Trinucleotide cap analogs and methods of use thereof

Abstract

Described herein are novel trinucleotide cap analogs and methods for making and using them.Also described herein are RNA molecules that include a 5' cap, where the 5' cap includes a trinucleotide cap analog described herein.Also described herein are methods of inducing a therapeutic effect in a subject, where the method includes administering to the subject an RNA molecule that includes a trinucleotide cap analog.

Description

[Technical field]

[0001] CROSS-REFERENCE TO PRIORITY APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 267,223, filed January 27, 2022, U.S. Provisional Application No. 63 / 382,956, filed November 9, 2022, and U.S. Provisional Application No. 63 / 476,787, filed December 22, 2022, the entire contents of which are incorporated by reference herein. [Background technology]

[0002] In vitro transcribed messenger RNA (mRNA) has numerous in vivo applications, such as vaccination, in which mRNA encoding a specific antigen is administered to induce protective immunity in a patient; cell therapy, in which mRNA is transfected into cells ex vivo to alter cell phenotype or function and these altered cells are then delivered to a patient; or replacement therapy, in which mRNA encoding a therapeutic protein is administered to a patient.

[0003] The primary structural element of mRNA molecules utilized in in vivo applications includes a cap structure at the 5' end of the mRNA. Naturally occurring cap structures consist of 7-methylguanosine (7M) linked via a 5'- to 5'-triphosphate chain at the 5' end of the mRNA molecule. 7m G or m7 G). The cap must be present for the mRNA to retain its template activity for protein synthesis. The chemical structure of the cap can regulate translation efficiency in the cell. Therefore, an effective cap structure is necessary. Summary of the Invention

[0004] This summary is a high-level overview of various aspects of the invention and introduces some of the concepts that are further described in the Detailed Description section below. This summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used alone to determine the scope of the claimed subject matter. The subject matter should be understood by reference to the entire specification, any or all drawings, and appropriate portions of each claim.

[0005] Described herein are trinucleotide cap analogs and methods of making and using same. Compounds of the formula: [ka] or any stereoisomer thereof provided herein, [ka] is a single or double bond; R1 is H or CH3; R2 is H and R3 is OCH3 or F, or R2 and R3 are covalently bonded to each other and together with the intermediate atom form a 2'-O,4'-C methylene bridge; X1 is O or CH; X2 is CH2 or CH; X3 is O or S; and each independent Y is H. + or a cation; n is 0, 1, 2, 3 or 4, and when X1 is CH, X2 is CH; [ka] is a double bond. Optionally, the compound has the structure: [ka] [ka] or any stereoisomer thereof.

[0006] A compound of the formula: [ka] or any stereoisomer thereof are provided herein, wherein R1 is H or CH3, X1 is O or S, and each independent Y is H + or a cation, n is 0, 1, 2, 3 or 4, and when X1 is S, R1 is CH3. Optionally, the compound has the following structure: [ka] or any stereoisomer thereof.

[0007] A compound of the formula: [ka] or any stereoisomer thereof are provided herein, wherein R1 is H or CH3; and each independent Y is H + or a cation; n is 0, 1, 2, 3, or 4. Optionally, the compound has the structure: [ka] or any stereoisomer thereof.

[0008] Additionally, compounds of the formula: [ka] or any stereoisomer thereof provided herein (wherein R1 is H or CH3; X1, X2, and X3 are each independently selected from O and S, and if one of X1, X2, or X3 is S, then the remainder of X1, X2, and X3 are O; and each independent Y is H). + or a cation; n is 0, 1, 2, 3, or 4. Optionally, the compound has the structure: [ka] [ka] or any stereoisomer thereof.

[0009] Additionally, compounds of the formula: [ka] or any stereoisomer thereof provided herein (wherein R1 is H or CH3; R2 is H and R3 is F, or R2 and R3 are covalently bonded to each other and together with the intermediate atom form a 2'-O,4'-C methylene bridge; each independent Y is H). + or a cation; n is 0, 1, 2, 3 or 4, and when R2 is H and R3 is F, then R1 is not H). Optionally, the compound has the following structure: [ka] or any stereoisomer thereof.

[0010] A compound of the formula: [ka] or any stereoisomer thereof are also provided herein, wherein R1 is H or CH3; X1, X2, X3, and X4 are each independently selected from O and S; and each independent Y is H. + or a cation; n is 0, 1, 2, 3 or 4, and one of X1, X2, X3 and X4 is S. Optionally, the compound has the following structure: [ka] [ka] or any stereoisomer thereof.

[0011] Additionally, compounds of the formula: [ka] or any stereoisomer thereof provided herein, wherein R and R are each independently selected from H and CH; and each independent Y is H. + or a cation; n is 0, 1, 2, 3 or 4, and at least one of R1 or R2 is CH3. Optionally, the compound has the following structure: [ka] or any stereoisomer thereof.

[0012] A compound of the formula: [ka] or any stereoisomer thereof are also provided herein, wherein R and R are each independently selected from H and CH; each independent Y is H + or a cation; n is 0, 1, 2, 3, or 4. Optionally, the compound has the structure: [ka] or any stereoisomer thereof.

[0013] Additionally, compounds of the formula: [ka] or any stereoisomer thereof provided herein, wherein R1 is H or CH3; R2 is OH, F, substituted or unsubstituted alkoxy, or thio; R3 is H or CH3; and each independent Y is H. + or a cation; n is 0, 1, 2, 3 or 4, and when R2 is OH, R1 is not H. Optionally, the compound has the following structure: [ka] [ka] [ka] or any stereoisomer thereof.

[0014] A compound of the formula: [ka] or any stereoisomer thereof are provided herein, wherein R1 is H and CH3; and each independent Y is H + or a cation; n is 0, 1, 2, 3, or 4. Optionally, the compound has the structure: [ka] or any stereoisomer thereof.

[0015] Additionally, compounds of the formula: [ka] or any stereoisomer thereof are provided herein, wherein R1 is H and CH3; and each independent Y is H + or a cation; n is 0, 1, 2, 3, or 4. Optionally, the compound has the structure: [ka] or any stereoisomer thereof.

[0016] A compound of the formula: [ka] or any stereoisomer thereof are also provided herein, wherein R and R are each independently selected from H and CH; each independent Y is H + or a cation; n is 0, 1, 2, 3, 4 or 5. Optionally, the compound has the structure: [ka] or any stereoisomer thereof.

[0017] Deuterated forms of the disclosed compounds are also described herein. For example, a compound selected from the group consisting of: [ka] or any stereoisomer thereof.

[0018] Also described herein is a pharmaceutical composition comprising a compound described herein and a pharma- ceutically acceptable carrier. Also provided is an RNA molecule comprising a 5' cap, the 5' cap comprising a compound described herein. Optionally, the RNA molecule is a messenger RNA (mRNA) molecule or a self-amplifying RNA (saRNA) molecule. Also provided is a method of inducing a therapeutic effect in a subject. The method comprises administering to a subject an RNA molecule (e.g., an mRNA molecule or a saRNA molecule) described herein. The administered RNA may contain some amount of immunogenic double-stranded RNA (dsRNA). The amount of dsRNA is calculated for each ug of RNA administered.

[0019] Further provided herein is a method of administering to an animal a therapeutic dose unit of an mRNA molecule comprising a 5' cap. In some examples, the 5' cap comprises a compound described herein. In some examples, the 5' cap comprises a compound selected from the group consisting of: [ka] [ka] Here, in some examples, the therapeutic dosage unit (e.g., therapeutic mRNA dosage unit) contains less than 7ng / μg of double-stranded RNA (dsRNA), and in some examples, the subject shows increased tolerance to the administered therapeutic dosage unit of the mRNA molecule compared to an equivalent therapeutic dosage unit of the mRNA molecule containing 7ng / μg or more of dsRNA. Optionally, the increased tolerance is determined by measuring one or more of body weight, organ weight, aspartate aminotransferase (AST) level, alanine transaminase (ALT) level, C-reactive protein (CRP) level, procalcitonin (PCT) level, interleukin-6 (IL-6) level, erythrocyte sedimentation rate (ESR), serum amyloid A level, and serum ferritin level before administration and before a period of time after administration. Optionally, the increased tolerance is measured by testing in a standard in vivo assay.In some examples, the increased tolerability is at least a 20% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least a 25% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least a 30% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least a 35% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least a 40% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least a 45% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least a 50% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least a 55% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay. Increased tolerability, at least a 60% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least a 65% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least a 70% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least a 75% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least an 80% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least an 85% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least a 90% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, or at least a 95% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay.

[0020] Optionally, the therapeutic dosage unit of the mRNA molecule is less than 6.9 ng / μg double stranded RNA (dsRNA), less than 6.8 ng / μg double stranded RNA (dsRNA), less than 6.7 ng / μg double stranded RNA (dsRNA), less than 6.6 ng / μg double stranded RNA (dsRNA), less than 6.5 ng / μg double stranded RNA (dsRNA), less than 6.4 ng / μg double stranded RNA (dsRNA), less than 6.3 ng / μg double stranded RNA (dsRNA), less than 6.2 ng / μg double stranded RNA (dsRNA), less than 6.1 ng / μg double stranded RNA (dsRNA), less than 6.0 ... / μg double stranded RNA (dsRNA), less than 5.9ng / μg double stranded RNA (dsRNA), less than 5.8ng / μg double stranded RNA (dsRNA), less than 5.7ng / μg double stranded RNA (dsRNA), less than 5.6ng / μg double stranded RNA (dsRNA), less than 5.5ng / μg double stranded RNA (dsRNA), less than 5.4ng / μg double stranded RNA (dsRNA), less than 5.3ng / μg double stranded RNA (dsRNA), less than 5.2ng / μg double stranded RNA (dsRNA), less than 5.1ng / μg double stranded RNA (dsRNA), 5.0ng less than 4.9ng / μg double stranded RNA (dsRNA), less than 4.8ng / μg double stranded RNA (dsRNA), less than 4.7ng / μg double stranded RNA (dsRNA), less than 4.6ng / μg double stranded RNA (dsRNA), less than 4.5ng / μg double stranded RNA (dsRNA), less than 4.4ng / μg double stranded RNA (dsRNA), less than 4.3ng / μg double stranded RNA (dsRNA), less than 4.2ng / μg double stranded RNA (dsRNA), less than 4.1ng / μg double stranded RNA (dsRNA), 4.0 less than 3.9ng / μg double stranded RNA (dsRNA), less than 3.8ng / μg double stranded RNA (dsRNA), less than 3.7ng / μg double stranded RNA (dsRNA), less than 3.6ng / μg double stranded RNA (dsRNA), less than 3.5ng / μg double stranded RNA (dsRNA), less than 3.4ng / μg double stranded RNA (dsRNA), less than 3.3ng / μg double stranded RNA (dsRNA), less than 3.2ng / μg double stranded RNA (dsRNA), less than 3.1ng / μg double stranded RNA (dsRNA), 3.Less than 0ng / μg double-stranded RNA (dsRNA), less than 2.9ng / μg double-stranded RNA (dsRNA), less than 2.8ng / μg double-stranded RNA (dsRNA), less than 2.7ng / μg double-stranded RNA (dsRNA), less than 2.6ng / μg double-stranded RNA (dsRNA), less than 2.5ng / μg double-stranded RNA (dsRNA), less than 2.4ng / μg double-stranded RNA (dsRNA), less than 2.3ng / μg double-stranded RNA (dsRNA) NA (dsRNA), double-stranded RNA (dsRNA) less than 2.2ng / μg, double-stranded RNA (dsRNA) less than 2.1ng / μg, double-stranded RNA (dsRNA) less than 2.0ng / μg, double-stranded RNA (dsRNA) less than 1.9ng / μg, double-stranded RNA (dsRNA) less than 1.8ng / μg, double-stranded RNA (dsRNA) less than 1.7ng / μg, double-stranded RNA (dsRNA) less than 1.6ng / μg, 1.5ng Less than 1.2ng / μg double stranded RNA (dsRNA), less than 1.1ng / μg double stranded RNA (dsRNA), less than 1.0ng / μg double stranded RNA (dsRNA), less than 0.9ng / μg double stranded RNA (dsRNA), less than 0.8ng / μg double stranded RNA (dsRNA), less than 0.7 ng / μg double stranded RNA (dsRNA), less than 0.6 ng / μg double stranded RNA (dsRNA), less than 0.5 ng / μg double stranded RNA (dsRNA), less than 0.4 ng / μg double stranded RNA (dsRNA), less than 0.3 ng / μg double stranded RNA (dsRNA), less than 0.2 ng / μg double stranded RNA (dsRNA), or less than 0.1 ng / μg double stranded RNA (dsRNA).

[0021] Further provided herein is a method of increasing in vivo translation of a polypeptide in a subject, comprising administering to the subject a therapeutic dose unit comprising an effective amount of an mRNA encoding the polypeptide, wherein the mRNA comprises a 5' cap having the following formula: [ka] wherein the therapeutic dosage unit administered is at least 20% lower than the therapeutic dosage unit required to elicit the same response in a subject when administered a comparison mRNA encoding the polypeptide, the comparison mRNA including a 5' cap having the following formula or any stereoisomer thereof: [ka] .

[0022] Also provided herein is a method of increasing in vivo translation of a polypeptide in a subject, comprising administering to the subject a therapeutic dose unit comprising an effective amount of an mRNA encoding the polypeptide, wherein the mRNA comprises a 5' cap having the following formula, or any stereoisomer thereof: [ka] wherein the therapeutic dosage unit administered is at least 20% lower than the therapeutic dosage unit required to elicit the same response in a subject when administered a comparison mRNA encoding the polypeptide, the comparison mRNA including a 5' cap having the following formula or any stereoisomer thereof: [ka] .

[0023] Further provided herein is a method of increasing in vivo translation of a polypeptide in a subject, comprising administering to the subject a therapeutic dose unit comprising an effective amount of an mRNA encoding the polypeptide, wherein the mRNA comprises a 5' cap having the following formula: [ka] wherein the therapeutic dosage unit administered is at least 20% lower than the therapeutic dosage unit required to elicit the same response in a subject when administered a comparison mRNA encoding the polypeptide, the comparison mRNA including a 5' cap having the following formula or any stereoisomer thereof: [ka] .

[0024] Also provided herein is a method of increasing in vivo translation of a polypeptide in a subject, comprising administering to the subject a therapeutic dose unit comprising an effective amount of an mRNA encoding the polypeptide, wherein the mRNA comprises a 5' cap having the following formula, or any stereoisomer thereof: [ka] wherein the therapeutic dosage unit administered is at least 20% lower than the therapeutic dosage unit required to elicit the same response in a subject when administered a comparison mRNA encoding the polypeptide, the comparison mRNA including a 5' cap having the following formula or any stereoisomer thereof: [ka] .

[0025] Optionally, the subject exhibits increased tolerance to the administered therapeutic dose unit as measured by testing in a standard in vivo assay, compared to the therapeutic dose unit required to elicit the same response in the subject when administered a comparison mRNA. In some cases, the increased tolerance is at least a 20% increase in tolerance to the administered therapeutic dose unit as measured by testing in a standard in vivo assay, at least a 25% increase in tolerance to the administered therapeutic dose unit as measured by testing in a standard in vivo assay, at least a 30% increase in tolerance to the administered therapeutic dose unit as measured by testing in a standard in vivo assay, at least a 35% increase in tolerance to the administered therapeutic dose unit as measured by testing in a standard in vivo assay, at least a 40% increase in tolerance to the administered therapeutic dose unit as measured by testing in a standard in vivo assay, at least a 45% increase in tolerance to the administered therapeutic dose unit as measured by testing in a standard in vivo assay, at least a 50% increase in tolerance to the administered therapeutic dose unit as measured by testing in a standard in vivo assay. % increase in tolerability to the therapeutic dose unit administered when measured by testing in a standard in vivo assay; at least a 55% increase in tolerability to the therapeutic dose unit administered when measured by testing in a standard in vivo assay; at least a 60% increase in tolerability to the therapeutic dose unit administered when measured by testing in a standard in vivo assay; at least a 65% increase in tolerability to the therapeutic dose unit administered when measured by testing in a standard in vivo assay; at least a 70% increase in tolerability to the therapeutic dose unit administered when measured by testing in a standard in vivo assay; at least a 75% increase in tolerability to the therapeutic dose unit administered when measured by testing in a standard in vivo assay; at least an 80% increase in tolerability to the therapeutic dose unit administered when measured by testing in a standard in vivo assay; at least an 85% increase in tolerability to the therapeutic dose unit administered when measured by testing in a standard in vivo assay.At least a 90% increase in tolerability of the administered therapeutic dosage unit as measured by testing in a standard in vivo assay, or at least a 95% increase in tolerability of the administered therapeutic dosage unit as measured by testing in a standard in vivo assay.

[0026] Optionally, the therapeutic dosage unit administered is less than 6.9 ng / μg double stranded RNA (dsRNA), less than 6.8 ng / μg double stranded RNA (dsRNA), less than 6.7 ng / μg double stranded RNA (dsRNA), less than 6.6 ng / μg double stranded RNA (dsRNA), less than 6.5 ng / μg double stranded RNA (dsRNA), less than 6.4 ng / μg double stranded RNA (dsRNA), less than 6.3 ng / μg double stranded RNA (dsRNA), less than 6.2 ng / μg double stranded RNA (dsRNA), less than 6.1 ng / μg double stranded RNA (dsRNA), less than 6.0 ... <μg double stranded RNA (dsRNA), <5.9ng / μg double stranded RNA (dsRNA), <5.8ng / μg double stranded RNA (dsRNA), <5.7ng / μg double stranded RNA (dsRNA), <5.6ng / μg double stranded RNA (dsRNA), <5.5ng / μg double stranded RNA (dsRNA), <5.4ng / μg double stranded RNA (dsRNA), <5.3ng / μg double stranded RNA (dsRNA), <5.2ng / μg double stranded RNA (dsRNA), <5.1ng / μg double stranded RNA (dsRNA), 5.0ng / μg double stranded RNA (dsRNA), less than 4.9ng / μg double stranded RNA (dsRNA), less than 4.8ng / μg double stranded RNA (dsRNA), less than 4.7ng / μg double stranded RNA (dsRNA), less than 4.6ng / μg double stranded RNA (dsRNA), less than 4.5ng / μg double stranded RNA (dsRNA), less than 4.4ng / μg double stranded RNA (dsRNA), less than 4.3ng / μg double stranded RNA (dsRNA), less than 4.2ng / μg double stranded RNA (dsRNA), less than 4.1ng / μg double stranded RNA (dsRNA), 4.0 less than 3.9ng / μg double stranded RNA (dsRNA), less than 3.8ng / μg double stranded RNA (dsRNA), less than 3.7ng / μg double stranded RNA (dsRNA), less than 3.6ng / μg double stranded RNA (dsRNA), less than 3.5ng / μg double stranded RNA (dsRNA), less than 3.4ng / μg double stranded RNA (dsRNA), less than 3.3ng / μg double stranded RNA (dsRNA), less than 3.2ng / μg double stranded RNA (dsRNA), less than 3.1ng / μg double stranded RNA (dsRNA), 3.Less than 0ng / μg double-stranded RNA (dsRNA), less than 2.9ng / μg double-stranded RNA (dsRNA), less than 2.8ng / μg double-stranded RNA (dsRNA), less than 2.7ng / μg double-stranded RNA (dsRNA), less than 2.6ng / μg double-stranded RNA (dsRNA), less than 2.5ng / μg double-stranded RNA (dsRNA), less than 2.4ng / μg double-stranded RNA (dsRNA), less than 2.3ng / μg double-stranded RNA (dsRNA) NA (dsRNA), double-stranded RNA (dsRNA) less than 2.2ng / μg, double-stranded RNA (dsRNA) less than 2.1ng / μg, double-stranded RNA (dsRNA) less than 2.0ng / μg, double-stranded RNA (dsRNA) less than 1.9ng / μg, double-stranded RNA (dsRNA) less than 1.8ng / μg, double-stranded RNA (dsRNA) less than 1.7ng / μg, double-stranded RNA (dsRNA) less than 1.6ng / μg, 1.5ng Less than 1.2ng / μg double stranded RNA (dsRNA), less than 1.1ng / μg double stranded RNA (dsRNA), less than 1.0ng / μg double stranded RNA (dsRNA), less than 0.9ng / μg double stranded RNA (dsRNA), less than 0.8ng / μg double stranded RNA (dsRNA), less than 0.7 ng / μg double stranded RNA (dsRNA), less than 0.6 ng / μg double stranded RNA (dsRNA), less than 0.5 ng / μg double stranded RNA (dsRNA), less than 0.4 ng / μg double stranded RNA (dsRNA), less than 0.3 ng / μg double stranded RNA (dsRNA), less than 0.2 ng / μg double stranded RNA (dsRNA), or less than 0.1 ng / μg double stranded RNA (dsRNA).

[0027] Further, a method for synthesizing a trinucleotide compound is described herein. In one example, the method for synthesizing a trinucleotide compound includes mixing N7-methyl-guanosine-5'-diphosphate with an activation reagent to form a reactive intermediate; reacting the reactive intermediate with imidazole to form an activated intermediate; and adding a salt reagent (e.g., a chloride salt such as magnesium chloride or zinc chloride) and a dinucleotide to the activated intermediate to form a trinucleotide compound, the method being a one-pot synthesis. In some examples, the method for synthesizing a trinucleotide compound includes mixing N7-methyl-guanosine-5'-diphosphate of the following structure: [ka] wherein X1 and X2 are each independently selected from the group consisting of O and S; X3 is O; R1 and R2 are each independently selected from H, OH, N3, F, substituted or unsubstituted alkoxy, and thio, and R1 and R2 are optionally joined to form a heterocycle, with an activating reagent and imidazole to form an activated intermediate; and a salt reagent (e.g., a chloride salt such as magnesium chloride or zinc chloride) and a dinucleotide of the structure: [ka] , (in the formula, [ka] is a single or double bond; X4 and X6 are each independently selected from the group consisting of O and S; X5 is O, S or CH; X7 is CH or CH2; R3 is OH, OMe, F and R4 is H, or R3 and R4 are covalently bonded to each other and together with the intermediate atom form a 2'-O,4'-C methylene bridge; B1 and B2 are each independently selected from the group consisting of a purine ring and a pyrimidine ring) to the activated intermediate to form a trinucleotide compound of the following structure: [ka] The method is a one-pot synthesis.

[0028] In another embodiment, the method of synthesizing a trinucleotide compound includes synthesizing a dinucleotide having the structure: [ka] , (in the formula, [ka] is a single or double bond; X4 is O; X6 is O or S; X5 is O, S or CH; X7 is CH or CH2; R3 is OH, OMe, F and R4 is H, or R3 and R4 are covalently bonded to each other and together with the intermediate atom form a 2'-O,4'-C methylene bridge; B1 and B2 are each independently selected from the group consisting of a purine ring and a pyrimidine ring) with an activating reagent and imidazole to form an activated phosphate imidazolide of the following structure: [ka] and A salt reagent (e.g., a chloride salt such as magnesium chloride or zinc chloride) and a compound of the structure: [ka] , (in the formula, X1 and X2 are each independently selected from the group consisting of O and S; X3 is S; R1 and R2 are each independently selected from H, OH, N3, F, substituted or unsubstituted alkoxy, and thio, and R1 and R2 are optionally joined to form a heterocycle) to an activated phosphate imidazolide to form a trinucleotide compound of the structure: [ka] .

[0029] In yet another embodiment, a method for synthesizing a trinucleotide compound includes the step of: [ka] , (in the formula, [ka] is a single or double bond; X3 is O; X4 and X6 are each independently selected from O and S; X5 is O, S or CH; X7 is CH or CH2; R3 is OH, OMe, F and R4 is H, or R3 and R4 are covalently bonded to each other and together with the intermediate atom form a 2'-O,4'-C methylene bridge; B1 and B2 are each independently selected from the group consisting of a purine ring and a pyrimidine ring) with an activating reagent and imidazole to form an activated phosphate imidazolide of the structure: [ka] and A salt reagent (e.g., a chloride salt such as magnesium chloride or zinc chloride) and a compound of the structure: [ka] , (in the formula, X1 and X2 are each independently selected from O and S; R1 and R2 are each independently selected from H, OH, N3, F, substituted or unsubstituted alkoxy, and thio, and R1 and R2 are optionally joined to form a heterocycle) to an activated phosphate imidazolide to form a trinucleotide compound of the structure: [ka] The method is a one-pot synthesis.

[0030] The details of one or more embodiments are set forth in the drawings and the description below. Other features, objects, and advantages will become apparent from the description and drawings, and from the claims. [Brief description of the drawings]

[0031] [Figure 1] Figure 1 is a bar graph showing in vitro translation of the cap analogs described herein using wheat germ extract, a cell-free translation system. The data demonstrate that protein expression of the cap analogs was comparable or enhanced compared to mRNA produced from a control cap. [Diagram 2] 2 is a bar graph showing in vivo luciferase expression in mice injected with LNPs containing mRNA generated from the cap analogs described herein along with mRNA generated from a comparative cap. The data demonstrate that in vivo luciferase expression was comparable or enhanced compared to mRNA generated from the comparative cap. [Diagram 3] Figure 3 shows a representative animal from a cohort of five animals shown across five imaging time points following LNP:mRNA and luciferin injection. Luminescence intensity scale is on the right. [Figure 4]Figure 4 contains plots showing that 3'OMe modification of the m7G portion of the cap analog results in increased in vivo translation. m7GpppA2'OMepG (control) was measured compared to m7G3'OmepppA2'OMepG (compound 29), which is m7GpppA2'OMepG (control) with a 3'OMe group on m7G (first column). m7Gpppm6A2'OMepG (compound 31) was measured compared to m7G3'OMepppm6A2'OMepG (compound 30), which is m7Gpppm6A2'OMepG (compound 31) with a 3'OMe group on m7G (second column). m7GpppA2',4'-LNApG (compound 1) was measured compared to m7G3'OMepppA2',4'-LNApG (compound 2), which is m7GpppA2',4'-LNApG (compound 1) with a 3'OMe group on m7G (third column). m7Gppp(diaminopurine)2'OMepG (compound 7) was measured compared to m7G3'OMeppp(diaminopurine)2'OMepG (compound 8), which is m7Gppp(diaminopurine)2'OMepG (compound 7) with a 3'OMe group on m7G (fourth column). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Described herein are novel trinucleotide cap analogs and compositions and methods of using them.Also described herein are RNA molecules that include a 5' cap, and the 5' cap comprises a trinucleotide cap analog as described herein.Also described herein are methods of inducing a therapeutic effect in a subject, and the method comprises administering a trinucleotide cap analog or an RNA molecule that includes a trinucleotide cap analog to a subject.

[0033] The following description lists various examples of the method. The specific examples are not intended to define the scope of the method. Rather, these are non-limiting exemplary methods. The description should be read from the perspective of those skilled in the art. Thus, it does not necessarily include information that is known to those skilled in the art.

[0034] I. Definition Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents, patent applications and publications mentioned throughout this disclosure are incorporated by reference in their entirety.

[0035] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to "a transcript" or "the transcript" may include a plurality of transcripts.

[0036] The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to better describe the invention and does not impose limitations on the scope of the invention unless otherwise claimed.

[0037] The terms "may," "may be," "can," and "can be," and related terms, unless the context clearly indicates otherwise, are intended to convey that the subject matter involved is optional (i.e., the subject matter is present in some instances and absent in other instances) and are not a reference to the ability or probability of the subject matter.

[0038] The terms "optional" and "optionally" mean that the subsequently described event, circumstance, or material may or may not occur or be present, and that the description includes instances where the event, circumstance, or material occurs or is present, as well as instances where it does not occur or is not present.

[0039] Use of the terms "including," "comprising," or "having" and variations herein is meant to encompass the elements listed thereafter, and equivalents thereof, as well as additional elements. Embodiments recited as "including," "comprising," or "having" particular elements are also contemplated as "consisting essentially of" and "consisting of" those particular elements. As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations interpreted as either / or.

[0040] As used herein, the transitional phrase "consisting essentially of" (and grammatical variations) should be construed to include the recited materials or steps of the claimed invention as well as "those which do not materially affect the basic and novel characteristics." See In re Herz, 537 F.2d 549,551-52,190 USPQ461,463 (CCPA 1976) (emphasis in original). See also MPEP §2111.03. Thus, the term "consisting essentially of" as used herein should not be construed as equivalent to "comprising."

[0041] Ranges may be expressed herein as from one particular value and / or to another particular value. When such ranges are expressed, the range from one particular value and / or to the other particular value is also considered to be specifically contemplated and disclosed, unless the context specifically indicates otherwise. It should be understood that each and every value and subrange of values ​​included within an explicitly disclosed range should also be considered to be specifically contemplated and disclosed, unless the context specifically indicates otherwise. Furthermore, all ranges should be understood to refer to the recited range both as a range and as a collection of individual numbers from and including the first endpoint, to and including the second endpoint. In the latter case, it should be understood that any of the individual numbers can be selected as one form of the quantity, value, or characteristic to which the range refers. Thus, a range represents a set of numbers or values ​​from a first endpoint, and including the first endpoint, to and including the second endpoint, from which a single member of the set (i.e., a single number) can be selected as the quantity, value or characteristic to which the range refers.

[0042] The term "about" as used herein in connection with numerical values ​​is intended to represent values ​​either above or below the stated value within about + / - 10%. In other examples, values ​​may range from values ​​either above or below the stated value within about + / - 5%. In other examples, values ​​may range from values ​​either above or below the stated value within about + / - 2%. In other examples, values ​​may range from values ​​either above or below the stated value within about + / - 1%.

[0043] As used herein, the term "cap analog" refers to a structural derivative of an RNA cap.

[0044] As used herein, the term "complement," "complementary," or "complementarity" refers to specific base pairing between nucleotides or nucleic acids. Complementary nucleotides are generally A and T (or A and U), and G and C. For example, the complementarity between a capped oligonucleotide primer and a DNA template can be "complete" or "total" when all of the nucleotide bases of the two nucleic acid strands match according to recognized base pairing rules, or "partial" when only a portion of the nucleotide bases of the capped initial oligonucleotide primer and the DNA template match according to recognized base pairing rules, or "absent" when none of the nucleotide bases of the two nucleic acid strands match according to recognized base pairing rules. Complementarity can also be "substantial complementarity" when the nucleotide bases of the two nucleic acids match according to recognized base pairing rules but include one or more mismatches (e.g., 1, 2, 3, 4) from complete complementarity.

[0045] As used herein, a "deoxyribonuclease (DNase)" is an enzyme that catalyzes the hydrolytic cleavage of phosphodiester bonds in the DNA backbone, thus degrading DNA.

[0046] As used herein, the term "hybridize" or "specifically hybridize" refers to the process in which an initiating trinucleotide primer anneals to a DNA template under appropriately stringent conditions during a transcription reaction. Hybridization to DNA is performed, in certain embodiments, with a capped initiating oligonucleotide primer that is 3-10 nucleotides in length and includes a 5'-5' inverted cap structure. Nucleic acid hybridization techniques are well known in the art (e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, NY (1989); Ausubel, FM, et al., Current Protocols in Molecular Biology, John Wiley & Sons, Secaucus, NJ (1994)).

[0047] As used herein, the term "impurities" refers to substances that differ from the chemical composition of the target material (e.g., mRNA transcripts). Impurities are also called contaminants.

[0048] "Inorganic pyrophosphatase" refers to an enzyme that catalyzes the conversion of one ion of pyrophosphate into two phosphate ions, thus inhibiting aggregation and possibly preventing the interaction of pyrophosphate with magnesium ions during the T7 transcription reaction.

[0049] As used herein, the term "internucleotide linkage" refers to the bond or bonds that connect two nucleosides of an oligonucleotide or nucleic acid, which may be a natural phosphodiester bond or a modified bond.

[0050] As used herein, the term "in vitro" refers to a process that is performed outside an organism (e.g., a multicellular organism such as a human or non-human animal), e.g., in a test tube, culture dish, or elsewhere outside the organism.

