Identification of Dihydropyrazolo[1,5- a ]pyrazin-4(5 H )-ones as Cyclic Products of β-Amidomethyl Vinyl Sulfone Alphavirus Cysteine Protease Inhibitors.

Optimized syntheses of ( E )-5-(2-ethoxyphenyl)- N -(3-(methylsulfonyl)allyl)-1 H -pyrazole-3-carboxamide (RA-0002034, 1 ), a promising antiviral covalent cysteine protease inhibitor lead, were developed. The syntheses avoid the contamination of 1 with the inactive cyclic dihydropyrazolo[1,5- a ]pyrazin-4(5 H )-one 2 , which is formed by the intramolecular aza-Michael reaction of the vinyl sulfone warhead under basic conditions and slowly at pH 7.4 in phosphate buffer. The pure cysteine protease inhibitor 1 could be synthesized using either modified amide coupling conditions or through the introduction of a MOM-protecting group and was stable as a TFA or HCl salt. Although acyclic 1 demonstrated poor pharmacokinetics with high in vivo clearance in mice, inactive cyclic 2 showed improved plasma exposure. The potential use of cyclic dihydropyrazolo[1,5- a ]pyrazin-4(5 H )-ones as prodrugs for the acyclic β-amidomethyl vinyl sulfone warhead was demonstrated by GSH capture experiments with an analog of 2 .