u o r o - 5 -
(trifluoromethyl)phenyl)-2-phenylethyl)-3-cyclopentylurea,
14f. 1-(1-(5-Bromopyridin-2-yl)-1-(3-fluoro-5-(trifluoromethyl)-
phenyl)-2-phenylethyl)-3-cyclopentylurea was obtained by using
general procedure B. This bromide (100 mg, 0.18 mmol), K4Fe(CN)6
(45 mg, 0.107 mmol), Pd(OAc)2 (catalytic amount, spatula tip), and
Na2CO3 (31 mg, 0.29 mmol) were stirred in DMAC (0.4 mL) at room
temperature. The reaction mixture was degassed several times and was
stirred and heated at 120 °C for 6 h. After the mixture was cooled,
EtOAc was added. The mixture was filtered through Celite, washed
with H2O, 5% NH4OH, dried over MgSO4, filtered, and concentrated
to dryness. The residue was purified by by flash chromatography (silica
gel, hexanes/EtOAc, came out 50% EtOAc) and was then further
purified by RP HPLC (20−100% CH3CN in H2O with 0.1 TFA in a
18 min run, came out at 13.38−13.88 min) to give pure racemic
mixture of the titled compound as white solids (16 mg, yield 18%).
LC−MS ESI 494.9 (M-H), retention time of 3.93 min (10−90%
MeOH in H2O with 10 mM NH4Cl in a 4 min run). The racemates
were separated by chiral preparative HPLC using AD column (20%
iPA/heptane/DEA, isocratic) to give (R)-1-(1-(5-cyanopyridin-2-yl)-
1-(3-fluoro-5-(trifluoromethyl)phenyl)-2-phenylethyl)-3-cyclopenty-
lurea as the fast eluting enantiomer (analytical chiral HPLC AD (25%
6170
dx.doi.org/10.1021/jm300611v | J. Med. Chem. 2012, 55, 6162−6175

Journal of Medicinal Chemistry
Article
enantiomer (analytical chiral AD, 10% isopropanol/heptane/0.1%
DEA, retention time of 4.60 min) and (S)-1-cyclopentyl-3-(1-(3-
fluoro-5-(trifluoromethyl)phenyl)-1-(6-methoxypyridin-3-yl)-2-
phenylethyl)urea 14j (18 mg, white solids) as the slower eluting
enantiomer (analytical chiral AD, 10%isopropanol/heptane/0.1%DEA,
retention time of 7.68 min). Data for the S-isomer: LC−MS ESI
502.18 (M + H), retention time of 3.99 min (10−90% MeOH in H2O
with 0.1% TFA in a 4 min run). 1H NMR (400 MHz, chloroform-d) δ
7.89 (d, J = 2.6 Hz, 1H), 7.30 (dd, J = 8.8, 2.5 Hz, 1H), 7.24 (s, 1H),
7.22−7.04 (m, 5H), 6.64 (m, 3H), 4.99 (s, 1H), 4.44 (br s, 1H), 3.86
(s, 3H), 3.81 (d, J = 12.9 Hz, 1H), 3.77 (m, 1H), 3.66 (d, J = 13.0 Hz,
1H), 1.79 (m, 2H), 1.61−1.33 (m, 4H), 1.22 (m, 2H). 13C NMR (101
MHz, chloroform-d) δ 163.81, 162.58 (d, J = 250 Hz), 156.62, 149.45,
144.71, 138.76, 135.33, 132.85, 132.38 (qd, J = 33, 8 Hz, 1C), 131.06,
128.25, 127.34, 123.42 (q, J = 274 Hz, 1C), 119.60, 118.20 (d, J = 23
Hz, 1C), 111.77 (d, J = 26 Hz, 1C), 110.64, 63.02, 54.20, 52.39, 45.04,
33.69, 33.64, 23.69. 19F NMR (376 MHz, chloroform-d) δ −62.46,
−109.84, −109.87. Orthogonal HPLC purity: 97.2%, retention time of
12.481 min (method A); 96.4%, retention time of 11.166 min
(method B). HRMS (M + H)+ calcd for C27H28F4N3O2 502.2112,
found 502.2100.
1 - ( 1 - ( 5 - C h l o r o p y r i d i n - 2 - y l ) - 2 - p h e n y l - 1 - ( 3 -
(trifluoromethoxy)phenyl)ethyl)-3-cyclopentylurea, 15a. Urea
15a was obtained using general procedure B, in 57% yield. LC−MS
ESI 504.07 (M + H), retention time of 3.59 min (10−90% MeOH in
H2O with 0.1% TFA in a 4 min gradient). 1H NMR (500 MHz,
chloroform-d) δ 8.26 (d, J = 1.94 Hz, 1H), 7.63 (dd, J = 2.50, 8.60 Hz,
1H), 7.42−7.48 (m, 1H), 7.37 (t, J = 8.05 Hz, 1H), 7.29 (s, 1H),
7.07−7.18 (m, 5H), 7.04 (br s, 1H), 6.61−6.68 (m, 2H), 4.49 (d, J =
12.76 Hz, 1H), 4.31 (d, J = 5.55 Hz, 1H), 3.88−4.00 (m, 1H), 3.55 (d,
J = 12.76 Hz, 1H), 1.96−2.07 (m, 1H), 1.82−1.93 (m, 1H), 1.52−1.71
(m, 4H), 1.37−1.47 (m, 1H), 1.23−1.35 (m, 1H). 13C NMR (126
MHz, chloroform-d) δ 159.7, 155.9, 149.4, 148.6, 145.4, 136.8, 136.0,
130.5, 130.3 (s, 2C), 129.7, 127.7 (s, 2C), 126.5, 124.9, 123.6, 119.5,
119.2, 120.4 (q, J = 256.5 Hz, 1C), 63.4, 52.2, 42.7, 33.8, 33.3, 23.6 (s,
2C). Orthogonal HPLC purity: 98%, retention time of 11.65 min
(HPLC method A); 91%, retention time of 13.17 min (HPLC method
B).
concentrated and used directly in the next step without further
purification. To a solution of 3-bromo-5-fluorophenol (104 mmol
crude) and iodo-1,1,2,2,-tetrafluoroethane (28.4 g, 125 mmol) in
DMSO (80 mL) was added K2CO3 (57.0 g, 420 mmol). The reaction
mixture was sealed in a thick walled glass pressure round-bottom flask
and heated at 70 °C for 18 h. The reaction mixture was allowed to cool
to room temperature, diluted with water (500 mL), and extracted with
ether (3 × 200 mL). The combined ether layers were washed with 1 N
NaOH (2 × 200 mL), water (2 × 200 mL), and brine (200 mL). The
organic layer was dried over sodium sulfate, filtered, and concentrated.
