Patent ID: 12221459

DETAILED DESCRIPTION OF THE EMBODIMENTS

Various embodiments of the compounds invention and intermediates therefore will now be illustrated by the following examples. The Examples are just intended to further illustrate certain embodiments of the invention and are by no means intended to limit the scope of the invention.

Chemistry Examples & Intermediates

As is well known to a person skilled in the art, reactions are performed in an inert atmosphere, including but not limited to nitrogen and argon atmosphere when necessary to protect reaction components from air or moisture. Temperatures are given in degrees Celsius (° C.). Solution percentages and ratios express a volume to volume relationship, unless stated otherwise. The reactants used in the examples below may be obtained from commercial sources or prepared from commercially available starting materials as described herein or by methods known in the art.

The compounds of the invention including intermediates are prepared as described in the Examples and in the general schemes herein. It will be apparent to a skilled person that analogous synthetic routes may be used, with appropriate modifications, to prepare the compounds of the invention as described herein. The progress of the reactions described herein were followed as appropriate by e.g. LC, GC or TLC, and as the skilled person will readily realise, reaction times and temperatures may be adjusted accordingly.

Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker spectrometer operating at 500 MHz for1H NMR and at 126 MHz for13C NMR using CDCl3(deuterated chloroform) or DMSO-d6(deuterated DMSO, dimethyl-d6sulfoxide) as solvent. Chemical shifts (δ) are reported in parts per million (ppm) relative to tetramethylsilane (TMS) which was used as internal reference, or to residual solvent peak. Coupling constants, J, are reported in Hertz. NMR shifts indicated were obtained using an automated process wherein residual solvent and/or impurities may be present, integrals and chemical shifts may not be completely accurate, signals may be broad with a low signal to noise ratio and may overlap with signals from residual solvents, and multiplicities may have been misinterpreted. Despite this, all spectra obtained by the automated process are supporting the structure of each of the analysed compounds.

The compound names were generated by ChemDraw Ultra software, Cambridgesoft, version 12.0.2.

Preparatory HPLC were conducted using any one of the Methods A-E

Prep HPLC Method A

Column: Kromosil C18 (25×150)mm 10μMobile phase: 10 mM ABC in H2O:MeCN (gradient)
Prep HPLC Method B:Column: Kromosil C18 (25×150) mm 10μMobile phase: 0.1% formic acid in H2O:MeCN (gradient)
Prep HPLC Method CColumn: X-Select C18 (19×150) mm 5μMobile phase: 10 mM ABC IN H2O:MeCN (gradient)
Prep HPLC Method DColumn: X-Select C18 (19×150) mm 5μMobile phase: 0.1% formic acid in H2O:MeCN (gradient)
Prep HPLC Method EColumn: X-Bridge C18 (30×250) mm 5μMobile phase: 0.1% formic acid in H2O:MeCN (gradient)
Prep HPLC Method FColumn: X-Bridge Amide C18 (19×250) mm 5μMobile phase: 10 mM ABC IN H2O:MeCN (gradient)
Prep HPLC Method GColumn: YMC TRIAT C18 (25×150) mm 10μMobile phase: 10 mM ABC IN H2O:MeCN (gradient)
Prep HPLC Method HColumn: YMC TRIAT C18 (25×150) mm 10μMobile phase: 0.1% formic acid in H2O:MeCN (gradient)

(S)-Methyl 4-methyl-2-(((S)-(perfluorophenoxy)(phenoxy)phosphoryl)amino)pentanoate (I-1)

Triethylamine (11 mL, 79 mmol) was added dropwise at −70° C. for 30 minutes under nitrogen to a stirred solution of (S)-methyl 2-amino-4-methylpentanoate hydrochloride (6.5 g, 35 mmol) in DCM (70 mL). Phenyl phosphodichloridate (5.4 ml, 36 mmol) in DCM (70 mL) was added dropwise at −70° C. over a period of 45 min, then reaction mixture was stirred at −70° C. to 0° C. for 3 h. To this mixture was added a solution of pentafluorophenol (6.3 g, 34 mmol) and triethylamine (5.5 mL, 40 mmol) in DCM (60 mL) during 45 minutes. The reaction mixture was stirred at 0° C. for 2 h, then concentrated and the residue was taken in tert-butyl methyl ether (100 mL) and insolubles were filtered off through Celite and the filtrated was concentrated under reduced pressure. The crude compound was purified by column chromatography on silica gel eluted with 15-20% EtOAc in p.ether. Pure fractions were pooled and concentrated under reduced pressure. The afforded racemic compound was dissolved in 20% EtOAc in p. ether (70 mL) and kept at refrigerator for 16 h and the thus formed crystals were filtered off which gave the title compound (3 g, 18%) as a solid. LC-MS showed 99.7% desired compound mass. MS (ES+) m/z 468.28 [M+H]+.

(S)-Isopropyl 4-methyl-2-(((S)-(perfluorophenoxy)(phenoxy)phosphoryl)amino)pentanoate (I-2)

To a stirred solution of (S)-isopropyl 2-amino-4-methylpentanoate (3.8 g, 18 mmol) in DCM (50 mL) at −78° C. was added Et3N (5.6 mL, 40 mmol) dropwise for 10 min. Phenyl phosphorodichloridate (2.7 mL, 18 mmol) in DCM (30 mL) was added dropwise for 30 min. The reaction mixture was stirred at −78° C. for additional 30 min, then allowed to warm to 0° C. during 2 h and stirred for additionally 1 h. A solution of 2,3,4,5,6-pentafluorophenol (3.0 g, 16 mmol) and Et3N (2.0 mL, 20 mmol) in DCM (20 mL) was added dropwise to the reaction mixture. The reaction mixture was stirred at rt for 2 h, then concentrated under reduced pressure. The afforded residue was dissolved in of MTBE (100 mL), insolubles were filtered off and the filtrate concentrated. The crude compound was purified by column chromatography on silica gel eluted with 10% EtOAc in p.ether. Pure fractions were pooled and concentrated under reduced pressure. The afforded racemic mixture (4.5 g, 44% undesired isomer and 55% desired isomer), was dissolved in 20% EtOAc in hexane (100 mL) and kept standing for 2 h at 0° C. The thus formed crystals were filtered off, washed with cold hexane (2×30 mL) and dried under reduced pressure which gave the title compound (2.0 g, 22%, chiral purity 98.9%). MS (ES+) m/z 496.15 [M+H]+. LCMS shown 99% of desired product.

Step a) (S)—(S)-Pentan-2-yl 2-((tert-butoxycarbonyl)amino)-3-methylbutanoate (I-3a)

To a stirred solution of Boc-Val-OH (7.0 g, 32 mmol) and (S)-pentan-2-ol (3.4 ml, 31 mmol) in DCM (100 mL) was added DMAP (400 mg, 3.2 mmol), EDCI (6.8 g, 35 mmol) at rt. The solution was stirred for 16 h, then diluted with DCM (100 mL), washed with water (2×50 mL), saturated sodium bicarbonate solution (2×50 mL), 2M HCl (2×50 mL) and brine (100 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The afforded crude compound was purified by column chromatography on silica gel, eluted with 5-10% ethyl acetate in p. ether which gave the title compound (7.5 g, 80%). MS (ES+) m/z 288.29 [M+H]+.

Step b) (S)—(S)-Pentan-2-yl 3-methyl-2-(((S)-(perfluorophenoxy)(phenoxy)phosphoryl)amino)butanoate (I-3b)

To a stirred solution of compound I-3a (7.5 g, 26 mmol) in dry 1,4-dioxane (30 mL) was added 4.0 M HCl in dioxane (34 mL, 135 mmol) at rt. The solution was stirred for 2 h, then concentrated under reduced pressure and co-distilled with toluene (2×50 mL). The residue was dissolved in DCM (50 mL), put under nitrogen and cooled to −70° C. Et3N (8.2 mL, 58 mmol) was added dropwise for 10 min followed by dropwise addition of a solution of phenyl phosphorodichloridate (3.8 mL, 26 mmol) in DCM (20 mL). The reaction mixture was stirred at −70° C. to 0° C. for 3 h, then a solution of pentafluoro phenol (4.8 g, 26 mmol) and Et3N (4.0 mL, 29 mmol) in DCM (30 mL) was added dropwise. The reaction mixture was stirred at 0° C. for 2 h, then concentrated. The residue was taken in tert. butyl methyl ether (100 mL) and insolubles were filtered off. The filter was washed with tert. butyl methyl ether (2×30 mL) and the combined filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel eluted with 10% EtOAc in hexane. Pure fractions were pooled and concentrated under reduced pressure. The afforded racemic mixture was dissolved in 15% EtOAc in p. ether (150 mL) and kept in refrigerator for overnight. The thus formed crystals were filtered off, washed with p. ether (100 mL) and dried under reduced pressure which gave the title compound (3.5 g, 26%, chiral purity 98.1%). MS (ES+) m/z 510.16 [M+H]+.

(S)-2-Propylpentyl 2-(((S)-(perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate (I-4)

Et3N (12.3 mL, 88 mmol) was added dropwise at −78° C. to a stirred solution of (S)-2-propylpentyl 2-aminopropanoate (10 g, 42 mmol) in DCM (100 mL) followed by dropwise addition of a solution of phenyl phosphorodichloridate (1.4 mL, 42 mmol) in DCM (70 mL). The reaction mixture was stirred at −78° C. for 30 min, then allowed to warm to 0° C. during 2 h and was then stirred for additionally 1 h at 0° C. A solution of 2,3,4,5,6-pentafluorophenol (7 g, 38 mmol) and Et3N (6.5 mL, 46 mmol) in DCM (80 mL) was added dropwise at 0° C. and the reaction mixture was then stirred for at rt 3 h, then concentrated to dryness under reduced pressure, The crude product was dissolved in MTBE (200 mL), insolubles were filtered off and the filtrate was concentrated to dryness under reduced pressure. The crude compound was purified by column chromatography on silica gel eluted with 10% EtOAc in hexane. Pure fractions were pooled and concentrated under reduced pressure. The afforded racemic compound was dissolved in 20% EtOAc in n-pentane (100 mL) and kept at rt for 2 h, the thus formed crystals were filtered off and washed with cold n-pentane (2×30 mL), and dried under vacuum which gave the title compound (3.1 g, 14%, chiral purity 99%). MS (ES+) m/z 524.17 [M+H]+.

Step a) (S)—(S)-Pentan-2-yl 2-((tert-butoxycarbonyl)amino)-4-methylpentanoate (I-7a)

To a stirred solution of (tert-butoxycarbonyl)-L-leucine (5.0 g, 22 mmol) and (S)-pentan-2-ol (2.1 mL, 19 mmol) in DCM (50 mL) were added DMAP (400 mg, 3.24 mmol) and EDC-HCl (5.0 g, 26 mmol). The solution was stirred at rt for 18 h, then diluted with DCM (200 mL) and washed with water (100 mL) and brine (100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The afforded crude compound was purified by column chromatography on silica gel eluted with a gradient of 5-10% EtOAc in p. ether, which gave the title compound (4.0 g, 61%).

Step b) (S)—(S)-Pentan-2-yl 4-methyl-2-(((S)-(perfluorophenoxy)(phenoxy) phosphoryl)amino)pentanoate (I-7b)

4 M HCl in dioxane (14 mL, 56 mmol) was added to a stirred solution of compound I-7a (4.0 g, 13 mmol) in 1,4-dioxane (40 mL). The reaction mixture was stirred at rt for 2 h, then concentrated. The residue was dissolved in DCM (50 mL), the solution was cooled to −78° C. and Et3N (3.7 mL, 26.50 mmol) was added dropwise followed by dropwise addition of a solution of phenyl phosphorodichloridate (1.9 mL, 13 mmol) in DCM (25 mL). The reaction mixture was stirred at −78° C. for 30 min, then allowed to warm to 0° C. during 2 h and stirred for additionally 1 h at 0° C. A solution of 2,3,4,5,6-pentafluorophenol (2.1 g, 11 mmol) and Et3N (1.9 mL, 14 mmol) in DCM (25 mL) was added dropwise at 0° C. to the reaction mixture. The mixture was stirred at rt for 5 h, then diluted with MTBE (100 mL) and stirred for 10 min. Precipitated insoluble were filtered off and the filtrate was concentrated under reduced pressure. The afforded crude compound was purified by column chromatography on silica gel eluted with 20% EtOAc in hexane. The afforded racemic mixture was dissolved in 10% EtOAc in p. ether (50 mL) and kept in refrigerator for 4 h. The thus formed crystals were filtered off, washed with p. ether (50 mL) and dried under vacuum which gave the title compound (1.5 g, 22%). MS (ES+) m/z 524.21 [M+H]+. Chiral HPLC showed 99.75% of the desired isomer.

Step a) (S)-2-Ethylbutyl 2-((tert-butoxycarbonyl)amino)-3-methylbutanoate (I-8a)

To a stirred solution of N-Boc-L-valine (4.68 g, 21.5 mmol) in DCM (20 mL) were added EDC-HCl (4.13 g, 21.5 mmol) and 4-dimethyl amino pyridine (0.24 g, 2.0 mmol) at 0° C. The mixture was stirred for 30 min at 0° C., then 2-ethylbutan-1-ol (2.41 mL, 19.6 mmol) was added dropwise at 0° C. The mixture was stirred for 16 h at rt, then diluted with DCM (150 mL) and washed with water (200 mL) and brine (200 mL), dried over sodium sulfate and concentrated. The crude compound was purified by column chromatography on silica gel and eluted with 10% EtOAc in hexane, which gave the title compound. (ES+) m/z 302.26 [M+H]+.

Step b) (S)-2-Ethylbutyl 2-((bis(4-nitrophenoxy)phosphoryl)amino)-3-methylbutanoate (I-8b)

pTSA monohydrate (1.8 g, 9.5 mmol) was added at rt to a stirred solution of I-8a (2.6 g, 8.6 mmol) in EtOAc (25 mL). The mixture was stirred at 70° C. for 4 h, then concentrated under reduced pressure, co-distilled with toluene (×2) and washed with n-pentane. The afforded residue and bis(4-nitrophenyl) phosphorochloridate (3.0 g, 8.3 mmol) were dissolved in DCM (60 mL) at 0° C., Et3N (2.3 mL, 17 mmol) was added dropwise. The solution was stirred at rt for 16 h, then extracted with DCM (2×20 mL) and water (20 mL). The organic layer was washed with NaHCO3aq. (20 mL) and brine (20 mL), dried over Na2SO4, filtered and concentrated which gave the title compound (2 g).

Step a) (S)-(S)-Pentan-2-yl 2-((tert-butoxycarbonyl)amino)propanoate (I-9a)

To a stirred solution of (tert-butoxycarbonyl)-L-alanine (4.5 g, 24 mmol) and (S)-pentan-2-ol (3.1 mL, 29 mmol) in DCM (50 mL) were added EDC-HCl (5.1 g, 26 mmol) and DMAP (0.3 g, 2 mmol) at 0° C. The reaction was stirred for 16 h at rt, then diluted with DCM (50 mL) and washed with water (2×50 mL) and brine (2×50 mL), dried over sodium sulfate, filtered and concentrated. The crude compound was purified by column chromatography on silica gel eluted with 10% EtOAc in hexane, which gave the title compound (3.5 g).

Step b) (S)-(S)-Pentan-2-yl 2-(((S)-(perfluorophenoxy)(phenoxy) phosphoryl)amino)propanoate (I-9b)

To a stirred solution of compound I-9a (3.5 g, 13.5 mmol) in 1,4-dioxane (15 mL) was added 4 M HCl in dioxane (14 mL, 57 mmol) at rt. The reaction mixture was stirred at rt for 2 h, then concentrated. The residue was dissolved in DCM (30 mL) and cooled to −78° C. Et3N (3.9 mL, 28 mmol) was added dropwise followed by dropwise addition of a solution of phenyl phosphorodichloridate (2.0 mL, 13 mmol) in DCM (20 mL). The reaction mixture was stirred at −78° C. for additional 30 min, then allowed to warm to 0° C. during 2 h and stirred for additionally 1 h at 0° C. A solution of 2,3,4,5,6-pentafluorophenol (2.2 g, 12 mmol) and Et3N (2.1 mL, mmol) in DCM (20 mL) was added dropwise at 0° C. to the reaction mixture. The reaction mixture was stirred at rt for 3 h. After 3 h, then concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel and eluted with 10% EtOAc in hexane. The afforded solid was dissolved in 10% EtOAc in p. ether (100 mL) and kept standing at rt for 2 h. The thus formed crystals were filtered off, washed with p. ether (2×20 mL) and dried under vacuum which gave the title compound (1.3 g, 20% yield, 99.86% chiral purity). LCMS (ES+) m/z 482.11 [M+H]+.

(Z)-(1-(Hydroxymethyl)-2-((2-isobutyramido-6-oxo-1H-purin-9(6H)-yl)methylene)cyclopropyl)methyl acetate (I-10)

The title compound was prepared as described By Li, Chengwei; Gentry, Brian G.; Drach, John C.; Zemlicka, Jiri in Nucleosides, Nucleotides & Nucleic Acids (2009), 28(9), 795-808.

(Z)-(2-((2-Amino-6-oxo-1H-purin-9(6H)-yl)methylene)-1-(hydroxymethyl)cyclopropyl)methyl isobutyrate (I-11)

To a stirred solution of (Z)-2-amino-9-((2,2-bis(hydroxymethyl)cyclopropylidene)methyl)-1H-purin-6(9H)-one (500 mg, 1.9 mmol) in DMF (30 mL) were added trimethyl ortho butyrate (1.2 mL, 7.6 mmol) and p-TSA (360 mg, 1.9 mmol) at rt. The reaction mixture was stirred at rt for 3 h, then concentrated. Acetic acid (80% in water, 50 mL) was added to the residue and the mixture was stirred at rt for 3 h, then concentrated. The crude compound was purified by column chromatography on silica gel eluted with 10-15% MeOH in DCM, which gave the title compound (350 mg, 5%) as a solid. LCMS (ES+) m/z 334.30 [M+H]+.

Step a) (Z)-(2-((2-Isobutyramido-6-oxo-1H-purin-9(6H)-yl)methylene)cyclopropane-1,1-diyl)bis(methylene) diacetate (I-12a)

To a stirred solution of compound I-10 (100 mg, 0.30 mmol) in DMF (10 mL) were added DMAP (7 mg, 0.06 mmol) and acetic anhydride (0.2 mL, 2.1 mmol) at rt. The mixture was stirred for 1 h at rt then concentrated under reduced pressure. The afforded crude compound was purified by column chromatography on silica gel eluted with 5% MeOH, which gave the title compound (110 mg, 82%) as a solid. LCMS (ES+) m/z 418.41[M+H]+.

Step b) (R,Z)-(1-(Hydroxymethyl)-2-((2-isobutyramido-6-oxo-1H-purin-9(6H)-yl)methylene)cyclopropyl)methyl acetate (I-12b)

To a stirred solution of compound 2a (110 mg, 0.26 mmol) in DMF (15 mL) were added 0.02 M phosphate buffer (pH 7, 110 mL) and porcine liver esterase (230 mg) at rt. The reaction mixture was stirred at rt for 1 h, then concentrated under reduced pressure. The afforded crude compound was purified by column chromatography on silica gel eluted with 10% MeOH in DCM. Appropriate fractions were pooled and concentrated under reduced pressure. Chiral HPLC showed 91.7% of the desired isomer. The residue was purified by chiral SFC, which gave the title compound (40 mg, 39%) as a solid. LCMS (ES+) m/z 376.32 [M+H]+. The afforded compound was analyzed by LCMS,1H NMR, chiral HPLC & specific optical rotation (SOR). SOR of the afforded compound was found to be +26.52° (c 0.5, DMSO) which is in accordance with reported literature values for the R-isomer (+21.7, c 1.0, DMSO,Nucleosides, Nucleotides and Nucleic Acids,28:795-808, 2009).

Preparative SFC Conditions:

Column/dimensions: Chiralpak IC (21×250 mm), 5μCO2:70.0%Co solvent: 30.0% (100% EtOH)Total Flow: 60.0 g/minBack Pressure: 90.0 barUV: 234 nmStack time: 6.0 minLoad/inj.: 3.5 mg

(Z)-(1-(Hydroxymethyl)-2-((2-isobutyramido-6-oxo-1H-purin-9(6H)-yl)methylene)cyclopropyl)methyl acetate (I-13)

The compound was prepared as a racemate as described By Li, Chengwei; Gentry, Brian G.; Drach, John C.; Zemlicka, Jiri in Nucleosides, Nucleotides & Nucleic Acids (2009), 28(9), 795-808. The two stereoisomers were separated by prep. NP HPLC.

Fraction-1 (200 mg, 14%) specific optical rotation (SOR)+21.10°,

Fraction-2, (230 mg, 16%), SOR −19.79°.

The chirality of the isomers was determined by comparison of SOR with the enzymatically prepared compound of Example 12 (+26.52°).

Preparative NP HPLC Conditions

Column: Chiralpak IC (150×4.6) mm: 3μMobile phase: Acetonitrile 100% (isocratic)Flowrate: 1.0 ml/minTemperature: Ambient

Step b) 2-Amino-9-((Z)-((2R)-2-(hydroxymethyl)-2-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)cyclopropylidene)methyl)-1H-purin-6(9H)-one (I-13b)

Methanesulfonic acid (0.15 mL, 2.4 mmol) in DMF (10 mL) was added dropwise at rt to a stirred solution of compound I-13S (900 mg, 2.4 mmol) and 3,4-dihydro-2H-pyran (4.3 mL, 48 mmol) in DMF (40 mL). The reaction mixture was stirred at rt for 5 h, then triethyl amine (2.5 mL) was added and the mixture was concentrated. The afforded crude was dissolved in MeOH (80 mL) and 25% aq ammonia (65 mL, 420 mmol) was added at rt. The reaction mixture was stirred at 50° C. for 12 h, then concentrated. The crude compound was purified by column chromatography on silica gel eluted with 15% MeOH in DCM, which gave the title compound (700 mg, 69%) as a solid. LCMS (ES+) m/z 348.38 [M+H]+.

Step a) (R,Z)-(2-((2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methylene)-1-(hydroxymethyl)cyclopropyl)methyl isobutyrate (I-14)

The racemate compound I-11 (450 mg) was purified by chiral SFC to obtain peak-1(150 mg, 23%) as a solid. The afforded compound was analysed by optical rotation (SOR). SOR of the afforded compound was found to be +10.7° (0.44%, DMSO) and was assumed as R-isomer.

(2S)-2-Ethylbutyl 3-methyl-2-(((perfluorophenoxy)(phenoxy)phosphoryl)amino)butanoate (I-15)

The title compound was prepared by reaction of I-8a according to the method described for I-4. Yield: 26%. LCMS (ES+) 524.29 [M+H]+. Chiral HPLC showed 99.11% of the desired isomer.

(2S)-Isopropyl 2-(((4-(1-methylcyclopropyl)phenoxy)(perfluorophenoxy)phosphoryl)amino)propanoate (I-16)

The title compound was prepared as described in WO2015/034420.

(2S)-methyl 2-(((Perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate (I-17)

The title compound was prepared by reaction of (S)-methyl 2-((tert-butoxycarbonyl)amino)propanoate according to the method described as described in I-3 step b. Yield: 25%. LCMS (ES+) m/z 426.13 [M+H]+. The chiral purity of the title compound was 99.5% according to chiral HPLC.

