Substituted oxazolidinones as novel NPC1L1 ligands for the inhibition of cholesterol absorption

Article abstract of DOI:10.1016/j.bmcl.2007.11.083

Cholesterol absorption inhibition (CAI) represents an important treatment option for hypercholesterolemia. Herein, we report the design and evaluation of a series of substituted oxazolidinones as ligands for the Niemann Pick C1 Like 1 (NPC1L1) protein, a

Full text of DOI:10.1016/j.bmcl.2007.11.083

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 546–553
Substituted oxazolidinones as novel NPC1L1 ligands
for the inhibition of cholesterol absorption
Jeffrey A. Pfefferkorn,* Scott D. Larsen, Chad Van Huis, Roderick Sorenson,
Tom Barton, Thomas Winters, Bruce Auerbach, Chenyan Wu,
Thaddeus J. Wolfram, Hongliang Cai, Kathleen Welch, Nadia Esmaiel, JoAnn Davis,
Richard Bousley, Karl Olsen, Sandra Bak Mueller and Thomas Mertz
Pfizer Global Research & Development, Michigan Laboratories, 2800 Plymouth Road, Ann Arbor, MI 48105, USA
Received 25 October 2007; revised 19 November 2007; accepted 20 November 2007
Available online 28 November 2007
Abstract—Cholesterol absorption inhibition (CAI) represents an important treatment option for hypercholesterolemia. Herein, we
report the design and evaluation of a series of substituted oxazolidinones as ligands for the Niemann Pick C1 Like 1 (NPC1L1)
protein, a key mediator of cholesterol transport. Novel analogs were initially evaluated in a brush border membrane NPC1L1 bind-
ing assay; subsequently, promising compounds were evaluated in vivo for acute inhibition of cholesterol absorption. These studies
identified analogs with low micromolar NPC1L1 binding affinity and acute in vivo efficacy of >50% absorption inhibition at
3 mg/kg.
Ó 2007 Elsevier Ltd. All rights reserved.
Coronary heart disease (CHD) is a leading cause of
death in the United States.1 Given that hypercholester-
olemia is a key risk factor for CHD, substantial efforts
have been undertaken to mitigate this condition.2 The
standard of care for treating hypercholesterolemia has
been the use of HMG-CoA reductase inhibitors, or stat-
ins, which block the rate-limiting step of cholesterol bio-
synthesis.3 However, since plasma cholesterol results
from a combination of biosynthesis and absorption of
dietary cholesterol, the approval of the cholesterol
absorption inhibitor ZetiaÒ (ezetimibe, 1) has offered a
complementary approach for the treatment of hypercho-
lesterolemia.4 Clinically, as a monotherapy ezetimibe
achieves an average 18% reduction in LDL.5 When used
in combination with a statin, ezetimibe affords an addi-
tional 15–18% reduction in LDL beyond that achieved
by the statin alone.5 This combination strategy is partic-
ularly valuable for achieving aggressive LDL lowering
as well as for treating patients that cannot tolerate high
dose statin therapy.
Ezetimibe (1) was originally discovered in the absence of
a known molecular target.5c However, researchers at
Schering-Plough/Merck recently demonstrated that it
blocks cholesterol absorption via interaction with Nie-
mann Pick C1 Like 1 (NPC1L1).6 This protein is highly
expressed in the proximal intestine and is a key mediator
of cholesterol transport.7 Given that ezetimibe (1) is rap-
idly converted in vivo to its equipotent glucuronide
derivative 2, early NPC1L1 binding experiments were
conducted using [3H]ezetimibe-glucuronide as the radio-
ligand.6 In these studies using brush border membrane
(BBM) preparations from rat intestine, Garcia-Calvo
and coworkers demonstrated that ezetimibe-glucuronide
(2) bound selectively to rat NPC1L1 with sub-micromo-
lar affinity (Ki = 0.35 lM).6 The parent ezetimibe (1) was
found to have similar binding affinity (Ki = 0.97 lM).8
Both 1 and 2 were also shown to bind to brush border
membrane preparations containing native or recombi-
nant NPC1L1 from various other species, including hu-
man, with binding affinities correlating with the
observed CAI efficacies in those species.6,8
To enable the evaluation of novel NPC1L1 ligands, we
established
a competition binding assay utilizing
Keywords: NPC1L1; Cholesterol absorption inhibitor; Hypercholes-
terolemia; Dyslipidemia.
