5`S-LNA nucleotides and oligonucleotides

Abstract

The invention relates to a compound of formula (I) wherein R2 and R4 are joined and together form a group, such a —CH2O—. The compound of formula (I) can be used in the manufacture of 5'S-LNA oligonucleotides as antisense drugs.

##STR00001##

Claims

1. A compound of formula (I) ##STR00034## wherein R.sup.1 is a phosphate protecting group; R.sup.2 and R.sup.4 together form —CH.sub.2O—, —CH.sub.2NH—, —CH.sub.2S—, —CH.sub.2N(OR′)—, —CHCH.sub.3O—, C(CH.sub.3).sub.2O, —CH.sub.2C(═CH.sub.2)—, —CHCH.sub.3C(═CH.sub.2)—, —CHCH.sub.3S—, —CH.sub.2NR.sup.p—, —CH.sub.2CH.sub.2O—, —CH.sub.2CH.sub.2CH.sub.2O—, —CH.sub.2OCH.sub.2—, —CH(CH.sub.2OCH.sub.3)O—, —CH(CH.sub.2CH.sub.3)O— or —CH.sub.2OCH.sub.2O—; R.sup.3 is dialkylamino; R.sup.5 is a thiohydroxyl protecting group; each R.sup.p is alkyl; and Nu is a nucleobase optionally comprising a protected primary amino group.

2. A compound according to claim 1, wherein R.sup.1 is cyanoethyl or methyl.

3. A compound according to claim 1, wherein R.sup.2 and R.sup.4 together form —CH.sub.2O—.

4. A compound according to claim 1, wherein R.sup.3 is diisopropylamino.

5. A compound according to claim 1, wherein R.sup.5 is trityl, 4-methoxytrityl, 4,4′-dimethoxytrityl or 4,4′,4″-trimethoxytrityl.

6. A compound according to claim 1, wherein each Rp is independently methyl, ethyl or propyl.

7. A compound according to claim 1, wherein Nu is adenine, guanine, cytosine, 5-methyl-cytosine, thymine or uracil.

8. A compound according to claim 1, wherein Nu is (A), (B), (C) or (D) ##STR00035## wherein R.sup.6 is hydrogen or a protecting group of the amino group to which it is attached.

9. A compound according to claim 8, wherein the protecting group of the amino group is benzoyl, dimethylformamide, acetyl or isobutyryl.

10. A compound according to claim 1, wherein Nu is (A1), (B1) or (C1) ##STR00036##

11. A process for the manufacture of a compound of formula (I) as defined in claim 1, comprising the reaction of a compound of formula (II) ##STR00037## in the presence of P(R.sup.3).sub.2OR.sup.1 and an oligonucleotide synthesis activator, wherein R.sup.1 is a phosphate protecting group; R.sup.2 and R.sup.4 together form —CH.sub.2O—, —CH.sub.2NH—, —CH.sub.2S—, —CH.sub.2N(OR′)—, —CHCH.sub.3O—, C(CH.sub.3).sub.2O, —CH.sub.2C(═CH.sub.2)—, —CHCH.sub.3C(═CH.sub.2)—, —CHCH.sub.3S—, —CH.sub.2NR.sup.p—, —CH.sub.2CH.sub.2O—, —CH.sub.2CH.sub.2CH.sub.2O—, —CH.sub.2OCH.sub.2—, —CH(CH.sub.2OCH.sub.3)O—, —CH(CH.sub.2CH.sub.3)O— or —CH.sub.2OCH.sub.2O—; R.sup.3 is dialkylamino; R.sup.5 is a thiohydroxyl protecting group; and each R.sup.p is alkyl.

12. A process according to claim 11, wherein the compound of formula (II) is obtained by the reaction of a compound of formula (III) ##STR00038## in the presence of R.sup.5X.sup.1 and a base, wherein R.sup.2 and R.sup.4 together form —CH.sub.2O—, —CH.sub.2NH—, —CH.sub.2S—, —CH.sub.2N(OR′)—, —CHCH.sub.3O—, C(CH.sub.3).sub.2O, —CH.sub.2C(═CH.sub.2)—, —CHCH.sub.3C(═CH.sub.2)—, —CHCH.sub.3S—, —CH.sub.2NR.sup.p—, —CH.sub.2CH.sub.2O—, —CH.sub.2CH.sub.2CH.sub.2O—, —CH.sub.2OCH.sub.2—, —CH(CH.sub.2OCH.sub.3)O—, —CH(CH.sub.2CH.sub.3)O— or —CH.sub.2OCH.sub.2O—; R.sup.3 is dialkylamino; R.sup.5 is a thiohydroxyl protecting group; each R.sup.p is alkyl; Nu is a nucleobase optionally comprising a protected primary amino group and wherein X.sup.1 is a leaving group.

