Advantageous combination anti-HCV therapy

Abstract

A synergistic pharmaceutical combination of compound 1 or a pharma- ceutically acceptable salt thereof (e.g., compound 1-A) and compound 2 or a pharma- ceutically acceptable salt thereof, and use thereof for treating hepatitis C or a condition associated with hepatitis C, and methods for preparing the pharmaceutical combinations: 1, 1-A, 2: TIFF2024525164000085.tif48170TIFF2024525164000086.tif60170TIFF2024525164000087.tif49170

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 212,047, filed June 17, 2021, the entirety of which is incorporated by reference herein for all purposes.

[0002] The present invention is a pharmaceutical combination of a specific NS5B polymerase inhibitor and a specific NS5A inhibitor for advantageous and synergistic HCV therapy. [Background technology]

[0003] Hepatitis C virus (HCV) is an RNA single-stranded virus and a member of the Hepacivirus genus. It is estimated that more than half of all cases of liver disease are caused by HCV. HCV infection can lead to cirrhosis and liver cancer, and if untreated, can lead to liver failure, which may require a liver transplant.

[0004] RNA polymerase is a major target in the development of drugs against RNA single-stranded viruses. The nonstructural protein NS5B RNA-dependent RNA polymerase of HCV is the main enzyme responsible for initiating and catalyzing viral RNA synthesis. There are two main subclasses of NS5B inhibitors: nucleoside analogs and non-nucleoside inhibitors (NNIs). Nucleoside analogs are anabolized to active triphosphates that act as alternative substrates for the polymerase. Non-nucleoside inhibitors (NNIs) bind to an allosteric region on the protein. Nucleoside or nucleotide inhibitors mimic natural polymerase substrates and act as chain terminators. They inhibit the initiation of RNA transcription and / or the elongation of the nascent RNA chain.

[0005] In addition to targeting RNA polymerase, other RNA viral proteins can also be targeted therapeutically. Examples of HCV proteins that are additional targets for therapeutic approaches include NS3 / 4A (a serine protease) and NS5A (a nonstructural protein that is an essential component of the HCV replicase and has multiple effects on cellular pathways).

[0006] The first nucleoside NS5B polymerase inhibitor, sofosbuvir (Sovaldi™, Gilead Sciences), was approved in December 2013. Sovaldi™ is a uridine phosphoramidate prodrug that is taken up by hepatocytes and undergoes intracellular activation to provide the active metabolite, 2'-deoxy-2'-α-fluoro-β-C-methyluridine-5'-triphosphate. [ka]

[0007] Sovaldi™ is the first drug with demonstrated safety and efficacy to treat certain types of HCV infection without the need for co-administration with interferon. Sovaldi™ is the third drug to receive Breakthrough Therapy Designation from the FDA.

[0008] A number of fixed-dose combinations have been approved for the treatment of HCV. In 2014, the US FDA approved Harvoni™ (ledipasvir, an NS5A inhibitor, and sovosbuvir) for the treatment of chronic Hepatitis C virus genotype 1 infection. Harvoni™ is the first combination pill approved for the treatment of chronic HCV genotype 1 infection. It is also the first approved regimen that does not require administration with interferon or ribavirin. In addition, the FDA approved simeprevir (Olysio™) in combination with sovosbuvir (Sovaldi™) as an all-oral, interferon- and ribavirin-free treatment once daily for adults with genotype 1 HCV infection.

[0009] Also in 2014, the US FDA approved AbbVie's Vikirapak™, a combination pack containing dasabuvir (a non-nucleoside NS5B polymerase inhibitor), ombitasvir (an NS5A inhibitor), paritaprevir (an NS3 / 4A inhibitor), and ritonavir. Vikirapak™ can be used with or without ribavirin to treat patients with genotype 1 HCV infection, including those with compensated cirrhosis. Vikirapak™ does not require co-therapy with interferon.

[0010] In July 2015, the US FDA approved Technivi™ and Daklinza™ for the treatment of HCV genotype 4 and HCV genotype 3, respectively. Technivi™ (ombitasvir / paritaprevir / ritonavir) is approved for use with ribavirin for the treatment of HCV genotype 4 in patients without scarring or cirrhosis and is the first-line treatment for HCV-4 infected patients without the need for co-administration with interferon. Daklinza™ is approved for use with Sovaldi™ to treat HCV genotype 3 infection. Daklinza™ is the first drug with demonstrated safety and efficacy in the treatment of HCV genotype 3 without the need for co-administration of interferon or ribavirin. In October 2015, the U.S. FDA warned that the HCV treatments Vikirapak and Technivi can cause serious liver damage, primarily in patients with underlying advanced liver disease, and requested that additional safety information be added to the labels.

[0011] In August 2017, MAVYRET™ (glecaprevir / pibrentasvir) was approved by the US FDA for the treatment of patients with all major genotypes of HCV (genotypes 1-6). The treatment was also approved for patients with no cirrhosis or mild cirrhosis, patients undergoing dialysis, and patients with genotype 1 infection who have been previously treated with a regimen containing an NS5A or NS3 / 4A inhibitor. MAVYRET™ is administered in an 8-week course to previously untreated non-cirrhotic patients. In 2019, the FDA approved an 8-week course in previously untreated patients with compensated cirrhosis.

[0012] In addition to Mavyret™, Epclusa™ is another therapeutic agent for the treatment of HCV. Epclusa is a fixed-dose combination therapy developed by Gilead that includes sovosbuvir (NS5B inhibitor) and velpatasvir (NS5A inhibitor). Epclusa™ was approved in 2016 for the treatment of adults with chronic HCV infection of all major HCV genotypes (genotypes 1-6) and is prescribed in 12-week courses to patients without cirrhosis or with compensated cirrhosis. For patients with decompensated cirrhosis, Epclusa™ is approved in combination with ribavirin.

[0013] Other approved treatments for HCV include interferon alfa-2b or pegylated interferon alfa-2b (PegIntron™), which may be administered with ribavirin (Rebetol™), NS3 / 4A telaprevir (Incivec™, Vertex and Johnson & Johnson), boceprevir (Victorelis™, Merck), simeprevir (Olysio™, Johnson & Johnson), paritaprevir (AbbVie), ombitasvir (AbbVie), and NNI dasabuvir (ABT-333).

[0014] U.S. patents and WO applications describing nucleoside polymerase inhibitors for the treatment of Flaviviridae, including HCV, include those filed by Idenix Pharmaceuticals (U.S. Pat. Nos. 6,812,219, 6,914,054, 7,105,493, 7,138,376, 7,148,206, 7,157,441, 7,163,929, 7,169,766, 7,192,936, 7,365,057, 7,384,924, 7,385,102, and 7,386,106, respectively). No. 7,456,155, No. 7,547,704, No. 7,582,618, No. 7,608,597, No. 7,608,600, No. 7,625,875, No. 7,635,6 No. 89, No. 7,662,798, No. 7,824,851, No. 7,902,202, No. 7,932,240, No. 7,951,789, No. 8,193,372, No. 8, Nos. 299,038, 8,343,937, 8,362,068, 8,507,460, 8,637,475, 8,674,085, 8,680,071, 8,691,788, 8,742,101, 8,951,985, 9,109,001, and 9,243,025; U.S. Patent Application Publication No. 2016 / 00022 81, U.S. Patent Application Publication No. 2013 / 0064794, WO 2015 / 095305, WO 2015 / 081133, WO 2015 / 061683, WO 2013 / 177219, WO 2013 / 039920, WO 2014 / 137930, WO 2014 / 052638, WO 2012 / 154321);Merck's applications (U.S. Patent Nos. 6,777,395, 7,105,499, 7,125,855, 7,202,224, 7,323,449, 7,339,054, 7,534,767, 7,632,821, 7,879,815, 7,0 Nos. 71,568, 8,148,349, 8,470,834, 8,481,712, 8,541,434, 8,697,694, 8,715,638, 9,061,041, 9,156,872, and WO 2013 / 009737; Emory University applications (U.S. Patent Nos. 6,348,587, 6,911,424, 7,307,065, 7,495,006, 7,662,938, 7,772,208, 8,114,994, 8,168,583, 8,609,627, U.S. Patent Application Publication No. 2014 / 0212382, and WO 2014 / 1244430);Applications filed by Gilead Sciences / Pharmasset Inc. (U.S. Patent Nos. 7,842,672, 7,973,013, 8,008,264, 8,012,941, 8,012,942, 8,318,682, 8,324,179, 8,415,308, 8,455,451, 8,563,530, 8,841,275, 8,853,171, 8,871,785 ...563,530, 8,841,275, 8,853,171, 8,871,785, 8,563,530, 8,563,530, 8,563,530, 8,563,530, 8,563,530, 8,563,530, 8, Nos. 8,877,733, 8,889,159, 8,906,880, 8,912,321, 8,957,045, 8,957,046, 9,045,520, 9,085,573, 9,090,642 and 9,139,604) and (U.S. Pat. Nos. 6,908,924, 6,949,522, 7,094,770, 7,211,570 , RE No. 7,429,572, RE No. 7,601,820, RE No. 7,638,502, RE No. 7,718,790, RE No. 7,772,208, RE No. 42,015, RE No. 7,919,247, RE No. No. 7,964,580, No. 8,093,380, No. 8,114,997, No. 8,173,621, No. 8,334,270, No. 8,415,322, No. 8,481,713, No. 8, No. 492,539, No. 8,551,973, No. 8,580,765, No. 8,618,076, No. 8,629,263, No. 8,633,309, No. 8,642,756, No. 8,71 6,262, 8,716,263, 8,735,345, 8,735,372, 8,735,569, 8,759,510 and 8,765,710);Hoffman those filed by La-Roche (U.S. Patent No. 6,660,721); those filed by Roche (U.S. Patent Nos. 6,784,166, 7,608,599, 7,608,601 and 8,071,567);Applications filed by Alios BioPharma Inc. (U.S. Patent Nos. 8,895,723, 8,877,731, 8,871,737, 8,846,896, 8,772,474, 8,980,865, 9,012,427, U.S. Patent Application Publication No. 2015 / 0105341, U.S. Patent Application Publication No. 2015 / 0011497, U.S. Patent Application Publication No. 2010 / 0249068 ... WO 2012 / 0070411, WO 2015 / 054465, WO 2014 / 209979, WO 2014 / 100505, WO 2014 / 100498, WO 2013 / 142159, WO 2013 / 142157, WO 2013 / 096680, WO 2013 / 088155, WO 2010 / 108135); Enanta those filed by Biotech Pharmaceuticals (U.S. Patent Nos. 8,575,119, 8,846,638, 9,085,599, WO 2013 / 044030, WO 2012 / 125900); those filed by Biota (U.S. Patent Nos. 7,268,119, 7,285,658, 7,713,941, 8,119,607, 8,415,309, 8,501,699, and 8,802,840); Applications filed by Alla Chem, LLC (U.S. Patent No. 8,889,701 and WO 2015 / 053662); applications filed by Inhibitex (U.S. Patent No. 8,759,318 and WO 2012 / 092484); applications filed by Janssen Pharmaceuticals (U.S. Patent No. 7,388,002, U.S. Patent No. 7,429,571, U.S. Patent No. 7,514,410, U.S. Patent No. 7,560,434, U.S. Patent No. 7,994,139, U.S. Patent No. 8,133,870, U.S. Patent No. 8,163,703, U.S. Patent No. 8,242,085 and U.S. Patent No. 8,440,813); applications filed by Alla Chem, LLC (U.S. Patent No. 8,889,701 and WO 2015 / 053662); applications filed by Inhibitex (U.S. Patent No. 8,759,318 and WO 2012 / 092484); Products (U.S. Patent Nos. 8,399,429, 8,431,588, 8,481,510, 8,552,021, 8,933,052, 9,006,29 and 9,012,428);Applications filed by the University of Georgia Foundation (U.S. Patent Nos. 6,348,587, 7,307,065, 7,662,938, 8,168,583, 8,673,926, 8,816,074, 8,921,384 and 8,946,244); RFS Pharma, LLC (U.S. Patent Nos. 8,895,531, 8,859,595, 8,815,829, 8,609,627, 7,560,550, U.S. Patent Application Publication No. 2014 / 0066395, U.S. Patent Application Publication No. 2014 / 0235566, U.S. Patent Application Publication No. 2010 / 0279969, WO 2010 / 091386 and WO 2012 / 158811); University College Cardiff Consultants Limited (WO 2014 / 076490, WO 2010 / 081082, WO 2008 / 062206); Achillion Pharmaceuticals, Inc. (WO 2014 / 169278 and WO 2014 / 169280); Cocrystal Pharma, Inc. (U.S. Pat. No. 9,173,893); Katholieke Universiteit Leuven (WO 2015 / 158913); Catabasis (WO 2013 / 090420); and the Regents of the University of Minnesota (WO 2006 / 004637).

[0015] Elbasvir is an HCV NS5A inhibitor approved by the FDA in 2016 as a component of a fixed-dose combination with grazoprevir, an NS3 / 4A protease inhibitor. This combination (Zepatia™) is approved for the treatment of treatment-naïve and previously treated patients, with a typical course of treatment of 12 weeks. However, in patients with HCV genotype 1a, harboring mutations at positions 28, 30, 31, and / or 93, Zepatia™ is only effective when administered with ribavirin for 16 weeks. Ribavirin carries several FDA boxed warnings, including the risk of hemolytic anemia and teratogenicity. [ka]

[0016] Ruzasvir (MK-8408) is an oral pan-genotype NS5A inhibitor that is being clinically evaluated for the treatment of chronic HCV infection. Ruzasvir has sub-picomolar to low-picomolar affinity for HCV NS5A across a broad range of HCV genotypes and common clinical mutants, including HCV genotype 1 with Y93H, Q30R, L31V, and Y93C mutations (Tong et al. "Discovery of Ruzasvir (MK-8408): A Potent, Pan-Genotype HCV NS5A Inhibitor with Optimized Activity against Common Resistance-Associated Polymorphisms" J. Med. Chem. 2017, 60, 290-306). International Publication No. WO 2014 / 110705 and U.S. Patent No. 9,555,038, assigned to Merck and Company, disclose ruzasvir, pharmaceutical compositions containing ruzasvir, and methods of use thereof. [ka]

[0017] In 2015, Merck, Sharp & Dohme sponsored a clinical trial to determine the efficacy of grazoprevir and uprifosbuvir in combination with elbasvir or ruzasvir for patients with various HCV genotypes. This triple combination regimen achieved sustained virologic responses after 12 weeks in 85%-100% of patients infected with a range of HCV genotypes (see, e.g., NCT02332707; NCT02332720; Lawitz, E. "Safety and efficacy of a fixed-dose combination regimen of grazoprevir, ruzasvir and uprifosbuvir with or without ribavirin in participants with and without cirrhosis with chronic hepatitis C virus genotype 1, 2, or 3 infection (C-CREST-1 and C-CREST-2, part B): two randomized, phase 2, open-label trials" 2017, Lancet Gastroenterol Hepatol, doi:10.1016 / S2468-1253(17)30163-2). Grazoprevir, one component of the triple combination regimen, is an NS3 / 4A protease inhibitor, a class of therapeutic agents that can cause drug-drug interactions and elevated hepatic transaminase levels.

[0018] In 2016, Merck initiated a Phase II open-label clinical trial of ruzasvir with the NS5B inhibitor uprifosbuvir in a two-drug combination regimen that did not include an NS3 / 4A inhibitor. At the study doses of 60 mg ruzasvir and 450 mg uprifosbuvir per day, the combination was well tolerated, but suboptimal efficacy was observed and the trial was discontinued (C-BREEZE 1; NCT02759315). The same combination, but at higher doses (180 mg ruzasvir, 450 mg uprifosbuvir), achieved sustained virologic responses in over 90% of participants infected with HCV GT1, GT2, GT4, GT5, and GT6. However, only 73.8% of participants infected with GT3 responded. Additionally, more than 30% of study participants experienced drug-related adverse events (C-BREEZE 2; NCT02956629; Lawitz, E. "Efficacy and Safety of Two-Drug Direct-Acting Antiviral Agent Regimen Ruzasvir 180mg and Uprifosbuvir 450mg for 12 Weeks in Adults with Chronic Hepatitis C Virus Genotype 1, 2, 3, 4, 5, or 6" 2019, J. Viral Hepat. 26, 9, 1127-1138). A second, higher-dose trial was also discontinued.

[0019] Atea Pharmaceuticals, Inc. has developed the structure β-D-2'-deoxy-2'-α-fluoro-2'-β-C-substituted-2-modified-N 6-Advantageous nucleotide phosphoramidates of (mono- and di-methyl)purine nucleosides have been discovered (U.S. Pat. Nos. 9,828,410, 10,000,523, 10,005,811, 10,239,911, 10,519,186, 10,815,266, 10,870,672, 10,870,673, 10,875,885, 10,894,804, and 10,906,928, as well as U.S. Patent Application Publication No. 2021-0015841 and U.S. Patent Application Publication No. 2020-0179415). In particular, isopropyl ((S)-(((2R,3R,4R,5R)-5-(2-amino-6-(methylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate hemisulfate (AT-527, bemnifosbuvir) has surprising advantages such as high bioavailability, target organ selectivity, and high potency against all genotypes of HCV, including GT3 (WO 2018 / 144640). [ka]

[0020] In the multiple-dose arm of the Phase I trial, bemnifosbuvir reduced viral load by 4.5 log in non-cirrhotic participants. 10 IU / mL, and 4.6 log in participants with cirrhosis. 10In patients with cirrhosis, AT-527 was equally effective against various genotypes, including the difficult-to-treat GT3 (GT1b: 4.0, 4.0, 4.5; GT2: 5.0; GT3: 4.8, 5.2) (NCT03219957; Berliba, E. et al. "Safety, Pharmacokinetics, and Antiviral Activity of AT-527, a Novel Purine Nucleotide Prodrug, in Hepatitis C Virus-Infected Subjects with or without Cirrhosis" 2019, Antimicrob Agents Chemother, 63 (12): e01201-19).

[0021] In a study of bemnifosbuvir in combination with the first-generation HCV NS5A inhibitor daclatasvir, all 10 subjects achieved HCV RNA below the lower limit of quantification. 90% of subjects experienced a sustained undetectable viral load after 12 weeks of treatment (SVR12). The one subject who did not experience SVR12 had a range of resistance-associated mutations (NS5A:R30Q, NS5B:L159F / A218S / C316N) (NCT04019717; Mungur, O. et al. "A combination of AT-527, a potent pan-genotypic guanosine nucleotide prodrug and daclatasvir was well-tolerated and effective in HCV-infected subjects", 2020, poster THU438 at The International Liver Congress meeting). However, the study was discontinued.

[0022] There is a strong medical need to develop safe, effective, and well-tolerated anti-HCV therapies. Potential drug resistance exacerbates this need. HCV RNA polymerase exhibits a high rate of replication that contributes to the generation of potentially resistant single and double point mutations throughout the genome and to the maintenance of viral polymorphism. Resistance mutations have been identified both in vitro and in vivo with nearly all monotherapy treatments.

[0023] It is therefore an object of the present invention to provide compounds, pharmaceutical compositions, methods and dosage forms for treating and / or preventing HCV infection. Summary of the Invention

[0024] The present invention provides a highly synergistic combination of compound 1, or a pharma- ceutically acceptable salt thereof, which is a potent pan-genotypic NS5B polymerase inhibitor (e.g., AT-527, bemnifosbuvir), and compound 2, or a pharma- ceutically acceptable salt thereof, which is an NS5A inhibitor (ruzasvir, MK-8408), for the treatment of Hepatitis C infection in a host, typically a human.

[0025] Compound 1 is isopropyl((S)-(((2R,3R,4R,5R)-5-(2-amino-6-(methylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate: [ka] It is.

[0026] Compound 1 was previously described in U.S. Patent Nos. 9,828,410, 10,000,523, 10,005,811, and 10,239,911, 10,815,266, 10,80,672, 10,870,673, and 10,875,885, all assigned to Atea Pharmaceuticals, as well as WO 2016 / 21276 and WO 2019 / 200005.

[0027] The hemisulfate salt of compound 1 is shown below as compound 1-A: [ka] It is shown as:

[0028] Compound 1-A is disclosed in U.S. Pat. Nos. 10,519,186, 10,906,928, and 10,894,804, assigned to Atea Pharmaceuticals, as well as WO 2018 / 144640 and WO 2019 / 200005.

[0029] Compound 2 is ruzasvir (dimethyl N,N'-([(6S)-6-(2-cyclopropyl-1,3-thiazol-5-yl)-1-fluoro-6H-indolo[1,2-c][1,3]benzoxazine-3,10-diyl]bis{1H-imidazole-5,2-diyl-(2S)-pyrrolidine-2,1-diyl[(2S)-3-methyl-1-oxobutane-1,2-diyl]}) dicarbamate): [ka] It is.

[0030] In one embodiment, compound 2 is an amorphous solid. In yet another embodiment, compound 2 is a crystalline solid. In one embodiment, compound 2 is administered as a pharma- ceutically acceptable salt thereof. Compound 2 is disclosed in WO 2014 / 110705 and U.S. Pat. No. 9,555,038.

[0031] Previous clinical trials of ruzasvir in combination with the NS5B inhibitor uplifosbuvir were discontinued due to lack of efficacy. Against this background, the combination of Compound 1 (e.g., AT-527) and ruzasvir was found to be highly synergistic when tested at multiple doses (see Example 30 and Figure 34). At each combined dose, the expected additive antiviral protection was subtracted from the experimentally observed antiviral activity, resulting in positive values ​​for synergy, zero values ​​for additivity, or negative values ​​for antagonism. -50 μM 2 %~50μM 2 A synergistic effect of 50 μM is considered additive. 2 %~100μM 2 % showed slight synergy, 100 μM 2 % is considered highly synergistic. The combination of Compound 1 and Compound 2 was evaluated at various doses, demonstrating high synergy. A plot of synergy versus dose for each compound is shown in Figure 34. At 40 nM Compound 1 and 0.008 nM Compound 2, the amount of synergy was 255 μM 2 %, which is more than 5-fold the cutoff for synergy and falls into the highly synergistic category.

[0032] This synergistic combination of two anti-HCV agents that work together by different mechanisms can be provided systemically, for example orally, as two or more separate dosage forms or combined dosage forms.When administered separately, the agents must be provided so that the host receives the benefit of both active agents that work together biologically, for example to achieve overlapping pharmacokinetics, plasma and / or AUC exposure.Combination drug therapy is particularly advantageous in limiting the emergence of drug resistance in addition to effectively treating the virus.

[0033] In one non-limiting embodiment, compound 1 is provided as a hemisulfate salt.

[0034] In one embodiment, the fixed dose combination is intended to achieve a sustained viral response in about 12 weeks or less, such as about 10 weeks or less, 8 weeks or less, or 6 weeks or less. In addition to effectively treating the virus, combination drug therapy is useful in limiting the development of drug resistance.

[0035] The weights of active compounds in the dosage forms described herein relate to either the free or salt form of the compound, unless otherwise indicated. For example, 600 mg of Compound 1-A is equivalent to 550 mg of Compound 1.

[0036] In typical embodiments, Compound 1 is administered in a dosage of about 300 mg to 1000 mg (with or without considering the weight of the salt), more typically 400 mg or 500 mg to 600 mg or 800 mg, or 500 mg to 750 mg. In certain embodiments, Compound 1 is administered in a dosage of about 500 mg to about 1500 mg. In one example, 550 mg of Compound 1 is administered in a dosage of about 600 mg of Compound 1-A. In an alternative embodiment, 1100 mg of Compound 1 is administered in a dosage of about 1200 mg of Compound 1-A.

[0037] In typical embodiments, Compound 2 or a pharma- ceutically acceptable salt thereof is administered in a dosage form at a dose of about 20 mg to 500 mg, more typically 40 mg to 250 mg, including, but not limited to, at least 60 mg, 70 mg, 75 mg, 100 mg, 125 mg, 150 mg, 180 mg, 200 mg, 225 mg, 250 mg, 270 mg, 300 mg, 350 mg, or 400 mg. In certain embodiments, Compound 2 is administered in a dosage form containing at least about 90 mg, 180 mg, 270 mg, or 360 mg.

[0038] In certain embodiments, the combination comprises 550 mg of Compound 1-A and at least about 90 mg, 180 mg, 270 mg, or 360 mg of Compound 2 or a pharma- ceutically acceptable salt thereof (e.g., 180 mg). In certain embodiments, the combination is provided once, twice, or three times daily.

[0039] The combination therapy may be administered once, twice, or three or more times per day as recommended by a health care professional. In certain embodiments, the combination therapy is provided once per day. In other embodiments, the combination therapy is provided twice per day. In yet other embodiments, the combination therapy is provided three times per day.

[0040] In certain embodiments, compound 1-A is provided in a solid dosage form of 600 mg per day and compound 2 is provided in a combined or separate dosage form of 180 mg / day, which may be given together once, twice, or three times per day.

[0041] In various embodiments, compound 1 or a pharma- ceutically acceptable salt thereof and compound 2 or a pharma- ceutically acceptable salt thereof are formulated together in a single dosage form or are provided in several dosage forms (e.g., two or more dosage forms, each with both active agents or one dosage form with one active agent and the other dosage form with the other active agent). In alternative embodiments, compound 1 or a pharma- ceutically acceptable salt thereof and compound 2 or a pharma- ceutically acceptable salt thereof are provided in separate dosage forms, but such that they can act in a coordinated, e.g., synergistic, manner in the host. For example, the separate dosage forms can be administered such that there is an overlapping AUC or other pharmacokinetic parameter that indicates that the active agents are working together against the virus.

[0042] In one embodiment of the invention, Compound 1 and Compound 2 are provided in separate pills and are administered at approximately the same time during the day, ie, concurrently.

[0043] The combination of Compound 1 (or a pharma- ceutically acceptable salt thereof, e.g., Compound 1-A) and Compound 2 (or a pharma-ceutically acceptable salt thereof) may also be used to treat associated conditions such as anti-HCV antibody-positive and antigen-positive states, chronic viral liver inflammation, liver cancer (hepatocellular carcinoma (HCC)) resulting from progressive hepatitis C, liver cirrhosis, chronic or acute hepatitis C, fulminant hepatitis C, chronic persistent hepatitis C, and anti-HCV fatigue.

[0044] In certain embodiments, compound 1 or a pharma- ceutically acceptable salt thereof, such as compound 1-A and compound 2 or a pharma- ceutically acceptable salt thereof, are administered for up to 24 weeks, up to 12 weeks, up to 10 weeks, up to 8 weeks, up to 6 weeks, or up to 4 weeks. In alternative embodiments, compound 1 or a pharma- ceutically acceptable salt thereof, such as compound 1-A and compound 2 or a pharma- ceutically acceptable salt thereof, are administered for at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks, at least 12 weeks, or at least 24 weeks. In certain embodiments, compound 1 or a pharma- ceutically acceptable salt thereof and compound 2 or a pharma- ceutically acceptable salt thereof are administered at least once a day or every other day. In alternative embodiments, compound 1 or a pharma- ceutically acceptable salt thereof and compound 2 or a pharma- ceutically acceptable salt thereof are administered twice a day. In an alternative embodiment, Compound 1, or a pharma- ceutically acceptable salt thereof, and Compound 2, or a pharma- ceutically acceptable salt thereof, are administered three or more times per day.

[0045] In certain embodiments, the patient is non-cirrhotic. In certain embodiments, the patient is cirrhotic. In further embodiments, the cirrhotic host has compensated cirrhosis. In alternative embodiments, the cirrhotic host has decompensated cirrhosis. In one embodiment, the host has Child-Pugh A cirrhosis. In alternative embodiments, the host has Child-Pugh B cirrhosis or Child-Pugh C cirrhosis.

[0046] The above combinations can also be used to treat a range of HCV genotypes. At least six different genotypes of HCV, each with multiple subtypes, have been identified worldwide. Genotypes 1-3 are widespread worldwide, while genotypes 4, 5, and 6 are more geographically restricted. Genotype 4 is prevalent in the Middle East and Africa. Genotype 5 is mostly found in South Africa. Genotype 6 is found primarily in Southeast Asia. The most common genotype in the United States is genotype 1, but defining the genotype and subtype can help determine the type and duration of treatment. For example, different genotypes respond differently to different drug therapies. Optimal treatment times vary depending on the genotype infection. Within the genotype range, subtypes such as genotype 1a and genotype 1b may respond differently to treatment as well. Infection with one genotype does not preclude later infection with a different genotype.

