WO2016044243A1 - Pyrimidine nucleoside phosphoramidate - Google Patents

Abstract

The disclosure describes methods of synthesis of phosphoramidate ester derivatives. Also disclosed are morphic forms of (S)-isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(5-deutero-2,4- dioxo-3,4-dihydropyrimidin- 1(2H)-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)dideuteromethoxy)-(phenoxy)phosphoryl)amino)propanoate and antiviral therapy using (S)- isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(5-deutero-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)dideuteromethoxy)(phenoxy)phosphoryl)amino)- propanoate (e.g., anti-HCV therapies).

Description

PYRIMIDINE NUCLEOSIDE PHOSPHORAMIDATE

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 62/051,290 filed September 16, 2014. The entirety of this application is hereby incorporated by reference for all purposes.

FIELD

This invention is in the area of isolated morphic forms of, and uses of, (S)-isopropyl 2- (((S)-(((2R,3R,4R,5R)-5-(5-deutero-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)-3,4-dihydroxy-4- methyltetrahydrofuran-2-yl)dideuteromethoxy)-(phenoxy)phosphoryl)amino)propanoate

(Structure (1A)), also named as 1-methylethyl N-[(S)-{[(2R,3R,4R,5R)-5-(2,4-dioxo-3,4- dihydro(5 -2H)pyrimidin- 1 (2H)-yl)-3 ,4-dihydroxy-4-methyltetrahydrofuran-2- yl] (2H2)methoxy } phenoxyphosphoryl] -L-alaninate.

BACKGROUND

An estimated 3% of the world's population is infected with the hepatitis C virus. The World Health Organization estimates that 150 million people are chronically infected worldwide. Of those exposed to HCV, 80% to 85% become chronically infected, at least 30%> develop cirrhosis of the liver and 1-4% develop hepatocellular carcinoma. Hepatitis C Virus (HCV) is one of the most prevalent causes of chronic liver disease in the United States, reportedly accounting for about 15 percent of acute viral hepatitis, 60 to 70%> of chronic hepatitis, and up to 50%) of cirrhosis, end-stage liver disease, and liver cancer. Chronic HCV infection is the most common cause of liver transplantation in the U.S., Australia, and most of Europe. Hepatitis C causes an estimated 10,000 to 12,000 deaths annually in the United States. While the acute phase of HCV infection is usually associated with mild symptoms, some evidence suggests that only about 15% to 20%> of infected people will spontaneously clear HCV.

U.S. 2014/0309189, titled "Highly Active Nucleoside Derivative for the Treatment of HCV" and assigned to Achillion Pharmaceuticals, describes a nucleotide NS5b inhibitor 2'- methyl 5'-deuterated uridine phosphoramidate (Formula II), and various derivatives thereof, having the structure:

According to U.S. Pub. No. 2014/0309189, it has surprisingly been discovered that deuterium in the 5 '-position of the nucleoside stabilizes the nucleoside derivative from dephosphorylation to the undesired 5'-OH, 5'-deuterated-nucleoside. This is surprising because the deuterium atom(s) are not cleaved during dephosphorylation and are not bound to an atom that is cleaved during dephosphorylation. The disclosure includes the use of 5 '-deuterium to produce a significant effect on metabolism and efficacy through a remote and unexpectedly important secondary deuterium isotope effect. Such an important secondary deuterium isotope effect on de-monophosphorylation at the 5 '-position had not been previously reported. By increasing the stability of the 5 '-monophosphate of the nucleoside against dephosphorylation, an increase in the active 5 '-triphosphate pool of the nucleoside can be achieved, which can result in increased efficacy at a given oral dosage or equal efficacy using a lower dose of the nucleoside in the clinic. It may also have a significant effect on the half-life, and thus pharmacokinetics, of the drug.

US 2014/0309164, titled "Deuterated Nucleoside Prodrugs Useful for Treating HCV" and assigned to Achillion Pharmaceuticals, describes 5 '-deuterated nucleosides and nucleotides and uses thereof for treating a host afflicted with a disorder that is treatable with a nucleoside or nucleotide, wherein one or both of the hydrogens at the 5 '-position of the nucleoside or nucleotide is substituted with a deuterium with at least 90% enrichment over protium (i.e., less than 10% 1H hydrogen) (and in other embodiments, 50, 95, 98 or 99% enrichment).

U.S. Application No. 14/805,421, titled "Stabilized Nucleotides for Medical Treatment" and assigned to Achillion Pharmaceuticals, discloses the use of 5 '-deuterium substitutions to stabilize phosphate prodrugs. Examples of stabilized 5 '-deuterated phosphate nucleotide prodrugs include phosphoramidates, phosphate esters, diesters, and triesters, 3 ',5 '-cyclic phosphates, SATE derivatives (bis-(S-acyl-2-thioester)s), 3 ',5 '-cyclic phosphoramidates, phospholipids, Bis-SATE and bis-DTE prodrugs, mixed phosphotriesters containing mono-aryl or mono-amidate moiety in combination with a mono-SATE, cyclic phosphates such as cyclosaligenyl (CycloSAL) and cyclic l-aryl-l ,3-propanyl ester (HepDirect) prodrugs, diphosphate and triphosphate prodrugs, acylphospholipid nucleoside derivatives of monophosphates, diphosphates, and triphosphates; ether lipid-nucleoside covalent conjugates, and protein conjugates.

It is known that effective treatment against hepatitis C includes combination therapy, due to the onset of viral resistance during monotherapy. Given the documented challenges of developing optimal hepatitis C agents, and the fact that multiple optimal agents are required for effective therapy, there is a strong need for additional hepatitis C agents.

SUMMARY OF THE INVENTION

The present invention generally provides isolated morphic forms of (S)-isopropyl 2-(((S)- (((2R,3R,4R,5R)-5-(5-deutero-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)-3,4-dihydroxy-4- methyltetrahydrofuran-2-yl)dideuteromethoxy)-(phenoxy)phosphoryl)amino)propanoate

(Structure (1A)), also named as 1-methylethyl N-[(5)- {[(2i?,3i?,4i?,5i?)-5-(2,4-dioxo-3,4- dihydro(5 -²H)pyrimidin- 1 (2H)-yl)-3 ,4-dihydroxy-4-methyltetrahydrofuran-2- yl](²H₂)methoxy}phenoxyphosphoryl]-L-alaninate, pharmaceutical compositions containing such morphic forms, methods of inhibiting or reducing the activity of a virus or neoplasm in a host using said isolated morphic forms, and treating a host having a viral infection such as, for example, a flavivirus including HCV or a neoplastic disorder using the morphic forms described herein, and methods of p

Structure 1A In one aspect, the present invention is directed to isolated morphic Form A of (S)- isopropyl 2-(((S)-(((2R,3R,4R,5R)-5 5-deutero-2,4-dioxo-3,4-di ydrop

dihydroxy-4-methyltetrahydrofuran-2- yl)dideuteromethoxy)(phenoxy)phosphoryl)amino)propanoate (Form A), and uses and methods of making thereof. In one embodiment, isolated Form A is characterized by an X-ray powder diffraction (XRPD) pattern comprising at least the 2theta values of 16.75+0.2° and 19.19+0.2°. In another embodiment, isolated morphic Form A is characterized by an X-ray powder diffraction (XRPD) pattern comprising the 2theta values of 6.83±0.2°, 10.12+0.2°, 13.72+0.2°, 16.75±0.2°, 17.68+0.2°, 19.19±0.2°, 19.53+0.2°, 21.90±0.2°, and 26.28+0.2°. In one embodiment, isolated morphic Form A is characterized as having a differential scanning calorimetry (DSC) onset endotherm of about 137 °C.

Also provided is a method for preparing isolated Form A using selective crystallization as described further below.

In another aspect, the present invention is directed to isolated morphic Form B of (S)- isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(5-deutero-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)dideuteromethoxy)(phenoxy)phosphoryl)- amino)propanoate (Form B), and uses and methods of making thereof. In one embodiment, isolated Form B is characterized by an X-ray powder diffraction (XRPD) pattern comprising at least the 2theta values of 16.41+0.2° and 19.05+0.2°. In another embodiment, isolated Form B is characterized by an X-ray powder diffraction (XRPD) pattern comprising the 2theta values of 9.64+0.2°, 10.57±0.2°, 13.89±0.2°, 16.41+0.2°, 16.77±0.2°, 19.05±0.2°, 19.55±0.2° and 22.37+0.2°. In one embodiment, isolated Form B is characterized as having a differential scanning calorimetry (DSC) onset endotherm of about 134 °C.

Also provided is a method for preparing isolated Form B using selective crystallization as described further below.

In another aspect, the present invention is directed to isolated morphic Form C of (S)- isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(5-deutero-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)dideuteromethoxy)(phenoxy)phosphoryl)- amino)propanoate (Form C), and uses and methods of making thereof. In one embodiment, isolated Form C is characterized by an XRPD pattern comprising 2theta values of 10.12+0.2°, 16.66±0.2°, 16.85±0.2°, 18.23±0.2°, 19.22+0.2°, 20.15±0.2°, 22.07±0.2° and 23.11+0.2°. Also provided is a method for preparing Form C using selective crystallization as described further below.

In another aspect, the present invention is directed to isolated morphic Form D of (S)- isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(5-deutero-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)dideuteromethoxy)(phenoxy)phosphoryl)- amino)propanoate (Form D), and uses and methods of making thereof. In one embodiment, isolated Form D is characterized by an XRPD pattern comprising 2theta values of 10.04+0.2°, 10.07±0.2°, 16.44±0.2°, 16.79±0.2°, 19.03±0.2°, 19.69±0.2°, 20.22±0.2°, 21.68±0.2°, and 22.60+0.2°. In one embodiment, isolated Form D is characterized by an XRPD pattern comprising at least four of the 2theta values selected from 10.04+0.2°, 10.07+0.2°, 16.44+0.2°, 16.79±0.2°, 19.03±0.2°, 19.69±0.2°, 20.22+0.2°, 21.68±0.2°, and 22.60±0.2°. In one embodiment, isolated Form D is characterized as having a DSC onset endotherm of about 137 °C.

Also provided is a method for preparing isolated Form D using selective crystallization as described further below.

In another aspect of the present invention, provided is an isolated morphic Form E of (S)- isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(5-deutero-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)dideuteromethoxy)-(phenoxy)phosphoryl)- amino)propanoate, and uses and methods of making thereof. In one embodiment, isolated Form E is characterized by an XRPD pattern comprising 2theta values of 7.88+0.2°, 10.60+0.2°, 12.35±0.2°, 17.15±0.2°, 17.79±0.2°, 17.86±0.2°, 19.60±0.2° and 20.07±0.2°. In one embodiment, isolated Form E is characterized by an XRPD pattern comprising at least four 2theta values selected from 7.88±0.2°, 10.60±0.2°, 12.35±0.2°, 17.15±0.2°, 17.79±0.2°, 17.86+0.2°, 19.60+0.2° and 20.07+0.2°. In one embodiment, isolated Form E is characterized by a DSC onset endotherm of about 124 °C.

Also provided is a method of preparing isolated Form E using selective crystallization as described further below.

In alternative embodiments, an isolated combination of two morphic forms is provided, such as Forms D and A; D and B; D and C; B and C; or A and C. In another alternative embodiment, an isolated combination of three morphic forms is provided, for example, Forms D, A and B; D, B and C; Forms D, A and C; and Forms A, B and C. In one embodiment, provided is an amorphous form of (S)-isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(5-deutero-2,4-dioxo-3,4- dihydropyrimidin- 1 (2H)-yl)-3 ,4-dihydroxy-4-methyltetrahydrofuran-2- yl)dideuteromethoxy)(phenoxy)phosphoryl)amino)propanoate.

In one embodiment a pharmaceutical composition is provided comprising isolated morphic Form A, B, C, D, or E and a pharmaceutically acceptable excipient. In another embodiment, the pharmaceutical composition further comprised one or more additional therapeutic agents, for example but not limited to, a caspase inhibitor, a cyclophilin inhibitor, a cytochrome P450 monooxygenase inhibitor, an entry inhibitor, a glucocorticoid, an HCV protease inhibitor, a hematopoietin, a homeopathic therapy, an immunomodulatory compound, an immunosuppressant, an interleukin, an interferon or interferon enhancer, an IRES inhibitor, a monoclonal or polyclonal antibody, a nucleoside or nucleotide analogue or prodrug, a non- nucleoside inhibitor, an NS4B inhibitor, an NS5A inhibitor, an NS5B inhibitor, a P7 protein inhibitor, a polymerase inhibitor, an RNAi compound, a therapeutic vaccine, a TNF agonist, a tubulin inhibitor, a sphingosine-1 -phosphate receptor modulator, or a TLR agonist. In one embodiment the one or more additional therapeutic agents include an HCV NS3 protease inhibitor and/or an NS5A inhibitor. In one embodiment, the one or more additional therapeutic agents are selected from the following:

Sovaprevir, NS3 inhibitor, or

NS5A inhibitor.

or a combination thereof.

In one aspect of the present invention, a method for treating a hepatitis C virus (HCV) infection is provided comprising administering to a host in need thereof a therapeutically effective amount of an isolated morphic form of (5)-isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(5- deutero-2,4-dioxo-3 ,4-dihydropyrimidin- 1 (2H)-yl)-3 ,4-dihydroxy-4-methyltetrahydrofuran-2- yl)dideuteromethoxy)(phenoxy)phosphoryl)amino)propanoate selected from isolated Form A, B, C, D, or E. In an alternative embodiment, a mixture limited to two morphic forms is used. In yet another embodiment, a mixture limited to three morphic forms is used.

