Nucleic acid polypeptide composition and its use

Abstract

To provide an oligonucleotide in production of pharmaceutical formulations for treating a subject suffering from cancer, neuromuscular disease, muscular dystrophy, muscular atrophy, muscle wasting, genetic disease, hereditary disease, or cardiovascular disease.SOLUTION: Provided is an oligonucleotide containing a compound of formula (IIa), and in the formula, R1 each are independently substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 fluoroalkyl, or substituted or unsubstituted C1-C6 heteroalkyl; L1, L2 and L3 are selected such that L1-L2-L3 may be equal to (a)-(CH2)3-, (b)-(CH2)4-, (c)-(CH2)5-, or (d)-(C2H2)(CH2)2-; and J is a binding group between nucleotides that binds to an adjacent nucleotide of the oligonucleotide.SELECTED DRAWING: None

Description

[Technical Field]

[0001] cross reference

[0002] This application claims the interests of U.S. Provisional Patent Application No. 62 / 858,285, filed on June 6, 2019, which is incorporated herein by reference in whole.

[0003] Sequence List This application includes a sequence listing, which was submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy said to have been created on June 6, 2020, is named 45532-734_601_SL.txt and is 3,143,382 bytes in size. [Background technology]

[0004] RNA-induced gene silencing provides multiple levels of control: transcriptional inactivation, small interfering RNA (siRNA)-induced mRNA reduction, and siRNA-induced transcriptional attenuation. In some cases, RNA interference (RNAi) can produce long-lasting effects across multiple cell divisions. Therefore, RNAi represents a viable method useful for drug target validation, gene function analysis, pathway analysis, and disease treatment. [Overview of the project]

[0005] In some embodiments, compositions and pharmaceutical formulations comprising polynucleic acid molecules and binding sites optionally conjugated to polymers are disclosed herein. In some embodiments, methods for treating diseases or illnesses (e.g., cancer) utilizing compositions and pharmaceutical formulations comprising polynucleic acid molecules and binding sites conjugated to polymers are also described herein.

[0006] In one embodiment, a compound of formula (II) is disclosed herein,

[0007] [ka] In the formula: R 1 are each independently substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 fluoroalkyl, or substituted or unsubstituted C1-C6 heteroalkyl; R 2 are each independently hydrogen, deuterium, substituted or unsubstituted C1-C6 alkyl, or substituted or unsubstituted C1-C6 heteroalkyl; alternatively, two R 2 together with the nitrogen atom to which they are bonded form a substituted or unsubstituted C2-C 10 heterocycloalkyl; L 1 is a bond, substituted or unsubstituted C1-C5 alkylene, substituted or unsubstituted C2-C5 alkenylene, or substituted or unsubstituted C2-C5 alkynylene; L 2 is a bond, O, S, NR 3 , substituted or unsubstituted C4-C7 cycloalkylene, substituted or unsubstituted C4-C7 heterocycloalkylene, substituted or unsubstituted C5-C8 arylene, or substituted or unsubstituted C4-C8 heteroarylene; R 3 , when present, is selected from hydrogen, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C1-C6 fluoroalkyl, unsubstituted or substituted C1-C6 heteroalkyl, unsubstituted or substituted monocyclic carbocycle, and unsubstituted or substituted monocyclic heterocycle; L 3 is a bond, substituted or unsubstituted C1-C5 alkylene, substituted or unsubstituted C2-C5 alkenylene, or substituted or unsubstituted C2-C5 alkynylene, and L 1 , L 2 , and L 3 are not bonds for at least two of them.

[0008] In some examples, L 2 is a bond, O, S, or NR3 These are substituted or unsubstituted C4-C7 cycloalkylenes, substituted or unsubstituted C5-C8 arylenes, phenylenes, or cyclohexyls. In some examples, L 1 is C1-C5 alkylene, C1-C3 alkenylene, or C1-C5 alkynylene, and L 3 These are C1-C5 alkylenes, C1-C3 alkenylenes, or C1-C5 alkynylenes. In some examples, L 1 It is a C1-C5 alkylene, and L 3 is a C1-C5 alkylene. In some embodiments, L 2 is methylene, bond, O, S, or NR 3 That is the case.

[0009] In some examples, R 1 Each of these is independently a substituted or unsubstituted C1-C6 alkyl group. In some examples, R 2 Each of these is independently a substituted or unsubstituted C1-C6 alkyl, CH3, -CH2CH3, -CH2CH2CH3, or -CH2(CH3)2. In some examples, the compound is selected from the group consisting of the following:

[0010] [ka]

[0011] [ka] R xThese are H, halogens, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C1-C6 fluoroalkyl, unsubstituted or substituted C1-C6 heteroalkyl, unsubstituted or substituted monocyclic carbocyclic, unsubstituted or substituted monocyclic heterocyclic, -CN, -OH, -O-alkyl, -CO2H, -CO2-alkyl, -CH2CO2H, -CH2CO2-alkyl, -C(=O)NH2, -C(=O)NH-alkyl, -CH2C(=O)NH2, -CH2C(=O)NH-alkyl, NH2, -NH-alkyl, -CH2NH2, -CH2NH-alkyl, -NHC(=O)alkyl, -CH2NHC(=O)alkyl, -SH, -S-alkyl, -S(=O)H, -S(=O)alkyl, -SO2H, -SO2-alkyl, -SO2NH2, or -SO2NH-alkyl.

[0012] An oligonucleotide containing the compound of formula (IIa) at one of its terminals is also disclosed.

[0013] [ka] During the ceremony, R 1 Each of these is independently a substituted or unsubstituted C1-C6 alkyl, a substituted or unsubstituted C1-C6 fluoroalkyl, or a substituted or unsubstituted C1-C6 heteroalkyl. L 1 These are bonded, substituted, or unsubstituted C1-C5 alkylenes, substituted or unsubstituted C2-C5 alkenylenes, or substituted or unsubstituted C2-C5 alkynylenes. L 2 is bond, O, S, NR 3 , substituted or unsubstituted C4-C7 cycloalkylene, substituted or unsubstituted C4-C7 heterocycloalkylene, substituted or unsubstituted C5-C8 arylene, or substituted or unsubstituted C4-C8 heteroarylene, R 3If present, it is selected from hydrogen, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C1-C6 fluoroalkyl, unsubstituted or substituted C1-C6 heteroalkyl, unsubstituted or substituted monocyclic carbocyclic, and unsubstituted or substituted monocyclic heterocyclic. L 3 These are bonded, substituted, or unsubstituted C1-C5 alkylenes, substituted or unsubstituted C2-C5 alkenylenes, or substituted or unsubstituted C2-C5 alkynylenes. J is an internucleotide binding group that binds to adjacent nucleotides in a polynucleotide, and also, L 1 , L 2 , and L 3 At least two of them are not combinations.

[0014] In some examples, the oligonucleotide is an RNA oligonucleotide. In some examples, the oligonucleotide further comprises at least one modification or at least one 2'-modified nucleotide. In some embodiments, the oligonucleotide further comprises at least one 2'-modified nucleotide selected from 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-deoxy, 2-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modified nucleotides. In some embodiments, the oligonucleotide further comprises at least one 2'-modified nucleotide selected from locked nucleic acid (LNA) or ethylene nucleic acid (ENA), or at least one modified nucleotide-nucleotide bond. In some embodiments, the oligonucleotide further comprises at least one modified internucleotide bond selected from phosphorothioate bonds, phosphorodithioate bonds, methylphosphonate bonds, phosphotriester bonds, or amide bonds.

[0015] In some examples, the compound of formula (IIa) is located at the 5'-terminus of the oligonucleotide. In some examples, the oligonucleotide is conjugated to the binding site. In some embodiments, the compound of formula (IIa) is located at the 5'-terminus of the oligonucleotide, and the binding site is conjugated to the 3'-terminus of the oligonucleotide.

[0016] In some examples, the binding site includes an antibody or its binding fragment. In some embodiments, the antibody or its binding fragment includes a humanized antibody or its binding fragment, a chimeric antibody or its binding fragment, a monoclonal antibody or its binding fragment, a monovalent Fab', a bivalent Fab2, a single-strand variable fragment (scFv), a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), or a camelid antibody, or its binding fragment. In some embodiments, the binding site includes a peptide, an aptamer, or a small molecule.

[0017] In some examples, oligonucleotides contain approximately 8 to 50 nucleotides, or approximately 10 to 30 nucleotides.

[0018] In some embodiments, the oligonucleotide is an RNA oligonucleotide, conjugated to a binding site, consisting of approximately 10 to approximately 30 nucleotides, containing at least one 2'-modified nucleotide, and containing at least one modified internucleotide bond. In some embodiments, the oligonucleotide hybridizes to at least eight adjacent bases of the target gene sequence. In some embodiments, the oligonucleotide mediates RNA interference. In some embodiments, the oligonucleotide is a sense strand. In some embodiments, the oligonucleotide hybridizes with a second oligonucleotide to form a double-stranded oligonucleotide molecule. In some embodiments, the second oligonucleotide is an antisense strand. In some embodiments, the second oligonucleotide is an RNA oligonucleotide. In some embodiments, the second oligonucleotide contains at least one modification. In some embodiments, the second oligonucleotide contains at least one 2'-modified nucleotide. In some embodiments, the second oligonucleotide comprises at least one 2'-modified nucleotide selected from nucleotides modified with 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA). In some embodiments, the second oligonucleotide comprises at least one 2'-modified nucleotide selected from locked nucleic acid (LNA) or ethylene nucleic acid (ENA). In some embodiments, the second oligonucleotide comprises at least one modified internucleotide bond. In some embodiments, the second oligonucleotide comprises at least one modified internucleotide bond selected from a phosphorothioate bond, a phosphorodithioate bond, a methylphosphonate bond, a phosphotriester bond, or an amide bond.

[0019] In some embodiments, the oligonucleotide comprises a polymer. In some embodiments, the oligonucleotide comprises polyethylene glycol.

[0020] In some embodiments, the oligonucleotide comprises a first strand and a second strand, where the first strand is a sense strand, an RNA oligonucleotide, conjugated to a binding site, polymer, or a combination thereof, comprising about 10 to about 30 nucleotides, comprising at least one 2'-modified nucleotide, and comprising at least one modified internucleotide bond, and the second strand is an antisense strand, an RNA oligonucleotide, comprising about 10 to about 30 nucleotides, comprising at least one 2'-modified nucleotide, and comprising at least one modified internucleotide bond.

[0021] In another embodiment, an oligonucleotide conjugate of formula (I) is disclosed herein, AB Equation (I) During the ceremony, A is the connecting part, B is a oligonucleotide compound of formula (IIa),

[0022] [ka] During the ceremony, R 1 Each of these is independently a substituted or unsubstituted C1-C6 alkyl, a substituted or unsubstituted C1-C6 fluoroalkyl, or a substituted or unsubstituted C1-C6 heteroalkyl. L 1 These are bonded, substituted, or unsubstituted C1-C5 alkylenes, substituted or unsubstituted C2-C5 alkenylenes, or substituted or unsubstituted C2-C5 alkynylenes. L 2 is bond, O, S, NR 3, substituted or unsubstituted C4-C7 cycloalkylene, substituted or unsubstituted C4-C7 heterocycloalkylene, substituted or unsubstituted C5-C8 arylene, or substituted or unsubstituted C4-C8 heteroarylene, R 3 If present, it is selected from hydrogen, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C1-C6 fluoroalkyl, unsubstituted or substituted C1-C6 heteroalkyl, unsubstituted or substituted monocyclic carbocyclic, and unsubstituted or substituted monocyclic heterocyclic. L 3 These are bonded, substituted, or unsubstituted C1-C5 alkylenes, substituted or unsubstituted C2-C5 alkenylenes, or substituted or unsubstituted C2-C5 alkynylenes. J is an internucleotide binding group that binds to adjacent nucleotides in a polynucleotide, and also, L 1 , L 2 , and L 3 At least two of them are not combinations.

[0023] In some examples, oligonucleotide conjugates further contain C to form formula ABC (formula IA).

[0024] In certain embodiments, a method for inhibiting the expression of a target gene in a patient's primary cells is disclosed herein, comprising the step of administering the above-mentioned molecule to the primary cells. In some embodiments, the above method is an in vivo method. In some embodiments, the patient is human. A method for treating a subject with a disease or illness characterized by a protein deficiency is disclosed herein, comprising the step of administering an oligonucleotide, such as those described herein, to modulate the expression of a protein-coding gene, thereby treating the disease or illness characterized by a protein deficiency. Furthermore, a method for treating a subject with a disease or illness characterized by a protein overexpression is disclosed herein, comprising the step of administering an oligonucleotide, such as those described herein, to modulate the expression of a protein-coding gene, thereby treating the disease or illness characterized by protein overexpression. In some examples, the disease or illness is a neuromuscular disease, genetic disease, muscular dystrophy, muscle atrophy, muscle wasting, cancer, hereditary disease, or cardiovascular disease in humans or mammals.

[0025] In one embodiment, an immuno-oncological therapy comprising the above-mentioned molecules for the treatment of a patient's disease or disorder is disclosed herein.

[0026] In one embodiment, a kit comprising such molecules, oligonucleotides, or oligonucleotide conjugates is disclosed herein. [Brief explanation of the drawing]

[0027] The following drawings form part of this specification and are included to further illustrate certain aspects of this disclosure. This disclosure can be better understood by referring to one or more of these drawings in conjunction with the detailed descriptions of the specific embodiments presented herein. [Figure 1]This diagram illustrates a caricature of a polynucleotide double helix, comprising a compound of formula (II) bonded to the 5' end of the guide (antisense) strand, and further comprising a binding portion A that can be directly bound to the polynucleotide or to the 5' end (top), 3' end (middle), or 3' end (bottom) of the passenger (sense) strand via a linker. [Figure 2A] The graphs show the relative %SSB mRNA levels in various cell types. Figure 2A shows a plot of concentration (nM) versus %SSB mRNA in HCT116 cells transfected with SSB-siRNA as described in Molecular Biology Example 1. [Figure 2B] Figure 2B shows graphs of relative %SSB mRNA levels in various cell types. Figure 2B plots concentration (nM) versus relative %SSB mRNA levels (%) of untreated control for DM1 control myoblasts transfected with SSB siRNA as described in Molecular Biology Example 2. [Figure 2C] Figure 2C shows graphs of relative %SSB mRNA levels in various cell types. Concentration (nM) versus relative %SSB mRNA levels (%) of untreated control for SJCRH30 transfection with SSB siRNA, as described in Molecular Biology Example 2. [Figure 3A] The graphs show the relative %MSTN mRNA levels in various cell types. Figure 3A shows a plot of concentration (nM) versus relative MSTN mRNA levels (%) compared to untreated control for DM1-controlled myoblasts treated with MSTN siRNA, as described in Molecular Biology Example 2. [Figure 3B] Figure 3B shows graphs of relative %MSTN mRNA levels in various cell types. Figure 3B plots concentration (nM) versus relative MSTN mRNA levels (%) compared to untreated control for SJCRH30 transfection with MSTN siRNA, as described in Molecular Biology Example 2. [Figure 3C]This graph shows the relative %MSTN mRNA levels upon introduction of MSTN siRNA conjugates with various modified nucleotides into SJCRH30 cells. [Figure 3D] This graph shows the relative %SSB mRNA levels when SSB siRNA conjugates with various modified nucleotides were introduced into SJCRH30 cells. [Figure 4] The images show in vivo MSTN mRNA downregulation in the gastrocnemius (upper left), quadriceps (lower left), diaphragm (upper right), and tibialis anterior (lower right) muscles one week after intravenous administration of antibody siRNA conjugates at 0.1, 0.3, 1.0, and 3.0 mg / kg, as described in Molecular Biology Example 3. [Figure 5] The following plots show siRNA concentration versus in vivo MSTN mRNA downregulation in the gastrocnemius (upper left), quadriceps (lower left), diaphragm (upper right), and tibialis anterior (lower right) muscles one week after intravenous administration of antibody-siRNA conjugates, as described in Molecular Biology Example 3. [Figure 6] This shows in vivo MSTN mRNA downregulation in the gastrocnemius (upper left), quadriceps (lower left), diaphragm (upper right), and tibialis anterior (lower right) muscles one week after intravenous administration of antibody siRNA conjugates, as described in Molecular Biology Example 3. [Figure 7] As described in Molecular Biology Example 3, another in vivo dose-response study demonstrates in vivo MSTN mRNA downregulation in the gastrocnemius (upper left), quadriceps (lower left), diaphragm (upper right), and tibialis anterior (lower right) muscles one week after intravenous administration of an antibody siRNA conjugate. [Figure 8A] Graphs of in vivo MSTN mRNA downregulation and MSTN siRNA concentrations in the gastrocnemius muscle from two separate studies are shown. Figures 8A and 8B show in vivo MSTN mRNA downregulation and MSTN siRNA concentrations in the gastrocnemius muscle from one in vivo study measuring mRNA concentrations on days 7, 14, 21, 28, and 35. [Figure 8B]Graphs of in vivo MSTN mRNA downregulation and MSTN siRNA concentrations in the gastrocnemius muscle from two separate studies are shown. Figures 8A and 8B show in vivo MSTN mRNA downregulation and MSTN siRNA concentrations in the gastrocnemius muscle from one in vivo study measuring mRNA concentrations on days 7, 14, 21, 28, and 35. [Figure 8C] Graphs of in vivo MSTN mRNA downregulation and MSTN siRNA concentrations in the calf muscle from two separate studies are shown. Figures 8C and 8D show in vivo MSTN mRNA downregulation and MSTN siRNA concentrations in the calf muscle from another in vivo study measuring mRNA concentrations on days 7, 14, 21, 28, and 35. [Figure 8D] Graphs of in vivo MSTN mRNA downregulation and MSTN siRNA concentrations in the calf muscle from two separate studies are shown. Figures 8C and 8D show in vivo MSTN mRNA downregulation and MSTN siRNA concentrations in the calf muscle from another in vivo study measuring mRNA concentrations on days 7, 14, 21, 28, and 35. [Figure 9] As shown in Molecular Biology Example 3, dose-response graphs of in vivo SSB mRNA downregulation in the liver by different types of SSB-siRNA are presented. [Modes for carrying out the invention]

[0028] Nucleic acid (e.g., RNAi) therapy is a targeted therapy boasting high selectivity and specificity. However, in some cases, nucleic acid therapy is also hindered by fragile intracellular uptake, limited blood stability, and nonspecific immune stimulation. To address these issues, various modifications of nucleic acid compositions are being explored, such as novel linkers for better stability and / or lower toxicity, optimization of binding sites for increased target specificity and / or target delivery, and nucleic acid polymer modifications for increased stability and / or reduced off-target effects.

[0029] In some embodiments, the arrangement or sequence of the various components constituting the nucleic acid composition further results in intracellular uptake, stability, toxicity, efficacy, and / or nonspecific immune stimulation. For example, if the nucleic acid components include a binding moiety, a polymer, and a polynucleic acid molecule (or polynucleotide), the sequence or arrangement of the binding moiety, polymer, and / or polynucleic acid molecule (or polynucleotide) (e.g., binding moiety-polynucleic acid molecule-polymer, binding moiety-polymer-polynucleic acid molecule, or polymer-binding moiety-polynucleic acid molecule) further results in intracellular uptake, stability, toxicity, efficacy, and / or nonspecific immune stimulation.

[0030] In some embodiments, oligonucleotide conjugates are described herein in which the arrangement of nucleic acid components results in intracellular uptake, stability, toxicity, efficacy, and / or nonspecific immunostimulation. In some examples, the oligonucleotide conjugate comprises a binding site conjugated to a polynucleic acid molecule and polymer. In some embodiments, the oligonucleotide conjugate comprises a compound of formula (II).

[0031] [ka]

[0032] In some embodiments, oligonucleotide conjugates, comprising a conjugated binding site on a polynucleic acid molecule and polymer, arranged as described herein, enhance intracellular uptake, stability, and / or efficacy. In some examples, oligonucleotide conjugates, comprising a conjugated binding site on a polynucleic acid molecule and polymer, arranged as described herein, reduce toxicity and / or nonspecific immunostimulation. In some cases, the oligonucleotide conjugate comprises a compound of formula (II).

[0033] [ka]

[0034] In additional embodiments, a kit comprising one or more of the molecules described herein is described herein.

[0035] Therapeutic molecular platform In some embodiments, the oligonucleotide conjugates described herein (e.g., therapeutic oligonucleotide conjugates) include a binding site conjugated to a polymer and a polynucleic acid molecule containing one or more modified nucleotides. In some embodiments, the oligonucleotide conjugate includes a compound of formula (I) or formula (IA), AB Equation (I) (In the formula, A is the connecting part, and, B is an oligonucleotide containing the compound of formula (II), ABC Formula (IA) (In the formula, A is the connecting part, and, B is a oligonucleotide containing a compound of formula (II) or (IIa), C is optionally a polymer.

[0036] In some embodiments, the oligonucleotide comprises a compound of formula (II).

[0037] [ka] During the ceremony, R 1 Each of these is independently a substituted or unsubstituted C1-C6 alkyl, a substituted or unsubstituted C1-C6 fluoroalkyl, or a substituted or unsubstituted C1-C6 heteroalkyl. R 2 Each of these is independently hydrogen, deuterium, a substituted or unsubstituted C1-C6 alkyl, or a substituted or unsubstituted C1-C6 heteroalkyl. Alternatively, two R's 2These, together with the nitrogen atom to which they are bonded, form a substituted or unsubstituted C2-C 10 Forms heterocycloalkyl groups, L 1 These are bonded, substituted, or unsubstituted C1-C5 alkylenes, substituted or unsubstituted C2-C5 alkenylenes, or substituted or unsubstituted C2-C5 alkynylenes. L 2 is bond, O, S, NR 3 , substituted or unsubstituted C4-C7 cycloalkylene, substituted or unsubstituted C4-C7 heterocycloalkylene, substituted or unsubstituted C5-C8 arylene, or substituted or unsubstituted C4-C8 heteroarylene, R 3 If present, it is selected from hydrogen, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C1-C6 fluoroalkyl, unsubstituted or substituted C1-C6 heteroalkyl, unsubstituted or substituted monocyclic carbocyclic, and unsubstituted or substituted monocyclic heterocyclic. L 3 These are bonded, substituted, or unsubstituted C1-C5 alkylenes, substituted or unsubstituted C2-C5 alkenylenes, or substituted or unsubstituted C2-C5 alkynylenes, and also, L 1 , L 2 , and L 3 At least two of them are not combinations.

[0038] In some embodiments of the compound of formula (II), L 2 is a combination, O, S, or NR 3 In some embodiments, L 2 is O, S, or NR 3 In some embodiments, L 2 is NR 3 In some embodiments, L 2 In some embodiments, L 2 is S. In some embodiments, L 2 It is a combination.

[0039] In some embodiments of the compound of formula (II), L 2 L is a substituted or unsubstituted C4-C7 cycloalkylene. In some embodiments, L 2 L is a substituted or unsubstituted C5-C8 arylene. In some embodiments, L 2 L is an unsubstituted C4-C7 cycloalkylene. In some embodiments, L 2 is phenylene. In some embodiments, L 2 L is methylene. In some embodiments, L 2 L is an unsubstituted C5-C8 arylene. In some embodiments, L 2 is phenylene. In some embodiments, L 2 L is methylene. In some embodiments, L 2 It is cyclohexyl.

[0040] In some embodiments of the compound of formula (II), L 1 L is a substituted or unsubstituted C1-C5 alkylene, a substituted or unsubstituted C2-C5 alkenylene, or a substituted or unsubstituted C2-C5 alkynylene, and 3 is a bonded, substituted, or unsubstituted C1-C5 alkylene, a substituted or unsubstituted C2-C5 alkenylene, or a substituted or unsubstituted C2-C5 alkynylene. In some embodiments, L 1 is C1-C5 alkylene, C1-C3 alkenylene, or C1-C5 alkynylene, and L 3 is a C1-C5 alkylene, a C1-C3 alkenylene, or a C1-C5 alkynylene. In some embodiments, L 1 It is a C1-C5 alkylene, and L 3 It is a C1-C5 alkylene.

[0041] In some embodiments of the compound of formula (II), L 1 , L 2 , and L 3 At least two of them are not bonded. In some embodiments, L 1 L is a bond. In some embodiments, L3 is a bond.

[0042] In some embodiments of the compound of formula (II), R 1 are each independently substituted or unsubstituted C1-C6 alkyl, or substituted or unsubstituted C1-C6 heteroalkyl. In some embodiments, R 1 are each independently substituted or unsubstituted C1-C6 alkyl. In some embodiments, R 1 are each independently -CH3, -CH2CH3, -CH2CH2CH3, or -CH2(CH3)2. In some embodiments, R 1 are each independently -CH3, -CH2CH2CH3, or -CH2(CH3)2. In some embodiments, R 1 is -CH3.

[0043] In some embodiments of the compound of formula (II), R 2 are each independently substituted or unsubstituted C1-C6 alkyl, or substituted or unsubstituted C1-C6 heteroalkyl. In some embodiments, R 2 are each independently substituted or unsubstituted C1-C6 alkyl. In some embodiments, R 2 are each independently -CH3, -CH2CH3, -CH2CH2CH3, or -CH2(CH3)2. In some embodiments, R 2 are each independently -CH3, -CH2CH2CH3, or -CH2(CH3)2. In some embodiments, R 2 are each -CH2(CH3)2.

[0044] In some embodiments of the compound of formula (II), two R 2 together with the nitrogen atom to which they are attached form a substituted or unsubstituted C2-C 10 heterocycloalkyl.

[0045] In some embodiments of the compound of formula (II), R 3is selected from hydrogen, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C1-C6 fluoroalkyl, and unsubstituted or substituted C1-C6 heteroalkyl. In some embodiments, R 3 is selected from unsubstituted or substituted monocyclic carbocycle and unsubstituted or substituted monocyclic heterocycle. In some embodiments, R 3 is hydrogen.

[0046] In some embodiments of the compound of formula (II), the compound is selected from the group consisting of:

[0047]

Chemical Formula

[0048]

Chemical Formula

[0049] In some embodiments, R x is halogen, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C1-C6 fluoroalkyl, unsubstituted or substituted C1-C6 heteroalkyl, unsubstituted or substituted monocyclic carbocycle, or unsubstituted or substituted monocyclic heterocycle. In some embodiments, Rx is H-CN, -OH, -O-alkyl, -CO2H, -CO2-alkyl, -CH2CO2H, -CH2CO2-alkyl, -C(=O)NH2, -C(=O)NH-alkyl, -CH2C(=O)NH2, -CH2C(=O)NH-alkyl, NH2, -NH-alkyl, -CH2NH2, -CH2NH-alkyl, -NHC(=O)alkyl, -CH2NHC(=O)alkyl, -SH, -S-alkyl, -S(=O)H, -S(=O)alkyl, -SO2H, -SO2-alkyl, -SO2NH2, or -SO2NH-alkyl. In some embodiments, R x is a halogen. In some embodiments, R x H is H.

[0050] In some embodiments, the oligonucleotide comprises a compound of formula (III),

[0051] [ka] During the ceremony, R 1 Each of these is independently a substituted or unsubstituted C1-C6 alkyl, a substituted or unsubstituted C1-C6 fluoroalkyl, or a substituted or unsubstituted C1-C6 heteroalkyl. R 2 Each of these is independently hydrogen, deuterium, a substituted or unsubstituted C1-C6 alkyl, or a substituted or unsubstituted C1-C6 heteroalkyl. Alternatively, two R's 2 These, together with the nitrogen atom to which they are bonded, form a substituted or unsubstituted C2-C 10 Forms heterocycloalkyl groups, L 1 These are bonded, substituted, or unsubstituted C1-C5 alkylenes, substituted or unsubstituted C2-C5 alkenylenes, or substituted or unsubstituted C2-C5 alkynylenes. L 2 is bond, O, S, NR 3, substituted or unsubstituted C4-C7 cycloalkylene, substituted or unsubstituted C4-C7 heterocycloalkylene, substituted or unsubstituted C5-C8 arylene, or substituted or unsubstituted C4-C8 heteroarylene, R 3 If present, it is selected from hydrogen, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C1-C6 fluoroalkyl, unsubstituted or substituted C1-C6 heteroalkyl, unsubstituted or substituted monocyclic carbocyclic, and unsubstituted or substituted monocyclic heterocyclic. L 3 These are bonded, substituted, or unsubstituted C1-C5 alkylenes, substituted or unsubstituted C2-C5 alkenylenes, or substituted or unsubstituted C2-C5 alkynylenes, and also, L 1 , L 2 , and L 3 At least two of them are not combinations.

[0052] In some embodiments, B is an oligonucleotide having a 3'-terminus and a 5'-terminus, one of which contains the compound of formula (IIa).

[0053] [ka] During the ceremony, R 1 Each of these is independently a substituted or unsubstituted C1-C6 alkyl, a substituted or unsubstituted C1-C6 fluoroalkyl, or a substituted or unsubstituted C1-C6 heteroalkyl. L 1 These are bonded, substituted, or unsubstituted C1-C5 alkylenes, substituted or unsubstituted C2-C5 alkenylenes, or substituted or unsubstituted C2-C5 alkynylenes. L 2 is bond, O, S, NR 3, substituted or unsubstituted C4-C7 cycloalkylene, substituted or unsubstituted C4-C7 heterocycloalkylene, substituted or unsubstituted C5-C8 arylene, or substituted or unsubstituted C4-C8 heteroarylene, R 3 If present, it is selected from hydrogen, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C1-C6 fluoroalkyl, unsubstituted or substituted C1-C6 heteroalkyl, unsubstituted or substituted monocyclic carbocyclic, and unsubstituted or substituted monocyclic heterocyclic. L 3 These are bonded, substituted, or unsubstituted C1-C5 alkylenes, substituted or unsubstituted C2-C5 alkenylenes, or substituted or unsubstituted C2-C5 alkynylenes. J is an internucleotide binding group that binds to adjacent nucleotides in a polynucleotide, and also, L 1 , L 2 , and L 3 At least two of them are not combinations.

[0054] In some embodiments of the compound of formula (IIa), L 2 is a combination, O, S, or NR 3 In some embodiments, L 2 is O, S, or NR 3 In some embodiments, L 2 is NR 3 In some embodiments, L 2 In some embodiments, L 2 is S. In some embodiments, L 2 It is a combination.

[0055] In some embodiments of the compound of formula (IIa), L 2 L is a substituted or unsubstituted C4-C7 cycloalkylene. In some embodiments, L 2 L is a substituted or unsubstituted C5-C8 arylene. In some embodiments, L 2 L is an unsubstituted C4-C7 cycloalkylene. In some embodiments, L2 is phenylene. In some embodiments, L 2 L is methylene. In some embodiments, L 2 L is an unsubstituted C5-C8 arylene. In some embodiments, L 2 is phenylene. In some embodiments, L 2 L is methylene. In some embodiments, L 2 It is cyclohexyl.

[0056] In some embodiments of the compound of formula (IIa), L 1 L is a substituted or unsubstituted C1-C5 alkylene, a substituted or unsubstituted C2-C5 alkenylene, or a substituted or unsubstituted C2-C5 alkynylene, and 3 is a bonded, substituted, or unsubstituted C1-C5 alkylene, a substituted or unsubstituted C2-C5 alkenylene, or a substituted or unsubstituted C2-C5 alkynylene. In some embodiments, L 1 is C1-C5 alkylene, C1-C3 alkenylene, or C1-C5 alkynylene, and L 3 is a C1-C5 alkylene, a C1-C3 alkenylene, or a C1-C5 alkynylene. In some embodiments, L 1 It is a C1-C5 alkylene, and L 3 It is a C1-C5 alkylene.

[0057] In some embodiments of the compound of formula (IIa), L 1 , L 2 , and L 3 At least two of them are not bonded. In some embodiments, L 1 L is a bond. In some embodiments, L 3 It is a combination.

[0058] In some embodiments of equation (IIa), R 1 Each of these is independently a substituted or unsubstituted C1-C6 alkyl, or a substituted or unsubstituted C1-C6 heteroalkyl. In some embodiments, R 1Each of these is independently a substituted or unsubstituted C1-C6 alkyl group. In some embodiments, R 1 These are independently -CH3, -CH2CH3, -CH2CH2CH3, or -CH2(CH3)2. In some embodiments, R 1 These are independently -CH3, -CH2CH2CH3, or -CH2(CH3)2. In some embodiments, R 1 It is -CH3.