(S)-(S)-Pentan-2-yl 2-(((S)-(4-bromophenoxy)(perfluorophenoxy)phosphoryl)amino)-4-methylpentanoate (I-18)

The title compound was prepared by reaction of I-7a according to the method described in 1-7 step b. LCMS (ES+) m/z 602.10 & 604.10 [M+H]+. The chiral purity of the title compound was 99% according to chiral HPLC.

Step a) (Z)-(2-((2-Isobutyramido-6-oxo-1H-purin-9(6H)-yl)methylene)-1-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)cyclopropyl)methyl acetate (I-19a)

3,4-Dihydro-2H-pyran (5 mL, 55.41 mmol) and CH3SO3H (0.2 mL, 3.5 mmol) were added at rt. to a stirred solution of compound I-11 (1.3 g, 3.5 mmol) in DMF (20 mL). The reaction mixture was stirred at rt for 5 h, then Et3N (5 mL) was added and the mixture was concentrated under reduced pressure. The crude compound (1.4 g) was used in next step without further purification. MS (ES+) m/z 460.29 [M+H]+.

Step b) (Z)-2-Amino-9-((2-(hydroxymethyl)-2-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)cyclopropylidene)methyl)-1H-purin-6(9H)-one (I-19b)

To a stirred solution of compound I-19a (1.4 g) in MeOH (100 mL) was added 25% aq ammonia (150 mL, 975 mmol) at rt. The reaction mixture was stirred at 50° C. for 12 h, then allowed to attain rt and concentrated. The afforded crude compound was purified by column chromatography on silica gel eluted with 15% MeOH in DCM, which gave the title compound (850 mg, 71% over two steps) as a solid. MS (ES+) m/z 348.26 [M+H]+.

Step c) (2S)—(S)-Pentan-2-yl 2-(((S)-(((Z)-2-((2-amino-6-oxo-1H-purin-9(6H)-yl)methylene)-1-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)cyclopropyl)methoxy)(4-bromophenoxy)phosphoryl)amino)propanoate (I-19c)

tert-butylmagnesium chloride (1M in THF, 4.3 mL, 4.3 mmol) was added dropwise to a solution of compound I-19b (300 mg, 0.86 mmol) in DMF (30 mL). The reaction mixture was stirred at rt for 30 min, then compound I-5 (580 mg, 1.04 mmol) in dry THE (15 mL) was added dropwise. The reaction mixture was stirred at rt for 2 h, then concentrated and the afforded crude compound was purified by column chromatography on silica gel eluted with 10% MeOH in DCM, which gave the title compound (350 mg, 40%) as a solid. MS (ES+) m/z 723.21 & 725.22 [M+H]+.

Step d) (S)-(S)-Pentan-2-yl 2-(((S)-(((S,Z)-2-((2-amino-6-oxo-1H-purin-9(6H)-yl)methylene)-1-(hydroxymethyl)cyclopropyl)methoxy)(4-bromophenoxy)phosphoryl)amino)propanoate (I-19d-1) & (S)-(S)-Pentan-2-yl 2-(((S)-(((R,Z)-2-((2-amino-6-oxo-1H-purin-9(6H)-yl)methylene)-1-(hydroxymethyl)cyclopropyl)methoxy)(4-bromophenoxy)phosphoryl)amino)propanoate (I-19d-2)

A solution of compound I-19c (400 mg, 0.553 mmol) in 80% acetic acid (40 mL, 558 mmol) was stirred at rt for 24 h, then concentrated under reduced pressure. The crude compound was purified by column chromatography on silica gel eluted with 12% MeOH in DCM, which gave a racemic mixture of the title compounds. The mixture was subjected to separation by prep. SFC, which gave isomer-1 (80 mg) as a solid and isomer-2 (100 mg).

Isomer 2 was further purified by prep HPLC using Method B, which gave the pure isomer (52 mg) as a solid.

Preparative SFC Conditions

Column/dimensions: Chiralpak AD-H (30×250 mm), 5μCO2:50.0%Co solvent: 50.0% (100% IPA)Total Flow: 90.0 g/minBack Pressure: 90.0 barUV: 214 nmStack time: 8.5 minLoad/Inj.: 19.0 mg

(S)-Isopropyl 2-(((S)-(((S,Z)-2-((2-amino-6-oxo-1H-purin-9(6H)-yl)methylene)-1-(hydroxymethyl)cyclopropyl)methoxy)(phenoxy)phosphoryl)amino)-4-methylpentanoate (I-20)

To a solution of I-13b (200 mg, 0.576 mmol) in DMF (12 mL) was added tert-butylmagnesium chloride (2.9 mL, 1M in THF, 2.9 mmol) dropwise. The mixture was stirred for 30 min at rt, then a solution of I-2 (343 mg, 0.691 mmol) in dry THF (8 mL) was added dropwise. The reaction mixture was stirred at rt for 24 h, then concentrated. The crude compound was purified by column chromatography on silica gel eluted with 4% MeOH. Appropriate fractions were pooled and concentrated and the residue dissolved in 80% acetic acid (37 mL, 509 mmol). The reaction mixture was stirred at rt for 24 h, then concentrated. The crude compound was purified by column chromatography on silica gel eluted with 8% MeOH in DCM. Pure fractions were pooled and concentrated and the residue was purified by prep HPLC using Method C, which gave the title compound (70 mg, 45%) as a solid. LCMS (ES+) m/z 575.31 [M+H]+.

(2S)—(S)-Pentan-2-yl 2-(((S)-(((1S,Z)-2-((2-amino-6-oxo-1H-purin-9(6H)-yl)methylene)-1-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)cyclopropyl)methoxy)(4-bromophenoxy)phosphoryl)amino)propanoate (I-21)

To a solution of I-13b (100 mg, 0.288 mmol) in DMF (10 mL) was added tert-butylmagnesium chloride (1.4 mL, 1M in THF, 1.4 mmol) dropwise for 5 min and stirred at rt for 1 h. Then added I-5 (190 mg, 0.345 mmol) in dry THF (5 mL) dropwise for 5 min, the reaction mixture was stirred at rt for 16 h, then concentrated. The crude compound was purified by column chromatography on silica gel eluted with 7% MeOH in DCM. Appropriate fractions were pooled and concentrated and the residue purified by prep HPLC using Method C, which gave the title compound (60 mg, 28%) as a white solid. LCMS (ES+) m/z 725.24 [M+H]+.

(S)-Methyl 2-(((S)-(((S,Z)-2-((2-amino-6-oxo-1H-purin-9(6H)-yl)methylene)-1-(hydroxymethyl)cyclopropyl)methoxy)(phenoxy)phosphoryl)amino)-4-methylpentanoate (I-22)

The title compound was prepared from I-13b and I-1 using the method described for Intermediate 20. Yield 21%. LCMS (ES+) m/z 631.44 [M+H]+.

(S)-Isopropyl 2-(((S)-(((S,Z)-2-((2-amino-6-oxo-1H-purin-9(6H)-yl)methylene)-1-(hydroxymethyl)cyclopropyl)methoxy)(4-(1-methylcyclopropyl)phenoxy)phosphoryl)amino)propanoate (I-25)

The title compound was prepared from I-13b and I-4 using the method described for Intermediate 20. The final compound was purified by prep HPLC using method D. 26% yield. LCMS (ES+) m/z 587.26 [M+H]+.

(S)-Methyl 2-(((S)-(((S,Z)-2-((2-amino-6-oxo-1H-purin-9(6H)-yl)methylene)-1-(hydroxymethyl)cyclopropyl)methoxy)(phenoxy)phosphoryl)amino)propanoate (I-26)

The title compound was prepared from I-13b and I-17 using the method described for Intermediate 20. The final compound was purified by prep SFC. 5.4% yield, LCMS (ES+) m/z 505.31 [M+H]+.

Preparative SFC Conditions

Column/dimensions: Chiralpak AD-H (30×250 mm), 5μCO2:70.0%Co solvent: 30.0% (100% MeOH)Total Flow: 70.0 g/minBack Pressure: 90.0 barUV: 214 nmStack time: 7.5 minLoad/inj.: 2.3 mg

(Z)-(2-((2-Amino-6-oxo-1H-purin-9(6H)-yl)methylene)-1-(hydroxymethyl)cyclopropyl)methyl butyrate (I-27)

To a stirred solution of (Z)-2-amino-9-((2,2-bis(hydroxymethyl)cyclopropylidene)methyl)-1H-purin-6(9H)-one (500 mg, 1.9 mmol) in DMF (30 mL) were added trimethyl orthobutyrate (0.5 mL, 2.8 mmol) and pTSA (37 mg, 0.19 mmol) at rt. The reaction mixture was stirred at rt for 3 h, then triethyl amine (0.5 mL) was added and the mixture was concentrated. The residue was taken in 80% acetic acid in water (75 mL), stirred at rt for 2 h, then concentrated. The crude compound was purified by column chromatography on silica gel eluted with 8% MeOH in DCM, which gave the title compound (350 mg, 55%) as a solid. LCMS (ES+) m/z 34.23 [M+H]+.

Step a) dibenzyl 1-((benzyloxy)methyl)-2-bromocyclopropane-1,2-dicarboxylate (I-28a)

n-BuLi (1.6M in hexane) (520 mL, 829 mmol) was added dropwise at −78° C. under argon to a solution of benzyl alcohol (86 mL, 829 mmol) in dry THE (1000 mL) at −78° C. over a period of 1 h. The solution was stirred for 3 h at −78° C., then a solution of compound benzyl 2-bromoacrylate (400 g, 1659 mmol) in THE (500 mL) was added at −78° C. over a period of 1 h and stirred at that temperature for 4 h and 16 h at rt. To the reaction mixture, saturated ammonium chloride solution (700 mL) was added at 0° C. and the mixture was extracted with EtOAc (2×1500 mL). The combined organic layers were washed with water (1000 mL), brine (1000 mL), dried (Na2SO4), filtered and concentrated. The crude compound was purified by column chromatography on silica gel and eluted with 15% EtOAc in pet ether, which gave the title compound (270 g). MS (ES+) 511.09 [M+H]+. The crude compound was used in next step without further purification.

Step b) (1-((benzyloxy)methyl)-2-bromocyclopropane-1,2-diyl)dimethanol (I-28b)

DIBAL-H (1M) (2120 mL, 2120 mmol) was added dropwise under argon to a solution of compound I-28a (270 g, 530.05 mmol) in dry THE (2300 mL) at 0° C. over a period of 2 h. The solution was stirred for 16 h at rt, then saturated ammonium chloride solution (700 mL) was added at 0° C. and precipitated solid was filtered and washed with EtOAc (2×1500 mL). The combined organic layers were washed with water (700 mL), brine (700 mL), dried (Na2SO4), filtered and concentrated. The crude compound was purified by column chromatography on silica gel and eluted with 50% EtOAc in pet ether, which gave the title compound (95 g, 54%). MS (ES+) 301.09 [M+H]+.

Step c) (1-((benzyloxy)methyl)-2-bromocyclopropane-1,2-diyl)bis(methylene) diacetate (I-28c)

Pyridine (128 mL, 1577 mmol) and acetic anhydride (68.5 mL, 725.5 mmol) were added dropwise over a period of 30 min at 0° C. to a stirred solution of compound I-28b (95 g, 315.43 mmol) in DCM (450 mL). The reaction mixture was stirred for 16 h at rt, then quenched with 2N HCl (500 mL) at 0° C. and the mixture was extracted with DCM (2×400 mL). The combined organic layers were washed with water (500 mL), brine (500 mL), dried (Na2SO4), filtered and concentrated, which gave the title compound (120 g, 88%). MS (ES+) 404.15 [M+H]+

Step d) (1-((benzyloxy)methyl)-2-methylenecyclopropyl)methanol (I-28d)

Zn powder (163 g, 2492 mmol) [activated by stirring commercial Zn powder (200 g) with 2M HCl (300 mL) for 1 h at rt, then Zn was filtered, washed with water (2×400 mL), acetone (2×400 mL) and dried under vacuum overnight] was added portionwise over a period of 15 min at rt to a stirred solution of compound I-28c (120 g, 311.5 mmol) in EtOH (800 mL). The reaction mixture was stirred at 80° C. for 16 h, then cooled to rt and filtered through the celite bed and washed with EtOAc (2×200 mL). The filtrate was concentrated to dryness under reduced pressure. The crude compound was purified by column chromatography on silica gel and eluted with 20% EtOAc in pet ether, which gave the title compound (34 g, 47%). MS (ES+) 205.25 [M+H]+.

Step e) (2-methylenecyclopropane-1,1-diyl)dimethanol (I-28e)

BCl3-DMS (2M in DCM) (100 mL, 200 mmol) was added dropwise over a period of 1H at 0° C. to a stirred solution of compound I-28d (34 g, 166.5 mmol) in DCM (400 mL). The reaction mixture was stirred for 5 h at rt, then filtered and washed with 10% MeOH in DCM (2×500 mL). The filtrate was concentrated to dryness under reduced pressure. The crude compound was purified by column chromatography on silica gel and eluted with 80% EtOAc in pet ether, which gave the title compound (18 g, 95%).

Step f) (2-methylenecyclopropane-1,1-diyl)bis(methylene) diacetate (I-28f)

Acetic anhydride (104.2 mL, 1104 mmol) were added dropwise over a period of 30 min at 0° C. to a stirred solution of compound I-28e (18 g, 157.7 mmol) in pyridine (45 mL, 552 mmol). The reaction mixture was stirred for 16 h at rt, then quenched with water (200 mL) at 0° C. and extracted with DCM (2×500 mL). The combined organic layers were washed 2N HCl (2×200 mL), water (200 mL), brine (200 mL), dried (Na2SO4), filtered and concentrated. The crude compound was purified by column chromatography on silica gel and eluted with 15% EtOAc in pet ether, which gave the title compound (27 g, 81%). MS (ES+) 216.27 [M+H]+as ammonium adduct.

Step a) (2-bromo-2-(bromomethyl)cyclopropane-1,1-diyl)bis(methylene) diacetate (I-28a)

Bromine (7 mL, 136.22 mmol) were added dropwise over a period of 30 min at 0° C. to a stirred solution of compound I-28f (27 g, 136.22 mmol) in carbon tetrachloride (250 mL). The reaction mixture was stirred for 1 h at 0° C., then diluted with DCM (300 mL) and washed with sodium thiosulphate solution (2×300 mL). The combined organic layers were washed saturated sodium bicarbonate solution (300 mL), water (300 mL), brine (300 mL), dried (Na2SO4), filtered and concentrated. The crude compound was purified by column chromatography on silica gel and eluted with 20% EtOAc in pet ether, which gave the title compound (40 g, 74%). MS (ES+) 376.02 [M+H]+as ammonium adduct.

Step h) (Z)-4-amino-1-((2,2-bis(hydroxymethyl)cyclopropylidene)methyl)pyrimidin-2(1H)-one (I-28h)

Dried K2CO3(9.3 g, 67 mmol) and N4-Benzoylcytosine (2.4 g, 11.2 mmol) were added to a stirred solution of compound I-28g (4 g, 11.2 mmol) in DMF (400 mL). The reaction mixture was stirred at 100° C. for 12 h, then cooled to 50° C. and MeOH (40 mL) was added. The resulting reaction mixture was stirred for 2 h at 50° C., then cooled to rt and filtered. The filtrate was concentrated to dryness under reduced pressure. The crude compound was purified by column chromatography on silica gel and eluted with 10% MeOH in DCM, which gave the title compound (2.4 g) as a mixture of E and Z isomers. MS (ES+) 224.2 [M+H]+.

Step i) (Z)—N-(1-((2,2-bis(hydroxymethyl)cyclopropylidene)methyl)-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (I-28i)

Benzoic anhydride (7.3 g, 32.3 mmol) was added to a stirred solution of compound I-28h (2.4 g, 10.8 mmol) in EtOH (300 mL) and the reaction mixture at was stirred at 100° C. for 3 h, The reaction mixture was cooled to rt, filtered, the filtrate was concentrated to dryness under reduced pressure. The crude compound was purified by column chromatography on silica gel and eluted with 5% MeOH in DCM. The residue was further purified by column chromatography on silica gel and eluted with 5% MeOH in DCM, which gave the title compound (500 mg, 14%). MS (ES+) 328.29 [M+H]+.

Step i) (Z)-4-amino-1-((2,2-bis(hydroxymethyl)cyclopropylidene)methyl)pyrimidin-2(1H)-one (I-2j)

A stirred solution of compound I-28i (500 mg, 1.53) in 7M NH3in MeOH (50.2 mL) was stirred at rt for 16 h, then the precipitated solid was filtered and dried. The residue was further purified by prep. HPLC on an X-bridge C18 column (30×250) mm 5u using a gradient of 10 mM NH4HCO3in H2O:MeCN as mobile phase, which gave the title compound (80 mg) as a solid. The filtrate was concentrated under reduced pressure and the residue was purified by column chromatography on silica gel and eluted with 15% MeOH in DCM, which gave another lot of the title compound (80 mg). MS (ES+) 224.27 [M+H]+.

Step k) (Z)-(2-((4-amino-2-oxopyrimidin-1(2H)-yl)methylene)-1-(hydroxymethyl)cyclopropyl)methyl isobutyrate (I-28k)

To a stirred solution of compound I-28j (130 mg, 0.6 mmol) in DMF (10 mL) were added trimethyl orthoisobutyrate (0.14 mL, 0.9 mmol) and pTSA (11 mg, 0.1 mmol) at rt. The reaction mixture was stirred at rt for 3 h, then Et3N (0.5 mL) was added and the mixture was concentrated. The residue was taken in 80% acetic acid in water (15 mL), stirred at rt for 2 h, then concentrated. The crude compound was purified by column chromatography on silica gel eluted with 7% MeOH in DCM, which gave the title compound (180 mg, 97%) as a solid. LCMS (ES+) m/z 294.26 [M+H]+.

Step a) pentan-3-yl(tert-butoxycarbonyl)-L-phenylalaninate (I-29a)

To a stirred solution of (tert-butoxycarbonyl)-L-phenylalanine (7.2 g, 27.2 mmol) and pentan-3-ol (2.5 mL, 22.7 mmol) in DCM (150 mL) were added DMAP (416 mg, 3.4 mmol), EDCI (4.8 g, 25 mmol) at 0° C. The solution was stirred for 18 h, then diluted with DCM (100 mL), washed with water (2×50 mL) and brine (100 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The afforded crude compound was purified by column chromatography on silica gel, eluted with 10% ethyl acetate in hexane which gave the title compound (6.5 g, 85%).

Step b) (S)-1-oxo-1-(pentan-3-yloxy)-3-phenylpropan-2-aminium chloride (I-29b)

4M HCl in 1,4 dioxane (22 mL, 87.2 mmol) was added at 0° C. to a solution of compound I-29a (6.5 g, 19.4 mmol) in 1,4-dioxane (60 mL). The reaction mixture was stirred at room temperature for 2 h, then concentrated under reduced pressure, which gave the title compound (5 g, HCl salt) as a solid. The crude product was used in the next step without further purification.

Step bc pentan-3-yl((S)-(perfluorophenoxy)(phenoxy)phosphoryl)-L-phenylalaninate (I-29c)

Et3N (5.4 mL, 38.6 mmol) was added dropwise over a period of 10 min at −78° C. to a stirred solution of compound I-29b (5 g, 18.4 mmol) in DCM (100 mL) followed by dropwise addition of a solution of phenyl phosphorodichloridate (2.8 mL, 18.4 mmol) in DCM (50 mL) over a period of 30 min. The reaction mixture was stirred at −78° C. for 30 min, then allowed to warm to 0° C. during 2 h and was then stirred for 1 h at 0° C. A solution of 2,3,4,5,6-pentafluorophenol (3 g, 16.6 mmol) and Et3N (2.8 mL, 20.2 mmol) in DCM (50 mL) was added dropwise at 0° C. and the reaction mixture was then stirred for at rt 3 h, then concentrated to dryness under reduced pressure, The crude product was dissolved in MTBE (100 mL), insolubles were filtered off and the filtrate was concentrated to dryness under reduced pressure. The crude compound was purified by column chromatography on silica gel eluted with 10% EtOAc in hexane. Pure fractions were pooled and concentrated under reduced pressure. The afforded racemic compound was dissolved in 30% EtOAc in pet ether (250 mL) and kept at rt for 24 h, the crystals formed were filtered off and washed with pet ether (2×100 mL), and dried under vacuum which gave the title compound (2 g, 18%, chiral purity 99%). MS (ES+) m/z 558.30 [M+H]+.

Step a) 2-ethylbutyl(tert-butoxycarbonyl)-L-phenylalaninate (I-30a)

To a stirred solution of (tert-butoxycarbonyl)-L-phenylalanine (7.8 g, 29.5 mmol) and 2-ethylbutan-1-ol (3.1 mL, 24.7 mmol) in DCM (80 mL) were added DMAP (450 mg, 3.7 mmol), EDCI (5.2 g, 27.1 mmol) at 0° C. The solution was stirred for 16 h at rt, then diluted with DCM (200 mL), washed with saturated sodium bicarbonate solution (100 mL) and brine (100 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The afforded crude compound was purified by column chromatography on silica gel, eluted with 20% ethyl acetate in hexane which gave the title compound (7.5 g, 87%). MS (ES+) m/z 350.47 [M+H]+.

Step b) (S)-1-(2-ethylbutoxy)-1-oxo-3-phenylpropan-2-aminium chloride (I-30b)

4M HCl in 1,4 dioxane (32 mL, 128 mmol) was added at rt to a solution of compound I-30a (7.5 g, 21.5 mmol) in 1,4-dioxane (100 mL). The reaction mixture was stirred at room temperature for 4 h at rt, then concentrated under reduced pressure, which gave the title compound (5.5 g, 88%, HCl salt) as a solid.

Step c) 2-ethylbutyl((S)-(perfluorophenoxy)(phenoxy)phosphoryl)-L-phenylalaninate (I-30c)

Et3N (5.6 mL, 40.1 mmol) was added dropwise over a period of 10 min at −78° C. to a stirred solution of compound I-30b (5.5 g, 19.2 mmol) in DCM (100 mL) followed by dropwise addition of a solution of phenyl phosphorodichloridate (2.9 mL, 19.4 mmol) in DCM (40 mL) over a period of 30 min. The reaction mixture was stirred at −78° C. for 30 min, then allowed to warm to 0° C. during 2 h and was then stirred for 1 h at 0° C. A solution of 2,3,4,5,6-pentafluorophenol (3.2 g, 17.4 mmol) and Et3N (3.4 mL, 24.3 mmol) in DCM (50 mL) was added dropwise at 0° C. over a period of 15 min and the reaction mixture was then stirred for at rt 5 h, then concentrated to dryness under reduced pressure, The crude product was dissolved in MTBE (100 mL) and stirred for 10 min, insolubles were filtered off and the filtrate was concentrated to dryness under reduced pressure. The crude compound was purified by column chromatography on silica gel eluted with 20% EtOAc in hexane. Pure fractions were pooled and concentrated under reduced pressure. The afforded racemic compound was dissolved in 20% EtOAc in pet ether (100 mL) and kept in refrigerator for 4 h, the crystals formed were filtered off and washed with 20% EtOAc in pet ether (30 mL), and dried under vacuum which gave the title compound (1.5 g, 13%, chiral purity 99%). MS (ES+) m/z 572.30 [M+H]+.

