Carboxyl proteases, protease inhibitors

As a direct appUcation a potent C2-symmetric HIV-1 protease inhibitor (with two tetrazoles as carboxyl group bioisosteres) was prepared in one pot [77]. The process involved microwave-promoted cyanation followed by conversion of the nitrile group in a tetrazole with azide (Scheme 64). It is notable that the fimctionahzation was achieved so smoothly without side reactions such as the ehmination of water. 

Further, Wasserman and coworkers developed a direct acylation of stabilized phosphonium ylides by carboxylic acids in presence of the EDCI/DMAP (way c). This last method allows the introduction of a-aminoacid structures into the resulting P-oxo phosphorus ylides [19-25],opening the way to the total synthesis of depsipeptide elastase inhibitors [22,24] or cyclic peptidic protease inhibitor EurystatinA [20]. 

Coumarincarboxylate derivatives are versatile, efficient, low molecular weight, nonpeptidic protease inhibitors. Both esters and amides behave as time-dependent inhibitors of a-chymotrypsin but the esters are clearly more efficient than the corresponding amides. The criteria for a suicide mechanism are met. The presence of a latent alkylating function at the 6-position (chloromethyl group) is required to produce to inactivation by a suicide mechanism (Scheme 11.3, pathway a). Aryl esters, in particular the meta-substituted phenyl esters are the best inhibitors. Thus, m-chlorophenyl 6-(chloromethyl)-2-oxo-27/-l-benzopyran-3-carboxylate is one of the well-known inactivator of a-chymotrypsin (kJK, = 76(),000M s 1 at pH 7.5 and 25 °C, Table 11.1). 

Several aryl esters of 6-chloromethyl-2-oxo-2//-l -benzopyran-3-carboxylic acid act as human Lon protease inhibitors (alternate substrate inhibitors)46 without having any effect on the 20S proteasome. Proteasomes are the major agents of protein turnover and the breakdown of oxidized proteins in the cytosol and nucleus of eukaryotic cells,47 whereas Lon protease seems to play a major role in the elimination of oxidatively modified proteins in the mitochondrial matrix. The coumarin derivatives are potentially useful tools for investigating the various biological roles of Lon protease without interfering with the proteasome inhibition. 

Recently, Jankowski et al. [80] reported the application of MW to the Diels-Alder synthesis of N-methyloctahydroisoquinoline adducts, which are important intermediates in the synthesis of medicinally important compounds, such as HIV protease inhibitor isoquinoline carboxylate pharmaceuticals. The reaction of the arecoline 67 or its isomer 70 with Danishefsky s diene 68 in toluene (Scheme 4.32) was studied under both conventional and MW heating in sealed tubes, i. e. at elevated pressure. 

Tipranavir is a unique, nonpeptidic protease inhibitor currently in phase 111 clinical trials for HIV treatment. In this compound a phenohc hydroxyl group behaves as a pseudo carboxylic acid group and the chiral hydrogenation step shown in Figure 1.11 gives reasonable enantioselectivity. Benincori et have... 

Additionally, 1,2-dihydroxyethylene dipeptide analogues without the C-terminal carboxylic acid have been used to obtain aspartyl proteases inhibitors.[641 These efforts include stereoselective alkylation of imines, one-pot reductive amination of epoxy ketones, ring opening of epoxides with sodium azide, diastereoselective dihydroxylation of allylic amines, and enzymatic resolution and stereocontrolled intramolecular amidation. 

The naturally occurring inhibitor pepstatin (structure 16.34) binds very tightly to carboxyl proteases Kt with porcine pepsin is 4.5 X 10-11 M.175 The statine residue has a tetrahedral carbon replacing the normal carboxyl carbon, and so... 

Finally, of note is the hydrogenation of a,/J-unsaturated carboxylic acids. This may be accomplished in a highly diastereoselective manner by the use of ruthenium(II)-BINAP complexes (equation 88)351. The chemical yields are high (83-99%) and the reaction occurs in up to 97% ee. This type of hydrogenation has been used as the key step in a synthesis of building blocks for protease inhibitors and has been performed on a 100 g scale352. 