The residue was dissolved in ether (150 mL) and filtered through a
plug of activated basic alumina. The filtrate was concentrated to give 1-
bromo-3-fluoro-5-(1,1,2,2-tetrafluoroethoxy)benzene as a pale yellow
oil (27.2 g, 88% for two steps) which was used without further
purification. HPLC: 3.76 min (4 min gradient, MeOH/H2O 0.2%
polyphosphoric acid), purity 100%. By use of this aryl bromide urea,
15c and 15d were obtained employing general procedure B, in 94%
yield. The penultimate primary amine was resolved by chiral SFC, and
the faster eluting antipode was used in the final acylation to obtain
urea 15c, while the slower eluting antipode was used to obtain urea
15d (analytical Chiralpak AD 10 μm, 4.6 mm × 250 mm column, 5%
MeOH/95% carbobdioxide/0.1% diethylamine, 0.5 mL/min, retention
time of 10.36 and 11.23 min). Data for urea 15d: LC−MS ESI 554.28
(M + H), retention time of 4.09 min (10−90% MeOH in H2O with
0.1% TFA in a 4 min gradient). 1H NMR (500 MHz, chloroform-d) δ
8.26 (d, J = 1.92 Hz, 1H), 7.66 (d, J = 6.41 Hz, 1H), 7.00−7.22 (m,
7H), 6.88 (d, J = 7.90 Hz, 1H), 6.63 (br s, 2H), 5.74−6.03 (m, 1H),
3.83−4.66 (m, 3H), 3.51 (br s, 1H), 2.05 (br s, 1H), 1.90 (br s, 1H),
1.69 (br s, 4H), 1.28−1.49 (m, 2H). 13C NMR (126 MHz,
chloroform-d) δ 162.8 (d, J = 248.0 Hz, 1C), 158.4, 157.2, 149.74−
149.07 (m, 1C), 145.5, 137.2, 135.2, 131.0, 130.1 (s, 2C), 127.9 (s,
2C), 126.8, 123.4, 115.67−115.36 (m, 1C), 119.55−113.31 (m, 1C),
112.0 (d, J = 23.7 Hz, 1C), 108.60−107.96 (m, 1C), 107.35−107.27
(m, 1C), 107.5 (tt, J = 252.5, 41.2 Hz, 1C), 63.4, 52.6, 42.6, 33.3, 32.9,
23.6, 23.6. Orthogonal HPLC purity: 94%, retention time of 11.69 min
(HPLC method A); 97%, retention time of 13.04 min (HPLC method
B).
( S ) - 1 - ( 1 - ( 5 - C h l o r o p y r i d i n - 2 - y l ) - 1 - ( 3 -fl u o r o - 5 -
(trifluoromethyl)phenyl)-2-(1H-1,2,4-triazol-5-yl)ethyl)-3-cy-
clopentylurea, 18a. Compound 17 (41 mg, 0.096 mmol) was
dissolved in MeOH (1 mL), and HCl (4.0 M in dioxanes, 1.5 mL) was
added. The solution was diluted with DCM, washed with saturated
NaHCO3, dried, and concentrated under reduced pressure to yield a
white residue that was taken on directly to the cyclopentylurea as
described for compound 18b. Intermediate 19 was isolated in 36%
yield by preparative HPLC (retention time of 3.75 min, purity 96%).
LC−MS: [M + 1] 473.2. Compound 19 (10 mg, 0.021 mmol) was
dissolved in DMF−DMA (0.5 mL) and the solution stirred at room
temperature for 1.5 h. The solvents were removed under reduced
pressure, and the residue was azeotroped with hexanes (10 mL). The
resulting oil was dissolved in AcOH (0.5 mL) and hydrazine hydrate
added (30 mg). The reaction mixture was heated to 55 °C for 30 min
and allowed to cool. MeOH was added and the solution filtered. The
filtrate was purified by preparative HPLC, yielding compound 18a as a
colorless oil (2.1 mg, 21% yield). 1H NMR (400 MHz,) δ 8.44 (d, J =
2.0 Hz, 1H), 8.19 (s, 1H), 7.70 (dd, J = 8.8, 2.4 Hz, 1H), 7.39 (s, 1H),
7.28 (m, 1H), 7.24 (s, 1H), 7.22 (s, 1H), 4.59 (d, J = 14.8 Hz, 1H),
4.33 (d, J = 14.4 Hz, 1H), 1.89 (m, 2H), 1.62 (m, 4H), 1.38 (m, 2H).
LCMS: [M + 1] 497.2. HPLC: method C, purity 90% retention time
3.69 min.
( S ) - 1 - ( 1 - ( 5 - C h l o r o p y r i d i n - 2 - y l ) - 1 - ( 3 -fl u o r o - 5 -
(trifluoromethyl)phenyl)-2-(pyridin-2-yl)ethyl)-3-cyclopenty-
lurea, 18b. To a solution of (S)-1-(5-chloropyridin-2-yl)-1-(3-fluoro-
5-(trifluoromethyl)phenyl)-2-(pyridin-2-yl)ethanamine15 (15 mg,
0.029 mmol) in DCE (0.5 mL) were added 4 N HCl (0.5 mL),
MeOH (0.5 mL), cyclopentyl isocyanate (17 μL, 0.15 mmol), and
citric acid (1 mg, catalytic). The reaction mixture was stirred at room
temperature for 18 h, then concentrated to a volume of 0.5 mL under
reduced pressure and the residue applied to a preparative silica gel
TLC plate (1 mm thickness, 25 mm × 25 mm). Elution was with
1-(1-(5-Chloropyridin-2-yl)-2-phenyl-1-(3-(1,1,2,2-
tetrafluoroethoxy)phenyl)ethyl)-3-cyclopentylurea, 15b. Urea
15b was obtained using general procedure B, in 21% yield. LC−MS
ESI 536.09 (M + H), retention time of 4.00 min (10−90% MeOH in
H2O with 0.1% TFA in a 4 min gradient). 1H NMR (500 MHz,
chloroform-d) δ 8.26 (d, J = 2.22 Hz, 1H), 7.62 (dd, J = 2.50, 8.60 Hz,
1H), 7.41 (d, J = 8.05 Hz, 1H), 7.34 (t, J = 7.91 Hz, 1H), 7.29 (s, 1H),
7.02−7.18 (m, 6H), 6.66 (d, J = 7.21 Hz, 2H), 5.88 (tt, J = 2.20, 53.00
Hz, 1H), 4.39−4.54 (m, 2H), 3.87−4.01 (m, 1H), 3.58 (d, J = 12.76
Hz, 1H), 1.94−2.04 (m, 1H), 1.82−1.92 (m, 1H), 1.50−1.70 (m, 4H),
1.35−1.45 (m, 1H), 1.23−1.33 (m, 1H). 13C NMR (126 MHz,
chloroform-d) δ 159.7, 156.0, 149.0, 148.3, 145.3, 136.7, 136.1, 130.4,
130.3 (s, 2C), 129.5, 127.6 (s, 2C), 126.4, 124.6, 123.5, 120.0, 119.8,
118.91−113.71 (m, J = 271.8, 271.8, 27.7, 27.7 Hz, 1C), 107.6 (tt, J =
252.7, 42.0 Hz, 1C), 63.5, 52.1, 42.7, 33.7, 33.3, 23.5, 23.5. Orthogonal
HPLC purity: 95%, retention time of 11.55 min (HPLC method A);
95%, retention time of 12.77 min (HPLC method B).