Step a) 2-ethylbutyl(tert-butoxycarbonyl)-L-leucinate (I-31a)

To a stirred solution of (tert-butoxycarbonyl)-L-leucine (6.8 g, 29.4 mmol) and 2-ethylbutan-1-ol (2.6 mL, 24.5 mmol) in DCM (100 mL) were added DMAP (449 mg, 3.7 mmol), EDCI (5.2 g, 26.9 mmol) at 0° C. The solution was stirred for 16 h at rt, then diluted with DCM (100 mL), washed with water (50 mL) and brine (50 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The afforded crude compound was purified by column chromatography on silica gel, eluted with 10% ethyl acetate in hexane, which gave the title compound (7.2 g, 93%). MS (ES+) m/z 316.43 [M+H]+.

Step b) (S)-1-(2-ethylbutoxy)-4-methyl-1-oxopentan-2-aminium chloride (I-31b)

4M HCl in 1,4 dioxane (36 mL, 143.8 mmol) was added at rt to a solution of compound I-31a (7.2 g, 22.8 mmol) in 1,4-dioxane (50 mL). The reaction mixture was stirred at room temperature for 2 h at rt, then concentrated under reduced pressure, which gave the title compound (5.5 g, HCl salt) as a solid. MS (ES+) m/z 216.28 [M+H]+. The crude product was used in the next step without further purification.

Step c) 2-ethylbutyl((S)-(perfluorophenoxy)(phenoxy)phosphoryl)-L-leucinate (I-31c)

Et3N (6.4 mL, 46 mmol) was added dropwise over a period of 10 min at −78° C. to a stirred solution of compound I-31b (5.5 g, 22 mmol) in DCM (125 mL) followed by dropwise addition of a solution of phenyl phosphorodichloridate (3.3 mL, 22 mmol) in DCM (125 mL) over a period of 30 min. The reaction mixture was stirred at −78° C. for 30 min, then allowed to warm to 0° C. during 2 h and was then stirred for 1 h at 0° C. A solution of 2,3,4,5,6-pentafluorophenol (3.6 g, 20 mmol) and Et3N (3.4 mL, 24 mmol) in DCM (50 mL) was added dropwise at 0° C. over a period of 15 min and the reaction mixture was then stirred for at rt 4 h, then concentrated to dryness under reduced pressure, The crude product was dissolved in MTBE (100 mL) and stirred for 10 min, insolubles were filtered off and the filtrate was concentrated to dryness under reduced pressure. The crude compound was purified by column chromatography on silica gel eluted with 20% EtOAc in hexane. Pure fractions were pooled and concentrated under reduced pressure. The afforded racemic compound was dissolved in 10% EtOAc in pet ether (100 mL) and kept in refrigerator for 12 h, the crystals formed were filtered off and washed with 5% EtOAc in pet ether (30 mL), and dried under vacuum. The obtained residue was further purified by chiral SFC, which gave the title compound (500 mg). MS (ES+) m/z 538.43 [M+H]+.

Preparative SFC Conditions:

Column/dimensions: Chiralpak IG (4.6×250 mm), 5μCO2:85.0%Co solvent: 15.0% (100% isopropanol)Total Flow: 3.0 g/minBack Pressure: 100.0 barUV: 214 nm

((Z)-2-((2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methylene)-1-(hydroxymethyl)cyclopropyl)methyl(tert-butoxycarbonyl)-L-valinate (I-32a)

DCC (549 mg, 2.7 mmol) and DMAP (33 mg, 0.27 mmol) were added at rt to a stirred solution of (Z)-2-amino-9-((2,2-bis(hydroxymethyl)cyclopropylidene)methyl)-1H-purin-6(9H)-one (350 mg, 1.3 mmol) and (tert-butoxycarbonyl)-L-valine (289 mg, 1.3 mmol) in DMF (20 mL). The resulting reaction mixture was stirred at rt for 16 h, then concentrated under reduced pressure. The crude compound was combined with another batch and purified by column chromatography on silica gel and eluted with 10% EtOAc in hexane. The pure compound was further purified by chiral SFC, which gave the title compound (65 mg, 10%) as a solid. MS (ES+) 463.43 [M+H]+.

Preparative SFC Conditions:

Column/dimensions: Chiralcel OX-H (250×30) mm, 5μCO2:65.0%Co solvent: 35.0% (MeOH)Total Flow: 70 g/minBack Pressure: 100.0 barUV: 214 nmLoad ability/inj.: 5.0 mgStack time: 14 minLoad/Inj.: 7 mg

Step a) (Z)-(2-((4-amino-2-oxopyrimidin-1(2H)-yl)methylene)-1-(hydroxymethyl)cyclopropyl)methyl butyrate (I-33a)

To a stirred solution of compound I-28j (300 mg, 1.34 mmol) in DMF (20 mL) were added trimethyl orthobutyrate (0.32 mL, 2.02 mmol) and pTSA (26 mg, 0.13 mmol) at rt. The reaction mixture was stirred at rt for 3 h, then Et3N (0.5 mL) was added and the mixture was concentrated. The residue was taken in 80% acetic acid in water (20 mL), stirred at rt for 2 h, then concentrated. The crude compound was purified by column chromatography on silica gel eluted with 8% MeOH in DCM, which gave the title compound (260 mg, 57%) as a solid. LCMS (ES+) m/z 294.34 [M+H]+.

Step a) 2-ethylbutyl 0-(2-ethylbutyl)-L-homoserinate (I-34a)

To a stirred solution of L-aspartic acid (5 g, 37.6 mmol in toluene (100 mL) was added pTSA (449 mg, 3.7 mmol), followed by 2-ethylbutan-1-ol (7.7 g, 75.1 mmol) at 0° C. The solution was stirred for 16 h at 110° C., then concentrated under reduced pressure. The residue was dissolved in water and extracted with EtOAc. The combined organic extracts were washed with saturated sodium bicarbonate solution, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure, which gave the title compound (7.15 g, 63%). MS (ES+) m/z 302.41 [M+H]+.

Step b) bis(2-ethylbutyl) L-aspartate hydrochloride (I-34b)

4M HCl in 1,4 dioxane (40 mL, 160 mmol) was added at rt to a solution of compound I-34a (7.15 g, 23.7 mmol) in 1,4-dioxane (35 mL). The reaction mixture was stirred at room temperature for 12 h, then concentrated under reduced pressure, which gave the title compound (8 g, HCl salt) as a solid. MS (ES+) m/z 302.28 [M+H]+.

Step c) bis(2-ethylbutyl) ((S)-(perfluorophenoxy)(phenoxy)phosphoryl)-L-aspartate (I-34c)

Et3N (6.9 mL, 49.3 mmol) was added dropwise over a period of 5 min at −70° C. to a stirred solution of compound I-34b (8 g, 23.5 mmol) in DCM (40 mL) followed by dropwise addition of a solution of phenyl phosphorodichloridate (3.5 mL, 23.5 mmol) in DCM (20 mL) over a period of 10 min. The reaction mixture was stirred at −70° C. for 30 min, then allowed to warm to 0° C. during 2 h and was then stirred for 1 h at 0° C. A solution of 2,3,4,5,6-pentafluorophenol (3.9 g, 21.12 mmol) and Et3N (3.6 mL, 25.8 mmol) in DCM (20 mL) was added dropwise at 0° C. and the reaction mixture was then stirred for at rt for 4 h, then concentrated to dryness under reduced pressure, The crude product was dissolved in MTBE (100 mL), insolubles were filtered off and the filtrate was concentrated to dryness under reduced pressure. The crude compound was purified by column chromatography on silica gel eluted with 10% EtOAc in hexane. Pure fractions were pooled and concentrated under reduced pressure. The afforded racemic compound was dissolved in 10% EtOAc in pet ether (90 mL) and kept at 0° C. for 12 h, the crystals formed were filtered off and dried under vacuum. The obtained compound was again dissolved in 10% EtOAc in pet ether (30 mL) and kept at 0° C. for 12 h, the crystals formed were filtered off and dried under vacuum. The obtained compound was again dissolved in 2% EtOAc in pet ether (50 mL) and kept at 0° C. for 4 h, the crystals formed were filtered off and dried under vacuum, which gave the title compound (2 g, 12%, chiral purity 98.6%). MS (ES+) m/z 624.60 [M+H]+.

Step a) (Z)-(2-((2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methylene)-1-(hydroxymethyl)cyclopropyl)methyl pivalate (I-35a)

DMAP (464 mg, 3.8 mmol) was added at rt to a stirred solution of (Z)-2-amino-9-((2,2-bis(hydroxymethyl)cyclopropylidene)methyl)-1H-purin-6(9H)-one (1 g, 3.8 mmol) in DMF (50 mL). The reaction mixture was cooled to 10° C., then pivaloyl chloride (920 mg, 7.6 mmol) was added. The resulting reaction mixture was stirred at rt for 1 h, then concentrated under reduced pressure. The crude compound was purified by column chromatography on silica gel and eluted with 8% MeOH in DCM. The pure compound was further purified by chiral SFC, which gave the title compound (100 mg) as a solid. MS (ES+) 348.41 [M+H]+.

(R,Z)-(2-((4-amino-2-oxopyrimidin-1(2H)-yl)methylene)-1-(hydroxymethyl)cyclopropyl)methyl isobutyrate (I-36a) & (S,Z)-(2-((4-amino-2-oxopyrimidin-1(2H)-yl)methylene)-1-(hydroxymethyl)cyclopropyl)methyl isobutyrate (I-36b)

To a stirred solution of compound I-28j (300 mg, 1.34 mmol) in DMF (15 mL) were added trimethyl orthoisobutyrate (0.64 mL, 4.03 mmol) and pTSA (128 mg, 0.7 mmol) at rt. The reaction mixture was stirred at rt for 3 h, then Et3N (1 mL) was added and the mixture was concentrated. The residue was taken in 80% acetic acid in water (20 mL), stirred at rt for 2 h, then concentrated. The crude compound was purified by column chromatography on silica gel eluted with 8% MeOH in DCM. The obtained compound was further purified by prep HPLC using Method G. The pure compound was further purified by chiral SFC, which gave the title compounds peak-1 (61 mg, 15%) and peak-2 (62 mg, 15%) as solids. MS (ES+) 294.30 [M+H]+.

Peak-1:

SOR: +46.14° (based on the SOR value of the guanine coupled compound, the stereochemistry is assumed to be R-isomer).
Peak-2:SOR: −46.18° (based on the SOR value of the guanine coupled compound, the stereochemistry is assumed to be S-isomer).
Preparative SFC ConditionsColumn/dimensions: Chiralcel OX-H (30×250 mm), 5μCO2:70.0%Co solvent: 30.0% (EtOH)Total flow: 70.0 g/minBack pressure: 100.0 barUV: 214 nmStack time: 6.0 minLoad/inj.: 10.0 mg

Step a) (Z)—N-(1-((2,2-bis(hydroxymethyl)cyclopropylidene)methyl)-5-fluoro-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (I-37a)

Dried K2CO3(7.1 g, 51.5 mmol) and compound I-28g (3 g, 8.6 mmol) were added to a stirred solution of N-(5-fluoro-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (2.0 g, 8.6 mmol) in DMF (100 mL). The reaction mixture was stirred at 100° C. for 48 h, then cooled to 50° C. and MeOH (6 mL) was added. The resulting reaction mixture was stirred for 2 h at 50° C., then cooled to rt and filtered. The filtrate was concentrated to dryness under reduced pressure. The crude compound was purified by column chromatography on silica gel and eluted with 12% MeOH in DCM. The obtained residue was further purified by column chromatography on silica gel and eluted with 5% MeOH in DCM, which gave the title compound (500 g, 16%) as Z isomer. MS (ES+) 346.28 [M+H]+.

Step b) (Z)-4-amino-1-((2,2-bis(hydroxymethyl)cyclopropylidene)methyl)-5-fluoropyrimidin-2(1H)-one (I-37b)

A stirred solution of compound I-37a (500 mg, 1.45 mmol) in 7M NH3in MeOH (20 mL, 14 mmol) was stirred at rt for 16 h, then concentrated. The residue was further purified by trituration with EtOAc (10 mL), filtered and dried, which gave the title compound (300 mg, 85%) as a solid. MS (ES+) 242.28 [M+H]+.

Step c) (Z)-(2-((4-amino-5-fluoro-2-oxopyrimidin-1(2H)-yl)methylene)-1-(hydroxymethyl)cyclopropyl)methyl isobutyrate (I-37c)

To a stirred solution of compound I-37b (250 mg, 1.04 mmol) in DMF (10 mL) were added trimethyl orthoisobutyrate (0.5 mL, 3.1 mmol) and pTSA (79 mg, 0.4 mmol) at rt. The reaction mixture was stirred at rt for 2 h, then Et3N (2 mL) was added and the mixture was concentrated. The residue was taken in 80% acetic acid in water (30 mL), stirred at rt for 2 h, then concentrated. The crude compound was combined with another batch and purified by column chromatography on silica gel eluted with 8% MeOH in DCM, which gave the title compound (250 mg, 77%) as a solid. LCMS (ES+) m/z 312.39 [M+H]+.

Step a) (Z)-(2-((4-amino-5-fluoro-2-oxopyrimidin-1(2H)-yl)methylene)-1-(hydroxymethyl)cyclopropyl)methyl butyrate (I-38a)

To a stirred solution of compound I-37b (300 mg, 1.24 mmol) in DMF (30 mL) were added trimethyl orthobutyrate (0.6 mL, 3.7 mmol) and pTSA (119 mg, 0.6 mmol) at rt. The reaction mixture was stirred at rt for 3 h, then Et3N (0.5 mL) was added and the mixture was concentrated. The residue was taken in 80% acetic acid in water (30 mL), stirred at rt for 2 h, then concentrated. The crude compound was purified by column chromatography on silica gel eluted with 7% MeOH in DCM, which gave the title compound (350 mg, 88%) as a solid. LCMS (ES+) m/z 312.31 [M+H]+.

Step a) (R,Z)-(2-((4-amino-5-fluoro-2-oxopyrimidin-1(2H)-yl)methylene)-1-(hydroxymethyl)cyclopropyl)methyl isobutyrate (I-39a)

Compound I-37a (3.25 g) was further purified by chiral SFC, which gave the title compound (peak-2) (1.21 g, 31%) as a solid. MS (ES+) 312.35 [M+H]+.

Peak-2:

SOR: +92.72° (based on the SOR value of the cytosine coupled compound, the stereochemistry is assumed to be R-isomer).
Preparative SFC Conditions:Column/dimensions: Chiralpak IG (30×250 mm), 5μCO2:70.0%Co solvent: 30.0% (100% MeOH)Total Flow: 90.0 g/minBack Pressure: 100.0 barUV: 214 nmStack time: 7.0 minLoad/inj.: 101.1 mg

Step b) ((1R,Z)-2-((4-amino-5-fluoro-2-oxopyrimidin-1(2H)-yl)methylene)-1-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)cyclopropyl)methyl isobutyrate (I-39b)

Methanesulfonic acid (0.53 mL, 8.1 mmol) in DMF (10 mL) was added dropwise at rt to a stirred solution of compound I-39a (1.1 g, 3.4 mmol) and 3,4-dihydro-2H-pyran (6.2 mL, 67.5 mmol) in DMF (30 mL). The reaction mixture was stirred at rt for 2 h, then Et3N (10 mL) was added and concentrated at reduced pressure. The obtained crude compound (1.25 g) was used in next step without further purification. LCMS (ES+) m/z 396.37 [M+H]+.

Step c) 4-amino-5-fluoro-1-((Z)-((2S)-2-(hydroxymethyl)-2-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)cyclopropylidene)methyl)pyrimidin-2(1H)-one (I-39c)

To a stirred solution of compound I-39b (1.25 g, 3.2 mmol) in MeOH (60 mL) was added 25% aq ammonia (60 mL, 389.5 mmol) at rt. The reaction mixture was stirred at rt for 36 h, then concentrated under reduced pressure. The obtained crude compound was purified by column chromatography on silica gel and eluted with 7% MeOH in DCM, which gave the title compound (910 mg, 88%) as a solid. LCMS (ES+) m/z 326.34 [M+H]+.

Step d) ((1R,Z)-2-((4-amino-5-fluoro-2-oxopyrimidin-1(2H)-yl)methylene)-1-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)cyclopropyl)methyl(tert-butoxycarbonyl)-L-valinate (I-39d)

A mixture of (tert-butoxycarbonyl)-L-valine (1.52 mg, 7.0 mmol) and DCC (866 mg, 4.2 mmol) in DMF (60 mL) was stirred at rt for 3 h, then the insoluble solids were filtered and the filtrate was concentrated under reduced pressure. The obtained residue was dissolved in DMF (60 mL) and compound I-39c (910 mg, 2.8 mmol) was added followed by addition of DMAP (103 mg, 0.84 mmol). The resulting reaction mixture was stirred at rt for 16 h, then concentrated under reduced pressure. The crude compound was purified by column chromatography on silica gel and eluted with 4% MeOH in DCM, which gave the title compound as a mixture of isomers (930 mg, 63%) as a solid. MS (ES+) 525.46 [M+H]+.

Step e)

((R,Z)-2-((4-amino-5-fluoro-2-oxopyrimidin-1(2H)-yl)methylene)-1-(hydroxymethyl)cyclopropyl)methyl(tert-butoxycarbonyl)-L-valinate (I-39e)

80% AcOH (100 mL, 1397.5 mmol) was added to compound I-39d (930 mg, 1.8 mmol) and the resulting reaction mixture was stirred for 36 h at rt, then concentrated under reduced pressure. The obtained crude compound was purified by column chromatography on silica gel eluted with 6% MeOH in DCM, which gave the title compound (520 mg, 64%) as a solid. LCMS (ES+) m/z 441.39 [M+H]+.

Example 1

Step a) ((1R,Z)-2-((2-Isobutyramido-6-oxo-1H-purin-9(6H)-yl)methylene)-1-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)cyclopropyl)methyl acetate (1a)

Methanesulfonic acid (0.3 mL, 4.3 mmol) in DMF (40 mL) was added dropwise at rt to a stirred solution of compound I-12b (800 mg, 2.13 mmol) in DMF (10 mL) and 3,4-dihydro-2H-pyran (3.9 mL, 43 mmol). The reaction mixture was stirred at rt for 4 h, then concentrated at reduced pressure. The obtained of crude compound (1.1 g) was used in next step without further purification. LCMS (ES+) m/z 460.26 [M+H]+.

Step b) 2-Amino-9-((Z)-((2S)-2-(hydroxymethyl)-2-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)cyclopropylidene)methyl)-1H-purin-6(9H)-one (1b)

To a stirred solution of compound 1a (1.1 g, 2.4 mmol) in MeOH (50 mL) was added 25% aq ammonia (55 mL, 360 mmol) at rt. The reaction mixture was stirred at 50° C. for 12 h then concentrated at reduced pressure. The obtained crude compound was purified by column chromatography on silica gel and eluted with 15% MeOH, which gave the title compound (750 mg) as a solid. LCMS (ES+) m/z 348.20 [M+H]+.

Step c) (2S)-((1R,Z)-2-((2-Amino-6-oxo-1H-purin-9(6H)-yl)methylene)-1-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)cyclopropyl)methyl 2-((tert-butoxycarbonyl)amino)-3-methylbutanoate (1c)

A mixture of N-Boc-L-valine (915 mg, 4.21 mmol) and dicyclohexylcarbodiimide (500 mg, 2.43 mmol) in DCM (40 mL) was stirred at rt for 3 h, then filtered and concentrated under reduced pressure. The obtained crude was dissolved in DMF (60 mL) and compound 1b (650 mg, 1.87 mmol) was added followed by addition of 4-dimethylaminopyridine (70 mg, 0.56 mmol). The reaction mixture was stirred at rt for 16 h, then concentrated under reduced pressure.

The afforded crude compound was purified by column chromatography on silica gel eluted with 7% MeOH in DCM, which gave the title compound (900 mg, 86%) as a solid. LCMS (ES+) m/z 547.37 [M+H]+.

Step d) (S)-((R,Z)-2-((2-Amino-6-oxo-1H-purin-9(6H)-yl)methylene)-1-(hydroxymethyl)cyclopropyl)methyl 2-((tert-butoxycarbonyl)amino)-3-methylbutanoate (1d)

A solution of compound 1c (900 mg, 1.6 mmol) in 80% acetic acid (90 mL, 1.3 mol) was stirred at rt for 16 h, then concentrated under reduced pressure. The crude compound was purified by column chromatography on silica gel eluted with 10-12% MeOH in DCM, which gave the title compound (600 mg, 67%) as a solid. LCMS (ES+) m/z 463.29 [M+H]+.

Step e) (S)-((S,Z)-2-((2-Amino-6-oxo-1H-purin-9(6H)-yl)methylene)-1-((((S)-(((S)-1-oxo-1-((2-propylpentyl)ox)propan-2-)amino)(phenoxy)phosphoryl)oxy)methyl)cyclopropyl)methyl 2-((tert-butoxycarbonyl)amino)-3-methylbutanoate (1e)

tert-Butylmagnesium chloride (1M in THF, 1.1 mL, 1.1 mmol) was added dropwise to a solution of compound 1d (100 mg, 0.22 mmol) in DMF (10 mL) the mixture was stirred at rt for 30 min, then a solution of 1-4 (140 mg, 0.26 mmol) in dry THE (5 mL) was added dropwise. The reaction mixture was stirred at rt for 12 h, then concentrated under reduced pressure. The crude compound was purified by column chromatography on silica gel and eluted with 10-12% MeOH in DCM, which gave the title compound (120 mg, 46%) as a solid. LCMS (ES+) m/z 802.42 [M+H]+.

Step f) (S)-((S,Z)-2-((2-Amino-6-oxo-1H-purin-9(6H)-yl)methylene)-1-((((S)-(((S)-1-oxo-1-((2-propylpentyl)oxy)propan-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl)cyclopropyl)methyl 2-amino-3-methylbutanoate (1f-1) & (R)-((S,Z)-2-((2-Amino-6-oxo-1H-purin-9(6H)-yl)methylene)-1-((((S)-(((S)-1-oxo-1-((2-propylpentyl)oxy)propan-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl)cyclopropyl)methyl 2-amino-3-methylbutanoate (1f-2)

A solution of compound 1e (120 mg, 0.14 mmol) in 60% acetic acid (12 mL, 126 mmol) was stirred for 5 h at 90° C., then allowed to attain rt and concentrated under reduced pressure. The obtained residue was purified by prep HPLC using HPLC Method A. The afforded residue was combined with another batch and purified by chiral NP HPLC.

which gave the two title compounds, 1f-1 (22 mg) & 1f-2 (8 mg).

Preparative NP-HPLC Conditions

Column/dimensions: Chiralpak IC (250×30) mm, 5μMobile Phase: 0.2% DEA in n-hexane:EtOH (30:70)Flow: 38.0 ml/minTemperature: AmbientWave length: 270 nmRun time: 17 minSolubility: EtOH+DCMLoad ability/inj.: 5.0 mg

1f-1: LCMS (ES+) m/z 702.39 [M+H]+.

1H NMR (500 MHz, DMSO) δ 10.71 (s, 1H), 7.93 (s, 1H), 7.33 (m, J=4.0 Hz, 2H), 7.19 (m, J=8.1 Hz, 4H), 6.54 (s, 2H), 6.06 (q, J=7.7 Hz, 1H), 4.44 (d, J=11.6 Hz, 1H), 4.37 (q, J=5.8 Hz, 1H), 4.07 (q, J=5.5 Hz, 1H), 3.97 (m, J=3.8 Hz, 2H), 3.87 (m, J=4.9 Hz, 2H), 3.06 (d, J=5.4 Hz, 1H), 1.64 (t, J=6.1 Hz, 4H), 1.22 (m, J=3.2 Hz, 12H), 0.82 (q, J=4.3 Hz, 6H), 0.75 (d, J=6.8 Hz, 3H), 0.70 (d, J=6.8 Hz, 3H).

