2012
B.-M. Swahn et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2009–2012
R.; Tu, H.; Luk, A.; Repa, J. J.; Medina, J. C.; Li, L.; Schwendner, S.; Wang, S.;
Cl
OH
CF3
H
N
Thoolen, M.; Mangelsdorf, D. J.; Lustig, K. D.; Shan, B. Gene. Dev. 2000, 14,
2831; (c) 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.; Beaudet, E. J.; Whitney, K. D.; Kliewer, S. A.; Wilson, T. M. J.
Med. Chem. 2002, 45, 1963.
a
10
R
O
R
S
O
N
SO2Cl
O
H
19
18
5. Chisholm, JW.; Hong, J.; Mills, S. A.; Lawn, R. M. J. Lipid Res. 2003, 44,
2039.
Cl
OH
CF3
6. (a) Molteni, V.; Li, X.; Nabakka, J.; Liang, F.; Wityak, J.; Koder, A.; Vargas, L.;
Romeo, R.; Mitro, N.; Mak, P. A.; Seidel, M.; Haslam, J. A.; Chow, D.;
Tuntland, T.; Spalding, T. A.; Brock, A.; Bradley, M.; Castrillo, A.; Tontonoz,
P.; Saez, E. J. Med. Chem. 2007, 50, 4255; (b) Hu, B.; Quinet, E.; Unwalla, R.;
Collini, M.; Jetter, J.; Dooley, R.; Andraka, D.; Nogle, L.; Savio, D.; Halpern,
A.; Goos-Nilsson, A.; Wilhelmsson, A.; Nambi, P.; Wrobel, J. Bioorg. Med.
Chem. Lett. 2008, 18, 54.
H
20 R= CN
21 R= CO2Me
23 R= CH2CH2CO2Me
N
b
R
O
S
O
N
O
O
7. LXR co-activator recruitment assay: The Ligand binding domain (LBD) of LXR
a
(amino acid 205–447) and LXRb (amino acid 216–461) was produced in
Escherichia coli. A fragment of the human Steroid Receptor Co-Activator-1 (SRC-
1) was produced as a synthetic peptide. An a-6xHis antibody coupled to Eu3+
was used to detect the His-tag on LXR-LBD and allophycocyanin (APC) coupled
to streptavidin was used to detect biotinylated SRC-1. Agonist binding to LXR
enhances affinity for SRC-1 and thereby brings Eu3+ in close proximity. Eu3+ is
exited at 337 nm and emits light at 620 nm; this emission in turn exits APC to
emit light at 655 nm. Time-resolved fluorescence readings were done in a
Cl
OH
H
22 R= CO2H
24 R= CH2CH2CO2H
N
c
R
CF3
S
O
N
O
Scheme 2. Reagents and conditions: (a) pyridine/CH2Cl2, rt, 45% for 20; (b) MeI,
K2CO3, acetone, rt, 82% for 20; (c) LiOH, H20/THF.
Wallac Victor2 reader. An assay mix with 0.06
1.15 g/ml streptavidin APC, 90 nM SRC-1 peptide and 0.4
(20 mM Tris/0.125% CHAPS (pH 7.5), 2 mM DTT, 0.05% BSA (FA free)) was used.
l
g/ml Eu-labeled anti-6x His Ab,
l
l
g/ml LXR in buffer
A control LXR agonist T0901317 at 1
level of inhibition.
8. Butlin, R. J.; Nowak, T.; Burrows, J. N.; Block, M. H. PCT Int. Appl., WO 9962506
A1, 1999.
lM was used as the 100% control to assess
Table 3
FRET and reporter EC50
(
l
M)a for compounds 20–24
Compound
FRET LXRb
LXRa
Reporter LXRb
LXRa
9. Bebernitz, G. R.; Aicher, T. D.; Stanton, J. L.; Gao, J.; Shetty, S. S.; Knorr, D. C.;
Strohschein, R. J.; Tan, J.; Brand, L. J.; Liu, C.; Wang, W. H.; Vinluan, C. C.; Kaplan,
E. L.; Dragland, C. J.; DelGrande, D.; Islam, A.; Lozito, R. J.; Liu, X.; Maniara, W.
M.; Mann, W. R. J. Med. Chem. 2000, 43, 2248.
10. (a) Ohnmacht, C. J.; Russell, K.; Empfield, J. R.; Frank, C. A.; Gibson, K. H.;
Mayhugh, D. R.; McLaren, F. M.; Shapiro, H. S.; Brown, F. J.; Trainor, D. A.;
Ceccarelli, C.; Lin, M. M.; Masek, B. B.; Forst, J. M.; Harris, R. J.; Hulsizer, J. M.;
Lewis, J. J.; Silverman, S. M.; Smith, R. W.; Warwick, P. J.; Kau, S. T.; Chun, A. L.;
Grant, L. G.; Howe, B. B.; Li, J. H.; Trivedi, S.; Halterman, T. J.; Yochim, C.; Dyroff,
M. C.; Kirkland, M.; Neilson, K. L. J. Med. Chem. 1996, 39, 4592; (b) Empfield, J.
R.; Mayhugh, D.; Ohnmacht, C. J.; Frank, C. A.; Grant, T.; Li, J. Bioorg. Med. Chem.
