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

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 mast 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.