1f-2: LCMS (ES+) m/z 702.39 [M+H]+.

1H NMR (500 MHz, DMSO) δ 10.70 (s, 1H), 7.95 (s, 1H), 7.34 (t, J=7.9 Hz, 2H), 7.18 (m, J=6.0 Hz, 4H), 6.55 (s, 2H), 6.07 (q, J=7.8 Hz, 1H), 4.34 (d, J=11.6 Hz, 1H), 4.20 (m, J=5.4 Hz, 2H), 4.02 (d, J=11.6 Hz, 1H), 3.96 (q, J=5.6 Hz, 1H), 3.77 (m, J=3.8 Hz, 2H), 3.05 (d, J=5.2 Hz, 1H), 1.79 (m, J=4.7 Hz, 1H), 1.58 (m, J=4.9 Hz, 3H), 1.20 (m, J=5.7 Hz, 11H), 0.82 (m, J=3.5 Hz, 9H), 0.74 (d, J=6.8 Hz, 3H).

Example 2

(S)-((S,Z)-2-((2-amino-6-oxo-1H-purin-9(6H)-yl)methylene)-1-((((S)-(((S)-3-methyl-1-oxo-1-((S)-pentan-2-yloxy)butan-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl)cyclopropyl)methyl 2-amino-3-methylbutanoate & (R)—((S,Z)-2-((2-amino-6-oxo-1H-purin-9(6H)-yl)methylene)-1-((((S)-(((S)-3-methyl-1-oxo-1-((S)-pentan-2-yloxy)butan-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl)cyclopropyl)methyl 2-amino-3-methylbutanoate (2-1 & 2-2)

The title compound was prepared from compound 1d and I-3b as Example 1 step e followed by removal of the Boc group by treatment of a solution of the Boc protected compound in dioxane with 4 M HCl in dioxane for 4h, then concentrate. The residue was purified by prep HPLC Method C followed by separation of the stereoisomers by chiral prep. NP HPLC.

Chiral Prep NP HPLC

Column/dimensions: Chiralcel OX-H (250×30) mm, 5μMobile Phase: 0.2% DEA in n-hexane:EtOH (50:50)Flow: 44.0 ml/minTemperature: AmbientWave length: 232 nmRun time: 22 minSolubility: EtOH:MeCN:IPALoad/inj.: 10.8 mg

2-1: (60 mg, 27% yield) LCMS (MS+) 688.34 [M+H]+, 99.4% chiral purity according to chiral NP HPLC.

1H NMR (500 MHz, DMSO) δ 10.70 (s, 1H), 7.92 (s, 1H), 7.30 (t, J=7.5 Hz, 2H), 7.22 (s, 1H), 7.13 (d, J=7.7 Hz, 3H), 6.54 (s, 2H), 5.90 (t, J=11.3 Hz, 1H), 4.77 (d, J=6.1 Hz, 1H), 4.42 (m, J=10.7 Hz, 2H), 4.08 (q, J=5.3 Hz, 1H), 3.98 (d, J=11.6 Hz, 1H), 3.51 (q, J=9.1 Hz, 2H), 3.04 (d, J=4.9 Hz, 1H), 1.91 (t, J=6.3 Hz, 1H), 1.64 (s, 3H), 1.48 (s, 1H), 1.39 (d, J=13.6 Hz, 1H), 1.26 (m, J=10.0 Hz, 3H), 1.11 (d, J=6.0 Hz, 3H), 0.82 (t, J=6.8 Hz, 9H), 0.71 (q, J=11.4 Hz, 6H).

2-2. (12 mg, 5.2% yield) LCMS (MS+) 688.38 [M+H]+, 97.8% chiral purity according to chiral NP HPLC.

1H NMR (500 MHz, DMSO) δ 10.73 (s, 1H), 7.93 (s, 1H), 7.31 (t, J=7.8 Hz, 2H), 7.17 (m, J=9.9 Hz, 5H), 6.56 (s, 1H), 5.89 (t, J=11.6 Hz, 1H), 4.75 (q, J=6.1 Hz, 1H), 4.36 (d, J=11.6 Hz, 1H), 4.21 (m, J=5.7 Hz, 2H), 4.00 (q, J=6.0 Hz, 2H), 3.47 (m, J=6.9 Hz, 1H), 3.04 (d, J=5.1 Hz, 1H), 1.91 (q, J=6.5 Hz, 2H), 1.77 (t, J=6.2 Hz, 1H), 1.61 (q, J=8.5 Hz, 2H), 1.42 (m, J=10.1 Hz, 3H), 1.23 (m, J=7.6 Hz, 5H), 1.08 (d, J=6.2 Hz, 3H), 0.78 (m, J=12.6 Hz, 17H).

Example 3

Step a) (2S)-2-propylpentyl 2-(((S)-(((Z)-2-((2-amino-6-oxo-1H-purin-9(6H)-yl)methylene)-1-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)cyclopropyl)methoxy)(phenoxy)phosphoryl)amino)-propanoate (3a)

tert-Butylmagnesium chloride (0.7 mL, 1M in THF, 0.72 mmol) was added dropwise at rt to a solution of compound I-19b (50 mg, 0.14 mmol) in DMF (5 mL). The reaction mixture was stirred at rt for 30 min. Then a solution of compound I-4 (90 mg, 0.17 mmol) in dry THE (2.5 mL) was added dropwise. The reaction mixture was stirred at rt for 2 h, then concentrated. The crude compound was purified by column chromatography on silica gel eluted with 10% MeOH in DCM, which gave the title compound (55 mg, 53%) as a solid. LCMS (ES+) m/z 687.39 [M+H]+.

Step b) (S)-2-Propylpentyl 2-(((S)-(((R,Z)-2-((2-amino-6-oxo-1H-purin-9(6H)-yl)methylene)-1-(hydroxymethyl)cyclopropyl)methoxy)(phenoxy)phosphoryl)amino)propanoate (S,Z)-2-Propylpentyl 2-((1-((2-amino-6-oxo-1H-purin-9(6H)-yl)methylene)-6-oxido-5,7-dioxa-6-phosphaspiro[2.5]octan-6-yl)amino)propanoate (3b-1 & 3b-2)

A solution of compound 3a (400 mg, 0.582 mmol) in 80% acetic acid (40 mL, 559 mmol) was stirred at rt for 24 h, then concentrated. The obtained crude compound was purified by column chromatography on silica gel eluted with 12% MeOH in DCM. The afforded residue was subjected to chiral prep SFC purification which gave two fractions, 3b-1 and 3b-2.

3b-1 was purified by prep HPLC using Method B with gradient 0/35, 9/67, 9.1/99, 10/99, 10.1/35, 12/35, which gave the title compound (35 mg). Purity according to LCMS: 96.6% & chiral HPLC 99.9%. LCMS (ES+) m/z 607.31 [M+H]+.

3b-2 was purified by chiral prep NP HPLC, followed by chiral prep SFC using the “IPA method”, which gave the cyclized compound 3b-2. LCMS (ES+) m/z 509.19 [M+H]+.

Preparative SFC Conditions “IPA Method”

Column/dimensions: Chiralpak AD-H (21×250 mm), 5μCO2:75.0%Co solvent: 25.0% (100% IPA)Total Flow: 60.0 g/minBack Pressure: 80.0 barUV: 229 nmStack time: 10.0 minLoad/inj.: 5.0 mg

Example 4

(S,Z)-2-Ethylbutyl 2-((1-((2-amino-6-oxo-1H-purin-9(6H)-yl)methylene)-6-oxido-5,7-dioxa-6-phosphaspiro[2.5]octan-6-yl)amino)propanoate (4)

A solution of 1,8-diazabicyclo[5.4.0]undec-7-ene (0.2 mL, 1.14 mmol) in DMF (2 mL) was added at rt dropwise over a period of 30 min to a stirred suspension of (Z)-2-amino-9-((2,2-bis(hydroxymethyl)cyclopropylidene)methyl)-1H-purin-6(9H)-one (150 mg, 0.57 mmol) and I-6 (0.31 g, 0.62 mmol) in DMF (3 mL). The mixture was stirred at rt for 3 h, then diluted with NaHCO3aq. (10 mL) and extracted with DCM (25 mL). The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The crude compound was purified by column chromatography on silica gel eluted 5% MeOH in DCM. Appropriate fractions were pooled, concentrated and purified by prep HPLC using Method C, which gave the title compound (18 mg, 6.6%) as a mixture of P-isomers. LCMS (ES+) m/z 481.25 [M+H]+.

Example 5

(S,Z)-2-Ethylbutyl 2-((1-((2-amino-6-oxo-1H-purin-9(6H)-yl)methylene)-6-oxido-5,7-dioxa-6-phosphaspiro[2.5]octan-6-yl)amino)-3-methylbutanoate (5)

To a stirred suspension of (Z)-2-amino-9-((2,2-bis(hydroxymethyl)cyclopropylidene)methyl)-1H-purin-6(9H)-one (0.1 g, 0.38 mmol) and I-8b (0.2 g, 0.38 mmol) in DMF (10 mL) was added a solution of 1,8-diazabicyclo[5.4.0]undec-7-ene (0.12 mL, 0.760 mmol) in DMF (5 mL) dropwise over a period of 30 min at rt. The mixture was stirred 3 h, then diluted with NaHCO3aq (5 mL) and extracted with DCM (3×20 mL). The organic layer was dried (Na2SO4), filtered and concentrated under reduced pressure. The crude compound was purified by column chromatography on silica gel eluted with 5% MeOH in DCM. The afforded residue was purified by prep HPLC using Method A, which gave the title compound (12 mg) as a mixture of P-stereoisomers. LCMS (ES+) 509.32 [M+H]+.

Example 6

((R,Z)-2-((2-amino-6-oxo-1H-purin-9(6H)-yl)methylene)-1-((((S)-(((S)-1-oxo-1-((S)-pentan-2-yloxy)propan-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl)cyclopropyl)methyl isobutyrate & ((S,Z)-2-((2-amino-6-oxo-1H-purin-9(6H)-yl)methylene)-1-((((S)-(((S)-1-oxo-1-((S)-pentan-2-yloxy)propan-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl)cyclopropyl)methyl isobutyrate (6-1 & 6-2)

tert-Butylmagnesium chloride (2.7 mL, 1M in THF, 2.700 mmol) was added dropwise to a solution of I-11 (180 mg, 0.540 mmol) in DMF (20 mL). The mixture was stirred at rt for 20 min, then a solution of I-9b (312 mg, 0.648 mmol) in dry THE (20 mL) was added dropwise. The mixture was stirred at rt for 2 h, then concentrated. The residue was combined with another batch and purified by column chromatography using a Combi flash instrument eluted with 7% DCM in MeOH. Appropriate fractions were pooled and concentrated and the residue was purified by prep HPLC using Method B. Appropriate fractions were pooled and concentrated. Chiral HPLC showed two enantiomers which were separated by prep. SFC.

6-1: LCMS (ES+) m/z 631.30 [M+H]+. Chiral HPLC showed 99.9% purity.

6-2: LCMS (ES+) m/z 631.30 [M+H]+. Chiral HPLC showed 99.8% purity.

Preparative SFC Conditions

Column/dimensions: Chiralcel OX-H (30×250 mm), 5μCO2:50.0%Co solvent: 50.0% (100% IPA)Total flow: 90.0 g/minBack pressure: 100.0 barUV: 214 nmStack time: 12.6 minLoad/inj.: 13.0 mg

Example 7

(S)-(S)-pentan-2-yl 2-(((S)-(((S,Z)-1-((((S)-2-amino-3-methylbutanoyl)oxy)methyl)-2-((2-amino-6-oxo-1H-purin-9(6H)-yl)methylene)cyclopropyl)methoxy)(phenoxy)phosphoryl)amino)-4-methylpentanoate & (S)-(S)-pentan-2-yl 2-(((S)-(((S,Z)-1-((((R)-2-amino-3-methylbutanoyl)oxy)methyl)-2-((2-amino-6-oxo-1H-purin-9(6H)-yl)methylene)cyclopropyl)methoxy)(phenoxy)phosphoryl)amino)-4-methylpentanoate (7-1 & 7-2)

The title compound was prepared from compound 1d and I-7b as Example 1 steps e and f with the difference that the purification/separation of the final compound was performed by1) Prep. HPLC Method C2) Separation of isomers by prep. chiral NP HPLC]3) Fraction 2 was purified by prep HPLC Method C
Prep Chiral NP HPLCColumn/dimensions: Chiralcel OX-H (250×30) mm, 5μMobile Phase: 0.2% DEA in n-hexane:EtOH (55:45)Flow: 42.0 ml/minTemperature: AmbientWave length: 232 nmRun time: 22 minSolubility: EtOHLoad/inj.: 8.1 mg

7-1: LCMS (ES+) m/z 702.35 [M+H]+, 99.96% chiral purity according to chiral NP HPLC.

1H NMR (500 MHz, DMSO) δ 10.83 (s, 1H), 7.90 (s, 1H), 7.32 (m, J=4.0 Hz, 2H), 7.22 (t, J=1.8 Hz, 1H), 7.14 (t, J=7.3 Hz, 3H), 6.60 (s, 2H), 6.01 (q, J=7.7 Hz, 1H), 4.77 (m, J=4.8 Hz, 1H), 4.42 (m, J=7.6 Hz, 2H), 4.04 (q, J=5.5 Hz, 1H), 3.96 (d, J=11.5 Hz, 1H), 3.71 (m, J=4.7 Hz, 1H), 3.03 (d, J=5.5 Hz, 1H), 1.63 (q, J=4.1 Hz, 4H), 1.43 (m, J=3.0 Hz, 4H), 1.24 (m, J=2.9 Hz, 2H), 1.11 (d, J=6.3 Hz, 3H), 0.83 (q, J=3.1 Hz, 5H), 0.79 (t, J=5.6 Hz, 4H), 0.74 (d, J=6.8 Hz, 3H), 0.69 (d, J=6.8 Hz, 3H).

7-2: LCMS (ES+) m/z 702.35 [M+H]+, 97.97% chiral purity according to chiral NP HPLC.

1H NMR (500 MHz, DMSO) δ 10.93 (s, 1H), 7.90 (s, 1H), 7.32 (q, J=5.3 Hz, 2H), 7.22 (t, J=1.8 Hz, 1H), 7.15 (m, J=6.7 Hz, 3H), 6.72 (s, 2H), 6.01 (q, J=7.8 Hz, 1H), 4.75 (q, J=6.4 Hz, 1H), 4.34 (d, J=11.5 Hz, 1H), 4.20 (m, J=6.1 Hz, 2H), 4.00 (d, J=11.5 Hz, 1H), 3.66 (m, J=8.7 Hz, 1H), 3.03 (d, J=5.2 Hz, 1H), 1.78 (m, J=5.4 Hz, 1H), 1.59 (m, J=4.4 Hz, 4H), 1.42 (m, J=4.1 Hz, 5H), 1.23 (m, J=3.8 Hz, 3H), 1.07 (t, J=5.6 Hz, 3H), 1.05 (s, 1H), 0.81 (m, J=4.1 Hz, 13H), 0.74 (d, J=6.8 Hz, 3H).

Example 8

(S)-(S)-Pentan-2-yl 2-(((S)-(((S,Z)-1-(acetoxymethyl)-2-((2-amino-6-oxo-1H-purin-9(6H)-yl)methylene)cyclopropyl)methoxy)(4-bromophenoxy)phosphoryl)amino) & (S)-(S)-Pentan-2-yl 2-(((S)-(((R,Z)-1-(acetoxymethyl)-2-((2-amino-6-oxo-1H-purin-9(6H)-yl)methylene)cyclopropyl)methoxy)(4-bromophenoxy)phosphoryl)amino)propanoate propanoate (8-1 & 8-2)

(Z)-(2-((2-Amino-6-oxo-1H-purin-9(6H)-yl)methylene)-1-(hydroxymethyl)cyclopropyl)methyl acetate and I-5 were reacted according to the method described in Example 1 step e. The afforded compound was purified by prep HPLC using Method A. The racemic mixture was separated by SFC, which gave the two isomers as separate compounds.

8-1: 2.7% yield, 98.82% chiral purity according to chiral HPLC, LCMS (ES+) m/z 681.16[M+H]+.

8-2: 3.2% yield, 99.7% chiral purity according to chiral HPLC, LCMS (ES+) m/z 681.16 [M+H]+.

Preparative SFC Conditions

Column/dimensions: Chiralpak AD-H (30×250 mm), 5μCO2:50.0%Co solvent: 50.0% (0.5% diethylamine in EtOH)Total Flow: 90.0 g/minBack Pressure: 90.0 barUV: 214 nmStack time: 9.4 minLoad/inj.: 11.0 mg

Example 9

(S)-(S)-Pentan-2-yl 2-(((S)-(((S,Z)-1-(acetoxymethyl)-2-((2-amino-6-oxo-1H-purin-9(6H)-yl)methylene)cyclopropyl)methoxy)(phenoxy)phosphoryl)amino)propanoate & (S)-(S)-Pentan-2-yl 2-(((S)-(((R,Z)-1-(acetoxymethyl)-2-((2-amino-6-oxo-1H-purin-9(6H)-yl)methylene)cyclopropyl)methoxy)(phenoxy)phosphoryl)amino)propanoate (9-1 & 9-2)

(Z)-(2-((2-Amino-6-oxo-1H-purin-9(6H)-yl)methylene)-1-(hydroxymethyl)cyclopropyl)methyl acetate and I-3b were reacted according to the method described in Example 1 step e. The afforded compound was purified by prep HPLC using Method D. The racemic mixture was separated by prep SFC, which gave the two isomers as separate compounds.

9-1: 6.0% yield, 98.07% chiral purity according to chiral HPLC, LCMS (ES+) m/z 631.30 [M+H]+.

9-2: 6.0% yield, 98.9% chiral purity according to chiral HPLC, LCMS (ES+) m/z 631.30 [M+H]+.

Preparative SFC Conditions

Column/dimensions: Chiralcel OX-H (30×250 mm), 5μCO2: 60.0%Co solvent: 40.0% (100% MeOH)Total Flow: 90.0 g/minBack Pressure: 100.0 barUV: 214 nmStack time: 5.5 minLoad/inj.: 3.0 mg

Example 10

(S)-(S)-Pentan-2-yl 2-(((S)-(((S,Z)-1-(acetoxymethyl)-2-((2-amino-6-oxo-1H-purin-9(6H)-yl)methylene)cyclopropyl)methoxy)(4-bromophenoxy)phosphoryl)amino)propanoate & (S)-(S)-Pentan-2-yl 2-(((S)-(((R,Z)-1-(acetoxymethyl)-2-((2-amino-6-oxo-1H-purin-9(6H)-yl)methylene)cyclopropyl)methoxy)(4-bromophenoxy)phosphoryl)amino)propanoate (10-1 & 10-2)

(Z)-(2-((2-Amino-6-oxo-1H-purin-9(6H)-yl)methylene)-1-(hydroxymethyl)cyclopropyl)methyl acetate and I-18b were reacted according to the method described in Example 1 step e. The afforded compound was purified by prep HPLC using Method D. The racemic mixture was separated by prep SFC, which gave the two isomers as separate compounds.

10-1: Yield 5.1%, 99.9% chiral purity according to analytical SFC, LCMS (ES+) m/z 723.22 & 725.20 [M+H]+.

10-2: Yield 8.0%, 99.34% chiral purity according to analytical SFC, LCMS (ES+) m/z 723.19 & 725.20 [M+H]+.

Preparative SFC Conditions

Column/dimensions: Chiralcel OX-H (30×250 mm), 5μCO2:60.0%Co solvent: 40.0% (100% EtOH)Total Flow: 90.0 g/minBack Pressure: 100.0 barUV: 214 nmStack time: 6.2 minLoad/inj.: 9.1 mg

Example 11

((S,Z)-2-((2-Amino-6-oxo-1H-purin-9(6H)-yl)methylene)-1-((((S)-(((S)-1-oxo-1-((2-propylpentyl)oxy)propan-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl) cyclopropyl)methyl butyrate &A ((R,Z)-2-((2-Amino-6-oxo-1H-purin-9(6H)-yl)methylene)-1-((((S)-(((S)-1-oxo-1-((2-propylpentyl)oxy)propan-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl) cyclopropyl)methyl butyrate (11-1 & 11-2)

I-27 and I-4 were reacted according to the method described in Example 1 step e. The afforded compound was purified by prep HPLC using Method A. The racemic mixture was separated by prep SFC, which gave the two isomers as separate compounds.

11-1 Yield 4.2%, chiral purity 99.95% according to SFC, LCMS (ES+) m/z 673.33 [M+H]+.1H NMR (500 MHz, DMSO) δ 10.79 (s, 1H), 7.93 (s, 1H), 7.33 (t, J=7.8 Hz, 2H), 7.22 (s, 1H), 7.15 (t, J=7.3 Hz, 3H), 6.58 (s, 2H), 6.05 (q, J=7.7 Hz, 1H), 4.35 (m, J=7.0 Hz, 2H), 4.04 (q, J=6.6 Hz, 2H), 3.97 (q, J=5.5 Hz, 1H), 3.88 (m, J=5.6 Hz, 2H), 2.18 (m, J=7.4 Hz, 2H), 1.60 (d, J=9.4 Hz, 3H), 1.45 (q, J=7.1 Hz, 2H), 1.24 (d, J=6.9 Hz, 11H), 0.81 (m, J=4.9 Hz, 9H).

11-2: Yield 4.0%, chiral purity 99.14% according to SFC, LCMS (ES+) m/z 673.29 [M+H]+.1H NMR (500 MHz, DMSO) δ 10.74 (s, 1H), 7.94 (s, 1H), 7.34 (t, J=7.8 Hz, 2H), 7.20 (d, J=7.1 Hz, 3H), 7.16 (t, J=7.4 Hz, 1H), 6.57 (s, 2H), 6.05 (q, J=7.8 Hz, 1H), 4.27 (d, J=11.6 Hz, 1H), 4.18 (m, J=6.0 Hz, 2H), 4.02 (d, J=11.6 Hz, 1H), 3.96 (q, J=5.6 Hz, 1H), 3.79 (m, J=5.0 Hz, 2H), 2.17 (m, J=7.7 Hz, 2H), 1.56 (q, J=10.5 Hz, 3H), 1.45 (q, J=7.3 Hz, 2H), 1.20 (m, J=7.1 Hz, 11H), 0.81 (q, J=7.8 Hz, 9H).

Preparative SFC Conditions

Column/dimensions: Chiralcel OX-H (30×250 mm), 5μCO2: 50.0%Co solvent: 50.0% (100% IPA)Total Flow: 90.0 g/minBack Pressure: 100.0 barUV: 214 nmStack time: 23.0 minLoad/inj.: 28.0 mg

Example 12

((S,Z)-2-((2-Amino-6-oxo-1H-purin-9(6H)-yl)methylene)-1-((((S)-(4-bromophenoxy)(((S)-1-oxo-1-((S)-pentan-2-yloxy)propan-2-yl)amino)phosphoryl)oxy)methyl)cyclopropyl)methyl butyrate & ((R,Z)-2-((2-Amino-6-oxo-1H-purin-9(6H)-yl)methylene)-1-((((S)-(4-bromophenoxy)(((S)-1-oxo-1-((S)-pentan-2-yloxy)propan-2-yl)amino)phosphoryl)oxy)methyl)cyclopropyl)methyl butyrate (12-1 & 12-2)

I-27 and I-5 were reacted according to the method described in Example 1 step e. The afforded compound was purified by prep HPLC using Method D. The racemic mixture was separated by prep SFC, which gave the two isomers as separate compounds.

