Serine proteases stable derivatives

SCHEME 11.3 Postulated mechanisms for the inhibition of serine proteases by coumarin derivatives. NuH nucleophile. Pathway a suicide-type inactivation (suicide substrate). Pathway b transient inactivation by formation of a stable acyl-enzyme (alternate substrate-inhibitor). 

Compound 51 was found to be unstable and difficult to purify, as described in the literature [93—95]. Therefore, 51 was not isolated, but was instead converted to the stable pinacol 1-acetamido-l-hexylboronate derivative 52. However, the acylated derivative 52 could not be purified by column chromatography as it was destroyed on silica gel and partially decomposed on alumina. Fortunately, we found that it dissolves in basic aqueous solution (pH > 11) and can then be extracted into diethyl ether when the pH of the aqueous layer is 5—6. Finally, pure 52 was obtained by repeated washing with weak acids and bases. It should be mentioned here that exposure to a strongly acidic solution, which also dissolves compound 51, results in its decomposition. Compared with other routes, the present two-step method involves mild reaction conditions (THF, ambient temperature) and a simple work-up procedure. It should prove very useful in providing an alternative access to a-aminoboronic esters, an important class of inhibitors of serine proteases. 

Ecotin (eco) is a potent inhibitor of serine proteases that is derived from Escherichia coli. It was originally named for its ability to inhibit trypsin (E. coli trypsin inhibitor), but it is known to interact with and inhibit virtually all characterized tryp-sin-fold serine proteases. It is insensitive to the active site PI preference of the protease (the amino acid N-terminal to the cleaved or scissile bond ) and inhibits proteases with specificity towards basic, large hydrophobic, small aliphatic and acidic amino acids [2]. This remarkable breadth of inhibition classifies eco as a fold-specific inhibitor. It forms a unique tetrameric complex consisting of two protease molecules and two inhibitor molecules (the E2P2 complex), binding in a bi-dentate manner with two surface loop regions known as the primary and secondary sites (3) (Fig. 7.1). Eco itself is a 142 amino acid protein that forms a stable... 

A final group of covalent small-molecule inhibitors of proteases are mechanism-based inhibitors. These inhibitors are enzyme-activated irreversible inhibitors, and they involve a two-hif mechanism that completely inhibits the protease. Some isocoumarins and -lactam derivatives have been shown to be mechanistic inhibitors of serine proteases. A classic example is the inhibition of elastase by several cephalosporin derivatives developed at Merck (Fig. 8). The catalytic serine attacks and opens the -lactam ring of the cephalosporin, which through various isomerization steps, allows for a Michael addition to the active site histidine and the formation of a stable enzyme-inhibitor complex (34). These mechanism-based inhibitors require an initial acylation event to take place before the irreversible inhibitory event. In this way, these small molecules have an analogous mechanism of inhibition to the naturally occurring serpins and a-2-macroglobin, which also act as suicide substrates. 

Yamamoto et al. [4] showed that 0.01% aprotinin (a serine protease inhibitor) reduced the metabolism of insulin and proinsuHn in homogenates of albino rabbit buccal mucosa, which otherwise would have occurred at 70-80% within 2.5 hours. Moreover, Lehr et al. suggest that polycar-bophil, a bioadhesive polymer, may protect some peptides from proteolysis, though the mechanism of this is unknown [5]. Others [6] have developed a series of pro-dmgs for peptides, with the aim of overcoming the metabohc barrier imposed by different peptidases. Stable prodrugs proved to be N-hydroxymethylated derivatives of the assessed dipeptides Gly-L-Leu and Gly-L-Ala [6]. 

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