CRYSTALLINE SOLVATES OF NUCLEOSIDE PHOSPHOROAMIDATES, THEIR STEREOSELECTIVE PREPARATION, NOVEL INTERMEDIATES THEREOF, AND THEIR USE IN THE TREATMENT OF VIRAL DISEASE

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. Provisional Application Ser. No. 61/553,543, filed October 31, 201 1, the entire disclosure of said application being incorporated herein by reference.

FIELD OF THE INVENTION

This application relates to novel crystalline solvates of the (S)-P diastereomer of the anti- HCV nucleoside phosphoroamidate, (25)-neopentyl 2-((((2R,3R,4R,5R)-5-(2-amino-6- methoxy-9H-purin-9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran -2-yl)methoxy)(naphthalen- l-yloxy)phosphorylamino)propanoate (also referred to as ΓΝΧ-08189 or ΓΝΧ-189), and to a recrystallization method for separating the two diastereomers of ΓΝΧ-08189. In addition, this application relates to a novel stereoselective method for the preparation of either of the two diastereomers, and to the novel synthetic intermediates used in this stereoselective synthesis. This application also relates to the use of the crystalline solvates to increase the liver exposure of 2'-C-methyl guanosine triphosphate from an oral dose.

BACKGROUND OF THE INVENTION

The published application WO 2010/081082, incorporated by reference herein, relates to the discovery and preparation of phosphoramidate derivatives of 2'-C-methyl 06-methyl guanosine nucleoside compounds, and their use for the treatment of viral infections including HCV (hepatitis C virus). One specific embodiment of WO 2010/081082, is designated herein as compound 1, or ΓΝΧ-08189, or ΓΝΧ-189 or (25)-neopentyl 2-((((2R,3R,4R,5R)-5-(2- amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy-4-methyltetrahy drofuran-2- yl)methoxy)(naphthalen- 1 -yloxy)phosphorylamino)propanoate

1 which, because of asymmetry about the phosphorous atom, includes diastereomers designated herein as la (ΓΝΧ-100005, ΓΝΧ-005, or (5)-P-neopentyl 2-((5)-(((2R,3R,4R,5R)- 5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy-4-methylte trahydrofuran-2- yl)methoxy)(naphthalen- 1 -yloxy)phosphorylamino)propanoate) :

(5)-P and lb (ΓΝΧ-100004, ΓΝΧ-004, or (R)-P-neopentyl 2-((5)-(((2R,3R,4R,5R)-5-(2-amino-6- methoxy-9H-purin-9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran -2-yl)methoxy)(naphthalen- l-yloxy)phosphorylamino)propanoate.

(R)-P The published application WO 2010/081082A teaches a process for separating the individual diastereomers la and lb, each substantially free of other diastereomeric forms, using chiral chromatography. However, the two diastereomers isolated by the method of WO 2010/081082A are amorphous solids. It is more desirable to have crystalline solids in order to better control the chemical, physical and biological behavior of each when they are used and formulated as medicaments for the treatment of viral disease. In addition, processes using chiral chromatography are generally not suitable for large scale manufacture. We have now also found crystallization conditions that allows separation of la from lb from a mixture containing both, without the use of chiral chromatography. In addition, we have now found that crystallized forms of la (ΓΝΧ 100005) as a solvate of formula (la) are of an acceptable final form and of sufficient quality to use in large scale manufacture and as medicaments.

It is recognized that the process of separation of an approximately 1 : 1 isomeric mixture in order to obtain a single compound , e.g., la or lb, inherently results in a 50% reduction in yield in the final step if only one compound is desired. Significant reduction in the cost of goods can be realized by employing methods which avoid wasting expensive intermediates and are stereoselective for the desired compound. We have now discovered a method for stereoselectively making a single compound la, starting from a less expensive key chiral intermediate 2a, and we have provided a method for the preparation of this chiral intermediate.

SUMMARY OF THE INVENTION

This invention relates to novel crystalline solvates of formula (la), i.e., crystalline solvates of the (5)-P diastereomer (la), an anti-HCV nucleoside phosphoroamidate, (25)-neopentyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4-d ihydroxy-4- methyltetrahydrofuran-2-yl)methoxy)(naphthalen- 1 -yloxy)phosphorylamino)propanoate (1), (also referred to as ΓΝΧ-08189 or ΓΝΧ-189), and to a recrystallization method for separating the two diastereomers la and lb which are present in 1 (ΓΝΧ-08189). In addition, this invention relates to a novel stereoselective method for the preparation of either of the two diastereomers, and to the novel synthetic intermediates used in this stereoselective synthesis. Finally, this application relates to the use of crystalline solvates of la to increase the liver exposure of 2'-C-methyl guanosine triphosphate from an oral dose. The crystalline solvates of the pure diastereomer la provide an unexpected advantage when used as a medicament, as compared to the amorphous mixture of diastereomers 1 (ΓΝΧ-08189) or as compared to either separated, amorphous diastereomers.

An aspect of the present invention provides for a crystalline solvate compound represented by formula (la)

(la)

(S)-J> wherein X represents a molecule of solvation, selected from p-xylene, anisole, 1.4-dioxane, cyclohexane, l-methoxy-2-propanol, MTB, 1-propanol, toluene, 2-butanone, 2- methoxyethanol, 4-methyl-2-pentanone, pentane, acetone, acetonitrile, hexane, chlorobenzene, chloroform, dichloromethane, ethanol, ethyl acetate, isopropyl acetate, methanol, methyl acetate, nitromethane, tetrahydrofuran, heptane, or combinations thereof, and n represents a numerical value ranging from about 0.01 to about 3.00.

Another aspect of the invention provides for a method isolating the diastereomeric compounds la and lb, each substantially free of other diastereomeric forms, from the diastereomeric mixture 1, by fractional crystallization using a suitable solvent.

1

(R)-P

Other aspects of the invention provide for a stereoselective method for preparation of the compounds la and lb, and for the novel chiral intermediates (,S)-neopentyl 2-((S)- (naphthalen-l -yloxy)(4-nitrophenoxy)phosphorylamino)propanoate 2a, and (5)-neopentyl 2-((R)-(naphthalen-l -yloxy)(4-nitrophenoxy)phosphorylamino)propanoate 2b used therein.

2b

An additional aspect of the invention provides for is the use of the compound of formula (la) in the treatment of a viral disease such as a virus from the family Flaviviridae e.g., the hepatitis C virus (HCV).

BRIEF DESCRIPTION OF THE DRAWING FIGURES

Figure 1 shows the Vibrational Spectra of Materials 1,1a and lb

Figure 2 shows the Polarized Light Microscopy and XRPD of Materials 1,1a and lb

Figure 3 shows the Thermal Analysis of Materials 1,1a and lb

Figure 4 shows the Moisture Sorption Data of Materials 1,1a and lb

Figure 5 shows the Chromatographic Data of Materials 1,1a and lb

Figure 6 shows the FT-Raman Spectra and XRPD Patterns of Selected Solvates of formula (la) Discovered in the Studies

Figure 7 shows the Chromatographic and XRPD Data for the ^-Xylene Solvate [formula (la), X = -Xylene]

Figure 8 shows the Single-crystal Structure Data, Photomicrograph, and XRPD Patterns for the ^-Xylene Solvate [formula (la), X = / Xylene]

Figure 9 shows the XRPD and Thermal Analyses for the Anisole Solvate [formula (la), X =Anisole]

Figure 10 shows the Moisture Sorption Data for the Anisole Solvate [formula (la), X

=Anisole]

Figure 11 shows the XRPD and Thermal Analyses for the Ethyl Acetate/Heptane Solvate [formula (la), X = Ethyl Acetate/Heptane

Figure 12 shows Moisture Sorption Data for the Ethyl Acetate/Heptane Solvate [formula (la), X = Ethyl Acetate/Heptane

Figure 13 shows the XRPD and Thermal Analyses for the Acetone/Pentane Solvate, [formula (la), X = Acetone/Pentane]

Figure 14 shows the Moisture Sorption and Thermal Analyses for the Acetone/Pentane Solvate, [formula (la), X = Acetone/Pentane]

Figure 15 shows the FT Raman of Compound 2

Figure 16 shows the HPLC of Compound 2 Figure 17 shows the PLM of Compound 2 Figure 18 shows the TGA of Compound 2 Figure 19 shows the ³¹ P NMR of Compound 2

Figure 20 shows the XRPD and DSC profiles of Compound 2, Crystalline Form A, assigned structure 2b.

Figure 21 shows the XRPD, DSC/TGA and ¹³ P-NMR profiles of Compound 2, Crystalline Form AB (Mixture of structures 2b and 2a)

Figure 22 shows the XRPD, DSC/TGA and ¹³ P-NMR profiles of Compound 2, Crystalline Form B, assigned structure 2a

Figure 23 shows XRPD and DSC overlaid profiles of Compound 2: Crystalline Form A, Crystalline Forms A+ B and Crystalline Form B

Figure 24 shows the ³¹ P NMR of Compound lb

Figure 25 shows the ³¹ P NMR of Compound la

Figure 26 shows the ³¹ P NMR of the Compound from Example 19

Figure 27 shows the ³¹ P NMR of an Admixture of Authentic Compound lb with the

Compound from Example 19

Figure 28 shows the ³¹ P NMR of the Compound Isolated from Example 19 (Recrystallized)

Figure 29 shows the ³¹ P NMR of an Admixture of Authentic Compound la with the

Compound from Example 19

Figure 30 shows the HPLC of the Diastereomeric Mixture, Compound 1

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

An aspect of the present invention provides for a crystalline solvate compound represented by formula (la)

(la)

wherein nX represents n molecules of solvation of (la) with a suitable solvent X.

The term "suitable solvate" refers to pharmaceutically acceptable solvates of a compound, which solvates are derived from a variety of solvents well known in the art and include, by way of example only, tetrahydrofuran, anisole, toluene, acetonitrile, ethyl acetate, benzonitrile, 1,2-dichloroethane, 1,2-dimethoxyethane, 1,4-dioxane, methyl ethyl ketone, methyl isobutyl ketone, methyl-i-butyl ketone, nitrobenzene, nitromethane, ^-xylene, tetrachloroethylene, trichloroethene, cyclohexane, heptane, hexane, methylcyclohexane, pentane, acetone, butyronitrile, chlorobenzene, chloroform, cyclohexanone, cyclopentyl methyl ether, 3-pentanonediethyl ether, diisobutyl ketone, diisopropyl ether, dimethyl carbonate, methyl acetate, methyl i-butyl ether, 4-methyl-2-penatanone, 2-butanone, 2-methyl tetrahydrofuran, dichloromethane, isopropyl acetate, isobutyl acetate and the like. Such solvates may exist in distinct crystalline forms in which the ratio of solvent to compound is from about 0.01 to about 3.00.

An embodiment of this aspect is the crystalline solvate compound represented by formula

(la)

(la) wherein nX represents n molecules of solvation of (la) with a suitable solvent X; wherein X is selected from p-xylene, anisole, 1.4-dioxane, cyclohexane, l-methoxy-2- propanol, MTB, 1-propanol, toluene, 2-butanone, 2-methoxyethanol, 4-methyl-2-pentanone, pentane, acetone, acetonitrile, hexane, chlorobenzene, chloroform, dichloromethane, ethanol, ethyl acetate, isopropyl acetate, methanol, methyl acetate, nitromethane, tetrahydrofuran, heptane, or combinations thereof, and n represents a numerical value ranging from about 0.01 to about 3.00.

Specific embodiments of this aspect include the crystalline forms of formula (la) wherein X is selected from 2-butanone/toluene, acetone, acetone/MTBE, acetone/pentane, acetone/toluene, anisole, ethyl acetate/cyclohexane, ethyl acetate/heptane, ethyl acetate/hexane, methyl acetate/cyclohexane, nitromethane/MTBE, -xylene, tetrahydrofuran, tetrahydrofuran/heptane, or tetrahydrofuran/p-xylene.

The crystalline forms are characterized by their Power Diffraction X-ray Crystallography, which produces a fingerprint that is unique to the crystalline form which distinguishes it from amorphous forms of compound la and exhibit physical, pharmaceutical, physiological and biological characteristics that may be significantly different from amorphous forms of compound la.

In accordance with the present invention there are provided the following specific embodiments of compounds of formula (la) and are listed in Table 1.

Table 1 Selected Crystalline Solvates of Formula (la)

Solvate (X) n Crystallization Solvent

2-Butanone / Toluene 0.5 2 -Butanone : Toluene

Acetone 0.5 Acetone:Water

Acetone/MTBE 0.5 Acetone TBE

Acetone / Pentane 0.5 Acetone:Pentane

Acetone / Toluene 0.5 Acetone:Toluene

Anisole 0.5 Anisole

EtOAc / Cyclohexane 0.5 EtOAc:Cyclohexane Solvate (X) n Crystallization Solvent

EtOAc / Heptane 0.5 EtOAc :heptane

EtOAc / Hexane 0.5 Ethyl Acetate:Hexane

MeOAc / Cyclohexane 0.5 Methyl acetate:Cyclohexane

Nitromethane / MTBE 0.5 Nitromethane:MTBE

(-Xylene 0.5 1 -Propanol:/?-Xylene

THF 0.5 THF:Water

THF / Heptane 0.5 T etrahy dro furan : H ep tan e

THF / /^-Xylene 0.5 T etrahy dro furan : p-Xy 1 en e

The solvates can generally prepared by suspending the diastereomeric mixture, compound 1 or the amorphous single diastereomer, compound la, in suitable solvent or solvent mixture and stirring the slurry for a sufficient amount of time, such as from about 12 to 48 hours to cause dissolution of one isomer while promoting crystallization of the other isomer as solvate of compound la, i.e., a compound of formula (la).