[0047] In one embodiment, a combination of Compound 1 or a pharma- ceutically acceptable salt thereof, e.g., Compound 1-A and Compound 2 or a pharma- ceutically acceptable salt thereof, is used to treat HCV genotype 1, HCV genotype 2, HCV genotype 3, HCV genotype 4, HCV genotype 5, or HCV genotype 6. In one embodiment, Compound 1 or a pharma- ceutically acceptable salt thereof and Compound 2 or a pharma- ceutically acceptable salt thereof are used to treat HCV genotype 1a. In one embodiment, Compound 1 or a pharma- ceutically acceptable salt thereof and Compound 2 or a pharma- ceutically acceptable salt thereof are used to treat HCV genotype 1b. In one embodiment, Compound 1 or a pharma- ceutically acceptable salt thereof and Compound 2 or a pharma- ceutically acceptable salt thereof are used to treat HCV genotype 2a. In one embodiment, Compound 1 or a pharma- ceutically acceptable salt thereof and Compound 2 or a pharma- ceutically acceptable salt thereof are used to treat HCV genotype 2b. In one embodiment, compound 1 or a pharma- ceutically acceptable salt thereof and compound 2 or a pharma- ceutically acceptable salt thereof are used to treat HCV genotype 3a. In one embodiment, compound 1 or a pharma- ceutically acceptable salt thereof and compound 2 or a pharma- ceutically acceptable salt thereof are used to treat HCV genotype 3b. In one embodiment, compound 1 or a pharma- ceutically acceptable salt thereof and compound 2 or a pharma- ceutically acceptable salt thereof are used to treat HCV genotype 4a. In one embodiment, compound 1 or a pharma- ceutically acceptable salt thereof and compound 2 or a pharma- ceutically acceptable salt thereof are used to treat HCV genotype 4d. In one embodiment, compound 1 or a pharma- ceutically acceptable salt thereof and compound 2 or a pharma- ceutically acceptable salt thereof are used to treat HCV genotype 5a. In one embodiment, compound 1 or a pharma- ceutically acceptable salt thereof and compound 2 or a pharma- ceutically acceptable salt thereof are used to treat HCV genotype 6a. In one embodiment, compound 1, or a pharma- ceutically acceptable salt thereof, and compound 2, or a pharma- ceutically acceptable salt thereof, are used to treat HCV genotypes 6b, 6c, 6d, 6e, 6f, 6g, 6h, 6i, 6j, 6k, 6l, 6m, 6n, 6o, 6p, 6q, 6r, 6s, 6t, or 6u.

[0048] The present invention also includes certain combinations and dosage forms in which compound 1-A can be in an amorphous or crystalline salt form and, independently, compound 2 can be crystalline or amorphous.

[0049] Thus, the present invention includes at least the following embodiments:

[0050] (a) an anti-HCV effective combination of Compound 1, or a pharma- ceutically acceptable salt thereof, and Compound 2, or a pharma- ceutically acceptable salt thereof;

[0051] (b) an effective solid dosage form of a combination of Compound 1, or a pharma- ceutically acceptable salt thereof, and Compound 2, or a pharma- ceutically acceptable salt thereof;

[0052] (c) embodiment (a) or (b), wherein compound 1 is compound 1-A;

[0053] (d) any of embodiments (a)-(c), wherein a third anti-HCV active agent is used in combination;

[0054] (e) embodiment (d), wherein the third anti-HCV active agent acts by a mechanism different from Compound 1, Compound 1-A, or Compound 2;

[0055] (f) A pharmaceutical composition comprising a combination of any one of embodiments (a)-(e) in a pharma- ceutically acceptable excipient.

[0056] (g) the embodiment (f), wherein the combination is in the form of a combined pharmaceutical composition;

[0057] (h) the embodiment (f), wherein the combination is present in separate pharmaceutical dosage forms for each active agent used in a coordinated manner;

[0058] (i) a pharmaceutical dosage form of (f) to (h) suitable for oral delivery;

[0059] (j) a dosage form of (i) present in the form of a pill, tablet, or gel;

[0060] (k) a pharmaceutical dosage form of (f) to (h) suitable for parenteral delivery;

[0061] (l) a pharmaceutical dosage form of (f) to (h) suitable for intravenous delivery;

[0062] (m) Use of an effective combination of any one of the embodiments (a) to (l) in the manufacture of a medicament for the treatment of Hepatitis C virus infection in a patient in need of such treatment.

[0063] (n) a method for the manufacture of a medicament intended for therapeutic use to treat a Hepatitis C virus infection in a patient in need thereof, characterized in that said manufacture uses an effective combination according to any one of the embodiments (a) to (l),

[0064] (o) a method for treating a Hepatitis C virus infection comprising administering to a patient in need of such treatment an effective combination of any one of the embodiments (a)-(l);

[0065] (p) a method for curing a Hepatitis C virus infection comprising administering to a patient in need thereof an effective combination of any one of the embodiments (a) to (l);

[0066] (q) a method for prophylactically treating a patient at risk for Hepatitis C virus infection, comprising administering to the patient in need of prophylactic treatment an effective combination of any one of embodiments (a)-(l);

[0067] (r) a method for treating a condition associated with Hepatitis C virus infection selected from chronic viral liver inflammation, liver cancer (hepatocellular carcinoma (HCC)) resulting from progressive Hepatitis C, liver cirrhosis, chronic or acute Hepatitis C, fulminant Hepatitis C, chronic persistent Hepatitis C, and anti-HCV fatigue, comprising administering to a patient in need of such treatment an effective combination of any one of the embodiments (a)-(l);

[0068] (s) Any of embodiments (m) to (r), wherein the patient has cirrhosis.

[0069] (t) Any of embodiments (m) to (r), wherein the patient is non-cirrhotic.

[0070] (u) Any of embodiments (m)-(t), wherein the HCV infection is genotype 1.

[0071] (v) Any of embodiments (m)-(t), wherein the HCV infection is genotype 2.

[0072] (w) Any of embodiments (m)-(t), wherein the HCV infection is genotype 3.

[0073] (x) Any of embodiments (m)-(t), wherein the HCV infection is genotype 4.

[0074] (y) Any of embodiments (m)-(t), wherein the HCV infection is genotype 5.

[0075] (z) Any of embodiments (m)-(t), wherein the HCV infection is genotype 6.

[0076] (aa) A combination of (a) to (l) for use in treating HCV or a condition associated with HCV.

[0077] In the present invention, compound 1-A can be provided as a crystalline form in synergistic combination therapy, or alternatively, the crystalline form can be used in spray-drying manufacturing procedures. Figures 1-15 show the characterization of the physical form of compound 1-A by XRPD, DSC, and TGA. Compound 1-A can be isolated in a crystalline form, which can facilitate synthesis and processing.

[0078] The pharmacokinetics of compound 1-A is important for the success of combination therapy. Figure 16 shows the favorable biodistribution profile of compound 1-A. The compound is concentrated in the liver, which is the target organ of HCV infection, rather than in the heart. The high liver concentration is favorable for hepatic combination therapy.

[0079] Similarly, Figures 17, 18, and 21-23 show the pharmacokinetic properties of compound 1-A and the major metabolites. Compound 1-A is metabolized within 8 hours, but active metabolites are present for up to 24 hours. Suppression of HCV viral RNA is observed during the 24-hour period in which the metabolites are present. At a dose of 300 mg, the plasma concentrations of metabolites of compound 1-A are greater than the EC50 of HCV GT1b. 95 These pharmacokinetics and pharmacodynamics are conducive to successful combination therapy.

[0080] Figures 19 and 20 show the pan-genotypic efficacy of compound 1-A. The pan-genotypic activity of this combination therapy may prevent the emergence of resistant HCV mutants.

[0081] Figures 25-31 show metabolites of Compound 1 and HCV viral RNA levels in cirrhotic hosts administered Compound 1. Figure 27 highlights that Compound 1 is as effective in cirrhotic patients as in non-cirrhotic patients in the host. A combination of Compound 1 or a pharma- ceutically acceptable salt thereof and Compound 2 or a pharma- ceutically acceptable salt thereof can be used to treat HCV in both cirrhotic and non-cirrhotic patients. A pharmaceutical composition comprising Compound 1 or a pharma- ceutically acceptable salt thereof and Compound 2 or a pharma- ceutically acceptable salt thereof can be formulated as an oral dosage form. Figure 32 shows an exemplary process by which tablets for oral administration can be prepared. Compound 1 or a pharma- ceutically acceptable salt thereof and Compound 2 or a pharma- ceutically acceptable salt thereof are combined, sieved, mixed and tableted to provide the combination as an oral dosage form. [Brief description of the drawings]

[0082] [Figure 1A] FIG. 1 shows an overlay of XRPD diffractograms of Sample 1-1 (amorphous Compound 1), Sample 1-2 (crystalline Compound 1), and Sample 1-3 (amorphous Compound 1-A) prior to stability studies for characterization purposes, as described in Examples 2 and 5. The x-axis is 2-theta measured in degrees and the y-axis is intensity measured in counts. [Figure 1B]HPLC chromatograph of amorphous Compound 1 (Sample 1-1) for determining purity as described in Example 2. The purity of the sample was 98.7%. The x-axis is time measured in minutes and the y-axis is intensity measured in counts. [Figure 2A] FIG. 1 is an HPLC chromatograph of crystalline Compound 1 (Sample 1-2) for determining purity as described in Example 2. The purity of the sample was 99.11%. The x-axis is time measured in minutes and the y-axis is intensity measured in counts. [Figure 2B] FIG. 1 is a DSC and TGA graph of crystalline Compound 1 (Sample 1-2) prior to any stability study for characterization purposes as described in Example 2. The x-axis is temperature measured in ° C., the left y-axis is heat flow measured in (W / g), and the right y-axis is weight measured in percent. [Diagram 3] 1 is an X-ray crystal image of Compound 1 showing the absolute stereochemistry, as described in Example 2. [Figure 4A] FIG. 1 shows an overlay of XRPD diffractograms of Sample 1-1 (amorphous Compound 1), Sample 1-2 (crystalline Compound 1), and Sample 1-3 (amorphous Compound 1-A) after 14 days of storage at 25° C. and 60% relative humidity, as described in Examples 2 and 5. The x-axis is 2-theta measured in degrees and the y-axis is intensity measured in counts. [Figure 4B] FIG. 1 shows an overlay of XRPD diffractograms of Samples 1-4, 1-5, 1-6, 1-7, and 1-9 after 7 days of storage at 25° C. and 60% relative humidity, as described in Example 4. The x-axis is 2-theta measured in degrees and the y-axis is intensity measured in counts. [Figure 5A] FIG. 1 shows an overlay of XRPD diffractograms of Samples 1-4, 1-6, 1-7, and 1-9 after 14 days of storage at 25° C. and 60% relative humidity, as described in Example 4. The x-axis is 2-theta measured in degrees and the y-axis is intensity measured in counts. [Figure 5B]FIG. 1 is an XRPD pattern of amorphous Compound 1-A (Sample 1-3), as described in Example 5. The x-axis is 2-theta measured in degrees and the y-axis is intensity measured in counts. [Figure 6A] HPLC chromatograph of amorphous Compound 1-A (Sample 1-3) for purity determination as described in Example 5. The purity of the sample was 99.6%. The x-axis is time measured in minutes and the y-axis is intensity measured in counts. [Figure 6B] 1 is a DSC and TGA graph for amorphous Compound 1-A (Sample 1-3) prior to any stability study for characterization purposes, as described in Example 5. The x-axis is temperature measured in ° C., the left y-axis is heat flow measured in (W / g), and the right y-axis is weight measured in percent. [Figure 7A] 1 shows an overlay of XRPD diffractograms of crystalline (samples 2-2, 2-6, and 2-7) and poorly crystalline (samples 2-3, 2-4, 2-5, and 2-8) samples identified from the crystallization of compound 1-A (Example 6). The x-axis is 2-theta measured in degrees and the y-axis is intensity measured in counts. [Figure 7B] 1 shows an overlay of XRPD diffractograms of amorphous samples (samples 2-9, 2-10, and 2-11) identified from the crystallization of compound 1-A (Example 6), where the x-axis is 2-theta measured in degrees and the y-axis is intensity measured in counts. [Figure 8A] 1 shows an overlay of XRPD diffractograms of samples (Samples 2-2, 2-3, 2-4, 2-5, 2-6, 2-7, and 2-8) after 6 days of storage at 25° C. and 60% relative humidity (Example 6). The x-axis is 2-theta measured in degrees and the y-axis is intensity measured in counts. [Figure 8B] 1 is a DSC and TGA graph for sample 2-2 (Example 6). The x-axis is temperature measured in ° C., the left y-axis is heat flow measured in (W / g), and the right y-axis is weight measured in percent. The experimental procedures for the DSC and TGA collection are given in Example 2. [Figure 9A] 1 is a DSC and TGA graph for sample 2-3 (Example 6). The x-axis is temperature measured in ° C., the left y-axis is heat flow measured in (W / g), and the right y-axis is weight measured in percent. The experimental procedures for the DSC and TGA collection are given in Example 2. [Figure 9B] 1 is a DSC and TGA graph for Samples 2-4 (Example 6). The x-axis is temperature measured in ° C., the left y-axis is heat flow measured in (W / g), and the right y-axis is weight measured in percent. The experimental procedures for the DSC and TGA collection are given in Example 2. [Figure 10A] 1 is a DSC and TGA graph for Samples 2-5 (Example 6). The x-axis is temperature measured in ° C., the left y-axis is heat flow measured in (W / g), and the right y-axis is weight measured in percent. The experimental procedures for the DSC and TGA collection are given in Example 2. [Figure 10B] 1 is a DSC and TGA graph for Samples 2-6 (Example 6). The x-axis is temperature measured in ° C., the left y-axis is heat flow measured in (W / g), and the right y-axis is weight measured in percent. The experimental procedures for the DSC and TGA collection are given in Example 2. [Figure 11A] 1 is a DSC and TGA graph for Samples 2-7 (Example 6). The x-axis is temperature measured in ° C., the left y-axis is heat flow measured in (W / g), and the right y-axis is weight measured in percent. The experimental procedures for the DSC and TGA collection are given in Example 2. [Figure 11B] 1 is a DSC and TGA graph for Samples 2-8 (Example 6). The x-axis is temperature measured in ° C., the left y-axis is heat flow measured in (W / g), and the right y-axis is weight measured in percent. The experimental procedures for the DSC and TGA collection are given in Example 2. [Figure 12A]1 is an XRPD pattern of amorphous Compound 1-B (Sample 3-12) as discussed in Example 7. The x-axis is 2-theta measured in degrees and the y-axis is intensity measured in counts. No crystallization of the malonate salt was observed regardless of the solvent used. [Figure 12B] 1 shows an overlay of XRPD diffractograms of amorphous samples (samples 3-6, 3-10, 3-11, and 3-12) identified from unsuccessful crystallizations of Compound 1 with the malonate salt (Example 7). The x-axis is 2-theta measured in degrees and the y-axis is intensity measured in counts. [Figure 13A] HPLC chromatograms of samples 3-12 from unsuccessful crystallization of Compound 1 with the malonate salt as described in Example 7. The samples were 99.2% pure. The x-axis is time measured in minutes and the y-axis is intensity measured in mAu. [Figure 13B] FIG. 1 shows an overlay of XRPD diffractograms of solid samples obtained from crystallization with LAG (samples 4-13, 4-12, 4-9, 4-3, and 4-1) compared to compound 1 (sample 1-2) as described in Example 8. The XRDPs all fit the pattern of crystalline acid counterions with no additional peaks. The x-axis is 2-theta measured in degrees and the y-axis is intensity measured in counts. [Figure 14A] FIG. 1 shows an overlay of XRPD diffractograms of samples (Samples 6-13, 6-12, 6-11, 6-10, 6-8, 6-7, 6-6, 6-5, 6-4, and 6-2) obtained from utilizing ethyl acetate as the crystallization solvent as described in Example 10 compared to crystalline Compound 1 (Sample 1-2). The XRPD patterns were generally found to match that of Compound 1, except for Samples 6-2, 6-4, and 6-5, which showed minor differences. The x-axis is 2-theta measured in degrees and the y-axis is intensity measured in counts. [Figure 14B]Figure 1 shows an overlay of XRPD diffractograms of sample 5-1 after a second dissolution in MEK and the addition of the anti-solvents cyclohexane and pamoic acid, as described in Example 9. Sample 5-1, crystallized with pamoic acid, was a solid after maturation, but the XRPD pattern matched that of pamoic acid. [Figure 15A] FIG. 1 shows an overlay of XRPD diffractograms of samples obtained from utilizing ethyl acetate as the crystallization solvent (Samples 6-5, 6-4, and 6-2) compared to crystalline Compound 1 (Sample 1-2), as described in Example 10. The XRPD patterns were generally found to match that of Compound 1, except for Samples 6-2, 6-4, and 6-5, which showed minor differences. The x-axis is 2-theta measured in degrees and the y-axis is intensity measured in counts, and refers to the acid used in crystallization. [Figure 15B] FIG. 1 is an XRPD pattern for compound 1-A, as described in Example 14. The x-axis is 2-theta measured in degrees and the y-axis is intensity measured in counts. [Figure 16A] 1 is a graph of active TP (metabolites 1-6) concentration levels in the liver and heart of rats, dogs, and monkeys (Example 18). The x-axis is the dose measured in mg / kg for each species, and the y-axis is the active TP concentration measured in ng / g. [Figure 16B] 1 is a graph of active TP (metabolites 1-6) concentration levels in the liver and heart of dogs (n=2) measured 4 hours after a single oral dose of Compound 1 or Compound 1-A (Example 19). The x-axis is the dose of each compound measured in mg / kg, and the y-axis is the active TP concentration measured in ng / g. [Figure 17] 2 shows the plasma profiles of Compound 1 and metabolites 1-7 in rats given a single 500 mg / kg oral dose of Compound 1-A measured up to 72 hours after dosing (Example 20). The x-axis is time measured in hours and the y-axis is plasma concentration measured in ng / mL. [Figure 18]FIG. 2 shows the plasma profiles of Compound 1 and metabolites 1-7 in monkeys given a single oral dose of 30 mg, 100 mg, or 300 mg of Compound 1-A measured up to 72 hours after dosing (Example 20). The x-axis is time measured in hours and the y-axis is plasma concentration measured in ng / mL. [Figure 19] 1 is a graph of EC95 values ​​measured in nM for sovosbuvir and compound 1 against HCV clinical isolates. The EC95 value for compound 1 is 7-33 fold lower than sovosbuvir (Example 22). On the x-axis are genotypes and on the y-axis are EC95 measured in nM. [Figure 20] FIG. 2 is a graph of EC50 values ​​measured in nM for sovosbuvir and Compound 1 against laboratory strains of HCV genotypes 1a, 1b, 2a, 3a, 4a, and 5a. Compound 1 is approximately 6-11 times more potent than sovosbuvir for genotypes 1-5 (Example 22). On the x-axis are the genotypes and on the y-axis are the EC50 measured in nM. [Figure 21] FIG. 2 is a graph of the mean plasma concentration-time profile of Compound 1 following administration of a single oral dose of Compound 1-A in all cohorts of Part B of the study as described in Example 24. Compound 1 was rapidly absorbed and rapidly metabolized within approximately 8 hours in all cohorts of Part B. The x-axis is time measured in hours and the y-axis is geometric mean plasma concentration measured in ng / mL. [Figure 22] FIG. 2 is a graph of the mean plasma concentration-time profile of metabolites 1-7 following administration of a single oral dose of Compound 1-A in all cohorts of Part B of the study as described in Example 24. Metabolites 1-7 showed sustained plasma concentrations in all cohorts of Part B. The x-axis is time measured in hours and the y-axis is geometric mean plasma concentration measured in ng / mL. [Figure 23A]FIG. 2 shows individual pharmacokinetic / pharmacodynamic analyses of subjects enrolled in the 1b cohort as described in Example 24. The graphs show plasma metabolite 1-7 exposure and HCV RNA reduction levels. The dashed line represents the minimum concentration of metabolite 1-7 required to sustain a viral response greater than the EC95 value for GT1b. The x-axis is time measured in hours. The left y-axis is metabolite 1-7 plasma concentration measured in ng / mL and the right y-axis is HCV RNA reduction measured in log10 IU / mL. [Figure 23B] FIG. 2 shows individual pharmacokinetic / pharmacodynamic analyses of subjects enrolled in the 1b cohort as described in Example 24. The graphs show plasma metabolite 1-7 exposure and HCV RNA reduction levels. The dashed line represents the minimum concentration of metabolite 1-7 required to sustain a viral response greater than the EC95 value for GT1b. The x-axis is time measured in hours. The left y-axis is metabolite 1-7 plasma concentration measured in ng / mL and the right y-axis is HCV RNA reduction measured in log10 IU / mL. [Figure 23C] FIG. 2 shows individual pharmacokinetic / pharmacodynamic analyses of subjects enrolled in the 1b cohort as described in Example 24. The graphs show plasma metabolite 1-7 exposure and HCV RNA reduction levels. The dashed line represents the minimum concentration of metabolite 1-7 required to sustain a viral response greater than the EC95 value for GT1b. The x-axis is time measured in hours. The left y-axis is metabolite 1-7 plasma concentration measured in ng / mL and the right y-axis is HCV RNA reduction measured in log10 IU / mL. [Figure 23D] FIG. 2 shows individual pharmacokinetic / pharmacodynamic analyses of subjects enrolled in the 3b cohort, as described in Example 24. Each graph shows plasma metabolite 1-7 exposure and HCV RNA reduction levels. The dashed line represents the minimum concentration of metabolite 1-7 required to sustain a viral response greater than the EC95 value for GT1b. The x-axis is time measured in hours. The left y-axis is metabolite 1-7 plasma concentration measured in ng / mL, and the right y-axis is HCV RNA reduction measured in log10 IU / mL. [Figure 23E] FIG. 2 shows individual pharmacokinetic / pharmacodynamic analyses of subjects enrolled in the 3b cohort, as described in Example 24. Each graph shows plasma metabolite 1-7 exposure and HCV RNA reduction levels. The dashed line represents the minimum concentration of metabolite 1-7 required to sustain a viral response greater than the EC95 value for GT1b. The x-axis is time measured in hours. The left y-axis is metabolite 1-7 plasma concentration measured in ng / mL, and the right y-axis is HCV RNA reduction measured in log10 IU / mL. [Figure 23F] FIG. 2 shows individual pharmacokinetic / pharmacodynamic analyses of subjects enrolled in the 3b cohort, as described in Example 24. Each graph shows plasma metabolite 1-7 exposure and HCV RNA reduction levels. The dashed line represents the minimum concentration of metabolite 1-7 required to sustain a viral response greater than the EC95 value for GT1b. The x-axis is time measured in hours. The left y-axis is metabolite 1-7 plasma concentration measured in ng / mL, and the right y-axis is HCV RNA reduction measured in log10 IU / mL. [Figure 24]Graph of EC95 values ​​of Compound 1 and Sovosbuvir against clinical isolates of GT1, GT2, GT3, and GT4 HCV infected patients. The dashed horizontal line (-----) represents the steady state trough concentration (C24,ss) of Sovosbuvir nucleoside after a dose of 400 mg QD of Sovosbuvir. The solid horizontal line (-) represents the steady state trough concentration (C24,ss) of Metabolites 1-7 after 600 mg of Compound 1-A (equivalent to 550 mg of Compound 1). The dotted horizontal line (---------) represents the steady state trough concentration (C24,ss) of Metabolites 1-7 after 450 mg of Compound 1-A (equivalent to 400 mg of Compound 1). As discussed in Example 25, the predicted steady-state trough plasma levels (C24,ss) of metabolites 1-7 after 600 mg and 450 mg of compound 1-A exceed the in vitro EC95 of compound 1 against all clinical isolates tested. The steady-state trough plasma levels (C24,ss) of sovosbuvir exceed the EC95 only for the GT2 clinical isolate. The x-axis lists the clinical isolates and the table below the x-axis lists the EC95 values ​​for compound 1 and sovosbuvir. The y-axis is the EC95 against the clinical isolates measured in ng / mL. The EC95 is expressed as nucleoside equivalents. Sovosbuvir and compound 1-A were administered daily (QD). [Diagram 25] 1 is a graph showing the mean HCV RNA change from baseline in subjects with non-cirrhotic GT1b HCV infection after a single dose of Compound 1-A equivalent to 92 mg, 275 mg, 368 mg, or 550 mg of Compound 1, as described in Example 26 and Example 27. The x-axis is time measured after dosing, and the y-axis is the mean HCV RNA change from baseline measured in log10 IU / mL. [Figure 26] 1 is a graph showing the mean HCV RNA change from baseline in subjects with non-cirrhotic GT1b HCV infection after 7 days of QD dosing with Compound 1-A, as described in Example 26 and Example 27. The x-axis is the number of days measured after the first dose, and the y-axis is the mean HCV RNA change from baseline measured in log10 IU / mL. [Figure 27]FIG. 2 is a graph comparing the mean HCV RNA change from baseline after 600 mg / day of Compound 1-A (equivalent to 550 mg of Compound 1) QD administration in subjects with non-cirrhotic GT1 HCV infection, subjects with non-cirrhotic GT3 HCV infection, and subjects with cirrhotic GT1 or GT3 HCV infection, as described in Example 26 and Example 27. As shown in the graph, subjects with cirrhosis showed similar mean HCV RNA change to non-cirrhotic subjects. The x-axis is the number of days measured after the first dose, and the y-axis is the mean HCV RNA change from baseline measured in log10 IU / mL. [Figure 28A] FIG. 2 is a graph of individual HCV RNA change from baseline in subjects with non-cirrhotic GT1b HCV infection after QD administration of 600 mg / day of Compound 1-A (corresponding to 550 mg of Compound 1), as described in Example 26 and Example 27. The dashed horizontal line (-----) is the limit of quantification (LOQ=15 IU / mL), where 50% of subjects achieved HCV RNA below the LOQ. The x-axis is the number of days measured after the first dose, and the y-axis is the HCV RNA change from baseline measured in log10 IU / mL. [Figure 28B] FIG. 2 is a graph of individual HCV RNA change from baseline in subjects with non-cirrhotic GT3 HCV infection after QD administration of 600 mg / day of Compound 1-A (corresponding to 550 mg of Compound 1), as described in Example 26 and Example 27. The dashed horizontal line (-----) is the limit of quantification (LOQ=15 IU / mL). The x-axis is the number of days measured after the first dose, and the y-axis is the HCV RNA change from baseline measured in log10 IU / mL. [Figure 28C]FIG. 2 is a graph of individual HCV RNA change from baseline in subjects with cirrhotic GT1 or GT3 HCV infection after QD administration of 600 mg / day of Compound 1-A (corresponding to 550 mg of Compound 1), as described in Example 26 and Example 27. The dashed horizontal line (-----) is the limit of quantification (LOQ=15 IU / mL). The x-axis is the number of days measured after the first dose, and the y-axis is the HCV RNA change from baseline measured in log10 IU / mL. [Figure 29] 1 shows the mean plasma concentration-time profile of metabolites 1-7 in GT1 / GT3 HCV-infected cirrhotic and non-cirrhotic subjects. As described in Example 26 and Example 27, GT1-infected non-cirrhotic subjects were given either 138 mg / day, 275 mg / day, or 550 mg / day of Compound 1 equivalent QD of Compound 1-A, GT3-infected non-cirrhotic subjects were given 600 mg / day of Compound 1-A (550 mg / day of Compound 1) QD, and GT1 / GT3-infected cirrhotic subjects were given 600 mg of Compound 1-A (550 mg / day of Compound 1) QD. The x-axis is time measured in hours, and the y-axis is mean plasma concentration measured in ng / mL. [Figure 30A] FIG. 2 is a graph plotting the mean metabolite 1-7 plasma concentration (left y-axis) and mean HCV RNA reduction (right y-axis) after 600 mg / day QD of Compound 1-A (corresponding to 550 mg of Compound 1) versus time for subjects with non-cirrhotic GT1b HCV infection as described in Example 26 and Example 27. The EC95 of Compound 1 in GT1b is shown as a dashed horizontal line (-----). The dots represent the steady-state plasma trough levels (Cτ) of metabolites 1-7, and as shown in the figure, (Cτ) consistently exceeds the EC95 at all time points studied. The left y-axis is the mean metabolite 1-7 plasma concentration measured in ng / mL, the right y-axis is the HCV RNA reduction after 550 mg QD of Compound 1 measured in log10 IU / mL, and the x-axis is time measured in hours. [Figure 30B]FIG. 2 is a graph plotting the mean metabolite 1-7 plasma concentration (left y-axis) and mean HCV RNA reduction (right y-axis) after 600 mg / day QD of Compound 1-A (corresponding to 550 mg of Compound 1) versus time for subjects with non-cirrhotic GT3 HCV infection as described in Example 26 and Example 27. The EC95 of Compound 1 in GT3 is shown as a dashed horizontal line (-----). The dots represent the steady-state plasma trough levels (Cτ) of metabolites 1-7, and as shown in the figure, (Cτ) consistently exceeds the EC95 at all time points studied. The left y-axis is the mean metabolite 1-7 plasma concentration measured in ng / mL, the right y-axis is the HCV RNA reduction after 550 mg QD of Compound 1 measured in log10 IU / mL, and the x-axis is time measured in hours. [Figure 30C] FIG. 2 is a graph plotting the mean metabolite 1-7 plasma concentration (left y-axis) and mean HCV RNA reduction (right y-axis) after 600 mg / day QD of Compound 1-A (corresponding to 550 mg of Compound 1) versus time for subjects with cirrhotic GT1b HCV infection as described in Example 26 and Example 27. The EC95 of Compound 1 in GT1b is shown as a dashed horizontal line (-----). The dots represent the steady-state plasma trough levels (Cτ) of metabolites 1-7, and as shown in the figure, (Cτ) consistently exceeds the EC95 at all time points studied. The left y-axis is the mean metabolite 1-7 plasma concentration measured in ng / mL, the right y-axis is the HCV RNA reduction after 550 mg QD of Compound 1 measured in log10 IU / mL, and the x-axis is time measured in hours. [Figure 30D]FIG. 2 is a graph plotting the mean metabolite 1-7 plasma concentration (left y-axis) and mean HCV RNA reduction (right y-axis) after 600 mg / day QD of Compound 1-A (corresponding to 550 mg of Compound 1) versus time for subjects with cirrhotic GT3 HCV infection as described in Example 26 and Example 27. The EC95 of Compound 1 in GT1b is shown as a dashed horizontal line (-----). The dots represent the steady-state plasma trough levels (Cτ) of metabolites 1-7, and as shown in the figure, (Cτ) consistently exceeds the EC95 at all time points studied. The left y-axis is the mean metabolite 1-7 plasma concentration measured in ng / mL, the right y-axis is the HCV RNA reduction after 550 mg QD of Compound 1 measured in log10 IU / mL, and the x-axis is time measured in hours. [Diagram 31] Figure 2 shows the Emax model plotting the HCV RNA reduction measured on day 7 for subjects with non-cirrhotic GT1b HCV infection, non-cirrhotic GT3 HCV infection, cirrhotic GT1b HCV, and cirrhotic GT3 HCV infection against the AUC of metabolites 1-7 after QD administration of compound 1-A. As described in Example 26 and Example 27, subjects with non-cirrhotic GT1b HCV were repeatedly dosed with compound 1-A equivalent to 138 mg / day, 275 mg / day, or 550 mg / day QD for 7 days. Subjects with non-cirrhotic GT3 infection and subjects with cirrhotic GT1 / GT3 infection were given 600 mg of compound 1-A (equivalent to 550 mg / day of compound 1) QD for 7 days. 95%CI interval ranges are shown for non-cirrhotic GT1b HCV administered 138 mg / day, 275 mg / day, or 550 mg / day. This model predicts that exposure to metabolites 1-7 of 2000 ng / mL×hour or greater will result in a maximum viral load reduction of at least 4 log after 7 days of dosing. Regardless of whether the subject exhibited cirrhosis or was non-cirrhotic, all subjects were able to achieve exposure to metabolites 1-7 of greater than 2000 ng / mL×hour after dosing with 550 mg of Compound 1. The x-axis is the AUC of metabolites 1-7 measured in ng / mL×hour, and the y-axis is the HCV RNA reduction on day 7 measured on a log10 scale. [Diagram 32] FIG. 2 is a flow diagram showing the manufacturing process of 50 mg and 100 mg tablets of Compound 1-A as described in Example 28. In step 1, microcrystalline cellulose, Compound 1-A, lactose monohydrate, and croscarmellose sodium are filtered through a 600 μM screen. In step 2, the contents from step 1 are placed in a V-blender and mixed at 25 rpm for 5 minutes. In step 3, magnesium stearate is filtered through a 600 μM screen. In step 4, magnesium stearate is placed in a V-blender with the contents from step 2 (microcrystalline cellulose, Compound 1-A, lactose monohydrate, and croscarmellose sodium) and mixed at 25 rpm for 2 minutes. The common blend is then divided for the production of 50 mg and 100 mg tablets. To produce 50 mg tablets, the blend from step 4 is compressed with a 6 mm round standard concave tooling. The blend from step 4 is compressed on 8 mm round standard concave tooling to produce 100 mg tablets. The tablets are then packaged in HDPE bottles induction sealed with PP caps along with desiccant. [Diagram 33] FIG. 1 shows compound 1-A, an NS5B polymerase inhibitor, and compound 2, an NS5A inhibitor. [Diagram 34]3 is a three-dimensional combination surface plot showing at which concentrations the combination of Compound 1 and Compound 2 is synergistic, additive, or antagonistic. As described in Example 30, the effect of drug combinations is calculated based on the activity of the two compounds when tested alone. The expected additive antiviral protection is subtracted from the experimentally determined antiviral activity at each combination concentration, resulting in a positive value (synergy, or potentiation), a negative value (antagonism), or zero (additivity). The results of the combination assay are displayed in three dimensions at each combination concentration, resulting in a surface of activity that extends above (synergy) or below (antagonism) the plane of additivity. The combination of Compound 1 and Compound 2 is synergistic over a wide range of concentrations of both Compound 1 and Compound 2. Synergy is observed at about 0.156 nM to about 1 nM and at 2.5 nM to 40 nM or higher of Compound 1. About 0.001 nM to about 0.008 nM of Compound 2 is the range of Compound 2 where the most synergistic effects are observed. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0083] The present invention provides a highly active and synergistic combination of a specific NS5B polymerase inhibitor and a specific NS5A inhibitor for the advantageous treatment of Hepatitis C infection in a host, typically a human.

