Dosage regimen for the treatment of dengue infection

Abstract

The present invention provides advantageous high-dose therapeutic compositions and combinations, as well as manufacturing processes, for treating or preventing dengue virus infections that can be serotype 1, 2, 3 or 4.

Description

Technical Field

[0001] [Cross - Reference to Related Applications] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 346,274, filed May 26, 2022; U.S. Provisional Patent Application No. 63 / 414,812, filed Oct. 10, 2022; and U.S. Provisional Patent Application No. 63 / 427,359, filed Nov. 22, 2022. These applications are hereby incorporated by reference in their entirety for all purposes.

[0002] The present invention provides advantageous therapeutic compositions, combinations, and uses thereof for the treatment or prevention of dengue virus infection.

Background Art

[0003] Dengue fever is caused by four related viruses known as dengue virus serotypes 1 - 4. Dengue virus (DENV) is a member of the Flavivirus genus in the Flaviviridae family. They are enveloped viruses with an 11 kb positive - strand RNA genome encoding ten proteins. These proteins are the membrane (M) protein, envelope (E) protein, capsid (C) protein, and non - structural (NS) proteins. The NS proteins are NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5A, and NS5. These proteins are translated as a single polyprotein, which is cleaved into individual proteins by proteases. The NS5 protein is the largest among the non - structural proteins and functions as both a viral RNA - dependent RNA polymerase and an RNA 2’ - O - methyltransferase. The RNA - dependent RNA polymerase (RdRp) activity is responsible for viral genome replication.

[0004] Dengue is an endemic disease in tropical regions including the Eastern Mediterranean, the American continent, Southeast Asia, the Western Pacific, and Africa. As the habitat of Aedes aegypti, which transmits dengue, expands, new cases have been confirmed in areas where dengue has not been seen before. The US Centers for Disease Control and Prevention has reported recent outbreaks of dengue infections in the United States in Hawaii (2015), Florida (2013, 2020), and Texas (2013) (https: / / www.cdc.gov / Dengue / areaswithrisk / in-the-us.html). Mosquitoes that transmit dengue are now commonly found in most of the southeastern United States (Non-Patent Document 1).

[0005] Previously, the distribution of individual dengue serotypes was geographically limited, but as the disease spreads worldwide, the distribution of serotypes has spread more widely (Non-Patent Document 2). This is of particular concern because if a person is infected with one serotype and then later infected with a second serotype, the risk of severe dengue increases. Severe dengue (or dengue hemorrhagic fever) is accompanied by severe plasma leakage, severe bleeding, shock, and organ impairment. Without treatment, the mortality rate of severe dengue is 13% (US Centers for Disease Control and Prevention, Clinical Findings for Healthcare Workers on Dengue, updated April 13, 2023).

[0006] In addition to the geographical spread of dengue outbreaks, recent outbreaks have become more severe. Since 2000, the incidence rate has increased eightfold worldwide. Not only has the frequency of outbreaks increased, but the scale has also grown. The World Health Organization estimates that half of the world's population is at risk of infection, and hundreds of millions of infections are recorded each year (https: / / www.who.int / news-room / fact-sheets / detail / Dengue-and-severe-Dengue). Nearly half a million cases of severe dengue have also been reported. Most people at risk of dengue infection are children (Non-Patent Documents 3 and 4).

[0007] Vaccines against dengue have been developed, but they have several drawbacks. The most serious one is the phenomenon known as antibody-dependent enhancement (ADE) of infection. The only approved vaccine, Dengvaxia, provides incomplete (https: / / www.fda.gov / news-events / press-announcements / first-fda-approved-vaccine-prevention-dengue-disease-endemic-regions) and heterogeneous protection against different serotypes, with a protection rate of only 50% against DENV1 and a lower rate against DENV2 (Non-Patent Document 5; Non-Patent Document 6). Therefore, vaccination may promote ADE and, as a result, severe infection in dengue-naive individuals (Non-Patent Document 7). Thus, vaccination is limited to individuals aged 9 to 16 years who have a record of at least one previous infection with the dengue virus. Despite the increasing importance of the dengue virus as a global health threat, the development of a safe and effective pan-dengue vaccine has not yet been achieved.

[0008] Therefore, a potential strategy for combating dengue worldwide is pharmaceuticals, such as direct-acting antivirals (DAAs). Unfortunately, there are no DAAs approved for dengue fever. In DENV, the viral polymerase is the non-structural protein NS5 (Non-Patent Document 8). The DENV RdRp has been the target of many investigational DAAs, including many nucleoside / nucleotide analogs (Non-Patent Document 9), but to date, the only nucleoside that has been clinically tested is balapiravir, a cytosine analog (Non-Patent Document 8; Non-Patent Document 10). Unfortunately, no difference was observed between compound treatment and placebo treatment with respect to the antiviral response, cytokine profile, and time to fever resolution (Non-Patent Document 10).

[0009] 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 (Patent Document 1). In 2020, the Manfroni laboratory described pyridobenzothiazolones that inhibit NS5 (Non-Patent Document 11). Other publications describing dengue inhibitors include, but are not limited to, Non-Patent Document 12; Non-Patent Document 13; Non-Patent Document 14; Non-Patent Document 15; Non-Patent Document 16; Non-Patent Document 17; Non-Patent Document 18; and Non-Patent Document 19.

[0010] Specific antiviral compounds for treating certain RNA viruses include Compound 1 and Compound 2. Compound 1, which is a free base, is disclosed, for example, in Patent Document 2.

Chemical formula

[0011] Compound 2 disclosed in Patent Document 3 is the hemisulfate salt of isopropyl ((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-alaninate, and is specifically disclosed for the treatment of viral infections such as hepatitis C virus. Compound 2 has been reported to be effective against SARS-CoV2, the causative agent of COVID-19 (see, for example, Patent Document 4). The S-phosphorus diastereomer of Compound 2 is currently in human clinical trials for the treatment of HCV and COVID-19.

Chemical formula

[0012] In Patent Document 5, Atea Pharmaceuticals, Inc. discloses the general use of Compound 1 or Compound 2 for the treatment of flaviviruses, along with activity data against yellow fever, dengue (serotype 2), West Nile virus, and Zika virus.

[0013] Despite these disclosures, due to the above-mentioned difficult problems, it has been found that it is difficult to effectively treat dengue. There remains a strong medical need to determine an appropriate protocol for the treatment of dengue that is safe, effective, and highly tolerable.

[0014] Accordingly, an object of the present invention is to provide an advantageous regimen for treating dengue infection. A further object of the present invention is to provide a novel method for producing Compounds 1 and 2, which are phosphorus R-diastereomers.

Prior Art Documents

Patent Documents

[0015]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Non-Patent Documents

[0016]

Non-Patent Document 1

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 18

Non-Patent Document 19

Summary of the Invention

[0017] When a high dose (i.e., at least about 700 mg or 750 mg per single dose, more typically 700 mg to 1000 mg (which can be delivered in one, two, or three separate dosage forms for convenience)) of Compound 1 or Compound 2 (where Compound 1 can be its pharmaceutically acceptable salt) is administered to a host such as a human in need of treatment for dengue virus, it has unexpectedly been found to be advantageous for the treatment of dengue virus. In a typical embodiment, Compound 1 or Compound 2 is provided in one or more pharmaceutical compositions so as to reach this effective amount.

[0018] Furthermore, it has been discovered that Compound 1 or Compound 2 is also active against dengue serotypes 1, 3, and 4. This new discovery confirms that the high-dose regimens of Compound 1 and Compound 2 described herein are effective against all serotypes of dengue virus (dengue virus serotypes 1, 2, 3, and 4). [Chemical formula]

[0019] As used herein, the term "Compound 1" can refer to either the free base or a pharmaceutically acceptable salt thereof. Compound 1 is isopropyl ((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-alaninate.

[0020] It has also been discovered that Compound 1 (and its hemisulfate Compound 2) has an advantageous dual mechanism of action. The active metabolite is an inhibitor of the RNA-dependent RNA polymerase (RdRp) domain of the viral protein NS5, and at the same time, it is also an inhibitor of the 2'-O-methyltransferase (MTase) domain of NS5. Both functions of the NS5 protein are required for replication. Inhibition of the two major protein functions involved in RNA replication reduces the likelihood of the formation of resistant mutants.

[0021] As described in Example 2 and shown in Table 1 and Figure 2, Compound 1 (AT-281) has an EC 50 = 0.57 μM against serotype 1, an EC 50 < 7 μM against serotype 2 (multiple strains), an EC 50 < 1.5 μM against serotype 3 (multiple strains), and an EC 50It has been found to exhibit in vitro efficacy against all four serotypes of dengue containing <1.2 μM). Pan-serotype activity is advantageous as it minimizes the risk of severe dengue and reduces or eliminates the need for serotype testing prior to administration of the compound.

[0022] Alternatively, Compound 1 can be used in the high-dose regimens described herein in the form of the free base or as any desired pharmaceutically acceptable salt. Compound 2, which is the hemisulfate salt of Compound 1, 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 virus strain) as described in Example 18. For example, the use of Compound 2 provides a significant difference in viremia observed on days 6 and 8 post-infection (pi) in the blood shown in FIG. 8A and the spleen shown in FIG. 8B. Furthermore, from day 3 to day 8 post-infection, a significant effect on the prevention of weight loss shown in FIG. 9A and the worsening of the health score shown in FIG. 9B was seen compared to animals treated with the vehicle control. Importantly, as shown in FIG. 10, there was a very significant extension of the survival period, which demonstrates the effectiveness of Compound 2 against dengue virus or alternative Compound 1 as the free base or its pharmaceutically acceptable salt.

[0023] In certain embodiments, Compound 2 or Compound 1 (as the free base or its pharmaceutically acceptable salt) is typically administered to a patient in the high-dose regimens described herein, where the patient is suffering from multiple flaviviruses, one of which is dengue, e.g., a patient suffering from dengue serotype 1, 2, 3, or 4 and another flavivirus. Compound 1 (AT-281) showed a potent effect in reducing viral titers against 11 different strains of 7 flaviviruses tested in vitro in 3 different cell types, achieving 50% inhibition of the virus-induced cytopathic effect (CPE) in the concentration range of 0.21 μM to 1.41 μM (Table 1 and FIG. 2).

[0024] As described in Example 10 and as shown in FIG. 1, the active metabolite of Compound 2 or Compound 1 (Compound 5) acts as a GTP analog, is incorporated into RNA by NS5 RNA-dependent RNA polymerase (RdRp), and synthesis is immediately terminated.

Chemical formula

[0025] The chain termination efficiency of Compound 5 was measured in a competitive nucleotide incorporation assay using all four DENV serotypes. Compound 5 is incorporated into RNA at 1 / 9 to 1 / 12 that of the natural substrate guanosine triphosphate (i.e., for every approximately 7 to 8 GTP incorporated, 1 AT-9010 is incorporated). In comparison, sofosbuvir triphosphate is incorporated into RNA less than once per 100 UTP (natural uridine triphosphate) incorporations. Discrimination assay data are provided below.

[0026] TIFF2025517992000006.tif20170

[0027] Compound 5 (and thus 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 alleviates the host immune response against the viral RNA. Inhibition of the MTase domain is the second mechanism of action of Compound 5.

[0028] The binding of Compound 5 to the MTase domain was measured by a thermal shift assay. Compound 5 stabilized the MTase domains of DENV1, DENV2, DENV3, and DENV4 nearly 2-fold more than the previously reported MTase inhibitor sinefungin (3.8 ± 0.1 °C for Compound 5 compared to 1.9 ± 0.1 °C for sinefungin).

[0029] Furthermore, Compound 5 stabilizes the RdRp domain more effectively than the natural substrate guanosine triphosphate (5.6 ± 0.1 °C for Compound 5 compared to 4.5 ± 0.2 °C for GTP), while sinefungin hardly stabilizes the RdRp domain (0.1 °C ± 0.1 °C).

[0030] To confirm the activity of Compound 5 against the MTase domain, an IC of 29.6 + 2 μM was measured in a filter binding assay. 50 Since it has been previously shown that Compound 5 reaches micromolar concentrations intracellularly (Good, S. et al. Preclinical evaluation of AT-527, a novel guanosine nucleotide prodrug with potent, pan-genotypic activity against hepatitis C virus, PLoS ONE 15(1): e0227104), an IC 50 of 29.6 μM indicates that Compound 5 is an effective inhibitor of the MTase domain.

[0031] Therefore, Compound 2 and Compound 5, the active metabolite of Compound 1, have multiple mechanisms of action against dengue virus. The multiple mechanisms of action can, inter alia, prevent the emergence of resistance against the highly conserved MTase domain.

[0032] In certain embodiments of the invention, a compound of formula I, optionally in a pharmaceutically acceptable carrier:

Chemical formula

[0033] In certain embodiments of the invention, a compound of formula II, optionally in a pharmaceutically acceptable carrier:

Chemical formula

[0034] All R groups are intended to be interpreted as non-redundant, i.e., as is known in the art, alkyl is not substituted by alkyl, but for example, alkoxy substituted by alkoxy is not redundant. Aryl substituted by aryl is included in the definition of aryl to a limited extent. R groups are not optionally substituted unless specifically indicated in the context.

[0035] C 1 ~C 6 Non-limiting examples of alkyl include methyl, ethyl, propyl, isopropyl, butyl, t-butyl, sec-butyl, isobutyl, -CH 2 C(CH 3 ) 3 , -CH(CH 2 CH 3 ) 2 , and -CH 2 CH(CH 2 CH 3 ) 2 . C 3 ~C 6 Non-limiting examples of cycloalkyl include cyclopropyl, CH 2 -cyclopropyl, cyclobutyl, and CH 2 -cyclobutyl.

[0036] Aryl(C 1 ~C 4 alkyl)- A non-limiting example is benzyl. Non-limiting examples of aryl are phenyl and naphthyl.

[0037] In certain embodiments, the compound is a compound of formula IIA:

Chemical formula

[0038] In certain embodiments, the compound is a compound of Formula IIB:

Chemical formula

[0039] In certain embodiments, the selected compounds described herein are used to treat dengue virus infections that can be serotype 1, 3, and / or 4. In some embodiments, the selected compounds described herein are administered to patients in need of treatment for dengue virus at a dosage of about 700 mg to 1000 mg per single dose, such as 700 mg to 850 mg, two or three times a day or more (and in some embodiments, 650 milligrams, 675 milligrams, 700 milligrams, 725 milligrams, 750 milligrams, 775 milligrams, 800 milligrams, 825 milligrams, 850 milligrams, 875 milligrams, 900 milligrams, 925 milligrams, 950 milligrams, 975 milligrams, or 1000 milligrams two or three times a day) to treat dengue virus infections that can be serotype 1, 2, 3, and / or 4. In certain embodiments, Compound 1 or Compound 2 is administered at a dosage of about 700 mg to 1000 mg per single dose, such as at least about 700 mg, 750 mg, or 800 mg (which can be delivered in multiple solid dosage forms to reach the total dose).

[0040] In a specific embodiment, Compound 2 is delivered at a dosage of three solid dosage forms of 250 mg of the free base weight to reach a total dosage of 750 mg per day, twice a day. In other embodiments, Compound 2 is delivered in two solid dosage forms of 375 mg to reach a total dosage of 750 mg per day, twice a day.

[0041] In certain embodiments, Compound 1 or Compound 2 is administered 1, 2, 3, or 4 times a day for 4, 5, 6, 7, 8, 9, or 10 days. In certain embodiments, this treatment regimen can treat dengue virus serotype 1, 3, and / or 4, or even 1, 2, 3, and / or 4. For clarity, unless otherwise specified, the milligram dosage of the active compound administered is based on the weight of the salt of the nucleotide phosphoramidate when a pharmaceutically acceptable salt is used. For conversion, a dosage of about 750 mg of Compound 2 corresponds to a dosage of about 692 mg of Compound 1 (which is within the range of "about 700 mg") in the form of the free base.

[0042] The present invention includes both treatment and prophylactic or preventive therapies. In some embodiments, an active NS5 RdRp inhibitory compound as described herein is administered as a prophylactic to a host such as a human who has been exposed to and is thus at risk of infection with a dengue virus such as dengue virus type 1, dengue virus type 2, dengue virus type 3, and / or dengue virus type 4 according to the high-dose methods provided herein. In another alternative embodiment, a sufficient period prior to exposure to a population at risk of infection, including during travel or during public events or meetings, for example, up to 3, 5, 7, 10, 12, 14 days or more prior to the infectious situation, is provided, which includes administering to a human a high-dose effective amount of one of the compounds as described herein.

[0043] The present invention also relates to R P A stereoselective process for the scalable production of Compound 1 is provided in which the diastereomers are produced in a substantially pure form. By substantially pure form of the diastereomers is typically meant at least about 90% or more of the R P with respect to the diastereomers PRefers to diastereomers. The reaction of the compounds of the formulas of Intermediate A and Intermediate B with a uronium-based activator, for example, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), is S P Preferentially forms phosphoramidate Intermediate C. A similar process is described in International Publication No. WO 2022 / 040473 assigned to Atea Pharmaceuticals, where the desired product was S P A phosphoramidate. [Chemical formula]

[0044] The aryloxy group on the phosphoramidate of Intermediate C is substituted with one, two, or three R 6 Groups that activate it for replacement, for example, by an electron-withdrawing function. In the second step, the activated phenoxy leaving group is replaced by phenol, and the stereochemistry of phosphorus is inverted to form Compound 1.

