Compositions and methods for delivering a macromolecule to a cell 2

Abstract

Compounds and methods are provided for facilitating delivery of an oligonucleotide, for example, into a nucleus and / or cytosol of a cell.

Description

[0001] COMPOSITIONS AND METHODS FOR DELIVERING A MACROMOLECULE TO A CELL 2

[0002] FIELD OF THE DISCLOSURE

[0003] The present disclosure relates to compositions and methods for delivering a macromolecule, e.g., an oligonucleotide, a polypeptide, or a combination thereof, to a cell, for instance, to compositions and methods for delivering a macromolecule, e.g., an oligonucleotide, a polypeptide, or a combination thereof, to the cytosol and / or nucleus of a cell.

[0004] BACKGROUND OF THE DISCLOSURE

[0005] Antisense oligonucleotides (ASOs) are short, exogenous, single stranded DNA or RNA with a sequence that is complementary to a nucleotide sequence of a target nucleic acid. Classic single stranded antisense oligonucleotides (ASOs) can act in the nucleus to cleave mRNAs via an RNase H dependent mechanism. (Juliano, RL (2016) Nuc. Acids. Res. 44(14):6518-6548). Many therapeutic ASOs are ‘gapmers’, which have a central DNA region that supports RNase H activity that is flanked by chemically modified ends to increase affinity and reduce susceptibility to nucleases. (Bennett et al. (2010) Annu. Rev. Pharmacol. Toxicol. 50:259-293).

[0006] Splice switching oligonucleotides (SSOs) are another form of antisense oligonucleotides that hybridize with pre-mRNA to disrupt transcript splicing by blocking RNA-RNA base pairing or protein-RNA binding interactions that occur between components of the splicing machinery (Havens and Hastings (2016) Nucleic Acids Res. 44(14);6549-63). SSOs can be designed to induce intron and exon inclusion or exclusion, ultimately restoring or inhibiting protein function or re-directing splicing to produce alternative protein isoforms. Additionally, SSOs can be used to mask aberrant splice sites, thereby restoring normal alternative splicing to produce functional proteins. Theoretically, any pre-mRNA sequence could be targeted with SSOs, but to date, only four SSOs have been FDA approved. (See e.g., Neil and Bisaccia (2019) J. Pediatr. Pharmacol. Ther. 24(3): 194-203; Kim et al. (2019) N. Engl. J. Med. 381:1644-1652). It is estimated that up to 70% of human genes undergo alternative splicing, and 50% of human genetic diseases arise from mutations that affect splicing. (Bauman et al., (2009) Oligonucleotides. 19(1): 1- 13) Several diseases, including Spinal Muscular Atrophy (SMA) and Duchenne Muscular Dystrophy (DMD), currently lack any efficacious treatments which target the underlying genetic defect. (Bestas et al. (2014) Nucleic Acid Ther. 24(1): 13-24). SSOs are a promising therapeutic approach to target the underlying causes of these diseases.

[0007] RNA interference (RNAi) is an endogenous regulatory pathway for control of gene expression in which short (approx. 15 - 22 bp) double-stranded RNA fragments, known as small interfering RNAs (siRNAs), are loaded into an RNA-induced silencing complex (RISC) to cleave target mRNA in a sequence-dependent manner. (Gavriolv and Saltzman (2012) Yale J. Biol. Med. 85(2): 187-200).

[0008] Although nucleic acid-based therapeutics are gaining attention as a promising approach for treatment of a variety of diseases and disorders, many have failed to meet therapeutic end points, often due to challenges with effective methods for in vivo delivery. (Juliano, RL (2016) Nuc. Acids. Res. 44(14):6518-6548). One hindrance to the widespread use of oligonucleotide therapeutics is the inability of the oligonucleotide to escape endosomal compartments and reach the cytosol or nucleus in sufficient concentrations. (Juliano et al. (2008) Nucleic Acids Res. 36(12):4158-4171).

[0009] Many recent developments focus on increasing cellular uptake and endosomal release of therapeutic oligonucleotides, for example, via chemical conjugation to ligands or encapsulation in synthetic nanoparticles. (Barton and Medzhitov (2002) Proc. Natl. Acad. Sci. U.S.A. 99(23): 14943-5; Johannes and Lucchino (2018) Nucleic Acid Ther. 28(3): 178- 193). Historically, use of nano-carriers was believed to be required to facilitate cellular uptake of polyanionic macromolecules such as ASOs. However, it has been discovered that single-stranded oligonucleotides are spontaneously endocytosed by cells, in the absence of carriers, by a process referred to as gymnosis. (Stein et al. (2009) Nucleic Acids Res. 38(1): 10.1093 / nar / gkp841).

[0010] Endosomolytic small molecule compounds (SMCs) are compounds that facilitate the release of gymnotically delivered oligonucleotides that might otherwise accumulate in endosomes or lysosomes. Some endosomolytic SMCs induce endosomal membrane destabilization by buffering the lumen of endosomes as the luminal pH decreases with endosomal maturation. The increase in luminal pH occurs quickly and can be reversible with proper dosing. (Maxfield, F.R. (1982) J. Cell Biol. 95(2):676-681). This buffering leads to an increase of luminal osmotic pressure, engorging the endosome and triggering membrane rupture, ultimately allowing the endosomal cargo to leak into the cytosol.

[0011] Chloroquine and derivatives thereof have been widely used to enhance activity of oligonucleotide-containing nanoparticles by promoting endosomal release. Although these compounds display great potency in vitro, high micromolar concentration ranges are typically required and there is a narrow window between effective and toxic concentrations. (Yang et al. (2015) Nucleic Acids Res. 43(4): 1987-96; Wang et al. (2017) ACS Chem. Biol.

[0012] 12(8): 1999-2007). For example, chloroquine induces leakage between 40-100 μM (Ldnn et al. (2016) Sci. Rep. 6:32301; Heath et al. (2019) Nanomedicine. 14(21):2799-2814).

[0013] SUMMARY OF THE DISCLOSURE

[0014] Compositions and methods for delivering a macromolecule, e.g., an oligonucleotide, a polypeptide, or a combination thereof, to a cell are provided. In one aspect, a macromolecule conjugated to one or more endosomal escape enhancer (EEE) compounds of Formula I is provided.

[0015] Formula I: one of Zi and Z2 is N, and the other is C; R1 is hydro, halo, C1-C4 alkyl, -OR4, -C(=O)NR5R5, -CO2R6, or cyano;

[0016] X is hydro, C1-C4 alkyl, or -OR2;

[0017] R2 is hydro or C1-C4 alkyl;

[0018] R3 is hydro, halo, C1-C4 alkyl, -(CH2)yOH, -OR4, -C(=O)NR5R5, -CO2R6, or cyano;

[0019] R4 is C1-C4 alkyl; each R5 is independently hydro or C1-C4 alkyl;

[0020] Re is hydro or C1-C4 alkyl;

[0021] R7, R8, R9, Rio, R11 are each independently CHR12, CR12R17 or NR13;

[0022] R12 is hydro, C1-C4 alkyl, -OR14, or -CO2R15;

[0023] R13 is hydro, C1-C4 alkyl, -(CH2)yOH, -OR14, -CO2R15, or -C(=O)RI6;

[0024] R14 is hydro or C1-C4 alkyl;

[0025] R15 is hydro or C1-C4 alkyl; R16 is hydro or C1-C4 alkyl;

[0026] R17 is hydro or C1-C4 alkyl; y is 0, 1, 2, or 3; and wherein one or more of the alkyl are optionally substituted with one or more halo, or a pharmaceutically acceptable salt thereof.

[0027] In one aspect, a macromolecule conjugated to one or more endosomal escape enhancer (EEE) compounds of Formula I is provided.

[0028] Formula I: one of Zi and Z2 is N, and the other is C; R1 is hydro, halo, C1-C4 alkyl, -OR4, -C(=O)NRsR8, -CO2R6, or cyano;

[0029] X is hydro, C1-C4 alkyl, or -OR2;

[0030] R2 is hydro or C1-C4 alkyl;

[0031] R3 is hydro, halo, C1-C4 alkyl, -(CthjyOH, -OR4, -C(=O)NR5R5, -CO2R6, or cyano;

[0032] R4 is C1-C4 alkyl; each R5 is independently hydro or C1-C4 alkyl;

[0033] Re is hydro or C1-C4 alkyl;

[0034] R7, R8, R9, Rio, R11 are each independently CHR12, CR12R17 or NR13;

[0035] R12 is hydro, C1-C4 alkyl, -OR14, or -CO2R15;

[0036] R13 is hydro, C1-C4 alkyl, -(CH2)yOH, -OR14, -CO2R15, or -C(=O)RI6;

[0037] R14 is hydro or C1-C4 alkyl;

[0038] R15 is hydro or C1-C4 alkyl;

[0039] R16 is hydro or C1-C4 alkyl;

[0040] R17 is hydro or C1-C4 alkyl; y is 0, 1, 2, or 3; and wherein one or more of the alkyl are optionally substituted with one or more halo, and wherein the compound of Formula I is conjugated via R7, R8, R9, Rio or Rn, or a pharmaceutically acceptable salt thereof.

[0041] In another aspect, a macromolecule conjugated to one or more endosomal escape enhancer (EEE) compounds of Formula la is provided.

[0042] Formula la: wherein one of Zi and Z2 is N, and the other is C; R1 is hydro, halo, C1-C4 alkyl, -OR4, -C(=O)NRsR8, -CO2R6, or cyano;

[0043] X is hydro, C1-C4 alkyl, or -OR2;

[0044] R2 is hydro or C1-C4 alkyl;

[0045] R3 is hydro, halo, C1-C4 alkyl, -(CH2)yOH, -OR4, -C(=O)NR5R5, -CO2R6, or cyano;

[0046] R4 is C1-C4 alkyl; each R5 is independently hydro or C1-C4 alkyl, e.g., methyl or ethyl;

[0047] Re is hydro or C1-C4 alkyl, e.g., methyl or ethyl;

[0048] R14 is hydro or C1-C4 alkyl;

[0049] R15 is hydro or C1-C4 alkyl;

[0050] R16 is hydro or C1-C4 alkyl;

[0051] - denotes the conjugation point; and wherein one or more of the alkyl are optionally substituted with one or more halo, or a pharmaceutically acceptable salt thereof. In another aspect, a macromolecule conjugated to one or more endosomal escape enhancer (EEE) compounds of Formula lb is provided.

[0052] Formula lb: wherein one of Zi and Z2 is N, and the other is C; R1 is hydro, halo, C1-C4 alkyl, -OR4, -C(=O)NR5R5, -CO2R6, or cyano;

[0053] X is hydro, C1-C4 alkyl, or -OR2;

[0054] R2 is hydro or C1-C4 alkyl;

[0055] R3 is hydro, halo, C1-C4 alkyl, -(CthjyOH, -OR4, -C(=O)NR5R5, -CO2R6, or cyano;

[0056] R4 is C1-C4 alkyl; each R5 is independently hydro or C1-C4 alkyl, e.g., methyl or ethyl;

[0057] Re is hydro or C1-C4 alkyl, e.g., methyl or ethyl;

[0058] R14 is hydro or C1-C4 alkyl;

[0059] R15 is hydro or C1-C4 alkyl;

[0060] R16 is hydro or C1-C4 alkyl;

[0061] - denotes the conjugation point; and wherein one or more of the alkyl are optionally substituted with one or more halo, or a pharmaceutically acceptable salt thereof.

[0062] In another aspect, a macromolecule conjugated to one or more endosomal escape enhancer (EEE) compounds of Formula Ic, Formula Id, Formula le or Formula If is provided. Formula Ic

[0063] Formula Id:

[0064] Formula le: wherein Ri, X, and R3 are as defined above and - denotes the conjugation point.

[0065] In another aspect, a macromolecule conjugated to one or more endosomal escape enhancer (EEE) compounds of Formula I, la, lb, Ic, Id, le or If is provided, wherein R1 is hydro, halo, C1-C4 alkyl, -OR4, -C(=O)NR5R5, -CO2R6, or cyano; X is hydro, C1-C4 alkyl, or -OR2,

[0066] R2 is hydro or C1-C4 alkyl;

[0067] R3 is hydro, halo, C1-C4 alkyl, -(CH2)yOH, -OR4, -C(=O)NR5R5, -CO2R6, or cyano;

[0068] R4 is C1-C4 alkyl;

[0069] Rs is hydro, methyl or ethyl, Re is hydro, methyl or ethyl, R14 is hydro or C1-C4 alkyl;

[0070] R15 is hydro or C1-C4 alkyl;

[0071] R16 is hydro or C1-C4 alkyl; wherein one or more of the alkyl are optionally substituted with one or more halo, or a pharmaceutically acceptable salt thereof.

[0072] In some aspects, R1 is cyano, halo, or C1-C4 alkyl optionally substituted with one or more chloro or fluoro. In one aspect, R1 is cyano, methyl, ethyl, trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, dichloromethyl, chloromethyl, fluoro, chloro, or bromo. In one aspect, R1 is cyano, bromo, chloro, fluoro, or trifluoromethyl. In one aspect, R1 is fluoro.

[0073] In one aspect, X is -OR2, and R2 is methyl, ethyl, or isopropyl. In one aspect, X is hydro.

[0074] In one aspect, R3 is hydro; cyano; C1-C4 alkyl optionally substituted with one or more chloro, fluoro, or hydroxy; or -OR4, wherein R4 is C1-C4 alkyl.

[0075] In one aspect, R7, R8, Rio, and R11 are each independently CHR12 or CR12R17, and R9 is NR13. In one aspect, each R12 is hydro. In one aspect, R13 is hydro, C1-C4 alkyl, -C(=O)R16, or -(CH2)yOH, wherein y is 1, 2 or 3. In one aspect, R13 is -(CH2)3OH. In one aspect, R13 is methyl.

[0076] In one aspect, R1 is halo, X is -OR2, R2 is ethyl or methyl, R3 is cyano, and R13 is hydro, C1-C4 alkyl, -C(=O)CH3, or -(CH2)3OH. In one aspect, R1 is haloalkyl, X is -OR2, R2 is ethyl or methyl, R3 is cyano, and R13 is hydro or methyl. In one aspect, R1 is halo, X is - OR2, R2 is ethyl or methyl, R3 is methyl or methoxy, and R13 is hydro. In one aspect, R1 is halo, X is -OR2, R2 is C1-C4 alkyl, R3 is hydro, and R13 is hydro. In one aspect, R1 is haloalkyl, X is -OR2, R2 is ethyl or methyl, R3 is hydro, and R13 is hydro or methyl.

[0077] In one aspect, R1 is halo, X is hydro, R3 is cyano, and R13 is hydro. In one aspect, Ri is cyano, X is -OR2, R2 is ethyl or methyl, R3 is haloalkyl, and R13 is hydro. In one aspect, Ri is ethyl or methyl, X is -OR2, R2 is ethyl or methyl, R3 is hydro, and R13 is hydro. In one aspect, R1 is halo, X is -OR2, R2 is ethyl or methyl, R3 is -(CH2)yOH a,nd R13 is hydro, wherein y is 0, 1, 2, or 3.

[0078] In some embodiments, disclosed are the compounds of Table 1, or a pharmaceutically acceptable salt thereof. Table 1. Exemplified endosomal escape enhancer (EEE) Compounds or a pharmaceutically acceptable salt thereof.

[0079] In one aspect, a composition is provided that includes a peptide conjugated to one or more compounds of any one of Formulae I, la, lb, Ic, Id, le, If or of Table 1, a pharmaceutically acceptable salt of one or more compounds of any one of Formulae I, la, lb, Ic, Id, le, If or of Table 1, or a combination thereof. In one aspect, a pharmaceutical composition is provided that includes an oligonucleotide conjugated to one or more compounds of any one of Formulae I, la, lb, Ic, Id, le, If or of Table 1, a pharmaceutically acceptable salt of one or more compounds of any one of Formulae I, la, lb, Ic, Id, le, If or of Table 1, or a combination thereof; and a pharmaceutically acceptable diluent or carrier.

[0080] In one aspect, a composition is provided that includes an oligonucleotide conjugated to one or more compounds of any one of Formulae I, la, lb, Ic, Id, le, If or of Table 1, a pharmaceutically acceptable salt of one or more compounds of any one of Formulae I, la, lb, Ic, Id, le, If or of Table 1, or a combination thereof. In one aspect, a pharmaceutical composition is provided that includes an oligonucleotide conjugated to one or more compounds of any one of Formulae I, la, lb, Ic, Id, le, If or of Table 1, a pharmaceutically acceptable salt of one or more compounds of any one of Formulae I, la, lb, Ic, Id, le, If or of Table 1, or a combination thereof; and a pharmaceutically acceptable diluent or carrier.

[0081] In one aspect, the oligonucleotide is single stranded. In one aspect, the oligonucleotide is double stranded. In one aspect, the oligonucleotide includes DNA. In one aspect, the oligonucleotide includes RNA. In one aspect, the oligonucleotide includes from about 8 to about 30 nucleotides. In one aspect, the oligonucleotide is an antisense oligonucleotide (ASO), a splice switching oligonucleotide (SSO), interfering RNA (RNAi), small interfering RNA (siRNA), micro RNA (miRNA), an antagomir, a decoy oligonucleotide, or a combination thereof.

[0082] In one aspect, the oligonucleotide includes one or more modified nucleotides. In one aspect, the one or more modified nucleotides comprise: phosphodiester (PO); phosphorothioate (PS); 2’0-methyl (2’OMe); 2’0-methoxyethyl (MOE); peptide nucleic acid (PNA); phosphoroamidate morpholino (PMO); locked nucleic acid (LNA); 2’-deoxy-2’- fluoro (2’-F); any other 2’ modified oligonucleotide; or a combination thereof.

[0083] In one aspect, the compound of any one of Formulae I, la, lb, Ic, Id, le, If or of Table 1, a pharmaceutically acceptable salt of one or more compounds of any one of Formulae I, la, lb, Ic, Id, le, If or of Table 1, or a combination thereof is attached to the macromolecule via a linker.

[0084] In one aspect, the compound of any one of Formulae I, la, lb, Ic, Id, le, If or of Table 1, a pharmaceutically acceptable salt of one or more compounds of any one of Formulae I, la, lb, Ic, Id, le, If or of Table 1, or a combination thereof is attached to the oligonucleotide via a linker.

[0085] In one aspect, the linker is cleavable.

[0086] In one aspect, a method of introducing an oligonucleotide into a nucleus and / or cytosol of a cell is provided.

[0087] In one aspect, the method includes: contacting the cell with an oligonucleotide conjugated to one or more compounds of any one of Formulae I, la, lb, Ic, Id, le, If or of Table 1, a pharmaceutically acceptable salt of one or more compounds of any one of Formulae I, la, lb, Ic, Id, le, If or of Table 1, or a combination thereof. In one aspect, the conjugated compound(s) facilitates entry of the oligonucleotide into the nucleus and / or cytosol of the cell. In one aspect, the oligonucleotide is internalized by the cell through endocytosis and encapsulated within an endosome and the compound facilitates release of the oligonucleotide from the endosome. In one aspect, the oligonucleotide is internalized by transient pore formation induced by the conjugated compound(s).

[0088] In one aspect, contacting the cell with the oligonucleotide conjugated to one or more compounds of Formulae I, la, lb, Ic, Id, le, If or of Table 1, a pharmaceutically acceptable salt of one or more compounds of any one of Formulae I, la, lb, Ic, Id, le, If or of Table 1, or a combination thereof is performed in a composition, wherein the composition includes about 0.025 μM to about 20 μM of the oligonucleotide. In one aspect, the composition includes about 0.1 μM to about 10 μM, about 0.1 μM to about 5 μM, or about 0.1 μM to about 1 μM of the oligonucleotide.

[0089] In one aspect, the oligonucleotide hybridizes to a target nucleic acid in the cell. In one aspect, the target nucleic acid is in the nucleus of the cell. In one aspect, the target nucleic acid is in the cytosol of the cell.

[0090] In one aspect, contacting the cell with an oligonucleotide conjugated to one or more compounds of any one of Formulae I, la, lb, Ic, Id, le, If or of Table 1, a pharmaceutically acceptable salt of one or more compounds of any one of Formulae I, la, lb, Ic, Id, le, If or of Table 1, results in endosomal membrane permeabilization as determined by mCherry-GAL9 recruitment assay and with reduced accumulation of the oligonucleotide of the oligonucleotide in endosomes and / or lysosomes.

[0091] In one aspect, the oligonucleotide alters activity of a gene expressed by the cell. In one aspect, the oligonucleotide increases activity of a gene expressed by the cell. In one aspect, the activity of the gene expressed by the cell is increased at least about lOx when the cell is contacted with the oligonucleotide conjugated to the compound(s) as compared to a cell that is contacted with the oligonucleotide not conjugated to the compound(s). In one aspect, the activity of the gene expressed by the cell is increased at least about lOOx when the cell is contacted with the oligonucleotide conjugated to the compound(s) as compared to a cell that is contacted with the oligonucleotide not conjugated to the compound(s).

[0092] In one aspect, the oligonucleotide decreases activity of a gene expressed by the cell. In one aspect, the activity of the gene expressed by the cell is decreased at least about lOx when the cell is contacted with the oligonucleotide conjugated to the compound(s) as compared to a cell that is contacted with the oligonucleotide not conjugated to the compound(s). In one aspect, the activity of the gene expressed by the cell is decreased at least about lOOx when the cell is contacted with the oligonucleotide conjugated to the compound(s) as compared to a cell that is contacted with the oligonucleotide not conjugated to the compound(s).

[0093] In one aspect, the method includes in vitro delivery of the oligonucleotide to the cell. In one aspect, the method includes in vivo delivery of the oligonucleotide to the cell.

[0094] In one aspect, the cell is a cultured cell. In one aspect, the cell is an isolated cell. In one aspect, the cell is an isolated cell from a subject in need of treatment. In one aspect, the cell is part of a tissue or organ. In one aspect, the organ or tissue is the brain, central nervous system (CNS) or peripheral nervous system (PNS), heart, liver, kidney, spleen, pancreas, lung, adipose, and / or muscle (e.g., skeletal muscle). In one aspect, the cell is a brain cell, a CNS cell, a PNS cell, a heart cell, a liver cell, a kidney cell, a spleen cell, a pancreas cell, a lung cell, a muscle cell, an adipose cell, an immune cell, or combination thereof.

[0095] In one aspect, the cell is a mammalian cell. In another aspect, the cell is a eukaryotic cell and / or a prokaryotic cell.

[0096] In one aspect, a method of releasing an oligonucleotide from an endosome is provided. In one aspect, the method includes: contacting the cell with the oligonucleotide conjugated to one or more compounds of any one of Formulae I, la, lb, or Ic, Id, le, If or of Table 1, a pharmaceutically acceptable salt of one or more compounds of any one of Formulae I, la, lb, or Ic, Id, le, If or of Table 1, or a combination thereof, wherein the oligonucleotide is internalized by the cell through endocytosis and encapsulated within the endosome, and wherein the compound(s) facilitates release of the oligonucleotide from the endosome.

[0097] In one aspect, a method for the treatment and / or prevention of a disorder in a subject is provided. In one aspect, the method includes: administering to the subject a therapeutically effective amount of an oligonucleotide conjugated to one or more compounds of any one of Formulae I, la, lb, or Ic, Id, le, If or of Table 1, or a combination thereof. In one aspect, administration includes parenteral administration. In one aspect, administration includes intravenous or subcutaneous administration. In one aspect, the subject is a mammal. In one aspect, the subject is a human.

[0098] In one aspect, a method for the treatment and / or prevention of a disorder in a subject is provided that includes: isolating a cell from the subject; contacting the isolated cell with a therapeutically effective amount of an oligonucleotide conjugated to one or more compounds of any one of Formulae I, la, lb, or Ic, Id, le, If or of Table 1, a pharmaceutically acceptable salt of one or more compounds of any one of Formulae I, la, lb, or Ic, Id, le, If or of Table 1, or a combination thereof to produce an engineered cell; and transplanting the engineered cell in the subject. In one aspect, the isolated cell is contacted with a composition that includes about 0.025 μM to about 20 μM of the oligonucleotide. In one aspect, the composition includes about 0.1 μM to about 10 μM, about 0.1 μM to about 5 μM, or about 0.1 μM to about 1 μM of the oligonucleotide.

[0099] In one aspect, the subject is a mammal. In one aspect, the subject is a human.

[0100] BRIEF DESCRIPTION OF THE FIGURES

[0101] FIG. 1 is a general schematic of the synthesis of the preparation of Compounds 2-7, 19-31.

[0102] FIG. 2 is a breakdown of the starting material “X” identified in FIG. 29 and the required intermediate for each of the Compounds 2-7, 19-31.

[0103] FIG. 3 is a schematic of the synthesis of Compound 2.

