The discovery of tertiary-amine LXR agonists with potent cholesterol efflux activity in macrophages

Article abstract of DOI:10.1016/j.bmcl.2009.08.036

The liver X receptors (LXR) play a key role in cholesterol homeostasis and lipid metabolism. SAR studies around tertiary-amine lead molecule 2, an LXR full agonist, revealed that steric and conformational changes to the acetic acid and propanolamine groups produce dramatic effects on agonist efficacy and potency. The new analogs possess good functional activity, demonstrating the ability to upregulate LXR target genes, as well as promote cholesterol efflux in macrophages.

Full text of DOI:10.1016/j.bmcl.2009.08.036

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5617–5621
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The discovery of tertiary-amine LXR agonists with potent cholesterol efflux
activity in macrophages
a
a
a
a
a
Joseph P. Marino Jr. a, , Lara S. Kallander , Chun Ma , Hye-Ja Oh , Dennis Lee , Dimitri E. Gaitanopoulos ,
*
John A. Krawiec b, Derek J. Parks c, Christine L. Webb b, Kelly Ziegler b, Michael Jaye b, Scott K. Thompson a
a Chemistry, Metabolic Pathways Centre for Excellence in Drug Discovery, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406, United States
b Biology, Metabolic Pathways Centre for Excellence in Drug Discovery, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406, United States
c Biochemical and Cellular Targets, Molecular Discovery Research, GlaxoSmithKline, Research Triangle Park, NC 27709, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The liver X receptors (LXR) play a key role in cholesterol homeostasis and lipid metabolism. SAR studies
around tertiary-amine lead molecule 2, an LXR full agonist, revealed that steric and conformational
changes to the acetic acid and propanolamine groups produce dramatic effects on agonist efficacy and
potency. The new analogs possess good functional activity, demonstrating the ability to upregulate
LXR target genes, as well as promote cholesterol efflux in macrophages.
Received 15 June 2009
Revised 7 August 2009
Accepted 7 August 2009
Available online 13 August 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
LXR
Liver X receptor
HDL-c
LDL-c
VLDL-c
Triglycerides
Adenosine triphosphate binding cassette
ABC transporter
ABCA1
ABCG1
SREBP1c
Epoxycholesterol
Atherosclerosis
LXR modulator
Mammalian cells participate in a diverse set of functions, and
central to this is the regulation of cellular cholesterol, which in-
cludes biosynthesis, internalization, transport, and metabolism. It
is widely recognized that elevated systemic levels of low density
lipoprotein cholesterol (LDLc) and reduced levels of high density
lipoprotein cholesterol (HDLc) represent major risk factors for cor-
cassette) transporters A1 and G1 (which drive cellular cholesterol
efflux),4–6 A5 and G8 (which promote hepatocyte-biliary and
enterocyte excretion of cholesterol),7,8 adipocyte and macrophage
ApoE (promote cholesterol efflux),9 and cholesterol ester transfer
protein CETP (transfer of cholesterol from HDL to non-HDL lipopro-
teins).10 LXRs inhibit atherosclerosis through reciprocal regulation
of macrophage genes involved in cellular cholesterol efflux and
inflammation.11 Furthermore, the upregulation of ABC transporters
by LXR promotes the process of reverse cholesterol transport,
whereby cholesterol from peripheral tissue including atheroscle-
rotic plaque is transported back to the liver where it is metabolized
and excreted into the bile. Indeed treatment of mice with synthetic
ligand 2 (Fig. 1) promoted reverse cholesterol transport, as mea-
sured by transport of cholesterol from macrophages to feces.13e
Therefore LXR agonists represent an attractive approach for the pre-
vention and treatment of atherosclerosis.
onary heart disease. The liver X receptors (LXR
ar hormone receptor super family serve as oxysterol activated
transcription factors.1 LXR
is expressed in high levels in the liver,
a and b) of the nucle-
a
but is also prevalent in macrophages, adipose, gut, and kidney tis-
sue. LXRb is ubiquitously expressed, widely occurring in most tis-
sue. The LXR’s operate as heterodimers with retinoid X receptors
(RXR), which are activated by ligands of either receptor. The primary
role of LXRs is to control the expression of genes which code for key
enzymes and transporters responsible for cholesterol homeostasis
and lipid metabolism in response to intracellular cholesterol.2,3
LXR target proteins include ABC (adenosine triphosphate binding
Small molecule agonists that mimic the natural LXR steroid li-
gand, 24(S),25-epoxycholesterol 1,1,12 have been found to increase
expression of target genes, promote cholesterol efflux, and as a
* Corresponding author.
