Withanolide Structural Revisions by 13C NMR Spectroscopic Analysis Inclusive of the γ-Gauche Effect

A classic withanolide is defined as a highly oxygenated C-28 ergostane-type steroid that is characterized by a C-22-hydroxy-C-26-oic acid delta-lactone in the nine-carbon side chain. Analysis of the reported C-13 NMR data of classic withanolides with hydroxy groups (C-14, C-17, and C-20) revealed that (1) a hydroxy (C-14 or C-17) substituent significantly alters the chemical shifts (C-7, C-9, C-12, and C-21) via the gamma-gauche effect; (2) the chemical shift values (C-9, C-12, and C-21) reflect the orientation (alpha or beta) of the hydroxy moiety (C-14 or C-17); (3) a double-bond positional change in ring A (Delta(2) to Delta(3)), or hydroxylation (C-27), results in a minuscule effect on the chemical shifts of carbons in rings C and D (from C-12 to C-18); and (4) the C-13 NMR gamma-gauche effect method is more convenient and reliable than the traditional approach (H-1 NMR shift comparisons in C5D5N versus CDCl3) to probe the orientation of the hydroxy substituent (C-14 and C-17). Utilization of these rules demonstrated that the reported C-13 NMR data of withanolides 1a-29a were inconsistent with their published structures, which were subsequently revised as 1-16 and 12 and 18-29, respectively. When combined, this strongly supports the application of these methods to determine the relative configuration of steroidal substituents.