SYNTHESIS OF PHOSPHATE DERIVATIVES

Abstract

The present invention is a process for the preparation of certain 5′-phosphoramidate nucleotide diastereoisomers. The phosphoramidates include those useful in the treatment of cancer such as NUC-3373 (5-fluoro-2′-deoxyuridine-5′-O-[1-naphthyl(benzyloxy-L-alaninyl)]phosphate].

Claims

1-44. (canceled)

45. A process for the diastereoisomeric enrichment of a compound of Formula IIa wherein R.sup.1 is selected from the group consisting of halogen, trifluoromethyl, cyano and nitro and a is an integer from 1 to 5: ##STR00061## the process comprising steps a), b), and c), wherein the X-diastereoisomer of the compound of Formula IIa is the (S.sub.p)-diastereoisomer and the Y-diastereoisomer of the compound of Formula IIa is the (R.sub.p)-diastereoisomer, except when the OPh(R.sup.1).sub.a leaving group has lower priority assignment under the Cahn-Ingold-Prelog rules than the naphthyloxy group, in which case the X-diastereoisomer of the compound of Formula IIa is the (R.sub.p)-diastereoisomer and the Y-diastereoisomer of the compound of Formula IIa is the (S.sub.p)-diastereoisomer: a) suspending or dissolving the Y-diastereoisomer of the compound of Formula IIa or a mixture of the Y- and X-diastereoisomers of the compound of Formula IIa in a solvent (S2), wherein S2 is a mixture of hexane or heptane and methyl tert butyl ether; b) treating the solution or suspension with an organic amine base for 24 hours to 100 hours at a temperature from 10° C. to 35° C. to obtain the X-diastereoisomer having a diastereoisomeric purity of greater than 95%, wherein B2 is an organic amine base; and c) isolating the X-diastereoisomer of Formula IIa.

46. The process of claim 45, wherein B2 is a trialkylamine.

47. The process of claim 46, wherein B2 is triethylamine.

48. The process of claim 46, wherein B2 is diisopropylethylamine.

49. The process of claim 45, wherein S2 is a mixture of hexane and methyl tert-butyl ether.

50. The process of claim 45, wherein S2 is a mixture of heptane and methyl tert-butyl ether.

51. The process of claim 45 wherein the solution or suspension of step (b) is stirred for at least 60 hours.

52. The process of claim 45, wherein the solution or suspension of step (b) is stirred for at least 72 hours.

53. The process of claim 45, wherein R.sup.1 is halogen.

54. The process of claim 53, wherein halogen is fluorine.

55. The process of claim 53, wherein halogen is chlorine.

56. The process of claim 53, wherein halogen is bromine.

57. The process of claim 53, wherein halogen is iodine.

58. The process of claim 53, wherein a is 1.

59. The process of claim 53, wherein a is 2.

60. The process of claim 53, wherein a is 5.

61. The process of claim 45, wherein R.sup.1 is fluorine and a is 5.

62. The process of claim 45, wherein R.sup.1 is trifluoromethyl.

63. The process of claim 45, wherein R.sup.1 is cyano.

64. The process of claim 45, wherein R.sup.1 is nitro.

65. The process of claim 64, wherein a is 1.

66. The process of claim 64, wherein a is 2.

67. The process of claim 45, wherein R.sup.1 is halogen and a is 1, 2, or 5.

68. The process of claim 45, wherein R.sup.1 is nitro and a is 1 or 2.

69. The process of claim 45, wherein R.sup.1 is cyano and a is 1.

70. The process of claim 45, wherein R.sup.1 is trifluoromethyl and a is 1.

Description

EXAMPLES

[0157] 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 5 (an Illustrative Example of a Compound of Formula IIb)

[0158] ##STR00048##

[0159] To a stirred mixture of L-alanine benzyl ester hydrochloride 1 (100 g) in methylene chloride (1 L) was added phenyl dichlorophosphate 2 (77 mL) at 25-35° C. and the resulting mixture was cooled to −70° C. to −78° C., triethylamine (130.5 mL) was added and the mixture was stirred for 1 hour at same temperature. Reaction mass temperature was raised to 25-35° C. and allowed to stir for 2 hours. 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 L) 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 3. The obtained compound was dissolved in methylene chloride (1 L) at 25-35° C. and cooled to −5° C. to −10° C. To the reaction mass pentafluorophenol 4 (85.5 g), triethylamine (65.2 mL) were 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 L) at 25-35° C. and stirred for 30 min at same temperature. Filtered the solids and washed with ethyl acetate (1 L). To the filtrate was given water (1 L), 10% sodium carbonate (2×1 L), brine (1 L) washings and dried the organic layer with anhydrous sodium sulphate, concentrated under vacuum at 35-45° C. to obtain diastereoisomeric mixture of title compound 5 as a white colored semi solid.

[0160] Yield: 210 g

[0161] 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 5 (an Illustrative Example of a Compound of Formula IIb)

[0162] ##STR00049##

[0163] To a diastereoisomeric mixture of compound 5 (210 g; R.sub.P:S.sub.P—33.74:66.26%) was charged 20% ethyl acetate in hexane (1.2 L) 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 diastereoisomeric mixture of compound 5.

[0164] Yield: 112 g

[0165] Chiral Purity by HPLC (% area): 22.13:77.87% (R.sub.P:S.sub.P)

[0166] Filtrate was concentrated under vacuum to obtain a diastereoisomeric mixture of compound of 5 (75 g; R.sub.P:S.sub.P—65.43:34.57%).

