Dosing regimens for treatment of dengue infections

Abstract

The present invention provides advantageous high dose therapeutic compositions and combinations for the treatment or prevention of dengue virus infections that may be serotype 1, 2, 3 or 4, and methods of making the same.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 346,274, filed May 26, 2022, U.S. Provisional Application No. 63 / 414,812, filed October 10, 2022, and U.S. Provisional Application No. 63 / 427,359, filed November 22, 2022. These applications are incorporated herein by reference for all purposes. Field of the Invention

[0003] The present invention provides advantageous therapeutic compositions, combinations thereof and uses thereof for treating or preventing dengue virus infection. Background of the Invention

[0005] Dengue fever is caused by four related viruses, called dengue virus serotypes 1-4. Dengue virus (DENV) is a member of the genus Flaviviridae. They are enveloped viruses with a positive-sense RNA genome of 11kb that encodes ten proteins. These proteins are membrane (M) protein, envelope (E) protein, capsid (C) protein, and non-structural (NS) protein. NS proteins are NS1, NS2A, NS2B, NS3, NS4A, NS5B, and NS5. These proteins are translated into a single polyprotein, which is cleaved into individual proteins by proteases. NS5 protein is the largest nonstructural protein, acting as a viral RNA-dependent RNA polymerase and RNA2'-O-methyltransferase. RNA-dependent RNA polymerase (RdRp) activity is responsible for the replication of the viral genome.

[0006] Dengue is endemic in tropical regions, including the eastern Mediterranean, the Americas, Southeast Asia, the western Pacific, and Africa. As the distribution of the Aedes mosquito that carries dengue expands, new cases are emerging in areas where dengue has never been found before. The Centers for Disease Control and Prevention has reported recent outbreaks of locally acquired dengue in Hawaii (2015), Florida (2013, 2020), and Texas (2013) (https: / / www.cdc.gov / Dengue / areaswithrisk / in-the-us.html). Mosquitoes that carry dengue are now common throughout much of the southeastern United States (Rivera et al. 2020. Travel-associated and Locally Acquired Dengue Cases-United States, 2010-2017. MMWR Morb Mortal Wkly Rep 69:149-154).

[0007] Although the distribution of dengue serotypes was previously limited to specific regions, as the disease has spread around the world, the distribution of serotypes has become more common (Nature, "Dengue virus"). This is particularly concerning because if a person is infected with one serotype and later becomes infected with a second serotype, they are at greater risk for severe dengue. Severe dengue (or dengue hemorrhagic fever) is accompanied by severe plasma leakage, severe bleeding, shock, and organ damage. If left untreated, severe dengue has a mortality rate of 13% (Centers for Disease Control and Prevention, Dengue for Healthcare Providers, Clinical Presentation, updated April 13, 2023).

[0008] In addition to the geographical distribution of dengue outbreaks, recent outbreaks are more severe. Since 2000, the global incidence has increased eightfold. Not only has the frequency of outbreaks increased, but the size has also increased. The World Health Organization estimates that half of the world's population is at risk of infection, and hundreds of millions of people are infected each year (https: / / www.who.int / news-room / fact-sheets / detail / Dengue-and-severe-Dengue). Nearly 500,000 cases of severe dengue have also been reported. Most people at risk of dengue are children (Aranda C et al., 2018. Arbovirus surveillance: first Denguevirus detection in local Aedes albopictus mosquitoes in Europe, Catalonia, Spain, 2015. Euro Surveill 23, Wilder-Smith et al., 2014. The 2012 Dengue outbreak in Madeira: exploring the origins. Euro Surveill 19: 20718).

[0009] Vaccines for dengue have been developed, but they have some drawbacks. The most notable is a phenomenon known as antibody-dependent enhancement (ADE). The only licensed vaccine, Dengvaxia, provides incomplete and uneven protection against different serotypes, with only 50% protection against DENV1 and even lower protection against DENV2 (Sabchareon et al., 2012. Protective efficacy of therecombinant, live-attenuated, CYD tetravalent Dengue vaccine in Thai schoolchildren: a randomized, controlled phase 2b trial. Lancet 380: 1559-67; Villar et al., 2015. Efficacy of a tetravalent Dengue vaccine in children in Latin America. N Engl J Med. 372: 113-23). Therefore, vaccination may promote ADE, resulting in severe infection in individuals who are not infected with dengue (Aguiar et al., 2016. The Impact of Newly Licensed Dengue Vaccine in Endemic Countries. PLoS Negl Trop Dis. 10: e0005179). Therefore, vaccination is limited to individuals aged 9-16 years who have at least one record of dengue virus infection. Although dengue virus has now become a global health threat and its importance is becoming increasingly prominent, a safe and effective pan-dengue vaccine has not yet been developed.

[0010] Therefore, a potential strategy to combat dengue fever worldwide is drugs, such as direct-acting antivirals (DAAs). Unfortunately, there are currently no DAAs approved for the treatment of dengue fever. In DENV, the viral polymerase is a nonstructural protein NS5 (Karuna et al., 2020, A Cyclic Phosphoramidate Prodrug of 2'-Deoxy-2'-Fluoro-2'C-Methylguanosine for the Treatment of Dengue Virus Infection. Antimicrob Agents Chemother 64). Although DENV RdRp has been the target of many investigational DAAs, including many nucleoside / nucleotide analogs (Troost et al., 2020. Recent advances in antiviral drug development towards Dengue virus. Curr Opin Virol 43:9-21), to date, only one nucleoside, the cytosine analog balapiravir, has been clinically tested (Karuna et al., 2020. A Cyclic Phosphoramidate Prodrug of 2'-Deoxy-2'-Fluoro-2'C-Methylguanosine for the Treatment of Dengue Virus Infection. Antimicrob Agents Chemother 64; Nguyen et al., 2013. A randomized, double-blind placebo-controlled trial of balapiravir, a polymerase inhibitor, in adult Dengue patients. J Infect Dis. 207:1442-50). Unfortunately, no differences were observed between compound and placebo treatment in terms of antiviral response, cytokine profile, and time to fever reduction (Nguyen et al., 2013. Arandomized, double-blind placebo-controlled trial of balapiravir, a polymerase inhibitor, in adult Dengue patients. J Infect Dis. 207: 1442-50).

[0011] Other compounds targeting NS5 or other viral proteins have also been reported in the literature. In 2017, Janssen disclosed a series of compounds that bind to the NS3 protein (WO 2017 / 167951). In 2020, the Manfroni laboratory described pyridobenzothiazolones that inhibit NS5 (Cannalire, R. et al. Pyridobenzothiazolones Exert Potent Anti-Dengue Activity by Hampering Multiple Functions of NS5 Polymerase, ACSMed Chem Lett, 2020 11, 773-782). Other publications describing dengue inhibitors include, but are not limited to, Moquin et al., NITD-688, a pan-serotype inhibitor of the Dengue virus NS4B protein, shows favorable pharmacokinetics and efficacy in preclinical animal models. Science Translational Medicine, 2021, 13, 579; Kaptein, S. et al. A pan-serotype Dengue virus inhibitor targeting the NS3-NS4B interaction. Nature, 2021, 598, 504-509; Shimizu, H. et al. Discovery of a small molecule inhibitor targeting Dengue virus NS5 RNA-dependent RNApolymerase. PLoS Negl Trop Dis. 2019, 13, e0007894; Yin, Z. et al. Anadenosine nucleoside inhibitor of Dengue virus, PNAS, 2009, 106, 20435-20439; Putra, H. et al. Identification of natural product compounds as NS5 RDRP inhibitor for Dengue virus serotype 1-4through in silico analysis.AIP ConferenceProceedings 2020,2237;Wang,Q.-Y.et al. A Small-Molecule Dengue Virus EntryInhibitor. Antimicrobial Agents and Chemotherapy, 2009, 53, 1823-1831; deOliviera, L. et al. The small molecule AZD6244 inhibits Dengue virus replication in vitro and protects against lethal challenges in a mouse model. 2020, Archives of Virology, doi:10.1007 / s00705-020-04524-7; and Celegato, M. et al. Small-Molecule Inhibitor of Flaviviral NS3-NS5 Interaction with Broad-SpectrumActivity and Efficacy In Vivo, Antimicrobial Chemotherapy, 2022 14(1)e03097-22. .

[0012] Specific antiviral compounds for treating certain RNA viruses include Compound 1 and Compound 2. For example, the free base Compound 1 is disclosed in U.S. Pat. No. 9,828,410.

[0013]

[0014] Compound 2 (disclosed in U.S. Pat. No. 10,519,186) is the hemisulfate salt of ((R)-(((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-alanine isopropyl ester and is specifically disclosed for use in treating viral infections, such as hepatitis C virus. Compound 2 is also reported to be active against SARS-CoV2 (the pathogen of COVID19 infection) (see, e.g., U.S. Pat. No. 10,874,687). The S-phosphorus diastereomer of Compound 2 is currently undergoing human clinical trials for the treatment of HCV and COVID19.

[0015]

[0016] In U.S. Pat. No. 10,946,033, Atea Pharmaceuticals, Inc. discloses the use of Compound 1 or Compound 2 for treating flaviviruses generally, with activity data against yellow fever, dengue (serotype 2), West Nile virus, and Zika virus.

[0017] Despite these disclosures, effective treatment of dengue fever remains difficult due to the above-mentioned challenging issues. There remains an urgent medical need to identify appropriate regimens for the treatment of dengue fever that are safe, effective, and well-tolerated.

[0018] Therefore, it is an object of the present invention to provide an advantageous regimen for treating dengue infection.Another object of the present invention is to provide a novel process for the preparation of Compound 1 and Compound 2, which are phosphorus R-diastereomers. Summary of the invention

[0019] It was unexpectedly found that when a high dose (i.e., at least about 700 mg or 750 mg per dose, more typically 700-1000 mg (for convenience, it can be delivered in one, two or three separate dosage forms), twice, three times or more daily) of Compound 1 or Compound 2 (wherein Compound 1 can be a pharmaceutically acceptable salt thereof) is administered to a host (e.g., a human) in need thereof, it is beneficial to treat dengue virus. In a typical embodiment, Compound 1 or Compound 2 is provided in one or more pharmaceutical compositions to achieve this effective amount.

[0020] In addition, it was found that compound 1 or compound 2 is also active against dengue serotypes 1, 3, and 4. Through this new discovery, it was confirmed that compound 1 and compound 2 in the high-dose regimen described herein are effective against all serotypes of dengue virus (dengue virus serotypes 1, 2, 3, and 4).

[0021]

[0022] The term "Compound 1" used in this specification may refer to its free base or a pharmaceutically acceptable salt thereof. Compound 1 is ((R)-(((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-alanine isopropyl ester.

[0023] Compound 1 (and its hemisulfate salt compound 2) was also found to have a favorable dual mechanism of action. The active metabolite is an inhibitor of the RNA-dependent RNA polymerase (RdRp) domain of the viral protein NS5, and also an inhibitor of the 2'-O-methyltransferase (MTase) domain of NS5. Both functions of the NS5 protein are essential for replication. Inhibition of two key protein functions involved in RNA replication may reduce the chance of forming resistant mutants.

[0024] As described in Example 2 and Tables 1 and Figure 2 As shown in , compound 1 (AT-281) was found to exhibit in vitro potency against all four dengue serotypes, including serotype 1 (EC 50 =0.57 μM), serotype 2 (EC 50 <7μM), serotype 3 (EC 50 <1.5 μM) and serotype 4 (EC 50 <1.2 μM). Pan-serotype activity is advantageous because it minimizes the risk of severe dengue and reduces or eliminates the need for serotype testing prior to administration of the compound.

[0025] Alternatively, Compound 1 can be used in the high-dose regimen described herein in free base form or in any desired pharmaceutically acceptable salt form. Compound 2 is a hemisulfate salt of Compound 1, which is an advantageous form of Compound 1 with improved in vivo properties. For example, Compound 2 is effective in vivo in a mouse model of dengue infection (D2Y98P strain), as described in Example 18. For example, Compound 2 can be used to observe 6 and 8 days post infection (pi). Fig. 8A Blood neutralization Figure 8B In addition, compared with animals treated with the vehicle control, significant differences in viremia were observed between days 3 and 8 post infection. Fig.9A Weight loss and Fig. 9B Significant preventive effect on health score deterioration was shown. Importantly, Fig.10 As shown, the survival time was significantly prolonged, which demonstrates the efficacy of Compound 2 against dengue virus, or the efficacy of Compound 1 as a free base or a pharmaceutically acceptable salt thereof.

[0026] In certain aspects, Compound 2 or Compound 1 (as a free base or a pharmaceutically acceptable salt thereof) is administered to a patient, typically at a high dose regimen as described herein, wherein the patient has multiple flaviviruses, one of which is dengue, e.g., the patient has dengue serotype 1, 2, 3, or 4 and another flavivirus. Compound 1 (AT-281) exhibited potent effects in reducing viral titers of 11 different strains of 7 flaviviruses tested in vitro in 3 different cell types, with a concentration range of 0.21 to 1.41 μM achieving 50% inhibition of virus-induced cytopathic effect (CPE) (Tables 1 and 2). Figure 2 ).

[0027] As described in Example 10 and Figure 1 As shown, compound 2 or the active metabolite of compound 1 (compound 5) acts as a GTP analog and is incorporated into RNA by the NS5 RNA-dependent RNA polymerase (RdRp), thereby immediately terminating synthesis.

[0028]

[0029] The chain termination efficiency of compound 5 was measured in a competitive nucleotide incorporation assay using all four DENV serotypes. Compared to the natural substrate guanosine triphosphate, compound 5 was incorporated into RNA 9 to 12 times less (i.e., one AT-9010 was incorporated for every approximately 7-8 GTPs incorporated). In contrast, sofosbuvir triphosphate was incorporated into RNA less than 1 time for every 100 UTPs (natural uridine triphosphates) incorporated.

[0030] The identification test data are as follows.

[0031] Preference DENV1 DENV2 DENV3 DENV4 GTP>AT-9010 7.7±0.5 9.3±0.1 11.6±1.3 11.2±0.7 GTP>AT-9002 19.4±2.8 18.2±0.2 17.5±1.5 41.5±7.3 UTP>SOF-TP >100 >100 >100 >100

[0032] Compound 5 (as well as compounds 1 and 2) is also an inhibitor of the 2'-O-methyltransferase (MTase) domain of dengue virus NS5. The MTase domain is responsible for capping the 2' end of the replicated viral RNA. The RNA cap can reduce the host's immune response to viral RNA. Inhibition of the MTase domain is the second mechanism of action of compound 5.

[0033] The binding of compound 5 to the MTase domain was measured by thermal shift analysis. Compound 5 showed almost twice the stability of the MTase domains of DENV1, DENV2, DENV3, and DENV4 as that of the previously reported MTase inhibitor sinefungin (1.9 ± 0.1 °C for sinefungin vs. 3.8 ± 0.1 °C for compound 5).

[0034] In addition, compound 5 stabilized the RdRp domain more effectively than the natural substrate GTP (5.6±0.1°C for compound 5 and 4.5±0.2°C for GTP), while sinefungin had a negligible stabilizing effect on the RdRp domain (0.1±0.1°C).

[0035] Confirmation of the activity of compound 5 against the MTase domain, IC measured in the filter binding assay 50 It has been previously demonstrated that the concentration of compound 5 in cells can reach hundreds of micromolar (Good, S. et al. Preclinicalevaluation of AT-527, a novel guanosine nucleotide prodrug with potent, pan-genotypic activity against hepatitis C virus, PLoS ONE 15(1):e0227104). 50 The result showed that compound 5 is a potent inhibitor of MTase domain.

[0036] Therefore, compound 5, which is an active metabolite of compound 2 and compound 1, has multiple mechanisms of action against dengue virus. Multiple mechanisms of action may prevent the development of drug resistance, especially targeting the highly conserved MTase domain.

[0037] In certain aspects of the present invention, a compound of formula I or a pharmaceutically acceptable salt thereof is optionally administered in a pharmaceutically acceptable carrier at a high dose (at least 700 or 750 mg per dose, for example 700-1000 mg per dose (which can provide 1, 2, 3 or 4 different dosage forms for convenient swallowing), twice, three times or more daily) to treat dengue virus infection in a host (usually a human) in need thereof:

[0038]

[0039] in:

[0040] R 2 C 1-6 Alkyl (including methyl, ethyl, propyl and isopropyl), C 3-7 cycloalkyl, aryl, aryl(C1-C4alkyl)-, heteroaryl, heteroaryl(C1-C4alkyl)- or heteroalkyl, each of which is optionally substituted by 1, 2, 3 or 4 substituents, the substituents being independently selected at each occurrence from R 22 ;

[0041] R 3 is hydrogen or C optionally substituted with 1, 2, 3 or 4 substituents1-6 Alkyl, the substituents are independently selected from R 33 ;

[0042] R 4a and R 4b are independently selected from hydrogen, C 1-6 Alkyl, aryl (C 1-6 Alkyl)-, C 3-7 Cycloalkyl, C 3-7 Cycloalkyl (C 1-6 Alkyl)-, heteroaryl (C 1-6 alkyl)- and aryl, each of which is optionally substituted by 1, 2, 3 or 4 substituents, said substituents being independently selected at each occurrence from R 44 ;

[0043] R 5 For hydrogen, C 1-6 Alkyl (including methyl, ethyl, propyl and isopropyl), C 1-6 Haloalkyl or C 3-7 cycloalkyl, and in an alternative embodiment, R 5 is aryl(C1-C4alkyl)-, aryl, heteroaryl or heteroalkyl, which is optionally substituted by 1, 2, 3 or 4 substituents, the substituents being independently selected in each case from R 55 ;

[0044] R 22 , R 33 , R 44 and R 55 is independently selected at each occurrence from hydrogen, C 1- C6 alkyl, C 3- C7 cycloalkyl, alkenyl, alkynyl, F, Cl, Br, I, heterocycle, heterocycloalkyl, haloalkoxy, haloalkyl, C(O)R 66 、C(O)OR 66 、C(O)N(R 66 )2 and cyano;

[0045] R 66 is independently selected at each occurrence from hydrogen, C1-C6-alkyl, aryl(C1-C6-alkyl)-, C3-C7-cycloalkyl, C3-C7-cycloalkyl(C1-C6-alkyl)-, heteroaryl(C1-C6-alkyl)- and aryl.

[0046] In certain aspects of the present invention, the compound of formula II or a pharmaceutically acceptable salt thereof is optionally administered in a pharmaceutically acceptable carrier at a high dose (at least 700-1000 mg per dose, for example 700-850 mg per dose, twice, three times or more daily) to treat dengue virus infection in a host (usually a human) in need thereof:

[0047]

[0048] in:

[0049] R 3 is hydrogen or C optionally substituted with 1, 2, 3 or 4 substituents 1-6 Alkyl, the substituents are independently selected from R 33 ;

[0050] R 4a and R 4b are independently selected from hydrogen, C 1-6 Alkyl, aryl (C 1-6 Alkyl)-, C 3-7 Cycloalkyl, C 3-7 Cycloalkyl (C 1-6 Alkyl)-, heteroaryl (C 1-6 alkyl)- and aryl, each of which is optionally substituted by 1, 2, 3 or 4 substituents, said substituents being independently selected at each occurrence from R 44 ;

[0051] R 5 For hydrogen, C 1-6 Alkyl (including methyl, ethyl, propyl and isopropyl), C 1-6 Haloalkyl or C 3-7 cycloalkyl, and in an alternative embodiment, R 5 is aryl(C1-C4alkyl)-, aryl, heteroaryl or heteroalkyl, which is optionally substituted by 1, 2, 3 or 4 substituents, the substituents being independently selected in each case from R 55 ;

[0052] R 22 , R 33 , R 44 and R 55 is independently selected at each occurrence from hydrogen, C 1- C6 alkyl, C 3- C7 cycloalkyl, alkenyl, alkynyl, F, Cl, Br, I, heterocycle, heterocycloalkyl, haloalkoxy, haloalkyl, C(O)R 66 、C(O)OR 66 、C(O)N(R 66 )2 and cyano;

[0053] R 66 is independently selected at each occurrence from hydrogen, C1-C6-alkyl, aryl(C1-C6-alkyl)-, C3-C7-cycloalkyl, C3-C7-cycloalkyl(C1-C6-alkyl)-, heteroaryl(C1-C6-alkyl)- and aryl.

[0054] All R groups should be interpreted in a manner that does not include redundancy, i.e., an alkyl group is not substituted by an alkyl group as is known in the art; however, for example, an alkoxy group substituted by an alkoxy group is not redundant. Aryl groups substituted by aryl groups fall within the definition of aryl groups to a limited extent. Unless the context clearly indicates otherwise, R groups are not optionally substituted.

[0055] Non-limiting examples of C1-C6 alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, sec-butyl, isobutyl, -CH2C(CH3)3, -CH(CH2CH3)2, and -CH2CH(CH2CH3)2. Non-limiting examples of C3-C6 cycloalkyl groups include cyclopropyl, CH2-cyclopropyl, cyclobutyl, and CH2-cyclobutyl.

[0056] A non-limiting example of an aryl (C1-C4 alkyl)- is benzyl. A non-limiting example of an aryl is phenyl and naphthyl.

[0057] In certain aspects, the compound is a compound of Formula IIA or a pharmaceutically acceptable salt thereof:

[0058]

[0059] wherein all variables are as defined herein.

[0060] In certain aspects, the compound is a compound of Formula IIB or a pharmaceutically acceptable salt thereof:

[0061]

[0062] wherein all variables are as defined herein.

[0063] In certain aspects, the selected compounds described herein are used to treat dengue virus infection, which may be serotype 1, 3, and / or 4. In some embodiments, the selected compounds described herein are used to treat dengue virus infection, which may be serotype 1, 2, 3, and / or 4, by administering to a patient in need thereof a dose of about 700-1000 mg (e.g., 700-850 mg per dose), twice, three times or more a day (and in some embodiments, administering a dose of 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975 or 1000 mg, twice or three times a day). In certain embodiments, compound 1 or compound 2 is administered at a dose of about 700-1000 mg, for example, at least about 700, 750, or 800 mg per administration (which can be delivered in a variety of solid dosage forms to achieve a full dose).

[0064] In a specific embodiment, compound 2 is delivered in a dosage of three 250 mg free base weight solid dosage forms twice daily to reach a total dosage of 750 mg. In other embodiments, compound 2 is delivered in a dosage of two 375 mg solid dosage forms twice daily to reach a total dosage of 750 mg.

[0065] In certain embodiments, compound 1 or compound 2 is administered twice, three times or four times daily for four days, five days, six days, seven days, eight days, nine days or ten days. In certain embodiments, the treatment regimen can treat dengue virus serotype 1, 3 and / or 4, or even 1, 2, 3 and / or 4. For clarity, unless otherwise stated, the milligram dose of the active compound used is based on the weight (if a pharmaceutically acceptable salt is used) of the salt of nucleotide phosphoramidate (nucleotide phosphoramidate). For conversion purposes, the compound 2 of about 750 mg dosage corresponds to the compound 1 of about 692 mg dosage (within the range of "about 700 mg"), and compound 1 is a free alkali form.

[0066] The present invention includes treatment and prophylactic or preventative therapy. In some embodiments, according to the high-dose method provided herein, the active NS5RdRp inhibitory compound described herein is applied as a prophylactic to a host (e.g., human) that has been exposed to dengue virus infection (e.g., dengue type 1 virus, dengue type 2 virus, dengue type 3 virus, and / or dengue type 4 virus) and is therefore at risk of infection with dengue virus infection. In another alternative embodiment, a method for preventing transmission is provided, including administering to humans one of the compounds described herein in a high-dose effective amount described herein before exposure to a potentially infected population (including during travel or public events or meetings) for a sufficiently long time, including, for example, up to 3, 5, 7, 10, 12, 14 days or more before an infectious situation occurs.

[0067] The present invention also provides a stereoselective method for scalable preparation of compound 1, wherein R P Diastereomers are produced in substantially pure form. Substantially pure forms of diastereomers are generally referred to as R P Diastereomers relative to S P The diastereoisomers are at least about 90% or more. The reaction of the compound of formula intermediate A, intermediate B with a urea-based activator (e.g., 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU)) preferentially forms S PA similar process is described in WO 2022 / 040473A1, assigned to Atea Pharmaceuticals, where the desired product is S P Aminophosphoric acid ester.

[0068]

[0069] The aryloxy group on the phosphoramidate of intermediate C is replaced by one, two or three R 6 In the second step, the activated phenoxy leaving group is replaced by a phenol, thereby inverting the phosphorus stereochemistry to form compound 1.

