3-Ethoxycarbonyl-5-phenyl-1, 3a, 4, 5, 6, 6a-hexahydropyrrolo[3,4-c]
pyrazole-4, 6-dione, 2, 2, 6, 6-tetraethyl-1H, 5H-
pyrazole[1, 2-a]
pyrazole-1, 3, 5, 7-[2H, 6H]-tetraone and 6-ethoxycarbonyl-3-phenyl-3-azabicyclo[3.1.0] hexane-2, 4-dione demonstrated potent cytotoxic activity in the human Tmolt3, Tmolt4 and HL-60
leukemia screens, HuT-78
lymphoma and HeLa suspended uterine
carcinoma cell lines. Most notable was the finding that these compounds were significantly more active than the standard
cytotoxic agents examined in the MCF-7 breast (ED50 0.2-1.0 microg/ml) and U87MG
glioma (ED50 1.3-2. 6 microg/ml)
tumor screens. The agents inhibited Tmolt4
leukemia DNA and RVA syntheses after 60 min at 100 microM Multiple
enzymes involved with
nucleic acid metabolism appeared to be targeted including inhibition of
RNA polymerases,
ribonucleotide reductase and
nucleoside kinase activities, however, inhibition of de novo
purine synthesis at the key regulatory
enzyme IMP dehydrogenase appeared to be the primary target. The predominant IMPDH
isoform (Type II) detected in a number of human
cancers, such as
leukemias, ovarian and breast, was inhibited by the compounds yielding IC50 values in the microM range. Furthermore, inhibition of
IMP dehydrogenase activity led to the selective depletion of
dGTP pool levels by two of the compounds. The
DNA molecule was not a target of the agents since no alkylation of the bases, cross-linking of the
DNA strands or intercalation between base pairs occurred. Yet, the compounds did cause DNA fragmentation after incubating at 100 microuM for 24 h which was consistent with the observed decrease in ct-
DNA viscosity.