Patent GB-878060-A (GB)
Title: Novel nucleotides and salts thereof and a process for the manufacture of same
IPC: C12P19/30
Filed: 1959-05-20
Abstract: The invention comprises 5-fluoro-uracil N-ribotides and N-deoxyribotides and salts thereof with pharmaceutically acceptable bases.  They may be obtained by incubating tumour cells in the presence of 5-fluoro-uracil nucleosides and nutrient material containing a source of phosphate.  Suitable tumour cells are EHRLICH ascites tumour cells and a preferred embodiment comprises incubating such cells in a nutrient medium containing a source of phosphate in the presence of 5-fluoro-21-deoxy uridine and glucose.  The nucleotides may also be obtained by reacting 5-fluoro-uracil ribosides or deoxyribosides with a phosphorochloridic acid (which is defined as being phosphoromonochloridic acid or phosphorodichloridic acid or mixture thereof), the reaction being preferably carried out in the presence of a tertiary organic base e.g. pyridine or quinoline. The nucleotides obtained by either method can be isolated by chromatographic methods and may be converted into their salts with pharmaceutically acceptable bases, e.g. alkali- or alkaline earth-metal hydroxides, ammonia or organic bases, e.g. diethanolamine or morpholine.  In an example of the biochemical method EHRLICH ascites tumour cells are incubated for 30 minutes at 37 DEG C. in an incubation medium consisting of ascites serum, chick embryo extract, isotonic saline solution containing small amounts of penicillin and streptomycin, glucose, and 5-fluoro-21-deoxy-uridine-2-C14.  At the end of the incubation the cells are centrifuged rapidly, washed with isotonic saline solution and then treated with aqueous perchloric acid.  The acid soluble extract obtained is neutralised with potassium hydroxide and the potassium chlorate formed removed by centrifuging.  The acid soluble fractions are then adjusted to pH 12 by the addition of KOH solution and then chromatographed by passing the liquid through a column of anion exchange resin and the resin washed with water until the washings are neutral and then eluted with water, 4N formic acid and water, 4N formic acid and 0.4M ammonium formate, and 4N formic acid and 1M ammonium formate successively. Radioactive examination of the tubes in which the eluates are collected shows the presence of five distinct fractions, these fractions being: (1) pyrimidine base and nucleosides, (2) 5-fluoro-uridine-51-monophosphate and 5-fluoro-21-deoxy-uridine-51-monophosphate, (3) 5-fluoro-uridine-51-diphosphate glucose, (4) 5-fluoro-uridine-51-diphosphate, and (5) 5 - fluoro - uridine - 51 - triphosphate.  Examples are also given for the production of (1) 5-fluoro-uridine polyphosphate sodium salts by reacting 5-fluoro-uridine dissolved in dry pyridine with a phosphoromonochloridic acid containing a minor amount of phosphorodichloridic acid (obtained by slowly adding technical grade phosphorus oxychloride to distilled water) followed by chromatography and conversion to the sodium salts; (2) 5-fluoro-21-deoxy-uridine-51-monophosphate from 5-fluoro-21-deoxy-uridine and the above referred to phosphoromonochloridic acid product, (3) the sodium salt of 5-fluoro-21-deoxy-uridine-31,51-diphosphate from 5 - fluoro-21-deoxy-uridine and the above referred to phosphoromonochloridic acid, and (4) 5-fluoro-21-deoxy-uridine-51-monophosphate together with some 31,51-diphosphate from 5-fluoro - 21 - deoxy-uridine and phosphorodichloridic acid obtained by slowly adding freshly distilled phosphorus oxychloride to an equimolar proportion of distilled water. The nucleotide products and salts thereof are useful as germicidal agents and as antimetabolites.  5-Fluoro-uridine is obtained as follows:-5-fluoro uracil is dissolved in aqueous NaOH solution and a solution of mercuric chloride in ethanol is added.  The pH of the mixture is adjusted to 5.1 by adding aqueous NaOH solution and mercury-di-(5-fluoro-uracil) which is precipitated is then separated and suspended in xylene.  The suspension is heated and a benzene solution of tri(O-benzoyl)-b -D-ribofuranosyl chloride is added and the mixture refluxed to yield 5-fluoro-21,31,51-tri (O-benzoyl)-uracil ribofuranoside which is then heated with ethanolic ammonia in a sealed tube at 100 DEG C. to form crude 5-fluoro-uridine which is then purified by ion-exchange chromatography.  The tri(O-benzoyl)-b -D-ribofuranosyl chloride is obtained by adding 1-(O-acetyl)-2,3,5-tri(O-benzoyl)-b -D-ribose to anhydrous ether previously saturated with hydrogen chloride at 0 DEG C. and allowing the mixture to remain at 4 DEG C. for 8 days. 5-Fluoro-21-deoxy-uridine is obtained as follows:- cells of Streptococcus fecalis (ATCC 8043) are grown in a folic acid assay medium supplemented with thymine and after 20 hours incubation at 37 DEG C. the cells are collected by centrifuge, washed with potassium phosphate buffer solution and finally suspended in the said buffer solution and ground in a glass tissue homogenizer. p  5-Fluoro-uracil and thymidine are then dissolved in potassium phosphate buffer solution and the solution mixed with the buffer solution containing the cells and the mixture incubated at 37 DEG C. for 18 hours, the enzyme action being then stopped by the addition of acetone and peroxide-free ether.  The precipitated solids are removed by filtration and the filtrate treated for the recovery of 5-fluoro-21-deoxy uridine.