Design, synthesis, and structural analysis of influenza neuraminidase inhibitors containing pyrrolidine cores

The discovery of (±)-(2S,3R,4R)-2-(trifluoroacetamido)methyl-3-amino-1- (N′-ethyl-N′-isopropylcarbamyl)pyrrolidine-4-carboxylic acid (A-192558, 20e) as a potent inhibitor of influenza neuraminidase (NA) is described. Efficient syntheses of two core structures, cis-3-(allyloxycarbonyl)amino-1-(9′-fluorenylmethoxycarbonyl) pyrrolidine-4-carboxylic acid (7) and tert-butyl (±)-(2S,3R,4R)-2-aminomethyl-3-bis(tert-butyloxycarbonyl)amino-1- (N′-ethyl-N′-isopropylcarbamyl)pyrrolidine-4-carboxylate (18b), were developed. Starting with these core structures and using available structural information of the NA active site as the guide, analogues were synthesized in both the tri- and tetrasubstituted pyrrolidine series by means of high-throughput parallel synthesis in solid or solution phase for expeditious SAR. These studies accelerated the identification of (±)-(2S,3R,4R)-2-(trifluoroacetamido)methyl-3-amino-1- (N-ethyl-N-isopropylcarbamyl)pyrrolidine-4-carboxylate (20e, A-192558) as the most potent NA inhibitor in this series (IC₅₀ = 0.2 μM against NA A and 8 μM against NA B). The X-ray crystallographic structure of A-192558 bound to NA revealed the predicted interaction of the carboxylic group with the positively charged pocket (Arg118, Arg292, Arg371) and interaction of the trifluoro-acetamino residue with the hydrophobic pocket (Ile222, Trp178) of the enzyme active site. Surprisingly, the ethyl and isopropyl groups of the urea functionality induced a conformational change of Glu276, turning the Glu276/Glu277 hydrophilic pocket, which normally accommodates the triglycerol side chain of substrate sialic acid, into an induced hydrophobic pocket.