Cyclopamine analogs bearing exocyclic methylenes are highly potent and acid-stable inhibitors of hedgehog signaling

The chemical synthesis and biological evaluation of new cyclopamine analogs bearing exocyclic methylenes in different positions is described. Bis-exo-cyclopamine 6 was identified as a potent inhibitor of the Gli1-dependent luciferase expression in Shh-LIGHTII cells. An extension of this study to F-ring-modified structures shows the necessity of a rigidly positioned nitrogen atom for bioactivity as well as the presence of the C21 methyl group for acid stability and bioactivity.

Cyclopamine (1, see Figure 1) was the first inhibitor of the hedgehog signaling pathway to be identified. As a highly selec-tive inhibitor of the transmembrane protein Smoothened (Smo), an integral component of hedgehog signaling, it presents an attractive target for medicinal and pharmaceutical research [29,30]. Unfortunately, its direct development into a drug is hampered by its low metabolic stability (decomposition at pH < 3) [31] and rather moderate potency (IC 50 ~ 5 µM). We previously reported the first chemical synthesis of cyclopamine (1) starting from dehydroepiandrosterone and utilizing the C-H-functionalization logic and a biomimetic skeleton rearrangement [32,33]. Furthermore, quantum mechanical calculations guided our design and synthesis of exocyclopamine (2, see Figure 1), a ten-fold more potent and acidstable analog with an exo-methylene unit at C13-C18 [34]. Herein, we describe a comprehensive study of cyclopamine analogs bearing exo-methylene units in different positions and extent this rational to F-ring-modified structures.
Given the negative test results of compounds 8 and 9 it becomes evident that the F-ring is necessary for bioactivity. The piperidine moiety provides a rather rigidly placed nitrogen atom. Nevertheless, a pyrrolidine as in compound 23 still provides the correct orientation of the nitrogen atom. Despite compounds 8 and 9 being inactive in the assay, both derivatives induced cytotoxicity in the concentration range tested. Very subtle changes of the conformation of the piperidine ring significantly change   bioactivity: While 25-epi-exo-cyclopamine 5 shows reduced activity in comparison to exo-cyclopamine 2, bis-exocyclopamine 6 is the most active compound tested in this study. Furthermore, the methyl group at C-20 seems to have a pronounced effect on the bioactivity, with 20-demethyl-bis-exocyclopamine 19 being completely inactive in the tested concentration range.
Finally, we studied the stability of all newly synthesized compounds towards acidic conditions. Therefore, they were exposed to a pH of approximately 1 (MeOH, 1 M HCl) for 24 h. After evaporation of all volatiles, 1 H NMR spectra were acquired and compared to the initially obtained spectra of the pure compounds. While compounds 5, 6, 8, and 9 remained unchanged, compounds 19 and 23 showed decomposition. This experiment emphasizes the importance of the C-21 methyl group for the stability of exo-cyclopamine derivatives. All synthesized compounds, the number of steps required, and the respective overall yield starting from 3 or 14, as well as their biological activity and stability under acidic conditions are summarized in Table 1.

Conclusion
In conclusion, we succeeded in identifying the new cyclopamine derivative bis-exo-cyclopamine 6 which surpasses the biological potency of the parent compound by the 25-fold and is stable at pH 1. Further insights were gained into the structure-activity relationship of F-ring-modified analogs of cyclopamine and the necessity of the C-21 methyl group for  bioactivity and acid stability was revealed. Our designed analogs of cyclopamine are accessible in noticeably shorter and higher yielding synthetic routes than the parent compound, a fact that will further contribute to their usefulness in biological and medicinal studies.

Supporting Information
Supporting Information File 1 Experimental details and analytical data of all synthesized compounds are provided.