The synthesis of two phosphocitrate analogues and their effectiveness as calcification inhibitors

The synthesis of two structural analogues of the naturally occurring compound phosphocitrate (PC) was investigated, with a view to producing compounds more enzymatically stable than PC, yet with similar anti-calcifying properties. It was envisaged that such compounds might prove to be suitable agents for preventing the deposition of insoluble calcium salts associated with pathological calcification, especially kidney stones. The analogues sought were the sulphamate and phosphoramidate derivatives of PC; namely N-phospho-2-amino tricarballylate (PAT) and N-sulpho-2-amino tricarballylate (SAT). The preparation of PAT, which was ultimately achieved after investigating a number of unsuccessful synthetic routes, involved two distinct stages: a) synthesis of a new precursor in trimethyl 2-amino tricarballylate, and b) coupling of the latter compound with 2-cyanoethyl phosphate, followed by alkaline hydrolysis. The preparation of SAT was effected by the coupling of 2-amino tricarballylate with pyridine-sulphur trioxide. The same synthetic route was also utilized to yield [\\(^{35}\\)S]-labelled SAT. Systems which were developed to aid in the ultimate purification and characterization of synthetic products included ion-exchange and thin layer chromatography, electrophoresis, isotachophoresis and chemical assays. Additional structural proof of the new compounds was obtained through [\\(^1\\)H]-NMR and infra-red spectroscopy. The ability of the PC analogues to inhibit calcification in vitro was assessed. PAT was as potent as PC in preventing hydroxyapatite formation, while SAT was less potent but still a strong inhibitor. Both compounds also inhibited calcium oxalate crystallization, the order of potency being PC > SAT >> PAT. The chemical and biological stabilities of the molecules were studied. In vitro, PAT was found to be readily hydrolyzed at acid pH, unstable at neutral pH and susceptible to the action of alkaline phosphatase. SAT was shown to be completely stable at neutral and alkaline pH, relatively stable in acid and totally resistant to the actions of the hydrolytic enzymes sulphamatase and sulphatase. The stability of SAT was confirmed in vivo from metabolic studies utilizing [\\(^{35}\\) S]-SAT. When given orally, SAT was well absorbed across the gut and rapidly cleared unchanged to the urine from blood and all tissues. The effectiveness of the compounds in arresting renal calcification in vivo was also studied using well established test systems. Comparisons were made with other inhibitors including PC. SAT was proven capable of inhibiting calcium oxalate crystallization, whereas PC and PAT were ineffective. With hydroxyapatite formation, different trends were observed; PAT and SAT had no effect whereas PC produced pronounced inhibition. Rationalization of these findings have been given. Results presented suggest that, in terms of future possible therapeutic value, PAT would be unsuitable, whereas PC and SAT might prove useful under certain situations. The studies help define further the structure-activity relationships of PC and its new analogues in terms of anti-calcifying potential and stability. On this basis, avenues for future research are discussed for the development of further inhibitor molecules that might have greater activity and hence ultimately prove more useful agents for stone prevention.