[0104] FIG. 4 is a schematic of the synthesis of Compound 3.

[0105] FIG. 5 is a schematic of the synthesis of Compound 4.

[0106] FIG. 6 is a general schematic of the synthesis of the preparation of Compounds 8-17 with the additional alkylation step.

[0107] FIG. 7 is a general schematic of the synthesis of the preparation of Compound 18.

[0108] FIG. 8 is a schematic of the synthesis of Compound 7.

[0109] FIG. 9a % knockdown (normalized to untreated) in HEK293T wt after 24 hours.

[0110] FIG. 9b % knockdown (normalized to untreatedjin HEK293 GAL9-mCherry cells after 24 hours.

[0111] DETAILED DESCRIPTION OF THE DISCLOSURE

[0112] A. Definitions

[0113] Unless otherwise defined, scientific and technical terms used herein shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular, for example, “a” or “an,” include pluralities, e.g., “one or more” or “at least one” and the term “or” can mean “and / or” unless stated otherwise. The terms “including,” “includes” and “included” are not limiting. Ranges provided herein, of any type, include all values within a particular range described and values about an endpoint for a particular range. “Alkyl” refers to a saturated, branched or straight-chain hydrocarbon group. “Lower alkyl” refers to an alkyl group having from 1 and up to about 8 carbon atoms, for example, 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. In one aspect, the alkyl group includes from 1 to 4 carbon atoms (C1-C4 alkyl). Lower alkyls include, but are not limited to, methyl, ethyl, propyl, isopropyl, and butyl, including n-butyl, sec-butyl, isobutyl, and tert-butyl.

[0114] The alkyl group may be substituted or unsubstituted. In some embodiments, the alkyl group is substituted with one or more halo groups, e.g., F, Cl, Br, I, At, etc. In some embodiments, the alkyl group is substituted with one or more F or Cl, e.g., a mono-, di- or tri- fluoro or chloro alkyl. An alkyl group in which one or more of the hydrogen atoms are replaced by halogen can be referred to as “halo alkyl”, e.g., “halo C1-C4 alkyl” refers to a Ci- C4 alkyl substituted by one or more of the same or different halogen atoms. Examples of C1- C4 alkyls substituted with one or more halo groups include, but are not limited to, trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, dichloromethyl, and chloromethyl.

[0115] “Alkoxy,” also represented as “-OR4” where R4 is an alkyl group, refers to a saturated or unsaturated branched or straight-chain hydrocarbon group attached to a parent molecule through an oxygen atom. In one aspect, the alkoxy group includes from 1 to 4 carbon atoms (C1-C4 alkoxy). Examples of C1-C4 alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, including n-butoxy, sec-butoxy iso-butoxy and t-butoxy. In some embodiments, the alkoxy group is substituted with one or more halo groups, e.g., F, Cl, Br, I, At, etc. In some embodiments, the alkoxy group is substituted with one or more F or Cl, e.g., a mono-, di-, or tri- fluoro or chloro alkoxy.

[0116] “Halogen” or “halo” can be used interchangeably to refer to a fluoro, chloro, bromo, or iodo group. In one aspect, halo refers to fluoro, chloro, or bromo. In one aspect, halo refers to fluoro or chloro.

[0117] “Amide “ refers to the group “-C(O)NR5R5” (also represented as “-C(=O)NR5R5”) where each R5 is independently hydro or alkyl (optionally substituted and / or interrupted) and includes primary, secondary, and tertiary amides. In one aspect, the alkyl in the amide includes a Ci to Cs alkyl. In one aspect, the alkyl substituent includes a Ci to C4 alkyl. In some embodiments, at least one R5 in the formula “-C(O)NR5R5” is a hydro.

[0118] “Ester” refers to the group “-C(0)0R6” group (also represented as (“-C(=0)0R6”) where Re is hydro or alkyl. In some embodiments, the ester is a “short chain ester,” wherein Re is C1-C4 alkyl. In some embodiments, Re is methyl or ethyl. In some embodiments, Re is propyl or isopropyl. “Cyano” refers to a group that includes a carbon atom triple-bonded to a nitrogen atom (-C=N).

[0119] “Hydro” refers to a hydrogen substituent and is also represented by “-H ” “Endosomolytic” refers to one or more compounds that facilitates release of an oligonucleotide from an endosome / lysosome / autophagosome / multivesicular body or other endosomal vesicle into the cytosol of a cell. In one aspect, the endosomolytic agent is one or more small molecule compounds (SMC). In one aspect, the endosomolytic agent has a structure represented by any of Formulae I, la, or of Table 1, or a pharmaceutically acceptable salt thereof.

[0120] “Small molecule compound” or “SMC” refers to an organic molecule with a molecular weight of less than 1000 g / Mol and includes compounds having a structure represented by any of Formulae I, la, or of Table 1.

[0121] “Nucleic acid” refers to an oligomer or polymer of nucleotides and includes naturally occurring or synthetically produced single stranded or double stranded deoxyribonucleotides (DNA) or ribonucleotides (RNA). A nucleic acid can include naturally occurring nucleic acid nucleobases such as adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U), as well base analogs or modified nucleobases that do not occur in nature.

[0122] “Target nucleic acid” refers to a nucleic acid to which an antisense oligonucleotide hybridizes. In one aspect, hybridization of an antisense oligonucleotide to a target nucleic acid in a cell alters activity of a gene expressed by the cell. In one aspect, hybridization of the antisense oligonucleotide to the target nucleic acid increases activity of a gene expressed by the cell. In one aspect, hybridization of the antisense oligonucleotide to the target nucleic acid decreases activity of a gene expressed by the cell.

[0123] “Oligonucleotide” refers to an exogeneous, naturally occurring, or non-naturally occurring single- stranded or double- stranded polymer of deoxyribonucleotides (DNA) or ribonucleotides (RNA). In one aspect, the oligonucleotide is about 2 to about 50 nucleotides in length. In one aspect, the oligonucleotide includes one or more nucleotide analogs or modified backbone residues or linkages, including, but not limited to, phosphodiester (PO); phosphorothioate (PS); 2’0-methyl (2’0Me); 2’0-methoxyethyl (MOE); peptide nucleic acid (PNA); phosphoroamidate morpholino (PMO); locked nucleic acid (ENA); 2’-deoxy-2’- fluoro (2’-F); or a combination thereof. “Locked nucleic acid nucleoside” or “LNA” refers a nucleoside that includes a bicyclic sugar moiety with a 4’-CH2-O-2’bridge. “Phosphorothioate” refers to an internucleotide linkage in which one of the non-bridging oxygens is replaced by sulfur. As “modified oligonucleotide” refers to an oligonucleotide that includes at least one modified nucleoside and / or at least one modified intemucleoside linkage.

[0124] An “antisense oligonucleotide” or “ASO” is an oligonucleotide that includes at least a portion of which is complementary to a target nucleic acid such that the ASO can hybridize to the target nucleic acid. An antisense oligonucleotide can increase or decrease expression of a target nucleic acid.

[0125] A “splice switching oligonucleotide” or “SSO” is a short, synthetic, antisense oligonucleotide that can hybridize to a pre-mRNA and disrupt splicing of the transcript, for example, by blocking the RNA-RNA base pairing or protein-RNA binding interactions that occur between components of the splicing machinery and the pre-mRNA. “Pre-mRNA” refers to an RNA transcript that includes one or more introns and has not been fully processed into mRNA.

[0126] A “small interfering RNA,” also known as “short interfering RNA,” “silencing RNA,” or “siRNA,” is a class of double-stranded RNA that is non-coding, typically between about 20 to about 25 base pairs, with hydroxylated 3’ and phosphorylated 5’ ends. In general, siRNA is part of the RNA interference pathway and interferes with expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription, thereby preventing translation. siRNA may be conjugated e.g., to sugars such as GalNAc or lipids such as cholesterol, to enhance delivery to a target cell, e.g., with improved pharmacokinetics and / or efficacy. See, e.g., Osborn et al., Nucleic Acid Ther. 28(3): 128-136 (2018). In some aspects, the present disclosure provides a method of enhancing delivery of unconjugated siRNA.

[0127] “Polypeptide,” used interchangeably herein with “peptide” or “protein,” refers to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. In some aspects, a polypeptide comprises about 2 to about 5000 amino acids. In some aspects, the polypeptide is capable of providing a site- specific modification in a target nucleic acid. In some embodiments, the polypeptide is a therapeutic polypeptide.

[0128] A “macromolecule” includes proteins, nucleic acids, carbohydrates, lipids, nanogels, and macrocycles. In some aspects, a macromolecule of the present disclosure comprises an oligonucleotide. In some aspects, a macromolecule of the present disclosure comprises a polypeptide. In some aspects, a macromolecule of the present disclosure comprises one or more components of a site-specific modification (SSM) system described herein, e.g., a CRISPR system, a Cre-Lox system, and / or a FLP-FRT system. Throughout the present disclosure, references to a SSM system can mean any one or more components of the system. In some aspects, a SSM system comprises a CRISPR system, a Cre-Lox system, a FLP-FRT system, any component thereof, or any combination thereof.

[0129] A “CRISPR” system is a SSM system capable of performing an SSM at a target nucleic acid. In some aspects, a CRISPR system includes (a) a protein capable of providing the SSM, e.g., a Cas protein; and (b) a guide RNA (also referred to herein as “gRNA”), which includes (i) a “crRNA” or “spacer” region that hybridizes to the target nucleic acid, and (ii) a “tracrRNA” or “scaffold” region that associates with the protein. In some aspects, the SSM comprises single-stranded cleavage of the target nucleic acid. In some aspects, the SSM comprises double- stranded cleavage of the target nucleic acid. In some aspects, the SSM comprises a deletion. In some aspects, the SSM comprises an insertion. In some aspects, the SSM comprises a mutation. In some aspects, the SSM comprises a base edit, e.g., conversion of a C-G base pair to a T-A base pair.

[0130] A “Cre-Lox” system is a SSM system capable of performing an SSM at a target nucleic acid. In some aspects, a Cre / Lox system includes a Cre recombinase, which recognizes a pair of Lox (also called LoxP) sequences flanking the target nucleic acid and catalyzes site- specific recombination at the target nucleic acid. An analogous system to the Cre-Lox system is the “FLP-FRT” system. A FLP-FRT system is a SSM system capable of performing an SSM at a target nucleic acid. In some aspects, a FLP-FRT system includes the FLP recombinase, which recognizes a pair of FRT sequences flanking the target nucleic acid and catalyzes site-specific recombination at the target nucleic acid. In some aspects, the SSM comprises an inversion. In some aspects, the SSM comprises an insertion. In some aspects, the SSM comprises a deletion. In some aspects, the SSM comprises a translocation. In some aspects, the location and orientation of the Lox sequences (or the FRT sequences) determines the type of SSM (e.g., inversion, deletion, or translocation) performed by the Cre recombinase (or the FLP recombinase).

[0131] “Hybridize” refers to the pairing of complementary oligomeric compounds, for example, pairing between an antisense oligonucleotide and its corresponding target nucleic acid. While not limited to any mechanism, the most common mechanism of pairing involves hydrogen bonding between complementary nucleobases, including, for example, Watson- Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding. For example, in Watson-Crick base pairing, guanine (G) is complementary to cytosine (C), adenine (A) is complementary to thymine (T) in DNA, and adenine (A) is complementary to uracil (U) in RNA. Additionally, some modified nucleobases maintain the ability to pair with a counterpart nucleobase. Hybridization can occur between two complementary DNA molecules (DNA-DNA hybridization), two RNA molecules (RNA-RNA hybridization), or between complementary DNA and RNA molecules (DNA-RNA hybridization). Hybridization can occur between a short nucleotide sequence that is complementary to a portion of a longer nucleotide sequence. Hybridization can occur between sequences that do not have 100% “sequence complementarity,” i.e., complementary sequences need not have nucleobase complementarity at each nucleoside, although sequences having less sequence complementarity are less stable and less likely hybridize than sequences having greater sequence complementarity.

[0132] “Specifically hybridizes” refers to the ability of an oligonucleotide to hybridize to a target nucleic acid with greater affinity than to a different nucleic acid. In one aspect, the antisense oligonucleotide specifically hybridizes to a target nucleic acid sequence under physiological conditions, for example, for in vivo or therapeutic use.

[0133] “Targeting” or “targeted to,” in the context of antisense oligonucleotides, refers to the association of an antisense oligonucleotide with a particular target nucleic acid or region of a target nucleic acid. An antisense oligonucleotide targets a target nucleic acid if it is sufficiently complementary to the target nucleic acid to allow hybridization under physiological conditions. “Targeting” or “targeted to,” in the context of SSM systems (e.g., CRISPR, Cre-Lox, and / or FLP-FRT systems described herein), refers to the association of the protein of the SSM system (e.g., Cas protein, Cre recombinase, or FLP recombinase) with a particular target nucleic acid or region of a target nucleic acid. In some aspects, the Cas protein of a CRISPR system targets a target nucleic acid upon hybridization of the guide RNA with the target nucleic acid. In some embodiments, the Cre recombinase of a Cre-Lox system targets a target nucleic acid upon recognition of the Lox sequences flanking the target nucleic acid. In some embodiments, the FLP recombinase of a FLP-FRT system targets a target nucleic acid upon recognition of the FRT sequences flanking the target nucleic acid.

[0134] “Alter” or “modulate” refer a change in an amount, function, or activity of a molecule, e.g., a macromolecule described herein, when compared to the amount, function, or activity prior to treatment. In one aspect, one or more compounds described herein increases or decreases an amount, function or activity of a gene expressed by a target nucleic acid sequence. In one aspect, the compound(s) increases the activity of an antisense oligonucleotide (ASO) that acts on pre-mRNA via RNase H in the nucleus. In one aspect the compound(s) increases the activity of an siRNA that acts via the RISC complex in the cytosol. In one aspect, the compound(s) increases the alteration of pre-mRNA splicing by a splice switching oligonucleotide (SSO), as reflected by an increase in the desired splice variant. In one aspect, the compound(s) results in reduced levels of the corresponding target mRNA and / or protein as compared to treatment with the ASO in the absence of the compound. In some aspects, the compound(s) increases the activity of a Cas protein in a CRISPR system. In some aspects, the compound(s) increases the activity of a Cre recombinase. In some aspects, the compound(s) increases the activity of a FLP recombinase. In some aspects, the compound(s) increases the frequency of the SSM at the target nucleic acid. In some aspects, the compound(s) increases the editing efficiency of a SSM system (e.g., the CRISPR, Cre-Lox, and / or FLP-FRT systems).

[0135] In one aspect, the compound(s) described herein “enhances the delivery” of a macromolecule provided herein, e.g., an oligonucleotide and / or polypeptide. In one aspect, the compound described herein “enhances the delivery” of an antisense oligonucleotide to increase cytosolic and / or nuclear concentration, accumulation, and / or half-life of the oligonucleotide as compared to that found without administration of the compound(s). In one aspect, the compound(s) described herein “enhances the delivery” of one or more components of a SSM system to increase cytosolic and / or nuclear concentration, accumulation, and / or half-life of the SSM system as compared to that found without administration of the compound. In some aspects, the SSM system comprises a CRISPR system, a Cre-Lox system, a FLP-FRT system, any component thereof, or any combination thereof.

[0136] “Expression” refers to a process by which a protein is produced in a host cell from a nucleic acid and includes, but is not limited to, transcription, translation, post-translational modification, and secretion. “Increased” expression is in the context of a comparison between a treated cell and an untreated control, for example, a cell treated with a macromolecule, e.g., an oligonucleotide such as an antisense oligonucleotide, a polypeptide such as a recombinase (e.g., Cre or FLP), or a combination thereof such as a Cas protein and a guide RNA, versus an untreated cell, or a cell treated with a macromolecule, e.g., an oligonucleotide such as an antisense oligonucleotide, a polypeptide such as a recombinase (e.g., Cre or FLP), or a combination thereof such as a Cas protein and a guide RNA, and conjugated to one or more compounds described herein versus a cell treated with only the macromolecule, e.g., the antisense oligonucleotide, recombinase (e.g., Cre or FLP), or Cas protein and guide RNA. Similarly, “decreased” expression is in the context of a comparison between a treated cell and an untreated control, for example, a cell treated with a macromolecule, e.g., an oligonucleotide such as an antisense oligonucleotide, a polypeptide such as a recombinase (e.g., Cre or FLP), or a combination thereof such as a Cas protein and a guide RNA, versus an untreated cell, or a cell treated with a macromolecule, e.g., an oligonucleotide such as an antisense oligonucleotide, a polypeptide such as a recombinase (e.g., Cre or FLP), or a combination thereof such as a Cas protein and a guide RNA, and conjugated to one or more compounds described herein versus a cell treated with only the macromolecule, e.g., the antisense oligonucleotide, recombinase (e.g., Cre or FLP), or Cas protein and guide RNA.

[0137] “Disease” refers to any disease, disorder, condition, symptom, or indication.

[0138] “Treating” or “treatment” refer to curative, symptomatic, preventive and prophylactic treatment and include, but are not limited to, arresting or ameliorating a disease or at least one clinical symptoms of a disease, reducing the risk of acquiring a disease or at least one clinical symptoms of a disease, reducing the development of a disease or at least one clinical symptoms of the disease, reducing the risk of developing a disease or at least one clinical symptoms of a disease, or delaying the onset of the disease or at least one clinical symptoms of a disease.

[0139] A “subject” and “patient” can be used interchangeably to refer to any animal subjects, for example, mammalian subjects such as humans, primates, cattle, pigs, horses, sheep, goats, rodents, cats, and / or dogs. In one aspect, the subject is human.

[0140] The term “cell” can include a single cell, a plurality of cells or a population of cells where context permits, unless otherwise specified. In one aspect, the cell is in vitro, for example, a cell explanted from a subject. In one aspect, the cell is a cell grown in batch culture or in tissue culture. In one aspect, the cell is in vivo, for example, located in a subject in need of treatment. In one aspect, the subject is a human subject.

[0141] “Pharmaceutically acceptable” as used herein means approved by a regulatory agency of a Federal or state government, or listed in the U.S. Pharmacopeia, European Pharmacopia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0142] A “pharmaceutical composition” includes one or more active agents, including, for example, a macromolecule, e.g., an oligonucleotide such as an antisense oligonucleotide, a polypeptide such as a recombinase (e.g., Cre or FLP), or a combination thereof such as a Cas protein and a guide RNA, conjugated to one or more compounds described herein, and a pharmaceutically acceptable carrier or diluent. In one aspect, the carrier or diluent is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration. “Pharmaceutically acceptable salt” refers to a salt of one or more compounds that is physiologically and pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound and includes a salt prepared from pharmaceutically acceptable non-toxic acid or base, including inorganic or organic acids and bases. “Pharmaceutically acceptable salts” of the compounds described herein may be prepared by methods well-known in the art. For a review of pharmaceutically acceptable salts, see Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection and Use (Wiley-VCH, Weinheim, Germany, 2002).

[0143] An “effective amount” of a macromolecule conjugated to one or more compounds refers to an amount sufficient to increase the efficacy of a macromolecule, e.g., an oligonucleotide such as an antisense oligonucleotide, a polypeptide such as a recombinase (e.g., Cre or FLP), or a combination thereof such as a Cas protein and a guide RNA. In one aspect, an “effective” amount of a macromolecule conjugated to one or more compounds refers to an amount sufficient to facilitate entry of a macromolecule, e.g., an oligonucleotide such as an antisense oligonucleotide, a polypeptide such as a recombinase (e.g., Cre or FLP), or a combination thereof such as a Cas protein and a guide RNA, into the nucleus and / or cytosol of a cell. In one aspect, an “effective” amount of a macromolecule conjugated to one or more compounds refers to an amount that facilitates the release of a macromolecule, e.g., an oligonucleotide such as an antisense oligonucleotide, a polypeptide such as a recombinase (e.g., Cre or FLP), or a combination thereof such as a Cas protein and a guide RNA, from an endosome into the cytosol of a cell.

[0144] A “therapeutically effective amount” of a macromolecule, e.g., an oligonucleotide such as an antisense oligonucleotide, a polypeptide such as a recombinase (e.g., Cre or FLP), or a combination thereof such as a Cas protein and a guide RNA, refers to an amount sufficient to provide a therapeutic benefit in the treatment of a disease, or to delay or reduce one or more symptoms associated with the disease. A “therapeutically effective amount” can vary depending on many factors, including, but not limited to, the macromolecule, e.g., an oligonucleotide such as an antisense oligonucleotide, a polypeptide such as a recombinase (e.g., Cre or FLP), or a combination thereof such as a Cas protein and a guide RNA, being administered, the disease, the severity of the disease, the age of the subject being treated, and / or the weight of the subject being treated.

[0145] “Dose” refers to a specified quantity of an active agent (for example, a macromolecule, e.g., an oligonucleotide such as an antisense oligonucleotide, a polypeptide such as a recombinase (e.g., Cre or FLP), or a combination thereof such as a Cas protein and a guide RNA conjugated to one or more compounds) provided in a single administration, or in a specified time -period. A dose can be administered in one, two, or more boluses or injections. In one aspect, the active agent is administered by infusion over an extended period of time or continuously. Doses can be stated as the amount of pharmaceutical agent per unit time (e.g., hour, day, week, or month). Doses can also be stated as the amount per unit weight of the subject (e.g., mg / kg or g / kg).

[0146] “Dosage unit” refers to a form in which an active agent, for example, a macromolecule, e.g., an oligonucleotide such as an antisense oligonucleotide, a polypeptide such as a recombinase (e.g., Cre or FLP), or a combination thereof such as a Cas protein and a guide RNA conjugated to one or more compounds, is provided. In one aspect, the dosage unit is a vial containing lyophilized active agent. In one aspect, a dosage unit is a vial containing reconstituted active agent. In one aspect, the active agent is a macromolecule. In one aspect, the active agent is an oligonucleotide. In one aspect, the active agent comprises a polypeptide. In one aspect, the active agent comprises an antisense oligonucleotide. In one aspect, the active agent comprises a Cre recombinase. In one aspect, the active agent comprises a FLP recombinase. In one aspect, the active agent comprises Cas protein. In one aspect, the active agent comprises Cas protein and a guide RNA. In one aspect, the active agent comprises a CRISPR system, a Cre-Lox system, a FLP-FRT system, any component thereof, or any combination thereof.

[0147] The methods and compositions described herein can be used in vitro on a sample (for example, on isolated cells, organs, or tissues) or in vivo in a subject (for example, in a living organism, such as a patient).

[0148] The compositions described herein may be administered in a number of ways depending upon whether local or systemic treatment is desired and the area to be treated. In one aspect, the composition is administered parenterally. Parenteral administration includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. Administration can be continuous, chronic, short, or intermittent.

[0149] B. Overview

[0150] Delivering macromolecules such as oligonucleotides or proteins into cells requires that the macromolecule, e.g., oligonucleotide or protein, traverse cellular membranes, including the plasma membrane and / or endosomal membranes. Use of macromolecules such as oligonucleotides or proteins can be hindered by their inability to effectively reach the cytosolic and / or nucleus of the cell, for example, due to their inability to cross the cell membrane or to escape from endosomal compartments following endocytosis. Provided herein are compounds, conjugated to a macromolecule, that can increase the activity of a macromolecule, e.g., an oligonucleotide, such as an antisense oligonucleotide or siRNA; a polypeptide, such as recombinase (e.g., Cre or FLP); or a combination thereof, such as a Cas protein and a guide RNA. In one aspect, the compound is a small molecule compound (SMC). In one aspect, the compound is an endosomolytic compound that facilitates the release of the macromolecule, e.g., oligonucleotide and / or polypeptide from an endosome into the cytosol of a cell. In one aspect, the compound disclosed herein increase transfection efficiency of a macromolecule, e.g., an oligonucleotide and / or polypeptide. Advantageously, the compounds described herein interfere with the normal cell trafficking machinery to a minimal extent, i.e., until leakage is induced and do not induce damage or toxicity which irreversibly impedes cell proliferation or results in cell death.

[0151] C. Oligonucleotides

[0152] In one aspect, compositions and methods are provided for delivering a macromolecule, e.g., an oligonucleotide, to the cytosol and / or nucleus of a cell. In one aspect, the oligonucleotide is single stranded. In one aspect, the oligonucleotide is double stranded. In one aspect, the oligonucleotide includes deoxyribonucleic acid (DNA). In one aspect, the oligonucleotide includes ribonucleic acid (RNA). In one aspect, the oligonucleotide is about 5 nucleotides to about 100 nucleotides, about 5 nucleotides to about 50 nucleotides, about 8 nucleotides to about 30 nucleotides, about 10 nucleotides to about 30 nucleotides, about 15 nucleotides to about 30 nucleotides, or about 18 to about 30 nucleotides in length. In one aspect, the oligonucleotide has a molecular weight from about 5 kDa to about 15 kDa.

