Synthesis and DAF-12 receptor activity of 4-dafachronic acid analogues

SAMAJA GA; DANSEY, V.; ALVAREZ, L; VELEIRO, A.; PECCI A.; BURTON, G.

Viena

Congreso; 14th Tetrahedron Symposium: Challenges in Organic & Bioorganic Chemistry; 2013

Elsevier y Tetrahedron Journals

In the nematode C. elegans (Ce), dafachronic acids (DAs) are cholesterol metabolites that regulate multiple physiological functions, such as developmental timing, fertility and longevity. Their action is exerted through the activation of the nuclear receptor DAF-12, a transcription factor involved in the regulation of key genes of animal life. A keto group at C-3, an unsaturated double bond at C-7 or C-4, and an acidic carboxyl group at the end of the side chain (C-26) are required for the efficient CeDAF-12 activation.1 Recently, using a combination of molecular modelling, synthetic chemistry and biological experiments we predicted and confirmed that the removal of the C-25 methyl in 4-DAs (i.e. 3) does not abolish the in vitro or in vivo activity.2 To further investigate the relation between the structure of the steroid side chain and the DAF-12 activity, we have now synthesized three 4-DA analogues: compound 4 exhibiting reduced side chain flexibility, compound 5 with a shorter but flexible side chain and compound 6 with a shorter but less flexible side chain. Compound 4 was prepared from hyodeoxycholic acid in 10 steps and 9.2% overall yield. The 3-carbon homologation was carried out on the intermediate C-24 aldehyde using a Wittig reaction with [Ph3PCH(CH3)CO2CH2CH3]Br and aqueous NaHCO3 in dichloromethane. Compounds 5 and 6 were prepared from20-carboxylaldehyde--pregnen-3-one in 5 (41.3% yield) and 4 steps (27.2%) respectively, via a Wittig reaction with [Ph3PCH2CO2CH2CH3]Br and aqueous NaHCO3 in dichloromethane. The results obtained show that while compounds 4 and 5 are able to induce DAF-12 activity in transient transfections of GAL4-DAF12 and GAL4-LUC reporter in HEK-293T cell cultures, compound 5 was inactive. Molecular dynamics simulations of the DAF-12 complexes with 4, 5 and 6 using our previously developed homology model of the receptor,3 were consistent with the observed activity differences of these compounds.