196
S. Hintermann et al. / Bioorg. Med. Chem. Lett. 17 (2007) 193–196
4. Chu, Y.; Le, W.; Kompoliti, K.; Jankovich, J.; Mufson, E.
J.; Kordower, J. H. J. Comp. Neurol. 2006, 494, 495.
5. Wang, Z.; Benoit, G.; Liu, J.; Prasad, S.; Aarnisalo, P.;
Liu, X.; Xu, H.; Walker, N. P. C.; Perlmann, T. Nature
2003, 423, 555.
6. Codina, A.; Benoit, G.; Gooch, J. T.; Neuhaus, D.;
Perlmann, T.; Schwabe, J. W. R. J. Biol. Chem 2004, 279,
53338.
accompanied by a slight drop in potency. Replacing the
phenyl- by a 4-pyridyl group (8a) did not improve the
solubility and caused a significant drop in potency.
Finally, complete removal of the hydrophobic aryl-sub-
stituent to lower the lipophilicity turned the resulting
isoxazolo-pyridinone 8b completely inactive, as was
originally postulated in our pharmacophore hypothesis.
7. Ordentlich, P.; Yan, Y.; Zhou, S.; Heyman, R. A. J. Biol.
Chem. 2003, 278, 24791.
8. Morita, K.; Kawana, K.; Sodeyama, M.; Shimomura, I.;
Kagechika, H.; Makishima, M. Biochem. Pharmacol.
2005, 71, 98.
9. Kagaya, S.; Ohkura, N.; Tsukada, T.; Maiyagawa, M.;
Sugita, Y.; Tsujimoto, G.; Matsumoto, K.; Saito, H.;
Hashida, R. Biol. Pharm. Bull. 2005, 28, 1603.
Due to its in vitro profile, compound 7e seemed to be a
promising candidate for further investigations. Indeed,
in a preliminary pharmacokinetic experiment14 it was
found to have an excellent oral bioavailability of 95%
in mice and a rapid and extensive brain uptake, reaching
levels more than two orders of magnitude higher than
the cellular EC50 within 30 min.
10. MN9D cells were stably transfected with
a Nurr1
expressing plasmid under the control of a CMV promoter.
A clone showing a 4-fold increase in tyrosine hydroxylase
activity was used to generate the screening cell line by
transfecting the cells with a reporter plasmid in which
firefly luciferase expression was controlled by multiple
copies of a Nurr1 specific DNA binding element.11 To
demonstrate that luciferase expression was under the
control of Nurr1, a cell line was established in which the
retinoic acid receptor RXRa was coexpressed. RXRa can
form heterodimers with Nurr1 so that Nurr1 responsive
elements can become retinoic acid inducible. In fact, the
luciferase reporter activity became strongly inducible by 9-
cis retinoic acid in this cell line while it was not in the
screening cell line expressing only Nurr1. As a negative
control to eliminate Nurr1 independent inducers, a plas-
mid, without any Nurr1 specific DNA binding sites, was
co-transfected. Compounds were tested by incubating the
MN9D cells with various concentrations in triplicates
(controls received the same amount of vehicle). After 24 h,
the cells were processed for luciferase activity. The EC50
values for the stimulation over the basal activity were
calculated after fitting of the curves.
In conclusion, this report describes the identification of
a novel class of activators of the Nurr1 signaling path-
way showing low-nanomolar activities in cellular assays.
The molecular mode of action of these compounds is at
the moment not clarified and work is in progress, to ana-
lyze a possible direct interaction with the newly de-
scribed hydrophobic pocket of the receptor. In
addition, efforts are ongoing to further optimize and
characterize this promising compound class in vitro
for later in vivo profiling in animal models.
Acknowledgments
We thank A. Foggetta-Lorenzi, S. Liverneaux, and A.
Sturzinger for excellent technical assistance, M. Tintel-
¨
not-Blomley and J. Kallen for their valuable input for
the pharmacophore hypothesis, I. Filipuzzi for perform-
ing the HTS, A. Enz for pharmacokinetic data, and A.
Walmsley for editorial assistance.
11. Zetterstro¨m, R. H.; Solomin, L.; Mitsiadis, T.; Olson, L.;
Perlmann, T. Mol. Endocrinol. 1996, 12, 1556.
12. Denzer, M.; Smith, J. A. U.S. Patent application
US4113727.
References and notes
13. Measured in analogy to: Lipinski, C. A.; Lombardo, L.;
Dominy, B. W.; Feeney, P. J. Adv. Drug Delivery Rev.
1997, 23, 3.
1. Zetterstro¨m, R. H.; Solomin, L.; Jansson, L.; Hoffer, B. J.;
Olson, L.; Perlmann, T. Science 1997, 276, 248.
2. Le, W. D.; Conneely, O. M.; He, Y.; Jankovic, J.; Appel,
S. H. J. Neurochem. 1999, 73, 2218.
3. Hermanson, E.; Joseph, B.; Castro, D.; Lindqvist, E.;
14. Mice were dosed with 7e (10 lmol/kg) dissolved in 10%
Tween 80/water iv and po. Plasma and brain samples were
collected at 7 time points between 5 min and 24 h (N = 3).
Samples were extracted and analyzed by HPLC, and
bioavailability calculated, following standard procedures.
´
Aarnisalo, P.; Wallen, A.; Benoit, G.; Hengerer, B.; Olson,
L.; Perlmann, T. Exp. Cell Res. 2003, 288, 324.