Chromanol derivatives - A novel class of CETP inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.10.110

Based on our former development candidate BAY 38-1315, optimization efforts led to the discovery of a novel chemical class of orally active cholesteryl ester transfer protein (CETP) inhibitors. The chromanol derivative 19b is a highly potent CETP inhibitor with favorable pharmacokinetic properties suitable for clinical studies. Chemical process optimization furnished a robust synthesis for a kilogram-scale process.

Full text of DOI:10.1016/j.bmcl.2010.10.110

Bioorganic & Medicinal Chemistry Letters 21 (2011) 488–491
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Chromanol derivatives—A novel class of CETP inhibitors
Alexandros Vakalopoulos a, , Carsten Schmeck a, Michael Thutewohl b, Volkhart Li a, Hilmar Bischoff a,
⇑
Klemens Lustig a, Olaf Weber a, Holger Paulsen a, Harry Elias a
a Bayer HealthCare AG, Bayer Schering Pharma, Global Drug Discovery, D-42096 Wuppertal, Germany
b Merck & Cie, Weisshausmatte, CH-6460 Altdorf, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on our former development candidate BAY 38-1315, optimization efforts led to the discovery of a
novel chemical class of orally active cholesteryl ester transfer protein (CETP) inhibitors. The chromanol
derivative 19b is a highly potent CETP inhibitor with favorable pharmacokinetic properties suitable for
clinical studies. Chemical process optimization furnished a robust synthesis for a kilogram-scale process.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 2 September 2010
Revised 20 October 2010
Accepted 21 October 2010
Available online 26 October 2010
Keywords:
CETP inhibitor
Torcertrapib
HDL
Chromanols
Despite the wide use of HMG-CoA reductase inhibitors
(statins) that lower low-density lipoprotein cholesterol (LDL-C),
cardiovascular disease is still the most common cause of morbidity
and mortality in developed countries.1 Guidelines for the
prevention of cardiovascular disease still focus on the management
of LDL-C,2,3 although a large portion of cardiovascular events can-
not be prevented by LDL-C lowering.4
ation of other CETP inhibitors, such as anacetrapib, MK-0859 (2)
and dalcetrapib, JTT-705 (3), which do not have the off-target ef-
fects of torcetrapib, for the treatment of cardiovascular disease is
still ongoing12,13 (see Fig. 1).
Our research program focused on identifying orally active CETP
inhibitors suitable for clinical development. Tetrahydronaphtalene
derivative BAY 38-1315 (4)14 and tetrahydrochinoline compound
BAY 60-5521 (5)15 were found to be highly active CETP inhibitors
and were selected as development candidates (see Fig. 2). Unfortu-
nately, BAY 38-1315 later revealed unfavorable pharmacokinetic
properties thus leading to termination of preclinical development
while BAY 60-5521 entered clinical phase I.
In addition, low levels of high-density lipoprotein cholesterol
(HDL-C) are an independent and important risk factor for
cardiovascular disease and HDL-C has been proposed to have po-
tential atheroprotective effects.5 Epidemiological studies suggest
that the risk for coronary heart disease is 2–3% lower for each
0.1 mg/l increase in HDL-C.6 Inhibition of cholesterol ester transfer
protein (CETP) still represents a promising mechanism for HDL-C
elevation, especially since currently available HDL-C-elevating
drugs based on different mechanisms have limited efficacy and
undesirable side effects.7,8 CETP inhibitors have achieved remark-
able elevations of HDL-C and marked decreases in LDL-C, thus
clinical trials are ongoing to validate the therapeutic potential of
CETP inhibition for the treatment of cardiovascular disease.9
More recently, phase III clinical studies of the CETP inhibitor
torcetrapib (1) were terminated because of an increase in cardio-
vascular events and mortality.10 Most likely, torcetrapib has some
compound-specific and off-target effects, such as raising blood
pressure and aldosterone, which could have led to an increase in
cardiovascular events and mortality.11 Nevertheless, clinical evalu-
In order to find a backup candidate for compound 4 we sought
derivatives with lowered lipophilicity and reduced synthetic
complexity. Replacing the tetrahydronaphtalene moiety by
a
chromanol system seemed to be a good starting point to fulfill both
criteria.
