DIASTEREOSELECTIVE SYNTHESIS OF PHOSPHATE DERIVATIVES

Abstract

Disclosed is a method for the preparation of intermediates useful in the synthesis of gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate. Also disclosed is a method of preparing gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate.

Claims

1. A process for the preparation of gemcitabine-[phenyl (benzoxy-L-alaninyl)] phosphate (Formula I) in substantially diastereoisomerically pure form: ##STR00028## the process comprising step a) and optionally step b): a) reacting a compound of Formula II; wherein R.sup.1 represents an electron withdrawing group and a is an integer from 1 to 5, with a compound of Formula III in presence of a base (B1) to provide a compound of Formula IV in substantially diastereomerically pure form; wherein P.sup.1, P.sup.2 and P.sup.3 independently represents hydrogen or a protecting group; and wherein the compound of formula II is in substantially diastereomerically pure form: ##STR00029## and b) where any one or more of P.sup.1, P.sup.2 and P.sup.3 are protecting groups, optionally removing the protecting groups P.sup.1, P.sup.2 and P.sup.3 from the compound of formula IV to provide gemcitabine-[phenyl (benzoxy-L-alaninyl)] phosphate in substantially diastereomerically pure form.

2. The process of claim 1, wherein P.sup.1 is C(O)OC.sub.1-C.sub.6-alkyl or optionally substituted C(O)OCH.sub.2-aryl.

3. The process of claim 2, wherein P.sup.1 is C(O)O.sup.tBu.

4. The process of claim 3, wherein step b) is achieved using by reacting the product of step a) with a mixture of a C.sub.1-C.sub.4-alcohol and water.

5. The process of claim 1, wherein P.sup.2 is H.

6. The process of claim 1, wherein P.sup.3 is H.

7. The process of claim 1, wherein B.sup.1 is a Grignard reagent.

8. The process of claim 7, wherein B.sup.1 is .sup.tBuMgCl.

9. The process of claim 1, wherein step a) is conducted in an ether solvent.

10. The process of claim 9, wherein step a) is conducted in THF.

11. The process of claim 1, wherein the compound of formula II is the (S)-diastereoisomer in substantially diastereomerically pure form and the process is a method of making the (S)-diastereoisomer of NUC-1031 in substantially diastereomerically pure form.

12. A process for the diastereoisomeric enrichment of a compound of Formula II, comprising the steps of: a) suspending or dissolving the R-diastereoisomer of the compound of Formula II or a mixture of the (R)- and (S)-diastereoisomers of the compound of Formula II in a solvent (S2), b) treating the solution or suspension with a base (B2) to obtain (S)-diastereoisomer in substantially diastereomerically pure form, and c) isolating the (S)-diastereoisomer of Formula II.

13. The process of claim 12, further comprising forming the compound of Formula II as a mixture of the (R)- and (S)-diastereoisomers; and wherein step c) comprises suspending or dissolving the mixture of the (R)- and (S)-diastereoisomers of the compound of Formula II in a solvent (S2).

14. The process of claim 12, wherein B2 is a tertiary amine.

15. The process of claim 14, wherein B2 is triethylamine.

16. The process of claim 12, wherein S2 is a hydrocarbon or a mixture comprising a hydrocarbon.

17. The process of claim 16, wherein S2 is hexane or heptane.

18. The process of claim 16 wherein S2 is a mixture of hexane or heptane and a polar organic solvent, and the mixture comprising over 50% by volume hexane or heptane.

19. The process of claim 18, wherein S2 is a mixture of heptane or hexane and ethyl acetate, and the mixture comprising over 50% by volume heptane or hexane.

20. The process of claim 12, wherein step d) comprises stirring the mixture of the compound of formula II and the base B2 for 6 h or longer.

21. The process of claim 12, wherein step d) comprises stirring the mixture of the compound of formula II and the base B2 at a temperature from 0 to 50 C.