[0153] In one aspect, the oligonucleotide reduces expression of a target nucleic acid, which can be referred to as “gene silencing.” In one aspect, the oligonucleotide increases expression of a target nucleic acid, which can be referred to as “gene activation.” In one aspect, the oligonucleotide alters the splicing of a target nucleic acid, which can be referred to as “splice switching.” In one aspect, the oligonucleotide interacts with a target protein. In one aspect, the oligonucleotide is an agonist or antagonist of a target protein.

[0154] In one aspect, the oligonucleotide is an antisense oligonucleotide (ASO), a small (-18-30 nucleotides), synthetic, single-stranded nucleic acid polymer which modulates gene expression via various mechanisms. In one aspect, the ASO includes DNA and forms an RNA-DNA heteroduplex that is recognized by endogenous RNase H enzyme which catalyzes the degradation of RNA, thereby decreasing expression of the gene. In one aspect, the ASO binds to a target nucleic acid but does not induce degradation. In one aspect, the oligonucleotide is a splice switching oligonucleotide (SSO) that masks sequences within a target nucleic acid and thereby interferes with transcript RNA-RNA and / or RNA-protein interactions. In one aspect, the oligonucleotide is a small-interfering RNA (siRNA), which has a characteristic 19 + 2mer structure (e.g., a duplex of two 21 -nucleotide RNA molecules with 19 complementary bases and terminal 2-nucleotide 3' overhangs). In one aspect, the oligonucleotide is a microRNA (miRNA). In one aspect, the oligonucleotide targets a non- coding RNA sequence associated with transcriptional repression to reverse the effects of this negative regulation thereby activating gene expression. Other oligonucleotides include, but are not limited to, interfering RNA (RNAi) and decoy oligonucleotides. In one aspect, the oligonucleotide is a gapmer. In one aspect, the oligonucleotide is an aptamer.

[0155] In some aspects, the oligonucleotide is part of a site-specific modification (SSM) system, e.g., a CRISPR system, described herein. In some aspects, the oligonucleotide is a guide RNA. In one aspect, the guide RNA comprises one or both of: (i) a scaffold region or tracrRNA capable of associating with a Cas protein (e.g., Cas nuclease); and (ii) a spacer region or crRNA capable of hybridizing to a specific target nucleic acid sequence, thereby directing the Cas protein to make a site-specific modification at the target nucleic acid. In general, the spacer region is about 15 to about 25 nucleotides in length. In some aspects, the guide RNA is a single guide RNA (sgRNA) comprising both the tracrRNA and the crRNA. In some aspects, the guide RNA comprises a tracrRNA and a crRNA as two separate oligonucleotides that, together with the Cas protein, are capable of forming a complex.

[0156] In one aspect, the oligonucleotide includes one or more modified nucleotides. In one aspect, the oligonucleotide includes one or more modifications to the oligonucleotide phosphate linkages. In one aspect, the oligonucleotide includes one or more modifications to the ribose sugar. In one aspect, the oligonucleotide includes one or more nucleotides that are covalently modified to limit conformation, i.e., locked nucleic acids (LNA). In one aspect, the oligonucleotide includes a peptide nucleic acid (PNA). In one aspect, the oligonucleotide includes a methylated cystosine at the 5’ position. In one aspect, the oligonucleotide includes one or more modified nucleotides selected from: phosphodiester (PO); phosphorothioate (PS); 2’0-methyl (2’0Me); 2’0-methoxyethyl (MOE); peptide nucleic acid (PNA); phosphoroamidate morpholino (PMO); locked nucleic acid (LNA); 2’-deoxy-2’-fluoro (2’-F); or a combination thereof. In embodiments, the oligonucleotide is an antisense oligonucleotide targeting metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). In embodiments, the oligonucleotide is an antisense oligonucleotide targeting metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) comprising at least one nucleic acid with an LNA. In such embodiments, the antisense oligonucleotide targets MALAT1 and has the sequence: GM5CAttm5ctaatagm5cAGM5C, where m5c is 5- methylcytidine and capital letters are LNA nucleosides (SEQ ID NO:1). In embodiments, the oligonucleotide is an antisense oligonucleotide that targets MALAT1 and reduces expression of MALAT1 in cells by about 1% to about 100%. In embodiments, the oligonucleotide is an antisense oligonucleotide that targets MALAT1 and reduces expression of MALAT1 in cells by about 1%, by about 10%, by about 20%, by about 30%, by about 40%, by about 50%, by about 60%, by about 70%, by about 80%, by about 90%, by about 95% or by about 100%.

[0157] D. Polypeptides

[0158] In one aspect, compositions and methods are provided for delivering a macromolecule, e.g., a polypeptide, to the cytosol and / or nucleus of a cell. In some aspects, the polypeptide comprises a therapeutic protein, which may be an antibody or a non-antibody protein. In one aspect, the polypeptide is a therapeutic peptide, e.g., as described in Wang et al., Sig Transduct Target Ther. 7:48 (2022).

[0159] In some aspects, the polypeptide is capable of providing a SSM at a target nucleic acid. In some aspects, the target nucleic acid is DNA. In some aspects, the target nucleic acid is RNA. In some aspects, the polypeptide is a nuclease. In some aspects, the polypeptide is a recombinase. In some aspects, the polypeptide is part of a SSM system described herein, e.g., a CRISPR system, a Cre-Lox system, or a FLP-FRT system. In some aspects, the polypeptide is a Cas protein. In some aspects, the polypeptide is Cas9. In some aspects, the polypeptide is Casl2a. In some aspects, the polypeptide is a recombinase. In some aspects, the polypeptide is Cre. In some aspects, the polypeptide is FEP.

[0160] In some aspects, the polypeptide comprises a modified Cas protein. In some aspects, the modified Cas protein is a Cas nickase, e.g., Cas9 nickase or Cas 12a nickase, which cleaves only one strand of a double- stranded target nucleic acid. In some aspects, the modified Cas protein is a catalytically inactivated Cas protein (dead Cas), e.g., dCas9 or dCasl2a, which does not comprise nuclease activity. In some aspects, the dead Cas is capable of binding to a target nucleic acid and preventing other enzymes such as transcription factors from binding to the target nucleic acid. Cas nickase and dead Cas proteins are further described, e.g., in Xu et al., J Mol Biol. 431(l):34-47 (2019); Qi et al., Cell 152(5): 1173-1183 (2013); and Liu et al., Microbial Cell Factories 19:172 (2020).

[0161] In some aspects, the polypeptide comprises a Cas fusion protein. In some aspects, the Cas fusion protein comprises a modified Cas protein, e.g., a Cas nickase or a dead Cas, fused to an effector domain. In some aspects, the polypeptide comprises a Cas nickase or a dead Cas, e.g., dCas9 or dCasl2, fused to a nucleotide deaminase, e.g., cytidine deaminase or adenosine deaminase, and optionally further fused to a DNA glycosylase inhibitor. In some aspects, the polypeptide comprises a Cas nickase, e.g., Cas9 nickase or Casl2 nickase, fused to a reverse transcriptase. Cas fusion proteins are further described, e.g., in Rees et al., Nat Rev Genet. 19(12):770-788 (2018); Anzalone et al., Nature 576(7785): 149- 157 (2019); and Liu et al., Microbial Cell Factories 19:172 (2020).

[0162] In some aspects, the polypeptide comprises a recombinase. In some aspects, the polypeptide comprises Cre. In some aspects, the polypeptide comprises FLP. In some aspects, the recombinase is a modified recombinase. In some aspects, the recombinase is an inducible recombinase. Modified (e.g., inducible) recombinases are further described, e.g., in Kaczmarcyk et al., Nucleic Acids Res. 29(12): e56 (2001); Badea et al., PLOS One 4(11): e7859 (2009); and Akbudak et al., Mol Biotechnol. 49(l):82-89 (2011).

[0163] E. Cellular entry

[0164] In some aspects, the macromolecule is delivered to a target cell. In one aspect, the target cell is a cultured cell. In one aspect, the target cell is an isolated cell. In one aspect, the target cell is an isolated cell from a subject in need of treatment. In one aspect, the target cell is a mammalian cell. In one aspect, the target cell is a eukaryotic cell. In one aspect, the target cell is a prokaryotic cell.

[0165] In one aspect, the target cell is part of a tissue or organ. In one aspect, the organ or tissue is the brain, central nervous system (CNS) or peripheral nervous system (PNS), heart, liver, kidney, spleen, pancreas, lung, adipose, and / or muscle (e.g., skeletal muscle). In one aspect, the target cell is a brain cell, a CNS cell, a PNS cell, a heart cell, a liver cell, a kidney cell, a spleen cell, a pancreas cell, a lung cell, a muscle cell, an adipose cell, an immune cell, or combination thereof.

[0166] In some aspects, the target cell is a CNS cell. In some aspects, the CNS cell comprises a glial cell and / or a neuron. Glial cells of the CNS include, e.g., astrocytes, oligodendrocytes, microglia, and ependymal cells. Neurons include, e.g., afferent neurons, efferent neurons, and interneurons. In some aspects, the target cell is a liver cell, e.g., a hepatocyte or a non-parenchymal cell. In some aspects, the target cell comprises a plateable metabolism qualified human hepatocyte, a plateable induction qualified human hepatocyte, plateable human hepatocyte, suspension qualified human hepatocyte (including 10-donor and 20-donor pooled hepatocytes), human hepatic kupffer cells, human hepatic stellate cells, dog hepatocytes (including single and pooled Beagle hepatocytes), mouse hepatocytes (including CD-I and C57BI / 6 hepatocytes), rat hepatocytes (including Sprague-Dawley, Wistar Han, and Wistar hepatocytes), monkey hepatocytes (including Cynomolgus or Rhesus monkey hepatocytes), cat hepatocytes (including Domestic Shorthair hepatocytes), and rabbit hepatocytes (including New Zealand White hepatocytes).

[0167] In some aspects, the target cell is a stem cell, e.g., a human stem cell. The stem cells can be, for example, pluripotent stem cells, including embryonic stem cells (ESCs), adult stem cells, induced pluripotent stem cells (iPSCs), tissue specific stem cells (e.g., hematopoietic stem cells) and mesenchymal stem cells (MSCs). In some aspects, the cell is a differentiated form of any of the cells described herein. In some aspects, the eukaryotic cell is a cell derived from any primary cell in culture.

[0168] In some aspects, the target cell is an immune cell. Non-limiting examples of immune cells include T cells, B cells, dendritic cells, NK cells, T helper cells, cytotoxic T cells, regulatory T cells, gamma delta T cells, neutrophils, mast cells, monocytes, antigen- presenting cells, lymphocytes, basophils, and phagocytes.

[0169] In some aspects, the macromolecule to be delivered to a target cell is an oligonucleotide. In some aspects, the oligonucleotide is an antisense oligonucleotide. In some aspects, the oligonucleotide is a siRNA. In some aspects, the siRNA is unconjugated, i.e., not conjugated to a lipid or sugar. In some aspects, the siRNA is conjugated to a lipid and / or a sugar. In some aspects, the oligonucleotide is a guide RNA of a CRISPR system. In some aspects, the macromolecule to be delivered to a target cell is an polypeptide. In some aspects, the polypeptide is a Cas protein, e.g., Cas9 or Casl2a. In some aspects, the Cas protein is a modified Cas protein or a Cas fusion protein as described herein. In some aspects, the polypeptide is a recombinase. In some aspects, the polypeptide is Cre. In some aspects, the polypeptide is FLP. Oligonucleotides are hydrophilic polyanions with a molecular weight ranging from about 5 kDa to about 15 kDa and do not readily pass through the plasma membrane. Polypeptides also typically do not passively diffuse across the plasma membrane. To be effective, macromolecules such as oligonucleotides and polypeptides must traverse the plasma membrane and enter the cytosol and / or nucleus of the cell. In one aspect, the macromolecule, e.g., oligonucleotide and / or polypeptide, is taken up by endocytosis, in which the macromolecule, e.g., oligonucleotide and / or polypeptide, is surrounded by the plasma membrane, which then buds off inside the cell to form a vesicle containing the ingested macromolecule, e.g., oligonucleotide and / or polypeptide. In another aspect, the macromolecule, e.g., oligonucleotide and / or polypeptide, is taken up by formation of pores in the plasma membrane.

[0170] The endocytic pathway of mammalian cells includes distinct membrane compartments which include: early endosomes, late endosomes and lysosomes.

[0171] Early endosomes (EE) are the first compartment of the endocytic pathway and are the main sorting station in the endocytic pathway (Huotari and Helenius (2011) EMBO J. 30(17):3481-3500). EEs recycle the majority of cargo internalized by endocytosis. Ees are heterogenous in terms of morphology, localization, composition, and function. Most Ees are relatively small and remain close to the plasma membrane, although the overall distribution of Ees is cell-type dependent.

[0172] Late endosomes (LE) receive endocytosed material, usually from early endosomes in the endocytic pathway and include proteins characteristic of nucleosomes, mitochondria and mRNAs including lysosomal membrane glycoproteins and acid hydrolases. They are acidic (35pprox.. pH 5.5) and are thought to mediate a final sorting of the internalized cargo prior to delivery of the cargo to the lysosomes.

[0173] Lysosomes are compartment of the endocytic pathway which sequester cargo for arrest or degradation. Their chief function is to break down cellular waste products, fats, carbohydrates, proteins, and other macromolecules and return them to the cytoplasm as new cell-building materials. To accomplish this, lysosomes include a variety of hydrolytic enzymes that function in an acidic environment (35 approx.. pH 4.8).

[0174] When taken up by endocytosis, macromolecules, e.g., oligonucleotides and / or polypeptides, often accumulate in endosomes, in particular, late endosomes or lysosomes, where they are pharmacologically inert. In order to be active, the macromolecule, e.g., oligonucleotide and / or polypeptide, must escape the endosomal compartment to access their cytosolic or nuclear targets before degradation or exportation via exocytosis.

[0175] F. Compounds

[0176] Provided herein are compounds that facilitate entry of a macromolecule, e.g., an oligonucleotide and / or polypeptide, into the cytosol and / or nucleus of a cell. In one aspect, a small molecule compound (SMC) is provided that facilitates entry of the macromolecule, e.g., oligonucleotide and / or polypeptide, into the cytosol of a cell. In one aspect, the compound facilitates entry of the macromolecule, e.g., oligonucleotide and / or polypeptide, into the cytosol by forming pores in the plasma membrane of the cell. In one aspect, the compound facilitates release of the macromolecule, e.g., oligonucleotide and / or polypeptide, from an endosomal compartment within the cell. In one aspect, the macromolecule is an oligonucleotide, and the compound facilitates the endosomal escape of a gymnotically delivered oligonucleotide. In one aspect, the compound engorges the endosomal compartment, physically inducing membrane rupture and concurrent macromolecule, e.g., oligonucleotide and / or polypeptide, escape into the cytosol during the early-to-late endosomal transition and / or late endosomal transition to lysosome.

[0177] In one aspect, the endosomal escape enhancer (EEE) compound has a structure represented by Formula I:

[0178] Formula I: one of Zi and Z2 is N, and the other is C; R1 is hydro, halo, C1-C4 alkyl, -OR4, -C(=O)NR5R5, -CO2R6, or cyano;

[0179] X is hydro, C1-C4 alkyl, or -OR2;

[0180] R2 is hydro or C1-C4 alkyl;

[0181] R3 is hydro, halo, C1-C4 alkyl, -(Cth / yOH, -OR4, -C(=O)NR5R5, -CO2R6, or cyano;

[0182] R4 is C1-C4 alkyl;

[0183] Rs is hydro or C1-C4 alkyl;

[0184] Re is hydro or C1-C4 alkyl;

[0185] R7, R8, R9, Rio, R11 are each independently CHR12, CR12R17 or NR13;

[0186] R12 is hydro, C1-C4 alkyl, -OR14, or -CO2R15;

[0187] R13 is hydro, C1-C4 alkyl, -(CH2)yOH, -OR14, -CO2R15, or -C(=O)R16; R14 is hydro or C1-C4 alkyl;

[0188] R15 is hydro or C1-C4 alkyl;

[0189] R16 is hydro or C1-C4 alkyl;

[0190] R17 is hydro or C1-C4 alkyl; y is 0, 1, 2, or 3; and wherein one or more of the alkyl are optionally substituted with one or more halo, or a pharmaceutically acceptable salt thereof.

[0191] In one aspect, R1 is hydro. In one aspect, R1 is halo, including, for example, chloro, fluoro, bromo or iodo. In one aspect, R1 is bromo, chloro, or fluoro. In one aspect, R1 is fluoro. In one aspect, R1 is alkyl. In one aspect, R1 is a saturated alkyl. In one aspect, R1 is an unsaturated alkyl, e.g., an alkenyl or alkynyl. In some aspects, R1 can be polyunsaturated. In one aspect, R1 is a straight chain alkyl. In one aspect, R1 is a branched alkyl. In one aspect, Ri is C1-C4 alkyl. In one aspect, R1 is methyl, ethyl, propyl, isopropyl, or butyl. In one aspect, butyl includes n-butyl, sec-butyl, isobutyl, and tert-butyl. In one aspect, R1 is a substituted alkyl, e.g., substituted with one or more halo. In some embodiments, R1 is a C1-C4 alkyl, wherein at least one carbon of the alkyl is substituted with one or more chloro or fluoro. In some aspects, R1 is substituted with a hydroxyl or a ketone. In one aspect, R1 is an unsubstituted alkyl. In one aspect, R1 is substituted with a halo group, i.e., a haloalkyl, e.g., a mono-haloalkyl, a di-haloalkyl, or a tri-haloalkyl. In one aspect, R1 is a halo C1-C4 alkyl. In one aspect, R1 is a halo C1-C4 alkyl such as trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, dichloromethyl, and chloromethyl.

[0192] In one aspect, R1 is alkoxy, as represented by -OR4. In one aspect, R1 is a saturated alkoxy. In one aspect, R1 is an unsaturated alkoxy. In some embodiments, R4 is C1-C4 alkyl, e.g., R4 is methyl, ethyl, propyl, isopropyl, or butyl, including n-butyl, sec -butyl, isobutyl, and tert-butyl. In some embodiments, the alkoxy can be optionally substituted with one or more halo. In some aspects, R1 is alkoxy substituted with a hydroxyl or a ketone. In one aspect, R1 is alkoxy substituted with a halo group, i.e., a haloalkoxy, e.g., a mono-haloalkoxy, a di-haloalkoxy, or a tri-haloalkoxy. In one aspect, R1 is a straight chain alkoxy. In one aspect, R1 is a branched alkoxy. In one aspect, R1 is C1-C4 alkoxy (-OR4), where R4 is C1-C4 alkyl, e.g., methyl or ethyl. In one aspect, R1 is an alkoxy that includes, but is not limited to, methoxy, ethoxy, propoxy, isopropoxy, or butoxy, including for example, n-butoxy, sec- butoxy iso-butoxy, and t-butoxy. In one aspect, R1 is amide, which can be represented by the group “-C(=O)NR5R5,” in which each R5 is independently hydro or alkyl. In some aspects, at least one R5 of -C(=O)NR5R5 is hydro. In one aspect, R5 is C1-C4 alkyl, e.g., methyl, ethyl, propyl, or butyl. In one aspect, R1 is a primary amide. In one aspect, R1 is a secondary amide. In one aspect, R1 is a tertiary amide. In one aspect, R1 is -C(=O)NHRs.

[0193] In one aspect, R1 is a short chain ester, which can be represented by the group “-C(O)OR6” or “-CO2R6” where R6 is hydro or C1-C4 alkyl, e.g., methyl, ethyl, propyl, or butyl.

[0194] In one aspect, R1 is cyano.

[0195] In one aspect, X is hydro. In some embodiments, X is an alkyl. In one aspect, X is a saturated alkyl. In one aspect, Xis an unsaturated alkyl. In one aspect, Xis a straight chain alkyl. In one aspect, X is a branched alkyl. In one aspect, X is C1-C4 alkyl. In one aspect, X is methyl, ethyl, propyl, isopropyl, or butyl. In one aspect, butyl includes n-butyl, sec-butyl, isobutyl, and tert-butyl. In one aspect, X is an unsubstituted alkyl. In one aspect, X is substituted with a halo group, i.e., a haloalkyl, e.g., a mono-haloalkyl, a di-haloalkyl, or a tri- haloalkyl. In one aspect, X is a halo C1-C4 alkyl. In one aspect, X is a halo C1-C4 alkyl such as trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, dichloromethyl, and chloromethyl. In some embodiments, X is -OR2, where R2 is defined below.

[0196] In one aspect, R2 is hydro. In some embodiments, R2is an alkyl. In one aspect, R2 is a saturated alkyl. In one aspect, R2 is an unsaturated alkyl. In one aspect, R2is a straight chain alkyl. In one aspect, R2 is a branched alkyl. In one aspect, R2 is C1-C4 alkyl. In one aspect, R2 is methyl, ethyl, propyl, isopropyl, or butyl. In one aspect, R2 is methyl. In one aspect, R2 is ethyl. In one aspect, butyl includes n-butyl, sec-butyl, isobutyl, and tert-butyl. In one aspect, R2 is an unsubstituted alkyl. In one aspect, R2 is substituted with a halo group, i.e., a haloalkyl, e.g., a mono-haloalkyl, a di-haloalkyl, or a tri-haloalkyl. In one aspect, R2 is a halo C1-C4 alkyl. In one aspect, R2 is a halo C1-C4 alkyl such as trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, dichloromethyl, and chloromethyl.

[0197] In one aspect, R3 is hydro. In some aspect, R3 is a halo, e.g., a chloro, fluoro, or bromo. In one aspect, R3 is alkyl. In one aspect, R3 is a saturated alkyl. In one aspect, R3 is an unsaturated alkyl. In one aspect, R3 is a straight chain alkyl. In one aspect, R3 is a branched alkyl. In one aspect, R3 is C1-C4 alkyl. In one aspect, R3 is methyl, ethyl, propyl, isopropyl, or butyl. In one aspect, butyl includes n-butyl, sec-butyl, isobutyl, and tert-butyl. In one aspect, R3 is a haloalkyl, e.g., a mono-haloalkyl, a di-haloalkyl, or a tri-haloalkyl. In one aspect, R3 is a C1-C4 alkyl optionally substituted with one or more halo. In one aspect, R3 is a halo C1-C4 alkyl such as trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, dichloromethyl, or chloromethyl. In one aspect, R3 is amide, which can be represented by the group “-C(O)NR5R5,” in which each R5 is independently hydro or alkyl, e.g., methyl or ethyl. In one aspect, the alkyl substituent includes from 1 and up to about 8 carbon atoms. In one aspect, the alkyl substituent includes from about 1 and up to about 4 carbon atoms. In one aspect, R3 is a primary amide. In one aspect, R3 is a secondary amide. In one aspect, R3 is a tertiary amide. In one aspect, R3 is a short chain ester, which can be represented by the group “-C(0)0R6” or “-CO2R6” where Re is hydro or C1-C4 alkyl, e.g., methyl or ethyl. In one aspect, R3 is cyano. In one aspect, R3 is alkyl substituted with one or more hydroxyl, as represented by “-(CH0H)nCH20H” wherein n is 0, 1, 2, or 3. In one aspect, R3 is methylhydroxyl, as represented by “-CH2OH.”

[0198] In one aspect, R3 is alkoxy, as represented by “-OR4.” In one aspect, R3 is a saturated alkoxy. In one aspect, R3 is an unsaturated alkoxy. In some embodiments, R4 is C1-C4 alkyl, e.g., R4 is methyl, ethyl, propyl, or butyl. In some embodiments, the alkoxy can be optionally substituted with one or more halo. In some aspects, R3 is alkoxy substituted with a hydroxyl or a ketone. In one aspect, R3 is alkoxy substituted with a halo group, i.e., a haloalkoxy, e.g., a mono-haloalkoxy, a di-haloalkoxy, or a tri-haloalkoxy. In one aspect, R3 is a straight chain alkoxy. In one aspect, R3 is a branched alkoxy. In one aspect, R3 is C1-C4 alkoxy (-OR4), where R4 is C1-C4 alkyl, e.g., methyl or ethyl. In one aspect, R3 is an alkoxy that includes, but is not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, including for example, n- butoxy, sec -butoxy iso-butoxy and t-butoxy. In one aspect, R3 is methoxy.