12-1: Yield 3.5%, 99.48% chiral purity according to analytical SFC, LCMS (ES+) m/z 709.41 & 711.35 [M+H]+.

1H nmr (500 MHz, DMSO) δ 10.74 (s, 1H), 7.91 (s, 1H), 7.51 (d, J=8.5 Hz, 2H), 7.22 (s, 1H), 7.13 (d, J=8.5 Hz, 2H), 6.56 (s, 2H), 6.09 (t, J=11.5 Hz, 1H), 4.78 (m, J=6.2 Hz, 1H), 4.36 (m, J=7.0 Hz, 2H), 4.04 (t, J=9.9 Hz, 2H), 3.81 (q, J=8.9 Hz, 1H), 2.18 (m, J=7.4 Hz, 2H), 1.63 (s, 2H), 1.44 (m, J=7.0 Hz, 4H), 1.23 (d, J=6.9 Hz, 5H), 1.12 (d, J=6.2 Hz, 3H), 0.81 (m, J=7.3 Hz, 6H).

12-2: Yield 4.0%, 98.93% chiral purity according to analytical SFC, LCMS (ES+) m/z 709.41 & 711.39 [M+H]+.

1H nmr (500 MHz, DMSO) δ 10.72 (s, 1H), 7.92 (s, 1H), 7.51 (d, J=8.5 Hz, 2H), 7.21 (s, 1H), 7.16 (d, J=8.4 Hz, 2H), 6.55 (s, 2H), 6.09 (t, J=11.6 Hz, 1H), 4.75 (q, J=6.1 Hz, 1H), 4.29 (d, J=11.6 Hz, 1H), 4.20 (d, J=5.8 Hz, 2H), 4.04 (d, J=11.5 Hz, 1H), 3.73 (q, J=9.0 Hz, 1H), 2.16 (m, J=7.8 Hz, 2H), 1.61 (s, 2H), 1.42 (m, J=6.7 Hz, 4H), 1.22 (t, J=7.1 Hz, 5H), 1.09 (d, J=6.3 Hz, 3H), 0.81 (m, J=6.8 Hz, 6H).

Preparative SFC Conditions

Column/dimensions: Chiralpak AD-H (30×250 mm), 5μCO2:60.0%Co solvent: 40.0% (100% EtOH)Total Flow: 70.0 g/minBack Pressure: 90.0 barUV: 219 nmStack time: 10.5 minLoad/inj.: 5.1 mg

Example 13

(S)-2-propylpentyl 2-(((S)-(((S,Z)-1-(acetoxymethyl)-2-((2-amino-6-oxo-1H-purin-9(6H)-yl)methylene)cyclopropyl)methoxy)(phenoxy)phosphoryl)amino)propanoate & (S)-2-propylpentyl 2-(((S)-(((R,Z)-1-(acetoxymethyl)-2-((2-amino-6-oxo-1H-purin-9(6H)-yl)methylene)cyclopropyl)methoxy)(phenoxy)phosphoryl)amino)propanoate (13-1 & 13-2)

(Z)-(2-((2-Amino-6-oxo-1H-purin-9(6H)-yl)methylene)-1-(hydroxymethyl)cyclopropyl)methyl acetate and I-4 were reacted according to the method described in Example 1 step e. The afforded compound was purified by prep HPLC. The racemic mixture was separated by prep SFC, which gave the two isomers as separate compounds.

13-1: Yield 14%, 96.88% chiral purity according to analytical SFC, LCMS (ES+) m/z 645.35 [M+H]+.

1H NMR (500 MHz, DMSO)_10.81 (s, 1H), 7.93 (s, 1H), 7.33 (t, J=7.7 Hz, 2H), 7.22 (s, 1H), 7.15 (t, J=7.9 Hz, 3H), 6.60 (s, 2H), 6.05 (t, J=11.5 Hz, 1H), 4.32 (m, J=6.7 Hz, 2H), 4.06 (t, J=9.7 Hz, 2H), 3.96 (q, J=5.5 Hz, 1H), 3.87 (m, J=7.1 Hz, 2H), 1.93 (s, 3H), 1.61 (s, 3H), 1.24 (d, J=6.8 Hz, 11H), 0.82 (t, J=6.2 Hz, 6H). 13-2: Yield 18%, 98.23% chiral purity according to analytical SFC LCMS (ES+) m/z 645.39 [M+H]+.

1H NMR (500 MHz, DMSO)_11.22 (s, 1H), 7.92 (s, 1H), 7.34 (t, J=7.6 Hz, 2H), 7.21 (d, J=7.0 Hz, 3H), 7.16 (t, J=7.4 Hz, 1H), 6.74 (s, 2H), 6.05 (t, J=11.7 Hz, 1H), 4.20 (t, J=10.9 Hz, 3H), 4.05 (d, J=11.6 Hz, 1H), 3.96 (q, J=5.5 Hz, 1H), 3.80 (q, J=5.3 Hz, 2H), 1.92 (s, 3H), 1.57 (d, J=21.6 Hz, 3H), 1.20 (m, J=7.3 Hz, 11H), 0.82 (t, J=6.7 Hz, 6H).

Preparative SFC Conditions

Column/dimensions: Chiralpak IC (21×250 mm), 5μCO2:75.0%Co solvent: 25% (100% IPA)Total Flow: 70.0 g/minBack Pressure: 100.0 barUV: 227 nmStack time: 7.0 minLoad/inj.: 3.0 mg

Example 14

Step a) (Z)-(2-((2-amino-6-(cyclopropylamino)-1H-purin-9(6H)-yl)methylene)-1-(hydroxymethyl)cyclopropyl)methyl acetate (14a)

To a stirred solution of (Z)-(2-((2-amino-6-(cyclopropylamino)-1H-purin-9(6H)-yl)methylene)cyclopropane-1,1-diyl)dimethanol (700 mg, 2.22 mmol) in DMF (70 mL) were added Trimethyl orthoacetate (1.15 ml, 8.90 mmol) and pTSA (425 mg, 2.23 mmol) at rt. The reaction mixture was stirred at rt for 3 h, then concentrated. The residue was taken in 80% acetic acid (aq., 100 mL), the mixture was stirred at rt for 3 h, then concentrated. The crude compound was purified by column chromatography on silica gel eluted with 4.5% MeOH in DCM. the afforded compound was purified by prep HPLC using Method C, which gave the title compound (180 mg, 24%) of as a solid. LCMS (ES+) m/z 345.31 [M+H]+.

Step b) (2S)-2-propylpentyl 2-(((S)-(((Z)-1-(acetoxymethyl)-2-((2-amino-6-cyclopropylamino-1H-purin-9(6H)-yl)methylene)cyclopropyl)methoxy)(phenoxy)phosphoryl)amino)propanoate (14b)

To a solution of 14a (50 mg, 0.14 mmol) in dry THE (4 mL) was added tert-butylmagnesium chloride (0.8 mL, 1M in THF, 0.8 mmol) dropwise for 5 min. The mixture was stirred for 30 min at rt, then 1-4 (92 mg, 0.18 mmol) in dry THE (2 mL) was added dropwise. The reaction mixture was stirred at rt for 2 h, then concentrated. The crude product was purified by column chromatography on silica gel eluted with 8% MeOH in DCM. Appropriate fractions were pooled and concentrated. The afforded residue was purified by prep HPLC using Method D, which gave the title compound (10 mg, 10%) as a solid. LCMS (ES+) m/z 684.45 [M+H]+.

1H NMR (500 MHz, DMSO) δ 7.98 (d, J=4.6 Hz, 1H), 7.44 (s, 1H), 7.31 (m, J=7.3 Hz, 3H), 7.16 (q, J=9.5 Hz, 3H), 6.02 (m, J=6.9 Hz, 3H), 4.36 (m, J=13.5 Hz, 1H), 4.18 (m, J=9.5 Hz, 2H), 3.97 (m, J=5.2 Hz, 1H), 3.83 (m, J=7.3 Hz, 2H), 3.03 (s, 1H), 1.92 (d, J=3.0 Hz, 3H), 1.59 (d, J=7.0 Hz, 3H), 1.20 (m, J=7.6 Hz, 12H), 0.81 (d, J=6.9 Hz, 6H), 0.66 (d, J=6.6 Hz, 2H), 0.60 (s, 2H).

Example 15

(S)-((S,Z)-2-((2-amino-6-oxo-1H-purin-9(6H)-yl)methylene)-1-((((S)-(((S)-1-oxo-1-((S)-pentan-2-yloxy)propan-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl)cyclopropyl)methyl 2-amino-3-methylbutanoate & (R)—((S,Z)-2-((2-amino-6-oxo-1H-purin-9(6H)-yl)methylene)-1-((((S)-(((S)-1-oxo-1-((S)-pentan-2-yloxy)propan-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl)cyclopropyl)methyl 2-amino-3-methylbutanoate (15-1 & (15-2)

The title compound was prepared from compounds 1d and I-9b as Example 1 steps e and f with the difference that the purification/separation of the final compounds was performed by1) Prep. chiral NP HPLC which gave a first (15-1) and second fraction (15-2).2-1) First fraction was combined with another batch of the same compound and purified by prep HPLC, Method C.2-2) Second fraction was combined with another batch of the same compound and purified by prep HPLC, Method A.3) The second fraction was purified by prep chiral NP HPLC.
Prep NP HPLCColumn/dimensions: Chiralcel OX-H (250×30) mm, 5μMobile Phase: 0.2% DEA in n-hexane:EtOH (45:55)Flow: 38.0 ml/minTemperature: AmbientWave length: 270 nmRun time: 24 minSolubility: EtOHLoad/inj.: 9.7 mg

15-1: LCMS (ES+) m/z 660.33 [M+H]+, 99.2% chiral purity according to chiral NP HPLC.

1H NMR (500 MHz, DMSO) δ 10.60 (s, 1H), 7.93 (s, 1H), 7.33 (m, J=3.2 Hz, 2H), 7.23 (t, J=1.8 Hz, 1H), 7.16 (d, J=7.9 Hz, 3H), 6.54 (s, 2H), 6.02 (q, J=7.7 Hz, 1H), 4.79 (m, J=3.8 Hz, 1H), 4.46 (d, J=11.6 Hz, 1H), 4.38 (q, J=5.8 Hz, 1H), 4.06 (q, J=5.5 Hz, 1H), 3.96 (d, J=11.5 Hz, 1H), 3.80 (m, J=5.1 Hz, 1H), 3.03 (d, J=5.5 Hz, 1H), 1.63 (q, J=4.0 Hz, 4H), 1.45 (m, J=3.5 Hz, 2H), 1.22 (d, J=7.1 Hz, 5H), 1.13 (d, J=6.3 Hz, 3H), 0.83 (t, J=7.4 Hz, 3H), 0.74 (d, J=6.8 Hz, 3H), 0.69 (d, J=6.8 Hz, 3H).

15-2: LCMS (ES+) m/z 660.33 [M+H]+, 99.0% chiral purity according to chiral NP HPLC.

1H NMR (500 MHz, DMSO) δ 11.01 (s, 1H), 7.93 (s, 1H), 7.33 (q, J=5.3 Hz, 2H), 7.18 (m, J=5.2 Hz, 4H), 6.70 (s, 2H), 6.03 (q, J=7.8 Hz, 1H), 4.76 (q, J=6.4 Hz, 1H), 4.35 (d, J=11.6 Hz, 1H), 4.20 (m, J=3.3 Hz, 2H), 4.03 (d, J=11.5 Hz, 1H), 3.73 (m, J=5.1 Hz, 1H), 3.04 (d, J=5.2 Hz, 1H), 1.78 (m, J=3.8 Hz, 1H), 1.61 (m, J=4.0 Hz, 3H), 1.42 (m, J=3.2 Hz, 3H), 1.28 (q, J=4.4 Hz, 1H), 1.22 (m, J=5.7 Hz, 5H), 1.09 (d, J=6.3 Hz, 3H), 0.82 (t, J=7.3 Hz, 6H), 0.74 (d, J=6.8 Hz, 3H).

Example 16

((Z)-2-((4-amino-2-oxopyrimidin-1(2H)-yl)methylene)-1-((((((S)-1-oxo-1-((2-propylpentyl)oxy)propan-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl)cyclopropyl)methyl isobutyrate

tert-Butylmagnesium chloride (1M in THF, 2.6 mL, 2.6 mmol) was added dropwise over a period of 10 min to a solution of compound I-28k (150 mg, 0.51 mmol) in DMF (15 mL). The reaction mixture was stirred at rt for 30 min, then compound I-4 (322 mg, 0.6 mmol) in dry THE (10 mL) was added dropwise over a period of 10 min. The reaction mixture was stirred at rt for 4 h, then concentrated and the afforded crude compound was combined with another batch and purified by column chromatography on silica gel eluted with 8-10% MeOH in DCM. The afforded product was further purified by prep HPLC using Method C. The pure compound was further purified by chiral SFC, which gave the title compound (peak-1)(9 mg) as a solid. LCMS (ES+) m/z 633.53 [M+H]+.

1H NMR (500 MHz, DMSO): δ 7.74 (t, J=5.5 Hz, 1H), 7.43 (t, J=1.7 Hz, 2H), 7.35 (t, J=8.0 Hz, 3H), 7.16 (q, J=3.7 Hz, 3H), 6.05 (q, J=7.7 Hz, 1H), 5.80 (d, J=7.4 Hz, 1H), 4.37 (d, J=11.5 Hz, 1H), 4.22 (q, J=5.6 Hz, 1H), 3.99 (m, J=5.4 Hz, 2H), 3.88 (m, J=5.7 Hz, 3H), 2.46 (t, J=3.5 Hz, 1H), 1.60 (t, J=5.7 Hz, 1H), 1.47 (d, J=1.8 Hz, 2H), 1.23 (m, J=6.3 Hz, 12H), 1.06 (t, J=3.5 Hz, 3H), 1.03 (q, J=3.1 Hz, 3H), 0.84 (m, J=2.5 Hz, 6H).

Preparative SFC Conditions:

Column/dimensions: Chiralpak IC (30×250 mm), 5μCO2:50.0%Co solvent: 50.0% (100% isopropanol)Total Flow: 90.0 g/minBack Pressure: 100.0 barUV: 214 nmStack time: 14.0 minLoad/inj.: 7.0 mg

Example 17

Step a) 2-propylpentyl((S)-(((1S,Z)-2-((2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methylene)-1-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)cyclopropyl)methoxy)(phenoxy)phosphoryl)-L-alaninate (17a)

tert-Butylmagnesium chloride (1M in THF, 5 mL, 5.0 mmol) was added dropwise over a period of 5 min to a solution of compound I-13b (350 mg, 1 mmol) in DMF (35 mL). The reaction mixture was stirred at rt for 30 min, then compound I-4 (633 mg, 1.2 mmol) in dry THE (17 mL) was added dropwise over a period of 5 min. The reaction mixture was stirred at rt for 16 h, then concentrated and the afforded crude compound was combined with was purified by column chromatography on silica gel eluted with 10% MeOH in DCM, which gave the title compound (250 mg) as a solid. LCMS (ES+) m/z 687.29 [M+H]+.

Step b) 2-propylpentyl((S)-(((S,Z)-2-((2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methylene)-1-(hydroxymethyl)cyclopropyl)methoxy)(phenoxy)phosphoryl)-L-alaninate (17b)

80% AcOH (104.2 mL, 1455.7 mmol) was added to compound 17a (250 mg, 0.4 mmol) and the resulting reaction mixture was stirred for 48 h at rt, then concentrated. The obtained crude compound was purified by column chromatography on silica gel eluted with 12% MeOH in DCM. The afforded product was further purified by prep HPLC using Method A, which gave the title compound (110 mg, 42%) as a solid. LCMS (ES+) m/z 603.48 [M+H]+.

Step c) ((((S,Z)-2-((2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methylene)-1-(hydroxymethyl)cyclopropyl)methoxy)(hydroxy)phosphoryl)-L-alanine (17c)

A solution of compound 17b (40 mg, 0.07 mmol) in Et3N (1 mL) and water (0.25 mL) was stirred at 50° C. for 46 h. The reaction mixture was lyophilised and purified by prep HPLC using Method G, which gave the title compound (15 mg) as a solid. LCMS (ES+) m/z 603.48 [M+H]+.

Step d) lithium ((((S,Z)-2-((2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methylene)-1-(hydroxymethyl)cyclopropyl)methoxy)oxidophosphoryl)-L-alaninate (17d)

Dowex 50 WX4-200, ion exchange resin was taken in a column (2×10 cm), washed with water: MeOH (1:1, 100 mL) until colorless eluent was obtained, then washed with Milli Q water (100 mL) to wash off MeOH. The ion exchange resin was again eluted with 0.5M sulphuric acid (50 mL) until acidic pH was attained and washed with water (200 mL) until neutral pH. The ion exchange resin was again eluted with 1M lithium hydroxide (50 mL) until basic pH was attained and washed with water (200 mL) until neutral pH. A solution of compound 17c (13 mg, 0.031 mmol) in Milli Q water (2 mL) was passed through the above freshly prepared Dowex Li+column. The appropriate fractions were lypholyised, which gave the title compound (8 mg) as a solid. LCMS (ES+) m/z 415.30 [M+H]+.

1H NMR (500 MHz, D2O) δ 8.31 (d, J=11.3 Hz, 1H), 7.23 (m, J=2.2 Hz, 1H), 4.01 (m, J=5.3 Hz, 1H), 3.77 (m, J=5.2 Hz, 3H), 3.49 (m, J=5.7 Hz, 1H), 1.54 (m, J=3.1 Hz, 2H), 1.19 (d, J=7.0 Hz, 3H).

Example 18

methyl((S)-(((Z)-2-((2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methylene)-1-((isobutyryloxy)methyl)cyclopropyl)methoxy)(phenoxy)phosphoryl)-L-leucinate (18)

tert-Butylmagnesium chloride (1M in THF, 1.5 mL, 1.5 mmol) was added dropwise over a period of 15 min to a solution of compound I-11a (210 mg, 0.63 mmol) in DMF (25 mL). The reaction mixture was stirred at rt for 20 min, then compound I-1 (308 mg, 0.66 mmol) in dry THE (10 mL) was added dropwise over a period of 5 min. The reaction mixture was stirred at rt for 2 h, then concentrated under reduced pressure and the afforded crude compound was combined with another batch and purified by column chromatography on silica gel eluted with 7% MeOH in DCM. The afforded product was further purified by prep HPLC using Method D. The pure compound was further purified by chiral SFC, which gave the title compound (peak-2)(67 mg, 17%) as a solid. LCMS (ES+) m/z 617.46 [M+H]+.

1H NMR (500 MHz, DMSO): δ 10.69 (s, 1H), 7.92 (s, 1H), 7.32 (t, J=7.9 Hz, 2H), 7.22 (s, 1H), 7.14 (q, J=6.7 Hz, 3H), 6.55 (s, 2H), 6.05 (q, J=7.8 Hz, 1H), 4.38 (m, J=6.9 Hz, 2H), 4.01 (m, J=7.3 Hz, 2H), 3.75 (q, J=5.2 Hz, 1H), 3.57 (s, 3H), 2.44 (m, J=7.0 Hz, 1H), 1.61 (d, J=1.4 Hz, 3H), 1.43 (m, J=4.7 Hz, 2H), 1.04 (d, J=7.0 Hz, 3H), 0.99 (d, J=7.0 Hz, 3H), 0.82 (d, J=6.7 Hz, 3H), 0.78 (d, J=6.6 Hz, 3H).

Preparative SFC Conditions:

Column/dimensions: Chiralpak IE (30×250 mm), 5μCO2:65.0%Co solvent: 35.0% (0.5% DEA in EtOH)Total Flow: 90.0 g/minBack Pressure: 100.0 barUV: 214 nmStack time: 8.0 minSolubility: 30 mL of acetonitrile+5 mL of MeOHLoad/inj.: 10.0 mg

Example 19

Pentan-3-yl((S)-(((Z)-2-((2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methylene)-1-((isobutyryloxy)methyl)cyclopropyl)methoxy)(phenoxy)phosphoryl)-L-phenylalaninate

tert-Butylmagnesium chloride (1M in THF, 2.3 mL, 2.3 mmol) was added dropwise over a period of 10 min at rt to a solution of compound I-11 (300 mg, 0.9 mmol) in DMF (15 mL). The reaction mixture was stirred at rt for 30 min, then compound I-29c (552 mg, 1.0 mmol) in dry THE (10 mL) was added dropwise over a period of 10 min. The reaction mixture was stirred at rt for 4 h, then concentrated under reduced pressure and the afforded crude compound was combined with another batch and purified by column chromatography on silica gel eluted with 8% MeOH in DCM. The afforded product was further purified by prep HPLC using Method D. The pure compound was further purified by chiral SFC, which gave the title compound (peak-2)(90 mg) as a solid. LCMS (ES+) m/z 707.70 [M+H]+.

1H NMR (500 MHz, DMSO): δ 10.82 (s, 1H), 7.88 (s, 1H), 7.28 (t, J=7.9 Hz, 2H), 7.20 (t, J=2.8 Hz, 5H), 7.11 (m, J=4.3 Hz, 2H), 7.06 (d, J=8.4 Hz, 2H), 6.62 (s, 2H), 6.13 (q, J=7.8 Hz, 1H), 4.58 (m, J=3.5 Hz, 1H), 4.25 (d, J=11.6 Hz, 1H), 3.96 (q, J=5.4 Hz, 1H), 3.87 (m, J=6.1 Hz, 2H), 3.69 (q, J=5.8 Hz, 1H), 3.25 (t, J=7.6 Hz, 1H), 2.98 (m, J=3.2 Hz, 1H), 2.77 (q, J=7.5 Hz, 1H), 2.41 (q, J=7.0 Hz, 1H), 1.53 (t, J=5.0 Hz, 1H), 1.41 (m, J=5.3 Hz, 5H), 1.02 (d, J=7.0 Hz, 3H), 0.97 (d, J=7.0 Hz, 3H), 0.75 (t, J=7.4 Hz, 3H), 0.64 (t, J=7.4 Hz, 3H).

Preparative SFC Conditions:

Column/dimensions: Lux Cellulose-2 (250×30) mm, 5μCO2:60.0%Co solvent: 40.0% (MeOH)Total Flow: 90.0 g/minBack Pressure: 100.0 barUV: 214 nmStack time: 13.0 minLoad/inj.: 15.62 mg

Example 20

2-ethylbutyl((S)-(((Z)-2-((2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methylene)-1-((isobutyryloxy)methyl)cyclopropyl)methoxy)(phenoxy)phosphoryl)-L-phenylalaninate (20)

tert-Butylmagnesium chloride (1M in THF, 2.1 mL, 2.1 mmol) was added dropwise over a period of 15 min at rt to a solution of compound I-11 (300 mg, 0.9 mmol) in DMF (25 mL). The reaction mixture was stirred at rt for 20 min, then compound I-30c (566 mg, 1.0 mmol) in dry THE (10 mL) was added dropwise over a period of 5 min. The reaction mixture was stirred at rt for 2 h, then concentrated under reduced pressure and the afforded crude compound was combined with another batch and purified by column chromatography on silica gel eluted with 8% MeOH in DCM. The afforded product was further purified by prep HPLC using Method D.

The pure compound was further purified by chiral SFC. The impure compound was again purified by chiral SFC, which gave the title compound (peak-2)(75 mg, 11%) as a solid. LCMS (ES+) m/z 721.64 [M+H]+.

