The first stereoselective total synthesis of the naturally occurring flavan myristinin A has been accomplished, as well as its biochemical evaluation. This synthesis verified the structural assignment and allowed for the determination of the absolute stereochemistry. Myristinin A exhibits biochemical activity both as a potent DNA-damaging agent and DNA polymerase beta inhibitor. Relaxation of supercoiled plasmid DNA was observed at picomolar concentrations, in addition to inhibition of polymerase beta at low micromolar concentrations.

A study of di- and trihydroxyalkylbenzenes and bis(dihydroxyalkylbenzenes) revealed that several compounds were capable of both mediating Cu(2+)-dependent DNA cleavage and strongly inhibiting DNA polymerase beta. The most potent DNA polymerase beta inhibitors were bis(dihydroxyalkylbenzenes) 5 and 6; compounds 3 and 4 were also reasonably potent. The length of the alkyl substituent was found to be a critical element for DNA polymerase beta inhibition, since compounds 1 and 2 had shorter substituents than 3 and were completely inactive. Lineweaver-Burk plots revealed that 3, 4, and 6 exhibited mixed inhibition of DNA polymerase beta with respect to both activated DNA and dTTP. Unsaturated bis(dihydroxyalkylbenzene) 5 was a pure noncompetitive inhibitor with respect to both substrates and associated avidly with the enzyme whether or not it was in complex with its substrate(s). Copper(II)-mediated DNA cleavage was the most pronounced for the trihydroxyalkylbenzene 3, consistent with an earlier report [Singh, U. S., Scannell, R. T., An, H., Carter, B. J., and Hecht, S. M. (1995) J. Am. Chem. Soc. 117, 12691-12699]. Unsaturated bis(dihydroxyalkylbenzene) 5 was the next most active DNA cleaving agent, followed by the dihydroxyalkylbenzene 4. The saturated bis(dihydroxyalkylbenzene) (6) did not cleave DNA well in a cell-free system under the conditions studied but nonetheless potentiated the effects of bleomycin to the greatest extent in cell culture studies. Interestingly, compound 5 produced a reduction in the numbers of viable cells when incubated in the presence of bleomycin and a further reduction in the numbers of viable cells in the presence of both bleomycin and Cu(2+). The same effect was noted to a lesser extent for compound 3 but not for 4 or 6.

A survey of crude plant extracts for DNA polymerase beta inhibitors resulted in the identification of a methyl ethyl ketone extract prepared from Knema elegans that strongly inhibited the enzyme. Subsequent bioassay-guided fractionation of the extract, using an assay to monitor the activity of DNA polymerase beta, led to the isolation of two potent inhibitors, (+)-myristinins A (1) and D (2), which are known flavans having unusual structures. (+)-Myristinins A and D exhibited IC50 values of 12 and 4.3 microM, respectively, as inhibitors of DNA polymerase beta in the presence of bovine serum albumin (BSA), and 2.7 and 1.2 microM in the absence of BSA. As such, they are the most potent DNA polymerase beta inhibitors reported to date. Compounds 1 and 2 potentiated the cytotoxicity of bleomycin toward cultured P388D1 cells, reducing the number of viable cells by at least 30% when employed at 9 microM concentration for 6 h in the presence of an otherwise nontoxic concentration of bleomycin (75 nM). Principles 1 and 2 also induced strong Cu2+-dependent DNA strand scission in a DNA cleavage assay. Accordingly, 1 and 2 exhibit two activities, namely, DNA polymerase beta inhibition and DNA damage.

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