Discovery of XEN445: A potent and selective endothelial lipase inhibitor raises plasma HDL-cholesterol concentration in mice

Article abstract of DOI:10.1016/j.bmc.2013.10.023

Endothelial lipase (EL) activity has been implicated in HDL metabolism and in atherosclerotic plaque development; inhibitors are proposed to be efficacious in the treatment of dyslipidemia related cardiovascular disease. We describe here the discovery of a novel class of anthranilic acids EL inhibitors. XEN445 (compound 13) was identified as a potent and selective EL inhibitor, that showed good ADME and PK properties, and demonstrated in vivo efficacy in raising plasma HDLc concentrations in mice.

Full text of DOI:10.1016/j.bmc.2013.10.023

Bioorganic & Medicinal Chemistry 21 (2013) 7724–7734
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of XEN445: A potent and selective endothelial lipase
inhibitor raises plasma HDL-cholesterol concentration in mice
a
a
a
a
a
a
Shaoyi Sun a, , Richard Dean , Qi Jia , Alla Zenova , Jing Zhong , Celene Grayson , Clark Xie ,
Andrea Lindgren a, Pritpaul Samra a, Luis Sojo a, Margaret van Heek b, Linus Lin b, David Percival a,
Jian-min Fu a, Michael D. Winther a, Zaihui Zhang a
⇑
a Xenon Pharmaceuticals Inc, 200-3650 Gilmore Way, Burnaby, BC V5G 4W8, Canada
b Merck Research Laboratories, Rahway, NJ 07065, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Endothelial lipase (EL) activity has been implicated in HDL metabolism and in atherosclerotic plaque
development; inhibitors are proposed to be efficacious in the treatment of dyslipidemia related cardio-
vascular disease. We describe here the discovery of a novel class of anthranilic acids EL inhibitors.
XEN445 (compound 13) was identified as a potent and selective EL inhibitor, that showed good ADME
and PK properties, and demonstrated in vivo efficacy in raising plasma HDLc concentrations in mice.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 31 August 2013
Revised 8 October 2013
Accepted 17 October 2013
Available online 28 October 2013
Keywords:
Anthranilic acid
Endothelial lipase inhibitors
HDL cholesterol
Cardiovascular disease
1. Introduction
anti-oxidant, anti-inflammatory, nitric oxide-inducing and anti-
microbial effects amongst others.7 Thus, the importance of HDL
Despite the high rate of adoption of therapeutics, improved
diagnostics and the understanding of the role of preventative mea-
sures, coronary artery disease (CAD) arising from atherosclerosis
remains the leading cause of death in the Western world and
CAD has one of the highest disease burdens on health globally.1
Elevated levels of plasma low density lipoprotein cholesterol
(LDLc) are associated with increased atherosclerotic plaque forma-
tion and CAD, and as such, therapies to reduce LDLc have become a
cornerstone of medicine. However, current therapies only reduce
CAD rates by 25–30%.2 Plasma high density cholesterol (HDLc) is
mainly known for its role in reverse cholesterol transport, in which
excess cholesterol supplied from the liver and gut to the tissues via
LDL, is returned to the liver for excretion via the bile and feces. As
plasma HDLc levels are negatively correlated with CAD,3 therapeu-
tics which raise HDLc have become a focus for development.2,4,5
HDLc concentration was long believed to be a surrogate for the effi-
ciency of the reverse cholesterol transport process. This concept is
being challenged, as ex vivo cholesterol efflux from macrophages
to HDL is a better predictor of CAD than HDLc concentration itself.6
Recently, other roles of HDL particles that may contribute to its
atheroprotective functionality have been identified. These include
functionality, beyond its ability to promote the efflux of cholesterol
from peripheral cells and to return it to the liver has gained recent
prominence.8,9
Endothelial lipase (EL) is a member of the triglyceride lipase
family which also includes hepatic lipase (HL), lipoprotein lipase
(LPL) and pancreatic lipase. Although these enzymes share consid-
erable homologies, including the catalytic triad common to serine
hydrolases, their structures do differ in the lid region that plays a
role in controlling substrate specificity.10,11 EL is secreted from vas-
cular endothelial cells and hepatocytes and preferentially hydro-
lyses the sn-1 (PLA1) ester bond of phosphatidylcholine (PC)
present in HDL particles, releasing Lyso-PC and resulting in a
reduction in HDL particle size. LPL predominantly hydrolyses
triglycerides in ApoB-containing particles and HL hydrolyses both
triglycerides and phospholipids in all classes of lipoproteins.12,13
The potential role of EL in HDL metabolism has been delineated
by a number of mouse genetic studies showing a gene-dose effect,
in which EL knockout mice have HDLc concentrations which are
51–140% higher than in wild-type mice.14–17 Transgenic mice
expressing human EL have reduced plasma HDLc, and treatment
of wild-type mice with an anti-EL antibody increased plasma
HDLc.18,19 As plasma EL mass and activity both correlate with
CAD risk in humans and human loss of function EL variants are
associated with increased plasma HDLc,20 EL has been highlighted
⇑
Corresponding author. Tel.: +1 604 484 3300; fax: +1 604 484 3459.
E-mail address: ssun@xenon-pharma.com (S. Sun).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.10.023

S. Sun et al. / Bioorg. Med. Chem. 21 (2013) 7724–7734
7725
OH
HO
HO
OBn
O
O
O
O
N
OMe
R
OMe
O
F3C
NH
F3C
O
a
F3C
F3C
N
N
1
18a
17a
EL IC50: 1.010 μM
O
OH
N
Figure 1. Design strategy.
b
OH
F3C
O
HO
HO
18
Scheme 3. Reagents: (a) H2, 10% Pd/C; (b) NaOH, THF, H2O.
O
F
O
N
R
a
R
HN
F3C
F3C
potent and highly selective inhibitor that raises plasma HDLc con-
centrations in vivo in wild-type mice.
2-10, 11a, 12, 13, 19
Scheme 1. Reagents: (a) 4-methylmorpholine (NMM), 1,4-dioxane.
2. Chemistry
as a therapeutic target to increase HDLc and prevent and treat
CAD.19,21,13,22–24
The synthesis of these anthranilic acids are outlined in Schemes
1–3. For the synthesis of analogues 2–10, 11a, 12, 13 and 19 a
simple and efficient aromatic nucleophilic substitution of 2-flu-
oro-5-(trifluoromethyl)benzoic acid with an appropriate amine
was applied (Scheme 1). This method was general, and a wide
range of amines could be used in a library fashion to prepare the
desired molecules.
The commercial availability of functionalized pyrrolidines is
limited; therefore, a short sequence starting from intermediate
11a was used to prepare analogs 11, 14–17, 20 and 21. As shown
in Scheme 2, the carboxylic acid group in 11a was masked as
methyl ester 11b with K2CO3 and MeI; subsequent treatment of
11b with NaH and 2-cyclopropylethyl 4-methylbenzenesulfonate
(for 11c) or alkyl bromides (for 14a–17a, 20a and 21a) led to the
corresponding ether. Finally, hydrolysis of the methyl ester with
NaOH in aqueous THF under reflux conditions led to the desired
compound.
Compound 18 was prepared from intermediate 17a (Scheme 3).
Deprotection of the benzyl group gave the alcohol 18a and subse-
quent saponification of the methyl ester gave the desired com-
pound 18.
To prepare analogs 22–25, a similar aromatic nucleophlic sub-
stitution was applied. Treatment of nitrile 22a with NaN3 and
NH4Cl generated tetrazole 22 (Scheme 4). Acylation of sulfonamide
23 with acetyl chloride gave acylsulfonamide 24 (Scheme 5).
Reduction of ketone 25a with NaBH4 led to alcohol 25 (Scheme 6).
Despite the important role of EL in HDLc metabolism, only a few
EL inhibitors have been reported25–27 with no published selectivity
or in vivo efficacy. Our efforts to identify novel EL inhibitors uti-
lized a high-throughput screening campaign against EL that identi-
fied compound 1 (Fig. 1) as an attractive inhibitor with moderate
inhibitory activity against human EL. Inhibitor 1 has high target
selectivity over LPL and good physical properties (MW = 259 Da,
clogP = 2.73). This profile led us to consider this hit as the starting
point for our discovery program. The 4-(trifluoromethyl) aniline
moiety present in 1 raised a safety concern due to the possible for-
mation of reactive metabolites28, although leflunomide carrying
this motif is used as a pyrimidine synthesis inhibitor for rheuma-
toid arthritis. Our initial SAR focused on replacing this motif which
proved to be critical for EL inhibitory activity. Replacement of CF3
group with other groups such as –OCF3, –Cl, –F, –CH3, –CN,
–SO2CH3, and 40-CF3-Ph or replacement –NH– with –O– abolishes
activity. Towards this end, we considered substituting the aniline
proton and evaluating tertiary anilines which are expected to be
less susceptible to potential degradation. This strategy eventually
led to the introduction of various nitrogen containing heterocycles
such as functionalized pyrrolidines, piperidines or azetidines at the
para position to the CF3 on the phenyl ring (Fig. 1). These efforts re-
sulted in the discovery of a class of novel, potent and selective EL
inhibitors, exemplified by compound 13 (XEN445), which is a
MeO
O
HO
O
a
F3C
N
F3C
N
OH
OH
11b
11a
b
HO
MeO
O
N
O
N
c
F3C
F3C
R'
R'
O
O
11, 14-17, 20, 21
Scheme 2. Reagents: (a) MeI, K2CO3, DMF; (b) NaH, XR0; (c) NaOH, THF, H2O.
11c, 14a-17a, 20a, 21a

7726
S. Sun et al. / Bioorg. Med. Chem. 21 (2013) 7724–7734
CN
complete lack of activity of the piperidinyl analog 2 was quite
CN
a
striking. To determine if there was any stereoisomeric preference
at position 3 of the pyrrolidine, the R isomer 5 was prepared
according to scheme 1. The potency of 5 suggested no strong pref-
erence for this chirality difference. Replacement of the oxygen lin-
ker with a carbon linker (compound 6, racemic) led to a sixfold
increase of potency relative to 4. However, further profiling of 6 re-
vealed an unwanted inhibitory effect against cytochrome P450 2C9
F
F3C
N
F3C
F
O
22a
N
N
N
NH
F
b
F3C
N
with 86% inhibiton at 10 lM. Thus, compound 4 was taken as a
lead compound for further optimization. Exploration of the ether
substitution at the 3S-position of the pyrrolidine by replacement
of the 4-fluorobenzene group of 4 revealed broad tolerances for po-
tency on EL and good selectivity versus LPL and HL. This modifica-
tion resulted in compounds 9 and 17 with modest improvement in
inhibitory potencies relative to 4.
