The similarity of spatial structure between radicicol and matrine urged us to perform conformation modification of matrine, followed by L-shaped matrine derivatives, 6, 12, 21a-h and 22a-h were originally designed, synthesized and evaluated for Hsp90<sup>N</sup> inhibitors as anticancer agents. TSA (Thermal Shift Assay) results indicated that 21e, 22a-c and 22e-g exhibited strong binding force against Hsp90<sup>N</sup> with∣ΔTm∣ > 3, meanwhile, MTT assay also revealed these compounds displayed potent anticancer activity with IC<sub>50</sub> values below 25 μM against HepG2, HeLa and MDA-MB-231 cells lines. Then, compound 22g with a high ΔTm = 10.92 was chosen as a representative to perform further mechanism study. It can induce cell apoptosis, arrest the cell cycle at the S phase and decrease the expression level of Hsp90 in Hela cell. These results originally provided targeted modification strategy for matrine derivatives to serve as Hsp90 inhibitors for cancer therapy.