Oligonucleotide delivery agents, pharmaceutical compositions and methods of use thereof

Abstract

Disclosed is an oligonucleotide delivery agent comprising at least one lipid moiety directly or indirectly coupled to a single-stranded oligonucleotide, the lipid moiety comprising at least one linear alkyl or linear alkenyl group, which oligonucleotide delivery agent can be delivered to various subjects, such as muscles, heart, liver, and the like, and has enhanced efficiency, and therefore, expression regulation of the target gene is improved. Also disclosed are pharmaceutical compositions comprising the oligonucleotide delivery agent and methods of using the pharmaceutical compositions to modulate expression of a target gene.

Description

Technical Field

[0001] The present disclosure relates to the technical field of gene expression regulation, and in particular to a unique oligonucleotide delivery agent for effectively delivering oligonucleotides. Background Art

[0002] Oligonucleotides are an emerging class of therapeutic agents currently under active development for the treatment of a variety of diseases through a variety of different mechanisms of action (MOA). The main categories of oligonucleotide therapeutics include single-stranded antisense oligonucleotides (ASOs) and duplex (double-stranded) RNA (dsRNA), such as siRNA and saRNA.

[0003] There are two main types of ASOs. One type is called a "gapmer" ASO, which degrades target mRNA through the RNase H mechanism, thereby inhibiting gene expression. Gapmer ASOs have a central DNA region required to support RNase H activity and two ribonucleotide wings to increase the binding affinity of the ASO to the target sequence. Another type of ASO is a steric hinderer, which is usually composed of a uniform composition of ribonucleotides with a phosphorothioate (PS) backbone and binds to pre-mRNA in the cell nucleus, altering mRNA processing (e.g., mRNA splicing, translation, and nuclear export) by preventing certain proteins from binding to mRNA (e.g., splicing factors). This type of ASO has been successful in treating diseases that can be improved by correcting abnormal splicing errors and has been approved by the U.S. Food and Drug Administration (FDA), including exon-containing ASO drugs for the treatment of spinal muscular atrophy (SMA). and exon 4 skipping ASO drugs for the treatment of Duchenne muscular dystrophy (DMD).

[0004] ASO is a self-deliverable molecule without the need for additional delivery facilitators, and has limited bioavailability in several types of organs and tissues. However, they usually require high systemic or tissue concentrations to achieve therapeutic effectiveness, and such high concentrations are usually associated with toxicity, resulting in a smaller therapeutic window. So far, the approved PS-ASO drugs are mainly administered locally, including the eyes and CNS (central nervous system), so high local concentrations can be achieved without worrying about systemic toxicity [Vitravene Research Group, Safety of intravitreous fomivirsen for treatment of cytomegalovirus retinitis in patients with AIDS (safety of intravitreous injection of fomivirsen for the treatment of cytomegalovirus retinitis in AIDS patients). Am J Ophthalmol (2002); US20180273943A1; and Stein, CA and D. Castanotto. FDA-Approved Oligonucleotide Therapies in 2017. Mol Thera (2017)]. Systemic ASO drugs are mainly morpholino-based oligomers (PMO) administered by IV for the treatment of Duchenne muscular dystrophy (DMD). Due to the neutral morpholino ring in its molecular structure, PMO generally has a wider safety margin than PS-ASO, and can be given to patients at very high doses. Nevertheless, therapeutic efficacy is still limited [P Duda, GA Laforet et al., Development of Phosphorodiamidate Morpholino Oligomers (PMOs) for the Treatment of Duchenne Muscular Dystrophy (DMD) (development of phosphorodiamidate morpholino oligomers (PMO) for the treatment of Duchenne muscular dystrophy (DMD)) (S42.008), Neurology (2017)]. Improved delivery mechanisms based on oligonucleotides have long been needed to address these challenges.

[0005] After unremitting exploration, the inventors have developed a unique oligonucleotide delivery agent that can enhance the in vivo activity of ASOs, especially in muscle where there is a huge unmet medical need. Summary of the invention

[0006] The present disclosure provides an oligonucleotide delivery agent comprising one or more lipid moieties coupled directly or indirectly to an oligonucleotide, wherein at least one lipid moiety comprises at least one C6-C 22Straight chain alkyl and / or alkenyl, preferably at least one C12, C14, C15, C16, C18 or C20 straight chain alkyl or alkenyl, and the oligonucleotide is a single-stranded oligonucleotide, preferably an antisense oligonucleotide (ASO). In a specific embodiment, an oligonucleotide delivery agent is provided, which has a structure represented by formula A:

[0007]

[0008] The lipid portion comprises at least one C6-C 22 A straight chain alkyl or alkenyl group, M is an integer from 1 to 5, the oligonucleotide is at least one single-stranded oligonucleotide, and N is an integer of 1, 2 or 3; and the delivery enhancing compound (DEC) portion is a divalent to hexavalent portion comprising at least one nitrogen-containing heterocycle, at least one nitrogen-containing straight chain group, at least one nitrogen-containing straight chain carbocyclic group or a combination thereof, and the lipid portion and the oligonucleotide are each directly or indirectly coupled to the delivery enhancing compound portion.