[0070] In certain embodiments, the substantially pure R-phosphorus diastereomer is about 93% pure or greater, about 95% pure or greater, about 98% pure or greater, or even 99% pure or greater.

[0071] In certain embodiments, a dose of 700 mg to 1,000 mg is administered to a patient in need of treatment of dengue fever in three solid dosage forms, wherein a total of about 700 mg to about 1,000 mg of compound 1 or 2 in solid dosage form is administered twice daily (a total of six capsules per day). In certain embodiments, a total of about 700 mg to about 1,000 mg of compound 1 or 2 in three solid dosage forms is administered for four, five, six, or seven days in a row. In certain embodiments, a total of about 700 mg to about 1,000 mg of compound 1 or 2 in three solid dosage forms is administered for eight, nine, ten, eleven, twelve, thirteen, or fourteen days in a row.

[0072] In certain embodiments, composition B is administered for four, five, six, or seven consecutive days. In certain embodiments, composition A is administered for eight, nine, ten, eleven, twelve, thirteen, or fourteen consecutive days.

[0073] In some embodiments, Compound 1 or a pharmaceutically acceptable salt thereof is used in Composition A or Composition B.

[0074] In certain embodiments, Compound 1 or Compound 2 is administered twice daily at a dosage of at least about 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, or 1000 mg, wherein as an illustrative example, the compound is administered every 8 + / - 2 hours.

[0075] In some aspects, the dosing regimen described herein is administered within four, three, two or one day after the onset of symptoms. In some embodiments, the dosing regimen described herein is administered within 48 hours after the onset of symptoms.

[0076] Therefore, the present invention includes the following features:

[0077] (a) The dosing regimen of Compound 1 or Compound 2 comprises administering at least about 675-1000 mg or 700-1000 mg, e.g., 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975 or 1000 mg of Compound 1 or Compound 2 twice or three times daily to a patient suffering from dengue serotype 1, 2, 3 and / or 4.

[0078] (b) Treatment of dengue serotype 1, 3 or 4 as further described herein.

[0079] (c) A dosing regimen of Compound 1 or Compound 2 comprises administering at least about 675-1000 mg, e.g., 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975 or 1000 mg of Compound 1 or Compound 2 twice or three times daily to a patient suffering from dengue serotype 1, 3 and / or 4.

[0080] (d) The dosing regimen of (a), (b) or (c), wherein Compound 1 or Compound 2 is administered continuously for three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen days.

[0081] (e) The dosing regimen of (a), (b) or (c), wherein Compound 1 or Compound 2 is administered three times daily for fourteen days at a dose of about 750 mg.

[0082] (f) The dosing regimen of (a), (b) or (c), wherein Compound 1 or Compound 2 is administered three times daily for five days at a dose of about 750 mg by weight of the free base.

[0083] (g) The dosing regimen of (a) or (b), wherein Compound 1 or Compound 2 is administered twice daily at a dose of about 1,000 mg by weight of the free base for five, six or seven days.

[0084] (h) The dosing regimen of any one of (a)-(g), wherein Compound 2 is administered to the patient.

[0085] (i) The dosing regimen of (h), wherein a dose of about 675 mg to 1,000 mg is administered in three solid dosage forms.

[0086] (j) The dosing regimen of any one of (a)-(g), wherein Compound 1 is administered to the patient.

[0087] (k) A method of treating dengue fever in a patient in need thereof, the method comprising administering Compound 1 or Compound 2 according to the dosing regimen of any one of (a) to (j).

[0088] (l) (k) The method of claim 1, wherein the dosing regimen comprises administering a pharmaceutical composition comprising Compound 1 or Compound 2.

[0089] (m) Use of the dosage regimen of any one of (a) to (k) in the preparation of a medicament for treating dengue fever.

[0090] (n) The use of (l) or (m), wherein the dosage regimen comprises administering a pharmaceutical composition comprising Compound 1 or Compound 2.

[0091] (o) A method of treating dengue fever in a patient in need thereof, the method comprising administering a compound of formula I according to the high dose regimen described herein.

[0092] (p) A method of treating dengue fever in a patient in need thereof, the method comprising administering a compound of Formula II, Formula IIA or Formula IIB according to the high-dose regimen described herein.

[0093] (q) a method for preparing compound 1 or 2, comprising reacting a compound of formula intermediate A or intermediate B with a urea-based activator (e.g., 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU)) to preferentially form S P The phosphoramidate intermediate C is then reacted in a second step by replacing the activated phenoxy leaving group with a phenol, thereby inverting the phosphorus stereochemistry to form compound 1 in the R-phosphorus diastereoisomeric configuration.

[0094] (r) The method of (q), wherein the substantially pure R-phosphorus diastereomer is about 93% or more, about 95% or more, about 98% or more, or even 99% or more. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Figure 1 The metabolic pathways of compound 2 (AT-752) and its pharmacologically active metabolite compound 5 (AT-9010) were elucidated.

[0097] Figure 2 The concentration-dependent inhibition curves of compound 1 (AT-281) against dengue virus type 2 (DENV), West Nile virus (WNV) and yellow fever virus (YFV) are shown. The x-axis is the logarithmic concentration of compound 1 (AT-281) and the y-axis measures the reduction in virus production, measured as a percentage of virus titer. The experimental procedure is provided in Example 2.

[0098] Figure 3 The in vitro formation of compound 5 (AT-9010) in human peripheral blood mononuclear cells is shown. PBMCs were incubated with 10 μM compound 1 (AT-281) for 6 hours. The prodrug was then removed, the cells were rinsed and incubated for another 28 hours (rinsing phase). At each time point, cells (n=3) were collected in ice-cold 60% MeOH and analyzed using LC-MS / MS assays for compound 5 (AT-9010), i.e., active intracellular TP. Data are expressed as mean + / - SD. The x-axis shows the time point measured in hours and the y-axis shows the time point measured in pmoles / 10 6 The experimental procedure is provided in Example 8.

[0099] Figure 4 Phosphorescence images of a 20% acrylamide:bisacrylamide (19:1), 7M urea sequencing gel are shown, representing the results of a nucleotide incorporation assay that observed the incorporation of compound 5 (AT-9010) in the presence and absence of GTP. The left half of the gel shows the incorporation of DENV-2 NS5 protein along T in the presence of ATP, UTP, and CTP (100 μM each) and increasing concentrations of compound 5 (AT-9010) (0-250 μM). 20 RNA template extension P 10 Time course of primers. The right half of the gel shows extension in the presence of all four NTPs (100 μM) when competing with compound 5 (AT-9010) (100–625 μM). The incorporation products of compound 5 (AT-9010) at positions +5 and +7 are represented by the lower dots, while the incorporation of native GTP at the same positions is represented by the upper dots. The experimental procedure is provided in Example 10.

[0100] Figure 5 Phosphorescence images of a 20% acrylamide:bisacrylamide (19:1), 7M urea sequencing gel are shown, showing the results of a nucleotide incorporation assay that observed the incorporation of the chain terminator 3'dGTP in the presence and absence of GTP. The left half of the gel shows the incorporation of the DENV-2 NS5 protein along the T in the presence of ATP, UTP, and CTP (100 μM each) and increasing concentrations of 3'dGTP (0-250 μM). 20 Time course of RNA template extension of P10 primer. The right half of the gel shows extension in the presence of all four NTPs (100 μM) in competition with 3'dGTP (100-625 μM). The incorporation products of 3'dGTP at the +5 and +7 positions are represented by the lower dots, while the incorporation of native GTP at the same positions is represented by the upper dots. The experimental procedure is provided in Example 10.

[0101] Fig. 6A A line graph showing the plasma concentrations of compound 1 (AT-281) and its metabolites measured after oral administration of compound 2 (AT-752) to mice. Male CD-1 mice were administered 420 mg / kg of compound 2 (AT-752). Blood samples were collected within 24 hours after administration, plasma was separated and analyzed by LC-MS / MS for compound 1 (AT-281), compound 3 (AT-551) and compound 6 (AT-273). The data are expressed as mean + / - SD (n = 3-5 / time point). The x-axis is the time after administration measured in hours, and the y-axis is the plasma concentration of metabolites measured in nanomoles / ml. The experimental procedure is provided in Example 14.

[0102] Figure 6B A line graph showing the plasma concentrations of compound 1 (AT-281) and its metabolites measured after oral administration of compound 2 (AT-752) to rats. Male SD rats were administered 300 mg / kg compound 2 (AT-752). Blood samples were collected within 24 hours after administration, plasma was separated and analyzed by LC-MS / MS for compound 1 (AT-281), compound 3 (AT-551) and compound 6 (AT-273). The data are expressed as mean + / - SD (n = 3-5 / time point). The x-axis is the time after administration measured in hours, and the y-axis is the plasma concentration of metabolites measured in nanomoles / ml. The experimental procedure is provided in Example 14.

[0103] Figure 6C A line graph showing the plasma concentrations of compound 1 (AT-281) and its metabolites measured after oral administration of AT-752 to monkeys. Male cynomolgus monkeys were administered 300 mg / kg of compound 2 (AT-752). Blood samples were collected within 24 hours after administration, plasma was separated and analyzed by LC-MS / MS for compound 1 (AT-281), compound 3 (AT-551) and compound 6 (AT-273). The data are expressed as mean + / - SD (n = 3-5 / time point). The x-axis is the time after administration measured in hours, and the y-axis is the plasma concentration of metabolites measured in nanomoles / ml. The experimental procedure is provided in Example 14.

[0104] Figure 7Results showing the in vivo formation of compound 5 (AT-9010) in PBMCs after oral administration of compound 2 (AT-752) to mice, rats and monkeys. Compound 1 (AT-281) was orally administered to mice and rats, and blood was collected at 4 and 12 hours (mice) and 24, 48 and 72 hours (rats). Monkeys were orally administered compound 2 (AT-752) at a dose equivalent to 55 mg / kg of compound 1 (AT-281), and blood was collected at 12, 24 and 48 hours. PBMCs were isolated, compound 5 (AT-9010) was extracted, and quantified using LC / MS / MS. Mouse PBMCs were collected from 3 animals at each time point; rat and monkey data are expressed as mean + / - SD (n = 2-3 per time point). The x-axis is the time after administration measured in hours, and the y-axis is picomoles / 10 6 The concentration of compound 5 (AT-9010) in PBMC was measured per cell. The experimental procedure is provided in Example 15.

[0105] Fig. 8A Viremia of DENV-infected mice treated with compound 2 (AT-752) is shown. AG129 mice challenged with DENV2 were treated orally with vehicle (control) or compound 2 (AT-752) (1000 mg / kg loading dose 4 hours before challenge, followed by 500 mg / kg BID dose for 7 consecutive days starting 1 hour after inoculation). Blood samples were collected on days 4, 6, 7, 8 and 10 after infection, and viral load was assessed by plaque assay. Dashed line = detection limit; data are expressed as mean + / - SD. The t-test results at each time point are *p<0.01. The x-axis represents the number of days after infection measured in days, and the y-axis is log 10 The amount of viremia measured in the blood is measured in PFU / mL. The experimental procedure is provided in Example 18.

[0106] Figure 8B Spleen viral loads of DENV-infected mice treated with compound 2 (AT-752) are shown. AG129 mice challenged with DENV2 were treated orally with vehicle (control) or compound 2 (AT-752) (1000 mg / kg loading dose 4 hours before attack, followed by 500 mg / kg BID dose for 7 consecutive days starting 1 hour after inoculation). Spleen samples were collected on days 4, 6, 7, 8, and 10 after infection, and viral load was assessed by plaque assay. Dashed line = detection limit; data are expressed as mean + / - SD. The t-test results at each time point are *p<0.01. The x-axis represents the number of days after infection measured in days, and the y-axis is log 10 The amount of virus in the spleen was measured in PFU / gram. The experimental procedure is provided in Example 18.

[0107] Fig.9A Show the weight changes of DENV infected mice treated with compound 2 (AT-752). AG129 mice attacked with DENV2 were orally administered with vehicle (control) or compound 2 (AT-752) for treatment (1000 mg / kg loading dose 4 hours before attack, followed by treatment with a BID dose of 500 mg / kg for 7 consecutive days starting 1 hour after inoculation). Body weight was recorded daily during the study. Body weight was compared with day 0 (baseline) and expressed as a percentage change. Data are expressed as mean + / - SD. The t-test results at each time point were *p<0.01. The x-axis represents the number of days after infection measured in days, and the y-axis is the weight change expressed as a percentage (relative to baseline). Experimental procedures are provided in Example 18.

[0108] Fig. 9B Show the health scores of DENV infected mice treated with compound 2 (AT-752). AG129 mice attacked with DENV2 were orally administered with vehicle (control) or compound 2 (AT-752) for treatment (1000 mg / kg loading dose 4 hours before attack, followed by treatment with a BID dose of 500 mg / kg for 7 consecutive days starting 1 hour after inoculation). Health scores were recorded daily during the study. Data are expressed as mean + / - SD. The t-test results at each time point were *p<0.01. The x-axis represents the number of days after infection measured in days, and the y-axis is the health score measured numerically, and the lower the score, the better the health status. The experimental procedure is provided in Example 18.

[0109] Fig.10 Kaplan-Meier survival curves of DENV-infected mice treated with compound 2 (AT-752) are shown. AG129 mice attacked with DENV2 were treated with vehicle (control) or compound 2 (AT-752) (1000 mg / kg loading dose 4 hours before attack, followed by treatment with a BID dose of 500 mg / kg for 7 consecutive days starting 1 hour after inoculation). Survival rates within 20 days after infection were calculated, excluding mice that were killed as planned for the collection of blood and spleen samples. The treatment group was significantly different from the control group, with p<0.001 according to the Mantel-Cox log-rank test. The x-axis represents the number of days after infection measured in days, and the y-axis is the survival rate. The experimental procedure is provided in Example 18.

[0110] Fig.11 is a line graph showing the plasma concentration of compound 1 determined by three daily dosings of 750 mg of compound 2. Throughout the treatment period, the plasma concentration was maintained at the EC value of compound 1 for dengue serotype 2. 90(approximately 200 ng / mL) or more. Six healthy participants were orally administered 13 doses of 750 mg of compound 2 every 8 hours (three times a day for 4 consecutive days, with one dose on the 5th day). Plasma samples were collected before dosing, within 8 hours after the first and eleventh doses, and within 120 hours after the last (thirteenth) dose for in-depth pharmacokinetic evaluation. Plasma samples were collected before each dose to determine trough concentrations. Plasma levels of compound 6, which is believed to be the intracellular active triphosphate metabolite of compound 5 ( Figure 1 ) is an alternative to .

[0111] Fig.12 : is a pharmacokinetic plot showing the concentration of Compound 1 in plasma over time. The doses tested were 250 mg, 500 mg, 500 mg (postprandial), 1000 mg, 1000 mg (for East Asian / Southeast Asian / South Asian patients), and 1500 mg. Plasma samples were collected at predetermined time points and quantitatively analyzed using LC-MS / MS. Data analysis can be found in Table 10 of Example 24. Plasma exposure of Compound 1 increased in a greater than dose-proportional manner over the dose range studied. A high-fat / high-calorie meal delayed the C of Compound 1. max , but had limited or no effect on total exposure. In fasting patients, Compound 1 was rapidly absorbed and eliminated.

[0112] Fig.13 It is a pharmacokinetic graph showing the concentration of compound 3 in plasma over time. The doses tested were 250 mg, 500 mg, 500 mg (after meal), 1000 mg, 1000 mg (for East Asian / Southeast Asian / South Asian patients) and 1500 mg. Plasma samples were collected at predetermined time points and quantitatively analyzed using LC-MS / MS. Data analysis can be found in Table 11 of Example 24. Similar to compound 1, the plasma exposure of compound 3 increased in a manner greater than dose proportional.

[0113] Fig.14It is a pharmacokinetic diagram showing the change in the concentration of compound 6 in plasma over time. The doses tested were 250mg, 500mg, 500mg (postprandial), 1000mg, 1000mg (for East Asian / Southeast Asian / South Asian patients) and 1500mg. Plasma samples were collected at predetermined time points and quantitatively analyzed using LC-MS / MS. Data analysis can be found in Example 24 Table 12. The plasma exposure of compound 6 increased in a manner less than dose proportional within the range of the study. The plasma level of compound 6 increased slowly and exhibited a longer elimination half-life (group average 23 hours). A high-fat / high-calorie diet slightly increased the exposure of compound 6. It was observed that the renal clearance of compound 6 exceeded the glomerular filtration rate, indicating that the active secretion process is involved in the renal clearance of this metabolite.

[0114] Fig.15 It is a pharmacokinetic graph showing the concentration of compound 7 in plasma over time. The doses tested were 250 mg, 500 mg, 500 mg (postprandial), 1000 mg, 1000 mg (for East Asian / Southeast Asian / South Asian patients) and 1500 mg. Plasma samples were collected at predetermined time points and quantitatively analyzed using LC-MS / MS. Data analysis can be found in Example 24 Table 13. The plasma exposure of compound 7 increased in a dose-dependent manner within the dose range studied. Compound 7 exhibited a slower elimination phase than compounds 2 or 3. The renal clearance of compound 7 was also higher than the glomerular filtration rate, indicating that active secretion processes are involved in the renal clearance of this metabolite.

[0115] Fig.16 . Demonstrate in vitro activity of AT-281 against DENV1-4 reporter genes, as described in Example 26. Serial dilutions of AT-281 and viruses modified with nanoluciferase reporter genes (MOI of 0.1 for DENV-1, DENV-2, and DENV-3; MOI of 0.001 for DENV-4) were added to each well inoculated with 1×10 4 Huh-7 cells were incubated in 96-well plates. After 48 hours of incubation, cells were washed and EC50 values ​​were determined by luciferase activity. Data were normalized to set DMSO-treated samples as 100%. Error bars represent mean ± SD. Results represent three independent experiments, each of which was analyzed in triplicate.

[0116] Fig.17 Thermostability shift experiments are shown, which show the stabilizing effect of GTP, AT-9010 or sinefungin on DENV serotypes 1 to 4 MTases, RdRp and NS5 proteins. The proteins were expressed and purified as described in Examples 27-30. Thermo-shift experiments were performed as described in Example 31. The protein itself was used as the baseline, and the values ​​were reported as ΔTm (ΔTm (°C)).

[0117] Fig.18 The functional and structural basis of AT-9010 inhibition of DENV MTase is shown. A) Dose response assays were performed using a filter binding assay (FBA). Reactions were incubated at 30°C for 30 minutes and enzyme activity was determined using FBA as described in Example 33. B) The overall structure of DENV3 MTase represented by a blue ribbon in complex with SAH and AT-9010 represented by sticks. AT-9010 is located in the Cap binding site. C) Omit map at 1σ of AT-9010. D) A close-up view of AT-9010 binding in the GTP binding site. The interacting residues are labeled and shown as sticks. E) Structural comparison of the positions where GTP (pink) and AT9010 (light brown) bind in the Cap binding site. The Cap binding site structure belongs to DENV2 (pink-PDB2P1D) and DENV3 (light brown) MTases and is conserved.

[0118] Fig.19A Incorporation and inhibition of AT-9010 in de novo initiation reactions of DENV2 NS5 are shown, as described in Example 34. A) PAGE analysis of de novo initiation reactions using a 10-nt template corresponding to the 3' end of the genome (AACAGGUUCU) resulted in the formation of pppApG, pppApGpA, and pppApGpApA over time. GTP was either replaced by AT9010 or AT9010 was added at increasing concentrations to reactions containing 100 μM GTP. B) Inhibition of de novo product formation by AT-9010 based on quantification of product bands from four (Mn) or two (Mg) independent reactions; corresponding IC50 values ​​are given.

[0119] Fig.19BDENV4 NS5 is shown to discriminate AT-9010, AT-9002 (2'-methyl-GTP) and SOF-TP from their natural nucleotide competitors GTP, GTP and UTP, respectively. A) Primer extension assay, as described in Example 34, allows multiple insertions of ATP, CTP and UTP in the absence, presence of GTP; or in the presence of a mixture of GTP and AT-9010, as shown below the autofluorescence plot of the gel. The properties of the conventionally inserted Watson-Crick nucleotides are shown on the right side of the gel. B) Primer extension assay, allowing the incorporation of single nucleotides GTP, AT-9010, AT-9002, UTP or SOF-TP. Using the Typhoon FluorImager, band products with sizes larger than the G / AT(+5) band were quantified and their sizes relative to the G / AT(+5) band were measured, allowing calculation of discrimination values. The incorporation rates of AT-901 and AT9002 were compared with that of GTP, and the incorporation rate of SOF-TP was compared with that of UTP. Fig.21A -D reported similar results for other DENV1-3 NS5.

[0120] Fig. 20A The RdRp active sites of HCV, DENV, and SARS-CoV-2 RdRp were compared. The HCV NS5b and sofosbuvir (PDB: 4WTG) cocrystal was superimposed on the DENV2 NS5 (PDB: 5ZQK). The operation of the modeling study is described in Example 35. The motif D and the amino acid side chains near the nucleotide phosphates are shown in the figure. The motif D region of HCV is shown. Metals A and B are represented by spheres near the catalytic aspartic acid of motifs A and C, respectively.

[0121] Fig. 20B The SARS-CoV-2nsp12 structure and AT-9010 are superimposed on DENV NS5 (PDB: 5ZQK) as described in Example 35. The motif D lysine amino acid side chains of SARS-CoV-2nsp12 and DENV NS5 are shown in the figure, respectively. The essential amino acids that contact the nucleotide phosphate are shown in the figure. The unique metal B is represented by spheres near the motif A and C catalytic aspartic acid, respectively (Shannon, A. et al., A second type of N7-guanine RNA capmethyltransferase in an unusual locus of a large RNA virus genome, Nucleic Acids Research, 2022, 50 (19) 11186-11198).

[0122] Fig.21A, 21B , 21C and 21D show primer extension assays that allow incorporation of single nucleotides GTP, AT-9010, AT-9002 (2'-methyl-GTP), UTP or SOF-TP as described in Example 34, but comparative reports are given for the four DENV NS5 serotypes. Measurement and quantification Fig.19B The incorporation rates of AT-9010 and 2'-methylguanosine triphosphate (AT-9002) were compared to that of GTP, and the incorporation rate of SOF-TP was compared to that of UTP.

[0123] Fig. 22 A primer extension assay is shown that allows for the incorporation of multiple nucleotides AT-9010 with all four NTPs or only with UTP, ATP and CTP as described in Example 34, but a comparison is reported for the four DENV NS5 serotypes.

[0124] Fig.23 Quantification of the gel shown in Figure 21 is shown, along with a graphical representation of the incorporation rates. The data provided in the graph can be found in Figure 21, and the procedure used to collect the data can be found in Example 34. The incorporation rates of AT-9010 and AT9002 are compared to that of GTP, and the incorporation rate of SOF-TP is compared to that of UTP.

[0125] Fig.24 The structures of Compound 1 and Compound 2 are shown. DETAILED DESCRIPTION OF THE INVENTION

[0127] It was unexpectedly found that when a high dose (i.e., at least 700-1000 mg per dose, twice, three times or more daily) of Compound 1 or Compound 2 (wherein Compound 1 may be a pharmaceutically acceptable salt thereof) is administered to a host (e.g., a human) in need thereof, it is beneficial for treating dengue virus. In certain embodiments, Compound 1 or Compound 2 may be provided in the form of a pharmaceutical composition.

[0128] In addition, it was found that compound 1 or compound 2 is also active against dengue serotypes 1, 3, and 4. Through this new discovery, it was confirmed that compound 1 and compound 2 in the high-dose regimen described herein are effective against all serotypes of dengue virus (dengue virus serotypes 1, 2, 3, and 4).

[0129]

[0130] The term "Compound 1" used in this specification may refer to its free base or a pharmaceutically acceptable salt thereof. Compound 1 is ((R)-(((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-alanine isopropyl ester.

[0131] Compound 1 (and its hemisulfate salt compound 2) was also found to have a favorable dual mechanism of action. The active metabolite is an inhibitor of the RNA-dependent RNA polymerase (RdRp) domain of the viral protein NS5, and also an inhibitor of the 2'-O-methyltransferase (MTase) domain of NS5. Both functions of the NS5 protein are essential for replication. Inhibition of two key protein functions involved in RNA replication may reduce the chance of forming resistant mutants.

[0132] Compounds 1 and 2 have a favorable dual mechanism of action. It is an inhibitor of the RNA-dependent RNA polymerase (RdRp) domain of the viral protein NS5, and also an inhibitor of the 2'-O-methyltransferase (MTase) domain of NS5. Both functions of the NS5 protein are required for replication. Inhibition of two key protein functions involved in RNA replication reduces the chances of resistant mutants forming.

