HS- CHj-CH-CONHCHJOOOH CHaCOS-CH2- CH - CONHChjCOOCHrH^^ (8,5): thiorphan (S.6); acetorphan

subsequently converted by brain enzymes to the active drug. The absorption of the antiviral acyclovir (8.7) has been improved as the valine ester, valaciclovir (8.8) and other analogues penciclovir (7.53) and famcyclovir (7.52) are futher improvements.

The oral administration of peptide-like enzyme inhibitors may lead to poor absorption due to the polar nature of the peptide backdown as well as degradation losses by intestinal proteases. Consequently high potency with an IC50 value in the low nanomolar range is required for such drugs. Saquinavir (8.82) an HIV protease inhibitor has a low oral absorption (c. 2%) but this is offset by a low IC50 of 0.4 nM.

8.4.2 Metabolism

For a reversible inhibitor to be a useful drug it must exist sufficiently long at the site of its target enzyme to exert its therapeutic effect. Since the level of the inhibitor at the site is a function of its plasma level, liver metabolism of the drug in the plasma to biologically inert product(s) leads to dissociation from the site and reversal of the inhibition.

The biological half life (t1/2) of an inhibitor in man is not directly related to that obtained from animal experiments although it is usually longer than that observed in the rat. A half life of c. 8 h is an acceptable figure in man although for cancer chemotherapy a longer half life 12-36 h is required to provide adequate drug cover in the event of patient non compliance with the dose regimen.

The metabolic processes by which drugs are modified have been considered in Chapter 1 and most of these processes will lead to a shortening of the t1/2 of the inhibitor. The most important of these involves hydroxylation by liver P450 enzymes. This general phenomenon has been previously described for aromatase inhibitors (Chapter 6) where hydroxylation can occur on a vulnerable imidazole nucleus and benzylic -CH2- group with loss of activity. Replacement of imidazole by triazole may lead to a loss in in vitro potency but this is reversed in the in vivo situation due to the greater metabolic stability of the triazole nucleus. Furthermore substitution of vulnerable "^^"and groups with electron withdrawing substituents decreases the chance of "^development and subsequent hydroxylation (see Chapter 6). This approach is also illustrated in the development of fluconazole (8.12). Ketoconazole (8.9), an antifungal agent, has a short t1/2 and is highly protein bound, due to its lipophilic nature, so that less than 1% of the unbound form exists at the site of action. Modification led to UK-46,245 (8.10) which had twice the potency in a murine candidosis model but further manipulation was required to improve metabolic stability and decrease lipophilicity. This was achieved in UK-47,265 (8.11) which has 100 times the potency of ketoconazole on oral dosing. Unfortunately this compound was hepatotoxic to mice and dogs and teratogenic to rats. Alteration of the aryl substituent to 2,4-difluorophenyl gave fluconazole (8.12) which is >90% orally absorbed and has t1/2=30 h. It is used for the treatment of candida infections and as a broad spectrum antifungal. The stability to metabolism of fluconazole could be attributed to possession of the stable triazole nucleus which is not hydroxylated unlike imidazole as well as protection of the groups to hydroxy lation by flanking electron withdrawing groups (hydroxyl, triazole, difluorophenyl).

Table 8.3 Toxic or side effects exhibited by some enzyme inhibitors as drugs or drug candidates.

ACE inhibitors

cough; due to build up of bradykinin

(controlling PGE2/PGI2) and substance P



renal syndromes; gastrointestinal effects

HMG CoA reductase



MAO inhibitors

hypertensive reaction with tyramine-containing




abdominal cramps, salivation, diarrhoea



adrenal hormone suppression

cytochrome P-450

enzyme inhibitors

Toxic effects may become apparent on chronic dosing during animal pharmacology studies, clinical trials or even after marketing. A well-known example is aminoglutethimide introduced as an anti-epileptic and subsequently withdrawn due to effects on steroidogenesis enzymes leading to a 'medical adrenalectomy'. It was later reintroduced as an anti-cancer agent for the treatment of breast cancer by oestrogen deprivation to capitalise on this toxic effect. The toxic side effects may merely be a matter of inconvenience or may be more severe (see Table 8.3). Many drugs e.g. cimetidine, erythromycin, ketoconazole, choramphenicol, isoniazid, verapamil, including enzyme inhibitors are non-specific inhibitors of liver cytochrome P-450 enzymes i.e. inhibit many iso-enzyme forms. They consequently affect the metabolism of other drugs given concurrently leading to enhanced levels of these drugs and appearance of toxic effects. Specific inhibitors of P450 isoezymes have a similar effect but this effect is restricted to specific substrates of the particular isoenzyme concerned. Examples include quinolone antibiotics (isoenzyme CYP1A2) and sulphaphenazole (CYP2C8/9).


The stereochemistry of enzyme inhibitors possessing a chiral centre (s) is usually important in determining their potency towards a specific enzyme and this is a problem to be addressed in the early stages of drug design since it can sometimes be avoided by limiting the studies to achiral compounds.

Drug Registration Authorities world-wide are moving towards a requirement that for all drugs the enantiomeric active form must be marketed unless for the racemate the activity of the separate enantiomers is available and enantioselective methods of chemical and biological analysis have been used in both animal and human studies. These requirements take into account the pharmacological consequences of the use of racemic drugs which has been previously described in Chapter 4.

Whereas the literature abounds with examples of activity residing mainly in one enantiomer following in vitro studies, very few of these compounds have, as yet, reached the clinic or been subjected to registration requirements and in vivo information is not available from animal studies.

