WO2019071105A1

WO2019071105A1 - Crystalline compounds and methods for the treatment of viral infections

Info

Publication number: WO2019071105A1
Application number: PCT/US2018/054574
Authority: WO
Inventors: Rayomand H. Gimi; Seetharamaiyer Padmanabhan; Dillon CLEARY; Radhakrishnan P. Iyer; Subramanian Baskaran
Original assignee: Spring Bank Pharmaceuticals, Inc.
Priority date: 2017-10-05
Filing date: 2018-10-05
Publication date: 2019-04-11

Abstract

This invention provides, in part, a crystalline compound of formula I that is diastereomerically pure and stable at about 25 °C and ambient humidity or a pharmaceutically acceptable salt thereof, for example, a hemi-tartrate salt, and methods of using same. Also provided herein are compositions comprising a crystalline compound of formula I or a pharmaceutically acceptable salt thereof, and methods of using same.

Description

CRYSTALLINE COMPOUNDS AND METHODS

FOR THE TREATMENT OF VIRAL INFECTIONS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of, and priority to, U.S. provisional patent application serial number 62/568,635, filed October 5, 2017, which is hereby incorporated by reference herein in its entirety.

BACKGROUND OF INVENTION

[0002] Compounds that activate the immune system of a subject can be used to treat a variety of diseases such as infections and cancer. A viral infection is a proliferation of a virus inside the body. Because a virus is incapable of reproducing without the assistance of a host, it invades a living cell to reproduce. A virus enters a host cell and introduces its DNA or RNA to the cell, which contains the genetic information to replicate the virus. The virus' genetic material controls the cell and makes it replicate the virus. Viruses affect many parts of the body including the respiratory, reproductive and gastrointestinal systems. They can also affect the skin, liver and brain. Commonly known viruses that can cause infectious diseases include rhinovirus, respiratory syncytial virus (RSV), hepatitis A virus, hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus, norovirus, rotavirus, influenza virus, human papillomavirus (HPV) and human immunodeficiency virus (HIV). Studies also reveal that viruses are implicated in many cancers. For example, HBV and HCV can cause long-term infections which can increase the patient's chance of developing liver cancer. Immune activating compounds can also be used to treat certain cancers, as seen in immuno-oncology target compounds.

SUMMARY OF INVENTION

[0003] In one aspect, the present invention provides a method of making a crystalline compound of formula I:

(Rp isomer),

the method comprising crystallization from a solubilized form of a compound of formula II:

or slurry conversion.

[0004] In another aspect, the present invention provides a crystalline compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein the compound is at least 90%

diastereomerically pure, e.g., as measured by ³¹P MR, wherein the compound is stable at about 25 °C at ambient humidity for at least 1 week (e.g., at least 2 weeks, at least 4 weeks, at least 2 months, 3 months, at least 6 months, at least 1 year).

[0005] In certain embodiments, the crystalline compound is produced by crystallization from a solubilized form of a compound of formula II:

or slurry conversion.

[0006] In another aspect, the present invention provides a compound of formula III:

(Bp isomer),

[0007] or a pharmaceutically acceptable salt thereof.

[0008] In another aspect, the present invention provides a composition compri

crystalline compound of formula I:

isomer),

[0009] In another aspect, the present invention provides a composition comprising a compound of formula III:

(R ? isomer),

or a pharmaceutically acceptable salt thereof.

[0010] In certain embodiments, the composition further comprises a compound of formula

stereochemistry at each phosphorus).

[0011] In one aspect, the present invention provides a composition comprising

a crystalline compound of formula I:

(Rp isomer),

or a pharmaceutically acceptable salt thereof, and

tenofovir or a prodrug thereof, such as tenofovir dipivoxil and tenofovir alafenamide, wherein the compound of formula I is at least 90% diastereomerically pure, e.g., as measured by ³¹P NMR, wherein the compound of formula I and tenofovir are stable at about 25 °C at ambient humidity for at least 1 week (e.g., at least 2 weeks, at least 4 weeks, at least 2 months, 3 months, at least 6 months, at least 8 months, at least 10 months). [0012] In another aspect, the present invention provides a particulate composition comprising:

a crystalline compound of formula I:

isomer),

or a pharmaceutically acceptable salt thereof, and

tenofovir or a prodrug thereof, such as tenofovir dipivoxil or tenofovir alafenamide, wherein the compound of formula I is at least 90% diastereomerically pure, e.g., as measured by ³¹P NMR, wherein the compound of formula I and tenofovir are stable at about 25 °C at ambient humidity for at least 1 week (e.g., at least 2 weeks, at least 4 weeks, at least 2 months, 3 months, at least 6 months, at least 8 months, at least 10 months).

[0013] In another aspect, the present invention provides a pharmaceutical composition comprising:

a crystalline compound of formula I:

(Rp isomer),

or a pharmaceutically acceptable salt thereof, and

tenofovir or a prodrug thereof, such as tenofovir dipivoxil or tenofovir alafenamide, wherein the compound of formula I is at least 90% diastereomerically pure, e.g., as measured by ³¹P NMR, wherein the compound of formula I and tenofovir are stable at about 25 °C at ambient humidity for at least 1 week (e.g., at least 2 weeks, at least 4 weeks, at least 2 months, 3 months, at least 6 months, at least 8 months, at least 10 months). [0014] In one aspect, the present invention provides a solid oral dosage form comprising a disclosed compound. In another aspect, the present invention provides a solid oral dosage form comprising a disclosed particulate composition.

[0015] In another aspect, the present invention provides a method of treating a viral infection in a patient in need thereof, the method comprising administering a therapeutically effective amount of a disclosed compound or a disclosed composition. In certain embodiments, the viral infection is caused by a virus selected from the group consisting of HBV, HCV, RSV, norovirus, and influenza virus.

[0016] In another aspect, the present invention provides a method of treating or preventing a proliferative disorder or cancer in a patient in need thereof, the method comprising administering a therapeutically effective amount of a disclosed compound or a disclosed composition. In some embodiments, the proliferative disorder or cancer is associated with a viral infection.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 A shows ³¹P MR of the compound of formula I after the first

crystallization.

[0018] Figure IB shows ³¹P NMR of the compound of formula I after the second

crystallization.

[0019] Figure 2 shows ³¹P NMR of the compound of formula V after the crystallization.

[0020] Figures 3 A and 3B show the HPLC chromatograms of samples of the compound of formula I stored at 25 °C at ambient humidity at t=0 and t=3 months respectively.

[0021] Figures 4A-4D show the HPLC chromatograms of samples of the compound of formula I mixed with tenofovir dipivoxyl stored at 25 °C at ambient humidity at t=0, t=16 weeks, t=45 weeks, and t=l year respectively.

[0022] Figures 5A-5C show the HPLC chromatograms of samples of the compound of formula I mixed with tenofovir dipivoxyl stored at 25 °C at ambient humidity at t=0, t=45 weeks, and t=l year respectively.

[0023] Figures 6A-6C show the HPLC chromatograms of samples of the compound of formula III stored at 25 °C at ambient humidity at t=0, t=3 months, and t=4 months respectively.

[0024] Figure 7 A shows the effect of test agents including the compound of formula I on liver HBV DNA using Southern blot hybridization.

[0025] Figure 7B shows the effect of test agents including the compound of formula I on liver HBV DNA using semi -quantitative PCR. [0026] Figure 8A shows therapeutic antiviral activity of the compound of formula II during human respiratory syncytial virus (RSV) infection.

[0027] Figure 8B shows antiviral activity of the compound of formula I against RSV.

[0028] Figure 8C shows anti-RSV activity of tartrate salts of the compound of formula I and the compound of formula IV.

[0029] Figure 9 shows XRPD pattern of a crystalline compound of formula I.

[0030] Figure 10 shows DSC thermogram of a crystalline compound of formula I.

[0031] Figure 1 1 shows TG thermogram of a crystalline compound of formula I. DETAILED DESCRIPTION OF THE INVENTION

[0032] In one aspect, the present invention provides a crystalline compound of formula I or a pharmaceutically acceptable salt thereof. In some embodiments, the crystalline compound of formula I is diastereomerically pure. In other embodiments, the crystalline compound of formula I is stable at about 25 °C at ambient humidity (e.g., between about 20% to about 80% relative humidity). Also provided herein is a compound of formula III and compositions comprising a compound of formula I or III. In another aspect, methods of making a crystalline compound of formula I and methods of treating a viral infection (e.g., infection with Hepatitis B virus) comprising administering a disclosed compound or composition are provided herein. Definitions

[0033] As used herein, the articles "a" and "an" refer to one or to more than one (e.g., to at least one) of the grammatical obj ect of the article.

[0034] "About" and "approximately" shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given value or range of values.

[0035] As used herein, the term "acquire" or "acquiring" as the terms are used herein, refer to obtaining possession of a physical entity (e.g., a sample, e.g., blood sample or liver biopsy specimen), or a value, e.g., a numerical value, by "directly acquiring" or "indirectly acquiring" the physical entity or value. "Directly acquiring" means performing a process (e.g., an analytical method) to obtain the physical entity or value. "Indirectly acquiring" refers to receiving the physical entity or value from another party or source (e.g., a third party laboratory that directly acquired the physical entity or value). Directly acquiring a value includes performing a process that includes a physical change in a sample or another substance, e.g., performing an analytical process which includes a physical change in a substance, e.g., a sample, performing an analytical method, e.g., a method as described herein, e.g., by sample analysis of bodily fluid, such as blood by, e.g., mass spectroscopy (e.g. LC-MS), or PCR (e.g., RT-PCR).

[0036] As used herein, an amount of a compound, conjugate, or substance effective to treat a disorder (e.g., a disorder described herein), "therapeutically effective amount," "effective amount" or "effective course" refers to an amount of the compound, substance, or composition which is effective, upon single or multiple dose administration(s) to a subject, in treating a subject, or in curing, alleviating, relieving or improving a subject with a disorder (e.g., an HBV infection or HBV/HDV co-infection) beyond that expected in the absence of such treatment.

[0037] As used herein, the terms "prevent" or "preventing" as used in the context of a disorder or disease, refer to administration of an agent to a subject, e.g., the administration of a compound of the present invention (e.g., compound of formula I) to a subject, such that the onset of at least one symptom of the disorder or disease is delayed as compared to what would be seen in the absence of administration of the said agent.

[0038] As used herein, the term "prodrug" refers to a compound which, when metabolized (e.g., in vivo or in vitro), yields an active compound. In some embodiments, the prodrug may be inactive, or possess less activity that the free drug, but may provide advantageous handling, administration, or metabolic properties. Exemplary prodrug moieties of the present invention may be linked to the free drug through the hydroxyl, amino, phosphate, or phosphorothioate backbone of the nucleotide, and may comprise an ester, a carbamate, a carbonyl, a thioester, amide, isocyanate, urea, thiourea, or other physiologically acceptable metabolically labile moiety. In some embodiments, a prodrug is activated through enzymatic hydrolysis.

[0039] As used herein, the term "resistant" or "resistance" refers to a strain of HBV that is not substantially diminished or inactivated upon administration with an anti-HBV agent. In some embodiments, a resistant HBV strain comprises a protein (e.g., an HBsAg, HBcAg,

HBeAg, L, M, P, or X protein) that substantially maintains its activity, function, or structure in the presence of an anti-HBV agent known to inhibit, bind to, or alter the said protein. In some embodiments, a resistant HBV strain comprises a protein bearing an amino acid mutation (e.g., an amino acid substitution, addition, or deletion) compared with a reference sequence of the said protein. In some embodiments, an HBV protein bearing an amino acid mutation (e.g., an amino acid substitution, addition, or deletion) may result in aberrant function of said protein or affect the inhibition of the said protein with an anti-HBV agent. In some embodiments, the level of resistance may be determined through a measurement of viral load or other biomarker in a sample (e.g., a serum sample), or through the determination of the IC₅₀ value of a specific antiviral agent or other agents beyond a compound of formula I in a sample (e.g., a serum sample).

[0040] As used herein, the terms "stable" or "stability" refer to chemical stability and/or physical stability of a compound. Chemical stability refers to the tendency of a compound to resist decomposition into other compounds such as degradants. Physical stability refers to the ability of a compound to maintain physical properties, such as crystalline forms. For example, a physically stable compound may have a minimal conversion to a different stereoisomer, a different crystalline form, or an amorphous form of the compound, or may have minimal changes in appearance, color, odor and so on. In some embodiments, the stability of a compound may be measured by HPLC. In certain embodiments, a stable compound may remain >90% pure (e.g., >95% pure, >97% pure, >98% pure, >99% pure) as measured by HPLC for at least 1 week (e.g., at least 2 weeks, at least 4 weeks, at least 2 months, 3 months, at least 6 months, at least 1 year) at about 25 °C at ambient humidity, e.g., at between about 20% to about 80%) relative humidity.

[0041] As used herein the term "ambient humidity" refers to the humidity of the room where a compound is stored. In some embodiments, the ambient humidity is between about 0% to about 100%) relative humidity. In other embodiments, the ambient humidity is between about 10%) to about 90%) relative humidity. In certain embodiments, the ambient humidity is between about 20% to about 80%> (e.g., between about 20% to about 70%, between about 30%> to about 70%), between about 30%> to about 60%>, between about 30%> to about 50%>) relative humidity.

[0042] As used herein, the terms "pure" and "purity" refer to chemical purity, stereoisomeric purity, and/or crystalline purity. Chemical purity refers to the degree to which a compound is unmixed with other compounds such as starting materials and degradants. For example, a chemically pure compound may be substantially free of degradants. Stereoisomeric purity refers to the degree to which a compound is unmixed with other stereoisomers such as diastereomers. For example, a stereoisomerically pure compound may be diastereomerically or chirally pure. For example, a diastereomerically pure compound may be substantially free of a specific diastereomer. In some embodiments, a crystalline compound of formula I may be

diastereomerically pure. In certain embodiments, a composition comprising a crystalline compound of formula I may be substantially free of a compound of formula IV. Crystalline purity refers to the degree to which a crystalline compound is unmixed with other crystalline forms or amorphous forms of the compound. For example, a compound with crystalline purity may be substantially free of other crystalline forms or amorphous forms of the compound. In some embodiments, the purity of a compound may be determined by HPLC and/or MR (e.g., 1H, ³¹P MR). In some embodiments, a compound may be >90% pure (e.g., >95% pure, >97% pure, >98% pure, >99% pure) as measured by HPLC. In some embodiments, a compound may be >80% pure (e.g., >85% pure, >90% pure, >95% pure, >97% pure, >98% pure, >99% pure) as measured by NMR (e.g., 1H, ³¹P MR). In certain embodiments, a crystalline compound of formula I may be at least 80% pure (e.g., at least 85% pure, at least 90% pure, at least 95% pure, at least 97% pure, at least 98% pure, at least 99% pure) as determined by ³¹P NMR.

