SALTS OF HIV INHIBITOR COMPOUNDS

FIELD OF THE INVENTION

The invention relates generally to salts of compounds with antiviral activity and more specifically with anti-HIV properties.

BACKGROUND OF THE INVENTION

Human immunodeficiency virus (HIV) is a retrovirus that can lead to acquired immunodeficiency syndrome (AIDS), a condition in humans in which the immune system is weakened, leading to life-threatening opportunistic infections. Inhibitors of HIV are useful to treat HIV infection in a mammal (e.g., reduce and limit the establishment and progression of infection by HIV) as well as in diagnostic assays for HIV. The usefulness of currently marketed inhibitors of HIV is to some extent limited by toxicity and other side effects. Thus, there is a need for new HIV therapeutic agents.

A pharmaceutical formulation of a therapeutic agent must reproducibly and consistently deliver the therapeutic agent to a patient in need thereof. This consistency of delivery can be achieved, at least in part, by incorporation of a stable, soluble, solid-state form of the therapeutic agent into the pharmaceutical composition. Moreover, the synthesis of the desired solid-state form of the therapeutic agent should be technically and econonioically feasible, and should be suitable for full-scale commercial production.

SUMMARY OF THE INVENTION

Ethyl N-[(S)( { [(2R,5R)-5-(6-amino-9H-puπn-9-yl)-4-fluoro -2,5-dihydrofuran-2- yl]oxy} methyl)phenoxyphosphinoyl]-L-alaninate is a reverse transcriptase inhibitor that blocks the replication of IiIV viruses, in vivo and in vitro, and has limited undesirable side effects when administered to human beings. The structure of Ethyl N- [(S){ { [(2R,5R)-5-(6-amino-9H-purin-9-yl)-4-fluoro -2,5-dihydrofuran -2- yi]oxy}methyl)phenoxyphosphinoyl]-L-alaninate is shown in Formula P :

Ethyl N-[(S)( { [(2R,5R)-5-(6-amino -9H-purin-9-yl)-4-fluoro -2, 5-dihydrofuran-2- yl]oxy} methyl )phenoxyphosphinoyl]-L-alaninate is a low melting point, amorphous, solid which is difficult to isolate, purify, store for an extended period, and formulate as a pharmaceutical composition. Ethyl N-[(S)( {[(2R,5R)-5-(6-amino-9H-purin-9-yl)-4- fluoro-2,5-dihydrofuran-2-yl]oxy}methyI)phenoxypliosphinoyl] -L-alaninate is also a weak base that is capable of forming salts with acids. Accordingly, in one aspect, the present invention provides stable salts of Ethyl N-[(S)( {[(2R,5R)-5-(6-amino-9H-purin- 9-yl)-4-fluoro -2,5-dihydrofuran-2-yl]oxy}methy])phenoxyphosphinoyl]-L-ala� �inate that are more physically stable, and are more readily isolated and formulated, than the free base form of the compound.

The salts of the present invention are useful, for example, for treating human patients infected with human immunodeficiency vims (strains of HIV-I or HIV-2) which causes AIDS. The salts of the present invention are also useful, for example, for preparing a medicament for treating HIV or an HIV associated disorder. The salts of the present invention are also useful, for example, for inhibiting the replication of HΪV viruses in vitro, and can be used, therefore, in biological assays as a control compound for identifying other reverse transcriptase inhibitors, or for investigating the mechanism of action of HIV reverse transcriptase and its inhibition.

Thus, in one aspect, the present invention provides citrate, succinate and malonate salts of Ethyl N-[(S)({[(2R,5R)-5-(6-amino-9H-purin-9-yl)-4-fluoro-2,5-dihy drofuran-2- yI]oxy}methyl)phenoxyphosphinoyl]-L-alaninate, and methods of making the foregoing salts. In some embodiments, the salts of the present invention are anhydrous, while in other embodiments the salts of the present invention are at least partially hydrated. In some embodiments, the salts of the present invention exist as crystalline forms.

Thus, the present invention comprises citrate, succinate and malonate salts of the compound of Formula P, as well as hydrates thereof. The inventive hydrates may be in a partial (e.g., hemi-hydrate) or full hydrated state (e.g., mono-hydrate). The present invention also comprises the subject salts in anhydrous or essentially anhydrous states. Similarly, the inventive salts and hydrates thereof comprise amorphous and crystalline states, as well as states comprising both amorphous and crystalline characteristics. As used herein, "crystalline" means a material that has an ordered, long range molecular structure. In contrast, "amorphous" materials do not possess long range order. It is understood that crystalline materials are generally more stable thermodynamically than amorphous forms of the same substance. Thus, with only a few notable exceptions, it is generally preferred to use crystalline materials in pharmaceutical applications. A measure of the degree of crystallinity of the inventive compounds can be seen, for example, in the sharpness of the DSC and XRPD absorption bands (peaks). The sharper the peak, the higher the degree of crystallinity. Conversely, the broader the peak, the lower the degree of crystallinity.

As described more fully in Example 12 herein, in specific embodiments, a citrate salt of Ethyl N-[(S)({[(2R,5R)-5-(6-amino-9H-ptiHn-9-yl)-4-fluoro-2,5-dihy drofuran-2- yl]oxy}methyI)phenoxyphosphinoyl]-L-aIaninate is characterized by absorption bands, obtained from an X-ray powder diffraction pattern, at spectral d-spacings of 4.48, 3.12 and 6.05 angstroms; a succinate salt of Ethyl N-[(S)({[(2R,5R )-4-fluoro-2,5-dihydrofuran-2- yl]oxy}methyl)phenoxyphosphinoyl]- by absorption bands, obtained from an X-ray powder diffraction pattern, at spectral d-spacings of 3.57, 4.80 and 4.99 angstroms; and a malonate salt of Ethyl N-[(SX{[(2R,5R)-5-{6-amino -9H-punn-9-y])-4-fiuoro-2,5-dihydrofuran -2- y!]oxy}methyl)phenoxypliosphinoyI]-L-alaninate is characterized by absorption bands, obtained from X-ray powder diffraction pattern, at spectral d-spacings of 4.99, 5.93 and 4.72 angstroms. In another specific embodiment, the present invention provides a citrate salt of Ethyl N-[(S)({[(2R.5R)-5-(6- amino-9H-purin-9-yl)-4-fluoro-2,5-dihydrofui-an-2-yl]oxy} methy])phenoxyphosphinoyl]-L-alaninate having a melting point of from 142°C to 150°C.

EthyJ N-[(S)({[(2R,5R)-5-(6-amino-9H-purin-9-yl)-4-fluoro-2,5-dthy drofuran-2- yl]oxy}methy!)phenoxyphosphinoyl]-L-alaninale is an amidate prodrug which undergoes reaction with and decomposition in protic solvents. The rate of the reaction depends on pH and temperature. Consequently, the formation of a stable salt between the amidate prodrug and citric acid, succinic acid and/or malonic acid, which each contain nucleophilic moieties capable of reacting with the prodrug (e.g., reaction with the amidate moiety of the prodrug) is a surprising and unexpected result.

In another aspect, the present invention provides pharmaceutical compositions that each include a therapeutically effective amount of a salt of the present invention and a pharmaceutically acceptable carrier or excipient. Pharmaceutical compositions of the present invention may also include an additional therapeutic agent, such as an antiviral, antibacterial, antifungal or anticancer agent. The pharmaceutical compositions of the invention may be in the form of unit dosage forms, such as tablets or capsules. The unit dosage forms typically provide an effective daily dose of a sait of the present invention to a human being in need thereof. Effective daily doses of the salts of the present invention are typically from 1 mg to 100mg, such as from 10mg to 30mg.

In another aspect, the present invention provides methods of making the citrate, succinate and maionate salts of the present invention. Thus, for example, the present invention provides a process for preparing a citrate salt of Ethyl N-[(S)( {K2R,5R)-5-(6-amino -9H-purin-9-yl)-4-fluoro-2,5- diliydrofuran-2-yI]oxy}methyI)phenoxyphosphinoyl]-L-alaninat e, wherein the process inciiides the step of contacting about one equivalent of Ethyl N-[(S)( {[(2R,5R)-5-(6-amino-9H-pιirin-9-yl)~4-fluoro -2,5-dihydrofuran-2-yl]oxy}methyl)phenoxyphosphmoy!]-L-alani nate free base in a suitable solvent (e.g., acetonitrile) with form about one equivalent to about 1.2 equivalents of citric acid at a temperature in the range of from about The present invention also provides a process for preparing a succinate salt of Ethyl N- [(S)({[(2R,5R)-5-(6-amino-9H-purin-9-yl)-4-fIuoiO-2 _J 5-dihydrofuran-2-yl)oxy} niethyl) phenoxyphosphinoyl]-L-alaninate, wherein the process includes the step of contacting about one equivalent of Ethyl N-[(S){{ [(2R,5R)-5-(6-amino -9H-ρurin-9-yI)-4-fiuoro-2,5-dihydrofuran-2- yI]σxy}methy])pheπoxyphosphinoy]]-L-alaninate free base in a suitable solvent (e.g., 2-butanone) with from about one equivalent to about 1 .2 equivalents of succinic acid at a temperature in the range of from about 60°C to about 70°C to form the succinate salt of Ethyl N-[(S)({[(2R,5R)-5-(6-amino - 9H-purin-9-yl)-4-fluoro-2,5-dihydrofuran-2-yl]oxy} methy!)phenoxyphosphinoyI]-L-alaninate,

Further, the present invention provides a process for preparing a malonatβ salt of Ethyl N- [(SX{[(2R,5R)-5-(6-amino-9H-purin-9-yl)-4-fluoro-2,5-dihydro furan-2-yl]oxy}methyl) phenoxyphosphinoyl]-L-a!aninate, wherein the process includes the step of contacting about one equivalent of Ethyl N-[(S)({[(2R,5R)-5-(6-amino -9H-purin-9-yl)-4-f!uoiO-2,5-dihydrofu ran-2- yI]oxy}methy])phenoxyphosphinoyl]-L-alaninate free base in a suitable solvent (e.g., 2-butanone) with from about one equivalent to about 1.2 equivalents of malorric acid at a temperature in the range of from about 50°C to about 70°C to form the malonate salt of Ethyl N-[(S)({[(2R,5R)-5-(6-amino - 9H-purin-9-yl)-4-fluoro-2,5-dihydrofuran-2-yl]oxy} methyl)phenoxyphosphinoyl]-L-alaninate.

