Synthesis of phosphorus containing pseudopeptide inhibitors of folylpolyglutamate synthetase

Folylpolyglutamate synthetase (FPGS) catalyzes the synthesis of poly (gamma-glutamyl) metabolites of folates and antifolates. The design and synthesis of inhibitors of FPGS are important for studying the significance of poly (gamma-glutamyl) metabolite synthesis and degradation in cellular regulation and could be an important lead in increasing the efficacy of current and future antifolates in use as anti-tumor agents.; Previous results from our laboratory have shown that phosphorus-containing pseudopeptides are excellent inhibitors of FPGS. The stereospecific methotrexate-based phosphonate has been reported (Ki = 46 nM against human FPGS). More recently the synthesis of racemic folate- and methotrexate-based phosphinate inhibitors of FPGS has been reported. The racemic methotrexate analog is a potent inhibitor of human FPGS (Ki = 3.1 nM). FPGS is known to accept only substrates of the L-amino acid configuration and thus it is likely that only one isomer (2'S, 2″S) of the racemic mixture actually inhibits the enzyme. To test this hypothesis a stereoselective route to two of the four diastereomers was devised.; This thesis describes an improved route to the fully protected phosphonate pseudopeptide and the stereoselective synthesis of the (2S, 2'S) and (2S, 2' R) isomers of the phosphinate pseudopeptide and their incorporation onto both the pteroyl and methotrexate heterocycles though an acyl azide coupling. Two improved methodologies for the synthesis of carbon-phosphorus bonds from PIII intermediates and unactivated alkyl halides were developed. During the course of this research, stereospecific routes to both enantiomers of 3-bromomethylcyclopentene were developed. A novel route to differentially protected methylene glutarate was also devised. Ultimately, the desired compounds were synthesized by a bis(trimethylsilyl)phosphite addition to methylene glutarate containing a chiral auxiliary giving a 1.2:1 ratio of the desired (2 S,2'S) phosphinate pseudodipeptide to the (2S, 2'R) diastereomer. These compounds were separated, deprotected and coupled to both the pteroyl azide and 4-amino-10-methyl pteroyl azide. The IC50 of the more potent methotrexate-based diastereomer, presumed to be 2' S, 2″S, was 20 nM at saturating substrate concentration. The synthesis of a tripeptide analog containing the pteroyl heterocycle was also completed.