In certain embodiments, the crystalline solvates of formula (la) are characterized by their X- ray Powder Diffraction patterns.

The crystalline characteristics of the formula (la) compounds can also be observed using other techniques such as thermogravimetric analysis (TGA) and Polarized Light Microscopy (PLM). The stereochemical purity and absolute configuration can be confirmed by comparison of isolated material to standards samples of la and lb obtained by HPLC separation methods, by ³¹ P-NMR spectroscopy, and by single crystal X-Ray diffraction.

The crystalline solvates of formula (la) have surprisingly unexpected enhanced properties, such as enhanced or greater stability, enhanced storage stability and enhanced stability against chemical degradation, as compared to the amorphous material la. In addition the crystalline solvates of formula (la) may have surprisingly unexpected enhanced biological properties, such as enhanced oral bioavailability from a solid dose.

Another aspect of the invention provides for a method isolating the diastereomeric compounds la and lb, each substantially free of other diastereomeric forms, from the diastereomeric mixture 1, by fractional crystallization using a suitable solvent.

la

(5)-P

More specifically this aspect of the invention provides for a method isolating the diastereomeric compounds la and lb, each substantially free of other diastereomeric forms, said method comprising the steps of

(1) combining an suitable solvent or solvent mixture with a sample containing a diastereomeric mixture of the compound 1,

(2) stirring the resulting mixture for a period of time until sufficient crystal formation occurs,

(3) isolation of the crystals A from the solution, and

(4) concentration of the solution to a residue B. The crystals A and the residue B represent samples of the diastereomers la and lb, respectively, which are substantially free of each other, and, whose identity can be confirmed by standard spectroscopic methods.

The term "substantially free" as used herein refers to samples of a compound, e.g., la or lb, which preferably contain less than 10% of other diastereomers, and more particularly, approximately 5% or less of other diastereomers.

An embodiment of this aspect comprises the optional addition of seed crystals in step (2)

A second embodiment of this aspect comprises the optional warming of the mixture prepared in step (2), followed by cooling prior to step (3).

A third embodiment of this aspect comprises the optional repetition of steps (1-4) using the residue obtained in step (4) as the sample containing the compound 1.

A fourth embodiment comprises the optional step of recrystallization of the crystals (A) isolated in step 3 with the suitable solvent or solvent mixture used in step (1).

In a fifth embodiment, the crystalline A is a crystalline solvate of formula (la).

In a further aspect, the invention provides for a method isolating the diastereomeric compound la substantially free of the diastereomeric compound lb and any other diastereomeric forms, from the diastereomeric mixture 1, by fractional crystallization using a suitable solvent.

More specifically this aspect of the invention provides for a method isolating crystalline forms of the diastereomeric compound la, substantially free of the diastereomeric compound lb and any other diastereomeric forms, said method comprising the steps of

(1) combining a suitable solvent or solvent mixture with a sample containing a diastereomeric mixture of the compound 1,

(2) stirring the resulting mixture for a period of time until sufficient crystal

formation occurs,

(3) isolation of the crystalline forms of compound la from the solution.

An embodiment of this aspect comprises the optional addition of seed crystals in step (2) A second embodiment of this aspect comprises the optional warming of the mixture prepared in step (2), followed by cooling prior to step (3).

\A third embodiment comprises the optional step of recrystallization of the crystals isolated in step 3 with the suitable solvent or solvent mixture used in step (1).

In a fourth embodiment, the crystalline material isolated in step 3 is a crystalline solvate of compound la represented by formula (la).

Non-limiting examples of suitable solvents useful in carrying out the method of separation of the diastereomeric compounds la and lb from the diastereomeric mixture 1 by fractional crystallization are ^-xylene, toluene, pentane, heptane, 1.4-dioxane, 1-butanol, 1 -propanol, 2- butanone, 2-propanol, acetone, acetonitrile, chloroform, dichloromethane, dimethyl carbonate, ethyl acetate, methyl acetate, tetrahydrofuran, and anisole or combinations thereof.

Preferred suitable solvents are anisole, anisole/heptane, 1,4-dioxane/p-xylene, 1-butanol/p- xylene, 1 -propanoic-xylene, 2-butanone/toluene, 2-propanolol/p-xylene, acetone, toluene, acetonitrile/p-xylene, chloroform/toluene, dichloromethane/toluene, dimethyl carbonate/p- xylene, ethyl acetate/p-xylene„ methyl acetate/toluene, tetrahydrofuran/p-xylene, acetone/pentane, and ethyl acetate/heptane.

The solvent mixtures may be in any proportion. The preferred solvents or solvent combinations are those that allow full dissolution of one of the diastereomers, therefore facilitating the filtration/isolation of the other diastereomer.

A further embodiment of this aspect is a solvent or solvent combination that allows full dissolution of the (R)-P diastereomer lb and crystallization of the (S)-P diastereomer la.

In another embodiment of this aspect, the crystalline diastereomer la isolated by filtration may be a solvate formed from one of the crystallizing solvents, as described hereinabove for the compound of formula (la).

Specific embodiments of solvent and solvent combinations that are useful for isolating the compounds la and lb are listed in Table 2: Table 2 Solvents and Solvent Combinations Useful for Isolating Compounds la and lb by Crystallization

Additional non-limiting examples of the above are described in more detail in the experimental section.

General techniques for performing the steps are well known in the art, some which are described below.

For crystallization techniques that employ solvent, the choice of solvent or solvents is typically dependent upon one or more factors, such as solubility of the compound, the crystallization technique, and the vapor pressure of the solvent. Combinations of solvents may be employed, for example, the compound may be solubilized into a first solvent to afford a solution, followed by the addition of an antisolvent to decrease the solubility of the compound in the solution and to afford the formation of crystals . An antisolvent is a solvent in which the compound has low solubility. Suitable solvents for preparing crystals include polar and nonpolar solvents. In one method to prepare crystals, a compound is suspended and/or stirred in a suitable solvent to afford a slurry, which may be heated to promote dissolution. The term "slurry", as used herein, means a saturated solution of the compound, which may also contain an additional amount of the compound to afford a heterogeneous mixture of the compound and a solvent at a given temperature.

Seed crystals may be added to any crystallization mixture to promote crystallization. Seeding may be employed to control growth of a particular polymorph or to control the particle size distribution of the crystalline product. Accordingly, calculation of the amount of seeds needed depends on the size of the seed available and the desired size of an average product particle as described, for example, in "Programmed Cooling of Batch Crystallizers ," J. W. Mullin and J. Nyvlt, Chemical Engineering Science, 1971, 26, 369-377. In general, seeds of small size are needed to control effectively the growth of crystals in the batch. Seed of small size may be generated by sieving, milling, or micronizing of large crystals , or by micro- crystallization of solutions. Care should be taken that milling or micronizing of crystals does not result in any change in crystallinity from the desired crystal form (i.e., change to amorphous or to another polymorph).

A cooled crystallization mixture may be filtered under vacuum, and the isolated solids may be washed with a suitable solvent, such as cold recrystallization solvent, and dried under a nitrogen purge to afford the desired crystalline form. The isolated solids may be analyzed by a suitable spectroscopic or analytical technique, such as solid state nuclear magnetic resonance, differential scanning calorimetry, x-ray powder diffraction, or the like, to assure formation of the preferred crystalline form of the product. The resulting crystalline form is typically produced in an amount of greater than about 70 weight % isolated yield, preferably greater than 90 weight % isolated yield, based on the weight of the compound originally employed in the crystallization procedure. The product may be comilled or passed through a mesh screen to delump the product, if necessary.

In further aspect the invention provides a stereoselective method for the preparation of compound of formula (la) comprising the steps of

(1) allowing the reaction of the intermediate of formula 2 with the nucleoside 3 in the presence of base;

(2) selective crystallization of the product obtained from the reaction using a suitable solvent to form a crystalline solvate of formula (la); and (3) isolation of the crystalline solvate of formula (la) produced thereby. This aspect is summarized in Reaction Scheme I, as shown below:

Reaction Scheme 1

In this scheme, X and n are as described hereinabove.

In an embodiment of this aspect, the product (la) may be further purified by recrystallization using solvent(s) and conditions described hereinabove for la.

The procedure illustrated in Reaction Scheme 1 uses the intermediate 2 as a diastereomeric mixture, and as a result, both diastereomers la and lb are produced in the reaction step. However, diastereomer la is separated from this mixture by the recrystallization step in which a crystalline solvate of formula (la) forms and is removed by filtration.

In further aspect the invention provides a stereoselective method for the preparation of compound of formula la comprising the steps of

(1) allowing the reaction of an essentially pure diastereomeric intermediate compound of formula 2a with the nucleoside 3 in the presence of base; and

(2) isolation of the pure diastereomer product la produced thereby.

This aspect is summarized in Reaction Scheme 2, as shown below:

Reaction Scheme 2

In an embodiment of this aspect, the product la may be further purified by recrystallization using solvent(s) and conditions described hereinabove for la.

In another embodiment, the product la may be further converted to a crystalline solvate of formula (la), by recrystallization of la produced by the process using a solvent in which said solvate forms.

In another aspect the present invention provides for a stereoselective method of preparation of the compound lb, comprising the steps of

(1) allowing the reaction of an essentially pure diastereomeric intermediate compound 2b with the nucleoside 3 in the presence of base; and

(2) isolation of the pure diastereomer product lb produced thereby. This embodiment is summarized in the Reaction Scheme 3, as shown below:

1 b In an embodiment of this aspect, the products lb may be further purified by recrystallization using solvent(s) and conditions described hereinabove.

In these Reaction Schemes 2 and 3, the essentially pure diastereomeric intermediates, i.e., compound 2a or compound 2b, are allowed to react with the nucleoside 3 in the presence of a strong base, such as i-butyl magnesium chloride, in an inert solvent such as THF at a temperature sufficient to facilitate complete reaction. The reaction carried out under these conditions likely produces inversion of the configuration about phosphorous. In contrast to the procedure of Reaction Scheme 1, each of these schemes illustrate that a single, specific diastereomeric product is produced which is predictable from the stereochemical configuration of the starting material. Therefore, it is anticipated that the phosphoroamidate intermediate 2a provides the product la, and similarly, the intermediate 2b provides the product of formula lb.

Another aspect of the invention provides the novel compounds 2a and 2b, useful as intermediates in the preparation of compounds la and lb.

2b

(5)-P

In a further embodiment, the present invention provides for a method of preparation of the compounds 2a and 2b in substantially diastereomerically pure form comprising the steps of (1) reaction of a chlorophosphosphoroamidate intermediate 4 with ntirophenol 5 in the presence of a base

(2) separation of the diastereomers of the product 2 formed by fractional

crystallization to provide 2a and 2b.

This embodiment is summarized in the Reaction Scheme 4, as shown below:

In this scheme, the chlorophosphosphoroamidate ester of formula 4 is allowed to react with 4-nitrophenol (compound 5) in the presence of a base such as triethylamine in an inert solvent such as THF to produce the phosphoramidate of formula 2 as a mixture of diastereomers. Preferably, the crude material isolated from the reaction is purified by column chromatographic methods in order to provide material which is more amendable to fractional recrystallization.

The diastereomeric phosphoramidate mixture of formula 2 is separated into pure diastereomers of formulae 2a and 2b by fractional crystallization using a suitable solvent or solvent mixture.

The terms "suitable solvent" or "suitable solvent mixtures" refer to a solvent or solvent mixtures which is capable of dissolving a compound, or useful in the purification of a mixture of compounds by fractional crystallization. Among the suitable solvents for this step are methyl cyclohexane, isobutyl acetate and mixtures thereof.

Techniques of fractional crystallization are well known in the art and include, by way of example only, the warming of the solvent, into which the compound mixture has been placed, up to a temperature until dissolution occurs, allowing the solution to cool at a controlled rate until crystallization occurs, and removal of the crystallized material by filtration. Crystallization is optionally initiated by scratching the surface of the solution-containing vessel or by addition of a seed crystal of the desired compound to the supersaturated solution. A mixture of solvents may also be used to control the solubility properties of the solvent. Typical acceptable solvents, which are suitable for this purpose, may be used alone or in a mixture include tetrahydrofuran, anisole, ethanol, methanol, isopropanol, toluene, acetonitrile, ethyl acetate, benzonitrile, 1,2-dichloroethane, 1,2-dimethoxyethane, 1,4- dioxane, methyl ethyl ketone, methyl isobutyl ketone, methyl-i-butyl ketone, nitrobenzene, nitromethane, ^-xylene, tetrachloroethylene, trichloroethene, cyclohexane, heptane, hexane, methylcyclohexane, pentane, acetone, butyronitrile, chlorobenzene, chloroform, cyclohexanone, cyclopentyl methyl ether, 3-pentanonediethyl ether, diisobutyl ketone, diisopropyl ether, dimethyl carbonate, methyl acetate, methyl i-butyl ether, 4-methyl-2- penatanone, 2-butanone, 2-methyl tetrahydrofuran, water, dichloromethane, isopropyl acetate, isobutyl acetate and the like. Acceptable mixtures include isopropyl ether/methyl cyclohexane, diethyl ether/hexane, methanol/anisole, ethanol/anisole, isopropanol/anisole, n- butanol/anisole, acetone/anisole, isobutyl acetate/methyl cyclohexane, 2-methyl- tetrahydrodfuran/hexane, ethyl acetate/heptane, tetrachloroethylene/ methylcyclohexane and the like. The mixture of solvents may be in any proportion. Non-limiting examples of such proportions are 50/50, 70/30, 60/40, and 95/5 volume %. Preferred solvents and "suitable solvent or solvent mixtures" include isobutyl acetate/methyl cyclohexane, 2-methyl-tetrahydrofuran/methyl cyclohexane, ethyl acetate/heptane, isopropyl ether/methyl cyclohexane and tetrachloroethylene/methyl cyclohexane. The preferred solvents for the formation of crystalline 2a may differ from the preferred solvents for the formation of crystalline 2b.

