WO2016140615A1

WO2016140615A1 - Nucleotide derivatives which are hcv inhibitors for use in the treatment of hepatitis c

Info

Publication number: WO2016140615A1
Application number: PCT/SE2016/050166
Authority: WO
Inventors: Gennadiy KALAYANOV; Pedro Pinho; Paul TARGETT-ADAMS; Staffan Torssell
Original assignee: Medivir Ab
Priority date: 2015-03-02
Filing date: 2016-03-02
Publication date: 2016-09-09
Also published as: HK1243712A1; EP3271371A1; CN107531739A; EP3271371A4

Abstract

Use of a compound represented by formula (1A), or a pharmaceutically acceptable salt thereof, in the therapy of HCV in a mammal or human, wherein the compound of formula 1A is administered in combination with a further HCV antiviral selected from: a) asunaprevir, daclatasvir and/or beclabuvir; or b) simeprevir and/or JNJ56914845; or c) an HCV protease inhibitor selected from paritaprevir or ABT493, and an NS5A inhibitor selected from ombitasvir or ABT-530; or d) alisporavir; or e) EDP-239; or f) odalasvir and/or sovaprevir; or g) grazoprevir and/or elbasvir.

Description

NUCLEOTIDE DERIVATIVES WHICH ARE HCV INHIBITORS FOR USE IN THE TREATMENT OF

HEPATITIS C

Technical Field

The present invention relates to a specific nucleotide derivative which is an inhibitor of the polymerase of hepatitis C virus (HCV) for use in the treatment or prophylaxis of hepatitis C, including HCV genotype 3 virus infection. The invention further relates to combination therapy for HCV GT3 and all other genotypes with the nucleotide derivative and certain specified antiviral agents. Background of the Invention

HCV is a single stranded, positive-sense RNA virus belonging to the Flaviviridae family of viruses in the hepacivirus genus. The NS5B region of the RNA polygene encodes an RNA dependent RNA polymerase (RdRp), which is essential to viral replication. Following the initial acute infection, a majority of infected individuals develop chronic hepatitis because HCV replicates preferentially in hepatocytes but is not directly cytopathic. In particular, the lack of a vigorous T-lymphocyte response and the high propensity of the virus to mutate appear to promote a high rate of chronic infection. Chronic hepatitis can progress to liver fibrosis, leading to cirrhosis, end-stage liver disease and HCC (hepatocellular carcinoma), making it the leading cause of liver transplantations.

There are six major HCV genotypes and more than 50 subtypes, which are differently distributed geographically. HCV genotype 1 is the predominant genotype in Europe and in the US. The extensive genetic heterogeneity of HCV has important diagnostic and clinical implications, perhaps explaining difficulties in vaccine development and the lack of response to current therapy.

Transmission of HCV can occur through contact with contaminated blood or blood products, for example following blood transfusion or intravenous drug use. The introduction of diagnostic tests used in blood screening has led to a downward trend in post-transfusion HCV incidence. However, given the slow progression to the end-stage liver disease, the existing infections will continue to present a serious medical and economic burden for decades.

The first generation HCV therapies were based on (pegylated) interferon-alpha (IFN-a) in combination with ribavirin. This combination therapy yields a sustained virologic response in more than 40% of patients infected by genotype 1 viruses and about 80% of those infected by genotypes 2 and 3. Beside the limited efficacy on HCV genotype 1 , this combination therapy has significant side effects and is poorly tolerated in many patients. Major side effects include influenza-like symptoms, hematologic abnormalities and neuropsychiatric symptoms. The second generation of HCV treatments added the HCV protease inhibitors telepravir or boceprevir, allowing treatment times to be shortened, but generating a significant number of serious side-effects. A major improvement in treatment was possible with the introduction of the protease inhibitor simeprevir and the HCV polymerase inhibitor sofosbuvir. These were initially co-administered with interferon and ribavirin, but more recently the co-administration of simeprevir (WO2007/014926) and sofosbuvir (WO2008/121634) has allowed interferon-free and ribavirin-free HCV treatment with further diminished treatment times and dramatically decreased side effects.

An advantage of nucleoside/nucleotide HCV polymerase inhibitors such as sofosbuvir, is that they tend to be active against several of the HCV genotypes. Sofosbuvir for example has been approved by the FDA and EMA for treatment of HCV genotypes 1 and 4. However, in the Fission phase III clinical trials reported in Lawitz et al, N. Eng. J. Med. 2013; 368:1878-87, it was noted "Response rates in the sofosbuvir - ribavirin group were lower among patients with genotype 3 infection than amongst those with genotype 2 infection (56% vs. 97%)". Hence there is a need for more effective, convenient and better-tolerated treatments.

Experience with HIV drugs, in particular with HIV protease inhibitors, has taught that sub- optimal pharmacokinetics and complex dosing regimes quickly result in inadvertent compliance failures. This in turn means that the 24 hour trough concentration (minimum plasma concentration) for the respective drugs in an HIV regime frequently falls below the IC₉₀ or ED₉₀ threshold for large parts of the day. It is considered that a 24 hour trough level of at least the IC₅₀, and more realistically, the IC₉₀ or ED₉₀, is essential to slow down the development of drug escape mutants. Achieving the necessary pharmacokinetics and drug metabolism to allow such trough levels provides a stringent challenge to drug design.

The NS5B RdRp is absolutely essential for replication of the single-stranded, positive sense HCV RNA genome which makes it an attractive target for the development of antiviral compounds. There are two major classes of NS5B inhibitors: non-nucleoside inhibitors (NNIs) and nucleoside analogues. The NNIs bind to allosteric regions of the protein whereas the nucleoside inhibitors are anabolized to the corresponding nucleotide and act as alternative substrate for the polymerase. The formed nucleotide is then incorporated in the nascent RNA polymer chain and can terminate the growth of the polymer chain. To date, both nucleoside and non-nucleoside inhibitors of NS5B are known.

As stated above, the inhibition mechanism of nucleoside inhibitors involves phosphorylation of the nucleoside to the corresponding triphosphate. The phosphorylation is commonly mediated by host cell kinases and is an absolute requirement for the nucleoside to be active as an alternative substrate for the NS5B polymerase. Typically, the first phosphorylation step, i.e. conversion of the nucleoside to the nucleoside 5'-monophosphate is the rate limiting step. Subsequent conversion of the monophosphate to the di- and tri-phosphate usually proceed facile and are usually not rate limiting. A strategy for increasing nucleoside triphosphate production is to use cell permeable nucleoside prodrugs of the monophosphate, i.e. a nucleoside carrying a masked phosphate moiety, a "prodrug moiety", which are susceptible to intracellular enzymatic activation leading to a nucleoside monophosphate. The thus formed monophosphate is subsequently converted to the active triphosphate by cellular kinases.

Chemical modifications of an active compound to afford a potential prodrug produces an entirely new molecular entity which can exhibit undesirable physical, chemical and biological properties, thus the identification of optimal prodrugs remains an uncertain and challenging task.

There is a need for HCV inhibitors that may overcome the disadvantages of current HCV therapy such as side effects e.g. toxicity, limited efficacy, lack of pan-genotypic coverage, the emerging of resistance, and compliance failures, as well as improve the sustained viral response.

The present invention provides a new HCV inhibiting compound for use in the treatment of hepatitis C genotype 3 virus infection and which has useful properties regarding one or more of the following parameters: antiviral efficacy; pan-genotypic coverage; favourable profile of resistance development; lack of toxicity and genotoxicity; favourable pharmacokinetics and pharmacodynamics; and ease of formulation and administration. The skilled person will appreciate that an HCV inhibiting compound for use in the treatment of hepatitis C genotype 3 virus infection of the present invention need not demonstrate an improvement in every respect over all known compounds but may instead provide a balance of properties which in combination mean that the HCV inhibiting compound is a valuable alternative pharmaceutical agent.

Compounds for use according the invention may also be attractive due to the fact that they lack activity against other viruses, i.e. are selective, in particular against HIV. HIV infected patients often suffer from co-infections such as HCV. Treatment of such patients with an HCV inhibitor that also inhibits HIV may lead to the emergence of resistant HIV strains. Description of the Invention

In one aspect, the present invention provides the diastereomeric Compound 1 A:

and pharmaceutically acceptable salts thereof.

A further aspect of the invention provides the diastereomeric Compound 1A for use in the treatment or prophylaxis of hepatitis C genotype 2a, 3a, 4a, 5a or 6a virus infection

In a specific embodiment, the invention relates to a diastereomeric compound of formula (1 A) for use in the treatment of hepatitis C genotype 3a.

The compound of formula (1 A) may optionally be provided in the form of a pharmaceutically acceptable salt and/or solvate. In one embodiment the compound for use according to the invention is provided in the form of a pharmaceutically acceptable salt. In a second embodiment the compound for use according to the invention is provided in the form of a pharmaceutically acceptable solvate. In a third embodiment the compound for use according to the invention is provided in its free form.

The active metabolite of the diastereomeric compound of formula (1 A) is believed to be the triphosphate,having the structure:

where U is O, R² is H and the base is uracil.

In a preferred embodiment, the invention provides a compound of formula (1 A)

for use in the treatment of hepatitis C genotype 2a, 3a, 4a, 5a or 6a infection. Preferably, the invention relates to a compound of formula (1 A) for use in the treatment of hepatitis C genotype 3a. Consequently, there is provided a compound of formula (1A) for use in the treatment or prophylaxis of HCV genotype 3 infection. In a preferred aspect, the invention provides compounds of formula (1 A) for use in the treatment of HCV genotype 3 infection in humans.

In an additional aspect, the invention provides compounds of formula (1 A) for use in the prophylaxis of HCV genotype 3 infection in humans.

In a preferred embodiment of this aspect, a compound of formula (1 A) is provided

Additionally, there is provided a compound of formula (1 A) for use as a medicament for the treatment or prophylaxis of HCV genotype 3 infection, especially the treatment of HCV genotype 3 infection.

Further provided is the use of a compound of formula (1A) in the manufacture of a medicament for the treatment or prophylaxis of HCV genotype 3 infection, especially a medicament for the treatment of HCV genotype 3 infection.

In a further aspect, the invention provides pharmaceutical compositions comprising a compound of formula (1 A) in association with a pharmaceutically acceptable adjuvant, diluent, excipient or carrier for use in the treatment of hepatitis C genotype 3 infection. The pharmaceutical composition will typically contain an antivirally effective amount (e.g. for humans) of the compound of formula (1A), although sub-therapeutic amounts of the compound of formula (1A) may nevertheless be of value when intended for use in combination with other agents or in multiple doses. One aspect of the invention relates to a pharmaceutical composition that comprises, in a pharmaceutically acceptable vehicle, carrier or diluent: a first compound, with the formula

Compound 1A

or a pharmaceutically acceptable salt thereof, and one to three additional HCV antivirals selected from:

a) asunaprevir;

b) daclatasvir; and

c) beclubavir.

The invention thus provides antiviral compositions and HCV antiviral treatment regimes comprising:

Compound 1 A and asunaprevir;

Compound 1 A, asunaprevir and daclatasvir;

Compound 1 A and daclatasvir;

Compound 1 A, asunaprevir and beclabuvir

Compound 1 A, asunaprevir, daclatasvir and beclabuvir

Compound 1 A, daclatasvir and beclabuvir

Compound 1 A and beclabuvir,

wherein Compound 1 A in each case includes its pharmaceutically acceptable salts.

Asunaprevir, (INN), has shown to be effective in inhibiting HCV replication. The aforementioned compound can be obtained using methods known to those skilled in the art, including, for example, those methods disclosed in WO2003/099274, and WO2009/085659, which are hereby incorporated by reference in its entirety. Although this invention is not limited by any particular theory, asunaprevir is believed to inhibit the HCV protease, in particular the NS3/4A protease. Pharmaceutically acceptable salts of asunaprevir can be utilized in the compositions described herein.

Daclatasvir (INN), has been demonstrated to be effective in inhibiting HCV replication. Daclatasvir can be obtained using methods known to those skilled in the art, such as those methods described in WO2008/021927 & 21928 and WO2009/20828, which is hereby incorporated by reference in its entirety. Although this invention is not limited by any particular theory, it is believed that daclatasvir is an NS5A inhibitor where NS5A is a protein involved in the replication of the hepatitis C virus.

Beclabuvir (INN) has been demonstrated to be effective in inhibiting HCV replication. Beclauvir can be obtained using methods known to those skilled in the art, such as those methods described in WO2014/014885 and WO2007/136982, which is hereby incorporated by reference in its entirety. Although this invention is not limited by any particular theory, it is believed that beclabuvir is a non-nucleoside inhibitor of the HCV NS5A polymerase.

In some embodiments, the composition can further include a pharmaceutically acceptable excipient, diluent and/or carrier, such as those described herein. In general it is contemplated that an antiviral effective daily amount of Compound 1 A within the combination treatment of the invention, would be from about 0.01 to about 700 mg/kg, or about 0.5 to about 400 mg/kg, or about 1 to about 250 mg/kg, or about 2 to about 200 mg/kg, or about 10 to about 150 mg/kg, or about 0.1 to about 50 mg/kg, or about 1 to about 20 mg/kg, or about 2 to about 10 mg/kg, or about 5 to about 8 mg/kg body weight. It may be appropriate to administer the required dose as one, two, three, four or more sub-doses at appropriate intervals throughout the day. Said sub-doses may be formulated as unit dosage forms, for example, containing about 1 to about 5000 mg Compound 1 , or about 50 to about 3000 mg Compound 1 , or about 100 to about 1000 mg Compound 1 , or about 200 to about 600 mg Compound 1 , or about 100 to about 400 mg Compound 1 , or about 7 to about 3500 mg Compound 1 , or about 70 to about 1400 mg Compound 1 A, or about 140 to about 700 mg Compound 1 , or about 340 to about 580 mg of Compound 1 A per unit dosage form. Preferably the unit dosage form is formulated for QD dosing with one of the above ranges.

Similarly, various amounts of asunaprevir or a pharmaceutically acceptable salt, can he included in the compositions. In some embodiments, the composition can include an amount of asunaprevir, or a pharmaceutically acceptable salt thereof in the range of about 50 mg to about 500 mg QD or BID. In other embodiments, the composition can include an amount of asunaprevir, or a pharmaceutically acceptable salt thereof in the range of about 100 mg to about 300 mg QD or BID. In still other embodiments, the composition can include an amount of asunaprevir, or a pharmaceutically acceptable salt thereof, at a dosage of about 100 mg 150 or 200 mg BID or preferably QD.

Similarly, various amounts of daclatasvir, or a pharmaceutically acceptable salt thereof can be included in the compositions. In some embodiments, the composition can include an amount of daclatasvir, or a pharmaceutically acceptable salt thereof in the range of about 10 mg to about 225 mg BID or QD. In other embodiments, the composition can include an amount of daclatasvir, or a pharmaceutically acceptable salt thereof, in the range of about 15 mg to about 100 mg BID or preferably QD. In still other embodiments, the composition can include an amount of daclatasvir or a pharmaceutically acceptable salt thereof in the range of about 30-60 mg QD. In yet still other embodiments, a loading dose of 30 - 100 mg such as 30, 50 or 60 mg declatasvir or pharmaceutically acceptable salt thereof can be administered for the initial 1 -3 days of therapy.

Similarly, various amounts of beclabuvir, or a pharmaceutically acceptable salt thereof can be included in the compositions. In some embodiments, the composition can include an amount of beclabuvir, or a pharmaceutically acceptable salt thereof in the range of about 25 mg to about 225 mg BID or QD. In other embodiments, the composition can include an amount of beclabuvir, or a pharmaceutically acceptable salt thereof, in the range of about 50 mg to about 100 mg QD or typically BID. In still other embodiments, the composition can include an amount of beclabuvir or a pharmaceutically acceptable salt thereof in the range of about 75 mg BID or QD. A potential advantage of making a combination of Compound 1 A; and asunaprevir, daclatasvir and/or beclabuvir or pharmaceutically acceptable salts thereof may be a reduction in the required amounts of one or more compounds that are effective in treating a disease condition disclosed herein (for example, HCV) as compared to monotherapy treatment of an otherwise comparable patient population using any one component alone needed to achieve the same viral load reduction when administered as a monotherapy.

In general however, it is often convenient to administer Compound 1A, and asunaprevir, daclatasvir and/or beclabuvir at dosages reflecting monotherapy dosage regimes for the respective component(s), and thereby provide an enhanced antiviral efficacy and/or onset of action. Additional advantages of utilizing a combination of Compound 1 A and asunaprevir, daclatasvir and/or beclabuvir or pharmaceutically acceptable salts thereof may include little to no cross resistance between the respective components; different routes for elimination of the respective components; little, to no significant effects on cytochrome P 450; and/or little to no pharmacokinetic interactions between the respective components. The percentages of Compound 1A, and asunaprevir, daclatasvir and/or beclabuvir or pharmaceutically acceptable salts thereof present in the composition can also vary. For example, in some embodiments, the composition can include an amount of Compound 1A, or a pharmaceutically acceptable salt thereof, in the range of about 1 % to about 98% (weight/weight) based on the sum of the amount of Compound 1 A, or a pharmaceutically acceptable salt thereof and the amount of and asunaprevir, daclatasvir and/or beclabuvir or pharmaceutically acceptable salts thereof, in the composition. Additional embodiments include, but are not limited to, an amount of Compound 1A, or a pharmaceutically acceptable salt thereof in the range of about 5% to about 80%, about 10% to about 70%, about 15% to about 60%, about 20% to about 50% and about 30% to about 40% (weight/weight) based on the sum of the amount of Compound 1A, or a pharmaceutically acceptable salt thereof, and the amount of and asunaprevir, daclatasvir and/or beclabuvir or pharmaceutically acceptable salts thereof in the composition.

As to asunaprevir, in an embodiment, the composition can include an amount of asunaprevir, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 1 % to about 98% (weight/weight) based on the sum of the amount of asunaprevir and the other components in the composition. Examples of additional embodiments, include, but are not limited to, an amount of asunaprevir, or a pharmaceutically acceptable salt thereof in the range of about 5% to about 80%, about 10% to about 70%, about 15% to about 60%, about 20% to about 50% and about 30% to about 40 (weight/ weight) based on the sum of the amount of asunaprevir and the other components in the composition.

As to daclatasvir, in an embodiment, the composition can include an amount of daclatasvir, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 1 % to about 98% (weight/weight) based on the sum of the amount of daclatasvir, or a pharmaceutically acceptable salt or prodrug thereof, and the amount of the other components in the composition. Examples of additional embodiments include, but are not limited, to, an amount of daclatasvir, or a pharmaceutical y acceptable salt thereof, in the range of about 5% to about 80%, about 10% to about 70%, about 15% to about 60%, about 20% to about 50% and about 30% to about 40% (weight/weight) based on the sum of the amount of daclatasvir, or a pharmaceutically acceptable salt thereof, and the amount of the other components in the composition.

As to beclabuvir, in an embodiment, the composition can include an amount of beclabuvir, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 1 % to about 98% (weight/weight) based on the sum of the amount of beclabuvir, or a pharmaceutically acceptable salt or prodrug thereof, and the amount of the other components in the composition. Examples of additional embodiments include, but are not limited, to, an amount of beclabuvir, or a pharmaceutically acceptable salt thereof, in the range of about 5% to about 80%, about 10% to about 70%, about 15% to about 60%, about 20% to about 50% and about 30% to about 40% (weight/weight) based on the sum of the amount of beclabuvir, or a pharmaceutically acceptable salt thereof, and the amount of the other components in the composition. In general it is contemplated that an antiviral effective daily amount of Compound 1 A within the combination treatment of the invention, would be from about 0.01 to about 700 mg/kg, or about 0.5 to about 400 mg/kg, or about 1 to about 250 mg/kg, or about 2 to about 200 mg/kg, or about 10 to about 150 mg/kg, or about 0.1 to about 50 mg/kg, or about 1 to about 20 mg/kg, or about 2 to about 10 mg/kg, or about 5 to about 8 mg/kg body weight. It may be appropriate to administer the required dose as one, two, three, four or more sub-doses at appropriate intervals throughout the day. Said sub-doses may be formulated as unit dosage forms, for example, containing about 1 to about 5000 mg Compound 1A, or about 50 to about 3000 mg Compound 1A, or about 100 to about 1000 mg Compound 1 A, or about 200 to about 600 mg Compound 1A, or about 100 to about 400 mg Compound 1A, or about 7 to about 3500 mg Compound 1A, or about 70 to about 1400 mg Compound 1 A, or about 140 to about 700 mg Compound 1 A, or about 340 to about 580 mg of Compound 1 A per unit dosage form. Preferably the unit dosage form is formulated for QD dosing with one of the above ranges.

Similarly, various amounts of asunaprevir or a pharmaceutically acceptable salt, can he included in the compositions. In some embodiments, the composition can include an amount of asunaprevir, or a pharmaceutically acceptable salt thereof in the range of about 50 mg to about 500 mg QD or BID. In other embodiments, the composition can include an amount of asunaprevir, or a pharmaceutically acceptable salt thereof in the range of about 100 mg to about 300 mg QD or BID. In still other embodiments, the composition can include an amount of asunaprevir, or a pharmaceutically acceptable salt thereof, at a dosage of about 100mg 150 or 200 mg BID or preferably QD.

In general however, it is often convenient to administer Compound 1 A, and other antivirals at dosages reflecting monotherapy dosage regimes for the respective component(s), and thereby provide an enhanced antiviral efficacy and/or onset of action.

The percentages of Compound 1A, and asunaprevir, daclatasvir and/or beclabuvir or pharmaceutically acceptable salts thereof present in the composition can also vary. For example, in some embodiments, the composition can include an amount of Compound 1A, or a pharmaceutically acceptable salt thereof, in the range of about 1 % to about 98% (weight/weight) based on the sum of the amount of Compound 1 A, or a pharmaceutically acceptable salt thereof and the amount of and asunaprevir, daclatasvir and/or beclabuvir or pharmaceutically acceptable salts thereof, in the composition. Additional embodiments include, but are not limited to, an amount of Compound 1A, or a pharmaceutically acceptable salt thereof in the range of about 5% to about 80%, about 10% to about 70%, about 15% to about 60%, about 20% to about 50% and about 30% to about 40% (weight/weight) based on the sum of the amount of Compound 1A, or a pharmaceutically acceptable salt thereof, and the amount of and asunaprevir, daclatasvir and/or beclabuvir or pharmaceutically acceptable salts thereof in the composition.

As to asunaprevir, in an embodiment, the composition can include an amount of asunaprevir, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 1 % to about 98% (weight/weight) based on the sum of the amount of asunaprevir and the other components in the composition. Examples of additional embodiments, include, but are not limited to, an amount of asunaprevir, or a pharmaceutically acceptable salt thereof in the range of about 5% to about 80%, about 10% to about 70%, about 15% to about 60%, about 20% to about 50% and about 30% to about 40 (weight/ weight) based on the sum of the amount of asunaprevir and the other components in the composition. As to daclatasvir, in an embodiment, the composition can include an amount of daclatasvir, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 1 % to about 98% (weight/weight) based on the sum of the amount of daclatasvir, or a pharmaceutically acceptable salt or prodrug thereof, and the amount of the other components in the composition. Examples of additional embodiments include, but are not limited, to, an amount of daclatasvir, or a pharmaceutical y acceptable salt thereof, in the range of about 5% to about 80%, about 10% to about 70%, about 15% to about 60%, about 20% to about 50% and about 30% to about 40% (weight/weight) based on the sum of the amount of daclatasvir, or a pharmaceutically acceptable salt thereof, and the amount of the other components in the composition. As to beclabuvir, in an embodiment, the composition can include an amount of beclabuvir, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 1 % to about 98% (weight/weight) based on the sum of the amount of beclabuvir, or a pharmaceutically acceptable salt or prodrug thereof, and the amount of the other components in the composition. Examples of additional embodiments include, but are not limited, to, an amount of beclabuvir, or a pharmaceutically acceptable salt thereof, in the range of about 5% to about 80%, about 10% to about 70%, about 15% to about 60%, about 20% to about 50% and about 30% to about 40% (weight/weight) based on the sum of the amount of beclabuvir, or a pharmaceutically acceptable salt thereof, and the amount of the other components in the composition.

Some embodiments described herein relate to a method for ameliorating or treating a disease condition that, can include administering an amount of Compound I, or a pharmaceutically acceptable salt thereof, and an amount of asunaprevir, daclatasvir and/or beclabuvir or pharmaceutically acceptable salts thereof wherein the disease condition can be a hepatitis C virus infection, liver fibrosis, and/or impaired liver function.

Various dosages forms of Compound IA or a pharmaceutically acceptable salt thereof, asunaprevir, daclatasvir and/or beclabuvir or pharmaceutically acceptable salts thereof can be used to ameliorate and/or treat a disease condition. In some instances, Compounds 1 A and asunaprevir, daclatasvir and/or beclabuvir or pharmaceutically acceptable salts thereof can be present in the same dosage form such as the compositions described herein. In other instances, Compound 1 A, asunaprevir, daclatasvir and/or beclabuvir or pharmaceutically acceptable salts thereof, can be administered as separate dosage forms. For example, Compound 1A, or a pharmaceutically acceptable salt thereof, can be administered in one tablet, asunaprevir (if present in the regime), can be administered in a second tablet, daclatasvir (if present) can be administered in a separate tablet and beclabuvir (if present) can be administeredin yet another tablet. When Compounds 1 A and asunaprevir, daclatasvir and/or beclabuvir or pharmaceutically acceptable salts thereof, are contained in separate dosage forms, the dosage forms can be the same (e.g., as both pills) or different (e.g., two compounds can be formulated in a pill and the other compound can fee formulated as art injectable).

