activity, however, it is not enterally absorbed and rapidly eliminated from the circulation. Variations of the basic structures were performed to improve the pharmacokinetic properties, however, most of them led to a drastic loss in inhibitory activity, without clear improvement in oral bioavailability.

From the X-ray crystal structure of the NAPAP-thrombin complex (Figure 8.5) it was obvious that the NAPAP molecule is bound nearly perfectly to thrombin, so that there is only limited space for additional substituents. Despite this, the NAPAP-thrombin crystal structure was used for modelling of several new derivatives. Changing Gly at P2 to Asp (CRC 220) enhanced the antithrombin potency but not bioavailability. A remarkable compound was developed by changing the benzamidine moiety to 3-amidinopiperidine. Ro 46-6240 (8.46) inhibits thrombin with a Ki of 0.3 nM and has outstanding selectivity, never found with benzamidines. However, (8.46) has no improved pharmacokinetic properties.

From the X-ray structure of its complex with thrombin, 3-TAPAP (8.43) was chosen as a promising model for the synthesis of new inhibitors allowing different substitutions without drastic influence on antithrombin activity. Introduction of a C-terminal piperazide opened the way to introducing quite different substituents on the second nitrogen.

Synthesis Benzamidine

Several derivatives of 4-guanidinophenylalanine were also synthesized. The Na-dansylated piperidide (S-2581; (8.47)) has moderate antithrombin activity, however, introduction of 4-guanidinophenylalanine into peptides does not result in potent inhibitors.

Two classes of synthetic peptides were designed after solving the X-ray crystal structure of the thrombin-hirudin complex. Hirudin exerts a concerted binding of its N-terminal and C-terminal domains to the active site and the anion-binding exosite of thrombin. Several analogues of the C-terminal anionic hirudin "tail" were synthesized. The so-called hirugens inhibit thrombin-induced fibrinogen clotting in micromolar concentrations but not the cleavage of synthetic peptide substrates. Also single-stranded DNA molecules (aptamers), selected from a pool of oligodeoxyribonucleotides, interact with the anion-binding exosite of thrombin. Very potent bifunctional thrombin inhibitors with nanomolar Ki values based on the C-terminal sequence of hirudin and the D-Phe-Pro-Arg sequence were designed using

Gly residues as spacer. Also introduction of NAPAP as N-terminal part was proposed. The so-called hirulog is very effective antithrombotically in animal models. The drug was well tolerated in man and entered Phase III trials. Hirugens and hirulogs composed of more than 10 amino acids do not show any enteral absorption as is also the case with most of the synthetic thrombin inhibitors designed so far.

The knowledge on thrombin inhibitors accumulated so far shows that the desirable goal in the development of new thrombin inhibitors for therapeutic use should not only be the design of more active compounds but the improvement of the pharmacokinetic properties of known types of inhibitor, especially with regard to oral bioavailability. Metallo-proteases

Metallo-proteases are a group of enzymes which possess a catalytically essential zinc atom at the active site. Much of the information regarding the catalytic mechanism of metalloproteases has been based on active site models of carboxypeptidase A and thermolysin. Here, four ligands—two histidine residues, a glutamic acid residue and a water molecule—are arranged in a tetrahedral fashion around the zinc atom. A catalytically functional glutamyl residue (Glu-270 for carboxypeptidase A and Glu-143 for thermolysin) is also present at the active site. In general all metalloproteases use the same basic mechanism for peptide bond cleavage. The water bound as the fourth zinc ligand is hydrogen bonded to the carboxylate of the glutamyl residue during the resting state of the enzyme. The water is displaced from the zinc by the carbonyl group of the amide bond to be cleaved in the peptide substrate, through a five coordinate transition state, during formation of the enzyme-substrate complex. The carbonyl group of the scissile bond of the substrate is then attacked by water to give a tetrahedral intermediate. The zinc atom polarizes the carbonyl making it more susceptible to nucleophilic attack and stabilizes the tetrahedral intermediate. Decomposition of the tetrahedral intermediate results in bond cleavage. The structures of the zinc-inhibitor complexes, determined using thermolysin-inhibitor complexes, have shown thiol (2-benzyl-3-mercaptopropanoyl-L-alanylglycinamide), carboxylate (L-benzylsuccinic acid) and phosphinyl (phosphoramidon) inhibitors to be tetrahedrally coordinated to zinc with displacement of a water molecule. The structure of the thermolysin-hydroxamic acid inhibitor complex differs in that the hydroxamate moiety (CONHOH) forms a bidentate complex with the zinc through the carbonyl oxygen and the hydroxyl group, so that a penta-coordinate complex is formed between three ligands from the enzyme and two from the hydroxamate group. The active sites of metallo-proteases also contain a varying number of residues responsible for substrate recognition which differ significantly between individual metallo-proteases.

Examples of metallo-proteases include leucocyte collagenase, membrane metalloendopeptidase (neutral endopeptidase) and angiotensin-converting enzyme (ACE). Approaches used in the design and development of inhibitors of zinc metallo-proteases include leads from biproduct inhibitors (incorporating features of both cleavage products from the enzyme catalysed reaction), introduction of a zinc-binding ligand (e.g. thiol, hydroxamate) and modification of substrates.

Angiotensin converting enzyme inhibitors

Angiotensin converting enzyme (ACE) cleaves a dipeptide from angiotensin I (a decapeptide) at the carboxyl terminal to generate angiotensin II (an octapeptide) which has hypertensive activity and stimulates the release of aldosterone (Equation [8.34]). ACE also catalyzes the hydrolysis of the vasodilator bradykinin (a nonapeptide). ACE inhibitors are therefore important antihypertensive agents.

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