Design,Synthesis,and Structure-Activity-Relationship Of New Hedgehog Pathway Inhibitors

The Hedgehog（Hh）signaling pathway plays a critical role in controlling differentiation,growth and cell migration during embryonic development.Aberrant Hh signaling during development can lead to abnormality.Hh pathway is much less active and its function is limited to tissue maintenance and stem cell regulation in adults.Aberrant activation of Hh signaling has been linked to tumorigenesis in various cancers,such as basal cell carcinoma（BCC）and medulloblastoma.Consequently,targeting the Hh pathway has become an attractive approach for the treatment of cancer.Smoothened（Smo）receptor is a key member of Hh pathway and belongs to G protein-coupled receptor（GPCR）.Vismodegib and Sonidegib are Smo inhibitors and have been approved by the US Food and Drug Administration for treatment of locally advanced or metastatic basal cell carcinoma.The clinical success of these two drugs provided strong support for the development of Hh signaling pathway inhibitors via targeting the Smo receptor.However,low aqueous solubility of Vismodegib leads to its nonlinear pharmacokinetic profiles at high doses,hampering its clinical utility.In addition,patients were found to develop resistance to Vismodegib and Sonidegib.Therefore,there is an urgent need to develop new scaffolds which can improve physicochemical properties and overcome acquired drug resistance.With the exception of the terminal methylgroup,the skeleton of Vismodegib was constructed with sp2-hybridized carbons,resulting in poor solubility.We introduced sp3-hybridized carbons to design and synthesize a series of new compounds.However,these compounds can be easily metabolized by live microsomes.We took two approaches to get more metabolic stable compounds:（1）adding small fuctional groups to the potential site of metabolism;（2）introducing acyl groups to slow oxidation by reducing electrondensity.We were able to obtain numerous compounds with improved activity(B3:IC₅₀=8 nM,B6:IC₅₀=10 nM and E4:IC₅₀=3.5 nM).These compounds will be profiled in metabolic stability and pharmacokinetics studies.Lastly,these compounds will be tested to inhibit Smo-D473 H mutant.Based on the Smo crystal complexes,the molecular docking based virtual screening was used to identify promising Smo antagonists from ChemDiv library.The scoring function of Schrodinger 9.0 was employed to carry out the sequential virtual screening strategy.Finally,1000 top-ranked chemicals for each Smo crystal complex were retained for the following analysis.These compounds were evaluated by "Rule-of Five".The molecules with toxic functional groups and more than two chiral centers were removed.Finally,21 structurally novel and diverse compounds were selected for experimental testing.Six of the 21 selected compounds exhibited significant inhibitory activity against Hh pathway activation(IC₅₀<10μM).Specifically,compound V20 has the most potent activity(IC₅₀=47 nM)and was further confirmed to be a Smo antagonist.We then studied the structure activity relationship（SAR）of compound V20 and got some more potent compounds(G2:IC₅₀=7.1nM,G25:IC₅₀=7.1nM and G26:IC₅₀=8.4 nM).This new scaffold can be used as a good starting point for developing new Smo antagonists.The compounds with good activity will be profiled in metabolic stability and pharmacokinetics studies.Lastly,these compounds will also be tested to inhibit Smo-D473 H mutant.