In a further aspect, the present invention provides methods of treating or prophylactically preventing AFDS, wherein the methods include the step of administering to a human being suffering from AIDS a therapeutically effective amount of a salt of the invention, or a hydrate thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1 shows a characteristic differential scanning calorimetry (DSC) trace for the malonate salt of Ethyl N-[(Sχ{[(2R,5R)-5-(6-amino -9H-purin-9-yl)-4-fluoro-2,5-dihydrofuran-2- yl]oxy} methyl)phenoxyphosphinoyI]-L-alaninate.

FIGURE 2 shows a characteristic DSC tr Ethyl N-[(S)({ [(2R,5R)-5-(6- amino -9H-ρurin-9-yl)-4-fluoro-2,5-dihyd henoxyphosphinoyl]-L-alaninate. FIGURE 3 shows a characteristic DSC trace for the citrate salt of Ethyl N-[(S)( [[(2R,5R)-5-(6-amino- 9H-purin-9-yl)-4-fluoro-2,5-dihydrofuran-2-yl]oxy} methyI)phenoxyphosphinoyl]-L-alaninate.

FIGURE 4 shows a characteristic X-ray powder diffraction (XRPD) pattern for the malonate salt of Ethyl N-[(S)( {[(2R,5R)-5-(6-amino -9H-purin-9-yl)-4-fluoro-2,5-dihydrofuran -2- yI]oxy} methy!)phenoxyphosphinoyI]-L-alaninate.

FIGURE 5 shows a characteristic XRPD pattern for the succinate salt of Ethyl N-[(S)({[(2R,5R)-5-(6- amino-9H-purin-9-yI)-4-fluoro-2,5-dihydrofuran -2-yl]oxy} methyl)phenoxyphosphinoy!3-L-alaninate.

FIGURE 6 shows a characteristic XRPD pattern for the citrate salt of Ethyl N-[(S)({[(2R,5R)-5-(6- amino-9H-purin-9-y!)-4-fluoro-2,5-dihydrofuran -2-yl]oxy} methyI)phenoxyphosphmoyl3-L-alaninate.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

When tradenames are used herein, applicants intend to independently include the tradename product and the active pharmaceutical ingredient(s) of the tradename product.

Salts of Ethyl N-[(S)( {[(2R,5R))-5-(6-amino-9H-purin-9-yl)-4-fluoro-2,5-dihydrorur --o-2- yl]oxy}methyl1phenoxyphosphinoyl]-L-alaninate

In one aspect, the present invention provides citrate, succinate and inalonate salts of Ethyl N-[(S)( {[(2R,5R)-5-(6-amino-9H-purin-9-yl}-4-fluoro-2,5-dihydrofαr an-2- yl]oxy} methyl)phenoxyphosphinoyl]-L-alaninate.

The citrate salt is shown in Formula I :

The succinate salt is shown in Formula II •

II

The malonate salt is shown in Formula III :

A method for synthesizing Ethyl N-[(S)( {[(2R,5R)-5-(6-amino-9H-purin-9-yl)-4- fluoro-2,5-dihydrofuran -2-yl]oxy} methyl)phenoxyphosphinoyl]-L-alaninate is described in Example I herein. Methods for making the malonate, succinate and citrate salts of Ethyl N-[(S)( { [(2R,5R)-5-(6-amino-9H-purin-9-yl)-4-fluoro-2,5-dihydrofuran .-2- yl]oxy}methyI)phenoxyphosphinoyl]-L-aIanmate are described in Examples 3, 4 and 5, respectively, herein. Some physical properties of the foregoing salts arc described in Example 6 herein and demonstrate, for example, that each of these salts is physically stable when stored at 40°C and a relative humidity of 75%.

The citrate, succinate and makmate salts of Ethyl N-[(S)( {[(2R,5R)-5-(6-amino- 9H-purin-9-yl)-4-fluoro-2,5-dihydrofΛiran-2-yl3oxy}methy])p henoxyphosphinoyl]-L- alaninate are useful, for example, for inhibiting the replication of HIV in vitro and in vivo. In this regard, as explained more fully in Example 8 herein, Ethyl N- [(SX{[(2R,5R)-5-(6.amino-9H-purin-9-yl)-4-fluoro-2.5-dihydro furan-2- yl]oxy}methyl)phenoxyphosphinoyl]-L-aIaninate is a prodrug that is metabolized in the human body to yield a parent compound which, in turn, is phosphorylated within the body to produce the active metabolite that inhibits HΪV replication. Example 8 herein shows that Ethyl N-[(S)({[(2R,5R)-5-(6-amino-9H-purin-9-yl)-4-fluoro-2,5- dihydrofuran-2-yl]oxy}methy!)phenoxyphosphinoyl]-L-alaninate causes a greater accumulation of the active metabolite in white blood cells, which are the cells that harbor the HIV virus, than does the parent compound. Further, Example 9 herein presents in vitro data showing that Ethyl N-[(S)({[(2R,5R)-5-(6-amino-9H-pιιrin-9-yl)-4-fluoro-2,5- dihydrofuran -2-yl]oxy}methyl}phenoxyphosphinoyl]-L-aIaninate is a more potent anti-HlV drug than the parent compound as assessed in an in vitro assay. Additionally. Example 10 herein provides data showing that a tablet containing the citrate salt of Ethyl N-[(S)( {[(2R,5R)-5-(6- amino -9H-purin-9-yI)-4-fluoro-2,5-dihydrofuran -2-y!]oxy}methyl)phenoxyphosphinoyl]-L- alaninate provides this drug to the bloodstream of Beagle dogs with similar pharmacokinetics to a liquid preparation of the drug administered orally. Thus, the citrate salt of Ethyl N- [(S)({[(2R,5R)-5-(6-amino-9H-pυrin-9-yl)-4-fluoro-2,5-dihyd rofuraπ-2- yl]oxy} methyl)phenoxyphosphinoyl]-L-alaninate is a physically and chemically stable composition of matter that can be administered orally to a living subject, to provide a therapeutically effective amount of Ethyl N-[(SX{ [(2R,5R)-5-(6-amino -9H-pιiι-in-9-yl)-4-fluoro- 2,5-dihydrofuran -2-yl]oxy}methyI)phenoxyphosphinoyI3-L-alantnate, which is a more effective anti-HlV agent than the parent compound.

Pharmaceutical Compositions

In another aspect, the present invention provides pharmaceutical compositions, also referred to as pharmaceutical formulations that include a therapeutically effective amount of one or more salts of the invention, and a pharmaceutically acceptable earner or excipient.

Whiie it is possible for the salts of the invention to be administered alone, it is typically preferable to administer them as pharmaceutical compositions. The pharmaceutical compositions of the invention are formulated with conventional carriers and excipients, which are selected in accord with ordinary practice. The carrier(s) must be "acceptable'' in the sense of being compatible with the other ingredients of the formuiation and physiologically innocuous to the recipient thereof. Tablets contain such components as excipients, glidants, fillers, binders and the like. Aqueous formulations are prepared in sterile form, and when intended for delivery by other than oral administration they are generally isotonic. AU formulations optionally contain excipients such as those set forth in the Handbook of Pharmaceutical Excipients (R.C. Rowe et al.,

Pharmaceutical Press, 5 ^th ed., 2006). Excipieπts include ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextrin, hydroxyalkylccllulose, hydroxyalkylraethylcellulose, stearic acid and the like. The pH of the fomvulations ranges from about 3 to about ] 1 , but is ordinarily about 7 to 10.

The formulations may conveniently be presented in unit dosage form (e.g., tablet) and may be prepared by any of the methods well known in the art of pharmacy. Techniques and formulations generally are found in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA). Such methods include the step of bringing into association the active ingredient with the earner which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly arid intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Pharmaceutical formulations containing the active ingredient may be in any form suitable for the intended method of administration, When used for oral use for example, tablets, troches, lozenges, aqueous or oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs may be prepared. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents including sweetening agents, flavoring agents, coloring agents and preserving agents, in order to provide a palatable preparation. Tablets containing the active ingredient in admixture with non-toxic pharmaceutically acceptable excipient which are suitable for manufacture of tablets are acceptable. These excipients may be, for example, inert diluents, such as calcium or sodium carbonate, lactose, lactose monohydrate, croscarmellose sodium, povidone, calcium or sodium phosphate; granulating and disintegrating agents, such as maize starch, or alginic acid; binding agents, such as cellulose, microcrystalline cellulose, starch, gelatin or acacia; and lubricating agents, such as magnesium stearate, stearic acid or talc. Tablets may be uncoated or may be coated by known techniques including microencapsulation to delay disintegration and adsorption in the gastrointestinal tract and thereby provide a

sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may be employed.

Formulations for oral use may be also presented as hard gelatin capsules where the active ingredient is mixed with an inert solid diluent, for example calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil.

Aqueous suspensions of salts of the invention contain the active materials in admixture with excipienls suitable for the manufacture of aqueous suspensions. Such cxcipients can include a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylccllulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide {e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride (e.g.. polyoxyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives such as ethyl or n-propyl p-hydroxy-benzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose or saccharin.

Oil suspensions may be formulated by suspending the active ingredient in a vegetable oil, such as arachis oii, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oral suspensions may contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents, such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.

