Pyrrolopyrimidine nucleosides and analogs thereof as useful antiviral agents

Abstract

Disclosed are pyrrolopyrimidine nucleoside analogs of Formula I, Formula IA, Formula IB, or Formula II, and phospholipid conjugates thereof, or pharmaceutical compositions thereof, wherein R c and A. The disclosure also provides methods of treating and / or preventing viral infections and / or diseases or conditions associated with viral infections with one or more compounds of Formula I, Formula IA, Formula IB, or Formula II.

Description

[0001] Related applications

[0002] This application claims priority and interest in U.S. Provisional Application No. 62 / 202010, filed August 6, 2015, and in the United Kingdom Application No. 1606645.8, filed April 15, 2016, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] This application relates to pyrrolopyrimidine nucleoside analogs and their phospholipid conjugates, as well as methods for their synthesis. Pyrrolopyrimidine nucleoside analogs and their phospholipid conjugates can be used as antiviral agents for treating viral infections. This application also relates to pharmaceutical compositions comprising pyrrolopyrimidine nucleoside analogs and their phospholipid conjugates. Background Technology

[0004] Viral infections can have severe adverse effects on individuals and society as a whole. Even non-fatal viral infections, such as Ebola, can have serious social and economic consequences. For example, human norovirus (NV) is the most common cause of epidemic acute gastroenteritis worldwide, with an estimated 19-21 million cases annually in the United States, including 56,000-71,000 hospitalizations and 570-800 deaths (Hall). wait , Emerg.Infect.Dis . 2013 Aug;19(8):1198-205).

[0005] Therefore, developing effective antiviral treatments that are effective against the virus is important for improving the health of infected individuals and as a public health measure to prevent outbreaks of other pathogenic viruses. Summary of the Invention

[0006] This disclosure provides pyrrolopyrimidine nucleoside analogs and their phospholipid conjugates. It also includes pharmaceutical compositions comprising the same substances and methods for their synthesis.

[0007] This disclosure also provides methods for treating and / or preventing viral infections and / or diseases or conditions associated with viral infections using one or more compounds according to embodiments of the present invention. This disclosure addresses the need for novel therapies for treating and / or preventing virus-induced diseases using novel antiviral agents and delivery vectors.

[0008] On the one hand, this disclosure relates to compounds of formula I:

[0009] (Formula I),

[0010] and its pharmaceutically acceptable salts, solvates, enantiomers, diastereomers, racemates and mixtures, wherein:

[0011] A is:

[0012] ,or ;

[0013] X1 is CR 11 R 12 Or OCH2CH2, where the oxygen atom is far from the R in A. I Part, of which R 11 and R 12 Independently hydrogen or substituted or unsubstituted C1-C4 alkyl;

[0014] X2 does not exist. O , C(O)O ,or OCH2 In which oxygen atoms are in A, R I The distal part;

[0015] Each R I Hydrogen alone, substituted or unsubstituted C1-C6 alkyl groups, ;

[0016] Or R I It is an amino acid residue that is bound by the carbonyl group of X2;

[0017] v is 0 or 1;

[0018] n is 0, 1, 2, or 3 and when X2 is C(O)O When n is 0;

[0019] p is 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11;

[0020] q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18;

[0021] R z It can be hydrogen, halogen, C1-C4 alkylthio, C1-C4 alkoxy, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C2-C4 alkenyl, substituted or unsubstituted C2-C4 alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, or substituted or unsubstituted non-aromatic heterocycle;

[0022] R a R b R x and R yEach is independently selected from hydrogen, halogen, OH, SH, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted aryloxy, substituted or unsubstituted C1-C6 alkylthio, substituted or unsubstituted arylthio, substituted or unsubstituted -O-carbonylalkyl, substituted or unsubstituted -O-carbonylaryl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl;

[0023] Or, any R a Or R b In (CR) a R b ) p In and another R a Or R b Together with the atoms it is attached to and any atoms in between, it forms C-C double or triple bonds, C6-C. 10 Aryl, 5- to 10-membered heteroaryl, C3-C 10 cycloalkyl, C4-C 10 Cycloalkenyl, or 5- to 10-membered non-aromatic heterocyclic structure; or any R x Or R y In (CR) x R y ) q In and another R x Or R y Together with the atoms it is attached to and any atoms in between, it forms C-C double or triple bonds, C6-C. 10 Aryl, 5- to 10-membered heteroaryl, C3-C 10 cycloalkyl, C4-C 10 Cycloalkenyl, or 5- to 10-membered non-aromatic heterocyclic structure; or any CR a R b or CR x R y Replaced by oxygen, sulfur, sulfinyl (SO) or sulfonyl (SO2);

[0024] R1 and R 45 Each of these can be independently hydrogen, halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C4-C8 cycloalkenyl, substituted or unsubstituted C2-C6 ynyl, substituted or unsubstituted C8-C 12 Cycloalkynyl, azide, OH, substituted or unsubstituted C1-C6 alkoxy groups, substituted or unsubstituted amino groups, SH, or substituted or unsubstituted C1-C6 alkylthio groups;

[0025] Each R2, R3, R4 and R 44 Independently, it is hydrogen, halogen, substituted or unsubstituted C1-C6 alkyl, N3, OH, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted amino, SH, or substituted or unsubstituted C1-C6 alkylthio; or, R3 and R4 and R 44 One of them, together with the atoms they are bonded to, forms a carbon-carbon double bond;

[0026] R5 is hydrogen, R I M + , substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted non-aromatic heterocycle, or substituted or unsubstituted heteroaryl; wherein M + It is a cation, and R5 is not an amino acid; and

[0027] R c Substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C4-C8 cycloalkenyl, substituted or unsubstituted C2-C6 ynyl, substituted or unsubstituted C8-C 12 Cycloalkynyl, or substituted or unsubstituted aryl.

[0028] On the other hand, this disclosure relates to compounds of formula IA:

[0029] (IA),

[0030] and its pharmaceutically acceptable salts, solvates, enantiomers, diastereomers, racemates and mixtures, wherein:

[0031] A is:

[0032] ,or ;

[0033] X1 is –CR 11 R 12 – or –OCH2CH2–, where the oxygen atom is far from the R in A. IA part;

[0034] R 11 and R 12 It is independently hydrogen or C1-C4 alkyl, wherein the alkyl group is optionally substituted by one or more halogens, –OH, –SH or –NH2;

[0035] X2 does not exist. O , C(O)O ,or OCH2 The oxygen atom is far from the R in A. IA part;

[0036] X3 can be either –O– or –NH–;

[0037] B can be independently –C(O)NH2, aryl, or heteroaryl;

[0038] C is independent of –OR, –NHR, or –N=CHN(R)2;

[0039] Each R IA Independently hydrogen or –C1-C6 alkyl, wherein the alkyl group is optionally converted by one or more –OH, –SH, or –NH2, oxo, or –OR. a replace;

[0040] ,

[0041] Or R IA These are amino acid residues bound by carbonyl groups;

[0042] v is 0 or 1;

[0043] n is 0, 1, 2, or 3 and when X2 is C(O)O When n is 0;

[0044] p is 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11;

[0045] q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18;

[0046] R z It is a hydrogen, halogen, -C1-C4 alkylthio, -C1-C4 alkoxy, –C1-C4 alkyl, –C2-C4 alkenyl, –C2-C4 alkynyl, aryl, heteroaryl, –C3-C8 cycloalkyl, –C4-C8 cycloalkenyl or 3- to 5-membered non-aromatic heterocycle, wherein each alkylthio, alkoxy, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl or heterocycle is optionally substituted by one or more halogens, –OH, –SH or –NH2;

[0047] R a R b R x and R yEach is independently selected from hydrogen, halogen, -OH, -SH, –C1-C6 alkoxy, aryloxy, –C1-C6 alkylthio, arylthio, –OC(O)C1-C6 alkyl, -OC(O) aryl, –C1-C6 alkyl, –C2-C6 alkenyl, –C2-C6 alkynyl, aryl, heteroaryl, –C3-C8 cycloalkyl, and –C4-C8 cycloalkenyl, wherein each alkoxy, aryloxy, alkylthio, aryl, alkenyl, alkynyl, heteroaryl, cycloalkyl, or cycloalkenyl is optionally separated by one or more halogens, –OR 11 –SR 11 Or –NR 11 R 12 replace;

[0048] Or any two R a Or R b Together with the two atoms they are attached to, they can combine to form C3-C8 spirocyclic alkyl groups or 3- to 8-membered spiroheterocyclic rings;

[0049] Or any two R a Or R b When on two adjacent atoms, they can combine to form cis- or trans-carbon-carbon double bonds or carbon-carbon triple bonds;

[0050] Or any two R a Or R b When on two adjacent atoms, they can combine to form oxo, aryl, heteroaryl, and –C3-C groups. 10 cycloalkyl, –C4-C 10 Cycloalkenyl or 5- to 10-membered heterocyclic rings;

[0051] or any CR a R b It can be replaced by –O–, –S–, –S(O)– or –SO2–;

[0052] Or any two R x Or R y Together with the two atoms they are attached to, they can combine to form C3-C8 spirocyclic alkyl groups or 3- to 8-membered spiroheterocyclic rings;

[0053] Or any two R x Or R y When on two adjacent atoms, they can combine to form cis- or trans-carbon-carbon double bonds or carbon-carbon triple bonds;

[0054] Or any two R x Or R y When on two adjacent atoms, they can combine to form oxo, aryl, heteroaryl, and –C3-C groups. 10 cycloalkyl, –C4-C 10Cycloalkenyl or 5- to 10-membered heterocyclic rings;

[0055] or any CR x R y It can be replaced by –O–, –S–, –S(O)– or –SO2–;

[0056] R1 and R 45 Each can be independently classified as hydrogen, halogen, -N3, OH, –NH2, SH, –C1-C6 alkyl, –C3-C6 cycloalkyl, –C2-C6 alkenyl, –C4-C8 cycloalkenyl, –C2-C6 ynyl, –C8-C 12 Cycloalkynyl, –C1-C6 alkoxy, or –C1-C6 alkylthioyl, wherein each alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, alkoxy, or alkylthioyl group is independently influenced by one or more halogens, –N3, or N2. OH, –NH2 or SH replaces;

[0057] R2, R3, R4 and R 44 Each is independently hydrogen, halogen, –N3, –OH, –NH2, –SH, –C1-C6 alkyl, –C1-C6 alkoxy or –C1-C6 alkylthio, wherein each alkyl, alkoxy or alkylthio is optionally substituted by one or more halogens, oxo, –OH, –NH2 or –SH.

[0058] Or R3, R4, and R 44 One of them can form a carbon-carbon double bond together with the atoms they are attached to;

[0059] Or R3, R4, and R 44 One of them, together with the atoms they are attached to, can combine to form 4- to 8-membered cycloalkyl or heterocyclic rings that are optionally substituted with C1-C6 alkyl groups;

[0060] R5 is hydrogen, R IA M + aryl, aralkyl, –C1-C6 alkyl, –C1-C6 heteroalkyl, cycloalkyl, non-aromatic heterocyclic or heteroaryl, wherein M + It is a cation, and each aryl, aralkyl, alkyl, heteroalkyl, cycloalkyl, heterocyclic, or heteroaryl group is optionally substituted with one or more halogens, N3, –OH, –NH2, or –SH, wherein R5 is not an amino acid; and

[0061] R 11 and R 12Each independently, in each case, is hydrogen, halogen, –OH, –SH, –C1-C6 alkoxy, aryloxy, –C1-C6 alkylthio, arylthio, –OC(O)C1-C6 alkyl, –OC(O) aryl, –C1-C6 alkyl, –C2-C6 alkenyl, –C2-C6 alkynyl, aryl, heteroaryl, –C3-C8 cycloalkyl and –C4-C8 cycloalkenyl, wherein each alkyl, aryl, alkenyl, alkynyl, heteroaryl, cycloalkyl and cycloalkenyl is optionally substituted by one or more halogens, –N3, –OH, –NH2 or –SH;

[0062] R c –C1-C6 alkyl, –C3-C6 cycloalkyl, –C2-C6 alkenyl, –C4-C8 cycloalkenyl, –C2-C6 ynyl, –C8-C 12 Cycloalkynyl or aryl, wherein each alkyl, cycloalkyl, alkenyl, cycloalkenyl or aryl group is optionally substituted by one or more halogens, –N3, –OH, –NH2 or –SH;

[0063] On the other hand, this disclosure relates to compounds of formula II:

[0064] (II)

[0065] and its pharmaceutically acceptable salts, solvates, enantiomers, diastereomers, racemates and mixtures, wherein:

[0066] Y is –C(O)– or , where X 1 Independent of O, NH or S, X 2 Independent of the bond, –O–, –S– or –NH–, and X 3 Independent of –OR, –NHR II Or –SR II ;

[0067] Each R II Independent as –H, –C1-C 20 Alkyl, –C2-C 20 alkenyl, –C2-C 20 Alkynyl, –C3-C8 cycloalkyl, –C4-C8 cycloalkenyl, aryl, heteroaryl, or heterocyclic, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocyclic group is optionally oxidized by one or more halogens, oxo groups, or alkyl groups. 1 –OR 1 –NR 1 R 2 –SR 1 –OC(O)R 1 –C(O)OR 1 –NHC(O)OR1 Or –NHC(O)R 1 replace;

[0068] R a and R b Each is independent, and in each case, it is –H, –C1-C 20 Alkyl, –C2-C 20 alkenyl, –C2-C 20 Alkynyl, –C3-C8 cycloalkyl, –C4-C8 cycloalkenyl, aryl, heteroaryl, or heterocyclic, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocyclic group is optionally oxidized by one or more halogens, oxo groups, or alkyl groups. 1 –OR 1 –NR 1 R 2 –SR 1 –OC(O)R 1 –C(O)OR 1 –NHC(O)OR 1 Or –NHC(O)R 1 replace;

[0069] R 1 and R 2 Each is independent, and in each case, it is –H, –C1-C 20 Alkyl, –C2-C 20 alkenyl, –C2-C 20 Alkynyl, –C3-C8 cycloalkyl, –C4-C8 cycloalkenyl, aryl, heteroaryl, or heterocyclic, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocyclic group is optionally oxidized by one or more halogens, oxo groups, or –R groups. 3 –R 4 –OR 3 –NR 3 R 4 –SR 3 –OC(O)R 3 –C(O)OR 3 –NHC(O)OR 3 Or –NHC(O)R 3 replace;

[0070] R 3 and R 4 Each is independent, and in each case, it is –H, –C1-C 20 Alkyl, –C2-C 20 alkenyl, –C2-C 20Alkynyl, –C3-C8 cycloalkyl, –C4-C8 cycloalkenyl, aryl, heteroaryl or heterocyclic, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl or heterocyclic group is optionally substituted by one or more halogens, oxo, aryl, heteroaryl, –OH, –NH2, –SH, –OC(O)H, –C(O)OH, –NHC(O)OH or –NHC(O)H;

[0071] R d Independent of –H or –D; and

[0072] n can be 0, 1, 2, or 3.

[0073] On the other hand, this disclosure relates to a pharmaceutical composition comprising a compound of formula I, IA, IB, or II, or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, racemate, or mixture thereof, and a pharmaceutically acceptable carrier. In some embodiments, this disclosure relates to a pharmaceutical composition comprising compound 1, or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, racemate, or mixture thereof, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition can be used to treat viral infections (e.g., norovirus).

[0074] On the other hand, this disclosure provides a method for treating a viral infection or a disease or condition related to a viral infection, wherein the method comprises administering to a subject in need an effective amount of a compound described herein (e.g., a compound of formula I, IA, IB, or II), or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, racemate, or mixture thereof. In some embodiments, the compound is compound 1 or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, racemate, or mixture thereof. In some embodiments, the virus is norovirus.

[0075] On the other hand, this disclosure also relates to a pharmaceutical formulation of the said compound or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, racemate, or mixture thereof, for use in methods for treating or preventing viral infections or diseases or conditions associated with viral infections (e.g., double-stranded DNA (dsDNA) or single-stranded RNA (ssRNA) viral infections). In some embodiments, the compound is compound 1. In some embodiments, the virus is norovirus.

[0076] On the other hand, this disclosure also relates to the use of the compounds or pharmaceutical formulations disclosed herein, or pharmaceutically acceptable salts, solvates, enantiomers, diastereomers, racemates, or mixtures thereof, in the preparation of medicaments for the treatment or prevention of viral infections and / or diseases or conditions associated with viral infections (e.g., ssRNA virus infection). The pharmaceutical formulations may comprise compounds of formula I, IA, IB, or II, or pharmaceutically acceptable salts, solvates, enantiomers, diastereomers, racemates, or mixtures thereof. In some embodiments, the compound is compound 1, or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, racemate, or mixture thereof. In some embodiments, the virus is norovirus.

[0077] This disclosure also relates to methods for treating or preventing viral infections and / or viral infection-related diseases or conditions (e.g., ssRNA virus infection). The method may include administering a compound of formula I, IA, IB, or II to a subject in need. In some embodiments, the compound is compound 1. In some embodiments, the virus is norovirus.

[0078] This disclosure also relates to the compounds described herein, or pharmaceutically acceptable salts, solvates, enantiomers, diastereomers, racemates, or mixtures thereof, for the treatment or prevention of viral infections or viral infection-related diseases or conditions. The compound may be a compound of formula I, IA, IB, or II. In some embodiments, the compound is compound 1, or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, racemate, or mixture thereof. In some embodiments, the virus is norovirus.

[0079] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In case of conflict, this specification (including definitions) will be used to limit the scope. In this specification, the singular form includes the plural form unless the context clearly requires otherwise. Although similar or equivalent methods and materials described herein may be used in the practice or testing of this disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference. References cited herein are not considered prior art to the claimed disclosure. Furthermore, these materials, methods, and embodiments are illustrative only and are not intended to be limiting.

[0080] Other features and advantages of this disclosure will become apparent from the following detailed description and claims. Attached Figure Description

[0081] Figure 1AThe results show mouse norovirus titers (number of plaque-forming units per mL) in tissues and feces harvested 3 days after infection as part of Study 1.

[0082] Figure 1B The results show mouse norovirus titers (number of tissue plaque-forming units per mg) in tissues and feces harvested 3 days after infection as part of Study 1.

[0083] Figure 2A The results show mouse norovirus titers (number of plaque-forming units per mL) in tissues and feces harvested 3 days after infection as part of Study 2.

[0084] Figure 2B The results show the mouse norovirus titers (number of plaque-forming units per mg of tissue) in tissues and feces harvested 3 days after infection as part of Study 2.

[0085] Figure 3A The number of plaque-forming units per gram of cecum from Study 1 is shown on a linear scale.

[0086] Figure 3B The number of plaque-forming units per gram of feces from Study 1 is shown on a linear scale.

[0087] Figure 4 The first copy of the results showing the in vitro efficacy of compound 1 in inhibiting human norovirus is presented compared to 2'-C-methylcytidine triphosphate and compound 2.

[0088] Figure 5 A second copy of the results showing the in vitro efficacy of compound 1 in inhibiting human norovirus was presented compared to 2'-C-methylcytidine triphosphate and compound 2.

[0089] Figure 6 The results show the first and second copies of the in vitro efficacy of compound 1 in inhibiting human norovirus compared to 2'-C-methylcytidine triphosphate and compound 2.

[0090] Figure 7a shows the HPLC curve of compound 1.

[0091] Figure 7b shows the HPLC curve of compound 1 after slurrying at room temperature for 3 hours.

[0092] Figure 7c shows the HPLC curve of compound 1 after slurrying at 50°C for 3 hours.

[0093] Figure 7d shows the HPLC curve of compound 1 after slurrying at room temperature for 24 hours.

[0094] Figure 8a shows the concentrations of compound 1 from about -2 to about 14 ppm. 1 HNMR spectrum.

[0095] Figure 8b shows the concentrations of compound 1 from about 2 to about 9 ppm. 1 HNMR spectrum.

[0096] Figure 8c shows the concentrations of compound 1 from approximately 0 to approximately 9 ppm. 1 HNMR spectrum.

[0097] Detailed description

[0098] Nucleoside phosphonates (e.g., ribonucleoside derivatives) represent antiviral drugs targeting specific types of viruses that rely on viral-encoding enzymes that use ribonucleotides or deoxyribonucleotides as substrates, such as viral polymerases for various RNA viruses and / or viral helicases for RNA (e.g., ssRNA) or DNA viruses. However, to avoid being bound by theory, one obstacle to the efficacy of such antiviral drugs is the requirement for biochemical modification of the agent administered intracellularly to form an active antiviral nucleoside triphosphate. In some embodiments, if the nucleoside is delivered, three phosphorylation steps are required to form the triphosphate. Delivery of nucleoside phosphonates effectively bypasses the first phosphorylation but may exacerbate the problem of clinically effective amounts of charged drug crossing the lipid bilayer surrounding cells.

[0099] Unwilling to be bound by theory, lipid conjugation can be used to disguise oral medications, including nucleoside phosphonates, as readily absorbed natural compounds. Specifically, in some embodiments, nucleoside phosphonates can be modified to partially resemble metabolic (monoacyl) phospholipids. In some embodiments, unlike normal diacyl phospholipids, monoacyl lipid-modified nucleosides can readily cross intestinal cells in the intestinal lumen, enter the circulating blood and / or lymph, and, unlike standard drugs, remain intact. Therefore, the lipid moiety not only delivers the nucleoside to the plasma, but it can also effectively promote absorption by target cells. The lipid can be cleaved in the cytoplasm of target cells, and in the case of nucleoside analogue conjugation, the corresponding monophospholipid can be produced. Overall, this approach can significantly increase the level of active antiviral drugs at the site of viral replication.

[0100] This disclosure provides compounds, pharmaceutical compositions, and methods of synthesizing and using said compounds to treat or prevent viral infections and viral infection-related diseases or conditions (e.g., single-stranded RNA virus infections).

[0101] In some embodiments, the compounds disclosed herein have an improved efficacy / toxicity ratio compared to similar compounds used in the art.

[0102] definition

[0103] The definitions of some compounds and specific functional groups disclosed herein are also described in more detail below.

[0104] It is understood that the compounds described herein, as well as those described herein, can be substituted with any number of substituents or functional moieties. Generally, the term "substituted," whether preceded by the term "optional" and the substituents contained in the general formula of this disclosure, refers to the substitution of a hydrogen group in a given structure by a specific substituent. When more than one position in any given structure can be substituted by more than one substituent selected from a particular group, the substituents at each position can be the same or different. As stated herein, the term "substituted" is intended to protect all permissible substituents of organic compounds. Broadly speaking, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. For the purposes of this invention, for example, the nitrogen heteroatom may have a hydrogen substituent and / or any permissible substituent of organic compounds satisfying the heteroatom valence state as described herein. Nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. Examples of substituents on the disclosed portions (e.g., alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, non-aromatic heterocyclic groups) include, but are not limited to, alkenyl, alkynyl, halogen, haloalkyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, heteroaryl, aryl, cycloalkyl, cycloalkenyl, non-aromatic heterocyclic, hydroxyl, carbamoyl, oxo, amino, nitro, azide, -SH, and -CN.

[0105] This disclosure is intended to include all isotopes of the atoms appearing in the compounds of the invention. Isotopes include those atoms having the same atomic number but different mass numbers. In particular, one, more, or all of the hydrogen atoms may be deuterium. Radioactive isotopes may be used, for example, for structural analysis or to facilitate tracking the fate of the compound or its metabolites after administration. By way of general example and not limitation, isotopes of hydrogen include deuterium and tritium, and isotopes of carbon include C-13 and C-14. Compounds of Formula I include those wherein R1 is H or D; R2 and R3 are independently H, D, OH, OD, CH3, or CD3 and / or R4 is H, D, CH3, or CD3.

