Design, synthesis and testing of purple acid phosphatase inhibitors for the development of anti-osteoporotic drugs

Abstract

Purple acid phosphatases (PAPs) are binuclear metallohydrolases that have been isolated from various mammals, plants, fungi and bacteria. They catalyse phosphoric ester hydrolysis under acidic to neutral conditions by utilising two closely spaced heterovalent metal ions in their active sites. In mammals, PAP activity is associated with bone resorption, which can lead to bone diseases such as osteoporosis. For instance, osteoporosis patients are observed to have elevated PAP serum levels. Furthermore, research has shown that transgenic mice that over-expressed the PAP gene developed mild osteoporosis. These observations therefore make human PAP an attractive target to develop anti-osteoporotic drugs. This project employed the strategy of using a substrate analogue, where the lead compound (as shown below) with Ki value of 8 μM against pig PAP was chosen as the basis in this rational drug design. Therefore acyl derivatives of α-aminoarylmethylphosphonic acids (as shown below) were designed as potential new potent PAP inhibitors. Docking studies suggested that the derivatives would have high binding affinity due to the phosphonate moiety (substrate mimic) binding to the binuclear metal centre, which are as intended. Furthermore, the long acyl chains make favourable interactions with the long hydrophobic groove adjacent to the dimetal centre, and depending on the size and substitutions on the aromatic ring, the ring might occupy the space in any of the small pockets in the vicinity of the active site; all of which contribute to the predicted high binding affinities of the derivatives to the enzyme. The lead compound: (naphthalene-1-yl(tetradecanamido)methyl)phosphonic acid R1 = , , , , R2 = Me(CH2)m-, Me(CH2)nOCH2-; m = 8, 10, 12, 14, 16; n = 7, 15, 17. Acyl derivatives of α-(amino(aryl)methyl)phosphonic acids The derivatives were synthesised in three or four steps method in good yields and then tested as pig PAP inhibitors. The kinetic studies in this project were performed using newly extracted and purified pig PAP. The extraction and purification of pig PAP from the allantoic fluid of a pregnant pig was successfully carried out using fast protein liquid chromatography (FPLC) by adapting a well-established protocol. Kinetic analysis showed that the derivatives have high inhibition potencies against pig PAP with Ki values in the low micromolar to nanomolar range. The most potent pig PAP inhibitor within this series was the octadecyl-derivative of α-(phenyl(amido)methyl)phosphonic acid with Ki value of 168 nM. This is the most potent pig PAP inhibitor to date. Screening tests on a hundred other compounds were also performed to identify new possible alternative PAP inhibitors. Most of the compounds have metal-binding moieties as they were developed as inhibitors of a metallo-β-lactamase, which is also a binuclear metallohydrolase. Therefore it was thought that they might have inhibition properties against PAPs. However, they were found to have little or no inhibition against pig PAP. Nonetheless, these results provide valuable information, where phosphonic acid is a better choice to be employed as the metal-binding component, compared with other metal-ligating motifs such as thiol, carboxylate, imidazole, nitrile, or pyrrole groups, in the future design of potential PAP inhibitors.