In one embodiment, the selected isolated morphic form of (5)-isopropyl 2-(((S)- (((2R,3R.4R.5R)-5 5-deutero-2,^

methyltetrahydrofuran-2-yl)dideuteromethoxy)(phenoxy)phosphoryl)amino)propanoate is administered in a pharmaceutical composition. In a further embodiments, the method comprises administering to the subject one or more additional therapeutic agents wherein the one or more additional therapeutic agents are selected from a caspase inhibitor, a cyclophilin inhibitor, a cytochrome P450 monooxygenase inhibitor, an entry inhibitor, a glucocorticoid, an HCV protease inhibitor, a hematopoietin, a homeopathic therapy, an immunomodulatory compound, an immunosuppressant, an interleukin, an interferon or interferon enhancer, an IRES inhibitor, a monoclonal or polyclonal antibody, a nucleoside or nucleotide analogue or prodrug, a non- nucleoside inhibitor, an NS4B inhibitor, an NS5A inhibitor, an NS5B inhibitor, a P7 protein inhibitor, a polymerase inhibitor, an RNAi compound, a therapeutic vaccine, a TNF agonist, a tubulin inhibitor, a sphingosine-1 -phosphate receptor modulator, or a TLR agonist. In one embodiment, the one or more additional therapeutic agents are selected from the following:

NS5A inhibitor.

or a combination thereof.

Also provided is the use of isolated morphic Form A, B, C, D, or E, in the manufacture of a medicament for treating a viral infection, such as a flavivirus, including an HCV infection, or a neoplasm or other abnormal cellular proliferation in a host.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical structure for Structure 1 A.

FIG. 2 illustrates the X-ray powder diffraction (XRPD) pattern for Structure 1A lot 3.

FIG. 3 illustrates the thermal data for Structure 1A lot 3.

FIG. 4 illustrates the modulated DSC data for Structure 1A lot 3.

FIG. 5 shows the 1H NMR spectrum for Structure 1 A, lot 7.

FIG. 6 shows the indexing results for the XRPD file for Form A, lot 285-183.

FIG. 7 shows the thermal data for Form A, lot 258-91-2.

FIG. 8 illustrates the thermal data for Form B, lot 285: 179-180.

FIG. 9 illustrates the XRPD pattern for Form B.

FIG. 10 illustrates the XRPD pattern for Form C.

FIG. 11 shows the XRPD indexing for Form D, lot 285-181-1. FIG. 12 illustrates the thermal data for Form D, lot 285-181-1. TGA data indicate that the sample is anhydrous. A sharp endothermic transition with an onset at about 137 °C was attributed to melting, based on hot stage microscopy.

FIG. 13 illustrates the DVS data for Form D, lot 285-181-1. The data indicate Form D is not hygroscopic, showing less than 0.1 wt% change throughout the entire experiment, between 5% and 95% RH. XRPD analysis of the sample following the DVS experiment indicated it remained Form D.

FIG. 14 shows the XRPD indexing data for Form E, Sample No. 5447-34-01.

FIG. 15 shows the 1H NMR spectrum for Form E, Sample No. 5447-34-01.

FIG. 16 illustrates the thermal data for Form E, Sample No. 5447-34-01.

DETAILED DESCRIPTION OF THE INVENTION

It has been discovered that Structure 1A can exist in different polymorphic forms. Polymorphism is the ability of a compound to exist in more than one crystal form. An amorphous material is a non-crystalline solid that lacks the long-range order of a crystal. An amorphous solid was historically referred to as a glass, yet more precisely can be a solid that exhibits a glass transition temperature.

Various crystal forms of the same compound can exhibit different properties. They can have different stabilities, melting points or solubilities (which can affect the dissolution rate of the drug and thus its pharmacokinetics). Crystallization can be affected by the process of manufacture such as speed of crystallization, temperature, solvent effects, impurities, super- saturation or even changes in agitation during the process. Polymorphic forms can also be varied if solvent molecules are incorporated into the packing, creating a polymorph solvate. It is sometimes true that the least stable polymorph is the first formed. Alternatively, a situation can occur that one morphic form is more stable at one temperature range and another morphic form is stable at a different temperature range.

Pharmaceutical drugs are often administered as a crystalline solid and thus whether a compound exists in polymorphic forms and, if so, the properties of the polymorphs, are quite important to drug delivery. As one extreme example, the drug ritonavir is active in one polymorphic form and inactive in another form, and the inactive form is the more stable and converts the active form to the inactive form. It cannot be predicted in advance whether a compound is a polymorph and can exist in various isolated crystal forms. It also cannot be predicted in advance which polymorph is best suited for drug delivery or provides the optimal pharmacokinetics.

Generally, different polymorphs can be characterized by analytical methods such as X- ray powder diffraction pattern (XRDP), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC), or by its melting point, or other techniques known in the art.

Morphic Form A

Isolated Morphic Form A of (S)-isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(5-deutero-2,4- dioxo-3 ,4-dihydropyrimidin- 1 (2H)-yl)-3 ,4-dihydroxy-4-methyltetrahydrofuran-2- yl)dideuteromethoxy)-(phenoxy)phosphoryl)amino)propanoate is provided in this invention.

In one embodiment, Form A is characterized by an XRPD pattern comprising 2theta values of 6.83+0.2°, 10.12+0.2°, 13.72+0.2°, 16.75+0.2°, 17.68+0.2°, 19.19+0.2°, 19.53+0.2°, 21.90+0.2°, and 26.28+0.2°.

In one embodiment, Form A is characterized by an XRPD pattern comprising at least four 2theta values selected from 6.83+0.2°, 10.12+0.2°, 13.72+0.2°, 16.75+0.2°, 17.68+0.2°, 19.19+0.2°, 19.53+0.2°, 21.90+0.2°, and 26.28+0.2°.

In one embodiment, Form A is characterized by an XRPD pattern substantially similar to that set forth in FIG. 6.

In one embodiment, Form A is characterized by a DSC onset endotherm of about 137 °C.

The compound (S)-isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(5-deutero-2,4-dioxo-3,4- dihydropyrimidin- 1 (2H)-yl)-3 ,4-dihydroxy-4-methyltetrahydrofuran-2-yl)dideuteromethoxy)- (phenoxy)phosphoryl)amino)propanoate, Structure 1A, see FIG.l, can be prepared as described in the Chemical Syntheses section found below. Form A can be prepared using selective crystallization. The method can be carried out by treating a solution comprising a suitable solvent(s) and (S)-isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(5-deutero-2,4-dioxo-3,4- dihydropyrimidin- 1 (2H)-yl)-3 ,4-dihydroxy-4-methyltetrahydrofuran-2-yl)dideuteromethoxy)- (phenoxy)phosphoryl)amino)propanoate to conditions that provide for the crystallization of Form A. The selective crystallization can be carried out in any suitable solvent. For example, it can be carried out in a protic solvent or a mixture thereof. In one embodiment, the solvent comprises the protic solvent water. In another embodiment, the solvent comprises the protic solvents water and methanol. The selective crystallization can be carried out at, for example, a temperature in the range of about 5o C to about 50o C.

Methods utilized in preparing Form A are further described in the Examples section below.

Morphic Form B

Isolated Morphic Form B of (S)-isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(5-deutero-2,4- dioxo-3 ,4-dihydropyrimidin- 1 (2H)-yl)-3 ,4-dihydroxy-4-methyltetrahydrofuran-2- yl)dideuteromethoxy)-(phenoxy)phosphoryl)amino)propanoate is also provided in this invention.

In one embodiment, Form B is characterized by an XRPD pattern comprising 2theta values of 9.64+0.2°, 10.57+0.2°, 13.89+0.2°, 16.41+0.2°, 16.77+0.2°, 19.05+0.2°, 19.55+0.2° and 22.37+0.2°. In one embodiment, Form B is characterized by an XRPD pattern comprising at least four 2theta values selected from 9.64+0.2°, 10.57+0.2°, 13.89+0.2°, 16.41+0.2°, 16.77+0.2°, 19.05+0.2°, 19.55+0.2° and 22.37+0.2°. In one embodiment, Form B is characterized by an XRPD pattern substantially similar to that set forth in FIG. 9.

In one embodiment, Form B is characterized by a DSC onset endotherm of about 134 °C.

Form B can be prepared using selective crystallization. The method can be carried out by treating a solution comprising a suitable solvent(s) and (S)-isopropyl 2-(((S)-(((2R,3R,4R,5R)-5- (5-deutero-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)dideuteromethoxy)-(phenoxy)phosphoryl)amino)propanoate optionally in the presence of one or more seeds comprising Form B to conditions that provide for the crystallization of Form B. The selective crystallization can be carried out in any suitable solvent. For example, it can be carried out in an aprotic solvent or a mixture thereof. In one embodiment, the solvent comprises the aprotic solvents dichloromethane and ethyl ether. The selective crystallization can be carried out at, for example, a temperature in the range of about 20o C to about 25o C.

In another embodiment, the solvent comprises the aprotic solvent methyl tert-butyl ether (MTBE). In one embodiment, the solvent comprises the aprotic solvent methyl tert-butyl ether (MTBE) and one or more seed crystals of Form B. The selective crystallization can be carried out at, for example, a temperature in the range of about 5o C to about 55o C.

Methods utilized in preparing Form B are further described in the Examples section below. Morphic Form C

Isolated morphic Form C of (S)-isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(5-deutero-2,4- dioxo-3 ,4-dihydropyrimidin- 1 (2H)-yl)-3 ,4-dihydroxy-4-methyltetrahydrofuran-2- yl)dideuteromethoxy)-(phenoxy)phosphoryl)amino)propanoate is further provided in this invention.

In one embodiment, Form C is characterized by an XRPD pattern comprising 2theta values of 10.12+0.2°, 16.66+0.2°, 16.85+0.2°, 18.23+0.2°, 19.22+0.2°, 20.15+0.2°, 22.07+0.2° and 23.11+0.2°. In one embodiment, Form C is characterized by an XRPD pattern comprising at least four 2theta values selected from 10.12+0.2°, 16.66+0.2°, 16.85+0.2°, 18.23+0.2°, 19.22+0.2°, 20.15+0.2°, 22.07+0.2° and 23.11+0.2°. In one embodiment, Form C is characterized by an XRPD pattern substantially similar to that set forth in FIG. 10.

Form C can be prepared using selective crystallization. The method can be carried out by treating a solution comprising a suitable solvent(s) and (S)-isopropyl 2-(((S)-(((2R,3R,4R,5R)-5- (5-deutero-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)dideuteromethoxy)-(phenoxy)phosphoryl)amino)propanoate optionally in the presence of one or more seeds comprising Form B to conditions that provide for the crystallization of Form C.

The selective crystallization can be carried out in any suitable solvent. For example, it can be carried out in a protic solvent or a mixture thereof. In one embodiment, the solvent comprises the protic solvents methanol and water. The selective crystallization can be carried out at, for example, a temperature in the range of about 5 oC to ambient temperature.

In another embodiment, the solvent comprises the protic solvents methanol and water and one or more seed crystals of Form B. The selective crystallization can be carried out at, for example, a temperature in the range of about 5 oC to ambient temperature.

Methods utilized in preparing Form C are further described in the Examples section below.

Morphic Form D

Isolated Morphic Form D of (S)-isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(5-deutero-2,4- dioxo-3 ,4-dihydropyrimidin- 1 (2H)-yl)-3 ,4-dihydroxy-4-methyltetrahydrofuran-2- yl)dideuteromethoxy)-(phenoxy)phosphoryl)amino)propanoate is additionally provided in this invention.

In one embodiment, Form D is characterized by an XRPD pattern comprising 2theta values of 10.04+0.2°, 10.07+0.2°, 16.44+0.2°, 16.79+0.2°, 19.03+0.2°, 19.69+0.2°, 20.22+0.2°, 21.68+0.2°, and 22.60+0.2°. In one embodiment, Form D is characterized by an XRPD pattern comprising at least four 2theta values selected from 10.04+0.2°, 10.07+0.2°, 16.44+0.2°, 16.79+0.2°, 19.03+0.2°, 19.69+0.2°, 20.22+0.2°, 21.68+0.2°, and 22.60+0.2°. In one embodiment, Form D is characterized by an XRPD pattern substantially similar to that set forth in FIG. 11.

In one embodiment, Form D is characterized by a DSC onset endotherm of about 137 °C.

Form D can be prepared using selective crystallization. The method can be carried out by treating a solution comprising a suitable solvent(s) and (S)-isopropyl 2-(((S)-(((2R,3R,4R,5R)-5- (5-deutero-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)dideuteromethoxy)-(phenoxy)phosphoryl)amino)propanoate, optionally in the presence of one or more seeds comprising Forms C and D to conditions that provide for the crystallization of Form D. The selective crystallization can be carried out in any suitable solvent. For example, it can be carried out in a protic solvent or a mixture thereof. In one embodiment, the solvent comprises the protic solvent water.

In one embodiment, the solvent comprises the protic solvent water in the presence of one or more seeds comprising Forms C and D. The selective crystallization can be carried out at, for example, a temperature in the range of about 5 oC to about 70 oC.

In another embodiment, the solvent comprises the protic solvent 2-propanol. The selective crystallization can be carried out at, for example, a temperature in the range of about ambient temperature to about 82 oC.

The selective crystallization can be carried out in any suitable solvent. For example, it can be carried out in a mixture of a protic solvent and an aprotic solvent. In one embodiment, the solvent comprises the protic solvent 2-propanol and the aprotic solvent heptane. In one embodiment, the solvent comprises the protic solvent 2-propanol and the aprotic solvent heptane in the presence of one or more seeds comprising Form D. The selective crystallization can be carried out at, for example, a temperature in the range of about 5 oC to about 82 oC. Methods utilized in preparing Form D are further described in the Examples section below.

Morphic Form E

Isolated Morphic Form E of (S)-isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(5-deutero-2,4- dioxo-3 ,4-dihydropyrimidin- 1 (2H)-yl)-3 ,4-dihydroxy-4-methyltetrahydrofuran-2- yl)dideuteromethoxy)-(phenoxy)phosphoryl)amino)propanoate is also described.

In one embodiment, Form E is characterized by an XRPD pattern comprising 2theta values of 7.88+0.2°, 10.60+0.2°, 12.35+0.2°, 17.15+0.2°, 17.79+0.2°, 17.86+0.2°, 19.60+0.2° and 20.07+0.2°. In one embodiment, Form E is characterized by an XRPD pattern comprising at least four 2theta values selected from 7.88+0.2°, 10.60+0.2°, 12.35+0.2°, 17.15+0.2°, 17.79+0.2°, 17.86+0.2°, 19.60+0.2° and 20.07+0.2°. In one embodiment, Form E is characterized by an XRPD pattern substantially similar to that set forth in FIG. 14.