[0106] In this paper, the term “independently” refers to variables that are applied independently, such as atoms or functional groups, which vary independently with the application. For example, when more than one substituent or atom (carbon or heteroatom, such as oxygen (O), sulfur (S), or nitrogen (N)) is present, each substituent or atom is independent of the other substituent or atom, and the substituent or atom may also be alternating.

[0107] In this document, the term "alkyl" refers to a saturated straight-chain or branched hydrocarbon group, in some embodiments containing 1 to 20, including 1 to 10, or 1 to 6 carbon atoms. Branching refers to one or more lower C1-C6 alkyl groups such as methyl, ethylene, or propyl linked to a straight-chain alkyl chain. Exemplary alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, and 3-pentyl. Examples of C1-C6 alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, neopentyl, and n-hexyl; examples of C1-C8 alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, neopentyl, n-hexyl, heptyl, and octyl. C1-C 20 Examples of alkyl groups include, but are not limited to, hexadecylmethyl, hexadecylethyl, hexadecylpropyl, octadecylmethyl, octadecylethyl, octadecylpropyl, etc.

[0108] In this document, the term "alkenyl" refers to a monovalent straight-chain or branched group derived from a hydrocarbon moiety containing, in some embodiments, 2 to 6, 2 to 8, or 2 to 20 carbon atoms having at least one carbon-carbon double bond. The double bond may or may not be the point of connection to another group. Examples of C2-C8 alkenyl groups include, but are not limited to, vinyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, heptenyl, octenyl, etc. As defined herein, an "alkenyl" group includes cis and trans isomers.

[0109] In this document, the term "alkynyl" refers to a monovalent straight-chain or branched group derived from a hydrocarbon moiety containing, in some embodiments, 2 to 6, 2 to 8, or 2 to 20 carbon atoms having at least one carbon-carbon triple bond. The triple bond may or may not be the point of connection to another group. Examples of C2-C8 alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, etc.

[0110] The term "alkoxy" refers to -O-alkyl.

[0111] The term "thioalkyl" or "alkylthio" refers to -S-alkyl. In some embodiments, the thio group may be replaced by a sulfinyl (SO) or sulfonyl (SO2) group.

[0112] As used in this article, the terms “halogen,” “halogenated,” or “halogen” refer to atoms selected from fluorine, chlorine, bromine, and iodine.

[0113] As used herein, the terms “haloalkyl,” “haloalkenyl,” or “haloalkynyl” refer to an alkyl, alkenyl, or alkynyl group substituted with one or more halogens or halogenated groups. Examples of haloalkyl groups include, but are not limited to, CF3, CH2CF3, and CCl3.

[0114] As used herein, the term "aryl" refers to a monocyclic or polycyclic carbocyclic system having one or more fused or unfused aromatic rings, including but not limited to phenyl, naphthyl, tetrahydronaphthyl, dihydroindene, and idenyl. The term aryl also includes dihydroindole.

[0115] As used herein, the term "cycloalkyl" refers to a monovalent group derived from a monocyclic or polycyclic saturated carbocyclic compound. Examples of C3-C8-cycloalkyl (3- to 8-membered cycloalkyl) include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopentyl, and cyclooctyl; C3-C 12 -Cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[2.2.1]heptyl and bicyclo[2.2.2]octyl, etc.

[0116] As used herein, the term "cycloalkenyl" refers to a monovalent group derived from a monocyclic or polycyclic partially unsaturated (i.e., non-aromatic) carbocyclic compound. In other words, it refers to a monovalent group derived from a monocyclic or polycyclic carbocyclic compound having at least one carbon-carbon double bond. Examples of such groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, etc.

[0117] As used herein, the term "cycloynyl" refers to a monovalent group derived from a partially unsaturated (i.e., non-aromatic) carbocyclic compound having at least one carbon-carbon triple bond, whether monocyclic or polycyclic. Examples include cyclooctyne.

[0118] As used herein, the term "heteroaryl" refers to a monocyclic or polycyclic (e.g., bicyclic, tricyclic, or more) fused or unfused group or ring system having at least one aromatic ring, comprising 5 to 10 ring atoms, wherein at least one ring atom is selected from S, O, P, and N. In other words, a heteroaryl is an aryl group containing at least one heteroatom. Examples of heteroaryl groups include, but are not limited to, pyridyl, furanyl, thiazolyl, imidazolyl, indolyl, benzofuranyl, etc.

[0119] The term "5- or 6-membered heteroaryl" refers to a ring having 5 to 12 ring atoms, wherein at least one ring atom is selected from S, O, P, and N. Heteroaryl groups include, but are not limited to, pyridyl, piperazinyl, pyrimidinyl, pyrroleyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolyl, quinoxalinyl, etc.

[0120] As used herein, the term "non-aromatic heterocycle" or "non-aromatic heterocyclic ring" refers to a saturated or unsaturated, non-aromatic monocyclic or polycyclic, fused or unfused system, wherein, for example, at least one ring contains one to four heteroatoms independently selected from oxygen, sulfur, phosphorus, and nitrogen. The nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatomium may optionally be quaternized. Representative non-aromatic heterocyclic groups include, but are not limited to, [1,3]dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolylyl, imidazolinyl, imidazolinyl, piperidinyl, piperazinyl, oxazolyl, isoxazolyl, morpholinyl, thiazolinyl, isothiazolyl, and tetrahydrofuranyl.

[0121] The term “oxo” as used in this article is understood to describe the carbonyl group (i.e., C(O)).

[0122] As described herein, the compounds of this disclosure may optionally be substituted with one or more substituents, such as those generally described above, or those exemplified by the specific classes, subclasses, and species of this disclosure. It is understood that the phrase “optionally substituted” is used interchangeably with the phrase “substituted or unsubstituted.” Generally, the term “substituted,” whether or not preceded by the term “optionally,” refers to the substitution of a hydrogen group in a given structure by a particular substituent. Unless otherwise stated, an optionally substituted group may have a substituent at each substituted position of the group, and when more than one position in any given structure may be substituted by more than one substituent selected from a particular group, the substituents at each position may be the same or different.

[0123] The term "protected" as used herein refers to a functional group or compound of this disclosure that has a protecting group in synthesis that temporarily masks the chemical characteristics of a functional group (e.g., hydroxyl, amino, carboxyl, etc.) because it interferes with another reaction. After the reaction is complete, these protecting groups are removed by conventional methods, or the protecting compound is used as a prodrug or as a compound of this disclosure.

[0124] As used in this article, the term "prodrug" or "pharmaceutically acceptable prodrug" refers to a compound that is rapidly converted in the body to produce the parent compound, for example, through hydrolysis in the blood (T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems , Vol. 14 of the ACS Symposium Series; Edward B.Roche, ed., Bioreversible Carriers in Drug Design (American Pharmaceutical Association and Pergamon Press, 1987, these two articles are cited here for reference).

[0125] When used as an adjective herein, the term “pharmaceutical” or “pharmaceutical acceptable” means substantially non-toxic and substantially harmless to the recipient. In this context, the phrase “pharmaceutical acceptable” refers to those compounds, materials, compositions, carriers, and / or dosage forms that, to a reasonable extent of medical judgment, are suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0126] "Pharmaceutical formulation" also refers to a formulation in which the carrier, solvent, excipient, and salt must be compatible with the active ingredient (e.g., the compounds disclosed herein). Those skilled in the art will understand that the terms "pharmaceutical formulation" and "pharmaceutical composition" are generally interchangeable and are therefore used for the purposes of this application, and include formulations suitable for administration to mammals (e.g., humans).

[0127] The term "pharmaceutical composition" as used herein refers to formulations containing the disclosed compound in a form suitable for administration to a subject. In one embodiment, the pharmaceutical composition is in bulk or unit dosage form. The unit dosage form is any of a variety of forms, including, for example, a capsule, an IV bag, a tablet, an aerosol inhaler, or a single pump in a medicine bottle. The amount of the active ingredient (e.g., a formulation of the disclosed compound or its salts, hydrates, solvates, or isomers) in a unit dosage composition is an effective amount and varies depending on the specific treatment involved. Those skilled in the art will understand that dose adjustments are sometimes necessary based on the patient's age and condition. The dose will also depend on the route of administration. "Pharmaceutically acceptable carriers" as used herein may include any and all solvents, diluents or other liquid carriers, dispersing or suspending agents, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, etc., depending on the desired specific dosage form. Remington's Pharmaceutical Sciences, Sixteenth Edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in the formulation of pharmaceutical compositions and known techniques for their preparation. Except for any common carrier media that are incompatible with the compound, for example by producing any undesirable biological effects or interacting with any other component of the pharmaceutical composition in a harmful manner, its use is considered to be within the scope of this disclosure. Examples of materials that can be used as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered astragalus gum; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil, sesame oil; olive oil; corn oil, and soybean oil; ethylene glycol; such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol and phosphate buffer solutions; and other non-toxic and compatible lubricants such as sodium dodecyl sulfate and magnesium stearate. Colorants, release agents, coating agents, sweeteners, flavoring agents, and aromatizers, preservatives, and antioxidants may also appear in the compositions at the formulator's discretion. "Pharmaceutically acceptable excipients or carriers" also refers to excipients or carriers used in the preparation of pharmaceutical compositions that are generally safe, non-toxic, and biologically or otherwise unadverse, and includes excipients acceptable for veterinary and human use. The term "pharmaceutical acceptable excipients" as used includes one or more such excipients in the specification and claims.

[0128] The compounds disclosed herein include the compounds themselves, their salts, their solvates, and their prodrugs, if applicable. Salts, for example, can be formed between an anion and a positively charged group (e.g., a protonated amino group) of the disclosed compound. Suitable anions include chloride, bromide, iodide, sulfate, hydrogen sulfate, aminosulfonate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, glutamate, glucuronide, glutarate, malate, maleate, succinate, fumarate, tartrate, toluenesulfonate, salicylate, lactate, naphthalenesulfonate, and acetate (e.g., trifluoroacetate). The term "pharmaceutically acceptable anion" refers to an anion suitable for forming a pharmaceutically acceptable salt. Similarly, salts can also be formed between a cation and a negatively charged group (e.g., a carboxyl group) on the disclosed compound. Suitable cations include sodium, potassium, magnesium, calcium, and ammonium cations such as tetramethylammonium ions. The compounds disclosed herein also include those salts containing a tetravalent nitrogen atom. Examples of prodrugs include esters and other pharmaceutically acceptable derivatives that, when administered to a subject, provide the active compound of this disclosure.

[0129] Additionally, physiologically acceptable, i.e. pharmaceutically compatible, salts can be salts of the disclosed compounds with inorganic or organic acids. Preferred are salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, or sulfuric acid, or salts formed with organic carboxylic acids or sulfonic acids such as acetic acid, trifluoroacetic acid, propionic acid, maleic acid, fumaric acid, malic acid, citric acid, tartaric acid, lactic acid, benzoic acid, or methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, or naphthalenedisulfonic acid.

[0130] Other pharmaceutically compatible salts that may be mentioned are those that form with common bases, such as alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., calcium or magnesium salts), or ammonium salts, derived from ammonia or organic amines, such as diethylamine, triethylamine, ethyl diisopropylamine, procaine, dibenzylamine, N-methylmorpholine, dihydrorosinamine, or methylpiperidine.

[0131] As used herein, "pharmaceutically acceptable salt" may refer to derivatives of the compounds disclosed herein, wherein the parent compounds are modified by preparing their acid or base salts. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines, and bases or organic salts of acidic residues such as carboxylic acids. Pharmaceutically acceptable salts include, for example, conventional nontoxic salts or quaternary ammonium salts of parent compounds formed from nontoxic inorganic or organic acids.

[0132] For example, such conventional non-toxic salts include, but are not limited to, those derived from 2-acetoxybenzoic acid, 2-hydroxyethanesulfonic acid, acetic acid, ascorbic acid, benzenesulfonic acid, benzoic acid, hydrocarbonic acid, carbonic acid, citric acid, acetic acid, ethanedisulfonic acid, 1,2-ethanesulfonic acid, fumaric acid, glucoheponic acid, gluconic acid, glutamic acid, glycolic acid, ethylene glycol silane, hexyl isophthalic acid, hydroxycarbamic acid, hydrobromic acid, hydroiodic acid, and hydroxymaleic acid. Hydroxynaphthyl carboxylic acid, hydroxyethanesulfonic acid, lactic acid, lactobionic acid, lauryl sulfonic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, naphthalenesulfonic acid, nitric acid, oxalic acid, dihydroxynaphthyl acid, pantothenic acid, phenylacetic acid, phosphoric acid, polygalacturonic acid, propionic acid, salicylic acid, stearic acid, acetic acid, succinic acid, aminosulfonic acid, sulfanilic acid, sulfuric acid, tannic acid, tartaric acid, toluenesulfonic acid, and commonly present amino acids such as glycine, alanine, phenylalanine, arginine, etc.

[0133] Other examples of pharmaceutically acceptable salts may include hexanoic acid, cyclopentanepropionic acid, pyruvic acid, malonic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo-[2.2.2]-oct-2-ene-1-carboxylic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, mucoconic acid, etc. The present invention also includes salts formed when acid protons present in the parent compound are replaced by metal ions or combined with organic bases, wherein the metal ions are, for example, alkali metal ions or alkaline earth metal ions, such as aluminum ions; and the organic bases are, for example, ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucosamine, diethylamine, diethylaminoethanol, ethylenediamine, imidazole, lysine, arginine, morpholine, 2-hydroxyethylmorpholine, dibenzylethylenediamine, trimethylamine, piperidine, pyrrolidine, benzylamine, tetramethylammonium hydroxide, etc.

[0134] It should be understood that all references to pharmaceutically acceptable salts include the same salt in its solvation form (solvent) or crystalline form (polymorph) as defined herein.

[0135] The compounds disclosed herein can also be formulated as prodrugs. In some embodiments, one or more compounds of the present disclosure are formulated as prodrugs. In some embodiments, upon administration in vivo, the prodrug is chemically converted into a biologically, pharmaceutically, or therapeutically more active form. In some embodiments, prodrugs are useful because they are easier to administer than the corresponding active form. For example, in some cases, a prodrug may be more bioavailable than the corresponding active form (e.g., by oral administration). In some cases, a prodrug may have improved solubility compared to the corresponding active form. In some embodiments, a prodrug is less water-soluble than the corresponding active form. In some cases, such a prodrug has excellent cell membrane crossing ability (where aqueous solutions are detrimental to fluidity). In some embodiments, the prodrug is an ester. In some such embodiments, the ester is metabolically hydrolyzed to a carboxylic acid upon administration. In some cases, the carboxylic acid-containing compound is the corresponding active form. In some embodiments, the prodrug comprises a short peptide (polyamino acid) bound to an acid group. In some such embodiments, the peptide is cleaved upon administration to form the corresponding active form.

[0136] In some embodiments, prodrugs are prepared by modifying pharmaceutically active compounds so that the active compounds can be regenerated upon administration in vivo. The prodrugs can be designed to alter the metabolic stability or permeability of the drug, mask side effects or toxicity, improve the flavor of the drug, or change other characteristics or properties of the drug. With knowledge of in vivo pharmacodynamic processes or drug metabolism, those skilled in the art can design prodrugs of a known active pharmaceutical compound (see, for example, Nogrady (1985)). Medicinal Chemistry A Biochemical Approach, OxfordUniversity Press, New York, pages 388-392).

[0137] Furthermore, the compounds disclosed herein, such as salts of said compounds, may exist in hydrated or non-hydrated (anhydrous) form or as solvates with other solvent molecules. Non-limiting examples of hydrates include monohydrates, dihydrates, etc. Non-limiting examples of solvates include ethanol solvates, acetone solvates, etc.

[0138] Some of the compounds disclosed herein can exist in both non-solventized and solvated forms (e.g., hydrates).

[0139] "Solvate" refers to a solvent addition form containing stoichiometric or non-stoichiometric amounts of solvent. Some compounds, in their crystalline solid state, tend to trap solvent molecules in a fixed molar ratio, thus forming a solvate. If the solvent is water, the formed solvate is a hydrate; when the solvent is an alcohol, the formed solvate is an alcohol. Hydrates are formed by the combination of one or more water molecules with one of the substances, wherein the water retains its molecular state H₂O; this combination can form one or more hydrates. In hydrates, water molecules are linked by secondary valence bonds through intermolecular forces (particularly hydrogen bridges). Solid hydrates contain a stoichiometric amount of so-called water of crystallization, wherein the water molecules need not be identical in terms of their binding state. Examples of hydrates are sesquihydrates, monohydrates, dihydrates, or trihydrates. Hydrates of salts of the compounds of this invention are also suitable.

[0140] This disclosure also includes metabolites of the compounds described herein. Metabolites from chemical compounds, whether inherent or pharmaceutical, are formed during the natural biochemical processes that degrade and eliminate the compound. The degradation rate of a compound is a significant determinant of its duration and potency of action. Analyzing the metabolites of pharmaceutical compounds, and understanding drug metabolism, is an important part of drug development, enabling the identification of any undesirable side effects.

[0141] As used herein, the terms “treatment (verb),” “treatment (gerund),” or “treatment (noun)” refer to any reduction, to any considerable degree, of symptoms, signs, and / or any side effects in a patient currently suffering from the condition. In some implementations, treatment may be administered only to recipients exhibiting early symptoms to reduce the risk of developing the disease, condition, and / or symptom.

[0142] As used herein, the terms “prevention (verb),” “prevention (noun),” or “prevention (gerund)” refer to any method of partially or completely preventing or delaying the onset of one or more symptoms or features of a disease, symptom, and / or condition. Preventive treatment may be administered to subjects who show no signs of disease, symptom, and / or condition.

[0143] As used herein, the term "therapeutic effective amount" refers to the amount of a pharmaceutical agent used to treat, alleviate, or prevent an identified disease or condition, or to exhibit a detectable therapeutic or inhibitory effect. This effect can be detected by any analytical method known in the art. "Therapeutic effective amount" as used herein may also refer to the amount required to produce a clinically observable improvement in a patient. In some embodiments, the composition is formulated such that it contains an amount that will not cause one or more undesirable side effects. An effective amount of a pharmaceutical agent may also refer to an objectively identifiable improvement as indicated by a clinician or other qualified observer.

[0144] As used herein, the term "therapeutic effective amount" refers to the amount of a drug agent used to treat, improve, or prevent an identified disease or condition, or to exhibit a detectable therapeutic or inhibitory effect. This effect can be detected by any assay method known in the art. As used herein, "therapeutic effective amount" can also mean the amount necessary to achieve clinically observed improvement in a patient. The exact effective amount for a subject depends on the subject's weight, build, and health status; the nature and severity of the condition; and the combination of the chosen therapeutic agent or treatment method. The therapeutic effective amount for a given situation can be determined through routine testing within the skill and judgment of a clinician.

[0145] As used herein, "subject" refers to a human or an animal (more typically a mammal in the case of an animal). In one aspect, the subject is a human. In one aspect, the subject is male. In one aspect, the subject is female.

[0146] The compounds disclosed herein can also be prepared as esters, for example, pharmaceutically acceptable esters. For example, the carboxylic acid functional group in the compound can be converted into its corresponding ester, such as methyl, ethyl, or other esters. Furthermore, the alcohol or hydroxyl group in the compound can be converted into its corresponding ester, such as acetate, propionate, or other esters.

[0147] This disclosure includes new compounds or pharmaceutically acceptable salts thereof, typically represented by Formula I, Formula IA, Formula IB or Formula II, as well as their preparation methods and uses.

[0148] Throughout this specification, when a composition is described as having, including, or containing a particular component, it is taken into consideration that the composition is also essentially composed of said component, or that the composition is composed of said component.

[0149] compound

[0150] In one aspect, this disclosure relates to compounds of formula I:

[0151] (I)

[0152] and its pharmaceutically acceptable enantiomers, diastereomers, racemates, mixtures, solvates or salts thereof, wherein R c As defined above, and A.

[0153] In one or more embodiments of Formula I, Formula IA, or Formula IB, A is selected from A1 to A14, wherein R can be R I R IA Or R IB :

[0154] A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12, A13, and A14.

[0155] In one or more embodiments of Formula I, Formula IA, or Formula IB, A is selected from A1 to A14, wherein R can be R I R IA Or R IB :

[0156] , , ,or .

[0157] In one or more embodiments of Formula I, Formula IA, or Formula IB, A is selected from A1 to A14, wherein R can be R I R IA Or R IB :

[0158] , , , , , , , , and ,

[0159] In one or more embodiments of Formula I, Formula IA, or Formula IB, A is

[0160] ,

[0161] Where R can be R I Or R IA .

[0162] In one or more embodiments of Formula I, Formula IA, or Formula IB, A is

[0163] Where R can be R I Or R IA .

[0164] In one or more embodiments, the compound of formula I may have one or more of the following characteristics: R4 is H, a substituted or unsubstituted C1-C6 alkyl group, NH2, OH, or SH. R4 may be a C1-C6 alkyl group optionally substituted with one or more halogens. R4 may be methyl, CH2X, CHX2, or CX3, where X is a halogen. R4 may be pentyl. R1 may be hydrogen. R2 and R3 may each be independently hydrogen, CH3, CH2X, CHX2, CX3, N3, OH, or NH2, where X is a halogen, and R5 may be H, M, a C1-C6 alkyl group, phenyl, or benzyl. + It could be Na + Li + K + Ca 2+ Mg 2+ or NR g R d R e R f + , where R g R d R e and R f Each is independently hydrogen or C 1-5 Alkyl, R I It can be H. When X2 does not exist, v can be 0. R1 can be hydrogen. 45 It could be hydrogen. R c It can be a C1-C6 alkyl group, such as methyl.

[0165] In one or more embodiments of the compound of formula I or IA, R1 is –H. In one or more embodiments, R2 is –OH. In one or more embodiments, R4 is –OH. In one or more embodiments, R2 and R4 are each –OH. In one or more embodiments, R3 is –H. In one or more embodiments, R… 44 For –H. In one or more embodiments, R3 and R 44 Each is –OH. In one or more embodiments, R IA For –H. In one or more embodiments, R c For –CH3. In one or more embodiments, v is 1, X2 is –O–, n is 0, R I For –H. In one or more embodiments, v is 1, X2 is –O–, n is 0, R IA For –H.

[0166] In one or more embodiments of the compound of formula II, R a and R b All are -H. In one or more embodiments, Rc -H. In one or more embodiments, n is 0. In one or more embodiments, R II For -H. In one or more embodiments, R a R b and R c For -H. In one or more embodiments, R a R b and R c It is -H and n is 0. In one or more embodiments, R a R b and R c For -H. In one or more embodiments, R a R b and R c =-H, n is 0 and R II It is -H.

