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 microM against NA A and 8 microM 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 trifluoroacetamino 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.

Synthesis of 2-substituted (+/-)-(2r,3r,5r)-tetrahydrofuran-3,5-dicarboxylic acid derivatives.

An efficient synthesis of 2-substituted (+/-)-(2R,3R,5R)-tetrahydrofuran-3,5-dicarboxylic acid derivatives has been developed. Starting from 5-norborne-2-ol, the key intermediate (+/-)-methyl 5,6-exo,exo-(isopropylidenedioxy)-2-oxabicyclo[2.2.1]heptane-3-exo-carboxylate (15) was synthesized in an efficient six-step sequence. The key transformation is the base-catalyzed methanolysis-rearrangement of (+/-)-6,7-exo,exo-(isopropylidenedioxy)-4-exo-iodo-2-oxabicyclo[3.2.1]octan-3-one (14). Further manipulation of the 3-substituent of (+/-)-methyl 5,6-exo,exo-(isopropylidenedioxy)-2-oxabicyclo[2.2.1]heptane-3-exo-carboxylate (15) followed by deprotection of the diol moiety and ring opening catalyzed by RuCl(3)/NaIO(4) gave the title compounds in good yield.

Isolation and characterization of oligodeoxynucleotides containing dG-N2-AAF and oxidation products of dG-C8-AF.

Decadeoxynucleotides containing N-(deoxyguanosine-N2-yl)-2-acetylaminofluorene (dG-N2-AAF) and three recently described products of oxidation of N-(deoxyguanosine-8-yl)-2-aminofluorene (dG-C8-AF) were isolated and characterized. dG-N2-AAF was synthesized; its structure was established by mass spectroscopic and 1H-NMR analysis. Decadeoxynucleotides containing dG-N2-AAF and dG-C8-AAF were prepared by permitting d(CACTAGTCAC) to react with N-acetoxy-AAF and separating the products by HPLC. The decamer containing dG-C8-AAF was incubated under aerobic alkaline conditions. In the presence of 2-mercaptoethanol, the adduct is deacetylated; in the absence of antioxidant, decamers bearing oxidation products are formed. Homogeneity of the modified oligomers was established by polyacrylamide gel electrophoresis. The modified oligodeoxy-nucleotides will be used to introduce dG-N2-aminofluorene adducts and oxidative lesions, site-specifically, into DNA, thereby to correlate these adducts with their mutagenic properties.