CF3
CF3
CF3
CF3
O
O
N
O
O
N
N
CF3
CF3
O
NH
S
O
O
F
O
O
Torcetrapib
1
Anacetrapib
2
Dalcetrapib 3
⇑
Corresponding author. Tel.: +49 202 365431; fax: +49 202 364061.
E-mail address: alexandros.vakalopoulos@bayer.com (A. Vakalopoulos).
Figure 1. Former and current CETP inhibitors in clinical development.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.110

A. Vakalopoulos et al. / Bioorg. Med. Chem. Lett. 21 (2011) 488–491
489
F
F
F
F
OH
F
OH
OH
R1
OH
O
OH
R1
OH
O
+
CF3
N
R2
X
R2
X
CF3
13
14
BAY 38-1315
4
BAY 60-5521
(phase I)
5
a
Figure 2. Tetrahydronaphtalene and tetrahydrochinoline derived CETP inhibitors.
F
F
b,c
For the synthesis of the complex pentasubstituted benzene ring
we chose phloroglucinol dihydrate as starting material to establish
a synthetic route to the highly substituted chromanols (Scheme 1).
Chromanones 6 were formed by Michael addition/Friedel–Crafts
acylation with the corresponding cycloalkylideneacetic acids in a
single step. The para-phenolic group reacted selectively in the
presence of the second ortho-phenolic group to form trifluorome-
thanesulfonates 7, which turned out to be suitable compounds
for palladium-catalyzed reactions using zinc reagents to introduce
branched alkyl residues. Selective ortho-formylation of the remain-
ing phenolic group was achieved by using dichloromethyl methyl
ether in the presence of the Lewis acid TiCl4, to give functionalized
pentasubstituted chromanones 9 in only four steps from an
inexpensive starting material. Activation of the phenols as triflates
10 followed by Suzuki cross-coupling reactions provided the
aldehydes 11, which were then subjected to Grignard reactions
at low temperatures to obtain alcohols 12 as racemic mixtures.
The intermediate chromanones turned out to be highly versatile
compounds for further derivatisation and SAR exploration
(Scheme 2). Thus, hydroxy ketones 12 undergo a CBS-type reduc-
tion, using (1R,2S)-1-aminoindan-2-ol as a source of chirality,16
with high diastereoselectivity (>85% de) to provide the syn-diols
13 and anti-diols 14 (both S-configuration in the chromanol
OH
R1
O
O
OH
O
R2
O
X
R2
R1
X
15
12
R1 =
R2 = CF3, CMe3, OCF3
X =
,
d
e
F
F
,
,
f
F
O
O
OH
O
R1
R2
X
f
R1
R2
X
16
17
F
F
F
OH
F
OH
O
+
R2
R1
O
X
R2
R1
X
18
19
Scheme 2. Synthesis of alcohols 13–15; 17–19. Reagents and conditions: (a)
0.10–0.15 equiv (1R,2S)-1-aminoindan-2-ol, 4 equiv N,N-diethylaniline–borane
(1:1), THF, 0 °C or rt, 10–30 min, then 12, rt, 6–18 h, 60–93% of 13 and 14; (b)
1.5 equiv Dess–Martin periodinane, 1 equiv pyridine, À30 °C to 0 °C, 1–6 h, 78–99%;
(c) 0.10–0.20 equiv (1R,2S)-1-aminoindan-2-ol, 4 equiv N,N-diethylaniline–borane
(1:1), THF, 0 °C or rt, 10–30 min, then ketone, rt, 4–18 h, 54–83% of 15; (d) for
R2 = CF3: (i) 1.1 equiv SOCl2, 1.6 equiv Et3N, THF, rt, 1.5 h; (ii) 0.10 equiv (1R,2S)-1-
aminoindan-2-ol, 4 equiv N,N-diethylaniline–borane (1:1), THF, rt, 30 min, then
ketone, rt, 18 h; (iii) 15 equiv LAH, rt, 5 h, 16%; (e) 1.5 equiv DAST, toluene, À78 °C
to À20 °C, 2 h, 61–83%; (f) 0.10–0.15 equiv (1R,2S)-1-aminoindan-2-ol, 4 equiv
N,N-diethylaniline–borane (1:1), THF, 0 °C or rt, 10–30 min, then ketone, rt, 3–18 h,
22–38% of 17 (for R2 = OCF3, CMe3) and 26–80% of 18 and 19 (for R2 = OCF3, CF3).