[0167] In one or more embodiments, this disclosure provides compounds of formula IB:

[0168] (Form IB),

[0169] and its pharmaceutically acceptable salts, solvates, enantiomers, diastereomers, racemates, or mixtures, wherein:

[0170] A is:

[0171] ,or ;

[0172] X1 is –CR 11 R 12 – or –OCH2CH2–, where the oxygen atom is far from the R in A. IB part;

[0173] R 11 and R 12 Independently hydrogen or C1-C4 alkyl, wherein the alkyl group is optionally substituted with one or more halogens, –OH, –SH or –NH2;

[0174] X2 does not exist. O , C(O)O ,or OCH2 The oxygen atom is far from the R in A. IB part;

[0175] Each RI B Independently hydrogen, –C1-C6 alkyl,

[0176] ,

[0177] Or R IB It is an amino acid residue bound by a carbonyl group, wherein the alkyl group is optionally substituted with one or more halogens, –OH, –SH or –NH2;

[0178] v is 0 or 1;

[0179] n is 0, 1, 2, or 3 and when X2 is C(O)O When n is 0;

[0180] p is 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11;

[0181] q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18;

[0182] R z It is a hydrogen, halogen, -C1-C4 alkylthio, -C1-C4 alkoxy, –C1-C4 alkyl, –C2-C4 alkenyl, –C2-C4 alkynyl, aryl, heteroaryl, –C3-C8 cycloalkyl, –C4-C8 cycloalkenyl or 3- to 5-membered non-aromatic heterocycle, wherein each alkylthio, alkoxy, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl or heterocycle is optionally substituted by one or more halogens, –OH, –SH or –NH2;

[0183] R a R b R x and R y Each of the following is independently selected from hydrogen, halogen, -OH, -SH, –C1-C6 alkoxy, aryloxy, –C1-C6 alkylthio, arylthio, –OC(O)C1-C6 alkyl, OC(O) aryl, –C1-C6 alkyl, –C2-C6 alkenyl, –C2-C6 alkynyl, aryl, heteroaryl, –C3-C8 cycloalkyl and –C4-C8 cycloalkenyl, wherein each alkoxy, aryloxy, alkylthio, aryl, alkenyl, alkynyl, heteroaryl, cycloalkyl or cycloalkenyl is optionally substituted by one or more halogens, –OH, –SH or –NH2;

[0184] Or any two R a Or R b Together with the two atoms they are attached to, they can combine to form C3-C8 spirocyclic alkyl groups or 3- to 8-membered spiroheterocyclic rings;

[0185] Or any two R a Or R bWhen on two adjacent atoms, they can combine to form cis- or trans-carbon-carbon double bonds or carbon-carbon triple bonds;

[0186] Or any two R a Or R b When on two adjacent atoms, they can combine to form aryl, heteroaryl, and –C3-C groups. 10 cycloalkyl, –C4-C 10 Cycloalkenyl or 5- to 10-membered heterocyclic rings;

[0187] or any CR a R b It can be replaced by –O–, –S–, –S(O)– or –SO2–;

[0188] Or any two R x Or R y Together with the two atoms they are attached to, they can combine to form -C3-C8 spirocyclic alkyl groups or 3- to 8-membered spiroheterocyclic rings;

[0189] Or any two R x Or R y When on two adjacent atoms, they can combine to form cis- or trans-carbon-carbon double bonds or carbon-carbon triple bonds;

[0190] Or any two R x Or R y When on two adjacent atoms, they can combine to form aryl, heteroaryl, and -C3-C groups. 10 cycloalkyl, –C4-C 10 Cycloalkenyl or 5- to 10-membered heterocyclic rings;

[0191] or any CR x R y It can be replaced by –O–, –S–, –S(O)– or –SO2–;

[0192] R1 and R 45 Each can be independently classified as hydrogen, halogen, -N3, OH, –NH2, SH, –C1-C6 alkyl, –C3-C6 cycloalkyl, –C2-C6 alkenyl, –C4-C8 cycloalkenyl, –C2-C6 ynyl, –C8-C 12 Cycloalkynyl, –C1-C6 alkoxy, or –C1-C6 alkylthioyl, wherein each alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, alkoxy, or alkylthioyl group is independently influenced by one or more halogens, –N3, or N2. OH, –NH2 or SH replaces;

[0193] R2, R3, R4 and R44 Each is independently hydrogen, halogen, –N3, –OH, –NH2, –SH, –C1-C6 alkyl, –C1-C6 alkoxy or –C1-C6 alkylthio, wherein each alkyl, alkoxy or alkylthio is optionally substituted by one or more halogens, –OH, –NH2 or –SH;

[0194] Or R3, R4, and R 44 One of them, together with the atoms they are attached to, can form a carbon-carbon double bond;

[0195] R5 is independently hydrogen, -R IB M + aryl, aralkyl, –C1-C6 alkyl, –C1-C6 heteroalkyl, cycloalkyl, non-aromatic heterocyclic or heteroaryl, wherein M + It is a cation, and each aryl, aralkyl, alkyl, heteroalkyl, cycloalkyl, heterocyclic, or heteroaryl group is optionally substituted with one or more halogens, N3, –OH, –NH2, or –SH, wherein R5 is not an amino acid; and

[0196] R c –C1-C6 alkyl, –C3-C6 cycloalkyl, –C2-C6 alkenyl, –C4-C8 cycloalkenyl, –C2-C6 ynyl, –C8-C 12 Cycloalkynyl or aryl, wherein each alkyl, cycloalkyl, alkenyl, cycloalkenyl or aryl group is optionally substituted by one or more halogens, –N3, –OH, –NH2 or –SH;

[0197] In one or more embodiments, R I R IA R B Or R II Selected from -H,

[0198] , , , , , , , , , , , , , , , , , , , , , , , ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， , , , , , , , , , , , , , and .

[0199] In one or more embodiments, the compound is of formula Ia:

[0200] (Ia),

[0201] Or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, racemate, or mixture thereof.

[0202] In one or more embodiments, the compound is of formula Ib:

[0203] (Ib),

[0204] Or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, racemate, or mixture thereof.

[0205] In one or more embodiments, the compound is of formula Ic:

[0206] (Ic),

[0207] Or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, racemate, or mixture thereof.

[0208] In one or more embodiments, the compound is of formula Id:

[0209] (Id),

[0210] Or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, racemate, or mixture thereof.

[0211] In one or more embodiments, the compounds disclosed herein are:

[0212] ,

[0213] Or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, racemate, or mixture thereof.

[0214] In one or more embodiments, the compound of this disclosure is compound 1:

[0215] (Compound 1; 4-amino-7-((2R, 3R, 4S, 5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-methyl-7H-pyrrolo[2, 3-d]pyrimidine-5-carboxamide), or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, racemate, or mixture thereof.

[0216] In one or more embodiments, the compound of this disclosure is a compound 1-triphosphate (compound 1-TP or compound 1-PPP), or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, racemate, or mixture thereof:

[0217] .

[0218] In one or more embodiments, the compounds disclosed herein are selected from:

[0219] or Or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, racemate, or mixture thereof.

[0220] In one or more embodiments, the compounds disclosed herein are selected from:

[0221] , , , and Or, a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, racemate, or mixture thereof.

[0222] In one or more embodiments, the compounds disclosed herein are selected from:

[0223] , , , and Or, a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, racemate, or mixture thereof.

[0224] In one or more embodiments, the compounds disclosed herein are selected from:

[0225] ,

[0226] ,

[0227] or Or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, racemate, or mixture thereof.

[0228] In one or more embodiments, the compounds disclosed herein are selected from:

[0229] ,

[0230] ,

[0231] ,or Or, a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, racemate, or mixture thereof.

[0232] In some embodiments, this disclosure provides the use of the compound or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, racemate, or mixture thereof in the preparation of a medicament for treating a disease.

[0233] In some embodiments, this disclosure provides compounds

[0234]

[0235] Use in the preparation of medicaments for treating diseases. In some embodiments, the disease is a viral infection. In some embodiments, the viral infection is norovirus.

[0236] In some embodiments, this disclosure provides the use of the compounds disclosed herein in the treatment of diseases.

[0237] In some embodiments, this disclosure provides compounds

[0238]

[0239] Used for treating diseases. In some embodiments, the disease is a viral infection. In some embodiments, the viral infection is a norovirus infection.

[0240] In some embodiments, the compounds of this disclosure are selected from compounds 1, 2, 3, 4, 5, 6, 71, 77, 76, 107, 111, 126, 133, 137, 139, 141, 143 or 145, or pharmaceutically acceptable salts, solvates, enantiomers, diastereomers, racemates or mixtures thereof, or combinations thereof.

[0241] Synthesis method

[0242] The compounds disclosed herein can be prepared using various methods, including standard chemistry. Suitable synthetic routes are described in the schemes given below.

[0243] The compounds described herein (e.g., compounds of formula I, IA, IB, or II) can be prepared by methods known in the field of organic synthesis, as in part by the synthetic schemes and examples described below. In the schemes described below, it will be readily understood that protective groups for sensitive or reactive groups are employed, as necessary, based on general principles or chemistry. Protective groups are treated according to standard methods of organic synthesis (TW Greene and PGM Wuts, "Protective Groups in Organic Synthesis", Third edition, Wiley, New York 1999). These groups are removed at a convenient stage of compound synthesis using methods readily apparent to those skilled in the art. The chosen procedures, reaction conditions, and sequence of execution should be consistent with the preparation of compounds of formula I, IA, IB, or II.

[0244] Those skilled in the art will recognize the presence of a stereocenter in compounds of Formula I, IA, IB, or II. Therefore, this disclosure includes two possible stereoisomers (unless specifically stated in the synthesis) and encompasses not only racemic compounds but also individual enantiomers and / or diastereomers. When a single enantiomer or diastereomer is required, it can be obtained through stereospecific synthesis or by isolating the final product or any convenient intermediate. The isolation of the final product, intermediate, or starting material may be affected by any suitable method known in the art. See, for example, "Stereochemistry of Organic Compounds" by E.L. Eliel, S.H. Wilen, and L.N. Mander (Wiley-Interscience, 1994).

[0245] The compounds described herein can be prepared from commercially available raw materials or synthesized using known organic, inorganic, and / or enzymatic methods.

[0246] Scheme 1: General synthesis of the compounds of the present invention

[0247]

[0248] As shown in Scheme 1 above, 4-chloro-2-methyl-7H-pyrrolo[2, 3-d]pyrimidine (a; Scheme 1 number) can be iodinated in the presence of N-iodosuccinimide (NIS). As shown in step 2, the resulting 4-chloro-5-iodo-2-methyl-7H-pyrrolo[2, 3-d]pyrimidine (b) can be treated with a protected furan (c) to give compound (d). Radical substitution of (d) yields the corresponding cyano derivative (e), which can be deprotected and nucleophilically substituted on the chlorinated carbon to give the amine derivative (f). Finally, hydration of the nitrile of (f) gives 4-amino-7-((2R, 3R, 4S, 5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-methyl-7H-pyrrolo[2, 3-d]pyrimidine-5-carboxamide (1).

[0249] Option 2: General synthesis of the compounds of the present invention

[0250]

[0251] Step 1 4-Amino-6-bromo-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-carboxylonitrile, (3R,4R,5R)-2-acetoxy-5-((benzoyloxy)methyl)tetrahydrofuran-3,4-dimethyldibenzoate, and DCE were charged into a reactor. Stirring was started and DBU was added. TMSOTf (8.01 kg) was slowly added. The reaction mixture was diluted with DCM and slowly quenched with water while cooling. The reaction product was extracted with DCM (19.90 kg) and washed with saturated NaHCO3. The aqueous phase was further extracted with DCM (19.71 kg) and washed with brine.

[0252] Step 2 (2R,3R,4R,5R)-2-(4-amino-6-bromo-5-cyano-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5-((benzoyloxy)methyl)tetrahydrofuran-3,4-dimethyldibenzoate was placed in a reactor with 10% Pd C and THF. Hydrogen was supplied to the reactor, and the mixture was stirred at approximately 31 psi at room temperature for about 4 hours.

[0253] The reaction mixture was filtered using diatomaceous earth (7.2 kg) and a polishing filter, and the residue was washed with THF. The combined filtrate and washes were transferred to a 100-L jacketed reactor by means of THF washing. The reactor contents were vacuum distilled at a maximum temperature of 30.0 °C for approximately 6 hours to obtain a final volume of 27 L. IPA was added to the reactor. The reactor contents were vacuum distilled. IPA was added to the reactor. The reactor contents were heated to approximately 60 °C, stirred, and slowly cooled to approximately 5 °C. Cooling and stirring were continued at a minimum temperature of approximately 1 °C for approximately 9 hours. The slurry was filtered and washed with IPA. The residue was dried under vacuum and supplied with a nitrogen stream to achieve a LOD of 0.36%. Yield: (73.9%). 1 The structure was determined by 1H NMR. Purity: 97.78% (HPLC, AUC).

[0254] Step 3 A solution of (2R,3R,4R,5R)-2-(4-amino-5-cyano-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5-((benzoyloxy)methyl)tetrahydrofuran-3,4-dimethylbenzoate and THF was heated, and NaOH was added. The initial addition produced a biphasic mixture and an endothermic effect, but as the addition continued, a single-phase, clear solution formed, accompanied by rapid exothermic reaction; the reaction temperature was maintained for approximately 2 1 / 2 hours during the remaining addition. IPC showed no remaining (2R,3R,4R,5R)-2-(4-amino-5-cyano-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5-((benzoyloxy)methyl)tetrahydrofuran-3,4-dimethylbenzoate).

[0255] The reaction mixture was cooled to 21°C and neutralized to a neutral pH with 3N HCl while being externally cooled. The mixture was further cooled, and the resulting neutral mixture was vacuum distilled until solids were observed in the vessel. The suspension was cooled and stirred at approximately 2°C for about 2 hours. The pale yellow suspension was filtered to obtain a dark filtrate; the grayish-white residue was washed once with cold water.

[0256] In some embodiments, the compounds of this disclosure, such as Formula I, IA, IB, or II, can be prepared as enantiomers, diastereomers, and racemates. In some embodiments, the compounds of this disclosure, such as Formula I, IA, IB, or II, include all isomeric (e.g., enantiomers, diastereomers, and geometric (or conformational) forms of the structure; for example, each asymmetric center R and S Configuration, （Z） and （E） Double bond isomers, and （Z） and（E） Conformational isomers. Therefore, single stereochemical isomers of the compounds of the present invention, as well as enantiomers, diastereomers, and geometric (or conformational) mixtures, are all within the scope of this disclosure. Unless otherwise stated, all tautomer forms of the compounds disclosed herein are within the scope of this disclosure.

[0257] The compounds of the present invention, such as those of formula I, IA, IB, or II, can be synthesized substantially without impurities. The compounds of this disclosure have a purity greater than or equal to about 99% w / w. In some embodiments, phosphonates can be prepared on a large scale, e.g., at an industrial production scale rather than a laboratory scale. For example, the batch method according to the present disclosure allows for the preparation of at least 1 g, or at least 5 g, or at least 10 g, or at least 100 g, or at least 1 kg, or at least 100 kg of phosphonate product. Furthermore, the method allows for the preparation of phosphonate products with a purity of at least 98%, or at least 98.5% (as measured by HPLC). In preferred embodiments, these products are obtained in a reaction sequence that does not involve purification by any form of chromatographic method (e.g., gas chromatography, HPLC, preparative LC, volumetric exclusion chromatography, etc.).

[0258] Pharmaceutical compositions and treatment methods

[0259] As described above, this document provides a pharmaceutical composition comprising a compound of the present disclosure (e.g., Formula I, Formula IA, Formula IB, or Formula II) or a pharmaceutically acceptable salt thereof. In some embodiments, this disclosure provides a pharmaceutical composition comprising a compound of Formula I, Formula IA, Formula IB, or Formula II or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and / or diluent. In some embodiments, this disclosure provides a compound of Formula I, Formula IA, Formula IB, or Formula II formulated as a pharmaceutical composition. In some embodiments, the compound of Formula I, Formula IA, Formula IB, or Formula II is formulated as a tablet. In another embodiment, the compound of Formula I, Formula IA, Formula IB, or Formula II is formulated as a suspension.

[0260] The formulation and application techniques of the compounds disclosed herein can be used to... Remington: the Science and Practice of Pharmacy ,twenty two nd The edition can be found in Pharmaceutical Press (2012).

[0261] In one embodiment, the compounds described herein, and their pharmaceutically acceptable salts, are used together with their pharmaceutically acceptable carriers or diluents in a pharmaceutical formulation. Suitable pharmaceutically acceptable carriers include inert solid fillers or diluents and sterile aqueous or organic solutions. The compounds will be present in the pharmaceutical composition in an amount sufficient to provide the desired dosage within the range described herein.

[0262] The compounds disclosed herein, such as compounds of formula I, IA, IB, or II described herein, or pharmaceutically acceptable salts thereof, can be combined with pharmaceutically acceptable carriers according to conventional pharmaceutical compounding techniques. Furthermore, the carrier can take various forms depending on the desired dosage form (e.g., oral, nasal, rectal, vaginal, parenteral (including intravenous injection or infusion). Any commonly used pharmaceutical medium can be used when preparing compositions for oral dosage forms. Commonly used pharmaceutical media for oral liquid dosage forms (e.g., suspensions, solutions, emulsions, and elixirs) include, for example, water, ethylene glycol, oils, alcohols, flavoring agents, preservatives, coloring agents, etc.; for oral solid dosage forms (e.g., powders, capsules, and tablets), these include aerosols, or carriers such as starch, sugar, microcrystalline cellulose, diluents, granulators, lubricants, binders, disintegrants, etc.

[0263] In another embodiment, this disclosure provides a method for therapeutically and / or prophylactically treating a viral infection in a subject (e.g., an immunocompromised subject), the method comprising administering any one of a compound of formula I, formula IA, formula IB, or a pharmaceutically acceptable salt thereof. In some embodiments, the salt has a purity equal to or greater than 91% w / w, for example, having impurities less than or equal to 9% w / w, to the subject.

[0264] Pharmaceutical compositions comprising compounds of this disclosure (e.g., compounds of formula I, IA, IB, or II) can be formulated to any desired concentration. In some embodiments, the composition is formulated such that it contains at least a therapeutically effective amount.

[0265] Pharmaceutical compositions include those suitable for oral, sublingual, nasal, rectal, vaginal, topical, oral, and parenteral (including subcutaneous, intramuscular, and intravenous) administration; however, the most suitable route will depend on the nature and severity of the condition being treated. The compositions can be conveniently provided in unit dosage forms and can be prepared by any method known in the pharmaceutical field. In some embodiments, the pharmaceutical compositions are formulated as pills, capsules, lozenges, or tablets for oral administration. In other embodiments, the pharmaceutical compositions are in suspension form.

[0266] When the compounds of this disclosure are administered as pharmaceuticals to mammals, such as humans, they can be administered alone or as pharmaceutical compositions comprising, for example, about 0.1% to 99.9%, about 0.2% to 98%, about 0.3% to 97%, about 0.4% to 96%, or about 0.5% to 95% of an active ingredient and a pharmaceutically acceptable carrier. In some embodiments, pharmaceutical compositions comprising about 0.5% to 90% of an active ingredient and a pharmaceutically acceptable carrier are suitable for administration to mammals, such as humans. Some embodiments of this disclosure provide preparation of a pharmaceutically acceptable salt of formula I, IA, IB, or II, or any of the pharmaceutically acceptable salts thereof, such as any one of the compounds in Table 7, comprising about 0.1% to 99.9%, about 0.2% to 98%, about 0.3% to 97%, about 0.4% to 96%, or about 0.5% to 95%, for the treatment, prevention, or prevention of viral infections or viral infection-related conditions. This disclosure provides the use of about 0.1% to 99.9%, about 0.2% to 98%, about 0.3% to 97%, about 0.4% to 96%, or about 0.5% to 95% of a pharmaceutically acceptable salt of Formula I, Formula IA, Formula IB or Formula II or thereof for the preparation of a medicament containing an effective amount of the compound, the medicament being used to treat, prevent or prevent treatment of viral infections and viral infection-related diseases.

[0267] This disclosure provides compounds of formula I, IA, IB, or II for treating viral infections or diseases or conditions related to viral infections. The compositions may be pharmaceutical formulations comprising about 0.1% to 99.9%, about 0.2% to 98%, about 0.3% to 97%, about 0.4% to 96%, or about 0.5% to 95% of the compound of formula I, IA, IB, or II.

[0268] For any compound, the therapeutically effective amount of the compound or composition can initially be estimated in cell culture assays (e.g., tumor cells) or animal models (typically rats, mice, rabbits, dogs, or pigs). The animal models can be used to determine appropriate concentration ranges or routes of administration. This information can then be used to determine the effective dose and route of administration for humans. Therapeutic / prophylactic efficacy and toxicity can be assessed using standard pharmaceutical procedures in cell cultures or laboratory animals, such as ED. 50 (Dose effective for 50% of the population) and LD 50 The toxicity-to-therapeutic-effect dose ratio is determined by the 50% group lethal dose. This ratio is called the therapeutic index, which can be expressed as a ratio, LD50. 50 / ED 50 Pharmaceutical compositions exhibiting a high therapeutic index are preferred. The dosage may vary within this range depending on the dosage form used, patient sensitivity, and route of administration.

[0269] Pharmaceutical compositions containing compounds of formula I, IA, IB, or II of this disclosure can be manufactured in a manner commonly known, such as by conventional mixing, dissolving, granulation, pelleting, grinding, emulsification, encapsulation, embedding, or lyophilization processes. The pharmaceutical compositions can be formulated in a conventional manner using one or more pharmaceutically acceptable carriers containing excipients and / or adjuvants that facilitate the processing of the active compound into a pharmaceutically acceptable formulation. A suitable dosage form depends on the chosen route of administration.

[0270] Suitable pharmaceutical compositions for injection include sterile aqueous solutions (wherein water-soluble) or dispersions and sterile powders for the ad hoc preparation of sterile injection solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, Cremophor EL... TM (BASF, Parsippany, NJ) or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and should be fluid for easy injection. It must be stable under production and storage conditions and must be protected from contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Suitable flowability can be maintained, for example, by using lecithin coating, by maintaining the desired particle size during dispersion, and by using surfactants. Microbial action can be prevented by various antimicrobial or antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, isotonic agents, such as sugars, polyols such as mannitol, sorbitol, and sodium chloride, are preferably included in the composition. Prolonged absorption of the injectable composition can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin, in the composition.

[0271] Sterile injection solutions can be prepared as required by mixing the desired amount of the active compound with one or a combination of the components listed above in a suitable solvent, followed by sterile filtration. Typically, dispersions are prepared by incorporating the active composition into a sterile medium containing a base dispersion medium and any other desired components selected from those listed above. In the case of sterile powders used to prepare sterile injection solutions, the preparation method is vacuum drying and freeze-drying to obtain powders of the active component and any other desired components from the preceding sterile filtration solution.

[0272] Oral compositions typically include an inert diluent or an edible, pharmaceutically acceptable carrier. They may be encapsulated in gelatin capsules or compressed into tablets. For oral therapeutic administration, the active compound may be mixed with excipients and administered in the form of tablets, lozenges, or capsules. Oral compositions may also be prepared using liquid carriers for use as mouthwashes, wherein the compound in the liquid carrier is administered orally and rinsed and spat out or swallowed. As part of the composition, pharmaceutically compatible binders and / or adjuvant materials may be included. Tablets, pills, capsules, lozenges, etc., may contain any of the following ingredients or compounds with similar properties: binders, such as microcrystalline cellulose, astragalus gum, or gelatin; excipients such as starch or lactose; disintegrants such as alginate, starch, or corn starch; lubricants such as magnesium stearate or sterotes; gliding agents such as colloidal silica; sweeteners such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavorings.

[0273] The active compound can be prepared using a pharmaceutically acceptable carrier that protects the compound from rapid loss from the body, such as controlled-release agents, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. In some embodiments, the materials are commercially available, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc., and liposome suspensions (including liposomes targeting infected cells with monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers.

[0274] Preparing oral or injectable pharmaceutical compositions in unit dose form is highly advantageous for ease of administration and uniform dosage. As used herein, unit dose form refers to a physically independent unit suitable for a subject to be treated; each unit contains a predetermined amount of the active compound, which is calculated to bind with a desired drug carrier to produce the desired therapeutic effect. The specifications of the unit dose form disclosed herein depend on and are directly dependent on the characteristics of the active compound and the specific therapeutic effect to be achieved.