[0009] Also provided herein is a pharmaceutical composition comprising the oligonucleotide delivery agent of the present disclosure, which may optionally include additional ingredients, such as pharmaceutically acceptable carriers, excipients, solvents, diluents, stabilizers, dispersants, buffers, extenders, preservatives, and combinations thereof.

[0010] Also provided herein is a method for regulating target gene expression in a subject, wherein the method comprises: in the case of assays or therapeutic treatments, a step of administering the pharmaceutical composition of the present disclosure to the subject, or in the case of assays, a step of contacting the pharmaceutical composition with cells.

[0011] It should be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

[0012] Brief description of the attached figure

[0013] The features and advantages of the present invention may be better understood with reference to the following detailed description and accompanying drawings which set forth exemplary embodiments in which the principles of the invention are employed and in which:

[0014] Figures 1A-1DThe splicing activity of an exemplary "lipid-DEC-ASO" (LDEC-ASO) and a non-conjugate control in converting SMN2 pre-mRNA to a full-length transcript (SMN2FL) relative to its exon 7 deleted isoform (SMN2Δ7) in skeletal muscle of Type III SMA mice was compared. The indicated doses (i.e., 10 mg / kg, 30 mg / kg, and 100 mg / kg) of LDEC-ASO (i.e., RD-14644) or a non-conjugate control (i.e., RD-10004) were administered to female Type III SMA mice (n=3 mice / dose) by subcutaneous (SC) injection at postnatal days 40-50 (PND 40-50). Treatment with saline alone (n=4 mice / group) was used as a vehicle control to establish baseline expression levels of SMN2FL and SMN2Δ7 mRNA isoforms. Mice were sacrificed 14 days after administration, and the levels of SMN2FL and SMN2Δ17 were quantified by RT-qPCR using isoform-specific primer sets in tissue samples from different skeletal muscles, including the biceps ( Figure 1A )、Semitendinosus( Figure 1B )、Platinum( Figure 1C ) and gluteal muscles ( Figure 1D ). Tbp was amplified as an internal reference for normalization of expression data. Data represent the mean expression levels of SMN2FL or SMN2Δ7 in muscle tissue of treated animals relative to saline control group. Statistical significance was calculated using one-way ANOVA.

[0015] Figure 2A-2B Compared with the liver ( Figure 2A ) and heart ( Figure 2B ) in peripheral tissues. Figure 2A In the present study, the indicated doses (i.e., 10 mg / kg, 30 mg / kg, and 100 mg / kg) of LDEC-ASO (i.e., RD-14644, RD-14645, and RD-14646) or non-conjugate control (i.e., RD-10004) were administered by subcutaneous (SC) injection to female type III SMA mice (n=2-3 mice / dose) at PND40-50. In the heart, only LDEC conjugate RD-14644 treatment was compared with RD-10004 ( Figure 2BTreatment with saline alone (n=4 mice / group) was used as a vehicle control to establish baseline expression levels of SMN2FL and SMN2Δ7 mRNA isoforms. Rats were sacrificed on day 14 post-dose and SMN2FL and SMN2Δ7 levels were quantified by RT-qPCR using isoform-specific primer sets. Tbp was amplified as an internal control for normalization of expression data. Data represent liver ( Figure 2A ) or heart ( Figure 2B ) The average expression levels of SMN2FL or SMN2ΔΔ7 in . Statistical significance was calculated using one-way ANOVA.

[0016] Figure 3A-3B The splicing activity of LDEC-ASO and non-conjugated ASO in converting SMN2 pre-mRNA to SMN2FL relative to the SMN2Δ7 mRNA isoform was compared in peripheral tissues and skeletal muscle of female type III SMA mice. 30 mg / kg of LDEC-ASO (i.e., RD-14644) or non-conjugated control (i.e., RD-10004) was administered to female type III SMA mice by SC injection at PND 40-50. Treatment with aCSF alone was used as a vehicle control to establish baseline expression levels of SMN2FL and SMN2Δ7 mRNA isoforms. Rats were sacrificed on day 14 after dosing, where SMN2FL and SMN2Δ17 levels were quantified by RT-qPCR using isoform-specific primer sets. Tbp was amplified as an internal control for normalization of expression data. Figure 3A-3B The mean expression levels of SMN2FL or SMN2Δ7 in peripheral tissues (i.e., heart, liver, adipose tissue, diaphragm, thoracic aorta with veins) and skeletal muscles (i.e., biceps, semitendinosus, platysma and gluteal muscles) of treated animals relative to aCSF control group are shown. Data are presented as mean ± SEM of 3 mice per group.

