HK40117526A - A 5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-2-cyano-3,4-dihydroxytetrahydrofurane derivative as antiviral agent - Google Patents

Description

5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-cyano-3,4-dihydroxytetrahydrofuran derivatives as antiviral agents

This application is a divisional application of Chinese invention patent application (application date: February 17, 2021; application number: 202180014297.5 (international application number: PCT/US2021/018415); invention title: 5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-cyano-3,4-dihydroxytetrahydrofuran derivative as an antiviral agent).

Cross-references to related applications

This application claims priority to U.S. Provisional Patent Application No. 62/977,969, filed February 18, 2020, entitled “Antiviral Compounds,” the entire contents of which are incorporated herein by reference.

sequence list

This application includes a sequence list, which is submitted electronically in ASCII format and incorporated herein by reference in its entirety. The ASCII copy, created on February 17, 2021, is named 1307-WO-PCT_SL.txt and is 1,537 bytes in size.

Background Technology

Pneumoviridae viruses are negative-sense, single-stranded RNA viruses that cause many epidemic diseases in humans and animals. The Pneumoviridae family includes human respiratory syncytial virus (HRSV) and human metapneumovirus. Almost all children will have an HRSV infection by age two. HRSV is a leading cause of lower respiratory tract infections in infancy and childhood, with 0.5% to 2% of infections requiring hospitalization.

Currently, there is no vaccine available to prevent HRSV infection. The monoclonal antibody palizumab can be used for immunization, but its use is limited to high-risk infants, such as premature infants or those with congenital heart or lung diseases, and its general cost is often very high. Additionally, the nucleoside analog ribavirin has been approved as the only antiviral agent for treating HRSV infection, but its efficacy is limited. Therefore, antiviral treatments targeting pulmonary viruses are needed.

Older adults and those with chronic heart disease, lung disease, or immunosuppression are also at high risk of developing severe HRSV disease (http://www.cdc.gov/rsv/index.html). Specifically, patients with chronic respiratory diseases such as chronic obstructive pulmonary disease (COPD) are at high risk of developing acute respiratory exacerbations. Acute respiratory exacerbations are a major cause of morbidity, mortality, and reduced quality of life in patients with COPD (Frickmann, Eur. J. Microbiol. Immun. 2012 Sep 2(3):176-185).

About half to two-thirds of respiratory exacerbations in COPD patients are due to viral infections. Some common viral pathogens that cause such respiratory exacerbations include, but are not limited to, HRSV, human metapneumovirus (HMPV), and human rhinovirus (HRV). COPD patients with infectious exacerbations typically experience longer hospital stays and suffer greater lung damage than those with non-infectious exacerbations (Frickmann, Eur. J. Microbiol. Immun. 2012 Sep 2(3):176-185).

There is still a need for new antiviral agents that are effective and have acceptable toxicity properties for treating infections caused by pulmonary viral viruses, such as HRSV.

WO2015/069939, published on May 14, 2015, discloses compounds that can be used to treat infections caused by viruses of the Pneumoviridae family. Among other things, WO2015/069939 relates to compounds of the following formula or pharmaceutically acceptable salts thereof:

in:

^R1 is H or F;

^R2 is H or F;

^R3 is OH or F;

^R4 is CN, _C1 - _C4 alkyl, _C2 - _C4 alkenyl, _C2 - _C4 alkynyl, _C3 - _C4 cycloalkyl, azide, halogen, or _C1 - _C2 haloalkyl;

^R6 is OH;

^R5 selects the group consisting of the following items: H and

in:

n' is selected from 1, 2, 3, and 4;

^R8 is selected from _C1 - _C8 alkyl, -OC1 _{- C8} alkyl, benzyl, -O-benzyl, _-CH2 - _C3 - _C6 cycloalkyl, -O- _CH2 - _C3 - _C6 cycloalkyl and _CF3 ;

^R9 is selected from phenyl, 1-naphthyl, 2-naphthyl,

^R10 is selected from H and _CH3 ;

R ¹¹ is selected from H or C1 _{- C6} alkyl groups;

^R12 is selected from H, _C1 - _C8 alkyl, benzyl, _C3 - _C6 cycloalkyl and _-CH2 - _C3 - _C6 cycloalkyl.

Summary of the Invention

In one embodiment, this disclosure provides compounds of formula I:

Or its pharmaceutically acceptable salt.

In another embodiment, this disclosure provides a pharmaceutical formulation comprising a therapeutically effective amount of a compound of the disclosure or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

In another embodiment, this disclosure provides a method for treating or preventing pulmonary viral infection in a person in need, the method comprising administering to the person a therapeutically effective amount of a compound of the disclosure or a pharmaceutically acceptable salt thereof.

In another embodiment, this disclosure provides a method for treating or preventing infection with a Picornaviridae virus in a person in need, the method comprising administering to a person a therapeutically effective amount of a compound of this disclosure or a pharmaceutically acceptable salt thereof.

In another embodiment, this disclosure provides a method for treating or preventing infection with a Flaviviridae virus in a person in need, the method comprising administering to the person a therapeutically effective amount of a compound of this disclosure or a pharmaceutically acceptable salt thereof.

In another embodiment, this disclosure provides a method for treating or preventing infection with a Filoviridae virus in a person in need, the method comprising administering to the person a therapeutically effective amount of a compound of this disclosure or a pharmaceutically acceptable salt thereof.

In another embodiment, this disclosure provides a method for manufacturing a medicament for treating or preventing pulmonary viral infections in persons in need, characterized by using a compound of this disclosure or a pharmaceutically acceptable salt thereof.

In another embodiment, this disclosure provides a method for manufacturing a medicament for treating or preventing infection with a microribonucleoviridae virus in a person in need, characterized by using a compound of this disclosure or a pharmaceutically acceptable salt thereof.

In another embodiment, this disclosure provides a method for manufacturing a medicament for treating or preventing flaviviral infections in persons in need, characterized by using a compound of this disclosure or a pharmaceutically acceptable salt thereof.

In another embodiment, this disclosure provides a method for manufacturing a medicament for treating or preventing filoviridae virus infection in persons in need, characterized by using a compound of this disclosure or a pharmaceutically acceptable salt thereof.

In another embodiment, this disclosure provides the use of the compounds of this disclosure or pharmaceutically acceptable salts thereof for the manufacture of medicaments for the treatment or prevention of human pulmonaryviridae virus infections.

In another embodiment, this disclosure provides the use of the compounds of this disclosure or pharmaceutically acceptable salts thereof for the manufacture of medicaments for the treatment or prevention of human microribonucleoviridae virus infections.

In another embodiment, this disclosure provides the use of the compounds of this disclosure or pharmaceutically acceptable salts thereof for the manufacture of medicaments for the treatment or prevention of human flaviviridae virus infections.

In another embodiment, this disclosure provides the use of the compounds of this disclosure or pharmaceutically acceptable salts thereof for the manufacture of medicaments for the treatment or prevention of human filoviridae virus infections.

In another embodiment, this disclosure provides a compound of the present disclosure or a pharmaceutically acceptable salt thereof for the treatment or prevention of pulmonary viral infections in persons in need.

In another embodiment, this disclosure provides a compound of the present disclosure or a pharmaceutically acceptable salt thereof for the treatment or prevention of microribonucleovir infections in persons in need.

In another embodiment, this disclosure provides a compound of the present disclosure or a pharmaceutically acceptable salt thereof for the treatment or prevention of flaviviridae virus infection in a person in need.

In another embodiment, this disclosure provides a compound of the present disclosure or a pharmaceutically acceptable salt thereof for the treatment or prevention of filoviridae virus infection in a person in need.

In another embodiment, this disclosure provides a method for treating or preventing the exacerbation of respiratory symptoms caused by a viral infection in a person in need, the method comprising administering to a person a therapeutically effective amount of a compound of this disclosure or a pharmaceutically acceptable salt thereof, wherein the respiratory symptoms are chronic obstructive pulmonary disease.

In another embodiment, this disclosure provides a method for treating or preventing the exacerbation of respiratory symptoms caused by a viral infection in a person in need, the method comprising administering to a person a therapeutically effective amount of a compound of this disclosure or a pharmaceutically acceptable salt thereof, wherein the respiratory symptom is asthma.

In another embodiment, this disclosure provides a method for manufacturing a treatment or prevention of exacerbation of respiratory symptoms caused by a viral infection in a person in need, characterized by using a compound of the disclosure or a pharmaceutically acceptable salt thereof, wherein the respiratory symptom is chronic obstructive pulmonary disease.

In another embodiment, this disclosure provides a method for manufacturing a treatment or prevention of exacerbation of respiratory symptoms caused by a viral infection in a person in need, characterized by using a compound of this disclosure or a pharmaceutically acceptable salt thereof, wherein the respiratory symptom is asthma.

In another embodiment, this disclosure provides the use of the disclosed compounds or pharmaceutically acceptable salts thereof for manufacturing for the treatment or prevention of exacerbation of respiratory symptoms caused by viral infection in humans, wherein the respiratory symptoms are chronic obstructive pulmonary disease.

In another embodiment, this disclosure provides the use of the disclosed compounds or pharmaceutically acceptable salts thereof for the manufacture of a treatment or prevention of exacerbation of respiratory symptoms caused by a viral infection in a human being, wherein the respiratory symptoms are asthma.

In another embodiment, this disclosure provides the compounds of this disclosure or pharmaceutically acceptable salts thereof for the treatment or prevention of exacerbation of respiratory symptoms caused by a viral infection in a person in need, wherein the respiratory symptoms are chronic obstructive pulmonary disease.

In another embodiment, this disclosure provides compounds of the present disclosure or pharmaceutically acceptable salts thereof for the treatment or prevention of exacerbation of respiratory symptoms caused by viral infection in persons in need, wherein the respiratory symptoms are asthma.

In another embodiment, this disclosure provides compounds of the present disclosure or pharmaceutically acceptable salts thereof for use in medical therapies.

In another embodiment, this disclosure provides a method for preparing a compound of formula I-11:

The method includes making:

(i) Compounds of Formula I-7:

and

(ii) Compounds of formula I-12:

The reaction takes place in the presence of _NdCl₃ and tetrabutylammonium chloride; wherein R is a hydroxyl protecting group. In some embodiments, R is a benzyl group. In some embodiments, R is a silyl protecting group. In some embodiments, R is a tert-butyldimethylsilyl (TBS) group.

In another embodiment, this disclosure provides a method for preparing compounds of formula I-6:

The method includes making:

(i) Compounds of Formula I-7:

and

(ii) Compounds of formula I-5:

The reaction takes place in the presence of _NdCl₃ and tetrabutylammonium chloride; wherein R is a hydroxyl protecting group. In some embodiments, R is a benzyl group. In some embodiments, R is a silyl protecting group. In some embodiments, R is a tert-butyldimethylsilyl (TBS) group.

Attached Figure Description

Figure 1. Shows the measurement of intracellular triphosphate formation in NHBE in vitro with three donors of compound I and compound 6.

Figure 2. Shows the measurement of intracellular triphosphate formation in PBMCs of compounds having formula I, as well as compounds 2 and 6.

Figure 3 shows the pharmacokinetic data of compounds of formula I and compound 6 in cynomolgus monkeys.

Detailed Implementation

It should be understood in the following description that this disclosure is considered illustrative of the claimed subject matter and is not intended to limit the appended claims to the specific embodiments illustrated. The headings used throughout this disclosure are provided for convenience and should not be construed as limiting the claims in any way. Embodiments exemplified under any heading may be combined with embodiments exemplified under any other heading.

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.

I. Overview

This disclosure provides a 2',3'-hydroxy-4'-cyanonucleotide analog for treating viral infections such as pulmonaryviridae virus infections, viral infections, and other viral infections, including but not limited to micronucleotideviridae virus infections, flaviviridae virus infections, filoviridae virus infections, and other viral infections.

II. Definition

"The compounds disclosed herein" refers to compounds of formula I.

"Pharmaceutically effective amount" means the amount of the compound of this disclosure in a formulation or combination thereof that provides the desired therapeutic or pharmaceutical outcome.

"Pharmaceutical acceptable excipients" include, but are not limited to, any adjuvants, carriers, excipients, glidants, sweeteners, diluents, preservatives, dyes/colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers that have been approved by the U.S. Food and Drug Administration for acceptable use in humans or livestock.

As used herein, “treatment” or “treat” or “treating” refers to a method for achieving a beneficial or desired outcome. For the purposes of this disclosure, beneficial or desired outcomes include, but are not limited to, the reduction of symptoms and/or the attenuation of the severity of symptoms and/or the prevention of the worsening of symptoms associated with a disease or condition. In one embodiment, “treatment” or “treating” includes one or more of the following: a) suppressing a disease or condition (e.g., reducing one or more symptoms caused by a disease or condition, and/or attenuating the severity of a disease or condition); b) slowing or preventing the development of one or more symptoms associated with a disease or condition (e.g., stabilizing a disease or condition, delaying the worsening or progression of a disease or condition); and c) alleviating a disease or condition, such as causing the disappearance of clinical symptoms, improving disease status, delaying disease progression, improving quality of life, and/or prolonging survival.

"Prevention" refers to preventing or delaying the progression of clinical disease in patients with viral infections.

"Respiratory symptoms" refers to diseases or conditions such as respiratory infections caused by viral infections, allergic rhinitis, nasal congestion, rhinorrhea, perennial rhinitis, rhinitis, all types of asthma, chronic obstructive pulmonary disease (COPD), chronic or acute bronchoconstriction, chronic bronchitis, small airway obstruction, emphysema, chronic eosinophilic pneumonia, adult respiratory distress syndrome, exacerbations of airway hyperresponsiveness due to other drug therapies, pulmonary vascular disease (including pulmonary hypertension), acute lung injury, bronchiectasis, sinusitis, allergic conjunctivitis, idiopathic pulmonary fibrosis, or atopic dermatitis, especially asthma, allergic rhinitis, atopic dermatitis, or allergic conjunctivitis.

"Exacerbation of respiratory symptoms" refers to exacerbation induced by viral infection. Representative viral infections include, but are not limited to, respiratory syncytial virus (RSV), rhinovirus, and metapneumovirus.

As used herein, "therapeutic effective amount" or "effective amount" means an amount that effectively elicits the desired biological or medical response, including amounts of compounds sufficient to achieve such treatment of a disease when administered to a subject. Effective amounts will vary depending on the compound being treated, the disease and its severity, and factors such as age and weight. Effective amounts may include a range of amounts. As understood in the art, an effective amount may be one or more doses; that is, a single dose or multiple doses may be required to achieve the desired therapeutic endpoint. Effective amounts may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered if, in combination with one or more other agents, a desired or beneficial outcome can be achieved. The appropriate dose of any co-administered compound may optionally be reduced due to the combined effects of the compounds (e.g., additive or synergistic effects).

As used herein, “co-administration” means administering a unit dose of the compound disclosed herein before or after administering a unit dose of one or more adjunctive therapeutic agents, for example, administering the compound disclosed herein within seconds, minutes, or hours after administering one or more adjunctive therapeutic agents. For example, in some embodiments, a unit dose of the compound disclosed herein is administered first, followed by a unit dose of one or more adjunctive therapeutic agents within seconds or minutes. Alternatively, in other embodiments, a unit dose of one or more adjunctive therapeutic agents is administered first, followed by a unit dose of the compound disclosed herein within seconds or minutes. In some embodiments, a unit dose of the compound disclosed herein is administered first, followed by a unit dose of one or more adjunctive therapeutic agents several hours later (e.g., 1 hour to 12 hours). In other embodiments, a unit dose of one or more adjunctive therapeutic agents is administered first, followed by a unit dose of the compound disclosed herein several hours later (e.g., 1 hour to 12 hours). Co-administration of the compound disclosed herein with one or more adjunctive therapeutic agents generally means administering the compound disclosed herein and one or more adjunctive therapeutic agents simultaneously or sequentially, such that a therapeutically effective amount of each agent is present in the patient.

Pharmaceutically acceptable salts, hydrates, solvates, tautomers, polymorphs, and prodrugs of the compounds described herein are also provided. "Pharmaceutically acceptable" or "physiologically acceptable" means compounds, salts, compositions, dosage forms, and other substances that can be used to prepare pharmaceutical compositions suitable for veterinary or human use.

The compounds described herein can be prepared and/or formulated as pharmaceutically acceptable salts, or, where appropriate, as free bases. A "pharmaceutically acceptable salt" is a non-toxic salt of a compound in its free base form, possessing the desired pharmacological activity of a free base. These salts can be derived from inorganic acids, organic acids, or bases. For example, compounds containing basic nitrogen can be prepared as pharmaceutically acceptable salts by contacting the compound with an inorganic or organic acid. Non-limiting examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, octanoates, acrylates, formates, isobutyrates, hexanoates, heptanoates, propargylates, oxalates, malonates, succinates, octanoates, sebacic acid salts, fumarates, maleates, etc. Alkyne-1,4-diacidate, hexyne-1,6-diacidate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, methanesulfonate, propylsulfonate, benzenesulfonate, xylenesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, γ-hydroxybutyrate, glycolate, tartrate, and mandelate. A list of other suitable pharmaceutically acceptable salts can be found in Remington: The Science and Practice of Pharmacy, 21st edition, Lippincott Wiliams and Wilkins, Philadelphia, Pa., 2006.

Examples of pharmaceutically acceptable salts of ^{the compounds disclosed herein also include salts derived from suitable bases such as alkali metals (e.g., sodium, potassium), alkaline earth metals (e.g., magnesium), ammonium, and NX 4+} ₍ where X is a C ₁ -C ₄ alkyl group). Base addition salts, such as sodium or potassium salts, are also included.

Also provided are compounds described herein, or pharmaceutically acceptable salts, isomers, or mixtures thereof, wherein one to n hydrogen atoms attached to a carbon atom may be replaced by a deuterium atom or D, where n is the number of hydrogen atoms in the molecule. As is known in the art, a deuterium atom is a non-radioactive isotope of a hydrogen atom. Such compounds can increase resistance to metabolism and are therefore used to increase the half-life of compounds described herein, or pharmaceutically acceptable salts, isomers, or mixtures thereof, when administered to mammals. See, for example, Foster, “Deuterium Isotope Effects in Studies of Drug Metabolism”, Trends Pharmacol. Sci., 5(12):524-527 (1984). Such compounds are synthesized by methods well known in the art, for example by using starting materials in which one or more hydrogen atoms have been replaced by deuterium.

Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, such as ^2H , ^3H , ^11C , ^13C , ^14C , ^13N , ^15N , ^15O , ^17O , ^18O , ^31P , ^32P , ^35S , ^18F , ^36Cl , ^123I , and ^125I , respectively. Substitution with positron-emitting isotopes such as ^11C , ^18F , ^15O , and ^13N can be used in positron emission tomography (PET) studies to examine substrate acceptor occupancy. Isotopically labeled compounds of formula I can generally be prepared using conventional techniques known to those skilled in the art, or by methods similar to those described in the examples listed below, using a suitable isotopically labeled reagent instead of the previously used unlabeled reagent.

The compounds of the embodiments disclosed herein, or their pharmaceutically acceptable salts, may contain one or more asymmetric centers, such as chiral carbon and phosphorus atoms, and thus may produce enantiomers, diastereomers, and other stereoisomers that can be defined by absolute stereochemistry as (R)- or (S)- or (D)- or (L)- for amino acids. This disclosure is intended to include all such possible isomers as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)- or (D)- and (L)- isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques such as chromatography and fractional crystallization. Conventional techniques for preparing/separating individual enantiomers include chiral synthesis from suitable optically pure precursors or resolution of racemates (or racemates of salts or derivatives) using, for example, chiral high-performance liquid chromatography (HPLC). When a compound is represented in its chiral form, it should be understood that the embodiments cover, but are not limited to, specific diastereomers or enantiomerically enriched forms. When chirality is not specified but is present, it should be understood that the embodiment relates to a specific diastereomer or enantiomer-enriched form; or a racemic or non-racemic mixture of such compounds. As used herein, a "non-racemic mixture" is a mixture of stereoisomers in a ratio not equal to 1:1.

A "racemate" is a mixture of enantiomers. The mixture may contain equal or unequal amounts of each enantiomer.

"One stereoisomer" and "multiple stereoisomers" refer to compounds that differ in chirality in one or more stereocenters. Stereoisomers include enantiomers and diastereomers. If a compound has one or more asymmetric centers or has double bonds with asymmetric substitution, the compound may exist in stereoisomeric forms and therefore may be produced as individual stereoisomers or as mixtures. Unless otherwise stated, the description is intended to include individual stereoisomers as well as mixtures. Methods for determining stereochemistry and isolating stereoisomers are well known in the art (see, for example, Advanced Organic Chemistry, Chapter 4, 4th Edition, J. March, John Wiley and Sons, New York, 1992).

"Tautomers" refer to alternative forms of compounds that differ in the position of the proton, such as enol-ketone and imine-enamine tautomers, or tautomers containing heteroaryl groups attached to both the ring-NH- and ring=N-, such as pyrazoles, imidazoles, benzimidazoles, triazoles, and tetraazoles.

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.

As used herein, "solvent" refers to the result of the interaction between the solvent and the compound. Solvents of salts of the compounds described herein are also provided. Hydrates of the compounds described herein are also provided.

As used in this article, a "prodrug" is a derivative of a drug that is converted into an active drug through some chemical or enzymatic pathways when applied to the human body.

III. Compounds

This disclosure provides compounds of formula I:

Or its pharmaceutically acceptable salt.

Compounds of Formula I described herein, or their pharmaceutically acceptable salts, and their in vivo metabolites also fall within the scope of this document, to some extent being novel and non-obvious relative to the prior art. Such products can be generated, for example, by oxidation, reduction, hydrolysis, amidation, esterification, etc., of the applied compound, primarily due to enzymatic processes. Thus, methods including the generation of novel and non-obvious compounds by means of exposing the compound to a mammal for a sufficient period to produce its metabolites are included. Such products are typically identified by preparing a radiolabeled (e.g., ^14C or ^3H ) compound, administering the radiolabeled compound parenterally at a detectable dose (e.g., greater than about 0.5 mg/kg) to an animal, such as a rat, mouse, guinea pig, monkey, or human, allowing sufficient time for metabolism (typically about 30 seconds to 30 hours), and isolating its metabolites from urine, blood, or other biological samples. These products are readily isolated because they are labeled (other products are isolated using antibodies capable of binding to epitopes that survive in the metabolites). The metabolite structure is determined in a conventional manner, for example by MS or NMR analysis. Typically, metabolite analysis is performed in the same manner as routine drug metabolism studies well known to those skilled in the art. Transformation products, even if they do not possess antiviral activity, can be used for diagnostic determination of therapeutic doses of compounds of Formula I, provided they are not otherwise detected in vivo.

The desired goal is to discover compounds with lower _EC50 values or their pharmaceutically acceptable salts. The _EC50 value refers to the concentration at which 50% of the compound achieves its maximum efficacy in an assay. Compounds with lower _EC50 achieve similar efficacy at lower concentrations compared to compounds with higher _EC50 . Therefore, lower _EC50 is generally preferred for drug development.

Another expectation is to discover compounds or their pharmaceutically acceptable salts with a high selectivity index (SI). SI is the ratio of the window between cytotoxicity and antiviral activity (AVA) measured by dividing a given AVA value by a TOX (toxicity) value (AVA/TOX). A higher SI ratio theoretically implies that the drug will be more effective and safer during in vivo treatment of a given viral infection. Ideally, the drug would exhibit cytotoxicity only at very high concentrations and antiviral activity only at very low concentrations, thus producing a high SI value (high AVA/low TOX) and thereby enabling the elimination of the target virus at concentrations far below its cytotoxic concentration (Human Herpesviruses HHV-6A, HHV-6B & HHV-7 (Third Edition), Diagnosis and Clinical Management, 2014, Chapter 19, pp. 311-331).

It is also desirable to discover compounds or their pharmaceutically acceptable salts that possess good physical and/or chemical stability. Increased overall stability of a compound can provide for increased circulation time in vivo. Because of less degradation, stable compounds can be administered at lower doses while still retaining their efficacy. Similarly, less degradation means fewer problems with byproducts generated from compound degradation. Higher drug stability means more drug is available to target cells without being metabolized.

Further research is expected to discover compounds or pharmaceutically acceptable salts thereof with improved pharmacokinetic and/or pharmacodynamic properties and long half-lives. Advantageously, drugs with moderate or low clearance and long half-lives are desirable, as this may result in good bioavailability and high systemic exposure. Reducing the clearance of a compound and increasing its half-life can reduce the daily dose required for efficacy, and thus produce better efficacy and safety characteristics. Therefore, improved pharmacokinetic and/or pharmacodynamic properties and long half-lives can provide better patient compliance.

There is also a desire to develop compounds with improved solubility. Compounds with low solubility are often characterized by poor adsorption and bioavailability. Compounds with low solubility are also generally difficult to formulate and face development challenges, leading to increased development costs and/or time.

Further development is expected to target prodrug compounds that can undergo selective metabolism in target cells and/or tissues. Selective metabolism in target cells/tissues ensures the delivery of the active metabolite to the target cells/tissues, resulting in increased efficacy. This can also lead to lower dose requirements and side effects.

Advantageously, the compounds of formula I exhibit improved properties compared to the structure-related compounds described in WO2015/069939 (here designated as compound 1 and compound 2).