[0167] To a diastereoisomeric mixture of the compound of formula IIb (112 g; R.sub.P:S.sub.P—22.13:77.87%) 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 substantially pure S.sub.p-diastereoisomer of compound 5.

[0168] Yield: 80 g

[0169] Chiral Purity by HPLC (% area): 0.20:99.80% (R.sub.P:S.sub.P)

[0170] .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)

[0171] ESI-MS (m/z): 524 (M+1)

[0172] Filtrate was concentrated under vacuum to obtain a diastereoisomeric mixture of compound 5 (28 g; R.sub.P:S.sub.P—80.77:19.23%).

Example 3: Enrichment of 2-[(2,3,4,5,6-pentafluorophenoxy)-phenoxy-phosphoryl amino] propionic Acid Benzyl Ester 5 S-Isomer (an Illustrative Example of a Compound of Formula IIb)

[0173] ##STR00050##

[0174] To a stirred solution of 2-[(2,3,4,5,6-pentafluorophenoxy)-phenoxy-phosphoryl amino] propionic acid benzyl ester 5 (75 g; R.sub.P:S.sub.P—65.43:34.57%) in 20% ethyl acetate in hexane (1.1 L), 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.

[0175] Yield: 45 g

[0176] Chiral Purity by HPLC (% area): 91.29:8.71% (S.sub.P:R.sub.P)

[0177] 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 5 (45 g; R.sub.P:S.sub.P—8.71:91.29%) was slurred in 20% ethyl acetate in hexane (1.1 L) 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.

[0178] Yield: 19 g

[0179] Chiral Purity by HPLC (% area): 99.92:0.08% (S.sub.P:R.sub.p)

Example 4: Preparation of Diastereoisomeric Mixture of 2-[(4-nitrophenoxy)-phenoxy-phosphorylamino] propionic Acid Benzyl Ester 7 (an Illustrative Example of a Compound of Formula IIb

[0180] ##STR00051##

[0181] To a stirred mixture of L-alanine benzyl ester hydrochloride 1 (50 g) in methylene chloride (500 mL) was added phenyl dichlorophosphate 2 (54 g) 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 hour 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 L) 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 3. 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 4-nitrophenol 6 (27.5 g), 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 (2×500 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 7 as a thick oily liquid.

[0182] Yield: 90 g

[0183] Chiral Purity by HPLC (% area): 45.6:54.94% (R.sub.P:S.sub.P)

[0184] The above obtained diastereoisomeric mixture of 2-[(4-nitrophenoxy)-phenoxy-phosphorylamino] propionic acid benzyl ester 7 (40 g; R.sub.p:S.sub.p—45.6:54.94%) 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.

[0185] Yield: S.sub.p-diastereoisomer: 8 g,

[0186] .sup.1H NMR (300 MHz, CDCl.sub.3): 8.15-8.19 (d, 2H), 7.15-7.37 (m, 12H), 5.12 (s, 2H), 4.02-4.24 (m, 2H), 1.39-1.42 (d, 3H)

[0187] ESI-MS (m/z): 479 (M+Na)

[0188] R.sub.p-diastereoisomer: 6 g,

[0189] .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)

[0190] ESI-MS (m/z): 457 (M+1)

[0191] S.sub.p and R.sub.p-diastereoisomers mixture: 20 g

Example 5—Preparation of (Sp)-2-[(2,3,4,5,6-pentafluorophenoxy)-phenoxy-phosphoryl amino] propionic Acid Benzyl Ester 5 (an Illustrative Example of a Compound of Formula IIb)

[0192] ##STR00052##

[0193] To a stirred mixture of L-Alanine Benzyl ester. HCl 1 (100 g) in 1000 mL of methylene dichloride was added phenyl dichlorophosphate 2 (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. (10±5) and stirred for a further 1.5 h.

[0194] A solution of pentafluorophenol 4 (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. (10±5) 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.

[0195] 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° C. 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 5-(Sp). Yields are in the range 40 to 80% and the diastereoisomeric purity is over 99%.

Example 6: Preparation of Diastereoisomeric Mixture of 2-[(2,3,4,5,6-pentafluorophenoxy)-naphth-1-oxy-phosphoryl amino] propionic Acid Benzyl Ester 12 (an Illustrative Example of a Compound of Formula IIa)