[0017] Figures 4A-4F We compared the splicing activity of LDEC-ASOs and unconjugated ASOs in converting SMN2 pre-mRNA to SMN2FL relative to the SMN2Δ7 mRNA isoform in various tissues from type III SMA mice. Figures 4A-4C In the present invention, 10 mg / kg of LDEC-ASO (ie, RD-14644 and RD-14645) or non-conjugate control (ie, RD-10004) was administered to female type III SMA mice by femoral vein (IV) injection at PND 40-50. Figures 4D-4FIn the present study, LDEC conjugate RD-14644 treatment alone was compared to RD-10004 in the heart, diaphragm, and lung. Treatment with saline alone was used as a vehicle control to establish baseline expression levels of SMN2FL and SMN2Δ7 mRNA isoforms. Rats were sacrificed on day 14 after dosing, and SMN2FL and SMN2ΔΔ7 levels were quantified by RT-qPCR using isoform-specific primer sets. Tbp was amplified as an internal control for normalization of expression data. Data represent the average expression levels of SMN2FL or SMN2Δ7 in the liver, adipose tissue (epididymal fat), thoracic aorta, heart, diaphragm, and lung of treated animals relative to saline controls (mean ± SEM of 2-3 mice per group).

[0018] Figure 5A-5B The splicing activity of LDEC-ASOs and non-conjugated ASOs in converting SMN2 pre-mRNA to SMN2FL relative to the SMN2Δ7 mRNA isoform in GM03813 cells was compared. The indicated LDEC-ASOs (i.e., RD-15865, RD-16070, RD-16071, RD-16072, RD-16073, RD-16074, RD-16075, RD-16076, RD-16077, RD-16078, RD-16079, RD-16080, RD-16081, and RD-16098) were transfected into GM03813 cells at 1 nM and 10 nM for 3 days. RD-10004 was transfected and used as a non-conjugated control. Mock treatment was transfection in the absence of oligonucleotides. dsCon2 served as a nonspecific duplex control. Figure 5A-5B The mRNA levels of SMN2FL and SMN2Δ7 quantified by RT-qPCR using gene-specific primer sets in each PCR reaction are shown. SDHA was amplified as an internal control for normalization of expression data. Data represent the average expression level values ​​(mean ± SEM of two replicate transfection wells) of SMN2FL or SMN2Δ7 relative to mock treatment after normalization to SDHA.

[0019] Figure 6A-6BThe splicing regulatory activity of LDEC-ASOs and unconjugated ASOs in converting SMN2 pre-mRNA to SMN2FL relative to the SMN2Δ7 mRNA isoform in GM09677 and GM03813 cells was compared. The indicated LDEC-ASOs (i.e., RD-15865, RD-16070, RD-16071, RD-16072, RD-16073, RD-16074, RD-16075, RD-16076, RD-16077, RD-16078, RD-16079, RD-16080, RD-16081, and RD-16098) were transfected into GM09677 cells at 0.1 nM and 1 nM. Fig. 6A ) and GM03813 cells ( Figure 6B ) for 3 days. RD-10004 was transfected and used as a non-conjugate control. Mock treatment was transfection in the absence of oligonucleotide. dsCon2 served as a non-specific duplex control. Figure 6A-6B The mRNA levels of SMN2FL and SMN2Δ7 quantified by RT-qPCR using gene-specific primer sets in each PCR reaction are shown. SDHA was amplified as an internal control for normalization of expression data. Data represent the average expression level values ​​(mean ± SEM of two replicate transfection wells) of SMN2FL or SMN2Δ7 relative to mock treatment after normalization to SDHA.