IV. Pharmaceutical Preparations

In some embodiments, this disclosure provides a pharmaceutical formulation comprising a therapeutically effective amount of a compound of the disclosed invention or a pharmaceutically acceptable salt thereof (active ingredient) and a pharmaceutically acceptable carrier or excipient. This document also provides a pharmaceutical formulation comprising a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt, solvate, and/or ester thereof, and a pharmaceutically acceptable carrier or excipient.

The compounds of Formula I described herein are formulated with conventional carriers and excipients, which will be selected according to conventional practice. Tablets will contain excipients, flow aids, fillers, binders, etc. Aqueous formulations are prepared aseptically and will generally be isotonic when intended for delivery by non-oral administration. All formulations will optionally contain excipients, such as those described in the "Handbook of Pharmaceutical Excipients" (1986). Excipients include ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextrin, hydroxyalkyl cellulose, hydroxyalkyl methyl cellulose, stearic acid, etc. The pH range of the formulation is from about 3 to about 11, but is typically from about 7 to 10.

While the active ingredients can be administered alone, it may be preferred to provide them as pharmaceutical formulations. Formulations for veterinary and human use contain the active ingredient as defined above, along with one or more carriers and optional other therapeutic ingredients, particularly those additional therapeutic ingredients discussed herein. The carrier must be "acceptable," meaning compatible with the other components of the formulation and physiologically harmless to the recipient.

Formulations are readily available in unit dosage forms and can be prepared using any of the methods well-known in the pharmaceutical field. Techniques and formulations are typically found in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA). Such methods involve the step of associating the active ingredient with a carrier that constitutes one or more auxiliary components. Generally, formulations are prepared by uniformly and tightly associating the active ingredient with a liquid carrier or a finely dispersed solid carrier, or both, and then, if desired, shaping the product.

Formulations suitable for oral administration may be provided as discrete units such as capsules, granules, or tablets, each containing a predetermined amount of the active ingredient; as powders or granules; as solutions or suspensions in aqueous or non-aqueous liquids; or as oil-in-water or water-in-oil liquid emulsions. The active ingredient may also be administered as pills, granules, or pastes.

Tablets are made by compression or molding, optionally using one or more excipients. Compressed tablets are prepared by compressing an active ingredient in a free-flowing form (such as powder or granules) in a suitable machine, optionally mixed with a binder, lubricant, inert diluent, preservative, surfactant, or dispersant. Molded tablets are prepared by molding a mixture of powdered active ingredients moistened with an inert liquid diluent in a suitable machine. Tablets may optionally be coated or scored, and optionally formulated to provide a slow or controlled release of the active ingredient therefrom.

For infections of the eyes or other external tissues (e.g., the mouth and skin), the formulation is preferably applied as a topical ointment or cream containing active ingredients in amounts, for example, from 0.075% w/w to 20% w/w (including active ingredients in increments of 0.1% w/w, such as 0.6% w/w, 0.7% w/w, etc.), preferably from 0.2% w/w to 15% w/w, and most preferably from 0.5% w/w to 10% w/w. When formulated as an ointment, the active ingredients may be used with a paraffin base or a water-miscible ointment base. Alternatively, the active ingredients may be formulated as a cream with an oil-in-water emulsion base.

If desired, the aqueous phase of the cream matrix may include, for example, at least 30% w/w polyols, i.e., alcohols having two or more hydroxyl groups such as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerin, and polyethylene glycol (including PEG400), and mixtures thereof. Topical formulations may ideally include compounds that enhance the absorption or penetration of the active ingredient through the skin or other affected areas. Examples of such skin penetration enhancers include dimethyl sulfoxide and related analogues.

The oil phase of an emulsion can be composed of known ingredients in a known manner. While this phase may consist only of emulsifiers (or simply emulsifiers), it ideally includes at least one emulsifier with fats or oils, or with a mixture of both. Preferably, hydrophilic emulsifiers are included together with lipophilic emulsifiers that act as stabilizers. In some embodiments, both oils and fats may be included. Emulsifiers, with or without stabilizers, together constitute a so-called emulsified wax, and the wax, together with the oils and fats, constitutes a so-called emulsified ointment matrix, which forms the oily dispersed phase of the ointment formulation.

Emulsifiers and emulsion stabilizers suitable for formulations include 60, 80, cetearyl alcohol, benzyl alcohol, myristicin, glyceryl monostearate, and sodium lauryl sulfate.

The appropriate oil or fat is selected for the formulation based on achieving the desired cosmetic properties. The cream should preferably be a non-greasy, non-staining, and washable product with a suitable consistency to prevent leakage from tubes or other containers. Straight-chain or branched monoalkyl or dialkyl esters can be used, such as diisohexyl adipate, isohexadecanoyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate, or mixtures of branched esters known as Crodamol CAP. These esters can be used alone or in combination, depending on the desired properties. Alternatively, high-melting-point lipids, such as white soft paraffin and/or liquid paraffin or other mineral oils, can be used.

The pharmaceutical formulations described herein comprise an active ingredient, together with one or more pharmaceutically acceptable carriers or excipients and optional other therapeutic agents. Pharmaceutical formulations containing an active ingredient can be in any form suitable for the intended method of administration. For example, when intended for oral use, they can be prepared as tablets, lozenges, tablets, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, solutions, syrups, or elixirs. Compositions intended for oral use can be prepared according to any method known in the art for manufacturing pharmaceutical compositions, and such compositions may contain one or more pharmaceutical agents, including sweeteners, flavoring agents, coloring agents, and preservatives, to provide a palatable formulation. Tablets containing an active ingredient mixed with non-toxic, pharmaceutically acceptable excipients suitable for manufacturing tablets are acceptable. These excipients may be, for example, inert diluents such as calcium carbonate or sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrants such as corn starch or alginic acid; binders such as starch, gelatin, or gum arabic; and lubricants such as magnesium stearate, stearic acid, or talc. Tablets may be uncoated or coated using known techniques, including microencapsulation, to delay disintegration and adsorption in the gastrointestinal tract, thereby providing sustained action over a longer period. For example, delaying materials such as glyceryl monostearate or glyceryl distearate may be used alone or in combination with waxes.

Formulations intended for oral use may also be provided as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent (such as calcium phosphate or kaolin) or as soft gelatin capsules in which the active ingredient is mixed with an aqueous or oily medium (such as peanut oil, liquid paraffin, or olive oil).

Aqueous suspensions contain active substances mixed with excipients suitable for manufacturing aqueous suspensions. Such excipients include suspending agents such as sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum, and gum arabic; and dispersing or wetting agents such as naturally occurring phospholipids (e.g., lecithin), condensation products of olefinic oxygen and fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide and long-chain fatty alcohols (e.g., heptadecanethoxycetyl alcohol), and condensation products of ethylene oxide and esters derived from fatty acids and hexyl anhydrides (e.g., polyoxyethylene sorbitan monooleate). Aqueous suspensions may also contain one or more preservatives, such as ethylparaben or n-propylparaben, one or more colorants, one or more flavoring agents, and one or more sweeteners such as sucrose or saccharin.

Oily suspensions can be prepared by suspending the active ingredients in vegetable oils (such as peanut oil, olive oil, sesame oil, or coconut oil) or mineral oils (such as liquid paraffin). Oral suspensions may contain thickeners such as beeswax, hard paraffin, or cetyl alcohol. Sweeteners (such as those mentioned above) and flavoring agents may be added to provide palatable oral formulations. These compositions may be preserved by adding antioxidants (such as ascorbic acid).

Dispersible powders and granules suitable for preparing aqueous suspensions by adding water provide an active ingredient that can be mixed with a dispersant or wetting agent, a suspending agent, and one or more preservatives. Suitable dispersants or wetting agents and suspending agents are exemplified by those disclosed above. Additional excipients, such as sweeteners, flavoring agents, and coloring agents, may also be present.

Pharmaceutical compositions may also be in the form of oil-in-water emulsions. The oil phase may be vegetable oils (such as olive oil or peanut oil), mineral oils (such as liquid paraffin), or mixtures thereof. Suitable emulsifiers include naturally occurring gums, such as gum arabic and tragacanth; naturally occurring phospholipids, such as soybean lecithin; esters or metaesters derived from fatty acids and hexitan anhydrides, such as sorbitan monooleate; and condensation products of these metaesters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. Emulsions may also contain sweeteners and flavoring agents. Syrups and elixirs may be formulated with sweeteners such as glycerin, sorbitol, or sucrose. Such formulations may also contain modifiers, preservatives, flavoring agents, or coloring agents.

The pharmaceutical composition may be in the form of a sterile injectable or intravenous formulation, such as a sterile injectable aqueous or oily suspension. This suspension may be formulated using suitable dispersants or wetting agents and suspending agents mentioned above, according to known techniques. The sterile injectable or intravenous formulation may also be a sterile injectable solution or suspension in a non-toxic, parenteral-acceptable diluent or solvent (such as a solution in 1,3-butanediol), or prepared as a lyophilized powder. Acceptable solvents and media are water, Ringer's solution, and isotonic sodium chloride solution. Furthermore, sterile non-volatile oils are commonly used as solvents or suspension media. For this purpose, any mild non-volatile oil may be used, including synthetic monoglycerides or diglycerides. Additionally, fatty acids such as oleic acid may also be used in the preparation of the injection.

The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the specific route of administration. For example, a sustained-release formulation intended for oral administration to humans may contain approximately 1 mg to 1000 mg of the active material, compounded with an appropriate and convenient amount of carrier material, which may vary between approximately 5% to approximately 95% (weight:weight) of the total composition. Pharmaceutical compositions can be prepared to provide easily measurable dosages. For example, an aqueous solution intended for intravenous infusion may contain approximately 3 to 500 μg of the active ingredient per milliliter to allow for the infusion of an appropriate volume at a rate of approximately 30 mL/hr.

Formulations suitable for topical application to the eyes also include eye drops, wherein the active ingredient is dissolved or suspended in a suitable carrier, particularly in an aqueous solution of the active ingredient. The active ingredient is preferably present in such formulations at a concentration of 0.5% to 20%, advantageously 0.5% to 10%, and particularly about 1.5% w/w.

Preparations suitable for topical application in the oral cavity include lozenges containing flavoring active ingredients, typically sucrose and gum arabic or tragacanth; tablets containing inert active ingredients, such as gelatin and glycerin, or sucrose and gum arabic; and mouthwashes containing the active ingredients in a suitable liquid carrier.

Formulations for rectal administration may be provided as suppositories with a suitable matrix, including, for example, cocoa butter or salicylates.

Formulations suitable for intrapulmonary or intranasal administration have particle sizes ranging from, for example, 0.1 micrometers to 500 micrometers, such as 0.5 micrometers, 1 micrometer, 30 micrometers, 35 micrometers, etc., and are administered by inhalation through the nasal passage or by inhalation into the mouth. Suitable formulations include aqueous or oily solutions of the active ingredient. Formulations suitable for aerosol or dry powder administration can be prepared according to conventional methods and can be delivered together with other therapeutic agents, such as compounds used to date for the treatment or prevention of pulmonary viral infections, as described below.

Another embodiment provides a novel, effective, safe, non-irritating, and physiologically compatible inhalable composition comprising a compound of formula I suitable for treating pulmonary viral infections and potentially related bronchitis, or a pharmaceutically acceptable salt thereof. Non-limiting exemplary pharmaceutically acceptable salts are inorganic acid salts, including hydrochlorides, hydrobromides, sulfates, or phosphates, as they can cause less lung irritation. In some embodiments, the inhalable formulation is delivered into the intrabronchial space in an aerosol comprising particles with an aerodynamic mass median diameter (MMAD) between about 1 μm and about 5 μm. In some embodiments, the compound of formula I is formulated for aerosol delivery using a nebulizer, a metered-dose inhaler (pMDI), or a dry powder inhaler (DPI).

Non-limiting examples of nebulizers include pulverizing, jetting, ultrasonic, pressurizing, vibrating perforated plate, or equivalent nebulizers, including those utilizing adaptive aerosol delivery technology (Denyer, J. Aerosol Medicine Pulmonary Drug Delivery 2010, 23 Supplement 1, S1-S10). Jet nebulizers use air pressure to break up a liquid solution into aerosol droplets. Ultrasonic nebulizers operate via piezoelectric crystals that shear the liquid into small aerosol droplets. Pressurized nebulization systems force a solution under pressure through a small orifice to generate aerosol droplets. Vibrating perforated plate devices utilize rapid vibration to shear a liquid stream into appropriate droplet sizes.

In some embodiments, the formulation for nebulization is in an aerosol containing particles with a main MMAD between about 1 μm and about 5 μm, and is delivered to the intrabronchial space using a nebulizer capable of atomizing the formulation of a compound of Formula I into particles with the desired MMAD. For optimal therapeutic efficacy and to avoid upper respiratory tract and systemic side effects, most nebulized particles should not have an MMAD greater than about 5 μm. If the aerosol contains a large number of particles with an MMAD greater than 5 μm, the particles deposit in the upper airways, thereby reducing the amount of drug delivered to the sites of inflammation and bronchoconstriction in the lower respiratory tract. If the aerosol has an MMAD less than about 1 μm, the particles tend to remain suspended in the inhaled air and are subsequently exhaled during exhalation.

When formulated and delivered according to the methods described herein, the aerosol formulation for nebulization delivers a therapeutically effective dose of a compound of Formula I to a site of pulmonary viral infection sufficient to treat the pulmonary viral infection. The amount of drug administered must be adjusted to reflect the efficiency of delivering a therapeutically effective dose of a compound of Formula I. In some embodiments, the aqueous aerosol formulation is combined with a pulverizing, spraying, pressurizing, vibrating perforated plate, or ultrasonic nebulizer, depending on the nebulizer, allowing approximately 20% to approximately 90%, for example, approximately 70% of the administered dose of a compound of Formula I to be delivered into the airways. In some embodiments, at least approximately 30% to approximately 50% of the active compound is delivered. In some embodiments, approximately 70% to approximately 90% of the active compound is delivered.

In another embodiment, the compound of Formula I or a pharmaceutically acceptable salt thereof is delivered in the form of a dry, inhalable powder. The compound is administered intrabronchially as a dry powder formulation to efficiently deliver fine particles of the compound into the bronchial space using a dry powder or metered-dose inhaler. For delivery via DPI, the compound of Formula I is processed into particles with a main MMAD size between about 1 μm and about 5 μm by means of milling spray drying, critical fluid processing, or precipitation from solution. Media milling, jet milling, and spray drying apparatus and procedures capable of producing particle sizes with MMAD sizes between about 1 μm and about 5 μm are well known in the art. In one embodiment, an excipient is added to the compound of Formula I prior to processing into particles of the desired size. In another embodiment, the excipient is blended with particles of the desired size to aid in the dispersion of the drug particles, for example, by using lactose as an excipient.

Particle size determination is performed using devices well known in the art. For example, a multi-stage Anderson cascade impactor or other suitable methods, such as those specifically described in Chapter 601 of the United States Pharmacopeia for the characterization of aerosols in metered dose and dry powder inhalers.

In some embodiments, a device such as a dry powder inhaler or other dry powder dispersing device is used to deliver the compound of Formula I in dry powder form. Non-limiting examples of dry powder inhalers and devices include those described in US 5,458,135; US 5,740,794; US 5775320; US 5,785,049; US 3,906,950; US 4,013,075; US 4,069,819; US 4,995,385; US 5,522,385; US 4,668,218; US 4,667,668; US 4,805,811 and US 5,388,572. There are two main designs for dry powder inhalers. One design is a metering device in which a drug reservoir is placed within the device, and the patient adds a dose of drug to the inhalation chamber. The second design is a factory metering device in which each individual dose has been manufactured in a separate container. Both systems rely on formulating the drug into small particles with a MMAD of 1 μm to approximately 5 μm, and often involve co-formulation with larger excipient particles (such as, but not limited to, lactose). The drug powder is placed in the inhalation chamber (dose metered by the device or by opening the factory), and the patient's inspiratory airflow accelerates the powder from the device into the oral cavity. The non-laminar characteristics of the powder path cause the excipient-drug aggregates to break down, and the clumps of large excipient particles cause them to compact against the back of the throat, while smaller drug particles deposit deep in the lungs. In some embodiments, compounds of Formula I or pharmaceutically acceptable salts thereof are delivered in dry powder form using any type of dry powder inhaler as described herein, wherein the MMAD of the dry powder (excluding any excipients) is primarily in the range of 1 μm to approximately 5 μm.

In another embodiment, the compound of Formula I is delivered in dry powder form using a metered-dose inhaler. Non-limiting examples of metered-dose inhalers and devices include those described in US 5,261,538; US 5,544,647; US 5,622,163; US 4,955,371; US 3,565,070; US 3,361,306 and US 6,116,234. In some embodiments, the compound of Formula I or a pharmaceutically acceptable salt thereof is delivered in dry powder form using a metered-dose inhaler, wherein the MMAD of the dry powder (excluding any excipients) is primarily in the range of 1 μm to 5 μm.

Preparations suitable for vaginal application may be provided in the form of pessaries, tampons, creams, gels, pastes, foams or sprays, and contain, in addition to the active ingredient, a suitable carrier known in the art.

Preparations suitable for parenteral administration include aqueous and non-aqueous sterile injectable solutions that may contain antioxidants, buffers, antibacterial agents, and solutes to make the preparation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickeners.

The formulation is present in single-dose or multi-dose containers, such as sealed ampoules and vials, and can be stored under lyophilized (freeze-dried) conditions, requiring only immediate addition of a sterile liquid carrier, such as water for injection, before use. Temporary injectable solutions and suspensions are prepared from sterile powders, granules, and tablets of the aforementioned types. Exemplary single-dose formulations are those containing a daily dose or a unit daily sub-dose or a suitable portion thereof of the active ingredient as described above.

It should be understood that, in addition to the ingredients specifically mentioned above, formulations may include other agents conventional in the art related to the type of formulation discussed, such as flavoring agents suitable for oral administration.

Furthermore, a veterinary drug composition comprising at least one active ingredient as defined above and its veterinary drug carrier is provided.

Veterinary drug carriers are materials that can be used to administer compositions and can be solid, liquid, or gaseous materials. They are otherwise inert or acceptable in the veterinary field and compatible with the active ingredient. These veterinary drug compositions can be administered orally, parenterally, or via any other desired route.

In some embodiments, compounds of Formula I are formulated to provide controlled-release pharmaceutical preparations (“controlled-release preparations”), wherein the release of compounds of Formula I is controlled and modulated to allow for less frequent administration or to improve the pharmacokinetic or toxic properties of a given active ingredient.

The effective dose of the active ingredient depends at least on the nature of the condition being treated, its toxicity, whether the compound is used prophylactically (at a lower dose) or to combat an active viral infection, the delivery method, and the pharmaceutical formulation, and will be determined by clinicians using routine dose-escalation studies. The dose is expected to be from about 0.0001 mg/kg body weight to about 100 mg/kg body weight daily; for example, from about 0.01 mg/kg body weight to about 10 mg/kg body weight daily. In some embodiments, the effective dose is from about 0.01 mg/kg body weight to about 5 mg/kg body weight daily; typically, for example, from about 0.05 mg/kg body weight to about 0.5 mg/kg body weight daily. For example, the candidate daily dose range for an adult weighing approximately 70 kg would be from 1 mg to 1000 mg, for example, from 5 mg to 500 mg, and may be in single or multiple dose form.

V. Route of application

Compounds of Formula I (also referred to herein as the active ingredient) can be administered via any suitable route. Suitable routes include oral, rectal, nasal, topical (including buccal and sublingual), transdermal, vaginal, and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal, and epidural). It should be understood that preferred routes may vary depending on, for example, the recipient's condition.

The compounds disclosed herein can be administered to an individual for a desired period of time or duration according to an effective dosing regimen, such as at least one week, at least about two weeks, at least about three weeks, one month, at least about two months, at least about three months, at least about six months, or at least about 12 months or longer. In one variant, the compounds are administered for the desired duration on a daily or intermittent schedule throughout the individual's life.

The dosage or frequency of administration of the compounds disclosed herein may be adjusted during treatment based on the judgment of the physician administering the medication.

The compound can be applied in an effective amount to an individual (e.g., a human). In some embodiments, the compound is administered once daily.

The compound may be administered by any available route and method, such as by oral or parenteral (e.g., intravenous) administration. Therapeutic amounts of the compound may include from about 0.00001 mg/kg body weight/day to about 10 mg/kg body weight/day, such as from about 0.0001 mg/kg body weight/day to about 10 mg/kg body weight/day, or such as from about 0.001 mg/kg body weight/day to about 1 mg/kg body weight/day, or such as from about 0.01 mg/kg body weight/day to about 1 mg/kg body weight/day, or such as from about 0.05 mg/kg body weight/day to about 0.5 mg/kg body weight/day, or such as from about 0.3 mg/day to about 30 mg/day, or such as from about 30 mg/day to about 300 mg/day.

The compounds disclosed herein may be combined with one or more additional therapeutic agents at any dose of the disclosed compounds (e.g., 1 mg to 1000 mg of the compound). Therapeuticly effective doses may include about 1 mg/dose to about 1000 mg/dose, such as about 50 mg/dose to about 500 mg/dose, or such as about 100 mg/dose to about 400 mg/dose, or such as about 150 mg/dose to about 350 mg/dose, or such as about 200 mg/dose to about 300 mg/dose. Other therapeutically effective doses of the compounds disclosed herein are about 100 mg/dose, about 125 mg/dose, about 150 mg/dose, about 175 mg/dose, about 200 mg/dose, about 225 mg/dose, about 250 mg/dose, about 275 mg/dose, about 300 mg/dose, about 325 mg/dose, about 350 mg/dose, about 375 mg/dose, about 400 mg/dose, about 425 mg/dose, about 450 mg/dose, about 475 mg/dose, or about 500 mg/dose. Other therapeutically effective amounts of the compounds disclosed herein are about 100 mg/dose, or about 125 mg/dose, about 150 mg/dose, about 175 mg/dose, about 200 mg/dose, about 225 mg/dose, about 250 mg/dose, about 275 mg/dose, about 300 mg/dose, about 350 mg/dose, about 400 mg/dose, about 450 mg/dose, or about 500 mg/dose. A single dose may be administered hourly, daily, or weekly. For example, a single dose may be administered every 1, 2, 3, 4, 6, 8, 12, or 16 hours, or every 24 hours. A single dose may also be administered every 1, 2, 3, 4, 5, or 6 days, or every 7 days. A single dose may also be administered every 1, 2, or 3 weeks, or every 4 weeks. In some embodiments, a single dose may be administered weekly. A single dose may also be administered monthly.

Other therapeutically effective doses of the compounds disclosed herein are about 20 mg/dose, 25 mg/dose, 30 mg/dose, 35 mg/dose, 40 mg/dose, 45 mg/dose, 50 mg/dose, 55 mg/dose, 60 mg/dose, 65 mg/dose, 70 mg/dose, 75 mg/dose, 80 mg/dose, 85 mg/dose, 90 mg/dose, 95 mg/dose, or about 100 mg/dose.

The frequency of dosage of the disclosed compound will be determined by the individual patient's needs and may be, for example, once daily or twice daily or more. Administration of the compound will continue as long as treatment of the viral infection is required. For example, the compound may be administered to a person with a viral infection for a period of 20 to 180 days, or for a period of, for example, 20 to 90 days, or for example, 30 to 60 days.

Administration may be intermittent, with the patient receiving a daily dose of the disclosed compound for periods of several days or more, followed by periods of several days or more without receiving a daily dose of the compound. For example, the patient may receive a dose of the compound every other day or three times a week. Again, by way of example, the patient may receive a daily dose of the compound for periods of 1 to 14 days, followed by periods of 7 to 21 days without receiving a dose of the compound, followed by periods of receiving a daily dose of the compound again in subsequent periods (e.g., 1 to 14 days). The alternating periods of administration followed by non-administration of the compound may be repeated as needed to treat the patient's clinical needs.

In one embodiment, pharmaceutical compositions are provided comprising a compound of the present disclosure or a pharmaceutically acceptable salt thereof in combination with one or more (e.g., one, two, three, four, one or two, one to three, or one to four) additional therapeutic agents and pharmaceutically acceptable excipients.

In one embodiment, kits are provided comprising compounds of the present disclosure or pharmaceutically acceptable salts thereof in combination with one or more (e.g., one, two, three, four, one or two, one to three, or one to four) additional therapeutic agents.

In some embodiments, the compound of this disclosure or a pharmaceutically acceptable salt thereof is combined with one, two, three, four, or more additional therapeutic agents. In some embodiments, the compound of this disclosure or a pharmaceutically acceptable salt thereof is combined with two additional therapeutic agents. In other embodiments, the compound of this disclosure or a pharmaceutically acceptable salt thereof is combined with three additional therapeutic agents. In still other embodiments, the compound of this disclosure or a pharmaceutically acceptable salt thereof is combined with four additional therapeutic agents. The one, two, three, four, or more additional therapeutic agents may be different therapeutic agents selected from the same class of therapeutic agents, and/or they may be selected from different classes of therapeutic agents.

In some embodiments, when the compounds of this disclosure are combined with one or more additional therapeutic agents as described herein, the components of the composition are administered simultaneously or sequentially. When administered sequentially, the combination may be administered in two or more doses.