[0059] In some embodiments of equation (IIa), R 2 Each of these is independently a substituted or unsubstituted C1-C6 alkyl, or a substituted or unsubstituted C1-C6 heteroalkyl. In some embodiments, R 2 Each of these is independently a substituted or unsubstituted C1-C6 alkyl group. In some embodiments, R 2 These are independently -CH3, -CH2CH3, -CH2CH2CH3, or -CH2(CH3)2. In some embodiments, R 2 These are independently -CH3, -CH2CH2CH3, or -CH2(CH3)2. In some embodiments, R 2 These are each -CH2(CH3)2.

[0060] In some embodiments of equation (IIa), two R 2 These, together with the nitrogen atom to which they are bonded, form a substituted or unsubstituted C2-C 10 It forms a heterocycloalkyl group.

[0061] In some embodiments of equation (IIa), R 3 R is selected from hydrogen, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C1-C6 fluoroalkyl, and unsubstituted or substituted C1-C6 heteroalkyl. In some embodiments, R 3 R is selected from unsubstituted or substituted monocyclic carbocyclic rings and unsubstituted or substituted monocyclic heterocyclic rings. In some embodiments, R 3 It is hydrogen.

[0062] In some embodiments, the oligonucleotide contains at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 30, or more compounds of formula (II) (e.g., formula IIa). In some cases, the oligonucleotide contains at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, or more compounds of formula (II) (e.g., formula IIa). In some embodiments, the compounds of formula (II) (e.g., formula IIa) are present in tandem within the oligonucleotide. In other embodiments, the compound of formula (II) (e.g., formula IIa) is dispersed within the oligonucleotide, and the nucleotide is modified by one or more additional modifications as described below.

[0063] In some embodiments, the oligonucleotide comprises at least one of the following modifications: approximately 5% to approximately 100%, approximately 10% to approximately 100%, approximately 20% to approximately 100%, approximately 30% to approximately 100%, approximately 40% to approximately 100%, approximately 50% to approximately 100%, approximately 60% to approximately 100%, approximately 70% to approximately 100%, approximately 80% to approximately 100%, and approximately 90% to approximately 100%, wherein the modification is a compound of formula (II) (e.g., formula (IIa)). For example, if the oligonucleotide has 20 nucleosides, the oligonucleotide having approximately 60% modification comprises approximately 12 nucleosides substituted with 12 compounds of formula (II) (e.g., formula IIa).

[0064] In some embodiments, the oligonucleotide conjugate is an example molecule.

[0065] [ka]

[0066] In some embodiments, the oligonucleotide conjugate is an example molecule.

[0067] [ka]

[0068] The above

[0069] [ka] This is for illustrative purposes only and includes humanized antibodies or their conjugated fragments, anti-human antibodies, anti-mouse antibodies (e.g., anti-mouse antibodies, anti-rat antibodies), chimeric antibodies or their conjugated fragments, monoclonal antibodies or their conjugated fragments, monovalent Fab', bivalent Fab2, single-chain variable fragments (scFv), diabodies, minibodies, nanobodies, single-domain antibodies (sdAb), or camelid antibodies or their conjugated fragments.

[0070] Additional modifications In some examples, the additional modifications include synthetic or artificial nucleotide analogs or bases that involve modifications to one or more of the ribose moiety, phosphate moiety, nucleoside moiety, or combinations thereof.

[0071] In some embodiments, the modification at the 2'-hydroxyl group includes 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA).

[0072] In some embodiments, the nucleotide analog includes, but is not limited to, a modified base, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N,N-dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine, and other nucleotides having a modification at the 5-position, 5-(2-amino) Ropyruridine, 5-halocytidine, 5-halolidine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine, deazanucleotide (7-deaza-adenosine, 6-azolidine, 6-azacytidine, or 6-azothymidine, etc.), 5-methyl-2- These include uridine, other thiobases (such as 2-thiouridine, 4-thiouridine, and 2-thiocytidine), dihydrouridine, pseudouridine, quosin, alkaeosin (archaeosine), naphthyl, and substituted naphthyl groups, any O-alkylated and N-alkylated purines and pyrimidines (such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridine-4-one, or pyridine-2-one), phenyl and modified phenyl groups, such as aminophenol or 2,4,6-trimethoxybenzene, modified cytosine that functions as a G-clamp nucleotide, 8-substituted adenine and guanine, 5-substituted uracil and thymine, azapyrimidine, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonylalkylated nucleotides. 5'-phosphonate modified nucleotides further include those modified at the sugar moiety, and 5'-phosphonate modified nucleotides having a non-ribosyl sugar or an analogue thereof.For example, the sugar portion may be mannose, arabinose, glucopyranose, galactopyranose, 4'-thioribose, and other sugars, heterocyclic or carbocyclic, or based on these. The term nucleotide further includes those known in the art as universal bases. Examples of universal bases include, but are not limited to, 3-nitropyrrole, 5-nitroindole, or nebularin.

[0073] In some embodiments, the nucleotide analog or artificial nucleotide base comprises a 5'-phosphonate modified nucleotide nucleic acid having a modification at the 5' hydroxyl group of the ribose moiety. In some embodiments, the nucleotide analog or artificial nucleotide base comprises a 5'-vinylphosphonate modified nucleotide nucleic acid having a modification at the 5' hydroxyl group of the ribose moiety.

[0074] In some embodiments, the modification at the 2' hydroxyl group is a 2'-O-aminopropyl modification in which an extended amine group containing a propyl linker bonds the amine group to the 2' oxygen. In some examples, this modification neutralizes the overall negative charge derived from the phosphate of the oligonucleotide molecule by introducing one positive charge from the amine group per sugar, thereby improving its cellular uptake properties due to its zwitterionic nature.

[0075] In some cases, 5'-phosphonate modified nucleotides are further modified with a 2' hydroxyl group in locked or cross-linked ribose modification (e.g., locked nucleic acid or LNA), where the oxygen molecule bonded at the 2' carbon is bonded to the 4' carbon by a methylene group, thereby forming a 2'-C,4'-C-oxymethylene bicyclic ribonucleotide monomer. An exemplary representation of the chemical structure of 5'-phosphonate modified LNA is shown below, where J is an internucleotide bond.

[0076] [ka]

[0077] In some embodiments, additional modifications further include morpholino, peptide nucleic acid (PNA), methylphosphonate nucleotide, thiolphosphonate nucleotide, 2'-fluoroN3-P5'-phosphoramidite, or 1',5'-anhydrohexitol nucleic acid (HNA). Morphorino or phosphorodiamidate morpholino oligos (PMOs) include synthetic molecules whose structures mimic natural nucleic acid structures but deviate from the normal sugar and phosphonate structures. In some examples, a five-membered ribose ring is replaced by a six-membered morpholino ring containing four carbons, one nitrogen, and one oxygen. In some cases, the ribose monomer is bonded by a phosphorodiamidate group instead of a phosphate group. In some cases, the skeletal modification removes all positive and negative charges, allowing the morpholino neutral molecule to cross the cell membrane without the help of cell delivery agents, such as those used in charged oligonucleotides. Non-restrictive examples of 5'-phosphonate-modified morpholino oligonucleotides are shown below.

[0078] [ka]

[0079] In some embodiments, the 5'-phosphonate-modified morpholino or PMO described above is a PMO containing a positive or cationic charge. In some examples, PMO is PMOplus(Sarepta). PMOplus refers to a phosphorodiamidate morpholino oligomer containing any number of (1-piperazino)phosphinylideneoxy, (1-(4-(omega-guanidino-alkanoyl))-piperazino)phosphinylideneoxy bonds (e.g., as described in PCT publication number WO2008 / 036127). In some examples, PMO is a PMO described in U.S. Patent No. 7,943,762.

[0080] In some embodiments, the morpholino or PMO described above is PMO-X (Sarepta). In some cases, PMO-X refers to a phosphorodiamidate morpholino oligomer containing at least one bond or at least one of the disclosed terminal modifications, such as those disclosed in PCT Publication WO2011 / 150408 and U.S. Patent Application Publication 2012 / 0065169.

[0081] In some embodiments, the morpholino or PMO described above is a PMO as described in Table 5 of U.S. Patent Application Publication No. 2014 / 0296321.

[0082] In some embodiments, the peptide nucleic acid (PNA) does not contain sugar rings or phosphonate bonds, the bases are bound and appropriately spaced by oligoglycine-like molecules, and thus the skeletal charge is removed.

[0083] [ka]

[0084] In some embodiments, one or more of the above modifications occur in internucleotide bonds. In some examples, the modified internucleotide bonds include, but are not limited to, phosphorothioates, phosphorodithioates, methylphosphonates, 5'-alkylenephosphonates, 5'-methylphosphonates, 3'-alkylenephosphonates, boron trifluoride, 3'-5' or 2'-5' linked boranophosphates and selenophosphates, phosphotriesters, thionoalkylphosphotriesters, hydrogen phosphonate bonds, alkylphosphonates, alkylphosphonothioates, arylphosphonothioates, phosphoroselenoates, phosphorodiselenoates, phosphinates, phosphoramidates, 3'-alkylphosphoramidates, aminoalkylphosphoramidates, thionophosphoramidates, and phosphoropipates. Examples include radiodates, phosphoranilothioates, phosphoranilidetes, ketones, sulfones, sulfonamides, carbonates, carbamates, methylenehydrazo, methylenedimethylhydrazo, formacetals, thioformacetals, oximes, methyleneiminos, methylenemethyliminos, thioamides, bonds with riboacetyl groups, aminoethylglycine, silyl or siloxane bonds, alkyl or cycloalkyl bonds containing or not containing 1 to 10 carbon heteroatoms, which may be saturated or unsaturated and / or substituted and / or contain heteroatoms, morpholino structures in which a base is directly or indirectly bonded to the aza nitrogen of the skeleton, bonds with amides or polyamides, and combinations thereof.

[0085] In some examples, the modifications are methyl or thiol modifications, such as methylphosphonate modifications or thiolphosphonate modifications. Exemplary thiolphosphonate nucleotides (left), phosphorodithioates (center), and methylphosphonate nucleotides (right) are illustrated below.

[0086] [ka]

[0087] In some examples, 5'-vinylphosphonate-modified nucleotides include, but are not limited to, phosphoramidites, as exemplified below.

[0088] [ka]

[0089] In some examples, the modified internucleotide bond is a phosphorodiamidate bond. Non-restrictive examples of phosphorodiamidate bonds with morpholino systems are shown below.

[0090] [ka]

[0091] In some examples, the modified internucleotide bond is a methylphosphonate bond. Non-restrictive examples of methylphosphonate bonds are shown below.

[0092] [ka]

[0093] In some examples, the modified nucleotide bond is an amide bond. Non-restrictive examples of amide bonds are shown below.

[0094] [ka]

[0095] In some embodiments, one or more modifications involve a modified phosphate skeleton in which the modification generates a neutral or uncharged skeleton. In some examples, the phosphate skeleton is modified by alkylation to generate an uncharged or neutral phosphate skeleton. As used herein, alkylation includes methylation, ethylation, and propylation. In some cases, alkyl refers to a linear or branched saturated hydrocarbon group containing 1 to 6 carbon atoms, as used herein in the context of alkylation. In some examples, exemplary alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, and 2-ethylbutyl groups. In some cases, the modified phosphate is a phosphate group described in U.S. Patent No. 9481905.

[0096] In some embodiments, the further modified phosphate skeleton includes methylphosphonate, ethylphosphonate, methylthiophosphonate, or methoxyphosphonate. In some cases, the modified phosphate is methylphosphonate. In some cases, the modified phosphate is ethylphosphonate. In some cases, the modified phosphate is methylthiophosphonate. In some cases, the modified phosphate is methoxyphosphonate.

[0097] In some embodiments, the additionally modified phosphate skeleton includes one of the following:

[0098] [ka]

[0099] In some embodiments, one or more modifications may further optionally include modifications to the ribose moiety, phosphate backbone, and nucleoside, or modifications to nucleotide analogs at the 3' or 5' terminus. For example, the 3' terminus may optionally include a 3' cationic group, or invert the nucleoside at the 3' terminus via a 3'-3' bond. In another alternative, the 3' terminus may optionally be conjugated with an aminoalkyl group, e.g., 3'C5-aminoalkyldT. In an additional alternative, the 3' terminus may optionally be conjugated with a debasic site, e.g., an aprinic acid site or an apyrimidine acid site.

[0100] In some embodiments, an oligonucleotide comprising a compound of formula (II) (e.g., formula IIa) further comprises one or more of the artificial nucleotide analogs described herein. In some embodiments, an oligonucleotide comprising a compound of formula (II) (e.g., formula IIa) further comprises one or more additional modifications, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more, e.g., 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2' These include 2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modifications, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoroN3-P5'-phosphoramidite, or combinations thereof. In some examples, oligonucleotides containing compounds of formula (II) (e.g., formula IIa) are further: 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2' Includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 20, 25, or more artificial nucleotide analogs selected from LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoroN3-P5' phosphoramidites, or combinations thereof, modified with -O-DMAEOE) or 2'-ON-methylacetamide (2'-O-NMA).In some examples, oligonucleotides containing the compound of formula (II) (e.g., formula IIa) contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more 2'-O-methyl modified nucleotides. In some examples, oligonucleotides containing compounds of formula (II) (e.g., formula IIa) contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more thiolphosphonate nucleotides.

[0101] In some examples, about 5% to about 100% of oligonucleotides containing compounds of formula (II) (e.g., formula IIa) contain the artificial nucleotide analogs described herein. In some examples, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of polynucleic acid molecules contain the artificial nucleotide analogs described herein. In some embodiments, the artificial nucleotide analogs include 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoroN3-P5'-phosphoramidites, or combinations thereof, modified with 2'-ON-methylacetamide (2'-O-NMA).

[0102] In some embodiments, the oligonucleotide (or B in formula AB) described herein comprises RNA or DNA. In some cases, the oligonucleotide comprises RNA. In some examples, RNA comprises small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), double-stranded RNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), or heteronuclear RNA (hnRNA). In some examples, RNA comprises shRNA. In some examples, RNA comprises miRNA. In some examples, RNA comprises dsRNA. In some examples, RNA comprises tRNA. In some examples, RNA comprises rRNA. In some examples, RNA comprises hnRNA. In some examples, RNA comprises siRNA. In some cases, the oligonucleotide comprises the sense strand (or passenger strand) of siRNA. In other cases, the oligonucleotide comprises the antisense strand (or guide strand) of siRNA.

[0103] In some embodiments, the oligonucleotide is about 10 to about 50 nucleotides long. In some examples, the oligonucleotide is about 10 to about 30, about 15 to about 30, about 18 to about 25, about 18 to about 24, about 19 to about 23, or about 20 to about 22 nucleotides long.

[0104] In some embodiments, the oligonucleotide is about 50 nucleotides long. In some examples, the oligonucleotide is about 45 nucleotides long. In some examples, the oligonucleotide is about 40 nucleotides long. In some examples, the oligonucleotide is about 35 nucleotides long. In some examples, the oligonucleotide is about 30 nucleotides long. In some examples, the oligonucleotide is about 25 nucleotides long. In some examples, the oligonucleotide is about 20 nucleotides long. In some examples, the oligonucleotide is about 19 nucleotides long. In some examples, the oligonucleotide is about 18 nucleotides long. In some examples, the oligonucleotide is about 17 nucleotides long. In some examples, the oligonucleotide is about 16 nucleotides long. In some examples, the oligonucleotide is about 15 nucleotides long. In some examples, the oligonucleotide is about 14 nucleotides long. In some examples, the oligonucleotide is about 13 nucleotides long. In some examples, the oligonucleotide is about 12 nucleotides long. In some examples, the oligonucleotide is about 11 nucleotides long. In some examples, the oligonucleotide is about 10 nucleotides long. In some examples, the oligonucleotide is about 10 to about 50 nucleotides long. In some examples, the oligonucleotide is about 10 to about 45 nucleotides long. In some examples, the oligonucleotide is about 10 to about 40 nucleotides long. In some cases, oligonucleotides are approximately 10 to 35 nucleotides long. In some cases, oligonucleotides are approximately 10 to 30 nucleotides long. In some cases, oligonucleotides are approximately 10 to 25 nucleotides long. In some cases, oligonucleotides are approximately 10 to 20 nucleotides long. In some cases, oligonucleotides are approximately 15 to 25 nucleotides long. In some cases, oligonucleotides are approximately 19 to 23 nucleotides long. In some cases, oligonucleotides are approximately 15 to 30 nucleotides long. In some cases, oligonucleotides are approximately 12 to 30 nucleotides long.

[0105] In some embodiments, oligonucleotides further hybridize with a second oligonucleotide to form a double helix. In some examples, the oligonucleotide is a sense strand or passenger strand. In some examples, the oligonucleotide is an antisense strand or guide strand.

[0106] In some embodiments, the second oligonucleotide is about 10 to about 50 nucleotides long. In some embodiments, the second oligonucleotide is about 10 to about 30, about 15 to about 30, about 18 to about 25, about 18 to about 24, about 19 to about 23, or about 20 to about 22 nucleotides long.

[0107] In some cases, the second oligonucleotide is approximately 50 nucleotides long. In some cases, the second oligonucleotide is approximately 45 nucleotides long. In some cases, the second oligonucleotide is approximately 40 nucleotides long. In some cases, the second oligonucleotide is approximately 35 nucleotides long. In some cases, the second oligonucleotide is approximately 30 nucleotides long. In some cases, the second oligonucleotide is approximately 25 nucleotides long. In some cases, the second oligonucleotide is approximately 20 nucleotides long. In some cases, the second oligonucleotide is approximately 19 nucleotides long. In some cases, the second oligonucleotide is approximately 18 nucleotides long. In some cases, the second oligonucleotide is approximately 17 nucleotides long. In some cases, the second oligonucleotide is approximately 16 nucleotides long. In some cases, the second oligonucleotide is approximately 15 nucleotides long. In some cases, the second oligonucleotide is approximately 14 nucleotides long. In some cases, the second oligonucleotide is approximately 13 nucleotides long. In some cases, the second oligonucleotide is approximately 12 nucleotides long. In some cases, the second oligonucleotide is approximately 11 nucleotides long. In some cases, the second oligonucleotide is approximately 10 nucleotides long. In some cases, the second oligonucleotide is approximately 10 to 50 nucleotides long. In some cases, the second oligonucleotide is approximately 10 to 45 nucleotides long. In some cases, the second oligonucleotide is approximately 10 to 40 nucleotides long. In some cases, the second oligonucleotide is approximately 10 to 35 nucleotides long. In some cases, the second oligonucleotide is approximately 10 to 30 nucleotides long. In some cases, the second oligonucleotide is approximately 10 to 25 nucleotides long. In some cases, the second oligonucleotide is approximately 10 to 20 nucleotides long. In some cases, the first oligonucleotide is approximately 15 to 25 nucleotides long. In some cases, the first oligonucleotide is approximately 15 to 30 nucleotides long.In some examples, the first oligonucleotide is approximately 19 to 23 nucleotides long. In some examples, the second oligonucleotide is approximately 12 to 30 nucleotides long.

[0108] In some embodiments, the polynucleotide molecule comprises a first oligonucleotide and a second oligonucleotide. In some examples, the polynucleotide molecule further comprises a blunt end, an overhang, or a combination thereof. In some examples, the blunt end is a 5' blunt end, a 3' blunt end, or both. In some cases, the overhang is a 5' overhang, a 3' overhang, or both. In some cases, the overhang contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-base-paired nucleotides. In some cases, the overhang contains 1, 2, 3, 4, 5, or 6 non-base-paired nucleotides. In some cases, the overhang contains 1, 2, 3, or 4 non-base-paired nucleotides. In some cases, the overhang contains 1 non-base-paired nucleotide. In some cases, the overhang contains 2 non-base-paired nucleotides. In some cases, the overhang contains 3 non-base-paired nucleotides. In some cases, the overhang contains 4 non-base-paired nucleotides.

[0109] In some embodiments, the sequence of the polynucleotide molecule is at least 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleotide molecule is at least 50% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleotide molecule is at least 60% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleotide molecule is at least 70% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleotide molecule is at least 80% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleotide molecule is at least 90% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleotide molecule is at least 95% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleotide molecule is at least 99% complementary to the target sequence described herein. In some cases, the sequences of polynucleic acid molecules are 100% complementary to the target sequences described herein.

[0110] In some embodiments, the sequence of the polynucleotide molecule has five or fewer mismatches with respect to the target sequence described herein. In some embodiments, the sequence of the polynucleotide molecule has four or fewer mismatches with respect to the target sequence described herein. In some cases, the sequence of the polynucleotide molecule has three or fewer mismatches with respect to the target sequence described herein. In some cases, the sequence of the polynucleotide molecule has two or fewer mismatches with respect to the target sequence described herein. In some cases, the sequence of the polynucleotide molecule has one or fewer mismatches with respect to the target sequence described herein.

[0111] In some embodiments, the specificity of the polynucleic acid molecule hybridizing to the target sequence described herein is 95%, 98%, 99%, 99.5%, or 100% sequence complementarity of the polynucleic acid molecule to the target sequence. In some examples, the hybridization is a highly stringent hybridization state.

[0112] In some embodiments, the polynucleic acid molecule hybridizes to at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more adjacent bases of the target sequence described herein. Exemplary target sequences include, but are not limited to, sequences of any DMD gene or its mRNA, any DMPK gene or its mRNA, any oncogene or its mRNA, any gene associated with hereditary disease or genetic disorder (e.g., GYS1), any gene associated with muscle atrophy, muscular dystrophy, or muscle wasting (e.g., DMD, DMPK, DUX4) and any of its mRNA sequences. In some embodiments, the polynucleic acid molecule hybridizes to at least 8 adjacent bases of the target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 9 adjacent bases of the target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 10 adjacent bases of the target sequence described herein. In some embodiments, the polynucleotide molecule hybridizes to at least 11 adjacent bases of the target sequence described herein. In some embodiments, the polynucleotide molecule hybridizes to at least 12 adjacent bases of the target sequence described herein. In some embodiments, the polynucleotide molecule hybridizes to at least 13 adjacent bases of the target sequence described herein. In some embodiments, the polynucleotide molecule hybridizes to at least 14 adjacent bases of the target sequence described herein. In some embodiments, the polynucleotide molecule hybridizes to at least 15 adjacent bases of the target sequence described herein. In some embodiments, the polynucleotide molecule hybridizes to at least 16 adjacent bases of the target sequence described herein. In some embodiments, the polynucleotide molecule hybridizes to at least 17 adjacent bases of the target sequence described herein. In some embodiments, the polynucleotide molecule hybridizes to at least 18 adjacent bases of the target sequence described herein.In some embodiments, the polynucleic acid molecule hybridizes to at least 19 adjacent bases of the target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 20 adjacent bases of the target sequence described herein.

[0113] In some embodiments, polynucleic acid molecules reduced off-target effects. In some examples, “off-target” or “off-target effect” refers to any instance in which a polynucleic acid polymer directed towards a given target causes an unintended effect by directly or indirectly interacting with another mRNA sequence, DNA sequence, or cellular protein or other part. In some examples, “off-target effects” occur when there is simultaneous degradation of the other transcript due to partial homology or complementarity between the other transcript and the sense and / or antisense strand of the polynucleic acid molecule.

[0114] In some cases, one or more of the artificial nucleotide analogs described herein may have resistance to nucleases such as ribonucleases like RNase H, deoxyribonucleases like DNase, or exonucleases like 5'-3' exonuclease and 3'-5' exonuclease, compared to natural polynucleic acid molecules. In some examples, synthetic nucleotide analogs including compounds of formula II (e.g., formula IIa), 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoroN3-P5'-phosphoramidite, or combinations thereof, are RNases. These molecules possess resistance to nucleases such as ribonucleases like RNaseH, deoxyribonucleases like DNase, or exonucleases such as 5'-3' exonucleases and 3'-5' exonucleases. In some cases, 2'-O-methyl modified polynucleic acid molecules exhibit nuclease resistance (e.g., resistance to RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, 2'O-methoxyethyl (2'-O-MOE) modified polynucleic acid molecules exhibit nuclease resistance (e.g., resistance to RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, 2'-O-aminopropyl-modified polynucleic acid molecules exhibit nuclease resistance (e.g., resistance to RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease).In some cases, 2'-deoxy-modified polynucleic acid molecules exhibit nuclease resistance (e.g., resistance to RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, 2'-deoxy-2'-fluoro-modified polynucleic acid molecules exhibit nuclease resistance (e.g., resistance to RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, 2'-O-aminopropyl (2'-O-AP)-modified polynucleic acid molecules exhibit nuclease resistance (e.g., resistance to RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, polynucleic acid molecules modified with 2'-O-dimethylaminoethyl (2'-O-DMAOE) exhibit nuclease resistance (e.g., resistance to RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, polynucleic acid molecules modified with 2'-O-dimethylaminopropyl (2'-O-DMAP) exhibit nuclease resistance (e.g., resistance to RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, polynucleic acid molecules modified with 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE) exhibit nuclease resistance (e.g., resistance to RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, polynucleotide molecules modified with 2'-ON-methylacetamide (2'-O-NMA) exhibit nuclease resistance (e.g., resistance to RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, LNA-modified polynucleotide molecules exhibit nuclease resistance (e.g., resistance to RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, ENA-modified polynucleotide molecules exhibit nuclease resistance (e.g., resistance to RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease).In some cases, HNA-modified polynucleic acid molecules are nuclease-resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). Morphorino is nuclease-resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, PNA-modified polynucleic acid molecules are nuclease-resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, methylphosphonate-modified polynucleic acid molecules are nuclease-resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, thiophosphonate-modified polynucleic acid molecules exhibit nuclease resistance (e.g., resistance to RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, polynucleic acid molecules containing 2'-fluoroN3-P5'-phosphoramidite exhibit nuclease resistance (e.g., resistance to RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, the 5' conjugate described herein inhibits 5'-3' exonuclease cleavage. In some cases, the 3' conjugate described herein inhibits 3'-5' exonuclease cleavage.

[0115] In some embodiments, one or more artificially modified nucleotide analogs, including compounds of formula II (e.g., formula IIa) described herein, exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotide acid molecules. One or more modified nucleotide analogs, including compounds of formula II, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA)-modified LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotide, thiolphosphonate nucleotide, or 2'-fluoroN3-P5' phosphoramidite, may have increased binding affinity to mRNA compared to equivalent native polynucleic acid molecules. In some cases, 2'-O-methyl modified polynucleic acid molecules exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleic acid molecules. In some cases, 2'-O-methoxyethyl (2'-O-MOE) modified polynucleic acid molecules exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleic acid molecules. In some cases, 2'-O-aminopropyl modified polynucleic acid molecules exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleic acid molecules. In some cases, 2'-deoxy modified polynucleic acid molecules exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleic acid molecules. In some cases, 2'-deoxy-2'-fluoro modified polynucleic acid molecules exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleic acid molecules. In some cases, 2'-O-aminopropyl (2'-O-AP) modified polynucleic acid molecules exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleic acid molecules.In some cases, polynucleotide molecules modified with 2'-O-dimethylaminoethyl (2'-O-DMAOE) exhibit increased binding affinity to their mRNA targets compared to equivalent naturally occurring polynucleotide molecules. In some cases, polynucleotide molecules modified with 2'-O-dimethylaminopropyl (2'-O-DMAP) exhibit increased binding affinity to their mRNA targets compared to equivalent naturally occurring polynucleotide molecules. In some cases, polynucleotide molecules modified with 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE) exhibit increased binding affinity to their mRNA targets compared to equivalent naturally occurring polynucleotide molecules. In some cases, polynucleotide molecules modified with 2'-ON-methylacetamide (2'-O-NMA) exhibit increased binding affinity to their mRNA targets compared to equivalent naturally occurring polynucleotide molecules. In some cases, LNA-modified polynucleotide molecules exhibited increased binding affinity to their mRNA targets compared to equivalent naturally occurring polynucleotide molecules. In some cases, ENA-modified polynucleotide molecules showed increased binding affinity to their mRNA targets compared to equivalent native polynucleotide molecules. In some cases, PNA-modified polynucleotide molecules showed increased binding affinity to their mRNA targets compared to equivalent native polynucleotide molecules. In some cases, HNA-modified polynucleotide molecules showed increased binding affinity to their mRNA targets compared to equivalent native polynucleotide molecules. In some cases, morpholino-modified polynucleotide molecules showed increased binding affinity to their mRNA targets compared to equivalent native polynucleotide molecules. In some cases, methylphosphonate nucleotide-modified polynucleotide molecules showed increased binding affinity to their mRNA targets compared to equivalent native polynucleotide molecules. In some cases, thiolphosphonate nucleotide-modified polynucleotide molecules showed increased binding affinity to their mRNA targets compared to equivalent native polynucleotide molecules. In some cases, polynucleotide molecules containing 2'-fluoroN3-P5'-phosphoramidites exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotide molecules.In some cases, increased affinity is exemplified by low Kd, high melting temperature (Tm), or a combination thereof.

[0116] In some embodiments, the modified nucleotide analogs comprising the compound of formula II (e.g., formula IIa) described herein are chiral-pure (or sterically-pure) polynucleotide molecules, or polynucleotide molecules comprising a single enantiomer. In some examples, the polynucleotide molecule contains an L-nucleotide. In some examples, the polynucleotide molecule contains a D-nucleotide. In some examples, the polynucleotide molecule composition contains 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of its enantiomers. In some cases, the polynucleotide molecule composition contains 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of a racemic mixture. In some examples, polynucleic acid molecules are those described in U.S. Patent Application Publications 2014 / 194610 and 2015 / 211006, and PCT International Publication WO2015107425.

[0117] In some embodiments, the polynucleic acid molecules described herein are further modified to include an aptamer-conjugate moiety. In some examples, the aptamer-conjugate moiety is a DNA aptamer-conjugate moiety. In some examples, the aptamer-conjugate moiety is an Alphamer (Centauri Therapeutics), which includes an aptamer moiety that recognizes a specific cell surface target and a moiety that exhibits a specific epitope for binding to a circulating antibody. In some examples, the polynucleic acid molecules described herein are further modified to include an aptamer-conjugate moiety as described in U.S. Patents 8,604,184, 8,591,910, and 7,850,975.

[0118] In further embodiments, the polynucleic acid molecules described herein are modified to increase their stability. In some embodiments, the polynucleic acid molecule is RNA (e.g., siRNA), and the polynucleic acid molecule is modified to increase its stability. In some examples, the polynucleic acid molecule is modified by one or more of the modifications described above to increase its stability. In some cases, polynucleic acid molecules are modified at the 2'-hydroxyl position by 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modification, or by locked or cross-linked ribose structures (e.g., LNA or ENA). In some cases, polynucleic acid molecules are modified by 2'-O-methyl and / or 2'-O-methoxyethylribose. In some cases, polynucleotide molecules further contain morpholino, PNA, HNA, methylphosphonate nucleotides, thiolphosphonate nucleotides, and / or 2'-fluoroN3-P5'-phosphoramidites to increase their stability. In some examples, the polynucleotide molecule is chiral-pure (or stereopure). In some examples, the chiral-pure (or stereopure) polynucleotide molecule is modified to increase its stability. Appropriate modifications of RNA to increase delivery stability will be apparent to those skilled in the art.

[0119] In some embodiments, the polynucleic acid molecules described herein have RNAi activity that modulates the expression of RNA encoded by the gene described above. In some examples, the polynucleic acid molecules described herein are double-stranded siRNA molecules that downregulate gene expression, wherein one strand of the double-stranded siRNA molecule contains a nucleotide sequence complementary to the nucleotide sequence of the gene, or RNA encoded by the gene or a portion thereof, and the second strand of the double-stranded siRNA molecule contains a nucleotide sequence substantially similar to the nucleotide sequence of the gene, or RNA encoded by the gene or a portion thereof. In some cases, the polynucleic acid molecules described herein are double-stranded siRNA molecules that downregulate gene expression, wherein each strand of the siRNA molecule contains about 15–25, 18–24, or 19–about 23 nucleotides, and each strand contains at least about 14, 17, or 19 nucleotides complementary to the nucleotides of the other strand. In some cases, the polynucleic acid molecules described herein are double-stranded siRNA molecules that downregulate gene expression, where each strand of the siRNA molecule contains approximately 19 to approximately 23 nucleotides, and each strand contains at least approximately 19 nucleotides complementary to the nucleotides of the other strand.