[0043] As used herein, the term "subject" is intended to include human and non-human animals. Exemplary human subjects include a human patient having a disorder, e.g., a disorder described herein, or a normal subject. The term "non-human animals" includes all vertebrates, e.g., non-mammals (such as chickens, amphibians, reptiles) and mammals, such as non-human primates, domesticated and/or agriculturally useful animals, e.g., sheep, dogs, cats, cows, pigs, etc. In exemplary embodiments of the invention, the subject is a woodchuck (e.g., an Eastern woodchuck (Marmota monax)).

[0044] As used herein, the terms "treat" or "treating" a subject having a disorder or disease refer to subjecting the subject to a regimen, e.g., the administration of a compound of formula I or a pharmaceutically acceptable salt thereof, or a composition comprising formula I or a pharmaceutically acceptable salt thereof, such that at least one symptom of the disorder or disease is cured, healed, alleviated, relieved, altered, remedied, ameliorated, or improved.

Treating includes administering an amount effective to alleviate, relieve, alter, remedy, ameliorate, improve or affect the disorder or disease, or the symptoms of the disorder or disease. The treatment may inhibit deterioration or worsening of a symptom of a disorder or disease.

[0045] Numerous ranges, e.g., ranges for the amount of a drug administered per day, are provided herein. In some embodiments, the range includes both endpoints. In other

embodiments, the range excludes one or both endpoints. By way of example, the range can exclude the lower endpoint. Thus, in such an embodiment, a range of 250 to 400 mg/day, excluding the lower endpoint, would cover an amount greater than 250 that is less than or equal to 400 mg/day.

[0046] Administered "in combination", as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject's affliction with the disorder, e.g., the two or more treatments are delivered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has ceased for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as "simultaneous" or "concurrent delivery". In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In some embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.

[0047] A "course" or "course of therapy," as referred to herein, comprises one or more separate administrations of a therapeutic agent (e.g., a compound of formula I or a

pharmaceutically acceptable salt thereof, in combination with tenofovir). A course of therapy can comprise one or more cycles of a therapeutic agent. In some embodiments, a therapeutic agent is administered to a subject at least once, at least twice, at least three times, at least four times, or more over a course of treatment. A subject may be administered with one or more courses of treatment. In some embodiments, rest periods may be interposed between courses of treatment. For example, a rest period may be about 1, about 2, about 4, about 6, about 8, about 10, about 12, about 16, about 20, or about 24 hours; or about 1, about 2, about 3, about 4, about 5, about 6, or about 7 days; or about 1, about 2, about 3, about 4 or more weeks in length.

[0048] A "cycle," as used herein in the context of a cycle of administration of a drug, refers to a period of time for which a drug is administered to a patient. For example, if a drug is administered for a cycle of 4 weeks days, the periodic administration, e.g., daily or twice daily, is given for 4 weeks. A drug can be administered for more than one cycle. In some

embodiments, the first and second or subsequent cycles are the same in terms of one or both of duration and periodic administration. In embodiments, a first and second or subsequent cycle differs in terms of one or both of duration and periodic administration. Rest periods may be interposed between cycles. A rest cycle may be about 1, about 2, about 4, about 6, about 8, about 10, about 12, about 16, about 20, or about 24 hours; or about 1, about 2, about 3, about 4, about 5, about 6, or about 7 days; or about 1, about 2, about 3, about 4 or more weeks in length.

[0049] The phrases "systemic administration," "administered systemically," "peripheral administration" and "administered peripherally" as used herein mean the administration of the compound other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.

[0050] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0051] The phrase "pharmaceutically acceptable carrier" as used herein means a

pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, stabilizing agent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject antagonists from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) ascorbic acid; (17) pyrogen-free water; (18) isotonic saline; (19) Ringer's solution; (20) ethyl alcohol; (21) phosphate buffer solutions; (22) cyclodextrins such as Captisol®; and (23) other non-toxic compatible substances such as antioxidants and antimicrobial agents employed in pharmaceutical formulations.

[0052] As set out above, certain embodiments of the compounds described herein may contain a basic functional group, such as an amine, and are thus capable of forming

pharmaceutically acceptable salts with pharmaceutically acceptable acids. The term

"pharmaceutically acceptable salts" in this respect, refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by separately reacting a purified compound of the invention in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like (see, for example, Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66: 1- 19).

[0053] In other cases, the compounds of the present invention may contain one or more acidic functional groups and thus, are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term "pharmaceutically acceptable salts" in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of the compound of the present invention (e.g., a compound of formula I or III). These salts can likewise be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine,

ethanolamine, diethanolamine, piperazine and the like (see, for example, Berge et al., supra).

Crystalline compounds

[0054] In another aspect, provided herein is a crystalline compound of formula I:

(Rp isomer),

diastereomerically pure, e.g., as measured by ³¹P MR, wherein the compound is stable at about 25 °C at ambient humidity for at least 1 week (e.g., at least 2 weeks, at least 4 weeks, at least 2 months, 3 months, at least 6 months, at least 1 year). [0055] In some embodiments, the ambient humidity is between about 20% to about 80% relative humidity.

[0056] In some embodiments, the compound is a pharmaceutically acceptable salt of a compound of formula I.

[0057] In other embodiments, the pharmaceutically acceptable salt is selected from the group consisting of a hemi-tartrate salt, an oxalate salt, a citrate salt, and a fumarate salt.

[0058] In certain embodiments, the pharmaceutically acceptable salt is a hemi-tartrate salt.

[0059] In other embodiments, the compound is at least about 95% diastereomerically pure, e.g., as measured by ³¹P MR.

[0060] In some embodiments, the compound has an XRPD pattern with characteristic peaks at the following values of 2Θ in degrees: from about 7.4 to about 7.8, from aboutl3.0 to about 13.4, from about 15.0 to about 15.4, from about 16.5 to about 16.9, from about 18.7 to about 19.1, and from about 21.1 to about 21.5.

[0061] In some embodiments, the compound has an XRPD pattern with characteristic peaks at the following values of 2Θ in degrees: 7.6, 13.2, 15.2, 16.7, 18.9, and 21.3.

[0062] In certain embodiments, the compound has an XRPD pattern with characteristic peaks at the following values of 2Θ in degrees: from about 7.4 to about 7.8, from about 10.5 to about 10.9, from about 12.0 to about 12.4, from about 13.0 to about 13.4, from about 14.0 to about 14.4, from about 15.0 to about 15.4, from about 16.5 to about 16.9, from about 17.6 to about 18.0, from about 17.9 to about 18.3, from about 18.7 to about 19.1, from about 20.1 to about 20.5, from about 20.8 to about 21.2, from about 21.1 to about 21.5, from about 21.8 to about 22.2, from about 23.2 to about 23.6, and from about 24.1 to about 24.5.

[0063] In certain embodiments, the compound has an XRPD pattern with characteristic peaks at the following values of 2Θ in degrees: 7.6, 10.7, 12.2, 13.2, 14.2, 15.2, 16.7, 17.8, 18.1, 18.9, 20.3, 21.0, 21.3, 22.0, 23.4, and 24.3.

[0064] In other embodiments, the compound has an XRPD pattern substantially as depicted in Figure 9.

[0065] In some embodiments, the crystalline compound is produced by crystallization from a solubilized form of a compound of formula II:

or slurry conversion.

[0066] In certain embodiments, the crystallization is performed using water.

[0067] In some embodiments, the compound of formula II is dissolved in water or in a slurry in water.

[0068] In some embodiments, the crystallization is performed using N-methylpyrrolidone.

[0069] In other embodiments, the crystallization is recrystallization.

Salt compounds

[0070] In another aspect, provided herein is a compound of formula III:

(Bp isomer),

[0071] or a pharmaceutically acceptable salt thereof.

[0072] In another aspect, also provided herein is a compound selected from a

consisting of:

(Sp isomer), isomer), isomer),

(Rp isomer) ,

stereochemistry at each phosphorus),

stereochemistry at each phosphorus),

[0073] In some embodiments, the compound is crystalline.

[0074] In certain embodiments, the compound is stable at about 25 °C at ambient humidity for at least 1 week (e.g., at least 2 weeks, at least 4 weeks, at least 2 months, 3 months, at least 6 months, at least 1 year).

[0075] In some embodiments, the ambient humidity is between about 20% to about 80% relative humidity.

[0076] In other embodiments, the ammonium ion of the compound of formula I:

(Rp isomer),

in the compound of formula III is at least 90% diastereomerically pure, e.g., as measured by ³¹P

NMR.

[0077] In some embodiments, the ammonium salt of the compound of formula I is at least 95% diastereomerically pure, e.g., as measured by ³¹P MR.

[0078] The compounds provided herein may contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomenc mixtures. Unless otherwise indicated, when a compound is named or depicted by a structure without specifying the stereochemistry and has one or more chiral centers, it is understood to represent all possible stereoisomers of the compound.

Compositions

[0079] In another aspect, provided herein is a composition comprising a crystalline compound of formula I:

(Rp isomer),

diastereomerically pure, e.g., as measured by ³¹P MR, wherein the compound is stable

25 °C at ambient humidity for at least 1 week (e.g., at least 2 weeks, at least 4 weeks, at least 2 months, 3 months, at least 6 months, at least 1 year).

[0080] In some embodiments, the ambient humidity is between about 20% to about 80% relative humidity.

[0081] In some embodiments, the composition further comprises a pharmaceutically acceptable excipient.

[0082] In certain embodiments, the composition is substantially free of chemical impurities.

[0083] In certain embodiments, the chemical impurity is

[0084] In other embodiments, the composition is substantially free of the compound of formula V:

(Sp isomer). [0085] In some embodiments, the composition is substantially free of non-crystalline or amorphous forms of the compound of formula I.

[0086] In certain embodiments, the purity of the compound remains over 95% pure as measured by HPLC for at least 1 week (e.g., at least 2 weeks, at least 4 weeks, at least 2 months, 3 months, at least 6 months, at least 1 year).

[0087] In other embodiments, the compound of formula I is at least 95% diastereomerically pure, e.g., as measured by ³¹P MR.

[0088] In some embodiments, the compound has an XRPD pattern with characteristic peaks at the following values of 2Θ in degrees: from about 7.4 to about 7.8, from about 13.0 to about 13.4, from about 15.0 to about 15.4, from about 16.5 to about 16.9, from about 18.7 to about 19.1, and from about 21.1 to about 21.5.

[0089] In some embodiments, the compound has an XRPD pattern with characteristic peaks at the following values of 2Θ in degrees: 7.6, 13.2, 15.2, 16.7, 18.9, and 21.3.

[0090] In certain embodiments, the compound has an XRPD pattern with characteristic peaks at the following values of 2Θ in degrees: from about 7.4 to about 7.8, from about 10.5 to about 10.9, from about 12.0 to about 12.4, from about 13.0 to about 13.4, from about 14.0 to about 14.4, from about 15.0 to about 15.4, from about 16.5 to about 16.9, from about 17.6 to about 18.0, from about 17.9 to about 18.3, from about 18.7 to about 19.1, from about 20.1 to about 20.5, from about 20.8 to about 21.2, from about 21.1 to about 21.5, from about 21.8 to about 22.2, from about 23.2 to about 23.6, and from about 24.1 to about 24.5.

[0091] In certain embodiments, the compound has an XRPD pattern with characteristic peaks at the following values of 2Θ in degrees: 7.6, 10.7, 12.2, 13.2, 14.2, 15.2, 16.7, 17.8, 18.1, 18.9, 20.3, 21.0, 21.3, 22.0, 23.4, and 24.3.

[0092] In other embodiments, the compound has an XRPD pattern substantially as depicted in Figure 9.

[0093] In another aspect, provided herein is a composition comprising a compound of formula III:

(Bp isomer), or a pharmaceutically acceptable salt thereof.

[0094] In some embodiments, the composition further comprises a compound of formula IV:

stereochemistry at each phosphorus).

[0095] In some embodiments, the compound of formula III is crystalline.

[0096] In some embodiments, the compound of formula IV is crystalline.

[0097] In certain embodiments, the ammonium ion of the compound of formula I:

(Rp isomer)

is at least 90% diastereomerically pure, e.g., as measured by ³¹P NMR, and the compound of formula I is stable at about 25 °C at ambient humidity for at least 1 week (e.g., at least 2 weeks, at least 4 weeks, at least 2 months, 3 months, at least 6 months, at least 1 year).

[0098] In some embodiments, the ambient humidity is between about 20% to about 80% relative humidity.

[0099] In some embodiments, the composition further comprises a pharmaceutically acceptable excipient.

[00100] In certain embodiments, the composition is substantially free of chemical impurities. [00101] In certain embodiments, the chemical impurity is

[00102] In other embodiments, the composition is substantially free of the compound of formula V:

(Sp isomer),

or a salt thereof.

[00103] In some embodiments, the composition is substantially free of non-crystalline or amorphous forms of the compound of formula I.

[00104] In some embodiments, the purity of the compound remains over 95% pure as measured by HPLC for at least 1 week (e.g., at least 2 weeks, at least 4 weeks, at least 2 months, 3 months, at least 6 months, at least 1 year).

[00105] In some embodiments, the compound of formula I is at least 95% diastereomerically pure, e.g., as measured by ³¹P MR.

[00106] Also provided herein is a composition comprising:

a crystalline compound of formula I:

(Rp isomer), or a pharmaceutically acceptable salt thereof, and

tenofovir or a prodrug thereof, such as tenofovir dipivoxil and tenofovir alafenamide,

wherein the compound of formula I is at least 90% diastereomerically pure, e.g., as measured by ³¹P NMR, wherein the compound of formula I and tenofovir are stable at about 25 °C at ambient humidity for at least 1 week (e.g., at least 2 weeks, at least 4 weeks, at least 2 months, 3 months, at least 6 months, at least 8 months, at least 10 months).

[00107] In some embodiments, the ambient humidity is between about 20% to about 80% relative humidity.

[00108] In some embodiments, the composition further comprises a pharmaceutically acceptable excipient.

[00109] In certain embodiments, the composition is substantially free of chemical impurities.

[00110] In certain embodiments, the chemical impurity is

[00111] In other embodiments, the composition is substantially free of the compound of formula V:

(Sp isomer).

[00112] In some embodiments, the composition is substantially free of non-crystalline or amorphous forms of the compound of formula I.

[00113] In certain embodiments, the purity of the compound remains over 95% pure as measured by HPLC for at least 1 week (e.g., at least 2 weeks, at least 4 weeks, at least 2 months, 3 months, at least 6 months, at least 1 year). [00114] In other embodiments, the compound of formula I is at least 95% diastereomerically pure, e.g., as measured by ³¹P NMR.

[00115] In some embodiments, the compound has an XRPD pattern with characteristic peaks at the following values of 2Θ in degrees: 7.6, 13.2, 15.2, 16.7, 18.9, and 21.3.