[0084] The anti-HCV compounds used in this combination therapy are 1) an NS5B inhibitor, isopropyl((S)-(((2R,3R,4R,5R)-5-(2-amino-6-(methylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate (Compound 1) or a pharma- ceutical acceptable salt thereof, and 2) an NS5A inhibitor, lusas Bis(dimethyl N,N'-([(6S)-6-(2-cyclopropyl-1,3-thiazol-5-yl)-1-fluoro-6H-indolo[1,2-c][1,3]benzoxazine-3,10-diyl]bis{1H-imidazole-5,2-diyl-(2S)-pyrrolidine-2,1-diyl[(2S)-3-methyl-1-oxobutane-1,2-diyl]})dicarbamate) (Compound 2) or a pharma- ceutically acceptable salt thereof. In an exemplary embodiment, Compound 1 is administered as the hemisulfate derivative (Compound 1-A). [ka]

[0085] In one embodiment, the drug combination is administered in a fixed dosage form, such as a pill or tablet. In an alternative embodiment, the two compounds are administered such that a host in need receives the benefit of both compounds in a coordinated manner as measured by standard pharmacokinetics.

[0086] Compound 1 and Compound 1-A Compound 1 (isopropyl ((S)-(((2R,3R,4R,5R)-5-(2-amino-6-(methylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate) was previously described in U.S. Pat. Nos. 9,828,410, 10,000,523, 10,005,811 and 10,239,911, all assigned to Atea Pharmaceuticals, and in WO 2016 / 21276 and WO 2019 / 200005. The synthesis of Compound 1 is described in Example 1 below.

[0087] Compound 1-A was previously disclosed in U.S. Patent Application Publication No. 2018-0215776, assigned to Atea Pharmaceuticals, and WO 2018 / 144640 and WO 2019 / 200005. The synthesis of compound 1-A (isopropyl((S)-(((2R,3R,4R,5R)-5-(2-amino-6-(methylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate hemisulfate salt) is described in Example 4 below. In one embodiment, compound 1-A is provided in a pharma- ceutically acceptable composition or solid dosage form thereof. In another embodiment, compound 1-A is an amorphous solid. In one embodiment, compound 1-A is a crystalline solid. 1-15 show the characterization of the physical form of compound 1-A by XRPD, DSC, and TGA.

[0088] As noted above, Compound 1-A has completed Phase 1b / 2a clinical trials in patients infected with HCV. The multi-part study evaluated the efficacy of single and multiple doses of Compound 1-A in healthy subjects, non-cirrhotic HCV-infected patients, and cirrhotic HCV-infected patients. Compound 1-A induced significant antiviral reductions when administered to all HCV-infected cohorts tested. Potent antiviral activity was observed when Compound 1-A was administered once daily (QD) for 7 days. In non-cirrhotic HCV-infected patients administered 600 mg Compound 1-A (equivalent to 550 mg Compound 1) QD, the mean maximum HCV RNA reduction was 4.4 log in HCV GT1-infected patients; 10 IU / mL, and 4.6 log 10 IU / mL. The effect of compound 1-A on antiviral reduction extended to patients with difficult-to-treat cirrhosis. In a cohort of patients with CPA cirrhosis infected with HCV GT1 or HCV GT3, the mean maximum HCV RNA reduction was 4.4 log 10 IU / mL (Zhou, X. et al., "AT-527, a pan-genotypic purine nucleotide prodrug, exhibits potent antiviral activity in subjects with chronic hepatitis C," presented at the 2018 International Liver Congress, April 13, 2018, Paris, France). Figures 25-31 show metabolites of Compound 1 and HCV viral RNA levels in cirrhotic patients administered Compound 1. Figure 27 highlights that Compound 1 is as effective in cirrhotic patients as in non-cirrhotic patients.

[0089] Unless otherwise specified, compound 1 or a pharma- ceutically acceptable salt thereof, such as compound 1-A, is provided in the β-D configuration. In alternative embodiments, compound 1 or a pharma- ceutically acceptable salt thereof, such as compound 1-A, may be provided in the β-L configuration. Compound 1 or a pharma- ceutically acceptable salt thereof, such as the phosphoramidate of compound 1-A, may be provided as a chiral phosphorus derivative in the R or S configuration, or mixtures thereof, including racemic or diastereomeric mixtures. All combinations of these configurations are alternative embodiments of the invention described herein.

[0090] These alternative configurations include, but are not limited to: [ka] Contains TIFF2024525164000011.tif200170.

[0091] Additional alternative configurations include: [ka] Includes:

[0092] In one embodiment, any one of the above stereoisomers or pharma- ceutically acceptable salts thereof is used in any aspect of the invention herein as compound 1 or a pharma- ceutically acceptable salt thereof. In another embodiment, any one of the above stereoisomers or a pharma- ceutically acceptable salts thereof is used in any aspect of the invention herein as compound 1-A.

[0093] In an alternative embodiment, compound 1-A is provided as a hemisulfate salt of a phosphoramidate other than the specific phosphoramidate described in the compound diagram.In another alternative embodiment, compound 1 or its pharmaceutically acceptable salt is provided as a phosphoramidate other than the specific phosphoramidate described in the compound diagram.A wide range of phosphoramidates are known to those skilled in the art and can be selected as desired to provide the active compounds described herein.For example, the phosphoramidate of compound 1 or its pharmaceutically acceptable salt can be represented by the formula A: [ka] (In the formula, R 7 is hydrogen, C 1~6 Alkyl (including methyl, ethyl, propyl, and isopropyl), C 3~7 cycloalkyl, or aryl (including phenyl and naphthyl); R 8 is hydrogen or C 1~6 alkyl (including methyl, ethyl, propyl, and isopropyl); R 9a and R 9b are independently hydrogen, C 1~6 Alkyl (including methyl, ethyl, propyl, and isopropyl), or C 3~7 cycloalkyl; R 10 is hydrogen, C 1~6 Alkyl (including methyl, ethyl, propyl, and isopropyl), C 1~6 Haloalkyl, or C 3~7 cycloalkyl) or a pharma- ceutically acceptable salt thereof.

[0094] In alternative non-limiting embodiments, the invention includes Compound 1 as an oxalate salt (Compound 1-B), an HCl salt (Compound 1-C), or a sulfate salt (Compound 1-D). [ka]

[0095] The metabolism of compound 1 and compound 1-A involves the production of 5'-monophosphate followed by N 6 The metabolic pathway includes the anabolism of 2-amino-9-((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methyl dihydrogen phosphate (1-4) as the 5'-monophosphate. The monophosphate is then further anabolized to the active triphosphate species: 5'-triphosphate (1-6). The 5'-triphosphate can be further metabolized to generate 2-amino-9-((2R,3R,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)-3-methyltetrahydrofuran-2-yl)-1,9-dihydro-6H-purin-6-one (1-7). Figures 17-24 show the pharmacokinetic properties of compound 1-A and the major metabolites. Compound 1-A is metabolized within 8 hours, but active metabolites are present for up to 24 hours. Suppression of HCV viral RNA is observed during the 24-hour period in which the metabolites are present. At a dose of 300 mg, the plasma concentration of metabolites of compound 1-A was greater than the EC50 of HCV GT1b. 95 Figure 16 shows the favorable biodistribution profile of compound 1-A. The compound is concentrated in the liver, the target organ of HCV infection, rather than in the heart. Due to the high liver concentration, lower doses may be used. The effect was not species specific and was observed in three different preclinical species. Figure 24 shows the EC values ​​for compound 1 and sofosbuvir against various HCV genotypes. 95 Sovosbuvir has various EC ratios based on genotype. 95 Compound 1 has an EC 95 varies little based on genotype.

[0096] Alternatively, the 5'-monophosphate 1-2 can be metabolized to produce the purine base 1-8. The metabolic pathway for isopropyl((S)-(((2R,3R,4R,5R)-5-(2-amino-6-(methylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate is shown in Scheme 1. [ka]

[0097] Atea Pharmaceuticals, Inc. is a registered trademark of U.S. Patent Nos. 9,828,410, 10,000,523, 10,005,811, 10,239,911, 10,815,266, 10,870,672, 10,870,673, 10,870,885, 10,519,186, 10,906,9 28, 10,894,804, and WO 2016 / 144918, WO 2018 / 048937, WO 2018 / 013937 and WO 2018 / 144640, the disclosures of which are incorporated herein by reference in their entirety, disclose β-D-2'-deoxy-2'-α-fluoro-2'-β-C-substituted-2-modified-N-acetylglucosamine derivatives for the treatment of HCV. 6 Atea also discloses β-D-2'-deoxy-2'-substituted-4'-substituted-2-N-β-D-p-4'-purine nucleotides for the treatment of paramyxovirus and orthomyxovirus infections in U.S. Pat. No. 10,202,412 and WO 2018 / 009623. 6 -substituted 6-aminopurine nucleotides are disclosed.

[0098] compound 2 Compound 2 is disclosed in International Publication No. WO 2014 / 110705 and U.S. Patent No. 9,555,038, assigned to Merck and Company. In one embodiment, compound 2 is administered as a pharma- ceutically acceptable salt thereof. In one embodiment, a solid form of compound 2 is used. In one embodiment, the solid form of compound 2 is a crystalline solid.

[0099] The synthesis of ruzasvir (dimethyl N,N'-([(6S)-6-(2-cyclopropyl-1,3-thiazol-5-yl)-1-fluoro-6H-indolo[1,2-c][1,3]benzoxazine-3,10-diyl]bis{1H-imidazole-5,2-diyl-(2S)-pyrrolidine-2,1-diyl[(2S)-3-methyl-1-oxobutane-1,2-diyl]}) dicarbamate, compound 2) is known in the art. Non-limiting examples of synthetic methods that can be used to synthesize compound 2 include the method shown in Example 29 and reported in WO 2016 / 196932, assigned to Merck.

[0100] definition The term "D configuration" as used in the context of the present invention refers to the principle configuration that mimics the natural configuration of the sugar moiety of non-naturally occurring nucleosides, i.e., the opposite of the "L" configuration. The term "β" or "β anomer" is used in reference to nucleoside analogs in which the nucleoside base is formed (positioned) above the plane of the furanose moiety in the nucleoside analog.

[0101] The terms "co-administer" and "co-administration" or combination therapy are used to describe the administration of compound 1 or a pharma- ceutically acceptable salt thereof in combination with compound 2 or a pharma- ceutically acceptable salt thereof according to the present invention. In certain embodiments, compound 1 or a pharma- ceutically acceptable salt thereof, such as compound 1-A and compound 2 or a pharma- ceutically acceptable salt thereof, are administered with at least one other active agent, for example, optionally at least one additional anti-HCV agent. The timing of the co-administration is best determined by the specialist treating the patient. It may be preferred to administer the agents simultaneously, or at least to allow for overlapping pharmacological effects of the two drugs in the patient being treated. Alternatively, the drugs selected for combination therapy may be administered to the patient at different times. Of course, when more than one virus or other infection or other condition is present, the compounds of the present invention may be combined with other agents to treat the other infections or conditions as necessary.

[0102] The term "host" as used herein refers to a unicellular or multicellular organism, including cell lines and animals, typically humans, in which the HCV virus can replicate. The term host specifically refers to infected cells, cells transfected with all or part of the HCV genome, and animals carrying the HCV genome or a part thereof, particularly primates (including chimpanzees) and humans, which can be treated with the combinations described herein. In most animal applications of the present invention, the host is a human patient, which includes, but is not limited to, dosing regimens with overlapping pharmacokinetics. However, veterinary applications are clearly anticipated by the present invention in certain indications (such as chimpanzees). The host may be one capable of carrying the above-mentioned viruses, such as, for example, bovine, equine, avian, canine, feline, etc.

[0103] "Pharmaceutically acceptable salts" are derivatives of the disclosed compounds, where the parent compound is modified into its inorganic and organic acid addition salts or base addition salts without undue toxicity. The salts of the compounds of the present invention can be synthesized from the parent compound having a basic or acidic moiety by conventional chemical methods. In general, such salts can be prepared by reacting the free acid forms of these compounds with a stoichiometric amount of a suitable base (e.g., hydroxides, carbonates, bicarbonates, etc. of Na, Ca, Mg, or K) or by reacting the free base forms of these compounds with a stoichiometric amount of a suitable acid. Such reactions are typically carried out in water or in an organic solvent, or in a mixture of the two. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are typical, where practicable. The salts of the compounds of the present invention can be provided in the form of an optional solvate.

[0104] Examples of pharma- ceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, etc. Pharmaceutically acceptable salts include conventional salts of the parent compound formed, for example, from inorganic or organic acids which are not overly toxic, and quaternary ammonium salts. For example, conventional acid salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like, as well as salts derived from acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, mesylic acid, esylic acid, besylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, HOOC-(CH2) nIncluded are salts prepared from organic acids such as -COOH, where n is 0 to 4, or using a different acid which produces the same counterion. Additional lists of suitable salts can be found, for example, in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., p. 1418 (1985).

[0105] The compound can be delivered in any molar ratio that gives the desired result.For example, the compound can be provided with less than the molar equivalent of counterion, for example in the form of hemisulfate.Alternatively, the compound can be provided with more than the molar equivalent of counterion, for example in the form of disulfate.Non-limiting examples of the molar ratio of compound to counterion include 1:0.25, 1:0.5, 1:1, and 1:2.

[0106] Isotope Substitution The present invention includes a combination of Compound 1 or a pharma- ceutically acceptable salt thereof, e.g., Compound 1-A, and Compound 2 or a pharma- ceutically acceptable salt thereof, where one or both of these compounds have the desired isotopic substitution of atoms at greater than natural abundance, i.e., enriched isotope amounts. Isotopes are atoms having the same atomic number but different mass numbers, i.e., the same number of protons but different numbers of neutrons. As a general example and without limitation, for example, deuterium ( 2 H) and tritium ( 3 H) may be used anywhere in the structures depicted. Alternatively, or in addition, isotopes of carbon, such as 13 C and 14C may be used. A preferred isotopic substitution is the replacement of hydrogen with deuterium at one or more positions on a molecule to improve drug performance. Deuterium can be attached at the position of bond cleavage during metabolism (α-deuterium kinetic isotope effect) or adjacent to or near the site of bond cleavage (β-deuterium kinetic isotope effect). Achillion Pharmaceuticals, Inc. (WO 2014 / 169278 and WO 2014 / 169280) describes the deuteration of nucleotides to improve their pharmacokinetics or efficacy, including at the 5-position of the molecule.

[0107] Substitution with isotopes such as deuterium can confer certain therapeutic advantages due to greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements. Substitution of hydrogen at a site of metabolic degradation with deuterium can reduce the rate of metabolism at that bond or eliminate metabolism at that bond. At any position in a compound where a hydrogen atom can be present, the hydrogen atom can be substituted with protium ( 1 H), deuterium ( 2 H) and tritium ( 3 Any isotope of hydrogen may be used, including H. Accordingly, unless the context clearly dictates otherwise, references herein to compounds include all possible isotopic forms.

[0108] The term "isotopically labeled" analogues refers to "deuterated analogues," 13 C-labeled analogues or deuterated / 13 The term "deuterated analog" refers to an analog that is a "C labeled analog". 1 H) is converted to the H-isotope, namely deuterium ( 2H). Deuterium substitution may be partial or complete. Partial deuterium substitution means that at least one hydrogen is replaced by at least one deuterium. In certain embodiments, the isotope is enriched to 90%, 95%, or 99% or more in the isotope at any desired position. In some embodiments, it is deuterium that is enriched to 90%, 95%, or 99% at the desired position. Unless otherwise specified, deuteration is at least 80% at the selected position. Deuteration of a nucleoside may occur at any replaceable hydrogen that produces the desired result.

[0109] Treatment methods As used herein, treatment refers to administering an effective amount of a combination of the present invention to a host, such as a human, infected or potentially infected with the HCV virus. In one embodiment, the method of treatment includes administering an effective amount of Compound 1 or a pharmaceutically acceptable salt thereof and Compound 2 or a pharmaceutically acceptable salt thereof to a host, such as a human, infected or potentially infected with the HCV virus. In another embodiment, the method of treatment includes administering Compound 1-A and Compound 2 to a host, such as a human, infected or potentially infected with the HCV virus.

[0110] The term "prophylactic" or preventative, when used, refers to the administration of the combinations of the invention to prevent or reduce the likelihood of viral disease occurring. The invention includes, in alternative embodiments, therapeutic and prophylactic or preventative therapies. In one embodiment, the combinations are administered to a host at risk of infection by Hepatitis C virus due to exposure thereto.

[0111] The present invention includes a method for treating Hepatitis C virus, including drug-resistant and multi-drug resistant forms of HCV, and associated disease states, symptoms or complications of HCV infection, including cirrhosis of the liver and associated liver toxicity, as well as other symptoms secondary to HCV infection, such as weakness, loss of appetite, weight loss, breast enlargement (especially in men), rash (especially on the palms), difficulty in clotting blood, spider veins in the skin, confusion, coma (encephalopathy), accumulation of fluid in the abdominal cavity (ascites), esophageal varices, portal hypertension, renal failure, splenomegaly, low blood counts, anemia, thrombocytopenia, jaundice, and hepatocellular carcinoma, among others, by administering an effective amount of a synergistic combination of Compound 1 (e.g., Compound 1-A) and Compound 2. The method includes administering an effective amount of the combination described herein, optionally in combination with at least one additional bioactive agent, such as an additional anti-HCV agent, and optionally further in combination with a pharma- ceutically acceptable carrier, additive, and / or excipient, to a host, typically a human, in need thereof. In another embodiment, the method includes administering an effective amount of the combination of the present invention to a patient potentially infected with HCV. In another embodiment, the combination is used in combination with a pharma- ceutically acceptable carrier, additive, or excipient, optionally in combination with a third anti-HCV agent. In another embodiment, the combination of the present invention can be administered to a patient to protect the new organ after hepatitis-related liver transplantation.

[0112] Combination therapy and dosage forms can also be used to treat pathologies associated with or resulting from HCV virus exposure.For example, active compounds can be used to treat HCV antibody positive and HCV antigen positive conditions, chronic liver inflammation caused by viruses, liver cancer (e.g., hepatocellular carcinoma) caused by progressive hepatitis C, liver cirrhosis, acute hepatitis C, fulminant hepatitis C, chronic persistent hepatitis C, and anti-HCV fatigue.The above combinations can also be used prophylactically to prevent or limit the progression of clinical disease in individuals who are anti-HCV antibody positive or antigen positive or who have been exposed to hepatitis C.

[0113] Pharmaceutical Compositions and Dosage Forms Administration of Compound 1 or its pharma- ceutically acceptable salt and Compound 2 or its pharma- ceutically acceptable salt may be carried out using any desired form, including, but not limited to, oral, topical, parenteral, intramuscular, intravenous, subcutaneous, transdermal (which may include a permeation enhancer), buccal, and suppository administration, among other routes of administration. In one embodiment, the active compound or combination of compounds is provided in a solid dosage form, which is well known in the art and further described below. Enteric coated oral tablets may also be used to enhance the bioavailability of the compounds for oral routes of administration. The most effective dosage form will depend on the severity of the disease in the patient as well as the bioavailability / pharmacokinetics of the particular agent selected. Oral dosage forms are particularly preferred due to ease of administration and the potential for favorable patient compliance.

[0114] In certain embodiments, pharmaceutical compositions according to the invention comprise an anti-HCV virus effective amount of Compound 1 or a pharma- ceutically acceptable salt thereof and Compound 2 or a pharma- ceutically acceptable salt thereof, each in separate or combined form, as described herein, optionally in combination with a pharma- ceutically acceptable carrier, excipient, or vehicle, and optionally in combination or alternation with at least one other active compound.

[0115] In one embodiment, the combination includes a solid dosage form of Compound 1 or a pharma- ceutically acceptable salt thereof, such as Compound 1-A, and Compound 2 or a pharma- ceutically acceptable salt thereof, in a pharma- ceutically acceptable carrier. The pharmaceutical composition can contain both Compound 1 or a pharma- ceutically acceptable salt thereof and Compound 2 or a pharma- ceutically acceptable salt thereof, or alternatively, the compounds can be in separate dosage forms that are administered such that the host receives the benefit of both compounds in a coordinated manner as measured by standard pharmacokinetics.

[0116] Those skilled in the art will recognize that the therapeutically effective amount will vary depending on the infection or condition being treated, its severity, the treatment regimen being employed, the pharmacokinetics of the agents used, as well as the patient or subject (animal or human) being treated, and that such therapeutic amount can be determined by the attending physician or specialist.

[0117] Compound 1 or its pharmaceutically acceptable salt, such as Compound 1-A and Compound 2 or its pharmaceutically acceptable salt, may be formulated as one or more mixtures with one or more pharmaceutically acceptable carriers. In general, it is desirable to administer one or more oral dosage forms of the pharmaceutical composition, particularly one or more solid dosage forms, such as pills or tablets. Certain formulations may be administered by other routes, including parenteral, intravenous, intramuscular, topical, transdermal, buccal, subcutaneous, suppository, or nasal spray. Intravenous and intramuscular formulations are often administered in sterile saline. Those skilled in the art may modify the formulation to make it more soluble in water or another vehicle, for example, this can be easily accomplished by minor modifications (salt formulations, esterification, etc.), which are well within the ordinary skill in the art. It is also well within the ability of one of ordinary skill in the art to modify the route of administration and dosing regimen of Compound 1 or a pharma- ceutically acceptable salt thereof, e.g., Compound 1-A and Compound 2 or a pharma- ceutically acceptable salt thereof, in order to manage the pharmacokinetics of the compounds of the invention for maximum beneficial effect in the patient.

[0118] In certain pharmaceutical dosage forms, prodrug forms of the compounds of the present invention, including acylated (acetylated or otherwise) and ether (alkyl and related) derivatives, phosphate esters, thiophosphoramidates, phosphoramidates, and various salt forms of the compounds of the present invention, may be used to achieve the desired effect.Those skilled in the art will recognize how to easily convert the compounds of the present invention into prodrug forms to facilitate the delivery of active compounds to the target site of the host organism or patient.Those skilled in the art will also take advantage of the advantageous pharmacokinetic parameters of prodrug forms, if applicable, in the delivery of the compounds of the present invention to the target site of the host organism or patient to maximize the intended effect of the compounds of the present invention.

[0119] The amounts given in this disclosure typically refer to the free form (i.e., non-salt, hydrated, or solvated form). The typical values ​​given herein represent the equivalent of the free form, i.e., the same amount as when the free form is administered. When a salt is administered, the amount must be calculated based on the molecular weight ratio between the salt and the free form.