[0045] In certain embodiments, the substantially pure R-phosphorus diastereomer is at least about 93% pure, at least about 95% pure, at least about 98% pure, or even at least 99% pure.

[0046] In certain embodiments, a dose of 700 mg to 1000 mg is administered to a patient in need of dengue treatment for the treatment of dengue in three solid dosage forms, where a total of about 700 mg to about 1000 mg of the solid dosage form of Compound 1 or Compound 2 is administered twice a day (6 tablets in total per day). In certain embodiments, a total of about 700 mg to about 1000 mg of the three solid dosage forms of Compound 1 or Compound 2 is administered continuously for 4 days, 5 days, 6 days, or 7 days. In certain embodiments, a total of about 700 mg to about 1000 mg of the three solid dosage forms of Compound 1 or Compound 2 is administered continuously for 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.

[0047] In certain embodiments, Composition B is administered for 4, 5, 6, or 7 consecutive days. In certain embodiments, Composition A is administered for 8, 9, 10, 11, 12, 13, or 14 consecutive days.

[0048] In some embodiments, Compound 1 or a pharmaceutically acceptable salt thereof is used in Composition A or Composition B.

[0049] In certain embodiments, Compound 1 or Compound 2 is administered twice a day at a dose of at least about 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, 825 mg, 850 mg, 875 mg, 900 mg, 925 mg, 950 mg, 975 mg, or 1000 mg, where the compound is administered, by way of illustrative example, every 8 ± 2 hours.

[0050] In certain aspects, the dosing regimens described herein are administered within 4 days, 3 days, 2 days, or 1 day of symptom onset. In certain embodiments, the dosing regimens described herein are administered within 48 hours of symptom onset.

[0051] Accordingly, the present invention includes the following features: (a) A dosing regimen of Compound 1 or Compound 2 comprising administering to a patient with dengue serotype 1, 2, 3, and / or 4, twice or three times a day, at least about 675 mg to 1000 mg or 700 mg to 1000 mg per dose, for example 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, 825 mg, 850 mg, 875 mg, 900 mg, 925 mg, 950 mg, 975 mg, or 1000 mg. (b) Treatment of dengue serotype 1, 3, or 4, as further described herein. (c) A dosing regimen of Compound 1 or Compound 2, which comprises administering to a patient with dengue fever serotype 1, 3, and / or 4 at least about 675 mg to 1000 mg, for example, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, 825 mg, 850 mg, 875 mg, 900 mg, 925 mg, 950 mg, 975 mg, or 1000 mg of Compound 1 or Compound 2 per dose, two or three times a day. (d) A dosing regimen of (a), (b), or (c), wherein Compound 1 or Compound 2 is administered continuously for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days. (e) A dosing regimen of (a), (b), or (c), wherein Compound 1 or Compound 2 is administered three times a day for 14 days at a dose of about 750 mg. (f) A dosing regimen of (a), (b), or (c), wherein Compound 1 or Compound 2 is administered three times a day for 5 days at a dose of about 750 mg based on the weight of the free base. (g) A dosing regimen of (a) or (b), wherein Compound 1 or Compound 2 is administered twice a day for 5, 6, or 7 days at a dose of about 1000 mg based on the weight of the free base. (h) A dosing regimen of any one of (a)-(g), wherein Compound 2 is administered to a patient. (i) A dosing regimen of (h), wherein a dose of about 675 mg to 1000 mg is administered in three solid dosage forms. (j) A dosing regimen of any one of (a)-(g), wherein Compound 1 is administered to a patient. (k) A method for treating dengue in a patient in need of treatment for dengue, which comprises administering Compound 1 or Compound 2 according to any one of the dosing regimens of (a)-(j). (l) The method of (k), wherein the dosing regimen comprises administering a pharmaceutical composition comprising Compound 1 or Compound 2. (m) Use of any one of the dosing regimens of (a)-(k) in the manufacture of a medicament for the treatment of dengue fever. (n) The use of (l) or (m), wherein the dosing regimen comprises administering a pharmaceutical composition comprising Compound 1 or Compound 2. (o) A method of treating dengue in a patient in need of treatment for dengue, comprising administering a compound of formula I according to the high-dose protocol described herein. (p) A method of treating dengue in a patient in need of treatment for dengue, comprising administering a compound of formula II, formula IIA or formula IIB according to the high-dose protocol described herein. (q) Reacting a compound of the formula of Intermediate A, Intermediate B with a uronium-based activator, such as 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), as described above in S P Reacting to preferentially form phosphoramidate intermediate C, and then, in a second step, replacing the activated phenoxy leaving group with phenol and inverting the stereochemistry of the phosphorus to form compound 1 in the R-phosphorus diastereomeric configuration, a process for the manufacture of compound 1 or compound 2. (r) The process of (q), wherein the substantially pure R-phosphorus diastereomer is at a purity of about 93% or greater, about 95% or greater, about 98% or greater or even 99% or greater.

Brief Description of the Drawings

[0052]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6A

Figure 6B

Figure 6C

Figure 7

Figure 8A

Figure 8B

Figure 9A

Figure 9B

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19A

Figure 19B

Figure 20A

Figure 20B

Figure 21A

Figure 21B

Figure 21C

Figure 21D

Figure 22

Figure 23

Figure 24

Mode for Carrying Out the Invention

[0053] When a high dose (i.e., at least 700 mg to 1000 mg per single dose, two, three or more times a day) of Compound 1 or Compound 2 (wherein Compound 1 may be its pharmaceutically acceptable salt) is administered to a host such as a human in need of treatment for dengue virus, it has unexpectedly been found to be advantageous for the treatment of dengue virus. In certain embodiments, Compound 1 or Compound 2 may be provided in a pharmaceutical composition.

[0054] Furthermore, it has been found that Compound 1 or Compound 2 is also active against dengue serotypes 1, 3 and 4. This new finding confirms that the high-dose regimens of Compound 1 and Compound 2 described herein are effective against all serotypes of dengue virus (dengue virus serotypes 1, 2, 3, and 4).

Chemical formula

[0055] As used herein, the term "Compound 1" can refer to either the free base or its pharmaceutically acceptable salt. Compound 1 is isopropyl ((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-alaninate.

[0056] Compound 1 (and its hemisulfate compound 2) was also found to have an advantageous dual mechanism of action. The active metabolite is an inhibitor of the RNA-dependent RNA polymerase (RdRp) domain of the viral protein NS5, and at the same time, it is also an inhibitor of the 2'-O-methyltransferase (MTase) domain of NS5. Both functions of the NS5 protein are required for replication. Inhibition of the two major protein functions involved in RNA replication reduces the likelihood of the formation of resistant mutants.

[0057] Compound 1 and Compound 2 have an advantageous dual mechanism of action. It is an inhibitor of the RNA-dependent RNA polymerase (RdRp) domain of the viral protein NS5, and at the same time, it is also an inhibitor of the 2'-O-methyltransferase (MTase) domain of NS5. Both functions of the NS5 protein are required for replication. Inhibition of the two major protein functions involved in RNA replication reduces the likelihood of the formation of resistant mutants.

[0058] Compound 2 (AT-752) is the hemisulfate salt of Compound 1 (AT-281), i.e., a phosphoramidate prodrug that forms the L-alanyl metabolite Compound 3 (AT-551) as an intermediate prodrug before being 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 here it has been shown to inhibit dengue serotypes 1, 2, 3, and 4. One mechanism is the inhibition of NS5, which is the RdRp of DENV-2.

Chemical formula

[0059] In certain embodiments, dosing regimens, methods, compositions, and manufacturing processes for treating a host infected with dengue virus are provided. For example, Compound 1 or Compound 2 or Formula I or Formula II can be administered at a dose of about 700 mg to 1000 mg, such as a dose of 750 mg, twice or three times a day, either alone or in combination with another anti-RNA virus agent, to treat an infected host in need of treatment for dengue virus. In certain embodiments, it is useful to administer in combination agents that modulate the same or different pathways or inhibit different targets within the virus. Since Compounds 1 / 2 are polymerase inhibitors, it may be advantageous to administer Compounds 1 / 2 to a host in combination with a protease inhibitor or an NS3-NS4B interaction inhibitor. Alternatively, Compounds 1 / 2 can also be administered in combination with structurally different polymerase inhibitors.

[0060] Compounds 1 / 2 can be administered orally, for example, in the form of pills or tablets, or via another route that the attending physician deems appropriate, including those via intravenous, transdermal, subcutaneous, topical, parenteral, or other suitable routes.

[0061] I. Definitions 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 this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice and testing of the present invention, the preferred methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0062] Unless the context specifically 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 specifically stated otherwise. Further, the use of the term "including" is not limiting, as are other forms such as "includes" and "included".

[0063] "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), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, birds, etc.

[0064] The terms "prophylactic" or "prevention", when used, refer to administering an active NS5 RNA-dependent RNA polymerase (RdRp) inhibitory compound to prevent the occurrence or recurrence of dengue virus infection, such as dengue type 2 virus or dengue type 3 virus, reduce the likelihood of its occurrence or recurrence, or minimize new infections or infections to others compared to infections that would occur without such treatment. The present invention includes both treatment and prophylactic or preventive therapies. In some embodiments, the active NS5 RdRp inhibitory compound is administered to a host that has been exposed to and is thus at risk of infection with a dengue virus, such as dengue type 1 virus, dengue type 2 virus, dengue type 3 virus, or dengue type 4 virus. In another alternative embodiment, a method of preventing transmission is provided that includes administering to a human an effective amount of one of the compounds described herein for a sufficient period prior to exposure to a population at risk of infection, including, for example, during travel or during a public event or meeting, including up to 3, 5, 7, 10, 12, 14 days or more prior to the infectious situation.

[0065] The terms "co-administer", "co-administration" or "combination" are used to describe administering an NS5 RdRp interfering compound in combination with at least one other antiviral active agent. The timing of co-administration is preferably determined by the healthcare professional treating the patient. In some cases, it may be desirable to administer the agents simultaneously. Alternatively, the drugs selected for combination therapy are administered to the patient at different times. Of course, if two or more viral infections or other symptoms are present, the compound can be combined with other agents as needed to treat the other infections or symptoms.

[0066] Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic acid salts of acidic residues such as carboxylic acids. Pharmaceutically acceptable salts include, for example, salts of parent compounds formed from inorganic or organic acids that are not overly toxic and quaternary ammonium salts. For example, acid salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, and nitric acid; and acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, mesylic acid, esylic acid, besylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, HOOC-(CH 2 ) n -COOH (wherein n is from 0 to 4) and salts prepared from organic acids such as these, or salts prepared using different acids that produce the same counterion. A list of additional suitable salts can be found, for example, in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., p. 1418 (1985).

[0067] The compound can be delivered in any molar ratio of salt that provides the desired result. For example, the compound can be provided with less than a molar equivalent of counterion, such as in the form of a hemisulfate. Alternatively, the compound can be provided with more than a molar equivalent of counterion, such as in the form of a disulfate. Non-limiting examples of the molar ratio of the compound to the counterion include 1:0.25, 1:0.5, 1:1, and 1:2.

[0068] In certain embodiments, the term "about" means plus or minus 5% of the recited value. In certain embodiments, the term "about" means plus or minus 10% of the recited value.

[0069] I. Advantageous Dosage Forms of Compound 2 for the Treatment of Dengue Virus Compositions, methods, and dosage forms for treating a host infected with dengue through administration of an effective amount of Compound 1 or Compound 2 are provided.

[0070] In one aspect of the invention, a pharmaceutical composition according to the invention comprises a high anti-dengue virus dose of Compound 1 or Compound 2 as described herein, optionally in combination with a pharmaceutically acceptable carrier, additive, or excipient, and further optionally in combination with or alternatively at least one other active compound. In one embodiment, the invention comprises a solid dosage form of Compound 2 in a pharmaceutically acceptable carrier.

[0071] In certain embodiments, Compound 1 or Compound 2 is administered to a patient in need of treatment for dengue virus in a pharmaceutical composition, such as Composition A or Composition B.

[0072] TIFF2025517992000014.tif34170

[0073] TIFF2025517992000015.tif68170

[0074] In one aspect of the invention, a pharmaceutical composition according to the invention comprises a high anti-dengue effective amount of Compound 1 or Compound 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, such as another anti-dengue agent.

[0075] The invention includes pharmaceutical compositions comprising an amount of Compound 1 or Compound 2 or prodrug of the invention effective to treat dengue virus infection in a pharmaceutically acceptable carrier or excipient. In alternative embodiments, the invention includes pharmaceutical compositions comprising an amount of Compound 1 or Compound 2 or prodrug of the invention effective to prevent dengue virus infection in a pharmaceutically acceptable carrier or excipient.

[0076] One of ordinary skill in the art will recognize that the therapeutically effective high dose amount will vary depending on the infection or condition being treated, its severity, the treatment regimen employed, the pharmacokinetics of the agent being used, and the patient or subject (animal or human) being treated, and that such a therapeutic amount can be determined by a treating physician or specialist.

[0077] Compound 1 or Compound 2 according to the present invention can be formulated in a mixture with a pharmaceutically acceptable carrier. Generally, it is typical to administer the pharmaceutical composition in an orally administrable form, particularly in a solid dosage form such as pills or tablets. Certain formulations can be administered via other routes including parenteral, intravenous, intramuscular, topical, transdermal, buccal, subcutaneous, suppository, or intranasal spray. Intravenous and intramuscular formulations are often administered in sterile saline. One of ordinary skill in the art can modify the formulation to enhance solubility in water or another vehicle, for example, this can be readily achieved by minor modifications (salt formulation, esterification, etc.) well within the skill level of one of ordinary skill in the art. Also, as described in more detail herein, it is well within the skill of one of ordinary skill in the art to change the route of administration and dosing regimen of Compound 2 to manage the pharmacokinetics of the present compound for maximum beneficial effect in the patient.

[0078] Compound 1 or Compound 2 can be provided, for example, for oral or parenteral delivery. In certain embodiments, Compound 1 or Compound 2 is provided in a solid, gel, or liquid dosage form. In certain embodiments, Compound 1 or Compound 2 is provided in a liquid dosage form for parenteral administration. In certain embodiments, Compound 1 or Compound 2 is provided in a liquid dosage form for intravenous administration. In certain embodiments, Compound 1 or Compound 2 is provided in a solid dosage form for oral administration. In certain embodiments, Compound 1 or Compound 2 can be provided as a soft shell capsule or tablet for oral administration.

[0079] The high dose amounts of Compound 1 or Compound 2 contained within a therapeutically active formulation according to the present invention are described in detail herein and are, for example, an amount effective to achieve the desired results according to the present invention for the treatment of DENV infection, reduction of the likelihood of DENV infection, or inhibition, reduction, and / or abrogation of DENV or its secondary effects (including disease states, symptoms, and / or complications subsequent to DENV).

[0080] The dosages described herein refer to the amount of the active pharmaceutical ingredient in the dosage with added weight by a pharmaceutically acceptable salt, unless otherwise indicated. For example, a dosage of 750 mg of Compound 2 contains approximately 692 mg of Compound 1 (which is within the range of "about 700 mg") when accounting for the molecular weight of the hemisulfate salt for measurement.

[0081] Generally, a therapeutically effective amount of Compound 2 in a pharmaceutical formulation can range from about 600 mg to about 1000 mg, from about 700 mg to about 1000 mg, or from about 700 mg to about 800 mg or 850 mg, when measured as the free base. In certain embodiments, Compound 1 is administered in the same range as the free base.

[0082] In certain embodiments, Compound 2 is administered two or three times a day in an amount of at least about 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, 1150 mg, 1200 mg, 1250 mg, 1300 mg, 1350 mg, 1400 mg, 1450 mg, or 1500 mg, when measured as the free base. In some embodiments, the dosage is provided in several dosage forms to reduce the size of solid dosage forms such as pills, tablets, or capsules.

[0083] In certain embodiments, about 700 mg to about 800 mg or 850 mg of Compound 1 or Compound 2 is administered once, twice, or three times a day (QD). In certain embodiments, about 700 mg to about 800 mg of Compound 1 or Compound 2 is administered once, twice, or three times a day (QD). In certain embodiments, about 800 mg to about 900 mg of Compound 2 is administered once a day (QD). In certain embodiments, about 900 mg to about 1000 mg of Compound 2 is administered once a day (QD).

[0084] In certain embodiments, about 700 mg to about 800 mg of Compound 2 is administered twice a day (BID). In certain embodiments, about 700 mg to about 900 mg of Compound 2 is administered twice a day (BID). In certain embodiments, about 800 mg to about 900 mg of Compound 2 is administered twice a day (BID). In certain embodiments, about 900 mg to about 1000 mg of Compound 2 is administered twice a day (BID).