[00116] In certain embodiments, the compound has an XRPD pattern with characteristic peaks at the following values of 2Θ in degrees: 7.6, 10.7, 12.2, 13.2, 14.2, 15.2, 16.7, 17.8, 18.1, 18.9, 20.3, 21.0, 21.3, 22.0, 23.4, and 24.3.

[00117] In other embodiments, the compound has an XRPD pattern substantially as depicted in Figure 9.

Particulate compositions

[00118] In another aspect, provided herein is a particulate composition comprising:

a crystalline compound of formula I:

(Rp isomer),

or a pharmaceutically acceptable salt thereof, and

[00119] In some embodiments, the ambient humidity is between about 20% to about 80% relative humidity.

[00120] In some embodiments, the purity of the compound of formula I and tenofovir remain over 95% pure as measured by HPLC for at least 1 week (e.g., at least 2 weeks, at least 4 weeks, at least 2 months, 3 months, at least 6 months, at least 8 months, at least 10 months).

[00121] In certain embodiments, the compound of formula I is at least 95% diastereomerically pure, e.g., as measured by ³¹P NMR. [00122] In some embodiments, the compound has an XRPD pattern with characteristic peaks at the following values of 2Θ in degrees: 7.6, 13.2, 15.2, 16.7, 18.9, and 21.3.

[00123] In certain embodiments, the compound has an XRPD pattern with characteristic peaks at the following values of 2Θ in degrees: 7.6, 10.7, 12.2, 13.2, 14.2, 15.2, 16.7, 17.8, 18.1, 18.9, 20.3, 21.0, 21.3, 22.0, 23.4, and 24.3.

[00124] In other embodiments, the compound has an XRPD pattern substantially as depicted in Figure 9.

Pharmaceutical compositions

[00125] While it is possible for the compound of the present invention (e.g., a compound of formula I or III) to be administered alone, it is preferable to administer said compound as a pharmaceutical composition or formulation, where the compounds are combined with one or more pharmaceutically acceptable diluents, excipients or carriers. The compounds according to the invention may be formulated for administration in any convenient way for use in human or veterinary medicine. In certain embodiments, the compounds included in the pharmaceutical preparation may be active itself, or may be a prodrug, e.g., capable of being converted to an active compound in a physiological setting (e.g., a compound of formula I). Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into a pharmaceutically acceptable dosage form such as described below or by other conventional methods known to those of skill in the art.

[00126] The amount and concentration of compounds of the present invention (e.g., a compound of formula I or III) in the pharmaceutical compositions, as well as the quantity of the pharmaceutical composition administered to a subject, can be selected based on clinically relevant factors, such as medically relevant characteristics of the subject (e.g., age, weight, gender, other medical conditions, and the like), the solubility of compounds in the

pharmaceutical compositions, the potency and activity of the compounds, and the manner of administration of the pharmaceutical compositions. For further information on Routes of Administration and Dosage Regimes the reader is referred to Chapter 25.3 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.

[00127] Thus, one aspect of the present invention provides pharmaceutically acceptable compositions comprising a therapeutically effective amount or prophylactically effective amount of a compound described herein (e.g., a compound of formula I or III), formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. As described in detail below, the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for oral or parenteral administration, for example, by oral dosage, or by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension. However, in certain embodiments the subject compounds may be simply dissolved or suspended in sterile water. In certain embodiments, the pharmaceutical preparation is non-pyrogenic, i.e., does not elevate the body temperature of a patient.

[00128] Wetting agents, emulsifiers, and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions. Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium

metabi sulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[00129] The pharmaceutically acceptable carriers, as well as wetting agents, emulsifiers, lubricants, coloring agents, release agents, coating agents, sweetening, flavoring agents, perfuming agents, preservatives, antioxidants, and other additional components may be present in an amount between about 0.001% and 99% of the composition described herein. For example, said pharmaceutically acceptable carriers, as well as wetting agents, emulsifiers, lubricants, coloring agents, release agents, coating agents, sweetening, flavoring agents, perfuming agents, preservatives, antioxidants, and other additional components may be present from about 0.005%, about 0.01%, about 0.05%, about 0.1%, about 0.25%, about 0.5%, about 0.75%, about 1%, about 1.5%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 85%), about 90%, about 95%, or about 99% of the composition described herein.

[00130] Pharmaceutical compositions of the present invention may be in a form suitable for oral administration, e.g., a liquid or solid oral dosage form. In some embodiments, the liquid dosage form comprises a suspension, a solution, a linctus, an emulsion, a drink, an elixir, or a syrup. In some embodiments, the solid dosage form comprises a capsule, tablet, powder, dragee, or powder. The pharmaceutical composition may be in unit dosage forms suitable for single administration of precise dosages. Pharmaceutical compositions may comprise, in addition to the compound described herein (e.g., a compound of formula I) or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier, and may optionally further comprise one or more pharmaceutically acceptable excipients, such as, for example, stabilizers (e.g., a binder, e.g., polymer, e.g., a precipitation inhibitor, diluents, binders, and lubricants.

[00131] In some embodiments, the composition described herein comprises a liquid dosage form for oral administration, e.g., a solution or suspension. In other embodiments, the composition described herein comprises a solid dosage form for oral administration capable of being directly compressed into a tablet. In addition, said tablet may include other medicinal or pharmaceutical agents, carriers, and or adjuvants. Exemplary pharmaceutical compositions include compressed tablets (e.g., directly compressed tablets), e.g., comprising a compound of the present invention (e.g., a compound of formula I or III) or a pharmaceutically acceptable salt thereof.

[00132] Formulations of the present invention include those suitable for parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about 99 percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent. Pharmaceutical compositions of this invention suitable for parenteral administration comprise compounds of the invention in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

[00133] In some embodiments, a compound of the present invention (e.g., a compound of formula I or III) is provided as a composition in combination with an additional agent (e.g., tenofovir). For example, a compound of formula I or III may be prepared as a fixed dose composition in combination with additional therapeutic agent or agents [e.g., tenofovir (e.g., tenofovir dipivoxil, tenofovir alafenamide), entecavir, telbivudine]. The fixed dose composition may be formulated for oral administration, e.g., as a solid dosage form or a liquid dosage form. In some embodiments, the liquid dosage form comprises a suspension, a solution, a linctus, an emulsion, a drink, an elixir, or a syrup. In some embodiments, the solid dosage form comprises a capsule, tablet, dragee, or powder.

[00134] Examples of suitable aqueous and non-aqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[00135] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

[00136] In some cases, in order to prolong the effect of a compound of the present invention (e.g., a compound of formula I or III), it may be desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally

administered form of the compound of the present invention is accomplished by dissolving or suspending the compound in an oil vehicle.

[00137] In some embodiments, it may be advantageous to administer the compound of the present invention (e.g., a compound of formula I or III) in a sustained fashion. It will be appreciated that any formulation that provides a sustained absorption profile may be used. In certain embodiments, sustained absorption may be achieved by combining a compound of the present invention with other pharmaceutically acceptable ingredients, diluents, or carriers that slow its release properties into systemic circulation.

[00138] In one aspect, provided herein is a pharmaceutical composition comprising a disclosed crystalline compound of formula I or formula III.

[00139] The present invention, in part, provides a pharmaceutical composition comprising: a crystalline compound of formula I:

(Rp isomer),

or a pharmaceutically acceptable salt thereof, and

[00140] In some embodiments, the ambient humidity is between about 20% to about 80% rel ati ve humi dity .

[00141] In some embodiments, the purity of the compound of formula I and tenofovir remain over 95% pure, e.g., as measured by HPLC for at least 1 week (e.g., at least 2 weeks, at least 4 weeks, at least 2 months, 3 months, at least 6 months, at least 8 months, at least 10 months).

[00142] In certain embodiments, the compound of formula I is at least 95% diastereomerically pure, e.g., as measured by ³¹P NMR.

[00143] In some embodiments, the compound has an XRPD pattern with characteristic peaks at the following values of 2Θ in degrees: from about 7.4 to about 7.8, from aboutl3.0 to about 13.4, from about 15.0 to about 15.4, from about 16.5 to about 16.9, from about 18.7 to about 19.1, and from about 21.1 to about 21.5.

[00144] In some embodiments, the compound has an XRPD pattern with characteristic peaks at the following values of 2Θ in degrees: 7.6, 13.2, 15.2, 16.7, 18.9, and 21.3.

[00145] In certain embodiments, the compound has an XRPD pattern with characteristic peaks at the following values of 2Θ in degrees: from about 7.4 to about 7.8, from about 10.5 to about 10.9, from about 12.0 to about 12.4, from about 13.0 to about 13.4, from about 14.0 to about 14.4, from about 15.0 to about 15.4, from about 16.5 to about 16.9, from about 17.6 to about 18.0, from about 17.9 to about 18.3, from about 18.7 to about 19.1, from about 20.1 to about 20.5, from about 20.8 to about 21.2, from about 21.1 to about 21.5, from about 21.8 to about 22.2, from about 23.2 to about 23.6, and from about 24.1 to about 24.5.

[00146] In certain embodiments, the compound has an XRPD pattern with characteristic peaks at the following values of 2Θ in degrees: 7.6, 10.7, 12.2, 13.2, 14.2, 15.2, 16.7, 17.8, 18.1, 18.9, 20.3, 21.0, 21.3, 22.0, 23.4, and 24.3.

[00147] In other embodiments, the compound has an XRPD pattern substantially as depicted in Figure 9.

[00148] In other embodiments, the composition further comprises a pharmaceutically acceptable excipient.

Methods of making a crystalline compound of formula I

[00149] In one aspect, provided herein is a method of making a crystalline compound of formula I:

(Rp isomer),

or slurry conversion.

[00150] In some embodiments, the crystallization is performed using water.

[00151] In certain embodiments, the compound of formula II is dissolved in water or in a slurry in water. [00152] In other embodiments, the crystallization is performed at a temperature from about 60 °C to about 90 °C (e.g., from about 70 °C to about 85 °C, from about 75 °C to about 80 °C) or even boiling.

[00153] In some embodiments, the crystallization is recrystallization.

Methods of treating a viral infection

[00154] The present invention, in part, is directed to a method of treating a viral infection in a patient in need thereof, the method comprising administering a therapeutically effective amount of a disclosed compound, salt, or composition. In some embodiments, the viral infection is selected from the group consisting of HBV, HCV, RSV, norovirus, and influenza virus. In some embodiments, the viral infection is HBV.

[00155] The present invention, in part, features methods for treating a subject infected with HBV, the methods comprising administering a compound of formula I or a pharmaceutically acceptable salt thereof, a compound of formula III, or a composition disclosed herein. An HBV infection may comprise infection with one or more resistant strains of HBV. The present invention further includes methods for treating a subject infected with HBV or HDV (e.g., a co- infection of HBV and HDV), the methods comprising administering a compound of formula I or a pharmaceutically acceptable salt thereof, a compound of formula III, or a composition disclosed herein, and/or in combination with tenofovir (e.g., tenofovir dipivoxil, tenofovir alafenamide).

HBV Infection

[00156] The present invention, in part, relates to methods for treating a subject infected with HBV through administration of a crystalline compound of formula I or a pharmaceutically acceptable salt thereof, a compound of formula III, or a composition disclosed herein. HBV is an enveloped DNA virus classified as the species type Orthohepadnavirus, which contains three other species, the woodchuck hepatitis virus (WHV), the woolly monkey hepatitis B virus, and the ground squirrel hepatitis virus. The virus is characterized into four major serotypes (adr, adw, ayr, ayw) based upon the antigenic epitopes present on the viral envelope proteins and eight genotypes (genotypes A-H) according to the overall nucleotide sequence of the viral genome. In some embodiments, the methods described herein are used to treat a subject suffering from any known form of HBV infection (e.g., any genotype or serotype of HBV or a combination thereof).

[00157] While effective antiviral therapy exists for chronic HBV infection, the infected patient often requires prolonged or lifelong therapy. There are five nucleoside and nucleotide analogs commercially available for treatment of HBV (e.g., lamivudine, adefovir, tenofovir, telbivudine, and entecavir), but their use is limited due to the emergence of drug resistant variants during treatment, the risk of relapse upon treatment discontinuation, and unwarranted side effects. A major challenge of current HBV therapy is to clear the viral, covalently closed circular (ccc) DNA molecule within the nucleus of hepatocytes, which is representing the HBV genome and that is used by the virus as a template for synthesizing the pre-genomic RNA needed for replication. Drugs that target directly HBV cccDNA are currently not available for use in patients. Indirect evidence for treatment-induced reduction of this viral molecule includes the loss of HBV surface antigen (HBsAg), but even after 5 years of therapy with currently available nucleoside and nucleotide analogs, clearance of HBsAg and subsequent seroconversion to antibodies against HBsAg (anti-HBs) are rare events and only achieved in less than 10% of treated patients. In addition, successfully treated patients with antiviral response still exhibit significant levels of HBV-induced liver disease above those in uninfected individuals.

[00158] Interferons (e.g., IFN-a) and alternate formulations (e.g., pegylated IFN-a) are also licensed for therapy of HBV but their use is limited because of unwanted side effects. In addition, variability in treatment response of chronic HBV carriers is still a common observation with IFN-a, administered alone or in combination with nucleoside and/or nucleotide analogs, but overall approximately 25-30% of such patients achieve a sustained antiviral response after 2 years of drug administration, including the loss of HBsAg. Therefore, one goal of current HBV therapy is to develop new antiviral compounds that can mimic the benefits of IFN-a therapy but induce suppression of HBV replication, clearance of HBsAg, and seroconversion to anti-HBs in more than one-third of treated patients.

HBV and Drug-Resistance

[00159] The present invention further relates to methods for treating a subject infected with a resistant variant of HBV through administration of a crystalline compound of formula I or a pharmaceutically acceptable salt thereof, a compound of formula III, or a composition disclosed herein. The HBV genome is comprised of circular, partially duplexed DNA that encodes four known genes termed C, X, P, and S. Multiple open-reading frames and/or proteolytic processing of the resulting gene products give rise to HBV proteins including the surface antigen (HBsAg), core protein (HBcAg or C), E antigen (HBeAg or pre-C), long surface protein (L), middle surface protein (M), polymerase (P), and X protein.

[00160] Naturally, HBV exists within a host as a population of genetically distinct but closely related virions, due in part to the low fidelity of the viral reverse transcriptase, or polymerase P (Locarnini, S. and Warner, N. Antivir Ther (2007) 12 Suppl 3 :H15-H23; Coleman, RE Emer Infect Dis (2006) 12: 198-203). Treatment with standard anti-HBV agents may eliminate some or nearly all of the HBV population, and readily select out a small and possibly undetectable HBV population that is resistant to said treatment and capable of developing into a chronic infection. Drug-resistance is further affected by other factors including, but not limited to, the viral mutation frequency, the mutability of the antiviral target site, the particular selective pressure applied by the antiviral agent, and the overall replication fitness of the resistant strain (Locarnini, S. and Warner, N. Antivir Ther (2007) 12 Suppl 3 :H15-H23). HBV strains resistant to a number of standard anti-HBV agents have been reported, including lamivudine and adefovir dipivoxil.

