M. Thor et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3565–3567
Table 1. In vitro bindingaffinity ( Ki) and potency (EC50) for selected
compounds
3567
Ki (mM)a,c
EC50 (mM)b,c,d
Compd
5-Substituent
PPARa PPARg PPARa PPARg
BVT.142
5a
5b
5c
5d
5e
5f
9a
9b
9c
9d
9e
10a
10b
10c
Methyl
30
25
20
>100
19
>100
>100
28
2.2
0.4
0.6
1
9.5
5
3.8
>100
2.5
>100
n.d.
12
10
>100
>100
1.6
0.3
0.8
0.7
0.6
0.7
3.2
0.7
1
2-Thienylmethoxy
2-(3-Thienyl)ethoxy
2-(2-Thienyl)ethoxy
OCH2CH2SCH3
4-Ethoxybenzyloxy
2-(2-Pyridinyl)ethoxy
3-Thienyl
1.6
3.6
1.5
0.9
0.5
1.2
0.7
1.4
0.18
0.17
0.1
0.16
2-Furyl
22
3-Ethoxyphenyl
8-Quinolinyl
3-Carboxyphenyl
5-Nitro-2-pyridinyloxy >100
3-Nitro-2-pyridinyloxy >100
>100
>100
67
0.3
0.3
>100 >100
>100
>100
>100
>100
0.4
0.4
1.3
0.45
2-Pyrimidinyloxy
6-Chloro-2-pyrazinyloxy
>100
21
10d
Figure 2. The bindingsite of 5a in the PPARg receptor.
aLigand binding affinities were determined by displacement of a tri-
tiated tracer by the unlabeled compound to a GST-PPAR fusion-pro-
tein, for PPARa, containingresidues 167–468 and 3H-GW2331 as
tracer, and for PPARg, containingresidues 204–477 and 3H-Rosigli-
tazone as tracer.
ligands such as rosiglitazone, which appear to activate
the PPARg receptor through interactions with amino
acids (e.g., His323, His449 and Tyr473) located deep
into the ligand binding pocket, compound 5a occupies a
region in proximity with helix 3. In Figure 2 the key
interaction between the carboxylic acid group of 5a and
the backbone nitrogen of Ser342, situated near the
entrance of the PPARg pocket, is shown.
bTransactivation potency was measured by luciferase activity in Caco-
2/TC7 cells transiently co-transfected with an expression vector for the
fusion-protein Gal4-PPARa (167–468) or Gal4-PPARg (204–477),
respectively, and a reporter vector containing4xGAL4RE-luciferase,
after 24 h of incubation with compounds.
cValues are the mean of at least two experiments, where nꢀ2, and
Xlfit version 2.0 (Business Solutions Limited) was used for curve fit-
tinganalysis.
dn.d.=not determined.
Other variations around BVT.142, such as carboxylic
acid isosters and various modifications of the
2,4-dichloro moiety, have also been investigated but are
not within the scope of this paper.
hit. Interestingly, compound 5c, which only differs from
5b in the position of the sulfur in the thiophene ring,
does not bind or activate the PPARa receptor but still
retains PPARg receptor activity. In general, analogues
containingan aromatic portion in the 5-substituent are
more active, however compound 5d shows that aliphatic
substituents could also be of interest. In the Suzuki
biaryl series, compounds 9a and 9b are dual PPARa/g
agonists while compounds such as 9c and 9d are potent
and highly selective PPARg agonists. From the same
series the PPARg antagonist 9e was also identified. The
analogues showing the highest binding affinities are the
diaryl ethers 10a–d. Interestingly, all four compounds,
prepared accordingto Scheme 3, are highly selective and
potent PPARg agonists. The data demonstrates the pos-
sibility to modify the effect on the PPARa and PPARg
receptors by variations in the 5-position of BVT.142.
References and Notes
1. Mangelsdorf, D. J.; Evans, R. M. Cell 1995, 83, 841.
2. King, A. B. Diabetes Care 2000, 23, 557.
3. Vu-Dac, N.; Chopin-Delannoy, S.; Gervois, P.; Bonnelye,
E.; Martin, G.; Fruchart, J. C.; Laudet, V.; Staels, B. J. Biol.
Chem. 1998, 273, 25713.
4. Hughes, D. L. In Organic Reactions; Paquette, L. A., Ed.;
John Wiley & Sons: New York, 1992; Vol. 42, pp 335–656.
5. Miyaura, N.; Suzuki, A. Chem. Rev. 1995, 95, 2457.
6. Nolte, R. T.; Wisely, G. B.; Westin, S.; Cobb, J. E.; Lam-
bert, M. H.; Kurokawa, R.; Rosenfeld, M. G.; Willson, T. M.;
Glass, C. K.; Milburn, M. V. Nature 1998, 395, 137.
7. Renaud, J. P.; Rochel, N.; Ruff, M.; Vivat, V.; Chambon,
P.; Gronemeyer, H.; Moras, D. Nature 1995, 378, 681.
8. The ligand binding domain of hPPARg was produced in E.
coli and the protein was purified to homogeneity and con-
centrated to a final concentration of 10 mg/mL.9 Crystals of
PPARg-LBD complexes were grown by the hanging drop dif-
fusion method. A GRIP-1 derived co-activator peptide was
co-crystallized with the receptor. All data were collected at
room temperature usinga Rigaku RU300 rotatinganode and
an Raxis4 image plate detector. The crystals diffracted to 2.9
A resolution. A detailed description of the structure will be
published elsewhere.
It has been suggested that PPARg and other nuclear
receptors are activated by a common mechanism in which
the ligand stabilizes helix 12.6,7 This in turn allows for the
recruitment of co-activator proteins and subsequent
stimulation of transcriptional activity. We have been
able to obtain the structure of human PPARg LBD in
complex with the PPARg agonists 5a–b and 9a.8
The X-ray crystallographic data shows that these com-
pounds bind to a site different from the reported bind-
ingsite of the thiazolidinediones. In contrast to TZD
9. Uppenberg, J.; Svensson, C.; Jaki, M.; Bertilsson, G.; Jen-
deberg, L.; Berkenstam, A. J. Biol. Chem. 1998, 273, 31108.