Administration of Compound IA or a pharmaceutically acceptable salt thereof; and asunaprevir, daclatasvir and/or beclabuvir or pharmaceutically acceptable salts thereof can vary. When Compounds IA, and the other components are contained in separate dosage forms, the dosage forms can be administered simultaneously or sequentially. In some embodiments, the dosage form that contains Compound 1A, or pharmaceutically acceptable salt thereof, can be administered before, after, in-between, concurrently or sequentially with asunaprevir (if present), delcatasvir (if present) and beclubavir (if present in the regime) In some embodiments, Compounds IA; and asunaprevir, daclatasvir and/or beclabuvir or pharmaceutically acceptable salts thereof, or pharmaceutically acceptable salts or prodrugs thereof can be administered concurrently. As used herein, the term "concurrently means effective concentrations of all two, three or four compounds are present in a subject. When being administered concurrently, Compound 1 A, and asunaprevir, daclatasvir and/or beclabuvir or pharmaceutically acceptable salts thereof, can be administered in the same dosage form or separate dosage forms. In other embodiments, Compound 1A, and asunaprevir, daclatasvir and/or beclabuvir or pharmaceutically acceptable salts thereof can be administered sequentially. As used herein, the term "sequentially" means administering one compound for a first time period, then administering a second compound for a second time period, optionally followed by administering a third compound for a third period, in which the first, second, and if applicable third time periods do not overlap.

One aspect of the invention relates to a pharmaceutical composition that comprises, in a pharmaceutically acceptable vehicle, carrier or diluent: a first compound, with the formula

or a pharmaceutically acceptable salt thereof, and a) a second compound, or a pharmaceutically acceptable salt or prodrug thereof wherein the second compound is simeprevir ("Compound 2"); and/or b) a third compound, or a pharmaceutically acceptable salt or prodrug thereof, wherein she third compound is GSK 2336805 (also known as JNJ56914845) ("Compound 3").

In another aspect the invention relates to a composition comprising, in a pharmaceutically acceptable vehicle, carrier or diluent Compound 1 , and Compound 2 and/or Compound 3, or pharmaceutically acceptable salts or prodrugs thereof, wherein the composition additionally comprises one or more therapeutic agents. In one embodiment the one or more therapeutic agents are ribavirin and ritonavir.

Simeprevir, (INN), (Compound 2) has shown to be effective in inhibiting HCV replication. The aforementioned compound can be obtained using methods known to those skilled in the art, including, for example, those methods disclosed in WO2013/061285, WO2008/092955 and WO2013/041655, which are hereby incorporated by reference in its entirety. Although this invention is not limited by any particular theory, Compound 2 is believed to inhibit the HCV protease, in particular the NS3/4A protease. Pharmaceutically acceptable salts and prodrugs of Compound 2 can be utilized in the compositions described herein. JNJ56914845 (also known as GSK2336805) (Compound 3) has been demonstrated to be effective in inhibiting HCV replication. Compound 3 is believed to have the structure:

and can be obtained using methods known to those skilled in the art, such as those methods described in WO201 1 /028596, which is hereby incorporated by reference in its entirety. Although this invention is not limited by any particular theory, it is believed that Compound 3 is an NS5A inhibitor where NS5A is a protein involved in the replication of the hepatitis C virus.

It will be appreciated that one aspect of the invention provides pharmaceutical compositions comprising, in a pharmaceutically acceptable vehicle, carrier or diluent, Compound 1 A and simeprevir. A further aspect of the invention provides pharmaceutical compositions comprising, in a pharmaceutically acceptable vehicle, carrier or diluent, Compound 1A and GSK2336805. A further aspect of the invention provides pharmaceutical compositions comprising, in a pharmaceutically acceptable vehicle, carrier or diluent, Compound 1 A and simeprevir and GSK2336805. In general it is contemplated that an antiviral effective daily amount of Compound 1 A within the combination treatment of the invention, would be from about 0.01 to about 700 mg/kg, or about 0.5 to about 400 mg/kg, or about 1 to about 250 mg/kg, or about 2 to about 200 mg/kg, or about 10 to about 150 mg/kg, or about 0.1 to about 50 mg/kg, or about 1 to about 20 mg/kg, or about 2 to about 10 mg/kg, or about 5 to about 8 mg/kg body weight. It may be appropriate to administer the required dose as one, two, three, four or more sub-doses at appropriate intervals throughout the day. Said sub-doses may be formulated as unit dosage forms, for example, containing about 1 to about 5000 mg Compound 1A, or about 50 to about 3000 mg Compound 1A, or about 100 to about 1000 mg Compound 1 A, or about 200 to about 600 mg Compound 1A, or about 100 to about 400 mg Compound 1A, or about 7 to about 3500 mg Compound 1A, or about 70 to about 1400 mg Compound 1 A, or about 140 to about 700 mg Compound 1 A, or about 340 to about 580 mg of Compound 1 A per unit dosage form. Preferably the unit dosage form is formulated for QD dosing with one of the above ranges.

Similarly, various amounts of Compound 2, or a pharmaceutically acceptable salt or prodrug thereof, can he included in the compositions. In some embodiments, the composition can include an amount of Compound 2, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 50 mg to about 300 mg. In other embodiments, the composition can include an amount of Compound 2, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 100 mg to about 200 mg. In still other embodiments, the composition can include an amount of Compound 2, or a pharmaceutically acceptable salt or prodrug thereof, in an amount of 100, 125 or 150 mg.

Similarly, various amounts of Compound 3, or a pharmaceutically acceptable salt or prodrug thereof can be included in the compositions. In some embodiments, the composition can include an amount of Compound 3, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 10 mg to about 100 mg. In other embodiments, the composition can include an amount of Compound 3, or a pharmaceutically acceptable salt or prodrug thereof, in the range of about 20 mg to about 60 mg. In still other embodiments, the composition can include an amount of Compound 3 or a pharmaceutically acceptable salt or prodrug thereof in the range of about 30-50 mg. A potential advantage of making a combination of Compound 1 A and Compound 2, and/or 3, or pharmaceutically acceptable salts or prodrugs thereof may be a reduction in the required amounts of one or more compounds that are effective in treating a disease condition disclosed herein (for example, HCV) as compared to monotherapy treatment of an otherwise comparable patient population using either Compound 1 A, 2 or 3 alone. In some embodiments, the amount of Compound 1A or a pharmaceutically acceptable salt thereof in the composition can be less compared to the amount of Compound I or a pharmaceutically acceptable salt thereof needed to achieve the same viral load reduction when administered as a monotherapy. In some embodiments, the amount of Compound 2 or a pharmaceutically acceptable salt or prodrug thereof in the composition can be less compared to the amount of Compound 2, or a pharmaceutically acceptable salt or prodrug thereof, needed to achieve the same viral load reduction when administered as a monotherapy. In some embodiments, the amount of Compound 3 or a pharmaceutically acceptable salt or prodrug thereof in the composition can be less compared to the amount of Compound 3 or a pharmaceutically acceptable salt or prodrug thereof needed to achieve the same viral load reduction when administered as a monotherapy. In an embodiment, the sum of the amount of Compound 1A or a pharmaceutically acceptable salt and the amount of Compound 2, or a pharmaceutically acceptable salt or prodrug thereof and/or the amount of Compound 3, or a pharmaceutically acceptable salt or prodrug thereof is less than expected or predicted based on the additive combination of Compound 1 A or a pharmaceutically acceptable salt alone, Compound 2, or a pharmaceutically acceptable salt or prodrug thereof alone, and/or Compound 3, or a pharmaceutically acceptable salt or prodrug thereof alone for treating the disease condition such as HCV.

In general however, it is often convenient to administer Compound 1A, and Compound 2 and/or Compound 3 at dosages reflecting monotherapy dosage regimes for the respective component(s), and thereby provide an enhanced antiviral efficacy and/or onset of action. Additional advantages of utilizing a combination of Compound 1A and Compound, 2, and/or 3, or pharmaceutically acceptable salts or prodrugs thereof may include little to no cross resistance between Compound 1 A, and Compounds 2 and/or 3, or pharmaceutically acceptable salts or prodrugs thereof; different routes tor elimination of Compound 1A, and Compounds 2, and/or 3 or pharmaceutically acceptable salts or prodrugs thereof; little to no overlapping toxicities between Compound 1 A, and Compound 2, and/or 3 or pharmaceutically acceptable salts or prodrugs thereof; little, to no significant effects on cytochrome P 450; and/or little to no pharmacokinetic interactions between Compound 1A, and Compound 2, and/or 3 or pharmaceutically acceptable salts or prodrugs thereof.

The percentages of Compound 1 A, and Compound 2, and/or 3 or pharmaceutically acceptable salts or prodrugs thereof present in the composition can also vary. For example, in some embodiments, the composition can include an amount of Compound 1 A, or a pharmaceutically acceptable salt thereof, in the range of about 1 % to about 98% (weight/weight) based on the sum of the amount of Compound 1 A, or a pharmaceutically acceptable salt thereof and the amount of Compounds 2 and/or 3 or pharmaceutically acceptable salts or prodrugs thereof, in the composition. Additional embodiments include, but are not limited to, an amount of Compound 1 A, or a pharmaceutically acceptable salt thereof in the range of about 5% to about 80%, about 10% to about 70%, about 15% to about 60%, about 20% to about 50% and about 30% to about 40% (weight/weight) based on the sum of the amount of Compound 1A, or a pharmaceutically acceptable salt thereof, and the amount of Compounds 2 and/or 3, or pharmaceutically acceptable safe or prodrugs thereof in the composition.

As to Compound 2, in an embodiment, the composition can include an amount of Compound 2, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 1 % to about 98% (weight/weight) based on the sum of the amount of Compound 2, or a pharmaceutically acceptable salt or prodrug thereof and the amount of Compounds 1 and/or 3, or pharmaceutically acceptable salts or prodrugs thereof, in the composition. Examples of additional embodiments, include, but are not limited to, an amount of Compound 2, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 5% to about 80%, about 10% to about 70%, about 15% to about 60%, about 20% to about 50% and about 30% to about 40 (weight/ weight) based on the sum of the amount of Compound 2, or a pharmaceutically acceptable salt or prodrug thereof, and the amount of Compounds 1 and/or 3, or pharmaceutically acceptable salts or prodrugs thereof in the composition.

As to Compound 3, in an embodiment, the composition can include an amount of Compound 3, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 1 % to about 98% (weight/weight) based on the sum of the amount of Compound 3, or a pharmaceutically acceptable salt or prodrug thereof, and the amount of Compounds 1 and/or 2, or pharmaceutically acceptable salts or prodrugs thereof, in the composition. Examples of additional embodiments include, but are not limited, to, an amount of Compound 3, or a pharmaceutical y acceptable salt or prodrug thereof, in the range of about 5% to about 80%, about 10% to about 70%, about 15% to about 60%, about 20% to about 50% and about 30% to about 40% (weight/weight) based on the sum of the amount of Compound 3, or a pharmaceutically acceptable salt or prodrug thereof, and the amount of Compounds 1 and/or 2, or pharmaceutically acceptable salts or prodrugs thereof in the composition.

In some embodiments, the composition can further include a pharmaceutically acceptable excipient, diluent and/or carrier, such as those described herein. In general it is contemplated that an antiviral effective daily amount of Compound 1 A within the combination treatment of the invention, would be from about 0.01 to about 700 mg/kg, or about 0.5 to about 400 mg/kg, or about 1 to about 250 mg/kg, or about 2 to about 200 mg/kg, or about 10 to about 150 mg/kg, or about 0.1 to about 50 mg/kg, or about 1 to about 20 mg/kg, or about 2 to about 10 mg/kg, or about 5 to about 8 mg/kg body weight. It may be appropriate to administer the required dose as one, two, three, four or more sub-doses at appropriate intervals throughout the day. Said sub-doses may be formulated as unit dosage forms, for example, containing about 1 to about 5000 mg Compound 1A, or about 50 to about 3000 mg Compound 1A, or about 100 to about 1000 mg Compound 1 A, or about 200 to about 600 mg Compound 1A, or about 100 to about 400 mg Compound 1A, or about 7 to about 3500 mg Compound 1A, or about 70 to about 1400 mg Compound 1 A, or about 140 to about 700 mg Compound 1 A, or about 340 to about 580 mg of Compound 1 A per unit dosage form. Preferably the unit dosage form is formulated for QD dosing with one of the above ranges.

Similarly, various amounts of Compound 3, or a pharmaceutically acceptable salt or prodrug thereof can be included in the compositions. In some embodiments, the composition can include an amount of Compound 3, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 10 mg to about 100 mg. In other embodiments, the composition can include an amount of Compound 3, or a pharmaceutically acceptable salt or prodrug thereof, in the range of about 20 mg to about 60 mg. In still other embodiments, the composition can include an amount of Compound 3 or a pharmaceutically acceptable salt or prodrug thereof in the range of about 30-50 mg.

A potential advantage of making a combination of Compound 1 A and Compound 2, and/or 3, or pharmaceutically acceptable salts or prodrugs thereof may be a reduction in the required amounts of one or more compounds that are effective in treating a disease condition disclosed herein (for example, HCV) as compared to monotherapy treatment of an otherwise comparable patient population using either Compound 1 A, 2 or 3 alone. In some embodiments, the amount of Compound 1 _; or a pharmaceutically acceptable salt thereof in the composition can be less compared to the amount of Compound 1 A or a pharmaceutically acceptable salt thereof needed to achieve the same viral load reduction when administered as a monotherapy. In some embodiments, the amount of Compound 2 or a pharmaceutically acceptable salt or prodrug thereof in the composition can be less compared to the amount of Compound 2, or a pharmaceutically acceptable salt or prodrug thereof, needed to achieve the same viral load reduction when administered as a monotherapy. In some embodiments, the amount of Compound 3 or a pharmaceutically acceptable salt or prodrug thereof in the composition can be less compared to the amount of Compound 3 or a pharmaceutically acceptable salt or prodrug thereof needed to achieve the same viral load reduction when administered as a monotherapy.

In an embodiment, the sum of the amount of Compound 1A or a pharmaceutically acceptable salt and the amount of Compound 2, or a pharmaceutically acceptable salt or prodrug thereof and/or the amount of Compound 3, or a pharmaceutically acceptable salt or prodrug thereof is less than expected or predicted based on the additive combination of Compound 1 A or a pharmaceutically acceptable salt alone, Compound 2, or a pharmaceutically acceptable salt or prodrug thereof alone, and/or Compound 3, or a pharmaceutically acceptable salt or prodrug thereof alone for treating the disease condition such as HCV.

In general however, it is often convenient to administer Compound 1A, and Compound 2 and/or Compound 3 at dosages reflecting monotherapy dosage regimes for the respective component(s), and thereby provide an enhanced antiviral efficacy and/or onset of action.

The percentages of Compound 1 A, and Compound 2, and/or 3 or pharmaceutically acceptable salts or prodrugs thereof present in the composition can also vary. For example, in some embodiments, the composition can include an amount of Compound 1 A, or a pharmaceutically acceptable salt thereof, in the range of about 1 % to about 98% (weight/weight) based on the sum of the amount of Compound 1 A, or a pharmaceutically acceptable salt thereof and the amount of Compounds 2 and/or 3 or pharmaceutically acceptable salts or prodrugs thereof, in the composition. Additional embodiments include, but are not limited to, an amount of Compound 1 A, or a pharmaceutically acceptable salt thereof in the range of about 5% to about 80%, about 10% to about 70%, about 15% to about 60%, about 20% to about 50% and about 30% to about 40% (weight/weight) based on the sum of the amount of Compound 1A, or a pharmaceutically acceptable salt or prodrug thereof, and the amount of Compounds 2 and/or 3, or pharmaceutically acceptable safe or prodrugs thereof in the composition.

As to Compound 2, in an embodiment, the composition can include an amount of Compound 2, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 1 % to about 98% (weight/weight) based on the sum of the amount of Compound 2, or a pharmaceutically acceptable salt or prodrug thereof and the amount of Compounds 1 A and/or 3, or pharmaceutically acceptable salts or prodrugs thereof, in the composition. Examples of additional embodiments, include, but are not limited to, an amount of Compound 2, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 5% to about 80%, about 10% to about 70%, about 15% to about 60%, about 20% to about 50% and about 30% to about 40 (weight/ weight) based on the sum of the amount of Compound 2, or a pharmaceutically acceptable salt or prodrug thereof, and the amount of Compounds 1 A and/or 3, or pharmaceutically acceptable salts or prodrugs thereof in the composition.

As to Compound 3, in an embodiment, the composition can include an amount of Compound 3, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 1 % to about 98% (weight/weight) based on the sum of the amount of Compound 3, or a pharmaceutically acceptable salt or prodrug thereof, and the amount of Compounds 1 A and/or 2, or pharmaceutically acceptable salts or prodrugs thereof, in the composition. Examples of additional embodiments include, but are not limited, to, an amount of Compound 3, or a pharmaceutically acceptable salt or prodrug thereof, in the range of about 5% to about 80%, about 10% to about 70%, about 15% to about 60%, about 20% to about 50% and about 30% to about 40% (weight/weight) based on the sum of the amount of Compound 3, or a pharmaceutically acceptable salt or prodrug thereof, and the amount of Compounds 1 A and/or 2, or pharmaceutically acceptable salts or prodrugs thereof in the composition in an appropriate container, and labelled for treatment of an indicated condition. Some embodiments described herein relate to a method for ameliorating or treating a disease condition that, can include administering an amount of Compound 1 A, or a pharmaceutically acceptable salt thereof, and an amount of Compound 2, or a pharmaceutically acceptable salt or prodrug thereof and/or an amount of Compound 3, or a pharmaceutically acceptable salt or prodrug thereof wherein the disease condition can be a hepatitis C virus infection, liver fibrosis, and/or impaired liver function.

Various dosages forms of Compound IA or a pharmaceutically acceptable salt thereof, and/or Compound 2, or a pharmaceutically acceptable salt or prodrug thereof and/or Compound 3, or a pharmaceutically acceptable salt or prodrug thereof can be used to ameliorate and/or treat a disease condition. In some instances, Compounds 1 A and 2 and/or 3 or pharmaceutically acceptable salts or prodrugs thereof can be present in the same dosage form such as the compositions described herein. In other instances, Compound 1A, and 2 and/or 3, or pharmaceutically acceptable salts or prodrugs thereof, can be administered as separate dosage forms. For example, Compound 1 A, or a pharmaceutically acceptable salt thereof, can be administered in one tablet, Compound 2, or a pharmaceutically acceptable salt or prodrug thereof, can be administered In a second tablet, and/or Compound 3, or a pharmaceutically acceptable salt or prodrug thereof, can be administered in a third tablet. When Compounds 1 A , 2, and 3 or pharmaceutically acceptable salts or prodrugs thereof, are contained in separate dosage forms, the dosage forms can be the same (e.g., as both pills) or different (e.g., two compounds can be formulated in a pill and the other compound can fee formulated as art injectable). Administration of Compound 1 A or a pharmaceutically acceptable salt thereof; and Compound

2, or a pharmaceutically acceptable salt or prodrug thereof, and/or Compound 3, or a pharmaceutically acceptable salt or prodrug thereof can vary. When Compounds 1 A, and 2 and/or 3, or pharmaceutically acceptable salts or prodrugs thereof are contained in separate dosage forms, the dosage forms can be administered simultaneously or sequentially. In same embodiments, the dosage form that contains Compound 1A, or pharmaceutically acceptable salt thereof, can be administered before, after, in-between, concurrently or sequentially with Compounds 2 and/or 3, or pharmaceutically acceptable salts or prodrugs thereof. In some embodiments, the dosage form that contains Compound 2, or a pharmaceutically acceptable salt or prodrug thereof, can be administered before, after, in-between, concurrently or sequentially with Compound 1 A, or Compounds IA and 3, or pharmaceutically acceptable salts or prodrugs thereof. In some embodiments, the dosage form that contains Compound 3, or a pharmaceutically acceptable salt or prodrug thereof, can be administered before, after, inbetween, concurrently or sequentially with Compound 1 A and 2, or pharmaceutically acceptable salts or prodrugs thereof.

In some embodiments, Compounds 1 A; and Compound 2 and/or 3, or pharmaceutically acceptable salts or prodrugs thereof can be administered concurrently. As used herein, the term "concurrently means effective concentrations of all three compounds are present in a subject. When being administered concurrently, Compound 1 A, and Compound 2 and/or 3, or pharmaceutically acceptable salts or prodrugs thereof, can be administered in the same dosage form or separate dosage forms. In other embodiments, CompoundI A, and Compound 2, and/or

3, or pharmaceutically acceptable salts or prodrugs hereof can be administered sequentially. As used herein, the term "sequentially" means administering one compound for a first time period, then administering a second compound for a second time period, optionally followed by administering a third compound for a third period, in which the first, second, and if applicable third time periods do not overlap.

Additional therapeutic agents can also be administered to the subject having the disease condition. A non-limiting list of additional therapeutic agents_: includes those previously described herein. When one or more additional therapeutic agents are utilized, the additional agent(s) can be administered in the same dosage form as Compound 1 A or a pharmaceutically acceptable salt and/or Compound 2, or a pharmaceutically acceptable salt or prodrug thereof and/or Compound 3 or a pharmaceutically acceptable salt or prodrug thereof. For example, the additional therapeutic agent(s) can be included in a composition that includes Compound 1A, or a pharmaceutically acceptable salt, without Compounds 2 and/or 3, or pharmaceutically acceptable salts or prodrugs thereof; or a composition that includes Compound 2 or a pharmaceutically acceptable salt or prodrug thereof, without Compounds 1 A or without Compounds 1A and 3, or pharmaceutically acceptable salts or prodrugs thereof; or a composition that includes Compound 3, or a pharmaceutically acceptable salt or prodrug thereof, without Compounds 1 A or without Compounds 1 A and 2, or pharmaceutically acceptable salts or prodrugs thereof. One aspect of the invention relates to a pharmaceutical composition that comprises, in a pharmaceutically acceptable vehicle, carrier or diluent: a first compound, with the formula

Compound 1A or a pharmaceutically acceptable salt thereof, and one or two further HCV antivirals selected from a) an HCV protease inhibitor selected from paritaprevir or ABT493; each a "Compound 2a" b) an NS5A inhibitor selected from ombitasvir or ABT-530; each a "Compound 3a"

Various aspects of the invention therefore provide pharmaceutical compositions or HCV treatment regimes comprising i) Compound 1 A, paritaprevir, ombitasvir

ϋ) Compound 1A, paritaprevir, ombitasvir,

iii) Compound 1A, paritaprevir,

iv) Compound 1A, ombitasvir,

v) Compound 1A, ABT-493, ombitasvir

vi) Compound 1A, ABT-493

vii) Compound 1A, ABT-493, ABT-530

viii) Compound 1 A, ABT-530

In another aspect, the invention relates to the use of such compositions for ameliorating or treating a disease condition in a patient population, and/or for the preparation of a medicament for ameliorating or treating such a disease condition. For example, the disease condition can be selected from a hepatitis C virus infection, liver fibrosis, and impaired liver function. In one embodiment the invention relates to the use of a composition comprising Compound 1 A, and a Compound 2a and/or a Compound 3a, or pharmaceutically acceptable salts or prodrugs thereof, for ameliorating or treating hepatitis C virus infection, liver fibrosis, and impaired liver function.

In another aspect the invention relates to a method for ameliorating or treating a disease condition in a patient population that comprises administering a therapeutically effective amount of a first compound, or a pharmaceutically acceptable salt or prodrug thereof wherein the first compound is Compound 1 A; and a therapeutically effective amount of a second compound, or a pharmaceutically acceptable salt or prodrug thereof, wherein the second compound is a Compound 2a; and/or a therapeutically effective amount of a third compound, or a pharmaceutically acceptable salt or prodrug thereof wherein the third compound is a Compound 3a; to a subject suffering from the disease conditions. In one embodiment, the disease condition can be selected from & hepatitis C virus infection, liver fibrosis, and impaired liver function.

In another aspect the invention relates to the use of Compound 1A or a pharmaceutically acceptable salt or prodrug thereof for ameliorating or treating a disease condition in a patient population and/or for the preparation of a medicament for ameliorating or treating such a disease condition, wherein Compound 1A or a pharmaceutically acceptable salt thereof is manufactured for use in combination with a Compound 2a or a pharmaceutically acceptable salt or prodrug thereof; and wherein Compound 1A and a Compound 2a or pharmaceutically acceptable salts or prodrugs thereof are manufactured for use in combination with a Compound 3a or a pharmaceutical acceptable salt or prodrug thereof wherein the disease condition is selected from hepatitis C virus infection, liver fibrosis, and impaired liver function,

In one embodiment of the Invention the method or use for ameliorating or treating a disease condition in a patient population comprises administering, one or more additional therapeutic agents. In another embodiment the one or more additional therapeutic agents are ribavirin and/or ritonavir. Paritaprevir, (INN), one of the possibilities for Compound 2a has shown to be effective in inhibiting HCV replication. The aforementioned compound can be obtained using methods known to those skilled in the art, including, for example, those methods disclosed in WO201 1/1 12558, and WO2013/106631 , which are hereby incorporated by reference in its entirety. Although this invention is not limited by any particular theory, Compound 2a are believed to inhibit the HCV protease, in particular the NS3/4A protease. Pharmaceutically acceptable salts and prodrugs of Compound 2a can be utilized in the compositions described herein. Paritaprevir is preferably co-dosed with a pharmacokinetic booster of ritonavir, and each recital of Compound 2a herein shall be understood as including an optional boosting dose, for example 25-100 mg, such as 50 mg of ritonavir. Ombitasvir (INN), one of the possibilities for Compound 3a has been demonstrated to be effective in inhibiting HCV replication. Compound 3a can be obtained using methods known to those skilled in the art, such as those methods described in WO2012/051361 , which is hereby incorporated by reference in its entirety. Each Compound 3a is an NS5A inhibitor where NS5A is a protein involved in the replication of the hepatitis C virus.