[0199] In one aspect, R7, R8, R9, Rio, or Rn, are each independently CHR12, CR12R17 or NR13. In at least one embodiment, all of R7, R8, R9, Rio, and Rn are CHR12, where R12 for each atom is independently chosen from hydro, C1-C4 alkyl, -OR14, or -CO2R15. In at least one embodiment, all of R7, R8, R9, Rio, and Rn are CHR12, where each R12 is hydro. In at least one embodiment, all of R7, R8, R9, Rio, and Rn are CHR12, where R12 is each independently chosen from hydro or an unsubstituted alkyl. In one aspect, R12 may be substituted with a halo group, i.e., a haloalkyl, e.g., a mono-haloalkyl, a di-haloalkyl, or a tri- haloalkyl. In one aspect, R12 may be a halo C1-C4 alkyl, for instance trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, dichloromethyl, and chloromethyl. In at least one embodiment, all of R7, R8, R9, Rio, and Rn are CHR12, where each R12 is independently chosen from hydro, C1-C4 alkyl, or -OR14, where R14 is further chosen from hydro or C1-C4 alkyl. In some embodiments, R12 is alkoxy, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, including for example, n-butoxy, sec -butoxy iso-butoxy and t-butoxy. In at least one embodiment, all of R7, R8, R9, Rio, and Rn are CHR12, where each R12 is independently chosen from hydro, C1-C4 alkyl, -OR14, or -CO2R15, where each of R14 and R15 is hydro or C1-C4 alkyl, e.g., methyl, ethyl, propyl, or butyl.

[0200] In one aspect, at least one of R7, R8, R9, Rio, and Rn is CR12R17, where R12 and R17 are each independently chosen from hydro or C1-C4 alkyl, e.g., methyl, ethyl, propyl, or butyl. In another embodiment, at least two of R7, R8, R9, Rio, and Rn are CR12R17, where R12 and R17 are each independently chosen from hydro or C1-C4 alkyl, e.g., methyl, ethyl, propyl, or butyl. In at least one aspect, R7 is CR12R17, where R12 is methyl and R17 is methyl, and R8, R9, Rio, and Rn are each independently chosen from CHR12 or NR13. In at least one aspect, Rs is CR12R17, where R12 is methyl and R17 is methyl, and R7, R9, Rio, and Rn are each independently chosen from CHR12 or NR13. In at least one aspect, R9 is CR12R17, where R12 is methyl and R17 is methyl, and R7, R8, Rio, and Rn are each independently chosen from CHR12 or NR13. In at least one aspect, Rio is CR12R17, where R12 is methyl and R17 is methyl, and R7, R8, R9, and Rn are each independently chosen from CHR12 or NR13. In at least one aspect, Rn is CR12R17, where R12 is methyl and R17 is methyl, and R7, R8, R9, and Rio are each independently chosen from CHR12 or NR13.

[0201] In one aspect, at least one of R7, R8, R9, Rio, and Rn is NR13, where R13 is hydro or C1-C4 alkyl, e.g., methyl, ethyl, propyl, or butyl. In another embodiment, at least two of R7, R8, R9, Rio, and Rn are NR13, where each of the two R13 groups are independently hydro or C1-C4 alkyl, e.g., methyl, ethyl, propyl, or butyl. In one aspect, R7 is NR13 and R8, R9, Rio, and Rn are CHR12, where R13 is hydro or C1-C4 alkyl, e.g., methyl, ethyl, propyl, or butyl and R12 is hydro. In one aspect, Rs is NR13 and R7, R9, Rio, and Rn are CHR12, where R13 is hydro or C1-C4 alkyl, e.g., methyl, ethyl, propyl, or butyl and R12 is hydro. In one aspect, R9 is NR13 and R7, R8, Rio, and Rn are CHR12, where R13 is hydro or C1-C4 alkyl, e.g., methyl, ethyl, propyl, or butyl and R12 is hydro. In one aspect, Rio is NR13 and R7, R8, R9, and Rn are CHR12 where R13 is hydro or C1-C4 alkyl, e.g., methyl, ethyl, propyl, or butyl and R12 is hydro. In one aspect, Rn is NR13 and R7, R8, R9, and Rio are CHR12 where R13 is hydro or C1-C4 alkyl, e.g., methyl, ethyl, propyl, or butyl and R12 is hydro.

[0202] In one aspect, R7 is NR13 and R8, R9, Rio, and Rn are CHR12, where R13 is hydro, C1-C4 alkyl, -(CH2)yOH, -OR14, -CO2R15, or -C(=O)Ri6, and R12 is hydro. In one aspect, Rs is NR13 and R7, R9, Rio, and Rn are CHR12, where R13 is hydro, C1-C4 alkyl, -(CH2)yOH, -OR14, -CO2R15, or -C(=O)Ri6, and R12 is hydro. In one aspect, R9 is NR13 and R7, R8, Rio, and Rn are CHR12, where R13 is hydro, C1-C4 alkyl, - -(CH2)yOH -,OR14, -CO2R15, or -C(=O)Ri6, and R12 is hydro. In one aspect, Rio is NR13 and R7, R8, R9, and Rn are CHR12, where R13 is hydro, C1-C4 alkyl, - -(CH2)yOH -,OR14, -CO2R15, or -C(=0)Ri6, and R12 is hydro. In one aspect, Rn is NR13 and R7, R8, R9, and Rio are CHR12, where R13 is hydro, C1-C4 alkyl, -(CH2)yOH, -OR14, -CO2R15, or -C(=O)Ri6, and R12 is hydro. In any of these instances, y is chosen from 0, 1, 2, or 3, and R14, R15, and Ri6 are each independently chosen from hydro and C1-C4 alkyl.

[0203] In one aspect, R9 is NR13 and R7, R8, Rio, and Rn are CHR12, where each R12 and R13 are hydro. In one aspect, R9 is NR13 and R7, R8, Rio, and Rn are CHR12, where each R12 is hydro, and R13 is C1-C4 alkyl. In one aspect, R9 is NR13 and R7, R8, Rio, and Rn are CHR12, where each R12 is hydro, and R13 is methyl.

[0204] In one aspect, R9 is NR13, where R13 is hydro; at least one of R7, R8, Rio, and Rn is CR12R17; where R12 and R17 are each C1-C4 alkyl; and the remaining of R7, R8, Rio, and Rn are CHR12, where each R12 is hydro, and R13 is hydro or C1-C4 alkyl. In one aspect, R9 is NR13, where R13 is hydro; R7, Rio, and Rn are CHR12, where R12 is hydro; and Rs is CR12R17, where each of R12 and R17 is methyl. In one aspect, R9 is NR13, where R13 is hydro; R7, R8, and Rn are CHR12; where R12 is hydro; and Rio is CR12R17, where each of R12 and R17 is methyl. In one aspect, R9 is NR13, where R13 is hydro; R7 and Rn are CHR12, where R12 is hydro; and Rs and Rio are CR12R17, where each of R12 and R17 is methyl.

[0205] In another aspect, the endosomal escape enhancer (EEE) compound has a structure represented by Formula la:

[0206] Formula la wherein one of Zi and Z2 is N, and the other is C; R1 is hydro, halo, C1-C4 alkyl, -OR4, -C(=O)NR5R5, -CO2R6, or cyano;

[0207] X is hydro, C1-C4 alkyl, or -OR2; R2 is hydro or C1-C4 alkyl;

[0208] R3 is hydro, halo, C1-C4 alkyl, -(CthjyOH, -OR4, -C(=O)NRsR8, -CO2R6, or cyano;

[0209] R4 is C1-C4 alkyl;

[0210] R5 is hydro or C1-C4 alkyl, e.g., methyl or ethyl;

[0211] R6 is hydro or C1-C4 alkyl, e.g., methyl or ethyl;

[0212] R16 is hydro or C1-C4 alkyl;

[0213] - denotes the conjugation point; and wherein one or more of the alkyl are optionally substituted with one or more halo, or a pharmaceutically acceptable salt thereof.

[0214] In another aspect, the endosomal escape enhancer (EEE) compound has a structure represented by Formula lb:

[0215] Formula lb wherein one of Z1 and Z2 is N, and the other is C; R1 is hydro, halo, C1-C4 alkyl, -OR4, -C(=O)NR5R5, -CO2R6, or cyano;

[0216] X is hydro, C1-C4 alkyl, or -OR2;

[0217] R2 is hydro or C1-C4 alkyl;

[0218] R3 is hydro, halo, C1-C4 alkyl, -(CthjyOH, -OR4, -C(=O)NR5R5, -CO2R6, or cyano;

[0219] R4 is C1-C4 alkyl;

[0220] Rs is hydro or C1-C4 alkyl, e.g., methyl or ethyl;

[0221] Re is hydro or C1-C4 alkyl, e.g., methyl or ethyl;

[0222] R16 is hydro or C1-C4 alkyl;

[0223] - denotes the conjugation point; and wherein one or more of the alkyl are optionally substituted with one or more halo, or a pharmaceutically acceptable salt thereof. In another aspect, the endosomal escape enhancer (EEE) compound has a structure represented by Formula Ic, Formula Id, Formula le or Formula If:

[0224] Formula le

[0225]

[0226] Formula If wherein R1 is hydro, halo, C1-C4 alkyl, -OR4, -C(=O)NR5R5, -CO2R6, or cyano;

[0227] X is hydro, C1-C4 alkyl, or -OR2;

[0228] R2 is hydro or C1-C4 alkyl;

[0229] R3 is hydro, halo, C1-C4 alkyl, -(CFhjyOH, -OR4, -C(=O)NR5R5, -CO2R6, or cyano;

[0230] R4 is C1-C4 alkyl;

[0231] Rs is hydro or C1-C4 alkyl, e.g., methyl or ethyl;

[0232] Re is hydro or C1-C4 alkyl, e.g., methyl or ethyl;

[0233] R16 is hydro or C1-C4 alkyl;

[0234] - denotes the conjugation point; and wherein one or more of the alkyl are optionally substituted with one or more halo, or a pharmaceutically acceptable salt thereof.

[0235] In another aspect, a compound of Formula la, Formula lb, Ic, Id, le or If is provided, wherein R1 is hydro, halo, C1-C4 alkyl, -OR4, -C(=O)NR5R5, -CO2R6, or cyano;

[0236] X is -OR2;

[0237] R2 is hydro or C1-C4 alkyl;

[0238] R3 is hydro, halo, C1-C4 alkyl, -OR4, -C(=O)NR5R5, -CO2R6, or cyano;

[0239] R4 is C1-C4 alkyl;

[0240] Rs is hydro, methyl or ethyl;

[0241] Re is hydro, methyl or ethyl; and

[0242] - denotes the conjugation point, wherein one or more of the alkyl are optionally substituted with one or more halo, or a pharmaceutically acceptable salt thereof.

[0243] In one aspect, R1 is hydro. In one aspect, R1 is halo, including, for example, chloro, fluoro, bromo, or iodo. In one aspect, R1 is bromo, chloro, or fluoro. In one aspect, R1 is fluoro. In one aspect, R1 is alkyl. In one aspect, R1 is a saturated alkyl. In one aspect, R1 is an unsaturated alkyl, e.g., an alkenyl or alkynyl. In some aspects, R1 can be polyunsaturated. In one aspect, R1 is a straight chain alkyl. In one aspect, R1 is a branched alkyl. In one aspect, Ri is C1-C4 alkyl. In one aspect, R1 is methyl, ethyl, propyl, isopropyl, or butyl. In one aspect, butyl includes n-butyl, sec-butyl, isobutyl, and tert-butyl. In one aspect, R1 is a substituted alkyl, e.g., substituted with one or more halo. In some embodiments, R1 is a C1-C4 alkyl, wherein at least one carbon of the alkyl is substituted with one or more chloro or fluoro. In some aspects, R1 is substituted with a hydroxyl or a ketone. In one aspect, R1 is an unsubstituted alkyl. In one aspect, R1 is substituted with a halo group, i.e., a haloalkyl, e.g., a mono-haloalkyl, a di-haloalkyl, or a tri-haloalkyl. In one aspect, R1 is a halo C1-C4 alkyl. In one aspect, R1 is a halo C1-C4 alkyl such as trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, dichloromethyl, and chloromethyl.

[0244] In one aspect, R1 is alkoxy, as represented by “-OR4”. In one aspect, R1 is a saturated alkoxy. In one aspect, R1 is an unsaturated alkoxy. In some embodiments, R4 is C1-C4 alkyl, e.g., R4 is methyl, ethyl, propyl, isopropyl, or butyl, including n-butyl, sec -butyl, isobutyl, and tert-butyl. In some embodiments, the alkoxy can be optionally substituted with one or more halo. In some aspects, R1 is alkoxy substituted with a hydroxyl, or a ketone. In one aspect, R1 is alkoxy substituted with a halo group, i.e., a haloalkoxy, e.g., a mono-haloalkoxy, a di-haloalkoxy, or a tri-haloalkoxy. In one aspect, R1 is a straight chain alkoxy. In one aspect, R1 is a branched alkoxy. In one aspect, R1 is C1-C4 alkoxy (-OR4), where R4 is C1-C4 alkyl, e.g., methyl or ethyl. In one aspect, R1 is an alkoxy that includes, but is not limited to, methoxy, ethoxy, propoxy, isopropoxy, or butoxy, including for example, n-butoxy, sec- butoxy iso-butoxy and t-butoxy.

[0245] In one aspect, R1 is amide, which can be represented by the group “-C(=O)NR5R5,” in which each R5 is independently hydro or alkyl. In some aspects, at least one R5 of “-C(=O)NR5R5” is hydro. In one aspect, R5 is C1-C4 alkyl, e.g., methyl, ethyl, propyl, or butyl. In one aspect, R1 is a primary amide. In one aspect, R1 is a secondary amide. In one aspect, R1 is a tertiary amide. In one aspect, R1 is -C(=O)NHR5.

[0246] In one aspect R1 is a short chain ester, which can be represented by the group “-C(O)OR6” or “-CO2R6” where Re is hydro or C1-C4 alkyl, e.g., methyl or ethyl. In one aspect, R1 is cyano.

[0247] In one aspect, X is hydro. In one aspect, X is C1-C4 alkyl, e.g., methyl, ethyl, propyl, or butyl. In one aspect, X is -OR2.

[0248] In one aspect, R2 is hydro. In some embodiments, R2is an alkyl. In one aspect, R2 is a saturated alkyl. In one aspect, R2 is an unsaturated alkyl. In one aspect, R2is a straight chain alkyl. In one aspect, R2 is a branched alkyl. In one aspect, R2 is C1-C4 alkyl. In one aspect, R2 is methyl, ethyl, propyl, isopropyl, or butyl. In one aspect, R2 is methyl. In one aspect, R2 is ethyl. In one aspect, butyl includes n-butyl, sec-butyl, isobutyl, and tert-butyl.

[0249] In one aspect, R3 is hydro. In some aspects, R3 is a halo, e.g., a chloro, fluoro or bromo. In one aspect, R3 is alkyl. In one aspect, R3 is a saturated alkyl. In one aspect, R3 is an unsaturated alkyl. In one aspect, R3 is a straight chain alkyl. In one aspect, R3 is a branched alkyl. In one aspect, R3 is C1-C4 alkyl. In one aspect, R3 is methyl, ethyl, propyl, isopropyl, or butyl. In one aspect, butyl includes n-butyl, sec-butyl, isobutyl, and tert-butyl. In one aspect, R3 is a haloalkyl, e.g., a mono-haloalkyl, a di-haloalkyl, or a tri-haloalkyl. In one aspect, R3 is a C1-C4 alkyl optionally substituted with one or more halo. In one aspect, R3 is a halo C1-C4 alkyl such as trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, dichloromethyl, or chloromethyl. In one aspect, R3 is amide, which can be represented by the group “-C(O)NR5R5,” in which each R5 is independently hydro or alkyl, e.g., methyl or ethyl. In one aspect, the alkyl substituent includes from 1 and up to about 8 carbon atoms. In one aspect, the alkyl substituent includes from about 1 and up to about 4 carbon atoms. In one aspect, R3 is a primary amide. In one aspect, R3 is a secondary amide. In one aspect, R3 is a tertiary amide. In one aspect, R3 is a short chain ester, which can be represented by the group “-C(O)OR6” or “-CO2R6” where R6 is hydro or C1-C4 alkyl, e.g., methyl or ethyl. In one aspect, R3 is cyano. In one aspect, R3 is alkyl substituted with one or more hydroxyl, as represented by “-(CH0H)11CH20H” wherein n is 0, 1, 2, or 3. In one aspect, R3 is methylhydroxyl, as represented by “-CH2OH.”

[0250] In one aspect, the endosomal escape enhancer (EEE) compounds of Formula la, lb, Ic, Id, le and If as described herein can include, by way of non-limiting example, any of the following as found in the Table 2A below:

[0251] Table 2A. Compounds of Formula la, lb, Ic, Id, le and If, where X is OR2

[0252] In one aspect, the endosomal escape enhancer (EEE) compound has a structure represented by Formula la, lb, Ic, Id, le or If, and wherein Ri, X and R3 are as found in Table 2B.

[0253] Table 2B.

[0254] In one aspect, the compound has a structure represented by Formula I, wherein Ri is halo, X is -OR2, Riis methyl, R3 is cyano, R7, R8, R10, and Rn are CHR12, where R12 is hydro, and R9 is NR13, where R13 is C1-C4 alkyl or -(CFDyOH, where y is 1-3. In one aspect, the compound has a structure represented by Formula I wherein Ri is fluoro, X is -OR2, R2is methyl, R3 is cyano, R7, R8, Rio, and Rn are CHR12, where R12 is hydro, and R9 is NR13, where R13 is C1-C4 alkyl.

[0255] In one aspect, the compound has a structure represented by Formula I wherein Ri is fluoro, X is -OR2, Riis methyl, R3 is cyano, R7, R8, Rio, and Rn are CHR12, where Rn is hydro, and R9 is NR13, where R13 is methyl.

[0256] In one aspect, the compound has a structure represented by Formula I wherein Zi is N, Z2 is C, R1 is fluoro, X is -OR2, R2 is methyl, R3 is cyano, R7, R8, R10, and R11 are CHR12, where R12 is hydro, and R9 is NR13, where R13 is hydro.

[0257] In one aspect, the compound has a structure represented by Formula I wherein Zi is N, Z2 is C, R1 is fluoro, X is -OR2, Riis ethyl, R3 is cyano, R7, R8, Rio, and Rn are CHR12, where R12 is hydro, and R9 is NR13, where R13 is methyl.

[0258] In one aspect, the compound has a structure represented by Formula I wherein Zi is N, Z2 is C, R1 is fluoro, X is -OR2, Riis methyl, R3 is cyano, R7, Rio, and Rn are CHR12, where R12 is hydro, Rs is CR12R17, where R12 and R17 are each methyl, and R9 is NR13, where R13 is methyl.

[0259] In one aspect, the compound has a structure represented by Formula I wherein Ri is fluoro, X is -OR2, R2is methyl, R3 is cyano, R7, R8, R10, and Rn are CHR12, where R12 is hydro, and R9 is NR13, where R13 is (CH2)yOH, where y is 1.

[0260] In one aspect, the compound has a structure represented by Formula I wherein Ri is fluoro, X is -OR2, R2is methyl, R3 is cyano, R7, R8, R10, and Rn are CHR12, where R12 is hydro, and R9 is NR13, where R13 is (CH2)yOH, where y is 2.

[0261] In one aspect, the compound has a structure represented by Formula I wherein Ri is fluoro, X is -OR2, R2 is methyl, R3 is cyano, R7, R8, Rio, and Rn are CHR12, where R12 is hydro, and R9 is NR13, where R13 is (CH2)yOH, where y is 3.

[0262] In one aspect, the compound has a structure represented by Formula la, lb, Ic, Id, le or If, wherein R1 is halo, X is -OR2, R2 is ethyl or methyl and R3 is cyano. In some embodiments, R1 is fluoro and R2 is ethyl. In some embodiments, R1 is fluoro and R2 is methyl. In some embodiments, R1 is chloro and R2 is ethyl. In some embodiments, Ri is chloro and R2 is methyl.

[0263] In one aspect, the compound has a structure represented by Formula la, lb, Ic, Id, le or If, wherein R1 is haloalkyl, X is -OR2, R2 is ethyl or methyl and R3 is cyano. In some embodiments, R1 is haloalkyl, e.g., trifluoromethyl, R2 is ethyl and R3 is cyano. In some embodiments, R1 is haloalkyl, e.g., trifluoromethyl, R2 is methyl and R3 is cyano In one aspect, the compound has a structure represented by Formula la, lb, Ic, Id, le or If, wherein R1 is halo, X is -OR2, R2 is ethyl or methyl and R3 is methyl or methoxy. In some embodiments, R1 is fluoro, R2 is ethyl and R3 is methyl. In some embodiments, R1 is fluoro, R2 is methyl and R3 is methoxy. In some embodiments, R1 is fluoro, R2 is methyl and R3 is methyl. In some embodiments, R1 is fluoro, R2 is ethyl and R3 is methoxy. In some embodiments, R1 is chloro, R2 is ethyl and R3 is methyl. In some embodiments, R1 is chloro, R2 is methyl and R3 is methoxy. In some embodiments, R1 is chloro, R2 is methyl and R3 is methyl. In some embodiments, R1 is chloro, R2 is ethyl and R3 is methoxy.

[0264] In one aspect, the compound has a structure represented by Formula la, lb, Ic, Id, le or If, wherein R1 is halo, X is -OR2, R2 is C1-C4 alkyl and R3 is hydro. In some embodiments, Ri is fluoro, R2 is methyl and R3 is hydro. In some embodiments, R1 is chloro, R2 is methyl and R3 is hydro. In some embodiments, R1 is bromo, R2 is methyl and R3 is hydro. In some embodiments, R1 is fluoro, R2 is ethyl and R3 is hydro. In some embodiments, R1 is chloro, R2 is ethyl and R3 is hydro. In some embodiments, R1 is bromo, R2 is ethyl and R3 is hydro. In some embodiments, R1 is fluoro, R2 is propyl, e.g., isopropyl and R3 is hydro. In some embodiments, R1 is chloro, R2 is propyl, e.g., isopropyl and R3 is hydro. In some embodiments, R1 is bromo, R2 is propyl, e.g., isopropyl and R3 is hydro.

[0265] In one aspect, the compound has a structure represented by Formula la, lb, Ic, Id, le or If, wherein R1 is haloalkyl, X is -OR2, R2 is ethyl or methyl and R3 is hydro. In some embodiments, R1 is trifluoromethyl, R2 is methyl and R3 is hydro. In some embodiments, Ri is trifluoromethyl, R2 is ethyl and R3 is hydro. In some embodiments, R1 is trifluoromethyl, R2 is methyl and R3 is hydro. In some embodiments, R1 is trifluoromethyl, R2 is ethyl and R3 is hydro.

[0266] In one aspect, the compound has a structure represented by Formula la, lb, Ic, Id, le or If, wherein R1 is halo, X is hydro and R3 is cyano. In some embodiments, R1 is fluoro. In some embodiments, R1 is chloro. In some embodiments, R1 is bromo.

[0267] In one aspect, the compound has a structure represented by Formula la, lb, Ic, Id, le or If, wherein R1 is cyano, X is -OR2, R2 is ethyl or methyl and R3 is haloalkyl. In some embodiments, R1 is cyano, R2 is methyl and R3 is trifluoromethyl. In some embodiments, Ri is cyano, R2 is ethyl and R3 is trifluoromethyl.

[0268] In one aspect, the compound has a structure represented by Formula la, lb, Ic, Id, le or If, wherein R1 is ethyl or methyl, X is -OR2, R2 is ethyl or methyl and R3 is hydro. In some embodiments, R1 is methyl, R2 is methyl and R3 is hydro. In some embodiments, R1 is methyl, R2 is ethyl and R3 is hydro. In some embodiments, R1 is ethyl, R2 is methyl and R3 is hydro. In some embodiments, R1 is ethyl, R2 is ethyl and R3 is hydro.

[0269] In one aspect, the compound has a structure represented by Formula la, lb, Ic, Id, le or If, wherein R1 is halo, X is -OR2, R2 is ethyl or methyl and R3 is -(CFDyOH, wherein y is 0, 1, 2, or 3. In some embodiments, R1 is fluoro, R2 is methyl and R3 is -OH. In some embodiments, R1 is fluoro, R2 is methyl and R3 is -CH2OH. In some embodiments, Ri is fluoro, R2 is methyl and R3 is -(CH2)2OH. In some embodiments, R1 is chloro, R2 is methyl and R3 is -OH. In some embodiments, R1 is chloro, R2 is methyl and R3 is -CH2OH. In some embodiments, R1 is chloro, R2 is methyl and R3 is -(CH2)2OH. In some embodiments, Ri is chloro, R2 is methyl and R3 is -(CH2)3OH. In some embodiments, R1 is fluoro, R2 is ethyl and R3 is -OH. In some embodiments, R1 is fluoro, R2 is ethyl and R3 is -CH2OH. In some embodiments, R1 is fluoro, R2 is ethyl and R3 is -(CH2)2OH. In some embodiments, Ri is fluoro, R2 is ethyl and R3 is -(CH2)3OH. In some embodiments, R1 is chloro, R2 is ethyl and R3 is -OH. In some embodiments, R1 is chloro, R2 is ethyl and R3 is -CH2OH. In some embodiments, R1 is chloro, R2 is ethyland R3 is -(CH2)2OH. In some embodiments, Ri is chloro, R2 is ethyl and R3 is -(CH2)3OH.