OH O
OH O
OH
a
b
OH
O
HO
O
X
TfO
O
HO
x 2 H2O
OH
X
X
C
6
7
c
OH O
O
OH O
O
OTfO
d
e
H
H
R1
O
R1
O
R1
O
X
X
X
moiety). The hydroxy ketones with interchanged functionalities
were prepared by oxidation with Dess–Martin periodinane to
afford the diketones, which subsequently were reduced in the de-
scribed usual manner with complete regioselectivity and moderate
to high enantioselectivities (>70% ee), to yield hydroxy ketones 15.
The removal of the hydroxyl group in the bis-benzylic position in
12 was achieved in two steps by two different methods, which
were employed depending on the electronic properties of the sub-
stituents R2. For an electron-withdrawing group like trifluoro-
methyl group (R2), lithium aluminum hydride was required for
the reduction of the bis-benzylic chloride prepared from the
hydroxyketone 12 with thionylchloride. On the other hand, elec-
tron-donating groups for R2 (t-Bu, OCF3) enabled a reduction of a
bis-benzylic fluoride by a milder reagent, such as the 1-aminoin-
dan-2-ol-borane complex. Both pathways led to the formation of
alcohols 17 (>80% ee, S-configuration) which were assembled in
two/three steps starting from the key intermediates 12. However,
in accordance with our previously observed SAR15 the fluoro alco-
hols 18 and 19 were of further interest, which were easily prepared
from the racemic ketones 16 by the described chiral reduction
8
9
10
f
F
F
R1
=
,
OH
R1
O
O
O
O
R2 = CF3, CMe3, OCF3
X =
g
H
R2
X
R1
O
X
,
,
11
12
Scheme 1. Synthesis of alcohols 12. Reagents and conditions: (a) 1.2 equiv acid,
4 equiv BF3–Et2O, 70 °C, 3 h, 43–82%; (b) 1.05 equiv PhNTf2, 1.1 equiv K2CO3, DMF,
À20 °C, 2–5 h, 60–80%; (c) 2 equiv Zn(i-Pr)2 or 4.5 equiv ZnBr(c-pentyl),
0.1–0.2 equiv PdCl2(dppf)–CH2Cl2, 3–6 equiv LiCl, DMF, 0 °C to rt, 4 h, 78–96%; (d)
1.1 equiv dichloromethyl methyl ether, 2.5 equiv TiCl4, CH2Cl2, À70 °C to 0 °C,
2.5–4 h, 56–96%; (e) 1.1 equiv PhNTf2, 1.1 equiv K2CO3, DMF, À20 °C to rt, 2–4 h,
76–93%; (f) 1.3 equiv 4-fluorophenyl-boronic acid, 0.11 equiv Pd(PPh3)4, 1.7 equiv
K3PO4, dioxane, 100 °C, 18 h, 62–86%; (g) 1.2–1.5 equiv R2-Ph–MgBr, THF, À78 °C to
0 °C, 0.5–1.5 h, 42–67%.

490
A. Vakalopoulos et al. / Bioorg. Med. Chem. Lett. 21 (2011) 488–491
Table 2
protocol to give both the syn- and anti-products 18 and 19 in mod-
erate to high diastereoselectivities (>77% de).