[0120] The amount of compound 1 or a pharma- ceutically acceptable salt thereof, e.g., compound 1-A and compound 2 or a pharma- ceutically acceptable salt thereof, contained in the therapeutically active formulation according to the invention is an amount effective to achieve the desired outcome according to the invention, e.g., to treat HCV infection, to reduce the likelihood of HCV infection, or to inhibit, reduce and / or eliminate HCV or its secondary effects, including disease states, pathologies, and / or complications secondary to HCV. In general, the therapeutically effective amount of the compound of the invention in a pharmaceutical dosage form may range, for example, from about 0.001 mg / kg to about 100 mg / kg or more per day. Compound 1 or compound 1-A may be administered in an amount ranging, for example, from about 0.1 mg / kg to about 15 mg / kg per day of a patient, depending on the pharmacokinetics of the agent in the patient.

[0121] In certain embodiments, 600 mg of Compound 1-A, which is 550 mg of Compound 1, is provided in the dosage form.

[0122] In certain embodiments, the pharmaceutical composition is in a dosage form containing about 1 mg to about 2000 mg, about 10 mg to about 1000 mg, about 100 mg to about 800 mg of compound 2 or an equivalent amount of a pharma- ceutically acceptable salt of compound 2, in addition to about 1 mg to about 2000 mg, about 10 mg to about 1000 mg, about 100 mg to about 800 mg, about 200 mg to about 600 mg, about 300 mg to about 500 mg, or about 400 mg to about 450 mg of compound 1 or an equivalent amount of compound 1-A in a unit dosage form.

[0123] In an alternative embodiment, the pharmaceutical composition is in a dosage form containing about 100 mg to about 800 mg, about 150 mg to about 200 mg, about 250 mg to about 300 mg, or about 350 mg to about 400 mg of compound 2 or a corresponding amount of a pharma- ceutically acceptable salt of compound 2, and about 500 mg to about 600 mg, about 550 mg to about 750 mg, about 600 mg to about 800 mg, or about 1000 mg to about 1300 mg of compound 1 or a corresponding amount of compound 1-A in a unit dosage form.

[0124] In certain embodiments, the pharmaceutical composition may be in a unit dosage form, with up to about 10 mg, about 50 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg , about 575 mg, about 600 mg, about 625 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, about 750 mg, about 775 mg, about 800 mg, about 825 mg, about 850 mg, about 875 mg, about 900 mg, about 925 mg, about 950 mg, about 975 mg, or about 1000 mg or more of Compound 1 or an equivalent amount of Compound 1-A, e.g., in a solid dosage form.

[0125] In certain embodiments, the pharmaceutical composition may comprise up to about 10 mg, about 50 mg, about 60 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, about 580 mg, about 590 mg, about 600 mg, about 625 mg, about 630 mg, about 640 mg, about 650 mg, about 660 mg, about 675 mg, about 680 mg, about 690 mg, about 700 mg, about 710 mg, about 725 mg, about 750 mg, about 765 mg, about 775 mg, about 780 mg, about 790 mg, about 800 mg, about 810 mg, about 825 mg, about 830 mg, about 840 mg, about 850 mg, about 865 mg, about 875 mg, about 880 mg, about 890 mg, about 900 mg, about 925 mg, about 930 mg, about 940 mg, about 950 mg, about 965 mg, about 975 mg, about 980 mg, about 990 mg, about 1000 mg, about 1025 mg, about 1040 mg, about 1050 mg, about 1060 mg, about 1070 mg, about 1080 mg, about 1090 mg, about 1100 mg, about 11 In some embodiments, the compound is present in a dosage form containing about 600 mg, about 625 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, about 750 mg, about 775 mg, about 800 mg, about 825 mg, about 850 mg, about 875 mg, about 900 mg, about 925 mg, about 950 mg, about 975 mg, or about 1000 mg or more of Compound 2, or a corresponding amount of a pharma- ceutically acceptable salt of Compound 2, e.g., in a solid dosage form.

[0126] In alternative embodiments, the pharmaceutical composition is in a dosage form, e.g., a solid dosage form, containing up to about 90 mg, about 180 mg, about 270 mg, or about 360 mg of Compound 2 or an equivalent amount of a pharma- ceutically acceptable salt of Compound 2.

[0127] In one embodiment, up to about 800 mg, up to about 700 mg, up to about 600 mg, up to about 550 mg, up to about 500 mg, up to about 400 mg, up to about 300 mg, up to about 200 mg, or up to about 100 mg of Compound 1 or an equivalent amount of Compound 1-A and up to about 360 mg, up to about 270 mg, up to about 180 mg, up to about 145 mg, up to about 130 mg, up to about 125 mg, up to about 110 mg, up to about 100 mg, up to about 90 mg A solid dosage form containing up to about 75 mg, up to about 70 mg, up to about 65 mg, up to about 60 mg, up to about 55 mg, up to about 50 mg, up to about 45 mg, up to about 40 mg, up to about 35 mg, up to about 30 mg, up to about 25 mg, up to about 20 mg, up to about 15 mg, up to about 10 mg, or up to about 5 mg of Compound 2 or an equivalent amount of a pharma- ceutically acceptable salt of Compound 2 is administered once daily to a host in need thereof for the treatment of HCV.

[0128] In one embodiment, at least about 100 mg, at least about 200 mg, at least about 300 mg, at least about 400 mg, at least about 500 mg, at least about 550 mg, at least about 600 mg, at least about 700 mg, at least about 750 mg, or at least about 1100 mg of Compound 1 or an equivalent amount of Compound 1-A and at least about 5 mg, at least about 10 mg, at least about 15 mg, at least about 20 mg, at least about 25 mg, at least about 30 mg, at least about 35 mg, at least about 40 mg, at least about 45 mg, at least about 50 mg, A solid dosage form containing at least about 100 mg, at least about 110 mg, at least about 125 mg, at least about 130 mg, at least about 145 mg, at least about 180 mg, at least about 270 mg, at least about 360 mg, at least about 400 mg, or at least about 500 mg of Compound 2, or an equivalent amount of a pharma- ceutically acceptable salt of Compound 2, is administered once daily to a host in need thereof for the treatment of HCV.

[0129] In alternative embodiments, a solid dosage form containing up to about 1100 mg, up to about 750 mg, or up to about 550 mg of Compound 1 or an equivalent amount of Compound 1-A and up to about 360 mg, up to about 270 mg, up to about 180 mg, or up to about 90 mg of Compound 2 or an equivalent amount of a pharma- ceutically acceptable salt of Compound 2 is administered once daily to a host in need thereof for the treatment of HCV.

[0130] In alternative embodiments, a solid dosage form containing at least about 500 mg, at least about 550 mg, at least about 750 mg, or at least about 1100 mg of Compound 1 or an equivalent amount of Compound 1-A and at least about 90 mg, at least about 180 mg, at least about 270 mg, or at least about 360 mg of Compound 2 or an equivalent amount of a pharma- ceutically acceptable salt of Compound 2 is administered twice daily to a host in need thereof for the treatment of HCV.

[0131] In one embodiment, the combination of compounds described herein is administered as a single tablet containing up to about 600 mg of Compound 1-A and up to about 90 mg, 180 mg, 270 mg, or 360 mg of Compound 2, or an equivalent amount of a pharma- ceutically acceptable salt of Compound 2.

[0132] In an alternative embodiment, the combination of compounds described herein is administered as a single tablet containing up to about 600 mg of Compound 1-A and up to about 180 mg of Compound 2 or an equivalent amount of a pharma- ceutically acceptable salt of Compound 2.

[0133] In an alternative embodiment, the combination of compounds described herein is administered as a single tablet containing up to about 600 mg of Compound 1-A and up to about 270 mg of Compound 2 or an equivalent amount of a pharma- ceutically acceptable salt of Compound 2.

[0134] In an alternative embodiment, the combination of compounds described herein is administered as a single tablet containing up to about 600 mg of Compound 1-A and up to about 360 mg of Compound 2 or an equivalent amount of a pharma- ceutically acceptable salt of Compound 2.

[0135] In an alternative embodiment, the combination of compounds described herein is administered as a single tablet containing up to about 750 mg of Compound 1-A and up to about 180 mg of Compound 2 or an equivalent amount of a pharma- ceutically acceptable salt of Compound 2.

[0136] In an alternative embodiment, the combination of compounds described herein is administered as a single tablet containing up to about 750 mg of compound 1-A and up to about 270 mg of compound 2 or an equivalent amount of a pharma- ceutically acceptable salt of compound 2.

[0137] In an alternative embodiment, the combination of compounds described herein is administered as a single tablet containing up to about 750 mg of Compound 1-A and up to about 360 mg of Compound 2 or an equivalent amount of a pharma- ceutically acceptable salt of Compound 2.

[0138] In an alternative embodiment, the combination of compounds described herein is administered as a single tablet containing up to about 1200 mg of Compound 1-A and up to about 180 mg of Compound 2 or an equivalent amount of a pharma- ceutically acceptable salt of Compound 2.

[0139] In an alternative embodiment, the combination of compounds described herein is administered as a single tablet containing up to about 1200 mg of Compound 1-A and up to about 270 mg of Compound 2 or an equivalent amount of a pharma- ceutically acceptable salt of Compound 2.

[0140] In an alternative embodiment, the combination of compounds described herein is administered as a single tablet containing up to about 1200 mg of Compound 1-A and up to about 360 mg of Compound 2 or an equivalent amount of a pharma- ceutically acceptable salt of Compound 2.

[0141] Alternatively, a solid dosage form of compound 1-A or an equivalent amount of compound 1 can be administered in combination with a separate solid dosage form containing compound 2 or a pharma- ceutically acceptable salt of compound 2. This combination can be administered once a day, twice a day, three times a day, or up to four times a day, as directed by a medical provider. In one embodiment, compound 1-A or compound 1 is administered on a separate schedule from compound 2. For example, compound 1 or an equivalent amount of compound 1-A can be administered twice a day, while compound 2 is administered only once a day, or vice versa. Compound 2 can be administered multiple times a day, while compound 1 or an equivalent amount of compound 1-A is administered only once a day.

[0142] In one embodiment, a solid dosage form containing up to about 800 mg, up to about 700 mg, up to about 600 mg, up to about 500 mg, up to about 400 mg, up to about 300 mg, up to about 200 mg, or up to about 100 mg of Compound 1 or an equivalent amount of Compound 1-A is administered once daily to a host in need thereof for treatment of HCV, and up to about 145 mg, up to about 130 mg, up to about 125 mg, up to about 110 mg, up to about A separate solid dosage form containing 100 mg, up to about 90 mg, up to about 75 mg, up to about 70 mg, up to about 65 mg, up to about 60 mg, up to about 55 mg, up to about 50 mg, up to about 45 mg, up to about 40 mg, up to about 35 mg, up to about 30 mg, up to about 25 mg, up to about 20 mg, up to about 15 mg, up to about 10 mg, or up to about 5 mg of Compound 2 or an equivalent amount of a pharma- ceutically acceptable salt of Compound 2 is administered once daily.

[0143] In one embodiment, a solid dosage form containing at least about 100 mg, at least about 200 mg, at least about 300 mg, at least about 400 mg, at least about 500 mg, at least about 600 mg, at least about 700 mg, or at least about 800 mg of Compound 1 or an equivalent amount of Compound 1-A is administered once daily to a host in need thereof for treatment of HCV, and at least about 5 mg, at least about 10 mg, at least about 15 mg, at least about 20 mg, at least about 25 mg, at least about 30 mg or at least about 35 mg, at least about 40 mg, at least about 45 mg, at least about 50 mg, at least about 55 mg, at least about 60 mg, at least about 65 mg, at least about 70 mg, at least about 75 mg, at least about 80 mg, at least about 90 mg, at least about 100 mg, at least about 110 mg, at least about 125 mg, at least about 130 mg, or at least about 145 mg of Compound 2 or a corresponding amount of a pharma- ceutically acceptable salt of Compound 2 is administered once daily.

[0144] In certain embodiments, the combination of compounds described herein comprises: (1) Single dosage form; (2) Two dosage forms, (3) Three or more dosage forms; (4) A kit containing two dosage forms; (5) Kits containing three or more dosage forms; (either as separate doses of the two active agents or as a combined dose of the two active compounds) to a host in need thereof, wherein the dosage form in each of embodiments (1) to (5) is (a) about 500 mg or 550 mg to about 1100 mg of compound 1-A and an effective amount of compound 2, (b) about 100 mg to about 800 mg of compound 1-A and an effective amount of compound 2, (c) about 550 mg to about 650 mg of compound 1-A and an effective amount of compound 2, (d) about 500 mg to about 750 mg of compound 1-A and an effective amount of compound 2, (e) about 1000 mg to about 1300 mg of compound 1-A and an effective amount of compound 2, (f) at least about 600 mg of Compound 1-A and an effective amount of Compound 2; (g) at least about 750 mg of Compound 1-A and an effective amount of Compound 2; (h) at least about 1100 mg of Compound 1-A and an effective amount of Compound 2; (i) any one of embodiments (a)-(h), wherein about 60 mg to about 500 mg of Compound 2 is present; (j) any one of embodiments (a)-(h), wherein from about 90 mg to about 360 mg of Compound 2 is present; (k) any one of embodiments (a)-(h), wherein about 250 mg to about 300 mg of Compound 2 is present; (l) any one of embodiments (a)-(h), wherein about 350 mg to about 400 mg of Compound 2 is present; (m) any one of embodiments (a)-(h), wherein at least about 90 mg of Compound 2 is present; (n) Any one of embodiments (a)-(h), wherein at least about 180 mg of Compound 2 is present; (o) Any one of embodiments (a)-(h), wherein at least about 270 mg of Compound 2 is present; (p) any one of embodiments (a)-(h), wherein at least about 360 mg of Compound 2 is present; (q) any one of embodiments (1)-(5) and (a)-(h), wherein the dosage form is administered once daily; (r) any one of embodiments (1)-(5) and (a)-(h), wherein the dosage form is administered twice daily; (s) any one of embodiments (1)-(5) and (a)-(h), wherein the dosage form is administered three or more times per day; (t) any one of embodiments (1)-(5) and (a)-(s), wherein the dosage form is administered orally; (u) any one of embodiments (1)-(5) and (a)-(t), wherein the host is a human; Includes.

[0145] In an alternative embodiment, compound 1 may be provided to a host in need thereof in an amount that results in a plasma concentration of compound 1 of about 0.15 nM to about 1 nM. In one embodiment, compound 1 may be provided to a host in need thereof in an amount that results in a plasma concentration of compound 1 of about 0.25 nM to about 40 nM. In one embodiment, compound 1 may be provided to a host in need thereof in an amount that results in a plasma concentration of compound 1 of about 40 nM to about 200 nM. In one embodiment, compound 1 may be provided to a host in need thereof in an amount that results in a plasma concentration of compound 2 of about 0.001 nM to about 0.008 nM.

[0146] The compounds of the combination of the present invention are often administered orally, but can also be administered parenterally, topically, or in the form of a suppository, and intranasally, as a nasal spray, or in other ways as described herein.More generally, these compounds can be administered in the form of one or more tablets, capsules, injections, intravenous preparations, suspensions, solutions, emulsions, implants, particles, spheres, creams, ointments, suppositories, inhalation forms, transdermal forms, buccal forms, sublingual forms, topical forms, gel forms, mucosal forms, etc.

[0147] In certain embodiments, the combination is administered at least once a day for up to 24 weeks. In certain embodiments, the combination is administered at least once a day for up to 12 weeks. In certain embodiments, the combination is administered at least once a day for up to 10 weeks. In certain embodiments, the combination is administered at least once a day for up to 8 weeks. In certain embodiments, the combination is administered at least once a day for up to 6 weeks. In certain embodiments, the combination is administered at least once a day for up to 4 weeks. In certain embodiments, the combination is administered at least once a day for at least 4 weeks. In certain embodiments, the combination is administered at least once a day for at least 6 weeks. In certain embodiments, the combination is administered at least once a day for at least 8 weeks. In certain embodiments, the combination is administered at least once a day for at least 10 weeks. In certain embodiments, the combination is administered at least once a day for at least 12 weeks. In certain embodiments, the combination is administered at least once a day for at least 24 weeks. In certain embodiments, the combination is administered at least every other day for up to 24 weeks, up to 12 weeks, up to 10 weeks, up to 8 weeks, up to 6 weeks, or up to 4 weeks. In certain embodiments, the combination is administered at least every other day for at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks, at least 12 weeks, or at least 24 weeks.

[0148] For purposes of this invention, a prophylactically or preventatively effective amount of a composition according to the invention falls within the same concentration ranges as set forth above for a therapeutically effective amount, and is usually the same as the therapeutically effective amount.

[0149] To prepare pharmaceutical compositions according to the invention, a therapeutically effective amount of Compound 1 or a pharma- ceutically acceptable salt thereof, such as Compound 1-A and Compound 2 or a pharma- ceutically acceptable salt thereof, can be essentially mixed with a pharma- ceutical acceptable carrier according to conventional pharmaceutical compounding techniques to produce a dosage. The carrier can take a variety of forms, depending on the dosage form desired, for example, for oral or parenteral administration. In preparing pharmaceutical compositions into oral dosage forms, any of the usual pharmaceutical media can be used. Thus, for liquid oral preparations such as suspensions, elixirs and solutions, suitable carriers and additives can be used, including water, glycols, oils, alcohols, flavorings, preservatives, colorings, and the like. For solid oral preparations such as powders, tablets, capsules, and solid preparations such as suppositories, suitable carriers and additives can be used, including sugar carriers such as starch, dextrose, manifolds, lactose, and related carriers, diluents, granulating agents, lubricants, binders, disintegrants, and the like. If desired, the tablets or capsules can be enteric coated or sustained release by standard techniques. The use of these dosage forms can significantly enhance the bioavailability of the compound in the patient.

[0150] For parenteral formulations, the carrier usually comprises sterile water or aqueous sodium chloride solution, but may contain other ingredients, including those that aid in dispersion. Where sterile water is to be used and maintained as sterile, the composition and carrier must, of course, also be sterilized. Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents, and the like may be utilized.

[0151] Liposomal suspensions (including liposomes targeted to viral antigens) may also be prepared by conventional methods to produce pharma- ceutically acceptable carriers, which may be suitable for delivery of the free nucleoside, acyl / alkyl nucleoside, or phosphate ester prodrug forms of the nucleoside compounds according to the invention.

[0152] In an exemplary embodiment according to the invention, the pharmaceutical composition is used to treat, prevent, or delay HCV infection, or secondary disease states, symptoms, or complications of HCV.

[0153] solid dosage form One aspect of the invention is a fixed dosage form (separately or combined) of the active compounds or pharma- ceutically acceptable salts thereof, optionally in a combined fixed dosage form.

[0154] Either or both compounds can be provided in crystalline or amorphous form, hi certain embodiments, they are provided in separate or the same oral dosage form, such as a gelcap, solid form, spray-dried dispersion, tablet, capsule, or other form of pill.

[0155] In one embodiment, the fixed dose combination comprises a spray-dried solid dispersion of at least one or both compounds, or a pharma- ceutically acceptable salt thereof, and the composition is suitable for oral delivery. In one aspect of this embodiment, the fixed dose combination comprises Compound 1, or a pharma- ceutically acceptable salt thereof, and Compound 2, or a pharma- ceutically acceptable salt thereof, wherein at least one compound is a spray-dried solid dispersion.

[0156] In another embodiment, the fixed dose combination is at least one granulo layered solid dispersion of a compound or a pharma- ceutically acceptable salt thereof, and the composition is suitable for oral delivery. An exemplary process for preparing a solid dosage form for oral delivery can be seen in Figure 32. In one aspect of this embodiment, the fixed dose combination is a granulo layered solid dispersion comprising Compound 1 or a pharma- ceutically acceptable salt thereof and Compound 2 or a pharma- ceutically acceptable salt thereof. In certain embodiments, the components of the spray-dried dispersion or granulo layered solid dispersion are prepared using crystalline Compound 1-A. In an alternative embodiment, Compound 1 or a pharma- ceutically acceptable salt thereof, such as Compound 1-A or Compound 2 or a pharma- ceutically acceptable salt thereof, can be delivered as an amorphous compound.

[0157] In other embodiments, the solid dispersion also includes at least one excipient selected from copovidone, poloxamer, and HPMC-AS. In one embodiment, the poloxamer is poloxamer 407 or a mixture of poloxamers that may include poloxamer 407. In one embodiment, the HPMC-AS is HPMC-AS-L.

[0158] In yet another embodiment, the fixed dose composition made from Compound 1 or a pharma- ceutically acceptable salt thereof and Compound 2 or a pharma- ceutically acceptable salt thereof includes one or more of the following excipients: phosphoglycerides; phosphatidylcholine; dipalmitoylphosphatidylcholine (DPPC); dioleoylphosphatidylethanolamine (DOPE); dioleoyloxypropyltriethylammonium (DOTMA); dioleoylphosphatidylcholine; cholesterol; cholesterol esters; diacylglycerol; diacylglycerol. rol succinate;diphosphatidylglycerol (DPPG);hexanedecanol;fatty alcohols such as polyethylene glycol (PEG);polyoxyethylene-9-lauryl ether;surface active fatty acids such as palmitic acid or oleic acid;fatty acids;fatty acid monoglycerides;fatty acid diglycerides;fatty acid amides;sorbitan trioleate (Span™ 85);glycocholate;sorbitan monolaurate (Span™ 20);polysorbate 20 (Tween™ 20);polysorbate 60 (Tween™ 20); Polysorbate 60; Polysorbate 65 (Tween 65); Polysorbate 80 (Tween 80); Polysorbate 85 (Tween 85); Polyoxyethylene monostearate; Surfactin; Poloxamer; Sorbitan fatty acid esters such as sorbitan trioleate; Lecithin; Lysolecithin; Phosphatidylserine; Phosphatidylinositol; Sphingomyelin; Phosphatidylethanolamine (cephalin); Cardiolipin; Phosphatidic acid; Cerebroside; Dicetyl phosphate phosphatidylglycerol;dipalmitoylphosphatidylglycerol;stearylamine;dodecylamine;hexadecyl-amine;acetyl palmitate;glycerol ricinoleate;hexadecyl stearate;isopropyl myristate;tyloxapol;poly(ethylene glycol) 5000-phosphatidylethanolamine;poly(ethylene glycol) 400-monostearate;phospholipids;synthetic and / or natural detergents with high surfactant properties;deoxycholate;cyclodextrin;chaotropic salts;ion pairing agents;Glucose, fructose, galactose, ribose, lactose, sucrose, maltose, trehalose, cellobiose, mannose, xylose, arabinose, glucuronic acid, galacturonic acid, mannuronic acid, glucosamine, galactosamine, and neuraminic acid;Pullulan, cellulose, microcrystalline cellulose, silicified microcrystalline cellulose, hydroxypropyl methylcellulose (HPMC), hydroxycellulose (HC), methylcellulose (MC), dextran, cyclodextran, glycogen, hydroxyethyl starch, carrageenan, glycon, amylose, chitosan, N,O-carboxymethyl chitosan, algin and alginic acid, starch, chitin, inulin, konjac, glucomannan, pastulan, heparin, hyaluronic acid, curdlan, and xanthan, mannitol, sorbitol, xylitol, erythritol, maltitol, and lactitol, Pluronic polymers, polyethylene, polycarbonates (e.g., poly(1,3-dioxane-2-one) )), polyanhydrides (e.g., poly(sebacic anhydride)), polypropyl fumarate, polyamides (e.g., polycaprolactam), polyacetals, polyethers, polyesters (e.g., polylactides, polyglycolides, polylactide-co-glycolides), polycaprolactones, polyhydroxy acids (e.g., poly((β-hydroxyalkanoates))), poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polyureas, polystyrenes, and polyamines, polylysine, polylysine-PEG copolymers, and poly(ethyleneimine), poly(ethyleneimine)-PEG copolymers, glycerol monocaprylocaprate, propylene glycol, vitamin E. TPGS (also known as d-α-tocopheryl polyethylene glycol 1000 succinate), gelatin, titanium dioxide, polyvinylpyrrolidone (PVP), hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), methylcellulose (MC), block copolymers of ethylene oxide and propylene oxide (PEO / PPO), polyethylene glycol (PEG), sodium carboxymethylcellulose (NaCMC), or hydroxypropyl methylcellulose acetate succinate (HPMCAS);

[0159] In another embodiment, the fixed dose composition made of Compound 1 or a pharma- ceutically acceptable salt thereof and Compound 2 or a pharma- ceutically acceptable salt thereof comprises one or more of the following surfactants: polyoxyethylene glycol, polyoxypropylene glycol, decyl glucoside, lauryl glucoside, octyl glucoside, polyoxyethylene glycol octylphenol, Triton X-100, glycerol alkyl ester, glyceryl laurate, cocamide MEA, cocamide DEA, dodecyl dimethylamine oxide, and poloxamer. Examples of poloxamers include poloxamers 188, 237, 338, and 407. These poloxamers are available under the trade name Pluronic™ (available from BASF, Mount Olive, NJ), and correspond to Pluronic™ F-68, F-87, F-108, and F-127, respectively. Poloxamer 188 (corresponding to Pluronic™ F-68) is a block copolymer having an average molecular weight of about 7000 Da to about 10000 Da, or about 8000 Da to about 9000 Da, or about 8400 Da. Poloxamer 237 (corresponding to Pluronic™ F-87) is a block copolymer having an average molecular weight of about 6000 Da to about 9000 Da, or about 6500 Da to about 8000 Da, or about 7700 Da. Poloxamer 338 (corresponding to Pluronic™ F-108) is a block copolymer having an average molecular weight of about 12000 Da to about 18000 Da, or about 13000 Da to about 15000 Da, or about 14600 Da. Poloxamer 407 (corresponding to Pluronic™ F-127) is a polyoxyethylene-polyoxypropylene triblock copolymer having a ratio of about E101 P56 E101 to about E106 P70 E106, or about E101 P56E101, or about E106 P70 E106, and an average molecular weight of about 10,000 Da to about 15,000 Da, or about 12,000 Da to about 14,000 Da, or about 12,000 Da to about 13,000 Da, or about 12,600 Da.

[0160] In yet another embodiment, the fixed dose composition made from Compound 1 or a pharma- ceutically acceptable salt thereof and Compound 2 or a pharma-ceutically acceptable salt thereof comprises one or more of the following surfactants: polyvinyl acetate, sodium cholic acid salt, sodium dioctyl sulfosuccinate, hexadecyltrimethyl ammonium bromide, saponin, sugar ester, Triton. X series, sorbitan trioleate, sorbitan monooleate, polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (20) sorbitan monooleate, oleyl polyoxyethylene (2) ether, stearyl polyoxyethylene (2) ether, lauryl polyoxyethylene (4) ether, block copolymers of oxyethylene and oxypropylene, diethylene glycol dioleate, tetrahydrofurfuryl oleate, ethyl oleate, isopropyl myristate, glyceryl monooleate, glyceryl monostearate, glyceryl monoricinoleate, cetyl alcohol, stearyl alcohol, cetylpyridinium chloride, benzalkonium chloride, olive oil, glyceryl monolaurate, corn oil, cottonseed oil and sunflower seed oil.

[0161] In an alternative embodiment, the fixed dose composition made from Compound 1, or a pharma- ceutically acceptable salt thereof, and Compound 2, or a pharma- ceutically acceptable salt thereof, is made by a process that includes solvent or dry granulation, optionally followed by compression or consolidation, spray drying, nanosuspension processing, hot melt extrusion, extrusion / spheronization, molding, spheronization, layering (e.g., spray layering suspension or solution), and the like. Examples of such techniques include direct compression using suitable punches and dies (e.g., where the punches and dies are fitted to a suitable tablet press); wet granulation using suitable granulation equipment such as a high shear granulator to form wet particles that are dried into granules; granulation followed by compression using suitable punches and dies (where the punches and dies are fitted to a suitable tablet press); extrusion of the wet mass to form a cylindrical extrudate that is cut to the desired length or broken into lengths under gravity and attrition; extrusion / spheronization (where the extrudate is rolled into spherical particles and densified by spheronization); spray layering of a suspension or solution onto an inert core using techniques such as a convention pan or Wurster column; injection or compression molding using a suitable mold adapted for the compression unit, etc.