[0133] Compound 2 (AT-752) is the hemisulfate salt of Compound 1 (AT-281), a phosphoramidate protein that serves as an intermediate prodrug to form the L-alanyl metabolite Compound 3 (AT-551), which is then converted to the 5'-monophosphate (MP) metabolite Compound 4 (AT-8001) of the nucleoside 2'-fluoro-2'-C-methylguanosine (Compound 6 (AT-273)). Compound 4 (AT-8001) is then phosphorylated to the active 5'-triphosphate (TP) metabolite Compound 5 (AT-9010). Compound 5 (AT-9010) has been shown to selectively inhibit the viral RNA-dependent RNA polymerase (RdRp) of HCV (Good et al., Preclinical evaluation of AT-527, a novel guanosine nucleotide prodrug with potent, pan-genotypic activity against hepatitis C virus. PLos One 15: e0227104), and is shown herein to inhibit dengue serotypes 1, 2, 3, and 4. One mechanism is inhibition of NS5, the RdRp of DENV-2.

[0134]

[0135]

[0136] In some aspects, a dosing regimen, method, composition and preparation process for treating a host infected with dengue virus are provided. For example, compound 1 or 2 or formula I or II can be administered at a dose of about 700-1000 mg, for example twice or three times a day, at a dose of 750 mg each time, used alone or in combination with another anti-RNA viral agent to treat an infected host in need thereof. In certain embodiments, it is useful to administer a drug combination that regulates the same or different pathways or inhibits different targets in the virus. Since compound 1 / 2 is a polymerase inhibitor, it is advantageous to administer compound 1 / 2 in combination with a protease inhibitor or an NS3-NS4B interaction inhibitor to the host. Alternatively, compound 1 / 2 can be administered in combination with a structurally different polymerase inhibitor.

[0137] Compound 1 / 2 may be administered orally, for example in the form of a pill or tablet, or by other routes deemed appropriate by the attending physician, including intravenous, transdermal, subcutaneous, topical, parenteral or other appropriate routes.

[0138] I. Definition

[0139] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and are not intended to be limiting.

[0140] Unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. In this application, the use of "or" means "and / or" unless otherwise stated. In addition, the use of the word "including" and other forms such as "includes" and "included" is not limiting.

[0141] A "patient" or "host" or "subject" is a human or non-human animal in need of treatment or prevention of dengue virus infection. Typically, the host is a human. "Patient" or "host" or "subject" also refers to, for example, mammals, primates (e.g., humans), cattle, sheep, goats, horses, dogs, cats, rabbits, rats, mice, birds, etc.

[0142] The term "preventative" or "prophylactic" when used refers to the administration of an active NS5 RNA-dependent RNA polymerase (RdRp) inhibitory compound to prevent, reduce the likelihood of the occurrence or recurrence of a dengue virus infection (e.g., dengue type 2 virus or dengue type 3 virus), or to minimize new infections compared to infections that would occur without such treatment, or to minimize transmission to others. The present invention includes therapeutic and prophylactic or preventative therapies. In some embodiments, an active NS5 RdRp inhibitory compound is administered to a host that has been exposed to a dengue virus infection (e.g., dengue type 1 virus, dengue type 2 virus, dengue type 3 virus, or dengue type 4 virus) and is therefore at risk of contracting a dengue virus infection. In another alternative embodiment, a method of preventing transmission is provided, comprising administering an effective amount of one of the compounds described herein to a human being for a sufficient period of time, including, for example, up to 3, 5, 7, 10, 12, 14 days or more before an infectious situation occurs, prior to exposure to a potentially infected population (including during travel or public events or meetings).

[0143] The terms "coadminister," "coadministration," or "combination" are used to describe the administration of an NS5 RdRp interfering compound in combination with at least one other antiviral agent. The timing of coadministration is best determined by the medical professional treating the patient. Sometimes it is desirable to administer the drugs simultaneously. Alternatively, the drugs selected for combination therapy are administered to the patient at different times. Of course, when more than one viral or other infection or other condition is present, the compounds of the invention can be combined with other agents as needed to treat the other infection or condition.

[0144] Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; alkali metal or organic salts of acidic residues such as carboxylic acids; and the like. Pharmaceutically acceptable salts include salts of the parent compound formed, for example, by inorganic or organic acids that are not toxic, and quaternary ammonium salts. For example, acid salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, aminosulfonic acid, phosphoric acid, nitric acid, and the like; and salts derived from organic acids such as 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, methanesulfonic acid, ethanesulfonic acid, besylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, HOOC-(CH2) n-COOH (wherein n is 0-4), etc., or salts prepared using different acids that produce the same counterion. Lists of other suitable salts can be found, for example, in Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Company, Easton, Pa., page 1418 (1985).

[0145] The compound can be delivered in the form of a salt in any molar ratio, as long as the desired effect is achieved. For example, the compound can be provided in the form of a counterion less than a molar equivalent, such as provided in the form of a hemisulfate salt. Alternatively, the compound can be provided in the form of a counterion greater than a molar equivalent, such as provided in the form of a disulfate salt. Non-limiting examples of molar ratios of the compound to the counterion include 1:0.25, 1:0.5, 1:1, and 1:2.

[0146] In certain embodiments, the term "about" means ± 5% of the listed value. In certain embodiments, the term "about" means ± 10% of the listed value.

[0147] I. Advantageous Dosage Forms of Compound 2 for Treatment of Dengue Virus

[0148] Compositions, methods and dosage forms for treating a host infected with dengue fever by administering an effective amount of Compound 1 or Compound 2 are provided.

[0149] In one aspect of the present invention, the pharmaceutical composition of the present invention comprises a high dose of compound 1 or 2 against dengue virus as described herein, optionally in combination with a pharmaceutically acceptable carrier, additive or excipient, and optionally in combination or alternating combination with at least one other active compound. In one embodiment, the present invention includes a solid dosage form of compound 2 in a pharmaceutically acceptable carrier.

[0150] In certain embodiments, Compound 1 or 2 is administered to a patient in need thereof in the form of a pharmaceutical composition (eg, Composition A or Composition B).

[0151] Composition A

[0152]

[0153]

[0154] Composition B

[0155]

[0156] In one aspect of the present invention, the pharmaceutical composition of the present invention comprises an anti-dengue high-dose effective amount of compound 1 or 2 as described herein, optionally in combination with a pharmaceutically acceptable carrier, additive or excipient, and further optionally in combination with at least one other antiviral agent (e.g., another anti-dengue agent).

[0157] The present invention includes a pharmaceutical composition comprising an effective amount of a compound 1 or 2 or a prodrug of the present invention for treating dengue virus infection, and a pharmaceutically acceptable carrier or excipient. In an alternative embodiment, the present invention includes a pharmaceutical composition comprising an effective amount of a compound 1 or 2 or a prodrug of the present invention for preventing dengue virus infection, and a pharmaceutically acceptable carrier or excipient.

[0158] One of ordinary skill in the art will recognize that the therapeutically effective upper dose will vary with the infection or condition being treated, its severity, the treatment regimen employed, the pharmacokinetics of the agent used, and the patient or subject (animal or human) being treated, and such therapeutic amount can be determined by the attending physician or specialist.

[0159] Compound 1 or 2 of the present invention can be mixed with a pharmaceutically acceptable carrier. In general, pharmaceutical compositions, particularly solid dosage forms, such as pills or tablets, are usually administered in oral form. Certain preparations can be administered parenterally, intravenously, intramuscularly, topically, transdermally, buccally, subcutaneously, suppositories or other routes, including intranasal sprays. Intravenous and intramuscular preparations are usually administered with sterile saline. Those of ordinary skill in the art can modify the preparation to make it more soluble in water or other carriers, such as this can be easily achieved by minor modifications (salt preparations, esterification, etc.) known to those of ordinary skill in the art. The technician is also fully capable of modifying the route of administration and the dosing regimen of compound 2 in order to manage the pharmacokinetics of the present compound, thereby producing the greatest beneficial effect on the patient, as described in more detail herein.

[0160] Compound 1 or 2 can be used, for example, for oral or parenteral administration. In certain embodiments, Compound 1 or 2 is provided in a solid, gel or liquid dosage form. In certain embodiments, Compound 1 or 2 is provided in a liquid dosage form for parenteral administration. In certain embodiments, Compound 1 or 2 is provided in a liquid dosage form for intravenous administration. In certain embodiments, Compound 1 or 2 is provided in a solid dosage form for oral administration. In certain embodiments, Compound 1 or 2 can be provided in a soft shell capsule or tablet for oral administration.

[0161] The high doses of Compound 1 or 2 contained in the therapeutically active formulations of the present invention are described in detail herein, which are effective amounts to achieve the desired results of the present invention, such as for treating DENV infection, reducing the likelihood of DENV infection, or inhibiting, reducing and / or eliminating DENV or its side effects, including disease states, conditions and / or complications caused by DENV.

[0162] Unless otherwise indicated, the dosages described herein refer to the amount of active pharmaceutical ingredient in the dosage plus the weight of the pharmaceutically acceptable salt. For example, when the molecular weight of the hemisulfate salt is taken into account in the measurement, a 750 mg dose of Compound 2 contains approximately 692 mg of Compound 1 (within the "about 700 mg" range).

[0163] Generally speaking, when measured as a free base, the therapeutically effective amount of Compound 2 in a pharmaceutical dosage form can range from about 600 mg to about 1,000 mg, about 700 mg to about 1,000 mg, or about 700 mg to about 800 or 850 mg. In certain embodiments, Compound 1 is administered in the same range as the free base.

[0164] In certain embodiments, the dosage of Compound 2 is at least about 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1,000 mg, 1,150 mg, 1,200 mg, 1,250 mg, 1,300 mg, 1,350 mg, 1,400 mg, 1,450 mg or 1,500 mg when measured in free base form, twice or three times daily. In some embodiments, the dosage is provided in several dosage forms to reduce the size of a solid dosage form (e.g., a pill, tablet or capsule).

[0165] In certain embodiments, about 700 mg to about 800 or 850 mg of Compound 1 or 2 is administered once, twice, or three times daily (QD). In certain embodiments, about 700 mg to about 800 mg of Compound 1 or 2 is administered once, twice, or three times daily (QD). In certain embodiments, about 800 mg to about 900 mg of Compound 2 is administered once daily (QD). In certain embodiments, about 900 mg to about 1,000 mg of Compound 2 is administered once daily (QD).

[0166] In certain embodiments, about 700 mg to about 800 mg of Compound 2 is administered twice daily (BID). In certain embodiments, about 700 mg to about 900 mg of Compound 2 is administered twice daily (BID). In certain embodiments, about 800 mg to about 900 mg of Compound 2 is administered twice daily (BID). In certain embodiments, about 900 mg to about 1,000 mg of Compound 2 is administered twice daily (BID).

[0167] In certain embodiments, about 600 mg to about 700 mg of Compound 2 is administered three times daily (TID). In certain embodiments, about 700 mg to about 800 mg of Compound 2 is administered three times daily (TID). In certain embodiments, about 800 mg to about 900 mg of Compound 2 is administered three times daily (TID). In certain embodiments, about 900 mg to about 1,000 mg of Compound 2 is administered three times daily (TID).

[0168] In certain embodiments, about 700 mg to about 900 mg of Compound 2 is administered four times a day (QID). In certain embodiments, about 600 mg to about 700 or 750 mg of Compound 2 is administered four times a day (QID). In certain embodiments, about 700 mg to about 800 mg of Compound 2 is administered four times a day (QID). In certain embodiments, about 800 mg to about 900 mg of Compound 2 is administered three or four times a day (QID). In certain embodiments, about 900 mg to about 1,000 mg of Compound 2 is administered four times a day (QID).

[0169] For purposes of the present invention, a prophylactically effective amount of the compositions of the present invention is within the same high dosage concentration range as the therapeutically effective amount described above, and is generally the same as the therapeutically effective amount.

[0170] In certain embodiments, the dengue virus is serotype 1, 3 or 4. In certain embodiments, the dengue virus is serotype 1, 3 or 4, and about 700 mg to about 1000 mg of compound 1 or 2 is administered once daily (QD). In certain embodiments, the dengue virus is serotype 1, 3 or 4, and about 550 mg to about 750 mg of compound 2 is administered once daily (QD). In certain embodiments, the dengue virus is serotype 1, 3 or 4, and about 600 mg of compound 1 or 2 is administered once daily (QD).

[0171] In certain embodiments, the dengue virus is serotype 1, 3 or 4, and about 500 mg to about 800 mg of compound 1 or 2 is administered twice daily (BID). In certain embodiments, the dengue virus is serotype 1, 3 or 4, and about 550 mg to about 750 mg of compound 1 or 2 is administered twice daily (BID). In certain embodiments, the dengue virus is serotype 1, 3 or 4, and about 600 mg of compound 1 or 2 is administered twice daily (BID).

[0172] In certain embodiments, the dengue virus is serotype 1, 3 or 4, and about 500 mg to about 800 mg of compound 1 or 2 is administered three times daily (TID). In certain embodiments, the dengue virus is serotype 1, 3 or 4, and about 550 mg to about 750 mg of compound 1 or 2 is administered three times daily (TID). In certain embodiments, the dengue virus is serotype 1, 3 or 4, and about 600 mg of compound 1 or 2 is administered three times daily (TID).

[0173] The administration of compound 1 or 2 can be continuous (intravenous drip) to several oral or intranasal administrations (e.g., twice, three times, four times or five times a day) or transdermal administration, and can include oral, topical, parenteral, intramuscular, intravenous, subcutaneous, transdermal (which may include permeation enhancers), buccal and suppository administration and other administration routes. Enteric-coated oral tablets can also be used to enhance the bioavailability of oral administration of compounds. The most effective dosage form will depend on the bioavailability / pharmacokinetics of the selected specific agent and the severity of the patient's disease. Oral dosage forms are particularly preferred because they are easy to administer and are expected to have good patient compliance.

[0174] In order to prepare the pharmaceutical composition of the present invention, the therapeutically effective amount of the compound 1 or 2 of the present invention can be fully mixed with a pharmaceutically acceptable carrier to generate a dosage according to conventional pharmaceutical formulation techniques. The carrier can take a variety of forms, depending on the desired form of the administration preparation, such as oral or parenteral administration. When preparing a pharmaceutical composition of an oral dosage form, any commonly used pharmaceutical excipients and components can be used. Therefore, for liquid oral preparations, such as suspensions, elixirs and solutions, suitable carriers and additives can be used, including water, ethylene glycol, oil, alcohol, flavoring agents, preservatives, colorants, etc. For solid oral preparations such as powders, tablets, capsules, and solid preparations such as suppositories, suitable carriers and additives can be used, including starch, sugar carriers such as glucose, sucrose, lactose and related carriers, diluents, granulating agents, lubricants, adhesives, disintegrants, etc. If necessary, tablets or capsules can be enteric coated or sustained-released by standard techniques. The use of these dosage forms can significantly improve the bioavailability of the compound in the patient.

[0175] For parenteral preparations, the carrier generally includes sterile water or sodium chloride aqueous solution, but may also contain other ingredients, including ingredients that aid dispersion. Of course, when sterile water is used and sterility is maintained, the composition and carrier must also be sterilized. Injectable suspensions can also be prepared, in which case appropriate liquid carriers, suspending agents, etc. can be used.

[0176] Liposomal suspensions (including liposomes targeted to viral antigens) may also be prepared by conventional methods to produce pharmaceutically acceptable carriers. This may be suitable for delivery of free nucleosides, acyl / alkyl nucleosides or phosphate prodrug forms of nucleoside compounds according to the invention.

[0177] In an exemplary embodiment according to the present invention, Compound 1 or 2 and the composition are used to treat, prevent or delay dengue infection or secondary disease states, symptoms or complications of dengue.

[0178] Isotope substitution

[0179] The present invention includes compounds and uses of Compound 1 or Compound 2 having a desired isotopic substituent having an atomic number greater than the natural abundance of the isotope (i.e., enriched). Isotopes are atoms having the same atomic number but different mass numbers, i.e., having the same number of protons but different numbers of neutrons. As a general example and not limitation, isotopes of hydrogen such as deuterium ( 2 H) and tritium ( 3 H) can be used at any position in the structure. Alternatively, or in addition, isotopes of carbon can be used, such as 13 C and 14 C. Preferred isotopic substitution is the replacement of hydrogen with deuterium at one or more positions on the molecule to improve the performance of the drug. Deuterium can be incorporated at a position where the bond breaks during metabolism (α-deuterium kinetic isotope effect) or near or adjacent to the bond break site (β-deuterium kinetic isotope effect). Achillion Pharmaceuticals, Inc. (WO / 2014 / 169278 and WO / 2014 / 169280) describes deuteration of nucleotides to improve their pharmacokinetics or pharmacodynamics, including at position 5 of the molecule.

[0180] Substitution with isotopes such as deuterium may provide certain therapeutic advantages due to greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements. Substitution of deuterium for hydrogen at a metabolic breakdown site may reduce the metabolic rate of that bond or eliminate the metabolism of that bond. At any position in a compound where a hydrogen atom may be present, the hydrogen atom may be any isotope of hydrogen, including protium ( 1 H), deuterium ( 2 H) and tritium ( 3 H). Therefore, compounds referred to herein encompass all potential isotopic forms, unless the context clearly dictates otherwise.

[0181] The term "isotopically labeled" analog refers to a "deuterated analog," 13 C-labeled analogues” or “deuterated / 13 The term "deuterated analog" refers to a compound described herein in which the H isotope (i.e., hydrogen / tritium (1 H)) is replaced by H isotopes (i.e. deuterium ( 2 In some embodiments, the deuterated nucleoside is substituted with at least one deuterium. The deuterated nucleoside may be substituted with at least one deuterium. The deuterated nucleoside may be substituted with at least one deuterium. The deuterated nucleoside may be substituted with at least one deuterium. The deuterated nucleoside may be substituted with at least one deuterium. The deuterated nucleoside may be substituted with at least one deuterium. The deuterated nucleoside may be substituted with at least one deuterium. In some embodiments, the isotopic enrichment of the isotope at any position of interest is 90%, 95%, or 99% or more. In some embodiments, the enrichment of deuterium at the desired position is 90%, 95%, or 99%. Unless otherwise stated, the deuteration degree at the selected position is at least 80%. The deuteration of a nucleoside may occur on any replaceable hydrogen to provide the desired result.

[0182] In certain aspects of the present invention, a compound of formula I or a pharmaceutically acceptable salt thereof, optionally in a pharmaceutically acceptable carrier, is administered in a high-dose effective amount as described herein to treat dengue virus infection in a host (usually a human) in need thereof;

[0183]

[0184] in:

[0185] R 2 C 1-6 Alkyl (including methyl, ethyl, propyl and isopropyl), C 3-7 cycloalkyl, aryl, aryl(C1-C4alkyl)-, heteroaryl, heteroaryl(C1-C4alkyl)- or heteroalkyl, each of which is optionally substituted by 1, 2, 3 or 4 substituents, the substituents being independently selected at each occurrence from R 22 ;

[0186] R 3 is hydrogen or C 1-6 alkyl, which is optionally substituted with 1, 2, 3 or 4 substituents, said substituents being independently selected at each occurrence from R 33 ;

[0187] R 4a and R 4b are independently selected from hydrogen, C 1-6 Alkyl, aryl (C 1-6 Alkyl)-, C 3-7 Cycloalkyl, C 3-7 Cycloalkyl (C 1-6 Alkyl)-, heteroaryl (C 1-6 alkyl)- and aryl, each of which is optionally substituted by 1, 2, 3 or 4 substituents, said substituents being independently selected at each occurrence from R 44 ;

[0188] R 5 For hydrogen, C 1-6 Alkyl (including methyl, ethyl, propyl and isopropyl), C 1-6Haloalkyl or C 3-7 cycloalkyl, and in an alternative embodiment, R 5 is aryl(C1-C4alkyl)-, aryl, heteroaryl or heteroalkyl, which is optionally substituted by 1, 2, 3 or 4 substituents, the substituents being independently selected in each case from R 55 ;

[0189] R 22 , R 33 , R 44 and R 55 is independently selected at each occurrence from hydrogen, C 1- C6 alkyl, C 3- C7 cycloalkyl, alkenyl, alkynyl, F, Cl, Br, I, heterocycle, heterocycloalkyl, haloalkoxy, haloalkyl, C(O)R 66 、C(O)OR 66 、C(O)N(R 66 )2 and cyano;

[0190] R 66 is independently selected at each occurrence from hydrogen, C1-C6-alkyl, aryl(C1-C6-alkyl)-, C3-C7-cycloalkyl, C3-C7-cycloalkyl(C1-C6-alkyl)-, heteroaryl(C1-C6-alkyl)- and aryl.

[0191] All R groups are to be interpreted in a manner that does not include redundancy, i.e., an alkyl group is not substituted by an alkyl group, as is known in the art; however, for example, an alkoxy group substituted by an alkoxy group is not redundant. Aryl groups substituted by aryl groups fall within the definition of aryl groups to a limited extent. Unless the context clearly indicates otherwise, R groups are not optionally substituted.

[0192] Non-limiting examples of C1-C6 alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, sec-butyl, isobutyl, -CH2C(CH3)3, -CH(CH2CH3)2, and -CH2CH(CH2CH3)2. Non-limiting examples of C3-C6 cycloalkyl groups include cyclopropyl, CH2-cyclopropyl, cyclobutyl, and CH2-cyclobutyl.

[0193] A non-limiting example of an aryl (C1-C4 alkyl)- is benzyl. A non-limiting example of an aryl is phenyl and naphthyl.

[0194] In certain embodiments, Formula I is:

[0195]

[0196] In certain aspects of the present invention, a compound of formula II or a pharmaceutically acceptable salt thereof, optionally in a pharmaceutically acceptable carrier, is administered in an effective amount to treat dengue virus infection in a host (usually a human) in need thereof; and all variables are as defined herein:

[0197]

[0198] In certain aspects, the compound is a compound of Formula IIA or a pharmaceutically acceptable salt thereof:

[0199]

[0200] wherein all variables are as defined herein.

[0201] In certain embodiments, the compound of Formula IIA is selected from:

[0202]

[0203] In certain aspects, the compound is a compound of Formula IIB or a pharmaceutically acceptable salt thereof:

[0204]

[0205] wherein all variables are as defined herein.

[0206] In certain embodiments, the compound of Formula IIB is selected from:

[0207]

[0208] In certain embodiments, the compound of Formula II, Formula IIA, or Formula IIB is used to treat infection by DENV1, DENV2, DENV3, or DENV4.

[0209] In certain embodiments, R 4a The side chains are selected to be naturally occurring amino acids, for example:

[0210]

[0211]

[0212] In certain alternative embodiments, the variable R in Formula II, IIA or IIB is 4a For C7-C 15 Alkyl, variable R 4b is hydrogen, and all other variables are as defined herein. In alternative embodiments, the compound is selected from

[0213]

[0214]

[0215] II. Treatment or prevention methods

[0216] As used herein, treatment refers to administering Compound 1 or Compound 2 to a host, such as a human who is infected or may be infected with dengue virus.

[0217] The term "preventive" or prophylactic, when used, refers to the administration of compound 1 or 2 to prevent or reduce the likelihood of viral disease. The present invention includes therapeutic and prophylactic or preventive therapies. In one embodiment, compound 1 or 2 is administered to a host that has been exposed to dengue virus infection and is therefore at risk of infection.

[0218] The present invention relates to a method for treating or preventing dengue virus, including drug-resistant and multidrug-resistant dengue virus and related disease states, conditions or complications. The method comprises administering an effective amount of compound 1 or 2 as described herein to a host (usually a human) in need thereof, optionally in combination with at least one additional bioactive agent (e.g., an additional anti-dengue virus agent), and further in combination with a pharmaceutically acceptable carrier additive and / or excipient.

[0219] In another aspect, the invention is a method of preventing or treating a dengue infection or a disease state of a dengue infection or a related or subsequent disease state, condition or complication.

[0220] (1) In certain embodiments, a dosing regimen for treating a human host infected with dengue virus in need thereof or for preventing dengue virus is provided, which comprises administering about 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1075, 1100, 1125, 1150, 1175, 1200, 1225 or 1250 mg of Compound 1 or Compound 2 twice, three times or four times a day.

[0221] (2) In certain embodiments, a dosing regimen for treating a human host infected with dengue virus in need thereof or for preventing dengue virus is provided, which comprises administering about 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1075, 1100, 1125, 1150, 1175, 1200, 1225 or 1250 mg of Compound 1 or 2 twice, three times or four times a day.