Dispersible powders and granules of the invention suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or welting agent, a suspending agent, and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those disclosed above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.

The pharmaceutical compositions of the invention may also be in the form of oil- in-water emulsions. The oily phase may be a vegetable oil, such as olive oil or arachis oil, a mineral oil, such as liquid paraffin, or a mixture of these. Suitable emulsifying agents include naturally-occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. The emulsion may also contain sweetening and flavoring agents. Syrups and elixirs maybe formulated with sweetening agents, such as glycerol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, a flavoring or a coloring agent.

The pharmaceutical compositions of the invention may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenlerally acceptable diluent or solvent, such as a solution in 1 ,3-butane-diol or prepared as a lyophilized powder. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils may conventionally be employed as a solvent or suspending medium. For this purpose any bland Fixed oil may be employed including synthetic mono- or diglycerides, In addition, fatty acids such as oleic acid may likewise be used in the preparation of injectables.

The amount of active ingredient that may be combined with the carrier material to produce a single dosage form will vary depending, for example, upon the particular mode of administration. For example, a time-release formulation intended for oral administration to humans may contain approximately 3 to 1000 mg of active material compounded with an appropriate and convenient amount of carrier material which may vary from about 5 to about 95% of the total composition (weightrweight).

Formulations suitable for administration to the eye include eye drops wherein the active ingredient Ls dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active ingredient.

Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, such as sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid earner.

Formulations for rectal administration may be presented as a suppository with a suitable base comprising for example cocoa butter or a salicylate.

Formulations suitable for intrapulmonary or nasal admimstration have a particle size for example in the range of 0.1 to 500 microns (including particle sizes in a range between 0.1 and 500 microns in increments of microns such as 0.5, 1 , 30 microns, 35 microns, etc.). which is administered by rapid inhalation through the nasal passage or by inhalation through the mouth so as to reach the alveolar sacs. Suitable formulations include aqueous or oϋy solutions of the active ingredient. Formulations suitable for aerosol or dry powder administration may be prepared according to conventional methods and may be delivered with other therapeutic agents such as compounds heretofore used in the treatment or prophylaxis of conditions associated with HlV activity.

Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous and nonaqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.

The formulations are presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dπed (Jyophϋized) condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets of the kind previously described. Preferred unit dosage formulations are those containing a daily dose or unit daily sub- dose or an appropriate fraction thereof, of the active ingredient. Thus, for example, a daily dose of a salt of the present invention can be provided in a single tablet, or in multiple tablets (e.g., two or three tablets).

It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.

Pharmaceutical formulations that are also within the scope of the invention provide controlled release of the active ingredient to allow less frequent dosing or to improve the pharmacokinetic or toxicity profile of the active ingredient. Accordingly, the invention also provides pharmaceutical compositions comprising one or more salts of the invention formulated for sustained or controlled release.

An effective dose of a salt of the present invention depends, for example, on whether the salt is being used prophylactically (typically a lower dose is required compared to therapeutic use of the same salt), the method of delivery, and the pharmaceutical formulation, and will be determined by the clinician using conventional dose escalation studies. An effective dose can be expected to be from about 0.0001 mg/kg body weight per day to about 100 mg/kg body weight per day. Typically, from about 0.0 J to about 10 mg/kg body weight per day. More typically, from about .01 to about 5 mg/kg body weight per day. More typically, from about .05 to about 0.5 mg/kg body weight per day. For example, the daily dose for an aduli human of approximately 70 kg body weight will typically range from 1 mg to 1000 rag, preferably between 5 mg and 500 mg, and may take the form of single or multiple doses. By way of example, the dose of a salt of the present invention in a unit dose formulation to be administered once per day may be from 1 mg to 100 mg, such as from 30mg to 60τng, such as a 30mg daily dose or a 60mg daily dose. Combination Therapy

Each of the citrate, succinate and malonate salts of Ethyl N-[(S)({[(2R,5R)-5-(6- amino-9H-puriii-9-yl)-4-fluoro-2.5-dihydrofuran-2-yl]oxy}met hyl)phenoxyphosphinoyl]- L-alaninate may be employed in combination with other therapeutic agents for the treatment or prophylaxis of AIDS and/or one or more other diseases present in a human subject suffering from AIDS (e.g., bacterial and/or fungal infections, other viral infections such as hepatitis B or hepatitis C, or cancers such as Kaposi's sarcoma). The additional therapeutic agent(s) may be coformulated with one or more salts of the invention (e.g., coformulaled in a tablet).

Examples of such additional therapeutic agents include agents that are effective for the treatment or prophylaxis of viral, parasitic or bacterial infections, or associated conditions, or for treatment of tumors or related conditions, include 3'-azido-3'- deoxythymidine (zidovudine, AZT), 2'-deoxy-3'-thiacytidine (3TC), 2',3'-dideoxy-2',3'- didehydroadenosine (D4A), 2',3'-dideoxy-2',3'-didehydrolhymidine (D4T), carbovir (carbocyciic 2',3'-dideoxy-2',3'-didehydroguanosine), 3'-azido-2',3'-dideoxyuridine, 5- fiuorotϊrymidinc, (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), 2- chlorodeoxyadenosine, 2-deoxycoforrnycin, 5-fluorouracil, 5-fiuorouridine, 5-fluoro-2'- deoxyuridine, 5~trifiuoromethyl-2'-deoxyuridme, 6-azauridine, 5-fluoroorotic acid, methotrexate, triacetyhiridine, 1 -(2'-deoxy-2'-fluoro - 1 -β-arabinosyI)-S-iodocytidine (FlAC), telrahydro-imidazo(4,5, I -Jk)-( 1,4)-benzodiazepin-2(l H)-thione (TIBO), 2'-nor- cyclicGMP, 6-methoxyρurine arabinoside (ara-M), 6-methoxypurine arabinoside 2'-O- vaJerate; cytosine arabinoside (ara-C). 2',3'-dideoxynucleosides such as 2\3'- dideoxycytidine (ddC), 2',3'-dideoxyadenosine (ddA) and 2',3'-dideoxyinosine (ddl); acyclic nucleosides such as acyclovir, penciclovir, famciclovir, ganciclovir, HPMPC, PMEA, PMEG, PMPA, PMPDAP, FPMPA, HPMPA, HPMPDAP, (2R, 5R)-9- >terrahydro-5-(phosphonomethoxy)-2-furanyladenine, (2R, 5R)-l ->tetrabydro-5-

(phosphonomethoxy)-2-furanyithymine; other antivirals including ribavirin (adenine arabinoside), 2-thio-6-azauridine, tubercidin, aurintricarboxylic acid, 3- deazaneoplanocin, neoplanocin, rimantidine, adamantine, and foscarnel (trisodium phosphonoformate); antibacterial agents including bactericidal fluoroquinolones (ciprofloxacin, pefloxacin and the like); aminoglycoside bactericidal antibiotics (streptomycin, gentamicin, amicacin and the like) ; β-lactamase inhibitors (cephalosporins, penicillins and tlie like); other antibacterials including tetracycline, isoniazid, rifampin, cefoperazone, claithromycin and azithromycin, antiparasite or antifungal agents including pentamidine (1,5-bis(4'-aminophenoxy)pentane), 9-deaza- inosine, sulfamethoxazole, sulfadiazine, quiπapyramine, quinine, fluconazole, ketoconazole, itraconazole. Amphotericin B, 5-fluorocytosine. clotrimazole, hexadecylphosphocholine and nystatin; renal excretion inhibitors such as probenicid; nucleoside transport inhibitors such as dipyridamole, dilazep and nitrobenzyithioinosine, imimmomodulators such as FK506, cyclosporin A, thymosin α-1; cytokines including TNF and TGF-β; interferons including IFN-α, IFN-β, and IFN-γ; interleukins including various interleukins, macrophage/granulocyte colony stimulating factors including GM- CSF, G-CSFt M-CSF, cytokine antagonists including anti-TNF antibodies, anti- interleukin antibodies, soluble iπterleukin receptors, protein kinase C inhibitors and the like.

Examples of suitable active therapeutic agents or ingredients which can be combined with the salts of the invention, and which have activity against HIV, include I) HIV protease inhibitors, e.g., amprenavir, atazanavir, fosamprenavir, indinavir, lopinavir, ritonavir, lopinavir + ritonavir, nelfinavir, saquinavir, tipranavir, brecanavir, daranavir, TMC- 126, TMC- 114, mozenavir (DMP-450), JE-2147 (AG1776), AG1859, DG35, L- 756423, RO0334649, KNI-272, DPC-681, DPC-684, and GW640385X, DG 17, PPL-I ϋϋ, 2) a HIV non-nucleoside inhibitor of reverse transcriptase, e.g., capravirine, emivirine, delaviridine, efavirenz, nevirapine, (+) calanolide A, etravirine, GW5634, DPC-083, DPC-961, DPC-963, MlV-150, and TMC-L20, TMC-278 (rilpivirine), efavirenz, BILR 355 BS, VRX 840773, UK-453,061 , RDEA8O6, 3) a HIV nucleoside inhibitor of reverse transcriptase, e.g., zidovudine, emtricitabine, didanosine, stavudine, zalcitabine, ianiivudrne, abacavir, amdoxovir, dvucitabine, alovudine, MTV-210, racivir ( -FTC), D- d4FC, emtricitabine, phosphatide, fozivudine tidoxil, fosalvudine tidox.il, apricitibine (AVX754), amdoxovir. KP- 1461, abacavir + lamivudine, abacavir +- lamivudine + zidovudine, zidovudine + lamivudine, 4) a HIV nucleotide inhibitor of reverse transcriptase, e.g., tenofovir, tenofovir disoproxil fumarate + emtricitabine, tenofovir disoproxil fumarate + emtricitabine -+ efavirenz, and adefovir, 5) a HIV integrase inhibitor, e.g., curcumin, derivatives of curcumin, chicoric acid, derivatives of chicoric acid, 3,5-dicaffeoylquinic acid, derivatives of 3,5-dicaffeoylquinic acid, aurintricarboxylic acid, derivatives of aurintricarboxylic acid, caffeic acid phenethyl ester, derivatives of caffeic acid phenethyl ester, tyrphostin, derivatives of tyrphostin, quercetin, derivatives of quercetin, S-1360, zintevir (AR- 177). L-870812, and L-870810, MK-0518 (raltegravir), BMS-707035, MK.-2048, BA-011 , BMS-538158, GSK364735C, 6) a gp41 inhibitor, e.g., enfuvirtide, sifuvulide, FB006M, TRI-1144, SPC3, DES6, Locus gp41, CovX, and REP 9, 7) a CXCR4 inhibitor, e.g., AMD-070, 8) an entry inhibitor, e.g., SPOl A, TNX-355, 9) a gp!20 inhibitor, e.g., BMS-488043 and BlockAide/CR, 10) a G6PD and NADH-oxidase inhibitor, e.g., immunitin, 10) a CCR5 inhibitor, e.g., aplaviroc, vicriviroc, INCB9471 , PRO-140, INCB] 5050, PF-232798, CCRSmA b004, and maraviroc, 1 1) an interferon, e.g., pegylated rIFN-alpha 2b, pegylated rIFN-alpha 2a. rIFN-alpha 2b, IFN alpha-2b XL, rIFN-alpha 2a, consensus IFN alpha, infergen, rebif, locteron, AVI-005, PEG-infergen. pegylated IFN-beta, oral