[0120] In some embodiments, the polynucleic acid molecules described herein are constructed using chemical synthesis and / or enzymatic ligation reactions with respect to procedures known in the art. For example, polynucleic acid molecules are chemically synthesized using naturally occurring nucleotides or using various modified nucleotides designed to increase the biostability of the molecule or to increase the physical stability of the double helix formed between the polynucleic acid molecule and the target nucleic acid. Exemplary methods include those described in U.S. Patents 5,142,047, 5,185,444, 5,889,136, 6,008,400, and 6,111,086, PCT International Publication WO2009099942, or European Patent Publication 1579015.Additional exemplary methods include Griffey et al., “2'-O-aminopropyl ribonucleotides: a zwitterionic modification that enhances the exonuclease resistance and biological activity of antisense oligonucleotides,” J. Med. Chem. 39(26):5100-5109 (1997)); Obika, et al. “Synthesis of 2'-O,4'-C-methyleneuridine and -cytidine. Novel bicyclic nucleosides having a fixed C3, -endo sugar puckering”. Tetrahedron Letters 38 (50): 8735 1997; Koizumi, M. “ENA oligonucleotides as therapeutics”. Current opinion in molecular therapeutics 8 (2): 144-149 (2006); and Abramova et al., “Novel oligonucleotide analogues based on morpholino nucleoside subunits-antisense technologies: new chemical possibilities,” This includes those described in Indian Journal of Chemistry 48B:1721-1726 (2009). Alternatively, polynucleotide molecules are biologically generated using expression vectors in which the polynucleotide molecule is subcloned in antisense orientation (i.e., the RNA transcribed from the inserted polynucleotide molecule is antisense-oriented relative to the desired target polynucleotide molecule).

[0121] One embodiment provides an oligonucleotide conjugate of formula (I) or formula (IA), AB Equation (I), ABC Formula (IA) During the ceremony, A is the connecting part, C is optionally a polymer, B is a nucleotide compound of formula (II) or an oligonucleotide compound derived from formula (II) (for example, the phosphoramidite group of formula (II) is converted to a phosphate group in the oligonucleotide structure),

[0122] [ka] During the ceremony, R 1 Each of these is independently a substituted or unsubstituted C1-C6 alkyl, a substituted or unsubstituted C1-C6 fluoroalkyl, or a substituted or unsubstituted C1-C6 heteroalkyl. R 2 Each of these is independently hydrogen, deuterium, a substituted or unsubstituted C1-C6 alkyl, or a substituted or unsubstituted C1-C6 heteroalkyl. Alternatively, two R's 2 These, together with the nitrogen atom to which they are bonded, form a substituted or unsubstituted C2-C 10 Forms heterocycloalkyl groups, L 1 These are bonded, substituted, or unsubstituted C1-C5 alkylenes, substituted or unsubstituted C2-C5 alkenylenes, or substituted or unsubstituted C2-C5 alkynylenes. L 2 is bond, O, S, NR 3 , substituted or unsubstituted C4-C7 cycloalkylene, substituted or unsubstituted C4-C7 heterocycloalkylene, substituted or unsubstituted C5-C8 arylene, or substituted or unsubstituted C4-C8 heteroarylene, R 3 If present, it is selected from hydrogen, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C1-C6 fluoroalkyl, unsubstituted or substituted C1-C6 heteroalkyl, unsubstituted or substituted monocyclic carbocyclic, and unsubstituted or substituted monocyclic heterocyclic. L 3is a bond, substituted or unsubstituted C1-C5 alkylene, substituted or unsubstituted C2-C5 alkenylene, or substituted or unsubstituted C2-C5 alkynylene, and L 1 , L 2 , and L 3 at least two are not a bond.

[0123] In some embodiments of the compound of formula (II), L 2 is a bond, O, S, or NR 3 . In some embodiments, L 2 is O, S, or NR 3 . In some embodiments, L 2 is NR 3 . In some embodiments, L 2 is O. In some embodiments, L 2 is S. In some embodiments, L 2 is a bond.

[0124] In some embodiments of the compound of formula (II), L 2 is substituted or unsubstituted C4-C7 cycloalkylene. In some embodiments, L 2 is substituted or unsubstituted C5-C8 arylene. In some embodiments, L 2 is unsubstituted C4-C7 cycloalkylene. In some embodiments, L 2 is phenylene. In some embodiments, L 2 is methylene. In some embodiments, L 2 is unsubstituted C5-C8 arylene. In some embodiments, L 2 is phenylene. In some embodiments, L 2 is methylene. In some embodiments, L 2 is cyclohexyl.

[0125] In some embodiments of the compound of formula (II), L 1L is a substituted or unsubstituted C1-C5 alkylene, a substituted or unsubstituted C2-C5 alkenylene, or a substituted or unsubstituted C2-C5 alkynylene, and 3 is a bonded, substituted, or unsubstituted C1-C5 alkylene, a substituted or unsubstituted C2-C5 alkenylene, or a substituted or unsubstituted C2-C5 alkynylene. In some embodiments, L 1 is C1-C5 alkylene, C1-C3 alkenylene, or C1-C5 alkynylene, and L 3 is a C1-C5 alkylene, a C1-C3 alkenylene, or a C1-C5 alkynylene. In some embodiments, L 1 It is a C1-C5 alkylene, and L 3 It is a C1-C5 alkylene.

[0126] In some embodiments of the compound of formula (II), L 1 , L 2 , and L 3 At least two of them are not bonded. In some embodiments, L 1 L is a bond. In some embodiments, L 3 It is a combination.

[0127] In some embodiments of equation (II), R 1 Each of these is independently a substituted or unsubstituted C1-C6 alkyl, or a substituted or unsubstituted C1-C6 heteroalkyl. In some embodiments, R 1 Each of these is independently a substituted or unsubstituted C1-C6 alkyl group. In some embodiments, R 1 These are independently -CH3, -CH2CH3, -CH2CH2CH3, or -CH2(CH3)2. In some embodiments, R 1 These are independently -CH3, -CH2CH2CH3, or -CH2(CH3)2. In some embodiments, R 1 It is -CH3.

[0128] In some embodiments of equation (II), R 2Each of these is independently a substituted or unsubstituted C1-C6 alkyl, or a substituted or unsubstituted C1-C6 heteroalkyl. In some embodiments, R 2 Each of these is independently a substituted or unsubstituted C1-C6 alkyl group. In some embodiments, R 2 These are independently -CH3, -CH2CH3, -CH2CH2CH3, or -CH2(CH3)2. In some embodiments, R 2 These are independently -CH3, -CH2CH2CH3, or -CH2(CH3)2. In some embodiments, R 2 These are each -CH2(CH3)2.

[0129] In some embodiments of equation (II), two R 2 These, together with the nitrogen atom to which they are bonded, form a substituted or unsubstituted C2-C 10 It forms a heterocycloalkyl group.

[0130] In some embodiments of equation (II), R 3 R is selected from hydrogen, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C1-C6 fluoroalkyl, and unsubstituted or substituted C1-C6 heteroalkyl. In some embodiments, R 3 R is selected from unsubstituted or substituted monocyclic carbocyclic rings and unsubstituted or substituted monocyclic heterocyclic rings. In some embodiments, R 3 It is hydrogen.

[0131] In some embodiments of the oligonucleotide conjugate of formula (I), B is a compound having the structure of formula (II).

[0132] One embodiment provides an oligonucleotide conjugate of formula (Xa), AX-B'-YC Equation (Xa) During the ceremony, A is the connecting part, B' is a polynucleotide compound of formula (II), C is optionally a polymer, X is a single bond or the first linker, and, Y is a single bond or a second linker. Polynucleotides further contain one or more additional non-natural nucleotides, and A and C do not bond to B at the same end.

[0133] Another embodiment provides an oligonucleotide of formula (I) or (Xa), where the polynucleotide further comprises at least one modified internucleotide bond or at least one inverted debase moiety.

[0134] Another embodiment provides an oligonucleotide of formula (I) or (Xa), where the compound of formula (IIa) is located in the internucleotide bond of a polynucleotide.

[0135] Another embodiment provides an oligonucleotide of formula (I) or (Xa), wherein the compound of formula (IIa) is further modified at the 2' position.

[0136] Another embodiment provides an oligonucleotide of formula (I) or (Xa), wherein one or more additional non-natural nucleosides comprise a 2'-modification selected from nucleotides modified with 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA).

[0137] Another embodiment provides an oligonucleotide of formula (I) or (Xa), where the compound of formula (IIa) is selected from locked nucleic acid (LNA) or ethylene nucleic acid (ENA).

[0138] Another embodiment provides an oligonucleotide of formula (I) or (Xa), wherein at least one inverted debase portion has at least one terminus.

[0139] Another embodiment provides an oligonucleotide of formula (I) or (Xa), where the oligonucleotide is single-stranded. Another embodiment provides an oligonucleotide of formula (Xa), where the oligonucleotide is double-stranded.

[0140] Another embodiment provides an oligonucleotide of formula (I) or (Xa), where the oligonucleotide is 2 to about 100 residues long. Another embodiment provides an oligonucleotide of formula (I) or (Xa), where the oligonucleotide is 2 to about 90 residues long. Another embodiment provides an oligonucleotide of formula (I) or (Xa), where the oligonucleotide is 2 to about 80 residues long. Another embodiment provides an oligonucleotide of formula (I) or (Xa), where the oligonucleotide is 2 to about 70 residues long. Another embodiment provides an oligonucleotide of formula (I) or (Xa), where the oligonucleotide is 2 to about 60 residues long. Another embodiment provides an oligonucleotide of formula (I) or (Xa), where the oligonucleotide is 2 to about 50 residues long. Another embodiment provides an oligonucleotide of formula (I) or (Xa), where the oligonucleotide is 2 to about 40 residues long. Another embodiment provides an oligonucleotide of formula (I) or (Xa), where the oligonucleotide is 2 to about 30 residues long. Another embodiment provides an oligonucleotide of formula (I) or (Xa), where the oligonucleotide is 2 to about 20 residues long. Another embodiment provides an oligonucleotide of formula (I) or (Xa), where the oligonucleotide is 2 to about 10 residues long. Another embodiment provides an oligonucleotide of formula (I) or (Xa), where the oligonucleotide is 8 to about 30 residues long. Another embodiment provides an oligonucleotide of formula (Xa), where the oligonucleotide is 10 to about 30 residues long. Another embodiment provides an oligonucleotide of formula (I) or (Xa), where the oligonucleotide is 14 to about 30 residues long. Another embodiment provides an oligonucleotide of formula (Xa), where the oligonucleotide is 18 to about 30 residues long. Another embodiment provides an oligonucleotide of formula (I) or (Xa), where the oligonucleotide is 22 to about 30 residues long. Another embodiment provides an oligonucleotide of formula (I) or (X), where the oligonucleotide is 26 to about 30 residues long.

[0141] One embodiment provides a compound suitable for the synthesis of oligonucleotides selected from the following group:

[0142] [Chem.]

[0143] [Chem.] In the formula, R x is H, halogen, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C1-C6 fluoroalkyl, unsubstituted or substituted C1-C6 heteroalkyl, unsubstituted or substituted monocyclic carbocycle, unsubstituted or substituted monocyclic heterocycle, -CN, -OH, -O-alkyl, -CO2H, -CO2-alkyl, -CH2CO2H, -CH2CO2-alkyl, -C(=O)NH2, -C(=O)NH-alkyl, -CH2C(=O)NH2, -CH2C(=O)NH-alkyl, NH2, -NH-alkyl, -CH2NH2, -CH2NH-alkyl, -NHC(=O)alkyl, -CH2NHC(=O)alkyl, -SH, -S-alkyl, -S(=O)H, -S(=O)alkyl, -SO2H, -SO2-alkyl, -SO2NH2, or -SO2NH-alkyl.

[0144] Conjugation Chemistry In some embodiments, the polynucleic acid molecule is conjugated to a binding moiety. In some examples, the binding moiety comprises amino acids, peptides, polypeptides, proteins, antibodies, antigens, toxins, hormones, lipids, nucleotides, nucleosides, sugars, carbohydrates, polymers such as polyethylene glycol and polypropylene glycol, as well as all analogs or derivatives of substances belonging to these classes. Additional examples of binding moieties further include steroids, for example, cholesterol, phospholipids, diacylglycerols and triacylglycerols, fatty acids, hydrocarbons (for example, saturated, unsaturated, or those containing substitutions), enzyme substrates, biotin, digoxigenin, and polysaccharides. In some examples, the binding moiety is an antibody or a binding fragment thereof. In some examples, the polynucleic acid molecule is further conjugated to a polymer, and optionally conjugated to an endosomolytic moiety.

[0145] In some embodiments, polynucleic acid molecules are conjugated to the binding site by a chemical ligation process. In some examples, polynucleic acid molecules are conjugated to the binding site by spontaneous ligation. In some examples, the conjugation is as described in Dawson, et al. “Synthesis of proteins by native chemical ligation,” Science 1994, 266, 776-779; Dawson, et al. “Modulation of Reactivity in Native Chemical Ligation through the Use of Thiol Additives,” J. Am. Chem. Soc. 1997, 119, 4325-4329; Hackeng, et al. “Protein synthesis by native chemical ligation: Expanded scope by using straightforward methodology.” Proc. Natl. Acad. Sci. USA 1999, 96, 10068-10073; or Wu, et al. “Building complex glycopeptides: Development of a cysteine-free native chemical ligation protocol,” Angew. Chem. Int. Ed. 2006, 45, 4116-4125. In some examples, the conjugation is as described in U.S. Patent No. 8,936,910. In some embodiments, polynucleic acid molecules conjugate to the binding site site-specifically or non-specifically via natural ligation chemistry.

[0146] In some cases, polynucleotide molecules are conjugated to the binding site using a site-specific method that utilizes "traceless" coupling technology (PhiloChem). In some cases, the "traceless" coupling technology utilizes the N-terminal 1,2-aminothiol group of the binding site to conjugate to polynucleotide molecules containing aldehyde groups. (See Casi et al., “Site-specific traceless coupling of potent cytotoxic drugs to recombinant antibodies for pharmacodelivery,” JACS 134(13): 5887-5892 (2012))

[0147] In some cases, polynucleic acid molecules are conjugated to a binding site by a site-specific method utilizing unnatural amino acids introduced into the binding site. In some cases, the unnatural amino acid includes p-acetylphenylalanine (pAcPhe). In some cases, the keto group of pAcPhe is selectively coupled to the alkoxyamine derivative conjugate site to form an oxime bond. (See Axup et al., “Synthesis of site-specific antibody-drug conjugates using unnatural amino acids,” PNAS 109(40): 16101-16106 (2012)).

[0148] In some cases, polynucleic acid molecules are conjugated to binding sites by site-specific methods utilizing enzyme-catalyzed processes. In some cases, site-specific methods utilize SMARTag® technology (Redwood). In some cases, SMARTag® technology involves the generation of formylglycine (FGly) residues from cysteine ​​by formylglycine-producing enzyme (FGE) via an oxidation process in the presence of an aldehyde tag, and the subsequent conjugation of FGly to alkylhydrazine-functionalized polynucleic acid molecules via hydrazino-Pictet-Spengler (HIPS) ligation. (Wu et al., “Site-specific chemical modification of recombinant proteins produced in mammalian cells by using the genetically encoded aldehyde tag,” PNAS 106(9): 3000-3005 (2009); Agarwal, et al., “A Pictet-Spengler ligation for protein chemical modification,” PNAS 110(1): 46-51 (2013)).

[0149] In some cases, the enzyme-catalyzed process involves microbial transglutaminase (mTG). In some cases, polynucleic acid molecules are conjugated to the binding site using a microbial transglutaminase-catalyzed process. In some cases, mTG catalyzes the formation of a covalent bond between the amide side chain of glutamine in the recognition sequence and the primary amine of the functionalized polynucleic acid molecule. In some cases, mTG is produced from Streptomyces mobarensis. (See Strop et al., “Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates,” Chemistry and Biology 20(2) 161-167(2013)).

[0150] In some cases, polynucleic acid molecules are conjugated to the binding site by methods such as those described in PCT International Publication WO2014 / 140317, which utilize sequence-specific transpeptidases.

[0151] In some examples, polynucleic acid molecules are conjugated to the binding site in a manner such as that described in U.S. Patent Publications 2015 / 0105539 and 2015 / 0105540.

[0152] joining part In some embodiments, the binding portion A is a polypeptide. In some examples, the polypeptide is an antibody or a fragment thereof. In some cases, the fragment is a binding fragment. In some examples, the antibody or its binding fragment includes a humanized antibody or its binding fragment, a human antibody or its binding fragment, an anti-mouse antibody (e.g., an anti-mouse antibody, an anti-rat antibody, etc.), an anti-human antibody (e.g., an anti-human transferrin receptor antibody), a mouse antibody or its binding fragment, a chimeric antibody or its binding fragment, a monoclonal antibody or its binding fragment, a monovalent Fab', a bivalent Fab2, an F(ab)'3 fragment, a single-strand variable fragment (scFv), a bis-scFv, (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide-stabilized Fv protein (dsFv), a single-domain antibody (sdAb), an Ig NAR, a camelid antibody or its binding fragment, a bispecific antibody or its binding fragment, or chemically modified derivatives thereof.

[0153] In some cases, A is an antibody or its binding fragment. In some cases, A is a humanized antibody or its binding fragment, a mouse antibody or its binding fragment, a chimeric antibody or its binding fragment, a monoclonal antibody or its binding fragment, a monovalent Fab', a bivalent Fab2, an F(ab)'3 fragment, a single-strand variable fragment (scFv), bis-scFv, (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide-stabilized Fv protein ("dsFv"), a single-domain antibody (sdAb), an Ig NAR, a camelid antibody or its binding fragment, a bispecific antibody or its binding fragment, or a chemically modified derivative thereof. In some cases, A is a humanized antibody or its binding fragment. In some cases, A is a mouse antibody or its binding fragment. In some cases, A is a chimeric antibody or its binding fragment. In some cases, A is a monoclonal antibody or its binding fragment. In some cases, A is a full-size antibody. In some cases, A is a monovalent Fab'. In some cases, A is a bivalent Fab2. In some examples, A is a single-stranded variable fragment (scFv).

[0154] In some embodiments, binding site A is a bispecific antibody or its binding fragment. In some examples, the bispecific antibody is a trifunctional antibody or a bispecific mini-antibody. In some cases, the bispecific antibody is a trifunctional antibody. In some examples, the trifunctional antibody is a full-length monoclonal antibody containing binding sites for two different antigens. Exemplary trifunctional antibodies include catumakisomab (targeting EpCAM and CD3; Fresenius Biotech / Trion Pharma), erzumakisomab (targeting HER2 / neu / CD3; Fresenius Biotech / Trion Pharma), and lymphomun FBTA05 (targeting CD20 / CD3; Fresenius Biotech / Trion Pharma). This includes Pharm, RG7221 (RO5520985; targeting angiopoietin 2 / VEGF; Roche), RG7597 (targeting Her1 / Her3; Genentech / Roche), MM141 (targeting IGF1R / Her3; Merrimack), ABT122 (targeting TNFα / IL17; Abbvie), ABT981 (targeting IL1α / IL1β; Abbott), LY3164530 (targeting Her1 / cMET; Eli Lilly), and TRBS07 (Ektomab; targeting GD2 / CD3; Trion Research GmbH). Further exemplary trifunctional antibodies include mAb2 from F-star Biotechnology Ltd. In some examples, A is a bispecific trifunctional antibody.In some embodiments, A is catumakisomab (targeting EpCAM and CD3; Fresenius Biotech / Trion Pharma), erzumakisomab (targeting HER2 / neu / CD3; Fresenius Biotech / Trion Pharm), lymphomun FBTA05 (targeting CD20 / CD3; Fresenius Biotech / Trion Pharm), RG7221 (RO5520985; angiopoietin These are bispecific trifunctional antibodies selected from mAb2 from F-star Biotechnology Ltd., including 2 / VEGF-targeting (Roche), RG7597 (Her1 / Her3-targeting (Genentech / Roche)), MM141 (IGF1R / Her3-targeting (Merrimack)), ABT122 (TNFα / IL17-targeting (Abbvie)), ABT981 (IL1α / IL1β-targeting (Abbott)), LY3164530 (Her1 / cMET-targeting (Eli Lilly)), TRBS07 (Ektomab; GD2 / CD3-targeting (Trion Research GmbH)), and mAb2.

[0155] In some cases, bispecific antibodies are bispecific mini-antibodies. In some examples, bispecific mini-antibodies include bivalent Fab2, F(ab)'3 fragments, bis-scFv, (scFv)2, diabodies, minibodies, triabodies, tetrabodies, or bispecific T cell engagers (BiTEs). In some embodiments, a bispecific T cell engager is a fusion protein containing two single-strand variable fragments (scFvs) in which two scFvs target epitopes of two different antigens. Exemplary bispecific mini-antibodies include, but are not limited to, DART (Biaffinity Retargeting Platform; MacroGenics), blinatumomab (MT103 or AMG103; targeting CD19 / CD3; Micromet), MT111 (targeting CEA / CD3; Micromet / Amegen), MT112 (BAY2010112; targeting PSMA / CD3; Micromet / Bayer), MT110 (AMG Includes 110 (targeting EPCAM / CD3; Amgen / Micromet), MGD006 (targeting CD123 / CD3; MacroGenics), MGD007 (targeting GPA33 / CD3; MacroGenics), BI1034020 (targeting two different epitopes on β-amyloid; Ablynx), ALX0761 (targeting IL17A / IL17F; Ablynx), TF2 (targeting CEA / heptene; Immunomedics), IL-17 / IL-34 biAb (BMS), AFM13 (targeting CD30 / CD16; Affimed), AFM11 (targeting CD19 / CD3; Affimed), and domain antibodies (dAbs from Domantis / GSK).

[0156] In some embodiments, the binding portion A is a bispecific mini-antibody. In some examples, A is a bispecific Fab2. In some examples, A is a bispecific F(ab)'3 fragment. In some cases, A is a bispecific bis-scFv. In some embodiments, A is a bispecific diabody, and in some cases, A is a bispecific (scFv)2. In some embodiments, A is a bispecific mini-body. In some embodiments, A is a bispecific triabody. In other embodiments, A is a bispecific tetrabody. In other embodiments, A is a bispecific T-cell engager (BiTE). In further embodiments, A is DART (Biaffinity Retargeting Platform; MacroGenics), blinatumomab (MT103, or AMG103; targeting CD19 / CD3; Micromet), MT111 (targeting CEA / CD3; Micromet / Amegen), MT112 (BAY2010112; targeting PSMA / CD3; Micromet / Bayer), MT110 (AMG These are bispecific mini-antibodies selected from 110 (targeting EPCAM / CD3; Amgen / Micromet), MGD006 (targeting CD123 / CD3; MacroGenics), MGD007 (targeting GPA33 / CD3; MacroGenics), BI1034020 (targeting two different epitopes on β-amyloid; Ablynx), ALX0761 (targeting IL17A / IL17F; Ablynx), TF2 (targeting CEA / heptene; Immunomedics), IL-17 / IL-34 biAb (BMS), AFM13 (targeting CD30 / CD16; Affimed), AFM11 (targeting CD19 / CD3; Affimed), and domain antibodies (dAbs from Domantis / GSK).

[0157] In some embodiments, binding site A is a trispecific antibody. In some examples, the trispecific antibody includes an F(ab)'3 fragment or a triabody. In some examples, A is a trispecific F(ab)'3 fragment. In some cases, A is a triabody. In some embodiments, A is a trispecific antibody as described in Dimas, et al., “Development of a trispecific antibody designed to simultaneously and efficiently target three different antigens on tumor cells,” Mol. Pharmaceutics, 12(9): 3490-3501 (2015).

[0158] In some embodiments, binding portion A is an antibody or its binding fragment that recognizes a cell surface protein. In some examples, the cell surface protein is an antigen expressed by cancer cells. Exemplary cancer antigens include, but are not limited to, alpha-fetoprotein, ASLG659, B7-H3, BAFF-R, Brevican, CA125 (MUC16), CA15-3, CA19-9, carcinoembryonic antigen (CEA), CA242, CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratoma-derived growth factor), CTLA-4, CXCR5, E16 (LAT1, SLC7A5), FcRH2 (IFGP4, IRTA4, S PAP1A (SH2 domain-containing phosphatase anchor protein 1a), SPAP1B, SPAP1C), epidermal growth factor, ETBR, Fc receptor-like protein (FCRH1), GEDA, HLA-DOB (β-subunit of MHC class II molecule (Ia antigen)), human chorionic gonadotropin, ICOS, IL-2 receptor, IL20Rα, immunoglobulin superfamily receptor translocation-related 2 (IRTA2), L6, Lewis Y, Lewis X, MAGE-1, MAGE-2, MAGE-3, MAGE 4, MART1, Mesoserine, MDP, MPF (SMR, MSLN), MCP1 (CCL2), Macrophage Inhibitor (MIF), MPG, MSG783, Mucin, MUC1-KLH, Napi3b (SLC34A2), Nectin-4, Neu Oncogene Product, NCA, Placental Alkaline Phosphatase, Prostate-Specific Membrane Antigen (PMSA), Prostatic Acid Phosphatase, PSCA hlg, p97, Purine Receptor P2X Ligand Open Ion Channel 5 (P2X5), LY64 (Lymphocyte Antigen 64 (RP105), gp100, P21, Six Transmembrane Epithelial Antigen of Prostate (STEAP1), STEAP2, Sema 5b) These include transferrin receptors, tumor-associated glycoprotein 72 (TAG-72), TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4), etc. In some examples, the binding site is an α-transferrin receptor antibody or its binding fragment.In some cases, the binding site is an α-human transferrin receptor antibody. In some cases, the binding site is an α-human transferrin receptor antibody, such as that described in PCT / US2019 / 068078, which is incorporated by reference herein.

[0159] In some cases, cell surface proteins contain clusters of differentiated (CD) cell surface markers. The CD cell surface markers mentioned are, but are not limited to, CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CDw12, CD13, CD14, CD15, CD15s, CD16, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD45RO, CD45RA, CD45RB, CD46, CD47, CD48, CD49a, CD49b, CD 49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CDw60, C D61, CD62E, CD62L (L-selectin), CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66d, CD66e, CD7 1. Includes CD79 (e.g., CD79a, CD79b), CD90, CD95 (Fas), CD103, CD104, CD125 (IL5RA), CD134 (OX40), CD137 (4-1BB), CD152 (CTLA-4), CD221, CD274, CD279 (PD-1), CD319 (SLAMF7), CD326 (EpCAM), etc.

[0160] In some cases, binding site A is an antibody or its binding fragment that recognizes a cancer antigen. In some cases, binding site A is alpha-fetoprotein, ASLG659, B7-H3, BAFF-R, Brevican, CA125 (MUC16), CA15-3, CA19-9, carcinoembryonic antigen (CEA), CA242, CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratoma-derived growth factor), CTLA-4, CXCR5, E16 (LAT1, SLC7A5), FcRH2 (IFGP4, IRTA4, S PAP1A (SH2 domain-containing phosphatase anchor protein 1a), SPAP1B, SPAP1C), epidermal growth factor, ETBR, Fc receptor-like protein (FCRH1), GEDA, HLA-DOB (β-subunit of MHC class II molecule (Ia antigen)), human chorionic gonadotropin, ICOS, IL-2 receptor, IL20Rα, immunoglobulin superfamily receptor translocation-related 2 (IRTA2), L6, Lewis Y, Lewis X, MAGE-1, MAGE-2, MAGE-3, MAGE 4, MART1, Mesothelin, MCP1 (CCL2), MDP, Macrophage migration inhibitory factor (MIF), MPF (SMR, MSLN), MPG, MSG783, Mucin, MUC1-KLH, Napi3b (SLC34A2), Nectin-4, Neu oncogene product, NCA, Placental alkaline phosphatase, Prostate-specific membrane antigen (PMSA), Prostatic acid phosphatase, PSCA hlg, p97, Purine receptor P2X ligand open ion channel 5 (P2X5), LY64 (Lymphocyte antigen 96 (RP105), gp100, P21, Six transmembrane epithelial antigens of the prostate (STEAP1), STEAP2, Sema 5b, an antibody or its binding fragment that recognizes tumor-associated glycoprotein 72 (TAG-72), TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4), or a combination thereof.

[0161] In some cases, binding site A is an antibody or its binding fragment that recognizes CD cell surface markers. In some cases, binding site A is CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CDw12, CD13, CD14, CD15, CD15s, CD16, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD 43, CD44, CD45, CD45RO, CD45RA, CD45RB, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD4 9f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CDw60, CD61, CD62E, CD62L (L-Select CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66d, CD66e, CD71, CD79 (e.g. CD79a, CD79b), C An antibody or fragment that recognizes D90, CD95 (Fas), CD103, CD104, CD125 (IL5RA), CD134 (OX40), CD137 (4-1BB), CD152 (CTLA-4), CD221, CD274, CD279 (PD-1), CD319 (SLAMF7), CD326 (EpCAM), or a combination thereof.

[0162] In some embodiments, the antibody or its binding fragment is saltumumab (HuMax-EFGr, Genmab), avagovomab (Menarini), abituzumab (Merck), adecatumumab (MT201), aracizumab pegol, alemtuzumab (Campath®, MabCampath or Campath-1H; Leukosite), AlloMune (BioTransplant), amatsuximab (Morphotek Inc.), anti-VEGF (Genetech), anatumomab mafenatox, apolizumab (hu1D10), ascrinbakumab (Pfizer Inc.), atezolizumab (MPDL3280A; Genentech / Roche), B43.13 (OvaRex, AltaRex) Novartis Corporation, basiliximab (Simulect®, Novartis), belimumab (Benlysta®, GlaxoSmithKline), bevacizumab (Avastin®, Genentech), blinatumomab (Blincyto, AMG103; Amgen), BEC2 (ImGlone Systems Inc.), carlumab (Janssen Biotech), catumakisomab (Removab, Trion Pharma), CEAcide (Immunomedics), cetuximab (Erbitux®, ImClone), sitatuzumab vogatox (VB6-845), cizutumumab (IMC-A12, ImClone Systems Inc.), konatumumab (AMG 655 (Amgen), Dasetuzumab (SGN-40, huS2C6; Seattle Genetics, Inc.), Daratumumab (Darzalex®, Janssen Biotech), Detumomab, Drojituzumab (Genentech), Durvalumab (MedImmune), Dusigitumab (dusigitumab) (MedImmune), Edrecolomab (MAb171A, Panorex, Glaxo Wellcome), Elotuzumab (Empliciti®, Bristol-Myers Squibb), Emibetuzumab (Eli Lilly), Enabatuzumab (Facet Biotech Corp.)), Enfortumab Vedotin (Seattle Genetics, Inc.), Enobrituzumab (MGA271, MacroGenics, Inc.), Encituzumab (ensituxumab) (Neogenix Oncology, Inc.), Epratuzumab (LymphoCide, Immunomedics, Inc.), Erzmakisomab (Rexomun®, Trion Pharma), Etalacizumab (Abegrin, MedImmune), Faretuzumab (MORAb-003, Morphotek Inc.), FBTA05 (Lymphomun, Trion Pharma), Ficlatuzumab (AVEO Pharmaceuticals), Figitumumab (CP-751871, Pfizer), Frambotumab (ImClone (Systems), Frezolimumab (GC1008, Aanofi-Aventis), Futuximab, Glaximab, Ganitumumab (Amgen), Dilentuximab (Rencarex®, Wilex AG), IMAB362 (Claudiximab, Ganymed Pharmaceuticals AG), Imarumab (Baxalta), IMC-1C11 (ImClone Systems), IMC-C225 (ImClone Systems Inc.), Imugatuzumab (Genentech / Roche), Intetumumab (Centocor), Ipilimumab (Yervoy®, Bristol-Myers) Squibb), Iratumumab (Medarex, Inc.), Isatuximab (SAR650984, Sanofi-Aventis), Labetuzumab (CEA-CIDE, Immunomedics), Lexatumumab (ETR2-ST01, Cambridge Antibody Technology), Lintuzumab (SGN-33, Seattle Genetics), Lucatumumab (Novartis), Lumiliximab, Mapatuzumab (HGS-ETR1, Human Genome Sciences), Matuzumab (EMD 72000, Merck), Miratuzumab (hLL1, Immunomedics, Inc.)), mitumomab (BEC-2, ImClone Systems), narnatumab (ImClone Systems), nesitumumab (Portrazza®, Eli Lilly), nesbacumab (Regeneron Pharmaceuticals), nimotuzumab (h-R3, BIOMAb EGFR, TheraCIM, Theraloc, or CIMAher; Biotech Pharmaceutical Co.), nivolumab (Opdivo®, Bristol-Myers Squibb), obinutuzumab (Gazyva or Gazyvaro; Hoffmann-La Roche), okalatuzumab (AME-133v, LY2469298; Mentrik Biotech, LLC), ofatumumab (Arzerra®, Genmab), onartuzumab (Genentech), ontuxizumab (Morphotek Inc.), olegovomab (OvaRex®, AltaRex, Corp.), otlertuzumab (Emergent BioSolutions), panitumumab (ABX-EGF, Amgen), pankomab (Glycotope) GMBH), parsatuzumab (Genentech), patritumab, pembrolizumab (Keytruda®, Merck), pemtumomab (Theragyn, Antisoma), pertuzumab (Perjeta, Genentech), pidilizumab (CT-011, Medivation), polatuzumab vedotin (Genentech / Roche), pritumumab, lacosumomab (Vaxira®, Recombio), ramucirumab (Cyramza®, ImClone Systems Inc.), rituximab (Rituxan®, Genentech), lobatumumab (Schering-Plough), ceribantomab (Sanofi / Merrimack Pharmaceuticals, Inc.)), sibrotuzumab, siltuximab (Sylvant®, Janssen Biotech), Smart MI95 (Protein Design Labs, Inc.), Smart ID10 (Protein Design Labs, Inc.), tabarmab (LY2127399, Eli Lilly), tapritumomab paptox, tenatumomab, teprotumumab (Roche), tetulomab, TGN1412 (CD28-SuperMAB or TAB08), tigatuzumab (CD-1008, Daiichi Sankyo), tositumomab, trastuzumab (Herceptin®), tremelimumab (CP-672,206; Pfizer), tucotsuzumab cermoloykin (EMD This includes pharmaceuticals such as ubrituximab, urelumab (BMS-663513, Bristol-Myers Squibb), borosiximab (M200, Biogen Idec), and zatuximab.

