Isolation, purification, and full NMR assignments of cyclopamine from Veratrum californicum

In this paper, we report a rapid and efficient isolation and purification of large quantities of cyclopamine from the roots and rhizomes of Veratrum californicum Dur. (the Corn Lily or Western false hellebore). We also provide unambiguous assignments of the carbon and proton resonances by using the multinuclear spectra and the spin coupling networks.This method could meet a very real need within diverse scientific communities by allowing cyclopamine to become more readily available.Cyclopamine is a naturally occurring steroidal alkaloid teratogen that interferes with the Hedgehog (Hh) signaling pathway by binding to Smoothened, a heptahelical transmembrane receptor that is a critical Hh response element [1,2]. Cyclopamine is thus a very useful reagent for probing and modifying the activity of the important and widely studied Hh pathway. As one important example, cyclopamine has been shown to be an extremely effective inhibitor of the growth and survival of specific tumors in vitro and in vivo [1,2], such that cyclopamine, cyclopamine analogs, and related alkaloids [3,4] have the potential to become important cancer chemotherapeutic agents. However, because it is very expensive commercially (~$10/mg), the large quantities required for research in animals, and especially in large mammals as required in our own cardiac research, would at present be prohibitively expensive.A totally synthetic approach to obtaining cyclopamine can be envisioned. First, the synthesis of 17-acetyl-5α-etiojerva-12,14,16-trien-3β-ol from simple chemicals has been reported by Johnson et al [5], and Masamune et al reported [6] its conversion into the cyclopamine relative jervine, where the carbon-11 methylene in cyclopamine is oxidized to ketone in jervine. Second, the Wolff-Kishner reduction of jervine has been reported to yield cyclopamine [7]. However, because this would in aggregate be a nontrivial complex multistep synthesis of very low net yield, and because the concentration of cyclopam