In some embodiments, the compounds of this disclosure are combined with one or more additional therapeutic agents in a single dosage form for simultaneous administration to a patient, such as as a solid dosage form for oral administration.

In some embodiments, the compounds of this disclosure are administered in combination with one or more additional therapeutic agents.

To prolong the effects of the compounds disclosed herein, it is often desirable to slow down the absorption of the compound from subcutaneous or intramuscular injection. This can be achieved by using liquid suspensions of poorly water-soluble crystalline or amorphous materials. The absorption rate of the compound depends on its dissolution rate, and subsequently on the crystal size and crystal form. Alternatively, delayed absorption of the compound in the form of a parenteral administration is achieved by dissolving or suspending the compound in an oil medium. Injectable long-acting forms are prepared by forming microcapsule matrices of the compound in biodegradable polymers, such as polylactide-polyglycolic acid. The release rate of the compound can be controlled depending on the ratio of compound to polymer and the properties of the specific polymer used. Examples of other biodegradable polymers include poly(orthoester) and poly(anhydride). Long-acting injectable formulations are also prepared by encapsulating the compound in liposomes or microemulsions that are compatible with body tissues.

VI. Combination Therapy

The compounds and compositions of Formula I provided herein can also be used in combination with other active therapeutic agents for the treatment of viral infections, such as those of the Pneumoviridae, Picornaviridae, Flaviviridae, or Filoviridae families.

Combination therapy for treating pulmonary virology

The compounds and compositions provided herein can also be used in combination with other active therapeutic agents. For the treatment of pulmonary viral infections, preferably, the other active therapeutic agents are active against pulmonary viral infections, particularly respiratory syncytial virus (RSV) and/or metapneumovirus (MRV) infections. Non-limiting examples of these other active therapeutic agents active against RSV include ribavirin, pallizumab, motavizumab, RSV-IGIVMEDI-557, A-60444 (also known as RSV604), MDT-637, BMS-433771, ALN-RSV0, ALX-0171, and mixtures thereof. Other non-limiting examples of other active therapeutic agents effective against respiratory syncytial virus (RSV) infection include RSV protein F inhibitors such as AK-0529; RV-521, ALX-0171, JNJ-53718678, BTA-585, and presatovir; RNA polymerase inhibitors such as lumicitabine and ALS-8112; anti-RSV G protein antibodies such as anti-G protein mAbs; and viral replication inhibitors such as nitazoxanide.

In some implementations, other active therapeutic agents may be vaccines for treating or preventing RSV, including but not limited to MVA-BN RSV, RSV-F, MEDI-8897, JNJ-64400141, DPX-RSV, SynGEM, GSK-3389245A, GSK-300389-1A, RSV-MEDIδM2-2 vaccine, VRC-RSVRGP084-00VP, Ad35-RSV-FA2, Ad26-RSV-FA2, and RSV fusion glycoprotein subunit vaccines.

Non-limiting examples of other active therapeutic agents that are active against metapneumovirus infection include sialidase modulators, such as DAS-181; RNA polymerase inhibitors, such as ALS-8112; and antibodies used to treat metapneumovirus infection, such as EV-046113.

In some implementations, other active therapeutic agents may be vaccines used to treat or prevent metapneumovirus infection, including but not limited to mRNA-1653 and rHMPV-Pa vaccines.

Combination therapy for treating Picornaviridae

The compounds and compositions provided herein can also be used in combination with other active therapeutic agents. For the treatment of microviral infections, preferably, the other active therapeutic agents are active against microviral infections, particularly enterovirus infections. Non-limiting examples of these other active therapeutic agents are capsid binding inhibitors, such as pleconaril, BTA-798 (vapendavir), and other compounds disclosed by Wu et al. (US 7,078,403) and Watson (US 7,166,604); fusion sialidase proteins, such as DAS-181; capsid protein VP1 inhibitors, such as VVX-003 and AZN-001; viral protease inhibitors, such as CW-33; phosphatidylinositol 4-kinase β inhibitors, such as GSK-480 and GSK-533; and anti-EV71 antibodies.

In some implementations, other active therapeutic agents may be vaccines used to treat or prevent infection with microribonucleoviridae viruses, including but not limited to EV 71 vaccines, TAK-021 and EV-D68 adenocarrier-based vaccines.

Combination therapy for respiratory infections

Many infections caused by viruses of the Pneumoviridae and Picornaviridae families are respiratory infections. Therefore, adjunctive active therapeutic agents for treating respiratory symptoms and post-infectious sequelae can be used in combination with compounds of Formula I. The adjunctive agents are preferably administered orally or by direct inhalation. For example, adjunctive therapeutic agents used in combination with compounds of Formula I for treating viral respiratory infections include, but are not limited to, bronchodilators and corticosteroids.

Glucocorticoids

First introduced as a treatment for asthma in 1950 (Carryer, Journal of Allergy, 21, 282-287, 1950), glucocorticoids remain the most effective and consistently effective treatment for this disease, but their mechanisms of action are not fully understood (Morris, J. Allergy Clin. Immunol., 75(1Pt)1-13, 1985). Unfortunately, oral glucocorticoid therapy is associated with serious adverse side effects such as truncal obesity, hypertension, glaucoma, impaired glucose tolerance, accelerated cataract formation, bone mineral loss, and psychogenic effects, all of which limit their use as long-term treatment agents (Goodman and Gilman, 10th ed., 2001). A solution to systemic side effects is to deliver steroid medications directly to the site of inflammation. Inhaled corticosteroids (ICS) have been developed to mitigate the serious adverse effects of oral steroids. Non-limiting examples of corticosteroids that can be used in combination with compounds of Formula I are dexamethasone, dexamethasone sodium phosphate, fluocinolone, fluocinolone acetate, clotepone, clotepone ecaponicate, hydrocortisone, prednisolone, fluocinolone acetonide, triamcinolone acetonide, betamethasone, beclomethasone dipropionate, methylprednisolone, fluocinolone acetonide, fluocinolone acetonide, flunisolone, fluocinolone acetonide-21-butyrate, flumethasone, fluocinolone valerate, budesonide, halobetasol propionate, mometasone furoate, fluticasone, AZD-7594, ciclesonide; or their pharmaceutically acceptable salts.

anti-inflammatory agents

Other anti-inflammatory agents that act through an anti-inflammatory cascade mechanism can also be used as adjunctive therapeutic agents in combination with compounds of Formula I for the treatment of viral respiratory infections. The application of “anti-inflammatory signal transduction modulators” (referred to as AISTMs in this text), such as phosphodiesterase inhibitors (e.g., PDE-4, PDE-5, or PDE-7 specific), transcription factor inhibitors (e.g., blocking NFκB via IKK inhibition), or kinase inhibitors (e.g., blocking P38 MAP, JNK, PI3K, EGFR, or Syk), is a logical approach to cutting off inflammation because these small molecules target a limited number of common intracellular pathways—those signal transduction pathways that are key points of intervention for anti-inflammatory therapies (see the review by P.J. Barnes, 2006). These non-restrictive adjunctive therapies include: 5-(2,4-difluoro-phenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (2-dimethylamino-ethyl)-amide (P38 Map kinase inhibitor ARRY-797); 3-cyclopropylmethoxy-N-(3,5-dichloro-pyridin-4-yl)-4-difluoromethoxy-benzamide (PDE-4 inhibitor Roflumilast); 4-[2-(3-cyclopentoxy-4-methoxyphenyl)-2-phenyl-ethyl]pyridine (PDE-4 inhibitor CDP- 840); N-(3,5-dichloro-4-pyridinyl)-4-(difluoromethoxy)-8-[(methanesulfonyl)amino]-1-dibenzofuran carboxamide (PDE-4 inhibitor Oglemilast); N-(3,5-dichloro-pyridin-4-yl)-2-[1-(4-fluorobenzyl)-5-hydroxy-1H-indole-3-yl]-2-oxo-acetamide (PDE-4 inhibitor AWD 12-281); 8-methoxy-2-trifluoromethyl-quinoline-5-carboxylic acid (3,5-dichloro-1-oxy-pyridinyl- 4-yl)-amide (PDE-4 inhibitor Sch 351591); 4-[5-(4-fluorophenyl)-2-(4-methylsulfinyl-phenyl)-1H-imidazol-4-yl]-pyridine (P38 inhibitor SB-203850); 4-[4-(4-fluorophenyl)-1-(3-phenyl-propyl)-5-pyridin-4-yl-1H-imidazol-2-yl]-but-3-yn-1-ol (P38 inhibitor RWJ-67657); 4-cyano-4-(3-cyclopentoxy-4-methoxy-phenyl)-cyclohexanecarboxylic acid 2-diethyl Aminoethyl ester (Cilomilast, a 2-diethyl-ethyl ester prodrug of the PDE-4 inhibitor); (3-chloro-4-fluorophenyl)-[7-methoxy-6-(3-morpholin-4-yl-propoxy)-quinazolin-4-yl]-amine (Gefitinib, an EGFR inhibitor); and 4-(4-methyl-piperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-benzamide (Imatinib, an EGFR inhibitor).

β2-adrenergic receptor agonists and bronchodilators

Combinations of inhaled β2-adrenergic receptor agonist bronchodilators (such as formoterol, abebuterol, or salmeterol) with compounds of formula I are also suitable, but not limiting, combinations for the treatment of respiratory viral infections.

Inhaled β2-adrenergic receptor agonist bronchodilators (such as formoterol or salmeterol) in combination with ICS are also used to treat bronchoconstriction and inflammation (respectively, bronchoconstriction and bronchoconstriction). Combinations of these ICS and β2-adrenergic receptor agonists, together with compounds of formula I, are also suitable, but not limiting, combinations for the treatment of respiratory viral infections.

Other examples of β2-adrenergic receptor agonists are bedoradrine, vilanterol, indacaterol, olodaterol, tulobuterol, formoterol, abetero, salmeterol, artoformoterol, levalbuterol, fenoterol, and TD-5471.

Anticholinergic drugs

Anticholinergic drugs have potential use in treating or preventing bronchoconstriction and can therefore be used as adjunctive therapeutic agents in combination with compounds of formula I for the treatment of viral respiratory infections. These anticholinergic drugs include, but are not limited to: muscarinic receptor (especially M3 subtype) antagonists that have shown therapeutic efficacy in controlling the cholinergic tone of COPD in humans (Witek, 1999); 1-{4-hydroxy-1-[3,3,3-tris-(4-fluoro-phenyl)-propionyl]-pyrrolidine-2-carbonyl}-pyrrolidine-2-carboxylic acid (1-methyl-piperidin-4-ylmethyl)-amide; 3-[3-(2-diethylamino-acetoxy)-2-phenyl-propionyloxy]-8-isopropyl-8-methyl-8-aza-bicyclo[3.2.1]octane (ipratropium-N,N-diethylglycinate); 1-cyclohexyl-3,4-dihydro-1H-isoquinoline-2-carboxylic acid-1-aza-bicyclo[2.2.2]oct-3-yl ester (Solifenacin); 2 1-Aza-bicyclo[2.2.2]oct-3-yl ester of hydroxymethyl-4-methylsulfinyl-2-phenyl-butyric acid (Revatropate); 2-{1-[2-(2,3-dihydro-benzofuran-5-yl)-ethyl]-pyrrolidine-3-yl}-2,2-diphenyl-acetamide (Darifenacin); 4-azacycloheptane-1-yl -2,2-Diphenyl-butyramide (Buzepide); 7-[3-(2-diethylamino-acetoxy)-2-phenyl-propionyloxy]-9-ethyl-9-methyl-3-oxa-9-aza-tricyclo[3.3.1.02,4]nonane (Ottoammonium bromide-N,N-diethylglycine salt); 7-[2-(2-diethylamino-acetoxy)-2,2-di-thiophene- 2-yl-acetoxy]-9,9-dimethyl-3-oxa-9-aza-tricyclo[3.3.1.02,4]nonane (tiotropium bromide-N,N-diethylglycine salt); dimethylaminoacetic acid 2-(3-diisopropylamino-1-phenyl-propyl)-4-methyl-phenyl ester (tolterodine-N,N-dimethylglycine salt); 3-[4,4-bis-(4-fluoro-phenyl)-2-oxo- [Imidazolin-1-yl]-1-methyl-1-(2-oxo-2-pyridin-2-ylethyl)-pyrrolidineonium; 1-[1-(3-fluoro-benzyl)-piperidin-4-yl]-4,4-bis-(4-fluoro-phenyl)-imidazolidine-2-one; 1-cyclooctyl-3-(3-methoxy-1-aza-bicyclo[2.2.2]oct-3-yl)-1-phenyl-prop-2-yn-1-ol; 3-[2-(2-di] [Ethylamino-acetoxy]-2,2-di-thiophene-2-yl-acetoxy]-1-(3-phenoxy-propyl)-1-aza-bicyclo[2.2.2]octane (acetyl bromide-N,N-diethylglycine salt); or (2-diethylamino-acetoxy)-di-thiophene-2-yl-acetic acid 1-methyl-1-(2-phenoxy-ethyl)-piperidin-4-yl ester; revefenacin, glycopyrronium bromide, umeclidinium bromide, tiotropium bromide, aclidinium bromide, bencycloquidium bromide.

Mucus Dissolving Agent

The compounds and compositions of Formula I provided herein can also be combined with mucolytics to treat symptoms of infections and respiratory tract infections. A non-limiting example of a mucolytic is ambroxol. Similarly, compounds of Formula I can be combined with expectorants to treat symptoms of infections and respiratory tract infections. A non-limiting example of an expectorant is guaifenesin.

Nebulized hypertonic saline is used to improve immediate and long-term airway clearance in patients with lung disease (Kuzik, J. Pediatrics 2007, 266). Therefore, compounds of Formula I can also be combined with nebulized hypertonic saline, particularly when pulmonary viral infections are complicated by bronchitis. The combination of compounds of Formula I with hypertonic saline may also include any additional agents discussed above. In one embodiment, approximately 3% nebulized hypertonic saline is used.

Combination therapy for the treatment of COPD

The compounds and compositions described herein can also be used in combination with other active therapeutic agents. Other active therapeutic agents for treating respiratory exacerbations of COPD include other active agents targeting COPD. Non-limiting examples of these other active therapeutic agents include anti-IL5 antibodies, such as benazolizumab and mepolizumab; dipeptidyl peptidase I (DPP1) inhibitors, such as AZD-7986 (INS-1007); DNA gyrase inhibitors/topoisomerase IV inhibitors, such as ciprofloxacin hydrochloride; MDR-associated protein 4/phosphodiesterase (PDE) 3 and 4 inhibitors, such as RPL-554; and CFTR stimulants, such as ivacathotoxin. ftor), QBW-251; MMP-9/MMP-12 inhibitors, such as RBx-10017609’; adenosine A1 receptor antagonists, such as PBF-680; GATA 3 transcription factor inhibitors, such as SB-010; muscarinic receptor modulators/nicotinic acetylcholine receptor agonists, such as ASM-024; MARCKS protein inhibitors, such as BIO-11006; kit tyrosine kinase/PDGF inhibitors, such as masitinib; phosphodiesterase (PDE) 4 inhibitors, such as roflumilast, CHF-6001; phosphoinositol-3 kinase δ Inhibitors, such as nemiralisib; 5-lipoxygenase inhibitors, such as TA-270; muscarinic receptor antagonists/β2-adrenergic receptor agonists, such as perphenerol succinate, AZD-887, and ipratropium bromide; TRN-157; elastase inhibitors, such as erdosteine; metalloproteinase-12 inhibitors, such as FP-025; interleukin-18 ligand inhibitors, such as tadekinig alfa; skeletal muscle troponin activators, such as CK-2127107; p38 MAP kinase inhibitors, such as acumamod (a cumapimod); IL-17 receptor modulators, such as CNTO-6785; CXCR2 chemokine antagonists, such as danirixin; leukocyte elastase inhibitors, such as POL-6014; epoxide hydrolase inhibitors, such as GSK-2256294; HNE inhibitors, such as CHF-6333; VIP agonists, such as avitadil; phosphatidylinositol-3 kinase δ/γ inhibitors, such as RV-1729; complement C3 inhibitors, such as APL-1; and G protein-coupled receptor-44 antagonists, such as AM-211.

Other non-limiting examples of active therapeutic agents include budesonide, adipocell, nitric oxide, PUR-1800, YLP-001, LT-4001, azithromycin, gamunex, QBKPN, sodium pyruvate, MUL-1867, mannitol, MV-130, MEDI-3506, BI-443651, VR-096, OPK-0018, TEV-48107, doxofylline, TEV-46017, OligoG-COPD-5/20, ZP-051, and lysine acetylsalicylate.

In some implementations, other active therapeutic agents may be vaccines active against COPD, including but not limited to MV-130 and GSK-2838497A.

Combination therapy for the treatment of dengue fever

The compounds and compositions provided herein can also be used in combination with other active therapeutic agents. For the treatment of flaviviridae virus infections, preferably, the other active therapeutic agents are active against flaviviridae virus infections, particularly dengue fever. Non-limiting examples of these other active therapeutic agents are host cytokine modulators, such as GBV-006; fenretinide ABX-220, BRM-211; α-glucosidase 1 inhibitors, such as celgosivir; platelet-activating factor receptor (PAFR) antagonists, such as modipafant; cadherin-5/factor Ia modulators, such as FX-06; NS4B inhibitors, such as JNJ-8359; viral RNA splicing modulators, such as ABX-202; NS5 polymerase inhibitors; NS3 protease inhibitors; and TLR modulators.

In some implementations, other active therapeutic agents may be vaccines used to treat or prevent dengue fever, including but not limited to TetraVax-DV, DPIV-001, TAK-003, live attenuated dengue vaccine, quadrivalent dengue vaccine, quadrivalent DNA vaccine, rDEN2delta30-7169; and DENV-1PIV.

Combination therapy for the treatment of Ebola

The compounds and compositions provided herein can also be used in combination with other active therapeutic agents. For the treatment of filoviral infections, preferably, the other active therapeutic agents are active against filoviral infections, particularly Marburg virus, Ebola virus, and Cueva virus infections. Non-limiting examples of these other active therapeutic agents include: ribavirin, palizumab, motuzumab, RSV-IGIVMEDI-557, A-60444, MDT-637, BMS-433771, amiodarone, dronedarone, verapamil, Ebola convalescent plasma (ECP), TKM-100201, and BCX4430 ((2S,3S,4R,5R)-2-(4-amino-5 ... H-pyrrolo[3,2-d]pyrimidin-7-yl)-5-(hydroxymethyl)pyrrolidine-3,4-diol), TKM-Ebola, T-705 monophosphate, T-705 diphosphate, T-705 triphosphate, FGI-106 (1-N,7-N-bis[3-(dimethylamino)propyl]-3,9-dimethylquinolino[8,7-h]quinolone-1,7-diamine), rNAPc2, OS-2966, brincidofovir, remdesivir;

RNA polymerase inhibitors, such as galidesivir, favipiravir (also known as T-705 or Avigan), and JK-05; host cytokine modulators, such as GMV-006; cadherin-5/factor Ia modulators, such as FX-06; and antibodies used to treat Ebola, such as REGN-3470-3471-3479 and ZMapp.

Other non-restricted active therapeutic agents against Ebola include alpha-glucosidase 1 inhibitors, cathepsin B inhibitors, CD29 antagonists, dendritic ICAM-3 inhibitors of non-integrin 1, estrogen receptor antagonists, factor VII antagonists, HLA class II antigen modulators, host cytokine modulators, interferon alpha ligands, neutral alpha-glucosidase AB inhibitors, Niemann-Pick C1 protein inhibitors, nucleoprotein inhibitors, polymerase cofactor VP35 inhibitors, serine protease inhibitors, tissue factor inhibitors, TLR-3 agonists, viral envelope protein inhibitors, and Ebola virus entry inhibitors (NPC1 inhibitors).

In some implementations, other active therapeutic agents may be vaccines used to treat or prevent Ebola, including but not limited to VRC-EBOADC076-00-VP, adenovirus-based Ebola vaccines, rVSV-EBOV, rVSVN4CT1-EBOVGP, MVA-BN Filo+Ad26-ZEBOV regimen, INO-4212, VRC-EBODNA023-00-VP, VRC-EBOADC069-00-VP, GamEvac-combi vaccine, SRC VB vector, HPIV3/EboGP vaccine, MVA-EBOZ, recombinant Ebola glycoprotein vaccine, Vaxart adenovirus vector 5-based Ebola vaccine, FiloVax vaccine, GOVX-E301, and GOVX-E302.

The compounds and compositions provided herein can also be used in combination with aminophosphate morpholino oligomers (PMOs), which are synthetic antisense oligonucleotide analogs designed to interfere with translation by forming base-pair duplexes with specific RNA sequences. Examples of PMOs include, but are not limited to, AVI-7287, AVI-7288, AVI-7537, AVI-7539, AVI-6002, and AVI-6003.

The compounds and compositions provided herein are also intended for use with general care provided to patients with filoviridae virus infections, including parenteral fluids (including glucose saline and Ringer's lactate) and nutrients, antibiotics (including metronidazole and cephalosporin antibiotics such as ceftriaxone and cefuroxime) and/or antifungal prophylaxis, fever and pain medications, antiemetics (such as metoclopramide) and/or antidiarrheals, vitamin and mineral supplements (including vitamin K and zinc sulfate), anti-inflammatory agents (such as ibuprofen), pain medications, and medications for other common illnesses in the patient population, such as antimalarial agents (including artemether and artemisinin-benfluridine combination therapy), typhoid vaccines (including quinolone antibiotics such as ciprofloxacin, macrolide antibiotics such as azithromycin, cephalosporin antibiotics such as ceftriaxone, or aminopenicillins such as ampicillin) or Shigella vaccines.

VII. Methods for treating viral infections

This disclosure provides methods for treating a variety of diseases, such as respiratory syncytial virus (RSV), HRV, hMPV, Ebola, Zika, West Nile, dengue fever, HCV, and HBV, using compounds of Formula I.

Pulmonary Virology

In some embodiments, this disclosure provides methods for treating pulmonary viral infections, which include administering a therapeutically effective amount of a compound of this disclosure or a pharmaceutically acceptable salt thereof to an individual (e.g., a human) infected with a pulmonary viral virus. Pneumoviridae viruses include, but are not limited to, respiratory syncytial virus (RSV) and other pulmonary viral viruses.

In some embodiments, this disclosure provides a method of treating a person in need of a pulmonaviridae virus infection, the method comprising administering to the person a therapeutically effective amount of a compound of this disclosure or a pharmaceutically acceptable salt thereof. Pulmonaviridae viruses include, but are not limited to, respiratory syncytial virus (RSV) and human metapneumovirus (HMV). In some embodiments, the pulmonaviridae virus infection is an RSV infection. In some embodiments, the pulmonaviridae virus infection is a HMV infection.

In some embodiments, this disclosure provides a method of manufacturing a medicament for treating pulmonary viral infections in persons of need, characterized by using a compound of this disclosure or a pharmaceutically acceptable salt thereof. In some embodiments, this disclosure provides the use of a compound of this disclosure or a pharmaceutically acceptable salt thereof for manufacturing a medicament for treating pulmonary viral infections in persons. In some embodiments, the pulmonary viral infection is respiratory syncytial virus infection. In some embodiments, the pulmonary viral infection is human metapneumovirus infection.

In some embodiments, this disclosure provides a compound of the present disclosure or a pharmaceutically acceptable salt thereof for the treatment of pulmonary viral infections in persons of need. In some embodiments, the pulmonary viral infection is respiratory syncytial virus infection. In some embodiments, the pulmonary viral infection is human metapneumovirus infection.

In some embodiments, this disclosure provides methods for treating RSV infection, which include administering a therapeutically effective amount of a compound of this disclosure or a pharmaceutically acceptable salt thereof to an individual infected with respiratory syncytial virus (RSV), e.g., a person. Typically, the individual has a chronic RSV infection, but individuals treated for acute RSV infection are also within the scope of this disclosure.

In some embodiments, a method for inhibiting RSV replication is provided, which includes administering to an individual (e.g., a human) a compound of the present disclosure or a pharmaceutically acceptable salt thereof.

In some embodiments, this disclosure provides a method for reducing viral load associated with RSV infection, wherein the method comprises administering to an individual infected with RSV (e.g., a human) a therapeutically effective amount of a compound of this disclosure or a pharmaceutically acceptable salt thereof, wherein the therapeutically effective amount is sufficient to reduce RSV viral load in the individual.

As described more fully herein, the compounds of this disclosure can be administered to an individual (e.g., a person) infected with RSV, together with one or more adjunctive therapeutic agents. The adjunctive therapeutic agent can be administered to the infected individual (e.g., a person) simultaneously with, before, or after the administration of the compounds of this disclosure.

In some embodiments, compounds of the present disclosure or pharmaceutically acceptable salts thereof are provided for treating or preventing RSV infection. In some embodiments, compounds of the present disclosure or pharmaceutically acceptable salts thereof are provided for manufacturing a medicament for treating or preventing RSV infection.