[0162] Exemplary disintegrants include alginic acid, calcium carboxymethylcellulose, sodium carboxymethylcellulose, cross-linked sodium carboxymethylcellulose (sodium croscarmellose), powdered cellulose, chitosan, croscarmellose sodium, crospovidone, guar gum, low substituted hydroxypropyl cellulose, methylcellulose, microcrystalline cellulose, sodium alginate, sodium starch glycolate, partially pregelatinized starch, pregelatinized starch, starch, sodium carboxymethyl starch, and the like, or combinations thereof.

[0163] Exemplary lubricants include calcium stearate, magnesium stearate, glyceryl behenate, glyceryl palmitostearate, hydrogenated castor oil, light mineral oil, sodium lauryl sulfate, magnesium lauryl sulfate, sodium stearyl fumarate, stearic acid, zinc stearate, silicon dioxide, colloidal silicon dioxide, dimethyldichlorosilane treated with silica, talc, or combinations thereof.

[0164] The dosage cores described herein may be coated to provide coated tablets. The dosage cores may be coated with functional or non-functional coatings, or a combination of functional and non-functional coatings. "Functional coatings" include tablet coatings that modify the release characteristics of the overall composition, such as sustained or delayed release coatings. "Non-functional coatings" include coatings that are not functional, such as cosmetic coatings. Non-functional coatings may have some effect on the release of the active agent, such as initial dissolution, hydration, perforation of the coating, etc., but are not considered to be a significant deviation from the uncoated composition. Non-functional coatings may also mask the taste of the uncoated composition containing the pharmacoactive ingredient. The coating may include a light-blocking material, a light-absorbing material, or a light-blocking material and a light-absorbing material.

[0165] Exemplary polymethacrylates include copolymers of acrylic and methacrylic acid esters, such as: a. amino methacrylate copolymers USP / NF such as poly(butyl methacrylate, (2-dimethylaminoethyl) methacrylate, methyl methacrylate) 1:2:1 (e.g., EUDRAGIT E 100, EUDRAGIT EPO, and EUDRAGIT E 12.5; CAS No. 24938-16-7); b. poly(methacrylic acid, ethyl acrylate) 1:1 (e.g., EUDRAGIT L30 D-55, EUDRAGIT L100-55, EASTACRYL 30D, KOLLICOAT MAE 30D, and 30DP; CAS No. 25212-88-8); c. poly(methacrylic acid, methyl methacrylate) 1:1 (e.g., EUDRAGIT L 100, EUDRAGIT L 12.5, and 12.5P; also known as methacrylic acid copolymers, Type A NF; CAS number 25806-15-1; d. poly(methacrylic acid, methyl methacrylate) 1:2 (e.g. EUDRAGIT S 100, EUDRAGIT S 12.5 and 12.5P; CAS number 25086-15-1); e. poly(methyl acrylate, methyl methacrylate, methacrylic acid) 7:3:1 (e.g. Eudragit FS 30 D; CAS number 26936-24-3); f. poly(ethyl acrylate, methyl methacrylate, trimethylammonioethyl methacrylate chloride) 1:2:0.2 or 1:2:0.1 (e.g. EUDRAGITS RL 100, RL PO, RL 30 D, RL 12.5, RS 100, RS PO, RS 30 D or RS 12.5; CAS No. 33434-24-1; g. Poly(ethyl acrylate, methyl methacrylate) 2:1 (e.g., EUDRAGIT NE 30 D, Eudragit NE 40D, Eudragit NM 30D; CAS No. 9010-88-2), and the like, or combinations thereof.

[0166] Suitable alkyl celluloses include, for example, methyl cellulose, ethyl cellulose, etc., or combinations thereof. Exemplary water-based ethyl cellulose coatings include AQUACOAT, a 30% dispersion that further includes sodium lauryl sulfate and cetyl alcohol, available from FMC, Philadelphia, Pennsylvania; SURELEASE, a 25% dispersion that further includes stabilizers or other coating ingredients (e.g., ammonium oleate, dibutyl sebacate, colloidal anhydrous silica, medium chain triglycerides, etc.), available from Colorcon, West Point, Pennsylvania; and Ethocel, available from Aqualon or Dow Chemical Co, Midland, Michigan. Those skilled in the art will appreciate that other cellulose polymers, including other alkyl cellulose polymers, can replace some or all of the ethyl cellulose.

[0167] Other suitable materials that can be used to make the functional coating include hydroxypropyl methylcellulose acetate succinate (HPMCAS); cellulose acetate phthalate (CAP); polyvinyl acetate phthalate; neutral or synthetic waxes, fatty alcohols (such as lauryl, myristyl, stearyl, cetyl, or specifically cetostearyl alcohol), fatty acid esters, fatty acids including fatty acid glycerides (mono-, di-, and tri-glycerides), hydrogenated fats and oils, hydrocarbons, regular waxes, stearic acid, stearyl alcohol, hydrophobic and hydrophilic materials with a hydrocarbon backbone, or combinations thereof. Suitable waxes include beeswax, glycowax, castor wax, carnauba wax, microcrystalline wax, candelilla, and wax-like substances, such as materials that are normally solid at room temperature and have a melting point of about 30° C. to about 100° C., or combinations thereof.

[0168] In other embodiments, the functional coating comprises a digestible long chain (e.g., C8-C 50 , specifically C 12 ~C 40) substituted or unsubstituted hydrocarbons, such as fatty acids, fatty alcohols, glyceryl esters of fatty acids, mineral and vegetable oils, waxes, or combinations thereof. Hydrocarbons having a melting point of about 25°C to about 90°C may be used. Specifically, fatty (aliphatic) alcohols, which are long-chain hydrocarbon materials, may be used.

[0169] The coating may optionally include additional pharma- ceutically acceptable excipients, such as plasticizers, stabilizers, water-soluble components (e.g., pore-forming agents), anti-tacking agents (e.g., talc), surfactants, and the like, or combinations thereof.

[0170] The functional coating may include a release modifier that affects the release characteristics of the functional coating. The release modifier may function, for example, as a pore former or matrix disintegrant. The release modifier may be organic or inorganic and includes materials that can be dissolved, extracted, or leached from the coating in the environment of use. The release modifier may include one or more hydrophilic polymers, including cellulose ethers and other cellulose compounds such as hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, methylcellulose, cellulose acetate phthalate, or hydroxypropyl methylcellulose acetate phthalate; povidone; polyvinyl alcohol; acrylic polymers such as gastrosoluble Eudragit FS 30D, pH sensitive Eudragit L30D 55, L 100, S 100, or L 100-55; or combinations thereof. Other exemplary release-modifying agents include povidone; saccharides (e.g., lactose, etc.); metal stearates; inorganic salts (e.g., dibasic calcium phosphate, sodium chloride, etc.); polyethylene glycols (e.g., polyethylene glycol (PEG) 1450, etc.); sugar alcohols (e.g., sorbitol, mannitol, etc.); alkali alkyl sulfates (e.g., sodium lauryl sulfate); polyoxyethylene sorbitan fatty acid esters (e.g., polysorbates); or combinations thereof. Exemplary matrix disintegrants include water-insoluble organic or inorganic materials. Organic polymers, including but not limited to cellulose, cellulose ethers such as ethyl cellulose, cellulose esters such as cellulose acetate, cellulose acetate butyrate, and cellulose acetate propionate, and starch, can function as matrix disintegrants. Examples of inorganic disintegrants include many calcium salts, such as monocalcium phosphate, dicalcium phosphate, and tricalcium phosphate, silica, and talc.

[0171] The coating agent may optionally contain a plasticizer to improve the physical properties of the coating. For example, since ethyl cellulose has a relatively high glass transition temperature and does not form a flexible film under normal coating conditions, it may be advantageous to add a plasticizer to the ethyl cellulose before use as a coating material. Generally, the amount of plasticizer included in the coating solution is based on the concentration of the polymer, and may be, for example, about 1% to about 200% depending on the polymer, but is often about 1% to about 100% by weight of the polymer. However, the concentration of the plasticizer can be determined by routine experimentation.

[0172] Examples of plasticizers for ethylcellulose and other celluloses include dibutyl sebacate, diethyl phthalate, triethyl citrate, tributyl citrate, triacetin, or combinations thereof, although other water insoluble plasticizers (acetylated monoglycerides, phthalates, castor oil, etc.) can be used.

[0173] Examples of plasticizers for acrylic polymers include citric acid esters such as triethyl citrate NF, tributyl citrate, dibutyl phthalate, 1,2-propylene glycol, polyethylene glycol, propylene glycol, diethyl phthalate, castor oil, triacetin, or combinations thereof, although other plasticizers (such as acetylated monoglycerides, phthalates, castor oil, etc.) can be used.

[0174] The coating material can be applied to the surface of the dosage form core using any suitable method, including processes such as simple or complex coacervation, interfacial polymerization, submerged drying, thermal and ionic gelation, spray drying, spray chilling, fluidized bed coating, pan coating, or electrostatic deposition.

[0175] In certain embodiments, an optional intermediate coating is used between the dosage form core and the outer coating. Such intermediate coatings can be used to protect the active agent or other components of the core subunit from the material used in the outer coating or to provide other properties. Exemplary intermediate coatings typically include water-soluble film-forming polymers. Such intermediate coatings may include film-forming polymers such as hydroxyethyl cellulose, hydroxypropyl cellulose, gelatin, hydroxypropyl methyl cellulose, polyethylene glycol, polyethylene oxide, and the like, or combinations thereof, and plasticizers. Plasticizers can be used to reduce brittleness and increase tensile strength and elasticity. Exemplary plasticizers include polyethylene glycol propylene glycol and glycerin.

[0176] Combination and Alternation Therapy Drug resistance may occur due to mutations in genes that code for enzymes used in viral replication. The efficacy of combination therapy against HCV infection may be extended, increased, or restored by adding additional compounds to the combination therapy. This additional combination therapy may be combined with or alternated with another (perhaps even two or three other) antiviral compound that induces different mutations or acts through a different pathway than the principle combination. Alternatively, the pharmacokinetics, biodistribution, half-life, or other parameters of the combination may be altered by such combination therapy (which may include alternation therapy if considered as coordinated).

[0177] The present invention already provides advantageous combination therapy for the treatment of HCV or disorders associated with HCV infection by administering a selected NS5B inhibitor together with an NS5A inhibitor. Additional therapeutic benefits can be achieved by adding a third, fourth, or even fifth active agent, either co-formulated or delivered separately.

[0178] Since compound 1 and compound 1-A are NS5B polymerase inhibitors and compound 2 is an NS5A inhibitor, compound 1 and compound 2 can be used in combination, for example, (1) Protease inhibitors (NS3 / 4A protease inhibitors, etc.), (2) another NS5A inhibitor, (3) another NS5B polymerase inhibitor, (4) NS5B non-substrate inhibitors (5) interferon alpha-2a, which may be pegylated or otherwise modified, and / or ribavirin; (6) non-substrate inhibitors, (7) helicase inhibitors, (8) antisense oligodeoxynucleotide (S-ODN), (9) aptamer, (10) nuclease-resistant ribozymes, (11) iRNA, including microRNA and siRNA, (12) An antibody, partial antibody, or domain antibody against a virus, or (13) A viral antigen or partial antigen that induces a host antibody response, It may be useful to administer to a host in combination with

[0179] Non-limiting examples of additional anti-HCV agents that may be further administered in combination or alternation with the combination of the present invention include: (i) protease inhibitors such as telaprevir (Incivec™), boceprevir (Victoris™), simeprevir (Olysio™), paritaprevir (ABT-450), glecaprevir (ABT-493), ritonavir (Norvir), ACH-2684, AZD-7295, BMS-791325, danoprevir, filibuster, GS-9256, GS-9451, MK-5172, setrobuvir, sovaprevir, tegobuvir, VX-135, VX-222, ALS-220 and voxilaprevir; (ii) NS5A inhibitors such as ACH-2928, ACH-3102, IDX-719, daclatasvir, resipasvir, velpatasvir (Epclusa), elbasvir (MK-8742), grazoprevir (MK-5172), and ombitasvir (ABT-267); (iii) NS5B inhibitors such as AZD-7295, clemizole, dasabuvir (Exviera), ITX-5061, PPI-461, PPI-688, sovosbuvir (Sovaldi™), MK-3682, and mericitabine; (iv) NS5B inhibitors such as ABT-333 and MBX-700, (v) antibodies such as GS-6624, (vi) Concomitant medications such as Harvoni (ledipasvir / sovosbuvir), Vikirapac (ombitasvir / paritaprevir / ritonavir / dasabuvir), Vikirax (ombitasvir / paritaprevir / ritonavir), G / P (paritaprevir and glecaprevir), Technivi™ (ombitasvir / paritaprevir / ritonavir), Epclusa (sovosbuvir / velpatasvir), Zepatia (elbasvir and grazoprevir), Maviret (glecaprevir and pibrentasvir), and Vosevi (sovosbuvir, velpatasvir and voxilaprevir); Examples include:

[0180] When the combination is administered to treat advanced Hepatitis C virus disease leading to liver cancer or cirrhosis, in one embodiment, the compound can be administered in combination or alternation with another drug typically used to treat hepatocellular carcinoma (HCC), e.g., as described by Andrew Zhu in "New Agents on the Horizon in Hepatocellular Carcinoma" Therapeutic Advances in Medical Oncology, V 5(1), (January 2013), 41-50. Examples of compounds suitable for combination therapy when the host has or is at risk for HCC include anti-angiogenic agents, sunitinib, brivanib, linifanib, ramucirumab, bevacizumab, cediranib, pazopanib, TSU-68, lenvatinib, antibodies against EGFR, mTor inhibitors, MEK inhibitors, and histone deacetylase inhibitors, capecitabine, cisplatin, carboplatin, doxorubicin, 5-fluorouracil, gemcitabine, irinotecan, oxaliplatin, topotecan, and other topoisomerase inhibitors.

[0181] Common methods 1 H, 19 F and 31 P NMR spectra were recorded on a 400 MHz Fourier transform Bruker spectrometer. Spectra were obtained in DMSO-d6 unless otherwise specified. Spin multiplicities are designated by the symbols s (singlet), d (doublet), t (triplet), m (multiplet) and br (broad). Coupling constants (J) are reported in Hz. Reactions were generally carried out under a dry nitrogen atmosphere using anhydrous solvents from Sigma-Aldrich. All common chemicals were purchased from commercial sources.

[0182] The following abbreviations are used in the examples: BID: Twice a day DCM: dichloromethane EtOAc: ethyl acetate EtOH: Ethanol GT: Genotype HPLC: High-performance liquid chromatography LD:Loading dose NaOH: Sodium hydroxide Na2SO4: Sodium sulfate (anhydrous) MeOH: Methanol Na2SO4: Sodium sulfate NH4Cl: Ammonium chloride PE: Petroleum ether Silica gel (230~400 mesh, Sorbent) t-BuMgCl: t-Butyl magnesium chloride THF: Tetrahydrofuran (THF), anhydrous TP: Triphosphate

[0183] Example 1. Synthesis of Compound 1 and Compound 1-A Part A: Synthesis of (2R,3R,4R,5R)-5-(2-amino-6-(methylamino)-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)-4-methyltetrahydrofuran-3-ol (1-7) [ka] In step 1, compound 1-1 is dissolved in DCM and the reaction is cooled to 10°C before adding benzyl chloroformate followed by NEt3. The reaction is warmed to room temperature and stirred for 12-14 hours. Compound 1-2 is isolated following appropriate work-up and purification conditions. In step 2, compound 1-2 is dissolved in acetonitrile and cooled to -15°C to 5°C before adding Morpho DAST. The reaction is stirred for 6 hours. Compound 1-3 is isolated following appropriate work-up and purification conditions. In step 3, compound 1-3 is dissolved in toluene and the reaction is cooled to 0°C to 10°C before adding Red Al. Compound 1-4 is isolated following appropriate work-up and purification conditions as a diastereomer with (R)-stereochemistry at the hydroxyl position. In step 4, compound 1-4 is dissolved in acetonitrile and cooled to -15°C to 5°C before adding CBr4 and PPh3. Compound 1-5 is isolated by following appropriate work-up and purification conditions. In step 5, compound 1-5 is dissolved in acetonitrile, and t-BuOH, t-BuOK, and 6-chloro-9H-purin-2-amine are added. The reaction is heated to 40°C to 50°C. Compound 1-6 is isolated by following appropriate work-up and purification conditions. In step 6, compound 1-6 is dissolved in MeOH, and MeNH2 is added. The reaction is heated to 20°C to 30°C. Compound 1-7 is isolated by following appropriate work-up and purification conditions.

[0184] Part B: Synthesis of dihydroquinine salts of isopropyl(hydroxy(phenoxy)phosphoryl)-L-alaninate (1-12) [ka] Phenyl dichlorophosphate (1-8, 150 g, 1.0 equiv.) was added to 1300 mL of isopropyl acetate. The solution was cooled to -10°C ± 5°C, and then a solution of benzyl alcohol (1-9, 80.6 g, 1.05 equiv.) and Et3N (86.3 g, 1.2 equiv.) was added. The mixture was stirred at -10°C ± 5°C for 3 h. The end point of the reaction was monitored by TLC.

[0185] L-alanine isopropyl ester hydrochloride (1-10, 125 g, 1.05 eq.) and EtN (152 g, 2.1 eq.) were added at −10° C.±5° C. The reaction mixture was stirred at −10±5° C. for 2 h. The end point of the reaction was monitored by TLC.

[0186] The reaction mixture was filtered and the filter cake was washed with 20 mL of isopropyl acetate. The filtrate was washed with 1N HCl, water, and aqueous sodium bicarbonate. The separated organic layer was dried over anhydrous Na2SO4 and then concentrated to dryness under vacuum at 40°C-50°C to give 240 g of crude product 1-11 as a diastereomeric mixture (approximately 1:1). (light yellow oil; yield: 89.6% (mol / mol); HPLC purity: 83.4 area %; HPLC assay: 86.2% w / w). The product contained around 6%-7% residual benzyl alcohol. The crude 1-4 was used directly in the next step.

[0187] Compound 1-11 (135 g, 1.0 equiv., assay 86.2%) and quinine (100 g, 1.0 equiv.) were added to 650 mL of i-PrOH. 5% Pd / C (19.2 g, 60% water by KF) was added and hydrogenation was carried out at 20°C-25°C for 8 hours in a closed system using a hydrogen bag. After completion of the reaction, the mixture was filtered through a Buchner funnel. The filtrate was concentrated under vacuum to remove the solvent.

[0188] To the above residue, 300 mL of TBME was added. The mixture was concentrated under vacuum at 40°C-45°C to remove the solvent, and the process was repeated with another 300 mL of MTBE. To the above, 600 mL of MTBE was added and the mixture was stirred at 40°C-45°C for 1 h, followed by another 1 h at 0°C-5°C. The mixture was filtered and the filter cake was washed with 100 mL of MTBE. The cake was dried at 45°C for 16 h without vacuum to give 152 g of dihydroquinine salt of isopropyl(hydroxy(phenoxy)phosphoryl)-L-alaninate (1-12, white solid; yield: 69.5% (mol / mol); HPLC purity: 97.91%).

[0189] Part C: Synthesis of Compound 1 [ka] The dihydroquinine salt of isopropyl(hydroxy(phenoxy)phosphoryl)-L-alaninate (1-12, 5.9 g, 1.5 eq.), compound 1-7 (2.0 g, 1.0 eq.), DIPEA (0.83 g, 1.0 eq.), and HATU (3.65 g, 1.5 eq.) were added to 100 mL of dichloromethane. The mixture was heated to 40° C. and stirred for 18 h. The reaction was monitored by TLC and HPLC.

[0190] After the reaction was completed, the reaction mixture was cooled to room temperature and washed with 1N hydrochloric acid (100 mL x 2), water (100 mL x 2), and 5% aqueous sodium bicarbonate solution (15 mL x 1). The separated organic phase was dried over 2 g of anhydrous sodium sulfate, filtered, and concentrated under vacuum at 40°C to 45°C to obtain a yellow oil.

[0191] Isopropyl acetate (10 mL) was added. After stirring, the mixture was concentrated under vacuum. Then, 25 mL of isopropyl acetate was added. The mixture was heated to 45° C. to obtain a clear solution. After stirring at room temperature for 2 h, the solid precipitate was filtered and dried at 45° C. for 15 h without vacuum to give 2.0 g of crude compound 1 (yield: 53.8% (mol / mol); HPLC purity: 93.1 area % (reverse R p The compound was obtained (containing 3.7% of the compound having the stereoselective configuration).

[0192] A mixture of crude compound 1 (2.0 g) and 15 mL of isopropyl acetate was heated to 80°C-85°C to obtain a solution. The solution was cooled to 20°C-25°C and stirred for 1 h. The precipitated solid was filtered, washed with isopropyl acetate (1 mL), and dried at 50°C for 16 h without vacuum to obtain 1.7 g of compound 1 (yield: 45.7% (mol / mol); HPLC purity: 98.99%). 1 H NMR spectrum, 19 F NMR spectrum, and 31 The structure of compound 1 was confirmed by P NMR spectrum.

[0193] Example 2. Characterization of amorphous Compound 1 and crystalline Compound 1 Amorphous Compound 1 and crystalline Compound 1 were first analyzed by XRPD, 1 The compounds were analyzed by HNMR and HPLC. The XRPD patterns for both compounds are shown in Figure 1A, and the HPLC traces for determining the purity are shown in Figures 1B and 2A, respectively. Table 1 lists the peaks from the XRPD of crystalline Compound 1, and Table 2 lists the relative retention times (RTT) from the HPLC trace. Amorphous Compound 1 was 98.61% pure, and crystalline Compound 1 was 99.11% pure. Both compounds were white solids. Figure 2B shows the TGA and DSC graphs of crystalline Compound 1. For crystalline Compound 1, an endotherm was observed at 88.6°C, and a mass loss of 7.8% was observed between 80°C and 110°C.

[0194] A sample of compound 1 was recrystallized from EtOAc / hexane and drawn using ORTEP. The absolute structure of compound 1 was confirmed by single crystal recrystallization. Figure 3 shows the ORTEP diagram of compound 1. The crystallographic and experimental data are shown in Table 3. The absolute stereochemistry of compound 1 based on X-ray crystallography is shown below: [ka]

[0195] DSC data were collected on a TA Instruments Q2000 equipped with a 50-position autosampler. Heat capacity calibration was performed using sapphire, and energy and temperature calibration was performed using certified indium. Typically, approximately 3 mg of each sample was heated from 25° C. to 200° C. at 10° C. / min in a pinhole aluminum pan. A purge of dry nitrogen at 50 ml / min was maintained over the samples. The instrument control software was Advantage for Q Series v2.8.0.394 and Thermal Advantage v5.5.3, and data were analyzed using Universal Analysis v4.5A.

[0196] TGA data were collected on a TA Instruments Q500 TGA equipped with a 16-position autosampler. The instrument was temperature calibrated using certified alumel and nickel. Typically, 5 mg to 10 mg of each sample was placed on a pre-tared aluminum DSC pan and heated at 10 °C / min from ambient to 350 °C. A nitrogen purge at 60 ml / min was maintained over the samples. The instrument control software was Advantage for Q Series v2.5.0.256 and Thermal Advantage v5.5.3, and data were analyzed using Universal Analysis v4.5.

[0197] Amorphous Compound 1 (1-1): 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.01 - 1.15 (m, 9 H), 1.21 (d, J=7.20 Hz, 3 H), 2.75 - 3.08 (m, 3 H), 3.71 - 3.87 (m, 1 H), 4.02 - 4.13 (m, 1 H), 4.22 - 4.53 (m, 3 H), 4.81 (s, 1 H), 5.69 - 5.86 (m, 1 H), 6.04 (br d, J=19.33 Hz, 4 H), 7.12 - 7.27 (m, 3 H), 7.27 - 7.44 (m, 3 H), 7.81 (s, 1H)

[0198] Crystalline compound 1(1-2): 1H NMR (400 MHz, DMSO-d6) δ ppm 0.97 - 1.16 (m, 16 H), 1.21 (d, J=7.07 Hz, 3 H), 2.87 (br s, 3 H), 3.08 (s, 2 H), 3.79 (br d, J=7.07 Hz, 1 H), 4.08 (br d, J=7.58 Hz, 1 H), 4.17 - 4.55 (m, 3 H), 4.81 (quin, J=6.25 Hz, 1 H), 5.78 (br s, 1 H), 5.91 - 6.15 (m, 4 H), 7.10 - 7.26 (m, 3 H), 7.26 - 7.44 (m, 3H), 7.81 (s, 1H)

[0199] TIFF2024525164000020.tif243170

[0200] TIFF2024525164000021.tif76170

[0201] TIFF2024525164000022.tif209170

[0202] Following this initial characterization, the samples were stored at 25° C. / 60% relative humidity (RH) for 14 days and analyzed by HPLC and XRPD after 7 and 14 days. Figure 4A shows the XRPD after 14 days at 25° C. / 60% (RH). Amorphous Compound 1 (Sample 1-1) was still poorly crystalline, whereas crystalline Compound 1 (Sample 1-2) retained its crystallinity, but both compounds were stable after 14 days at 25° C. / 60% (RH).

[0203] Example 3. Formation of oxalate compound 1-B Compound 1-B, the oxalate salt of compound 1, was first formed by mixing the oxalate salt with solvent (5 volumes, 100 μL) and allowing all the solution to evaporate at room temperature. Any suspension was allowed to mature (room temperature to 50 °C) for 3 h to assess the degree of crystallinity. [ka]

[0204] Table 4 shows the various solvents used in the production of compound 1-B. All solvents except two (cyclohexane and n-heptane) yielded crystalline products. Despite the high crystallinity and solubility of compound 1-B, the oxalate salt was not acceptable for clinical development due to the potential for kidney stone formation, and other salts of compound 1 were investigated.

[0205] TIFF2024525164000024.tif72170

[0206] Example 4. Amorphous Compound 1 Salt Compound Since oxalate Compound 1-B (Example 3) could not proceed in clinical trials due to its potential to form kidney stones, the amorphous salt of Compound 1 was formed using the counterions listed in Table 5. Compound 1 was dissolved in t-butanol (20 volumes, 6 ml) and the solution was treated with acid counterions (1 equivalent for each sample, except Sample 1-9, which had 0.5 equivalents of sulfate). The samples were then frozen and the solvent was removed by lyophilization. The residual solids in Samples 1-4, 1-5, 1-6, 1-7, 1-8, and 1-9 were first analyzed by XRPD and HPLC.

[0207] TIFF2024525164000025.tif119170

[0208] 1 HNMR spectra were collected for all samples.

