Pyranoxanthenones, pyranothioxanthenones, and their pyrazole-fused derivatives, which bind to DNA, block the G₂ + M phase of the cell cycle and inhibit the proliferation of ascitic and solid tumor cell lines in vitro, were tested for their ability to induce apoptosis in HL-60 cells.

            _______Compounds_______

            Number    X    NR₁R₂      __  R₃

                 7         O    N(CH₂CH₃)₂    H

                 8         O    N(CH₃)₂          H

            _ 10         O    N(CH₂)₄          H  

The pyrazole-fused pyranoxanthenones 7, 8 and 10 are more potent than the pyranoxanthenones and reduce the proliferation of HL-60 cells at 2-4 days in the 3-5 μM range. These drugs inhibit DNA synthesis at 2 h in relation with their ability to block the cellular uptake of purine and pyrimidine nucleosides within 15 min. Internucleosomal DNA fragmentation, a late marker of apoptosis, is induced by 7 and 10 at 24 h. Since caspase-mediated poly(ADP-ribose) polymerase-1 (PARP-1) cleavage is an early event required for apoptosis, the activations of initiator caspase-2 and -9 and effector caspase-3 and the cleavage of PARP-1 are induced by 7 and 10 within 9-12 h. Mitochondrial cytochrome c, the apoptotic trigger that activates caspase-9, is also released within 9 h in HL-60 cells treated with 7 and 10. However, 7 and 10 do not cause the rapid mitochondrial depolarization and swelling linked to mitochondrial permeability transition. Hence, pyrazole-fused pyranoxanthenones, which bear structural similarity to both the acridone alkaloid acronycine and the anthrapyrazoles, are DNA-interacting antiproliferative drugs that do not directly target mitochondria in cell and cell-free systems to induce the intrinsic pathway of apoptosis.