E-mail address: joseph.p.marino@gsk.com (J.P. Marino).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.08.036

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J. P. Marino et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5617–5621
ester, followed displacement by amine 4 (described in Scheme 1)
H
O
afforded secondary alcohol 9. Mitsunobu alkylation (inversion) of
either enantiomer of alcohol 9 with phenol 5, followed by ester
hydrolysis, provided target molecules 10a or 10b. Incorporation
of a methyl substituent at the 3-position of the propanolamine lin-
ker is described in Scheme 3. Mono-p-toluene-sulfonylation of
either (R) or (S)-1,3-butanediol, and subsequent alkylation by phe-
nol 5, yielded intermediate 12. Alcohol 12 was converted to the
corresponding p-toluenesulfonate, and alkylated (inversion) with
2,2-diphenylethylamine to afford secondary amine 13. Reductive
amination of 2-chloro-3-trifluoromethylbenzaldehyde with either
enantiomer of amine 13, followed by ester hydrolysis afforded tar-
get molecules 14a or 14b.
O
1
3
HO
O
N
2
HO
Cl
1
2
CF3
Figure 1. 24(S),25-Epoxycholesterol, 1, and tertiary-amine GSK lead 2.
result, elevate plasma lipids in multiple animal species.13,14 How-
ever, the upregulation of lipogenic genes and elevation of LDL-c ob-
served in CETP (cholesteryl ester transfer protein) species (hamster
and monkey) is a major obstacle for the development of current
LXR agonists such as 2 and 14b.13d
More severe conformational constraints on the propanolamine
linker of 2 were also examined. This was accomplished by con-
structing a ring between the ortho-position of the aryl group
(ether) and the ether methylene carbon. Based on this approach,
two benzofuran isomers were synthesized according to Scheme
4. Iodination of 3-hydroxy and 4-hydroxy- phenylacetic acid 15
provided the corresponding ortho-iodophenol intermediate.16
Sonogashira coupling followed by intramolecular cyclization affor-
ded benzofurans 16a and 16b. The alcohol 16a was converted to
the corresponding amine. The amine was then subjected to two
successive reductive aminations, and the resulting tertiary-
amine-ester was hydrolyzed to yield the target 5-benzofuran acetic
acid 17a. Alcohol 16b was first converted to the mesylate, and then
reacted with (2,2-diphenylethyl)amine to form a secondary amine.
Reductive amination of the secondary amine using 2-chloro-3-tri-
fluoromethylbenzaldehyde followed by ester hydrolysis provided
the target 6-benzofuran acetic acid 17b.
The impact of steric and conformational changes to the acetic
acid group of 2 was also studied. Substitution of the acetic acid
methylene group, and cyclization to the ortho-position of the aryl
group (ether) were examined. Mono-alkylation/dialkylation of 2,
and dimethylation of 14b was accomplished following Scheme 5
below. Treatment of methyl esters 18a and 18b (from 2 and 14b)
with LDA (or sodium hydride), followed by addition of excess
methyl-iodide or ethyl-iodide afforded the dimethyl ester interme-
diates. Hydrolysis using lithium chloride in DMF provided carbox-
ylic acids 19a, 19b, 19c, and 20.
Previously, GSK revealed the discovery of tertiary-amine 2, a no-
vel LXR agonist identified as a result of high through-put screening
and parallel array synthesis.14 Collins and co-workers demon-
strated that the tertiary-amine 2, was selective against a panel of
nuclear hormone and steroid receptors. When administered to
wild-type mice, compound 2 upregulated ABCA1 expression in
macrophages and in the intestine, and increased HDL-c levels.14
Further, 2, inhibited atherosclerosis in both apoEꢀ/ꢀ and LDLrꢀ/
ꢀ knockout mice models,13b as well as in double apoEꢀ/ꢀ &
LXRa
ꢀ/ꢀ knockout mice.15
Early lead optimization goals around tertiary-amine 2 focused
on improving functional activity as measured by greater upregula-
tion of LXR target genes, and enhanced cholesterol efflux in macro-
phages. Towards this end exploratory chemistry efforts around
tertiary-amine 2 were initiated in an attempt to probe the effects
of (1) substitution on the propanolamine linker, and (2) steric
and conformational constraints placed on the acetic acid moiety.
Initial SAR studies focused on the effects of methyl substitution
on the propanolamine linker of 2. The installation of a methyl
group at the 2-position of the propanolamine linker is described
in Scheme 1. Reductive amination of 2-chloro-3-trifluoromethyl-
benzaldehyde using 2,2,-diphenylethylamine afforded secondary
amine 4. Treatment of 3-hydroxy-phenyl acetic acid methyl ester
with (S)-(ꢀ)-3-bromo-2-methyl-1-propanol under Mitsunobu con-
ditions gave bromide 6. Alkylation of bromide 6 with amine 4, fol-
lowed by ester hydrolysis afforded 3-(R)-methyl-analog 7.24
Analogs with methyl substitution at the 1-position of the propa-
nolamine chain were synthesized according to the procedure
shown in Scheme 2. Conversion of the primary alcohol group of
(R) or (S)-1,3-butanediol to the corresponding p-toluene-sulfonate
Synthesis of constrained tetrahydronaphthylene carboxylic acid
25 was carried out from commercially available 5-hydroxy-tetra-
lone 23 (Scheme 6). The tetralone 23 was converted to 24 using
straight-forward methodology. Alkylation of phenol 24 with bro-
mide 22, and subsequent saponification afforded the naphthylene
ether 25.