[3H]ezetimibe-glucuronide according to the method re-
ported.8 Using NPC1L1 in brush border membranes
isolated from rat, we reproduced the reported binding
*
Corresponding author. Tel.: +1 860 686 3421; fax: +1 860 715
4608; e-mail: jeffrey.a.pfefferkorn@pfizer.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.083

J. A. Pfefferkorn et al. / Bioorg. Med. Chem. Lett. 18 (2008) 546–553
547
R1
the A-ring and oxazolidinone core as well as alternative
substituents on the A-, B-, and C-aryl rings.
1: R1 = OH
HO
OH
OH
Schemes 1–5 outline the preparation of representative
oxazolidinone-based NPC1L1 ligands. Scheme 1 illus-
trates the enantioselective synthesis of analog 12 where-
in the secondary hydroxyl of the linker unit has been
transposed two carbons closer to the oxazolidinone
(relative to compound 4).11 Hydrocinnamaldehyde (5)
was initially converted to a-hydroxyketone 6 via treat-
ment with formaldehyde and N-ethylbenzothiazolium
bromide.12 Utilizing an asymmetric three-component
Mannich reaction reported by List and coworkers,13 a-
hydroxyketone 6 was subsequently reacted with 4-fluo-
roaniline (7), 4-benzyloxybenzaldehyde (8) in the pres-
ence of catalytic L-proline to afford 1,2-amino alcohol
9 in moderate yield and excellent stereoselectivity
(>15:1). Treatment of 9 with triphosgene and triethyl-
amine resulted in formation of oxazolidinone 10. Noyori
reduction14 of the side chain ketone provided alcohol 11
as a single diastereomer which was subsequently deben-
zylated using transfer hydrogenation conditions to af-
ford compound 12. In order to confirm the relative
stereochemistry of 12, a portion of intermediate 11
was reacted with acetic anhydride and pyridine to give
crystalline derivative 13 which was subjected to small
molecule X-ray analysis as shown.15
2: R1 =
N
F
O
O
OH
CO2H
O
F
Figure 1. Structure of ezetimibe (1) and ezetimibe-glucuronide (2).
affinities of both ezetimibe-glucuronide 2 (Ki = 0.18 lM)
and ezetimibe 1 (Ki = 0.20 lM) (Fig. 1).
In an effort to identify novel cholesterol absorption
inhibitors, we and others have been interested in replace-
ment of the b-lactam core of ezetimibe with alternative
heterocycles.9 A preliminary evaluation of various 4-,
5-, and 6-membered heterocycles suggested that the
3,4,5-trisubstituted oxazolidinone was a promising core
replacement. Previously, Dugar and coworkers reported
oxazolidinone 3 (Fig. 2) as having modest cholesterol
absorption inhibition efficacy in a cholesterol-fed animal
model.9b Additionally, Carrierea and coworkers de-
scribed the synthesis of oxazolidinone 4 bearing a substi-
tution pattern analogous to that of ezetimibe.9c,d In our
hands, resynthesis of these two oxazolidinones and eval-
uation in the radioligand rat NPC1L1 binding assay re-
vealed that 4 had an affinity (Ki = 9.9 lM) about 50-fold
less than ezetimibe (Ki = 0.20 lM), whereas 3 had very
weak affinity (Ki > 50 lM).10 Based on these observa-
tions, we anticipated that a diverse survey of the SAR
around this oxazolidinone core might afford ligands
with increased NPC1L1 binding potency. In addition,
these SAR studies would be useful in further under-
standing the relationship between NPC1L1 binding
and CAI efficacy since, to date, correlation data have
only been published for a small number of ezetimibe
analogs.6,8
Scheme 2 illustrates the preparation of ligands that con-
tain ether or amine functionalities in the linker back-
bone.16 Utilizing the same asymmetric Mannich
reaction described above, hydroxyacetone (14), 4-fluoro-
aniline (7), and 4-benzyloxybenzaldehyde (8) were con-
densed in the presence of catalytic L-proline to
stereoselectively provide 1,2-amino alcohol 15 which
cyclized to oxazolidinone 16 upon treatment with tri-
phosgene and triethylamine.