Depending on the recrystallization conditions, crystalline material of varying degrees of isomeric purity can be obtained. In order to provide higher diastereomeric purity, one embodiment of this aspect of the invention is the optional repetition of the recrystallization using the crystalline material isolated in step (2), using a suitable solvent/solvent mixture. The recrystallization may be carried out at a temperature suitable for controlling the rate of crystallization. Preferred temperatures are from about 5 °C to about 50 °C.

Once isolated, the compounds are substantially pure diastereomers, i.e., are samples of compound 2a or 2b which preferably contain less than 10% of other diastereomers, and more particularly, approximately 5% or less of other diastereomers.

According to another aspect, the present invention provides for the use of the crystalline solvent compound of formula (la) in the treatment of viral diseases and for the preparation of medicaments useful in the treatment of viral diseases.

General Methods of Preparation of Compounds

The starting materials used are known in the art or are prepared by well-known general methods and procedures. For example, many of the starting materials are available from commercial suppliers such as Aldrich Chemical Co. (Milwaukee, Wis., USA), Bachem (Torrance, Calif, USA), Emka-Chemce or Sigma (St. Louis, Mo., USA). Others may be prepared by procedures, or obvious modifications thereof, described in standard reference texts such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-15 (John Wiley and Sons, 1991), Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplemental (Elsevier Science Publishers, 1989), Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), March's Advanced Organic Chemistry, (John Wiley and Sons, ^ Edition), and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989). Specifically, the compounds of this invention may be prepared by various methods known in the art of organic chemistry in general and nucleoside and nucleotide analogue synthesis in particular. General reviews of the preparation of nucleoside and nucleotide analogues include 1) Michelson A. M., "The Chemistry of Nucleosides and Nucleotides, ^' " Academic Press, New York, 1963; 2) Goodman L., "Basic Principles in Nucleic Acid Chemistry, " Academic Press, New York, 1974, vol. 1, Ch. 2; and 3) "Synthetic Procedures in Nucleic Acid Chemistry," Eds. Zorbach W. & Tipson R., Wiley, New York, 1973, vol. 1 & 2.

In particular, the preparation of compound 1, and the separation of its diastereomers la and lb by chiral column chromatography, as well as the nucleoside 3 have been described in WO 2010/081082.

It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.

Additionally, as will be apparent to those skilled in the art, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions. Suitable protecting groups for various functional groups as well as suitable conditions for protecting and deprotecting particular functional groups are well known in the art. For example, numerous protecting groups are described in T. W. Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, Third Edition, Wiley, New York, 1999, and references cited therein.

The crystalline solvates of formula (la) prepared by the methods described above may be used to prepare formulated compositions and medicaments, useful for administration of therapeutically acceptable amounts of compound. The crystalline solvates provide advantageous properties such as enhanced or greater stability, enhanced storage stability and enhanced stability against chemical degradation, as compared to the amorphous material of formula (la). In addition the crystalline forms of (la) may have surprisingly unexpected enhanced biological properties, such as enhanced biological activity, enhanced metabolic behavior in vivo, and a reduction in undesirable side effects, including toxicity.

Dosages and routes of administration.

In general, the crystalline solvates of formula (la) will be administered in a therapeutically effective amount by any of the accepted modes of administration for agents that serve similar utilities. The effective amount will be that amount of the compound of this invention that would be understood by one skilled in the art to provide therapeutic benefits, i.e., the active ingredient, and will thus depend upon numerous factors such as the severity of the disease to be treated, the age and relative health of the subject, the potency of the compound used, the route and form of administration, and other factors. The drug can be administered more than once a day, and in the preferred mode the drug is administered once or twice a day. As indicated above, all of the factors to be considered in determining the effective amount will be well within the skill of the attending clinician or other health care professional.

For example, therapeutically effective amounts of compounds of formula (la) may range from approximately 0.05 to 50 mg per kilogram body weight of the recipient per day; preferably about 0.1-25 mg/kg/day, more preferably from about 0.5 to 10 mg/kg/day. Thus, for administration to a 70 kg person, the dosage range would most preferably be about 35-700 mg per day. In general, compounds of this invention will be administered as pharmaceutical compositions by any one of the following routes: oral, systemic (e.g., transdermal, intranasal or by suppository), or parenteral (e.g., intramuscular, intravenous or subcutaneous) administration. The preferred manner of administration is oral using a convenient daily dosage regimen that can be adjusted according to the degree of affliction. Compositions can take the form of tablets, pills, capsules, semisolids, powders, sustained release formulations, solutions, suspensions, elixirs, aerosols, or any other appropriate compositions. Another preferred manner for administering compounds of this invention is inhalation. This is an effective method for delivering a therapeutic agent directly to the respiratory tract (see U.S. Pat. No. 5,607,915, said patent incorporated herein by reference).

The choice of formulation depends on various factors such as the mode of drug administration and bioavailability of the drug substance. For delivery via inhalation the compound can be formulated as liquid solution, suspensions, aerosol propellants or dry powder and loaded into a suitable dispenser for administration. There are several types of pharmaceutical inhalation devices-nebulizer inhalers, metered dose inhalers (MDI) and dry powder inhalers (DPI). Nebulizer devices produce a stream of high velocity air that causes the therapeutic agents (which are formulated in a liquid form) to spray as a mist that is carried into the patient's respiratory tract. MDI's typically are formulation packaged with a compressed gas. Upon actuation, the device discharges a measured amount of therapeutic agent by compressed gas, thus affording a reliable method of administering a set amount of agent. DPI dispenses therapeutic agents in the form of a free flowing powder that can be dispersed in the patient's inspiratory air-stream during breathing by the device. In order to achieve a free flowing powder, the therapeutic agent is formulated with an excipient such as lactose. A measured amount of the therapeutic agent is stored in a capsule form and is dispensed with each actuation.

Recently, pharmaceutical formulations have been developed especially for drugs that show poor bioavailability based upon the principle that bioavailability can be increased by increasing the surface area i.e., decreasing particle size. For example, U.S. Pat. No. 4, 107,288 describes a pharmaceutical formulation having particles in the size range from 10 to 1,000 nm in which the active material is supported on a cross-linked matrix of macromolecules. U.S. Pat. No. 5, 145,684 describes the production of a pharmaceutical formulation in which the drug substance is pulverized to nanoparticles (average particle size of 400 nm) in the presence of a surface modifier and then dispersed in a liquid medium to give a pharmaceutical formulation that exhibits remarkably high bioavailability. These patents are incorporated herein by reference.

As indicated above, the compositions in accordance with the invention generally comprise a compound of formula (la) in combination with at least one pharmaceutically acceptable vehicle, carrier, excipient or diluent. Some examples of acceptable excipients are those that are non-toxic, will aid administration, and do not adversely affect the therapeutic benefit of the compound of the invention. Such excipient may be any solid, liquid, semi-solid or, in the case of an aerosol composition, gaseous excipient that is generally available to one of skill in the art.

Solid pharmaceutical excipients useful in the invention may include starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk and the like. Liquid and semisolid excipients may be selected from glycerol, propylene glycol, water, ethanol and various oils, including those of petroleum, animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc. Preferred liquid carriers, particularly for injectable solutions, include water, saline, aqueous dextrose, and glycols. Compressed gases may be used to disperse a compound of this invention in aerosol form. Inert gases suitable for this purpose are nitrogen, carbon dioxide, etc. Other suitable pharmaceutical excipients and their formulations are described in Remington's Pharmaceutical Sciences, edited by E. W. Martin (Mack Publishing Company, 18th ed., 1990). The amount of the compound in a formulation can vary within the full range employed by those skilled in the art. For example, the formulation will contain, on a weight percent (wt %) basis, from about 0.01-99.99 wt % wherein the compound is a compound of formula (la) based on the total formulation, with the balance being one or more suitable pharmaceutical excipients. Preferably, the compound is present at a level of about 1-80 wt %. Pharmaceutical formulations containing a compound in accordance with the invention are described further below.

Additionally, the present invention is directed to a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present invention in combination with a therapeutically effective amount of another active agent against RNA-dependent RNA virus and, in particular, against HCV. Agents active against HCV include, but are not limited to, ribavirin, levovirin, viramidine, thymosin alpha- 1, an inhibitor of HCV NS3 serine protease, interferon-a, pegylated interferon-a (peginterferon-a), a combination of interferon-a and ribavirin, a combination of peginterferon-a and ribavirin, a combination of interferon-α and levovirin, and a combination of peginterferon-α and levovirin. Interferon-α includes, but is not limited to, recombinant interferon-a2a (such as Roferon interferon available from Hoffman-LaRoche, Nutley, N.J.), interferon-a2b (such as Intron-A interferon available from Schering Corp., Kenilworth, N.J., USA), a consensus interferon, and a purified interferon-a product. For a discussion of ribavirin and its activity against HCV, see J. O. Saunders and S. A. Raybuck, "Inosine Monophosphate Dehydrogenase: Consideration of Structure, Kinetics and Therapeutic Potential," Ann. Rep. Med. Chem., 35:201-210 (2000).

Even further, the present invention is directed to a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present invention in combination with a therapeutically effective amount of another agent active against hepatitis C virus. Such agents include those that inhibit HCV proteases, HCV polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCV egress, HCV NS5A protein, and inosine 5'- monophosphate dehydrogenase. Other agents include nucleoside analogs for the treatment of an HCV infection. Still other compounds include those disclosed in WO 2004/014313 and WO 2004/014852 and in the references cited therein. The patent applications WO 2004/014313 and WO 2004/014852 are hereby incorporated by references in their entirety. Specific antiviral agents include Omega IFN (BioMedicines Inc.), BILN-2061 (Boehringer Ingelheim), Summetrel (Endo Pharmaceuticals Holdings Inc.), Roferon A (F. Hoffman-La Roche), Pegasys (F. Hoffman-La Roche), Pegasys/Ribaravin (F. Hoffman-La Roche), CellCept (F. Hoffman-La Roche), Wellferon (GlaxoSmithKline), Albuferon-a (Human Genome Sciences Inc.), Levovirin (ICN Pharmaceuticals), IDN-6556 (Idun Pharmaceuticals), IP-501 (Indevus Pharmaceuticals), Actimmune (InterMune Inc.), Infergen A (InterMune Inc.), ISIS 14803 (ISIS Pharmaceuticals Inc.), JTK-003 (Japan Tobacco Inc.), Pegasys/Ceplene (Maxim Pharmaceuticals), Ceplene (Maxim Pharmaceuticals), Civacir ( abi Biopharmaceuticals Inc.), Intron A/Zadaxin (RegeneRx), Levovirin (Ribapharm Inc.), Viramidine(Ribapharm Inc.), Heptazyme (Ribozyme Pharmaceuticals), Intron A (Schering- Plough), PEG-Intron (Schering-Plough), Rebetron (Schering-Plough), Ribavirin (Schering- Plough), PEG-Intron/Ribavirin (Schering-Plough), Zadazim (SciClone), Rebif (Serono), IFN- β/ΕΜΖ701 (Transition Therapeutics), T67 (Tularik Inc.), VX-497 (Vertex Pharmaceuticals Inc.), VX-950/LY-5703 10 (Vertex Pharmaceuticals Inc.), Omniferon (Viragen Inc.), XTL- 002 (XTL Biopharmaceuticals), SCH 503034 (Schering-Plough), isatoribine and its prodrugs ANA971 and ANA975 (Anadys), R1479 (Roche Biosciences), Valopicitabine (Idenix), NIM81 1 ( ovartis), and Actilon (Coley Pharmaceuticals).

In some embodiments, the compositions and methods of the present invention contain a compound of formula (la) and interferon. In some aspects, the interferon is selected from the group consisting of interferon alpha 2B, pegylated interferon alpha, consensus interferon, interferon alpha 2A, and lymphoblastiod interferon tau.

In other embodiments the compositions and methods of the present invention utilize a combination of a compound of formula (la) and a compound having anti-HCV activity such as those selected from the group consisting of interleukin 2, interleukin 6, interleukin 12, a compound that enhances the development of a type 1 helper T cell response, interfering RNA, anti-sense RNA, Imiqimod, ribavirin, an inosine 5' monophospate dehydrogenase inhibitor, amantadine, and rimantadine.

Anti-hepatitis C Activity Assays

Compounds can exhibit anti-hepatitis C activity by inhibiting HCV polymerase, by inhibiting other enzymes needed in the replication cycle, or by other pathways. A number of assays have been published to assess these activities. A general method that assesses the gross increase of HCV virus in culture was disclosed in U.S. Pat. No.5, 738,985 to Miles et al. In vitro assays have been reported in Ferrari et al. J. of Vir., 73 : 1649-1654, 1999; Ishii et al, Hepatology, 29: 1227-1235, 1999; Lohmann et al, J. Bio. Chem., 274: 10807-10815, 1999; and Yamashita et al, J. of Bio. Chem., 273: 15479-15486, 1998. WO 97/12033 relates to HCV polymerase assay that can be used to evaluate the activity of the of the compounds described herein. Another HCV polymerase assay has been reported by Bartholomeusz, et al., Hepatitis C Virus (HCV) RNA polymerase assay using cloned HCV non-structural proteins; Antiviral Therapy 1996: 1 (Supp 4) 18-24.

Screens that measure reductions in kinase activity from HCV drugs were disclosed in U.S. Pat. No. 6,030,785, to Katze et al., U.S. Pat. No. 6,228,576, Delvecchio, and U.S. Pat. No.5, 759,795 to Jubin et al. Screens that measure the protease inhibiting activity of proposed HCV drugs were disclosed in U.S. Pat. No. 5,861,267 to Su et al, U.S. Pat. No. 5,739,002 to De Francesco et al, and U.S. Pat. No. 5,597,691 to Houghton et al.

