Synthesis of medronic acid monoesters and their purification by high-performance countercurrent chromatography or by hydroxyapatite

We achieved the synthesis of important medronic acid monoalkyl esters via the dealkylation of mixed trimethyl monoalkyl esters of medronic acid. Two methods were developed for the purification of medronic acid monoesters: 1) small scale (10–20 mg) purification by using hydroxyapatite and 2) large scale (tested up to 140 mg) purification by high-performance countercurrent chromatography (HPCCC).


S3
a mixture of ethyl acetate and methanol as the eluent and TLC staining being done with potassium permanganate.
General procedure: the silylation of trimethyl monoalkyl esters. The reaction was performed under argon atmosphere. The ester was dissolved in dry acetonitrile and the mixture was cooled to −5 to 0 °C and subsequently 3.5-5 equiv of trimethylsilylbromide were added. The reaction temperature was kept at approximately −5 °C and the reaction was monitored by 1 H NMR (disappearance of methyl signals) and stopped after 2-5 hours by evaporation to dryness. The silyl esters were hydrolyzed by mixing the crude product with methanol for 15 minutes.
Finally, the methanol was evaporated and the crude product was purified by HPCCC.
General procedure: the purification of monoalkyl esters by HPCCC with HEMWat solvent system 1. Samples were dissolved in 1 mL of the lower phase prior to the separation. Stationary phase retention was typically 33%. The flow rate was kept at 0.5 mL/min until the dynamic equilibrium was reached and for the first 10 minutes of the separation. After 10 minutes the flow was set to 1 mL/min. The fraction volume was 0.5-1.0 mL. A typical separation lasted for 40 minutes.
Procedure example: the purification of compound 3g by hydroxyapatite. The monoester 3g (10 mg) as its disodium salt form containing 10% of medronate as an impurity was dissolved in dry methanol (12 mL). Hydroxyapatite (50 mg) was added and the mixture was stirred for 20 minutes at room temperature. Hydroxyapatite was filtered off and the mixture was evaporated to dryness, giving 7 mg of the pure product. The amount of hydroxyapatite was estimated prior to the experiment by determining the amount of medronate that the hydroxyapatite could bind [6]. The

[(Dimethoxyphosphino)methyl]phosphonic acid monomethyl ester N,N,N-
tributyl-N-methyl ammonium salt (1) was prepared by following the procedure described by Vepsäläinen et al. [7]. from tetramethyl methylenebisphosphonate and tributylamine. Equimolar amounts of tetramethyl methylenebisphosphonate (3.0 g, 0.0129 mol) and tributylamine (2.40 g, 0.0129 mol) were refluxed in acetonitrile for seven days. The reaction mixture was evaporated to dryness and washed with a mixture of ether and n-hexane (60 mL, 1:1) to remove traces of unreacted tetramethyl methylenebisphosphonate. Compound 1 was obtained as a viscous oil.