[0020] Figure 7A-7B The splicing regulatory activity of LDEC-ASOs and non-conjugated ASOs in converting SMN2 pre-mRNA to SMN2FL relative to the SMN2Δ7 mRNA isoform in GM03813 cells was compared. The indicated LDEC-ASOs (i.e., RD-16935, RD-16936, and RD-16937) were transfected into GM03813 cells at 1 nM and 10 nM for 3 days. RD-10004 was transfected and used as a non-conjugated control. Mock treatment was transfection in the absence of oligonucleotides. Figure 7A-7B The mRNA levels of SMN2FL and SMN2Δ7 quantified by RT-qPCR using gene-specific primer sets in each PCR reaction are shown. SDHA was amplified as an internal control for normalization of expression data. Data represent the average expression level values ​​(mean ± SEM of four replicate transfection wells) of SMN2FL or SMN2Δ7 relative to mock treatment after normalization to SDHA.

[0021] Figures 8A-8BShown are changes in the expression level of human SCN9A mRNA mediated by ASO in a human neuroblastoma cell line (SK-N-AS). 119 ASOs on human SCN9A transcripts were transfected into SK-N-AS cells at 25nM and 100nM for 24 hours. Mock treatment was transfected in the absence of oligonucleotides (not shown). RD-10750 was used as a nonspecific control (not shown). RD-12746 was transfected as a silencing dsRNA control (not shown). SCN9A mRNA levels were quantified by one-step RT-qPCR using a gene-specific primer set in each PCR reaction. The geometric mean of the mRNA levels of HPRT1 and HMBS was used as an internal reference. Fig. 8A Mean expression levels of SCN9A mRNA at 25 nM and 100 nM relative to mock treatment after normalization to HPRT1 and HMBS are shown. ASOs are ranked on the x-axis in descending order by their activity in knocking down SCN9A mRNA expression (mean ± SEM of duplicate transfection wells). Figure 8B The E of ASO to SCN9A mRNA between 25 and 100 nM is shown. 最大 The top 32 ASOs are marked with solid rectangles.

[0022] Figures 9A-9D Shown in SK-N-AS cells, ASOs knock down activity of SCN9A mRNA in a dose-dependent manner. Based on the screening results of SCN9A knockdown activity, the first 32 functional ASOs were selected and transfected into SK-N-AS cells with a specified concentration gradient (i.e., 0.02nM, 0.1nM, 0.39nM, 1.56nM, 6.25nM, 25nM, 100nM, and 400nM) for 24 hours. Simulated treatment was transfected in the absence of oligonucleotides (not shown). RD-10750 was used as a nonspecific control (not shown). RD-12746 was transfected as a silencing dsRNA control (not shown). Figures 9A-9D Quantification of SCN9A mRNA levels by one-step RT-qPCR using gene-specific primer sets in each PCR reaction is shown. The geometric mean of HPRT1 and HMBS mRNA levels was used as an internal control. Data (y-axis) represent the SCN9A mRNA expression in dose response relative to mock treatment after normalization to HPRT1 and HMBS (mean ± SEM of two replicate transfection wells).

[0023] Fig.10The tail-flick reaction latency of SD rats after intrathecal (IT) injection of LDEC-ASO is shown. By IV injection, 0.5mg and 1mg of the indicated LDEC-ASO (i.e., RD-13767 and RD-15069) and uncoupled ASO (i.e., RD-12835 and RD-15064) are injected into adult female SD rats. RD-15873 is processed with 1mg and used as a positive control. Only the processing of aCSF is used as a vehicle control, thereby establishing the baseline of the tail-flick reaction latency. As described in the materials and methods section, the tail-flick reaction latency is tested by the hot water tail immersion test on the 27th day. Data represent the average tail-flick reaction latency (mean ± SEM of 4-5 mice per group) of the treated group relative to the aCSF group.

[0024] Figures 11A to 11B The knockdown activity of LDEC-ASO and non-conjugated ASO on rat Scn9a mRNA expression in SD rats was compared. 0.5 mg and 1 mg of the indicated LDEC-ASO (i.e., RD-13767 and RD-15069) and non-conjugated ASO (i.e., RD-12835 and RD-15064) were given by IV injection. RD-15873 was treated with 1 mg and used as a positive control. Treatment with aCSF alone was used as a vehicle control to establish baseline expression of Scn9a mRNA. Fig.11A Shown is the remaining Scn9a mRNA quantified by RT-qPCR in tissues of the brain (ie, cerebrum), spinal cord (ie, cervical, thoracic, and lumbar vertebrae). Fig. 11B The remaining Scn9a mRNA quantified in dorsal root ganglia (DRG) by RT-qPCR is shown. Rats were sacrificed on day 27 after administration, and Scn9a mRNA levels were quantified by RT-qPCR. The geometric mean of the mRNA levels of Tbp and B2m was used as an internal reference to normalize the expression data. The data represent the average expression of Scn9a mRNA in treated animals relative to the aCSF control group (mean ± SEM of 4-5 mice per group).