[0222] (3) In certain embodiments, a dosing regimen for treating a human host infected with dengue virus in need thereof or for preventing dengue virus is provided, which comprises administering about 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1075, 1100, 1125, 1150, 1175, 1200, 1225 or 1250 mg of Compound 1 or 2 twice, three times or four times a day.

[0223] (4) Any one of embodiments (1) to (3), wherein Compound 1 or Compound 2 is administered twice daily.

[0224] (5) Any one of embodiments (1) to (3), wherein Compound 1 or Compound 2 is administered three times a day.

[0225] (6) Any one of embodiments (1) to (3), wherein Compound 1 or Compound 2 is administered four times a day.

[0226] (7) Any one of embodiments (1)-(6), wherein about 600 mg of Compound 1 or Compound 2 per dose is administered to a patient in need thereof.

[0227] (8) Any one of embodiments (1)-(6), wherein about 650 mg of Compound 1 or Compound 2 per dose is administered to a patient in need thereof.

[0228] (9) Any one of embodiments (1)-(6), wherein about 700 mg of Compound 1 or Compound 2 per dose is administered to a patient in need thereof.

[0229] (10) Any one of embodiments (1)-(6), wherein about 750 mg of Compound 1 or Compound 2 per dose is administered to a patient in need thereof.

[0230] (11) Any one of embodiments (1)-(6), wherein about 800 mg of Compound 1 or Compound 2 per dose is administered to a patient in need thereof.

[0231] (12) Any one of embodiments (1)-(6), wherein about 850 mg of Compound 2 or Compound 1 per dose is administered to a patient in need thereof.

[0232] (13) Any one of embodiments (1)-(6), wherein about 900 mg of Compound 1 or Compound 2 per dose is administered to a patient in need thereof.

[0233] (14) Any one of embodiments (1)-(6), wherein about 950 mg of Compound 1 or Compound 2 per dose is administered to a patient in need thereof.

[0234] (15) Any one of embodiments (1)-(6), wherein about 1000 mg of Compound 1 or Compound 2 per dose is administered to a patient in need thereof.

[0235] (16) Any one of embodiments (1) to (15), wherein composition A is administered.

[0236] (17) Any one of embodiments (1) to (15), wherein composition B is administered.

[0237] (18) Any one of embodiments (1) to (17), wherein Compound 1 or Compound 2 is administered for three consecutive days.

[0238] (19) Any one of embodiments (1) to (17), wherein Compound 1 or Compound 2 is administered for four consecutive days.

[0239] (20) Any one of embodiments (1) to (17), wherein Compound 1 or Compound 2 is administered for five consecutive days.

[0240] (21) Any one of embodiments (1) to (17), wherein Compound 1 or Compound 2 is administered for six consecutive days.

[0241] (22) Any one of embodiments (1) to (17), wherein Compound 1 or Compound 2 is administered for seven consecutive days.

[0242] (23) Any one of embodiments (1) to (17), wherein Compound 1 or Compound 2 is administered for eight consecutive days.

[0243] (24) Any one of embodiments (1) to (17), wherein Compound 1 or Compound 2 is administered for nine consecutive days.

[0244] (25) Any one of embodiments (1) to (17), wherein Compound 1 or Compound 2 is administered continuously for ten days.

[0245] (26) Any one of embodiments (1) to (17), wherein Compound 1 or Compound 2 is administered continuously for eleven days.

[0246] (27) Any one of embodiments (1) to (17), wherein Compound 1 or Compound 2 is administered continuously for twelve days.

[0247] (28) Any one of embodiments (1) to (17), wherein Compound 1 or Compound 2 is administered continuously for thirteen days.

[0248] (29) Any one of embodiments (1) to (17), wherein Compound 1 or Compound 2 is administered continuously for fourteen days.

[0249] (30) Any one of embodiments (1)-(29), wherein the method is used prophylactically in healthy patients to prevent infection.

[0250] (31) Any one of embodiments (1)-(29), wherein the method is used to treat a patient infected with dengue virus.

[0251] (32) Any one of embodiments (1)-(29), wherein the human is suffering from dengue serotype 1.

[0252] (33) Any one of embodiments (1)-(29), wherein the human is suffering from dengue serotype 2.

[0253] (34) Any one of embodiments (1)-(29), wherein the human is suffering from dengue serotype 3.

[0254] (35) Any one of embodiments (1)-(29), wherein the human is suffering from dengue serotype 4.

[0255] In an alternative embodiment, compound 2 is provided as a phosphoramidate of compound 1 (rather than the specific phosphoramidate described in the compound description), such as a hemisulfate salt of Formula I or II. A variety of phosphoramidates, including various esters and phosphates, are known to those skilled in the art, and any combination of these can be used to provide the active compounds described herein in the form of a hemisulfate salt.

[0256] VI. Combination and alternation therapy

[0257] It is well known that resistant viral variants will emerge after long-term treatment with antiviral drugs. Resistance is sometimes caused by mutations in genes encoding enzymes used by the virus to replicate. The efficacy of the drug against DENV infection can be prolonged, enhanced, or restored by combining or alternating with another antiviral compound (or even two or three other antiviral compounds) that induces different mutations or acts through different pathways than the main drug. Alternatively, the pharmacokinetics, biodistribution, half-life, or other parameters of the drug may be altered by such combination therapy (which may include alternation therapy if it is considered synergistic). Since the disclosed compounds 2 and 1 are NS5 polymerase inhibitors, it may be useful to administer the compound to the host in combination with, for example, the following substances:

[0258] (1) Protease inhibitors, such as NS2B-NS3 protease inhibitors;

[0259] (2) NS3 inhibitors;

[0260] (3) NS3-NS4B interaction inhibitors;

[0261] (4) NS4B inhibitors;

[0262] (5) NS3-NS5 interaction inhibitors;

[0263] (6) another NS5 polymerase inhibitor;

[0264] (7) interferon α-2a (may be pegylated or otherwise modified) and / or ribavirin;

[0265] (8) Non-substrate inhibitors;

[0266] (9) Helicase inhibitors;

[0267] (10) antisense oligodeoxynucleotide (S-ODN);

[0268] (11) Aptamer;

[0269] (12) Nuclease-resistant ribozymes;

[0270] (13) iRNA, including microRNA and siRNA;

[0271] (14) Antibodies, partial antibodies or domain antibodies to viruses, or

[0272] (15) Viral antigens or parts of antigens that induce host antibody responses.

[0273] Non-limiting examples of dengue DAA drugs that can be administered in combination with high doses of Compound 1 or 2 or Formula I or II of the present invention (alone or with multiple drugs from these lists) are:

[0274] (i) Protease inhibitors, such as Telaprevir Boceprevir (Victrelis TM ), simeprevir (Olysio TM ), paritaprevir (ABT-450), glecaprevir (ABT-493), ritonavir (Norvir), ACH-2684, AZD-7295, BMS-791325, danoprevir, feribuvir, GS-9256, GS-9451, MK-5172, Setrobuvir, Sovaprevir, Tegobuvir, VX-135, VX-222, and ALS-220;

[0275] (ii) NS3 inhibitors, such as suramin, dabrafenib, idelalisib, nintedanib, ivermectin, or BP13944

[0276] (iii) NS3-NS4B interaction inhibitors, such as JNJ-1802 and JNJ-A07;

[0277] (iv) NS4B inhibitors, such as NITD-688 or lycorine;

[0278] (v) NS5 inhibitors, such as balaprevir;

[0279] (vi) HCV NS5B inhibitors, such as AZD-7295, clemizole, dasabuvir (Exviera), ITX-5061, PPI-461, PPI-688, sofosbuvir MK-3682, ABT-333, MBX-700, and mecitabine;

[0280] (vii) Antiviral combination drugs, such as Harvoni (ledipasvir / sofosbuvir); Viekira Pak (ombitasvir / paritaprevir / ritonavir / dasabuvir); Viekirax (ombitasvir / paritaprevir / ritonavir); G / P (paritaprevir and glecaprevir); Technivie (ombitasvir / paritaprevir / ritonavir) and Epclusa (sofosbuvir / velpatasvir) and Zepatier (elbasvir and grazoprevir). Example

[0281] Example 1. Cell lines, viruses and test compounds

[0282] The cell lines, viruses, and compounds described herein were used to generate the data shown in Examples 2, 3, 4, 5, 6, 7, and 8. Huh-7 (human hepatoma; AcceGen Biotechnology, Fairfield, NJ) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 mg / mL penicillin and 100 mg / mL streptomycin (Lonza, Walkersville, MD). Vero 76 cells (American Type Culture Collection (ATCC), Manassas, VA) used for virus yield reduction experiments were maintained in a similar manner. BHK-21 (baby hamster kidney; ATCC, Manassas, VA) cells were maintained in Earle's salts minimal essential medium (EMEM) containing 1 mM sodium pyruvate and 25 μg / mL kanamycin and supplemented with 10% FBS. All cell cultures were maintained at 37°C, in 5% CO2 and > The cells were maintained at 95% humidity. Infections were performed in EMEM supplemented with 5% FBS and 50 μg / mL gentamicin.

[0283] Dengue viruses (DENV-2NGC and DENV-3H87) were obtained from ATCC (Manassas, VA), and DENV-2 used in human PBMC and BHK-21 experiments was a clinical isolate. Japanese encephalitis virus (JEV SA-14), West Nile virus (WN02 Kern515), yellow fever virus (YFV 17D), and Zika virus (ZIKV MR766) were obtained from the University of Texas Medical Branch (Galveston, TX), while Powassan virus (POWV Spooner and LB strains) and Usutu virus (USUV TC-508) were obtained from the World Reference Center for Emerging Viruses and Arboviruses at the University of Texas Medical Branch. For studies using fresh human PBMCs, cells from a single male donor (lot LS-88-45477C) were obtained from BioIVT (Westbury, NY) or isolated from blood collected from healthy donors using Ficoll-Paque (Pham et al., 2008. Hepatitis C virus replicates in the same immune cell subsets in chronic hepatitis C and occult infection. Gastroenterology. 134: 812-22, Pham et al., 2004. Hepatitis C virus persistence after spontaneous or treatment-induced resolution of hepatitis C. J Virol 78: 5867-74). DENV-2 studies in PBMCs and BHK21 cells were completed at the University of Cagliari (Monserrato, Italy). Other viral efficacy and specificity studies were performed by Utah State University (Logan, UT) and ImQuest BioSciences (Frederick, MD). The latter laboratory's protocol for different RNA and DNA viruses (HCV, HIV-1, influenza A and B viruses, RSV, rhinovirus, herpes simplex-1, and adenovirus) has been previously described (Good et al., 2020. Preclinical evaluation of AT-527, a novel guanosine nucleotide prodrug with potent, pan-genotypic activity against hepatitis C virus. PLos One 15:30227104).Compound 2 (AT-752) and its free base Compound 1 (AT-281) were synthesized using a stereospecific process and prepared for Atea Pharmaceuticals by Topharman Shanghai Co., Ltd. (Shanghai, China) along with its plasma metabolite Compound 6 (AT-273). Compound 5 (AT-9010) and the TP internal standard used to quantify Compound 5 (AT-9010) were synthesized by NuBlocks (Oceanside, CA). Stock solutions were prepared in DMSO and stored at -20°C. Frozen plasma from humans and SD rats (Bioreclamation IVT, Westbury, NY) and cynomolgus monkeys (Xishan, Suzhou, China) were purchased to test the stability of Compound 1 (AT-281) at 37°C.

[0284] Example 2. Compound 1 inhibits dengue virus (DENV) and other flaviviruses.

[0285] The antiviral activity of Compound 1 (AT-281) against flaviviruses DENV-2NGC, DENV-3, JEV, POWV, USUV, WNV, YFV and ZIKV was evaluated using the neutral red dye uptake assay (described in Example 3) to determine the inhibition of virus-induced and compound-induced cytopathic effect (CPE), and the virus yield reduction (VYR) assay (described in Example 4) was used as a second independent assay for inhibition of viral replication. Human hepatoma (Huh-7), baby hamster kidney (BHK-21) or human peripheral blood mononuclear cells (PBMC) were acutely infected with different viruses and exposed to serial dilutions of the drug (described in Example 5). The activity of Compound 1 (AT-281) in infected cells was measured using the neutral red assay and / or the virus yield reduction (VYR) assay to determine the time required to achieve 50% inhibition (EC) of virus-induced cytopathic effect (CPE). 50 ) The effective concentration required to reduce the virus production by 1 log 10 The concentration (EC 90 ) and the cytotoxic concentration of the drug that causes death of 50% of the viable cells not infected with the virus (CC 50 ) (see Table 1). After 3 to 6 days of culture, compound 1 (AT-281) (2'-fluoro-2'-C-methylguanosine nucleotide prodrug) achieved 50% inhibition of virus-induced cytopathic effect (CPE) (EC 50 ) The effective concentration range required is 0.19 to 1.41 μM (see table on the next page).

[0286] Table 1A. Antiviral activity of compound 1 (AT-281) in cells infected with various flaviviruses

[0287]

[0288]

[0289] a Neutral red assay b VYR assay c The highest concentration detected was not determined.

[0290] Table 1B. Antiviral activity of compound 1 (AT-281) in cells infected with various serotypes of dengue virus

[0291]

[0292] a Neutral red assay

[0293] Although the growth kinetics of different flaviviruses in cell culture may vary, compound 1 (AT-281) showed similarly potent effects in reducing the viral titers of all seven flaviviruses (including 11 different strains tested in vitro) (Table 1A) and also maintained antiviral activity against dengue serotypes 1, 2, 3, and 4 of all tested strains (Table 1B). Inhibition curves for DENV, WNV, and YFV ( Figure 2 ) demonstrates this consistency. The concentration of compound 1 (AT-281) required to kill 50% of cells exposed to the drug alone (CC 50 ) exceeded the highest concentration tested (85 to 170 μM), and the antiviral selectivity index (SI; CC 50 / EC 50 ) ranged from >120 to >650. Specifically, compound 1 (AT-281) exhibited potent multi-serotype anti-DENV activity in vitro. In Huh-7 cells infected with DENV-2 (New Guinea C strain), compound 1 (AT-281) reduced the production of infectious virus by 90% at a concentration of 0.64M (EC 90 Similarly, compound 1 (AT-281) exhibited EC values ​​of 0.60, 0.63, and 0.77 μM in inhibiting virus-induced CPE in human PBMCs and BHK-21 cells infected with DENV2 (clinical isolate) and Huh-7 cells infected with DENV-3 (H87 strain), respectively. 50 In each case, the SI was >210.

[0294] To evaluate the antiviral specificity of compound 1 (AT-281), serial dilutions were incubated with various host cell lines infected with a panel of DNA and RNA viruses other than flaviviruses. The activity of compound 1 (AT-281) in infected cells was measured using a neutral red assay to determine the effective concentration (EC 50 ) (as described in Example 3) and the threshold concentration required to produce a significant effect on 50% of cells not infected with the virus (CC 50 Compound 1 (AT-281) had no activity against the DNA viruses tested and had weak or no activity against several RNA viruses (Table 2). However, it was highly selective for HCV (>2,000), inhibiting the virus at nanomolar levels (Table 2). Compound 1 (AT-281) was evaluated for its potential cytotoxicity in multiple cell lines, and the results showed that CC 50 The values ​​were greater than 100 μM (Table 2 below).

[0295] Table 2. Antiviral selectivity of compound 1 (AT-281) against a panel of RNA and DNA viruses

[0296]

[0297] The lack of cytotoxicity was further confirmed in human induced pluripotent stem (iPS) cardiomyocytes (described in Example 6) and granulocyte macrophage (GM) and erythroid (E) human bone marrow progenitor cells (described in Example 7). When incubated with Compound 1 (AT-281), the CC of these cells 50 The values ​​were also greater than 100 μM, while the positive control compound had the expected cytotoxic effect (in the GM and E cell assays, doxazosin had no significant effect on CC of cardiomyocytes). 50 The value is 7 μM, and the CC of AZT on cardiomyocytes 50 In addition, it has been previously shown that active TP, compound 5 (AT-9010), does not inhibit the in vitro enzymatic activity of human cell DNA-dependent DNA polymerases α, β, or γ (estimated IC 50 Value>100 μM), nor does it affect mitochondrial integrity or inhibit human mitochondrial DNA-directed RNA polymerase (POLRMT; (Good et al., Preclinical evaluation of AT-527, a novel guanosine nucleotide prodrug with potent, pan-genotypic activity against hepatitis C virus. PLos One 15:e0227104).

[0298] Example 3. Neutral red assay to measure 50% inhibition rate (EC 50 ) 。

[0299] The neutral red assay herein was used to generate the data labeled "neutral red assay" or "NR" in Tables 1A and 1B of Example 2. AT-281 was dissolved in DMSO at a concentration of 10 mg / mL and serially diluted using eight half-log dilutions to give a maximum test concentration of 100 μg / mL (172 μM). Each dilution was added to 5 wells of a 96-well plate with 80-100% confluency of Huh-7 cells. Three wells of each dilution were infected with the virus, and two wells remained uninfected as toxicity controls. Six untreated wells were infected as virus controls, and six untreated wells remained uninfected for use as virus controls. The virus was diluted to approximately 0.001 CCID per cell. 50 The MOI (50% cell culture infection dose) of POWVLB strain and USUV were 0.037 and 0.028 CCID 50 ). The plates were incubated at 37°C in a humidified atmosphere containing 5% CO2. On day 3 (ZIKV, EEEV, and POWV Spooner), day 4 (CHIKV), day 5 (YFV, RVFV, MERS, POWV LB, and USUV), or day 6 (WNV, JEV, DENV-2NGC, and DENV-3) post-infection, when the untreated virus control wells reached maximum CPE, the plates were stained with neutral red dye for approximately 2 hours (±15 minutes). The supernatant dye was removed, the wells were rinsed with PBS, and the incorporated dye was extracted in 50:50 Sorensen citrate buffer / ethanol for >30 minutes. The optical density was read at 540 nm using a spectrophotometer and converted to a percentage of the control. The concentration of test compound required to prevent 50% of virus-induced CPE (EC 50 ) and the concentration of test compound required to cause 50% cell death in the absence of virus (CC 50)(Smee et al., 2017. Evaluation of cell viability dyes in antiviral assays with RNAviruses that exhibit different cytopathogenic properties. J Virol Methods 246:51-57; Repetto et al., 2008. Neutral red uptake assay for the estimation of cell viability / cytotoxicity. Nature Protoc 3:1125-1131). Unless otherwise stated, the selection index is CC 50 Divide by EC 50 Selectivity index (SI) values ​​between 0-3.9 indicate inactive compounds; SI values ​​between 4-9.9 indicate minimally active compounds; SI values ​​between 10-49.9 indicate moderately active compounds; and SI values ​​>50 indicate highly active compounds. Compounds with SI values ​​>100 are indistinguishable from one another.

[0300] Example 4. Virus yield reduction assay to measure 1 log reduction in virus yield 10 The concentration (EC 90 )

[0301] The virus yield reduction assay herein was used to generate the data labeled "VYR" or "VYR assay" in Tables 1A and 1B of Example 2. As previously published (Prichard et al., 1990. A microtiter virus yield reduction assay for the evaluation of antiviral compounds against human cytomegalovirus and herpes simplex virus. J Virol Methods 246: 51-57), Vero 76 cells were seeded in 96-well plates and grown overnight (37°C) to confluence. Supernatant samples (combined from 3 wells) were collected for each compound concentration on day 3 post infection (day 4 for POWV and day 7 for USUV) and the CCID values ​​were calculated using standard endpoint dilution. 50The virus titer was measured and calculated using the Reed-Muench equation (Reed et al., 1938. A simple method of estimating fifty percent endpoints. Am J Hygiene 27: 493-497). The concentration of compound required to reduce virus production by 90% (EC 90 ).

[0302] Example 5. DENV-2 infection and treatment of human PBMC and BHK-21 cells

[0303] The PBMC assay herein was used to generate the data labeled "PBMC" or "human PBMC" in Tables 1A and 1B of Example 2. Compound 1 (AT-281) was dissolved in DMSO at 100 mM and then diluted in growth medium to final concentrations of 100, 20, 4, and 0.8 μM. PBMCs were suspended in RPMI 1640 medium containing 2 mM L-glutamine, 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin (PBMC growth medium) and activated with PHA (5 μg / mL) for 3 days. After infection with DENV-2 for 1 hour, cells were seeded in 96-well plates (1x10 5 Cells were seeded into 96-well plates (1x10 5 Cells / well), in PBMC growth medium, with or without serial dilutions of compound 1 (AT-281). Similarly, BHK-21 cells were grown to confluence in 96-well plates, and then the growth medium was replaced with fresh maintenance medium (growth medium containing 1% inactivated FBS instead of 10% FBS), which contained serial dilutions of compound 1 (AT-281) and DENV-2, with a multiplicity of infection (MOI) of 0.01. In the presence of serially diluted compounds, uninfected cells were used to evaluate the cytotoxicity of the compounds. After incubation for 3 days in a humid 5% CO2 atmosphere at 37°C, cell viability was determined by MTT method (Pauwels et al., 1988. Rapid and automated tetrazolium-based colorimetric assay for the detection of anti-HIV compounds. J Virol Methods 20: 309-21). The effective concentration of AT-281 required to prevent 50% of the virus-induced cytopathic effect (CPE) was calculated by regression analysis. 50) and the effective concentration of AT-281 required to cause 50% cell death in the absence of virus (CC 50) .

[0304] Example 6. Cardiomyocyte assay to determine the cytotoxic concentration of drug that causes death of 50% of viable cells not infected with the virus (CC50)

[0305] The cardiomyocyte toxicity test herein was used to generate the CC in Table 2 of Example 2. 50 Data. Cytotoxicity was assessed in human iPS cardiomyocytes (Cellular Dynamics; Madison, WI) and primary human bone marrow progenitor cells (Invitrogen, Grand Island, NY). Doxazosin mesylate and AZT, used as positive controls, were purchased from Sigma Aldrich. iPS cardiomyocytes were plated in Cellular Dynamics medium in 96-well plates precoated with 0.1% gelatin (Sigma) at a density of 1.5 × 10 4 Cells / well in a final volume of 100 μL were incubated at 37°C under 5% CO2 for 48 hours. Cells were washed with DPBS, and AT-281 diluted in the same medium (100 μL) was added to the monolayer in triplicate and incubated at 37°C under 5% CO2 for 3 days. Staining to measure cell viability. Remove the medium from the test plate and replace it with fresh medium (100 μL) and CellTiter Glo reagent (100 μL per well), then incubate at room temperature for 10 minutes. Transfer the well contents to a white 96-well plate and measure luminescence within 15 minutes on a Wallac 1450Microbeta Trilux liquid scintillation counter.

[0306] Example 7. Bone marrow progenitor cell assay

[0307] The cardiomyocyte toxicity test herein is used to confirm the CC in Table 2 of Example 2 50 Data. Bone marrow progenitor cells were suspended in Iscove's modified Dulbecco's medium containing 15% heat-inactivated FBS, 10% giant cell tumor conditioned medium (Bone Marrow Plus, Sigma), 10 ng / mL recombinant human IL-6, 10 ng / mL recombinant human IL-3, and 25 ng / mL recombinant human granulocyte macrophage colony-stimulating factor (GM-CSF, R&D Systems) and a final concentration of 1% methylcellulose and added to a 6-well plate (1x10 5AT-281 (100 μL) at a 10-fold higher test concentration was added to each well in triplicate, incubated at 37° C., 5% CO 2 for 14 days, and colonies (greater than 30 cells) were counted.

[0308] Example 8. Compound 1 (AT-281) forms active metabolite Compound 5 (AT-9010) in human PBMC in vitro

[0309] Since viral infection in the blood is an important component of dengue fever, it is important to determine the stability of compound 1 (AT-281) in the blood and the ability of PBMCs to phosphorylate compound 1 (AT-281) to form the active triphosphate metabolite compound 5 (AT-9010). Compound 2 (AT-752) was stable in cynomolgus monkey and human plasma for up to 120 minutes (98.2% and 93.5% of the initial 2 μM concentration were retained after 120 minutes of incubation, respectively), but was extremely unstable in Sprague Dawley (SD) rat plasma, with no drug remaining at the first time point (10 minutes of incubation). The half-life in rat plasma at 37°C (T 1 / 2 ) is <3 minutes.

[0310] Fresh unstimulated human PBMCs were treated with 10 μM compound 1 (AT-281) (as described in Example 9) for up to 6 hours. The concentration of compound 5 (AT-9010) increased over time throughout the exposure period and continued to increase after the drug was washed out. The average peak concentration was 1.88 ± 0.01 pmol / 10 at 10 hours after the start of exposure. 6 cell( Figure 3 ). When compound 1 (AT-281) was removed for 28 hours (washout period), the level of compound 5 (AT-9010) in PBMC decreased over time ( Figure 3 ), estimate T 1 / 2 It is 21.6 hours.