[0275] Compounds of Formula I, IA, IB, or II, or pharmaceutically acceptable salts thereof, are formulated into pharmaceutical compositions or used to prepare medicaments for treating viral infections and / or diseases and / or conditions associated with viral infections. Additionally, this disclosure provides compounds of Formula I, IA, IB, or II, or compositions comprising compounds of Formula I, IA, IB, or II, for treating viral infections and / or diseases or conditions associated with viral infections. Compositions and / or medicaments comprising the compounds of Formula I, IA, IB, or II, or pharmaceutically acceptable salts thereof, can be formulated into tablets or suspensions. Tablets of compounds of formula I, IA, IB or II or their pharmaceutically acceptable salts are formulated to contain a pharmacologically acceptable buffer, excipient, carrier including emulsifier, enhancer (e.g., absorption enhancer), disintegrant (e.g., polyvinylpyrrolidone (PVPP, cross-linked pyrrolidone or E1202, which is a highly cross-linked modified polyvinylpyrrolidone (PVP)), and / or polymers disclosed in this disclosure and well known in the art.

[0276] In one embodiment, this disclosure provides tablet formulations of Formula I, IA, IB, or II and compounds or pharmaceutically acceptable salts thereof for the treatment or prevention of viral infections and / or diseases and conditions associated with viral infections. This disclosure provides tablet formulations of Formula I, IA, IB, or II and compounds or pharmaceutically acceptable salts thereof for the treatment of subjects requiring such treatment, including, but not limited to, immunocompromised subjects, and subjects before or after organ and / or tissue transplantation. This disclosure provides the use of Formula I, IA, IB, or II and compounds or pharmaceutically acceptable salts thereof in the preparation of medicaments for the treatment of subjects requiring such treatment, including, but not limited to, immunocompromised subjects, and subjects before or after organ and / or tissue transplantation.

[0277] In one embodiment, this disclosure provides suspension formulations of Formula I, Formula IA, Formula IB, or Formula II and compounds thereof, or pharmaceutically acceptable salts thereof, for the prevention, treatment, or prevention of viral infections and / or virus-related diseases and / or conditions. This disclosure also provides suspension formulations of compounds of Formula I, Formula IA, Formula IB, or Formula II, or pharmaceutically acceptable salts thereof, for the treatment of subjects requiring such treatment, including but not limited to immunocompromised subjects, and subjects before or after organ and / or tissue transplantation.

[0278] In another embodiment, additional excipients include, but are not limited to, sodium phosphate, dibasic citric acid (monohydrate) (about 0.01-5 wt%), sodium citrate (about 0.01-5 wt%), xanthan gum (about 0.01-5 wt%), methylparaben (sodium salt) (about 0.01-5 wt%), propylparaben (sodium salt) (about 0.01-5 wt%), sucralose (about 0.01-5 wt%), sodium microcrystalline cellulose and carboxymethyl cellulose (VivaPur MCG 591) (about 0.5-10 wt%), high fructose corn syrup (about 10-70 wt%), lemon lime flavoring (WONF220J15) (about 0.01-5 wt%), sodium hydroxide pellets, sodium hydroxide / hydrochloric acid and purified water (about 68.93 wt%).

[0279] The formulation disclosed herein is used to prepare a medicine for the prevention, treatment and / or prevention of viral infections and / or virus-related diseases and / or conditions.

[0280] In another embodiment, this disclosure provides compositions (e.g., pharmaceutical compositions) having desirable pharmacokinetic characteristics. For example, compositions of this disclosure may provide blood concentrations of compounds of formula I, IA, IB, or II, or pharmaceutically acceptable salts thereof, which, upon metabolism into a therapeutically active form (e.g., a diphosphate equivalent), produce blood concentrations of non-toxic metabolites.

[0281] In some embodiments, this disclosure provides pharmaceutical compositions comprising the compounds described herein (e.g., compounds 1, 2, 3, 4, 5, 6, 71, 77, 76, 107, 111, 126, 133, 137, 139, 141, 143 or 145, or pharmaceutically acceptable salts, solvates, enantiomers, diastereomers, racemates or mixtures thereof, or any combination thereof).

[0282] Disease indications

[0283] In some embodiments, this disclosure provides a method of treating a viral infection or a disease or condition related to a viral infection, comprising administering to a subject in need a compound of the disclosure (e.g., compound 1, 2, 3, 4, 5, 6, 71, 77, 76, 107, 111, 126, 133, 137, 139, 141, 143 or 145, or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, racemate or mixture thereof, or any combination thereof).

[0284] In some embodiments, this disclosure provides for the use of the compounds of this disclosure (e.g., compounds 1, 2, 3, 4, 5, 6, 71, 77, 76, 107, 111, 126, 133, 137, 139, 141, 143 or 145, or pharmaceutically acceptable salts, solvates, enantiomers, diastereomers, racemates or mixtures thereof, or any combination thereof) in the preparation of a medicament for treating a disease (e.g., a viral infection or a disease or condition related to a viral infection).

[0285] In some embodiments, this disclosure provides the use of the compounds of this disclosure (e.g., compounds 1, 2, 3, 4, 5, 6, 71, 77, 76, 107, 111, 126, 133, 137, 139, 141, 143 or 145, or pharmaceutically acceptable salts, solvates, enantiomers, diastereomers, racemates or mixtures thereof, or any combination thereof) to treat diseases (e.g., viral infections or diseases or conditions associated with viral infections).

[0286] This disclosure provides for the treatment and / or prevention of viral infections using the compounds disclosed herein or pharmaceutically acceptable salts thereof. Compounds represented by Formula I, IA, IB, or II are used for the treatment, prevention, and / or preparation of medicaments for the treatment and / or prevention of at least one virus selected from, but not limited to, ssRNA viruses. In some embodiments, the virus may be norovirus, human cytomegalovirus (HCMV), BK virus (BKV), Epstein-Barr virus (EBV), adenovirus, JC virus (JCV), SV40, MC virus (MCV), KI virus (KIV), WU virus (WUV), vaccinia virus, herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), human herpesvirus 6 (HHV-6), human herpesvirus 8 (HHV-8), hepatitis B virus, hepatitis C virus, varicella-zoster virus (VZV). Smallpox, smallpox, cowpox, camelpox, monkeypox, poliovirus, picoronaviridae (e.g., rhinovirus), paramyxoviridae (e.g., respiratory syncytial virus, RSV), Ebola virus, Marburg virus, Epstein-Barr virus (EBV), influenza virus, enterovirus (e.g., EV68 and EV71), papillomavirus, West Nile virus, yellow fever virus, foot-and-mouth disease virus, Rift Valley fever virus and other flaviviruses, arenavirus, Bunyavirus, alphavirus and human immunodeficiency virus (HIV) infection and any combination thereof.

[0287] This disclosure also provides a viral infection or a disease or condition related to a viral infection (e.g., norovirus, human cytomegalovirus (HCMV), BK virus (BKV), Epstein-Barr virus (EBV), adenovirus, JC virus (JCV), SV40, MC virus (MCV), KI virus KIV), WU virus (WUV), vaccinia, herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), human herpesvirus 6 (HHV-6), human herpesvirus 8 (HHV-8), hepatitis B virus, hepatitis C virus, varicella-zoster virus (VZV), smallpox, smallpox, cowpox, camelpox, monkeypox, poliovirus, picornaviridae (e.g., rhinovirus). Methods for treating, preventing, or delaying the onset of infections with paramyxoviridae viruses (e.g., respiratory syncytial virus, RSV), Ebola virus, Marburg virus, Epstein-Barr virus (EBV), influenza virus, enteroviruses (e.g., EV68 and EV71, papillomavirus, West Nile virus, yellow fever virus, foot-and-mouth disease virus, Rift Valley fever virus and other flaviviruses, arenavirus, Bunyavirus, alphavirus and human immunodeficiency virus (HIV) infection and any combination thereof), by oral administration of a subject pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I, IA, IB or II or a pharmaceutically acceptable salt thereof, and one or more compounds or compositions selected from immunotherapeutic agents and / or antiviral agents.

[0288] This disclosure also provides compounds of formula I, IA, IB, or II for use in treating viral infections or diseases or conditions associated with viral infections (e.g., norovirus, human cytomegalovirus (HCMV), BK virus (BKV), Epstein-Barr virus (EBV), adenovirus, JC virus (JCV), SV40, MC virus (MCV), KI virus (KIV), WU virus (WUV), vaccinia, herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), human herpesvirus 6 (HHV-6), human herpesvirus 8 (HHV-8), hepatitis B virus, hepatitis C virus, varicella-zoster virus (VZV), smallpox, smallpox, cowpox, camelpox, monkeypox, poliovirus, picornaviridae). Use in the preparation of a medicament for the treatment, prevention, or delay of onset of infections with rhinoviruses, paramyxoviridae viruses (e.g., respiratory syncytial virus, RSV), Ebola virus, Marburg virus, Epstein-Barr virus (EBV), influenza virus, enteroviruses (e.g., EV68 and EV71, papillomavirus, West Nile virus, yellow fever virus, foot-and-mouth disease virus, Rift Valley fever virus and other flaviviruses, arenaviruses, Bunyaviruses, alphaviruses, and human immunodeficiency virus (HIV) infections, and any combination thereof), by oral administration of a subject pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I, IA, IB, or II, or a pharmaceutically acceptable salt thereof, in combination with one or more compounds or compositions selected from immunotherapeutic agents and / or antiviral agents.

[0289] In some embodiments, this disclosure also provides treatment for viral infections or diseases or conditions associated with viral infections (e.g., norovirus, human cytomegalovirus (HCMV), BK virus (BKV), Epstein-Barr virus (EBV), adenovirus, JC virus (JCV), SV40, MC virus (MCV), KI virus (KIV), WU virus (WUV), vaccinia, herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), human herpesvirus 6 (HHV-6), human herpesvirus 8 (HHV-8)). Hepatitis B virus, hepatitis C virus, varicella-zoster virus (VZV), smallpox, smallpox, cowpox, camelpox, monkeypox, poliovirus, picoronaviridae (e.g., rhinovirus), paramyxoviridae (e.g., respiratory syncytial virus, RSV), Ebola virus, Marburg virus, Epstein-Barr virus (EBV), influenza virus, enterovirus (e.g., EV68 and EV71, papillomavirus, West Nile virus, yellow fever virus, foot-and-mouth disease virus, Rift Valley fever virus and other flaviviruses, arenavirus, Bunyavirus, alpha virus and human immunodeficiency virus (HIV) infection and any combination thereof), for the treatment, prevention or delay of onset of these infections, by oral administration of a drug composition to a subject, said drug composition comprising a therapeutically effective amount of a compound of formula I, IA, IB or II or a pharmaceutically acceptable salt thereof and one or more compounds or compositions selected from immunotherapeutic agents and / or antiviral agents.

[0290] In some embodiments, this disclosure provides a method for treating, preventing, or delaying the onset of Marburg virus infection or a disease or condition associated with Marburg virus infection by oral administration of a therapeutically effective amount of a compound of formula I, IA, IB, or II, or a pharmaceutically acceptable salt thereof, to a subject in need.

[0291] In some embodiments, this disclosure provides the use of compounds of formula I, IA, IB or II in the manufacture of medicaments for the treatment, prevention or delay of the onset of Marburg virus infection or a disease or condition associated with Marburg virus infection, by oral administration to a subject in need.

[0292] In some embodiments, this disclosure provides a compound of formula I, IA, IB, or II for the treatment, prevention, or delay of the onset of Marburg virus infection or a disease or condition associated with Marburg virus infection, administered orally to a subject in need.

[0293] In some embodiments, this disclosure provides a method for treating, preventing, or delaying the onset of Marburg virus infection or a disease or condition associated with Marburg virus infection by oral administration of a therapeutically effective amount of a compound of formula I, IA, IB, or II, or a pharmaceutically acceptable salt thereof, to a subject in need.

[0294] In some embodiments, this disclosure provides the use of compounds of formula I, IA, IB or II in the manufacture of medicaments for the treatment, prevention or delay of the onset of diseases or conditions related to Ebola virus infection, by oral administration to a subject in need.

[0295] In some embodiments, this disclosure provides a compound of formula I, IA, IB, or II for the treatment, prevention, or delay of the onset of Ebola virus infection or an Ebola virus infection-related disease or condition, administered orally to a subject in need.

[0296] In some embodiments, this disclosure provides a method for treating, preventing, or delaying the onset of Ebola virus infection or an Ebola virus infection-related disease or condition by oral administration of a therapeutically effective amount of a compound of formula I, IA, IB, or II, or a pharmaceutically acceptable salt thereof, to a subject in need.

[0297] In some embodiments, this disclosure provides the use of compounds of formula I, IA, IB or II in the manufacture of medicaments for the treatment, prevention or delay of the onset of influenza virus infection or an influenza virus infection-related disease or condition, by oral administration to a subject in need.

[0298] In some embodiments, this disclosure provides a compound of formula I, IA, IB, or II for the treatment, prevention, or delay of the onset of influenza virus infection or an influenza virus infection-related disease or condition, administered orally to a subject in need.

[0299] In some embodiments, this disclosure provides a method for treating, preventing, or delaying the onset of influenza virus infection or an influenza virus infection-related disease or condition by oral administration of a therapeutically effective amount of a compound of formula I, IA, IB, or II, or a pharmaceutically acceptable salt thereof, to a subject in need.

[0300] In some embodiments, this disclosure provides the use of compounds of formula I, IA, IB or II in the manufacture of medicaments for treating, preventing or delaying the onset of norovirus infection or norovirus-related diseases or symptoms, by oral administration to a subject in need.

[0301] In some embodiments, this disclosure provides a compound of formula I, IA, IB, or II for the treatment, prevention, or delay of norovirus infection or norovirus-related diseases or conditions, administered orally to a subject in need.

[0302] In some embodiments, this disclosure provides a method for treating, preventing, or delaying the onset of norovirus infection or a disease or condition associated with norovirus infection by oral administration of a therapeutically effective amount of a compound of formula I, IA, IB, or II, or a pharmaceutically acceptable salt thereof, to a subject in need.

[0303] In some embodiments, this disclosure provides the use of compounds of formula I, IA, IB or II in the manufacture of medicaments for the treatment, prevention or delay of onset of diseases or conditions associated with orphanaviviridae virus infection, administered orally to a subject in need.

[0304] In some embodiments, this disclosure provides a compound of formula I, IA, IB, or II for the treatment, prevention, or delay of onset of a disease or condition associated with oral administration to a subject in need.

[0305] In some embodiments, this disclosure provides a method for treating, preventing, or delaying the onset of a disease or condition associated with a paramyxoviridae virus infection by oral administration of a therapeutically effective amount of a compound of formula I, IA, IB, or II, or a pharmaceutically acceptable salt thereof, to a subject in need.

[0306] In some embodiments, this disclosure provides the use of compounds of formula I, IA, IB or II in the manufacture of medicaments for the treatment, prevention or delay of onset of diseases or conditions associated with paramyxoviridae virus infection or paramyxoviridae virus infection, by oral administration to a subject in need.

[0307] In some embodiments, this disclosure provides a compound of formula I, IA, IB, or II for the treatment, prevention, or delay of onset of a disease or condition associated with a paramyxoviridae virus infection, administered orally to a subject in need.

[0308] In some embodiments, this disclosure provides a method for treating, preventing, or delaying the onset of enterovirus infection or enterovirus-related diseases or conditions by oral administration of a therapeutically effective amount of a compound of formula I, IA, IB, or II, or a pharmaceutically acceptable salt thereof, to a subject in need.

[0309] In some embodiments, this disclosure provides the use of compounds of formula I, IA, IB or II in the manufacture of medicaments for the treatment, prevention or delay of the onset of enterovirus infection or enterovirus-related diseases or conditions, by oral administration to a subject in need.

[0310] In any of the above embodiments, the compound may be compound 1.

[0311] In some embodiments, this disclosure provides a compound of formula I, IA, IB, or II for the treatment, prevention, or delay of the onset of enterovirus infection or enterovirus-related diseases or conditions, administered orally to a subject in need.

[0312] This disclosure also provides a method for the preventive treatment, prevention, or delay of the onset of norovirus infection or norovirus-related diseases or conditions, by orally administering a pharmaceutical composition to a subject, said pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I, IA, IB, or II (e.g., compound 1) or a pharmaceutically acceptable salt thereof, combined with one or more antiviral agents. In some embodiments, the method of preventive treatment includes treating the subject with a compound of this disclosure prior to norovirus infection.

[0313] This disclosure also provides the use of compounds of formula I, IA, IB or II (e.g., compound 1) in the manufacture of medicaments for the preventive treatment, prevention or delay of onset of norovirus infection or norovirus-related diseases or conditions, by oral administration of a pharmaceutical composition to a subject, said pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I, IA, IB or II or a pharmaceutically acceptable salt thereof in combination with one or more antiviral agents.

[0314] This disclosure also provides compounds of formula I, IA, IB, or II (e.g., compound 1) for the preventive treatment, prevention, or delay of onset of norovirus infection or norovirus-related diseases or conditions, by oral administration of the pharmaceutical composition to a subject, said pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I, IA, IB, or II, or a pharmaceutically acceptable salt thereof, combined with one or more antiviral agents. This disclosure also provides a method for the preventive treatment, prevention, or delay of onset of norovirus infection or norovirus-related diseases or conditions by oral administration of the pharmaceutical composition to a subject, said pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I, IA, IB, or II, or a pharmaceutically acceptable salt thereof, combined with one or more antiviral agents.

[0315] This disclosure also provides the use of compounds of formula I, IA, IB, or II in the manufacture of medicaments for the preventive treatment, prevention, or delay of onset of enterovirus infection or enterovirus-related diseases or conditions, by oral administration of a pharmaceutical composition to a subject, said pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I, IA, IB, or II or a pharmaceutically acceptable salt thereof, combined with one or more antiviral agents.

[0316] This disclosure also provides compounds of formula I, IA, IB, or II for the preventive treatment, prevention, or delay of onset of enterovirus infection or enterovirus-related diseases or conditions, by oral administration of a pharmaceutical composition to a subject, said pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I, IA, IB, or II or a pharmaceutically acceptable salt thereof, combined with one or more antiviral agents.

[0317] In one embodiment, the compound of formula I, formula IA, formula IB, or formula II is used to treat norovirus. In another embodiment, the compound of formula I, formula IA, formula IB, or formula II is used to treat norovirus associated with a specific genotype, for example, those known to infect humans in genotypes I, II and IV, VI and VII (Phan et al., J. Med. Virol. 2007 Sep; 79(9): 1388-1400).

[0318] Dosing regimen

[0319] The administration regimen can affect the composition of a pharmaceutically effective dose. Compounds of Formula I, IA, IB, or II, or pharmaceutically acceptable salts thereof, may be administered to the subject before or after the onset of disease. Additionally, several separate and staggered doses may be administered daily or sequentially, or the doses may be administered continuously by injection or intravenously. Furthermore, the dose may be increased or decreased proportionally depending on the urgency of the treatment or prophylactic situation. Additionally, the dose may be administered in combination with other antiviral agents.

[0320] Dosing regimens using these compounds can be chosen based on a variety of factors, including patient type, age, weight, and medical condition; the severity of the condition to be treated; route of administration; patient's renal and hepatic function; and the specific composition or salt thereof used. A physician or veterinarian with general skills can easily determine and limit the effective amount of the drug needed to prevent, combat, or halt the progression of the condition.

[0321] In some embodiments, a compound of formula I, IA, IB, or II, or a pharmaceutically acceptable salt thereof, is administered to a subject with a viral infection once or twice weekly, at doses of about 40 mg, 50 mg, 75 mg, 100 mg, 150 mg, 175 mg, 200 mg, or 250 mg. This disclosure provides treatment of norovirus or norovirus-related diseases or conditions by administering a compound of formula I, IA, IB, or II, or a pharmaceutically acceptable salt thereof, to a subject once weekly (QW) or twice weekly (BIW). In one embodiment, the subject is treated twice weekly (BIW) with about 100 mg of the compound. In another embodiment, the subject is treated once weekly (QW) with about 200 mg of the compound or twice weekly (BIW) with about 100 mg of the compound.

[0322] In one embodiment, a compound of formula I, IA, IB or II, or a pharmaceutically acceptable salt thereof, with a purity equal to or greater than about 91% w / w, is administered orally to a subject, for example, at about 0.01 mg / kg to about 10 mg / kg or more, such as up to 100 mg / kg, up to 400 mg / kg or up to 1000 mg / kg.

[0323] In another embodiment, a compound of formula I, IA, IB, or II, or a pharmaceutically acceptable salt thereof, with a purity equal to or greater than about 91% w / w, is orally administered to a subject at a dose of about 0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, 5 mg / kg, 5.5 mg / kg, 6 mg / kg, 6.5 mg / kg, 7 mg / kg, 7.5 mg / kg, 8 mg / kg, 8.5 mg / kg, 9 mg / kg, 9.5 mg / kg, or 10 mg / kg or more, or any range thereof.

[0324] In one preferred aspect, the disease or ailment to be treated is a viral infection.

[0325] Dosage and administration should be adjusted to provide adequate levels of active agent or maintain the desired effect. Factors that may be considered include the severity of the disease state, the subject's general health condition, the subject's age, weight and sex, diet, timing and frequency of administration, concomitant medications, sensitivity to response, and tolerance / response to treatment. Depending on the half-life and clearance rate of the specific formulation, long-acting drug compositions may be administered every 3 to 4 days, weekly, every two weeks, or monthly.

[0326] In some embodiments, the compound is administered for a total of 10 consecutive doses. For example, a compound of formula I, IA, IB, or II may be administered twice weekly for 5 weeks at a dose of about 100 mg (i.e., 10 total doses). Alternatively, a compound of formula I, IA, IB, or II may be administered as a loading dose of about 200 mg, followed by continuous administration of a dose of about 100 mg twice weekly. In some embodiments, the compound is administered for a total of 10 consecutive doses. For example, a compound of formula I, IA, IB, or II may be administered as a loading dose of about 200 mg, followed by nine additional doses of about 100 mg twice weekly, for a total of 10 doses. In one embodiment, a compound of formula I, IA, IB, or II may be administered in the range of 20-200 mg / day or at a dose of about 200 mg-3000 mg / week.

[0327] In one or more embodiments, the compounds of this disclosure are useful in treating viral infections such as norovirus infection or diseases or conditions associated with norovirus infection. In some embodiments, treatment of an infection, such as norovirus infection, may include daily administration or multiple daily administration. In some embodiments, the overall treatment regimen continues as long as the norovirus infection is active (e.g., 1-3 days). In some embodiments, the compounds of this disclosure may be administered multiple times daily for 1-3 days to treat norovirus infection.

[0328] In another embodiment, tablets or suspensions of compounds of formula I, IA, IB, or II, or pharmaceutically acceptable salts thereof, are administered at a dose of about 40-3000 mg daily, by dose (BID), by dose (TID), once weekly (QW), or twice weekly (BIW). In another embodiment, tablets or suspensions of compounds of formula I, IA, IB, or II, or pharmaceutically acceptable salts thereof, are administered at a dose of 40-400 mg daily, by dose (BID), by dose (TID), once weekly (QW), or twice weekly (BIW).