interferon alpha, feron, reaferon, intermax alpha, r-IFN-beta, infergen + actimmune, IFN- otnega with DUROS, and albuferon, 12) ribavirin analogs, e.g.. rebetol, copegus, lcvovirin, VX-497, and viramidine (taribavirin) 13) NS 5a inhibitors, e.g., A-831 and A- 689. 14) NS5b polymerase inhibitors, e.g., NM-283, valopicitabine, R1626, PSI-6130 (Rl 656), HrV-796, BILB 1941. MK-0608, NM-107, R7128. VCH-759, PF-S68554, GSK625433, and XTL-2125, 15) N S3 protease inhibitors, e.g., SCH-503034 (SCH-7), VX-950 (Telaprcvir), ITMN-I 91. and BILN-2065, 16) alpha-glucosidasc 1 inhibitors, e.g., MX-3253 (celgosivir) and UT-231B, 17) hepatoprotectants, e.g., IDN-6556, ME 3738. MitoQ, and LB-84451 , 3 8) non-nucleoside inhibitors of HIV, e.g., benzimidazole derivatives, beπzo-1,2,4-thiadiazine derivatives, and phenylalanine derivatives, 19) other drugs for treating HIV, e.g., zadaxin, nitazoxanide (alinea), BIVN-401 (virostat), DEBIO-025, VGX-4] 0C, EMZ-702, AVI 4065, bavituximab, oglufanide, PYN-17, KPE02O03002, actilon (CPG-10101 ), KRN-7000, civacir, GI-5005, ANA-975 (isatoribine), XTL-6865, ANA 971 , NOV-205, tarvacin, EHC-18, and NIM 81 1 , 1 9) pharmacokinetic enhancers, e.g., BAS-100 and SPI452, 2O)RN Ase H inhibitors, e.g., ODN-93 and ODN-1 12, 21) other anti-HlV agents, e.g., VGV-I , PA-457 (bevirimat), ampligen, HRG214, cytolin, polymun, VGX-410, KD247, AM2 0026, CYT 99007. A- 221 HIV, BAY 50-4798, MDXOl O (iplimumab), PBSl 19, ALG889. and PA-1050040.

Again by way of example, the Following list discloses exemplary HIV antivirals, with their corresponding U.S. Patent numbers that can be combined with the salts of the present invention.

Lexiva (Fosamprenavir calcium, US 6,436,989)

Virudiπ; Triapteα; Foscavir (Foscarnet sodium, US 6,476,009)

Crixivan (Indinavir sulfate, US 5,413,999)

Epivir (Lamivudine. US 5 047,407) Combivir (Lamivudine/Zidovudine, US 4,724,232)

Aluviran (Lopinavir)

Kaletra (Lopinavir/ritonavir, US 5,541,206)

Viracept (NeLfinavir mesilate, US 5,484,926)

Viramune (Nevirapine, US 5,366,972) Norvir (Ritonavir, US 5,541.206)

Invirase; Fortovase (Saquinavir mesilate, US 5,196,438)

Zerit (Stavudine, US 4,978,655)

Truvada (Tenofovir disoproxil fumarate/emtricitabiπe, US 5,210,085)

Aptivus (Tipranavir) Retrovir (Zidovudine; Azidothymidine, US 4,724,232)

Methods of Inhibition of HIV

In a further aspect, the present invention provides methods of treating Acquired Immune Deficiency Syndrome (AIDS), wherein each method includes the step of administering to a human being suffering from AIDS a therapeutically effective amount of a salt of the invention, or a hydrate of a salt of the invention. Treatment of AIDS includes the amelioration of at least one symptom of AEDS, and/or slowing or preventing the progression of the disease. Typically the therapeutically effective amount of the salt is administered to a human being in the form of a pharmaceutical composition, as described under the heading "Pharmaceutical Compositions". Typically, the pharmaceutical composition is administered orally, for example in the form of a tabiet. Examples of therapeutically effective daily doses of one or more salts of the invention, or hydrates thereof, are from 1 mg to IOO mg, such as from 10 mg to 30 mg. The salts of the invention can be administered daily, for example in the form of one or more tablets that include an amount of salt that provides an effective amount, such as 1 Omg or 30mg or 60 mg, of the free base when the salt dissociates in an aqueous medium within the human body.

Routes of Administration

One or more salts of the invention are administered by any route appropriate to the condition to be treated. Suitable routes include oral, rectal, nasal, topical (including

buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural), and the like. It will be appreciated that the preferred route may vary with, for example, the condition of the recipient. An advantage of the salts of the present invention is that they are orally bioavailable and can be dosed orally.

Examples and Exemplary Embodiments:

See also WO 2006/1 10157, the disclosure of which is herein incorporated by reference in its entirety, particularly, pages 167-1 74.

Example 1. Synthesis of Ethyl N-[(S)({[(2R,5R)-5-(6-amino -9H-purin-9-yl)-4-fluoro-2,5- dihydrofuran -2-yl]oxy}methyl)phenoxyphosphinoyl]-L-alaninate.

2-deoxy-2-fluoro-3,5-di-0-benzoyl-a-D-arabinofuranosyIbro mide (2)

Compound (2) was synthesized according to the synthetic schemes disclosed in Tann et. al., JOC, 1985, Vol. 50 pg. 3644 and HoweJI et. al., JOC, 1988, VoL 53, pg. 85.

To a solution of 1.3.5-lri-O-benzoyl-2-deoxy-2-fluoro-a-D-arabinofuraiiose ( 1 ) ( 120 g, 258 mmol), commercially available from Davos or CMS chemicals, in CH ₂ CI ₂ (1 L) was added 33% HBr/ Acetic acid (80 inL). The mixture was stirred at room temperature for 16 h, cooled with ice- water, and slowiy neutralized over 1 -2 h with NaHCO ₃ ( 150 g/1.5 L solution).

The CH ₂ CI ₂ phase was separated and concentrated under reduced pressure. The residue was dissolved in ethyl acetate and washed with NaHC0 ₃ until no acid was present. The organic phase was dried over MgSO ₄ , filtered and concentrated under reduced pressure to give product 2 as a yellow oil (~1 15 g).

2-deoxy-2-fluorα-3, 5-di-O-benzoyI-β-D-arabinofuranosyl-9H-6-chloropurine (3) Compound (3) was synthesized according to the synthetic schemes disclosed in

Ma et. al., J. Med. Chem., 1997, Vol. 40, pg. 2750; Marquez et. aL, J. Med. Chem., 1990, Vol. 33, pg. 978; Hildebrand et. al., J. Org. Chem., 1992, VoL 57, pg. 1808 and Kazimierczuk et. al., JACS, 1984, Vol. 106, pg. 6379. To a suspension of NaH (14 g, 60%} in Acetonitrile (900 mL), 6-chloropurine

(52.6 g) was added in 3 portions. The mixture was stirred at room temperature for i .5 h. A solution of 2 (258 mmol) in Acetonitrile (300 mL) was added dropwise. The resulting mixture was stirred at room temperature for 16 h. The reaction was quenched with Acetic acid (3.5 mL), filtered and concentrated under reduced pressure. The residue was partitioned between CH ₂ CI ₂ and water. The organic phase was dried over MgSO ₄ , filtered and concentrated. The residue was treated with CH ₂ CI ₂ and then EtOH (~1 :2 overall) to precipitate out the desired product 3 as a yellowish solid (83 g, 65% from 1 ).

2-deoxy-2-fluoro -β-D-arabinofuranosyl-6-methoxyadenine (4) To a suspension of 3 (83 g, 167 mmol) in Methanol (1 L) al 0°C, NaOMe (25% wt, 76 mL) was added. The mixture was stirred at room temperature for 2 h, and then quenched with Acetic acid (~ 11 mL, pH~7). The mixture was concentrated under reduced pressure and the resultant residue partitioned between hexane and water (approximately 500 mL hexane and 300 mL water). The aqueous layer was separated and the organic layer mixed with water once again (approximately 300 mL). The water fractions were combined and concentrated under reduced pressure to ~100 mL. The product, 4, precipitated out and was collected by filtration (42 g, 88%).

