Growth inhibition of hepatocellular carcinoma cells in vitro and in vivo by the 8-methoxy analog of WMC79.

HKH40A (RTA 502), an optimized 8-methoxy analog of the unsymmetrical bifunctional antitumor agent WMC79, was found to be potently active against liver cancer cell growth in vitro and in vivo. Studies on selected human hepatocellular carcinoma (HCC) cell lines with differing p53 status (HepG2, Hep3B, and PLC/PRF/5), revealed that drug-mediated growth inhibition was independent of p53 status. FACS analysis showed an accumulation of cells in S-phase within 24 h of treatment with 100 nM HKH40A. Subsequent incubation of cells, either in the presence of drug or without, caused cell cycle block at the S and G2/M checkpoints, which was consistent with the observed up-regulation of p21, cyclin A, cyclin B1, sustained phosphorylation of Cdk1, and down-regulation of Cdc6, Cdc7, and RRM2. This irreversible growth arrest eventually led to apoptosis. HKH40A completely suppressed growth of the rat transplantable HCC cell line (JM-1) in an orthotopic model in Fisher 344 rats in vivo, without evidence of toxicity. HKH40A may be a useful agent for new therapeutic strategies focusing on inhibition of HCC cell proliferation.

Optimization of naphthalimide-imidazoacridone with potent antitumor activity leading to clinical candidate (HKH40A, RTA 502).

Unsymmetrical bifunctional antitumor agent WMC79 was further optimized to generate compound 7b that not only inhibited the growth of many tumor cell lines, but caused rapid apoptosis. Unlike the parent compound, 7b is toxic to both p53 positive and negative cancer cells. It has potent in vivo activity against xenografts of human colon and pancreatic tumors in athymic mice.

A new synthetic agent with potent but selective cytotoxic activity against cancer.

The synthesis of novel unsymmetrical bifunctional antitumor agents was accomplished by linking an imidazoacridone moiety to another polycyclic heteroaromatic moiety via linkers of various length and rigidity. These compounds bind to cellular DNA, but it is hypothesized that biological effects become manifested when the drug-DNA complexes interact with critical DNA binding proteins that are involved in repair and transcription. The most promising compound of the series, 4ad (WMC79), consists of an imidazoacridone linked to a 3-nitronaphthalimide moiety via a 1,4-dipropanopiperazine linker. It was found to be potently, but selectively, cytotoxic against colon cancers (GI(50) = 0.5 nM, LC(50) = 32 nM) and leukemias (GI(50) = 3.5 nM, LC(50) = 33 nM). Compound 4ad, which appears to be a candidate for further development as an anticancer drug, kills sensitive cells by induction of apoptosis. It also showed significant in vivo activity against HCT-116 colon cancer xenografts in nude mice. Other compounds in the series also exhibited antitumor properties, but they were significantly lower than that of 4ad.

Bisimidazoacridones: 2. Steady-state and time-resolved fluorescence studies of their diverse interactions with DNA.

Several bisimidazoacridones (BIA) are potent, selective antineoplastic agents, whereas others have potent anti-human immunodeficiency virus activity. BIA are bifunctional agents that consist of two imidazoacridone (IA) chromophores held together by various linkers. Interaction of BIA with DNA has been postulated to be required for their biological activity. Fluorescence data on free and bound BIA suggest that the binding of BIA and similar drugs to DNA is driven by a transfer of hydrophobic molecules from aqueous media to the more amphiphilic DNA environment. Binding to DNA was sensitive to sequence and depended on the length and rigidity of the linker. Time-resolved fluorescence measurements showed that BIA adopt an extended conformation upon binding and that all of the molecules are tightly associated with DNA. Gel-shift and melting assays indicated that one of the aromatic residues of BIA is intercalated, whereas the other, together with a linker, resides in a groove, probably the minor groove. A continuum of structures may be possible where intercalation and classical minor groove binding are limiting structures. In general, the hypothesis for the mechanism of action of BIA wherein the unintercalated residue, accessible for additional interactions, captures a critical protein involved in repair or transcription, is consistent with this model.