[0163] In some embodiments, the binding portion A is saltumumab (HuMax-EFGr, Genmab), avagovomab (Menarini), abituzumab (Merck), adecatumumab (MT201), aracizumab pegol, alemtuzumab (Campath®, MabCampath or Campath-1H; Leukosite), AlloMune (BioTransplant), amatsuximab (Morphotek Inc.), anti-VEGF (Genetech), anatumomab mafenatox, apolizumab (hu1D10), askrinbakumab (Pfizer Inc.), atezolizumab (MPDL3280A; Genentech / Roche), B43.13 (OvaRex, AltaRex) Novartis Corporation, basiliximab (Simulect®, Novartis), belimumab (Benlysta®, GlaxoSmithKline), bevacizumab (Avastin®, Genentech), blinatumomab (Blincyto, AMG103; Amgen), BEC2 (ImGlone Systems Inc.), carlumab (Janssen Biotech), catumakisomab (Removab, Trion Pharma), CEAcide (Immunomedics), cetuximab (Erbitux®, ImClone), sitatuzumab vogatox (VB6-845), cizutumumab (IMC-A12, ImClone Systems Inc.), konatumumab (AMG 655 (Amgen), Dasetuzumab (SGN-40, huS2C6; Seattle Genetics, Inc.), Daratumumab (Darzalex®, Janssen Biotech), Detumomab, Drojituzumab (Genentech), Durvalumab (MedImmune), Dusigitumab (dusigitumab) (MedImmune), Edrecolomab (MAb171A, Panorex, Glaxo Wellcome), Elotuzumab (Empliciti®, Bristol-Myers Squibb), Emibetuzumab (Eli Lilly), Enabatuzumab (Facet Biotech Corp.)), Enfortumab Vedotin (Seattle Genetics, Inc.), Enobrituzumab (MGA271, MacroGenics, Inc.), Encituzumab (ensituxumab) (Neogenix Oncology, Inc.), Epratuzumab (LymphoCide, Immunomedics, Inc.), Erzmakisomab (Rexomun®, Trion Pharma), Etalacizumab (Abegrin, MedImmune), Faretuzumab (MORAb-003, Morphotek Inc.), FBTA05 (Lymphomun, Trion Pharma), Ficlatuzumab (AVEO Pharmaceuticals), Figitumumab (CP-751871, Pfizer), Frambotumab (ImClone) (Systems), Frezolimumab (GC1008, Aanofi-Aventis), Futuximab, Glaximab, Ganitumab (Amgen), Dilentuximab (Rencarex®, Wilex AG), IMAB362 (Claudicimab, Ganymed Pharmaceuticals AG), Imarumab (Baxalta), IMC-1C11 (ImClone Systems), IMC-C225 (ImClone Systems Inc.), Imugatuzumab (Genentech / Roche), Intetumumab (Centocor), Ipilimumab (Yervoy®, Bristol-Myers) Squibb), Iratumumab (Medarex, Inc.), Isatuximab (SAR650984, Sanofi-Aventis), Labetuzumab (CEA-CIDE, Immunomedics), Lexatumumab (ETR2-ST01, Cambridge Antibody Technology), Lintuzumab (SGN-33, Seattle Genetics), Lucatumumab (Novartis), Lumiliximab, Mapatuzumab (HGS-ETR1, Human Genome Sciences), Matuzumab (EMD 72000, Merck), Miratuzumab (hLL1, Immunomedics, Inc.)), mitumomab (BEC-2, ImClone Systems), narnatumab (ImClone Systems), necitumumab (Portrazza®, Eli Lilly), nesbacumab (Regeneron Pharmaceuticals), nimotuzumab (h-R3, BIOMAb EGFR, TheraCIM, Theraloc, or CIMAher; Biotech Pharmaceutical Co.), nivolumab (Opdivo®, Bristol-Myers Squibb), obinutuzumab (Gazyva or Gazyvaro; Hoffmann-La Roche), okalatuzumab (AME-133v, LY2469298; Mentrik Biotech, LLC), ofatumumab (Arzerra®, Genmab), onartuzumab (Genentech), ontuxizumab (Morphotek Inc.), olegovomab (OvaRex®, AltaRex, Corp.), otlertuzumab (Emergent BioSolutions), panitumumab (ABX-EGF, Amgen), pankomab (Glycotope) GMBH), parsatuzumab (Genentech), patritumab, pembrolizumab (Keytruda®, Merck), pemtumomab (Theragyn, Antisoma), pertuzumab (Perjeta, Genentech), pidilizumab (CT-011, Medivation), polatuzumab vedotin (Genentech / Roche), pritumumab, lacosumomab (Vaxira®, Recombio), ramucirumab (Cyramza®, ImClone Systems Inc.), rituximab (Rituxan®, Genentech), lobatumumab (Schering-Plough), ceribantomab (Sanofi / Merrimack Pharmaceuticals, Inc.)), sibrotuzumab, siltuximab (Sylvant®, Janssen Biotech), Smart MI95 (Protein Design Labs, Inc.), Smart ID10 (Protein Design Labs, Inc.), tabarmab (LY2127399, Eli Lilly), tapritumomab paptox, tenatumomab, teprotumumab (Roche), tetulomab, TGN1412 (CD28-SuperMAB or TAB08), tigatuzumab (CD-1008, Daiichi Sankyo), tositumomab, trastuzumab (Herceptin®), tremelimumab (CP-672,206; Pfizer), tucotsuzumab cermoloykin (EMD This includes pharmaceuticals such as ubrituximab, urelumab (BMS-663513, Bristol-Myers Squibb), borosiximab (M200, Biogen Idec), or zatuximab. In some embodiments, the binding site A is zaltumumab (HuMax-EFGr by Genmab).

[0164] Additional connection In some embodiments, the binding site is a plasma protein. In some examples, the plasma protein includes albumin. In some examples, binding site A is albumin. In some examples, albumin is conjugated to a polynucleic acid molecule by one or more of the conjugation chemistry described herein. In some examples, albumin is conjugated to a polynucleic acid molecule by a natural ligation chemistry. In some examples, albumin is conjugated to a polynucleic acid molecule by a lysine linkage.

[0165] In some examples, binding site A is a steroid. Exemplary steroids include cholesterol, phospholipids, diacylglycerols and triacylglycerols, fatty acids, hydrocarbons (saturated, unsaturated, substituted, or combinations thereof). In some examples, the steroid is cholesterol. In some examples, the binding site is cholesterol. In some examples, cholesterol is conjugated to a polynucleic acid molecule by one or more of the conjugation chemistry described herein. In some examples, cholesterol is conjugated to a polynucleic acid molecule by a natural ligation chemistry. In some examples, cholesterol is conjugated to a polynucleic acid molecule by lysine conjugation.

[0166] In some examples, the binding site is a polymer containing a polynucleotide molecular aptamer that binds to a specific surface marker on a cell, though not limited to this example. In this example, the binding site does not hybridize to a target gene or mRNA, but instead is a polynucleotide that can selectively bind to the cell surface marker, similar to an antibody that binds to that specific epitope of the cell surface marker.

[0167] In some cases, binding site A is a peptide. In some cases, the peptide contains approximately 1 to 3 kDa. In some cases, the peptide contains approximately 1.2 to 2.8 kDa, approximately 1.5 to 2.5 kDa, or approximately 1.5 to 2 kDa. In some examples, the peptide is a bicyclic peptide. In some cases, the bicyclic peptide is a constrained bicyclic peptide. In some examples, the peptide portion is a bicyclic peptide (e.g., the two rings in Bicycle Therapeutics).

[0168] In further cases, the binding portion is a small molecule. In some examples, the small molecule is an antibody-recruiting small molecule. In some cases, the antibody-recruiting small molecule includes a target binding end and an antibody binding end, where the target binding end recognizes cell surface receptors and can interact with them. For example, in some examples, a target binding end containing a glutamate urea compound enables interaction with PSMA, thereby enhancing antibody interactions with cells expressing PSMA (e.g., cancer cells). In some cases, the binding site is a small molecule described in Zhang et al., “A remote arene-binding site on prostate specific membrane antigen revealed by antibody-recruiting small molecules,” J Am Chem Soc. 132(36): 12711-12716 (2010); or McEnaney, et al., “Antibody-recruiting molecules: an emerging paradigm for engaging immune function in treating human disease,” ACS Chem Biol. 7(7):1139-1151 (2012).

[0169] Polynucleic acid molecular targets In some embodiments, polynucleotide molecule B is a polynucleotide molecule (or polynucleotide) that hybridizes to a target region on the oncogene. In some examples, the oncogene is further classified into several categories: growth factors or mitogens, receptor tyrosine kinases, cytoplasmic tyrosine kinases, cytoplasmic serine / threonine kinases, regulatory GTPases, and transcription factors. Exemplary growth factors include c-Sis. Exemplary receptor tyrosine kinases include epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), and HER2 / neu. Exemplary cytoplasmic tyrosine kinases include Src family tyrosine kinases, the Syk-ZAP-70 family of tyrosine kinases, the BTK family of tyrosine kinases, and the Abl gene in CML. Exemplary cytoplasmic serine / threonine kinases include Raf kinases and cyclin-dependent kinases. Exemplary regulatory GTPases include the Ras family of proteins such as KRAS. Exemplary transcription factors include MYC genes. In some examples, the oncogenes described herein include oncogenes selected from growth factors or mitogens, receptor tyrosine kinases, cytoplasmic tyrosine kinases, cytoplasmic serine / threonine kinases, regulatory GTPases, or transcription factors. In some embodiments, the polynucleic acid molecule is a polynucleic acid molecule that hybridizes to the target region of the oncogene selected from growth factors or mitogens, receptor tyrosine kinases, cytoplasmic tyrosine kinases, cytoplasmic serine / threonine kinases, regulatory GTPases, or transcription factors.

[0170] In some embodiments, the oncogenes described herein include Abl, AKT-2, ALK, AML1 (or RUNX1), AR, AXL, BCL-2,3,6, BRAF, c-MYC, EGFR, ErbB-2 (Her2,Neu), Fms, FOS, GLI1, HPRT1, IL-3, INTS2, JUN, KIT, KS3, K-sam, LBC (AKAP13), LCK, LMO1 , LMO2, LYL1, MAS1, MDM2, MET, MLL(KMT2A), MOS, MYB, MYH11 / CBFB, NOTCH1(TAN1), NTRK1(TRK), OST(SLC51 B), including PAX5, PIM1, PRAD-1, RAF, RAR / PML, HRAS, KRAS, NRAS, REL / NRG, RET, ROS, SKI, SRC, TIAM1, or TSC2. In some embodiments, the polynucleic acid molecule is Abl, AKT-2, ALK, AML1 (or RUNX1), AR, AXL, BCL-2,3,6, BRAF, c-MYC, EGFR, ErbB-2 (Her2,Neu), Fms, FOS, GLI1, HPRT1, IL-3, INTS2, JUN, KIT, KS3, K-sam, LBC (AKAP13), LCK, LMO1, LMO2, LYL1, MAS 1. A polynucleic acid molecule that hybridizes to the target region of MDM2, MET, MLL (KMT2A), MOS, MYB, MYH11 / CBFB, NOTCH1 (TAN1), NTRK1 (TRK), OST (SLC51B), PAX5, PIM1, PRAD-1, RAF, RAR / PML, HRAS, KRAS, NRAS, REL / NRG, RET, ROS, SKI, SRC, TIAM1, or TSC2.

[0171] In some embodiments, the oncogenes described herein include KRAS, EGFR, AR, HPRT1, CNNTB1 (β-catenin), or β-catenin-related genes. In some embodiments, polynucleotide molecule B is a polynucleotide molecule that hybridizes to the target region of KRAS, EGFR, AR, HPRT1, CNNTB1 (β-catenin), or β-catenin-related genes. In some embodiments, polynucleotide molecule B is a polynucleotide molecule that hybridizes to the target region of KRAS. In some embodiments, polynucleotide molecule B is a polynucleotide molecule that hybridizes to the target region of EGFR. In some embodiments, polynucleotide molecule B is a polynucleotide molecule that hybridizes to the target region of AR. In some embodiments, polynucleotide molecule B is a polynucleotide molecule that hybridizes to the target region of CNNTB1 (β-catenin). In some embodiments, polynucleotide molecule B is a polynucleotide molecule that hybridizes to the target region of CNNTB1 (β-catenin)-related genes. In some cases, β-catenin-related genes include PIK3CA, PIK3CB, and Myc. In some cases, polynucleotide molecule B is a polynucleotide molecule that hybridizes to the target region of HPRT1.

[0172] Polynucleic acid molecules targeting the Karsten rat sarcoma virus oncogene homolog (KRAS). The Carsten rat sarcoma virus oncogene homolog (GTPase KRas, V-Ki-ras Carsten rat sarcoma virus oncogene homolog, or KRAS) is involved in regulating cell division. The K-Ras protein is a GTPase belonging to the Ras superfamily. In some cases, K-Ras regulates cell cycle progression and induces growth arrest, apoptosis, and replication senescence under various environmental triggers (e.g., cellular stress, ultraviolet radiation, heat shock, or ionizing radiation). In some cases, wild-type KRAS genes have been shown to be frequently lost during tumor progression in various types of cancer, but mutations in the KRAS gene are associated with cancer development. In some cases, KRAS amplification is also involved in cancer development (see, e.g., Valtorta et al. "KRAS gene amplification in colorectal cancer and impact on response to EGFR-targeted therapy," Int. J. Cancer 133: 1259-1266 (2013)). In such cases, the cancer is associated with refractory cancer in which the patient has developed resistance to a particular inhibitor or class of inhibitors.

[0173] In some embodiments, the KRAS gene is wild-type or contains mutations. In some examples, the KRAS mRNA is wild-type or contains mutations. In some examples, the polynucleic acid molecule is a polynucleic acid molecule that hybridizes to a target region of wild-type KRAS DNA or RNA. In some examples, the polynucleic acid molecule is a polynucleic acid molecule that hybridizes to a target region of KRAS DNA or RNA containing mutations (e.g., substitutions, deletions, or additions).

[0174] In some embodiments, the KRAS DNA or RNA contains one or more mutations. In some embodiments, the KRAS DNA or RNA contains one or more mutations at codon 12 or 13 of exon 1. In some examples, the KRAS DNA or RNA contains one or more mutations at codon 61, 63, 117, 119, or 146. In some examples, the KRAS DNA or RNA contains one or more mutations at positions corresponding to amino acid residues 12, 13, 18, 19, 20, 22, 24, 26, 36, 59, 61, 63, 64, 68, 110, 116, 117, 119, 146, 147, 158, 164, 176, or combinations thereof, of the KRAS polypeptide. In some embodiments, the KRAS DNA or RNA contains one or more mutations at positions corresponding to amino acid residues selected from the KRAS polypeptides G12V, G12D, G12C, G12A, G12S, G12F, G13C, G13D, G13V, A18D, L19F, T20R, Q22K, I24N, N26K, I36L, I36M, A59G, A59E, Q61K, Q61H, Q61L, Q61R, E63K, Y64D, Y64N, R68S, P110S, K117N, C118S, A146T, A146P, A146V, K147N, T158A, R164Q, K176Q, or combinations thereof.

[0175] In some embodiments, the polynucleic acid molecule hybridizes to a target region of KRAS DNA or RNA containing one or more mutations. In some embodiments, the polynucleic acid molecule hybridizes to a target region of KRAS DNA or RNA containing one or more mutations at codon 12 or 13 in exon 1. In some embodiments, the polynucleic acid molecule hybridizes to a target region of KRAS DNA or RNA containing one or more mutations at codon 61, 63, 117, 119, or 146. In some embodiments, the polynucleic acid molecule hybridizes to a target region of KRAS DNA or RNA containing one or more mutations at positions corresponding to amino acid residues 12, 13, 18, 19, 20, 22, 24, 26, 36, 59, 61, 63, 64, 68, 110, 116, 117, 119, 146, 147, 158, 164, 176, or combinations thereof of the KRAS polypeptide. In some embodiments, a polynucleic acid molecule hybridizes to a target region of KRAS DNA or RNA containing one or more mutations corresponding to amino acid residues selected from KRAS polypeptides G12V, G12D, G12C, G12A, G12S, G12F, G13C, G13D, G13V, A18D, L19F, T20R, Q22K, I24N, N26K, I36L, I36M, A59G, A59E, Q61K, Q61H, Q61L, Q61R, E63K, Y64D, Y64N, R68S, P110S, K117N, C118S, A146T, A146P, A146V, K147N, T158A, R164Q, K176Q, or combinations thereof.

[0176] In some embodiments, the binding portion A is conjugated to a polynucleic acid molecule (B) and optionally a polymer (C) according to formula (I). In some examples, polymer C comprises a polyalkylene oxide (e.g., polyethylene glycol).

[0177] In some embodiments, binding portion A conjugates to a polynucleic acid molecule (B) and optionally to a polymer (C). In some examples, binding portion A is an antibody or its binding fragment.

[0178] In some embodiments, binding portion A conjugates non-specifically to the polynucleic acid molecule (B). In some examples, binding portion A conjugates to the polynucleic acid molecule (B) via a lysine residue or a cysteine ​​residue in a non-site-specific manner. In some examples, binding portion A conjugates to the polynucleic acid molecule (B) via a lysine residue in a non-site-specific manner. In some cases, binding portion A conjugates to the polynucleic acid molecule (B) via a cysteine ​​residue in a non-site-specific manner. In some examples, binding portion A is an antibody or its binding fragment.

[0179] In some embodiments, binding portion A is conjugated to the polynucleotide molecule (B) by a site-specific method. In some examples, binding portion A is conjugated to the polynucleotide molecule (B) by a site-specific method via a lysine residue, a cysteine ​​residue, at the 5'-terminus, at the 3'-terminus, a non-natural amino acid, or an enzyme-modified or enzyme-catalyzed residue. In some examples, binding portion A is conjugated to the polynucleotide molecule (B) by a site-specific method via a lysine residue. In some examples, binding portion A is conjugated to the polynucleotide molecule (B) by a site-specific method via a cysteine ​​residue. In some examples, binding portion A is conjugated to the polynucleotide molecule (B) at the 5'-terminus by a site-specific method. In some examples, binding portion A is conjugated to the polynucleotide molecule (B) at the 3'-terminus by a site-specific method. In some examples, binding portion A is conjugated to the polynucleotide molecule (B) by a site-specific method via a non-natural amino acid. In some cases, binding site A is conjugated to a polynucleic acid molecule (B) via site-specific enzymatic modification or enzymatically catalyzed residues. In some cases, binding site A is an antibody or its binding fragment.

[0180] In some embodiments, one or more regions of binding region A (e.g., an antibody or its binding fragment) are conjugated to a polynucleic acid molecule (B). In some examples, one or more regions of binding region A include the N-terminus, C-terminus, or Fc-terminus within the constant region, hinge region, or Fc region of binding region A. In some examples, the polynucleic acid molecule (B) is conjugated to the N-terminus of binding region A (e.g., the N-terminus of an antibody or its binding fragment). In some examples, the polynucleic acid molecule (B) is conjugated to the C-terminus of binding region A (e.g., the N-terminus of an antibody or its binding fragment). In some examples, the polynucleic acid molecule (B) is conjugated to the constant region of binding region A (e.g., the constant region of an antibody or its binding fragment). In some examples, the polynucleic acid molecule (B) is conjugated to the hinge region of binding region A (e.g., the constant region of an antibody or its binding fragment). In some examples, the polynucleic acid molecule (B) is conjugated to the Fc-terminus of binding region A (e.g., the constant region of an antibody or its binding fragment).

[0181] In some embodiments, one or more polynucleotide molecules (B) conjugate to binding site A. In some examples, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more polynucleotide molecules conjugate to one binding site A. In some examples, about 1 polynucleotide molecule conjugates to one binding site A. In some examples, about 2 polynucleotide molecules conjugate to one binding site A. In some examples, about 3 polynucleotide molecules conjugate to one binding site A. In some examples, about 4 polynucleotide molecules conjugate to one binding site A. In some examples, about 5 polynucleotide molecules conjugate to one binding site A. In some examples, about 6 polynucleotide molecules conjugate to one binding site A. In some examples, about 7 polynucleotide molecules conjugate to one binding site A. In some cases, approximately 8 polynucleotide molecules conjugate to one binding site A. In some cases, approximately 9 polynucleotide molecules conjugate to one binding site A. In some cases, approximately 10 polynucleotide molecules conjugate to one binding site A. In some cases, approximately 11 polynucleotide molecules conjugate to one binding site A. In some cases, approximately 12 polynucleotide molecules conjugate to one binding site A. In some cases, approximately 13 polynucleotide molecules conjugate to one binding site A. In some cases, approximately 14 polynucleotide molecules conjugate to one binding site A. In some cases, approximately 15 polynucleotide molecules conjugate to one binding site A. In some cases, approximately 16 polynucleotide molecules conjugate to one binding site A. In some cases, one or more polynucleotide molecules are the same. In other cases, one or more polynucleotide molecules are different. In some cases, binding site A is an antibody or its binding fragment.

[0182] In some embodiments, the number of polynucleotide molecules (B) conjugated to binding site A (e.g., an antibody or its binding fragment) forms a certain ratio. In some examples, the ratio is called the DAR (drug-to-antibody) ratio, and the drug as referred herein is a polynucleotide molecule (B). In some examples, the DAR ratio of polynucleotide molecules (B) to binding site A is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more. In some examples, the DAR ratio of polynucleotide molecules (B) to binding site A is about 1 or more. In some examples, the DAR ratio of polynucleotide molecules (B) to binding site A is about 2 or more. In some examples, the DAR ratio of polynucleotide molecules (B) to binding site A is about 3 or more. In some examples, the DAR ratio of polynucleotide molecules (B) to binding site A is about 4 or more. In some examples, the DAR ratio of polynucleotide molecules (B) to binding site A is about 5 or more. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 6 or higher. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 7 or higher. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 8 or higher. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 9 or higher. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 10 or higher. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 11 or higher. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 12 or higher.

[0183] In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A (e.g., antibody or its binding fragment) is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 1. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 2. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 3. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 4. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 5. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 6. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 7. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 8. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 9. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 10. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 11. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 12. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 13. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 14. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 15. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 16.

[0184] In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is 1. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is 2. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is 4. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is 6. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is 8. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is 12.

[0185] In some embodiments, the antibody or its binding fragment is further modified using conventional techniques known in the art, for example, by amino acid deletion, insertion, substitution, and addition, either alone or in combination, and by recombination and / or other modifications known in the art (e.g., post-translational modifications such as glycosylation and phosphorylation, and chemical modifications). In some examples, the modifications further include modifications to modulate the interaction with the Fc receptor. In some examples, one or more modifications include, for example, those described in International Publication No. WO97 / 34631, which discloses amino acid residues involved in the interaction between the Fc domain and the FcRn receptor. Methods for introducing such modifications into the nucleic acid sequence underlying the amino acid sequence of the antibody or its binding fragment are well known to those skilled in the art.

[0186] In some examples, the antibody-bound fragment further contains its derivatives and includes a polypeptide sequence containing at least one CDR.

[0187] In some examples, the term “single-stranded” as used herein means that the first and second domains of a bispecific single-stranded construct are covalently linked in the form of a collinear amino acid sequence, which can preferably be encoded by a single nucleic acid molecule.

[0188] In some examples, bispecific single-chain antibody constructs relate to constructs containing binding domains derived from two antibodies. In such embodiments, the bispecific single-chain antibody construct is a tandem bi-scFv or diabody. In some examples, the scFv contains VH and VL domains linked by a linker peptide. In some examples, the linker is of sufficient length and sequence to allow each of the first and second domains to maintain its differential binding specificity independently of each other.

[0189] In some embodiments, as used herein, binding to or interaction by an antigen-interacting site defines the binding / interaction of at least two antigen-interacting sites to each other. In some examples, an antigen-interacting site defines a motif of a polypeptide that exhibits the ability to have a specific interaction with a specific antigen or a specific group of antigens. In some cases, binding / interaction is also understood to define a specific recognition. In such cases, specific recognition refers to the fact that an antibody or its binding fragment can specifically interact with and / or bind to at least two amino acids of each of the target molecule. For example, specific recognition relates to the specificity of the antibody molecule or to its ability to distinguish a specific region of the target molecule. In additional examples, a specific interaction of an antigen-interacting site with a specific antigen results in the initiation of a signal, such as by inducing a conformational change of the antigen or oligomerization of the antigen. In further embodiments, binding is illustrated by the specificity of the "key-lock principle." Thus, in some examples, antigen-interacting sites and specific motifs in the amino acid sequence of the antigen bind to each other as a result of their primary, secondary, or tertiary structure, as well as as a result of secondary modifications of the above structure. In such cases, the specific interaction of the antigen interaction site with its specific antigen results in the site's easy binding to the antigen. In some examples, the specific interaction further refers to a reduction in the cross-reactivity of the antibody or its binding fragment, or a reduction in off-target effects. For example, an antibody or its binding fragment that binds to a target polypeptide / protein but does not essentially bind to any other polypeptide is considered specific to the target polypeptide / protein. Examples of specific interactions of an antigen interaction site with its specific antigen include the specificity of a ligand with its receptor, e.g., the interaction of an antigenic determinant (epitope) with the antigen-binding site of an antibody.

[0190] Production of antibodies or their conjugated fragments In some embodiments, the polypeptides described herein (e.g., antibodies and their conjugated fragments) are produced, in particular, by chemical synthesis or by recombinant expression using any method known in the art to facilitate the synthesis of polypeptides (e.g., antibodies), and preferably by recombinant expression techniques.

[0191] In some cases, the antibody or its binding fragment is recombinantly expressed, and the nucleic acid encoding the antibody or its binding fragment is assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which involves the synthesis of duplicate oligonucleotides containing a portion of the sequence encoding the antibody, annealing and ligation of those oligonucleotides, and subsequent amplification of the ligated oligonucleotides by PCR.

[0192] Alternatively, nucleic acid molecules encoding antibodies can be selectively generated from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from any tissue or cell expressing immunoglobulins) by PCR amplification using synthetic primers that can hybridize to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific to a particular gene sequence.

[0193] In some cases, the antibody or its binding is optionally generated by immunizing animals such as rabbits to produce polyclonal antibodies, or more preferably by producing monoclonal antibodies, as described, for example, by Kohler and Milstein (1975, Nature 256:495-497), or by Kozbor et al. (1983, Immunology Today 4:72), or by Cole et al. (1985 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Alternatively, clones encoding at least the Fab portion of an antibody can be selectively obtained by screening a Fab expression library (e.g., Huse et al., 1989, Science 246:1275-1281) for clones of FAb fragments that bind to a specific antigen, or by screening an antibody library (see Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937).

[0194] In some embodiments, techniques developed for the production of "chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) are used by splicing genes from mouse antibody molecules with appropriate antigen specificity together with genes from human antibody molecules with appropriate biological activity. Chimeric antibodies are molecules in which the different parts are derived from various animal species, such as the variable region derived from a mouse monoclonal antibody and the constant region of human immunoglobulin, for example, animal species that have humanized antibodies.

[0195] In some embodiments, techniques described for the production of single-chain antibodies (USPat. No. 4, 694, 778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54) are adapted for the production of single-chain antibodies. Single-chain antibodies are formed by linking heavy and light chain fragments of the Fv region via amino acid bridges, resulting in single-chain polypeptides. Techniques for assembling functional Fv fragments in E. coli are also used selectively (Skerra et al., 1988, Science 242:1038-1041).

[0196] In some embodiments, an expression vector containing the antibody nucleotide sequence or the antibody nucleotide sequence itself is introduced into host cells by conventional techniques (e.g., electroporation, liposome transfection, and calcium phosphate precipitation), and the transfected cells are subsequently cultured by conventional techniques to produce the antibody. In certain embodiments, antibody expression is regulated by constitutive, inducible, or tissue-specific promoters.

[0197] In some embodiments, various host expression vector systems are used to express the antibodies or their conjugated fragments described herein. Such host expression systems represent not only a vehicle from which the antibody coding sequence is generated and subsequently purified, but also cells that express the antibody or its conjugated fragment in insights when transformed with or transfected with the appropriate nucleotide coding sequence. These include, but are not limited to, microorganisms such as bacteria (e.g., Escherichia coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibodies or their binding fragment coding sequences; yeast (e.g., Saccharomyces picia) transformed with recombinant yeast expression vectors containing antibodies or their binding fragment coding sequences; insect cell lines infected with recombinant virus expression vectors (e.g., baculovirus) containing antibodies or their binding fragment coding sequences; plant cell lines infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibodies or their binding fragment coding sequences; or mammalian cell lines (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) possessing recombinant expression constructs containing the genome of mammalian cells (e.g., metallothionein promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter).

[0198] For long-term, high-yield production of recombinant proteins, stable expression is preferred. In some cases, cell lines that stably express antibodies are selectively manipulated. Rather than using expression vectors containing viral replication origins, host cells are transformed with DNA controlled by appropriate expression regulators (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selectable markers. After introduction of exogenous DNA, the manipulated cells are grown in enriched medium for 1-2 days, and then switched to selective medium. The selectable markers in the recombinant plasmid provide resistance to selection, allowing the cells to stably integrate the plasmid into their chromosomes, grow, and form a foci, which are then cloned and expanded into a cell line. This method can be advantageously used to manipulate cell lines that express antibodies or their binding fragments.

[0199] In some cases, but not limited to, many selection systems are used, including the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes, which are utilized in TK cells, HGPRT cells, or APRT cells, respectively. Similarly, resistance to antimetabolites is attributed to the following genes: dhfr (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527), which gives resistance to methotrexate; gpt (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072), which gives resistance to mycophenolate; and neo (Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, It is used as a selection criterion for hygro (Santerre et al., 1984, Gene 30:147), which provides resistance to hygromycin, as reported in Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIB TECH 11(5):155-215).The commonly known methods in the field of available recombinant DNA technology are described in Ausubel et al. (eds., 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1).

[0200] In some cases, antibody expression levels increase with vector amplification (see Bebbington and Hentschel, *The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning*, Vol. 3 (Academic Press, New York, 1987) for consideration). If the marker in the antibody-expressing vector system is amplified, an increase in the level of the inhibitor present in the host cell culture increases the number of copies of the marker gene. Since the amplified region is related to the antibody's nucleotide sequence, antibody production also increases (Crouse et al., 1983, Mol. Cell Biol. 3:257).

[0201] In some cases, any method for purifying antibodies known in the art is used, for example, by chromatography (e.g., ion exchange, affinity, in particular affinity of protein A to specific antigens, and sizing column chromatography), centrifugation, differential solubility, or other standard techniques for protein purification.

[0202] Polymer conjugate portion In some embodiments, polymer portion C is further conjugated to a polynucleic acid molecule, a binding moiety, or a combination thereof as described herein. In some examples, polymer portion C is conjugated to a polynucleic acid molecule. In some cases, polymer portion C is conjugated to a binding moiety. In other cases, polymer portion C is conjugated to a polynucleic acid molecule-binding moiety molecule. In further cases, polymer portion C is conjugated as discussed in the section on therapeutic molecular platforms.

