University of Tasmania
whole_BrownMalcolmRoy1984_thesis.pdf (8.89 MB)

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

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posted on 2023-05-27, 07:55 authored by Brown, MR
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.


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Copyright 1984 the author - The University is continuing to endeavour to trace the copyright owner(s) and in the meantime this item has been reproduced here in good faith. We would be pleased to hear from the copyright owner(s). An in press version of a published article is included in the back of the thesis. Material from: Brown M. R., Sallis J. D., 1985. N-sulfo-2-amino tricarballylate, a new analog of phosphocitrate: metabolism and inhibitory effects on renal calcification. In: Schwille P. O., Smith L. H., Robertson W. G., Vahlensieck W. (eds), Urolithiasis and related clinical research. Springer, Boston, MA. An article included in the back of the thesis appears to be the equivalent of the peer reviewed version of the following article: Brown, M. R., Shankar, R., Sallis, J. D., 1984. Synthesis of N-[\\(^{35}\\)S]-sulpho-2-amino tricarballylate, Journal of labelled compounds and radiopharmaceuticals, 21(10), 905-911, which has been published in final form at This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley's version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited. A published article is included in the back of the thesis. It is: Brown M. R., Sallis J. D., 1983. The synthesis and characterization of N-sulfo-2-amino tricarballylate: An analog of phosphocitrate and inhibitor of calcification, Analytical biochemistry, 132(1), 115-123

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