[0051] As used herein, the term "in vivo" refers to events that take place within an organism.

[0052] As used herein, the term "in vivo assay" refers to a method used to detect and / or measure the ability of one or more of a compound, or a molecule containing a compound (e.g., an mRNA molecule in a therapeutic dose), to increase or decrease a property compared to a control (e.g., a biomarker level). Optionally, the in vivo assay described herein can be used to determine a subject's level of tolerance to a given compound or molecule. Exemplary measurements for evaluating tolerance include one or more of body weight, organ weight, aspartate aminotransferase (AST) level, alanine transaminase (ALT) level, C-reactive protein (CRP) level, procalcitonin (PCT) level, interleukin-6 (IL-6) level, erythrocyte sedimentation rate (ESR), serum amyloid A level, and serum ferritin level. Optionally, the in vivo assay can be characterized herein as a "standard in vivo assay" and can be optionally performed using the conditions outlined in Examples 2 and 3 of the present application.

[0053] As used herein, the term "label" or "detectable label" refers to any compound or combination of compounds that can be bound to or associated with a molecule such that the molecule can be directly or indirectly detected by detecting the label. Detectable labels include radioisotopes (e.g., carbon, phosphorus, iodine, indium, sulfur, tritium, etc.), mass isotopes (e.g., H 2 , C 13 Or N 15 ), a dye or fluorophore (eg, cyanine, fluorescein or coumarin), a hapten (eg, biotin), or any other agent that can be detected directly or indirectly.

[0054] As used herein, "locked nucleic acid" (LNA) refers to a ribonucleotide having a bridge between the 2'O and 4'C methylene bicyclonucleotide monomers. The LNA portion can have the following structure: [ka] .

[0055] As used herein, a "messenger RNA transcript" or "mRNA transcript" is a transcript transcribed from a DNA template that encodes a desired polypeptide. An mRNA transcript may contain coding and non-coding regions. For example, the DNA template may include an RNA polymerase promoter sequence, a 5'UTR sequence, an open reading frame, and a 3'UTR sequence. In some instances, the DNA template also includes a nucleic acid sequence that encodes a poly(A) tail.

[0056] As used herein, the term "modified NTP" refers to a nucleoside 5'-triphosphate having a chemical moiety attached or substituted at any position, including the sugar, the base, the triphosphate chain, or any combination of these three positions. Optionally, the chemical moiety can be of any nature compatible with the process of transcription. Examples of such NTPs include inosine triphosphate, dihydrouridine triphosphate, 2'-fluoro-2'-deoxycytidine triphosphate, pseudouridine triphosphate, N1-methylpseudouridine triphosphate, and 5-methyluridine triphosphate, which can be found, for example, in "Nucleoside Triphosphates and Their Analogs: Chemistry, Biotechnology and Biological Applications," Vaghefi, M., ed., Taylor and Francis, Boca Raton (2005).

[0057] As used herein, the term "modified oligonucleotide" or "modified trinucleotide" includes, for example, oligonucleotides that contain modified nucleosides, modified internucleotide bonds, or have any combination of modified nucleosides and modified internucleotide bonds. Examples of internucleotide bond modifications include phosphorothioate derivatives, phosphotriester derivatives, and methylphosphonate derivatives (Stec, WJ, et al., Chem. Int. Ed. Engl., 33: 709-722 (1994); Lebedev, AV, et al., E., Perspect. Drug Discov. Des., 4: 17-40 (1996); and Zon, et al., US Patent Application No. 20070281308). Other examples of internucleotide bond modifications can be found in Waldner, et al., Bioorg. Med. Chem. Letters 6: 2363-2366 (1996).

[0058] As used herein, the term "nucleoside" refers to a nitrogenous base linked to a five-carbon sugar (e.g., ribose or deoxyribose). The term includes all nucleosides, including all forms of nucleoside bases and furanoses. Base rings include purine and pyrimidine rings. Purine rings include, for example, adenine (also referred to herein as "A"), guanine (also referred to herein as "G"), and N6-methyladenine (also referred to herein as "N6-methyladenine"). m6 Pyrimidine rings include, for example, cytosine (also referred to herein as "C"), thymine (also referred to herein as "T"), 5-methylcytosine (also referred to herein as "A"). m5 Other nucleosides include, but are not limited to, ribo, 2'-O-methyl or 2'-deoxyribo derivatives of adenosine, guanosine, cytidine, thymidine, uridine, inosine, 7-methylguanosine or pseudouridine.

[0059] As used herein, the term "nucleoside analogs," "modified nucleosides," or "nucleoside derivatives" includes synthetic nucleosides described herein. Nucleoside derivatives also include nucleosides with modified base moieties or / and modified sugar moieties, with or without protecting groups, including, for example, 2'-deoxy-2'-fluorouridine, 5-fluorouridine, and the like. The compounds and methods provided herein include such base rings and their synthetic analogs, as well as non-natural heterocyclic and acyclic substituted base sugars. Other nucleoside derivatives that may be utilized with the present disclosure include, for example, LNA nucleosides, halogen-substituted purines (e.g., 6-fluoropurine), halogen-substituted pyrimidines, N-substituted nucleosides, and the like. 6 -Ethyladenine, N 4 -(alkyl)-cytosine, 5-ethylcytosine, and the like (U.S. Pat. No. 6,762,298).

[0060] As used herein, the term "nucleoside triphosphate", "nucleoside 5' triphosphate" or "NTP" refers to a nucleoside linked to three phosphate groups. The term encompasses natural NTPs (e.g., adenosine triphosphate (ATP), uridine triphosphate (UTP), guanine triphosphate (GTP) and cytosine triphosphate (CTP)) and modified NTPs.

[0061] As used herein, "oligo dT purification" is an affinity chromatography method for purifying mRNA comprising or including a polyA tail.

[0062] As used herein, "phosphorothioate linkage" refers to a linkage between nucleosides in which the phosphorodiester bond has been modified by replacing one of the oxygen atoms connected to the phosphorus atom with a sulfur atom.

[0063] "Primary RNA" or "primary RNA transcript" means an RNA molecule synthesized de novo by RNA polymerase in vitro and in which the RNA molecule has a triphosphate on the 5' carbon of its 5'-most nucleotide.

[0064] As used herein, the term "prematurely terminated RNA transcript" refers to an incomplete product of an in vitro transcription reaction. A prematurely terminated RNA sequence can be of any length that is shorter than the intended length of the desired transcript.

[0065] The term "promoter" as used herein refers to a nucleotide sequence within a DNA template that induces and controls the initiation of transcription of a particular DNA sequence. A promoter is typically directly adjacent to (or partially overlaps with) the DNA sequence to be transcribed. The promoter sequence is typically located immediately upstream or 5'-end of the transcription start site. Nucleotide positions within a promoter are designated relative to the transcription start site where transcription of DNA begins (position +1).

[0066] As used herein, the term "purified" or "to purify" refers to the separation of a material from at least some of the components (e.g., impurities or contaminants) with which it was originally produced. For example, RNA transcripts are purified by removal of contaminating proteins or other undesirable nucleic acid species (e.g., double-stranded RNA, DNA, and / or incomplete or interrupted RNA transcripts). Purified materials (e.g., capped mRNA transcripts) can be separated from 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% of other components with which they were originally associated.

[0067] As used herein, the term "RNase inhibitor" or "ribonuclease inhibitor" refers to a protein that inhibits RNAse activity, for example, during an in vitro transcription reaction.

[0068] As used herein, the term "RNA polymerase" refers to an enzyme that uses a DNA template to synthesize RNA. In vitro transcription methods typically use single subunit phage RNA polymerases derived from T7, T3, SP6, K1-5, K1E, K1F or K11 bacteriophage or their mutants. This family of polymerases does not require accessory proteins and has a simple minimal promoter sequence of about 17 nucleotides with minimal constraints on the start nucleotide sequence.

[0069] In the disclosed methods, salts of one or more compounds (e.g., primers) described herein can be used. The term "salt" as used herein refers to derivatives of the compounds described herein prepared by reaction of an acidic or basic moiety of the compound with a mineral or organic acid or base. Optionally, the salts can be pharmaceutically acceptable salts. As used herein, the term "pharmaceutically acceptable salts" refers to salts of the compounds described herein or derivatives thereof, and where possible zwitterionic forms of the compounds described herein, that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio, and are effective for their intended use. These salts can be prepared in situ during isolation and purification of the compounds, or by separately reacting the purified compounds in free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate salts mesylate, glucoheptonate, lactobionate, methanesulfonate and laurylsulfonate salts, etc. Salts may include cations based on alkali and alkaline earth metals such as sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium cations, quaternary ammonium cations and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.(See S. M. Barge et al., J. Pharm. Sci. (1977) 66, 1; and Remington: The Science and Practice of Pharmacy, 23d Edition, Adejare et al. eds., Academic Press (2020), which are incorporated by reference in their entireties.)

[0070] As used herein, "self-amplifying RNA" or "saRNA" is a linear, single-stranded RNA molecule that codes for a gene of interest. saRNA is a type of mRNA, but also contains nonstructural proteins that code for viral replicase. Viral replicase allows the RNA to self-replicate when delivered into a cell.

[0071] As used herein, the term "specific" when used in reference to an initial trinucleotide primer sequence and its ability to hybridize to a DNA template is a sequence that has at least 50% sequence identity with a portion of the DNA template when the initial trinucleotide primer is aligned with the DNA strand. Higher levels of sequence identity include at least 66%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, and optionally 100% sequence identity.

[0072] As used herein, the term "substantially free" refers to a condition in which there is a relatively small amount of undesired material (e.g., prematurely terminated RNA sequences, DNA and / or double-stranded RNA) in a sample, or there is no amount of undesired material (e.g., prematurely terminated RNA sequences, DNA and / or double-stranded RNA). "Substantially free of impurities" means that impurities are present in a sample at a level of about 5%, about 4%, about 3%, about 2%, about 1.0%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, about 0.1% or less (w / w). For example, "substantially free of double-stranded RNA" means that double-stranded RNA is present in a sample at levels of about 5%, about 4%, about 3%, about 2%, about 1.0%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, about 0.1% or less (w / w).

[0073] As used herein, "tangential flow filtration (TFF)" is a type of filtration in which the material to be filtered passes tangentially through the filter, rather than through it. In TFF, the undesired permeate passes through the filter, while the desired retentate passes along the filter and is collected downstream. In TFF, the desired material is typically contained in the retentate, which is the opposite of what is encountered when performing traditional membrane filtration or dead-end filtration.

[0074] As used herein, the term "transcription" refers to the enzymatic production or synthesis of RNA complementary to a DNA template, thereby generating multiple RNA copies of a DNA sequence. The RNA molecule synthesized in a transcription reaction is an "RNA transcript", "primary transcript" or "transcript". The transcription reaction involving the compositions and methods provided herein uses a capped initiation oligonucleotide primer as described herein. The transcription of the DNA template can be exponential, nonlinear or linear. The DNA template can be double-stranded linear DNA, partially double-stranded linear DNA, circular double-stranded DNA, DNA plasmid, PCR amplification product, or a modified nucleic acid template compatible with RNA polymerase.

[0075] As used herein, the terms "universal base," "degenerate base," "universal base analog," and "degenerate base analog" include, for example, in certain embodiments, nucleoside analogs that have an artificial base recognizable by RNA polymerase in place of one of the natural NTPs (e.g., ATP, UTP, CTP, and GTP) or other specific NTPs. Universal or degenerate bases are disclosed in Loakes, D., Nucleic Acids Res., 29:2437-2447 (2001); Crey-Desbiolles, C., et. al., Nucleic Acids Res., 33:1532-1543 (2005); Kincaid, K., et. al., Nucleic Acids Res., 33:2620-2628 (2005); Preparata, FP, Oliver, JS, J. Comput. Biol. 753-765 (2004); and Hill, F., et. al., Proc Natl Acad. Sci. USA, 95:4258-4263 (1998)).

[0076] As used herein, the terms "unsubstituted" or "unmodified" in reference to capped initiating oligonucleotide primers and NTPs refer to capped initiating oligonucleotide primers and NTPs that are not modified.

[0077] In this specification and in the eventual claims, a reference to parts by weight of a particular element or component in a composition indicates the weight relationship between that element or component and any other element or component in the composition or article for which the parts by weight are expressed. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight of component Y, X and Y are present in a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.

[0078] Weight percentage (wt %) of a component is based on the total weight of the formulation or composition in which the component is included, unless specifically stated otherwise.

[0079] As used herein, the terms "optional" or "optionally" mean that the subsequently described event or circumstance may or may not occur, and that the description includes cases where the event or circumstance occurs and cases where the event or circumstance does not occur.

[0080] As used herein, the term "subject" may be a vertebrate, such as a mammal, fish, bird, reptile, or amphibian. Thus, the subject of the methods disclosed herein may be a human, a non-human primate, a horse, a pig, a rabbit, a dog, a sheep, a goat, a cow, a cat, a guinea pig, or a rodent. The term does not indicate a particular age or sex. Thus, it is intended to cover adult and newborn subjects, as well as fetuses, of either sex. In one aspect, the subject is a mammal. A patient refers to a subject suffering from a disease or disorder. The term "patient" includes human and veterinary subjects.

[0081] As used herein, the term "treatment" refers to the medical management of a patient with the intention of curing, improving, stabilizing, or preventing a disease, pathological condition, or disorder. This term includes active treatment, i.e., treatment specifically aimed at improving a disease, pathological condition, or disorder, as well as causal treatment, i.e., treatment aimed at eliminating the cause of associated disease, pathological condition, or disorder. In addition, this term includes symptomatic treatment, i.e., treatment designed to relieve symptoms, but not cure, of a disease, pathological condition, or disorder; preventive treatment, i.e., treatment aimed at minimizing, or partially or completely suppressing the onset of associated disease, pathological condition, or disorder; and supportive treatment, i.e., treatment used to supplement another specific therapy aimed at improving associated disease, pathological condition, or disorder. In various embodiments, the term encompasses any treatment of a subject, including a mammal (e.g., a human), including (i) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed as having it; (ii) inhibiting the disease, i.e., arresting its development; or (iii) alleviating the disease, i.e., causing the disease to regress. In one embodiment, the subject is a mammal, such as a primate, and in a further embodiment, the subject is a human. The term "subject" also includes domestic animals (e.g., cats, dogs, etc.), livestock animals (e.g., cows, horses, pigs, sheep, goats, etc.) and laboratory animals (e.g., mice, rabbits, rats, guinea pigs, fruit flies, etc.).

[0082] As used herein, the term "prevent" or "preventing" refers to eliminating, avoiding, preventing, forestalling, stopping, or impeding something from happening, especially by prior action. It is understood that where reduce, inhibit, or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed.

[0083] As used herein, the term "diagnosed" means having undergone a physical examination by a person skilled in the art, e.g., a physician, and found to have a condition that can be diagnosed or treated by the compounds, compositions, or methods disclosed herein.

[0084] As used herein, the terms "administer" and "administration" refer to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, intranasal administration, topical administration, intravaginal administration, eye drop administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, such as injectables, such as intravenous administration, intraarterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. In various aspects, the preparation can be administered therapeutically, i.e., administered to treat an existing disease or condition. In further various aspects, the preparation can be administered prophylactically, i.e., administered for the prevention of a disease or condition.

[0085] As used herein, the terms "effective amount" and "effective amount" refer to an amount sufficient to achieve a desired outcome or to affect an undesired condition. For example, a "therapeutically effective amount" refers to an amount sufficient to achieve a desired therapeutic outcome or to affect an undesired condition, but generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend on a variety of factors, including the disorder being treated, and the severity of the disorder; the particular composition used; the patient's age, weight, general health, sex, and diet; the time of administration; the route of administration; the rate of excretion of the particular compound used; the duration of treatment; drugs used in combination with or simultaneously with the particular compound used, and similar factors well known in the medical arts. For example, it is well within the skill of the artisan to start a dose of the compound at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for administration. As a result, a single dose composition can contain such amounts or fractions thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage may vary and may be administered in one or more doses per day for one or several days.Guidance can be found in the literature for appropriate dosages of a given class of pharmaceutical.Furthermore, in various embodiments, the preparation may be administered in a "prophylactically effective amount", i.e., an amount effective for preventing a disease or condition.

[0086] As used herein, "dosage form" means a pharmacologically active material in a medium, carrier, vehicle or device suitable for administration to a subject. The dosage form can include the disclosed compounds of the present invention, the products of the disclosed methods of making, or salts, solvates or polymorphs thereof, in combination with pharma- ceutically acceptable excipients, such as preservatives, buffers, saline or phosphate buffered saline. The dosage form can be made using conventional pharmaceutical manufacturing and compounding techniques. The dosage form can contain inorganic or organic buffers (e.g., sodium or potassium salts of phosphate, carbonate, acetate or citrate) and pH adjusting agents (e.g., hydrochloric acid, sodium or potassium hydroxide, citrate or acetate salts, amino acids and their salts), antioxidants (e.g., ascorbic acid, alpha-tocopherol), surfactants (e.g., polysorbate 20, polysorbate 80, polyoxyethylene 9-10 nonylphenol, sodium deoxycholate), solution and / or cryogenic / lyophilization stabilizers (e.g., sucrose, glycerol, sorbitol ... The dosage form may include an injectable agent, such as glycerol, lactose, mannitol, trehalose, osmolality adjusters (e.g., salts or sugars), antibacterial agents (e.g., benzoic acid, phenol, gentamicin), antifoaming agents (e.g., polydimethylsilozone), preservatives (e.g., thimerosal, 2-phenoxyethanol, EDTA), polymeric stabilizers and viscosity adjusters (e.g., polyvinylpyrrolidone, poloxamer 488, carboxymethylcellulose), and cosolvents (e.g., glycerol, polyethylene glycol, ethanol). Dosage forms formulated for injection use may include a suspension of the disclosed compound, the product of the disclosed method of making, or a salt, solvate, or polymorph thereof, in sterile saline for injection with a preservative.

[0087] As used herein, terms such as "elevated," "increased," "decreased," and "reduced" are generally considered relative to a control or normal state. However, when terms such as "reduce" or "lower" are used herein relative to a treatment, they refer to normalizing levels or amounts toward a control or normal state, and can include partial or complete normalization.

[0088] As used herein, a "kit" refers to an assembly of at least two components that constitute a kit. The components together constitute a functional unit for a given purpose. The individual component parts may be physically packaged together or separately. For example, a kit that includes instructions for using the kit may or may not physically include the instructions with the other individual component parts. Alternatively, the instructions may be provided as a separate component part, either in paper or electronic form that can be provided on a computer-readable memory device or downloaded from an internet website, or as a recorded presentation.

[0089] As used herein, "instructions" refers to documents that describe materials or methods associated with the kit. These materials may include any combination of background information, a list of components and their availability information (such as purchasing information), simple or detailed protocols for using the kit, troubleshooting, references, technical support, and any other related documents. Instructions may be provided with the kit or as a separate component, either in paper or electronic form, which may be provided on a computer-readable memory device or downloaded from an Internet website, or as recorded presentations. Instructions may include one or more documents, and are meant to include future updates.

[0090] As used herein, the term "therapeutic agent" includes any synthetic or naturally occurring biologically active compound or composition that induces a desired pharmacological, immunogenic and / or physiological effect by local and / or systemic action when administered to an organism (human or non-human animal). Thus, the term encompasses compounds or chemicals that are traditionally considered drugs, vaccines and biologics, including molecules such as proteins, peptides, hormones, nucleic acids, gene constructs, etc. Examples of therapeutic agents are described in well-known texts such as the Merck Index (14th Edition), Physicians' Desk Reference (64th Edition) and Pharmacological Basis of Therapeutics (12th Edition), and include, but are not limited to, pharmaceuticals; vitamins; mineral supplements; substances used to treat, prevent, diagnose, cure or mitigate disease or illness; substances that affect the structure or function of the body, or prodrugs that become biologically active or more active after being placed in a physiological environment.For example, the term "therapeutic agent" includes, but is not limited to, adjuvants; anti-infectives, such as antibiotics and antivirals; analgesics and analgesic combinations, anorexics, anti-inflammatory agents, anti-epileptics, local and general anesthetics, hypnotics, sedatives, antipsychotics, neuroleptics, antidepressants, anxiolytics, antagonists, neuron blocking agents, anticholinergics and cholinergic drugs, antimuscarinic agents and muscarinic drugs, antiadrenergics, antiarrhythmics, antihypertensives, hormones and nutrients, antiarthritic agents, antiasthmatics, anticonvulsants, antihistamines, antiemetics, antineoplastic agents, antipruritic agents, antipyretics; antispasmodics, cardiovascular preparations (including calcium channel blockers, beta blockers, beta agonists and antiarrhythmics), antihypertensives, diuretics, vasodilators; central nervous system These include compounds or compositions for use in any major therapeutic area, including stimulants; cough and cold preparations; decongestants; diagnostics; hormones; bone growth stimulants and bone resorption inhibitors; immunosuppressants; muscle relaxants; psychostimulants; sedatives; tranquilizers; proteins, peptides and fragments thereof (whether naturally occurring, chemically synthesized or recombinantly produced); and nucleic acid molecules (polymeric forms of two or more nucleotides, either ribonucleotides (RNA) or deoxyribonucleotides (DNA), including both double-stranded and single-stranded molecules, gene constructs, expression vectors, antisense molecules, etc.), small molecules (e.g., doxorubicin), and other biologically active macromolecules, such as proteins and enzymes. The drugs may be biologically active agents used in veterinary medicine, medicine including applications, and agriculture with plants, etc., as well as other areas. The term "therapeutic agent" also includes, but is not limited to, a medicine; a vitamin; a mineral supplement; a substance used in the treatment, prevention, diagnosis, cure, or mitigation of a disease or disorder; or a substance that affects the structure or function of the body; or a prodrug that becomes biologically active or more active after being placed in a given physiological environment.

[0091] The term "pharmacologically acceptable" refers to a material that is not biologically or otherwise undesirable, i.e., that does not cause unacceptable levels of undesirable biological effects or interact in a deleterious manner.

[0092] As used herein, the term "derivative" refers to a compound having a structure derived from that of a parent compound (e.g., a compound disclosed herein) and whose structure is sufficiently similar to that disclosed herein that a person skilled in the art would predict, based on that similarity, that it will exhibit the same or similar activity and utility as the claimed compound, or, as a precursor, induce the same or similar activity and utility as the claimed compound. Exemplary derivatives include salts, esters, amides, salts of esters or amides, and N-oxides of the parent compound.

[0093] As used herein, the term "pharmaceutical acceptable carrier" refers to sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions immediately prior to use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters such as ethyl oleate. For example, proper fluidity can be maintained by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms can be ensured by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, etc. Prolonged absorption of injectable pharmaceutical forms can be brought about by the inclusion of agents such as aluminum monostearate and gelatin which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters), and poly(anhydrides). The rate of drug release can be controlled depending on the ratio of drug to polymer and the nature of the particular polymer used. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues. Injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating a sterilizing agent in the form of a sterile solid composition which can be dissolved or dispersed in sterile water or other sterile injectable medium immediately prior to use. A suitable inert carrier can include a sugar such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size within the range of 0.01 to 10 micrometers.

[0094] As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds. In a broad aspect, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. Exemplary substituents include, for example, those described below. Permissible substituents can be one or more of the same or different for appropriate organic compounds. For purposes of this disclosure, heteroatoms such as nitrogen can have hydrogen substituents and / or any permissible substituents of organic compounds described herein that satisfy the valence of the heteroatom. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. The terms "substituted" or "substituted by" also include the implicit proviso that such substitution is in accordance with the permissible valences of the substituted atom and substituent, as well as the implicit proviso that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation by rearrangement, cyclization, elimination, and the like. In certain embodiments, unless expressly stated to the contrary, it is also contemplated that individual substituents may be further optionally substituted (i.e., may be further substituted or unsubstituted).

[0095] In defining various terms, this specification refers to "A 1 ","A 2 ","A 3 " and "A 4 " is used as a general symbol to represent various specific substituents. These symbols can be any substituent, including but not limited to those disclosed herein, and if one instance is defined as a specific substituent, another instance may be defined as some other substituent. The term "aliphatic" or "aliphatic group" as used herein refers to a hydrocarbon moiety that may be straight-chained (i.e., unbranched), branched, or cyclic (including fused, bridged, and spiro-fused polycyclic), may be fully saturated, or may contain one or more units of unsaturation, but is not aromatic. Unless otherwise specified, an aliphatic group contains 1-20 carbon atoms. Aliphatic groups include, but are not limited to, straight-chained or branched alkyl, alkenyl, and alkynyl groups, as well as hybrids thereof, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkyl)alkenyl.

[0096] The term "alkyl" as used herein refers to a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. The alkyl group can be cyclic or acyclic. The alkyl group can be branched or unbranched. The alkyl group can also be substituted or unsubstituted. For example, the alkyl group can be substituted with one or more groups, including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, thio, or thiol, as described herein. A "lower alkyl" group is an alkyl group containing 1 to 6 (e.g., 1 to 4) carbon atoms. The term alkyl group can also be C1 alkyl, C1-C2 alkyl, C1-C3 alkyl, C1-C4 alkyl, C1-C5 alkyl, C1-C6 alkyl, C1-C7 alkyl, C1-C8 alkyl, C1-C9 alkyl, C1-C10 alkyl, etc. up to C1-C24 alkyl.

[0097] Throughout this specification, "alkyl" is used generally to refer to both unsubstituted and substituted alkyl groups. However, substituted alkyl groups are also specifically referred to herein by identifying the particular substituents on the alkyl group. For example, the term "halogenated alkyl" or "haloalkyl" specifically refers to an alkyl group substituted with one or more halides, such as fluorine, chlorine, bromine, or iodine. Alternatively, the term "monohaloalkyl" specifically refers to an alkyl group substituted with a single halide, such as fluorine, chlorine, bromine, or iodine. The term "polyhaloalkyl" specifically refers to an alkyl group independently substituted with two or more halides, i.e., each halide substituent need not be the same halide as another halide substituent, nor do multiple instances of a halide substituent have to be on the same carbon. The term "alkoxyalkyl" specifically refers to an alkyl group substituted with one or more alkoxy groups, as described below. The term "aminoalkyl" specifically refers to an alkyl group substituted with one or more amino groups. The term "hydroxyalkyl" specifically refers to an alkyl group substituted with one or more hydroxy groups. When "alkyl" is used in one instance and a specific term such as "hydroxyalkyl" is used in another instance, this does not mean that the term "alkyl" does not also refer to the specific term such as "hydroxyalkyl".

[0098] This practice is also used for other groups described herein. That is, a term such as "cycloalkyl" refers to both unsubstituted and substituted cycloalkyl moieties, but substituted moieties may additionally be specifically identified herein. For example, a specific substituted cycloalkyl may be referred to, for example, as an "alkylcycloalkyl". Similarly, a substituted alkoxy may be specifically referred to, for example, as a "halogenated alkoxy", a specific substituted alkenyl may be, for example, as an "alkenylalcohol", and the like. Again, the practice of using a general term such as "cycloalkyl" and a specific term such as "alkylcycloalkyl" does not mean that the general term does not also include the specific term.

[0099] The term "cycloalkyl" as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, etc. The term "heterocycloalkyl" is a type of cycloalkyl group defined above and falls within the meaning of the term "cycloalkyl" where at least one of the ring carbon atoms is replaced by a heteroatom, such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. Cycloalkyl and heterocycloalkyl groups can be substituted or unsubstituted. Cycloalkyl and heterocycloalkyl groups can be substituted by one or more groups, including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, thio, or thiol, as described herein.

[0100] The terms "alkoxy" and "alkoxyl" as used herein to refer to an alkyl or cycloalkyl group bonded through an ether linkage, i.e., an "alkoxy" group is an -OA 1 where A 1 is alkyl or cycloalkyl as defined above. "Alkoxy" also includes polymeric alkoxy groups as just described. That is, alkoxy includes -OA, -O, -O-C, -O-H ... 1 -OA 2 , or -OA 1 -(OA 2 ) a -OA 3 where "a" is an integer from 1 to 200, and A 1 , A 2 and A 3 is an alkyl group and / or a cycloalkyl group.

[0101] As used herein, the term "alkenyl" refers to a hydrocarbon group of 2 to 24 carbon atoms having a structural formula containing at least one carbon-carbon double bond. 1 A 2 )C=C(A 3 A 4 Asymmetric structures such as aryl, aryl, aryl and aryl groups are intended to include both the E and Z isomers. This can be inferred in the structural formulas herein where an asymmetric alkene is present, or can be explicitly indicated by the bond symbol C=C. Alkenyl groups can be substituted with one or more groups, including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, thio, or thiol, as described herein.

[0102] The term "cycloalkenyl" as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms and containing at least one carbon-carbon double bond, i.e., C=C. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, norbornenyl, and the like. The term "heterocycloalkenyl" is a type of cycloalkenyl group defined above, and is included within the meaning of the term "cycloalkenyl" in which at least one of the ring carbon atoms is replaced by a heteroatom, such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. Cycloalkenyl and heterocycloalkenyl groups can be substituted or unsubstituted. The cycloalkenyl and heterocycloalkenyl groups can be substituted with one or more groups, including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, thio, or thiol, as described herein.

[0103] The term "alkynyl" as used herein is a hydrocarbon group of 2 to 24 carbon atoms having a structural formula containing at least one carbon-carbon triple bond. Alkynyl groups can be unsubstituted or substituted with one or more groups, including alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, thio, or thiol, as described herein.

[0104] The term "cycloalkynyl" as used herein is a non-aromatic carbon-based ring composed of at least 7 carbon atoms and containing at least one carbon-carbon triple bond. Examples of cycloalkynyl groups include, but are not limited to, cycloheptynyl, cyclooctynyl, cyclononynyl, etc. The term "heterocycloalkynyl" is a type of cycloalkenyl group defined above, and falls within the meaning of the term "cycloalkynyl" in which at least one of the carbon atoms of the ring is replaced by a heteroatom, such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. Cycloalkynyl and heterocycloalkynyl groups can be substituted or unsubstituted. The cycloalkynyl and heterocycloalkynyl groups may be substituted with one or more groups, including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, thio, or thiol, as described herein.

[0105] The term "aryl" as used herein is a group containing any carbon-based aromatic group, including, but not limited to, benzene, naphthalene, phenyl, biphenyl, anthracene, and the like. The aryl group can be substituted or unsubstituted. The aryl group can be substituted by one or more groups, including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, -NH2, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, thio, or thiol, as described herein. The term "biaryl" is a specific type of aryl group and is included in the definition of "aryl". In addition, the aryl group can be a single ring structure, or it can be a fused ring structure, or it can contain multiple ring structures linked through one or more bridging groups, such as carbon-carbon bonds. For example, a biaryl can be two aryl groups linked together through a fused ring structure, such as naphthalene, or linked through one or more carbon-carbon bonds, such as biphenyl.

[0106] The term "aldehyde" as used herein is represented by the formula -C(O)H. Throughout this specification, "C(O)" is an abbreviation for a carbonyl group, i.e., C=O.

[0107] As used herein, the term "amine" or "amino" refers to a group of the formula -NA 1 A 2 In the formula, A 1 and A 2 may independently be hydrogen or an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl or heteroaryl group, as described herein. A specific example of amino is -NH2.

[0108] The term "alkylamino" as used herein is represented by the formula -NH(-alkyl), where alkyl is described herein. Representative examples include, but are not limited to, methylamino, ethylamino, propylamino, isopropylamino, butylamino, isobutylamino, (sec-butyl)amino, (tert-butyl)amino, pentylamino, isopentylamino, (tert-pentyl)amino, hexylamino, and the like.

[0109] The term "dialkylamino" as used herein is represented by the formula -N(-alkyl)2, where alkyl is described herein. Representative examples include, but are not limited to, dimethylamino, diethylamino, dipropylamino, diisopropylamino, dibutylamino, diisobutylamino, di(sec-butyl)amino, di(tert-butyl)amino, dipentylamino, diisopentylamino, di(tert-pentyl)amino, dihexylamino, N-ethyl-N-methylamino, N-methyl-N-propylamino, N-ethyl-N-propylamino, and the like.

