As part of our ongoing silybin project, this study aims to introduce a basic nitrogen-containing group to 7-OH of 3,5,20-O-trimethyl-2,3-dehydrosilybin or 3-OH of 5,7,20-O-trimethyl-2,3-dehydrosilybin via an appropriate linker for in vitro evaluation as potential anti-prostate cancer agents. The synthetic approaches to 7-O-substituted-3,5,20-O-trimethyl-2,3-dehydrosilybins through a five-step procedure and to 3-O-substituted-5,7,20-O-trimethyl-2,3- dehydrosilybins via a four-step transformation have been developed. Thirty-two nitrogen-containing derivatives of silybin have been achieved through these synthetic methods for the evaluation of their antiproliferative activities towards both androgen-sensitive (LNCaP) and androgen-insensitive prostate cancer cell lines (PC-3 and DU145) using the WST-1 cell proliferation assay. These derivatives exhibited greater in vitro antiproliferative potency than silibinin. Among them, 11, 29, 31, 37, and 40 were identified as five optimal derivatives with IC50 values in the range of 1.40–3.06 µM, representing a 17- to 52-fold improvement in potency compared to silibinin. All these five optimal derivatives can arrest the PC-3 cell cycle in the G0/G1 phase and promote PC-3 cell apoptosis. Derivatives 11, 37, and 40 are more effective than 29 and 31 in activating PC-3 cell apoptosis.