[0196] ##STR00053##

[0197] Alpha-naphthol 8 (100 g) was dissolved in DCM (1 L) at 25° C. and POCl.sub.3 9 (1.1 eq) was added at 25° C. and stirred for 10 min before the mixture was cooled to −70° C. and stirred for 10 min. Triethylamine (1.1 eq.) was added slowly maintaining the temperature at below −70° C. and the mixture was stirred for 1 h at −70° C. The mixture was warmed to 25° C. and stirred for 1 h before being cooled to −50° C. L-alanine benzyl ester 1 (HCl salt; 1 eq.) was added to the mixture which stirred for 10 min before triethylamine (2.2 eq) in DCM (200 mL) was added at −50° C. over 30 minutes. The mixture was stirred for 1 h at −50° C. before being warmed to 25° C. and stirred for a further 1 h. The mixture was cooled to −10° C. and stirred for 10 min before pentafluorophenol 4 in DCM (200 mL) was added to the reaction mass slowly at below −10° C. The mixture was stirred at −10° C. for 10 min before triethylamine (1.1 eq.) was added over 30 min at −10° C. The mixture was stirred at −10° C. for 1 h before the mixture was warmed to 0° C. Water (1 L) was added and the mixture was stirred for 30 min at 0° C. The mixture was warmed to 25° C. and stirred for 5-10 min before the organic layer was separated. The aqueous layer was extracted with DCM (500 mL). The combined organic layers were washed with 7% sodium bicarbonate solution (2×1 L) and the organic layer was dried over anhydrous sodium sulphate before being concentrated in vacuo. 50% IPA/water (2.4 L) was added to the crude compound and stirred for 1 h at 25° C. The solid compound was filtered and the wet cake was washed with 50% IPA/water (500 mL) before being dried in vacuo. Again 50% IPA/water (2.4 L) was added to the crude compound and stirred for 1 h at 25° C. before being filtered and the wet cake was again washed with 50% IPA/water (500 mL) before being dried in vacuo. The semi-dried compound was washed with cyclohexane (10 v/w) at 25-30° C. for 1 h before the solid compound was washed with cyclohexane (2 L) and the wet compound 12 was dried under vacuum at 55-60° C. ° C. for 12 h

[0198] Results: [0199] Weight of the compound: 252 g [0200] Overall yield: 66% [0201] HPLC purity: 98.31% (diastereoisomeric ratio is 1:1)

[0202] .sup.31P NMR (202 MHz, CDCl.sub.3): δ.sub.P−1.35, −1.41; .sup.1H NMR (500 MHz, CDCl.sub.3): δ.sub.H 8.13-8.10 (1H, m, H—Ar), 7.90-7.88 (1H, m, H—Ar), 7.73 (1H, apparent d, J=8.5 Hz, H—Ar), 7.62-7.55 (3H, m, H—Ar), 7.45-7.41 (1H, m, H—Ar), 7.36-7.28 (5H, m, H—Ar), 5.01 (1H, apparent s, CH.sub.2Ph), 5.12 (1H, q, J=12.5 Hz, CH.sub.2Ph), 4.38-4.31 (1H, m, NHCHCH3), 4.17-4.08 (1H, m, NHCHCH.sub.3), 1.49, 1.47 (3H, 2×d, J=3.5 Hz, NHCHCH.sub.3); MS (ES+) m/z: 574 (M+Na.sup.+, 100%), Accurate mass: C.sub.26H.sub.19F.sub.5NO.sub.5P required 551.40 found 574.05 (M+Na.sup.+); Reverse-phase HPLC, eluting with H.sub.2O/MeOH in 20/80 in 35 min, F=1 mL/min, λ=254, two peaks for two diastereoisomers with t.sub.R=12.96, 14.48 min.

[0203] The diastereoisomers of compound 12 were separated by HPLC with Biotage Isolera using C18 SNAP Ultra (30 g) cartridge with a mixture of MeOH/H.sub.2O (70%/30%) as an eluent to give: the fast eluting isomer (believed to be the Rp diastereoisomer) and the slow eluting isomer (believed to be the Sp diastereoisomer)

[0204] Note: Isomers are named as fast eluting (FE) and slow eluting (SE) based on retention time on C18 (reversed phase) cartridge and HPLC analytical column.

[0205] Fast eluting isomer (believed to be the Rp diastereoisomer): .sup.31P NMR (202 MHz, CDCl.sub.3): δ.sub.P−1.41; .sup.1H NMR (500 MHz, CDCl.sub.3): δ.sub.H 8.02 (1H, dd, J=7.0, 2.0 Hz, H—Ar), 7.79 (1H, dd, J=6.5, 3.0 Hz, H—Ar), 7.64 (1H, d, J=8.5 Hz, H—Ar), 7.53-7.45 (3H, m, H—Ar), 7.33 (1H, t, J=8.0 Hz, H—Ar), 7.28-7.23 (5H, m, H—Ar), 5.09 (s, 2H, CH.sub.2Ph), 4.29-4.21 (1H, m, NHCHCH.sub.3), 4.02-3.97 (1H, m, NHCHCH.sub.3), 1.38 (3H, d, J=7.0 Hz, NHCHCH.sub.3); MS (ES+) m/z: MS (ES+) m/z: 574 (M+Na.sup.+, 100%), Accurate mass: C.sub.26H.sub.19F.sub.5NO.sub.5P required 551.40 found 574.05 (M+Na.sup.+); Reverse-phase HPLC, eluting with H.sub.2O/MeOH in 20/80 in 35 min, F=1 mL/min, λ=254, t.sub.R=12.96.

[0206] Slow eluting isomer (believed to be the Sp diastereoisomer): .sup.31P NMR (202 MHz, CDCl.sub.3): δ.sub.P−1.36; .sup.1H NMR (500 MHz, CDCl.sub.3): δ.sub.H 8.14-8.11 (1H, m, H—Ar), 7.90-7.87 (1H, m, H—Ar), 7.74 (1H, d, J=8.0 Hz, H—Ar), 7.60 (1H, d, J=8.0 Hz, H—Ar), 7.58-7.55 (2H, m, H—Ar), 7.44 (1H, t, J=8.0 Hz, H—Ar), 7.34-7.30 (5H, m, H—Ar), 5.12 (2H, q, J=12.5 Hz, CH.sub.2Ph), 4.35-4.29 (1H, m, NHCHCH.sub.3), 4.04-4.00 (1H, m, NHCHCH3), 1.48 (3H, d, J=7.0 Hz, NHCHCH3); MS (ES+) m/z: MS (ES+) m/z: 574 (M+Na.sup.+, 100%), Accurate mass: C.sub.26H.sub.19F.sub.5NO.sub.5P required 551.40 found 574.05 (M+Na.sup.+); Reverse-phase HPLC, eluting with H.sub.2O/MeOH in 20/80 in 35 min, F=1 mL/min, λ=254, t.sub.R=14.48.