13. A process according to claim 12, wherein the compound of formula (III) is obtained by the hydrolysis of a compound of formula (IV) ##STR00039## wherein R.sup.7 is alkyl, aryl, arylalkyl, substituted aryl or substituted arylalkyl, wherein substituted aryl and substituted arylalkyl are aryl and arylalkyl substituted on the aryl with one to three substitutents independently selected from alkyl, alkoxy and halogen.

14. A process according to claim 13, wherein R.sup.7 is phenyl.

15. A process according to claim 13, wherein the compound of formula (IV) is obtained by the reaction of a compound of formula (V) ##STR00040## in the presence of R.sup.7COSH, a phosphine and a dehydrating agent, wherein R.sup.7 is alkyl, aryl, arylalkyl, substituted aryl or substituted arylalkyl, wherein substituted aryl and substituted arylalkyl are aryl and arylalkyl substituted on the aryl with one to three substitutents independently selected from alkyl, alkoxy and halogen.

16. A process according to claim 15, wherein the compound of formula (V) is obtained by the removal of the hydroxyl protecting group R.sup.5 of a compound of formula (VI) ##STR00041## wherein R.sup.5 is a hydroxyl protecting group.

17. A process according to claim 11, wherein the compound of formula (II) is obtained by the reaction of a compound of formula (VII) ##STR00042## in the presence of a base, a nucleophile and a compound of formula (VIII) ##STR00043## wherein R.sup.8 is a leaving group; R.sup.9 is alkyl.

18. A process according to claim 17, wherein the compound of formula (VII) is obtained by the reaction of a compound of formula (V) ##STR00044## in the presence of R.sup.8X.sup.2 and a non-nucleophilic base, wherein X.sup.2 is a leaving group and R.sup.8 is a group capable of forming a leaving group together with the 5′ hydroxyl oxygen atom of the compound of formula (V).

19. A process according to claim 17, wherein the compound of formula (VIII) is obtained by the reaction of R.sup.5X.sup.3 in the presence of R.sup.9C(O)SH X.sup.3 is a leaving group.

20. A process according to claim 11, wherein the oligonucleotide synthesis activator is an azole.

21. A process according to claim 11, wherein the oligonucleotide synthesis activator is 1H-tetrazole, 5-nitrophenyl-1H-tetrazole (NPT), 5-ethylthio-1H-tetrazole (ETT), 5-benzylthio-1H-tetrazole (BTT), 5-methylthio-1H-tetrazole (MTT), 5-mercapto-tetrazoles (MCT) or 4,5-dicyanoimidazole (DCI).

22. A process according to claim 17, wherein the nucleophile is NaOH, KOH, NaOMe, KOMe, methylamine or NH.sub.3.

23. A process according to claim 15, wherein the dehydrating agent is diethyl azodicarboxylate or diisopropyl azodicarboxylate.

24. A process according to claim 12, wherein the phosphine is triphenylphosphine or trimethylphosphine.

25. A process according to claim 16, wherein the removal of the hydroxyl protecting group R.sup.5 of a compound of formula (VI) is done by the reaction of a compound of formula (VI) in the presence of acid.

26. A process according to claim 25, wherein the acid is perchloroacetic acid, acetic acid, chloroacetic acid, dichloroacetic acid or trifluoroacetic acid.

27. (canceled)

28. (canceled)

29. (canceled)

30. A compound according to claim 1 selected from N-{9-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-({[bis(propan-2-yl)amino](2-cyanoethoxy)phosphanyl}oxy)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-9H-purin-6-yl}benzamide; 3-[({[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-3-(5-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl]oxy}[bis(propan-2-yl)amino]phosphanyl)oxy]propanenitrile; N′-{9-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-({[bis(propan-2-yl)amino](2-cyanoethoxy)phosphanyl}oxy)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}-N,N-dimethylmethanimidamide; and N-{1-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-({[bis(propan-2-yl)amino](2-cyanoethoxy)phosphanyl}oxy)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl}benzamide.