Oral Bioavailability Studies

The compounds 1, la, lb and the solvates of Compound la [Formula (la) compounds] are studied in oral bioavailability experiments in cynomolgus monkeys. The compounds are dosed as solids in capsules (25 mg/kg) and the resulting plasma concentrations of a selection of metabolites are measured. These results are compared to values obtained when compounds 1, la, lb, and the solvates of Compound la are formulated in 95:5 CapmukTween and dosed orally.

Experimental Examples

Embodiments of the present invention will now be described by way of example only with respect to the following non-limiting examples.

General Procedures

Instrumentation

Proton, Carbon, and Phosphorus Nuclear Magnetic Resonance (¾, ¹³ C, ³¹ P NMR): Spectra were recorded on Bruker Avance spectrometers operating either at 500, 125, and 202 MHz or at 300, 75, and 121 MHz or a Varian Unity Inova instrument operating at 400, 100, and 161.9 MHz. The solvents used are indicated for each compound. All ¹³ C and ³¹ P spectra were recorded proton decoupled. Chemical shifts for ^l R and ¹³ C spectra are in parts per million downfield from tetramethylsilane. Coupling constants are referred to as J values. Signal splitting patterns are described as singlet (s), doublet (d), triplet (t), quartet (q), broad signal (br), doublet of doublet (dd), doublet of triplet (dt), or multiplet (m). Chemical shifts for ³¹ P spectra are in parts per million relative to an external phosphoric acid standard. Some of the proton and carbon NMR signals were split because of the presence of (phosphate) diastereoisomers in the samples.

FT-Raman Spectroscopy. Raman spectra were collected with a Nicolet NXR9650 or NXR 960 spectrometer (Thermo Electron) equipped with 1064 nm Nd:YV04 excitation laser,

InGaAs and liquid N2 cooled Ge detectors, and a MicroStage. All spectra were acquired at 4

-1

cm resolution, 64-128 scans, using Happ-Genzel apodization function and 2 level zero- filling.

Mass Spectrometry: The mode of ionization was fast atom bombardment (FAB) using MNOBA (m-nitrobenzyl alcohol) as matrix for some compounds. Electrospray mass spectra were obtained using a Waters LCT time-of-flight mass spectrometer coupled to a Waters M600 HPLC pump. Samples were dissolved in methanol and injected into the solvent stream via a Rheodyne injector. The mobile phase used was methanol at a flow rate of 200 ^L/min. The electrospray source was operated at a temperature of 130 °C with a desolvation temperature of 300 °C, a capillary voltage of 3 kV, and cone voltage of 30 V. Data were collected in the continuum mode over the mass range 100-2000 amu and processed using Masslynx 4.1 software. Accurate mass measurements were facilitated by the introduction of a single lockmass compound of known elemental composition into the source concurrently with sample.

Polarized-light Microscopy (PLM). The photomicrographs were collected using Olympus BX60 polarized-light microscope equipped with Olympus DP70 camera, or Olympus BX51 polarized-light microscope with Olympus DP71 camera.

Powder X-Ray Diffraction (XRPD) XRPD diffractograms were acquired using PANalytical X'Pert Pro diffractometer on Si zero-background wafers. All diffractograms were collected using a monochromatic Cu Ka (45 kV/40 mA) radiation and a step size of 0.02 ^° 2Θ. Differential Scanning Calorimetry (DSC). DSC was conducted with a TA Instruments Q100 differential scanning calorimeter equipped with an autosampler and a refrigerated cooling system under 40 mL/min 2 purge. DSC thermograms were obtained at 15 °C/min in Pt or Al pans.

Thermogravimetric Analysis with IR off-gas detection (TGA-IR). TGA-IR was conducted with a TA Instruments Q5000 thermogravimetric analyzer interfaced to a Nicolet 6700 FT-IR spectrometer (Thermo Electron) equipped with an external TGA-IR module with a gas flow cell and DTGS detector. TGA was conducted with 60 mL/min N ₂ flow and heating rate of 15°C/min in Pt or Al pans. IR spectra were collected at 4 cm ^"1 resolution and 32 scans at each time point.

High-performance Liquid Chromatography (HPLC). HPLC analyses were conducted with an HP 1 100 system equipped with a Gl 131 Quad pump, G1367A autosampler, and G1315B diode array detector. Column: Luna CI 8(2) (50 x 2.0 mm, 3 μιη). Mobile phase: 100% water (0.05%TFA) to 95% ACN (0.05% TFA) over 8 min and 2 min re-equilibration. Flow rate: 1 HPLC. HPLC analyses were conducted with an HP 1 100 system equipped with a Gl 131 Quad pump, G1367A autosampler, and G1315B diode array detector. Column: Luna CI 8(2) (50 x 2.0 mm, 3 μιη). Mobile phase: 100% water (0.05% TFA) to 95% ACN (0.05% TFA) over 8 min and 2 min re-equilibration. Flow rate: 1 mL/min. Detection: 254 nm.

Dynamic Vapor Sorption (DVS). DVS experiments were conducted on a Surface Measurement Systems DVS-HT at 25 °C. The instrument was operated in step mode and the relative humidity was increased in 10% RH increments from 40% RH to 90% RH, then decreased from 90% RH to 0% RH, then increased from 0% RH to 90% RH, then decreased from 90% RH to 0% RH. An extra step at 75% RH was included in each cycle. The mass equilibrium criterion was set at 0.005% change in mass over time (dm/dt) prior to each humidity level. A minimum step time of 10 minutes and a maximum step time of 240 minutes were specified. mL/min. Detection: 254 nm.

Example 1 - (2/?,3/?,4/?,5/?)-2-(2-Amino-6-chloro-9H-purin-9-yl)-5-(benz oyloxymethyl)- 3-methyltetrahydrofuran-3,4-diyl Dibenzoate

The compound is prepared as described in WO 2010/081082.

To a pre-cooled (0 °C) solution of (2S,3R,4R,5R)-5-(benzoyloxymethyl)-3- methyltetrahydrofuran-2,3,4-triyl tribenzoate (or 2,3,4,5-tetra-0-benzoyl-2-C-methyl- ?-D- ribofuranose) (CarboSynth Ltd, 10.0 g, 17.22 mmol), 2-amino-6-chloropurine (Aldrich, 3.2 g, 18.87 mmol), and l,8-diazabicycl[5.4.0]undec-7-ene (DBU) (7.7 mL, 51 mmol) in anhydrous acetonitrile (200 mL), was added trimethysilyl triflate (12.5 mL, 68.8 mmol) dropwise. The reaction mixture was then heated at 65 °C for 4 to 6 h, allowed to cool down to room temperature, poured into saturated aqueous sodium bicarbonate (300 mL), and extracted with dichloromethane (3x150 mL). The combined organic phase was dried over sodium sulfate and evaporated under reduced pressure. The residue was precipitated from dichloromethane and methanol, filtrated, the solid was washed 2 times with methanol and dried to give the desired compound (8.5 g, 79 %) as a white solid (yields are from 65% (column) up to 90% (precipitation)).

^L H NMR (500 MHz, CDC1 ₃ ) δ 8.13 (dd, J = 1.2, 8.3, 2H), 8.02 - 7.94 (m, 5H), 7.65 - 7.60 (m, 1H), 7.58 - 7.45 (m, 4H), 7.35 (q, J = 7.7, 4H), 6.65 (s, 1H), 6.40 (d, J = 6.7, 1H), 5.31 (s, 2H), 5.08 (dd, J = 4.2, 1 1.6, 1H), 4.79 (dd, J = 6.4, 1 1.6, 1H), 4.74 (td, J = 4.2, 6.5, 1H), 1.60 (s, 3H).

¹³ C NMR (126 MHz, CDC1 ₃ ) δ 166.31(C=0), 165.38(C=0), 165.32(C=0), 159.13(C2), 152.87(C6), 152.06(C4), 141.42(C8), 133.77(C-H), 133.69 (C-H), 133.28(C-H), 129.90(C- H), 129.82(C-H), 129.78 (C), 129.70(C-H), 129.41(C), 128.78(C), 128.61(C-H), 128.50(C- H), 128.41(C-H), 126.00(C5), 88.84(C1 '), 85.68(C2'), 79.43(C4'), 76.07(C3 '), 63.57(C5'), 17.77(2'-Me).

Example 2 - (2/?,3/?,4/?,5/?)-2-(2-Amino-6-methoxy-9H-purin-9-yl)-5-(hyd roxymethyl)-3- methyltetrahydrofuran-3,4-diol (Compound 3).

The compound is prepared as described in WO 2010/081082.

To a suspension of (2R,3R,4R,5R)-2-(2-amino-6-chloro-9H-purin-9-yl)-5- (benzoyloxymethyl)-3-methyltetrahydrofuran-3,4-diyl dibenzoate (3.0 g, 4.78 mmol) in methanol (36 mL) at 0 °C was added NaOMe in methanol (5.4 mL, 25% w/w). The mixture was stirred at room temperature for 24 h then quenched by addition of amberlite (H ⁺ ). The mixture was then filtrated and methanol was removed under reduced pressure. The resultant residue was dissolved in water (50 mL) and extracted with hexane (50 mL). The organic layer was then extracted with water (50 mL), and the combined water fractions were concentrated under reduced pressure. The residue was purified by silica gel chromatography (CHCl ₃ /MeOH 85: 15) to give the pure compound (1.125 g, 76 %) as a white solid. ^l H NMR (500 MHz, MeOH-i¾) δ 8.26 (s, 1H), 5.99 (s, 1H), 4.24 (d, J = 9.1, 1H), 4.08 (s, 3H), 4.04 (ddd, J = 2.3, 5.7, 8.6, 2H), 3.87 (dd, J = 3.0, 12.4, 1H), 0.96 (s, 3H).

¹³ C NMR (126 MHz, MeOH-i/ ₄ ) δ 162.75(C6), 161.86(C2), 154.50(C4), 139.35(C8), 115.36(C5), 93.00(C1 '), 84.15(C4'), 80.34(C2'), 73.57(C3'), 61.17(C5'), 54.25(6-OMe), 20.35(2'-Me).

Example 3 (Compound 1)

2,2-Dimethylpropyl 2(5)-((((2/?,3/?,4/?,5/?)-5-(2-amino-6-methoxy- 9H-purin-9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy(naphthalene-l-yloxy) phosphorylamino)propanoate

The compound can be prepared as described in WO 2010/081082. Alternatively the compound can be prepared as follows:

Step 1: (25)-neopentyl 2-(chloro(naphthalen-l-yloxy)phosphorylamino)-propanoate

L-Alanine neopentyl ester tosylate salt (1.5 g), was combined with naphthalene- 1-yl phosphorodichloridate (1.18 g), TEA (1.26 mL), and DCM (20 mL) to give (2S)-neopentyl 2- (chloro(naphthalen-l-yloxy)phosphorylamino)-propanoate in an 81% yield (1.4 g) as a pale yellow thick oil: ^l H NMR (500 MHz, CDC1 ₃ ) δ 8.11 (m, 1H, H ₈ -napht), 7.89 (m, 1H, H ₅ -napht), 7.76 (m, 1H, H ₄ -napht), 7.66 - 7.53 (m, 3H, H ₆ , H ₇ , H ₂ -napht), 7.46 (td, J = 2.0, 8.0, 1H, H ₃ -napht), 4.54 (m, 0.5H, NH), 4.46 (m, 0.5H, NH), 4.41 - 4.30 (m, 1H, Ha), 3.99-3.84 (m, 2H, CH ₂ ester), 1.61 (d, J = 7.1, 1.5H, CH ₃ ala), 1.59 (d, J = 7.0, 1.5H, CH ₃ ala), 1.00 (s, 4.5H, tBu), 0.98 (s, 4.5H, tBu).

³¹ P NMR (202 MHz, CDC1 ₃ ) δ 8.25, 7.96.