O
22
Scheme 4. Reagents: (a) (S)-3-(4-fluorophenoxy)pyrrolidine, Cs2CO3; (b) NaN3,
NH4Cl, DMF.
EL hydrolyses HDL phospholipids both as a free plasma enzyme
and associated with the surface of vascular endothelial cells via
proteoglycan binding. Therefore, the in vivo activities of EL inhibi-
tors are determined by free, un-bound drug plasma levels as well
as intrinsic inhibitory potency. Not surprisingly, like most acidic
drugs, compounds 4–7, 9–12 and 17 displayed high in vitro affin-
ities for rat plasma proteins. We sought to identify new molecules,
which maintained or improved in vitro inhibitory potency versus
EL were less bound to plasma protein. Therefore, new designs were
focused on molecules with less hydrophobic groups or basic groups
as exemplified by 13–16 and 18–21. These compounds were ex-
pected to have less interaction with plasma protein. In general,
the inhibitory activity in the FELA assay was closely correlated
with the lipophilicity of molecules, decreasing lipophilicity had a
negative effect on potency and compounds 18–21 were more than
fivefold less potent than 17; however, the high selectivity was
maintained and plasma protein binding profile was significantly
improved.
O
O
H2N
H2N
S
S
O
O
a
F
F3C
N
F3C
F
O
23
O
O
HN
S
b
O
F
F3C
N
O
24
Scheme 5. Reagents: (a) (S)-3-(4-fluorophenoxy)pyrrolidine, NMM; (b) AcCl,
pyridine.
To examine the effects of the carboxylic acid group, we investi-
gated a variety of replacements of the carboxylic acid moiety. As
indicated in Table 2, the acidic group proved to be important for
activity, as a more than 10-fold drop in potency was observed
when the carboxylic acid moiety was replaced by an alcohol as
indicated in 25; while the tetrazole analogue 22 maintained the
activity with a slightly improvement in plasma protein binding.
In addition, replacement of the carboxylic acid group with sulfon-
amide group (23) and an acylsulfonamide group (24) resulted in a
dramatic loss in inhibitory activity.
O
O
a
F
F3C
N
F3C
F
O
25a
HO
b
F
F3C
N
Based on their good inhibitory activity, selectivity and plasma
protein binding, the pyridinyl derivatives 13–16 were evaluated
in the functional HDL particle assay (Table 3), which uses purified
human HDL particles as a more physiologically relevant substrate,
with the release of free fatty acids as the readout of lipase activity.
The potency observed in this assay confirmed that 13, 14 and 15
were equally potent, while 16 was about twofold less active. 13
(XEN445) can be readily prepared in good yield from commercial
available chemicals, which was selected for further evaluation of
this series of EL inhibitors.
The relative importance of cell-associated EL and plasma free EL
to HDL particle cleavage in vivo is not clear and furthermore, EL is
believed to be active as a dimer. EL catalyses the sn-1 cleavage of
phosphatidylcholine at the lipid interface, and as such fluorogenic
and other EL substrate assays generally employ mixed micelle or
particle-based formats. To mimic the in vivo environment in an
in vitro assay as closely as possible, we used the recently-described
soluble EL fluorogenic substrate PED-A1 to determine the potency
of XEN445 using EL-transfected HEK cells.29 After a 30 min prein-
cubation of EL-expressing HEK cells with XEN445, the IC50 value
O
25
Scheme 6. Reagents: (a) (S)-3-(4-fluorophenoxy)pyrrolidine, NMM; (b) NaBH4,
MeOH.
3. Results and discussion
Compounds prepared in this study were evaluated in a mixed
micelle fluorogenic phosphatidylcholine substrate (FELA, bis-BOD-
IPY-FL C11-PC) assay against isolated human and mouse EL; se-
lected inhibitors were also tested against other members of the
triglyceride lipase family, mainly human LPL and HL. Taking 1 as
the starting point, our first set of compounds examined the effect
of a change of ring size by the synthesis of piperidinyl analog 2,
azidinyl analog 3 and pyrrolidinyl analog 4; The structure–activity
relationship (SAR) indicated that the pyrrolidine ring played a sig-
nificant role in both EL potency and selectivity (Table 1). Com-
pound 4 had an almost fivefold increase in potency compared to
1 and importantly demonstrated more than 500-fold and 40-fold
selectivity over LPL and HL, respectively. Switching pyrrolidinyl
to azidinyl (3) resulted in a decrease in potency; while the
for XEN445 of 0.25 lM was obtained. This value was very similar
to that determined in the cell-free assay (Table 1) and provides
more evidence that XEN445 will have in vivo activity, provided

S. Sun et al. / Bioorg. Med. Chem. 21 (2013) 7724–7734
7727
Table 1
SAR of anthranilic acids
HO
O
R
F3C
N
R
PPBb (%)
clogPe
a
a
a
Compound
hEL IC50
(lM)
hLPL IC50
(
lM)
hHL IC50
(l
M)
N
1
2
cyclopropanemethylamine
1.010
>10
>10
nd
6.4
nd
99.5
nd
2.73
4.57
N
N
O
F
3
4
1.96
nd
nd
nd
4.35
4.46
O
F
F
0.184
>100
7.6
99.8/99.0c/97.9d
N
N
O
F
5
0.259
>33
nd
99.5
4.46
O
F
6
7
8
0.025
0.372
0.236
2.56
>20
nd
nd
nd
99.9
99.7
98.7
5.27
4.60
3.87
N
N
CF3
O
N
Cl
31.59
N
N
O
O
9
0.131
0.236
0.156
0.366
>33
nd
99.4
99.8
99.6
99.5
4.37
3.21
3.56
3.72
F
N
N
O
10
11
12
49.90
>33
nd
O
nd
N
O
>100
24.11
N
N
O
13 (XEN445)
0.237
0.219
20
9.5
97.4/88.8c/95.4d
98.0
3.30
3.46
N
O
O
F
14
76.3
16.92
N
N
N
F
15
16
0.252
0.214
77.2
45.2
19.5
23.3
97.9
93.5
3.46
3.04
N
F
O
N
N
O
O
17
18
0.088
0.57
>33
nd
nd
nd
99.3
nd
4.06
1.97
N
O
OH
N
(continued on next page)

7728
S. Sun et al. / Bioorg. Med. Chem. 21 (2013) 7724–7734
Table 1 (continued)
R
PPBb (%)
clogPe
a
a
a
Compound
hEL IC50
(
lM)
hLPL IC50
(l
M)
hHL IC50 (lM)
N
O
19
20
0.499
0.551
>100
>100
nd
85.5
85.3
2.67
2.17
N
N
OEt
O
O
88.31
O
O
O
N
21
0.659
nd
nd
nd
2.08
N
a
IC50s are an average of at least two independent determinations.
PPB refers to rat plasma protein binding assessed by equilibrium dialysis using 10% rat plasma, 90% PBS and 2.5
Mouse plasma protein binding.
Human plasma protein binding.
Calculated by ChemBioDraw Ultra 12.0 (CambridgeSoft, Cambridge, MA) with the option of ‘LogP’. nd: not determined.
b
c
lM compound for 4 h at 37 °C, unless otherwise indicated.
d
e
to be an appropriate candidate for testing in a mouse HDLc raising
efficacy model.
Table 2
SAR on replacement of carboxylic acid group
Since the pharmacokinetic parameters (e.g., inhibitor AUC or
trough plasma concentration) associated with the HDLc raising
efficacy of EL inhibition are not known, male C57BL6 mice where
administered XEN445 by oral gavage at a dose that would provide
a high degree of EL inhibition throughout the duration of the
experiment. Wild-type mice were orally dosed with XEN445 at
30 mg/kg b.i.d. for 3 days and blood was taken on the morning of
day 4, 16 h post final dose. At termination, the average plasma lev-
W
F3C
N
F
O
a
Compound
W
hEL IC50
(lM)
Rat PPB (%)
99.0
N
N
N
22
0.209
>10
∗
els of XEN445 was 9.9 lM and the drug caused an 18% and 16% in-
crease in total plasma cholesterol and HDLc, respectively (Table 5,
Fig. 2).
N
H
O
O
23
24
S
ndb
ndb
∗
NH2
An HDLc increase of ꢀ10% was observed when mice were trea-
ted for the same length of time with XEN445 at a dose of 10 mg/kg
b.i.d. (data not shown). To determine whether the HDL-raising effi-
cacy was due to on-target inhibition, XEN445, as well as vehicle,
were dosed to EL knockout mice using the same paradigm. In
agreement with previously published studies using the same foun-
der mice,17 the plasma HDLc concentration from vehicle-treated EL
knockout mice was increased (121% in this study) compared to
vehicle-treated wild-type mice (Table 5). However, XEN445 treat-
ment for 3 days did not significantly alter the total plasma choles-
terol or HDLc concentrations in EL knockout mice compared to
vehicle alone (Table 5, Fig. 3). To determine whether a longer drug
treatment paradigm would result in further increase in plasma
O
O
∗
S
>10
N
H
O
OH
25
1.946
ndb
∗
a
IC50 values are an average of at least two independent determinations.
nd: not determined.
b
sufficient plasma drug levels are achieved. The pharmacokinetic
parameters of XEN445 in mice are shown in Table 4. XEN445 has
sufficiently high oral bioavailability and low clearance rates for it
Table 3
In vitro potency as determined in functional HDL particle assay
Compound
13
14
15
16
a,b
hEL IC50
(
lM)
0.11
0.13
0.12
0.33
a
IC50s are an average of at least two independent determinations.
HDL particle assay.
b
Table 4
Mouse Pharmacokinetics of XEN445a
Cmax
M)
Tmax
(h)
Cmin24 h
M)
AUClast
M h)
AUClast
M h)
T1/2
(h)
CL
(L/h/kg)
F
(%)
(
l
(
l
(
l
(
l
62b
0.5b
4.5b
47c
253b
11.9b
0.06c
54
a
b
c
All data from non-fasted, male, C57BL/6 mice (n = 3).
