Carboxylic acid based quinolines as liver X receptor modulators that have LXRβ receptor binding selectivity

Article abstract of DOI:10.1016/j.bmcl.2007.11.013

A series of potent and binding selective LXRβ agonists was developed using the previously reported non-selective LXR ligand WAY-254011 as a structural template. With the aid of molecular modeling, it was found that 2,3-diMe-Ph, 2,5-diMe-Ph, and naphthalene substituted quinoline acetic acids (such as quinoline 33, 37, and 38) showed selectivity for LXRβ over LXRα in binding assays.

Full text of DOI:10.1016/j.bmcl.2007.11.013

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 54–59
Carboxylic acid based quinolines as liver X receptor
modulators that have LXRb receptor binding selectivity
Baihua Hu,a,* Elaine Quinet,b Rayomand Unwalla,a Mike Collini,a James Jetter,a
Rebecca Dooley,a Diane Andraka,a Lisa Nogle,a Dawn Savio,b Anita Halpern,b
Annika Goos-Nilsson,c Anna Wilhelmsson,c Ponnal Nambib and Jay Wrobela
aChemical and Screening Sciences, Wyeth Pharmaceuticals, Collegeville, PA 19426, USA
bCardiovascular and Metabolic Diseases, Wyeth Pharmaceuticals, Collegeville, PA 19426, USA
cKaro-Bio, Huddinge, Sweden
Received 11 October 2007; accepted 6 November 2007
Available online 9 November 2007
Abstract—A series of potent and binding selective LXRb agonists was developed using the previously reported non-selective LXR
ligand WAY-254011 as a structural template. With the aid of molecular modeling, it was found that 2,3-diMe-Ph, 2,5-diMe-Ph, and
naphthalene substituted quinoline acetic acids (such as quinoline 33, 37, and 38) showed selectivity for LXRb over LXRa in binding
assays.
Ó 2007 Elsevier Ltd. All rights reserved.
Liver X receptors (LXRa and LXRb) are members of
the nuclear hormone receptor superfamily and are in-
volved in the regulation of cholesterol and lipid metab-
olism.1 They are also recently reported to be glucose
sensors involved in liver carbohydrate metabolism.2
LXRs are ligand-activated transcription factors and
bind to DNA as obligate heterodimers with retinoid X
receptors. In macrophages, liver, and intestine, activa-
tion of LXRs induces the expression of several genes in-
volved in lipid metabolism and reverse cholesterol
transport including ATP binding cassette transporter
A1 (ABCA1), ABCG1, and apolipoprotein E (ApoE).
The potential to prevent or even reverse atherosclerotic
process by increasing the expression of these genes
makes LXR an attractive drug target in the treatment
of atherosclerosis, which is one of the leading health
concerns in the United States.3 Several LXR agonists
(Fig. 1), such as a natural ligand 24(S), 25-epoxycholes-
terol (1, EPC)4 as well as two structurally distinct syn-
cholesterol transport including ABCA1, ABCG1, and
ApoE. These compounds reduced or even reversed ath-
erosclerotic processes in mouse models of atherosclero-
sis. Currently available synthetic LXR agonists,
however, also activated triglyceride (TG) synthesis in
the liver by the upregulation of sterol regulatory element
binding protein 1c (SREBP-1c) and fatty acid synthase
(FAS) which limits the utility of these LXR synthetic
agonists. Since the liver contains predominantly LXRa,
LXRb-specific agonists or tissue-specific LXR modula-
tors may have less impact on TG synthesis, but may
be effective in macrophage reverse cholesterol transport.
Thus, our new efforts were focused on the identification
of LXRb selective modulators. The concept of LXR
subtype selective agonists suitable for pharmacologic
evaluation has been supported by a number of LXR
knockout (KO) animal studies.1 Treatment of LXRa
KO mice with a potent and highly selective LXRa ago-
nist did not have any effect on plasma TG, liver choles-
terol, liver TG, whereas in wild-type mice an increase in
liver TG was noted, indicating that LXRa is the subtype
controlling hepatic SREBP-1c transcription.7 In con-
trast, in peripheral tissues from these LXRa KO mice,
LXRb activation increased ABCA1 gene expression.8
Therefore, selective LXRb activation is expected to
stimulate ABCA1 expression in macrophages, while
having no or little effort on hepatic LXRa-dominated
lipogenesis. A recent study has shown that ligand
thetic non-steroidal ligands
2 3
(GW3965)5 and
(TO901317),6 have been shown to increase expression
of several genes involved in lipid metabolism and reverse
Keywords: LXR agonists; Selectivity in binding assays; Quinolines;
Carboxylic acids.
*
Corresponding author. Tel.: +1 484 865 7867; fax: +1 484 865
9399; e-mail: hub@wyeth.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.013