[00161] Without being bound by any particular theory, a drug-resistant strain of HBV may comprise an amino acid mutation (e.g., an amino acid substitution, addition, or deletion) in a particular protein that may result in a structural change, e.g., a conformational or steric change, that affects the ability of an anti-HBV agent from binding to said protein and modulating its activity, e.g., through inhibiting HBV replication or pathogenicity. Particularly, amino acids in and around the active site or close to the inhibitor binding site may be mutated such that the activity of the protein is impacted. In some instances, the amino acid mutation (e.g., an amino acid substitution, addition, or deletion) may be conservative and may not substantially impact the structure or function of a protein. For example, in certain cases, the substitution of a serine residue with a threonine residue may not significantly impact the function of a protein. In other cases, the amino acid mutation may be more dramatic, such as the substitution of a charged amino acid (e.g., aspartic acid or lysine) with a large, nonpolar amino acid (e.g., phenylalanine or tryptophan) and therefore may have a substantial impact on protein function. The nature of the mutations that render the HBV strain resistant to one or more antiviral agents can be readily identified using standard sequencing techniques, e.g., deep sequencing techniques, that are well known in the art.

[00162] In some embodiments, the drug-resistant HBV strain comprises a variant or mutant form of the HBsAg, HBcAg, HBeAg, L, M, P, or X proteins. In some embodiments, the drug- resistant HBV strain comprises a variant or mutant form of the HBsAg, HBcAg, HBeAg, L, M, P, or X proteins compared with the accepted consensus sequence of said proteins.

[00163] In some embodiments, the drug-resistant HBV variant comprises an amino acid mutation (e.g., an amino acid substitution, addition, or deletion) in the sequence of the HBsAg, HBcAg, HBeAg, L, M, P, or X proteins, e.g., as compared to a reference or consensus sequence. In some embodiments, the amino acid mutation in the sequence of the HBsAg, HBcAg, HBeAg, L, M, P, or X proteins is an amino acid substitution. In some embodiments, the amino acid mutation in the sequence of the HBsAg, HBcAg, HBeAg, L, M, P, or X proteins is an amino acid addition. In some embodiments, the amino acid mutation in the sequence of the HBsAg, HBcAg, HBeAg, L, M, P, or X proteins is an amino acid deletion.

[00164] In some embodiments, the amino acid mutation in the sequence of the HBsAg, HBcAg, HBeAg, L, M, P, or X proteins comprises an amino acid substitution of the wild type amino acid residue present at a particular position in the sequence with another amino acid selected from one of the naturally occurring amino acids. In some embodiments, the amino acid mutation in the sequence of the HBsAg, HBcAg, HBeAg, L, M, P, or X proteins comprises an amino acid substitution of the wild type amino acid residue present at a particular position in the sequence with an alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine residue.

[00165] In some embodiments, the amino acid mutation in the sequence of the HBsAg, HBcAg, HBeAg, L, M, P, or X proteins comprises an amino acid addition to the wild type sequence at a particular position of an amino acid selected from one of the naturally occurring amino acids. In some embodiments, the amino acid mutation in the sequence of the HBsAg, HBcAg, HBeAg, L, M, P, or X proteins comprises an amino acid addition to the wild type sequence at a particular position selected from an alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine residue.

[00166] In some embodiments, the amino acid mutation in the sequence of the HBsAg, HBcAg, HBeAg, L, M, P, or X proteins comprises an amino acid deletion at a particular position of the wild type sequence. In some embodiments, the amino acid deletion in the sequence of the HBsAg, HBcAg, HBeAg, L, M, P, or X proteins comprises an amino acid deletion of an alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine residue.

[00167] In some embodiments, the drug-resistant HBV variant comprises an amino acid mutation (e.g., an amino acid substitution, addition, or deletion) in the sequence of the HBsAg protein, e.g., as compared to a reference or consensus sequence. In some embodiments, the amino acid mutation (e.g., an amino acid substitution, addition, or deletion) in the HBsAg protein sequence comprises a mutation from amino acid position 100 to amino acid position 200, e.g., as compared to a reference or consensus sequence. In some embodiments, the amino acid mutation (e.g., an amino acid substitution, addition, or deletion) in the HBsAg protein sequence comprises a mutation from amino acid position 105 to amino acid position 160 e.g., as compared to a reference or consensus sequence. In some embodiments, the amino acid mutation (e.g., an amino acid substitution, addition, or deletion) in the HBsAg protein sequence comprises a mutation from amino acid position 115 to amino acid position 155, e.g., as compared to a reference or consensus sequence. In some embodiments, the amino acid mutation (e.g., an amino acid substitution, addition, or deletion) in the HBsAg protein sequence comprises a mutation at amino acid positions 115, 118, 120, 123, 126, 129, 131, 133, 134, 142, 143, 144, 145, or 154, e.g., as compared to a reference or consensus sequence. In some embodiments, the amino acid mutation (e.g., an amino acid substitution, addition, or deletion) in the HBsAg protein sequence comprises a T115N, T118V, P120L, P120Q, T126S, Q129H, T131K, M133I, M133L, F134N, F134H, P142L, P142S, T143L, D144A, D144V, G145R, or S154P mutation.

[00168] In some embodiments, the amino acid mutation (e.g., an amino acid substitution, addition, or deletion) in the HBsAg protein sequence comprises a mutation from amino acid position 150 to amino acid position 200 e.g., as compared to a reference or consensus sequence. In some embodiments, the amino acid mutation (e.g., an amino acid substitution, addition, or deletion) in the HBsAg protein sequence comprises a mutation from amino acid position 160 to amino acid position 200, e.g., as compared to a reference or consensus sequence. In some embodiments, the amino acid mutation (e.g., an amino acid substitution, addition, or deletion) in the HBsAg protein sequence comprises a mutation at amino acid positions 161, 172, 173, 175, 176, 193, 194, or 196, e.g., as compared to a reference or consensus sequence. In some embodiments, the amino acid mutation (e.g., an amino acid substitution, addition, or deletion) in the HBsAg protein sequence comprises a F161H, F161L, W172L, W172*, L173F, L175F, L176V, L176*, S193L, V194F, V194S, I195M, W196L, W196S, or W196* mutation, e.g., as compared to a reference or consensus sequence, wherein "*" represents a stop codon.

[00169] In some embodiments, the drug-resistant HBV variant comprises an amino acid mutation (e.g., an amino acid substitution, addition, or deletion) in the sequence of the P protein, e.g., as compared to a reference or consensus sequence. In some embodiments, the amino acid mutation (e.g., an amino acid substitution, addition, or deletion) in the P protein sequence comprises a mutation from amino acid position 60 to amino acid position 275, e.g., as compared to a reference or consensus sequence. In some embodiments, the amino acid mutation (e.g., an amino acid substitution, addition, or deletion) in the P protein sequence comprises a mutation from amino acid position 80 to amino acid position 250, e.g., as compared to a reference or consensus sequence. In some embodiments, the amino acid mutation (e.g., an amino acid substitution, addition, or deletion) in the P protein sequence comprises a mutation at amino acid positions 80, 169, 173, 180, 181, 184, 169, 202, 204, 215, 233, 236, or 250, e.g., as compared to a reference or consensus sequence. In some embodiments, the amino acid mutation (e.g., an amino acid substitution, addition, or deletion) in the P protein sequence comprises a mutation at amino acid positions 180, 204, or 236, e.g., as compared to a reference or consensus sequence.

[00170] In some embodiments, the amino acid mutation (e.g., an amino acid substitution, addition, or deletion) in the P protein sequence comprises a N169T, I169T, V173L, L180M, A181T, A181V, T184A, T184C, T184G, T184I, T184L, T184M, T184S, S202C, S202G, S202I, M204I, M204V, N236T, M250I, or M250V mutation. In some embodiments, the amino acid mutation (e.g., an amino acid substitution, addition, or deletion) in the P protein sequence comprises a L180M, M204I, M204V, or N236T mutation. In some embodiments, the amino acid mutation (e.g., an amino acid substitution, addition, or deletion) in the P protein sequence comprises an L180M mutation. In some embodiments, the amino acid mutation (e.g., an amino acid substitution, addition, or deletion) in the P protein sequence comprises an M204I mutation. In some embodiments, the amino acid mutation (e.g., an amino acid substitution, addition, or deletion) in the P protein sequence comprises an M204V mutation. In some embodiments, the amino acid mutation (e.g., an amino acid substitution, addition, or deletion) in the P protein sequence comprises an L180M and an M204V mutation. In some embodiments, the amino acid mutation (e.g., an amino acid substitution, addition, or deletion) in the P protein sequence comprises an N236T mutation.

[00171] In some embodiments, the amino acid mutation (e.g., an amino acid substitution, addition, or deletion) in the P protein sequence comprises an L180M, M204V/I, I169T, V173L, and M250V mutation. In some embodiments, the amino acid mutation (e.g., an amino acid substitution, addition, or deletion) in the P protein sequence comprises an L180M, M204V/I, T184G, and S202I/G mutation.

[00172] In some embodiments, the drug-resistant HBV variant comprises an amino acid mutation (e.g., an amino acid substitution, addition, or deletion) in the sequences of both the HBsAg and P proteins, e.g., as compared to reference or consensus sequences.

[00173] In some embodiments, the drug-resistant HBV variant comprises an amino acid mutation (e.g., an amino acid substitution, addition, or deletion) in the sequence of the HBcAg protein. In some embodiments, the drug-resistant HBV variant comprises an amino acid mutation (e.g., an amino acid substitution, addition, or deletion) in the sequence of the HBeAg protein. In some embodiments, the drug-resistant HBV variant comprises an amino acid mutation (e.g., an amino acid substitution, addition, or deletion) in the sequence of the L protein. In some embodiments, the drug-resistant HBV variant comprises an amino acid mutation (e.g., an amino acid substitution, addition, or deletion) in the sequence of the M protein. In some embodiments, the drug-resistant HBV variant comprises an amino acid mutation (e.g., an amino acid substitution, addition, or deletion) in the sequence of the X protein.

[00174] In some embodiments, the drug-resistant HBV variant comprises more than one amino acid mutation (e.g., an amino acid substitution, addition, or deletion) in the sequence of the HBsAg, HBcAg, HBeAg, L, M, P, or X proteins. In some embodiments, the drug-resistant HBV variant comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 or more amino acid mutations (e.g., an amino acid substitution, addition, or deletion) in the sequence of the HBsAg, HBcAg, HBeAg, L, M, P, or X proteins. In some embodiments, the drug-resistant HBV variant comprises an amino acid mutation (e.g., an amino acid substitution, addition, or deletion) in the sequence of the only one of the HBsAg, HBcAg, HBeAg, L, M, P, or X proteins. In some embodiments, the drug-resistant HBV variant comprises an amino acid mutation (e.g., an amino acid substitution, addition, or deletion) in the sequence of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or all of the HBsAg, HBcAg, HBeAg, L, M, P, or X proteins. In some embodiments, the drug-resistant HBV variant may comprise an amino acid mutation in a protein other than the HBsAg, HBcAg, HBeAg, L, M, P, or X proteins.

[00175] In the above embodiments, the amino acid mutation (e.g., an amino acid substitution, addition, or deletion) in the drug-resistant HBV strain comprises a variant or mutant form of the HBsAg, HBcAg, HBeAg, L, M, P, or X proteins compared with the accepted consensus sequence or a reference sequence of said proteins.

[00176] In some embodiments, the drug-resistant variant of HBV is resistant to an anti-HBV agent other than a compound other than formula I or a pharmaceutically acceptable salt thereof. In some embodiments, the drug-resistant variant of HBV is resistant to an interferon, a nucleoside analog, a non-nucleoside antiviral, a non-interferon immune enhancer, or a direct- acting antiviral, each of which does not include a compound of formula I or a pharmaceutically acceptable salt thereof. In some embodiments, the drug-resistant variant of HBV is resistant to interferon (e.g., peg-interferon), ribavirin, lamivudine, adefovir dipivoxil, entecavir, telbivudine, clevudine, tenofovir, tenofovir alafenamide, besifovir, or AGX-1009 or a combination thereof.

[00177] In some embodiments, the drug-resistant variant of HBV is resistant to an interferon (e.g., peg-interferon). In some embodiments, the drug-resistant variant of HBV is resistant to ribavirin. In some embodiments, the drug-resistant variant of HBV is resistant to an interferon (e.g., peg-interferon) and ribavirin. In some embodiments, the drug-resistant variant of HBV is resistant to lamivudine, adefovir dipivoxil, entecavir, telbivudine, clevudine, tenofovir, tenofovir alafenamide, besifovir. In some embodiments, the drug-resistant variant of HBV is resistant to lamivudine, adefovir dipivoxil, or entecavir. In some embodiments, the drug-resistant HBV variant is resistant to more than one anti-HBV agent.

[00178] In some embodiments, the IC₅₀ of an anti-HBV agent other than a compound of formula I or a pharmaceutically acceptable salt thereof in a sample infected with a drug-resistant variant of HBV is higher than the IC₅₀ of a compound of formula I or a pharmaceutically acceptable salt thereof. In some embodiments, the IC₅₀ of an anti-HBV agent other than a compound of formula I or a pharmaceutically acceptable salt thereof is more than about 5%, more than about 10%, more than about 15%, more than about 20%, more than about 25%, more than about 30%, more than about 35%, more than about 40%, more than about 45%, more than about 50%), more than about 55%, more than about 60%, more than about 65%, more than about 70%), more than about 75%, more than about 80%, more than about 85%, more than about 90%, or more than about 95% higher than the IC₅₀ of a compound of formula I or a pharmaceutically acceptable salt thereof. In some embodiments, the IC₅₀ of an anti-HBV agent other than a compound of formula I or a pharmaceutically acceptable salt thereof is more than about 1.5 fold, about 2 fold, about 2.5 fold, about 3 fold, about 3.5 fold, about 4 fold, about 4.5 fold, about 5 fold, about 10 fold, about 15 fold, about 20 fold, about 25 fold, about 35 fold, or about 50 fold higher than the IC₅₀ of a compound of formula I or a pharmaceutically acceptable salt thereof.

HDV Infection

[00179] The present invention further relates to methods for treating a subject suffering from a HDV (e.g., a co-infection with HBV and HDV) through administration of formula I or a pharmaceutically acceptable salt thereof, in combination with tenofovir (e.g., tenofovir dipivoxil, tenofovir alafenamide). Hepatitis D (HDV) is small circular enveloped RNA virus and is the sole member of the Delta virus genus. The circular genome comprises 1,700 nucleotides and encodes only a single protein, the HDV surface antigen (HDAg). As HDV does not produce envelope proteins, the virus is unable to generate progeny viral particles on its own and requires the co-infection of the host cell with HBV to complete the viral replication. The viral replication machinery utilizes the HBV-derived envelope proteins to produce and package mature virions to propagate virulence. HDV is characterized into eight major serotypes (HDV-1, HDV-2, HDV-3, HDV-4, HDV-5, HDV-6, HDV-7, and HDV-8) according to the overall nucleotide sequence of the viral genome. In some embodiments, the methods described herein are used to treat a subject suffering from a co-infection of HBV and HDV in combination with tenofovir (e.g., tenofovir dipivoxil, tenofovir alafenamide). The HBV and HDV may comprise any genotype of HBV or HDV, or a combination of varying gentotypes of HBV and HDV.

