Identification of a potent and metabolically stable series of fluorinated diphenylpyridylethanamine-based cholesteryl ester transfer protein inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.08.011

A novel series of diphenylpyridylethanamine-based inhibitors of cholesteryl ester transfer protein is described. Optimization of the urea moiety, particularly by incorporation of fluorine, is explored to balance in vitro metabolic stability with CETP pote

Full text of DOI:10.1016/j.bmcl.2012.08.011

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6503–6508
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of a potent and metabolically stable series of fluorinated
diphenylpyridylethanamine-based cholesteryl ester transfer protein inhibitors
Michael M. Miller a, , Yalei Liu a, Ji Jiang a, James A. Johnson a, Muthoni Kamau a, David S. Nirschl b,
⇑
Yufeng Wang a, Lalgudi Harikrishnan a, David S. Taylor c, Alice Ye A. Chen c, Xiaohong Yin c,
Ramakrishna Seethala d, Tara L. Peterson d, Tatyana Zvyaga e, Jun Zhang b, Christine S. Huang f,
Ruth R. Wexler a, Michael A. Poss a, R. Michael Lawrence a, Leonard P. Adam c, Mark E. Salvati a
a Discovery Chemistry, Research and Development, Bristol-Myers Squibb, PO Box 4000, Princeton, NJ 08543-4000, USA
b Synthesis & Analysis Technology Team, Research and Development, Bristol-Myers Squibb, PO Box 4000, Princeton, NJ 08543-4000, USA
c Discovery Biology, Research and Development, Bristol-Myers Squibb, PO Box 4000, Princeton, NJ 08543-4000, USA
d Lead Evaluation, Research and Development, Bristol-Myers Squibb, PO Box 4000, Princeton, NJ 08543-4000, USA
e Lead Profiling, Research and Development, Bristol-Myers Squibb, PO Box 4000, Princeton, NJ 08543-4000, USA
f PCO MAP, Research and Development, Bristol-Myers Squibb, PO Box 4000, Princeton, NJ 08543-4000, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of diphenylpyridylethanamine-based inhibitors of cholesteryl ester transfer protein is
described. Optimization of the urea moiety, particularly by incorporation of fluorine, is explored to bal-
ance in vitro metabolic stability with CETP potency in the whole plasma assay.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 5 June 2012
Revised 25 July 2012
Accepted 1 August 2012
Available online 9 August 2012
Keywords:
CETP inhibitor
Cholesteryl ester transfer protein
Reverse cholesterol transport
High-density lipoprotein cholesterol
Low-density lipoprotein cholesterol
Atherosclerosis
Coronary heart disease
Atherosclerosis is characterized by the accumulation of lipid-
rich, rupture-prone plaques within arterial walls, and can lead to
coronary heart disease (CHD) which afflicts over 17.6 million indi-
viduals in the United States.1 Current clinical strategies of lipid
lowering for the treatment of atherosclerosis have primarily fo-
cused on lowering circulating levels of low-density lipoprotein
cholesterol (LDL-C)2 via the use of agents such as statins.3 It is also
recognized that high-density lipoprotein cholesterol (HDL-C) is a
powerful independent inverse predictor of CHD risk.4 Importantly,
elevation of plasma HDL-C has been shown to decrease CHD risk,5,6
in large part by facilitating the transport of cholesterol from the
arterial wall to the liver for excretion (a process termed ‘reverse
cholesterol transport’, or RCT). Current approaches to increase
HDL-C include the use of fibrates7 and niacin8–10 that have limited
effects on increasing HDL-C levels11 (i.e., 10% and 16%, respec-
tively). Due to the limited efficacy of current HDL-C increasing
therapies,12 and the role of HDL-C in reducing CHD risk, additional
approaches to increase HDL-C are being investigated.
Cholesteryl ester transfer protein (CETP) is a plasma glycoprotein
that mediates the net transfer of cholesterol ester (CE) from HDL to
LDL and very low-density lipoprotein (VLDL) in exchange for triglyc-
eride (TG).13 Inhibition of CETP activity leads to increased plasma
HDL-C levels in both animals and humans.13 Despite its role in
HDL-C regulation, CETP has been proposed to have both pro- and
anti-atherosclerotic properties, and defining the absolute role of
CETP in atherosclerosis has been a subject of debate.14–20 The
beneficial HDL raising and anti-atherosclerotic effects of CETP
inhibition have been supported by animal studies in hamsters21
and rabbits.22–24 However, despite increasing HDL-C in humans25
to a similar extent as in animals, the first CETP inhibitor did not pro-
gress beyond phase III clinical trials, although it is likely this was
due, at least in part, to off-target toxicities.26 Despite the ongoing
controversies surrounding CETP, widespread interest in discovering
and developing inhibitors of this protein as a potential new thera-
peutic approach to treat CHD is evident by the numerous reports
describing preclinical and clinical lead compounds (Fig. 1).27–33
⇑
Corresponding author. Tel.: +1 609 252 3238.