B. Hu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 54–59
55
H
O
CO2H
O
N
Cl
HO
CF3
1
2
CO2H
O
O
S
F3C
F3C
HO
O
N
N
F3C
CF3
4 (WAY-254011)
3
Figure 1. Reported LXR agonists.
activation of LXRb reversed atherosclerosis and cellular
cholesterol overload in mice lacking LXRa and ApoE.9
This observation provided a strong in vivo support for
LXRb as a drug target for the treatment of
atherosclerosis.
Recently, we reported the identification and X-ray
structures of phenyl acetic acid substituted quinolines
as potent LXR pan agonists.10 These quinolines dis-
played good binding affinity for LXRb and were po-
tent agonists in Gal4 transactivation assays.
However, the compounds also increased plasma TG
levels. In order to identify selective LXR modulators
that would have the potential to stimulate ABCA1
gene expression without the lipogenic activity, we used
a structure-based approach to improve selectivity. The
design of LXR selective ligands is quite challenging
since the ligand binding domains of LXRa/b share a
high sequence identity (78%) and residue differences
are located far away from the ligand binding pocket.11
All residues in close contact to known ligands (i.e.,
Figure 2. X-ray crystal structure of WAY-254011 bound to hLXRb.
The red arrow draws attention to the corresponding region for which
there is amino acid difference between LXRa and LXRb. The blue
dotted line indicates opportunities to improve selectivity from the
phenyl group.
obu reaction. Synthesis of anilines 11 is described in
Scheme 2. Treatment of 2,3 or 2,5-dimethyl substituted
acetanilide 8 with acetyl chloride in the presence of alu-
minum chloride gave the acetamidoacetophenones 9.
The latter were oxidized to the thioamides 10 using sul-
fur in boiling morpholine and the amides were subse-
quently hydrolyzed in hydrochloric acid.12 In Scheme
3 nitration of 1-naphthaleneacetic acid with fuming ni-
tric acid in acetic acid gave a mixture of position isomers
which were separated into 4-nitro- and 5-nitro-1-naph-
thaleneacetic acids (1313 and 14). The regio-chemistry
of 14 was assigned by 2D NMR studies (COSY and
NOESY). Compounds 13 and 14 were then reduced to
anilines 15 and 16 under palladium-catalyzed hydroge-
nation. a-Substituted phenyl acetic acids 18 were pre-
pared by mono or dialkylation of 1710a with
substituted alkyl bromides followed by basic hydrolysis
of the methyl ester (Scheme 4).
˚
5 A) are identical, and there is only one conservative
amino acid difference within the helix-3 region (i.e.,
LXRbIle277/LXRaVal263). Examination of the X-ray
structure of compound 4 (WAY-254011) suggested
that this residue could be accessed by modification
of the phenyl acetic acid head group (Fig. 2), although
we were aware that this approach would have to posi-
tion functional groups in the right orientation to take
advantage of the small differences within these 2 ami-
no acid residues. Using this observation we proposed
the synthesis of 2,3 and 2,5-disubstituted phenyl acetic
acids or a-substituted acetic acids to probe this selec-
tivity pocket.
General synthesis of carboxylic acid based quinolines 7
is shown in Scheme 1 using methods described in the
previous papers.10 Benzylamine bond (X–Y = NHCH2
or CH2NH) was formed by a reductive amination reac-
tion between aldehydes and anilines and benzyl ether
bond (X–Y = OCH2 or CH2O) was formed by a Mitsun-
The LXR binding activity of the newly synthesized com-
pounds was evaluated in binding assays as reported.10
As a reference, TO-901317 (compound 3) was tested in