[0329] In therapeutic applications, the dosage of the pharmaceutical composition of this disclosure is varied based on the recipient patient's medication, age, weight, and clinical condition, as well as the experience and judgment of the clinician or practicing physician administering the treatment, and other factors affecting the selected dosage. The dosage can vary from about 0.01 mg / kg to about 100 mg / kg. In a preferred aspect, the dosage can vary from about 0.1 mg / kg to about 10 mg / kg. On one hand, the dosage is in the range of about 1 mg to about 1 g; about 10 mg to about 500 mg; about 20 mg to about 400 mg; about 40 mg to about 400 mg; or about 50 mg to about 400 mg, in a single dose, fractional dose, or continuous dose (which may be based on the patient's weight (kg), body surface area (m²)). 2(and age (years) adjustment). In some embodiments, the amount of each dosage form can be from about 0.1 mg to about 3000 mg, for example about 0.1 mg, about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg. The dosage is approximately 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1250 mg, 1500 mg, 1750 mg, 2000 mg, 2500 mg, or 3000 mg. In one embodiment, the dosage may be approximately 20 mg. In one embodiment, the dosage may be approximately 50 mg. In another embodiment, the dosage may be approximately 100 mg. In yet another embodiment, the dosage may be approximately 500 mg.

[0330] In another embodiment, a compound of formula I, IA, IB, or II, or a pharmaceutically acceptable salt thereof, is administered to a subject in a single dose. In another embodiment, a compound of formula I, IA, IB, or II, or a pharmaceutically acceptable salt thereof, is administered to a subject in multiple doses. Multiple doses may be administered periodically, for example, every 12 hours, once daily, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, every 8 days, every 9 days, every 10 days, every 11 days, every 12 days, every 13 days, every 14 days, or every 15 days. For example, the dose may be administered twice weekly. Furthermore, each individual dose may be administered at the same or different dosages.

[0331] For example, any one of the compounds of formula I, IA, IB, or II, or a pharmaceutically acceptable salt thereof, may be administered to the subject, wherein the first dose is about 1-20 mg / kg (e.g., about 1-1.1 mg / kg, about 1.1-1.2 mg / kg, about 1.2-1.3 mg / kg, about 1.3-1.4 mg / kg, about 1.4-1.5 mg / kg, about 1.5-1.6 mg / kg, about 1.6-1.7 mg / kg, about 1.7-1.8 mg / kg, about 1.8-1.9 mg / kg, about 1.9-2.0 mg / kg, about 2.0-2.1 mg / kg, about 2.1-2.2 mg / kg, about 2.2-2.3 mg / kg, about 2.3-2.4 mg / kg, about 2.4-2.5 mg / kg, about 2.5-2.6 mg / kg). mg / kg, approximately 2.6-2.7 mg / kg, approximately 2.7-2.8 mg / kg, approximately 2.8-2.9 mg / kg, approximately 2.9-3.0 mg / kg, approximately 3.0-3.1 mg / kg, approximately 3.1-3.2 mg / kg, approximately 3.2-3.3 mg / kg, approximately 3.3-3.4 mg / kg, approximately 3.4-3.5 mg / kg, approximately 3.5-3.6 mg / kg, approximately 3.6-3.7 mg / kg, approximately 3.7-3.8 mg / kg, approximately 3.8-3.9 mg / kg, approximately 3.9-4.0 mg / kg, approximately 4.0-5.0 mg / kg, approximately 5.0-6.0 mg / kg, approximately 6.0-7.0 mg / kg, approximately 7.0-8.0 mg / kg, approximately 8.0-9.0 mg / kg. In the same week or the following week, at a dose of about 1-4 mg / kg (e.g., about 1-1.1 mg / kg, about 1.1-1.2 mg / kg, about 1.2-1.3 mg / kg, about 1.3-1.4 mg / kg, about 1.4-1.5 mg / kg, about 1.5-1.6 mg / kg, about 1.6-1.7 mg / kg, about 1.7-1.8 mg / kg, about 1.8-1.9 mg / kg, about 1.9-2.0 mg / kg, about 2.0-2.1 mg / kg, about 2.1-2.2 mg / kg, about 2.2-2.3 mg / kg, about 2.3-2.4 mg / kg, about 2.4-2.5 mg / kg, or ...5-2.4 mg / kg, about 1.5-1.6 mg / kg, about 1.7-1.8 mg / kg, about 1.8-1.9 mg / kg, about 1.9-2.0 mg / kg, about 2.9-2.0 mg / kg, about 2.0-2.1 mg / kg, about 2 mg / kg, approximately 2.5-2.6 mg / kg, approximately 2.6-2.7 mg / kg, approximately 2.7-2.8 mg / kg, approximately 2.8-2.9 mg / kg, approximately 2.9-3.0 mg / kg, approximately 3.0-3.1 mg / kg, approximately 3.A compound of formula I, IA, IB, or II (or a pharmaceutically acceptable salt thereof) may be administered once or more in doses of about 3.2 mg / kg, about 3.2-3.3 mg / kg, about 3.3-3.4 mg / kg, about 3.4-3.5 mg / kg, about 3.5-3.6 mg / kg, about 3.6-3.7 mg / kg, about 3.7-3.8 mg / kg, about 3.8-3.9 mg / kg, or about 3.9-4.0 mg / kg. For example, a subject may be given a first dose of about 3 mg / kg followed by one or more additional doses of about 1 mg / kg. For example, a subject may be given a first dose of about 2 mg / kg followed by one or more additional doses of about 3 mg / kg. For example, a subject may be given a first dose of about 4 mg / kg followed by one or more additional doses of about 4 mg / kg.

[0332] Multiple doses can be administered at varying time intervals. For example, the first 2, 3, 4, 5, 6, 7, or 8 or more doses can be administered at 6-day intervals, followed by additional doses at 7-day intervals. Alternatively, the first 2, 3, 4, 5, 6, 7, or 8 or more doses can be administered at 7-day intervals, followed by additional doses at 3-day intervals.

[0333] In one embodiment, a compound of formula I, IA, IB or II, or a pharmaceutically acceptable salt thereof, is administered to the subject at a dose of about 40-3000 mg once weekly or at a dose of about 40-3000 mg twice weekly.

[0334] In some embodiments, a compound of formula I, IA, IB or II, or a pharmaceutically acceptable salt thereof, is administered in doses of about 40-3000 mg daily, by dose, by dose, once weekly (QW), or twice weekly (BIW). The pharmaceutical compositions disclosed herein are administered at doses of about 40 mg, 50 mg, 75 mg, 100 mg, 150 mg, 175 mg, 200 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 450 mg, 500 mg, 500-600 mg, 600-700 mg, 700-800 mg, 800-900 mg, or 900-1000 mg daily, twice daily (BID), once weekly (QW), or twice weekly (BIW), or at doses of about 40 mg, 50 mg, 75 mg, 100 mg, 150 mg, 175 mg, 200 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, or 400 mg, 450 mg, 500 mg, 500-600 mg daily. Compounds of Formula I, IA, IB or II or their pharmaceutically acceptable salts, administered twice weekly (BIW), at doses of 600-700 mg, 700-800 mg, 800-900 mg, or 900-1000 mg.

[0335] This disclosure provides compounds of formula I, IA, IB or II, administered at a dose of about 1-100 mg / kg (e.g., 10-20 mg / kg, 20-50 mg / kg, 50-75 mg / kg, 75-100 mg / kg).

[0336] Application route

[0337] The compounds disclosed herein, or their pharmaceutically acceptable salts, esters, or derivatives, can be administered orally, nasally, intranasally, transdermally, pulmonaryly, by inhalation, orally, sublingually, intraperitoneally, subcutaneously, intramuscularly, intravenously, rectally, intrapleurally, intrathecally, and parenterally. In one embodiment, the compounds are administered orally. Those skilled in the art will understand the advantages of specific routes of administration.

[0338] Dosage forms for topical or transdermal administration of the compounds disclosed herein include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. In one embodiment, the compound is mixed under sterile conditions with a pharmaceutically acceptable carrier and any desired preservative, buffer, or propellant.

[0339] For inhalation administration, the compound is delivered as an aerosol from a pressure vessel or dispenser containing a suitable propellant, such as a gas like carbon dioxide, or a nebulizer.

[0340] Systemic administration can be achieved via mucin-transfer or transdermal routes. For mucin-transfer or transdermal routes, a penetrant suitable for penetrating the barrier is used in the formulation. Such penetrants are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives for mucin-transfer administration. Mucin-transfer administration can be achieved using nasal sprays or suppositories. For transdermal administration, the active compound is formulated into ointments, creams, gels, or creams generally known in the art.

[0341] The pharmaceutical compositions disclosed herein are formulated to match the intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), and mucinous conversion administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous use may include the following components: sterile diluents, such as water for injection, saline solution, fixative oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetate, citrate, or phosphate; and tonic agents such as sodium chloride or glucose. The pH may be adjusted with acid or base, hydrochloric acid, or sodium hydroxide. Parenteral formulations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0342] Combination therapy

[0343] This disclosure provides a method for preventing or treating a viral infection (e.g., norovirus infection) in a subject. The method includes administering a therapeutically effective amount of a compound of this disclosure to the subject. The compound can be used in a monotherapy or combination therapy regimen.

[0344] As used herein, "monotherapy" means administering a single active or therapeutic compound (e.g., a compound of formula I, IA, IB, or II) to a subject in need. Preferably, monotherapy comprises administering a therapeutically effective amount of an active compound. For example, norovirus monotherapy uses one of the compounds disclosed herein, or a pharmaceutically acceptable salt, prodrug, metabolite, analogue, or derivative thereof, to a subject in need of treatment for norovirus. Monotherapy can be contrasted with combination therapy, in which a combination of multiple active compounds is administered, preferably each component of the composition being present in a therapeutically effective amount. On the one hand, monotherapy with the compounds disclosed herein, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph, or solvate thereof, is more effective than combination therapy in producing the desired biological effect.

[0345] As used herein, “combination therapy” or “synergistic therapy” includes the administration of a compound of this disclosure, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph, or solvate thereof, and a second agent as part of a particular treatment regimen, intended to provide a beneficial effect in synergy with these therapeutic agents. The beneficial effect of the combination includes, but is not limited to, pharmacokinetic or pharmacodynamic synergies resulting from the combination of therapeutic agents. The combination of these therapeutic agents is typically administered over a defined time period (typically minutes, hours, days, or weeks, depending on the chosen combination). “Combination therapy” may, but is not typically, intended to include the administration of two or more of these therapeutic agents as part of separate single-therapy regimens, which arbitrarily and deliberately result in a combination of these therapeutic agents as part of the present disclosure.

[0346] "Combination therapy" is intended to include the administration of these therapeutic agents in a sequential manner, wherein each therapeutic agent is administered at different times, and in a nearly simultaneous manner, or at least two therapeutic agents. Nearly simultaneous administration can be achieved, for example, by administering to the subject a single capsule of each therapeutic agent in a fixed ratio, or multiple single capsules of each therapeutic agent. Sequential or nearly simultaneous use of each therapeutic agent can be achieved via any suitable route, including but not limited to, oral administration, intravenous administration, intramuscular administration, and direct absorption through mucosal tissue. The therapeutic agents can be administered via the same or different routes. For example, the first therapeutic agent of a selected combination can be administered intravenously, while the other therapeutic agents in the combination can be administered orally. Alternatively, for example, all therapeutic agents can be administered orally, or all therapeutic agents can be administered intravenously. The order of administration of the therapeutic agents is not critical.

[0347] "Combination therapy" also includes further administration of the aforementioned therapeutic agents in combination with other bioactive ingredients or non-pharmacological therapies. When the combination therapy further includes non-pharmacological treatment, the non-pharmacological treatment can be administered at any appropriate time, as long as the beneficial effects of the synergistic effect of the combination of therapeutic agents and non-pharmacological treatments are achieved. For example, where appropriate, the beneficial effects can still be achieved when the non-pharmacological treatment is temporarily removed from the administration of the therapeutic agent (perhaps for a few days or even a few weeks).

[0348] In some embodiments, this disclosure provides a method for treating, preventing, or delaying the onset of a viral infection (e.g., norovirus infection or norovirus-related disease or condition, influenza virus infection or influenza virus-related disease or condition) by administering to a subject in need a therapeutically effective amount of a pharmaceutical composition of a compound of formula I, IA, IB, or II, or a pharmaceutically acceptable salt thereof, in combination with one or more antiviral agents.

[0349] This disclosure also provides the use of a compound of formula I, IA, IB or II, or a pharmaceutically acceptable salt thereof, in combination with one or more antiviral agents in the manufacture of a medicament for the treatment, prevention or delay of the onset of a viral infection (e.g., norovirus infection or norovirus-related disease or condition, influenza virus infection or influenza virus-related disease or condition), and a pharmaceutical composition comprising administering a therapeutically effective amount of a compound of formula I, IA, IB or II, or a pharmaceutically acceptable salt thereof, in combination with one or more antiviral agents to a subject in need.

[0350] This disclosure also provides compounds of formula I, IA, IB, or II for the treatment, prevention, or delay of viral infections (e.g., norovirus infection or norovirus-related disease or condition, influenza virus infection or influenza virus-related disease or condition), and pharmaceutical compositions thereof, by administering a therapeutically effective amount of a compound of formula I, IA, IB, or II, or a pharmaceutically acceptable salt thereof, to a subject in need, in combination with one or more antiviral agents.

[0351] In some embodiments, this disclosure provides a method for treating, preventing, or delaying the onset of a microRNA infection or a disease or condition associated with microRNA infection by orally administering to a subject in need a therapeutically effective amount of a compound of formula I, IA, IB, or II, or a pharmaceutically acceptable salt thereof, in combination with one or more antiviral agents.

[0352] This disclosure also provides the use of compounds of formula I, IA, IB or II in the manufacture of medicaments for the treatment, prevention or delay of onset of diseases or conditions associated with microRNA infection or microRNA infection, and pharmaceutical compositions comprising a combination of a therapeutically effective amount of a compound of formula I, IA, IB or II or a pharmaceutically acceptable salt thereof with one or more antiviral agents, administered orally to a subject in need.

[0353] This disclosure also provides compounds of formula I, IA, IB, or II for the treatment, prevention, or delay of onset of piconereonine virus infection or diseases or conditions associated with piconereonine virus infection, and pharmaceutical compositions thereof, by oral administration to a subject in need of a therapeutically effective amount of a compound of formula I, IA, IB, or II, or a pharmaceutically acceptable salt thereof, in combination with one or more antiviral agents.

[0354] In some embodiments, this disclosure provides a method for treating, preventing, or delaying the onset of a disease or condition associated with a paramyxoviridae virus infection by orally administering to a subject in need a therapeutically effective amount of a pharmaceutical composition consisting of a compound of formula I, IA, IB, or II, or a pharmaceutically acceptable salt thereof, in combination with one or more antiviral agents.

[0355] This disclosure also provides the use of compounds of formula I, IA, IB or II in the manufacture of medicaments for the treatment, prevention or delay of onset of diseases or conditions associated with paramyxoviridae virus infection or paramyxoviridae virus infection, and pharmaceutical compositions comprising a combination of a therapeutically effective amount of a compound of formula I, IA, IB or II or a pharmaceutically acceptable salt thereof with one or more antiviral agents, administered orally to a subject in need.

[0356] This disclosure also provides compounds of formula I, IA, IB, or II for the treatment, prevention, or delay of onset of diseases or conditions associated with paramyxoviridae virus infection, and pharmaceutical compositions thereof, by oral administration to a subject in need of a therapeutically effective amount of a compound of formula I, IA, IB, or II, or a pharmaceutically acceptable salt thereof, in combination with one or more antiviral agents.

[0357] In one embodiment, the method of treating a viral infection (e.g., influenza virus infection or norovirus infection) further includes administering at least one additional antiviral agent. In one embodiment, a compound of formula I, IA, IB, or II is used in combination with an additional antiviral agent. In one or more embodiments, a compound of formula I, IA, IB, or II is used to manufacture a medicament for use in combination with an additional antiviral agent. In one embodiment, the additional antiviral agent is adamantane. In another embodiment, the additional antiviral agent is adamantane or ramantadine. In another embodiment, the additional antiviral agent is a neuraminidase inhibitor (e.g., oseltamivir, zanamivir, lanimivir, and peramivir). In yet another embodiment, the additional antiviral agent is oseltamivir or zanamivir.

[0358] In some embodiments, the pharmaceutical compositions of this disclosure (compounds of formula I, IA, IB, or II) are administered in combination with one or more compounds or compositions selected from midazolam, cyclosporine A, tacrolimus, ganciclovir, valganciclovir, foscavir, cidofovir, second-line anti-CMV drugs, second-line anti-HCV drugs, foscarnet, filgrastim, pegfilgrastim, corticosteroids such as budesonide, beclomethasone, and broad-spectrum CYP inhibitors such as aminobenzotriazole, or combinations thereof.

[0359] In another embodiment, the compound is used in combination with at least one other immunosuppressant. In one embodiment, the immunosuppressants are administered simultaneously or sequentially. The immunosuppressants include, but are not limited to, dacrolimus, baliximab, tacrolimus, sirolimus, rapamycin, mycophenolate mofetil, cyclosporine A, glucocorticoids, anti-CD3 monoclonal antibodies, anti-thymocyte globulin, anti-CD52 monoclonal antibodies, azathioprine, everolimus, actinomycin D, cyclophosphamide, platinum, nitrosourea (Nitrosurea), methotrexate, mercaptopurine, muromonab, IFNγ, infliximab, etanercept, adalimumab, nateliximab, fingolimod, and combinations thereof.

[0360] The compounds or compositions provided herein may be used in combination with enhancers, other active ingredients, or immunosuppressants. In some embodiments, the compounds may be administered in combination with or sequentially with another therapeutic agent or enhancer. The other therapeutic agents include those known for treating, preventing, or improving one or more symptoms associated with viral infection. It is understood that any suitable combination of the compounds provided herein with one or more of the above-described compounds and optionally one or more additional pharmacologically active substances is considered to be within the scope of this disclosure. In another embodiment, the compounds provided herein are administered before or after one or more additional active ingredients. In one embodiment, two or more antiviral agents disclosed herein are administered sequentially or in combination. The amount of some enhancers may be selected using methods known in the art to enhance the bioavailability of the antiviral agents. Any amount may be used to provide the desired effect of some enhancer. The dosage may be varied, from 0.001 mg to about 3000 mg of compound per kilogram of body weight per day in one non-limiting embodiment, for example, 0.01 to 500 mg / kg or, for example, 0.1-20 mg / kg.

[0361] The pharmacokinetic behavior of the compositions may vary among subjects within a population. The figures described above for the compositions disclosed herein are based on average behavior within a population. This disclosure is intended to cover compositions that average within the disclosed range, but it should be understood that some subjects may fall outside that range.

[0362] The pharmaceutical composition may be included in a container, package, or dispenser along with instructions for use. This disclosure provides a kit that, in addition to handling any of the pharmaceutical compositions of this disclosure, includes a container, package, or dispenser, and instructions for use.

[0363] The compounds disclosed herein, or pharmaceutically acceptable salts, prodrugs, metabolites, analogs, or derivatives thereof, may be administered in combination with a second antiviral compound. For example, as described above, the compositions of this disclosure may include the compounds described above, as well as one or more additional active agents as described in this section, in a similar manner known in the art. Other antiviral active agents that may be used with the compositions of this disclosure in carrying out the methods of this disclosure include, but are not limited to, those antiviral active agents that target the M2 ion channel in influenza A virus (e.g., adamantane, such as amantadine and rimantadine); those that inhibit viral uncoating after entering cells, preventing newly formed viral particles from being released from the surface of infected cells (e.g., neuraminidase inhibitors, such as oseltamivir and zanamivir).

[0364] Methods to prevent diseases or symptoms caused by viral reactivation

[0365] This disclosure also provides a method for preventing disease or symptom in a subject at risk of viral reinfection by orally administering to the subject a therapeutically effective amount of a compound of formula I, IA, or IB, or a pharmaceutically acceptable salt thereof. In some embodiments, the virus at risk of reinfection may be influenza virus, norovirus, EBV, Ebola virus, paroxaviridae, paramyxoviridae, and Marburg virus.

[0366] This disclosure also provides a method for preventing disease or condition in a subject at risk of viral reactivation by orally administering a pharmaceutical composition of a compound of formula I, IA, or IB, in a therapeutically effective dose to a subject, or formula II or a pharmaceutically acceptable salt thereof. In some embodiments, the virus at risk of reactivation may be influenza virus, norovirus, EBV, Ebola virus, picornaviridae virus, paramyxoviridae virus, and Marburg virus. In some preferred embodiments, the virus at risk of reactivation may be influenza virus.

[0367] The effects of food

[0368] In some embodiments, the pharmaceutical composition of the present embodiments, such as tablets or suspensions, may be administered to the subject under fasting or eating conditions. In one embodiment, a composition comprising a compound of formula I, IA, IB, or II (or a pharmaceutically acceptable salt thereof) may be administered to a fasting subject, for example, after fasting for less than 24 hours but more than 12 hours, more than 11 hours, more than 10 hours, more than 8 hours, or more than 5 hours.

[0369] In other embodiments, compositions comprising compounds of formula I, IA, IB, or II (or pharmaceutically acceptable salts thereof) are administered to the subject with or after food. In one embodiment, compounds of formula I, IA, IB, or II (or pharmaceutically acceptable salts thereof) may be taken by the subject on an empty stomach.

[0370] patient group

[0371] In some embodiments, a compound of formula I, IA, IB, or II (or a pharmaceutically acceptable salt thereof), a composition comprising a compound of formula I, IA, IB, or II, or a combination therapy comprising a composition of formula I, IA, IB, or II is administered to a mammal (e.g., a human) of interest for approximately 1 to 6 months, 6 to 12 months, 1 to 5 years, 5 to 10 years, 10 to 15 years, 15 to 20 years, 20 to 25 years, 25 to 30 years, 30 to 35 years, 35 to 40 years, 40 to 45 years, 45 to 50 years, 50 to 55 years, 55 to 60 years, 60 to 65 years, 65 to 70 years, 70 to 75 years, 75 to 80 years, 80 to 85 years, 85 to 90 years, 90 to 95 years, or 95 to 100 years. In some embodiments, the mammal has a viral infection (e.g., ssRNA infection such as norovirus infection).

[0372] In some embodiments, a compound of formula I, formula IA, formula IB, or formula II, a composition comprising a compound of formula I, formula IA, formula IB, or formula II, or a combination therapy comprising formula I, is administered to a person at risk of developing a viral infection. In some embodiments, a compound of formula I, formula IA, formula IB, or formula II, a composition comprising a compound of formula I, formula IA, formula IB, or formula II, or a combination therapy comprising formula I, is administered to a person suffering from a viral infection. In some implementation schemes, patients are individuals aged approximately 1 to 6 months, 6 to 12 months, 1 to 5 years, 5 to 10 years, 5 to 12 years, 10 to 15 years, 15 to 20 years, 13 to 19 years, 20 to 25 years, 25 to 30 years, 20 to 65 years, 30 to 35 years, 35 to 40 years, 40 to 45 years, 45 to 50 years, 50 to 55 years, 55 to 60 years, 60 to 65 years, 65 to 70 years, 70 to 75 years, 75 to 80 years, 80 to 85 years, 85 to 90 years, 90 to 95 years, or 95 to 100 years.

[0373] In some embodiments, a compound of formula I, formula IA, formula IB, or formula II, a composition comprising a compound of formula I, formula IA, formula IB, or formula II, or a combination therapy comprising formula I, or a combination therapy comprising a compound of formula IA, formula IB, or formula II, is administered to a human infant. In another embodiment, a compound of formula I, formula IA, formula IB, or formula II, or a combination therapy comprising a compound of formula I, formula IA, formula IB, or formula II, is administered to a human child. In other embodiments, a compound of formula I, formula IA, formula IB, or formula II, a composition comprising a compound of formula I, formula IA, formula IB, or formula II, or a combination therapy comprising formula I, or a combination therapy comprising a compound of formula IA, formula IB, or formula II, is administered to an elderly person.