[0270] In one aspect, the compound has a structure represented by Formula la, lb, Ic, Id, le or If, wherein R1 is fluoro, X is -OR2, R2is methyl and R3 is cyano.

[0271] In some embodiments, the compound comprises a structure as shown in Table 1. In some embodiments, the compound is any one of Compounds 2-7 as shown in Table 1. In some embodiments, the compound is Compound 4 as shown in Table 1. In some embodiments, the compound is Compound 8 as shown in Table 1. In some embodiments, the compound is Compound 32 as shown in Table 1. In some embodiments, the compound is any one of Compounds 32a or 32b as shown below.

[0272] In one aspect, the compounds represented by Formula I, la, lb, Ic, Id, le or Ifmay have one or more stereocenters and may exist as racemates or racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. Stereoisomers may be separated using conventional techniques, e.g., chromatography or fractional crystallization, or the stereoisomers may be made by stereoselective synthesis. For instance, Compounds 32-35 contain at least one stereocenter and their individual isomers (further depicted below) are all disclosed herein. Compound 32a:

[0273] (S)-l-(2-((l-(2,2-dimethylpiperidin-4-yl)-3-methoxy-lH-pyrazol-4-yl)amino)-5- fluoropyrimidin-4-yl)- 1 H-indolc-4-carbonitrilc

[0274] (7?)-l-(2-((l-(2,2-dimethylpiperidin-4-yl)-3-methoxy-lH-pyrazol-4-yl)amino)-5- fluoropyrimidin-4-yl)-lH-indole-4-carbonitrile;

[0275] Compound (S)- l-(5-fluoro-2-((3-methoxy- l-(piperidin-3-yl)- lH-pyrazol-4-yl)amino)pyrimidin-4-yl)- lH-indole-4-carbonitrile

[0276]

[0277] (R)-l-(5-fluoro-2-((3-methoxy-l-(piperidin-3-yl)-lH-pyrazol-4-yl)amino)pyrimidin-4-yl)- lH-indole-4-carbonitrile; l-(5-fluoro-2-((3-methoxy-l-((2R,4R)-2-methylpiperidin-4-yl)-lH-pyrazol-4- yl)amino)pyrimidin-4-yl)-lH-indole-4-carbonitrile l-(5-fluoro-2-((3-methoxy-l-((2S,4R)-2-methylpiperidin-4-yl)-lH-pyrazol-4- yl)amino)pyrimidin-4-yl)-lH-indole-4-carbonitrile

[0278] l-(5-fluoro-2-((3-methoxy-l-((2R,4S)-2-methylpiperidin-4-yl)-lH-pyrazol-4- yl)amino)pyrimidin-4-yl)-lH-indole-4-carbonitrile l-(5-fluoro-2-((3-methoxy-l-((2S,4S)-2-methylpiperidin-4-yl)-lH-pyrazol-4- yl)amino)pyrimidin-4-yl)-lH-indole-4-carbonitrile;

[0279] Compound l-(5-fluoro-2-((3-methoxy-l-((3R,4S)-3-methylpiperidin-4-yl)-lH-pyrazol-4- yl)amino)pyrimidin-4-yl)-lH-indole-4-carbonitrile Compound l-(5-fluoro-2-((3-methoxy-l-((3S,4S)-3-methylpiperidin-4-yl)-lH-pyrazol-4- yl)amino)pyrimidin-4-yl)-lH-indole-4-carbonitrile Compound l-(5-fluoro-2-((3-methoxy-l-((3R,4R)-3-methylpiperidin-4-yl)-lH-pyrazol-4- yl)amino)pyrimidin-4-yl)-lH-indole-4-carbonitrile l-(5-fluoro-2-((3-methoxy-l-((3S,4R)-3-methylpiperidin-4-yl)-lH-pyrazol-4- yl)amino)pyrimidin-4-yl)-lH-indole-4-carbonitrile. G. Compositions

[0280] In one aspect, a composition is provided that includes a macromolecule conjugated to one or more compounds described herein. In some aspects, the macromolecule comprises an oligonucleotide. In some aspects, the macromolecule is an antisense oligonucleotide. In some aspects, the macromolecule is a siRNA. In some aspects, the siRNA is conjugated to a lipid and / or a sugar. In some aspects, the macromolecule comprises a polypeptide. In some aspects, the macromolecule comprises one or more components of a SSM system described herein. In some aspects, the SSM system is a CRISPR system. In some aspects, the SSM system is a Cre-Lox system. In some aspects, the SSM system is a FLP-FRT system. In some aspects, the macromolecule comprises a Cas protein and / or a guide RNA. In some aspects, the macromolecule comprises Cre. In some aspects, the macromolecule comprises FLP.

[0281] In one aspect, a composition is provided that includes an oligonucleotide and / or polypeptide conjugated to one or more compounds described herein. In one aspect, a composition is provided that includes an oligonucleotide and / or polypeptide conjugated to one or more compounds represented by Formula I, or a pharmaceutically acceptable salt thereof. In one aspect, a composition is provided that includes an oligonucleotide and / or polypeptide conjugated to one or more compounds represented by Formula la, or a pharmaceutically acceptable salt thereof. In one aspect, a composition is provided that includes an oligonucleotide and / or polypeptide conjugated to one or more compounds represented by Formula lb, or a pharmaceutically acceptable salt thereof. In one aspect, a composition is provided that includes an oligonucleotide and / or polypeptide conjugated to one or more compounds represented by Formula Ic, or a pharmaceutically acceptable salt thereof. In one aspect, a composition is provided that includes an oligonucleotide and / or polypeptide conjugated to one or more compounds represented by Formula Id, or a pharmaceutically acceptable salt thereof. In one aspect, a composition is provided that includes an oligonucleotide and / or polypeptide conjugated to one or more compounds represented by Formula le, or a pharmaceutically acceptable salt thereof. In one aspect, a composition is provided that includes an oligonucleotide and / or polypeptide conjugated to one or more compounds represented by Formula If, or a pharmaceutically acceptable salt thereof. In one aspect, a composition is provided that includes an oligonucleotide and / or polypeptide conjugated to one or more of Compounds 2-35, or a pharmaceutically acceptable salt thereof. In one aspect, a composition is provided that includes an oligonucleotide and / or polypeptide conjugated to one or more of Compounds 4, 32, and 33, or a pharmaceutically acceptable salt thereof. In one aspect, a pharmaceutical composition is provided that includes an oligonucleotide and / or polypeptide conjugated to one or more compounds described herein, and a pharmaceutically acceptable carrier or diluent. In one aspect, a pharmaceutical composition is provided that includes an oligonucleotide and / or polypeptide conjugated to one or more compounds represented by any one of Formulae I, la, lb, Id, le, andlf, or one or more compounds listed in Table 1, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier or diluent. In one aspect, a pharmaceutical composition is provided that includes an oligonucleotide and / or polypeptide conjugated to one or more of Compounds 2-35, a pharmaceutically acceptable salt thereof, or combinations thereof; and a pharmaceutically acceptable carrier or diluent. In one aspect, a pharmaceutical composition is provided that includes an oligonucleotide and / or polypeptide conjugated to one or more of Compounds 4, 32, and 33, a pharmaceutically acceptable salt thereof, or combinations thereof; and a pharmaceutically acceptable carrier or diluent. The pharmaceutical composition can be formulated to be compatible with the intended route of administration, including, but not limited to, parenteral administration, such as intravenous (IV), or subcutaneous (SC or SQ) administration; intraperitoneal, intramuscular, oral, transdermal, or transmucosal administration.

[0282] In one aspect, a composition is provided that includes an oligonucleotide. In one aspect, a composition is provided that includes an antisense oligonucleotide (ASO). In one aspect, a composition is provided that includes a splice switching oligonucleotide (SSO). In one aspect, a composition is provided that includes siRNA. In some aspects, the siRNA is conjugated to a lipid and / or a sugar. In one aspect, a composition is provided that includes a guide RNA. In one aspect, a composition is provided that includes about 0.025 μM to about 20 μM oligonucleotide. In one aspect, the composition includes at least about 0.1 μM and up to about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 15 μM, or about 20 μM oligonucleotide. In one aspect, the composition includes from about 0.1 μM to about 10 μM, about 0.1 μM to about 5 μM, or about 0.1 μM to about 1 μM oligonucleotide.

[0283] In one aspect, a composition is provided that includes a polypeptide. In one aspect, a composition is provided that includes a Cas protein, e.g., a Cas9 or Casl2a protein. In some aspects, the Cas protein is a modified Cas protein or a Cas fusion protein as described herein. In one aspect, a composition is provided that includes Cre. In one aspect, a composition is provided that includes FLP. In one aspect, a composition is provided that includes about 0.001 μM to about 20 μM polypeptide. In one aspect, the composition includes at least about 0.01 μM and up to about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 15 μM, or about 20 μM polypeptide. In one aspect, the composition includes from about 0.01 μM to about 10 μM, about 0.05 μM to about 5 μM, or about 0.1 μM to about 1 μM polypeptide.

[0284] In one aspect, a composition is provided that includes an oligonucleotide and a polypeptide. In one aspect, a composition is provided that includes a Cas protein and a guide RNA. In some aspects, the Cas protein is Cas9. In some aspects, the Cas protein is Cas 12a. In some aspects, the Cas protein is a modified Cas protein or a Cas fusion protein as described herein. In one aspect, a composition is provided that includes about 0.001 μM to about 20 μM of each of the oligonucleotide and the polypeptide. In one aspect, the composition includes at least about 0.1 μM and up to about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 15 μM, or about 20 μM of each of the oligonucleotide and the polypeptide. In one aspect, the composition includes from about 0.01 μM to about 10 μM, about 0.05 μM to about 5 μM, or about 0.1 μM to about 1 μM of each of the oligonucleotide and the polypeptide.

[0285] In one aspect, a composition is provided that includes one or more compounds described herein. In one aspect, a composition is provided that includes a small molecule compound described herein. In one aspect, a composition is provided that includes one or more compounds represented by Formulae I, la, lb, Ic, Id, le or If, or the compounds listed in Table 1, or a pharmaceutically acceptable salt thereof. In one aspect, a composition is provided that includes one or more compounds represented by any one of Formulae I, la, lb, Ic, Id, le or If, or the compounds listed in Table 1, a pharmaceutically acceptable salt thereof, or combinations thereof. In one aspect, a composition is provided that includes one or more of Compounds 4, 32, and 33, or a pharmaceutically acceptable salt thereof. In one aspect, a composition is provided that includes one or more compounds of Compounds 4, 32, and 33, a pharmaceutically acceptable salt thereof, or combinations thereof.

[0286] The “compositions comprising the oligonucleotides and / or polypeptides” and / or the “compositions comprising the compounds of Formulae I, la, lb, Ic, Id, le or If, or the compounds listed in Table 1” can include a biological cell, e.g., an in vitro cell. For example, the compositions comprising the oligonucleotides, polypeptides, and / or the compounds of Formula I, Formula la, lb, Ic, Id, le or If, or the compounds listed in Table 1 can include an in vitro cell culture, e.g., a mammalian cell culture. In another aspect, the compositions comprising the oligonucleotides, polypeptides, and / or the compounds of Formula I, Formula la, lb, Ic, Id, le or If, or the compounds listed in Table 1 can include an in vitro isolated cell from a subject, e.g., a mammalian subject, e.g., a human subject.

[0287] H. Methods of treatment

[0288] In one aspect, a method is provided for introducing a macromolecule into a cell, e.g., a target cell described herein. In one aspect, the cell is a cultured cell. In one aspect, the cell is an isolated cell. In one aspect, the cell is an isolated cell from a subject in need of treatment. In one aspect, the cell is a mammalian cell. In one aspect, the cell is a eukaryotic cell and / or a prokaryotic cell.

[0289] In one aspect, the cell is part of a tissue or organ of a mammal, e.g., a human. In one aspect, the organ or tissue is the brain, central nervous system (CNS) or peripheral nervous system (PNS), heart, liver, kidney, spleen, pancreas, lung, adipose, and / or muscle (e.g., skeletal muscle). In one aspect, the cell is a brain cell, a CNS cell, a PNS cell, a heart cell, a liver cell, a kidney cell, a spleen cell, a pancreas cell, a lung cell, a muscle cell, an adipose cell, an immune cell, or combination thereof.

[0290] In some aspects, the cell is a CNS cell. In some aspects, the CNS cell comprises a glial cell and / or a neuron. Glial cells of the CNS include, e.g., astrocytes, oligodendrocytes, microglia, and ependymal cells. Neurons include, e.g., afferent neurons, efferent neurons, and interneurons.

[0291] In some aspects, the cell is a liver cell, e.g., a hepatocyte or a non-parenchymal cell. In some aspects, the cell comprises a plateable metabolism qualified human hepatocyte, a plateable induction qualified human hepatocyte, plateable human hepatocyte, suspension qualified human hepatocyte (including 10-donor and 20-donor pooled hepatocytes), human hepatic Kupffer cells, human hepatic stellate cells, dog hepatocytes (including single and pooled Beagle hepatocytes), mouse hepatocytes (including CD-I and C57BI / 6 hepatocytes), rat hepatocytes (including Sprague-Dawley, Wistar Han, and Wistar hepatocytes), monkey hepatocytes (including Cynomolgus or Rhesus monkey hepatocytes), cat hepatocytes (including Domestic Shorthair hepatocytes), and rabbit hepatocytes (including New Zealand White hepatocytes).

[0292] In some aspects, the cell is a human stem cell. The stem cells can be, for example, pluripotent stem cells, including embryonic stem cells (ESCs), adult stem cells, induced pluripotent stem cells (iPSCs), tissue specific stem cells (e.g., hematopoietic stem cells) and mesenchymal stem cells (MSCs). In some aspects, the cell is a differentiated form of any of the cells described herein. In some aspects, the eukaryotic cell is a cell derived from any primary cell in culture.

[0293] In some aspects, the cell is an immune cell. Non-limiting examples of immune cells include T cells, B cells, dendritic cells, NK cells, T helper cells, cytotoxic T cells, regulatory T cells, gamma delta T cells, neutrophils, mast cells, monocytes, antigen-presenting cells, lymphocytes, basophils, and phagocytes.

[0294] In one aspect, a method is provided for introducing an oligonucleotide into a cell. In one aspect, a method is provided for introducing a polypeptide into a cell. In one aspect, a method is provided for introducing an oligonucleotide into a nucleus and / or cytosol of a cell. In one aspect, a method is provided for introducing a polypeptide into a nucleus and / or cytosol of a cell. In one aspect, the oligonucleotide is ASO, SSO, and / or siRNA. In one aspect, the oligonucleotide is siRNA, wherein the siRNA is unconjugated. In one aspect, the oligonucleotide is siRNA, wherein the siRNA is conjugated to a lipid and / or a sugar. In one aspect, the oligonucleotide is a guide RNA. In one aspect, the oligonucleotide hybridizes to a target nucleic acid in the cell. In one aspect, the polypeptide is capable of providing a SSM in a target nucleic acid in the cell. In one aspect, the target nucleic acid is in the nucleus of the cell. In one aspect, the target nucleic acid is in cytosol of the cell.

[0295] In one aspect, the method includes contacting the cell with the oligonucleotide and / or polypeptide conjugated to one or more compounds described herein. In one aspect, the oligonucleotide is an antisense oligonucleotide (ASO). In one aspect, the oligonucleotide is a splice switching oligonucleotide (SSO). In one aspect, the oligonucleotide is siRNA. In some aspects, the siRNA is conjugated to a lipid and / or a sugar. In one aspect, the cell is contacted with an oligonucleotide. In one aspect, the cell is contacted with a polypeptide. In one aspect, the cell is contacted with both an oligonucleotide and a polypeptide. In one aspect, the oligonucleotide is a guide RNA. In one aspect, the polypeptide is a recombinase. In one aspect, the polypeptide is Cre. In one aspect, the polypeptide is FLP. In one aspect, the polypeptide is a Cas protein. In one aspect, the polypeptide is a Cas protein and the oligonucleotide is a guide RNA. In one aspect, the compound is a small molecule compound (SMC). In one aspect, the compound is an endosomolytic compound. In one aspect, the compound has a structure represented by Formula I, or a pharmaceutically acceptable salt thereof. In one aspect, the compound has a structure represented by Formula la, or a pharmaceutically acceptable salt thereof. In one aspect, the compound has a structure represented by Formula lb, or a pharmaceutically acceptable salt thereof. In one aspect, the compound has a structure represented by Formula Ic, or a pharmaceutically acceptable salt thereof. In one aspect, the compound has a structure represented by Formula Id, or a pharmaceutically acceptable salt thereof. In one aspect, the compound has a structure represented by Formula le, or a pharmaceutically acceptable salt thereof. In one aspect, the compound has a structure represented by Formula If, or a pharmaceutically acceptable salt thereof. In one aspect, the compound has a structure represented by Formula I, Formula la, lb, Ic, Id, le or If, or the compounds listed in Table 1, or a pharmaceutically acceptable salt thereof. In one aspect, the compound is any one of Compounds 4, 32, and 33, or a pharmaceutically acceptable salt thereof.

[0296] In one aspect, the method includes in vitro delivery of the macromolecule, e.g., oligonucleotide and / or polypeptide conjugated to one or more of the compounds to the cell. In one aspect, the method includes in vivo delivery of the macromolecule, e.g., oligonucleotide and / or polypeptide conjugated to one or more of the compounds to the cell. In one aspect, the cell is a cultured cell. In one aspect, the cell is an isolated cell from a patient in need of treatment. In one aspect, the cell is part of a tissue or organ. In one aspect, the cell is a mammalian cell. In one aspect, the cell is a human cell. In one aspect the cell is a eukaryotic and / or a prokaryotic cell.

[0297] In one aspect, the macromolecule, e.g., oligonucleotide and / or polypeptide, is internalized by the cell through endocytosis and encapsulated within an endosome. In one aspect, the conjugated compound(s) facilitates release of the macromolecule, e.g., oligonucleotide and / or polypeptide, from the endosome.

[0298] In one aspect, the macromolecule, e.g., oligonucleotide and / or polypeptide, is internalized by the cell through transient pore formation. In one aspect, the conjugated compound facilitates transient pore formation in the plasma membrane.

[0299] In one aspect, the cell is contacted with a composition that includes an oligonucleotide. In one aspect, the cell is contacted with a composition that includes an antisense oligonucleotide (ASO). In one aspect, the cell is contacted with a composition that includes a splice switching oligonucleotide (SSO). In one aspect, the cell is contacted with a composition that includes a siRNA. In some aspects, the siRNA is conjugated to a lipid and / or a sugar. In some aspects, the siRNA is unconjugated. In one aspect, the cell is contacted with a composition that includes a guide RNA. In one aspect, the cell is contacted with a composition that includes oligonucleotide in an amount sufficient to provide a therapeutic effect. In one aspect, the cell is contacted with a composition that includes oligonucleotide in an amount sufficient to provide a SSM at a target nucleic acid. In one aspect, the cell is contacted with a composition that includes about 0.025 μM to about 20 μM oligonucleotide. In one aspect, the cell is contacted with a composition that includes at least about 0.1 μM and up to about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 15 μM, or about 20 μM, oligonucleotide. In one aspect, the cell is contacted with a composition that includes from about 0.1 μM to about 10 μM, about 0.1 μM to about 5 μM, or about 0.1 μM to about 1 μM oligonucleotide.

[0300] In one aspect, the cell is contacted with a composition that includes a polypeptide. In one aspect, the cell is contacted with a composition that includes a Cas protein. In one aspect, the cell is contacted with a composition that includes a recombinase, e.g., Cre or FLP. In one aspect, the cell is contacted with a composition that includes polypeptide in an amount sufficient to provide a therapeutic effect. In one aspect, the cell is contacted with a composition that includes polypeptide in an amount sufficient to provide a SSM at a target nucleic acid. In one aspect, the cell is contacted with a composition that includes about 0.3 μM to about 20 μM polypeptide. In one aspect, the cell is contacted with a composition that includes at least about 0.01 μM and up to about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 15 μM, or about 20 μM, polypeptide. In one aspect, the cell is contacted with a composition that includes from about 0.01 μM to about 10 μM, about 0.05 μM to about 5 μM, or about 0.1 μM to about 1 μM polypeptide.

[0301] In one aspect, the cell is contacted with a composition that includes an oligonucleotide and a polypeptide. In one aspect, the cell is contacted with a composition that includes a Cas protein and a guide RNA. In one aspect, the cell is contacted with a composition that includes oligonucleotide and polypeptide in an amount sufficient to provide a therapeutic effect. In one aspect, the cell is contacted with a composition that includes oligonucleotide and polypeptide in an amount sufficient to provide a SSM at a target nucleic acid. In one aspect, the cell is contacted with a composition that includes about 0.001 μM to about 20 μM of each of an oligonucleotide (e.g., guide RNA) and a polypeptide (e.g., Cas protein). In one aspect, the cell is contacted with a composition that includes at least about 0.01 μM and up to about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 15 μM, or about 20 μM of each of the oligonucleotide and polypeptide. In one aspect, the cell is contacted with a composition that includes from about 0.01 μM to about 10 μM, about 0.05 μM to about 5 μM, or about 0.1 μM to about 1 μM of each of the oligonucleotide and polypeptide. In one aspect, the cell is contacted with a composition that includes one or more compounds described herein. In one aspect, the cell is contacted with a composition that includes one or more compounds represented by Formula I or a pharmaceutically acceptable salt thereof. In one aspect, the cell is contacted with a composition that includes one or more compounds represented by Formula I, Formula la, lb, Ic, Id, le or If, or the compounds listed in Table 1, a pharmaceutically acceptable salt thereof, or combinations thereof. In one aspect, the cell is contacted with a composition that includes any one of Compounds 4, 32, and 33, a pharmaceutically acceptable salt thereof, or combinations thereof. In one aspect, the cell is contacted with a composition at a concentration sufficient to (i) reduce accumulation of the oligonucleotide in endosomes and / or lysosomes, (ii) induce physical rupture of endosomes, and / or (iii) facilitate transient pore formation in the plasma membrane, thereby facilitating release of a macromolecule, e.g., an oligonucleotide and / or polypeptide, into the cytosol or nucleus.

[0302] In one aspect, contacting the cell with one or more compounds described herein results in endosomal membrane permeabilization as determined by mCherry-GAL9 recruitment assay. In one aspect, contacting the cell with one or more compounds with a structure shown in Formula I, or a pharmaceutically acceptable salt thereof results in endosomal membrane permeabilization as determined by mCherry-GAL9 recruitment assay. In one aspect, contacting the cell with one or more compounds with a structure shown in Formula I, Formula la, lb, Ic, Id, le or If, or the compounds listed in Table 1, or a pharmaceutically acceptable salt thereof, results in membrane permeabilization as determined by mCherry-GAL9 recruitment assay. In one aspect, contacting the cell with one or more compounds of any one of Compounds 2-35, or a pharmaceutically acceptable salt thereof, results in membrane permeabilization as determined by mCherry-GAL9 recruitment assay. In one aspect, contacting the cell with one or more compounds with the structure of Compound 32, or a pharmaceutically acceptable salt thereof, results in membrane permeabilization as determined by mCherry-GAL9 recruitment assay. In one aspect, contacting the cell with one or more compounds with the structure of Compound 33, or a pharmaceutically acceptable salt thereof, results in membrane permeabilization as determined by mCherry-GAL9 recruitment assay. In one aspect, contacting the cell with one or more compounds with a structure shown in Formula I, Formula la, lb, Ic, Id, le or If, or the compounds listed in Table 1, or a pharmaceutically acceptable salt thereof, results in endosomal membrane permeabilization as determined by mCherry-GAL9 recruitment assay. In one aspect, the macromolecule, e.g., oligonucleotide and / or polypeptide, alters activity of a gene expressed by the cell. In one aspect, the gene is an endogenous gene. In one aspect, the gene is an exogenous gene. In one aspect, the macromolecule, e.g., oligonucleotide and / or polypeptide, increases activity of a gene expressed by the cell. In one aspect, the activity of the gene expressed by the cell is increased at least about lOx when the cell is contacted with the macromolecule, e.g., oligonucleotide and / or polypeptide conjugated to the compound(s) as compared to a cell that is contacted with the macromolecule, e.g., oligonucleotide and / or polypeptide not conjugated to the compound(s). In one aspect, the activity of the gene expressed by the cell is increased at least about lOx, about 20x, about 30x, about 40x or about 50x and up to about lOOx, 200x, 300x, 400x, 500x or lOOOx. In one aspect, the activity of the gene expressed by the cell is increased from about lOx to about lOOOx, about lOx to about 500x, about lOx to about 400x, about lOx to about 300x, about lOx to about 200x, about lOx to about lOOx, about lOOx to about 300x, or about lOOx to about 200x. In one aspect, the activity of the gene expressed by a cell is increased from about lOx to about lOOOx when the cell is contacted with the macromolecule, e.g., oligonucleotide and / or polypeptide conjugated to the compound(s) as compared to a cell that is contacted with the macromolecule, e.g., oligonucleotide and / or polypeptide not conjugated to the compound(s). In one aspect, the activity of the gene expressed by a cell is increased from about lOOx to about 500x. In one aspect, the activity of the gene expressed by a cell is increased from about lOOx to about 300x. In one aspect, the activity of the gene expressed by a cell is increased from about lOOx to about 200x. In one aspect, the activity of the gene expressed by a cell is increased from about lOx to about 500x. In one aspect, the activity of the gene expressed by a cell is increased from about lOx to about 300x. In one aspect, the activity of the gene expressed by a cell is increased from about lOx to about 200x. In one aspect, the activity of the gene expressed by a cell is increased from about lOx to about lOOx. In one aspect, the activity of the gene expressed by a cell is increased from about lOx to about 50x.