E-mail address: michael.miller@bms.com (M.M. Miller).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.011

6504
M. M. Miller et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6503–6508
F
Initial SAR optimization of the urea moiety was carried out with
both enantiomers and confirmed that the activity resided predom-
inantly in the S-antipode (8a compared to 8b) as shown previ-
O
CF3
O
MeO
F3C
N
N
F
OH
N
H
Me
Me
ously.33 Subsequent exploration utilized the chiral amine
9
CF3
S
Et
prepared from the corresponding chiral sulfinylimine.38 Profiling
of 8a demonstrated the analogue had poor in vitro metabolic sta-
bility in human, mouse and rat liver microsomes.39 To investigate
whether the urea was responsible for this liability, additional SAR
of the urea alkyl group was desired.
Me
Me
O
F3C
N
Me Me
EtO
O
1
3
2
Pfizer
Bayer
Japan Tobacco / Roche
JTT 705
Torcetrapib
BAY 194789
Cl
Starting with 9, a robust method to prepare urea analogues was
utilized wherein an in situ generated para-nitrophenyl carbamate
was directly treated with a variety of amines (Scheme 1, Table 1).
From this study it was shown that incorporation of a heteroatom
into the cyclopentyl ring was tolerated with respect to potency
(11a), but resulted in a further decrease in metabolic stability (8a
vs 11a). However, the corresponding lactone analogue 12a,
although showing reduced CETP inhibitory potency, did provide
some indications of improved in vitro metabolic stability. This
observation coupled with a biotransformation analysis of the par-
ent compound 8 suggested that the cyclopentyl ring system was
the principal site of oxidative metabolism for these analogues
and a potential area for continued optimization. In order to resolve
this liability, a strategy was implemented to not only modulate
metabolic stability,40 but also to increase biological activity.
The profound effect and role of fluorine in drug discovery has
been well documented.41 Selective installation of fluorine into a
target molecule has been shown to favorably influence a number
of pharmacokinetic, physicochemical, and physical properties,
including binding affinity (potency, selectivity), metabolic stability,
ADME, molecular conformation, and safety issues. In particular, it
was recently reported that fluorine substitution of CETP inhibitors
during the development of 1 provided a beneficial effect in refining
the activity of this series by influencing the lipophilicity of the mol-
ecules.42 In addition, it was observed that although a variety of
structurally diverse scaffolds have been developed as small mole-
cule CETP inhibitors (Fig. 1), these are generally highly fluorinated
lipophilic compounds.
Me
CF3
CF3
Me
OMe
O
F
N
CF3
N
O
O
OCF2CF2H
Me Me
CF3
HO
5
4
Pharmacia
SC 795
Merck
Anacetrapib
Me
Me
Me
O
O
Me
O
O
O
N
NH Me
N
Me
F3C
6
7
BMS hit
BMS hit
SPA IC50 = 280 nM
SPA IC50 = 6.5 µM
WPA IC50 = 26 µM
WPA IC50 = 10 µM
Figure 1. Reported CETP inhibitors.
In previous communications we31 and others32 described a no-
vel series of 2-arylbenzoxazole-based (6) and diphenylpyridyl eth-
anol-based (7)33 CETP inhibitors identified by leveraging the BMS
compound collection via a screening campaign.34 These two scaf-
folds are architecturally unique from other reported chemotypes
in this target area and were deemed to have suitable SAR to war-
rant follow up exploration.
To this end, fluorine incorporation was explored extensively in
the alkyl urea functionality of 8. Examination of fluorination at the
Early SAR exploration of screening hit 7 led to the identification of
compound 8, wherein the ester moiety was converted to an urea
linkage with refined substitution of the groups off the quaternary
center. These changes resulted in a lead exhibiting increased activity
in the program’s scintillation proximity assay (SPA)35 with a deter-
mined IC50 of 20 nM. Although 8 demonstrated that SPA potency
could be improved in this chemotype, other parameters also re-
quired attention: potency in the human whole plasma assay
(WPA),36 solubility, metabolic stability, PXR activation, hERG inhibi-
tion, and oral bioavailability. To improve on these properties, dis-
crete areas of exploration (i.e., modifications of the A-, B-, and/or
C-rings, varying the amine capping fragments, and constraining
the scaffold’s molecular framework) were defined as outlined in Fig-
ure 2. This communication describes the optimization of the urea
fragmentofthe diphenylpyridylethanamine (DPPE)basedlead(8).37
3-position of the cyclopentyl ring (13a) provided
a modest
enhancement in WPA activity (13a vs 8a), while exhibiting an
overall improvement in metabolic stability (Table 1). Fluorine
incorporation in the corresponding cyclohexylurea (14–17 vs 8a)
failed to improve WPA activity, but 17 showed an improvement
in its in vitro liver microsomal stability compared to 14.