In general it is contemplated that an antiviral effective daily amount of Compound 1 A within the combination treatment of the invention, would be from about 0.01 to about 700 mg/kg, or about 0.5 to about 400 mg/kg, or about 1 to about 250 mg/kg, or about 2 to about 200 mg/kg, or about 10 to about 150 mg/kg, or about 0.1 to about 50 mg/kg, or about 1 to about 20 mg/kg, or about 2 to about 10 mg/kg, or about 5 to about 8 mg/kg body weight. It may be appropriate to administer the required dose as one, two, three, four or more sub-doses at appropriate intervals throughout the day. Said sub-doses may be formulated as unit dosage forms, for example, containing about 1 to about 5000 mg Compound 1A, or about 50 to about 3000 mg Compound 1A, or about 100 to about 1000 mg Compound 1 A, or about 200 to about 600 mg Compound 1A, or about 100 to about 400 mg Compound 1A, or about 7 to about 3500 mg Compound 1A, or about 70 to about 1400 mg Compound 1 A, or about 140 to about 700 mg Compound 1 A, or about 340 to about 580 mg of Compound 1 A per unit dosage form. Preferably the unit dosage form is formulated for QD dosing with one of the above ranges.

Similarly, various amounts of a Compound 2a, or a pharmaceutically acceptable salt or prodrug thereof, can he included in the compositions. In some embodiments, the composition can include an amount of a Compound 2a, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 50 mg to about 300 mg. In other embodiments, the composition can include an amount of a Compound 2a, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 75 mg to about 200 mg. In still other embodiments, the composition can include an amount of a Compound 2a, or a pharmaceutically acceptable salt or prodrug thereof, in the range of about 100-150 mg. Especially when compound 2a is paritaprevir, the each dosage unit can include a booster of ritonavir, for example 25-100 mg, such as 50 mg.

Similarly, various amounts of a Compound 3a, or a pharmaceutically acceptable salt or prodrug thereof can be included in the compositions. In some embodiments, the composition can include an amount of a Compound 3a, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 5 mg to about 100 mg. In other embodiments, the composition can include an amount of a Compound 3a, or a pharmaceutically acceptable salt or prodrug thereof, in the range of about 12.5 mg to about 75 mg. In still other embodiments, the composition can include an amount of a Compound 3a or a pharmaceutically acceptable salt or prodrug thereof in the range of about 25 mg. A potential advantage of making a combination of Compound 1A and a Compound 2a, and/or a compound 3a, or pharmaceutically acceptable salts or prodrugs thereof may be a reduction in the required amounts of one or more compounds that are effective in treating a disease condition disclosed herein (for example, HCV) as compared to monotherapy treatment of an otherwise comparable patient population using either Compound 1A, 2a or 3a alone. In some embodiments, the amount of Compound 1A_; or a pharmaceutically acceptable salt thereof in the composition can be less compared to the amount of Compound 1A or a pharmaceutically acceptable salt thereof needed to achieve the same viral load reduction when administered as a monotherapy. In some embodiments, the amount of a Compound 2a or a pharmaceutically acceptable salt or prodrug thereof in the composition can be less compared to the amount of a Compound 2a, or a pharmaceutically acceptable salt or prodrug thereof, needed to achieve the same viral load reduction when administered as a monotherapy. In some embodiments, the amount of a Compound 3a or a pharmaceutically acceptable salt or prodrug thereof in the composition can be less compared to the amount of a Compound 3a or a pharmaceutically acceptable salt or prodrug thereof needed to achieve the same viral load reduction when administered as a monotherapy.

In an embodiment, the sum of the amount of Compound 1A or a pharmaceutically acceptable salt and the amount of a Compound 2a, or a pharmaceutically acceptable salt or prodrug thereof and/or the amount of a Compound 3a, or a pharmaceutically acceptable salt or prodrug thereof is less than expected or predicted based on the additive combination of Compound 1 A or a pharmaceutically acceptable salt or prodrug thereof, alone, a Compound 2a, or a pharmaceutically acceptable salt or prodrug thereof alone, and/or a Compound 3a, or a pharmaceutically acceptable salt or prodrug thereof alone for treating the disease condition such as HCV. In general however, it is often convenient to administer Compound 1A, and Compound 2a and/or Compound 3a at dosages reflecting monotherapy dosage regimes for the respective component(s), and thereby provide an enhanced antiviral efficacy and/or onset of action.

The percentages of Compound 1A, and Compound 2a, and/or 3a or pharmaceutically acceptable salts or prodrugs thereof present in the composition can also vary. For example, in some embodiments, the composition can include an amount of Compound 1 A, or a pharmaceutically acceptable salt thereof, in the range of about 1 % to about 98% (weight/weight) based on the sum of the amount of Compound 1 A, or a pharmaceutically acceptable salt thereof and the amount of Compounds 2a and/or 3a or pharmaceutically acceptable salts or prodrugs thereof, in the composition. Additional embodiments include, but are not limited to, an amount of Compound 1A, or a pharmaceutically acceptable salt thereof in the range of about 5% to about 80%, about 10% to about 70%, about 15% to about 60%, about 20% to about 50% and about 30% to about 40% (weight/weight) based on the sum of the amount of Compound 1A, or a pharmaceutically acceptable salt or prodrug thereof, and the amount of Compounds 2a and/or 3a, or pharmaceutically acceptable safe or prodrugs thereof in the composition. As to Compound 2a, in an embodiment, the composition can include an amount of a Compound 2a, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 1 % to about 98% (weight/weight) based on the sum of the amount of Compound 2a, or a pharmaceutically acceptable salt or prodrug thereof and the amount of Compounds 1 A and/or 3a, or pharmaceutically acceptable salts or prodrugs thereof, in the composition. Examples of additional embodiments, include, but are not limited to, an amount of a Compound 2a, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 5% to about 80%, about 10% to about 70%, about 15% to about 60%, about 20% to about 50% and about 30% to about 40 (weight/ weight) based on the sum of the amount of Compound 2a, or a pharmaceutically acceptable salt or prodrug thereof, and the amount of Compounds 1 A and/or 3a, or pharmaceutically acceptable salts or prodrugs thereof in the composition.

As to Compound 3a, in an embodiment, the composition can include an amount of a Compound 3a, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 1 % to about 98% (weight/weight) based on the sum of the amount of Compound 3a, or a pharmaceutically acceptable salt or prodrug thereof, and the amount of Compounds 1 A and/or 2a, or pharmaceutically acceptable salts or prodrugs thereof, in the composition. Examples of additional embodiments include, but are not limited, to, an amount of a Compound 3a, or a pharmaceutically acceptable salt or prodrug thereof, in the range of about 5% to about 80%, about 10% to about 70%, about 15% to about 60%, about 20% to about 50% and about 30% to about 40% (weight/weight) based on the sum of the amount of Compound 3a, or a pharmaceutically acceptable salt or prodrug thereof, and the amount of Compounds 1A and/or 2a, or pharmaceutically acceptable salts or prodrugs thereof in the composition.

Various dosages forms of Compound 1A or a pharmaceutically acceptable salt thereof, and/or a Compound 2a, or a pharmaceutically acceptable salt or prodrug thereof and/or Compound 3a, or a pharmaceutically acceptable salt or prodrug thereof can be used to ameliorate and/or treat a disease condition. In some instances, Compounds 1 A and 2a and/or 3a or pharmaceutically acceptable salts or prodrugs thereof can be present in the same dosage form such as the compositions described herein. In other instances, Compound 1 A, and 2a and/or 3a, or pharmaceutically acceptable salts or prodrugs thereof, can be administered as separate dosage forms. For example, Compound 1 A, or a pharmaceutically acceptable salt thereof, can be administered in one tablet, a Compound 2a, or a pharmaceutically acceptable salt or prodrug thereof, can be administered In a second tablet, and/or a Compound 3a, or a pharmaceutically acceptable salt or prodrug thereof, can be administered in a third tablet. When Compounds 1 A , 2a, and 3a or pharmaceutically acceptable salts or prodrugs thereof, are contained in separate dosage forms, the dosage forms can be the same (e.g., both or all as pills) or different (e.g., two compounds can be formulated in a pill and the other compound can be formulated as art injectable).

Administration of Compound 1 A or a pharmaceutically acceptable salt thereof; and a Compound 2a, or a pharmaceutically acceptable salt or prodrug thereof, and/or a Compound 3a, or a pharmaceutically acceptable salt or prodrug thereof can vary. When Compounds 1 A, and 2a and/or 3a, or pharmaceutically acceptable salts or prodrugs thereof are contained in separate dosage forms, the dosage forms can be administered simultaneously or sequentially. In same embodiments, the dosage form that contains Compound 1A, or pharmaceutically acceptable salt thereof, can be administered before, after, in-between, concurrently or sequentially with Compounds 2a and/or 3a, or pharmaceutically acceptable salts or prodrugs thereof. In some embodiments, the dosage form that contains Compound 2a, or a pharmaceutically acceptable salt or prodrug thereof, can be administered before, after, in-between, concurrently or sequentially with Compound 1A, or Compounds 1 A and 3a, or pharmaceutically acceptable salts or prodrugs thereof. In some embodiments, the dosage form that contains Compound 3a, or a pharmaceutically acceptable salt or prodrug thereof, can be administered before, after, inbetween, concurrently or sequentially with Compound 1A or Compound 1 A and 2a, or pharmaceutically acceptable salts or prodrugs thereof.

In some embodiments, Compounds 1 A; and Compound 2a and/or 3a, or pharmaceutically acceptable salts or prodrugs thereof can be administered concurrently. As used herein, the term "concurrently means effective concentrations of all three compounds are present in a subject. When being administered concurrently, Compound 1 A, and Compound 2a and/or 3a, or pharmaceutically acceptable salts or prodrugs thereof, can be administered in the same dosage form or separate dosage forms. In other embodiments, Compound 1A, and Compound 2a, and/or Compound 3a, or pharmaceutically acceptable salts or prodrugs hereof can be administered sequentially. As used herein, the term "sequentially" means administering one compound for a first time period, then administering a second compound for a second time period, optionally followed by administering a third compound for a third period, in which the first, second, and if applicable third time periods do not overlap.

Compound 1A or a pharmaceutically acceptable salt thereof, and a second compound, or a pharmaceutically acceptable salt or prodrug thereof wherein the second compound is alisporavir ("Compound 2b"); In another aspect the invention relates to a composition comprising, in a pharmaceutically acceptable vehicle, carrier or diluent Compound 1 A, and Compound 2b, or pharmaceutically acceptable salts or prodrugs thereof, wherein the composition additionally comprises one or more therapeutic agents. In one embodiment the one or more therapeutic agents are ribavirin and ritonavir. In another aspect, the invention relates to the use of such compositions for ameliorating or treating a disease condition in a patient population, and/or for the preparation of a medicament for ameliorating or treating such a disease condition. For example, the disease condition can be selected from a hepatitis C virus infection, liver fibrosis, and impaired liver function. In one embodiment the invention relates to the use of a composition comprising Compound 1A, and Compound 2b, or pharmaceutically acceptable salts or prodrugs thereof, for ameliorating or treating hepatitis C virus infection, liver fibrosis, and impaired liver function.

In another aspect the invention relates to a method for ameliorating or treating a disease condition in a patient population that comprises administering a therapeutically effective amount of a first compound, or a pharmaceutically acceptable salt or prodrug thereof wherein the first compound is Compound 1 A; and a therapeutically effective amount of a second compound, or a pharmaceutically acceptable salt or prodrug thereof, wherein the second compound is Compound 2b; to a subject suffering from the disease conditions. In one embodiment, the disease condition can be selected from & hepatitis C virus infection, liver fibrosis, and impaired liver function. In another aspect the invention relates to the use of Compound 1A or a pharmaceutically acceptable salt or prodrug thereof for ameliorating or treating a disease condition in a patient population and/or for the preparation of a medicament for ameliorating or treating such a disease condition, wherein Compound 1 A or a pharmaceutically acceptable salt thereof is manufactured for use in combination with Compound 2b or a pharmaceutically acceptable salt or prodrug thereof; wherein the disease condition is selected from hepatitis C virus infection, liver fibrosis, and impaired liver function,

In one embodiment of the invention the method or use for ameliorating or treating a disease condition in a patient population comprises administering, one or more additional therapeutic agents. In another embodiment the one or more additional therapeutic agents are ribavirin and/or ritonavir,

Alisporavir (INN) (Compound 2b) is a host cyclophilin A inhibitor (otherwise known as peptidyl prolylc/sfransisomerase inhibitors) which has been demonstrated to be effective in HCV treatment in phase II clinical trials. Alisporavir belongs to the cyclosporin class of macromolecules.

Compound 2b can be obtained using methods known to those skilled in the art, such as those methods described in WO2013/167703 and WO2006/038088 which are hereby incorporated by reference in its entirety. In general it is contemplated that an antiviral effective daily amount of Compound 1A within the combination treatment of the invention, would be from about 0.01 to about 700 mg/kg, or about 0.5 to about 400 mg/kg, or about 1 to about 250 mg/kg, or about 2 to about 200 mg/kg, or about 10 to about 150 mg/kg, or about 0.1 to about 50 mg/kg, or about 1 to about 20 mg/kg, or about 2 to about 10 mg/kg, or about 5 to about 8 mg/kg body weight. It may be appropriate to administer the required dose as one, two, three, four or more sub-doses at appropriate intervals throughout the day. Said sub-doses may be formulated as unit dosage forms, for example, containing about 1 to about 5000 mg Compound 1 , or about 50 to about 3000 mg Compound 1 , or about 100 to about 1000 mg Compound 1A, or about 200 to about 600 mg Compound 1A, or about 100 to about 400 mg Compound 1 A, or about 7 to about 3500 mg Compound 1 A, or about 70 to about 1400 mg Compound 1 A, or about 140 to about 700 mg Compound 1A, or about 340 to about 580 mg of Compound 1 A per unit dosage form. Preferably the unit dosage form is formulated for QD dosing with one of the above ranges.

Various amounts of Compound 2b, or a pharmaceutically acceptable salt or prodrug thereof can be included in the compositions. In some embodiments, the composition can include an amount of Compound 2b, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 100-800 mg BID or QD In other embodiments, the composition can include an amount of Compound 2b, or a pharmaceutically acceptable salt or prodrug thereof, in the range of about 200-400 mg. In still other embodiments, the composition can include an amount of Compound 2b or a pharmaceutically acceptable salt or prodrug thereof in the range of about 200, 250, 300, 350 or 400 mg.

A potential advantage of making a combination of Compound 1A and Compound 2b, or pharmaceutically acceptable salts or prodrugs thereof may be a reduction in the required amounts of one or more compounds that are effective in treating a disease condition disclosed herein (for example, HCV) as compared to monotherapy treatment of an otherwise comparable patient population using either Compound 1A, or 2b alone. In some embodiments, the amount of Compound 1A_; or a pharmaceutically acceptable salt thereof in the composition can be less compared to the amount of Compound 1 A or a pharmaceutically acceptable salt thereof needed to achieve the same viral load reduction when administered as a monotherapy. In some embodiments, the amount of Compound 2b or a pharmaceutically acceptable salt or prodrug thereof in the composition can be less compared to the amount of Compound 2b, or a pharmaceutically acceptable salt or prodrug thereof, needed to achieve the same viral load reduction when administered as a monotherapy. In an embodiment, the sum of the amount of Compound 1A or a pharmaceutically acceptable salt and the amount of Compound 2b, or a pharmaceutically acceptable salt or prodrug thereof is less than expected or predicted based on the additive combination of Compound 1A or a pharmaceutically acceptable salt or prodrug thereof, alone, or Compound 2b, or a pharmaceutically acceptable salt or prodrug thereof alone, for treating the disease condition such as HCV.

In general however, it is often convenient to administer Compound 1 A, and Compound 2b at dosages reflecting monotherapy dosage regimes for the respective component(s), and thereby provide an enhanced antiviral efficacy and/or onset of action.

Additional advantages of utilizing a combination of Compound 1A and Compound, 2b, or pharmaceutically acceptable salts or prodrugs thereof may include little to no cross resistance between Compound 1 A, and Compound 2b, or pharmaceutically acceptable salts or prodrugs thereof; different routes tor elimination of Compound 1 , and Compound 2b, or pharmaceutically acceptable salts or prodrugs thereof; little to no overlapping toxicities between Compound 1 A, and Compound 2b, or pharmaceutically acceptable salts or prodrugs thereof; little, to no significant effects on cytochrome P 450; and/or little to no pharmacokinetic interactions between Compound 1 A, and Compound 2b, or pharmaceutically acceptable salts or prodrugs thereof.

The percentages of Compound 1 A, and Compound 2b, or pharmaceutically acceptable salts or prodrugs thereof present in the composition can also vary. For example, in some embodiments, the composition can include an amount of Compound 1 A, or a pharmaceutically acceptable salt thereof, in the range of about 1 % to about 98% (weight/weight) based on the sum of the amount of Compound 1 A, or a pharmaceutically acceptable salt thereof and the amount of Compounds 2b or pharmaceutically acceptable salts or prodrugs thereof, in the composition. Additional embodiments include, but are not limited to, an amount of Compound 1 A, or a pharmaceutically acceptable salt thereof in the range of about 5% to about 80%, about 10% to about 70%, about 15% to about 60%, about 20% to about 50% and about 30% to about 40% (weight/weight) based on the sum of the amount of Compound 1 A, or a pharmaceutically acceptable salt or prodrug thereof, and the amount of Compounds 2b, or pharmaceutically acceptable safe or prodrugs thereof in the composition. As to Compound 2b, in an embodiment, the composition can include an amount of Compound 2b, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 1 % to about 98% (weight/weight) based on the sum of the amount of Compound 2b, or a pharmaceutically acceptable salt or prodrug thereof and the amount of Compounds 1 A, or pharmaceutically acceptable salts or prodrugs thereof, in the composition. Examples of additional embodiments, include, but are not limited to, an amount of Compound 2b, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 5% to about 80%, about 10% to about 70%, about 15% to about 60%, about 20% to about 50% and about 30% to about 40 (weight/ weight) based on the sum of the amount of Compound 2b, or a pharmaceutically acceptable salt or prodrug thereof, and the amount of Compounds 1 A, or pharmaceutically acceptable salts thereof in the composition.

Some embodiments described herein relate to a method for ameliorating or treating a disease condition that, can include administering an amount of Compound 1 A, or a pharmaceutically acceptable salt thereof, and an amount of Compound 2b, or a pharmaceutically acceptable salt or prodrug thereof wherein the disease condition can be a hepatitis C virus infection, liver fibrosis, and/or impaired liver function.

Various dosages forms of Compound 1A or a pharmaceutically acceptable salt thereof, and/or Compound 2b, or a pharmaceutically acceptable salt or prodrug thereof can be used to ameliorate and/or treat a disease condition. In some instances, Compounds 1 A and 2b or pharmaceutically acceptable salts or prodrugs thereof can be present in the same dosage form such as the compositions described herein. In other instances, Compound 1 A, and 2b, or pharmaceutically acceptable salts or prodrugs thereof, can be administered as separate dosage forms. For example, Compound 1 A, or a pharmaceutically acceptable salt thereof, can be administered in one tablet, Compound 2b, or a pharmaceutically acceptable salt or prodrug thereof, can be administered In a second tablet. When Compounds 1 A and 2b, or pharmaceutically acceptable salts or prodrugs thereof, are contained in separate dosage forms, the dosage forms can be the same(e.g., as both pills) or different (e.g. one can be formulated in a pill and the other compound can fee formulated as an injectable).

Administration of Compound 1 A or a pharmaceutically acceptable salt thereof; and Compound 2b, or a pharmaceutically acceptable salt or prodrug thereof, can vary. When Compounds 1 A, and 2b, or pharmaceutically acceptable salts or prodrugs thereof are contained in separate dosage forms, the dosage forms can be administered simultaneously or sequentially. In same embodiments, the dosage form that contains Compound 1A, or pharmaceutically acceptable salt thereof, can be administered before, after, in-between, concurrently or sequentially with Compounds 2b, or pharmaceutically acceptable salts or prodrugs thereof. In some embodiments, the dosage form that contains Compound 2b, or a pharmaceutically acceptable salt or prodrug thereof, can be administered before, after, in-between, concurrently or sequentially with Compound 1 A, or pharmaceutically acceptable salts or prodrugs thereof.

In some embodiments, Compounds 1 A; and Compound 2b, or pharmaceutically acceptable salts or prodrugs thereof can be administered concurrently. As used herein, the term "concurrently means effective concentrations of both compounds are present in a subject. When being administered concurrently, Compound 1A, and Compound 2b, or pharmaceutically acceptable salts or prodrugs thereof, can be administered in the same dosage form or separate dosage forms. In other embodiments, Compound 1 A, and Compound 2b, or pharmaceutically acceptable salts or prodrugs hereof can be administered sequentially. As used herein, the term "sequentially" means administering one compound for a first time period, then administering a second compound for a second time period, in which the first and second time periods do not overlap.

Compound 1 A or a pharmaceutically acceptable salt thereof, and a second compound, or a pharmaceutically acceptable salt or prodrug thereof wherein the second compound is EDP-239 ("Compound 2c");

In another aspect the invention relates to a composition comprising, in a pharmaceutically acceptable vehicle, carrier or diluent Compound 1 A, and Compound 2c, or pharmaceutically acceptable salts or prodrugs thereof, wherein the composition additionally comprises one or more therapeutic agents. In one embodiment the one or more therapeutic agents are ribavirin and ritonavir.

In another aspect, the invention relates to the use of such compositions for ameliorating or treating a disease condition in a patient population, and/or for the preparation of a medicament for ameliorating or treating such a disease condition. For example, the disease condition can be selected from a hepatitis C virus infection, liver fibrosis, and impaired liver function. In one embodiment the invention relates to the use of a composition comprising Compound 1A, and Compound 2c, or pharmaceutically acceptable salts or prodrugs thereof, for ameliorating or treating hepatitis C virus infection, liver fibrosis, and impaired liver function. In another aspect the invention relates to a method for ameliorating or treating a disease condition in a patient population that comprises administering a therapeutically effective amount of a first compound, or a pharmaceutically acceptable salt or prodrug thereof wherein the first compound is Compound 1 A; and a therapeutically effective amount of a second compound, or a pharmaceutically acceptable salt or prodrug thereof, wherein the second compound is Compound 2c; to a subject suffering from the disease conditions. In one embodiment, the disease condition can be selected from & hepatitis C virus infection, liver fibrosis, and impaired liver function.

In another aspect the invention relates to the use of Compound 1A or a pharmaceutically acceptable salt or prodrug thereof for ameliorating or treating a disease condition in a patient population and/or for the preparation of a medicament for ameliorating or treating such a disease condition, wherein Compound 1A or a pharmaceutically acceptable salt thereof is manufactured for use in combination with Compound 2c or a pharmaceutically acceptable salt or prodrug thereof; wherein the disease condition is selected from hepatitis C virus infection, liver fibrosis, and impaired liver function, In one embodiment of the Invention the method or use for ameliorating or treating a disease condition in a patient population comprises administering, one or more additional therapeutic agents. In another embodiment the one or more additional therapeutic agents are ribavirin and/or ritonavir. EDP-239 (Compound 2c) has been demonstrated to be effective in inhibiting HCV replication in phase II clinical trials. The formula of Compound 2c is believed to be

Compound 2c can be obtained using methods known to those skilled in the art, such as those methods described in WO2013/059281 and WO2010/099527, which are hereby incorporated by reference in its entirety. Although this invention is not limited by any particular theory, it is believed that Compound 2c is an NS5A inhibitor where NS5A is a protein involved in the replication of the hepatitis C virus.

In some embodiments, the composition can further include a pharmaceutically acceptable exipient, diluent and/or carrier, such as those described herein.

In general it is contemplated that an antiviral effective daily amount of Compound 1 A within the combination treatment of the invention, would be from about 0.01 to about 700 mg/kg, or about 0.5 to about 400 mg/kg, or about 1 to about 250 mg/kg, or about 2 to about 200 mg/kg, or about 10 to about 150 mg/kg, or about 0.1 to about 50 mg/kg, or about 1 to about 20 mg/kg, or about 2 to about 10 mg/kg, or about 5 to about 8 mg/kg body weight. It may be appropriate to administer the required dose as one, two, three, four or more sub-doses at appropriate intervals throughout the day. Said sub-doses may be formulated as unit dosage forms, for example, containing about 1 to about 5000 mg Compound 1A, or about 50 to about 3000 mg Compound 1A, or about 100 to about 1000 mg Compound 1 A, or about 200 to about 600 mg Compound 1 A, or about 100 to about 400 mg Compound 1 A, or about 7 to about 3500 mg Compound 1 A, or about 70 to about 1400 mg Compound 1 A, or about 140 to about 700 mg Compound 1 A, or about 340 to about 580 mg of Compound 1 A per unit dosage form. Preferably the unit dosage form is formulated for QD dosing with one of the above ranges.

Various amounts of Compound 2c, or a pharmaceutically acceptable salt or prodrug thereof can be included in the compositions. In some embodiments, the composition can include an amount of Compound 2c, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 25 mg to about 225 mg. In other embodiments, the composition can include an amount of Compound 2c, or a pharmaceutically acceptable salt or prodrug thereof, in the range of about 50 mg to about 150 mg. In still other embodiments, the composition can include an amount of Compound 2c or a pharmaceutically acceptable salt or prodrug thereof in the range of about 50- 100 mg. In yet still other embodiments, the a loading dose of 100-200 mg, such as 150 mg can be administered for the initial 1 -3 days of therapy.

A potential advantage of making a combination of Compound 1 A and Compound 2c, or pharmaceutically acceptable salts or prodrugs thereof may be a reduction in the required amounts of one or more compounds that are effective in treating a disease condition disclosed herein (for example, HCV) as compared to monotherapy treatment of an otherwise comparable patient population using either Compound 1 A, or 2c alone. In some embodiments, the amount of Compound 1A_; or a pharmaceutically acceptable salt thereof in the composition can be less compared to the amount of Compound 1 A or a pharmaceutically acceptable salt thereof needed to achieve the same viral load reduction when administered as a monotherapy. In some embodiments, the amount of Compound 2c or a pharmaceutically acceptable salt or prodrug thereof in the composition can be less compared to the amount of Compound 2c, or a pharmaceutically acceptable salt or prodrug thereof, needed to achieve the same viral load reduction when administered as a monotherapy.