Per oral (po), dose 10 mg/kg, 50% PEG 400 and 50% D5W.
Intravenous (iv), dose 1 mg/kg, 10% ethanol 25% PEG 400 and 65% D5W.

S. Sun et al. / Bioorg. Med. Chem. 21 (2013) 7724–7734
7729
Table 5
In vivo efficacy of XEN445 in wild-type and EL knockout mice
Strain
Treatment
Plasma total cholesterol
Plasma HDLc
Percentage change (%)
Concentration
Percentage change (%)
Concentration
(SEM) (mg/dL)
(SEM) (mg/dL)
Wild-type
Wild-type
Wild-type
EL KO
EL KO
Wild-type
Wild-type
3 d Vehicle
3 d XEN445
3 d Vehicle
3 d Vehicle
3 d XEN445
9 d Vehicle
9 d XEN445
106.1 (3.9)
125.2 (7.2)*
103.5 (4.4)
219.6 (5.9)*
212.0 (11.7)à
108.3 (3.9)
131.2 (3.4)*
—
+18
—
+112
ꢂ3
—
74.7 (2.2)
86.5 (4.2)*
57.1 (4.8)
126.4 (4.5)*
117.0 (8.5)à
68.2 (3.9)
88.7 (4.3)*
—
+16
—
+121
ꢂ7
—
+21
+30
*
p < 0.05 versus vehicle-treated wild-type mice within each experiment.
p = not significant versus vehicle-treated EL knockout mice.
à
HDL levels, XEN445 was dosed to wild-type mice for 9 days at a
dose of 30 mg/kg b.i.d. Longer treatment with XEN445 resulted in
a numerically greater plasma HDLc increase (+30%, Table 5,
Fig. 4) than that observed with 3 days dosing.
4. Conclusion
We discovered a class of novel, potent and selective endothelial
lipase (EL) inhibitors through the optimization of anthranilic acid
hit 1 with focus on safety, potency, selectivity and protein binding
properties. XEN445 demonstrated good in vitro potency against EL
with high selectivity over LPL and HL. Most importantly, XEN445
demonstrated in vivo efficacy in raising plasma HDLc in wild-type
mice. This class of compounds will be useful for further defining
the catalytic and non-catalytic roles of EL in HDL metabolism and
are promising starting points for the development of EL-directed
therapeutics. Further optimization is needed to identify a more po-
tent EL inhibitor suitable for potential therapeutic evaluation in
humans.
Figure 2. In vivo efficacy of XEN445 in wild-type mice dosed for 3 days at 30 mg/kg
b.i.d.
5. Experimental section
5.1. General method
All chemicals, reagents and solvents were purchased from com-
mercial sources and were either used as supplied or purified using
reported methods. All final compounds reported herein exhibited
spectral data consistent with their proposed structure by nuclear
magnetic resonance spectra (1H NMR and 13C NMR) and mass
spectra data. NMR spectra were recorded on a Bruker Avance 300
spectrometer with chemical shifts (d) reported in parts-per-million
(ppm) relative to the residual signal of the deuterated solvent. 1H
NMR data are tabulated in the following order: multiplicity (s, sin-
glet; d, doublet; t, triplet; m, multiplet; br, broad), coupling con-
stants in Hertz and number of protons. Mass spectra were
obtained using a Waters 2795/ZQ LC/MS system (Waters Corpora-
tion, Milford, MA). All final compounds were greater than 95% a/a
pure as determined by analytical HPLC on Agilent 1200 systems
(Agilent Technologies, Santa Clara, CA) using an EMD Chromolith
SpeedROD RP-18e column (4.6 mm i.d. ꢁ 50 mm length) (Merck
KGaA, Darmstadt, Germany). The mobile phase consisted of a gra-
dient of component ‘A’ (0.1% v/v aqueous trifluoroacetic acid) and
component ‘B’ (acetonitrile) at a flow rate of 1 mL/min. The gradi-
ent program used was as follows: initial conditions 5% B, hold at 5%
B for 1 min., linear ramp from 5% to 95% B over 5 min, 100% B for
3 min., return to initial conditions for 1 min. Peaks were detected
at a wavelength of 254 nm with an Agilent photodiode array detec-
tor. Melting points were determined on a Fisher–Johns melting
point apparatus and are uncorrected. Chemical names were gener-
ated using ChemBioDraw version 12.0 (CambridgeSoft, Cambridge,
Figure 3. In vivo efficacy of XEN445 in LIPG knockout mice dosed for 3 days at
30 mg/kg b.i.d.
Figure 4. In vivo efficacy of XEN445 in wild-type mice dosed for 9 days at 30 mg/kg
b.i.d.

7730
S. Sun et al. / Bioorg. Med. Chem. 21 (2013) 7724–7734
MA.) The fluorogenic substrates bis-BODIPY-FL C11-PC and PED-A1
were purchased from Invitrogen. The EL knockout mice (on a pure
C57BL6 background) were obtained from the laboratory of Dr. T.
Quertermous (Standford University). The wild-type male C57BL6
mice were obtained from Taconic Farms Inc.
5.1.5. 2-(3-(4-Fluorobenzyl)pyrrolidin-1-yl)-5-
(trifluoromethyl)benzoic acid (6)
By a similar procedure described for 4, 6 was obtained as a col-
orless solid (0.76 g, 43%). mp 111–113 °C (EtOAc/hexanes); 1H
NMR (300 MHz, CDCl3) d 13.7 (br s, 1H), 8.23 (s, 1H), 7.66–7.58
(m, 1H), 7.20–6.83 (m, 5H), 3.46–3.05 (m, 4H), 2.82–3.53 (m,
3H), 2.22–2.08 (m, 1H), 1.85–1.67 (m, 1H); 13C NMR (75 MHz,
CDCl3) d 170.3, 161.5 (d, JCF = 242.7 Hz), 150.7, 135.4, 130.0,
129.9, 129.7 (q, JCF = 3.9 Hz), 129.6 (q, JCF = 3.8 Hz), 124.0 (q,
JCF = 260.9 Hz), 117.5, 115.5, 115.2, 58.2, 52.6, 40.6, 38.5, 31.2;
MS (ES+) m/z 368.0 (M+1).
5.1.1. (S)-2-(3-(4-Fluorophenoxy)pyrrolidin-1-yl)-5-
(trifluoromethyl)benzoic acid (4)
To
a solution of 2-fluoro-5-(trifluoromethyl)benzoic acid
(6.26 g, 30.0 mmol) and (S)-3-(4-fluorophenoxy)pyrrolidine (7.0 g,
38.62 mmol) in 1,4-dioxane (100 mL) was added 4-methylmor-
pholine (10.0 mL, 91.04 mmol). The resulting mixture was heated
at 110 °C for 18 h, concentrated in vacuo, and the residue cooled
in an ice bath and acidified with 5% hydrochloric acid solution to
pHꢀ3; extracted with EtOAc (100 mL ꢁ 3) and washed with water
and brine. The organic solution was dried over anhydrous Na2SO4
and filtered. The filtrate was concentrated in vacuo, the residue
was subjected to column chromatography eluting with a gradient
of 20–50% ethyl acetate in hexanes to give 4 as a colorless solid
(6.84 g, 61%). mp 98–99 °C (EtOAc/hexanes); 1H NMR (300 MHz,
CDCl3) d 8.36–8.21 (m, 1H), 7.72–7.65 (m, 1H), 7.25–7.20 (m,
1H), 6.99–6.92 (m, 2H), 6.85–6.77 (m, 2H), 4.98 (br s, 1H), 3.82–
3.73 (m, 1H), 3.66–3.54 (m, 1H), 3.44–3.28 (m, 2H), 2.37–2.24
(m, 2H); 13C NMR (75 MHz, CDCl3) d 170.0, 157.6 (d, JCF = 238.0 Hz),
152.8, 150.8, 130.0, 129.7, 123.8 (q, JCF = 269.9 Hz), 123.4 (q,
JCF = 31.1 Hz), 120.1, 118.9, 116.9, 116.8, 116.3, 116.0, 76.6, 58.7,
51.3, 31.4; MS (ES+) m/z 370.0 (M+1). Anal. Calcd for C18H15F4NO3:
C, 58.54; H, 4.09; N, 3.79. Found: C, 58.52; H, 4.06; N, 3.89.
5.1.6. (S)-5-(Trifluoromethyl)-2-(3-(5-(trifluoromethyl)-pyridin-
2-yloxy)pyrrolidin-1-yl)benzoic acid (7)
By a similar procedure described for 4, 7 was obtained as a col-
orless solid (0.37 g, 87%). mp 46–47 °C (EtOAc/hexanes); 1H NMR
(300 MHz, CDCl3) d 8.39 (s, 1H), 8.27 (s, 1H), 7.80–7.73 (m, 1H),
7.70–7.64 (m, 1H), 7.21–7.14 (m, 1H), 6.85–6.80 (m, 1H), 5.75 (s,
1H), 3.89–3.80 (m, 1H), 3.69–3.60 (m, 1H), 3.43–3.29 (m, 2H),
2.48–2.31 (m, 2H); 13C NMR (75 MHz, CDCl3) d 164.6, 150.7,
144.7, 136.1, 130.1, 129.7, 122.0 (q, JCF = 269.4 Hz), 120.5 (q,
JCF = 32.8 Hz), 111.8, 75.0, 58.8, 51.2, 31.6; MS (ES+) m/z 420.9
(M+1).
5.1.7. (S)-2-(3-(6-Chloropyridin-3-yloxy)pyrrolidin-1-yl)-5-
(trifluoromethyl)benzoic acid (8)
By a similar procedure described for 4, 8 was obtained as a col-
orless solid (0.79 g, 50%). mp 42–43 °C (EtOAc/hexanes); 1H NMR
(300 MHz, CDCl3) d 10.28 (br s, 1H), 8.23 (s, 1H), 8.09–8.04 (m,
1H), 7.69–7.62 (m, 1H), 7.27–7.10 (m, 3H), 5.10–5.04 (m, 1H),
3.89–3.82 (m, 1H), 3.68–3.57 (m, 1H), 3.46–3.37 (m, 1H), 3.32–
3.25 (m, 1H), 2.38–2.32 (m, 2H); MS (ES+) m/z 386.9 (M+1).