[0110] The term "carboxylic acid" as used herein is represented by the formula --C(O)OH.

[0111] As used herein, the term "ester" refers to an ester of the formula -OC(O)A 1 , or -C(O)OA 1 In the formula, A 1 may be an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an alkynyl group, a cycloalkynyl group, an aryl group, or a heteroaryl group, as described herein. As used herein, the term "polyester" refers to a compound of the formula -(A 1 O(O)CA 2 -C(O)O) a - or -(A 1 O(O)CA 2 -OC(O) a In the formula, A 1and A 2 may independently be an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an alkynyl group, a cycloalkynyl group, an aryl group, or a heteroaryl group, as described herein, and "a" is an integer from 1 to 500. "Polyester" is a term used to describe a group produced by the reaction between a compound having at least two carboxylic acid groups and a compound having at least two hydroxyl groups.

[0112] As used herein, the term "ether" refers to a compound of formula A 1 Office Automation 2 In the formula, A 1 and A 2 may be independently an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an alkynyl group, a cycloalkynyl group, an aryl group, or a heteroaryl group as described herein. As used herein, the term "polyether" refers to a group having the formula -(A 1 Office Automation 2 O) a In the formula, A 1 and A 2 can independently be an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an alkynyl group, a cycloalkynyl group, an aryl group, or a heteroaryl group as described herein, and "a" is an integer from 1 to 500. Examples of polyether groups include polyethylene oxide, polypropylene oxide, and polybutylene oxide.

[0113] As used herein, the terms “halo”, “halogen” or “halide” may be used interchangeably and refer to F, Cl, Br or I.

[0114] The term "heteroalkyl" as used herein refers to an alkyl group containing at least one heteroatom. Suitable heteroatoms include, but are not limited to, O, N, Si, P and S, with nitrogen, phosphorus and sulfur atoms being optionally oxidized, and nitrogen heteroatoms being optionally quaternized. Heteroalkyl can be substituted as defined above for alkyl groups.

[0115] The term "heteroaryl" as used herein refers to an aromatic group having at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus, with N-oxides, sulfur oxides, and dioxides being acceptable heteroatom substitutions. Heteroaryl groups can be substituted or unsubstituted. Heteroaryl groups can be substituted with one or more groups, including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, thio, or thiol, as described herein. Heteroaryl groups can be monocyclic or fused ring systems. Heteroaryl groups include, but are not limited to, furyl, imidazolyl, pyrimidinyl, tetrazolyl, thienyl, pyridinyl, pyrrolyl, N-methylpyrrolyl, quinolinyl, isoquinolinyl, pyrazolyl, triazolyl, thiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, isothiazolyl, pyridazinyl, pyrazinyl, benzofuranyl, benzodioxolyl, benzothiophenyl, indolyl, indazolyl, benzimidazolyl, imidazopyridinyl, pyrazolopyridinyl, and pyrazolopyrimidinyl. Further non-limiting examples of heteroaryl groups include, but are not limited to, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, thiophenyl, pyrazolyl, imidazolyl, benzo[d]oxazolyl, benzo[d]thiazolyl, quinolinyl, quinazolinyl, indazolyl, imidazo[1,2-b]pyridazinyl, imidazo[1,2-a]pyrazinyl, benzo[c][1,2,5]thiadiazolyl, benzo[c][1,2,5]oxadiazolyl, and pyrido[2,3-b]pyrazinyl.

[0116] The term "heterocycle" or "heterocyclyl" as used herein can be used interchangeably and refers to monocyclic and polycyclic aromatic or non-aromatic ring systems in which at least one of the ring members is other than carbon. Thus, the term includes, but is not limited to, "heterocycloalkyl", "heteroaryl", "bicyclic heterocycle" and "polycyclic heterocycle". Heterocycles include pyridine, pyrimidine, furan, thiophene, pyrrole, isoxazole, isothiazole, pyrazole, oxazole, thiazole, imidazole, oxazole, such as 1,2,3-oxadiazole, 1,2,5-oxadiazole, and 1,3,4-oxadiazole, thiadiazole, such as 1,2,3-thiadiazole, 1,2,5-thiadiazole, and 1,3,4-thiadiazole, triazole, such as 1,2,3-thiadiazole, 1,2,5-thiadiazole, and 1,3,4-thiadiazole, and ... ,2,3-triazole, 1,3,4-triazole, tetrazoles such as 1,2,3,4-tetrazole and 1,2,4,5-tetrazole, pyridazine, pyrazine, triazines such as 1,2,4-triazine and 1,3,5-triazine, tetrazines such as 1,2,4,5-tetrazine, pyrrolidine, piperidine, piperazine, morpholine, azetidine, tetrahydropyran, tetrahydrofuran, dioxane, and the like. The term heterocyclyl group can also be C2 heterocyclyl, C2-C3 heterocyclyl, C2-C4 heterocyclyl, C2-C5 heterocyclyl, C2-C6 heterocyclyl, C2-C7 heterocyclyl, C2-C8 heterocyclyl, C2-C9 heterocyclyl, C2-C10 heterocyclyl, C2-C11 heterocyclyl, etc. For example, C2 heterocyclyl includes groups having two carbon atoms and at least one heteroatom, including, but not limited to, aziridinyl, diazetidinyl, dihydrodiazetyl, oxiranyl, thiiranyl, etc. Alternatively, for example, C5 heterocyclyl includes groups having five carbon atoms and at least one heteroatom, including, but not limited to, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, diazepanyl, pyridinyl, etc.It is understood that a heterocyclyl group may be attached, if chemically possible, either through a heteroatom within the ring or through one of the carbons comprising the heterocyclyl ring.

[0117] The term "bicyclic heterocycle" or "bicyclic heterocyclyl" as used herein refers to a ring system in which at least one of the ring members is other than carbon. Bicyclic heterocyclyl includes ring systems in which an aromatic ring is fused to another aromatic ring or an aromatic ring is fused to a non-aromatic ring. Bicyclic heterocyclyl includes ring systems in which a benzene ring is fused to a 5- or 6-membered ring containing 1, 2, or 3 ring heteroatoms, or a pyridine ring is fused to a 5- or 6-membered ring containing 1, 2, or 3 ring heteroatoms. Bicyclic heterocyclic groups include, but are not limited to, indolyl, indazolyl, pyrazolo[1,5-a]pyridinyl, benzofuranyl, quinolinyl, quinoxalinyl, 1,3-benzodioxolyl, 2,3-dihydro-1,4-benzodioxinyl, 3,4-dihydro-2H-chromenyl, 1H-pyrazolo[4,3-c]pyridin-3-yl; 1H-pyrrolo[3,2-b]pyridin-3-yl; and 1H-pyrazolo[3,2-b]pyridin-3-yl.

[0118] The term "heterocycloalkyl" as used herein refers to an aliphatic partially unsaturated or fully saturated 3-14 membered ring system, including monocyclic, bicyclic and tricyclic ring systems of 3-8 atoms. Heterocycloalkyl ring systems contain 1-4 heteroatoms independently selected from oxygen, nitrogen and sulfur, where the nitrogen and sulfur heteroatoms can be optionally oxidized and the nitrogen heteroatom can be optionally substituted. Representative heterocycloalkyl groups include, but are not limited to, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl and tetrahydrofuryl.

[0119] The term "hydroxyl" or "hydroxyl" as used herein is represented by the formula --OH.

[0120] As used herein, the term "ketone" refers to a compound of formula A 1 C(O)A 2 In the formula, A 1 and A 2 can independently be an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an alkynyl group, a cycloalkynyl group, an aryl group, or a heteroaryl group, as described herein.

[0121] The term "azide" or "azido" as used herein is represented by the formula --N3.

[0122] The term "nitro" as used herein is represented by the formula --NO.sub.2.

[0123] The term "nitrile" or "cyano" as used herein is represented by the formula --CN.

[0124] As used herein, the term "silyl" refers to a group of the formula -SiA 1 A 2 A 3 In the formula, A 1 , A 2 and A 3 may be independently hydrogen or an alkyl group, a cycloalkyl group, an alkoxy group, an alkenyl group, a cycloalkenyl group, an alkynyl group, a cycloalkynyl group, an aryl group, or a heteroaryl group, as described herein.

[0125] As used herein, the term "sulfo-oxo" refers to a group of the formula -S(O)A 1 , -S(O)2A 1 , -OS(O)2A 1 , or -OS(O)2OA 1 In the formula, A 1may be hydrogen or an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl or heteroaryl group, as described herein. Throughout this specification, "S(O)" is an abbreviation for S=O. The term "sulfonyl" refers to a group of the formula -S(O)A 1 is used herein to refer to a sulfo-oxo group represented by the formula: 1 can be hydrogen or an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an alkynyl group, a cycloalkynyl group, an aryl group, or a heteroaryl group, as described herein. 1 S(O)2A 2 In the formula, A 1 and A 2 may be independently an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an alkynyl group, a cycloalkynyl group, an aryl group, or a heteroaryl group, as described herein. 1 S(O)A 2 In the formula, A 1 and A 2 can independently be an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an alkynyl group, a cycloalkynyl group, an aryl group, or a heteroaryl group, as described herein.

[0126] As used herein, the term "thio" refers to a group of the formula -SA 1 In the formula, A 1 can be an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an alkynyl group, a cycloalkynyl group, an aryl group, or a heteroaryl group, as described herein.

[0127] The term "thiol" as used herein is represented by the formula --SH.

[0128] As used herein, "R 1 ", "R2 ", "R 3 ", "R n " (where n is an integer) can independently have one or more of the groups listed above. For example, R 1 When is a straight chain alkyl group, one of the hydrogen atoms of the alkyl group can be optionally replaced by a hydroxyl group, an alkoxy group, an alkyl group, a halide, etc. Depending on the group selected, the first group can be incorporated within the second group, or the first group can be pendant to the second group (i.e., bonded to the second group). For example, in the phrase "an alkyl group comprising an amino group," the amino group can be incorporated within the backbone of the alkyl group. Alternatively, the amino group can be bonded to the backbone of the alkyl group. The nature of the group selected will determine whether the first group is embedded or bonded to the second group.

[0129] As described herein, the compounds of the present disclosure may contain "optionally substituted" moieties. In general, the term "substituted", whether preceded by the term "optionally", means that one or more hydrogens of the specified moiety are replaced by a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when multiple positions in any given structure may be substituted by multiple substituents selected from a specified group, the substituents may be the same or different at all positions. Combinations of substituents envisioned by the present disclosure are preferably those that result in the formation of stable or chemically feasible compounds. In certain embodiments, it is also contemplated that individual substituents may be further optionally substituted (i.e., may be further substituted or unsubstituted) unless expressly indicated otherwise.

[0130] As used herein, the term "stable" refers to compounds that do not substantially change when subjected to conditions that permit their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.

[0131] The term "leaving group" refers to an atom (or group of atoms) with electron-withdrawing capability that can be displaced as a stable species with the bonding electrons. Examples of suitable leaving groups include, but are not limited to, halides and sulfonate esters, including triflates, mesylates, tosylates, and brosylates.

[0132] The terms "hydrolyzable group" and "hydrolyzable moiety" refer to functional groups that can undergo hydrolysis, for example, under basic or acidic conditions. Examples of hydrolyzable residues include, but are not limited to, acid halides, activated carboxylic acids, and various protecting groups known in the art (see, for example, "Greene's Protective Groups in Organic Synthesis," PGM Wuts, Wiley, 2014).

[0133] The compounds described herein may contain one or more double bonds and therefore may potentially give rise to cis / trans (E / Z) isomers, as well as other conformational isomers. Unless otherwise stated, the compounds described herein include all such possible isomers, as well as mixtures of such isomers.

[0134] Unless otherwise stated, formulas in which chemical bonds are shown only as solid lines and not as wedges or dashed lines contemplate each possible isomer, e.g., each enantiomer and diastereomer, as well as mixtures of isomers, e.g., racemic or scalemic mixtures. Compounds described herein may contain one or more asymmetric centers and thus potentially give rise to diastereomers and optical isomers. Unless otherwise stated, the present disclosure includes all such possible diastereomers, as well as their racemic mixtures, their substantially pure resolved enantiomers, all possible geometric isomers, and their pharma- ceutically acceptable salts. Mixtures of stereoisomers, and isolated specific stereoisomers are also included. During the course of the synthetic procedures used to prepare such compounds, or when using racemization or epimerization procedures known to those skilled in the art, the products of such procedures may be mixtures of stereoisomers.

[0135] Many organic compounds exist in optically active forms that have the ability to rotate the plane of plane-polarized light. In describing optically active compounds, the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule around its chiral center. The prefixes d and l or (+) and (-) are used to indicate the indication of the rotation of plane-polarized light by the compound, with (-) or meaning that the compound is levorotatory. Compounds prefixed with (+) or d are dextrorotatory. For a given chemical structure, these compounds, called stereoisomers, are identical except that they are non-superimposable mirror images of one another. Certain stereoisomers are also called enantiomers, and mixtures of such isomers are often called enantiomeric mixtures. A 50:50 mixture of enantiomers is called a racemic mixture. Many of the compounds described herein can have one or more chiral centers and therefore can exist in various enantiomeric forms. When the bond to a chiral atom is shown as a straight line in the disclosed formula, it is understood that the formula encompasses both the (R) and (S) configurations of the chiral atom, and therefore both enantiomers, and mixtures thereof.As used in the art, when it is desired to specify the absolute configuration for a chiral atom, one of the bonds to the chiral atom can be shown as a wedge (a bond to an atom above the plane), and the other can be shown as a series of short parallel lines or a wedge (a bond to an atom below the plane).The Cahn-Ingold-Prelog system can be used to assign the (R) or (S) configuration to a chiral atom.

[0136] When the disclosed compounds contain one chiral center, the compounds exist in two enantiomeric forms.Unless otherwise specifically stated, the disclosed compounds include both enantiomers and mixtures of enantiomers, for example, a particular 50:50 mixture called racemic mixture.Enantiomers can be resolved by methods known to those skilled in the art, for example, by forming diastereomeric salts that can be separated by crystallization (see CRC Handbook of Optical Resolutions via Diastereomeric Salt Formation by David Kozma (CRC Press, 2001)); by forming diastereomeric derivatives or complexes that can be separated by, for example, crystallization, gas-liquid or liquid chromatography; by selective reaction of one enantiomer with an enantiomer-specific reagent, for example, enzymatic esterification; or by gas-liquid or liquid chromatography in a chiral environment, for example, on a chiral support, for example, silica with a chiral ligand attached, or in the presence of a chiral solvent. It will be appreciated that if the desired enantiomer is converted to another chemical entity by one of the above separation procedures, a further step can liberate the desired enantiomeric form. Alternatively, a particular enantiomer can be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by converting one enantiomer into the other by asymmetric transformation.

[0137] It is understood that the designation of a particular absolute configuration at a chiral atom in a disclosed compound means that the designated enantiomeric form of the compound can be provided in enantiomeric excess (ee). Enantiomeric excess, as used herein, is the presence of a particular enantiomer at greater than 50%, for example, greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 98% or greater than 99%. In one aspect, the designated enantiomer is substantially free of other enantiomers. For example, the "R" form of the compound may be substantially free of the "S" form of the compound, and thus is in enantiomeric excess of the "S" form. Conversely, the "S" form of the compound may be substantially free of the "R" form of the compound, and thus is in enantiomeric excess of the "R" form.

[0138] When a disclosed compound has two or more chiral atoms, it can have more than two optical isomers and can exist in diastereoisomeric forms. For example, when two chiral atoms are present, the compound can have up to four optical isomers and two pairs of enantiomers ((S,S) / (R,R) and (R,S) / (S,R)). Enantiomeric pairs (e.g., (S,S) / (R,R)) are mirror image stereoisomers of each other. Stereoisomers that are not mirror images (e.g., (S,S) and (R,S)) are diastereomers. Diastereoisomeric pairs can be separated by methods known to those skilled in the art, such as chromatography or crystallization, and individual enantiomers within each pair can be separated as described above. Unless otherwise specifically excluded, the disclosed compounds include each diastereoisomer of such compounds and mixtures thereof.

[0139] Compounds according to the present disclosure may form prodrugs at hydroxyl or amino functional groups using groups such as alkoxy, amino acids, etc. as prodrug-forming moieties. For example, hydroxymethyl positions may form monophosphates, diphosphates, or triphosphates, and these phosphates may also form prodrugs. The preparation of such prodrug derivatives is described in various publications (examples include Alexander et al., J.Med.Chem.1988,31,318; Aligas-Martin et al., PCT International Publication No. 2000 / 041531, p.30). The nitrogen functional group that is converted in preparing these derivatives is one (or more) of the nitrogen atoms of the compounds of the present disclosure. The "derivative" of the compound disclosed herein is pharmaceutically acceptable salt, prodrug, deuterated form, radioactively labeled form, isomer, solvate and combinations thereof.In this context, the "combination" refers to a derivative that is included in at least two of the following groups: pharmaceutically acceptable salt, prodrug, deuterated form, radioactively labeled form, isomer and solvate.Examples of radioactively labeled forms include compounds labeled with tritium, phosphorus-32, iodine-129, carbon-11, fluorine-18, etc.

[0140] The compounds described herein include atoms in their natural isotopic abundance and non-natural abundance.The disclosed compounds may be the same isotopically labeled compounds or isotopically substituted compounds as described, but due to the fact that one or more atoms are replaced by atoms with atomic mass or mass number different from the atomic mass or mass number typically found in nature.The examples of isotopes that can be incorporated into the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, for example, 2 H, 3 H, 13 C. 14 C. 15 N, 18 O. 17 O. 35 S, 18 F and 36The compounds further include prodrugs thereof, and pharma- ceutically acceptable salts of the compounds or prodrugs that contain the aforementioned isotopes, and / or other isotopes of other atoms, are within the scope of this disclosure. Certain isotopically labeled compounds of the present disclosure, such as 3 H and 14 Incorporating a radioactive isotope, such as C, is useful in drug and / or substrate tissue distribution assays. 3 H, and carbon-14, i.e. 14 C isotopes are particularly preferred for their ease of preparation and detectability. Additionally, deuterium, i.e., 2 Substitution with heavier isotopes, such as H, can confer certain therapeutic advantages resulting from greater metabolic stability, e.g., increased in vivo half-life, or reduced dosage requirements, and therefore may be preferred in some circumstances. Isotopically labeled compounds of the present disclosure and prodrugs thereof can generally be prepared by carrying out the following procedures by substituting a readily available isotopically labeled reagent for the non-isotopically labeled reagent.

[0141] The compounds described herein can exist as solvates. In some cases, the solvent used to prepare solvates is an aqueous solution, and solvates are often referred to as hydrates. Compounds can exist as hydrates, and hydrates can be obtained, for example, by crystallization from a solvent or aqueous solution. In this regard, one, two, three or any number of solvents or water molecules can be combined with a compound according to the present disclosure to form solvates and hydrates.

[0142] It is also understood that certain compounds described herein may exist in equilibrium between tautomers, for example, a ketone having an α-hydrogen may exist in equilibrium between the keto and enol forms. [ka]

[0143] Similarly, amides with an N-hydrogen can exist in equilibrium between the amide form and the imidic acid form. [ka]

[0144] As another example, pyrazole exists in two tautomeric forms, N 1 unsubstituted, 3-A 3 and N 1 unsubstituted, 5-A 3 It can exist in. [ka]

[0145] Unless otherwise stated, the present disclosure includes all such possible tautomers.

[0146] Chemical substances are known to form solids that exist in different ordered states, called polymorphic forms or modifications. Different modifications of polymorphic substances can have significantly different physical properties. Compounds according to the present disclosure can exist in different polymorphic forms, and certain modifications can be metastable. Unless otherwise stated, the present disclosure includes any such possible polymorphic forms.

[0147] The specific materials, compounds, compositions and components disclosed herein can be obtained commercially or readily synthesized using techniques generally known to those of skill in the art. For example, starting materials and reagents used in preparing the disclosed compounds and compositions are available from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), Acros Organics (Morris Plains, NJ), Strem Chemicals (Newburyport, MA), Fisher Scientific (Pittsburgh, Pa.) or Sigma (St. Louis, Mo.), or may be readily obtained from such sources as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and supplemental volumes (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March's Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition); and Larock's Comprehensive Organic Transformations (VCH Publishers, 1999). They can be prepared by methods known to those skilled in the art following procedures described in references such as (E. G., Inc., 1989).

[0148] Unless expressly stated otherwise, it is never intended that any method described herein be interpreted as requiring that its steps be performed in a particular order. Thus, unless a method claim actually recites the order in which its steps are followed, or unless the claim or description specifically states otherwise that the steps are to be limited to a particular order, no order is intended to be inferred in any respect. This applies to any possible non-express basis for interpretation, including matters of logic regarding the arrangement of steps or operational flow; plain meaning derived from grammatical construction or punctuation; and the number or type of embodiments described herein.

[0149] II. Compounds One class of compounds described herein has formula I: [ka] or any stereoisomer thereof.

[0150] In formula I: [ka] is a single bond or a double bond.

[0151] Also in formula I, R1 is H or CH3.

[0152] Additionally, in formula I, R2 is H and R3 is OCH3 or F, or R2 and R3 are covalently bonded to each other and together with the intermediate atom form a 2'-O,4'-C methylene bridge.

[0153] Further, in formula I, X1 is O or CH.

[0154] Also in formula I, X2 is CH2 or CH.

[0155] Additionally, in formula I, X3 is O or S.

[0156] Further, in formula I, each independent Y is H + or a cation, where n is 0, 1, 2, 3 or 4. Optionally, the cation is a pharma- ceutically acceptable cation (e.g., Na + , K + , Li + or TEAH + ).

[0157] In formula I, when X1 is CH, X2 is CH; [ka] is a double bond.

[0158] As will be understood by those skilled in the art, the structure shown of the compound described herein is representative of one form of the compound.Such compound may be depicted or described in protonated (free acid) form, ionized (anionic) form, or ionized and associated with one or more cations (salt form), but the aqueous solution of such compound exists in equilibrium between such forms.For example, the phosphate bond of the compound described herein in aqueous solution exists in equilibrium between free acid form, anionic form and salt form.

[0159] Optionally, Formula I may be shown in a protonated (free acid) form, as shown below: [ka] , in the formula, [ka] , R1, R2, R3, X1, X2 and X3 are as defined above for formula I.

[0160] Optionally, Formula I may be shown in an ionized (anionic) form, as shown below: [ka] , in the formula, [ka] , R1, R2, R3, X1, X2 and X3 are as defined above for formula I.

[0161] Optionally, Formula I may be depicted in an ionized form and associated with one or more cations (salts), as shown below, where the compound of this structure is associated with a sodium cation (for representational purposes only, and not by way of limitation): [ka] , in the formula, [ka] , R1, R2, R3, X1, X2 and X3 are as defined above for formula I. As will be appreciated by one of skill in the art, cationic salt forms can exist in which one, two, three or four cations are present.

[0162] Unless otherwise indicated, the compounds described herein are intended to include all such forms. Moreover, certain compounds have several such bonds, each of which is in equilibrium. Thus, any compound in solution exists as a collection of forms in multiple positions in equilibrium. A depicted structure necessarily shows a single form. Nevertheless, such drawings are also intended to include the corresponding forms unless otherwise indicated. Herein, structures showing the free acid of a compound followed by the term "or a salt thereof" expressly include all such forms, which may be fully or partially protonated / deprotonated / associated with a cation. In certain cases, one or more specific cations are identified. In certain methods, the compounds disclosed herein are in an aqueous solution containing sodium. In certain methods, the compounds are in an aqueous solution containing potassium. In certain methods, the compounds are in an aqueous solution containing lithium. In certain methods, the compounds are in an aqueous solution containing triethylammonium.

[0163] As noted above, Formula I can include compounds in which R2 and R3 are covalently linked to each other and together with the intermediate atom form a 2'-O,4'-C methylene bridge. The 2'-O,4'-C methylene bridge moiety may also be referred to herein as a linked nucleic acid (LNA) moiety. Optionally, a compound of Formula I having an LNA moiety is represented by the structure IA shown below, or a salt thereof: [ka] .

[0164] In structure IA, R1 and X3 are as defined above for formula I. Examples of structure IA include the following compounds and salts thereof: [ka] .

[0165] As noted above, formula I can include compounds where X1 is CH. In these examples, X2 is also CH, such that X1 and X2 form a vinyl moiety (-CH=CH-). Optionally, compounds of formula I having a vinyl moiety are represented by structure IB, as shown below, or a salt thereof: [ka] .

[0166] In structure IB, R1 is as defined above for formula I. Examples of structure IB include the following compounds and salts thereof: [ka] .

[0167] Additional examples of Formula I include compound 6, shown below, and salts thereof: [ka] .

[0168] One class of compounds described herein has formula II: [ka] or any stereoisomer thereof.

[0169] In formula II, R1 is H or CH3.

[0170] Also in formula II, X1 is O or S.

[0171] Additionally, in formula II, each independent Y is H + or a cation, where n is 0, 1, 2, 3 or 4. Optionally, the cation is a pharma- ceutically acceptable cation (e.g., Na + , K + , Li + or TEAH + ).

[0172] Optionally, in formula II, when X1 is S, R1 is CH3.

[0173] Optionally, Formula II may be shown in a protonated (free acid) form, as shown below: [ka] , in the formula, R1 and X1 are as defined above for formula II.

[0174] Optionally, Formula II may be shown in an ionized (anionic) form, as shown below: [ka] , in the formula, R1 and X1 are as defined above for formula II.

[0175] Optionally, Formula II may be depicted in an ionized form and associated with one or more cations (salts), as shown below, where the compound of this structure is associated with a sodium cation (for representational purposes only, and not by way of limitation): [ka] , in the formula, R1 and X1 are as defined above for formula II.

[0176] Examples of Formula II include the following compounds and salts thereof: [ka] .

[0177] One class of compounds described herein has formula III: [ka] or any stereoisomer thereof.

[0178] In formula III, R1 is H or CH3.

[0179] Additionally, in formula III, each independent Y is H + or a cation, where n is 0, 1, 2, 3 or 4. Optionally, the cation is a pharma- ceutically acceptable cation (e.g., Na + , K + , Li + or TEAH + ).

[0180] Optionally, Formula III may be shown in a protonated (free acid) form, as shown below: [ka] , in the formula, R 1 is as defined above for formula III.

[0181] Optionally, Formula III may be shown in an ionized (anionic) form, as shown below: [ka] , in the formula, R 1 is as defined above for formula III.

[0182] Optionally, formula III may be depicted in an ionized form and associated with one or more cations (salts), as shown below, where the compound of this structure is associated with a sodium cation (for representational purposes only, and not by way of limitation): [ka] , in the formula, R 1 is as defined above for formula III.

[0183] Examples of Formula III include the following compounds and salts thereof: [ka] .

[0184] One class of compounds described herein has formula IV: [ka] or any stereoisomer thereof.

[0185] In formula IV, R1 is H or CH3.

[0186] Also in formula IV, X1, X2, and X3 are each independently selected from O and S, and when one of X1, X2, or X3 is S, the remaining of X1, X2, and X3 are O. Optionally, X1 is S, and X2 and X3 are both O. Optionally, X2 is S, and X1 and X3 are both O. Optionally, X3 is S, and X1 and X2 are both O.

[0187] Additionally, in formula IV, each independent Y is H + or a cation, and n is 0, 1, 2, 3 or 4.

[0188] Optionally, formula IV may be shown in a protonated (free acid) form, as shown below: [ka] , in the formula, R1, X1, X2 and X3 are as defined above for formula IV.

[0189] Optionally, Formula IV may be shown in ionized (anionic) form, as shown below: [ka] , in the formula, R1, X1, X2 and X3 are as defined above for formula IV.

[0190] Optionally, formula IV may be depicted in an ionized form and associated with one or more cations (salts), as shown below, where the compound of this structure is associated with a sodium cation (for representational purposes only, and not by way of limitation): [ka] , in the formula, R1, X1, X2 and X3 are as defined above for formula IV.

[0191] Examples of Formula IV include the following compounds and salts thereof: [ka] [ka] .

[0192] One class of compounds described herein has formula V: [ka] or any stereoisomer thereof.

[0193] In formula V, R1 is H or CH3.

[0194] Also in formula V, R2 is H and R3 is F, or R2 and R3 are covalently bonded to each other and together with the intermediate atom form a 2'-O,4'-C methylene bridge.

[0195] Additionally, in formula V, each independent Y is H + or a cation, where n is 0, 1, 2, 3 or 4. Optionally, the cation is a pharma- ceutically acceptable cation (e.g., Na + , K + , Li + or TEAH + ).

[0196] Further, in formula V, when R2 is H and R3 is F, then R1 is not H.

[0197] Optionally, formula V may be shown in a protonated (free acid) form, as shown below: [ka] , in the formula, R1, R2 and R3 are as defined above for formula V.

[0198] Optionally, Formula V may be shown in an ionized (anionic) form, as shown below: [ka] , in the formula, R1, R2 and R3 are as defined above for formula V.

[0199] Optionally, formula V may be depicted in an ionized form and associated with one or more cations (salts), as shown below, where the compound of this structure is associated with a sodium cation (for representational purposes only, and not by way of limitation): [ka] , in the formula, R1, R2 and R3 are as defined above for formula V.

[0200] As described above, formula V can include compounds in which R2 and R3 are covalently linked to each other and together with the intermediate atom form a 2'-O,4'-C methylene bridge (also referred to herein as a linked nucleic acid (LNA) moiety). Optionally, a compound of formula V having an LNA moiety is represented by the structure VA shown below, or a salt thereof: [ka] .

[0201] In structure VA, R1 is as defined above for formula V. Examples of structure VA include the following compounds and salts thereof: [ka] .

[0202] Additional examples of formula V include compound 17, shown below, and salts thereof: [ka] .

[0203] One class of compounds described herein has formula VI: [ka] or any stereoisomer thereof.

[0204] In formula VI, R1 is H or CH3.

[0205] Also in Formula VI, X1, X2, X3, and X4 are each independently selected from O and S.

[0206] Additionally, in formula VI, each independent Y is H + or a cation, where n is 0, 1, 2, 3 or 4. Optionally, the cation is a pharma- ceutically acceptable cation (e.g., Na + , K + , Li + or TEAH + ).

[0207] In formula VI, one of X1, X2, X3 and X4 is S.

[0208] Optionally, formula VI may be shown in a protonated (free acid) form, as shown below: [ka] , in the formula, R1, X1, X2, X3 and X4 are as defined above for formula VI.

[0209] Optionally, Formula VI may be shown in ionized (anionic) form, as shown below: [ka] , in the formula, R1, X1, X2, X3 and X4 are as defined above for formula VI.

[0210] Optionally, formula VI may be depicted in an ionized form and associated with one or more cations (salts), as shown below, where the compound of this structure is associated with a sodium cation (for representational purposes only, and not by way of limitation): [ka] , in the formula, R1, X1, X2, X3 and X4 are as defined above for formula VI.

[0211] Examples of Formula VI include the following compounds and salts thereof: [ka] [ka] .

[0212] One class of compounds described herein has formula VII: [ka] or any stereoisomer thereof.

[0213] In formula VII, R1 is H or CH3.

[0214] Also in formula VII, R2 is H or CH3.

[0215] In formula VII, at least one of R1 or R2 is CH3. In other words, R1 and R2 are not H at the same time.

[0216] Additionally, in formula VII, each independent Y is H + or a cation, where n is 0, 1, 2, 3 or 4. Optionally, the cation is a pharma- ceutically acceptable cation (e.g., Na + , K + , Li + or TEAH + ).

[0217] Optionally, Formula VII may be shown in a protonated (free acid) form, as shown below: [ka] , in the formula, R1 and R2 are as defined above for formula VII.