In an embodiment, the sum of the amount of Compound 1A or a pharmaceutically acceptable salt and the amount of Compound 2c, or a pharmaceutically acceptable salt or prodrug thereof is less than expected or predicted based on the additive combination of Compound 1A or a pharmaceutically acceptable salt thereof, alone, or Compound 2c, or a pharmaceutically acceptable salt or prodrug thereof alone, for treating the disease condition such as HCV.

In general however, it is often convenient to administer Compound 1 A, and Compound 2c at dosages reflecting monotherapy dosage regimes for the respective component(s), and thereby provide an enhanced antiviral efficacy and/or onset of action.

The percentages of Compound 1 A, and Compound 2c, or pharmaceutically acceptable salts or prodrugs thereof present in the composition can also vary. For example, in some embodiments, the composition can include an amount of Compound 1 A, or a pharmaceutically acceptable salt thereof, in the range of about 1 % to about 98% (weight/weight) based on the sum of the amount of Compound 1A, or a pharmaceutically acceptable salt thereof and the amount of Compound 2c or pharmaceutically acceptable salts or prodrugs thereof, in the composition. Additional embodiments include, but are not limited to, an amount of Compound 1 A, or a pharmaceutically acceptable salt thereof in the range of about 5% to about 80%, about 10% to about 70%, about 15% to about 60%, about 20% to about 50% and about 30% to about 40% (weight/weight) based on the sum of the amount of Compound 1 A, or a pharmaceutically acceptable salt or prodrug thereof, and the amount of Compound 2c, or pharmaceutically acceptable safe or prodrugs thereof in the composition. As to Compound 2c, in an embodiment, the composition can include an amount of Compound 2c, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 1 % to about 98% (weight/weight) based on the sum of the amount of Compound 2c, or a pharmaceutically acceptable salt or prodrug thereof and the amount of Compounds 1A, or pharmaceutically acceptable salts or prodrugs thereof, in the composition. Examples of additional embodiments, include, but are not limited to, an amount of Compound 2c, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 5% to about 80%, about 10% to about 70%, about 15% to about 60%, about 20% to about 50% and about 30% to about 40 (weight/ weight) based on the sum of the amount of Compound 2c, or a pharmaceutically acceptable salt or prodrug thereof, and the amount of Compounds 1 , or pharmaceutically acceptable salts or prodrugs thereof in the composition.

Some embodiments described herein relate to a method for ameliorating or treating a disease condition that, can include administering an amount of Compound 1 A, or a pharmaceutically acceptable salt thereof, and an amount of Compound 2c, or a pharmaceutically acceptable salt or prodrug thereof wherein the disease condition can be a hepatitis C virus infection, liver fibrosis, and/or impaired liver function.

Various dosages forms of Compound 1A or a pharmaceutically acceptable salt thereof, and/or Compound 2c, or a pharmaceutically acceptable salt or prodrug thereof can be used to ameliorate and/or treat a disease condition. In some instances, Compounds 1 A and 2c or pharmaceutically acceptable salts or prodrugs thereof can be present in the same dosage form such as the compositions described herein. In other instances, Compound 1A, and 2c, or pharmaceutically acceptable salts or prodrugs thereof, can be administered as separate dosage forms. For example, Compound 1 A, or a pharmaceutically acceptable salt thereof, can be administered in one tablet, Compound 2c, or a pharmaceutically acceptable salt or prodrug thereof, can be administered In a second tablet. When Compounds 1 A and 2c, or pharmaceutically acceptable salts or prodrugs thereof, are contained in separate dosage forms, the dosage forms can be the same(e.g., both as pills) or different (e.g. one can be formulated in a pill and the other compound can be formulated as an injectable).

Administration of Compound 1 AI or a pharmaceutically acceptable salt thereof; and Compound 2c, or a pharmaceutically acceptable salt or prodrug thereof, can vary. When Compounds 1 A, and 2c, or pharmaceutically acceptable salts or prodrugs thereof are contained in separate dosage forms, the dosage forms can be administered simultaneously or sequentially. In same embodiments, the dosage form that contains Compound 1A, or pharmaceutically acceptable salt thereof, can be administered before, after, in-between, concurrently or sequentially with Compounds 2c, or pharmaceutically acceptable salts or prodrugs thereof. In some embodiments, the dosage form that contains Compound 2c, or a pharmaceutically acceptable salt or prodrug thereof, can be administered before, after, in-between, concurrently or sequentially with Compound 1 A, or pharmaceutically acceptable salts or prodrugs thereof.

In some embodiments, Compounds 1 A; and Compound 2c, or pharmaceutically acceptable salts or prodrugs thereof can be administered concurrently. As used herein, the term "concurrently means effective concentrations of both compounds are present in a subject. When being administered concurrently, Compound 1 A, and Compound 2c, or pharmaceutically acceptable salts or prodrugs thereof, can be administered in the same dosage form or separate dosage forms. In other embodiments, Compound 1 A, and Compound 2c, or pharmaceutically acceptable salts or prodrugs hereof can be administered sequentially. As used herein, the term "sequentially" means administering one compound for a first time period, then administering a second compound for a second time period, in which the first and second time periods do not overlap.

Compound 1A or a pharmaceutically acceptable salt thereof, and a) a second compound, or a pharmaceutically acceptable salt or prodrug thereof wherein the second compound is sovaprevir ("Compound 2d");

b) a third compound, or a pharmaceutically acceptable salt or prodrug thereof, wherein she third compound is odalasvir (also known as ACH-3102) ("Compound 3d").

In another aspect the invention relates to a composition comprising, in a pharmaceutically acceptable vehicle, carrier or diluent Compound 1 A, and Compound 2d and/or Compound 3d, or pharmaceutically acceptable salts or prodrugs thereof, wherein the composition additionally comprises one or more therapeutic agents. In one embodiment the one or more therapeutic agents are ribavirin and ritonavir.

It will be appreciated that one aspect of the invention provides pharmaceutical compositions comprising, in a pharmaceutically acceptable vehicle, carrier or diluent, Compound 1 A and sovaprevir. A further aspect of the invention provides pharmaceutical compositions comprising, in a pharmaceutically acceptable vehicle, carrier or diluent, Compound 1A and odalasvir. A further aspect of the invention provides pharmaceutical compositions comprising, in a pharmaceutically acceptable vehicle, carrier or diluent, Compound 1 A and sovaprevir and odalasvir. In another aspect, the invention relates to the use of such compositions for ameliorating or treating a disease condition in a patient population, and/or for the preparation of a medicament for ameliorating or treating such a disease condition. For example, the disease condition can be selected from a hepatitis C virus infection, liver fibrosis, and impaired liver function. In one embodiment the invention relates to the use of a composition comprising Compound 1A, and Compound 2d and/or Compound 3d, or pharmaceutically acceptable salts or prodrugs thereof, for ameliorating or treating hepatitis C virus infection, liver fibrosis, and impaired liver function.

In another aspect the invention relates to a method for ameliorating or treating a disease condition in a patient population that comprises administering a therapeutically effective amount of a first compound, or a pharmaceutically acceptable salt or prodrug thereof wherein the first compound is Compound 1 A; and a therapeutically effective amount of a second compound, or a pharmaceutically acceptable salt or prodrug thereof, wherein the second compound is Compound 2d; and/or a therapeutically effective amount of a third compound, or a pharmaceutically acceptable salt or prodrug thereof wherein the third compound is Compound 3d; to a subject suffering from the disease conditions. In one embodiment, the disease condition can be selected from & hepatitis C virus infection, liver fibrosis, and impaired liver function.

In another aspect the invention relates to the use of Compound 1A or a pharmaceutically acceptable salt or prodrug thereof for ameliorating or treating a disease condition in a patient population and/or for the preparation of a medicament for ameliorating or treating such a disease condition, wherein Compound 1A or a pharmaceutically acceptable salt thereof is manufactured for use in combination with Compound 2d or a pharmaceutically acceptable salt or prodrug thereof; and wherein Compound 1 A and Compound 2d or pharmaceutically acceptable salts or prodrugs thereof are manufactured for use in combination with Compound 3d or a pharmaceutical acceptable salt or prodrug thereof wherein the disease condition is selected from hepatitis C virus infection, liver fibrosis, and impaired liver function, In one embodiment of the Invention the method or use for ameliorating or treating a disease condition in a patient population comprises administering, one or more additional therapeutic agents. In another embodiment the one or more additional therapeutic agents are ribavirin and/or ritonavir, Sovaprevir, (INN), (Compound 2d) has shown to be effective in inhibiting HCV replication. The aforementioned compound can be obtained using methods known to those skilled in the art, including, for example, those methods disclosed in WO2008/008502, and WO2014/152928, which are hereby incorporated by reference in its entirety. Although this invention is not limited by any particular theory, Compound 2d is believed to inhibit the HCV protease, in particular the NS3/4A protease. Pharmaceutically acceptable salts and prodrugs of Compound 2d can be utilized in the compositions described herein.

Odalasvir (PINN, also known as ACH-3102) (Compound 3d) has been demonstrated to be effective in inhibiting HCV replication. Compound 3d can be obtained using methods known to those skilled in the art, such as those methods described in WO2012/166716, which is hereby incorporated by reference in its entirety. Although this invention is not limited by any particular theory, it is believed that Compound 3d is an NS5A inhibitor where NS5A is a protein involved in the replication of the hepatitis C virus.

In some embodiments, the composition can further include a pharmaceutically acceptable exipient, diluent and/or carrier, such as those described herein. In general it is contemplated that an antiviral effective daily amount of Compound 1 A within the combination treatment of the invention, would be from about 0.01 to about 700 mg/kg, or about 0.5 to about 400 mg/kg, or about 1 to about 250 mg/kg, or about 2 to about 200 mg/kg, or about 10 to about 150 mg/kg, or about 0.1 to about 50 mg/kg, or about 1 to about 20 mg/kg, or about 2 to about 10 mg/kg, or about 5 to about 8 mg/kg body weight. It may be appropriate to administer the required dose as one, two, three, four or more sub-doses at appropriate intervals throughout the day. Said sub-doses may be formulated as unit dosage forms, for example, containing about 1 to about 5000 mg Compound 1A, or about 50 to about 3000 mg Compound 1A, or about 100 to about 1000 mg Compound 1 A, or about 200 to about 600 mg Compound 1A, or about 100 to about 400 mg Compound 1A, or about 7 to about 3500 mg Compound 1A, or about 70 to about 1400 mg Compound 1 A, or about 140 to about 700 mg Compound 1 A, or about 340 to about 580 mg of Compound 1 A per unit dosage form. Preferably the unit dosage form is formulated for QD dosing with one of the above ranges.

Similarly, various amounts of Compound 2d, or a pharmaceutically acceptable salt or prodrug thereof, can he included in the compositions. In some embodiments, the composition can include an amount of Compound 2d, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 50 mg to about 1000 mg. In other embodiments, the composition can include an amount of Compound 2d, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 100 mg to about 800 mg. In still other embodiments, the composition can include an amount of Compound 2, or a pharmaceutically acceptable salt or prodrug thereof, in the range of about 200 mg to about 400 mg. In an embodiment, the composition can include an amount of Compound 2d, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 250-350 mg.

Similarly, various amounts of Compound 3d, or a pharmaceutically acceptable salt or prodrug thereof can be included in the compositions. In some embodiments, the composition can include an amount of Compound 3d, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 25 mg to about 225 mg. In other embodiments, the composition can include an amount of Compound 3, or a pharmaceutically acceptable salt or prodrug thereof, in the range of about 50 mg to about 100 mg. In still other embodiments, the composition can include an amount of Compound 3d or a pharmaceutically acceptable salt or prodrug thereof in the range of about 50 mg. In yet still other embodiments, the a loading dose of 100-200 mg, such as 150 mg can be administered for the initial 1 -3 days of therapy.

A potential advantage of making a combination of Compound 1 A and Compound 2d, and/or 3d, or pharmaceutically acceptable salts or prodrugs thereof may be a reduction in the required amounts of one or more compounds that are effective in treating a disease condition disclosed herein (for example, HCV) as compared to monotherapy treatment of an otherwise comparable patient population using either Compound 1 A, 2d or 3d alone. In some embodiments, the amount of Compound 1A_; or a pharmaceutically acceptable salt thereof in the composition can be less compared to the amount of Compound 1A or a pharmaceutically acceptable salt thereof needed to achieve the same viral load reduction when administered as a monotherapy. In some embodiments, the amount of Compound 2d or a pharmaceutically acceptable salt or prodrug thereof in the composition can be less compared to the amount of Compound 2d, or a pharmaceutically acceptable salt or prodrug thereof, needed to achieve the same viral load reduction when administered as a monotherapy. In some embodiments, the amount of Compound 3d or a pharmaceutically acceptable salt or prodrug thereof in the composition can be less compared to the amount of Compound 3d or a pharmaceutically acceptable salt or prodrug thereof needed to achieve the same viral load reduction when administered as a monotherapy.

In an embodiment, the sum of the amount of Compound 1A or a pharmaceutically acceptable salt and the amount of Compound 2d, or a pharmaceutically acceptable salt or prodrug thereof and/or the amount of Compound 3d, or a pharmaceutically acceptable salt or prodrug thereof is less than expected or predicted based on the additive combination of Compound 1 A or a pharmaceutically acceptable salt, alone, Compound 2d, or a pharmaceutically acceptable salt or prodrug thereof alone, and/or Compound 3d, or a pharmaceutically acceptable salt or prodrug thereof alone for treating the disease condition such as HCV.

In general however, it is often convenient to administer Compound 1A, and Compound 2d and/or Compound 3d at dosages reflecting monotherapy dosage regimes for the respective component(s), and thereby provide an enhanced antiviral efficacy and/or onset of action.

The percentages of Compound 1A, and Compound 2d, and/or 3d or pharmaceutically acceptable salts or prodrugs thereof present in the composition can also vary. For example, in some embodiments, the composition can include an amount of Compound 1 A, or a pharmaceutically acceptable salt thereof, in the range of about 1 % to about 98% (weight/weight) based on the sum of the amount of Compound 1 A, or a pharmaceutically acceptable salt thereof and the amount of Compounds 2d and/or 3d or pharmaceutically acceptable salts or prodrugs thereof, in the composition. Additional embodiments include, but are not limited to, an amount of Compound 1A, or a pharmaceutically acceptable salt thereof in the range of about 5% to about 80%, about 10% to about 70%, about 15% to about 60%, about 20% to about 50% and about 30% to about 40% (weight/weight) based on the sum of the amount of Compound 1A, or a pharmaceutically acceptable salt, and the amount of Compounds 2d and/or 3d, or pharmaceutically acceptable safe or prodrugs thereof in the composition.

As to Compound 2d, in an embodiment, the composition can include an amount of Compound 2d, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 1 % to about 98% (weight/weight) based on the sum of the amount of Compound 2d, or a pharmaceutically acceptable salt or prodrug thereof and the amount of Compounds 1 A and/or 3d, or pharmaceutically acceptable salts or prodrugs thereof, in the composition. Examples of additional embodiments, include, but are not limited to, an amount of Compound 2d, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 5% to about 80%, about 10% to about 70%, about 15% to about 60%, about 20% to about 50% and about 30% to about 40 (weight/ weight) based on the sum of the amount of Compound 2d, or a pharmaceutically acceptable salt or prodrug thereof, and the amount of Compounds 1 A and/or 3d, or pharmaceutically acceptable salts or prodrugs thereof in the composition.

As to Compound 3d, in an embodiment, the composition can include an amount of Compound 3d, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 1 % to about 98% (weight/weight) based on the sum of the amount of Compound 3d, or a pharmaceutically acceptable salt or prodrug thereof, and the amount of Compounds 1 A and/or 2d, or pharmaceutically acceptable salts or prodrugs thereof, in the composition. Examples of additional embodiments include, but are not limited, to, an amount of Compound 3A, or a pharmaceutically acceptable salt or prodrug thereof, in the range of about 5% to about 80%, about 10% to about 70%, about 15% to about 60%, about 20% to about 50% and about 30% to about 40% (weight/weight) based on the sum of the amount of Compound 3d, or a pharmaceutically acceptable salt or prodrug thereof, and the amount of Compounds 1 A and/or 2d, or pharmaceutically acceptable salts or prodrugs thereof in the composition.

Some embodiments described herein relate to a method for ameliorating or treating a disease condition that, can include administering an amount of Compound 1 A, or a pharmaceutically acceptable salt thereof, and an amount of Compound 2d, or a pharmaceutically acceptable salt or prodrug thereof and/or an amount of Compound 3d, or a pharmaceutically acceptable salt or prodrug thereof wherein the disease condition can be a hepatitis C virus infection, liver fibrosis, and/or impaired liver function.

Various dosages forms of Compound 1A or a pharmaceutically acceptable salt thereof, and/or Compound 2d, or a pharmaceutically acceptable salt or prodrug thereof and/or Compound 3d, or a pharmaceutically acceptable salt or prodrug thereof can be used to ameliorate and/or treat a disease condition. In some instances, Compounds 1 A and 2d and/or 3d or pharmaceutically acceptable salts or prodrugs thereof can be present in the same dosage form such as the compositions described herein. In other instances, Compound 1 A, and 2d and/or 3d, or pharmaceutically acceptable salts or prodrugs thereof, can be administered as separate dosage forms. For example, Compound 1 A, or a pharmaceutically acceptable salt thereof, can be administered in one tablet, Compound 2d, or a pharmaceutically acceptable salt or prodrug thereof, can be administered in a second tablet, and/or Compound 3d, or a pharmaceutically acceptable salt or prodrug thereof, can be administered in a third tablet. When Compounds 1 A, 2d, and 3d or pharmaceutically acceptable salts or prodrugs thereof, are contained in separate dosage forms, the dosage forms can be the same (e.g., as both pills) or different (e.g., two compounds can be formulated in a pill and the other compound can fee formulated as art injectable).

Administration of Compound 1 A or a pharmaceutically acceptable salt thereof; and Compound 2d, or a pharmaceutically acceptable salt or prodrug thereof, and/or Compound 3d, or a pharmaceutically acceptable salt or prodrug thereof can vary. When Compounds 1A, and 2d and/or 3d, or pharmaceutically acceptable salts or prodrugs thereof are contained in separate dosage forms, the dosage forms can be administered simultaneously or sequentially. In same embodiments, the dosage form that contains Compound 1A, or pharmaceutically acceptable salt thereof, can be administered before, after, in-between, concurrently or sequentially with Compounds 2d and/or 3d, or pharmaceutically acceptable salts or prodrugs thereof. In some embodiments, the dosage form that contains Compound 2d, or a pharmaceutically acceptable salt or prodrug thereof, can be administered before, after, in-between, concurrently or sequentially with Compound 1A, or Compounds 1 A and 3d, or pharmaceutically acceptable salts or prodrugs thereof. In some embodiments, the dosage form that contains Compound 3d, or a pharmaceutically acceptable salt or prodrug thereof, can be administered before, after, inbetween, concurrently or sequentially with Compound 1A or Compound 1 A and 2d, or pharmaceutically acceptable salts or prodrugs thereof.

In some embodiments, Compounds 1 A; and Compound 2d and/or 3d, or pharmaceutically acceptable salts or prodrugs thereof can be administered concurrently. As used herein, the term "concurrently means effective concentrations of all three compounds are present in a subject. When being administered concurrently, Compound 1 A, and Compound 2d and/or 3d, or pharmaceutically acceptable salts or prodrugs thereof, can be administered in the same dosage form or separate dosage forms. In other embodiments, Compound 1A, and Compound 2d, and/or 3d, or pharmaceutically acceptable salts or prodrugs hereof can be administered sequentially. As used herein, the term "sequentially" means administering one compound for a first time period, then administering a second compound for a second time period, optionally followed by administering a third compound for a third period, in which the first, second, and if applicable third time periods do not overlap. One aspect of the invention relates to a pharmaceutical composition that comprises, in a pharmaceutically acceptable vehicle, carrier or diluent: a first compound, with the formula

Compound 1 A or a pharmaceutically acceptable salt thereof, and a) a second compound, or a pharmaceutically acceptable salt or prodrug thereof wherein the second compound is grazoprevir ("Compound 2e"); and/or b) a third compound, or a pharmaceutically acceptable salt or prodrug thereof, wherein she third compound is elbasvir ("Compound 3e").

In another aspect the invention relates to a composition comprising, in a pharmaceutically acceptable vehicle, carrier or diluent Compound 1 A, and Compound 2e and/or Compound 3e, or pharmaceutically acceptable salts or prodrugs thereof, wherein the composition additionally comprises one or more therapeutic agents. In one embodiment the one or more therapeutic agents are ribavirin and ritonavir.

It will be appreciated that one aspect of the invention provides pharmaceutical compositions comprising, in a pharmaceutically acceptable vehicle, carrier or diluent, Compound 1 A and grazoprevir. A further aspect of the invention provides pharmaceutical compositions comprising, in a pharmaceutically acceptable vehicle, carrier or diluent, Compound 1A and elbasvir. A further aspect of the invention provides pharmaceutical compositions comprising, in a pharmaceutically acceptable vehicle, carrier or diluent, Compound 1 A and grazoprevir and elbasvir. In another aspect, the invention relates to the use of such compositions for ameliorating or treating a disease condition in a patient population, and/or for the preparation of a medicament for ameliorating or treating such a disease condition. For example, the disease condition can be selected from a hepatitis C virus infection, liver fibrosis, and impaired liver function. In one embodiment the invention relates to the use of a composition comprising Compound 1A, and Compound 2e and/or Compound 3e, or pharmaceutically acceptable salts or prodrugs thereof, for ameliorating or treating hepatitis C virus infection, liver fibrosis, and impaired liver function.

In another aspect the invention relates to a method for ameliorating or treating a disease condition in a patient population that comprises administering a therapeutically effective amount of a first compound, or a pharmaceutically acceptable salt or prodrug thereof wherein the first compound is Compound 1 A; and a therapeutically effective amount of a second compound, or a pharmaceutically acceptable salt or prodrug thereof, wherein the second compound is Compound 2e; and/or a therapeutically effective amount of a third compound, or a pharmaceutically acceptable salt or prodrug thereof wherein the third compound is Compound 3e; to a subject suffering from the disease conditions. In one embodiment, the disease condition can be selected from & hepatitis C virus infection, liver fibrosis, and impaired liver function.

In another aspect the invention relates to the use of Compound 1A or a pharmaceutically acceptable salt thereof for ameliorating or treating a disease condition in a patient population and/or for the preparation of a medicament for ameliorating or treating such a disease condition, wherein Compound 1 A or a pharmaceutically acceptable salt thereof is manufactured for use in combination with Compound 2e or a pharmaceutically acceptable salt or prodrug thereof; and wherein Compound 1 A and Compound 2e or pharmaceutically acceptable salts or prodrugs thereof are manufactured for use in combination with Compound 3e or a pharmaceutical acceptable salt or prodrug thereof wherein the disease condition is selected from hepatitis C virus infection, liver fibrosis, and impaired liver function,

In one embodiment of the Invention the method or use for ameliorating or treating a disease condition in a patient population comprises administering, one or more additional therapeutic agents. In another embodiment the one or more additional therapeutic agents are ribavirin and ritonavir, Grazoprevir, (Compound 2e) has shown to be effective in inhibiting HCV replication. The aforementioned compound can be obtained using methods known to those skilled in the art, including, for example, those methods disclosed in WO2010/01 1566 and WO2013/024465, which are hereby incorporated by reference in its entirety. Although this invention is not limited by any particular theory, Compound 2e is believed to inhibit the HCV protease, in particular the NS3/4A protease. Pharmaceutically acceptable salts and prodrugs of Compound 2e can be utilized in the compositions described herein.

Elbasvir (Compound 3e) has been demonstrated to be effective in inhibiting HCV replication. Compound 3e can be obtained using methods known to those skilled in the art, such as those methods described in WO2012/040923, which is hereby incorporated by reference in its entirety. Although this invention is not limited by any particular theory, it is believed that Compound 3e is an NS5A inhibitor a protein involved in the replication of the hepatitis C virus.

Similarly, various amounts of Compound 2e, or a pharmaceutically acceptable salt or prodrug thereof, can he included in the compositions. In some embodiments, the composition can include an amount of Compound 2e, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 10 mg to about 250 mg. In other embodiments, the composition can include an amount of Compound 2e, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 25 mg to about 150 mg. In still other embodiments, the composition can include an amount of Compound 2e, or a pharmaceutically acceptable salt or prodrug thereof, in the range of about 50 mg to about 100 mg. In an embodiment, the composition can include an amount of Compound 2e, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 100 mg.

Similarly, various amounts of Compound 3e, or a pharmaceutically acceptable salt or prodrug thereof can be included in the compositions. In some embodiments, the composition can include an amount of Compound 3e, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 10 mg to about 250 mg. In other embodiments, the composition can include an amount of Compound 3e, or a pharmaceutically acceptable salt or prodrug thereof, in the range of about 25 mg to about 100 mg. In still other embodiments, the composition can include an amount of Compound 3e or a pharmaceutically acceptable salt or prodrug thereof in the range of about 40 mg to about 60 mg. In yet still other embodiments, the composition can include an amount of Compound 3e, or a pharmaceutically acceptable salt or prodrug thereof in the amount of about 50 mg.

A potential advantage of making a combination of Compound 1 A and Compound 2e, and/or 3e, or pharmaceutically acceptable salts or prodrugs thereof may be a reduction in the required amounts of one or more compounds that are effective in treating a disease condition disclosed herein (for example, HCV) as compared to monotherapy treatment of an otherwise comparable patient population using either Compound 1 A, 2e or 3e alone. In some embodiments, the amount of Compound 1A_; or a pharmaceutically acceptable salt thereof in the composition can be less compared to the amount of Compound 1A or a pharmaceutically acceptable salt thereof needed to achieve the same viral load reduction when administered as a monotherapy. In some embodiments, the amount of Compound 2e or a pharmaceutically acceptable salt or prodrug thereof in the composition can be less compared to the amount of Compound 2e, or a pharmaceutically acceptable salt or prodrug thereof, needed to achieve the same viral load reduction when administered as a monotherapy. In some embodiments, the amount of Compound 3e or a pharmaceutically acceptable salt or prodrug thereof in the composition can be less compared to the amount of Compound 3e or a pharmaceutically acceptable salt or prodrug thereof needed to achieve the same viral load reduction when administered as a monotherapy.