O
i
HN
Cl
i,ii
Cl
HO
N
HO
OH
CF3
CF3
3
4
8
Cl
O
O
ii
CF3
O
OH
O
O
Br
9
6
5
O
O
iii,iv
iii, iv
HO
O
N
HO
O
N
Cl
Cl
CF3
CF3
10a; 2-(S)
10b; 2-(R)
7
Scheme 1. Reagents and conditions: (i) NaB(OAc)3H, (2,2-diphenylethyl)amine,
AcOH, CH2Cl2, 76%; (ii) (S)-(ꢀ)-3-Br-2-methyl-1-propanol, polymer-P(C6H5)3, DIAD,
toluene, 63%; (iii) amine 4, NaI, K2CO3, CH3CN, reflux, 14%; (iv) LiOH, 3:1 THF/H2O,
78%.
Scheme 2. Reagents and conditions: (i) TsCl, Et3N, CH2Cl2, 89–96%; (ii) amine 4,
NaI, K2CO3, CH3CN, reflux, 58–76%; (iii) phenol 5, polymer-P(C6H5)3, DIAD, toluene,
38–42%; (iv) LiOH, 3:1 THF/H2O, 98%.

J. P. Marino et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5617–5621
5619
O
i, ii
HO
OH
O
O
OH
i
11
HN
Cl
12
Br
N
Cl
22
CF3
CF3
OH
O
iii, iv
21
O
O
N
H
O
ii-vii
13
O
OH
O
23
24
O
v, vi
HO
O
N
O
viii-ix
HO
O
N
Cl
CF3
14a; 2-(S)
14b; 2-(R)
Cl
25
CF3
Scheme 3. Reagents and conditions: (i) TsCl, Et3N, CH2Cl2, 82–89%; (ii) phenol 5,
Cs2CO3, DMF, reflux, 37–68%; (iii) TsCl, Et3N, CH2Cl2, 63–67%; (iv) (2,2-diphenyl-
ethyl)amine, K2CO3, CH3CN, reflux, 54–63%; (v) NaB(OAc)3H, 2-Cl–3CF3–benzalde-
hyde, AcOH, CH2Cl2, 44–64%; (vi) LiOH, 3:1 THF/H2O, 89–98%.
Scheme 6. Reagents and conditions: (i) 1,3-Br-propane, K2CO3, CH3CN, reflux, 70%;
(ii) benzylbromide, K2CO3, CH3CN, 88%; (iii) (PPh3)3PCH2OMe, t-Bu-OꢀK+, 1,4-
dioxane, 23%; (iv) p-TsOH, H2O, 1,4-dioxane, 58%; (v) CrO3, H2SO4, H2O, acetone,
57%; (vi) H2SO4, MeOH, 85%; (vii) H2, 10% Pd/C, EtOAc, 97%; (viii) bromide 22, NaI,
K2CO3, acetone, 95%; (ix) LiCl, DMF, 15%.
A cell-free ligand sensing assay based on fluorescence reso-
nance energy transfer (FRET) was used as a primary screen to
determine LXR activity.14,17 Ligand dependent recruitment of a
24-amino acid fragment of the steroid receptor coactivator 1
(SRC1) to the LXR binding domain was measured. Percent efficacy
was also determined to compare efficacy relative to the maximal
agonist response observed for compound 2 (previously determined
to be similar to endogenous ligand, 24(S),25-epoxycholesterol14).
i (16a)
or ii (16b), iii
5
6
O
OH
OH
RO2C
MeO2C
16a; 5-CO2Me
16b; 6-CO2Me
15
O
O
iv - ix
All analogs showed no selectivity preference for LXRa versus LXRb,
and most compounds demonstrated comparable agonism or effi-
O
OH
O
OH
O
N
16a
cacy between the two receptor subtypes.
17a
Cl
In the methyl-propanolamine linker series, methyl substitution
at the 2-position, exemplified by compound 7, maintained LXR po-
tency however agonist efficacy dropped by 30% relative to lead 2.
