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"Reverse" analogues (RAD's) of actinomycin D (AMD) and their antitumor activity against mouse and human colon tumor cells are reported. RAD's are tetracyclic, and they have an oxazole ring fused on the tricyclic phenoxazine chromophore of AMD. The oxazole ring in RAD is substituted at the C-2 carbon with either a CH3 (in RAD I), a C6H5 (in RAD II), or a CH2CONH(CH2)4NH2 (in RAD III) group. In tumor cells and rat hepatic microsomes, RAD's are metabolized to a tricyclic "symmetrical" analogue of AMD (SAD) with the loss of the oxazole ring and its substituents. RAD and SAD are very active in priming superoxides in the presence of microsomal enzymes as well as in inhibiting the synthesis of DNA and the growth of human colon tumor HT-29 cells in vitro. RAD III and SAD efficiently cleave closed circular plasmid pBR322 DNA like the antitumor agent bleomycin. In addition to their strong inhibitory activity against P388 and B16 tumors in vitro and in vivo, RAD III and SAD demonstrate high levels of activity against primary C26 and advanced C38 colon tumors in mice and against a xenograft of human colon adenocarcinoma CX-1 in athymic mice. In all these biological activities, the analogues demonstrate superiority to AMD in several experimental tumors. Also, the analogues, in contrast to AMD, show reduced toxicity in tumor-free mice, which is possibly due to the metabolic deactivation of SAD in host organs.

1,4-Oxazinone derivatives of the phenoxazinone chromophore in actinomycin D (AMD) have been synthesized by condensation of AMD with alpha-keto acids. By varying the starting alpha-keto acid, the substitutions on the oxazinone ring and, consequently, the lipophilicity of the molecule could be altered. These oxazinone derivatives revert to AMD in physiological media and it appears that these oxazinones are "depot" forms of AMD and possess physicochemical and DNA-binding properties which are significantly different from those of AMD. The oxazinones, which have bulky and lipophilic substituents at position 3, demonstrate more pronounced antitumor activity against P388 mouse leukemia and are less toxic than AMD.

Two different tetracyclic chromophoric analogues of actinomycin D have been synthesized by engaging two chromophoric DNA-binding functions in actinomycin D, i.e., 2-amino and 3-oxo, into either a 1,4-oxazin-2-one or an oxazole ring system. A third analogue has an extra quinone function at C-8 of the oxazole analogue. In all the analogues the chemical integrity of the peptide lactones of the parent antibiotic is kept intact, but their sterochemistry is altered. The analogues are designed as transport-modified prodrug forms of either the tricyclic active analogues of actinomycin D or actinomycin D itself. All analogues exhibit cytotoxicity that is several-fold less potent than AMD; they also have no binding affinity toward extracellular DNA. Nonetheless, the analogues of the first and the third series show improved antitumor activities (P388 leukemia, CDF1 mice). In fact, two of these analogues having a phenyl substituent at the C-3 site of the oxazinone ring or the C-2 position of the 8-oxo-8H-oxazole ring exhibit the highest antitumor effects. Most of the analogues are active over a broader dose range than actinomycin D and are 6- to 16-fold less cytotoxic to human lymphoblastic leukemia (CCFR-CEM) cells in vitro. The analogues with the most pronounced antitumor activity are those that retain most elements in the peptide stereochemistry of actinomycin D and have a quinone function or demonstrate susceptibility of their chromophores to biotransformation.

DNA-binding strengths of three anthracene-9,10-dicarboxaldehyde hydrazones were examined by spectral shifts of the drug-DNA combinations, spectral titration by Scatchard plots, elevation of DNA melting temperature during complexation and comparison of spectral shifts in the presence of DNA's having variable base contents. Dissociation of the DNA-complexes was also observed. The results showed a strong degree of binding by the three compounds. They did not exhibit noticeable base-pair specificity, but both associated with and dissociated rapidly from DNA. Scatchard plots for DNA association indicated two types of binding; the stronger was most likely due to intercalation of the planar anthracene ring into the DNA double helix. No direct correlation can be drawn between the observed anti-cancer activities and DNA binding affinities of these compounds in vitro.