In an embodiment, the sum of the amount of Compound 1A or a pharmaceutically acceptable salt and the amount of Compound 2e, or a pharmaceutically acceptable salt or prodrug thereof and/or the amount of Compound 3e, or a pharmaceutically acceptable salt or prodrug thereof is less than expected or predicted based on the additive combination of Compound 1 A or a pharmaceutically acceptable salt thereof, alone, Compound 2e, or a pharmaceutically acceptable salt or prodrug thereof alone, and/or Compound 3e, or a pharmaceutically acceptable salt or prodrug thereof alone for treating the disease condition such as HCV.

In general however, it is often convenient to administer Compound 1A, and Compound 2e and/or Compound 3e at dosages reflecting monotherapy dosage regimes for the respective component(s), and thereby provide an enhanced antiviral efficacy and/or onset of action.

The percentages of Compound 1A, and Compound 2e, and/or 3e or pharmaceutically acceptable salts or prodrugs thereof present in the composition can also vary. For example, in some embodiments, the composition can include an amount of Compound 1 A, or a pharmaceutically acceptable salt thereof, in the range of about 1 % to about 98% (weight/weight) based on the sum of the amount of Compound 1 A, or a pharmaceutically acceptable salt thereof and the amount of Compounds 2e and/or 3e or pharmaceutically acceptable salts or prodrugs thereof, in the composition. Additional embodiments include, but are not limited to, an amount of Compound 1A, or a pharmaceutically acceptable salt thereof in the range of about 5% to about 80%, about 10% to about 70%, about 15% to about 60%, about 20% to about 50% and about 30% to about 40% (weight/weight) based on the sum of the amount of Compound 1A, or a pharmaceutically acceptable salt or prodrug thereof, and the amount of Compounds 2e and/or 3e, or pharmaceutically acceptable safe or prodrugs thereof in the composition.

As to Compound 2e, in an embodiment, the composition can include an amount of Compound 2e, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 1 % to about 98% (weight/weight) based on the sum of the amount of Compound 2e, or a pharmaceutically acceptable salt or prodrug thereof and the amount of Compounds 1 A and/or 3e, or pharmaceutically acceptable salts or prodrugs thereof, in the composition. Examples of additional embodiments, include, but are not limited to, an amount of Compound 2e, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 5% to about 80%, about 10% to about 70%, about 15% to about 60%, about 20% to about 50% and about 30% to about 40 (weight/ weight) based on the sum of the amount of Compound 2e, or a pharmaceutically acceptable salt or prodrug thereof, and the amount of Compounds 1 A and/or 3e, or pharmaceutically acceptable salts or prodrugs thereof in the composition.

As to Compound 3e, in an embodiment, the composition can include an amount of Compound 3e, or a pharmaceutically acceptable salt or prodrug thereof in the range of about 1 % to about 98% (weight/weight) based on the sum of the amount of Compound 3e, or a pharmaceutically acceptable salt or prodrug thereof, and the amount of Compounds 1 A and/or 2e, or pharmaceutically acceptable salts or prodrugs thereof, in the composition. Examples of additional embodiments include, but are not limited, to, an amount of Compound 3e, or a pharmaceutically acceptable salt or prodrug thereof, in the range of about 5% to about 80%, about 10% to about 70%, about 15% to about 60%, about 20% to about 50% and about 30% to about 40% (weight/weight) based on the sum of the amount of Compound 3e, or a pharmaceutically acceptable salt or prodrug thereof, and the amount of Compounds 1 A and/or 2e, or pharmaceutically acceptable salts or prodrugs thereof in the composition.

Some embodiments described herein relate to a method for ameliorating or treating a disease condition that, can include administering an amount of Compound 1 A, or a pharmaceutically acceptable salt thereof, and an amount of Compound 2e, or a pharmaceutically acceptable salt or prodrug thereof and/or an amount of Compound 3e, or a pharmaceutically acceptable salt or prodrug thereof wherein the disease condition can be a hepatitis C virus infection, liver fibrosis, and/or impaired liver function. Various dosages forms of Compound 1A or a pharmaceutically acceptable salt thereof, and/or Compound 2e, or a pharmaceutically acceptable salt or prodrug thereof and/or Compound 3e, or a pharmaceutically acceptable salt or prodrug thereof can be used to ameliorate and/or treat a disease condition. In some instances, Compounds 1 A and 2e and/or 3e or pharmaceutically acceptable salts or prodrugs thereof can be present in the same dosage form such as the compositions described herein. In other instances, Compound 1 A, and 2e and/or 3e, or pharmaceutically acceptable salts or prodrugs thereof, can be administered as separate dosage forms. For example, Compound 1 A, or a pharmaceutically acceptable salt thereof, can be administered in one tablet, Compound 2e, or a pharmaceutically acceptable salt or prodrug thereof, can be administered in a second tablet, and/or Compound 3e, or a pharmaceutically acceptable salt or prodrug thereof, can be administered in a third tablet. When Compounds 1 , 2, and 3 or pharmaceutically acceptable salts or prodrugs thereof, are contained in separate dosage forms, the dosage forms can be the same (e.g., both or all as pills) or different (e.g., two compounds can be formulated in a pill and the other compound can fee formulated as art injectable). Administration of Compound 1 A or a pharmaceutically acceptable salt thereof; and Compound 2e, or a pharmaceutically acceptable salt or prodrug thereof, and/or Compound 3e, or a pharmaceutically acceptable salt or prodrug thereof can vary. When Compounds 1 A, and 2e and/or 3e, or pharmaceutically acceptable salts or prodrugs thereof are contained in separate dosage forms, the dosage forms can be administered simultaneously or sequentially. In same embodiments, the dosage form that contains Compound 1A, or pharmaceutically acceptable salt thereof, can be administered before, after, in-between, concurrently or sequentially with Compounds 2e and/or 3e, or pharmaceutically acceptable salts or prodrugs thereof. In some embodiments, the dosage form that contains Compound 2e, or a pharmaceutically acceptable salt or prodrug thereof, can be administered before, after, in-between, concurrently or sequentially with Compound 1A, or Compounds 1 A and 3e, or pharmaceutically acceptable salts or prodrugs thereof. In some embodiments, the dosage form that contains Compound 3e, or a pharmaceutically acceptable salt or prodrug thereof, can be administered before, after, inbetween, concurrently or sequentially with Compound 1A or Compound 1 A and 2e, or pharmaceutically acceptable salts or prodrugs thereof.

In some embodiments, Compounds 1 A; and Compound 2e and/or 3e, or pharmaceutically acceptable salts or prodrugs thereof can be administered concurrently. As used herein, the term "concurrently means effective concentrations of all three compounds are present in a subject. When being administered concurrently, Compound 1 A, and Compound 2e and/or 3e, or pharmaceutically acceptable salts or prodrugs thereof, can be administered in the same dosage form or separate dosage forms. In other embodiments, Compound 1A, and Compound 2e, and/or 3e, or pharmaceutically acceptable salts or prodrugs hereof can be administered sequentially. As used herein, the term "sequentially" means administering one compound for a first time period, then administering a second compound for a second time period, optionally followed by administering a third compound for a third period, in which the first, second, and if applicable third time periods do not overlap.

The invention relates to compounds for use in the treatment of HCV genotype 3a infection. Representative genotypes in the context of treatment or prophylaxis in accordance with the invention include genotype 3a, such as wild type genotype 3a and mutant strains of the genotype 3a, for example the S282T and L159/320F mutants. Typically, the invention provides a method for the treatment of HCV infection, in particular of the genotype 3a, such as wild type genotype 3a and mutant strains of the genotype 3a, for example the S282T and L159/320F mutants Additional representative HCV genotypes in the context of the invention include the major HCV genotypes, i.e. genotype 1 a, 1 b, 2a, 4a, 5a and 6a. In particular genotype 1 b (prevalent in Europe) and 1 a (prevalent in North America). The invention further relates to the treatment or prophylaxis of HCV infection caused by the genotypes 2a, 4a, 5a, 6a, especially the treatment of HCV infection caused by the genotypes 2a, 4a, 5a, 6a. The invention also provides a method for the treatment or prophylaxis of HCV infection, of the genotypes 2a, 4a, 5a, 6a, especially the treatment of HCV infection caused by the genotypes 2a, 4a, 5a, 6a.

The good activity of the compounds of the invention against genotype 3a is noteworthy given the poor performance of previous generations of nucleotides. Preferably the compositions of the invention have pan-genotypic coverage against each of the 6 genotypes, that is the EC₅₀ of the compound of the invention does not differ markedly between genotypes, thereby simplifying treatment.

As disclosed above, the combination therapies of the invention are generally applicable to all HCV genotypes, especially genotype 1 a and genotype 1 b. The compound of the invention have several chiral centers and may exist and be isolated in optically active and racemic forms, andmay exhibit polymorphism. It is to be understood that any racemic, optically active, diastereomeric, polymorphic or stereoisomeric form or mixtures thereof, of a compound provided herein is within the scope of this invention. The absolute configuration of such compounds can be determined using methods known in the art such as, for example, X-ray diffraction or NMR and/or implication from starting materials of known stereochemistry and/or stereoselective synthesis methods. Pharmaceutical compositions in accordance with the invention will preferably comprise substantially stereoisomerically pure preparations of the indicated stereoisomer. Pure stereoisomeric forms of the compounds and intermediates as mentioned herein are defined as isomers substantially free of other enantiomeric or diastereomeric forms of the same basic molecular structure of said compounds or intermediates. In particular, the term

"stereoisomerically pure" concerns compounds or intermediates having a stereoisomeric excess of at least 80% (i.e. minimum 90% of one isomer and maximum 10% of the other possible isomers) up to a stereoisomeric excess of 100% (i.e. 100% of one isomer and none of the other), more in particular, compounds or intermediates having a stereoisomeric excess of 90% up to 100%, even more in particular having a stereoisomeric excess of 94% up to 100% and most in particular having a stereoisomeric excess of 97% up to 100%. The terms "enantiomerically pure" and "diastereomerically pure" should be understood in a similar way, but then having regard to the enantiomeric excess, and the diastereomeric excess, respectively, of the mixture in question.

Pure stereoisomeric forms of the compounds and intermediates of this invention may be obtained by using procedures well known in the art. For instance, enantiomers may be separated from each other by resolution of the racemic mixture, i.e. formation of a

diastereomeric salt effected by reaction with an optically active acid or base followed by selective crystallization of the formed diastereomeric salt. Examples of such acids are tartaric acid, dibenzoyltartaric acid, ditoluoyltartaric acid and camphorsulfonic acid. Alternatively, enantiomers may be separated by chromatographic techniques using chiral stationary phases. Pure stereochemically isomeric forms may also be obtained by synthesis from stereochemically pure forms of the appropriate starting materials, provided that the reaction occurs

stereospecifically, by chiral synthesis or by utilisation of a chiral auxiliary. If a specific stereoisomer is desired, the preparation of that compound is preferably performed using stereospecific methods. These methods will advantageously employ enantiomerically pure starting materials. Diastereomeric racemates of the compounds of the invention can be separated by conventional methods. Appropriate physical separation methods that may advantageously be employed are, for example, selective crystallization and chromatography, e.g. column chromatography.

When a phosphorus atom is present in compounds of the invention comprising, the phosphorus atom may represent a chiral centre. The chirality at this centre is designated "R" or "S" according to the Cahn-lngold-Prelog priority rules. When the chirality is not indicated, it is contemplated that both the R- and S-isomers are meant to be included as well as a mixture of both stereoisomers. In preferred embodiments of the invention compounds of formula (I) or any subgroup of formula (I) are pure stereoisomers at the phosphorus atom. Preferred are compounds having the S- configuration at the phosphorus atom. These stereoisomers are designated S_P.

The present invention also includes isotope-labelled compounds of formula (I) or any subgroup of formula (I), wherein one or more of the atoms is replaced by an isotope of that atom, i.e. an atom having the same atomic number as, but an atomic mass different from, the one(s) typically found in nature. Examples of isotopes that may be incorporated into the compounds of formula (I) or any subgroup of formula (I), include but are not limited to isotopes of hydrogen, such as ²H and ³H (also denoted D for deuterium and T for tritium, respectively), carbon, such as ¹¹C, ¹³C and ¹⁴C, nitrogen, such as ¹³N and ¹⁵N, oxygen, such as ¹⁵0, ¹⁷0 and ¹⁸0, phosphorus, such as ³¹ P and ³²P, sulfur, such as ³⁵S, fluorine, such as ¹⁸F, chlorine, such as ³⁶CI, bromine such as ⁷⁵Br, ⁷⁶Br, ⁷⁷Br and ⁸²Br, and iodine, such as ¹²³l, ¹²⁴l, ¹²⁵l and ¹³¹1. The choice of isotope included in an isotope-labelled compound will depend on the specific application of that compound. For example, for drug or substrate tissue distribution assays, compounds wherein a radioactive isotope such as ³H or ¹⁴C is incorporated will generally be most useful. For radio-imaging applications, for example positron emission tomography (PET) a positron emitting isotope such as ¹¹C, ¹⁸F, ¹³N or ¹⁵0 will be useful. The incorporation of a heavier isotope, such as deuterium, i.e. ²H, may provide greater metabolic stability to a compound of formula (I) or any subgroup of formula (I), which may result in, for example, an increased in vivo half life of the compound or reduced dosage requirements.

Isotope-labelled compounds of formula (I) or any subgroup of formula (I) can be prepared by processes analogous to those described in the Schemes and/or Examples herein below by using the appropriate isotope-labelled reagent or starting material instead of the corresponding non-isotope-labelled reagent or starting material, or by conventional techniques known to those skilled in the art.

The pharmaceutically acceptable addition salts comprise the therapeutically active non-toxic acid and base addition salt forms of the compounds of formula (I). Of interest are the free, i.e. non-salt forms of the compounds of formula (I).

The pharmaceutically acceptable acid addition salts can conveniently be obtained by treating the base form with such appropriate acid. Appropriate acids comprise, for example, inorganic acids such as hydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric, nitric, phosphoric and the like acids; or organic acids such as, for example, acetic, propionic, hydroxyacetic, lactic, pyruvic, oxalic (i.e. ethanedioic), malonic, succinic (i.e. butanedioic acid), maleic, fumaric, malic (i.e. hydroxylbutanedioic acid), tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic, p-aminosalicylic, pamoic and the like acids. Conversely said salt forms can be converted by treatment with an appropriate base into the free base form. The compounds of formula (I) containing an acidic proton may also be converted into their nontoxic metal or amine addition salt forms by treatment with appropriate organic and inorganic bases. Appropriate base salt forms comprise, for example, the ammonium salts, the alkali and earth alkaline metal salts, e.g. the lithium, sodium, potassium, magnesium, calcium salts and the like, salts with organic bases, e.g. the benzathine, A/-methyl-D-glucamine, hydrabamine salts, and salts with amino acids such as, for example, arginine, lysine and the like. The terms and expressions used herein throughout the abstract, specification and claims shall be interpreted as defined below unless otherwise indicated. The meaning of each term is independent at each occurrence. These definitions apply regardless of whether a term is used by itself or in combination with other terms, unless otherwise indicated. A term or expression used herein which is not explicitly defined, shall be interpreted as having its ordinary meaning used in the art. Chemical names, common names, and chemical structures may be used interchangeably to describe the same structure. If a chemical compound is referred to using both a chemical structure and a chemical name and an ambiguity exists between the structure and the name, the structure predominates. The term "monophosphate, diphosphate and triphosphate ester" refers to groups:

Whenever used herein, the term "compounds of formula (I)", or "the compounds of the invention" or similar terms, it is meant to include the compounds of formula (I) and subgroups of compounds of formula (I), including the possible stereochemical^ isomeric forms, and their pharmaceutically acceptable salts and solvates.

The term "solvates" covers any pharmaceutically acceptable solvates that the compounds of formula (I) as well as the salts thereof, are able to form. Such solvates are for example hydrates, alcoholates, e.g. ethanolates, propanolates, and the like, especially hydrates. In general, the names of compounds used in this application are generated using ChemDraw Ultra 12.0. In addition, if the stereochemistry of a structure or a portion of a structure is not indicated with for example bold or dashed lines, the structure or portion of that structure is to be interpreted as encompassing all stereoisomers of it. General synthetic methods

Compounds of the present invention may be prepared by a variety of methods e.g. as disclosed in WO2015/034420and exemplified herein.

A route to the active metabolite of the compounds of formula (1 A) wherein B' is uracil is is illustrated in Scheme 15. r (b) which

is optionally protected.

15c

Scheme 15

A suitable phosphorylating agent for the preparation of the triphosphate of the compounds of formula (I) wherein B is the group (a) or (b) is 5-nitrocyclosalgenylchlorophosphite (I-6), which is prepared by reaction of phosphorous trichloride and 2-hydroxy-5-nitrobenzyl alcohol as detailed in the experimental part herein below.

Reaction of a suitably 3'-0-protected derivative of the nucleoside of the invention (15a) with nitrocyclosalgenylchlorophosphite (1-1 ) in the presence of Et₃N in an inert solvent like DCM or MeCN, followed by oxidation using for instance Oxone^®, provides the cyclic phosphate tri-ester (15b). The triphosphate (15c) is then achieved by reaction with a pyrophosphate for instance tributylamine pyrophosphate followed by treatment with ammonia. In order to get the desired salt form, the triphosphate is subjected to the appropriate ion exchange procedure, for instance, if the potassium salt form is desired, the residue is passed through a column Dowex^®-K⁺.

As will be readily apparent to one skilled in the art, the useful in vivo dosage to be administered and the particular mode of administration will vary depending upon the age, weight, the severity of the affliction, the mammalian species treated, the particular compounds employed, and the specific use for which these compounds are employed. (See e.g. Fingl et al 1975, in "The Pharmacological Basis of Therapeutics ^", which is hereby incorporated herein by reference in its entirety, with particular reference to Ch. 1 , p. 1 ). The determination of effective dosage levels, that is the dosage levels necessary to achieve the desired result, can be accomplished by one skilled in the art using routine pharmacological methods. Typically, human clinical applications of products are commenced at lower dosage levels, with dosage level being increased until the desired effect is achieved. Alternatively, acceptable in vitro studies can be used to establish useful doses and routes of administration of the compositions identified by the present methods using established pharmacological methods.

Although the exact dosage will be determined on a drug-by-drug basis, in most cases, some generalizations regarding &e dosage can be made. The dosage may be a single one or a series of two or more given in the course of one or more days, as is needed by the subject. In some embodiments, the compounds will be administered tor a period of continuous therapy, for example for a week or more, or for months or years,

In instances where human dosages for compounds have been established for at least some conditions, those same dosages, or dosages that are between about 10% and 200%, more preferably between about 50% and 150% of the established human dosage will be used. Where no human, dosage is established, as will be the case or newly-discovered pharmaceutical compositions, a suitable human dosage can be inferred from ED₅₀ or ID₅₀ values, or other appropriate values derived from in vitro or in vivo studies, as qualified by toxicity studies and efficacy studies in animals. It will often be convenient to use the same dose as monotherapy for the respective components of the combination, with the aim of achieving a more profound antiviral effect, onset of action, reduction in side effects or reduction in resistance.

In cases of administration of a pharmaceutically acceptable salt, dosages may be calculated as the free base or the free acid as the case may be. As will be understood by those of skill in the art, in certain situations it may be necessary to administer the compounds disclosed herein in amounts that exceed, or even far exceed, the above-stated, preferred dosage range in order to effectively and aggressively treat particularly aggressive diseases or infections.

Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the modulating effects, or minimal effective concentration (MEC). The MEC will vary for each compound but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, conventional MS or HPLC assays or bioassays can be used to determine plasma concentrations.

Dosage intervals can also be determined using MEC value. Compositions should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.

It should he noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity or organ dysfunctions. Conversely, the attending physician would also know to adjust treatment, to higher levels if the clinical response was not adequate (precluding toxicity). The magnitude of an administrated dose in the management of the disorder of interest will vary with the severity of the condition to be treated and the route of administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.

In non-human animal studies, applications of potential products are commenced at higher dosage levels, with dosage being decreased until the desired effect is no longer achieved or adverse side effects disappear. The dosage may range broadly, depending upon the desired effects and the therapeutic indication. Alternatively dosages may he based and calculated upon the surface area of the patient, as understood by those of skill in the art.

Compounds disclosed herein can be evaluated for efficacy and toxicity using know methods. For example, the toxicology of a particular compound, or of a subset of the compounds, sharing certain chemical moieties, may be established by determining in vitro toxicity towards a cell line, such as a mammalian, and preferably human, cell line_v The results of such studies are often predictive of toxicity in animals, such as mammals, or more specifically, humans. Alternatively the toxicity of particular compounds in an animal model, such as mice, rats, rabbits, or monkeys, may he determined using known methods. The efficacy of a particular compound may be established using several recognized methods, such as in vitro methods, animal models, or human clinical trials. Similarly, acceptable animal models may be used to establish efficacy of chemicals to treat such conditions. When selecting a model to determine efficacy, the skilled artisan can be guided by the state of the art to choose an appropriate model, dose, and route of administration, and regime. Of course, human clinical trials can also be used to determine the efficacy of a compound or composition in humans.

Any of the compositions and methods described herein can be administered to individuals who have been diagnosed with an HCV infection. Any of the compositions and methods described herein can be administered to individuals who have failed previous treatment for HCV infection (treatment failure patients, including non-responders and relapsers). Individuals who have been clinically diagnosed as infected with HCV are of particular interest in many embodiments. Individual who are infected with HCV are identified as having HCV RNA in their blood, and/or having anti-HCV antibody in their serum. Such individuals include anti HCV ELISA-positive individuals, and individuals with a positive recombinant immunoblot assay (RIBA). Such individuals may also, but need not, have elevated serum ALT levels. Individuals who are clinically diagnosed as infected with HCV include naive- individuals (e.g. individuals not previously treated for HCV, particularly those who have not previously received IFN-alpha based and/ribavirin-based therapy) and individuals who have failed prior treatment for HCV ("treatment failure" patients). Treatment failure patients include non-responders (i.e. individuals in whom the HCV titer was not significantly or sufficiently reduced by a previous treatment for HCV, e.g. a previous IFN-alpha monotherapy, a previous IFN-alpha and ribavirin combination therapy, or a previous pegylated IFN-alpha and ribavirin combination therapy); and relapsers (i.e. individual who were previously treated for HCV, e.g., who received a previous IFN-alpha monotherapy a previous IFN-alpha and ribavirin combination therapy, or previous pegylated IFN-alpha and ribavirin combination therapy, whose HCV titer decreased, and subsequently increased).

In an embodiment, HCV-positive individuals have an HCV titer of at least about 10⁵, at least about 5 x 10⁵ or at least about 10⁶ or at least about 2x10⁶ genome copies of HCV per milliliter of serum. The patient may be infected with any HCV genotype (genotype I, including la and lb, 2, 3, 4, 6, etc, and subtypes (e.g. 2a, 2b, 3a etc)), particularly difficult to treat genotypes such as HCV genotype 1 , and particular HCV subtypes and quasispecies. In one embodiment the patient is infected with HCV genotype 1 a. In another embodiment, the patient is infected with genotype 1 b.

In some embodiments, the HCV-positive individuals (as described above) are those who exhibit severe fibrosis or early cirrhosis (non-decompensated Childs-Pugh class A or less), or more advanced cirrhosis (decompensated, Childs-Pugh class B or C) due to chronic HCV infection and who are viremic despite prior anti-viral treatment with IFN-alpha-based therapies or who cannot tolerate IFN-based therapies, or who have a contraindication to such therapies. In an embodiment, HCV-positive individuals with stage 3 or 4 liver fibrosis according to the METAVIR scoring system are suitable for treatment with the compositions and methods described herein. In other embodiments, individuals suitable for treatment with the compositions and methods described herein are patients with decompensated cirrhosis with clinical manifestations, including patients with far-advanced liver cirrhosis, including those awaiting liver transplantation. In still other embodiments, individuals suitable for treatment with the compositions md methods described herein include patients with milder degrees of fibrosis including those with early fibrosis (stages 1 and 2 in the METAVIR, Ludwig, and Schemer scoring systems; or stages 1 , 2, or in the Ishak scoring system).

In one aspect, the present invention concerns a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (1 A), and a pharmaceutically acceptable carrier. A therapeutically effective amount in this context is an amount sufficient to stabilize or to reduce viral infection, and in particular HCV infection, in infected subjects (e.g. humans). The "therapeutically effective amount" will vary depending on individual requirements in each particular case. Features that influence the dose are e.g. the severity of the disease to be treated, age, weight, general health condition etc. of the subject to be treated, route and form of administration. In one aspect, the invention relates to the use of a compound of formula (1 A), for the treatment of "treatment naive" patients, i.e. patients infected with HCV that are not previously treated against the infection.

In another aspect the invention relates to the use of a compound of formula (1 A), the treatment of "treatment experienced" patients, i.e. patients infected with HCV that are previously treated against the infection and have subsequently relapsed. In another aspect the invention relates to the use of a compound of formula (1 A), the treatment of "non-responders", i.e. patients infected with HCV that are previously treated but have failed to respond to the treatment.