31. A compound selected from N-(9-{1-[(benzoylsulfanyl)methyl]-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl}-9H-purin-6-yl)benzamide; N-{9-[7-hydroxy-1-(sulfanylmethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-9H-purin-6-yl}benzamide; N-{9-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-9H-purin-6-yl}benzamide; 1-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-5-methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione; N′-(9-{1-[(benzoylsulfanyl)methyl]-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl}-6-oxo-6,9-dihydro-1H-purin-2-yl)-N,N-dimethylmethanimidamide; N′-{9-[7-hydroxy-1-(sulfanylmethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}-N,N-dimethylmethanimidamide; N′-{9-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}-N,N-dimethylmethanimidamide; N-(1-{1-[(benzoylsulfanyl)methyl]-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl}-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide; N-{1-[7-hydroxy-1-(sulfanylmethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl}benzamide and; N-{1-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl}benzamide.

32. (canceled)

33. An oligonucleotide comprising a fragment of formula (IX) ##STR00045## wherein R.sup.2 and R.sup.4 together form —CH.sub.2O—, —CH.sub.2NH—, —CH.sub.2S—, —CH.sub.2N(OR′)—, —CHCH.sub.3O—, —C(CH.sub.3).sub.2O—, —CH.sub.2C(═CH.sub.2)—, —CHCH.sub.3C(═CH.sub.2)—, —CHCH.sub.3S—, —CH.sub.2NR.sup.p—, —CH.sub.2CH.sub.2O—, —CH.sub.2CH.sub.2CH.sub.2O—, —CH.sub.2OCH.sub.2—, —CH(CH.sub.2OCH.sub.3)O—, —CH(CH.sub.2CH.sub.3)O— or —CH.sub.2OCH.sub.2O—; wherein each R′ is alkyl; and Nu is a nucleobase optionally comprising a protected primary amino group.

34. A method for the manufacture of an oligonucleotide comprising a fragment of formula (IX) as defined in claim 33 comprising the following steps: (a) Providing a solid support comprising: a hydroxyl group; a nucleotide comprising a hydroxyl group; or an oligonucleotide comprising a hydroxyl group; (b) Coupling a compound according to claim 1, a nucleotide, a locked nucleic acid nucleotide, a 2′-sugar modified nucleotide, a 3'S-DNA or a 3'S-LNA to the hydroxyl group of said solid support; (c) Oxidizing or thiooxydizing the product obtained from (b); (d) optionally capping unreacted hydroxyl groups of the product obtained from step (c); (e) Optionally removing hydroxyl protecting groups or thiohydroxyl protecting groups from the product obtained from step (c) or (d); (f) Optionally repeating steps (b) to (e); (g) Optionally removing any remaining protecting groups from the product obtained from any one of steps (c) to (f); and (h) Optionally cleaving the oligonucleotide from the solid support.

35. (canceled)

Description

EXAMPLES

Abbreviations:

[0128] AcOH=acetic acid, CAS RN=chemical abstracts registration number, DMAP=4-dimethylaminopyridine, DME=dimethoxyethane, DMF=N,N-dimethylformamide, DIPEA=N,N-diisopropylethylamine, dppf=1,1 bis(diphenylphosphino)ferrocene, EI=electron impact, ESI=electrospray ionization, h=hour, HATU=1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate, HPLC=high performance liquid chromatography, ISP=ion spray positive (mode), ISN=ion spray negative (mode), min=minutes, LAH=lithium aluminium hydride, LiHMDS=lithium bis(trimethylsilyl)amide, MPLC=medium performance liquid chromatography, MS=mass spectrum, PG=protecting group, Pd-C=palladium on activated carbon, PdCl.sub.2(dppf)-CH.sub.2Cl.sub.2=1,1′-bis(diphenylphosphino)ferrocene-palladium(II)dichloride dichloromethane complex, RT=room temperature, S-PHOS=2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl, TFA=trifluroacetic acid, THF=tetrahydrofuran, TMEDA=N,N,N′,N′-tetramethylethylenediamine, HBTU=O-Benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate, DMEDA=N,N′-dimethylethylenediamine, ACN=acetonitrile, TBAI=tetra butyl ammonium iodile, DME=di methoxy ethane, DEAD=diethyl azodicarboxylate, DMTrCl=4,4′-dimethoxytrityl chloride.
All examples and intermediates were prepared under argon atmosphere if not specified otherwise.