Parallel SAR development on the B-ring identified a 3,5-disub-
stituted aryl fragment which was subsequently utilized for the
optimization of fluorinated ureas (Table 2).33 Specifically, it was
observed that in some instances the addition of a tetrafluorinated
H
N
(a)
Cl
O
Cl
Cl
NH2
F
NO2
N
N
O
F3C
F3C
F
Ureas
Amine
Caps
Amides
9
10
Carbamates
Alkyl / Aryl
3
(b)
R
2
n
1
H
N
R
N
H
N
H
Constrained
H3N
Cl
Cl
O
1
2
Analogues
n
A
N
N
H
N
3
O
Asymmetric
Synthesis
50-95%
C
F3C
F
B
F3C
F
8, 11-17
8
Scheme 1. General synthetic route to DPPE analogues. Reagents (a) para-nitro-
Figure 2. DPPE lead and strategy for optimization.
phenyl chloroformate, K2CO3, CH2Cl2; (b) amine, DIPEA, dioxane.

M. M. Miller et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6503–6508
6505
Table 1
SAR for substituted cycloalkyl ring against human CETP
H
N
H
N
Cl
R
Cl
R
N
N
O
O
F3C
F
F3C
F
S
R
R
Cmpd #
Core
SPA, IC50
(lM)
WPA, IC50
(lM)
MTS (% remaining)
8a
8b
S
R
0.02
0.48
1.57
33.33
9(h), 8(m), 10(r)
N
H
11a
11b
S
R
0.05
–––
4.88
44.59
1(h), 1(m), 9(r)
O
N
H
12a
12b
S
R
0.27
14.46
7.10
–––
28(h), 29(m), 61(r)
61(h), 59(m), 47(r)
O
O
N
H
13a
13b
S
R
0.006
0.34
0.67
32.75
F
F
N
H
14a
14b
S
R
0.09
–––
8.89
91.82
9(h), 4(m), 6(r)
N
H
15a
16a
S
S
0.02
0.14
4.23
F
N
F
H
F
F
17.15
N
H
F
F
17a
S
0.13
13.07
37(h), 57(m), 62(r)
N
H
MTS: 3 lM metabolic stability (human, mouse, rat), n = 4 (Ref. 39).
ethoxy group demonstrated an enhancement in potency (i.e., 18 vs
8a). Combination of the 3-fluoro-5-(1,1,2,2-tetrafluoro-eth-
oxy)benzene B-ring fragment of 18 and the 3,3-difluoro cyclopen-
tanamine unit of 13 resulted in 19 as a mixture of diastereomers.
Separation of the corresponding isomers showed that the activity
resided in the S,R-isomer 20 which, with a WPA IC50 of 76 nM,
was 36-fold more potent than R,S-isomer 21 and 21-fold more po-
tent that the initial starting point 8.
Although 20 proved to be quite active in whole plasma, it still
was found to be rapidly metabolized by liver microsomes across
species (h, m, r). While the difluorocyclopropyl analogue 22 pro-
vided excellent metabolic stability, a 20-fold drop off in WP activ-
ity was observed versus 20. However, the corresponding
difluorocyclobutyl variant 23 addressed metabolic stability in
addition to maintaining an excellent level of CETP inhibitory po-
tency. This 3,3-difluoro-substituted cyclobutyl fragment was of
interest, not only because of its increased potency but for its sym-
metry which resulted in a simplified structure possessing only one
chiral center.
responding cyclopentyl urea 18. Incorporation of fluorine to pre-
pare 25, provided an increase in potency relative to the parent
24 but remained significantly less potent than the corresponding
19. Selective placement of additional fluorine atoms provided ana-
logues, such as 28, with enhanced potency and improved meta-
bolic stability over initial pyrrolidine compounds (i.e., 24 vs 28).
In addition, there was an observed stereochemical preference for
substitution as observed upon comparison of 26 with 27. While
(trifluoromethyl)pyrrolidine systems (i.e., 29) were also tolerated,
stereochemical considerations still played a role in affecting activ-
ity (30 vs 31). In general, although modest improvement was found
within the series, compounds 20 and 23 showed superior WPA po-
tency and metabolic stability compared to the pyrrolidine
analogues.
The SAR of the fluoro-substituted alkyl urea was further ex-
tended by incorporation of acyclic fluorinated amine variants (Ta-
ble 4). As observed previously, addition of fluorine into the alkyl
chain of 32 provided an enhancement of CETP inhibition (i.e.,
33). Inclusion of two additional fluorine atoms into this urea frag-
ment (34) enhanced potency and provided very good metabolic
stability. Ultimately, truncation of 34 to analogue 36 afforded a
compound that was equivalently potent in whole plasma to the
best of the cyclic systems, while also demonstrating good meta-
bolic stability.
An alternate structural variation explored in an attempt to re-
solve the poor metabolic stability exhibited by 8 involved deter-
mining whether the urea nitrogen could be moved into the
cycloalkyl ring (Table 3). These pyrrolidine-based urea analogues
were furnished utilizing the route developed for the initial tem-
plate represented by 9.37 The parent pyrrolidinyl urea 24 was
found to have significantly reduced potency compared to the cor-
In order to determine their effectiveness as inhibitors of CETP
activity in vivo, compounds were evaluated in a CETP/apoB100

6506
M. M. Miller et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6503–6508
Table 2
SAR for optimized fluorinated cycloalkyl rings against human CETP
H
N
Cl
R
N
O
F F
F
O
F
F
R
Cmpd #
SPA, IC50
0.006
(l
M)
WPA, IC50
0.63
(l
M)
MTS (% remaining)
18
N
H
F
F
19
20
0.005
0.001
0.62
N
H
F
F
(R)
H
0.076
9(h), 20(m), 26(r)
N
H
F
F
H
21
22
0.007
0.01
2.72
1.65
N
(S)
H
F
F
100(h), 84(m), 81(r)
99(h), 99(m), 81(r)
N
H
F
F
23
0.007
0.18
N
H
MTS: 3
lM metabolic stability (human, mouse, rat), n = 4 (Ref. 39).