Substitution at the 1-position of the linker, adjacent to oxygen, re-
sulted in a slight decrease in potency for the S-enantiomer (10a)
and a partial agonist response (39–59%) relative to lead 2. In con-
trast to 2, the R-enantiomer (10b) displayed comparable potency
and almost a full agonist response. Placement of the methyl at
the 3-position of the linker led to similar activity to lead 2 in the
case of the S-enantiomer 14a, however a slight improvement in po-
tency was observed with R-enantiomer 14b. Both stereoisomers
exhibited comparable agonism to the lead 2. The benzofurans
17a and 17b displayed partial agonist activity, and a small decrease
in activity relative to the lead 2. Given these observations it ap-
pears the flexibility of the linker group plays a key role in agonist
response. In addition, methyl substitutions on the linker were also
discovered to improve PXR selectivity21 over lead 2, and thus may
be preferred in order to avoid blockade of PXR-mediated xenobi-
otic metabolism.22 Substitution on the acetic acid moiety had a sig-
nificant impact on potency. Mono-methyl and dimethyl analogs
19a, 19b, and 20 demonstrated a 5–10-fold improvement in po-
tency, with all three exhibiting full agonist activity compared to
the lead 2. This SAR is consistent with structural observations
which can be made from the published co-crystal structure of LXRb
and 2, suggesting improved affinity may be gained from more
favorable hydrophobic contacts in the pocket where the methylene
group resides.20 However this pocket is finite in terms of the size of
group which may be accommodated given the loss in potency and
efficacy observed for diethyl analog 19c relative to its methylated
counterparts. The constrained acetic acid, tetrahydroquinoline 25
(racemic), was comparable in potency and agonism to the lead 2.
CF3
O
O
x - xiii
O
OH
O
O
N
HO
16b
17b
Cl
CF3
Scheme 4. Reagents and conditions: (i) R = Me; chloroamine-T, NaI, DMF, 54%; (ii)
R = H; (a) MeOH, cat. H2SO4, 100%; (b) I2, KI, aq NH2OH, 32%; (iii) 3-butyne-1-ol, CuI,
Pd(PPh3)2Cl2, PPh3, 3:1 toluene/Et3N; (iv) MsCl, Et3N, CH2Cl2, 54–78%; (v) NaN3,
DMF, 75%; (vi) H2, 10% Pd/C, MeOH; (vii) diphenylacetaldehyde, p-TsOH, NaBH4,
MeOH, 22% for two steps; (viii) 2-Cl–3-CF3–benzaldehyde, NaB(OAc)3H, AcOH,
CH2Cl2, 100%; (ix) LiOH, 3:1 THF/H2O, 43%; (x) MsCl, Et3N, CH2Cl2, 100%; (xi) (2,2-
diphenylethyl)amine, K2CO3, CH3CN, reflux, 50%; (xii) 2-Cl–3-CF3–benzaldehyde,
NaB(OAc)3H, AcOH, CH2Cl2, 74%; (xiii) LiOH, 3:1 THF/H2O, 65%.
R3
R3
O
O
i,ii
HO
O
N
O
O
N
R1 R2
R1
R2
Cl
Cl
CF3
CF3
19a; R1,R2 = Me; R3 = H
19b; R1 = Me; R2,R3 = H
19c; R1,R2 = Et; R3 = H
20; R1,R2,R3 = Me
18a; R1,R2,R3 = H
18b; R1,R2 = H, R3 = Me
Scheme 5. Reagents and conditions: (i) LDA or NaH, MeI or EtI, THF, 2–42%
(variable); (ii) LiCl, DMF, ꢁ20%.

5620
J. P. Marino et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5617–5621
Table 3
The new tertiary-amine analogs were also evaluated in a func-
In vivo pharmacokinetic data for compound 14b
tional cellular assay to gauge their effectiveness in promoting cho-
lesterol efflux.18,19 This experiment measures a compounds ability
to efflux 3H-cholesterol from mouse RAW 264.7 macrophage cells.
All compounds in Table 1 demonstrated cholesterol efflux activity
comparable or superior to the potency observed in the FRET assay.
Similar to the trend observed in the FRET assay, analogs 2, 14b,
19a, 19b, 20, and 25 displayed the greatest potency in the efflux as-
say, exhibiting a 2–10-fold increase in potency relative to lead 2.
As further confirmation that the cholesterol efflux effects ob-
served in mouse macrophages was due to LXR activity, real-time
PCR gene expression studies18 in primary human macrophages
were conducted on 2 and three new analogs, 14b, 19a, and 25 (Ta-
ble 2). All compounds increased expression of target genes for the
ABCA1 and ABCG1 transporters which play integral roles in macro-
phage cholesterol efflux, in a concentration-dependent manner.
However, expression of SREBP1c, a lipogenic target gene believed
to play a role in triglyceride synthesis, was also increased. It is
important to note that the cholesterol efflux assay discussed earlier
measures cholesterol efflux to the apolipoprotein ApoA-I mediated
by ABCA1, however ABCG1 mediates cholesterol efflux to HDL par-
ticles rather than apoA-I.5 Since 19a produced the greatest effect
on ABCG1 expression, it will be of interest to examine the activity
of this compound in cholesterol efflux assays to HDL particles.