2-deoxy-2-fluoro-5-carboxy-β-D-arabinofuranosyl-6-methoxyad enine (5)

Compound (5) was synthesized according to the synthetic schemes disclosed in Moss et, al., J, Chem. Soc, 1963, pg. 1149. A mixture of Pt/C (10%, 15 g (20-30% mol eqpuiv.) as a water slurry) and

NaHCOj (1.5 g, 17.94 mmol) in H ₂ O (500 mL) was stirred at 65°C under H ₂ for 0.5 h. The reaction mixture was then allowed to cool, placed under a vacuum and flushed with N ₂ several times to completely remove all H2. Compound 4 (5.1 g, 17.94 mmol) was then added at room temperature. The reaction mixture was stirred at 65°C under Oj (balloon) until the reaction was complete by LC-MS (typically 24-72h). The mixture was cooled to room temperature and filtered. The Pt/C was washed with H ₂ O extensively. The combined filtrates were concentrated to ~ 30 mL, and acidified (pH 4) by the addition of HCl (4N) at 0°C, A black solid precipitated out which was collected by filtration. The crude product was dissoh'ed in a minimum amount of Methanol and

filtered through a pad of silica gel (eluting with Methanol). The filtrate was concentrated and crystallized from water to give compound S (2,5 g) as an off-white solid.

(

Compound (6) was synthesized according to the synthetic schemes disclosed in

Zemlicka et. al., J. Amer. Chem., Soc, 1972, Vol. 94, pg. 3213.

To a solution of 5 (22 g, 73.77 inmol) in DMF (400 mL), DMF dineopentyl acetal (150 mL, 538 nunoi) and metlianesulfonic acid (9.5 rnL, 146.6 mmol) were added. The reaction mixture was stirred at 80-93°C (interna! temperature) for 30 min, then cooled to room temperature and concentrated under reduced pressure. The residue was partitioned between ethyl acetate and water. The organic phase was separated and washed with NaHC0 ₃ followed by brine, dried over MgSO ₄ , filtered and concentrated under reduced pressure. The residue and diethyl (hydroxymethyl)phosphonale (33 mL, 225 mmol) were dissolved in CH ₂ Cl ₂ (250 mL) and cooled down to -4O°C. A solution of iodine monobromide (30.5 g, 1.1 moi) in CH ₂ Cl ₂ (100 mL) was added dropwise. The mixture was stirred at -20 to -5 °C for 6 h. The reaction was then quenched with NaHCOj and Na ₂ S ₂ O ₃ . The organic phase was separated and the water phase was extracted with CH ₂ Cl ₂ . The combined organic phases were washed with brine, dried over MgSO ₄ . filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography to give product 6 (6 g, 15.3%).

Alternative Procedure for the Preparation of 6

A soϊution of 5 (2.0 g, 6.7 mmol) in THF (45 mL) was treated with triphenyl phosphine (2.3 g, 8,7 mmol) under N ₂ . Diisopropyl azodicarboxylate (1 .8 g, S.7 mmol) was added slowly. The resultant mixture was stirred at room temperature for 1 h and then concentrated under reduced pressure to dryness. The residue was dissolved in CH ₂ Cl ₂ (20 ml), and then treated with diethyl(hydroxymethyl)phosphonate (4.5 g, 27 mmol). The mixture was cooled lo -60°C and then a cold solution of iodine monobromide 2 g, 9.6 mmot) in CH ₂ Cl ₂ (10 ml) was added. The reaction mixture was warmed to -10°C and then kept at -10°C for 1 h. The reaction mixture was diluted with CH ₂ Cl ₂ , washed with saturated aqueous NaHCO ₃ , and then with aqueous sodium thiosulfate. The organic phase was separated, dried over MgSO<t, and concentrated under reduced pressure to dryness. The reaction mixture was purified by silica gel chromatography (eluting with 25% ethyl acetate in CH2CI ₂ , then switching to 3% methanol in CH ₂ Cl ₂ ) to afford product 6 (0.9 g, 33 %).

(2'R, 5'R)-6-Methoxy-9-[3-fluoro-2,5-dihydro-5-(diethoxyphosphinyl )methoxy-2- furanyl]purine (7)

To a solution of compound 6 (6 g, 11.3 mmol) in acetic acid (2.5 mL) and methanol (50 mL), NaClO ( 10-13%) (50 mL) was added dropwise. The reaction mixture was then stirred for 0.5 h and concentrated under reduced pressure. The residue was treated with ethyl acetate and then filtered to remove solids. The filtrate was concentrated and the residue was purified by silica gel chromatography to give product 7 (4 g, 88%).

(2'R, 5'R)-9-{3-fluoro-2,5-dihydro-5-phosphonomethoxy-2-furany])ad enine di sodium salt (8)

A solution of compound 7 (2.3 g, 5.7 mmol) in methanol (6 mL) was mixed with aminonium hydroxide (28-30%) (60 mL). The resultant mixture was stirred at 120°C for 4 h, cooled, and then concentrated under reduced pressure. The residue was dried under vacuum for 12 h. The residue was dissolved in DMF (40 mL) and bromotrimethylsilane (3.5 mL} was added. The mixture was stirred at room temperature for 16 h, and then concentrated under reduced pressure. The residue was dissolved in aqueous NaHCO ₃ (2.3 g in 100 mL of water). The solution was evaporated and the residue was purified on C-18 (40 μm) column, eluting with water. The aqueous fractions were freeze dried to give di-sodium salt 8 ( 1.22 g, 57%).

Example of Monoamidate Preparation (9)

Di sodium salt 8 (25 mg, 0.066 mmol), (S)-Ala-O-cyclobutyl ester hydrochloride (24 mg, 2 eq.. 0.133 mmol) and phenol (31 mg, 0.333 mmol) were mixed in anhydrous pyridine (I mL) Triethylamine (11 1 μL, 0.799 mmol) was added and the resultant mixture was stirred at 60°C under nitrogen. In a separate flask, 2'-Aldrithiol (122 mg,

0.466 mmol) and triphenylphosphine (103 mg, 0.466 mmol) were dissolved in anhydrous

pyridine (0.5mL) and the resulting yellow solution was stirred for 15-20 min. The solution was then added to the solution of 8 in one portion. The combined mixture was stirred at 60°C under nitrogen for 16 h to give a clear yellow to light brown solution. The mixture was then concentrated under reduced pressure. The resultant oil was dissolved in CH ₂ Cl ₂ and purified by silica gel chromatography (eluling with a linear gradient of 0 to 5% MeOH in CH ₂ Cl ₂ ) to give an oil. The resulting oil was dissolved in acetonitrile and water and purified by preparative HPLC (linear gradient, 5-95% acetonitrile in water). Pure fractions were combined and freeze-dried to give mono amidate 9 as a white powder.

Example of bis amidate preparation (10)

Di sodium salt 8 (12 mg, 0.032 mmol) and (S)-Ala-O-n-Pr ester hydrochloride (32 mg, 6 eq., 0.192 mmol} were mixed in anhydrous pyridine (1 niL). Triethylamine (53 μL, 0.384 mmol) was added and the resultant mixture was stirred at 60°C under nitrogen. In a separate flask, 2'-Aldrithiol (59 mg, 0.224 mmol) and triphenylphosphine (49 mg, 0.224 mmol) were dissolved in anhydrous pyridine (0.5 mL) and the resulting yellow solution was stirred for 15-20 min. The solution was then added to the solution of 8 in one portion. The combined mixture was stirred at 60°C under nitrogen for 16 h to give a clear yellow to light brown solution. The mixture was then concentrated under reduced pressure. The resultant oil was dissolved in CH2CI2 and purified by silica gel chromatography (eluting with a linear gradient of 0 to 5% MeOH in CH ₂ CI ₂ ) to give an oil. The resulting oil was dissolved in acetonitrile and water and purified by preparative HPLC (linear gradient, 5-95% acetonitrile in water). Pure fractions were combined and freeze-dried to give bis amidate as a white powder.

Example of Monoamidate Preparation (I I)

Compound 8 (1.5g, 4 mmoi) was mixed with ethyl alanine ester HCl salt (1.23g, S mmol) and phenol (1.88 g, 20 mmol). Anhydrous pyridine (35 mL) was added followed by TEA (6.7 mL, 48 mmol). The mixture was stirred at 60°C under nitrogen for 15-20 min. 2'-Aldrithiol (7.3 g) was mixed in a separate flask with triphenylphosphine (6.2 g) in anhydrous pyridine (5 mL) and the resultant mixture was stirred for 10-15 min to give a clear light yellow solution. The solution was then added to the above mixture and stirred overnight at 6O°C. The mixture was concentrated under reduced pressure to remove pyridine. The resultant residue was dissolved in ethyl acetate and washed with, saturated sodium bicarbonate solution (2x) and then with saturated sodium chloride solution. The organic layer was dried over sodium sulfate, filtered and then concentrated under reduced pressure. The resultant oil was dissolved in dichloromethane and loaded onto a dry CombiFlash column, 4Og, elutiπg with a linear gradient of 0-5% methanol in dichloromethane over 10 min and then 5% methanol in dichloromethane for 7-10 min. Fractions containing the desired product were combined and concentrated under reduced pressure to give a foam. The foam was dissolved in acetonitrile and purified by prep HPLC to give 11 (0.95 g).