Table 3
SAR for substituted pyrrolidines against human CETP
H
N
R
Cl
F
N
O
F F
O
F
F
R
Cmpd #
SPA, IC50
1.27
(lM)
WPA, IC50
92.53
(l
M)
MTS (% remaining)
3(h), 0.4(m), 1(r)
N
N
N
N
N
N
24
F
F
F
F
25
26
27
0.34
3.30
0.97
19.14
66.64
41.30
F
11(h), 0.2(m), 3(r)
2(h), 0.4(m), 1(r)
H
H
F
F
28
0.009
1.68
84(h), 7(m), 90(r)
73(h), 5(m), 26(r)
F
29
30
0.13
8.15
–––
CF3
N
24.04
H
F3C
N
31
0.05
4.63
H
F3C
MTS: 3 lM metabolic stability (human, mouse, rat), n = 4 (Ref. 39).
transgenic mouse model (Table 5).43 Compound 20 was found to
have activity in the transgenic mouse WPA comparable to its activ-
ity in the human WPA. At an oral dose of 30 mpk, 20 was found to
have plasma exposures that exceeded or were nearly equivalent to
its WPA IC50 for up to 8 h. This resulted in significant reduction of
in vivo CETP activity at all time points examined with a 44% reduc-

M. M. Miller et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6503–6508
6507
Table 4
SAR for acyclic fluorinated alkyl ureas against human CETP
H
N
R
Cl
F
F F
N
O
F
F
R =
F F
n
F
N
N
O
F
cyclic
acyclic
H
H
F
R
Cmpd #
SPA, IC50
0.008
(l
M)
WPA, IC50
1.74
(l
M)
MTS (% remaining)
1(h), 4(m), 7(r)
Me
32
N
H
CF3
33
34
0.002
0.006
0.69
0.22
88(h), 43(m), 51(r)
N
H
CF3
F
100(h), 100(m), 85(r)
N
F
H
Me
N
35
36
0.027
0.003
7.03
0.17
13(h), 6(m), 20(r)
H
CF3
N
100(h), 91(m), 100(r)
H
MTS: 3 lM metabolic stability (human, mouse, rat), n = 4 (Ref. 39).
Table 5
Comparison of CETP pharmacodynamics and pharmacokinetics for compound 20 in transgenic mouse model (30 mpk, po)
Cmpds #
CETP
CETP
AUC
2 h
CETP
4 h
CETP
8 h
CETP
hWPA
TgWPA
IC50
(0–8)
nM h
Levels
% of pre-dose
Levels
% of pre-dose
Levels
% of pre-dose
IC50
,
l
Ma
, l
Ma
nMb
2 hb
nMb
4 hb
nMb
8 hb
20
0.076
0.11
3201
1070 ( 222)
34 ( 1)
249 ( 38)
38 ( 2)
112 ( 21)
44 ( 2)
a
2–4 Point determinations.
Values are means of five experiments, standard deviation is given in parentheses.
b
Heart Protection Study Collaborative Group. MRC/BHF Heart Protection Study
of cholesterol lowering with simvastatin in 20,536 high-risk individuals: a
randomised placebo-controlled trial. Lancet 2002, 360, 7; (e) Sever, P. S.;
Dahlof, B.; Poulter, N. R.; Wedel, H.; Beevers, G.; Caulfield, M.; Collins, R.;
Kjeldsen, S. E.; Kristinsson, A.; McInnes, G. T.; Mehlsen, J.; Nieminen, M.;
O’Brien, E.; Ostergren, J. Lancet 2003, 361, 1149; (f) Cannon, C. P.; Braunwald, E.;
McCabe, C. H.; Rader, D. J.; Rouleau, J. L.; Belder, R.; Joyal, S. V.; Hill, K. A.;
Pfeffer, M. A.; Skene, A. M. N. Engl. J. Med. 2004, 350, 1495.
tion of plasma CETP activity (versus pre-dose levels) observed 8 h
after the oral dose.
In conclusion, although the addition of fluorine did not uni-
versally improve inhibitory potency, selective fluorination on
the cyclopentyl urea fragment of 8 did provide CETP inhibitors
with excellent potency in whole plasma (i.e., 20 and 23). Optimi-
zation of the cyclopentyl moiety resulted in the preparation of
acyclic variants (i.e., 34 and 36) which retained CETP inhibitory
potency with improved liver microsome stability. Finally, treat-
ment of CETP/apoB100 dual transgenic mice with 20 led to po-
tent suppression of CE transfer activity demonstrating in vivo
efficacy in this chemotype. The effect of this CETP inhibition
on modulation of plasma lipoprotein levels will be reported in
due course.
