Proteases Isocoumarins

Some of these heterocycles can also be suicide inhibitors if a new reactive structure is unmasked during acylation and this reactive species can further react with an active site nucleophile. For example, the presence of a 7-amino substituent (compounds 14, Fig. 11.9) makes the formation of a QIM possible. 

Some new 3-alkoxy-7-amino-4-chloroisocoumarins have been found recently to be poor inhibitors of a-chymotrypsin, trypsin, caspase 3, and HIV protease and to 

SCHEME 11.5 Mechanism of the inactivation of a protease with a 3-alkoxy-7-amino-4-chloro-isochromen-l-one 14.57 

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The acyl-enzyme can eliminate the 4-chlorine atom to generate this reactive intermediate that can then react with a nearby nucleophile such as His57 to give an alkylated acyl-enzyme derivative in which the inhibitor moiety is bound to the enzyme by two covalent bonds (Scheme 11.5). Inhibition is irreversible.59 The mechanism has been confirmed by X-ray structural analysis of protease-isocoumarin complexes. There is a cross-link between the inhibitor and the Serl95 and His57 residues of PPE.60 Human leukocyte elastase is also very efficiently inactivated.61... 

Bihel, F. Quelever, G. Lelouard, H. Petit, A. Alves de Costa, C. Pourqie, O. Checler, F. Thellend, A. Pierre, P Kraus, J.-L. Synthesis of new 3-alkoxy-7-amino-4-chloro-isocoumarin derivatives as new beta-amyloid peptide production inhibitors and their activities on various classes of protease. Bioorg. Med. Chem. 2003, 11, 3141-3152. 

Isocoumarins inactivate many serine proteases. For example, 7-amino-4-chloro-3-methoxyisocoumarin acylates serine 195 of elastases as follows.s... 

Powers JC, Kam C-M, Narasimhan L, Oleksyszyn J, Hernandez MA, Ueda T (2004) Mechanism-based isocoumarin inhibitors for serine proteases use of active site structure and substrate specificity in inhibitor design. J Cell Biochem 39 33—46... 

Harper, J.W., Hemmi, K., and Powers, J.C., Reaction of serine proteases with substituted isocoumarins discovery of 3,4-dichloroisocoumarin, a new general mechanism-based serine protease inhibitor. Biochemistry 24, 1831-1841, 1985. 

DCI was developed by James C. Powers and coworkers at Georgia Institute of Technology (Harper, J.W., Hemmi, K., and Powers, J.C., Reaction of serine proteases with substituted isocoumarins discovery of 3,4-dichloroisocoumatin, a new general mechanism-based serine protease inhibitor, Biochemistry 24,1831-1841,1985). This inhibitor is reasonably specific, although side reactions have been described. As with the sulfonyl fluorides and DFR the modification is slowly reversible and enhanced by basic solvent conditions and/or nucleophiles. DCI has been used as a proteosome inhibitor. See Rusbridge, N.M. and... 

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. 

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