Spontaneous butenolide ring formation of pregnane-21-O-malonyl hemiesters under mild reaction conditions is facilitated by the 14β-hydroxy group present in all natural cardenolides

Abstract Cardenolides are composed of a steroid nucleus and a five-membered unsaturated lactone (butenolide) ring at C-17β. It is assumed that the butenolide ring is formed from a pregnane 21- O -malonyl hemiester after a condensation reaction between the methylenic group of the malonic acid residue and the C-20 carbonyl of the pregnane scaffold. Here, the formation of the butenolide ring was studied starting from pregnane 21- O -malonyl hemiesters with and without a hydroxyl group in position C-14β. Efficient lactonization was only achieved in the case of the 14β-hydroxylated derivative. The reaction involves: 1) formation of an intramolecular hydrogen bond between the C-20 carbonyl group and the 14β-hydroxyl group which stabilizes a negatively charged intermediate and/or increases the positive character of the C-20 carbonyl carbon, 2) nucleophilic attack that enables efficient lactonization by release of water, and 3) subsequent decarboxylation. We provide chemical and mechanistic evidence that 14β-hydroxylation would precede lactonization in cardenolide biosynthesis.