Example 7: Enrichment of S.SUB.p.-Diastereoisomer of 2-[(2,3,4,5,6-pentafluorophenoxy)-naphth-1-oxy-phosphoryl amino] propionic Acid Benzyl Ester 12 Sp Isomer (an Illustrative Example of a Compound of Formula IIb)

[0207] ##STR00054##

[0208] A 1:1 diastereoisomeric mixture of compound 12 (25 g) was dissolved in 10% MTBE/n-Hexane (500 mL) and triethylamine (2.5 mL) was added to the reaction mass at 25° C. The mixture was stirred for 80 h at 30° C. The mixture was filtered and the wet cake was washed with 10% MTBE/n-hexane (75 mL) before being dried in vacuo 30 min. 50% IPA/water (200 mL) was added to above crude compound and stirred for 1 h at 25-35° C. before being filtered. The wet cake was washed with 50% IPA/water (100 mL) before being dried in vacuo at 55-60° C. ° C. for 12 h

Result:

[0209] Wt. of the compound: 17 g [0210] Yield: 68% [0211] HPLC purity: 97.66%

[0212] Slow eluting isomer (believed to be Sp-diastereoisomer): .sup.31P NMR (202 MHz, CDCl.sub.3): δ.sub.P−1.36; .sup.1H NMR (500 MHz, CDCl.sub.3): δ.sub.H 8.14-8.11 (1H, m, H—Ar), 7.90-7.87 (1H, m, H—Ar), 7.74 (1H, d, J=8.0 Hz, H—Ar), 7.60 (1H, d, J=8.0 Hz, H—Ar), 7.58-7.55 (2H, m, H—Ar), 7.44 (1H, t, J=8.0 Hz, H—Ar), 7.34-7.30 (5H, m, H—Ar), 5.12 (2H, q, J=12.5 Hz, CH.sub.2Ph), 4.35-4.29 (1H, m, NHCHCH.sub.3), 4.04-4.00 (1H, m, NHCHCH.sub.3), 1.48 (3H, d, J=7.0 Hz, NHCHCH.sub.3); MS (ES+) m/z: MS (ES+) m/z: 574 (M+Na.sup.+, 100%), Accurate mass: C.sub.26H.sub.19F.sub.5NO.sub.5P required 551.40 found 574.05 (M+Na.sup.+); Reverse-phase HPLC, eluting with H.sub.2O/MeOH in 20/80 in 35 min, F=1 mL/min, λ=254, t.sub.R=14.48.

[0213] The stereochemistry (Rp vs Sp) of the two compound 12 isomers described above has been assigned tentatively on the basis of comparison of .sup.31P chemical shift, 1H NMR spectra, and HPLC retention times of the NUC-3373 isomers made using the compound 12 isomers with those of other ProTides known in the literature. As mentioned above, the stereochemistry of phosphate stereocentre is inverted during the process of the invention so the (Sp)-diastereoisomer of the compound of formula 12 will form the (Sp)-diastereoisomer of NUC-3373 and likewise the (R)-diastereoisomer of the compound of formula 12 will form the (R)-diastereoisomer of NUC-3373. The stereochemical assignment is supported by powder X-ray diffraction and differential scanning calorimetry that has been carried out on the two compound 12 isomers, but this is not in itself definitive.

Example 8—Formation of Sp and Rp Isomers of NUC-3373

[0214] 3′-BOC protected FUDR 16 can be made according to the following scheme.

##STR00055##

[0215] Compound 16 can then be coupled with a compound of formula IIa.

##STR00056##

[0216] Compound 16 (1 g) and the Sp isomer of compound 12 (1.2 eq) were dissolved in THF (10 mL) and the mixture was cooled to 0° C. t-Butyl magnesium chloride (2.5 eq, 2.0 M in THF) was added to the mixture over 15 min. The mixture was warmed and stirred at 25° C. for 4 h. The mixture was cooled to 10° C. and sat. ammonium chloride solution (10 mL) was added. Ethyl acetate (10 mL) was added to the mixture and the organic layer was separated. The aqueous layer was extracted with ethyl acetate (5 mL). The combined organic layers were washed with deionised water (5 mL) followed by 20% sodium chloride solution (5 mL). The organic layers were dried over anhydrous sodium sulphate before being concentrated in vacuo to provide 2.16 g of compound 17 (100% crude yield).

[0217] Crude compound 17 (1 g) was dissolved in DCM (5 mL) and cooled to 10° C. TFA (2 mL) was added slowly to the mixture, maintaining the temperature at below 20° C. The mixture was warmed to 30° C. and the stirred for 6 h. The mixture was cooled to 10° C. and deionized water (5 mL) was added slowly, maintaining the temperature at below 20° C. After stirring for 10 min the organic layer was separated and the aqueous layer was extracted with DCM (5 mL). The combined organic layers were washed with deionised water (2×5 mL), 7% sodium bicarbonate solution (2×5 mL) and 20% sodium chloride solution (5 mL) before being dried with anhydrous sodium sulphate (1 w/w) and concentrated in vacuo. Crude compound was purified with column chromatography in ethyl acetate/DCM using silica gel (100-200 mesh). Pure compound was eluted in 50% Ethyl acetate/DCM to 100% ethyl acetate. The combined pure fractions were concentrated in vacuo before the compound slurry was washed with cyclohexane (5 mL).