56
B. Hu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 54–59
R2
R2
Ar R3
X
X
Y
Ar
R3
X-Y = OCH2, NHCH2, CH2O, CH2NH
Ar = phenyl or naphthalene
R1 = H, Me, C(O)Ph, Bn
R2 = H, 2,3-diMe, 2,5-diMe,
R3 = H, CO2R4,CR5R6CO2R4
R4 = H, Me, or Et
Y
R1
6
R1
N
a, b
N
CF3
CF3
52-95%
7
5
R5, R6 = H or alkyl
Scheme 1. Reagents: (a) benzyl bromides, K2CO3 or benzyl amines, NaBH(OAc)3, DMF, or PPh3/DEAD; (b) LiOH, THF/MeOH/H2O.
R2
R2
R2
O
O
N
a
b
S
AcHN
AcHN
AcHN
10a, 36%
10b, 69%
c
9a, 76%
9b, 100%
8a, b
R2 = 2,3-diMe for a or 2,5-diMe for b
R2
OH
O
H2N
11a, 38%
11b, 68%
Scheme 2. Reagents: (a) AcCl, AlCl3; (b) S/morpholine; (c) 6 N HCl.
NO2
NO2
a
+
10% for 13
9% for 14
COOH
COOH
b, 48%
14
COOH
12
13
b, 100%
NH2
NH2
15
COOH
16
COOH
Scheme 3. Reagents: (a) HNO3, CH3COOH; (b) H2, Pd/C, EtOH.
R1
R2
CO2H
CO2Me
O
O
O
O
R1, R2 = H or
substituted alkyl
a, b
N
N
40-90%
CF3
CF3
18
17
Scheme 4. Reagents: (a) substituted alkyl bromides, Cs2CO3; (b) LiOH, THF/MeOH/H2O.
our binding assays and was found to be a potent LXR
pan agonist (Table 1). Initial docking studies suggested
that R1/R2 group of a-substituted acid analogs 18 would
perturb the LXRbIle277/LXRaVal263 pocket to achieve
the desired binding selectivity. Compared to the unsub-
stituted compound 1910a the binding activity for a small
mono-substitution a to the carboxylic acid functionality
(23–25) was tolerated while a large substitution (20–22)
decreased LXR binding affinity. No major binding selec-
tivity for LXRb over LXRa was observed. For example,
2-allyl substituted compound 23 had the same binding
affinity as 19. Increasing the size of the substituent from
benzyl (20) to substituted benzyls (21 and 22) reduced
LXRb binding activity with no improvement on the