[0203] In some examples, polymer moiety C is a natural or synthetic polymer consisting of long chains of branched or unbranched monomers and / or a crosslinked network of two-dimensional or three-dimensional monomers. In some examples, polymer moiety C includes polysaccharides, lignin, rubber, or polyalkylene oxides (e.g., polyethylene glycol). In some examples, at least one polymer moiety C may be, but are not limited to, alpha-dihydroxyl polyethylene glycol, omega-dihydroxyl polyethylene glycol, biodegradable lactone-based polymers such as polyacrylic acid, polylactidic acid (PLA), poly(glycolic acid) (PGA), polypropylene, polystyrene, polyolefin, polyamide, polycyanoacrylate, polyimide, polyethylene terephthalate (PET, PETG), polyethylene terephthalate (PETE), polytetramethylene glycol (PTG), or polyurethane, and mixtures thereof. As used herein, mixture refers to the use of various polymers within the same compound, as is the case with respect to block copolymers. In some cases, a block copolymer is a polymer in which at least one part of the polymer is constructed from monomers of another polymer. In some examples, polymer moiety C contains polyalkylene oxide. In some examples, polymer moiety C contains PEG. In some examples, polymer moiety C contains polyethyleneimide (PEI) or hydroxyethyl starch (HES).

[0204] In some examples, C is the PEG portion. In some examples, the PEG portion conjugates to the 5' end of the polynucleotide molecule, while the binding portion conjugates to the 3' end. In some examples, the PEG portion conjugates to the 3' end of the polynucleotide molecule, while the binding portion conjugates to the 5' end. In some examples, the PEG portion conjugates to an internal region of the polynucleotide molecule. In some examples, the PEG portion, the binding portion, or a combination thereof conjugates to an internal region of the polynucleotide molecule. In some examples, the conjugation is direct. In some examples, the conjugation is via a natural ligation.

[0205] In some embodiments, the polyalkylene oxide (e.g., PEG) is a polydisperse or monodisperse compound. In some examples, the polydisperse material includes a dispersion distribution of materials of different molecular weights, characterized by an average weight (weight-average) size and degree of dispersion. In some examples, monodisperse PEG contains molecules of one size. In some embodiments, C is a polydisperse or monodisperse polyalkylene oxide (e.g., PEG), and the indicated molecular weight represents the average molecular weight of the polyalkylene oxide, e.g., PEG molecules.

[0206] In some embodiments, the molecular weight of the polyalkylene oxide (e.g., PEG) is approximately 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 27 The values ​​are 00, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000Da.

[0207] In some embodiments, C is a polyalkylene oxide (e.g., PEG), and the values ​​are approximately 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700. It has a molecular weight of 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da. In some embodiments, C is PEG, and the values ​​are approximately 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 290 It has molecular weights of 0, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da. In some examples, the molecular weight of C is approximately 200 Da. In some examples, the molecular weight of C is approximately 300 Da. In some examples, the molecular weight of C is approximately 400 Da. In some examples, the molecular weight of C is approximately 500 Da. In some examples, the molecular weight of C is approximately 600 Da. In some examples, the molecular weight of C is approximately 700 Da. In some cases, the molecular weight of C is approximately 800 Da. In some cases, the molecular weight of C is approximately 900 Da. In some cases, the molecular weight of C is approximately 1000 Da. In some cases, the molecular weight of C is approximately 1100 Da. In some cases, the molecular weight of C is approximately 1200 Da. In some cases, the molecular weight of C is approximately 1300 Da. In some cases, the molecular weight of C is approximately 1400 Da. In some cases, the molecular weight of C is approximately 1450 Da.In some cases, the molecular weight of C is approximately 1500 Da. In some cases, the molecular weight of C is approximately 1600 Da. In some cases, the molecular weight of C is approximately 1700 Da. In some cases, the molecular weight of C is approximately 1800 Da. In some cases, the molecular weight of C is approximately 1900 Da. In some cases, the molecular weight of C is approximately 2000 Da. In some cases, the molecular weight of C is approximately 2100 Da. In some cases, the molecular weight of C is approximately 2200 Da. In some cases, the molecular weight of C is approximately 2300 Da. In some cases, the molecular weight of C is approximately 2400 Da. In some cases, the molecular weight of C is approximately 2500 Da. In some cases, the molecular weight of C is approximately 2600 Da. In some cases, the molecular weight of C is approximately 2700 Da. In some cases, the molecular weight of C is approximately 2800 Da. In some cases, the molecular weight of C is approximately 2900 Da. In some cases, the molecular weight of C is approximately 3000 Da. In some cases, the molecular weight of C is approximately 3250 Da. In some cases, the molecular weight of C is approximately 3350 Da. In some cases, the molecular weight of C is approximately 3500 Da. In some cases, the molecular weight of C is approximately 3750 Da. In some cases, the molecular weight of C is approximately 4000 Da. In some cases, the molecular weight of C is approximately 4250 Da. In some cases, the molecular weight of C is approximately 4500 Da. In some cases, the molecular weight of C is approximately 4600 Da. In some cases, the molecular weight of C is approximately 4750 Da. In some cases, the molecular weight of C is approximately 5000 Da. In some cases, the molecular weight of C is approximately 5500 Da. In some cases, the molecular weight of C is approximately 6000 Da. In some cases, the molecular weight of C is approximately 6500 Da. In some cases, the molecular weight of C is approximately 7000 Da. In some cases, the molecular weight of C is approximately 7500 Da. In some cases, the molecular weight of C is approximately 8000 Da. In some cases, the molecular weight of C is approximately 10,000 Da. In some cases, the molecular weight of C is approximately 12,000 Da. In some cases, the molecular weight of C is approximately 20,000 Da. In some cases, the molecular weight of C is approximately 35,000 Da. In some cases, the molecular weight of C is approximately 40,000 Da. In some cases, the molecular weight of C is approximately 50,000 Da.In some cases, the molecular weight of C is approximately 60,000 Da. In other cases, the molecular weight of C is approximately 100,000 Da.

[0208] In some embodiments, the polyalkylene oxide (e.g., PEG) is dispersed PEG, and dispersed PEG is a polymer PEG containing more than one repeating ethylene oxide unit. In some examples, dispersed PEG (dPEG) contains 2 to 60, 2 to 50, or 2 to 48 repeating ethylene oxide units. In some examples, dPEG contains about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 35, 40, 42, 48, 50, or more repeating ethylene oxide units. In some examples, dPEG contains about 2 or more repeating ethylene oxide units. In some examples, dPEG contains about 3 or more repeating ethylene oxide units. In some examples, dPEG contains about 4 or more repeating ethylene oxide units. In some examples, dPEG contains about 5 or more repeating ethylene oxide units. In some examples, dPEG contains about 6 or more repeating ethylene oxide units. In some examples, dPEG contains approximately 7 or more repeated ethylene oxide units. In some examples, dPEG contains approximately 8 or more repeated ethylene oxide units. In some examples, dPEG contains approximately 9 or more repeated ethylene oxide units. In some examples, dPEG contains approximately 10 or more repeated ethylene oxide units. In some examples, dPEG contains approximately 11 or more repeated ethylene oxide units. In some examples, dPEG contains approximately 12 or more repeated ethylene oxide units. In some examples, dPEG contains approximately 13 or more repeated ethylene oxide units. In some examples, dPEG contains approximately 14 or more repeated ethylene oxide units. In some examples, dPEG contains approximately 15 or more repeated ethylene oxide units. In some examples, dPEG contains approximately 16 or more repeated ethylene oxide units. In some examples, dPEG contains approximately 17 or more repeated ethylene oxide units. In some examples, dPEG contains approximately 18 or more repeated ethylene oxide units. In some examples, dPEG contains approximately 19 or more repeated ethylene oxide units. In some examples, dPEG contains approximately 20 or more repeated ethylene oxide units. In some examples, dPEG contains approximately 22 or more repeated ethylene oxide units.In some cases, dPEG contains approximately 24 or more repeated ethylene oxide units. In some cases, dPEG contains approximately 26 or more repeated ethylene oxide units. In some cases, dPEG contains approximately 28 or more repeated ethylene oxide units. In some cases, dPEG contains approximately 30 or more repeated ethylene oxide units. In some cases, dPEG contains approximately 35 or more repeated ethylene oxide units. In some cases, dPEG contains approximately 40 or more repeated ethylene oxide units. In some cases, dPEG contains approximately 42 or more repeated ethylene oxide units. In some cases, dPEG contains approximately 48 or more repeated ethylene oxide units. In some cases, dPEG contains approximately 50 or more repeated ethylene oxide units. In some cases, dPEG is synthesized stepwise from pure (e.g., approximately 95%, 98%, 99%, or 99.5%) starting materials as a single molecular weight compound. In some cases, dPEG has a specific molecular weight rather than an average molecular weight. In some cases, the dPEG described herein is dPEG from Quanta Biodesign, LMD.

[0209] In some embodiments, polymer portion C comprises a cationic mucin-based polymer (cMAP). In some examples, the cMAP comprises one or more subunits of at least one repeating subunit, the subunit structure of which is represented by formula (III),

[0210] [ka] In the formula, m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 4-6, or 5, at each occurrence, and n is independently 1, 2, 3, 4, or 5 at each occurrence. In some embodiments, m and n are, for example, about 10.

[0211] In some cases, cMAP is further conjugated to the PEG portion to produce cMAP-PEG copolymers, mPEG-cMAP-PEGm triblock polymers, or cMAP-PEG-cMAP triblock polymers. In some cases, the PEG portion is in the range of approximately 500 Da to approximately 50,000 Da. In some cases, the PEG portion is in the range of approximately 500 Da to approximately 1000 Da, over 1000 Da to approximately 5000 Da, over 5000 Da to approximately 10,000 Da, over 10,000 to approximately 25,000 Da, over 25,000 Da to approximately 50,000 Da, or any combination of two or more of these ranges.

[0212] In some cases, polymer portion C is a cMAP-PEG copolymer, an mPEG-cMAP-PEGm triblock polymer, or a cMAP-PEG-cMAP triblock polymer. In some cases, polymer portion C is a cMAP-PEG copolymer. In other cases, polymer portion C is an mPEG-cMAP-PEGm triblock polymer. In further cases, polymer portion C is a cMAP-PEG-cMAP triblock polymer.

[0213] Endosomal lytic or transmembrane portion In some embodiments, the molecule of formula (Xa):A-X1-B'-X2-C further comprises an additional conjugate moiety. In some examples, the additional conjugate moiety is an endosomal soluble moiety and / or a transmembrane moiety. In some cases, the endosomal soluble moiety is a cellular compartment-releasing component, a compound that can be released from any of the cellular compartments known in the art, such as endosomes, lysosomes, endoplasmic reticulum (ER), Golgi apparatus, microtubules, peroxisomes, or other endoplasmic reticulum having cells. In some cases, the endosomal soluble moiety comprises an endosomal soluble polypeptide, an endosomal soluble polymer, an endosomal soluble lipid, or an endosomal soluble small molecule. In some cases, the endosomal soluble moiety comprises an endosomal soluble polypeptide. In other cases, the endosomal soluble moiety comprises an endosomal soluble polymer. In some cases, the transmembrane moiety comprises a cell-permeable peptide (CPP). In other cases, the transmembrane moiety comprises a cell-permeable lipid. In other cases, the transmembrane moiety comprises a cell-permeable small molecule.

[0214] Endosomal-soluble polypeptides and transmembrane polypeptides In some embodiments, the molecule of formula (Xa):A-X1-B-X2-C is further conjugated with an endosomal soluble polypeptide. In some cases, the endosomal soluble polypeptide is a pH-dependent membrane-active peptide. In some cases, the endosomal soluble polypeptide is an amphiphilic polypeptide. In further cases, the endosomal soluble polypeptide is a peptide mimetic. In some examples, the endosomal soluble polypeptide comprises INF, melittin, mucin, or derivatives thereof. In some examples, the endosomal soluble polypeptide comprises INF or its derivatives. In other cases, the endosomal soluble polypeptide comprises melittin or its derivatives. In further cases, the endosomal soluble polypeptide comprises mucin or its derivatives. In some examples, endosomal lytic polypeptides include Pep-1 (derived from NLS from Simian Virus 40 large antigen and HIV reverse transcriptase), Pvec (derived from VE-cadherin), VT5 (derived from synthetic peptide), C105Y (derived from 1-antitrypsin), transportan (derived from galanin and mastoparan), TP10 (derived from galanin and mastoparan), MPG (derived from the hydrophobic domain of the fusion sequence of HIV gp41 and SV40 T antigen NLS), and GH625 (HSV It contains (derived from type I glycoprotein gH), CADY (PPTG1 peptide), GALA (synthetic peptide), INF (influenza HA2 fusion peptide), HA2E5-TAT (influenza HA2 subunit of influenza virus X31 strain fusion peptide), HA2-Penetratin (influenza HA2 subunit of influenza virus X31 strain fusion peptide), HA-K4 (influenza virus X31 strain fusion peptide HA2 subunit), HA2E4 (influenza virus X31 strain fusion peptide HA2 subunit), H5WYG (HA2 analog), GALA-INF3-(PEG)6-NH (INF3 fusion peptide), or CM18-TAT11 (cecropine A-meritotin 2-12 (CM18) fusion peptide).

[0215] In some cases, the endosomal lytic moiety contains a Bak BH3 polypeptide that induces apoptosis through antagonism of repressor targets such as Bcl-2 and / or Bcl-xL. In some examples, the endosomal lytic moiety contains a Bak BH3 polypeptide described in Albarran, et al., “Efficient intracellular delivery of a pro-apoptotic peptide with a pH-responsive carrier,” Reactive & Functional Polymers 71: 261-265 (2011).

[0216] In some cases, the endosomal lytic portion includes polypeptides (e.g., cell-permeable polypeptides) as described in PCT International Publication No. WO2013 / 166155 or No. WO2015 / 069587.

[0217] Endosomal soluble lipids In some embodiments, the endosomal soluble portion is a lipid (e.g., a fusion lipid). In some embodiments, the molecule of formula (Xa): A-X1-B'-X2-C is further conjugated to an endosomal soluble lipid (e.g., a fusion lipid). Exemplary fusion lipids include 1,2-dileoyl-sn-3-phosphoethanolamine (DOPE), phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (Di-Lin), N-methyl(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)methaneamine (DLin-k-DMA), and N-methyl-2-(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)ethaneamine (XTC). In some cases, the endosomal soluble portion is a lipid (e.g., a fusion lipid) as described in PCT Publication No. WO09 / 126,933.

[0218] Endosomal soluble small molecules In some embodiments, the endosomal soluble portion is a small molecule. In some embodiments, the molecule of formula (Xa): A-X1-B'-X2-C is further conjugated to an endosomal soluble small molecule. Suitable exemplary small molecules as the endosomal soluble portion include, but are not limited to, quinine, chloroquine, hydroxychloroquine, amodiaquine (carnoquines), amopyroquine, primaquine, mefloquine, nivaquines, halophanthrin, quinone imines, or combinations thereof. In some examples, the quinoline endosome-soluble portion may include, but is not limited to, 7-chloro-4-(4-diethylamino-1-methylbutylamino)quinoline (chloroquine), 7-chloro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline (hydroxychloroquine), 7-fluoro-4-(4-diethylamino-1-methylbutylamino)quinoline, 4-(4-diethylamino-1-methylbutylamino)quinoline, 7-hydroxy-4-(4-diethylamino-1-methylbutylamino)quinoline, 7-chloro-4-(4-diethylamino-1-butylamino)quinoline (desmethylchloroquine), 7-fluoro-4-(4-diethylamino-1-butylamino)quinoline, 4-(4-diethylamino-1-butylamino)quinoline, and 7-hydroxy -4-(4-diethylamino-1-butylamino)quinoline, 7-chloro-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline, 7-fluoro-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline, 4-(1-carboxy-4-diethylamino-1-butylamino)quinoline, 7-hydroxy-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline, 7-chloro-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline, 7-fluoro-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline, 4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline, 7-hydroxy-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline,7-Fluoro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline, 4-(4-ethyl-(2-hydroxy-ethyl)-amino-1-methylbutylamino-)quinoline, 7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline, hydroxychloroquine phosphate, 7-chloro-4-(4-ethyl-(2-hydroxyethyl-1)-amino-1-butylamino)quinoline (desmethylhydroxychloroquine), 7-Fluoro-4-(4-ethyl-( 2-hydroxyethyl)-amino-1-butylamino)quinoline, 4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline, 7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline, 7-chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline, 7-fluoro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline, 4-(1-carboxy-4-ethyl-(2 (-hydroxyethyl)-amino-1-butylamino)quinoline, 7-hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline, 7-chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline, 7-fluoro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline, 4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino) Quinoline, 7-hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline, 8-[(4-aminopentyl)amino-6-methoxydihydrochloridequinoline, 1-acetyl-1,2,3,4-tetrahydroquinoline, 8-[(4-aminopentyl)amino]-6-methoxyquinoline dihydrochloride, 1-butyryl-1,2,3,4-tetrahydroquinoline, 3-chloro-4-(4-hydroxy-α,α'-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline,4-[(4-diethyl-amino)-1-methylbutyl-amino]-6-methoxyquinoline, 3-fluoro-4-(4-hydroxy-α,α'-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline, 4-[(4-diethylamino)-1-methylbutyl-amino]-6-methoxyquinoline, 4-(4-hydroxy-α,α'-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline, 4-[(4-diethyl Examples include (1-methylbutyl-amino)-6-methoxyquinoline, 3,4-dihydro-1-(2H)-quinoline carboxyaldehyde, 1,1'-pentamethylenequinolinium diiodide, 8-quinolinol sulfate, and their amino, aldehyde, carboxylic acid, hydroxyl, halogen, keto, sulfhydryl, and vinyl derivatives or analogs. In some examples, the endosomal soluble portion is a small molecule described in Naisbitt et al (1997, J Pharmacol Exp Therapy 280:884-893) and U.S. Patent No. 5,736,557. Cell-permeable polypeptide (CPP) In some embodiments, the cell-permeable polypeptide comprises a short, positively charged peptide having 5 to 30 amino acids. In some embodiments, the cell-permeable polypeptide comprises an arginine or lysine-rich amino acid sequence. In some embodiments, the cell-permeable polypeptide comprises any polypeptide or combination thereof, including Antennapedia Penetratin (43-58), HIV-1 TAT protein (48-60), pVEC cadherin (615-632), transportan galanine / mastoparan, MPG HIV-gp41 / SV40 T-antigen, Pep-1 HIV-reverse transcriptase / SV40 T-antigen, polyarginine, MAP, R6W3, NLS, 8-lysine, ARF (1-22), and azurin-p28.

[0219] Linker In some embodiments, the linkers described herein are cleavable or incleavable linkers. In some examples, the linker is a cleavable linker. In some examples, the linker is an acid-cleavable linker. In some examples, the linker is an incleavable linker. In some examples, the linker comprises a C1-C6 alkyl group (e.g., C5, C4, C3, C2, or C1 alkyl group). In some examples, the linker comprises a homobifunctional crosslinked linker, a heterobifunctional crosslinked linker, and the like. In some examples, the linker is a traceless linker (or a linker with zero length). In some examples, the linker is a nonpolymeric linker. In some cases, the linker is a non-peptide linker or a linker that does not contain amino acid residues.

[0220] In some examples, the linker includes homobifunctional linkers. Exemplary homobifunctional linkers include, but are not limited to, Lomant's reagents: dithiobis(succinimidylpropionate) DSP, 3'3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP), disuccinimidylsverate (DSS), bis(sulfosuccinimidyl)sverate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfoDST), ethylene glycobis(succinimidylsuccinate) (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidylcarbonate (DSC), dimethyladipimidate (DMA), dimethylpimerimidate (DMP), dimethylsverimidate (DMS), dimethyl-3,3'-dithiobispropionimidate (DTBP), 1,4-di-3'-(2'-pyridyldithio (O) Propionamide) butane (DPDPB), bismaleimide hexane (BMH), aryl halide-containing compounds (DFDNB), for example, 1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'-dinitrophenyl sulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl]disulfide (BASE D) Contains formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-p-diaminodiphenyl, diiodo-p-xylenesulfonic acid, N,N'-ethylene-bis(iodoacetamide), or N,N'-hexamethylene-bis(iodoacetamide).

[0221] In some embodiments, the linker includes a heterobifunctional linker. Exemplary heterobifunctional linkers include, but are not limited to, amine-reactive and sulfhydryl-bridged linkers, such as N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (LC-sPDP), and water-soluble long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamide]hexanoate (sulfo-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimid ester (MBs), m- Maleimidobenzoyl-N-hydroxysulfosuccinimide (sulfo-MB), N-succinimidyl (4-iodoacetyl)aminobenzoate (sIAB), sulfosuccinimidyl (4-iodoacetyl)aminobenzoate (sulfo-sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(γ-maleimidobutyryloxy)succinimide (GMB), N-(γ-maleimidobutyryloxy)sulfosuccinimide (sulfo-GMB), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (sIAXX), succinimidyl 4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl6-((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyliodoacetate (NPIA), carbonyl-reactive and sulfhydryl-reactive crosslinking linkers, e.g., 4-(4-N-maleimidophenyl)butyrate hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8 (M2C2H), 3-(2-pyridyldithio (o) Propionyl hydrazide (PDPH), amine-reactive and photoreactive crosslinking linkers, e.g., N-hydroxysuccinimidyl-4-azidosalicylic acid (NH-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylic acid (sulfo-NH-AsA), sulfosuccinimidyl-(4-azidosalicylamide)hexanoate (sulfo-NH-LC-AsA), sulfosuccinimidyl-2-(ρ-azidosalicylamide)ethyl-1 ,3'-dithiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB), N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-sANPAH), N -5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOs), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamide)-ethyl-1,3'-dithiopropionate (sAND), N-succinimidyl-4(4-azidophenyl)1,3'-dithiopropionate (sADP), N-succinimidyl(4-azidophenyl)-1,3'-dithiopropionate (sulfo-sADP), sulfosuccinimidyl 4-(ρ-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3'-dithiopropionate (sAED), sulfosuccinimidyl7-Azido-4-methylcoumarin-3-acetate (sulfo-sAMCA), ρ-nitrophenyldiazopirubate (ρNPDP), ρ-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), sulfhydryl-reactive and photoreactive crosslinking linkers, e.g., 1-(ρ-azidosalicylamide)-4-(iodoacetamide)butane (AsIB), N-[4-(ρ-azidosalicylamide)butyl]-3'-(2'-pyridyldithio) Examples include ropionamide (APDP), benzophenone-4-iodoacetamide, benzophenone-4-maleimide carbonyl-reactive and photoreactive crosslinkers, such as ρ-azidobenzoylhydrazide (ABH), carboxylate-reactive and photoreactive crosslinkers, such as 4-(ρ-azidosalicylamide)butylamine (AsBA), and arginine-reactive and photoreactive crosslinkers, such as ρ-azidophenylglyoxal (APG).

[0222] In some examples, the linker contains a reactive functional group. In some cases, the reactive functional group contains a nucleophile that is reactive to an electrophile present in the bonding portion. Exemplary electrophiles include carbonyl groups such as aldehydes, ketones, carboxylic acids, esters, amides, enones, acyl halides, or acid anhydrides. In some embodiments, the reactive functional group is an aldehyde. Exemplary nucleophiles include hydrazides, oximes, aminos, hydrazines, thiosemicarbazones, hydrazine carboxylates, and aryl hydrazides.

[0223] In some embodiments, the linker contains a maleimide group. In some examples, the maleimide group is also called a maleimide spacer. In some examples, the maleimide group further contains caproic acid to form maleimidocaproyl (mc). In some cases, the linker contains maleimidocaproyl (mc). In some cases, the linker is maleimidocaproyl (mc). In other examples, the maleimide group contains a maleimidomethyl group such as succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC) or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC) as described above.

[0224] In some embodiments, the maleimide group is a self-stabilizing maleimide. In some examples, the self-stabilizing maleimide utilizes diaminopropionic acid (DPR) to incorporate a basic amino group adjacent to the maleimide to provide intramolecular catalytic action of thiosuccinimide ring hydrolysis, thereby preventing the maleimide from undergoing elimination via the retromichael reaction. In some examples, the self-stabilizing maleimide is the maleimide group described in Lyon, et al., “Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates,” Nat. Biotechnol. 32(10):1059-1062 (2014). In some examples, the linker contains a self-stabilizing maleimide. In some examples, the linker is a self-stabilizing maleimide.

[0225] In some embodiments, the linker includes a peptide moiety. In some examples, the peptide includes at least 1, 2, 3, 4, 5, 6, 7, 8, or more amino acid residues. In some examples, the peptide moiety is cleavable (e.g., enzymatically or chemically). In some examples, the peptide moiety is incleavable. In some examples, the peptide moiety includes Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly (SEQ ID NO: 14223), Phe-Lys, Val-Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Ala-Leu-Ala-Leu (SEQ ID NO: 14224), or Gly-Phe-Leu-Gly (SEQ ID NO: 14225). In some cases, the linker contains peptide moieties such as Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly (SEQ ID NO: 14223), Phe-Lys, Val-Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Ala-Leu-Ala-Leu (SEQ ID NO: 14224), or Gly-Phe-Leu-Gly (SEQ ID NO: 14225). In some cases, the linker contains Val-Cit. In some cases, the linker is Val-Cit.

[0226] In some embodiments, the linker comprises a benzoic acid group or a derivative thereof. In some examples, the benzoic acid group or a derivative thereof comprises para-aminobenzoic acid (PABA). In some examples, the benzoic acid group or a derivative thereof comprises gamma-aminobutyric acid (GABA).

[0227] In some embodiments, the linker comprises one or more maleimide groups, peptide moieties, and / or benzoic acid groups in any combination. In some embodiments, the linker comprises a combination of maleimide groups, peptide moieties, and / or benzoic acid groups. In some examples, the maleimide group is maleimidocaproyl (mc). In some examples, the peptide group is val-cit. In some examples, the benzoic acid group is PABA. In some examples, the linker comprises an mc-val-cit group. In some cases, the linker comprises a val-cit-PABA group. In further cases, the linker comprises an mc-val-cit-PABA group.

[0228] In some embodiments, the linker is a self-destructing linker or a self-destructing linker. In some cases, the linker is a self-destructing linker. In other cases, the linker is a self-destructing linker (e.g., a cyclized self-destructing linker). In some examples, the linker includes linkers described in U.S. Patent No. 9,089,614 or PCT Publication No. WO2015038426.

[0229] In some embodiments, the linker is a dendritic linker. In some examples, the dendritic linker includes a branched, multifunctional linker moiety. In some examples, the dendritic linker is used to increase the molar ratio of polynucleotide B pairs to binding moiety A. In some examples, the dendritic linker includes a PAMAM dendrimer.

[0230] In some embodiments, the linker is a traceless linker, or a linker that does not leave a linker portion (e.g., an atom or linker group) on the binding portion A, polynucleotide B, polymer C, or endosomal soluble portion D after cleavage. Examples of traceless linkers include, but are not limited to, germanium linkers, selenium linkers, sulfur linkers, selenium linkers, nitrogen linkers, phosphorus linkers, boron linkers, chromium linkers, or phenylhydrazide linkers. In some cases, the linker is a traceless aryl-triazene linker as described in Hejesen, et al., “A traceless aryl-triazene linker for DNA-directed chemistry,” Org Biomol Chem 11(15):2493-2497(2013). In some cases, the linker is a traceless linker as described in Blaney, et al., “Traceless solid-phase organic synthesis,” Chem. Rev. 102:2607-2024 (2002). In some cases, the linker is a traceless linker as described in U.S. Patent No. 6,821,783.

[0231] In some cases, the linker contains functional groups that exhibit steric hinderance at the binding site between the linker and the conjugate moiety (e.g., A, B, C, or D as described herein). In some cases, the steric hinderance is around a disulfide bond. Exemplary linkers exhibiting steric hinderance include heterobifunctional linkers such as the heterobifunctional linkers described above. In some cases, linkers exhibiting steric hinderance include SMCC and SPDB.

[0232] In some cases, the linker is an acid-cleavable linker. In some cases, the acid-cleavable linker contains a hydrazone bond that is susceptible to hydrolysis. In some cases, the acid-cleavable linker contains a thiomaleiamic acid linker. In some cases, the acid-cleavable linker is a thiomaleiamic acid linker as described in “Acid-cleavable thiomaleamic acid linker for homogeneous antibody-drug conjugation,” Chem.Commun. 49:8187-8189 (2013).”

[0233] In some cases, the linker is related to U.S. Patent Nos. 6,884,869, 7,498,298, 8,288,352, 8,609,105, or 8,697,688, U.S. Patent Publications 2014 / 0127239, 2013 / 028919, 2014 / 286970, and 2013 / 030 Linkers listed in No. 9256, No. 2015 / 037360, or No. 2014 / 0294851, or PCT Publication Nos. WO2015057699, WO2014080251, WO2014197854, WO2014145090, or WO2014177042.

[0234] In some embodiments, X, Y, and L are independently single bonds or linkers. In some examples, X, Y, and L are independently single bonds. In some cases, X, Y, and L are independently linkers.

[0235] In some examples, X is a single bond or a linker. In some examples, X is a single bond. In some examples, X is a linker. In some examples, the linker is a C1-C6 alkyl group. In some cases, X is a C1-C6 alkyl group, such as a C5, C4, C3, C2, or C1 alkyl group. In some cases, the C1-C6 alkyl group is an unsubstituted C1-C6 alkyl group. When used in the context of a linker, and especially in the context of X, alkyl means a saturated linear or branched hydrocarbon radical containing up to six carbon atoms. In some examples, X is a nonpolymer linker. In some examples, X includes a homobifunctional or heterobifunctional linker as described above. In some cases, X includes a heterobifunctional linker. In some cases, X includes an sMCC. In other examples, X includes a heterobifunctional linker optionally conjugated to a C1-C6 alkyl group. In other examples, X includes an sMCC optionally conjugated to a C1-C6 alkyl group. In some additional examples, X does not contain the homobifunctional or heterobifunctional linkers described above.

[0236] In some examples, Y is a single bond or a linker. In some examples, Y is a single bond. In other cases, Y is a linker. In some embodiments, Y is a C1-C6 alkyl group. In some examples, Y is a homobifunctional or heterobifunctional linker as described above. In some examples, Y is a homobifunctional linker as described above. In some examples, Y is a heterobifunctional linker as described above. In some examples, Y comprises a maleimide group such as maleimidocaproyl (mc) as described above, or a self-stabilizing maleimide group. In some examples, Y comprises a peptide moiety such as Val-Cit. In some examples, Y comprises a benzoic acid group such as PABA. In further examples, Y comprises a combination of a maleimide group, a peptide moiety, and / or a benzoic acid group. In further examples, Y comprises an mc group. In further examples, Y comprises an mc-val-cit group. In further examples, Y comprises a val-cit-PABA group. In a further example, Y contains an mc-val-cit-PABA group.

[0237] In some examples, L is a single bond or a linker. In some cases, L is a single bond. In some cases, L is a linker. In some embodiments, L is a C1-C6 alkyl group. In some examples, L is a homobifunctional or heterobifunctional linker as described above. In some examples, L is a homobifunctional linker as described above. In some examples, L is a heterobifunctional linker as described above. In some examples, L comprises a maleimide group such as maleimidocaproyl (mc) as described above, or a self-stabilizing maleimide group. In some examples, L comprises a peptide moiety such as Val-Cit. In some examples, L comprises a benzoic acid group such as PABA. In further examples, L comprises a combination of a maleimide group, a peptide moiety, and / or a benzoic acid group. In further examples, L comprises an mc group. In further examples, L comprises an mc-val-cit group. In further examples, L comprises a val-cit-PABA group. In a further example, L contains an mc-val-cit-PABA group.

[0238] How to use In some embodiments, compositions or pharmaceutical formulations described herein, comprising conjugated binding portions and polymers to polynucleic acid molecules, are used to treat diseases or disorders. In some examples, the diseases or disorders are muscular dystrophy, muscular atrophy, and / or muscle wasting. Muscular dystrophy refers to a decrease in muscle mass and / or progressive weakness and degeneration of muscles. In some cases, the decrease in muscle mass and / or progressive weakness and degeneration of muscles results from a high rate of proteolysis, a low rate of protein synthesis, or a combination of both. In some cases, a high rate of muscle proteolysis is due to muscle protein catabolism (i.e., the breakdown of muscle proteins to use amino acids as substrates for gluconeogenesis). In some examples, the diseases or disorders are cancer. In some embodiments, compositions or pharmaceutical formulations described herein are used as immunotherapy for the treatment of diseases or disorders. In some examples, the immunotherapy is cancer immunotherapy.

[0239] cancer In some embodiments, the compositions or pharmaceutical formulations described herein are used to treat cancer. In some examples, the cancer is a solid tumor. In some examples, the cancer is a hematological malignancy. In some examples, the cancer is a recurrent or refractory cancer or a metastatic cancer. In some examples, the solid tumor is a recurrent or refractory solid tumor or a metastatic solid tumor. In some cases, the hematological malignancy is a recurrent or refractory hematological malignancy or a metastatic hematological malignancy.