As described more fully herein, the compounds of this disclosure can be administered to an individual (e.g., a human) infected with RSV in combination with one or more additional therapeutic agents. Further, in some embodiments, when used for the treatment or prevention of RSV, the compounds of this disclosure can be administered in combination with one or more (e.g., one, two, three, four, or more) additional therapeutic agents selected from the group consisting of: RSV combination drugs, RSV vaccines, RSV DNA polymerase inhibitors, immunomodulators, Toll-like receptor (TLR) modulators, interferon α receptor ligands, hyaluronidase inhibitors, respiratory syncytial antigen inhibitors, cytotoxic T-lymphocyte-associated protein 4 (ipi4) inhibitors, cyclic protein inhibitors, RSV viral entry inhibitors, antisense oligonucleotides targeting viral mRNA, short interfering RNA (siRNA), and d dRNAi endonuclease modulators, ribonucleotide reductase inhibitors, RSV E antigen inhibitors, covalently closed circular DNA (cccDNA) inhibitors, farnesol X receptor agonists, RSV antibodies, CCR2 chemokine antagonists, thymosin agonists, cytokines, nucleoprotein modulators, retinoic acid-induced gene 1 stimulators, NOD2 stimulators, phosphatidylinositol 3-kinase (PI3K) inhibitors, indoleamine-2,3-dioxygenase (IDO) pathway inhibitors, PD-1 inhibitors, PD-L1 inhibitors, recombinant thymosin α-1, Bruton's tyrosine kinase (BTK) inhibitors, KDM inhibitors, RSV replication inhibitors, arginase inhibitors, and other RSV drugs.

Microribonucleoviridae

In some embodiments, this disclosure provides a method for treating a person in need of a picornaviridae virus infection, the method comprising administering to the person a therapeutically effective amount of the disclosed compound or a pharmaceutically acceptable salt thereof. Picornaviridae viruses are a heterogeneous class of enteroviruses that cause infections including herpetic pharyngitis, aseptic meningitis, common cold-like syndrome (human rhinovirus infection), nonparalytic myelitis-like syndrome, epidemic chest pain (an acute, febrile, infectious disease typically occurring during epidemics), hand-foot-mouth disease, pancreatitis in children and adults, and severe myocarditis. In some embodiments, the picornaviridae virus infection is a human rhinovirus infection.

In some embodiments, this disclosure provides a method for manufacturing a medicament for treating picoriviral infections in people of need, characterized by using a compound of this disclosure or a pharmaceutically acceptable salt thereof. In some embodiments, this disclosure provides the use of a compound of this disclosure or a pharmaceutically acceptable salt thereof for manufacturing a medicament for treating picoriviral infections in humans. In some embodiments, the picoriviral infection is a human rhinovirus infection.

In some embodiments, this disclosure provides compounds of the present disclosure or pharmaceutically acceptable salts thereof for the treatment of microRNAviridae virus infections in persons of need. In some embodiments, the microRNAviridae virus infection is a human rhinovirus infection.

Flaviviridae

In some embodiments, this disclosure provides a method for treating a person in need of a flaviviridae virus infection, the method comprising administering to the person a therapeutically effective amount of a compound of this disclosure or a pharmaceutically acceptable salt thereof. Representative flaviviridae viruses include, but are not limited to, dengue virus, yellow fever virus, West Nile virus, Zika virus, Japanese encephalitis virus, hepatitis C virus (HCV), and hepatitis B virus (HBV). In some embodiments, the flaviviridae virus infection is dengue virus infection. In some embodiments, the flaviviridae virus infection is yellow fever virus infection. In some embodiments, the flaviviridae virus infection is West Nile virus infection. In some embodiments, the flaviviridae virus infection is Zika virus infection. In some embodiments, the flaviviridae virus infection is Japanese encephalitis virus infection. In some embodiments, the flaviviridae virus infection is hepatitis C virus infection. In some embodiments, the flaviviridae virus infection is hepatitis B virus infection.

In some embodiments, this disclosure provides a method of manufacturing a medicament for treating flaviviridae virus infections in persons of need, characterized by using a compound of this disclosure or a pharmaceutically acceptable salt thereof. In some embodiments, this disclosure provides the use of a compound of this disclosure or a pharmaceutically acceptable salt thereof for manufacturing a medicament for treating flaviviridae virus infections in persons. In some embodiments, the flaviviridae virus infection is dengue virus infection. In some embodiments, the flaviviridae virus infection is yellow fever virus infection. In some embodiments, the flaviviridae virus infection is West Nile virus infection. In some embodiments, the flaviviridae virus infection is Zika virus infection. In some embodiments, the flaviviridae virus infection is hepatitis C virus infection. In some embodiments, the flaviviridae virus infection is hepatitis B virus infection.

In some embodiments, this disclosure provides compounds of the present disclosure or pharmaceutically acceptable salts thereof for treating flaviviridae virus infection in persons of need. In some embodiments, the flaviviridae virus infection is dengue virus infection. In some embodiments, the flaviviridae virus infection is yellow fever virus infection. In some embodiments, the flaviviridae virus infection is West Nile virus infection. In some embodiments, the flaviviridae virus infection is Zika virus infection. In some embodiments, the flaviviridae virus infection is hepatitis C virus infection. In some embodiments, the flaviviridae virus infection is hepatitis B virus infection.

Filoviridae

In some embodiments, this disclosure provides a method for treating a person in need of a filoviridae virus infection, the method comprising administering to the person a therapeutically effective amount of a compound of this disclosure or a pharmaceutically acceptable salt thereof. Representative filoviridae viruses include, but are not limited to, Ebola virus and Marburg virus. In some embodiments, the filoviridae virus infection is an Ebola virus infection.

In some embodiments, this disclosure provides a method of manufacturing a medicament for treating filoviridae virus infections in persons of need, characterized by using a compound of this disclosure or a pharmaceutically acceptable salt thereof. In some embodiments, this disclosure provides the use of a compound of this disclosure or a pharmaceutically acceptable salt thereof for manufacturing a medicament for treating filoviridae virus infections in persons. In some embodiments, the filoviridae virus infection is Ebola virus infection.

In some embodiments, this disclosure provides compounds of the present disclosure or pharmaceutically acceptable salts thereof for the treatment of filoviridae virus infections in persons of need. In some embodiments, the filoviridae virus infection is Ebola virus infection.

VIII. Methods for treating or preventing the worsening of respiratory symptoms caused by viral infection

Compounds of Formula I can also be used to treat or prevent the worsening of respiratory symptoms caused by viral infections in people in need.

In some embodiments, this disclosure provides a method for treating or preventing the exacerbation of respiratory symptoms caused by a viral infection in a person of need, the method comprising administering to a person a therapeutically effective amount of a compound of this disclosure or a pharmaceutically acceptable salt thereof, wherein the respiratory symptom is chronic obstructive pulmonary disease. In some embodiments, the viral infection is caused by respiratory syncytial virus, rhinovirus, or metapneumovirus.

In some embodiments, this disclosure provides a method for treating or preventing the exacerbation of respiratory symptoms caused by a viral infection in a person of need, the method comprising administering to a person a therapeutically effective amount of a compound of this disclosure or a pharmaceutically acceptable salt thereof, wherein the respiratory symptom is asthma. In some embodiments, the viral infection is caused by respiratory syncytial virus, rhinovirus, enterovirus, or metapneumovirus.

In some embodiments, this disclosure provides a method for manufacturing a treatment or prevention of exacerbation of respiratory symptoms caused by a viral infection in a person of need, characterized by the use of a compound of this disclosure or a pharmaceutically acceptable salt thereof, wherein the respiratory symptom is chronic obstructive pulmonary disease. In some embodiments, the viral infection is caused by respiratory syncytial virus, rhinovirus, or metapneumovirus.

In some embodiments, this disclosure provides a method for manufacturing a treatment or prevention of exacerbation of respiratory symptoms caused by a viral infection in a person of need, characterized by the use of a compound of this disclosure or a pharmaceutically acceptable salt thereof, wherein the respiratory symptom is asthma. In some embodiments, the viral infection is caused by respiratory syncytial virus, rhinovirus, enterovirus, or metapneumovirus.

In some embodiments, this disclosure provides for the use of the disclosed compounds or pharmaceutically acceptable salts thereof for the manufacture of a treatment or prevention of exacerbation of respiratory symptoms caused by a viral infection in a human body, wherein the respiratory symptoms are chronic obstructive pulmonary disease (COPD). In some embodiments, the viral infection is caused by respiratory syncytial virus (RSV), rhinovirus, or metapneumovirus.

In some embodiments, this disclosure provides for the use of the disclosed compounds or pharmaceutically acceptable salts thereof for the manufacture of a treatment for or prevention of exacerbation of respiratory symptoms caused by a viral infection in humans, wherein the respiratory symptoms are asthma. In some embodiments, the viral infection is caused by respiratory syncytial virus, rhinovirus, enterovirus, or metapneumovirus.

In some embodiments, this disclosure provides compounds of the present disclosure or pharmaceutically acceptable salts thereof for the treatment or prevention of exacerbations of respiratory symptoms caused by a viral infection in a person in need, wherein the respiratory symptoms are chronic obstructive pulmonary disease. In some embodiments, the viral infection is caused by respiratory syncytial virus, rhinovirus, or metapneumovirus.

In some embodiments, this disclosure provides compounds of the present disclosure or pharmaceutically acceptable salts thereof for the treatment or prevention of exacerbations of respiratory symptoms caused by viral infection in persons in need, wherein the respiratory symptoms are asthma. In some embodiments, the viral infection is caused by respiratory syncytial virus, rhinovirus, enterovirus, or metapneumovirus.

IX. Implementation Examples

abbreviation

Certain abbreviations and acronyms are used to describe experimental details. While most of these are understandable to those skilled in the art, the table below contains a list of acronyms and acronyms that have many of these.

Table 1. List of Abbreviations and Acronyms

Preparative HPLC can be performed on the compounds and intermediates (Phenomenex Gemini 10u C18AXIA 250×21.2mm column, gradient of 30-70% acetonitrile/water containing 0.1% TFA). Following this preparative HPLC process, some compounds are obtained as TFA salts.

A. Intermediate

Intermediate 1.

3-O-benzyl-4-(hydroxymethyl)-1,2-O-isopropylidene-α-D-ribofuranoside (125 g, 402 mmol, 1.0 equivalent) was added to reactor 1. THF (625 mL, 5 volumes) was then added, followed by benzyl bromide (106 mL, 2.2 equivalent). The jacket was set to 0°C. NaHMDS (40% wt% THF solution, 450 mL, 2.2 equivalent) was added while maintaining T _{_int} <10°C. After the addition was complete, the jacket was set to 15°C and stirred for 60 min. The reaction was monitored by TLC in 20% ethyl acetate/80% hexane with staining with cerium ammonium molybdate (CAM). The jacket was set to 5°C. Acetic acid (70 mL, 2.5 equivalent) was dissolved in water (1 L, 8 volumes). The aqueous solution was added to the reaction mixture while maintaining T _{_int} <15°C, allowing the phases to separate. The lower aqueous layer was drained into reactor 2. Concentrate the contents of reactor 1 to approximately 50%. Add MTBE (1.25 L, 10 volumes) to reactor 2. Stir for 15 minutes and separate the layers. Discard the lower aqueous layer. Add the contents of reactor 2 to reactor 1. Add a 14% brine solution (1 L, 8 volumes) to reactor 1. Stir for 15 minutes and discard the lower aqueous layer. Concentrate the organic matter to approximately 50%. Co-evaporate the contents with methanol (3 x 8 volumes). Concentrate intermediate 1 to approximately 4 volumes and use it as is for the next step.

Intermediate 2.

To reactor 1 (197 g, 402 mmol, 1 equivalent), methanol (1 L, 5 volumes), a 4 M HCl solution in dioxane (120 mL, 1.2 equivalents), and concentrated sulfuric acid (1.1 mL, 0.05 equivalents) were added. The mixture was stirred at ambient temperature for 2 hours. The reaction was monitored by TLC using 30% ethyl acetate/70% hexane and CAM staining agent. 5 M KOH solution was slowly added until pH > 7 (100 mL, 1.25 equivalents). The reaction mixture was concentrated to approximately 2 volumes. Ethyl acetate (1 L, 5 volumes) was added. Water (1 L, 5 volumes) was added. The mixture was stirred for 15 minutes. The lower aqueous layer was discharged into reactor 2. Ethyl acetate (1 L, 5 volumes) was added into reactor 2 and stirred for 15 minutes. The lower aqueous layer was discharged and discarded. The remaining contents of reactor 2 were transferred into reactor 1. The contents of reactor 1 were concentrated to approximately 3 volumes. MTBE (400 mL, 2 volumes) was added to reactor 1. Sodium sulfate (400 g, 2S) was added and stirred for 15 minutes. The solid was filtered off and the filter cake was washed with MTBE (200 mL, 1 volume). The organic matter was added to reactor 1. It was concentrated to ~3 volumes. THF (400 mL, 2 volumes) was added and concentrated to ~3 volumes. THF (400 mL, 2 volumes) was added and concentrated to ~3 volumes to obtain intermediate 2. In 30% ethyl acetate/70% hexane, _Rf was ~0.1 and 0.4.

Intermediate 3.

THF (1 L, 5 volumes) and benzyl bromide (60 mL, 1.25 equivalents) were charged into reactor 1 containing crude intermediate 2 (186 g, 402 mmol). The jacket was set to 5 °C. 40% by weight of NaHMDS (245 mL, 1.25 equivalents) was added while maintaining T _{_int} <20 °C. The jacket was set to 15 °C and stirred for 60 min. The reaction progress was monitored by TLC using 30% ethyl acetate/70% hexane and CAM staining agent. The jacket was set to 0 °C. Acetic acid (46 mL, 2 equivalents) was added to water (1 L, 5 volumes). The aqueous solution was added into reactor 1 while maintaining T _{_int} <15 °C. The jacket was set to 15 °C and stirred for 15 min. The phases were separated, and the lower aqueous layer was discharged into reactor 2. MTBE (1 L, 5 volumes) was added to reactor 2 and stirred for 15 min. Reactor 1 was concentrated to approximately 50%. Separate the phases in reactor 2 and discard the lower aqueous layer. Transfer the contents of reactor 2 into reactor 1. Add a 14% brine solution (1 L, 5 volumes). Stir for 15 minutes. Discard the lower aqueous layer. Concentrate to approximately 1 volume in 30% ethyl acetate/70% hexane, concentrating the crude intermediate 3 to an _Rf of approximately 0.8, and use it as is for the next step.

Intermediate 4.

Water (222 mL, 1 volume) was added to reactor 1, which contained intermediate 3 (222 g, 401 mmol) and was jacketed at 20 °C. TFA (667 mL, 3 volumes) was added to maintain T _int < 30 °C. The mixture was stirred at 20 °C for 24 hours. Monitoring was performed by TLC with 30% ethyl acetate/70% hexane and CAM staining agent. The contents of reactor 1 were concentrated to ~2 volumes (removing 550 mL of solvent). MTBE (1.5 L, 7 volumes) was added. The jacket was set to 10 °C. 5M NaOH (600 mL, 7.5 volumes) was added to maintain T _int < 25 °C until pH > 6. 5% _NaHCO3 (1.1 L, 5 volumes) was added to minimize gas release. The mixture was stirred for 15 minutes. The lower aqueous layer was drained and discarded. A 14% brine solution (1.1 L, 5 volumes) was added. The mixture was stirred for 15 minutes. The lower aqueous layer was drained and discarded. The MTBE layer was concentrated to ~4.5 volumes and used as is in the next step. In 30% ethyl acetate/70% hexane, the _Rf of intermediate 4 was ~0.5.

Intermediate 5.

TEMPO (0.6 g, 0.01 equivalent) and KBr (4.53 g, 0.1 equivalent) were added to reactor 1, which was set to -5°C and contained intermediate 4 (216 g, 401 mmol) in 4.5 volumes of MTBE. _{K₂HPO₄ · 3H₂O} (87 g, 1 equivalent) was dissolved in water (1.5 volumes). This solution was added to the reactor. 8.25% bleach solution (425 mL, 1.35 equivalent) was added while maintaining _Tint <10°C (approximately 50 minutes). The jacket was set to 5°C and stirred for 1 hour. The reaction was monitored by TLC with 30% ethyl acetate/70% hexane and CAM staining agent. Sodium thiosulfate (30 g, 0.5 equivalent) was dissolved in water (310 mL, 1.5 volumes). This solution was added to reactor 1 while maintaining _Tint <15°C. The jacket was set to 15°C. The mixture was stirred for 15 minutes. The consumption of bleach was determined using KI banding. The lower aqueous layer was drained and discarded. 1S sodium sulfate was added and stirred for 15 minutes. The solid was filtered off and the filter cake was washed with 1 volume of MTBE. The mixture was concentrated in oil and purified by silica gel chromatography over 45 minutes using a hexane solution of 25S silica/0-50% ethyl acetate to give 3R,4S)-3,4-bis(benzyloxy)-5,5-bis((benzyloxy)methyl)dihydrofuran-2(3H)-one (intermediate 5). ^¹H NMR (400MHz, DMSO- _d⁶ ) δ 7.40–7.24 (m, 18H), 7.24–7.19 (m, 2H), 4.87–4.74 (m, 2H), 4.74–4.68 (m, 2H), 4.61–4.39 (m, 6H), 3.83–3.66 (m, 4H).

Intermediate 6.

Add intermediate 7 (50.83 g, 195.5 mmol, 1.11 equivalents) to reactor 1, followed by THF (5 volumes). Set the jacket of reactor 1 to 0°C. Add intermediate 5 (94.84 g, 176.1 mmol, 1.0 equivalents), THF (5 volumes), and a 0.6 M LaCl3* _2LiCl THF solution (290 mL, 170 mmol, 1 equivalent) to reactor 2. Stir reactor 2 at ambient temperature for 30 minutes. Add TMS-Cl (25.1 mL, 197.2 mmol, 1.12 equivalents) to reactor 1 while maintaining _Tint < 5°C. Stir for 15 minutes. Set the jacket of reactor 1 to -10°C and add a 2.0 M PhMgCl THF solution (185 mL, 370 mmol, 2.1 equivalents) while maintaining _Tint < 0°C. Stir for 15 minutes. Set the jackets of reactors 1 and 2 to -20°C. Add 100 mL (199 mmol, 1.13 equivalents) of 2.0 M iPrMgCl THF solution to reactor 1 while maintaining T _{_int} < -15°C. Stir at -15°C for 15 minutes. Transfer the contents of reactor 1 to reactor 2 while maintaining T _{_int} < -15°C. Stir at -15°C for 60 minutes. Add 66 mL (1145 mmol, 6.5 equivalents) of acetic acid in water (5 volumes) to reactor 2 while maintaining T _{_int} < 20°C. Set the jacket of reactor 2 to 20°C. Stir for 15 minutes. Separate the layers. Add 4 volumes of isopropyl acetate and 3 volumes of water to reactor 2. Stir for 5 minutes. Separate the layers and wash the organic matter with 0.5 M HCl (2 volumes). Separate the layers and wash the organic matter with 2 x 5 volumes of 10% KHCO_₃ (aqueous solution). The organic matter was washed with a 14% saline solution (5 volumes). The layers were separated and the organic matter was dried over sodium sulfate. The solid was filtered off and the liquid was concentrated to give intermediate 6, which proceeded to the next step.

UPLC/MS t _R = 3.759 and 3.825 minutes, MS m/z = 673.33 [M+1];

UPLC/MS system: Waters Acquity Class H

Column: Waters Acquity BEH 1.7μM C18 2.1×50mm

Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid. Gradient: 2% ACN 0-0.5 min. 2% ACN - 98% ACN 0.5 min-3.0 min. 98% ACN 3 min-4 min. 98% ACN - 2% ACN 4 min to 4.5 min. 2% ACN 4.5 min-5 min.

Flow rate: 0.5 mL/min

Quality range: 100-1200

Alternative synthesis of intermediate 6 .

Intermediate 7 (5.90 g, 22.7 mmol, 1.11 equivalents) was added to reactor 1, followed by THF (5 volumes). The jacket of reactor 1 was set to 0°C. Anhydrous _NdCl3 (5.1 g, 20.4 mmol, 1 equivalent), TBACl (6.1 g, 22.1 mmol, 1.08 equivalents), and THF (10 volumes) were added to reactor 2. The jacket of reactor 2 was set to 90°C. ~50% of the THF was distilled off to azeotropically dry the contents. Intermediate 5 (11 g, 20.4 mmol, 1.0 equivalent) was added to reactor 2 and stirred at ambient temperature for 30 minutes. TMS-Cl (2.9 mL, 22.9 mmol, 1.12 equivalents) was added to reactor 1 while maintaining T _{_int} < 5°C. The mixture was stirred for 15 minutes. Set the jacket of reactor 1 to -10°C and charge a 2.0M PhMgCl THF solution (22.2 mL, 44.3 mmol, 2.1 equivalents) while maintaining T_{_int}< 0°C. Stir for 15 minutes. Set the jackets of reactors 1 and 2 to -20°C. Charge a 2.0M MiPrMgCl THF solution (11.5 mL, 199 mmol, 1.13 equivalents) into reactor 1 while maintaining T _{_int} < -15°C. Stir at -15°C for 15 minutes. Transfer the contents of reactor 1 to reactor 2 while maintaining T _{_int}< -15°C. Stir at -15°C for 60 minutes. Charge reactor 2 with acetic acid in water (5 volumes) (66 mL, 1145 mmol, 6.5 equivalents) while maintaining T _{_int} < 20°C. Set the jacket of reactor 2 to 20°C. Stir for 15 minutes. Separate the layers. Isopropyl acetate (4 volumes) and water (3 volumes) were added to reactor 2. The mixture was stirred for 5 minutes. The layers were separated and the organic matter was washed with 0.5 M HCl (2 volumes). The layers were then separated and the organic matter was washed with 2 × 5 volumes of 10% _KHCO3 (aqueous solution). The organic matter was washed with 14% brine (5 volumes). The layers were separated and the organic matter was dried over sodium sulfate. The solid was filtered off and the liquid was concentrated to give intermediate 6, which proceeded to the next step. UPLC/MS t _R = 3.759 and 3.825 min, MS m/z = 673.33 [M+1]

UPLC/MS system: Waters Acquity Class H

Column: Waters Acquity BEH 1.7μM C18 2.1×50mm

Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid. Gradient: 2% ACN 0-0.5 min. 2% ACN - 98% ACN 0.5 min-3.0 min. 98% ACN 3 min-4 min. 98% ACN - 2% ACN 4 min to 4.5 min. 2% ACN 4.5 min-5 min.

Flow rate: 0.5 mL/min

Quality range: 100-1200

Intermediate 8.

Intermediate 6 (~118 g, 176 mmol) in DCM (10 volumes) was charged into the reactor. The jacket was set to -20°C. Triethylsilane (73 mL, 456 mmol, 2.6 equivalents) was added. A 46.5% boron trifluoride solution in diethyl ether (72 mL, 263.1 mmol, 1.5 equivalents) was added while maintaining T _int < -15°C. The mixture was stirred for 30 minutes. The jacket was set to 0°C. 5M NaOH (175 mL, 877 mmol, 5 equivalents) was added while maintaining T _int < 20°C. The jacket was set to 20°C. Water (10 volumes) was added. The layers were separated. The organic layer was concentrated. The aqueous layer was back-extracted with ethyl acetate (2 x 5 volumes). The organic matter was combined and washed with 14% brine (8 volumes). The organic matter was dried over sodium sulfate, filtered, and concentrated. Intermediate 8 was separated by silica gel chromatography using a 50-100% ethyl acetate solution in hexane. ¹ H NMR (400MHz, DMSO-d ₆ )δ7.83(s,1H),7.71(brs,2H),7.37-7.14(m,20H),6.83(d,J＝4.5Hz,1H),6.61(d,J＝4.4Hz,1H),5.47(d,J＝7.0Hz ,1H),4.68(d,J＝11.6Hz,1H),4.61-4.43(m,8H),4.34(d,J＝4.8Hz,1H),3.81-3.64(m,3H),3.62(d,J＝10.0Hz,1H).

UPLC/MS t _R = 3.919 minutes, MS m/z = 657.32 [M+1]

UPLC/MS system: Waters Acquity Class H

Column: Waters Acquity BEH 1.7μM C18 2.1×50mm

Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid. Gradient: 2% ACN 0-0.5 min. 2% ACN - 98% ACN 0.5 min-3.0 min. 98% ACN 3 min-4 min. 98% ACN - 2% ACN 4 min to 4.5 min. 2% ACN 4.5 min-5 min.

Flow rate: 0.5 mL/min

Quality range: 100-1200

Intermediate 9.

Intermediate 8 (21.7 g, 33 mmol, 1 equivalent) was loaded into a round-bottom flask purged with nitrogen. THF (3 volumes), 2,2-dimethoxypropane (3 volumes), and pTsOH (6.6 g, 34.6 mmol, 1.05 equivalent) were added. The flask was cooled in a dry ice bath. 10% Pd/C was added. The flask was evacuated and backfilled with hydrogen three times. The mixture was stirred at ambient temperature and pressure. The mixture was quenched with saturated _NaHCO3 (aqueous solution) to pH > 7. The catalyst was filtered off and the filter cake was washed with methanol (2.5 volumes). The mixture was partitioned between ethyl acetate (10 volumes) and brine (10 volumes). The layers were separated and the organic matter was dried over sodium sulfate. The solid was filtered off and concentrated to give intermediate 9, which proceeded to the next step.