Aminoglutethimide (AG) (8.13), a long-established aromatase inhibitor, is used clinically as the racemate in the treatment of breast cancer in post-menopausal women (after surgery) to decrease their tumour oestrogen levels. The (+) (R)- form is about 38 times more potent as an inhibitor than the (-) (S)- form. AG is also an inhibitor of the side-chain cleavage enzyme (CSCC) which converts cholesterol to pregnenolone in the adrenal steroidogenic pathway. Depletion of corticosteroids in this manner requires adjuvant hydrocortisone administration with the drug. Here the (+) (R)- form is about 2.5 times more potent than the (-) (S)- form.

For pyridoglutethimide (rogletimide) (8.14), an analogue of AG without the undesirable depressant effect, the inhibitory potency resides mainly in the (+) (R)- form (20 times that of the (-)(S)- form). 1-Alkylation improves potency in vitro but the activity for the most potent inhibitor in the series, the 1-octyl, resides in the (-)(S)-form owing to a change in the mode of binding of inhibitor to enzyme.

A more selective inhibitor of aromatase than AG is the triazole vorozole (8.15) which is about 1000-fold more potent as an inhibitor. The (+)S)- form is 32 times more active than the (-) R)- form, but the very small inhibitory activity of the racemate towards other steroidogenic pathway enzymes, 11 P-hydroxylase and 17,20-lyase, originates in the (-)-and (+)- forms respectively.

It is of interest that in the benzofuranyl methyl imidazoles (8.16), some of which are 1000 times more potent as aromatase inhibitors in the racemic form than AG, comparable activity lies in both enantiomers.

MAO occurs in two forms, MAO-A and MAO-B. The use of MAO inhibitors as antidepressents is complicated by a dangerous hypertensive reaction with tyramine-

containing foods (the 'cheese-effect') which is due to inhibition of MAO-A located in the gastro-intestinal tract which would otherwise remove the tyramine. L-(-) deprenyl (selegiline) (8.17), a selective inhibitor of MAO-B, is widely employed to limit dopamine breakdown in Parkinson's disease in selective inhibitory dosage. The (-)-isomer is much more potent than the (+)-isomer and, since the products of metabolism are (-)-metamphetamine and (+)-metamphetamine respectively, the more potent (+)-metamphetamine side-effects are removed from the racemate by use of L-deprenyl.

y-Aminobutyric acid (GABA) transaminase inhibitors allow a build-up of the inhibitory neurotransmitter GABA and are potential drugs in the treatment of epilepsy. The inhibitory action of y-vinyl GABA (vigabatrin), a drug used clinically in the treatment of this disease, resides mainly in the (5^-enantiomer (see Section


8.6.1 Protease inhibitors

Proteases have been classified according to substrate specificity (enkephalinase, collagenase, elastase), substrate size (peptidases, proteinases) or cleavage site on the substrate (aminopeptidases, carboxypeptidases) and localisation (human neutrophil elastase, pancreatic elastase, HIV-protease). However for the purpose of developing protease inhibitors based on the mechanism of the respective enzyme, a classification based on a knowledge of the catalytic function at the active site has proved to be more useful and four subclasses (serine, cysteine, aspartic and metalloproteases) have been identified which catalyze the cleavage of the amide bond linking two amino acids by nucleophilic attack on the scissile carbonyl carbon atom.

Little was known of the actual structure of these various enzymes during the early development of enzyme inhibitors. The approach of first identifying a 'lead' compound, using models of active sites based on knowledge of substrate specificity, and then optimising its structure has been highly successful in the design and development of potent and selective enzyme inhibitors. Crystal structures are now available for many enzymes. Information from X-ray crystallography studies of enzyme-inhibitor complexes and computer assisted molecular modelling is becoming an important part of the design and development process. Serine proteases

Serine proteases form the largest group and occur in the plasma (as coagulation factors and complement components), and the intestine (as cellular proteases). Examples include chymotrypsin, trypsin, elastase, cathepsin G, thrombin and plasminogen activator. The key catalytic element is a serine hydroxyl group and the nucleophile is an intergral part of the enzyme structure and therefore substrates for these enzymes undergo covalent catalysis.

The primary sequences of individual serine proteases vary but the active site consists of:

(i) a catalytic site where the covalent bond making-bond breaking reactions take place involving three amino acid residues (His-57, Asp-102, Ser-195 for chymotrypsin) known as the 'catalytic triad' and the oxyanion hole' comprised of NH groups of serine and glycine (Ser-195, Gly-193 for chymotrypsin) which stablise the oxyanion of the tetrahedral adduct and,

(ii) an extended binding site where noncovalent binding, through hydrogen bonding and hydrophobic forces, occurs between the enzyme and the substrate through the amino acid residues extending on either side of the scissile bond. The most important of these is the interaction between the S1 subsite and the P1 residue (see Figure 8.1 for definitions) as it determines substrate specificity for serine proteases. Modification of the P1 residue may alter the enzyme selectivity of the substrate or inhibitor.

The stages of peptide bond hydrolysis illustrated using a-chymotrypsin include (see Figure 8.2):

(i) complex formation between the substrate and the extended binding site of the enzyme

(ii) formation of a tetrahedral intermediate (a high energy transition state-like intermediate between the substrate and acyl-enzyme) formed by nucleophilic addition of the serine hydroxyl group (Ser-195) to the carbonyl carbon atom of the scissile peptide bond. Hydrogen bond formation of the serine hydroxyl group with the imidazole of His-57 (which is also interacting with Asp-102), increases the nucleophilicity of Ser-195 hydroxyl group.

(iii) the proton on the serine hydroxyl group which was transferred to His-57 is shuttled to the nitrogen atom of the C-terminal amine product so aiding the collapse of the tetrahedral intermediate and formation of the acyl enzyme.

Sj1 s2l

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