5.1.2. 2-(4-(4-Fluorophenoxy)piperidin-1-yl)-5-
(trifluoromethyl)benzoic acid (2)
By a similar procedure described for 4, 2 was obtained as a col-
orless solid (0.07 g, 10%). mp 146–147 °C (EtOAc/hexanes); 1H
NMR (300 MHz, CDCl3) d 8.63–8.59 (m, 1H), 7.91–7.83 (m, 1H),
7.68–7.59 (m, 1H), 7.08–6.87 (m, 4H), 4.58 (br s, 1H), 3.51–3.26
(m, 2H), 3.09–2.90 (m, 2H), 2.27–2.12 (m, 4H); 13C NMR
(75 MHz, CDCl3) d 165.6, 157.6 (d, JCF = 239.9 Hz), 153.7, 152.7 (d,
JCF = 2.2 Hz), 130.5 (q, JCF = 3.2 Hz), 130.1 (q, JCF = 33.2 Hz), 129.7
(q, JCF = 3.5 Hz), 126.0, 123.2 (q, JCF = 272.6 Hz), 123.1, 117.5 (d,
JCF = 8.0 Hz), 116.2 (d, JCF = 23.1 Hz), 69.6, 50.0, 30.3; MS (ES+)
m/z 384.1 (M+1).
5.1.8. (S)-2-(3-((4-Fluorobenzyl)oxy)pyrrolidin-1-yl)-5-
(trifluoromethyl)benzoic acid (9)
By a similar procedure described for 4, 9 was obtained as a col-
orless foam (0.23 g, 49%). 1H NMR (300 MHz, CDCl3) d 12.9 (br s,
1H), 8.24 (s, 1H), 7.68–7.60 (m, 1H), 7.31–7.22 (m, 2H), 7.19–
7.13 (m, 1H), 7.03–6.95 (m, 2H), 4.48 (ABq, 2H), 4.33–4.24 (m,
1H), 3.61–3.45 (m, 2H), 3.39–3.28 (m, 1H), 3.25–3.18 (m, 1H),
2.30–2.08 (m, 2H); 13C NMR (75 MHz, CDCl3) d 169.7, 162.3 (d,
JCF = 244.5 Hz), 151.0, 133.5, 129.9, 129.4, 124.3 (q, JCF = 269.9 Hz),
122.7 (q, JCF = 32.1 Hz), 119.7, 118.6, 115.5, 115.2, 77.6, 70.3, 58.6,
51.2, 31.3; MS (ES+) m/z 384.0 (M+1).
5.1.3. 2-(3-(4-Fluorophenoxy)azetidin-1-yl)-5-
(trifluoromethyl)benzoic acid (3)
By a similar procedure described for 4, 3 was obtained as a col-
orless solid (1.00 g, 39%). mp 175–177 °C (EtOAc/hexanes); 1H
NMR (300 MHz, DMSO-d6) d 13.02 (s, 1H), 7.83–7.82 (m, 1H),
7.65–7.61 (m, 1H), 7.18–7.13 (m, 2H), 6.92–6.89 (m, 2H), 6.74–
6.72 (m, 1H), 5.11–5.04 (m, 1H), 4.40–4.34 (m, 2H), 3.98–3.93
5.1.9. (S)-2-(3-(Cyclopropylmethoxy)pyrrolidin-1-yl)-5-
(trifluoromethyl)benzoic acid (10)
By a similar procedure described for 4, 10 was obtained as a col-
orless foam (0.32 g, 83%). 1H NMR (300 MHz, CDCl3) d 8.33–8.30
(m, 1H), 7.72–7.65 (m, 1H), 7.34–7.27 (m, 1H), 4.27–4.20 (m,
1H), 3.59–3.14 (m, 6H), 2.26–2.07 (m, 2H), 1.10–0.96 (m, 1H),
0.58–0.49 (m, 2H), 0.23–0.14 (m, 2H); 13C NMR (75 MHz, CDCl3)
d 168.7, 151.5, 130.1, 129.4, 124.7, 123.8 (q, JCF = 269.9 Hz),
121.6, 120.2, 77.6, 73.8, 59.3, 52.1, 31.7, 10.6, 3.2, 3.1; MS (ES+)
m/z 330.0 (M+1).
(m, 2H); 13C NMR (75 MHz, DMSO-d6)
d 167.0, 156.9 (d,
JCF = 236.6 Hz), 152.7 (d, JCF = 1.9 Hz), 151.8, 128.3 (q, JCF = 3.3 Hz),
128.0 (q, JCF = 3.6 Hz), 124.7 (q, JCF = 270.3 Hz), 116.3 (d, JCF = 23.1
Hz), 116.2 (q, JCF = 32.7 Hz), 115.9 (d, JCF = 8.6 Hz), 115.3, 114.5,
66.4, 60.0; MS (ES+) m/z 355.7 (M+1).
5.1.4. (R)-2-(3-(4-Fluorophenoxy)pyrrolidin-1-yl)-5-
(trifluoromethyl)benzoic acid (5)
5.1.10. (S)-2-(3-Butoxypyrrolidin-1-yl)-5-(trifluoromethyl)-
benzoic acid (12)
By a similar procedure described for 4, 5 was obtained as a col-
orless solid (0.57 g, 60%). mp 97–99 °C (EtOAc/hexanes); 1H NMR
(300 MHz, CDCl3) d 8.36–8.21 (m, 1H), 7.72–7.65 (m, 1H), 7.25–
7.20 (m, 1H), 6.99–6.92 (m, 2H), 6.85–6.77 (m, 2H), 4.98 (br s,
1H), 3.82–3.73 (m, 1H), 3.66–3.54 (m, 1H), 3.44–3.28 (m, 2H),
2.37–2.24 (m, 2H); 13C NMR (75 MHz, CDCl3) d 169.2, 159.2,
156.1, 152.7, 151.8 (d, JCF = 140.2 Hz), 130.1, 129.7, 123.8 (q,
JCF = 269.9 Hz), 123.4 (q, JCF = 31.1 Hz), 120.1, 118.9, 116.9, 116.8,
116.3, 116.0, 58.7, 51.3, 31.4; MS (ES+) m/z 370.0 (M+1).
By a similar procedure described for 4, 12 was obtained as a col-
orless oil (1.0 g, 94%). 1H NMR (300 MHz, CDCl3) d 8.38–8.37 (m,
1H), 7.74–7.72 (m, 1H), 7.38–7.35 (m, 1H), 4.20 (br s, 1H), 3.57–
3.31 (m, 5H), 3.20–3.17 (m, 1H), 2.21–2.14 (m, 2H), 1.62–1.53
(m, 2H), 1.44–1.32 (m, 2H), 0.92 (t, J = 7.3 Hz, 3H); 13C NMR
(75 MHz, CDCl3) d 168.2, 151.6, 130.2 (q, JCF = 3.3 Hz), 129.3 (q,
JCF = 3.8 Hz), 125.0, 123.7 (q, JCF = 271.8 Hz), 122.3, 120.8, 77.9,

S. Sun et al. / Bioorg. Med. Chem. 21 (2013) 7724–7734
7731
68.9, 59.5, 52.5, 31.8, 31.7, 19.3, 13.8; MS (ES+) m/z 332.1 (M+1),
MS (ES-) m/z 330.2.
5.1.16. (S)-2-(3-(2-Cyclopropylethoxy)pyrrolidin-1-yl)-5-
(trifluoromethyl) benzoic acid (11)
To a solution of 11c (1.70 g, 4.75 mmol) in THF (30 mL) and
water (10 mL) was added NaOH (1.90 g, 11.0 mmol) at ambient
temperature. The reaction mixture was heated to reflux for 18 h.
The solvent was removed in vacuo, and the residue was neutral-
ized to pH ꢀ3 with 5% hydrochloric acid solution’ extracted with
EtOAc (50 mL ꢁ 3) and washed with water and brine. The organic
solution was dried over anhydrous Na2SO4 and filtered. The filtrate
was concentrated in vacuo and the residue was subjected to col-
umn chromatography eluting with a gradient of 10–50% ethyl ace-
tate in hexanes to give 11 as a colorless oil (1.28 g, 78%). 1H NMR
(300 MHz, CDCl3) d 8.36–8.22 (m, 1H), 7.70–7.63 (m, 1H), 7.28–
7.23 (m, 1H), 4.22–4.17 (m, 1H), 3.59–3.29 (m, 5H), 3.20–3.14
(m, 1H), 2.26–2.05 (m, 2H), 1.44 (q, J = 6.9 Hz, 2H), 0.77–0.63 (m,
1H), 0.43–0.35 (m, 2H), 0.45–0.37 (m, 2H); MS (ES+) m/z 344.1
(M+1).
5.1.11. (S)-2-(3-(Pyridin-2-ylmethoxy)pyrrolidin-1-yl)-5-
(trifluoromethyl)benzoic acid (13)
By a similar procedure described for 4, 13 was obtained as a col-
orless foam (1.20 g, 45%). 1H NMR (300 MHz, CDCl3) d 12.75 (br s,
1H), 8.60–8.55 (m, 1H), 8.15 (s, 1H), 7.75–7.66 (m, 1H), 7.61–7.54
(m, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.28–7.20 (m, 1H), 7.07 (d,
J = 8.7 Hz, 1H), 4.73 (s, 2H), 4.39–4.34 (m, 1H), 3.67–3.49 (m, 2H),
3.39–3.30 (m, 1H), 3.28–3.20 (m, 1H), 2.34–2.10 (m, 2H); 13C
NMR (75 MHz, CDCl3) d 169.0, 157.4, 150.5, 147.9, 137.9, 129.2,
123.8 (q, JCF = 269.4 Hz), 123.0, 122.1, 121.6, 120.5 (q, JCF = 33.4 -
Hz), 117.9, 117.6, 78.6, 70.7, 58.2, 50.5, 31.4; MS (ES+) m/z 366.9
(M+1). Anal. Calcd for C18H17F3N2O3: C, 59.02; H, 4.68; N, 7.65.
Found C, 58.97; H, 5.01; N, 7.62.
