Linker length in podophyllotoxin-acridine conjugates determines potency in vivo and in vitro as well as specificity against MDR cell lines.

Abstract

We have synthesized two podophyllotoxin-acridine conjugates-pACR6 and pACR8. In these compounds an 9-acridinyl moiety is beta linked to the C4 carbon of the four ring system in 4'-demethylepipodophyllotoxin (epiDPT) via eighter an N-6-aminohexanylamide linker (pACR6) or via an N-8-aminooctanylamide linker containing two more carbon atoms (pACR8). The acridine-linker moiety occupies the position where different glucoside moieties, dispensable for activity, are normally linked to epiDPT in the well known epipodophyllotoxins VP-16 and VM-26. As with VP-16 and VM-26, pACR6 and pACR8 show evidence of being topoisomerase II poisons as they stimulate topoisomerase II mediated DNA cleavage in vitro and induce DNA damage in vivo. This in vivo DNA damage, as well as pACR6/pACR8 mediated cytotoxicity, is antagonized by the catalytic topoisomerase II inhibitors ICRF-187 and aclarubicin, demonstrating that topoisomerase II is a functional biological target for these drugs. Despite their structural similarities, pACR6 was more potent than pACR8 in stimulating topoisomerase II mediated DNA cleavage in vitro as well as DNA damage in vivo and pACR6 was accordingly more cytotoxic towards various human and murine cell lines than pACR8. Further, marked cross-resistance to pACR6 was seen among a panel of multidrug-resistant (MDR) cell lines over-expressing the MDR1 (multidrug resistance protein 1) ABC drug transporter, while these cell lines remained sensitive towards pACR8. pACR8 was also capable of circumventing drug resistance among at-MDR (altered topoisomerase II MDR) cell lines not over-expressing drug transporters, while pACR6 was not. Two resistant cell lines, OC-NYH/pACR6 and OC-NYH/pACR8, were developed by exposure of small cell lung cancer (SCLC) OC-NYH cells to gradually increasing concentrations of pACR6 and pACR8, respectively. Here, OC-NYH/pACR6 cells were found to over-express MDR1 and, accordingly, displayed active transport of 3H-labeled vincristine, while OC-NYH/pACR8 cells did not, further suggesting that pACR6, but not pACR8, is a substrate for MDR1. Our results show that the spatial orientation of podophyllotoxin and acridine moieties in hybrid molecules determine target interaction as well as substrate specificity in active drug transport.