4. (a) Rhoads, G. G.; Gulbrandsen, C. L.; Kagan, A. N. Engl. J. Med. 1976, 294, 293;
(b) Gordon, T.; Castelli, W. P.; Hjortland, M. C.; Kannel, W. B.; Dawber, T. R. Am.
J. Med. 1977, 62, 707; (c) Castelli, W. P.; Garrison, R. J.; Wilson, P. W.; Abbott, R.
D.; Kalousdian, S.; Kannel, W. B. J. Am. Med. Assoc. 1986, 256, 2835; (d) Kannel,
W. B. Am. J. Cardiol. 1995, 76, 69c; (e) Despres, J.-P.; Lemieux, I.; Dagenais, G.-R.;
Cantin, B.; Lamarche, B. Atherosclerosis 2000, 153, 263.
5. Robins, S. J.; Collins, D.; Wittes, J. T.; Papademetriou, V.; Deedwania, P. C.;
Schaefer, E. J.; McNamara, J. R.; Kashyap, M. L.; Hershman, J. M.; Wexler, L. F.;
Rubins, H. B.; Braun, B. JAMA 2001, 285, 1585.
6. Manninen, V.; Elo, M. O.; Frick, M. H.; Haapa, K.; Heinonen, O. P.; Heinsalmi, P.;
Helo, P.; Huttunen, J. K.; Kaitaniemi, P.; Koskinen, P.; Maenppa, H.; Malkonen,
M.; Manttari, M.; Norola, S.; Pasternack, A.; Pikkarainen, J.; Romo, M.; Sjoblom,
T.; Nikkila, E. A. JAMA 1988, 260, 641.
References and notes
7. Fibrates: Gotto, A. M., Jr. Circulation 2001, 103, 2213.
8. Niacin: King, J. M.; Crouse, J. R.; Terry, J. G.; Morgan, T. M.; Spray, B. J.; Miller, N.
E. Am. J. Med. 1994, 97, 323.
1. AHA: (a) World Health Organization. Atlas of Heart Disease and Stroke; World
Health Organization: Geneva, Switzerland, 2004. (b) National Center for Health
Statistics. Health, United States, 2005 with Chartbook on Trends in the Health of
Americans; National Center for Health Statistics: Hyattsville, MD, 2005, p 178.
(c) Lloyd-Jones, D.; Adams, R.; Brown, T.M.; Carnethon, M.; Dai, S.; De Simone,
G.; Ferguson, T.B.; Furie, K.; Gillespie, C.; Go, A.; Greenlund, K.; Haase, N.;
Hailpern, S.; Ho, M.; Howard, V.; Kissela, B.; Kittner, S.; Lackland, D.; Lisabeth,
L.; Marelli, A.; McDermott, M.; Meigs, J.; Mozaffarian, D.; Mussolino, M.; Nichol,
G.; Roger, V.; Rosamond, W.; Sacco, R.; Sorlie, P.; Stafford, R.; Thom, T.;
Wasserthiel-Smoller, S.; Wong, N.; Wylie-Rosett, J. Heart Disease and Stroke
Statistics-2010 Update; A Report from the American Heart Association: Dallas,
TX, 2008. Circulation 2010, 121, e1.
9. Adverse flushing effects of niacin can be mitigated by the use of extended
release niacin (ERN) formulations: (a) Chapman, M. J.; Assmann, G.; Fruchart, J.
C.; Shepherd, J.; Sirtori, C. Curr. Med. Res. Opin. 2004, 20, 1253; (b) McGovern, M.
Eur. Heart J. Suppl. 2005, 7, F41; (c) Gille, A.; Bodor, E. T.; Ahmed, K.;
Offermanns, S. Annu. Rev. Pharmacol. Toxicol. 2008, 48, 79.
10. Flushing effect of niacin has also been reduced with the combination of ERN
and laropiprant, see: Paolini, J. F.; Mitchel, Y. B.; Reyes, R.; Kher, U.; Lai, E.;
Watson, D. J.; Norquist, J. M.; Meehan, A. G.; Bays, H. E.; Davidson, M.;
Ballantyne, C. M. Am. J. Cardiol. 2008, 101, 625.
11. Birjmohun, R. S.; Hutten, B. A.; Kastelein, J. J.; Stroes, E. S. J. Am. Coll. Cardiol.
2005, 45, 185.
12. Barter, P.; Kastelein, J.; Nunn, A.; Hobbs, R. Atherosclerosis 2003, 168, 195.
13. (a) Tall, A. R. J. Lipid Res. 1993, 34, 1255; (b) Barter, P. J.; Brewer, H. B.; Chapman,
M. J.; Hennekens, C. H.; Rader, D. J.; Tall, A. R. Arterioscler. Thromb. Vasc. Biol.
2003, 23, 160.
2. Bucher, H. C.; Griffith, L. E.; Guyatt, G. H. Arterioscler. Thromb. Vasc. Biol. 1999,
19, 187.