[0374] Unless otherwise stated, all percentages and ratios used herein are by weight. Other features and advantages of this disclosure will become apparent in the various examples. The provided embodiments illustrate different components and methods useful for implementing this disclosure. The embodiments are not limiting of the disclosure. Based on this disclosure, those skilled in the art can identify and use other useful components and methods to implement this disclosure.

[0375] All patents, patent applications, and publications mentioned herein are incorporated herein by reference in their entirety. However, where a patent, patent application, or publication contains express definitions, those express definitions should be understood to apply to the patent, patent application, or publication in which they appear, and not to the rest of this application, particularly the claims of this application.

[0376] In some embodiments, the compound of formula II is:

[0377]

[0378] Or its pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, racemate or mixture.

[0379] In some embodiments, compounds of formulas I, II, IA, IB, or their analogues are:

[0380]

[0381] Or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, racemate, or mixture thereof.

[0382] HPLC chromatograms were recorded on a Zorbax Eclipse Plus C18 column (4.6 × 50 mm × 1.8 μm). The first mobile phase was 97.5% water: 2.5% acetonitrile: 0.05% trifluoroacetic acid. The second mobile phase was 97.5% acetonitrile: 2.5% water: 0.05% trifluoroacetic acid.

[0383] Unless otherwise stated, recording at 300MHz 1 HNMR.

[0384] Antiviral and cytotoxicity analysis

[0385] Cell Culture and Viral Strains: Human foreskin fibroblasts (HFF) were prepared from human foreskin tissue. The tissue was incubated in a cell culture medium consisting of minimally essential medium (MEM) supplemented with Earle's salts containing 10% fetal bovine serum (FBS) and standard concentrations of L-glutamine, fungizone, and vancomycin. The tissue was then placed in phosphate-buffered saline (PBS), minced, rinsed to remove red blood cells, and resuspended in trypsin / EDTA solution. The tissue suspension was incubated at 37°C with gentle agitation to disperse the cells, followed by collection by centrifugation. The cells were resuspended in the medium and placed in tissue culture flasks, incubated at 37°C in a humidified CO2 incubator. Cell growth was then monitored daily with fresh medium until a confluent cell monolayer was formed. HFF cells were then expanded by continuous passage in standard MEM growth medium supplemented with Earle's salts containing 10% FBS, L-glutamine, penicillin, and gentamicin. Cells were routinely passaged and used for analysis at passage 10 or below.

[0386] Influenza virus: Influenza virus was passaged in canine kidney cells to produce working material for antiviral analysis.

[0387] Antiviral assays: Each assay evaluating the antiviral activity of compounds included both positive and negative control compounds to ensure the performance of each assay. If possible, cytotoxicity was assessed simultaneously under the same compound exposure levels. Methods: Effective concentrations that reduced viral replication by 50% in vitro, and cell viability (CC) were used. 50 ) and the selectivity index (SI, derived by CC) 50 Divide by EC 50 The calculations were performed at a concentration reduction of 50%. When sufficient material was available, multiple analyses were conducted for each compound to obtain statistical data.

[0388] Cytotoxicity assays: Each antiviral assay included parallel cytotoxicity analyses using the same cells, cell numbers, drug concentrations, and latency times as for each virus to provide identical drug exposure. To ensure that the cytotoxicity of all compounds could be directly compared, standard neutral red uptake cytotoxicity assays were performed on all compounds in confluent HFF cells with a latency time of 7 days.

[0389] Neutral red uptake cytotoxicity assay: Each compound was evaluated in a standard cytotoxicity assay using standard methods. Briefly, HFF cells were seeded into tissue culture plates in standard tissue medium. After incubation for 24 hours, maintenance cell culture medium was replaced with the compound, followed by a series of 5-fold serial dilutions to generate a series of compound concentrations. The assay plates were incubated for 7 days, and neutral red solution in PBS was added to each well, followed by incubation for 1 hour. The plates were then cleaned, rinsed with PBS, and the dye for live cell internalization was dissolved in PBS supplemented with 50% ethanol and 1% glacial acetic acid. The optical density was then determined, and CC values ​​were interpolated from the experimental data. 50 value.

[0390] For all plaque reduction assays in HFF cells, a neutral erythrocytotoxicity assay was performed on a set of parallel 6-well plates containing uninfected HFF cells, receiving the same compound concentration as used for the antiviral assay. On the same day as each antiviral assay, the cytotoxicity plate was removed from the incubator and the cell monolayer was stained with neutral red solution in PBS. The dye was then removed, residual dye was rinsed from the cells with PBS, and the cell monolayer was visually inspected for any signs of toxicity. Cell viability was determined using a cell proliferation assay according to the manufacturer's instructions.

[0391] Cell proliferation assay: The cytotoxicity of several compounds was precisely assessed using the inhibition of HFF cell proliferation, and the procedure was performed according to standard laboratory procedures. Cells were seeded at low density onto plates using standard culture medium. After 24 hours, the medium was aspirated, and a series of compound solutions were prepared for growth. The plates were incubated at 37°C for 72 hours, and then the cells were removed with trypsin and counted. The concentration of the compound that reduced cell proliferation by 50% was interpolated from the experimental data.

[0392] Lymphocyte cytotoxicity assay: Cell viability in all assays performed with lymphocytes was assessed using a chemiluminescent cell viability assay. Briefly, the assay plate was incubated at ambient temperature for 30 minutes, then the chemiluminescent reagent was added to each well, and the plate was mixed to lyse the cells. The plate was then incubated at ambient temperature, and quantitative chemiluminescence was measured on a chemiluminometer. The drug concentration (CC) required to inhibit cell proliferation by 50% was calculated using standard methods. 50 ).

[0393] Mouse norovirus (MNV) assay: RAW cells were incubated at 37°C in cell culture medium with 5% CO2. Mouse norovirus isolates used for this assay were isolated from wild-type mice.

[0394] DMEM solutions of serially diluted test compounds were added to tissue culture plates. Cell suspensions were added to the compound diluents in the plates and incubated at 37°C with 5% CO2 for 2 days. Each assay plate included uninfected cell controls and untreated virus control wells. After 2 days of incubation, MTS reagent aqueous solution was added to each well according to the manufacturer's instructions, and the plates were incubated at 37°C until the untreated cell controls reached an absorbance between 1.1 and 1.8 at 490 nm. The final absorbance readings were recorded, and software was used to calculate the concentration of RAW cells that protected against MNV infection to 50% compared to the uninfected cell controls (EC50).

[0395] Human Norovirus Assay: Antiviral activity against human norovirus (NoV) was assessed using a cell line stably expressing human norovirus replicons in a 3-day assay maintained as a subconfluent culture. In some embodiments, four doses in triplicate (10-fold or 3-fold steps) were used. Antiviral activity was determined by Western blot analysis of intracellular NoV RNA (normalized to the level of cellular β-actin RNA in each culture sample). Cytotoxicity was assessed by the uptake of neutral red dye in the culture maintained on parallel plates (Korba and Gerin, 1992, Antivir. Res. 19:55).

[0396] EC was calculated using linear regression analysis of data from all treatment culture combinations. 50 and CC 50 value (Korba & Gerin, 1992, Antivir. Res. 19:55; Okuse, et al (Antivir. Res. 65:23, 2005). EC is calculated from the standard error generated by regression analysis. 50 and EC 90 Standard deviation of the value. EC 50 and EC 90 These are drug concentrations that, respectively, showed 2-fold or 10-fold inhibition of intracellular NoV RNA (relative to the average level in untreated cultures). 50 This refers to drug concentrations where neutral red dye uptake levels (relative to the average level in untreated cultures) were observed to be 2-fold lower. The selectivity index (SI) was calculated in CC... 50 / EC 50 Calculations were performed. Recombinant human interferon 2b (PBL laboratories, Inc.) was used as a control.

[0397] BKV EC in VERO cells 50Tissue culture plates were seeded with Vero cells in DMEM containing 2% Hyclone standard fetal bovine serum and 1% Hyclone penicillin and streptomycin. Cells were seeded with BKV. Serial dilutions of the test compounds were added to the cells, and the plates were incubated at 37°C with 5% CO2 for 10 days. After 10 days of incubation, the supernatant was mixed with lysis buffer. Each plate was incubated at 37°C. BKV DNA in the supernatant was measured by quantitative polymerase chain reaction (qPCR) using forward and reverse BKV PCR primers and FAM-labeled probes. Absolute quantification of viral copy number was performed using a standard curve containing dilutions of BKV DNA amplicons containing sequences homologous to the amplified fragment.

[0398] Plaque reduction assays for HSV-1, HSV-2, VZV, and HCMV: Monolayers of cells were prepared in six-well plates and incubated at 37°C to allow cell confluence. Culture medium was then aspirated from the wells, and the virus was added to each of three wells, producing 20–30 plaques per well. The plates were allowed to adsorb the cells for 1 hour, with gentle shaking every 15 minutes to redistribute the culture medium. The compound was diluted in maintenance cell medium consisting of MEM and Earl's salts supplemented with 2% FBS, L-glutamine, penicillin, and gentamicin. The solution was added to the replication wells, and the plates were incubated for varying times depending on the virus used. Plaque reduction assays for HSV-1 strain F were performed in a similar manner, but Vero cells were infected one day after inoculation. For HSV-1 and -2, monolayers were stained with a 20% methanol solution of 1% crystal violet, and unbound dye was removed by washing with dH₂O. For HCMV and VZV assays, cell monolayers were stained with 1% neutral red solution for 4 hours, then aspirated and washed with PBS. For all assays, plaques were counted using a stereomicroscope, and the concentrations of compounds that reduced plaque formation by 50% (EC50) were interpolated from the experimental data. 50 ).

[0399] Plaque reduction assay using MCMV: Mouse embryonic fibroblasts were prepared from mouse embryos using a procedure similar to that outlined above for HFF cells, suspended in tissue culture medium as described above, and seeded onto plates and incubated. The culture medium was aspirated and the cell monolayers were infected with MCMV. The infected cells were then incubated at 37°C for 1 hour, occasionally shaking the culture plate to ensure the medium covered the entire monolayer. The compound was serially diluted in the tissue maintenance cell culture medium described above, and the solution was added to the infected monolayers. The infected monolayers were cultured for 7 days and then stained with neutral red solution as described above. Plaques were counted, and EC values ​​were interpolated from the experimental data using standard methods. 50 value.

[0400] Quantitative DNA assays for EBV and HHV-6B: EBV assays were performed in Akata cells induced by standard methods and subjected to lysing infection with goat anti-human IgG antibodies. The experimental compounds were diluted in plates. Akata cells were added to the plates and incubated. For HHV-6, the compounds were serially diluted, and uninfected HSB-2 or Molt-3 cells were added to each well. Infection was initiated by adding HHV-6A-infected HSB-2 cells or HHV-6B-infected Molt-3 cells and incubating at 37°C for 7 days. For all assays, denaturing buffer was added to each well to denature the DNA, and aliquots were aspirated through a nylon membrane. The membranes were then allowed to dry before equilibration in DIG. Specific digoxigenin (DIG)-labeled probes were prepared for each virus according to the manufacturer's protocol. Detection of specifically bound DIG probes was performed using anti-DIG antibodies with the manufacturer's protocol. Images from photographic film were captured and quantified, and the concentrations of compounds sufficient to reduce viral DNA accumulation by 50% (EC) were inserted from the experimental data. 50 ).

[0401] HHV-8 DNA quantification: The test compound was diluted in two wells. BCBL-1 cells were lysed by the addition of phorbol 12-myristate 13-acetate, and the cells were added to each well in the plate. The cells were incubated at 37°C in a humidified CO2 incubator for 7 days, and then total DNA was prepared. Viral DNA was quantified by real-time PCR. The compound concentration sufficient to reduce the genome copy number by 50% was calculated from the experimental data.

[0402] Cell-based assay for influenza: For dose-response profiling, the drug was individually added to MDCK cells in 96-well microplates. Each test plate included untreated wells containing infected cells (virus control) and untreated cells (cell control). Three days after infection, the virus control wells showed 100% cytopathology. The extent of viral cytopathology in each well was determined by examination and microscopic staining with neutral red (NR). Briefly, cells were stained with NR diluted in MEM to determine cell viability. Two hours later, the plates were treated to quantify NR uptake in viable cells. The amount of NR absorbed by cells was determined spectrophotometrically.

[0403] qPCR assays for BKV and JCV: Preliminary assays of BKV were performed in 96-well plates containing a monolayer of HFF cells. Compound dilutions were prepared in cells containing the cells, which were then infected with Gardnerella vaginalis strain of BKV. After 7 days of incubation, total DNA was prepared using a purification kit, and genomic copy number was quantified by real-time PCR. Positive compounds in this assay were confirmed in a similar assay added 1 hour post-infection to identify compounds that inhibit early stages of replication, including adsorption and penetration. Genomic copy number was determined using the methods described above.

[0404] Preliminary assessments of the compounds against JC virus were performed using methods similar to those used for the preliminary assays against BK virus. Secondary assays against JCV were then performed in COS7 cells using methods similar to those used for BK virus to identify compounds that inhibit viral adsorption or penetration.

[0405] Hepatitis C Virus Assay: Luciferase Reporter Gene (Replicon) / Cytoxin-1 (Toxicity). The anti-HCV activity of compounds was screened using the luciferase (Luc) reporter gene endpoint in the initial HCV assay. The Luc reporter gene was used as an indirect measure of HCV replication because its activity is proportional to HCV RNA levels. Cytotoxicity assessments were performed in parallel. Unless otherwise specified, drug stocks were prepared in DMSO and diluted with tissue culture medium to the desired high test concentration. For each assay, the compound was then further diluted in tissue culture medium as needed. After incubation, cells were processed to obtain suitable EC50. 50 and EC 90 (Reducing the concentration of compounds that replicate replicons by 50% and 90%, respectively). Measurement and reporting of CC. 50 (Concentration that reduces cell viability by 50%) and SI 50 (CC) 50 / EC 50 The replicon (genotype 1b or 2a) or HCVcc virus-derived Luc activity was used as a reading to assess anti-HCV activity; while the cytotoxic concentration that reduced cell number was assessed using a CytoTox-1 cell proliferation assay according to the manufacturer's protocol. Recombinant interferon-α was used as a positive control to validate assay performance.

[0406] Assays for influenza virus, RSV, and SARS-CoV were performed using CellTiter-Glo's cytopathic effect / toxicity-based assays. The antiviral cytoprotective assay examined the effect of a compound at a specified dose-response concentration in a specific cell type to test the compound's efficacy in preventing virus-induced cytopathic effects. Ribavirin was used as a positive control for influenza and RSV, while a calpain IV inhibitor was used for the SARS antiviral assay. Subconfluent cell cultures were plated in 96-well plates to analyze cell viability (cytotoxicity) and antiviral activity (CPE). For standard assays, the drug was added to the cells 24 hours later. CPE wells were also titrated with 100 tissue culture infectious doses (100 TCID50s) of virus. Cell viability was measured 72 hours later.

[0407] The measurement of virus-induced cell cytotoxicity (CPE) is based on the quantification of ATP, an indicator of metabolically active cells. CPE assays use a commercially available luminescent cell viability kit and are a reliable method for determining cytotoxicity and cell proliferation in cultures. The procedure involves the direct addition of a single reagent to pre-cultured, subconfluent cells in the culture medium. This induces cell lysis and the generation of a bioluminescent signal (half-life greater than 5 hours, depending on cell type), which is proportional to the amount of ATP present (a biomarker of viability).

[0408] Assays for dengue fever (DENV), West Nile virus (WNV), yellow fever virus (YFV), Rift Valley fever virus (RVFV), and Venezuelan equine encephalitis virus (VEEV): Primary cytopathic effect (CPE) reduction assays were performed. Four concentrations of CPE inhibition assays were conducted. Confluent or near-confluent cell culture monolayers were prepared in microplates. Cells were maintained in MEM or DMEM supplemented with FBS as needed for each cell line. For antiviral assays, the same medium was used, but the FBS was reduced to 2% or less and supplemented with gentamicin. Different concentrations of the test compound were prepared. Virus control and cell control wells were prepared in each microplate. In parallel, known active drugs were tested as positive controls using the same method as for the test compounds. Positive control assays were performed in each assay. Assays were established by first removing the growth medium from the 96-well plate of cells. The test compound was then applied to the wells. The virus was placed in those wells designated for viral infection. Virus-free medium was placed in the virulence control wells and cell control wells. The virus control wells were treated similarly to the virus. Plates were incubated at 37°C with 5% CO2 until maximum CPE was observed in the virus control wells. Then, the plates were stained with neutral red for approximately 2 hours in a 37°C, 5% CO2 incubator. The neutral red medium was thoroughly aspirated to remove the cells, and the cells were washed with phosphate-buffered saline (PBS) to remove residual dye. The PBS was thoroughly removed, and any incorporated neutral red was eluted with buffer. The dye concentration in each well was quantified using a spectrophotometer. The dye concentration in each group of wells was converted into the percentage of dye present in the untreated control wells using a Microsoft Excel-based spreadsheet. The 50% effective rate (EC50) was then calculated using linear regression analysis. 50 ) concentration and 50% cytotoxicity (CC) 50 Concentration. CC 50 Divide by EC 50 The quotient yields the Selectivity Index (SI) value.

[0409] Assays for adenovirus (AdV), measles virus (MEV), poliovirus (POV), and enterovirus (ENTV): The initial screening is an assay for reduction in cytopathic effect (CPE). In short, cell cultures are infected with the virus in the presence of the test compound and incubated for 4–7 days (depending on the specific virus / cell). Each virus is pre-titrated so that control wells show approximately 95% loss of cell viability due to viral replication. Therefore, antiviral or cytoprotective effects are observed when the compound inhibits viral replication. Each assay plate contains cell control wells (cells only), virus control wells (cells plus virus), compound toxicity control wells (cells and compound only), compound colorimetric control wells (compound only, no cells or virus), and experimental wells (compound plus cells plus virus). Cell protection and compound cytotoxicity are assessed by MTS dye reduction. The percentage decrease in viral CPE (antiviral activity) and the percentage of cell viability (cytotoxicity) are measured and reported.

[0410] Vacciniocytosis Virus (VACV) Assay: The initial assay was a reduction in cytopathic effect (CPE). Low-throughput HFF cells were trypsinized, counted, and seeded into tissue culture plates. Cells were then incubated at 37°C for 24 hours. The culture medium was then removed, and MEM containing 2% FBS was added to all wells except the first row. In the first row, culture medium containing the experimental drug (e.g., compound 1) was added to three-well plates. Culture medium was added separately to the cell and virus control wells. The drug in the first row was then serially diluted 1:5 in the remaining wells. The plates were then incubated for 60 minutes, and the virus suspension was added to each well except the cell control wells receiving MEM. The plates were then incubated at 37°C in a CO2 incubator. After incubation, the culture medium was aspirated, and cells were stained with crystal violet in formalin for 4 hours. The dye was then removed, and the plates were washed until all excess dye was removed. The plates were dried for 24 hours, and the amount of CPE in each row was measured. EC was determined by comparing drug-treated and untreated cells using a computer program. 50 and CC 50 value.

[0411] As described in Example 1 below, compound 1 exhibits anti-mouse norovirus activity in vitro. In some embodiments, compound 1 has an EC50 activity against mouse norovirus. 50 Value approximately 2.1, CC 50 The value is approximately 114. Furthermore, compound 1 exhibits activity against various DNA and RNA viruses.

[0412] As described in Example 2 below, compound 1 exhibits anti-mouse norovirus activity in vivo. As described in Example 2, compound 1 was able to reduce the viral load in mice infected with norovirus in a dose-dependent manner. Results are as follows... Figure 1A and Figure 1B As shown, this illustrates the decrease in viral titers in the feces and tissues of mice in Study 1. Additionally, Figure 2A and 2B The results showed a decrease in viral titers in the feces and tissues of mice in Study 2. Figure 3A and 3B The decrease in viral titers in tissues and feces of mice in Study 1 is shown separately. Results are presented on a linear scale. In Study 1, mice were treated with compound 1 at 30 mg / kg / day, 100 mg / kg / day, or 300 mg / kg / day. In Study 2, mice were treated with compound 1 at 150 mg / kg / day or 300 mg / kg / day.

[0413] Example 3 demonstrates that compound 1 can inhibit norovirus polymerase in vitro. Without being bound by theory, it is proposed that compound 1 can prevent and treat norovirus by inhibiting norovirus polymerase.

[0414] As described in Example 4, compound 1 was converted to a triphosphate in vitro. As shown, when cells were incubated with compound 1, the corresponding triphosphate (i.e., compound 1-TP) was produced. After the incubation period, the level of compound 1-TP was 12 to 23 times higher than that of compound 1.

[0415] Example 5 below demonstrates that compound 1 inhibits mouse norovirus more effectively than compound 2 or 2'-C-methylcytidine triphosphate. A comparison with DMSO as a control is provided for reference. Experiments were repeated, and results are as follows. Figure 4 (First repetition) and Figure 5 (The second repetition) is shown. Figure 6 This shows the superposition of the results from the first and second replicates of the experiment. (Example) Figure 4 As shown in Figures 5 and 6, “A” is DMSO, “B” is compound 2, “C” is 2'-C-methylcytidine triphosphate (2'CmeC TP), and “D” is compound 1. Figure 4-6 It was demonstrated that treatment with DMSO or 2'CmeC TP alone increased the viral titer by nearly two orders of magnitude. However, in the presence of compound 1, the increase in viral titer was less than one order of magnitude.

[0416] Example 6 shows a comparison of compound 1 and other compounds of formula II (Table 7) and their analogues. Not wishing to be bound by theory, in vitro, even compounds with slightly different structures from formula I exhibited a significant decrease in activity. For example, compound 7 has a hydroxyl group on an arylamine and EC... 50 Value >38 μM; Compound 35 shows an analogue of Compound 1 without an arylamine group, and EC 50Value >100 μM. Additionally, compound 11 has a cyano group on an aryl amide, and EC... 50 Value >121 μM. Similarly, compounds 36 and 37 show methyl-substituted amines and EC 121 μM. 50 Value > 100 μM. Those skilled in the art will understand other comparisons.

[0417] Figure 7a shows the HPLC chromatogram of compound 1.

[0418]

[0419] Not wanting to be bound by theory, peak 2 was identified as benzoic acid.

[0420] Figure 7b shows the HPLC chromatogram of compound 1 after slurrying at room temperature for 3 hours.

[0421]

[0422] Figure 7c shows the HPLC chromatogram of compound 1 after slurrying at 50°C for 3 hours.

[0423]

[0424] Figure 7d shows the HPLC chromatogram of compound 1 after slurry preparation at room temperature for 24 hours.

[0425]

[0426] Figure 8a shows the concentrations of compound 1 from about -2 to about 14 ppm. 1 HNMR spectrum.

[0427] Figure 8b shows the concentrations of compound 1 from about 2 to about 9 ppm. 1 HNMR spectrum.

[0428] Figure 8c shows the concentrations of compound 1 from approximately 0 to approximately 9 ppm. 1 HNMR spectrum.

[0429] Example

[0430] General Procedure

[0431] HPLC chromatograms were recorded on a Zorbax Eclipse Plus C18 column (4.6 × 50 mm × 1.8 μm). The first mobile phase was 97.5% water: 2.5% acetonitrile: 0.05% trifluoroacetic acid. The second mobile phase was 97.5% acetonitrile: 2.5% water: 0.05% trifluoroacetic acid.

[0432] Unless otherwise specified, recording is performed at 500 MHz. 1 HNMR.