[0311] Example 9. In vitro formation of compound 5 (AT-9010) in unstimulated human PBMCs treated with compound 1 (AT-281)

[0312] Fresh human PBMCs (single male donor, lot number LS-88-45477C) were received and centrifuged at 400 × g for 5 min. The resulting cell pellet was suspended in 20 mL of warm PBMC growth medium. After staining with trypan blue, the cell density of the cell suspension was counted using an automated cell counter (Cellometer K2, Nexcelom) and adjusted to 2 × 10 6 Live cells / mL. Human PBMC suspension was diluted to 0.5 mL / well (1×10 6Cells) were transferred to a 24-well tissue culture treated plate at a density of 10 μM and cultured in a humidified incubator at 37°C and 5% CO2 for 20-24 hours. Then, compound 1 (AT-281) stock solution was added to each well to reach a final concentration of 10 μM, and the stimulated cells were incubated at 37°C and 5% CO2 for 0, 2, 4 and 6 hours. At each time point, the samples were processed in triplicate to analyze compound 5 (AT-9010). For the rinse samples, the incubated PBMCs were collected and centrifuged at 400 × g for 5 minutes. The supernatant was discarded, the cells were washed with 0.5 mL of culture medium, resuspended in 0.5 mL of culture medium and transferred to each well of a 24-well plate. Human PBMCs were further placed in a humidified incubator at 37°C and 5% CO2 for 0, 2, 4, 16, 20, 24 and 28 hours (i.e., 6, 8, 10, 22, 26, 30 and 34 hours after the start of test article exposure). At each time point, triplicate samples were processed for compound 5 (AT-9010) analysis as follows: The samples were transferred to 2 mL tubes and centrifuged at 800 × g for 5 min. After aspirating the supernatant, each sample was vortexed after adding 0.1 mL of water. It was then quenched with 0.3 mL of ice-cold 60% MeOH and stored at -70 °C until analysis by LC / MS / MS ( Figure 3 ).

[0313] Example 10. Compound 5 (AT-9010) disrupts RNA extension caused by DENV NS5

[0314] To confirm the mechanism of action and antiviral target of compound 5 (AT-9010), incorporation and extension experiments (described in Example 13) were performed using full-length DENV NS5 protein (described in Example 11) (serotype 2) and annealed primer-template RNA pairs (described in Example 12) that mimic the 3' end of the DENV-2 genome. In the absence of GTP, compound 5 (AT-9010) was easily incorporated into the +5 position of the RNA primer as a surrogate ( Figure 4 , left side of the gel). Despite the presence of the next correct nucleotide (UTP), no further extension was observed, indicating that compound 5 (AT-9010) immediately terminates RNA chain synthesis regardless of the presence of the 3'OH group. In the presence of all four NTPs, compound 5 (AT-9010) competes with GTP for incorporation into the +5 and +7 positions ( Figure 4, right). In fact, the incorporation of GTP and compound 5 (AT-9010) at the same position can be distinguished on the gel, and the calculated discrimination factor (based on the intensity of the band product, the preference of GTP is greater than that of compound 5 (AT-9010)) is 12.6 ± 4.3. Comparison with the incorporation of the mandatory chain terminator 3'-dGTP shows an equivalent curve, supporting chain termination as the mechanism of action of compound 5 (AT-9010) ( Figure 5 ).

[0315] Example 11. DENV NS5 protein expression and purification

[0316] The DENV NS5 protein used in Example 10 was prepared by the method described herein. The expression and purification of full-length DENV NS5 (serotype 2, New Guinea C) were as described previously (Potisopon et al., 2017. Substrate selectivity of Dengue and Zika virus NS5 polymerase towards 2'-modified nucleotide analogues. Antiviral Res 140: 25-36). In brief, the gene encoding NS5 with an N-terminal 6-His tag was expressed by a pQE30 vector in Escherichia coli NEBExpress cells (New England Biolabs, Ipswich, MA) transformed with pRare2-LacI (Novagen, Madison, WI) and induced with 50 μM IPTG and 2% EtOH until OD 600 The value reached 0.6. The cells were lysed by ultrasound and the NS5 protein was isolated using TALON metal affinity resin slurry (Clontech, Mountain View, CA) according to the manufacturer's instructions, followed by washing and elution. Size exclusion chromatography was performed as a second step of purification, where the protein was loaded onto a Superdex 200 HR 16 / 20 column (GE Healthcare) and eluted in a buffer containing 50 mM HEPES pH 7.5, 300 mM NaCl, 10% glycerol and 1 mM DTT.

[0317] Example 12. Oligonucleotides and nucleotides.

[0318] The RNA oligonucleotides used in Example 10 were prepared as described herein. RNA oligonucleotides were purchased from Biomers.net (Ulm / Donau, Germany). The 20 nt template sequence corresponding to the 3' end of the DENV-2 antigenome (T 20) was annealed to a 10 nt complementary primer (P10) containing a fluorescent 6-FAM moiety at the 5' end. Annealing was performed in the presence of 110 mM KCl using a primer-template molar ratio of 1:1.5, incubated at 70°C for 10 minutes, and then slowly cooled to room temperature. NTPs were purchased from GE Healthcare (Chicago, IL).

[0319] Example 13. Nucleotide analog incorporation assay

[0320] DENV NS5 was annealed with P 10 / T 20 RNA was pre-incubated for 10 min at 30°C in assembly buffer containing 20 mM HEPES pH 7.5, 10% glycerol, 5 mM MgCl2, and 5 mM DTT to generate active RNA extension complexes. Reactions were initiated by the addition of AT-9010 and all four NTPs or only UTP, ATP, and CTP. Final concentrations were 0.5 μM NS5, 0.25 μM 10 / T 20 , 100 μM each NTP and 10-625 μM AT-9010, the final buffer contained 20 mM HEPES pH7.5, 15% glycerol, 5 mM MgCl2 and 5 mM DTT. The reaction was quenched with 2X volume FBD stop solution (formamide, 10 mM EDTA) at the specified time point and analyzed on a 20% acrylamide: bisacrylamide (19:1), 7M urea sequencing gel. RNA products were visualized using TyphoonFluorImager and analyzed using ImageQuant software.

[0321] Example 14. Compound 2 (AT-752) exhibits good pharmacokinetics in preclinical species

[0322] The plasma pharmacokinetics (PK) of compound 1 (AT-281) and its metabolite compound 3 (AT-551) (intermediate prodrug) and compound 6 (AT-273) (a plasma surrogate for the intracellular levels of the active triphosphate metabolite compound 5 (AT-9010)) were determined in CD-1 mice, SD rats and cynomolgus monkeys after single oral doses of compound 2 (AT-752) at 420, 300 and 300 mg / kg, respectively (Table 3).

[0323] Table 3. Pharmacokinetic parameters after a single oral dose of AT-752

[0324]

[0325]

[0326] In rodents, the parent prodrug compound 1 (AT-281) is rapidly metabolized and its metabolites compound 3 (AT-551) and compound 6 (AT-273) rapidly appear in plasma ( Fig. 6A and 6B For rats, the plasma concentration of compound 1 (AT-281) was below the limit of quantification at 1 hour (the earliest time point measured). Figure 6B ), which is consistent with the metabolic instability of compound 1 (AT-281) in rat plasma. In monkeys, compound 1 (AT-281) was present in plasma for more than 4 hours ( Figure 6C ), C at 1 hour max The prodrug was converted into intermediate plasma metabolites, compound 3 (AT-551) and compound 6 (AT-273), whose PK was comparable to the previously reported results of the analog AT-511 (Good et al., Preclinicalevaluation of AT-527, a novel guanosine nucleotide prodrug with potent, pan-genotypic activity against hepatitis C virus. PLos One 15: e0227104).

[0327] Example 15. Formation of active metabolite Compound 5 (AT-9010) in PBMCs in vivo

[0328] The concentration of compound 5 (AT-9010) isolated from PBMCs of mice and rats administered a single oral dose of 50 and 300 mg / kg of compound 1 (AT-281) and monkeys administered a single oral dose of compound 2 (AT-281) equivalent to 55 mg / kg of compound 1 (AT-281) was measured at various time points after administration ( Figure 7 ). Similar concentrations of compound 5 (AT-9010) were observed in mice and monkeys at 12 h after administration of comparable doses (0.092 and 0.070 ± 0.046 pmol / 10 6 cells). Compound 5 (AT-9010) (0.439±0.218 pmol / 10 6The concentrations of compound 5 (AT-9010) in PBMCs were 0.297 and 0.322 pmol / 10 4 and 12 hours after the second dose, respectively. 6 cells (not shown).

[0329] To test the tolerability of compound 1 (AT-281) and determine the steady-state concentration of active TP in PBMCs, mice were given a loading dose of 1000 mg / kg and, starting 4 hours later, twice daily (12 hours apart) at a dose of 500 mg / kg for 3 days. 12 hours after the first and sixth 500 mg / kg doses, the concentrations of compound 5 (AT-9010) in the pooled (n=3) PBMCs were 0.421 and 0.575 pmol / 10 6 cells, while the plasma compound 6 (AT-273) concentrations were 0.67±0.12 and 1.35±0.86 nmol / mL at the same time points. No adverse clinical symptoms were observed in mice, so multiple doses of the prodrug were well tolerated.

[0330] Example 16. Animal welfare

[0331] The studies using CD-1 mice, SD rats and cynomolgus monkeys in Example 17 were conducted at WuXi AppTec (Suzhou, China) in strict compliance with AAALAC International, NIH guidelines and the Regulations on the Administration of Laboratory Animals of the Ministry of Science and Technology of the People's Republic of China, 2017. Before the start of the study, WuXi AppTec's IACUC reviewed and approved the protocol, and all animals were evaluated by WuXi AppTec veterinary staff throughout the study. All animals were housed in a room with controlled temperature (18 to 26°C), relative humidity (40 to 70%) and light cycle (12 hours of artificial light and 12 hours of darkness), with an air circulation rate of 100%. Teaching aids / educational toys were provided for the animals. The AG129 mouse studies described herein were conducted at IBT Bioservices (Rockville, MD) in strict compliance with the USDA Animal Welfare Act and in accordance with the PHS and NIH Laboratory Animal Humane Care and Use Policy and the National Research Council-ILAR Laboratory Animal Care and Use Guide (2011 Revised Edition). This study adhered fully to a protocol that was reviewed and approved by IBT's Institutional Animal Care and Use Committee (IACUC) prior to study initiation, and mice were evaluated and monitored by IBT veterinary staff throughout the study in accordance with PHS policy and the USDA.

[0332] Example 17. PK studies and in vivo development of compound 5 (AT-9010) in mice, rats and monkeys

[0333] Male young CD-1 mice (Shanghai Sippr BK Laboratory Animal Co., Ltd., China) were administered a 420 mg / kg dose of compound 2 (AT-752) (dissolved in 40% PEG400, 10% solutol HS15, 50% 100 mM citrate buffer, pH 4.5 (v / v)) by oral gavage. Blood samples (N=3) were collected at 0.5, 1, 2, 4, 8, 12 and 24 hours after administration, and plasma was separated in EDTA and 1 μL dichlorvos (2 mg / mL; stabilizer, used to prevent the ester group of compound 1 (AT-281) from being hydrolyzed by blood esterase in vitro) and stored at -70°C. The concentrations of compound 1 (AT-281) and metabolites compound 3 (AT-551) and compound 6 (AT-273) in plasma were determined by LC / MS / MS. For PBMC, six male juvenile CD-1 mice were administered one dose of compound 2 (AT-752) in 60% PEG400 by oral gavage (compound 1 (AT-281) equivalent dose was 50 mg / kg). Blood samples were collected from three people in each group 4 and 12 hours after dosing, PBMCs were isolated, and the concentration of compound 5 (AT-9010) was measured by LC / MS / MS.

[0334] AT-752 (dissolved in 40% PEG400, 10% solutol HS15, 50% 100mM citrate buffer, pH 4.5 (v / v)) was administered to male juvenile SD rats (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., China) at a dose of 300 mg / kg by oral gavage. Blood samples (n=4) were collected 1, 2, 4, 8, 12 and 24 hours after administration, and plasma was separated and stored at -70°C. The concentrations of compound 1 (AT-281) and metabolites compound 3 (AT-551) and compound 6 (AT-273) were determined by LC / MS / MS. For PBMC, a dose of compound 2 (AT-752) (equivalent dose of compound 1 (AT-281) was 300 mg / kg) was administered to nine male juvenile SD rats by oral gavage. At 24, 48, and 72 hours after administration, blood samples were collected from three people in each group, PBMCs were isolated, and the concentration of compound 5 (AT-9010) was determined by LC / MS / MS.

[0335] Male non-juvenile cynomolgus monkeys weighing at least 2 kg (Hainan Jingang Experimental Animal Co., Ltd., China) were housed in stainless steel cages during the study, with free access to reverse osmosis water, fed twice a day (except once on the day of administration), each feeding of about 60 grams of certified monkey food (Beijing Vital Keao Feed Co., Ltd., Beijing, China), and fresh fruit daily. They (n = 5) were administered a 300 mg / kg dose of compound 2 (AT-752) (dissolved in 40% PEG400, 10% solutol HS15, 50% 100 mM citrate buffer, pH 4.5 (v / v)) by oral gavage, and blood samples (~0.5 mL) were collected at 1, 2, 4, 8, 12 and 24 hours after administration. Plasma was separated at each time point for pharmacokinetic analysis and stored at -70 ° C. The concentrations of compound 1 (AT-281) and metabolites compound 3 (AT-551) and compound 6 (AT-273) were determined by LC / MS / MS. Compound 2 (AT-752) powder capsules were orally administered to three monkeys at a dose of 60 mg / kg (the equivalent dose of compound 2 (AT-281) was 55 mg / kg), and blood samples were collected 12, 24 and 48 hours after administration. PBMCs were separated and the concentration of compound 5 (AT-9010) was measured by LC / MS / MS. All animals were observed for any abnormal or adverse clinical symptoms before and immediately after administration and before each blood collection time point, but no such symptoms were found.

[0336] In addition, two multiple-dose mouse studies were conducted. First, compound 1 (AT-281) in 100 mM citrate buffer (pH 4.5) was administered to 9 young CD-1 mice by oral gavage, with a first dose of 500 mg / kg, and 4 hours later, a second dose of 250 mg / kg. Blood samples (3 animals at each time point) were collected 4 hours after the first dose (before the second dose), 8 hours, and 16 hours after the first dose, and PBMCs were isolated. Secondly, compound 1 (AT-281) was administered to 9 young CD-1 mice by oral gavage, with a first dose of 1000 mg / kg, and 4 hours later, twice a day for 3 consecutive days, with an interval of 12 hours, at a dose of 500 mg / kg. Blood samples (3 animals at each time point) were collected 16 hours after the first dose (received one 500 mg / kg dose), 40 hours (received three 500 mg / kg doses), and 76 hours (received six 500 mg / kg doses), and PBMCs were isolated. The concentrations of Compound 1 (AT-281) and Compound 6 (AT-273) in plasma and Compound 5 (AT-9010) in PBMCs were determined by LC / MS / MS (described in Example 21).

[0337] Example 18. Compound 2 (AT-752) can reduce viremia and improve survival rate of AG129 mice infected with DENV2 D2Y98P

[0338] To test the efficacy of compound 2 (AT-752) against DENV infection, DENV-2D2Y98P (a non-mouse-adapted strain with similar pathogenesis to humans) was subcutaneously inoculated into AG129 mice (Tan et al., 2010. A nonmouse-adapted Dengue virus strain as a new model of severe Dengue infection in AG129 mice. PLoS Negl Trop Dis 4: 3672). The prodrug was orally administered with a loading dose (1000 mg / kg) 4 hours before the virus attack, followed by continuous administration of 500 mg / kg twice daily (BID) for 7 days, starting at 1 hour after infection (pi). Viremia and spleen viral load (i.e., the primary endpoint) were assessed on days 4, 6, 7, 8, and 10 after infection. On day 6 after infection, viremia ( Fig. 8A) was significantly lower than that in the vehicle-only group (t-test, p=0.000014), and virus was cleared in all treated animals on day 8 post-infection, while virus was still measurable in all surviving control groups (t-test, p=0.0045). Although compound 2 (AT-752) treatment reduced spleen viral load on day 7 post-infection compared with the control group ( Figure 8B ), but this change was not statistically different between the two groups. This result may be because the virus is eventually cleared from the spleen in this mouse disease model.

[0339] The mice were assessed daily for weight changes, appearance, activity, and alertness, and were euthanized when they lost 20% of their body weight or had a health score >5. There was a significant difference in body weight between the treatment group and the vehicle control group from day 4 to day 8 after infection (t-test, p<0.001). None of the mice treated with the vehicle survived on day 8 after infection. Animals treated with compound 2 (AT-752) lost significantly less weight on days 5, 7, and 8 after infection compared to vehicle control mice ( Fig.9A ; t-test, p<0.001). In addition, the health scores of mice treated with compound 2 (AT-752) were significantly better than those of the vehicle control group ( Fig. 9B ; t test, p<0.001). Kaplan-Meier survival curve ( Fig.10 ) presents the mortality data derived therefrom indicating that Compound 2 (AT-752) significantly increased the survival of infected mice compared to the vehicle control group (p=0.0004, Mantel-Cox log-rank test). All mice in the control group that were not scheduled to be sacrificed for blood and spleen samples died or were euthanized on days 5 to 8 after infection. In contrast, animals treated with Compound 2 (AT-752) survived until days 11 to 18 after infection.

[0340] Example 19. AG129 Mouse Study

[0341] The tissue samples used in Example 20 were collected according to the protocol described herein. 55 female AG129 mice (α-, β-, γ-interferon knockout), 6-8 weeks old, were divided into two groups, 25 mice in group 1 (vehicle) and 30 mice in group 2. On day 0, all mice were injected subcutaneously with 1×10 5PFU of DENV-2D2Y98P for challenge. Group 1 animals were given vehicle [(PEG400 (40%, v / v) / SolutolHS15 (10%, v / v) / 100mM citrate buffer pH 4.5±0.2 (50%, v / v)] by oral gavage twice daily for 7 consecutive days starting from 1 hour after infection. Group 2 received a single dose of 1000mg / kg of compound 2 (AT-752) 4 hours before challenge, and then received a dose of 500mg / kg 1 hour after challenge (pi). Compound 2 (AT-752) was administered at a dose of 500mg / kg twice daily for 7 consecutive days. According to the protocol, 5 mice were sacrificed in each group, and terminal serum and spleen were collected on days 4, 6, 7, 8 and 10 after infection. On day 20 after infection, all surviving mice in Group 2 were sacrificed to collect terminal serum and Spleen. Serum samples were stored at -80°C until further analysis. Harvested spleen samples were weighed, snap frozen in an ethanol / dry ice mixture, and immediately stored at -80°C. All samples were processed and viral load was determined by plaque assay. All animals were monitored daily for weight loss, morbidity, mortality, and neurological decline. Mice with severe disease such as weight loss >20%, health scores greater than 5 (fur appearance, activity and alertness scores), extreme lethargy and / or paralysis were euthanized and terminal serum and spleen were harvested. Any mouse found dead was used only for spleen collection.

[0342] Example 20. Plaque assay for determination of spleen viral load

[0343] The spleen samples collected according to Example 19 were homogenized in 250 μL DPBS using a TissueRuptor. The homogenate was then centrifuged and the supernatant was collected and immediately frozen in two different aliquots until the plaque assay was performed. The results of the plaque assay are shown in Fig. 8A and 8B shown.

[0344] One day before the experiment, Vero cells were plated at 1x10 5 Cells were seeded at a density of 100 cells / well in 1 mL of 1% Hi-FBS medium in a 24-well tissue culture plate and cultured at 37°C in a 5% CO2 atmosphere. The next day, 10-fold serial dilutions of serum and spleen samples were prepared (10 -2 Up to 10 -5), titrated in triplicate. Remove the cell culture medium of Vero cells, add 100 μL of diluted sample and 100 μL of MEM medium supplemented with 2mM L-glutamine and 1X Pen / Strep. The cells were cultured for 1 hour at 37°C and 5% CO2. Then, 1 mL of 0.8% methylcellulose (containing 2% FBS, supplemented with 2mM L-glutamine and 1X Pen / Strep) was added to the cells (without removing the inoculum) and incubated for another 3 days at 37°C and 5% CO2 to form plaques. At -20°C, fix and permeabilize the cells with a cold 80% ethanol / 20% methanol mixture for 30 minutes. 200 μL of anti-dengue monoclonal antibody (diluted 1:2000 in 5% skim milk) was added to each well and incubated overnight at 4°C. The wells were washed 3 times with 1xDPBS, 200 μL of HRP-labeled goat anti-mouse antibody (diluted 1:2000 in 5% skim milk) was added, and each well was incubated for 1 hour at room temperature. Viral plaques were decomposed and counted using an insoluble peroxidase substrate (TrueBlue) and a plaque counter. The data of spleen viral load were standardized according to the spleen weight of each mouse.

[0345] Example 21. LC-MS / MS analysis of compound 1 (AT-281), compound 3 (AT-551), compound 6 (AT-273) and compound 5 (AT-9010)

[0346] LC-MS / MS analysis was performed according to the protocol described in this article. Fig.11 , 12 , 13, 14 and 15. Plasma samples were prepared for MS analysis by adding internal standards and extracting with 20 volumes of frozen ACN. After vortexing (800 rpm, 10 min) and centrifugation (4,000 rpm, 15 min, 4 ° C), the supernatant (25 μL) was diluted with an equal volume of H2O, mixed and spun again. After adding internal standards, PBMC samples (10 6Cells), vortexed and centrifuged (12,000 rpm, 15 minutes, 4°C). An aliquot of the supernatant (35 μL) was dried under nitrogen and redissolved in H2O, then mixed and spun again. To measure compound 1 (AT-281) and plasma metabolites compound 3 (AT-551) and compound 6 (AT-273), 4 μL of sample was injected into an Acquity Gemini C18 (50x4.6mm), 5 μm UPLC column (equipped with a Sciex Triple Quad 6500 mass spectrometer) (ESI positive ion, MRM mode). The sample was eluted at a rate of 0.8 mL / min using a binary nonlinear gradient of mobile phase A (0.1% formic acid in water) and B (0.1% formic acid in ACN) with a run time of 5 minutes. For AT-9010 in cells, 8 μL was injected into an Acquity BEH C18 (50x2.1 mm), 1.7 μm UPLC column (equipped with an API 14000 mass spectrometer) (ESI negative ion, MRM mode). The sample was eluted at a flow rate of 0.5 mL / min using a binary nonlinear gradient of mobile phase A (0.001% NH3·H2O, 0.18 mM DBAA in H2O) and B (10 mM N,N-dimethylhexylamine, 3 mM NH4OAc in ACN / H2O (50:50, v / v)) with a run time of 3 min. Calibration was performed using a 50% MeOH standard. The m / z of the monitored ions were 538.2 / 440.0 (AT-9010), 582.3 / 330.1 (AT-281), 464.2 / 165.1 (AT-551), and 300.2 / 152.2 (AT-273). A previously described internal standard (ISS) (Good et al., Preclinical evaluation of AT-527, a novel guanosine nucleotide prodrug with potent, pan-genotypic activity against hepatitis C virus. PLos One 15: e02271045) was used to correct for variations in the recovery process.

[0347] Example 22. Pharmacokinetic Data Analysis

[0348] According to the method described herein, the method generated according to Example 21 and displayed on Fig.11 , 12Data analysis was performed on the pharmacokinetic data of , 13, 14 and 15. Non-compartmental pharmacokinetic analysis of plasma concentrations of compound 1 (AT-281), compound 3 (AT-551) and compound 6 (AT-273) was performed using Phoenix WinNonlin software (version 6.3 or higher, Pharsight, Mountain View, CA). PK parameters were obtained using the linear / logarithmic trapezoidal rule.

[0349] Example 23. Human Safety Study

[0350] Healthy men and women aged between 18 and 65 years were enrolled in the single ascending dose (SAD) and multiple ascending dose (MAD) groups and randomized to receive oral Compound 2 or placebo according to Tables 4-7.

[0351] Safety assessments included adverse events, standard clinical laboratory tests, vital sign measurements, electrocardiograms, and physical examinations.