3. (a) Statins: Scandinavian Simvastatin Survival Study Group Lancet 1994, 344,
1383; (b) Shepherd, J.; Cobbe, S. M.; Ford, I.; Isles, C. G.; Lorimer, A. R.;
MacFarlane, P. W.; McKillop, J. H.; Packard, C. J. N. Engl J. Med. 1995, 333, 1301;
(c) Downs, J. R.; Clearfield, M.; Weis, S.; Whitney, E.; Shapiro, D. R.; Beere, P. A.;
Langendorfer, A.; Stein, E. A.; Kruyer, W.; Gotto, A. M. JAMA 1998, 279, 1615; (d)
14. Chapman, M. J.; Le Goff, W.; Guerin, M.; Kontush, A. Eur. Heart J. 2010, 31, 149.
15. Vourvouhaki, E.; Dedoussis, G. V. Expert Opin. Ther. Targets 2008, 12, 937.

6508
M. M. Miller et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6503–6508
16. Cuchel, M.; Rader, D. J. J. Am. Coll. Cardiol. 1956, 2007, 50.
34. Screening of the BMS compound collection was carried out using a BODIPY
fluorescence assay based on: Bisgaier, C.L.; Minton, L.L.; Essenburg, A.D.;
White, A.; Homan, R.J. Lipid Res. 1993, 34, 1625. After positive retests were
complete, compounds were reconfirmed in a dose–response mode using a
scintillation proximity assay (SPA). Potent compounds in SPA were then
evaluated for activity in a human whole plasma assay (WPA).
17. (a) Sikorski, J. A.; Connolly, D. T. Curr. Opin. Drug Discovery Dev. 2001, 4, 602; (b)
Sikorski, J. A.; Glenn, K. C. Annu. Rep. Med. Chem. 2000, 35, 251.
18. Hirano, K.-I.; Yamashita, S.; Matsuzawa, Y. Curr. Opin. Lipidol. 2000, 11, 589.
19. Inazo, A.; Koizumi, J.; Mabuchi, H. Curr. Opin. Lipidol. 2000, 11, 389.
20. Lagrost, L. Adv. Vasc. Biol. 1999, 5, 217 (Plasma Lipids and Their Role in Disease).
21. Hamster study: Evans, G. F.; Bensch, W. R.; Apelgren, L. D.; Bailey, D.;
Kauffman, R. F.; Bumol, T. F.; Zuckerman, S. H. J. Lipid Res. 1994, 35, 1634.
22. Rabbit (antisense) study: Sugano, M.; Makino, N.; Sawada, S.; Otsuka, S.;
Watanabe, M.; Okamoto, H.; Kamada, M.; Mizushima, A. J. Biol. Chem. 1998,
273, 5033.
23. Rabbit studies: (a) Okamoto, H.; Yonemori, F.; Wakitani, K.; Minowa, T.; Maeda,
K.; Shinkai, H. Nature 2000, 406, 203; (b) Shinkai, H.; Maeda, K.; Yamasaki, T.;
Okamoto, H.; Uchida, I. J. Med. Chem. 2000, 43, 3566.
24. Rabbit (JTT-705) study: Kobayashi, J.; Okamoto, H.; Otabe, M.; Bujo, H.; Saito, Y.
Atherosclerosis 2002, 162, 131.
25. Human study: Clark, R. W.; Sutfin, T. A.; Ruggeri, R. B.; Willauer, A. T.;
Sugarman, E. D.; Magnus-Aryitey, G.; Cosgrove, P. G.; Sand, T. M.; Wester, R. T.;
Williams, J. A.; Perlman, M. E.; Bamberger, M. J. Arterioscler. Thromb. Vasc. Biol.
2004, 24, 490.
26. Barter, P. J.; Caulfield, M.; Eriksson, M.; Grundy, S. M.; Kastelein, J. J. P.;
Komajda, M.; Lopez-Sendon, J.; Mosca, L.; Tardif, J.-C.; Waters, D. D.; Shear, C.
L.; Revkin, J. H.; Buhr, K. A.; Fisher, M. R.; Tall, A. R.; Brewer, B. N. Engl. J. Med.
2007, 357, 2109.
27. For reviews of clinical and pre-clinical CETP inhibitors, see: (a) Hunt, J. A.; Lu, Z.
Curr. Top. Med. Chem. 2009, 9, 419; (b) Kappelle, P. J. W. H.; van Tol, A.;
Wolffenbuttel, B. H. R.; Dullaart, R. P. F. Cardiovascular Therapeutics 2011, 29,
e89; (c) Toth, P. P. Curr. Cardiol. Rep. 2011, 13, 559.
28. Bayer (BAY 194789): (a) Schmeck, C.; Gielen-Haertwig, H.; Vakalopoulos, A.;
Bischoff, H.; Li, V.; Wirtz, G.; Weber, O. Bioorg. Med. Chem. Lett. 2010, 20, 1740;
(b) Boettcher, M.-F.; Heinig, R.; Schmeck, C.; Kohlsdorfer, C.; Ludwig, M.;
Schaefer, A.; Gelfert-Peukert, S.; Wensing, G.; Weber, O. Br. J. Clin. Pharmacol.