[0085] In certain embodiments, about 600 mg to about 700 mg of Compound 2 is administered three times a day (TID). In certain embodiments, about 700 mg to about 800 mg of Compound 2 is administered three times a day (TID). In certain embodiments, about 800 mg to about 900 mg of Compound 2 is administered three times a day (TID). In certain embodiments, about 900 mg to about 1000 mg of Compound 2 is administered three times a day (TID).

[0086] 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 mg 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 1000 mg of Compound 2 is administered four times a day (QID).

[0087] For the purposes of the present invention, a prophylactic or prophylactically effective amount of the composition according to the present invention is included within the same high-dose concentration range as defined above for a therapeutically effective amount and is usually the same as the therapeutically effective amount.

[0088] 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 Compound 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 Compound 2 is administered once daily (QD).

[0089] In certain embodiments, the dengue virus is serotype 1, 3, or 4 and about 500 mg to about 800 mg of Compound 1 or Compound 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 Compound 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 Compound 2 is administered twice daily (BID).

[0090] In certain embodiments, the dengue virus is serotype 1, 3, or 4 and about 500 mg to about 800 mg of Compound 1 or Compound 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 Compound 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 Compound 2 is administered three times daily (TID).

[0091] The administration of Compound 1 or Compound 2 can range from continuous administration (intravenous drip) to oral administration several times a day or intranasal administration (e.g., 2, 3, 4, or 5 times a day) or transdermal administration. Among other administration routes, it can include oral administration, topical administration, parenteral administration, intramuscular administration, intravenous administration, subcutaneous administration, transdermal administration (which may include a penetration enhancer), buccal administration, and suppository administration. In order to enhance the bioavailability of the compound via the oral administration route, enteric-coated oral tablets can also be used. The most effective dosage form depends on the bioavailability / pharmacokinetics of the specific agent selected and the severity of the patient's disease. Oral dosage forms are particularly preferred because administration is easy and good patient compliance can be expected.

[0092] To prepare the pharmaceutical composition according to the present invention, a therapeutically effective amount of Compound 1 or Compound 2 according to the present invention can be intimately mixed with a pharmaceutically acceptable carrier according to conventional pharmaceutical compounding techniques to provide a dosage. The carrier can take a wide variety of forms depending on the desired dosage form for administration, e.g., oral or parenteral. When preparing a pharmaceutical composition in an oral dosage form, any of the usual pharmaceutical excipients and ingredients can be used. Thus, for liquid oral dosage forms such as suspensions, elixirs, and solutions, appropriate carriers and additives including water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, etc. can be used. For solid oral formulations such as powders, tablets, capsules, etc., and solid formulations such as suppositories, appropriate carriers and additives including sugar carriers such as starch, dextrose, mannitol, lactose, etc., and related carriers, diluents, granulating agents, lubricants, binders, disintegrants, etc. can be used. If desired, tablets or capsules can be enteric-coated or sustained-release by standard techniques. By using these dosage forms, the bioavailability of the compound in the patient can be significantly increased.

[0093] In the case of a parenteral preparation, the carrier usually contains sterile water or an aqueous sodium chloride solution, but may also contain other components including components that assist dispersion. Of course, when using sterile water and maintaining a sterile state, the composition and the carrier must also be sterilized. It is also possible to prepare an injectable suspension, in which case an appropriate liquid carrier, suspending agent, etc. can be employed.

[0094] Liposome suspensions (including liposomes targeting viral antigens) can also be prepared by conventional methods to produce pharmaceutically acceptable carriers. This may be suitable for the delivery of the free nucleoside, acyl / alkyl nucleoside or phosphate ester prodrug form of the nucleoside compound according to the present invention.

[0095] In a typical embodiment according to the present invention, Compound 1 or Compound 2 and the described composition are used to treat, prevent or delay dengue infection or secondary disease states, symptoms or complications of dengue.

[0096] Isotope substitution The present invention includes the use of compounds and Compound 1 or Compound 2 involving substitution of the desired isotope of an atom with an amount of isotope above natural abundance, i.e., enriched. Isotopes are atoms that have the same atomic number but different mass numbers, i.e., the same number of protons but different numbers of neutrons. As a general example, but not limited to, for example, deuterium ( 2 H) and tritium ( 3 H), which are isotopes of hydrogen, can be used at any position of the described structure. Alternatively, or additionally, isotopes of carbon, such as 13 C and 14C may also be used. Preferred isotope substitutions are the replacement of hydrogen with deuterium at one or more positions on the molecule to improve the performance of the drug. Deuterium can be attached at the position of bond cleavage during metabolism (α-deuterium kinetic isotope effect), or adjacent to or near the bond cleavage site (β-deuterium kinetic isotope effect). Achillion Pharmaceuticals, Inc. (International Publication Nos. WO 2014 / 169278 and WO 2014 / 169280) describes the deuteration of nucleotides to improve their pharmacokinetics or pharmacodynamics, including deuteration at the 5-position of the molecule.

[0097] Substitution with isotopes such as deuterium can provide certain therapeutic advantages due to greater metabolic stability, such as an increase in the in vivo half-life or a decrease in the required dosage. Replacement of hydrogen with deuterium at the site of metabolic breakdown can reduce the rate of metabolism in that bond or eliminate metabolism in that bond. At any position in a compound where a hydrogen atom can be present, the hydrogen atom can be any isotope of hydrogen, including protium ( 1 H), deuterium ( 2 H), and tritium ( 3 H). Thus, unless the context clearly indicates otherwise, references to compounds herein encompass all possible isotopic forms.

[0098] The term "isotopically labeled" analog refers to an analog that is a "deuterated analog", a " 13 C-labeled analog", or a "deuterated / 13 C-labeled analog". The term "deuterated analog" means that an H-isotope, i.e., hydrogen / protium ( 1 H), has been replaced by an H-isotope, i.e., deuterium ( 2Means a compound described herein substituted by (H). Deuterium substitution may be partial or complete. Partial deuterium substitution means that at least one hydrogen is substituted by at least one deuterium. In certain embodiments, the isotope is enriched to 90%, 95% or more than 99% in the isotope at any desired position. In some embodiments, it is deuterium that is enriched to 90%, 95% or 99% at the desired position. In the absence of a contrary designation, deuteration is at least 80% at the selected position. Deuteration of the nucleoside may occur at any replaceable hydrogen that provides the desired result.

[0099] In certain aspects of the invention, a compound of formula I, optionally in a pharmaceutically acceptable carrier:

Chemical formula

[0100] All R groups are intended to be interpreted without redundancy, i.e., as known in the art, alkyl is not substituted by alkyl, but for example, alkoxy substituted by alkoxy is not redundant. Aryl substituted by aryl is included in the definition of aryl to a limited extent. R groups are not arbitrarily substituted unless specifically indicated in the context.

[0101] C 1 ~C 6 Non-limiting examples of alkyl include methyl, ethyl, propyl, isopropyl, butyl, t-butyl, sec-butyl, isobutyl, -CH 2 C(CH 3 ) 3 , -CH(CH 2 CH 3 ) 2 , and -CH 2 CH(CH 2 CH 3 ) 2 . C 3 ~C 6 Non-limiting examples of cycloalkyl include cyclopropyl, CH 2 -cyclopropyl, cyclobutyl, and CH 2 -cyclobutyl.

[0102] A non-limiting example of aryl(C 1 ~C 4 (alkyl)- is benzyl. Non-limiting examples of aryl are phenyl and naphthyl.

[0103] In certain embodiments, Formula I is

Chemical Formula

[0104] In certain embodiments of the present invention, a compound of formula II, optionally in a pharmaceutically acceptable carrier:

Chemical formula

[0105] In certain embodiments, the compound is a compound of formula IIA:

Chemical formula

[0106] In certain embodiments, the compound of formula IIA is

Chemical formula

[0107] In certain embodiments, the compound is a compound of formula IIB:

Chemical formula

[0108] In certain embodiments, the compound of formula IIB is

Chemical formula

[0109] In certain embodiments, the compounds of formula II, formula IIA, or formula IIB are used in the treatment of infections by DENV1, DENV2, DENV3, or DENV4.

[0110] In certain embodiments, R 4a is a side chain of a naturally occurring amino acid, e.g.,

Chem.

[0111] In certain alternative embodiments, the variable moiety R of Formula II, IIA, or IIB 4a is C 7 ~C 15 alkyl, the variable moiety R 4b is hydrogen, and all other variable moieties are as defined herein. In alternative embodiments, the compound is

Chem.

[0112] II. Methods of Treatment or Prevention Treatment, as used herein, refers to the administration of Compound 1 or Compound 2 to a host, e.g., a human infected or at risk of infection with dengue virus.

[0113] The term "prophylactic" or prophylaxis, when used, refers to the administration of Compound 1 or Compound 2 to prevent or reduce the likelihood of the occurrence of a viral disorder. The present invention includes both treatment and prophylactic or preventive therapies. In one embodiment, Compound 1 or Compound 2 is administered to a host that has been exposed to dengue virus and is thus at risk of infection.

[0114] The present invention is directed to methods for treating or preventing dengue virus, including drug-resistant and multi-drug resistant forms of DENV, and related disease states, symptoms, or complications. The method involves administering an effective amount of Compound 1 or Compound 2 as described herein to a host, typically a human, in need of treatment for dengue virus, optionally in combination with at least one additional bioactive agent, such as an additional anti-DENV agent, and further in combination with a pharmaceutically acceptable carrier additive and / or excipient.

[0115] In yet another aspect, the present invention is a method of preventing or precluding dengue infection, or a disease state of dengue infection or related or subsequent disease states, symptoms or complications.

[0116] (1) In certain embodiments, there is provided a dosing regimen for use in treating a human host in need of treatment for dengue virus or preventing dengue virus, comprising administering Compound 1 or Compound 2 in an amount of about 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, 825 mg, 850 mg, 875 mg, 900 mg, 925 mg, 950 mg, 975 mg, 1000 mg, 1025 mg, 1050 mg, 1075 mg, 1100 mg, 1125 mg, 1150 mg, 1175 mg, 1200 mg, 1225 mg, or 1250 mg, two, three, or four times a day.

[0117] (2) In certain embodiments, there is provided a dosing regimen for use in treating a human host in need of treatment for dengue virus or preventing dengue virus, comprising administering Compound 1 or Compound 2 in an amount of about 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, 825 mg, 850 mg, 875 mg, 900 mg, 925 mg, 950 mg, 975 mg, 1000 mg, 1025 mg, 1050 mg, 1075 mg, 1100 mg, 1125 mg, 1150 mg, 1175 mg, 1200 mg, 1225 mg, or 1250 mg, two, three, or four times a day.

[0118] (3) In certain embodiments, a dosing regimen for use in treating a human host in need of treatment of dengue virus or preventing dengue virus, which comprises administering Compound 1 or Compound 2 at about 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, 825 mg, 850 mg, 875 mg, 900 mg, 925 mg, 950 mg, 975 mg, 1000 mg, 1025 mg, 1050 mg, 1075 mg, 1100 mg, 1125 mg, 1150 mg, 1175 mg, 1200 mg, 1225 mg, or 1250 mg, twice, three times, or four times a day, is provided.

[0119] (4) Any one of embodiments (1)-(3), wherein Compound 1 or Compound 2 is administered twice a day.

[0120] (5) Any one of embodiments (1)-(3), wherein Compound 1 or Compound 2 is administered three times a day.

[0121] (6) Any one of embodiments (1)-(3), wherein Compound 1 or Compound 2 is administered four times a day.

[0122] (7) Any one of embodiments (1)-(6), wherein about 600 mg of Compound 1 or Compound 2 is administered to a patient in need of treatment of dengue virus at each dose.

[0123] (8) Any one of embodiments (1)-(6), wherein about 650 mg of Compound 1 or Compound 2 is administered to a patient in need of treatment of dengue virus at each dose.

[0124] (9) Any one of embodiments (1)-(6), wherein about 700 mg of Compound 1 or Compound 2 is administered to a patient in need of treatment of dengue virus at each dose.

[0125] (10) Any one of embodiments (1)-(6), wherein about 750 mg of Compound 1 or Compound 2 is administered to a patient in need of treatment of dengue virus at each dose.

[0126] (11) Any one of embodiments (1)-(6), wherein about 800 mg of Compound 1 or Compound 2 is administered at each dose to a patient in need of treatment for dengue virus.

[0127] (12) Any one of embodiments (1)-(6), wherein about 850 mg of Compound 1 or Compound 2 is administered at each dose to a patient in need of treatment for dengue virus.

[0128] (13) Any one of embodiments (1)-(6), wherein about 900 mg of Compound 1 or Compound 2 is administered at each dose to a patient in need of treatment for dengue virus.

[0129] (14) Any one of embodiments (1)-(6), wherein about 950 mg of Compound 1 or Compound 2 is administered at each dose to a patient in need of treatment for dengue virus.

[0130] (15) Any one of embodiments (1)-(6), wherein about 1000 mg of Compound 1 or Compound 2 is administered at each dose to a patient in need of treatment for dengue virus.

[0131] (16) Any one of embodiments (1)-(15), wherein Composition A is administered.

[0132] (17) Any one of embodiments (1)-(15), wherein Composition B is administered.

[0133] (18) Any one of embodiments (1)-(17), wherein Compound 1 or Compound 2 is administered continuously for 3 days.

[0134] (19) Any one of embodiments (1)-(17), wherein Compound 1 or Compound 2 is administered continuously for 4 days.

[0135] (20) Any one of embodiments (1)-(17), wherein Compound 1 or Compound 2 is administered continuously for 5 days.

[0136] (21) Any one of embodiments (1)-(17), wherein Compound 1 or Compound 2 is administered continuously for 6 days.

[0137] (22) Any one of embodiments (1) to (17) in which compound 1 or compound 2 is administered continuously for 7 days.

[0138] (23) Any one of embodiments (1) to (17) in which compound 1 or compound 2 is administered continuously for 8 days.

[0139] (24) Any one of embodiments (1) to (17) in which compound 1 or compound 2 is administered continuously for 9 days.

[0140] (25) Any one of embodiments (1) to (17) in which compound 1 or compound 2 is administered continuously for 10 days.

[0141] (26) Any one of embodiments (1) to (17) in which compound 1 or compound 2 is administered continuously for 11 days.

[0142] (27) Any one of embodiments (1) to (17) in which compound 1 or compound 2 is administered continuously for 12 days.

[0143] (28) Any one of embodiments (1) to (17) in which compound 1 or compound 2 is administered continuously for 13 days.

[0144] (29) Any one of embodiments (1) to (17) in which compound 1 or compound 2 is administered continuously for 14 days.

[0145] (30) Any one of embodiments (1) to (29) in which the method is prophylactically used in a healthy patient to prevent infection.

[0146] (31) Any one of embodiments (1) to (29) in which the method is used to treat a patient having dengue virus.

[0147] (32) Any one of embodiments (1) to (29) in which a human has dengue serotype 1.

[0148] (33) Any one of embodiments (1) to (29), wherein the human has dengue serotype 2.

[0149] (34) Any one of embodiments (1) to (29), wherein the human has dengue serotype 3.

[0150] (35) Any one of embodiments (1) to (29), wherein the human has dengue serotype 4.

[0151] In an alternative embodiment, Compound 2 is provided as the hemisulfate of a phosphoramidate of Compound 1 other than the specific phosphoramidates depicted in the illustration of compounds of Formula I or Formula II, etc. A wide variety of phosphoramidates, including various esters and phosphoesters, are known to those skilled in the art, and any combination thereof can be used to provide the active compound in the form of a hemisulfate as described herein.

[0152] VI. Combination Therapy and Alternating Therapy It is well known that viral drug-resistant variants can emerge after long-term treatment with antiviral agents. Drug resistance can occur due to mutations in the genes encoding the enzymes used in viral replication. The efficacy of a drug against DENV infection can be extended, enhanced, or restored by administering the compound in combination with or alternating with another antiviral compound that induces mutations different from the main drug or acts through a different pathway, and in some cases, two or three additional antiviral compounds. Alternatively, the pharmacokinetics, biodistribution, half-life, or other parameters of the drug can be altered by such combination therapy (which can include alternating therapy when considered synergistic). Since the disclosed Compound 2 and Compound 1 are NS5 polymerase inhibitors, it may be useful to administer this compound to a host in combination with, for example: (1) Protease inhibitors, such as NS2B-NS3 protease inhibitors; (2) NS3 inhibitors; (3) NS3-NS4B interaction inhibitors; (4) NS4B inhibitor; (5) NS3-NS5 interaction inhibitor; (6) Another NS5 polymerase inhibitor; (7) Interferon alpha-2a, which may be pegylated or modified by other methods, and / or ribavirin; (8) Non-substrate-based inhibitor; (9) Helicase inhibitor; (10) Antisense oligodeoxynucleotide (S-ODN); (11) Aptamer; (12) Nuclease-resistant ribozyme; (13) iRNA including microRNA and siRNA; (14) Antibody, partial antibody or domain antibody against the virus, or (15) Virus antigen or partial antigen that induces a host antibody response.