Combination Therapies

[00180] In some embodiments, additional therapeutic agents may be administered with compositions of the present invention for the treatment of a viral infection, e.g., HBV, or any symptom or associated condition thereof. When combination therapy is employed, the additional therapeutic agent(s) can be administered as a separate formulation or may be combined with any of the compositions described herein.

[00181] For example, any of the methods described herein may further comprise the administration of a therapeutically effective amount of an additional agent [e.g., tenofovir (e.g., tenofovir dipivoxil, tenofovir alafenamide), entecavir, telbivudine] in conjunction with a compound of formula I or a pharmaceutically acceptable salt thereof. In some embodiments, the additional agent is an antiviral agent or an anticancer agent. In some embodiments, the antiviral agent comprises an interferon, a nucleoside analog, a non-nucleoside antiviral, or a non- interferon immune enhancer. In some embodiments, the interferon comprises interferon alfa-2a, interferon alfa-2b, interferon alfa-nl, interferon alfacon-1, or a pegylated interferon (e.g., peginterferon alfa-2a, peginterferon alfa-2b). In some embodiments, the nucleoside analog comprises lamivudine, adefovir dipivoxil, entecavir, telbivudine, clevudine, ribavarin, tenofovir, tenofovir dipivoxil, tenofovir alafenamide, besifovir, or AGX-1009. In some embodiments, the antiviral agent is entecavir. In some embodiments, the antiviral agent is tenofovir (e.g., tenofovir dipivoxil or tenofovir alafenamide). In some embodiments, the antiviral compound comprises NOV-225, BAM 205, Myrcludex B, ARC-520, BAY 41-4109, REP 9AC, Alinia (nitazoxanide), Dd-RNAi, NVR-121 (NVR 3-778), BSBI-25, NVP-018, TKM-HBV, or ALN-HBV. In some embodiments, the non-interferon immune enhancer comprises zadaxin (thymosin alpha-1), GS- 4774, CYT107 (interleukin-7), Dv-601, HBV core antigen vaccine, or GS-9620. In some embodiments, the antiviral agent is a capsid inhibitor, an entry inhibitor, a secretion inhibitor, a microRNA, an antisense RNA agent, an RNAi agent, or other agent designed to inhibit viral RNA. In some embodiments, the anticancer agent is selected from methotrexate, 5-fluorouracil, doxorubicin, vincristine, bleomycin, vinblastine, dacarbazine, toposide, cisplatin, epirubicin, and sorafenib tosylate.

[00182] Administration in combination can proceed by any technique apparent to those of skill in the art including, for example, separate, sequential, concurrent, and alternating administration. As used herein, "administered in combination" or a combined administration of two or more agents means that two or more agents (e.g., compounds described herein) are administered to a subject at the same time or within an interval such that there is overlap of an effect of each agent on the patient. Preferably they are administered within 15, 10, 5, or 1 minute of one another. In some embodiments, the combination of a compound of formula I and the additional agent has a synergistic or additive effect. In some embodiments, the term

"additive" refers to an outcome wherein when two agents are used in combination, the combination of the agents acts in a manner equal to but not greater than the sum of the individual anti-HBV activities of each agent.

[00183] In some embodiments, the terms "synergy" or "synergistic" refer to an outcome wherein when two agents are used in combination, the combination of the agents acts so as to require a lower concentration of each individual agent than the concentration required to be efficacious in the absence of the other agent. In some embodiments, a synergistic effect results in a reduced minimum inhibitory concentration of one or both agents, such that the effect is greater than the sum of the effects. A synergistic effect is greater than an additive effect. In some embodiments, the agents in the composition herein may exhibit a synergistic effect, wherein the anti-HBV activity at a particular concentration is greater than at least about 1.25, 1.5, 1.75, 2, 2.5, 3, 4, 5, 10, 12, 15, 20, 25, 50, or 100 times the anti-HBV activity or anti-HDV activity of either agent alone. Preferably the administrations of the agents are spaced sufficiently close together such that a combinatorial (e.g., a synergistic) effect is achieved.

[00184] The combinations can have synergistic effect when used to treat a subject suffering from an HBV infection, a resistant HBV infection, or an HBV/HDV co-infection. The agents can be administered simultaneously, for example in a combined unit dose (providing

simultaneous delivery of both agents). Alternatively, the agents can be administered at a specified time interval, for example, an interval of minutes, hours, days or weeks. Generally, the agents are concurrently bioavailable, e.g., detectable, in the subject.

[00185] In another aspect, the present invention features methods for treating a subject infected with HBV or an HBV/HDV through administration of a compound of compound of formula I or a pharmaceutically acceptable salt thereof, in combination with tenofovir (e.g., tenofovir dipivoxil or tenofovir alafenamide). In some embodiments, the combination of a compound of formula I or III and tenofovir has a synergistic or additive effect. In some embodiments, the term "additive" refers to an outcome wherein when two agents are used in combination, the combination of the agents acts in a manner equal to but not greater than the sum of the individual anti-HBV or anti-HDV activities of each agent. [00186] The methods of combination therapy include co-administration of a single formulation containing a compound of formula I or III or a pharmaceutically acceptable salt thereof and additional therapeutic agent or agents [e.g., tenofovir (e.g., tenofovir dipivoxil, tenofovir alafenamide), entecavir, telbivudine], essentially contemporaneous administration of more than one formulation comprising a compound of formula I or III or a pharmaceutically acceptable salt thereof and additional therapeutic agent or agents [e.g., tenofovir (e.g., tenofovir dipivoxil, tenofovir alafenamide), entecavir, telbivudine], and consecutive administration of a compound of formula I or a pharmaceutically acceptable salt thereof and additional therapeutic agent or agents [e.g., tenofovir (e.g., tenofovir dipivoxil, tenofovir alafenamide), entecavir, telbivudine], in any order, wherein preferably there is a time period where a compound of formula I or a pharmaceutically acceptable salt thereof and additional therapeutic agent or agents [e.g., tenofovir (e.g., tenofovir dipivoxil, tenofovir alafenamide), entecavir, telbivudine] simultaneously exert their therapeutic effect. Cancer Immunotherapy

[0001] Cancer immunotherapy refers to a diverse set of therapeutic strategies designed to induce the patient's own immune system to fight the tumor. Cancer immunotherapy is a paradigm in cancer treatment that instead of targeting cancer cells focuses on the activation of the immune system. Its principle is to rearm the host's immune response, especially the adaptive T cell response, to provide immune surveillance to kill the cancer cells, in particular, the minimal residual disease that has escaped other forms of treatment, hence achieving long-lasting protective immunity.

[00187] The present invention provides, in part, a method of treating or preventing a condition selected from a proliferative or hyperproliferative disease, for example, cancer. For example, the invention provides for the treatment of diseases, disorders, and conditions characterized by excessive or abnormal cell proliferation. Such diseases include a proliferative or

hyperproliferative disease. Examples of proliferative and hyperproliferative diseases include cancer and myeloproliferative disorders. In some embodiments, the proliferative disease or cancer is associated with a viral infection.

[00188] The invention provides, in part, a method of treating or preventing a proliferative disease or a cancer in a subject in need thereof. The method comprises administering to the subject a therapeutically effective amount of a crystalline compound of formula I or a pharmaceutically acceptable salt thereof, a compound of formula III, or a composition disclosed herein. [00189] In some embodiments, the proliferative disorder or cancer is associated with a viral infection.

[00190] In certain embodiments, the viral infection is caused by HBV or HCV.

[00191] In other embodiments, the cancer is a liver cancer.

[00192] In particular, it is contemplated that the compounds or compositions of the invention can be used to treat one or more of the following cancers: Oral: head and neck, including buccal cavity, lip, tongue, mouth, pharynx; Cardiac: sarcoma (angiosarcoma, fibrosarcoma,

rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung: Non-small cell lung carcinoma including adenocarcinoma, bronchioalveolar, squamous cell carcinoma (basal oid, clear cell, papillary, small cell), large cell carcinoma, large cell neuroendocrine carcinoma (LCNEC); small cell lung cancer including small cell (oat cell) carcinoma, combined small cell; adenoid cystic carcinoma; hamartoma; lymphoma;

neuroendocrine/carcinoid; sarcoma; Gastrointestinal: esophagus (squamous cell carcinoma, larynx, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel or small intestines (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel or large intestines (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma), colon, colon-rectum, colorectal, rectum; Genitourinary tract: kidney

(adenocarcinoma, Wilm's tumor, lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver:

hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma, biliary passages; Bone: osteogenic sarcoma

(osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma, glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal cord neurofibroma, meningioma, glioma, sarcoma); Female/Gynecological: uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervical dysplasia), ovaries (ovarian carcinoma, granulosa-thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tubes (carcinoma), breast; Hematologic: blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma, hairy cell; lymphoid disorders; Skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, keratoacanthoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis, Thyroid gland: papillary thyroid carcinoma, follicular thyroid carcinoma,

undifferentiated thyroid cancer, medullary thyroid carcinoma, multiple endocrine neoplasia type 2A, multiple endocrine neoplasia type 2B, familial medullary thyroid cancer,

pheochromocytoma, paraganglioma; and Adrenal glands: neuroblastoma.

[00193] Also provided herein is a method treatment, as described above, further comprising an additional step of administering to the subject an additional therapeutic agent selected from a chemotherapeutic or anti-proliferative agent.

[00194] In certain embodiments, the additional therapeutic agent is administered together with a disclosed compound or composition as a single dosage form. In certain embodiments, the additional therapeutic agent is administered separately from the compound/composition disclosed herein as part of a multiple dosage form.

[00195] In certain embodiments, the invention is directed towards a method of treating cancer in a subject in need thereof, comprising administration of a compound or composition disclosed herein and an additional therapeutic agent. In certain embodiments, the additional therapeutic agent is administered together with the compound or composition disclosed herein as a single dosage form. In certain embodiments, the additional therapeutic agent is administered separately from a compound or composition disclosed herein as part of a multiple dosage form.

[00196] In certain embodiments, the additional therapeutic agent is an anti-cancer agent, an anti-proliferative agent, or a chemotherapeutic agent.

[00197] Other cancer therapies or anticancer agents that may be used in combination with a compound or composition of the present invention include surgery, radiotherapy (e.g., gamma- radiation, neutron beam radiotherapy, electron beam radiotherapy, proton therapy,

brachytherapy, low-dose radiotherapy, and systemic radioactive isotopes), immune response modifiers such as chemokine receptor antagonists, chemokines and cytokines (e.g., interferons, interleukins, tumour necrosis factor (TNF), and GM-CSF)), hyperthermia and cryotherapy, agents to attenuate any adverse effects (e.g. antimetics, steroids, anti-inflammatory agents), and other approved chemotherapeutic drugs.

Routes of Administration

[00198] The compounds and compositions used in the methods described herein may be administered to a subject in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art. Exemplary routes of administration of the compositions used in the methods described herein include topical, enteral, or parenteral applications. Topical applications include but are not limited to epicutaneous, inhalation, enema, eye drops, ear drops, and applications through mucous membranes in the body. Enteral applications include oral administration, rectal administration, vaginal administration, and gastric feeding tubes. Parenteral administration includes intravenous, intraarterial, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrastemal, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, rectal, and topical modes of administration. Parenteral administration may be by continuous infusion over a selected period of time. In certain embodiments of the invention, the compositions described herein comprising a compound of formula I or III is administered orally. In other embodiments of the invention, the compositions described herein comprising a compound of formula I or III is administered intravenously.

[00199] In an embodiment, the compositions described herein comprising a compound of formula I or III is administered orally in combination with tenofovir (e.g., tenofovir dipivoxil or tenofovir alafenamide). In an embodiment, the compositions described herein comprising a compound of formula I or III is administered orally prior to or after oral administration of tenofovir (e.g., tenofovir dipivoxil or tenofovir alafenamide).

[00200] In other embodiments of the invention, the compositions described herein comprising a compound of formula I or III is administered parenterally (e.g., intraperitoneally). In an embodiment, the compositions described herein comprising a compound of formula I or III is administered parenterally in combination with tenofovir (e.g., tenofovir dipivoxil or tenofovir alafenamide). In an embodiment, the compositions described herein comprising a compound of formula I or III is administered parenterally prior to or after oral administration of tenofovir (e.g., tenofovir dipivoxil or tenofovir alafenamide).

[00201] For intravenous, intraperitoneal, or intrathecal delivery or direct injection, the composition must be sterile and fluid to the extent that the composition is deliverable by syringe. In addition to water, the carrier can be an isotonic buffered saline solution, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion and by use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition. Long-term absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

[00202] The choice of the route of administration will depend on whether a local or systemic effect is to be achieved. For example, for local effects, the composition can be formulated for topical administration and applied directly where its action is desired. For systemic, long term effects, the composition can be formulated for enteral administration and given via the digestive tract. For systemic, immediate and/or short-term effects, the composition can be formulated for parenteral administration and given by routes other than through the digestive tract.

Dosages

[00203] The compositions of the present invention are formulated into acceptable dosage forms by conventional methods known to those of skill in the art. For example, the present invention provides a solid oral dosage form comprising a disclosed compound (e.g., a crystalline compound of formula I) or a disclosed composition (e.g., a disclosed particulate composition). Actual dosage levels of the active ingredients in the compositions of the present invention (e.g., a compound of formula I or III) may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of absorption of the particular agent being employed, the duration of the treatment, other drugs, substances, and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well known in the medical arts. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the composition required. For example, the physician or veterinarian can start doses of the substances of the invention employed in the composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of a composition of the invention will be that amount of the substance which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

Preferably, the effective daily dose of a therapeutic composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.

[00204] Preferred therapeutic dosage levels are between about 0.1 mg/kg to about 1000 mg/kg (e.g., about 0.2 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 350 mg/kg, 400 mg/kg, 450 mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, or 1000 mg/kg) of the composition per day administered (e.g., orally or intraperitoneally) to a subj ect afflicted with the disorders described herein (e.g., HBV infection). Preferred prophylactic dosage levels are between about 0.1 mg/kg to about 1000 mg/kg (e.g., about 0.2 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 350 mg/kg, 400 mg/kg, 450 mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, or 1000 mg/kg) of the composition per day administered (e.g., orally or intraperitoneally) to a subject. The dose may also be titrated (e.g., the dose may be escalated gradually until signs of toxicity appear, such as headache, diarrhea, or nausea).

