Inhibition of serine proteases by a new class of cyclosulfamide-based carbamylating agents

Abstract

A new class of carbamylating agents based on the cyclosulfamide scaffold is reported. These compounds were found to be efficient time-dependent inhibitors of human neutrophil elastase (HNE). Exploitation of the three sites of diversity present in the cyclosulfamide scaffold yielded compounds which inhibited HNE but not proteinase 3 (PR 3) or bovine trypsin. The findings reported herein suggest that the introduction of appropriate recognition elements into the cyclosulfamide scaffold may lead to highly selective agents of potential value in the design of activity-based probes suitable for investigating proteases associated with the pathogenesis of chronic obstructive pulmonary disease.

Figure 1

General structure of inhibitor (I).

Figure 2

Progress curves for the inhibition of human neutrophil elastase (HNE) by inhibitor 7g. Absorbance was monitored at 410 nm for reaction solutions containing 10 nM HNE, 105 μM MeOSuc-AAPV p-nitroanilide, and the inhibitor at the indicated inhibitor to enzyme ratios in 0.1 M HEPES buffer containing 0.5 M NaCl, pH 7.25, and 2.5% DMSO. The temperature was maintained at 25°C, and reactions were initiated by the addition of enzyme. Inset shows the linear dependence of k_obs with increasing [I].

Figure 3

Time dependent loss of enzymatic activity. Percent remaining activity versus time plot obtained by incubating inhibitor 7e (37 μM) with human neutrophil elastase (700 nM) in 0.1 M HEPES buffer containing 0.5 M NaCl, pH 7.25, and 1% DMSO. Aliquots were withdrawn at different time intervals and assayed for enzymatic activity using MeOSuc-AAPV p-NA by monitoring the absorbance at 410 nm.

Figure 4

Effect of hydroxylamine on enzyme reactivation. Human neutrophil elastase (700 nM) was totally inactivated by incubating the enzyme with a 20-fold excess of inhibitor 7e (35 μM) for 15 minutes in 0.1 M HEPES buffer/0.5 M NaCl, pH 7.25. Excess hydroxylamine (0.7 mM) was then added, and aliquots were removed at different time intervals and assayed for enzyme activity using MeOSuc-Ala-Ala-Pro-Val pNA (o). Enzyme activity was determined by comparison with a control (absence of hydroxylamine) at the same time point (•).

Figure 5

Postulated mechanism of action of (I).

Scheme 1

Synthesis of compounds 7a-g