5.1.12. (S)-2-(3-(2-Ethoxyethoxy)pyrrolidin-1-yl)-5-
(trifluoromethyl)benzoic acid (19)
Compounds 14–17 and 20–21 were prepared following the gen-
eral procedure for 11.
By a similar procedure described for 4, 19 was obtained as a col-
orless oil (0.32 g, 66%). 1H NMR (300 MHz, CDCl3) d 10.84 (br s, 1H),
8.14 (s, 1H), 7.58–7.54 (m, 1H), 7.14–7.09 (m, 1H), 4.20 (br s, 1H),
3.59–3.14 (m, 10H), 2.17–2.05 (m, 2H), 1.12 (t, J = 6.9 Hz, 3H); 13C
NMR (75 MHz, CDCl3) d 169.6, 150.8, 129.6, 129.2, 124.0 (q,
JCF = 269.6 Hz), 121.9 (q, JCF = 34.0 Hz), 119.5, 118.2, 78.4, 69.7,
68.3, 66.7, 58.4, 51.0, 31.2, 15.0; MS (ES+) m/z 348.0 (M+1).
5.1.17. (S)-Methyl 2-(3-((5-fluoropyridin-2-
yl)methoxy)pyrrolidin-1-yl)-5-(trifluoromethyl)benzoate (14a)
By a similar procedure described for 11c, 14a was obtained as a
colorless oil (0.74 g, 61%). 1H NMR (300 MHz, CDCl3) d 8.36–8.34
(m, 1H), 7.82 (s, 1H), 7.51–7.46 (m, 1H), 7.34–7.30 (m, 2H), 6.81–
6.77 (m, 1H), 4.66–4.52 (m, 2H), 4.28 (br s, 1H), 3.87 (s, 3H),
3.63–3.49 (m, 2H), 3.38–3.30 (m, 1H), 3.11–3.06 (m, 1H), 2.26–
2.03 (m, 2H).
5.1.13. (S)-2-(3-Hydroxypyrrolidin-1-yl)-5-(trifluoromethyl)
benzoic acid (11a)
By a similar procedure described for 4, 11a was obtained as a
colorless foam (10.70 g, 85%). 1H NMR (300 MHz, CDCl3) d 8.35
(s, 1H), 7.76–7.70 (m, 1H), 7.45–7.39 (m, 1H), 4.69 (s, 1H), 3.63–
3.50 (m, 2H), 3.79–3.28 (m, 1H), 3.24–3.16 (m, 1H), 2.35–2.10
(m, 2H); MS (ES+) m/z 276.1 (M+1).
5.1.18. (S)-2-(3-((5-Fluoropyridin-2-yl)methoxy)pyrrolidin-1-
yl)-5-(trifluoromethyl)benzoic acid (14)
By a similar procedure described for 11, 14 was obtained as a
colorless foam (0.52 g, 73%). 1H NMR (300 MHz, CDCl3) d 8.39 (s,
1H), 8.16 (s, 1H), 7.62–7.57 (m, 1H), 7.41–7.32 (m, 2H), 7.10–7.06
(m, 1H), 4.65 (s, 2H), 4.37–4.33 (m, 1H), 3.63–3.49 (m, 2H), 3.37–
3.27 (m, 1H), 3.25–3.18 (m, 1H), 2.32–2.10 (m, 2H); 13C NMR (75
MHz, CDCl3) d 169.1, 158.8 (d, JCF = 253.8 Hz), 153.7, 150.6, 137.0,
136.7, 129.4, 124.1 (q, JCF = 269.7 Hz), 124.0 (d, JCF = 18.2 Hz),
123.9, 122.9 (d, JCF = 4.4 Hz), 121.7 (q, JCF = 35.6 Hz), 119.5, 117.6,
78.4, 70.7, 60.4, 50.5, 31.3; MS (ES+) m/z 384.9 (M+1).
5.1.14. (S)-Methyl 2-(3-hydroxypyrrolidin-1-yl)-5-
(trifluoromethyl)benzoate (11b)
To a solution of 11a (8.00 g, 29.06 mol) in DMF (30 mL) was
added K2CO3 (4.40 g, 31.83 mol). The mixture was stirred at ambi-
ent temperature for 0.5 h, MeI (2.40 mL, 38.55 mmol) was added,
and stirring was continued for 18 h at ambient temperature, di-
luted with EtOAc (300 mL), washed with water and brine, dried
over anhydrous Na2SO4 and filtered. The filtrate was concentrated
in vacuo to give 11b as a colorless oil (8.37 g, 99%). 1H NMR
(300 MHz, CDCl3) d 7.83–7.80 (m, 1H), 7.51–7.46 (m, 1H), 6.79–
6.75 (m, 1H), 4.52 (br s, 1H), 3.87 (s, 3H), 3.65–3.53 (m, 2H),
3.31–3.23 (m, 1H), 2.99–2.92 (m, 1H), 2.11–1.95 (m, 2H).
5.1.19. (S)-Methyl 2-(3-((3-fluoropyridin-2-
yl)methoxy)pyrrolidin-1-yl)-5-(trifluoromethyl)benzoate (15a)
By a similar procedure described for 11c, 15a was obtained as a
colorless oil (0.44 g, 36%). 1H NMR (300 MHz, CDCl3) d 8.28–8.25
(m, 1H), 7.74–7.73 (m, 1H), 7.40–7.36 (m, 1H), 7.29–7.22 (m,
1H), 7.17–7.10 (m, 1H), 6.70–6.66 (m, 1H), 4.67–4.53 (m, 2H),
4.24 (br s, 1H), 3.78 (s, 3H), 3.54–3.38 (m, 2H), 3.28–3.20 (m,
1H), 3.05–3.01 (m, 1H), 2.18–1.94 (m, 2H).
5.1.15. (S)-Methyl 2-(3-(2-cyclopropylethoxy)pyrrolidin-1-yl)-5-
(trifluoromethyl)-benzoate (11c)
To a solution of 11b (1.50 g, 5.18 mmol) in DMF (20 mL) was
added NaH (60% in mineral oil w/w, 0.23 g, 5.75 mmol) in small
portions at 0 °C. The reaction mixture was stirred at 0 °C for 1 h,
2-cyclopropylethyl 4-methylbenzenesulfonate, (1.60 g, 6.65 mmol)
was added; stirring was continued for 18 h and then heated at
50 °C for 4 h. The reaction mixture was quenched by addition of
aqueous saturated NH4Cl solution (10 mL) at 0 °C and extracted
with ethyl acetate (100 mL ꢁ 3), washed with water and brine’
dried over anhydrous Na2SO4 and filtered. The filtrate was concen-
trated in vacuo, the residue was subjected to column chromatogra-
phy with a gradient of 10–30% EtOAc in hexanes to give 11c as a
colorless oil (1.76 g, 95%). 1H NMR (300 MHz, CDCl3) d 7.83–7.79
(m, 1H), 7.50–7.45 (m, 1H), 6.80–6.75 (m, 1H), 4.14–4.08 (m,
1H), 3.88 (s, 3H), 3.58–3.38 (m, 4H), 3.36–3.28 (m, 1H), 3.08–3.00
(m, 1H), 2.16–2.00 (m, 2H), 1.43–1.35 (m, 2H), 0.69–0.59 (m, 1H),
0.41–0.35 (m, 2H), 0.19–0.03 (m, 2H); MS (ES+) m/z 358.1 (M+1).
5.1.20. (S)-2-(3-((3-Fluoropyridin-2-yl)methoxy)pyrrolidin-1-
yl)-5-(trifluoromethyl)benzoic acid (15)
By a similar procedure described for 11, 15 was obtained as a
colorless foam (0.41 g, 96%). 1H NMR (300 MHz, CDCl3) d 8.43–
8.40 (m, 1H), 8.21–8.19 (m, 1H), 7.62–7.57 (m, 1H), 7.45–7.37
(m, 1H), 7.33–7.27 (m, 1H), 7.20–7.14 (m, 1H), 4.75 (ABq, 2H),
4.41–4.38 (m, 1H), 3.65–3.59 (m, 1H), 3.52–3.42 (m, 1H), 3.39–
3.31 (m, 1H), 3.24–3.19 (m, 1H), 2.34–2.10 (m, 2H); 13C NMR
(75 MHz, CDCl3) d 168.8, 158.2 (d, JCF = 257.5 Hz), 151.1, 145.1,
144.9, 129.7, 129.2, 125.1, 124.0, 123.7, 119.0, 78.5, 66.4, 58.8,
51.3, 31.4; (ES+) m/z 384.9 (M+1).
5.1.21. (S)-Methyl 2-(3-((5-fluoropyridin-3-
yl)methoxy)pyrrolidin-1-yl)-5-(trifluoromethyl)benzoate (16a)
By a similar procedure described for 11c, 16a was obtained as a
colorless oil (0.08 g, 6%). 1H NMR (300 MHz, CDCl3) d 8.35–8.30 (m,

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S. Sun et al. / Bioorg. Med. Chem. 21 (2013) 7724–7734
2H), 7.82 (s, 1H), 7.50–7.46 (m, 1H), 7.35–7.31 (m, 1H), 6.80–676
(m, 1H), 4.51 (ABq, 2H), 4.22 (br s, 1H), 3.87 (s, 3H), 3.63–3.48
(m, 2H), 3.37–3.29 (m, 1H), 3.11–3.00 (m, 1H), 2.22–2.05 (m, 2H).
5.1.28. (S)-2-(3-(2-(2-Methoxyethoxy)ethoxy)pyrrolidin-1-yl)-5-
(trifluoromethyl)benzoic acid (20)
By a similar procedure described for 11, 20 was obtained as a
colorless oil (0.40 g, 59%). 1H NMR (300 MHz, CDCl3) d 8.31–8.29
(m, 1H), 7.71–7.66 (m, 1H), 7.34–7.30 (m, 1H), 4.28–4.20 (m,
1H), 3.68–3.58 (m, 6H), 3.54–3.47 (m, 3H), 3.45–3.38 (m, 1H),
3.34–3.26 (m, 4H), 3.25–3.19 (m, 1H), 2.27–2.06 (m, 2H); 13C
NMR (75 MHz, CDCl3) d 168.2, 151.5, 130.0, 129.2, 124.9, 124.5,
123.8 (q, JCF = 266.3 Hz), 122.1, 120.6, 78.5, 71.8, 70.6, 70.5, 68.4,
59.2, 58.9, 52.6, 31.7; MS (ES+) m/z 378.1 (M+1).