Compound 14b was found to exhibit excellent in vivo PK char-
acteristics in the rat such as a reasonable half-life, low clearance,
and good oral bioavailability (Table 3). Consistent with the
in vitro gene expression studies in macrophages discussed above,
14b (also known as SB742881) has previously demonstrated ro-
bust effects on LXR target genes in the hamster.13d Once again in-
creases in ABCA1, ABCG1, and SREBP1c gene expression was
observed in tissue and macrophages taken from these animals,
however 14b to failed to produce significant increases in plasma
HDL-c in the hamster. In fact, significant increases in plasma tri-
glycerides and VLDL-c were observed, which is also consistent with
14b
Parametera
Dose (mg/kg), iv
Clp (ml/min/kg)
Vdss (L/kg)
2.6
23.7 2.5
1.9 0.2
109 15
2126 152
4.5
T1/2 (min), iv
Cmax (ng/mL), iv
Dose (mg/kg), po
T1/2 (min), po
Oral F (%)
144 39
71 10
a
Sprague-dawley rats (n = 3) were used in this experiment.
the increased expression of the lipogenic gene SREBP1c.13d Moving
forward these data suggest that an LXR modulator capable of tar-
geting selective genes (ex. ABCA1 vs SREBP1c) is essential in order
to achieve the desirable effects on HDL-c without the undesirable
effects on triglycerides. Although unproven, it has been postulated
that modulation could be achieved through the differential recruit-
ment of LXR transcription cofactor peptides.13d,23
In summary, substitutions which impart conformational
changes around the linker group of tertiary-amine lead 2 had a sig-
nificant effect on LXR agonist efficacy but not potency. Interest-
ingly, conformational and/or hydrophobic effects imparted by
acetic acid gem-dimethyl substitution had a profound effect on
LXR potency. Several compounds also demonstrated potent choles-
terol efflux activity in macrophages and robust effects on LXR tar-
get gene expression as confirmation of functional LXR agonist
activity in cells. Analogs such as 14b and 19a represent potent
molecular tools for benchmarking the potential therapeutic bene-
fits as well as liabilities of LXRa/b full agonists.
References and notes
1. (a) Lehmann, J. M.; Kliewer, S. A.; Moore, L. B.; Smith-Oliver, T. A.; Oliver, B. B.;
Su, J. L.; Sundseth, S. S.; Winegar, D. A.; Blanchard, D. E.; Spencer, T. A.; Willson,
T. M. J. Biol. Chem. 1997, 272, 3137; (b) Janowski, B. A.; Willy, P. J.; Devi, T. R.;
Falck, J. R.; Mangelsdorf, D. J. Nature 1996, 383, 728.
Table 1
LXR activity and cell assay results for compounds 2, 7, 10, 14, 17, 19, 20 and 25
2. Repa, J. J.; Mangelsdorf, D. J. Nat. Med. 2002, 8, 1243.
Compd LXR
a
%
LXRb
%
Cholesterol efflux,
a
a
b
3. Edwards, P. A.; Kast, H. R.; Anisfeld, A. M. J. Lipid Res. 2002, 43, 2.
4. Venkateswaran, A.; Laffitte, B. A.; Joseph, S. B.; Mak, P. A.; Wilpitz, D. C.;
Edwards, P. A.; Tontonoz, P. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 12097.
5. Wang, N.; Lan, D.; Chen, W.; Matsuura, F.; Tall, A. R. Proc. Natl. Acad. Sci. U.S.A.
2004, 9774.
6. Nakamura, K.; Kennedy, M. A.; Baldan, A.; Bojanic, D. D.; Lyons, K.; Edwards, P.
A. J. Biol. Chem. 2004, 279, 45980.
7. Berge, K. E.; Tian, H.; Graf, G. A.; Yu, L.; Grishin, N. V.; Schultz, J.; Kwiterovich,
P.; Shan, B.; Barnes, R.; Hobbs, H. H. Science 2000, 290, 1771.
8. Yu, L.; Hammer, R. E.; Li-Hawkins, J.; Von Bergmann, K.; Lutjohann, D.; Cohen, J.
C.; Hobbs, H. H. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 16237.
9. Laffitte, B. A.; Repa, J. J.; Joseph, S. B.; Wilpitz, D. C.; Kast, H. R.; Mangelsdorf, D.
J.; Tontonoz, P. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 507.
10. Luo, Y.; Tall, A. R. J. Clin. Invest. 2000, 105, 513.
11. Joseph, S. B.; Castrillo, A.; Laffitte, B. A.; Mangelsdorf, D. J.; Tontonoz, P. Nat
Med. 2003, 9, 213.
EC50 (nM) Efficacy EC50 (nM) Efficacy EC50 (nM)
2
7
200
280
365
200
211
74
305
331
43
100
67
39
78
91
100
20
53
100
100
69
100
100
40
86
145
41
87
25
145
63
15
15
174
11
100
70
59
76
80
89
26
75
95
90
68
100
100
29
76
NT
34
54
17
NT
NT
7
15
NT
<3
18
10a
10b
14a
14b
17a
17b
19a
19b
19c
20
46
380
9
25
56
26
12. Janowski, B. A.; Willy, P. J.; Falck, J. R.; Mangelsdorf, D. J. Nature 1997, 383, 728.
13. (a) Schultz, J. R.; Tu, H.; Luk, A.; Repa, J. J.; Medina, J. C.; Li, L.; Schwendner, S.;
Wang, S.; Thoolen, M.; Mangelsdorf, D. J.; Lustig, K. D.; Shan, B. Gene Dev. 2000,
14, 2831; (b) Joseph, S. B.; Mckilligin, L.; Pei, M. A.; Watson, A. R.; Collins, B. A.;
Laffitte, M.; Chen, G.; Noh, J.; Goodman, G. N.; Hagger, G. N.; Tran, J.; Tippin, T.
