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(Fig. 4b). Binding of compound 8g to GR binding pocket induces
similar conformational changes, wherein the 2,4-difluoro phenoxy
benzyl moiety of 8g overlays with the C11-dimethylaniline of
RU486 and compare to C11-dimethylaniline group, 2,4-difluoro
phenoxy group of 8g protrude-out more from the binding pocket
and strongly displaces H-12 to antagonist position, which may
account for its strong passive in vitro antagonistic activity (both
in GRAF and TAT functional assays). Together, docking studies
results illustrate favorable interactions of compound 8g with key
residues of GR binding pocket, which supports its potent in vitro
binding affinity and functional antagonistic activity.
In summary, we report series of benzyl-phenoxybenzyl amino-
phenyl acid derivatives (8a–q) as non-steroidal GR antagonist.
Compound 8g showed excellent h-GR binding, selectivity over
closely associated NR and potent antagonistic activity (in-vitro).
VP16 assay and docking studies confirms passive antagonistic
activity of 8g. The lead compounds 8g showed no impact upon
the HPA axis, exhibited significant oral antidiabetic and antihyper-
lipidemic effects (in vivo), along with liver selectivity. Thus
preliminary study results confirm discovery of potent and liver
selective GR antagonist, which could be viable approach for the
safe and effective treatment of T2DM.
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18. NR binding activities of test compounds were assessed using radioligand
binding assays. For h-GR binding assay, [3H]-dexamethasone (3H-dex; Sigma)
was used as a radioligand. The h-GR was extracted from Sf9 cells and IC50
values were determined for test compounds. Similar protocols were employed
to measure affinity/selectivity of test compounds over other NR (PR, MR, AR, ER
Acknowledgments
Authors are thankful to management of Zydus Group for
encouragement and analytical department for analytical support.
(a/b) and TR (a/b)).The reporter gene of GRAF cells expressing h-GR, encodes a
secreted form of alkaline phosphatase (ALP). Antagonists are evaluated by their
inhibition of dexamethasone-induced ALP expression in these cells.
Compounds are also tested in freshly isolated rat hepatocytes for their
effects on dexamethasone induced expression of the GR regulated enzyme
tyrosine. In both the functional assays, IC50 values were determined.
19. Link, J. T.; Sorensen, B.; Patel, J.; Arendsen, D.; Li, G.; Swanson, S.; Nguyen, B.;
Emercy, M.; Grynfarb, M.; Goos-Nilsson, A. Bioorg. Med. Chem. Lett. 2004, 14,
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Supplementary data
Supplementary data associated with this article can be found,
20. Wang, J.; Harvey, R. G. Tetrahedron 2002, 58, 5927.
References and notes
21. A third functional assay (VP16) is used to assess the active or passive nature of
test compound. VP16 is a transcriptional activation domain that if present in
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a reporter (GRE-Lue). In this assay passive antagonist show little or no response
while active antagonists robustly stimulate luciferase expression.
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weight basis (20 mpk) to fasted male SD rats (n = 6). Serial blood samples were
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