IC50 data from CETP SPA assay and from CETP fluorescence assay
in human plasma for selected compounds
SAR trends comparing the derivatives 13–15 and 17–19 are
summarized in Table 1. CETP inhibition was determined using a
CETP fluorescence assay.17a A rather steep SAR was observed for
the syn-diols 13 and anti-diols 14. While the syn-stereochemistry
of the alcohol moieties is not favorable (compound 13a), the cho-
sen substituents at R1 and R2 seemed to be well tolerated in this
assay. In particular, the combination of R1 = CF3, R2 = iPr and
X = sprirocyclopropyl (compound 14d) revealed a reduction of
lipophilicity indicated by a decreased c log P value by three units
comparing to compounds 4 and 5. In addition, the replacement
of the hydroxyl group in the bis-benzylic position by a ketone or
a methylene group in the alcohols 15 and 17 showed similar
activity. The spirocyclobutyl substitution adds additional potency
compared to the gem-dimethyl pattern (IC50 = 19–59 nM). Finally,
the bis-benzylic position could be substituted by a fluorine atom
in 18 and 19 with a similar trend to the diols. The syn-stereochem-
istry of the fluorine and hydroxyl groups in 18a–c resulted in
compounds that were less potent when compared to the anti-
stereochemistry of compounds 19a–c, while the lipophilicity was
slightly reduced on comparison with 4 and 5.
Additional in vitro assays were performed to characterize all
subseries (see Table 2). While CETP inhibition of the selected com-
pounds in the scintillation proximity assay were comparable, the
fluoro substituted compounds 19a–c could be distinguished in
the human plasma assay showing the best overall profile for com-
pound 19b.
The pharmacokinetic profile of 19b was investigated in rats and
dogs and exhibited good half-lives (t1/2 = 3.0 and 6.6 h, respec-
tively) and oral bioavailability (F = 79% in rats and 43% in dogs).
Compds
SPA assay17c
IC50 (nM)
Human plasma
assay17b IC50 (nM)
1
5
3
7
22
4
16
4
20
12
6
20
50
14c
14d
15b
17d
19a
19b
19c
100
449
1000
437
216
41
74
In order to provide a reliable large scale synthesis for the prep-
aration of 19b, avoiding the use of a sensitive zinc reagent, we
switched to an alternative synthetic route starting from methyl es-
ter 20, which was transformed to an isopropyl group in a two step
procedure (Scheme 3). Reaction with methylmagnesium bromide,
hydrogenation of the tertiary alcohol under acidic conditions and
cleavage of the methoxy groups gave the diphenol 21 in excellent
yields. The chromanone 22 was formed by the aforementioned Mi-
chael addition/Friedel–Crafts acylation with cyclobutylidenacetic
acid; however it became clear that the sequence to introduce the
side chain had to be modified in order to ensure high reaction
yields. The 4-fluorophenyl head group was installed and the subse-
quent reduction using (1R,2S)-1-aminoindan-2-ol gave excellent
yields of 25 with a very high enantioselectivity (95% ee). Alcohol
OH
OH O
O
a
b
O
OH
O
O
OH
O