[0240] In some cases, compositions or pharmaceutical formulations described herein, comprising oligonucleotides, polymers, or combinations thereof optionally conjugated to a binding site, are used for the treatment of solid tumors. In some cases, compositions or pharmaceutical formulations described herein, comprising oligonucleotides, polymers, or combinations thereof optionally conjugated to a binding site, are used for the treatment of anal cancer, appendiceal cancer, bile duct cancer (i.e., intrahepatic bile duct cancer), bladder cancer, brain tumors, breast cancer, cervical cancer, colon cancer, cancer of unknown primary origin (CUP), esophageal cancer, eye cancer, ductal cancer, gastrointestinal cancer, kidney cancer, liver cancer, lung cancer, medulloblastoma, melanoma, oral cancer, ovarian cancer, pancreatic cancer, parathyroid disease, penile cancer, pituitary tumors, prostate cancer, rectal cancer, skin cancer, gastric cancer, testicular cancer, laryngeal cancer, thyroid cancer, uterine cancer, vaginal cancer, or vulvar cancer. In some cases, the solid tumors are recurrent or refractory solid tumors, or metastatic solid tumors.

[0241] In some cases, cancer is a hematological malignancy. In some cases, compositions or pharmaceutical formulations described herein, comprising oligonucleotides, polymers, or combinations thereof optionally conjugated to a binding site, are used to treat hematological malignancies. In some cases, compositions or pharmaceutical formulations described herein, comprising oligonucleotides, polymers, or combinations thereof optionally conjugated to a binding site, are used to treat leukemia, lymphoma, myeloma, non-Hodgkin lymphoma, or Hodgkin lymphoma. In some cases, hematological malignancies include chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high-risk CLL, non-CLL / SLL lymphoma, prolymphocytic leukemia (PLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenström macroglobulinemia, multiple myeloma, extranomial marginal zone B-cell lymphoma, and nodular marginal zone B-cell lymphoma. This includes pamas, Burkitt lymphoma, non-Burkitt high-grade B-cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, progenitor B-lymphoblastic lymphoma, B-cell pre-lymphoblastic leukemia, lymphoplasmacytic lymphoma, perisplenic zone lymphoma, plasmacytotic myeloma, plasmacytoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary coelocellular lymphoma, or lymphomatoid granulomatosis. In some cases, hematological malignancies are relapsed or refractory hematological malignancies, or metastatic hematological malignancies.

[0242] In some cases, the cancer is KRAS-related, EGFR-related, AR-related, HPRT1-related, or β-catenin-related. In some cases, compositions or pharmaceutical formulations described herein, comprising oligonucleotides, polymers, or combinations thereof optionally conjugated to a binding site, are used to treat KRAS-related, EGFR-related, AR-related, HPRT1-related, or β-catenin-related cancers. In some cases, compositions or pharmaceutical formulations described herein, comprising oligonucleotides, polymers, or combinations thereof optionally conjugated to a binding site, are used to treat KRAS-related cancers. In some cases, compositions or pharmaceutical formulations described herein, comprising oligonucleotides, polymers, or combinations thereof optionally conjugated to a binding site, are used to treat EGFR-related cancers. In some cases, compositions or pharmaceutical formulations described herein, comprising oligonucleotides, polymers, or combinations thereof optionally conjugated to a binding site, are used to treat AR-related cancers. In some cases, compositions or pharmaceutical formulations described herein, comprising oligonucleotides, polymers, or combinations thereof optionally conjugated to a binding site, are used to treat HPRT1-associated cancers. In some cases, compositions or pharmaceutical formulations described herein, comprising oligonucleotides, polymers, or combinations thereof optionally conjugated to a binding site, are used to treat β-catenin-associated cancers. In some cases, the cancer is a solid tumor. In some cases, the cancer is a hematological malignancy. In some cases, the solid tumor is a recurrent or refractory solid tumor, or a metastatic solid tumor. In some cases, the hematological malignancy is a recurrent or refractory hematological malignancy, or a metastatic hematological malignancy. In some cases, the cancer includes bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, glioblastoma multiforme, head and neck cancer, kidney cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, thyroid cancer, acute myeloid leukemia, CLL, DLBCL, or multiple myeloma. In some cases, β-catenin-related cancers further include PIK3C-related cancers and / or MYC-related cancers.

[0243] immunotherapy In some embodiments, the compositions or pharmaceutical formulations described herein are used as immunotherapies for the treatment of a disease or disorder. In some examples, the immunotherapy is cancer immunotherapy. In some examples, cancer immunotherapy is classified into active, passive, or combined (active and passive) methods. In active cancer immunotherapy, for example, tumor-associated antigens (TAAs) are presented to the immune system, triggering an attack on cancer cells that present these TAAs. In some examples, active cancer immunotherapy includes tumor-targeting and / or immunotargeting agents (e.g., checkpoint inhibitors such as monoclonal antibodies), and / or vaccines, for example, in situ vaccines and / or cell-based or non-cell-based (e.g., dendritic cell-based, tumor cell-based, antigen, anti-idiotype, DNA, or vector-based) vaccines. In some examples, cell-based vaccines are vaccines produced using activated immune cells, which are obtained from the patient's own immune system and subsequently activated by the patient's own cancer. In some examples, active cancer immunotherapy is further subdivided into non-specific active immunotherapy and specific active immunotherapy. In some cases, non-specific active immunotherapy utilizes cytokines and / or other cellular signaling components to induce a general immune system response. In other cases, specific active immunotherapy utilizes specific TAAs to induce an immune response.

[0244] In some embodiments, the compositions or pharmaceutical formulations described herein are used as active cancer immunotherapy for the treatment of a disease or disorder (e.g., cancer). In some embodiments, the compositions or pharmaceutical formulations described herein include a tumor targeting agent. In some examples, the tumor targeting agent is encompassed by binding moiety A. In other examples, the tumor targeting agent is an additional agent used in combination with the molecule of formula (I). In some examples, the tumor targeting agent is a tumor-directed polypeptide (e.g., a tumor-directed antibody). In some examples, the tumor targeting agent is a tumor-directed antibody that exerts its antitumor activity through mechanisms such as direct killing (e.g., signal-signal-induced apoptosis), complement-dependent cytotoxicity (CDC), and / or antibody-dependent cell-mediated cytotoxicity (ADCC). In further examples, the tumor targeting agent induces an adaptive immune response, which involves the induction of antitumor T cells.

[0245] In some embodiments, binding moiety A is a tumor-targeting polypeptide (e.g., a tumor-targeting antibody). In some examples, binding moiety A is a tumor-targeting antibody that exerts its antitumor activity through mechanisms such as direct killing (e.g., signal-signal-induced apoptosis), complement-dependent cytotoxicity (CDC), and / or antibody-dependent cytotoxicity (ADCC). In further examples, binding moiety A induces an adaptive immune response, including the induction of antitumor T cells.

[0246] In some embodiments, the compositions or pharmaceutical formulations described herein include an immunotargeting agent. In some examples, the immunotargeting agent is encompassed by binding moiety A. In other examples, the immunotargeting agent is an additional agent used in combination with the molecule of formula (I). In some examples, the immunotargeting agent includes cytokines, checkpoint inhibitors, or combinations thereof.

[0247] In some embodiments, the immunotargeting agent is a checkpoint inhibitor. In some cases, the immune checkpoint molecule is a molecule presented on the cell surface of CD4 T cells and / or CD8 T cells. Exemplary immune checkpoint molecules include, but are not limited to, programmed death ligand 1 (PD-L1 (also known as B7-H1, CD274)), programmed death 1 (PD-1), CTLA-4, B7H1, B7H4, OX-40, CD137, CD40, 2B4, IDO1, IDO2, VISTA, CD27, CD28, PD-L2 (B7-DC, CD273), LAG3, CD80, CD86, PDL2, B7H3, HVEM, BTLA, KIR, GAL9, TIM3, A2aR, MARCO (a macrophage receptor with a collagen structure), PS (phosphatidylserine), ICOS (an inducer T cell costimulatory molecule), HAVCR2, CD276, VTCN1, CD70, and CD160.

[0248] In some examples, an immune checkpoint inhibitor refers to any molecule that modulates or inhibits the activity of an immune checkpoint molecule. In some examples, immune checkpoint inhibitors include antibodies, antibody derivatives (e.g., FAb fragments, scFv, minobodies, diabodies), antisense oligonucleotides, siRNA, aptamers, or peptides. In some embodiments, Immune checkpoint inhibitors are inhibitors of programmed death ligand 1 (PD-L (also known as B7-H1, CD274)), programmed death 1 (PD-1), CTLA-4, PD-L2 (B7-DC, CD273), LAG3, TIM3, 2B4, A2aR, B7H1, B7H3, B7H4, BTLA, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137, CD160, CD226, CD276, DR3, GAL9, GITR, HAVCR2, HVEM, IDO1, IDO2, ICOS (induced T cell costimulatory molecule), KIR, LAIR1, LIGHT, MARCO (macrophage receptor with collagen structure), PS (phosphatidylserine), OX-40, SLAM, TIGHT, VISTA, VTCN1, or any combination thereof.

[0249] In some embodiments, exemplary checkpoint inhibitors include:

[0250] PD-L1 inhibitors, for example, Genentech's MPDL3280A (RG7446), BioXcell's anti-mouse PD-L1 antibody clone 10F.9G2 (Cat# BE0101), Bristol-Myers Squibb's anti-PD-L1 monoclonal antibody MDX-1105 (BMS-936559) and BMS-935559, MSB0010718C, mouse anti-PD-L1 clone 29E.2A3, and AstraZeneca's MEDI4736;

[0251] PD-L2 inhibitors, such as GlaxoSmithKline's AMP-224 (Amplimmune) and rHIgM12B7;

[0252] PD-1 inhibitors, such as BioXcell's anti-mouse PD-1 antibody clone J43 (Cat# BE0033-2), BioXcell's anti-mouse PD-1 antibody clone RMP1-14 (Cat# BE0146), mouse anti-PD-1 antibody clone EH12, Merck's MK-3475 anti-mouse PD-1 antibody (Keytruda, pembrolizumab, lambrolizumab), AnaptysBio's anti-PD-1 antibody known as ANB011, antibody MDX-1 106 (ONO-4538), Bristol-Myers Squibb's human IgG4 monoclonal antibody nivolumab (Opdivo®, BMS-936558, MDX1106), AstraZeneca's AMP-514 and AMP-224, and CureTech Ltd's pidilizumab (CT-011);

[0253] CTLA-4 inhibitors, such as Bristol Meyers Squibb's anti-CTLA-4 antibody ipilimumab (also known as Yervoy®, MDX-010, BMS-734016, and MDX-101), anti-CTLA4 antibodies, Millipore clone 9H10, Pfizer's tremelimumab (CP-675,206, tisilimmab), and Abcam's anti-CTLA4 antibody clone BNI3;

[0254] LAG3 inhibitors, for example, eBioscience's anti-Lag-3 antibody clone eBioC9B7W (C9B7W), LifeSpan Biosciences' anti-Lag-3 antibody LS-B2237, Immutep's IMP321 (ImmuFact), anti-Lag-3 antibody BMS-986016, and LAG-3 chimeric antibody A9H12;

[0255] B7-H3 inhibitors, e.g., MGA271;

[0256] KIR inhibitors, e.g., Lirilumab (IPH2101);

[0257] CD137 (41BB) inhibitors, such as urelumab (BMS-663513 Bristol-Myers Squibb), PF-05082566 (anti-4-1BB, PF-2566, Pfizer), or XmAb-5592 (Xencor);

[0258] PS inhibitors, e.g., Bavituximab; and inhibitors, e.g., antibodies or their fragments (e.g., monoclonal antibodies, human antibodies, humanized antibodies, or chimeric antibodies), RNAi molecules, or small molecules against TIM3, CD52, CD30, CD20, CD33, CD27, OX40 (CD134), GITR, ICOS, BTLA (CD272), CD160, 2B4, LAIR1, TIGHT, LIGHT, DR3, CD226, CD2, or SLAM.

[0259] In some embodiments, binding portion A, which contains an immune checkpoint inhibitor, is used to treat a disease or disorder (e.g., cancer). In some examples, binding portion A is a bispecific antibody containing an immune checkpoint inhibitor or its binding fragment. In some examples, programmed death-ligand 1 (PD-L1 (also known as B7-H1, CD274)), programmed death 1 (PD-1), CTLA-4, PD-L2 (B7-DC, CD273), LAG3, TIM3, 2B4, A2aR, B7H1, B7H3, B7H4, BTLA, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137, CD160, CD226, CD276, DR 3. Binding moiety A, which includes inhibitors of GAL9, GITR, HAVCR2, HVEM, IDO1, IDO2, ICOS (induced T cell costimulatory molecule), KIR, LAIR1, LIGHT, MARCO (macrophage receptor with collagen structure), PS (phosphatidylserine), OX-40, SLAM, TIGHT, VISTA, VTCN1, or any combination thereof, is used to treat diseases or disorders (e.g., cancer).

[0260] In some embodiments, molecules of formula (I) combined with immune checkpoint inhibitors are used to treat diseases or disorders (e.g., cancer). In some examples, the immune checkpoint inhibitors are programmed death-ligand 1 (PD-L1, also known as B7-H1, CD274), programmed death 1 (PD-1), CTLA-4, PD-L2 (B7-DC, CD273), LAG3, TIM3, 2B4, A2aR, B7H1, B7H3, B7H4, BTLA, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD1 37, CD160, CD226, CD276, DR3, GAL9, GITR, HAVCR2, HVEM, IDO1, IDO2, ICOS (induced T cell costimulatory molecule), KIR, LAIR1, LIGHT, MARCO (macrophage receptor with collagen structure), PS (phosphatidylserine), OX-40, SLAM, TIGHT, VISTA, VTCN1, or any combination thereof. In some cases, the molecule of formula (I) is used in combination with ipilimumab, tremelimumab, nivolumab, pemrolizumab, pidilizumab, MPDL3280A, MEDI4736, MSB0010718C, MK-3475, or BMS-936559 for the treatment of a disease or disorder (e.g., cancer).

[0261] In some embodiments, immunotargeting agents are cytokines. In some cases, cytokines are further subgrouped into chemokines, interferons, interleukins, and tumor necrosis factors. In some embodiments, chemokines act as chemotaxis that guide cell migration and are classified into four subfamilies: CXC, CC, CX3C, and XC. Exemplary chemokines include the CC subfamily: CCL1, CCL2 (MCP-1), CCL3, CCL4, CCL5 (RANTES), CCL6, CCL7, CCL8, CCL9 (or CCL10), CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL Examples include 27, and CCL28; CXC subfamily: CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, and CXCL17; XC subfamily: XCL1 and XCL2; and CX3C subfamily; chemokines from CX3CL1.

[0262] Interferons (IFNs) include type I interferons (e.g., IFN-α, IFN-β, IFN-ε, IFN-κ, and IFN-ω), type II interferons (e.g., IFN-γ), and type III interferons. In some embodiments, IFN-α is further classified into about 13 subtypes, including IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, and IFNA21.

[0263] Interleukins are expressed by leukocytes or leukocytes and promote the development and differentiation of T lymphocytes, B lymphocytes, and hematopoietic cells. Exemplary interleukins include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, and IL-36.

[0264] Tumor necrosis factor (TNF) is a group of cytokines that regulate apoptosis. In some cases, the TNF family includes, but is not limited to, about 19 members, including TNFα, lymphotoxin-α (LT-α), lymphotoxin-β (LT-β), T cell antigen gp39 (CD40L), CD27L, CD30L, FASL, 4-1BBL, OX40L, and TNF-associated apoptosis-inducing ligand (TRAIL).

[0265] In some embodiments, the molecule of formula (I) in combination with a cytokine is used to treat a disease or disorder (e.g., cancer). In some cases, the molecule of formula (I) in combination with a chemokine is used to treat a disease or disorder (e.g., cancer). In some cases, the molecule of formula (I) in combination with an interferon is used to treat a disease or disorder (e.g., cancer). In some cases, the molecule of formula (I) in combination with an interleukin is used to treat a disease or disorder (e.g., cancer). In some cases, the molecule of formula (I) in combination with tumor necrosis factor is used to treat a disease or disorder (e.g., cancer). In some cases, molecules of formula (I) combined with IL-1β, IL-2, IL-7, IL-8, IL-15, MCP-1 (CCL2), MIP-1α, RANTES, MCP-3, MIP5, CCL19, CCL21, CXCL2, CXCL9, CXCL10, or CXCL11 are used to treat diseases or disorders (e.g., cancer).

[0266] In some embodiments, the compositions or pharmaceutical formulations described herein include a vaccine. In some examples, the vaccine is an in situ vaccine. In some examples, the vaccine is a cell-based vaccine. In some examples, the vaccine is a non-cell-based vaccine. In some examples, a molecule of formula (I) combined with a dendritic cell-based vaccine is used to treat a disease or disorder (e.g., cancer). In some examples, a molecule of formula (I) combined with a tumor cell-based vaccine is used to treat a disease or disorder (e.g., cancer). In some examples, a molecule of formula (I) combined with an antigen vaccine is used to treat a disease or disorder (e.g., cancer). In some examples, a molecule of formula (I) combined with an anti-idiotype vaccine is used to treat a disease or disorder (e.g., cancer). In some examples, a molecule of formula (I) combined with a DNA vaccine is used to treat a disease or disorder (e.g., cancer). In some examples, a molecule of formula (I) combined with a vector-based vaccine is used to treat a disease or disorder (e.g., cancer).

[0267] In some embodiments, the compositions or pharmaceutical formulations described herein are used as passive cancer immunotherapy for the treatment of a disease or disorder (e.g., cancer). In some examples, the passive method utilizes adaptive immune system components, such as exogenously generated T cells, natural killer (NK) T cells, and / or chimeric antigen receptor (CAR) T cells, to attack cancer cells.

[0268] In some embodiments, a molecule of formula (I), combined with a T cell-based therapeutic agent, is used to treat a disease or disorder (e.g., cancer). In some cases, the T cell-based therapeutic agent is an activated T cell agent that recognizes one or more of the CD cell surface markers listed above. In some examples, the T cell-based therapeutic agent includes an activated T cell agent that recognizes one or more of CD2, CD3, CD4, CD5, CD8, CD27, CD28, CD80, CD134, CD137, CD152, CD154, CD160, CD200R, CD223, CD226, CD244, CD258, CD267, CD272, CD274, CD278, CD279, or CD357. In some cases, a molecule of formula (I), combined with an activated T-cell agent that recognizes one or more of the following cells: CD2, CD3, CD4, CD5, CD8, CD27, CD28, CD80, CD134, CD137, CD152, CD154, CD160, CD200R, CD223, CD226, CD244, CD258, CD267, CD272, CD274, CD278, CD279, or CD357, is used to treat a disease or disorder (e.g., cancer).

[0269] In some embodiments, a molecule of formula (I) combined with a natural killer (NK) T cell-based therapeutic agent is used to treat a disease or disorder (e.g., cancer). In some examples, the NK-based therapeutic agent is an activated NK agent that recognizes one or more of the CD cell surface markers listed above. In some cases, the NK-based therapeutic agent is an activated NK agent that recognizes one or more of CD2, CD11a, CD11b, CD16, CD56, CD58, CD62L, CD85j, CD158a / b, CD158c, CD158e / f / k, CD158h / j, CD159a, CD162, CD226, CD314, CD335, CD337, CD244, or CD319. In some cases, molecules of formula (I) combined with activated NK agents that recognize one or more of the following: CD2, CD11a, CD11b, CD16, CD56, CD58, CD62L, CD85j, CD158a / b, CD158c, CD158e / f / k, CD158h / j, CD159a, CD162, CD226, CD314, CD335, CD337, CD244, or CD319, are used to treat diseases or disorders (e.g., cancer).

[0270] In some embodiments, the molecule of formula (I), combined with a CAR-T cell-based therapeutic agent, is used to treat a disease or disorder (e.g., cancer).

[0271] In some embodiments, molecules of formula (I) are used to treat diseases or disorders (e.g., cancer) in combination with additional agents that destabilize the endosomal membrane (or interfere with endosomal-lysosome membrane transport). In some embodiments, the additional agents include mitotic inhibitors. Exemplary mitotic inhibitors include, but are not limited to, taxanes such as paclitaxel and docetaxel; vinca alkaloids such as vinblastine, vincristine, vindesine, and vinorelbine; cabazitaxel; colchicine; eribulin; estramustine; etoposide; ixabepyrone; podophyllotoxin; teniposide; or griseofulvin. In some cases, the additional medication includes paclitaxel, docetaxel, vinblastine, vincristine, vindesine, vinorelbine, cabazitaxel, colchicine, eribulin, estramustine, etoposide, ixabepyrone, podophyllotoxin, teniposide, or griseofulvin. In some cases, the additional medication includes taxol. In some cases, the additional medication includes paclitaxel. In some cases, the additional medication includes etoposide. In other cases, the additional medication includes vitamin K3.

[0272] In some embodiments, the compositions or pharmaceutical formulations described herein are used as combination methods (including both active and passive methods) in the treatment of a disease or disorder (e.g., cancer).

[0273] Muscular dystrophy, muscular atrophy, muscle wasting In one embodiment, muscular dystrophy refers to a significant decrease in muscle strength. Significant decrease in muscle strength means a decrease in the strength of the diseased, injured, or unused muscle tissue of a subject compared to the same muscle tissue of a control subject. In one embodiment, significant decrease in muscle strength is a decrease of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or more, compared to the same muscle tissue of a control subject. In another embodiment, significant decrease in muscle strength means a decrease in the strength of the unused muscle tissue compared to the muscle strength of the same muscle tissue of the same subject before the period of non-use. In one embodiment, significant decrease in muscle strength is a decrease of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or more, compared to the muscle strength of the same muscle tissue of the same subject before the period of non-use.

[0274] In another embodiment, muscular dystrophy refers to a significant decrease in muscle mass. Significant decrease in muscle mass means a reduction in muscle volume in the diseased, damaged, or unused muscle tissue of a subject compared to the same muscle tissue of a control subject. In some embodiments, a significant decrease in muscle volume is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or more, compared to the same muscle tissue of a control subject. In another embodiment, a significant decrease in muscle mass means a reduction in muscle volume in unused muscle tissue compared to the muscle volume in the same muscle tissue of the same subject before the period of non-use. In some embodiments, a significant decrease in muscle volume is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or more, compared to the muscle volume in the same muscle tissue of the same subject before the period of non-use. Muscle volume is selectively measured by evaluating the cross-sectional area of ​​the muscle using methods such as magnetic resonance imaging (e.g., muscle volume / cross-sectional area (CSA) MRI).

[0275] Myotonic dystrophy is a multisystemic neuromuscular disease that includes two main types: myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2). DM1 is caused by the dominant repetition elongation of "CTG" in the DM protein kinase (DMPK) gene, which, when transcribed into mRNA, forms a hairpin that binds with high affinity to the Muscleblind-like (MBNL) family of proteins. MBNL proteins are involved in regulating post-transcriptional splicing and polyadenylation (polyadenylatin) sites, and loss of MBNL protein function leads to downstream accumulation of nuclear lesions, increasing missplicing events and subsequently causing myotonia and other clinical symptoms. In some embodiments, methods for treating a target muscular dystrophy, muscular atrophy, and / or muscle wasting are described herein, the methods comprising the steps of providing a polynucleic acid molecule or a polynucleic acid molecule conjugate as described herein, and administering a therapeutically effective amount of the polynucleic acid molecule or polynucleic acid molecule conjugate to a target for treating muscular dystrophy, muscular atrophy, and / or muscle wasting. In some embodiments, the polynucleic acid molecule or polynucleic acid molecule conjugate targets a gene transcript which is mutated or upregulated so that downregulation, deletion, or exon skipping of the transcript is desired to treat the disease. In some embodiments, the polynucleic acid molecule or polynucleic acid molecule conjugate targets DMPK mRNA, DMD mRNA, or GYS1 mRNA.

[0276] Pharmaceutical preparations In some embodiments, the pharmaceutical formulations described herein are administered to a target by multiple routes of administration, including, but not limited to, parenteral (e.g., intravenous, subcutaneous, intramuscular), oral, intranasal, buccal, rectal, or transdermal administration routes. In some examples, the pharmaceutical compositions described herein are formulated for parenteral (e.g., intravenous, subcutaneous, intramuscular) administration. In other examples, the pharmaceutical compositions described herein are formulated for oral administration. In yet another example, the pharmaceutical compositions described herein are formulated for intranasal administration.

[0277] In some embodiments, the pharmaceutical composition may include, but is not limited to, aqueous dispersions, self-emulsifying dispersions, solid solutions, liposome dispersions, aerosols, solid dosage forms, powders, immediate-release formulations, controlled-release formulations, rapidly dissolving formulations, tablets, capsules, pills, delayed-release formulations, sustained-release formulations, pulsed-release formulations, multi-particle formulations (e.g., nanoparticle formulations), and mixed formulations of immediate-release and controlled-release.

[0278] In some cases, the pharmaceutical formulation includes multi-particle formulations. In some cases, the pharmaceutical formulation includes nanoparticle formulations. In some cases, the nanoparticles include cMAP, cyclodextrin, or lipids. In some cases, the nanoparticles include solid lipid nanoparticles, polymer nanoparticles, self-emulsifying nanoparticles, liposomes, microemulsions, or micelle solutions. Further exemplary nanoparticles include, but are not limited to, paramagnetic nanoparticles, superparamagnetic nanoparticles, metallic nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers (such as those having covalently bonded metal chelates), nanofibers, nanohorns, nanoonions, nanorods, nanoropes, and quantum dots. In some examples, nanoparticles are nanoparticles of metals, such as scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium, niobium, molybdenum, ruthenium, rhodium, palladium, silver, cadmium, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, gadolinium, aluminum, gallium, indium, tin, thallium, lead, bismuth, magnesium, calcium, strontium, barium, lithium, sodium, potassium, boron, silicon, phosphorus, germanium, arsenic, antimony, and combinations thereof, their alloys, or oxides.

[0279] In some examples, nanoparticles consist of a core, or a core and a shell, as in core-shell nanoparticles.

[0280] In some examples, the nanoparticles are further coated with molecules (e.g., one or more polynucleic acid molecules or binding sites as described herein) for the binding of functional elements. In some examples, the coating includes chondroitin sulfate, dextran sulfate, carboxymethyl dextran, alginic acid, pectin, carrageenan, fucoidan, agaropectin, porphyran, karaya gum, gelan gum, xanthan gum, hyaluronic acid, glucosamine, galactosamine, chitin (or chitosan), polyglutamic acid, polyaspartic acid, lysozyme, cytochrome C, ribonuclease, trypsinogen, chymotrypsinogen, α-chymotrypsin, polylysine, polyarginine, histone, protamine, ovalbumin, dextrin, or cyclodextrin. In some examples, the nanoparticles include graphene-coated nanoparticles.

[0281] In some cases, the nanoparticles have at least one dimension of approximately 500 nm, 400 nm, 300 nm, 200 nm, or less than 100 nm.

[0282] In some examples, nanoparticle formulations include paramagnetic nanoparticles, superparamagnetic nanoparticles, metallic nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers (such as those having covalently bonded metal chelates), nanofibers, nanohorns, nanoonions, nanorods, nanoropes, or quantum dots. In some examples, the polynucleic acid molecules or binding sites described herein are directly or indirectly conjugated to the nanoparticles. In some examples, at least 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more polynucleic acid molecules or binding sites described herein are directly or indirectly conjugated to the nanoparticles.

[0283] In some embodiments, the pharmaceutical formulation comprises a carrier or carrier material selected based on compatibility with the compositions disclosed herein and the release profile characteristics of the desired dosage form. Exemplary carrier materials include, for example, binders, suspending agents, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, humectants, and diluents. Pharmaceutically compatible carrier materials include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerin, magnesium silicate, polyvinylpyrrolidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurocholic acid, phosphatidylcholine, sodium chloride, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan, monoglycerides, diglycerides, and pregelatinized starch. For example, see Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999).

[0284] In some examples, pharmaceutical formulations further include pH adjusters or buffers, such as acids including acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; bases including sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and tris-hydroxymethylaminomethane; and buffers including citrate / dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in amounts necessary to maintain the pH of the composition within an acceptable range.

[0285] In some cases, a pharmaceutical formulation contains one or more salts in amounts necessary to bring the osmotic pressure of the composition into an acceptable range. Such salts include sodium cations, potassium cations, or ammonium cations, and chloride anions, citrate anions, ascorbate anions, borate anions, phosphate anions, bicarbonate anions, sulfate anions, thiosulfate anions, or bisulfite anions. Suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

[0286] In some cases, pharmaceutical formulations further include diluents used to stabilize the compound, as these diluents can provide a more stable environment. Salts dissolved in buffers (which may further result in pH control or maintenance) are used as diluents in the art, but are not limited to phosphate-buffered saline solutions. In some cases, the diluent increases the volume of the composition to facilitate compression or to create a sufficient volume for a homogeneous blend for capsule filling. Such compounds may include, for example, microcrystalline cellulose such as lactose, starch, mannitol, sorbitol, dextrose, and Avicel®; calcium hydrogen phosphate, calcium phosphate dihydrate; tricalcium phosphate, calcium phosphate; anhydrous lactose, spray-dried lactose; pregelatinized starch, compressible sugars such as Di-Pac® (Amstar); mannitol, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate stearate, sucrose-based diluents, powdered sugar; mononucleotide calcium sulfate monohydrate, calcium sulfate dihydrate; calcium lactate trihydrate, dextrates; hydrolyzed cereal solids, amylose; powdered cellulose, calcium carbonate; glycine, kaolin; mannitol, sodium chloride; inositol, bentonite, etc.

[0287] In some cases, pharmaceutical formulations contain disintegrants or breakdown agents that promote the breakdown or disintegration of substances. The term "break down" includes both the disintegration and dispersion of the dosage form upon contact with gastrointestinal fluids. Examples of breakdown agents include starch, e.g., natural starch, e.g., corn starch or potato starch; pregelatinized starch, e.g., National 1551 or Amijel®; or sodium starch glycolate, e.g., Promogel® or Explotab®; cellulose, e.g., wood product; methylcrystalline cellulose, e.g., Avicel®, Avicel® PH101, Avicel® PH102, Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, Min Examples include Tia® and Solka-Floc®, methylcellulose, croscarmellose, or cross-linked cellulose, such as cross-linked carboxymethylcellulose sodium (Ac-Di-Sol®), cross-linked carboxymethylcellulose, or cross-linked croscarmellose sodium, cross-linked starch, such as sodium starch glycolate, cross-linked polymers, such as crospovidone, cross-linked polyvinylpyrrolidone, alginates, such as alginic acid, or salts of alginic acid, such as sodium alginate, clay, such as Veegum® HV (magnesium aluminum silicate), rubber, such as agar, gaua, carob, karaya, pectin, or tragacanth, sodium starch glycolate, bentonite, sponges, surfactants, resins, such as cation exchange resins, citrus pulp, sodium lauryl sulfate, and sodium lauryl sulfate in combination with starch.

[0288] In some examples, pharmaceutical formulations contain fillers such as lactose, calcium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrate, dextran, starch, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, and polyethylene glycol.

[0289] Lubricants and lubricants are optionally included in the pharmaceutical formulations described herein to prevent, reduce, or inhibit adhesion or friction between materials. Examples of lubricants include, for example, stearic acid, calcium hydroxide, talc, sodium stearyl fumarate, hydrocarbons such as mineral oil or hydrogenated vegetable oils such as hydrogenated soybean oil (Sterotex®), higher fatty acids and their alkali metal and alkaline earth metal salts such as aluminum, calcium, magnesium, zinc, stearic acid, sodium stearate, glycerol, talc, wax, Stearowet®, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, polyethylene glycol (e.g., PEG-4000) or methoxypolyethylene glycol such as Carbowax®, sodium oleate, sodium benzoate, glyceryl behenate, polyethylene glycol, magnesium or sodium lauryl sulfate, Syloid®, colloidal silica such as Cab-O-Sil®, starch such as corn starch, silicone oil, and surfactants.

[0290] Plasticizers include compounds used to reduce brittleness by softening microencapsulated materials or film coatings. Suitable plasticizers include, for example, polyethylene glycol such as PEG300, PEG400, PEG600, PEG1450, PEG3350, and PEG800, stearic acid, propylene glycol, oleic acid, triethylcellulose, and triacetin. Plasticizers may further act as dispersants or wetting agents.