22. The process of claim 11, wherein the compound of Formula II is made according to the process of claim 12.

23. The process of claim 1, wherein the compound of Formula II is a compound selected from: ##STR00030## ##STR00031## ##STR00032## ##STR00033## ##STR00034## ##STR00035## ##STR00036## ##STR00037## ##STR00038## ##STR00039## ##STR00040## ##STR00041##

24. The process of claim 1, wherein the compound of formula II is: ##STR00042##

25. The process of claim 1, wherein the compound of formula II is: ##STR00043##

26. The process of claim 12, wherein the compound of Formula II is a compound selected from: ##STR00044## ##STR00045## ##STR00046## ##STR00047## ##STR00048## ##STR00049## ##STR00050## ##STR00051## ##STR00052## ##STR00053## ##STR00054## ##STR00055##

27. The process of claim 12, wherein the compound of formula II is: ##STR00056##

28. The process of claim 12, wherein the compound of formula II is: ##STR00057##

Description

EXAMPLES

[0109] The present invention is further illustrated by the following examples, which are provided by way of illustration only and should not be construed to limit the scope of the invention.

Example 1

Preparation of Diastereoisomeric Mixture of 2-[(2,3,4,5,6-pentafluorophenoxy)-phenoxy-phosphoryl amino] propionic acid benzyl ester (Formula IIa)

[0110] ##STR00019##

[0111] To a stirred mixture of L-alanine benzyl ester hydrochloride (100 gms) in methylene chloride (1 lit) was added phenyl dichlorophosphate (77 ml) at 25-35 C. and the resulting mixture was cooled to 70 C. to 78 C., added triethyl amine (130.5 ml) and stirred for 1 hrs at same temperature. Reaction mass temperature was raised to 25-35 C. and allowed to stir for 2 hrs. After reaction completion, concentrated the reaction mass under vacuum at below 35 C. to obtain residue. To the obtained residue was added diisopropyl ether (2 lit) at 25-35 C. and stirred for 30 min at same temperature. Filtered the reaction mass and washed with diisopropyl ether (500 ml) followed by concentrating the filtrate under vacuum at below 35 C. to obtain phenyl-(benzoxy-L-alaninyl)-phosphorochloridate. The obtained compound was dissolved in methylene chloride (1 lit) at 25-35 C. and cooled to 5 C. to 10 C. To the reaction mass Pentafluorophenol (85.5 gms), triethyl amine (65.2 ml) was added at same temperature and stirred for 2 hrs. After reaction completion, concentrated the reaction mass under vacuum at below 35 C. and charged ethyl acetate (1 lit) at 25-35 C. and stirred for 30 min at same temperature. Filtered the solids and washed with ethyl acetate (1 lit). To the filtrate was given water (1 lit), 10% sodium carbonate (21 lit), brine (1 lit) washings and dried the organic layer with anhydrous sodium sulphate, concentrated under vacuum at 35-45 C. to obtain diastereoisomeric mixture of title compound as a white colored semi solid.

[0112] Yield: 210 gms

[0113] Chiral Purity by HPLC (% area): 33.74:66.26% (R.sub.P: S.sub.P)

Example 2

Separation of S.SUB.p.-diastereoisomer of 2-[(2,3,4,5,6-pentafluorophenoxy)-phenoxy-phosphoryl amino] propionic acid benzyl ester (Formula IIa)

[0114] ##STR00020##

[0115] To the above obtained diastereoisomeric mixture of Formula IIa (R.sub.P: S.sub.P33.74:66.26%) was charged 20% ethyl acetate in hexane (1.2 lit) at 25-35 C. and stirred for 1 hrs. Filtered the solids and washed with 20% ethyl acetate in hexane (300 ml) to obtain a mixture of compound of Formula IIa.

[0116] Yield: 112 gms

[0117] Chiral Purity by HPLC (% area): 22.13:77.87% (R.sub.P: S.sub.P)

[0118] Filtrate was concentrated under vacuum to obtain a diastereoisomeric mixture of Formula IIa (75 gm; R.sub.P: S.sub.P65.43:34.57%).

[0119] To the above obtained diastereoisomeric mixture of Formula IIa (R.sub.P: S.sub.P22.13:77.87%) was charged 20% ethyl acetate in hexane (1.2 lit) at 25-35 C. and stirred for 1 hrs. Filtered the solids and washed with 20% ethyl acetate in hexane (300 ml) to obtain a pure S.sub.p-diastereoisomer of compound of Formula IIa.