K.; Wang, X.; Lusis, A. J.; Hsueh, W. A.; Law, R. E.; Collins, J. L.; Willson, T. M.;
Tontonoz, P. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 7604; (c) Terasaka, N.;
Hiroshima, A.; Koieyama, T.; Ubukata, N.; Morikawa, Y.; Nakai, D.; Inaba, T.
FEBS Lett. 2003, 536, 6; (d) Groot, P. H. E.; Pearce, N. J.; Yates, J. W.; Stocke, C.;
Sauermelch, C.; Doe, C. P.; Willette, R. N.; Olzinski, A.; Peters, T.; d’Epagnier, D.;
Morasco, K. O.; Krawiec, J. A.; Webb, C. L.; Aravindhan, K.; Jucker, B.; Burgert,
M.; Ma, C.; Marino, J. P.; Collins, J. L.; Macphee, C. H.; Thompson, S. K.; Jaye, M. J.
Lipid Res. 2005, 46, 2182; (e) Naik, S. U.; Wang, X.; Da Silva, J. S.; Jaye, M.;
Macphee, C. H.; Reilly, M. P.; Billheimer, J. T.; Rothblat, G. H.; Rader, D. J.
Circulation 2006, 113, 90.
a
Values are means of at least two experiments, standard deviation 610%.
b
Mouse 264.7 macrophages; (NT = not tested).
Table 2
Gene expression data from macrophagesa for 2, 14b, 19a, and 25
Compd Fold " in
Fold " in
Fold " in
Fold " in
Fold " in
ABCA1b
ABCA1b
ABCG1b
ABCG1b
SREBP1cb
(@ 30 nM) (@ 100 nM) (@ 30 nM) (@ 100 nM) (@ 100 nM)
2
2.0
3.6
3.0
1.4
3.1
6.3
4.0
2.6
3.2
4.2
7.5
2.0
5.4
8.0
11.2
3.6
4.1
5.5
5.8
2.6
14b
19a
25
14. (a) Collins, J. L.; Fivush, A. M.; Watson, M. A.; Galardi, C. M.; Lewis, M. C.; Moore,
L. B.; Parks, D. J.; Wilson, J. G.; Tippin, T. K.; Binz, J. G.; Plunket, K. D.; Morgan, D.
G.; Beaudet, E. J.; Whitney, K. D.; Kliewer, S. A.; Willson, T. M. J. Med. Chem.
2002, 45, 1963; (b) Ratni, H.; Blum-Kaelin, D.; Dehmlow, H.; Hartman, P.;
a
Primary human macrophages.
Values represent a single experiment.
b

J. P. Marino et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5617–5621
5621
Jablonski, P.; Masciadri, R.; Maugeais, C.; Patiny-Adam, A.; Panday, N.; Wright,
M. Bioorg. Med. Chem. Lett. 2009, 19, 1654.
15. Bradley, M. N.; Hong, C.; Chen, M.; Joseph, S. B.; Wilpitz, D. C.; Wang, X.; Lusis,
A. J.; Collins, A.; Hseuh, W. A.; Collins, J. L.; Tangirala, R. K.; Tontonoz, P. J. Clin.
Invest. 2007, 117, 2337.
minutes. The reaction mixture was then cooled to 0 °C and
diisopropylazodicarboxylate (1.1 g, 0.00560 mol) was added in a dropwise
fashion. After stirringat room temperature overnight, the crude reaction mixture
wasfiltered, and thesolidwashedwith toluene. After concentrationofthefiltrate
in vacuo, the crude product was purified by column chromatography over silica
gel (silica gel 60, EM Science) using 15% EtOAc/hexane as eluent to afford 0.86 g
(63% yield) of the title compound as an oil: 1H NMR (400 MHz, CDCl3): d 7.23 (m,
1H), 6.87 (m, 3H), 3.92 (d, 2H, J = 6.0 Hz), 3.72 (s, 3H), 3.62 (s, 2H), 3.59 (d, 2H,
J = 5.4 Hz), 2.33 (m, 1H), and 1.17 (d, 3H, J = 6.8 Hz); MS (ESI) 303.0 (M+2H+).