Example 1

N-{9-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-({[bis(propan-2-yl)amino](2-cyanoethoxy)phosphanyl}oxy)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-9H-purin-6-yl}benzamide

[0129] ##STR00015##

A solution of 5-ethylmercapto-1H-tetrazole (1.3 g, 9.97 mmol, 0.25 M solution in 38.4 mL dry ACN CAS RN 89797-68-2) was added to a stirred solution of N-{9-[1-({[bis(4-methoxyphenyl) (phenyl)methyl]sulfanyl}methyl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-9H-purin-6-yl}benzamide (3.5 g, 4.99 mmol) in dry DCM (120 mL) under argon at room temperature followed by addition of 2-cyanoethyl tetraisopropylphosphorodiamodite (3.17 mL, 9.98 mmol, CAS RN 102691-36-1). The reaction mixture was stirred at room temperature for 4 h. Then the reaction mixture was diluted with DCM (300 mL) and poured onto a sat. NaHCO.sub.3 solution (100 mL). The organic layer was separated off and the aqueous layer was extracted with DCM (70 mL×2). The combined organic layer was dried over sodium sulfate, filtered and evaporated under reduced pressure. The resulting crude compound was purified by combiflash (10-20% ACN in DCM) to get (2.7 g) impure compound. Using the same protocol another 1 g batch was performed to get 0.6 g impure compound. Using the same protocol another 2.5 g were carried out to get 1 g (pure) compound and 1.5 g impure compound. The impure compound thus obtained from different batches was mixed and repurified to get the title product (3.0 g) which was mixed with pure compound (1 g) to get the title compound (4.0 g, 44%) as a white solid. MS: (ESI): m/z=901.6 [M+H].sup.+.

Example 1.1

N-{9-[7-hydroxy-1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-9H-purin-6-yl}benzamide

[0130] ##STR00016##

To a solution of Cl.sub.3CCOOH (2.98 g, 18.23 mmol, CAS RN 76-03-9) in DCM (150 ml) was added N-[9-(1-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl)-9H-purin-6-yl]benzamide (10 g, 14.58 mmol) at 25° C. Then the reaction mixture was stirred for 3 h at 25° C. Volatiles were removed under reduced pressure and the resulting crude was purified by combiflash (10% MeOH in DCM) to get the title product (2) (5 g, 89%) as a white solid. MS: (ESI): m/z=383.8 [M+H].sup.+.

Example 1.2

N-(9-{1-[(benzoylsulfanyl)methyl]-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl}-9H-purin-6-yl)benzamide

[0131] ##STR00017##

[0132] To an ice cooled solution of PPh.sub.3 (10.26 g, 39.13 mmol CAS RN 603-35-0) in anhydrous THF (150.0 mL) was added DEAD (6.14 mL, 39.13 mmol, CAS RN 1972-28-7) and the reaction mixture was stirred at 0° C. for 30 min. PhCOSH (4.62 mL, 39.13 mmol, CAS RN 98-91-9) was added drop-wise to the reaction mixture and it was stirred at 0° C. for another 30 min. N-{9-[7-hydroxy-1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-9H-purin-6-yl}benzamide (5.0 g, 13.04 mmol) was added to the stirred reaction mixture at 0° C. for 2 h. The reaction mixture was then stirred at room temperature for 2 h. The reaction mixture was diluted with water (200 mL) and extracted with ethyl acetate (120 mL×3). The combined organic part was washed with NaHCO.sub.3 (100 mL), dried over Na.sub.2SO.sub.4, filtered and evaporated under reduced pressure to get the title product (25 g, crude) as a yellow viscous oil. MS: (ESI): m/z=504.3 [M+H].sup.+.