(R)-1-(1-(5-Chloropyridin-2-yl)-1-(3-fluoro-5-(1,1,2,2-
tetrafluoroethoxy)phenyl)-2-phenylethyl)-3-cyclopentylurea,
15c, and (S)-1-(1-(5-Chloropyridin-2-yl)-1-(3-fluoro-5-(1,1,2,2-
tetrafluoroethoxy)phenyl)-2-phenylethyl)-3-cyclopentylurea,
15d. A solution of 1-bromo-3,5-difluorobenzene (20.0 g, 104 mmol)
was cooled in a water bath, and 2-(methylsulfonyl)ethanol (26.0 g, 207
mmol) in DMSO (100 mL) was added. KOtBu (29.0 g, 260 mmol)
was added to this reaction mixture in portions. The reaction mixture
turned dark. After the addition was complete, the water bath was
removed and the mixture was stirred at room temperature for 1 h. The
pH was adjusted to 1 using 1 N HCl, and extraction was with ether (3
× 200 mL). The combined organic portions were washed with
aqueous 1 N NaOH (2 × 200 mL). The NaOH layer was acidified to
pH 1 and extracted with ether (3 × 200 mL). The combined organic
layers were dried over sodium sulfate and filtered. The filtrate was
6171
dx.doi.org/10.1021/jm300611v | J. Med. Chem. 2012, 55, 6162−6175

Journal of Medicinal Chemistry
Article
hexane/EtOAc, 1:1. Compound 18b, Rf = 0.3, was isolated from the
silica as a white solid (9.5 mg, 65% yield). 1H NMR (400 MHz,
CD3OD) δ 8.38 (s, 1H), 8.23 (s, 1H), 7.76 (d, J = 8.4 Hz, 1H), 7.65
(m, 2H), 7.54 (d, J = 11.2 Hz, 2H), 7.34 (s, 2H), 7.16 (s, 1H), 7.08 (d,
J = 6.5 Hz, 1H), 4.40 (d, J = 12.7 Hz, 1H), 3.99 (d, J = 13.3 Hz, 1H),
3.94 (s, 1H), 2.03−1.81 (m, 2H), 1.73 (s, 2H), 1.61 (s, 2H), 1.54−
was isolated as the minor diastereomer (88 mg), Rf = 0.6 (hexane/
EtOAc, 1:1). Analytical data for (S)-N-((S)-1-(5-chloropyridin-2-yl)-2-
cyano-1-(3-fluoro-5-(trifluoromethyl)phenyl)ethyl)-2-methylpropane-
1
2-sulfinamide: H NMR (400 MHz, CDCl3) δ 8.61 (d, J = 2.6 Hz,
1H), 7.73 (dd J = 8.8 Hz and J = 2.6 Hz, 1H), 7.37 (s, 1H), 7.32 (d, J
= 7.9 Hz, 1H), 7.21 (s, 1H), 7.19 (s, 1H), 5.87 (s, 1H), 3.75 (d, J =
16.7 Hz, 1H), 3.67 (d, J = 16.7 Hz, 1H), 3.45 (br s, 1H), 1.36 (s, 9H).
LC−MS: [M + 1] 448.0. HPLC: purity 97%, retention time of 3.57
min (method C).
1.28 (m, 2H). 19F NMR (376 MHz, CD3OD) δ −60.56, −109.00. 13
C
NMR (101 MHz, CD3OD) δ 165.21, 162.76, 160.18, 158.82, 158.10,
151.97, 149.32, 146.77, 138.03, 137.54, 131.86, 126.80, 125.30, 123.09,
120.35 (m), 118.86, 118.75 (d, J = 22.9 Hz), 111.95 (dd, J = 24.9, 3.5
Hz), 65.13, 52.80, 46.09, 34.39, 34.39, 34.09, 24.54, 24.51. LC−MS:
[M + 1] 507.1. HPLC: method A, purity 97% retention time of 6.65
min; method B, purity 99%, retention time of 7.29 min. HRMS (ESI)
calcd for C25H24N4ClF4O (M + H)+ = 507.156 926, found 507.156 42.
(S)-Methyl 3-Amino-3-(5-chloropyridin-2-yl)-3-(3-fluoro-5-
(trifluoromethyl)phenyl)propanoate, 18c. Compound 18c was
prepared from (S)-methyl 3-amino-3-(5-chloropyridin-2-yl)-3-(3-fluo-
ro-5-(trifluoromethyl)phenyl)propanoate15 using the procedure de-
scribed for compound 18b as a white solid in 70% isolated yield. Rf =
0.3, hexanes/EtOAc, 4:1. 1H NMR (400 MHz, CD3OD) δ 8.51 (d, J =
2.4 Hz, 1H), 7.74 (dd, J = 8.7, 2.5 Hz, 1H), 7.59 (s, 1H), 7.51 (s, 1H),
7.45 (d, J = 10.1 Hz, 1H), 7.38 (d, J = 8.6 Hz, 1H), 7.27 (d, J = 8.4 Hz,
1H), 6.74 (s, 1H), 4.06 (dd, J = 14.9, 2.0 Hz, 1H), 3.87 (m, 1H), 3.80
(d, J = 14.9 Hz, 1H), 3.51 (s, 3H), 1.85 (m, 2H), 1.70 (m, 2H), 1.56
(m, 2H), 1.40 (m, 2H). 19F NMR (376 MHz, CD3OD) δ −64.39,
−112.25. 13C NMR (101 MHz, MeOD) δ 172.21, 160.65, 158.89,
147.51, 138.28, 132.07, 131.90, 123.89, 119.73 (m), 118.05 (d, J = 33.9
Hz), 117.88, 63.30, 52.79, 52.06, 42.40, 34.25, 24.60. LCMS: [M + 1]
488.2. HPLC: method A, purity 97% retention time of 10.13 min;
method B, purity 96%, retention time of 9.04 min. HRMS (ESI) calcd
for C22H23N3ClF4O3 (M + H)+ = 488.135 856, found 488.135 51.
