Methods of separating gemcitabine-phosphate diastereoisomers

1. A method of providing at least one diastereoisomer of gemcitabine-[phenyl-(benzoxy-L-alaninyl)]-phosphate in a diastereoisomeric purity of greater than 85%, the method comprising the steps of:

a) suspending a mixture comprising gemcitabine-[phenyl-(benzoxy-L-alaninyl)]-(R)-phosphate:

and gemcitabine-[phenyl-(benzoxy-L-alaninyl)]-(S)-phosphate:

in a first solvent or mixture of solvents to form a slurry, wherein said first solvent or mixture of solvents consists of one or more solvents selected from the group consisting of a C 2 -C 4 alcohol, acetonitrile, toluene, acetone, and methyl ethyl ketone; and

b) filtering the slurry to provide solid gemcitabine-[phenyl-(benzoxy-L-alaninyl)]-phosphate and a filtrate comprising gemcitabine-[phenyl-(benzoxy-L-alaninyl)]-phosphate dissolved in the first solvent or mixture of solvents;

wherein the solid gemcitabine-[phenyl-(benzoxy-L-alaninyl)]-phosphate is gemcitabine-[phenyl-(benzoxy-L-alaninyl)]-(R)-phosphate in a diastereoisomeric purity of greater than 85%, or the filtrate comprises gemcitabine-[phenyl-(benzoxy-L-alaninyl)]-(S)-phosphate in a diastereoisomeric purity of greater than 85%.

2. The method of claim 1 , further comprising heating the slurry to a temperature from about 30° C. to about 80° C. prior to filtration.

3. The method of claim 2 , wherein the slurry is not cooled prior to filtration.

4. The method of claim 1 , further comprising the step of washing the solid gemcitabine-[phenyl-(benzoxy-L-alaninyl)]-phosphate either with the first solvent or mixture of solvents or, if the first solvent or mixture of solvents is a mixture of solvents, with one of the solvents in the mixture.

5. The method of claim 1 , further comprising the step of removing the first solvent or mixture of solvents from the filtrate to obtain solid gemcitabine-[phenyl-(benzoxy-L-alaninyl)]-(S)-phosphate in a diastereoisomeric purity of greater than 85%.

6. The method of claim 5 , wherein the step of removing the first solvent or mixture of solvents comprises:

removing a portion of the first solvent or mixture of solvents from the filtrate by evaporation to provide a concentrated filtrate comprising solid gemcitabine-[phenyl-(benzoxy-L-alaninyl)]-(S)-phosphate;

optionally agitating the concentrated filtrate; and

filtering the concentrated filtrate to obtain solid gemcitabine-[phenyl-(benzoxy-L-alaninyl)]-(S)-phosphate in a diastereoisomeric purity of greater than 85%.

7. The method of claim 5 , wherein the step of removing the first solvent or mixture of solvents comprises:

cooling the filtrate to provide a cooled filtrate comprising solid gemcitabine-[phenyl-(benzoxy-L-alaninyl)]-(S)-phosphate;

optionally agitating the cooled filtrate; and

filtering the cooled filtrate to obtain solid gemcitabine-[phenyl-(benzoxy-L-alaninyl)]-(S)-phosphate in a diastereoisomeric purity of greater than 85%.

8. The method of claim 7 , further comprising the step of adding seed material to the filtrate.

9. The method of claim 7 , further comprising the step of adding additional solvent to the filtrate.

10. The method of claim 7 , further comprising washing the solid gemcitabine[phenyl-(benzoxy-L-alaninyl)]-(S)-phosphate in a diastereoisomeric purity of greater than 85% with the first solvent or mixture of solvents or, if the first solvent or mixture of solvents is a mixture of solvents, with one of the solvents in the mixture.

11. The method of claim 1 , wherein the method provides gemcitabine-[phenyl-(benzoxy-L-alaninyl)]-(R)-phosphate in a diastereoisomeric purity of greater than 85%.

12. The method of claim 11 , further comprising the steps of:

suspending the solid gemcitabine-[phenyl-(benzoxy-L-alaninyl)]-phosphate obtained from step b) in a second solvent or mixture of solvents to form a second slurry, said second solvent or mixture of solvents being different to the first solvent or mixture of solvents; wherein the second solvent or mixture of solvents consists of one or more solvents selected from the group consisting of a C 2 -C 4 alcohol, acetonitrile, toluene, acetone, and methyl ethyl ketone; and

filtering the second slurry to provide solid gemcitabine-[phenyl-(benzoxy-L-alaninyl)]-(R)-phosphate in a diastereoisomeric purity of greater than 85%.

13. The method of claim 1 , wherein the first solvent or mixture of solvents comprises isopropanol.

14. The method of claim 13 , wherein the first solvent or mixture of solvents is isopropanol.

15. The method of claim 1 , wherein the first solvent or mixture of solvents comprises acetonitrile.

16. A crystalline form of gemcitabine-[phenyl-(benzoxy-L-alaninyl)]-(S)-phosphate, wherein the crystalline form is form I, wherein said crystalline form has an XRPD pattern comprising at least two peaks at 20 selected from the group consisting of 5.0±0.2, 6.7±0.2, 8.0±0.2, 11.3±0.2, 20.2±0.2 and 21.4±0.2 when measured with Cu radiation with a K α2 /K α1 ratio of 0.5.

17. The crystalline form of claim 16 , wherein said crystalline form has an XRPD pattern comprising at least four peaks at 2θ selected from the group consisting of 5.0±0.2, 6.7±0.2, 8.0±0.2, 11.3±0.2, 20.2±0.2 and 21.4±0.2 when measured with Cu radiation with a K α2 /K α1 ratio of 0.5.

18. The crystalline form of claim 17 , wherein said crystalline form has an XRPD pattern comprising peaks at 2θ 5.0±0.2, 6.7±0.2, 8.0±0.2, 11.3±0.2, 20.2±0.2 and 21.4±0.2 when measured with Cu radiation with a K α2 /K α1 ratio of 0.5.

19. The crystalline form of claim 16 , wherein said crystalline form has an XRPD pattern comprising peaks at 2θ 5.0±0.2, 6.7±0.2, 8.0±0.2, 11.3±0.2, 12.3±0.2, 12.8±0.2, 13.1±0.2, 13.3±0.2, 14.2±0.2, 14.5±0.2, 15.1±0.2, 16.1±0.2, 16.4±0.2, 17.3±0.2, 17.6±0.2, 19.4±0.2, 20.2±0.2 and 21.4±0.2 when measured with Cu radiation with a K α2 /K α1 ratio of 0.5.

20. The crystalline form of claim 16 wherein said crystalline form has an FTIR pattern comprising peaks at cm −1 426±2, 449±2, 464±2, 526±2, 550±2, 586±2, 601±2, 627±2, 645±2, 689±2, 699±2, 725±2, 754±2, 770±2, 791±2, 817±2, 838±2, 894±2, 904±2, 943±2, 1010±2, 1032±2, 1066±2, 1093±2, 1149±2, 1196±2, 1250±2, 1292±2, 1335±2, 1410±2, 1489±2, 1521±2, 1620±2, 1655±2, 1734±2, 3203±2, 3426±2, and 3468±2 when measured as a suspension in Nujol.