[0352] Table 4. Patients participating in the study

[0353] SAD Group MAD Group Average age 28.6 35.3 gender 51.2% male, 48.8% female 62.5% male, 37.5% female Weight (kg) 67.3 74.7

[0354] Table 5: Distribution of patients in each group in the study

[0355] SAD Group Patient number MAD Group Patient number A / B / C / D / E 31 F / G / H 18 Placebo 10 Placebo 6

[0356] Table 6. Summary of studies on SAD population

[0357] group Dosage (mg) Drug administration A 250 Day 1 (Fasting) B(Food Effect) 500 Day 1 (fasting); Day 7 (eating)* C 1000 Day 1 (Fasting) D (race)** 1000 Day 1 (Fasting) E 1500 Day 1 (Fasting)

[0358] *High fat / high calorie test meal. **Healthy participants of South / Southeast / East Asian descent

[0359] Table 7. Summary of studies in the MAD population

[0360] group Dosage (mg) Drug administration F 1000 QD x 7 days G 750 BID x 4 days, plus one dose on day 5 H 750 TID x 4 days, plus one dose on day 5

[0361] Compound 2 was well tolerated, with no serious adverse events or discontinuations due to adverse events reported. Non-serious adverse events were mild or moderate in severity and resolved by the end of the study (Table 8). Occasional gastrointestinal-related events (including mild to moderate vomiting) occurred mostly at higher doses.

[0362] Table 8. Adverse events in the SAD population reported by more than two subjects

[0363] dose Placebo 250 500 500* 1000 1000** 1500 total Number of patients 10 6 7 7 6 6 6 41 Headache 1 0 2 4 1 0 2 8 nausea 0 0 0 1 1 0 1 3 Vomit 0 0 0 0 1 0 2 3

[0364] *Eating; **Patients of South / Southeast / East Asian descent

[0365] Table 9. Adverse events in the MAD population reported by more than two subjects

[0366]

[0367] Example 24. Pharmacokinetic Study in SAD Population

[0368] Healthy men and women aged between 18 and 65 years participated in a single ascending dose (SAD) trial and were randomized to receive oral Compound 2 or placebo according to Tables 4-6.

[0369] Plasma and urine samples were collected at predetermined time points and quantified using LC-MS / MS for compound 1 and its metabolites, compounds 3, 6, and 7. Pharmacokinetic analysis was performed using a non-compartmental approach.

[0370] After oral administration under fasting conditions, the parent prodrug Compound 1 was rapidly absorbed and cleared, followed by Compound 3, which also showed transient exposure (Tables 10-14 and Figure 12-15 ). 6 -methyl 2'-methyl, 2'-fluoro nucleoside metabolite compound 7 peaked later and exhibited a slower elimination phase ( Fig.15 Compound 6 appeared more slowly in plasma and exhibited a longer elimination half-life (population mean up to 23 h), reflecting the sustained intracellular exposure of the active triphosphate metabolite, compound 5.

[0371] Dose proportionality analysis

[0372] Plasma exposure of compound 1 and its metabolites increased over the dose range studied (250 to 1500 mg). Compound 7 increased in a dose-proportional manner. Compound 1 and compound 3 increased in a greater-than-dose-proportional manner, while compound 6 increased in a slightly less-than-dose-proportional manner.

[0373] Food Effect

[0374] A high-fat / high-calorie diet delayed and reduced the peak levels of compounds 1, 3, and 7, but had limited or no effect on their total exposure and slightly increased the plasma exposure of compound 6.

[0375] Table 10. Summary of plasma pharmacokinetic parameters of compound 1

[0376]

[0377] *Eating; **Patients of South / Southeast / East Asian descent

[0378] Table 11. Summary of plasma pharmacokinetic parameters of compound 3

[0379]

[0380] *Eating; **Patients of South / Southeast / East Asian descent

[0381] Table 12. Summary of plasma pharmacokinetic parameters of compound 6

[0382]

[0383] *Eating; **Patients of South / Southeast / East Asian descent

[0384] Table 13. Summary of plasma pharmacokinetic parameters of compound 7

[0385]

[0386]

[0387] *Eating; **Patients of South / Southeast / East Asian descent

[0388] The pharmacokinetic profiles of South / Southeast / East Asian and mostly Caucasian populations were similar, suggesting that there is no pharmacokinetic ethnic sensitivity. Therefore, no dose adjustment is required for Compound 2.

[0389] Urine excretion

[0390] After a single oral administration of 250 to 1500 mg of compound 2, the urinary excretion of compound 1 and compound 3 was low, and the urinary excretion of compound 7 and compound 6 was moderate (Table 9). The total urine recovery rate at different doses ranged from about 20% to about 30%. The renal clearance of nucleoside metabolites compound 6 and compound 7 exceeded the estimated glomerular filtration rate, which indicates that there is active secretion in the renal environment.

[0391] Table 14. Urinary excretion of compound 2 after single oral administration

[0392] Compound Name Urine excretion Compound 1 <3％ Compound 3 <1％ Compound 6 6.7-8.8％ Compound 7 10.7-17.9％

[0393] Example 25. Pharmacokinetic Study in MAD Population

[0394] Healthy men and women aged between 18 and 65 years participated in the multiple ascending dose (MAD) trial and were randomized according to Tables 4 and 7 to receive oral Compound 2 or placebo.

[0395] Plasma and urine samples were collected at predetermined time points and quantified using LC-MS / MS for compound 1 and its metabolites, compounds 3, 6, and 7. Pharmacokinetic analysis was performed using a non-compartmental approach.

[0396] Due to the long plasma half-life of compound 6, the plasma exposure of compound 6 increased by approximately 25%, 60%, and 80% with QD, BID, and TID, respectively, which reflects the rapid accumulation and sustained intracellular exposure of the active metabolite compound 5.

[0397] The 750 mg TID dose resulted in a rapid increase in the plasma concentration of compound 6, exceeding the 90% effective concentration (EC 90 )(0.64μM). These levels remained constant throughout the treatment period ( Fig.11 ).

[0398] Table 15. Summary of steady-state plasma pharmacokinetic parameters of compound 1

[0399] Compound 1 <![CDATA[C max (ng / mL)]]> <![CDATA[T max (h)]]> <![CDATA[AUC tau (ng / mL*h)]]> 1000mg QD 3810±1670 0.5(0.5-0.5) 3090±1530 750 mg BID 3490±1280 0.5(0.5-0.8) 2609±640 750 mg 1 time 3010±754 0.5(0.5-1.0) 2540±572

[0400] Table 16. Summary of steady-state plasma pharmacokinetic parameters of compound 3

[0401] Compound 1 <![CDATA[C max (ng / mL)]]> <![CDATA[T max (h)]]> <![CDATA[AUC tau (ng / mL*h)]]> 1000mg QD 392±143 1.0(0.8-2.0) 1650±498 750 mg BID 365±159 1.0(0.8-2.0) 1330±503 750 mg 1 time 245±30 1.5(0.8-2.0) 944±173

[0402] Table 17. Summary of steady-state plasma pharmacokinetic parameters of compound 6

[0403] Compound 1 <![CDATA[C max (ng / mL)]]> <![CDATA[T max (h)]]> <![CDATA[AUC tau (ng / mL*h)]]> 1000mg QD 241±56 4.0(3.0-4.0) 3120±694 750 mg BID 288±58 3.5(3.0-4.0) 2680±730 750 mg 1 time 330±98 3.5(3.0-6.0) 2370±743

[0404] Table 18. Summary of steady-state plasma pharmacokinetic parameters of compound 7

[0405] Compound 1 <![CDATA[C max (ng / mL)]]> <![CDATA[T max (h)]]> <![CDATA[AUC tau (ng / mL*h)]]> 1000mg QD 1200±284 0.9(0.8-2.0) 8290±2290 750 mg BID 1470±404 1.5(0.8-2.2) 7960±2970 750 mg 1 time 1600±640 1.5(1.0-2.0) 22300±14100

[0406] In Table 15-18, C max and AUC tau The data are expressed as mean ± standard deviation. max Expressed as median (minimum - maximum). AUC tau It represents the area under the plasma concentration-time curve during the dosing interval.

[0407] Example 26. Antiviral test

[0408] As previously described (Baker et al., 2020b, 2020a), transcripts of dengue viruses (DENV1 WP, DENV2 NGC, DENV3 VN32, DENV4 MY01) were modified with a nanoluciferase reporter gene between the 5'UTR and the capsid gene to create labeled viruses that were stable when passaged in cell culture while producing a strong luciferase signal after inoculation for efficient screening of antiviral activity. Eight 2-fold serial dilutions of AT-281 were mixed with each reporter virus (MOI of 0.1 for DENV-1, DENV-2, and DENV-3; MOI of 0.001 for DENV-4) and added to plates containing Huh-7 cells (RRID: CVCL0336) that had been cultured the day before at 1 × 10 4 Cells were seeded at a density of 100 cells / well in 96-well plates in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) containing 2% fetal bovine serum (Hyclone, Logan, UT). 48 hours after infection, cells were washed three times with phosphate-buffered saline and then added to The 5-well plate was diluted 1:100 in assay buffer (Promega, Madison, WI). Substrate. After 3 minutes, luciferase activity was read using a BioTek Cytation 5 plate reader. Data were analyzed using GraphPad Prism 9 software. Luciferase activity was normalized to DMSO-treated samples (set to 100%). Error bars represent mean ± SD. Results represent three independent experiments, each analyzed in triplicate. Fig.16 The antiviral activity against the tested viruses was shown in .

[0409] Example 27. Expression of DENV NS5, RdRp and MTase domains

[0410] The proteins used in the thermal shift assay (Example 31) were prepared as described herein. The full-length DENV NS5 and RdRp domains used in this study were expressed under the control of the T7 promoter in the pET28a vector in E. coli NEB C2566 cells (New England Biolabs) carrying the pRARE2LacI (Novagen) plasmid. The proteins were expressed overnight at 17°C in TB (containing 25 μg / mL kanamycin and 17 μg / mL chloramphenicol), and then induced with 100 μM IPTG and 2% EtOH (% v / v), with an OD600 of 0.8-1.

[0411] DENVMTases were expressed under the control of the T7 promoter in the pDEST17 vector in E. coli NEB C2566 cells (New England Biolabs) carrying the pRARE2LacI (Novagen) plasmid. The protein was expressed overnight at 17°C in TB (containing 100 μg / mL ampicillin and 17 μg / mL chloramphenicol), and then induced with 200 μM IPTG and 2% EtOH (% v / v), with an OD600 of 0.8-1.

[0412] Example 28. Purification of DENV NS5

[0413] DENV NS5 used in the thermal shift assay (Example 31) was purified as described herein. Cells were disrupted by sonication on ice in lysis buffer (50 mM NaP pH 8, 1 M NaCl, 20% glycerol) supplemented with 1.0 mg / mL lysozyme, 22 μg / mL DNase, 1.6% igepal, 0.5 mM TCEP, and complete protease inhibitor cocktail (COC) from Roche. Proteins in the soluble fraction were loaded into Superflow TM The samples were washed 5 times with lysis buffer, 10 times with lysis buffer (without igepal) and then eluted with lysis buffer supplemented with 250 mM imidazole and 250 mM glycine. The imidazole was removed by overnight dialysis in dialysis buffer (50 mM NaP pH 8, 20% glycerol, 150 mM NaCl, 250 mM glycine and 0.5 mM TCEP) and then analyzed by size exclusion chromatography (SEC). 16 / 600 The final purification step was performed on a 200 pg column in buffer (10 mM HEPES pH 8, 300 mM NaCl, 10% glycerol and 0.5 mM TCEP). The protein was then concentrated to 10 to 15 mg.mL -1 , and stored at −80 °C after the final dialysis in SEC buffer supplemented with 40% glycerol.

[0414] Example 29. Purification of DENV RdRp domain

[0415] DENV RdRp used in the thermal shift assay (Example 31) was purified as described herein. Cells were disrupted by sonication on ice in lysis buffer (50 mM NaP pH 8, 1 M NaCl, 20% glycerol) supplemented with 1.0 mg / mL lysozyme, 22 μg / mL DNase, 1.6% igepal, 0.5 mM TCEP, and complete protease inhibitor cocktail (COC) from Roche. Proteins in the soluble fraction were loaded into Superflow TM On a cobalt-based IMAC resin (Cytiva), wash 5 times with lysis buffer, wash 10 times with lysis buffer (without igepal), and then elute with lysis buffer supplemented with 250 mM imidazole and 250 mM glycine. Dialyze overnight in dialysis buffer (50 mM NaP pH 8, 20% glycerol, 150 mM NaCl, 250 mM glycine, and 0.5 mM TCEP) to remove imidazole, and then use a GE Hi-trap Heparine column for the final purification step. The protein was then concentrated to 10 to 15 mg.mL -1 , and stored at -80°C after a final dialysis in a final buffer containing 20 mM HEPES pH 8, 300 mM NaCl, 40% glycerol and 0.5 mM TCEP.

[0416] Example 30. Purification of DENV MTase domain

[0417] DENV MTase used in the thermal shift assay (Example 31) was purified as described herein. Cells were disrupted by sonication on ice in a lysis buffer (50 mM HEPES pH 7.5, 10% glycerol, 500 mM NaCl, and 5 mM imidazole) supplemented with 0.2 mM benzamidine, 1 mg / mL lysozyme, 22 μg / mL DNase, and 0.5 mM TCEP. Proteins in the soluble fraction were loaded into Superflow TM The samples were loaded onto a cobalt-based IMAC resin (Cytiva), washed with lysis buffer (1 M NaCl), and then eluted with 250 mM imidazole. 16 / 600 The protein was purified in a buffer of 50 mM HEPES pH 8, 300 mM NaCl and 0.5 mM TCEP (200 pg). The protein was then concentrated to 10 to 15 mg / mL -1, and stored at -80°C after a final dialysis in a final buffer containing 20 mM HEPES pH 8, 300 mM NaCl, 40% glycerol, and 0.5 mM TCEP.

[0418] Example 31. Thermal migration test

[0419] The thermal stability of flavivirus proteins and their interactions with compounds were assessed using a thermal shift assay. Reactions were performed in white framed star PCR 96-well plates (reference number 044705) in a final volume of 20 μL. For DENV NS5, polymerase, and MTase, optimal conditions were obtained using a buffer containing 20 mM Hepes pH 7.5, 50 mM NaCl, 5 mM DTT, 10% glycerol, and 1 M sorbitol.

[0420] 0.4 μL of GTP, Sinefungin or AT-9010 was placed in the plate (final concentration was 100 μM). Water without compound was used as a control. 2 μM NS5, 2 μM polymerase or 6 μM MTase were mixed with 2.5X, 0.75X or 1.5X dye (final concentration) in buffer, respectively. A mixture of protein and the protein thermal shift kit from ThermoFisher (reference number 4461146) was prepared and 19.6 μl was dispensed into each well.

[0421] The plate was sealed with adhesive film and then run on a CFX96 RT-PCR (Bio-Rad). The program started with a 1-min equilibration phase at 20°C, followed by a temperature gradient of 0.5°C / 30 seconds from 20°C to 95°C, with fluorescence recorded every 0.5°C. Fluorescence was recorded using the FRET channel. Each experiment was performed in triplicate. The melting temperature was determined by plotting and analyzing the curves using the Boltzmann sigmoidal equation using GraphPad Prism software. Graphical representation of the data in the table below is as follows Fig.17 shown.

[0422] Table 19. Data of thermal migration test

[0423]

[0424]

[0425] Example 32. pppApG Primer Synthesis

[0426] Reactions were performed in the presence of 2 mM MnCl2 or 5 mM MgCl2, which are the optimal catalytic ion concentrations for RNA synthesis by DENV2 RdRp (Selisko et al., 2006).

[0427] The discrimination of AT-9010 from GTP was measured in four independent reactions, and the results were consistent with those of Fig.19B and 21A , 21B, 21C, and 21D show similar results.

[0428] Example 33. MTase activity test

[0429] The transfer of tritiated methyl groups from [3H]-SAM to RNA substrates was monitored by a filter binding assay (FBA) according to previously described methods (Paesen et al., 2015). The recombinant 2'-O-methyltransferase domain of the nonstructural protein NS5 of DENV serotype 2 (DENV NS5 MTase) corresponding to residues 1-296 of NS5 was expressed and purified as previously described (Egloff et al., 2002). FBA was performed in the presence of 0.7μM mGpppAC4 synthetic RNA and DENV2 NS5MTase protein (500nM) in a reaction mixture [40mM Tris-HCl (pH 8.0), 1mM DTT, 2μM SAM and 0.1μM 3H-SAM (Perkin Elmer)]. The enzyme was first mixed with the compound suspended in water (0.5 to 1000μM), then the RNA substrate and SAM were added, and then incubated at 30°C. After 30 minutes, the reaction mixture was diluted 10-fold in ice water to stop the reaction. The samples were transferred to diethylaminoethyl (DEAE) filter pads (Perkin Elmer) using a Filtermat Harvester (Packard Instruments). The RNA retention pads were washed twice with 10 mM ammonium formate (pH 8.0), twice with water, and once with ethanol. They were soaked with scintillation fluid (Perkin Elmer) and the 3H-methyl transfer to the RNA substrate was measured using a Wallac MicroBeta TriLux liquid scintillation counter (PerkinElmer). For IC 50 The values ​​were normalized and fitted using Prism (GraphPad Software) using the following equation: Y = 100 / (1 + ((X / IC 50 )^Hillslope)). IC 50 It is defined as the concentration of inhibitory compound that causes a 50% decrease in enzyme activity. The data curve can be found in Fig.18 Found in.

[0430] Example 34. Incorporation of nucleotide analogs into RNA

[0431] DENV NS5 was preincubated with annealed P10 / T20 RNA in assembly buffer containing 20 mM HEPES pH 7.5, 10% glycerol, 5 mM MgCl2, and 5 mM DTT at 30°C for 10 min to generate active RNA extension complexes.

[0432] The polynucleotide incorporation was tested as follows, and the results were as follows Fig.19A , 21, 22 and 23. Reactions were initiated by the addition of AT-9010 and all four NTPs or only UTP, ATP and CTP. Final concentrations were 1 μM NS5, 0.25 μM P10 / T20, 100 μM of each NTP, and 10 to 625 μM AT-9010 in a final buffer containing 20 mM HEPES pH 7.5, 15% glycerol, 5 mM MgCl2 and 5 mM DTT.

[0433] The single nucleotide incorporation assay is as follows. The reaction is started by adding the corresponding NTP or analog. The final concentration is 1 μM NS5, 0.25 μM P10 / T20, 100 μM NTP or analog in a final buffer containing 20 mM HEPES pH 7.5, 15% glycerol, 5 mM MgCl2 and 5 mM DTT.

[0434] At the indicated time points, the reactions were quenched with 3 volumes of FBD stop solution (formamide, 10 mM EDTA) and analyzed on a 20% acrylamide-bisacrylamide (19:1) 7M urea sequencing gel. RNA products were visualized using a Typhoon FluorImager and then analyzed and quantified using ImageQuant software.

[0435] Example 35. Crystallographic and modeling studies

[0436] Fig. 20A and 20BCrystallographic images generated by the protocol described herein are shown. The construct used for crystallization was the same as described in (Barral et al., 2013), with a cleavable hexahistidine-thioredoxin tag at the N-terminus of the DENV3 NS5 coding sequence (amino acid region 1-277) in the plasmid pMcox20A. Transformed E. coli T7 expression cells (New England BioLabs) were grown in LB medium supplemented with 100 μg / mL ampicillin at 37°C with an OD600nm of 0.6-0.8. Expression of recombinant protein was induced by the addition of 100 μM IPTG and incubation was continued at 17°C for another 16 hours. Cells were harvested by centrifugation at 8000 g for 10 minutes at 4°C and stored at -80°C. Protein purification and tag removal were performed under non-denaturing conditions as described in (Lantez et al., 2011) using Superflow TM Affinity chromatography was performed using cobalt-based IMAC resin (Cytiva).The final size exclusion chromatography step was performed using a Superdex S200 HiLoad 16 / 60 column and a buffer containing 20 mM HEPES pH 7.5, 200 mM NaCl, 10% glycerol and 2 mM DTT.

[0437] Crystallization conditions were adapted from (Lim et al., 2011) using the sitting drop vapor diffusion method with crystallization buffer. All crystals were grown at 293.15 K using a 1:2 ratio of protein (10 mg / mL) to precipitant solution (22% PEG 8000, 200 mM NaCl, 20 mM trisodium citrate, 100 mM Tris pH 8.5). Crystals were grown over 4 days and soaked overnight with 1 mM AT-9010 (final concentration). All crystals were cryoprotected with a reservoir solution supplemented with 10% glycerol and flash frozen in liquid nitrogen at 100 K.

[0438] Data collection and diffraction data were collected at the Soleil synchrotron. The original native data set was collected on Proxima2. The data set was processed and analyzed using the autoPROC toolbox (Vonrhein et al., 2011). The structure was solved by molecular replacement using PHASER (McCoy et al., 2007) and PDB 4CTK as a reference model. As mentioned before, the crystallographic asymmetric unit consists of 2 chains, but the extra positive density spots found in the two chains are not equivalent. In one chain, the extra density corresponds to a single SAM molecule, while in the other chain the positive density corresponds to one SAH and one AT-9010 molecule. The model was built using COOT (Emsley and Cowtan, 2004) and compiled using PHENIX (Adams et al., 2010) on The structure was refined at 1.30 Å. The structure was checked according to MOLPROBITY (Chen et al., 2010) and confirmed to have good stereochemistry. Data collection and refinement statistics are listed in Table 21. Structural analysis and graphics were completed using UCSF CHIMERA (Pettersen et al., 2004).

[0439] Table 20. Conservation and identification of essential amino acids in viral RdRps HCV NS5b, DENV2 NS5, and SARS-CoV2 nsp12 that contact the phosphate of the incoming nucleotide

[0440]

[0441] Table 21. Crystallographic parameters

[0442]

[0443]

[0444]

[0445] Example 36. Synthesis of Compound 1

[0446] Compound 1 (the free base form of Compound 2) can be synthesized, for example, by reacting a compound of formula Intermediate A with Intermediate B and an activating agent to form a compound of formula Intermediate C, which is then reacted with phenol to form Compound 1:

[0447]

[0448] Where R 6 is independently selected at each occurrence from fluoro, chloro, bromo, cyano, haloalkyl (eg, trifluoromethyl, difluoromethyl, fluoromethyl), azido, and nitro; and

[0449] x is 1, 2, or 3.

[0450] The reaction of the compound of formula intermediate A, intermediate B, activator and trialkylamine base preferably forms S P Phosphoramidates (as described in WO2022 / 040473A1 assigned to Atea Pharmaceuticals).

[0451] As used herein, “activating agents” are uronium-based peptide coupling reagents, including, but not limited to, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate (HATU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylammonium tetrafluoroborate (TBTU), and 1-cyano-2-ethoxy-2-oxoethylenaminooxy)dimethylamino-morpholino-carbonium hexafluorophosphate (COMU).

[0452] As used herein, a "trialkylamine base" is a 1-6 Alkyl amine bases include, but are not limited to, triethylamine or diisopropylethylamine.

[0453] As used herein, a "pyridine base" is an optionally substituted pyridine molecule including, but not limited to, pyridine, 2,6-dimethylpyridine (lutidine), or 2,4,6-trimethylpyridine (collidine).

[0454] If S P The phosphoramidate has an electron-withdrawing substituent on the aryl ester (intermediate C), so S P The compounds can be further reacted. Phosphorus stereochemistry can be determined by S P The phosphoramidate (intermediate C) is then reacted with phenol and a base to form R P Phosphoramidate (Compound 1).

[0455] Synthesis of Intermediate A

[0456] Intermediate A can be synthesized by multi-step phosphorus chloride displacement (Intermediate E) followed by reductive deprotection of the benzyl protecting group.

[0457]

[0458] In certain embodiments, the electron withdrawing group on the phenol of Intermediate A is an ortho-fluorine substituent (Intermediate A-1):

[0459]

[0460]

[0461] In certain embodiments, intermediate A has one or more electron-withdrawing substituents on phenol. Electron-withdrawing substituents include, but are not limited to, fluorine, chlorine, cyano, azido, nitro, trifluoromethyl, difluoromethyl, fluoromethyl, esters, and amides. In certain embodiments, intermediate A is used in the form of a salt. In certain embodiments, intermediate A is used in the form of a dihydroquinine salt.

[0462] Example 37. Phenol stereoinversion reaction

[0463]

[0464] Amine base

[0465] The reaction of intermediate C with phenol to form compound 1 can be carried out using a base (e.g., an amine base, such as a guanidine base). Examples of guanidine bases are bases A, B, C, D, E, F, G, and J. Bases A, C, D, and J provide the highest yields of compound 1. Intermediate C-1 is used as a model substrate for this reaction.