Epoxide 17 (Scheme 2) was prepared from ketone 16 via
a three-step sequence commencing with a-bromination
followed by carbonyl reduction (NaBH4) and finally
base-mediated closure of the intermediate halohydrin
to give a mixture (ca. 3:1) of diastereomeric epoxides.
Chromatographic separation provided epoxide 17 as
the major diastereomer (43% yield over three steps).
With 17 in hand, analogs bearing an ether linkage were
accessible via base-mediated addition of phenols as illus-
trated for compound 18. In parallel, analogs bearing an
amine linkage could be accessed via treatment of epox-
ide 17 with anilines in the presence of LiClO4 as illus-
trated for compound 20. Finally, 18 and 20 were
debenzylated utilizing standard transfer hydrogenation
conditions to provide 19 and 21, respectively.
As shown in Figure 3, our SAR strategy focused on the
evaluation of analogs with alternative linkers between
R3
R2
R1 R2
R3
R4
OMe
F
3
4
H
F
H
OMe
OH OH
O
N
R1
R4
O
Figure 2. Previous oxazolidinone cholesterol absorption inhibitors.
Linker
R2
Analogs containing amide functionalities in the linker
unit were prepared as outlined in Schemes 3 and 4. As
shown in Scheme 3, ketone 16 (from Scheme 2) was ini-
tially subjected to a haloform reaction (Br2, NaOH) to
generate carboxylic acid 22. EDAC-mediated coupling
of acid 22 with 4-fluorobenzyl amine afforded amide
23 which was subsequently debenzylated to provide
compound 24 as shown. The reverse amide was subse-
quently prepared as outlined in Scheme 4 by initial
B
O
X
Y
R1
A
O
N
C
R3
O
Figure 3. SAR strategy for evaluation of oxazolidinone-based
inhibitors.

548
J. A. Pfefferkorn et al. / Bioorg. Med. Chem. Lett. 18 (2008) 546–553
OBn
OBn
CHO
OH
OR
O
NH2
O
b
+
+
R
N
O
HN
F
OBn
F
7
O
5: R = H
6: R = CH2OH
11: R = H
13: R = OAc
9
F
a
f
8
c
ORTEP Structure of 13:
OR
OBn
d
OH
O
O
N
O
N
F
F
O
O
10
11: R = Bn
e
12: R = H
Scheme 1. Synthesis of oxazolidinone 12. Reagents and conditions: (a) (CHO)n, Et3N, N-ethylbenzothiazolium bromide, EtOH, 80 °C, 36 h, 25%; (b)
L-proline (10 mol%), DMSO, 25 °C, 72 h, 75%; (c) triphosgene, Et3N, CH2Cl2, ꢀ20 ! 25 °C, 12 h, 63%; (d) RuCl2-(S)-(DM-BINAP)-(S)-DAIPEN,
KOt-Bu, H2 (50 psi), i-PrOH:THF, 25 °C, 6 h, 92%; (e) Pd/C, cyclohexene, EtOH:EtOAc, 80 °C, 5 h, 91%; (f) Ac2O, pyridine, MeCN, 25 °C, 16 h,
85%.
OBn
F
OBn
OBn
CHO
NH2
OH
a
O
O
O
a
+
+
OH
O
HN
15
HO
14
O
N
O
N
OBn
8
F
7
F
F
O
O
16
22
b
OR
OBn
OBn
b
O
O
c,d, e
O
N
H
O
N
O
N
O
N
F
F
O
F
F
O
O
23: R = Bn
24: R = H
17
16
c
OR
Scheme 3. Synthesis of oxazolidinone amide 24. Reagents and
conditions: (a) Br2, NaOH, H2O:dioxane, 0 °C, 2 h, 80%; (b) 4-
fluorobenzyl amine, EDAC–HCl, HOBt, CH2Cl2, 25 °C, 4 h, 50%; (c)
Pd/C, cyclohexene, EtOH:EtOAc, 80 °C, 3 h, 79%.