( S ) - 1 - ( 1 - ( 5 - C h l o r o p y r i d i n - 2 - y l ) - 1 - ( 3 - fl u o r o - 5 -
(trifluoromethyl)phenyl)-2-(4-methylthiazol-2-yl)ethyl)-3-cy-
clopentylurea, 18d. Compound 18d was prepared from (S)-1-(5-
chloropyridin-2-yl)-1-(3-fluoro-5-(trifluoromethyl)phenyl)-2-(4-meth-
ylthiazol-2-yl)ethanamine15 using the procedure described for
compound 18b as a white solid in 70% isolated yield. Rf = 0.5,
To (S)-N-((S)-1-(5-chloropyridin-2-yl)-2-cyano-1-(3-fluoro-5-
(trifluoromethyl)phenyl)ethyl)-2-methylpropane-2-sulfinamide (30
mg, 0.067 mmol) solution in EtOH (1 mL) was added NH2OH (1
mL, 50% solution in water). The solution was heated to 70 °C for 3 h,
allowed to cool, and diluted with DCM, and the layers were separated.
The aqueous layer was extracted with DCM, and the combined
extracts were dried and concentrated to yield a tan solid which was
dissolved directly in DCM (1 mL) and cooled to 0 °C. Acetic
anhydride (11 μL) and TEA (25 μL) were added. The reaction
mixture was stirred for 14 h. Then additional acetic anhydride (20 μL)
and TEA (50 μL) were added. After 30 min, the reaction mixture was
diluted with DCM, washed with saturated NaCl, dried, and
concentrated under reduced pressure. LCMS [M + 1] 523.1. The
residue was dissolved in THF (2 mL), and Cs2CO3 (63 mg, mmol)
was added. The slurry was heated to 65 °C under reflux for 14 h,
whereupon the solvent evaporated leaving a residual solid which was
redissolved in methanol and purified using preparative HPLC.
Intermediate (S)-N-((S)-1-(5-chloropyridin-2-yl)-1-(3-fluoro-5-
(trifluoromethyl)phenyl)-2-(5-methyl-1,2,4-oxadiazol-3-yl)ethyl)-2-
methylpropane-2-sulfinamide was isolated as a colorless oil (15 mg).
HPLC: retention time of 3.80 min, purity 96%, method C. LC−MS:
[M + 1] 505.1. The sulfinamide was deprotected using HCl/MeOH15
and converted directly to compound 18e as described for compound
18b and isolated as a white solid in 43% isolated yield. Rf = 0.2
1
hexanes/EtOAc, 2:1. H NMR (400 MHz, CDCl3) δ 8.37 (d, J = 1.7
Hz, 1H), 7.61 (dd, J = 8.6, 2.4 Hz, 1H), 7.48 (s, 1H), 7.36 (d, J = 9.8
Hz, 1H), 7.16 (s, 1H), 7.09 (d, J = 8.6 Hz, 1H), 4.64 (d, J = 14.0 Hz,
1H), 4.46 (d, J = 2.4 Hz, 1H), 4.07−3.86 (m, 1H), 3.80 (d, J = 14.0
Hz, 1H), 2.45 (s, 3H), 1.96 (m, 2H), 1.61 (m, 4H), 1.42 (m, 2H). 19F
NMR (376 MHz, CD3OD) δ −62.23, −110.03. 13C NMR (101 MHz,
CDCl3) δ 175.79, 166.32, 158.07, 156.09, 146.27, 137.56, 131.70,
122.95, 118.79, 118.78 (m), 117.39, 117.02 (d, J = 37.4 Hz), 62.60,
52.36, 34.20, 33.42, 22.86, 11.78. LCMS: [M + 1] 512.2. HPLC:
method A, purity 95%, retention time of 9.98 min; method B, purity
95%, retention time of 8.93 min. HRMS (ESI) calcd for
C23H23N5ClF4O2 (M + H)+ = 512.147 096, found 512.146 76.
( S ) - 1 - ( 1 - ( 5 - C h l o r o p y r i d i n - 2 - y l ) - 1 - ( 3 -fl u o r o - 5 -
(trifluoromethyl)phenyl)-2-(4-fluorophenyl)ethyl)-3-cyclopen-
tylurea, 18f. Urea 18f was obtained using general procedure B, in
65% yield. LC−MS ESI 524.2 (M + H), retention time of 4.2 min
1
hexanes/EtOAc, 1:1. H NMR (400 MHz, CD3OD) δ 8.44 (s, 1H),
7.76 (dd, J = 8.6, 1.4 Hz, 1H), 7.52 (s, 2H), 7.49−7.44 (m, 1H), 7.39
(d, J = 8.7 Hz, 1H), 7.31 (d, J = 7.9 Hz, 1H), 6.91 (s, 1H), 4.68 (d, J =
13.6 Hz, 1H), 4.35 (d, J = 14.2 Hz, 1H), 3.98−3.78 (m, 1H), 2.24 (s,
3H), 1.88 (m, 2H), 1.71 (m, 2H), 1.58 (m, 2H), 1.48 (m, 1H), 1.39
(m, 1H). 19F NMR (376 MHz, CD3OD) δ −64.40, −112.19. 13C
NMR (101 MHz, CD3OD) δ 166.41, 159.77, 152.46, 147.30, 138.29,
132.22 (m), 124.66, 120.13, 115.76, 64.48, 53.05, 40.37, 34.48, 33.74,
24.64, 15.95. LC−MS: [M + 1] 527.2. HPLC: method A, purity 99%
retention time of 9.40 min; method B, purity 96%, retention time of
8.65 min. HRMS (ESI) calcd for C25H24N4ClF4OS (M + H)+
527.128 996, found 527.128 55.
=
1
(10−90% MeOH in H2O with 0.1% TFA in a 4 min gradient). H
(1-(5-Chloropyridin-2-yl)-1-(3-fluoro-5-(trifluoromethyl)-
phenyl)-2-(5-methyl-1,2,4-oxadiazol-3-yl)ethyl)-3-cyclopenty-
lurea, 18e. To a solution of acetonitrile (150 mg, 3.7 mmol) in ether
(10 mL) at −78 °C under argon was added LDA (2.0 M in
cyclohexane, 1.8 mL, 3.7 mmol). The yellow solution was stirred for
20 min. In a separate flask at 0 °C under argon, (S,E)-N-((5-
chloropyridin-2-yl)(3-fluoro-5-(trifluoromethyl)phenyl)methylene)-2-
methylpropane-2-sulfinamide15 (0.75 g, 1.8 mmol) was dissolved in
ether (2 mL), and BF3·Et2O (0.26 mg, 1.8 mmol) was added. This
solution was taken up via syringe and added to a solution of
acetonitrile pretreated with LDA. After 30 min, 1.0 M HCl (5 mL) was
added to the solution at −78 °C and the reaction mixture allowed to
reach room temperature, transferred to a separatory funnel, and
extracted with EtOAc. The combined extracts were dried over Na2SO4,
decanted, and concentrated, and the diastereomers were separated by
ISCO chromatography, 120 g from elution with hexane/EtOAc, 1:1.