[0291] Examples of solubilizing agents include compounds such as triacetin, triethyl citrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium doxate, vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cyclodextrin, ethanol, n-butanol, isopropyl alcohol, cholesterol, bile salts, polyethylene glycol 200-600, glycoflor, transktol, propylene glycol, and dimethyl isosorbide.

[0292] Stabilizers include mixtures of various antioxidants, buffers, acids, and preservatives.

[0293] The suspending agent is, for example, polyvinylpyrrolidone such as polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, vinylpyrrolidone / vinyl acetate copolymer (S630), polyethylene glycol (for example, polyethylene glycol may have a molecular weight of about 300 to about 6000, about 3350 to about 4000, or about 7000 to about 5400), sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, hydroxyethylcellulose acetate stearate, poly This product contains compounds such as sorbate 80, hydroxyethylcellulose, sodium alginate, rubber (e.g., tragacanth gum and acacia gum), guar gum, xanthan gum, sugars (e.g., cellulose compounds such as sodium carboxymethylcellulose), methylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, hydroxyethylcellulose, polysorbate 80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, and povidone.

[0294] Surfactants include compounds such as sodium lauryl sulfate, sodium doxate, or Tween 60 or 80, triacetin, vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbate, polaxomers, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, e.g., Pluronic® (BASF). Additional surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil; as well as polyoxyethylene alkyl ethers and alkylphenyl ethers, e.g., octoxynol 10, octoxynol 40. Often, surfactants are included to enhance physical stability or for other purposes.

[0295] Examples of viscosity enhancers include methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, hydroxypropylmethylcellulose phthalate, carbomer, polyvinyl alcohol, alginate, acacia, chitosan, and combinations thereof.

[0296] The humectants include compounds such as oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium doxate, sodium oleate, sodium lauryl sulfate, sodium docusate, triacetin, Tween 80, vitamin E TPGS, and ammonium salts.

[0297] Treatment regimen In some embodiments, the pharmaceutical compositions described herein are administered for therapeutic purposes. In some embodiments, the pharmaceutical compositions are administered once daily, twice daily, three times daily, or more frequently. The pharmaceutical compositions are administered daily, every other day, five days a week, once a week, every other week, two weeks a month, three weeks a month, once a month, twice a month, three times a month, or more frequently. The pharmaceutical compositions are administered for at least one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, eighteen months, two years, three years, or longer.

[0298] In some embodiments, one or more pharmaceutical compositions are administered simultaneously, sequentially, or at intervals of time. In some embodiments, one or more pharmaceutical compositions are administered simultaneously. In some cases, one or more pharmaceutical compositions are administered sequentially. In further cases, one or more pharmaceutical compositions are administered at intervals of time (for example, the first administration of the first pharmaceutical composition is on day 1, followed by at least one, two, three, four, five days or more before the administration of at least a second pharmaceutical composition).

[0299] In some embodiments, two or more different pharmaceutical compositions are administered simultaneously. In some cases, two or more different pharmaceutical compositions are administered simultaneously in sequence without any interval between administrations. In other cases, two or more different pharmaceutical compositions are administered simultaneously in sequence with intervals of approximately 0.5 hours, 1 hour, 2 hours, 3 hours, 12 hours, 1 day, 2 days, or longer between administrations.

[0300] If the patient's condition improves, the physician may, at their discretion, continue administering the composition, temporarily reduce the dose of the administered composition, or temporarily discontinue it for a specific period (i.e., a “drug-free period”). In some cases, the length of the drug-free period varies between 2 days and 1 year, and includes, but are not limited to, 2, 3, 4, 5, 6, 7, 10, 12, 15, 20, 28, 35, 50, 70, 100, 120, 150, 180, 200, 250, 280, 300, 320, 350, or 365 days. The dose reduction during drug-free days ranges from 10% to 100%, and includes, but are not limited to, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0301] Once the patient's condition improves, a maintenance dose is administered as needed. Subsequently, the dose, frequency, or both are selectively reduced, depending on the symptoms, to a level at which the improved disease, impairment, or illness is maintained.

[0302] In some embodiments, the amount of a given drug corresponding to such a quantity depends on factors such as the specific compound, the severity of the disease, and the characteristics of the subject or host requiring treatment (e.g., body weight), but nevertheless, it is routinely determined in methods known in the art according to the specific circumstances surrounding the case, including, for example, the specific drug being administered, the route of administration, and the subject or host being treated. In some examples, the desired dose is conveniently presented as a single dose, or as divided doses administered simultaneously (or over a short period of time), or at appropriate intervals, e.g., two, three, four or more times per day as sub-doses.

[0303] Due to the large number of variables in each treatment regimen, the ranges mentioned above are merely suggestive, and deviations from these recommendations are not uncommon. Such dosages are not limited but will vary depending on many variables, including the activity of the compound used, the disease or illness being treated, the mode of administration, the requirements of the individual subject, the severity of the disease or illness being treated, and the physician's judgment.

[0304] In some embodiments, the toxicity and therapeutic efficacy of such treatment regimens are determined by standard pharmaceutical procedures in cell culture or experimental animals, which include, but are not limited to, the determination of the LD50 (lethal dose up to 50% of the population) and the ED50 (therapeutably effective dose for 50% of the population). The dose-to-toxicity ratio is the therapeutic index, which is expressed as the ratio between the LD50 and the ED50. Compounds exhibiting a high therapeutic index are preferred. Data obtained from cell culture assays and animal studies are used when formulating a set of dosages for human use. Doses of such compounds are preferably within the range of blood concentrations containing the ED50 with the lowest toxicity. Doses vary within this range depending on the dosage form used and the route of administration utilized.

[0305] Kit / Product In some embodiments, kits and products used in conjunction with one or more compositions and methods described herein are disclosed herein. Such kits include a partitioned carrier, packaging, or container for housing one or more containers such as vials, tubes, etc., each container comprising one of the distinct elements for using the methods described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the containers are formed from a variety of materials such as glass or plastic.

[0306] Products provided herein include packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, bottles, and any packaging materials suitable for the selected formulation and the intended mode of administration and treatment.

[0307] For example, the container contains the molecule of formula (Xa):AX-B'-YC, as disclosed herein. Such a kit optionally includes an identifying statement or label, or instructions for its use as described herein.

[0308] A kit typically includes a label listing the contents and / or instructions for use, and accompanying documentation containing the instructions for use. A set of instructions is also typically included.

[0309] In one embodiment, the label is on or attached to the container. In one embodiment, the label is on the container if the letters, numbers, or other markings forming the label are affixed, molded, or engraved onto the container itself. The label is attached to the container if, for example, the label is present in the receptacle or transport device that holds the container as an accompanying document. In one embodiment, the label is used to indicate that the contents are to be used for a specific therapeutic purpose. The label also indicates how to use the contents, for example, in the method described herein.

[0310] In one embodiment, the pharmaceutical composition is presented in a pack or dispenser device containing one or more unit dosage forms comprising the compounds provided herein. For example, the pack may include metal foil or plastic foil, such as a blister pack. In one embodiment, the pack or dispenser device is accompanied by instructions for administration. In one embodiment, the pack or dispenser is accompanied by a notice attached to a container in a form specified by a government agency that regulates the manufacture, use, or sale of pharmaceuticals, the notice reflecting the government agency's approval of the form of the drug for administration to humans or animals. Such a notice may be, for example, a label approved by the U.S. Food and Drug Administration with respect to a prescription drug or approved package insert. In one embodiment, compositions comprising the compounds provided herein, formulated in a suitable pharmaceutical carrier, are also prepared, placed in a suitable container, and labeled for the treatment of the indicated disease.

[0311] Specific terms Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in the subject matter. It will be understood that the above general statements and the following detailed statements are illustrative and descriptive only and do not limit any claimed subject matter. In this application, the use of the singular includes the plural unless otherwise specified. Where used in the specification and appended claims, the singular forms "a," "an," and "the" include multiple references unless the context explicitly indicates otherwise. In this application, the use of "or" means "and / or" unless otherwise specified. Furthermore, the use of the term "including" is not limited to other forms such as "include," "includes," and "included."

[0312] As used herein, ranges and quantities can be expressed as "approximately" a specific value or range. "Approximately" also includes exact quantities. Therefore, "approximately 5 μL" also means "approximately 5 μL" and "5 μL". Generally, the term "approximately" includes quantities that are expected to be within experimental error.

[0313] The paragraph headings used in this specification are for organizational purposes only and should not be construed as limiting the subject matter described herein.

[0314] As used herein, the terms “individual,” “subject,” and “patient” mean any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human. No term is limited to situations characterized by supervision (e.g., constant or intermittent) of a healthcare worker (e.g., a physician, registered nurse, clinical nurse, physician’s assistant, nursing assistant, or hospice staff).

[0315] chemical definition The abbreviations used herein have their conventional meanings within the fields of chemistry and biology. The chemical structures and formulas described herein are constructed according to the standard rules of chemical bond valency known in the fields of chemistry.

[0316] If substituents are identified by their conventional chemical formulas written from left to right, then those substituents equally encompass chemically identical substituents that would result from writing the structure from right to left; for example, -CH2O- is equal to -OCH2-.

[0317] The term "alkyl" means, unless otherwise specified, a straight (i.e., unbranched) carbon chain or a branched carbon chain (or carbon), or a combination thereof, either by itself or as part of another substituent, which may be fully saturated, monounsaturated, and polyunsaturated, and may include monovalent, divalent, and polyvalent radicals having a specified number of carbon atoms (i.e., C1-C 10(where n means 1 to 10 carbon atoms). Alkyls are uncyclized chains. Examples of saturated hydrocarbon radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, and congeners and isomers such as n-pentyl, n-hexyl, n-heptyl, and n-octyl. Unsaturated alkyl groups have one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, clotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1-propynyl and 3-propynyl, 3-butynyl, and more highly congeners and isomers. Alkoxys are alkyl groups that are bonded to the remainder of the molecule via an oxygen linker (-O-).

[0318] The term "alkylene" means, unless otherwise specified, a divalent radical derived from an alkyl group, either by itself or as part of another substituent, such as -CH2CH2CH2CH2-. Typically, alkyl (or alkylene) groups have 1 to 24 carbon atoms, and in this specification, those groups having 10 or fewer carbon atoms are preferred. "Lower alkyl" or "lower alkylene" generally refers to short-chain alkyl or alkylene groups having 8 or fewer carbon atoms. The term "alkenylene" means, unless otherwise specified, a divalent radical derived from an alkene, either by itself or as part of another substituent.

[0319] The term "heteroalkyl," either by itself or in combination with other terms, means, unless otherwise specified, a stable linear or branched chain, or a combination thereof, comprising at least one carbon atom and at least one heteroatom (e.g., O, N, P, Si, and S), where the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroatom (e.g., N, S, Si, or P) may be located at any internal position of the heteroalkyl group, or at a position where the alkyl group is bonded to the remainder of the molecule. Heteroalkyl groups are non-cyclized chains. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2, -S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, -O-CH3, -O-CH-2-CH3, and -CN. Up to two or three heteroatoms may be consecutive, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. The heteroalkyl moiety may contain one heteroatom (e.g., O, N, S, Si, or P). The heteroalkyl moiety may contain two optionally distinct heteroatoms (e.g., O, N, S, Si, or P). The heteroalkyl moiety may contain three optionally distinct heteroatoms (e.g., O, N, S, Si, or P). The heteroalkyl moiety may contain four optionally distinct heteroatoms (e.g., O, N, S, Si, or P). The heteroalkyl moiety may contain five optionally distinct heteroatoms (e.g., O, N, S, Si, or P). The heteroalkyl moiety may contain up to eight optionally distinct heteroatoms (e.g., O, N, S, Si, or P).

[0320] Similarly, the term “heteroalkylene” means, without limitation, a divalent radical derived from a heteroalkyl group, such as -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-, either by itself or as part of another substituent, unless otherwise specified. In the case of a heteroalkylene group, the heteroatom can also occupy either or both ends of the chain (e.g., alkylene oxy, alkylenedioxy, alkylene amino, alkylenediamino, etc.). Furthermore, in the case of linking groups of alkylenes and heteroalkylenes, the orientation of the linking group is not indicated by the direction in which the formula of the linking group is written. For example, the formula -C(O)2R'- represents both -C(O)2R'- and -R'C(O)2-. As stated above, heteroalkyl groups as used herein include groups bonded to the remainder of the molecule via heteroatoms, such as -C(O)R', -C(O)NR', ​​-NR'R'', -OR', -SR', and / or -SO2R'. When "heteroalkyl" is mentioned after a specific heteroalkyl group such as -NR'R'', it should be understood that the terms "heteroalkyl" and -NR'R'' are neither redundant nor mutually exclusive. Rather, specific heteroalkyl groups are mentioned for clarity. Therefore, the term "heteroalkyl" should not be interpreted herein as excluding specific heteroalkyl groups such as -NR'R''.

[0321] The terms "cycloalkyl" and "heterocycloalkyl," either by themselves or in combination with other terms, mean the cyclic versions of "alkyl" and "heteroalkyl," respectively, unless otherwise specified. Cycloalkyls and heterocycloalkyls are not aromatic. In addition, in the case of heterocycloalkyls, the heteroatom can occupy a position where the heterocycle is bonded to the remainder of the molecule. Examples of cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, and cycloheptyl. Examples of heterocycloalkyls include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, and 2-piperazinyl. "Cycloalkylene" and "heterocycloalkylene" refer to divalent radicals derived from cycloalkyl and heterocycloalkyl, respectively, either alone or as part of another substituent. "Cycloalkyl" also refers to bicyclic and polycyclic hydrocarbon rings, such as bicyclo[2.2.1]heptane and bicyclo[2.2.2]octane.

[0322] The terms "halo" or "halogen" mean, unless otherwise specified, atoms of fluorine, chlorine, bromine, or iodine, either by themselves or as part of another substituent. In addition, terms such as "haloalkyl" are intended to include monohaloalkyl and polyhaloalkyl. For example, the term "halo(C1-C4)alkyl" includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, and 3-bromopropyl.

[0323] The term "acyl" means -C(O)R unless otherwise specified, where R is a substituted or unsubstituted alkyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heteroalkyl, a substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl.

[0324] The term "aryl," unless otherwise specified, means a polyunsaturated, aromatic, hydrocarbon substituent, which may be one or more rings (preferably 1 to 3 rings) that are condensed together (i.e., a fused ring aryl) or covalently bonded. A fused ring aryl refers to multiple rings condensed together, where at least one of the fused rings is an aryl group. The term "heteroaryl" refers to an aryl group (or ring) containing at least one heteroatom such as N, O, or S, where the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom is optionally quaternized. Thus, the term "heteroaryl" includes a fused ring heteroaryl group (i.e., multiple rings condensed together, where at least one of the fused rings is an aromatic heterocycle). A 5,6-fused ring heteroarylene refers to two rings condensed together, where one ring has 5 members and the other ring has 6 members, where at least one of the rings is a heteroaryl ring. Similarly, a 6,6-fused heteroarylene refers to two rings fused together, one having 6 members and the other having 6 members, where at least one ring is a heteroaryl ring. And a 6,5-fused heteroarylene refers to two rings fused together, one having 6 members and the other having 5 members, where at least one ring is a heteroaryl ring. The heteroaryl group may be bonded to the remainder of the molecule via carbon or heteroatoms.Non-limiting examples of aryl and heteroaryl groups include phenyl, naphthyl, pyrrolyl, pyrazolyl, pyridadinyl, triazinyl, pyrimidinyl, imidazolyl, pyrazinyl, purinyl, oxazolyl, isoxazolyl, thiazolyl, furyl, thienyl, pyridyl, pyrimidyl, benzothiazolyl, benzoxazolylbenzimidazolyl, benzofuran, isobenzofuranyl, indolyl, isoindolyl, benzothiophenyl, isoquinolyl, quinoxalinyl, quinolyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrzolyl, 3-pyrazolyl, 2-imidazolyl Examples include lyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. The substituents for each of the above-mentioned aryl and heteroaryl ring systems are selected from the group of acceptable substituents listed below. "Arylene" and "heteroarylene" refer to divalent radicals derived from aryl and heteroaryl groups, respectively, either alone or as part of another substituent. The substituents on the heteroaryl group can be -O- groups bonded to the ring heteroatom nitrogen.

[0325] A spiroring is a group of two or more rings linked by one atom. The individual rings within a spiroring may be identical or distinct. Each ring in a spiroring may be substituted or unsubstituted and may have different substituents than the other individual rings within a set of spirorings. Possible substituents for an individual ring within a spiroring are also possible substituents for the same ring if it is not part of the spiroring (e.g., substituents for a cycloalkyl ring or a heterocycloalkyl ring). A spiroring may be a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted cycloalkylene, a substituted or unsubstituted heterocycloalkylene, or a substituted or unsubstituted heterocycloalkylene, and each individual ring within the spiroring group may be one of those in the preceding list, including all rings of one type (e.g., all rings are substituted heterocycloalkylenes, and each ring may be the same or a different substituted heterocycloalkylene). When referring to spiro ring systems, a heterocyclic spiro ring means a spiro ring in which at least one ring is heterocyclic and each ring is distinct. When referring to spiro ring systems, a substituted spiro ring means in which at least one ring is substituted and each substituent can be arbitrarily distinct.

[0326] The symbols below

[0327] [ka] The symbol indicates the location where a chemical part of a molecule or chemical formula is bonded to the rest of the molecule or chemical formula.

[0328] As used herein, the term "oxo" refers to oxygen double-bonded to a carbon atom.

[0329] The term "alkylarylene" as an arylene moiety refers to a covalent bond to an alkylene moiety (also known herein as an alkylene linker). In some embodiments, the alkylarylene group has the following formula:

[0330] [ka]

[0331] The alkylarylene moiety may be substituted on the alkylene moiety or arylene linker (e.g., at carbon 2, 3, 4, or 6) with halogens, oxo, -N3, -CF3, -CCl3, -CBr3, -CI3, -CN, -CHO, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO2CH3-SO3H, -OSO3H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, substituted or unsubstituted C1-C5 alkyls, or substituted or unsubstituted 2- to 5-membered heteroalkyls (e.g., by substituents). In some embodiments, the alkylarylene is not substituted.

[0332] Each of the terms mentioned above (e.g., "alkyl," "heteroalkyl," "cycloalkyl," "heterocycloalkyl," "aryl," and "heteroaryl") includes both substituted and unsubstituted forms of the radicals shown. Preferred substituents for each type of radical are provided below.

[0333] Substituents for alkyl and heteroalkyl radicals (including those groups often called alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) are not limited to -OR', =O, =NR', =N-OR', -NR'R'', -SR', -halogen, -SiR'R''R''', -OC(O)R', -C(O)R', -CO2R', -CONR'R'', -OC(O)NR'R'', -NR' It can be one or more of various groups selected from 'C(O)R', -NR'-C(O)NR''R''', -NR''C(O)2R', -NR-C(NR'R''R''')=NR'''', -NR-C(NR'R'')=NR''', -S(O)R', -S(O)2R', -S(O)2NR'R'', -NRSO2R', -NR'NR''R''', -ONR'R'', -NR'C(O)NR''NR'''R'''', -CN, -NO2, -NR'SO2R'', -NR'C(O)R'', -NR'C(O)-OR'', -NR'OR'', where m' is the total number of carbon atoms in such radicals. R, R', R'', R''', and R'''' each preferably independently refers to hydrogen, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted heterocycloalkyl group, a substituted or unsubstituted aryl group (e.g., aryls substituted with 1 to 3 halogens), a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted alkyl group, an alkoxy or thioalkoxy group, or an arylalkyl group. If the compounds described herein contain more than one R group, for example, each of the R groups is independently selected as the R', R'', R''', and R'''' groups, respectively, when more than one of these groups are present. When R' and R'' are bonded to the same nitrogen atom, they can combine with the nitrogen atom to form a 4-membered, 5-membered, 6-membered, or 7-membered ring. For example, -NR'R'' includes, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl.From the above description of substituents, those skilled in the art will understand that the term "alkyl" is intended to include groups containing carbon atoms bonded to groups other than hydrogen groups, such as haloalkyls (e.g., -CF3 and -CH2CF3) and acyls (e.g., -C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, etc.).

[0334] Similar to the substituents described for alkyl radicals, substituents for aryl and heteroaryl groups are diverse, ranging from zero to the total number of open valences on the aromatic ring system, for example: -OR', -NR'R'', -SR', -halogen, -SiR'R''R''', -OC(O)R', -C(O)R', -CO2R', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'-C(O)NR''R''', -NR''C(O)2R', -NR-C(NR'R''R''')=NR'''', -NR-C(NR'R'')=NR''', -S(O)R', -S(O)2R', -S(O)2NR'R'', -NRSO2R', -NR'NR''R''', -ONR'R'', -NR'C(O)NR''NR R'''' is selected from -CN, -NO2, -R', -N3, -CH(Ph)2, fluoro(C1-C4)alkoxy, and fluoro(C1-C4)alkyl, -NR'SO2R'', -NR'C(O)R'', -NR'C(O)-OR'', and -NR'OR'', and R', R'', R''', and R'''' are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. If the compounds described herein contain more than one R group, for example, each of the R groups is independently selected as the R', R'', R''', and R'''' group, respectively, if more than one of these groups are present.

[0335] Substituents for a ring (e.g., cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene) may be represented as substituents on the ring rather than on specific atoms of the ring (commonly called floating substituents). In such cases, the substituent may be bonded to any of the ring atoms (according to the rules of chemistry), and in the case of a fused ring or spiro ring, a substituent represented as relating to a member of the fused ring or spiro ring (a floating substituent on one ring) may be a substituent on either the fused ring or spiro ring (a floating substituent on multiple rings). If the substituent is bonded to the ring rather than a specific atom (a floating substituent), and the substitution subscript is an integer greater than 1, then multiple substituents may be on the same atom, the same ring, different atoms, different fused rings, or different spiro rings, and each substituent may be arbitrarily different. If the site of the ring's attachment to the remainder of the molecule is not limited to a single atom (a floating substituent), the attachment site is any atom of the ring, and in the case of a fused or spiro-ring, any atom of either the fused or spiro-ring, subject to the rules of chemical supply. If a ring, fused or spiro-ring contains one or more ring heteroatoms, and that ring, fused or spiro-ring is shown with one or more floating substituents (including, but not limited to, attachment sites to the remainder of the molecule), the floating substituents may be attached to the heteroatoms. If a ring heteroatom is shown to be attached to one or more hydrogens (e.g., a nitrogen atom with two bonds to a ring atom and a third bond to a hydrogen) in a structure or formula with floating substituents, then when the heteroatom is attached to a floating substituent, the substituent is understood to replace the hydrogen, subject to the rules of chemical supply.

[0336] Two or more substituents may be optionally bonded to form an aryl group, a heteroaryl group, a cycloalkyl group, or a heterocycloalkyl group. Such so-called ring-forming substituents are typically, but not always, found to be bonded to a cyclic base structure. In one embodiment, the ring-forming substituent is bonded to an adjacent member of the base structure. For example, two ring-forming substituents bonded to adjacent members of a cyclic base structure form a fused ring structure. In another embodiment, the ring-forming substituent is bonded to a single member of the base structure. For example, two ring-forming substituents bonded to a single member of a cyclic base structure form a spiro-ring structure. In yet another embodiment, the ring-forming substituent is bonded to a non-adjacent member of the base structure.

[0337] Two of the substituents on adjacent atoms of an aryl ring or heteroaryl ring can be optionally defined as -TC(O)-(CRR') q A -U- ring may be formed, where T and U are independently -NR-, -O-, -CRR'-, or a single bond, and q is an integer from 0 to 3. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may be optionally replaced with substituents of the formula -A-(CH2)rB-, where A and B are independently -CRR'-, -O-, -NR-, -S-, -S(O)-, -S(O)2-, -S(O)2NR'-, or a single bond, and r is an integer from 1 to 4. One of the single bonds of the new ring thus formed may be optionally replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may be optionally replaced with substituents of the formula -(CRR')s-X'-(C''R''R''') d The substituents R, R', R'', and R'''' are preferably selected independently from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.

[0338] As used herein, the terms “heteroatom” or “ring heteroatom” are intended to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).

[0339] "Substituent" as used herein means a group selected from the following: (a) oxo, halogen, -CF3, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC=(O)NHNH2, -NHC=(O)NH2, -NHSO2H, -NHC=(O)H, -NHC(O)-OH, -NHOH, -OCF3, -OCHF2, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and (b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, substituted with at least one substituent selected from the following: (i) oxo, halogen, -CF3, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC=(O)NHNH2, -NHC=(O)NH2, -NHSO2H, -NHC=(O)H, -NHC(O)-OH, -NHOH, -OCF3, -OCHF2, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and (ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, substituted with at least one substituent selected from the following: (a) Oxo, halogen, -CF3, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC=(O)NHNH2, -NHC=(O)NH2, -NHSO2H, -NHC=(O)H, -NHC(O)-OH, -NHOH, -OCF3, -OCHF2, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and (b) Alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl: oxo, halogen, -CF3, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC=(O)NHNH2, -NHC=(O)NH2, -NHSO2H, -NHC=(O)H, -NHC(O)-OH, -NHOH, -OCF3, -OCHF2, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl.

[0340] When used herein, “size-limited substituent” or “size-limited substituent group” means a group selected from all of the substituents described above for “substituent,” where each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C 20 Alkyl, substituted or unsubstituted heteroalkyls are each substituted or unsubstituted 2-20 member heteroalkyls, substituted or unsubstituted cycloalkyls are each substituted or unsubstituted C3-C8 cycloalkyls, substituted or unsubstituted heterocycloalkyls are each substituted or unsubstituted 3-8 member heterocycloalkyls, substituted or unsubstituted aryls are each substituted or unsubstituted C6-C 10An aryl is, and a substituted or unsubstituted heteroaryl is, respectively, a substituted or unsubstituted 5- to 10-membered heteroaryl.

[0341] When used herein, “lower substituent” or “lower substituent group” means a group selected from all of the substituents described above for “substituent,” where each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2- to 8-membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C7 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3- to 7-membered heterocycloalkyl, and each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C 10 An aryl is, and a substituted or unsubstituted heteroaryl is, respectively, a substituted or unsubstituted 5- to 9-member heteroaryl.

[0342] In some specific embodiments, each substituent described in the compounds herein is substituted with at least one substituent. More specifically, in some specific embodiments, each of the substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene described in the compounds herein is substituted with at least one substituent. In other specific embodiments, at least one or all of such groups are substituted with at least one size-limiting substituent. In other specific embodiments, at least one or all of such groups are substituted with at least one lower substituent.

[0343] In other embodiments of the compounds described herein, the substituted or unsubstituted alkyl groups are, respectively, substituted or unsubstituted C1-C20 Alkyl, substituted or unsubstituted heteroalkyls are each substituted or unsubstituted 2-20 member heteroalkyls, substituted or unsubstituted cycloalkyls are each substituted or unsubstituted C3-C8 cycloalkyls, substituted or unsubstituted heterocycloalkyls are each substituted or unsubstituted 3-8 member heterocycloalkyls, substituted or unsubstituted aryls are each substituted or unsubstituted C6-C 10 The aryl and / or substituted or unsubstituted heteroaryls are each substituted or unsubstituted 5- to 10-membered heteroaryls. In some embodiments of the compounds herein, the substituted or unsubstituted alkylenes are each substituted or unsubstituted C1-C 20 Alkylenes, substituted or unsubstituted heteroalkylenes are substituted or unsubstituted 2-20 member heteroalkylenes, substituted or unsubstituted cycloalkylenes are substituted or unsubstituted C3-C8 cycloalkylenes, substituted or unsubstituted heterocycloalkylenes are substituted or unsubstituted 3-8 member heterocycloalkylenes, substituted or unsubstituted arylenes are substituted or unsubstituted C6-C 10 An arrine and / or a substituted or unsubstituted heteroarrine is a substituted or unsubstituted 5- to 10-membered heteroarrine, respectively.

[0344] In some specific embodiments, each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2- to 8-membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C7 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3- to 7-membered heterocycloalkyl, and each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C 10Each aryl and / or substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5- to 9-membered heteroaryl. In some specific embodiments, each substituted or unsubstituted alkylene is a substituted or unsubstituted C1-C8 alkylene, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2- to 8-membered heteroalkylene, each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C3-C7 cycloalkylene, each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3- to 7-membered heterocycloalkylene, and each substituted or unsubstituted arylene is a substituted or unsubstituted C6-C 10 The arylenes and / or substituted or unsubstituted heteroarylenes are each substituted or unsubstituted 5- to 9-membered heteroarylenes. In some specific embodiments, the compounds are the chemical species described in the following Examples section, figures, or tables.

[0345] Certain compounds of the present invention have an asymmetric carbon atom (optical or chiral center) or a double bond, and can be defined in terms of absolute stereochemistry as (R)- or (S)- or (D)- or (L)- for amino acids. Enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisometric forms, and individual isomers are included within the scope of the present invention. The compounds of the present invention do not include those known in the art to be too unstable to be synthesized and / or separated. The present invention is intended to include compounds in racemic and optically pure forms. Optically active (R)- and (S)- or (D)- and (L)- isomers can be prepared using chiral synthons or chiral reagents, or separated using conventional techniques. Where a compound described herein contains an olefin bond or another center of geometric asymmetricity, and unless otherwise specified, the compound is intended to include both E and Z geometric isomers.

[0346] As used herein, the term "isomer" refers to compounds having the same number and type of atoms, and therefore the same molecular weight, but differing in terms of the structural arrangement or composition of the atoms.

[0347] As used herein, the term "tautomer" refers to one of two or more structural isomers that exist in equilibrium and are readily convertible from one isomeric form to another.

[0348] It will be obvious to those skilled in the art that certain compounds of the present invention exist in tautomeristic forms, and that all such tautomeristic forms of the above compounds fall within the scope of the present invention.

[0349] Unless otherwise specified, the structures shown herein also mean all stereochemical forms of the structure, i.e., the R and S configurations of each chiral center. Therefore, single stereoisomers, as well as mixtures of enantiomers and astereomers of the compound, are within the scope of the present invention.

[0350] Unless otherwise specified, the structures described herein also mean that the compounds differ only in the presence of one or more isotopically enriched atoms, for example, the substitution of hydrogen with deuterium or tritium, or 13 C or 14 Apart from the substitution of carbon by carbon-enriched carbon, compounds having the structure of the present invention are within the scope of the present invention.

[0351] The compounds of the present invention may also contain isotopes of atoms in an unnatural ratio in one or more of the atoms constituting such compounds. For example, the compound may contain tritium ( 3 H), iodine 125 ( 125 I), or carbon 14 ( 14 They can be radiolabeled with radioactive isotopes such as C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are included within the scope of the present invention.

[0352] Throughout this application, it should be noted that options are described in Markush groups, such as each amino acid position containing more than one possible amino acid. It is particularly intended that each member of a Markush group be considered separately, thereby including other embodiments, and that a Markush group is not to be read as a single unit.

[0353] The terms "analog" or "analogous" are used according to their simple, ordinary meaning in chemistry and biology, and refer to a chemical compound that is structurally similar to another compound (i.e., a so-called "reference" compound), but differs in composition, for example, by the substitution of one atom with an atom of a different element, or the presence of a particular functional group, or the substitution of one functional group with another functional group, or the absolute stereochemistry of one or more chiral centers of the reference compound. Thus, an analog is a compound that is similar to or equivalent to a reference compound in function and appearance, rather than in structure or origin.

[0354] The terms “one (a)” or “one (an)” as used herein mean one or more. In addition, the phrase “substituted with one (a[n])” as used herein means that a particular group may be substituted with one or more of any or all of the specified substituents. For example, a group such as an alkyl group or heteroaryl group may be substituted with an unsubstituted C1-C 20 If substituted with an alkyl group or an unsubstituted 2-20 member heteroalkyl group, the group consists of one or more unsubstituted C1-C 20 It may contain alkyl and / or one or more unsubstituted 2- to 20-membered heteroalkyl groups.

[0355] Furthermore, if a portion is substituted with an R substituent, the group may be referred to as "R-substituted." When a portion is R-substituted, it is substituted with at least one R substituent, each R substituent being arbitrarily different. If a particular R group is present in the description of a chemical genus (e.g., formula (I)), a Romanization symbol may be used to distinguish each occurrence of that particular R group. For example, multiple R 13If substituents are present, each R 13 The substituent is R 13A , R 13B , R 13C , R 13D They can be distinguished as such, and here, R 13A , R 13B , R 13C , R 13D Each of these is R 13 Within the scope of the definition, and optionally defined differently.