OH
O
X
N
F
reduction of carboxylic acid 22 to the corresponding
alcohol 25. Conversion of alcohol 25 to the correspond-
ing mesylate 26 followed by reaction with NaN3 resulted
in formation of azide 27. Treatment of 27 with Pd/C un-
der transfer hydrogenation conditions resulted in reduc-
tion of the azide functionality to a primary amine with
concomitant removal of the O-benzyl protecting group
to afford amino-phenol 28. Finally, 28 was reacted with
4-fluorobenzoyl chloride in the presence of triethylamine
to generate amide 29.
F
O
f
18: X = O; R = Bn
19: X = O; R = H
h
h
g
20: X = NH; R = Bn
21: X = NH; R = H
Scheme 2. Synthesis of oxazolidinones 19 and 21. Reagents and
conditions: (a) L-proline (20 mol%), DMSO, 25 °C, 12 h, 95%; (b)
triphosgene, Et3N, CH2Cl2, ꢀ20 ! 25 °C, 12 h, 41%; (c) trimethylph-
enylammonium tribromide, THF, 0 °C, 22 h; (d) NaBH4, EtOH,
0 ! 25 °C, 3 h; (e) NaOt-Bu, THF, 0 ! 25 °C, 2 h, 43% over three
steps; (f) 4-fluorophenol, Cs2CO3, DMSO, 70 °C, 3 h, 78%; (g) 4-
fluoroaniline, LiClO4, Et2O, 25 °C, 16 h, 50%; (h) Pd/C, cyclohexene,
EtOH:EtOAc, 80 °C, 5 h, 75–90%.
Finally, to enable comparison with the known ezetim-
ibe-glucuronide 2, the glucuronide conjugate of an oxa-

J. A. Pfefferkorn et al. / Bioorg. Med. Chem. Lett. 18 (2008) 546–553
549
OBn
OBn
MeO2C
AcO
O
Br
MeO2C
AcO
O
OH
a
O
OAc
OAc
a
OAc
30
OAc
31
HO
HO
b
O
N
O
c
N
F
F
O
O
OR1
b
22
25: R = H
26: R = Ms
OAc
NH
CCl3
OAc
MeO2C
AcO
O
+
O
N
OH
OBn
F
O
OAc
32
13: R1 = Bn
33: R1 = H
c
d
H2N
N3
O
e
N
O
N
d
F
F
O
28
O
27
R2O
OR2
O
OR2
O
OH
OR2
CO2R3
O
O
N
N
H
O
N
F
O
F
34: R2 = Ac; R3 = Me
F
O
29
e
35: R2, R3 = H
Scheme 4. Synthesis of oxazolidinone amide 29. Reagents and
conditions: (a) BH3ÆTHF, THF, 0 ! 25 °C, 16 h, 68%; (b) methane-
sulfonic anhydride, Et3N, CH2Cl2, 25 C, 5 h, 75%; (c) NaN3, DMF,
60 °C, 16 h, 72%; (d) Pd/C, cyclohexene, EtOH:EtOAc, 80 °C, 1.5 h;
(e) 4-fluorobenzoyl chloride, Et3N, CH2Cl2, 25 C, 1.5 h, 31% over two
steps.
Scheme 5. Synthesis of oxazolidinone–glucuronide 35. (a) CdCO3,
H2O, MeCN, 70 °C, 4 h, 78%; (b) Cl3CCN, DBU, CH2Cl2, 0 °C, 1 h,
50%; (c) Pd/C, cyclohexene, EtOH:EtOAc, 80 °C, 3 h, 73%; (d)
BF3ÆOEt2, CH2Cl2:THF, ꢀ20 ! 25 °C, 2.5 h, 23%; (e) Et3N, H2O,
MeOH, 25 °C, 16 h, 31%.
