Potential Inhibitors of Dihydrofolate Reductase

: Synthesis and NMR Spectroscopy

Abstract

Our aim was to investigate differences in the manner various dihydrofolate reductases (DHFRs) bind folic acid analogues. These studies were hoped to eventually lead to the design of antifolates capable of discriminating between closely related enzymes based on small differences in their primary structures.

Several folates were synthesized and assayed against bacterial and vertebrate DHFRs to determine reduction rates and Kₘ values. NMR spectra were recorded to determine how minor modifications at N(10) and the benzene ring affected proton and ¹³C chemical shifts. Analysis of primary structure and X-ray crystallography of DHFRs containing bound antifolate suggested the presence of potential nucleophiles in the binding pockets of many reductases. Folates with reduced electron density at the benzene ring were hoped to bind preferentially to these enzymes and discriminate against others.

NMR spectroscopy suggested a ‘bent’ conformation for folates in 2:1 dimethylsulphoxide. Benzene and pteridine rings are thought to be in close proximity in space, with π-cloud interactions presumably stabilizing the molecule. Small modifications produced large chemical shift changes, mainly attributable to conformational change. Since they could not be separated into ‘through-bond’ effects (from the substituents) and ‘through-space’ effects (from the anisotropic benzene and pteridine rings), we were unable to correlate the electronic properties of substituents with changing electron density at the benzene ring. We have speculated on possible reasons behind different reduction rates and Kₘ values.

Benzene-ring fluorinated folates were synthesized with several studies in mind, including:

(i) DHFR-folate interaction in solution using ¹⁹F NMR,
(ii)Folate metabolism by analyzing urine samples, and
(iii)Positron emission tomography using ¹⁸F.

Time limitations, non-reducibility of fluorofolates, publication of a paper (Clore et al., 1984) describing some of the intended ¹⁹F NMR experiments, and inability to prepare ¹⁸F-folate have forced the cancellation of these studies.

Date of Award	Dec 1985
Original language	English
Awarding Institution	Aston University