5.1.22. (S)-2-(3-((5-Fluoropyridin-3-yl)methoxy)pyrrolidin-1-
yl)-5-(trifluoromethyl)benzoic acid (16)
By a similar procedure described for 11, 16 was obtained as a
colorless foam (0.07 g, 93%). 1H NMR (300 MHz, DMSO-d6) d
8.46–8.44 (m, 1H), 8.38 (s, 1H), 7.64–7.57 (m, 1H), 7.29–7.27 (m,
1H), 7.22–7.17 (m, 1H), 6.57–6.53 (m, 1H), 4.54 (ABq, 2H), 4.22–
4.16 (m, 1H), 3.71–3.64 (m, 1H), 3.44–3.32 (m, 3H), 2.10–1.89
(m, 2H); 13C NMR (75 MHz, DMSO-d6)
d
173.1, 159.5 (d,
5.1.29. (S)-Methyl 2-(3-(2-morpholinoethoxy)-pyrrolidin-1-yl)-
5-(trifluoromethyl)benzoate (21a)
JCF = 252.7 Hz), 147.3, 145.3, 137.2, 136.8, 131.6, 126.1 (q,
JCF = 269.0 Hz), 125.6, 123.6, 122.5, 122.3, 114.6 (q, JCF = 29.7 Hz),
112.4, 78.3, 66.9, 55.0, 47.2, 30.8; MS (ES-) m/z 383.1 (Mꢂ1).
By a similar procedure described for 11c, 21a was obtained as a
colorless oil (0.24 g, 13%). 1H NMR (300 MHz, CDCl3) d 7.77–7.75
(m, 1H), 7.47–7.39 (m, 1H), 6.74–6.70 (m, 1H), 4.07–4.02 (m,
1H), 3.82 (s, 3H), 3.64–3.36 (m, 8H), 3.27–3.19 (m, 1H), 3.02–
3.97 (m, 1H), 2.48–2.35 (m, 6H), 2.09–1.95 (m, 2H); MS (ES+) m/z
403.1 (M+1).
5.1.23. (S)-Methyl 2-(3-(2-(benzyloxy)ethoxy)-pyrrolidin-1-yl)-
5-(trifluoromethyl)benzoate (17a)
By a similar procedure described for 11c, 17a was obtained as a
colorless oil (3.40 g, 29%). 1H NMR (300 MHz, CDCl3) d 7.82 (s, 1H),
7.50–7.46 (m, 1H), 7.35–7.22 (m, 5H), 6.79–6.75 (m, 1H), 4.51 (s,
2H), 4.15–4.20 (m, 1H), 3.87 s, 3H), 3.66–3.43 (m, 6H), 3.38–3.28
(m, 1H), 3.09–3.03 (m, 1H), 2.09–1.98 (m, 2H).
5.1.30. (S)-2-(3-(2-Morpholinoethoxy)pyrrolidin-1-yl)-5-
(trifluoromethyl)benzoic acid (21)
By a similar procedure described for 11, 21 was obtained as a
colorless solid (0.13 g, 55%). mp 77–79 °C; 1H NMR (300 MHz,
CDCl3) d 7.82 (s, 1H), 7.44–7.40 (m, 1H), 6.78–6.74 (m, 1H), 4.09
(br s, 1H), 3.94–3.67 (m, 6H), 3.54–3.43 (m, 2H), 3.32–3.19 (m,
2H), 3.03–2.82 (m, 6H), 2.17–1.99 (m, 2H); 13C NMR (75 MHz,
CDCl3) d 172.1, 149.0, 128.1, 127.7, 124.6 (q, JCF = 269.1 Hz),
121.7, 118.9 (q, JCF = 34.9 Hz), 114.5, 78.6, 64.5, 64.2, 57.4, 56.0,
53.1, 48.5, 31.0; MS (ES+) m/z 389.1 (M+1).
5.1.24. (S)-2-(3-(2-(Benzyloxy)ethoxy)pyrrolidin-1-yl)-
5-(trifluoromethyl)benzoic acid (17)
By a similar procedure described for 11, 17 was obtained as a
colorless oil (0.27 g, 56%). 1H NMR (300 MHz, CDCl3) d 8.27 (s,
1H), 7.53–7.50 (m, 1H), 7.38–7.22 (m, 6H), 4.53 (s, 2H), 4.23 (s,
1H), 3.64–3.15 (m, 8H), 2.27–2.03 (m, 2H); 13C NMR (75 MHz,
CDCl3) d 169.0, 151.3, 137.9, 130.0, 129.2, 128.4, 127.8, 124.0,
123.9 (q, JCF = 265.7 Hz), 123.5, 121.0, 119.9, 78.5, 73.3, 69.5,
68.4, 59.0, 52.0, 31.7; MS (ES+) m/z 410.0 (M+1).
5.1.31. (S)-2-(3-(4-Fluorophenoxy)pyrrolidin-1-yl)-
5-(trifluoromethyl)benzonitrile (22a)
To
a suspension of 2-fluoro-5-(trifluoromethyl)benzonitrile
5.1.25. (S)-Methyl 2-(3-(2-hydroxyethoxy)pyrrolidin-1-yl)-
5-(trifluoromethyl)benzoate (18a)
(3.80 g, 20.00 mmol) and (S)-3-(4-fluorophenoxy)pyrrolidine
(3.80 g, 20.97 mmol) in 1,4-dioxane (100 mL) was added cesium
carbonate (9.82, 30.13 mmol). The reaction mixture was heated
at reflux for 19 h. The reaction mixture was colded to ambient tem-
perature and filtered. The filtrate was concentrated in vacuo, the
residue was subjected to column chromatography (10% EtOAc in
hexanes) to give 22a as a pale yellow oil (4.70 g, 67%): 1H NMR
(300 MHz, CDCl3) d 7.66 (s, 1H), 7.52–7.47 (m, 1H), 7.00–6.93 (m,
2H), 6.83–6.78 (m, 2H), 6.70–6.66 (m, 1H), 4.91–4.98 (m, 1H),
4.05–4.01 (m, 1H), 3.90–3.72 (m, 3H), 2.39–2.12 (m, 2H); (ES+)
m/z 351.0 (M+1).
A solution of 17a (2.90 g, 6.84 mmol) in methanol (100 mL) was
hydrogenated with 10% Pd/C (0.5 g) under atmosphere hydrogen at
ambient temperature for 20 h. The catalyst was removed by filtra-
tion, and the filtrate was concentrated in vacuo, the residue was
subjected to column chromatography eluating with 30% ethyl ace-
tate in hexanes to give 18a as a colorless oil (2.00 g, 87%). 1H NMR
(300 MHz, CDCl3) d 7.83–7.82 (m, 1H), 7.51–7.46 (m, 1H), 6.80–
6.76 (m, 1H), 4.17–4.14 (m, 1H), 3.88 (s, 3H), 3.68–3.44 (m, 6H),
3.33–3.27 (m, 1H), 3.08–3.03 (m, 1H), 2.18–1.94 (m, 3H); MS
(ES+) m/z 333.9 (M+1).
5.1.32. (S)-5-(2-(3-(4-Fluorophenoxy)pyrrolidin-1-yl)-
5.1.26. (S)-2-(3-(2-Hydroxyethoxy)pyrrolidin-1-yl)-
5-(trifluoromethyl)phenyl)-1H-tetrazole (22)
5-(trifluoromethyl)benzoic acid (18)
To a solution of 22a (0.49 g, 1.39 mmol) in DMF (20 mL) was
added NaN3 (1.80 g, 27.68 mmol) and NH4Cl (1.48 g, 27.66 mmol).
The resulting mixture was heated at 120 °C for 72 h. The reaction
was cooled to ambient temperature, followed by addition of water
(10.0 mL) and extraction with EtOAc (50 mL ꢁ 3). The combined
organic phase was washed with water and brine, dried over anhy-
drous sodium sulfate and concentrated in vacuo, the residue was
subjected to column chromatography (50% EtOAc in hexanes) to
give 22 as a colorless solid (0.14 g, 25%). mp 97–99 °C (EtOAc/hex-
anes); 1H NMR (300 MHz, DMSO-d6) d 7.68–7.23 (m, 1H), 7.59–
7.57 (m, 1H), 7.11–7.00 (m, 3H), 6.91–6.84 (m, 2H), 4.94 (br s,
1H), 3.33–3.15 (m, 2H), 3.07–3.00 (m, 1H), 2.86–2.81 (m, 1H),
2.15–1.94 (m, 2H); 13C NMR (75 MHz, DMSO-d6)) d 158.7, 155.5,
155.0, 151.7 (d, JCF = 259.5 Hz), 130.0, 128.5, 125.1 (q, JCF = 268.6 -
Hz), 117.3, 117.2, 116.5, 116.2, 115.2, 109.1, 76.6, 55.9, 48.3,
30.6; MS (ES+) m/z 394.0 (M+1).
By a similar procedure described for 11, 18 was obtained as a
colorless oil (0.26 g, 54%). 1H NMR (300 MHz, CDCl3) d 8.15 (s,
1H), 7.61–7.57 (m, 1H), 7.15–7.11 (m, 1H), 4.20 (br s, 1H), 3.72–
3.66 (m, 2H), 3.61–3.43 (m, 4H), 3.30–3.18 (m, 2H), 2.21–2.04
(m, 2H); 13C NMR (75 MHz, CDCl3) d 169.1, 150.3, 129.7, 129.2,
123.9 (q, JCF = 269.7 Hz), 122.7 (q, JCF = 33.7 Hz), 120.3, 118.5,
78.3, 70.2, 61.6, 58.5, 51.0, 31.0; (ES+) m/z 320.0 (M+1).
5.1.27. (S)-Methyl 2-(3-(2-(2-methoxyethoxy)ethoxy)-
pyrrolidin-1-yl)-5-(trifluoromethyl)benzoate (20a)
By a similar procedure described for 11c, 20a was obtained as a
colorless oil (0.70 g, 76%). 1H NMR (300 MHz, CDCl3) d 7.83–7.82
(m, 1H), 7.50–7.45 (m, 1H), 6.79–6.76 (m, 1H), 4.20–4.15 (m,
1H), 3.88 (s, 1H), 3.64–3.42 (m, 10H), 3.40–3.26 (m, 4H), 3.07–
3.02 (m, 1H), 2.19–2.00 (m, 2H); (ES+) m/z 392.0 (M+1).