Our structure–activity studies focused largely on modifi-
cation of the linker region along with evaluation of
alternative A-, B-, and C-ring substitution patterns. As
shown in Table 1, initial transposition11 of the second-
ary hydroxyl group from the aryl side (reference com-
zolidinone analog, 35, was prepared as outlined in
Scheme 5. Commercially available 1-bromo-2,3,4-tri-
O-acetyl-a-D-glucuronic acid methyl ester (30) was
hydrolyzed with aqueous CdCO3 and then treated with
trichloroacetonitrile in the presence of DBU to generate
trichloroacetimidate 32.17 Reaction of 32 with phenol 33
in the presence of catalytic BF3ÆOEt2 selectively gener-
ated b-glucuronide 34 which underwent subsequent glo-
bal deprotection upon treatment with triethylamine/
MeOH/H2O to provide oxazolidinone glucuronide 35.18
Table 1. NPC1L1 binding and acute CAI efficacy of oxazolidinones 12
and 35–4019,20
R3
R2
OH
By appropriate substitution of starting materials, the
routes outlined in Schemes 1–5 were subsequently uti-
lized to prepare the compounds utilized in the SAR
studies described below.
R1
O
N
F
O
Compound R1
R2
R3
BBM Acute CAI
NPC1L1 at 3 mg/kg
All analogs were initially screened in the NPC1L1 BBM
binding assay using native rat enterocytes and [3H]eze-
timibe-glucuronide (2) as the radioligand.19 Compounds
with observed binding activity (Ki < 50 lM) were then
evaluated in an acute in vivo cholesterol absorption
inhibition assay. In this efficacy model, chow-fed ham-
sters were dosed orally with analogs at 3 mg/kg. Two
hours post-dose, the animals were administered a bolus
of [14C]cholesterol, and after an additional two hours,
serum cholesterol samples were taken and analyzed. Per-
cent reduction of total cholesterol was determined rela-
tive to the vehicle-treated control group.20
Ki (lM)
1
—
—
—
—
H
—
—
0.20
9.9
ꢀ89%
ꢀ64%
NT
4
12
35
36
37
38
39
40
H
OH
O-Gluc
H
>50
>50
33
H
H
NT
H
OH
H
ꢀ4%
0%
OH
OH
OH
OMe
H
OH
12
9.2
H
ꢀ55%
ꢀ33%
NT
H
OMe
OMe
4.8
>50
H
NT, not tested; Gluc, glucuronide.

550
J. A. Pfefferkorn et al. / Bioorg. Med. Chem. Lett. 18 (2008) 546–553
Table 3. NPC1L1 binding and acute CAI efficacy of oxazolidinones
43–4919,20
pound 4) of the linker to the oxazolidinone side (com-
pound 12) resulted in a loss of binding activity. The cor-
responding glucuronide of phenol 12, compound 35, was
also inactive. While maintaining the transposed linker,
various phenol isomers were then evaluated. Relocating
the B-ring phenol from the 4-position (12, Ki = 50 lM)
to the 3-position (36, Ki = 33 lM) and then to the 2-po-
sition (37, Ki = 12 lM) resulted in increased binding
activity albeit without in vivo efficacy. Encouragingly,
combination of the 2-OH substituent with either a 4-
OH (38, Ki = 9.2 lM) or a 4-OMe (39, Ki = 4.8 lM)
afforded increased NPC1L1 binding and also acute
in vivo efficacy. This increase in efficacy with the 2-OH
substituent was consistent with previous observations
in the ezetimibe series.21 The importance of the 2-OH
substituent to binding affinity was further demonstrated
by comparison of compounds 39 (2-OH, 4-OMe) and 40
(2,4-OMe) with NPC1L1 binding affinities of 4.8 and
>50 lM, respectively.
R3
R2
R1
OH
OH
F
O
N
O
Compound
R1
R2
R3
BBM
NPC1L1
Ki (lM)
Acute CAI
at 3 mg/kg
43
44
45
46
47
48
49
H
OMe
H
H
H
2.3
5.7
>50
19
ꢀ33%
NT
NT
OMe
H
H
OMe
H
H
F
0%
0%
H
H
F
18
F
H
Me
H
>50
16
0%
ꢀ19%
H
H
NT, not tested.
