9-nitrocamptothecin (9-NC), an active derivative of camptothecin (CPT), demonstrated antitumor effect on experimental tumors in mice by topoisomerase I (Topo I) inhibition. However, under human physiological conditions, the rapid opening of lactone ring of 9-NC resulting in the formation of inactive and high toxic carboxylate limited its clinical efficacy. Therefore, strategies aimed to maintain the active closed-lactone form of 9-NC in the plasma were developed, such as prodrugs. In our study, 9-nitro-20(S)-carbonate-camptothecin (NCP4), a novel prodrug of 9-NC, was designed and synthesized. A preclinical evaluation of the chemotherapeutic potential of NCP4 was performed in vitro and in vivo. In cytotoxicity assay against six human cancer cells, the cytotoxic effect of NCP4 was slightly weaker than 9-NC. In addition, our data showed that 9-NC can be converted from NCP4 in vivo, and that the intracellular conversion of NCP4 to its active metabolites was correlated well with its cytotoxicity, demonstrating that NCP4 could serve as a prodrug of 9-NC. In human hepatoma Bel-7402 xenografts, NCP4 by intravenous injection showed more potent antitumor efficacy than 9-NC. Mechanistically, NCP4 induced cell apoptosis by increasing the expressions of caspase-3 and Bax in tumor tissues. In human hepatoma Hep G2 xenografts, NCP4 by oral administration significantly inhibited tumor growth. Importantly, the toxic effect of NCP4 on mice was much lower than 9-NC, demonstrating improved safety of NCP4. Overall, our study indicated that NCP4 would be a promising anticancer candidate and worthy of further investigation. CI - Copyright (c) 2022. Published by Elsevier B.V.