Dissolved J 1 (950 mg) in small amount of acetonitrile and let stand at room temperature overnight. Collected solid by filtration and washed with small amount of acetonitrile. Filtrate was reduced under vacuum and then loaded onto Chiralpak AS-H column equilibrated in Buffer A, 2% ethanol in acetonitrile. Isomer A, 12, was eluted out with Buffer A at 1 OmlVmin for 17mins. After which Buffer B, 50% methanol in acetonitrile, was used to ehite isomer B, 13, separately out from the column in δrains. Removed all solvent and then separately re-dissolved in acetonitrile and water. Separately freeze-dried the samples (Mass — 348 mg). Isomer 12 is shown below

Example 2- Screening Salt Forms of Ethyl N-f(S)({[(2R,5R)-5-(6-ainino-9H-purin-9- yl)-4-fluoro-2,5-dihydrofuran-2-yl]oxy}methy[)phenoxyphosphm oyI]-L-alaninate

The following acids were screened to determine whether they formed suitable crystalline salts of Ethyl N-[{SX{[(2R,5R)-5-(6-amino-9H-purin-9-yl)-4-fIuoro-2,5- dihydrofiiran-2-yl]oxyj-methyI)phenoxyphosphinoyl]-L-alanina te: Mineral acids (HX, wherein X = halogen; H ₃ PO ₄ ); organic sulfonic acids (RSO ₃ H, wherein R = Me, Et, Ph, (+)-camphor-10-suifonic acid; naphthalene-2-sulfonic acid; naphthalene-l ,5-disulfonic acid); mono carboxylic acids (RCO ₂ H, wherein R = H, Me, Et, Ph, trans-PhCH=CH,

CIiCH, PhCONHCHz), and dicarboxylic acids (malonic. succinic, fumaric, adipic, oxalic, maleic).

Solids were obtained with three of the foregoing acids when mixed with Ethyl N- [(S)( {t(2R,5R)-5-(6-amino-9H-purin-9-yl)-4-fluoro-2,5-dihydrorura n-2- yl]oxy] methyl)phenoxyphosphinoyl]-L-alaninate: trifluoroacetic acid, malonic acid, and succinic acid. Trifluoroacetic acid is not considered to be pharmaceutically acceptable.

Ethyl N-[(SX{[(2R,5R)-5-(6-amino-9H-purin-9-yl)-4-iluoiO-2,5-dihyd rofuran -2- yl]oxy}metliyl)phenoxyphosphinoyl]-L-aIaninate is an atnidate prodrug which undergoes decomposition in protic solvents under acidic or basic conditions. For this reason, acids with potentially nucleophilic moieties were not included in the initial round of screening. Subsequently, citric acid, giycoϋc acid, (S)-(+) -lactic acid, salicylic acid, (S)-(-)-malic acid, {5)-(+)-mandelic acid and (S)-(+)-glutamic acid were evaluated. Ethyl N- [(S)({[(2R,5R)-5-(6-amino-9H-purin-9-yl>-4-fluoro-2,5-dih ydrofuran-2- yl]oxy}methyl)phenoxyphosphinoyl]-L-alaninate unexpectedly formed a stable crystalline salt with citric acid. This was an unexpected result because citric acid includes a hydroxyl group which can act as a nucleophile toward the amidatc moiety of Ethyl N- [(S)({[(2R,5R)-5-(6-amiπo-9H-purin-9-yI)-4-fluoro-2,5-dihyd rofuran-2- yl]oxy} methyl)phenoxyphosphinoy]]-L-alaninate and potentially undergo a reaction therewith in which a covalent bond is foπned between Ethyl N-[(S)( ([{2R,5R)-5-(6- amino-9H-purin-9-yl)-4-fluoro-2,5-dihydrofuran-2-yl]oxy}meth yl)phenoxyphosphinoyl]- L-alaninate and the hydroxyl group of citric acid with the cxpuLsion of either phenol or alanine ethyl ester.

Example 3. Synthesis of Malonate Salt of Ethyl N-[(S)( {[(2R,5R)-5-(6-amino-9H- purin _' 9-yI)-4-fluoro-2,5-dihydrofuran-2-yl]oxy}inethyl)pheno xyphosphiiioyl]-L- alaninate

The free base form of Ethyl K-[(S)( {[(2R,5R)-5-(6-amino-9H-ρurin-9-yl)-4-fluoro-2,5-dihydrofur an-2-yl]oxy}methyl>phenoxyphosphinoyI]-L-alaninate was dissolved in warm 2-butanone (15 parts), and malonic acid (0.26 parts) was added to form a solution with agitation. Heptane (5 parts) was added to the solution which was slowly cooled to about 5°C, collected and rinsed with cold 2-butanone/heptane. The resulting malonate salt of Ethyl N-[(SX {[(2R,5R)-5-(6-amino-9H-purin-9-yl)-4-fluoro- 2,5-dihydrofuran-2-yl]oxy) methyl)phenoxyphosphinoyl]-L-alaninate was thereby produced with a yield of about 80% ,

The NMR spectrum of the malonate salt had the following characteristics: ¹ H NMR (400MHz ,CHLOROFORM-d) ppm = 8.27 (s, 1 H), 8.26 (s, 1 H), 7.32 - 7.20 (m, 3 H), 7.15 (d. J - 7.8 Hz, 2 H), 6.7S (m, 1 H), 5.88 (br. s., 1 H), 5.78 (s, 1 H), 4.17 (m. 2 H), 4.15 - 4.07 (m, 3 H), 3.85 (dd, J =8.0, 8.0, 1 H), 3.38 (s, 2 H), 1.31 (d, J = 7.0 Hz, 3 H), 1.23 (i, J = 7.0 Hz, 3 H) 1 ¹ P NMR (162 MHz,CHLOROFORM-d) ppm = 20.64 (s) ¹⁹ F NMR (376 MHz, ACETONITR]LE-d3) ppm = -335. ] 9 (s)

Example 4. Synthesis of Succinate Salt of Ethyl N-[(S)( {[(2R,5R)-5-(6-amino-9H- purin-9-yl)-4-fluoro-2,5-dihydrofuran-2-y]]oxy}methyl)phenox yphosphinoyl]-L- aianinate.

The free base form of Ethyl N-[(S)(ft(2R,5R)-5-(6-amino-9H-purin-9-yl)-4- fluoro-2,5-dihydrofuran-2-yl]oxy}methyl)phenoxyphosphinoyl]- L-alaninafe was dissolved in warm 2-butanαne (15 parts), and succinic acid (0.28 parts) was added to form a solution with agitation. The solution was sJowly cooled lo about 5°C, collected, and rinsed with cold 2-butanone, thereby yielding the succinate salt of Ethyl N- [(S)({[(2R,5R)-5-(6-amino-9H-purin-9-yI)-4-fluoro-2,5-dihydr ofliran-2- yl]oxy}methyl)phenoxyphosphinoyl]-L-alaninate at a yield of 80%. The NMR spectrum of the succinate salt had the following characteristics: ¹ H

NMR (400MHz .ACETONITRILE-d3) ppm = 8.26 (s, I H), 8.15 (s, 1 H), 7.29 (dd, J = 7.6, 7.6 Hz, 2 H). 7.16 (d, 7 = 7.6 Hz, 1 H), 7.12 (d, J= 8.0 Hz, 2 H), 6.78 (m, 1 H), 6.17 (br s, 1H), 5.89 (m, 2 H), 4.12 - 3.95 (overlapped multiplets, 6 H), 2.53 (s, 4 H), 1.24 (d, J= 6.8, 3 H), 1.18 (t, J= 7.2, 3 H). 3 ¹ P NMR (162 MHz, Acetonitrile-d3) ppm = 21.60 (s) 1 ⁹ F NMR (376 MHz, Aceionitrile-d3) ppm = -135.19 (s)

Example 5. Synthesis of Citrate Salt of Ethyl N-[(S)({[(2R,5R)-5-(6-amino-9H-purin-9- yI)-4-fluoro-2,5-dihydrofuran-2-y]]oxy}methyl)phenoxyphospni noy]]-L-alaninate.

The free base form of Ethyl N-[(S)({[(2R,5R)-5-(6-amino-9H-purin-9-yl)-4- fluoro-2.5-dihydrofuran-2-yl]oxy}methyl)phenoxyphosphinoyI]- L-alaninate (about 3Og) was dissolved in hot acetonitrile (16 parts) and citric acid (0.38 parts) were added with agitation. The resulting solution was slowly cooled to about 5°C, collected and nnsed with cold acetonitrile, and dried, thereby affording the citrate sait of Ethyl N-

[(S)({[(2R,5R)-5-(6-amino-9H-purin-9-yI)-4-fluoro-2,5-dih ydrofuran-2- yl]oxy}methyl)phenoxyphosphinoy]]-L-alaninate with a yield of about 84%.

The NMR. spectrum of the citrate salt had the following characteristics; ¹ H NMR {400MHz ,DMSO-d6) ppm = 8.20 (s, 2H), 7.45 (2H, br s), 7.29 (dd, J = 7.6, 7.6 Hz. 2

H), 7.13 (dd, J - 7.2 Hz, J = 7.2 Hz 1 H), 7.12 (dd, J- 8.0 Hz, J= 8.0 Hz 2 H), 6.86 (d, J

= 2.4 Hz, 1 H), 6.14 (s, I H). 5.97 (d, J- 3.6 Hz, I H), 5.78 (dd, J = 12.2, 10.4 Hz, 1H),

4.05 (m, 1H), 4.02 (m, 2H), 3.98 (m, 1H), 3.89 (m, 1H), 2.76 (d, J - 15.6 Hz, 2H), 2.66

(d, J= 15.6 Hz, 2H), 1.16 (d, J= 7.2 Hz, 1H). 3 ¹ P NMR (162 MHz, DMSO-d6) ppm = 22.29 (s)

¹⁹ F NMR (376 MHz, ACETONITRTLE-d3) ppm = - 133.88 (s)

HRMS: m/-z. 507.1561 ; Calcd for C ₂₁ H ₂₄ FN ₆ O ₆ P: 507.1557.

Anal. Calcd for C ₂₁ H ₂₄ EN ₆ O ₆ P: C: 46.42; H: 4.62; N: 12.03; P: 4.43; F: 2.72; Found: C:

45.83; H: 4.74; N: 1 1.81 : P: 4.45; F: 2.80.