[0303] In one aspect, the macromolecule, e.g., oligonucleotide and / or polypeptide, decreases the activity of a gene expressed by the cell. In one aspect, the activity of the gene expressed by the cell is decreased at least about lOx when the cell is contacted with the macromolecule, e.g., oligonucleotide and / or polypeptide conjugated to the compound(s) as compared to a cell that is contacted with the macromolecule, e.g., oligonucleotide and / or polypeptide not conjugated to the compound(s). In one aspect, the activity of the gene expressed by the cell is decreased at least about lOx, about 20x, about 30x, about 40x or about 50x and up to about lOOx, 200x, 3OOx, 400x, 5OOx or lOOOx. In one aspect, the activity of the gene expressed by the cell is decreased from about lOx to about lOOOx, about lOx to about 500x, about lOx to about 400x, about lOx to about 300x, about lOx to about 200x, about lOx to about lOOx, about lOOx to about 300x, or about lOOx to about 200x. In one aspect, the activity of the gene expressed by a cell is decreased from about lOx to about lOOOx when the cell is contacted with the macromolecule, e.g., oligonucleotide and / or polypeptide conjugated to the compound(s) as compared to a cell that is contacted with the macromolecule, e.g., oligonucleotide and / or polypeptide not conjugated to the compound(s). In one aspect, the activity of the gene expressed by a cell is decreased from about lOOx to about 500x. In one aspect, the activity of the gene expressed by a cell is decreased from about lOOx to about 300x. In one aspect, the activity of the gene expressed by a cell is decreased from about lOOx to about 200x. In one aspect, the activity of the gene expressed by a cell is decreased from about lOx to about 500x. In one aspect, the activity of the gene expressed by a cell is decreased from about lOx to about 300x. In one aspect, the activity of the gene expressed by a cell is decreased from about lOx to about 200x. In one aspect, the activity of the gene expressed by a cell is decreased from about lOx to about lOOx. In one aspect, the activity of the gene expressed by a cell is decreased from about lOx to about 50x.

[0304] In one aspect, a method is provided for altering expression of a target nucleic acid in a cell. In one aspect, the method includes: contacting the cell with an oligonucleotide that is capable of hybridizing to the target nucleic acid, wherein the oligonucleotide is conjugated to one or more compounds described herein and is internalized by the cell through endocytosis and encapsulated within an endosome, wherein the compound facilitates release of the oligonucleotide from the endosome, wherein the hybridization of the oligonucleotide to the target nucleic acid alters expression of the target nucleic acid. In one aspect, hybridization of the oligonucleotide to the target nucleic acid increases expression of the target nucleic acid. In one aspect, hybridization of the oligonucleotide to the target nucleic acid decreases expression of the target nucleic acid. In some embodiments, the oligonucleotide is an ASO. In some embodiments, the oligonucleotide is an SSO. In some embodiments, the oligonucleotide is an siRNA. In some embodiments, the oligonucleotide is conjugated to a lipid and / or a sugar and / or a peptide. In some embodiments, the compound has a structure represented by Formula I, Formula la, lb, Ic, Id, le or If, or is a compound listed in Table 1, or a pharmaceutically acceptable salt thereof, or combinations thereof. In one aspect, the compound is any one of Compounds 4, 32, and 33, a pharmaceutically acceptable salt thereof, or combinations thereof. In one aspect, a method is provided for altering expression of a target nucleic acid in a cell. In one aspect, the method includes: contacting the cell with a polypeptide, conjugated to one or more compounds described herein, that is capable of performing a SSM at the target nucleic acid, wherein the polypeptide is internalized by the cell through endocytosis and encapsulated within an endosome, wherein the compound facilitates release of the polypeptide from the endosome, wherein the SSM at the target nucleic acid alters expression of the target nucleic acid. In one aspect, the SSM increases expression of the target nucleic acid. In one aspect, the SSM decreases expression of the target nucleic acid. In some embodiments, the polypeptide is a Cas protein. In some embodiments, the polypeptide is a recombinase / meganuclease. In some embodiments, the polypeptide is Cre or FLP. In some embodiments, the compound has a structure represented by Formula I, Formula la, lb, Ic, Id, le or If, or is one or more compounds listed in Table 1, or a pharmaceutically acceptable salt thereof, or combinations thereof. In one aspect, the compound is one or more of Compounds 4, 32, and 33, a pharmaceutically acceptable salt thereof, or combinations thereof.

[0305] In one aspect, a method is provided for releasing a macromolecule, e.g., an oligonucleotide and / or a polypeptide, from an endosome. In one aspect, the method includes: contacting the cell with the macromolecule, e.g., oligonucleotide and / or polypeptide conjugated to one or more compounds described herein, wherein the macromolecule, e.g., oligonucleotide and / or polypeptide, is internalized by the cell through endocytosis and encapsulated within the endosome, wherein the compounds facilitates release of the macromolecule, e.g., oligonucleotide and / or polypeptide, from the endosome. In some embodiments, the oligonucleotide is an ASO. In some embodiments, the oligonucleotide is an SSO. In some embodiments, the oligonucleotide is a siRNA. In some embodiments, the oligonucleotide is a guide RNA. In some embodiments, the polypeptide is a Cas protein. In some embodiments, the polypeptide is a recombinase. In some embodiments, the polypeptide is Cre. In some embodiments, the polypeptide is FLP. In some embodiments, the polypeptide is a Cas protein, and the polynucleotide is a guide RNA. In some embodiments, the one or more compounds has a structure represented by Formula I, Formula la, lb, Ic, Id, le or If, or are compounds listed in Table 1, or a pharmaceutically acceptable salt thereof, or combinations thereof. In some embodiments, the compound is one or more of Compounds 4, 32, and 33, a pharmaceutically acceptable salt thereof, or combinations thereof.

[0306] In one aspect, a method is provided for the treatment and / or prevention of a disorder in a subject, such as a genetic disorder. In one aspect, administration includes parenteral administration. In one aspect, parenteral administration includes intravenous (IV) or subcutaneous (SC) administration. In one aspect, the subject is a mammal. In one aspect, the subject is a human. In some embodiments, the oligonucleotide is an ASO. In some embodiments, the oligonucleotide is an SSO. In some embodiments, the oligonucleotide is a siRNA. In some embodiments, the oligonucleotide is a guide RNA. In some embodiments, the polypeptide is a Cas protein. In some embodiments, the polypeptide is a recombinase. In some embodiments, the polypeptide is Cre. In some embodiments, the polypeptide is FLP. In some embodiments, the polypeptide is a Cas protein, and the polynucleotide is a guide RNA. In some embodiments, the one or more compounds has a structure represented by Formula I, Formula la, lb, Ic, Id, le or If, or are one or more compounds compounds listed in Table 1, or a pharmaceutically acceptable salt thereof, or combinations thereof. In some embodiments, the compound is one or more of Compounds 4, 32, and 33, a pharmaceutically acceptable salt thereof, or combinations thereof.

[0307] In one aspect, a method is provided for the treatment and / or prevention of a disorder in a subject. In one aspect, the method includes: isolating a cell from the subject; contacting the isolated cell with a therapeutically effective amount of a macromolecule, e.g., an oligonucleotide and / or polypeptide, conjugated to one or more compounds described herein to produce an engineered cell; and transplanting the engineered cell in the subject. In one aspect, the isolated cell is contacted with a composition comprising about 0.025 μM to about 20 μM of the macromolecule, e.g., oligonucleotide and / or polypeptide. In one aspect, the isolated cell is contacted with about 0.1 μM to about 10 μM, about 0.1 μM to about 5 μM, or about 0.1 μM to about 1 μM of the macromolecule, e.g., oligonucleotide and / or polypeptide. In some embodiments, the oligonucleotide is an ASO. In some embodiments, the oligonucleotide is an SSO. In some embodiments, the oligonucleotide is a siRNA. In some embodiments, the oligonucleotide is a guide RNA. In some embodiments, the polypeptide is a Cas protein. In some embodiments, the polypeptide is a recombinase. In some embodiments, the polypeptide is Cre. In some embodiments, the polypeptide is FLP. In some embodiments, the polypeptide is a Cas protein, and the polynucleotide is a guide RNA. In some embodiments, the compound has a structure represented by Formula I, Formula la, lb, Ic, Id, le or If, or is one or more compounds listed in Table 1, or a pharmaceutically acceptable salt thereof, or combinations thereof. In some embodiments, the compound is any one of Compounds 4, 8, and 32, a pharmaceutically acceptable salt thereof, or combinations thereof. Provided herein is a use of one or more compounds described herein in the manufacture of a medicament for gene therapy. Provided herein is a use of one or more compounds of Formula I conjugated to a macromolecule, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for gene therapy. Provided herein is a use of one or more compounds of Formula la conjugated to a macromolecule, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for gene therapy. Provided herein is a use of one or more compounds of Formula lb conjugated to a macromolecule, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for gene therapy. Provided herein is a use of one or more compounds of Formula Ic conjugated to a macromolecule, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for gene therapy. Provided herein is a use of one or more compounds of Formula Id conjugated to a macromolecule, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for gene therapy. Provided herein is a use of one or more compounds of Formula le conjugated to a macromolecule, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for gene therapy. Provided herein is a use of one or more compounds of Formula If conjugated to a macromolecule, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for gene therapy. Provided herein is a use of one or more compounds of Formula I, Formula la, lb, Ic, Id, le or If, or the compounds listed in Table 1 conjugated to a macromolecule, or a pharmaceutically acceptable salt thereof, or a combination thereof, in the manufacture of a medicament for gene therapy. Provided herein is a use of one or more of Compounds 4, 32, and 33 conjugated to a macromolecule, a pharmaceutically acceptable salt thereof, or a combination thereof, in the manufacture of a medicament for gene therapy.

[0308] The entire contents of all publications, patents, and patent applications referenced herein are hereby incorporated herein by reference.

[0309] The specific examples included herein are for illustrative purposes only and are not to be considered as limiting to this disclosure. Moreover, the compositions and methods provided herein have been described in relation to certain embodiments thereof, and many details have been set forth for purposes of illustration. It will be apparent to those skilled in the art that the disclosure is susceptible to additional embodiments and that certain details described herein may be varied without departing from the basic principles of the disclosure. WORKING EXAMPLES

[0310] Example 1. Preparation of Small Molecule Compound (SMC) Library

[0311] Small-molecule compounds (SMC) were screened for their ability to increase the activity of a gapmer antisense oligonucleotide (ASO) by co-treatment of cells with the SMC and ASO as shown in the subsequent examples. Representative procedures for synthesizing the SMCs are provided below.

[0312] General procedure for preparation of Compounds 2-7 and 19-31

[0313] The general procedure for the preparation of Compounds 2-7 and 19-31 is shown schematically in FIG. 1-2 where FIG. 1 provides the overall generic scheme and includes starting material X shown in step 3. FIG. 2 describes the definition of starting material X and required intermediate for each of the depicted compounds (e.g., Compound II depicted in FIG. 2 is the starting material “X” in the preparation described in FIG. 1 for Compound 2). Further descriptions of exemplified intermediates and Compounds are provided below. The amino pyrazole and amino isopropoxypyrazole derivative intermediates identified in FIG. 2 that are used to make Compounds 19 and 30, respectively, were prepared according to the same procedures described below for intermediate 3 (amino methoxypyrazole) and 7 (amino ethoxypyrazole), respectively.

[0314] General procedure for preparation of intermediate 2

[0315] 1. Add Cpd.l (9.00 g, 62.8 mmol, 1.00 eq) and Cpd.lA (28.8 g, 81.1 mmol, 1.29 eq) into a flask charged with DMF (63 mL).

[0316] 2. Add CS2CO3 (30.5 g, 93.7 mmol, 1.49 eq) to the mixtures.

[0317] 3. Degas with N2 for 3 times.

[0318] 4. Stir at 120 °C for 2 hrs.

[0319] 5. TLC show Cpd.l consumed, a new spot formed.

[0320] 6. Adjust the PH of the mixture to 7~8 with HC1 (1 M).

[0321] 7. Extract with mixture with DCM (200 mL x 3). 8. Wash the organic layer with brine (100 mLx 2) and dried over magnesium sulfate.

[0322] 9. Concentrate the organic layer in vacuo to remove DCM.

[0323] 10. The residue was purified by column chromatography (SiCh, Petroleum ether / Ethyl acetate=30 / l to 3 / 1).

[0324] 11. Obtain Cpd.2 (18.1 g, 70.5% yield, 80.0% purity) as yellow solid.

[0325] 'H NMR: 400 MHz DMSO-de

[0326] <5 8.746 (s, 1H), 7.94 (s, 1H), 4.69-4.68 (m, 1H), 4.31-4.26 (m, 2H), 4.05 (s, 3H), 3.33 (s, 4H), 2.88 (s, 3H), 2.72 (s, 1H), 2.01-1.98 (m, 2H), 1.81-1.74 (m, 2H), 1.40 (s, 9H)

[0327] General procedure for preparation of intermediate 3

[0328] 1. Add Cpd.2 (17.0 g, 52.0 mmol, 1.00 eq) in a flask charged with EtOAc (110 mL).

[0329] 2. Add Pd / C (1.70 g, 52.0 mmol, 10% purity, 1.00 eq) to the mixture.

[0330] 3. Add H2 (15 Psi) to the mixture.

[0331] 4. Stir at 25 °C for 10 hrs.

[0332] 5. TLC (Petroleum ether / Ethyl acetate = 3 / 1, product Rf = 0.42) shows Cpd.3 formed.

[0333] 6. Filtered through celites.

[0334] 7. Wash filter cake with EtOAc(150 mL x 3).

[0335] 8. Concentrate the filtrate in vacuo to remove EtOAc.

[0336] 9. The residue was purified by column chromatography (SiCE, Petroleum ether / Ethyl acetate=30 / l to 3 / 1).

[0337] 10. Obtain Cpd.3 (10.9 g, 70.6% yield) as black-blue oil.

[0338] 'H NMR: 400 MHz DMSO-de

[0339] <5 7.00 (s, 1H), 4.01-3.94 (m, 3H), 3.52 (s, 3H), 2.88-2.73 (m, 2H), 1.88-1.84 (m, 2H), 1.66-

[0340] 1.60 (m, 2H), 1.40 (s, 9H)

[0341] Alternate method for preparation of intermediate compound 3

[0342] A solution of compound 2 and iron(iii)chloride (3.41 g, 1.05 mmol) in MeOH) (21.14 ml) was treated with hydrazine (2.197 ml, 70.00 mmol) and brought to reflux overnight. Reaction was filtered. Contraction of filtrate afforded residue. The residue was redissolved in DCM / MeOH (10:1) and washed with water. Concentration of organic layer yielded tertObuytyl 4-(4-amino-3methoxy-lH-pyrazol-l-yl)piperidine-l-carboxylate (2.000 g, 96%).

[0343] General procedure for preparation of intermediate 4-1

[0344] 1. Add Cpd.3 (0.50 g, 1.69 mmol, 1.00 eq) and Cpd.3b-10 (444 mg, 1.54 mmol, 0.91 eq) charged with i-PrOH (10.0 mL) to a microwave vial.

[0345] 2. Add TsOH (529 mg, 3.08 mmol, 1.82 eq) to the mixture.

[0346] 3. Stir at 120 °C for 2 hrs.

[0347] 4. LCMS (ET37912-40-Pla) shows Cpd.4-1 formed.

[0348] 5. Adjust the pH Of the mixture to 7~8 with Sat. NaHCOa

[0349] 6. Extract the mixture with EtOAc (50 mL x 3).

[0350] 7. Wash the organic layer with brine (30 mL x 2).

[0351] 8. Concentrate in vacuo to remove EtOAc.

[0352] 9. Obtain Cpd.4-l(0.40 g, crude) as yellow solid.

[0353] Example procedure for preparation of Compound 2

[0354] The procedure for the preparation of Compound 2 is shown schematically in FIG. 3.

[0355] 1. Dissolve Cpd.4-l(0.90 g, 1.64 mmol, 1.00 eq) in HCl / dioxane (4 M, 9.00 mL, 21.9 eq).

[0356] 2. Stir for 3 h at 25 °C.

[0357] 3. LCMS (product Rt = 0.582 min) shows Compound 2 formed.

[0358] 4. Add sat. NaHCOa (50.0 mL) to the solution, pH = 8.

[0359] 5. Extract with EtOAc (100.0 mL x 2), separate the organic layer and dry over NaiSCL, concentrate in vacuum. 6. Purity by prep- HPLC(column: Phenomenex Gemini-NX 80*40mm*3um;mobile phase: [water(10mM NH4HCO3)-ACN];B%: 20%-40%,8min).

[0360] 7. Obtain Compound 2 (0.062 g, 8.34% yield) as white solid.

[0361] 'H NMR: 400 MHz DMSO-de

[0362] <5 8.44(s, 1H), 8.08 (s, 1H), 7.85 (d, J= 8.0 Hz, 1H), 7.73 (s, 1H), 7.61 (d, J= 8.0 Hz, 1H), 7.34 (t, J = 8.0 Hz ,1H), 3.90 (s, 1H), 3.77 (m, 3H), 2.96 (s, 2H), 1.84 (s, 2H), 1.67-1.60 (m, 2H).

[0363] Example procedure for preparation of Compound 6

[0364] The procedure for the preparation of Compound 6 is the same as described above for Compound 2 but starting from intermediate 1, where the methoxy substitutent is replaced with an ethoxy substituent and a modified Cpd.3b-10 (i.e. indole intermediate) lacking the cyano substituent.

[0365] General procedure for preparation of intermediate 3b- 10

[0366] 1. Add Cpd.3C (4.84 g, 26.3 mmol, 1.50 eq) charged with DCE (100 mL) into a flask.

[0367] 2. Add A1CE (3.52 g, 26.3 mmol, 1.44 mL, 1.50 eq) to the mixture, the color becomes yellow.

[0368] 3. Stir at 80 °C for 0.5 hrs.

[0369] 4. Add Cpd.3b_Int (2.5 g, 17.59 mmol, 1.00 eq) to the mixture, the color becomes orange red.

[0370] 5. Stir at 80 °C for 12 hrs.

[0371] 6. LCMS show Cpd.3b_10 was formed.

[0372] 7. Add the mixture to ice- water mixture (200 mL).

[0373] 8. Extract the mixture with EtOAc (250 mL x 3).

[0374] 9. Wash the organic layer with brine (100 mL x 2).

[0375] 10. Concentrate in vacuo to remove EtOAc. 11. The residue was purified by column chromatography (SiCL, Petroleum ether / Ethyl acetate=30 / l to 1 / 1).

[0376] 12. Obtain Cpd.3b_10 (0.50 g, 9.83% yield) as orange solid.

[0377] 'H NMR: 400 MHz DMSO-de

[0378] <5 8.95 (s, 1H), 8.37 (s, 1H), 7.91 (d, J= 8.0 Hz, 1H), 7.69 (d, J= 8.0 Hz, 1H), 7.41 (t, J= 8 Hz, 1H).

[0379] General procedure for preparation of intermediate 4-2

[0380] 1. Add Cpd.3 (0.50 g, 1.69 mmol, 1.00 eq) and Cpd.7a-10 (545 mg, 1.69 mmol, 1.00 eq) charged with i-PrOH (10.0 mL) to a microwave vial.

[0381] 2. Add TsOH (529.65 mg, 3.08 mmol, 1.82 eq) to the mixture.

[0382] 3. Stir at 120 °C for 2 hrs.

[0383] 4. LCMS shows Cpd.4-2 formed.

[0384] 5. Adjust the pH of the mixture to 7~8 with Sat. NaHCCE.

[0385] 6. Extract the mixture with EtOAc (30 mL x 3).

[0386] 7. Wash the organic layer with brine (15 mL x 2).

[0387] 8. Concentrate in vacuo to remove EtOAc.

[0388] 9. Obtain Cpd.4-2 (0.40 g, crude) as yellow solid.

[0389] General procedure for preparation of Compound 3

[0390] The procedure for the preparation of Compound 3 is shown schematically in FIG. 4.

[0391] Dissolve Cpd.4-2 (0.90 g, 1.54 mmol, 1.00 eq) in HCl / dioxane (4 M, 9.00 mL, 23.3 eq).

[0392] 1. Stir for 3 h at 25 °C.

[0393] 2. LCMS (product Rt = 0.602 min) shows Compound 3 formed.

[0394] 3. Add sat. NaHCOa (10.0 mL) to the solution, pH = 8. 4. Extract with EtOAc (50.0 mL x 2), separate the organic layer and dry over NaiSCU, concentrate in vacuum.

[0395] 5. Purity by prep- HPLC (column: Phenomenex Gemini-NX 80*40mm*3um; mobile phase: [water (lOmM NH4HCO3)-ACN];B%: 15%-35%,8min).

[0396] 6. Obtain Compound 3 (0.08 g, 10.7% yield, 100% purity) as yellow solid.

[0397] 'H NMR: 400 MHz DMSO-de

[0398] 3 8.84 (brs, 1H), 7.84 (d, J= 8.0 Hz, 1H), 7.72 (s, 1H), 7.56 (d, J= 7.2 Hz), 7.33 (t, J= 8 Hz, 1H), 3.91 (brs, 1H), 3.90 (s, 3H), 2.99 (s, 2H), 2.53 - 2.50 (m, 1H), 1.85 (s, 1H), 1.62 (brs, 1H).

[0399] General procedure for preparation of intermediate 7a- 10

[0400] 1. Add Cpd.7a_l (5.72 g, 26.3 mmol, 1.50 eq) charged with DCE (100 mL) into a flask.

[0401] 2. Add A1CE (2.34 g, 17.5 mmol, 961 uL, 1.00 eq) to the mixture, the color becomes yellow.

[0402] 3. Stir at 80 °C for 0.5 hrs.

[0403] 4. Add Cpd.3C (2.50 g, 17.5 mmol, 1.00 eq) to the mixture, the color becomes orange red.

[0404] 5. Stir at 80 °C for 12 hrs.

[0405] 6. LCMS shows Cpd.7a_10 is formed.

[0406] 7. Add the mixture to ice- water mixture (200 mL).

[0407] 8. Extract the mixture with EtOAc (200 mL x 3).

[0408] 9. Wash the organic layer with brine (100 mL x 2).

[0409] 10. Concentrate in vacuo to remove EtOAc.

[0410] 11. The residue was purified by column chromatography (SiOi, Petroleum ether / Ethyl acetate=20 / l to 1 / 1)

[0411] 12. Obtain Cpd.7a_10 (1.60 g, 28.2% yield) as yellow solid

[0412] 'H NMR: 400 MHz DMSO-de d 9.26 (s, 1H), 8.03 (s, 1H), 7.91 (d, J= 8.0 Hz, 1H), 7.73 (d, J= 8.0 Hz, 1H), 7.41 (t, J= 8 Hz, 1H).

[0413] General procedure for preparation of intermediate 4-3

[0414] 1. Add tert-butyl Cpd.3 (399 mg, 1.47 mmol, 0.91 eq) charged with i-PrOH (7.00 mL) to a micro wave vial.

[0415] 2. Add TsOH (504 mg, 2.93 mmol, 1.82 eq) to the mixture.

[0416] 3. Stir at 120 °C for 2 hrs.

[0417] 4. LCMS (product Rt = 0.857 min) shows Cpd.4-3. formed.

[0418] 5. Adjust the PH Of the mixture to 7~8 with Sat. NaHCOa.

