C–H-Functionalization logic guides the synthesis of a carbacyclopamine analog

Summary The chemical synthesis of carbacyclopamine analog 2, a cyclopamine analog with an all-carbon E-ring, is reported. The use of C–H-functionalization logic and further metal-catalyzed transformations allows for a concise entry to this new class of acid-stable cyclopamine analogs.


H and 13 C NMR spectra for new compounds
. Multiplicities are classified by the following abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, p = quintet, and combinations thereof, or m = multiplet, or br = broad signal.
Where 2D-spectra were recorded and allowed complete assignment of all hydrogen and carbon-atoms of a compound, spectral data include this assignment using common steroid numbering. Where this is not the case, all hydrogen signals below 2 ppm are omitted and only methyl groups and isolated signals in this range are listed. All spectra can be found as copies at the end of the experimental section.
High resolution mass spectra were obtained on a Bruker Daltonics ESI-FT-ICR-MS APEX II. IR spectra were obtained on an ATI/MATTSON Genesis FT-IR and JASCO FT/IR-4100typeA as thin film (in CCl 4 ) or KBr disk. Absorbance frequencies are reported in reciprocal centimeters (cm −1 ).
Melting points were measured on a Boetius-micro hot stage and are uncorrected.
Optical rotations were obtained on a Schmidt+Haensch Polartronic MHZ-8 at the sodium-D line (589 nm) using a 50 mm path-length cell and solvent and concentration as indicated.

Acid Stability
To a solution of carbacyclopamine analog 2 (2.0 mg, 5.5 mol) in THF (0.5 mL) was dropwise added aqueous HCl (2.0 M) until a pH value of approx. 0.3 was reached. The mixture was stirred for 1 h at room temperature after which the solution was neutralized with saturated NaHCO 3 -solution, extracted with CH 2 Cl 2 (3 x 5 mL), dried (MgSO 4 ) and filtered. All volatiles were removed under reduced pressure and the so-obtained white solid (2.0 mg, 5.5 mol) was used directly for H-NMR measurements. The 1 H-NMR spectra recorded before and after treatment with acid are shown in Figure 1.

Biochemistry
The interference of the carbacyclopamine analog 2 with the hedgehog signaling pathway was tested in an established reporter gene assay [1]

Luciferase reporter assay
Incubation of cells. For performing the assay, Shh-LIGHTII cells were grown to reach 80% confluence, washed twice with Versene, detached with 2 mL trypsin for not longer than 2 min and resuspended in 8 mL of growth medium. After centrifugation for 3 min, the supernatant was removed and the cell pellet dissolved in approximately 10 mL of growth medium. Finally, 20,000 to 100,000 cells per well were cultured in a 24-well plate for 48 h. For exposure to the analog the growth medium was removed and the cells were exposed to the compound in 500 μL/well incubation medium for 48 h. Stock solutions were prepared in EtOH and introduced in different concentrations into the incubation medium to reach a final solvent concentration of 0.05%. Due to the assay principle, Shh-LIGHTII cells had to be co-exposed to 100 nM of SAG (Smoothened agonist) for determining the inhibitory activity of the compounds. A SAG stock solution was prepared in EtOH and introduced into the medium. The final EtOHconcentrationintroduced by SAG and the test compoundwas 0.1%. As a positive control a 100 nM SAG solution in incubation medium (0.1% EtOH) was used, negative controls were treated with EtOH only.