UPLC/MS t _R = 2.580 minutes, MS m/z = 477.14 [M+1]

UPLC/MS system: Waters Acquity Class H

Column: Waters Acquity BEH 1.7μM C18 2.1×50mm

Solvents: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid

Gradient: 2% ACN 0-0.5 minutes. 2% ACN - 98% ACN 0.5-3.0 minutes. 98% ACN 3-4 minutes. 98% ACN - 2% ACN 4-4.5 minutes. 2% ACN 4.5-5 minutes.

Flow rate: 0.5 mL/min

Quality range: 100-1200

Intermediate 10.

Add intermediate 9 (12.3 g, 32.7 mmol, 1 equivalent), THF (10 volumes), and di-tert-butyl dicarbonate (14.4 g, 65.4 mmol, 2.0 equivalent) to a round-bottom flask. Add DMAP (10.1 g, 81.7 mmol, 2.5 equivalent) fractionally to minimize gas release and exothermic reactions. Stir for 60 minutes to generate a mixture of mono- and di-Boc compounds. Concentrate the reaction to approximately 50%. Add MTBE (10 volumes) and 2.0 M HCl (3.5 volumes). Separate the layers. Back-extract the aqueous layer with an aqueous solution of ethyl acetate (10 volumes). Combine the organic compounds and wash with saturated _NaHCO3 (aqueous solution). Concentrate the organic compounds. Add methanol (10 volumes) to the crude mixture, followed by KOH (3.67 g, 2.0 equivalent). Stir until di-Boc compounds are converted to mono-Boc compounds. Concentrate the reaction. Partitioned with ethyl acetate (10 v/v) and water (10 v/v). The layers were separated and concentrated. Methanol (8 v/v) was added to the crude product, followed by pTsOH (6.5 g, 34.2 mmol, 1.05 equivalents). The mixture was stirred at ambient temperature. Quenched with 5.25% _NaHCO3 (aqueous solution) (80 mL, 52 mmol, 1.5 equivalents). The mixture was concentrated to approximately 25% and stirred overnight. The solid was filtered off and the filter cake was washed with MTBE (8 v/v). The mixture was dried in a vacuum oven to give intermediate 10. ¹ H NMR (400MHz, DMSO-d ₆ )δ10.45(s,1H),8.20(s,1H),7.19(d,J＝4.3Hz,1H),6.95(d,J＝4.7Hz,1H),5.35(d,J＝5.2Hz,1H),5.06(t,J＝5.7H z,1H),4.79-4.74(m,2H),4.45(t,J=5.8Hz,1H),3.73-3.46(m,3H),3.40-3.30(m,1H),1.50(s,12H),1.27(s,3H).

UPLC/MS t _R = 2.767 minutes, MS m/z = 437.17 [M+1]

UPLC/MS system: Waters Acquity Class H

Column: Waters Acquity BEH 1.7μM C18 2.1×50mm

Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid. Gradient: 2% ACN 0-0.5 min. 2% ACN - 98% ACN 0.5 min-3.0 min. 98% ACN 3 min-4 min. 98% ACN - 2% ACN 4 min to 4.5 min. 2% ACN 4.5 min-5 min.

Flow rate: 0.5 mL/min

Quality range: 100-1200

Intermediate 11.(3R,4R,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-3,4-bis(benzyl) 5-((benzyloxy)methyl)tetrahydrofuran-2-ol

The product is prepared according to WO2015/069939. For example, pages 43-45 of WO2015/069939 provide a method for preparing the compound (identified as compound 1d in WO2015/069939). Alternatively, it is prepared as follows.

A cylindrical reactor equipped with a reprocessing curve top stirrer, thermocouples, and an _N2 bubbler was charged with anhydrous _NdCl3 (60.00 g, 239 mol, 1.00 equivalent), n- _Bu4 NCl (71.51 g, 239 mmol, 1.0 equivalent), and THF (900 g). The resulting mixture was concentrated to approximately 450 mL under ambient pressure using a jacket temperature of 90 °C under an _N2 pad. THF (500 g) was added, and the distillation was repeated (twice). The mixture was cooled to 22 °C and intermediate 12 ((3R,4R,5R)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)dihydrofuran-2(3H)-one) (100.02 g, 239 mmol, 1.00 equivalent) was added. After 30 minutes, the mixture was cooled to -20 °C and held. Iodopyrrolidinyl triazine intermediate 7 (68.52 g, 264 mmol, 1.10 equivalents) and THF (601 g) were combined in a separate reaction flask and cooled to 0 °C. TMSCl (28.64 g, 264 mmol, 1.10 equivalents) was slowly added, and after approximately 30 minutes, the mixture was cooled to 10 °C. PhMgCl (2.0 M THF solution, 270.00 g, 5.18 mmol, 2.17 equivalents) was slowly added, and the mixture was stirred for approximately 30 minutes and cooled to -20 °C. i-PrMgCl (2.0 M THF solution, 131.13 g, 269 mmol, 1.13 equivalents) was slowly added. After approximately 2 hours, the Grignard reaction mixture was transferred via a sleeve to a lactone/ _NdCl3 / _nBu4 NCl/THF mixture, and the mixture was stirred at approximately 20 °C. After approximately 16 hours, a solution of acetic acid (100 g) in water (440 g) was added, and the mixture was warmed to 22°C. ⁱ PrOAc (331 g) was added, and the layers were separated. The organic layer was washed with 10% _KHCO3 (aqueous solution) (2 × 500 g) and 10% NaCl (aqueous solution) (500 g). The organic layer was concentrated to approximately 450 mL and loaded with ⁱ PrOAc (870 g). The organic mixture was washed with water (2 × 500 g) and concentrated to approximately 450 mL. ⁱ PrOAc (435 g) was loaded, and the mixture was concentrated to approximately 450 mL. The mixture was filtered, and the residue was rinsed with 129 g of ⁱ PrOAc beforehand. The filtrate was concentrated to approximately 250 mL and MTBE (549 g) was loaded, and the mixture was adjusted to 22°C. Seed crystals (0.15 g) were loaded, followed by n-heptane (230 mL), and the mixture was cooled to 0°C. The solid was separated by filtration and pre-washed with a mixture of MTBE/n-heptane (113 g/30 g). The solid was dried under vacuum at 35 °C to give intermediate 11 (79% yield and 99.92% LC purity).

Intermediate 13.(3aS,4R,6S,6aS)-6-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-4- (((tert-butyldimethylsilyl)oxy)methyl)-2,2-dimethyltetrahydrofurano[3,4-d][1,3]dioxane-pentane 4-carboxynitrile

The product was prepared according to WO2015/069939. For example, pages 127-138 of WO2015/069939 provide a method for preparing the compound (identified as compound 14k in WO2015/069939).

Alternatively, intermediate 10 is prepared as described above and then converted to intermediate 13 as described in WO2015/069939 (compound 14f in WO2015/069939 is converted to compound 14k in WO2015/069939, as described on pages 133-138 of WO2015/069939).

Alternatively, intermediate 11 is prepared as described above, and then converted to intermediate 13 as described in WO2015/069939 (compound 1d in WO2015/069939 is converted to compound 14k in WO2015/069939, as described on pages 45-46 and 127-138 of WO2015/069939).

Intermediate 14.(3aS,4R,6S,6aS)-6-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-4- (hydroxymethyl)-2,2-dimethyltetrahydrofurano[3,4-d][1,3]dioxacyclopentene-4-carboxynitrile

Intermediate 13 (8.41 g, 18.87 mmol) was placed in 100 mL of THF. A single addition of 1.0 M TBAF in THF (28.31 mL, 28.31 mmol) was made at ambient temperature. Stirring was allowed for 10 minutes at ambient temperature. The reaction was confirmed to be complete by LC-MS. The reaction mixture was quenched with water and the organic matter was removed under reduced pressure. The crude product was partitioned between EtOAc and water. The layers were separated and the aqueous layer was washed with EtOAc. The organic matter was combined and dried over sodium sulfate. The solids were filtered off and the solvent was removed under reduced pressure. The crude product was purified by silica gel chromatography using a 120 g column with 0–10% _{CH3OH in CH2Cl2} solution. LC/MS: t _R = 0.76 min, MS m/z = 332.14 [M+1]; LC system: Thermo Accela 1250U HPLC. MS system: Thermo LCQFleet; Column: 2.6 μX B-C18 100A, 50 × 3.00 mm. Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid. Gradient: 0 min - 2.4 min 2 - 100% ACN, 2.4 min - 2.80 min 100% ACN, 2.8 min - 2.85 min 100% - 2% ACN, 2.85 min - 3.0 min 2% ACN, 1.8 mL/min. ¹ HNMR (400MHz, DMSO-d ₆ )δ7.87-7.80(m,3H),6.85(d,J＝4.5Hz,1H),6.82(d,J＝4.5Hz,1H),5.74(t,J＝5.8Hz,1H),5.52(d,J＝4.2Hz,1 H), 5.24 (dd, J = 6.8, 4.2Hz, 1H), 4.92 (d, J = 6.8Hz, 1H), 3.65 (dd, J = 6.1, 1.7Hz, 2H), 1.61 (s, 3H), 1.33 (s, 3H).

Intermediate 15.(S)-Cyclohexyl-2-aminopropionate

TMSCl (20 mL) was added to a mixture of L-alanine (5 g, 56.12 mmol) and cyclohexanol (56 g, 561 mmol). The resulting mixture was stirred at about 70 °C for about 15 hours and concentrated under vacuum at about 80 °C, co-evaporated with toluene, dissolved in hexane, and stirred at about room temperature, during which time a solid precipitated. The solid was collected by filtration, and the filter cake was washed several times with a 5% EtOAc solution in hexane and dried under high vacuum for about 15 hours to give the product. ^1H NMR (400MHz, chloroform-d) δ 8.76 (s, 3H), 4.85 (tt, J = 8.7, 3.8Hz, 1H), 4.17 (p, J = 6.5Hz, 1H), 1.84 (dd, J = 9.9, 5.5Hz, 2H), 1.70 (d, J = 7.3Hz, 5H), 1.57–1.42 (m, 3H), 1.32 (m, 3H).

Intermediate 16.(2S)-2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)cyclohexyl propionate

Intermediate 15 (3.4 g, 16.37 mmol) was dissolved in methylene chloride (45 mL), cooled to -78 °C, and phenyl phosphate dichloride (2.45 mL, 16.37 mmol) was rapidly added. Triethylamine (4.54 mL, 32.74 mmol) was added over 60 minutes at -78 °C, followed by a single addition of 4-nitrophenol (2277 mg, 16.37 mmol). Triethylamine (2.27 mL, 16.37 mmol) was added over 60 minutes at -78 °C. The resulting mixture was stirred at -78 °C for 2 hours, diluted with methylene chloride (100 mL), washed twice with water and then with brine, dried over sodium sulfate, and concentrated under vacuum. The residue was purified by silica gel column chromatography (0 to 20% EtOAc in hexane solution) to give the product. ^¹H NMR (400MHz, chloroform-d) δ 8.22 (m, 2H), 7.46–7.30 (m, 4H), 7.29–7.09 (m, 3H), 4.76 (m, 1H), 4.20–4.02 (m, 1H), 3.92 (m, 1H), 1.87–1.64 (m, 4H), 1.54 (m, 2H), 1.46–1.18 (m, 7H). ^³¹P NMR (162MHz, chloroform-d) δ -2.94, -3.00. MS m/z = 449 (M+H) ⁺ .

B. Compounds

Example 1 Preparation of (2S)-2-(((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)cyclohexyl propionate (Formula Ia)

At room temperature, N,N-diisopropylethylamine (0.13 mL, 0.75 mmol) was added dropwise to a mixture of intermediate 14 (99 mg, 0.30 mmol), intermediate ₁₆ (201 mg, 0.45 mmol), and MgCl₂ (43 mg, 0.45 mmol) in DMF (4 mL). The resulting mixture was stirred at room temperature for 15 hours and purified by preparative HPLC (Phenominex Synergi 4u Hydro-RR 150 × 30 mm column, 10%–100% acetonitrile/water gradient) to obtain an intermediate, which was dissolved in ACN (3 mL) and c-HCl (0.1 mL). The resulting mixture was stirred at 50 °C for 2 hours, cooled, and purified by preparative HPLC (Phenominex Synergi 4u Hydro-RR 150 × 30 mm column, 10%–80% acetonitrile/water gradient) to obtain the product. ^¹H NMR (400 MHz, methanol-d⁴) δ 7.80 (s, 0.5H), 7.78 (s, 0.5H), 7.42–7.05 (m, 5H), 6.84 (m, 1H), 6.73 (m, 1H), 5.50 (m, 1H), 4.64 (m, 2H), 4.57–4.25 (m, 3H), 3.86 (m, 1H), 1.91–1.61 (m, 4H), 1.61–1.09 (m, 9H). ^³¹P NMR (162 MHz, methanol-d⁴) δ 3.3. MS m/z = 601 (M+H) ^⁺ .

Separation of diastereomers. The product was purified by chiral preparative HPLC (Chiralpak IA, 150 × 4.6 mm, 70% heptane, 30% ethanol).

Example 2. Preparation of ((R)-(((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine cyclohexyl ester (Formula Ib)

First eluted diastereomer of Example 1: ^¹H NMR (400MHz, methanol- _d4) )δ7.78(s,1H),7.34-7.23(m,2H),7.19-7.10(m,3H),6.85(d,J＝4.5Hz, 1H),6.73(d,J＝4.5Hz,1H),5.51(d,J＝5.0Hz,1H),4.69(td,J＝8.8,4.2Hz ,1H),4.62(t,J＝5.3Hz,1H),4.53-4.44(m,2H),4.36(dd,J＝10.9,5.2Hz ,1H),3.86(dq,J=9.4,7.1Hz,1H),1.85-1.62(m,4H),1.58-1.20(m,9H). ³¹ P NMR (162MHz, Methanol-d ₄ ) δ3.31.

Example 3. Preparation of ((S)-(((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine cyclohexyl ester (Formula I)

The second eluted diastereomer of Example 1: ^¹H NMR (400 MHz, methanol- _d⁴ ) δ 7.80 (s, 1H), 7.37–7.27 (m, 2H), 7.26–7.13 (m, 3H), 6.84 (d, J = 4.5 Hz, 1H), 6.73 (d, J = 4.5 Hz, 1H), 5.49 (d, J = 5.0 Hz, 1H), 4.71–4.56 (m, 2H), 4.46 (d, J = 5.6 Hz, 1H), 4.45–4.30 (m, 2H), 3.97–3.77 (m, 1H), 1.80–1.61 (m, 4H), 1.55–1.21 (m, 9H). ^¹¹P NMR (162 MHz, methanol- _d⁴ ) δ 3.31.

Example 4. Synthesis of ((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine cyclohexyl ester (compound 3)

(2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-3,4-hydroxy-2-(hydroxymethyl)-tetrahydrofuran-2-carboxynitrile was prepared according to WO2015/069939. For example, pages 43-54 of WO2015/069939 provide a method for preparing the compound identified as compound 1 in WO2015/069939.

(2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-3,4-dihydroxy-2-(hydroxymethyl)tetrahydrofuran-2-carboxynitrile (0.149 g, 0.512 mmol) was placed in anhydrous THF and concentrated. The resulting residue was placed under high vacuum for 1.5 h. The residue was then dissolved in NMP (4 mL), followed by the addition of THF (1 mL). The solution was cooled in an ice bath, and a 1 M solution of tert-BuMgCl in THF (0.767 mL, 0.767 mmol) was added, causing the formation of a white precipitate. After 5 min, the cold bath was removed, the mixture was sonicated to disperse the precipitate, and the reaction was stirred at room temperature for 10 min. A solution of intermediate ((4-nitrophenoxy)(phenoxy)phosphoryl)-L-alanine isopropyl ester (0.251 g, 0.614 mmol; WO2011123668) in THF (0.9 mL) was added. The reaction was stirred at room temperature and the progress was monitored by LC/MS. After 1 hour and 45 minutes, the reaction was cooled in an ice bath and quenched by adding ice-cold AcOH (0.25 mL). The ice bath was removed and stirring was continued at room temperature for 5 minutes. Volatiles were removed by evaporation and the product was separated from the residue by HPLC. ^¹H NMR (400 MHz, methanol- _d⁴⁻ , chemical shifts marked with an asterisk (*) indicate the shifts of the relevant protons in the present second isomer) δ 7.81 (s, 0.41H), 7.79* (s, 0.59H), 7.36–7.12 (m, 5H), 6.85 (m, 1H), 6.74 (m, 1H), 5.50 (m, 1H), 4.97–4.85 (m, 1H), 4.63 (m, 1H), 4.54–4.32 (m, 3H), 3.85 (m, 1H), 1.25 (d, J = 7.1 Hz, 2H), 1.20* (d, J = 6.3 Hz, 4H), 1.16 (t, J = 6.3 Hz, 3H). ^¹¹P NMR (162 MHz, methanol- _d⁴⁻ ) δ 3.30 (s). MS m/z = 561.03 [M+1].

Example 5. Synthesis of (S)-2-(((S)-(((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)isopropyl propionate (compound 4)

(2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-3,4-hydroxy-2-(hydroxymethyl)-tetrahydrofuran-2-carboxynitrile was prepared as described in Example 4.

((S)-(4-nitrophenoxy)(phenoxy)phosphoryl)-L-alanine isopropyl ester was prepared as described by Cho et al., J. Med. Chem. 2014, 57, 1812-1825.

(2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-3,4-dihydroxy-2-(hydroxymethyl)tetrahydrofuran-2-carboxynitrile (50 mg, 0.172 mmol) and ((S)-(4-nitrophenoxy)(phenoxy)phosphoryl)-L-alanine isopropyl ester (84 mg, 0.206 mmol) were mixed in anhydrous N,N-dimethylformamide (2 mL). Magnesium chloride (36 mg, 0.378 mmol) was added in a single addition. The reaction mixture was heated at 50 °C. N,N-diisopropylethylamine (75 μL, 0.43 mmol) was added, and the reaction mixture was stirred at 50 °C for 4.5 h. The reaction mixture was cooled, diluted with ethyl acetate (30 mL), washed with 5% citric acid aqueous solution (10 mL), and then washed with brine (10 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by _SiO2 column chromatography (4g _SiO2 combined with a rapid HP gold column, 0-2%-5% methanol/dichloromethane) to obtain the final product. ¹ H NMR (400MHz, methanol-d ₄ )δ7.79(s,1H),7.36-7.25(m,2H),7.25-7.12(m,3H),6.84(d,J＝4.5Hz, 1H),6.73(d,J＝4.5Hz,1H),5.49(d,J＝5.1Hz,1H),4.91-4.84(m,1H),4. 62(dd,J＝5.6,5.0Hz,1H),4.47(d,J＝5.6Hz,1H),4.45-4.30(m,2H),3.85(dq,J＝10.0,7.1Hz,1H),1.25(d,J＝7.2Hz,3H),1.15(t,J＝6.4Hz,6H). ³¹ P NMR (162MHz, Methanol-d ₄ ) δ3.31. MS m/z=561.0[M+1],559.0[M-1].

Example 6. Synthesis of (2S)-pentane-3-yl 2-(((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-2-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propionate (compound 5)

(S)-Pentane-3-yl-2-aminopropionate salt. TMSCl (20 mL) was added to a mixture of L-alanine salt (5 g, 56.12 mmol) and 3-hydroxypentane (50 mL). The resulting mixture was stirred at 70 °C for 15 h and concentrated at 80 °C in a rotary evaporator. The resulting solid was ground with a 5% hexane solution of EtOAc, filtered, washed several times with a 5% hexane solution of EtOAc, and dried overnight under high vacuum to give the intermediate. ^¹H NMR (400 MHz, chloroform-d) δ 8.79 (s, 3H), 4.83 (p, J = 6.2 Hz, 1H), 4.19 (p, J = 6.5 Hz, 1H), 1.72 (d, J = 7.2 Hz, 3H), 1.67–1.52 (m, 4H), 0.88 (td, J = 7.5, 1.7 Hz, 6H).

(2S)-pentan-3-yl 2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)propionate. (S)-pentan-3-yl 2-aminopropionate (1.00 g, 5.11 mmol) was suspended in methylene chloride (15 mL), cooled to -78 °C, and phenyl phosphate dichloride (0.76 mL, 5.11 mmol) was rapidly added. Triethylamine (1.42 mL, 10.22 mmol) was added over 30 minutes at -78 °C, and the resulting mixture was stirred at -78 °C for 30 minutes. Then, 4-nitrophenol (711 mg, 5.11 mmol) was added in a single addition, followed by triethylamine (0.71 mL, 5.11 mmol) over 30 minutes at -78 °C. The mixture was stirred at -78 °C for 30 minutes, washed with water and brine, dried over sodium sulfate, and concentrated under vacuum. The residue was purified by silica gel column chromatography (0 to 20% EtOAc in hexane solution) to give (2S)-pentane-3-yl 2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)propionate. ^¹H NMR (400 MHz, chloroform-d) δ 8.22 (m, 2H), 7.46–7.30 (m, 4H), 7.31–7.14 (m, 3H), 4.78 (m, 1H), 4.27–4.04 (m, 1H), 3.98–3.77 (m, 1H), 1.72–1.45 (m, 4H), 1.42 (m, 3H), 0.84 (m, 6H). ^¹¹P NMR (162 MHz, chloroform-d) δ -2.99, -3.06. MS m/z = 437 (M+H) ⁺ .

(2S)-pentane-3-yl 2-(((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-2-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propionate. At room temperature, N,N-diisopropylethylamine (0.087 mL, 0.50 mmol) was added dropwise to a mixture of intermediate 14 (66 mg, 0.30 mmol), (2S)-pentane-3-yl 2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)propionate (170 mg, 0.39 mmol) and _MgCl2 (28 mg, 0.30 mmol) in DMF (3 mL). The resulting mixture was stirred at 60°C for 15 hours and purified by HPLC (0 to 100% aqueous solution of ACN) to obtain an intermediate. This intermediate was dissolved in ACN (3 mL) and C-HCl (0.1 mL) was added. The resulting mixture was stirred at 50°C for 2 hours and purified by preparative HPLC (Phenominex Synergi 4uHydro-RR 150×30 mm column, 5%–100% acetonitrile/water gradient) to obtain the product. ¹ H NMR(400MHz,Methanol-d4)δ7.79(m,1H),7.36-7.07(m,5H),6.84(m,1H),6.73(m,1H),5.50(m,1H),4.76-4 .59(m,2H),4.54-4.40(m,2H),4.34(m,1H),3.89(m,1H),1.63-1.42(m,4H),1.27(m,3H),0.91-0.75(m,6H). ³¹ P NMR (162MHz, methanol-d4) δ 3.37, 3.29. MS m/z=589(M+H) ⁺ .

Example 7. Synthesis of 2-((S)-(((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)alanine ethyl butyl ester (compound 6)

Prepare 2-((S)-(4-nitrophenoxy)(phenoxy)(phosphoryl)-L-alanine ethyl butyl ester as described in WO 2016/069825.

At room temperature, tetrahydrofuran (8.5 mL) was added to a mixture of intermediate 14 (700 mg, 2.113 mmol), 2-((S)-(4-nitrophenoxy)(phenoxy)(phosphoyl)-L-alanine ethyl butyl ester (998 mg, 2.218 mmol), and magnesium chloride (302 mg, 3.169 mmol), followed by N,N-diisopropylethylamine (0.92 mL, 5.282 mmol). The resulting mixture was stirred at 50 °C for 3 hours. The reaction mixture was then concentrated under reduced pressure, and the resulting residue was diluted with saturated sodium chloride solution and dichloromethane. The layers were separated, and the organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude residue was subjected to chromatography on _{a SiO2} column (80 g SiO2). Purification was performed using a combination of _two rapid HP gold columns (100% dichloromethane - 14% methanol in dichloromethane solution as eluent). The obtained pure substance was dissolved in anhydrous acetonitrile (10 mL) and cooled in an ice bath, followed by the dropwise addition of concentrated hydrochloric acid (4 mL, 48 mmol). The reaction mixture was stirred at room temperature for 1 hour. After ₁ hour, the reaction mixture was cooled in an ice bath and diluted with water. The solution was neutralized with 3N sodium hydroxide and extracted with dichloromethane. The organic layer was separated, dried over sodium sulfate, filtered, and concentrated. The obtained residue was purified by SiO2 column chromatography (40 g _SiO2 combination rapid HP gold column, 100% dichloromethane - 20% methanol in ^{dichloromethane} solution) to obtain the product. NMR (400 MHz, methanol-d4) δ 7.80 (s, 1H), 7.38–7.29 (m, 2H), 7.27–7.13 (m, 3H), 6.84 (d, J = 4.5 Hz, 1H), 6.74 (d, J = 4.5 Hz, 1H), 5.49 (d, J = 5.0 Hz, 1H), 4.61 (t, J = 5.3 Hz, 1H), 4.49–4.29 (m, 3H), 4.04–3.82 (m, 3H), 1.43 (dq, J = 12.5, 6.1 Hz, 1H), 1.37–1.23 (m, 7H), 0.84 (td, J = 7.5, 1.1 Hz, 6H). ³¹ P NMR (162 MHz, acetonitrile-d3) δ 2.73. MS m/z = 603[M+1].