Example 1.3

N-{9-[7-hydroxy-1-(sulfanylmethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-9H-purin-6-yl}benzamide

[0133] ##STR00018##

Argon was bubbled through NaOH (0.5 M, 238 mL) as well as solution of THF-MeOH (6:4, 250 mL) for 30 min. N-(9-{1-[(benzoylsulfanyl)methyl]-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl}-9H-purin-6-yl)benzamide (20 g, crude) was dissolved in an argon purged solution of THF-MeOH (6:4, 250 mL) under argon and cooled at 0° C. to −5° C. To this solution was added NaOH solution (0.5 M, 238 mL, 119.16 mmol) and the reaction mixture was stirred at 0° to −5° C. for 30 min. Then a solution of citric acid (30.04 g, 142.98 mmol) was added at 0° C. A saturated NaHCO.sub.3 solution (300 mL) was added to the reaction mixture and the mixture was extracted with ethyl acetate (200 mL×3). The combined organic layer was washed with brine, dried over sodium sulfate, filtered and evaporated under reduced pressure to get the title product (20 g, crude) as an off white viscous oil. MS: (ESI): m/z=400.2 [M+H].sup.+.

Example 1.4

N-{9-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-9H-purin-6-yl}benzamide

[0134] ##STR00019##

To a solution of N-{9-[7-hydroxy-1-(sulfanylmethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-9H-purin-6-yl}benzamide (20 g, crude) in anhydrous pyridine (20 mL, argon purged) was added DMTrCl (5.09 g, 15.02 mmol CAS RN 40615-36-9) at 25° C. and the reaction mixture was stirred at 25° C. for 4 h. Volatiles were removed under reduced pressure and the reaction mixture was diluted with DCM (300 mL). The DCM layer was washed with a NaHCO.sub.3 solution (100 mL×2) followed by brine (100 mL). The organic layer was dried over Na.sub.2SO.sub.4, filtered and evaporated under reduced pressure. The resulting crude was purified by combiflash (2% MeOH in DCM containing 0.5% TEA) to get the title product (5 g, 68% over 3 steps) as a pale yellow solid. MS: (ESI): m/z=702.14 [M+H].sup.+.

Example 2

3-[({[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-3-(5-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl]oxy}[bis(propan-2-yl)amino]phosphanyl)oxy]propanenitrile

[0135] ##STR00020##

A solution of 5-ethylmercapto-1H-tetrazole (1.77 g, 13.59 mmol, 0.25 M solution in 55.3 mL dry ACN, CAS RN 89797-68-2) and 2-cyanoethyl tetraisopropylphosphorodiamodite (4.31 mL, 13.59 mmol, CAS RN 102691-36-1) were added sequentially to a stirred solution of 1-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-5-methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione (4.0 g, 6.80 mmol) in dry DCM (150 mL) under argon at 25° C. and the reaction mixture was stirred at 25° C. for 4 h. Then the reaction mixture was diluted with DCM (200 mL) and poured onto a sat. NaHCO.sub.3 solution (200 mL). The organic layer was separated off and the aq. layer was extracted with DCM (70 mL×2). The combined organic layer was dried over sodium sulfate, filtered and evaporated under reduced pressure. The resulting crude compound was purified by FCC under argon atmosphere (10-20% ACN in DCM) to get title compound (TM-6) (2.45 g, 46%) as an off white solid. MS: (ESI): m/z=789.4 [M+H].sup.+

Example 2.1

[7-hydroxy-3-(5-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)-2,5-dioxabicyclo[2.2.1]heptan-1-yl]methyl methanesulfonate

[0136] ##STR00021##

[0137] To a stirred solution of 1-[7-hydroxy-1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-5-methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione (10 g, 37.00 mmol) in anhydrous pyridine (100.0 mL, CAS RN 110-86-1) were added DMAP (0.452 g, 3.7 mmol) and methane sulfonyl chloride (3.15 mL, 40.71 mmol) at 0° C. to −5° C. sequentially. Then the reaction mixture was stirred at 0° C. for another 3 h. Volatiles were removed under reduced pressure and the resulting crude was purified by combiflash (5% MeOH in DCM) to get the title product (2) (8 g, 62%) as a pale white solid. MS: (ESI): m/z=348.97 [M+H].sup.+.