In one embodiment, Form E is characterized by a DSC onset endotherm of about 124 °C.

Form E can be prepared using selective crystallization. The method can be carried out by treating a solution comprising a suitable solvent(s) and (S)-isopropyl 2-(((S)-(((2R,3R,4R,5R)-5- (5-deutero-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)dideuteromethoxy)-(phenoxy)phosphoryl)amino)propanoate to conditions that provide for the crystallization of Form E. The selective crystallization can be carried out in any suitable solvent. For example, it can be carried out in an aprotic solvent or a mixture thereof. In one embodiment, the solvent comprises the aprotic solvents dichloromethane and ethyl ether. The selective crystallization can be carried out at any suitable temperature, for example, at about ambient temperature.

Methods utilized in preparing Form E are further described in the Examples section below.

Highly Active Nucleotide Phosphoramidate Structures

In one embodiment, isolated morphic Forms A, B, C, D and E of Structure 1 A (provided below), or a pharmaceutically acceptable salt thereof, wherein deuterium (D) as defined in the Structure 1A has an enrichment over protium of at least 90% (i.e., less than 10% 1H hydrogen). It has been found that isolated morphic Forms A, B, C, D and E of Structure 1A are superior NS5B inhibitors for the treatment of hepatitis C.

Structure 1A

In one embodiment a method for the treatment is provided for a host infected with a Flavivirus, hepatitis C or a related or other disorder involving abnormal cellular proliferation that includes the administration of an effective amount of an isolated morphic Form A, B, C, D or E of Structure 1 A of at least 90% purity, or a pharmaceutically acceptable salt thereof, optionally in a pharmaceutically acceptable carrier.

In an alternative embodiment, both of the 5 '-deuteriums independently represent at least 50% enrichment. In another embodiment, the enrichment is independently at least 75% or 80%. In another embodiment, both of the 5 '-deuteriums independently represent at least 90%, 95% 96%, 97%, 98% or 99% enrichment. In another embodiment, the deuterium in the 5-position of the pyrimidine represents at least 50% enrichment. In another embodiment, the enrichment is independently at least 75% or 80%. In another embodiment, both of the 5 '-deuteriums independently represent at least 90%>, 95%, 96%, 97%, 98% or 99% enrichment. In the absence of an indication to the contrary, the deuterium is at least 90% at that position.

In another embodiment, an isolated morphic Form A, B, C, D or E of Structure 1A is administered as a phosphorus S stereoisomer, wherein the phosphorus stereoisomer is at least in 90%) pure form, and typically, 95%, 98%, or 99% pure form.

In another embodiment, an effective amount of an isolated morphic Form A, B, C, D or E of Structure 1A or its pharmaceutically acceptable salt, optionally in a pharmaceutically acceptable carrier, is provided to a host in need of hepatitis C therapy, or another therapy as disclosed herein. Chemical Description and Terminology

Compounds are described using standard nomenclature. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs.

The terms "a" and "an" do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced item. The term "or" means "and/or". Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The endpoints of all ranges are included within the range and independently combinable.

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as"), is intended merely for illustration and does not pose a limitation on the scope of the invention unless otherwise claimed.

An "active agent" is a compound (including a compound disclosed herein), element, or mixture that when administered to a patient, alone or in combination with another compound, element, or mixture, confers, directly or indirectly, a physiological effect on the patient. The indirect physiological effect may occur via a metabolite or other indirect mechanism.

"Deuteration" and "deuterated" means that a hydrogen is replaced by a deuterium such that the deuterium exists over natural abundance and is thus "enriched". An enrichment of 50% means that rather than hydrogen at the specified position the deuterium content is 50%. For clarity, it is confirmed that the term "enriched" as used herein does not mean percentage enriched over natural abundance. In other embodiments, there will be at least 80%, at least 90%, or at least 95% deuterium enrichment at the specified deuterated position or positions. In other embodiments there will be at least 96%>, at least 97%, at least 98%>, or at least 99% deuterium enrichment at the specified deuterated position or positions indicated. In the absence of indication to the contrary, the enrichment of deuterium in the specified position of the compound described herein is at least 90%>.

A "dosage form" means a unit of administration of an active agent. Non-limiting examples of dosage forms include tablets, capsules, injections, suspensions, liquids, intravenous fluids, emulsions, creams, ointments, suppositories, inhalable forms, transdermal forms, and the like.

"Pharmaceutical compositions" are compositions comprising at least one active agent, such as a compound or salt of one of the active compounds disclosed herein, and at least one other substance, such as a carrier. Pharmaceutical compositions optionally contain more than one active agent. "Pharmaceutical combinations" or "combination therapy" refers to the administration of at least two active agents, and in one embodiment, three or four or more active agents which may be combined in a single dosage form or provided together in separate dosage forms optionally with instructions that the active agents are to be used together to treat a disorder, such as but not limited to a viral disease such as hepatitis C, or a disorder associated with hepatitis C, or another viral infection as described herein.

"Pharmaceutically acceptable salts" includes derivatives of the disclosed compounds in which the parent compound is modified by making inorganic and organic, suitably non-toxic, acid or base addition salts thereof. The salts of the present compounds can be synthesized from a parent compound that contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting free acid forms of these compounds with a stoichiometric amount of the appropriate base (such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate, or the like), or by reacting free base forms of these compounds with a stoichiometric amount of the appropriate acid. Such reactions are typically carried out in water or in an organic solvent, or in a mixture of the two. The pharmaceutically acceptable salt can be in the form of a pure crystal, or single polymorphic form, or can be used in non-crystalline or amorphic, glassy, or vitreous form, or a mixture thereof. In an alternative embodiment, the active compound can be provided in the form of a solvate.

Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts and the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, conventional non-toxic acid salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, mesylic, esylic, besylic, sulfanilic, 2- acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, HOOC-(CH2)n-COOH where n is 0-4, and the like. Lists of additional suitable salts may be found, e.g., in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., p. 1418 (1985).

The term "carrier" means a diluent, excipient, or vehicle with which an active compound is provided.

A "pharmaceutically acceptable excipient" means an excipient that is useful in preparing a pharmaceutical composition/combination that is generally safe, is sufficiently non-toxic, and neither biologically nor otherwise undesirable. A "pharmaceutically acceptable excipient" as used in the present application includes both one and more than one such excipient.

A "patient" or "host" is a human or non-human animal, including, but not limited to, simian, avian, feline, canine, bovine, equine or porcine in need of medical treatment. Medical treatment can include treatment of an existing condition, such as a disease or disorder, or a prophylactic or diagnostic treatment. In a particular embodiment, the patient or host is a human patient. In an alternative embodiment, the patient such as a host is treated to prevent a disorder or disease described herein.

The term "isolated" as used herein refers to the material in substantially pure form. An isolated compound does not have another component that materially affects the properties of the compound. In a particular embodiment, an isolated form is at least 95, 98 or 99% pure.

Methods of Treatment

The disclosure provides a method to treat a host, typically a human, infected with any disorder that can be treated with a nucleoside or nucleotide, including but not limited to, a viral disease, tumor, cancer or other neoplastic or abnormal cellular proliferation, hyperuricaemia, a disorder treated with an immunosuppressive agent, a disorder treatable with an anti-methylating agent or a phosphodiesterase inhibitor, a disorder treated with an epigenetic modulator, or a neural or cardiovascular disease using an effective amount of an isolated morphic Form of A, B, C, D or E, optionally as a pharmaceutically acceptable salt and optionally in a pharmaceutically acceptable carrier. In alternative embodiments, an isolated combination of two morphic forms is provided, such as Forms D and A; D and B; D and C; B and C; or A and C. In another alternative embodiment, an isolated combination of three morphic forms is provided, for example, Forms D, A and B; Forms D, B and C; Forms D, A and C; and Forms A, B and C. In an alternative embodiment, the amorphous form of (S)-isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(5-deutero-2,4- dioxo-3 ,4-dihydropyrimidin- 1 (2H)-yl)-3 ,4-dihydroxy-4-methyltetrahydrofuran-2- yl)dideuteromethoxy)(phenoxy)phosphoryl)amino)propanoate is provided.

In one embodiment, the viral infection is selected from a Flaviviridae (such as flavivirus, hepacivirus (HCV), and pestivirus); a respiratory virus (such as adenovirus, avian influenza, Influenza virus type A and B, respiratory syncytial virus, rhinovirus, and SARS); a gastro-enteric virus (such as coxsackie, enterovirus, poliovirus, and rotavirus); herpes simplex 1 and 2; cytomegalovirus; varicella; and a Caliciviridae (such as norovirus).

In one embodiment the disease is hepatitis C.

In another embodiment, an effective amount of one of the isolated morphic Forms A, B, C, D or E as described herein, optionally as a pharmaceutically acceptable salt and optionally in a pharmaceutically acceptable carrier can be used to treat a host, typically a human, with a secondary condition associated with hepatitis C, or another disorder described herein, including but not limited to those disorders described below in (i) through (viii).

This disclosure provides methods of treating a viral infection in a patient, including a hepatitis C infection, by providing an effective amount of a morphic Form A, B, C, D or E or pharmaceutically acceptable salt thereof, to the patient infected with a hepatitis C virus. A an isolated morphic Form A, B, C, D or E described herein or salt may be provided as the only active agent or may be provided together with one or more additional active agents. In certain embodiments the compound or salt is administered together with a NS3 protease inhibitor, a NS5A inhibitor, a NS5B inhibitor, or a combination of these.

An effective amount of a pharmaceutical composition/combination of the disclosure may be an amount sufficient to (a) inhibit the progression of hepatitis C or other disorder described herein; (b) cause a regression of the hepatitis C infection or other disorder described herein; or (c) cause a cure of a hepatitis C infection, or other disorder described herein, for example such that HCV virus or HCV antibodies can no longer be detected in a previously infected patient's blood or plasma. An amount of a pharmaceutical composition/combination effective to inhibit the progress or cause a regression of hepatitis C, or other disorder described herein, includes an amount effective to stop the worsening of symptoms of hepatitis C, or other disorder described herein, or reduce the symptoms experienced by a patient infected with the hepatitis C virus, or other disorder described herein. Alternatively a halt in progression or regression of a disorder described herein, for example hepatitis C, may be indicated by any of several markers for the disease. For example, a lack of increase or reduction in the hepatitis C viral load or a lack of increase or reduction in the number of circulating HCV antibodies in a patient's blood are markers of a halt in progression or regression of hepatitis C infection. Other hepatitis C disease markers include aminotransferase levels, particularly levels of the liver enzymes AST and ALT. Normal levels of AST are from 5 to 40 units per liter of serum (the liquid part of the blood) and normal levels of ALT are from 7 to 56 units per liter of serum. These levels will typically be elevated in a HCV infected patient. Disease regression is usually marked by the return of AST and ALT levels to the normal range.

In yet another embodiment, an effective amount of one of the morphic Forms A, B, C, D or E described herein, optionally as a pharmaceutically acceptable salt and optionally in a pharmaceutically acceptable carrier can be used as a prophylaxis to ward off or prevent a host, typically a human, from having a disorder described herein, for example the hepatitis C infection. In an alternative embodiment, an effective amount of one of the morphic Forms A, B, C, D or E described herein, optionally as a pharmaceutically acceptable salt and optionally in a pharmaceutically acceptable carrier can be used to treat a secondary condition associated with a disorder described herein, for example hepatitis C, including but not limited to those disorders described below in (i) through (viii).

(i) Cryoglobulinemia which is abnormal antibodies (called cryoglobulins) that come from hepatitis C virus stimulation of lymphocytes. These antibodies can deposit in small blood vessels, thereby causing inflammation of the vessels (vasculitis) in tissues throughout the body including the skin, joints and kidneys (glomerulonephritis).

(ii) B-cell non-Hodgkin's lymphoma associated with hepatitis C, which is considered to be caused by excessive stimulation by hepatitis C virus of B-lymphocytes, resulting in abnormal reproduction of the lymphocytes.

(iii) Skin conditions such as lichen planus and porphyria cutanea tarda have been associated with hepatitis C infection. (iv) Cirrhosis, which is a disease in which normal liver cells are replaced with scar or abnormal tissue. Hepatitis C is one of the most common causes of liver cirrhosis.

(v) Ascites, which is the accumulation of fluid in the abdominal cavity commonly caused by cirrhosis of the liver, which can be caused by hepatitis C infection.

(vi) Hepatocellular carcinoma, of which 50% of the cases in the U.S. are currently caused by chronic hepatitis C infection.

(vii) Hepatitis C related jaundice, which is a yellowish pigmentation caused by increased bilirubin.

(viii) Thrombocytopenia is often found in patients with hepatitis C and may be the result of bone marrow inhibition, decrease in liver thrombopoietin production and/or an autoimmune mechanism. In many patients, as hepatitis C advances, the platelet count decreases and both bone marrow viral inhibition and antiplatelet antibodies increase.

Other symptoms and disorders associated with hepatitis C that may be treated by an effective amount of a pharmaceutical composition/combination of the disclosure include decreased liver function, fatigue, flu-like symptoms: fever, chills, muscle aches, joint pain, and headaches, nausea, aversion to certain foods, unexplained weight loss, psychological disorders including depression, and tenderness in the abdomen.

The active compounds presented herein can also be used to enhance liver function generally associated with hepatitis C infection, for example, synthetic function including synthesis of proteins such as serum proteins (e.g., albumin, clotting factors, alkaline phosphatase, aminotransferases (e.g., alanine transaminase, aspartate transaminase), 5 '-nucleosidase, y glutammyltranspeptidase, etc.), synthesis of bilirubin, synthesis of cholesterol, and synthesis of bile acids; a liver metabolic function, including carbohydrate metabolism, amino acid and ammonia metabolism, hormone metabolism, and lipid metabolism; detoxification of exogenous drugs; and a hemodynamic function, including splanchnic and portal hemodynamics.