In a further aspect, the present invention concerns a pharmaceutical composition comprising a prophylactically effective amount of a compound of formula (1A) as specified herein, and a pharmaceutically acceptable carrier. A prophylactically effective amount in this context is an amount sufficient to act in a prophylactic way against HCV infection, in subjects being at risk of being infected. In still a further aspect, this invention relates to a process of preparing a pharmaceutical composition as specified herein, which comprises intimately mixing a pharmaceutically acceptable carrier with a therapeutically or prophylactically effective amount of a compound of formula (1 A), as specified herein. Therefore, the compounds of the present invention may be formulated into various pharmaceutical forms for administration purposes. As appropriate compositions there may be cited all compositions usually employed for systemically administering drugs. To prepare the pharmaceutical compositions of this invention, an effective amount of the particular compound, optionally in addition salt form or solvate, as the active ingredient is combined in intimate admixture with a pharmaceutically acceptable carrier, which carrier may take a wide variety of forms depending on the form of preparation desired for administration. These pharmaceutical compositions are desirable in unitary dosage form suitable, particularly, for administration orally, rectally, percutaneously, or by parenteral injection. For example, in preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed such as, for example, water, glycols, oils, alcohols and the like in the case of oral liquid preparations such as suspensions, syrups, elixirs, emulsions and solutions; or solid carriers such as starches, sugars, kaolin, lubricants, binders, disintegrating agents and the like in the case of powders, pills, capsules, and tablets. Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit forms, in which case solid pharmaceutical carriers are obviously employed. For parenteral compositions, the carrier will usually comprise sterile water, at least in large part, though other ingredients, for example, to aid solubility, may be included. Injectable solutions, for example, may be prepared in which the carrier comprises saline solution, glucose solution or a mixture of saline and glucose solution. Injectable suspensions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed. Also included are solid form preparations intended to be converted, shortly before use, to liquid form preparations. In the compositions suitable for percutaneous administration, the carrier optionally comprises a penetration enhancing agent and/or a suitable wetting agent, optionally combined with suitable additives of any nature in minor proportions, which additives do not introduce a significant deleterious effect on the skin. The compounds of the present invention may also be administered via oral inhalation or insufflation in the form of a solution, a suspension or a dry powder using any art-known delivery system.

It is especially advantageous to formulate the aforementioned pharmaceutical compositions in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form as used herein refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Examples of such unit dosage forms are tablets (including scored or coated tablets), capsules, pills, suppositories, powder packets, wafers, injectable solutions or suspensions and the like, and segregated multiples thereof.

In some embodiments, the compounds of a combination treatment, or pharmaceutically acceptable salts or prodrugs thereof are formulated in an aqueous buffer. Suitable aqueous buffers include, but are not limited to, acetate, succinate, citrate, and phosphate buffers varying in strengths from about 5 mM to about 100 mM. In some embodiments, the aqueous buffer includes reagents that provide for an isotonic solution. Such reagents include, but are not limited to, sodium chloride; and sugars e.g., mannitol, dextrose, sucrose, and the like, In some embodiments, the aqueous buffer further includes a non-ionic surfactant such a polysorbate 20 or 80,

Alternatively, the compounds, or pharmaceutically acceptable salts or prodrugs thereof of a combination therapy, are formulated in a conventional SEDDS (Self emulsifying Drug Delivery System) vehicle, in particular those of Pouten Type IIIA, NIB or IV.

Optionally the formulations may further include a preservative. Suitable preservatives include, but are not limited to, a benzyl alcohol, phenol, chlorobutanol, benzalkonium chloride, and the like. In many cases, the formulation is stored at about 4^. Formulations may also be lyophilized, in which case they generally include cryoprotectants such as sucrose, trehalose, lactose, maltose, mannitol, and the like. Lyophilized formulations can be stored over extended periods of time, even at ambient temperatures. The compounds of formula (1 A) show activity against HCV and can be used in the treatment and/or prophylaxis of HCV infection or diseases associated with HCV. Typically the compounds of formula (1A) can be used in the treatment of HCV infection or diseases associated with HCV. Diseases associated with HCV include progressive liver fibrosis, inflammation and necrosis leading to cirrhosis, end-stage liver disease, and HCC. A number of the compounds of this invention may be active against mutated strains of HCV. Additionally, many of the compounds of this invention may show a favourable pharmacokinetic profile and have attractive properties in terms of bioavailability, including an acceptable half-life, AUC (area under the curve) and peak values and lacking unfavourable phenomena such as insufficient quick onset and tissue retention.

The in vitro antiviral activity against HCV of the compounds of formula (I) can be tested in a cellular HCV replicon system based on Lohmann et al. (1999) Science 285:1 10-1 13, with the further modifications described by Krieger et al. (2001 ) Journal of Virology 75: 4614-4624 (incorporated herein by reference), which is further exemplified in the examples section. This model, while not a complete infection model for HCV, is widely accepted as the most robust and efficient model of autonomous HCV RNA replication currently available. It will be appreciated that it is important to distinguish between compounds that specifically interfere with HCV functions from those that exert cytotoxic or cytostatic effects in the HCV replicon model, and as a consequence cause a decrease in HCV RNA or linked reporter enzyme concentration. Assays are known in the field for the evaluation of cellular cytotoxicity based for example on the activity of mitochondrial enzymes using fluorogenic redox dyes such as resazurin. Furthermore, cellular counter screens exist for the evaluation of non-selective inhibition of linked reporter gene activity, such as firefly luciferase. Appropriate cell types can be equipped by stable transfection with a luciferase reporter gene whose expression is dependent on a constitutively active gene promoter, and such cells can be used as a counter-screen to eliminate non-selective inhibitors.

Due to their antiviral properties, particularly their anti-HCV properties, the compounds of formula (1 A), the pharmaceutically acceptable addition salts or solvates thereof, are useful in the treatment of warm-blooded animals, in particular humans, infected with HCV. The compounds of formula (1A) are further useful for the prophylaxis of HCV infections. The present invention furthermore relates to a method of treating a warm-blooded animal, in particular human, infected by HCV, or being at risk of infection by HCV, said method comprising the administration of an anti-HCV effective amount of a compound of formula (I).

The compounds of the present invention may therefore be used as a medicine, in particular as an anti HCV medicine. Said use as a medicine or method of treatment comprises the systemic administration to HCV infected subjects or to subjects susceptible to HCV infection of an amount effective to combat the conditions associated with HCV infection.

The present invention also relates to the use of the present compounds in the manufacture of a medicament for the treatment or the prevention of HCV infection.

In a preferred embodiment, the present invention relates to the use of the compounds of formula (1 A) in the manufacture of a medicament for the treatment of HCV infection. In general it is contemplated that an antiviral effective daily amount would be from about 0.01 to about 700 mg/kg, or about 0.5 to about 400 mg/kg, or about 1 to about 250 mg/kg, or about 2 to about 200 mg/kg, or about 10 to about 150 mg/kg, or about 0.1 to about 50 mg/kg, or about 1 to about 20 mg/kg, or about 2 to about 10 mg/kg, or about 5 to about 8 mg/kg body weight. It may be appropriate to administer the required dose as one, two, three, four or more sub-doses at appropriate intervals throughout the day. Said sub-doses may be formulated as unit dosage forms, for example, containing about 1 to about 5000 mg, or about 50 to about 3000 mg, or about 100 to about 1000 mg, or about 200 to about 600 mg, or about 100 to about 400 mg , or about 7 to about 3500 mg, or about 70 to about 1400 mg, or about 140 to about 700 mg, or about 340 to about 580 mg of active ingredient per unit dosage form.

The invention also relates to a combination of a compound of formula (1A), a pharmaceutically acceptable salt or solvate thereof, and another antiviral compound, in particular another anti- HCV compound. The term "combination" may relate to a product containing (a) a compound of formula (1A) and (b) optionally another anti-HCV compound, as a combined preparation for simultaneous, separate or sequential use in treatment of HCV infections.

Anti-HCV compounds that can be used in such combinations include HCV polymerase inhibitors, HCV protease inhibitors, inhibitors of other targets in the HCV life cycle, and an immunomodulatory agents, and combinations thereof. HCV polymerase inhibitors include, NM283 (valopicitabine), R803, JTK-109, JTK-003, HCV-371 , HCV-086, HCV-796 and R-1479, R-7128, MK-0608, VCH-759, PF-868554, GS9190, XTL-2125, NM-107, GSK625433, R-1626, BILB-1941 , ANA-598, IDX-184, IDX-375, INX-189, MK-3281 , MK-1220, ABT-333, PSI-7851 , PSI-6130, GS-7977 (sofosbuvir), VCH-916. Inhibitors of HCV proteases (NS2-NS3 inhibitors and NS3-NS4A inhibitors) include BILN-2061 , VX-950 (telaprevir), GS-9132 (ACH-806), SCH- 503034 (boceprevir), TMC435350 (simeprevir), TMC493706, ITMN-191 , MK-7009, BI-12202, BILN-2065, BI-201335, BMS-605339, R-7227, VX-500, BMS650032, VBY-376, VX-813, SCH-6, PHX-1766, ACH-1625, IDX-136, IDX-316. An example of an HCV NS5A inhibitor is BMS790052, A-831 , A-689, NIM-81 1 and DEBIO-025 are examples of NS5B cyclophilin inhibitors.

Inhibitors of other targets in the HCV life cycle, including NS3 helicase; metalloprotease inhibitors; antisense oligonucleotide inhibitors, such as ISIS-14803 and AVI-4065; siRNA's such as SIRPLEX-140-N; vector-encoded short hairpin RNA (shRNA); DNAzymes; HCV specific ribozymes such as heptazyme, RPI.13919; entry inhibitors such as HepeX-C, HuMax-HepC; alpha glucosidase inhibitors such as celgosivir, UT-231 B and the like; KPE-02003002; and BIVN 401 .

Immunomodulatory agents include, natural and recombinant interferon isoform compounds, including a-interferon, β-interferon, γ-interferon, and ω-interferon, such as Intron A®, Roferon- A®, Canferon-A300®, Advaferon®, Infergen®, Humoferon®, Sumiferon MP®, Alfaferone®, IFN- beta®, and Feron®; polyethylene glycol derivatized (pegylated) interferon compounds, such as PEG interferon-a-2a (Pegasys®), PEG interferon-a-2b (PEG-lntron®), and pegylated IFN- a-con1 ; long acting formulations and derivatizations of interferon compounds such as the albumin-fused interferon albuferon a; compounds that stimulate the synthesis of interferon in cells, such as resiquimod; interleukins; compounds that enhance the development of type 1 helper T cell response, such as SCV-07; TOLL-like receptor agonists such as CpG-10101 (actilon), and isatoribine; thymosin a-1 ; ANA-245; ANA-246; histamine dihydrochloride; propagermanium; tetrachlorodecaoxide; ampligen; IMP-321 ; KRN-7000; antibodies, such as civacir and XTL-6865; and prophylactic and therapeutic vaccines such as InnoVac C and HCV E1 E2/MF59. Other antiviral agents include, ribavirin, amantadine, viramidine, nitazoxanide; telbivudine; NOV- 205; taribavirin; inhibitors of internal ribosome entry; broad-spectrum viral inhibitors, such as IMPDH inhibitors, and mycophenolic acid and derivatives thereof, and including, but not limited to, VX-497 (merimepodib), VX-148, and/or VX-944); or combinations of any of the above. Particular agents for use in said combinations include interferon-a (IFN-a), pegylated interferon- α or ribavirin, as well as therapeutics based on antibodies targeted against HCV epitopes, small interfering RNA (Si RNA), ribozymes, DNAzymes, antisense RNA, small molecule antagonists of for instance NS3 protease, NS3 helicase and NS5B polymerase.

In another aspect there are provided combinations of a compound of formula (I) as specified herein and an anti-HIV compound. The latter preferably are those HIV inhibitors that have a positive effect on drug metabolism and/or pharmacokinetics that improve bioavailability. An example of such an HIV inhibitor is ritonavir. As such, this invention further provides a combination comprising (a) a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof; and (b) ritonavir or a pharmaceutically acceptable salt thereof. The compound ritonavir, its pharmaceutically acceptable salts, and methods for its preparation are described in WO 94/14436. US 6,037,157, and references cited therein: US 5,484,801 , US 08/402,690, WO 95/07696, and WO 95/09614, disclose preferred dosage forms of ritonavir.

The invention also concerns a process for preparing a combination as described herein, comprising the step of combining a compound of formula (I) and another agent, such as an antiviral, including an anti-HCV or anti-HIV agent, in particular those mentioned above.

The said combinations may find use in the manufacture of a medicament for treating HCV infection in a mammal infected therewith, said combination in particular comprising a compound of formula (I), as specified above and interferon-a (IFN-a), pegylated interferon-a, or ribavirin. Or the invention provides a method of treating a mammal, in particular a human, infected with HCV comprising the administration to said mammal of an effective amount of a combination as specified herein. In particular, said treating comprises the systemic administration of the said combination, and an effective amount is such amount that is effective in treating the clinical conditions associated with HCV infection.

In one embodiment the above-mentioned combinations are formulated in the form of a pharmaceutical composition that includes the active ingredients described above and a carrier, as described above. Each of the active ingredients may be formulated separately and the formulations may be co-administered, or one formulation containing both and if desired further active ingredients may be provided. In the former instance, the combinations may also be formulated as a combined preparation for simultaneous, separate or sequential use in HCV therapy. The said composition may take any of the forms described above. In one embodiment, both ingredients are formulated in one dosage form such as a fixed dosage combination. In a particular embodiment, the present invention provides a pharmaceutical composition comprising (a) a therapeutically effective amount of a compound of formula (I), including a possible stereoisomeric form thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, and (b) a therapeutically effective amount of ritonavir or a pharmaceutically acceptable salt thereof, and (c) a carrier.

The individual components of the combinations of the present invention can be administered separately at different times during the course of therapy or concurrently in divided or single combination forms. The present invention is meant to embrace all such regimes of simultaneous or alternating treatment and the term "administering" is to be interpreted accordingly. In a preferred embodiment, the separate dosage forms are administered simultaneously. In one embodiment, the combinations of the present invention contain an amount of ritonavir, or a pharmaceutically acceptable salt thereof, that is sufficient to clinically improve the bioavailability of the compound of formula (I) relative to the bioavailability when said compound of formula (I) is administered alone. Or, the combinations of the present invention contains an amount of ritonavir, or a pharmaceutically acceptable salt thereof, which is sufficient to increase at least one of the pharmacokinetic variables of the compound of formula (I) selected from t_1/2, Cmin, C_max, C_ss, AUC at 12 hours, or AUC at 24 hours, relative to said at least one pharmacokinetic variable when the compound of formula (I) is administered alone.

The combinations of this invention can be administered to humans in dosage ranges specific for each component comprised in said combinations, e.g. the compound of formula (I) as specified above, and ritonavir or a pharmaceutically acceptable salt, may have dosage levels in the range of 0.02 to 5.0 g/day.

The weight ratio of the compound of formula (I) to ritonavir may be in the range of from about 30:1 to about 1 :15, or about 15: 1 to about 1 : 10, or about 15: 1 to about 1 : 1 , or about 10: 1 to about 1 : 1 , or about 8: 1 to about 1 : 1 , or about 5: 1 to about 1 : 1 , or about 3: 1 to about 1 :1 , or about 2:1 to 1 :1 . The compound formula (I) and ritonavir may be co-administered once or twice a day, preferably orally, wherein the amount of the compound of formula (I) per dose is as described above; and the amount of ritonavir per dose is from 1 to about 2500 mg, or about 50 to about 1500 mg, or about 100 to about 800 mg, or about 100 to about 400 mg, or 40 to about 100 mg of ritonavir.

Detailed Description of the Embodiments

Various embodiments of the invention and intermediates therefore will now be illustrated by the following examples. The Examples are just intended to further illustrate the invention and are by no means limiting the scope of the invention. The compound names were generated by ChemDraw Ultra software, Cambridgesoft, version 12.0.2. In addition to the definitions above, the following abbreviations are used in the examples and synthetic schemes below. If an abbreviation used herein is not defined, it has its generally accepted meaning

Bn Benzyl

Bz Benzoyl

BOP-CI Bis(2-oxo-3-oxazolidinyl)phosphinic chloride

Bz Benzoyl

DCC Dicyclohexylcarbodiimide

DCM Dichloromethane

DIEA Diisopropylethylamine

DMAP 4-Dimethylaminopyridine

DMF A/,A/-Dimethylformamide

DMPU 1 ,3-Dimethyl-3,4,5,6-tetrahydro-2-pyrimidinone

EDC 1 -(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

ES Electrospray

Et₃N Triethylamine

EtOAc Ethyl acetate

EtOH Ethanol

Et₂0 Diethyl ether

LC Liquid chromatography

HOAc Acetic acid

HPLC High performance liquid chromatography

MeCN Acetonitrile

MeOH Methanol

MS Mass spectrometry

NT 3-nitro-1 ,2,4-triazole

NTP Nucleoside triphosphate

Pg Protecting group

Ph Phenyl

SEM Standard error of the mean

TEST bis(triethoxysilyl)propyl-tetrasulfide

THF Tetrahydrofuran

TFA Trifluoroacetic acid

TFAA Trifluoroacetic anhydride

TIPS Triisopropylsilyl ntermediate 52

Step a) L-Alanine isopropylester hydrochloride (l-52a)

Thionylchloride (80.2 g, 0.674 mol,1 .5 eq) was added with cooling to 2-propanol (400 mL) at -7 to 0 °C over a period of 30 minutes, followed by addition of L-alanine (40.0 g, 0.449 mol) at 0 °C. A flow indicator and a scrubber with a mixture of 27.65% sodium hydroxide (228 g) and water (225 g) were attached to the outlet. The reaction mixture was stirred at 67°C for two hours, then at 70 °C for one hour and at 20 - 25 °C over night. The reaction mixture was distilled at 47-50 °C under reduced pressure (250 - 50 mBar) from a 60 °C bath. When the distillation became very slow, toluene (100 mL) was added to the residual oil, and the distillation at 48-51 °C under reduced pressure (150 - 50 mBar) from a 60 °C bath was continued until it became very slow, t- butylmethylether (tBME)(400 mL) was added to the residual oil, and the two-phase system ws seeded under efficient stirring at 34 - 35 °C. When crystallization was observed the mixture was cooled to 23 °C over a period of one hour, and the precipitate isolated by filtration. The filter cake was washed with tBME (100 mL) and dried to constant weight under reduced pressure without heating, which gave the title compound (67.7 g, 90%) as white solids.

Step b) (S)-lsopropyl 2-(((S)-(perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate (I-52) Phenyl dichlorophosphate (62.88 g, 0.298 mol, 1 .0 eq) was added under nitrogen to a solution of L-alanine isopropylester hydrochloride (50.0 g, 0.298 mol) in DCM (310 mL) at 0 °C - the addition was completed by wash with DCM (39 mL). The mixture was cooled and triethylamine (63.35 g, 0.626 mol, 2.1 eq) was added over a period of 70 minutes with cooling keeping the temperature not higher than -14 °C, the addition was completed by wash with DCM (39 mL). The mixture was stirred for one hour at -15 to -20 °C, then heated to -8 °C and a solution of pentafluorophenol (60.38 g, 0.328 mol, 1 .1 eq) and triethylamine (33.19 g, 0.328 mol, 1 .1 eq) in DCM (78 mL) was added over a period of 42 minutes with cooling keeping the temperature not higher than 0 °C - the addition was completed by wash with DCM (39 mL). The mixture was stirred for one hour at 0 °C and then over night at +5 °C. The formed precipitate was removed by filtration, and the filter cake washed with DCM (95 mL). The combined filtrates were washed at 5 °C with water (2x190 mL). The organic phase was distilled at 32 - 38°C at reduced pressure (650 - 600 mBar), and distillation was continued until a residual volume of approx. 170 mL partly crystallized mass was obtained. Ethyl acetate (385 mL) was added, and the resulting clear solution was distilled at 43 - 45 °C under reduced pressure (300 - 250 mBar). Distillation was continued until a residual volume of approx. 345 mL was obtained. The clear solution was cooled to 36 °C, and crystallization is induced by addition of seed crystals of (S)-isopropyl 2- (((S)-(perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate (20 mg) prepared as described in J. Org. Chem., 201 1 , 76, 831 1 - 8319. The mixture was cooled to 27 °C over a period of one hour, then n-heptane (770 mL) was added over a period of 47 minutes, and the mixture was stirred for an additional period of 37 minutes. Triethylamine (6.03 g, 0.2 eq) was added, and the mixture was stirred at 23 - 25 °C over night. The precipitate was isolated by filtration. The filter cake was washed with ethyl acetate:n-heptane (1 :9, 80 mL) and dried to constant under reduced pressure (below 0.1 mBar) without heating, which gave the title compound (75.64 g, 56%) as a white crystalline material.

¹ H NMR (CDCI₃, 300 MHz) δ 7.38-7.32 (m, 2 H), 7.27-7.24 (m, 2 H), 7.23-7.19 (m, 1 H), 5.10- 4.98 (m, 1 H), 4.20-4.08 (m, 1 H), 4.03-3.96 (m, 1 H), 1 .46 (dd, 7.2, 0.6 Hz, 3 H), 1 .26-1 .23 (2xd, 6 H);

¹³CNMR (CDCI₃, 100 MHz) δ 172.7 (d, J = 8.8 Hz), 150.4 (d, J = 7.1 Hz), 143.4-143.0 (m), 141 .0-140.2 (m), 140.0-139.8 (m), 137.6-137.2 (m), 136.8-136.2 (m), 130.0 (d, J = 0.82 Hz),

125.8 (d, J = 1 .4 Hz), 120.3 (d, J = 5.0 Hz), 69.8, 50.6, (d, J = 1 .9 Hz), 21 .8 (d, J = 1 .9 Hz), 21 .2 (d, J = 4.4 Hz);

The crystallization properties and NMR spectral data of the title compound were in agreement with published data (J. Org. Chem., 201 1 , 76, 831 1 -8319), thus confirming the S

stereochemistry of the phosphorus atom of the title compound.

Example 1

1a 1b

^TIPSO ° DIBAI ^TIPS° ^<°i

TIPSO H F-^Cl

TIPSOH F*^CI ^c id 1e

if

Step a) (4S,5R)-4-((triisopropylsilyl)oxy)-5-(((triisopropylsilyl)oxy)methyl)dihvdrofuran-2(3H)-one iia) TIPS-chloride (16.4 g, 85 mmol) was added drop wise to an ice cooled stirred solution of (4S,5R)-4-hydroxy-5-(hydroxymethyl)dihydrofuran-2(3H)-one (3.30 g, 25.0 mmol) and imidazole (10.2 g, 150 mmol) in DMF (35 mL). The mixture was stirred for 1 h at 0 °C then at rt for 40 h. The reaction was quenched with water and the mixture extracted three times with EtOAc. The organic phase was dried (Na₂S0₄), filtered and concentrated, and the product was isolated by silica gel column chromatography eluted with a gradient of isohexane and 0 to 10 % EtOAc. Mixed fractions were purified again by silica gel column chromatography eluted with toluene, which gave the title compound (1 1 .1 g, 94%). Step b) (3S,4R,5R)-3-fluoro-4-((triisopropylsilyl)oxy)-5-(((triisopropylsilyl)oxy)methyl)- dihvdrofuran-2(3H)-one (1 b)

A 1 M solution of lithium bis(trimethylsilyl) amide (2.18 g, 13.0 mmol) was added dropwise during 10 min to a solution at -70 °C of 1 a (4.45 g, 10.0 mmol) and NFSI (4.73 g, 15.0 mmol) in dry THF (50 mL). The mixture was stirred for 90 min at -70 °C, then added to a saturated solution of ammonium chloride and cracked ice. The mixture was extracted three times with EtOAc, the organic phase was dried (Na₂S0₄), filtered and concentrated, and the product was isolated by silica gel chromatography eluted with a gradient of isohexane and 0 to 5% EtOAc. Yield 4.63 g, 67%. Step c) (3S,4R,5R)-3-chloro-3-fluoro-4-((triisopropylsilyl)oxy)-5-(((triisopropylsilyl)oxy)methyl)- dihvdrofuran-2(3H)-one (1 c)

A 1 M solution of lithium bis(trimethylsilyl) amide was added drop wise during 10 min to a solution at -70 °C of 1 b (3.08 g, 6.65 mmol) and N-chlorosuccinimide (1 .07 g, 7.99 mmol) in dry THF (25 mL). The mixture was stirred for 90 min at -70 °C, then added to a saturated solution of ammonium chloride and cracked ice. The mixture was extracted three times with EtOAc, the organic phase was dried (Na₂S0₄), filtered and concentrated, and the product was isolated by silica gel chromatography eluted with a gradient of isohexane and 0 to 5% EtOAc. Yield 2.40 g, 73%. Step d) (3S,4R,5R)-3-chloro-3-fluoro-4-((triisopropylsilyl)oxy)-5-(((triisopropylsilyl)oxy)methyl)- tetrahydrofuran-2-ol (1 d)

A 1 M solution of DIBAL (2.23 g, 15.7 mmol) in DCM was added dropwise under argon to solution at -70 °C of 1 c (5.20 g, 10.5 mmol) in dry toluene (50 mL). The mixture was stirred for 2 h at -70 °C, then the temperature was raised to -30 °C and the reaction was quenched with 2mL MeOH and then added to a mixture of Rochelle salt and crashed ice. The mixture was stirred for 30 minutes and then extracted three times with EtOAc. The organic phase was dried (Na₂S0₄), filtered and concentrated under reduced pressure. The product was isolated by silica gel column chromatography eluted with a gradient of isohexane and 0 to 10% EtOAc. Yield 5.22 g, 85%.

Step e) (2S,3S,4R,5R)-3-chloro-3-fluoro-4-((triisopropylsilyl)oxy)-5- (((triisopropylsilyl)oxy)methyl)tetrahvdrofuran-2-yl methanesulfonate (1 e)

Mesyl chloride (688 mg, 6.00 mmol) was slowly added to a cooled solution of 1 d (2.00 g, 4.01 mmol) and TEA (608 mg, 6.00 mmol) in DCM (20 ml_). The mixture was stirred for three hours at RT, then diluted with EtOAc (80 ml_), washed with saturated NaHC0₃ (aq), HCI , water and with brine. The organic phase was dried (Na₂S0₄), filtered and concentrated. The crude product was dried in vacuo and then was used in the next step without further purification.