(S)-N-((R)-1-(5-Chloropyridin-2-yl)-2-cyano-1-(3-fluoro-5-
(trifluoromethyl)phenyl)ethyl)-2-methylpropane-2-sulfinamide was
isolated as the major diastereomer (230 mg), Rf = 0.8 (hexane/
EtOAc, 1:1). (S)-N-((S)-1-(5-Chloropyridin-2-yl)-2-cyano-1-(3-fluo-
ro-5-(trifluoromethyl)phenyl)ethyl)-2-methylpropane-2-sulfinamide
NMR (500 MHz, chloroform-d) δ 8.29 (d, J = 2.19 Hz, 1H), 7.66 (dd,
J = 2.44, 8.52 Hz, 1H), 7.52 (s, 1H), 7.37 (d, J = 9.74 Hz, 1H), 7.21 (d,
J = 8.04 Hz, 1H), 7.11 (br s, 1H), 7.08 (d, J = 8.52 Hz, 1H), 6.75−6.83
(m, J = 8.60, 8.60 Hz, 2H), 6.58 (dd, J = 5.60, 8.52 Hz, 2H), 4.50 (d, J
= 12.91 Hz, 1H), 4.44 (d, J = 6.57 Hz, 1H), 3.87−3.98 (m, J = 6.50,
6.50, 6.50, 12.60 Hz, 1H), 3.51 (d, J = 12.91 Hz, 1H), 1.98−2.08 (m,
1H), 1.84−1.94 (m, 1H), 1.51−1.76 (m, 4H), 1.38−1.48 (m, 1H),
1.29 (qd, J = 6.72, 13.70 Hz, 1H). 13C NMR (126 MHz, chloroform-
d) δ 163.91−161.36 (m, 1C), 161.8 (d, J = 245.2 Hz, 1C), 158.7,
155.8, 150.3 (d, J = 6.4 Hz, 1C), 145.7, 137.1, 132.4 (qd, J = 33.6, 8.2
Hz, 1C), 131.7 (d, J = 8.2 Hz, 2C), 131.3 (d, J = 2.7 Hz, 1C), 131.0,
123.51−123.23 (m, 1C), 126.64−119.88 (m, J = 272.5, 272.5, 272.5,
1.8 Hz, 1C), 119.0 (br s, 1C), 117.5 (d, J = 22.7 Hz, 1C), 114.82−
114.44 (m, 2C), 111.83−111.44 (m, 1C), 63.3, 52.2, 41.8, 33.8, 33.3,
23.6 (s, 2C). Orthogonal HPLC purity: 95%, retention time of 11.69
min (HPLC method A); 95%, retention time of 13.20 min (HPLC
method B).
( S ) - 1 - ( 1 - ( 5 - C h l o r o p y r i d i n - 2 - y l ) - 1 - ( 3 -fl u o r o - 5 -
(trifluoromethyl)phenyl)but-3-enyl)-3-cyclopentylurea, 18g.
Urea 18g was obtained using general procedure B, in 21% yield.
6172
dx.doi.org/10.1021/jm300611v | J. Med. Chem. 2012, 55, 6162−6175

Journal of Medicinal Chemistry
Article
LC−MS ESI 456.53 (M + H), retention time of 4.00 min (10−90%
(m, 1C), 162.82−160.17 (m, J = 248.0 Hz, 1C), 158.0, 149.7 (d, J =
11.4 Hz, 1C), 148.5 (d, J = 7.6 Hz, 1C), 145.8, 137.5, 134.9, 132.5 (d, J
= 9.5 Hz, 1C), 131.5 (d, J = 3.8 Hz, 1C), 131.4, 130.0 (s, 2C), 128.0
(s, 2C), 127.0, 126.92−126.74 (m, J = 4.3, 2.4 Hz, 1C), 123.4, 122.1
(q, J = 272.8 Hz, 1C), 119.61−118.19 (m, J = 32.4, 32.4, 32.4 Hz, 1C),
117.3 (d, J = 21.9 Hz, 1C), 115.5 (d, J = 3.8 Hz, 1C), 112.0 (d, J = 21.9
Hz, 1C), 108.5 (d, J = 24.8 Hz, 2C), 107.5 (tt, J = 252.7, 40.1 Hz, 1C),
63.7 (d, J = 1.9 Hz, 1C), 41.6. Orthogonal HPLC purity: 98%,
retention time of 12.59 min (HPLC method A); 100%, retention time
of 14.25 min (HPLC method B).
1
MeOH in H2O with 0.1% TFA in a 4 min gradient). H NMR (500
MHz, chloroform-d) δ 8.49 (dd, J = 0.55, 2.50 Hz, 1H), 7.61 (dd, J =
2.50, 8.60 Hz, 1H), 7.46 (s, 1H), 7.28−7.36 (m, 2H), 7.18 (d, J = 8.05
Hz, 1H), 6.97 (dd, J = 0.55, 8.60 Hz, 1H), 5.39 (tdd, J = 7.14, 9.99,
17.06 Hz, 1H), 4.97−5.08 (m, 2H), 4.36 (d, J = 6.94 Hz, 1H), 3.90−
4.02 (m, 2H), 3.04 (dd, J = 6.94, 13.32 Hz, 1H), 1.92−2.07 (m, 2H),
1.57−1.70 (m, 4H), 1.32−1.45 (m, 2H). 13C NMR (126 MHz,
chloroform-d) δ 162.6 (d, J = 248.0 Hz, 1C), 159.1, 155.7, 150.0 (d, J
= 6.7 Hz, 1C), 146.0, 137.3, 132.89−131.80 (m, J = 32.4, 32.4, 32.4,
7.6 Hz, 1C), 132.0, 131.0, 122.9, 126.92−119.76 (m, J = 270.8, 270.8,
270.8, 3.8 Hz, 1C), 119.5, 119.14−118.79 (m, J = 3.8 Hz, 1C), 117.4
(d, J = 21.9 Hz, 1C), 111.82−111.22 (m, 1C), 62.6, 52.4, 41.4, 33.8,
33.6, 23.7 (s, 2C). Orthogonal HPLC purity: 97%, retention time of
11.12 min (HPLC method A); 100%, retention time of 12.75 min
(HPLC method B).