[0218] Optionally, Formula VII may be shown in ionized (anionic) form, as shown below: [ka] , in the formula, R1 and R2 are as defined above for formula VII.

[0219] Optionally, formula VII may be depicted in an ionized form and associated with one or more cations (salts), as shown below, where the compound of this structure is associated with a sodium cation (for representational purposes only, and not by way of limitation): [ka] , in the formula, R1 and R2 are as defined above for formula VII.

[0220] Examples of Formula VII include the following compounds and salts thereof: [ka] .

[0221] One class of compounds described herein has formula VIII: [ka] or any stereoisomer thereof.

[0222] In formula VIII, R1 and R2 are each independently selected from H and CH3.

[0223] Also in formula VIII, each independent Y is H + or a cation, where n is 0, 1, 2, 3 or 4. Optionally, the cation is a pharma- ceutically acceptable cation (e.g., Na + , K + , Li + or TEAH + ).

[0224] Optionally, Formula VIII may be shown in a protonated (free acid) form, as shown below: [ka] , in the formula, R1 and R2 are as defined above for formula VIII.

[0225] Optionally, Formula VIII may be shown in ionized (anionic) form, as shown below: [ka] , in the formula, R1 and R2 are as defined above for formula VIII.

[0226] Optionally, formula VIII may be depicted in an ionized form and associated with one or more cations (salts), as shown below, where the compound of this structure is associated with a sodium cation (for representational purposes only, and not by way of limitation): [ka] , in the formula, R1 and R2 are as defined above for formula VIII.

[0227] Examples of Formula VIII include the following compounds and salts thereof: [ka] [ka] .

[0228] Other compounds described herein for use in certain methods include the following compounds or any stereoisomers thereof: [ka] [ka] .

[0229] Optionally, the compound for use in a particular method is not a compound according to Compound 29, Compound 30, Compound 31, Compound 32 and / or Compound 33.

[0230] One class of compounds described herein has formula IX: [ka] or any stereoisomer thereof.

[0231] In formula IX, R1 is H or CH3.

[0232] Also in formula IX, R2 is OH, F, substituted or unsubstituted alkoxy, or thio. Optionally, the substituted or unsubstituted alkoxy group can be methoxy, methoxyethoxy, or azidomethoxy.

[0233] Also, in formula IX, R3 is H or CH3.

[0234] In formula IX, when R2 is OH, R1 is not H.

[0235] Also in formula IX, each independent Y is H + or a cation, where n is 0, 1, 2, 3 or 4. Optionally, the cation is a pharma- ceutically acceptable cation (e.g., Na + , K + , Li + or TEAH + ).

[0236] Optionally, formula IX may be shown in a protonated (free acid) form, as shown below: [ka] , in the formula, R1, R2 and R3 are as defined above for formula IX.

[0237] Optionally, Formula IX may be shown in ionized (anionic) form, as shown below: [ka] , in the formula, R1, R2 and R3 are as defined above for formula IX.

[0238] Optionally, formula IX may be depicted in an ionized form and associated with one or more cations (salts), as shown below, where the compound of this structure is associated with a sodium cation (for representational purposes only, and not by way of limitation): [ka] , in the formula, R1, R2 and R3 are as defined above for formula IX.

[0239] Examples of Formula IX include the following compounds and salts thereof: [ka] [ka] [ka] .

[0240] One class of compounds described herein has the formula X: [ka] or any stereoisomer thereof.

[0241] In formula X, R1 is H or CH3.

[0242] Additionally, in formula X, each independent Y is H + or a cation, and n is 0, 1, 2, 3 or 4.

[0243] Optionally, formula X may be shown in a protonated (free acid) form, as shown below: [ka] , in the formula, R1 is as defined above for formula X.

[0244] Optionally, formula X may be shown in ionized (anionic) form, as shown below: [ka] , in the formula, R1 is as defined above for formula X.

[0245] Optionally, formula X may be depicted in an ionized form and associated with one or more cations (salts), as shown below, where the compound of this structure is associated with a sodium cation (for representational purposes only, and not by way of limitation): [ka] , in the formula, R1 is as defined above for formula X.

[0246] Examples of Formula X include the following compounds and salts thereof: [ka] .

[0247] One class of compounds described herein is represented by formula XI: [ka] .

[0248] In formula XI, R1 is H or CH3.

[0249] Also, in formula XI, each independent Y is H+ or a cation, where n is 0, 1, 2, 3 or 4. Optionally, the cation is a pharma- ceutically acceptable cation (e.g., Na + , K + , Li + or TEAH + ).

[0250] Optionally, formula XI may be shown in a protonated (free acid) form, as shown below: [ka] , in the formula, R1 is as defined above for formula XI.

[0251] Optionally, formula XI may be shown in ionized (anionic) form, as shown below: [ka] , in the formula, R1 is as defined above for formula XI.

[0252] Optionally, formula XI may be depicted in an ionized form and associated with one or more cations (salts), as shown below, where the compound of the structure is associated with a sodium cation (for representational purposes only, and not by way of limitation): [ka] , in the formula, R1 is as defined above for formula XI.

[0253] Examples of Formula XI include the following compounds and salts thereof: [ka] .

[0254] One class of compounds described herein is represented by Formula XII: [ka] .

[0255] In formula XII, R1 and R2 are each independently selected from H and CH3.

[0256] Also, in formula XII, each independent Y is H + or a cation, where n is 0, 1, 2, 3, 4 or 5. Optionally, the cation is a pharma- ceutically acceptable cation (e.g., Na + , K + , Li + or TEAH + ).

[0257] Optionally, formula XII may be shown in a protonated (free acid) form, as shown below: [ka] , in the formula, R1 and R2 are as defined above for formula XII.

[0258] Optionally, Formula XII may be shown in an ionized (anionic) form, as shown below: [ka] , in the formula, R1 and R2 are as defined above for formula XII.

[0259] Optionally, formula XII may be depicted in an ionized form and associated with one or more cations (salts), as shown below, where the compound of this structure is associated with a sodium cation (for representational purposes only, and not by way of limitation): [ka] , in the formula, R1 and R2 are as defined above for formula XII.

[0260] Examples of Formula XII include the following compounds and salts thereof: [ka] .

[0261] In some examples, the compounds described herein include deuterated forms of the compounds in which at least one (e.g., one or more, two or more, three or more, four or more, or five or more) hydrogen atom has been replaced with deuterium. Exemplary deuterated forms include, for example, the following compounds, which are provided as representative examples: [ka] .

[0262] III. Methods for preparing compounds The compounds described herein can be prepared in a variety of ways. The compounds can be synthesized using a variety of synthetic methods. At least some of these methods are known in the field of synthetic organic chemistry. The compounds described herein can be prepared from readily available starting materials. Although optimal reaction conditions may vary depending on the specific reactants or solvents used, such conditions can be determined by those skilled in the art by routine optimization procedures.

[0263] The variations of formula I, formula II, formula III, formula IV, formula V, formula VI, formula VII, formula VIII, formula IX, formula X, formula XI and formula XII include the addition, subtraction or movement of various components as described for each compound. Similarly, when one or more chiral centers are present in a molecule, all possible chiral variants are included. Furthermore, compound synthesis may involve the protection and deprotection of various chemical groups. The use of protection and deprotection, as well as the selection of suitable protecting groups, can be determined by those skilled in the art. The chemistry of protecting groups can be found, for example, in Wuts, Greene's Protective Groups in Organic Synthesis, 5th.Ed., Wiley&Sons, 2014, which is incorporated herein by reference in its entirety.

[0264] The reactions producing the compounds described herein may be carried out in a solvent that may be selected by one skilled in the art of organic synthesis. The solvent may be substantially non-reactive with the starting materials (reactants), intermediates, or products under the conditions, i.e., temperature and pressure, at which the reaction is carried out. The reaction may be carried out in one solvent, or a mixture of multiple solvents. The formation of the product or intermediate may be monitored according to any suitable method known in the art. For example, the formation of the product may be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C) It may be monitored by infrared spectroscopy, spectrophotometry (eg, UV-visible) or mass spectrometry, or by chromatography, for example, high performance liquid chromatography (HPLC) or thin layer chromatography.

[0265] The compounds described herein may be prepared by a two-step process that includes an activation step followed by a coupling step. In the activation step, modified N7-methyl-guanosine-5'-diphosphate, where R1 is H or CH3, (optionally with a triethylammonium salt (TEAH) + ) (N7-Me-GDP) compound is dissolved in a solvent mixture to achieve the desired concentration. An activating reagent such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl salt) is then added to form a reactive intermediate in situ, which is then reacted with imidazole. After a period of time, the activated intermediate is formed and prepared for use in the coupling step. See Scheme A below.

[0266] Scheme A: Activation [ka]

[0267] In the coupling step, the activated intermediate is reacted with a desired dinucleotide (wherein R2 is H and R3 is OCH3 or F, or R2 and R3 are covalently bonded to each other and together with the intermediate atom form a 2'-O,4'-C methylene bridge) to couple to the activated intermediate, and the mixture is allowed to react for a period of time. The final product is then obtained as shown in Scheme B below.

[0268] Scheme B: Coupling [ka]

[0269] Schemes A and B shown above are provided for representative purposes only, and one of skill in the art will understand that, with modifications within the understanding of one of skill in the art, together with the disclosure in the Examples section, these schemes can be applied to any of the formulas described herein (i.e., Formula I, Formula II, Formula III, Formula IV, Formula V, Formula VI, Formula VII, Formula VIII, Formula IX, Formula X, Formula XI, and Formula XII). By way of example, Scheme B reflects a dinucleotide containing adenosine and guanosine (pApG dinucleotide), although the methods described herein can be used to convert other dinucleotides (e.g., pApG dinucleotide). m6 ApG dinucleotide, pp m6 The method can be applied to ApG dinucleotides, ppApG dinucleotides, pDAP (diaminopurine) pG dinucleotides, pApA dinucleotides, pApU dinucleotides, and others described herein. Similarly, although the two-step method shown above shows phosphoro linkages, the two-step method can be applied to phosphorothioate linkages according to Formula I, Formula II, Formula IV, and Formula VI using appropriate reagents.

[0270] The synthetic methods described herein can be carried out as one-pot synthesis, such that all steps are carried out in a single reactor without isolating intermediate products during the course of the synthetic method. In some examples, the method of synthesizing a trinucleotide compound comprises reacting N7-methyl-guanosine-5'-diphosphate of the following structure: [ka] wherein X1 and X2 are each independently selected from the group consisting of O and S; X3 is O; R1 and R2 are each independently selected from H, OH, N3, F, substituted or unsubstituted alkoxy, and thio, and R1 and R2 are optionally joined to form a heterocycle) with an activating reagent and imidazole to form an activated intermediate; and a salt reagent (e.g., a chloride salt such as MgCl2 or ZnCl2) with a dinucleotide of the structure: [ka] , (in the formula, [ka] is a single or double bond; X4 and X6 are each independently selected from the group consisting of O and S; X5 is O, S or CH; X7 is CH or CH2; R3 is OH, OMe, F and R4 is H, or R3 and R4 are covalently bonded to each other and together with the intermediate atom form a 2'-O,4'-C methylene bridge; B1 and B2 are each independently selected from the group consisting of a purine ring and a pyrimidine ring) to the activated intermediate to form a trinucleotide compound of the following structure: [ka] The method is a one-pot synthesis.

[0271] In another embodiment, the method of synthesizing a trinucleotide compound includes synthesizing a dinucleotide having the structure: [ka] , (in the formula, [ka] is a single or double bond; X4 is O; X6 is O or S; X5 is O, S or CH; X7 is CH or CH2; R3 is OH, OMe, F and R4 is H, or R3 and R4 are covalently bonded to each other and together with the intermediate atom form a 2'-O,4'-C methylene bridge; B1 and B2 are each independently selected from the group consisting of a purine ring and a pyrimidine ring) with an activating reagent and imidazole to form an activated phosphate imidazolide of the following structure: [ka] and A salt reagent (e.g., a chloride salt such as magnesium chloride or zinc chloride) and a compound of the structure: [ka] , (in the formula, X1 and X2 are each independently selected from the group consisting of O and S; X3 is S; R1 and R2 are each independently selected from H, OH, N3, F, substituted or unsubstituted alkoxy, and thio, and R1 and R2 are optionally joined to form a heterocycle) to an activated phosphate imidazolide to form a trinucleotide compound of the structure: [ka] .

[0272] In yet another embodiment, the method of synthesizing a trinucleotide compound comprises synthesizing a dinucleotide diphosphate having the following structure: [ka] , (in the formula, [ka] is a single or double bond; X3 is O; X4 and X6 are each independently selected from O and S; X5 is O, S or CH; X7 is CH or CH2; R3 is OH, OMe, F and R4 is H, or R3 and R4 are covalently bonded to each other and together with the intermediate atom form a 2'-O,4'-C methylene bridge; B1 and B2 are each independently selected from the group consisting of a purine ring and a pyrimidine ring) with an activating reagent and imidazole to form an activated phosphate imidazolide of the structure: [ka] and A salt reagent (e.g., a chloride salt such as magnesium chloride or zinc chloride) and a compound of the structure: [ka] , (in the formula, X1 and X2 are each independently selected from O and S; R1 and R2 are each independently selected from H, OH, N3, F, substituted or unsubstituted alkoxy, and thio, and R1 and R2 are optionally joined to form a heterocycle) to an activated phosphate imidazolide to form a trinucleotide compound of the structure: [ka] The method is a one-pot synthesis.

[0273] Exemplary procedures for synthesizing the compounds described herein are provided in Example 1 below.

[0274] IV. Pharmaceutical Preparations The compounds described herein or derivatives thereof can be provided in pharmaceutical compositions.Depending on the intended mode of administration, the pharmaceutical compositions can be in solid, semi-solid or liquid dosage forms, such as tablets, suppositories, pills, capsules, powders, liquids or suspensions, preferably in unit dosage forms suitable for single administration of precise dosages.The compositions comprise a therapeutically effective amount of the compounds described herein or derivatives thereof in combination with a pharma-ceutically acceptable carrier, and may additionally comprise other medicinal agents, pharmaceuticals, carriers or diluents.Pharmaceutically acceptable means a material that is not biologically or otherwise undesirable, and that can be administered to an individual together with the selected compound without causing unacceptable biological effects or interacting in a deleterious manner with other components of the pharmaceutical composition in which it is contained.

[0275] As used herein, the term carrier includes any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material known in the art for use in pharmaceutical preparations.The selection of carrier for use in composition depends on the intended route of administration of the composition.The preparation of pharmaceutical acceptable carriers and formulations containing these materials is described, for example, in Remington: The Science and Practice of Pharmacy, 22d Edition, Loyd et al.eds., Pharmaceutical Press and Philadelphia College of Pharmacy at University of the Sciences (2012). Examples of physiologically acceptable carriers include buffers, such as phosphate buffers, citrate buffers, and buffers including other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and / or non-ionic surfactants, such as TWEEN® (ICI, Inc.; Bridgewater, New Jersey), polyethylene glycol (PEG), and PLURONICS™ (BASF; Florham Park, NJ).

[0276] The composition containing the compound or its derivative described herein suitable for parenteral injection can include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable or dispersion solutions.Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, etc.), suitable mixtures thereof, vegetable oils (olive oil, etc.), and injectable organic esters such as ethyl oleate.Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by using surfactants.

[0277] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers and dispensing agents. Prevention of microbial action can be promoted by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Isotonic agents, such as sugars, sodium chloride, etc., can also be included. Prolonged absorption of injectable pharmaceutical forms can be brought about by using agents that delay absorption, such as aluminum monostearate and gelatin.

[0278] The solid dosage form for oral administration of the compound described herein or its derivatives includes capsules, tablets, pills, powders and granules.In such solid dosage forms, the compound described herein or its derivatives is mixed with at least one inert conventional excipient (or carrier), such as sodium citrate or dicalcium phosphate, or (a) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol and silicic acid, (b) binders, such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia, (c) humectants, such as glycerol, (d) disintegrants, such as agar, calcium carbonate. , potato starch or tapioca starch, alginic acid, certain complex silicates and sodium carbonate, (e) solution retarders such as paraffin, (f) absorption accelerators such as quaternary ammonium compounds, (g) wetting agents such as cetyl alcohol and glycerol monostearate, (h) adsorbents such as kaolin and bentonite, and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also include buffering agents.

[0279] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols.

[0280] Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and other coatings known in the art.They can contain opacifiers and can be of a composition that releases active compound or compounds in a delayed manner in a certain part of the intestinal tract.Examples of embedding compositions that can be used include polymeric substances and waxes.Active compound can also be in microencapsulated form, if necessary, with one or more of the above-mentioned excipients.

[0281] The liquid dosage form for oral administration of the compound described herein or its derivatives includes pharmaceutically acceptable emulsion, solution, suspension, syrup and elixir.In addition to active compound, liquid dosage form can contain inert diluent commonly used in the art, such as water or other solvent, solubilizer and emulsifier, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oil, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid ester of sorbitan, or mixture of these substances.

[0282] Besides such inert diluents, compositions can also include additional agents, such as wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, or perfuming agents.

[0283] Suspensions may contain additional agents in addition to the active compound, such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances.

[0284] Compositions of the compounds described herein or derivatives thereof for rectal administration are optionally suppositories, which can be prepared by mixing the compound with a suitable non-irritating excipient or carrier such as cocoa butter, polyethylene glycol or a suppository wax which is solid at ordinary temperatures but liquid at body temperature and thus will melt in the rectum or vaginal cavity and release the active ingredient.

[0285] The dosage form for topical administration of the compound or its derivatives described herein includes ointment, powder, spray, inhalant and skin patch.The compound or its derivatives described herein is mixed with physiologically acceptable carrier and any preservative, buffer or propellant as required under sterile conditions.Ophthalmic preparations, ointments, powders and solutions are also considered to be within the scope of the composition.

[0286] Optionally, the compounds described herein may be contained in a drug depot. The drug depot comprises a physical structure to facilitate implantation and retention at a desired site (e.g., a patient's synovial joint, intervertebral disc space, spinal canal, abdominal region, tissue, etc.). The drug depot may provide an optimal concentration gradient of the compound at a distance of up to about 0.1 cm to about 5 cm from the implant site. As used herein, depots include, but are not limited to, capsules, microspheres, microparticles, microcapsules, microfiber particles, nanospheres, nanoparticles, coatings, matrices, wafers, pills, pellets, emulsions, liposomes, micelles, gels, antibody-compound conjugates, protein-compound conjugates, or other pharmaceutical delivery compositions. Materials suitable for the depot include pharma- ceutically acceptable biodegradable materials, preferably approved by the FDA, or GRAS materials. These materials may be polymeric or non-polymeric, and synthetic or naturally occurring, or a combination thereof. The depot may optionally include a drug pump.

[0287] The composition may include one or more of the compounds described herein and a pharma- ceutically acceptable carrier. As used herein, the term pharma- ceutically acceptable salt refers to salts of the compounds described herein or derivatives thereof, and, where possible, zwitterionic forms of the compounds described herein, that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio, and are effective for their intended use. The term salt refers to the relatively non-toxic inorganic and organic acid addition salts of the compounds described herein. These salts may be prepared in situ during the isolation and purification of the compounds, or by separately reacting the purified compounds in free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate salts mesylate, glucoheptonate, lactobionate, methanesulfonate and laurylsulfonate salts, etc. These may include cations based on alkali and alkaline earth metals such as sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium cations, quaternary ammonium cations and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. (See S. M. Barge et al., J. Pharm. Sci. (1977) 66, 1, which is incorporated by reference in its entirety, at least for the compositions taught therein.)

[0288] Administration of the compounds and compositions described herein or their pharma- ceutically acceptable salts can be performed using a therapeutically effective amount of the compounds and compositions described herein or their pharma- ceutically acceptable salts described herein for a period of time effective to treat the disorder. The effective amount of the compounds and compositions described herein or their pharma- ceutically acceptable salts described herein can be determined by one skilled in the art.

[0289] Those skilled in the art will understand that the specific dose level and frequency of administration for any particular subject may vary and will depend on a variety of factors, including the activity of the particular compound used, the metabolic stability and length of action of that compound, the subject's species, age, weight, general health, sex and diet, mode and time of administration, rate of excretion, drug combination, and the severity of the particular condition.

[0290] The exact dosage used in the formulation also depends on the route of administration and the severity of the disease or disorder, and should be determined according to the judgment of the practitioner and the circumstances of each subject.Effective dosage can be extrapolated from the dose-response curve obtained from in vitro or animal model test system.In addition, depending on the route of administration, a person skilled in the art will know how to determine the dosage that will bring about the plasma concentration for the desired level of response in the cells, tissues and / or organs of the subject.

[0291] V.RNA synthesis Provided herein is an in vitro method for synthesizing capped RNA transcripts, including capped messenger RNA (mRNA) transcripts. The method described herein includes (a) forming a reaction mixture including a compound described herein (also referred to herein as a cap analog), a DNA template, and an RNA polymerase, and (b) incubating the reaction mixture under conditions that allow transcription of the DNA template to produce a capped mRNA transcript. In the method described herein, the reaction mixture includes NTPs, including ATP, CTP, GTP, and UTP. One or more of the NTPs in the in vitro transcription reaction mixture can be modified NTPs. Exemplary nucleosides with modified bases include, but are not limited to, inosine, 7-deazaguanosine, 7-methylguanosine, dihydrouridine, 2'-O-methylguanosine, 2'-fluoro-2'-deoxycytidine, pseudouridine, N1-methylpseudouridine, and 5-methyluridine. In some methods, one or more uridines in the in vitro transcribed RNA are replaced with a modified nucleoside. Optionally, some methods include adding a reaction mixture containing the capped mRNA transcripts to a Ca 2+ and incubating with a DNase I buffer containing DNase I.

[0292] Optionally, some methods further include subjecting the DNase-treated reaction mixture to elimination of proteins from the in vitro transcription reaction. Optionally, some methods further include subjecting the DNase-treated reaction mixture to phosphatase treatment. Optionally, the method further includes subjecting the DNase-treated reaction mixture to one or more purification steps. The mRNA transcripts produced by the methods described herein can be purified using one or more purification techniques known to those of skill in the art. See Baronti et al. "A guide to large-scale RNA sample preparation," Anal. Bioanal. Chem. 410(14):3239-33252 (2018). For example, the mRNA can be purified by liquid chromatography (e.g., HPLC, reversed-phase ion-pair HPLC (RP-IP-HPLC), anion exchange chromatography, cation exchange chromatography, affinity chromatography, size exclusion chromatography), precipitation, diafiltration, tangential flow filtration, oligo dT chromatography, silica membrane purification, and hydrophobic interaction chromatography, to name a few. The synthesized capped mRNA transcripts can be substantially free of impurities such as DNA, protein, double-stranded RNA and / or incomplete mRNA transcripts.

[0293] Also described herein is the resulting RNA molecule comprising a 5' cap, wherein the 5' cap comprises a compound described herein. Optionally, the RNA molecule is a messenger RNA (mRNA) molecule. Optionally, the RNA molecule is a self-amplifying RNA (saRNA) molecule.

[0294] Also provided herein is a method of inducing a therapeutic effect in a subject, comprising administering to a subject the RNA molecule described herein.The administered RNA can contain a certain amount of immunogenic double-stranded RNA (dsRNA).The amount of dsRNA is calculated for each ug of administered RNA.

[0295] Any of the 5' cap analogs described herein are useful for optimized in vivo translation of mRNA, as further detailed herein.

[0296] Further provided herein is a method of administering to an animal a dose of an mRNA molecule comprising a 5' cap as described herein. Optionally, the 5' cap comprises a compound selected from the group consisting of: [ka] [ka] .

[0297] Optionally, the mRNA dose comprises less than 7ng double-stranded RNA (dsRNA) per μg of mRNA, and the subject shows increased tolerance to the administered mRNA dose compared to an equivalent dose of mRNA comprising 7ng / μg or more of dsRNA.Optionally, the increased tolerance is determined by measuring one or more of body weight, organ weight, aspartate aminotransferase (AST) level, alanine transaminase (ALT) level, C-reactive protein (CRP) level, procalcitonin (PCT) level, interleukin-6 (IL-6) level, erythrocyte sedimentation rate (ESR), serum amyloid A level and serum ferritin level before administration and for a period of time after administration.

[0298] In some cases, the increased tolerance is measured by testing in a standard in vivo assay. As mentioned above, the term "in vivo assay" refers to a method used to detect and / or measure the ability of one or more of a compound or molecule, including a compound (e.g., an mRNA molecule in a therapeutic dose) to increase or decrease a characteristic compared to a control (e.g., a biomarker level). Optionally, the in vivo assay described herein can be used to determine a subject's tolerance level to a given compound or molecule. Exemplary measurements for evaluating tolerance include one or more of body weight, organ weight, aspartate aminotransferase (AST) level, alanine transaminase (ALT) level, C-reactive protein (CRP) level, procalcitonin (PCT) level, interleukin-6 (IL-6) level, erythrocyte sedimentation rate (ESR), serum amyloid A level, and serum ferritin level. Optionally, the in vivo assay may be characterized herein as a "standard in vivo assay" and may optionally be performed using the conditions outlined in Examples 2 and 3 of the present application.

[0299] In some cases, the increased tolerability is at least a 20% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least a 25% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least a 30% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least a 35% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least a 40% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least a 45% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least a 50% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least a 55% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay. Increased tolerability, at least a 60% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least a 65% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least a 70% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least a 75% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least an 80% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least an 85% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least a 90% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, or at least a 95% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay.

[0300] Optionally, the therapeutic dosage unit of the mRNA molecule is less than 6.9 ng / μg double stranded RNA (dsRNA), less than 6.8 ng / μg double stranded RNA (dsRNA), less than 6.7 ng / μg double stranded RNA (dsRNA), less than 6.6 ng / μg double stranded RNA (dsRNA), less than 6.5 ng / μg double stranded RNA (dsRNA), less than 6.4 ng / μg double stranded RNA (dsRNA), less than 6.3 ng / μg double stranded RNA (dsRNA), less than 6.2 ng / μg double stranded RNA (dsRNA), less than 6.1 ng / μg double stranded RNA (dsRNA), less than 6.0 ... / μg double stranded RNA (dsRNA), less than 5.9ng / μg double stranded RNA (dsRNA), less than 5.8ng / μg double stranded RNA (dsRNA), less than 5.7ng / μg double stranded RNA (dsRNA), less than 5.6ng / μg double stranded RNA (dsRNA), less than 5.5ng / μg double stranded RNA (dsRNA), less than 5.4ng / μg double stranded RNA (dsRNA), less than 5.3ng / μg double stranded RNA (dsRNA), less than 5.2ng / μg double stranded RNA (dsRNA), less than 5.1ng / μg double stranded RNA (dsRNA), 5.0ng less than 4.9ng / μg double stranded RNA (dsRNA), less than 4.8ng / μg double stranded RNA (dsRNA), less than 4.7ng / μg double stranded RNA (dsRNA), less than 4.6ng / μg double stranded RNA (dsRNA), less than 4.5ng / μg double stranded RNA (dsRNA), less than 4.4ng / μg double stranded RNA (dsRNA), less than 4.3ng / μg double stranded RNA (dsRNA), less than 4.2ng / μg double stranded RNA (dsRNA), less than 4.1ng / μg double stranded RNA (dsRNA), 4.0 less than 3.9ng / μg double stranded RNA (dsRNA), less than 3.8ng / μg double stranded RNA (dsRNA), less than 3.7ng / μg double stranded RNA (dsRNA), less than 3.6ng / μg double stranded RNA (dsRNA), less than 3.5ng / μg double stranded RNA (dsRNA), less than 3.4ng / μg double stranded RNA (dsRNA), less than 3.3ng / μg double stranded RNA (dsRNA), less than 3.2ng / μg double stranded RNA (dsRNA), less than 3.1ng / μg double stranded RNA (dsRNA), 3.Less than 0ng / μg double-stranded RNA (dsRNA), less than 2.9ng / μg double-stranded RNA (dsRNA), less than 2.8ng / μg double-stranded RNA (dsRNA), less than 2.7ng / μg double-stranded RNA (dsRNA), less than 2.6ng / μg double-stranded RNA (dsRNA), less than 2.5ng / μg double-stranded RNA (dsRNA), less than 2.4ng / μg double-stranded RNA (dsRNA), less than 2.3ng / μg double-stranded RNA (dsRNA) NA (dsRNA), double-stranded RNA (dsRNA) less than 2.2ng / μg, double-stranded RNA (dsRNA) less than 2.1ng / μg, double-stranded RNA (dsRNA) less than 2.0ng / μg, double-stranded RNA (dsRNA) less than 1.9ng / μg, double-stranded RNA (dsRNA) less than 1.8ng / μg, double-stranded RNA (dsRNA) less than 1.7ng / μg, double-stranded RNA (dsRNA) less than 1.6ng / μg, 1.5ng Less than 1.2ng / μg double stranded RNA (dsRNA), less than 1.1ng / μg double stranded RNA (dsRNA), less than 1.0ng / μg double stranded RNA (dsRNA), less than 0.9ng / μg double stranded RNA (dsRNA), less than 0.8ng / μg double stranded RNA (dsRNA), less than 0.7 ng / μg double stranded RNA (dsRNA), less than 0.6 ng / μg double stranded RNA (dsRNA), less than 0.5 ng / μg double stranded RNA (dsRNA), less than 0.4 ng / μg double stranded RNA (dsRNA), less than 0.3 ng / μg double stranded RNA (dsRNA), less than 0.2 ng / μg double stranded RNA (dsRNA), or less than 0.1 ng / μg double stranded RNA (dsRNA).

[0301] Also described herein is a method for increasing in vivo translation of a polypeptide in a subject. The method includes administering to the subject a therapeutic dose unit that includes an effective amount of an mRNA encoding the polypeptide, the mRNA including a 5' cap having a formula according to any of the compounds described herein.

[0302] In some examples, the method includes administering to the subject a therapeutic dosage unit comprising an effective amount of an mRNA encoding a polypeptide, the mRNA having the formula: m7 G 3’OMe pppA 1 2’OMe G (where A 1 adenosine, N6-methyladenosine, N6,N6-dimethyladenosine). 1 For use in a method for increasing translation of a polypeptide in a subject, m7 G 3’OMe pppA 1 2’OMe The G compound may include one or more of the following compounds: [ka] .

[0303] According to the methods described herein, the therapeutic dosage unit administered is at least 20% lower than the therapeutic dosage unit required to induce the same response in a subject when a comparison mRNA encoding the polypeptide is administered, the comparison mRNA having the formula m7 GpppA 1 2’OMe G (where A 1 adenosine, N6-methyladenosine, 6,6-dimethyladenosine). 1 can optionally contain an LNA portion. m7 GpppA 1 2’OMe The G compound may include one or more of the following compounds: [ka] [ka] .

[0304] Comparison of mRNA m7 GpppA 12’OMe G compound is compared with mRNA m7 The compound was administered, except that it lacked a 3'OMe moiety on the G nucleotide. m7 G 3’OMe pppA 1 2’OMe It may be selected to be identical to G.

[0305] The therapeutic dosage unit administered may be at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower, or at least 95% lower than the therapeutic dosage unit required to induce the same response in a subject when a comparative mRNA encoding the polypeptide outlined above is administered.

[0306] The subject may demonstrate increased tolerance to the administered therapeutic dose unit as compared to the therapeutic dose unit required to elicit the same response in the subject when a comparative mRNA is administered, as measured by testing in standard in vivo assays described above, for example, by using the conditions described in the Examples herein.