[0153] Non-limiting examples of dengue DAA agents that can be administered alone or in combination with a plurality of agents from these lists, together with the high-dose compound 1 or compound 2 or formula I or formula II of the present invention, are as follows: (i) Protease inhibitors, such as telaprevir (Incivek (trademark)), boceprevir (Victrelis (trademark)), simeprevir (Olysio (trademark)), paritaprevir (ABT-450), glecaprevir (ABT-493), ritonavir (Norvir), ACH-2684, AZD-7295, BMS-791325, danoprevir, filibuvir, GS-9256, GS-9451, MK-5172, cerdulatinib, sofosbuvir, tegobuvir, VX-135, VX-222, and ALS-220; (ii) NS3 inhibitors, such as simeprevir, dabrafenib, idelalisib, nintedanib, ivermectin, or BP13944; (iii) NS3-NS4B interaction inhibitors, such as JNJ-1802 and JNJ-A07; (iv) NS4B inhibitors, such as NITD-688 or rilociguat; (v) NS5 inhibitor, for example, balapiravir; (vi) HCV NS5B inhibitor, for example, AZD-7295, clemizole, dasabuvir (Exviera), ITX-5061, PPI-461, PPI-688, sofosbuvir (Sovaldi (trademark)), MK-3682, ABT-333, and MBX-700 and mericitabine; (vii) Antiviral combination drugs, for example, 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).

Examples

[0154] Example 1. Cell line, virus and test compound Using the cell lines, viruses and compounds described herein, the data shown in Examples 2, 3, 4, 5, 6, 7, and 8 were generated. Huh-7 (human liver cancer; AcceGen Biotechnology, Fairfield, NJ) cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 μg / mL penicillin and 100 μg / mL streptomycin (Lonza, Walkersville, MD). Vero 76 cells (American Type Culture Collection (ATCC), Manassas, VA) used in the virus yield reduction assay were maintained similarly. BHK-21 (baby hamster kidney; ATCC, Manassas, VA) cells were maintained in Minimal Essential Medium with Earle's salts (EMEM) containing 1 mM sodium pyruvate and 25 μg / mL kanamycin and supplemented with 10% FBS. All cell cultures were maintained in an atmosphere of 37 °C, 5% CO 2 and humidity of 95% or more. Infection was carried out in EMEM supplemented with 5% FBS and 50 μg / mL gentamicin.

[0155] Dengue viruses (DENV-2 NGC and DENV-3 H87) were obtained from ATCC (Manassas, VA) and the DENV-2 used in human PBMC and BHK-21 assays was a clinical isolate. Japanese encephalitis (JEV SA-14), West Nile (WN02 Kern 515), yellow fever (YFV 17D), and Zika (ZIKV MR766) viruses were obtained from the University of Texas Medical Branch (Galveston, TX), and Powassan (POWV Spooner and LB strains) and Usutu (USUV TC-508) viruses were obtained from the World Reference Center for Emerging Viruses and Arboviruses at the University of Texas Medical Branch. In studies using fresh human PBMC, 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 PBMC and BHK21 cells were completed at the University of Cagliari ( Monserrato, Italy). Other virus studies regarding efficacy and specificity were conducted at Utah State University (Logan, UT) and ImQuest BioSciences (Frederick, MD).Protocols for various RNA and DNA viruses, such as HCV, HIV-1, influenza A and B, RSV, rhinovirus, herpes simplex type 1, and adenovirus, at the latter institute have been described previously (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). Compounds 2 (AT-752) and its free base, compound 1 (AT-281), were synthesized by a stereospecific process and, together with its plasma metabolite, compound 6 (AT-273), were prepared for Atea Pharmaceuticals by Topharman Shanghai Co., Ltd. (Shanghai, China) (compound 5 (AT-9010) and the TP internal standard used for the quantification of compound 5 (AT-9010) were synthesized by NuBlocks (Oceanside, CA)). Stock solutions were prepared in DMSO and stored at -20°C. To test the stability of compound 1 (AT-281) at 37°C, frozen plasma from humans and Sprague-Dawley rats (BioreclamationIVT, Westbury, NY) and cynomolgus monkeys (Xishan, Suzhou, China) was purchased.

[0156] Example 2. Compound 1 inhibits dengue virus (DENV) and other flaviviruses. The antiviral activity of compound 1 (AT-281) against flaviviruses DENV-2 NGC, 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 effects (CPE), and the virus yield reduction (VYR) assay (described in Example 4) as a second independent determination of virus replication inhibition. Human liver cancer (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) was measured in infected cells using the neutral red assay and / or the virus yield reduction (VYR) assay to determine the effective concentration (EC 50 ) required to achieve 50% inhibition of virus-induced cytopathic effect (CPE), the concentration (EC 10 ) that reduces the virus yield by 1 log 90 , and the cytotoxic concentration (CC 50 ) of the drug that kills 50% of the viable cells incubated without virus (see Table 1). After 3 to 6 days of incubation, the effective concentration (EC 50 ) of compound 1 (AT-281), a 2'-fluoro-2'-C-methylguanosine nucleotide prodrug, required to achieve 50% inhibition of virus-induced cytopathic effect (CPE) was in the range of 0.19 μM to 1.41 μM (see the following table).

[0157] TIFF2025517992000025.tif76170

[0158] TIFF2025517992000026.tif90170

[0159] The growth kinetics of different flaviviruses may vary in cell culture, but compound 1 (AT-281) showed a similar potent effect in reducing viral titers against all seven flaviviruses, including 11 different strains tested in vitro (Table 1A). The antiviral activity was also maintained against dengue serotypes 1, 2, 3, and 4 in all strains tested (Table 1B). The inhibition curves for DENV, WNV, and YFV (Figure 2) demonstrate this consistency. The concentration of compound 1 (AT-281) required to kill 50% of the cells exposed only to the drug (CC 50 ) exceeded the highest concentration tested (85 μM - 170 μM), and the antiviral selectivity index (SI; CC 50 / EC 50 ) ranged from greater than 120 to greater than 650. In particular, compound 1 (AT-281) showed 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 yield of infectious virus by 90% (EC 90 ) at a concentration of 0.64 μM. Similarly, in human PBMC and BHK-21 cells infected with DENV2 (clinical isolate) and Huh-7 cells infected with DENV-3 (H87 strain), compound 1 (AT-281) showed EC 50 values of 0.60 μM, 0.63 μM, and 0.77 μM, respectively, in the inhibition of virus-induced CPE. The SI was greater than 210 in all cases.

[0160] 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) was measured in infected cells using the neutral red assay, and the effective concentration required to inhibit 50% of viral replication (EC 50 ) (described in Example 3) and the threshold concentration required to obtain a perceptible effect in 50% of the cells incubated without virus (CC 50) was determined. Compound 1 (AT-281) was not active against the DNA viruses tested and was either slightly active or not active against some RNA viruses (Table 2). However, it had high selectivity (over 2000) against HCV and inhibited the virus at the nanomolar level (Table 2). When the potential cytotoxicity of Compound 1 (AT-281) was evaluated in multiple cell lines, the CC 50 value exceeded 100 μM (Table 2 below).

[0161] TIFF2025517992000027.tif70170

[0162] Furthermore, in human induced pluripotent stem cell (iPS) cardiomyocytes (described in Example 6) and granulocyte macrophage (GM) and erythrocyte (E) human bone marrow progenitor cells (described in Example 7), the absence of cytotoxicity was shown. When incubated with Compound 1 (AT-281), these cells also had CC 50 values exceeding 100 μM, while the positive control compounds showed the expected cytotoxic effects (the CC 50 value of doxazosin in cardiomyocytes was 7 μM, and the CC 50 values of AZT in GM and E cell assays were 2 μM and 3 μM, respectively). Furthermore, Compound 5 (AT-9010), which is the active TP, did not inhibit the in vitro enzyme activity of human cell DNA-dependent DNA polymerases α, β, or γ, and it has previously been shown that the estimated IC 50 value does not exceed 100 μM, and there is also no possibility of affecting mitochondrial integrity or inhibiting 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).

[0163] Example 3. 50% inhibition of virus-induced cytopathic effect (CPE) (EC50 Neutral red assay for measuring Using the neutral red assay procedure described herein, the data shown as "Neutral red assay" or "NR" in Tables 1A and 1B of Example 2 were generated. AT-281 was dissolved in DMSO at a concentration of 10 mg / mL and serially diluted using eight-step log dilution such that the highest test concentration was 100 μg / mL (172 μM). Each dilution was added to five wells of a 96-well plate containing 80% - 100% confluent Huh-7 cells. Three wells of each dilution were infected with virus and two wells were left uninfected as toxicity controls. Six untreated wells were infected as virus controls and six untreated wells were left uninfected for use as virus controls. The virus was diluted to achieve an MOI of approximately 0.001 CCID 50 (50% cell culture infective dose) (0.037 CCID for POWV LB strain and USUV respectively 50 and 0.028 CCID 50 ). The plates were incubated at 37 °C in a humidified atmosphere containing 5% CO 2 . On day 3 post-infection (ZIKV, EEEV and POWV Spooner), day 4 (CHIKV), day 5 (YFV, RVFV, MERS, POWV LB and USUV) or day 6 (WNV, JEV, DENV-2 NGC and DENV-3), 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 with 50:50 sorensen citrate buffer / ethanol for over 30 minutes. The optical density was read at 540 nm using a spectrophotometer and converted to percent of control. The concentration of the test compound required to prevent 50% of virus-induced CPE (EC 50 ), and the concentration of the test compound required to cause 50% cell death in the absence of virus (CC 50) were calculated (Smee et al., 2017. Evaluation of cell viability dyes in antiviral assays with RNA viruses 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). The selectivity index, unless otherwise specified, is CC 50 divided by EC 50 . Selectivity index (SI) values of 0 to 3.9 indicate inactive compounds, SI values of 4 to 9.9 indicate minimally active compounds, SI values of 10 to 49.9 indicate moderately active compounds, and SI values greater than 50 indicate highly active compounds. Compounds with SI values greater than 100 cannot be distinguished from each other.

[0164] Example 4. Virus yield reduction assay for measuring the concentration (EC 10 ) that reduces the virus yield by 1 log 90 . Using the viral yield reduction assay procedure described herein, the data presented as "VYR" or "VYR assay" in Tables 1A and 1B of Example 2 were generated. 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) until confluent. On day 3 post-infection (day 4 for POWV, day 7 for USUV), samples of the supernatant from each compound concentration were collected (pooling three wells) and the viral titer was tested using a standard endpoint dilution CCID 50 assay, and the titer was calculated using the Reed-Muench formula (Reed et al., 1938. A simple method of estimating fifty percent endpoints. Am J Hygiene 27:493-497). The concentration of the compound required to reduce the viral yield by 90% (EC 90 ) was determined by regression analysis.

[0165] Example 5. DENV-2 infection and treatment of human PBMC and BHK-21 cells. Using the PBMC assay procedure described herein, the data presented as "PBMC" or "human PBMC" in Tables 1A and 1B of Example 2 were generated. Compound 1 (AT-281) was dissolved in DMSO to 100 mM and then diluted in growth medium to final concentrations of 100 μM, 20 μM, 4 μM, and 0.8 μM. PBMC were resuspended in RPMI1640 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 1 hour of infection with DENV-2, the cells were seeded in 96-well plates (1×10 5(Cells / well) were seeded in the absence or presence of serial dilutions of compound 1 (AT-281) in PBMC growth medium. 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) containing serially diluted compound 1 (AT-281) and DENV-2 at a multiplicity of infection (MOI) of 0.01. The cytotoxicity of the compound was evaluated using uninfected cells in the presence of serially diluted compound. After incubation at 37 °C for 3 days in a humidified 5% CO 2 atmosphere, cell viability was determined by the 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 (EC 50 ) required to prevent 50% of the virus-induced cytopathic effect (CPE) and the effective concentration of AT-281 (CC 50 ) required to cause 50% cell death in the absence of virus were calculated by regression analysis.

[0166] Example 6. Cardiomyocyte assay to determine the cytotoxic concentration (CC 50 ) of a drug that causes the death of 50% of viable cells incubated without virus. Using the cardiomyocyte cytotoxicity assay procedure described herein, the CC 50 data in Table 2 of Example 2 were generated. Cytotoxicity was evaluated 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, respectively. The iPS cardiomyocytes in the medium from Cellular Dynamics were plated at a final volume of 100 μL at 1.5×10 in 96-well plates pre-coated with 0.1% gelatin (Sigma)4 Seeded in cells / wells and incubated at 37°C in 5% CO 2 for 48 hours. The cells were washed with DPBS and AT-281 diluted in the same medium (100 μL) was added to the monolayer in three portions, and incubated at 37°C in 5% CO 2 for 3 days. Cell viability was measured by staining with CellTiter Glo™. The medium was removed from the test plates and replaced with fresh medium (100 μL) and CellTiter Glo reagent (100 μL per well), and then incubated at room temperature for 10 minutes. The contents of the wells were transferred to a white 96-well plate and luminescence was measured using a Wallac 1450 Microbeta Trilux liquid scintillation counter within 15 minutes.

[0167] Example 7. Bone marrow progenitor cell assay. Using the cardiomyocyte cytotoxicity assay procedure herein, the CC in Table 2 of Example 2 50 data was confirmed. Bone marrow progenitor cells suspended in Iscove's modified Dulbecco's medium containing 15% heat-inactivated FBS, 10% macrophage-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 methylcellulose at a final concentration of 1% were added to 6-well plates (1×10 5 cells / well) in a volume of 900 μL. AT-281 (100 μL) was added to each well three times at 10-fold the test concentration, and incubated at 37°C in 5% CO 2 for 14 days, and colonies (more than 30 cells) were counted.

[0168] Example 8. Compound 1 (AT-281) forms the active metabolite compound 5 (AT-9010) in human PBMCs in vitro. Since viral infection in the blood is an important component of dengue fever, it was 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 found to be stable for up to 120 minutes in cynomolgus monkey and human plasma (98.2% and 93.5% of the initial 2 μM concentration remained after 120 minutes of incubation, respectively), but was very unstable in Sprague Dawley (SD) rat plasma and no drug remained at the first time point (10 minutes of incubation). The half-life (T 1 / 2 ) in rat plasma at 37 °C was estimated to be less than 3 minutes.

[0169] Fresh unstimulated human PBMCs treated with 10 μM of compound 1 (AT-281) (described in Example 9) for up to 6 hours showed a time-dependent increase in compound 5 (AT-9010) concentration throughout the exposure phase and continued to increase after drug washout, with an average peak concentration of 1.88 ± 0.01 pmol / 10 6 cells at 10 hours after the start of exposure (Figure 3). Removal of compound 1 (AT-281) for 28 hours (washout phase) resulted in a time-dependent decrease in the concentration of compound 5 (AT-9010) in PBMCs (Figure 3), and the T 1 / 2 was estimated to be 21.6 hours.

[0170] Example 9. In vitro formation of compound 5 (AT-9010) in unstimulated human PBMCs treated with compound 1 (AT-281). After receiving fresh human PBMCs (one male donor, lot LS-88-45477C), they were centrifuged at 400 × g for 5 minutes. The resulting cell pellet was resuspended in 20 mL of warm PBMC growth medium. The cell density of the cell suspension was counted using an automated cell counter (Cellometer K2, Nexcelom) after staining with trypan blue and adjusted to 2 × 10 6 viable cells / mL. The human PBMC suspension was added at 0.5 mL / well (1 × 10 6Transfer to a 24-well tissue culture treatment plate with (cells), and culture in a humidified incubator maintained at 37 °C and 5% CO 2 for 20 to 24 hours. Then, add the compound 1 (AT-281) stock solution to each well to a final concentration of 10 μM, and culture the stimulated cells at 37 °C and 5% CO 2 atmosphere for 0 hour, 2 hours, 4 hours, and 6 hours. At each time point, three replicate samples were processed for the analysis of compound 5 (AT-9010). For the washout 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 medium, resuspended in 0.5 mL of medium, and transferred to each well of a 24-well plate. The human PBMCs were further incubated in a humidified incubator maintained at 37 °C and 5% CO 2 for 0 hour, 2 hours, 4 hours, 16 hours, 20 hours, 24 hours, and 28 hours (i.e., 6 hours, 8 hours, 10 hours, 22 hours, 26 hours, 30 hours, and 34 hours after the start of the exposure to the test substance). At each time point, three samples for the analysis of compound 5 (AT-9010) were processed as follows: The samples were transferred to 2 mL tubes and centrifuged at 800 × g for 5 minutes. After aspirating the supernatant, 0.1 mL of water was added and each sample was vortexed. Then, they were quenched with 0.3 mL of ice-cold 60% MeOH and stored at -70 °C until analysis by LC / MS / MS (Figure 3).

