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Volatile organophosphorus compounds such as sarin (8.101) and tabun (8.102) have been prepared for use as nerve gases in war and other less volatile compounds have been used as insecticides for the spraying of crops. Inhibition of the mammalian or insect enzyme leads to a build-up of acetylcholine and death from accumulated acetylcholine poisoning.

Much research has been carried out to find antidotes, for nerve gas poisoning, which could be distributed to the population in the event of war. One of these discoveries, pyridine-2-aldoxime mesylate (pralidoxime (8.103)) has been successfully used, in conjunction with atropine to block the action of acetylcholine on receptors, in the treatment of accidental poisoning during crop spraying. Pralidoxime is considered to complex at the anionic site where it is firmly held by electrostatic attraction in the correct spacial configuration for attack by the oxime anion on the phosphorus atom with displacment of the inhibitor residue from the enzyme.

There is evidence that Alzheimer's disease and senile dementia of the Alzheimer's type (SDAT) are associated with dysfunction of normal cholinergic neurotransmission in the brain leading to learning and memory deficiencies. Examination of patients with these diseases has shown reduced levels of ChAT (acetyl-Co A: choline O-transferase), acetylcholinesterase and the muscarinic receptor sub type M1. ChAT is responsible for the synthesis of acetylcholine in the cerebral cortex and it has been postulated that by inhibiting acetylcholinesterase in the brain the associated build up of acetylcholine will enhance the cognitive function. Three main types of inhibitors have been used which lead to improvement in SDAT patients. The carbamate inhibitors exemplified by physostigmine (8.96) which itself has a poor pharmacokinetic profile but this is improved in the N-heptyl derivative heptylphysostigmine (eptastigmine). Tacrine has been approved for use for Alzheimer's patients but is non-specific in its action by also inhibiting plasma butyrylcholinesterase leading to adverse peripheral effects. Its use also leads to hepatotoxicity as a significant side effect. Recent work has centred on the third group of inhibitors, the benzylpiperidines. These are not quaternised and so are able to penetrate the blood brain barrier as the unionised base form and are potent selective inhibitors of acetylcholinesterase with good pharmacokinetic properties. Compounds in clinical trial are E-2020 (8.104) and TAK-147 (8.105).

8.6.3 Aromatase inhibitors

Aromatase belongs to a group of cytochrome P-450 enzymes responsible for hydroxylation processes in the body. It contains a Fe3+ -haem catalytic site which, after reduction to Fe2+, binds and activates oxygen, leading to initial insertion of two hydroxyl groups on the C-19 (methyl) carbon of its substrates androstenedione and testosterone. A further hydroxylation occurs, followed by aromatization to oestrone and oestradiol, respectively, accompanied by elimination of water and formate by a mechanism only partially understood.

The steroidogenic pathway (see Figure 8.6) from cholesterol to the substrates of aromatase commences in the adrenals with the action of the cytochrome P-450 enzyme, cholesterol side chain cleavage enzyme (CSCC), producing pregnenolone which is then isomerized by another enzyme to progesterone. Progesterone is converted by 17 a-hydroxy: 17,20-lyase (P450 17), another P-450 enzyme, to androstenedione which can be reduced by a dehydrogenase to testosterone. Aromatase is located mainly in fatty tissue in postmenopausal women and mainly in ovarian tissue in premenopausal women.

After diagnosis of a breast tumour, it is removed by surgery and this is followed by a course of chemotherapy to reduce new tumour growth or suppress metastasis in other parts of the body. Mammary tissue contains oestrogen receptors, and depending on their concentration the patient can be categorized as either oestrogen receptor-positive (ER+) or negative (ER-). About one-third of the cases of breast cancer in women are hormone-dependent, the major hormone involved in supporting the growth of the tumours being oestradiol. The categorization can determine the type of chemotherapeutic treatment employed.

The first line drug for use in the treatment of mammary cancer in postmenopausal women with (ER+) and (ER-) tumours is tamoxifen. This is an oestrogen receptor antagonist which, by competing with oestradiol for the receptor, can reduce the ability of oestradiol to stimulate tumour growth. Tamoxifen has weak oestrogenic activity and compounds ICI 164,384 and ICI 182,780 without this effect are now in clinical trial.

Figure 8.6 Steroidogenesis pathway.

Oeslrone Oestradul

Figure 8.6 Steroidogenesis pathway.

Tamoxifen-resistant tumours (ER+) are sometimes amenable to treatment with a second line drug which is an aromatase inhibitor. This reduces the plasma level of circulating oestradiol available to the tumour tissue by inhibiting the action of aromatase, present in the fatty tissue, on androstenedione.

The non-steroidal aromatase inhibitor aminoglutethimide (8.106) is in current clinical use for the treatment of (ER+) breast cancer in postmenopausal women. On chronic administration of the drug, the already low plasma oestrogen level present in elderly women is further rapidly lowered and maintained, enabling a success rate in terms of remission or stabilization of about 33% (unselected patients) or 52% (ER+ patients). Aminoglutethimide was initially introduced into therapy as an anti-epileptic drug, but after initial withdrawal due to noted side-effects of adrenal insufficiency it was reintroduced into cancer chemotherapy due to its potential effect for interrupting the steroidogenic pathway to oestrogen production. Subsequent work showed that it was a potent, competitive, reversible inhibitor of aromatase with a weaker effect on the CSCC enzyme (which accounts for its effects on adrenal hormone production).