Limited examination of C-ring substitution was also
undertaken as shown in Table 2. Comparison of the 4-
F (12, Ki >50 lM), 3-F (41, Ki = 11 lM), and 2-F (42,
Ki >50 lM) substitution patterns revealed that 3-F sub-
stitution was preferred on this ring. Interestingly, 41 also
had very good acute in vivo efficacy (ꢀ69% at 3 mg/kg).
and had unexpectedly good acute efficacy (ꢀ55% at
3 mg/kg). Unfortunately, installation of the previously
optimized B- and C-ring substitution pattern to give
50 did not afford improved binding affinity. Addition-
ally, increasing the size of the R1 substituent on the A-
Ring (i.e., 51 and 52) resulted in loss of binding activity.
A parallel chemistry array scanning alternative A-ring
substitution (complete data not shown) identified the
3-OMe group as a preferred substituent on this ring as
illustrated by 53 (Ki = 15 lM) and 54 (Ki = 8.7 lM). De-
spite increased binding affinity however, both 53 and 54
had inferior acute efficacy relative to 18. Lastly, incorpo-
ration of a secondary amine into the linker (i.e., 21) did
not offer improved efficacy.
Table 3 illustrates the combination of the A- and B-ring
improvements described above along with further refine-
ment of the B-ring SAR. Analog 43, with the optimized B-
ring (2-OH, 4-OMe) and C-ring (3-F) combination, had
improved binding activity (Ki = 2.3 lM) relative to either
compound 39 or 41; however, despite increased affinity
for NPC1L1, 44 had only modest acute efficacy (ꢀ33%
at 3 mg/kg). Maintaining the B-ring 2-OH and examining
additional substitution including methoxy (44–45), fluo-
rine (46–48) or methyl (49) to the B-ring did not offer
improvements in binding affinity relative to 43.
A final set of modifications, as highlighted in Table 5, in-
volved installation of secondary and tertiary amides into
the linker unit. While 4-fluorobenzyl amide 24 had mod-
est affinity (Ki = 30 lM), evaluation of isomeric 29 re-
vealed that introduction of an amide functionality on
opposite side of the linker unit was not tolerated. Fol-
lowing up on amide 24, N-methylation to afford the cor-
responding tertiary amide 55 resulted in a 2-fold
increase in binding affinity (Ki = 14 lM) with modest
acute efficacy. Encouragingly, combination of this 4-flu-
orobenzyl-N-methyl tertiary amide with the previously
optimized B- and C-ring substitution patterns gave com-
pound 56 which had a 10-fold increase in binding affin-
ity (Ki = 1.4 lM). Disappointingly, despite this
improved binding affinity, 56 did not have in vivo effi-
cacy. As shown for 57, incorporation of the tertiary
amide into a ring system retained binding affinity
(Ki = 3.6 lM) and modest efficacy. Lastly, 58, bearing
(3-OMe)-benzyl-N-methyl amide, was found to have
activity similar to its 4-F counterpart 55. The fact that
these oxaolidinones with amide linkers such as 56 and
57 have low micromolar NPC1L1 binding affinity and
modest efficacy is encouraging given that to our knowl-
edge, such modifications have not been reported on the
extensively studied ezetimibe template. This suggests
Next, the introduction of heteroatoms into the linker
unit was examined as outlined in Table 4.22 Ether analog
18 (Ki = 28 lM) had somewhat improved binding affin-
ity relative to the all carbon comparator 12 (Ki > 50 lM)
Table 2. NPC1L1 binding and acute CAI efficacy of oxazolidinones 12
and 41–4219,20
OH
OH
O
N
R3
O
R1
R2
Compound
R1
R2
R3
BBM
NPC1L1
Acute CAI
at 3 mg/kg
Ki (lM)
12
41
42
H
H
F
H
F
F
>50
11
NT
ꢀ69%
NT
H
H
H
>50
NT, not tested.