Example 8. Synthesis of 2-((R)-(((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine ethyl butyl ester (compound 7)

This compound was prepared by the decomposition of the Sp and Rp diastereomers from Example 34 of WO 2015/069939. Compound 7 was purified from Example 34 of WO 2015/069939 via chiral preparation of SFC (Chiralpak AD-H, 30% ethanol isocratic), as the first eluted diastereomer from Example 34 of WO 2015/069939: ^¹H NMR (400 MHz, methanol- _d4) )δ7.78(s,1H),7.32-7.24(m,2H),7.19-7.10(m,3H),6.84(d,J＝4.5Hz,1H),6.72(d,J＝4.5Hz,1H),5.51(d,J＝5.0Hz,1H),4.63( t,J＝5.3Hz,1H),4.54-4.43(m,2H),4.36(m,1H),4.07-3.84(m,3H),1.53-1.42(m,1H),1.38-1.24(m,7H),0.86(t,J＝7.5Hz,6H). ³¹ P NMR (162 MHz, methanol-d ₄ ) δ 3.26 (s). HPLC: t _R = 5.068 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6u 110A, 100×4.6mm; solvent: A: water containing 0.1% TFA; B: acetonitrile containing 0.1% TFA; gradient: 2%-98% B, 8.5 min gradient, 1.5 mL/min.

Example 9. Synthesis of ((S)-(((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine ethyl ester (compound 8)

((S)-(perfluorophenoxy)(phenoxy)phosphoyl)-L-alanine ethyl ester

At -78°C, phenoxyphosphoryl dichloride (0.368 mL, 2.465 mmol) was added in a single addition to a solution of L-alanine ethyl ester-HCl (631 mg, 2.465 mmol) in DCM (15 mL), followed by dropwise addition of triethylamine (0.68 mL, 4.93 mmol) over 5 minutes at -78°C. After removing the dry ice bath, the resulting mixture was stirred for 30 minutes and then cooled to -78°C. Pentafluorophenol (454 mg, 2.465 mmol) was added in a single addition, followed by dropwise addition of triethylamine (0.34 mL, 2.465 mmol) over 5 minutes at -78°C. After removing the dry ice bath, the resulting mixture was stirred for 1 hour, then diluted with DCM, washed with brine, concentrated under vacuum, and the resulting residue was purified by silica gel column chromatography (0 to 60% EtOAc in hexane solution) to give a diastereomeric mixture, to which diisopropyl ether (4 mL) was added. The suspension was sonicated and filtered. ^1H NMR analysis of the filter cake showed it to be a 3:1 mixture. Diisopropyl ether (5 mL) was added to the filter cake, and the suspension was heated at 70 °C to a clear solution. After removing the heating bath, needle-like crystals began to form, and after 10 minutes, the mixture was filtered, and the filter cake was dried under high vacuum for 30 minutes to obtain the Sp isomer.

Diastereomer mixture: ^1H NMR (400MHz, chloroform-d) δ 7.43-7.30 (m, 2H), 7.32-7.17 (m, 3H), 4.29-4.11 (m, 3H), 3.94 (m, 1H), 1.52-1.42 (m, 3H), 1.28 (q, J = 7.0Hz, 3H).

Sp isomers: ^¹H NMR (400 MHz, acetonitrile-d³) δ 7.50–7.36 (m, 2H), 7.32–7.21 (m, 3H), 4.75 (t, J = 11.5 Hz, 1H), 4.17–3.98 (m, 3H), 1.37 (dd, J = 7.1, 1.1 Hz, 3H), 1.22 (t, J = 7.1 Hz, 3H). ^¹¹P NMR (162 MHz, acetonitrile-d³) δ -0.51. ^¹⁹F NMR (376 MHz, acetonitrile-d³) δ -155.48–155.76 (m), -162.73 (td, J = 21.3, 3.7 Hz), -165.02–165.84 (m). LCMS m/z = 440.5 (M-ethyl + H), t _R = 1.57 min; LC system: Thermo Accela 1250U HPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μXB-C18 100A, 50×3.0mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min–1.8 min 2%–100% acetonitrile, 1.8 min–1.85 min 100%–2% acetonitrile, 1.85 min–2.00 min 2% ACN, 1800 μl/min.

N,N-diisopropylethylamine (0.20 mL, 1.13 mmol) was added dropwise to a mixture of intermediate 14 (150 mg, 0.45 mmol), ((S)-(perfluorophenoxy)(phenoxy)phosphoryl)-L-alanine ethyl ester (298 mg, 0.68 mmol), and _MgCl₂ (65 mg, 0.68 mmol) in THF (6 mL). The resulting mixture was stirred at 50 °C for 2 hours, cooled, diluted with EtOAc (150 mL), washed with brine (50 mL x 2), dried, concentrated under vacuum, and then dissolved in acetonitrile (6 mL), with c-HCl (0.3 mL) added in an ice bath. The resulting mixture was stirred in an ice bath for 1 hour and then at room temperature for 1 hour. It was treated with saturated NaHCO₃ ( ₂ mL) and purified by HPLC (Phenomenex Gemini-NX 10 μC18 110°A 250 × 30 mm column, 5%–70% acetonitrile/water gradient over 25 minutes) to obtain the product. ^¹H NMR (400 MHz, methanol-d⁴) δ 7.80 (s, 1H), 7.31 (d, J = 7.7 Hz, 2H), 7.25–7.14 (m, 3H), 6.84 (d, J = 4.5 Hz, 1H), 6.73 (d, J = 4.6 Hz, 1H), 5.49 (d, J = 5.1 Hz, 1H), 4.62 (t, J = 5.3 Hz, 1H), 4.46 (d, J = 5.3 Hz, 1H). ,J＝5.6Hz,1H),4.40(dd,J＝10.9,6.2Hz,1H),4.33(dd,J＝10.9,5.4Hz,1H),4.11-3.98( m, 2H), 3.87 (dd, J = 9.9, 7.1 Hz, 1H), 1.25 ( dd, J = 7.1, 1.0 Hz, 3H), 1.16 ( t, J = 7.1 Hz, 3H). ³¹ P NMR (162 MHz, methanol-d4) δ 3.26. LCMS: MS m/z = 547.12 [M+1]; t _R = 0.76 min; LC system: Thermo Accela 1250U HPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μXB-C18 100A, 50×3.0mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min-1.8 min 2%-100% acetonitrile, 1.8 min-1.85 min 100%-2% acetonitrile, 1.85 min-2.00 min 2% ACN, 1800 μl/min. HPLC: t _R = 4.03 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6u 110A, 100×4.6mm; solvent: A: water containing 0.1% TFA; B: acetonitrile containing 0.1% TFA; gradient: 2%-98% B, 8.5 min gradient, 1.5 mL/min.

C. Biological Examples

Example 10. DENV Pol IC ₅₀

In the DENV2-NS5 polymerase assay, a 244-nucleotide heterologous polymeric RNA (sshRNA) without secondary structure, having the sequence 5'-(UCAG)20(UCCAAG)14(UCAG)20-3' (SEQ ID NO:1), was used as a template with a 5'-CUG-3' primer. Six 2-fold dilutions of the compound, starting at 200 nM and without an inhibitor control, were seeded into 96-well plates. 100 nM DENV2 NS5 was pre-incubated for 5 minutes at room temperature in a reaction mixture containing 40 mM Tris-HCl (pH 7.5), 10 mM NaCl, 3 mM DTT, 0.2 units/μL RNasin Plus RNase inhibitor, 200 ng/μL sshRNA, 20 μM CUG, and 2 mM MgCl2. The enzyme mixture was added to the compound dilution buffer, and the reaction was initiated by adding 20 μM of three natural NTPs plus ₂ μM of analogues: a base-matching competitive natural NTP mixture (containing 1:100 α-33P-NTPs). After 90 min at 30 °C, 5 μL of the reaction mixture was spotted onto DE81 anion exchange paper. The filter paper was washed with _Na₂HPO₄ (125 mM, pH 9) for 5 min, rinsed with water and ethanol, then air-dried and exposed to a phosphorus imager. The synthesized RNA was quantified using a Typhoon Trio imager and ImageQuant TL software, and the reaction rate was calculated by linear regression using GraphPad Prism 5.0. The _IC₅₀ value was calculated in Prism by nonlinear regression analysis using a dose-response (variable slope) equation (four-parameter logistic equation): Y = Bottom + (Top - Bottom) / (1 + 10^(( _LogEC₅₀ - X) * HillSlope)).

Example 11. Preparation of RSV RNP

The RSV ribonucleoprotein (RNP) complex was prepared using a modified method from Mason et al. (Mason, S., Lawetz, C., Gaudette, Y., Do, F., Scouten, E., Lagace, L., Simoneau, B., and Liuzzi, M. (2004) Polyadenylation-dependent screening assay for respiratory syncytial virus RNAtranscriptase activity and identification of an inhibitor. Nucleic Acids Research, 32, 4758-4767). HEp-2 cells were seeded at a density of 7.1 × ^10⁴ cells/ ^cm² in MEM + 10% fetal bovine serum (FBS) and allowed to attach overnight at 37°C (5% _CO₂ ). After attachment, the cells were infected with RSV A2 (MOI = 5) in 35 mL MEM + 2% FBS. Twenty hours post-infection, the culture medium was replaced with MEM supplemented with 2 μg/mL actinomycin D and 2% FBS, and the cells were restored to 37°C for one hour. Cells were then washed once with PBS and treated with 35 mL PBS + 250 μg/mL lysophosphatidylcholine for one minute, followed by aspiration of all fluid. Cells were harvested by displacing them into 1.2 mL of buffer A [50 mM MTT, RIS (pH 8.0), 100 mM potassium acetate, 1 mM DTT, and 2 μg/mL actinomycin D] and lysed by repeatedly passing the cells through an 18-gauge needle (10 times). The cell lysate was placed on ice for 10 minutes and then centrifuged at 2400 g for 10 minutes at 4°C. Remove the supernatant (S1) and dissolve the clumps (P1) by repeatedly passing the sample through an 18-gauge needle 10 times in 600 μL of buffer B [10 mM TRIS acetate (pH 8.0), 10 mM potassium acetate, and 1.5 mM MgCl2] supplemented with 1% Triton _X -100. Place the resuspended clumps on ice for 10 min and then centrifuge at 2400 g for 10 min at 4 °C. Remove the supernatant (S2) and dissolve the clumps (P2) in 600 μL of buffer B supplemented with 0.5% deoxycholate and 0.1% Tween 40. Place the resuspended clumps on ice for 10 min and then centrifuge at 2400 g for 10 min at 4 °C. Collect the supernatant fraction (S3) containing the enriched RSV RNP complex and determine the protein concentration by UV absorbance at 280 nm. Store the aliquots of the RSV RNP S3 fraction at -80 °C.

Example 12. RSV RNP Measurement

The transcription reaction contained 25 μg of crude RSV RNP complex in 30 μL of reaction buffer [50 mM TRIS-acetate (pH 8.0), 120 mM potassium acetate, 5% glycerol, _4.5 mM MgCl₂, 3 mM DTT, 2 mM ethylene glycol-bis(2-aminoethyl ether)-tetraacetic acid (EGTA), 50 μg/mL BSA, 2.5 U RNasin (Promega), ATP, GTP, UTP, CTP, and 1.5 uCi [α-32P]NTP (3000 Ci/mmol)]. Radiolabeled nucleotides used in the transcription assay were selected to match the nucleotide analogs used to evaluate their inhibition of RSV RNP transcription. Cold competitive NTPs were added at half their final concentration (ATP = 20 μM, GTP = 12.5 μM, UTP = 6 μM, and CTP = 2 μM). The remaining three nucleotides were added at a final concentration of 100 μM.

To determine whether the nucleotide analogues inhibited RSV RNP transcription, the compound was added in 5-fold increments using a 6-step serial dilution. After incubation at 30°C for 90 min, the RNP reaction was terminated with 350 μL of Qiagen RLT dissolution buffer, and RNA was purified using the Qiagen RNeasy 96 kit. The purified RNA was denatured in RNA loading buffer (Sigma) at 65°C for 10 min and run on a 1.2% agarose/MOPS gel containing 2M formaldehyde. The agarose gel was dried and exposed to a Storm phosphorus imaging screen, and developed using a Storm phosphorus imaging system (GE Healthcare). The concentration ( _IC50 ) of the compound that reduced total radiolabeled transcripts by 50% was calculated by nonlinear regression analysis of two replicate samples.

Example 13. DENV-2moDC EC ₅₀

Human monocyte-derived dendritic cells (moDCs) were derived from CD14+ monocytes (AllCells) cultured in human Mo-DC differentiation medium (Miltenyi Biotec) containing GM-CSF and IL-4. On day 7, moDCs were harvested by mechanical disruption, washed, and resuspended in serum-free RPMI. The moDCs were then infected in serum-free RPMI with Vero-derived dengue virus 2 New Guinea strain (NGC) at MOI = 0.1 for two hours with gentle agitation at 37°C. Cells were washed and resuspended in RPMI (Gibco, supplemented with sodium pyruvate, NEAA, and penicillin-streptomycin) containing 10% serum. Cells were seeded in triplicate in 96-well plates, with compound dispensed in fractionated doses (Hewlett-Packard D300 digital dispenser). All wells were normalized to 0.25% DMSO. At 48 hours, cells were washed with 1x PBS and all supernatant was removed. Total RNA was extracted using RNEasy 96 plates (Qiagen) and used to generate first-strand cDNA using XLT cDNA 5xSupermix (QuantaBio). The cDNA was used as a template in Taqman qPCR double-stranded reactions specific for DENV2 virus and GAPDH gene expression. _EC50 values were determined using Prism Graphpad software and normalized to positive control wells and negative control wells without the compound.

Example 14. DENV-2Huh-7 EC ₅₀

Huh7 (human hepatocellular carcinoma 7) cells were kept in complete DMEM medium containing 10% FCS. On the day of assay, cells were trypsinized (0.1% trypsin-EDTA), washed, and infected with dengue serotype 2 New Guinea C (NGC) virus at MOI = 0.1 for 2 hours in serum-free DMEM with gentle agitation at 37°C. After 2 hours, cells were washed with serum-free medium and resuspended in DMEM (Gibco, supplemented with sodium pyruvate, NEAA, and penicillin-streptomycin) containing 10% FCS. Cells were seeded in triplicate in 96-well plates, and the compound was dispensed in fractionated doses (Hewlett-Packard D300 digital dispenser). All wells were normalized to 0.25% DMSO. At 48 hours, cells were washed with 1x PBS and all supernatant was removed. Total RNA was extracted using RNEasy 96 plates (Qiagen) and used to generate first-strand cDNA using XLT cDNA 5xSupermix (QuantaBio). The cDNA was used as a template in Taqman qPCR double-stranded reactions specific for DENV2 virus and GAPDH gene expression. _EC50 values were determined using Prism Graphpad software and normalized to positive control wells and negative control wells without the compound.

Example 15. DENV-2Huh-7 Rep EC ₅₀

In 384-well plates (Greiner, catalog number 781091), compounds were acoustically transferred at 200 nmol per well in a dose-response format of 8 compounds (4 replicates) or 40 compounds (3 replicates). For all plates tested, Balapiravir, GS-5734, and NITD008 were included as positive inhibition controls along with negative control wells containing 0% inhibition and DMSO only. After compound addition, Huh-7 cells containing the DENV2 replicon construct were harvested following a standard cell culture procedure and adjusted to a concentration of 1.25E5 cells/mL in cDMEM culture medium without gentamicin. 40 μL of cell stock solution was then added to each well, resulting in a final cell density of 5,000 cells/well. The mixture of cells and compounds was incubated at 37°C/5% _CO2 for 48 h. Prior to cell harvesting, EnduRen live cell substrate (Promega, catalog number E6481) was prepared by suspending 3.4 mg in 100 μL of DMSO to generate a 60 mM stock solution. The stock solution was then diluted 1:200 in pre-warmed cDMEM, and 10 μL of this diluted solution was added to each well of a 384-well plate. The plate was then briefly centrifuged at 500 rpm and placed on a plate shaker for 2 minutes. After mixing, the plate was incubated at 7 °C/5% _CO2 for 1.5 h, after which luminescence was measured on an Envision photometer. The percentage of replicon signal inhibition was calculated relative to the 0% and 100% inhibition controls for each test concentration, and the _EC50 value for each compound was determined by 4-parameter nonlinear regression according to the effective concentration of the compound that inhibited 50% of the replicon signal.

Example 16. RSV HEp-2 EC ₅₀

Antiviral activity against RSV was determined in HEp-2 cells using an infectious cytopathic cell protection assay. In this assay, compounds that inhibit viral infection and/or replication exerted a cytoprotective effect against virus-induced cell killing, which was quantified using a cell viability reagent. The technique used here is a novel adaptation of a method described in the published literature (Chapman et al., Antimicrob Agents Chemother. 2007, 51(9):3346-53.).

HEp-2 cells were obtained from ATCC (Manassas, VI) and maintained in MEM medium supplemented with 10% fetal bovine serum and penicillin/streptomycin. Cells were passaged twice weekly and maintained in the subconvergent phase. A commercial stock solution of RSV strain A2 (Advanced Biotechnologies, Columbia, MD) was titrated prior to compound assays to determine the appropriate dilution factor of the viral stock solution to produce the desired cytopathic effect in HEp-2 cells.

For the antiviral assay, HEp-2 cells were grown in large cell culture flasks to near-convergence, but not complete confluence. The test compounds were pre-diluted in DMSO in 384-well compound dilution plates using a standardized dose-response format of 8 or 40 samples per plate. Three-fold serial dilution increments of each test compound were prepared in the plate, and test samples were transferred to the 384-well plates for cell culture assays at 100 nL per well via an acoustic transfer device (Echo, Labcyte). Each compound dilution was transferred to a dried assay plate as a single sample or in quadruplicate, and the assay plate was stored for assay preparation. Positive and negative controls were placed opposite each other at the ends of the vertical blocks (1 column) of the plate.

Subsequently, an infectious mixture was prepared using an appropriate dilution factor of the viral stock solution previously determined by titration with cells at a density of 50,000/mL, and 20 μL/well was added to the test plates containing the compounds via automation (uFlow, Biotek). Each plate included a negative control and a positive control (16 replicates each) to generate 0% and 100% viral inhibition standards, respectively. After RSV infection, the test plates were incubated in a 37°C cell culture incubator for 4 days. After incubation, Cell TiterGlo (Promega, Madison, WI) was added to the test plates, the plates were briefly incubated, and luminescence readings were measured in all test plates (Envision, Perkin Elmer). RSV-induced cytopathic effect, i.e., the percentage of inhibition, was determined by the level of residual cell viability. These figures were calculated for each test concentration relative to the 0% and 100% inhibition controls, and the _EC50 value for each compound was determined by nonlinear regression at the concentration that inhibited 50% of RSV-induced cytopathic effect. Various effective anti-RSV tool compounds were used as positive controls for antiviral activity.

Example 17. RSV NHBE EC ₅₀

Normal human bronchial epithelial (NHBE) cells (Walkersville, MD, catalog number CC-2540) were purchased from Lonza and cultured in bronchial epithelial growth medium (BEGM) (Lonza, Walkersville, MD, catalog number CC-3170). Cells were passaged 1-2 times per week to maintain <80% confluence. NHBE cells were discarded after 6 passages in culture.

To perform the RSV A2 antiviral assay, NHBE cells were grafted into BEGM in 96-well plates at a density of 7,500 cells per well and incubated overnight at 37°C. After attachment, 100 μL of cell culture medium was removed and a 3-fold serially diluted compound was added using a Hewlett-Packard D300 digital dispenser. The final concentration of DMSO was normalized to 0.05%. Following the addition of the compound, NHBE cells were infected by adding 100 μL of RSV A2 to BEGM at a titer of 1 × 10⁴ ^4.5 tissue culture infection doses/mL and then incubated at 37°C for 4 days. NHBE cells were then equilibrated to 25°C, and cell viability was determined by removing 100 μL of culture medium and adding 100 μL of Cell-Titer Glo viability reagent. The mixture was incubated at 25°C for 10 minutes, and the luminescence signal was quantified using an Envision luminescent microplate reader.

Example 18. RSV HAE EC ₅₀

HAE cells were cultured at the air-liquid interface with a top side exposed to air and a base side in contact with the culture medium. Prior to the experiment, HAEs were removed from their agar-based transport packages and acclimated overnight at 37°C/5% CO2 in 1 ml of HAE assay medium (AIR-100-MM, Mattek Corp). HAEs were prepared for infection by washing the top surface twice with 400 μL of PBS (using direct pipetting or by running each transwell through a PBS-containing trough) to remove the mucus layer. The PBS in the top chamber was drained and gently tapped onto absorbent material to remove as much PBS as possible. After washing, cells were transferred to fresh HAE maintenance medium containing 4-fold serially diluted compound, delivered to the basal side of a cell monolayer, and top-infected at 37°C in 5% CO2 for 3 h with 100 μL of a 1:600 dilution of RSV A strain A2 1000x stock solution (ABI, Columbia, MD, catalog number 10-124-000). The viral inoculum was removed, and the top surface of the cells was washed three times with PBS using the previously described method. Cells were then cultured at 37°C for 3 days in the presence of the compound. Following incubation, total RNA was extracted from HAE cells using the MagMAX-96 Virus RNA Isolation Kit (Applied Biosystems, Foster City, CA, catalog number AM1836), and intracellular RSV RNA was quantified by real-time PCR. Approximately 25 ng of purified RNA was added to a PCR reaction mixture (Applied Biosystems, Foster City, CA, catalog number 4392938) containing 0.9 μM RSV N forward and reverse primers, 0.2 μM RSV N probe, and 1x Taqman RNA-to-Ct 1-step kit. RNA levels were normalized using the Taqman GAPDH control primer set (Applied Biosystems, Foster City, CA, catalog number 402869). The real-time PCR primers and probes used in the RSV A2 HAE antiviral assay are: RSV N forward primer CATCAGCAAATACACCATCCA (SEQ ID NO:2), RSV N reverse primer TTCTGCACATCATAATTAGGAGTATCAA (SEQ ID NO:3), and RSV N probe FAM-CGGAGCACAGGAGAT-BHQ (SEQ ID NO:4).

Example 19. HRV16 HeLa EC ₅₀

One day prior to compound administration and infection, H1-HeLa cells cultured in complete DMEM medium containing 10% heat-inactivated FBS and 1% penicillin/streptomycin were seeded at 3000 cells/well in 96-well plates. Antiviral activity of each compound was measured in triplicate. Immediately prior to infection, the compounds were added directly to the cell culture in serially 3-fold dilutions using an HP300 digital dispenser (Hewlett Packard, Palo Alto, CA). The plates were transferred to BSL-2 restrictions, and an appropriate dilution of the viral stock solution previously determined by titration and prepared in cell culture medium was added to the test plates containing the cells and serially diluted compounds. Each plate contained 6 wells of infected, untreated cells and 6 wells of uninfected cells, serving as 0% and 100% viral inhibition controls, respectively. After infection, the test plates were incubated for 96 hours in a tissue culture incubator set to 33°C/5% _CO2 . After incubation, the H1-HeLa cells were removed from the incubator and equilibrated to 25°C. Cell viability was determined by adding 100 μL of Cell-Titer Glo viability reagent to 100 μL of culture medium. The mixture was incubated at 25°C for 10 minutes on a shaker, and the luminescence signal was quantified using an Envision luminescent microplate reader. The percentage of viral infection inhibition was calculated for each test concentration relative to the 0% and 100% inhibition controls, and the _EC50 value for each compound was determined by 4-parameter nonlinear regression according to the effective concentration of the compound that inhibited cytopathic effects by 50%.

Example 20. HRV1A HeLa EC ₅₀

One day prior to compound administration and infection, H1-HeLa cells cultured in complete RPMI 1640 medium containing 10% heat-inactivated FBS and 1% penicillin/streptomycin were seeded at 5000 cells/well in 96-well plates. Antiviral activity of each compound was measured in triplicate. Immediately prior to infection, the compounds were added directly to the cell culture at serially 3-fold dilutions using an HP300 digital dispenser (Hewlett Packard, Palo Alto, CA). The plates were transferred to BSL-2 confinement plates, and 100 μL of a 1/4000 dilution of HRV1a virus stock solution was added to each well containing cells and the serially diluted compound. Each plate contained 6 wells of infected, untreated cells and 6 wells containing 5 μM rupintrivir, serving as 0% and 100% virus inhibition controls, respectively. After infection, the test plates were incubated for 96 hours in a tissue culture incubator set to 37°C/5% _CO2 . After incubation, H1-HeLa cells were removed from the incubator and equilibrated to 25°C. Cell viability was determined by removing 100 μL of culture medium and adding 100 μL of Cell-Titer Glo viability reagent. The mixture was incubated on a shaker at 25°C for 10 minutes, and the luminescence signal was quantified using an Envision luminescent microplate reader. The percentage of viral infection inhibition was calculated relative to the 0% and 100% inhibition controls for each test concentration, and the _EC50 value for each compound was determined by 4-parameter nonlinear regression according to the effective concentration of the compound that inhibited cytopathic effects by 50%.