Since the discovery of cis-1 -amino-2-indanol as a ligand for human immunodeficiency virus protease inhibitors and the development of a practical industrial process for the synthesis of either ris-isomers in enantiopure form, the remarkable properties of the rigid indane platform have been used extensively in an ever-increasing number of asymmetric methodologies. In addition to the use of this amino alcohol as a chiral auxiliary and ligand for asymmetric synthesis, it has found application as a useful resolution agent. Applications include amines, carboxylic acids, and alcohols. 

Protease Classification. In order to rationally design an inhibitor for a protease it is first necessary to place it into one of four families of proteases (see Table V). For a new enzyme, a study of its inhibition profile with a series of general protease inhibitors is sufficient to classify it into one of the four families. The inhibitors usually used are diiso-propylphosphofluoridate (DFP) or phenylmethane sulfonyl fluoride (PMSF) for serine proteases, 1,10-phenanthroline for metalloproteases, thiol reagents such as iodoacetate or N-ethylmaleimide for thiol proteases, and pepstatin or diazo compounds such as diazoacetyl-norleucine methyl ester for carboxyl proteases. 

Inhibitors of Thiol and Carboxyl Proteases. Thiol proteases are inactivated by peptide chloromethyl ketones (30) and other alkylating agents. Peptide diazomethyl ketones are much more selective reagents since they do not react with serine proteases as do chloromethyl ketones. Diazoketones have been applied to papain and cathepsin B (48) thus far and it appears that they should be applicable to most thiol proteases. Specificity should be obtainable by changing the peptide sequence of the inhibitor to match that of the enzyme being studied. 

No selective irreversible inhibitors for carboxyl proteases have been developed yet. Reagents such as diazoacetyl-DL-norleucine methyl ester and l,2-epoxy-3-(-p-nitrophenoxyl) propane will inhibit many of the carboxyl proteases that have been examined, but little specificity is likely to be observed. 

Rich DH, Bernatowicz MS. Synthesis of analogues of the carboxyl protease inhibitor pepstatin. Effect of structure in subsite P3 on inhibition of pepsin. J. Med. Chem. 1982 25 791-795. 

A Rh catalyst with (/ ,/ )-MeDuPHOS is effective for the asymmetric hydrogenation of sterically congested functionalized olefins, a key intermediate in the synthesis of Tipranavir, an HIV protease inhibitor [32]. The cationic Rh catalyst is found to enable an highly efficient and enantioselective hydrogenation of a unique carboxylate for the 12-kg reaction scale production of candoxatril, a cardiovascular agent [33]. 

Various solid-phase syntheses have been reported including the phosphinic acid dipeptide analogues 209 which were constructed by coupling the appropriate Wang Resin-bound amino acid to the phosphinate carboxylic acid 210 using Fmoc chemistry. Similar phosphinate carboxylic acid derivatives (211) protected at phosphorus as their 1-adamanyl esters have also been used in solid-phase synthesis directed towards zinc metallo protease inhibitors. ... 

Reported bioanalytical applications that use SS-LLE products include both tlie cartridge (tube) format and the microplate format. Some of these applications include the determination of mexiletine [44], amiodarone [45] and other antiarrhythmic drugs [46], proxyphylline [47], 16(3-hydroxystanozolol [48], dextromethorphan [49], and simvastatin [50] from biological fluids. Microplate applications for SS-LLE include a crude purification of crude combinatorial library samples [51], carboxylic acid-based matrix metallo-protease inhibitors [52], and a p3-adrenergic receptor agonist [53]. 

Other drugs such as a hypolipidermic drug, 4-trifluoromethyl-2-biphe-nyl carboxylic acid (79), fluconazole (80), SC-52151 (an experimental FlIV protease inhibitor drug) (81), propanolol, and tamoxifen (82) have also been extracted from animal feeds using supercritical CO2. However, the potential use of a SCF other than supercritical CO2 for extraction of pharmaceutical... 

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