ASSOCIATED CONTENT
* Supporting Information
■
S
Determination of absolute stereochemistry, crystal structure
analysis, and crystal data. This material is available free of
charge via the Internet at http://pubs.acs.org.
( S ) - 1 - ( 1 - ( 5 - C h l o r o p y r i d i n - 2 - y l ) - 1 - ( 3 - fl u o r o - 5 -
(trifluoromethyl)phenyl)-2-p-tolylethyl)-3-cyclopentylurea,
18h. Urea 18h was obtained using general procedure B, in 60% yield.
LC−MS ESI 520.48 (M + H), retention time of 4.21 min (10−90%
AUTHOR INFORMATION
Corresponding Author
*Phone: (609) 252-3806. Fax: (609) 252-7446. E-mail:
Lalgudi.Harikrishnan@bms.com.
■
1
MeOH in H2O with 0.1% TFA in a 4 min gradient). H NMR (500
MHz, chloroform-d) δ 8.28 (d, J = 2.14 Hz, 1H), 7.66 (dd, J = 2.24,
8.43 Hz, 1H), 7.54 (br s, 1H), 7.38 (d, J = 9.39 Hz, 1H), 7.21 (d, J =
7.69 Hz, 1H), 7.08 (d, J = 8.54 Hz, 1H), 6.91 (d, J = 6.19 Hz, 2H),
6.50 (d, J = 6.83 Hz, 2H), 4.47 (d, J = 8.75 Hz, 1H), 4.34 (br s, 1H),
3.99 (br s, 1H), 3.50 (d, J = 10.89 Hz, 1H), 2.27 (s, 3H), 2.04 (br s,
1H), 1.90 (br s, 1H), 1.60−1.73 (m, 4H), 1.46 (br s, 1H), 1.30 (br s,
1H). 13C NMR (126 MHz, chloroform-d) δ 162.6 (d, J = 248.0 Hz,
1C), 159.0, 150.6, 150.56−150.51 (m, 1C), 145.6, 137.0, 136.2,
132.44−132.32 (m, 1C), 132.74−131.90 (m, J = 31.0, 31.0, 31.0 Hz,
1C), 130.8, 130.2 (s, 2C), 128.5 (s, 2C), 123.5, 123.3 (q, J = 273.7 Hz,
1C), 119.26−118.96 (m, J = 3.8 Hz, 1C), 117.81−117.35 (m, 1C),
111.77−111.28 (m, 1C), 63.3, 52.2 (br s, 1C), 42.3, 33.9 (br s, 1C),
33.4 (br s, 1C), 23.6, 23.6, 21.0. Orthogonal HPLC purity: 94%,
retention time of 11.92 min (HPLC method A); 95%, retention time
of 13.55 min (HPLC method B).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank Dr. James Johnson, Dr. Wu Yang, and
Yufeng Wang for the resynthesis of amide 20, Sarah C. Traeger
for the NMR support, Atsu Apedo for resolution of
enantiomers by chiral HPLC and SFC, and Dr. Jonathan
Josephs and Ming Chang for conducting the biotransformation
studies. The authors also thank the Discovery Analytical
Sciences, Compound Management, Lead Evaluation, and Lead
Profiling groups for their contributions.
( S ) - 1 - ( 1 - ( 5 - C h l o r o p y r i d i n - 2 - y l ) - 1 - ( 3 - fl u o r o - 5 -
(trifluoromethyl)phenyl)-2-(2-fluorophenyl)ethyl)-3-cyclopen-
tylurea, 18i. Urea 18i was obtained using general procedure B, in
40% yield. LC−MS ESI 524.20 (M + H), retention time of 4.10 min
ABBREVIATIONS USED
■
CETP, cholesteryl ester transfer protein; DPPE, diphenylpyr-
idylethanamine; RCT, reverse cholesterol transport; SPA,
scintillation proximity assay; WPA, whole plasma assay; ND,
not determined; MABP, mean arterial blood pressure; HR,
heart rate
1
(10−90% MeOH in H2O with 0.1% TFA in a 4 min gradient). H
NMR (500 MHz, chloroform-d) δ 8.22 (d, J = 2.14 Hz, 1H), 7.60 (dd,
J = 2.46, 8.65 Hz, 1H), 7.55 (s, 1H), 7.36−7.43 (m, J = 9.80 Hz, 1H),
7.20−7.25 (m, J = 8.10 Hz, 1H), 7.11−7.19 (m, 2H), 7.07 (dt, J =
1.81, 7.53 Hz, 1H), 6.97−7.04 (m, 2H), 6.72−6.80 (m, J = 9.30, 9.30
Hz, 1H), 4.31−4.41 (m, 2H), 3.90−4.01 (m, 1H), 3.76 (d, J = 12.81
Hz, 1H), 2.00−2.10 (m, 1H), 1.87−1.96 (m, 1H), 1.54−1.73 (m, 4H),
1.42−1.50 (m, 1H), 1.28−1.37 (m, 1H). 13C NMR (126 MHz,
chloroform-d) δ 162.5 (d, J = 248.9 Hz, 1C), 161.1 (d, J = 246.1 Hz,
1C), 158.4, 155.8, 150.3 (d, J = 6.4 Hz, 1C), 145.2, 136.8, 133.2 (d, J =
4.5 Hz, 1C), 132.90−131.77 (m, J = 32.7, 32.7, 32.7, 8.2 Hz, 1C),
131.0, 128.6 (d, J = 8.2 Hz, 1C), 124.0 (d, J = 2.7 Hz, 1C), 123.6 (d, J
= 3.6 Hz, 1C), 122.5 (d, J = 15.4 Hz, 1C), 126.80−119.95 (m, J =
272.5, 272.5, 272.5, 2.7 Hz, 1C), 119.23−118.92 (m, J = 3.6 Hz, 1C),
117.6 (d, J = 21.8 Hz, 1C), 114.8 (d, J = 23.6 Hz, 1C), 111.93−111.44
(m, 1C), 63.0, 52.2, 35.7, 33.8, 33.3, 23.6 (s, 2C). Orthogonal HPLC
purity: 96%, retention time of 11.60 min (HPLC method A); 95%,
retention time of 13.16 min (HPLC method B).
REFERENCES
■
(1) (a) Sacks, F. M. Lipid and lipoprotein metabolism, and risk for
cardiovascular disease. Metab. Risk Cardiovasc. Dis. 2011, 18−40.
(b) Goldenberg, N.; Glueck, C. Statins efficacy, effectiveness and real
life goal attainment of statins in managing cardiovascular risk. Vasc.
Health Risk Manage. 2009, 5, 369−376.