The pharmaceutical compositions/combinations disclosed herein are also useful for treating viral infections in patients other than a hepatitis C infection. In an alternative embodiment, the infection may be an RNA viral infection, such as Togaviridae, Picornaviridae, Coronaviridae, or Flaviviridae viral infection. The disclosure includes a method of treating a Togaviridae, Picornaviridae, Coronaviridae, or Flaviviridae viral infection by administering an effective amount of one of the active compounds disclosed herein, to a subject infected with a togavirus, picomavirus, coronavirus, or flavivirus. Flaviviridae viral infections include infections with viruses of the genera Flavivirus, Pestivirus, and Hepacivirus. Flavivirus infections include yellow fever, Dengue fever, West Nile virus, encephalitis, including St. Louis encephalitis, Japanese B encephalitis, California encephalitis, central European encephalitis, Russian spring- summer encephalitis, and Murray Valley encephalitis, Wesselsbron disease, and Powassan disease. Pestivirus infections include primarily livestock diseases, including swine fever in pigs, BVDV (bovine viral diarrhea virus) in cattle, and Border Disease virus infections. Hepacivirus infections includes Hepatitis C and canine Hepacivirus. Togavirus infections include Sindbis virus, Eastern equine encephalitis virus, Western equine encephalitis virus, Venezuelan equine encephalitis virus, Ross River virus, O'nyong'nyong virus, Chikungunya virus, Semliki Forest virus, and Rubella virus. Picomavirus infections include infections with viruses of the genuses Aphthovirus, Aquamavirus, Avihepatovirus, Cardiovirus, Cosavirus, Dicipivirus, Enterovirus, Erbovirus, Hepatovirus, Kobuvirus, Megrivirus, Parechovirus, Salivirus, Sapelovirus, Senecavirus, Teschovirus, and Tremovirus. Coronavirus infections include infections with vims of the genuses Alphacoronavirus, Betacoronavirus (which includes severe acute respiratory coronavims (SARS)), Gammacoronavirus, and Deltacoronavirus. The disclosure includes compositions comprising a compound of the present disclosure useful in an effective amount for treating Dengue fever, West Nile fever, yellow fever, or BVDV (bovine viral diarrhea vims) and methods of treating these infections by administering a morphic Form A, B, C, D or E described herein to a patient infected with the vims.

The active compounds and methods described herein are useful for the treatment of cancer or other abnormal proliferative disorders. As contemplated herein, the cancer treated can be a primary tumor or a metastatic tumor. In one aspect, the methods described herein are used to treat a solid tumor, for example, melanoma, lung cancer (including lung adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, large cell carcinoma, bronchioloalveolar carcinoma, bronchiogenic carcinoma, non-small-cell carcinoma, small cell carcinoma, mesothelioma); breast cancer (including ductal carcinoma, lobular carcinoma, inflammatory breast cancer, clear cell carcinoma, mucinous carcinoma, serosal cavities breast carcinoma); colorectal cancer (colon cancer, rectal cancer, colorectal adenocarcinoma); anal cancer; pancreatic cancer (including pancreatic adenocarcinoma, islet cell carcinoma, neuroendocrine tumors); prostate cancer; prostate adenocarcinoma; ovarian carcinoma (ovarian epithelial carcinoma or surface epithelial-stromal tumor including serous tumor, endometrioid tumor and mucinous cystadenocarcinoma, sex-cord-stromal tumor); liver and bile duct carcinoma (including hepatocellular carcinoma, cholangiocarcinoma, hemangioma); esophageal carcinoma (including esophageal adenocarcinoma and squamous cell carcinoma); oral and oropharyngeal squamous cell carcinoma; salivary gland adenoid cystic carcinoma; bladder cancer; bladder carcinoma; carcinoma of the uterus (including endometrial adenocarcinoma, ocular, uterine papillary serous carcinoma, uterine clear-cell carcinoma, uterine sarcomas and leiomyosarcomas, mixed mullerian tumors); glioma, glioblastoma, medulloblastoma, and other tumors of the brain; kidney cancers (including renal cell carcinoma, clear cell carcinoma, Wilm's tumor); cancer of the head and neck (including squamous cell carcinomas); cancer of the stomach (gastric cancers, stomach adenocarcinoma, gastrointestinal stromal tumor); testicular cancer; germ cell tumor; neuroendocrine tumor; cervical cancer; carcinoids of the gastrointestinal tract, breast, and other organs; signet ring cell carcinoma; mesenchymal tumors including sarcomas, fibrosarcomas, haemangioma, angiomatosis, haemangiopericytoma, pseudoangiomatous stromal hyperplasia, myofibroblastoma, fibromatosis, inflammatory myofibroblastic tumor, lipoma, angiolipoma, granular cell tumor, neurofibroma, schwannoma, angiosarcoma, liposarcoma, rhabdomyosarcoma, osteosarcoma, leiomyoma, leiomysarcoma, skin, including melanoma, cervical, retinoblastoma, head and neck cancer, pancreatic, brain, thyroid, testicular, renal, bladder, soft tissue, adrenal gland, urethra, cancers of the penis, myxosarcoma, chondrosarcoma, osteosarcoma, chordoma, malignant fibrous histiocytoma, lymphangiosarcoma, mesothelioma, squamous cell carcinoma; epidermoid carcinoma, malignant skin adnexal tumors, adenocarcinoma, hepatoma, hepatocellular carcinoma, renal cell carcinoma, hypernephroma, cholangiocarcinoma, transitional cell carcinoma, choriocarcinoma, seminoma, embryonal cell carcinoma, glioma anaplastic; glioblastoma multiforme, neuroblastoma, medulloblastoma, malignant meningioma, malignant schwannoma, neurofibrosarcoma, parathyroid carcinoma, medullary carcinoma of thyroid, bronchial carcinoid, pheochromocytoma, Islet cell carcinoma, malignant carcinoid, malignant paraganglioma, melanoma, Merkel cell neoplasm, cystosarcoma phylloide, salivary cancers, thymic carcinomas, lymphoma, leukemia, and cancers of the vagina among others. Combination Therapy

The present disclosure also includes pharmaceutical compositions and combinations comprising an isolated morphic Form A, B, C, D or E described herein and at least one additional active agent, as well as methods of treatment comprising administering such compositions to a patient infected with hepatitis C, or another disorder described herein. In certain embodiments the additional active agent is an HCV NS3 protease inhibitor or an HCV NS5A or another NS5B inhibitor.

In alternative embodiments, the combination therapy is provided with an isolated combination of two morphic forms, such as Forms D and A; D and B; D and C; B and C or A and C. In another alternative embodiment, the combination therapy is provided with an isolated combination of three morphic forms, for example, Forms D, A and B; D, B and C; or Forms D, A and C; and Forms A, B and C. In an alternative embodiment, the combination therapy is provided with amorphous (S)-isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(5-deutero-2,4-dioxo-3,4- dihydropyrimidin- 1 (2H)-yl)-3 ,4-dihydroxy-4-methyltetrahydrofuran-2- yl)dideuteromethoxy)(phenoxy)phosphoryl)amino)propanoate.

In nonlimiting embodiments, the isolated morphic Forms A, B, C, D or E (or a specified combination thereof, as described above) of the present disclosure can be administered in combination or alternation with one or more of the active compounds that are a caspase inhibitor, a cyclophilin inhibitor, a cytochrome P450 monooxygenase inhibitor, an entry inhibitor, a glucocorticoid, an HCV protease inhibitor, a hematopoietin, a homeopathic therapy, an immunomodulatory compound, an immunosuppressant, an interleukin, an interferon or interferon enhancer, an IRES inhibitor, a monoclonal or polyclonal antibody, a nucleoside or nucleotide analogue or prodrug, a non-nucleoside inhibitor, an NS4B inhibitor, an NS5A inhibitor, an NS5B inhibitor, a P7 protein inhibitor, a polymerase inhibitor, an RNAi compound, a therapeutic vaccine, a TNF agonist, a tubulin inhibitor, a sphingosine-1 -phosphate receptor modulator, or a TLR agonist.

Nonlimiting examples of active agents in these categories include:

Caspase Inhibitors: IDN-6556 (Idun Pharmaceuticals);

Cyclophilin Inhibitors: for example, NIM811 (Novartis), SCY-635 (Scynexis), and DEBIO-025 (Debiopharm); Cytochrome P450 monooxygenase inhibitors: ritonavir, ketoconazole, troleandomycin, 4- methyl pyrazole, cyclosporin, clomethiazole, cimetidine, itraconazole, fluconazole, miconazole, fluvoxamine, fluoxetine, nefazodone, sertraline, indinavir, nelfinavir, amprenavir, fosamprenavir, saquinavir, lopinavir, delavirdine, erythromycin, and VX-497 (Merimebodib). Preferred CYP inhibitors include ritonavir, ketoconazole, troleandomycin, 4-methyl pyrazole, cyclosporin, and clomethiazole;

Entry Inhibitors: ITX-5061 (iTherX);

Glucocorticoids: hydrocortisone, cortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, paramethasone, betamethasone, and dexamethasone;

HCV Protease Inhibitors: for example Sovaprevir and ACH-2684, ABT-450 (Abbott), ACL-181 and AVL-192 (Avila), BMS-032 (Bristol Myers Squibb), Boceprevir (Merck), danoprevir (Hoffman-La Roche and Genentech), GS-9256 (Gilead), GS-9451 (Gilead), Telaprevir (VX-950, Vertex), VX-985 (Vertex), Simeprevir (TMC435, Tibotec), Fosamprenavir (prodrug of Amprenavir, Glaxo /Vertex), indinavir (Crixivan, Merck), TMC435350 (Tibotec/Medivir), Faldaprevir (BI 201335. Boehringer Ingelheim), PHX-1766 (Phenomix), Vaniprevir (MK-7009, Merck), narlaprevir (SCH900518, Schering), MK-5172 (Merck);

Hematopoietins: hematopoietin-1 and hematopoietin-2. Other members of the hematopoietin superfamily such as the various colony stimulating factors (e.g. G-CSF, GM-CSF, M-CSF), Epo, and SCF (stem cell factor);

Homeopathic Therapies: Milk Thistle, silymarin, ginseng, glycyrrhizin, licorice root, schisandra, vitamin C, vitamin E, beta carotene, and selenium;

Immunomodulatory compounds: thalidomide, IL-2, hematopoietins, IMPDH inhibitors, for example Merimepodib (Vertex Pharmaceuticals Inc.), interferon, including natural interferon (such as OMNIFERON, Viragen and SUMIFERON, Sumitomo, a blend of natural interferons), natural interferon alpha (ALFERON, Hemispherx Biopharma, Inc.), interferon alpha-nl from lymphblastoid cells (WELLFERON, Glaxo Wellcome), oral alpha interferon, Peg-interferon, Peg-interferon alfa 2a (PEGASYS, Roche), recombinant interferon alfa 2a (ROFERON, Roche), inhaled interferon alpha 2b (AERX, Aradigm), Peg-interferon alpha 2b (ALBUFERON, Human Genome Sciences/ Novartis, PEGINTRON, Schering), recombinant interferon alfa 2b (INTRON A, Schering), pegylated interferon alfa 2b (PEG-INTRON, Schering, VIRAFERONPEG, Schering) interferon beta- la (REBIF, Ares-Serono, Inc. and Pfizer), consensus interferon alpha (INFERGEN, Intermune), interferon gamma- lb (ACTIMMUNE, Intermune, Inc.), un-pegylated interferon alpha, alpha interferon, and its analogs, and synthetic thymosin alpha 1 (ZADAXIN, SciClone Pharmaceuticals Inc.), and lamdba interferon (BMS);

Immunosupressants: sirolimus (RAPAMUNE, Wyeth);

Interleukins: (IL-1, IL-3, IL-4, IL-5, IL-6, IL-10, IL-11, IL-12), LIF, TGF-beta, TNF- alpha) and other low molecular weight factors (e.g. AcSDKP, pEEDCK, thymic hormones, and minicytokines);

Interferon Enhancers: EMZ702 (Transition Therapeutics);

IRES inhibitors: VGX-410C (VGX Pharma);

Monoclonal and Polyclonal antibodies: XTL-6865 (HEPX-C, XTL), HuMax-HepC (Genmab), Hepatitis C Immune Globin (human) (CIVACIR, Nabi Biopharmceuticals), XTL-002 (XTL), Rituximab (RITUXAN, Genentech/ IDEC), GS-6624 (Gilead);

Nucleoside analogues: Sofosbuvir (PSI-7977, Pharmasset and Gilead), PSI-7851 (Pharmasset), PSI-7977 (Pharmasset), R7128 (mericitabine, Roche), R7348 (Roche), NM283 (valopicitabine, Idenix), GS-6620 (Gilead), TMC-649 (Tibotec), VX-135 (Vertex, Alios), ALS- 2200 (Alios), IDX184 (Idenix), IDX21437 (Idenix), IDX21459 (Idenix), Lamivudine (EPIVIR, 3TC, GlaxoSmithKline), MK-0608 (Merck), zalcitabine (HIVID, Roche US Pharmaceuticals), ribavirin (including COPEGUS (Roche), REBETOL (Schering), VILONA (ICN Pharmaceuticals, and VIRAZOLE (ICN Pharmaceuticals), isatoribine (Anadys Pharmaceuticals), ANA245 (Anadys Pharmaceuticals), and viramidine (ICN), an amidine prodrug of ribavirin. Combinations of nucleoside analogues may also be employed;

Non-nucleoside inhibitors: PSI-6130 (Roche/ Pharmasset), ABT-333 and ABT-072 (Abbott), delaviridine (RESCRIPTOR, Pfizer), PF-868554 (Pfizer), GSK-852 (GlaxoSmithKline), Setrobuvir (ANA-598, Anadys), VX-222 (Vertex), BI-127 (Boehringer Ingelheim), and BMS-325 (Bristol Meyers);

NS4B inhibitors: clemizole (Eiger BioPharmaceuticals, Inc.);

NS5A inhibitors: Daclatasvir (BMS-790052, BMS), AZD-729 (Astra Zeneca); PPI-461 (Presidio), PPI-688 (Presidio), samatasvir (IDX719, Idenix), ledipasvir (GS-5885, Gilead), GS- 5816 (Gilead), ombitasvir (ABT-267, AbbVie), GSK2336805 (GlaxoSmithKline), and elbasvir (MK-8742, Merck); NS5B inhibitors: MBX-700 (Microbotix/ Merck), RG-9190, VX-222 (Vertex), and BMS- 791325 (Bristol Meyers Squibb);

P7 protein inhibitor: amantadine (SYMMETREL, Endo Pharmaceuticals, Inc.);

Polymerase inhibitors: ANA598 (Anadys), Tegobuvir (GS 9190, Gilead);

RNA interference: SIRNA-034 RNAi (Sirna Therapeutics);

Therapeutic Vaccines: IC41 (Intercell), GI 5005 (Globeimmune), Chronvac-C (Tripep/ Inovio);

TNF agonists: adalimumab (HUMIRA, Abbott), entanercept (ENBREL, Amgen and Wyeth), infliximab (REMICADE, Centocor, Inc.);

Tubulin inhibitors: Colchicine;

Sphingosine-1 -phosphate receptor modulators: FTY720 (Novartis);

TLR agonists: TLR7 agonist (Anadys Pharmaceuticals), CPGIOIOI (Coley), andTLR9 agonists including CPG 7909 (Coley); and,

Vaccines: HCV/MF59 (Chiron), IC41 (Intercell).