Table 1
O
20
21
IC50 data from CETP fluorescence assay for compounds 13–15, 17–19
22
R1 =
,
c
F
F
F
R2 = CF3, CMe3, OCF3
Y
OH
O
OTfO
O
OH
O
d
e
X =
,
,
R1
R2
X
O
O
Y = OH, =O, H, F
23
24
25
Compds R1=
R2=
X=
Y=
IC50 (nM) c log P
a
1
4
5
18
24
25
800
14
77
26
27
176
59
42
36
35
30
28
90
19
373
32
53
7.55
8.63
8.77
7.11
7.11
6.17
6.80
5.61
8.64
8.48
8.40
7.77
7.63
9.50
8.87
8.09
7.93
7.72
7.72
7.55
7.55
6.99
6.99
f
F
F
F
13a
14a
14b
14c
14d
15a
15b
15c
15d
15e
17a
17b
17c
17d
18a
19a
18b
19b
18c
19c
tBu
tBu
CF3
CF3
CF3
tBu
tBu
iPr
iPr
iPr
Spirocyclobutyl
Spirocyclobutyl
Spirocyclobutyl
OH, syn
OH, anti
OH, anti
OH, anti
OH
OTBS
O
F
OH
O
OTBS
O
g
h
cPent Spirocyclobutyl
Br
iPr
iPr
iPr
Spirocyclopropyl OH, anti
Dimethyl
Spirocyclobutyl
F3C
F3C
@O
@O
@O
@O
@O
H
H
H
H
F, syn
F, anti
F, syn
F, anti
O
26
27
+ diastereomer 28
19b
H
OCF3 cPent Spirocyclobutyl
OCF3 iPr
F3C
Spirocyclobutyl
Spirocyclobutyl
CF3
tBu
tBu
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
iPr
cPent Spirocyclobutyl
Scheme 3. Synthesis of alcohol 19b. Reagents and conditions: (a) (i) 2.4 equiv
MeMgBr, THF, rt, 3 h, 93%; (ii) H2, 1 bar, Pd/C (10%), 1.35 eqiuv H3PO4, rt, 3 d, 92%;
(iii) HBr (50%), AcOH, rt, 2 h, 87%; (b) 1.2 equiv cyclobutylideneacetic acid, 0.1 equiv
CF3SO3H, o-xylene, 140 °C, –H2O, 2 h, 64%; (c) 1.1 equiv (Tf)2O, 1.25 equiv i-Pr2EtN,
CH2Cl2, 0 °C, 3.5 h, 86%; (d) 1.8 equiv (4-fluorophenyl)boronic acid, 0.04 equiv
Pd(PPh3)4, 1.7 equiv K3PO4, dioxane, 100 °C, 18 h, 78%; (e) 0.2 equiv (1R,2S)-1-
aminoindan-2-ol, 2 equiv N,N-diethylaniline–borane (1:1), THF, rt, 15 min, then
ketone, rt, 2.5 h, 97%; (f) (i) 1.05 equiv N-bromosuccinimide, CH2Cl2, 10 °C to rt,
1.5 h, 94%; (ii) 3.5 equiv TBSCl, 1.75 equiv DMAP, 3.5 equiv Et3N, DMF, 110 °C, 24 h,
87%; (g) 1.25 equiv n-BuLi, THF, À78 °C, 1 h, then 1.25 equiv 4-(trifluoromethyl)-
benzaldehyde, THF, À78 °C to rt, 18 h, 36% of 27 and 35% of diastereomer 28; (h) (i)
2 equiv DAST, toluene, À78 °C to 10 °C, 5 h, 88%; (ii) 4 equiv TBAF, THF, rt, 18 h, 85%.
iPr
iPr
iPr
iPr
iPr
iPr
iPr
iPr
iPr
Spirocyclobutyl
Dimethyl
Spirocyclobutyl
Dimethyl
Dimethyl
Spirocyclobutyl
Spirocyclobutyl
18
300
14
Spirocyclopropyl F, syn
Spirocyclopropyl F, anti
a
Chiral chromatography was employed to purify all compounds prior to bio-
logical testing (>95% ee or de).

A. Vakalopoulos et al. / Bioorg. Med. Chem. Lett. 21 (2011) 488–491
491
10. (a) 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; (b) Nissen, S. E.; Tardif, J.-C.; Nicholls, S. J.; Revkin, J. H.; Shear,
C. L.; Duggan, W. T.; Ruzyllo, W.; Bachinsky, W. B.; Lasala, G. P.; Tuzcu, E. M. N.
Engl. J. Med. 2007, 356, 1304.
11. It was suggested that torcetrapib induces more proinflammatory lesions in
mice of a less stable phenotype than atorvastatin: De Haan, W.; Vries-van, De.;
der Weij, J.; Van der Hoorn, J. W. A.; Gautier, T.; Van der Hoogt, C. C.;
Westerterp, M.; Romijn, J. A.; Jukema, J. W.; Havekes, L. M.; Princen, H. M. G.;
Rensen, P. C. N. Circulation 2008, 117, 2515.