[0171] Example 10. Compound 5 (AT-9010) inhibits RNA elongation by DENV NS5. To confirm the mechanism of action and antiviral target of compound 5 (AT-9010), an incorporation and extension assay (described in Example 13) was performed using the full-length DENV NS5 protein (described in Example 11) (serotype 2) and an annealed primer-template RNA pair that mimics the 3' end of the DENV-2 genome (described in Example 12). In the absence of GTP, compound 5 (AT-9010) was readily incorporated as a substituent at the +5 position of the RNA primer (Figure 4, left side of the gel). Since no further extension was observed despite the presence of the next correct nucleotide (UTP), compound 5 (AT-9010) is shown to cause immediate chain termination of RNA synthesis regardless of the presence of its 3'OH group. In the presence of all four NTPs, compound 5 (AT-9010) competed with GTP for incorporation at the +5 and +7 positions (Figure 4, right side). Indeed, the incorporation of GTP and compound 5 (AT-9010) at the same positions could be distinguished on the gel, and the discrimination factor (priority of GTP > compound 5 (AT-9010) as judged by band product intensity) was calculated to be 12.6 ± 4.3. A comparable profile was also shown in the comparison of incorporation with the essential chain terminator 3'-dGTP, supporting that chain termination is the mechanism of action of compound 5 (AT-9010) (Figure 5).

[0172] Example 11. Expression and purification of DENV NS5 protein. The DENV NS5 protein used in Example 10 was prepared by the procedure described herein. Full-length DENV NS5 (serotype 2, New Guinea C) was expressed and purified as previously described (Potisopon et al., 2017. Substrate selectivity of Dengue and Zika virus NS5 polymerase towards 2'-modified nucleotide analogues. Antiviral Res 140:25-36). Briefly, the gene encoding NS5 with an N-terminal 6-His tag was expressed from the pQE30 vector in Escherichia coli NEB Express cells (New England Biolabs, Ipswich, Massachusetts) transformed with pRare2-LacI (Novagen, Madison, Wisconsin), and induced with 50 μM IPTG and 2% EtOH until the OD 600 value reached 0.6. Cells were lysed by sonication, and the NS5 protein was separated, washed, and eluted using a TALON metal affinity resin slurry (Clontech, Mountain View, California) according to the manufacturer's instructions. Size exclusion chromatography was performed as a second purification step, and 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.

[0173] Example 12. Oligonucleotides and Nucleotides. The RNA oligonucleotides used in Example 10 were prepared as described herein. The RNA oligonucleotides were purchased from Biomers.net (Ulm / Donau, Germany). A 20 nt template sequence (T 20 ) corresponding to the 3' end of the DENV-2 antigenome was annealed to a 10 nt complementary primer (P 10) was annealed. Annealing was performed using a primer-template molar ratio of 1:1.5 in the presence of 110 mM KCl, incubated at 70 °C for 10 minutes, and slowly cooled to room temperature. NTPs were purchased from GE Healthcare (Chicago, IL).

[0174] Example 13. Nucleotide Analogue Incorporation Assay. DENV NS5 was pre-incubated with annealed P 2 / T 10 / T 20 RNA in assembly buffer containing 20 mM HEPES pH 7.5, 10% glycerol, 5 mM MgCl 2 and 5 mM DTT at 30 °C for 10 minutes to generate an active RNA elongation complex. The reaction was initiated by adding AT-9010 with either all four NTPs or UTP, ATP, and CTP only. The final concentrations were 0.5 μM NS5, 0.25 μM P 10 / T 20 , 100 μM each NTP, and 10 μM - 625 μM AT-9010 in a final buffer containing 20 mM HEPES pH 7.5, 15% glycerol, 5 mM MgCl

[0175] Example 14. Compound 2 (AT-752) Demonstrates Good Pharmacokinetics in Preclinical Species. The plasma pharmacokinetics (PK) of the intermediate prodrug, compound 1 (AT-281) and its metabolite compound 3 (AT-551), and the plasma surrogate of the active triphosphate metabolite, compound 6 (AT-273), of compound 5 (AT-9010) were measured in CD-1 mice, Sprague-Dawley rats, and cynomolgus monkeys after single oral administration of compound 2 (AT-752) at 420 mg / kg, 300 mg / kg, and 300 mg / kg, respectively (Table 3).

[0176] TIFF2025517992000028.tif57170

[0177] In rodents, the parent prodrug, compound 1 (AT-281), was rapidly metabolized, and its metabolites, compound 3 (AT-551) and compound 6 (AT-273), rapidly appeared in plasma (Figures 6A and 6B). In 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 reported above. In monkeys, compound 1 (AT-281) was present in plasma for more than 4 hours (Figure 6C), and the C max at 1 hour was 3.7 ± 1.5 nmol / mL. This prodrug was converted to the plasma intermediate metabolites, compound 3 (AT-551) and compound 6 (AT-273), and the PK was comparable to the results of its homolog, AT-511, reported previously (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).

[0178] Example 15. The active metabolite compound 5 (AT-9010) is formed in PBMC in vivo. The concentration of compound 5 (AT-9010) in PBMC isolated from mice and rats administered compound 1 (AT-281) at single oral doses of 50 mg / kg and 300 mg / kg, and from monkeys after single oral administration of compound 2 (AT-752) corresponding to 55 mg / kg of compound 1 (AT-281), was measured at various time points after dosing (Figure 7). Similar concentrations of compound 5 (AT-9010) were observed in mice and monkeys at 12 hours after equivalent doses (0.092 and 0.070 ± 0.046 pmol / 10 6 cells). At 6-fold higher doses, rat PBMC at 24 hours had 5- to 6-fold more compound 5 (AT-9010) (0.439 ± 0.218 pmol / 10 6 cells) than the 12-hour values observed in mice and monkeys, suggesting that exposure to compound 1 (AT-281) in rat systemic circulation was very low and even lower in mice, yet active TP formation in PBMC was comparable in all three species. When mice were given a loading dose of 500 mg / kg of compound 1 (AT-281) and a second dose of 250 mg / kg 4 hours later, the concentrations of compound 5 (AT-9010) in PBMC 4 and 12 hours after the second administration were 0.297 pmol / 10 6 cells and 0.322 pmol / 10 6 cells, respectively (not shown).

[0179] To test the tolerance of compound 1 (AT-281) and determine the steady-state concentration of active TP in PBMC, mice were given a loading dose of 1000 mg / kg and then 500 mg / kg twice daily (12-hour intervals) for 3 days starting 4 hours later. The concentrations of compound 5 (AT-9010) in pooled (n = 3) PBMC 12 hours after the first and sixth 500 mg / kg doses were 0.421 pmol / 10 6 cells and 0.575 pmol / 10 6The cells, and the concentrations of compound 6 (AT-273) in plasma at the same time point were 0.67 ± 0.12 nmol / mL and 1.35 ± 0.86 nmol / mL, respectively. No harmful clinical signs were observed in the mice, and thus multiple administrations of the prodrug were well tolerated.

[0180] Example 16. Animal welfare. The studies using CD-1 mice, SD rats and cynomolgus monkeys in Example 17 were conducted at WuXi AppTec (Suzhou, China) in strict accordance with AAALAC International, NIH guidelines and the "Regulations on the Administration of Affairs Concerning Experimental Animals" of the Ministry of Science and Technology of the People's Republic of China, 2017. The protocol was reviewed and approved by the IACUC of WuXi AppTec prior to the start of the study, and all animals were evaluated by the veterinary staff of WuXi AppTec throughout the study. All animals were housed in rooms with controlled temperature (18°C to 26°C), relative humidity (40% to 70%) and light cycle (12 hours of artificial light and 12 hours of darkness), and the ventilation rate was 100%. The animals were provided with manipulable toys / enrichment toys. The AG129 mouse study described herein was conducted at IBT Bioservices (Rockville, Maryland) in strict accordance with the USDA Animal Welfare Act, the PHS and NIH Policy of Humane Care and Use of Laboratory Animals, and the National Research Council-ILAR Guide for the Care and Use of Laboratory Animals (revised 2011). This study was conducted in full compliance with a protocol reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of IBT prior to the start of the study, and the mice were evaluated and monitored throughout the study by members of the veterinary staff of IBT in accordance with PHS policy and the USDA.

[0181] Example 17. PK study and in vivo formation of Compound 5 (AT-9010) in mice, rats, and monkeys. Male naive CD-1 mice (Shanghai Sippr BK Laboratory Animals Co., Ltd., China) were orally administered Compound 2 (AT-752) in 40% PEG400, 10% solutol HS15, 50% 100 mM citrate buffer (pH 4.5 (v / v)) at a dose of 420 mg / kg. Blood samples (N = 3) were collected at 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h after administration, and plasma was separated with EDTA and 1 μL dichlorvos (2 mg / mL; a stabilizer to prevent in vitro hydrolysis of the ester moiety of Compound 1 (AT-281) by blood esterases) 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 in plasma by LC / MS / MS. For PBMC, six male naive CD-1 mice were administered a dose of Compound 2 (AT-752) in 60% PEG400 (at a 50 mg / kg Compound 1 (AT-281) equivalent dose) by oral gavage. Blood samples were collected in three groups at 4 h and 12 h after administration, PBMC were isolated, and the concentration of Compound 5 (AT-9010) was measured by LC / MS / MS.

[0182] Male naive Sprague-Dawley rats (Beijing Vital River Laboratory Animals Co., Ltd., China) were orally administered AT-752 at a dose of 300 mg / kg in 40% PEG400, 10% solutol HS15, 50% 100 mM citrate buffer (pH 4.5 (v / v)). Blood samples (n = 4) were collected at 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours after administration, 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 measured by LC / MS / MS. For PBMCs, nine male naive Sprague-Dawley rats were administered compound 2 (AT-752) by oral gavage at a dose (equivalent dose of 300 mg / kg of compound 1 (AT-281)). Blood samples were collected in three groups at 24 hours, 48 hours, and 72 hours after administration, PBMCs were isolated, and the concentration of compound 5 (AT-9010) was measured by LC / MS / MS.

[0183] Male non-naïve cynomolgus monkeys (Hainan Jingang Laboratory Animal Co., Ltd., China) weighing at least 2 kg were individually housed in stainless steel mesh cages during the study period, allowed free access to RO water, and fed approximately 60 g of certified monkey diet (Beijing Vital Keao Feed Co., Ltd., Beijing, China) twice a day (except on the dosing day when fed once), and fresh fruits were given daily as snacks. These monkeys (n = 5) were administered compound 2 (AT-752) in 40% PEG400, 10% solutol HS15, 50% 100 mM citrate buffer (pH 4.5 (v / v)) by forced oral gavage at a dose of 300 mg / kg, and blood samples (about 0.5 mL) were collected at 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours after dosing. Plasma was separated at each time point for pharmacokinetics and stored at -70 °C. The concentrations of compound 1 (AT-281), and metabolites compound 3 (AT-551) and compound 6 (AT-273) were measured by LC / MS / MS. Separately, three monkeys were given compound 2 (AT-752) as a powdered capsule at an oral dose of 60 mg / kg (equivalent dose of 55 mg / kg of compound 2 (AT-281)), and blood samples were collected at 12 hours, 24 hours, and 48 hours after dosing. PBMCs were separated and the concentration of compound 5 (AT-9010) was measured by LC / MS / MS. In all animals, any abnormal or adverse clinical signs were observed immediately before and after dosing, and prior to each blood sampling time point, but no such signs were noted.

[0184] Separately from this, two mouse studies involving multiple administrations were conducted. First, nine naive CD-1 mice were orally gavaged with Compound 1 (AT-281) in 100 mM citrate buffer (pH 4.5) at a dose of 500 mg / kg for the first administration and 250 mg / kg for the second administration 4 hours later. Blood samples were collected from three animals at each time point 4 hours (before the second administration), 8 hours, and 16 hours after the first administration, and PBMCs were isolated. Next, nine naive CD-1 mice were orally gavaged with Compound 1 (AT-281) at a dose of 1000 mg / kg for the first administration and then 500 mg / kg twice daily at 12-hour intervals for 3 days 4 hours later. Blood samples were collected from three animals at each time point 16 hours (one administration of the 500 mg / kg dose), 40 hours (three administrations of the 500 mg / kg dose), and 76 hours (six administrations of the 500 mg / kg dose) after the first administration, and PBMCs were isolated. The plasma concentrations of Compound 1 (AT-281) and Compound 6 (AT-273), and the concentration of Compound 5 (AT-9010) in PBMCs were measured by LC / MS / MS (described in Example 21).

[0185] Example 18. Compound 2 (AT-752) reduced viremia and improved survival in AG129 mice after DENV2 D2Y98P infection. To test the efficacy of compound 2 (AT-752) against DENV infection, AG129 mice were subcutaneously inoculated with DENV-2 D2Y98P. DENV-2 D2Y98P is a non-mouse-adapted strain that mimics the disease dynamics similar to those described in humans (Tan et al., 2010. A non mouse-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 as a loading dose (1000 mg / kg) 4 hours before virus challenge and then orally administered twice a day (BID) at a dose of 500 mg / kg from 1 hour post-infection (pi) for 7 consecutive days. Viral load and spleen viral load, which are the primary evaluation items, were evaluated on days 4, 6, 7, 8, and 10 post-infection. Viral load in the compound 2 (AT-752) treatment group (Figure 8A) was significantly lower than that in the vehicle-only group on day 6 post-infection (p = 0.000014 by t-test) and disappeared in all treated animals by day 8 post-infection, while the virus was still measurable in all surviving controls (p = 0.0045 by t-test). Treatment with compound 2 (AT-752) reduced the spleen viral load by day 7 post-infection compared to the control group (Figure 8B), but this change was not statistically different between the groups. This result is thought to be because the virus ultimately disappears from the spleen in this disease model mouse.

[0186] Mice were evaluated daily for changes in body weight, appearance, motility, and attention, and were euthanized when their body weight decreased by 20% or their health score exceeded 5. From day 4 to day 8 after infection, a significant difference in body weight was observed between the treatment group and the vehicle control group (p < 0.001 by t-test). Mice treated with the vehicle did not survive after day 8 post-infection. Animals treated with compound 2 (AT-752) had a significantly attenuated weight loss compared to vehicle control mice on days 5, 7, and 8 post-infection (Figure 9A; p < 0.001 by t-test). Furthermore, mice treated with compound 2 (AT-752) had a significantly better health score from day 4 to day 9 post-infection compared to the vehicle control group (Figure 9B; p < 0.001 by t-test). The resulting mortality data are presented as Kaplan-Meier survival curves (Figure 10), demonstrating that compound 2 (AT-752) significantly improved the survival rate of infected mice compared to the vehicle control group (p = 0.0004, Mantel-Cox log-rank test). All control group mice that were not sacrificed as scheduled for blood and spleen sample collection died or were euthanized between days 5 and 8 post-infection. In contrast, animals treated with compound 2 (AT-752) survived until days 11 to 18 post-infection.

[0187] Example 19. AG129 mouse study. The tissue samples used in Example 20 were collected according to the protocol described herein. Fifty-five 6- to 8-week-old female AG129 mice (α-, β-, γ-interferon knockout) were divided into two groups, with 25 mice in group 1 (vehicle) and 30 mice in group 2. On day 0, all mice were inoculated with 1×10 5PFU of DENV-2 D2Y98P was challenged by subcutaneous injection. Animals in Group 1 were administered BID by forced oral administration with vehicle [(PEG400 (40%, v / v) / Solutol HS15 (10%, v / v) / 100 mM citrate buffer pH 4.5 ± 0.2 (50%, v / v)] continuously from 1 hour after infection to 7 days. In Group 2, 1000 mg / kg of Compound 2 (AT-752) was administered as a single dose 4 hours before challenge, followed by a single dose of 500 mg / kg 1 hour after challenge (pi). BID administration of 500 mg / kg of Compound 2 (AT-752) was continued continuously for 7 days. In the protocol, on the 4th, 6th, 7th, 8th, and 10th days after infection, 5 mice per group were sacrificed, and terminal serum and spleen were collected. On the 20th day after infection, the surviving mice in Group 2 were sacrificed, and terminal serum and spleen were collected. Serum samples were stored at -80 °C until further analysis. The collected spleen samples were weighed, rapidly frozen in a mixture of ethanol / dry ice, and immediately stored at -80 °C. All samples were processed, and the virus amount was assayed via plaque assay. All animals were monitored daily for weight loss, morbidity, mortality, and neurological weakness. Mice showing severe illness determined by more than 20% weight loss, a health score exceeding 5 (evaluation of coat appearance, motility, and attention), extreme lethargy, and / or paralysis were euthanized, and terminal serum and spleen were collected. All mice found dead were collected only for spleen collection.

[0188] Example 20. Plaque assay for determination of spleen virus amount. The spleen samples collected according to Example 19 were homogenized in 250 μL of DPBS using a TissueRuptor. The homogenate was then centrifuged, the supernatant was collected, and immediately frozen in two different aliquots until plaque assay was performed. The results of the plaque assay are shown in FIGS. 8A and 8B.