[0466]

[0467]

[0468] Table 22. Alkali analysis

[0469]

[0470]

[0471] Activated phenol leaving group

[0472] In certain embodiments, Compound 1 is composed of S P Preparation of phosphoramidate intermediate C (e.g., intermediate C-1, C-2, C-3, C-4, C-5, C-6, C-7, and C-8). Intermediates C-1 to C-8 have different electron withdrawing groups on phenol. These compounds can react with phenol and a base to form compound 1:

[0473]

[0474]

[0475] Table 23. Comparison of leaving groups

[0476]

[0477]

[0478] Table 24. Stoichiometric study of guanidine

[0479]

[0480] Table 25. Stoichiometric study of tetramethylguanidine

[0481]

[0482]

[0483] Example 38. Synthesis of Compound 1 Synthesis method of intermediate A-1

[0484]

[0485] Dichloromethane (2.0L, 10vol) was added to a 10°C three-necked flask. The flask was evacuated and re-injected with nitrogen three times. Phosphorus oxychloride (200g, 1.0 equivalent) was added to the flask, and the mixture was then cooled to -55°C. Dichloromethane (200mL, 1vol), benzyl alcohol (141g, 1 equivalent) and triethylamine (139g, 1.05 equivalent) were added to a 10°C mixing flask, and the resulting mixture was then added to the phosphorus oxychloride solution under nitrogen protection for 40 minutes. The reaction mixture was stirred at -55°C for 1 hour, during which time a white solid precipitated. HPLC analysis of a sample of the reaction mixture derived with benzylamine showed that the reaction appeared to be 99.6% complete. L-alanine isopropyl ester hydrochloride (219g, 1 equivalent) was added to the reaction mixture, and triethylamine (271g, 2.05 equivalent) was then added over 30 minutes.

[0486] The resulting reaction mixture was stirred for 1-2 hours and then analyzed by HPLC analysis of a sample derivatized with benzylamine for consumption of the first intermediate. The reaction appeared to be 97.7% complete.

[0487] Dichloromethane (400 mL, 2 vol), 2-fluorophenol (146 g, 1 eq.), and triethylamine (139 g, 1.05 eq., added over 20 min.) were mixed in a mixing flask at 10°C and then added to the reaction mixture over 43 min. The material was warmed from -55°C to 10°C over 3 h and then stirred for 2-4 h. HPLC analysis of a sample derived from benzylamine indicated a 2.0% area of ​​the second intermediate. The material was filtered and the filter cake was rinsed with dichloromethane (400 mL, 2 vol.). Water (600 mL, 3 vol.) was added to the filtrate and the mixture was stirred at 10°C for 5 min. The mixture was allowed to stand and the layers were separated. Aqueous hydrochloric acid solution (3.7% w / w, 1 L, 5 vol.) was added to the organics and the mixture was stirred at 10°C for 5 min. The mixture was allowed to settle and the layers were separated. Aqueous sodium bicarbonate solution (5% w / w, 1 L, 5 vol) was added to the organics and the mixture was stirred at 5°C for 5 minutes. The mixture was allowed to settle and the layers were separated. Water (600 mL, 3 vol) was added to the organics and the mixture was stirred at 10°C for 5 minutes. The mixture was allowed to settle and the layers were separated. Charcoal (10 g, 5% w / w) was added to the organics and the resulting suspension was stirred at 10°C for 2 hours. The suspension was filtered to remove the charcoal and the filter was rinsed with dichloromethane (100 mL). The filtrate was concentrated under vacuum at 45°C to a volume of about 600 mL (3 vol). Isopropanol (600 mL, 3 vol) was added to the mixture and concentrated again to about 600 mL (3 vol). Isopropanol (600 mL, 3 vol) was added to the mixture and the mixture was adjusted to 25°C and a sample was taken to quantify the amount of dichloromethane remaining (0.25% remaining).

[0488] Isopropanol (1.9 L, 9.5 vol) was added to the mixture, followed by dihydroquinine (DHQ, 387 g, 0.908 eq). The mixture was stirred until a clear solution was formed. Palladium on carbon (10% w / w, KF=63%, 13.7 g) was added to the mixture. The resulting suspension was degassed twice with nitrogen and then three times with hydrogen. The mixture was stirred at 25 ° C under 1 atm hydrogen for 18-20 hours. The sample was analyzed by HPLC to show that 0.05% of the third intermediate remained.

[0489] The suspension was filtered and the filter cake was rinsed with isopropanol (100 mL, 0.5 vol). The solvent was vacuum distilled at 55°C to form a mixture with a volume of about 1200 mL (6 vol), and acetonitrile (1.5 L, 7.5 vol) was then added to the mixture to provide a suspension. The distillation and addition of acetonitrile were repeated twice more. A sample of the resulting suspension was diluted with N-methylpyrrolidone and analyzed to check for residual isopropanol (measured 0.7%). The suspension was stirred at 80-90°C for 1-2 hours to provide a clear solution. The mixture was cooled to 5°C at a cooling rate of 20°C per hour and then stirred for 2-3 hours. The suspension was filtered and the filter cake was washed with cold acetonitrile (400 mL, 2 vol, 5°C). The filter cake was dried at 55°C non-vacuum for 18-20 hours to obtain 591.7 g of intermediate A-1 as a white powder (AUC purity of 97.9%, 4-step yield of 71.8%).

[0490] Characterization data of intermediate A-1:

[0491] 1 H NMR (400MHz, DMSO) δ12.50(s,1H),8.75(d,J=4.5Hz,1H),7.96(d,J=9.2Hz,1H),7.70–7.48(m,3H),7.41(dd,J=9.2,2.5Hz,1H ),7.12(dd,J=11.0,8.2Hz,1H),7.00(t,J=7.5Hz,1H),6.97–6.86(m,1H),6.59(s,1H),6.05(s,1H),4.77(dq,J=12.5,6.2Hz, 1H),3.97(s,4H),3.87–3.66(m,2H),3.57–3.34(m,2H),3.07(s,1H),2.84(d,J=6.4Hz,1H),1.97(d,J=36.8Hz,3H),1.87–1.6 1(m,2H),1.38(t,J=11.8Hz,1H),1.33–1.18(m,2H),1.16(d,J=6.6Hz,3H),1.09(dd,J=6.2,1.2Hz,6H),0.75(t,J=7.3Hz,3H).

[0492] 13C NMR (101MHz, DMSO) δ174.69(d,J=5.9Hz),158.27(s),154.97(s),152.58(d,J=6.7Hz),147.78(s),146.04(s) ),144.19(s),142.55–142.06(m),131.72(s),126.16(s),124.38(d,J=3.4Hz),122.73(s),122.67–122.04( m),119.39(s),116.16(d,J=18.9Hz),102.09(s),67.71(s),66.45(s),9.32(s),56.87(s),55.27(s),50.96 (s), 43.12 (s), 35.28 (s), 26.04 (s), 24.89 (s), 21.83 (d, J = 2.3Hz), 21.21 (d, J = 5.4Hz), 17.74 (s), 11.86 (s).

[0493] 31 P NMR (162MHz, DMSO) δ1.04 (s).

[0494] 19 F NMR (377MHz, DMSO) δ-132.65 (s).

[0495] Step 2: Coupling reaction

[0496]

[0497] Under nitrogen, dichloromethane (1.33 L, 20 vol), intermediate B (50.0 g, 1.0 eq., 0.160 mol), dihydroquinine salt of intermediate A (222 g, 2.2 eq.) and (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbonium hexafluorophosphate (82.3 g, 1.2 eq.) were charged into a 2 L three-necked flask equipped with a reflux condenser and a thermometer.

[0498] The resulting suspension was heated to 30-35°C. 2,6-Dimethylpyridine (20.6 g, 1.2 equivalents) was added to the reaction mixture over 2 hours, during which the suspension gradually dissolved. The mixture was stirred at 30-35°C for 5-6 hours to obtain a clear solution. The reactants were sampled, and HPLC analysis showed that the remaining intermediate B was less than 5%. The solution yield calculated by sample determination was 64.2 g (yield 66.9%). The reaction mixture was cooled to 0-10°C over 1 hour.

[0499] Water (500 mL, 10 vol) was added to the mixture and the batch was stirred for 2 minutes. Stirring was stopped and the batch was allowed to settle into two layers and the aqueous layer was removed. Aqueous hydrochloric acid (20% w / w, 133 g, 4.5 eq) was added over 1 hour during which time the dihydroquinine salt precipitated. The solution was filtered and the filter cake was rinsed with dichloromethane (50 mL, 1 vol) and the rinse was combined with the filtrate. Aqueous hydrochloric acid (7% w / w, 167 g, 2.0 eq) was added to the filtrate and the batch was stirred for 2 minutes. Stirring was stopped and the batch was allowed to settle into two layers and the aqueous layer was removed. The organics were washed three times with aqueous sodium bicarbonate (5% w / w, 500 g, 1.9 eq) and then with water (500 mL, 10 vol). The analytical yield of the organic layer was 59.4 g (61.9%). The organic layer was concentrated in vacuo at 30-50°C to a total volume of about 200 mL (4 vol). Toluene (500 mL, 10 vol) was added to the mixture and the mixture was concentrated under vacuum at a temperature of 40-55°C to a total volume of about 250 mL (5 vol). The addition and concentration of toluene were repeated once more. Ethyl acetate (250 mL, 5 vol) was added to the batch and the mixture was heated to 65-75°C. While stirring, the mixture was cooled to 5°C over 3 hours, during which time a crystalline solid was formed. The batch was kept for 4 hours and then filtered to obtain a yellow crystalline solid. The solid and ethyl acetate (250 mL, 5 vol) were added to a flask and heated to 65-75°C to obtain a clear solution. As the crystalline solid formed, the solution was cooled to 5°C over 3 hours. The batch was kept at 5°C for 4 hours and then filtered to obtain a yellow solid. The yellow solid was dried at 45°C for 15-16 hours to obtain intermediate C-1 (79 g, 56.2% corrected yield) as a yellow crystalline solid.

[0500] Characterization data of intermediate C-3:

[0501] 1 H NMR (400MHz, DMSO) δ7.80(s,1H),7.53-7.40(m,1H),7.40-7.26(m,2H),7.25-7.11(m,2H),6.14(dd,J=12.9,10.4Hz,1H),6.11-5.91(m,3H),5. 78(d,J=6.4Hz,1H),4.91-4.71(m,H),4.58-4.23(m),4.15-4.05(m,1H) ,3.91-3.72(m,1H),2.88(s,3H),1.32-1.20(m,3H),1.17-0.97(m,9H).

[0502] 19F NMR (377MHz, DMSO) δ -131.41 (s), -159.74 (s).

[0503] 31 P NMR (162MHz, DMSO) δ4.42 (s).

[0504] MS (ES+): expected: 600.2; found: 600.3

[0505] Step 3: Aryl oxide replacement

[0506]

[0507] Under nitrogen protection, acetonitrile (255 mL, 8.5 vol), dimethyl sulfoxide (45 mL, 1.5 vol), intermediate C-1 (30 g, 0.05 mol, 1.0 eq.), phenol (94.1 g, 20.0 eq.) and 1,1,3,3-tetramethylguanidine (17.3 g, 3.0 eq.) were charged into a 350 mL flask equipped with a thermometer. The batch was heated to 30 ° C for 22 hours while stirring.

[0508] The reaction was sampled and less than 2% of the intermediate C-1 remained. The reaction mixture was cooled to 0–10°C over 1 hour and the batch was poured into a reactor with water (600 mL, 20 vol) under stirring. Methyl tert-butyl ether (600 mL, 20 vol) was added to the mixture. Aqueous hydrochloric acid (20% w / w, 90 g, 10 eq) was added over 0.5 hour to a pH of 3–4 while maintaining the batch temperature at 0–10°C. The mixture was stirred for an additional 2 minutes, stirring was stopped, the aqueous and organic layers were separated, and the aqueous layer was collected. Water (300 mL, 10 vol) was added to the organic layer, and then aqueous hydrochloric acid (20% w / w, 32 g, 3.5 eq) was added over 0.5 hour while maintaining the batch temperature at 0-10°C.

[0509] The mixture was stirred for an additional 2 minutes, stirring was stopped, the aqueous and organic layers were separated, and the aqueous layer was collected. The two aqueous layers were combined and cooled to 0–10°C. Aqueous sodium bicarbonate solution (5% w / w, 85 g, 1.0 eq) was added over 0.5 hours to adjust the pH to 8–9, followed by the addition of dichloromethane (300 mL, 10 vol) and water (300 mL, 10 vol). The mixture was stirred for 2 minutes to allow it to settle into two phases, and the aqueous phase was removed. The resulting organics were concentrated in vacuo at 30–40°C to a volume of approximately 60 mL (2 vol).

[0510] Acetone (300 mL, 10 vol) was added to the mixture. The batch was concentrated under vacuum at 30–40 °C to a volume of approximately 60 mL (2 vol). Acetone (180 mL, 6 vol) was added to the mixture to provide a clear solution, which was cooled to 0–5 °C. Aqueous hydrochloric acid (36.5% w / w, 10 g, 2.0 eq) was added in one portion, and the mixture was stirred at 0–5 °C for 12 hours. The resulting suspension was filtered and the recovered solid was dried to give the dihydrochloride salt of compound 1 (19.2 g, 59% yield) as an off-white solid.

[0511] Characterization data of the dihydrochloride salt of compound 1:

[0512] 1 H NMR (400MHz, DMSO) δ9.68(d,J=4.7Hz,1H),8.14(s,3H),7.36(t,J=7.9Hz,2H),7.18(dd,J=17.8,7.9Hz,3H),6.11(d,J=18.6Hz,2H ),4.84(hept,J=6.2Hz,1H),4.47(dd,J=10.4,5.9Hz,1H),4.43–4.2,1H),3.77(s,1H),3.15(d,J=4.8Hz,3H),1.28–1.07(m,12H).

[0513] 13 C NMR (101MHz, DMSO) δ173.14(d,J=4.3Hz),153.46(s),151.11(d,J=6.0Hz),149.58(s),130.03(s),1 24.98(s),120.62(d,J=4.6Hz),112.08(s),102.03(s),100.24(s),80.59(s),72.00(d,J=18.2Hz), 68.43(s),65.85(s),50.42(s),40.49(d,J=13.9Hz),40.35(s),40.14(s),39.93(s),39.72(s),39. 51 (s), 39.30 (s), 31.16 (s), 30.07 (s), 21.84 (d, J = 7.2Hz), 20.08 (d, J = 7.2Hz), 17.15 (s), 16.91 (s).

[0514] 19 F NMR (377MHz, DMSO) δ-160.09 (s).

[0515] 31P NMR (162MHz, DMSO) δ3.66 (s).

[0516] MS (ES+): Expected: 582.22; Result: 582.35

[0517] General synthesis of intermediates C-1 to C-8

[0518]

[0519] General method for intermediates A-2, A-3 and A-7:

[0520] A mixture of benzyl alcohol (10.81 g, 0.1 mol) and TEA (10.62 g, 0.105 mol) was added dropwise to a solution of phosphorus oxychloride (15.3 g, 0.1 mol) in DCM (150 mL) under nitrogen at -60° C. After addition, the reaction was stirred at -60° C. for 1 hour.

[0521] L-alanine isopropyl ester hydrochloride (16.76 g, 0.1 mol) was added to the reaction, and then TEA (20.74 g, 0.205 mol) was added dropwise at -60°C. The reaction mixture was stirred at -60°C for 1 hour. At -60°C, the substituted phenol and TEA (10.62 g, 0.105 mol) and DCM (20 mL) were added dropwise to the reaction.

[0522] After addition, the reaction was stirred at room temperature overnight and then quenched with water. The organic layer was dried over Na2SO4, filtered, and then the solvent was removed in vacuo. The residue was purified by silica gel column chromatography to give a colorless oil (benzyl-protected intermediates A-2, A-3, and A-7).

[0523] Colorless oil (0.1 mol) and isopropanol (200 mL, 5 vol) were added to a 1000 mL three-necked glass flask. Dihydroquinine (32.64 g, 0.1 mol) was added and stirred to give a clear solution. To this solution was added 5% wet Pd / C (60% water, 5% w / w dry basis as determined by KF) and hydrogenolysis was performed at 20-25° C. for 18-20 hours using 1 atm hydrogen. When IPC showed that the starting material was completely consumed, the mixture was filtered through a Buchner funnel and the filtrate was stirred with 4.0 g of charcoal at 25-30° C. for 2 hours. The mixture was filtered and the filter cake was washed with isopropanol (40 mL). The filtrate was concentrated at 50-60° C. under vacuum conditions below 0.09 MPa to give a crude product.

[0524] Isopropyl acetate (200 mL, 5 vol) was added and the mixture was concentrated again at 50°C-60°C under a vacuum of less than 0.09 MPa. This step was repeated once more with 5 vol of isopropyl acetate. Fresh isopropyl acetate (200 ml, 5 vol) was added to the residue and the mixture was stirred at 80°C-90°C for 2-3 hours to obtain a clear solution. It was then cooled to 0°C-10°C at a cooling rate of 20°C / 1 hour and stirred at this temperature for 2-3 hours. The solid was collected by filtration and rinsed with cold isopropyl acetate (40 mL, 1 vol) to obtain a wet cake, which was dried at 50°C non-vacuum for 18 hours to obtain the dihydroquinine salt of intermediate A-2, A-3 or A-7 as an off-white solid.

[0525] Characterization data of intermediate A-2 dihydroquinine salt:

[0526]

[0527] 1 H NMR (400MHz, DMSO) δ12.30(s,1H),8.74(d,J=4.5Hz,1H),7.96(d,J=9.2Hz,1H),7.64(d,J=4.6Hz,1H),7.62-7.56(m,1H),7.52(d,J =2.5Hz,1H),7.41(dd,J=9.2,2.6Hz,1H),7.25-7.03(m,1H),6.97-6.76(m,1H),6.57(s,1H),6.02(s,1H),4.77(dt,J=12.5,6.2Hz,1 H),3.97(s,4H),3.75(dd,J=12.1,7.7Hz,2H),3.59-3.28(m,3H),3.09(d,J=5.3Hz,1H),2.86(s,1H),2.01(s,2H),1.92(s,1H),1.8 8-1.64(m,2H),1.38(t,J=11.8Hz,1H),1.33-1.18(m,2H),1.14(d,J=5.6Hz,3H),1.09(dd,J=6.2,0.8Hz,6H),0.75(t,J=7.3Hz,3H).

[0528] MS (ES-): Expected: 322.07; Found: 322.00

[0529] Characterization data of intermediate A-3 dihydroquinine salt:

[0530]

[0531] 1H NMR (400MHz, DMSO) δ12.36(s,1H),8.74(d,J=4.5Hz,1H),7.96(d,J=9.2Hz,1H),7.64(d,J=4.5Hz,1H),7.54(d,J=2.5Hz,1H),7.41(dd, J=9.2,2.6Hz,1H),7.21(dd,J=15.7,8.1Hz,1H),7.07(dd,J=11.5,2.0Hz,1H),6.94(d,J=8.2Hz,1H),6.74(td,J=8.5,2.4Hz,1H),6.57 (s,1H),6.04(s,1H),4.77(dt,J=12.5,6.2Hz,1H),3.97(s,4H),3.76(d,J=5.4Hz,2H),3.63-3.25(m,2H),3.09(d,J=4.7Hz,1H),2.85( s,1H),1.97(d,J=34.0Hz,3H),1.88-1.62(m,2H),1.38(t,J=11.8Hz,1H),1.33-1.18(m,2H),1.18-1.03(m,9H),0.75(t,J=7.3Hz,3H).

[0532] MS (ES-) expected: 304.08; measured: 304.04

[0533] Characterization data of intermediate A-7 dihydroquinine salt:

[0534]

[0535] 1H NMR (400MHz, DMSO) δ12.56(s,1H),8.74(d,J=4.5Hz,1H),7.96(d,J=9.2Hz,1H),7.64(d,J=4.5Hz,1H),7.54(d,J=2. 6Hz,1H),7.41(dd,J=9.2,2.6Hz,1H),7.15(dt,J=8.4,4.3Hz,2H),7.09-6.88(m,2H),6.60(s,1H),6.05(s,1H),4.9 5-4.60(m,1H),3.97(s,4H),3.81-3.58(m,2H),3.47(d,J=7.4Hz,1H),3.41(s,1H),3.05(s,1H),2.82(s,1H),2.11- 1.86 (m, 3H), 1.75 (d, J = 36.1Hz, 2H), 1.28 (ddt, J = 20.8, 13.9, 9.0Hz, 3H), 1.17-1.04 (m, 9H), 0.75 (t, J = 7.3Hz, 3H).

[0536] MS (ES-) expected: 304.08; measured: 304.18

[0537] General procedure for intermediates A-4 and A-5:

[0538] Under nitrogen, a mixture of benzyl alcohol (10.81 g, 0.1 mol) and TEA (10.62 g, 0.105 mol) was added dropwise to a DCM (150 mL) solution of phosphorus oxychloride (15.3 g, 0.1 mol) at -60 ° C. After addition, the reaction was stirred at -60 ° C for 1 hour. L-alanine isopropyl ester hydrochloride (16.76 g, 0.1 mol) was added to the reaction, and then TEA (20.74 g, 0.205 mol) was added dropwise at -60 ° C. The reaction mixture was stirred at -60 ° C for 1 hour. At -60 ° C, substituted phenol (0.1 mol) corresponding to phenyl substitution and TEA (10.62 g, 0.105 mol) and DCM (20 mL) were added dropwise to the reaction. After addition, the reaction was stirred to room temperature overnight and then quenched with water. The organic layer was dried over Na2SO4, filtered, and the solvent was removed in vacuo. The residue was purified by silica gel column chromatography to give a colorless oil.

[0539] Colorless oil (0.1 mol) and isopropanol (200 mL, 5 vol) were charged into a 1000 mL three-necked glass flask. Dihydroquinine (32.64 g, 0.1 mol) was added and stirred to obtain a clear solution. To this solution was added 5% wet Pd / C (60% water, 5% w / w dry basis as determined by KF) and hydrogenolysis was performed at 20°C-25°C using 1 atm hydrogen for 18-20 hours. When IPC showed that the starting material had been completely consumed, the mixture was filtered through a Buchner funnel and the filtrate was stirred with 4.0 g of charcoal for 2 hours at 25°C-30°C. The mixture was filtered and the filter cake was washed with isopropanol (40 mL).

[0540] The filtrate was concentrated under vacuum at 50°C-60°C to less than 0.09 MPa to obtain a crude product. Toluene (200 mL, 5 vol) was added and the mixture was concentrated again under vacuum at 50°C-60°C less than 0.09 MPa. This step was repeated twice with 5 vol of toluene to obtain an oily residue. The residue was used in the next step without purification.

[0541] Synthesis method of intermediate A-6:

[0542] A mixture of 3-chlorophenol (12.86 g, 0.1 mol) and TEA (10.62 g, 0.105 mol) was added dropwise to a solution of phosphorus oxychloride (15.3 g, 0.1 mol) in DCM (150 mL) at -60°C under nitrogen. After addition, the reaction was stirred at -60°C for 1 hour. L-alanine isopropyl ester hydrochloride (16.76 g, 0.1 mol) was added to the reaction, and then TEA (20.74 g, 0.205 mol) was added dropwise at -60°C. The reaction was stirred to room temperature overnight. Filtered, then the solvent was removed in vacuo, MTBE (128 mL, 10 v) was added, the mixture was stirred at 20°C for 1 hour, filtered, and the filtrate was stirred at 0°C. A solution of citric acid monohydrate (42 g, 0.2 mol) in H2O (200 mL) was then added dropwise to the filtrate at 0°C. The reaction was stirred to room temperature overnight. The reaction was allowed to stand for stratification. The organic layer was stirred with H2O (100 mL) and then allowed to stand for stratification. DIPEA (12.9 g, 0.1 mol) was added to the organic phase. The mixture was concentrated at 40 °C under a vacuum of less than 0.09 MPa to give a crude product. The purified product was obtained by preparative HPLC.

[0543] Characterization data of intermediate A-6 diisopropylethylamine salt

[0544]

[0545] 1H NMR (400MHz, DMSO) δ10.25(s,1H),7.23(dd,J=18.1,10.0Hz,2H),6.99(td,J=8.0,1.3Hz,2H),4.80(dt,J=12.5,6.2Hz,1H ),3.81-3.62(m,2H),3.60-3.43(m,2H),3.04(q,J=6.9Hz,2H),2.17-1.95(m,1H),1.32-1.19(m,13H),1.18-1.08(m,9H).

