Synthesis of phosphorus containing pseudopeptide inhibitors of folylpolyglutamate synthetase.

Abstract

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 (K_i = 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 (K_i = 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<super>'</super><italic>S</italic>, 2<super>&Prime;</super><italic>S</italic>) 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 (2<italic>S</italic>, 2<super>'</super>S) and (2<italic>S</italic>, 2<super>'</super><italic> R</italic>) 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 P<super>III</super> 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<italic> S</italic>,2<super>'</super><italic>S</italic>) phosphinate pseudodipeptide to the (2<italic>S</italic>, 2<super>'</super><italic>R</italic>) 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<super>'</super><italic> S</italic>, 2<super>&Prime;</super><italic>S</italic>, was 20 nM at saturating substrate concentration. The synthesis of a tripeptide analog containing the pteroyl heterocycle was also completed.