Lett. 1997, 7, 775.
20
21
22
23
24
2.60
2.32
>100
3.50
>100
3.40
3.10
>100
5.00
>100
2.70
3.81
>30
>30
>30
5.00
3.61
>30
>30
>30
a
Values are means of n P 2 determinations, standard deviation 6 10%.
In summary we found some compounds with selectivity for
LXRb, but more interestingly we discovered a variation of activity
in different cell lines, reflecting that secondary effects such as
co-activator recruitment might vary between cell-lines. An
attempt to optimize the activity of compound 5 was described
and one compound with good DMPK profile was evaluated for
ABCA1 and ABCG1 mRNA up regulation in vivo.
11. LXR-Gal4 reporter assay: SH-SY5Y cells were cultured in Dulbecco’s modified
Eagle’s medium/F12 (1:1) with glutamax, 1% NEAA, 1% PEST and 10% Fetal calf
serum (FCS) at 5% CO2. At confluence, cells were transiently transfected with a
pSGAL human LXRa (amino acid 205–447) or pSGAL human LXRb (amino acid
216–461) expression plasmid and
a 5xUSA Luc reporter plasmid using
Lipofectamin 2000 transfection. 10.5 ng pSGAL-hLXRa or b were mixed with
10.5 ng pGL3 5xUSA Luc reporter plasmid containing a minimal SV40 promotor
and 5 copies of the USA GAL4 recognition site in transfection buffer (DMEM/
F12 (1:1) with glutamine, 2% FCS, 1% NEAA, 1% PEST) containing approximately
6 million SH-SY5Y cells. Directly after transfection, LXR ligands or vehicle
(DMSO, 0.1%) were added to the cells. After 24 h, cells were analysed for
luciferase activities according to the manufacturers instructions. A control LXR
Acknowledgements
agonist T0901317 at 1
inhibition.
lM was used as the 100% control to assess level of
The authors would like to thank Dr. Eva-Lotte Lindstedt, Depart-
ment of Medicinal Chemistry AstraZeneca R&D Mölndal, for valu-
able assistance in facilitating testing in U2OS cells and 3 day
in vivo PD studies.
12. (a) Alberti, S.; Steffensen, K. R.; Gustafsson, J. A. Gene 2000, 243, 93; (b)
Williams, S.; Bledsoe, R. K.; Collins, J. L.; Boggs, S.; Lambert, M. H.; Miller, A. B.;
Moore, J.; McKee, D. D.; Moore, L.; Nichols, J.; Parks, D.; Watson, M.; Wisely, B.;
Willson, M. J. Biol. Chem. 2003, 278, 27138.
13. (a) Färnegårdh, M.; Bonn, T.; Sun, S.; Ljunggren, J.; Ahola, H.; Wilhelmsson, A.;
Gustafsson, J. Å.; Carlquist, M. J. Biol. Chem. 2003, 278, 38821; (b) Hoerer, S.;
Schmid, A.; Heckel, A.; Budzinski, R. M.; Nar, H. J. Mol. Biol. 2003, 334, 853.
14. Rarey, M.; Kramer, B.; Lengauer, T.; Klebe, G. J. Mol. Biol. 1996, 261, 470.
15. Figure 3 was generated using SYBYL 6.9.2, Tripos, 1699 South Hanley Rd., St.
Louis, Missouri, USA.
References and notes
1. For recent reviews, see: (a) Bennett, D. J.; Carswell, E. L.; Cooke, A. J.; Edwards,
A. S.; Nimz, O. Curr. Med. Chem. 2008, 15, 195; (b) Goodwin, B. J.; Zuercher, W.
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A. PNAS 2002, 99, 13878.
16. Solubility was measured as screen solubility: Incubation of 100 lM DMSO
solution in 0.1 M phosphate buffer pH 7.4, shaking for 24 h at rt Solubility
quantification on the bases of the UV-TIC.
17. The method for mRNA determination of ABCA1, ABCG1 and SREBP-1c will be
published elsewhere (J. Lindquist et al).
4. (a) Lehmann, J. M.; Kliewer, S. A.; Moore, L. B.; Smith-Oliver, T. A.; Blanchard,
D. E.; Spencer, T. A.; Wilson, T. M. J. Biol. Chem. 1997, 272, 3137; (b) Schultz, J.