(2) (a) Rhoads, G. G.; Gulbrandsen, C. L.; Kagan, A. Serum
lipoproteins and coronary heart disease in a polulation study of Hawaii
Japanese men. N. Engl. J. Med. 1976, 294, 293−298. (b) Castelli, W. P.;
Garrison, R. J.; Wilson, P. W.; Abbott, R. D.; Kalousdian, S.; Kannel,
W. B. Incidence of coronary heart disease and lipoprotein cholesterol
levels. The Framingham Study. JAMA, J. Am. Med. Assoc. 1986, 256,
2835−2838. (c) Kannel, W. B. Range of serum cholesterol values in
the population developing coronary artery disease. Am. J. Cardiol.
1995, 76, 69c−77c.
(3) Virani, S. S.; Ballantyne, C. M. Therapy and clinical trials: HDL-
cholesterol and niacin therapy: past, present, and future. Curr. Opin.
Lipidol. 2010, 21, 165−166.
(4) Barter, P. J.; Brewer, H. B.; Chapman, M. J.; Hennekens, C. H.;
Rader, D. J.; Tall, A. R. Cholesteryl ester transfer protein. Arterioscler.,
Thromb., Vasc. Biol. 2003, 23, 160−167.
(5) For a recent review see the following: Weber, O.; Bischoff, H.;
Schmeck, C.; Bottcher, M. Cholesteryl ester transfer protein and its
inhibition. Cell. Mol. Life Sci. 2010, 67, 3139−3149.
(S)-N-(1-(5-Chloropyridin-2-yl)-1-(3-fluoro-5-(1,1,2,2-
tetrafluoroethoxy)phenyl)-2-phenylethyl)-4-fluoro-3-
(trifluoromethyl)benzamide, 20. Amide 20 was obtained using
general procedure C in 49% yield. LC−MS ESI 633.3 (M + H),
retention time of 2.21 min (10−90% MeOH in H2O with 0.1% TFA in
a 2 min gradient). 1H NMR (500 MHz, chloroform-d) δ 9.11 (s, 1H),
8.36 (d, J = 2.14 Hz, 1H), 8.01−8.06 (m, 1H), 7.88 (ddd, J = 2.24,
4.70, 8.43 Hz, 1H), 7.75 (dd, J = 2.46, 8.65 Hz, 1H), 7.25−7.31 (m,
1H), 7.22 (d, J = 8.54 Hz, 1H), 7.11−7.17 (m, 3H), 7.04 (t, J = 7.69
Hz, 2H), 6.93 (d, J = 8.75 Hz, 1H), 6.55 (d, J = 7.05 Hz, 2H), 5.89 (tt,
J = 2.80, 53.00 Hz, 1H), 4.56 (d, J = 13.02 Hz, 1H), 3.62 (d, J = 12.81
Hz, 1H). 13C NMR (126 MHz, chloroform-d) δ 163.6, 164.03−161.74
6173
dx.doi.org/10.1021/jm300611v | J. Med. Chem. 2012, 55, 6162−6175

Journal of Medicinal Chemistry
Article
3
(6) (a) Chapman, M. J.; LeGoff, W.; Guerin, M.; Kontush, A.
Cholesteryl ester transfer protein: at the heart of the action of lipid-
modulating therapy with statins, fibrates, niacin, and cholesteryl ester
transfer protein inhibitors. Eur. Heart J. 2010, 31, 149−164. (b) Linsel-
Nitschke, P.; Tall, A. R. HDL as a target in the treatment of
atherosclerotic cardiovascular disease. Nat. Rev. Drug Discovery 2005, 4,
193−205 and references therein..
plasma containing H-cholesterol ester in HDL (3H-CE/HDL). The
mixture was incubated for 2.5 h at 37 °C and terminated with the
addition of 6 μL of water/1 M MgCl2/2% Dextralip 50 (2:1:1) to
precipitate LDL and VLDL. After 10 min at room temperature, the
mixture was transferred to filter plates (Millipore, no. MHVBN45)
that were prewet with phosphate buffered saline. The plates were
centrifuged (1800 rpm) at room temperature for 10 min. Scintillation
fluid was added and the amount of radioactivity determined using a
scintillation counter. Background activity was determined with plasma
samples incubated for 2.5 h at 4 °C. All concentrations of compounds
were tested in duplicate and the IC50 values calculated from a curve fit
of the data.
(7) (a) Boettcher, M.-F.; Heinig, R.; Schmeck, C.; Kohlsdorfer, C.;
Ludwig, M.; Schaefer, A.; Gelfert-Peukert, S.; Wensing, G.; Weber, O.
Single dose pharmacokinetics, pharmacodynamics, tolerability and
safety of BAY 60-5521, a potent inhibitor of cholesteryl ester transfer
protein. Br. J. Clin. Pharmacol. 2012, 73, 210−218. (b) Shinkai, H.
Cholesteryl ester transfer protein inhibitors as high-density lipoprotein
raising agents. Expert Opin. Ther. Pat. 2009, 19, 1229−1237.
(c) Ruggeri, R. B. Cholesteryl ester transfer protein: pharmacological
inhibition for the modulation of plasma cholesterol levels and
promising target for the prevention of atherosclerosis. Curr. Top.
Med. Chem. 2005, 5, 257−264 and references therein. (d) Sikorski, J.
A. Oral cholesteryl ester transfer protein (CETP) inhibitors: a
potential new approach for treating coronary artery disease. J. Med.
Chem. 2006, 49, 1−22. (e) Krishna, R.; Anderson, M. S.; Bergman, A.
J.; Jin, B.; Fallon, M.; Cote, J.; Rosko, K.; Chavez-Eng, C.; Lutz, R.;
Bloomfield, D. M.; Gutierrez, M.; Doherty, J.; Bieberdorf, F.;
Chodakewitz, J.; Gottesdiener, K. M.; Wagner, J. A. Effect of the
cholesteryl ester transfer protein inhibitor, anacetrapib, on lipoproteins
in patients with dyslipidemia and on 24-h ambulatory blood pressure
in healthy individuals: two double-blind, randomized placebo-
controlled phase I studies. Lancet 2007, 370, 1907−1914.
(8) Virtual screening details are as follows. A single low energy
structure for each of the reference compounds 1, 3, and 4 was
generated using Macromodel, version 7.2008; Schrodinger, LLC: New
York, 2002 , followed by SAM1 quantumchemical energy calculations
(AMPAC, version 6.55; Semichem (P.O. Box 1649, Shawnee Mission,
KS 66222), 1997 ).For each reference compound, a four-point
pharmacophore search (ChemX,Chemical Design, Ltd., flexible option
on) was performed against theBMS in-house deck, and the top 1000
(reference molecules 1 and 3) and 3000 (reference molecule 4)hits
were retained. The lowest energy conformation of reference molecule
3 was also used as input to an in-house molecule shape-matchingtool
in which BMS deck compounds were scored based on 3D matchingto
specific features including hydrophobes, aromatic rings, fluorineatoms,
and an acceptor atom. The assay yielded an initial hit thatcould be
traced back to the four-point similarity search around referencecom-
pound 3, and subsequent similarity searching aroundthis hit yielded
compound 7.