[0189] One day before the assay, Vero cells were seeded at a density of 1 × 10 5 cells / well in 1 mL of 1% Hi-FBS medium in a 24-well tissue culture plate and incubated in 5% CO2 It was cultured overnight at 37°C in an atmosphere. The next day, serial 10-fold dilutions (10 -2 ~10 -5 ) of the serum and spleen samples were prepared in triplicate for titration. The cell culture medium was removed from Vero cells, and 100 μL of MEM medium supplemented with 2 mM L-glutamine and 1X Pen / Strep was added together with 100 μL of the diluted sample. The cells were incubated at 37°C for 1 hour in a 5% CO 2 atmosphere. Then, 1 mL of 0.8% methylcellulose containing 2% FBS supplemented with 2 mM L-glutamine and 1X Pen / Strep was added to the cells without removing the inoculum, and the cells were incubated at 37°C for an additional 3 days in a 5% CO 2 atmosphere to form plaques. The cells were fixed and permeabilized with a cold 80% ethanol / 20% methanol mixture at -20°C for 30 minutes. 200 μL of anti-dengue monoclonal antibody diluted 1:2000 with 5% skim milk was added to each well and incubated overnight at 4°C. The wells were washed 3 times with 1×DPBS, and 200 μL of HRP-conjugated goat anti-mouse antibody diluted 1:2000 with 5% skim milk was added, and the wells were incubated at room temperature for 1 hour. Virus plaques were lysed and counted using an insoluble peroxidase substrate (TrueBlue) and a plaque counter. Data on the amount of virus in the spleen were normalized by the spleen weight of each mouse.

[0190] Example 21. LC-MS / MS analysis of Compound 1 (AT-281), Compound 3 (AT-551), Compound 6 (AT-273), and Compound 5 (AT-9010). The pharmacokinetic measurements in Figures 11, 12, 13, 14, and 15 were analyzed using LC-MS / MS according to the protocol described herein. Plasma samples were prepared for MS analysis by adding an internal standard and extracting with 20 volumes of chilled ACN. After vortexing (800 rpm, 10 minutes) and centrifugation (4000 rpm, 15 minutes, 4°C), the supernatant (25 μL) was diluted with an equal volume of H 2 O, mixed, and spun again. After adding the internal standard, PBMC samples (10 6The cells were dissolved in 300 μL of MeOH / H 2 O (70:30, v / v) and 40 mM dibutylammonium acetate (DBAA), mixed by vortexing, and centrifuged (12,000 rpm, 15 min, 4 °C). An aliquot (35 μL) of the supernatant was dried under nitrogen, reconstituted with H 2 O, then mixed and spun again. To measure compound 1 (AT-281) and plasma metabolite compounds 3 (AT-551) and 6 (AT-273), 4 μL of the sample was injected into an Acquity Gemini C18 (50 × 4.6 mm), 5 μm UPLC column equipped with a Sciex Triple Quad 6500 mass spectrometer (ESI positive ion, MRM mode). Using a binary non-linear gradient of mobile phase A (0.1% formic acid in water) and B (0.1% formic acid in ACN), the sample was eluted at 0.8 mL / min with a runtime of 5 min. For intracellular AT-9010, 8 μL was injected into an Acquity BEH C18 (50 × 2.1 mm), 1.7 μm UPLC column equipped with an API 14000 mass spectrometer (ESI negative ion, MRM mode). Mobile phase A (0.001% NH 3 ·H 2 O, 0.18 mM DBAA in H 2 O) and B (10 mM N,N-dimethylhexylamine, 3 mM NH 2 in ACN / H 4Using a binary non-linear gradient with OAc), the sample was eluted at 0.5 mL / min and the runtime was 3 minutes. Standard substances in 50% MeOH were used for calibration. The monitored ions were m / z 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). The coefficient of variation of the recovery rate was corrected using the above-mentioned internal standard substance (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).

[0191] Example 22. Pharmacokinetic data analysis. The data analysis of the pharmacokinetic data generated according to Example 21 and shown in FIGS. 11, 12, 13, 14, and 15 was analyzed according to the procedures described herein. The plasma concentrations of Compound 1 (AT-281), Compound 3 (AT-551), and Compound 6 (AT-273) were subjected to non-compartmental pharmacokinetic analysis using Phoenix WinNonlin software (version 6.3 or higher, Pharsight, Mountain View, CA). The linear / logarithmic trapezoidal rule was applied when obtaining PK parameters.

[0192] Example 23. Safety study in humans Healthy men and women aged 18 to 65 years were enrolled in the escalating single-dose (SAD) and escalating multiple-dose (MAD) cohorts and randomly assigned to receive Compound 2 or placebo orally according to Tables 4 to 7.

[0193] A safety assessment was performed, including adverse events, standard clinical tests, vital sign measurements, electrocardiograms, and physical examinations.

[0194] TIFF2025517992000029.tif25170

[0195] TIFF2025517992000030.tif20170

[0196] TIFF2025517992000031.tif39170

[0197] TIFF2025517992000032.tif25170

[0198] Compound 2 had good tolerability, and no serious adverse events or dose interruptions due to adverse events were reported. Non-serious adverse events were mild or moderate in severity and resolved by the end of the study (Table 8). Isolated cases of gastrointestinal-related events, including mild to moderate vomiting, occurred mainly at high doses.

[0199] TIFF2025517992000033.tif34170

[0200] TIFF2025517992000034.tif34170

[0201] Example 24. Pharmacokinetic study in the SAD cohort Healthy men and women aged 18 to 65 years were enrolled in a single ascending dose (SAD) study and randomly assigned to receive Compound 2 or placebo orally according to Tables 4 to 6.

[0202] Plasma and urine samples were collected at predetermined time points, and compounds 1 and its metabolites, compounds 3, 6, and 7, were quantified using LC-MS / MS. Pharmacokinetic analysis was performed using a non-compartmental approach.

[0203] When administered orally under fasting conditions, the parent prodrug, compound 1, was rapidly absorbed and disappeared, followed by the appearance of compound 3, which also showed transient exposure (Tables 10 to 14, and Figures 12 to 15). N 6Compound 7, a metabolite of 2'-methyl-2'-fluoro nucleoside, reached a peak thereafter and showed a slower elimination phase (Figure 15). Compound 6 appeared more gradually in plasma and showed a long elimination half-life (up to a cohort average of 23 hours), reflecting the sustained intracellular exposure of compound 5, an active triphosphate metabolite.

[0204] Dose-proportionality analysis Plasma exposure of compound 1 and its metabolites increased across the studied dose range of 250 mg to 1500 mg. Compound 7 increased in a dose-proportional manner. Compound 1 and compound 3 showed increases greater than dose-proportionality, while compound 6 was slightly less than dose-proportional.

[0205] Effect of diet A high-fat / high-calorie diet delayed and decreased the peak levels of compound 1, compound 3, and compound 7, but had a limited or no effect on their total exposure, and slightly increased the plasma exposure of compound 6.

[0206] TIFF2025517992000035.tif68170

[0207] TIFF2025517992000036.tif86170

[0208] TIFF2025517992000037.tif68170

[0209] TIFF2025517992000038.tif86170

[0210] The pharmacokinetic profiles were similar between the South Asian / Southeast Asian / East Asian and mainly Caucasian subject cohorts, suggesting no ethnic sensitivity to pharmacokinetics. Therefore, compound 2 does not require dose adjustment.

[0211] Urinary excretion rate After a single oral administration of 250 mg to 1500 mg of Compound 2, the urinary excretion rates were low for Compound 1 and Compound 3, and moderate for Compound 7 and Compound 6 (Table 9). The total urinary recovery rates were in the range of about 20% to about 30% over the entire dose range. Since the renal clearances of the nucleoside metabolites, Compound 6 and Compound 7, exceeded the estimated glomerular filtration rate, the involvement of active secretion in the renal environment was suggested.

[0212] TIFF2025517992000039.tif30170

[0213] Example 25. Pharmacokinetic Study in the MAD Cohort Healthy men and women aged 18 to 65 years were enrolled in a multiple ascending dose (MAD) study and randomly assigned to receive oral administration of Compound 2 or placebo according to Tables 4 and 7.

[0214] Plasma and urine samples were collected at predetermined time points, and Compound 1 and its metabolites, Compound 3, Compound 6, and Compound 7, were quantified using LC-MS / MS. Pharmacokinetic analysis was performed using a non-compartmental approach.

[0215] The plasma exposure of Compound 6 increased by approximately 25%, 60%, and 80% for QD, BID, and TID, respectively, due to its long plasma half-life. This reflects the rapid accumulation and sustained intracellular exposure of the active metabolite, Compound 5.

[0216] The plasma concentration of Compound 6 increased rapidly with a TID dose of 750 mg, exceeding the 90% effective concentration (EC 90 )(0.64 μM) of the agent that inhibits dengue virus replication in vitro. These levels were maintained throughout the treatment period (Figure 11).

[0217] TIFF2025517992000040.tif31170

[0218] TIFF2025517992000041.tif31170

[0219] TIFF2025517992000042.tif31170

[0220] TIFF2025517992000043.tif31170

[0221] In Tables 15 to 18, C max and AUC tau are expressed as mean ± standard deviation. T max is expressed as median (minimum value - maximum value). AUC tau indicates the area under the plasma concentration-time curve over the dosing interval.

[0222] Example 26. Antiviral assay Transcripts of dengue viruses (DENV1 WP, DENV2 NGC, DENV3 VN32, DENV4 MY01) were modified with a nanoluciferase reporter between the 5’UTR and the capsid gene as previously described (Baker et al., 2020b, 2020a) to generate stable tagged viruses in cell culture passages while producing a robust 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 was 0.1 for DENV-1, DENV-2 and DENV-3, and 0.001 for DENV-4), and 1×10 per well in a 96-well plate in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) containing 2% fetal bovine serum (Hyclone, Logan, UT) the day before. 4It was added to plates containing Huh-7 cells (RRID:CVCL 0336) seeded with cells. At 48 hours post-infection, the cells were washed three times with phosphate-buffered saline, and then NanoGlo™ substrate diluted 1:100 in NanoGlo™ assay buffer (Promega, Madison, Wisconsin) was added. Luciferase activity was read after 3 minutes using a BioTek Cytation 5 plate reader. Data were analyzed using GraphPad Prism 9 software. Luciferase activity was normalized by setting the DMSO-treated sample to 100%. Error bars represent mean ± SD. Results are representative of three independent experiments, each analyzed in triplicate. Antiviral activity against the tested viruses can be found in FIG. 16.

[0223] Example 27. Expression of DENV NS5, RdRp, and MTase Domains 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 Escherichia coli (E. coli) NEB C2566 cells (New England Biolabs) harboring the pRARE2LacI (Novagen) plasmid. After induction with 100 μM IPTG and 2% EtOH (% v / v) to an OD600 of 0.8 - 1, the proteins were expressed overnight at 17 °C in TB (containing 25 μg / mL kanamycin and 17 μg / mL chloramphenicol).

[0224] The DENV MTase was expressed under the control of the T7 promoter in the pDEST17 vector in Escherichia coli (E. coli) NEB C2566 cells (New England Biolabs) harboring the pRARE2LacI (Novagen) plasmid. After induction with 200 μM IPTG and 2% EtOH (% v / v) to an OD600 of 0.8 - 1, the proteins were expressed overnight at 17 °C in TB (containing 100 μg / mL ampicillin and 17 μg / mL chloramphenicol).

[0225] Example 28. Purification of DENV NS5 The 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 a complete protease inhibitor cocktail (COC) from Roche. Proteins from the soluble fraction were loaded onto TALON™ Superflow™ cobalt-based IMAC resin (Cytiva) and washed 5 times with lysis buffer and 10 times with lysis buffer (without Igepal) before eluting with lysis buffer supplemented with 250 mM imidazole and 250 mM glycine. Imidazole was removed by dialysis overnight in dialysis buffer (50 mM NaP pH 8, 20% glycerol, 150 mM NaCl, 250 mM glycine and 0.5 mM TCEP), and then the final step of purification was performed using a size exclusion chromatography (SEC) HiLoad™ 16 / 600 Superdex™ 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 mg / mL - 15 mg / mL and stored at -80 °C after the final dialysis in SEC buffer supplemented with 40% glycerol.

[0226] Example 29. Purification of the DENV RdRp domain The 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 a complete protease inhibitor cocktail (COC) from Roche. Proteins from the soluble fraction were loaded onto TALON™ Superflow™ cobalt-based IMAC resin (Cytiva) and washed 5 times with lysis buffer and 10 times with lysis buffer (without Igepal) before eluting with lysis buffer supplemented with 250 mM imidazole and 250 mM glycine. 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 the final step of purification was performed using a GE Hi-trap heparin column. The protein was then concentrated to 10 mg / mL - 15 mg / mL and subjected to a final dialysis in a final buffer containing 20 mM HEPES pH 8, 300 mM NaCl, 40% glycerol and 0.5 mM TCEP before storage at -80°C.

[0227] Example 30. Purification of the DENV MTase domain The DENV MTase used in the thermal shift assay (Example 31) was purified as described herein. Cells were lysed by sonication on ice in 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 from the soluble fraction were loaded onto TALON™ Superflow™ cobalt-based IMAC resin (Cytiva) and washed with lysis buffer (1 M NaCl) before elution with 250 mM imidazole. Proteins were finally purified using SEC (HiLoad™ 16 / 600 Superdex™ 200 pg) in buffer containing 50 mM HEPES pH 8, 300 mM NaCl, and 0.5 mM TCEP. The proteins were then concentrated to 10 mg / mL - 15 mg / mL and subjected to a final dialysis in a final buffer containing 20 mM HEPES pH 8, 300 mM NaCl, 40% glycerol, and 0.5 mM TCEP, and then stored at -80 °C.

[0228] Example 31. Thermal Shift Assay The thermal stability of flavivirus proteins and their interaction with compounds were evaluated by using the thermal shift assay. The reactions were carried out in a white frame star PCR96 well plate (reference number 044705) with a final volume of 20 μL. Optimal conditions were found by using a buffer containing 20 mM Hepes pH 7.5, 50 mM NaCl, 5 mM DTT, 10% glycerol, and 1 M sorbitol for DENV NS5, polymerase, and MTase.

[0229] 0.4 μL of GTP, sinefungin, or AT-9010 was placed in the plate (final concentration 100 μM). Water was used as a control without the compound. 2 μM of NS5, 2 μM of polymerase, or 6 μM of MTase was mixed in a buffer containing 2.5-fold, 0.75-fold, or 1.5-fold (final concentration) of the dye, respectively. A mixture of the protein and the ThermoFisher protein thermal shift kit (reference number 4461146) was prepared, and 19.6 μl was dispensed into each well.

[0230] The plate was sealed with an adhesive film and then subjected to operation using a CFX96 RT-PCR (Bio-Rad). The program started with an equilibration step at 20°C for 1 minute, followed by a temperature gradient of 0.5°C / 30 seconds from 20°C to 95°C, and fluorescence was recorded every 0.5°C. Fluorescence was recorded using the FRET channel. All experiments were performed in triplicate. The curves were plotted and analyzed to determine the melting temperature by using the Boltzmann sigmoid equation with GraphPad Prism software. The graphical representation of the data in the following table is shown in Figure 17.

[0231] TIFF2025517992000044.tif81170

[0232] Example 32. pppApG Primer Synthesis The reaction was carried out in the presence of 2 mM MnCl 2 or 5 mM MgCl 2 which are the optimal catalytic ion concentrations for RNA synthesis by DENV2 RdRp (Selisko et al., 2006). The discrimination of AT-9010 against GTP was measured in four independent reactions and similar results to those shown in Figures 19B and 21A, 21B, 21C, and 21D were obtained.

[0233] Example 33. MTase Activity Assay The transfer of tritiated methyl from [3H]-SAM onto the RNA substrate was monitored by filter binding assay (FBA) and performed according to the previously described method (Paesen et al., 2015). The recombinant 2'-O-methyltransferase domain of non-structural protein NS5 from dengue serotype 2 corresponding to residues 1 - 296 of NS5 (DENV NS5 MTase) was expressed and purified as previously described (Egloff et al., 2002). FBA was carried out in a reaction mixture [40 mM Tris-HCl (pH 8.0), 1 mM DTT, 2 μM SAM and 0.1 μM 3H-SAM (Perkin Elmer)] in the presence of 0.7 μM mGpppAC4 synthetic RNA and DENV2 NS5MTase protein (500 nM). The enzyme was first mixed with the compound (0.5 μM - 1000 μM) suspended in water and then incubated at 30 °C before adding the RNA substrate and SAM. The reaction mixtures were stopped after 30 minutes by diluting them 10-fold with ice-cold water. Samples were transferred to diethylaminoethyl (DEAE) filter mats (Perkin Elmer) using a Filtermat Harvester (Packard Instruments). The RNA retaining mats were washed twice with 10 mM ammonium formate pH 8.0, twice with water and once with ethanol. They were immersed in scintillation fluid (Perkin Elmer) and the 3H-methyl transfer onto the RNA substrate was measured using a Wallac MicroBeta TriLux liquid scintillation counter (Perkin Elmer). IC 50 In the measurement, the values were normalized using the following equation and fitted with Prism (GraphPad software): Y = 100 / (1 + ((X / IC 50 ))). IC Hillslope is defined as the inhibitor compound concentration that causes a 50% decrease in enzyme activity. The data curve can be found in Figure 18. 50

[0234] Example 34. Incorporation of nucleotide analogs into RNA ​DENV NS5 was pre-incubated with annealed P10 / T20 RNA for 10 minutes at 30°C in an assembly buffer containing 20 mM HEPES pH 7.5, 10% glycerol, 5 mM MgCl 2

[0235] The incorporation of multiple nucleotides was tested as follows and the results are shown in FIGS. 19A, 21, 22, and 23. The reaction was initiated by adding AT-9010 with either all four NTPs or only UTP, ATP, and CTP. The final concentrations were 1 μM NS5, 0.25 μM P10 / T20, 100 μM each NTP, and 10 μM - 625 μM AT-9010 in a final buffer containing 20 mM HEPES pH 7.5, 15% glycerol, 5 mM MgCl 2

[0236] The incorporation of single nucleotides was tested as follows. The reaction was initiated by adding the corresponding NTP or analog. The final concentrations were 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 MgCl 2

[0237] The reactions were quenched at the indicated time points with 3 volumes of FBD stop solution (formamide, 10 mM EDTA) and analyzed on a 20% acrylamide-bisacrylamide (19:1) 7 M urea sequencing gel. The RNA products were visualized using a Typhoon FluorImager and then analyzed and quantified using ImageQuant software.