2012, 73, 210.
29. Johnson & Johnson: (a) Rano, T. A.; Sieber-McMaster, E.; Pelton, P. D.; Yang, M.;
Demarest, K. T.; Kuo, G.-H. Bioorg. Med. Chem. Lett. 2009, 19, 2456; (b) Kuo, G.-
H.; Rano, T.; Pelton, P.; Demarest, K. T.; Gibbs, A. C.; Murray, W. V.; Damiano, B.
P.; Connelly, M. A. J. Med. Chem. 2009, 52, 1768; (c) Wang, A.; Prouty, C. P.;
Pelton, P. D.; Yong, M.; Demarest, K. T.; Murray, W. V.; Kuo, G.-H. Bioorg. Med.
Chem. Lett. 2010, 20, 1432.
30. Late stage clinical compounds: (a) Smith, C. J.; Ali, A.; Hammond, M. L.; Li, H.;
Lu, Z.; Napolitano, J.; Taylor, G. E.; Thompson, C. F.; Anderson, M. S.; Chen, Y.;
Eveland, S. S.; Guo, Q.; Hyland, S. A.; Milot, D. P.; Sparrow, C. P.; Wright, S. D.;
Cumiskey, A.-M.; Latham, M.; Peterson, L. B.; Rosa, R.; Pivnichny, J. V.; Tong, X.;
Xu, S. S.; Sinclair, P. J. J. Med. Chem. 2011, 54, 4880 and references therein; (b)
Cannon, C. P.; Shah, S.; Dansky, H. M.; Davidson, M.; Brinton, E. A.; Gotto, A. M.;
Stepanavage, M.; Liu, S. X.; Gibbons, P.; Ashraf, T. B.; Zafarino, J.; Mitchel, Y.;
Barter, P. N. Engl. J. Med. 2010, 363, 2406; (c) Nicholls, S. J.; Brewer, H. B.;
Kastelein, J. J. P.; Krueger, K. A.; Wang, M.-D.; Shao, M.; Hu, B.; McErlean, E.;
Nissen, S. E. J. Amer. Med. Assoc. 2011, 306, 2099; Fayad, Z. A.; Mani, V.;
Woodward, M.; Kallend, D.; Abt, M.; Burgess, T.; Fuster, V.; Ballantyne, C. M.;
Stein, E. A.; Tardif, J.-C.; Rudd, J. H. F.; Farkouh, M. E.; Tawakol, A. Lancet 2011,
378, 1547.
31. Harikrishnan, L. S.; Kamau, M. G.; Herpin, T. F.; Morton, G. C.; Liu, Y.; Cooper, C.
B.; Salvati, M. E.; Qiao, J. X.; Wang, T. C.; Adam, L. P.; Taylor, D. S.; Chen, A. Y. A.;
Yin, X.; Seethala, R.; Peterson, T. L.; Nirschl, D. S.; Miller, A. V.; Weigelt, C. A.;
Appiah, K. K.; O’Connell, J. C.; Lawrence, R. M. Bioorg. Med. Chem. Lett. 2008, 18,
2640.
32. Independently the 2-arylbenzoxazole chemotype represented by 6 was also
identified by researchers at Merck: (a) Smith, C. J.; Ali, A.; Chen, L.; Hammond,
M. L.; Anderson, M. S.; Chen, Y.; Eveland, S. S.; Guo, Q.; Hyland, S. A.; Milot, D.
P.; Sparrow, C. P.; Wright, S. D.; Sinclair, P. J. Bioorg. Med. Chem. Lett. 2010, 20,
346; (b) Hunt, J. A.; Gonzalez, S.; Kallashi, F.; Hammond, M. L.; Pivnichny, J. V.;
Tong, X.; Xu, S. S.; Anderson, M. S.; Chen, Y.; Eveland, S. S.; Guo, Q.; Hyland, S.
A.; Milot, D. P.; Sparrow, C. P.; Wright, S. D.; Sinclair, P. J. Bioorg. Med. Chem.
Lett. 2010, 20, 1019.
35. SPA: To the test compound in DMSO (1
l
l) was added 20
l
l of a mixture
containing 3H-CE/HDL (0.15
l
l), biotinylated LDL (ꢀ5 lg
protein/ml final
concentration) and unlabeled HDL (16 ug/ml final concentration) in a buffer
containing 50 mM HEPES, pH 7.4, 150 mM NaCl and 0.05% sodium azide.
Reactions were initiated by the addition of 10
human recombinant CETP, and incubated at 37 °C. At the end of the reaction,
60 l of LEADseeker beads (#RPNQ0261, 2 mg/ml in buffer containing 1 mg/ml
ll of buffer containing purified
l
BSA and 0.05 mg protein/ml HDL) were added, the plates were covered and
subsequently read. Background activity was determined in a set of wells that
received buffer but no CETP. The level of inhibition was determined by
comparing the readings in wells that contain compound to the readings in
control wells containing DMSO.
