Method of preparing vincristine by oxidation of vinblastine

This invention relates to a novel process for preparing a vinca dimer, more particularly to an improved method of preparing vincristine. . Vincristine has been previously prepared from vinblastine. For example, in U.S. Patent No. 3,899,493, there is described a process which utilizes oxidation with chromic ^acid in glacial acetic acid and acetone at -60°C. In this process vincristine yields of 50% based on recovered VLB are reported. This oxidation process is, however, not without, drawbacks. The maintenance of a low reaction temperature is difficult in a manufacturing plant; but higher temperatures are found to produce increased quantities of undesirable by-products. 5'-Acetonyl VLB and a related dehydration product constitute undesirable by-products of the chromic acid oxidation procedure in acetone since the 5'-acetonyl compound must be separated from vincristine by chromatography, with consequent mechanical loss of desired products. Furthermore, substantial quantities of N-desformyl vincristine are produced by chromic oxide oxidation even at low temperatures. This desformyl material must be reformylated in order to maximize vincristine yields. Reformylation is an added, necessary procedure where oxidation with chromic acid results in deformylation of the oxidized product.

According to the present invention there is provided a process for preparing vincristine which comprises oxidizing a solution of vinblastine sulfate at a temperature in the range from about -20°C to about -4O'°C with an acidic ferrous salt-hydrogen peroxide

X-5328 oxidizing mixture, making the reaction mixture strongly basic, extracting therefrom the vincristine thus produced.

^v A preferred reagent is a perchloric acidferrous perchlorate hydrogen peroxide oxidation reagent which is described in a series of papers by Groves et al., J. Am. Chem. Soc., 96 5274 (1974); 97 7118 (1975); and 98 859, 5290 (1976) .

The reaction is carried out in an inert mutual solvent, preferably acetonitrile, at a temperature in the range -20° to -40°C. The preferred iron salt is ferrous perchlorate.

The following specific examples illustrate the process of this invention.

Example 1

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°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-perchlorateperchloric acid solution over a 25 minute period while maintaining the temperature to about -20°C. After the addition had been completed, the reaction mixture was stirred for an additional 20 minutes and the reaction then quenched by the addition of 1 ml. of 15% aqueous sodium bisulfite. The resulting mixture was stirred for an

X-5328 -3* additional 10 minutes and then added to 10 ml. of 14M aqueous ammonium hydroxide chilled to about 0°C. The volume of the mixture was increased to 30 ml. by the addition of methylene dichloride. The organic layer was 5 separated and the aqueous layer extracted with 15 ml.

of methylene dichloride. 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 x 20 x 0.025 cm.). The chromatogram was developed at 0°C. with methylene dichloride 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. (11% yield)

Example 2

One hundred four milligrams of VLB sulfate were dissolved in 5 ml. of acetonitrile. The reaction ₂₅ mixture was cooled to about -20°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°C., .22 ml. of 30% hydrogen peroxide and 1 ml.

-._n of acetonitrile were added over a 20 minute period.

X-5328 -4Thp reaction mixture was stirred for an additional 15 minutes at -20°C. and, after cooling to about -60°C.

with a Dry Ice-acetone bath, the reaction was then quenched 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 methylene dichloride. 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. A crude red oil, 65 mg., comprising vincristine was obtained. The crude vincristine was applied to three 20 x 20 x 0.025 cm. preparative thin layer chromatographic plates coated with silica. The chromatogram was developed at 0°C with methylene dichloride containing 10% methanol and 3% isopropanol. Vincristine, free base, 0.6 mg., (0.7% yield) was 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.

Example 3

A solution of 100 mg. of VLB free base in

1.5 ml. of acetonitrile was placed in a 25 ml. round25 bottom flask, and cooled to a temperature in the range -30°C to -35°C. The flask was degassed and flushed with Ng. Ferrous perchlorate hexahydrate, 268 mg., was added and the flask again degassed and flushed with ₃θ Ng. Glacial acetic acid, 0.18 ml., was added followed

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X-5328 ־5־ by the addition over a 15 minute period of one ml. of a 10% hydrogen peroxide in acetonitrile solution (prepared by placing 0.13 ml. of 30% H₂O₂ in water in a 10 ml. graduate and adding acetonitrile to 10 ml.). The reaction mixture was stirred for 3 hours at a reaction temperature of about -32°C. One gram of HYFLO SUPERCEL® (a diatomaceous earth filter aid) was added followed by 1.5 ml. of 14N aqueous ammonium hydroxide and 1.5 ml. of 15% aqueous solution bisulfite. The reaction mix10 ture was allowed to warm to ambient temperature with stirring and was then filtered. The filtrate was extracted twice with 50 ml. portions of CH₂C1₂ and once with 40 ml. of CH₉C1₉. The organic extracts were combined and the combined extracts washed with 30 ml.

of saturated aqueous sodium chloride. The organic layer was dried and the volatile constituents removed by evaporation in vacuo, leaving a brownish-red residue comprising vincristine produced in the above oxidation; weight = 88.3 mg. chromatography of the residue over

5.1 g. of silica gel was carried out. The chromatogram was developed with CH₂C1₂ containing increasing amounts (1-8%) of methanol. Fractions shown to contain vincristine by TLC were combined. Evaporation of the solvent yielded 36.4 mg. of vincristine (free base) fractions 25 assayed to be about 75% pure. (about 26% yield)

Following the above procedure, yields as high as 35% of vincristine based upon VLB free base have been obtained.

The fact that the oxidation carried out at a 30 temperature range of -20°C to -40^eC rather than at -60°C as with the chromic .acid-acetic acid oxidation,

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ל ל® confers a definite advantage addition, under the reaction acetonitrile, does not react tine, as does acetone in the sulfuric acid oxidation.

Vincristine prepared by the can be purified to that degree necessary for intravenous drug by chromatography over alumina or a combination of both or by on the npvel process. In conditions, the solvent, with the standard

VLB or vincrischronic acidabove oxidation _Pff .־&

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procedure use as an silica or crystallization.

The iron salt-hydrogen peroxide mixture is employed in conjunction with an acid such as perchloric, acetic or s'ulfuric^acids. ^We prefer to use glacial acetic acid for this purpose.

We. have preferred to use acetonitrile as the with a suitably . . ✓ iron J -atteh^ao.-— preferred to reaction solvent. However, any solvent low coordination or chelating power for acetonitrile, can be used. We have also use ferrous perchlorate as the iron salt.