1H NMR (500 MHz, DMSO): δ 10.73 (s, 1H), 7.86 (s, 1H), 7.27 (q, J=5.3 Hz, 2H), 7.20 (d, J=4.4 Hz, 5H), 7.10 (m, J=5.1 Hz, 2H), 7.00 (d, J=8.6 Hz, 2H), 6.56 (s, 2H), 6.13 (q, J=7.7 Hz, 1H), 4.35 (d, J=11.6 Hz, 1H), 4.07 (q, J=5.8 Hz, 1H), 4.00 (q, J=5.5 Hz, 1H), 3.87 (m, J=5.3 Hz, 3H), 3.58 (q, J=5.4 Hz, 1H), 2.97 (m, J=3.3 Hz, 1H), 2.78 (q, J=7.5 Hz, 1H), 2.43 (m, J=7.0 Hz, 1H), 1.55 (t, J=5.1 Hz, 1H), 1.50 (q, J=3.6 Hz, 1H), 1.36 (t, J=6.2 Hz, 1H), 1.20 (m, J=4.5 Hz, 4H), 1.03 (d, J=7.0 Hz, 3H), 0.98 (d, J=7.0 Hz, 3H), 0.77 (m, J=3.5 Hz, 6H).

Preparative SFC Conditions-1:

Column/dimensions: Chiralpak IE (30×250 mm), 5μTotal Flow: 40.0 mL/minStack time: 11.0 minSolubility: 30 mL of acetonitrile+5 mL of MeOHLoad/inj.: 17.3 mg
Preparative SFC Conditions-2:Column/dimensions: Chiralpak IA (30×250 mm), 5μCO2:70.0%Co solvent: 30.0% (100% EtOH)Total Flow: 90.0 g/minBack Pressure: 100.0 barUV: 214 nmStack time: 6.5 minLoad/inj.: 20 mg

Example 21

2-ethylbutyl((S)-(((Z)-2-((2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methylene)-1-((isobutyryloxy)methyl)cyclopropyl)methoxy)(phenoxy)phosphoryl)-L-leucinate (21)

tert-Butylmagnesium chloride (1M in THF, 1.4 mL, 2.1 mmol) was added dropwise over a period of 3 min at rt to a solution of compound I-11 (200 mg, 0.6 mmol) in DMF (15 mL). The reaction mixture was stirred at rt for 20 min, then compound I-31c (360 mg, 0.7 mmol) in dry THE (8 mL) was added dropwise over a period of 5 min. The reaction mixture was stirred at rt for 2 h, then concentrated under reduced pressure and the afforded crude compound was combined with another batch and purified by column chromatography on silica gel eluted with 8% MeOH in DCM. The afforded product was further purified by prep HPLC using Method F. The pure compound was further purified by chiral SFC. The impure isomer was again purified by chiral SFC, which gave the title compound (peak-2) (7.5 mg) as a solid. LCMS (ES+) m/z 687.71 [M+H]+.

1H NMR (500 MHz, DMSO): δ 7.89 (s, 1H), 7.32 (t, J=7.9 Hz, 2H), 7.22 (s, 1H), 7.14 (t, J=8.6 Hz, 3H), 6.69 (s, 2H), 6.02 (t, J=11.6 Hz, 2H), 4.38 (t, J=10.1 Hz, 2H), 4.00 (q, J=5.2 Hz, 2H), 3.92 (t, J=5.8 Hz, 2H), 3.76 (q, J=8.3 Hz, 1H), 2.44 (q, J=7.0 Hz, 1H), 1.60 (d, J=9.6 Hz, 3H), 1.43 (t, J=5.9 Hz, 3H), 1.27 (m, J=7.2 Hz, 4H), 1.04 (d, J=7.0 Hz, 3H), 0.99 (d, J=7.0 Hz, 3H), 0.81 (m, J=6.8 Hz, 12H).

Preparative SFC Conditions-1

Column/dimensions: Chiralpak AD-H (30×250 mm), 5μCO2:80.0%Co solvent: 20.0% (100% EtOH)Total Flow: 60.0 g/minBack Pressure: 100.0 barUV: 214 nmStack time: 13 minLoad/Inj. 4.1 mg
Preparative SFC Conditions-2Column/dimensions: Chiralpak AD-H (30×250 mm), 5μCO2:75.0%Co solvent: 25.0% (100% EtOH)Total Flow: 70.0 g/minBack Pressure: 90.0 barUV: 230 nmStack time: 6.5 minLoad/Inj.: 2 mg

Example 22

Step a) 2-propylpentyl((S)-(((Z)-2-((2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methylene)-1-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)cyclopropyl)methoxy)(phenoxy)phosphoryl)-L-alaninate (22a)

tert-Butylmagnesium chloride (1M in THF, 4.3 mL, 4.3 mmol) was added dropwise over a period of 5 min at rt to a solution of compound I-19b (300 mg, 0.9 mmol) in DMF (33 mL). The reaction mixture was stirred at rt for 45 min, then compound I-4 (545 mg, 1.01 mmol) in dry THE (17 mL) was added dropwise over a period of 10 min. The reaction mixture was stirred at rt for 16 h, then concentrated under reduced pressure and the afforded crude compound was combined with another batch and purified by column chromatography on silica gel eluted with 7% MeOH in DCM, which gave the title compound (250 mg) as a solid. LCMS (ES+) m/z 687.71 [M+H]+. The crude product was used in the next step without further purification.

Step b) 2-propylpentyl((S)-(((Z)-2-((2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methylene)-1-(hydroxymethyl)cyclopropyl)methoxy)(phenoxy)phosphoryl)-L-alaninate (22b)

80% AcOH (105 mL, 1455.7 mmol) was added to 22a (250 mg, 0.4 mmol) and the resulting reaction mixture was stirred for 24 h at rt, then concentrated. The obtained crude compound was purified by column chromatography on silica gel eluted with 12% MeOH in DCM. The crude product was used in the next step without further purification.

Step c) ((Z)-2-((2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methylene)-1-((((S)-(((S)-1-oxo-1-((2-propylpentyl)oxy)propan-2yl)amino)(phenoxy)phosphoryl)oxy)methyl)cyclopropyl)methyl stearate (22c)

DCC (154 mg, 0.75 mmol) and DMAP (6 mg, 0.1 mmol) were added at rt to a stirred solution of compound 22b (100 mg, 0.17 mmol) and stearic acid (142 mg, 0.5 mmol) in DMF (10 mL). The resulting reaction mixture was stirred at rt for 20 h, then concentrated under reduced pressure. The crude compound was purified by column chromatography on silica gel and eluted with 5% MeOH in DCM. The obtained compound was further purified by prep HPLC using Method D. The pure compound was further purified by chiral SFC, which gave the title compound (peak-1) (7 mg, 5%) as a solid. MS (ES+) 869.77 [M+H]+.

1H NMR (500 MHz, DMSO): δ 10.68 (s, 1H), 7.94 (s, 1H), 7.32 (m, J=3.2 Hz, 2H), 7.22 (d, J=1.7 Hz, 1H), 7.16 (d, J=7.8 Hz, 3H), 6.53 (s, 2H), 6.05 (q, J=7.7 Hz, 1H), 4.34 (m, J=7.1 Hz, 2H), 4.04 (m, J=6.5 Hz, 2H), 3.97 (q, J=5.6 Hz, 1H), 3.87 (m, J=4.2 Hz, 2H), 2.19 (m, J=7.3 Hz, 2H), 1.61 (s, 3H), 1.42 (d, J=3.9 Hz, 2H), 1.21 (t, J=20.3 Hz, 40H), 0.84 (m, J=4.1 Hz, 9H).

Note: (S) Stereochemistry of the amino acid side chain was based on the chirality defined by starting material used for the synthesis of 1-4.

(S) Stereochemistry of the phosphorous centre was defined based on the literature report No loss of chiral purity is assumed although not verified (or) proven.

Preparative SFC Conditions

Column/dimensions: Chiralpak AD-H(30×2 50 mm), 5μCO2:75.0%Co solvent: 25.0% (IPA)Total Flow: 60.0 g/minBack Pressure: 100.0 barUV: 214 nmStack time: 10 minLoad/Inj.: 3.5 mg

Example 23

Step a) ((Z)-2-((2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methylene)-1-((((S)-(((S)-1-(2-ethylbutoxy)-1-oxo-3-phenylpropan-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl)cyclopropyl)methyl (tert-butoxycarbonyl)-L-valinate (23a)

tert-Butylmagnesium chloride (1M in THF, 0.7 mL, 0.7 mmol) was added dropwise over a period of 15 min at rt to a solution of compound I-32 (65 mg, 0.14 mmol) in DMF (10 mL). The reaction mixture was stirred at rt for 40 min, then compound I-30c (97 mg, 0.17 mmol) in dry THE (5 mL) was added dropwise over a period of 15 min. The reaction mixture was stirred at rt for 16 h, then concentrated under reduced pressure and the afforded crude compound was purified by column chromatography on silica gel eluted with 5% MeOH in DCM, which gave the title compound (65 mg) as a solid. LCMS (ES+) m/z 850.85 [M+H]+. The crude product was used in the next step without further purification.

Step b) ((Z)-2-((2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methylene)-1-((((S)-(((S)-1-(2-ethylbutoxy)-1-oxo-3-phenylpropan-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl)cyclopropyl)methyl L-valinate (23b)

60% AcOH (13 mL, 129 mmol) was added to compound 23a (130 mg, 0.15 mmol) and the resulting reaction mixture was stirred for 5 h at 90° C., then concentrated under reduced pressure. The obtained crude compound was purified by prep HPLC using Method D. The pure compound was further purified by chiral SFC, which gave the title compound (18 mg) as a solid. MS (ES+) 750.71 [M+H]+.

1H NMR (500 MHz, DMSO): δ 10.72 (s, 1H), 7.84 (s, 1H), 7.27 (t, J=7.9 Hz, 2H), 7.20 (t, J=2.4 Hz, 5H), 7.11 (m, J=5.8 Hz, 2H), 6.99 (d, J=8.6 Hz, 2H), 6.54 (s, 2H), 6.14 (q, J=7.7 Hz, 1H), 4.40 (d, J=11.6 Hz, 1H), 4.09 (q, J=5.6 Hz, 1H), 4.00 (t, J=8.3 Hz, 1H), 3.85 (q, J=5.5 Hz, 3H), 3.63 (q, J=5.4 Hz, 1H), 3.03 (d, J=5.5 Hz, 1H), 2.95 (t, J=3.2 Hz, 1H), 2.78 (q, J=7.5 Hz, 1H), 1.57 (m, J=7.6 Hz, 4H), 1.35 (q, J=6.2 Hz, 1H), 1.20 (m, J=5.4 Hz, 5H), 0.77 (m, J=3.5 Hz, 6H), 0.71 (q, J=10.0 Hz, 6H).

Preparative SFC Conditions:

Column/dimensions: Chiralcel OX-H (250×30) mm, 5μMobile Phase A: 0.2% DEA in n-hexaneMobile Phase B: EtOHFlow: 42.0 ml/min% Of Mobile phase A:Mobile phase B: 55:45Temperature: AmbientWave length: 230 nmStack time: 16 minNo of Injections: 5Load ability/inj.: 10.0 mg

Example 24

Step a) (S)-pentan-2-yl((S)-(((Z)-2-((2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methylene)-1-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)cyclopropyl)methoxy)(phenoxy)phosphoryl)-L-alaninate (24a)

tert-Butylmagnesium chloride (1M in THF, 2.9 mL, 2.9 mmol) was added dropwise over a period of 5 min at rt to a solution of compound I-19b (200 mg, 0.6 mmol) in DMF (30 mL). The reaction mixture was stirred at rt for 45 min, then compound I-9b (333 mg, 0.7 mmol) in dry THE (15 mL) was added dropwise over a period of 10 min. The reaction mixture was stirred at rt for 16 h, then concentrated under reduced pressure and the afforded crude compound was purified by column chromatography on silica gel eluted with 5% MeOH in DCM, which gave the title compound (200 mg) as a solid. LCMS (ES+) m/z 645.64 [M+H]+. The crude product was used in the next step without further purification.

Step b) (S)-pentan-2-yl((S)-(((Z)-2-((2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methylene)-1-(hydroxymethyl)cyclopropyl)methoxy)(phenoxy)phosphoryl)-L-alaninate (24b)

80% AcOH (88.8 mL, 1240.6 mmol) was added to compound 25a (200 mg, 0.3 mmol) and the resulting reaction mixture was stirred for 24 h at rt, then concentrated under reduced pressure. The obtained crude compound was purified by column chromatography on silica gel eluted with 12% MeOH in DCM, which gave the title compound (85 mg, 46%) as a solid. MS (ES−) 559.58 [M−H]−.

Step c) ((Z)-2-((2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methylene)-1-((((S)-(((S)-1-oxo-1-(((S)-pentan-2-yl)oxy)propan-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl)cyclopropyl)methyl stearate (24c)

DCC (141 mg, 0.7 mmol) and DMAP (6 mg, 0.05 mmol) were added at rt to a stirred solution of compound 24b (85 mg, 0.15 mmol) and stearic acid (130 mg, 0.5 mmol) in DMF (3 mL). The resulting reaction mixture was stirred at rt for 16 h, then concentrated under reduced pressure. The crude compound was purified by column chromatography on silica gel and eluted with 5% MeOH in DCM. The pure compound was further purified by chiral SFC, which gave the title compound (8 mg) as a solid. MS (ES+) 827.71 [M+H]+.

1H NMR (500 MHz, DMSO): δ 10.73 (s, 1H), 7.93 (s, 1H), 7.32 (q, J=5.3 Hz, 2H), 7.18 (m, J=7.2 Hz, 4H), 6.56 (s, 2H), 5.99 (m, J=7.2 Hz, 1H), 4.79 (m, J=6.4 Hz, 1H), 4.36 (m, J=6.6 Hz, 2H), 4.04 (q, J=5.8 Hz, 2H), 3.81 (m, J=3.4 Hz, 1H), 2.19 (m, J=7.4 Hz, 2H), 1.62 (s, 2H), 1.44 (m, J=4.5 Hz, 4H), 1.18 (m, J=13.4 Hz, 37H), 0.84 (m, J=4.7 Hz, 6H).

Preparative SFC Conditions:

Column/dimensions: Lux Cellulose-2 (250×30) mm, 5μCO2: 55.0%Co solvent: 45.0% (MeOH)Total Flow 70.0 g/minBack Pressure 90.0 barUV 227 nmStack time 13.1 minLoad/inj. 7.1 mg

Step a) 2-ethylbutyl((S)-(((Z)-2-((4-amino-2-oxopyrimidin-1 (2H)-yl) methylene)-1-((isobutyryloxy)methyl)cyclopropyl)methoxy)(phenoxy)phosphoryl)-L-phenylalaninate (25)

tert-Butylmagnesium chloride (1M in THF, 1.4 mL, 1.4 mmol) was added dropwise over a period of 10 min at rt to a solution of compound I-28k (80 mg, 0.27 mmol) in DMF (10 mL). The reaction mixture was stirred at rt for 30 min, then compound I-30c (187 mg, 0.33 mmol) in dry THE (5 mL) was added dropwise over a period of 10 min. The reaction mixture was stirred at rt for 4 h, then concentrated under reduced pressure and the afforded crude compound was purified by column chromatography on silica gel eluted with 6% MeOH in DCM. The obtained compound was combined with another batch and further purified by prep HPLC using Method D. The pure compound was further purified by chiral SFC (twice), which gave the title compound (peak-2) (26 mg, 14%) as a solid. MS (ES+) 681.66 [M+H]+.

1H NMR (500 MHz, DMSO): δ 7.67 (d, J=7.5 Hz, 1H), 7.42 (q, J=5.1 Hz, 2H), 7.36 (s, 1H), 7.27 (m, J=6.1 Hz, 4H), 7.17 (m, J=7.0 Hz, 4H), 7.00 (d, J=8.6 Hz, 2H), 6.15 (q, J=7.8 Hz, 1H), 5.79 (d, J=7.4 Hz, 1H), 4.20 (d, J=11.5 Hz, 1H), 3.95 (m, J=4.1 Hz, 1H), 3.89 (q, J=5.6 Hz, 1H), 3.79 (m, J=5.1 Hz, 4H), 2.96 (m, J=3.3 Hz, 1H), 2.77 (q, J=7.5 Hz, 1H), 2.46 (m, J=7.0 Hz, 1H), 1.36 (m, J=5.3 Hz, 4H), 1.21 (m, J=3.7 Hz, 4H), 1.03 (q, J=7.5 Hz, 6H), 0.77 (q, J=4.8 Hz, 6H).

Preparative SFC Conditions-1

Column/dimensions Chiralpak AS-H (30×250 mm), 5μCO270.0%Co solvent 30.0% (EtOH)Total Flow 60.0 g/minBack Pressure 90.0 barUV 214 nmStack time 8.8 minLoad/Inj. 10.9 mg
Preparative SFC Conditions-2:Column/dimensions: Lux Cellulose-2 (250×30) mm, 5μCO2:75.0%Co solvent: 25.0% (MeOH)Total Flow: 70.0 g/minBack Pressure: 90.0 barUV: 214 nmStack time: 5.0 minLoad/inj.: 1.5 mg

Example 26

Step a) ((S,Z)-2-((2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methylene)-1-(hydroxymethyl)cyclopropyl)methyl tetrahydrogen triphosphate (26a)

POCl3(0.5 mL, 0.53 mmol) was added to a suspension of I-13R (100 mg, 0.27 mmol) in triethyl phosphate (0.07 mL, 3.8 mmol) at 0° C. and stirred at 0° C. for 4 h. To the above reaction mixture, tributylamine (0.07 mL, 0.3 mmol) was added followed by addition of a solution of tributyl ammonium pyrophosphate (731 mg, 1.33 mmol) in DMF (2 mL) and stirred at 0° C. for 30 min and 1 h at rt. Ammonium hydroxide solution (10 mL, 65 mmol) was added and continued stirring for 16 h at rt. The reaction mixture was concentrated under reduced pressure, then lyophilised. The crude compound was purified by prep HPLC using method H. The residue contained di and tri phosphate compounds and was purified again by prep HPLC using method I. The impure compound was further purified by prep HPLC using method I. The obtained impure title compound was further purified by prep HPLC using method I, which gave the title compound (25 mg) as a solid. MS (ES+) 504.31 [M+H]+.

Step b) lithium(S,Z)-(2-((2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methylene)-1-(hydroxymethyl)cyclopropyl)methyl triphosphate (26b)

Dowex 50 WX8 hydrogen form, ion exchange resin was taken in a column (2×10 cm), washed with water:MeOH (1:1, 100 mL) until colorless eluent was obtained, then washed with Milli Q water (100 mL) to wash off MeOH. The ion exchange resin was again eluted with 0.5M sulphuric acid (50 mL) until acidic pH was attained and washed with water (200 mL) until neutral pH was observed. The ion exchange resin was again eluted with 1M lithium hydroxide (50 mL) until basic pH was attained and washed with water (200 mL) until neutral pH. A solution of compound 26a (25 mg, 0.05 mmol) in Milli Q water (3 mL) was passed through the above freshly prepared Dowex LI+column. The appropriate fractions were lypholyised, which gave the title compound (15 mg) as a solid. LCMS (ES+) m/z 504.27 [M+H]+.

1H NMR (500 MHz, D2O): δ 8.23 (s, 1H), 7.18 (s, 1H), 4.14 (q, J=5.3 Hz, 1H), 4.02 (q, J=5.3 Hz, 1H), 3.70 (d, J=4.2 Hz, 2H), 3.27 (d, J=1.0 Hz, 1H), 1.58 (d, J=9.4 Hz, 1H), 1.49 (d, J=9.3 Hz, 1H).

Example 27

Step a) ((S,Z)-2-((2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methylene)-1-(hydroxymethyl)cyclopropyl)methyl trihydrogen diphosphate (27a)

POCl3(0.5 mL, 0.53 mmol) was added to a suspension of I-13R (100 mg, 0.27 mmol) in triethyl phosphate (0.07 mL, 3.8 mmol) at 0° C. and stirred at 0° C. for 4 h. To the above reaction mixture, tributylamine (0.07 mL, 0.3 mmol) was added followed by addition of a solution of tributyl ammonium pyrophosphate (731 mg, 1.33 mmol) in DMF (2 mL) and stirred at 0° C. for 30 min and 1 h at rt. Ammonium hydroxide solution (10 mL, 65 mmol) was added and continued stirring for 16 h at rt. The reaction mixture was concentrated under reduced pressure, then lyophilised. The crude compound was purified by prep HPLC using method H. The residue contained di and tri phosphate compounds and was purified again by prep HPLC using method I. The impure compound was further purified by prep HPLC using method I. The obtained impure title compound was further purified by prep HPLC using method I, which gave the title compound (15 mg) as a solid. MS (ES+) 424.30 [M+H]+.

Step b) lithium(S,Z)-(2-((2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methylene)-1-(hydroxymethyl)cyclopropyl)methyl diphosphate (27b)

Dowex 50 WX8 hydrogen form (50-100 mesh), ion exchange resin was taken in a column (2×10 cm), washed with water:MeOH (1:1, 100 mL) until colorless eluent was obtained, then washed with Milli Q water (100 mL) to wash off MeOH. The ion exchange resin was again eluted with 0.5M sulphuric acid (50 mL) until acidic pH was attained and washed with water (200 mL) until neutral pH was observed. The ion exchange resin was again eluted with 1M lithium hydroxide (50 mL) until basic pH was attained and washed with water (200 mL) until neutral pH. A solution of compound 27a (15 mg, 0.04 mmol) in Milli Q water (3 mL) was passed through the above freshly prepared Dowex Li+column. The appropriate fractions were lypholyised, which gave the title compound (13 mg) as a solid. LCMS (ES+) m/z 424.34 [M+H]+.1H NMR (500 MHz, D2O): δ 8.36 (s, 1H), 7.26 (s, 1H), 4.21 (q, J=5.2 Hz, 1H), 4.05 (q, J=5.3 Hz, 1H), 3.83 (d, J=12.2 Hz, 1H), 3.69 (m, J=9.4 Hz, 2H), 1.64 (q, J=3.5 Hz, 1H), 1.57 (d, J=9.0 Hz, 1H).

Example 28

Step a) ((Z)-2-((4-amino-2-oxopyrimidin-1(2H)-yl)methylene)-1-((((S)-(((S)-1-oxo-1-(((S)-pentan-2-yl)oxy)propan-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl)cyclopropyl)methyl isobutyrate (28)

tert-Butylmagnesium chloride (1M in THF, 3.4 mL, 3.4 mmol) was added dropwise over a period of 10 min at rt to a solution of compound I-28k (200 mg, 0.7 mmol) in DMF (15 mL). The reaction mixture was stirred at rt for 30 min, then compound I-9b (394 mg, 0.82 mmol) in dry THE (10 mL) was added dropwise over a period of 10 min. The reaction mixture was stirred at rt for 4 h, then concentrated under reduced pressure and the afforded crude compound was purified by column chromatography on silica gel eluted with 4% MeOH in DCM. The obtained compound was further purified by prep HPLC using Method G. The pure compound was further purified by chiral SFC, which gave the title compound (peak-2) (36 mg, 9%) as a solid. MS (ES+) 591.60 [M+H]+.