[0307] Optionally, the increased tolerability is at least a 20% increase in tolerability for the therapeutic dose unit administered when measured by testing in a standard in vivo assay, at least a 25% increase in tolerability for the therapeutic dose unit administered when measured by testing in a standard in vivo assay, at least a 30% increase in tolerability for the therapeutic dose unit administered when measured by testing in a standard in vivo assay, at least a 35% increase in tolerability for the therapeutic dose unit administered when measured by testing in a standard in vivo assay, at least a 40% increase in tolerability for the therapeutic dose unit administered when measured by testing in a standard in vivo assay, at least a 45% increase in tolerability for the therapeutic dose unit administered when measured by testing in a standard in vivo assay, at least a 50 ... at least a 55% increase in tolerability to the therapeutic dosage unit administered when measured by testing in a standard in vivo assay; at least a 60% increase in tolerability to the therapeutic dosage unit administered when measured by testing in a standard in vivo assay; at least a 65% increase in tolerability to the therapeutic dosage unit administered when measured by testing in a standard in vivo assay; at least a 70% increase in tolerability to the therapeutic dosage unit administered when measured by testing in a standard in vivo assay; at least a 75% increase in tolerability to the therapeutic dosage unit administered when measured by testing in a standard in vivo assay; at least an 80% increase in tolerability to the therapeutic dosage unit administered when measured by testing in a standard in vivo assay; at least an 85% increase in tolerability to the therapeutic dosage unit administered when measured by testing in a standard in vivo assay; at least a 90% increase in tolerability to the therapeutic dosage unit administered when measured by testing in a standard in vivo assay;An increase in tolerability of at least 95% for the administered therapeutic dose unit.

[0308] In some cases, the therapeutic mRNA dosage unit administered is less than 6.9 ng / μg double stranded RNA (dsRNA), less than 6.8 ng / μg double stranded RNA (dsRNA), less than 6.7 ng / μg double stranded RNA (dsRNA), less than 6.6 ng / μg double stranded RNA (dsRNA), less than 6.5 ng / μg double stranded RNA (dsRNA), less than 6.4 ng / μg double stranded RNA (dsRNA), less than 6.3 ng / μg double stranded RNA (dsRNA), less than 6.2 ng / μg double stranded RNA (dsRNA), less than 6.1 ng / μg double stranded RNA (dsRNA). dsRNA), less than 6.0ng / μg double stranded RNA (dsRNA), less than 5.9ng / μg double stranded RNA (dsRNA), less than 5.8ng / μg double stranded RNA (dsRNA), less than 5.7ng / μg double stranded RNA (dsRNA), less than 5.6ng / μg double stranded RNA (dsRNA), less than 5.5ng / μg double stranded RNA (dsRNA), less than 5.4ng / μg double stranded RNA (dsRNA), less than 5.3ng / μg double stranded RNA (dsRNA), less than 5.2ng / μg double stranded RNA (dsRNA), less than 5.1ng / μg double stranded RNA (dsRNA). double stranded RNA (dsRNA), less than 5.0ng / μg double stranded RNA (dsRNA), less than 4.9ng / μg double stranded RNA (dsRNA), less than 4.8ng / μg double stranded RNA (dsRNA), less than 4.7ng / μg double stranded RNA (dsRNA), less than 4.6ng / μg double stranded RNA (dsRNA), less than 4.5ng / μg double stranded RNA (dsRNA), less than 4.4ng / μg double stranded RNA (dsRNA), less than 4.3ng / μg double stranded RNA (dsRNA), less than 4.2ng / μg double stranded RNA (dsRNA), 4.1ng / μ g double stranded RNA (dsRNA), less than 4.0 ng / μg double stranded RNA (dsRNA), less than 3.9 ng / μg double stranded RNA (dsRNA), less than 3.8 ng / μg double stranded RNA (dsRNA), less than 3.7 ng / μg double stranded RNA (dsRNA), less than 3.6 ng / μg double stranded RNA (dsRNA), less than 3.5 ng / μg double stranded RNA (dsRNA), less than 3.4 ng / μg double stranded RNA (dsRNA), less than 3.3 ng / μg double stranded RNA (dsRNA), less than 3.2 ng / μg double stranded RNA (dsRNA), 3.Less than 1ng / μg double-stranded RNA (dsRNA), less than 3.0ng / μg double-stranded RNA (dsRNA), less than 2.9ng / μg double-stranded RNA (dsRNA), less than 2.8ng / μg double-stranded RNA (dsRNA), less than 2.7ng / μg double-stranded RNA (dsRNA), less than 2.6ng / μg double-stranded RNA (dsRNA), less than 2.5ng / μg double-stranded RNA (dsRNA), less than 2.4ng / μg double-stranded RNA (dsRNA) NA), less than 2.3ng / μg double-stranded RNA (dsRNA), less than 2.2ng / μg double-stranded RNA (dsRNA), less than 2.1ng / μg double-stranded RNA (dsRNA), less than 2.0ng / μg double-stranded RNA (dsRNA), less than 1.9ng / μg double-stranded RNA (dsRNA), less than 1.8ng / μg double-stranded RNA (dsRNA), less than 1.7ng / μg double-stranded RNA (dsRNA), less than 1.6ng / μg double-stranded RNA (dsRNA). A(dsRNA), less than 1.5ng / μg double stranded RNA (dsRNA), less than 1.4ng / μg double stranded RNA (dsRNA), less than 1.3ng / μg double stranded RNA (dsRNA), less than 1.2ng / μg double stranded RNA (dsRNA), less than 1.1ng / μg double stranded RNA (dsRNA), less than 1.0ng / μg double stranded RNA (dsRNA), less than 0.9ng / μg double stranded RNA (dsRNA), less than 0.8ng / μg Double stranded RNA (dsRNA), less than 0.7 ng / μg double stranded RNA (dsRNA), less than 0.6 ng / μg double stranded RNA (dsRNA), less than 0.5 ng / μg double stranded RNA (dsRNA), less than 0.4 ng / μg double stranded RNA (dsRNA), less than 0.3 ng / μg double stranded RNA (dsRNA), less than 0.2 ng / μg double stranded RNA (dsRNA), or less than 0.1 ng / μg double stranded RNA (dsRNA).

[0309] VI. Kit Kits for carrying out transcription are also provided herein. The kits can include any of the compounds or compositions described herein. For example, the kits can include one or more compounds of formula I, formula II, formula III, formula IV, formula V, formula VI, formula VII, formula VIII, formula IX, formula X, formula XI and / or formula XII. The kits can further include one or more additional reagents, such as reagents used for synthesizing RNA. Optionally, the kits can contain a compound described herein (also referred to herein as a cap analog), a container, and one or more reagents selected from one or more unmodified NTPs, one or more modified NTPs, an RNA polymerase, a reaction buffer, magnesium, and a DNA template. The kits can further include instructions for using the kit (e.g., instructions for carrying out RNA synthesis), a means for administering the transcribed mRNA (e.g., a syringe), and / or a carrier. EXAMPLES

[0310] Example 1: Compound synthesis The compounds described herein were prepared using the general procedures outlined below. The structures shown herein may represent one particular isomer (e.g., one diastereomer). However, as noted above, the structures shown may represent a mixture of isomers. By way of example, the thiolated compounds described herein (e.g., Compound 3, Compound 6, Compound 9, Compound 13, Compound 18, Compound 19, Compound 20, Compound 21, and Compound 74) contain two diastereomers. In some cases, the two diastereomers were separated and tested separately (as shown in the examples below). However, unless otherwise indicated, the diastereomers were not separated prior to testing.

[0311] Synthesis of Compounds According to Formulas I, II, IV, VI, VIII, IX, X, and XI Compounds according to formulae I, II, IV and VI, which terminate with guanosine nucleotides, were prepared according to the general method shown in Scheme 1. Compounds according to formulae IX, X and XI, as well as compounds according to formula VIII, are similarly prepared (the position corresponding to R2 in Scheme 1 can be OCH3). [ka]

[0312] General Step 1: To a stirred solution of N7-Me-GDP 1 (triethylammonium (TEA) salt, 1.5 mol equivalents based on pApG dinucleotide 2) in 10% water / DMSO solution (10 mL per each gram of N7-Me-GDP 1), at room temperature, was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl salt; 2.4 mol equivalents based on pApG dinucleotide 2), followed by imidazole (4.5 mol equivalents based on pApG dinucleotide 2). The resulting mixture was stirred overnight (approximately 16-24 hours) at room temperature. On the second day, magnesium chloride (3.15 M in water, 2.0 mol equivalents based on pApG dinucleotide 2) was added to the above solution, followed by pApG dinucleotide 2 (TEA salt, 1 mol equivalent). The resulting solution was stirred overnight (approximately 16-24 hours) at room temperature. The crude reaction mixture was then diluted with 10x water and purified by anion exchange chromatography (Q Sepharose Fast Flow (QFF) Resin, using 20% ​​acetonitrile in water as buffer A and 1.5 M trimethylamine acetate (TEAA) in water as buffer B, a linear gradient of 25% to 45% buffer B for 4 column volumes (CV), held at 45% for 1.5 CV). The desired product was pooled, concentrated under vacuum, and the final product was precipitated as the sodium salt with an aqueous solution of sodium acetate and 95% absolute ethanol.

[0313] The synthesis of the following compounds followed the procedure described above and illustrated in Scheme 1. m7 Gppp m6 A 2’OMe pG (compound 31): [ka]

[0314] The compound shown above was prepared from N7-Me-GDP (0.40 g, 0.75 mmol), EDC (0.19 g, 0.975 mmol) and imidazole (0.07 g, 1.0 mmol) in 4.88 mL of 10% water in DMSO, 3.15 M magnesium chloride (0.32 mL, 1 mmol), and p(m6)ApG (0.46 g, 0.5 mmol) as described according to general procedure 1. m7 Gppp m6 A 2’OMe pG (Compound 31) was synthesized. A solid product (0.421 g) was obtained (yield: 68.6%) NMR (500 MHz, D2O) δ 8.25(s,1H),7.99(s,1H),7.88(s,1H),5.96(d,J=5.2 Hz,1H),5.80(d,J=3.5 Hz,1H),5.78(d,J=5.3 Hz,1H),4.87-4.91(m,1H),4.66-4.68(m,1H),4.45-4.49(m,3H),4.38-4.41(m,3 H),4.28-4.36(m,4H),4.17-4.25(m,4H),3.97(s,3H),3.46(s,3H),2.98(s,3H). 31 P NMR(200 MHz,D2O)0.38(s,1P),-10.85(d,J=12.1,1P),-10.94(d,J=12.1,1P),-22.22(t,J=18.2,1P).MS m / z=1159.2.

[0315] m7 G 3’Ome ppp m6 A 2’OMe pG (compound 30): [ka]

[0316] The compound shown above was prepared from N7-Me-3'-Ome-GDP (2.86 g, 4.5 mmol), EDC (1.12 g, 5.85 mmol) and imidazole (0.41 g, 6.0 mmol) in 28.6 mL of 30% water in DMSO, 3.15 M magnesium chloride (1.90 mL, 6.0 mmol), and p(m6)ApG (2.76 g, 3.0 mmol) as described according to general procedure 1. m7 G 3’Ome ppp m6 A 2’OMe pG (compound 30) was synthesized. 3.092 g of solid product was obtained (yield: 83%). 1H NMR (500 MHz, DO) δ 8.30 (s, 1H), 8.09 (s, 1H), 7.94 (s, 1H), 6.00 (d, J = 5.5 Hz, 1H), 5.80-5.83 (m, 2H), 4.91-4.94 (m, 1H), 4.65-4.66 (m, 1H), 4.49-4.52 (m, 2H), 4.36-4.42 (m, 5H), 4.18-4.33 (m, 4H), 4.08-4.10 (m, 1H), 4.01 (s, 3H), 3.48-3.47 (m, 6H), 3.08 (s, 3H). 31 P NMR(200 MHz,D2O)0.38(s,1P),-10.96(d,J=4.0,1P),-11.04(d,J=8.1,1P),-22.27(t,J=18.2,1P).MS m / z=1173.2.

[0317] m7 GpppA 2’,4’-LNA pG (compound 1): [ka]

[0318] Compound 1 was synthesized from N7-Me-GDP (0.30 g, 0.49 mmol), EDC (0.151 g, 0.79 mmol) and imidazole (0.101 g, 1.48 mmol) in 3.0 ml of 10% water in DMSO, magnesium chloride (62.4 mg, 0.66 mmol in 0.21 mL water), and p(LNA)ApG (0.298 g, 0.328 mmol) as described according to general procedure 1. A solid product (0.283 g) was obtained (yield: 71%). 1 H NMR(500 MHz,D2O):δ 8.07(s,1H),7.95(s,1H),7.75(s,1H),5.85(d,J=4.2 Hz,1H),5.69(d,J=5.5 Hz,2H),4.69(m,1H),4.53-4.28(m,15H),4.08(s,3H). 31 P NMR(200 MHz,D2O):δ-1.47(1P),-10.98(2P),-22.41(1P).C 32 H 42 N 15 O 24 MS(ESI) calculated value for P4 is 1144.66, found value is 1142.9 [MH] - .

[0319] m7 G 3’Ome pppA 2’,4’-LNA pG (compound 2): [ka]

[0320] Compound 2 was synthesized from N7-Me-3'-Ome-GDP (0.30 g, 0.459 mmol), EDC (0.141 g, 0.734 mmol) and imidazole (0.0938 g, 1.377 mmol) in 3.0 ml of 10% water in DMSO, magnesium chloride (58.3 mg, 0.612 mmol in 0.19 mL water), and p(LNA)ApG (0.277 g, 0.306 mmol) as described according to general procedure 1. A solid product (0.2573 g) was obtained (yield: 69%). 1H NMR(500 MHz,D2O):δ 8.06(s,1H).7.95(s,1H),7.75(s,1H),5.82(d,J=4.7 Hz,1H),5.68(m,2H),4.90(m,1H),4.69-4.11(m,13H),4.09(s,3H),3.49(s,3H). 31 P NMR(200 MHz,D2O):δ-1.46(1P),-10.98(2P),-22.30(1P).C 33 H 44 N 15 O 24 MS(ESI) calculated for P4: 1158.69, observed: 1156.4 [MH] - .

[0321] m7 GpppA 2’-4’-LNA p(s)G (compound 3): [ka]

[0322] Compound 3 was synthesized from N7-Me-GDP (0.2 g, 0.327 mmol), EDC (0.1 g, 0.523 mmol) and imidazole (0.067 g, 0.981 mmol) in 2.0 ml of 10% water in DMSO, magnesium chloride (50.9 mg, 0.536 mmol), and p(LNA)Ap(s)G (0.2 g, 0.218 mmol) as described according to general procedure 1. Solid product (149.5 mg) was obtained (yield: 56%). The two diastereomers were purified by RP-HPLC. The NMR / MS data are shown below. m7 GpppA 2’-4’-LNA p(s)G:D1 Final yield -137.3μmol;NMR(500MHz,D2O)δ 8.12(s,1H),7.98(s,1H),7.83(s,1H),5.86(d,J=4.1,1H),5.76(s,1H),5.72(d,J=4.6,1 31P NMR(200 MHz,D2O)55.13(1P),-11.02(2P),-22.39(1P),MS m / z=1159.8(MH) m7 GpppA 2’-4’-LNA p(s)G:D2 Final yield -130.4μmol;NMR(500MHz,D2O)δ 8.11(s,1H),7.95(s,1H),7.86(s,1H),5.84(d,J=4.2,1H),5.68(m,2H),4.90(m,2H),4.5 0-4.57(m,3H),4.41-4.46(m,3H),4.25-4.33(m,6H),4.13-4.19(m,2H),4.09(s,3H);31P NMR(200 MHz,D2O)56.19(1P),-11.00(2P),-22.42(1P),MS m / z=1157.9(MH)

[0323] m7 GpppA 2’OMe-5’ビニル pG (compound 4): [ka]

[0324] Compound 4 was synthesized from N7-Me-GDP (0.0716 g, 0.117 mmol), EDC (0.0358 g, 0.187 mmol) and imidazole (0.024 g, 0.351 mmol) in 0.7 ml of 10% water in DMSO, 3.15 M magnesium chloride (0.05 mL, 0.156 mmol), and 5'-vinylphosphonate AG (0.07 g, 0.078 mmol) as described according to general procedure 1. A solid product (67 mg) was obtained (yield: 71%). 1H NMR(500 MHz,D2O):δ 8.26(s,1H),8.11(s,1H),7.92(s,1H),6.62(m,1H),6.21(t,J=17.6 Hz,1H),6.00(d,J=5.9 Hz,1H),5.79(m,2H),4.54-4.35(m,7H),4.16(m,3H),4.02(s,3H),3.39(s,3H). 31 P NMR(200 MHz,D2O):δ 3.74(1P),-0.40(1P),-10.94(2P),-22.36(1P).C 33 H 43 N 15 O 23 MS(ESI) calculated for P4: 1141.68, found: 1140.7 [MH] - .

[0325] m7 G 3’-Ome pppA 2’OMe-5’ビニル pG (compound 5): [ka]

[0326] Compound 5 was synthesized from N7-Me-3'-Ome-GDP (0.076 g, 0.117 mmol), EDC (0.0358 g, 0.187 mmol) and imidazole (0.024 g, 0.351 mmol) in 0.7 ml of 10% water in DMSO, 3.15 M magnesium chloride (0.05 mL, 0.156 mmol), and 5'-vinylphosphonate AG (0.07 g, 0.078 mmol) as described according to general procedure 1. A solid product (70 mg) was obtained (yield: 73%). 1H NMR(500 MHz,D2O):δ 8.27(s,1H),8.11(s,1H),7.93(s,1H),6.62(m,1H),6.21(t,J=17.5 Hz,1H),6.00(d,J=5.9 Hz,1H),5.82(d,J=5.45 Hz,1H),5.71(d,J=4.25 Hz,1H),4.64(m,1H),4.48(m,1H),4.43(m,1H),4.35-4.23(m,3H),4.15(m,3H),4.08(m,1H),4.02(s,3H),3.46(s,3H),3.40(s,3H). 31 P NMR(200 MHz,D2O):δ 3.85(1P),-0.39(1P),-10.91(2P),-22.30(1P).C 34 H 45 N 15 O 23 MS(ESI) calculated for P4: 1155.71, found: 1155.5 [MH] - .

[0327] m7 GpppA 2’F p(s)G (compound 6): [ka]

[0328] Prepare 2.0 ml of 10% water in DMSO, magnesium chloride (50.9 mg, 0.536 mmol), and pA 2’F Compound 6 was synthesized from N7-Me-GDP (0.2 g, 0.327 mmol), EDC (0.100 g, 0.523 mmol) and imidazole (0.067 g, 0.981 mmol) in p(s)G (0.198 g, 0.218 mmol). A solid product (155 mg) was obtained (yield: 62%). The two diastereomers were purified by RP-HPLC. The NMR / MS data are shown below. m7 GpppA 2’F p(s)G:D1 Final yield -24.4 μmol; NMR (500 MHz, D2O) δ 8.24 (s, 1H), 7.99 (s, 1H), 7.81 (s, 1H), 6.09 (d, J = 15.9 Hz, 1H), 5.88 (d, J = 3.9 Hz, 1H), 5.70 (d, J = 6.5 Hz, 1H), 5.49-5.38 (m, 1H), 5.05-5.01 (m, 1H), 5.56 (t, J = 4.5 Hz, 2H), 4.49-4.33 (m, 7H), 4.26-4.20 (m, 4H), 4.04 (s, 3H) 31P NMR (200 MHz, D2O)55.72(1P),-10.93(1P),-10.05(1P),-22.40(1P), MS m / z=1149.0(MH). m7 GpppA 2’F p(s)G:D2 Final yield -29.8 μmol; NMR (500 MHz, D2O) δ 8.24 (s, 1H), 7.99 (s, 1H), 7.86 (s, 1H), 6.10 (d, J = 16.0 Hz, 1H), 5.88 (d, J = 3.9 Hz, 1H), 5.70 (d, J = 5.5 Hz, 1H), 5.42-5.31 (m, 1H), 5.07-4.99 (m, 1H), 4.57-4.44 (m, 6H), 4.42-4.21 (m, 7H), 4.19-4.16 (m, 1H), 4.04 (s, 3H) 31P NMR (200 MHz, D2O)57.23(1P),-10.97(2P),-22.38(1P), MS m / z=1148.8(MH).

[0329] m7 Gppp(ジアミノノリン) 2’OMe pG (Compound 7):

change

[0330] Compound 7 was synthesized from N7-Me-GDP (0.219 g, 0.358 mmol), EDC (0.11 g, 0.574 mmol) and imidazole (0.073 g, 1.076 mmol) in 2.2 ml of 10% water in DMSO, 3.15 M magnesium chloride (0.15 mL, 0.478 mmol), and DAP-G (0.22 g, 0.239 mmol) as described according to general procedure 1. 191 mg of solid product was obtained (yield: 65%). 1 H NMR (500 MHz, D2O): δ 8.02(s,1H),7.98(s,1H),5.82(m,3H),4.90(m,1H),4.55(m,1H),4.52-4.20(m,13H),4.01(s,3H),3.45(s,3H). 31 P NMR(200 MHz,D2O): δ-0.35(1P),-10.84(2P),-22.32(1P).

[0331] m7 G 3’Ome ppp (diaminopurine) 2’OMe pG (compound 8): [ka]

[0332] Compound 8 was synthesized from N7-Me-3'-Ome-GDP (0.233 g, 0.358 mmol), EDC (0.11 g, 0.574 mmol) and imidazole (0.073 g, 1.076 mmol) in 2.2 ml of 10% water in DMSO, 3.15 M magnesium chloride (0.15 mL, 0.478 mmol), and DAP-G (0.22 g, 0.239 mmol) as described according to general procedure 1. A solid product (202 mg) was obtained (yield: 68%). 1 H NMR(500 MHz,D2O):δ 8.02(s,1H),7.98(s,1H),5.82(m,3H),4.93(m,1H),4.69(m,1H),4.49(m,1H),4.42-4.10(m,11H),3.68(s,3H),3.48(s,3H),3.46(s,3H). 31P NMR(200 MHz,D2O): δ-0.34(1P),-10.94(2P),-22.28(1P).

[0333] m7 G 3’OMe ppp (diaminopurine) 2’OMe p(s)G (compound 9): [ka]

[0334] Compound 9 was synthesized from N7-Me-3'-OMe-GDP (0.201 g, 0.327 mmol), EDC (0.100 g, 0.523 mmol) and imidazole (0.067 g, 0.981 mmol) in 2.0 ml of 10% water in DMSO, magnesium chloride (50.9 mg, 0.536 mmol), and pDAPm p(s)G (0.204 g, 0.218 mmol) as described according to general procedure 1. The solid product (65.4 mg) obtained after anion exchange (Q Sepharose Fast Flow) and anion exchange (Q Sepharose High Performance) purification was obtained (yield: 25.2%). 1 H NMR(500 MHz,D2O):δ 8.05(s,1H),8.01-8.00(m,2H),5.86-5.79(m,3H),5.11-5.06(m,1H),4.64(t,J=4.5 Hz,1H),4.52-4.15(m,11H),4.08-4.05(m,1H),4.02(d,J=8.25 Hz,3H),3.46-3.44(m,4H),3.40(s,2H).). 31 P NMR(200 MHz,D2O):δ 56.76(1P),56.06(1P),-10.91 _ -11.09(4P),-22.27 _ -22.49(2P).).MS m / z=1189.8(MH)

[0335] m7 G 3’Ome pppA 2’OMe p(s)G (compound 21): [ka]

[0336] Compound 21 (two diastereomers) was synthesized as described following general procedure 1. The two diastereomers were prepared from pA(ps)G dinucleotide, which was made as a mixture of two diastereomers, D1 and D2, as shown below (the chiral P atoms generating the D1 and D2 diastereomers are indicated by an asterisk in the structures below). The diastereomers were resolved by reversed-phase chromatography using a Daisogel 150 x 20 mm SP-100-15-ODS-P column (Torrance, CA) using the following separation conditions: Buffer A: 100 mM triethylammonium bicarbonate (TEAB), Buffer B: acetonitrile (ACN). Gradient: 0% B x 1.5 column volumes (CV), 0 to 12.5% ​​B x 10 CV. The D1 diastereomer (referred to as pA(ps)G D1) eluted first from the reversed-phase chromatography column, and the D2 diastereomer (referred to as pA(ps)G D2) eluted second from the reversed-phase chromatography column. The final diastereomeric purity of each was >99%.

[0337] [ka]

[0338] The resulting diastereomers of compound 21 are designated as compound 21 D1 and compound 21 D2.

[0339] Compound 21 D1 was prepared from N7-Me-3'-OMe-GDP (0.119 g, 0.202 mmol), EDC (0.05 g, 0.262 mmol) and imidazole (0.018 g, 0.269 mmol) in 6.6 ml of 10% water in DMSO, magnesium chloride (0.085 mL, 0.269 mmol), and pA(ps)G D1 (0.1346 g, 0.134 mmol). The resulting compound was isolated and recovered as a solid product (61.7 mg; yield: 37%). 1H NMR(500 MHz,D2O)δ 8.38(s,1H),8.12(s,1H),8.05(s,1H),6.00(d,J=6.0 Hz,1H),5.86(d,J=5.9 Hz,1H),5.83(d,J=4.3 Hz,1H),5.11-5.08(m,1H),4.82-4.73(m,1H),4.56-4.52(m,2H),4.48(t,J=5.5 Hz,1H),4.38-4.35(m,3H),4.32-4.23(m,4H),4.20-4.17(m,1H),4.10(t,J=4.6 Hz,1H),4.03(s,3H),3.47(m,6H).31P NMR(200 MHz,D2O)56.05(s,1P),-11.01,-11.10(overlapping doublet, 2P),-22.33(t,J=18.2,1P).MS m / z=1173.8(MH).

[0340] This process was repeated using pA(ps)G D2 to isolate compound 21 D2. Specifically, the synthesis was carried out using N7-Me-3'-OMe-GDP (0.187g, 0.317mmol), EDC (0.079g, 0.413mmol) and imidazole (0.029g, 0.423mmol) in 1.87ml of 10% water in DMSO, magnesium chloride (0.134mL, 0.423mmol), and pA(ps)G D2 (0.2173g, 0.212mmol). A solid product (0.124mg) was obtained (yield: 47%). NMR(500 MHz,D2O)δ 8.38(s,1H),8.11(s,1H),8.01(s,1H),6.03(d,J=5.8 Hz,1H),5.84(m,2H),5.08-5.06(m,1H),4.66(m,1H),4.58(m,2H),4.52-4.50(t,J=4.9Hz,1H),4.45-4 .43(T,J=5.4Hz,1H),4.40-4.20(m,8H),4.11-4.09(m,1H),4.04(s,3H),3.47(s,3H),3.44(m,3H).31P NMR(200 MHz, DO) 56.68 (s, 1P), -10.98, -11.07 (overlapping doublet, 2P), -22.29 (t, J = 20.2, 1P). MS m / z = 1174.0 (MH).

[0341] m7 G 2’OMe3’OMe pppA 2’OMe pG (compound 25): [ka]

[0342] Prepare 5 mL of 30% water in DMSO, magnesium chloride (0.12 mL of a 3.15 MgCl solution, 0.48 mmol), and pH 7.0 as described in General Procedure 1. 2’OMe Compound 25 was synthesized from N-7-Me-2',3'-di-OMe-GDP (0.5 g, 0.36 mmol), EDC (0.12 g, 0.6 mmol) and imidazole (0.10 g, 1.4 mmol) in pG (0.2 g, 0.24 mmol). A solid product (0.16 g) was obtained (yield 57%).1 H NMR δ 8.32(s,1),8.06(s,1),7.92(s,1),5.94(d,1,J=10 Hz),5.80(d,1,J=10 Hz),5.79(d,1,J=10Hz),4.89(m,1),4.47(m,2),4.37(t,1,J=5 MS m / z=1172.0(MH).

[0343] m7 G 2’OMe pppA 2’OMe pG (compound 26): [ka]

[0344] Prepare 2.2 mL of 10% water in DMSO, magnesium chloride (0.13 mL of a 3.15 MgCl solution, 0.42 mmol), and pH 7.0 as described according to General Procedure 1. 2’OMe Compound 26 was synthesized from N-7-Me-2'-OMe-GDP (0.20 g, 3 mmol), EDC (0.091 g, 0.47 mmol) and imidazole (0.06 g, 0.89 mmol) in pG (0.19 g, 0.21 mmol). A solid product (0.21 g) was obtained (yield 78%). 1H NMR δ 8.34(s,1H),8.09(s,1H),7.92(s,1H),5.96(d,1H,J=9 Hz),5.86(d,1H,J=5 Hz),5.80(d,1H,J=9 Hz),4.90(m,1H),4.51(d,1H,J=5 Hz),4.48(m,2H),4.38(m,2H),4.33(m,1H),4.27(m,2H),4.23-4.16(m,5H),4.02(s,3H),3.52(s,3H),3.41(s,3H).31P δ-0.43(1P),-11.06(2P),-22.39(1P);MS m / z=1157.9(MH).

[0345] m7 G 2’OMe3’OMe ppp m6 A 2’OMe pG (compound 27): [ka]

[0346] As described according to General Procedure 1, 5 mL of 30% water in DMSO, magnesium chloride (0.12 mL of a 3.15 MgCl2 solution, 0.48 mmol), and p m6 A 2’OMe Compound 27 was synthesized from N-7-Me-2',3'-di-OMe-GDP (0.5 g, 0.36 mmol), EDC (0.12 g, 0.6 mmol) and imidazole (0.10 g, 1.4 mmol) in pG (0.22 g, 0.24 mmol). A solid product (0.18 g) was obtained (yield 64%). 1 H NMR δ 8.26(s,1),8.07(s,1),7.70(s,1)5.94(d,1,J=10 Hz),5.80(d,1,J=10 Hz),5.76(d,1 j=5 Hz),4.89(m,1),4.73,(m,1),4.21(m,1),4.18(m,2),4.14(t,2,J=5 Hz),3.99(s,3)3.49(s,3),3.42(s,3),3.08(br s,3).31P δ-0.42(1P),-11.12(2P),-22.43(1P);MS m / z=1172.0(MH).

[0347] m7 G 2’OMe ppp m6 A 2’OMe pG (compound 28): [ka]

[0348] As described according to General Procedure 1, 2.2 mL of 10% water in DMSO, magnesium chloride (0.13 mL of a 3.15 MgCl2 solution, 0.42 mmol), and p m6 A 2’OMeCompound 28 was synthesized from N-7-Me-2'-OMe-GDP (0.20 g, 9.3 mmol), EDC (9.091 g, 0.47 mmol) and imidazole (0.06 g, 0.89 mmol) in pG (0.19 g, 0.21 mmol). A solid product (0.17 g) was obtained (yield 68%). 1 H NMR δ 8.28(s,1H),8.09(br s,1H),7.92(s,1H),5.96(d,1H,J=5 Hz),5.84(d,1H,J=5 Hz),5.82(d,1,J=9 Hz),4.91(m,1H),4.49(m,3H),4.39(m,2H),4.25(m,2H),4.20-4.15(m,6H),4.00(s,3H),3.54(s,3H),3.46(s,3H),3.09(br s,3H).31P δ-0.40(1P),-11.06(2P),-22.37(1P);MS m / z=1174(M+H).

[0349] m7 G 3’SMe pppA 2’OMe pG (compound 42): [ka]

[0350] As described according to General Procedure 1, 1.8 mL of 10% water in DMSO, magnesium chloride (0.123 mL of 3.15 M MgCl2 solution, 0.39 mmol), and pA m Compound 42 was synthesized from N7-Me-3'-SMe-GDP (0.18 g, 0.29 mmol), EDC (0.09 g, 0.47 mmol) and imidazole (0.06 g, 0.90 mmol) in pG (0.18 g, 0.19 mmol). A solid product (0.161 g, 0.130 mmol) was obtained (yield: 67%). 1H NMR(500MHz,D2O)δ 8.37(s,1H),8.10(s,1H),7.92(s,1H),5.98(d,J=5.8,1H),5.81(m,2H),4.92(m,2H),4.57(d,J=4.7,2H),4.47 -4.51(m,4H),4.41(t,J=5.2,1H),4.29-4.40(m,3H),4.15-4.24(m,5H),4.01(s,3H),3.42(s,3H),2.12(s,3H). 31 P NMR(200 MHz,D2O)-0.41(1P),-11.04(1P),-11.35(1P),-22.32(1P).MS m / z=1173.9(MH).