12. Cannon, C. P.; Dansky, H. M.; Davidson, M.; Gotto, A. M., Jr.; Brinton, E. A.;
Gould, A. L.; Stepanavage, M.; Liu, S. X.; Shah, S.; Rubino, J.; Gibbons, P.;
Hermanowski-Vosatka, A.; Binkowitz, B.; Mitchel, Y.; Barter, P. Am. Heart J.
2009, 158, 513.e3.
13. Stein, E. A.; Stroes, E. S. G.; Steiner, G.; Buckley, B. M.; Capponi, A. M.; Burgess,
T.; Niesor, E. J.; Kallend, D.; Kastelein, J. J. P. Am. J. Cardiol. 2009, 104, 82.
14. (a) Paulsen, H.; Schmeck, C.; Brandes, A.; Schmidt, G.; Stoltefuss, J.; Wirtz, S. N.;
Lögers, M.; Naab, P.; Bremm, K.-D.; Bischoff, H.; Schmidt, D.; Zaiss, S.; Antons, S.
Chimia 2004, 58, 123; (b) Paulsen, H.; Antons, S.; Brandes, A.; Lögers, M.;
Müller, S. N.; Naab, P.; Schmeck, C.; Schneider, S.; Stoltefuss, J. Angew. Chem.,
Int. Ed. 1999, 38, 3373.
15. Schmeck, C.; Gielen-Haertwig, H.; Vakalopoulos, A.; Bischoff, H.; Li, V.; Wirtz,
G.; Weber, O. Bioorg. Med. Chem. Lett. 2010, 20, 1740.
16. (a) Di Simone, B.; Savoia, D.; Tagliavini, E.; Umani-Ronchi, A. Tetrahedron:
Asymmetry 1995, 6, 301; (b) Ghosh, A. K.; Fidanze, S.; Senanayake, C. H.
Synthesis 1998, 937.
17. (a) To assess CETP activity a microemulsion based assay according to Bisgaier
et al., (J. Lipid R., 1993, 34, 1625) was used with the following modification: (1)
Donor liposomes were prepared applying 1 mg cholesteryl 4,4-difluoro-5,7-
dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoate (cholesteryl BODIPYÒ
FL C12, from Molecular Probes), 5.35 mg triolein (Sigma–Aldrich), and 6.67 mg
25 could be regioselectively brominated in the para-position of the
ether function with N-bromosuccinimide and treatment with
t-butyldimethylsilyl-chloride in a basic mixture of DMAP and
triethylamine at high temperature furnished key intermediate 26.
Metal-halogen exchange at low temperature followed by quench-
ing with 4-(trifluoromethyl)benzaldehyde gave a mixture of the
corresponding alcohols which were easily separated by chroma-
tography to provide the desired protected anti-diol 27 in 36%
isolated yield. Diastereoselective fluorination with diethylamino-
sulfur trifluoride (DAST) in toluene at low temperature proceeded
with complete retention of the configuration at this sterically
encumbered site. In contrast to the usual SN2 pathway, the
observed SNi mechanism has also been reported in the literature.18
Final deprotection with TBAF provided the desired target com-
pound 19b in high overall yield.
In summary, the structures of our previous development com-
pounds 4 and 5 were successfully modified to afford chromanol
derivatives with reduced lipophilicity. Compound 19b displays
good overall in vitro properties, a favorable pharmacokinetic pro-
file and a remarkable in vivo profile in human CETP-transgenic
mice by increasing HDL-cholesterol dose dependently,19 and was
therefore selected as a candidate for further development. The
optimized synthesis of 19b comprises 12 robust chemical steps
applicable for a kilogram-scale process.
