Macrocyclic Hedgehog Pathway Inhibitors: Optimization of Cellular Activity and Mode of Action Studies

Macrocyclic Hedgehog (Hh) pathway inhibitors have been discovered with improved potency and maximal inhibition relative to the previously reported macrocycle robotnikinin. Analogues were prepared using a modular and efficient build-couple-pair (BCP) approach, with a ring-closing metathesis step to form the macrocyclic ring. Varying the position of the macrocycle nitrogen and oxygen atoms provided inhibitors with improved activity in cellular assays; the most potent analogue was <b>29</b> (BRD-6851), with an IC₅₀ of 0.4 μM against C3H10T1/2 cells undergoing Hh-induced activation, as measured by <i>Gli1</i> transcription and alkaline phosphatase induction. Studies with Patched knockout (<i>Ptch</i>^–/–) cells and competition studies with the Smoothened (Smo) agonists SAG and purmorphamine demonstrate that in contrast to robotnikinin, select analogues are Smo antagonists