(12) Corte, J.; Hangeland, J.; Quan, M.; Smallheer, J. M.; Fang, T.
Preparation of Pyridine Carboxamides as Serine Protease Inhibitors.
WO2005123680, 2005.
(13) Biel, J. H.; Warawa, E. J. Substituted Pyridines. US 3413298,
1968. The first eluting enantiomer was the more potent antipode.
(14) Satoh, T.; Taylor, P.; Bosron, W. F.; Sanghani, S. P.; Hosokawa,
M.; La Du, B. N. Current progress on esterases: from molecular
structure to function. Drug Metab. Dispos. 2002, 30, 488−493.
(15) Kamau, M. G.; Harikrishnan, L. S.; Finlay, H. J.; Qiao, J. X.;
Jiang, J.; Salvati, M. E.; Poss, M. A.; Wexler, R. R.; Lawrence, R. M.
Synthesis of tertiary carbinamines. Tetrahedron. 2012, 68, 2696−2703.
(16) Absolute stereochemistry of active antipode was determined by
X-ray crystal structure of Mosher’s amide. See Supporting Information
for details.
(17) For the transgenic mouse PD study, 3H-CE transfer activity was
measured in plasma obtained from hCETP/apoB-100 dual transgenic
mice before and at several time points after dosing. Briefly, H-CE/
3
HDL (refs 10 and 11) was added to the samples, and the amount of
radioactivity incorporated into LDL/VLDL after precipitation with
water/1 M MgCl2/2% Dextralip 50 (2:1:1) was determined. The
activities measured in samples obtained after dosing were normalized
to the activity in the plasma sample taken from the same animal before
dosing.
(18) Liver microsomal stability was determined by incubating
compound (3 μM) with microsomal protein (1 mg/mL) for 10 min
followed by quantitative determination of the remaining compound by
UV absorbance.
(19) Biotransformation studies were carried out by incubating the
compound (30 μM) with microsomal protein (1 mg/mL) for 60 min
followed by metabolite identification by LC/MS/MS using a Thermo
LTQ mass spectrometer.
(20) For the transgenic mouse pharmacodynamic assay, transgenic
mice expressing human CETP and apoB-100 were used. Predose
blood samples were collected by retro-orbital bleed. Compounds were
formulated in ethanol/Cremophor/water at a 1:1:8 ratio and dosed at
30 mg/kg, po. At 2, 4, and 8 h after dosing, blood samples were
collected. Cholesterol ester transfer activity was measured in all blood
samples and normalized by the activity measured in the same animal at
baseline (mean of four or five mice per dose).
(9) Based on the following: Bisgaier, C. L.; Minton, L. L.; Essenburg,
A. D.; White, A.; Homan, R. Use of fluorescent cholesteryl ester
microemulsions in cholesteryl ester transfer protein assays. J. Lipid Res.
1993, 34, 1625−1634 . Vesicles containing BODIPY-cholesterol ester
(Molecular Probes) as CE donor were incubated with CETP and LDL
(as CE acceptor), and fluorescence was monitored. Hits were
identified as compounds that inhibited fluorescence greater than
∼50%..
(21) For the hamster study, plasma HDL-C was measured in male
Golden Syrian hamsters initially fed a moderately fat diet (2.5%
coconut oil, 0.25% cholesterol) for 2 weeks. The hamsters were then
dosed with vehicle, torcetrapib, or amide 20, po, q.d. At 2 h after
dosing on day 3, plasma samples were obtained and HDL-C was
measured and compared to the values measured before dosing.
(22) Barter, P. J.; Caulfield, M.; Eriksson, M.; Grundy, S. M.;
Kastelein, J. J. P.; Komajda, M.; Lopez-Sendon, J.; Mosca, L.; Tardif, J.;
Waters, D. D.; Shear, C. L.; Revkin, J. H.; Buhr, K. A.; Fisher, M. R.;
Tall, A. R.; Brewer, B. Effects of torcetrapib in patients at high risk for
coronary events. N. Engl. J. Med. 2007, 357, 2109−2122.
(23) Stroes, E. S. G.; Kastelein, J. J. P.; Benardeau, A.; Kuhlmann, O.;
Blum, D.; Campos, L. A.; Clerc, R. G.; Niesor, E. J. Dalcetrapib: no off-
target toxicity on blood pressure or on genes related to the renin−
angiotensin−aldosterone system in rats. Br. J. Pharmacol. 2009, 158,
1763−1770.
(10) CETP activity was measured by scintillation proximity assay
3
(SPA) as the amount of H-cholesteryl ester transferred from HDL
(3H-CE/HDL) to biotinylated LDL and captured on avidin-SPA
beads. Reactions were initiated by the addition of purified human
recombinant CETP (∼5.5 nM final concentration) and incubated at
37 °C. Reactions were terminated by the addition of LEADseeker
beads (GE Healthcare, no. RPNQ0261, 2 mg/mL in buffer containing
1 mg/mL BSA and 0.05 mg protein/mL HDL). The beads were
allowed to settle overnight at room temperature before reading.
Compounds were tested at multiple concentrations, and the
concentration required to inhibit 50% of the activity (IC50) was
determined from a curve fit of the data with each concentration tested
in duplicate. Background activity was determined in a set of wells that
received buffer but not CETP. All data were background-subtracted.
(11) CE transfer activity was measured in a “whole plasma assay”
(WPA) as follows. Compound (1 μL) was added to 29 μL of human
(24) Clerc, R. G.; Stauffer, A.; Weibel, F.; Hainaut, E.; Perez, A.;
Hoflack, J.; Benardeau, A.; Pflieger, P.; Garriz, J. M. R.; Funder, J. W.;
6174
dx.doi.org/10.1021/jm300611v | J. Med. Chem. 2012, 55, 6162−6175

Journal of Medicinal Chemistry
Article
Capponi, A. M.; Niesor, E. J. Mechanisms underlying off-target effects
of the cholesteryl ester transfer protein inhibitor torcetrapib involve L-
type calcium channels. J. Hypertens. 2010, 28, 1676−1686.
(25) The compound was too hydrophobic to elute in the gradient
under HPLC method B.
6175
dx.doi.org/10.1021/jm300611v | J. Med. Chem. 2012, 55, 6162−6175