[0238] Example 35. Crystallography and Modeling Studies Crystallographic images generated by the protocol described in this specification are shown in FIGS. 20A and 20B. The construct used for crystallization was the same as that described in (Barral et al., 2013), having a cleavable hexahistidine-thioredoxin tag at the N-terminus of the DENV3 NS5 coding sequence (amino acid regions 1-277) in plasmid pMcox20A. Transformed E. coli T7 expression cells (New England BioLabs) were cultured in LB medium supplemented with 100 μg / mL ampicillin at 37 °C until the OD600nm reached 0.6 - 0.8. Expression of the recombinant protein was induced by the addition of 100 μM IPTG, and further incubation was continued at 17 °C for 16 hours. Cells were harvested by centrifugation at 8000g 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) by using TALON™ Superflow™ cobalt-based IMAC resin (Cytiva) for affinity chromatography. The final size exclusion chromatography step was performed using a Superdex S200 HiLoad 16 / 60 column with a buffer containing 20 mM HEPES pH 7.5, 200 mM NaCl, 10% glycerol and 2 mM DTT.

[0239] Crystallization conditions were adopted 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% PEG8000, 200 mM NaCl, 20 mM trisodium citrate, 100 mM Tris pH 8.5). Crystals grew in 4 days and were soaked overnight in 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.

[0240] Data collection and diffraction data were collected at the Soleil synchrotron. The original native data set was collected at Proxima2. The data set was processed and analyzed with 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 previously observed, the crystal asymmetric unit is composed of two chains, but the additional positive density blobs seen in both chains are not equivalent. In one chain, the extra density corresponds to a single molecule of SAM, and in the other chain, the positive density corresponds to one molecule of SAH and one molecule of AT-9010. The model was built using COOT (Emsley and Cowtan, 2004) and refined at 1.90 Å using PHENIX (Adams et al., 2010). The structure was checked according to MOLPROBITY (Chen et al., 2010) and confirmed to have good stereochemistry. Statistics for data collection and refinement are listed in Table 21. Structure analysis and figures were performed using UCSF CHIMERA (Pettersen et al., 2004).

[0241] TIFF2025517992000045.tif68170

[0242] TIFF2025517992000046.tif208170

[0243] Example 36. Synthesis of Compound 1 Compound 1 (the free base form of Compound 2) can be synthesized, for example, by reacting a compound of the formula of Intermediate A with a compound of the formula of Intermediate B and an activator to form a compound of the formula of Intermediate C, and then reacting this with phenol to form Compound 1:

Chemical formula

[0244] By reacting the compounds of the formulas of Intermediate A and Intermediate B with an activator and a trialkylamine base, S P phosphoroamidate is preferentially formed (described in International Publication No. WO 2022 / 040473 assigned to Atea Pharmaceuticals).

[0245] As used herein, "activator" is a uronium-based peptide coupling reagent, 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-tetramethylaminium tetrafluoroborate (TBTU), and 1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU).

[0246] As used herein, "trialkylamine base" is an amine base having 3 C 1~6 alkyl groups, including but not limited to triethylamine or diisopropylethylamine.

[0247] As used herein, "pyridine base" is an optionally substituted pyridine molecule, including but not limited to pyridine, 2,6-dimethylpyridine (lutidine), or 2,4,6-trimethylpyridine (collidine).

[0248] S PWhen the phosphoramidate has an electron-withdrawing substituent on the aryl ester (Intermediate C), the SP compound can be further reacted. S P React the phosphoramidate (Intermediate C) with phenol and a base to form R P By forming the phosphoramidate (Compound 1), the stereochemistry of phosphorus can be inverted.

[0249] Synthesis of Intermediate A Intermediate A can be synthesized by multi-step substitution of phosphorus chloride (Intermediate E), followed by reductive deprotection of the benzyl protecting group.

Chemical Structure

[0250] In certain embodiments, the electron-withdrawing group on the phenol of Intermediate A is an ortho-fluoro substituent (Intermediate A-1):

Chemical Structure

[0251] In certain embodiments, Intermediate A has one or more electron-withdrawing substituents on the phenol. Examples of electron-withdrawing substituents include, but are not limited to, fluorine, chlorine, cyano, azide, nitro, trifluoromethyl, difluoromethyl, fluoromethyl, ester, and amide. In certain embodiments, Intermediate A is used in the form of a salt. In certain embodiments, Intermediate A is used as a dihydroquinine salt.

[0252] Example 37. Stereoinversion Reaction of Phenol

Chemical Structure

[0253] Amine Base The formation of Compound 1 by the reaction of Intermediate C with phenol can be carried out using a base such as an amine base, for example, a guanidine base. Examples of guanidine bases include Bases A, B, C, D, E, F, G, and J. Bases A, C, D, and J provided the highest yields of Compound 1. Intermediate C-1 was used as the model substrate for the reaction.

Chemical formula

[0254] TIFF2025517992000052.tif241170TIFF2025517992000053.tif111170

[0255] Activated phenol leaving group In certain embodiments, Compound 1 is S P Phosphoroamidate Intermediate C, for example, Intermediate C-1, C-2, C-3, C-4, C-5, C-6, C-7, and C-8, is prepared. Intermediates C-1 to C-8 have different electron-withdrawing groups on the phenol. These compounds can be reacted with phenol and a base to form Compound 1:

Chemical formula

[0256] TIFF2025517992000057.tif86170

[0257] TIFF2025517992000058.tif77170

[0258] TIFF2025517992000059.tif150170

[0259] Example 38. Synthesis of Compound 1 Synthesis procedure of Intermediate A-1

Chemical formula

[0260] Dichloromethane (2.0 L, 10 volumes) was charged into a three-necked flask at 10 °C. The flask was evacuated three times and refilled with nitrogen gas. Phosphorus oxychloride (200 g, 1.0 equivalent) was charged into the flask, and then the mixture was cooled to -55 °C. Dichloromethane (200 mL, 1 volume), benzyl alcohol (141 g, 1 equivalent), and triethylamine (139 g, 1.05 equivalents) were added to the mixing flask at 10 °C, and then the resulting mixture was charged into the phosphorus oxychloride solution over 40 minutes under nitrogen protection. The reaction mixture was stirred at -55 °C for 1 hour, during which a white solid precipitated. HPLC analysis of a sample of the reaction mixture derivatized with benzylamine indicated that the reaction was apparently 99.6% complete. L-Alanine isopropyl ester hydrochloride (219 g, 1 equivalent) was charged into the reaction mixture, followed by the addition of triethylamine (271 g, 2.05 equivalents) over 30 minutes.

[0261] The resulting reaction mixture was stirred for 1 to 2 hours, and then the consumption of the first intermediate was analyzed by HPLC analysis of a sample derivatized with benzylamine. The reaction was apparently 97.7% complete.

[0262] Dichloromethane (400 mL, 2 volumes), 2-fluorophenol (146 g, 1 equivalent), and triethylamine (139 g, 1.05 equivalents, added over 20 minutes) were combined in a mixing flask at 10 °C and then added to the reaction mixture over 43 minutes. The batch was warmed from -55 °C to 10 °C over a 3-hour period and then stirred for 2 to 4 hours. HPLC analysis of the sample derivatized with benzylamine showed that the area of the second intermediate was 2.0%. The batch was filtered and the filter cake was washed with dichloromethane (400 mL, 2 volumes). Water (600 mL, 3 volumes) was charged to the filtrate and the mixture was stirred at 10 °C for 5 minutes. The mixture was allowed to settle and the layers were separated. Hydrochloric acid in water (3.7% w / w, 1 L, 5 volumes) was charged to the organic matter and the mixture was stirred at 10 °C for 5 minutes. The mixture was allowed to settle and the layers were separated. Sodium bicarbonate in water (5% w / w, 1 L, 5 volumes) was charged to the organic matter 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 volumes) was charged to the organic matter 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 charged to the organic matter 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 at 45 °C under vacuum to a volume of approximately 600 mL (3 volumes). Isopropanol (600 mL, 3 volumes) was charged to the mixture and this was again concentrated to approximately 600 mL (3 volumes). Isopropanol (600 mL, 3 volumes) was charged to the mixture, the mixture was adjusted to 25 °C, and a sample was taken to quantify the amount of residual dichloromethane (0.25% remaining).

[0263] Isopropanol (1.9 L, 9.5 volumes) was charged to the mixture, followed by dihydroquinine (DHQ, 387 g, 0.908 equivalents). The mixture was stirred until a clear solution was formed. Palladium on carbon (10% w / w, KF = 63%, 13.7 g) was charged to the mixture. The resulting suspension was degassed twice with nitrogen and then three times with hydrogen. The mixture was stirred at 25 °C for 18 to 20 hours under 1 atm of hydrogen gas. Analysis of the sample by HPLC showed that 0.05% of the third intermediate remained.

[0264] The suspension was filtered and the cake was rinsed with isopropanol (100 mL, 0.5 volume). The solvent was distilled from the mixture to about 1200 mL (6 volumes) under vacuum at 55 °C, and then acetonitrile (1.5 L, 7.5 volumes) was charged to the mixture such that a suspension was provided. The distillation and acetonitrile addition were repeated two more times. A sample of the resulting suspension was diluted with N-methylpyrrolidone and analyzed to confirm residual isopropanol (0.7% detected). The suspension was stirred at 80 °C to 90 °C for 1 hour to 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 hours to 3 hours. The suspension was filtered and the cake was washed with cold acetonitrile (400 mL, 2 volumes, 5 °C). The cake was dried at 55 °C for 18 hours to 20 hours without vacuum to provide 591.7 g of Intermediate A-1 as a white powder (AUC purity 97.9%, yield 71.8% through four steps).

[0265] Characteristic evaluation data of Intermediate A-1: 11H NMR (400 MHz, DMSO) δ 12.50 (s, 1H), 8.75 (d, J = 4.5 Hz, 1H), 7.96 (d, J = 9.2 Hz, 1H), 7.70 - 7.48 (m, 3H), 7.41 (dd, J = 9.2, 2.5 Hz, 1H), 7.12 (dd, J = 11.0, 8.2 Hz, 1H), 7.00 (t, J = 7.5 Hz, 1H), 6.97 - 6.86 (m, 1H), 6.59 (s, 1H), 6.05 (s, 1H), 4.77 (dq, J = 12.5, 6.2 Hz, 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 = 6.4 Hz, 1H), 1.97 (d, J = 36.8 Hz, 3H), 1.87 - 1.61 (m, 2H), 1.38 (t, J = 11.8 Hz, 1H), 1.33 - 1.18 (m, 2H), 1.16 (d, J = 6.6 Hz, 3H), 1.09 (dd, J = 6.2, 1.2 Hz, 6H), 0.75 (t, J = 7.3 Hz, 3H). 1313C NMR (101 MHz, DMSO) δ 174.69 (d, J = 5.9 Hz), 158.27 (s), 154.97 (s), 152.58 (d, J = 6.7 Hz), 147.78 (s), 146.04 (s), 144.19 (s), 142.55 - 142.06 (m), 131.72 (s), 126.16 (s), 124.38 (d, J = 3.4 Hz), 122.73 (s), 122.67 - 122.04 (m), 119.39 (s), 116.16 (d, J = 18.9 Hz), 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 = 2.3 Hz), 21.21 (d, J = 5.4 Hz), 17.74 (s), 11.86 (s). 31 31P NMR (162 MHz, DMSO) δ 1.04 (s). 19 19F NMR (377 MHz, DMSO) δ -132.65 (s).

[0266] Step 2: Coupling reaction [Chemical formula]

[0267] Dichloromethane (1.33 L, 20 volumes), Intermediate B (50.0 g, 1.0 equivalent, 0.160 mol), the dihydroquinine salt of Intermediate A (222 g, 2.2 equivalents), and (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (82.3 g, 1.2 equivalents) were charged under nitrogen into a 2 L three-necked flask equipped with a reflux condenser and a thermometer.

[0268] The resulting suspension was heated to 30 °C to 35 °C. 2,6-Lutidine (20.6 g, 1.2 equivalents) was charged to the reaction mixture over 2 hours, and the suspension gradually dissolved during the addition. The mixture was stirred at 30 °C to 35 °C for 5 to 6 hours to provide a clear solution. The reaction product was sampled and analyzed by HPLC, which showed that the residual ratio of Intermediate B was less than 5%. The solution yield calculated by assay of the sample was 64.2 g (yield 66.9%). The reaction mixture was cooled to 0 °C to 10 °C over 1 hour.

[0269] The mixture was charged with water (500 mL, 10 volumes) and the batch was stirred for 2 minutes. Stirring was stopped and after allowing the batch to settle in layers, the aqueous layer was removed. Hydrochloric acid aqueous solution (20% w / w, 133 g, 4.5 equivalents) was charged over 1 hour during which dihydroquinine salt precipitated. The solution was filtered and the filter cake was rinsed with dichloromethane (50 mL, 1 volume) and the rinse was combined with the filtrate. Hydrochloric acid aqueous solution (7% w / w, 167 g, 2.0 equivalents) was charged 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 organic matter was washed three times with sodium hydrogen carbonate aqueous solution (5% w / w, 500 g, 1.9 equivalents) and then with water (500 mL, 10 volumes). The assay yield of the resulting organic layer was 59.4 g (61.9%). The organic layer was concentrated under vacuum at a temperature of 30 °C to 50 °C to a total volume of approximately 200 mL (4 volumes). Toluene (500 mL, 10 volumes) was charged to the mixture and the mixture was concentrated under vacuum at a temperature of 40 °C to 55 °C to a total volume of approximately 250 mL (5 volumes). Toluene addition and concentration were repeated one more time. Ethyl acetate (250 mL, 5 volumes) was charged to the batch and the mixture was heated to 65 °C to 75 °C. While stirring, the mixture was cooled to 5 °C over 3 hours during which a crystalline solid formed. The batch was held for 4 hours and then filtered to give a yellow crystalline solid. This solid and ethyl acetate (250 mL, 5 volumes) were charged to a flask and heated to 65 °C to 75 °C to give a clear solution. The solution was cooled to 5 °C over 3 hours such that a crystalline solid formed. The batch was held at 5 °C for 4 hours and then filtered to give a yellow solid. The yellow solid was dried at 45 °C for 15 to 16 hours to give Intermediate C-1 (79 g, corrected yield 56.2%) as a yellow crystalline solid.

[0270] Characteristic evaluation data of Intermediate C-3: 11H NMR (400 MHz, DMSO) δ 7.80 (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, 10.4 Hz, 1H), 6.11 - 5.91 (m, 3H), 5.78 (d, J = 6.4 Hz, 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). 19 19F NMR (377 MHz, DMSO) δ -131.41 (s), -159.74 (s). 31 31P NMR (162 MHz, DMSO) δ 4.42 (s). MS(ES+): Calculated value: 600.2; Measured value: 600.3

[0271] Step 3: Aryloxide substitution

Chemical formula

[0272] Acetonitrile (255 mL, 8.5 volumes), dimethyl sulfoxide (45 mL, 1.5 volumes), Intermediate C-1 (30 g, 0.05 mol, 1.0 equivalent), phenol (94.1 g, 20.0 equivalents), 1,1,3,3-tetramethylguanidine (17.3 g, 3.0 equivalents) were charged into a 350 mL flask equipped with a thermometer under nitrogen. The batch was heated to 30 °C for 22 hours while stirring.