[0433] Example 1: Antiviral and Cytotoxicity Tests

[0434] Cell Cultures and Viral Strains: Human foreskin fibroblasts (HFF) cells were prepared from human foreskin tissue obtained from the University of Alabama at Birmingham, with IRB approval. The tissue was incubated at 4°C for 4 hours in cell culture medium consisting of minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Inc. Logan UT), standard concentrations of L-glutamine, amphotericin B, and vancomycin in Earle's salt. The tissue was then placed in phosphate-buffered saline (PBS), minced, rinsed to remove red blood cells, and resuspended in trypsin / EDTA solution. The tissue suspension was incubated at 37°C with gentle agitation to disperse the cells, followed by collection by centrifugation. The cells were resuspended in 4 mL of culture medium and incubated at 25 cm⁻¹. 2 HFF cells were incubated in tissue culture flasks at 37°C in a humidified CO2 incubator for 24 hours. The medium was then replaced with fresh medium, and cell growth was monitored daily until a confluent cell monolayer was formed. HFF cells were then expanded by continuous passage in standard MEM medium supplemented with Earle's salts containing 10% FBS, L-glutamine, amphotericin B, and vancomycin. Cells were routinely passaged and used for assays at passage 10 or lower.

[0435] Akata cells latently infected with EBV were obtained from John Sixbey (Louisiana State University, Baton Rouge, LA). The GS strain of HHV-6A was obtained through the NIH AIDS Research and Reference Reagent Program. HSB-2 and BCBL-1 cells were obtained through the NIH AIDS Research and Reference Reagent Program, AIDS, NIAID, and NIH departments. Molt-3 cells were obtained from Scott Schmid, Center for Control and Prevention, Atlanta, GA. All lymphocyte cultures were routinely preserved in RPMI 1640 (Mediatech, Inc., Herndon, VA) with 10% FBS, L-glutamine, and antibiotics, and passaged twice weekly. Vero cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and preserved in MEM standard growth medium supplemented with Earl's salts containing 10% FBS, L-glutamine, penicillin, and streptomycin.

[0436] Influenza viruses: The Centers for Disease Control and Prevention (Atlanta, GA) provided A / New Caledonia / 20 / 99 (H1N1) and A / Sydney / 05 / 97 (H3N2) viruses. The viruses were passaged in Madin-Darby canine kidney (MDCK) cells (American Center for Type Culture Collection, Manassas, VA) to produce working primary strains for antiviral assays.

[0437] HSV-1 strains E-377 and DM2.1, and HSV-2 strains MS and 13078, were used. HSV-1 strain F was obtained from ATCC. HCMV strains AD169 and Merlin were obtained from the American Type Culture Collection (ATCC, Manassas, VA), and C8805 / 37-1-1 and 759RD100 were provided by Karen Biron. VZV, Ellen strain, was obtained from ATCC. HHV-6B strain Z29 was provided by Scott Schmid from the Centers for Disease Control and Prevention in Atlanta, Georgia. HHV-8 was obtained from latently infected BCBL-1 cells using the NIH AIDS Research and Reference Reagent Procedure.

[0438] Antiviral assays: Each assay evaluating the antiviral activity of the compounds included both positive and negative control compounds to ensure the performance of each test. If possible, cytotoxicity was assessed simultaneously at the same compound exposure levels. Based on the list data, the method achieved an effective concentration (EC50) that reduced viral replication by 50% in vitro. 50 ), cell viability (CC) 50 ) and the selectivity index (SI, in CC) 50 Divide by EC 50 (Calculations) were performed at a concentration reduced by 50%. When sufficient material was available, multiple determinations were performed for each compound to obtain statistical data.

[0439] Cytotoxicity assays: Each antiviral assay included parallel cytotoxicity assays using the same cells, cell numbers, drug concentrations, and incubation times used for each virus to provide identical drug exposure. To ensure that the cytotoxicity of all compounds could be directly compared, a standard neutral red uptake cytotoxicity assay of all compounds in confluent HFF cells was performed during a 7-day incubation period.

[0440] Neutral red uptake cytotoxicity assay: Each compound was evaluated using standard methods in a standard cytotoxicity assay. Briefly, HFF cells were subjected to uptake at 2.5 × 10⁻⁶. 4Cells / well were seeded into 96-well tissue culture plates in standard tissue medium. After incubation for 24 hours, the medium was replaced with maintenance cell medium, and the compound was added to the first row. Then, a series of compound concentrations with a maximum concentration of 300 μM were generated using 5-fold serial dilutions. The plates were incubated for 7 days, and 100 μL of 0.66 mg / mL neutral red PBS solution was added to each well, followed by incubation for 1 hour. The dye was then removed, and the plates were washed with PBS. The dye, internalized into live cells, was dissolved in PBS supplemented with 50% ethanol and 1% glacial acetic acid. Density was then measured at 550 nm, and CC values ​​were interpolated from the experimental data. 50 value.

[0441] For all plaque reduction assays of HFF cells, the neutrophil cytotoxicity assay was performed on 6-well plates containing parallel groups of uninfected HFF cells, receiving the same compound concentration as used for the antiviral assay. On the same day as each antiviral assay, the cytotoxicity plate was removed from the incubator and the cell monolayer was stained for 6 hours with 2 mL of 0.165 mg / mL neutral red solution in PBS. The dye was then removed, and any remaining dye was washed off the cells with PBS. The cell monolayer was visually examined for any signs of toxicity. Cytotoxicity assays were performed using Vero cells at drug concentrations ranging from 1 μM to 1 mM. CellTiter 96 was used according to the manufacturer's instructions. ® The Promega assay measures cell viability.

[0442] Cell proliferation assay: Inhibition of HFF cell proliferation was used to precisely assess the cytotoxicity of certain compounds, and the assay was performed according to standard procedures used in the laboratory. 2.5 × 10⁻⁶ cells were used. 4 Cells were seeded at a low density into six-well plates using standard culture medium. After 24 hours, the medium was aspirated, and a series of compound solutions in the growth medium, starting at 300 μM, were prepared and added to the replication wells. The plates were incubated at 37°C for 72 hours, then cells were removed with trypsin and counted using a Beckman Coulter counter. The concentration of the compound that reduced cell proliferation by 50% was interpolated from the experimental data.

[0443] Lymphocyte cytotoxicity assay: Cell viability in all assays performed with lymphocytes was assessed using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). Briefly, the assay plates were incubated at ambient temperature for 30 min, then 50 μL of CellTiter-Glo reagent was added to each well, and the plates were mixed on a track-shaker for 2 min to lyse the cells. The plates were then incubated at ambient temperature for another 10 min, and quantitative luminescence was measured on a luminometer. The drug concentration (CC) required to inhibit the proliferation of Akata, HSB-2, BCLB-1, or Molt-3 cells at 50% was calculated using standard methods. 50 ).

[0444] Table 1. Activity of compound 1 against mouse norovirus in RAW cells.

[0445]

[0446] Table 2. Activity of Compound 1 against various DNA and RNA viruses

[0447]

[0448] Mouse norovirus (MNV) assay: RAW cells (mouse macrophages, ATCC TIV-71) were obtained from the American Type Culture Collection (ATCC). Cells were incubated at 37°C in 5% CO2 cell culture medium consisting of Dulbecco's minimum essential medium (DMEM) (ATCC) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Inc., Logan UT), 100 U / mL penicillin and 100 μg / mL streptomycin (Hyclone), 1% MEM NEAA (Gibco), 1% GlutaMAX (Gibco), and 1% HEPES (Hyclone). The mouse norovirus isolate used in this experiment was isolated from wild-type mice, cell culture adapted, plaque purified, and had its genetic sequence confirmed by Chimerix.

[0449] Serial dilutions of the test compound in DMEM were added to Costar 96-well tissue culture preparation plates. 100 μL of a cell suspension containing 50,000 RAW cells / well and MNV (MOI = 0.0005) was added to the compound dilutions in the 96-well plates, and the plates were incubated at 37°C with 5% CO2 for 2 days. Each assay plate included uninfected cell controls and untreated virus control wells. After 2 days of incubation, the untreated virus control wells showed 100% CPE. After 2 days of incubation, 40 μL of Cell Titer 96 Mqueous MTS reagent (Promega, G111) was added to 200 μL of culture medium in each well according to the manufacturer's instructions, and the plates were incubated at 37°C until the untreated cell controls showed an absorbance between 1.1 and 1.8 at 490 nm. The final absorbance readings were read using a BioTek Synergy 2, and the concentration of RAW cells protecting against MNV infection by 50% was calculated using Gen 5 software (BioTek Instruments, Inc.) compared to the uninfected cell control.

[0450] Human Norovirus Assay: Antiviral activity against human norovirus (NoV) was assessed in a 3-day assay using a stable human norovirus replicon cell line HG23 (genome I, genome length; parental cell line, HuH7) maintained as a subconfluence culture in 96-well plates. Typically, four doses (10-fold or 3-fold steps) were used in triplicate. Antiviral activity was determined by Western blot analysis of intracellular NoV RNA (normalized to the level of cellular β-actin RNA in each culture sample). Cytotoxicity was assessed by the uptake of neutral red dye in the culture maintained on parallel plates (Korba and Gerin, 1992, Antivir. Res. 19:55).

[0451] EC was calculated by linear regression analysis using data from combinations of cultures treated (Korba & Gerin, 1992, Antivir. Res. 19:55; Okuse, et al., 2005, Antivir. Res. 65:23). 50 and CC 50 EC value. EC is calculated from the standard error generated by regression analysis. 50 and EC 90 Standard deviation of the value. EC 50 and EC 90 These are the drug concentrations at which intracellular NoVRNA was observed to be 2-fold or 10-fold inhibited (relative to the average level in untreated cultures).50 This refers to drug concentrations where neutral red dye uptake levels (relative to the average level in untreated cultures) were observed to be 2-fold lower. The selectivity index (SI) was calculated in CC... 50 / EC 50 Calculations were performed. Recombinant human interferon 2b (PBL laboratories, Inc.) was used as a control.

[0452] BKV EC50 in VERO cells: Costar 96-well tissue culture plates were seeded at 10,000 Vero cells / well in DMEM containing 2% Hyclone standard fetal bovine serum (FBS, Cat SH30088.03) and 1% Hyclone penicillin and streptomycin. Outer wells were not used to minimize edge effects from prolonged incubation. Cells were seeded with 115 BKV DNA replicates / cells (ATCC, Gardner strain). Serial dilutions of the test compounds were added to the cells, and the plates were incubated at 37°C, 5% CO2 for 10 days. After 10 days of incubation, 50 μL of supernatant was mixed with 50 μL of 2X lysis buffer and dissolved in DEPC-treated water to a final concentration of 0.5 mg / mL proteinase K, 50 mM KCl, 10 mM Tris-Cl (pH 8.0), 2.5 mM MgCl2, 0.45% IGEPA, and 0.45% Tween-20. Each plate was incubated at 55°C for 2 hours. BKV DNA in the supernatant was measured by quantitative polymerase chain reaction (qPCR) using forward and reverse BKV PCR primers and FAM-labeled probes. Absolute quantification of viral copy number was performed using a standard curve containing dilutions of BKV DNA amplicons homologous to the amplified fragment. The following qPCR amplification conditions were used: 95°C for 10 minutes per cycle, followed by 45 cycles of 95°C for 15 seconds and 60°C for 60 seconds. qPCR reactions were performed using an Applied Biosystems 7500 real-time PCR system. Gen 5 software (BioTek Instruments, Inc.) is used to calculate the reduction of viral DNA levels in BKV-infected Vero cells by 50% (EC50). 50 The concentration of ).

[0453] Plaque reduction assays for HSV-1, HSV-2, VZV, and HCMV: A monolayer of HFF cells was prepared in a six-well plate and incubated at 37°C for 2 days to allow cell confluence. The culture medium was then aspirated from the wells, and 0.2 mL of virus was added to each of three wells to generate 20–30 plaques per well. The plates were allowed to adsorb to the cells for 1 hour, with the plates gently shaken every 15 minutes to redistribute the medium. The compounds were diluted in maintenance cell medium consisting of MEM supplemented with Earl salts containing 2% FBS, L-glutamine, penicillin, and gentamicin. Solutions ranging from 300 μM to 0.1 μM were added to the replication wells, and the plates were incubated for varying times depending on the virus used. Plaque reduction assays for HSV-1 strain F were performed in a similar manner, but Vero cells were infected one day after inoculation. The final FBS concentration in this assay was 5%. For HSV-1 and -2, monolayers were stained with 1% crystal violet in 20% methanol, and unbound dye was removed by washing with dH2O. For HCMV and VZV assays, cell monolayers were stained with 1% neutral red solution for 4 hours, then aspirated and washed with PBS. For all analyses, plaques were counted using a stereomicroscope, and the concentrations of compounds that reduced plaque formation by 50% (EC50) were interpolated from the experimental data. 50 ).

[0454] Plaque reduction assay of MCMV: Mouse embryonic fibroblasts were prepared from mouse embryos using a procedure similar to that described above for HFF cells. They were suspended in tissue culture medium as described above and seeded into 12-well plates, incubated at 37°C for 24 hours. The culture medium was aspirated, and a cell monolayer was infected with MCMV Smith strain at a final volume of 0.2 mL in each well of a 3-well plate. The infected cells were then incubated at 37°C for 1 hour, with occasional shaking of the culture plate to ensure the medium covered the entire monolayer. The compound was serially diluted 1:5 in the tissue maintenance cell culture medium described above, and the solution was added to the infected monolayer. The infected monolayer cells were incubated for 7 days and then stained with 2 mL of 1% neutral red solution as described above. Plaques were counted, and EC values ​​were interpolated from the experimental data using standard methods. 50 value.

[0455] EBV and HHV-6B DNA quantification: EBV was measured in Akata cells induced by a standard method and subjected to lytic infection with 50 μg / mL goat anti-human IgG antibody. The experimental compounds were diluted in round-bottom 96-well plates to produce concentrations from 20 to 0.016 μM. Akata cells were cultured at 4 × 10⁶ cells per well. 4 The concentration of the compound was added to the plate and incubated for 72 hours. For HHV-6, the compound was serially diluted in 96-well plates, and then 1×10⁻⁶ cells were added. 4Uninfected HSB-2 or Molt-3 cells were added to each well. Infection was initiated by adding HHV-6A-infected HSB-2 cells or HHV-6B-infected Molt-3 cells at a ratio of approximately 1 infected cell for every 10 uninfected HSB-2 or Molt-3 cells, and incubated at 37°C for 7 days. For all assays, 100 μL of denaturing buffer (1.2 M NaOH, 4.5 M 80 NaCl) was added to each well to denature the DNA, and 50 μL aliquots were aspirated through an Immobilon nylon membrane (Millipore, Bedford, MA) using a Biodot instrument (Bio-Rad, Hercules, CA). The membrane was then dried before equilibration at 56°C for 30 minutes in a DIG Easy Hyb (Roche Diagnostics, Indianapolis, IN). According to the manufacturer's protocol (Roche Diagnostics), specific digoxigenin (DIG)-tagged probes were prepared for each virus. For EBV, primers 5'-CCC AGG AGT CCC AGT AGT CA-3' and 5'-CAG TTC CTC GCC TTA GGT TG-3' amplified the fragment corresponding to coordinates 96802-97234 in the EBV genome (AJ507799). HHV-6 DIG-tagged probes were prepared using primers 5'-CCT TGA TCA TTC GAC CGTTT-3' and 5'-TGG GAT TGG GAT TAG AGC TG-3' to amplify the ORF2 fragment (coordinates 37820-38418 in X83413). Membranes containing EBV DNA were hybridized overnight at 56°C, followed by sequential washing at the same temperature in 0.2×SSC containing 0.1% SDS and 0.1×SSC containing 0.1% SDS. For HHV-6A and HHV-6B blots, probes were hybridized overnight at 42°C, and the blots were washed at the same temperature in 0.2×SSC containing 0.1% SDS and 0.1×SSC containing 0.1% SDS. Specific binding of DIG probes was detected using anti-DIG antibodies according to the manufacturer's protocol (Roche Diagnostics). Images of the photographic film were captured and quantified using QuantityOne software (Bio-Rad), and compound concentrations sufficient to reduce viral DNA accumulation by 50% (EC) were interpolated from the experimental data. 50 ).

[0456] HHV-8 DNA quantification: The test compound was diluted in two wells of a 96-well plate with a final concentration up to 20 μM. BCBL-1 cells were lysed and infected by adding phorbol 12-myristate 13-acetate (Promega, Madison WI) to a final concentration of 100 ng / mL, and 2 × 10⁻⁶ cells were then inoculated. 4 Cells were added to each well of the plate. Cells were incubated for 7 days in a humidified CO2 incubator at 37°C, and total DNA was then prepared using the Wizard SV 96-well purification kit (Promega). Viral DNA was quantified by real-time PCR using the forward primer 5'-TTC CCC AGA TAC ACG ACA GAA TC-3', the reverse primer 5'-CGG AGCGCA GGC TAC CT-3', and the probe 5'-(FAM)CCT ACG TGT TCG TCG AC(TAMRA)-3'. Absolute quantification of viral DNA was provided using plasmid pMP218 containing the DNA sequence corresponding to nucleotides 14120-14182 (AF148805.2). Compound concentrations sufficient to reduce the genome copy number by 50% were calculated from experimental data.

[0457] Cell-based assays for influenza: For dose-effect curves, individual drugs were added to MDCK cells in 96-well microplates (8 × 10⁴ cells / well) using three-well plates for each concentration. The compounds were added at the following concentrations: 0, 0.000032, 0.0001, 0.00032, 0.001, 0.0032, 0.01, 0.032, 0.1, 1.0, 10.0, and 100 μg / mL of oseltamivir carboxylate; and 0, 0.001, 0.0032, 0.01, 0.032, 0.1, 0.32, 1.3, 2, 10, 32, and 100 μg / mL of amantadine and ribavirin. Each test plate included untreated wells containing infected cells (virus control) and uninfected cells (cell control). Three days after infection, the virus control wells showed 100% cytopathology. The extent of viral cell pathology in each well was determined by examination and microscopic staining with neutral red (NR). Briefly, cells were stained with 0.011% NR diluted in MEM to determine cell viability. Two hours later, the plates were processed for quantification of NR-uptake activity in the cells. The amount of NR absorbed by the cells was determined spectrophotometrically.

[0458] qPCR assays for BKV and JCV: Preliminary assays of BKV were performed in 96-well plates containing a monolayer of HFF cells. Compound dilutions were prepared in cells that were subsequently infected with Gardner strain of BKV. After 7 days of incubation, total DNA was prepared using the Wizard SV 96-well purification kit, and genome copy number was quantified by real-time PCR using primers 5'-AGT GGA TGG GCA GCC TATGTA-3', 5'-TCA TAT CTG GGT CCC CTG GA-3', and probes 5'-6-FAM AGG TAG AAG AGG TTA GGGTGT TTG ATG GCA CAG TAMRA-3'. Plasmid pMP526 was used as a standard for quantifying DNA. Compounds that were positive in this assay were confirmed in a similar assay in 96-well plates, where the compounds were added 1 hour post-infection to identify compounds that inhibited the early stages of replication, including adsorption and penetration. Genome copy number was determined as described above.

[0459] Preliminary assessment of the compounds against JC virus was performed using a method similar to that used for the preliminary assays against BK virus, but in 293TT cells, using strains 1–4 of JCV. Viral DNA was quantified using primers 5'-CTG GTC ATG TGG ATGCTG TCA-3' and 5'-GCC AGC AGG CTG TTG ATA CTG-3', and probe 5'-6-FAM-CCC TTT GTT TGGCTG CT-TAMRA-3, along with plasmid pMP508, to provide a standard curve for absolute quantification. Secondary assays against JCV were performed in COS7 cells using a method similar to that used for BK virus to identify compounds that inhibit viral adsorption or penetration.

[0460] Hepatitis C Virus Assay: Luciferase Reporter Gene (Replicon) / Cytoxin-1 (Toxicity). The anti-HCV activity of compounds was screened using the luciferase (Luc) reporter gene endpoint in the initial HCV assay. Luc is reported as an indirect measure of HCV replication because its activity is proportional to HCV RNA levels. Cytotoxicity assessments were performed in parallel. Unless otherwise specified, drug stocks were prepared in DMSO and diluted with tissue culture medium to the desired high test concentration. For each assay, the compound was then further diluted in tissue culture medium as needed. After incubation, cells were processed to obtain suitable EC50. 50 and EC 90 (Replicator replication with compound concentrations reduced by 50% and 90%, respectively). Measurement and reporting of CC. 50 (Reduces cell viability by 50%) and SI 50 (CC)50 / EC 50 The anti-HCV activity was assessed using replicon (genotype 1b or 2a) or HCVcc virus-derived Luc activity readings; while the cytotoxic concentration that reduced cell numbers was assessed using the CytoTox-1 cell proliferation assay (Promega, Madison, WI) according to the manufacturer's protocol. Recombinant interferon α was used as a positive control to validate the assay performance.

[0461] Assays of influenza, respiratory syncytial virus (RSV), and SARS-CoV: The primary viruses and cells used were influenza A strain / California / 7 / 2009 (H1N1) in MDCK cells, respiratory syncytial virus strain A-2 in Hep2 cells, and SARS-CoV strain Toronto-2 in VeroE6 cells.

[0462] Assays for influenza virus, RSV, and SARS-CoV were performed using CellTiter-Glo's cytopathic effect / toxicity-based assays. The antiviral cytoprotective assay examined the effect of compounds at specified dose-response concentrations in specific cell types to test their efficacy in preventing virus-induced cytopathic effects. Ribavirin was used as a positive control for influenza and RSV, while a calpain IV inhibitor was used for the SARS antiviral assay. Subconfluent cell cultures were plated in 96-well plates to analyze cell viability (cytotoxicity) and antiviral activity (CPE). For the standard assay, the drug was added to the cells 24 hours later. The CPE wells were also titrated with 100 tissue culture infectious doses (100 TCID50s) of virus. Cell viability was measured 72 hours later.

[0463] The measurement of virus-induced CPE is based on the quantification of ATP, an indicator of metabolically active cells. CPE assays were performed using commercially available CellTiter-Glo. TM The CellViability Kit (Promega, Madison, WI) is a reliable method for determining cytotoxicity and cell proliferation in cultures. The procedure involves applying a single reagent (CellTiter-Globe) to the cell viability kit. TM The reagent is added directly to the culture medium of pre-cultured, subconfluent cells. This induces cell lysis and the generation of bioluminescent signals (half-life greater than 5 hours, depending on cell type), which are proportional to the amount of ATP present (a biomarker of viability).

