Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21 6153
Table 2. Gene Expression Activity of 15 and 16 and Selectivity in Gal4 Human Transactivation Assaysa
compd
ABCA1b
SREBP1cb
45 (106%)
Gal4 hLXRâb
Gal4 hLXRRb
Gal4 PPARRc
1312 ( 225
(19 ( 5%) (n ) 8)
1300 (71%)
Gal4 PPARγc
Gal4 PPARδc
15
55 (172%)
90 (63%)
240 (90%)
680 ( 230 (47 ( 11%)
(n ) 9)
320 (20%)
627 ( 365 (24 ( 5%)
(n ) 8)
1320 (37%)
16
33 (151%)
15 (73%)
87 (70%)
160 (82%)
a EC50 in nM, % eff.13-15 b Results are given as the mean of two independent experiments. The standard deviations for these assays were typically (50%
of mean or less. The % of efficacy is relative to 3. c Results are given as the mean of two independent experiments unless otherwise indicated. The % of
efficacy is relative to references: PPARR, [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid (WY-14643); PPARγ, 5-[[4-[2-(methyl-2-pyridinylami-
no)ethoxy] phenyl]methyl]-2,4-thiazolidinedione (Rosiglitazone); PPARδ, 3-chloro-4-[[3-[(3-phenyl-7-propyl-6-benzofuranyl)oxy]propyl]thio]-phenylacetic
acid (L-796449).
substituted quinoline, compound 15, was prepared. The removal
of the CdO greatly improved the stability of 15 in human liver
microsomes, giving an in vitro t1/2 of 107 min. Quinoline 15
maintained its potency in the binding assays as well as the
cellular assays. It showed a binding affinity of 2.1 nM against
hLXRâ, an EC50 of 71 nM with 97% efficacy in LAFâ, and an
EC50 of 90 nM with 63% efficacy in Gal4-DBD-hLXRâ LBD
transactivation assay.12a It also had potent affinity to LXRR
(binding IC50 ) 9.5 nM) and was nearly a full agonist in the
Gal4-DBD-hLXRR LBD transactivation assay (EC50 ) 240 nM,
90% efficacy). In addition, 15 increased expression of ABCA1
in THP-1 cells with an EC50 of 55 nM and an efficacy of 172%
relative to 3.12b It further stimulated cholesterol efflux in a
concentration-dependent manner in THP-1 macrophages pre-
incubated with acetylated LDL and [1,2-3H]cholesterol. An
estimated EC50 value of 20 nM was observed for 15 in these
lipid-laden cells resembling atherosclerotic foam cells.12b How-
ever, in the Huh7 liver cell line, 15 increased expression of
SREBP-1c with an EC50 value of 45 nM and an efficacy of
106% relative to 3, showing that this compound did not have
the desired gene selectivity.
cells, with LXRâ IC50 value of 115 nM and LAFâ EC50 value
of 374 nM.
Quinoline 15 and 16 were selective against a few closely
related nuclear receptors (data not shown). However, in human
Gal4-PPAR transactivation assays, both compounds were shown
to be PPAR active (Table 2), with an EC50 value of 0.3 µM or
higher.
After oral dosing at 10 mg/kg in male C57 mice, the
bioavailability of 16 was 67%, with a Cmax of 3.74 µg/mL,
AUC(0f∞) of 4.44 µg hr/mL, terminal t1/2 of 1.3 h and a Tmax of
0.5 h. An accelerated atherosclerotic lesion study17 was con-
ducted in high fat/high cholesterol (1.25%)-fed LDLr knockout
mice and administration of quinoline 16 for 8 weeks at 10 mg/
kg/day orally resulted in a significant reduction in lesion burden
by 45 ( 22% (n ) 8) compared to the control group. In the
same experiment, the literature standard 2 also significantly
reduced the lesion burden by 27 ( 7% (n ) 8). The details of
these studies will be reported in due course.
In summary, quinoline 16 has been identified using structure-
based design as a potent LXR agonist that upregulates ABCA1
binding cassette transporter in macrophage cells. The compound
also has good oral bioavailability in mice and displayed in vivo
efficacy in LDLr knockout mice for lesions. We are continuing
to investigate the SAR and biological properties of this new
series of LXR agonists.
An X-ray structure of the LBD domain of hLXRâ complexed
with 15 (Figure 3, resolution 2.15 Å)16 was subsequently solved,
and as predicted, the acid group did form a network of well-
ordered hydrogen bond interactions with the NH backbone of
Leu330 and Arg319. The quinoline nitrogen was involved in a
hydrogen bond interaction with His435, while the 3-benzyl
group protruded into the hydrophobic pocket formed by residues
Phe340, Phe349, and Phe271. The overall folding of this
structure is similar to those previously reported9,10 and will not
be described in detail here.
Acknowledgment. We thank the Wyeth Discovery Analytic
Chemistry Department for the analytical data. We also thank
Drs. Ron Magolda, Magid Abou-Gharbia, Steve Gardell, and
George Vlasuk for their support of this work.
1
Supporting Information Available: Experimental details, H
Further modifications on the linker region were undertaken,
and the benzylamine based quinoline 16 was found to have the
same LXR activity in the LXR binding and Gal4 transactivation
assays (LXRâ binding IC50 value of 1.9 nM, EC50 of 87 nM
with 70% efficacy in Gal4-DBD-hLXRâ assay) as 15. X-ray
structure of the LBD domain of LXRâ complexed with 16
showed the bound conformation of this ligand is identical to
that of 15, and the acid group makes similar hydrogen bond
interactions with the NH backbone of Leu330 and Arg319
residues (the X-ray structure of 16 is not shown). However, 16
showed a 2-fold increase in LAFâ with an EC50 value of 33
nM. Quinoline 16 increased expression of ABCA1 in THP-1
cells as well with an EC50 of 33 nM and an efficacy of 151%
relative to 3, which is slightly more potent than 15. It also
stimulated cholesterol efflux with an EC50 value of 37 nM.
However, in the liver cell line, 16 increased expression of
SREBP-1c with an EC50 value of 15 nM and an efficacy of
73% relative to 3, again demonstrating a lack of desired gene
selectivity. Compared to 16, the reversed benzylamine analogue
17 displayed a 3-fold reduction in binding affinity with LXRâ
IC50 value of 7 nM. The N-methylated derivative 18, however,
showed much weaker LXR activities in both binding and LAF
NMR, MS, and analytical data for all compounds. This material is
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