Aminoglutethimide is co-administered with hydrocortisone to supplement decreased production of 1ip-hydroxysteroids due to its effect on CSCC. Side-effects associated with use of the drug are ataxia, dizziness and lethargy, due to its sedative nature. These effects, which can lead to patient non-compliance, decrease after several weeks' administration of the drug. Consequently, more specific inhibitors without these side-effects have been sought.

Several anti-fungal agents based on imidazole e.g. ketoconazole, econazole were known at this time which inhibit the fungal P450 14a-demethylase enzyme. They are inhibitors of aromatase but have a wide spectrum of activity against other P450 enzymes in the steroidogenic chain. Several potent specific inhibitors of aromatase containing an imidazole or triazole nucleus (increased in vivo stability) have subsequently been developed. Fadrozole (8.107), (+)- vorozole (8.108), letrozole (8.109) and arimidex (6.27) (achiral) are now in clinical trial. These compounds are 400-1000 fold more potent than aminoglutethimide and have no CNS effects. Fadrozole also inhibits the 18-hydroxylase enzyme responsible for aldosterone production at doses much higher than used clinically; this side effect has been designed out in the more selective letrozole.

Mechanism-based inactivators of aromatase are known and these are based on the androstenedione (substrate) skeleton. 4-Hydroxyandrostenedione (8.110) is in clinical trial as a intramuscular injection (formestane) given once weekly and is a specific irreversible inhibitor of the enzyme although the mechanism is not clear. It has to be administered parenterally since it is rapidly metabolised by first-pass metabolism following oral administration. It has been reported to produce a 30% complete or partial tumour regression with disease stabilisation in a further 15% of patients. Other steroidal irreversible inhibitors in trial include plomestane (8.111) and exemestane (8.112).

Recent views are that breast tissue is capable of synthesising oestrogens mainly from the action of a sulphatase on oestrone sulphate. The oestrone produced provides oestradiol by the action of a 17p-hydroxysteroid dehydrogenase. Inhibitors of steroid sulphatase are being developed as potential adjuvants to aromatase inhibitors to further deplete oestrogen levels. One of these compounds emate (8.113) is an irreversible inhibitor of the sulphatase.

8.6.4 Pyridoxal phosphate-dependent enzyme inhibitors

Enzymes using pyridoxal phosphate as coenzyme catalyse several types of reactions of amino acid substrates, such as (1) transamination to the corresponding a-ketoacid, (2) racemization, (3) decarboxylation to an amine, (4) elimination of groups on the P-and y-carbon atoms, (5) oxidative deamination of œ-amino acids. The coenzyme is bound to the enzyme by formation of an aldimine (Schiff-base) with the œ-amino group

of a lysine residue. The first step in the reaction with the amino acid substrate is an exchange reaction to form an aldimine with the a-amino group of the amino acid (see Equation [8.38]. Either by hydrogen abstraction (transamination, racemization) or by decarboxylation, a negative charge is developed on the a-carbon atome and this is distributed over the whole conjugated cofactor system. Protonation then occurs on either the a-carbon atom (decarboxylation, racemization) or on the carbon atom adjacent to the pyridine ring (transamination) as shown in Equation [8.38]. The direction of the fission which occurs is dictated by the nature of the protein at the active site so that a specific enzyme catalyses a particular type of reaction. Information has recently become available on the crystal structure of several of these enzymes and the role of their active site residues.


At one time, several irreversible inhibitors of several pyridoxal phosphate-dependent were known but their mechanism of action was not clear since they did not possess the electrophilic centres present in the active site directed irreversible inhibitors known at that time. Later, when a new class of inhibitor, the mechanism-based enzyme inactivator, became known their inhibition mechanism became predictable from the well established mechanism of action of these enzymes. The next step for design was to manipulate the amino acid substrate structure of a suitable target enzyme in such a manner as to obtain maximal exploitation of the enzyme's machinery.

The inhibitors act as substrates of the enzyme but their structure is such that they either (1) divert the electron flux from the a-carbanion formed away from the coenzyme moiety, or (2) using the normal electron flux either give rise to reactive species or generate a stable substrate-cofactor which binds strongly to the enzyme active site. All these mechanisms can lead to irreversible inhibition of the enzyme.

Mechanism-based inactivators of many pyridoxal phosphate-dependent enzymes are known but only a few target enzymes and their inactivators of therapeutic interest will be discussed here. GABA ¡Transaminase (GABA-T) inhibitors y-Aminobutyric acid (GABA) is considered as the main inhibitory neurotransmitter in the mammalian central nervous system. There has been much interest recently in the design of inhibitors of the pyridoxal phosphate-dependent enzyme, a-ketoglutarate-GABA transaminase. This enzyme governs the levels of GABA in the brain (see Equation [8.39]). Inhibitors of the enzyme would allow a build-up of GABA and could be used as anticonvulsant drugs for the treatment of epilepsy.

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