S. Sun et al. / Bioorg. Med. Chem. 21 (2013) 7724–7734
7733
5.1.33. (S)-2-(3-(4-Fluorophenoxy)pyrrolidin-1-yl)-
5-(trifluoromethyl)benzenesulfonamide (23)
vacuo, the residue was subjected to column chromatography (30%
EtOAc in hexanes) to give 25 as a colorless oil (3.65 g, 98%): 1H
NMR (300 MHz, CDCl3) d 7.64–7.59 (m, 1H), 7.48–7.43 (m, 1H),
7.18–7.12 (m, 1H), 7.02–6.89 (m, 2H), 6.85–6.77 (m, 2H), 5.22–
5.15 (m, 1H), 4.95–4.91 (m, 1H), 4.21 (s, 0.6H), 3.93 (s, 0.4H),
3.70–3.37 (m, 2.4H), 3.31–3.24 (m, 1H), 3.15–3.07 (m, 0.6H),
2.38–2.15 (m, 2H), 1.61–1.50 (m, 3H); MS (ES+) m/z 370.0 (M+1).
To
a solution of 2-fluoro-5-(trifluoromethyl)benzenesulfon-
amide (3.80 g, 14.63 mmol) and (S)-3-(4-fluorophenoxy)pyrroli-
dine (3.00 g, 16.55 mmol) in 1,4-dioxane (40 mL) was added
4-methylmorpholine (8.0 mL, 72.76 mmol). The resulting mixture
was heated at 110 °C for 44 h, concentrated in vacuo, and the res-
idue cooled in an ice bath and acidified with 5% hydrochloric acid
solution to pH ꢀ3; extracted with EtOAc (100 mL ꢁ 3) and washed
with water and brine. The organic solution was dried over anhy-
drous Na2SO4 and filtered. The filtrate was concentrated in vacuo,
the residue was subjected to column chromatography (30% ethyl
acetate in hexanes) and recrystalization from EtOAc to give 23 as
a colorless solid (5.35 g, 90%). mp 122–124 °C (EtOAc); 1H NMR
(300 MHz, DMSO-d6) d 8.15–8.13 (m, 1H), 7.64–7.59 (m, 3H),
7.12–7.06 (m, 3H), 6.96–6.91 (m, 2H), 5.06 (br s, 1H), 4.08–4.02
(m, 1H), 3.73–3.51 (m, 3H), 2.26–2.03 (m, 2H); MS (ES+) m/z
405.0 (M+1).
5.1.37. Preparation of recombinant EL, HL and LPL
Recombinant human and mouse EL, human LPL and human HL
were each obtained by transient transfection of HEK-293 cells with
full length codon-optimised sequences in pcDNA3.1 plasmid vec-
tors. Cells were grown in 225 cm2 flasks in DMEM plus 10% FBS
to 50–80% confluency and transfected with 95 lg of plasmid
DNA using Lipofectamine (Invitrogen). After 5 h, the media was re-
placed by Opti-MEM media (invitrogen) and the cells cultured for
72 h before the cell-associated lipase was displaced by the addition
of heparin to a final concentration of 50 U/mL. After 30 min incuba-
tion with cells, the media was removed and concentrated 100-fold
using Amicon Ultra-15 concentrators (30 kDa cutoff) and frozen at
ꢂ80 °C until used. For each assay of enzyme activity (below), con-
centrated media from HEK-293 cells following mock-transfection
was used to determine the background activity. In each case, this
was less-than 5% that using media from lipase-transfected cells.
The active site concentration of each lipase in the concentrated
media was determined using the irreversible inhibitor orlistat. A
serial dilution of orlistat was added to the concentrated media
and incubated for 1 h. The mixture was then diluted into assay buf-
fer containing substrate (bis-BODIPY-FL C11-PC) as described below
and the initial reaction rate monitored. The lipase active site con-
centrations for EL, HL and LPL were 137, 207 and 700 nM, respec-
tively, and represent the lowest concentrations of orlistat in the
preincubation mix that resulted in complete inhibition.
5.1.34. (S)-N-((2-(3-(4-Fluorophenoxy)pyrrolidin-1-yl)-
5-(trifluoromethyl)phenyl)-sulfonyl)acetamide (24)
To a solution of (S)-2-(3-(4-fluorophenoxy)pyrrolidin-1-yl)-5-
(trifluoromethyl)-benzenesulfonamide 23 (0.70 g, 1.73 mmol) in
pyridine (20 mL) was added acetyl chloride (0.15 mL, 2.11 mmol)
at 0 °C. The reaction mixture was stirred at ambient temperature
for 19 h and then concentrated in vacuo. To the residue was added
aqueous saturated NH4Cl solution (50 mL) and extracted with
EtOAc (50 mL ꢁ 3). The combined organics were washed brine’
dried over anhydrous Na2SO4 and filtered. The filtrate was concen-
trated in vacuo, the residue was subjected to column chromatogra-
phy (50% EtOAc in hexanes) to give 24 as a colorless solid (0.41 g,
54%). mp 52–54 °C (EtOAc/hexanes); 1H NMR (300 MHz, CDCl3) d
9.57 (br s, 1H), 8.31 (s, 1H), 7.32–7.28 (m, 1H), 6.99–6.93 (m,
2H), 6.86–6.80 (m, 2H), 4.91 (br s, 1H), 3.76–3.55 (m, 3H), 3.32–
3.24 (m, 1H), 2.40–2.12 (m, 2H), 2.01 (s, 3H); 13C NMR (75 MHz,
CDCl3) d 169.0, 157.7 (d, JCF = 238.2 Hz), 152.7, 150.5, 131.2,
129.0, 124.0 (q, JCF = 34.2 Hz), 123.4 (q, JCF = 270.5 Hz), 121.6,
116.8, 116.7, 116.4, 116.1, 77.6, 59.1, 51.2, 31.3, 23.4; MS (ES+)
m/z 447.0 (M+1).
5.1.38. Measurement of lipase activities
A mixed micelle fluorogenic phosphatidylcholine/Triton X-100
assay was used to titrate inhibitors against human and mouse EL,
LPL and HL activities.30 EL was diluted in PBS (1.4 nM final con-
centration) containing 14% DMEM, plus 2% glycerol and 40
was added to each well of a clear bottom 96-well plate containing
inhibitors or vehicle alone (5 L in 10% DMSO). The enzyme-
inhibitor mix was incubated at room temperature for 30 min prior
to the addition of substrate (5 L of 10 M bis-BODIPY-FL C11-PC
lL
5.1.35. (S)-1-(2-(3-(4-Fluorophenoxy)pyrrolidin-1-yl)-
5-(trifluoromethyl)phenyl)ethanone (25a)
l
To a solution of 1-(2-fluoro-5-(trifluoromethyl)phenyl)etha-
none (4.99 g, 24.0 mmol) and (S)-3-(4-fluorophenoxy)pyrrolidine
(4.4 g, 24.28 mmol) in 1,4-dioxane (60 mL) was added 4-methyl-
morpholine (3.5 mL, 31.83 mmol). The reaction mixture was
heated at 110 °C for 23 h, concentrated in vacuo, and the residue
was dissolved in EtOAc (250 mL) and washed with water and brine.
The organic solution was dried over anhydrous Na2SO4 and filtered.
The filtrate was concentrated in vacuo, the residue was subjected
to column chromatography (15% EtOAc in hexanes) to give 25a
as a pale yellow oil (5.70 g, 64%). 1H NMR (300 MHz, CDCl3) d
7.76–7.75 (m, 1H), 7.56–7.51 (m, 1H), 6.98–6.91 (m, 2H), 6.88–
6.84 (m, 1H), 6.79–6.74 (m, 2H), 4.93–4.90 (m, 1H), 3.70–3.52
(m, 2H), 3.39–3.31 (m, 1H), 2.92–2.87 (m, 1H), 2.60 (s, 3H), 2.31–
2.18 (m, 2H); MS (ES+) m/z 368.0 (M+1).
l
l
in 0.1% Triton X-100) and enzyme activity determined using a
fluorescence plate reader at excitation and emission wavelengths
of 490 and 520 nm, respectively. The activity of LPL and HL were
determined in the same manner, except that the LPL assay (4 nM
final concentration) required addition of ApoCII (MJS Biolynx Inc,
1 lg/uL final concentration). The HL assay (6 nM final concentra-
tion) employed Tris buffer (50 mM Tris, pH 8.5, 1 M NaCl, no
DMDM or glycerol).
The potencies of EL inhibitors were also determined using a
more physiological human HDL particle substrate in an assay sim-
ilar to the method reported by Keller.31 In brief, 35
lL human EL
(diluted to 3.7 nM in PBS) in 96-well plates, in the presence or ab-
sence of test compounds (diluted from 100ꢁ stocks in DMSO), was
preincubated at room temperature for 30 min. Orlistat, at 10 lM fi-
5.1.36. 1-(2-((S)-3-(4-Fluorophenoxy)pyrrolidin-1-yl)-5-
(trifluoromethyl)phenyl)ethanol (25)
nal concentration, was used as a 100% inhibition control. HDL sub-
strate (10 L) isolated from fresh human donor plasma (Intracel
l
To a solution of 25a (3.67 g, 10.0 mmol) in methanol (80 mL)
was added sodium borohydride (0.5 g, 13.20 mmol) in small por-
tions at 0 °C. The reaction mixture was stirred at ambient tempera-
ture for 30 min, brine (20 mL) was added and stirred for another
30 min. The mixture was extracted with EtOAc (50 mL ꢁ 3) and
washed with water and brine. The organic solution was dried over
anhydrous Na2SO4 and filtered. The filtrate was concentrated in
Frederick, MD) was added to the enzyme mix and incubated at
37 °C for 2 h. The final concentration of phospholipids in the assay
was 1 mM which was determined using a Phospholipids C kit
(Wako Diagnostics, Richmond VA). EL-mediated HDL hydrolysis
was determined by measuring released free fatty acids in the final
reaction using a NEFA-HR(2) kit (Wako Diagnostics, Richmond, VA)
according to the manufacturer’s instructions.