(c) N-(2,2-Diphenylethyl)-N-(2-chloro-trifluoromethylbenzyl)amine: To a stirring
solution of 2,2-diphenethylamine (2.0 g, 0.010 mol) and 2-chloro-3-
trifluoromethylbenzaldehyde (2.33 g, 0.011 mol) in dichloromethane
(20 mL) was added sodium triacetoxyboro hydride (2.36 g, 0.011 mol) and
acetic acid (2.0 mL). The reaction mixture was stirred overnight. Solvent was
removed, the residue was washed with saturated NaHCO3, and extracted
three times with EtOAc. The organic extracts were dried over Na2SO4,
filtered, and concentrated. The crude mixture was subjected to column
chromatography over silica gel (Silica Gel 60, EM Science) using 30% EtOAc/
16. Kometani, T.; Watt, D. S.; Ji, T. Tetrahedron Lett. 1985, 26, 2043.
17. Spencer, T. A.; Li, D.; Russel, J. S.; Collins, J. L.; Bledsoe, R. K.; Consler, T. G.;
Moore, L. B.; Galardi, C. M.; McKee, D. D.; Moore, J. T.; Watson, M. A.; Parks, D.
J.; Lambert, M. H.; Willson, T. M. J. Med. Chem. 2001, 44, 886.
18. Cholesterol efflux measurement: 3H-cholesterol was incorporated into
acetylated LDL (acLDL, Biomedical Technologies, Stoughton, MA) as described
(Chen, W.; Sun, Y.; Welch, C.; Gorelik, A.; Leventhal, A. R.; Tabas, A.; Tall, A. R. J.
Biol. Chem. 2001, 276, 43564). Following overnight growth of macrophages in
96 well plates, 50
concentrations 50 g/mL acLDL, 5
loading for 24 h, wells were washed with PBS, placed in 100
l
L
of this mixture was added to each well (final
Ci 3H-cholesterol/mL). After cholesterol
L regular growth
l
l
l
media minus phenol red and serum, containing 1% fatty acid-free BSA, and
compounds in DMSO solution added as desired. After 24 h, wells were washed,
100
l
L media (as above) containing 0.1% fatty acid-free BSA and 5
l
g/mL
hexane as eluent to afford 3.0 g (76% yield) of the title compound as
a
apolipoprotein A-I (Intracel, MD USA), drug treatments replenished and cells
were incubated for an additional 24 h. To determine the 3H-cholesterol
effluxed to apoA-I, cellular debris and detached cells were filtered from the
conditioned media with a 96 well multiscreen vacuum manifold (Millipore
Corp., Bedford, MA) into opaque white 96 well plates (Packard). Cells were
lysed in 50 lL 0.1 M NaOH with gentle shaking. After addition of Microscint-20
scintillant (Packard), plates were sealed, shaken overnight, and radioactivity
quantitated in a TopCount instrument (Packard).
yellow oil: MS (ESI) 390.0 (M+H+).
(d) (R)-2-(3-{3-[[2-Chloro-3-(trifluoromethyl)benzyl](2,2-diphenylethyl)amino]-
2-methyl-propoxy}-phenyl)acetic acid methyl ester: To a stirring solution of
(S)-[3-(2-methyl-3-bromopropoxy)phenyl]acetic acid methyl ester (100 mg,
0.33 mmol) and N-(2,2-diphenylethyl)-N-(2-chloro-3-trifluoromethyl)amine
(130 mg, 0.33 mmol) in acetonitrile (5 mL) was added solid K2CO3
(138 mg, 1.0 mmol) and NaI (149 mg, 1.0 mmol). The reaction was heated
to reflux and stirred overnight. Upon cooling to room temperature, the
reaction was filtered, washed with acetonitrile, and the filtrate was
concentrated. The crude product was purified by preparative HPLC (TMC
CombiPrep PDS, 75 ꢂ 30 mm, 25 mL/min, acetonitrile/H2O, UV detection at
254 nm) to give 29 mg (14% yield) of title compound as a viscous oil. 1H
NMR (400 MHz, CDCl3): d 7.52 (d, 1H, J = 7.6 Hz), 7.30 (d, 1H, J = 7.2 Hz),
7.24–7.13 (m, 11H), 6.96 (m, 1H), 6.84 (d, 1H, J = 8 Hz), 6.61–6.57 (m, 2H),
4.11 (m, 1H), 3.92 (d, 1H, J = 14.8 Hz), 3.76 (d, 1H, J = 14.8 Hz), 3.70 (s, 3H),
3.62 (s, 2H), 3.57 (m, 1H), 3.51 (m, 1H), 3.32 (m, 1H), 3.02 (m, 1H), 2.63 (m,
1H), 2.37 (m, 1H), 2.10 (m, 1H), 0.94 (d, 3H, J = 6.8 Hz); MS(ESI) 610.2 (M+).
(e) (R)-2-(3-{3-[[2-Chloro-3-(trifluoromethyl)benzyl](2,2-diphenylethyl)amino]-
2-methyl-propoxy}-phenyl)acetic acid hydrochloride salt: A stirring solution of
(R)-2-(3-{3-[[2-chloro-3-(trifluoromethyl)benzyl](2,2-diphenylethyl)amino]-
2-methyl-propoxy}-phenyl)acetic acid methyl ester (22 mg, 0.0361 mmol) in
THF (0.75 mL) and water (0.25 mL) was treated with LiOH (3.0 mg, 0.072 mmol).