Example 21. HRV14 HeLa EC ₅₀

One day prior to compound administration and infection, H1-HeLa cells cultured in complete RPMI 1640 medium containing 10% heat-inactivated FBS and 1% penicillin/streptomycin were seeded at 5000 cells/well in 96-well plates. Antiviral activity of each compound was measured in triplicate. Immediately prior to infection, the compounds were added directly to the cell culture at serially 3-fold dilutions using an HP300 digital dispenser (Hewlett Packard, Palo Alto, CA). The plates were transferred to BSL-2 confinement plates, and 100 μL of a 1/4000 dilution of HRV14 virus stock solution was added to each well containing cells and the serially diluted compound. Each plate contained 6 wells of infected, untreated cells and 6 wells containing 5 μM rupintrivir, serving as 0% and 100% virus inhibition controls, respectively. After infection, the test plates were incubated for 96 hours in a tissue culture incubator set to 37°C/5% _CO2 . After incubation, H1-HeLa cells were removed from the incubator and equilibrated to 25°C. Cell viability was determined by removing 100 μL of culture medium and adding 100 μL of Cell-Titer Glo viability reagent. The mixture was incubated on a shaker at 25°C for 10 minutes, and the luminescence signal was quantified using an Envision luminescent microplate reader. The percentage of viral infection inhibition was calculated relative to the 0% and 100% inhibition controls for each test concentration, and the _EC50 value for each compound was determined by 4-parameter nonlinear regression according to the effective concentration of the compound that inhibited cytopathic effects by 50%.

Example 22. HRVc15 and HRVc25 HeLa EC ₅₀

First, HRV replicon RNA was prepared. 5 μg of DNA template (HRVc15 or HRVc25) was linearized at 37°C for 3 hours with 2 μL of MluI enzyme in NEB buffer-3 to a final volume of 25 μL. After incubation, the linearized DNA was purified on a PCR purification column, and in vitro transcription was performed under the following conditions: 10 μL RiboMAX Express T7 2x buffer, 1 μL–8 μL of linear DNA template (1 μg), 0 μL–7 μL of nuclease-free water, and 2 μL of enzyme mixture T7express. A final volume of 20 μL was mixed and incubated at 37°C for 30 minutes. After incubation, 1 μL of RQ1 RNase-free DNase was added, and the mixture was incubated at 37°C for 15 minutes. The resulting RNA was then purified using a MegaClear kit (Gibco Life Technologies catalog number 11835-030) and eluted twice with 50 μL of elution buffer at 95°C. One day prior to transfection, H1-HeLa cells cultured in complete RPMI 1640 medium containing 10% heat-inactivated FBS and 1% penicillin/streptomycin were seeded into T-225 flasks at a concentration of 2E6 cells/flask and incubated overnight at 37°C/5% _CO2 . On the day of transfection, cells were trypsinized according to standard cell culture protocols and washed twice with PBS. After washing, cells were resuspended in PBS at a concentration of 1E7 cells/mL, and the suspension was stored on moist ice. Replicon RNA was introduced into H1-HeLa cells using electroporation. A final volume of 10 μL containing either 10 μg of replicon c15 or 1 μg of C25 replicon RNA was pipetted into 4 mm electroporation cuvettes. The H1-HeLa cell stock was mixed by gentle swirl, and a previously prepared 0.5 mL cell stock was transferred to the cuvette containing the replicon RNA. The combined solutions were gently tapped to mix. Immediately after mixing, the cells were electroporated using the following settings: 900V, 25uF, infinite resistance, 1 pulse. The vials were incubated on ice for 10 minutes. After 10 minutes of incubation, 19 mL of RPMI 1640 containing 10% heat-inactivated FBS at ambient temperature, free of phenol red and antibiotics, was added to each electroporation well. 150 μL (4E4 cells) of the electroporated cell suspension was seeded into each well of a 96-well clear-bottomed white cell culture plate and incubated at 25°C for 30 minutes. The compound was added directly to the cell culture in serial 3-fold dilutions using an HP300 digital dispenser (Hewlett Packard, Palo Alto, CA) and tested in triplicate. After adding the compound, the plates were incubated at 33°C for 48 hours. Replicon activity was then measured using a Renilla-Glo luciferase assay system. Before signal quantification, the plate was removed from the incubator, and after removing 50 μL from each well, the plate was equilibrated to 25°C. Following the manufacturer's protocol, a 1:100 dilution of Renilla-Glo substrate with buffer was prepared, and 100 μL of the Renilla-Glo luciferase mixture was added to each well. The plate was then incubated at 25°C with gentle agitation for 20 minutes, and the luciferase signal was determined using an EnVision luciferase quantification reader with a 0.1-second detection setting. The percentage of replicon inhibition was calculated for each test concentration relative to the 0% and 100% inhibition controls included in the experiment, and the _EC50 value for each compound was determined by 4-parameter nonlinear regression based on the effective concentration of the compound that inhibited the luciferase signal by 50%.

Example 23. HCV Rep 1B and 2A EC ₅₀ and CC ₅₀

The compounds were serially diluted in 384-well plates in ten 1:3 dilution steps. All serial dilutions were performed in quadruple replicates for each compound within the same 384-well plate. 100 μM of the HCV protease inhibitor ITMN-191 was added as a control for 100% inhibition of HCV replication, while 10 mM of puromycin was added as a control for 100% cytotoxicity. 90 μL of cell culture medium (without geneticin) containing 2000 suspended HCV replicons was added to each well of a black polystyrene 384-well plate (Greiner Bio-one, Monroe, NC) using a Biomek μFlow workstation. For compound transfer to cell culture plates, 0.4 μL of the compound solution from the serial dilution plate was transferred to the cell culture plate using a Biomek FX workstation. The final DMSO concentration in the wells was determined to be 0.44%. The plates were incubated at 37°C with 5% _CO2 and 85% humidity for 3 days. The HCV replicon assay is a multiplex assay that can assess both cytotoxicity and antireplicon activity from the same well. First, the CC ₅₀ assay is performed. Culture medium is aspirated from the 384-well cell culture plate, and each well is washed four times with 100 μL of PBS using a Biotec ELX405 plate washer. Using a Biotec μFlow workstation, 50 μL of a solution containing 400 nM calcein AM (Anaspec, Fremont, CA) in 1×PBS is added to each well. The plate is incubated at room temperature for 30 minutes, after which the fluorescence signal (excitation 490 nm, emission 520 nm) is measured using a Perkin-Elmer Envision microplate reader. The EC ₅₀ assay is performed in the same wells as the CC ₅₀ assay. Calcein-PBS solution is aspirated from the 384-well cell culture plate using a Biotec ELX405 plate washer. Using a Biotec μFlow workstation, 20 μL of Dual-Glo luciferase buffer (Promega, Madison, WI) is added to each well. Incubate the plate at room temperature for 10 minutes. Using a Biotek μFlow workstation, add 20 μL of a 1:100 mixture of Dual-Glo Stop & Glo substrate (Promega, Madison, Wi) and Dual-Glo Stop & Glo buffer (Promega, Madison, Wi) to each well of the plate. Then incubate the plate at room temperature for 10 minutes, after which the luminescence signal is measured using a Perkin-Elmer Envision microplate reader.

Example 24. Inhibition of human mitochondrial RNA polymerase (POLRMT)

All reaction mixtures contained 50 mM Tris-HCl buffer (pH 8.0), 0.2 mg/ml BSA, 2 mM DTT, 0.05 mg/ml activated sperm DNA, 10 mM MgCl2, 1.3 μCi [α- ^33P ]dTTP (3,000 Ci/mmol), and 2 μM each of dATP, dGTP, and TTP. Optimal enzyme concentrations were selected within the linear range of enzyme concentration ([E]) relative to activity, and reaction times were chosen to ensure 10% substrate consumption. All reactions were run at 37 °C. The inhibition of mitochondrial RNA polymerase (POLRMT) was evaluated using 20 nM POLRMT pre-incubated with 20 nM POLRMT containing the POLRMT light chain promoter region template plasmid (pUC18-LSP) and mitochondrial transcription factor A (mtTFA) (100 nM) and mt-TFB2 (20 nM) in a buffer containing ₁₀ mM HEPES (pH 7.5), 20 mM NaCl, 10 mM DTT, 0.1 mg/ml BSA, and 10 mM MgCl2. The reaction was heated to 32 °C and initiated by adding 2.5 μM of each of the four native NTPs and 1.5 μCi of [ ^33P ]GTP. After incubation at 32 °C for 30 min, the reaction was spotted onto DE81 paper and then processed for quantification.

Example 25. Single nucleotide incorporation via human mitochondrial RNA polymerase (POLRMT)

A mixture of MTCN buffer (50 mM MES, 25 mM Tris-HCl, 25 mM CAPS, and 50 mM NaCl, pH 7.5), 200 nM 5′- ³² P-R12/D18, ₁₀ mM MgCl2, 1 mM DTT, and 376 nM POLRMT was pre-incubated at 30 °C for 1 min. The reaction was initiated by adding 500 μM (final) of a natural NTP or NTP analog. At selected time points, the reaction mixture was removed and quenched with a gel loading buffer containing 100 mM EDTA, 80% formamide, and bromophenol blue, and heated at 65 °C for 5 min. Samples were run on 20% polyacrylamide gels (8 M urea), and product formation was quantified using a Typhoon Trio imager and ImageQuant TL software (GE Healthcare, Piscataway, NJ). The rate of single nucleotide incorporation was calculated by fitting the formation of the product using a single exponential equation: [R13] = A(1-e^ ^(-kt )), where [R13] represents the amount of elongated product formed (in nM), t represents the reaction time, k represents the observed rate, and A represents the exponential amplitude.

Table 2. Activities of compounds of Formula I against RSV and HRV .

All values are in nM.

Table 3. Activity of compounds of Formula I against dengue virus .

All values are in nM.

Table 4. Activities of compounds of Formula I against HCV .

All values are in nM.

Table 5. Comparison of compounds of formula I and compounds 1 and 2 RSV efficacy

All values are in nM.

Table 6. Compounds of Formula I and a comparison of HRV efficacy between Compound 1 and Compound 2

All values are in nM.

Table 7. Comparison of compounds of Formula I and compounds 1 and 2 against dengue virus and HCV.

compound <![CDATA[DENV huh7 Rep EC₅₀]]> <![CDATA[HCV Rep 1B EC₅₀]]> <![CDATA[HCV Rep 2A EC₅₀]]> Formula I 2937 793 1268 1 73429 -- -- 2 5535 2776 1826

All values are in nM.

As shown in Tables 5-7, the compound of Formula I was more potent than compound 1 in RSV antiviral assays (Hep-2 and NHBE) (approximately 4.0 and 4.4 times more potent, respectively). The compound of Formula I was also more potent than compound 1 against HRV (in HRV16 HeLa, HRV1A HeLa, and HRV14 HeLa assays) (approximately 4.2, 51.1, and 12.8 times more potent, respectively). Similarly, the compound of Formula I was more potent than compound 1 against dengue virus (in the Denv huh7 Rep assay) (approximately 25.0 times more potent).

Similarly, the compound of formula I also showed higher anti-RSV activity compared to compound 2 in the HAE assay (Mirabelli, C. et al., J. Antimicrob. Chemother. 2018, 73, 1823-1829) (approximately 8.1-fold higher potency). The compound of formula I was also more potent than compound 2 in multiple HRV antiviral assays (in HRV1A HeLa, HRV14 HeLa, and HRV15 Rep assays) (approximately 12.1-fold higher potency in the HRV1A HeLa assay, approximately 2.2-fold higher potency in the HRV14 HeLa assay, and approximately 2.3-fold higher potency in the HRV15 Rep assay). The compound of formula I also showed higher potency in the dengue antiviral assay (approximately 1.9-fold higher potency in the DENV huh7 Rep assay). Similarly, the compound of formula I was more potent than compound 2 in HCV antiviral assays (approximately 3.5 times more potent in HCV Rep 1B and 1.4 times more potent in HCV Rep 2A).

Example 26. RSV efficacy of the compound of Formula I compared with structure-related compounds 3-5.

Among other things, compounds of Formula I are characterized by a cyclohexyl group at the ester group (the position indicated by * in the following structure).

Based on the determinations described above, the potency of compounds of Formula I and compounds 3-5 (structures shown below) was measured. The structures of compounds 3-5 are equivalent to those of compounds of Formula I, except that they lack the cyclic ring at the branched ester (the position indicated by * in the structures below). The results of these experiments are summarized in Table 8 below.

Table 8. RSV comparison power of compounds of formula I and compounds 1-3

All values are in nM.

As shown in Table 8 above, compound I was more potent than compound 3 in RSV and HRV antiviral assays (approximately 5 times more potent in RSV Hep-2 assay, approximately 9.3 times more potent in RSV NHBE assay, approximately 6.2 times more potent in HRV16 HeLa assay, approximately 91.9 times more potent in HRV1A HeLa assay, and approximately 23.2 times more potent in HRV14 HeLa assay), and more potent than compound 4 (approximately 3.9 times more potent in RSV Hep-2 assay, and approximately 23.2 times more potent in RSV NHBE assay). The concentration power is approximately 7.1 times higher in the assay (and approximately 4.3 times higher in the HRV16 HeLa assay), and even higher than compound 5 (approximately 19.8 times higher in the RSV Hep-2 assay, approximately 10.0 times higher in the RSV NHBE assay, approximately 13.8 times higher in the HRV16 HeLa assay, approximately 203.1 times higher in the HRV1A HeLa assay, and approximately 44.0 times higher in the HRV14 HeLa assay). Compounds 3, 4, and 5 each lack a cyclic cyclohexyl group at the branched ester group. Therefore, the compounds of formula I exhibit improved properties compared to compounds 3-5, which have branched alkyl groups at the ester group but lack a cyclohexyl group.

Example 27. The potency of the compound of formula I compared with the compounds of formula Ia, formula Ib, and compounds 2, 6, 7 and 8.

Based on the determinations described above, the potency of compounds of formula I, as well as structurally related compounds of formulas Ia and Ib, and compounds 2, 6, 7, and 8, was measured. The structures of compounds of formulas Ia and Ib, and compounds 6, 7, and 8 are shown below, and the results are summarized in Table 9 below.

Table 9: RSV and HRV of compounds of formula I, Ia, Ib, and compounds 2, 6, 7 and 8 Comparative effectiveness

All values are in nM.

The EC ₅₀ data in Table 9 above show that, in RSV and HRV assays, compounds of formula I with the S stereochemistry at P were significantly more potent than compounds of formula Ib with the R stereocenter at P. Specifically, compounds of formula I were 31.1 times more potent than compounds of formula Ib in RSV HEp-2 assays and 5.1 times more potent in RSV NHBE assays. Similarly, compounds of formula I were 2.9 times more potent than compounds of formula Ib in HRV16 HeLa assays.

In contrast, compounds 6 and 7, which differ only in stereochemistry at P (compound 6 has an S stereochemistry while compound 7 has an R stereochemistry at P), do not exhibit the same potency as compounds of formulas I and Ib. Compound 6 is 4.8 times more potent than compound 7 in RSV HEp-2 assays and 1.4 times more potent in HRV16 HeLa assays. In RSV NHBE assays, compound 6 is only 0.7 times more potent than compound 7.

Example 28. HEp-2 and MT-4 Cytotoxicity Assay

The cytotoxicity of compounds of formula I, as well as compounds 1, 2, and 6, was determined in uninfected cells using a cell viability reagent in a manner similar to that described previously for other cell types (Cihlar et al., Antimicrob Agents Chemother. 2008, 52(2):655-65.). HEp-2 cells (1.5 × 10³ cells/well) and MT-4 cells (2 × 10³ cells/well) were seeded in 384-well plates and incubated with appropriate medium containing 3-fold serial dilutions of the compounds (ranging from 15 nM to 100,000 nM). Cells were cultured at 37°C for 4–5 days. After incubation, cells were equilibrated to 25°C, and cell viability was determined by adding Cell-Titer Glo viability reagent. The mixture was incubated for 10 minutes, and the luminescent signal was quantified using an Envision microplate reader. Untreated cells and cells treated with 2 μM puromycin (Sigma, St. Louis, MO) served as 100% and 0% cell viability controls, respectively. Cell viability percentages were calculated relative to the 0% and 100% controls for each compound concentration tested, and CC ₅₀ values were determined by nonlinear regression based on the compound concentration that reduced cell viability by 50%.

Example 29. NHBE and SAEC Cytotoxicity Assays

Normal human bronchial epithelial (NHBE) cells (Walkersville, MD, catalog number CC-2540) were purchased from Lonza and cultured in bronchial epithelial growth medium (BEGM) (Lonza, Walkersville, MD, catalog number CC-3170). Cells were passaged 1-2 times per week according to the manufacturer's protocol to maintain <80% confluence. NHBE cells were discarded after 5 passages in culture.

Human small airway epithelial cells (SAECs) were purchased from Lonza (Walkersville, MD, catalog number CC-2547) and cultured in supplemented small airway epithelial cell growth medium (SAGM) (Lonza, Walkersville, MD, catalog number CC-3118). Cells were passaged 1-2 times per week according to the manufacturer's protocol to maintain <80% confluence. SAECs were discarded after 5 passages in culture.

To determine the 50% cytotoxic concentration (CC ₅₀ ) of compounds of Formula I and compounds 1, 2, and 6, NHBE or SAEC cells were seeded at a density of 10,000 cells per well in 200 μL of BEGM or SAGM in 96-well plates with clear bottoms and black walls and allowed to attach overnight at 37°C. After attachment, 3-fold serially diluted compounds were added in triplicate using a Hewlett-Packard D300 digital dispenser (Hewlett Packard, Palo Alto, CA). The final concentration of DMSO was normalized to 1.0%. After adding the compounds, NHBE or SAEC cells were incubated at 37°C for 5 days. The NHBE or SAEC cells were then equilibrated to 25°C, and cell viability was determined by removing 100 μL of culture medium and adding 100 μL of Cell-Titer Glo viability reagent (Promega, Madison, WI). The mixture was incubated at 25°C for 10 minutes, and the luminescence signal was quantified using an Envision luminescent microplate reader (PerkinElmer, Waltham, MA). Percentage activity values were determined by normalizing to control wells containing only 1.0% DMSO, after subtracting background luminescence signal.

Example 30. PHH Cytotoxicity Assay

Prepare compound I and three-fold serial dilutions of compounds 1, 2, and 6 in duplicate in 96-well plates, starting at a concentration of 50 μM or 100 μM. Order fresh human hepatocytes in 96-well plates from BioIVT (Baltimore, Maryland, catalog number F/M91565) with Matrigel coverings or from Invitrogen (Durham, North Carolina, catalog number HMFY96) with Geltrex coverings. Donor profiles should be limited to individuals aged 4–65 years with minimal alcohol consumption. PHH cells should be recovered in complete medium supplemented with the supplier's recommended supplements at 37°C in a 5% _CO2 incubator at 90% humidity for 4–24 hours, followed by compound treatment. Replace the serially diluted compounds and complete medium daily (130 μL/well) for 5 days, with a final DMSO volume of 0.5%. On day 5, the culture medium was removed from the assay plate and cell viability was determined by adding 100 μL of Cell-Titer Glo viability reagent (Promega, Madison, WI, catalog number G7573) to each well. After incubation at room temperature for 5–10 minutes, the luminescence signal was quantified using a Victor luminescent microplate reader (Perkin-Elmer, Waltham, MA).

Example 31. PRPT Cytotoxicity Assay

The following protocol was used to perform PRPT cytotoxicity assays on compounds of Formula I, as well as compounds 1, 2, and 6.

Cryopreserved human primary proximal tubular epithelial cells (PRPTEC) were obtained from LifeLine Cell Technology (Frederick, MD, catalog number FC-0013) and isolated from human kidney tissue. Cells were removed from cryopreserved vials and cultured in RenaLife complete medium (LifeLine, Frederick, MD, catalog number LL-0025) in T75 flasks for 3–4 days. After reaching 90% confluence, the cells were seeded into assay plates. PRPTEC cells were seeded at a density of 5 × 10³ cells per well in collagen-coated 96-well plates, resulting in a final volume of 160 mL per well. The next day, the compound was added directly to the cell plate using an HP D300 dispenser (Hewlett-Packard, Palo Alto, CA), with the program starting at a concentration 200 times lower than the compound stock solution concentration and diluted 1:3 times with 0.5% quantitative DMSO in duplicate. After 5 days of incubation, the culture medium was removed, and cell viability was measured by adding 100 mL of CellTiter Glo viability reagent (Promega, Madison, WI, catalog number G7573) to each well. The luminescence signal was quantified on a luminescent microplate reader (Perkin-Elmer, Waltham, MA).

Example 32. GALHEPG2 Cytotoxicity Assay

The cytotoxicity of compounds of Formula I, as well as compounds 1, 2 and 6, in galactose-adapted HepG2 cells (human hepatocellular carcinoma cell line) was tested using a high-throughput 384-well assay.

Cells were diluted to 16.6 K cells/mL in DMEM (11966), 10% FBS, 1% NEAA, 0.2% galactose, 1% pyruvate, 1% Glutamax, and 1% PSG, and seeded at 90 μL/well in 384-well poly-D-lysine-coated assay plates and incubated at 37°C and 5% CO2. The compound was serially diluted (1:3) in 100% DMSO in 384-well plates, in quadruplicate. DMSO and 2 mM puromycin were included as negative and positive controls, respectively. 24 hours after seeding, 0.4 μL was transferred from the compound plate to the assay plate using a 384-channel pipette. The assay plate was returned to the incubator. After 5 days, the assay plate was washed with 80 μL/well of PBS, followed by the addition of 20 μL of CellTiter Glo. The assay plate was read on an Envision microplate reader. The CC ₅₀ value is defined as the concentration of a compound that, as measured in the luminescence signal, induces 50% inhibition of growth. The CC ₅₀ value is calculated using a unit point dose-response model in Accord (an online tool) to generate an S-shaped curve fit.

Example 33. GALPC3 Cytotoxicity Assay

The cytotoxicity of the compound in galactose-adapted PC3 cells (human prostate cancer cell line) was tested using a high-throughput 384-well assay. Cells were diluted to 16.6 K cells/mL in DMEM (11966), 10% FBS, 1% NEAA, 0.2% galactose, 1% pyruvate, 1% Glutamax, and 1% PSG, and seeded at 90 μL/well in 384-well poly-D-lysine-coated assay plates and incubated at 37°C and 5% _CO2 . The compound was serially diluted (1:3) in 100% DMSO in 384-well plates, in quadruplicate. DMSO and 2 mM puromycin were included as negative and positive controls, respectively. 24 hours after cell seeding, 0.4 μL was transferred from the compound plate to the assay plate using a 384-channel pipette. The assay plate was then returned to the incubator. Five days later, the assay plate was washed with 80 μL/well of PBS, followed by the addition of 20 μL of Cell Titer Glo. The assay plate was read on an Envision microplate reader. The _{CC 50} value is defined as the concentration of a compound that induces 50% inhibition of growth, as measured in the luminescent signal. The CC ₅₀ value was calculated using a unit point dose-response model in Accord (an online tool) to generate an S-shaped curve fit.

Example 34. Huh-7 Cytotoxicity Assay

The cytotoxicity of the compound in Huh7 cells (hepatocellular carcinoma cell line) was tested using a high-throughput 384-well assay. Cells were diluted to 16.6 K cells/mL in DMEM (15-018-CM), 10% FBS, 1% NEAA, and 1% PSG, and seeded at 90 μL/well in 384-well poly-D-lysine-coated assay plates. The plates were incubated at 37°C and 5% CO2. The compound was serially diluted (1:3) in 100% DMSO in the 384-well plates, in quadruplicate. DMSO and 2 mM puromycin were included as negative and positive controls, respectively. 24 hours after cell seeding, 0.4 μL was transferred from the compound plate to the assay plate using a 384-channel pipette. The assay plate was returned to the incubator. After 5 days, the assay plate was washed with 80 μL/well of PBS, followed by the addition of 20 μL of Cell Titer Glo. The assay plate was read using an Envision microplate reader. The CC ₅₀ value is defined as the concentration of a compound that, as measured in the luminescence signal, induces 50% inhibition of growth. The CC ₅₀ value is calculated using a unit point dose-response model in Accord (an online tool) to generate an S-shaped curve fit.

Example 35. MRC5 Cytotoxicity Assay

The cytotoxicity of the compound in MRC5 cells (human fetal lung fibroblast cell line) was tested using a high-throughput 384-well assay. Cells were diluted to 16.6 K cells/mL in medium (MEM (10-010-CM), 10% FBS, 1% PSG) and seeded at 90 μL/well in 384-well poly-D-lysine-coated assay plates, which were then incubated at 37°C and 5% 2. The compound was serially diluted (1:3) in 100% DMSO in the 384-well plates, in quadruplicate. DMSO and 2 mM puromycin were included as negative and positive controls, respectively. 24 hours after cell seeding, 0.4 μL was transferred from the compound plate to the assay plate using a 384-channel pipette. The assay plate was returned to the incubator. After 5 days, the assay plate was washed with 80 μL/well PBS, followed by the addition of 20 μL Cell Titer Glo. The assay plate was read on an Envision microplate reader. The CC ₅₀ value is defined as the concentration of a compound that, as measured in the luminescence signal, induces 50% inhibition of growth. The CC ₅₀ value is calculated using a unit point dose-response model in Accord (an online tool) to generate an S-shaped curve fit.