[0273] The reactants were sampled and it was found that the remaining intermediate C-1 was less than 2%. The reaction mixture was cooled to 0 °C - 10 °C over 1 hour and poured into a reactor containing water (600 mL, 20 volumes) while stirring the batch. Methyl-tert-butyl ether (600 mL, 20 volumes) was charged to the mixture. Aqueous hydrochloric acid solution (20% w / w, 90 g, 10 equivalents) was charged over 0.5 hour while maintaining the temperature of the batch at 0 °C - 10 °C so that the pH value became 3 - 4. The mixture was stirred for an additional 2 minutes, stirring was stopped, and the aqueous and organic layers were separated, and the aqueous layer was collected. Water (300 mL, 10 volumes) was charged to the organic layer, and then aqueous hydrochloric acid solution (20% w / w, 32 g, 3.5 equivalents) was charged over 0.5 hour while maintaining the temperature of the batch at 0 °C - 10 °C.

[0274] The mixture was stirred for an additional 2 minutes, stirring was stopped, and the aqueous and organic layers were separated, and the aqueous layer was collected. The two aqueous layers were combined and cooled to 0 °C - 10 °C. Aqueous sodium hydrogen carbonate solution (5% w / w, 85 g, 1.0 equivalent) was added over 0.5 hour to adjust the pH to 8 - 9, and then dichloromethane (300 mL, 10 volumes) and water (300 mL, 10 volumes) were added. The mixture was stirred for 2 minutes, precipitated into two phases, and the aqueous phase was removed. The resulting organic matter was concentrated under vacuum to a volume of about 60 mL (2 volumes) at 30 °C - 40 °C.

[0275] Acetone (300 mL, 10 volumes) was charged to the mixture. The batch was concentrated under vacuum to a volume of about 60 mL (2 volumes) at 30 °C - 40 °C. Acetone (180 mL, 6 volumes) was charged to the mixture to provide a clear solution, which was cooled to 0 °C - 5 °C. Aqueous hydrochloric acid solution (36.5% w / w, 10 g, 2.0 equivalents) was charged all at once, and the mixture was stirred at 0 °C - 5 °C for 12 hours. The resulting suspension was filtered, and the recovered solid was dried to provide the bis hydrochloride salt of Compound 1 (19.2 g, yield 59%) as an off-white solid.

[0276] Characteristic evaluation data of the bis hydrochloride salt of Compound 1: 11H NMR (400 MHz, DMSO) δ 9.68 (d, J = 4.7 Hz, 1H), 8.14 (s, 3H), 7.36 (t, J = 7.9 Hz, 2H), 7.18 (dd, J = 17.8, 7.9 Hz, 3H), 6.11 (d, J = 18.6 Hz, 2H), 4.84 (hept, J = 6.2 Hz, 1H), 4.47 (dd, J = 10.4, 5.9 Hz, 1H), 4.43 - 4.2, 1H), 3.77 (s, 1H), 3.15 (d, J = 4.8 Hz, 3H), 1.28 - 1.07 (m, 12H). 13 13C NMR (101 MHz, DMSO) δ 173.14 (d, J = 4.3 Hz), 153.46 (s), 151.11 (d, J = 6.0 Hz), 149.58 (s), 130.03 (s), 124.98 (s), 120.62 (d, J = 4.6 Hz), 112.08 (s), 102.03 (s), 100.24 (s), 80.59 (s), 72.00 (d, J =18.2 Hz), 68.43 (s), 65.85 (s), 50.42 (s), 40.49 (d, J = 13.9 Hz), 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 = 7.2 Hz), 20.08 (d, J = 7.2 Hz), 17.15 (s), 16.91 (s). 19 19F NMR (377 MHz, DMSO) δ -160.09 (s). 31 31P NMR (162 MHz, DMSO) δ 3.66 (s). MS(ES+): Calculated value: 582.22; Measured value: 582.35

[0277] General synthesis of Intermediates C-1 to C-8

Chemical Structure

[0278] General procedures for intermediates A-2, A-3, and A-7: 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) at -60 °C under nitrogen. After the addition, the reaction mixture was stirred at -60 °C for 1 hour.

[0279] L-Alanine isopropyl ester hydrochloride (16.76 g, 0.1 mol) was added to the reaction mixture, 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. The substituted phenol, TEA (10.62 g, 0.105 mol), and DCM (20 mL) were added dropwise to the reaction mixture at -60 °C.

[0280] After the addition, the reaction mixture was stirred overnight until it reached room temperature and then quenched with water. The organic layer was dried over Na 2 SO 4 filtered, and then the solvent was removed in vacuo. The residue was purified by silica gel column chromatography to obtain a colorless oil (benzyl-protected intermediates A-2, A-3, and A-7).

[0281] A colorless oil (0.1 mol) and isopropanol (200 mL, 5 volumes) 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. 5% wet Pd / C (60% water by KF, 5% w / w on a dry basis) was charged into this solution, and hydrogenolysis was carried out at 20 °C - 25 °C for 18 hours - 20 hours using hydrogen at 1 atm. When it was shown by IPC 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 °C - 30 °C for 2 hours. The mixture was filtered, and the cake was washed with isopropanol (40 mL). The filtrate was concentrated under a vacuum of less than 0.09 MPa at 50 °C - 60 °C to obtain a crude product.

[0282] Isopropyl acetate (200 mL, 5 volumes) was added, and the mixture was concentrated again at 50 °C to 60 °C under a vacuum of less than 0.09 MPa. This step was repeated once more with isopropyl acetate (5 volumes). Fresh isopropyl acetate (200 ml, 5 volumes) was added to the residue, and the mixture was stirred at 80 °C to 90 °C for 2 to 3 hours to obtain a clear solution. Then, it was cooled to 0 °C to 10 °C at a cooling rate of 20 °C / 1 h and stirred at this temperature for 2 to 3 hours. The solid was collected by filtration, washed with cold isopropyl acetate (40 mL, 1 volume) to obtain a wet cake, which was dried at 50 °C for 18 hours without vacuum to obtain the dihydroquinine salt of intermediate A-2, A-3 or A-7 as an off-white solid.

[0283] Characteristic evaluation data of intermediate A-2 (dihydroquinine salt):

Chemical formula

[0284] Characteristic evaluation data of Intermediate A-3 (dihydroquinine salt):

Chemical formula

[0285] Characteristic evaluation data of Intermediate A-7 (dihydroquinine salt):

Chemical formula

[0286] General procedure for intermediates A-4 and A-5: 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) at -60 °C under nitrogen. After addition, the reaction mixture was stirred at -60 °C for 1 h. L-alanine isopropyl ester hydrochloride (16.76 g, 0.1 mol) was added to the reaction mixture, 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 h. The substituted phenol corresponding to the phenyl substitution (0.1 mol), TEA (10.62 g, 0.105 mol) and DCM (20 mL) were added dropwise to the reaction mixture at -60 °C. After addition, the reaction mixture was stirred overnight at room temperature and then quenched with water. The organic layer was washed with Na 2 SO 4It was dried above, filtered, and then the solvent was removed in vacuo. The residue was purified by silica gel column chromatography to obtain a colorless oil.

[0287] A colorless oil (0.1 mol) and isopropanol (200 mL, 5 volumes) 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. 5% wet Pd / C (60% water by KF, 5% w / w on a dry basis) was charged into this solution, and hydrogenolysis was carried out at 20 °C to 25 °C for 18 hours to 20 hours using hydrogen at 1 atm. When it was shown by IPC 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 °C to 30 °C for 2 hours. The mixture was filtered, and the cake was washed with isopropanol (40 mL).

[0288] The filtrate was concentrated at 50 °C to 60 °C under a vacuum of less than 0.09 MPa to obtain a crude product. Toluene (200 mL, 5 volumes) was added, and the mixture was concentrated again at 50 °C to 60 °C under a vacuum of less than 0.09 MPa. This step was repeated two more times with 5 volumes of toluene, and the residue was obtained as an oil. The residue was used in the next step without purification.

[0289] Synthesis procedure of intermediate A-6: A mixture of 3-chlorophenol (12.86 g, 0.1 mol) and TEA (10.62 g, 0.105 mol) was added dropwise at -60 °C under nitrogen to a solution of phosphorus oxychloride (15.3 g, 0.1 mol) in DCM (150 mL). After the addition, the reaction mixture was stirred at -60 °C for 1 hour. L-alanine isopropyl ester hydrochloride (16.76 g, 0.1 mol) was added to the reaction mixture, and then TEA (20.74 g, 0.205 mol) was added dropwise at -60 °C. The reaction mixture was stirred overnight to room temperature. It was filtered, then the solvent was removed in vacuo, MTBE (128 mL, 10 volumes) was added, the mixture was stirred at 20 °C for 1 hour, filtered, and the filtrate was stirred at 0 °C. Then, H 2Citric acid monohydrate (42 g, 0.2 mol) in O (200 mL) was added dropwise to the filtrate at 0 °C. The reaction mixture was stirred overnight to room temperature. The reaction mixture was allowed to stand for phase separation. The organic layer was stirred with H 2 O (100 mL), then allowed to stand for phase separation. DIPEA (12.9 g, 0.1 mol) was added to the organic phase. The mixture was concentrated under vacuum at 40 °C to less than 0.09 MPa to obtain the crude product. The purified product was obtained by preparative HPLC.

[0290] Characteristic evaluation data of Intermediate A-6 (diisopropylethylamine salt)

Chemical formula

[0291] Synthesis procedure of Intermediate A-8 To a solution of 4-nitrophenyldichlorophosphate (25.6 g, 0.1 mol) and L-alanine isopropyl ester hydrochloride (16.76 g, 0.1 mol) in DCM (256 mL, 10 volumes) was added dropwise TEA (20.74 g, 0.205 mol) at -10 °C under nitrogen. After the addition, the reaction mixture was stirred overnight to room temperature. The mixture was concentrated under vacuum at 40 °C to less than 0.09 MPa. MTBE (256 mL, 10 volumes) was added and the mixture was stirred at 20 °C for 1 hour. It was filtered and the filtrate was stirred at 0 °C. Tetrabutylammonium hydroxide (208 g, 0.2 mol, H 225% w / w of O was added dropwise to the filtrate at 0 °C. After the addition, the reaction mixture was stirred overnight until it reached room temperature. The reaction mixture was allowed to stand for layer separation. The aqueous layer was stirred with IPAC (200 mL) and then allowed to stand for layer separation. The IPAC layer was stirred with an aqueous HCl solution (200 mL, 1 mol / L) and then allowed to stand for layer separation. The organic layer was dried over Na 2 SO 4 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 volumes) was added, and the mixture was stirred at 20 °C for 1 hour and then filtered. The wet cake was dried at 60 °C for 16 hours without applying vacuum. Intermediate A-8 was obtained.

[0292] Characteristic evaluation data of intermediate A-8 (dihydroquinine salt)

Chemical formula

[0293] General procedures for Intermediates C-1 to C-5 and C-7 Salts of Intermediates A-1 to A-8 (1.7 equivalents), Intermediate B (6.25 g, 20 mmol, 1.0 equivalent), diisopropylethylamine (2.6 g, 20 mmol, 1.0 equivalent) and HATU (12.9 g, 34 mmol, 1.7 equivalents) were added to 188 mL of dichloromethane (30 volumes). The mixture was heated to 40 °C and stirred under nitrogen for 18 hours. The reaction was monitored by TLC and HPLC.

[0294] After completion of the reaction, the reaction mixture was cooled to 0 °C - 10 °C, washed twice with 2N HCl (100 mL) and with 5% aqueous sodium hydrogen carbonate solution (100 mL), and the separated organic phase was dried over Na 2 SO 4 and filtered. The filtrate was concentrated under vacuum at 40 °C - 45 °C to obtain a residue. The residue was purified by silica gel column chromatography to obtain a crude product. The crude product was recrystallized from EA (30 mL), and the precipitate was dried at 45 °C - 50 °C for 16 hours without vacuum to obtain Intermediates C-1 to C-5 or C-7.

[0295] Characteristic evaluation data of Intermediate C-2

Chemical Structure

[0296] Characteristic evaluation data of Intermediate C-3:

Chemical Structure

[0297] Characteristic evaluation data of Intermediate C-4:

Chemical Structure

[0298] Characteristic evaluation data of Intermediate C-5: 1 1H NMR (400 MHz, DMSO) δ 7.79 (s, 1H), 7.75 - 7.62 (m, 2H), 7.62 - 7.53 (m, 2H), 7.27 (s, 1H), 6.16 (dd, J = 12.7, 10.2 Hz, 1H), 6.10 - 5.93 (m, 3H), 5.76 (d, J = 6.8 Hz, 1H), 4.81 (dt, J = 12.5, 6.2 Hz, 1H), 4.53 - 4.27 (m, 3H), 4.08 (dd, J = 13.2, 6.3 Hz, 1H), 3.83 (ddd, J = 17.1, 7.2, 2.9 Hz, 1H), 2.88 (s, 3H), 1.23 (d, J = 7.1 Hz, 3H), 1.15 - 1.04 (m, 9H).

Chemical formula

[0299] Characteristic evaluation data of Intermediate C-7: [Chemical formula] 1 H NMR (400 MHz, DMSO) δ 7.80 (s, 1H), 7.22 (ddd, J = 20.8, 13.3, 7.0 Hz, 5H), 6.23 - 5.86 (m, 4H), 5.73 (d, J = 7.1 Hz, 1H), 4.81 (dt, J = 12.5, 6.2 Hz, 1H), 4.54 - 4.20 (m, 3H), 4.06 (dd, J = 12.8, 5.8 Hz, 1H), 3.90 - 3.67 (m, 1H), 2.89 (s, 3H), 1.21 (t, J = 6.2 Hz, 3H), 1.15 - 1.03 (m, 9H). MS(ES+): Calculated value: 600.21; Measured value: 600.36

[0300] Synthesis of Intermediate C-6 [Chemical formula]

[0301] Characteristic evaluation data of Intermediate C-6: [Chemical formula] 11H NMR (400 MHz, DMSO) δ 7.80 (s, 1H), 7.39 (t, J = 8.1 Hz, 1H), 7.33 (s, 1H), 7.26 (d, J = 7.9 Hz, 2H), 7.22 - 7.16 (m, 1H), 6.23 - 5.89 (m, 4H), 5.76 (d, J = 6.8 Hz, 1H), 4.81 (dt, J = 12.5, 6.2 Hz, 1H), 4.53 - 4.23 (m, 3H), 4.16 - 4.00 (m, 1H), 3.81 (ddd, J = 17.1, 7.2, 2.9 Hz, 1H), 2.88 (s, 3H), 1.22 (d, J = 7.1 Hz, 3H), 1.17 - 1.00 (m, 9H). MS(ES+): Calculated value: 616.18; Measured value: 616.15

[0302] Synthesis of Intermediate C-8

Chemical Structure

[0303] Characteristic evaluation data of Intermediate C-8:

Chemical Structure

[0304] This specification is described with reference to embodiments of the present invention. However, it will be understood by those skilled in the art that various modifications and changes can be made without departing from the scope of the present invention as set forth in the appended claims. Therefore, this specification is to be considered in an illustrative rather than a limiting sense, and it is intended that all such modifications be included within the scope of the present invention.

Claims

1. Approximately 700 mg to approximately 1000 mg of compound 1 in a pharmaceutically acceptable carrier. 【Chemistry 1】 A pharmaceutical composition for treating dengue virus infection in a person requiring treatment for dengue virus infection, comprising a pharmaceutically acceptable salt thereof or the same.

2. The aforementioned compound, 【Chemistry 2】 The pharmaceutical composition according to claim 1.

3. The pharmaceutical composition according to claim 1, comprising approximately 700 mg to approximately 850 mg of the compound.

4. The pharmaceutical composition according to claim 1, wherein approximately 750 mg of compound 1 or a pharmaceutically acceptable salt thereof is administered.

5. The pharmaceutical composition according to claim 2, wherein approximately 750 mg of compound 2 or a pharmaceutically acceptable salt thereof is administered.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the compound is administered twice a day.

7. The pharmaceutical composition according to any one of claims 1 to 5, wherein the compound is administered three times a day.

8. The pharmaceutical composition according to any one of claims 1 to 5, wherein the compound is administered four times a day.

9. The pharmaceutical composition according to any one of claims 1 to 5, wherein approximately 750 mg of the compound is administered three times a day.

10. The pharmaceutical composition according to any one of Claim 2, wherein approximately 750 mg of compound 2 is administered three times a day.

11. The pharmaceutical composition according to any one of claims 1 to 5, wherein the compound is administered for four consecutive days.

12. The pharmaceutical composition according to any one of claims 1 to 5, wherein the compound is administered for five consecutive days.

13. The pharmaceutical composition according to any one of claims 1 to 5, wherein the compound is administered for six consecutive days.

14. The pharmaceutical composition according to any one of claims 1 to 5, wherein the compound is administered for seven consecutive days.

15. The pharmaceutical composition according to any one of claims 1 to 5, wherein the compound is administered for eight consecutive days.

16. The pharmaceutical composition according to any one of claims 1 to 5, wherein the human has dengue virus serotype 1.

17. The pharmaceutical composition according to any one of claims 1 to 5, wherein the human has dengue virus serotype 2.

18. The pharmaceutical composition according to any one of claims 1 to 5, wherein the human being has dengue virus serotype 3.

19. The pharmaceutical composition according to any one of claims 1 to 5, wherein the human being has dengue virus serotype 4.

Images