For example, in some embodiments, the additional active agent is sovaprevir, ACH-2684

(HCV NS3 protease inhibitors) and/or an NS5A inhibitor.

The disclosure includes compositions in which the additional active

NS5A inhibitor.

or combinations thereof. NS3 protease inhibitors, useful in the pharmaceutical compositions and combinations described here have been disclosed previously, for example in U.S. Patent No. 7,906,619, issued March 15, 2011, which is hereby incorporated by reference in its entirety for its teachings regarding 4-amino-4-oxobutanoyl peptides. The '619 patent is particularly incorporated by reference at the Examples section beginning in column 50 and extending to column 85 which discloses compounds useful in compositions/combination with a morphic Form A, B, C, D or E described herein.

U.S. Publication No. 2010/0216725, published August 26, 2010, is hereby incorporated by reference in its entirety for its teachings regarding 4-amino-4-oxobutanoyl peptides. The '725 application is particularly incorporated by reference at the Examples section beginning at page 22 and extending to page 100 which discloses compounds useful in compositions/combination with a morphic Form A, B, C, D or E described herein.

U.S. Publication No. 2010/0152103, published June 17, 2010, is hereby incorporated by reference in its entirety for its teachings regarding 4-amino-4-oxobutanoyl peptide cyclic analogues. The ' 103 application is particularly incorporated by reference at the Examples section beginning at page 19 and extending to page 60 which discloses compounds useful in compositions/combination with a morphic Form A, B, C, D or E described herein. Particularly the compounds disclosed herein may be used in combination with an NS3 protease inhibitor of the formulae shown below.

NS5A inhibitors, useful in the pharmaceutical compositions and combinations described here have been disclosed previously. U.S. Publication No. 2012/0302528, published November 29, 2012, is hereby incorporated by reference in its entirety for its teachings regarding NS5A inhibitors.

In certain embodiments the NS3 protease inhibitor is chosen from

In certain embodiments, the NS5A inhibitor is chosen from

Pharmaceutical Compositions

Isolated morphic Forms A, B, C, D or E as disclosed herein can be independently administered as the neat chemical, but are preferably independently administered as a pharmaceutical composition. The disclosure provides pharmaceutical compositions comprising any of the isolated morphic Forms A, B, C, D or E described herein together with at least one pharmaceutically acceptable carrier, or alternatively the amorphous (S)-isopropyl 2-(((S)- (((2R,3R,4R,5R)-5-(5-deutero-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)-3,4-dihydroxy-4- methyltetrahydrofuran-2-yl)dideuteromethoxy)(phenoxy)phosphoryl)amino)propanoate, for use to treat any of the disclosed indications. The pharmaceutical composition/combination may contain a compound or salt of any of the active compounds described herein as the only active agent, but in another embodiment may also contain at least one additional active agent. In certain embodiments for the treatment of HCV it is preferred that the additional active agent is an NS3 protease inhibitor, NS5A, and/or NS5B inhibitor.

In alternative embodiments, the pharmaceutical composition includes an isolated combination of two morphic forms, such as Forms D and A; D and B; D and C; B and C or A and C. In another alternative embodiment, the pharmaceutical composition includes an isolated combination of three morphic forms, for example, Forms D, A and B; D, B and C; or Forms D, A and C; and Forms A, B and C.

In certain embodiments the pharmaceutical composition is in a dosage form that contains from about 0.1 mg to about 2000 mg, from about 10 mg to about 1000 mg, from about 100 mg to about 800 mg, or from about 200 mg to about 600 mg of a morphic Form A, B, C, D or E described herein and optionally from about 0.1 mg to about 2000 mg, from about 10 mg to about 1000 mg, from about 100 mg to about 800 mg, or from about 200 mg to about 600 mg of an additional active agent in a unit dosage form. In certain embodiments the morphic Form A, B, C, D or E is delivered in an oral dosage form such as a pill, tablet or capsule in an effective amount, which may in some embodiments be at least 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 mg, or any dosage falling in between these dosages. The pharmaceutical composition may also include a molar ratio of the morphic Form A, B, C, D or E and an additional active agent. For example, for the treatment of HCV, the pharmaceutical composition may contain a molar ratio of about 0.5:1, about 1 : 1, about 2: 1, about 3: 1 or from about 1.5: 1 to about 4: 1, and the other active agent may be, for example, an NS3 protease inhibitor, an NS5A inhibitor, and/or another NS5B inhibitor.

Compounds disclosed herein may be administered by any suitable means, including orally, topically, parenterally, by inhalation or spray, sublingually, transdermally, via buccal or sublingual transmucosal administration, rectally, as an ophthalmic solution or injection, or by other means, in dosage unit formulations containing conventional pharmaceutically acceptable carriers. The pharmaceutical composition may be formulated as any pharmaceutically useful form, e.g., as an aerosol, a cream, a gel, a pill, a capsule, a tablet, a syrup, a transdermal patch, or an ophthalmic solution. Some dosage forms, such as tablets and capsules, are subdivided into suitably sized unit doses containing appropriate quantities of the active components, e.g., an effective amount to achieve the desired purpose.

Carriers include excipients and diluents and should be of sufficiently high purity and sufficiently low toxicity to render them suitable for administration to the patient being treated. The carrier can be inert or it can possess pharmaceutical benefits of its own. The amount of carrier employed in conjunction with the compound is sufficient to provide a practical quantity of material for administration per unit dose of the compound.

Classes of carriers include, but are not limited to binders, buffering agents, coloring agents, diluents, disintegrants, emulsifiers, flavorants, glidents, lubricants, preservatives, stabilizers, surfactants, tableting agents, and wetting agents. Some carriers may be listed in more than one class, for example vegetable oil may be used as a lubricant in some formulations and a diluent in others. Exemplary pharmaceutically acceptable carriers include sugars, starches, celluloses, powdered tragacanth, malt, gelatin; talc, and vegetable oils. Optional active agents may be included in a pharmaceutical composition, which do not substantially interfere with the activity of the compound of the present disclosure.

The pharmaceutical compositions/combinations can be formulated for oral administration. These compositions typically contain between 5 or 10 to 99 weight % (wt.%) of any of the selected morphic forms described herein, for example, the morphic Form A, B, C, D or E and usually at least about 5 wt.% of the morphic Form A, B, C, D or E. Some embodiments contain from about 25 wt.% to about 50 wt.% or from about 5 wt.% to about 75 wt.% of the active material.

Specifically for HCV, an effective amount of a pharmaceutical composition/combination of the disclosure may be an amount sufficient, for example, to (a) inhibit the progression of hepatitis C or other disorder described herein; (b) cause a regression of the hepatitis C infection or other disorder described herein; (c) cause a cure of a hepatitis C infection, or other disorder described herein, for example such that HCV virus or HCV antibodies can no longer be detected in a previously infected patient's blood or plasma, or (d) treat an HCV-associated disorder. An amount of a pharmaceutical composition/combination effective to inhibit the progress or cause a regression of a disorder described herein, for example hepatitis C, includes an amount effective to stop the worsening of symptoms of the disorder or reduce the symptoms experienced by a patient with the disorder. Alternatively a halt in progression or regression of the disorder may be indicated by any of several markers for the disease. For example, in the case of HCV, a lack of increase or reduction in the hepatitis C viral load or a lack of increase or reduction in the number of circulating HCV antibodies in a patient's blood can be markers of a halt in progression or regression of hepatitis C infection. Other hepatitis C disease markers include aminotransferase levels, particularly levels of the liver enzymes AST and ALT.

The compound or pharmaceutically acceptable salt of any of the compounds described herein, including the isolated morphic Forms A, B, C, D, or E described herein and at least one additional active agent may be: (1) co-formulated and administered or delivered simultaneously in a combined formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by any other combination therapy regimen known in the art. When delivered in alternation therapy, the methods of the disclosure may comprise administering or delivering the compound or salt of any of the morphic forms described herein, and an additional active agent sequentially, e.g., in separate solution, emulsion, suspension, tablets, pills or capsules, or by different injections in separate syringes. In general, during alternation therapy, an effective dosage of each active ingredient is administered sequentially, i.e., serially, whereas in simultaneous therapy, effective dosages of two or more active ingredients are administered together. Various sequences of intermittent combination therapy may also be used.

Frequency of dosage may also vary depending on the compound used and the particular disease treated. However, for treatment of most infectious disorders, a dosage regimen of 4 times daily or less is preferred and a dosage regimen of 1 or 2 times daily is particularly preferred.

It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease in the patient undergoing therapy.

The pharmaceutical packaging may include an active compound or salt as described herein in a container together with instructions for using the compound to treat a patient suffering from a disorder described herein, for example Hepatitis C infection, are included herein.

Packaged pharmaceutical compositions/combinations are also included herein. Such packaged combinations include any of the compounds described herein, including the isolated morphic Forms A, B, C, D, or E described herein in a container together with instructions for using the combination to treat or prevent a viral infection, such as a hepatitis C infection, in a patient.

For HCV, the packaged pharmaceutical composition/combination may include one or more additional active agents. In certain embodiments the additional active agent is an NS3 protease inhibitor, an NS5A, and/or another NS5B inhibitor.

The packaged pharmaceutical combination may include any of the isolated morphic Forms A, B, C, D, or E described herein or pharmaceutically acceptable salts thereof and the additional active agent can be provided simultaneously in a single dosage form, concomitantly in separate dosage forms, or provided in separate dosage forms for administration for separated delivery by some amount of time that is within the time in which both the compound such as the morphic Form A, B, C, D, or E described herein and the additional active agent are within the bloodstream of the patient.

The packaged pharmaceutical combination may include the isolated morphic Form A, B, C, D, or E described herein or pharmaceutically acceptable salt thereof provided in a container with one or more additional active agents provided in the same or separate container, with instructions for using the combination to treat an HCV infection in a patient.

Chemical Syntheses Schemes

SCH

CH₂CI₂/TEA

Synthesis of (S)-isopropyl 2-(((S)-(perfluorophenoxy)(phenoxy)phosphoryl)amino) propanoate (Compound 1).

SC

Synthesis of (S)-isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(5-deutero-2,4-dioxo-3,4- dihydropyrimidin- 1 (2H)-yl)-3 ,4-dihydroxy-4-methyltetrahydrofuran-2-yl)dideuteromethoxy)- (phenoxy)phosphoryl)amino)propanoate. Chemical Syntheses

Synthesis of (S)-isopropyl 2-(((S)-(perfluorophenoxy)(phenoxy)phosphoryl)amino) propanoate (Compound 1)

1

CH₂CI₂/TEA

L-Alanine isopropyl ester HC1 salt (160 g) is charged in a 5 L four-necked flask equipped with a mechanical stirrer, thermometer and dropping funnel. To the flask, dichloromethane (1 L) is added and the suspension is cooled to -70 °C, followed by the addition of triethylamine (200 g, 276 mL) over 45 min. To the mixture is added a solution of phenyl dichlorophosphate (200 g) in dichloromethane (1 L) over 2.5 h. The reaction mixture is stirred at this temperature for an additional 90 min and then allowed to warm up to 0 °C over a period of 2 h and stirred for 2 h at 0 °C. To the mixture a solution of 2,3,4,5,6-pentafluorophenol (174.4 g) in dichloromethane (400 mL) and a solution of triethylamine (105.4 g) in dichloromethane (200 mL) are added dropwise simultaneously over a period of 1.2 h. The mixture is warmed to room temperature and stirred overnight.* The solid, triethylamine HC1 salt, is filtered off and the cake is washed with dichloromethane (3 x 150 mL). The filtrate is concentrated under reduced pressure and the residue triturated with MTBE (3.0 L). The white solid is removed by filtration. The cake is washed with MTBE (3 x 150 mL). The filtrate is concentrated and the resulting crude solid triturated with 20% ethyl acetate in hexane (2.0 L). The solid is collected by filtration and washed with 10%> NaHC03 until the aqueous phase reached pH 7, the solid is then washed with water and dried in a vacuum oven (55 °C) for 28 h. The dried solid is mixed with 500 mL heptane -EtO Ac (5 :1) and stirred for 1 h. The solid is collected by filtration and washed with heptane -EtO Ac (5: 1, 2 x 80 mL) to afford a >99%> single isomer. The solid is dried to give compound 1. *In an alternative work-up procedure, the reaction mixture is filtered and the DCM layer is washed with an aq. 0.1N NaOH solution, followed by water, dried, and evaporated to dryness. The residue is suspended in heptane/EtOAc (5: 1) and the solid is filtered. The solid is resuspended in heptane/toluene (85: 15) to isolate the pure single isomer.