1H NMR (500 MHz, DMSO): δ 7.73 (d, J=7.5 Hz, 1H), 7.43 (s, 1H), 7.35 (q, J=5.3 Hz, 1H), 7.16 (q, J=4.7 Hz, 1H), 6.00 (q, J=7.7 Hz, 1H), 5.80 (d, J=7.5 Hz, 1H), 4.80 (m, J=4.8 Hz, 1H), 4.38 (d, J=11.5 Hz, 1H), 4.23 (q, J=5.7 Hz, 1H), 3.99 (q, J=5.3 Hz, 1H), 3.88 (d, J=11.5 Hz, 1H), 3.79 (m, J=3.5 Hz, 1H), 2.47 (d, J=7.0 Hz, 1H), 1.47 (t, J=6.6 Hz, 1H), 1.23 (t, J=6.0 Hz, 1H), 1.13 (d, J=6.3 Hz, 1H), 1.04 (q, J=7.9 Hz, 1H), 0.84 (t, J=7.4 Hz, 1H).

Preparative SFC Conditions

Column/dimensions: Chiralpak AD-H (30×250 mm), 5μCO2:80.0%Co solvent: 20.0% (IPA)Total Flow: 70.0 g/minBack Pressure: 90.0 barUV: 214 nmStack time: 9.7 minLoad/Inj.: 7.0 mg

Example 29

Step a) ((Z)-2-((4-amino-2-oxopyrimidin-1(2H)-yl)methylene)-1-((((S)-(((S)-1-oxo-1-(((S)-pentan-2-yl)oxy)propan-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl)cyclopropyl)methyl butyrate (29)

tert-Butylmagnesium chloride (1M in THF, 4.3 mL, 4.3 mmol) was added dropwise over a period of 10 min at rt to a solution of compound I-33a (250 mg, 0.9 mmol) in DMF (20 mL). The reaction mixture was stirred at rt for 30 min, then compound I-9b (492 mg, 1.02 mmol) in dry THF (10 mL) was added dropwise over a period of 10 min. The reaction mixture was stirred at rt for 4 h, then concentrated under reduced pressure and the afforded crude compound was purified by column chromatography on silica gel eluted with 4% MeOH in DCM. The obtained compound was further purified by prep HPLC using Method G. The pure compound was further purified by chiral SFC, which gave the title compound (peak-1) (45 mg, 9%) as a solid. MS (ES+) 591.57 [M+H]+.

1H NMR (500 MHz, DMSO): δ 7.73 (d, J=7.5 Hz, 1H), 7.53 (s, 1H), 7.41 (t, J=1.8 Hz, 1H), 7.36 (m, J=4.6 Hz, 3H), 7.17 (d, J=7.4 Hz, 3H), 6.01 (q, J=7.7 Hz, 1H), 5.82 (d, J=7.4 Hz, 1H), 4.79 (m, J=4.2 Hz, 1H), 4.35 (d, J=11.5 Hz, 1H), 4.23 (q, J=5.7 Hz, 1H), 3.99 (q, J=5.4 Hz, 1H), 3.91 (d, J=11.5 Hz, 1H), 3.80 (m, J=3.5 Hz, 1H), 2.22 (m, J=5.7 Hz, 2H), 1.48 (m, J=3.7 Hz, 6H), 1.22 (t, J=3.6 Hz, 6H), 1.13 (d, J=6.3 Hz, 3H), 0.84 (m, J=3.1 Hz, 6H).

Preparative SFC Conditions

Column/dimensions: Chiralpak AD-H (30×250 mm), 5μCO2:90.0%Co solvent: 10.0% (MeOH)Total Flow: 70.0 g/minBack Pressure: 90.0 barUV: 214 nmStack time: 10.5 minLoad/Inj.: 6.9 mg

Example 30

Step a) ((Z)-2-((2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methylene)-1-((((S)-(((S)-1-oxo-1-(((S)-pentan-2-yl)ox)propan-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl)cyclopropyl)methyl butyrate (30)

tert-Butylmagnesium chloride (1M in THF, 3.8 mL, 3.8 mmol) was added dropwise over a period of 10 min at rt to a solution of compound I-27 (250 mg, 0.8 mmol) in DMF (20 mL). The reaction mixture was stirred at rt for 40 min, then compound I-9b (433 mg, 0.9 mmol) in dry THE (10 mL) was added dropwise over a period of 15 min. The reaction mixture was stirred at rt for 16 h, then concentrated under reduced pressure and the afforded crude compound was purified by column chromatography on silica gel eluted with 5% MeOH in DCM. The obtained compound was further purified by prep HPLC using Method G. The pure compound was further purified by chiral SFC, which gave the title compound (peak-1)(28 mg) as a solid. MS (ES+) 631.62 [M+H]+.1H NMR (500 MHz, DMSO): δ 10.71 (s, 1H), 7.93 (s, 1H), 7.33 (m, J=3.2 Hz, 2H), 7.22 (t, J=1.9 Hz, 1H), 7.16 (d, J=7.9 Hz, 3H), 6.55 (s, 2H), 6.02 (q, J=7.7 Hz, 1H), 4.79 (m, J=3.2 Hz, 1H), 4.37 (m, J=7.5 Hz, 2H), 4.03 (q, J=5.4 Hz, 2H), 3.84 (s, 1H), 2.18 (m, J=6.1 Hz, 2H), 1.62 (d, J=1.2 Hz, 2H), 1.45 (m, J=3.0 Hz, 4H), 1.22 (t, J=7.0 Hz, 5H), 1.13 (d, J=6.3 Hz, 3H), 0.81 (m, J=7.6 Hz, 6H).

Preparative SFC Conditions:

Column/dimensions: Chiralcel-OX-H (250×30) mm, 5μCO2: 65.0%Co solvent: 35.0% (EtOH)Total Flow: 70.0 g/minBack Pressure: 90.0 barUV: 214 nmStack time: 9.2 minLoad/inj.: 6.3 mg

Example 31

Step a) (S,Z)-(2-((2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methylene)-1-((phosphonooxy)methyl)cyclopropyl)methyl isobutyrate (31a)

POCl3(0.1 mL, 0.84 mmol) was added to a suspension of 1-14 (140 mg, 0.42 mmol) in triethyl phosphate (1.6 mL, 9.3 mmol) at 0° C. and stirred at 0° C. for 2 h. The excess POCl3was quenched by adding triethyl ammonium bicarbonate buffer (1 M, pH=8) (8 mL) at 0° C. The reaction mixture was concentrated under reduced pressure. The crude compound was purified by prep HPLC using method A, which gave the title compound (65 mg, 37%) as a solid. MS (ES+) 414.44 [M+H]+.

Step b) lithium (S,Z)-(2-((2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methylene)-1-((isobutyryloxy)methyl)cyclopropyl)methyl phosphate (31b)

Dowex 50 WX8 hydrogen form (50-100 mesh), ion exchange resin was taken in a column (2×10 cm), washed with water:MeOH (1:1, 100 mL) until colorless eluent was obtained, then washed with Milli Q water (100 mL) to wash off MeOH. The ion exchange resin was again eluted with 0.5M sulphuric acid (50 mL) until acidic pH was attained and washed with water (200 mL) until neutral pH was observed. The ion exchange resin was again eluted with 1M lithium hydroxide (50 mL) until basic pH was attained and washed with water (200 mL) until neutral pH. A solution of compound 31a (55 mg, 0.13 mmol) in Milli Q water (10 mL) was passed through the above freshly prepared Dowex Li+column. The appropriate fractions were lypholyised, which gave the title compound (55 mg, 93%) as a solid. LCMS (ES+) m/z 414.37 [M+H]+.

1H NMR (500 MHz, D2O): δ 8.07 (s, 1H), 7.22 (s, 1H), 4.63 (d, J=11.5 Hz, 1H), 4.20 (q, J=5.4 Hz, 1H), 3.92 (m, J=6.4 Hz, 2H), 2.31 (m, J=4.7 Hz, 1H), 1.80 (d, J=9.6 Hz, 1H), 1.67 (d, J=9.6 Hz, 1H), 0.97 (q, J=2.6 Hz, 3H), 0.88 (q, J=2.6 Hz, 3H).

Example 32

Step a) ((Z)-2-((4-amino-2-oxopyrimidin-1(2H)-yl)methylene)-1-((((S)-(((S)-1-oxo-1-((2-propylpentyl)oxy)propan-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl)cyclopropyl)methyl butyrate (32)

tert-Butylmagnesium chloride (1M in THF, 1.7 mL, 1.7 mmol) was added dropwise over a period of 10 min at rt to a solution of compound I-33a (100 mg, 0.34 mmol) in DMF (20 mL). The reaction mixture was stirred at rt for 30 min, then compound I-4 (214 mg, 0.4 mmol) in dry THE (10 mL) was added dropwise over a period of 10 min. The reaction mixture was stirred at rt for 4 h, then concentrated under reduced pressure and the afforded crude compound was combined with another batch and purified by column chromatography on silica gel eluted with 5% MeOH in DCM. The obtained compound was further purified by prep HPLC using Method G. The pure compound was further purified by chiral SFC, which gave the title compound (peak-1) (20 mg) as a solid. MS (ES+) 633.72 [M+H]+.

1H NMR (500 MHz, DMSO): δ 7.72 (d, J=7.5 Hz, 1H), 7.42 (s, 2H), 7.35 (q, J=5.3 Hz, 3H), 7.17 (t, J=6.8 Hz, 3H), 6.04 (q, J=7.7 Hz, 1H), 5.81 (d, J=7.4 Hz, 1H), 4.34 (d, J=11.5 Hz, 1H), 4.21 (q, J=5.6 Hz, 1H), 3.98 (m, J=3.6 Hz, 2H), 3.87 (m, J=8.1 Hz, 3H), 2.21 (m, J=7.9 Hz, 2H), 1.60 (s, 1H), 1.49 (m, J=5.0 Hz, 4H), 1.25 (q, J=5.7 Hz, 11H), 0.84 (m, J=3.0 Hz, 9H).

Preparative SFC Conditions:

Column/dimensions: Chiralpak IC (30×250 mm), 5μCO2: 50.0%Co solvent: 50.0% (isopropanol)Total Flow: 70.0 g/minBack Pressure: 100.0 barUV: 214 nmStack time: 9.3 minLoad/inj.: 4.95 mg

Example 33

Step a) 2-ethylbutyl((S)-(((Z)-2-((2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methylene)-1-((((tert-butoxycarbonyl)-L-valyl)oxy)methyl)cyclopropyl)methoxy)(phenoxy)phosphoryl)-L- leucinate (33a)

tert-Butylmagnesium chloride (1M in THF, 1 mL, 1 mmol) was added dropwise over a period of 15 min at rt to a solution of compound I-32 (90 mg, 0.2 mmol) in DMF (20 mL). The reaction mixture was stirred at rt for 40 min, then compound I-4 (126 mg, 0.23 mmol) in dry THE (10 mL) was added dropwise over a period of 15 min. The reaction mixture was stirred at rt for 16 h, then concentrated under reduced pressure and the afforded crude compound was purified by column chromatography on silica gel eluted with 5% MeOH in DCM, which gave the title compound (70 mg, 37%) as a solid. MS (ES+) 816.89 [M+H]+.

Step b) 2-ethylbutyl((S)-(((Z)-1-(((L-valyl)oxy)methyl)-2-((2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methylene)cyclopropyl)methoxy)(phenoxy)phosphoryl)-L-leucinate (33b)

60% AcOH (9 mL, 92.7 mmol) was added to compound 33a (90 mg, 0.11 mmol) and the resulting reaction mixture was stirred for 5 h at 90° C., then concentrated under reduced pressure. The obtained compound was further purified by prep HPLC using Method G. The pure compound was further purified by chiral SFC, which gave the title compound (peak-1)(10 mg, 12%) as a solid. MS (ES+) 716.51 [M+H]+.

1H NMR (500 MHz, DMSO): δ 10.69 (s, 1H), 7.91 (s, 1H), 7.33 (t, J=7.9 Hz, 2H), 7.23 (t, J=1.8 Hz, 1H), 7.15 (m, J=4.6 Hz, 3H), 6.55 (s, 2H), 6.05 (q, J=7.8 Hz, 1H), 4.42 (m, J=7.9 Hz, 2H), 4.06 (q, J=5.5 Hz, 1H), 3.94 (m, J=5.4 Hz, 3H), 3.75 (m, J=8.8 Hz, 1H), 1.62 (m, J=6.2 Hz, 5H), 1.43 (q, J=5.9 Hz, 4H), 1.27 (m, J=7.3 Hz, 6H), 0.77 (m, J=9.4 Hz, 20H).

Preparative SFC Conditions:

Column/dimensions: Chiralpak IG (30×250 mm), 5μCO2: 70.0%Co solvent: 30.0% (0.5% DEA in EtOH)Total Flow: 70.0 g/minBack Pressure: 100.0 barUV: 214 nmStack time: 15 minLoad/inj.: 3.5 mg

Example 34

Step a) 2-ethylbutyl((S)-(((Z)-2-((4-amino-2-oxopyrimidin-1(2H)-yl)methylene)-1-((isobutyryloxy)methyl)cyclopropyl)methoxy)(phenoxy)phosphoryl)-L-leucinate (34)

tert-Butylmagnesium chloride (1M in THF, 3.4 mL, 3.4 mmol) was added dropwise over a period of 10 min at rt to a solution of compound I-28k (200 mg, 0.7 mmol) in DMF (15 mL). The reaction mixture was stirred at rt for 30 min, then compound I-4 (403 mg, 0.8 mmol) in dry THF (8 mL) was added dropwise over a period of 10 min. The reaction mixture was stirred at rt for 4 h, then concentrated under reduced pressure and the afforded crude compound was purified by column chromatography on silica gel eluted with 8% MeOH in DCM. The obtained compound was further purified by prep HPLC using Method G. The pure compound was further purified by chiral SFC, which gave the title compound (peak-2)(56 mg, 12%) as a solid. MS (ES+) 647.69 [M+H]+.

1H NMR (500 MHz, DMSO): δ 7.72 (d, J=7.4 Hz, 1H), 7.43 (t, J=1.7 Hz, 2H), 7.34 (m, J=4.0 Hz, 3H), 7.15 (m, J=4.1 Hz, 3H), 6.03 (q, J=7.8 Hz, 1H), 5.80 (d, J=7.4 Hz, 1H), 4.36 (d, J=11.5 Hz, 1H), 4.23 (q, J=5.6 Hz, 1H), 3.92 (m, J=5.7 Hz, 4H), 3.73 (m, J=5.0 Hz, 1H), 2.46 (t, J=7.0 Hz, 1H), 1.59 (m, J=6.7 Hz, 1H), 1.44 (m, J=3.8 Hz, 5H), 1.28 (m, J=3.3 Hz, 4H), 1.04 (q, J=8.0 Hz, 6H), 0.81 (m, J=4.7 Hz, 12H).

Preparative SFC Conditions:

Column/dimensions: Chiralpak-IA (250×30) mm, 5μMobile Phase A: n-hexaneMobile Phase B: isopropanolFlow: 42.0 ml/min% Of Mobile phase A: Mobile phase B: 50:50Temperature: AmbientWave length: 298 nmStack time: 10 minNo of Injections: 6Load ability/inj.: 24.16 mg

Example 35

Step a) bis(2-ethylbutyl) ((S)-(((Z)-2-((2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methylene)-1-((isobutyryloxy)methyl)cyclopropyl)methoxy)(phenoxy)phosphoryl)-L-aspartate (35)

tert-Butylmagnesium chloride (1M in THF, 1.9 mL, 1.9 mmol) was added dropwise over a period of 5 min at rt to a solution of compound I-11 (125 mg, 0.4 mmol) in DMF (8 mL). The reaction mixture was stirred at rt for 20 min, then compound I-34c (257 mg, 0.4 mmol) in dry THF (4 mL) was added dropwise over a period of 5 min. The reaction mixture was stirred at rt for 3 h, then concentrated under reduced pressure and the afforded crude compound was combined with another batch and purified by column chromatography on silica gel eluted with 5% MeOH in DCM. The obtained compound was further purified by prep HPLC using Method H. The pure compound was further purified by chiral SFC, which gave the title compound (peak-1) (51 mg) as a solid. MS (ES+) 773.80 [M+H]+.

1H NMR (500 MHz, DMSO): δ 10.72 (s, 1H), 7.93 (s, 1H), 7.33 (q, J=5.3 Hz, 2H), 7.22 (t, J=1.8 Hz, 1H), 7.15 (m, J=4.3 Hz, 3H), 6.56 (s, 2H), 6.11 (q, J=7.6 Hz, 1H), 4.40 (m, J=7.7 Hz, 2H), 4.16 (m, J=3.5 Hz, 1H), 3.94 (m, J=4.1 Hz, 6H), 2.75 (q, J=8.0 Hz, 1H), 2.62 (m, J=5.6 Hz, 1H), 2.45 (m, J=7.0 Hz, 1H), 1.61 (d, J=1.6 Hz, 2H), 1.42 (m, J=4.2 Hz, 2H), 1.25 (m, J=3.2 Hz, 8H), 1.01 (q, J=11.4 Hz, 6H), 0.80 (q, J=7.0 Hz, 12H).

Preparative SFC Conditions:

Column/dimensions: Chiralcel OX-H (250×30) mm, 5μCO2: 65.0%Co solvent: 35.0% (EtOH)Total Flow: 70 g/minBack Pressure: 90.0 barUV: 214 nmStack time: 13.7 minLoad/Inj.: 5.6 mg

Example 36

Step a) isopropyl((S)-(((Z)-2-((2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methylene)-1-((isobutyryloxy)methyl)cyclopropyl)methoxy)(phenoxy)phosphoryl)-L-leucinate (36)

tert-Butylmagnesium chloride (1M in THF, 4.5 mL, 4.5 mmol) was added dropwise over a period of 5 min at rt to a solution of compound I-11 (300 mg, 0.9 mmol) in DMF (18 mL). The reaction mixture was stirred at rt for 20 min, then compound I-2 (490 mg, 1.0 mmol) in dry THF (9 mL) was added dropwise over a period of 5 min. The reaction mixture was stirred at rt for 3 h, then concentrated under reduced pressure and the afforded crude compound was combined with another batch and purified by column chromatography on silica gel eluted with 7% MeOH in DCM. The obtained compound was further purified by prep HPLC using Method D. The pure compound was further purified by chiral SFC, which gave the title compound (Peak-2) (62 mg) as a solid. MS (ES+) 645.64 [M+H]+.

1H NMR (500 MHz, DMSO): δ 10.70 (s, 1H), 7.92 (s, 1H), 7.32 (t, J=7.9 Hz, 2H), 7.22 (t, J=1.8 Hz, 1H), 7.14 (t, J=7.8 Hz, 3H), 6.55 (s, 2H), 5.99 (q, J=7.8 Hz, 1H), 4.84 (m, J=6.3 Hz, 1H), 4.39 (m, J=7.5 Hz, 2H), 4.01 (m, J=7.1 Hz, 2H), 3.69 (m, J=5.0 Hz, 1H), 2.44 (m, J=7.0 Hz, 1H), 1.61 (d, J=1.3 Hz, 3H), 1.41 (m, J=4.3 Hz, 2H), 1.14 (t, J=6.2 Hz, 6H), 1.01 (q, J=11.1 Hz, 6H), 0.81 (q, J=10.0 Hz, 6H).

Preparative SFC Conditions

Column/dimensions: Chiralpak AD-H (30×250 mm), 5μCO2:70.0%Co solvent: 30.0% (EtOH)Total Flow: 70.0 g/minBack Pressure: 90.0 barUV: 214 nmStack time: 11.8 minLoad/inj.: 12 mg

Example 37

Step a) (S,Z)-(2-((4-amino-2-oxopyrimidin-1(2H)-yl)methylene)-1-((phosphonooxy)methyl)cyclopropyl)methyl isobutyrate (37a)

POCl3(0.03 mL, 0.34 mmol) was added to a suspension of 1-36a (50 mg, 0.17 mmol) in triethyl phosphate (0.64 mL, 3.8 mmol) at 0° C. and stirred at 0° C. for 2 h. To the above reaction mixture, triethyl ammonium bicarbonate buffer (1 M, pH=8) (2 mL) was added at 0° C. and lyophilised. The crude compound was purified by prep HPLC using method C, which gave the title compound (15 mg, 23%) as a solid. MS (ES+) 374.27 [M+H]+.

Step b) Lithium(S,Z)-(2-((4-amino-2-oxopyrimidin-1(2H)-yl)methylene)-1-((isobutyryloxy)methyl)cyclopropyl)methyl phosphate (37b)

Dowex 50 WX8 hydrogen form (50-100 mesh), ion exchange resin was taken in a column (2×10 cm), washed with water:MeOH (1:1, 100 mL) until colorless eluent was obtained, then washed with Milli Q water (100 mL) to wash off MeOH. The ion exchange resin was again eluted with 0.5M sulphuric acid (25 mL) until acidic pH was attained and washed with water (100 mL) until neutral pH was observed. The ion exchange resin was again eluted with 1M lithium hydroxide (25 mL) until basic pH was attained and washed with water (100 mL) until neutral pH. A solution of compound 37a (15 mg, 0.04 mmol) in Milli Q water (1 mL) was passed through the above freshly prepared Dowex LI+column. The appropriate fractions were lypholyised, which gave the title compound (15 mg, 96%) as a solid. LCMS (ES+) m/z 374.42 [M+H]+.

1H NMR (500 MHz, D2O): δ 7.86 (q, J=2.8 Hz, 1H), 7.25 (s, 1H), 6.14 (d, J=7.5 Hz, 1H), 4.61 (d, J=11.5 Hz, 1H), 4.08 (q, J=5.3 Hz, 1H), 3.96 (q, J=5.0 Hz, 1H), 3.88 (d, J=11.5 Hz, 1H), 2.47 (m, J=5.6 Hz, 1H), 1.68 (q, J=3.8 Hz, 1H), 1.59 (d, J=9.4 Hz, 1H), 1.05 (m, J=4.2 Hz, 6H).

Example 38

Step a) ((Z)-2-((2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methylene)-1-((((S)-(((S)-1-oxo-1-((2-propylpentyl)oxy)propan-2-yl)amino)(phenoxy)phosphoryl)ox)methyl)cyclopropyl)methyl pivalate (38)

tert-Butylmagnesium chloride (1M in THF, 1.44 mL, 1.44 mmol) was added dropwise over a period of 2 min at rt to a solution of compound I-35 (100 mg, 0.3 mmol) in DMF (10 mL). The reaction mixture was stirred at rt for 30 min, then compound I-4 (181 mg, 0.35 mmol) in dry THE (5 mL) was added dropwise over a period of 5 min. The reaction mixture was stirred at rt for 16 h, then concentrated under reduced pressure and the afforded crude compound was combined with another batch and purified by column chromatography on silica gel eluted with 10% MeOH in DCM. The obtained compound was further purified by prep HPLC using Method A, which gave the title compound (15 mg) as a solid. MS (ES+) 687.75 [M+H]+.

1H NMR (500 MHz, DMSO): δ 10.68 (s, 1H), 7.93 (s, 1H), 7.33 (m, J=3.9 Hz, 2H), 7.18 (m, J=4.5 Hz, 4H), 6.54 (s, 2H), 6.06 (m, J=4.4 Hz, 1H), 4.29 (m, J=10.1 Hz, 3H), 3.99 (m, J=6.5 Hz, 2H), 3.86 (m, J=4.1 Hz, 1H), 3.77 (m, J=3.9 Hz, 1H), 1.57 (m, J=4.6 Hz, 3H), 1.19 (m, J=5.3 Hz, 12H), 1.08 (d, J=1.1 Hz, 9H), 0.82 (m, J=2.6 Hz, 6H).