Example 6. Physicocheraical Properties of Salts of Ethyl N-[(S)({[(2R,5R)-5-(6-amino-

9H-purin-9-yI)-4-fluoro-2,5-dihydrofuran-2-yl]oxy}methyl) phenoxyphosphinoyl]-L- alaninate

Representative batches of the malonate, succinate, and citrate salts of Ethyl N-

[(S)({[(2R,5R)-5-(6-amino-9H-purm-9-yl)-4-fluoro-2,5-dihy drofuran-2- yl]oxy}methyl)phenoxyphosphinoyI]-L-alaninate were prepared. The melting points of these salts were determined and serve as a rough measure of stability with a higher meϊtiτig point indicating a higher level of stability. As shown in Table 1 , the citrate salt had the highest melting point. Additionally, the heat of fusion {ΔH _(fusion) ) of each of the three salts is shown in Table 1. The citrate salt had the highest heat of fusion indicating a higher degree of solid-state crystallinity than the other two salts.

Table 1. Melting Temperatures and Heat of Fusion of Succinate, Malonate and Citrate Salts of Ethyl N-[(S)( {[(2R,5R)-5-(6-ainino-9H-purin-9-y])-4-fluoro-2,5- dihydrofuran-2~yl]oxy } methyl)phenαxyphosphinoyl] -L-alaninate

The free base form of Ethyl N-[(S)( {[{2R,5R)-5-(6-amino-9H-purin-9-yl)-4- fiuoro-2,5-dihydrofuran -2-yl]oxy}inethyl)phenoxyphosphmoyl]-L-alanir»aie is amorphous, hygroscopic and chemically unstable when stored under open condition at 40°C and 75% relative humidity (RH). As shown in Table 2, the corresponding succinate, malonate and citrate salts were not hygroscopic at room temperature when exposed to a relative humidity of 92% for several days.

Table 2. Solid State Hygroscopicity of Salt Forms of Ethyl N-[(S)( {[(2R,5R)-5-f6- amino - 9H-purin-9-yl )-4-fluoro-2,5 -dih ydrofiαran-2- yl]oxy}methyl)phenoxyphosphinoyl]-L-Blantnate at Room Temperature

The solid-state chemical stability of the free base, succinate, malonate and citrate salts of Ethyl N-[(S)({[(2R,5R)-5-(6-araino-9H-purin-9-yI)-4-nuoro-2,5-dihy drofuran-2- yl]oxy}methyI)phenoxyphosphinoyl]-L-alaninate were examined under open conditions at 40°C and 75% relative humidity. As shown in Table 3, the citrate salt displayed superior chemical stability compared to the succinate and malonate salts.

Table 3. Solid State Stability of Free Base form of Ethyl N-[(S)({{(2R,5R)-5-(6- amino-9H-purin-9-yl)-4-fluoro-2,5~dihydrofuran-2- yl]oxy}methyl)phenoxyphosphinoyl]-L-alaninate , and the corresponding Succinate, Citrate and Malonate Salts at 40 °C/75% Relative Humidity Under

Open Conditions

Example 7. Composition of Tablets Providing Equivalent of 10 mg and 30 rag of Ethyl N-[(S)( {[{2R,5R)-5-(6-amino-9H-purin-9-yI)-4-fluoro-2,5-dihydrofura n-2- yI]oxy}methyl)phenoxyphosphinoy]]-L-aIaninate Free Base

The citrate salt of Ethyl N-[(S)({[{2R,5R)-5-(6-amJno-9H-purin-9-y3)-4-fluoro- 2,5-dihydrofuran-2-yI]oxy) methyl)phenoxyphosphinoyl]-L-alaninate was foiτnulated into 10 mg and 30 mg tablets using the roller compaction process. The active ingredient, lactose anhydrous, microcrystalline cellulose and croscarmellose sodium were first blended _* the mixture was then lubricated with one third of the total amount of magnesium stearate, then roller compacted, followed by milling. The resulting granules were lubricated with the remaining amount of magnesium stearate and pressed into tablets.

Table 4 shows the compositon of tablets that include the citrate salt of Ethyl N- [(S){{[(2R,5R)-5-(6-amino-9H-purin-9-y!)-4-fluoro-2,5-dihydr ofuran-2- yl]oxy} methyl)phenoxyphosphinoyl3-L-alamnate, and that provide either 10 mg or 30 mg of the free base form of the compound when the citrate salt dissociates in an aqueous medium.

Table 4.

a Equivalent to 10% 2/2 of free base form of compound. Actual drug substance weight wiϋ be adjusted to account for drug substance purity. b Equivalent to 10 mg of free base form of compound. c Equivalent to 30 mg of free base form of compound. d The adjusted drug substance quantity will be subtracted from the quantity of anhydrous iactose. e The abbreviation NF means national formulary, and the abbreviation HSE means House

Compendial Reference which is the internal standard used at Gilead Sciences

Example 8. Comparison of Lymphocyte Loading of Active Metabolite After Administration to Lymphocytes of Ethyl N-[(S)({[(2R,5R)-5-(6-amino -9H-purin-9-yl)-4-fIuoro-2,5-dihydrofuran - 2-y!]oxy}methyl)phenoxyphosphinoyi]-L-alaninate (Prodrug) or Parent Compound.

Ethyl N-[(S){{ [(2R,5R)-5-(6-amino -9H-puπn-9-yl)-4-fluoro-2,5-dihydrofuran -2- yi]oxy}methyl)phenoxyphosphinoyl]-L-alaπiπate is a prodrug that is hydrolyzed in the human body to yield a hydroiysis product that is hereinafter referred Io as "the parent compound". The parent compound is phosphorylated within the human body to produce a biologically active phosphorylated product (hereinafter "the active metabolite") which inhibits the activity of reverse transcriptase enzyme.

To characterize the intracellular metabolism of the prodrug and parent compound, lymphocyte cells were treated with either 1 uM prodrug or 100 μM parent compound for 2, 6, and 24 hours. Peripheral blood mononuclear ceils (PBMCs) were isolated from human buffy coats (Stanford Blood Bank, Palo Alto, CA) using centrifugation in Ficoll Paque Plus (GE Healthcare, Piscataway, NJ) according to manufacturer's procedure. PBMCs isolated from 3 to 4 independent donors were maintained in RPMI- 1640 media with 20% fetal bovine serum and antibiotics (quiescent state) or were activated in the presence of interleukin 2 (20 units/mL, Roche Biochemicals, Indianapolis, IN) and phytohemaggiutinin PHA-P (1 μg/mL, Sigma) for 3 to 4 days before the initiation of experiments.

Human transformed CCRF-CEM T-cells were obtained from The American Type Culture Collection (Manassas, VA) and cultured in RPMI-1640 medium supplemented with 10% FBS and antibiotics. An aliquot of cells (2-3x10 ⁶ cells) was collected at each time point, counted, pelleted by centrifugation, resuspended in 0.5 mL of the original treatment media and layered onto 0.5 mL of Nyosil M25 oil. The samples were spun in a microcentrifuge for 20 seconds at the maximum speed (approximately 800 x g). The top layer of media was removed and the oil layer was washed twice with 0.8 mL phosphate- buffered saline. Washing buffer and the oil layer were carefully removed, the cell pellet was resuspended in 0.5 mL 70% methanol and incubated overnight at — 70°C to facilitate cell lysis. Cell lysates were centrifuged, supematants collected, dried by vacuum and resuspended in 10 μL tetrabutyl aminonium acetate containing the diphosphate of [5-(6- Amino-ρurin-9-yI}-2,5-dihydro-furan-2-yloxvmethyl]-phosphon ic acid (the non- fluorinated analog of the active metabolite) as an internal standard.

Transient ion-pairing high-performance liquid chromatography coupled to positive ion electrospray tandem mass spectrometery (LC/MS/MS) was used to quantitate intracellular nucleotides. Methods were adapted from those described for the acyclic phosphonate nucleotide analog adefovir, its phosphorylated metabolites and natural nucleotides (Vela, .1.E. et al. Simultaneous quantitation of the nucleotide analog adefovir, its phosphorylated anabolites and 2'-deoxyadenosine triphosphate by ion-pairing

LC/MS/MS. Journal of Chromatography B Anat. Technol. Biomed. Life Sci., vol. 848, 2007, pp 335-343). Standard curves and quality control samples were generated for all anaiytes using extracts from untreated cells. Seven point standard curves generally ranged from 0.03 to 20 pmol/million cells and had linearity in excess of r equal to 0.99 for all anaiytes. The lower limits of quantitations for all anaiytes ranged from 0.05 to 0.1 pmol/million cells. Low and high concentration quality control samples (typically 0.2 and 10 pmol/million cells, respectively) were ran with each analyte at the beginning and end of each analytical run to assure accuracy and precision within 20%.

The parent compound was incubated at a 100-fold higher concentration (100 μM) than the prodrug ( 1 μM) to facilitate accurate analysis of the much lower intracellular accumulation of metabolites observed following incubation of lymphocytes with the parent compound. As shown in Table 5, the prodrug induced 76-, 290- and 140-fold increased levels of the active metabolite relative to the parent compound following incubation with CEM-CCRF, quiescent PBMCs and activated PBMCs, respectively. Active metabolite levels were normalized based on extracellular concentration following incubations with 1 μM prodrug or with 100 μM parent compound.

Table 5.

Example 9. Comparison of Anti-HIV Activity of the Prodrug and the Parent Compound.

The terms ''prodrug" and "parent compound" have the meanings set forth in Example 8.

MT- 2 cells were maintained in RPMl-1640 medium supplemented with antibiotics and 10% fetal bovine serum (FBS). MT-2 cells were infected with HlV-I IIEB at a multiplicity of infection (moi) of 0.01 and added to 96-well plates with serial dilutions of tested compounds at a density of 20,000 cells/well. After a 5-day incubation, the virus-induced cytopathic effect was determined using a CellTiter-GloTM cell viability assay (Promega, Madison, WI) and expressed as a percentage of the signal from samples with fully suppressed virus replication after the subtraction of signal from the untreated control. The concentration of each drag that inhibited the virus-induced cylopathic effect by 50% (EC ₅ 0) was determined by non-linear regression. Activity against NRTl-resistant mutants was determined in parallel with wild-type control virus and fold change in EC so was calculated.