B. Hu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 54–59
57
Table 1. a-Substitutiona
R1 R2
OH
O
O
O
N
CF3
Compound
R1
R2
hLXRb IC50 (nM)
hLXRa IC50 (nM)
Ratio a/b (b/a)
3
9
13
17
112
65
528
16
6
1.4
3.4
19
20
21
22
23
24
25
26
27
H
H
5
H
Bn
67
81
473
5
1.7
0.8
H
4-ButBn
4-Ph–Bn
Allyl
H
1.1
3.2
H
H
2-Butynyl
2-Pentynyl
2-Pentynyl
Propargyl
2
3.0
2.1
H
10
219
26
21
147
2
2-Pentynyl
Propargyl
0.67 (1.5)
0.075 (13)
a Results are given as means of at least two independent experiments. The standard deviations for these assays were typically 30% of mean or less.
binding selectivity. Apparently mono-alkyl was not ori-
ented through the narrow cavity toward the LXRbIle277
might enhance the lipogenic activity.1 Therefore, no fur-
ther analogs were pursued in this line.
/
LXRaVal263 pocket. Interestingly, a bispentynyl group
a to the carboxylic acid functionality (26) gave minor
LXRa binding selectivity (1.5-fold) although the binding
affinity on LXRa was weak (IC50 147 nM). A moderate
selectivity for LXRa over LXRb (13-fold) was observed
for the bispropargyl substituted compound 27 and the
compound also had excellent binding affinity (IC50
2 nM for LXRa). However, LXRa selective agonists
The contribution of the 2,3 or 2,5-disubstituted phenyl
acetic acid to the LXRb binding selectivity was then
investigated (Table 2) as an alternative approach to ori-
ent groups toward the LXRbIle277/LXRaVal263 selectiv-
ity pocket. Similar to unsubstituted compound 410a the
2,5-dimethyl analog 28 showed no difference on binding
affinity for both LXRa and LXRb. Modifications on the
Table 2. C3, linker, and phenyl modificationsa
COOH
COOH
COOH
COOH
X
B
Y
B3
B1
B4
B2
Z
COOH
COOH
N
CF3
B5
B6
Compound
X–Y
Z
B
hLXRb IC50 (nM)
hLXRa IC50 (nM)
Ratio a/b
4
OCH2
OCH2
CH2O
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Me
H
B1
B2
B2
B2
B1
B3
B4
B4
B5
B6
B4
B4
2
10
11
5.0
3.7
7.8
11
28
29
30
31
32
33
34
35
36
37
38
3
4
31
21
CH2NH
CH2NH
CH2NH
CH2NH
CH2O
2
7
47
58
6.7
15
4
5
123
161
21
25
15
11
2
CH2NH
CH2NH
CH2NH
CH2NH
11
35
10
15
157
211
745
4.5
21
50
a Results are given as means of at least two independent experiments. The standard deviations for these assays were typically 30% of mean or less.

58
B. Hu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 54–59
linker region were undertaken and it was found that the
analog with a CH2NH linker (30, a/b 11-fold) was
slightly more LXRb selective than the corresponding
analogs with a CH2O (29, a/b 7.8-fold) or OCH2 linker
(28, a/b 3.7-fold). The corresponding 2,3-dimethyl ana-
log with the CH2NH linker, namely 32, showed a similar
selectivity profile (a/b 15-fold). A slightly better selectiv-
ity was observed for the 2-(naphthalen-1-yl)acetic acid
analog 33, which had the binding selectivity a/b of 25-
fold, and binding affinity on LXRb of 5 nM. As seen
earlier (29 vs 30), the benzyl amine 33 (a/b 25-fold)
was slightly more selective than the corresponding ben-
zyl ether analog 34 (a/b 15-fold). Compared to 33 the
positional isomer 35 showed similar binding affinity
(IC50 for LXRb = 2 nM) but reduced selectivity (a/b
11-fold). The benzoic acid analog 36 also showed re-
duced selectivity (a/b 4.5-fold).
Replacements for the C-3 benzyl substituent were also
evaluated. Compared to C-3 benzyl analog 33, the C-3
methyl analog 37 offered no advantage in terms of bind-
ing affinity or binding selectivity. Interestingly, the most
selective analog in the series was found with the C-3 H
analog 38, which was 50-fold selective for LXRb against
LXRa, and with LXRb binding IC50 of 15 nM.
A few binding selective LXR agonists were tested in
Gal4 functional transactivation assays (Table 3, for as-
say condition see Ref. 10a). Although compound 33,
37, and 38 showed good binding affinity (IC50 6 15 nM)
for LXRb and good binding selectivity for LXRb over
LXRa (a/b > 20-fold), the compounds showed much
less selectivity (ꢀ2-fold in potency against LXRa and
some differences in efficacy) in the Gal4 transactivation
assays. Nevertheless, compared to the reference com-
pound 3, these three compounds showed reduced po-
tency with partial agonism on LXRa (13 ꢀ 57%
efficacy) in Gal4, suggesting potentially less lipogenic
impact on the TG synthesis. In fact, relative to 3 and
4 compounds 33, 37, and 38 showed reduced potency
and efficacy in SREBP-1c gene expression and TG accu-
mulation in liver cells (HegG2 cells).14 However, these
LXRb binding selective compounds also had reduced
potency and efficacy relative to 3 and 4 on the LXRb-
Gal4 activity. The lower LXR-Gal4 activity translated
into reduced potency and efficacy in ABCA1 activation
in the human macrophage cell line (THP-1).15 To better
assess the cellular activity for LXRb these compounds
were also evaluated in murine J774 macrophages which
express LXRb predominantly with little or no LXRa
expression8,16 in contrast to differentiated THP-1 cells17
which have roughly equivalent LXRa and LXRb
expression. In the J774 macrophages, binding selective
compounds 33, 37, and 38 showed enhanced potency
and efficacy for stimulating ABCA1 gene expression
over THP-1 cells whereas a pan agonist such as 3 did
not show any difference in these two cell lines. Unfortu-
nately, compounds 33, 37, and 38 also had unwanted
peroxisome proliferator-activated receptor (PPAR)
activity (data for PPARa and PPARc not shown), espe-
cially PPARc activity18 (EC50 < 100 nM, effi-
cacy > 50%) which precluded further development of
the series.