Preparation of Compound 4

4

2,2-Dimethoxypropane (140 mL) is added to commercially available 2'-C-methyluridine 2 (100 g) in acetone (700 mL). The resulting mixture is cooled in an ice bath for 30 min, then p- toluenesulfonic acid (11 g) is added and the reaction mixture is stirred at room temperature for 24 h. After completion of the reaction (monitored by HPLC), the reaction mixture is cooled in an ice bath for 30 min and neutralized using cold potassium carbonate (12 g in 13 mL water, pH 7-8). The solvent is removed under reduced pressure until dryness. THF (~500 mL) is added to the residue and the solids are removed by filtration. The filtrate is co-evaporated with silica gel and purified by chromatography over silica gel (5-15% MeOH in CHC13) to give compound 3.

Compound 4 is prepared following the procedure reported by Corey et al. (J. Org. Chem. 1984, 49, 4735) with modifications described below. To acetonide 3 (50 g) in CH2C12 (1 L) is added PDC (126.1 g) at room temperature followed by Ac20 (171 g) and t-BuOH (248 g). The reaction temperature is maintained below 35 oC during the addition of reagents and then stirred at room temperature for 5 h. The reaction mixture is poured into aq. K2C03 (250 g K2C03 in 600 mL H20) and the organic layer is washed with CuS04 (100 g in 1 L H20). Activated charcoal (10 g) and silica gel (100 g) are added to the organic layer and stirred for 30 min and filtered. The filtrate is evaporated and the residue is purified by chromatography over silica gel (0-50% EtOAc in CHC13) to afford 4.

Preparation of (S)-isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(5-deutero-2,4-dioxo-3,4- dihydropyrimidin- 1 (2H)-yl)-3 ,4-dihydroxy-4-methyltetrahydrofuran-2-yl)dideuteromethoxy)- (phenoxy)phosphoryl)amino)propanoate

4 8

NaBD4 (7.96 g) is added in portions to a cooled (5 °C) 70:30 v/v mixture of EtOD/D20 (350 mL, 99% D) in a 1 L flask, followed by the addition of acetonide ester 4 (35 g) in portions (slowly bubbles). The resulting reaction mixture is stirred at room temperature for 3 h, and then heated at 80 °C for 1 d (1H NMR spectroscopic analysis indicates >85% deuterium incorporation at the 5 -uracil position). The reaction mixture is filtered to remove solids and concentrated under reduced pressure to remove EtOD. Additional D20 is added and the resulting mixture reheated at 95 °C to increase the deuterium incorporation at the 5 position to >98% (D-incorporation monitored by 1H NMR spectroscopy). After completion of the reaction, half of the solvent is removed under reduced pressure, the mixture is cooled in an ice bath, AcOD (59 g) is added, and the resulting mixture is stirred for 15-20 min. EtOAc (300 mL) and brine (100 mL) are added, the organic layer is separated, and the aqueous layer is again extracted with EtOAc (150 mL), followed by THF (150 mL). The combined organic layers are concentrated, the resulting residue is dissolved in 10% MeOH and CHC13 (300 mL), filtered, concentrated, and purified by chromatography over silica gel (ISCO, eluent DCM/MeOH) to give the deuterated acetonide 8.

Deuterated acetonide 8 (50 g) is added to a cooled (5 °C) 4N HC1 (250 mL) solution and stirred at room temperature for 3 h, during which time a white precipitate forms. The solvent is evaporated to dryness and to the residue is added to water (100 mL) with stirring. The suspension is cooled to 5 °C, stirred for 1 h. and the white precipitate is collected by filtration. The solid is washed with cold water (75 mL) and dried to afford the deuterated nucleoside 9.

Nucleoside 9 (37.3 g) in THF (750 mL) is cooled to -5 °C. t-BuMgCl (1M in THF, 430 mL) is added and the mixture is stirred for 30 min at the same temperature. The reaction mixture is stirred for another 30 minutes at room temperature, then cooled again to -5 °C, and a solution of 1 (129.5 g) in THF (650 mL) is added slowly. The reaction mixture is stirred at room temperature for 24 h., cooled to -5 °C, and to it added cold 2N HC1 (200 mL), followed by stirring for 10 min, and the addition of a saturated aq. solution of NaHC03 (~250 mL, pH ~8) and solid NaCl (50 g). The resulting mixture is stirred for 1 h and the organic layer is separated. The aq. layer is extracted with THF (2 X 150 mL). All organic layers are combined and evaporated to dryness. The residue is purified partially over a short silica gel (500 mL) column (10-20% MeOH in CHC13), then purified additionally by chromatography over silica gel (ISCO, 4 x 300 g cartridge, eluted with 0-10% MeOH in CH2C12) to afford the product.

Methods

XRD

XRPD figures were prepared using PatternMatch v2.3.6.XRPD.

Most XRPD patterns were 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 640d) was analyzed to verify the observed position of the Si 111 peak is consistent with the NIST-certified position. A specimen of the sample was sandwiched between 3^m-thick films and analyzed in transmission geometry. A beam-stop, short antiscatter extension, and antiscatter 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. 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.

Four XRPD patterns were collected with a PANalytical X'Pert PRO MPD diffractometer using an incident beam of Cu Ka radiation produced using a long, fine-focus source and a nickel filter. The diffractometer was configured using the symmetric Bragg-Brentano geometry. Prior to the analysis, a silicon specimen (NIST SRM 640d) was analyzed to verify the observed position of the Si 111 peak is consistent with the NIST-certified position. A specimen of the sample was prepared as a thin, circular layer centered on a silicon zero-background substrate. Antiscatter slits (SS) 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. Diffraction patterns were collected using a scanning position-sensitive detector (X'Celerator) located 240 mm from the sample and Data Collector software v. 2.2b.

Two XRPD patterns were collected with an Inel XRG-3000 diffractometer. An incident beam of Cu Ka radiation was produced using a fine-focus tube and a parabolically graded multilayer mirror. Prior to the analysis, a silicon specimen (NIST SRM 640d) was analyzed to verify the observed position of the Si 111 peak is consistent with the NIST-certified position. A specimen of the sample was packed into a thin-walled glass capillary, and a beam-stop was used to minimize the background from air. Diffraction patterns were collected in transmission geometry using Windif v. 6.6 software and a curved position-sensitive Equinox detector with a 2Θ range of 120°.

DSC

DSC was performed using a TA Instruments 2920 differential scanning calorimeter. Temperature calibration was performed using NIST-traceable indium metal. The sample was placed into an aluminum DSC pan, covered with a lid, and the weight was accurately recorded. A weighed aluminum pan configured as the sample pan was placed on the reference side of the cell. The sample was heated from -30 to 250 °C, at 10 °C/min. The method code on the thermogram is an abbreviation for the start and end temperature as well as the heating rate: (- 30)-250-10. The Tzero crimped pan configuration is abbreviated "TOC" in the comments field.

DSC data were obtained on a TA Instruments Q2000 differential scanning calorimeter equipped with a refrigerated cooling system (RCS). Temperature calibration was performed using NIST-traceable indium metal. The sample was placed into an aluminum DSC pan, and the weight was accurately recorded. The pan was covered with a lid, and the lid was crimped. A weighed, crimped aluminum pan was placed on the reference side of the cell. Data were obtained using a modulation amplitude of ± 1.0 °C and a 60 second period with an underlying heating rate of 2 °C/minute from 0 to 120 °C.

TGA

TGA analyses were performed using a TA Instruments 2950 thermogravimetric analyzer. Temperature calibration was performed using nickel and Alumel™. Each sample was placed in a platinum pan and inserted into the TG furnace. The furnace was heated under a nitrogen purge from ambient temperature to 350 °C, at 10 °C/min (abbreviated 00-350-10 in the method field on the thermogram).

Hot Stage Microscopy

Hot stage microscopy was performed using a Linkam hot stage (FTIR 600) mounted on a Leica DM LP microscope equipped with a SPOT Insight™ color digital camera. Temperature calibrations were performed using USP melting point standards. Samples were placed on a cover glass, and a second cover glass was placed on top of the sample. As the stage was heated, each sample was visually observed using a 20x 0.40 N.A. long working distance objective with crossed polarizers and a first order red compensator. Images were captured using SPOT software (v. 4.5.9).

Dynamic Vapor Sorption (DVS)

DVS data were collected on a VTI SGA-100 Vapor Sorption Analyzer. NaCl and PVP were used as calibration standards. The sample was not dried prior to analysis. Sorption and desorption data were collected over a range from 5% to 95% RH at 10% RH increments under a nitrogen purge. The equilibrium criterion used for analysis was less than 0.0100%) weight change in five minutes with a maximum equilibration time of three hours. Data were not corrected for the initial moisture content of the samples.

Proton NMR Spectroscopy

The solution proton NMR spectrum was acquired with an Agilent DD2-400 spectrometer. The sample was prepared by dissolving a portion in DMSO-d6 containing TMS.

XRPD Indexing

The XRPD patterns of Structure 1A were indexed using X'Pert High Score Plus [2.2a (2.2.1)].

Agreement between the allowed peak positions, marked with red bars in the figures, and the observed peaks indicates a consistent unit cell determination. Space groups consistent with the assigned extinction symbol, unit cell parameters, and derived quantities are tabulated below the figure. To confirm the tentative indexing solution, the molecular packing motifs within the crystallographic unit cells must be determined. No attempts at molecular packing were performed.

EXAMPLES

Example 1 : Synthesis of (S)-isopropyl 2-(((S)-(perfluorophenoxy)(phenoxy)phosphoryl)amino) propanoate (Compound 1)

CH₂CI₂/TEA

L-Alanine isopropyl ester HC1 salt (160 g) is charged in a 5 L four-necked flask equipped with mechanical stirrer, thermometer and dropping funnel. To the flask, dichloromethane (1 L) is added and the suspension is cooled to -70 °C, followed by addition of triethylamine (200 g, 276 mL) over 45 min. To the mixture is added a solution of phenyl dichlorophosphate (200 g) in dichloromethane (1 L) over 2.5 h. The reaction mixture is stirred at this temperature for an additional 90 min and then allowed to warm up to 0 °C over a period of 2 h and stirred for 2 h at 0 °C. To the mixture a solution of 2,3,4,5,6-pentafluorophenol (174.4 g) in 400 mL dichloromethane and a solution of triethylamine (105.4 g) in 200 mL dichloromethane are added dropwise simultaneously over a period of 1.2 h. The mixture is warmed to room temperature and stirred overnight.* The solid, triethylamine HC1 salt, is filtered off and the cake is washed with dichloromethane (3 x 150 mL). The filtrate is concentrated under reduced pressure and the residue triturated with MTBE (3.0 L). The white solid is removed by filtration. The cake is washed with MTBE (3 x 150 mL). The filtrate is concentrated and the resulting crude solid triturated with 20% ethyl acetate in hexane (2.0 L). The solid is collected by filtration and washed with 10%> NaHC03 until the aqueous phase reached pH 7, the solid is then washed with water and dried in a vacuum oven (55 °C) for 28 h. The dried solid is mixed with 500 mL heptane -EtO Ac (5 :1) and stirred for 1 h. The solid is collected by filtration and washed with heptane -EtO Ac (5: 1, 2 x 80 mL) to afford a >99%> single isomer. The solid is dried to give compound 1.

*In an alternative work-up procedure, the reaction mixture is filtered and the DCM layer is washed with an aq. 0.1 N NaOH solution, followed by water, dried, and evaporated to dryness. The residue is suspended in heptane/EtOAc (5: 1) and the solid is filtered. The solid is resuspended in heptane/toluene (85: 15) to isolate the pure single isomer.

Example 2: Preparation of Compound 4

4

2,2-Dimethoxypropane (140 mL) is added to commercially available 2'-C-methyluridine 2 (100 g) in acetone (700 mL). The resulting mixture is cooled in an ice bath for 30 min, then p- toluenesulfonic acid (11 g) is added and the reaction mixture is stirred at room temperature for 24 h. After completion of the reaction (monitored by HPLC), the reaction mixture is cooled in an ice bath for 30 min and neutralized using cold potassium carbonate (12 g in 13 mL water, pH 7-8). The solvent is removed under reduced pressure until dryness. THF (~500 mL) is added to the residue and the solids are removed by filtration. The filtrate is co-evaporated with silica gel and purified by chromatography over silica gel (5-15% MeOH in CHC13) to give compound 3. 1H NMR (400 MHz, DMSO-d6, 300 K): δ 1.22 (s, 3H), 1.34 (s, 3H), 1.49 (s, 3H), 3.63 (dd, J = 12.0 Hz, 2.8 Hz, 1H), 3.69 (dd, J = 12.0 Hz, 3.1 Hz, 1H), 4.15 (m, 1H), 4.47 (d, J = 2.8 Hz, 1H), 5.25 (br s, 1H), 5.63 (dd, J = 8.2 Hz, 2.3 Hz), 6.01 (s, 1H), 7.85 (d, J = 8.2 Hz, 1H), 11.37 (s, 1H); LC-MS: 299 amu (M + 1).

Compound 4 is prepared following the procedure reported by Corey et al. (J. Org. Chem. 1984, 49, 4735) with modifications described below. To acetonide 3 (50 g) in CH2C12 (1 L) is added PDC (126.1 g) at room temperature followed by Ac20 (171 g) and t-BuOH (248 g). The reaction temperature is maintained below 35 oC during the addition of reagents and then stirred at room temperature for 5 h. The reaction mixture is poured into aq. K2C03 (250 g K2C03 in 600 mL H20) and the organic layer is washed with CuS04 (100 g in 1 L H20). Activated charcoal (10 g) and silica gel (100 g) are added to the organic layer and stirred for 30 min and filtered. The filtrate is evaporated and residue purified by chromatography over silica gel (0-50% EtOAc in CHC13) to afford 4. 1H NMR (400 MHz, DMSO-d6, 300 K): δ 1.25 (s, 3H), 1.41 (s, 3H), 1.46 (s, 9H), 1.48 (s, 3H), 3.31 (s, 1H), 4.61 (s, 1H), 4.79 (s, 1H), 5.70 (dd, J = 8.1 Hz, 2.0 Hz, 1H), 5.93 (br s, 1H), 7.97 (d, J = 8.1 Hz, 1H), 11.41 (s, 1H); LC-MS: 369 amu (M + 1).

