DEFINICIÒN DE TERMINOS BÀSICOS

Boric Acid Boronic Acids Borinic Acids Figure 2.3: The Boron Acids

Serine proteinases operate by a different mechanism to any of those proposed for HIV-1 PR; in the proteolysis that they catalyse, the scissile peptide bond is directly attacked by an active site serine residue. A significant feature of the inhibition of serine proteinases by boronic and borinic acids is the formation of a tetrahedral boronates and borinates ("ate" complexes) between the electrophilic boron atom and nucleophilic serine residue (Fig. 2.4 ) .2 0 2 it is thought that "ate" complexes are good mimics of the transition state in amide bond hydrolysis.

HO. OHI.

Ser-OH + B-R ► Ser-O-B-R

X ' X

X = OH: boronic acid X = OH : boronate X = R' : borinic acid X = R' : borinate

Figure 2.4: Formation of "ate" complexes between boronic and borinic acids and a serine residue

Whilst there aie very few reports of borinic acids acting as inhibitors of serine p r o te in a se s ,2 0 0- 2 0 3 possibly as a result of their susceptibility to atmospheric oxidation,204 they are generally better than their boronic acid counterparts. For example, compared to phenyl boronic acid, diphenyl borinic acid is 30-times more potent an inhibitor of a-chymotrypsin and 60-times more potent as an inhibitor of bovine t r y p s i n .2 0 5 This difference has been attributed, at least in part, to the higher electrophilicity of the boron centre in borinic than in boronic acids. This is reflected in their pKa values (Fig. 2.5).

Ph OH X = Ph, pKg = 6.2

'B-OH + 2H2O ph-B-OH H3O+ X = OH, p K a =8 . 8

X' X X = Me, pK^ = 8.1

As the boron atom in diphenyl borinic acid is more electrophilic, the equilibrium of the reaction between it and water lies further to the right and hence the pKa is lower. If methyl phenyl borinic acid (X = Me) and phenyl boronic acid (X = OH) are compared, it appears that the replacement of a methyl with a hydroxyl group results in greater stabilisation of the acid than its conjugate base. It might have been thought that a greater stabilisation of the negative charge in the conjugate base by the hydroxyl group wquld make phenyl boronic acid the stronger acid.

2.1.4 Incorporation of the Borinic Acid Functionality into the Template for the Target Molecules

Although the serine and aspartic proteinases operate by different mechanisms, we hoped that the electrophilicity of the boron centre in borinic acids particularly would enable such compounds to function as inhibitors by forming "ate" adducts with the catalytic aspartic acids of HIV-1 PR and we sought to test this hypothesis. In order to do this we wished to synthesise a series of cyclohexanols based on structure D in Fig. 2.2. After testing them against the proteinase enzyme in an in vitro assay, it was aimed to establish what effect the borinic acid had on inhibition by incorporating it in exchange for the hydroxyl group in a directly comparable series of compounds.

2 .2 Synthesis of Cyclic Heteroatom-substituted Cyclohexanols

It was envisaged that the desired alcohols could be prepared by the reduction of the C=C bonds present in cyclic alcohols 61-63 (see Scheme 2.1, overleaf). The ketones 58-60 were all synthesised using the simple base-catalysed aldol reaction (Scheme 2.1) developed by C a m p , 2 0 6 a procedure which gave better yields than the acid-catalysed syntheses previously r e p o r t e d .202.208 Novel alcohols 62 and 63, which had previously been made by C a m p ,2 0 6 through reduction of the carbonyl groups in 59 and 60 with sodium borohydride in ethanol, were re-prepared and fully characterised. It was not

possible, however, to isolate the only known alcohol in this series, 61,209 without it decomposing almost immediately. It could be synthesised by reducing ketone 58 [the reduction of all three ketones could be monitored by both colour change (bright yellow to colourless) and by TLC] with subsequent precipitation from ethanol by the addition of water. However, when the white precipitate was left to dry for less than 5 minutes, either at the pump or under reduced pressure (the procedure used to produce 62 and 63), it was found to decompose consistently. ^H and NMR spectroscopic analysis of the partially dry solid revealed that it was the desired product. The spectra contained, amongst other resonances, a NMR signal at ô = 81.04 ppm, indicative of the hydroxyl-bearing carbon atom, and a ^H NMR signal corresponding to the phenylmethylene protons, upfield from its position in the spectrum of its parent ketone 58 [Ôh (CH=C) = 7.84 ppm in 58 and 6.61 ppm in 61]. The failure to isolate alcohol 61 was a disappointment as it reduced our ability to determine how effective the heteroatom present in other analogues was at interacting with the flap isoleucine residues.

(i)_

O O OH

X = CHz: 55 58 61

X = 0: 56 59 62

X = S: 57 60 63

Reagents and Conditions: (i) Benzaldehyde, NaOH, H2 0-EtOH, 0->25 °C, 1-16 h, 62-68%; (::) NaBH4, EtOH, 0->25 °C, then H2O, 92-94%