[0351] m7 G 2’OH,2’Me pppA 2’OMe pG (compound 43): [ka]

[0352] Compound 43 was synthesized from N7-Me-2'Me2'OH-GDP (0.502 g, 0.85 mmol), EDC (0.230 g, 1.2 mmol) and imidazole (0.153 g, 2.25 mmol) in 5 mL of 10% water in DMSO, magnesium chloride (0.317 mL of 3.15 M MgCl2 solution, 1 mmol), and pAmpG (0.454 g, 0.5 mmol) according to general procedure 1. A solid product (0.437 g) was obtained (yield 75%). 1 H NMR(500 MHz,D2O):δ 8.36(S,1H),8.02(s,1H),7.93(s,1H),5.96(d,J=5.7 Hz,1H),5.82(s,1H),5.81(s,1H),4.91(m,1H),4.49(m,3H),4.39(t,J=5.1Hz,1H),4.3 4(m,1H),4.29(m,3H),4.20(m,3H),4.14(m,1H),4.03(s,3H),3.41(s,3H),1.02(s,3H). 31P NMR(200 MHz,D2O):δ-0.39(1P),-11.0(2P),-22.3(1P).MS m / z=1158.9(MH).

[0353] m 7 G 3’AZM ppp m6 A 2’OMe pG (compound 45): [ka]

[0354] As described according to General Procedure 1, 3.0 mL of 30% water in DMSO, magnesium chloride (0.16 mL of a 3.15 MgCl solution, 0.5 mmol), and pA 2’OMe In pG (0.23 g, 0.25 mmol), N-7-Me-3'-AZM-GDP (0.20 g, 0.3 mmol), EDC (0.098 g, 0.51 mmol) and imidazole (0.082 g, 1.2 mmol) were added to m 7 G 3’AZM ppp m6 A 2’OMe pG was synthesized. A solid product (0.086 g) was obtained (yield 24%). 1 H NMR:δ 8.28(s,1H),8.08(s,1H),7.91(s,1H),5.97(d,1H,J=10 Hz),5.82-5.80(m,2H),4.91-4.88(m,2H),under D2O(3H),4.63(t,1H,J=5 Hz),4.50-4.46(m,2H),4.43(s,1H),4.39-4.30(m,5H),4.24-4.17(m,4H),3.99(s,3H),3.45(s,3H),3.08(s,3H). 31 P NMR δ-0.40(1P),-11.08(2P),-22.42(1P).[M-3H+4Na] + Expected mass: 1303.12;[M-3H+4Na] + Detection: m / z=1302.5 (M-3H+4Na).

[0355] m7 G 3’F pppm6 A 2’OMe pG (compound 49): [ka]

[0356] As described according to General Procedure 1, 2.2 mL of 10% water in DMSO, magnesium chloride (0.12 mL of a 3.15 MgCl2 solution, 0.38 mmol), and p m6 A 2’OMe Compound 49 was synthesized from N-7-Me-3'-F-GDP (0.20 g, 27 mmol), EDC (0.083 g, 0.43 mmol) and imidazole (0.055 g, 0.81 mmol) in pG (0.176 g, 0.19 mmol). A solid product (0.12 g) was obtained (yield 55%). 1 H NMR δ 8.27(s,1H),8.09(br s,1H),7.92(s,1H),5.96(d,1H,J=9 Hz),5.82(d,1,J=5 Hz),5.80(d,1H,J=9 Hz),5.32(d,1H,J=5 Hz),5.22(d,1,J=5 Hz),4.89(m,1H),4.72(m,1H),4.68(m,1H),4.48(m,2H),4.38(t,1H,J=5 Hz),4.30-4.20(M,7H),4.02(s,3H),3.45(s,3H),3.10(br s,3H).MS m / z=1159.7(M+H).

[0357] m7 G 3’MOE ppp m6 A 2’OMe pG (compound 50): [ka]

[0358] Prepare 3.5 mL of 10% water in DMSO, magnesium chloride (0.15 mL of 3.15 MgCl solution, 0.24 mmol), and pA M6 2’OMeCompound 50 was synthesized from N-7-Me-3'-(2-methoxyethoxy)-GDP (.030 g, 0.37 mmol), EDC (0.12 g, 0.6 mmol) and imidazole (0.10 g, 1.5 mmol) in pG (0.11 g, 0.12 mmol). A solid product (0.11 g) was obtained (yield 65%). 1 H NMR δ 8.21(s,1),8.06(br s,1),7.89(s,1),5.96(d,1,J=5.5 Hz),5.82(s,1),5.81(d,1,J=5.5 Hz),4.88(m,1),4.73(t,1,J=5.5 Hz),4.60(t,1,J=4.7),4.47(m,1),4.46(m,1),4.36(m,2),4.35(M,2),4.32(M,1),4.2 2(m,1),4.17(m,4),3.98(s,3),3.76(m,2),3.62(m,2),3.43(s,3),3.37(s,3),3.07(br s,3);31P δ-.40(1P),-11.12(2P),-22.45(1P);MS m / z=1215.8(MH).

[0359] m7 G 3’SMe ppp m6 A 2’OMe pG (compound 52): [ka]

[0360] As described according to General Procedure 1, 1.8 mL of 10% water in DMSO, magnesium chloride (0.123 mL of 3.15 M MgCl2 solution, 0.39 mmol), and p m6 A mCompound 52 was synthesized from N7-Me-3'-SMe-GDP (0.18 g, 0.29 mmol), EDC (0.09 g, 0.47 mmol) and imidazole (0.06 g, 0.90 mmol) in pG (0.18 g, 0.19 mmol). Solid product (0.109 g, 0.087 mmol) was obtained. Final yield - 86.5 μmol, starting scale 194 μmol, 44.6%. NMR(500MHz,D2O)δ 8.30(s,1H),8.07(s,1H),7.89(s,1H),5.97(d,J=5.4,1H),5.79(d,J=5.8),5.75(s,1H),4.91(m,2H),4.54(d,J=4.7,1H),4.45 -4.50(m,3H),4.40(t,J=5.0,1H),4.24-4.33(m,2H),4.15-4.23(m,5H),3.97(s,3H),3.45(s,3H),3.06(bs,3H),2.1(s,3H).31P NMR(200 MHz,D2O)-0.42(1P),-11.02(1P),-11.37(1P),-22.31(1P).MS m / z=1187.8(MH)

[0361] m7 (L-sugar isomer)GpppA 2’OMe pG (compound 75): [ka]

[0362] Compound 75 was synthesized from N7-Me-L-GDP (0.31 g, 0.5 mmol), EDC (0.153 g, 0.8 mmol) and imidazole (0.106 g, 1.550 mmol) in 3.1 mL of 10% water in DMSO, magnesium chloride (0.212 g, 0.667 mmol), and pAmpG (0.30 g, 0.333 mmol) according to general procedure 1. 218.07 umol of product was obtained (yield: 65.5%). 1H NMR(500 MHz,D2O):δ 8.30(s,1H),8.06(s,1H),7.87(s,1H),6.01(d,J=4.4,1H),5.77(m,2H),4.86(m,1H),4.73(m,1H),4.48-4.46(m, 2H),4.46-4.45(m,3H),4.43-4.42(m,1H)4.38-4.19(m,4H)4.00(s,3H),3.47(s,3H),1.90(s,1H),1.17(t,J=7.1 Hz,1H).31P NMR(200 MHz,D2O):δ 0.47(s,1P),-10.87(m,1P),-22.06(m,1P).C 32 H 42 N 15 O 24 The calculated value of P4 is 1144.66, and the measured value is 1144.0[MH]ネガティブモード.

[0363] m7 G 2’OH,2’Me ppp m6 A 2’OMe pG (Compound 76):

change

[0364] Compound 76 was synthesized from N7-Me-2'Me2'OH-GDP (0.502 g, 0.85 mmol), EDC (0.230 g, 1.2 mmol) and imidazole (0.153 g, 2.25 mmol) in 5 mL of 10% water in DMSO, magnesium chloride (0.317 mL of 3.15 M MgCl2 solution, 1 mmol), and pAmpG (0.406 g, 0.5 mmol) according to general procedure 1. A solid product (0.522 g) was obtained (yield 89%). 1H NMR(500 MHz,D2O):δ 8.28(S,1H),8.05(s,1H),7.89(s,1H),5.95(d,J=5.4Hz,1H),5.80(d,J=5.75Hz,1H),5.77(S,1H),4.91-4.88(m,1H),4.81-4.72(m,3H) ,4.50-4.46(m,2H),4.37(t,J=4.95Hz,1H),4.33-4.26(m,3H),4.25-4.18(m,3H),4.12-4.10(m,1H),4.00(s,3H),3.43(s,3H),3.06(br s,3H),1.00(s,3H).31P NMR(200 MHz,D2O):δ-0.40(1P),-10.99(2P),-22.31(1P).MS m / z=1195(M+Na).

[0365] m7 (L-sugar isomer)Gppp m6 A 2’OMe pG (compound 77): [ka]

[0366] According to general procedure 1, 3.1 mL of 10% water in DMSO, magnesium chloride (0.212 g, 0.667 mmol), and p m6 Compound 77 was synthesized from N7-Me-L-GDP (0.31 g, 0.5 mmol), EDC (0.153 g, 0.8 mmol) and imidazole (0.106 g, 1.550 mmol) in AmpG (0.31 g, 0.336 mmol). 257.86 umoles of product was obtained (yield: 76.7%). 1H NMR(500 MHz,D2O):δ 8.26(s,1H),8.05(s,1H),7.83(s,1H),6.03(d,J=3.6 Hz,1H),5.76(d,J=6.05 Hz,1H)5.67(d,2.8 Hz,1H),4.86(m,1H),4.69-4.67(t,J=5.5 Hz,1H),4.46-4.41(m,5H),4.39-4.37(m,3H),4.34-4.31(m,5H),3.98(s,3H),4.52(s,3H),3.00(bs,3H),1.90(s,1H),1.18(t,J=7.05 Hz,1H). 31P NMR(200 MHz,D2O):δ 0.47(s,1P),-10.92(m,1P),-22.11(m,1P)C 33 H 44 N 15 O 24 Calculated value for P4 is 1158.69, measured values ​​are 1158.0 and 1159.9 [MH] in negative mode.

[0367] m7 (Acyclo)Gppp m6 A 2’OMe pG: Compound 78 [ka]

[0368] According to general procedure 1, 1.6 mL of 10% water in DMSO, magnesium chloride (0.110 mL of 3.15 M MgCl2 solution, 1 mmol), and p m6 Compound 78 was synthesized from N7-methyl acyclic GDP (0.100 g, 0.251 mmol), EDC (0.061 g, 320 mmol) and imidazole (0.054 g, 0.80 mmol) in AmpG (0.125 g, 0.173 mmol). A solid product (0.105 g) was obtained (yield 55.0%). 1H NMR(500 MHz,D2O):δ 8.30(s,1H),8.08(s,1H),7.92(s,1H),6.00(d,J=5.8Hz,1H),5.82(d,J=5.7Hz,1 H),5.52-5.45(q,J=10.95Hz,2H),4.95-4.91(m,1H),4.78-4.72(m,2H),4.53(br s,1H),4.48-4.44(m,2H),4.34-4.29(m,2H),4.25-4.21(m,3H),4.20-4.09(m,2H),3.99(s,3H),3.79(m,2H),3.45(s,3H),3.03(br s,3H). 31 P NMR(200 MHz,D2O):δ-0.35(1P),-10.68(2P),-22.23(1P).MS m / z=1168.6(M+3Na).

[0369] Synthesis of compounds according to formula III: Compounds according to formula III, terminated with adenosine nucleotides, were prepared according to the general method depicted in Scheme 2. [ka]

[0370] General Procedure 2 (pApA dinucleotide 3 as p m6 A 2’OMe using pA dinucleotide): Modified N7-Me-GDP 1 (TEA salt, p m6 A 2’OMe To a stirred solution of EDC.HCl salt (pA dinucleotide, 1.5 mol equivalents) at room temperature, m6 A 2’OMe 2.4 molar equivalents based on pA dinucleotide 3), followed by imidazole (p m6 A 2’OMe On the second day, magnesium chloride (3.15 M in water, p m6 A 2’OMepA dinucleotide 3) was added to the above solution, followed by the modification p m6 A 2’OMe pA dinucleotide 3 (TEA salt, 1 molar equivalent) was added. The resulting solution was stirred at room temperature overnight (approximately 16-24 hours). The crude reaction mixture was then diluted with 10x water and purified by anion exchange chromatography (QFF Resin, using an aqueous solution of 20% acetonitrile as buffer A and 1.5 M TEAA as buffer B, a linear gradient of 25% to 45% buffer B for 4 CV, held at 45% for 1.5 CV). The desired product was pooled, concentrated under vacuum, and the final product was precipitated as the sodium salt with an aqueous solution of sodium acetate and 95% absolute ethanol.

[0371] The synthesis of the following compounds followed the procedure described above and illustrated in Scheme 2. m7 Gppp m6 A 2’OMe pA (compound 10): [ka]

[0372] As described according to General Procedure 2, 13 mL of DMSO, magnesium chloride (0.190 g, 2 mmol in 0.64 mL of water), and p m6 A 2’OMe Compound 10 was synthesized from N7-Me-GDP (0.88 g, 1.5 mmol), EDC (0.46 g, 2.4 mmol) and imidazole (0.31 g, 4.5 mmol) in pA (0.906 g, 1 mmol). A solid product (0.355 g) was obtained (yield: 29%). 1 H NMR(500 MHz,D2O):δ 8.26(s,1H),8.23(s,1H),8.15(s,1H),7.91(s,1H),6.008(d,J=4.5 Hz,1H),5.881(d,J=4.5 Hz,1H),5.761(d,J=3.5 Hz,1H),4.84-4.88(m,1H),4.25-4.49(m,14H),3.96(s,3H),3.53(s,3H). 31P NMR(200 MHz,D2O):δ-0.59(1P),-10.99,-11.04,-11.09,-11.13(overlapping doublet,2P),-22.50(t,J=16.2Hz,1P).C 33 H 45 N 15 O 23 MS(ESI) calculated for P4: 1143.70, found: 1144.7 [M+H] - .

[0373] m7 G 3’OMe ppp m6 A 2’OMe pA (compound 11): [ka]

[0374] As described according to General Procedure 2, 6.3 ml of 10% DMSO in water, magnesium chloride (122 mg, 1.28 mmol in 0.41 mL water), and p m6 A 2’OMe Compound 11 was synthesized from N7-Me-3'-OMe-GDP (0.63 g, 0.96 mmol), EDC (0.294 g, 1.536 mmol) and imidazole (0.196, 2.88 mmol) in pA (0.58 g, 0.64 mmol). A solid product (0.46 g) was obtained (yield: 53%). 1 H NMR(500 MHz,D2O):δ 8.28(s,1H),8.24(s,1H),8.14(s,1H),7.93(s,1H),6.013(d,J=4.5 Hz,1H),5.911(d,J=5 Hz,1H),5.741(d,J=4.5 Hz,1H),4.87-4.90(m,1H),4.59(t,J=4.5 Hz,1H),4.45-4.52(m,3H),4.32-4.40(m,5H),4.16-4.25(m,4H),4.04(t,J=5Hz,1H),3.96(s,3H),3.52(s,3H),3.44(s,3H). 31P NMR(200 MHz,D2O):δ-0.49(1P),-10.95,-11.02,-11.04,-11.11(overlapping doublet,2P),-22.37(t,J=16.2Hz,1P).C 34 H 47 N 15 O 23 The calculated value for P4 is 1157.73, and the measured value is 1156.7 [MH].

[0375] Synthesis of Compounds According to Formula V, Formula VII and Formula VIII: Compounds according to formula V, terminated with a uridine nucleotide, were prepared according to the general method shown in Scheme 3. Compounds according to formula VII and VIII are prepared similarly. [ka]

[0376] General procedure 3: To a stirred solution of N7-Me-GDP 1 (TEA salt, 1.5 mol equivalents based on pApU dinucleotide) in 10% water / DMSO (volume is 10 times the weight of N7-Me-GDP 1) solution, at room temperature, EDC.HCl salt (2.4 mol equivalents based on pApU dinucleotide 4) was added, followed by imidazole (4.5 mol equivalents based on pApU dinucleotide 4). The resulting mixture was stirred overnight (approximately 16-24 h) at room temperature. On the second day, magnesium chloride (3.15 M in water, 2.0 mol equivalents based on pApU dinucleotide 4) was added to the above solution, followed by the addition of modified pApU dinucleotide 4 (TEA salt, 1 mol equivalent). The resulting solution was stirred overnight (approximately 16-24 h) at room temperature. The crude reaction mixture was then diluted with 10x water and purified by anion exchange chromatography (QFF Resin, using 20% ​​acetonitrile in water as buffer A, 1.5 M TEAA in water as buffer B, a linear gradient of 25% to 45% buffer B for 4 CV, held at 45% over 1.5 CV). The desired product was pooled, concentrated under vacuum, and the final product was precipitated as the sodium salt with an aqueous solution of sodium acetate and 95% absolute ethanol.

[0377] m7 GpppA 2’4’LNA pU (compound 15): [ka]

[0378] Compound 15 was synthesized from N7-Me-GDP (0.341 g, 0.52 mmol), EDC (0.161 g, 0.84 mmol) and imidazole (0.109 g, 1.6 mmol) in 3.4 ml of 10% water in DMSO, magnesium chloride (0.254 mL of 3.15 M MgCl2 solution, 0.8 mmol), and p(LNA)ApU (0.355 g, 0.4 mmol) as described according to general procedure 3. A solid product (0.2976 g) was obtained (yield: 63%). 1 H NMR(500 MHz,D2O):δ 8.13(s,2H),7.63(d,J=8.15 Hz,1H),5.90(d,J=4.25 Hz,1H),5.78(s,1H),5.65(d,J=2.5 Hz,1H),5.30(d,J=8.1 Hz,1H),4.64(m,1H),4.56-4.25(m,12H),4.12(s,3H),4.07(m,1H). 31 P NMR(200 MHz,D2O):δ-1.85(1P),-11.02(2P),-22.37(1P).MS m / z=1102.7(MH).

[0379] m7 G 3’OMe pppA 2’4’LNA pU (compound 16): [ka]

[0380] Compound 16 was synthesized from N7-Me-3'-OMe-GDP (0.42 g, 0.60 mmol), EDC (0.184 g, 0.96 mmol) and imidazole (0.122 g, 1.8 mmol) in 4.2 ml of 10% water in DMSO, magnesium chloride (0.254 mL of 3.15 M MgCl2 solution, 0.8 mmol), and p(LNA)ApU (0.355 g, 0.4 mmol) as described according to general procedure 3. A solid product (0.3281 g) was obtained (yield: 69%). 1 H NMR(500 MHz,D2O):δ 8.14(s,2H),7.64(d,J=8.15 Hz,1H),5.88(d,J=4.7 Hz,1H),5.80(s,1H),5.66(d,J=2.55 Hz,1H),5.31(d,J=8.15 Hz,1H),4.82(m,2H),4.71-4.67(m,2H),4.54-4.4.51(m,7H),4.48-4.27(m,3H),4.07(s,3H),4.05(m,2H),3.50(s,3H). 31 P NMR(200 MHz,D2O):δ-1.83(1P),-10.98(2P),-22.32(1P).MS m / z=1117.9(MH).

[0381] m7 G 3’OMe pppA 2’F pU (compound 17): [ka]

[0382] As described according to General Procedure 3, 4.2 ml of 10% water in DMSO, magnesium chloride (0.254 mL of 3.15 M MgCl2 solution, 0.8 mmol), and pA 2’F Compound 17 was synthesized from N7-Me-3'-OMe-GDP (0.42 g, 0.60 mmol), EDC (0.184 g, 0.96 mmol) and imidazole (0.122 g, 1.8 mmol) in pU (0.348 g, 0.4 mmol). A solid product (0.2788 g) was obtained (yield: 59%). 1H NMR(500 MHz,D2O):δ 8.33(s,1H),8.16(s,1H),7.70(d,J=8.1 Hz,1H),6.23(dd,J=2.3 Hz,14.8 Hz,1H),5.84(m,2H),5.60(d,J=8.15 Hz,1H),5.48-5.36(m,1H),4.88(m,1H),4.78-4.11(m,13H),4.07(s,3H),3.48(s,3H). 31 P NMR(200 MHz,D2O):δ-0.85(1P),-10.97(2P),-22.31(1P).MS m / z=1106.7(MH).

[0383] m7 G 3’OMe pppA 2’OMe pU (compound 22): [ka]

[0384] Prepare 30 mL of 10% water in DMSO, magnesium chloride (1.9 mL of 3.15 M MgCl2 solution, 6.0 mmol), and pH 7.0 as described according to General Procedure 3. 2’OMe Compound 22 was synthesized from N7-Me-3'-OMe-GDP (2.81 g, 4.5 mmol), EDC (1.38 g, 7.2 mmol) and imidazole (0.95 g, 13.95 mmol) in pU (2.61 g, 3.0 mmol). A solid product (1.95 g, 1.64 mmol) was obtained (yield: 55%). NMR(500MHz,D2O)δ 8.40(s,1H),8.13(s,1H),7.85(d,J=8.2,1H),6.04(d,J=5.8,1H),5.92(d,J=4.7),5.78(m,2H),4.61(t,J=4.6,1H), 4.55(m,1H),4.45(t,J=5.2,1H),4.17-4.38(m,10H),4.07(t,J=4.9,1H),4.03(s,3H),3.49(s,3H),3.46(s,3H).31P NMR(200 MHz,D2O)-0.56(1P),-11.05(2P),-22.38(1P).MS m / z=1118.9(MH).

[0385] m7 Gppp m6 A 2’OMe pU (compound 23): [ka]

[0386] As described according to General Procedure 3, 30 mL of 10% water in DMSO, magnesium chloride (1.9 mL of 3.15 M MgCl2 solution, 6.0 mmol), and p m6 A 2’OMe Compound 23 was synthesized from N7-Me-GDP (2.93 g, 4.5 mmol), EDC (1.38 g, 7.2 mmol) and imidazole (0.95 g, 13.95 mmol) in pU (2.65 g, 3.0 mmol). A solid product (1.11 g, 0.94 mmol) was obtained (yield: 31%). NMR(500MHz,D2O)δ 8.34(s,1H),8.13(s,1H),7.81(d,J=8.2,1H),6.04(d,J=5.7,1H),5.89(d,J=4.6,1H),5.81(d,J=3.7,1H),5.72(d,J=8.1,1H), 4.55(m,1H),4.40-4.46(m,2H),4.32-4.39(m,4H),4.23-4.29(m,5H),4.17(m,1H),3.99(s,3H),3.50(s,3H),3.08(bs,3H).31P NMR(200 MHz,D2O)-0.57(1P),-11.01(2P),-22.41(1P).MS m / z=1118.7(MH).

[0387] m7 G 3’OMe ppp m6 A 2’OMe pU (compound 24): [ka]

[0388] As described according to General Procedure 3, 58 mL of 10% water in DMSO, magnesium chloride (4.0 mL of 3.15 M MgCl2 solution, 12.4 mmol), and p m6 A 2’OMe Compound 24 was synthesized from N7-Me-3'-OMe-GDP (5.8 g, 9.4 mmol), EDC (2.9 g, 15.0 mmol) and imidazole (2.0 g, 28.9 mmol) in pU (5.5 g, 6.3 mmol). A solid product (3.71 g, 3.08 mmol) was obtained (yield: 49%). Final yield - 3084 μmol, 49.5%. NMR (500 MHz, DO) δ 8.34(s,1H),8.14(bs,1H),7.82(d,J=8.2,1H),6.04(d,J=5.8,1H),5.90(d ,J=4.6,1H),5.79(d,J=4.2,1H),5.73(d,J=8.2,1H),4.95(m,1H),4.60(t,J =4.5,1H),4.55(m,1H),4.36-4.44(m,4H),4.33(m,2H),4.15-4.29(m,7H),4 .05(t,J=4.9,1H),3.99(s,3H),3.49(s,3H),3.45(s,3H),3.07(bs,3H).31P NMR(200 MHz,D2O)-0.56(1P),-11.08(2P),-22.40(1P).MS m / z=1132.8(MH).

[0389] m7 GpppA 2’F pU (compound 33): [ka]

[0390] N7-Me-GDP imidazolide was purified by pA chromatography as described in General Procedure 3. 2’F Compound 33 was synthesized by coupling to pU dinucleotide. 1H NMR(500 MHz,D2O):δ 8.30(s,1H),8.14(s,1H),7.68(d,J=8.1 Hz,1H),6.18(dd,J=2.1 Hz,14.7 Hz,1H),5.82(dd,J=3.8 Hz,11.2 Hz,2H),5.58(d,J=8.1 Hz,1H),5.45(m,1H),5.35(m,1H),4.56-4.11(m,11H),4.06(s,3H). 31 P NMR(200 MHz,D2O): δ-0.89(1P),-11.01(2P),-22.36(1P).

[0391] Synthesis of additional compounds according to formula IV: p.p. m6 Certain compounds according to formula IV were prepared according to the following general method (General Procedure 4), which involves the synthesis of an activated imidazolide of the ApG dinucleotide and the synthesis of N7-Me 3'OMe guanosine 5'-thiophosphate.

[0392] General procedure 4: p.p. m6 Synthesis of activated imidazolides of ApG dinucleotides: Prepare the peptide using a 20 mL 90% DMSO 10% HO solution of imidazole (2.20 g, 32.27 mmol) and EDC (2.59 g, 16.66 mmol) by combining the reagents and stirring at room temperature for 22 h. m6 Large scale activation of ApG dinucleotide (5.0 g, 6.94 mmol) was performed. After activation, addition of the β-phosphate was achieved by adding 1 M TBAP in DMF (85 mL, 85 mmol) with mixing for 18 h at room temperature. The reaction solution was then double purified by reversed phase and anion exchange chromatography. m6 ApG dinucleotide (0.5 g, 0.625 mmol) was activated with EDC (0.146 g, 0.94 mmol) and imidazole (0.128 g, 1.88 mmol) and used in the subsequent coupling reaction.

[0393] Synthesis of N7-Me 3'OMe guanosine 5'-thiophosphate: 5'OH 3'OMe guanosine was converted to 5'iodo-3'OMe guanosine using the Appel reaction. Specifically, the starting material 5'OH 3'OMe guanosine (1.00 g, 3.36 mmol) was dissolved in anhydrous NMP (10 mL), followed by the addition of imidazole (1.38 g, 20.2 mmol), TPP (2.65 g, 10.1 mmol) and iodine (2.55 g, 10.1 mmol). The reaction was stirred at 25° C. for 3 h and then transferred to 120 mL dichloromethane / water mixture (3:1). The mixture was kept at 4° C. overnight, at which point a white precipitate formed. The precipitate was filtered off under reduced pressure and dried overnight in a vacuum desiccator to obtain 5'iodo-3'OMe guanosine. The material was used without further purification.

[0394] 5'-Thiophosphorylation of 5'-iodo-3'OMe-guanosine was achieved using sodium thiophosphate. 5'-iodo-3'OMe-guanosine (1.00 g, 2.45 mmol) was dissolved in 15 mL of 100 mM NaOH, followed by the addition of sodium thiophosphate (2.21 g, 12.30 mmol). The reaction mixture was heated at 50° C. for 3 h. The product was purified by reverse phase HPLC.

[0395] N-7 methylation of 5'thiophosphate 3'OMe guanosine was achieved by using dimethyl sulfate. 5'thiophosphate 3'OMe guanosine (1.00 g, 2.54 mmol) was dissolved in 10 mL of 0.5 M NaOAc pH 4.0 buffer, followed by the addition of dimethyl sulfate (0.64 g, 5.09 mmol) in portions over 1 h. The pH was maintained at approximately 4.0 using 1 M NaOH as needed. The reaction volume was diluted 10-fold using deionized water and then washed twice with an equal volume (100 mL) of ethyl acetate. The reaction was purified by anion exchange chromatography (QFF Resin) to obtain the product as the triethylamine (TEA) salt.

[0396] m7 G 3’OMe5’-チオ pppm6 A 2’OMe Synthesis of pG (compound 12): [ka]

[0397] N7-Me 3'OMe guanosine 5'-thiophosphate was reacted with pp in the presence of MgCl2 as described in General Procedure 4. m6 A 2’OMe Compound 12 was synthesized by coupling with the activated imidazolide of pG dinucleotide. As above, N7-Me 3'OMe guanosine 5' thiophosphate (150.0 mg, 367 umol) and magnesium chloride 3.15 M (232 uL, 734 umol) were added, and pp m6 A 2’OMe The activated imidazolide of pG dinucleotide was utilized as a solution in 3.44 mL of 90% DMSO 10% H2O at [0.625 mmol / 5 mL]. The components were mixed and stirred overnight at room temperature. The product was purified by anion exchange chromatography using QFF resin followed by QHP anion exchange chromatography. The final yield after precipitation was 45.6%, giving 196.53 mg of white solid product. 1 H NMR(500 MHz,D2O):δ 8.29(s,1H),8.04(s,1H),7.93(s,1H),5.996(d,J=6.2 Hz,1H),5.821(d,J=5.9 Hz,1H),5.704(d,J=5.5 Hz,1H),4.89-4.92,(m,1H),4.74(t,J=3.6 Hz),4.48-4.52,(m,1H),4.43-4.47(m,1H),4.38-4.40(m,2H),4.30-4.34(m,2H),4.21-4.24(m,1H),4.18-4.2 0(m,2H),4.00(t,J=3.75Hz,1H),3.97(s,3H),3.47(s,3H),3.39(s,3H),3.26,(q,J=6.4Hz,2H),3.05,(s,3H). 31P NMR(200 MHz,D2O):δ 7.85(d,J=16.36Hz,1P),0.34(s,1P),-11.17,(d,J=18.18Hz,1P),-23.22(d,J=18.12Hz,1P).C 34 H 48 N 15 O 23 The calculated value for P4S is 1190.17, and the measured value is 1189.17 [MH].

[0398] m7 G 3’OMe p(s)p m6 A 2’OMe Synthesis of pG* (compound 13): [ka] * m7 G 3’OMe p(s)p m6 A 2’OMe pG contains a chiral phosphorothioate moiety and exists as two diastereomers: m7 G 3’OMe pp(s Rp )p m6 A 2’OMe pG and m7 G 3’OMe pp(s sp )p m6 A 2’OMe It is separated into pG.

[0399] A stirred suspension of β-S-3'-OMe-GDP (1.11 g, 1.61 mmol) in DMSO (8 mL) was added at room temperature. m6 A mpG-imidazolide (0.62 g, 0.81 mmol) was added, followed by magnesium chloride (0.51 mL of 3.15 M MgCl2 solution, 1.61 mmol). The reaction proceeded and was purified by anion exchange chromatography as described in general procedure 1. A pure mixture of diastereomers was obtained and concentrated under vacuum to remove acetonitrile. The resulting solution was diluted 5x with water and purified using reverse phase chromatography (Waters Nova-Pak C18 Prep Column, 60 Å, 6 μm, 19 mmX300 mm, WAT025822, using 50 mM ammonium acetate, pH 6.0 as buffer A, acetonitrile as buffer B, a linear gradient of 0% to 5% buffer B for 10 column volumes). The desired product was pooled, concentrated under vacuum, and the final product was precipitated as the sodium salt with an aqueous solution of sodium acetate and 95% absolute ethanol. The solid products were obtained as pure diastereomers (D1: 0.04 mmol, D2: 0.05 mmol, total yield of D1+D2 11.5%). m7 G 3’OMe p(s)p m6 A 2’OMe pG D1 1 H NMR(500MHz,D2O)δ 8.37(s,1H),8.11(s,1H),7.94(s,1H),6.02(d,J=5.7,1H),5.86(d,J=4.1,1H),5.83(d,J=5.6,1H),4.96(m,1H),4.71(m,1H),4.47-4. 53(m,3H),4.42-4.44(m,2H),4.36(m,1H),4.21-4.30(m,5H),4.13(t,J=4.6,1H),4.02(s,3H),3.47(s,3H),3.46(s,3H),3.08(bs,3H); 31 P NMR(200 MHz,D2O)30.49(1P),-0.37(1P),-11.92(2P);MS m / z=1187.5(MH). m7 G 3’OMe p(s)p m6 A 2’OMe pG D2 1H NMR(500MHz,D2O)δ 8.34(s,1H),8.12(s,1H),7.95(s,1H),6.01(d,J=5.8,1H),5.83(m,2H),4.96(m,1H),4.42-4.55(m,3H),4.3 2-4.39(m,4H),4.19-4.28(m,4H),4.14(t,J=4.6,1H),4.03(s,3H),3.48(s,3H),3.47(s,3H),3.08(bs,3H). 31 P NMR(200 MHz,D2O)30.38(1P),-0.41(1P),-11.95(2P);MS m / z=1187.6(MH).