[0546] MS (ES-): expected: 320.05; found: 320.10

[0547] Synthesis method of intermediate A-8

[0548] TEA (20.74 g, 0.205 mol) was added dropwise to a solution of 4-nitrophenyl dichlorophosphonate (25.6 g, 0.1 mol) and L-alanine isopropyl ester hydrochloride (16.76 g, 0.1 mol) in DCM (256 mL, 10 v) at -10 °C under nitrogen. After addition, the reaction was stirred to room temperature overnight. The mixture was concentrated at 40 °C under a vacuum of less than 0.09 MPa. MTBE (256 mL, 10 v) was added and the mixture was stirred at 20 °C for 1 hour. The filtrate was filtered and stirred at 0 °C. Tetrabutylammonium hydroxide (208 g, 0.2 mol, 25% w / w in H2O) was added dropwise to the filtrate at 0 °C. After addition, the reaction was stirred to room temperature overnight. The reactants were allowed to stand for stratification. The aqueous layer was stirred with IPAC (200 mL) and allowed to stand for stratification. The IPAC layer was stirred with HCl aqueous solution (200 mL, 1 mol / L) and then allowed to stand for stratification. The organic layer was dried over Na2SO4 and filtered. Dihydroquinine (19.6 g, 0.06 mol) was added to the filtrate. The mixture was concentrated at 50°C under a vacuum of less than 0.09 MPa to obtain a crude product, MTBE (256 mL, 10 v) was added, and the mixture was stirred at 20°C for 1 hour and filtered. The wet cake was dried at 60°C non-vacuum for 16 hours. The product intermediate A-8 was obtained.

[0549] Characterization data of intermediate A-8 dihydroquinine salt

[0550]

[0551] 1H NMR (400MHz, DMSO) δ11.89(s,1H),8.74(d,J=4.5Hz,1H),8.15-8.04(m,2H),7.96(d,J=9.2Hz,1H),7.64(d,J =4.4Hz,1H),7.49(d,J=2.3Hz,1H),7.45-7.32(m,3H),6.59(s,1H),6.00(s,1H),4.75(dt,J=12.5,6.2Hz,1H ),4.07-3.86(m,5H),3.85-3.68(m,1H),3.62-3.22(m,4H),3.10(s,1H),2.86(s,1H),1.97(d,J=30.9Hz,3H) ,1.76(d,J=35.8Hz,2H),1.40(t,J=11.7Hz,1H),1.34-1.18(m,2H),1.18-1.00(m,9H),0.75(t,J=7.3Hz,3H).

[0552] MS (ES-): expected: 331.07; found: 331.06

[0553] General method for intermediates C-1 to C-5 and C-7

[0554] The salt of intermediates A-1 to A-8 (1.7 eq.), intermediate B (6.25 g, 20 mmol, 1.0 eq.), diisopropylethylamine (2.6 g, 20 mmol, 1.0 eq.) and HATU (12.9 g, 34 mmol, 1.7 eq.) were added to 188 mL of dichloromethane (30 volumes). The mixture was heated to 40 ° C under nitrogen protection and stirred for 18 hours. The reaction was monitored by TLC and HPLC.

[0555] After the reaction is completed, the reaction mixture is cooled to 0-10°C, washed twice with 2N HCl (100 mL), washed with 5% sodium bicarbonate aqueous solution (100 mL), and the separated organic phase is dried over Na2SO4 and filtered. The filtrate is concentrated in vacuo at 40-45°C to obtain a residue. The residue is purified by silica gel column chromatography to obtain a crude product. The crude product is recrystallized from EA (30 mL) and the precipitate is dried at 45-50°C non-vacuum for 16 hours to obtain intermediates C-1 to C-5 or C-7.

[0556] Characterization data of intermediate C-2

[0557]

[0558] 1H NMR (400MHz, DMSO) δ7.79 (s, 1H), 7.54-7.35 (m, 2H), 7.28 (s, 1H), 7.06 (t, J= 8.6Hz,1H),6.27-5.87(m,4H),5.75(d,J=6.8Hz,1H),4.81(dt,J=12.5,6.3H z,1H),4.37(ddd,J=18.3,14.3,8.5Hz,3H),4.16-3.96(m,1H),3.79(ddd,J= 17.1, 7.2, 2.9Hz, 1H), 2.88 (s, 3H), 1.24 (d, J = 7.1Hz, 3H), 1.20-1.01 (m, 9H).

[0559] MS (ES+): Expected: 618.20; Measured: 618.24

[0560] Characterization data of intermediate C-3:

[0561]

[0562] 1 H NMR (400MHz, DMSO) δ7.79 (s, 1H), 7.40 (dd, J = 15.3, 8.0Hz, 1H), 7.27 (s, 1H), 7.16-6.99(m,3H),6.19-5.87(m,4H),5.75(d,J=6.6Hz,1H),4.81(dt,J=12.5 ,6.2Hz,1H),4.36(ddd,J=18.4,14.2,8.3Hz,3H),4.07(dd,J=15.2,8.0Hz,1 H), 3.93-3.70 (m, 1H), 2.88 (s, 3H), 1.21 (d, J = 7.0Hz, 3H), 1.17-1.02 (m, 9H).

[0563] MS (ES+): Expected: 600.21; Measured: 600.24

[0564] Characterization data of intermediate C-4:

[0565]

[0566] 1H NMR (400MHz, DMSO) δ7.79(s,1H),7.28(s,1H),7.15-6.90(m,3H),6.20(dd,J=12.6,10.4Hz,1H),6.13-5.88(m,3H),5.77(d,J=6.3Hz,1H),4.80(d q,J=12.5,6.2Hz,1H),4.54-4.24(m,3H),4.07(dd,J=17.6,10.7Hz,1H), 3.92-3.69(m,1H),2.87(s,3H),1.22(d,J=7.1Hz,3H),1.15-1.03(m,9H).

[0567] MS (ES+): Expected: 618.20; Measured: 618.34

[0568] Characterization data of intermediate C-5:

[0569] 1 H NMR (400MHz, DMSO) δ7.79(s,1H),7.75-7.62(m,2H),7.62-7.53(m,2H),7.27(s,1 H),6.16(dd,J=12.7,10.2Hz,1H),6.10-5.93(m,3H),5.76(d,J=6.8Hz,1H),4.81( dt,J=12.5,6.2Hz,1H),4.53-4.27(m,3H),4.08(dd,J=13.2,6.3Hz,1H),3.83(dd d,J=17.1,7.2,2.9Hz,1H),2.88(s,3H),1.23(d,J=7.1Hz,3H),1.15-1.04(m,9H).

[0570]

[0571] MS (ES+): Expected: 607.22; Measured: 607.19

[0572] Characterization data of intermediate C-7:

[0573]

[0574] 1H NMR (400MHz, DMSO) δ7.80(s,1H),7.22(ddd,J=20.8,13.3,7.0Hz,5H),6.23-5.86(m,4H),5.73(d,J=7.1Hz,1H),4.81(dt,J=12.5,6. 2Hz,1H),4.54-4.20(m,3H),4.06(dd,J=12.8,5.8Hz,1H),3.90-3.67(m,1H),2.89(s,3H),1.21(t,J=6.2Hz,3H),1.15-1.03(m,9H).

[0575] MS (ES+): Expected: 600.21; Measured: 600.36

[0576] Synthesis of intermediate C-6

[0577]

[0578] Characterization data of intermediate C-6:

[0579]

[0580] 1 H NMR (400MHz, DMSO) δ7.80 (s, 1H), 7.39 (t, J = 8.1Hz, 1H), 7.33 (s, 1H), 7.26 (d, J=7.9Hz,2H),7.22-7.16(m,1H),6.23-5.89(m,4H),5.76(d,J=6.8Hz,1H),4. 81(dt,J=12.5,6.2Hz,1H),4.53-4.23(m,3H),4.16-4.00(m,1H),3.81(ddd,J =17.1,7.2,2.9Hz,1H),2.88(s,3H),1.22(d,J=7.1Hz,3H),1.17-1.00(m,9H).

[0581] MS (ES+): Expected: 616.18; Measured: 616.15

[0582] Synthesis of intermediate C-8

[0583]

[0584] Characterization data of intermediate C-8:

[0585]

[0586] 1H NMR (400MHz, DMSO) δ8.31-8.18(m,2H),7.79(s,1H),7.44(dd,J=14.3,9.1H z,2H),7.18(d,J=46.5Hz,1H),6.35-6.15(m,1H),6.12-5.88(m,3H),5.75(s ,1H),4.88-4.73(m,1H),4.45(dd,J=14.1,7.1Hz,3H),4.18-4.01(m,1H),3. 81(s,1H),2.89(s,3H),1.24(d,J=7.3Hz,3H),1.10(dd,J=14.0,7.5Hz,9H).

[0587] MS (ES+): Expected: 627.21; Measured: 627.37

[0588] This specification has been described with reference to embodiments of the present invention. However, it will be appreciated by those skilled in the art that various modifications and variations may be made without departing from the scope of the present invention as described in the following claims. Therefore, this specification should be regarded as illustrative rather than restrictive, and all such modifications are intended to be included within the scope of the present invention.

Claims

1. A method of treating dengue virus infection in a human host in need thereof, the method comprising administering about 700 mg to about 1,000 mg of Compound 1 or a pharmaceutically acceptable salt thereof, optionally in a pharmaceutically acceptable carrier, two, three or four times a day, 2. The method according to claim 1, wherein the compound is 3. The method according to claim 1, wherein a pharmaceutically acceptable salt of Compound 1 is provided.

4. The method according to claim 1 or 2, wherein from about 700 mg to about 850 mg of the compound is administered.

5. The method according to claim 4, wherein from about 700 mg to about 800 mg of the compound is administered.

6. The method according to claim 2, wherein about 700 to 900 mg of Compound 2 is administered.

7. The method according to claim 3, wherein about 700 mg to about 900 mg of a pharmaceutically acceptable salt thereof is administered.

8. The method according to claim 7, wherein about 700 mg to about 800 mg of a pharmaceutically acceptable salt thereof is administered.

9. The method according to claim 3, wherein about 800 to 1000 mg of Compound 1 or a pharmaceutically acceptable salt thereof is administered.

10. The method according to any one of claims 1 to 9, wherein the compound is administered twice daily.

11. The method according to any one of claims 1 to 9, wherein the compound is administered three times a day.

12. The method according to any one of claims 1 to 9, wherein the compound is administered four times a day.

13. The method according to any one of claims 1 to 12, wherein the compound is administered for three consecutive days.

14. The method according to any one of claims 1 to 12, wherein the compound is administered for four consecutive days.

15. The method according to any one of claims 1 to 12, wherein the compound is administered for five consecutive days.

16. The method according to any one of claims 1 to 12, wherein the compound is administered for six consecutive days.

17. The method according to any one of claims 1 to 12, wherein the compound is administered for seven consecutive days.

18. The method according to any one of claims 1 to 12, wherein the compound is administered for eight consecutive days.

19. The method according to any one of claims 1 to 12, wherein the compound is administered for nine consecutive days.

20. The method according to any one of claims 1 to 12, wherein the compound is administered for ten consecutive days.

21. The method according to any one of claims 1 to 20, wherein the human is suffering from dengue virus serotype 1.

22. The method according to any one of claims 1 to 20, wherein the human is suffering from dengue virus serotype 2.

23. The method according to any one of claims 1 to 20, wherein the human is suffering from dengue virus serotype 3.

24. The method according to any one of claims 1 to 20, wherein the human is suffering from dengue virus serotype 4.

25. The method according to any one of claims 1 to 24, wherein the compound is administered in combination with another antiviral compound.

26. The method of claim 25, wherein the other antiviral compound is an NS2B-NS3 protease inhibitor.

27. The method of claim 25, wherein the other antiviral compound is an NS3 inhibitor.

28. The method according to claim 25, wherein the other antiviral compound is an inhibitor of NS3-NS4B interaction.

29. The method according to claim 28, wherein the NS3B-NS4B interaction inhibitor is JNJ-1802 or JNJ-A07.

30. The method of claim 25, wherein the another antiviral compound is another NS5 inhibitor.

31. The compound according to any one of claims 1-30, wherein the pharmaceutically acceptable carrier comprises silicified microcrystalline cellulose.

32. The compound according to any one of claims 1-31, wherein the pharmaceutically acceptable carrier further comprises croscarmellose sodium.

33. The compound according to any one of claims 1-32, wherein the pharmaceutically acceptable carrier further comprises magnesium stearate.

34. The compound according to any one of claims 1-32, wherein the pharmaceutically acceptable carrier further comprises colloidal silicon dioxide.

35. A compound according to any one of claims 1-34, wherein the pharmaceutically acceptable carrier comprises sodium stearyl fumarate.

36. A method of treating dengue virus infection in a human host in need thereof, the method comprising administering about 700 to about 1000 mg of a compound of formula I or a pharmaceutically acceptable salt thereof: in: R 2 C 1-6 Alkyl (including methyl, ethyl, propyl and isopropyl), C 3-7 cycloalkyl, aryl, aryl(C1-C4alkyl)-, heteroaryl, heteroaryl(C1-C4alkyl)- or heteroalkyl, each of which is optionally substituted by 1, 2, 3 or 4 substituents, the substituents being independently selected at each occurrence from R 22 ; R 3 is hydrogen or C optionally substituted with 1, 2, 3 or 4 substituents 1-6 Alkyl, the substituents are independently selected from R 33 ; R 4a and R 4b are independently selected from hydrogen, C 1-6 Alkyl, aryl (C 1-6 Alkyl)-, C 3-7 Cycloalkyl, C 3-7 Cycloalkyl (C 1-6 Alkyl)-, heteroaryl (C 1-6 alkyl)- and aryl, each of which is optionally substituted by 1, 2, 3 or 4 substituents, said substituents being independently selected at each occurrence from R 44 ; R 5 For hydrogen, C 1-6 Alkyl (including methyl, ethyl, propyl and isopropyl), C 1-6 Haloalkyl or C 3-7 cycloalkyl, and in an alternative embodiment, R 5 is aryl(C1-C4alkyl)-, aryl, heteroaryl or heteroalkyl, which is optionally substituted by 1, 2, 3 or 4 substituents, the substituents being independently selected in each case from R 55 ; R 22 , R 33 , R 44 and R 55 is independently selected at each occurrence from hydrogen, C 1- C6 alkyl, C 3- C7 cycloalkyl, alkenyl, alkynyl, F, Cl, Br, I, heterocycle, heterocycloalkyl, haloalkoxy, haloalkyl, C(O)R 66 、C(O)OR 66 、C(O)N(R 66 )2 and cyano; R 66 is independently selected at each occurrence from hydrogen, C1-C6-alkyl, aryl(C1-C6-alkyl)-, C3-C7-cycloalkyl, C3-C7-cycloalkyl(C1-C6-alkyl)-, heteroaryl(C1-C6-alkyl)- and aryl.

37. A compound according to claim 36, wherein the compound is or a pharmaceutically acceptable salt thereof.

38. A compound according to claim 36, wherein the compound is or a pharmaceutically acceptable salt thereof.

39. A compound according to claim 36, wherein the compound is or a pharmaceutically acceptable salt thereof.

40. A compound according to claim 37, wherein the compound is or a pharmaceutically acceptable salt thereof.

41. A compound according to claim 37, wherein the compound is or a pharmaceutically acceptable salt thereof.

42. A compound according to claim 36, wherein the compound is or a pharmaceutically acceptable salt thereof.

43. A compound according to claim 38, wherein the compound is or a pharmaceutically acceptable salt thereof.

44. A compound according to claim 38, wherein the compound is or a pharmaceutically acceptable salt thereof.

45. A method of treating dengue virus infection in a human host in need thereof, the method comprising administering from about 700 to about 1000 mg of a compound of formula II or a pharmaceutically acceptable salt thereof, optionally in a pharmaceutically acceptable carrier, in R 3 is hydrogen or C optionally substituted with 1, 2, 3 or 4 substituents 1-6 Alkyl, the substituents are independently selected from R 33 ; R 4a and R 4b are independently selected from hydrogen, C 1-6 Alkyl, aryl (C 1-6 Alkyl)-, C 3-7 Cycloalkyl, C 3-7 Cycloalkyl (C 1-6 Alkyl)-, heteroaryl (C 1-6 alkyl)- and aryl, each of which is optionally substituted by 1, 2, 3 or 4 substituents, said substituents being independently selected at each occurrence from R 44 ;and R 5 For hydrogen, C 1-6 Alkyl (including methyl, ethyl, propyl and isopropyl), C 1-6 Haloalkyl or C 3-7 cycloalkyl, and in an alternative embodiment, R 5 is aryl(C1-C4alkyl)-, aryl, heteroaryl or heteroalkyl, which is optionally substituted by 1, 2, 3 or 4 substituents, the substituents being independently selected in each case from R 55 .

46. ​​The method according to claim 45, wherein the compound is a compound of formula IIA or a pharmaceutically acceptable salt thereof:

47. A method according to claim 46, wherein the compound is selected from 48. The method according to claim 45, wherein the compound is a compound of formula IIB or a pharmaceutically acceptable salt thereof:

49. The method according to claim 48, wherein the compound is selected from 50. The method according to any one of claims 45-49, wherein the compound is administered once daily.

51. The method according to any one of claims 45-49, wherein the compound is administered twice daily.

52. The method according to any one of claims 45-49, wherein the compound is administered three times a day.

53. The method according to any one of claims 45-49, wherein the compound is administered four times per day.

54. The method according to any one of claims 45-53, wherein the compound is administered for three, four, five, six, seven, eight, nine or ten consecutive days.

55. The method according to any one of claims 45-54, wherein the dengue virus is serotype 1.

56. The method according to any one of claims 45-54, wherein the dengue virus is serotype 2.

57. The method according to any one of claims 45-54, wherein the dengue virus is serotype 3.

58. The method according to any one of claims 45-54, wherein the dengue virus is serotype 4.

59. A method of treating dengue virus infection in a human host in need thereof, the method comprising administering about 700 to about 800 mg of a compound of formula II, IIA or IIB, wherein R 4a For C7-C 15 Alkyl, variable R 4b is hydrogen, and all other variables are as defined herein.

60. The method according to claim 59, wherein the compound is selected from 61. The method according to claim 59 or 60, wherein the compound is administered once daily.

62. The method according to claim 59 or 60, wherein the compound is administered twice daily.

63. The method according to claim 59 or 60, wherein the compound is administered three times a day.

64. The method according to claim 59 or 60, wherein the compound is administered four times per day.

65. The method according to any one of claims 59-64, wherein the compound is administered for three, four, five, six, seven, eight, nine or ten consecutive days.

66. The method according to any one of claims 59-65, wherein the dengue virus is serotype 1.

67. The method according to any one of claims 59-65, wherein the dengue virus is serotype 2.

68. The method according to any one of claims 59-65, wherein the dengue virus is serotype 3.

69. The method according to any one of claims 59-65, wherein the dengue virus is serotype 4.

70. A method for preparing compound 1, comprising: a. reacting a compound of formula intermediate A with intermediate B in the presence of an activator and a trialkylamine or pyridine base to form a compound of formula intermediate C; and b. reacting a compound of formula intermediate C with phenol in the presence of a guanidine base.

71. The method according to claim 70, wherein the compound of formula Intermediate A is Intermediate A-1 or a salt thereof:

72. The method according to claim 70, wherein the compound of formula Intermediate A is Intermediate A-3 or a salt thereof:

73. The method according to claim 70, wherein the compound of formula Intermediate A is Intermediate A-5 or a salt thereof:

74. The method according to claim 70, wherein the compound of formula Intermediate A is Intermediate A-6 or a salt thereof:

75. A method according to any one of claims 70 to 74, wherein the activator is selected from 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) and 1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbonium hexafluorophosphate (COMU).

76. A method according to any one of claims 70-75, wherein the activator is COMU.

77. A method according to any one of claims 70-75, wherein the activating agent is HATU.

78. A process according to any one of claims 70 to 77, wherein a trialkylamine base is used in step (a).

79. A process according to claim 78, wherein the trialkylamine base is triethylamine.

80. The process according to claim 78, wherein the trialkylamine base is diisopropylethylamine.

81. A process according to any one of claims 70 to 77, wherein a pyridine base is used in step (a).

82. The method according to claim 81, wherein the pyridine base is pyridine.

83. The method according to claim 81, wherein the pyridine base is lutidine.

84. The method according to claim 81, wherein the pyridine base is collidine.

85. A method of treating serotype 1, 3 or 4 of dengue virus in a human host in need thereof, the method comprising administering about 700 mg to about 1,000 mg of Compound 1, or a pharmaceutically acceptable salt thereof, optionally in a pharmaceutically acceptable carrier, two, three or four times a day, 86. The method according to claim 85, wherein the compound is Compound 2:

87. The method according to claim 85, wherein the compound is Compound 1 or a pharmaceutically acceptable salt thereof.

88. The method according to claim 86, wherein about 750 mg of Compound 2 is administered.

89. The method according to claim 87, wherein about 750 mg of Compound 1 or a pharmaceutically acceptable salt thereof is administered.

90. The method according to any one of claims 85-89, wherein the compound is administered twice daily.

91. The method according to any one of claims 85-89, wherein the compound is administered three times a day.

92. The method according to any one of claims 85-89, wherein the compound is administered four times a day.

93. The method according to any one of claims 85-92, wherein the compound is administered for three consecutive days.

94. The method according to any one of claims 85-92, wherein the compound is administered for four consecutive days.

95. The method according to any one of claims 85-92, wherein the compound is administered for five consecutive days.

96. The method according to any one of claims 85-92, wherein the compound is administered for six consecutive days.

97. The method according to any one of claims 85-92, wherein the compound is administered for seven consecutive days.

98. The method according to any one of claims 85-92, wherein the compound is administered for eight consecutive days.

99. The method according to any one of claims 85-92, wherein the compound is administered for nine consecutive days.

100. The method according to any one of claims 85-92, wherein the compound is administered for ten consecutive days.

101. The method according to any one of claims 85-100, wherein the human is suffering from dengue virus serotype 1.

102. The method according to any one of claims 85-100, wherein the human has dengue virus serotype 3.

103. The method according to any one of claims 85-100, wherein the human has dengue virus serotype 4.

104. The method according to any one of claims 85-103, wherein the compound is administered in combination with another antiviral compound.

105. The method of claim 104, wherein the other antiviral compound is an NS2B-NS3 protease inhibitor.

106. The method according to claim 104, wherein the other antiviral compound is an NS3 inhibitor.

107. The method according to claim 104, wherein the other antiviral compound is an inhibitor of NS3-NS4B interaction.

108. The method according to claim 107, wherein the NS3B-NS4B interaction inhibitor is JNJ-1802 or JNJ-A07.

109. The method of claim 104, wherein the other antiviral compound is another NS5 inhibitor.

110. A compound of the formula: or a pharmaceutically acceptable salt thereof, optionally in a pharmaceutically acceptable carrier: It is useful for treating dengue virus serotype 1, 3 or 4 infection in a human host in need thereof, wherein about 700 mg to about 1,000 mg of the compound is administered.

111. The compound for use according to claim 110, wherein the compound is 112. The compound for use according to claim 110 or 111, wherein the dengue virus is serotype 1.

113. The compound for use according to claim 110 or 111, wherein the dengue virus is serotype 3.

114. The compound for use according to claim 110 or 111, wherein the dengue virus is serotype 4.

115. Use of a compound of the formula: or a pharmaceutically acceptable salt thereof, optionally in a pharmaceutically acceptable carrier, for treating dengue virus serotype 1, 3 or 4 infection in a human host in need thereof, comprising administering from about 700 mg to about 1,000 mg of the compound 116. The use according to claim 115, wherein the compound is 117. The compound for use according to claim 115 or 116, wherein the dengue virus is serotype 1.

118. The compound for use according to claim 115 or 116, wherein the dengue virus is serotype 3.

119. The compound for use according to claim 115 or 116, wherein the dengue virus is serotype 4.

120. Use of a compound of the formula: or a pharmaceutically acceptable salt thereof, optionally in a pharmaceutically acceptable carrier, in the preparation of a medicament comprising from about 700 mg to about 1,000 mg of the compound for treating dengue virus 121. The use according to claim 120, wherein the compound is 122. Use according to claim 120 or 121, wherein the dengue virus is serotype 1.

123. Use according to claim 120 or 121, wherein the dengue virus is serotype 2.

124. Use according to claim 120 or 121, wherein the dengue virus is serotype 3.

125. Use according to claim 120 or 121, wherein the dengue virus is serotype 4.

126. A process according to claims 70-74, wherein the salt of the compound of formula Intermediate A is a dihydroquinine salt.

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