Design and synthesis of 4H-3,1-Benzoxazin-4-ones as potent alternate substrate inhibitors of human leukocyte elastase

Article abstract of DOI:10.1021/jm00164a002

4H-3,1-Benzoxazin-4-ones are alternate substrate inhibitors of the serine proteinase human leukocyte elastase (HL elastase) and form acyl enzyme intermediates during enzyme catalysis. We have synthesized a large variety of benzoxazinones using specific methods that have been adapted to achieve the pattern of ring substitution dictated by theoretical considerations. The results of the inhibition of HL elastase by 175 benzoxazinones are reported herein with reference to hydrophobicity constants D, alkaline hydrolysis rates κ(OH^-), inhibition constants K(i), and their component acylation and deacylation rate constants, κ(on) and κ(off), respectively. The ranges for the compounds are considerable; alkaline hydrolysis rates and κ(on) span 6, κ(off) covers 5, and K(i) spans 8 orders of magnitude. Multiple regression on this large data set has been used to isolate the contributions of electronic and steric effects, as well as other factors specific to compound stability and elastase inhibition. Essentially, a simple electronic parameter is sufficient to account for almost all the variance in the alkaline hydrolysis data, indicating that electronic factors are the major determinants of this type of benzoxazinone reactivity. Factors that significantly enhance the potency of benzoxazinones I are R₅ alkly groups and electron withdrawal by R₂. Bulk in R₇ and R₈ and compound hydrophobicity are not significant, but substitution in R₆ is highly unfavorable as are substituents linked via carbon to C₂. The physicochemical factors that underlie these trends in K(i) are further analyzed in terms of equations that describe κ(on) and κ(off). A conclusion that emerges is that chemically stable, potent benzoxazinone inhibitors of HL elastase with inhibition constants in the nanomolar range can be designed with (1) R₅ alkyl groups to inhibit enzyme-catalyzed deacylation, (2) small alkyl substituents linked via heteroatoms to C₂ to enhance acylation and limit deacylation rates, and (3) strongly electron-donating groups at C₇ to stabilize the oxazinone ring to nucleophilic attack. Thus, 2-(isopropylamino)-5-n-propyl-7-(dimethylamino)benzoxazinone 95 has κ(OH^-) = 0.01 M^-1 s^-1, which extrapolates to a half-life at pH 7.4 of over 8.5 years, and 2-ethoxy-5-ethylbenzoxazinone 38 has K(i) = 42 pM.