[0218] Results: [0219] Weight of NUC-3373 (Sp isomer): 9.3 g [0220] Overall yield: 70% [0221] HPLC purity: 96.86%

[0222] .sup.1H-NMR (500 MHz, MeOD): δ.sub.H 8.16-8.14 (m, 1H, H—Ar), 7.90-7.80 (m, 1H, H—Ar), 7.72-7.70 (m, 2H, H—Ar), 7.54-7.49 (m, 3H, H—Ar, H-6), 7.43 (apparent t, 1H, J=8.0 Hz, H—Ar), 7.35-7.27 (m, 5H, H—Ar), 6.16-6.13 (m, 1H, H-1′), 5.11 (AB system, J=12.0 Hz, 2H, OCH.sub.2Ph), 4.35-4.33 (m, 2H, 2×H-5′), 4.30-4.28 (m, 1H, H-3′), 4.14-4.08 (m, H, CHCH.sub.3), 4.07-4.04 (m, 1H, H-4′), 2.14-2.09 (m, 1H, H-2′), 1.74-1.68 (m, 1H, H-2′), 1.35 (d, J=7.0 Hz, 3H, CHCH.sub.3);

[0223] .sup.13C-NMR (125 MHz, MeOD): δ.sub.C 174.92 (d, .sup.3J.sub.C-P=3.75 Hz, C═O, ester), 159.37 (d, .sup.2J.sub.C-F=25.9 Hz, C═O, base), 150.54 (d, .sup.4J.sub.C-F=4.0 Hz, C═O, base), 147.99 (d, .sup.2J.sub.C-P=7.1 Hz, C—Ar, Naph), 141.75 (d, .sup.1J.sub.C-F=232.1 Hz, CF-base), 137.18, 136.29 (C—Ar), 129.59, 129.36, 128.90, 127.91 (CH—Ar), 127.83 (d, .sup.3J.sub.C-P=5.4 Hz, C—Ar, Naph), 127.59, 126.52, 126.50, 126.18 (CH—Ar), 125.54 (d, .sup.2J.sub.C-F=34.1 Hz, CH-base), 122.64 (CH—Ar), 116.29 (d, .sup.3J.sub.C-P=2.75 Hz, CH—Ar, Naph), 86.95 (C-1′), 86.67 (d, .sup.3J.sub.C-P=8.1 Hz, C-4′), 72.12 (C-3′), 68.05 (OCH.sub.2Ph), 67.85 (d, .sup.2J.sub.C-P=5.3 Hz, C-5′), 51.96 (CHCH.sub.3), 40.84 (C-2′), 20.52 (d, .sup.3J.sub.C-P=7.5 Hz, CHCH.sub.3).

[0224] .sup.31P-NMR (202 MHz, MeOD): δ.sub.P 4.62;

[0225] .sup.19F NMR (470 MHz, MeOD): δ.sub.F−167.19;

[0226] (ES+) m/z: Found: (M+Na.sup.+) 636.1520. C.sub.29H.sub.29N.sub.3O.sub.9FNaP required: (M.sup.+), 613.15.

[0227] Reverse HPLC (Varian Pursuit XRs 5 C18, 150×4.6 mm) eluting with (H.sub.2O/AcCN from 90/10 to 0/100) in 35 min., t.sub.R 16.61 min.

[0228] The Rp isomer of NUC-3373 can be accessed by performing the above process but starting with the Rp diastereomer of compound 12:

[0229] .sup.1H-NMR (500 MHz, MeOD): δ.sub.H 8.17-8.15 (m, 1H, H—Ar), 7.91-7.88 (m, 1H, H—Ar), 7.72-7.69 (m, 2H, H—Ar), 7.56-7.52 (m, 2H, H—Ar, H-6), 7.50-7.48 (m, 1H, H—Ar), 7.39 (apparent t, J=8.0 Hz, 1H, H—Ar), 7.35-7.28 (m, 5H, H—Ar), 6.16-6.09 (m, 1H, H-1′), 5.13 (s, 2H, OCH.sub.2Ph), 4.35-4.25 (m, 3H, 2×H-5′, H-3′), 4.14-4.08 (m, 1H, CHCH.sub.3), 4.05-4.03 (m, 1H, H-4′), 2.15-2.10 (m, 1H, H-2′), 1.74-1.68 (m, 1H, H-2′), 1.36 (d, J=7.0 Hz, 3H, CHCH.sub.3);

