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147.6 mg. of vincristine sulfate were slowly added to 877.1 mg. of cold 18 M sulfuric acid. After the addition had been completed, the reacton mixture was stirred (at ambient temperature) for one-half hour. 13 ml. of dry methanol were then added followed by 2.8193 g. of solid sodium carbonate. The resulting mixture was stirred for about 45 minutes. 40 ml. of saturated aqueous sodium chloride were then added, and the volume made up to 80 ml. with distilled water. The resulting aqueous solution was extracted four times with equal volumes of benzene. The benzene extracts were separated, combined, dried and the benzene removed by evaporation. A residue weighing 57.2 mg. was obtained containing a mixture of three anhydro derivatives shown as three separate spots on thin layer chromatography. The reaction mixture was separated by preparative thin layer chromatography on silica gel using methanol as the eluant. The preparative chromatogram was divided into three zones manually and the anhydro derivative in each zone removed from the silica adsorbent by two washes with an ethyl acetate-methylene dichloride solvent mixture followed by two washes with methanol. The mixture of three anhydro compounds obtained from vincristine by the above reaction was thus separated and purified. During the sulfuric acid treatment and subsequent isolation procedures, the C-4 acetyl group hydrolyzed and the resulting anhydro derivatives were all derivatives of C-4 desacetyl vincristine. Each of these compounds had the structure of Formula II above in which R is methoxy, R' is hydroxy and R" is formyl. 19.2 mg. of the 3',4'-isomer (Z is IIIa) were obtained having the following physical characteristics: Mass spectrograph: Molecular ion at 764, other peaks at 750, 736, 688, 676, 660, 554, 537, 494, 478, 336, 337, 279, 261, 247, 249, 219, 167, 149, 136, 120, 121, 111, 106 and a transmethylation peak at 778. (Partial) NMR spectrum (in deuterated chloroform) δ's (ppm) at 0.96, 3.58, 3.63, 3.74, 3.81, 3.87, and 5.41 (multiplet).

1. additionAfter the addition
2. stirringThe resulting mixture was stirred for about 45 minutes
3. extractionThe resulting aqueous solution was extracted four times with equal volumes of benzene
4. customThe benzene extracts were separated
5. customdried
6. customthe benzene removed by evaporation
7. customwas obtained
8. additioncontaining
9. customseparate spots on thin layer chromatography
10. customThe reaction mixture was separated by preparative thin layer chromatography on silica gel
11. customthe anhydro derivative in each zone removed from the silica adsorbent by two washes with an ethyl acetate-methylene dichloride solvent mixture
12. additionThe mixture of three anhydro compounds