Example 2.2

1-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-5-methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione

[0138] ##STR00022##

To an argon purged solution of 1-{[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}ethan-1-one (10.43 g, 27.56 mmol) in dry DMF (80 mL) was added a freshly prepared solution of NaOMe (prepared by adding sodium (0.758 g, 34.45 mmol) to 24 mL of anhydrous methanol followed by the dilution of the resulting solution with DMF (8 mL)) in a drop-wise manner at 25° C. under argon. To the resulting reaction mixture was added a solution of [7-hydroxy-3-(5-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)-2,5-dioxabicyclo[2.2.1]heptan-1-yl]methyl methanesulfonate (4.0 g, 11.48 mmol) and 1,1,3,3 tetramethyl guanidine (3.03 mL, 24.12 mmol, CAS RN 80-70-6) in DMF (32 mL) rapidly. The reaction mixture was stirred at 25° C. for 3 h. Then the reaction mixture was diluted with DCM (400 mL) and washed with a sat. NaHCO.sub.3 solution (200 mL). The organic layer was dried over sodium sulfate, filtered and evaporated under reduced pressure. The crude compound was purified by combiflash (ethyl acetate containing 0.5% TEA) to get the title product (3.3 g, 49%) as a pale brown solid. MS: (ESI): m/z=588.89 [M+H].sup.+.

Example 2.3

1-{[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}ethan-1-one

[0139] ##STR00023##

To a solution of DMTrCl (10.0 g, 29.51 mmol, CAS RN 40615-36-9) in anhydrous DCM (100.0 mL) was added CH.sub.3COSH (6.24 ml, 88.54 mmol, CAS RN 507-09-5) drop-wise at 0° C. to −5° C. and the reaction mixture was stirred at 0° C. for another 30 min. After completion of reaction, the reaction mixture was neutralized with TEA (12.2 mL, 88.54 mmol) drop-wise at 0° C. Then the reaction mixture was washed with a saturated NaHCO.sub.3 solution (200 mL×2) and brine (100 mL), dried over sodium sulfate, filtered and concentrated under vacuum to get the title compound (11 g, 98%) as pale brown waxy solid.

Example 3

N′-{9-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-({[bis(propan-2-yl)amino](2-cyanoethoxy)phosphanyl}oxy)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}-N,N-dimethylmethanimidamide

[0140] ##STR00024##

A solution of 5-ethylmercapto-1H-tetrazole (2.65 g, 20.34, 0.25 M solution in 87.5 mL dry ACN, CAS RN 89797-68-2) and 2-cyanoethyl tetraisopropylphosphorodiamodite (6.45 mL, 20.34 mmol, CAS RN 102691-36-1) were added sequentially to a stirred solution of N′-{9-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}-N,N-dimethylmethanimidamide (6.8 g, 10.17 mmol) in dry DCM (200 mL) at 25° C. and the reaction mixture was stirred at 25° C. for 4 h. Then the reaction mixture was diluted with DCM (250 mL) and poured onto a sat. NaHCO.sub.3 solution (200 mL). The organic layer was separated off and the aq. layer was extracted with DCM (2×100 mL). The organic layer was dried over sodium sulfate, filtered and evaporated under reduced pressure. The resultant crude compound was purified by combiflash (90% ACN in DCM) to get title compound (4.2 g, 48%).MS: (ESI): m/z=869.0 [M+H].sup.+.

Example 3.1

N′-{9-[7-hydroxy-1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}-N,N-dimethylmethanimidamide

[0141] ##STR00025##

To a solution of 2-amino-9-[7-hydroxy-1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-6,9-dihydro-1H-purin-6-one (10 g, 33.87 mmol) in DMF (100 mL) was added DMF-DMA (9.0 mL, 67.74 mmol, CAS RN 4637-24-5) at 25° C. The mixture was stirred for 4 h at 25° C. Volatiles were removed under reduced pressure and the resultant crude compound was washed with n-hexane (50 mL×3) and dried to get the title compound (13 g, crude) as white solid. MS: (ESI): m/z=350.7 [M+H].sup.+.

