Synthesis and evaluation of diphenyl phosphonate esters as inhibitors of the trypsin-like granzymes A and K and mast cell tryptase

Article abstract of DOI:10.1021/jm970543s

Thirty-six new amino acid and peptidyl diphenyl phosphonate esters were synthesized and evaluated to identify potent and selective inhibitors for four trypsin-like proteases: lymphocyte granzymes A and K, human mast cell tryptase, and pancreatic trypsin. Among five Cbz derivatives of Lys and Arg homologues, Z-(4-AmPhe)(P)(OPh)₂ is the most potent inhibitor for granzyme A, and Z-Lys(P)(OPh)₂ is the best inhibitor for granzyme K, mast tryptase, and trypsin. The amidino P1 residue D,L-(4-AmPhGly)(P)(OPh)₂ was utilized in a series of compounds with several different N-protecting groups and systematic substitutions at P2 in Cbz-AA derivatives and at P3 in Cbz-AA-Ala derivatives. Generally, these phosphonates inhibit granzyme A and trypsin more potently than granzyme K and tryptase. The P2 Thr and Ala dipeptide phosphonates, Cbz-AA-(4-AmPhGly)(P)(OPh)₂, are the most potent inhibitors for granzyme A, and Cbz-Thr-(4-AmPhGly)(P)(OPh)₂ (k(obs)[I] = 2220 M^-1 s^{- 1}) was quite specific with much lower inhibition rates for granzyme K and trypsin (k(obs)[I] = 3 and 97 M^-1 s^-1, respectively) and no inhibition with tryptase. The most effective inhibitor of granzyme A was Ph-SO₂-Gly- Pro-(4-AmPhGly)(P)(OPh)₂ with a second-order rate constant of 3650 M^-1 s^{- 1}. The most potent inhibitor for granzyme K was 3,3-diphenylpropanoyl-Pro- (4-AmPhGly)(P)(OPh)₂ with a k(obs)/[I] = 1830 M^-1 s^-1; all other phosphonates inhibited granzyme K weakly (k(obs)/[I] < 60 M^-1 s^-1). Human mast cell tryptase was inhibited slowly by these phosphonates with Cbz- Lys(P)(OPh)₂ as the best inhibitor (k(obs)/[I] = 89 M^-1 s^-1). The overall results suggest that scaffolds of Phe-Thr-(4-AmPhe) and Phe-Pro-Lys will be useful to create selective phosphonate inhibitors for granzymes A and K, respectively, and that P4 substituents offer opportunities to further enhance selectivity and reactivity.