B. Hu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 54–59
59
Rex-Rabe, S. E.; Rosa, R. L.; Tian, J. Y.; Wright, S. D.;
Sparrow, C. P. Biochem. Pharmacol. 2006, 71, 453.
8. Quinet, E. M.; Savio, D. A.; Halpern, A. R.; Chen, L.;
Schuster, G. U.; Gustafsson, J.; Basso, M. D.; Nambi, P.
Mol. Pharmacol. 2006, 70, 1340.
9. 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.
10. (a) Hu, B.; Collini, M.; Unwalla, R.; Miller, C.; Singhaus,
R.; Quinet, E.; Savio, D.; Halpern, A.; Basso, M.; Keith,
J.; Clerin, V.; Chen, L.; Resmini, C.; Liu, Q.-Y.; Feingold,
I.; Huselton, C.; Azam, F.; Farnegardh, M.; Enroth, C.;
Bonn, T.; Goos-Nilsson, A.; Wilhelmsson, A.; Nambi, P.;
Wrobel, J. J. Med. Chem. 2006, 49, 6151; (b) Hu, B.;
Jetter, J.; Kaufman, D.; Singhaus, R.; Bernotas, R.;
Unwalla, R.; Quinet, E.; Savio, D.; Halpern, A.; Basso,
M.; Keith, J.; Clerin, V.; Chen, L.; Liu, Q.; Feingold, I.;
Huselton, C.; Azam, F.; Goos-Nilsson, A.; Wilhelmsson,
A.; Nambi, P.; Wrobel, J. Bioorg. Med. Chem. 2007, 15,
3321.
In summary, modifications on previously reported
WAY-254011 via molecular modeling guided SAR study
produced a series of novel carboxylic acid based quino-
lines that showed some selectivity for LXRb over LXRa
in binding assays and reduced efficacy in the TG accu-
mulation assay, however, compounds in this series dis-
played more modest selectivity in the Gal4 functional
assays. Unwanted PPAR agonist activity was also ob-
served in this quinoline carboxylic acid series.
Acknowledgments
We thank the Wyeth Discovery Analytical Chemistry
Department for the analytical data. We also thank
Drs. Ron Magolda, Magid Abou-Gharbia, Steve Gar-
dell, and George Vlasuk for their support of this work.
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