Chemical synthesis and biological properties of novel fluorescent antifolates in Pgp- and MRP-overexpressing tumour cell lines.
We have synthesised a series of fluorescent analogues of methylbenzoprim, a diaminopyrimidine antifolate which we have previously shown to exhibit in vivo antitumour activity in a methotrexate (MTX) "transport-resistant" tumour cell line. The analogues bear the dansyl, nitrobenzoxodiazole or methoxycoumarin fluorophores. The cytotoxicity of the compounds was evaluated using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay against two human lung cancer cell lines, together with their multidrug resistant (MDR) sublines. H69/P is a small cell line and its multidrug resistant subline H69/LX4 overexpresses P-glycoprotein (Pgp). COR-L23/P is a large cell line and its multidrug resistant subline COR-L23/R overexpresses the multidrug resistance associated protein (MRP). IC50 values for the compounds (i.e. concentration to reduce cell growth by 50%) in the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay ranged from 0.20 to 0.81 microM in the H69 parental line and from 0.83 to 5.10 microM in the COR-L23 parent line. The MDR sublines both showed clear cross-resistance to each of the compounds, with resistance factors (ratio of IC50 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) where drug accumulation was studied using flow cytometry, resistance was associated with an approximately 10-fold reduction in cellular drug accumulation over a period of 30 min. The drug resistance modifiers verapamil (used at 6.6 microM) and cyclosporin A (used at 4.2 microM) were tested for their ability to sensitise the resistant lines. Whereas verapamil showed little activity, cyclosporin A partially restored the activity 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 deficit 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 alternative mechanisms.
Department of Chemistry, University of Newcastle, Newcastle upon Tyne, UK.
C
C
Robson
K A
KA
Wright
P R
PR
Twentyman
P A
PA
Lambert
R J
RJ
Griffin
D009327:4-Chloro-7-nitrobenzofurazan / Q000737:chemistry; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis*; D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis*; D000970:Antineoplastic Agents / Q000138:chemical synthesis / Q000494:pharmacology; D002289:Carcinoma, Non-Small-Cell Lung; D018288:Carcinoma, Small Cell; D003619:Dansyl Compounds / Q000737:chemistry; D004354:Drug Screening Assays, Antitumor; D005456:Fluorescent Dyes / Q000737:chemistry*; D005493:Folic Acid Antagonists / Q000138:chemical synthesis* / Q000494:pharmacology*; D006131:Growth Inhibitors / Q000494:pharmacology; D006801:Humans; D008175:Lung Neoplasms; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011743:Pyrimidines / Q000737:chemistry / Q000494:pharmacology; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D016597:Trimetrexate / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014468:Umbelliferones / Q000737:chemistry
D009327:4-Chloro-7-nitrobenzofurazan; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D000970:Antineoplastic Agents; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D005493:Folic Acid Antagonists; D006131:Growth Inhibitors; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011743:Pyrimidines; D013762:Tetrahydrofolate Dehydrogenase; D016597:Trimetrexate; D014468:Umbelliferones; C064625:methylbenzoprim; C114462:multidrug resistance-associated protein 3; C000317:4-bromomethyl-7-methoxycoumarin