[0209] Sample 1-4, HCl (1:1) salt: 1H NMR (400 MHz, DMSO-d6) δ ppm 0.93 - 1.39 (m, 16 H), 2.97 (br s, 2 H), 3.70 - 3.88 (m, 1 H), 4.10 (br s, 1 H), 4.18 - 4.49 (m, 3 H), 4.70 - 4.88 (m, 1 H), 5.71 - 5.94 (m, 1 H), 6.07 (br d, J=19.07 Hz, 2 H), 7.14 - 7.27 (m, 3 H), 7.29 - 7.44 (m, 2 H), 7.83 - 8.19 (m, 1 H)

[0210] Sample 1-5, sulfuric acid (1:1): 1 H NMR (400 MHz, DMSO-d6) δ ppm 0.97 - 1.38 (m, 15 H), 2.96 (br s, 2 H), 4.06 - 4.18 (m, 1 H), 4.19 - 4.49 (m, 3 H), 4.66 - 4.91 (m, 1 H), 5.70 - 5.95 (m, 1 H), 5.96 - 6.16 (m, 2 H), 7.10 - 7.27 (m, 3 H), 7.30 - 7.43 (m, 2 H), 7.88 - 8.19 (m, 1 H)

[0211] Sample 1-6, fluoric acid (1:1): 1H NMR (400 MHz, DMSO-d6) δ ppm 0.95 - 1.31 (m, 21 H), 2.87 (br s, 3 H), 3.79 (br d, J=7.20 Hz, 1 H), 4.01 - 4.13 (m, 1 H), 4.16 - 4.23 (m, 1 H), 4.16 - 4.24 (m, 1 H), 4.20 (s, 1 H), 4.18 - 4.23 (m, 1 H), 4.24 - 4.52 (m, 1 H), 4.24 - 4.52 (m, 1 H), 4.24 - 4.49 (m, 1 H), 4.72 - 4.88 (m, 1 H), 5.68 - 5.86 (m, 1 H), 6.04 (br d, J=19.33 Hz, 4 H), 6.63 (s, 1 H), 6.61 - 6.66 (m, 1 H), 7.12 - 7.27 (m, 3 H), 7.27 - 7.45 (m, 3 H), 7.81 (s, 1 H), 13.16 (br s, 2 H)

[0212] Sample 1-7, Benzoic acid (1:1) salt: 1 H NMR (400 MHz, DMSO-d6) δ ppm 0.96 - 1.30 (m, 15 H), 2.87 (br s, 3 H), 3.79 (br d, J=7.07 Hz, 1 H), 4.07 (br s, 1 H), 4.20 (s, 1 H), 4.25 - 4.52 (m, 3 H), 4.81 (s, 1 H), 5.71 - 5.85 (m, 1 H), 6.04 (br d, J=19.33 Hz, 4 H), 7.08 - 7.27 (m, 3 H), 7.27 - 7.43 (m, 3 H), 7.45 - 7.57 (m, 2H), 7.63 (s, 1H), 7.81 (s, 1 H), 7.95 (dd, J=8.27, 1.33 Hz, 2 H), 12.98 (br s, 1 H)

[0213] Sample 1-8, succinic acid (1:1) salt: 11H NMR (400 MHz, DMSO-d6) δ ppm 0.98 - 1.28 (m, 15 H), 2.42 (s, 5 H), 2.87 (br s, 3 H), 3.57 - 3.62 (m, 1 H), 3.70 - 3.86 (m, 1 H), 4.02 - 4.14 (m, 1 H), 4.20 (s, 1 H), 4.24 - 4.51 (m, 3 H), 4.70 - 4.88 (m, 1 H), 5.69 - 5.86 (m, 1 H), 6.04 (br d, J=19.33 Hz, 4 H), 7.12 - 7.27 (m, 3 H), 7.27 - 7.44 (m, 3 H), 7.81 (s, 1 H), 11.95 - 12.58 (m, 2 H)

[0214] Samples 1 - 9, sulfuric acid (0.5:1) salt: 1 1H NMR (400 MHz, DMSO-d6) δ ppm 1.02 - 1.31 (m, 15 H), 2.94 (br s, 3 H), 3.79 (br d, J=7.20 Hz, 2 H), 4.09 (br s, 1 H), 4.22 - 4.48 (m, 3 H), 4.72 - 4.90 (m, 1 H), 5.71 - 5.92 (m, 1 H), 6.07 (br d, J=19.07 Hz, 2 H), 7.12 - 7.28 (m, 3 H), 7.31 - 7.44 (m, 2 H), 7.75 - 8.19 (m, 1 H).

[0215] The samples were then stored at 25° C. / 60% relative humidity (RH) for 14 days and analyzed by HPLC and XRPD after 7 days (FIG. 4B) and 14 days (FIG. 5A). All prepared salts remained amorphous and the observations are shown in Table 6. The monosulfate salt (Sample 1-5) and succinate salt (Sample 1-8) were found to be physically unstable and deliquescent or gummed over the course of the study. The fumarate salt (Sample 1-6) and benzoate salt (Sample 1-7) were found to be glassy solids. The HCl salt (Sample 1-4) was found to retain its physical appearance. Surprisingly, the hemisulfate salt (Sample 1-9) also retained its physical appearance as a white solid in contrast to the monosulfate compound (Sample 1-5), which was a sticky gum. The results are shown in Table 6. The mono-HCl salt (sample 1-4) and the hemisulfate salt (sample 1-9) were found to be physically and chemically stable after two weeks of storage at 25° C. / 60% relative humidity (RH). Both salts were stable over the two week period, but the hemisulfate salt was superior to the HCl salt because the HCl salt is hygroscopic and therefore less useful for long term storage or use compared to the hemisulfate salt.

[0216] TIFF2024525164000026.tif78170

[0217] Example 5. Characterization of amorphous compound 1-A Amorphous compound 1-A was first analyzed by XRPD, 1 HNMR, DSC, TGA, and HPLC were used to analyze the amorphous compound 1-A. The XRPD patterns for amorphous compound 1 and amorphous compound 1-A overlaid with crystalline compound 1 are shown in FIG. 1A, and the XRPD pattern for amorphous compound 1-A alone is shown in FIG. 5B. Table 7 lists the peaks from the XRPD pattern shown in FIG. 5B. The HPLC trace for determining purity is shown in FIG. 6A. Table 8 lists the relative retention times (RTT) from the HPLC trace shown in FIG. 6A. Amorphous compound 1-A was 99.68% pure. FIG. 6B shows the TGA and DSC graphs of amorphous compound 1-A. Details regarding the TGA and DSC experiments are given in Example 2.

[0218] TIFF2024525164000027.tif29170

[0219] TIFF2024525164000028.tif50170

[0220] Amorphous Compound 1-A: 1 H NMR (400 MHz, DMSO-d6) δ ppm 0.93 - 1.29 (m, 13 H), 2.94 (br s, 3 H), 3.79 (td, J=10.04, 7.07 Hz, 2 H), 4.05 - 4.19 (m, 1 H), 4.19 - 4.50 (m, 3 H), 4.81 (quin, J=6.25 Hz, 1 H), 5.71 - 5.94 (m, 1 H), 5.97 - 6.16 (m, 2 H), 7.14 - 7.28 (m, 3 H), 7.31 - 7.44 (m, 2 H), 7.82 - 8.09 (m, 1 H)

[0221] Example 6. Crystallization of amorphous compound 1-A Since the hemisulfate salt was found to remain as a solid after the 14-day stability study as shown in Table 6, preliminary studies were conducted to study the crystallization conditions using 11 different solvents. Amorphous compound 1-A was suspended in 5 volumes of solvent at 25° C. (samples 2-1, 2-2, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, and 2-11). An additional 5 volumes of solvent was added to the non-flowable samples (2-1, 2-2, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, and 2-10). The samples were then aged at 25° C. to 50° C. (1° C. / min between temperatures and 4 hours at each temperature) for 6 days and allowed to evaporate under ambient conditions, except for sample 2-1, which was observed to be a clear solution after 1 day. The results are shown in Table 9. Crystalline patterns were generated from crystallization using isobutanol (sample 2-1), acetone (sample 2-2), EtOAc (sample 2-6), and iPrOAc (sample 2-7). Two difficult-to-crystallize samples were also identified from crystallization using MEK (sample 2-4) and MIBK (sample 2-5). The XRPD patterns are shown in Figure 7A.

[0222] TIFF2024525164000029.tif117170

[0223] Seven samples (samples 2-2, 2-3, 2-4, 2-5, 2-6, 2-7, and 2-8) were analyzed by DSC, TGA, and NMR after storage at 25°C / 60% relative humidity (RH) for 6 days. 1 The amorphous samples were analyzed by H-NMR and IC (Table 10, Figures 8A, 8B, 9A, 9B, 10A, 10B, 11A, and 11B) and by XRPD (all samples remained crystalline / poorly crystalline after stabilization). All samples retained approximately half the equivalent amount of sulfate salt but contained relatively large amounts of residual solvent. An overlay of the X-ray diffractograms of amorphous samples 2-9, 2-10, and 2-11 is shown in Figure 7B.

[0224] TIFF2024525164000030.tif151170

[0225] 1 HNMR spectra were collected for all samples and are listed below.

[0226] Sample 2-2: 1 H NMR (400 MHz, DMSO-d6) δ ppm 0.83 (d, J=6.69 Hz, 7 H), 0.99 - 1.26 (m, 14 H), 1.61 (dt, J=13.26, 6.63 Hz, 1 H), 3.73 - 3.87 (m, 2 H), 4.03 - 4.18 (m, 1 H), 4.18 - 4.51 (m, 4 H), 4.66 - 4.92 (m, 1 H), 4.70 - 4.90 (m, 1 H), 4.72 - 4.88 (m, 1 H), 5.81 (br s, 1 H), 5.93 - 6.11 (m, 2 H), 7.10 - 7.26 (m, 3 H), 7.14 - 7.26 (m, 1 H), 7.30 - 7.41 (m, 2 H), 7.94 (br s, 1 H)

[0227] Sample 2-3: 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.00 - 1.26 (m, 13 H), 2.09 (s, 3 H), 3.74 - 3.87 (m, 2 H), 4.10 (br d, J=7.70 Hz, 1 H), 4.22 - 4.50 (m, 3 H), 4.81 (quin, J=6.28 Hz, 1 H), 5.71 - 5.90 (m, 1 H), 5.96 - 6.15 (m, 2 H), 7.12 - 7.26 (m, 3 H), 7.31 - 7.41 (m, 2 H), 7.79 - 8.07 (m, 1 H)

[0228] Sample 2-4: 1 H NMR (400 MHz, DMSO-d6) δ ppm 0.91 (t, J=7.33 Hz, 3 H), 1.01 - 1.28 (m, 13 H), 2.08 (s, 2 H), 3.72 - 3.89 (m, 2 H), 4.10 (br d, J=8.08 Hz, 1 H), 4.23 - 4.47 (m, 3 H), 4.81 (quin, J=6.25 Hz, 1 H), 5.69 - 5.89 (m, 1 H), 5.94 - 6.13 (m, 2 H), 7.14 - 7.25 (m, 3 H), 7.32 - 7.41 (m, 2 H), 7.79 - 8.11 (m, 1 H)

[0229] Sample 2-5: 11H NMR (400 MHz, DMSO-d6) δ ppm 0.86 (d, J=6.69 Hz, 1 H), 0.98 - 1.33 (m, 13 H), 2.02 - 2.09 (m, 1 H), 4.03 - 4.17 (m, 1 H), 4.22 - 4.50 (m, 3 H), 4.81 (quin, J=6.25 Hz, 1 H), 5.81 (br s, 1 H), 5.93 - 6.15 (m, 2 H), 7.11 - 7.27 (m, 3 H), 7.31 - 7.41 (m, 2 H), 7.77 - 8.21 (m, 1 H)

[0230] Sample 2-6: 1 1H NMR (400 MHz, DMSO-d6) δ ppm 0.98 - 1.28 (m, 15 H), 2.00 (s, 3 H), 3.99 - 4.14 (m, 3 H), 4.21 - 4.49 (m, 3 H), 4.81 (quin, J=6.22 Hz, 1 H), 5.82 (br s, 1 H), 5.93 - 6.14 (m, 2 H), 7.11 - 7.26 (m, 3 H), 7.29 - 7.42 (m, 2 H), 7.79 - 8.17 (m, 1 H)

[0231] Sample 2-7: 1 1H NMR (400 MHz, DMSO-d6) δ ppm 0.92 - 1.28 (m, 17 H), 1.97 (s, 2 H), 4.04 - 4.16 (m, 1 H), 4.20 - 4.51 (m, 3 H), 4.71 - 4.93 (m, 2 H), 5.82 (br s, 1 H), 5.95 - 6.14 (m, 2 H), 7.11 - 7.28 (m, 3 H), 7.31 - 7.43 (m, 2 H), 7.75 - 8.21 (m, 1 H)

[0232] Sample 2-8: 1H NMR (400 MHz, DMSO-d6) δ ppm 0.81 - 1.11 (m, 13 H), 1.19 (s, 1 H), 1.53 - 1.66 (m, 1 H), 3.87 - 4.01 (m, 1 H), 4.06 - 4.32 (m, 3 H), 4.64 (quin, J=6.25 Hz, 1 H), 5.55 - 5.75 (m, 1 H), 5.77 - 5.97 (m, 2 H), 6.94 - 7.10 (m, 3 H), 7.13 - 7.26 (m, 2 H), 7.66 - 7.96 (m, 1 H)

[0233] Example 7. Inability to crystallize amorphous malonate salt (compound 1-B) As shown in Example 3, a crystalline oxalate salt was identified in determining a suitable salt for Compound 1, but the oxalate Compound 1-B could not be advanced in clinical trials due to its potential to cause kidney stones. Therefore, crystallization of the chemically related malonate salt (Compound 1-E) was attempted using the same 11 solvents as those for the hemisulfate salt. Compound 1 (12 x 50 mg, samples 3-1, 3-2, 3-3, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-11, and 3-12) was dissolved in t-butanol (20 volumes), and the solution was subsequently treated with 1 equivalent of malonic acid stock solution (1M in THF). The samples were then frozen and the solvent was removed by lyophilization. To samples 3-1, 3-2, 3-3, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, and 3-11, the relevant solvent (5 volumes) was added at room temperature. All the resulting solutions were allowed to evaporate under ambient conditions while the gums or solids were aged at 25° C. to 50° C. (1° C. / min between temperatures and 4 hours at each temperature) for 5 days. The solids were analyzed by XRPD (FIG. 12B) and all the samples were found to form gums or were amorphous (FIG. 12B). The results are shown in Table 11. One of the solid (amorphous) samples (3-12) was 1It was analyzed by H-NMR and HPLC and found to contain approximately 1 equivalent of malonic acid (peak overlap) and 0.6 equivalents of t-BuOH. The compound was 99.2% pure (Figure 13A). Figure 12A is the XRDP of sample 3-12 and Figure 13A is the HPLC chromatograph of sample 3-12.

[0234] Sample 3-12: 1 H NMR (400 MHz, DMSO-d6) δ ppm 0.81 - 1.11 (m, 13 H), 1.19 (s, 1 H), 1.53 - 1.66 (m, 1 H), 3.87 - 4.01 (m, 1 H), 4.06 - 4.32 (m, 3 H), 4.64 (quin, J=6.25 Hz, 1 H), 5.55 - 5.75 (m, 1 H), 5.77 - 5.97 (m, 2 H), 6.94 - 7.10 (m, 3 H), 7.13 - 7.26 (m, 2 H), 7.66 - 7.96 (m, 1 H)

[0235] TIFF2024525164000031.tif85170

[0236] Example 8. Inability to Form Proper Salt Using Liquid Assisted Grinding (LAG) Liquid assisted grinding (LAG) studies were performed using the 14 acidic counterions in Table 12 to determine suitable salts other than the hemisulfate salt.

[0237] TIFF2024525164000032.tif71170

[0238] Compound 1 (30 mg) was placed in an HPLC vial equipped with two 3 mm ball bearings. The material was wetted with solvent (15 μl of ethanol, samples 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-11, 4-12, 4-13, and 4-14) and 1 equivalent of acid counterion was added. The samples were then milled for 2 hours at 650 rpm using a Fritsch milling system equipped with an Automaxion adapter. Most of the samples after milling were found to be transparent gums and were not further analyzed (Table 13). Those that were observed to contain solids were analyzed by XRPD and in all cases the patterns obtained were found to match that of crystalline acid counterions without additional peaks (Figure 13B).

[0239] TIFF2024525164000033.tif77170

[0240] Example 9. Inability to obtain proper salt formation using methyl ethyl ketone (MEK) Next, suitable salts other than the hemisulfate salt were studied utilizing methyl ethyl ketone (MEK) as the solvent. The study was carried out by dissolving compound 1 (50 mg) in MEK (20 volumes) at room temperature using the 14 acidic counterions in Table 12. The solution was treated with 1 equivalent of the selected counterion (Table 12). The samples were then cooled to 5° C. at 0.1° C. / min and stirred at this temperature overnight. All samples were allowed to evaporate under ambient conditions and any solids observed were analyzed by XRPD. This evaporation mainly produced gums, except for the samples with stearic acid (samples 4-12) and palmitic acid (samples 5-13) which produced glassy solvents. These solids were amorphous by XRPD and no crystalline form of the salt was obtained. The results are shown in Table 14.

[0241] TIFF2024525164000034.tif146170

[0242] Since all samples were amorphous, all samples were redissolved in MEK (5 volumes) and cyclohexane was added at room temperature (20 volumes of antisolvent) followed by stirring at 25° C. for 1 hour. Samples were then aged from 50° C. to 5° C. (1° C. / min between temperatures, 4 hours at each temperature) for 2 days, after which the cycle was changed from 50° C. to 25° C. for an additional 4 days. After aging, samples were visually observed. Results are shown in Table 15. After aging, all samples except 5-1 (with pamoic acid) were found to be gums. Sample 5-1, a yellow solid, was analyzed by XRPD and the pattern was found to match the known form of pamoic acid (FIG. 14B), thus not yielding a crystalline form of the salt.

[0243] TIFF2024525164000035.tif107170

[0244] Example 10. Inability to obtain proper salt formation using ethyl acetate Ethyl acetate was then used to study suitable salts other than the hemisulfate salt. Using the 14 acidic counterions in Table 12, studies were performed by dissolving compound 1 (50 mg) in ethyl acetate (20 volumes) at 50° C. The solution was treated with 1 equivalent of the selected counterion (Table 12). The sample was then cooled to 5° C. at 0.1° C. / min and stirred at this temperature for 4 days. The solution was allowed to evaporate under ambient conditions and any solids were analyzed by XRPD. The results of crystallization using ethyl acetate are shown in Table 16. In contrast to Example 8, where MEK is the solvent, most of the samples were observed to be suspensions after cooling of the acid:compound mixture (those that were solutions were allowed to evaporate under ambient conditions). However, the XRPD diffractograms were generally found to be consistent with crystalline compound 1. Samples 6-2, 6-4, and 6-5 are somewhat slightly different (FIGS. 14A and 15A). No crystalline forms of the salt were obtained.

[0245] TIFF2024525164000036.tif132170

[0246] Example 11. Determination of Chemical Purity by HPLC Purity analyses in Examples 2 and 4 were carried out on an Agilent HP1100 series system equipped with a diode array detector using ChemStation software vB.04.03, using the method shown in Table 17.

[0247] TIFF2024525164000037.tif94170

[0248] Example 12. X-ray Powder Diffraction (XRPD) Techniques XRPD patterns in Examples 2, 3, 4, 5, 6, 7, 8, and 9 were collected on a PANalytical Empyrean diffractometer using Cu Kα radiation (45 kV, 40 mA) in transmission geometry. A 0.5° slit with a collecting mirror, a 4 mm mask, and a 0.4 rad Soller slit were used in the incident beam. A PIXcel placed on the diffracted beam 3D The detector was fitted with a receiving slit and a 0.04 rad Soller slit. The instrument was checked for performance weekly using silicon powder. The software used for data collection was X'Pert Data Collector v.5.3 and data was analysed and displayed using Diffrac Plus EVA v.15.0.0.0 or Highscore Plus v.4.5. Samples were prepared and analysed in transmission mode in either metal or Millipore 96-well plates. X-ray transparent film was used between the metal sheet on the metal well-plate and the powder (approximately 1 mg to 2 mg) was used neat. Using Millipore plates, solids were isolated from the suspension and analysed by adding a small amount of the suspension directly to the plate followed by filtration under slight vacuum.

[0249] The scan mode for the metal plate used the gonioscan axis, while for the Millipore plate, 2θ scan was utilized. Performance testing was performed using silicon powder (metal well-plate). Data collection details were: angular range 2.5°2θ to 32.0°2θ, step width 0.0130°2θ, and total collection time 2.07 min.

[0250] Samples were also collected on a Bruker D8 diffractometer using Cu Kα radiation (40 kV, 40 mA), a goniometer with θ-2θ, and V4 divergence and receiving slits, a Ge monochromator, and a Lynxeye detector. A certified corundum standard (NIST 1976) was used to check the performance of the instrument. The software used for data collection was DiffracPlus XRD Commander v2.6.1, and data were analyzed and displayed using Diffrac Plus EVA v15.0.0.0.

[0251] Using the as-is powder, samples were processed under ambient conditions as flat plate specimens. The samples were gently packed into cavities cut into polished, zero background (510) silicon wafers. The samples were rotated in their own plane during analysis. Data collection details were: angular range 2°2θ to 42°2θ, step width 0.05°2θ, and collection time 0.5 sec / step.

[0252] Example 13. Synthesis of amorphous compound 1-A [ka] In a 250 mL flask, MeOH (151 mL) was placed and the solution was cooled to 0°C-5°C. Concentrated H2SO4 solution was added dropwise over 10 min. In a separate flask, compound 1 (151 g) and acetone (910 mL) were placed and H2SO4 / MeOH solution was added dropwise over 2.5 h at 25°C-30°C. A large amount of solid precipitated. After stirring the solution at 25°C-30°C for 12-15 h, the mixture was filtered, washed with MeOH / acetone (25 mL / 150 mL) and dried in vacuum at 55°C-60°C to give compound 1-A (121 g, 74%). 1 HNMR: (400 MHz, DMSO-d6): δ 8.41 (br, 1H), 7.97 (s, 1H), 7.36 (t, J = 8.0 Hz, 2H), 7.22 (d, J = 8.0 Hz, 2H ), 7.17 (t, J = 8.0 Hz, 1H), 6.73 (s, 2H), 6.07 (d, J = 8.0 Hz, 1H), 6.00 (dd, J = 12.0, 8.0 Hz, 1H), 5.81(br, 1H), 4.84-4.73 (m, 1H), 4.44-4.28 (m, 3H), 4.10 (t, J = 8.0 Hz, 2H), 3.85-3.74 (m, 1H), 2.95 (s, 3H), 1.21 (s, J = 4.0 Hz, 3H), 1.15-1.10 (m, 9H).

[0253] Analytical method for compound 1-A: The purity of compound 1-A was measured using a Waters XTerra Phenyl 5μm 4.6 * An Agilent 1100 HPLC system equipped with a 250 mm column was used to obtain the following conditions: flow rate 1 mL / min, reading at 254 nm, column temperature 30° C., injection volume 10 μL, run time 30 min. Samples were dissolved in ACN:water (90:10, v / v). The gradient method for separation is shown below. R t (min) was approximately 12.0 min.

[0254] TIFF2024525164000039.tif24170

[0255] Example 14. Characterization of Compound 1-A Compound 1-A was visually 1 HNMR, 13 CNMR, 19 Further characterization was performed by FNMR, MS, HPLC, and XRPD (Figure 15B). Residual solvent was measured by GC. Water content was measured by Karl Fischer titration, and was only 0.70%. The data are summarized in Table 18.

[0256] TIFF2024525164000040.tif72170

[0257] Example 15. Solubility of Compound 1 and Compound 1-A Both Compound 1 and Compound 1-A were tested for solubility in biorelevant test media including simulated gastric fluid (SGF), fasted state simulated gastric fluid (FaSSIF), and fed state gastric fluid (FeSSIF). Results for Compound 1 are shown in Table 19, and results for Compound 1-A are shown in Table 20. Samples were stirred at room temperature (20°C-25°C). Compound 1-A was over 40 times more soluble than Compound 1 in water at 2 hours and over 25 times more soluble at 24 hours. In SGF conditions, Compound 1 had a solubility of 15.6 mg / mL at 24 hours, while Compound 1-A had a solubility of 84.2 mg / mL at the same time point. Compound 1-A was also more soluble than Compound 1 at 2 hours in SGF conditions, and was sufficiently soluble even after 48 hours to allow experiments, whereas experiments at 48 hours were not performed with Compound 1.

[0258] TIFF2024525164000041.tif86170

[0259] TIFF2024525164000042.tif73170

[0260] Example 16. Chemical stability of compound 1-A Organic purity, water content, 1Compound 1-A was tested for chemical stability at 25°C and 40°C over a period of 6 months by monitoring HNMR, DSC, and Raman IR. The container closure system for the study was a composite medical valve bag with a pharmaceutical laminate film on the pouch and desiccant silica gel between the two layers. Compound 1-A (1 g) was weighed into each container. The bags were then stored at 25°C / 60% RH (relative humidity) and 40°C / 75% RH (relative humidity). Organic purity, water content, 1 HNMR, DSC and Raman were measured at time 0, 1 month, 2 months, 3 months and 6 months.

[0261] The purity of compound 1-A was obtained using a Shimadzu LC-20AD system equipped with a Waters XTerra Phenyl, 5 μm, 4.6×250 mm column under the following conditions: flow rate 1 mL / min, read at 254 nm, column temperature 35° C., and injection volume 10 μL. Samples were dissolved in acetonitrile-water (90:10) (v / v). The gradient method is shown below.

[0262] TIFF2024525164000043.tif24170

[0263] The water content of compound 1-A (250 mg) was determined by a water titrator using the Karl Fischer titration method.

[0264] The results are shown in Tables 21 and 22. Compound 1-A had minimal decomposition rates when stored at 25°C and 40°C for 6 months. At 3 months, Compound 1-A was 99.75% pure at 25°C and 99.58% pure at 40°C. At 6 months, Compound 1-A was still 99.74% pure at 25°C and 99.30% pure at 40°C. At 25°C, the percentage of decomposition products increased from 0.03% at day 0 to 0.08% after 6 months. At 40°C, the percentage of decomposition products increased from 0.03% to 0.39%. Over 6 months, the percentage of water increased by approximately 0.6% at 25°C and approximately 0.7% at 40°C.

[0265] Compound 1-A at 1, 2, 3, and 6 months 1 H NMR, Raman, and DSC characterization was identical to that of compound 1-A at day 0 at both temperature conditions (Table 22), highlighting the long-term stability of compound 1-A.

[0266] TIFF2024525164000044.tif105170

[0267] TIFF2024525164000045.tif101170

[0268] Further chemical stability studies of compound 1-A were measured to determine the impurity and water levels. Three conditions were tested: accelerated stability (40°C ± 2°C / 75% ± 5% RH) for 6 months, ambient stability (25°C ± 2°C / 60% ± 5% RH) for 9 months, and stability under refrigerated conditions (5°C ± 3°C) for 9 months. The results for accelerated stability, ambient stability, and refrigerated conditions are shown in Table 23, Table 24, and Table 25, respectively. Based on the results of these studies, compound 1-A is very chemically stable.

[0269] In the accelerated stability study (Table 23), at each time point Compound 1-A was measured (1 month, 3 months, and 6 months), the appearance of Compound 1-A was always a white solid, and the IR matched the reference standard. After 6 months, the total related substance 1 impurity was only 0.08%, and related substance 2 and isomers were not detected.

[0270] TIFF2024525164000046.tif149170

[0271] In an ambient stability study that measured appearance, IR, water and impurity levels over a 9-month period, Compound 1-A always appeared as a white solid and the IR always matched the reference sample. The results (Table 24) highlight how chemically stable Compound 1-A is. After 9 months, the percentage of water in the sample was only 0.20% and the total Related Substance 1 impurity was only 0.02%. As with the accelerated stability study, Related Substance 2 and any isomers of Compound 1-A were not detected.

[0272] TIFF2024525164000047.tif192170

[0273] Stability measurements under refrigerated conditions are shown in Table 25. Even after 9 months, the only impurities detected were from Related Substance 1 and water. After 9 months, the water content was 0.32%, and the total Related Substance 1 impurity made up only 0.01% of the sample. Compound 1-A is very chemically stable under refrigerated conditions.

[0274] TIFF2024525164000048.tif192170

[0275] Example 17. Plasma levels of metabolites following a single oral dose of Compound 1-A A single oral dose of compound 1-A was administered to rats, dogs, and monkeys and the plasma levels of certain metabolites shown in Scheme 1 were measured.

[0276] The conversion of compound 1-A to compound 1 and metabolite 1-7 is shown in Table 26, and the results for metabolites 1-8 and 1-2 are shown in Table 27. In rats, low levels of compound 1 exposure were observed, but high levels of metabolite 1-7, a nucleoside metabolite of the active triphosphate (metabolite 1-6), were observed. In monkeys, nearly dose-proportional exposure to compound 1 was measured. In dogs, more than proportional exposure to compound 1 was measured, indicating first-pass metabolic clearance in the liver. More significant vomiting was observed in dogs (5 / 5 of the high-dose group) than in monkeys (1 / 5 of the high-dose group) throughout the study.

[0277] TIFF2024525164000049.tif67170

[0278] TIFF2024525164000050.tif62170

[0279] Example 18. Tissue exposure of active triphosphate following an oral dose of compound 1-A Heart and liver tissue levels of active triphosphate (TP) (metabolites 1-6) of compound 1-A were measured 4 hours after an oral dose of compound 1-A. Liver and heart samples were taken 4 hours after a single dose of compound 1-A, flash frozen, homogenized, and analyzed by LC-MS / MS for intracellular levels of active TP. As shown in Figure 16A, tissue levels were measured in rats, dogs, and monkeys. High levels of active TP were measured in the liver of all species tested. Relatively low levels of active TP were measured in dog hearts due to saturation of initial hepatic metabolism, and unquantifiable levels of TP were measured in rat and monkey hearts, indicating liver-specific formation of active TP. Although not shown, administration of compound 1-A improved TP distribution compared to administration of compound 1.