J. A. Pfefferkorn et al. / Bioorg. Med. Chem. Lett. 18 (2008) 546–553
551
Table 4. NPC1L1 binding and acute CAI efficacy of oxazolidinones 18, 21, and 50–5419,20
R4
OH
R2
R1
X
R3
O
N
R5
O
Compound
R1
R2
X
R3
R4
R5
BBM NPC1L1 Ki (lM)
Acute CAI at 3 mg/kg
18
50
51
52
53
54
21
F
H
O
H
OH
4-F
3-F
4-F
4-F
4-F
3-F
4-F
28
28
ꢀ55%
0%
NT
F
H
O
OH
H
OMe
OH
OH
Cl
OMe
H
H
O
49
H
O
H
>50
15
NT
OMe
OMe
H
O
H
OH
OMe
OH
ꢀ13%
ꢀ26%
ꢀ5%
H
O
NH
OH
H
8.7
19
F
NT, not tested.
Table 5. NPC1L1 binding and acute CAI efficacy of oxazolidinones 24, 29, and 55–5719,20
R3
R1
R2
O
N
X
O
Compound
R1
R2
H
R3
X
BBM NPC1L1 Ki (lM)
Acute CAI at 3 mg/kg
NT
O
24
OH
4-F
4-F
30
N
H
F
F
O
29
H
OH
>50
NT
N
H
55
56
57
H
OH
4-F
3-F
3-F
14
ꢀ26%
0%
O
N
F
OH
OH
OMe
OMe
1.4
3.6
O
ꢀ21%
N
O
MeO
58
H
OH
4-F
11
ꢀ24%
N
NT, not tested.
that the novel substituent vectors provided by the cur-
rent oxazolidinone core may offer further additional
opportunity in this region of the template.
ing relationship between NPC1L1 binding and CAI effi-
cacy.6,8 Figure 4 highlights a retrospective analysis
wherein acute in vivo efficacy is plotted against NPC1L1
binding for analogs evaluated in both assays. While the
impact of pharmacokinetic differences amongst the ana-
logs cannot be discounted, binning analogs by color
In addition to analog optimization, the current studies
also offered an opportunity to evaluate the still develop-

552
J. A. Pfefferkorn et al. / Bioorg. Med. Chem. Lett. 18 (2008) 546–553
that molecule is glucuronidated. As shown in Table
0
-10
-20
-30
-40
-50
-60
-70
-80
-90
6, ezetimibe (1) had the shortest half-life (T1/
2 = 43 min) suggesting that it is very efficiently con-
verted to its glucuronide adduct. By this same mea-
sure, oxazolidinones 18 and 41 were converted to
their corresponding glucuronides more efficiently than
either 38 or 39. Interestingly, despite the fact that 39
(Ki = 4.8 lM) is 5-fold more potent than 18
(Ki = 28 lM), the latter is more efficacious in vivo,
perhaps due to its ability to be efficiently converted
to its glucuronide. Similarly, while 38 (Ki = 9.2 lM)
and 41 (Ki = 11 lM) have similar binding potencies,
41 is glucuronidated more efficiently as suggested by
its lower hepatocyte T1/2 (71 vs 136 min) perhaps
accounting for its somewhat greater in vivo efficacy.
B-Ring Substitution
4-phenol
Non 4-phenol
Ezetimibe (1)
0
5
10
15
20
25
30
NPC1L1 Ki (uM)
Figure 4. Relationship between acute efficacy and NPC1L1 affinity.
The suggested importance of glucuronidation to effi-
cacy in this oxazolidinone series is interesting and
may parallel the situation for ezetimibe (1). It is
known that rapid glucuronidation of ezetimibe
in vivo facilitates enterohepatic recirculation of the
drug thereby increasing its half-life and facilitating
localization (via bile delivery) of the equipotent glucu-
ronidated drug to its intestinal site of action. Addi-
tionally, one might propose that the formation of
equipotent glucuronide conjugates might protect the
ligands from oxidative metabolism that would other-
wise render the compounds inactive.23 While under-
standing the role of glucuronidation in the efficacy
of this oxazolidinone series will require additional
experimentation, the preliminary data suggest that fur-
ther structure–activity optimization may require atten-
tion to both NPC1L1 binding and glucuronidation
efficiency.
with regard to whether they contained (blue) or did not
contain (red) a 4-OH substituent on the B-ring revealed
an interesting trend. Specifically, among the analogs
tested, NPC1L1 binding affinity and the presence of a
4-OH substituted B-ring were necessary for significant
efficacy (>50% reduction at 3 mg/kg).