[0400] Synthesis of additional compounds according to formula VI: pP A 2’OMe Certain compounds according to formula VI were prepared according to the following general method (General Procedure 5), which involves the synthesis of an activated imidazolide of pG dinucleotide and the synthesis of N7-Me 3'OMe guanosine 5'-thiophosphate.

[0401] General procedure 5: pP A 2’OMe Synthesis of activated imidazolides of pG dinucleotides: Prepare a pH-compatible 10.0 solution of imidazole (696.5 mg, 10.23 mmol) and EDC (1.01 g, 5.28 mmol) in 10 mL of 90% DMSO 10% HO by combining the reagents and stirring at room temperature for 2 h. 2’OMe Activation of pG dinucleotide (2.0 g, 2.20 mmol) was performed. After activation, addition of the β-phosphate was achieved by adding 1 M TBAP in DMF (38.20 mL, 38.20 mmol) with mixing at room temperature for 18 h. 2’OMe The reaction solution was then double purified by reverse phase chromatography and anion exchange chromatography to obtain pG. 2’OMe pG (1 g, 0.917 mmol) was activated with EDC (0.214 g, 1.375 mmol) and imidazole (0.187 g, 2.75 mmol) and used in the subsequent coupling reaction.

[0402] Synthesis of N7-Me 3'OMe guanosine 5'-thiophosphate: 5'OH 3'OMe guanosine was converted to 5'iodo-3'OMe guanosine using the Appel reaction. The starting material 5'OH 3'OMe guanosine (1.00 g, 3.36 mmol) was dissolved in anhydrous NMP (10 mL) followed by the addition of imidazole (1.38 g, 20.2 mmol), TPP (2.65 g, 10.1 mmol) and iodine (2.55 g, 10.1 mmol). The reaction was stirred at 25°C for 3 h and then transferred to 120 mL dichloromethane / water mixture (3:1). The mixture was kept at 4°C overnight, at which point a white precipitate formed. The precipitate was filtered off under reduced pressure and dried overnight in a vacuum desiccator to give 5'iodo-3'OMe guanosine. The material was used without further purification.

[0403] 5'-Thiophosphorylation of 5'-iodo-3'OMe-guanosine was achieved using sodium thiophosphate. 5'-iodo-3'OMe-guanosine (1.00 g, 2.45 mmol) was dissolved in 15 mL of 100 mM NaOH, followed by the addition of sodium thiophosphate (2.21 g, 12.30 mmol). The reaction mixture was heated at 50° C. for 3 h. The product was purified by reverse phase HPLC.

[0404] N-7 methylation of 5'thiophosphate 3'OMe guanosine was achieved by using dimethyl sulfate. 5'thiophosphate 3'OMe guanosine (1.00 g, 2.54 mmol) was dissolved in 10 mL of 0.5 M NaOAc pH 4.0 buffer, followed by the addition of dimethyl sulfate (0.64 g, 5.09 mmol) in portions over 1 h. The pH was maintained at approximately 4.0 using 1 M NaOH as needed. The reaction volume was diluted 10-fold using deionized water and then washed twice with an equal volume (100 mL) of ethyl acetate. The reaction was purified by anion exchange chromatography (QFF Resin) to obtain the product as the triethylamine (TEA) salt.

[0405] m7 G3’OMe p(s)ppA 2’OMe Synthesis of pG (compound 18): [ka]

[0406] N7-Me 3'OMe guanosine 5'-thiophosphate was reacted with ppA in the presence of MgCl as described in General Procedure 5. 2’OMe Compound 18 was synthesized by coupling with the activated imidazolide of pG dinucleotide. As above, N7-Me 3'OMe guanosine 5' thiophosphate (43 mg, 67 umol) and magnesium chloride 3.15 M (43 uL, 134 umol) were added, and ppA 2’OMe The activated imidazolide of pG dinucleotide was utilized as a solution in 1.1 mL of 90% DMSO 10% H2O at [0.917 mmol / 10 mL]. The components were mixed and stirred overnight at room temperature. The product was purified by anion exchange chromatography using QFF resin followed by QHP anion exchange chromatography. The final yield after precipitation was 43.3%, giving 29 umol of product. 1 H NMR(500 MHz,D2O): δ 8.40(s,1H),8.17(d,J=3.1Hz,1H),7.98(d,J=5.8 Hz,1H),6.00(d,J=5.85 Hz,1H),5.86-5.81(m,2H),4.97-4.88(m,1H),4.69-4.68(m,2H),4.66-4.65(m,2H),4.45-4.37(m,4H),4.33- 4.30(m,2H),4.28-4.22(m,2H),4.15-4.10(m,1H),4.05-4.03(d,J=8.15,3H),3.48-3.42(m,6H),1.93(s,1H). 31 P NMR(200 MHz,D2O):δ 43.90-43.28(dd,1P)0.39(s,1P),-11.23(t,1P),-23.66(m,1P).C 33 H 44 N 15 O 23Calculated value for P4S is 1174.75, measured values ​​are 1174 and 1175.7 [MH] in negative mode.

[0407] m7 G 3’OMe pp(s)pA 2’OMe Synthesis of pG* (compound 19): [ka] * m7 G 3’OMe pp(s)pA 2’OMe pG contains a chiral phosphorothioate moiety and exists as two diastereomers: m7 G 3’OMe pp(s Rp )pA 2’OMe pG and m7 G 3’OMe pp(s Sp )pA 2’OMe It is separated into pG.

[0408] A stirred suspension of β-S-3'-OMe-GDP (0.98 g, 1.42 mmol) in DMSO (7 mL) was added at room temperature with 2’OMe pG-imidazolide (0.54 g, 0.71 mmol) was added followed by magnesium chloride (0.45 mL of a 3.15 M MgCl2 solution, 1.42 mmol). The reaction was worked up and purified by anion exchange chromatography as described in general procedure 1. A pure mixture of diastereomers was obtained and concentrated under vacuum to remove acetonitrile. The resulting solution was diluted 5 times with water and purified using reverse phase chromatography (Waters Nova-Pak C18 Prep Column, 60 Å, 6 μm, 19 mmX300 mm, WAT025822, using 50 mM ammonium acetate as buffer A, pH 6.0, acetonitrile as buffer B, a linear gradient of 0% to 5% buffer B for 10 column volumes). The desired products were pooled and concentrated under vacuum, and the final products were precipitated as sodium salts with an aqueous solution of sodium acetate and 95% absolute ethanol. The solid products were obtained as pure diastereomers (D1: 0.06 mmol, D2: 0.07 mmol, final yield -19% (D1+D2)). m7 G 3’OMe pp(s)pA 2’OMe pD1 1 H NMR(500MHz,D2O)δ 8.45(s,1H),8.14(s,1H),7.96(s,1H),6.04(d,J=6.0,1H),5.88(d,J=4.2,1H),5.85(d,J=5.8,1H),4.98(m,1H),4.49-4. 53(m,3H),4.41-4.45(m,2H),4.36(m,1H),4.19-4.28(m,5H),4.15(t,J=4.8,1H),4.04(s,3H),3.48(s,3H),3.44(s,3H); 31 P NMR(200 MHz,D2O)30.46(1P),-0.35(1P),-11.90(2P);MS m / z=1173.8(MH). m7 G 3’OMe pp(s)pA 2’OMe pG D2: 1 H NMR(500MHz,D2O)δ 8.42(s,1H),8.14(s,1H),7.97(s,1H),6.03(d,J=6.0,1H),5.85(m,2H),4.98(m,1H),4.51-4.55(m,3 H),4.36-4.48(m,3H),4.19-4.30(m,5H),4.16(t,J=4.7,1H),4.06(s,3H),3.49(s,3H),3.45(s,3H); 31 P NMR(200 MHz,D2O)30.36(1P),-0.41(1P),-11.91(2P);MS m / z=1173.8(MH).

[0409] m7 G 3’OMe ppp(s)A 2’OMe Synthesis of pG (compound 20):

change

[0410] N7-methyl-3'-O-methyl-GDP (480 μmol) was suspended in 10% water in DMSO in a clean glass vessel with vigorous stirring at 25°C. EDC-HCl was then added, followed immediately by imidazole. The reaction was then stirred at 25°C and monitored by analytical anion exchange ultra-high performance liquid chromatography (AX-UHPLC) and was complete in 8-18 hours. To the activated N7-methyl-3'-O-methyl-GDP was added 5'-O-thiophosphoryl-2'-O-methyladenylyl-(3'-O-phosphoryl-O-5')-guanosine (240 μmol) and aqueous magnesium chloride. The coupling reaction was stirred at 25°C and monitored by analytical AX-UHPLC. Completion was determined by less than 5% of the dinucleotide starting material remaining. The resulting crude reaction was diluted to 20x crude volume with water and purified by anion exchange chromatography using QFF Sepharose resin and 30-60% 1.5M TEAA in 20% acetonitrile with water. Fractions with purity >80% by analytical AX-UHPLC were pooled and partially evaporated to remove acetonitrile. The combined fractions were then diluted with 6-8 volumes of water and further purified by reversed-phase chromatography using C18 resin in 100mM TEAA, pH 6-7 and 0-10% acetonitrile. Fractions with purity >97% by analytical AX-UHPLC were pooled and evaporated, reducing the product to a viscous oil. The trinucleotides were converted to the sodium salt form by first adding 3M sodium acetate to the post-RP oil and gradually adding 95% aqueous ethanol to precipitate the trinucleotides as sodium salts. The precipitate was washed twice with 95% ethanol, the supernatant was decanted, and the pellet was resuspended in nuclease-free water. Residual ethanol was removed by rotary evaporation and the solution was adjusted to a final concentration of 100 mM (130 μmol, 54% yield) in water. 1H NMR δ(D2O,500 MHz)8.44(s,1H),8.08(s,1H),7.93(s,1H),6.00(m,1H),5.83(m,2H),4.95(m,1H),4.68(m,1H),4.50(m,2H), 4.42(m,1H),4.37(m,1H),4.33-4.25(m,3H),4.20(m,3H),4.12(m,1H),4.01(s,3H),3.45(s,3H),3.41(s,3H). 31 P NMR δ(D2O,200 MHz)43.70(d,J=24.2 Hz,1P),-0.41(s,1P),-11.12(d,J=24.2 Hz,1P),-23.56(dd,J=24 Hz,24 Hz,1P).

[0411] m7 G 3’OMe pppA 2’OMe5’チオ Synthesis of pG (compound 73): [ka]

[0412] The synthesis and processing of α-phosphorothioate trinucleotide capped compound 73 was carried out similarly to the procedure described for the α-phosphorothioate (α-PS) analogue - compound 20. 1 H NMR δ(D2O,500 MHz)8.21(s,1H),8.02(s,1H),7.93(s,1H),5.93(d,J=5 Hz,1H),5.79(m,2H),4.81(m,1H),4.70(m,1H),4.64(m,1H),4.50(m,2H),4.42(m,3H),4.33(m,1H),4.21(m,3H),4.08(t,J=5 Hz,1H),4.00(s,3H),3.46(s,3H),3.42(s,3H). 31 P NMR δ(D2O,200 MHz)7.66(d,J=26.2 Hz,1P),-0.56(s,1P),-11.08(d,J=18.2 Hz,1P),-23.15(dd,J=20 Hz,26.2 Hz,1P).

[0413] m7 G3’OMe ppp m6 A 2’OMe Synthesis of p(s)G* (Compound 74) [ka] * m7 G 3’OMe ppp m6 A 2’OMe p(s)G contains a chiral phosphorothioate moiety and is available as two diastereomers: m7 G 3’OMe ppp m6 A 2’OMe p(s Rp )G and m7 G 3’OMe ppp m6 A 2’OMe p(s Sp )G.

[0414] p m6 A 2’OMe p S The G-dinucleotide was purified by reverse phase chromatography to isolate the diastereomers (Daisogel SP-100-15-ODS-P, 10.0 mM TEAB as buffer A, acetonitrile as buffer B, using a linear gradient of 0% to 11% buffer B for 11 column volumes).

[0415] As described according to General Procedure 1, 1.9 mL of 10% water in DMSO, magnesium chloride (0.13 mL of 3.15 M MgCl2 solution, 0.40 mmol), and p m6 A 2’OMe p S Compound 74 (D1) was synthesized from N7-Me-3'-OMe-GDP (0.19 g, 0.30 mmol), EDC (0.07 g, 0.39 mmol) and imidazole (0.03 g, 0.40 mmol) in G (0.2 g, 0.20 mmol). A solid product (0.12 g, 0.09 mmol) was obtained. Final yield - 94.8 μmol, 47.7%. 1H NMR(500MHz,D2O)δ 8.29(s,1H),8.07(s,1H),8.02(s,1H),5.98(d,J=5.8,1H),5.83(d,J=5.8,1H),5.7 9(d,J=4.2,1H),5.08(m,1H),4.70(t,J=5.5,1H),4.65(t,J=4.6,1H),4.55(m,1H), 4.51(t,J=4.1,1H),4.46(t,J=5.3,1H),4.37(m,3H),4.34(m,1H),4.25(m,3H),4.1 8(m,1H),4.06(t,J=4.7,1H),3.98(s,3H),3.48(s,3H),3.45(s,3H),3.05(bs,3H). 31 P NMR(200 MHz,D2O)55.91(1P),-11.09(2P),-22.42(1P),MS m / z=1188.8(MH)

[0416] As described according to General Procedure 1, 1.9 mL of 10% water in DMSO, magnesium chloride (0.13 mL of 3.15 M MgCl2 solution, 0.40 mmol), and p m6 A 2’OMe p S Compound 74 (D2) was synthesized from N7-Me-3'-OMe-GDP (0.19 g, 0.30 mmol), EDC (0.07 g, 0.39 mmol) and imidazole (0.03 g, 0.40 mmol) in G (0.2 g, 0.20 mmol). A solid product (0.14 g, 0.11 mmol) was obtained. Final yield - 112.3 μmol, 55.9%. NMR(500MHz,D2O)δ 8.29(s,1H),8.06(s,1H),7.96(s,1H),6.00(d,J=5.5,1H),5.79(m,2H), 5.06(m,1H),4.69(t,J=5.2,1H),4.64(t,J=4.6,1H),4.56(m,1H),4.47(t ,J=4.5,1H),4.29(m,1H),4.39(m,1H),4.35(m,3H),4.18-4.27(m,4H),4 .07(t,J=4.7,1H),4.00(s,3H),3.46(s,3H),3.44(s,3H),3.054(bs,3H). 31P NMR(200 MHz,D2O)56.79(1P),-11.05(2P),-22.40(1P).MS m / z=1187.8(MH).

[0417] m7 G 3’OMe ppppA 2’OMe Synthesis of pG (compound 79): [ka]

[0418] Compound 79 was synthesized according to this procedure: Prepare the compound 79 using 8 mL of a 90% DMSO 10% HO solution of imidazole (696.50 mg, 10.23 mmol) and EDC (1.01 g, 5.28 mmol) by combining the reagents and stirring at room temperature for 22 h. m6 A 2’OMe Large scale activation of pG dinucleotide (2.0 g, 2.20 mmol) was performed. After activation, addition of the β-phosphate was achieved by adding 1 M TBAP in DMF (38.20 mL, 38.20 mmol) with mixing for 18 h at room temperature. The reaction solution was then double purified by reverse phase and anion exchange chromatography. If necessary, diphosphate was added for subsequent coupling reactions. m6 A 2’OMe pG dinucleotides were used.

[0419] N7-Me3'-OMe GDP (369.60 mg, 0.764 mmol) was activated with imidazole (161.35 mg, 2.37 mmol) and EDC (234 mg, 1.22 mmol) in 90% DMSO 10% HO (3.70 mL). The activation reaction was allowed to proceed for approximately 18 h, giving 97% conversion of the imidazole-activated N7-Me3'-OMe GDP. m6 A 2’OMepG dinucleotide (400 mg, 0.500 mmol) and magnesium chloride (324 uL, 3.15 M) were added to the same solution. The coupling reaction was stirred for about 48 hours. The reaction mixture was purified using anion exchange chromatography. 1 H NMR(500 MHz,D2O): δ 8.40(s,1H),8.10(s,1H),7.88(s,1H),5.97(d,J=6Hz,1H),5.85(d,J=4.25Hz, 1H),5.80(d,J=5.8Hz,1H)4.90(m,1H)4.86(t,J=5.5Hz,1H)4.69(t,J=4.65Hz,1 H)4.48(m,2H),4.49-4.40(m,3H)4.38(bs,1H)4.32-4.26(m,1H)4.23-4.16(m, 4H)4.15-4.11(m,1H)4.04(s,3H)3.46(s,3H)3.36(s,3H)3.34(s,3H)1.9(s,1H) 31 P NMR(200 MHz,D2O):δ 0.40(s,1P),10.84-1.98(t,1P),22.15-22.47(m,1P) C 33 H 45 N 15 O 27 Calculated value for P5 is 1238.67, measured values ​​are 1237.3 and 1238.6 [MH] in negative mode.

[0420] m7 G 3’OMe pppp m6 A 2’OMe Synthesis of pG (compound 80): [ka]

[0421] Prepare a solution of imidazole (2.20 g, 32.27 mmol) and EDC (2.59 g, 16.66 mmol) in 20 mL of 90% DMSO 10% HO with stirring at room temperature for 22 h. m6 A 2’OMeStarting with large-scale activation of pG dinucleotide (5.0 g, 6.94 mmol), N7-Me-3'Ome guanosine pppp m6 A 2’OMe pG was synthesized. After activation, addition of the β-phosphate was achieved by adding 1 M TBAP in DMF (85 mL, 85 mmol) with mixing at room temperature for 18 hours. The reaction solution was then double purified by reverse phase and anion exchange chromatography. If necessary, diphosphate was added. m6 A 2’OMe A pG dinucleotide is used in the coupling reaction to give the final compound.

[0422] N7-Me3'-OMe GDP (354.2 mg, 0.750 mmol) was activated with imidazole (153.6 mg, 2.325 mmol) and EDC (230 mg, 1.2 mmol) in 90% DMSO 10% HO (3.54 mL). The activation reaction was left for approximately 18 h, giving 97% conversion of the imidazole-activated N7-Me3'-OMe GDP. m6 A 2’OMe pG dinucleotide (400 mg, 0.500 mmol) and magnesium chloride (317.46 uL, 3.15 M) were added to the same solution. The coupling reaction was left for about 48 hours. The reaction mixture was purified using anion exchange chromatography. 1 H NMR(500 MHz,D2O):δ 8.32(s,1H),8.08(s,1H),7.86(s,1H),6.44(s,1H),5.97(d,J=5.5 Hz,1H)5.83(d,J=4.15 Hz,1H),5.78(d,J=5.75 Hz,1H)4.48-4.45(m,4H),4.42-4.32(m,2H)4.10-4.09(m,1H)4.01(s,3H),3.45(s,3H)3.40(s,2H),3.06(bs,1H),1.9(s,1H). 31 P NMR(200 MHz,D2O):δ 0.43(s,1P),-10.85(m,1P),-22.21(m,1P).C 34 H 47 N 15 O27 Calculated value for P5 is 1252.70, measured value is 1251.6 [MH] in negative mode.

[0423] m7 G 3’OMe p(s)pp m6 A 2’OMe Synthesis of pG (compound 81): [ka]

[0424] p.p. m6 Synthesis of activated imidazolides of ApG dinucleotides: Prepare the peptide using a 20 mL 90% DMSO 10% HO solution of imidazole (2.20 g, 32.27 mmol) and EDC (2.59 g, 16.66 mmol) by combining the reagents and stirring at room temperature for 22 h. m6 A 2’OMe Large scale activation of pG dinucleotide (5.0 g, 6.94 mmol) was performed. After activation, addition of the β-phosphate was achieved by adding 1 M TBAP in DMF (85 mL, 85 mmol) with mixing at room temperature for 18 h. The reaction solution was then double purified by reverse phase chromatography and anion exchange chromatography. The material was then precipitated using 2% volume of starting material NaClO4 (100 mg) and 500 mL acetone. The precipitate was a white solid (6 g). If necessary, the diphosphate was added for subsequent coupling reactions. m6 A 2’OMe pG dinucleotides were used.

[0425] Synthesis of N7-Me 3'OMe guanosine 5'-thiophosphate: N7-methylation of 5'thiophosphorylguanosine was carried out using 5'OH 3'Omeguanosine (1 g, 3.36 mmol). The starting material was methylated using a solution of CH3I (1.25 mL, 20.07 mmol) in 10 mL of DMF. The reaction was stirred at room temperature for 4 days. The reaction mixture changed from a cloudy suspension to a clear yellow liquid over the course of 4 days. The reaction mixture was then concentrated to 2.5 mL and triturated with 50 mL of dichloromethane to precipitate a white / yellow solid. The solid was vacuum filtered until no visible liquid was seen.

[0426] 5'-Thiophosphorylation of N7-Me 3'OMe guanosine was achieved using N7-Me 3'OMe guanosine (1 g, 3.2 mmol). The reaction was carried out under argon using dry materials. A solution of PSCl3 (487 uL, 4.8 mmol) and 2,6 lutidine (1118 uL, 4.6 mmol) in TMP was added at 0°C. The reaction was complete after 8-9 hours. The reaction was then quenched with 1.5 M TEAA to obtain a pH of 5-6. The reaction was purified by anion exchange chromatography using QFF resin to obtain the product as the TEA salt.

[0427] In the presence of MgCl2, N7-Me 3'OMe guanosine 5'-thiophosphate was reacted with pp m6 A 2’OMe Compound 81 was synthesized by coupling with the activated imidazolide of pG dinucleotide. N7-Me 3'OMe guanosine 5' thiophosphate (184 mg, 287 umol) and magnesium chloride 3.15 M (182 uL, 574 umol) were added and pp m6 A 2’OMe The activated imidazolide of pG dinucleotide was utilized as a solution in 3.44 mL of 90% DMSO 10% H2O at [0.625 mmol / 5 mL]. The components were mixed and stirred at room temperature for 4.5 days. The product was purified by anion exchange chromatography using QFF resin followed by QHP anion exchange chromatography. The final yield after precipitation was 1.805 mL (100 mM), 177 umol (5494.42 OD).1 H NMR(500 MHz,D2O): δ 8.30(s,1H),8.05(s,1H),7.92(d,J=4.4 Hz,1H),5.96(d,J=5.75Hz,1H),5.81(t,J=4.91Hz,1H)5.77-5.76(dd,J=3.9Hz,3.15Hz,1H)4.94-4.89(m,1H),4.63-4.58(m,1H),4.58-4.52(m ,2H)4.46-4.34(m,4H),4.3-4.2(m,4H),4.09-4.05(m,1H),3.97-3.95( d,J=9.95Hz,3H),3.44-42(m,J=2.35Hz,6H),3.05(bs,1H),1.9(s,1H). 31 P NMR(200 MHz,D2O):δ 43.86-43.27(dd,1P)0.40(s,1P),-11.16(t,1P),23.51(m,1P).C 34 H 47 N 15 O 23 Calculated value for P4S is 1189.79, measured values ​​are 1189 and 1187.8 [MH] in negative mode.

[0428] Example 2: mRNA synthesis For further testing, mRNAs were synthesized containing each of the cap analogs including the compounds described herein, along with comparative examples (see Table 1 for the analogs synthesized). [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13] [Table 1-14] [Table 1-15]

[0429] Example 3: In vitro performance of cap analogs In vitro testing was performed on the cap analogs described herein using a commercially available wheat germ extract cell-free translation system (PROMEGA L4380; Promega Corporation, Madison, WI). For the assay, 20 ng / μL of luciferase mRNA capped using the cap analogs listed in Table 1 was mixed with the translation components of the wheat germ extract system. The components were allowed to react for 2 hours at room temperature to allow protein production. The crude protein mixture was subjected to luciferin substrate and incubated in the dark for 10 minutes. Total luciferase signal was collected in a 96-well plate format as relative luminescence units (RLU). Blank wells containing luciferase alone and wheat germ extract controls without luciferase mRNA were used as system controls. The measured relative luminescence results are shown in Figure 1, with RLU values ​​indicated.

[0430] Example 4: In vivo performance of cap analogs Female CD-1 mice aged 8–9 weeks were randomized into groups of 5 animals based on their body weight at the start of the study (Charles River Laboratories, Discovery Research Services North Carolina). Lipid nanoparticle formulations containing mRNA (N1-methylpseudouridine-5'-triphosphate (N1-me-PsU) modified firefly luciferase (Fluc) mRNA, capped using the cap analogs listed in Table 1) (LNP:mRNA) test articles were diluted in PBS to achieve delivery of 1 mg / kg in a single bolus via tail vein injection. During the study period, each mouse was weighed once daily and blood was drawn twice for serum analysis by Mouse Cytokine / Chemokine 26-Plex ProcartaPlex Panel 1 on the Luminex platform (Thermo Fisher Scientific; Waltham, MA). Luciferase activity was measured by whole-body bioluminescence imaging using an IVIS Spectrum CT system by Perkin Elmer (Greenville, SC) at six time points after mRNA injection (3, 6, 9, 12, 24 and 48 hours after injection). D-luciferin was delivered by intraperitoneal injection (150 mg / kg total) 10 min prior to each imaging session. Measured luciferase expression results are shown in Figure 2, showing integrated flux from 3 to 48 hours. Figure 3 shows a representative animal from each cohort of five animals, shown across five imaging time points after LNP:mRNA and luciferin injection (3, 6, 9, 12 and 24 hours after injection) for a representative compound. Luminescence intensity scale is on the left.

[0431] When mice were injected with lipid nanoparticle formulations containing mRNA and luciferin produced according to Example 2, in vivo luciferase expression was significantly higher than in mice injected with LNPs containing mRNA containing the novel cap analogs described herein. m7 GpppA 2’OMewas comparable or increased compared to mRNA containing pG (control), see FIG.

[0432] m7 G 3’OMe ppp m6 A 2’OMe pG (compound 30), m7 G 3’Ome pppA 2’OMe pG (compound 29), m7 G 3’OMe pppA 2’,4’-LNA pG (compound 2), m7 G 2’4’-LNA pppA 2’OMe pG (compound 32), m7 G 3’OMe ppp (diaminopurine) 2’OMe pG (compound 8), m7 GpppA 2’,4’-LNA pG (Compound 1) and m7 Gppp m6 A 2’OMe pG (compound 31) is m7 GpppA 2’OMe The results showed increased in vivo luciferase expression compared to pG (control). m7 Gppp m6 A 2’OMe pG (compound 31) showed comparable in vivo luciferase expression. The data show that the analogs described herein m7 GpppA 2’OMe These results demonstrate increased or equivalent in vivo translation compared to pG (control).

[0433] The selected data obtained from the luciferase expression experiments above were sorted into m7 A compound having a 3'OH group in the G portion; m7 A direct comparison with the same compound having a 3'OMe modification at the G moiety is shown, see Figure 4. Surprisingly, in each of the comparisons tested and analyzed, m7 The 3'OMe modification to G results in increased in vivo translation. As shown in Figure 4, m7 Has a 3'OMe group on G m7 GpppA 2’OMe pG (control)m7 G 3’Ome pppA 2’OMe Compared to pG (compound 29), m7 GpppA 2’OMe pG (control) was measured (first column). m7 Has a 3'OMe group on G m7 Gppp m6 A 2’OMe pG (Compound 31) m7 G 3’OMe ppp m6 A 2’OMe Compared to pG (compound 30), m7 Gppp m6 A 2’OMe pG (compound 31) was measured (second column). m7 Has a 3'OMe group on G m7 GpppA 2’,4’-LNA pG (Compound 1) m7 G 3’OMe pppA 2’,4’-LNA Compared to pG (compound 2), m7 GpppA 2’,4’-LNA pG (Compound 1) was measured (third column). m7 Has a 3'OMe group on G m7 Gppp (diaminopurine) 2’OMe pG (compound 7) m7 G 3’OMe ppp (diaminopurine) 2’OMe Compared to pG (compound 8), m7 Gppp (diaminopurine) 2’OMe pG (compound 7) was measured (fourth column). In each of these examples, m7 The 3'OMe modification to G results in increased luciferase expression in vivo and therefore increased translation.

[0434] m 7 G 3’-OMe pppA 2’OMe-5’ビニル For pG (compound 5), m 7 GpppA 2’OMe-5’ビニル pG (compound 4; m7 G) compared to 7 G 2’OMe3’OMe pppA 2’OMeFor pG (compound 25), m 7 G 2’OMe pppA 2’OMe pG (compound 26; m7 G) compared to 7 G 2’OMe3’OMe ppp m6 A 2’OMe For pG (compound 27), m 7 G 2’OMe ppp m6 A 2’OMe pG (compound 28; m7 A similar increase in luciferase expression was obtained in vivo compared to the control (lacking a 3'OMe group on G). See FIG. 2.

[0435] The compounds and methods of the appended claims are not limited in scope by the specific compounds and methods described herein, which are intended as examples of some aspects of the claims, and any compounds and methods that are functionally equivalent are within the scope of this disclosure. In addition to those shown and described herein, various modifications of the compounds and methods are intended to be included within the scope of the appended claims. Furthermore, although only certain representative compounds, methods and aspects of these compounds and methods are specifically described, other compounds and methods are intended to be included within the scope of the appended claims. Thus, although a combination of steps, elements, components or elements may be explicitly referred to herein, all other combinations of steps, elements, components and elements are included, even if not explicitly stated.

Claims

1. A compound of the formula: 【Chemistry 1】 or a stereoisomer thereof, 【Chemistry 2】 is a single bond or a double bond; R １ is H or CH ３ and R ２ is H, and R ３ OCH ３ or F, or R ２ and R ３ are covalently bonded to each other and together with the intermediate atom form a 2'-O,4'-C methylene bridge; X １ is O or CH; X ２ is CH ２ or CH; X ３ is O or S; Each independent Y is H ＋ or a cation; n is 0, 1, 2, 3 or 4; X １ When is CH, X ２ is CH, 【Chemistry 3】 is a double bond).

2. The compound of claim 1 having the following structure: 【Chemistry 4】 Or a stereoisomer thereof.

3. The compound of claim 1 having the following structure: 【Chemistry 5】 Or a stereoisomer thereof.

4. The compound of claim 1 having the following structure: 【Chemistry 6】 Or a stereoisomer thereof.

5. The compound of claim 1 having the following structure: 【Chemistry 7】 Or a stereoisomer thereof.

6. The compound of claim 1 having the following structure: 【Chemistry 8】 Or a stereoisomer thereof.

7. The compound of claim 1 having the following structure: 【Chemistry 9】 Or a stereoisomer thereof.

8. A compound of the formula: 【Chemistry 10】 or a stereoisomer thereof, R １ is H or CH ３ and X １ is O or S; Each independent Y is H ＋ or a cation; n is 0, 1, 2, 3 or 4; X １ If S, then R １ is CH ３ (It is.)