[0230] .sup.13C-NMR (125 MHz, MeOD): δ.sub.C 174.58 (d, .sup.3J.sub.C-P=5.0 Hz, C═O, ester), 159.38 (d, .sup.2J.sub.C-F=26.3 Hz, C═O), 150.48 (C═O base), 147.80 (d, .sup.2J.sub.C-P=6.5 Hz, C—Ar, Naph), 141.67 (d, .sup.1J.sub.C-P=232.5 Hz, CF-base), 137.15, 136.26 (C—Ar), 129.62, 129.40, 129.36, 128.96, 127.89 (CH—Ar), 127.84 (d, .sup.3J.sub.C-P=5.5 Hz, C—Ar, Naph), 127.59, 126.57, 126.55, 126.21 (CH—Ar), 125.61 (d, .sup.2J.sub.C-F=34.0 Hz, CH-base), 122.62 (CH—Ar), 116.55 (d, .sup.3J.sub.C-P=3.75 Hz, CH—Ar, Naph), 86.97 (C-1′), 86.66 (d, .sup.3J.sub.C-P=7.5 Hz, C-4′), 72.01 (C-3′), 68.07 (OCH.sub.2Ph), 67.84 (d, .sup.2J.sub.C-P=5.0 Hz, C-5′), 51.83 (CHCH.sub.3), 40.89 (C-2′), 20.42 (d, .sup.3J.sub.C-P=7.5 Hz, CHCH.sub.3).

[0231] .sup.31P-NMR (202 MHz, MeOD): δ.sub.P 4.27;

[0232] .sup.19F NMR (470 MHz, MeOD): O.sub.F−167.27;

[0233] (ES+) m/z: Found: (M+Na.sup.+) 636.1520. C.sub.29H.sub.29N.sub.3O.sub.9FNaP required: (M.sup.+), 613.15.

[0234] Reverse HPLC (Varian Pursuit XRs 5 C18, 150×4.6 mm) eluting with (H.sub.2O/MeOH from 90/10 to 0/100) in 35 min., t.sub.R 16.03 min.

[0235] The stereochemistry (Rp vs Sp) of the two NUC-3373 isomers described above has been assigned tentatively on the basis of comparison of .sup.31P chemical shift, .sup.1H NMR spectra, and HPLC retention times with those of other ProTides known in the literature. The stereochemistry of compound 12 has been tentatively assigned based on which isomer of NUC-3373 that isomer of compound 12 forms.

Example 9—Formation of Sp and Rp Isomers of NUC-7738

[0236] 2′-TBDMS protected 3′-deoxyadenosine 21 can be made according to the following scheme.

##STR00057##

[0237] Compound 21 can then be coupled with a compound of formula IIb using the coupling process conditions described in Example 8. To form NUC-7738, the TBDMS group can be removed using TFA in THF.

##STR00058##

[0238] Adenosine (18) to Epoxide 19

[0239] One equivalent adenosine (18) was dissolved in 10 V acetonitrile and the mixture was cooled to 15° C. 3.0 molar equivalents acetoxy isobutyryl bromide was added slowly at 15° C. The mixture was warmed to room temperature and stirred for 8 hours. The reaction was quenched with sodium bicarbonate solution and extracted with ethyl acetate. The combined organic layers were washed with 5% sodium chloride solution and the organic layers were concentrated in vacuo.

[0240] The product was dissolved in 15 V methanol and 1 weight equivalent of potassium carbonate was added before stirring for 2 hours. The mixture was concentrated in vacuo and the product was washed with water before drying the product under vacuum at 60° C. to provide 2′,3′-anhydro adenosine in a yield of 70-85%.

[0241] 1 equivalent of 2′,3′-anhydro adenosine and 1.6 equivalents imidazole were dissolved in 5V DMF. The mixture was cooled to 15° C. and 0.8 equivalents TBDMSCI was added. The mixture was stirred for 1 to 2 hours at 30° C. before a further 0.4 equivalents imidazole and 0.4 equivalents TBDMSCI were added. The mixture was stirred for a further 1 to 2 hours at 30° C. before water was added (5V). The mixture was extracted with ethyl acetate. The combined organic layers were sequentially washed with 7% sodium bicarbonate solution, water and 5% sodium chloride solution before being concentrated in vacuo. The product was washed with heptane before being dried under vacuum at 50° C. to obtain epoxide 19 in 75-90% yield.

Epoxide 19 to 5′-Silyl Cordycepin 21

[0242] 1 Equivalent of epoxide 19 was dissolved in a mixture of DMSO (5V) and THF (5V). The mixture was cooled to 0° C. and the mixture was purged with nitrogen gas. 1M Lithium triethylborohydride (1 eq) in THF was added at 0(±5)° C. over a period of 1-2 hours. The mixture was stirred at 0° C. for 30 minutes, warmed to 30° C. and stirred for 2 hours before methanol (10 V) was slowly added at 5° C. 10V 10% sodium hydroxide and then 10V 10% hydrogen peroxide solution were added drop wise at 5° C. The mixture was extracted with ethyl acetate and the combined organic layers were washed sequentially with 10% sodium metabisulfite solution, water in to reactor, 7% sodium bicarbonate solution and 10% sodium chloride solution before being concentrated in vacuo. The product was washed with heptane before being dried under vacuum at 50° C. to obtain 2′-silyl cordycepin in 70-100% yield.

[0243] The 2′-silyl cordycepin, 2.5 equivalents imidazole and 0.15 equivalents DMAP were dissolved in 5V DMF. The mixture was cooled to 15° C. before 2.5 equivalents TBDMSCI were added portionwise. The reaction was stirred for 4 hours at 30° C. before being cooled to 15° C. 10V water was added and the mixture was extracted with ethyl acetate. The organic layers were washed with 7% sodium bicarbonate solution, water and 5% sodium chloride before being concentrated in vacuo.

