Method of preparing vincristine

#The alkaloids obtainable from Vinca rosea represent one of the most productive research areas among those groups of drugs which adversely affect the growth of experimental malignancies in mammals. Initially, only some of the alkaloids obtainable from the leaves of the plant by extraction and chromatography were found to be active as oncolytic agents. It was determined that these active anti-neoplastic vinca alkaloids obtained directly from the plant were dimeric indole-dihydroindole alkaloids representable by the formula: ##STR1##

#In the above formula where R.sup.1 is acetoxy, R.sup.2 is methyl, R.sup.3 is hydroxyl, R.sup.4 is ethyl and R.sup.5 is H, VLB (vincaleucoblastine, vinblastine) is represented; where R.sup.1 is acetoxy, R.sup.2 is formyl, R.sup.3 is hydroxyl, R.sup.4 is ethyl and R.sup.5 is H, vincristine (VCR, leurocristine) is represented; where R.sup.1 is acetoxy, R.sup.2 is methyl, R.sup.3 is ethyl, R.sup.4 is hydroxyl, and R.sup.5 is H, leurosidine (vinrosidine) is represented; where R.sup.1 is acetoxy, R.sup.2 is methyl or formyl, R.sup.3 is ethyl and R.sup.4 and R.sup.5 taken together form an .alpha.-epoxide ring, leurosine and leuroformine, respectively are represented. Literature references to the above alkaloids are as follows: leurosine (U.S. Pat. No. 3,370,057), VLB (U.S. Pat. No. 3,097,137), leuroformine (Belgian Pat. No. 811,110); leurosidine and leurocristine (both in U.S. Pat. No. 3,205,220).

#Two of the above alkaloids, VLB and vincristine, are now marketed for the treatment of malignancies, particularly the leukemias and related diseases in humans. The two marketed alkaloids are customarily administered by the i.v. route. Two others, leurosidine and leuroformine, have been on clinical trial, either in the U.S. or in Europe.

#The following specific examples illustrate the process of this invention.

#A stirred solution of 170 mg. of ferrous perchlorate hexahydrate in 1.5 ml. of acetonitrile was degassed with nitrogen while cooling to about -20.degree. C. A mixture of 142 mg. of VLB sulfate in 1 ml. of acetonitrile was mixed with 0.135 ml. of 70% perchloric acid at ambient temperature. This solution was added to the chilled solution of ferrous perchlorate. The reaction mixture turned a clear greenish brown. A degassed solution of 1.22 ml. of 30% hydrogen peroxide in 10 ml. of acetonitrile was added to the VLB sulfate-perchlorate-perchloric acid solution over a 25 minute period while maintaining the temperature to about -20.degree. C. After the addition had been completed, the reaction mixture was stirred for an additional 20 minutes and then quenched by the addition of 1 ml. of 15% aqueous sodium bisulfite. The resulting mixture was stirred for an additional 10 minutes and then added to 10 ml. of 14M aqueous ammonium hydroxide chilled to about 0.degree. C. The volume of the mixture was increased to 30 ml. by the addition of methylenedichloride. The organic layer was separated and the aqueous layer extracted with 15 ml. of methylenedichloride. The organic extracts were combined and the combined extracts washed once with 10 ml. of 14M aqueous ammonium hydroxide and then four times with 10 ml. of a pH 7 phosphate buffer followed by one wash with 10 ml. of saturated aqueous sodium chloride. The organic layer was dried and the solvents evaporated therefrom in vacuo. The residue, consisting of 12 mg. of a dark red oil, was purified by preparative thin layer chromatography using four silica-coated plates (20.times.20.times.0.025 cm.). The chromatogram was developed at 0.degree. C. with methylenedichloride containing 10% methanol. Bands corresponding to vincristine were removed manually and yielded 14 mg. of a pink glass which had the physical characteristics of vincristine free base.

#One hundred four milligrams of VLB sulfate were dissolved in 5 ml. of acetonitrile. The reaction mixture was cooled to about -20.degree. C. while being degassed with nitrogen. A mixture of 249 mg. of ferrous perchlorate and 0.1 ml. of 70% perchloric acid was added thereto. Next, to the stirred solution maintained at -20.degree. C., 0.22 ml. of 30% hydrogen peroxide and 1 ml. of acetonitrile were added over a 20 minute period. The reaction mixture was stirred for an additional 15 minutes at -20.degree. C. and, after cooling to about -60.degree. C. with a dry ice-acetone bath, was then quenced by the addition of 5 ml. of 14M aqueous ammonium hydroxide. Next, 1 ml. of 15% aqueous sodium bisulfite was added. The mixture was filtered and the filter pad washed with 40 ml. of methylenedichloride. The organic filtrate was washed twice with 20 ml. portions of 14M ammonium hydroxide and three times with a pH 7 phosphate buffer. The organic layer was then dried and the solvents evaporated therefrom in vacuo. 65 mg. of a crude red oil comprising vincristine were obtained. The crude vincristine was applied to three 20.times.20.times.0.025 cm. preparative thin layer chromatographic plates coated with silica. The chromatogram was developed at 0.degree. C. with methylenedichloride containing 10% methanol and 3% isopropanol. 0.6 mg. of vincristine were isolated from one of these plates plus 3.0 mg. of vincristine N-oxide. The N-oxide can be converted to vincristine by an additional sodium metabisulfite wash.