Example 3.2

N′-(9-{1-[(benzoylsulfanyl)methyl]-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl}-6-oxo-6,9-dihydro-1H-purin-2-yl)-N,N-dimethylmethanimidamide

[0142] ##STR00026##

To an ice cooled solution of PPh.sub.3 (29.20 g, 111.32 mmol, CAS RN 603-35-0) in anhydrous THF (400 mL) was added DEAD (17.46 mL, 111.32 mmol, CAS RN 1972-28-7) and the reaction mixture was stirred at 0° C. for 30 min. PhCOSH (13.15 mL, 37.11 mmol, CAS RN 98-91-9) was added drop-wise to the reaction mixture and stirring continued at 0° C. for another 30 min. N′-{9-[7-hydroxy-1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}-N,N-dimethylmethanimidamide (13 g, crude) was added and stiffing continued at 0° C. for 2 h. Then the reaction mixture was stirred at room temperature for 2 h. Volatiles were removed under reduced pressure and the resultant crude compound was purified by combiflash (5% MeOH in DCM) get the title product (18 g, crude) as a pale yellow solid. MS: (ESI): m/z=470.83 [M+H].sup.+.

Example 3.3

N′-{9-[7-hydroxy-1-(sulfanylmethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}-N,N-dimethylmethanimidamide

[0143] ##STR00027##

To an ice cooled solution of N′-(9-{1-[(benzoylsulfanyl)methyl]-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl}-6-oxo-6,9-dihydro-1H-purin-2-yl)-N,N-dimethylmethanimidamide (18 g, crude) in anhydrous MeOH (300 mL) was added K.sub.2CO.sub.3 (42.30 g, 306.05 mmol, CAS RN 584-08-7) and the reaction mixture was stirred at 0° C. for 2 h and at 25° C. for 2 h. The solvent was evaporated under reduced pressure to get the title compound (60 g, crude) as a pale brown solid. MS: (ESI): m/z=366.6 [M+H].sup.+.

Example 3.4

N′-{9-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}-N,N-dimethylmethanimidamide

[0144] ##STR00028##

To a solution of N′-{9-[7-hydroxy-1-(sulfanylmethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}-N,N-dimethylmethanimidamide (60 g, crude) in anhydrous pyridine (80 mL, CAS RN 110-86-1) degassed with argon was added DMTrCl (19.42 g, 57.32 mmol, CAS RN 40615-36-9) at 25° C. and the reaction mixture was stirred at 25° C. for 4 h. Volatiles were removed under reduced pressure and the crude reaction mixture was diluted with water (200 mL). The aq. phase was extracted with ethyl acetate (3×250 mL). The combined organic part was dried over Na.sub.2SO.sub.4, filtered and evaporated under reduced pressure. The resultant crude was purified by combiflash (5% MeOH in DCM containing 0.5% TEA) to get the title product (6.8 g, 30% over 4 steps) as a pale yellow solid. MS: (ESI): m/z=668.6 [M+H].sup.+.

Example 4

N-{1-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-({[bis(propan-2-yl)amino](2-cyanoethoxy)phosphanyl}oxy)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl}benzamide

[0145] ##STR00029##

To an ice-cooled solution of N-{1-[1-({[bis(4-methoxyphenyl) (phenyl) methyl] sulfanyl}methyl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl}benzamide (4.8 g, 6.938) in DCM (50 mL) was added a solution of 5-ethylthio-1H-tetrazole (1.8 g, 13.87 mmol, CAS RN 89797-68-2) in acetonitrile (35 mL) followed by 2-cyanoethyl tetra isopropyl phosphorodiamidite (6.6 mL, 20.18 mmol, CAS RN 102691-36-1) under argon atmosphere at 25° C. and the reaction mixture was stirred for 4 h at 25° C. Then the reaction mixture was poured onto a saturated NaHCO.sub.3 solution (50 mL) and the organic layer was separated off. The aq. layer was extracted with DCM (25 mL×2). The combined organic layer was dried over sodium sulfate, filtered and evaporated under reduced pressure. The resulting crude was purified by column chromatography over silica gel (20% acetonitrile in DCM) to get the title product (3.0 g, 48% yield) as an off white solid. Using the above protocol, another 3 g batch was carried out and mixed with this batch to get the deliverable amount. MS: (ESI): m/z=892.1 [M+H].sup.+.