7734
S. Sun et al. / Bioorg. Med. Chem. 21 (2013) 7724–7734
7. Soran, H.; Hama, S.; Yadav, R.; Durrington, P. N. Curr. Opin. Lipidol. 2012, 23,
For the cell-associated EL activity assay, HEK-293 cells were
353.
transfected with 2 l
g full length human EL plasmid in 35 mm2
8. Otocka-Kmiecik, A.; Mikhailidis, D. P.; Nicholls, S. J.; Davidson, M.; Rysz, J.;
Banach, M. Prog. Lipid Res. 2012, 51, 314.
wells containing DMEM plus 10% FBS as described above, except
that fugene (Roche) replaced lipofectamine. At 24 h post transfec-
tion, the cells were harvested and seeded in 96-well plates
(1 ꢁ 106 cells/well) and cultured in DMEM for a further 24 h. The
media was removed and replaced with HBSS containing 25 mM
HEPES, pH 7.4 and inhibitors added as 200-fold stocks in DMSO.
After an additional 30 min at 37 °C, substrate PED-A1 was added
9. Rosenson, R. S.; Brewer, H. B., Jr; Davidson, W. S.; Fayad, Z. A.; Fuster, V.;
Goldstein, J.; Hellerstein, M.; Jiang, X. C.; Phillips, M. C.; Rader, D. J.; Remaley, A.
T.; Rothblat, G. H.; Tall, A. R.; Yvan-Charvet, L. Circulation 2012, 125, 1905.
10. Choi, S. Y.; Hirata, K.; Ishida, T.; Quertermous, T.; Cooper, A. D. J. Lipid Res. 2002,
43, 1763.
11. Griffon, N.; Budreck, E. C.; Long, C. J.; Broedl, U. C.; Marchadier, D. H.; Glick, J.
M.; Rader, D. J. J. Lipid Res. 1803, 2006, 47.
12. Annema, W.; Tietge, U. J. Curr. Atheroscler., Rep. 2011, 13, 257.
13. Yasuda, T.; Ishida, T.; Rader, D. J. Circ. J. 2010, 74, 2263.
14. Ishida, T.; Choi, S. Y.; Kundu, R. K.; Spin, J.; Yamashita, T.; Hirata, K.; Kojima, Y.;
Yokoyama, M.; Cooper, A. D.; Quertermous, T. J. Biol. Chem. 2004, 279, 45085.
15. Ishida, T.; Choi, S. Y.; Ramendra, K.; Kundu, R. K.; Hirata, K.; Rubin, E. M.;
Cooper, A. D.; Quertermous, T. J. Clin Invest. 2003, 111, 347.
16. Ma, K.; Cilingiroglu, M.; Otvos, J. D.; Ballantyne, C. M.; Marian, A. J.; Chan, J.
Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 2748.
to a final concentration of 4 lM and the reaction monitored contin-
uously using a FlexStation 3 fluorescence plate reader (Molecular
Devices) at 37 °C to obtain the initial velocity. The titrations were
performed twice and IC50 values obtained differed by a factor of
2 or less.
17. Brown, R. J.; Lagor, W. R.; Sankaranaravanan, S.; Yasuda, T.; Quertermous, T.;
Rothblat, G. H.; Rader, D. J. Circ. Res. 2010, 107, 357.
5.1.39. In vivo models
18. Jin, W.; Millar, J. S.; Broedl, U.; Glick, J. M.; Rader, D. J. J. Clin. Invest. 2003, 111,
357.
19. Ishida, T.; Choi, S.; Ramendra, K.; Kundu, R. K.; Hirata, K.; Rubin, E. M.; Cooper,
D. D.; Quertermous, T. J. Clin. Invest. 2003, 111, 347.
Male EL knockout mice (12–16 weeks of age) and age-matched
wild-type male C57BL6 mice were acclimatized for 1 week before
experimentation, during which they were fed 7012 Teklad LM-
485 Mouse diet (Harlan). During the drug efficacy studies the mice
were fed the same diet and given b.i.d. oral doses (t = 8 h) of vehi-
cle, 30 mg/kg XEN455 (as a suspension in 0.2% Tween-20/1% car-
boxymethyl-cellulose) for either 3 d or for 9 d. At 16 h post the
final dose, approximately 150 lL blood was removed by sub-man-
dibular vein bleed and EDTA-plasma prepared for analysis of total
cholesterol, HDLc and drug concentrations. Further blood
20. Singaraja, R. R.; Sivapalaratnam, S.; Hovingh, K.; Dubé, M. P.; Castro-Perez, J.;
Collins, H. L.; Adelman, S. J.; Riwanto, M.; Manz, J.; Hubbard, B.; Tietjen, I.;
Wong, K.; Mitnaul, L. J.; van Heek, M.; Lin, L.; Roddy, T. A.; McEwen, J.; Dallinge-
Thie, G.; van Vark-van der Zee, L.; Verwoert, G.; Winther, M.; van Duijn, C.;
Hofman, A.; Trip, M. D.; Marais, A. D.; Asztalos, B.; Landmesser, U.; Sijbrands,
E.; Kastelein, J. J.; Hayden, M. R. Circ. Cardiovasc. Genet. 2013, 6, 54.
21. Annema, W.; Tietge, U. J. Curr. Atheroscler. Rep. 2011, 13, 257.
22. Badellino, K. O.; Wolf, M. L.; Reilly, M. P.; Rader, D. J. Circulation 2008, 117, 678.
23. Brown, R. J.; Edmondson, A. C.; Griffon, N.; Hill, T. B.; Fuki, I. V.; Badellino, K. O.;
Li, M.; Wolf, M. L.; Reilly, M. P.; Rader, D. J. J. Lipid Res. 1910, 2009, 50.
24. Edmondson, A. C.; Brown, R. J.; Kathiresan, S.; Cupples, L. A.; Demissie, S.;
Manning, A. K.; Jensen, M. K.; Rimm, E. B.; Wang, J.; Rodrigues, A.; Bamba, V.;
Khetarpal, S. A.; Wolf, M. L.; DerOhannessian, S.; Li, M.; Reilly, M. P.; Aberlr, J.;
Evans, D.; Hegele, R. A.; Rader, D. J. J. Clin. Invest. 2009, 119, 1042.
25. Goodman, K. B.; Bury, M. J.; Cheung, M.; Cichy-Knight, M. A.; Dowdell, S. E.;
Dunn, A. K.; Lee, D.; Lieby, J. A.; Moore, M. L.; Scherzer, D. A.; Sha, D.; Suarez, D.
P.; Murphy, D. J.; Harpel, M. R.; Manas, E. S.; McNulty, D. E.; Annan, R. S.;
Matico, R. E.; Schwartz, B. K.; Trill, J. J.; Sweitzer, T. D.; Wang, D. Y.; Keller, P. M.;
Krawiec, J. A.; Jaye, M. C. Bioorg. Med. Chem. Lett. 2009, 19, 27.
(ꢀ400
lL) was then immediately obtained by cardiac puncture at
euthanasia and serum prepared following the 9 d study. Total plas-
ma cholesterol and plasma HDLc concentrations were determined
using the HDL-cholesterol E kit (Wako Diagnostics, Richmond VA).
Acknowledgments
We thank Tarek Mansour and Robert N. Young for their sugges-
tions, Jay Cadieux for his assistance in preparing this manuscript,
as well as Hongzhi Liu, Monica Mork, Matthew Waldbrook, Abdol-
lah Diarra, Karen Nelkenbrecher and Lee Robinette for their techni-
cal assistance.
26. O’Connell, D. P.; LeBlanc, D. F.; Cromley, D.; Billheimer, J.; Rader, D. J.;
Bachovchin, W. W. Bioorg. Med. Chem. Lett. 2012, 22, 1397.
27. Greco, M. N.; Connelly, M. A.; Leo, G. C.; Olson, M. W.; Powell, E.; Huang, Z.;
Hawkins, M.; Smith, C.; Schalk-Hihi, C.; Darrow, A. L.; Xin, H.; Lang, W.;
Damiano, B. P.; Hlasta, D. J. Bioorg. Med. Chem. Lett. 2013, 23, 2595.
28. Tickner, D.; Kasthurikrishnan, N. Org. Process Res. Dev. 2001, 5, 270.
29. Darrow, A. L.; Olson, M. W.; Xin, H.; Burke, S. L.; Smith, C.; Schalk-Hihi, C.;
Williams, R.; Bayoumy, S. S.; Deckman, I. C.; Todd, M. J.; Damiano, B. P.;
Margery, A.; Connelly, M. A. J. Lipid Res. 2011, 52, 374.
References and notes
30. Mitnaul, L. J.; Tian, J.; Burton, C.; Lam, M. H.; Zhu, Y.; Olson, S. H.; Schneeweis, J.
E.; Zuck, P.; Pandit, S.; Anderson, M.; Maletic, M. M.; Waddell, S. T.; Wright, S.
D.; Sparrow, C. P.; Lund, E. G. J. Lipid Res. 2007, 48, 472.
31. Keller, P. M.; Rust, T.; Murphy, D. J.; Matico, R.; Trill, J. J.; Krawiec, J. A.;
Jurewicz, A.; Jaye, M.; Harpel, M.; Thrall, S.; Schwartz, B. J. Biomol. Screen. 2008,
13, 468.
1. Weber, C.; Noels, H. Nat. Med. 2011, 17, 1410.
2. Joy, T. R. Pharmacol. Ther. 2012, 135, 31.
3. Castelli, W. P.; Garrison, R. J.; Wilson, P. W.; Abbott, R. D.; Kalousdian, S.;
Kannel, W. B. JAMA 1986, 256, 2835.
4. deGoma, E. M.; Rader, D. J. Nat. Rev. Cardiol. 2011, 8, 277.
5. Khera, A. V.; Rader, D. J. Curr. Atheroscler. Rep. 2010, 12, 73.
6. Khera, A. V.; Cuchel, M.; Llera-Moya, M.; Rodriques, A.; Burke, M.; Jafri, K.;
French, B. C.; Phillips, J. A.; Mucksavage, M. L.; Wilensky, R. L.; Mohler, E. R.;
Rothblat, G. H.; Rader, D. J. N. Engl. J. Med. 2011, 364, 127.