Synthesis and crystal structures of D-annulated pentacyclic steroids: looking within and beyond AR signalling in prostate cancer

Carbocyclic steroids D-annulated at 16α and 17α positions with a 5-membered ring E are easily accessible via the interrupted Nazarov cyclization. Three steroid series have been structurally studied: chlorine-containing D-annulated pentacyclic steroids 2, their reduced derivatives 3 and their synthetic precursors – benzylidines of tetracyclic steroids 1. The presence of two conformers for pentacyclic steroids 2 and 3 have been found in crystals, where formation is evidently caused by the conformational flexibility of the ring E. Geometries obtained from X-ray diffraction experiments have been used as the initial ones for semi-empirical and ab initio quantum chemical calculations to confirm the reproducibility of the steroids' geometries by these methods and further used for docking studies against the human androgen receptor ligand-binding domain. The docking results have confirmed that the geometries of the studied D-annulated pentacyclic steroids perfectly match the human androgen receptor active site. Geometry analysis of docking results allowed us to explain the influence of subtle structural differences in series 2 and 3 on the ligand binding energy. Compounds with the highest binding energies have been studied in experiments on AR-positive 22Rv1 prostate cancer cells. Selected steroids revealed antiproliferative potency and inhibited AR pathways in 22Rv1 prostate cancer cells, including downregulation in NKX3.1 and PSA expression. A combination of pentacyclic steroid 2d with non-steroidal antiandrogen bicalutamide exhibited significant antiproliferative effects in 22Rv1 cells. The obtained results for this family of pentacyclic steroids provides insights for further structure–activity relationship studies towards novel drugs targeting nuclear receptors in cancer cells. Pentacyclic steroids are of great interest for the development of innovative therapeutic approaches in the treatment of prostate cancer.