Example 39

Step a) ((Z)-2-((4-amino-5-fluoro-2-oxopyrimidin-1(2H)-yl)methylene)-1-((((S)-(((S)-1-oxo-1-((2-propylpentyl)oxy)propan-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl)cyclopropyl)methyl isobutyrate (39)

tert-Butylmagnesium chloride (1M in THF, 4 mL, 4.0 mmol) was added dropwise over a period of 5 min at rt to a solution of compound I-37c (250 mg, 0.8 mmol) in DMF (12 mL). The reaction mixture was stirred at rt for 20 min, then compound I-4 (462 mg, 0.9 mmol) in dry THE (6 mL) was added dropwise over a period of 5 min. The reaction mixture was stirred at rt for 3 h, then concentrated under reduced pressure and the afforded crude compound was combined with another batch and purified by column chromatography on silica gel eluted with 4% MeOH in DCM. The obtained compound was further purified by prep HPLC using Method B. The pure compound was further purified by chiral SFC, which gave the title compound (Peak-2) (70 mg) as a solid. MS (ES+) 651.69 [M+H]+.

1H NMR (500 MHz, DMSO): δ 8.02 (s, 1H), 7.97 (d, J=6.8 Hz, 1H), 7.79 (s, 1H), 7.39 (d, J=2.0 Hz, 1H), 7.34 (t, J=8.0 Hz, 2H), 7.16 (q, J=3.9 Hz, 3H), 6.05 (q, J=7.8 Hz, 1H), 4.46 (d, J=11.6 Hz, 1H), 4.17 (q, J=5.5 Hz, 1H), 4.05 (q, J=5.2 Hz, 1H), 3.97 (q, J=5.5 Hz, 1H), 3.89 (q, J=5.5 Hz, 1H), 3.82 (m, J=5.0 Hz, 2H), 2.47 (d, J=7.0 Hz, 1H), 1.60 (t, J=5.9 Hz, 1H), 1.49 (d, J=1.6 Hz, 2H), 1.24 (m, J=5.6 Hz, 12H), 1.06 (q, J=8.8 Hz, 6H), 0.84 (q, J=4.4 Hz, 6H).

Preparative SFC Conditions

Column/dimensions (R,R) WHELK-01 (250×30)mm, 5μCO2: 75.0%Co solvent: 25.0% (EtOH)Total Flow: 100.0 g/minBack Pressure 100.0 barUV: 214 nmStack time: 8.5 minLoad/inj.: 22 mg

Example 40

Step a) ((Z)-2-((4-amino-5-fluoro-2-oxopyrimidin-1(2H)-yl)methylene)-1-((((S)-(((S)-1-oxo-1-((2-propylpentyl)oxy)propan-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl)cyclopropyl)methyl butyrate (40)

tert-Butylmagnesium chloride (1M in THF, 3.2 mL, 3.2 mmol) was added dropwise over a period of 10 min at rt to a solution of compound I-38 (200 mg, 0.64 mmol) in DMF (20 mL). The reaction mixture was stirred at rt for 30 min, then compound I-4 (403 mg, 0.8 mmol) in dry THE (10 mL) was added dropwise over a period of 10 min. The reaction mixture was stirred at rt for 4 h, then concentrated under reduced pressure and the afforded crude compound was combined with another batch and purified by column chromatography on silica gel eluted with 4% MeOH in DCM. The obtained compound was further purified by prep HPLC using Method A. The pure compound was further purified by chiral SFC, which gave the title compound (36 mg) as a solid. MS (ES+) 651.73 [M+H]+.

1H NMR (500 MHz, DMSO): δ 8.02 (d, J=7.0 Hz, 2H), 7.80 (s, 1H), 7.35 (m, J=4.1 Hz, 3H), 7.17 (t, J=4.3 Hz, 3H), 6.06 (q, J=7.8 Hz, 1H), 4.40 (d, J=11.6 Hz, 1H), 4.20 (q, J=5.5 Hz, 1H), 4.01 (q, J=5.5 Hz, 1H), 3.95 (q, J=5.6 Hz, 1H), 3.82 (m, J=5.2 Hz, 3H), 2.22 (q, J=7.1 Hz, 2H), 1.58 (t, J=6.0 Hz, 1H), 1.49 (m, J=5.1 Hz, 4H), 1.22 (m, J=4.5 Hz, 12H), 0.83 (m, J=3.7 Hz, 9H).

Preparative SFC Conditions

Column/dimensions Chiralpak AD-H (30×250 mm), 5μCO285.0%Co solvent 15.0% (100% IPA)Total Flow 70.0 g/minBack Pressure 90.0 barUV 214 nmStack time 6.0 minLoad/Inj. 8.9 mg.

Example 41

Step a) 2-ethylbutyl((S)-(((S,Z)-2-((4-amino-5-fluoro-2-oxopyrimidin-1 (2H)-yl)methylene)-1-((((tert-butoxycarbonyl)-L-valyl)oxy)methyl) cyclopropyl)methoxy)(phenoxy)phosphoryl)-L-leucinate (41a)

tert-Butylmagnesium chloride (1M in THF, 1.8 mL, 1.8 mmol) was added dropwise over a period of 5 min at rt to a solution of compound I-39e (150 mg, 0.34 mmol) in DMF (10 mL). The reaction mixture was stirred at rt for 20 min, then compound I-31c (201 mg, 0.4 mmol) in dry THE (5 mL) was added dropwise over a period of 5 min. The reaction mixture was stirred at rt for 3 h, then concentrated under reduced pressure and the afforded crude compound was combined with another batch and purified by column chromatography on silica gel eluted with 4% MeOH in DCM, which gave the title compound (170 mg) as a semi-solid. MS (ES+) 794.92[M+H]+.

Step b) 2-ethylbutyl((S)-(((S,Z)-1-(((L-valyl)oxy)methyl)-2-((4-amino-5-fluoro-2-oxopyrimidin-1(2H)-yl)methylene)cyclopropyl)methoxy)(phenoxy)phosphoryl)-L-leucinate (41b)

4M HCl in 1,4 dioxane (1 mL, 4 mmol) was added at 0° C. to a solution of compound 41a (150 mg, 0.2 mmol) in 1,4-dioxane (20 mL). The reaction mixture was stirred at room temperature for 1 h, then concentrated under reduced pressure. The obtained compound was purified twice by prep HPLC using Method A. The pure compound was further purified by chiral SFC and lyophilised, which gave the title compound (26 mg) as a solid. MS (ES+) 694.78 [M+H]+.

1H NMR (500 MHz, DMSO): δ 8.02 (s, 1H), 7.89 (d, J=6.7 Hz, 1H), 7.81 (s, 1H), 7.39 (d, J=2.0 Hz, 1H), 7.33 (m, J=4.0 Hz, 1H), 7.14 (q, J=5.8 Hz, 1H), 6.04 (q, J=7.8 Hz, 1H), 4.52 (d, J=11.6 Hz, 1H), 4.22 (q, J=5.6 Hz, 1H), 4.04 (q, J=5.3 Hz, 1H), 3.92 (m, J=5.1 Hz, 1H), 3.75 (m, J=7.6 Hz, 1H), 3.08 (d, J=5.2 Hz, 1H), 1.74 (q, J=6.4 Hz, 1H), 1.59 (m, J=6.7 Hz, 1H), 1.50 (s, 1H), 1.43 (q, J=6.1 Hz, 1H), 1.27 (m, J=3.9 Hz, 1H), 0.79 (m, J=5.1 Hz, 1H).

Preparative SFC Conditions:

Column/dimensions Chiralcel OX-H (250×30) mm, 5μCO2:80.0%Co solvent: 20.0% (MeOH)Total Flow: 70 g/minBack Pressure: 90.0 barUV: 214 nmStack time: 10.5 minLoad/Inj. 8.0 mg

Example 42

Step a) (S,Z)-(2-((4-amino-5-fluoro-2-oxopyrimidin-1(2H)-yl)methylene)-1-((phosphonooxy)methyl)cyclopropyl)methyl isobutyrate (42a)

Distilled POCl3(0.1 mL, 1.0 mmol) was added to a suspension of I-39a (150 mg, 0.5 mmol) in triethyl phosphate (1.9 mL, 11.2 mmol) at 0° C. and stirred at 0° C. for 3 h. To the above reaction mixture, triethyl ammonium bicarbonate buffer (1 M, pH=8) (7 mL) was added at 0° C. and concentrated under reduced pressure. The crude compound was purified twice by prep HPLC using method A and lyophilised, which gave the title compound (40 mg, 19%) as a solid. MS (ES+) 392.36 [M+H]+.

Step b) lithium (S,Z)-(2-((4-amino-5-fluoro-2-oxopyrimidin-1(2H)-yl)methylene)-1-((isobutyryloxy)methyl)cyclopropyl)methyl phosphate (42b)

Dowex 50 WX8 hydrogen form (50-100 mesh), ion exchange resin was taken in a column (2×10 cm), washed with water:MeOH (1:1, 100 mL) until colourless eluent was obtained, then washed with Milli Q water (100 mL) to wash off MeOH. The ion exchange resin was again eluted with 0.5M sulphuric acid (50 mL) until acidic pH was attained and washed with water (200 mL) until neutral pH was observed. The ion exchange resin was again eluted with 1M lithium hydroxide (50 mL) until basic pH was attained and washed with water (200 mL) until neutral pH. A solution of compound 41a (40 mg, 0.1 mmol) in Milli Q water (5 mL) was passed through the above freshly prepared Dowex LI+column. The appropriate fractions were lyophilised, which gave the title compound (40 mg, 92%) as a solid. LCMS (ES+) m/z 392.36 [M+H]+.

1H NMR (500 MHz, D2O): δ 8.14 (d, J=6.1 Hz, 1H), 7.26 (d, J=1.6 Hz, 1H), 4.67 (d, J=11.5 Hz, 1H), 4.04 (q, J=5.2 Hz, 1H), 3.96 (q, J=5.0 Hz, 1H), 3.92 (d, J=11.4 Hz, 1H), 2.51 (m, J=7.0 Hz, 1H), 1.66 (q, J=3.7 Hz, 1H), 1.56 (d, J=9.2 Hz, 1H), 1.08 (d, J=7.0 Hz, 3H), 1.03 (d, J=7.0 Hz, 3H).

Example 43

Step a) ((S,Z)-2-((4-amino-2-oxopyrimidin-1(2H)-yl)methylene)-1-(hydroxymethyl)cyclopropyl)methyl tetrahydrogen triphosphate (43a)

A solution of 2-chloro-4H-benzo[d][1,3,2]dioxaphosphinin-4-one (54 mg, 0.3 mmol) was added at rt to a solution of compound I-36a (75 mg, 0.3 mmol) in dry DMF (2 mL) and dry pyridine (0.6 mL) and stirred at rt for 30 min. A solution of tributylammonium pyrophosphate (130 mg, 0.23 mmol) and dry tributylamine (0.15 mL, 0.64 mmol) in dry DMF (1.2 mL) was added and stirred at RT for 45 min. A solution of iodine (84 mg, 0.33 mmol) in pyridine/water: 98:2 (5.25 mL/0.15 mL) was added and the reaction stirred for 15 min at rt. 5% NaHSO3(1.5 mL) was added and concentrated under reduced pressure. Ammonium hydroxide (1.5 mL) was added to the residue and stirred at rt for 16 h, then concentrated under reduced pressure. The obtained crude was combined with another batch and purified twice by prep HPLC using method F and lyophilised, which gave the title compound (23 mg, 18%) as a solid. MS (ES+) 464.32[M+H]+.

Step b) lithium (S,Z)-(2-((4-amino-2-oxopyrimidin-1(2H)-yl)methylene)-1-(hydroxymethyl)cyclopropyl)methyl triphosphate (43b)

Dowex 50 WX8 hydrogen form (50-100 mesh), ion exchange resin was taken in a column (2×10 cm), washed with water:MeOH (1:1, 50 mL) until colourless eluent was obtained, then washed with Milli Q water (50 mL) to wash off MeOH. The ion exchange resin was again eluted with 0.5M sulphuric acid (25 mL) until acidic pH was attained and washed with water (100 mL) until neutral pH was observed. The ion exchange resin was again eluted with 1M lithium hydroxide (25 mL) until basic pH was attained and washed with water (100 mL) until neutral pH. A solution of compound 43a (23 mg, 0.05 mmol) in Milli Q water (1 mL) was passed through the above freshly prepared Dowex LI+column. The appropriate fractions were lyophilised. The obtained residue was purified by prep HPLC using method F and lyophilised, which gave the title compound (7 mg, 23%) as a solid. LCMS (ES+) m/z 464.29 [M+H]+.

1H NMR (500 MHz, D2O): δ 8.11 (d, J=7.5 Hz, 1H), 7.33 (s, 1H), 6.16 (d, J=7.5 Hz, 1H), 4.32 (q, J=5.2 Hz, 1H), 3.94 (q, J=5.3 Hz, 1H), 3.82 (d, J=12.1 Hz, 1H), 3.61 (d, J=12.1 Hz, 1H), 1.51 (m, J=7.0 Hz, 2H).

Example 44-1 & 44-2

Step a) ((Z)-2-((2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methylene)-1-((((S)-(((S)-1-oxo-1-((2-propylpentyl)oxy)propan-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl)cyclopropyl)methyl pivalate (44-1 & 44-2)

Compound 38 (55 mg) was further purified by normal phase HPLC.

44-1:

Peak-1 was concentrated and lyophilised, which gave the title compound (13.6 mg, 35) as a solid.

MS (ES+) m/z 687.75 [M+H]+.

1H NMR (500 MHz, DMSO): δ 10.69 (s, 1H), 7.93 (s, 1H), 7.33 (m, J=4.0 Hz, 2H), 7.18 (m, J=4.8 Hz, 4H), 6.54 (s, 25H), 6.06 (q, J=7.8 Hz, 1H), 4.28 (d, J=11.5 Hz, 1H), 4.18 (m, J=6.1 Hz, 2H), 3.95 (q, J=5.6 Hz, 2H), 3.77 (m, J=3.9 Hz, 2H), 1.57 (m, J=4.3 Hz, 3H), 1.20 (m, J=4.4 Hz, 11H), 1.08 (s, 9H), 0.81 (t, J=7.0 Hz, 6H).

44-2:

Peak-2 was concentrated and lyophilised, which gave the title compound (11.6 mg, 3%) as a solid. MS (ES+) m/z 687.75 [M+H]+.

1H NMR (500 MHz, DMSO): δ 10.70 (s, 1H), 7.93 (s, 1H), 7.32 (t, J=8.0 Hz, 2H), 7.22 (t, J=1.8 Hz, 1H), 7.15 (m, J=3.2 Hz, 3H), 6.55 (s, 2H), 6.06 (q, J=7.7 Hz, 1H), 4.43 (d, J=11.6 Hz, 1H), 4.35 (q, J=5.7 Hz, 1H), 4.02 (q, J=5.4 Hz, 1H), 3.96 (m, J=5.7 Hz, 2H), 3.86 (m, J=4.1 Hz, 2H), 1.60 (s, 3H), 1.22 (m, J=5.0 Hz, 11H), 1.08 (s, 9H), 0.82 (m, J=3.1 Hz, 6H).

Normal Phase HPLC Conditions:

Column/dimensions: Chiralpak IA (30×250 mm), 5μMobile Phase: n-hexane:EtOH (30:70)Flow: 40.0 ml/minTemperature: AmbientWave length: 236 nmRun time: 13 minLoad ability/inj.: 9.2 mg

Example 45-1 & 45-2

Step a) ((Z)-2-((4-amino-2-oxopyrimidin-1(2H)-yl)methylene)-1-((((S)-(((S)-1-oxo-1-((2-propylpentyl)ox)propan-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl)cyclopropyl)methyl butyrate (45-1 & 45-2)

Compound 32 (1.5 g) was further purified by chiral SFC.

45-1:

The residue obtained from Peak-1 was further purified by prep HPLC using method A. The impure compound was further purified by chiral SFC and lyophilised, which gave the title compound (215 mg) as a solid. LCMS (ES+) m/z 633.72 [M+H]+

1H NMR (500 MHz, DMSO): δ 7.72 (d, J=7.5 Hz, 1H), 7.42 (t, J=1.8 Hz, 2H), 7.35 (m, J=4.0 Hz, 3H), 7.17 (m, J=3.5 Hz, 3H), 6.04 (q, J=7.7 Hz, 1H), 5.80 (d, J=7.4 Hz, 1H), 4.34 (d, J=11.5 Hz, 1H), 4.21 (q, J=5.6 Hz, 1H), 3.98 (m, J=3.6 Hz, 2H), 3.87 (m, J=8.1 Hz, 3H), 2.22 (m, J=6.7 Hz, 2H), 1.61 (q, J=5.8 Hz, 1H), 1.49 (m, J=4.4 Hz, 4H), 1.25 (m, J=4.3 Hz, 11H), 0.84 (m, J=2.2 Hz, 9H).

45-2:

The residue obtained from Peak-2 was further purified by prep HPLC using method A and lyophilised, which gave the title compound (280 mg) as a solid. LCMS (ES+) m/z 633.72 [M+H]+1H NMR (500 MHz, DMSO): δ 7.74 (d, J=7.4 Hz, 1H), 7.42 (d, J=1.8 Hz, 2H), 7.35 (m, J=3.2 Hz, 3H), 7.17 (t, J=7.8 Hz, 3H), 6.06 (q, J=7.8 Hz, 1H), 5.80 (d, J=7.4 Hz, 1H), 4.24 (d, J=11.5 Hz, 1H), 4.17 (q, J=5.5 Hz, 1H), 4.06 (q, J=5.5 Hz, 1H), 3.97 (m, J=3.8 Hz, 2H), 3.86 (q, J=5.5 Hz, 1H), 3.80 (m, J=3.4 Hz, 1H), 2.22 (q, J=7.1 Hz, 2H), 1.60 (q, J=6.0 Hz, 1H), 1.48 (m, J=6.1 Hz, 4H), 1.23 (m, J=5.1 Hz, 11H), 0.84 (m, J=3.3 Hz, 9H).
Preparative SFC Conditions:Column/dimensions: Chiralpak IC (30×250 mm), 5μCO2: 50.0%Co solvent: 50.0% (isopropanol)Total Flow: 110.0 g/minBack Pressure: 100.0 barUV: 214 nmStack time: 13 minLoad/inj.: 50 mg

Preparative SFC Conditions (example 45-1):Column/dimensions: Chiralpak IG (30×250 mm), 5μCO2:70.0%Co solvent: 30.0% (100% isopropanol)Total Flow: 100 mg/minBack Pressure: 100 barUV: 214 nmStack time: 12.2 minLoad/inj.: 24.5 mg

Comparative Example 1

Instability of Triester Phosphoroalaninates

Intermediate compounds I-19 d2 and I-25 synthesised above are triesterphosphoro-alaninates resembling the prior art compound described in Yan et al, J Med Chem 2005 48 91-99:

The structural integrity of these model compounds was assessed by LC MS over a 22-29 day period, as follows:

LCMS purity,LCMS purity,DayIntermediate I-19 d2Intermediate I-25096.86%95.56%594.32%88.14%1488.76%82.67%2273.76%77.76%29na72.84%

It is thus concluded that about a quarter of the respective triesterphosphoralinate has decomposed in a little over three weeks.

Biology Example 1

Compounds of the invention were evaluated for activity against the leukemia cell lines THP-1, EOL-1 and MV4-11, using the following assay:

Materials

Cells and Cell Culture:

THP-1 (human acute monocytic leukemia) from ATCC Cat. no TIB-202 was grown in complete cell medium: RPMI-1640 medium Gibco Cat. no. 11835-063 (Fisher Scientific), 10% Fetal Bovine serum (FBS), HyClone Cat. no. SV30160.03, lot no RAB35924 (GE Healthcare Life Sciences), Penicillin 50 u/ml/Streptomycin 0.05 mg/ml PAA Cat. no. P11-010 from Fisher Scientific.MV4-11 cells, human B-myelomonocytic leukemia, from ATCC Cat no. CRL-9591 was grown in complete cell medium: IMDM (w. GLUTAMAX-1) Cat no. 31980022 (Fisher Scientific), 10% Fetal Bovine serum (FBS) HyClone Cat. no. SV30160.03, lot no RAB35924 (GE Healthcare Life Sciences), Penicillin 50 u/ml/Streptomycin 0.05 mg/ml PAA Cat. no. P11-010 from Fisher Scientific.EOL-1 (human acute monocytic leukemia) from DSMZ Cat. no ACC 386 was grown in complete cell medium: RPMI-1640 medium Gibco Cat. no. 11835-063 (Fisher Scientific), 10% Fetal Bovine serum (FBS), HyClone Cat. no. A15102, lot no 10211-2117 (GE Healthcare Life Sciences), Penicillin 50 u/ml/Streptomycin 0.05 mg/ml PAA Cat. no. P11-010 from Fisher Scientific.Cell culture flask 75 cm2, Cat. no. 83.1813 from Sarstedt AB.Compound dilution plate, 96-well, V-bottom PP plate, Nunc Cat. no. 249944 from Thermo Scientific.Cell assay plate, 96-well, Cat. no. 128009296 from Fisher ScientificCell Counting Kit-8 CK04 from Dojindo.

Test compounds were made up to 10 mM stock solution in DMSO

Method

Leukemia cells (THP-1, EOL-1 and MV4-11) were grown in a cell culture flask 75 cm2with approximately 100 ml complete cell medium. The cells were counted using a Scepter-hand held automated cell counter, using 60 μm sensors (Millipore) and suspended in complete cell medium to 2×105cells. 100 μl of the cell suspension were seeded to all wells (2×104cells/well).

Test Compound Dilutions:

The compounds were tested in twelve concentrations, 10-fold serial dilutions, 50 μM-5×10−10μM.

100 μl from a compound dilution plate were transferred to the cell assay plate=200 μl/well total volume and incubated for 5 days, at 37° C., 5% CO2incubator.

After 5 days, 10l of KIT-8 was added and the culture was incubated for 3-4 hours, at 37° C., 5% CO2incubator.

Plate were read in the spectrophotometer at wavelength 450 nm with a reference filter of 620 nm.

Data Analysis

CC50values are calculated by plotting the degree of inhibition (compared to the vehicle ctrl) against the logarithm of the compound concentration. Result values in the dilution series are fitted to a 4-parameter sigmoidal dose-response curve described by the expression:
F(x)=D+(A−D)/(1+(x/C){circumflex over ( )}B)
where:A=MinimumB=Slope.C=Inflection point. The inflection point is defined as the point on the curve where the curvature changes direction or signs.D=MaximumF=fraction inhibition

The CC50value is the x-value giving F=0.5, as tabulated in Table 4 below: Table 4

TABLE 4THP-1MV4-11EOL.1THP-1MV4-11EOL.1ExampleCC50(μM)CC50(μM)CC50(μM)ExampleCC50(μM)CC50(μM)CC50(μM)1f-10.670.460.126-20.740.430.198-10.26na0.3411-10.190.0710.1412-10.29na0.1713-10.871.115-11na16-10.350.1918-21.80.8020-20.900.383.621-20.390.51232.40.85243.82.125-20.410.0860.128-20.88129-10.711.230-10.460.430.9432-10.190.180.05934-20.340.440.1735-10.290.18380.190.310.2139-20.62.6403.145-10.190.180.05945-222.2Cyclo-5.11111propavir

It will be apparent that the compounds of the invention are substantially more active in this leukemia cell line models than the cyclopropavir nucleoside of the prior art.