The reaction mixture was stirred overnight at rt. The reaction mixture was
concentrated and 3 N HCl (aq) was added until the pH was less than two. The
aqueous layer was extracted three times with EtOAc, the combined organic
layers were dried over sodium sulfate, filtered, and concentrated. The resulting
amine/carboxylic acid was dissolved in Et2O (diethylether) and acidified with
1.0 M HCl/ Et2O. The reaction mixture was concentrated in vacuo and dried
under reduced pressure to give 18 mg (78% yield) of the title compound as a
white solid: 1H NMR (400 MHz, MeOD): d 7.51 (d, 1H, J = 7.8 Hz), 7.30 (d, 1H,
7.1 Hz), 7.14–7.27 (m, 11 H), 6.96 (t, 1H, J = 7.7 Hz), 6.85 (d, 1H, J = 7.6 Hz), 6.61
(m, 1H), 6.57 (m, 1H), 4.19 (dd, 1H, J = 5.7 and 9.7 Hz), 3.92 (d, 1H, J = 14.9 Hz),
3.76 (d, 1H, J = 14.9 Hz), 3.58 (s, 2H), 3.56 (m, 1H), 3.48 (dd, 1H, J = 5.2 and
8.8 Hz), 3.33 (m, 1H), 3.01 (dd, 1H, J = 5.8 and 12.9 Hz), 2.64 (dd, 1H, J = 9.8 and
12.8 Hz), 2.35 (dd, 1H, J = 4.9 and 12.8 Hz), 2.02 (m, 1H), 0.94 (d, 3H, J = 6.8 Hz);
(MS(ESI) 596.0 (M+). Anal. Calcd for C34H33ClF3NO3ꢃ0.1EtOAc: C, 68.31; H, 5.63;
N, 2.32. Found: C, 67.96; H, 5.46; N, 2.37.
19. Isolation of human macrophages and Taqman studies are described in:
Johnston, T. P.; Jaye, M.; Webb, C. L.; Krawiec, J. A.; Alom-Ruiz, S. P.; Sachs-
Barrable, C.; Wasan, K. M. Eur. J. Pharm. 2006, 536, 232.
20. Fänegårdh, M.; Bonn, T.; Sun, S.; Ljunggren, J.; Ahola, H.; Wilhelmsson, J.;
Carlquist, M. J. Biol. Chem. 2003, 278, 38821.
21. Lead 2 exhibits PXR activity (Ki = 2
lM) however examples 10b and 14b did not
activate PXR (Ki >10 M). For references describing the PXR scintillation
l
proximity assay see: Jones, S. A.; Moore, L. B.; Shenk, J. L.; Wisely, G. B.;
Hamilton, G. A.; McKee, D. D.; Tomkinson, N. C. O.; LeCluyse, E. L.; Lambert, M.
H.; Willson, T. M.; Kliewer, S. A.; Moore, J. T. Mol. Endocrinol. 2000, 14, 27–39)
and WO 2000025134A1.
22. Moore, J. T.; Willson, T. M.; Kliewer, S. A. Comp. Toxicol. 2002, 159.
23. Miao, B.; Zondlo, S.; Gibbs, S.; Cromley, D.; Hosagrahara, V. P.; Kirchgessner, T.
G.; Billheimer, J.; Mukerjee, R. J. Lipid Res. 2004, 45, 1410.
24. Preparation of example 7: (R)-2-(3-{3-[[2-Chloro-3-(trifluoromethyl)benzyl]-
(2,2-diphenylethyl)amino]-2-methyl-propoxy}-phenyl)acetic acid
(a) (3-Hydroxy-phenyl)-acetic acid methyl ester: To a stirring solution of (3-
hydroxy-phenyl)-acetic acid (4.3 g, 0.028 mol) in methanol (30 mL) was added
H2SO4 (1 mL) and the mixture was heated to reflux for 2 h. The solvent was
removed, the residue was washed with H2O, and extracted three times with
EtOAc (ethyl acetate). The combined organic layers were dried over Na2SO4,
filtered, and concentrated to give 4.7 g (99% yield) of the title compound as an oil.
1H NMR (400 MHz, CDCl3): d 7.21 (1H, t, J = 7.8 Hz), 6.86 (1H, d, J = 7.6 Hz), 6.76
(m, 2H), 4.90 (1H, br s), 3.72 (3H, s), and 3.60 (s, 2H); MS (ESI) 167.0 (M+H+).
(b) (S)-[3-(2-Methyl-3-bromopropoxy)phenyl]acetic acid methyl ester: To a stirring
solution of (3-hydroxy-phenyl) acetic acid methyl ester (0.75 g, 0.0045 mol) in
anhydrous toluene (30 mL) was added (S)-(+)-3-bromo-2-methyl-1-propanol
(0.90 g, 0.0059 mol). Polymer bound triphenylphosphine (2.4 g, 0.0072 mol,
3 mmol/g, Fluka Chemie) was then added, and the mixture was stirred for 15