Human peripheral blood mononuclear cells (PBMC) were isolated from donor buffy coals using centrifugation in Ficoll Paque Plus and activated for 4-5 days in RPMI- 1640 medium with 20% FBS, antibiotics, inιerieukiri-2 (20 units/mL) and phytohemaggluriπine PMA-P (2 μg/mL). Activated PBMC were infected with HIV-] BaL for 3 hours, washed, seeded into 96-well plates (250,000 cells/well) and incubated with serial dilutions of tested compounds for 5 days, at which point cell supematanls were collected and virus production was determined using commercial HIV-I p24 ELISA (Beckman Coulter, Miami, FL). The concentration of each drug inhibiting the p24 antigen production by 50% (EC50) was determined by regression analysis.

The effect of the addition of pro-moieties on the anti-HIV activity was assessed in

MT-2 and stimulated PBMC infected with HIV- 1. As shown in Table 6, the prodrug was 71 - and 2,300-fold more potent than the parent compound in MT-2 and activated PBMC, respectively.

Table 6. Anti-HIV- 1 activity of the prodrug and the parent compound in a lymphoid derived cell line and primary lymphoid cells.

EC50 (μM)

MT-2 PBMC

Parent 10.6 ± 2.4 8.5 ± 7.7 Compound

Prodrug 0.15 ± 0.04 0.0037 ± 0.0001

Values represent the mean ± standard deviation from at least two dependent experiments performed in triplicate.

Example 10. Oral Bioavailability of the Citrate Salt of Ethyl N-[(S)( {[(2R,5R>5-(6- amino-9H-purin-9-yl)-4-flυoro-2,5-dihydrofuran -2-y!]oxy} raethyI)phenoxyphosphinoyl]- L-aianinate After Administration in Tablet Form to Beagle Dogs The dosing group consisted of 3 non-naϊve male beagle dogs. The animals were fasted overnight prior to dose administration and up to 4 hr after dosing. Dogs were administered a single tablet containing 41.38 mg of the citrate salt of the prodrug (providing 30 mg of the prodrug per tablet). The tablet consisted of 13.79% of the citrate salt of the prodrug, 66% anhydrous lactose, 15.21 % microcrystalline cellulose, 3.5% sodium croscamellose and 1.5% magnesium stearate on a weight per weight basis.

Plasma samples were obtained prior to dosing (0 hr) and at 0.083, 0.25, 0.50, 1.0, 2.0, 4,0, 8.0, 12, 24 hr. The blood samples were collected into Vacutainer™ tubes containing EDTA-K ₃ . The blood samples were centrifuged at 4°C to separate plasma. An aliquot of 100 μl of each plasma sample was first diluted with 300 μl 80% acetonitrile/water containing 200 nM internal standard. Following centrifugation of the protein precipitate, 100 μl of the supernatant was removed and used for analysis. Standard curves and quality control samples were prepared in dog plasma from animals not dosed with the prodrug. Samples were analyzed by a partially validated liquid chromatography couple to triple quadrupole mass spectrometry method.

Administration of the citrate salt of the prodrug as a tablet resulted in. rapid absorption of the prodrug and the parent compound derived from the prodrug. As summarized in Table 7. plasma exposure to the prodrug and the parent compound was observed following administration. The orai bioavailability of intact prodrug was 1 1.4%. These results were not markedly different from those observed following oral administration of the prodrug in a solution formulation, illustrating the effectiveness of lhe tablets containing the citrate salt of the prodrug to deliver the prodrug and its metabolites into the systemic circulation.

Table 7. Mean Plasma Pharmacokinetic Parameters for the Prodrug and the Parent Compound Following Oral Administration of a Tablet Formulation of the Citrate Salt of the Prodrug at a mean dose of 3.05 mg/kg equivalents (Mean, n=3)

Example 11. Differential Scanning Calorimetry (DSC) of Citrate, Malonate and Succinate Salts of Ethyl rJ-[(S)({[{2R _; 5R)-5-(6-amino-9H-purin-9-yl)-4-fl ιioro-2,5-dihydrofuran -2- ylJoxy} methyl)phenoxyphosphinoyl]-L-alaninate.

Differentia! scanning calorimetry accurately measures temperatures and heat flow associated with thermal transitions in a material. The DSC traces of the inventive salts of Ethyl N- [(S)({ [(2R,5R)-5-(6-amino-9H-purin-9-yl)-4-fluoro-2,5-dihydrofuran -2- yi]oxy}methyl)phenoxyphosphinoylJ-L-alaninale were generated using a TA Instrument (New Castie, DE) DSC 2010 at a scan rate of 5°C min ^~ \ Figure 1 shows the characteristic DSC trace for the maionate salt of Ethyl N-[(S)({[(2R,5R)-5-(6-amino-9H-puπn-9-yl)-4-fluoro-2,5- dihydrofuran -2-yl]oxy}methyl)phenoxyphosphinoyl]-L-aianinate. The DSC thermogram revealed a single endotherm corresponding to th Hr= 73.72 J/g) followed by a single exotherm corresponding to the d e salt. Figure 2 shows the characteristic DSC trace for the succinate salt of Ethyl N- [(S)({[(2R ₅ 5R)-5-(6-amino -9H-purin-9-yϊ)-4-fluoro-2,5-dihydrofuran-2- yl]oxy}methy])phenoxyphosphiπoyl]-L-alaninate. The DSC thermogram of the succinate salt of GS-9 I31 revealed a single emdotherm corresponding to the melting point (137.44°C, ΔH _f = 66.18 J/g) followed by a single exotherm corresponding to the decomposition of the sait.

Figure 3 shows the characteristic DSC trace for the citrate salt of Ethyl N-[(S)((l(2R,5R)- 5-(6-amino-9H-purin-9-yl)-4-fluoro-2,5-dihydrofuran-2-yl]oxy }methyl)phenoxyphosphinoyl]-L- alaninate. The DSC thermogram of the citrate salt revealed a single endotherm corresponding to the melting point (]49.41 °C, ΔH _f = 85.72 J/g) followed by a single exotherm corresponding to the decomposition of the citrate salt.

The citrate salt has a significantly greater heat of fusion than the maionate and succinate salts, indicating a higher degree of solid-state crystal unity.

Example 12. X-Ray Diffraction (XRD) Analysis of the Citrate, Maionate and Succinate Salts of Ethyl N-[(S)({[(2R,5R)-5-(6-amino-9H-ρurin-9-yl)-4-nuoro-2,5-dihy drofuran-2- yl]oxy}methyI)phenoxyphosphinoyl]-L-alaninate.

The X-ray powder diffraction (XRPD) patterns of the inventive salts of Ethyl N-

[(SX{[(2R,5R)-5-(6-amino-9H-purin-9-y1H-fluoro-2,5-dihydr ofuran-2- yl]oxy}methyl)phenoxyphosphinoyI]-L-alaninate were generated using two methods. In the first method, a Shimadzu XRD 6000 instrument with the following attributes was used: Cu X-ray tube, 2.2KW, NF (normal focus); monochromatσr curved graphite; goniometer vertical, 1 85 mm radius; divergence slits: 0.5°, 1.0°, 0.05 mm; soller slits: 0.05°, 1°, 2°; receiving slits: 0.15 mm, 0.3 mm; scintillation detector (NaI) HV 500-1200.

In the second method, a Shimadzu XRD 6000 instrument with the following attributes was used: Cu target X-ray tube, 35 kv, current 40 ma; continuous scan, monochromator, divergence slit, 1 °: soller slit 1", receiving slit 0.3 mm.

XRPD data for the malonate and succinate salts were obtained using method 1. XRPD data for the citrate salt was obtained using method 2.

It is understood that experimental deviations can slightly change the XRPD absorption band (peak) information. Thus, the numbers reported in this patent resulting from XRPD patterns of the inventive salts wil l be the same or essentially the same as the numbers which wiil occur upon repeating the tests. "Essentially the same" in this context means that typical peak position and intensity variability are taken into account (as they normally would be for any analytical technique). For example, one skilled in the ait will appreciate that the peak positions wili show some inter-apparatus variability. For example, the 2-thela peak positions typically deviate by as much as 0.1 degrees. Further, one skilled in the art will appreciate that relative peak intensities wil! also show variability due to the degree of crystallinity, preferred orientation, prepared sample surface, and other factors known in the art. Thus, the relative peak intensities should be taken as qualitative measure only.

' Figure 4 shows the characteristic XRPD pattern for the malonate salt. The principal and characteristic peaks that define this crystalline form of the malonate salt are shown in Tables 8A and 8B .

Table 8 A

Malonate Salt PXRD Peaks

Strongest Peaks 2Theta (deg) d (A) FWHM (deg) Integrated Int

I 17.76 4.9912 0.3190 16049

2 14.92 5.9342 0.3023 14513

3 ^Γ LS.SO 4.7163 0.6572 21597

Figure 5 shows the characteristic XRPD pattern for the succinate salt. The principal and characteristic peaks that define this crystalline form of the succinate sail are shown in Tables 9A and 9B .

Figure 6 shows the characteristic XRPD pattern for the citrate salt. The principal and characteristic peaks that define this crystalline form of the citrate salt are shown in Tables 1 OA and 1 OB .

AU literature and patent citations above are hereby expressly incorporated by reference at the locations of their citation. Specifically cited sections or pages of the above cited works are incorporated by reference with specificity. While the invention has been described in detail sufficient to allow one of ordinary skill in the art to make and use the subject matter of the following claims, it is also contemplated that certain modifications of the following claims can be made and yet remain within the scope and spirit of the invention.