Example 3: Preparation of (S)-isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(5-deutero-2,4-dioxo-3,4- dihydropyrimidin- 1 (2H)-yl)-3 ,4-dihydroxy-4-methyltetrahydrofuran-2- yl)dideuteromethoxy)(phenoxy)phosphoryl)amino)propanoate

4 8

NaBD4 (7.96 g) is added in portions to a cooled (5 °C) 70:30 v/v mixture of EtOD/D20 (350 mL, 99% D) in a 1 L flask, followed by the addition of acetonide ester 4 (35 g) in portions (slowly bubbles). The resulting reaction mixture is stirred at room temperature for 3 h, and then heated at 80 °C for 1 d (1H NMR spectroscopic analysis indicates >85% deuterium incorporation at the 5 -uracil position). The reaction mixture is filtered to remove solids and concentrated under reduced pressure to remove EtOD. Additional D20 is added and the resulting mixture reheated at 95 °C to increase the deuterium incorporation at the 5 position to >98% (D-incorporation monitored by 1H NMR spectroscopy). After completion of the reaction, half the solvent is removed under reduced pressure, the mixture is cooled in an ice bath, AcOD (59 g) is added, and the resulting mixture is stirred for 15-20 min. EtOAc (300 mL) and brine (100 mL) are added, the organic layer is separated, and the aqueous layer is again extracted with EtOAc (150 mL), followed by THF (150 mL). The combined organic layers are concentrated, the resulting residue is dissolved in 10% MeOH and CHC13 (300 mL), filtered, concentrated, and purified by chromatography over silica gel (ISCO, eluent DCM/MeOH) to give the deuterated acetonide 8. 1H NMR (400 MHz, DMSO-d6, 300 K): δ 1.22 (s, 3H), 1.36 (s, 3H), 1.49 (s, 3H), 3.31 (s, 2H), 4.14 (d, J = 2.8 Hz, 1H), 4.47 (d, J = 2.8 Hz, 1H), 5.21 (s, 1H), 6.01 (s, 1H), 7.85 (s, 1H), 11.36 (s, 1H); LC-MS: 302 amu (M + 1).

Deuterated acetonide 8 (50 g) is added to a cooled (5 °C) 4N HC1 (250 mL) solution and stirred at room temperature for 3 h, during which time a white precipitate forms. The solvent is evaporated to dryness and to the residue is added water (100 mL) with stirring. The suspension is cooled to 5 °C, stirred for 1 h. and the white precipitate is collected by filtration. The solid is washed with cold water (75 mL) and dried to afford the deuterated nucleoside 9. 1H NMR (400 MHz, CD30D, 300 K): 5 1.15 (s, 3H), 3.84 (d, J = 9.2 Hz, 1H), 3.91 (d, J = 9.2 Hz, 1H), 5.96 (s, 1H), 8.14 (s, 1H); 13C NMR (100 MHz, CD30D, 300 K): δ 20.2, 73.4, 80.0, 83.8, 93.2, 142.4, 152.5, 166.0 (ribose C-5' and uracil C-5 not observed); LC-MS: 262 amu (M + 1).

Nucleoside 9 (37.3 g) in THF (750 mL) is cooled to -5 °C. t-BuMgCl (1M in THF, 430 mL) is added and the mixture is stirred for 30 min at the same temperature. The reaction mixture is stirred for another 30 minutes at room temperature, then cooled again to -5 °C, and a solution of 1 (129.5 g, from Example 1) in THF (650 mL) is added slowly. The reaction mixture is stirred at room temperature for 24 h., cooled to -5 °C, and to it added cold 2N HC1 (200 mL), followed by stirring for 10 min, and the addition of a saturated aq. solution of NaHC03 (~250 mL, pH ~8) and solid NaCl (50 g). The resulting mixture is stirred for 1 h and the organic layer is separated. The aq. layer is extracted with THF (2 X 150 mL). All organic layers are combined and evaporated to dryness. The residue is purified partially over a short silica gel (500 mL) column (10-20% MeOH in CHC13), then purified additionally by chromatography over silica gel (ISCO, 4 x 300 g cartridge, eluted with 0-10% MeOH in CH2C12) to afford the product. 1H NMR (400 MHz, CD30D, 300 K): 5 1.15 (s, 3H), 1.21 (2 x d, J = 6.3 Hz, 6H), 1.35 (dd, J = 7.2 Hz, JH,P = 0.9 Hz, 3H), 3.79 (d, J = 9.2 Hz, 1H), 3.91 (dq, JH,P = 10.0 Hz, J = 7.2 Hz, 1H), 4.08 (dd, J = 9.2 Hz, JH,P = 2.3 Hz, 1H), 4.96 (septet, J = 6.3 Hz, 1H), 5.96 (s, 1H), 7.20 (m, 1H), 7.26 (m, 2H), 7.37 (m, 2H), 7.67 (s, 1H); 3 IP NMR (162 MHz, CD30D, 300 K): δ 3.8; LC-MS: 531 amu (M + 1)·

The X-ray powder diffraction (XRPD) pattern for Structure 1 A, lot 3 is illustrated in FIG. 2. FIG. 3 illustrates the thermal data for Structure 1A, lot 3. FIG. 4 illustrates the modulated DSC data for Structure 1A, lot 3. FIG. 5 shows the 1H NMR spectrum for Structure 1A, lot 7.

Example 4: Preparation of Form A, Lots 285-183 and 258-91-2

Lot 285-183: Structure 1A (1 g) was dissolved in methanol (1 mL) and added dropwise to water (20 mL) (containing a seed of Form B); a white precipitate was formed. The suspension was stirred for 1 h at room temperature and for an additional 2 h at 5 °C. The solid was collected by vacuum filtration and dried under vacuum to afford Form A (0.850 g). Note: Although Form B seed was used, Form A was obtained.

Lot 258-91-2: Structure 1A (1 g) was heated in water (20 mL) at 50 °C until most of the material had dissolved; a small oily droplet remained at the bottom of the tube. The mixture was cooled and decanted from the oily droplet and left standing at room temperature for 4 h and an additional 2 h at 5 °C. The solid was filtered and dried under vacuum to afford Form A (0.650 g). Note: The lot 258-91-2 of Form A did contain some Form D material.

The XRPD pattern of Form A was obtained using the following experimental settings: voltage 45KV, amperage 40 mA, Cu(l .54059 A), scan range, 1.00 - 39.99°, step size 0.017, collection time: 718 s, scan speed 3.3°/min.

The XRPD pattern and indexing results for Form A are shown in FIG. 6. The characteristic peaks include: 6.83+0.2°, 10.12+0.2°, 13.72+0.2°, 16.75+0.2°, 17.68+0.2°, 19.19+0.2°, 19.53+0.2°, 21.90+0.2°, and 26.28+0.2°.

Indexing results

a[A] = 5.371; b[A] = 9.245; c[A] = 25.809. Volume [A3] = 1,280.6.

Thermal data

Thermal data were generated with Lot 258-91-2. DSC Analysis

Form A was heated at 10 °C/min over the range of -30 to 250 °C. The onset endotherm was found to be at about 137 °C, see, FIG.7 with a peak maximum at about 139 °C.

TGA Data

Form A was heated at 10 °C/min over the range of ambient temperature to 350 °C. The TGA data indicated that the Lot, which is predominantly Form A, is neither solvated nor hydrated: 0.2% wt% loss up to 140 °C.

Example 5: Preparation of Form B, Lots 258-108-3 and 285-179-180

Lot 258-108-3: Structure 1A (0.5 g) was dissolved in dichloromethane (5 mL) and ethyl ether (45 mL) was added to the solution. The mixture was stirred for 1 week at room temperature. The product was collected by vacuum filtration and dried to afford Form B (0.35 g).

Lot 285: 179-180: Structure 1A (12 g) was diluted with MTBE (120 mL), seed crystals of Form B and heated at reflux for 2 h. The mixture was cooled to room temperature and then held at 5 °C for 2 h. The product was collected using vacuum filtration and dried under vacuum to afford Form B (11.5 g).

The XPvPD pattern of Form B was obtained using the following experimental settings: 45KV, 40 niA, Cu(l .54059 A), scan range, 1.00 - 39.99°, step size 0.017, collection time: 719 s, scan speed 3.3°/min.

The XPvPD pattern for Form B is shown in Figure 9. The characteristic peaks include: 9.64+0.2°, 10.57+0.2°, 13.89+0.2°, 16.41+0.2°, 16.77+0.2°, 19.05+0.2°, 19.55+0.2° and 22.37+0.2°.

Thermal data

Thermal data were generated with Lot 258-179-180.

DSC Analysis

The sample was heated at 10 °C/min over the range of -30 to 250 °C. The onset endotherm was found to be about 134 °C, see, FIG. 8, onset, max at about 137 °C. TGA Data

Form B was heated at 10 °C/min over the range of ambient temperature to 350

FIG. 8; 0.2 wt% gain up to 140 °C.

Table 1 : Characterization of Form B, Lot 285: 179-180

Example 6: Preparation of Form C, Lot 258-99-1

Structure 1A (5 g) was dissolved in methanol (5 mL) and added to water (10 mL) containing seed crystals of Form B. Immediately after addition, some oily material was observed in the mixture which solidified on stirring. The mixture was stirred for 5 h at room temperature and for 2 h at 5 °C. The product was collected by vacuum filtration and dried under vacuum to afford Form C (3.7 g). Note: Although Form B seed was used Form C was obtained.

The XRPD pattern of Form C was obtained using the following experimental settings: 45KV, 40 niA, Cu(l .54059 A), scan range, 1.01 - 39.98°, step size 0.017, collection time: 721 s, scan speed 3.2°/min.

The XRPD pattern for Form C is shown in Figure 10. The characteristic peaks include: 10.12+0.2°, 16.66+0.2°, 16.85+0.2°, 18.23+0.2°, 19.22+0.2°, 20.15+0.2°, 22.07+0.2° and 23.11+0.2°.

Indexing results

a[A] = 5.322; b[A] = 9.251; c[A] = 26.447. Volume [A3] = 1,300.9. Example 7: Preparation of Form D, Lots 285-181-1 and 258-118-1

Lot 285-181-1 : Structure 1A (11.5 g) was heated in water (200 mL) at 70 °C for about 1 hour. The reaction was cooled to 55 °C, seed crystals (Forms C+D) were added and the reaction was stirred for 1 h. The reaction was cooled to and maintained at 5 oC for 1 h. The product was collected by vacuum filtration and dried to afford Form D (8.8 g).

Lot 258-118-1 : Structure 1A (5 g) was dissolved in hot 2-propanol (25 mL). The warm solution was filtered, cooled to room temperature and stirred overnight (-18 h). The product was collected by vacuum filtration and dried to afford Form D (3.0 g).

This procedure has been slightly modified for larger scale synthesis. After dissolving Structure 1A in hot 2-propanol, the solution is cooled, seeded with Form D and stirred at room temperature for 4 h. An equal volume of heptane is added and after stirring for 2 h at room temperature and 2 h at 5 °C the product is filtered and dried.

The XRPD pattern of Form D was obtained using the following experimental settings: 45KV, 40 niA, Cu(1.54059 A), scan range, 3.00 - 39.98°, step size 0.008, collection time: 1859 s, scan speed 1.2°/min.

The XRPD pattern and indexing results for Form D are shown in FIG. 11. The characteristic peaks include: 10.04+0.2°, 10.07+0.2°, 16.44+0.2°, 16.79+0.2°, 19.03+0.2°, 19.69+0.2°, 20.22+0.2°, 21.68+0.2°, and 22.60+0.2°.

Indexing results

a[A] = 9.366; b[A] = 50.921; c[A] = 26.447. Volume [A3] = 1,300.9.

Thermal data

Thermal data were generated with Lot 258-181-1

DSC Analysis

Form D was heated at 10 °C/min over the range of -30 to 250 °C. The onset endotherm was found to be about 137 °C, see FIG 12.

TGA Data

The Form D sample was heated at 10 °C/min over the range of ambient temperature to 350 °C; 0.2 wt% gain up to 140 oC. DVS Analysis

DVS analysis was conducted using Form D. The material was kept at 25 °C in nitrogen at humidities ranging from 5% to 95% relative humidity; each step was equilibrated for 120 min.

The sorption isotherm is shown in Figure 13. The material was found to be nonhygroscopic, showing less than 0.1 wt% change throughout the entire experiment. XRPD analysis of the sample following the DVS experiment indicated the sample remained Form D.

Example 8: Preparation of Form E, Lot 5447-34-01

The sample was prepared by adding ethyl ether to a solution of Structure 1A in dichloromethane, resulting in precipitation. The sample was left in the mother liquor, with agitation for part of the time. The crystalline material was observed 72 days after the experiment was initiated; however, it is not known at what point the sample actually crystallized. The material was designated Form E.

Form E is a crystalline, anhydrous material with a possible, although unconfirmed, melt at about 124 °C. Characterization data for Form E are summarized in Table 2.

The XRPD pattern of Form E was successfully indexed, as displayed in Figure 14. The successful indexing of the pattern indicates the sample is composed primarily of a single phase. The material was determined to be composed of Structure 1 A using proton NMR spectroscopy, see Figure 15. Based on the XRPD indexing and NMR data, the "Form" designation is used.

The XRPD pattern of Form E was obtained using the following experimental settings: 45KV, 40 niA, Cu(1.54059 A), scan range, 3.51 - 39.99°, step size 0.017, collection time: 1824 s, scan speed 1.2°/min. The characteristic peaks include: 7.88+0.2°, 10.60+0.2°, 12.35+0.2°, 17.15+0.2°, 17.79+0.2°, 17.86+0.2°, 19.60+0.2° and 20.07+0.2°.