Example 36. Cytotoxicity assay of neonatal rat cardiomyocytes by NRVM

The cytotoxicity of the compound against newly harvested neonatal rat cardiomyocytes (NRVMs) was tested using a high-throughput 384-well assay. Cells were diluted to 25,000 cells/mL in DMEM + 10% FBS + 1% PSG + 1% NEAA and seeded at 90 μL per well in 384-well cytometry plates. The plates were incubated overnight at 37°C and 5% _CO2 , followed by addition of the compound. The compound was prepared by sequentially diluting (1:3) in 100% DMSO in the 384-well plates in quadruplicate. 400 nL/well of the compound was transferred to the cytometry plates via Biocel (Agilent Technologies). DMSO and 2 mM puromycin were included as negative and positive controls, respectively. After 5 days, the plates were washed once with 100 μL/well of PBS using a Biotec plate washer, and 20 μL of Cell Titer Glo was added to each well. The plate was incubated for 10 minutes and read on an EnVision reader (Perkin Elmer). The CC ₅₀ value is defined as the concentration of the compound that causes 50% inhibition of growth, as measured in the luminescence signal. The CC ₅₀ value was calculated using a unit point dose-response model in Accord (an online tool) to generate an S-shaped curve fit.

Example 37. PBMC Cytotoxicity Assay

The cytotoxicity of compounds in cryopreserved human PBMCs was tested using a high-throughput 384-well assay. Compounds were serially diluted (1:3) in 100% DMSO in quadruplicates in 384-well plates. 310 nL of the compound was transferred to the assay plate using an acoustic dispenser. DMSO and 2 mM puromycin were included as negative and positive controls, respectively. Cells were diluted to 72 kC/mL in medium (RPMI + 10% FBS + 1% PSG + 10 mM Hepes + 1% pyruvate + 0.1% BMe) and incubated at 37°C and 5% _CO2 for 4 hours, followed by seeding at 70 μL/well into pre-spotted assay plates. After 5 days, 25 μL of Cell Titer Glo was added to the assay plate. The _{CC 50} value was defined as the concentration of the compound that caused 50% inhibition of growth, as measured in the luminescent signal. Calculate the CC ₅₀ value using the unit point dose response model in Accord (an online tool) to generate an S-shaped curve fit.

The table above indicates that, compared with compounds 1, 2, 6 and 8, compounds of formula I exhibited better secondary cytotoxicity properties in multiple cell lines (NHBE, SAEC, Huh-7, NRVM, PBMC, PHH and PRPT).

Example 38. Plasma stability assay

For plasma stability, the compound was incubated in cynomolgus monkey or human plasma at 2 μM at 37 °C for up to 4 hours. At the desired time point, the incubated aliquots were quenched by adding 9 volumes of 100% acetonitrile containing the internal standard. After the final collection, the samples were centrifuged at 3000 g for 30 min, and the supernatant was transferred to a new plate containing an equal volume of water for analysis by liquid chromatography combined with triple quadrupole mass spectrometry (LC-MS/MS). The data (ratio of analyte to internal standard peak areas) were plotted on a semi-logarithmic scale and fitted using exponential fitting. Assuming first-order kinetics, the half-life (T _{_1/2} ) was determined.

Example 39. Stability determination in S9 fraction

For S9 stability, the compound was incubated at 37°C for up to 90 min at the desired time point after compound addition in cynomolgus monkey or human liver S9 fractions with NADPH and UDPGA (phase I and II cofactors, Sigma-Aldrich). The sample was quenched with 9 volumes of an aqueous solution containing an internal standard, 50% acetonitrile, and 25% methanol. The sample plate was centrifuged at 3000 g for 30 min, and 10 μL of the resulting solution was analyzed by LC-MS/MS. Data (the ratio of analyte peak area to internal standard peak area) were plotted on a semi-logarithmic scale and fitted using exponential fitting. Assuming first-order kinetics, the half-life (T _{_1/2} ) was determined.

Table 11. Stability of compounds of formula I compared to compounds 1, 2, 6, 7 and 8 (HepS9 and plasma).

The data in the table above show that the compound of formula I has a longer half-life than compounds 2, 6 and 7 (human and cynomolgus monkey hepS9 and human and cynomolgus monkey plasma).

Example 40. Determination of thermodynamic solubility

The thermodynamic solubility of the compounds was determined at room temperature in phosphate-buffered saline (pH 7.4) and 10 mM hydrochloric acid (pH 2.0). An aqueous sample of the compound was saturated with an excess of solid. The tubes were placed on a stirrer set to 1000 rpm and kept at constant agitation for four days. After stirring, excess solid was confirmed in all tubes. The tubes were centrifuged at 10,000 rpm for 5 minutes to remove excess solid, and the supernatant was transferred to a new vial. Concentration analysis was performed by UPLC, and quantification was performed against an internal standard.

Table 12. Thermodynamic solubility of compounds of formula I and compounds 5 and 6 .

compound pH 2 solubility (μg/mL) pH 7 solubility (μg/mL) Formula I 4115 131 2 3720 18 6 3793 16

As can be seen in the table above, the compound of formula I has higher solubility at pH 2 and pH 7 than compounds 2 and 6.

Example 41. In vitro triphosphate formation in cells of NHBE

The following protocol was used to measure intracellular triphosphate formation in vitro for compounds of formula I and compound 6. Normal human bronchial airway epithelial cells (NHBE) (250,000 cells/well) were continuously incubated with 10 μM of the compound for 26 h. At selected time points (2 h, 4 h, 6 h, and 26 h), the extracellular culture medium was removed from the wells, and the cells were washed twice with 2 mL of ice-cold 0.9% physiological saline and extracted into 0.5 mL of ice-cold 70% methanol containing 100 nM 2-chloro-adenosine-5'-triphosphate (Sigma-Aldrich, St. Louis, MO) as an internal standard. The samples were stored overnight at -20 °C to promote nucleotide extraction, centrifuged at 15,000 × g for 15 min, and the supernatant was transferred to clean tubes for drying in a MiVac Duo concentrator (Genevac, Gardiner, NY). The dried sample was then reconstructed in mobile phase A containing 3 mM ammonium formate (pH 5.0) and 10 mM dimethylhexylamine (DMHA) in water for analysis by LC-MS/MS. The results of these experiments are shown in Figure 1.

Example 42. Determination of Triphosphate Formation in In Vitro Cells of PBMCs

The following protocol was used to measure intracellular triphosphate formation in vitro for compounds of Formula I, as well as compounds 2 and 6. Freshly isolated PBMCs were derived from healthy donors and suspended in medium (RPMI 1164 containing L-glutamine) to a concentration of 5 million cells/mL prior to the experiment. 10 mL aliquots of PBMCs were transferred to loosely capped 50 mL conical tubes, and the compounds were added to a final concentration of 2 μM. Then, 1 mL aliquots of each sample were transferred to the wells of a 24-well plate. The PBMC-compound mixture was incubated at 37 °C/5% CO2 for ₂ h with gentle agitation. After incubation, the PBMCs were rotated at 5000 RPM for 3 min, and the supernatant was aspirated without disturbing cell clumps. For samples to be analyzed immediately, the samples were resuspended in pre-cooled 1x Tris-buffered saline and transferred to 1.5 mL conical tubes containing 0.5 mL of nyosil M25. The sample/oil aliquots were then centrifuged at 13,000 RPM for 1 minute. After centrifugation, all culture medium was aspirated from the tube without disturbing the oil layer. Water was added to the oil layer, and the centrifugation/aspiration process was repeated, followed by another water wash. After the second wash, all oil and water were removed, and the cell clumps were rapidly frozen on dry ice and stored at -80°C until further processing. Samples not immediately analyzed were washed twice with serum-free medium, resuspended in 1 mL of medium, and _incubated at 37°C/5% CO2 until they were processed according to the aforementioned protocol. Each PBMC sample was treated with 500 μL of dry ice-cold extraction buffer (70% methanol, containing 0.5 μM chloro-adenosine triphosphate as an internal standard). The solution was vortexed for 5 minutes and then centrifuged at 20,000 × g for 20 minutes. The supernatant was transferred to a clean 1.5 mL Eppendorf vial and loaded onto a centrifuge evaporator. Once dried, the sample was reconstructed with 80 μL of mobile phase A, centrifuged at 20,000 × g for 20 min, and the supernatant was transferred to an HPLC vial for analysis. 10 μL aliquots were injected into a Sciex 6500 LC/MS/MS system. A standard calibration curve for PBMCs was constructed based on the pmol of the compound in each sample. The values from each sample were then divided by the total number of cells in the sample to obtain pmol per million cells. The micromolar concentration was then derived using an intracellular volume of 0.2 pL per cell. The results of these experiments are shown in Figure 2.

As shown in Figures 1 and 2, compounds of Formula I exhibited equal or better in vitro intracellular NTP (nucleoside triphosphate) formation in NHBE compared to compounds 2 and/or 6, but lower in PBMCs. This suggests that compounds of Formula I undergo more selective metabolism in NHBE (target cell type) compared to PBMCs, compared to compounds 2 and/or 6.

Example 43. Animal Pharmacokinetic Determination

Animal pharmacokinetic studies of compounds of formula I and compound 6 were conducted using the following protocol. Live animals weighing 3 kg to 6 kg were used for the study. The test product was administered intravenously to female cynomolgus monkeys at a constant rate of 10 mg/kg body weight as an aqueous solution of 12% captisol in water (pH 3) over 30 minutes. Plasma samples were collected at 0.25 h, 0.5 h, 1 h, 1.5 h, 2 h, 4 h, 8 h, and 24 h after administration, and PBMC samples were collected at 2 h and 24 h after administration.

Blood samples (approximately 1 mL) were collected into pre-cooled collection tubes containing _K₂EDTA and centrifuged at 4°C to separate isoplasma. For PBMC collection, approximately 8 mL of blood sample was collected at room temperature into CPT vacuum blood collection tubes containing heparin sodium for separation. At each terminal collection, the animal was anesthetized and the lungs were harvested while the animal was still alive. The collected lungs were rapidly frozen in liquid nitrogen immediately after retrieval.

Protein precipitation was performed on plasma samples from pharmacokinetic studies using acetonitrile containing 5-iodotuberculin as an internal standard at a final concentration of 75%. Analytes in plasma samples were separated over 7 minutes at a flow rate of 250 μL/min using a mobile phase containing 0.2% formic acid and a linear gradient of 2% to 100% acetonitrile on a 4 μm 150 × 2 mm Synergi Max-RP column (Phenomenex, Torrance, CA). An eight-point standard curve prepared in blank plasma covered concentrations from 5.1 nM to 5000 nM and showed linearity exceeding R² of 0.99. Quality control samples prepared separately in plasma at 120 nM and 3000 nM were analyzed at the beginning and end of each sample group to ensure accuracy and precision within 20%.

Each PBMC sample was treated with 500 μL of extraction buffer containing 67 mM EDTA in 70% methanol, with 0.5 μM chloro-adenosine triphosphate as an internal standard. The extraction buffer was cooled on dry ice. The solution was vortexed for 5 min and then centrifuged at 20,000 × g for 20 min. The supernatant was transferred to a clean 1.5 mL Eppendorf vial and loaded onto a centrifuge evaporator. Once dried, the sample was reconstituted with 80 μL of 1 mM ammonium phosphate buffer (pH = 7), centrifuged at 20,000 × g for 20 min, and the supernatant was transferred to an HPLC vial for analysis. 10 μL aliquots were injected into an API 5000 LC/MS/MS system. To calculate intracellular metabolite concentrations, the total number of cells in each sample was determined using a total DNA counting method (Benech et al., Peripheral Blood Mononuclear Cell Counting Using a DNA-detection-based Method. July 1, 2004; 330(1):172-4). Standard calibration curves for PBMCs were constructed based on the pmol of the compounds in each sample. The values from each sample were then divided by the total number of cells in the sample to obtain pmol per million cells. The micromolar concentrations were then derived using an intracellular volume of 0.2 pL per cell.

Lung samples were prepared by cutting into small pieces and aliquoting into pre-weighed 15 mL conical tubes, which were then held on dry ice. ~2 mL of ice-cold extraction buffer (0.1% KOH and 67 mM EDTA in 70% methanol containing 0.5 μM chloro-adenosine triphosphate as an internal standard) was added to each ~0.5 g lung sample. The mixture was rapidly homogenized using an Omni-Tip TH ^™ (Omni International) probe with a disposable hard tissue homogenizer. The homogenate was filtered through a 0.2 μm 96-well polypropylene filter plate (Varian Captiva ^™ ). The filtrate was evaporated to dryness and reconstituted with an equal volume of 1 mM ammonium phosphate buffer (pH = 7) prior to LC-MS/MS analysis.

Quantitative analysis of nucleoside triphosphates was performed using an ion-paired nucleotide detection LC-MS/MS method. Analytes were separated within 11 minutes at a flow rate of 160 μL/min using a 2.5 μm 2.0 × 50 mm Luna C18 column (Phenomenex, Torrance, CA) with an ion-pairing buffer containing 3 mM ammonium phosphate (pH 5) and 10 mM dimethylhexylamine (DMH) and a multi-level linear gradient of 10% to 50% acetonitrile. A seven-point standard curve prepared in a blank matrix covered concentrations from 24.0 nM to 17,500 nM and showed linearity exceeding ^R² of 0.99.

The results of these experiments are shown in Figure 3 and the table below.

Table 13. Average concentrations of triphosphate in the lungs and PBMCs of compounds of formula I and 6 after intravenous infusion of 10 mg/kg into cynomolgus monkeys 30 minutes later (mean, n=2).

Infused compound organize 2 hours 24 hours Formula I Lung tissue (nmol/g) 2.88 2.01 Formula I PBMC (μM) 61.3 27.1 6 Lung tissue (nmol/g) 2.25 1.29 6 PBMC (μM) 169 22.4

As observed, compounds of formula I exhibited higher lung NTP concentrations and lower PBMC NTP concentrations in cynomolgus monkey PK studies. This indicates that, compared to compound 6, compounds of formula I undergo more selective metabolism in lung tissue relative to PBMCs.

This application specifically involves the following implementation plan:

1. A compound of formula I:

Or its pharmaceutically acceptable salt.

2. A pharmaceutical formulation comprising a therapeutically effective amount of the compound according to embodiment 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

3. A method for treating a person in need of a pulmonary viral infection, the method comprising administering to the person a therapeutically effective amount of the compound according to embodiment 1, or a pharmaceutically acceptable salt thereof.

4. The method according to implementation scheme 3, wherein the pulmonary virus infection is a respiratory syncytial virus infection.

5. The method according to implementation scheme 3, wherein the pulmonary virus infection is human metapneumovirus infection.

6. A method for treating a person in need of a microribonucleovir infection, the method comprising administering to the person a therapeutically effective amount of the compound according to embodiment 1, or a pharmaceutically acceptable salt thereof.

7. The method according to embodiment 6, wherein the microribonucleoviridae virus infection is a human rhinovirus infection.

8. A method for treating a person in need of a flaviviral infection, the method comprising administering to the person a therapeutically effective amount of the compound according to embodiment 1, or a pharmaceutically acceptable salt thereof.

9. The method according to embodiment 8, wherein the flaviviridae virus infection is dengue virus infection.

10. The method according to embodiment 8, wherein the flaviviridae virus infection is yellow fever virus infection.

11. The method according to embodiment 8, wherein the flaviviridae virus infection is a West Nile virus infection.

12. The method according to implementation scheme 8, wherein the flaviviridae virus infection is Zika virus infection.

13. The method according to embodiment 8, wherein the flaviviridae virus infection is hepatitis C virus infection.

14. The method according to implementation scheme 8, wherein the flaviviridae virus infection is hepatitis B virus infection.

15. A method for treating a person in need of a filoviridae virus infection, the method comprising administering to the person a therapeutically effective amount of the compound according to embodiment 1, or a pharmaceutically acceptable salt thereof.

16. The method according to embodiment 15, wherein the filoviridae virus infection is an Ebola virus infection.

17. A method of manufacturing a medicament for treating pulmonary viral infections in people in need, characterized in that a compound according to embodiment 1, or a pharmaceutically acceptable salt thereof, is used.

18. The method according to embodiment 17, wherein the pulmonary virus infection is a respiratory syncytial virus infection.

19. The method according to embodiment 17, wherein the pulmonary virus infection is human metapneumovirus infection.

20. A method for manufacturing a medicament for treating microribonucleovir infections in people in need, characterized in that a compound according to embodiment 1, or a pharmaceutically acceptable salt thereof, is used.

21. The method according to embodiment 20, wherein the microribonucleoviridae virus infection is a human rhinovirus infection.

22. A method for manufacturing a medicament for treating flaviviral infections in people in need, characterized in that a compound according to embodiment 1, or a pharmaceutically acceptable salt thereof, is used.

23. The method according to implementation scheme 22, wherein the flaviviridae virus infection is dengue virus infection.

24. The method according to implementation scheme 22, wherein the flaviviridae virus infection is yellow fever virus infection.

25. The method according to embodiment 22, wherein the flaviviridae virus infection is a West Nile virus infection.

26. The method according to implementation scheme 22, wherein the flaviviridae virus infection is Zika virus infection.

27. The method according to embodiment 22, wherein the flaviviridae virus infection is hepatitis C virus infection.

28. The method according to embodiment 22, wherein the flaviviridae virus infection is hepatitis B virus infection.

29. A method for manufacturing a medicament for treating filoviridae virus infections in people in need, characterized in that a compound according to embodiment 1, or a pharmaceutically acceptable salt thereof, is used.

30. The method according to embodiment 29, wherein the filoviridae virus infection is an Ebola virus infection.

31. Use of the compound or a pharmaceutically acceptable salt thereof according to embodiment 1 for the manufacture of a medicament for the treatment of human pulmonary virus infections.

32. The use according to embodiment 31, wherein the pulmonary virus infection is a respiratory syncytial virus infection.

33. The use according to embodiment 31, wherein the pulmonary virus infection is human metapneumovirus infection.

34. Use of the compound described in embodiment 1 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of human microribonucleoviridae virus infections.

35. The use according to embodiment 34, wherein the microribonucleoviridae virus infection is a human rhinovirus infection.

36. Use of the compound or a pharmaceutically acceptable salt thereof according to embodiment 1 for the manufacture of a medicament for the treatment of human flaviviridae virus infections.

37. The use according to embodiment 36, wherein the flaviviridae virus infection is dengue virus infection.

38. The use according to embodiment 36, wherein the flaviviridae virus infection is yellow fever virus infection.

39. The use according to embodiment 36, wherein the flaviviridae virus infection is a West Nile virus infection.

40. The use according to embodiment 36, wherein the flaviviridae virus infection is a Zika virus infection.

41. The use according to embodiment 36, wherein the flaviviridae virus infection is hepatitis C virus infection.

42. The use according to embodiment 36, wherein the flaviviridae virus infection is hepatitis B virus infection.

43. Use of the compound or a pharmaceutically acceptable salt thereof according to embodiment 1 for the manufacture of a medicament for the treatment of human filoviridae virus infections.

44. The use according to embodiment 43, wherein the filoviridae virus infection is an Ebola virus infection.

45. The compound or a pharmaceutically acceptable salt thereof according to embodiment 1, wherein the compound or a pharmaceutically acceptable salt thereof is used to treat pulmonary viral infections in persons in need.

46. The compound according to embodiment 45, wherein the pulmonary virus infection is a respiratory syncytial virus infection.

47. The compound according to embodiment 45, wherein the pulmonaryviridae virus infection is human metapneumovirus infection.

48. The compound or a pharmaceutically acceptable salt thereof according to embodiment 1, wherein the compound or a pharmaceutically acceptable salt thereof is used to treat microribonucleovir infections in persons in need.

49. The compound according to embodiment 48, wherein the microribonucleoviridae virus infection is a human rhinovirus infection.

50. The compound or a pharmaceutically acceptable salt thereof according to embodiment 1, the compound or a pharmaceutically acceptable salt thereof for treating flaviviridae virus infection in persons in need.

51. The compound according to embodiment 50, wherein the flaviviridae virus infection is dengue virus infection.

52. The compound according to embodiment 50, wherein the flaviviridae virus infection is yellow fever virus infection.

53. The compound according to embodiment 50, wherein the flaviviridae virus infection is a West Nile virus infection.

54. The compound according to embodiment 50, wherein the flaviviridae virus infection is a Zika virus infection.

55. The compound or a pharmaceutically acceptable salt thereof according to embodiment 1, wherein the compound or a pharmaceutically acceptable salt thereof is used to treat filoviridae virus infection in persons in need.

56. The compound according to embodiment 55, wherein the filoviridae virus infection is an Ebola virus infection.

57. The compound according to embodiment 55, wherein the flaviviridae virus infection is hepatitis C virus infection.

58. The compound according to embodiment 55, wherein the flaviviridae virus infection is a hepatitis B virus infection.

59. A method for treating or preventing the exacerbation of respiratory symptoms caused by a viral infection in a person in need, the method comprising administering to the person a therapeutically effective amount of the compound according to embodiment 1 or a pharmaceutically acceptable salt thereof, wherein the respiratory symptoms are chronic obstructive pulmonary disease.

60. A method for treating or preventing the exacerbation of respiratory symptoms caused by a viral infection in a person in need, the method comprising administering to the person a therapeutically effective amount of the compound according to embodiment 1 or a pharmaceutically acceptable salt thereof, wherein the respiratory symptoms are asthma.

61. The method according to embodiment 59 or 60, wherein the viral infection is caused by respiratory syncytial virus, rhinovirus or metapneumovirus.

62. A method for manufacturing a treatment or prevention of exacerbation of respiratory symptoms caused by a viral infection in a person in need, characterized in that a compound according to embodiment 1 or a pharmaceutically acceptable salt thereof is used, wherein the respiratory symptoms are chronic obstructive pulmonary disease.

63. A method for manufacturing a product for treating or preventing the worsening of respiratory symptoms caused by a viral infection in a person in need, characterized in that a compound or a pharmaceutically acceptable salt thereof is used according to embodiment 1, wherein the respiratory symptoms are asthma.

64. The method according to embodiment 62 or 63, wherein the viral infection is caused by respiratory syncytial virus, rhinovirus or metapneumovirus.

65. Use of the compound according to embodiment 1 or a pharmaceutically acceptable salt thereof for the manufacture of a treatment or prevention of exacerbation of respiratory symptoms caused by viral infection in humans, wherein the respiratory symptoms are chronic obstructive pulmonary disease.

66. Use of the compound or a pharmaceutically acceptable salt thereof according to embodiment 1 for the manufacture of a treatment or prevention of exacerbation of respiratory symptoms caused by viral infection in humans, wherein the respiratory symptoms are asthma.

67. The use according to embodiment 65 or 66, wherein the viral infection is caused by respiratory syncytial virus, rhinovirus or metapneumovirus.

68. The compound or a pharmaceutically acceptable salt thereof according to embodiment 1, the compound or a pharmaceutically acceptable salt thereof for the treatment or prevention of exacerbation of respiratory symptoms caused by viral infection in a person in need, wherein the respiratory symptoms are chronic obstructive pulmonary disease.

69. The compound or a pharmaceutically acceptable salt thereof according to embodiment 1, the compound or a pharmaceutically acceptable salt thereof being used to treat or prevent the exacerbation of respiratory symptoms caused by viral infection in a person in need, wherein the respiratory symptoms are asthma.

70. The compound according to embodiment 68 or 69, wherein the viral infection is caused by respiratory syncytial virus, rhinovirus or metapneumovirus.

71. A method for preparing a compound of formula I-11:

The method includes making:

(i) Compounds of Formula I-7:

and

(ii) Compounds of formula I-12:

The reaction occurs in the presence of _NdCl3 and tetrabutylammonium chloride; where R is a hydroxyl protecting group.

72. The method according to embodiment 71, wherein R is a benzyl group.

73. The method according to embodiment 71, wherein R is a silyl protecting group.

74. The method according to embodiment 73, wherein R is tert-butyldimethylsilyl (TBS).

75. A method for preparing compounds of formula I-6:

The method includes making:

(i) Compounds of Formula I-7:

(ii) Compounds of formula I-5:

The reaction occurs in the presence of _NdCl3 and tetrabutylammonium chloride; where R is a hydroxyl protecting group.

76. The method according to embodiment 75, wherein R is a benzyl group.

77. The method according to embodiment 75, wherein R is a silyl protecting group.

78. The method according to embodiment 77, wherein R is tert-butyldimethylsilyl (TBS).

While the foregoing invention has been described in considerable detail by way of illustration and examples for clarity of understanding, those skilled in the art will understand that certain variations and modifications may be practiced within the scope of the appended claims. Furthermore, each reference provided herein is incorporated by way of citation as if it were individually incorporated. In the event of any conflict between this application and the references provided herein, this application shall prevail.

Claims (8)

1. A method for preparing a compound of formula I-11: The method includes making: (i) Compounds of Formula I-7: (ii) Compounds of formula I-12: The reaction occurs in the presence of _NdCl3 and tetrabutylammonium chloride; where R is a hydroxyl protecting group. 2. The method according to claim 1, wherein R is a benzyl group. 3. The method according to claim 1, wherein R is a silyl protecting group. 4. The method of claim 3, wherein R is tert-butyldimethylsilyl (TBS). 5. A method for preparing compounds of formula I-6: The method includes making: (i) Compounds of Formula I-7: (ii) Compounds of formula I-5: The reaction occurs in the presence of _NdCl3 and tetrabutylammonium chloride; where R is a hydroxyl protecting group. 6. The method according to claim 5, wherein R is a benzyl group. 7. The method of claim 5, wherein R is a silyl protecting group. 8. The method of claim 7, wherein R is tert-butyldimethylsilyl (TBS).