[00205] The frequency of treatment may also vary. The subject can be treated one or more times per day (e.g., once, twice, three, four or more times) or every so-many hours (e.g., about every 2, 4, 6, 8, 12, or 24 hours). The composition can be administered 1 or 2 times per 24 hours. The time course of treatment may be of varying duration, e.g., for two, three, four, five, six, seven, eight, nine, ten, or more days, two weeks, 1 month, 2 months, 4 months, 6 months, 8 months, 10 months, or more than one year. For example, the treatment can be twice a day for three days, twice a day for seven days, twice a day for ten days. Treatment cycles can be repeated at intervals, for example weekly, bimonthly or monthly, which are separated by periods in which no treatment is given. The treatment can be a single treatment or can last as long as the life span of the subject (e.g., many years). Patient Selection and Monitoring

[00206] The methods of the present invention described herein entail administration of a compound of formula I or a pharmaceutically acceptable salt thereof for the treatment of HBV infection (e.g., a resistant HBV infection). The methods described herein further entail administration of a compound of formula I or a pharmaceutically acceptable salt thereof for the treatment of a subject infected with HBV or HDV (e.g., a co-infection of HBV and HDV) in combination with tenofovir (e.g., tenofovir dipivoxil or tenofovir alafenamide). Accordingly, a patient and/or subject can be selected for treatment using a compound of formula I or a pharmaceutically acceptable salt thereof by first evaluating the patient and/or subject to determine whether the subject is infected with HBV or HDV and determination of the serotypic and genotypic classification of the virus. A subject can be evaluated as infected with HBV or HDV using methods known in the art. The subject can also be monitored, for example, subsequent to administration of a compound described herein (e.g., a compound of formula I) or a pharmaceutically acceptable salt thereof.

[00207] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is an adult. In some embodiments, the subject is suffering from an acute form of HBV infection. In some embodiments, the subject is suffering from a chronic form of HBV infection. In some embodiments, the subject has been diagnosed with hepatitis B (e.g., acute or chronic hepatitis B).

[00208] In some embodiments, the genotype of the HBV infection is known. In some embodiments, the subject is infected with HBV genotype A (e.g., HBV-Al-7), HBV genotype B (e.g., HBV-B2-5), HBV genotype C (e.g., HBV-Cl-16), HBV genotype D (e.g., HBV-D1-7), HBV genotype E, HBV genotype F (e.g., HBV-F1-4), HBV genotype G, HBV genotype H, HBV genotype I, or HBV genotype J. In some embodiments, the subject is infected with HBV genotype A (e.g., HBV-Al-7), HBV genotype B (e.g., HBV-B2-5), HBV genotype C (e.g., HBV-Cl-16), HBV genotype D (e.g., HBV-D1-7), HBV genotype E, HBV genotype F (e.g., HBV-Fl-4), HBV genotype G, or HBV genotype H. In some embodiments, the subject is infected with HBV genotype A (e.g., HBV-Al-7). In some embodiments, the subject is infected with HBV genotype B (e.g., HBV-B2-5). In some embodiments, the subject is infected with HBV genotype C (e.g., HBV-Cl-16). In some embodiments, the subject is infected with HBV genotype D (e.g., HBV-Dl-7). In some embodiments, the subject is infected with HBV genotype E. In some embodiments, the subject is infected with HBV genotype F (e.g., HBV-F1- 4). In some embodiments, the subject is infected with HBV genotype G. In some embodiments, the subject is infected with HBV genotype H. In some embodiments, the subject is infected with HBV genotype I. In some embodiments, the subject is infected with HBV genotype J.

[00209] In some embodiments, the drug-resistant strain of HBV comprises HBV genotype A, (e.g., HBV-A1-7), HBV genotype B (e.g., HBV-B2-5), HBV genotype C (e.g., HBV-Cl-16), HBV genotype D (e.g., HBV-D1-7), HBV genotype E, HBV genotype F (e.g., HBV-F1-4), HBV genotype G, HBV genotype H, HBV genotype I, or HBV genotype J.

[00210] In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a woodchuck, e.g., the eastern woodchuck. The eastern woodchuck {Marmota monax) is naturally infected with the woodchuck hepatitis virus (WHV), a hepadnavirus which is genetically closely related to human HBV. Neonatal infection of woodchucks with WHV parallels the main route of human (vertical) transmission for chronic HBV infection and displays a disease course similar to that in HBV-infected patients. Thus, chronic WHV infection in woodchucks is a fully immunocompetent model for studying CHB and HBV-induced HCC, and chronic WHV carriers have extensively been used to evaluate efficacy and safety of current and new HBV therapeutics. The recent comparison of hepatic transcriptional profiles in woodchucks and humans with acute self-limiting and chronic hepadnaviral infections identified important parallels in the antiviral immune responses and demonstrated molecular similarities in HCC induced by WHV and HBV. As these studies have established the translational value of this animal model for CHB, woodchucks with chronic WHV infection may be used to evaluate antiviral efficacy, safety and pharmacodynamics associated with treatment.

[00211] In some embodiments, the subject is treatment naive. In some embodiments, the subject has previously been treated for HBV infection. In some embodiments, the subject is suffering from a relapsed HBV infection. In some embodiments, the subject has been treated with an anti-HB V agent other than a compound of formula I or a pharmaceutically acceptable salt thereof and is suffering from a relapsed HBV infection. In some embodiments, the subject has been treated with an interferon, a nucleoside analog, a non-nucleoside antiviral, or an immune enhancer and is suffering from a relapsed HBV infection. In some embodiments, the subject has been treated with an interferon, e.g., peg-interferon alfa (e.g., peg-interferon alfa-2a or peg-interferon alfa-2b) and is suffering from a relapsed HBV infection. In some

embodiments, the subject has been treated with ribavirin and is suffering from a relapsed HBV infection. In some embodiments, the subject has been treated with a nucleoside analog, e.g., lamivudine, adefovir dipivoxil, entecavir, telbivudine, clevudine, ribavarin, tenofovir, tenofovir alafenamide, besifovir, or AGX-1009, and is suffering from a relapsed HBV infection. In some embodiments, the subject has been treated with a non-nucleoside antiviral agent, e.g., NOV-225, BAM 205, Myrcludex B, ARC-520, BAY 41-4109, REP 9 AC, Alinia (nitazoxanide), Dd-RNAi, NVR-121 (NVR 3-778), BSBI-25, NVP-018, TKM-HBV, or ALN-HBV, and is suffering from a relapsed HBV infection. In some embodiments, the subject has been treated with an immune enhancer, e.g., zadaxin (thymosin alpha-1), GS-4774, CYT107 (interleukin-7), Dv-601, HBV core antigen vaccine, or GS-9620, and is suffering from a relapsed HBV infection.

[00212] In some embodiments, the subject has been diagnosed with cirrhosis of the liver. In some embodiments, the subject has been diagnosed with hepatocellular carcinoma. In some embodiments, the subject has been diagnosed with hepatocellular carcinoma and is awaiting liver transplantation.

[00213] In some embodiments, the subject has been further diagnosed with an HIV infection. In some embodiments, the strain of HIV infection is known. In some embodiments, the subject is infected with HIV-1 or HIV-2 (e.g., strain 1 or strain 2).

[00214] In some embodiments, the subject is suffering from an HBV infection and an HDV infection (e.g., an HBV and HDV co-infection). In some embodiments, the subject is suffering from a chronic form of HBV or HDV infection. In some embodiments, the subject has been diagnosed with hepatitis B (e.g., acute or chronic hepatitis B, e.g., a resistant variant of acute or chronic hepatitis B). In some embodiments, the subject has been diagnosed with hepatitis D (e.g., acute or chronic hepatitis D). In some embodiments, the genotype of the HDV infection is known. In some embodiments, the subject is treatment naive. In some embodiments, the subject has received previous treatment for HDV.

EXAMPLES

Example 1. Preparation of the compound of formula I

[00215] Isolation of the compound of formula I and the compound of formula V (see Scheme 1) was achieved taking advantage of solubility differences in water. Highly diastereomerically pure compound of formula I was isolated from the compound of formula II in two

crystallizations from hot water.

[00216] Experimental Procedure: In an Erlenmeyer flask, water (HPLC, 4000 ml) was heated slowly under stirring to 75-80 °C, or even to boiling, and the hot water was transferred to the compound of formula II (200 g, _^,65:35) in a 5L glass bottle in portions. After each addition, the contents were mixed well. At the end of addition, the bottle was closed with a cap and the hot contents of the bottle were thoroughly mixed by shaking. The contents of the flask, covered with aluminum foil, was left on the bench overnight. The solid settled down on standing, was filtered and dried. A small sample solid was dried using a lyophilizer for 48-72 h and the isomeric purity was determined by ³¹P- MR in DMSO-d6 to be _~ 89% (Figure 1 A).

[00217] The isolated dried sample, after first crystallization, was subjected to another hot water crystallization (1 :20 g/ml) under similar conditions. The solid separated was filtered, and dried using a lyophilizer for 3 days. The purity of the sample was found to be 96% by 31P- NMR (Figure IB).

[00218] After the isolation of the compound of formula I, NaCl (25% of volume) was added to the filtrate. White solid separated and the contents were left at 0-5 °C overnight. Filtration followed by washing with small amount of ice cold water led to the isolation of the compound of formula in -90% pure by ³¹P- MR. (Figure 2).

formula V

Scheme 1. Isolation of the compound of formula I from the compound of formula II Example 2. Preparation of (L)-(+)-hemi-tartrate salt of the compound of formula I

[00219] Experimental procedure: compound of formula I (2 g, 2.842 mmol) was weighed and transferred to a 100 mL IN RB flask containing a stir bar. Acetonitrile (20 mL) was added and the heterogeneous mixture was stirred for 2 min. Pre-dissolved (L)-(+)-tartaric acid (427 mg, 2.842 mmol, 1 equiv) in FIPLC grade water (20 mL) was then added to the heterogeneous mixture via a glass pipette. The flask was capped and the mixture was stirred to give a clear, colorless solution. This colorless solution was stirred for 65 h at room temperature. After 65 h of stirring time, additional FIPLC grade water (10 mL) was added and the clear solution was stirred for another 30 h. The stirring was stopped and the acetonitrile was carefully evaporated in vacuo to obtain a nearly clear aqueous solution. The solution was transferred to a 500 mL pear-shaped flask, freeze-dried and lyophilized to obtain the hemi-tartrate salt (compound of fomula III) as a white fluffy solid (2.4 g; 98.9%).

[00220] Water solubility of the hemi-tartrate salt was 12-15 mg/mL.

(2R'3R).(+)._Tartaric ^acid

( somer

L-(+)-Tartaric ^acid

formula I

H₂0/CH₃CN

rt, 4 days

Hemi tartrate salt

Compound of Formula III

Scheme 2. Synthesis of the compound of formula III

Example 3. Preparation of (L)-(+)-tartrate salt of the compound of formula I

[00221] Experimental procedure: Compound of formula I (2 g, 2.842 mmol) was weighed and transferred to a 100 mL IN RB flask containing a stir bar. (L)-(+)-tartaric acid (213.5 mg, 1.421 mmol, 0.5 equiv) was weighed and added to the vial. Acetonitrile (20 mL) followed by HPLC grade water (20 mL) was added. After stirring for 20 min. THF (5 mL) was then added and the solution turned clear. The flask was capped and the clear, colorless solution was stirred for 6 d at room temperature. After 72 h of stirring time, additional HPLC grade water (20 mL) was added and the clear solution was stirred for another 3 d. After a total of 6 d of stirring time, the solvent was carefully evaporated in vacuo to obtain an aqueous cloudy solution with some amount of white precipitate. Water (20 mL) was added to the flask to extract the left over solid in the flask. The heterogeneous mixture which was white and turbid, was transferred to a 500 mL pear- shaped flask and freeze-dried and lyophilized to obtain the tartrate salt (compound of formula

IV) as a white fluffy solid (2.1 g; 95%).

compound o ormu a H

Scheme 3. Synthesis of the compound of formula IV

Example 3. Stability of the compound of formula I when stored at room temperature (25 °C, ambient humidity)

[00222] The stability of the compound of formula I was evaluated by Reversed-phase HPLC analysis of samples stored at different temperature conditions. The chromatographic conditions are depicted below:

Mobile Phase A: 0.1 M H₄OAc; Mobile Phase B: 100% Acetonitrile

Injector Rinse Solution: 50% Acetonitrile in water

Reference Solution (RS) Preparation: Sample Preparation: About 5 mg of the compound RS of formula II is dissolved in 1 mL of 3 : 1 acetonitrile: water. From this, 120 uL is taken and further diluted in 880 uL of 1 : 1 acetonitrile: water. From this sample, 5 uL is injected into the HPLC for analysis.

Experimental Parameters: A Waters Alliance 2695 separations module with 2998 UV detection.

Column: Luna Phenyl Hexyl 100 A C8 column, 4.6x150mm, 3 μιη

Flow: 1.0 mL/min, Column Temp: 30 ± 2 °C, Injection volume: 20 μL, Extracted wavelength: 260 nm, Sample Temp: 5 ± 3 °C [00223] Table 1. HPLC gradient

[00224] Figures 3 A (T=0) and 3B (T=3months) depict the HPLC chromatograms at selected time points of samples stored over a period of 1 year.

[00225] Table 2. HPLC analysis at T=0

[00226] Table 3. HPLC analysis at T=3 months

Example 4. Chemical compatibility of the compound of formula I mixed with Tenofovir dipivoxyl and stored at 25 °C and ambient humidity

[00227] Figures 4 A (T=0), 4B (T=16 weeks), 4C (T=45 weeks), 4D (T=l year) depict the FIPLC chromatograms at selected time points of samples stored over a period of 1 year.

[00228] Table 4. HPLC analysis at T= 0 (May 26, 2016)

[00229] Table 5. HPLC analysis at T = 16 wks (September 15, 2016)

[00230] Table 6. HPLC analysis at T = 45 wks (April 6, 2017)

[00231] Table 7. HPLC analysis at T = lyear

Example 5. Chemical compatibility of the compound of formula I mixed with Tenofovir dipivoxyl and stored at 5 °C and ambient humidity

[00232] Figures 5A (T=0), 5B (T=45 weeks), and 5C (T=l year) depict the HPLC chromatograms at selected time points of samples stored over a period of 1 year.

[00233] Table 8. HPLC analysis at T=0

[00234] Table 9. HPLC analysis at T= 45 weeks

[00235] Table 10. HPLC analysis at T=l year

Example 6. Stability studies of the compound of formula III at 25 °C and ambient humidity

[00236] Figures 6A-6C display the HPLC traces of a sample of the compound of formula III stored at 25 °C and ambient humidity at T=0 (Figure 6A), T=3 months (Figure 6B), and T=4 months (Figure 6C).