[0419] 6. Extract the mixture with EtOAc (100 mL x 3).

[0420] 7. Wash the organic layer with brine (100 mL x 2).

[0421] 8. Concentrate the organic in vacuo to remove EtOAc.

[0422] 9. Obtain Cpd.4-3 (0.6 g, crude) as white solid.

[0423] General procedure for preparation of Compound 4 and Compounds 32-35

[0424] The procedure for the preparation of Compound 4 and Compounds 32-35 is shown schematically in FIG. 5, using Compound 4 as an example.

[0425] 1. Dissolve Cpd.4-3 (0.60 g, 1.13 mmol, 1.00 eq) in HCl / dioxane (4 M, 4.20 mL, 14.9 eq).

[0426] 2. Stir at 25 °C for 2 hrs.

[0427] 3. LCMS (product Rt = 0.348 min) shows Compound 4 formed.

[0428] 4. Add sat. NaHCOa (50.0 mL) to the solution, pH = 8.

[0429] 5. Extract with EtOAC (100.0 mL x 2), separate the organic layer and dry over Na2SO4. concentrate in vacuum.

[0430] 6. Purity by prep- HPLC (column: Phenomenex Gemini-NX 80*40mm*3um; mobile phase:

[0431] [water (lOmM NH4HCO3)-ACN];B%: 20%-50%,8min). Obtain Compound 4 (0.05 g, 10.0% yield, 98.3% purity) as yellow solid. Note that Compounds 32-35 follow the same procedure as described above for Compound 4, but replacing intermediate 1A (in the preparation of Intermediate 2— ► Intermediate 3 Intermediate 4-3) with the following compounds:

[0432] TsO -Boc (to result in Compound 32, 0.03 g, 4% yield, 96.5% purity as yellow solid); (to result in Compound 33, 0.05 g, 5% yield, 99.6% purity as yellow solid);

[0433] TsO — ( N-Boc

[0434] ' — f (to result in Compound 34, 0.03 g, 2% yield, 97% purity as yellow solid); and

[0435] TsO — ( N-Boc

[0436] (to result in Compound 35, 0.03 g, 3% yield, 98.5% purity as yellow solid)

[0437] Compounds 32-35 can be further separated into their isomeric parts by supercritical fluid chromatography (SFC). For example, Compound 32 was separated into its enantiomers by SFC with use of a mobile phase consisting of ethanol / DEA / CO2 and a YMC SA (IA) column (particle size 5 um). The preparative conditions consisted of a flow of 3.5 ml / min at 40 degC and the compounds were detected at 272 nm. The enantiomeric excess of both enantiomers exceeded 99%ee according to LC / MS analysis. The fractions containing the separated enantiomers were evaporated which yielded the final compounds as solids.

[0438] 1H NMR: 400 MHz DMSO-d6

[0439] Compound 4: 3 8.81 (brs, 1H), 8.60 (d, J= 4.0 Hz, 1H), 8.08 (s, 1H), 7.73 (s, 1H), 7.32 (brs, 1H), 6.95 (d, J= 4.0 Hz, 1H), 3.99-3.94 (m, 1H), 3.79 (s, 1H), 3.01 (d, J= 8.0 Hz, 1H), 2.57- 2.54 (m, 2H), 1.90-1.87 (m, 2H), 1.71-1.68 (m, 2H).

[0440] Compound 32: 5 8.82 (br s, 1H), 8.77 (s, 1H), 8.09 (s, 1H), 7.74 (m, 2H), 7.34 (br s, 1H), 6.96 (s, 1H), 4.17 (m, 1H), 3.79 (s, 3H), 2.80 (m, 2H), 1.75-1.87 (dd, 2H), 1.51-1.61 (m, 3H), 1.10 (s, 3H), 1.06 (s, 3H).

[0441] Compound 33: 5 8.81 (br s, 1H), 8.60 (s, 1H), 8.09 (s, 1H), 7.75 (m, 2H), 7.33 (br s, 1H), 6.96 (s, 1H), 3.90 (m, 1H), 3.79 (s, 3H), 3.12 (d, 1H), 2.84 (d, 1H), 2.65 (t, 1H), 2.44 (t, 1H), 2.01 (m, 1H), 1.70-1.81 (m, 2H), 1.45 (m, 1H). Compound 34: 5 8.56 (m, 3H), 8.07 (m, 1H), 7.70 (m, 2H), 7.35 (t, 1H), 6.94 (m, 1H), 3.99 (m, 1H), 3.82 (s, 3H), 3.05 (m, 2H), 2.66 (m, 2H), 1.93 (t, 2H), 1.68 (m, 1H), 1.36 (m, 1H), 1.05 (d, 3H).

[0442] Compound 35: 5 8.55 (br s, 3H), 8.05 (s, 1H), 7.64-7.71 (m, 2H), 7.33 (m, 1H), 6.93 (br s, 1H), 4.16 (br s, 1H), 3.83 (br s, 3H), 3.15 (br s, 1H), 2.80 (m, 2H), 2.65 (m, 1H), 2.21 (br s, 1H), 1.97 (m, 1H), 1.74 (m, 1H), 0.71 (d, 3H).

[0443] General procedure for preparation of Compounds 8-17

[0444] The procedure for the preparation of Compounds 8-17 follows the procedure above for Compound 4 plus an additional alkylation step as shown in FIG. 6:

[0445] 1. Add small molecule (i.e., either Compound 2-4, 21-22 or 24) with free NH (1.00 eq) to a flask charged with DMF (3 mL).

[0446] 2. Add triethylamine (2.0 eq) to the mixture.

[0447] 3. Add alkyl bromide or alkyl iodide (1.00 to 2.00 eq) to the mixture.

[0448] 4. Stir at 20 °C for 18 hours.

[0449] 5. LCMS shows starting material consumed and the reaction completed.

[0450] 6. Add aqueous ammonia 30% (three drops) to quench excess alkyl halide.

[0451] 7. Concentrate reaction mixture in vacuo to remove DMF.

[0452] 8. The residue was purified by column chromatography (SiO2, Heptane / Ethyl acetate= 100 / 0 to 0 / 100 followed by DCM / MeOH= 100 / 0 to 60 / 40).

[0453] 9. Obtain Compounds 8-17 as yellow solid.

[0454] 1H NMR: 500 MHz DMF-d 7

[0455] Compound 8: 5 1.94 - 2.1 (m, 6H), 2.24 (s, 3H), 2.89 (d, J = 10.7 Hz, 2H), 3.89 (s, 3H), 3.96 (dd, J = 11.1, 5.4 Hz, 1H), 7.02 (d, J = 3.6 Hz, 1H), 7.44 (s, 1H), 7.79 (d, J = 7.4 Hz, 1H),

[0456] 7.89 (s, 1H), 8.18 (dd, J = 3.6, 2.2 Hz, 1H), 8.63 (d, J = 4.3 Hz, 1H).

[0457] Compound 9: 5 1.10 (s, 6H), 1.95 - 2.5 (m, 6H), 3.06 (bs, 2H), 3.89 (s, 3H), 4.03 (m, 1H), 7.03 (d, J = 3.6 Hz, 1H), 7.45 (t, J = 7.7 Hz, 1H), 7.78 (d, J = 7.4 Hz, 1H), 7.90 (s, 1H), 8.16 - 8.21 (m, 1H), 8.64 (d, J = 4.3 Hz, 1H).

[0458] Compound 10: 5 0.91 (t, J = 7.4 Hz, 3H), 1.52 (s, 2H), 2.04 (s, 6H), 2.32 (s, 2H), 2.99 (s, 2H),

[0459] 3.89 (s, 3H), 4.01 (s, 1H), 7.03 (d, J = 3.6 Hz, 1H), 7.45 (s, 1H), 7.78 (d, J = 7.4 Hz, 1H), 7.90 (s, 1H), 8.17 - 8.21 (m, 1H), 8.64 (d, J = 4.2 Hz, 1H). Compound 11: 5 1.05 (t, J = 7.2 Hz, 3H), 1.93 - 2.09 (m, 6H), 2.38 (q, J = 7.2 Hz, 2H), 3.01 (d, J = 10.4 Hz, 2H), 3.89 (s, 3H), 3.98 (dt, J = 10.8, 5.8 Hz, 1H), 7.02 (d, J = 3.7 Hz, 1H), 7.45 (d, J = 8.4 Hz, 1H), 7.79 (d, J = 7.4 Hz, 1H), 7.89 (s, 1H), 8.18 (dd, J = 3.6, 2.2 Hz, 1H), 8.63 (d, J = 4.3 Hz, 1H).

[0460] Compound 12: 5 1.99 (s, 2H), 2.29 - 2.49 (m, 4H), 3.00 - 3.27 (m, 4H), 3.64 (m, 4H), 3.90 (s, 3H), 4.39 (m, 1H), 7.03 (s, 1H), 7.48 (t, J = 8.0 Hz, 1H), 7.78 (d, J = 7.3 Hz, 1H), 7.95 (s, 1H), 8.19 (s, 1H), 8.64 (d, J = 4.2 Hz, 1H).

[0461] Compound 13: 5 1.29 (t, J = 7.0 Hz, 3H), 1.91 - 2.14 (m, 6H), 2.23 (s, 3H), 2.88 (d, J = 11.3 Hz, 2H), 3.95 (m, 1H), 4.24 (q, J = 7.0 Hz, 2H), 7.03 (d, J = 3.6 Hz, 1H), 7.44 (s, 1H), 7.79 (d, J = 7.4 Hz, 1H), 7.90 (s, 1H), 8.18 - 8.2 (m, 1H), 8.64 (d, J = 4.2 Hz, 1H).

[0462] Compound 14: 5 1.95 - 2.07 (m, 6H), 2.22 (s, 3H), 2.87 (d, J = 10.7 Hz, 2H), 3.90 (s, 3H), 3.97 (s, 1H), 7.03 (d, J = 3.6 Hz, 1H), 7.63 (dd, J = 8.1, 1.4 Hz, 1H), 7.91 (d, J = 8.1 Hz, 2H), 8.18 (s, 1H), 8.62 (d, J = 4.4 Hz, 1H).

[0463] Compound 15: 5 1.29 (t, J = 7.0 Hz, 3H), 1.90-2.05 (m, 6H), 2.22 (s, 3H), 2.87 (d, J= 2.8 Hz, 2H), 4.01 (m, 1H), 4.24 (q, J = 7.0 Hz, 2H), 7.02 (s, 1H), 7.12 (s, 1H), 7.94 (s, 1H), 8.26 (s, 1H), 8.47 (s, 1H), 8.57 (s, 1H).

[0464] Compound 16: 5 2.19 (s, 2H), 2.40 (s, 4H), 2.95 - 3.17 (m, 6H), 3.77 (bs, 1H), 3.83 - 3.94 (m, 6H), 4.23 (m, 1H), 4.48 (m, 1H), 7.41 (t, J = 7.8 Hz, 2H), 7.66 (d, J = 7.3 Hz, 2H), 7.98 (d, J = 8.2 Hz, 2H), 8.04 (m, 2H), 8.77 (s, 2H), 9.20 (bs, 1H), 9.27 (bs, 1H).

[0465] Compound 17: 5 2.11 (s, 3H), 2.57 (m, 6H), 3.17 (bs, 2H), 3.89 (s, 3H), 4.08 (m, 1H), 7.42 (t, J = 7.8 Hz, 1H), 7.69 (d, J = 7.3 Hz, 1H), 7.96 (s, 1H), 7.97 (dd, J = 8.3, 1.0 Hz, 1H), 8.33 (s, 1H), 8.49 (s, 1H).

[0466] Procedure for preparation of Compound 18

[0467] The procedure for the preparation of Compound 18 is described below and shown schematically in FIG. 7:

[0468] 1. Add small molecule Compound 4 with free NH (1.00 eq) to a flask charged with DMF (3 mL).

[0469] 2. Add acetyl chloride (1.05 eq) to the mixture.

[0470] 3. Stir at 20 °C for 18 hours.

[0471] 4. LCMS shows starting material consumed and the reaction completed.

[0472] 5. Concentrate reaction mixture in vacuo to remove DMF. 6. The residue was purified by column chromatography (SiO2, Heptane / Ethyl acetate= 100 / 0 to 0 / 100 followed by DCM / MeOH= 100 / 0 to 60 / 40).

[0473] 7. Obtain acetylated molecule Compound 18 as yellow solid.

[0474] 1H NMR: 500 MHz DMF-t / 7

[0475] Compound 18: 5 1.80 (qd, J = 12.3, 4.5 Hz, 1H), 1.94 (qd, J = 12.2, 11.5, 3.8 Hz, 1H), 2.05 (m, 2H), 2.10 (s, 3H), 2.72 - 2.8 (m, 1H), 3.27 (td, J = 13.8, 13.1, 2.8 Hz, 1H), 3.89 (s, 3H), 4.03 (d, J = 13.9 Hz, 1H), 4.30 (tt, J = 11.3, 4.1 Hz, 1H), 4.58 (d, J = 13.2 Hz, 1H), 7.02 (d, J = 3.6 Hz, 1H), 7.45 (d, J = 7.6 Hz, 1H), 7.78 (d, J = 7.4 Hz, 1H), 7.93 (s, 1H), 8.17 - 8.2 (m, 1H), 8.64 (d, J = 4.2 Hz, 1H).

[0476] General procedure for preparation of intermediate 3b- 11

[0477] 1. Add Cpd.3b_6 (6.81 g, 47.9 mmol, 1.00 eq) to a flask charged with THF (300 mF).

[0478] 2. Add iodo(methyl)magnesium (3 M, 15.97 mF, 1.00 eq) to the mixture.

[0479] 3. Stir at 30 min for 0 °C.

[0480] 4. Add Cpd.3C (8.00 g, 47.9 mmol, 1.00 eq) to the mixture.

[0481] 5. Stir at 70 °C for 15 hrs

[0482] 6. TEC (Petroleum ether: Ethyl acetate=3:l, Rf = 0.60) shows Cpd.3b_6 consumed and the reaction completed.

[0483] 7. Wash the residue with Sat. NH4CI until pH = 7-8.

[0484] 8. Extract the mixture with EtOAc (300 mF x 3).

[0485] 9. Wash the organic layer with brine (200 mF x 2).

[0486] 10. Concentrate the organic layer in vacuo to remove EtOAc.

[0487] 11. The residue was purified by column chromatography (SiOi, Petroleum ether / Ethyl acetate=30 / l to 3 / 1).

[0488] 12. Obtain Cpd.3b_ll (5.00 g, 19.1% yield, 50% purity) as yellow solid. General procedure for preparation of intermediate 6

[0489] N-BOC

[0490] 1. Add Cpd.5 (1.00 g, 6.36 mmol, 1.00 eq) and Cpd.lA (2.92 g, 8.21 mmol, 1.29 eq) into a flask charged with DMF (7.00 mL).

[0491] 2. Add CS2CO3 (3.09 g, 9.48 mmol, 1.49 eq) in the mixtures.

[0492] 3. Degas with N2 for 3 times.

[0493] 4. Stir at 120 °C for 2 hrs.

[0494] 5. TLC (Petroleum ether / Ethyl acetate = 3 / 1, product Rf = 0.42) shows Cpd.6 formed.

[0495] 6. Adjust the PH of the mixture to 7~8 with HC1 (1 M).

[0496] 7. Extract with mixture with DCM (50 mL x 3).

[0497] 8. Concentrate in vacuo to remove DCM.

[0498] 9. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=15 / l to 2 / 1).

[0499] 10. Obtain Cpd.6 (1.50 g, 69.2% yield) as yellow solid.

[0500] 'H NMR: 400 MHz DMSO-t / e

[0501] <5 8.73 (s, 1H), 4.32-4.26 (m, 2H), 4.06-4.00 (m, 2H), 2.04-1.98 (m, 2H), 1.77-1.76 (m, 2H), 1.39 (s, 9H), 1.35-1.33 (m, 3H).

[0502] General procedure for preparation of intermediate 7

[0503] 1. Add Cpd.6 (1.00 g, 2.94 mmol, 1.00 eq) to a flask charged with EtOAc (5 mL).

[0504] 2. Add Pd / C (0.10 g, 10% purity, 1.00 eq) to the mixture.

[0505] 3. Add H2 (15 Psi) to the mixture.

[0506] 4. Stir at 25 °C for 2 hrs.

[0507] 5. TLC (Petroleum ether: Ethyl acetate=2:l, Rf = 0.47) show Cpd.6 consumed and Cpd.7 formed. 6. Filter through celites.

[0508] 7. Wash filter cake with EtO Ac.

[0509] 8. Concentrate the filtrate in vacuo to remove EtO Ac.

[0510] 9. The residue was purified by column chromatography (SiOi, Petroleum ether / Ethyl acetate=15 / l to 2 / 1)

[0511] 10. Obtain Cpd.7 (0.77 g, 84.4% yield) as light-yellow oil.

[0512] General procedure for preparation of intermediate 8

[0513] 1. Add Cpd.7 (0.30 g, 966 umol, 1.00 eq) and Cpd.7a (261 mg, 879 umol, 0.91 eq) charged with i-PrOH (7 mL) to a microwave vial.

[0514] 2. Add TsOH (302 mg, 1.76 mmol, 1.82 eq) to the mixture.

[0515] 3. Stir at 120 °C for 2 hrs.

[0516] 4. LCMS (ET37912-42-pla) shows Cpd.8 formed.

[0517] 5. Adjust the pH of the mixture to 7~8 with Sat. NaHCOa.

[0518] 6. Extract the mixture with EtOAc (60 mL x 3).

[0519] 7. Wash the organic layer with brine (30 mL x 2).

[0520] 8. Concentrate the organic in vacuo to remove EtOAc.

[0521] 9. Obtain Cpd.8 (0.3 g, crude) as yellow solid.

[0522] General procedure for preparation of Compound 7

[0523] The procedure for the preparation of Compound 7 is shown schematically in FIG. 8.

[0524] 1. Dissolve Cpd.8 (0.50 g, 874 umol, 1.00 eq) in HCl / dioxane (4 M, 5.00 mL, 22.8 eq).

[0525] 2. Stir for 3h at 20 °C.

[0526] 3. TLC (Petroleum ether / Ethyl acetate = 5 / 1, product Rf = 0.1) shows Compound 7 formed.

[0527] 4. Add sat. NaHCOa (50.0 mL) to the solution, pH = 8. 5. Extract with EtOAc (50.0 mL x 2), separate the organic layer and dry over NaiSCU, concentrate in vacuum.

[0528] 6. The crude product was triturated with EtOAc (100 mL) at 25 °C for 30 min.

[0529] 7. Filter and collect the cake.

[0530] 8. Obtain Compound 7 (0.068 g 16.0% yield) as white solid.

[0531] 1H NMR: 400 MHz DMSO-d6

[0532] <5 9.03-8.88 (s, 1H), 8.60 (s, 1H), 8.52-7.96 (s, 1H), 7.85 (s, 1H), 7.74 (s, 1H), 7.45-7.43 (s, 1H), 7.10-6.96 (m, 2H), 4.30 (s, 2H), 3.85 (s, 1H), 3.02-2.99 (m, 2H), 2.66-2.55 (m, 2H), 1.88 (m, 2H), 1.70-1.67 (m, 2H), 1.26-1.17 (m , 3H).

[0533] General procedure for preparation of Intermediate A

[0534] 3M MgICH3 (1.781 mL, 5.34 mmol) was added to a yellow solution of lJ-indole-4- carbonitrile (0.760 g, 5.34 mmol) in TFH (30 mL). The yellow suspension turned to a while solid, which was formed indole Mg complex. After 15 min, 2,4,5 -trichloropyrimidine (1 g, 5.34 mmol) was added at 0°C, the mixture was warmed to room temperature. LCMS showed slight product. The mixture was heated to 70 °C for 2 hours, the reaction progressive. After stirring overnight, the reaction completed. Product mass was [M+l }: 288.9 at retention time RT=3.05 min. The reaction was kept overnight at 70 °C. The reaction wasn’t completed. Filtered off solid to give a crude product 3-(2,5-dichloropyrimidin-4-yl)-lH-indole-4- carbonitrile (0.546 g, 35.0%). 25 mg crude compound was taken, purified by Gilson, UV lamp was 222 nm, mobile phase by 25-80% CAN in 0.1 TFA in water to give 17 mg while solid product was a TFA salt. LCMS and NMR confirmed the target compound. 1H NMR (300 MHz, DMSO-76): 5 6.94 (d, 7-3.58 Hz, 1H) 7.32-7.53 (m, 1H) 7.72 (d, 7=7.54 Hz, 1H) 8.09 (dd, 7=5.93, 2.35 Hz, 2H) 9.09 (s, 1H).

[0535] Example 2. EEE enhancer conjugates

[0536] MALAT1 ASO: 5’-GCATTCTAATAGCAGC-3’ (SEQ ID NO: 2)

[0537] HELM MALAT1:

[0538] RNAl{[LR](G)[sP].[LR]([5meC])[sP].[LR](A)[sP].[dR](T)[sP].[dR](T)[sP].[dR]([5meC])[s P].[dR](T)[sP].[dR](A)[sP].[dR](A)[sP].[dR](T)[sP].[dR](A)[sP].[dR](G)[sP].[dR]([5meC])[ sP].[LR](A)[sP].[LR](G)[sP].[LR]([5meC]) (SEQ ID NO: 3)}$$$$

[0539] PPIB siRNA: 5’ - CAGCAAAUUCCAUCGUGA-3 ’ (SEQ ID NO: 4), 5’- UCACGAUGGAAUUUGCUGUU-3’ (SEQ ID NO: 5)

[0540] HELM PPIB:

[0541] RNA 1 { [mR] (C) [sP] . [mR] (A)[sP] . [mR](G)P. [mR] (C)P. [mR](A)P. [mR] (A)P. [mR](A)P. [mR] (U)P. [mR](U)P. [mR] (C)P. [mR] (C)P. [mR](A)P. [mR] (U)P. [mR] (C)P. [mR] (G)P. [mR] (U)[sP] . [mR](G)[sP].[mR](A) (SEQ ID NO:

[0542] 6) } |RNA2{ P. [mR] (U) [sP] . [fR] (C) [sP] . [mR] (A)P. [fR] (C)P. [mR] (G)P. [fR] (A)P. [mR](U)P. [fR ] (G)P. [mR] (G)P. [fR] (A)P. [mR](A)P. [fR] (U)P. [mR](U)P. [fR] (U)P. [mR] (G)P. [fR] (C)P. [mR]( U)P.[fR](G)[sP].[mR](U)[sP].[fR](U) (SEQ ID NO: 7)}$RNAl,RNA2,2:pair- 54:pair|RNAl,RNA2,5:pair-51:pair|RNAl,RNA2,8:pair-48:pair|RNAl,RNA2,l l:pair- 45:pair|RNAl,RNA2,14:pair-42:pair|RNAl,RNA2,17:pair-39:pair|RNAl,RNA2,20:pair- 36:pair|RNAl,RNA2,23:pair-33:pair|RNAl,RNA2,26:pair-30:pair|RNAl,RNA2,29:pair- 27:pair|RNAl,RNA2,32:pair-24:pair|RNAl,RNA2,35:pair-21:pair|RNAl,RNA2,38:pair- 18 :pair|RNA 1 ,RN A2,41 :pair- 15 :pair|RNA 1 ,RNA2,44:pair- 12:pair|RNA 1 ,RNA2,47 :pair- 9:pair|RNAl,RNA2,50:pair-6:pair|RNAl,RNA2,53:pair-3:pair$$$V2.0

[0543] [sP]: phosphorothioate

[0544] P: phosphate

[0545] [mR]:2 '-methoxyribose

[0546] [fR]: 2'-fluoro-2'-deoxyribose

[0547] [LR]: locked nucleic acid (LNA)

[0548] [dR]: deoxyribose (DNA) [5meC]: 5'-methylcytidine

[0549] Conjugate structure A:

[0550] Conjugate structure B:

[0551] ( - denotes attachment point to oligonucleotide)

[0552] Conjugate structure C (SEQ ID NO: 8):

[0553] ( - denotes attachment point to oligonucleotide), peptide as disclosed in WO2022056286. Conjugate structure D:

[0554] ( - denotes attachment point to oligonucleotide) Conjugate structure E:

[0555] ( - denotes attachment point to oligonucleotide)

[0556] Conjugate structure F (SEQ ID NO: 9): ( - denotes attachment point to oligonucleotide), peptide as disclosed in

[0557] WO2022101633A1.