POPC (Sigma–Aldrich), respectively, dissolved in a total volume of 600 ll
Acknowledgments
dioxane and were slowly injected into 63 ml buffer (50 mM Tris/HCl pH 7.3,
150 mM NaCl, 2 mM EDTA) in a water bath sonicator. This suspension is then
sonicated with 50 W (Branson Sonifier 450 with a cup-horn resonator) for
30 minutes under a nitrogen atmosphere at room temperature. (2) Acceptor
liposomes were prepared in the same manner as donor liposomes using 86 mg
cholesteryl-oleate, 20 mg triolein, and 100 mg POPC dissolved in 1.2 ml
We thank Dr. Rolf Grosser and his lab for HPLC support and
Dr. Delf Schmidt for assay development.
dioxane and injected into 114 ml buffer. (3) In a total test volume of 100
test compounds dissolved in DMSO (2 l) were incubated at 37 °C for 4 h with
50 l of a CETP containing sample (1–3 g CETP, enriched from human plasma)
and 48 l of a liposome emulsion (one volume donor, one volume buffer, and
two volumes acceptor, respectively). The increase of the fluorescence intensity
(excitation 485 nm, emission 535 nm) is proportional to the cholesterol ester
transfer. The inhibition of the transfer is followed in comparison with a DMSO
ll
References and notes
l
l
l
1. Bays, H.; Stein, E. A. Expert Opin. Pharmacother. 2003, 4, 1901.
l
2. Third Report of the National Cholesterol Education Program (NCEP) Expert
Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in
Adults 1. (Adult Treatment Panel III) final report Circulation 2002, 106, 3143.
3. Grundy, S. M.; Cleeman, J. I.; Merz, C. N. B.; Brewer, H. B., Jr.; Clark, L. T.;
Hunninghake, D. B.; Pasternak, R. C.; Smith, S. C., Jr.; Stone, N. J. Circulation
2004, 110, 227 [Erratum: Circulation 2004, 110, 763].
control. (b) To assess CETP activity in the presence of human plasma 6
v/v) of donor liposomes and 1 l (2% v/v) of a solution of the substance to be
tested in DMSO are added to 42 l (86% v/v) of human plasma (Sigma P 9523).
ll (12%
l
l
4. Doggrell, S. A. Expert Opin. Investig. Drugs 2001, 10, 1755.
The mixture is incubated at 37 °C for 24 h. The change in the fluorescence at
510/520 nm is a measure of the cholesterol ester transfer. The inhibition of the
transfer is followed in comparison with a DMSO control. (c) To assess CETP
activity a scintillation proximity assay the transfer of 3H-cholesterol ester from
human HD lipoproteins to biotinylated LD lipoproteins is measured. In the test
5. (a) Chapman, M. J.; Assmann, G.; Fruchart, J.-C.; Shepherd, J.; Sirtori,
C.European Consensus Panel Curr. Med. Res. Opin. 2004, 20, 1253. and
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6. Boden, W. E. Am. J. Cardiol. 2000, 86, 19L.
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J. Med. 1999, 341, 410; (b) Shepherd, J.; Betteridge, J.; Van Gaal, L.European
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8. (a) 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;
(b) Brousseau, M. E.; Schaefer, E. J.; Wolfe, M. L.; Bloedon, L. T.; Digenio, A. G.;
Clark, R. W.; Mancuso, J. P.; Rader, D. J. N. Engl. J. Med. 2004, 350, 1505.
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batch, 10
l
l of HDL-3H-cholesterol ester (ꢀ50,000 cpm) are incubated at 37 °C
for 18 h with 10 ll of biotin-LDL (Amersham) in 50 nM Hepes/0.15 M
NaCl/0.1% bovine serum albumin/0.05% NaN3 pH 7.4 containing 10
ll of
CETP (1 mg/ml) and 3 l of a solution of the substance to be tested (dissolved
l
in 10% DMSO/1% BSA). Two hundred microliters of the SPA-streptavidin bead
solution (TRKQ 7005) are then added, incubated further with shaking for 1 h
and then measured in a scintillation counter. Corresponding incubations with
10 ll of buffer, 10 ll of CETP serve as controls.
18. (a) Mori, Y.; Morishima, N. Bull. Soc. Chem. Jpn. 1994, 67, 236; (b) Castillon, S.;
Dessinges, A.; Faghih, R.; Lukacs, G.; Olesker, A.; Thang, T. T. J. Org. Chem. 1985,
50, 4913.
19. Data published in WO2007/107243.