It is well established that the 4-phenol of ezetimibe is
rapidly glucuronidated in vivo and the resulting glu-
curonide adduct has activity comparable to the par-
ent molecule (vida supra).6 To understand how such
glucuronidation might relate to efficacy in the current
oxazolidinone series, we compared the metabolism of
ezetimibe and a representative set of oxazolidinone
analogs (Table 6: 18, 38, 39, and 41) in a rat hepa-
tocyte assay and performed metabolite identification
studies on each compound. For ezetimibe (1) and
oxazolidinones (18, 38, 41) all bearing a B-ring 4-
OH (Table 6, R2 = OH), the corresponding 4-O-b-glu-
curonide was the predominant if not the exclusive
metabolite. By contrast, 39, lacking a B-ring 4-OH
(Table 6, R2 = OMe), underwent oxidative metabo-
lism. For compounds where glucuronidation was the
We have described the optimization of a series of oxa-
zolidinone-based NPC1L1 ligands. These efforts re-
sulted in the identification of analogs with low
micromolar NPC1L1 affinity and acute in vivo effi-
cacy. The unique substituent vectors offered by the
oxazolidinone enabled the identification of novel mod-
ifications such as amide linkers. Finally, we highlighted
the impact that glucuronidation may have on analog
efficacy.
predominant metabolic pathway, the half-life (T1/2
)
of the parent molecule in the hepatocyte assay may
be taken as an estimate of the efficiency with which
Table 6. Comparison of relative rates of glucuronidation for 1, 18, and 38–39
R2
B
OH
X
R3
O
N
R1
R4
O
Compound
R1
X
R2
R3
R4
BBM NPC1L1
Ki (lM)
Acute CAI at
3 mg/kg
R2 = O-Gluc major
metabolite (%)
Rat hepatocyte
T1/2 (min)
1
—
F
—
O
—
—
H
—
0.20
28
ꢀ89
ꢀ55
ꢀ69
ꢀ55
ꢀ33
Yes
Yes
Yes
Yes
No
43
67
18
41
38
39
OH
OH
OH
OMe
4-F
3-F
4-F
4-F
H
H
H
CH2
CH2
CH2
H
11
71
OH
OH
9.2
4.8
136
249

J. A. Pfefferkorn et al. / Bioorg. Med. Chem. Lett. 18 (2008) 546–553
553
Kvaerno, L.; Ritter, T.; Werder, M.; Hauser, H.; Carreira,
E. M. Angew. Chem. Int. Ed. 2004, 43, 4653.
Acknowledgments
10. The fact that oxazolidinone 3 was active in vivo (Ref. 9b)
yet had no observable NPC1L1 binding affinity up to
50 lM may suggest the formation of an active metabolite
during the in vivo study.
We thank Jon Bordner and Ivan Samardjiev for assis-
tance with the X-ray structure in Scheme 1.
11. For precedent from ezetimibe SAR, see: McKittrick, B.
A.; Ke, M.; Dugar, S.; Clader, J. W.; Havis, H.;
Czarniecki, M.; McPhail, A. T. Bioorg. Med. Chem. Lett.
1996, 6, 1947.
12. Taylor, E. C.; Patel, H. H.; Jun, J.-G. J. Org. Chem. 1995,
60, 6684.
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20
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23. For example, evaluation of both ezetimibe (1) and its
glucuronide (2) in a rat liver microsomal stability assay
(lacking the cofactors required for glucuronidation)
revealed that the glucuronide conjugate was substantially
more resistant to oxidative metabolism 1, T1/2 = 9.5 min;
2, T1/2 > 60 min.