Step f) 1 -((2R.3S,4R.5R)-3-chloro-3-fluoro-4-((triisopropylsilvnoxy)-5- (((triisopropylsilyl)oxy)methyl)tetrahvdrofuran-2-yl)pyrimidine-2,4(1 H,3H)-dione (1 f)

A suspension of uracil (699 mg, 6.24 mmol) and ammonium sulfate (25.8 mg, 0.195 mmol) in hexamethyldisilazane (HDMS) (40 mL) was refluxed overnight. The solvent was removed in vacuo and the residue was dissolved in DCM (60 ml_).The 1 e (2.25 g, 3.90 mmol) was added under argon and then the TMS triflate was added slowly. The mixture was stirred for 10 minutes at RT and then refluxed for 4 hours. The mixture was added to cooled sodium hydrogen carbonate solution and extracted three times with EtOAc . The organic phase was washed with brine and dried over sodium sulfate. The solution was evaporated under reduced pressure and the mixture was purified by silica gel chromatography with isohexane and 20 to 50% ethyl acetate, which gave the two compounds diTIPS (1 .29 g, 56%) and monoTIPS (390 mg, 23%).

Step g) 1 -((2R,3S,4R,5R)-3-chloro-3-fluoro-4-hvdroxy-5-(hvdroxymethyl)tetrahvdrofuran-2- yl)pyrimidine-2,4(1 H,3H)-dione (1 a)

A solution of 1 f (1 .27 g, 2.14 mmol) in 80% acetic acid was stirred at 80 °C for 18 h, then concentrated and co-evaporated with toluene. The residue was dissolved in dry THF (10 mL), triethylamine trihydrofluoride was added (1 .38 g, 8.56 mmol) and the mixture was evaporated onto silica and purified by silica gel column chromatography eluted with DCM including 0 to 10% MeOH. The mixed fractions were purified by HPLC on a Hypercarb column eluted with 10 to 20% acetonitrile and 10 mmol ammonium acetate, which gave the title compound (19 mg, 3.2%). MS 281 .2 [M+H]⁺.

¹ H NMR (500 MHz, DMSO) δ 10.39 (s, 1 H), 7.87 (d, J = 8.1 Hz, 1 H), 6.74 (s, 1 H), 6.22 (d, J = 16.1 Hz, 1 H, 7), 5.73 (d, J = 8.1 Hz, 1 H), 5.52 (s, 1 H), 4.21 (dd, J = 19.6, 9.2 Hz, 1 H), 3.87 - 3.77 (m, 2H), 3.64 (dd, J = 12.7, 2.8 Hz, 1 H).

1³C NMR (126 MHz, DMSO) δ 162.76, 150.26, 139.06, 1 15.71 , 1 13.71 , 102.28, 86.98, 86.69, 81 .01 , 73.28, 73.14, 58.19. Example

(2S)-isopropyl 2-(((((2R,3R^S,5R)-4-chloro-5-(2,4-dioxo-3,4-dihvdropyrimidin-1 (2H)-yl)-4-fluoro- 3-hvdroxytetrahvdrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate(2)

A 1 M solution of tert-butyl magnesium chloride (0.22 mL, 0.22 mmol) was slowly added under argon to a solution of sugar 1 g (28 mg, 0.1 mmol) in THF (1 .5 mL). The suspension was stirred for one h at 0 °C, then DMPU (0.5 mL) was added followed by addition of a solution of

(2S)-isopropyl 2-(((perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate (57 mg, 0.12 mmol) (prepared as described in WO201 1/123672) in THF (0.5 mL) at 0 °C during ~5 min. The mixture was stirred for 5h at 0 °C, then allowed to attain RT and was quenched with saturated ammonium chloride solution. The mixture was extracted three times with EtOAc. The organic phase was dried (Na₂S0₄), concentrated under reduced pressure and the product was isolated by HPLC. (Gemini NX 30mm 20 to 60% acetonitrile 10 mmol ammonium acetate Gradient 17 minutes and flow 40 ml per minute. The title compound was isolated as a single stereoisomer at the phosphorus atom. Yield 22 mg, 40%. MS ES+ 550.4 [M+H]⁺, ES- 547.9 [M-H]\

¹ H NMR (500 MHz, DMSO) δ 1.15 (d, 6H), 1.23 (d, 3H), 3.80 (tq, 1 H), 4.04 (m, 1 H), 4.31 (m, 3H), 4.86 (hept, 1 H), 5.63 (dd, 1 H), 6.09 (dd, 1 H), 6.24 (d, 1 H), 6.66 (d, 1 H), 7.21 (m, 3H), 7.38 (m, 2H), 7.58 (d, 1 H), 1 1 .63 (m, 1 H).

¹³C NMR (126 MHz, DMSO) δ 19.64 (d), 21.26, 21.30, 49.67, 64.32, 67.89, 74.42 (d), 78.81 , 87.60 (m), 102.27, 1 13.96 (d), 1 19.96 (d), 124.52, 129.56, 139.91 , 150.01 , 150.53 (d), 162.52, 172.45 (d).

³¹ P NMR (162 MHz, DMSO) δ 3.76.

1⁹F NMR (376 MHz, DMSO) δ -1 19.05.

Example 3

Step a) (2R,3R,4S,5R)-4-chloro-5-(2,4-dioxo-3,4-dihvdropyrimidin-1 (2H)-yl)-4-fluoro-2- (hvdroxymethvntetrahvdrofuran-3-yl acetate (3a)

4-methoxytrityl chloride (133 mg, 0.43 mmol) was added to a solution of compound 1 f (81 mg, 0.29 mmol) in pyridine (25 mL). The resulting mixture was stirred at room temperature for 40 h, the diluted with DCM and washed with NaHC0₃. The organic phase was concentrated and the residue purified by column chromatography on silica gel, which gave the title compound (144 mg, 90%).

The afforded compound was dissolved in dry pyridine (1 .4 mL), Ac₂0 (29 μΐ, 0.31 mmol) was added and the solution was stirred at rt. After 2h, MeOH was added, the mixture was concentrated and extracted with DCM (x3) and the combined organic layers were washed with sat. aq. NaHC0₃, Na₂S0₄, concentrated and co-evaporated once with THF.

The residue was taken up in 80% HOAc (35 mL) and stirred at 45 °C for 3h, then concentrated. The residue was purified by column chromatography on silica gel, which gave the title compound (69 mg, 33%).

Step b) Lithium ((2R,3R,4S,5R)-4-chloro-5-(2,4-dioxo-3,4-dihvdropyrimidin-1 (2H)-yl)-4-fluoro-3- hvdroxytetrahvdrofuran-2-yl)methyl triphosphate (3b)

A freshly prepared solution of 2-chloro-1 ,3,2-benzodioxaphosphin-4-one (64 mg, 0.31 mmol) in anhydrous THF (280 μΐ) was added under nitrogen to a stirred solution of compound 3a (78 mg, 0.24 mmol) in a mixture of anhydrous pyridine (560 μΐ) and anhydrous THF (560 μΐ). The mixture was stirred at room temperature under nitrogen for 1 5 minutes, then a previously prepared solution of tributylammonium P₂0₇ (146 mg, 0.27 mmol) and tributylamine (127 μΐ, 0.53 mmol) in anhydrous DMF (560 μΐ) was added under nitrogen. The afforded solution was stirred for additional 15 minutes at room temperature under nitrogen, then l₂ (1 23 mg, 0.48 mmol) was added as a solution in pyridine/water (98/2, v/v) (1 .1 mL) and the reaction mixture was stirred for 1 5 minutes. Excess iodine was destroyed by addition of - 19 drops of a 5% aqueous solution of Na₂S0₃ and the reaction solution was concentrated. The residue was taken in water/acetonitrile (95:5) (5 mL) and left shaking at room temperature for 30 minutes.

Concentrated ammonia (10 mL) was added and the reaction mixture stirred for 1 ½h at room temperature, then concentrated and the residue dissolved in water/acetonitrile (95:5, 5 mL) and freeze dried.

The crude material -430 mg, was dissolved in 10% MeCN/water (3 mL) and filtered and purified by HPLC on a Gilson instrument using a Phenomenex Luna 5μ NH₂ (150x21 .2mm) column,

Solvent A: 95%water:5%acetonitrile: 0.05M ammonium bicarbonate

Solvent B: 95%water:5%acetonitrile: 0.8M ammonium bicarbonate

Gradient: 0% B to 50% B in 30 min.

The NTP fractions were pooled and concentrated, the residue was dissolved in 10%

MeCN/water and freeze dried. The afforded solids were taken up in 10% MeCN/water, insolubles were filtered off through 0.45 μηι frit filters and the clear filtrate was evaporated to dryness, dissolved in water/acetonitrile (95:5), passed through Dowex-Li⁺ and freeze-dried which gave the title compound (39.3 mg, 28%).

1 H NMR (500 MHz, D₂0) δ 7.87 (d, J = 8.2 Hz, 1 H), 6.41 (d, J = 15.9 Hz, 1 H, 1), 5.98 (d, J = 8.2

Hz, 1 H), 4.56 (dd, J = 19.1 , 9.4 Hz, 1 H, 5), 4.35 (dddd, J = 42.1 , 12.3, 5.1 , 2.2 Hz, 3H), 4.19 (d,

J = 9.4 Hz, 1 H,8).

¹³C NMR (126 MHz, D₂0) δ 165.94, 151.67, 140.78, 114.54, 1 12.55, 103.12, 87.95, 87.62, 79.45, 79.38, 73.16, 73.02, 62.60, 62.56.

Example 19, alternative route to compound 1 q

1c

Step b ( 1 i93aa,, R == T I I IPSS S _ott„e„p d^ ( / 19c, R = Ac

19b, R = H 19d, R = H

Step a) (3S,4R,5R)-3-Chloro-3-fluoro-4-((triisopropylsilyl)oxy)-5-(((triisopropylsilyl)oxy)methyl)- tetrahvdrofuran-2-yl acetate (19a)

A 1 M solution of Li(0-t-Bu)₃AIH in THF (39 mL, 39 mmol) was added dropwise under argon at - 35 °C to solution of compound 1 c (16.3 g, 32.8 mmol) in THF (120 mL). The mixture was stirred for 1 h at -35 °C, then at rt for 1 h. The mixture was cooled to -25 °C, DMAP (4.00 g, 32.8 mmol) was added and the mixture was stirred for 15 minutes, then acetic anhydride (33.5 g, 328 mmol) was added drop wise and the mixture was stirred 2h. The mixture was allowed to come to 0 °C and EtOAc (200 mL) and water (200 mL) were added. The phases were separated and the water phase was extracted with EtOAc (x2). The combined organic phases were washed with water (x2) and with brine (x1 ). The organic phase was dried (Na₂S0₄), filtered and concentrated under reduced pressure. The residue was co-evaporated twice with toluene and the product was purified by chromatography on silica gel with eluted with isohexane and 2 to 6% EtOAc, which gave the title compound (17.1 g, 96%).

Step b) (3S,4R,5R)-3-Chloro-3-fluoro-4-hvdroxy-5-(hvdroxymethyl)tetrahvdrofuran-2-yl acetate il9b)

Triethylamine tri hydrofluoride (20.5 g, 126 mmol) was added to a stirred solution of compound 19a (17.0 g, 31 .4 mmol) in acetonitrile (1 15 mL) and THF (23 mL). The mixture was stirred for 72 h at rt, 20 h at 50 °C and then at rt overnight. The solution was concentrated on silica (60 g) and purified by silica gel chromatography eluted with a gradient of isohexane and EtOAc, which gave the title compound (68.0 g, 85%).

Step c) (2R,3R,4S)-5-Acetoxy-2-((benzoyloxy)methyl)-4-chloro-4-fluorotetrahvdrofuran-3-yl benzoate (19c)

Triethylamine (10.8 g, 107 mmol) was added to a stirred solution of compound 19b (6.80 g, 26.8 mmol) under ice cooling followed by drop wise addition of benzoyl chloride (9.41 g, 66.9 mmol). The mixture was allowed to attain rt and stirred overnight. EtOH (5 mL) was added and the mixture was stirred for 30 minutes, then concentrated in vacuo. Water was added and the mixture was extracted with EtOAc (x3). The organic phase was washed with water and brine, dried (Na₂S0₄), filtered and concentrated under reduced pressure. The product was purified by silica gel chromatography eluted with a gradient of isohexane and EtOAc, which gave the title compound (10.1 g, 86%).

Step d) ((2R,3R,4S)-3-(Benzoyloxy)-4-chloro-4-fluoro-5-hvdroxytetrahvdrofuran-2-yl)methyl benzoate (19d)

Ethanolamine (1.55 g, 25.4 mmol) was added to a stirred solution of compound 19c (10.1 g, 23.0 mmol) in EtOAc (100 mL) and DMSO (50 mL). The mixture was stirred at rt for 72 h, then diluted with diethyl ether (300 mL) and EtOAc (300 mL) and washed with water (x4). The combined water phases were extracted with EtOAc then the EtOAc phase was washed with brine (x2). The combined organic phases were dried (Na₂S0₄), filtered and concentrated under reduced pressure. The product was purified by silica gel chromatography eluted with a gradient of DCM with and EtOAc, which gave the title compound (7.50 g, 82%).

Step e) ((2R,3R,4S)-3-(benzoyloxy)-4-chloro-4-fluoro-5-((methylsulfonyl)oxy)tetrahvdrofuran-2- vDmethyl benzoate (19e) Et₃N (3.54 mL, 25.4 mmol) was added at -15 °C under N₂ to a solution of compound 19d (8.36 g, 21 .2 mmol) in dry DCM (100 mL) followed by addition of MsCI (1 .97 mL, 25.4 mmol). The reaction mixture was stirred at -15 °C for 2 h, then poured into HCI (80 mL, 1 M, aq.). The phases were separated and the aqueous layer was extracted with DCM. The combined organic extracts were washed with NH₄CI (sat. aq.) dried (MgS0₄) and concentrated under reduced pressure to give the title compound (9.86 g, 98%) as a clear oil.

Step f) ((2R,3R,4S,5R)-3-(benzoyloxy)-4-chloro-5-(2,4-dioxo-3,4-dihvdropyrimidin-1 (2H)-yl)-4- fluorotetrahvdrofuran-2-yl)methyl benzoate (19f)

Uracil (3.09 g, 27.5 mmol) and ammonium sulfate (48.5 mg, 0.367 mmol) was heated to reflux under N₂ in HMDS (49.3 mL, 236 mmol) for 16 h. The reaction mixture was cooled to rt, concentrated under reduced pressure and dried in vacuo. The residue in dry DCE (50 mL) was added under N₂ to a solution of compound 19e (8.68 g, 18.4 mmol) in dry DCE (75 mL).

TMSOTf (6.12 g, 27.5 mmol) was slowly added under N₂ to the solution. After the addition, the reaction mixture was heated to 80 °C for 5 h and then at 65 °C for 16 h.

The reaction mixture was cooled to rt, quenched with NaHC0₃ (sat. aq.), filtered and extracted twice with DCM. The combined organic extracts were dried (MgS0₄) and concentrated under reduced pressure. EtOAc and DCM was added and the formed precipitate was collected by filtration which gave the pure β -isomer (660 mg, 7.4%). The filtrate was evaporated onto silica and purified by flash chromatography (hex:EtOAc 2:1 - 1 :1 ), which gave the title compound as a mixture with the a-isomer, α:β>5:95 (942 mg, 11 %).

Step e) 1 -((2R,3S,4R,5R)-3-chloro-3-fluoro-4-hvdroxy-5-(hvdroxymethyl)tetrahvdrofuran-2- yl)pyrimidine-2,4(1 H,3l-l)-dione (19a)

Compound 19f (670 mg, 1 .37 mmol) was suspended in NH₃ (7N in MeOH). After 30 min, EtOH (5 mL) was added and the suspension was stirred at rt. After an additional hour, the suspension went into solution and then reaction mixture was stirred at rt for 15 h. The solvents were evaporated under reduced pressure and the afforded residue was purified by flash

chromatography (DCM:MeOH 10:1 ) which gave the title compound (380 mg, 99%) as a white solid. LC-MS ES- 279.31 [M-H]\ xample 28, alternative route to Compound 2

Step a) (4S,5R)-4-Hvdroxy-5-(hvdroxymethyl)dihvdrofuran-2(3H)-one (28a)

Deoxy-D-ribose (400.0 g, 2.98 mol) was dissolved in water (1 .6 kg) under nitrogen and the solution cooled to 3 - 7 °C. Bromine (800 g, 10.0 mol, 3.36 eq.) was added at 3 - 7 °C while stirring over a period of approximately 2 hours and the stirring was continued at 3 - 7 °C for approximately 1 hour. The reaction mixture was gently warmed to 20 - 25^ and then stirred for approximately 20 hours.

The reaction mixture was cooled to -5 to -7°C and a solution of sodium hydroxide (27.65%, 720 g, 1 .67 eq.) was added while keeping the reaction temperature at -3 to -7 °C. The temperature was then adjusted to 0 - 5 °C and aqueous sodium hydroxide (9%, 470 g, 1 .06 mol, 0.35 eq. was added at 0 - 5 °C to obtain a final pH = 1 .40.

The water was distilled off at reduced pressure using a scrubber (cooled, 14% sodium hydroxide, 0.9 L), finally at p < 5 mbar and 50 °C. In order to remove residual water from the product, 2-propanol was added portion wise to the residue followed by azeotropic distillation at reduced pressure. The final water content was determined by KF titration to be less than 1 %. 2-Propanol (400 mL) was added to the residue and the mixture followed by filtration. The filter cake was washed with 2-propanol (1 L). The solvent was distilled off at reduced pressure. Toluene (400 mL) was added and distillation was resumed in order to remove residual 2- propanol and possibly more water. A residue of 474.6g (120% yield) was obtained. Step b) (4S,5R)-4-((Triisopropylsilvnoxy)-5-(((triisopropylsilvnoxy)methvndihvdrofuran-2(3H)-one (28b)

Compound 28a (470.9 g, 2.97 mol) was dissolved in DMF (1 .2 L) and cooled to 10 - 15 °C. Imidazole (707.0 g, 10.4 mol, 3.5 eq.) was added and the temperature of the mixture was adjusted to 3 - 7 °C. TIPS-CI (1 145 g, 5.94 mol, 2.0 eq.) was added with cooling to 3 - 7 °C over a period of 2 hours. The reaction mixture was stirred at 3 - 7 °C for another 1/2 h, then gently warmed to 20 - 25 °C and stirred for 20 h. The progress of the reaction was monitored as follows: A sample of the reaction mixture was diluted 10 times with dry DMF, N;0- bis(trimethylsilyl)trifluoroacetamide (0.25 mL) was added to 0.5 mL of the sample in DMF and analyzed by GC. If the reaction was not complete the necessary amount of TIPS-CI was calculated and added and the stirring continued for another 20 hours.

When the reaction was completed, methanol (50 mL) was added and the mixture was stirred for ½ - 1 hour at 20 - 25 °C. Water (1 .2 kg) was added and the temperature of the mixture was adjusted to 15 - 25 °C. pH was adjusted to pH 2.0 - 2.5 by careful addition of 36% hydrochloric acid (491 g, 4.7 mol). Toluene (0.9 kg) was added and the phases were separated. The organic phase was washed twice with 5% aqueous sodium chloride (1 kg), the aqueous phases were washed with toluene (0.9 kg). The organic phases were combined and dried with sodium sulfate (150 g) for minimum 1 hour. The suspension was filtered on a column prepared from silica Gel 60 (210 g) and toluene and the column was washed with toluene (1 .1 kg). The combined filtrate was concentrated to dryness at reduced pressure at 50 °C which gave the title compound (1338 g, 84.4% from crude 2a). Purity (GC): 93.9%.

Step c) (3S,4R,5R)-3-fluoro-4-((triisopropylsilyl)oxy)-5-(((triisopropylsilyl)oxy)methyl)- dihvdrofuran-2(3H)-one (28c)

Compound 28b (450.0 g, 1 .01 mol,) and NFSI (348.0 g, 1 .10 mol) were dissolved in Me-THF (2.2 L) under argon. The solution was cooled to below -75 °C and lithium bis(trimethylsilyl)amide (20.2% in THF, 1 .190 kg, 1 .42 eq.) was added over a period of 3-4 hours. The progress of the reaction was monitored by GC, and when deemed completed, methylsulfide (6 g, 0.1 mol) was added to quench residual NFSI and the stirring continued for another 20-30 minutes.

The reaction mixture was transferred into aqueous 12.5% ammonium chloride (1 .7 kg) and the mixture was warmed to room temperature. The aqueous layer (Aq. 1 ) was separated and the organic phase was washed with purified water (1 L). The aqueous wash (Aq. 2) was separated and the organic phase was secured. Aq.1 was washed with heptanes (0.6 kg). The aqueous phase was separated and then discarded. Aq. 2 was added to the organic phase and the mixture was stirred for 1 minute. The aqueous phase was separated and discarded. The two organic phases were combined and concentrated at reduced pressure at 50 °C. Heptanes (0.7 kg) was added to the residue and the resulting suspension was filtered. The filter cake was washed with heptanes (0.2 kg), the combined filtrate was concentrated at reduced pressure at 50 °C, which gave 506 g crude product. The crude product was dissolved in a mixture of heptanes and toluene (0.5 L, 3:1 ) and purified by column chromatography on silica gel (silica gel 60, 2.5 kg and heptanes/toluene 3:1 v/v). The column was eluted with heptanes/toluene (3:1 , 5.0 L), heptanes/toluene (2:1 , 2.5 L), heptanes/toluene (3:1 , 2.5 L) and toluene (7.5 L). Fractions of ~1 L were collected and fractions holding pure compound 2c were combined and concentrated and fractions holding mixtures of compound 2c and di-fluoro compound were combined and re-purified.

The above procedure was repeated twice, starting with 450 g and 525 g of compound 2b. Total yield of the title compound was 877.1 g (59.2%) + 104.1 g (7.0%) from reworked material. Purity (GC): 92.4%.

Step d) (3S,4R,5R)-3-Chloro-3-fluoro-4-((triisopropylsilyl)oxy)-5-(((triisopropylsilyl)oxy)methyl)- dihvdrofuran-2(3H)-one (28d)

Compound 28c (400.0 g, 0.86 mol) and NCS (138.0 g, 1 .04 mol, 1 .2 eq.) were stirred in THF (2.0 L) under argon at -20 °C. The suspension was cooled to below -70 °C and then lithium bis(trimethylsilyl)amide (20.2% in THF, 1 .150 kg, 1 .6 eq.) was added over a period of 1 - 1 .5 hours. The reaction was monitored by GC and when deemed completed, the mixture was transferred into a 12.5% aqueous solution of ammonium chloride (1 .5 kg). The mixture was warmed to room temperature. The stirring was stopped and the aqueous layer was separated, washed with heptanes (0.8 L) and then discarded.

The mother organic phase was concentrated to dryness at reduced pressure at 55 °C and then added to the heptane wash. The thus combined organic phases were washed with 5% aqueous sodium chloride. The phases were separated and the aqueous phase washed with heptanes (0.2 L), then discarded. The organic phase was concentrated at reduced pressure which gave 440 g of crude product.

The procedure was repeated starting with 426.5 g of compound 2c which gave 473 g of crude product.

The combined crude products were dissolved in a mixture of heptanes and toluene (1 .0 L, 2:1 ) and purified on a silica gel column prepared from silica gel 60 (2.25 kg) and heptanes/toluene 2:1 v/v. The column was eluted with: heptanes/toluene (2:1 , 15 L). Fractions of ~1 L were collected and pure fractions of compound 2d were combined and concentrated at reduced pressure which gave the title compound (667.3 g, 75.1 %). Step e) (3S,4R,5R)-3-Chloro-3-fluoro-4-hvdroxy-5-(hvdroxymethyl)dihvdrofuran-2(3H)-one (28e) Compound 28d (613.0 g, 1 .1 1 mol) was added to a 3 L glass reactor filled with nitrogen and methanol (1 .2 L) and. To the stirred emulsion was added 37% hydrochloric acid (368.0 g, 3.73 mol, 3.4 eq.) and the mixture was heated to gentle reflux (73 °C). The mixture was kept at reflux for 20 hours then cooled to Ι δ^Ο'Ό and extracted with heptanes (4 x 600 mL). The residual methanolic solution was concentrated to dryness at reduced pressure using a water bath of 80 - 90°C, finally at p < 35 mbar. Dioxane (600 mL) was added and distilled again as above, which gave the title compound (200.7 g, 98%) .

Step f (2R,3R,4S)-4-chloro-4-fluoro-2-(((4-methylbenzoyl)oxy)methyl)-5-oxotetrahvdrofuran-3-yl 4-methylbenzoate (28f)

A solution of compound 28e (200.7 g, 1 .1 1 mol) in dioxane (1 .4 L) in a 3 L glass reactor filled with nitrogen and equipped with mechanical stirring, thermometer and an addition funnel was heated to 40 to 45 Ό on a water bath. p-Toluoyl chloride (360.5 g, 2.33 mol, 2.1 eq.) was added whereafter triethylamine (258.3 g, 2.55 mol, 2.3 eq.) was added during 35 minutes so as to keep the reaction temperature below 70 °C. The resulting suspension was then stirred at 65 °C for 2 hours, then cooled to 15 °C and filtered. The 800 mL filter cake was washed with dioxane (800 mL, 15 °C), leaving a white filter cake which was discarded. The filtrate was concentrated at reduced pressure, finally at 35 mbar using a water bath of 65 °C. 2-Propanol (1 .50 L) was added to the residual oil (510 g) so as to keep the temperature of the solution at 40-45 °C. The solution was seeded and carefully allowed to cool to room temperature. During the cooling process samples of 0.25 mL were taken and mixed with 0.25 mL of water for pH measurements. Triethylamine (15 g) was added until pH 2.5 - 3.5 was obtained. Once room temperature was reached (one hour), the crystal suspension was cooled to 10 ± 1 °C and kept at this temperature for 15 hours. The title product was isolated by filtration, washed with 2-propanol (600 mL, 5 - 10 °C) and then dried at 30 - 50 °C in an air vented oven. Yield: 374.2 g, 80%. Purity (HPLC): 99.4 %. Melting point: 88.0 - 89.5°C (1 Ό/min) crystal form change and then melts at 97 - 98 °C.