[0120] Yield: 80 gms

[0121] Chiral Purity by HPLC (% area): 0.20:99.80% (R.sub.P: S.sub.P)

[0122] .sup.1H NMR (300 MHz, DMSO-d.sub.6): 7.18-7.41 (m, 10H), 6.91-6.99 (d, 1H), 5.10 (s, 2H), 4.01-4.11 (m, 1H), 1.30-1.32 (d, 3H)

[0123] ESI-MS (m/z): 524 (M+1)

[0124] Filtrate was concentrated under vacuum to obtain a diastereoisomeric mixture of Formula IIa (28 gm; R.sub.P: S.sub.P80.77:19.23%).

Example 3

Isomerization of 2-[(2,3,4,5,6-pentafluorophenoxy)-phenoxy-phosphoryl amino] propionic acid benzyl ester (Formula IIa)

[0125] To a stirred solution of above obtained 2-[(2,3,4,5,6-pentafluorophenoxy)-phenoxy-phosphoryl amino] propionic acid benzyl ester of Formula IIa (75 gms; R.sub.P: S.sub.P65.43:34.57%) in 20% ethyl acetate in hexane (1.1 lit), triethyl amine (7.5 ml) was added at 25-35 C. and stirred for 6 hrs at same temperature. After reaction completion, reaction mass was quenched in to a water (750 ml) and extracted with ethyl acetate (750 ml). Organic layer was dried with anhydrous sodium sulphate and concentrated under vacuum to afford title compound as a solid.

[0126] Yield: 45 gms

[0127] Chiral Purity by HPLC (% area): 91.29: 8.71% (S.sub.P: R.sub.P)

[0128] To the above obtained R.sub.p and S.sub.p-diastereoisomeric mixture of 2-[(2,3,4,5,6-pentafluorophenoxy)-phenoxy-phosphoryl amino] propionic acid benzyl ester of Formula IIa (45 gms; R.sub.P: S.sub.P8.71:91.29%) was slurred in 20% ethyl acetate in hexane (1.1 lit) at 25-30 C. and stirred for 1 hr at same temperature. Filtered the solid and washed with 20% ethyl acetate in hexane (225 ml) to obtain S.sub.p-diastereoisomer of the title compound as a solid.

[0129] Yield: 19 gms

[0130] Chiral Purity by HPLC (% area): 99.92: 0.08% (S.sub.P: R.sub.p)

Example 4

Preparation of S.SUB.p.-diastereoisomer of NUC-1031 (using S.SUB.p.-diastereoisomer of Formula IIa)

[0131] ##STR00021##

[0132] To a stirred mixture of 3-O-(tert-butoxycarbonyl) gemcitabine (5 gms) in tetrahydrofuran (75 ml), was added tert-butyl magnesium chloride (15.2 ml of 2.0 M in tetrahydrofuran) and S.sub.p-diastereoisomer of 2-[(2,3,4,5,6-pentafluorophenoxy)-phenoxy-phosphoryl amino] propionic acid benzyl ester of Formula IIa (8.3 gms diluted in 50 ml of tetrahydrofuran)) at 0 C. to 5 C. and temperatures was raised to 25-30 C. and stirred for 30 min at same temperature. After reaction completion, reaction mass was quenched in to 0.5 N hydrochloric acid (50 ml) and extracted with ethyl acetate (275 ml). To the organic layer was given 10% sodium carbonate (250 ml), brine solution (50 ml) washings sequentially. The organic layer separated, dried over sodium sulfate and concentrated under vacuum to obtain a residue.

[0133] The obtained residue was taken up in methylene chloride (50 ml) and added trifluoro acetic acid (18.5 ml) at 0 C. to 5 C. Maintained the reaction mass at 25-35 C. for 2 hrs and quenched in to 20% sodium carbonate solution (125 ml). Extracted with ethyl acetate (165 ml), dried the organic layer over sodium sulfate and evaporated under vacuum at 40-45 C. The obtained residue was slurried in 50% ethyl acetate in heptane (150 ml) mixture to obtain the title compound.