Step 2: (25)-neopentyl 2-((naphthalen-l-yloxy)(4-nitrophenoxy)phosphorylamino)propa noate (Compound 2)

To a mixture of -nitrophenol (7.0 g, 0.05 mol) and (2S)-neopentyl 2-(chloro(naphthalen-l- yloxy)phosphorylamino)propanoate (from Step 1) (19.27 g, 0.05 mol) in THF (100 mL) was added triethylamine (7.01 mL, 0.05 mol) at 0 °C. The reaction mixture was stirred at 0 °C for 1.5 h and the progress of the reaction monitored by ³¹ P NMR spectroscopy. The solids were filtered and the filtrate was concentrated and purified by column chromatography using gradient mixture of hexane and ethyl acetate (Hexane:EtOAc, 80:20) to give 19.0 g (78%) of Compound 2 (mixture of diastereomers). ^l H NMR (200 MHz, CDC1 ₃ ): δ 8.4 (bs, 0.31H), 8.2 (d, J = 10 Hz, 1.78H), 8.02 (m, 1.81 H), 7.85 (m, 0.97H), 7.7 (d, J =9.8 Hz, 0.96H), 7.6-7.3 (m, 5.65H), 6.75 (m, 0.95H), 4.4-4.1 (m, 2.04H), 3.9-3.65 (m, 2H), 1.4 (dd, J = 4.4 Hz, J = 4.4 Hz, 3H), 0.9 (d, J = 5.2 Hz, 9H)

³¹ P MR (80 MHz, CDC1 ₃ ): δ -1.810, -1.840

Step 3: (25)-Neopentyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(naphthalen- 1 - yloxy)phosphorylamino)propanoate

To a suspension of (2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9-yl)-5- (hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol, compound 3 (200 mg, 0.64 mmol) in THF (7 mL) at 0 °C, was slowly added i-BuMgCl (1M in THF) (1.92 mL, 1.92 mmol). After stirring 15 min at 0 °C, (25)-neopentyl 2-((naphthalen-l-yloxy)(4- nitrophenoxy)phosphorylamino)propanoate, compound 2 from step 2 (1.09 g, 2.24 mmol) in THF (3 mL) was slowly added to the reaction mixture. The reaction mixture was stirred at RT overnight, diluted with EtOAc (15 mL). The solution was successively washed with 0.2 N HC1 (10 mL), saturated NaHC0 ₃ solution (10 mL), and brine, dried with anhydrous Na ₂ S0 ₄ , filtered and concentrated. The residue was purified by column chromatography using gradient mixture of CH ₂ C1 ₂ and MeOH (MeOH:CH ₂ Cl ₂ , 5:95) to give 415 mg of 2,2-dimethylpropyl 2(5)-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3, 4-dihydroxy-4- methyltetrahydrofuran-2-yl)methoxy(naphthalene- 1 -yloxy) phosphorylamino)propanoate, Compound 1, in 98% yield. ^l H NMR (200 MHz, CDC1 ₃ ) δ 8.18 - 8.10 (m, 1H), 7.93 (d, J = 5.0 Hz, 1H), 7.87 - 7.62 (m, 1H), 7.65 (t, J= 8.2 Hz, 1H), 7.52 - 7.31 (m, 4H), 5.96 (d, J = 3.0 Hz, 1H), 4.63 - 4.53 (m, 2H), 4.33 - 4.19 (m, 2H), 4.07-3.96 (m, 1H), 4.02 (s, 3H), 3.76 - 3.52 (m, 2H), 1.30 (d, J = 7.2 Hz, 3H), 0.95 - 0.91 (2 s, 3H), 0.84 -0.82 (2 s, 9H).

³¹ P NMR (50 MHz, CDC1 ₃ ) δ 5.37, 5.32. Example 4 (Compound lb)

(R)-P lb

As described in WO 2010/081082, a 1 : 1 mixture of diastereomers of 2,2-dimethylpropyl 2(5")-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3 ,4-dihydroxy-4- methyltetrahydrofuran-2-yl)methoxy(naphthalene- 1 -yloxy) phosphorylamino)propanoate (10.0 g, Compound 1, Example 3) was taken up in 1 : 1 ethanol : hexanes and loaded onto a Chiral Pak AD chiral column and eluted with the same solvent. UV detection was done at 300 nm. Analysis of the fractions was done on a Chiral Pak AD (5 um, 4.6 mm ID x 250 mm; S/N ADHOCE-LDOOl) chiral column at a flow rate of 1 mL/min and detection at 300 nM. Complete separation of the two diastereomers was obtained (see chromatographic trace Figure 30.

Peak 1 (Compound lb) with a retention time of 6 min on the analytical column, and provided 4.75 g, (95.9% yield) of a single diastereomer with 99.9% ee (enantiomeric excess).

The following are NMR results analyzing Peak 1 (Compound lb):

'H NMR (500 MHz, MeOD) δ 8.16 (d, J= 8.50 Hz, 1H, H ₈ -naph), 7.97 (s, 1H, H ₈ ), 7.85 (d, J = 7.50 Hz, 1H, Hs-napht), 7.67 (d, J = 8.00 Hz, 1H, H ₄ -napht), 7.52 - 7.45 (m, 3H, H ₂ , H ₇ , H ₆ -napht), 7.38 (t, J = 8.00 Hz, 1H, H ₃ -napht), 6.00 (s, 1H, H _r ), 4.68- 4.57 (m, 1H, ¾·), 4.31- 4.25 (m, 2H, Η ₃ ·, Η ₄ ·), 4.09 - 4.03 (m, 4H, Ha, 60CH ₃ ), 3.76, 3.64 (AB, JAB= 10.50 Hz, 2H, CH ₂ ester), 1.33 (d, J = 7.50 Hz, 3H, CH ₃ Ala), 0.96 (s, 3H, 2'CCH ₃ ), 0.87 (s, 9H, 3 x CH ₃ ester)

¹³ C NMR (126 MHz, MeOD) δ 175.05 (d, ³ J C-C-N-P = 5.00 Hz, C=0 ester), 162.73 (C6), 161.87 (C2), 154.55 (C4), 147.97 (d, ² J _C -o-p = 6.30 Hz, ipso Naph), 139.09 (CH8), 136.26 (ClO-Naph), 128.78 (CH-Naph), 127.89 (d, ³ J _C -c-o-p = 6.30 Hz, C9-Naph), 127.71, 127.46, 126.46, 125.93, 122.80 (CH-Naph), 1 16.17 (d, ³ J c-c-o-p = 2.50 Hz, C2-Naph), 115.58 (C5), 93.21 (CI '), 82.16 (d, ³ J _C -c-o-p = 8.80 Hz, C4'), 79.97 (C2'), 75.36 (CH ₂ ester), 74.69 (C3'), 67.64 (d, ² Jc-o-p = 5.00 Hz, C5'), 54.23 (60CH ₃ ), 51.78 (Ca Ala), 32.24 (C ester), 26.69 (3 x CH ₃ ester), 20.60 (d, ³ J _C -C-N-P = 6.30 Hz, CH ₃ Ala), 20.30 (2'CCH ₃ )

³¹ P NMR (202 MHz, CDC1 ₃ ) δ 4.22

Example 5 (Compound la)

As described above, and in WO 2010/081082, Peak 2, (Compound la), with a retention time of 10.7 min on the analytical column, provided 5.29 g (100% yield) of a single diastereomer with 99.8% diastereomeric excess. (95.9% yield) of a single diastereomer with 99.9% ee (enantiomeric excess).

YThe following are the NMR results analyzing the synthesized compound: lR NMR (500 MHz, MeOD) δ 8.20 - 8.18 (m, 1H, H ₈ -naph), 7.95 (s, 1H, H ₈ ), 7.89 - 7.87 (m, 1H, Hj-napht), 7.70 (d, J = 8.50 Hz, 1H, H ₄ -napht), 7.54 - 7.50 (m, 3H, H ₂ , H ₇ , H ₆ - napht), 7.41 (t, J = 7.00 Hz, 1H, H ₃ -napht), 5.99 (s, 1H, H _r ), 4.63- 4.55 (m, 1H, ¾·), 4.34 (d, J = 9.00 Hz, 1H, ¾ , 4.28 - 4.23 (m, 1H, Η ₄ ·), 4.08 - 4.03 (m, 4H, Ha, 60CH ₃ ), 3.72, 3.59 (AB, J _AB = 10.50 Hz, 2H, CH ₂ ester), 1.33 (d, J = 7.50 Hz, 3H, CH ₃ Ala), 0.98 (s, 3H, 2'CCH ₃ ), 0.85 (s, 9H, 3 x CH ₃ ester)

¹³ C NMR (126 MHz, MeOD) δ 174.78 (d, ³ J C-C-N-P = 6.00 Hz, C=0 ester), 162.72 (C6), 161.89 (C2), 154.51 (C4), 148.00 (d, ² J _C -o-p = 7.00 Hz, ipso Naph), 139.38 (CH8), 136.30 (ClO-Naph), 128.84 (CH-Naph), 127.88 (d, ³ J _C -c-o-p = 6.30 Hz, C9-Naph), 127.74, 127.46, 126.51, 125.95, 122.76 (CH-Naph), 1 16.21 (d, ³ J c-c-o-p = 3.20 Hz, C2-Naph), 115.60 (C5), 93.37 (CI '), 82.32 (d, ³ J _C -c-o-p = 8.00 Hz, C4'), 79.91 (C2'), 75.34 (CH ₂ ester), 74.94 (C3'), 68.12 (d, ^J J _C -o-p = 5.00 Hz, C5'), 54.21 (60CH ₃ ), 51.71 (Ca Ala), 32.21 (C ester), 26.66 (3 x CH ₃ ester), 20.81 (d, ³ J _C -C-N-P = 6.30 Hz, CH ₃ Ala), 20.27 (2'CCH ₃ )

³¹ P NMR (202 MHz, CDC1 ₃ ) δ 4.28

Analysis of Solid State Attributes of Compounds 1, la and lb

The three materials: Compound 1, Compound lb and Compound la (Examples 3, 4 and 5 respectively) were analyzed by the following analytical techniques: FT-Raman spectroscopy, FT-IR spectroscopy, conventional and modulated differential scanning calorimetry (DSC and mDSC, respectively), thermogravimetric analysis (TGA), TGA-IR, high-performance liquid chromatography (HPLC), dynamic vapor sorption (DVS), polarized light microscopy (PLM), and X-ray Powder Diffraction (XRPD). A comparison of the selected physicochemical data of the three materials is presented in Figures 1 -5.

PLM and XRPD analyses of the supplied batches confirm that the materials are amorphous white powders. FT-IR and FT-Raman spectra of the three batches are nearly indistinguishable. TGA analyses of 1, la, and lb reveal weight-loss of 2.3% wt, 2.6% wt, 0.6% wt up to 170 °C, corresponding to a loss of water/2-propanol, water/2-propanol, and water, respectively. The conventional DSC analysis indicated endothermic transitions at 66.6 °C, 60.6 °C, and 86.1 °C, correspondingly. Modulated DSC confirmed that these transitions closely correlate to the glass transition temperatures: 70.5 °C, 69.3 °C, and 86.3 °C, respectively. DVS analysis revealed that the amorphous batches are hygroscopic, absorbing up to 7% wt, 6.5% wt, and 4.5% wt of water between 0-90% RH, respectively. HPLC analyses indicated high purity of all three materials (>99%) and highlighted the ability of the method to resolve the two diastereomers present in Compound 1

Crystalline Solvates of Formula (la)

(la)

Crystallization attempts utilizing la (Example 5) yielded a series of crystalline solvates (>40) of formula (la). A list of solvents (X) producing these solvates and the corresponding approximate crystallization rates are provided in Table 3. Generally, the formula (la) compound crystallizes relatively slowly. In particular, in solvents in which (la) exhibits solubility lower than 10 mg/mL (e.g., heptane) spontaneous crystallization may occur within several weeks. However, the crystallization rates can be significantly increased in the presence of a small amount of solvent in which the (la) product exhibits high solubility. For instance, in a toluene suspension, (la) crystallizes within a week; whereas addition of 10%vol of acetone to the toluene suspension decreases the crystallization rate to 1 day. Furthermore, the type of antisolvent used in crystallization mixture influences the crystallization rate and the relative ranking of several "poor" solvents is presented, according to their ability to facilitate crystallization: anisole>p-xylene>toluene>cyclohexane >MTBE>hexane>heptane

Table 3 - Solvents That Yielded Crystalline Solvates in Experiments Involving la and Approximate Rates of Crystallization

Ratio Approximate

Solvent 1 Solvent 2 Solventl : Crystallization

Solvent2 Rate [days]

Varied during

<6

1,4-Dioxane Cyclohexane experiment

Varied during

1

1,4-Dioxane (-Xylene experiment l-Methoxy-2- Varied during

<6 propanol MTBE experiment

Varied during

1

1-Propanol p-Xylene experiment

Varied during

<6

1-Propanol Toluene experiment

Varied during

7

2-Butanone Cyclohexane experiment

Varied during

1

2-Butanone Toluene experiment

2- Varied during

1

Methoxyethanol (-Xylene experiment

Varied during

3

2-Propanol (-Xylene experiment

4-Methyl-2- Varied during

<6 pentanone (-Xylene experiment

Acetone Anisole Varied during 1 Ratio Approximate

Solvent 1 Solvent 2 Solventl : Crystallization

Solvent2 Rate [days] experiment

Varied during

7

Acetone Cyclohexane experiment

Varied during

2

Acetone Heptane experiment

Varied during

2

Acetone MTBE experiment

Varied during

Acetone <3

Pentane experiment

Varied during

1

Acetone Toluene experiment

Acetone Water 20:80 14

Varied during

<6

Acetonitrile (-Xylene experiment

Anisole n/a 1

Varied during

7

Chlorobenzene Hexane experiment

Varied during

14

Chlorobenzene (-Xylene experiment

Varied during

1

Chloroform Toluene experiment

Varied during

7

Cyclohexanone Hexane experiment

Varied during

7

Dichloromethane Cyclohexane experiment

Varied during

14

Dichloromethane Toluene experiment

Varied during

<6

Ethanol Toluene experiment

Varied during

<6

Ethyl Acetate Hexane experiment

Ethyl Acetate Cyclohexane 1 :2 <6

Varied during

2

Ethyl Acetate Heptane experiment

Varied during

14

Isopropyl acetate (-Xylene experiment

Varied during

1

Methanol (-Xylene experiment

Varied during

<6

Methyl acetate Cyclohexane experiment

Varied during

1

Methyl acetate /(-Xylene experiment

Varied during

<6

Nitromethane MTBE experiment

Varied during

8

Tetrahydrofuran Heptane experiment

Varied during

1

Tetrahydrofuran /(-Xylene experiment

Tetrahydrofuran Water 5:95 8

Toluene n/a 7 A high number of solvated crystal-forms were isolated (>40), which indicates a high propensity of la to form solvated states (la) . Comparison of FT-Raman spectra of the respective solvates indicates slight differences in the peak positions, mainly in the 1200-1350 cm ^"1 region. The XRPD patterns of the solvates exhibit close similarity, indicating that the solvates are closely related with respect to the arrangement of molecules in the crystal packing. Overlays of the FT-Raman spectra and XRPD patters of several solvates are presented in Figure 6.