[0558] Conjugate structure G:

[0559] ( - denotes attachment point to oligonucleotide)

[0560] Synthesis of Conjugate structure G

[0561] Synthesis of II: A solution of l-azido-4-bromobutane (0.5 M, 6.0 ml, 3.02 mmol) in 2-methoxy methylpropane was concentrated on rotovap, then diluted with DMF (6.0 mL). To a microwave vial was added I (0.5 g, 2.74 mmol) and K2CO3 (1.14 g, 8.23 mmol) followed by the azide solution and additonal DMF (7.7 ml). The vial was capped, purged with Argon and stirred at 60°C overnight in an aluminium heating block. The reaction mixture was cooled, diluted with EtOAc and washed twice with water. The combined aqueous phase was extracted once with EtOAc. The combined organic phase was passed through a phase separator filter and concentrated to a light yellow oil. Purified with flash chromatography on a 10 g silica column (gradient 0-30% EtOAC in Heptane). Isolated product II as a colorless oil (716 mg, 93%). 'H NMR (500 MHz, DMSO, 25°C) δ 1.64-1.78 (m, 4H), 3.39 (t, J = 6.5 Hz, 2H), 3.86 (s, 6H), 3.98 (t, J = 5.9 Hz, 2H), 7.26 (s, 2H), 9.88 (s, 1H).

[0562] Synthesis of III: II (0.715 g, 2.56 mmol) was dissolved in EtOH (99.5%, 12.8 ml) and cooled to 0°C. NaBH4 (0.145 g, 3.84 mmol) was added. The reaction was quenched with water after 15 min and the mixture was poured into EtOAc. The phases were separated and the aqueous phase was extracted twice more. The combined organic phase was passed through a phase separator filter and concentrated. Product III was isolated as a colorless oil (664 mg, 92%). The product was used without additional purification.

[0563] 'H NMR (500 MHz, DMSO, 25°C) δ 1.6-1.77 (m, 4H), 3.39 (t, J= 6.8 Hz, 2H), 3.75 (s, 6H), 3.82 (t, J= 6.1 Hz, 2H), 4.42 (d, J= 5.7 Hz, 2H), 5.14 (t, J= 5.8 Hz, 1H), 6.62 (s, 2H). Synthesis of IV: A dry microwave vial was charged with III (0.346 g, 1.23 mmol) and purged with Argon. Anhydrous THF (611 pl), DIEA (0.643 ml, 3.69 mmol) and anhydrous pyridine (2.89 pl, 0.04 mmol)) was added. 4-Nitrophenyl chloroformate (0.496 g, 2.46 mmol) was added, the vial was purged and the resulting suspension was stirred vigorously for 2h. The THF was evaporated and the residue was purified with flash chromatography on a 10 g silica column (slow gradient 0-30%, EtOAC in Heptane). Isolated product IV as a light yellow oil (523 mg, 95%).

[0564] 'H NMR (500 MHz, DMSO, 25°C) δ 1.62-1.78 (m, 4H), 3.40 (t, J= 6.8 Hz, 2H), 3.78 (s, 6H), 3.87 (t, J= 6.0 Hz, 2H), 5.23 (s, 2H), 6.80 (s, 2H), 7.56-7.62 (m, 2H), 8.3-8.35 (m, 2H). Synthesis of V: To a solution of IV (69.5 mg, 0.16 mmol) in DCM (1.51 ml) was added Cmpd 4 (71 mg, 0.16 mmol) and EtaN (49.9 pl, 0.36 mmol). The resulting suspension was stirred for 3h. The reaction mixture was injected on a 5 g silica column purified (gradient 0-100% EtOAC in Heptane). Product V was isolated as a light yellow oil that foamed to a yellow solid under high vacuum (82 mg, 71%)

[0565] 'H NMR (500 MHz, DMSO, 25°C) δ 1.6-1.83 (m, 6H), 1.93-2.05 (m, 2H), 2.99 (s, 2H), 3.38 (t, J= 6.8 Hz, 2H), 3.76 (s, 6H), 3.79 (s, 3H), 3.83 (t, J= 6.0 Hz, 2H), 4.11 (d, J = 13.3 Hz, 2H), 4.14-4.24 (m, 1H), 5.03 (s, 2H), 6.68 (s, 2H), 6.96 (d, J= 3.6 Hz, 1H), 7.31 (s, 1H), 7.65-7.81 (m, 2H), 8.07-8.11 (m, 1H), 8.60 (d, J = 4.1 Hz, 1H), 8.83 (s, 1H). (One proton was exchanged) LCMS Expected: 739.3 Found: 739.3

[0566] Synthesis of VI: Synthesis was carried out on OP 10 synthesizer using Polystyrene (90 mg) as solid support using traditional conditions with the following improvements: The trebler was coupled with 3 cycles and the BCN with 4 cycles. The compound was oxidized 3 times after the last BCN coupling. The product was removed from the solid support in ammonium hydroxide at 55°C overnight. The suspension was filtered thorugh a 0.45 pm syringe filter and the solution was concentrated on a speedvac, followed by freeze-drying. Isolated a white solid that was dissolved in water (2 mL) and precipitated with NaOAc / EtOH solution (40 mL). The suspension was kept in freezer for 30 min then centrifuged. The supernatant was decanted off and the pellet was dissolved in water (2 mL) and freeze-dried to yield an unpure white solid. The crude material was dissolved in water (1 mL) and purified by HPLC (gradient 20-80% ACN in Ammonium bicarbonate) over 10 min. The fractions containing product were dried on speedvac overnight. Dissolved in water (2 mL) and freeze-dried. Product VI was isolated as a white solid (46 mg, 95% UV purity)

[0567] LCMS Expected: 2126 (3 charges), 1594 (4 charges), 1275 (5 charges). Found: 2127, 1595, 1276.

[0568] Synthesis of Conjugate structure G: VI (10 mg, 1.57 pmol) was dissolved in water (131 pl) in a falcon tube. 5 (10.43 mg, 0.01 mmol) was dissolved in DMF (261 pl) and added to the water solution. The suspension was stirred overnight. The product was precipitated with NaOAc / EtOH solution (12 mL), kept in freezer for Ih and centrifuged. The supernatant was decanted off and the pellet suspended in water (1 ml), reprecipitated, cooled and centrifuged. The resulting pellet was suspended in water (1 mL) and freeze-dried. Isolated a light yellow solid that was further purified by HPLC (gradient 20-80% ACN in Ammonium bicarbonate) over 10 min. Isolated product Conjugate structure G as a white solid (3.3 mg, 25%, 98% UV purity)

[0569] LCMS Expected: 2149 (4 charges), 1719 (5 charges). Found: 2149, 1719.

[0570] General procedure for solid phase oligo synthesis

[0571] PPIB (passenger strand) and Malatl sequences were synthesized on an AKTA OligoPilot 10 using routine methods and starting either 3’ amino functionalized controlled-pore glass (CPG) solid support at 8 pmol scale (for ASO1-4, sense strands for siRNA3-8) or regular PS solid support at 32 pmol scale (ASO5, ASO6, sense strand for siRNAl, siRNA2 and antisense strand for all siRNA).

[0572] Standard phosphoramidite chemistry was used. Dimethoxytrityl (DMT) -protected amidites (2’-fluoro, 2’-O-methyl, LNA, DNA) were purchased from Wuxi or Sigma-Aldrich / Merck. Ancillary synthesis reagents and solvents were purchased from Sigma-Aldrich / Merck, TCI Chemicals or other commercial suppliers.

[0573] Phosphoramidites were prepared as 0.1 M solutions in either dry MeCN or 15% DMF in dry MeCN. Couplings were carried out using 0.25 M 5-[3,5-Bis(trifluoromethyl)phenyl]-lH- tetrazole (Activator 42®) in MeCN for 3x6 min or 2x8min. Thiolation was performed using 0.2 M Xanthane hydride in pyridine for 6 min. Oxidation was carried out with 0.02 M iodine in tetrahydrofuran / water / pyridine 90.54 / 9.05 / 0.41 (v / v / v) for 1 min. Unreacted 5’hydroxyls were capped using a mixture of acetic anhydride / tetrahydrofuran 9.1 / 90.9 (v / v) (Cap A) and THF / NMI / pyridine 8 / 1 / 1 (v / v) (Cap B) for 1 min. All syntheses except ASO2, ASO4, ASO6, antisense strand, sense strands for siRNA2, siRNA4, siRNA6, siRNA8 were finalized by coupling 5'-trcblcr using 0.2M concentrated phosphoramidite under normal coupling conditions. For ASO2 and sense strand for siRNA2 BCN phosphoramidite had been coupled with subsequent thiolation.

[0574] Trityl groups were removed using 3% dichloroacetic acid (DCA) in toluene. After completed synthesis, final detritylation was not performed. The solid support with oligos attached were dried under high vacuum, and then further functionalized on 3 'or 5 'end.

[0575] Procedure for azide functionalization of endosomal escape enhancer (EEE) compound

[0576] Previously synthesized EEE compound (320mg, 0.69mmol), potassium carbonate (230 mg, 1.67 mmol) and DMF (12 mL) were placed into MW vial, the vial was capped and purged with N2, followed by the dropwise addition of l-azido-4-bromobutane (1.668 mL, 0.83 mmol). The reaction was heated at 80 °C in the MW reactor for 24h, monitored by LCMS resulting extra addition of ImL of l-azido-4-bromobutane, total time of reaction was 36h. The residue was poured into ice and the precipitated product was filtered out and purified using Hept:Acetone (6:4).

[0577] 1H NMR (500 MHz, CDC13) δ 8.52 (s, 1H), 8.39 (d, J = 3.9 Hz, 1H), 7.90 (dd, J = 3.7, 2.5 Hz, 1H), 7.70 (s, 1H), 7.59 (d, J = 7.4 Hz, 1H), 7.33 (t, J = 7.9 Hz, 1H), 6.98 (dd, J = 3.7, 0.8 Hz, 1H), 6.67 (s, 1H), 4.09 - 4.15 (m, 1H), 3.97 (s, 3H), 3.31 (t, 7 = 6.8 Hz, 2H), 2.87 (d, J = 12.4 Hz, 1H), 2.74 (dd, 7 = 14.3, 6.5 Hz, 1H), 2.36 - 2.42 (m, 1H), 2.09 (d, 7 = 12.2 Hz, 1H), 2.02 (td, 7 = 8.0, 3.9 Hz, 1H), 1.68 - 1.86 (m, 4H), 1.42 - 1.53 (m, 2H), 1.28 (d, 7 = 12.3 Hz, 3H), 1.15 (s, 3H), 1.02 (s, 3H).

[0578] General procedure for endosomal escape enhancer (EEE) compound conjugation to oligonucleotides

[0579] For synthesis of all oligonucleotides except ASO4, ASO6, siRNA4, siRNA6, siRNA8 the oligo-BCN loaded solid support was placed in a tight closed vial and 2ml of solution of EEE- azide (0.1M in 1,4-dioxane) were added. The solid supports were shaken overnight at room temperature followed by fdtration and solid support wash. The 5 ’-EEE functionalized oligonucleotides were cleaved from solid support by treatment of aq. ammonia (26%) at 55 °C overnight. The crude obtained 5 ’-EEE oligonucleotides were purified by HPLC and used for further 3’ functionalization.

[0580] General procedure for 3’ peptide conjugation

[0581] For synthesis of oligonucleotides ASO1, ASO2 and siRNA5-8 corresponding 3’-amino modified oligonucleotides were dissolved in MQ water to obtain 1.5mM solutions. A solution of BCN-PEG-NHS ester in MeCN (10 eq) was added to a solution of oligonucleotide and the mixtures were shaken at 40 °C for 18 h. The solvent was evaporated, and the conjugate was precipitated by addition of NaOAc and EtOH. The precipitate was redissolved in water and the corresponding peptide was added to the same vial as a lOmM DMSO solution. The mixture was shaken at rt overnight. The crude mixture was purified by HPLC. For siRNA, the guide strand and the obtained passenger strand were annealed by mixing the two strands in a 1 : 1 ratio and heating the water solution to 95 °C for 5 min. The reaction was allowed to cool to room temperature where it was shaken for 1 hour. The solvent was evaporated, and the solid product was redissolved in PBS.

[0582] General procedure for 3’ lipid conjugation

[0583] For synthesis of oligonucleotides ASO3 and ASO4 corresponding 3 ’-amino modified oligonucleotides were dissolved in MQ water to obtain 1.5mM solutions. Palmitic NHS ester dissolved in DMF was added (10-30 eq.), and the pH adjusted to 9 with triethylamine. The mixtures were shaken at 40°C until full conversion had been confirmed by LCMS. After reaction completion the excess of solvent was removed and the residues were redissolved in MQ water, the final products were precipitated by addition of NaOAc and EtOH.

[0584] General procedure for 3’ GalNac conjugation

[0585] For synthesis of oligonucleotides siRNA3 and siRNA4 corresponding 3 ’-amino modified oligonucleotides were dissolved in 0.5 M borate buffer pH 9.6. An equal volume of a solution of GalNAc PFP ester (1.5 equiv.) in DMSO was added and the resulting slightly cloudy mixture was stirred at room temperature for 3 hours. The mixture was subsequently diluted with 3 mL of 25% aqueous ammonia solution and left to stand at room temperature overnight. The next day, the mixture was diluted with water, freeze-dried and the dry residue was purified by reverse phase HPLC. To form double- stranded siRNA the guide strand and the obtained passenger strand were annealed by mixing the two strands in a 1:1 ratio and heating the water solution to 95 °C for 5 min. The reaction was allowed to cool to room temperature where it was shaken for 1 hour. The solvent was evaporated, and the solid product was redissolved in PBS.

[0586] Example 3. Cells and Culture Methods

[0587] HuH7 cells were obtained from Riken, 16HBE cells were gratefully received from Karl Staples (University of Southampton). Cells were modified to stably express mCherry-GAL9 as earlier described1. All cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with high glucose and GlutaMAX (Gibco: 31966-021), all growth media was supplemented with 10% FBS, and 1 pg / ml Puromycin (Gibco: Al 1138-03) to maintain mCherry-GAL9 reporter expression. Cells were maintained in a humidified incubator at 37 °C with 5% CO2. Cells were routinely tested and mycoplasma negative. Knockdown in human primary hepatocytes (GalNAc conjugates)

[0588] An assay was carried out to assess the ability of the siRNA (PPIB) constructs to 20 yield knockdown gene expression in human primary hepatocytes.

[0589] The following cell culture protocol was used:

[0590] Human hepatocytes were obtained from a commercial supplier (BioIVT, QNT lot) and cultured in rat tail collagen I coated plates. The hepatocytes were maintained in William's E Medium, supplemented with hepatocyte supplemented media and 5C supplements (DAPT, 25 SB431542, Forskolin, IWP2 and LDN193189). The cells were cultured at 37°C in a humidified incubator with 5% CO2. Next after plating (Day 1), cells were treated with different siRNA conjugates and PBS in maintenance media. After 24 h of treatment, media was changed and cells were harvested on Day 4 (72 hr from start of the treatment) for qPCR. Total RNA was extracted according to standard protocol setup in the lab. cDNA synthesis 5 and qPCR were also run according to standard validated protocol. PPIB, and GAPDH TaqMan primers were purchased from Thermo Fischer.

[0591] PPIB and MALAT1 knockdown in HEK293s ASGPR1 cells (Naked oligos and GalNAc conjugates)

[0592] An assay was carried out to assess the ability of the siRNA (PPIB) and ASO (MAEAT1) constructs to knockdown gene expression in HEK293 cells overexpressing ASGPR.

[0593] 15 Cryopreserved HEK293s-ASGPRl cells were thawed and seeded directly in assay-ready plates containing siRNA or ASO compounds as PBS serial dilutions (Day 0). Cells were incubated for 48h at 37 °C, 5% CO2 and 95% humidity. Two days after plating (Day 2), cells were lysed and RNA was extracted according to standard protocol. cDNA synthesis and qPCR were also run according to standard validated protocol. PPIB, MAEAT1 and RACK1 20 TaqMan primers were purchased from Thermo Fisher.

[0594] MALAT1 knockdown in HEK293T (wt) and HEK293 (Gal9-mCherry) cells (Naked oligo and ASO7)

[0595] An assay was carried out to assess the ability of the ASO constructs to knockdown gene expression in HEK293T (wt) and HEK293 (Gal9-mCherry) cells. Cultured HEK293 cells were seeded directly in assay-ready plates containing ASO compounds as PBS serial dilutions of 1.5 steps. Cells were incubated for 24h at 37°C, 5% CO2 and 95% humidity. One day after plating, cells were lysed and RNA was extracted according to standard protocol. cDNA synthesis and qPCR were also run according to standard validated protocol. MALAT1 and RACK1 TaqMan primers were purchased from Thermo Fisher.

[0596] C16-Malatl ASO qPCR method

[0597] Huh7 cells stably expressing mCherry-Gal9 (Huh7-mCherry-Gal9 cells) were cultured according to standard procedures in DMEM with GlutaMax, 4.5g / L glucose, 10% FBS and I p.g / ml puromycin. Cells were trypsinised, resuspended in standard culture medium and plated at 4000 cells per well in 384 well culture plates. 24h later the media was removed from the cells and replaced with serum free DMEM with Glutamax. 10 point dilution series (concentration range 0.0015-30 μM) were prepared for both naked and C16 lipid conjugated ASOs. These were dosed into the medium and cells were incubated at 37°C, under 5% CO2 for

[0598] 4 hours. After 4h, the media was removed and replaced with DMEM with GlutaMax, 4.5g / L glucose, Ipg / ml puromycin and 10% FBS. The cells were then left to incubate another 20h at 37°C, under 5% CO2.

[0599] After incubation, medium was removed, cells were washed in PBS and lysed in lOpL Cells to CT lysis buffer (Life Technologies) + 1% DNAse per well for 5 minutes at room temperature with shaking. 2ul of Cells to CT stop solution (Life Technologies) was then added per well. 4pL of each cell lysate was used a a template in a 9pL reverse transcription (RT) reactions (50% RT buffer and 5% enzyme mix from Invitrogen’s Cells-to-CT Bulk RT Reagents), and RT was performed at 37°C for 30 min, followed by RT inactivation at 95°C for

[0600] 5 min.

[0601] Real-Time PCR reactions were set up using 2pL cDNA, TaqMan™ Fast Advanced Master Mix, and Malatl and Hprtl TaqMan™ Gene Expression Assays (Hs00273907_sl and Hs02800695_ml, all Applied Biosystems) in a total volume of lOpL. Amplifications were performed on a QuantStudio™ 7 Flex Real-Time PCR System (Applied Biosystems) and were conducted at 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Quantification cycle (Cq) values were determined by the software using the Auto Baseline and Auto Threshold options and were then used to calculate relative Malatl expression (2A-dCq) normalized against the reference gene Hprtl. 2A-dCq values for Malatl were then normalized to values obtained from H2O treated samples. mCherry-GAL9 recruitment Assay

[0602] All compounds and conjugates were subjected to a mCherry-GAL9 recruitment assay. Under normal conditions, mCherry-GAL9 is homogenously dispersed in the cytosol. When an endosomal leakage event is detected, mCherry-GAL9 translocates to the damaged endosome2. The resulting localization pattern of discrete puncta can be visualized and quantitated.

[0603] Cell lines stably expressing mCherry-GAL9 were seeded into 384- well Phenoplates (PerkinElmer: 6007558) at 3500 (HuH7) or 6500 (16HBE) cells / well, ~24 h before experimental usage.

[0604] Cells were washed thrice with PBS prior to replacing with DMEM + 10% FBS, or in the case of C16 conjugates, with DMEM only using a liquid handling robot (Agilent Bravo). Dose-response curves of conjugates were generated using an Echo 655T acoustic dispenser (Labcyte) to dispense the indicated compounds direct to cell plates prior to incubation for noted time periods.

[0605] At assay endpoints, cells were washed 3x PBS at RT and fixed in 4% PFA (VWR: 9713.1000) for 15 mins / RT. Cells were washed a further 3x PBS before the addition of PBS + 1 pg / ml Hoechst 33342 (ThermoFisher Scientific: H21492) for a minimum of 1 h before imaging.

[0606] Plates were imaged using a spinning-disk confocal microscope (Yokogawa: CV8000) with a 20x water objective (NA 0.75). Images were processed utilizing Columbus image-analysis software (PerkinElmer: v2.9.1) to identify and quantify cells and mCherry-GAL9 structures. The resulting data were processed and normalized in Spotfire (Tibco: vl 1.4) and plotted in Prism (Graphpad: v9.1). Cell Image panels were assembled using the Figure! plugin for FIJI3.

[0607] 1. Munson, M. J. el al. A high-throughput Galectin-9 imaging assay for quantifying nanoparticle uptake, endosomal escape and functional RNA delivery. Commun Biol 4, 211 (2021).

[0608] 2. Du Rietz, H., Hedlund, H., Wilhelmson, S., Nordenfelt, P., Wittrup, A. Imaging small molecule-induced endosomal escape of siRNA. Nat Commun (2020) doi:10.1038 / s41467- 020-15300-1.

[0609] 3. Mutterer, J. & Zinck, E. Quick- and-clean article figures with FigureJ. J Microsc 252, 89-91 (2013).

[0610] I: IC50 and % knockdown at maximum concentration (10 |aM) in Hek293 overexpressing ASGPR (M) after 48h II: IC50 and % knockdown at maximum concentration (10 μM) in primary human hepatocytes (M) after 48h

[0611] III: IC50 and maximum % knockdown in Hek293T wt (M) after 24h

[0612] IV: IC50 in Hek293 Gal9 mCherry (M) after 24h

[0613] V: % knockdown at 10 μM in Huh7 cells VI: Galectin9 response (as fold-change compared to negative control) at 10 uM in 16HBE cells

[0614] VII: Galectin9 response (as fold-change compared to negative control) at 10 uM in Huh7 cells

Claims

CLAIMS1. A macromolecule conjugated to one or more endosomal escape enhancer (EEE) compounds comprising a structure represented by Formula I:wherein one of Z1 and Z2 is N, and the other is C; R1 is hydro, halo, C1-C4 alkyl, -OR4, -C(=O)NR5R5, -CO2R6, or cyano;X is hydro, C1-C4 alkyl, or -OR2;R2 is hydro or C1-C4 alkyl;R3 is hydro, halo, C1-C4 alkyl, -(CthjyOH, -OR4, -C(=O)NR5R5, -CO2R6, or cyano;R4 is C1-C4 alkyl; each R5 is independently hydro or C1-C4 alkyl,Re is hydro or C1-C4 alkyl,R7, R8, R9, Rio, R11 are each independently CHR12, CR12R17 or NR13;R12 is hydro, C1-C4 alkyl, -OR14, or CO2R15;R13 is hydro, C1-C4 alkyl, -(CH2)yOH, -OR14, -CO2R15, or -C(=O)R16;R14 is hydro or C1-C4 alkyl;R15 is hydro or C1-C4 alkyl;R16 is hydro or C1-C4 alkyl;R17 is hydro or C1-C4 alkyl; y is 0, 1, 2, or 3; and wherein one or more of the alkyl are optionally substituted with one or more halo; or a pharmaceutically acceptable salt thereof.

2. The macromolecule of claim 1, wherein the endosomal escape enhancer (EEE) compound comprises a structure represented by Formula la:Formula la., wherein - denotes the conjugation point.

3. The macromolecule of claim 1, wherein the endosomal escape enhancer (EEE) compound comprises a structure represented by Formula lb or Formula Ic or Formula Id or Formula le or Formula If:Formula lbFormula leFormula If, wherein - denotes the conjugation point.

4. The macromolecule of any one of claims 1 to 3, wherein R1 is cyano, bromo, chloro, fluoro, or trifluoromethyl.

5. The macromolecule of any one of claims 1 to 4, wherein X is -OR2, and R2 is methyl, ethyl, or isopropyl.

6. The macromolecule of claim 1, wherein R7, R8, Rio, and Rn are each independently CH2, and R9 is NR13.

7. The macromolecule of any one of claims 1 to 3, wherein the endosomal escape enhancer (EEE) compound is represented by any one of the following Compounds 2-8 or 32:Compound 2:Compound 3:Compound 6:Compound 7 :or a pharmaceutically acceptable salt thereof.

8. A pharmaceutical composition comprising: an oligonucleotide and / or a polypeptide conjugated to one or more compounds according to any of claims 1 to 7; and a pharmaceutically acceptable diluent or carrier.

9. A method of introducing an oligonucleotide and / or a polypeptide into a nucleus and / or cytosol of a cell, the method comprising: contacting the cell with the oligonucleotide and / or the polypeptide conjugated toone or more compounds according to any one of claims 1 to 7, wherein the compound facilitates entry of the oligonucleotide and / or the polypeptide into a nucleus and / or cytosol of the cell.

10. A method for the treatment and / or prevention of a disorder in a subject comprising: administering to the subject a therapeutically effective amount of an oligonucleotide and / or a polypeptide conjugated one or more compounds of any of claims 1 to 7.

11. The method of claim 10, wherein the subject is a human.

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