The design and synthesis of mechanism-based inhibitors of serine proteases
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
Serine proteases are involved in a number of physiological processes and have proved to be a valuable therapeutic target in the treatment of disease states resulting when the above processes move beyond homeostasis. The investigation of low molecular weight compounds as irreversible and reversible inhibitors of serine proteases has been fuelled by the possibility of rational drug design and their use as mechanistic probes of enzyme action. As introduced in Chapter one particular attention has been focused on mechanism-based inactivators as these elicit clinically desirable specific, efficient and irreversible inhibition. The synthesis and assay of funtionalised imide mechanism-based inhibitors of the serine protease α-chymotrypsin is the subject of this thesis. N-[(Sulfonyl)oxy]succinimides of type 1.41 (L=SO₂R') are known mechanism-based inhibitors of α-chymotrypsin operating via a Lossen rearrangement that unmasks an inactivating isocyanate species. Inhibitory activity has been found to be mediated by the nature of the R substituent and the R' substituent of the L group. Structure activity relationships were investigated by preparing a number of derivatives of type 1.41. The design of the derivatives prepared focused on modulating the R' substituent to interact with extended binding sites of α-chymotrypsin, a strategy that would enhance inhibitory activity. Chapter 2 describes the synthesis of 1.41 and 2.1. Retrosynthetic analysis identified a route involving N-hydroxyimides to be favoured. A synthesis of 1.41 and 2.1 via this key intermediate required reaction between hydroxylamine and succinic and glutaric acid derivatives respectively. These derivatives were prepared using literature methods employing Guareschi, Michael and malonate ester reactions. A systematic study of the synthesis of succinic acids found the optimum route to involve the Stobbe condensation however a short synthesis employing amide base alkylation of succinimide was undertaken and this methodology may prove to be the ideal general route. An aromatic series of derivatives, 1.41f-h was therefore synthesised using the methodology above as were "dimeric" inhibitors 1.41m and n capable of releasing two equivalents of inactivating species during inhibition. Succinimides with 3-C phenyl substituent rather than the benzyl substituent of the derivatives above, were prepared and a series of N-[(sulfonyl)oxy]glutarimides 2.1a, c-e where the extent and type of substitution were varied were prepared. N-[(Acyl)oxy]imides 1.41r-o and 2.1b (L=C(O)R') may also inhibit α-chymotrypsin and these too were investigated. Chapter 3 discusses the assay of inhibitory activity of compounds of type 1.41 and 2.1 against α-chymotrypsin. All the synthesised derivatives, excepting a series of N[(acyl)oxy]succinimides 1.41o-r, were found to be active to such a degree that all but one of the active compounds could not be assayed using sampling techniques. These potent inhibitors were then assayed using the progress curve method. Three compounds 1.41g and hand 2.1c were of such potency that the rate at which they inhibited achymotrypsin could not be measured even with the progress curve method. All three of these compounds possessed a benzyl substituent which was found to be a requiremnent for the exhibition of mechanism-based inhibition in the succinimide series. Compounds 1.41g and 1.41h owed their potency to being able to interact favourably with the Sn' subsites of α-chymotrypsin by containing aromatic substitution. Chapter 4 discusses the use of Evan's oxazolidinone chemistry in the preparation of chiral succinates from which an enantiopure inhibitor of type 1.41 was prepared. Preliminary inhibition studies showed that (R)-1.41f was less active than its racemate indicating more activity resided in the (S)-enantiomer. Chapter 5 discusses the design and synthesis of a potential novel imide mechanism-based inhibitor thought to act by unmasking a quinone imine methide in the active site of α-chymotrypsin. Although the compound released reactivity on hydrolysis it was not found to inhibit α-chymotrypsin significantly.