The design, synthesis and protease inhibitor properties of latent reactive amino acid analogues
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
This thesis examines the synthesis and proteolytic properties of a new class of latent reactive amino acid analogue, eg: 1-(N-Phthaloyl-Lphenylalanyl)-2-hydroxymethylpyrrole 2.52, designed to inhibit serine proteases and the HN protease. Chapter One discusses the possible mode of protease inhibition by these derivatives. The substrate specificity of chymotrypsin, and HN -1 protease suggests that the pyrrole occupies the P₁’ subsite, and the N-acyl group the P₁ subsite. Acceptance into the active site, and subsequent cleavage of the amide bond releases the highly reactive 1-azafulvene 1.3 that can alkylate an active site nucleophilic residue. Chapter Two presents procedures for the N-acylation of pyrrole derivatives. Pyrrole-2-carboxaldehyde was successfully N-acylated with non-amino acid acylating agents using either, NaH, MeLi, or BuLi. A new mild procedure was developed for the N-acylation of pyrroles using a DMAP/organic base combination. The DMAP/organic base (TEA or Hünigs base) methodology was extended to the N-acylation of pyrrole-2-carboxaldehyde with N-Pth protected amino acid acid chlorides. The subsequent reduction of the N-acylated formyl pyrrole, 1-(N-Phthaloyl-L-phenylalanyl)-2-formylpyrrole 2.37, with Zn(BH₄)₂ produced high yields of the N-acylated hydroxymethyl pyrrole 2.52. A ¹H NMR analysis of the camphanate prepared from 2.37 demonstrated that racemisation had not occurred. A detailed ¹H and ¹³C NMR spectral analysis of the formyl and hydroxymethyl pyrroles is discussed in this chapter. Chapter Three extends the synthesis to incorporate potential hydrogen bonding sites in the Px’ sub site direction. The imine N-(2-Pyrrolylmethylene)-L-alanine Ethyl Ester 3.12 was prepared from pyrrole-2-carboxaldehyde, by reaction with H₂N-Ala-OEt and pyrrole-2-carboxaldehyde with removal of water. The analogous imine formation using the N-acylated formyl pyrrole 1-(3-Phenylpropionyl)-2-formylpyrrole 2.31 produced high yields of the non-acylated imine 3.12. The N-acylation of 3.12 under the DMAP/Hünigs base conditions using hydroclnnamoyl chloride did not proceed. Esterification of the hydroxymethyl pyrrole 2.52 under Mitsunobu conditions with the dipeptide N-Cbz-Val-Val-OH produced the tetrapeptide, N-benzyloxycarbonyl-L-valinyl-L-valine-l-(Nphthaloyl-L-phenylalanyl)pyrrol-2-ylmethyl Ester 3.39. Chapter Four presents a hydrolysis and mechanistic study on the deacylation of 1-(3-Phenylpropionyl)-2-hydroxymethylpyrrole 2.32 with HO-. The deacylation of 2.32 in CD₃CN, containing one equivalent of KOH, produced 2-(3-Phenylpropionyl)-2-methylpyrrole Ester 4.5. This species was shown to form via an intramolecular mechanism. Chiral, deuterium labelling of the methylene position showed that racemisation at this position had not occurred in the formation of 4.5. An azafulvene intermediate is therefore precluded. A kinetic analysis of the subsequent conversion of 4.5 to 2-Butylaminomethylpyrrole 4.6, in the presence of n-butylamine, showed the reaction to be first order with respect to HO-. The hydrolysis of deuterium labelled [6-d₁]-(6S)-1-(N-Phthaloyl-L-leucyl)-2-hydroxymethylpyrrole 4.14 with HO- in CD₃CN, gave the corresponding O-acetylated pyrrole 4.9 without racemisation. The addition of (S)-(+)-sec-butylamine to the reaction, trapped the azafulvene as [6-d₁]-(8S)-(+)-2-(1-methylpropionylamino)methylpyrrole 4.36.N-acylation was also shown to suppress azafulvene chemistry and therefore increase the stability of the hydroxymethyl pyrrole system. The camphanate from [6-d₁]-(6S)-1-(3-Phenylpropionyl)-2-hydroxy methylpyrrole 4.10 formed under the Mitsunobu conditions proceeded with 62% inversion of configuration of the methylene position. Chapter Five deals with the α-chymotrypsin and HN-1 protease inhibitor properties of the pyrrole derivatives 2.32, 2.52, 1-(NPhthaloyl-D-phenylalanyl)-2-hydroxymethylpyrrole 2.55, 3.39, 1-(Undecyl)-2-hydroxymethylpyrrole 3.53 and 1-((5-Methoxycarbonyl)hexanyl)-2-hydroxymethylpyrrole 3.55. These compounds were found to be modest inhibitors of α-chymotrypsin, but poor inhibitors of HIV-1 protease. The stability of the hydroxymethyl pyrrole 2.52 was shown to be low under the HN protease assay conditions. A discussion on molecular modelling and docking the proposed inhibitors into the active site of HIV-1 protease is presented.