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Development of an assay for the estimation of N¹⁰-propargyl- 5,8-dideazafolic acid polyglutamates in tumor cells

Lookup NU author(s): Professor Herbie Newell, Professor Alan Calvert

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Abstract

A method is described herein for the isolation and quantitation of polyglutamates of the thymidylate synthase (TS) inhibitor N¹⁰-propargyl-5,8-dideazafolic acid (CB3717) in tumor cells exposed to the drug in vitro. Cells were incubated with 50 μ ³H-CB3717 for 12 h and then disrupted by sonication. CB3717 and its polyglutamates were extracted by boiling in 0.01 Tris-HCl pH 10. The extract was concentrated by lyophilization and analyzed by reverse phase HPLC (10 × 0.46-cm Polygosil 5-μm C₁₈ column) using linear gradient elution (5–16% acetonitrile in 0.1 sodium acetate, pH 5, over 15 min, 2 ml/min). Recovery of radioactivity at each stage of the method was >70%. CB3717 and its polyglutamates were identified by co-chromatography with synthetic standards and by inhibition of partially purified TS. Quantitation was by means of radiochemical analysis. The ³H-CB3717 used in these studies was prepared by catalytic tritiation of diethyl-(2-chloro-4-nitrobenzoyl)- -glutamate followed by consecutive alkylation with propargyl bromide and 2-amino-6-bromomethyl-3,4-dihydro-4-oxoquinazoline hydrobromide. The free diacid was prepared as required by hydrolysis in sodium hydroxide and purified by HPLC. Tritiation in only one position was confirmed by ³H NMR. Following the exposure of L1210 leukemia cells to 50 μ ³H-CB3717 for 12 h the total cellular radioactivity level was approximately 7 μ , of which 27% was present as polyglutamated metabolites with four and five glutamate residues. Author Keywords: folic acids; drug metabolism; organic synthesis, radioactive; cancer chemotherapy; HPLC, drug metabolites; isotope analysis

Publication metadata

Author(s): Sikora E, Newell DR, Jackman AL, Simmonds AJ, Jones TR, Calvert AH

Publication type: Article

Publication status: Published

Journal: Analytical Biochemistry

Year: 1988

Volume: 172

Issue: 2

Pages: 344-355

Print publication date: 01/08/1988

ISSN (print): 0003-2697

ISSN (electronic): 1096-0309

URL: http://dx.doi.org/10.1016/0003-2697(88)90454-X

DOI: 10.1016/0003-2697(88)90454-X

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