[0464] Assays for dengue fever (DENV), West Nile virus (WNV), yellow fever virus (YFV), Rift Valley fever virus (RVFV), and Venezuelan equine encephalitis virus (VEEV): Initial cytopathic effect (CPE) reduction assays were performed. Four concentrations of CPE inhibition assays were conducted. Confluent or near-confluent cell culture monolayers were prepared in 96-well disposable microplates. Cells were maintained in MEM or DMEM supplemented with FBS as needed for each cell line. For antiviral assays, the same medium was used, but the FBS was reduced to 2% or lower and supplemented with 50 μg / mL gentamicin. Test compounds were prepared at four log10 final concentrations, typically 0.1, 1.0, 10, and 100 μg / mL or μM. Virus control and cell control wells were included in each microplate. In parallel, known active drugs were tested as positive controls using the same method as for the test compounds. Positive control assays were performed in each assay. Assays were established by first removing the growth medium from the 96-well plate of cells. The test compound was then added to the wells at a 2X concentration in 0.1 mL volumes. Virus was typically added in 0.1 mL volumes at <100 50% cell culture infection dose (CCID50) to those wells designated for viral infection. Virus-free medium was placed in the virulence control wells and cell control wells. Virus control wells were treated similarly to those for virus infection. Plates were incubated at 37°C, 5% CO2 until maximum CPE was observed in the virus control wells. The plates were then stained with 0.011% neutral red in a 37°C, 5% CO2 incubator for approximately 2 hours. The neutral red medium was thoroughly aspirated to remove it, and the cells were washed 1X with phosphate-buffered saline (PBS) to remove residual dye. The PBS was completely removed, and the incorporated neutral red was eluted with 50% Sorensen's citrate buffer / 50% ethanol (pH 4.2) for at least 30 minutes. Neutral red dye permeates into living cells; therefore, the stronger the red, the greater the number of living cells present in the wells. The dye concentration in each well was quantified at 540 nm using a 96-well spectrophotometer. The dye concentration in each well was converted into the percentage of dye present in the untreated control wells using a Microsoft Excel-based spreadsheet. The 50% efficacy (EC50) was then calculated using linear regression analysis. 50 ) concentration and 50% cytotoxicity (CC) 50 Concentration. CC 50 Divide by EC 50 The quotient yields the Selectivity Index (SI) value.

[0465] Assays for adenovirus (AdV), measles virus (MEV), poliovirus (POV), and enterovirus (ENTV): Primary screening is an assay for reduction in cytopathic effect (CPE). Briefly, 96-well cell cultures are infected with the virus in the presence of the test compound and incubated for 4–7 days (depending on the specific virus / cell). Each virus is pre-titrated so that control wells show approximately 95% loss of cell viability due to viral replication. Therefore, antiviral or cytoprotective effects are observed when the compound inhibits viral replication. Each assay plate contains cell control wells (cells only), virus control wells (cells plus virus), compound toxicity control wells (cells only plus compound), compound colorimetric control wells (compound only, no cells or virus), and experimental wells (compound plus cells plus virus). Cytoprotection and compound cytotoxicity are assessed by MTS (CellTiter® 96 Promega, Madison WI) dye reduction. The percentage decrease in viral CPE (antiviral activity) and cell viability (cytotoxicity) is measured and reported.

[0466] Vacciniocytosis Virus (VACV) Assay: The preliminary assay was a reduction in cytopathic effect (CPE). Low-pass (3-10) HFF cells were trypsinized, counted, and seeded into 96-well tissue culture plates in 0.1 mL of MEM supplemented with 10% FBS. The cells were then incubated at 37°C for 24 hours. The medium was then removed, and 100 μL of MEM containing 2% FBS was added to all wells except the first row. In the first row, 125 μL of medium containing the experimental drug (i.e., compound 1) was added to three copies of the wells. Medium was added separately to the cell and virus control wells. The drug from the first row was then serially diluted 1:5 in the remaining wells. The plates were then incubated for 60 minutes, and 100 μL of virus suspension was added to each well except for the cell control wells receiving 100 μL of MEM. The plates were then incubated at 37°C in a CO2 incubator for 3 days for VACV assay. After the incubation period, the culture medium was aspirated, and the cells were stained with crystal violet in formalin for 4 hours. The dye was then removed, and the plates were rinsed until all excess dye was removed. The plates were allowed to dry for 24 hours, and the amount of CPE in each row was determined using a BioTek Multiplate Autoreader. EC was determined by comparing drug-treated and untreated cells using a computer program. 50 and CC 50 value.

[0467] Example 2 - Determination of the efficacy of compound 1 against mouse norovirus

[0468] method

[0469] Two studies (Study 1 and Study 2) examined the ability of compound 1 to protect mice from norovirus or reduce norovirus infection in mice:

[0470] Study 1 evaluated 10 mice using mouse norovirus (MNV) CR3. 6 The efficacy of compound 1 administered twice daily in the range of 30 mg / kg to 300 mg / kg, starting before infection with plaque-forming units (PFU), was assessed in mice. Control mice treated with the vector were also included. All doses were initiated 2 days (39 hours) prior to infection. Study groups are shown in Table 3.

[0471] The compound was delivered orally twice daily at a specified dose via strong feeding. A control group of mice treated with the carrier alone was also included. Two days after the first administration, mice were infected with norovirus by inhaling the virus orally. The mice used in group 5 were... 20-gram, 8-12 week old female BALB / c mice (study groups shown in Table 3). Mice were placed on a metal grid, and aggregated fecal products were collected every 24 hours. Tissue (distal ileum and cecum) and individual fecal pellets were harvested on day 3 post-inoculation. All samples were titrated by plaque assay.

[0472] Table 3: Study 1: Study design for proof-of-concept efficacy evaluation of compound 1 against norovirus infection in mice

[0473]

[0474] (1) 5 mice per group

[0475] (2) The compound was administered 51, 39, 27, 15 and 3 hours before infection, and then every 12 hours thereafter.

[0476] Note: 30 mg / kg / day (15 mg / kg / dose, bid); 100 mg / kg / day (50 mg / kg / dose, bid); 300 mg / kg / dose (150 mg / kg / dose, bid). Oral administration begins 2 days prior to injection; orally administered via gavage once daily.

[0477] Study 2 evaluated the efficacy of twice-daily administration of compound 1 at 150 mg / kg or 300 mg / kg, initiated before infection with MNV CR3 at 104 pfu. Control mice treated with the vector were also included. The 150 mg / kg dose was initiated 2 days (39 hours) before inoculation, 1 day (15 hours) before inoculation, or 3 days (3 hours) before inoculation; the 300 mg / kg dose was initiated 2 days (39 hours) before infection. Study groups are shown in Table 4.

[0478] This study tested the ability of compound 1 to protect mice from norovirus infection or reduce norovirus infection in mice. The compound was administered twice daily via oral bolus feeding at the specified dose, starting 2 days before vaccination, 1 day before vaccination, or at the time of vaccination. A control group of mice treated only with the carrier was also included. Three hours after the day 0 dose, mice were fed 10 mg of the compound orally via inhalation of the virus. 4 PFU-induced murine norovirus infection. The mice used in the 5 groups were... 20g, 8-12 week old female BALB / c mice (study group shown in Table 4). All samples were titrated by plaque assay.

[0479] Table 4: Study 2: Study design for evaluating the efficacy of compound 1 against norovirus infection in mice.

[0480]

[0481] (1) Five mice per group

[0482] (2) The compound was administered 51, 39, 27, 15 and 3 hours before infection, and then every 12 hours thereafter.

[0483] (3) The compound was administered 27, 15 and 3 hours before infection, and then every 12 hours thereafter.

[0484] (4) The compound was administered 3 hours before infection, and then every 12 hours thereafter.

[0485] Note: 150 mg / kg twice daily = 300 mg / kg total daily dose; 300 mg / kg twice daily = 600 mg / kg daily dose. Administer orally 2 days, 1 day, or 0 days before infection. On day 0, administer via strong oral feeding at 10... 4 Mice were infected with PFU MNV. They were administered oral feed twice daily for 12 hours.

[0486] For both studies, the compounds were delivered orally via forced feeding at specified doses and times prior to infection. Three hours after administration of compound 1 at dose 0 on day 1, mice were infected with mouse norovirus CR3 by inhaling the virus orally. Post-infection, the compounds were administered twice daily starting on day 3.

[0487] In the 5 groups, the mice used were 20g female BALB / c mice, 8-12 weeks old. Mice were housed on metal fences, and combined fecal production was collected every 24 hours starting from day 1. Tissue samples were collected on day 3 post-infection. 1 cm of distal ileum and cecum) and individual fecal particles were collected and weighed. All samples were titrated by plaque assay (qRT-PCR was used as a backup if the titer was too low); titers were normalized to grams of tissue or feces. On day 3, duplicate tissue samples were collected ( 1 cm distal ileum and cecum were rinsed with PBS and flash-frozen; serum and duplicate fecal samples were also collected on day 3 and flash-frozen; this set of samples was submitted for MS analysis to assess drug bioavailability.

[0488] result

[0489] Results from Study 1 showed that twice-daily administration of Compound 1, starting two days before infection, effectively reduced murine norovirus titers in tissues and feces. Figure 1A and Figure 1B The data showed that increasing the drug concentration resulted in a more significant reduction in viral titer. The data also indicated that animals treated with 300 mg / kg compound 1 twice daily exhibited significantly lower viral titers in tissues and feces compared to the vector.

[0490] like Figure 1A and 1B As shown in "Study No. 1", when using 10 6 Two days prior to PFU norovirus infection, mice were treated with a specified dose of compound 1 twice daily via oral intensive feeding.

[0491] The results of Study 2 indicate that, Figure 2A and Figure 2B As shown, treatment of mice with compound 1 at 300 mg / kg twice daily, starting two days before infection, significantly reduced norovirus titers in tissues and feces. These data confirm the findings of Study #1 and demonstrate the effectiveness of compound 1 in reducing norovirus infection in mice.

[0492] like Figure 2A and 2B As shown in the example, as described in "Study No. 1", when using 10 4 Mice were treated with a specified dose of compound 1 twice daily via oral intensive feeding starting on a designated day prior to PFU rodent norovirus infection.

[0493] Figure 3A and 3B The number of plaque-forming units per gram from Study 1 is displayed on a linear scale rather than a logarithmic scale. Figure 3A As shown, oral administration of compound 1 (twice daily) on day 2 before infection (n = 5 / group) showed a decrease in PFU / g with increasing dose. Figure 3A and 3B As shown, compound 1 reduced mouse norovirus in tissues and feces.

[0494] Example 3 - Norovirus Polymerase Inhibition Assay

[0495] Polymerase reactions (10 μL) were performed at 37 °C for 60 min. Nucleoside triphosphates (NTPs) were added to the polymerase reaction system at 100 μM each, along with 0.05 μCi α32P-UTP (800 Ci / mmol). With or without viral protein genome ligation (VPg), the compounds were incubated with polymerase (Pol) or prepolymerase (ProPol) on ice for 10 min in reaction buffer without NTPs. The reaction was initiated by adding NTPs and terminated by adding an equal volume of 2×Tris / borate / EDTA (TBE) loading dye / buffer (Invitrogen, Inc.). RNA products (100 nt) were separated by electrophoresis on a 6% TBE-urea gel (Invitrogen, Inc.). Semi-quantitative analysis of the RNA products was performed by exposing the dry gel to a Healthcare Phosphor screen (GE), followed by measuring the relative band intensity using GelQuant.NET software (BiochemLabSolutions, Inc.). Using linear regression to obtain IC 50 and IC 90 Calculation results. The results are given in Table 5 below. A comparison with 2'-C-methylcytidine triphosphate (2'CmeC TP) is provided for reference.

[0496] Table 5 Norovirus Polymerase Assay

[0497]

[0498] Example 4 - Conversion of compound 1 to triphosphate in RAW cells

[0499] RAW cells were incubated together with compound 1 at concentrations shown in Table 6a (ng / cell) and Table 6b (pmol / cell).

[0500] Table 6a. Conversion of Compound 1 to triphosphate at ng / cell

[0501]

[0502] Table 6b. Conversion of Compound 1 to Triphosphate at pmol / cell

[0503]

[0504] RAW cells were mixed with four different concentrations of compound 1 in a T75 flask at a concentration of 1.2 × 10⁻⁶. 7Cells were incubated at a density of 1 cell / flask for 48 hours. After incubation, cells were washed twice with cold PBS and counted. The cell pellet was resuspended in 1000 μL of cold methanol:distilled water (70:30), vortexed, and frozen at -80°C to the time of analysis. As shown in Tables 6a and 6b, compounds 1 and 1-TP were detectable in RAW cells treated with compound 1 at concentrations ranging from 0.5 μM to 10 μM. The concentration of compound 1-TP was 12 to 23 times higher than that of compound 1.

[0505] Example 5 - Efficacy of Compound 1 against Human Norovirus

[0506] The inhibitory efficacy of compound 1 against norovirus was compared with that of DMSO (as a control), compound 2, and 2'-C-methylcytidine triphosphate (2'CmeC TP). Cells were pretreated with 25 μM of the experimental compounds for 2 hours. Viral inoculum was added for 2 hours, unbound virus was washed away, and fresh culture medium and fresh experimental compounds were added.

[0507] The experiment was conducted in duplicate, and the results were displayed on [date / time]. Figure 4 (First repetition) and Figure 5 (Second repetition) Figure 6 This shows the superposition of the results from the first and second replicates of the experiment. (Example) Figure 4 , 5 As shown in Figure 6, “A” is DMSO, “B” is compound 2, “C” is 2'-C-methylcytidine triphosphate (2'CmeC TP), and “D” is compound 1. Figure 4-6 It was demonstrated that treatment with DMSO or 2'CmeC TP alone increased the viral titer by nearly two orders of magnitude. However, in the presence of compound 1, the increase in viral titer was less than one order of magnitude.

[0508] Example 6 - Effective Concentration of the Disclosed Compounds and Their Analogs

[0509] Table 7 below shows the effects of some of the compounds and analogues of this disclosure on the EC50 of rodent norovirus. 50 and CC 50 Value. In determining the compound but not the EC value. 50 In the case of values, EC 50 The value is represented as N / A.

[0510] Table 7 EC values ​​of Formula II compounds 50 value

[0511]

[0512] Table 8 EC of the disclosed compounds and their analogues 50 value

[0513]

[0514] Example 7 - Synthesis of Compound 1

[0515]

[0516] Step 1 (Option #1) : Add 3.00 kg of 4-amino-6-bromo-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-carboxylon, 6.60 kg of (3R,4R,5R)-2-acetoxy-5-((benzoyloxy)methyl)tetrahydrofuran-3,4-dimethyldibenzoate, and 18.89 kg of DCE to a 100-L jacketed reactor. Start stirring and add 3.61 kg of DBU. Add 8.01 kg of TMSOTf between 30.6 °C and 37.3 °C over a period of 0.3 hours and 14 minutes. After approximately 1 hour and 30 minutes, the IPC at 32°C showed a remaining 4% of 4-amino-6-bromo-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-carboxylon (3.00 kg), (3R,4R,5R)-2-acetoxy-5-((benzoyloxy)methyl)tetrahydrofuran-3,4-dimethylbenzoate. After approximately 3 hours and 16 minutes, the IPC at 32°C showed a remaining 2% of 4-amino-6-bromo-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-carboxylon (3.00 kg), (3R,4R,5R)-2-acetoxy-5-((benzoyloxy)methyl)tetrahydrofuran-3,4-dimethylbenzoate (specification: ≤3%). The reaction mixture was diluted with DCM (39.81 kg) and quenched with drinking water (15.02 kg) for 11 minutes between 9.5 °C and 15.6 °C. Post-extraction treatment was completed (at approximately 22 °C) by back-extraction of the aqueous phase with DCM (19.90 kg), back-extraction of the bicarbonate phase with DCM (19.71 kg) with saturated NaHCO3 (1.3 kg NaHCO3 in 14.9 kg drinking water), and washing with brine (4.5 kg NaCl in 14.9 kg drinking water). Note: The reactor was washed with drinking water, acetone, and DCM after each wash / back-extraction.

[0517] The organic phase containing the product, in drum form, was added to a 100-L jacketed reactor through an online filter. The drum was then washed with DCM and filtered using DCM (2.48 kg). The reactor contents were vacuum distilled at a maximum temperature of 50.1 °C for 06 h 04 min to a final volume of 31 L. At this point, a thick suspension had formed. Next, after 39 min, IPAc (41.88 kg) was added between 44.5 °C and 49.5 °C, and the reactor contents were heated to 76.9 °C and held for 01 h 25 min. The reactor contents were then cooled to 9.9 °C and held for 04 h 21 min, and stirred at a minimum temperature of 1.6 °C for 12 h 26 min.

[0518] Step 1 (Option #2) Add 3.00 kg of 4-amino-6-bromo-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-carboxylonitrile, 6.60 kg of (3R,4R,5R)-2-acetoxy-5-((benzoyloxy)methyl)tetrahydrofuran-3,4-dimethyldibenzoate, and 18.80 kg of DCE to a 100-L jacketed reactor. Start stirring and add 3.59 kg of DBU. Add 7.90 kg of TMSOTf between 30.4°C and 34.2°C over a period of 0.1 h 46 min. After approximately 0.2 h 49 min IPC, 34°C shows 1% remaining 4-amino-6-bromo-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-carboxylonitrile (specification: ≤3%). The reaction mixture was diluted with DCM (40 / 70 kg) and quenched for 0.4 min with drinking water (14.97 kg) between 9.9 °C and 18.0 °C. Post-extraction treatment was completed (at approximately 22 °C) by back-extraction of the aqueous phase with DCM (20.34 kg), washing with saturated NaHCO3 (1.30 kg NaHCO3 in 14.90 kg drinking water), back-extraction with DCM (20.65 kg) bicarbonate, and washing with brine (4.50 kg NaCl in 14.96 kg drinking water). Note: The reactor was washed with drinking water, acetone, and DCM after each wash / back-extraction.

[0519] The organic phase containing the product in a drum was added to a 100-L jacketed reactor through an online filter. The drum was then rinsed with DCM and filtered with DCM (1.49 kg). The reactor contents were vacuum distilled at a maximum temperature of 45.6 °C for 4 hours and 49 minutes. At this point, a thick suspension had formed. Next, after 27 minutes, IPAc (41.70 kg) was added between 45.6 °C and 48.2 °C, and the reactor contents were heated to 75.7 °C and held for 1 hour and 20 minutes. The reactor contents were then cooled to 9.4 °C and held for 4 hours and 15 minutes, and stirred overnight at a minimum temperature of 2.3 °C.

[0520] Step 2 Add (2R,3R,4R,5R)-2-(4-amino-6-bromo-5-cyano-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5-((benzoyloxy)methyl)tetrahydrofuran-3,4-dimethyldibenzoate (10.0 kg), 10% Pd / C (Degussa, model E101NE / W), trimethylamine (7.3 kg), and THF (44.5 kg) to the reactor. Pass hydrogen into the reactor and stir the mixture at 30.8 psig for 03 hours and 54 minutes, between approximately 24.7°C and 19.6°C. IPC (HPLC) showed that (2R,3R,4R,5R)-2-(4-amino-6-bromo-5-cyano-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5-((benzoyloxy)methyl)tetrahydrofuran-3,4-dimethylbenzoate was undetectable.

[0521] The reaction mixture was filtered using diatomaceous earth (7.2 kg) and a polishing filter, and the residue was washed with THF (5.2 kg). The combined filtrate and washes were transferred to a 100-L jacketed reactor using THF washing (2.12 kg). The reactor contents were vacuum distilled to a final volume of 27 L at a maximum batch temperature of 30.0 °C for 0.5 h 38 min. IPA (31.48 kg) was added to the reactor between 39.7 °C and 53.2 °C over 40 min. The reactor contents were vacuum distilled to a final volume of 33 L at a maximum batch temperature of 53.2 °C for 0.3 h 0.2 min. IPA (48.99 kg) was added to the reactor between 53.1 °C and 57.1 °C over 43 min. The reactor contents were heated to 60.2 °C, stirred for 12 min, and cooled to 5.4 °C for 0.4 h 28 min. Cooling and stirring were continued at a minimum temperature of 1.1 °C for 0.8 h 55 min. The slurry was filtered and washed with IPA (9.41 kg, at approximately 4.5 °C). The residue was vacuum dried under a nitrogen flow for 11 hours and 44 minutes at a maximum temperature of 44.0 °C to provide a LOD of 0.36%. Yield: 6.58 kg (73.9%). 1 Structure determined by 1H NMR. Purity: 97.78% (HPLC, AUC).

[0522] Step 3 :

[0523]

[0524] 1 Dissolve 100g of NaOH in drinking water to a total volume of 1L; 2 Dilute 500 ml of concentrated HCl with drinking water to a total volume of 2 L.

[0525] A solution of (2R,3R,4R,5R)-2-(4-amino-5-cyano-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5-((benzoyloxy)methyl)tetrahydrofuran-3,4-dimethyldibenzoate and THF was heated to 54°C, and 2.5M NaOH was then added. Initially, the addition produced a biphase mixture and an endothermic response (temperature dropped to 50°C), but with further addition, a single-phase, clear solution was formed, accompanied by rapid exothermic heating to 61°C. During subsequent additions, the reaction temperature was maintained at 60°C to 61°C for another 2.5 hours. IPC showed no remaining (2R,3R,4R,5R)-2-(4-amino-5-cyano-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5-((benzoyloxy)methyl)tetrahydrofuran-3,4-dimethyldibenzoate.

[0526] The reaction mixture was cooled to 21°C and neutralized with 3N HCl to pH = 7.06 under external cooling (using a Denver Instrument UB-10 pH meter equipped with a Sartorius P-P11 pH electrode, calibrated with buffer solution at pH = 4.00 and pH = 7.00); the mixture was further cooled to 8°C. The resulting neutralized mixture was vacuum distilled at a jar temperature of 45°C to 50°C until solids were observed in the jar. The suspension was cooled and stirred at 2°C for 2 hours. The beige suspension was filtered to give a dark filtrate; the off-white residue was washed once with cold water (500 mL, 5°C). The first LOD value after 16 hours was 18.73%. HPLC analysis of the dried material showed the presence of 1.6% benzoate.

[0527] A brief reprocessing study of compound 1 (containing 1.6% benzoic acid per AUC, HPLC) was conducted in 10 volumes of water (1 g in 10 mL):

[0528] Pulping at room temperature for 3 hours

[0529] Pulping at 50℃ for 3 hours

[0530] Pulping at room temperature for 24 hours

[0531] All three experiments yielded compound 1 containing less than 0.1% benzoic acid (UAC, HPLC). The slurry was fluid, easily stirred, and filtered rapidly. Short-term drying on the filter yielded a powdery solid, indicating that displacement washing with organic solvents was unnecessary. Not wanting to be bound by theory, a loss of more than 1% NMT (solubility 1 mg / mL) was expected. HPLC data for compound 1 were obtained using a Zorbax EclipsePlus C18 column (water / ACN / TFA, 97.5 / 2.5 / 0.05) employing methods suitable for polar compounds. This was the same column used for steps 1 and 2.

[0532] The cold product suspension was filtered, and the reactor and residue were washed with cold IPAc (approximately 7.5°C, 13.16 kg and 13.62 kg) until a colorless filtrate was obtained. The residue was dried under vacuum with nitrogen release at ≤45°C for 65 hours and 19 minutes to achieve 0% LOD. Yield: 5.87 kg (70.7%). 1 Identification was verified by ¹H NMR; purity was 98.84% (AUC) by HPLC.

[0533] Equivalent

[0534] This disclosure may be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the above embodiments are to be considered illustrative in all respects and not to limit the disclosure herein. The scope of this disclosure is thus defined by the appended claims rather than by the foregoing description, and all variations falling within the meaning and scope of equivalents of the claims are included therein.

Claims

1. A compound, ， Or its pharmaceutically acceptable salt.

2. A compound, ， Or its pharmaceutically acceptable salt.

3. A compound, ， Or its pharmaceutically acceptable salt.

4. A compound, ， Or its pharmaceutically acceptable salt.

5. A pharmaceutical composition comprising the compound of any one of claims 1-4 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

6. A pharmaceutical composition comprising: ， Or its pharmaceutically acceptable salts, and pharmaceutically acceptable carriers.

7. The use of any compound of claims 1-4 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating norovirus, EBV or JCV infection or diseases or conditions associated with norovirus, EBV or JCV infection.

8. The use of the following compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of norovirus, EBV, or JCV infection or diseases or conditions associated with norovirus, EBV, or JCV infection: 。 9. The application of claim 8, wherein it is used for norovirus infection.

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