Example 7. Activity of single isomer against HBV in the transgenic mouse model

Materials and Methods

[00237] Animals: Homozygous male transgenic HBV mice were used (21.6 ± 2.8 g). The mice were originally obtained from Dr. Frank Chisari (Scripps Research Institute, LaJolla, CA) (1) and were subsequently raised in the Biosafety Level 2 (BL-2) area of the USU Laboratory Animal Research Center (LARC). The animals were derived from founder 1.3.32.

[00238] Animal Welfare: This study was conducted in accordance with the approval of the Institutional Animal Care and Use Committee of Utah State University, which approval has an expiration date of 6/30/17. The work was done in the AAALAC-accredited Laboratory Animal Research Center of Utah State University. The U.S. Government (National Institutes of Health) approval was renewed 8 June, 2011 (Assurance no. A3801-01) in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Revision; 2010).

Test article: Compounds ready for injection at the appropriate concentration were administered 0.1 mL/30-gram mouse by oral gavage.

[00239] Adefovir dipivoxil (ADV) was used as a control in our HBV transgenic mouse experiments. A solution of 2 mg/mL was made in 0.025 M sodium citrate, wherein 0.1 mL was administered by oral gavage (per os, p.o.) per 30 g mouse for a dosage of 10 mg/kg/day. The drug solutions were stored at 4°C.

[00240] Experimental design: Female and male HBV transgenic mice were block-randomized to the treatment groups. Treatment was initiated at day 1. After the last treatment on day 14, necropsy was performed to obtain tissues to assay liver HBV DNA and sera for HBe and HBs. Weights were obtained on days -1, 1 (day of treatment initiation), 3, 7, and 14. Data were analyzed by one-way analysis of variance.

[00241] Conclusion: The single isomer showed potent anti-HBV activity in the HBV transgenic mouse model.

[00242] Figures 7A and 7B show the effect of test agents on liver HBV DNA using A) Southern blot hybridization, and B) semi-quantitative PCR. ***P < 0.001 using one-way analysis of variance compared to vehicle group. ##P < 0.01 using unpaired two-tail t test compared to the hemi-tartrate salt of the compound of formula I.

Example 8. Prophylactic and therapeutic antiviral activity of the compound of formula I during RSV infection of human lung epithelial cells

[00243] In in vitro studies, the compound of formula I and the compound of formula II demonstrated potent anti-RSV activity (Figure 8A and 8B). The two individual isomers, the compound of formula I and the compound of formula V for anti-RSV activity in human lung epithelial (HLE) A549 cells (Figure 8 A). When the cells were infected with RSV in the absence or presence of the compounds of formula I and formula V as their respective hemi-tartrate sodium salts, significant reduction in infectious viral titer was observed in the presence of the compound of formula I and the compound of formula V (Figure 8C). Overall, these studies have demonstrated potent prophylactic anti-RSV activity of these compounds as well as their hemi-tartrate salts.

Therapeutic antiviral activity of the compound of formula II during human respiratory syncytial virus (RSV) infection

[00244] Figure 8 A displays RSV infectivity in untreated (UT) and cells treated with the compound of formula II. Human lung epithelial A549 cells were infected with RSV (2 MOI). At 8h post-infection, the medium was removed and the cells were washed twice with DMEM. Fresh DMEM was added to the cells in the presence of DMSO (denoted as UT or untreated) or the compound of formula II (100 nm or 250 nm). At 16h post-addition of DMSO or the compound of formula II, the medium supernatant was collected and the viral titer in the supernatant was assayed by plaque assay analysis using CV-1 cell monolayer. 100% RSV infection represents viral titer from cells incubated with DMSO only (UT cells). The values represent mean ± S.D. S.D. is shown as error bars.

Anti-viral activity of the compound of formula I against RSV

[00245] Figure 8B displays anti-viral activity of the compound of formula I against RSV. Human lung epithelial A549 cells were treated with DMSO (vehicle control), a compound of negative control (10 μΜ) or the compound of formula I (10 μΜ) for 12h. After pre-treatment, cells were infected with RSV in the presence of DMSO or the corresponding compounds. At 36h post-infection, RSV infection efficiency was assessed by performing plaque assay with medium supernatant. 100% RSV infection corresponds to virus infection observed in control (i.e. DMSO treated cells) cells. The data represents mean of three independent experiments with similar results. The compound of formula I inhibited RSV infection by 80%-75%.

Anti-RSV activity of tartrate salts of the compound of formula I and the compound of formula V in vitro in HLE (A549) cells.

[00246] Figure 8C displays anti-RSV activity of tartrate salts of the compound of formula I and the compound of formula V in vitro in HLE (A549) cells. HLE cells were pre-treated with vehicle or tartrate salt of the compound of formula I or tartrate salt of the compound of formula V at (20 μΜ) for 16h. The cells were then infected with human RSV for 16h in the presence of either vehicle or the compounds. Infectious viral titer was calculated by plaque assay analysis of the medium supernatant from infected cells. *p and **p <0.05 were calculated using Student's t- test.

Example 9. XRPD, TGA, and DSC analyses of the compound of formula I

XRPD

[00247] The XRPD pattern of a sample of the compound of formula I was collected with a PANalytical X'Pert PRO MPD diffractometer using an incident beam of Cu radiation produced using an Optix long, fine-focus source. An elliptically graded multilayer mirror was used to focus Cu Ka X-rays through the specimen and onto the detector. Prior to the analysis, a silicon specimen (NIST SRM 640e) was analyzed to verify the observed position of the Si 111 peak is consistent with the NIST-certified position. A specimen of each sample was sandwiched between 3^m-thick films and analyzed in transmission geometry. A beam-stop, short anti- scatter extension, and an anti-scatter knife edge were used to minimize the background generated by air. Soller slits for the incident and diffracted beams were used to minimize broadening from axial divergence. The diffraction patterns were collected using a scanning position-sensitive detector (X'Celerator) located 240 mm from the specimen and Data Collector software v. 2.2b. Pattern Match version 2.3.6 was used to prepare Figure 9.

[00248] The XRPD pattern of the compound of formula I (Figure 9) consists of sharp peaks, suggesting the sample is a crystalline phase or mixture of crystalline phases.

DSC and TGA

[00249] DSC was performed using a TA Instruments Q2000 differential scanning calorimeter. Temperature calibration was performed using NIST-traceable indium metal. The samples were placed into an aluminum DSC pan, covered with a lid, and the weight was accurately recorded. The temperature was from -30°C to 250 °C and the heat rate was at 10 °C/min.

[00250] TG analysis was performed using a TA Instruments Q5000 IR thermogravimetric analyzer. Temperature calibration was performed using nickel and Alumel™. The samples were placed in an aluminum pan. The sample was hermetically sealed, the lid pierced, then inserted into the TG furnace. The furnace was heated under nitrogen. The temperature was from the ambient temperature to 350 °C and the heat rate was at 10 °C/min.

[00251] The DSC thermogram of the compound of formula I (Figure 10) displays a sharp endotherm at approximately 121 °C (onset~90 °C), which may be due to loss of volatile components, and another possible broad endotherm at approximately 164 °C which may be due to melt/decomposition. The TG thermogram of the compound of formula I (Figure 11) displays a weight loss of approximately 3.6% from 27 to 150 °C.

[00252] The results are summarized in the below table.

[00253] Table 11. Results of analyses

Example 10. Determination of absolute configuration of Formula I and Formula V

[00254] For the determination of absolute configuration of the Rp,<Sp isomer mix of formula II, the strategy involved initial conversion of the ^-alkyl phosphorothiolate group in formula II via stereospecific hydrolysis by serum esterase to generate the corresponding Rp formula VI, and Sp formula VII. Subsequently, the absolute configuration of the isomer of Rp formula VI and Sp formula VII was determined based upon the known susceptibility to hydrolytic degradation of Rp -dinucleotide phosphorothioate (Burgers, P. M. and Eckstein, F.

Proc.Natl.Acad.Sci., USA, 75, 4798-4800, 1978) towards snake venom phosphodiesterase (PDE) and <Sp-isomer by PI Nuclease. The hydrolysis can be conveniently monitored by UPLC.

formula II ₍Sp isomer)

(Rp/Sp mix) formula VI ₍Rp isomer) formula VII

phosphodiesterase

Degrades quickly Minimal degradation under assay conditions

[00255] Procedure: A solution of Rp,Sp isomer mix of formula II was incubated in mouse serum (Sigma) (1 mg in 100 uL of serum in 0.1 M phosphate buffer, pH 7.0) at 37 °C. After several hours, when complete hydrolysis to Rp formula VI and Sp formula VII occurred (as monitored by HPLC of aliquots), MeOH was added to precipitate the proteins. The supernatant was evaporated in vacuo to obtain a mix of Rp formula VI and Sp formula VII which was used for the next step.

[00256] A solution of Rp formula VI and Sp formula VII obtained as above was incubated with PDE at 37 °C (10: 1, substrate: enzyme) (Phosphodiesterase I from Crotalus adamanteus 2 mg/ml Sigma) in 0.1 M Tris buffer, 0.02 M CaCl₂, pH 8.9). Aliquots of the reaction mixture was processed at different time points and analyzed by reversed-phase HPLC. Under these

conditions, the peak, which disappears, was expected to be the Rp-isomer formula VI. In HPLC, the early eluting peak is significantly reduced in area by 72 h due to enzymatic digestion and therefore is the Rp-isomer formula VI. Accordingly, the corresponding analog formula I should have the Rp-configuration. By analogy, the later eluting peak is therefore the Sp isomer formula VII.

[00257] The Rp isomer of a dinucleoside phosphorothioate is known to be susceptible towards PDE digestion. Example 11. Preparation of suitable salts of Formula I

Salt of formula I

formula I

FT = CI , HS0₄ , Me-S0₃ , pTol-SOs , ^PhS0₃ '

[00258] Method 1 : lg (1.42 mmol) of formula I was weighed out in a 250mL round bottomed flask containing a stir bar. Acetonitrile (40 mL) was added and stirred for lOmin. N- methyl 2-pyrrolidone (NMP, 4 mL) was slowly added and stirred for 30min to form a clear solution. In a lOmL vial para toluene sulfonic acid (leq, 270mg, 1.42 mmol) was taken in 4mL acetonitrile and 0.5mL NMP was added and stirred for 10 min to form a clear solution. T his solution was added to the above formula I solution and formation of clear solution was noticed. The solution was stirred for an additional 2h. After that time MTBE (40 mL) was slowly added to the above solution. Precipitation was noticed, and the solution was stirred for lh. Then the solution was kept in the refrigerator for 12h (-30°C). The clear supernatant solution was decanted, and the remaining precipitate was kept under high vacuum for 12h. The solid formed was scratched, and the solid was powdery and crystalline in nature. Yield : 74% (920mg) , NMR shows 1 : 1 ratio of drug:salt formation. Final pTSA salt appears as semi crystalline.

[00259] HC1 and H₂S0₄ salts are synthesized utilizing this method. ³¹P NMR of all the salts are consistent with the structure. ³¹P-NMR, DMSO_d6, £>, 26 ppm. The purity of isolated salt was determined by ³¹P and FIPLC and most of them found to be > 91%. Solubility of the salt in water was greater than 20 mg/ml and pH of this water solution was found to be around 3-3.5.

[00260] Method 2 : 250mg (0.3 mmol) of formula I was weighed out in a 100 mL round bottomed flask containing a stir bar. Acetonitrile (20 mL) and acetone (20 mL) were added and stirred for lh. After forming a clear solution, 1M H₂S0₄ in acetonitrile [leq, freshly prepared] was added slowly into the above solution. The solution was stirred for 12h. After that time

MTBE (20 mL) was slowly added to the above solution. Precipitation was noticed, and the solution was stirred for lh. The clear supernatant solution was decanted, and the remaining precipitate was washed with ethyl acetate (15mL). After decanting, the precipitate was kept under vacuum for 12h. The solid formed was free flowing powder. Yield 85%.

[00261] Benzene sulfonic acid salt was prepared utilizing this method. ³¹P NMR of all the salts are consistent with the structure. ³¹P- MR, DMSO_d6,£>,26 ppm. The purity of isolated salt was determined by ³¹P and HPLC and most of them found to be > 91%. Solubility of the salt in water was greater than 20 mg/ml and the pH of this water solution was found to be around 3-3.5.

[00262] Method 3 : 250mg (0.3 mmol) of formula I was weighed out in a 100 mL round bottomed flask containing a stir bar. Acetonitrile (20 mL) and tetrahydrofuran(20 mL) were added and stirred for lh. After forming a clear solution, 1M H₂SO₄ in tetrahydrofuran [leq, freshly prepared] was added slowly into the above solution. The solution was stirred for 12h. After that time MTBE (20 mL) was slowly added to the above solution. Precipitation was noticed, and the solution was stirred for lh. The clear supernatant solution was decanted, and the remaining precipitate was washed with ethyl acetate (15mL). After decanting, the precipitate was kept under vacuum for 12h. The solid formed was free flowing powder and not crystalline in nature. Yield 85%. ³¹P NMR is consistent with the structure. Solubility: lOmg in 0.5mL forms a clear solution with pH of around 3-3.5.

[00263] Method 4 : 300mg (0.42 mmol) of formula I was weighed out in a 10 mL vial containing a stir bar. N-methyl 2-pyrrolidone (1 mL) was added and the mixture was stirred for lh to get clear solution. Tetrahydrofuran (3 mL) was added. 1M H₂SO₄ in tetrahydrofuran

[leq,freshly prepared] was added slowly into the above solution. The solution was stirred for 12h. The solution was concentrated and was slowly added to MTBE (20 mL) and stirred for 20min. The white solid precipitated out was filtered immediately and washed with lOmL MTBE. The precipitate was kept under vacuum for 12h. The solid formed was free flowing powder, semi crystalline and slightly hygroscopic in nature. Yield 90%. [00264] Para-toluene sulfonic acid and benzene sulfonic acid salts were prepared in a similar way utilizing this procedure. ³¹P NMR of all the salts are consistent with the structure. ³¹P- NMR, DMSO_d6,<5;26 ppm. The purity of isolated salt was determined by ³¹P and HPLC and most of them found to be > 91%. Solubility of the salt in water was greater than 20 mg/ml and the pH of this water solution was found to around 3-3.5 [00265] Method 5: A solution of formula I (1 g, 1.42 mmol) in N-methyl 2-pyrrolidone (NMP, 4 ml) was diluted with THF:acetone (1 : 1, 15ml) and was cooled in ice cold water (0- 5°C). Methane sulfonic acid (150 mg, 1.49 mmol, 1.05 eq) was added as a solution in

THF:acetone mixture (1.5 ml). Additional solvent mixture (THF -acetone) was added as needed to obtain a clear solution. The reaction mixture, in the cold-water bath, was stirred as such for 6 h. At the end of the period, the solvent was removed in a rotavap. The residual oil, containing the salt formed, was washed with EtOAc (3X 10 ml) and stirred overnight with EtOAc (20 ml). The white solid which separated was filtered, washed with EtOAc (5 ml) and dried under high vacuum overnight (0.8 g). The purity of isolated salt was determined by ³¹P and HPLC and found to be ca. 96%. Solubility of this salt in water was greater than 20 mg/ml. ³¹P- MR, DMSO_d6, 26 ppm.