A diverse set of fifteen of the solvates (la) were studied in more details to evaluate their solid state characteristics. The key attributes of these solvates are shown in Table 4 and can be collectively described as follows:

Solvates are highly crystalline by PLM and XRPD.

Desolvation on-set occurs above 100°C, even for solvates containing low boiling point solvents (e.g. pentane or acetone; analysis based on DSC measurement of non-hermetically sealed pans)

Desolvation onsets are highest for solvates containing high-boiling solvents (e.g., anisole, p- xylene, and toluene)

Solvates may contain a single solvent (e.g., acetone solvate) or a mixture of solvents (e.g., acetone/pentane solvate) as confirmed by IR analysis of the gas evolved upon heating (TGA- IR).

In most cases, loss of solvent on heating proceeds via two steps, as indicated by TGA traces. Whereas the first weight loss step is relatively distinct and can be integrated, the second step is gradual and overlaps with the decomposition of 1. While such desolvation mechanism did not allow for the determination of the exact solvent content for majority of solvates, evidence suggest that the included solvent amounts are sub-stoichiometric (<0.5 molar equivalent)

Solvates melt upon desolvation via heating and remain amorphous after the subsequent cooling, as confirmed by variable temperature XRPD analysis or ^-xylene and toluene solvates Solvates are physically stable (no change in FT-Raman and/or XRPD data over time) for at least four weeks of storage in an enclosed container or on drying under vacuum and elevated temperatures (e.g., 40 °C)

No hydrates were isolated from water-containing solvent systems

Table 4 - Selected Crystalline Solvates (la) and Their Properties

ND-not determined

RT- room temperature

Vac - vacuum

Crystallization attempts of compound 1, (Example 3) resulted in optical resolution of the R- P and S-P diastereomers. Specifically, the separation was based on precipitation of S-P diastereomer as solvated crystalline solid and dissolution of the amorphous R-P diastereomer. The optical separation was confirmed by HPLC analysis of the produced solids and indicated absence of R-P diastereomer. The optical purity of the isolated S-P diastereomer can be achieved using a suitable crystallization solvent system that allows full dissolution of the amorphous R-P diastereomer, therefore facilitating the filtration/isolation of S-P diastereomer. A list of solvents that facilitated crystallization is provided in Table 5. Table 5. Solvents That Yielded Crystalline Products (la) in Experiments Involving Compound 1 (Example 3).

Further details on preparation and physicochemical attributes of the Solvates (la), prepared either from Compound 1 (Example 3) or Compound la (Example 5) are presented in Examples 6-14 below.

Example 6 - Preparation of the /J-Xylene Solvate of Compound la

This solvate was isolated from a wide range of (-xylene containing solvent systems. Selected physicochemical data of the (-xylene solvate are presented in Figure 7. A sample of amorphous la (160 mg) was weighed into a 2-mL vial and combined with p- xylene (1.0 mL). The suspension was stirred for 15 min at 25 °C. 1-Propanol (0.45 mL) was added. The initially free-flowing powder turned into a gum. The gum was subjected to stirring at 25 °C for 20 h, over which time a white powder formed. The obtained powder was isolated by vacuum filtration, air-dried for 1 h, and dried at 50 °C under vacuum for 6 h.

Example 7 - Preparation of the /J-Xylene solvate of Compound la from Compound 1

The compound 1 (from Example 3) (160 mg) was weighed into a 2 mL vial and combined p- xylene (1.0 mL). The suspension was stirred for 15 min at 25 °C. 1-Propanol (0.45 mL) was added. The initially free-flowing powder turned into a gum. The gum was subjected to stirring at 25 °C for 20 h, over which time a white powder formed. The obtained powder was isolated on by vacuum filtration and air-dried for 4 h.

Characterization of the powder confirmed it to be the ^-xylene solvate . FT-Raman, XRPD, and HPLC data collected on the product were consistent with data of the solvate obtained using Compound (la) alone ((5)-P diastereomer; Figures 6, 7) together with the results for other solvates obtained (see Examples 10-19 below) indicated that separation of the diastereomers can be achieved due to the difference in solubility between the crystalline solvate and the amorphous material. This indicates that optical resolution of the respective diastereomers occurs via solvate formation of the («S)-P diastereomer. The result indicates that diastereomeric purity ((R)-P vs. (5)-P) can be achieved because of the difference in solubility between the crystalline solvate of (la) and the amorphous diastereomer (lb) that remains in solution.

Example 8 - Preparation of /J-Xylene Solvate of Compound la for Single Crystal X-ray Diffraction Analysis

A sample of amorphous (la) (80 mg) was weighed into a 2 mL vial and combined with p- xylene (1.0 mL). The suspension was stirred for 15 min at 25 °C. Ethyl acetate (0.3 mL) was added. The initially free-flowing powder turned into a gum. The gum was subjected to stirring at 25 °C for 48 h. The suspension was filtered using a syringe equipped with 0.22 μιη filter disk. The solution was cooled to 22 °C and held for 24 h. The obtained single crystals, immersed in the mother liquor, were submitted for SCXRD analysis.

Physicochemical Details of ^-Xylene Solvate The ^-xylene solvate obtained in Examples 6-8 is a white solid that is crystalline, as indicated by XRPD and PLM. (see Figure 6) Thermal analysis indicated that the solvent content (3.8 + 2.8 = 6.6% total weight loss) closely corresponds to a hemi-solvate composition (theoretical % wt. loss of 1 equiv of -xylene = 6.9% wt). The presence of -xylene was confirmed by IR analysis of the gas evolved upon heating. DSC analysis indicates desolvation endotherm between 130-145 °C, followed by decomposition. The absence of another crystalline form after heating the solvate past desolvation temperature and the subsequent cooling to room temperature was confirmed by variable-temperature XRPD. The ^-xylene solvate is physically stable for at least four weeks when stored in an enclosed container. Drying studies conducted at 50 °C and under vacuum for 72 h led to a ~1% solvent loss; however this operation did not induce a change in the crystal-form. Washing the ^-xylene solvate with pentane did not remove ^-xylene from the compound nor lead to a form change.

Suitable crystals of ^-xylene solvate were grown using la as the input material and characterized by single crystal X-ray diffraction. The single crystal structure of this solvate confirmed the following:

1. The assigned atomic connectivity for formula la

2. The 1 :2 ^-xylene: API stoichiometry (formula (la) n =

0.5

3. (S)-P stereochemistry around the chiral phosphorous

atom (i.e., the absolute configuration)

4. Consistency between the experimental and calculated

XRPD patterns, i.e. phase purity of the bulk ^-xylene

solvate material.

The selected crystallographic data are presented in Figure 8.

Example 9 - \Preparation of the Anisole Solvate of Compound la

A sample of la (1.02 g) was weighed into a 20-mL vial and combined with anisole (2.0 mL). The initially free-flowing powder turned into a gum. The gum was seeded with anisole solvate (~2 mg) and was subjected to stirring at 20 °C for 48 h, over which time a white powder formed. The obtained free-flowing suspension was cooled to 5 °C and stirred for 2 h. The white solid was isolated on Buchner funnel by filtration, washed with pre-cooled anisole (5 °C; 3 mL), air-dried for 1 h, and dried at 40 °C under vacuum for 24 h. Yield was approximately 77%.

Example 10

Preparation of the Anisole solvate of Compound la from Compound 1

The sample of 1 (0.83 g) was weighed into a 20-mL vial and combined with anisole (3.0 mL). The initially free-flowing powder turned into a gum. The gum was seeded with anisole solvate (~2 mg) and was subjected to stirring at 20 °C for 5 days, over which time a white powder formed. The obtained powder was isolated on Buchner funnel by filtration, air-dried for 1 h, and dried at 40 °C under vacuum for 12 h. Yield was approximately 25%. HPLC analysis of the isolated solid confirmed absence of (5)-P diastereomer.

Physicochemical Details of Anisole Solvate

Anisole solvate obtained from either of the prior two Examples 11 or 12 is a white solid that is crystalline, as indicated by XRPD and PLM. TGA analysis indicated that the solvent content (2.6 + 1.4= 4.0% total weight loss) corresponds to a non-stoichiometric composition (theoretical % wt. loss of 1 equiv. of anisole = 14.0% wt). The presence of anisole was confirmed by IR analysis of the gas evolved upon heating. DSC analysis indicates a sharp desolvation endotherm at 137.2 °C, followed by decomposition. Dynamic vapor sorption analysis indicates the solvate is non-hygroscopic (-0.3% water uptake between 0-90%RH). Anisole solvate maintains its integrity and crystallinity after drying at 40 °C under vacuum for 72 h and in aqueous suspension at 25 °C for 24 h.

Selected physicochemical data of this solvate are illustrated in Figures 9-10.

Example 11 - Preparation of the Ethyl Acetate/Heptane Solvate of Compound la

A sample of la (0.96 g) was weighed into a 20-mL vial and combined with heptane (4.0 mL). The suspension was stirred for 15 min at 25 °C. Ethyl acetate (1.0 mL) was added. The initially free-flowing powder turned into a gum. The gum was seeded with ethyl acetate/heptane solvate (~2 mg and was subjected to stirring at 20 °C for 5 days, over which time a white powder formed. The obtained free-flowing suspension was cooled to 5 °C and stirred for 3 h. The white solid was isolated on Buchner funnel by filtration, washed with pre- cooled heptane (5 °C; 5 niL), air-dried for 1 h, and dried at 40 °C under vacuum for 12 h. Yield was approximately 75%.

Example 12 - Preparation of the Ethyl Acetate/Heptane Solvate of Compound la from Compound 1

The sample of 1 (0.96 g;) was weighed into a 20-mL vial and combined with heptane (4.0 mL). The suspension was stirred for 15 min at 25 °C. Ethyl acetate (8.0 mL) was added. The initially free-flowing powder turned into a gum. The gum was seeded with ethyl acetate/heptane solvate (~2 mg) and was subjected to stirring at 25 °C for 5 days, over which time a white powder formed. The obtained free-flowing suspension was cooled to 5 °C and stirred for 3 h. The white solid was isolated on Buchner funnel by filtration, air-dried for 1 h, and dried at 40 °C under vacuum for 12 h. Yield was approximately 25%. HPLC analysis of the isolated solid confirmed absence of the (S)-P diastereomer.

Physicochemical Details of Ethyl Acetate/Heptane Solvate

Ethyl acetate/heptane solvate obtained in either of the Examples 13 or 14 is a white solid that is crystalline, as indicated by XRPD and PLM. TGA analysis indicated that the solvent content (1.4 + 0.5 = 1.9% total weight loss) corresponds to a non-stoichiometric composition (theoretical % wt. loss of 1 equiv. of ethyl acetate = 1 1.8% wt or 1 equiv of heptane = 13.0% wt). The presence of both solvents was confirmed by IR analysis of the gas evolved upon heating. DSC analysis indicated desolvation endotherm between 1 10-125 °C, followed by decomposition. Dynamic vapor sorption analysis indicates the solvate is non-hygroscopic (-0.7% water uptake between 0-90% RH). Ethyl acetate/heptane solvate remains crystalline after drying at 40 °C under vacuum for 72 h. The solvate maintains its integrity and crystallinity in aqueous suspension at 25 °C for 24 h.

Selected physicochemical data of this solvate are illustrated in Figures 1 1-12.

Example 13 - Preparation theAcetone/Pentane Solvate of Compound la

The sample of la (1.01 g) was weighed into a 20-mL vial and combined with pentane (4.5 mL). The suspension was stirred for 15 min at 25 °C. Acetone (0.5 mL) was added. The initially free-flowing powder turned into a gum. The gum was seeded with acetone/pentane solvate (~2 mg) and subjected to stirring at 25 °C for 72 h, over which time a white powder formed. The obtained suspension was cooled to 5 °C and stirred for 3 h. The white solid was isolated on Buchner funnel by filtration, washed with pre-cooled pentane (5 °C; 5 mL), air- dried for 1 h, and dried at 40 °C under vacuum for 72 h. Yield was approximately 78%.

Example 14 - Preparation Acetone/Pentane Solvate of Compound la from Compound 1

The sample of 1 (1.01 g) was weighed into a 20-mL vial and combined with pentane (4.5 mL). The suspension was stirred for 15 min at 25 °C. Acetone (5.5 mL) was added. The initially free-flowing powder turned into a gum. The gum was seeded with acetone/pentane solvate (~2 mg) and was subjected to stirring at 20 °C for 5 days, over which time a white powder formed. The obtained free-flowing suspension was cooled to 5 °C and stirred for 3 h. The white solid was isolated on Buchner funnel by filtration, air-dried for 1 h, and then dried at 40 °C under vacuum for 12 h. Yield was approximately 27%. HPLC analysis of the isolated solid confirmed absence of the (5)-P diastereomer.

Physicochemical Details of Acetone/Pentane Solvate

Acetone/pentane solvate obtained from either of the Examples 15 or 16 is a white solid that is crystalline, as indicated by XRPD and PLM. TGA analysis indicated that the solvent content (1.0 + 1.7 = 2.7% total weight loss) closely corresponds to a non-stoichiometric composition (theoretical % wt loss of 1 equiv. of acetone = 8.1% wt or 1 equiv of pentane = 9.8% wt). The presence of both solvents was confirmed by IR analysis of the gas evolved upon heating. DSC analysis indicates desolvation endotherm between 100-125 °C, followed by decomposition. Dynamic vapor sorption analysis indicates the solvate is non-hygroscopic (-0.9% water uptake between 0-90% RH). Acetone/pentane solvate maintains its integrity and crystallinity after drying at 40 °C under vacuum for 72 h and in aqueous suspension at 25 °C for 24 h. Grinding the solvate material for 15 min using mortar and pestle causes a partial loss of solvent and crystallinity, as indicated by TGA and XRPD analyses of the ground material.