Step q) (2R,3R,4S)-4-Chloro-4-fluoro-5-hvdroxy-2-(((4-methylbenzoyl)oxy)methyl)- tetrahvdrofuran-3-yl 4-methylbenzoate (28q)

A 3 L reaction flask set up with mechanical stirrer, thermometer and an addition funnel was filled with nitrogen. The flask was charged with ethyl acetate (1000 g) and cooled to 10 °C. Lithium tri-ferf-butoxyaluminium hydride (30% solution in THF, 35 g, 0.05 eq.) was added. Stirring at 10 °C was continued for 5-10 minutes and then compound 28f (370.0 g, 0.88 mol) was added. Further lithium tri-ferf-butoxyaluminium hydride (30% solution in THF, 933.8 g, 1 .10 mol, 1.25 eq.) was added over a period of 70 minutes while keeping the reaction temperature at 10 °C. The reaction was quenched by pouring the reaction mixture onto a quench mixture (1 .45 kg (10% NaCI - 10% NH₄CI in 3M HCI)) keeping the temperature at 10 - 15 °C. The resulting suspension was warmed to 20 - 25 °C. The aqueous was separated and discarded and the organic phase was washed with acidic water (1 .0 L + 10 mL of 3M HCI) followed by a wash with 25% sodium chloride (250 ml_). The organic phase was concentrated to dryness, finally at p < 35 mbar and 45 °C. The residue was re-dissolved in toluene (0.45 kg) and the solution was again concentrated, at p < 35 mbar and 45 °C, which gave the title compound as an oil containing a little solid sodium chloride (412.6 g, 1 1 1 %). Purity (HPLC) 97.5%.

Step h) (2R,3R,4S)-4,5-dichloro-4-fluoro-2-(((4-methylbenzoyl)oxy)methvntetrahvdrofuran-3-yl 4-methylbenzoate (28h)

A 2000 mL reaction flask set up for mechanical stirring, temperature measurement and condenser was filled with nitrogen and charged with toluene (740 mL), compound 28g (41 1 .5 g, 0.88 mol) and thionyl chloride (174.0 g, 1 .46 mol, 1 .66 equivalents). The reaction flask was placed on a water bath, pre-heated to 50 °C and DMF (0.50 mL) was added. The top of the condenser was connected a cooled scrubber (700 g of 27.65% sodium hydroxide) and a steady flow of nitrogen was applied. The reaction started shortly after the DMF was added and it was followed by HPLC. After approximately three hours, the gas evolution has decreased and the temperature was increased to 60 - 65 °C. Heating at 60 - 65 °C was continued for further 4.5 hours after which time the sulfite esters had vanished. The solvent and residual thionyl chloride was distilled off (500 mL) at reduced pressure using a water bath of 60 - 65 °C. Toluene (650 mL) was added to the residual oil and the mixture was cooled to 5 °C. Water (650 mL) was added and the pH was adjusted to 2.0 - 3.0 by addition of 3M sodium hydroxide (40 mL) at a temperature below 10 °C. The temperature was adjusted to 20 - 22 °C and the aqueous phase was separated. The organic phase was washed with 25% sodium chloride (250 mL). The aqueous phases were back washed with toluene (250 mL). The combined organic phase was dried with magnesium sulfate (25 g) and filtered. Evaporation of the solvent (finally at p < 35 mbar and 60 °C) provided the title compound as a light brown oil (378.5 g, 97% yield).

Chlorobenzene (200 g) was added to the residue and the mixture was concentrated using the above conditions. The residue was again dissolved in chlorobenzene (200.0 g) and the mixture concentrated.

Step i) (2R,3R,4S,5R)-5-(4-Benzamido-2-oxo-3,4-dihvdropyrimidin-1 (2H)-yl)-4-chloro-4-fluoro-2- (((4-methylbenzoyl)oxy)methyl)tetrahvdrofuran-3-yl 4-methylbenzoate (28i)

A 500 mL round bottom flask was charged with A/-benzoylcytosine (36.6 g, 170 mmol, 1 .5 eq.), chlorobenzene (165 g, 150 mL) and ammonium sulfate (0.45 g, 3.4 mmol, 0.03 eq.), to this suspension was added HMDS (29.3 g, 181 .3 mmol, 1 .6 eq.). The suspension was heated to reflux. When the reaction mixture became a clear solution, it was refluxed for additional 1 h and then concentrated by distillation in vacuo at 60 °C (distillate: 150 mL). Chlorobenzene (125 mL) was added to the residue. Residual toluene in Compound 28h (50 g, 1 13.3 mmol) was removed by distillation in vacuo from chlorobenzene. The residue from this co-evaporation was dissolved in 1 ,2-dichloroethane (200 mL), and this solution was charged to the solution of silylated nucleoside in chlorobenzene. Tin(IV)chloride (59.0 g, 226.6 mmol, 2 eq.) was added and the mixture was heated to reflux under nitrogen. The reaction mixture was stirred at reflux for 65 h. The reaction mixture was cooled to 5 °C, and ethyl acetate (99.8 g, 10 eq.) was added while keeping the temperature at 10-12 ^<C. Total weight of mixture: 601 .7 g._A quarter of this mixture (150.4 g, in theory 28.3 mmol) was charged to a 250 mL 3 necked round bottom flask, cooled to 5 °C, and

dichloromethane (147.5 g, 4 x vol. of EtOAc) was added together with Celite (6.25 g). A warm (approx. 60 °C) 50% NaOH solution (17.6 g, 7.76 eq.) was added to the mixture in such a rate that the temperature was kept at 5 - 12°C. The mixture was stirred for 20 min at 10 °C, then the temperature was adjusted to 25 °C and the mixture was stirred at this temperature for 30 min. The suspension was filtered on a pad of Celite (12.5 g) and the filter cake was washed with dichloromethane (190 mL). The combined filtrate and washings were concentrated to dryness by distillation in vacuo at 60 °C. Dichloromethane (86 mL) was added to the residue then toluene (62 mL). The content of dichloromethane was removed by distillation in vacuo at 50 °C. The resulting suspension was stirred at room temperature for 17 h whereafter the crude title compound was isolated by filtration. The filter cake was washed with toluene (25 mL) and the wet product was dried in an air ventilated dryer at 40 °C, which gave title compound as a solid (5.56 g, 31 .7%).

Step i) (2R.3R.4S.5R)-4-Chloro-5-(2.4-dioxo-3.4-dihvdropyrimidin-1 (2H)-yl)-4-fluoro-2-(((4- methylbenzoyl)oxy)methyl)tetrahvdrofuran-3-yl 4-methylbenzoate (28D

Compound 28i (15.2 g 24.5 mmol) was suspended in 65% AcOH/water (152 mL, v/v), and the suspension was heated to reflux for 20 h. The reaction mixture was allowed to cool to room temperature, then water (53 mL) was added and the mixture was stirred at room temperature for 1 .5 h. The suspension was filtrated and the filter cake washed with water (2 x 25 mL). The wet filter cake was dried in an air ventilated dryer at 40 °C for 20 h, which gave the title compound as a solid (10.8 g, 85%).

Step k) 1 -((2R,3S,4R,5R)-3-Chloro-3-fluoro-4-hvdroxy-5-(hvdroxymethyl)tetrahvdrofuran-2- yl)pyrimidine-2,4(1 H,3H)-dione (28k)

Compound 28j (8.0 g, 15.5 mmol) was suspended in MeOH (80 mL), n-propylamine (9.1 g, 154.8 mmol, 10 eq.) was added and the mixture was heated to 30 °C and stirred at this temperature for 24 h. The solvents were removed by distillation in vacuo at 40 °C. The residue was taken up in water (20 mL), the aqueous phase was washed with DCM (3 x 40 mL) and the combined organic phases were washed with water (5 mL). The two aqueous phases were combined, and pH adjusted to 1 .0 with 3 M HCI (approx. 7 mL). The acidic aqueous phase was extracted with Me-THF (4 x 40 mL), and the combined organic phases were concentrated to dryness by distillation in vacuo at 40 °C. Isopropyl acetate (80 mL) was added to the residue, and the turbid mixture was concentrated in vacuo at 60 °C. Isopropyl acetate (40 mL) was added and the distillation in vacuo was continued. Isopropyl acetate (10 mL) was added to the resulting thick suspension. The suspension was cooled to room temperature and stirred for 30 min. Crude title compound was collected by filtration, and the filter cake was washed with isopropyl acetate (2 x 4 mL). The afforded crude was dissolved in Me-THF (35 mL), isopropyl acetate (70 mL) was added and the mixture was concentrated by distillation in vacuo at 60 °C (distillate: 70 mL). Additional isopropyl acetate (30 mL) was added, and the distillation was continued (distillate: 30 mL). The suspension was cooled to room temperature, stirred at for 45 min and then filtered. The filter cake was washed with isopropyl acetate (2 x 4 mL) then dried in vacuo at room temperature. The title compound was isolated in 70% yield (3.0 g). Purity (HPLC) 98.5%.

Step I) (S)-lsopropyl 2-(((S)-(((2R.3R.4S.5R)-4-chloro-5-(2.4-dioxo-3.4-dihvdropyrimidin-1 (2H)- yl)-4-fluoro-3-hvdroxytetrahvdrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (28) THF (0.07% water, 12 mL) was added to Compound 28k (500 mg, 1 .78 mmol) and the solution was cooled to -10 °C under nitrogen. Terf-butylmagnesium chloride, 20%wt in THF (2.20 g, 3.74 mmol, 2.1 eq.) was added by syringe over 20 min at -10 °C. The syringe was rinsed with 500 μΐ THF and the rinse was added to the reaction mixture. The formed suspension was stirred at -10 °C for 40 min. A solution of (S)-isopropyl 2-(((S)-(perfluorophenoxy)(phenoxy)phosphoryl)- amino)propanoate (1 .01 g, 2.23 mmol, 1 .25 eq.) in THF (10 mL) and DMPU (2.0 mL, 16.9 mmol, 9.5 eq.) was added with a syringe at -10 °C over a period of 87 min whereafter the reaction mixture was stirred at -10 °C for 22 h. The reaction was quenched by addition of 1 M HCI (4.6 mL, 2.6 eq.) while keeping the temperature below 5 °C. Toluene (20 mL) was added and the mixture was heated to 25 °C and stirred at this temperature for 5 min. The phases were separated and the aqueous phase was extracted with toluene/THF (1 :1 , 10 mL). The organic phases were washed with 1 M HCI (2 x 10 mL) and 5% Na₂C0₃ (2 x 10 mL). The combined basic aqueous phases were extracted with toluene (1 x 10 mL) and toluene/THF (1 :1 , 2 x10 mL) and the combined organic phases were washed with 25% NaCI (15 mL). All organic phases were then combined and the solvents removed by distillation in vacuo at 60 °C. 2-Propanol (20 mL) and n-heptane (30 mL) was added to the residue and the suspension was cooled to 5 °C overnight. The suspension was filtered and the filtrate was concentrated by distillation in vacuo at 50 °C. The residue was dried on a pump for 3 h which gave the title compound as a foam (874 mg, 89%). Purity (HPLC) of crude 91 .8%. NMR spectra obtained for compound 28 prepared in Example 28 were in agreement with spectral data of compound 2 prepared in Example 2.

Biological Examples

Replicon assay

The compounds of formula (I) may be examined for activity in the inhibition of HCV RNA replication in a cellular assay aimed at identifying compounds that inhibit a HCV functional cellular replicating cell line, also known as HCV replicons. A suitable cellular assay is based on a bicistronic expression construct, as described by Lohmann et al. (1999), Science vol. 285 pp. 1 10-1 13 with modifications described by Krieger et al. (2001 ), Journal of Virology 75: 4614- 4624, in a multi-target screening strategy.

The assay utilizes the stably transfected cell line Huh-7 luc/neo (hereafter referred to as Huh- Luc). This cell line harbors an RNA encoding a bicistronic expression construct comprising the wild type NS3-NS5B regions of HCV type 1 b translated from an Internal Ribosome Entry Site (IRES) from encephalomyocarditis virus (EMCV), preceded by a reporter portion (FfL- luciferase), and a selectable marker portion (neo^R, neomycine phosphotransferase). The construct is bordered by 5' and 3' NTRs (non-translated regions) from HCV type 1 b. Continued culture of the replicon cells in the presence of G418 (neo^R) is dependent on the replication of the HCV RNA. The stably transfected replicon cells that express HCV RNA, which replicates autonomously and to high levels, encoding inter alia luciferase, are used for screening the antiviral compounds.

The replicon cells are plated in 384 well plates in the presence of the test and control compounds which are added in various concentrations. Following an incubation of three days, HCV replication is measured by assaying luciferase activity (using standard luciferase assay substrates and reagents and a Perkin Elmer ViewLux™ ultraHTS microplate imager). Replicon cells in the control cultures have high luciferase expression in the absence of any inhibitor. The inhibitory activity of a compound on luciferase activity is monitored on the Huh-Luc cells, enabling a dose-response curve for each test compound. EC₅₀ values are then calculated, which value represents the amount of the compound required to decrease the level of detected luciferase activity by 50%, or more specifically, the ability of the genetically linked HCV replicon RNA to replicate. Enzyme assay

As may be demonstrated in the replicon assay, the compounds of the invention are metabolised by cellular kinases in target tissues to the 5'-trisphosphate. It is this triphosphate which is believed to be the antivirally active species. The enzyme assay described here may be used to confirm that compound of the invention are antivirally active as the 5'-triphosphate metabolite.

The enzyme assay measures the inhibitory effect of triphosphate compounds in an HCV NS5B- 21 (21 -aminoacid C-terminally truncated version) SPA assay (scintillation proximity assay). The assay is performed by evaluating the amount of radiolabeled ATP incorporated by HCV NS5B- 21 into newly synthesized RNA using an heterogeneous biotinylated RNA template.

To determine IC₅₀ values the compounds are tested at various concentrations in a final volume of 100 μΙ of reaction mixture. The reaction is stopped by addition of 0.5M EDTA solution.

The samples are transferred into flashplates precoated with streptavidin. The incorporated radioactivity is quantified using a scintillation counter (Wallac Microbeta Trilux).

Materials & Supplier

Flashplate coated with streptavidin PerkinElmer Life Sciences

96 well polypropylene plate Corning

Biotinylated RNA template: with a sequence of

5^'-UUU UUU UUU UAG UCA GUC GGC CCG

GUU UUC CGG GCC-3' and biotinylated at the

5^'-primer end made up to 83 μΜ in 10 mM Tris-HCI,

100 mM NaCI, pH= 8.0 Medprobe

Enzyme: HCV NS5B-21 , made up to 500μg/ml in water. Replizyme

Nucleotides: GTP, CTP, UTP Invitrogen

Radiolabeled ³H-ATP (cat. no TRK747) GE Healthcare

0.5 M EDTA, pH=8.0 Life Technologies

Tris-HCI Sigma

MnCI₂ Sigma

Ammonium acetate Sigma

DTT (dithiothreitol) Sigma

CHAPS Sigma

RNase Out (cat. No 10777-019) Invitrogen

DMSO Carlo Erba Reactifs

Equipment

Wallac Microbeta Trilux Perkin Elmer Life Sciences

Method

Assay conditions

Buffer: 20 mM tris-HCI, 100 mM ammonium acetate, 20 mM NaCI, 2.5 mM MnCI₂,

pH 7.5 10 mM DTT, 2 mM CHAPS, RNase Out

GTP 50 μΜ

CTP 2 μΜ

UTP 2 μΜ

ATP 2 μΜ

³H-ATP (47 Ci/mmol) 0.5μΜ

Template: RNA-H3 83 ηΜ

Enzyme: NS5B-21 (500 μg/m\) 2 μg/ml

Assay volume 1 00 μΙ

The assay should include enzyme controls (about four, containing 1 μΙ DMSO instead of inhibitor) and background control containing all ingredients except template. Compounds are serially diluted in DMSO on a separate dilution plate to 1 0Ox the final desired assay concentrations.

Sufficient reaction mixture for the number of wells to be used is made up according to the table below and 90 μΙ/well is added to a 96 well polyproylene plate. 1 μΙ of compound in DMSO from the dilution plate is added to each well, except the enzyme control wells and background control wells to which 1 μΙ DMSO is added.

Reaction mixture

Template buffer: 10 mM tris-HCI, 100 mM NaCI pH=8.0 28.25

Prepare an ATP cocktail containing 1 .5 μΙ/well of ³H-ATP(45Ci/mmol), 2.0μΙ/ννβΙΙ of 100 μΜ ATP and 6.5μΙ/ννβΙΙ of H₂0 and start the reaction by adding 10 μΙ/well of this cocktail.

Incubate at 22 ^<C for 120 min.

Stop the reaction with the addition of 10ΟμΙ/well of 0.5M EDTA, pH=8.0.

Transfer 185 μΙ/well to the streptavidin flash plate.

Incubate the plate over night and read the flash plate in the Microbeta Trilux using the protocol Flash plates H3. Treatment of results

Calculation for inhibition:

„_τ CompoundCPM - Backer oundCPM

% Inhibition ^&

AverageEnzymeControlCPM - BackgroundCPM Background = Reaction buffer without template.

IC₅₀ is determined using Graphpad Prism. Plot Compound concentration in Log versus percentage inhibition. Fit the curve with nonlinear regression to the Log (Inhibitor) versus Response equation.

Top - Bottom

Y = Bottom +

1 + 10 ;X-log(lC₅₀)) Where Y is % Inhibition, X is log (inhibitor) and top and bottom are the upper and lower limits of the % Inhibition.

Biological Example 1

The inhibition of HCV replication exhibited by the compounds of the invention were tested in the above described replicon assay. The compounds showed sub micromolar activity, with a cell toxicity in the Huh-Luc cell line being in excess of 50 μΜ. The EC₅₀ values are presented in Table 1 .

Table 1

Biological Example 2

The nucleotide of Examples 3 were tested in the above described enzyme assay and the IC₅₀ values determined to be 0.72 μΜ. Biological Example 3

Replicon assay

The compositions of the invention may be examined for activity in the inhibition of HCV RNA replication in a cellular assay aimed at identifying compounds that inhibit a HCV functional cellular replicating cell line, also known as HCV replicons. A suitable cellular assay is based on a bicistronic expression construct, as described by Lohmann et al. (1999), Science vol. 285 pp. 1 10-1 13 with modifications described by Krieger et al. (2001 ), Journal of Virology 75: 4614- 4624, in a multi-target screening strategy.

The assay utilizes the stably transfected cell line Huh-7 luc/neo (hereafter referred to as Huh- Luc). This cell line harbors an RNA encoding a bicistronic expression construct comprising the wild type NS3-NS5B regions of HCV type 1 b translated from an Internal Ribosome Entry Site (IRES) from encephalomyocarditis virus (EMCV), preceded by a reporter portion (FfL- luciferase), and a selectable marker portion (neo^R, neomycine phosphotransferase). The construct is bordered by 5' and 3' NTRs (non-translated regions) from HCV type 1 b. Continued culture of the replicon cells in the presence of G418 (neo^R) is dependent on the replication of the HCV RNA. The stably transfected replicon cells that express HCV RNA, which replicates autonomously and to high levels, encoding inter alia luciferase, are used for screening the antiviral compounds. The replicon cells are plated in 384 well plates in the presence of the test and control compounds which are added in various concentrations. Following an incubation of three days, HCV replication is measured by assaying luciferase activity (using standard luciferase assay substrates and reagents and a Perkin Elmer ViewLux™ ultraHTS microplate imager). Replicon cells in the control cultures have high luciferase expression in the absence of any inhibitor. The inhibitory activity of a compound on luciferase activity is monitored on the Huh-Luc cells, enabling a dose-response curve for each test compound. EC₅₀ values are then calculated, which value represents the amount of the compound required to decrease the level of detected luciferase activity by 50%, or more specifically, the ability of the genetically linked HCV replicon RNA to replicate.

Compound 1A shows an EC₅₀ value of 0.055 uM (n >10), with a cell toxicity in the Huh-Luc cell line being in excess of 50 μΜ. The above described replicon assay can further be used to assess synergistic, additive or antagonistic effects of HCV antiviral on each other using the CI isobol method, which method provides a quantitative assessment of synergism between drugs. A CI is estimated from dose- effect data of single and combined drug treatments. A value of CI less than 1 indicates synergism; CI = 1 indicates additive effect; and CI > 1 indicates antagonism. Drug interaction (synergism or antagonism) is more pronounced the farther a CI value is from 1 . Formally, the combination index (CI) of a combined drug treatment is defined as:

CI = D₁/Dx_{i +} D₂/Dx₂

Here D₁ and D₂ are the doses of drug 7 and drug 2, respectively, in the combination; Dx₁ and

Dx₂ each is the dose of a treatment with only drug 7 and drug 2 that would give the same effect as that of the combination, respectively. The doses Dx_l and Dx₂ need to be estimated from the dose-effect data of single drug treatments. In practice the two drugs under assessment are presented in serial dilution series on the different axes of a well plate - see the attached Figure 1 . Commercially available computer software such as MacSynergy is used to calculate and Bonferroni-adjust the synergy volumes

As can be seen from the table above Compound 1 A shows antiviral synergy with a variety of direct acting and host acting antivirals of diverse mechanistic classes-Comparative Example 1 Sofosbuvir is marketed in several countries for the treatment of HCV, predominantly against genotypes 1 and 4. The structure of sofosbuvir is:

As can been seen, sofosbuvir differs from the compound of present Example 2, in that it possesses a beta-methyl group at the 2'-position, whereas the compounds of the invention have a beta-chloro substituent at this position. In the Fission phase III clinical trials reported in Lawitz et al., N. Eng. J. Med., 2013; 368:1878-87, "Response rates in the sofosbuvir -ribavirin group were lower among patients with genotype 3 infection than amongst those with genotype 2 infection (56% vs. 97%)".

The antiviral activity of commercially available sofosbuvir and the compound of Example 2 were compared in a genotype 3a transient replicon assay described in Kylefjord et al., J Virol.

Methods 2014 195:156-63.

The EC₅₀ of sofosbuvir against genotype 3a is 0.230 μΜ +/- 0.067, n = 1 1 , compared to an EC₅₀ of 0.072 μΜ +/-0.024, n= 9 for the compound of Example 2. A threefold better potency for the compound of the invention relative to sofosbuvir is expected to markedly improve viral response rates in the clinic.

The several-fold improvement in potency of the compounds of the invention relative to sofosbuvir was maintained in transient replicons of genotype 3a bearing the troublesome S282T mutation (conferring resistance to the HCV nucleoside mericitabine) where sofosbuvir had an EC₅₀ of 0.48 μΜ (n=1 ) and the compound of Example 2 had an EC₅₀ of 0.13 μΜ (n=1 ). Similarly, the L159F/L320F double mutant generated by exposure to the nucleoside mericitabine and conferring cross resistance to sofosbuvir (Tong et al 2013 J. Infect. Dis., 209 (5), 668-75) was prepared in a genotype 3a transient replicon as described above in Kylefjord et al. ibid. In the this double mutant, sofosbuvir had an EC₅₀ of 0.190 (n=1 ) whereas the compound of Example 2 shows an EC₅₀ of 0.062 (n=1 ).

The compound of Example 2 was further evaluated to assess the antiviral activity against genotypes 1 -6 of HCV, both wild type and a number of clinically relevant mutant strains. The result of the evaluation together with the average EC₅₀ of the genotypes and the corresponding values for sofosbuvir are summarised in Tables 2 and 3. Table 2

as the arithmetic means +/- SEM.

"Chimeric replicons containing stated GT NS5B genes in coni background.

References: Coni (Lohmann et al 2003); H77 (Blight et al 2003); GT2a (Wakita et al 2005); GT3a (Kylefjord et al 2013); GT4-6 (Wong et al 2012); L159F/L320F (Tong et al 2013).

Table 3

as the arithmetic means +/- SEM.

"Chimeric replicons containing stated GT NS5B genes in coni background. References: Con1 (Lohmann et al 2003); H77 (Blight et al 2003); GT2a (Wakita et al 2005); GT3a (Kylefjord et al 2013); GT4-6 (Wong et al 2012); L159F/L320F (Tong et al 2013).

From these two tables it is evident that the compound of present Example 2 has a significant improved potency as compared to sofosbuvir against HCV GT3a both in the wild type strain and in two clinically relevant mutant strains, while keeping the good potency against the other genotypes.

All documents referred to herein, including patents and patent applications, are incorporated by reference in their entirety.

Claims

Use of a compound represented by formula (1 A),

or a pharmaceutically acceptable salt thereof, in the therapy of HCV in a mammal or human, wherein the compound of formula (1 A) is administered in combination with a further HCV antiviral selected from:

a) asunaprevir, daclatasvir and/or beclabuvir; or

b) simeprevir and/or JNJ56914845; or

c) an HCV protease inhibitor selected from paritaprevir or ABT493, and an NS5A inhibitor selected from ombitasvir or ABT-530; or

d) alisporavir; or

e) EDP-239; or

f) odalasvir and/or sovaprevir; or

g) grazoprevir and/or elbasvir. 2. Use according to claim 1 , wherein the HCV is genotype 1 a or 1 b. 3. A compound represented by formula (1 A), or a pharmaceutically acceptable salt thereof for use in the treatment or prophylaxis of hepatitis C genotype 2a, 3a, 4a, 5a or 6a virus infection

The use according to claim 3 wherein the hepatitis C virus infection is of genotype 3a. A diastereomeric compound represented by formula (1A),

or a pharmaceutically acceptable salt thereof.

A pharmaceutical composition comprising a compound of formula (I) in association with a pharmaceutically acceptable adjuvant, diluent or carrier for use in the treatment or prophylaxis of hepatitis C genotype 2a, 3a, 4a, 5a or 6a virus infection.

The composition according to claim 21 wherein the hepatitis C virus infection is of genotype 3a.