36. WPA: To the test compound in DMSO (1
containing 0.15
3H-CE/HDL. The reaction was incubated at 37 °C and
terminated by the addition of of precipitation reagent (2:1:1 of
water:1 M MgCl2:2% Dextralip 50), to precipitate LDL and VLDL. After 10 min
at room temperature, 15 l of the reaction was transferred to filter plates
(Millipore, #MHVBN45) pre-wetted with 100 l phosphate buffered saline. The
plates were centrifuged (1800 rpm) at room temperature for 10 min, and 50 ll
ll) was added 29 ll of human plasma
ll
6 ll
l
l
Microscint-20 was added. The plates were then sealed and read. Background
activity was determined with plasma samples incubated at 4 °C. The level of
inhibition was determined by comparing the readings in wells that contain
compound to the readings in control wells containing DMSO.
37. Salvati, M.E.; Finlay, H.; Harikrishnan, L.S.; Jiang, J.; Johnson, J.A.; Kamau, M.G.;
Lawrence, R.M.; Miller, M.M.; Qiao, J.X.; Wang, T.C.; Wang, Y.; Yang, W. PCT Int.
Appl. WO 2007062314, 2007.
38. Kamau, M. G.; Harikrishnan, L. S.; Finlay, H. J.; Qiao, J. X.; Jiang, J.; Poss, M. A.;
Salvati, M. E.; Wexler, R. R.; Lawrence, R. M. Tetrahedron 2012, 68, 2696.
39. In vitro metabolic stability of compounds was determined using the following
protocol: Kieltyka, K.; Zhang, J.; Li, S.; Vath, M.; Baglieri, C.; Ferraro, C.; Zvyaga,
T. A.; Drexler, D. M.; Weller, H. N.; Shou, W. Z. Rapid Commun. Mass Spectrom.
2009, 23, 1579 and references therein.
40. The application of in vitro metabolic stability assays to predict in vivo
metabolism and metabolic clearance of compounds in the early stages of the
drug discovery process has been reported: (a) McNaney, C. A.; Drexler, D. M.;
Hnatyshyn, S. Y.; Zvyaga, T. A.; Knipe, J. O.; Belcastro, J. V.; Sanders, M. Assay
Drug Dev. Technol. 2008, 6, 121; (b) Obach, R. S. Drug Metab. Dispos. 1999, 27,
1350; (c) Obach, R. S.; Baxter, J. G.; Liston, T. E.; Silber, B. M.; Jones, B. C.;
Macintyre, F.; Rance, D. J.; Wastall, P. J. J. Pharmacol. Exp. Ther. 1997, 283, 46.
41. Observed beneficial effects of fluorine in medicinal chemistry have been
documented and summarized in the following references: (a) Hagmann, W. K.
J. Med. Chem. 2008, 51, 4359; (b) Mueller, K.; Faeh, C.; Diederich, F. Science
1881, 2007, 317; (c) Bohm, H.-J.; Banner, D.; Bendels, S.; Kansy, M.; Kuhn, B.;
Muller, K.; Obst-Sander, U.; Stahl, M. Chem. Biol. Chem. 2004, 5, 637.
42. Independently and subsequent to our work, others have observed the
beneficial effects of fluorine substitution: Ruggeri, R.B. Abstracts of Papers,
235th National Meeting of the American Chemical Society, New Orleans, LA,
April 6–10, 2008; American Chemical Society: Washington, DC, 2008, MEDI-
149.
43. In vivo CE transfer activity: Measurement of cholesterol ester transfer activity
in plasma samples obtained from compound-treated human CETP/apoB100
dual transgenic mice (Taconic Laboratories) was obtained following the
described variation of the hWPA methodology. To 9
diluted 3H-CE/HDL was added. The reaction was incubated at 37 °C, and LDL/
VLDL precipitated with 3 L of precipitation reagent (4:1:1 of water:0.5 M
MgCl2:1% Dextralip 50). The tubes were centrifuged for 15–30 min at 10,000Âg
(10 °C), the supernatants were discarded and the pellets dissolved in 140 L of
2% SDS. Half of the SDS solution (70 L) was transferred to scintillation tubes,
5 mL Optifluor was added, and radioactivity measured in scintillation
lL of plasma, 1 lL of
l
l
l
a
33. Harikrishnan, L. S.; Finlay, H. J.; Qiao, J. X.; Kamau, M. G.; Jiang, J.; Wang, T. C.;
Li, J.; Cooper, C. B.; Poss, M. A.; Adam, L. P.; Taylor, D. S.; Chen, A. Y. A.; Yin, X.;
Sleph, P. G.; Yang, R. Z.; Sitkoff, D. F.; Galella, M. A.; Nirschl, D. S.; Van Kirk, K.;
Miller, A. V.; Huang, C. H.; Chang, M.; Chen, X.-Q.; Salvati, M. E.; Wexler, R. R.;
Lawrence, R. M. J. Med. Chem. 2012, 55, 6162.
counter. Background activity was determined for each sample with an
aliquot incubated for 2.5 h at 4 °C. Results are reported as the amount of
radioactivity transferred to endogenous LDL/VLDL, normalized by the transfer
measured in the plasma sample obtained from the same animal before dosing
with compound. All data are background subtracted.