Selected physicochemical data of this solvate are presented in Figures 13 and 14

Example 15 - Preparation of Compound 2 (25)-neopentyl 2-((naphthalen-l-yloxy)(4- nitrophenoxy)phosphorylamino)propanoate sufficiently pure for crystallization

To a mixture of -nitrophenol (5) (7.0 g, 0.05 mol) and (2S)-neopentyl 2-(chloro(naphthalen- l-yloxy)phosphorylamino)propanoate (4) (19.27 g, 0.05 mol) in THF (100 mL) was added triethylamine (7.01 mL, 0.05 mol) at 0 °C. The reaction mixture was stirred at 0 °C for 1.5 h and the progress of the reaction monitored by ³¹ P NMR spectroscopy. The solids were filtered and the filtrate was concentrated and purified by column chromatography using gradient mixture of hexane and ethyl acetate (Hexane:EtOAc, 80:20) to give 19.0 g of Compound 2, as an amorphous oil.

'H NMR (200 MHz, CDC1 ₃ )*: δ 8.4 (bs, 0.31H), 8.2 (d, J = 10 Hz, 1.78H), 8.02 (m, 1.81 H), 7.85 (m, 0.97H), 7.7 (d, J =9.8 Hz, 0.96H), 7.6-7.3 (m, 5.65H), 6.75 (m, 0.95H), 4.4-4.1 (m, 2.04H), 3.9-3.65 (m, 2H), 1.4 (dd, J = 4.4 Hz, J = 4.4 Hz, 3H), 0.9 (d, J = 5.2 Hz, 9H). This ^L H NMR data has some impurities along with -nitrophenol.

³¹ P MR (80 MHz, CDC1 ₃ ): δ -1.810, -1.840

It was observed that Compound 2 contained p-nitrophenol (5) along with two other impurities. Impure 2 was purified again by column chromatography using a gradient mixture of hexane and ethyl acetate (Hexane:EtOAc, 90: 10) to (Hexane :EtOAc, 85: 15) to give 5.8 g (0.012 mol) of pure Compound 2.

The residue after evaporation of total solvent was characterized using FT-Raman spectroscopy, HPLC PLM, DSC, TGA, and ³¹ P NMR. The data are provided in Figures 15- 19. The material was shown to be a mixture of the two diastereomers 2a and 2b, as determined by ³¹ P NMR (Figure 19). HPLC analysis of the material showed relatively high purity (>98% AUC), but the two diastereomers were not resolved using current chromatographic conditions. TGA analysis of the material shows 3.7% weight loss up to 150 °C, which could be due to the presence of residual solvent.

Example 16

Crystallization of Compound 2

Preparation of Crystalline Form A

Methyl cyclohexane (500 μ was added to amorphous 2 obtained in by the purification procedure described in Examplel5 (20 mg) and the suspension was stirred at RT for 5 min. Isobutyl acetate (25 μΐ,) was added. The suspension was stirred while cycling the temperature between 25 °C and 5 °C for 12 h. The suspension was cooled to 5 °C and stored at 5 °C for 48 h. The solids were isolated by vacuum filtration and air dried for 15 min to provide a sample of Crystalline Form A.

Crystalline Form A is a crystalline solid form of 2 as confirmed by XRPD. It is mixture of diastereomers 2a and 2b. PLM image shows crystalline, rod-like habit. DSC curve exhibits two endothermic events having maxima at 86 °C and 93 °C. Characterizing data for Form A are provided in Figure 20.

Preparation of Crystalline Form AB.

Methyl cyclohexane (10 mL) was added to the amorphous Compound 2 (995.7 mg) followed by addition of seed crystals (~10 mg). The suspension was stirred at RT for 5 min followed by addition of isobutyl acetate (0.5 mL). The suspension was stirred at RT during which time precipitation occurred (within 30 min). The suspension was stirred at RT for 12 h, heated to 50 °C, and stirred for 2 h during which time partial dissolution of the solids occurred. The suspension was cooled to 5 °C (0.1 °C/min) and stirred overnight. The solids were isolated by vacuum filtration and air dried for 45 min. The yield of crystallization was -74%.

The product is a mixture of diastereomers 2a and 2b and is a crystalline solid as confirmed by XRPD. PLM data indicate a crystalline rod-like habit. DSC data show two endothermic events with a major event occurring at 73.8 °C. TGA data shows no significant weight loss below 200 °C, indicating that the products are not solvated. Characterizing data for Crystalline Form AB are provided in Figure 21.

Alternatively a larger scale preparation of Crystalline Form AB could be carried out as follows:

Methylcyclohexane (56.55 mL) was added to the liquid compound 2 (5.8 g) followed by addition of seed crystals (10 mg). The suspension was stirred at RT for 5 min followed by addition of isobutyl acetate (2.88 mL). The suspension was stirred at RT during which precipitation occurred and stirring was continued at RT overnight, then heated to 45 °C for 2 h. The suspension was cooled to 5 °C and stirred overnight. The solids were filtered and dried under vacuum to obtain 4.6 g (79%) of Crystalline Form AB. ^l H NMR (200 MHz, CDC1 ₃ ): δ 8.23 - 8.17 (m, 2H), 8.06 - 8.01 (m, 1H), 7.88 - 7.84 (m, 1H), 7.71 - 7.67 (m, 1H), 7.57 - 7.49 (m, 3H), 7.45 - 7.37 (m, 3H), 4.35 - 4.16 (m, 1H), 4.07 - 3.93 (m, 1H), 3.80 (q, J = 10.6 Hz, 1H), 3.76 (q, J = 10.6 Hz, 1H), 1.42 (d, J = 7.0 Hz, 1.5H), 1.40 (d, J = 7.0 Hz, 1.5H), 0.91 (s, 4.5H), 0.88 (s, 4.5H).

³¹ P NMR (80 MHz, CD ₃ OD): δ -1.83, -1.89

Example 17 - Preparation of Crystalline 2a as a Single Diastereomer

2b

Isobutyl acetate/Methylcyclohexane (9:91, 23 niL) was added to the Crystalline Form AB solids (obtained in Example 16) (4.6 g). The suspension was stirred at 40 °C overnight. The solids were filtered and dried under vacuum to obtain 1.5 g (32%) of crystalline material labeled Crystalline Form B.

^L H NMR (200 MHz, CDC1 ₃ ): δ 8.23 - 8.17 (m, 2H), 8.06 - 8.01 (m, 1H), 7.90 - 7.82 (m, 1H), 7.71 - 7.67 (m, 1H), 7.57 - 7.49 (m, 3H), 7.45 - 7.37 (m, 3H), 4.35 - 4.15 (m, 1H), 4.08 - 3.98 (m, 1H), 3.76 (q, J = 10.6 Hz, 2H), 1.41 (d, J = 7.0 Hz, 3H), 0.89 (s, 9H).

³¹ P MR (80 MHz, CD ₃ OD): δ -1.81

The crystallinity of Crystalline Form B is shown by XRPD in Figure 22. The ³¹ P NMR of Crystalline Form B shows that only one diastereomer is present. The absolute stereochemistry was not determined directly; however experiments with this material, described below, demonstrate that it is the phosphorous diastereomer 2a. The DSC curve for Crystalline Form B, shows a sharp single endothermic event occurring at 104.2 °C (onset value) with a peak at 107.0 °C. TGA data for the material shows no significant weight loss below 200 °C, indicating that it is non-solvated. Characterizing data for Crystalline Form B of Compound 2a are provided in Figure 22.

Comparison of Crystalline Forms A, AB and B The distinct nature of Crystalline Forms A, AB and B is illustrated in the overlay of their XRPD and DSC profiles in Figure 23. Crystalline Form B displays a single sharp endotherm at 107 °C, which appears advantageous over Crystalline Forms A or AB which show multiple, broad endotherms. The XRPD data for Crystalline Form B shows that it is peaks are a subset of the Crystalline Form AB peaks and that they are substantially different from Crystalline Form A.

Example 18

Synthesis of the Crystalline Anisole Solvate of Compound la starting from Compounds 2 and 3.

[(la) (X = anisole, n = 0.5)] To a suspension of Compound 2 (a diastereomeric mixture, 500 mg, 1.6 mmol) in THF (20 mL) at 0 °C, /-BuMgCl (1M in THF) (4.8 mL, 4.8 mmol) was slowly added. After stirring 15 min at 0 °C, Compound 3 (2.72 g, 5.6 mmol) in THF (7 mL) was slowly added to the reaction mixture. The reaction mixture was stirred at RT overnight, diluted with EtOAc (20 mL). The solution was successively washed with 0.2 N HC1 (15 mL), saturated aHC0 ₃ solution (15 mL), and brine, dried with anhydrous Na ₂ S0 ₄ , filtered and concentrated to obtain 3.2 g (300%) of crude compound la. The crude material la was purified and recrystallized in the following steps.

Step 1 : The crude material la (3.2 g) was stirred with isobutyl acetate/methylcyclohexane (5:95, 40 mL) at RT overnight. The solution was decanted and the residue dried in high vacuum to obtain 2.6 g (246%) of crude la.

Step 2: The above residue (2.6 g) was again stirred with isobutyl acetate/methylcyclohexane (9:91, 40 mL) at 40 °C overnight. The solution was decanted and the residue dried in high vacuum to obtain 1.5 g (142%) of brown solid la.

Step 3 : The above brown solid material (2.6 g, from step 2) was dissolved in anisole (25 mL) and heated to 80 °C for 15 min; the solution was allowed to cool to RT and left overnight at RT. The precipitated solid was filtered, dried in high vacuum to obtain 780 mg of the anisole solvate of Compound la as a white solid and single diastereomer. ^l H NMR (200 MHz, CD ₃ OD): δ 8.18 - 8.14 (m, 1H), 7.92 (s, 1H), 7.89 - 7.84 (m, 1H), 7.68 (d, J = 7.8 Hz, 1H), 7.53 - 7.48 (m, 2H), 7.40 (q, J = 7.8 Hz, 1H), 7.25 (t, J = 7.8 Hz, 1H), 5.95 (s, 1H), 4.58 - 4.54 (m, 2H), 4.29 (t, J = 10 Hz, 1H), 4.28 - 4.19 (m, 1H), 4.07 - 3.92 (m, 1H), 4.02 (s, 3H), 3.64 (q, J = 10 Hz, 2H), 1.30 (d, J = 7.1 Hz, 3H), 0.95 (s, 3H), 0.83 (s, 9H).

³¹ P NMR (80 MHz, CD ₃ OD): δ 5.372

Example 19 - Diastereoselective Synthesis of the Anisole Solvate of Compound la Starting from Compounds 2a and 3

(la), X = anisole

To a suspension of (2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9-yl)-5- (hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (Compound 3) (300 mg, 0.96 mmol) in THF (10 mL) at 0 °C was added /-BuMgCl (1M in THF) (2.88 mL, 2.88 mmol). After stirring 15 min at 0 °C, (25)-neopentyl 2-((naphthalen-l-yloxy)(4-nitrophenoxy) phosphorylamino) propanoate (Compound 2a) (1.5 g, 3.08 mmol) in THF (3 mL) was slowly added to the reaction mixture. The reaction mixture was stirred at RT overnight, diluted with EtOAc (20 mL). The solution was successively washed with 0.2 N HC1 (15 mL), saturated NaHC(¾ solution (15 mL), and brine, dried with anhydrous Na ₂ S0 ₄ , filtered and concentrated to obtain 1.78 g of crude compound la. ³¹ P NMR of this material showed only a single peak, indicating only one diastereomer had formed (See figures 24-26). The crude material la was purified in the following steps.

Step 1: The crude (2S neopentyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)- 3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(naphthal en-l- yloxy)phosphorylamino)propanoate (la) (1.78 g) was stirred with isobutyl acetate/methylcyclohexane (9:91, 40 mL) at 40 °C overnight. The solution was decanted and the residue dried in high vacuum to obtain 0.9 g (142%) of crude la which contains some p- nitro phenol and -nitro phenol phosphoramide. Step 2: The above residue (0.9 g) was again stirred with isobutyl acetate/methylcyclohexane (9:91, 25 mL) at 40 °C for 5 h. The solution was decanted and the residue dried in high vacuum to obtain 0.825 g (130%) of la.

Step 3: The above light yellow solid material (0.825 g, from step 2) was dissolved in anisole (5 mL) and heated to 80 °C for 15 min; the solution was allowed to cool to RT and left overnight at RT. The precipitated solid was filtered, washed with anisole (2 mL), dried in high vacuum to obtain 530 mg (0.80 mmol, 84%) single diastereomer as the crystalline anisole solvate of Compound la. The ³¹ P NMR is shown in Figure 28. The ability of this material to crystallize is consistent with its structural assignment as the anisole solvate of Compound la, since no crystalline solvates of Compound lb had been observed up to this time. Its absolute configuration was confirmed by examining the ³¹ P NMR of an admixture of a sample of the obtained compound with authentic la (Figure 29) and the ³¹ P MR of an admixture of authentic lb (Figure 27). The single peak of its ³¹ P NMR spectrum is superimposable with authentic la, and clearly distinct from the single peak of authentic lb. ^l H NMR (200 MHz, CD ₃ OD): δ 8.18 - 8.14 (m, 1H), 7.92 (s, 1H), 7.89 - 7.84 (m, 1H), 7.68 (d, J = 7.8 Hz, 1H), 7.53 - 7.48 (m, 2H), 7.40 (q, J = 7.8 Hz, 1H), 7.25 (t, J = 7.8 Hz, 1H), 5.95 (s, 1H), 4.58 - 4.54 (m, 2H), 4.29 (t, J = 10 Hz, 1H), 4.28 - 4.19 (m, 1H), 4.07 - 3.92 (m, 1H), 4.02 (s, 3H), 3.64 (q, J = 10 Hz, 2H), 1.30 (d, J = 7.1 Hz, 3H), 0.95 (s, 3H), 0.83 (s, 9H).

