In this study, a series of C-28 amides derivatives of hederagenin with or without the presence of an acetyl group at positions 3 and 23 in ring A, were synthetized aiming to develop potent cytotoxic agents. Their structures were confirmed by MS, IR, 1H NMR and 13C NMR spectroscopic analyses and their cytotoxic activities were screened in SRB assays using a panel of six human cancer cell lines. The majority of the amide derivatives were cytotoxic for a variety of human tumor cell lines. In general, the hydroxylated derivatives (1a-1d; EC50 in the range 1.2-22.5 μM) were less active than the acetylated derivatives (2a-2n; EC50 in the range 0.4-9.0 μM). Hydroxylated derivative bearing pyrrolidinyl substituent 1c, was the most active for HT29 human line cells (EC50 = 1.2 μM), however their acetylated derivative 2c was the most potent and selective against A2780, FaDu, SW1736 cells, showing EC50 values between 0.4 and 1.7 μM and SI between 5.6 and 24. Staining experiments combined with fluorescence microscopy indicate that the cell membrane became permeable, and finally a process of secondary necrosis was observed. In addition, the docking results showed that acetylated compounds display more affinity to HER2 than to USP7, indicating that HER2 is a most probable receptor, both proteins found in tumor cell line A2780.