[0134] Yield: 4.8 gms

[0135] Chiral Purity by HPLC (% area): 99.4% (S.sub.P-diastereoisomer)

Example 5

Preparation of Diastereoisomeric Mixture of 2-[(4-nitrophenoxy)-phenoxy-phosphorylamino] propionic acid benzyl ester of Formula IIb

[0136] ##STR00022##

[0137] To a stirred mixture of L-alanine benzyl ester hydrochloride (50 gms) in methylene chloride (500 ml) was added phenyl dichlorophosphate (54 gms) at 25-35 C. and the resulting mixture was cooled to 70 C. to 78 C., added triethyl amine (65.2 ml) and stirred for 1 hrs at same temperature. Reaction mass temperature was raised to 25-35 C. and allowed to stir for 2 hrs. After reaction completion, concentrated the reaction mass under vacuum at below 35 C. to obtain residue. To the obtained residue was added diisopropyl ether (1 litre) at 25-35 C. and stirred for 30 min at same temperature. Filtered the reaction mass and washed with diisopropyl ether (250 ml) followed by concentrating the filtrate under vacuum at below 35 C. to obtain phenyl-(benzoxy-L-alaninyl)-phosphorochloridate. The obtained compound was dissolved in methylene chloride (500 ml) at 25-35 C. and cooled to 5 C. to 10 C. To the reaction mass pentafluorophenol (27.5 gms), triethyl amine (65.2 ml) was added at same temperature and stirred for 2 hrs. After reaction completion, concentrated the reaction mass under vacuum at below 35 C. and charged ethyl acetate (500 ml) at 25-35 C. and stirred for 30 min at same temperature. Filtered the solids and washed with ethyl acetate (500 ml). To the filtrate was given water (500 ml), 10% sodium carbonate (2500 ml), brine (500 ml) washings and dried the organic layer with anhydrous sodium sulphate, concentrated under vacuum at 35-40 C. to obtain diastereoisomeric mixture of title compound as a thick oily liquid.

[0138] Yield: 90 gms

[0139] Chiral Purity by HPLC (% area): 45.6: 54.94% (R.sub.P: S.sub.P)

[0140] The above obtained diastereoisomeric mixture of 2-[(4-nitrophenoxy)-phenoxy-phosphorylamino] propionic acid benzyl ester of Formula IIb (40 gms; R.sub.p: S.sub.p45.6: 54.94%) was separated in to pure S.sub.p and R.sub.p diastereoisomers by preparative HPLC and concentrated the pure fractions under vacuum to obtain S.sub.p and R.sub.p diastereoisomers separately.

[0141] Yield: S.sub.p-diastereoisomer: 8 gms,

[0142] .sup.1H NMR (300 MHz, CDCl.sub.3): 8.15-8.19 (d, 2H), 7.15-8.37 (m, 12H), 5.12 (s, 2H), 4.02-4.24 (m, 2H), 1.39-1.42 (d, 3H)

[0143] ESI-MS (m/z): 479 (M+Na)

[0144] R.sub.p-diastereoisomer: 6 gms,

[0145] .sup.1H NMR (300 MHz, CDCl.sub.3): 8.08-8.13 (d, 2H), 7.15-7.34 (m, 12H), 5.10 (s, 2H), 4.48-4.56 (m, 1H), 4.11-4.20 (m, 1H), 1.39-1.41 (d, 3H)

[0146] ESI-MS (m/z): 457 (M+1).sup.+

[0147] S.sub.p & R.sub.p-diastereoisomer mixture: 20 gms

Example 6

Preparation of S.SUB.p.-diastereoisomer of NUC-1031 (using S.SUB.p.-diastereoisomer of Formula IIb)

[0148] ##STR00023##

[0149] To a stirred mixture of 3-O-(tert-butoxycarbonyl) gemcitabine (2 gms) in tetrahydrofuran (30 ml), was added N-methyl pyridine (2 ml), tert-butyl magnesium chloride (5.5 ml of 2.0 M in tetrahydrofuran) and S.sub.p-diastereoisomer of 2-[(4-nitrophenoxy)-phenoxy-phosphorylamino] propionic acid benzyl ester of Formula IIb (4 gms diluted in 20 ml of tetrahydrofuran) at 0 C. to 5 C. and temperatures was raised to 25-30 C. and stirred for 30 min at same temperature. After reaction completion, reaction mass was quenched in to 0.5 N hydrochloric acid (20 ml) and extracted with ethyl acetate (230 ml). To the organic layer was given 10% sodium carbonate (220 ml), brine solution (20 ml) washings sequentially. The organic layer separated, dried over sodium sulfate and concentrated under vacuum to obtain a residue.