Indexing results

a[A] = 5.279; b[A] = 14.542; c[A] = 16.730. Volume [A3] = 1,265.1.

Thermal data

Thermal data for Form E are displayed in Figure 16. TGA data indicated the sample is anhydrous. The DSC data revealed an endothermic transition with an onset at about 124 °C. This transition is similar to and likely a melt; however, a melt has not been confirmed experimentally. If this transition is a melt, it is noted as being both at a lower temperature and less energetic than the Form D melt. Therefore, these data suggest Form E is monotonically related and likely less stable.

Table 2: Characterization of Form E, Sample No. 5447-34-01

Claims

What is claimed is:

1. A morphic form of (S)-isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(5-deutero-2,4-dioxo-3,4- dihydropyrimidin- 1 (2H)-yl)-3 ,4-dihydroxy-4-methyltetrahydrofuran-2- yl)dideuteromethoxy)(phenoxy)phosphoryl)amino)propanoate (Form A) characterized by an X- ray powder diffraction (XRPD) pattern comprising the 2theta values of 16.75+0.2° and 19.19+0.2°.

2. The morphic form of claim 1, wherein the XRPD pattern comprises the 2theta values of 6.83+0.2°, 10.12+0.2°, 13.72+0.2°, 16.75+0.2°, 17.68+0.2°, 19.19+0.2°, 19.53+0.2°, 21.90+0.2°, and 26.28+0.2°.

3. The morphic form of claim 1 that has a differential scanning calorimetry (DSC) onset endotherm of about 137 °C.

4. The morphic form of claim 1 produced by a purification process comprising recrystallizing a crude preparation of (S)-isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(5-deutero-2,4- dioxo-3 ,4-dihydropyrimidin- 1 (2H)-yl)-3 ,4-dihydroxy-4-methyltetrahydrofuran-2- yl)dideuteromethoxy)(phenoxy)phosphoryl)amino)propanoate from water.

5. The morphic form of claim 1 produced by a purification process comprising recrystallizing a crude preparation of (S)-isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(5-deutero-2,4- dioxo-3 ,4-dihydropyrimidin- 1 (2H)-yl)-3 ,4-dihydroxy-4-methyltetrahydrofuran-2- yl)dideuteromethoxy)(phenoxy)phosphoryl)amino)propanoate from the solvent comprising the protic solvents water and methanol.

6. A pharmaceutical composition comprising the morphic form of claim 1 and a pharmaceutically acceptable excipient.

7. The pharmaceutical composition of claim 6, further comprising one or more therapeutic agents.

8. The pharmaceutical composition of claim 7, wherein the one or more additional therapeutic agents are selected from a caspase inhibitor, a cyclophilin inhibitor, a cytochrome P450 monooxygenase inhibitor, an entry inhibitor, a glucocorticoid, an HCV protease inhibitor, a hematopoietin, a homeopathic therapy, an immunomodulatory compound, an immunosuppressant, an interleukin, an interferon or interferon enhancer, an IRES inhibitor, a monoclonal or polyclonal antibody, a nucleoside or nucleotide analogue or prodrug, a non- nucleoside inhibitor, an NS4B inhibitor, an NS5A inhibitor, an NS5B inhibitor, a P7 protein inhibitor, a polymerase inhibitor, an RNAi compound, a therapeutic vaccine, a TNF agonist, a tubulin inhibitor, a sphingosine-1 -phosphate receptor modulator, or a TLR agonist.

9. A morphic form of (S)-isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(5-deutero-2,4-dioxo-3,4- dihydropyrimidin- 1 (2H)-yl)-3 ,4-dihydroxy-4-methyltetrahydrofuran-2- yl)dideuteromethoxy)(phenoxy)phosphoryl)amino)propanoate (Form B) characterized by an X- ray powder diffraction (XRPD) pattern comprising the 2theta values of 16.41+0.2° and 19.05+0.2°.

10. The morphic form of claim 9, wherein the XRPD pattern comprises the 2theta values of 9.64+0.2°, 10.57+0.2°, 13.89+0.2°, 16.41+0.2°, 16.77+0.2°, 19.05+0.2°, 19.55+0.2° and 22.37+0.2°.

11. The morphic form of claim 9 that has a differential scanning calorimetry (DSC) onset endotherm of about 134 °C.

12. The morphic form of claim 9 produced by a purification process comprising recrystallizing a crude preparation of (S)-isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(5-deutero-2,4- dioxo-3 ,4-dihydropyrimidin- 1 (2H)-yl)-3 ,4-dihydroxy-4-methyltetrahydrofuran-2- yl)dideuteromethoxy)(phenoxy)phosphoryl)amino)propanoate from the solvent comprising the aprotic solvents dichloromethane and ethyl ether.

13. The morphic form of claim 9 produced by a purification process comprising recrystallizing a crude preparation of (S)-isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(5-deutero-2,4- dioxo-3 ,4-dihydropyrimidin- 1 (2H)-yl)-3 ,4-dihydroxy-4-methyltetrahydrofuran-2- yl)dideuteromethoxy)(phenoxy)phosphoryl)amino)propanoate from methyl tert-butyl ether.

14. A pharmaceutical composition comprising the morphic form of claim 9 and a pharmaceutically acceptable excipient.

15. The pharmaceutical composition of claim 14, further comprising one or more additional therapeutic agents.

16. The pharmaceutical composition of claim 15, wherein the one or more additional therapeutic agents are selected from a caspase inhibitor, a cyclophilin inhibitor, a cytochrome P450 monooxygenase inhibitor, an entry inhibitor, a glucocorticoid, an HCV protease inhibitor, a hematopoietin, a homeopathic therapy, an immunomodulatory compound, an immunosuppressant, an interleukin, an interferon or interferon enhancer, an IRES inhibitor, a monoclonal or polyclonal antibody, a nucleoside or nucleotide analogue or prodrug, a non- nucleoside inhibitor, an NS4B inhibitor, an NS5A inhibitor, an NS5B inhibitor, a P7 protein inhibitor, a polymerase inhibitor, an RNAi compound, a therapeutic vaccine, a TNF agonist, a tubulin inhibitor, a sphingosine-1 -phosphate receptor modulator, or a TLR agonist.

17. A morphic form of (S)-isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(5-deutero-2,4-dioxo-3,4- dihydropyrimidin- 1 (2H)-yl)-3 ,4-dihydroxy-4-methyltetrahydrofuran-2- yl)dideuteromethoxy)(phenoxy)phosphoryl)amino)propanoate (Form C) characterized by an X- ray powder diffraction (XRPD) pattern comprising the 2theta values of 10.12+0.2° and 19.22+0.2°.

18. The morphic form of claim 17, wherein the XRPD pattern comprises the 2theta values of 10.12+0.2°, 16.66+0.2°, 16.85+0.2°, 18.23+0.2°, 19.22+0.2°, 20.15+0.2°, 22.07+0.2° and 23.11+0.2°.

19. The morphic form of claim 17 produced by a purification process comprising recrystallizing a crude preparation of (S)-isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(5-deutero-2,4- dioxo-3 ,4-dihydropyrimidin- 1 (2H)-yl)-3 ,4-dihydroxy-4-methyltetrahydrofuran-2- yl)dideuteromethoxy)(phenoxy)phosphoryl)amino)propanoate from the solvent comprising the protic solvents methanol and water.

20. A pharmaceutical composition comprising the morphic form of claim 17 and a pharmaceutically acceptable excipient.

21. The pharmaceutical composition of claim 20, further comprising one or more additional therapeutic agents.

22. The pharmaceutical composition of claim 21, wherein the one or more additional therapeutic agents are selected from a caspase inhibitor, a cyclophilin inhibitor, a cytochrome P450 monooxygenase inhibitor, an entry inhibitor, a glucocorticoid, an HCV protease inhibitor, a hematopoietin, a homeopathic therapy, an immunomodulatory compound, an immunosuppressant, an interleukin, an interferon or interferon enhancer, an IRES inhibitor, a monoclonal or polyclonal antibody, a nucleoside or nucleotide analogue or prodrug, a non- nucleoside inhibitor, an NS4B inhibitor, an NS5A inhibitor, an NS5B inhibitor, a P7 protein inhibitor, a polymerase inhibitor, an RNAi compound, a therapeutic vaccine, a TNF agonist, a tubulin inhibitor, a sphingosine-1 -phosphate receptor modulator, or a TLR agonist.

23. A morphic form of (S)-isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(5-deutero-2,4-dioxo-3,4- dihydropyrimidin- 1 (2H)-yl)-3 ,4-dihydroxy-4-methyltetrahydrofuran-2- yl)dideuteromethoxy)(phenoxy)phosphoryl)amino)propanoate (Form D) characterized by an X- ray powder diffraction (XRPD) pattern comprising the 2theta values of 16.44+0.2° and 19.03+0.2°.

24. The morphic form of claim 23, wherein the XRPD pattern comprises the 2theta values of 10.04+0.2°, 10.07+0.2°, 16.44+0.2°, 16.79+0.2°, 19.03+0.2°, 19.69+0.2°, 20.22+0.2°, 21.68+0.2°, and 22.60+0.2°.

25. The morphic form of claim 23 that has a differential scanning calorimetry (DSC) onset endotherm of about 137 °C.

26. The morphic form of claim 23 produced by a purification process comprising recrystallizing a crude preparation of (S)-isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(5-deutero-2,4- dioxo-3 ,4-dihydropyrimidin- 1 (2H)-yl)-3 ,4-dihydroxy-4-methyltetrahydrofuran-2- yl)dideuteromethoxy)(phenoxy)phosphoryl)amino)propanoate from water

27. The morphic form of claim 23 produced by a purification process comprising recrystallizing a crude preparation of (S)-isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(5-deutero-2,4- dioxo-3 ,4-dihydropyrimidin- 1 (2H)-yl)-3 ,4-dihydroxy-4-methyltetrahydrofuran-2- yl)dideuteromethoxy)(phenoxy)phosphoryl)amino)propanoate from 2-propanol.

28. The morphic form of claim 23 produced by a purification process comprising recrystallizing a crude preparation of (S)-isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(5-deutero-2,4- dioxo-3 ,4-dihydropyrimidin- 1 (2H)-yl)-3 ,4-dihydroxy-4-methyltetrahydrofuran-2- yl)dideuteromethoxy)(phenoxy)phosphoryl)amino)propanoate from the solvent comprising the solvents 2-propanol and heptane.

29. A pharmaceutical composition comprising the morphic form of claim 23 and a pharmaceutically acceptable excipient.

30. The pharmaceutical composition of claim 29, further comprising one or more additional therapeutic agents.

31. The pharmaceutical composition of claim 30, wherein the one or more additional therapeutic agents are selected from a caspase inhibitor, a cyclophilin inhibitor, a cytochrome P450 monooxygenase inhibitor, an entry inhibitor, a glucocorticoid, an HCV protease inhibitor, a hematopoietin, a homeopathic therapy, an immunomodulatory compound, an immunosuppressant, an interleukin, an interferon or interferon enhancer, an IRES inhibitor, a monoclonal or polyclonal antibody, a nucleoside or nucleotide analogue or prodrug, a non- nucleoside inhibitor, an NS4B inhibitor, an NS5A inhibitor, an NS5B inhibitor, a P7 protein inhibitor, a polymerase inhibitor, an R Ai compound, a therapeutic vaccine, a TNF agonist, a tubulin inhibitor, a sphingosine-1 -phosphate receptor modulator, or a TLR agonist.

32. A morphic form of (S)-isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(5-deutero-2,4-dioxo-3,4- dihydropyrimidin- 1 (2H)-yl)-3 ,4-dihydroxy-4-methyltetrahydrofuran-2- yl)dideuteromethoxy)(phenoxy)phosphoryl)amino)propanoate (Form E) characterized by an X- ray powder diffraction (XRPD) pattern comprising the 2theta values of 7.88+0.2° and 12.35+0.2°.

33. The morphic form of claim 32, wherein the XRPD pattern comprises the 2theta values of 7.88+0.2°, 10.60+0.2°, 12.35+0.2°, 17.15+0.2°, 17.79+0.2°, 17.86+0.2°, 19.60+0.2° and 20.07+0.2°.

34. The morphic form of claim 32 that has a differential scanning calorimetry (DSC) onset endotherm of about 124 °C.

35. The morphic form of claim 32 produced by a purification process comprising recrystallizing a crude preparation of (S)-isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(5-deutero-2,4- dioxo-3 ,4-dihydropyrimidin- 1 (2H)-yl)-3 ,4-dihydroxy-4-methyltetrahydrofuran-2- yl)dideuteromethoxy)(phenoxy)phosphoryl)amino)propanoate from the solvent comprising the aprotic solvents dichloromethane and ethyl ether.

36. A pharmaceutical composition comprising the morphic form of claim 32 and a pharmaceutically acceptable excipient.

37. The pharmaceutical composition of claim 36, further comprising one or more additional therapeutic agents.

38. The pharmaceutical composition of claim 37, wherein the one or more additional therapeutic agents are selected from a caspase inhibitor, a cyclophilin inhibitor, a cytochrome P450 monooxygenase inhibitor, an entry inhibitor, a glucocorticoid, an HCV protease inhibitor, a hematopoietin, a homeopathic therapy, an immunomodulatory compound, an immunosuppressant, an interleukin, an interferon or interferon enhancer, an IRES inhibitor, a monoclonal or polyclonal antibody, a nucleoside or nucleotide analogue or prodrug, a non- nucleoside inhibitor, an NS4B inhibitor, an NS5A inhibitor, an NS5B inhibitor, a P7 protein inhibitor, a polymerase inhibitor, an RNAi compound, a therapeutic vaccine, a TNF agonist, a tubulin inhibitor, a sphingosine-1 -phosphate receptor modulator, or a TLR agonist.

39. The use of a compound or composition of any of claims 1-38 in the manufacture of a medicament for treatment of a hepatitis C virus infection in a host.

40. The use of a compound or composition of claim 39, wherein the host is a human.