Example 4.1

N-{1-[7-hydroxy-1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl}benzamide

[0146] ##STR00030##

To a stirred solution of CCl.sub.3CO.sub.2H (4.5 g, 27.74 mmol, CAS RN 76-03-9) in DCM (100 mL) was added N-[1-(1-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl)-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl]benzamide (15 g, 22.19 mmol) at 25° C. and the reaction mixture was stirred for 2 hours at 25° C. After completion, volatiles were removed under reduced pressure to get the crude residue which was purified by combiflash (10% MeOH in DCM) to get the title product (5.8 g, 69%) as an off-white solid. MS: (ESI): m/z=373.9 [M+H].sup.+.

Examples 4.2

N-(1-{1-[(benzoylsulfanyl)methyl]-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl}-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide

[0147] ##STR00031##

To an ice-cooled solution of PPh.sub.3 (11.1 g, 42.58 mmol, CAS RN 603-35-0) in dry THF (150 mL) was added DEAD (6.7 mL, 42.58 mmol, CAS RN 1972-28-7) drop-wise and the reaction mixture was stirred at 0° C. under argon atmosphere for 30 min. To this was added PhCOSH (5.03 mL, 42.58 mmol, CAS RN 98-91-9) and stirring continued at 0° C. for 30 min. To the resultant reaction mixture was added N-{1-[7-hydroxy-1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl}benzamide (5.3 g, 14.19 mmol) and stirring continued at 0° C. for 2 h and 2 h at 25° C. Then the reaction mixture was diluted with water (200 mL) and extracted with ethyl acetate (100 mL×3). The combined organic layer was dried over Na.sub.2SO.sub.4 and evaporated under reduced pressure. The resulting crude was purified by combiflash chromatography (50% ethyl acetate in hexane) to get the title product (7.2 g, crude, contaminated with PPh.sub.3O) as an off white solid which was used as such in the next step without further purification MS: (ESI): m/z=493.6 [M+H].sup.+.

Example 4.3

N-{1-[7-hydroxy-1-(sulfanylmethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl}benzamide

[0148] ##STR00032##

[0149] To a degassed solution of N-(1-{1-[)benzoylsulfanyl)methyl]-7-hydroxy-2,5-dioxabicyclo [2.2.1]heptan-3-yl}-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (2.0 g, 4.052 mmol) in THF/MeOH (6:4) (40 mL) under argon was added a solution of NaOH (0.5 M, 24.3 mL) at −5° C. and the reaction mixture was stirred at 0 to −5° C. for 30 min. To the resultant reaction mixture was added a 10% aq. solution of citric acid (30 mL) at 0° C. A saturated sodium bicarbonate solution (50 mL) was added to the reaction mixture and the compound was extracted with ethyl acetate (70 mL×3). The combined organic layer was washed with brine (100 mL×2), dried over sodium sulfate and concentrated under reduced pressure to get the title compound (1.7 g, crude) as a white solid which was used as such in the next step without further purification. MS: (ESI): m/z=390.2 [M+H].sup.+.

Example 4.4

N-{1-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl}benzamide

[0150] ##STR00033##

To an argon purged solution of crude N-{1-[7-hydroxy-1-(sulfanylmethyl)-2,5-dioxabicyclo [2.2.1]heptan-3-yl]-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl}benzamide (5.5 g, 14.123 mmol) in dry pyridine (20 mL, CAS RN 110-86-1) was added DMTrCl (7.1 g, 21.18 mmol, CAS RN 40615-36-9) at 25° C. the reaction mixture was stirred at 25° C. for 16 h. Volatiles were removed under reduced pressure and the crude reaction mixture thus obtained was diluted with DCM (200 mL). The organic part was washed with a sat. NaHCO.sub.3 solution (50 mL×2) followed by brine (50 mL×2), dried over sodium sulfate and evaporated under reduced pressure. The resultant crude was purified by combiflash chromatography (35% ethyl acetate in hexane containing 0.5% TEA) to get impure an compound which was repurified by combiflash (30% ethyl acetate/hexane) to get the title compound (4.8 g, 49% yield). MS: (ESI): m/z=690.7 [M+H].sup.+.