[0150] The obtained residue was taken up in methylene chloride (20 ml) and added trifluoro acetic acid (7.4 ml) at 0 C. to 5 C. Maintained the reaction mass at 25-35 C. for 2 hrs and quenched in to 20% sodium carbonate solution (30 ml). Extracted with ethyl acetate (66 ml), dried the organic layer over sodium sulfate and evaporated under vacuum at 40-45 C. The obtained residue (3 gms; S.sub.p85.98%) was purified by column chromatography method by eluting with 2-10% isopropanol in methylene chloride mixture. The product containing fractions were collected and concentrated under vacuum to obtain a title compound as a solid.

[0151] Yield: 1.1 gms

[0152] Chiral Purity by HPLC (% area): 97.88: 0.48% (S.sub.p: R.sub.p)

Example 7

Preparation of R.SUB.p.-diastereoisomer of gemcitabine-[phenyl (benzoxy-L-alaninyl)] phosphate of Formula I (using R.SUB.p.-diastereoisomer of Formula IIb)

[0153] ##STR00024##

[0154] To a stirred mixture of 3-O-(tert-butoxycarbonyl) gemcitabine (2 gms) in tetrahydrofuran (30 ml), was added N-methyl pyridine (2 ml), tert-butyl magnesium chloride (5.5 ml of 2.0 M in tetrahydrofuran) and R.sub.p-diastereoisomer of 2-[(4-nitrophenoxy)-phenoxy-phosphorylamino] propionic acid benzyl ester of Formula IIb (4 gms diluted in 20 ml of tetrahydrofuran) at 0 C. to 5 C. and temperatures was raised to 25-30 C. and stirred for 30 min at same temperature. After reaction completion, reaction mass was quenched in to 0.5 N hydrochloric acid (20 ml) and extracted with ethyl acetate (230 ml). To the organic layer was given 10% sodium carbonate (220 ml), brine solution (20 ml) washings sequentially. The organic layer separated, dried over sodium sulfate and concentrated under vacuum to obtain a residue.

[0155] The obtained residue was taken up in methylene chloride (20 ml) and added trifluoro acetic acid (7.4 ml) at 0 C. to 5 C. Maintained the reaction mass at 25-35 C. for 2 hrs and quenched in to 20% sodium carbonate solution (30 ml). Extracted with ethyl acetate (66 ml), dried the organic layer over sodium sulfate and evaporated under vacuum at 40-45 C. The obtained residue (2.9 gms; R.sub.p-84.05%) was purified by column chromatography method by eluting with 2-10% isopropanol in methylene chloride mixture. The product containing fractions were collected and concentrated under vacuum to obtain a title compound as a solid.

[0156] Yield: 1.4 gms

[0157] Chiral Purity by HPLC (% area): 97.99: 0.86% (R.sub.p: S.sub.p)

Example 8

Preparation of S.SUB.p.-diastereoisomer of gemcitabine-[phenyl (benzoxy-L-alaninyl)] phosphate of Formula I (using isomerised S.SUB.p.-diastereoisomer of Formula IIa from example-3)

[0158] ##STR00025##

[0159] To a stirred mixture of 3-O-(tert-butoxycarbonyl) gemcitabine (5 gms) in tetrahydrofuran (75 ml), was added tert-butyl magnesium chloride (15.2 ml of 2.0 M in tetrahydrofuran) and S.sub.p-diastereoisomer of 2-[(2,3,4,5,6-pentafluorophenoxy)-phenoxy-phosphoryl amino] propionic acid benzyl ester of Formula IIa (8.3 gms from example-3 (99.92%); diluted in 50 ml of tetrahydrofuran) at 0 C. to 5 C. and temperatures was raised to 25-30 C. and stirred for 30 min at same temperature. After reaction completion, reaction mass was quenched in to 0.5 N hydrochloric acid (50 ml) and extracted with ethyl acetate (275 ml). To the organic layer was given 10% sodium carbonate (250 ml), brine solution (50 ml) washings sequentially. The organic layer separated, dried over sodium sulfate and concentrated under vacuum to obtain a residue.