9. The compound of claim 8 having the following structure: 【Chemistry 11】 Or a stereoisomer thereof.

10. The compound of claim 8 having the following structure: 【Chemistry 12】 Or a stereoisomer thereof.

11. The compound of claim 8 having the following structure: 【Chemistry 13】 Or a stereoisomer thereof.

12. A compound of the formula: 【Chemistry 14】 or a stereoisomer thereof, R １ is H or CH ３ and Each independent Y is H ＋ or a cation; n is 0, 1, 2, 3 or 4).

13. The compound of claim 12 having the following structure: 【Chemistry 15】 Or a stereoisomer thereof.

14. The compound of claim 12 having the following structure: 【Chemistry 16】 Or a stereoisomer thereof.

15. A compound of the formula: 【Chemistry 17】 or a stereoisomer thereof, R １ is H or CH ３ and X １ , X ２ and X ３ are each independently selected from O and S; X １ , X ２ or X ３ If one of is S, then X １ , X ２ and X ３ the remainder is O; Each independent Y is H ＋ or a cation; n is 0, 1, 2, 3 or 4).

16. The compound of claim 15 having the following structure: 【Chemistry 18】 Or a stereoisomer thereof.

17. The compound of claim 15 having the following structure: 【Chemistry 19】 Or a stereoisomer thereof.

18. The compound of claim 15 having the following structure: 【Chemistry 20】 Or a stereoisomer thereof.

19. The compound of claim 15 having the following structure: 【Chemistry 21】 Or a stereoisomer thereof.

20. A compound of the formula: 【Chemical 22】 or a stereoisomer thereof, R １ is H or CH ３ and R ２ is H, and R ３ is F or R ２ and R ３ are covalently bonded to each other and together with the intermediate atom form a 2'-O,4'-C methylene bridge; Each independent Y is H ＋ or a cation; n is 0, 1, 2, 3 or 4; R ２ is H, and R ３ If F, then R １ is not H).

21. 21. The compound of claim 20 having the structure: 【Chemistry 23】 Or a stereoisomer thereof.

22. 21. The compound of claim 20 having the structure: 【Chemistry 24】 Or a stereoisomer thereof.

23. 21. The compound of claim 20 having the structure: 【Chemistry 25】 Or a stereoisomer thereof.

24. A compound of the formula: 【Chemistry 26】 or a stereoisomer thereof, R １ is H or CH ３ and X １ , X ２ , X ３ and X ４ are each independently selected from O and S; Each independent Y is H ＋ or a cation; n is 0, 1, 2, 3 or 4; X １ , X ２ , X ３ and X ４ (one of which is S).

25. 25. The compound of claim 24 having the structure: 【Chemical 27】 Or a stereoisomer thereof.

26. 25. The compound of claim 24 having the structure: 【Chemistry 28】 Or a stereoisomer thereof.

27. 25. The compound of claim 24 having the structure: 【Chemical 29】 Or a stereoisomer thereof.

28. 25. The compound of claim 24 having the structure: 【Chemistry 30】 Or a stereoisomer thereof.

29. 25. The compound of claim 24 having the structure: 【Chemistry 31】 Or a stereoisomer thereof.

30. 25. The compound of claim 24 having the structure: 【Chemistry 32】 。

31. A compound of the formula: 【Chemical 33】 or a stereoisomer thereof, R １ and R ２ are each independently H and CH ３ Selected from: Each independent Y is H ＋ or a cation; n is 0, 1, 2, 3 or 4; R １ Or R ２ At least one of them is CH ３ (It is.)

32. 32. The compound of claim 31 having the structure: 【Chemical 34】 Or a stereoisomer thereof.

33. 32. The compound of claim 31 having the structure: 【Chemistry 35】 Or a stereoisomer thereof.

34. 32. The compound of claim 31 having the structure: 【Chemical 36】 Or a stereoisomer thereof.

35. A compound of the formula: 【Chemical 37】 or a stereoisomer thereof, R １ and R ２ are each independently H and CH ３ Selected from: Each independent Y is H ＋ or a cation; n is 0, 1, 2, 3 or 4).

36. 36. The compound of claim 35 having the structure: 【Chemical 38】 Or a stereoisomer thereof.

37. 36. The compound of claim 35 having the structure: 【Chemical 39】 Or a stereoisomer thereof.

38. 36. The compound of claim 35 having the structure: 【Chemistry 40】 Or a stereoisomer thereof.

39. 36. The compound of claim 35 having the structure: 【Chemistry 41】 Or a stereoisomer thereof.

40. A compound of the formula: 【Chemistry 42】 or a stereoisomer thereof, R １ is H or CH ３ and R ２ is OH, F, substituted or unsubstituted alkoxy, or thio; R ３ is H or CH ３ and Each independent Y is H ＋ or a cation; n is 0, 1, 2, 3 or 4; R ２ When is OH, R １ is not H).

41. 41. The compound of claim 40 having the structure: 【Chemistry 43】 Or a stereoisomer thereof.

42. 41. The compound of claim 40 having the structure: 【Chemistry 44】 Or a stereoisomer thereof.

43. 41. The compound of claim 40 having the structure: 【Chemistry 45】 。

44. 41. The compound of claim 40 having the structure: 【Chemistry 46】 Or a stereoisomer thereof.

45. 41. The compound of claim 40 having the structure: 【Chemistry 47】 Or a stereoisomer thereof.

46. 41. The compound of claim 40 having the structure: 【Chemistry 48】 Or a stereoisomer thereof.

47. 41. The compound of claim 40 having the structure: 【Chemistry 49】 Or a stereoisomer thereof.

48. A compound of the formula: 【Chemistry 50】 or a stereoisomer thereof, R １ is H and CH ３ and Each independent Y is H ＋ or a cation; n is 0, 1, 2, 3 or 4).

49. 49. The compound of claim 48 having the structure: 【Chemistry 51】 Or a stereoisomer thereof.

50. 49. The compound of claim 48 having the structure: 【Chemistry 52】 Or a stereoisomer thereof.

51. A compound of the formula: 【Chemistry 53】 or a stereoisomer thereof, R １ is H and CH ３ and Each independent Y is H ＋ or a cation; n is 0, 1, 2, 3 or 4).

52. 52. The compound of claim 51 having the structure: 【Chemical 54】 Or a stereoisomer thereof.

53. 52. The compound of claim 51 having the structure: 【Chemistry 55】 Or a stereoisomer thereof.

54. A compound of the formula: 【Chemistry 56】 or a stereoisomer thereof, R １ and R ２ are each independently H and CH ３ Selected from: Each independent Y is H ＋ or a cation; n is 0, 1, 2, 3, 4 or 5).

55. 55. The compound of claim 54 having the structure: 【Chemistry 57】 Or a stereoisomer thereof.

56. 55. The compound of claim 54 having the structure: 【Chemistry 58】 Or a stereoisomer thereof.

57. 57. The compound of any one of claims 1 to 56, which is a deuterated form of said compound.

58. A compound selected from the group consisting of: 【Chemistry 59】 Or a stereoisomer thereof.

59. 60. A pharmaceutical composition comprising a compound according to any one of claims 1 to 58 and a pharma- ceutically acceptable carrier.

60. 60. An RNA molecule comprising a 5' cap, wherein the 5' cap comprises a compound according to any one of claims 1 to 58.

61. 61. The RNA molecule of claim 60, which is a messenger RNA (mRNA) molecule or a self-amplifying RNA (saRNA) molecule.

62. 62. A method of inducing a therapeutic effect in a subject comprising administering to the subject an RNA molecule described in claim 60 or 61.

63. 60. A method of administering to an animal a therapeutic dosage unit of an mRNA molecule comprising a 5' cap, said 5' cap comprising a compound according to any one of claims 1 to 58.

64. 64. The method of claim 63, wherein the therapeutic dose unit of the mRNA molecule comprises less than 7 ng / μg double-stranded RNA (dsRNA).

65. 65. The method of claim 64, wherein the subject exhibits increased tolerance to an administered therapeutic dose unit of the mRNA molecule compared to an equivalent therapeutic dose unit of the mRNA molecule comprising 7 ng / μg or more of dsRNA.

66. 66. The method of claim 65, wherein the increased tolerability is determined by measuring one or more of body weight, organ weight, aspartate aminotransferase (AST) levels, alanine transaminase (ALT) levels, C-reactive protein (CRP) levels, procalcitonin (PCT) levels, interleukin-6 (IL-6) levels, erythrocyte sedimentation rate (ESR), serum amyloid A levels, and serum ferritin levels before and for a period of time after administration.

67. 67. The method of claim 65 or 66, wherein the increased tolerability is measured by testing in a standard in vivo assay.

68. The increased tolerability may be at least a 20% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least a 25% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least a 30% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least a 35% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least a 40% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least a 45% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least a 50% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least a 55% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, 68. The method of any one of claims 65 to 67, wherein the increased tolerance of the administered dosage unit is at least 60% when measured by testing in an assay, at least 65% when measured by testing in a standard in vivo assay, at least 70% when measured by testing in a standard in vivo assay, at least 75% when measured by testing in a standard in vivo assay, at least 80% when measured by testing in a standard in vivo assay, at least 85% when measured by testing in a standard in vivo assay, at least 90% when measured by testing in a standard in vivo assay, or at least 95% when measured by testing in a standard in vivo assay.

69. The therapeutic dosage unit of the mRNA molecule is less than 6.9 ng / μg double stranded RNA (dsRNA), less than 6.8 ng / μg double stranded RNA (dsRNA), less than 6.7 ng / μg double stranded RNA (dsRNA), less than 6.6 ng / μg double stranded RNA (dsRNA), less than 6.5 ng / μg double stranded RNA (dsRNA), less than 6.4 ng / μg double stranded RNA (dsRNA), less than 6.3 ng / μg double stranded RNA (dsRNA), less than 6.2 ng / μg double stranded RNA (dsRNA), less than 6.1 ng / μg double stranded RNA (dsRNA), less than 6.0 ng / μg double stranded RNA (dsRNA), less than 6.2 ... less than 5.9 ng / μg double stranded RNA (dsRNA), less than 5.8 ng / μg double stranded RNA (dsRNA), less than 5.7 ng / μg double stranded RNA (dsRNA), less than 5.6 ng / μg double stranded RNA (dsRNA), less than 5.5 ng / μg double stranded RNA (dsRNA), less than 5.4 ng / μg double stranded RNA (dsRNA), less than 5.3 ng / μg double stranded RNA (dsRNA), less than 5.2 ng / μg double stranded RNA (dsRNA), less than 5.1 ng / μg double stranded RNA (dsRNA), 5.0 less than 4.9 ng / μg double stranded RNA (dsRNA), less than 4.8 ng / μg double stranded RNA (dsRNA), less than 4.7 ng / μg double stranded RNA (dsRNA), less than 4.6 ng / μg double stranded RNA (dsRNA), less than 4.5 ng / μg double stranded RNA (dsRNA), less than 4.4 ng / μg double stranded RNA (dsRNA), less than 4.3 ng / μg double stranded RNA (dsRNA), less than 4.2 ng / μg double stranded RNA (dsRNA), less than 4.1 ng / μg double stranded RNA (dsRNA), 4 0 ng / μg double-stranded RNA (dsRNA), 3.9 ng / μg double-stranded RNA (dsRNA), 3.8 ng / μg double-stranded RNA (dsRNA), 3.7 ng / μg double-stranded RNA (dsRNA), 3.6 ng / μg double-stranded RNA (dsRNA), 3.5 ng / μg double-stranded RNA (dsRNA), 3.4 ng / μg double-stranded RNA (dsRNA), 3.3 ng / μg double-stranded RNA (dsRNA), 3.2 ng / μg double-stranded RNA (dsRNA), 3.1 ng / μg double-stranded RNA (dsRNA),Less than 3.0 ng / μg double stranded RNA (dsRNA), less than 2.9 ng / μg double stranded RNA (dsRNA), less than 2.8 ng / μg double stranded RNA (dsRNA), less than 2.7 ng / μg double stranded RNA (dsRNA), less than 2.6 ng / μg double stranded RNA (dsRNA), less than 2.5 ng / μg double stranded RNA (dsRNA), less than 2.4 ng / μg double stranded RNA (dsRNA), less than 2.3 ng / μg double stranded RNA (dsRNA) sRNA), less than 2.2 ng / μg double stranded RNA (dsRNA), less than 2.1 ng / μg double stranded RNA (dsRNA), less than 2.0 ng / μg double stranded RNA (dsRNA), less than 1.9 ng / μg double stranded RNA (dsRNA), less than 1.8 ng / μg double stranded RNA (dsRNA), less than 1.7 ng / μg double stranded RNA (dsRNA), less than 1.6 ng / μg double stranded RNA (dsRNA), less than 1.5 ng / μg double stranded RNA (dsRNA), RNA (dsRNA), less than 1.4 ng / μg double stranded RNA (dsRNA), less than 1.3 ng / μg double stranded RNA (dsRNA), less than 1.2 ng / μg double stranded RNA (dsRNA), less than 1.1 ng / μg double stranded RNA (dsRNA), less than 1.0 ng / μg double stranded RNA (dsRNA), less than 0.9 ng / μg double stranded RNA (dsRNA), less than 0.8 ng / μg double stranded RNA (dsRNA), 0.7 ng / μg 69. The method of any one of claims 65 to 68, comprising less than 0.05 ng / μg double stranded RNA (dsRNA), less than 0.6 ng / μg double stranded RNA (dsRNA), less than 0.5 ng / μg double stranded RNA (dsRNA), less than 0.4 ng / μg double stranded RNA (dsRNA), less than 0.3 ng / μg double stranded RNA (dsRNA), less than 0.2 ng / μg double stranded RNA (dsRNA), or less than 0.1 ng / μg double stranded RNA (dsRNA).

70. 1. A method of administering to an animal a therapeutic dosage unit of an mRNA molecule comprising a 5′ cap, said 5′ cap being a compound selected from the group consisting of: 【Chemistry 60-1】 【Chemistry 60-2】 or a stereoisomer thereof, the therapeutic dosage unit of the mRNA molecule comprises less than 7 ng / μg double-stranded RNA (dsRNA); The method, wherein the subject exhibits increased tolerance to an administered therapeutic dosage unit of the mRNA molecule compared to an equivalent therapeutic dosage unit of the mRNA molecule comprising 7 ng / μg or more of dsRNA.

71. 71. The method of claim 70, wherein the increased tolerance is determined by measuring one or more of body weight, organ weight, aspartate aminotransferase (AST) levels, alanine transaminase (ALT) levels, C-reactive protein (CRP) levels, procalcitonin (PCT) levels, interleukin-6 (IL-6) levels, erythrocyte sedimentation rate (ESR), serum amyloid A levels, and serum ferritin levels before administration and for a period of time after administration.

72. 72. The method of claim 70 or 71, wherein the increased tolerability is measured by testing in a standard in vivo assay.

73. The increased tolerability may be at least a 20% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least a 25% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least a 30% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least a 35% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least a 40% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least a 45% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least a 50% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, at least a 55% increase in tolerability for the administered dosage unit when measured by testing in a standard in vivo assay, 73. The method of any one of claims 70 to 72, wherein the increased tolerability of the administered dosage unit is at least 60% when measured by testing in an assay, at least 65% when measured by testing in a standard in vivo assay, at least 70% when measured by testing in a standard in vivo assay, at least 75% when measured by testing in a standard in vivo assay, at least 80% when measured by testing in a standard in vivo assay, at least 85% when measured by testing in a standard in vivo assay, at least 90% when measured by testing in a standard in vivo assay, or at least 95% when measured by testing in a standard in vivo assay.

74. The therapeutic dosage unit of the mRNA molecule is less than 6.9 ng / μg double stranded RNA (dsRNA), less than 6.8 ng / μg double stranded RNA (dsRNA), less than 6.7 ng / μg double stranded RNA (dsRNA), less than 6.6 ng / μg double stranded RNA (dsRNA), less than 6.5 ng / μg double stranded RNA (dsRNA), less than 6.4 ng / μg double stranded RNA (dsRNA), less than 6.3 ng / μg double stranded RNA (dsRNA), less than 6.2 ng / μg double stranded RNA (dsRNA), less than 6.1 ng / μg double stranded RNA (dsRNA), less than 6.0 ng / μg double stranded RNA (dsRNA), less than 6.2 ... less than 5.9 ng / μg double stranded RNA (dsRNA), less than 5.8 ng / μg double stranded RNA (dsRNA), less than 5.7 ng / μg double stranded RNA (dsRNA), less than 5.6 ng / μg double stranded RNA (dsRNA), less than 5.5 ng / μg double stranded RNA (dsRNA), less than 5.4 ng / μg double stranded RNA (dsRNA), less than 5.3 ng / μg double stranded RNA (dsRNA), less than 5.2 ng / μg double stranded RNA (dsRNA), less than 5.1 ng / μg double stranded RNA (dsRNA), 5.0 less than 4.9 ng / μg double stranded RNA (dsRNA), less than 4.8 ng / μg double stranded RNA (dsRNA), less than 4.7 ng / μg double stranded RNA (dsRNA), less than 4.6 ng / μg double stranded RNA (dsRNA), less than 4.5 ng / μg double stranded RNA (dsRNA), less than 4.4 ng / μg double stranded RNA (dsRNA), less than 4.3 ng / μg double stranded RNA (dsRNA), less than 4.2 ng / μg double stranded RNA (dsRNA), less than 4.1 ng / μg double stranded RNA (dsRNA), 4 0 ng / μg double-stranded RNA (dsRNA), 3.9 ng / μg double-stranded RNA (dsRNA), 3.8 ng / μg double-stranded RNA (dsRNA), 3.7 ng / μg double-stranded RNA (dsRNA), 3.6 ng / μg double-stranded RNA (dsRNA), 3.5 ng / μg double-stranded RNA (dsRNA), 3.4 ng / μg double-stranded RNA (dsRNA), 3.3 ng / μg double-stranded RNA (dsRNA), 3.2 ng / μg double-stranded RNA (dsRNA), 3.1 ng / μg double-stranded RNA (dsRNA),Less than 3.0 ng / μg double stranded RNA (dsRNA), less than 2.9 ng / μg double stranded RNA (dsRNA), less than 2.8 ng / μg double stranded RNA (dsRNA), less than 2.7 ng / μg double stranded RNA (dsRNA), less than 2.6 ng / μg double stranded RNA (dsRNA), less than 2.5 ng / μg double stranded RNA (dsRNA), less than 2.4 ng / μg double stranded RNA (dsRNA), less than 2.3 ng / μg double stranded RNA (dsRNA) sRNA), less than 2.2 ng / μg double stranded RNA (dsRNA), less than 2.1 ng / μg double stranded RNA (dsRNA), less than 2.0 ng / μg double stranded RNA (dsRNA), less than 1.9 ng / μg double stranded RNA (dsRNA), less than 1.8 ng / μg double stranded RNA (dsRNA), less than 1.7 ng / μg double stranded RNA (dsRNA), less than 1.6 ng / μg double stranded RNA (dsRNA), less than 1.5 ng / μg double stranded RNA (dsRNA), RNA (dsRNA), less than 1.4 ng / μg double stranded RNA (dsRNA), less than 1.3 ng / μg double stranded RNA (dsRNA), less than 1.2 ng / μg double stranded RNA (dsRNA), less than 1.1 ng / μg double stranded RNA (dsRNA), less than 1.0 ng / μg double stranded RNA (dsRNA), less than 0.9 ng / μg double stranded RNA (dsRNA), less than 0.8 ng / μg double stranded RNA (dsRNA), 0.7 ng / μg 74. The method of any one of claims 70 to 73, comprising less than 0.05 ng / μg double stranded RNA (dsRNA), less than 0.6 ng / μg double stranded RNA (dsRNA), less than 0.5 ng / μg double stranded RNA (dsRNA), less than 0.4 ng / μg double stranded RNA (dsRNA), less than 0.3 ng / μg double stranded RNA (dsRNA), less than 0.2 ng / μg double stranded RNA (dsRNA), or less than 0.1 ng / μg double stranded RNA (dsRNA).

75. 1. A method of increasing in vivo translation of a polypeptide in a subject, comprising administering to the subject a therapeutic dosage unit comprising an effective amount of an mRNA encoding a polypeptide, wherein the mRNA has a 5′ cap having the following formula: 【Chemistry 61】 or a stereoisomer thereof, wherein the therapeutic dosage unit administered is at least 20% lower than the therapeutic dosage unit required to induce the same response in said subject when a comparison mRNA encoding said polypeptide is administered, said comparison mRNA having a 5' cap having the following formula: 【Chemistry 62】 Or a stereoisomer thereof.

76. 1. A method of increasing in vivo translation of a polypeptide in a subject, comprising administering to the subject a therapeutic dosage unit comprising an effective amount of an mRNA encoding a polypeptide, wherein the mRNA has a 5′ cap having the following formula: 【Chemistry 63】 or a stereoisomer thereof, wherein the therapeutic dosage unit administered is at least 20% lower than the therapeutic dosage unit required to induce the same response in said subject when a comparison mRNA encoding said polypeptide is administered, said comparison mRNA having a 5' cap having the following formula: 【Chemistry 64】 Or a stereoisomer thereof.

77. 1. A method of increasing in vivo translation of a polypeptide in a subject, comprising administering to the subject a therapeutic dosage unit comprising an effective amount of an mRNA encoding a polypeptide, wherein the mRNA has a 5′ cap having the following formula: 【Chemistry 65】 or a stereoisomer thereof, wherein the dose administered is at least 20% lower than the therapeutic dose unit required to elicit the same response in said subject when a comparison mRNA encoding said polypeptide is administered, said comparison mRNA having a 5' cap having the following formula: 【Chemistry 66】 Or a stereoisomer thereof.

78. 1. A method of increasing in vivo translation of a polypeptide in a subject, comprising administering to the subject a therapeutic dosage unit comprising an effective amount of an mRNA encoding a polypeptide, wherein the mRNA has a 5′ cap having the following formula: 【Chemistry 67】 or a stereoisomer thereof, wherein the therapeutic dosage unit administered is at least 20% lower than the therapeutic dosage unit required to induce the same response in said subject when a comparison mRNA encoding said polypeptide is administered, said comparison mRNA having a 5' cap having the following formula: 【Chemistry 68】 Or a stereoisomer thereof.

79. The method of claims 75-78, wherein the subject exhibits increased tolerance to the administered therapeutic dosage unit as compared to the therapeutic dosage unit required to elicit the same response in the subject when administered the comparison mRNA, as measured by testing in a standard in vivo assay.

80. The increased tolerability may be at least a 20% increase in tolerability for the therapeutic dosage unit administered when measured by testing in a standard in vivo assay, at least a 25% increase in tolerability for the therapeutic dosage unit administered when measured by testing in a standard in vivo assay, at least a 30% increase in tolerability for the therapeutic dosage unit administered when measured by testing in a standard in vivo assay, at least a 35% increase in tolerability for the therapeutic dosage unit administered when measured by testing in a standard in vivo assay, at least a 40% increase in tolerability for the therapeutic dosage unit administered when measured by testing in a standard in vivo assay, at least a 45% increase in tolerability for the therapeutic dosage unit administered when measured by testing in a standard in vivo assay, at least a 50% increase in tolerability for the therapeutic dosage unit administered when measured by testing in a standard in vivo assay, at least a 50% increase in tolerability for the therapeutic dosage unit administered when measured by testing in a standard in vivo assay, at least a 55% increase in tolerability to the therapeutic dose unit administered, when measured by testing in a standard in vivo assay; at least a 60% increase in tolerability to the therapeutic dose unit administered, when measured by testing in a standard in vivo assay; at least a 65% increase in tolerability to the therapeutic dose unit administered, when measured by testing in a standard in vivo assay; at least a 70% increase in tolerability to the therapeutic dose unit administered, when measured by testing in a standard in vivo assay; at least a 75% increase in tolerability to the therapeutic dose unit administered, when measured by testing in a standard in vivo assay; at least an 80% increase in tolerability to the therapeutic dose unit administered, when measured by testing in a standard in vivo assay; at least an 85% increase in tolerability to the therapeutic dose unit administered, when measured by testing in a standard in vivo assay; at least a 90% increase in tolerability to the therapeutic dose unit administered, when measured by testing in a standard in vivo assay80. The method of claim 79, wherein the increase in tolerability of the administered therapeutic dose unit is at least 95%.

81. The therapeutic dosage unit administered is less than 6.9 ng / μg double stranded RNA (dsRNA), less than 6.8 ng / μg double stranded RNA (dsRNA), less than 6.7 ng / μg double stranded RNA (dsRNA), less than 6.6 ng / μg double stranded RNA (dsRNA), less than 6.5 ng / μg double stranded RNA (dsRNA), less than 6.4 ng / μg double stranded RNA (dsRNA), less than 6.3 ng / μg double stranded RNA (dsRNA), less than 6.2 ng / μg double stranded RNA (dsRNA), less than 6.1 ng / μg double stranded RNA (dsRNA), less than 6.0 ... full double-stranded RNA (dsRNA), less than 5.9 ng / μg double-stranded RNA (dsRNA), less than 5.8 ng / μg double-stranded RNA (dsRNA), less than 5.7 ng / μg double-stranded RNA (dsRNA), less than 5.6 ng / μg double-stranded RNA (dsRNA), less than 5.5 ng / μg double-stranded RNA (dsRNA), less than 5.4 ng / μg double-stranded RNA (dsRNA), less than 5.3 ng / μg double-stranded RNA (dsRNA), less than 5.2 ng / μg double-stranded RNA (dsRNA), less than 5.1 ng / μg double-stranded RNA (dsRNA), less than 5.0 ng / μg less than 4.9 ng / μg double stranded RNA (dsRNA), less than 4.8 ng / μg double stranded RNA (dsRNA), less than 4.7 ng / μg double stranded RNA (dsRNA), less than 4.6 ng / μg double stranded RNA (dsRNA), less than 4.5 ng / μg double stranded RNA (dsRNA), less than 4.4 ng / μg double stranded RNA (dsRNA), less than 4.3 ng / μg double stranded RNA (dsRNA), less than 4.2 ng / μg double stranded RNA (dsRNA), less than 4.1 ng / μg double stranded RNA (dsRNA), 4.0 less than ng / μg double stranded RNA (dsRNA), less than 3.9 ng / μg double stranded RNA (dsRNA), less than 3.8 ng / μg double stranded RNA (dsRNA), less than 3.7 ng / μg double stranded RNA (dsRNA), less than 3.6 ng / μg double stranded RNA (dsRNA), less than 3.5 ng / μg double stranded RNA (dsRNA), less than 3.4 ng / μg double stranded RNA (dsRNA), less than 3.3 ng / μg double stranded RNA (dsRNA), less than 3.2 ng / μg double stranded RNA (dsRNA), less than 3.1 ng / μg double stranded RNA (dsRNA),Less than 3.0 ng / μg double stranded RNA (dsRNA), less than 2.9 ng / μg double stranded RNA (dsRNA), less than 2.8 ng / μg double stranded RNA (dsRNA), less than 2.7 ng / μg double stranded RNA (dsRNA), less than 2.6 ng / μg double stranded RNA (dsRNA), less than 2.5 ng / μg double stranded RNA (dsRNA), less than 2.4 ng / μg double stranded RNA (dsRNA), less than 2.3 ng / μg double stranded RNA (dsRNA) sRNA), less than 2.2 ng / μg double stranded RNA (dsRNA), less than 2.1 ng / μg double stranded RNA (dsRNA), less than 2.0 ng / μg double stranded RNA (dsRNA), less than 1.9 ng / μg double stranded RNA (dsRNA), less than 1.8 ng / μg double stranded RNA (dsRNA), less than 1.7 ng / μg double stranded RNA (dsRNA), less than 1.6 ng / μg double stranded RNA (dsRNA), less than 1.5 ng / μg double stranded RNA (dsRNA), RNA (dsRNA), less than 1.4 ng / μg double stranded RNA (dsRNA), less than 1.3 ng / μg double stranded RNA (dsRNA), less than 1.2 ng / μg double stranded RNA (dsRNA), less than 1.1 ng / μg double stranded RNA (dsRNA), less than 1.0 ng / μg double stranded RNA (dsRNA), less than 0.9 ng / μg double stranded RNA (dsRNA), less than 0.8 ng / μg double stranded RNA (dsRNA), 0.7 ng / μg 81. The method of any one of claims 75 to 80, comprising less than 0.05 ng / μg double stranded RNA (dsRNA), less than 0.6 ng / μg double stranded RNA (dsRNA), less than 0.5 ng / μg double stranded RNA (dsRNA), less than 0.4 ng / μg double stranded RNA (dsRNA), less than 0.3 ng / μg double stranded RNA (dsRNA), less than 0.2 ng / μg double stranded RNA (dsRNA), or less than 0.1 ng / μg double stranded RNA (dsRNA).

82. (1) N7-methyl-guanosine-5′-diphosphate, of the following structure: 【Chemistry 69】 , (in the formula, X １ and X ２ are each independently selected from the group consisting of O and S; X ３ is O; R １ and R ２ are each independently H, OH, or N ３ , F, substituted or unsubstituted alkoxy, and thio; R １ and R ２ are optionally joined to form a heterocycle with an activating reagent and imidazole to form an activated intermediate; and (2) a salt reagent and a dinucleotide of the structure: 【Chemistry 70】 , (in the formula, 【Chemistry 71】 is a single bond or a double bond; X ４ and X ６ are each independently selected from the group consisting of O and S; X ５ is O, S or CH; X ７ is CH or CH ２ and R ３ is OH, OMe, F, R ４ is H or R ３ and R ４ are covalently bonded to each other and together with the intermediate atom form a 2'-O,4'-C methylene bridge; B １ and B ２ are each independently selected from the group consisting of a purine ring and a pyrimidine ring. and adding to said activated intermediate to form a trinucleotide compound of the structure: 【Chemical 72】 The method is a one-pot synthesis.

83. (1) A dinucleotide having the following structure: 【Chemical 73】 , (in the formula, 【Chemical 74】 is a single bond or a double bond; X ４ is O; X ６ is O or S; X ５ is O, S or CH; X ７ is CH or CH ２ and R ３ is OH, OMe, F, R ４ is H or R ３ and R ４ are covalently bonded to each other and together with the intermediate atom form a 2'-O,4'-C methylene bridge; B １ and B ２ are each independently selected from the group consisting of a purine ring and a pyrimidine ring. with an activating reagent and imidazole to form an activated imidazolide phosphate of the following structure: 【Chemistry 75】 and (2) A salt reagent and a compound having the following structure: 【Chemical 76】 , (in the formula, X １ and X ２ are each independently selected from the group consisting of O and S; X ３ is S; R １ and R ２ are each independently H, OH, or N ３ , F, substituted or unsubstituted alkoxy, and thio; R １ and R ２ are optionally joined to form a heterocycle and to said activated imidazolide phosphate to form a trinucleotide compound of the structure: 【Chemical 77】 。

84. (1) a dinucleotide diphosphate having the following structure: 【Chemical 78】 , (in the formula, 【Chemical 79】 is a single bond or a double bond; X ３ is O; X ４ and X ６ are each independently selected from O and S; X ５ is O, S or CH; X ７ is CH or CH ２ and R ３ is OH, OMe, F, R ４ is H or R ３ and R ４ are covalently bonded to each other and together with the intermediate atom form a 2'-O,4'-C methylene bridge; B １ and B ２ are each independently selected from the group consisting of a purine ring and a pyrimidine ring. with an activating reagent and imidazole to form an activated imidazolide phosphate of the following structure: 【Chemistry 80】 and (2) A salt reagent and a compound having the following structure: 【Chemistry 81】 , (in the formula, X １ and X ２ are each independently selected from O and S; R １ and R ２ are each independently H, OH, or N ３ , F, substituted or unsubstituted alkoxy, and thio; R １ and R ２ are optionally joined to form a heterocycle to the activated imidazolide phosphate to form a trinucleotide compound of the structure: 【Chemistry 82】 The method is a one-pot synthesis.

Images