[0244] The mixture was dissolved in 8V and 2V water was added before the mixture was cooled to 0° C. 2.5 Eq. trifluoroacetic acid was added to the reaction mixture at 0° C. over a period of 30-60 min. The mixture was warmed to 10° C. and stirred for 4 to 6 hours at 10° C. Water was added and the mixture was extracted with ethyl acetate. The combined organic layers were washed with 7% sodium bicarbonate solution, water (twice) and 5% sodium chloride solution before being concentrated in vacuo. The product was washed with heptane and dried under vacuum to provide 5′-silyl cordycepin 21 in 40-70% yield.

5′-Silyl Cordycepin 21 to S.SUB.p.-NUC-7738

[0245] 5′-silyl cordycepin 21 was dissolved in 10 V THF and cooled to 0° C. 2.0M t-BuMgCl (2.5 equivalents) was added and the mixture was stirred for 15 min. The S.sub.p isomer of compound 5 (2.5 eq) was dissolved in 5 V THF and was added to the reaction at 0° C. The mixture was stirred at 0° C. for 15 min before being warmed to 25° C. and stirred for a further 2 hours. The reaction was quenched into 10% ammonium chloride solution (10 vol) and extracted with ethyl acetate. The combined organic layers were washed with water and 10% brine solution before being concentrated in vacuo.

[0246] The product was dissolved in THF (10V) before being cooled to 0° C. A 10 V TFA and water (1:1) mixture was added to the reaction over a period of 30 min before the mixture was stirred for 45 min, warmed to 30° C. and stirred for a further 16 h. The reaction was quenched in to 7% NaHCO.sub.3 solution (90 V) at 0° C. before being extracted with ethyl acetate. The combined organic layers were washed with water, 7% sodium bicarbonate solution and 10% brine solution before being concentrated in vacuo.

[0247] The product was purified by column chromatography by using silica gel (100-200 mesh), the column was eluted by 2 to 10% MeOH in DCM to provide S.sub.p-NUC-7738 in 40% yield. The HPLC purity of the product was 99.50% and Chiral HPLC showed the S.sub.p isomer to be present in 99.90% and the R.sub.p isomer to be present in 0.10%.

[0248] The same procedure can be carried out to provide R.sub.p-NUC-7738.

Rp-NUC-7738:

[0249] .sup.1H NMR (500 MHz, CDCl.sub.3) δ.sub.H 8.26 (s, 1H, H8), 8.22 (s, 1H, H2), 7.37-7.25 (m, 7H, Ar), 7.22-7.12 (m, 3H, Ar), 6.01 (d, J=1.5 Hz, 1H, H1′), 5.12 (AB q, J.sub.AB=12.0 Hz, Δδ.sub.AB=0.04, 2H, CH.sub.2Ph), 4.74-4.70 (m, 1H, H2′), 4.69-4.62 (m, 1H, H4′), 4.44-4.38 (m, 1H, H5′), 4.28-4.21 (m, 1H, H5′), 3.99-3.90 (m, 1H, CHCH.sub.3 L-Ala), 2.35-2.27 (m, 1H, H3′), 2.09-2.02 (m, 1H, H3′), 1.29 (d, J=7.0 Hz, 3H, CHCH.sub.3 L-Ala).

[0250] .sup.31P NMR (202 MHz, CD.sub.3OD) Op 3.91.

[0251] MS (ES.sup.+) m/z found 569.2 [M+H.sup.+], 591.2 [M+Na.sup.+], 1159.4 [2M+Na.sup.+] C.sub.26H.sub.29N.sub.6O.sub.7P required m/z 568.2 [M].

[0252] HPLC Reverse-phase HPLC (Varian Pursuit XRs 5 C18, 150×4.6 mm) eluting with H.sub.2O/CH.sub.3CN from 90/10 to 0/100 in 30 minutes, F: 1 mL/min, λ=200 nm, shows one peak with t.sub.R 14.02 min.

Sp-NUC-7738:

[0253] .sup.1H NMR (500 MHz, CDCl.sub.3) δ.sub.H 8.24 (s, 1H, H8), 8.22 (s, 1H, H2), 7.36-7.26 (m, 7H, Ar), 7.22-7.13 (m, 3H, Ar), 6.01 (d, J=1.5 Hz, 1H, H1′), 5.08 (AB q, J.sub.AB=12.0 Hz, Δδ.sub.AB=0.01, 2H, CH.sub.2Ph), 4.70-4.67 (m, 1H, H2′), 4.66-4.60 (m, 1H, H4′), 4.41-4.35 (m, 1H, H5′), 4.26-4.19 (m, 1H, H5′), 4.02-3.94 (m, 1H, CHCH.sub.3 L-Ala), 2.36-2.27 (m, 1H, H3′), 2.08-2.01 (m, 1H, H3′), 1.34-1.30 (m, 3H, CHCH.sub.3 L-Ala).

[0254] .sup.31P NMR (202 MHz, CD.sub.3OD) δ.sub.P 3.73. MS (ES.sup.+) m/z found 569.2 [M+H.sup.+], 591.2 [M+Na.sup.+], 1159.4 [2M+Na.sup.+] C.sub.26H.sub.29N.sub.6O.sub.7P required m/z 568.2 [M].

[0255] HPLC Reverse-phase HPLC (Varian Pursuit XRs 5 C18, 150×4.6 mm) eluting with H.sub.2O/CH.sub.3CN from 90/10 to 0/100 in 30 minutes, F: 1 mL/min, λ=200 nm, shows one peak with t.sub.R 14.26 min.