[0356] The description of the compounds of the present invention is limited by the principles of chemical bonding known to those skilled in the art. Therefore, where a group can be substituted by one or more substituents, such substitutions are selected to result in a compound that follows the principles of chemical bonding and is not inherently unstable, and / or is known to those skilled in the art as possibly unstable under ambient conditions such as aqueous, neutral, and various known physiological conditions. For example, heterocycloalkyl or heteroaryl groups are bonded to the remainder of the molecule via ring heteroatoms according to the principles of chemical bonding known to those skilled in the art, thereby avoiding an inherently unstable compound.

[0357] The term "pharmaceutically acceptable salt" means that salts of the active compound prepared with a relatively non-toxic acid or base, depending on the specific substituents found on the compounds described herein. If the compounds of the present invention have relatively acidic functionality, a base addition salt can be obtained by contacting the neutral form of such compound with a sufficient amount of the desired base in a suitable (neat) or suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include salts of sodium, potassium, calcium, ammonium, organic amino, or magnesium, or similar salts. If the compounds of the present invention have relatively basic functionality, an acid addition salt can be obtained by contacting the neutral form of such compound with a sufficient amount of the desired acid in a suitable (neat) or suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid, or phosphorous acid, as well as those derived from relatively non-toxic organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, corticic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, oxalic acid, and methanesulfonic acid. The present invention also includes salts of amino acids such as arginine, and salts of organic acids such as glucuronic acid or galacturonic acid (see, for example, Berge et al., “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain compounds of the present invention possess both basic and acidic functionalities, which allow the compound to be converted into either a base-addition salt or an acid-addition salt.

[0358] Therefore, the compounds of the present invention may exist as salts with pharmaceutically acceptable acids, etc. The present invention includes such salts. Non-limiting salts of such salts include hydrochlorides, hydrobroms, phosphates, sulfates, methanesulfons, nitrates, maleates, acetates, citrates, fumarates, propions, tartrates (e.g., (+)-tartrate, (-)-tartrate, or mixtures thereof including racemic mixtures), succinates, benzoates, and salts having amino acids such as glutamic acid, and quaternary ammonium salts (e.g., methyl iodide, ethyl iodide, etc.). Such salts can be prepared by methods known to those skilled in the art.

[0359] The neutral form of the compound is preferably regenerated by contacting the salt with a base or acid and isolating the parent compound by conventional means. The parent form of the compound may differ from the various salt forms in certain physical properties, such as solubility in polar solvents. In some embodiments, the compounds of the present invention possess both basic and acidic functionalities, allowing the compound to be converted into either a base or an acid addition salt. The neutral form of the compound can be regenerated by contacting the salt with a base or acid and isolating the parent compound by conventional means. The parent form of the compound may differ from the various salt forms in certain physical properties, such as solubility in polar solvents, but unless otherwise specified, the salts disclosed herein are equivalent to the parent form of the compound for the purposes of the present invention.

[0360] In addition to salt forms, the present invention provides compounds in prodrug form. The prodrugs of the compounds described herein are compounds that readily undergo chemical transformation under physiological conditions in order to provide the compounds of the present invention. The prodrugs of the compounds described herein can be converted in vivo after administration. In addition, the prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment, for example, by contact with a suitable enzyme or chemical reagent.

[0361] Certain compounds of the present invention can exist in both solvated and solvated forms, including hydrated forms. Typically, the solvated forms are equivalent to the solvated forms and are included within the scope of the present invention. Certain compounds of the present invention can exist in multiple crystalline or amorphous forms. Typically, all physical forms are intended to be suitable for the applications envisioned by the present invention and are intended to be within the scope of the present invention.

[0362] "To bring into contact" is used in its simple, ordinary sense, referring to a process that allows at least two different kinds (e.g., chemical compounds including biomolecules or cells) to be close enough to react, interact, or physically come into contact. However, it should be understood that the resulting reaction products may be produced directly from the reaction between additional reagents, or from intermediates from one or more additional reagents that may be produced in the reaction mixture.

[0363] The term "to bring into contact" includes enabling two species to react, interact, or make physical contact, where the two species may be the compounds described herein and proteins or enzymes. In some embodiments, contact includes enabling the compounds described herein to interact with proteins or enzymes involved in signaling pathways.

[0364] As defined herein, with respect to proteins, terms such as “activation,” “to activate,” and “to activate” refer to the conversion of a protein from an initial inactive or inactive state to a biologically active derivative. These terms refer to the activation of signaling or enzymatic activity, or the activation, sensitization, or upregulation of reduced protein levels in disease.

[0365] The terms "agonist," "activator," and "upregulator" refer to substances that can increase the expression or activity of a given gene or protein to a detectable level. An agonist can increase expression or activity by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more compared to a control without the agonist. In some cases, the expression or activity may be 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times, or more than the expression or activity without the agonist. In some embodiments, an agonist is a molecule that interacts with a target to cause or promote increased activation of the target. In some embodiments, an activator is a molecule that increases, activates, promotes, enhances, sensitizes, or upregulates, for example, a gene, protein, ligand, receptor, or cell.

[0366] As defined herein, with respect to interactions of protein inhibitors, terms such as “inhibit,” “inhibit,” and “to inhibit” mean that the inhibitor negatively affects (e.g., reduces) the activity or function of a protein compared to the activity or function of the protein in the absence of the inhibitor. In embodiments, inhibition means that the inhibitor negatively affects (e.g., reduces) the concentration or level of a protein compared to the concentration or level of the protein in the absence of the inhibitor. In embodiments, inhibition means a reduction in disease or symptoms of disease. In some embodiments, inhibition means a reduction in the activity of a particular protein target. Thus, inhibition includes, at least partially, partially, or completely, blocking a stimulus, reducing, preventing, or delaying activation, or inactivating, desensitizing, or downregulating signaling, or enzyme activity, or the amount of a protein. In some embodiments, inhibition means a reduction in the activity of a target protein resulting from a direct interaction (e.g., the inhibitor binding to the target protein). In some embodiments, inhibition means a reduction in the activity of a target protein resulting from an indirect interaction (e.g., the inhibitor binding to a protein that activates the target protein, thereby preventing the activation of the target protein).

[0367] The terms "inhibitor," "suppressor," "antagonist," or "downregulator" interchangeably refer to substances that can detectably reduce the expression or activity of a given gene or protein. Antagonists can reduce expression or activity by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more compared to a control without the antagonist. In some cases, expression or activity may be 1.5, 2, 3, 4, 5, 10 times, or less than that without the antagonist. Antagonists can prevent, reduce, inhibit, or neutralize the activity of an agonist, and antagonists can also prevent, inhibit, or reduce the constitutive activity of a target, such as a target receptor, even in the absence of an identified agonist. In some embodiments, an inhibitor is a molecule that reduces, blocks, prevents, delays, inactivates, desensitizes, or downregulates, for example, a gene, protein, ligand, receptor, or cell. Inhibitors can also be defined as molecules that reduce, block, or inactivate homeostatic activity. An "antagonist" is a molecule that counteracts the action of an agonist.

[0368] The terms “disease” or “illness” refer to a condition or health state of a patient or subject that can be treated with the compounds or methods provided herein. A disease may be cancer.

[0369] The terms “treating” or “treatment” refer to any sign of success in treating or achieving remission of an injury, disease, condition, or illness, including any objective or subjective parameters, such as remission; reducing symptoms or making the injury, condition, or illness more tolerable to the patient; slowing the rate of degeneration or decay; preventing the final point of degeneration from becoming so debilitating; or improving the patient’s physical or mental health. Treatment or remission of symptoms may be based on objective or subjective parameters, including the results of a physical examination, neuropsychiatric examination, and / or psychiatric evaluation. The term “treating” and its conjugations may include the prevention of injury, condition, disease, or illness. In some embodiments, treating is prevention. In some embodiments, treating does not include prevention.

[0370] "To treat" or "treatment," as used herein (and as is well understood in the art), also broadly includes any approach to obtain beneficial or desired outcomes in a condition of interest, including clinical outcomes. Beneficial or desired clinical outcomes include, but are not limited to, relief or remission of one or more symptoms or conditions, whether partial or whole, and whether detectable or undetectable; reduction of the scope of the disease; stabilization of the disease condition (i.e., no worsening); prevention of transmission or spread of the disease; delay or slowing of disease progression; improvement or reduction of the disease condition; and remission. In other words, "treatment," as used herein, includes any treatment, remission, or prevention of a disease. Treatment may prevent the onset of the disease; inhibit the spread of the disease; adequately alleviate the symptoms of the disease, completely or partially eliminate the underlying cause of the disease, shorten the duration of the disease, or a combination of these.

[0371] "To treat" and "treatment" as used in the specification may also include prophylactic treatment. A treatment method includes the step of administering a therapeutically effective amount of the compound described herein to a subject. The administration step may consist of a single dose or include a series of doses. The length of the treatment period depends on various factors, such as the severity of the disease, the patient's age, the concentration of the compound, the activity of the composition used in the treatment, or a combination thereof. It will be understood that the effective amount of the agent used for treatment or prevention may increase or decrease during a particular treatment or prophylactic regimen. Changes in dosage may result in and be evident by standard diagnostic assays known in the art. In some cases, long-term administration may be required. For example, the composition is administered to a subject in an amount and for a duration sufficient to treat the patient.

[0372] The term “prevent” refers to a reduction in the occurrence of symptoms in a patient. As shown above, prevention can be complete (no detectable symptoms) or partial, so that fewer symptoms are observed compared to the symptoms that might occur if no treatment is given. In some embodiments, prevention refers to slowing the progression of a disease, illness, or condition, or preventing its progression to a harmful, otherwise undesirable condition. [Examples]

[0373] These examples are provided for illustrative purposes only and do not limit the scope of the claims provided herein. Examples of chemical synthesis The compounds in Table 1 are prepared as described in the following examples.

[0374] [Table 1-1]

[0375] [Table 1-2]

[0376] [Table 1-3]

[0377] [Table 1-4]

[0378] Example 1: 2-Cyanoethyl(3-(dimethoxyphosphoryl)propyl)diisopropylphosphorumidite

[0379] [ka]

[0380] Step 1A: 3-((tert-butyldimethylsilyl)oxy)propan-1-ol

[0381] [ka]

[0382] To a solution of propane-1,3-diol (1.00 equivalent) in DMF, DIPEA (10.0 equivalent) and TBSCl (1.05 equivalent) were added. The mixture was stirred at 25°C for 16 hours. TLC (petroleum ether / ethyl acetate = 5 / 1, R f The reaction was shown to be completely consumed by propane-1,3-diol (=0.43). The reaction mixture was concentrated under reduced pressure, diluted with ethyl acetate (100 mL), and washed with water (200 mL x 3). The combined organic layer was washed with brine (200 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue, which was purified by column chromatography (SiO2, PMA petroleum ether / ethyl acetate = 100 / 1~1 / 1).

[0383] Step 1B: tert-butyl(3-iodopropoxy)dimethylsilane

[0384] [ka]

[0385] Imidazole (1.50 equivalents) and iodine (1.35 equivalents) were added to a solution of PPh3 (1.20 equivalents) in DCM. 3-((tert-butyldimethylsilyl)oxy)propan-1-ol (1.00 equivalent) in DCM was added dropwise to the mixture and stirred at 25°C for 16 hours. TLC (petroleum ether / ethyl acetate = 5 / 1, compound 3B:R f The result (=0.89) indicated that tert-butyl(3-iodopropoxy)dimethylsilane was completely consumed. The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by column chromatography (SiO2, PMA petroleum ether / ethyl acetate = 1 / 0~100 / 1).

[0386] Step 1C: Dimethyl(3-((tert-butyldimethylsilyl)oxy)propyl)phosphonate

[0387] [ka]

[0388] Sodium hydride (1.30 equivalents) was added to a solution of dimethylphosphonate (1.30 equivalents) in THF at 0°C. The mixture was stirred at 0°C for 0.5 hours, and then stirred at 70°C for 1.5 hours. The mixture was then cooled to 0°C, and tert-butyl(3-iodopropoxy)dimethylsilane (1.00 equivalent) in THF was added to the mixture and stirred at 25°C for 24 hours. TLC (petroleum ether:ethyl acetate = 1:1, product:R fA reaction (0.38) indicated that the starting material had been completely consumed. The reaction mixture was quenched by the addition of NH4Cl, then diluted with ethyl acetate and washed with NH4Cl. The combined organic layer was washed with brine, dried over Na2SO4, filtered, and purified by column chromatography (SiO2, PMA, petroleum ether:ethyl acetate = 1:0 to 1:1) by concentration under reduced pressure.

[0389] Step 1D: Dimethyl(3-hydroxypropyl)phosphonate

[0390] [ka]

[0391] To a solution of dimethyl(3-((tert-butyldimethylsilyl)oxy)propyl)phosphonate (1.00 equivalent) in MeOH, NH4F (3.00 equivalent) was added. The mixture was stirred at 65°C for 3 hours. TLC (petroleum ether / ethyl acetate = 0 / 1, product R f A reaction (=0.09) indicated that the starting material was completely consumed. The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by column chromatography (SiO2, KMnO4 petroleum ether / ethyl acetate = 10 / 1~0:1).

[0392] Step 1E: 2-Cyanoethyl(3-(dimethoxyphosphoryl)propyl)diisopropylphosphorumidite

[0393] [ka]

[0394] To a solution of dimethyl(3-hydroxypropyl)phosphonate (1.00 equivalent) in DCM, DIPEA (4.00 equivalent) and 3-((chloro(diisopropylamino)phosphaneyl))oxy)propanenitrile (1.20 equivalent) were added at 0°C. The mixture was stirred at 25°C for 1 hour. TLC (petroleum ether / ethyl acetate = 0 / 1, product R f The compound was completely consumed by a reaction (0.43). The reaction mixture was quenched by adding NaHCO3, diluted with DCM, and extracted with DCM (5 mL x 2). The combined organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue, which was purified by column chromatography (SiO2, petroleum ether / ethyl acetate / TEA / DCM = 10 / 1 / 0.5% / 25%~3 / 1 / 0.5% / 25%). 1 H NMR:400MHz,CD3CN:δ ppm 3.51-3.75(m,2H),3.55-3.70(m,10H),2.64(t,J=6.02 Hz,2H),1.76-1.83(m,4H),1.16-1.18(m,12H) 31 P NMR:162MHz,CD3CN:δ ppm 147.19(s,1P),34.03(s,1P).

[0395] Example 2: 2-Cyanoethyl (4-(dimethoxyphosphoryl)butyl)diisopropylphosphorumidite

[0396] [ka]

[0397] Step 2A: 4-((tert-butyldimethylsilyl)oxy)butan-1-ol

[0398] [ka]

[0399] A solution of butane-1,4-diol (10.0 g, 110 mmol, 9.80 mL, 1.00 equivalent) in DMF (70.0 mL) was mixed with DIPEA (143 g, 1.11 mol, 193 mL, 10.0 equivalent) and TBSCl (17.5 g, 116 mmol, 14.3 mL, 1.05 equivalent). The mixture was stirred at 25°C for 16 hours. TLC (petroleum ether / ethyl acetate = 5 / 1, R) f A reaction ratio of 0.43 indicated that the starting material had been completely consumed. The reaction mixture was concentrated under reduced pressure to obtain a residue, which was then diluted with ethyl acetate (100 mL) and washed with water (200 mL x 3). The combined organic layer was washed with brine (200 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, PMA petroleum ether / ethyl acetate = 100 / 1~1 / 1). 4-((tert-butyldimethylsilyl)oxy)butan-1-ol (112 g, 58.7 mmol, 52.9% yield) was obtained as a white solid. 1 H NMR:400MHz, CDCl3:δ ppm 3.51-3.65(m,4H),1.43-1.66(m,4H),0.80-0.86(m,9H),-0.06-0.03(m,6H).

[0400] Step 2B: tert-butyl(4-iodobutoxy)dimethylsilane

[0401] [ka]

[0402] To a solution of PPh3 (7.70 g, 29.3 mmol, 1.20 equivalents) in DCM (20.0 mL), imidazole (2.50 g, 36.7 mmol, 1.50 equivalents) and iodine (8.38 g, 33.0 mmol, 6.65 mL, 1.35 equivalents) were added. 4-((tert-butyldimethylsilyl)oxy)butan-1-ol (5.00 g, 24.4 mmol, 1.00 equivalent) in DCM (15.0 mL) was added dropwise to the mixture. The mixture was stirred at 25°C for 16 hours. TLC (petroleum ether / ethyl acetate = 5 / 1, R) f A reaction ratio of 0.89 indicated that the starting material had been completely consumed. The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by column chromatography (SiO2, PMA petroleum ether / ethyl acetate = 1 / 0~100 / 1). Tert-butyl(4-iodobutoxy)dimethylsilane (6.70 g, 21.32 mmol, 87.1% yield) was obtained as a colorless oil. 1 H NMR:400MHz,CDCl3:δ ppm 3.64(t,J=6.2 Hz,2H),3.23(t,J=7.0 Hz,2H),1.92(q,J=7.2 Hz,2H),1.58-1.68(m,2H),0.89-0.93(m,9H),0.03-0.08(m,6H).

[0403] Step 2C: Dimethyl(4-((tert-butyldimethylsilyl)oxy)butyl)phosphonate

[0404] [ka]

[0405] To a solution of dimethylphosphonate (1.46 g, 13.2 mmol, 1.21 mL, 1.30 equivalents) in THF (15.0 mL), NaH (529 mg, 13.2 mmol, 60.0% purity, 1.30 equivalents) was added at 0°C, and the mixture was stirred at 0°C for 0.5 hours and at 70°C for 1.5 hours. The reaction mixture was then cooled to 0°C, and tert-butyl(4-iodobutoxy)dimethylsilane (3.20 g, 10.2 mmol, 2.64 mL, 1.00 equivalent) in THF (5.00 mL) was added to the mixture, and the mixture was stirred at 25°C for 24 hours. TLC (petroleum ether:ethyl acetate = 1:1, product:R) was performed. f A reaction (0.38) indicated that the starting material had been completely consumed. The reaction mixture was quenched by the addition of NH4Cl (100 mL), then diluted with ethyl acetate (100 mL), and washed with NH4Cl (100 mL). The combined organic layer was washed with brine (100 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue, which was purified by column chromatography (SiO2, PMA, petroleum ether:ethyl acetate = 1:0 to 1:1). Dimethyl (4-((tert-butyldimethylsilyl)oxy)butyl)phosphonate (2.20 g, 7.42 mmol, 72.9% yield) was obtained as a colorless oil. 1 H NMR:400MHz,CDCl3:δ ppm 3.74(d,J=10.8 Hz,6H),3.62(t,J=6.0 Hz,2H),1.57-1.83(m,7H),0.89(s,9H),0.05(s,6H) 31 P NMR:162MHz CDCl3:δ ppm 34.93(s,1P).

[0406] Step 2D: Dimethyl(4-hydroxybutyl)phosphonate

[0407] [ka]

[0408] To a solution of dimethyl(4-((tert-butyldimethylsilyl)oxy)butyl)phosphonate (2.10 g, 7.08 mmol, 1.00 equivalent) in MeOH (21.0 mL), NH4F (787 mg, 21.2 mmol, 3.00 equivalent) was added. The mixture was stirred at 65°C for 3 hours. TLC (petroleum ether / ethyl acetate = 0 / 1, R f A reaction (0.09) indicated that the starting material had been completely consumed. The reaction mixture was concentrated under reduced pressure and purified by column chromatography (SiO2, KMnO4 petroleum ether / ethyl acetate = 10 / 1~0:1). Dimethyl(4-hydroxybutyl)phosphonate (1.3 g, crude) was obtained as a colorless oil. 1 H NMR:400MHz, CDCl3:δ ppm 3.74(d,J=10.8 Hz,6H),3.66(t,J=6.0 Hz,2H),1.98(s,1H),1.62-1.85(m,6H).

[0409] Step 2E: 2-Cyanoethyl (4-(dimethoxyphosphoryl)butyl)diisopropylphosphorumidite

[0410] [ka]

[0411] To a solution of dimethyl(4-hydroxybutyl)phosphonate (1.30 g, 7.14 mmol, 1.00 equivalent) in DCM (13.0 mL), DIPEA (3.69 g, 28.5 mmol, 4.97 mL, 4.00 equivalent) and 3-((chloro(diisopropylamino)phosphanyl)oxy)propanenitrile (2.03 g, 8.56 mmol, 1.20 equivalent) were added at 0°C. The mixture was stirred at 25°C for 1 hour. TLC (petroleum ether / ethyl acetate = 0 / 1, product: R) fThe compound was completely consumed by a reaction (0.43). The reaction mixture was quenched by the addition of NaHCO3 (20 mL), then diluted with DCM (10 mL), and extracted with DCM (5 mL x 2). The combined organic layer was washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate / TEA / DCM = 10 / 1 / 0.5% / 25%~3 / 1 / 0.5% / 25%). 2-Cyanoethyl (4-(dimethoxyphosphoryl)butyl)diisopropylphosphorumidite (0.173 g, 452 umol, 6.34% yield) was obtained as a colorless oil. 1 H NMR:400MHz,CD3CN:δ ppm 3.71-3.84(m,2H),3.56-3.70(m,10H),2.64(t,J=6.02 Hz,2H),1.58-1.79(m,7H),1.13-1.20(m,14H). 31 P NMR:162MHz,CD3CN:δ ppm 147.03(s,1P),34.12(s,1P).

[0412] Example 3: (Dimethyl(E)-(5-((2-cyanoethoxy)(diisopropylamino)phosphanyl)pento-1-en-1-yl)phosphonate)

[0413] [ka]

[0414] Step 3A: 3-((tert-butyldiphenylsilyl)oxy)propan-1-ol

[0415] [ka]

[0416] To a solution of propane-1,3-diol (9.0 g, 100 mmol, 1 equivalent) in DMF (90.0 mL), DIPEA (143 g, 1.11 mol, 193 mL, 10.0 equivalents) and TBDPSCl (30.4 g, 110 mmol) were added. The mixture was stirred at 25°C for 16 hours. TLC (petroleum ether / ethyl acetate = 5 / 1, product R) was performed. f A reaction ratio of 0.55 indicated that the starting material had been completely consumed. The reaction mixture was concentrated under reduced pressure, diluted with ethyl acetate (100 mL), and washed with H₂O (200 mL x 3). The combined organic layer was washed with brine (200 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO₂, PMA petroleum ether / ethyl acetate = 100 / 1~1 / 1). 3-((tert-butyldiphenylsilyl)oxy)propan-1-ol (16.4 g, 50% yield) was obtained as a white solid.

[0417] Step 3B: 3-((tert-butyldiphenylsilyl)oxy)propanal

[0418] [ka]

[0419] A solution of 3-((tert-butyldiphenylsilyl)oxy)propan-1-ol (16.4 g, 50 mmol, 1 equivalent) in dichloromethane (200 mL) was cooled in an ice bath under an inert atmosphere. PCC (11.8 g, 55 mmol) was added in five portions, and the reaction mixture was heated to RT for 1 hour, followed by stirring at RT for a further 2 hours. The reaction mixture was filtered, and the filtrate was washed with ether. The combined organic layers were washed with brine (200 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue, which was used in the next reaction without further purification.

[0420] Step 3C: Dimethyl-(4-((tert-butyldiphenylsilyl)oxy)buto-1-en-1-yl)phosphonate

[0421] [ka]

[0422] A solution of tetramethylmethylenediphosphonate in dichloromethane (200 mL) was cooled in an ice bath under an inert atmosphere, and NaH was added in several portions under an argon atmosphere. 3-((tert-butyldiphenylsilyl)oxy)propanal was introduced via syringe, and the reaction mixture was slowly brought to RT over 2 hours, and stirred at RT for a further 3 hours. The reaction mixture was quenched with NH4Cl solution, and then washed with NH4Cl solution (3 × 50 ml). The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a sticky residue, which was purified on silica gel chromatography (petroleum ether / ethyl acetate = 100 / 1~1 / 1). Dimethyl(E)-(4-((tert-butyldiphenylsilyl)oxy)buto-1-en-1-yl)phosphonate (4.1 g, 22% yield from compound 2) was obtained as a white solid.

[0423] Process 3D: Dimethyl-(4-hydroxybuto-1-en-1-yl)phosphonate

[0424] [ka]

[0425] To a solution of dimethyl(E)-(4-((tert-butyldiphenylsilyl)oxy)buto-1-en-1-yl)phosphonate (4 g) in methanol (20.0 mL), NH4F (644 mg, 2 equivalents) was added. The mixture was stirred at 70°C for 2 hours. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by column chromatography (SiO2, KMnO4 petroleum ether / ethyl acetate = 10 / 1~0:1). Dimethyl-(4-hydroxybuto-1-en-1-yl)phosphonate (1.06 g) was obtained as a colorless oil.

[0426] Step 3E: 2-Cyanoethyl (4-(dimethoxyphosphoryl)buto-3-en-1-yl)diisopropylphosphorumidite

[0427] [ka]

[0428] To a solution of dimethyl-(4-hydroxybuto-1-en-1-yl)phosphonate (0.7 g, 3.8 mmol, 1 equivalent) in DCM (10 mL), diisopropylammonium tetrazolide (0.84 g) and 2-cyanoethyl N,N,N,N-tetraisopropylphosphodiamidite (1.9 g) were added. The mixture was stirred at 0°C for 1 hour, quenched with NaHCO3 (20 mL), diluted with DCM (10 mL), and then extracted with DCM (10 mL x 2). The combined organic layer was washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue, which was purified by column chromatography (SiO2, petroleum ether / ethyl acetate / TEA / DCM = 10 / 1 / 0.5% / 25%~3 / 1 / 0.5% / 25%). 2-Cyanoethyl(4-(dimethoxyphosphoryl)buto-3-en-1-yl)diisopropylphosphorumidite (0.3) was obtained as a colorless oil. 1 H NMR:400MHz,CD3CN:δ ppm 6.7-7.9(m,1H),5.75(dd,1H),4.1-4.25(m,2H),3.8-3.95(m,4H),3.7 0-3.85(m,6H),3.50-3.70(m,4H),2.55-2.85(m,6H),1.20-1.50(m,6H) 31 P NMR:162MHz, CDCl3:δ ppm 147.81(s,1P),31.39(s,1P).

[0429] Example 4: 2-Cyanoethyl (5-(dimethoxyphosphoryl)pentyl)diisopropylphosphorumidite

[0430] [ka]

[0431] Step 4A: 5-((tert-butyldimethylsilyl)oxy)pentan-1-ol

[0432] [ka]

[0433] To a solution of pentane-1,5-diol (10.0 g, 96.0 mmol, 10.1 mL, 1.00 equivalent) in DMF (100 mL), DIPEA (124 g, 960 mmol, 167 mL, 10.0 equivalent) and TBSCl (15.2 g, 100 mmol, 12.3 mL, 1.05 equivalent) were added. The mixture was stirred at 25°C for 16 hours. TLC (petroleum ether / ethyl acetate = 5 / 1, product: R) f A reaction (0.43) indicated that the starting material had been completely consumed. The reaction mixture was concentrated under reduced pressure, diluted with ethyl acetate (200 mL), and washed with water (200 mL x 3). The combined organic layer was washed with brine (400 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue, which was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 100 / 1~1 / 1). 5-((tert-butyldimethylsilyl)oxy)pentan-1-ol (11.5 g, 52.6 mmol, 54.8% yield) was obtained as a colorless oil. 1 H NMR:400MHz, CDCl3:δ ppm 3.55-3.61(m,4H),1.88(s,1H),1.53-1.56(m,4H),1.46-1.51(m,2H),0.82-0.85(m,9H),0.00-0.01(m,6H).

[0434] Step 4B: tert-butyl((5-iodopentyl)oxy)dimethylsilane

[0435] [ka]

[0436] To a solution of Ph3P (7.64 g, 29.1 mmol, 1.20 equivalents) in DCM (20.0 mL), imidazole (2.48 g, 36.4 mmol, 1.50 equivalents) and iodine (8.31 g, 32.7 mmol, 6.60 mL, 1.35 equivalents) were added at 0°C. 5-((tert-butyldimethylsilyl)oxy)pentan-1-ol (5.30 g, 24.2 mmol, 1 equivalent) in DCM (15.0 mL) was added dropwise to the mixture. The mixture was stirred at 25°C for 16 hours. TLC (petroleum ether / ethyl acetate = 10 / 1, product: R f A reaction (0.89) indicated that the starting material had been completely consumed. The reaction mixture was concentrated under reduced pressure and purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0). Tert-butyl((5-iodopentyl)oxy)dimethylsilane (7.00 g, 21.3 mmol, 87.8% yield) was obtained as a colorless oil. 1 H NMR:400MHz,CDCl3:δ ppm 3.62(t,J=6.2 Hz,2H),3.20(t,J=7.0 Hz,2H),1.85(q,J=7.2 Hz,2H),1.51-1.58(m,2H),1.41-1.49(m,2H),0.88-0.92(m,9H),0.06(d,J=0.8 Hz,6H).

[0437] Step 4C: Dimethyl(5-((tert-butyldimethylsilyl)oxy)pentyl)phosphonate

[0438] [ka]

[0439] Under argon conditions, NaH (792 mg, 19.8 mmol, 60% dispersion, 1.30 equivalents), THF (30.0 mL), and dimethylphosphonate (2.18 g, 19.8 mmol, 1.82 mL, 1.30 equivalents) were added to a three-...

Claims

1. An oligonucleotide comprising a compound of formula (IIa), wherein the compound of formula (IIa) is located at the 5'-terminus of the oligonucleotide. 【Chemistry 1】 During the ceremony, R １ Each of these independently represents a substitution or non-substitution of C. １ -C ６ Alkyl, substituted, or unsubstituted C １ -C ６ Fluoroalkyl, or substituted or unsubstituted C １ -C ６ It is heteroalkyl, L １ , L ２ , and L ３ is L １ -L ２ -L ３ such that (a)-(CH ２ ) ３ -, (b)-(CH ２ ) ４ -, (c)-(CH ２ ) ５ -, or (d)-(C ２ H ２ )(CH ２ ) ２ is selected to be equal to-, J is an oligonucleotide, which is an internucleotide binding group that binds to an adjacent nucleotide of an oligonucleotide.

2. The oligonucleotide according to claim 1, wherein the oligonucleotide is an RNA oligonucleotide.

3. The oligonucleotide according to claim 1 or 2, further comprising at least one 2'-modified nucleotide.

4. The oligonucleotide according to claim 3, wherein the at least one 2'-modified nucleotide is selected from 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-deoxy, 2-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-O-N-methylacetamide (2'-O-NMA) modified nucleotides, locked nucleic acid (LNA), or ethylene nucleic acid (ENA).

5. The oligonucleotide according to any one of claims 1 to 4, further comprising at least one modified internucleotide bond.

6. The oligonucleotide according to claim 5, wherein the at least one modified internucleotide bond is selected from a phosphorothioate bond, a phosphorodithioate bond, a methylphosphonate bond, a phosphotriester bond, or an amide bond.

7. The oligonucleotide according to any one of claims 1 to 6, wherein the compound of formula (IIa) can increase the stability of the oligonucleotide.

8. The oligonucleotide according to any one of claims 1 to 7, wherein the oligonucleotide is conjugated at the binding site.

9. The oligonucleotide according to claim 8, wherein the binding portion is conjugated to the 3'-terminus of the oligonucleotide.

10. The oligonucleotide according to claim 8 or 9, wherein the binding portion comprises an antibody or a binding fragment thereof.

11. The oligonucleotide according to claim 10, wherein the antibody or its binding fragment comprises a humanized antibody or its binding fragment, a chimeric antibody or its binding fragment, a monoclonal antibody or its binding fragment, a monovalent Fab', a bivalent Fab2, a single-strand variable fragment (scFv), a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), or a camelid antibody, or its binding fragment.

12. The oligonucleotide according to any one of claims 1 to 11, comprising 10 to 30 nucleotides.

13. The oligonucleotide according to any one of claims 1 to 12, wherein the oligonucleotide hybridizes to at least eight adjacent bases of the target gene sequence.

14. The oligonucleotide according to any one of claims 1 to 13, wherein the oligonucleotide mediates RNA interference.

15. The oligonucleotide according to any one of claims 1 to 14, wherein the oligonucleotide further comprises a polymer.

16. The oligonucleotide according to claim 15, wherein the polymer comprises polyethylene glycol.

17. Use of the oligonucleotide according to claim 1 in the manufacture of a drug for treating a disease or condition characterized by a deficiency of protein expression.

18. Use of the oligonucleotide according to claim 1 in the manufacture of a drug for treating a disease or condition characterized by protein overexpression.

19. The use according to claim 17 or 18, wherein the disease or illness is cancer, neuromuscular disease, muscular dystrophy, muscle atrophy, muscle wasting, genetic disease, hereditary disorder, or cardiovascular disease.

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