[0280] Example 19. Pharmacological comparison of Compound 1 and Compound 1-A in dogs A direct comparison was made between dogs administered Compound 1 and Compound 1-A. The study measured plasma levels of Compound 1 and metabolites 1-7 (from Scheme 1) 4 hours after administration of Compound 1 (25 mg / kg) and Compound 1-A (30 mg / kg) (Table 28), and the AUC (0時間~4時間) was approximately 2-fold greater with Compound 1-A compared to Compound 1. Dose-normalized exposures for Compound 1 and metabolites 1-7 are shown in Table 28. AUC for Compound 1, Metabolites 1-7, and the sum of Compound 1 + Metabolites 1-7 (0時間~4時間) Values ​​for were higher after administration of compound 1-A.

[0281] TIFF2024525164000051.tif50170

[0282] As shown in Table 29, the liver / heart ratios of triphosphate concentrations indicate that treatment with Compound 1-A increases the selective delivery of triphosphate to the liver when compared to Compound 1. AUC of active guanine metabolite (1-6) after administration of Compound 1 measured in the heart (0時間~4時間) is 174μM * AUC of active guanine metabolite (1-6) measured in the heart after administration of compound 1-A versus time. (0時間~4時間) is 28μM * The liver / heart ratio for compound 1 was 3.1, whereas the liver / heart ratio for compound 1-A was 20.

[0283] TIFF2024525164000052.tif38170

[0284] The effect of increased selectivity for liver over heart when compound 1-A is administered compared to compound 1 is also shown in Figure 16B. Heart and liver tissue levels of active triphosphate after administration of compound 1-A (30 mg / kg) were compared to tissue levels of active triphosphate after administration of compound 1 (25 mg / kg). Although the concentration of active TP was higher in liver than heart for both compound 1 and compound 1-A, active TP was more selective for liver than heart when compound 1-A was administered compared to compound 1.

[0285] Example 20. Plasma profile of metabolites of compound 1-A in rats and monkeys Male Sprague-Dawley rats and cynomolgus monkeys (3 animals per dose) were given a single oral dose of Compound 1-A. Aliquots of plasma prepared from dichlorvos-treated blood samples were analyzed by LC-MS / MS for concentrations of Compound 1 and metabolites 1-7 (nucleoside metabolites of the active triphosphate of Compound 1-A shown in Scheme 1), and pharmacokinetic parameters were determined using WinNonlin. Results for a single 500 mg / kg dose in rats are shown in Figure 17, and results for a single 30 mg / kg, 100 mg / kg, or 300 mg / kg dose in monkeys are shown in Figure 18. Results are also summarized in Table 30.

[0286] The high plasma levels of metabolites 1-7, nucleoside metabolites of the active triphosphate (TP) of compound 1-A, indicate the formation of high levels of TP even in rats where very low plasma levels of the parent nucleotide prodrug are observed due to the short half-life (<2 min) of compound 1 in rat blood. The sustained plasma levels of metabolites 1-7 reflect the long half-life of TP.

[0287] In monkeys, plasma exposure (AUC) of compound 1 was approximately dose proportional, whereas exposure to metabolites 1-7 was not nearly dose proportional, although AUC values ​​for both the parent drug and the active nucleoside metabolite of TP continued to increase up to the highest dose tested (300 mg / kg).

[0288] Oral administration of compound 1-A in rats and monkeys resulted in high and dose-dependent plasma exposure to metabolite 1-7, a nucleoside metabolite of the intracellularly active triphosphate of compound 1-A, and metabolite 1-7 exposure continued to increase up to the highest dose tested, reflecting substantial formation of active TP in these species.

[0289] TIFF2024525164000053.tif64170

[0290] Example 21. Effect of the active triphosphates of Compound 1 and Compound 1-A on mitochondrial integrity The relative efficiency of incorporation of the active triphosphates (TP) of Compound 1 and Compound 1-A, metabolites 1-6 (Scheme 1) by human mitochondrial RNA polymerase was compared to the relative efficiency of the active TP of sovosbuvir and the active TP of INX-189. Compound 1 and Compound 1-A are unlikely to affect mitochondrial integrity because their active triphosphates are poorly incorporated by human mitochondrial RNA polymerase with an efficiency similar to that of the triphosphate of sovosbuvir, and the relative efficiency of incorporation of the triphosphate of INX-189 was up to 55-fold higher. The results are shown in Table 31. Incorporation of these analogs by human mitochondrial RNA-dependent polymerase (POLRMT) was determined according to Arnold et al. (Sensitivity of Mitochondrial Transcription and Resistance of RNA Polymerase II Dependent Nuclear Transcription to Antiviral Ribonucleotides. PLoS Pathog., 2012, 8, e1003030).

[0291] TIFF2024525164000054.tif83170

[0292] Example 22. Activity of Compound 1 against replicons containing NS5B sequences The potency of compound 1 and sofosbuvir was determined using a panel of replicons containing NS5B sequences from various HCV genotypes obtained from six laboratory reference strains (GT1a, 1b, 2a, 3a, 4a and 5a) (Figure 19) and from eight HCV patient plasma samples (GT1a, 1b, 2a, 2b, 3a-1, 3a-2, 4a and 4d) (Figure 20).

[0293] Compound 1 was more potent than sofosbuvir against clinical and laboratory strains of HCV. Compound 1 had an EC 95Compound 1 showed potent pan-genotypic antiviral activity in vitro against wild-type clinical isolates, which was 4- to 14-fold more potent than sofosbuvir. As shown in FIG. 95 The EC values ​​for compound 1 were 7-33 fold lower than those for sofosbuvir against clinical isolates of all HCV genotypes tested. 50 Values ​​were 6-11 fold lower than sovosbuvir against laboratory strains of HCV genotypes 1-5 (Figure 19).

[0294] Example 23. Single Ascending Dose (SAD) Study of Compound 1-A in Healthy Volunteers (Part A) and GT1-HCV Infected Patients (Part B) Compound 1-A was tested in a single ascending dose (SAD) study to determine its safety, tolerability, and pharmacokinetics in healthy subjects (part A). Part A was a randomized, double-blind, placebo-controlled SAD study. Healthy subjects in part A were given a single dose of compound 1-A or placebo in the fasting state. Subjects were confined to the clinic from day -1 to day 6.

[0295] Dosing in each cohort was adjusted so that two subjects (1 active: 1 placebo) were evaluated 48 hours after dosing, followed by dosing for the remainder of the cohort. Each cohort received Compound 1-A in ascending order. Dosing of subsequent cohorts was based on review of available safety data (up to day 5) and plasma pharmacokinetic data (up to 24 hours) from previous cohorts.

[0296] Dose escalation was initiated following thorough review of these data. Upon availability of pharmacokinetic and safety data from previous cohorts, doses evaluated in cohorts 3a-4a were adjusted in increments of 100 mg or less. The total maximum dose evaluated in Part A did not exceed 800 mg. The dosing regimen for Part A is shown in Table 32.

[0297] TIFF2024525164000055.tif39170

[0298] Healthy volunteers in Part A of the study were male and female subjects aged 18 to 65 years. Active and placebo recipients were pooled within each Part A cohort to maintain study blinding.

[0299] Compound 1-A was also tested in a single ascending dose (SAD) study to determine its safety, tolerability, pharmacokinetics, and antiviral activity in GT1-HCV infected patients (Part B). Subjects in Part B received a single dose of Compound 1-A in the fasting state. Patients were confined to the clinic from day -1 to day 6.

[0300] Part B began after review of safety data (through Day 5) and plasma pharmacokinetic data (through 24 hours) from Cohort 3a in Part A. Subsequent Part B cohorts were enrolled after review of available safety data (through Day 5) and plasma pharmacokinetic data (through 24 hours) for the first cohort in Part B (Cohort 1b). Subsequent Part B cohorts were dosed only after review of available safety and pharmacokinetic data from each dose in Part A and available safety data (through Day 5) from the prior Part B cohort.

[0301] Dose escalation up to 600 mg in HCV-infected patients was initiated after full consideration of these data. The dosing regimen for Part B is shown in Table 33.

[0302] TIFF2024525164000056.tif39170

[0303] Patients infected with HCV have a 5log 10 Treatment-naïve, non-cirrhotic, GT1-infected subjects were included, with viral loads of ≥ 1 IU / mL.

[0304] No serious adverse events were recorded and no premature discontinuation was required in either Part A or Part B. All adverse events were mild to moderate in intensity, and no dose-related pattern was evident, including in laboratory parameters, vital signs, and ECGs.

[0305] Example 24. Results of a Single Ascending Dose (SAD) Study of Compound 1-A The pharmacokinetics of compound 1 and nucleoside metabolites 1-7 were measured after a single dose of compound 1-A. C 24 Trough plasma concentration (C 24時間 ) was 25.8 ng / mL, which is more than twice the plasma concentration dose after a 300 mg dose of compound 1-A. Metabolite 1-7 (shown in Scheme 1) can only be generated by dephosphorylation of the intracellular phosphate metabolite 1-4, metabolite 1-5, and the active species, metabolite 1-6. Therefore, metabolite 1-7 can be considered as a surrogate for the active species. Pharmacokinetic data for all cohorts are shown in Tables 34 and 35. Values ​​are reported as mean ± SD, except for T, where median (range) is reported. max Pharmacokinetic parameters were comparable in healthy and HCV-infected patients.

[0306] TIFF2024525164000057.tif113170

[0307] TIFF2024525164000058.tif123170

[0308] The mean plasma concentration-time profiles of Compound 1 and metabolites 1-7 were also calculated for all cohorts in Part A and Part B of the study. Figure 21 shows the mean plasma concentrations of Compound 1 after a single dose of Compound 1-A, and Figure 22 shows the mean plasma concentrations of metabolites 1-7 after a single dose of Compound 1-A. As shown in Figure 21, Compound 1 was rapidly absorbed and rapidly / extensively metabolized in all cohorts from Part B. As shown in Figure 22, metabolites 1-7 were the major metabolites and showed sustained plasma concentrations. Plasma exposure of Compound 1 was dose-related, whereas exposure of metabolites 1-7 was dose-proportional.

[0309] For HCV-infected subjects in Part B, HCV RNA quantification measurements were performed before, during, and after dosing with Compound 1-A. Plasma HCV RNA determinations were performed using a validated commercially available assay. Baseline was defined as the mean of days -1 and 1 (pre-dose). A single 300 mg dose of Compound 1-A (equivalent to 270 mg of Compound 1) provided significant antiviral activity in GT1b-HCV-infected subjects. The mean maximum HCV RNA reduction 24 hours after dosing following a single 300 mg dose was 1.7 log 10 IU / mL, which represents -2log 1 day after 400 mg sovosbuvir monotherapy in GT1a HCV-infected subjects 10 The mean maximum HCV RNA reduction 24 hours after administration following a single 100 mg dose was 0.8 log 10 IU / mL. The mean maximum HCV RNA reduction after a single 400 mg dose was 2.2 log 10 IU / mL. Individual pharmacokinetic / pharmacodynamic analyses for individual subjects from Part B of the study are shown in Figures 23A-F. Metabolite 1-7 concentrations were plotted against HCV RNA reduction concentrations, and as shown in Figures 23A-F, plasma HCV RNA reduction correlates with plasma metabolite 1-7 exposure. Viral responses were measured using EC 95The plasma concentrations of metabolite 1-7 were sustained at values ​​greater than 0.01 and 0.02, respectively. The correlation between plasma concentrations and HCV RNA reduction levels indicates that higher responses are achievable with higher doses of compound 1-A.

[0310] Example 25. Predicted steady-state trough levels of metabolites 1-7 correlate with the EC of Compound 1 against clinical isolates of HCV GT1-4 95 Exceeds the value As shown in FIG. 24, the steady-state trough plasma levels (C) of metabolites 1-7 following administration of compound 1-A in humans (600 mg QD (equivalent to 550 mg of free base) and 450 mg QD (equivalent to 400 mg of free base)). 24,ss ) predicted the in vitro EC of compound 1 against all clinical isolates tested. 95 Compared with the EC 95 The EC for compound 1 was determined to be consistently higher than that of compound 2 and to provide high efficacy against any or all clinical isolates tested in vivo. 95 is the EC of compound 1-A 95 For compound 1-A to be effective, steady-state trough plasma levels of metabolites 1-7 must be greater than or equal to the EC 95 It should exceed this.

[0311] As shown in Figure 24, the EC 95 was in the range of approximately 18 nM to 24 nM.

[0312] As shown in FIG. 24, Compound 1-A at a dose of 450 mg QD (equivalent to 400 mg of the free base) in humans increased steady-state trough plasma concentrations (C 24,ss A dose of 600 mg QD (equivalent to 550 mg of the free base) of Compound 1-A in humans results in a steady-state trough plasma concentration (C 24,ss ) yielding approximately 50 ng / mL.

[0313] Thus, steady-state plasma concentrations of the surrogate metabolites 1-7 were nearly twice as high as the EC 95 and shows excellent performance.

[0314] In contrast, the EC 95 The EC ranged from 50 nM to 265 nM against all HCV clinical isolates tested. 95 is less than the predicted steady-state concentration at the 400 mg commercial dose for only two isolates, GT2a and GT2b. EC 95 is higher than the predicted steady-state concentrations for the other clinical isolates GT1a, GT1b, GT3a, GT4a, and GT4d.

[0315] Steady-state trough plasma concentration (C 24,ss ) at steady-state trough plasma concentration (C 24,ss ) was used to predict the mean steady-state trough plasma concentration at 300 mg (C 24,ss ) is 26.4 ng / mL, so the calculated value is 26.4 * 450 / 300=39.6ng / mL.

[0316] 600mg steady-state trough plasma concentration (C 24,ss ) was predicted using three approaches: 1) C at 600 mg day 1 24 The mean value was 25.8 ng / mL, and a 60% increase was assumed to reach steady state. Thus, the calculated value was 25.8 * 1.6 = 41.3 ng / mL; 2) C on day 1 at 400 mg 24 The mean value was 22.5 ng / mL, with a 60% increase assumed to reach steady state. Considering dose-proportional PK, the calculated value was 22.5 * 1.6 * 600 / 400 = 54 ng / mL; and 3) the steady-state trough plasma concentration (C 24,ss) was 26.4 ng / mL and proportional PK was assumed. Therefore, the calculated value was 26.4 * The steady-state trough plasma concentration (C 24,ss ) is the average of three data points ((41.3 + 54 + 52.8) / 3 = 49.3 ng / mL). C after a single dose 24 In comparison, C at steady state is generally 24 There has been an increase of about 60% in

[0317] The data comparing efficacy and pharmacokinetic steady-state parameters in Figure 24 demonstrate the unexpected therapeutic importance of compound 1-A for the treatment of Hepatitis C. Indeed, the predicted steady-state plasma levels following administration of compound 1-A were greater than EC 95 The EC50 of sofosbuvir at GT1, GT3, and GT4 is predicted to be at least 2-fold higher than that at GT1 and 3-5-fold more potent against GT2. This data indicates that compound 1-A has potent pan-genotypic antiviral activity in humans. As shown in Figure 24, the EC50 of sofosbuvir at GT1, GT3, and GT4 is 95 is greater than 100 ng / mL. Thus, surprisingly, compound 1-A is active against HCV in a formulation that delivers a lower steady-state trough concentration (40 ng / mL to 50 ng / mL) than the steady-state trough concentration (approximately 100 ng / mL) achieved by sovosbuvir in a similar formulation.

[0318] Example 26. Three-part study evaluating the safety / tolerability, pharmacokinetics (PK), and antiviral activity of compound 1-A A three-part study was conducted with compound 1-A to evaluate safety / tolerability, pharmacokinetics (PK), and antiviral activity. The three parts included 1) multiple dosing of up to 600 mg compound 1-A (equivalent to 550 mg compound 1) once daily (QD) for 7 days in NC (non-cirrhotic) GT1 HCV-infected patients (Part C); 2) dosing of 600 mg compound 1-A (equivalent to 550 mg compound 1) QD for 7 days in NC GT3 HCV-infected patients (Part D); and 3) dosing of 600 mg compound 1-A (equivalent to 550 mg compound 1) QD for 7 days in a cohort of Child-Pugh A (CPA) cirrhotic patients with either GT1, GT2, or GT3 HCV infection (Part E). Doses were administered as compound 1-A salt basis. The equivalent amount of free base compound 1 is often indicated in parentheses.

[0319] Part C was a randomized, double-blind, placebo-controlled MAD study divided into three cohorts. Subjects received 150 mg, 300 mg, or 600 mg of Compound 1-A or placebo in a fasted state for 7 days. Dose escalation was initiated only after full review of the data. Parts D and E were open-label studies in which patients received 600 mg of Compound 1-A (equivalent to 550 mg of Compound 1) in a fasted state for 7 days.

[0320] HCV-infected patients had HCV RNA ≥ 5log 10The patients were treatment-naïve with a mean HCV RNA of 15 IU / mL. HCV RNA was quantified using COBAS™ AmpliPrep TaqMAN™ v2.0 with a LLQ of 15 IU / mL. Plasma drug levels were measured using LC-MS / MS. Baseline HCV RNA averaged >6 log in all cohorts of patients receiving 500 mg of Compound 1-A. Cirrhosis was confirmed by prior liver biopsy or fibroscan >12.5 kPa. Mean baseline fibroscans were 6.3 kPa, 6.8 kPa, and 17.6 kPa in patients receiving 600 mg equivalent of Compound 1-A in Parts C, D, and E, respectively. The mean ages of enrolled subjects were 44, 39, and 56 years in the non-cirrhotic GT1b 600 mg dose cohort, non-cirrhotic GT3 cohort, and cirrhosis cohort, respectively.

[0321] Parts A and B were previously conducted and described in WO 2018 / 144640. Parts A and B were single ascending dose (SAD) studies. In Part A, healthy subjects received up to 400 mg of Compound 1-A (equivalent to 367 mg of Compound 1), and in Part B, GT1 NC HCV-infected subjects received a single dose of up to 600 mg of Compound 1-A (equivalent to 550 mg of Compound 1).

[0322] Example 27. Results of the study of compound 1-A No severe adverse events (AEs), dose-limiting toxicities, or premature discontinuations were reported. Compound 1-A was well tolerated over 7 days up to the highest dose tested (600 mg salt form). The only pattern observed was a higher incidence of primarily low-grade lipid abnormalities (increased cholesterol and triglycerides) in subjects receiving Compound 1-A compared to placebo. However, this observation is consistent with previously published data showing that lipids increase rapidly with HCV clearance when DAA therapy is initiated in HCV-infected subjects. In addition, no findings suggestive of liver injury were observed. ALT / AST values ​​decreased over time during the treatment period in subjects receiving Compound 1-A. Finally, no other clinically relevant dose-related patterns were observed during analysis of AEs, laboratory parameters, ECGs, and vital signs.

[0323] In Part B, single doses of Compound 1-A equivalent to 92 mg, 275 mg, 368 mg, or 550 mg of Compound 1 were administered to non-cirrhotic GT1b HCV infected subjects divided into dosing cohorts (n=3 for each cohort) to determine the mean maximum reduction in HCV RNA, the results of which are shown in Figure 25 and Table 36. A single dose of 600 mg of Compound 1-A (equivalent to 550 mg of Compound 1) was administered to non-cirrhotic GT1b HCV infected subjects (n=3), resulting in a mean maximum reduction in HCV RNA of 2.3 log 10 IU / mL, and the maximum individual HCV RNA reduction in this cohort was 2.1 log 10 IU / mL, 2.3log 10 IU / mL, and 2.6 log 10 It was IU / mL.

[0324] TIFF2024525164000059.tif52170

[0325] In Part C, dose-related antiviral activity in non-cirrhotic GT1b HCV-infected subjects (n=6) was observed 7 days after dosing, with mean maximum HCV RNA reduction of up to 4.4 log 10IU / mL. 50% of subjects achieved HCV RNA below the LOQ. Figure 26 is a graph of the mean HCV RNA change from baseline in subjects receiving placebo, 150 mg, 300 mg, or 600 mg of Compound 2 once daily (QD). The mean maximum reduction was observed 7 days after dosing in the three cohorts receiving 150 mg, 300 mg, or 600 mg of Compound 1-A once daily (QD).

[0326] In Part D, robust antiviral activity was observed in non-cirrhotic GT3 HCV-infected subjects (n=6), with a mean maximum HCV RNA reduction of 4.5 log 10 IU / mL. The mean HCV RNA reduction after the first dose of 600 mg of Compound 1-A (equivalent to 550 mg of Compound 1) was 2.4 log 10 IU / mL, with one subject achieving HCV RNA below the LOQ within 4 days after the first dose.

[0327] Antiviral activity in CPA cirrhotic HCV-infected subjects in Part E was similar to non-cirrhotic GT1b and GT3 cohorts. In Part E, the mean maximum HCV RNA reduction in HCV-infected patients with cirrhosis was 4.6 log 10 IU / mL. The mean HCV RNA change from baseline in these populations is shown in Figure 27. For comparison, the curves for the escalating dose cohort (Part C, non-cirrhotic GT1b HCV infected patients) are shown in Figure 26, and the curves for all 600 mg QD cohorts (Parts C / D / E) are included in Figure 27. The antiviral activity of metabolites 1-7 observed in each cohort is summarized in Tables 39A, 39B, and 39C.

[0328] The mean maximum HCV RNA change in Parts C, D, and E is shown in Table 37. Figures 28A-C are graphs comparing the mean maximum reduction in non-cirrhotic subjects with GT1 HCV infection from Part C, non-cirrhotic subjects with GT3 HCV infection from Part D, and cirrhotic subjects with GT1 / GT2 / GT3 HCV from Part E. The mean maximum reduction 7 days after dosing was similar for subjects whether they were infected with GT1 or GT3 HCV and whether they were cirrhotic or non-cirrhotic. A summary of the antiviral activity in all these cohorts is shown in Tables 37 and 38. A severe early viral response was observed in cirrhotic subjects, with a 2.4 log decrease within the first 24 hours for GT1 and GT3 HCV subjects, respectively. 10 and 2.2log 10 Five subjects who received 600 mg QD of metabolites 1-7 (3 subjects (50%) in Part C and 1 subject (17%) each in Parts D and E) achieved HCV RNA levels below the lower limit of quantitation in the study.

[0329] TIFF2024525164000060.tif132170

[0330] TIFF2024525164000061.tif92170

[0331] Compound 1, the free base of compound 1-A, was rapidly and well absorbed, with an estimated absorption rate of approximately 50% based on urinary recovery. After repeated QD administration for 7 days under fasting conditions, compound 1 was rapidly absorbed, followed by rapid metabolic activation.

[0332] After 7 days of daily dosing in Part C, compound 1 exhibited a short half-life and did not accumulate over time. Plasma exposure of compound 1 was slightly greater than dose proportional from 150 mg to 300 mg and nearly dose proportional thereafter. Plasma peak and total exposure of metabolites 1-7 was dose proportional from 150 mg to 300 mg and less than dose proportional from 300 mg to 600 mg, while trough levels of metabolites 1-7 were nearly dose proportional in the dose range studied. Based on metabolite 1-7 trough levels, steady-state PK was essentially reached after the third or fourth dose. Formation of metabolites 1-7 peaked approximately 6 hours after dosing, and metabolites 1-7 exhibited a long half-life (approximately 13 hours to 30 hours), supporting once-daily (QD) dosing. The long half-life resulted in the desired higher trough of metabolite 1-7 (50%-60%) upon reaching steady state (active triphosphate 1-6 is not measurable in plasma since it is not released from cells, therefore 1-7 measurable in plasma acts as a surrogate for triphosphate 1-6 and reflects the active triphosphate within cells).

[0333] Steady state of metabolite 1-7 concentrations was reached by day 3 or 4 in NC subjects and by day 5 in subjects with cirrhosis. Overall, mild liver impairment did not significantly affect the PK of compound 1-A based on plasma exposure. No effect of food was observed on total and trough exposure of metabolite 1-7.

[0334] 29 is a graph of the mean plasma concentration-time profile of metabolite 1-7 at steady state comparing non-cirrhotic subjects with GT1 HCV infection receiving 138 mg / day of compound 1-A equivalent to QD of compound 1, non-cirrhotic subjects with GT1 HCV infection receiving 275 mg / day of compound 1-A equivalent to QD of compound 1, non-cirrhotic subjects with GT3 HCV infection receiving 600 mg of compound 1-A (equivalent to 550 mg of compound 1), and cirrhotic subjects with GT1 or GT3 HCV infection receiving 600 mg of compound 1-A (equivalent to 550 mg of compound 1). Plasma levels of metabolites 1-7 were measured using LC-MS / MS.

[0335] Tables 39A, 39B, and 39C show the mean PK results for subjects enrolled in the study. As shown in Tables 39A-39C and Figure 29, the PK of metabolites 1-7 is similar in non-cirrhotic and cirrhotic subjects.

[0336] TIFF2024525164000062.tif121170

[0337] TIFF2024525164000063.tif126170

[0338] TIFF2024525164000064.tif86170

[0339] Figures 30A-D are PK / PD analyses of non-cirrhotic subjects with GT1 HCV infection (Figure 30A), non-cirrhotic subjects with GT3 HCV infection (Figure 30B), cirrhotic subjects with GT1 HCV infection (Figure 30C), and cirrhotic subjects with GT3 HCV infection (Figure 30D). The left y-axis is the mean metabolite 1-7 concentration and the right y-axis is the mean HCV RNA reduction. The dashed horizontal line (-----) represents the EC 95 The dots represent the C τ, i.e., the steady-state plasma trough levels of metabolites 1-7 after 600 mg of compound 1-A (corresponding to 550 mg of compound 1). As shown in Figures 30A-30D, the steady-state plasma trough levels of metabolites 1-7 were consistent with the EC 95 The steady-state plasma trough levels of metabolites 1-7 in patients with cirrhosis were 45.7 ng / mL, and the EC 95 represents approximately 21.7 ng / mL, 11.6 ng / mL, and 17.5 ng / mL of metabolites 1-7, respectively. Figures 30A-30D also show that antiviral activity correlates with plasma exposure.

[0340] E generated by plotting the AUC of metabolites 1-7 against HCV RNA reduction max Using the model, we predicted that exposure to metabolite 1-7 at 2000 ng / mL x h or more would result in a maximum viral load reduction of at least 4 log units 7 days after QD administration of compound 1-A (Figure 31). A 600 mg dose of compound 1-A (equivalent to 550 mg of compound 1) consistently reached this threshold in non-cirrhotic and cirrhotic subjects, indicating that 550 mg QD of compound 1 (equivalent to 600 mg of compound 1-A) would result in the maximum viral load reduction.

[0341] Example 28. Description of the formula and preparation of compound 1-A Representative non-limiting batch formulations for Compound 1-A tablets (50 mg and 100 mg) are presented in Table 40. Tablets were produced from the common blend using a direct compression process as shown in FIG.

[0342] TIFF2024525164000065.tif92170

[0343] Compound 1-A was adjusted based on the as-is assay, and adjustments were made to the percentage of microcrystalline cellulose. Compound 1-A and excipients (microcrystalline cellulose, lactose monohydrate, and croscarmellose sodium) were sieved into a V-blender (PK Blendmaster, 0.5 L bowl) and mixed at 25 rpm for 5 minutes. Magnesium stearate was then sieved and added, and the blend was mixed for an additional 2 minutes. The common blend was split for use in producing 50 mg and 100 mg tablets. The lubricated blend was then compressed at a rate of 10 tablets / min using a single punch research tablet press (Korsch XP1) and a gravity powder feeder. 50 mg tablets were produced using round standard concave 6 mm tooling and a force of 3.5 kN. 100 mg tablets were produced using 8 mm round standard concave tooling and a force of 3.9 kN to 4.2 kN. The specifications for the 50 mg and 100 mg tablets are shown in Table 41.

[0344] TIFF2024525164000066.tif47170

[0345] The 50 mg and 100 mg tablets produced as described above were subjected to a 6-month stability study under three conditions: 5° C. (refrigerated), 25° C. / 60% RH (ambient), and 40° C. / 75% (accelerated). Both the 50 mg and 100 mg tablets were chemically stable under all three conditions tested.

[0346] Under refrigerated conditions (5°C), both the 50mg and 100mg tablets remained white solids with no change in appearance from T=0 to T=6 months. No impurities greater than 0.05% were reported in either the 50mg or 100mg tablets throughout the 6 month study. Moisture content after 6 months was also less than 3.0% w / w for both tablets. Similar results were reported when the tablets were subjected to ambient conditions (25°C / 60% RH), with no impurities greater than 0.05% reported for either tablets throughout the 6 months, and moisture content not exceeding 3.0% w / w at 6 months. When the tablets were subjected to accelerated conditions (40°C / 75% RH), the appearance of the 50mg and 100mg tablets did not change from white round tablets. One impurity was reported after 3 months, but the impurity was only 0.09%. A second impurity was reported after 6 months, but the overall impurity percentage was only 0.21% for both the 50 mg and 100 mg tablets. The moisture content was 3.4% w / w for the 50 mg tablets and 3.2% w / w for the 100 mg tablets at 6 months.

