CHEMICAL SYNTHESIS AND BIOLOGICAL PROPERTIES OF NOVEL FLUORESCENT ANTIFOLATES IN PGP-OVEREXPRESSING AND MRP-OVEREXPRESSING TUMOR-CELL LINES

We have synthesised a series of fluorescent analogues of methylbenzopr im, a diaminopyrimidine antifolate which we have previously shown to e xhibit in vivo antitumour activity in a methotrexate (MTX) ''transport -resistant'' tumour cell line. The analogues bear the dansyl, nitroben zoxodiazole or methoxycoumarin fluorophores. The cytotoxicity of the c ompounds was evaluated using the [4,5-dimethylthiazol-2-yl]-2,5-diphen yltetrazolium bromide (MTT) colorimetric assay against two human lung cancer cell lines, together with their multidrug resistant (MDR) subli nes. H69/P is a small cell line and its multidrug resistant subline H6 9/LX4 overexpresses P-glycoprotein (Pgp). COR-L23/P is a large cell li ne and its multidrug resistant subline COR-L23/R overexpresses the mul tidrug resistance associated protein (MRP). IC50 values for the compou nds (i.e. concentration to reduce cell growth by 50%) in the [4,5-dime thylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay ranged from 0. 20 to 0.81 mu M in the H69 parental line and from 0.83 to 5.10 mu M in the COR-L23 parent line. The MDR sublines both showed clear cross-res istance to each of the compounds, with resistance factors (ratio of IC 50 value in resistant vs parental cell line) ranging from 16 to 137 in H69/LX4 and from 5 to 16 in COR-L23/R. For compounds (10) and (11) wh ere drug accumulation was studied using flow cytometry, resistance was associated with an approximately 10-fold reduction in cellular drug a ccumulation over a period of 30 min. The drug resistance modifiers ver apamil (used at 6.6 mu M) and cyclosporin A (used at 4.2 mu M) were te sted for their ability to sensitise the resistant lines. Whereas verap amil showed little activity, cyclosporin A partially restored the acti vity of compound (10), and fully restored the activity of compound (11 ) in H69/LX4 cells. This sensitisation of H69/LX4 by cyclosporin A was associated with a partial restoration of the drug accumulation defici t in this line. Hence, these novel lipophilic antifolates appear to be substrates for both the P-glycoprotein and MRP resistance mechanisms. Therefore, although they have been designed to overcome one mechanism of methotrexate resistance, namely impaired drug transport, this has been achieved only at the cost of rendering them susceptible to altern ative mechanisms. BIOCHEM PHARMACOL 56;7:807-816, 1998. Crown Copyrigh t (C) 1998. Published by Elsevier Science Inc.