1,2-BENZISOTHIAZOL-3-ONE 1,1-DIOXIDE INHIBITORS OF HUMAN MAST-CELL TRYPTASE

A library of compounds were prepared by reacting 2-(bromomethyl)-1,2-b enzisothiazol-3(2N) one 1,1-dioxide (5) with commercially available ca rboxylic acids in the presence of potassium carbonate or a tertiary am ine base. From this library, 1-dioxido-3-oxo-1,2-benzisothiazol-2(SH)- yl)methyl N-[(phenylmethoxy)carbonyl]-beta-alanate (7b) emerged as a p otent inhibitor of human mast cell tryptase (IC50 = 0.85 mu M) Extensi on of the side chain of 7b by two carbons gave 1-dioxido-3-oxo-1,2-ben zisothiazol-2(3H)-yl)methyl 5-[[(phenylmethoxy)carbonyl]amino]-pentano ate (7d) which was an 8-fold more potent inhibitor (IC50 = 0.1 mu M). Further modification of this series produced benzoic acid derivative - dioxido-3-oxo-1,2-benzisothiazol-2(3H)-yl)-methyl 4-[[(phenylmethoxy)c arbonyl] amino]benzo ate (7n) which is the most potent inhibitor ident ified in this series (IC50 = 0.064 mu M). These compounds exhibit time -dependent inhibition consistent with mechanism-based inhibition. For 7b, the initial enzyme velocity is not a saturable function of the inh ibitor concentration and the initial Ki could not be determined (K-i > 10 mu M). The steady-state rate constant, K-i, was determined to be 396 nM. On the other hand, compounds 7d and 7n are time-dependent inhi bitors with a saturable initial complex. From these studies, an initia l rate constant, K-i, for 7d and 7n was found to be 345 and 465 nM, re spectively. The steady-state inhibition constants, K-i, for 7d and 7n were calculated to be 60 and 52 nM, respectively. Compound 7n is a 13 -fold more potent inhibitor than 7b, and these kinetic studies indicat e that the increase in inhibitory activity is due to an increase in in itial affinity toward the enzyme and not an increase in chemical react ivity. These inhibitors generally show high selectivity for tryptase, being 40-fold weaker inhibitors of elastase, being 100-fold weaker aga inst trypsin, and showing no inhibition against thrombin. These compou nds are not inhibitors of thrombin, plasmin t-PA, urokinase and factor Xa (IC50 > 33 mu M). In the delayed-type hypersensitivity (DTH) mouse model, a model of skin inflammation, a 5% solution of 7d reduced edem a by 69% compared to control animals.