[0347] In a separate study, the stability of 50mg and 100mg tablets of Compound 1-A at ambient conditions (25°C / 60%RH) was measured over 9 months. The appearance of the 50mg and 100mg tablets did not change from a white round tablet over 9 months. The impurities in the 50mg tablets were less than 0.10% after 9 months, and the impurities in the 100mg tablets were less than 0.05%. The moisture content of the 50mg and 100mg tablets after 9 months was only 2.7%w / w and 2.6%w / w, respectively.

[0348] Example 29: Synthesis of Compound 2 [ka]

[0349] Synthesis of Compounds 2-9 [ka] A 250 mL round bottom flask was charged with 3-chloro-5-fluorophenol (compound 2-1) and 2-(2-bromo-5-chlorophenyl)acetic acid (compound 2-2 20.02 g, 80.0 mmol), mixed with TfOH (91 mL), and heated to 60° C. under nitrogen atmosphere. After stirring at this temperature for 16 h, the mixture was cooled to room temperature and poured into isopropanol (500 mL) cooled in an ice / water bath over 20 min. The resulting slurry was diluted with water (125 mL) added over 10 min. After aging in an ice / water bath for 30 min, the mixture was filtered and the collected solid was washed with 4:1 isopropanol / water (50 mL). The solid was dried in vacuum to give compound 2-3 (20.14 g, 53.3 mmol, 80% yield).

[0350] Compound 2-3 (2.03 g, 5.37 mmol) was taken up in 2-methyltetrahydrofuran (20.3 mL, 10 volumes) and to this solution was added ammonia in methanol (11.51 mL of 7N, 81 mmol, 15 equiv). The resulting solution was aged at room temperature for 16 hours, then concentrated by removing 25 mL of solvent and the slurry was treated with toluene (70 mL). The resulting solution was then redistilled to remove an additional 35 mL of solvent to give a final solution of compound 2-5 in 20 volumes of toluene. This solution was used without further purification.

[0351] [ka] A 1 L round bottom flask equipped with an air condenser was charged with compound 2-5 (25 g, 163 mmol), 4-methoxyaniline (22.1 g, 180 mmol), and isopropanol (250 mL). The resulting slurry was warmed to 50° C. and stirred for 3.5 h, during which time a precipitate formed. The resulting slurry was cooled to 0° C., aged for 1 h, and filtered. The flask and pad were rinsed twice with isopropanol (84 mL) at 0° C., and the solid was dried to constant weight in a vacuum oven at 50° C. to give compound 2-6 (39.0 g, 93% yield). 1H NMR (DMSO-d6, 400 MHz): δ 8.77 (s, 1H), 8.06 (s, 1H), 7.26 (d, J=8.8 Hz, 2H), 6.96 (d, J=8.8 Hz, 2H), 3.77 (s, 3H), 2.45 (m, 1H), 1.19 (m, 2H), 1.05 (m, 2H). The imine configuration was not determined and was set to (E) for convenience.

[0352] Compound 2-6 (7.54 g, 29.2 mmol) was placed in a three-necked round-bottom flask equipped with a magnetic stir bar, temperature probe, and nitrogen inlet, and a solution of compound 2-4 in toluene (5.72 wt%, 174.8 g, 26.5 mmol) was added. The resulting suspension was stirred at room temperature until the solids dissolved, and the resulting solution was cooled using an ice / water bath. TFA (2.45 mL, 31.8 mmol) was added while maintaining the internal temperature below 5° C. The resulting solution was stirred in an ice / water bath for 16 h while warming to room temperature. The resulting slurry was filtered, the flask and pad were washed with toluene (27 mL), and the organic solution was washed with aqueous NaHCO3 (4 wt%, 54 mL) and water (54 mL). The organic layer was concentrated in vacuo to approximately 25 mL, diluted with isopropanol (110 mL), and concentrated in vacuo to a total volume of approximately 50 mL. The resulting slurry was warmed to 40° C., diluted with water (10 mL, added over 30 min), aged at 0° C. for 1 h, and filtered. The flask and pad were washed with 4:1 isopropanol / water (25 mL) and the solid was dried to constant weight in a vacuum oven at 50° C. to give compound 2-7 (10.1 g, 74% yield).

[0353] Pd(OAc)2 (219 mg, 0.98 mmol) and (R)-QuinoxP in a 100 mL round-bottom flask *To a solution of (compound 2-8) (343 mg, 1.03 mmol) was added degassed toluene (45 mL). The solution was subjected to three cycles of evacuation and nitrogen backfill, followed by purging with nitrogen above the surface for 5 min. The catalyst solution was then aged at 20 °C for 2 h. Next, compound 2-7 (25 g, 48.8 mmol), K3PO4 (41.4 g, 195 mmol) and toluene (700 mL) were placed in a 1 L three-neck round-bottom flask equipped with an overhead stirrer. The mixture was subjected to three cycles of evacuation and nitrogen backfill, followed by purging with nitrogen above the surface for 5 min. Next, degassed water (0.88 mL, 48.8 mmol) was added dropwise, followed by the ready-made catalyst solution, and the resulting reaction was heated to 50 °C-55 °C and stirred at this temperature for 11 h. Additional water (5.28 mL, 293 mmol) was added in six equal portions every hour during the first 6 hours of reaction time. After a total of 11 hours at 50° C.-55° C., the reaction mixture was cooled to 20° C. and charged with 75 mL of water and 5 mL of 50% (w / v) KOH (approximately 9 N). The aqueous layer was removed and the organic layer was washed with 100 mL of water. The organic layer was then filtered and concentrated in vacuo, and the resulting residue was purified using flash column chromatography to provide compound 2-9.

[0354] The ee was determined using SFC under the following conditions: Column: ChiralCel OJ-3; 4.6mm x 150mm; particle size 3μm Temperature: 40℃ Pressure: 200 bar Modifier: IPA with 25 mM isobutylamine Flow rate: 3.0mL / min Conditions: 1% modifier / 99% CO2 over 5 min to 40% modifier / 60% CO2, hold at 40% modifier for 1 min Purified compound 2-9: (23 mg, 90% yield, 91% ee). 1H NMR (CDCl3, 500 MHz): δ 7.663 (d, J=2.0 Hz, 1 H), 7.407 (d, J=0.4 Hz, 1H), 7.200 (dd, J=2.0, 8.8 Hz, 1H), 7.092 (d, J=0.4 8.4 Hz, 1H), 7.048-7.039 (m, 2H), 6.958-6.910 (m, 2H), 2.194-2.153 (m, 1H), 1.275-1.075 (m, 2H), 1.018-0.991 (m, 2H).

[0355] The crude product compound 2-9 was dissolved in about 50 mL of toluene and 128 mL of iPAC at 45° C. (S)-Camphorsulfonic acid (10.8 g, 46.4 mmol) was added in three portions over 2.5 h at 45° C. It was cooled to room temperature and additional (S)-camphorsulfonic acid (0.57 g, 2.4 mmol) was added. The mixture was aged at room temperature for 16 h and then filtered. The solid was washed with 50 ml of 1 / 2.5 toluene / isopropyl acetate followed by 50 ml of isopropyl acetate and dried under vacuum to give 27.1 g of compound 2-9 as the camphorsulfonic acid salt in 96% ee to >99% ee.

[0356] Synthesis of compound 2 [ka] Compound 2-9 ((S)-CSA salt, 10.0 g, or equivalent amount of free base), bis(pinacolato)diboron (8.50 g), potassium acetate (8.78 g), and 5-chloroindole (0.46 g) were placed under nitrogen in a 500 mL three-neck round bottom flask equipped with an overhead stirrer. Degassed 2-Me-THF (130 mL) and water (0.54 mL) were added. In a separate vessel, palladium acetate (0.067 g) and Xphos (2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl) (0.293 g) and degassed 2-Me-THF (20 mL) were placed under nitrogen, and the mixture was stirred for 30 minutes before adding compound 2-9 to the flask. The mixture was then heated to 75° C. and aged at this temperature for 1 hour or until complete conversion, and then cooled to room temperature. Water (30 mL) was added to the mixture and the layers were separated. The organic layer was washed with 10% brine (30 mL) and then treated with Cuno-3-carbon (1.0 g) for about 15 h. The mixture was filtered through a pad of Celite to remove the carbon. The solution was concentrated in vacuo to a mixture of approximately 35 mL. Seed crystals were added to initiate crystallization. The mixture was aged at room temperature for 10 min, after which acetonitrile (105 mL) was added slowly. The resulting slurry was filtered and the collected solid was washed with a mixture of acetonitrile / 2-Me-THF (3:7, 30 mL) and then dried under a stream of nitrogen to give compound 2-10. 1 H NMR (CDCl3, 500 MHz), 8.23 ​​(s, 1H), 7.70 (d, J=8.3 Hz, 1H), 7.50 (s, 1H), 7.31 (d, J=9.6 Hz, 1H), 7.29 (d, J=7.1 Hz, 1H), 7.23 (d, J=8.3 Hz, 1H), 7.18 (d, J=3.3 Hz, 1H), 7.10 (s, 1H), 2.15-2.10 (m, 1H), 1.39 (s, 6H), 1.37 (s, 3H), 1.37 (s, 3H), 1.06-1.02 (m, 4H), 1.01-0.95 (m, 4H).

[0357] A high pressure vessel was charged with compound 2-10 (10.0 g, 16.28 mmol, 1.0 equiv), compound 2-11 (11.5 g, 2.15 equiv), 2-Me-THF (90 mL), and K2CO3 (98 mL, 1 M, 6 equiv). The vessel was degassed. A second reaction vessel was charged with Pd(OAc)2 (0.11 g, 3%) and Xphos (0.58 g, 7.5%), then degassed, followed by the addition of degassed 2-Me-THF (20 mL). The resulting catalyst / ligand slurry was aged at room temperature under nitrogen for 2 hours. It was then transferred to the reaction vessel containing compound 2-11 and rinsed with degassed 2-Me-THF (10 mL). The resulting reaction mixture was degassed again, and the reaction vessel was sealed and heated to 85°C-90°C for approximately 8 hours until a conversion of >99.5% was reached. The reaction was cooled to room temperature and the organic layer was washed successively with 10% NaCl solution (18 mL) and 3% NaCl solution (18 mL) The organic layer was then concentrated in vacuo and azeotropically dried by distillation to give the crude product (14.66 g).

[0358] The crude product (14.66 g) in 2-Me-THF (135 mL) was added to a solution of tri-n-butylphosphine (2.32 mL) in MeOH (19.4 mL). The mixture was heated to 70° C., followed by the addition of a solution of (S)-mandelic acid (0.94 g) dissolved in 2-Me-THF (3.87 mL). After aging at 70° C. for several hours, the reaction mixture was cooled to 60° C. and another portion of (S)-mandelic acid (3.06 g) in 2-Me-THF (12.58 mL) was added. The batch was seeded with mandelate compound 2-12. The final portion of S-mandelic acid (35.41 g) in 2-Me-THF (22.25 mL) was charged at 60° C. over 4 hours. The reaction mixture was gradually cooled to 20° C. over 8 hours and aged at 20° C. for 1 hour. The slurry was filtered and rinsed with 2-Me-THF (containing 2 wt % (S)-mandelic acid). The collected solid was dried at 60° C. to give the bis-mandelate compound 2-12 as a solid (18.74 g).

[0359] Bis-mandelate compound 2-12 (6 g) was mixed with ethyl acetate (48 mL) and water (25.7 mL). To the biphasic mixture was added 2M potassium carbonate solution (6 mL, 2.5 equiv.) over 10 min, during which a biphasic solution was obtained. The lower aqueous layer was removed and the organic layer was washed successively with 8% brine solution (30 mL) and water (2×30 mL). The organic layer was azeotropically dried by distillation (final solution volume=30 mL). Heptane (66 mL) was charged to an inactivated flask. The ethyl acetate stream containing the product was added to the heptane over 2 h. After aging for an additional 2 h, the product slurry was filtered and the wet filter cake was washed with a mixture of heptane (10.8 mL) and EtOAc (2 mL). The solid was dried in vacuum at 60° C. for approximately 15 h to give compound 2 as the free base (4.40 g, MS: M+H 947.4047). 1H NMR (d6-DMSO, 500 MHz) δ (ppm) 8.30 (s, 1 H), 8.22 (br s, 1 H), 8.10 (br s, 1 H), 8.00 (s, 1 H), 7.78 (d, J=8.7 Hz, 1 H), 7.67-7.65 (m, 2 H), 7.52 (br s, 1 H), 7.38 (s, 1 H), 7.31-7.28 (m, 2 H), 7.19 (d, J=3.2 Hz, 1 H), 5.16 (t, J=7.4 Hz, 1 H), 5.14 (t, J=7.4 Hz, 1 H), 4.15-4.11 (m, 2 H), 3.91-3.81 (m, 4 H), 3.55 (s, 6 H), 2.45-2.36 (m, 2 H), 2.26 (m, 1 H), 2.20-2.13 (m, 2 H), 2.13-2.06 (m, 2 H), 2.06-2.00 (m, 4 H), 0.99 (m, 2 H), 0.85-0.77 (m, 14 H). 13C NMR (d6-DMSO, 126 MHz) δ (ppm) 175.20, 171.21, 171.15, 158.61 (d, J=251.0 Hz), 156.95, 156.94, 150.05, 149.54 (d, J=7.3 Hz), 148.88, 141.37, 133.88, 133.00, 131.11, 130.68, 129.14, 128.78, 125.35, 121.40, 120.38, 118.15, 117.01, 114.18, 111.04, 110.75, 107.30 (d, J=23.4 Hz), 106.67 (d, J=18.2 Hz), 102.94 (d, J=8.6 Hz), 78.46, 57.95, 57.93, 53.06, 52.91, 51.51, 47.16, 47.11, 31.02, 30.95, 29.08, 24.80, 24.75, 19.35, 19.32, 17.74, 13.88, 11.17, 11.05.

[0360] Example 30: Multiplicative anti-HCV activity of Compound 1 and Compound 2 Cell culture - The reporter cell line Huh-luc / neo-ET harbors a persistently replicating 13sgluc-ubi-neo / NS3-3' / ET replicon containing a firefly luciferase gene-ubiquitin-neomycin phosphotransferase fusion protein and an EMCV IRES-driven NS3-5B HCV coding sequence with ET tissue culture adaptive mutations (E1202G, T1208I, and K1846T). Stock cultures of Huh-luc / neo-ET were grown in DMEM supplemented with 10% FCS, 2 mM glutamine, penicillin (100 IU / ml) / streptomycin (100 μg / ml), and 1x non-essential amino acids + 1 mg / ml G418. Cells were split 1:4 and cultured for 2 passages in the same medium supplemented with 250 μg / ml G418. Cells were trypsinized, stained with trypan blue, counted, and plated in 96-well tissue culture plates at 7.5 × 10 3 Cells were seeded at a cell culture density of 100 cells / well and incubated at 37° C., 5% CO 2 for 24 hours.

[0361] Compound addition - After 24 hours of incubation, the medium was removed and replaced with the same medium, minus G418, to which the diluted test compounds were added in triplicate. Six wells of each plate contained medium alone as untreated controls. Cells were incubated for an additional 72 hours at 37°C, 5% CO2, after which anti-HCV activity was measured by luciferase endpoint. Duplicate plates were treated and incubated in parallel to assess cytotoxicity by XTT staining.

[0362] Cell viability - Cell culture monolayers from treated cells were stained with the tetrazolium dye XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide) after 72 hours of incubation to assess cell viability of the Huh-luc / neo-ET reporter cell line in the presence of compounds. Cells were stained with the tetrazolium dye XTT. XTT-tetrazolium is metabolized by mitochondrial enzymes in metabolically active cells to a soluble formazan product, allowing rapid quantitative analysis of inhibition of virus-induced cell death by antiviral test substances. XTT solution was prepared daily as a stock of 1 mg / ml in RPMI 1640. Phenazine methosulfate (PMS) solution was prepared at 0.15 mg / ml in PBS and stored at -20°C in the dark. XTT / PMS stock was prepared immediately prior to use by adding 40 μl of PMS per ml of XTT solution. 50 μl of XTT / PMS was added to each well of the plate and the plate was reincubated for 4 hours at 37° C. The plate was sealed with an adhesive plate sealer, gently shaken or inverted several times to mix the soluble formazan product, and the plate was read spectrophotometrically at 450 / 650 nm using a Molecular Devices Vmax plate reader.

[0363] Measurement of viral replication - HCV replication from the replicon assay system was measured by luciferase activity after 72 hours of incubation using the britelite plus luminescent reporter gene kit according to the manufacturer's instructions (Perkin Elmer, Shelton, CT). Briefly, one vial of britelite plus lyophilized substrate was dissolved in 10 ml of britelite reconstitution buffer and mixed by gentle inversion. After 5 minutes of incubation at room temperature, britelite plus reagent was added to a 96-well plate at 100 μL per well. The plate was sealed with adhesive film and incubated at room temperature for approximately 10 minutes to allow cells to lyse. The contents of the wells were transferred to a white 96-well plate and luminescence was measured within 15 minutes using a Wallac 1450 Microbeta Trilux liquid scintillation counter. Data were imported into a proprietary Microsoft Excel 2010 spreadsheet and the 50% viral inhibitory concentration (EC) for single concentration evaluation was calculated. 50 ) was decided.

[0364] Combination therapy assay Compound 2 was evaluated in combination with nine concentrations of compound 1 in the anti-HCV assay described above, using a high test concentration of 0.008 nM and four serial two-fold dilutions. 50 μl of each compound at four times (4×) the desired concentration was added to a 96-well microtiter plate containing cells for the antiviral assay. 50 μl of assay medium was added to wells where the antiviral activity of the test compound was being evaluated as a single compound.

[0365] Data Analysis - Raw data were collected from Softmax Pro and imported into Prichard and Shipman's MacSynergy II software template (Prichard et al. 1993. Antiviral Research 14: 181-206). The effect of drug combinations is calculated based on the activity of the two compounds when tested alone. The expected additive antiviral protection was subtracted from the experimentally determined antiviral activity at each combination concentration, yielding a positive value (synergism, or potentiation), negative value (antagonism), or zero (additivity). The results of the combination assays are displayed in three dimensions at each combination concentration, resulting in a surface of activity that extends above (synergism) or below (antagonism) the plane of additivity. The volume of the surface was calculated and the amount of synergy (μM 2 %.

[0366] For these studies, synergy was greater than 50 μM with a 95% confidence interval. 2 % synergistic activity. Slightly synergistic and highly synergistic activity were defined as drug combinations that produced an amount of synergy greater than 50 μM 2 %~100μM 2 % and 100μM 2 Synergy was defined as a level that produced a synergistic effect of >50 μM. 2 %~50μM 2 % synergistic effect is considered additive, -50μM 2 Amounts of synergy less than 10% are considered antagonistic.

[0367] Evaluation of anti-HCV combination therapy: Compound 2 was evaluated in combination with Compound 1 for inhibition of HCV replication in Huh-luc / neo-ET replicon cells. The percentage of viral replication inhibition above expectation at each concentration for each two-drug combination was calculated with 95%, 99% and 99.9% confidence intervals. The data obtained with 95% confidence values ​​were plotted three-dimensionally to calculate the amount of synergy. The amount of synergy for the combination with 95% confidence is summarized in Table 42 below.

[0368] TIFF2024525164000071.tif43170

[0369] The results of the HCV replicon assay show that within the range of concentrations used in the combination antiviral assay, compound 1 at 20 nM and 40 nM produced a synergistic interaction with compound 2 at concentrations of 0.004 nM and 0.008 nM. No antagonistic or synergistic toxic interactions were observed at the concentrations evaluated.

[0370] The specification has been described with reference to embodiments of the present invention. However, those skilled in the art will appreciate that various modifications and changes can be made without departing from the scope of the present invention as set forth in the appended claims. Accordingly, the specification is to be regarded in an illustrative rather than a restrictive sense, and all such modifications are intended to be included within the scope of the present invention.

Claims

1. Compound 1: 【Chemical Formula 1】 or a pharmaceutically acceptable salt thereof, and Compound 2: [Chemical 2] or a pharmaceutically acceptable salt thereof, A pharmaceutical composition for treating HCV infection.

2. The pharmaceutically acceptable salt of Compound 1 is Compound 1-A: 【Chemical Formula 3】 The pharmaceutical composition according to Claim 1.

3. The pharmaceutical composition according to Claim 1, wherein the HCV infection is non-cirrhotic.

4. The pharmaceutical composition according to Claim 1, which is administered orally.

5. The pharmaceutical composition according to Claim 1, which is administered parenterally.

6. The pharmaceutical composition according to Claim 5, which is administered intravenously.

7. The pharmaceutical composition according to Claim 1, which is administered once a day.

8. The pharmaceutical composition according to Claim 1, which is administered twice a day.

9. The pharmaceutical composition according to Claim 1, which is administered for a maximum of 12 weeks.

10. The pharmaceutical composition according to Claim 1, which is administered for a maximum of 8 weeks.

11. The pharmaceutical composition according to Claim 1, which is administered for a maximum of 6 weeks.

12. The pharmaceutical composition according to Claim 1, wherein the HCV infection is genotype 1.

13. The pharmaceutical composition according to Claim 1, wherein the HCV infection is genotype 2.

14. The pharmaceutical composition according to Claim 1, wherein the HCV infection is genotype 3.

15. The pharmaceutical composition according to Claim 1, wherein the HCV infection is genotype 4.

16. The pharmaceutical composition according to Claim 1, wherein the HCV infection is genotype 5.

17. The pharmaceutical composition according to Claim 1, wherein the HCV infection is genotype 6.

18. The pharmaceutical composition according to Claim 1, wherein the HCV is a resistance-related mutant.

19. The pharmaceutical composition according to any one of Claims 1 to 18, comprising about 100 mg to about 800 mg of Compound 1 or an equivalent amount of a pharmaceutically acceptable salt thereof.

20. The pharmaceutical composition according to any one of Claim 19, comprising about 90 mg to about 360 mg of Compound 2 or an equivalent amount of a pharmaceutically acceptable salt thereof.

21. The pharmaceutical composition according to Claim 20, which is used in a single dosage form.

22. The pharmaceutical composition according to Claim 21, comprising about 550 mg of Compound 1 or an equivalent amount of a pharmaceutically acceptable salt thereof.

23. The pharmaceutical composition according to Claim 21, comprising about 180 mg of Compound 2 or an equivalent amount of a pharmaceutically acceptable salt thereof. **Claim 24**: The pharmaceutical composition according to claim 20, which is used in two dosage forms. **Claim 25**: The pharmaceutical composition according to claim 24, wherein one dosage form contains compound 1 or a pharmaceutically acceptable salt thereof, and the second dosage form contains compound 2 or a pharmaceutically acceptable salt thereof. **Claim 26**: The pharmaceutical composition according to claim 24, wherein the two dosage forms each contain compound 1 or a pharmaceutically acceptable salt thereof and compound 2 or a pharmaceutically acceptable salt thereof. **Claim 27**: The pharmaceutical composition according to claim 24, which contains about 275 mg of compound 1 or an equivalent amount of a pharmaceutically acceptable salt thereof. **Claim 28**: The pharmaceutical composition according to claim 24, which contains about 90 mg of compound 2 or an equivalent amount of a pharmaceutically acceptable salt thereof. **Claim 29**: Compound 1: 【Chemical Formula 4】 A pharmaceutical composition for treating HCV infection, which contains Compound 2: 【Chemical Formula 5】 Or a pharmaceutically acceptable salt thereof, and is used in combination with a second pharmaceutical composition. **Claim 30**: The pharmaceutically acceptable salt of compound 1 is compound 1-A: [Chemical Formula 6] The pharmaceutical composition according to claim 29. **Claim 31**: The pharmaceutical composition according to claim 29, wherein the HCV infection is non-cirrhotic. **Claim 32**: The pharmaceutical composition according to claim 29, which is administered orally. **Claim 33**: The pharmaceutical composition according to claim 29, which is administered parenterally. **Claim 34**: The pharmaceutical composition according to claim 33, which is administered intravenously. **Claim 35**: The pharmaceutical composition according to any one of claims 29 to 34, which contains about 100 mg to about 800 mg of compound 1 or an equivalent amount of a pharmaceutically acceptable salt thereof. **Claim 36**: The pharmaceutical composition according to any one of claims 29 to 34, which contains about 550 mg of compound 1 or an equivalent amount of a pharmaceutically acceptable salt thereof. **Claim 37**: The pharmaceutical composition according to any one of claims 29 to 34, which contains about 275 mg of compound 1 or a pharmaceutically acceptable salt thereof. **Claim 38**: The pharmaceutical composition according to claim 35, wherein the second pharmaceutical composition contains about 90 mg to about 360 mg of compound 2 or an equivalent amount of a pharmaceutically acceptable salt. **Claim 39**: The pharmaceutical composition according to claim 35, wherein the second pharmaceutical composition contains about 180 mg of compound 2 or an equivalent amount of a pharmaceutically acceptable salt thereof. **Claim 40**: The pharmaceutical composition according to claim 35, wherein the second pharmaceutical composition contains about 90 mg of compound 2 or a pharmaceutically acceptable salt thereof. **Claim 41**: Compound 2: 【Chemical Formula 7】 A pharmaceutical composition for treating HCV infection, comprising Compound 1: 【Chemical Formula 8】 Or a pharmaceutical composition used in combination with a second pharmaceutical composition comprising a pharmaceutically acceptable salt thereof.

42. The pharmaceutically acceptable salt of Compound 1 is Compound 1-A: 【Chemical Formula 9】 The pharmaceutical composition according to Claim 41.

43. The pharmaceutical composition according to Claim 41, wherein the HCV infection is non-cirrhotic.

44. The pharmaceutical composition according to Claim 41, which is administered orally.

45. The pharmaceutical composition according to Claim 41, which is administered parenterally.

46. The pharmaceutical composition according to Claim 45, which is administered intravenously.

47. The pharmaceutical composition according to any one of Claims 41 to 46, wherein the second pharmaceutical composition comprises about 100 mg to about 800 mg of Compound 1 or an equivalent amount of its pharmaceutically acceptable salt.

48. The pharmaceutical composition according to any one of Claims 41 to 46, wherein the second pharmaceutical composition comprises about 550 mg of Compound 1 or an equivalent amount of its pharmaceutically acceptable salt.

49. The pharmaceutical composition according to any one of Claims 41 to 46, wherein the second pharmaceutical composition comprises about 275 mg of Compound 1 or its pharmaceutically acceptable salt.

50. The pharmaceutical composition according to Claim 47, comprising about 90 mg to about 360 mg of Compound 2 or an equivalent amount of a pharmaceutically acceptable salt.

51. The pharmaceutical composition according to Claim 47, comprising about 180 mg of Compound 2 or an equivalent amount of its pharmaceutically acceptable salt.

52. The pharmaceutical composition according to Claim 47, comprising about 90 mg of Compound 2 or an equivalent amount of its pharmaceutically acceptable salt.

53. Compound 1: 【Chemical Formula 10】 Or a pharmaceutical form of its pharmaceutically acceptable salt and Compound 2: 【Chemical 11】 Or a pharmaceutical form of its pharmaceutically acceptable salt, for use in treating HCV in a patient in need of treatment.

54. The pharmaceutically acceptable salt of the Compound 1 is Compound 1-A: 【Chemical Formula 12】 The kit according to Claim 53.

55. The kit according to Claim 53 or 54, comprising about 100 mg to about 800 mg of Compound 1 or an equivalent amount of its pharmaceutically acceptable salt.

56. The kit according to Claim 53 or 54, comprising at least about 550 mg of Compound 1 or an equivalent amount of its pharmaceutically acceptable salt.

57. The kit according to Claim 53 or 54, comprising about 180 mg of Compound 2 or its pharmaceutically acceptable salt.

58. The kit according to claim 53 or 54, comprising two dosage forms.

59. The kit according to claim 58, wherein about 275 mg of Compound 1 or a pharmaceutically acceptable salt in an equivalent amount is administered.

60. The kit according to claim 58, comprising about 90 mg of Compound 2 or a pharmaceutically acceptable salt in an equivalent amount.

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