Topoisomerase I-mediated DNA cleavage as a guide to the development of antitumor agents derived from the marine alkaloid lamellarin D: triester derivatives incorporating amino acid residues

Abstract

The marine alkaloid lamellarin D (LAM-D) has been recently characterized as a potent poison of human topoisomerase I endowed with remarkable cytotoxic activities against tumor cells. We report here the first structure–activity relationship study in the LAM-D series. Two groups of triester compounds incorporating various substituents on the three phenolic OH at positions 8, 14 and 20 of 6 H-[1]benzopyrano[4′,3′:4,5]pyrrolo[2,1- a]isoquinolin-6-one pentacyclic planar chromophore typical of the parent alkaloid were tested as topoisomerase I inhibitors. The non-amino compounds in group A showed no activity against topoisomerase I and were essentially non cytotoxic. In sharp contrast, compounds in group B incorporating amino acid residues strongly promoted DNA cleavage by human topoisomerase I. LAM-D derivatives tri-substituted with leucine, valine, proline, phenylalanine or alanine residues, or a related amino side chain, stabilize topoisomerase I–DNA complexes. The DNA cleavage sites detected at T↓G or C↓G dinucleotides with these molecules were identical to that of LAM-D but slightly different from those seen with camptothecin which stimulates topoisomerase I-mediated cleavage at T↓G only. In the DNA relaxation and cleavage assays, the corresponding Boc-protected compounds and the analogues of the non-planar LAM-501 derivative lacking the 5–6 double bond in the quinoline B-ring showed no effect on topoisomerase I and were considerably less cytotoxic than the corresponding cationic compounds in the LAM-D series. The presence of positive charges on the molecules enhances DNA interaction but melting temperature studies indicate that DNA binding is not correlated with topoisomerase I inhibition or cytotoxicity. Cell growth inhibition by the 41 lamellarin derivatives was evaluated with a panel of tumor cells lines. With prostate (DU-145 and LN-CaP), ovarian (IGROV and IGROV-ET resistant to ecteinascidin-743) and colon (LoVo and LoVo-Dox cells resistant to doxorubicin) cancer cells (but not with HT29 colon carcinoma cells), the most cytotoxic compounds correspond to the most potent topoisomerase I poisons. The observed correlation between cytotoxicity and topoisomerase I inhibition strongly suggests that topoisomerase I-mediated DNA cleavage assays can be used as a guide to the development of superior analogues in this series. LAM-D is the lead compound of a new promising family of antitumor agents targeting topoisomerase I and the amino acid derivatives appear to be excellent candidates for a preclinical development.