Leinamycin (1) is a novel antitumor antibiotic with a unique structure featuring a 1-oxo-1,2-dithiolan-3-one moiety fused in a spiro fashion to an 18-membered lactam with a conjugated thiazole ring, exhibiting thiol-dependent DNA-cleaving activity. However, its in vivo antitumor activity is compromised by poor stability (half-life of ~6 h in pH 7 aqueous solution at 37 °C). As part of a program aimed at discovering clinically useful leinamycin analogues, we synthesized novel thioester derivatives of leinamycin via chemical modification: leinamycin reacted with iodomethane and other reagents to form thioester derivatives (2a-d) containing a 3-isothiazolidinone 1-oxide moiety, and further modification of the C-8 hydroxy group yielded derivatives 3a-e. The structures of these derivatives were determined by extensive 2D NMR studies and confirmed by X-ray crystallographic analysis (e.g., for 2d and 3c). The thioester derivatives showed enhanced stability (e.g., 3c had a half-life of 30 h vs. 6 h for leinamycin). Introduction of prodrug moieties (e.g., pivaloyloxymethyl in 2c, R-configured tetrahydropyranyl (THP) in 3c) resulted in derivatives with potent in vitro antiproliferative activity against HeLa S3 cells (IC50 as low as 0.67 nM for 3c) and significant in vivo antitumor activity against mouse sarcoma 180 (e.g., T/C of 0.26 for 3c). The configuration of the THP group was crucial for activity, with the R-isomer (3c) being more potent than the S-isomer (3d). Mechanistic studies suggested these derivatives may act as prodrugs that release dithiolanone compounds in biological media. In summary, we discovered novel, stable thioester derivatives of leinamycin with potent in vitro antiproliferative activity and in vivo antitumor activity. Compound 3c (KF22678), which showed potent activity against human tumor xenografts (e.g., lung, liver, ovary, prostate, and colon carcinomas), was selected for further evaluation as a promising antitumor agent.