EQUIVALENTS

[00266] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this disclosure has been described with reference to specific aspects, it is apparent that other aspects and variations may be devised by others skilled in the art without departing from the true spirit and scope of the disclosure. The appended claims are intended to be construed to include all such aspects and equivalent variations. Any patent, publication, or other disclosure material, in whole or in part, that is said to be incorporated by reference herein is incorporated herein only to the extent that the incorporated material does not conflict with existing definitions, statements, or other disclosure material set forth in this disclosure. As such, and to the extent necessary, the disclosure as explicitly set forth herein supersedes any conflicting material incorporated herein by reference.

[00267] While this disclosure has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the disclosure encompassed by the appended claims.

Claims

What is claimed is:

1. A method of making a crystalline compound of formula I:

(Rp isomer), the method comprising crystallization from a solubilized form of a compound of formula II:

or slurry conversion.

2. The method of claim 1, wherein the crystallization is performed using water.

3. The method of claim 1 or 2, wherein the compound of formula II is dissolved in water or in a slurry in water.

4. The method of any one of claims 1-3, wherein the crystallization is performed at a temperature from about 60 °C to about 90 °C (e.g., from about 70 °C to about 85 °C, from about 75 °C to about 80 °C).

5. The method of any one of claims 1-4, wherein the crystallization is recrystallization.

6. A crystalline compound of formula I:

isomer), or a pharmaceutically acceptable salt thereof, wherein the compound is at least 90%

7. The crystalline compound of claim 6, wherein the ambient humidity is between about 20% to about 80%) relative humidity.

8. The crystalline compound of claim 6 or 7, wherein the compound is a pharmaceutically acceptable salt of a compound of formula I.

9. The crystalline compound of any one of claims 6-8, wherein the pharmaceutically acceptable salt is selected from the group consisting of: a hemi-tartrate salt, an oxalate salt, a citrate salt, and a fumarate salt.

10. The crystalline compound of claim 9, wherein the pharmaceutically acceptable salt is a hemi-tartrate salt.

1 1. The crystalline compound of any one of claims 6-10, wherein the compound is at least about 95% diastereomerically pure, e.g., as measured by ³¹P NMR.

12. The crystalline compound of any one of claims 6-1 1, wherein the compound has an XRPD pattern with characteristic peaks at the following values of 2Θ in degrees: 7.6, 13.2, 15.2, 16.7, 18.9, and 21.3.

13. The crystalline compound of any one of claims 6-12, wherein the compound has an XRPD pattern with characteristic peaks at the following values of 2Θ in degrees: 7.6, 10.7, 12.2, 13.2, 14.2, 15.2, 16.7, 17.8, 18.1, 18.9, 20.3, 21.0, 21.3, 22.0, 23.4, and 24.3.

14. The crystalline compound of any one of claims 6-13, wherein the compound has an XRPD pattern substantially as depicted in Figure 9.

15. The crystalline compound of any one of claims 6-14, wherein the crystalline compound is produced by crystallization from a solubilized form of a compound of formula II:

or slurry conversion.

16. The crystalline compound of claim 15, wherein the crystallization is performed using water.

17. The crystalline compound of claim 15, wherein the compound of formula II is dissolved in water or in a slurry in water.

18. The crystalline compound of any one of claims 15-17, wherein the crystallization is recrystallization.

19. A compound of formula III:

(Rp isomer), or a pharmaceutically acceptable salt thereof.

20. The compound of claim 19, wherein the compound is crystalline.

21. The compound of claim 19 or 20, wherein the compound is stable at about 25 °C at ambient humidity for at least 1 week (e.g., at least 2 weeks, at least 4 weeks, at least 2 months, 3 months, at least 6 months, at least 1 year).

22. The compound of claim 21, wherein the ambient humidity is between about 20% to about 80%) relative humidity.

23. The compound of any one of claims 19-22, wherein the ammonium ion of the compound of formula I:

(Rp isomer), in the compound of formula III is at least 90% diastereomerically pure, e.g., as measured by ³¹P NMR.

24. The compound of claim 23, wherein the ammonium salt of the compound of formula I is at least 95% diastereomerically pure, e.g., as measured by ³¹P NMR.

25. A composition comprising a crystalline compound of formula I:

(Rp isomer), or a pharmaceutically acceptable salt thereof, wherein the compound is at least 90%

diastereomerically pure, e.g., as measured by ³¹P NMR, wherein the compound is stable at about 25 °C at ambient humidity for at least 1 week (e.g., at least 2 weeks, at least 4 weeks, at least 2 months, 3 months, at least 6 months, at least 1 year).

26. The composition of claim 25, wherein the ambient humidity is between about 20% to about 80%) relative humidity.

27. The composition of claim 25 or 26, wherein the composition further comprises a pharmaceutically acceptable excipient.

28. The composition of any one of claims 25-27, wherein the composition is substantially free of chemical impurities.

29. The composition of claim 28, wherein the chemical impurity is

30. The composition of any one of claims 25-29, wherein the composition is substantially free of the compound of formula V :

31. The composition of any one of claims 25-30, wherein the composition is substantially free of non-crystalline or amorphous forms of the compound of formula I.

32. The composition of any one of claims 25-31, wherein the purity of the compound remains over 95% pure as measured by HPLC for at least 1 week (e.g., at least 2 weeks, at least 4 weeks, at least 2 months, 3 months, at least 6 months, at least 1 year).

33. The composition of any one of claims 25-32, wherein the compound of formula I is at least 95% diastereomerically pure, e.g., as measured by ³¹P MR.

34. The composition of any one of claims 25-33, wherein the compound has an XRPD pattern with characteristic peaks at the following values of 2Θ in degrees: 7.6, 13.2, 15.2, 16.7, 18.9, and 21.3.

35. The composition of any one of claims 25-34, wherein the compound has an XRPD pattern with characteristic peaks at the following values of 2Θ in degrees: 7.6, 10.7, 12.2, 13.2, 14.2, 15.2, 16.7, 17.8, 18.1, 18.9, 20.3, 21.0, 21.3, 22.0, 23.4, and 24.3.

36. The composition of any one of claims 25-35, wherein the compound has an XRPD pattern substantially as depicted in Figure 9.

37. A composition comprising a compound of formula III:

(Bp isomer), or a pharmaceutically acceptable salt thereof.

38. The composition of claim 37, wherein the composition further comprises a compound of formula IV:

stereochemistry at each phosphorus).

39. The composition of claim 37 or 38, wherein the compound of formula III is crystalline.

40. The composition of claim 38 or 39, wherein the compound of formula IV is crystalline.

41. The composition of any one of claims 37-40, wherein the ammonium ion of the compound of formula I:

42. The composition of claim 41, wherein the ambient humidity is between about 20% to about 80%) relative humidity.

43. The composition of any one of claims 37-42, wherein the composition further comprises a pharmaceutically acceptable excipient.

44. The composition of any one of claims 37-43, wherein the composition is substantially free of chemical impurities.

45. The composition of claim 44, wherein the chemical impurity is

and/or

46. The composition of any one of claims 37-45, wherein the composition is substantially free of the compound of formula V:

(Sp isomer) or a salt thereof.

47. The composition of any one of claims 37-46, wherein the composition is substantially free of non-crystalline or amorphous forms of the compound of formula I.

48. The composition of any one of claims 37-47, wherein the purity of the compound remains over 95% pure as measured by HPLC for at least 1 week (e.g., at least 2 weeks, at least 4 weeks, at least 2 months, 3 months, at least 6 months, at least 1 year).

49. The composition of any one of claims 37-48, wherein the compound of formula I is at least 95% diastereomerically pure, e.g., as measured by ³¹P NMR.

50. A composition comprising: a crystalline compound of formula I:

(Rp isomer), or a pharmaceutically acceptable salt thereof, and tenofovir or a prodrug thereof, such as tenofovir dipivoxil and tenofovir alafenamide, wherein the compound of formula I is at least 90% diastereomerically pure, e.g., as measured by ³¹P NMR, wherein the compound of formula I and tenofovir are stable at about 25 °C at ambient humidity for at least 1 week (e.g., at least 2 weeks, at least 4 weeks, at least 2 months, 3 months, at least 6 months, at least 8 months, at least 10 months).

51. The composition of claim 50, wherein the ambient humidity is between about 20% to about 80%) relative humidity.

52. The composition of claim 50 or 51, wherein the composition further comprises a pharmaceutically acceptable excipient.

53. The composition of any one of claims 50-52, wherein the composition is substantially free of chemical impurities.

54. The composition of claim 53, wherein the chemical impurity is

and/or

55. The composition of any one of claims 50-54, wherein the composition is substantially free of the compound of formula V:

(Sp isomer).

56. The composition of any one of claims 50-55, wherein the composition is substantially free of non-crystalline or amorphous forms of the compound of formula I.

57. The composition of any one of claims 50-56, wherein the purity of the compound remains over 95% pure as measured by HPLC for at least 1 week (e.g., at least 2 weeks, at least 4 weeks, at least 2 months, 3 months, at least 6 months, at least 1 year).

58. The composition of any one of claims 50-57, wherein the compound of formula I is at least 95%) diastereomerically pure, e.g., as measured by ³¹P MR.

59. The composition of any one of claims 50-58, wherein the compound has an XRPD pattern with characteristic peaks at the following values of 2Θ in degrees: 7.6, 13.2, 15.2, 16.7, 18.9, and

21.3.

60. The composition of any one of claims 50-59, wherein the compound has an XRPD pattern with characteristic peaks at the following values of 2Θ in degrees: 7.6, 10.7, 12.2, 13.2, 14.2, 15.2, 16.7, 17.8, 18.1, 18.9, 20.3, 21.0, 21.3, 22.0, 23.4, and 24.3.

61. The composition of any one of claims 50-60, wherein the compound has an XRPD pattern substantially as depicted in Figure 9.

62. A particulate composition comprising: a cr stalline compound of formula I:

isomer), or a pharmaceutically acceptable salt thereof, and tenofovir or a prodrug thereof, such as tenofovir dipivoxil or tenofovir alafenamide, wherein the compound of formula I is at least 90% diastereomerically pure, e.g., as measured by ³¹P MR, wherein the compound of formula I and tenofovir are stable at about 25 °C at ambient humidity for at least 1 week (e.g., at least 2 weeks, at least 4 weeks, at least 2 months, 3 months, at least 6 months, at least 8 months, at least 10 months).

63. The particulate composition of claim 62, wherein the ambient humidity is between about 20% to about 80%) relative humidity.

64. The particulate composition of claim 62 or 63, wherein the purity of the compound of formula I and tenofovir remain over 95% pure as measured by HPLC for at least 1 week (e.g., at least 2 weeks, at least 4 weeks, at least 2 months, 3 months, at least 6 months, at least 8 months, at least 10 months).

65. The particulate composition of any one of claims 62-64, wherein the compound of formula I is at least 95% diastereomerically pure, e.g., as measured by ³¹P NMR.

66. The particulate composition of any one of claims 62-65, wherein the compound has an XRPD pattern with characteristic peaks at the following values of 2Θ in degrees: 7.6, 13.2, 15.2, 16.7, 18.9, and 21.3.

67. The particulate composition of any one of claims 62-66, wherein the crystalline compound has an XRPD pattern with characteristic peaks at the following values of 2Θ in degrees: 7.6, 10.7, 12.2, 13.2, 14.2, 15.2, 16.7, 17.8, 18.1, 18.9, 20.3, 21.0, 21.3, 22.0, 23.4, and 24.3.

68. The particulate composition of any one of claims 62-67, wherein the compound has an XRPD pattern substantially as depicted in Figure 9.

69. A pharmaceutical composition comprising: a crystalline compound of formula I:

(Rp isomer), or a pharmaceutically acceptable salt thereof, and tenofovir or a prodrug thereof, such as tenofovir dipivoxil or tenofovir alafenamide, wherein the compound of formula I is at least 90% diastereomerically pure, e.g., as measured by ³¹P MR, wherein the compound of formula I and tenofovir are stable at about 25 °C at ambient humidity for at least 1 week (e.g., at least 2 weeks, at least 4 weeks, at least 2 months, 3 months, at least 6 months, at least 8 months, at least 10 months).

70. The pharmaceutical composition of claim 69, wherein the ambient humidity is between about 20% to about 80% relative humidity.

71. The pharmaceutical composition of claim 69 or 70, wherein the purity of the compound of formula I and tenofovir remain over 95% pure, e.g., as measured by HPLC for at least 1 week (e.g., at least 2 weeks, at least 4 weeks, at least 2 months, 3 months, at least 6 months, at least 8 months, at least 10 months).

72. The pharmaceutical composition of any one of claims 69-71, wherein the compound of formula I is at least 95% diastereomerically pure, e.g., as measured by ³¹P NMR.

73. The pharmaceutical composition of any one of claims 69-72, wherein the compound has an XRPD pattern with characteristic peaks at the following values of 2Θ in degrees: 7.6, 13.2, 15.2, 16.7, 18.9, and 21.3.

74. The pharmaceutical composition of any one of claims 69-73, wherein the compound has an XRPD pattern with characteristic peaks at the following values of 2Θ in degrees: 7.6, 10.7, 12.2, 13.2, 14.2, 15.2, 16.7, 17.8, 18.1, 18.9, 20.3, 21.0, 21.3, 22.0, 23.4, and 24.3.

75. The pharmaceutical composition of any one of claims 69-74, wherein the compound has an XRPD pattern substantially as depicted in Figure 9.

76. The pharmaceutical composition of any one of claims 69-75, further comprising a pharmaceutically acceptable excipient.

77. A solid oral dosage form comprising the compound of any one of claims 6-24.

78. A solid oral dosage form comprising the particulate composition of any one of claim 62-68.

79. The solid oral dosage form of claim 77 or 78, wherein the solid oral dosage form comprises a pharmaceutically acceptable excipient.

80. A method of treating a viral infection in a patient in need thereof, the method comprising administering a therapeutically effective amount of the compound of any one of claims 6-24 or the composition of any one of claims 25-76.

81. The method of claim 80, wherein the viral infection is caused by a virus selected from the group consisting of HBV, HCV, RSV, norovirus, and influenza virus.

82. The method of claim 80 or 81, wherein the virus is HBV.

83. A method of treating or preventing a proliferative disorder or cancer in a patient in need thereof, the method comprising administering a therapeutically effective amount of the compound of any one of claims 6-24 or the composition of any one of claims 25-76.

84. The method of claim 83, wherein the proliferative disorder or cancer is associated with a viral infection.

85. The method of claim 84, wherein the viral infection is caused by HBV or HCV.

86. The method of any one of claims 83-85, wherein the cancer is a liver cancer.

87. The crystalline compound of claim 15, wherein the crystallization is performed using N- methylpyrrolidone.