³¹ P NMR (80 MHz, CD30D): δ 5.387

Further to the above Examples, representative compounds, prepared according to the examples were tested for potency in an HCV replicon assay (Genotype lb) for activity against the virus (EC50) and toxicity to the cells (CC50). These results are set forth below.

Huh7 Replicon Cell Lines and Cell Culture Conditions. A luciferase-reporter genotype lb subgenomic replicon cell line was obtained from Apath, LLC, Brooklyn, NY: Cells were passaged twice a week by splitting 3 or 4 fold. Cells were maintained in DMEM-high glucose medium (HyClone, Logan, UT) supplemented with 9% FBS (HyClone), 2 mM glutamine (Invitrogen, Carlsbad, CA), 100 U/mL PenStrep (Invitrogen). Media also contained 0.25 mg/mL of the antibiotic G-418 to maintain stable expression of the replicon (Invitrogen). Incubation was performed at 37 °C in 5% CO ₂ atmosphere. Replicon cell lines were used until they accumulated 15-to-18 passages, after which cells were restarted from the frozen stock. Seeding cell counts were routinely determined using an automatic Cedex HiRes cell counter (Flownomics Analytical Instruments, Madison, WI) or manually using INCYTO C-Chip™ Disposable Hemacytometers (Fisher Scientific, Pittsburg, PA). The anti-HCV assays were done accordingly:

Luciferase Genotype lb Replicon Potency Assay. Replicon cells were seeded into white 96-well plates (Nunc/VWR) at a density of 2x10 ⁴ cells/well in medium without G-418. 18-24 h after cell plating, inhibitors were added and cells were incubated for additional 72 hours. Compounds were tested in triplicates and quadruplicates at 3X or 4X serial dilutions over a range of 0.0001 -to- 100 μΜ concentrations. HCV replication was monitored by Renilla luciferase reporter activity assay using Renilla luciferase reporter (Promega, Madison, WI) and a Veritas Luminometer (Turner Biosystems, Sunnyvale, CA). 50% effective concentration (EC ₅₀ ) values were calculated as the concentration of compound that resulted in a 50% decrease in the reporter expression as compared to untreated cells. The values were determined by non-linear regression (four-parameter sigmoidal curve fitting) analysis.

The cell cytotoxicity assay data was obtained as described below:

Cytotoxicity Assay. Cells were seeded into 96-well plates at a density of 2x10 ⁴ cells per well. 24 h after cell plating, serial 2X or 3X compound dilutions, starting with 100 μΜ, were applied to the testing plates (3 repeats per compound dilution). Each testing plate was run with a "no-compound" control. Incubation with compounds was continued at 37°C in 5% CO ₂ atmosphere for 72 hours. To determine cell viability, the CellTiter-Glo® assay (Promega, Madison, WI) was performed according to the manufacturer's protocol. The compound concentration resulting in 50% luminescent signal loss was reported as the CC5 ₀ concentration.

Table 6

Oral bioavailability experiments in cynomolgus monkeys. The compounds I, la, lb and (la) (X = anisole) were studied in oral bioavailability experiments in cynomolgus monkeys. The four compounds were dosed as solid in capsule (25 mg/kg) and the resulting plasma concentrations of a selection of metabolites were measured. The metabolites A-E were synthesized by methods familiar to one skilled in the art, or as described herein. These results were compared to values obtained when compounds 1,1a, and lb, were formulated in 95:5 CapmuhTween and dosed orally. The data is presented in Table 7.

Table 7. Oral dosing of Different Solid Forms of Compounds 1, la and lb.

Pharmacokinetic studies in cynomolgus monkeys. Primate studies were conducted at Avanza Laboratories, of 15 Firstfield Road, Gaithersburg, MD 20878. Compounds 1, la, lb, and (la) (X = anisole) were dosed as powder in capsule (25 mg/kg), in size zero gelatin capsules, and were administered as a single oral dose, in overnight fasted male cynomolgus monkeys. In addition, compounds 1, la and lb, were dosed IV (5 mg/kg) formulated in 10% ethanol in (5% Tween 80 / 95% Saline w/v). Blood samples (approximately 3 mL) were collected into 1 K ₂ EDTA tube from the femoral vein. Blood samples were stored on wet ice and samples were centrifuged at 5 ± 3°C within 30 minutes of collection. The resultant plasma was recovered and split into two 0.5 mL aliquots and placed in polypropylene tubes. All plasma samples were quick frozen over dry ice and stored under conditions set to maintain -75 ± 15°C until analysis.

Measurement of Metabolite A and Metabolite B (3) in Plasma Samples

Plasma samples were prepared for analysis as follows. 400 μϊ _^ of 50 mM ammonium acetate containing 2 ng/niL of internal standard Nl-Methyl-2'-deoxyguanosine; Berry &Associates, Dexter, MI; Cat# PR3748) was added to 100 of each sample. Calibration curves were prepared by serial dilution of a stock solution of Metabolite A (2'-C-methyl guanosine) or Metabolite B (6-O-methyl 2'-C-methyl guanosine) into blank media. Solid phase extraction of the samples was performed with H^O-Philic DVB speed disk columns (J.T. Baker Philipsburg, NJ) which were previously solvated with 1 mL of methanol followed by equilibration with 1 mL of 50mM ammonium acetate. The columns were rinsed with 1 mL of 50mM ammonium acetate: methanol (95:5, v/v) and samples were eluted with 1 mL of methanol: ammonium hydroxide (95:5, v/v). The samples were dried under nitrogen and reconstituted in 80μ1 of H ₂ O. 10 μϊ _^ of each test sample was analyzed for Metabolite A or Metabolite B concentrations by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Liquid chromatography was performed with an Agilent 1100 Series HPLC system equipped with a Betasil C18 2.1 x 100 mm 5μιη column (Thermo Fisher Scientific Waltham, MA). Samples were eluted using a linear gradient from 100% Solvent A (0.1% formic acid in H ₂ O) to 50% Solvent B (0.1% formic acid in acetonitrile) applied over 6min at 0.5 mL/min flow rate. The HPLC system was coupled to an API 4000 triple quadrupole mass spectrometer (Applied Biosystems, Framingham, MA). Mass spectrometry was performed in positive ion mode and data was analyzed using Analyst® vl.4.2 software (Applied Biosystems, Framingham, MA).

Measurement of Parent Compounds l,la,lb ^an d (la) (X = anisole) in Plasma Samples

Plasma samples were prepared for analysis as follows. 50 μΐ of each test sample were distributed in a 96-well V-bottom plate. 200 μΐ of acetonitrile containing 10 ng/mL Internal Standard was added to each Plasma sample. The precipitated samples were centrifuged at 3000 rpm, 4 °C for 20 minutes in a Sorvall RT6000S centrifuge (Thermo Scientific, Waltham MA) and 50 of Plasma from each sample was transferred into a 96 deep well plate followed by the addition of 50 μί 50:50 acetonitrile:H ₂ 0 to each sample. Samples were covered, mixed well by vortexing and maintained at 2-8 °C before and during analysis. Calibration curves were constructed by spiking varying concentrations of analyzing protide (Compounds 1,1a or lb) into blank media. 15 μί of each test sample was analyzed for analyte concentrations by liquid chromatography coupled to tandem mass spectrometry (LC- MS/MS). Liquid chromatography was performed with an Agilent 1100 Series HPLC system equipped with a Synergi 4 μιη Polar-RP, 30x2.0 mm column (Phenomenex, Torrance, CA). Samples were eluted using a linear gradient from 100% Solvent A (0.1% formic acid in H ₂ O) to 100% Solvent B (0.1% formic acid in acetonitrile) applied over 3 min at 1.0 mL/min flow rate. The HPLC system was as described above.

Measurement of Metabolite C in Plasma Samples

50μ1 of each test sample were distributed in a 96-well V-bottom plate. 200 μϊ _^ of methanol containing 25 ng/mL of Internal Standard was added to each sample. The precipitated samples were centrifuged at 3000 rpm, 4°C for 20 minutes in a Sorvall RT6000S centrifuge (Thermo Scientific, Waltham MA) and 50 μϊ _^ of Plasma from each sample was transferred into a 96 deep well plate followed by the addition of 50 μί H ₂ 0 to each sample. Samples were covered, mixed well by vortexing and maintained at 2-8°C before and during analysis. Calibration curves were constructed by spiking varying concentrations of analytes into blank media. 10 μϊ _^ of each test sample was analyzed for analyte concentrations by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Liquid chromatography was performed with an Agilent 1 100 Series HPLC system equipped with a Betasil CI 8, 2.1 x 100 mm 5 μιη column (Thermo Fisher Scientific Waltham, MA). Samples were eluted using a linear gradient from 100% Solvent A (0.1% formic acid in H ₂ 0) to 50% Solvent B (0.1% formic acid in acetonitrile) applied over 6min at 0.5 mL/min flow rate. The HPLC system was as described above.

Measurement of Metabolites D and E in Plasma Samples

50μ1 of each test sample were distributed in a 96-well V-bottom plate. 200 μϊ _^ of methanol containing 50 ng/mL Internal Standard was added to each sample. The precipitated samples were centrifuged at 3000rpm, 4°C for 20 minutes in a Sorvall RT6000S centrifuge (Thermo Scientific, Waltham MA), 100 μϊ _^ of Plasma from each sample was transferred into a 96 deep well plate, and samples were dried under N ₂ , overnight at room temperature. The dried samples were reconstituted in 50 ΙΟηΜ ammonium acetate in ¾0 per sample. Samples were covered, mixed well by vortexing and maintained at 2-8 °C before and during analysis. Calibration curves were constructed by spiking varying concentrations of analytes into blank media. 15 of each test sample was analyzed for analyte concentrations by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Liquid chromatography was performed with an Agilent 1 100 Series HPLC system equipped with a Agilent/ Varian Polaris 5 C18-A 50x2.0mm; Part# A2000050X020. Samples were eluted using a linear gradient from 100% Solvent A (ΙΟηΜ ammonium acetate in H ₂ 0) to 60% Solvent B (lOnM ammonium acetate in acetonitrile) applied over 6 min at 0.5 mL/min flow rate. The HPLC system was as described above.

Structure of Compounds studied in monkey PK (Parent Molecules):

Metabolites monitored in monkey PK:

Metabolite A Metabolite B, Compound 3

Metabolite D Metabolite E

The cynomolgus monkey PK data demonstrates that amorphous compound lb shows 3 -fold better oral bioavailability as powder in capsule, than amorphous la. However, the crystalline anisole solvate (la), (X = anisole) has improved oral bioavailability (as powder in capsule) over either lb or la amorphous, and similar to compound 1 as a liquid formulation in CapmuhTween (95:5).

While the invention has been described with reference to particularly preferred embodiments and examples, those skilled in the art recognize that various modifications may be made to the invention without departing from the spirit and scope thereof.

All of the above U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety.

Abbreviations and Acronyms

A number of abbreviations and acronyms are used herein, and a full description of these are provided as follows:

ACN acetonitrile

amu atomic mass unit

anhy anhydrous

AUC area under the curve

br broad signal (NMR)

Boc benzyloxycarbonyl

Bu butyl

i-BuMgCl tert-butyl magnesium chloride

CDCI ₃ deuterochloroform

¹³ C NMR carbon- 13 nuclear magnetic resonance spectroscopy cone concentrated

d doublet (NMR)

dd doublet of doublets (NMR)

ddd double doublet of doublets (nmr

DBU diaza( 1 ,3 )bicyclo[5.4.0]undecane

DCM dichloromethane

DMAP 4-dimethylaminopyridine

DSC differential scanning calorimetry

dt doublet of triplets (NMR)

DVS dynamic vapor sorption

EDCI 1 -(3 -dimethylamnopropyl)-3 -ethylcarbodiimide

hydrochloride

ee enantiomeric excess

equiv equivalent(s)

EtOAc ethyl acetate

EtOH ethanol

FT Fourier Transform

FAB fast atom bombardment g gram(s)

GC-MS gas chromatography-mass spectrometry h hour

HCV hepatitis C virus

lH NMR proton nuclear magnetic resonance spectroscopy

HPLC high performance liquid chromatography

IR infrared

J NMR coupling constant

kV kilovolt(s)

LC/MS liquid chromatography-mass spectrometry m multiplet (NMR)

MDI metered dose inhalers

Me methyl

MeOH methanol

MeOH-i/4 methanol-^ _?

mg milligram

MHz megahertz

min minute(s)

mL milliliter

μΐ _^ microliter

mmol millimole

MNOBA m-nitrobenzyl alcohol

mp melting point

MTBE methyl t-butyl ether

NaOMe sodium methoxide

NBS N-Bromo succinimide

NCS N-Chloro succinimide

NIS N-Iodo succinimide

NMI N-methylimidazole

ΝΜΟ N-methylmorpholine-N-oxide

NMR nuclear magnetic resonance

PLM polarized light microscopy

³¹ P NMR phosphorous-31 nuclear magnetic resonance spectroscopy

ppm part per million

PTSA ^-toluene sulfonic acid q quartet (NMR)

RH relative humidity

R _f retention factor (TLC)

RT room temperature

s singlet (NMR)

SCXRD single crystal X-ray diffraction t triplet (NMR)

TEA triethylamine

TFA trifluoroacetic acid

TGA thermogravimetric analysis

TGA-IR thermogravimetric analysis with IR

THF tetrahydrofuran

TMS tetramethylsilane

?R retention time

TLC thin layer chromatography

UV ultraviolet

V volt(s)

XRPD X-ray powder diffraction