Doctor of Philosophy
Marvin R. Boots
Atherosclerotic disease is an almost universal phenomenon and increases in severity and frequency with increasing age. Atherosclerosis may contribute to several disorders including bursting of an artery, blockage of an artery, or induction of arterial clotting. The culmination of these diseases is usually premature since at the time of death the unaffected organs are in reasonably satisfactory condition and could have operated for several more years. Among the many factors which act in concert to produce the disease, serum cholesterol levels play a central role. It has been suggested that the lowering of cholesterol levels will provide an effective means of treatment and prophylaxis of atherosclerosis.
The purpose of this investigation was to rationally design, synthesize, and evaluate agents to lower serum cholesterol levels by the inhibition of cholesterol biosynthesis at the site of the reduction of β-hydroxy-β-methylglutaryl coenzyme A (HMG CoA) to mevalonic acid. This reaction, mediated by HMG CoA reductase, was chosen as the inhibition target because it is the first irreversible reaction, the rate limiting step for the pathway, and the site of physiological regulation of cholesterol biosynthesis. Rat liver HMG CoA reductase provided a convenient test system for these agents.
Using the previously reported compound, 1-(4-biphenylyl)-n-pentyl hydrogen succinate as parent inhibitor, the present study accomplished two goals: first, a contribution toward elucidation of reversible binding sites for these inhibitors and second, probing of the suggested nonpolar n-pentyl binding area of the enzyme by introduction of a functional group capable of alkylating the enzyme and providing irreversible inhibition. The first objective was approached by replacing the ester of the parent inhibitor with an amide functional group. The resulting glutarimide exhibited inhibition comparable to that of the parent inhibitor. This may be taken as evidence that isosteric replacement of the parent ester group with the amide N-H did not seriously alter the ability of the inhibitor to bind to the enzyme and that an additional binding site in this region is not available. The data also support the suggestion that the ester group of the parent agent is not necessary for binding.
A similar inhibition study was made possible by synthesis of 1-(4-biphenylyl)-n-pentyl hydrogen 3-methyl-3-methoxyglutarate. With respect to the corresponding 3-methyl-3-hydroxyglutarate this compound showed an eleven fold decrease of activity. This considerable activity loss indicates that the 3-methoxy group interferes with reversible binding of inhibitor to the enzyme. The inhibition data indicate that the 3-hydroxy group of the 3-methyl-3-hydroxy compound contributes to reversible binding by participating as a hydrogen donor in hydrogen bonding with the enzyme.
The major portion of this investigation was designed to probe a region of the enzyme which is nonpolar and binds the n-alkyl moiety of the parent inhibitor. The purpose was to determine the feasibility of incorporating a functional group into this region of the inhibitor which could act as an acceptor for an enzymic nucleophile located in proximity to the reversible binding area. If successful, this could provide irreversible inhibition of the enzyme. A series of compounds was synthesized which bore a terminal alkenyl group two to four carbon atoms removed from the ester moiety. Testing showed that, with respect to the parent inhibitor, no appreciable loss of binding took place. Similarly, a series of epoxyalkyl esters was prepared, the epoxide group being the portion of the inhibitor susceptible to nucleophilic attack. Reversible binding of these compounds was found to be equal to that of the parent inhibitor and it was therefore concluded that the enzyme does accommodate this alkylating group with no loss of reversible binding. This provided the necessary preliminary work upon which subsequent irreversible binding studies will be based.
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