[0256] The stereochemistry (R.sub.p vs S.sub.p) of the two NUC-7738 isomers described above has been confirmed by conventional X-ray crystallographic analysis.

Example 10—Formation of NUC-9701

[0257] Dimethyl acetal 25 of 8-chloro-adenosine 24 can be made according to the following scheme (also described in WO2017/207989).

##STR00059##

[0258] Compound 25 can then be coupled with a compound of formula IIa using the coupling process conditions described in Example 8. To form NUC-9701, dimethyl acetal can be removed using 1:1 TFA: water at 0° C. for 5 h.

##STR00060##

[0259] Compound 25 (30 g; 1 equivalent) and the desired isomer of compound 12 (58.08 g; 1.2 equivalents) were dissolved in 300 ml (10 V) of THF. The mixture was cooled to 0° C. before t-butyl magnesium chloride (76.8 ml of 2.0 M in THF; 1.75 equivalents) was added slowly, maintaining the temperature at 0° C., and the mixture was stirred for 4 hours. 300 mL (10V) of 10% Ammonium chloride solution was added to the reaction mixture, maintaining the temperature at below 15° C. The mixture was extracted with ethyl acetate and the combined organic layers were washed with 7% sodium bicarbonate solution (twice), water (twice) and 20% sodium chloride before being dried with anhydrous sodium sulphate and filtered. The ethyl acetate was removed in vacuo.

[0260] To the resultant product as added 600 ml (20V) of 60% formic acid in water and the reaction was stirred for 65-70 h at 25° C. before ethyl acetate (600 mL; 20V) was added slowly. 600 ml (20 V) of 20% sodium chloride solution was added and the layers were separated. The aqueous layer was extracted with ethyl acetate before 600 ml (20 V) 10% Ammonia solution was added dropwise to the combined organic layers and the layers were separated. The organic layer was washed with water (three times) and 20% sodium chloride solution, dried with 30 g (1 w/w) of anhydrous sodium sulphate and filtered before being concentrated in vacuo. The crude product was purified by column chromatography to provide 20-45 g NUC-9701.

Sp-NUC-9701:

[0261] .sup.1H-NMR (500 MHz; MeOD-d4): δ.sub.H 8.07 (1H, d J=8.5 Hz, H-Napht), 8.05 (1H, s, 2-H), 7.87 (1H, d J=8.5 Hz, H-Napht), 7.67 (1H, d J=8.5 Hz, H-Napht) 7.54-7.48 (2H, m, H-Napht), 7.41-7.30 (1H, m, H-Napht), 7.36-7.33 (1H, m, H-Napht), 7.26-7.22 (5H, m, —CH.sub.2Ph), 6.03 (1H, d J=5.0 Hz, H-1′), 5.33 (1H, t J=5.0 Hz, H-2′), 5.01, 4.98 (AB, J.sub.AB=12.3 Hz, CH.sub.2Ph), 4.65 (1H t J=5.5 Hz, H-3′), 4.49-4.45 (1H, m, H.sub.a-5′), 4.41-4.36 (1H, m, H.sub.b-5′), 4.22-4.20 (1H, m, H-4′), 3.94-3.90 (1H, m, —CHCH.sub.3), 1.17 (1H, d J=7.0 Hz, CH.sub.3).

[0262] .sup.31P NMR (202 MHz, MeOD-d4): δ.sub.P 3.93 (1P, s).

[0263] Reverse-phase HPLC, eluting with H.sub.2O/CH.sub.3CN from 90/10 to 0/100 in 30 min; 1 mL/min, λ=254 nm, showed a peak with t.sub.R=16.43 min

Rp-NUC-9701:

[0264] .sup.1H-NMR (500 MHz; MeOD-d4): δ.sub.H 8.10 (1H, s, H-2), 8.08 (1H, d J=8.5 Hz, H-Napht), 7.87 (1H, d J=8.5 Hz, H-Napht), 7.67 (1H, d J=8.5 Hz, H-Napht), 7.53-7.50 (1H, m, H-Napht), 7.48-7.44 (1H, m, H-Napht), 7.40-7.38 (1H, m, H-Napht), 7.33-7.27 (6H, m, H-Napht and —CH.sub.2Ph), 6.02 (1H, d J=5.0 Hz, H-1′), 5.28 (1H, t J=5.0 Hz, H-2′), 5.04, 5.02 (AB, J.sub.AB=12.2 Hz, CH.sub.2Ph), 4.63 (1H t J=5.5 Hz, H-3′), 4.48-4.46 (1H, m, H.sub.a-5′), 4.38-4.35 (1H, m, H.sub.b-5′), 4.23-4.20 (1H, m, H-4′), 4.05-4.01 (1H, m, —CHCH.sub.3), 1.17 (1H, d J=7.0 Hz, CH.sub.3).

[0265] .sup.31P NMR (202 MHz, MeOD-d4): δ.sub.P 3.83 (1P, s).

[0266] Reverse-phase HPLC, eluting with H.sub.2O/CH.sub.3CN from 90/10 to 0/100 in 30 min; 1 mL/min, λ=254 nm, showed a peak with t.sub.R=16.59 min

[0267] The stereochemistry (R.sub.p vs S.sub.p) of the two NUC-9701 isomers described above has been assigned tentatively on the basis of comparison of .sup.31P chemical shift, 1H NMR spectra, and HPLC retention times with those of other ProTides known in the literature.