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

J Med Chem. 1990 Feb;33(2):464-79. 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 kOH-, inhibition constants Ki, and their component acylation and deacylation rate constants, kon and koff, respectively. The ranges for the compounds are considerable; alkaline hydrolysis rates and kon span 6, koff covers 5, and ki 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 alkyl 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 physiochemical factors that underlie these trends in Ki are further analyzed in terms of equations that describe kon and koff. 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 kOH = 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 Ki = 42 pM.

MeSH terms

  • Acylation
  • Binding Sites
  • Chemical Phenomena
  • Chemistry
  • Drug Design
  • Humans
  • Kinetics
  • Leukocytes / enzymology*
  • Oxazines / chemical synthesis*
  • Oxazines / pharmacology
  • Pancreatic Elastase / antagonists & inhibitors*
  • Protease Inhibitors / chemical synthesis*
  • Regression Analysis
  • Structure-Activity Relationship


  • Oxazines
  • Protease Inhibitors
  • Pancreatic Elastase