[0160] The obtained residue was taken up in methylene chloride (50 ml) and added trifluoro acetic acid (18.5 ml) at 0 C. to 5 C. Maintained the reaction mass at 25-35 C. for 2 hrs and quenched in to 20% sodium carbonate solution (125 ml). Extracted with ethyl acetate (165 ml), dried the organic layer over sodium sulfate and evaporated under vacuum at 40-45 C. The obtained residue was slurried in 50% ethyl acetate in heptane (150 ml) mixture to obtain the title compound.

[0161] Yield: 4.9 gms

[0162] Chiral Purity by HPLC (% area): 99.72% (S.sub.P-diastereoisomer)

[0163] It will be understood that various modifications may be made to the embodiments disclosed herein. Therefore the above description should not be construed as limiting, but merely as exemplifications of preferred embodiments. For example, the functions described above and implemented as the best mode for operating the present invention are for illustration purposes only. Other arrangements and methods may be implemented by those skilled in the art without departing from the scope and spirit of this invention. Moreover, those skilled in the art will envision other modifications within the scope and spirit of the specification appended hereto.

Example 9

Preparation of S.SUB.p.-diastereoisomer of NUC-1031 (using S.SUB.p.-diastereoisomer of Formula IIb) using IPA/water for Deprotection

[0164] ##STR00026##

[0165] To a stirred mixture of 3-O-(tert-butoxycarbonyl) gemcitabine (100 gms) in tetrahydrofuran (1 L), tert-butyl magnesium chloride (292 mL of 2.0 M in tetrahydrofuran) and S.sub.p-diastereoisomer of 2-[(4-nitrophenoxy)-phenoxy-phosphorylamino] propionic acid benzyl ester of Formula IIb (166 gms diluted in 700 mL of tetrahydrofuran) at 5 C. to 0 C. and temperatures was raised to 25-30 C. and stirred for 3 h at the same temperature. After reaction completion, reaction mass was quenched in to 0.5 N hydrochloric acid (1 L) and extracted with ethyl acetate. The organic layer was sequentially washed with 10% sodium carbonate, water and brine solution washings sequentially. The organic layer was separated and concentrated under vacuum to obtain a residue. Isopropyl alcohol (IPA; 850 mL) and water (2.5 L) were added to the residue and the mixture was heated to 75 C. for 3 h before more water was added (2.5 L) and the mixture was cooled to 25 C. and filtered. The resultant solid was washed with ethyl acetate and dried. 124 g of product was obtained (78%). Chiral Purity by HPLC (% area): 99.95% (S.sub.P-diastereoisomer)

Example 10Preparation of (Sp)-2-[(2,3,4,5,6-pentafluorophenoxy)-phenoxy-phosphoryl amino] propionic acid benzyl ester (Formula IIa)

[0166] ##STR00027##

[0167] To a stirred mixture of L-Alanine Benzyl ester. HCl (100 g) in 1000 mL of methylene dichloride was added phenyl dichlorophosphate (97.8 g) into reaction mass at 30 C. The mixture was cooled to 20 C. and triethylamine (93.8 g) was added slowly, maintaining the temperature at 20 C. The reaction was stirred for 1 h at 20 C., then warmed to 10 C. (105) and stirred for a further 1.5 h.

[0168] A solution of pentafluorophenol (85.3 g) in 100 mL of methylene dichloride was slowly added at 10 C. followed by trimethylamine (46.8 g) which is added slowly, maintaining the temperature at 10 C. Slowly add 46.9 g of triethylamine into reaction mass at 10 C. (105) under nitrogen atmosphere. The mixture was stirred for 2 h at 10 C. before being quenched by slow addition of 0.5 N HCl solution, maintaining the temperature at 10 C. After warming to room temperature the mixture was separated and the organics was washed with a saturated bicarbonate solution, distilled water and brine before being concentrated in vacuo.

[0169] The crude mixture was suspended in 1500 mL of 20% ethyl acetate in n-heptane at 25 C. Triethylamine (12.2 g) was added and the mixture was stirred at 25. The mixture was filtered and the solid dissolved in 2500 mL ethyl acetate which was washed with water and brine and concentrated in vacuo. The solid was suspended in 1200 mL of 20% ethyl acetate in n-heptane, stirred for 45-60 min and filtered. The material was dried under vacuum to provide the desired product. Yields are in the range 40 to 80% and the diastereoisomeric purity is over 99%.