[0025] Fig.12Shown is the exon 51 skipping level of the human DMDmRNA mediated by LDEC-ASO in the human malignant embryonic rhabdoid tumor cell line (RD).With 25nM, 50 human LDEC-ASO are transfected into RD cells separately, continue 48 hours.Simulation treatment is to carry out transfection in the absence of oligonucleotide.Transfection RD-16271 and RD-16272 is also used as non-specific control.Transfection RD-15946 is also used as LDEC-ASO positive control.Use two gene specific primer sets to carry out quantitative (shown in table 21) to exon 51 skipping level by nested PCR.Data represent the average expression level (mean ± SEM of two repeated transfection wells) of exon 51 skipping relative to simulation treatment.

[0026] Fig.13 Shown is the dose-dependent exon 51 skipping level of human DMD mRNA mediated by LDEC-ASO in RD cells.With 10nM, 25nM and 100nM, indicated LDEC-ASO (i.e., RD-15905, RD-15935, RD-15936, RD-15941, RD-15942, RD-15943 and RD-15944) is transfected separately into RD cells, and continues 48 hours.Simulation treatment is to carry out transfection in the absence of oligonucleotide.RD-16271 is used as non-specific control.Transfection RD-15945 is also used as non-coupling ASO positive control.Transfection RD-15946 is also used as LDEC-ASO positive control.Use two gene-specific primer sets to quantify (shown in table 21) to exon 51 skipping level by nested PCR.Data represent the average expression level (mean ± SEM of two repeated transfection wells) of exon 51 skipping relative to simulation treatment. DETAILED DESCRIPTION

[0027] Compared with the oligonucleotide delivery technology of the prior art, the newly developed oligonucleotide delivery agent can deliver the required amount or higher levels of oligonucleotide to the site of action at the target tissue indicated above more effectively, and therefore can improve biological activity and pharmacological properties (for example, biodistribution, bioavailability, pharmacokinetics, activity, effectiveness, etc.). In some embodiments, by improving the passage of biological barriers, higher protein binding, better cellular uptake, less entrapment in endosomes, less lysosomal degradation, higher effectiveness and / or longer duration / half-life, it is possible to cause the improvement of biological activity characteristics. In some embodiments, the improvement of pharmacological properties can also obtain lower toxicity, lower dosage, lower administration frequency and less undesirable immune response. In some embodiments, the oligonucleotide delivery agent of the present disclosure relates to a simpler synthesis process, and therefore has better processability and expansibility in manufacturing. In some embodiments, the oligonucleotide delivery agents of the present disclosure have the inherent pharmacological properties of free benzimidazole or benzimidazole derivatives, which are proton pumps or target proton pumps (as reversible and irreversible proton pump inhibitors, PPIs), dopamine receptors (e.g., DRD), angiotensin II type 1 receptor (AT1), histamine receptor (HRH), dual specificity mitogen-activated protein kinase (e.g., MEK) and / or cyclin-dependent kinase (CDK), etc. In some embodiments, the inherent pharmacological properties enable the oligonucleotide delivery agents of the present disclosure to be potentially used in medicine, including peptic ulcers, hypertension, schizophrenia, parasitic infections, bacterial infections, viral infections, cancer, etc.

[0028] In some embodiments, the newly developed oligonucleotide delivery agents disclosed herein can facilitate the delivery of an effective amount of oligonucleotides to achieve functions in target tissues or cells, for example, in the central nervous system (e.g., brain and spinal cord), heart, eye, ear, liver, lung, kidney, spleen, adipose tissue, intestine, pancreas, gallbladder, muscle (e.g., smooth muscle, skeletal muscle and myocardium), diaphragm, thoracic aorta and bone. In some embodiments, compared with the technology of the prior art, the regulatory activity of the oligonucleotide delivery agent, such as up-regulating or down-regulating the expression of the target gene in the cell, can be improved. Therefore, according to the findings provided herein, it will be understood by those skilled in the art that the use of the oligonucleotide delivery agents described herein can promote the therapeutic oligonucleotides to have improved biodistribution in various targets or cells as described above, to prevent, treat and / or delay the onset of various diseases, disorders and / or conditions.

[0029] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. In addition, all publications, patent applications, patents and other references mentioned herein are incorporated by reference.

[0030] As disclosed herein, terms such as "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0031] As disclosed herein, the terms "approximately," "about," "substantially," and similar terms are intended to have the broadest meaning consistent with commonly accepted usage by one of ordinary skill in the art to which the disclosed subject matter pertains.

[0032] As disclosed herein, "and / or" means "and, or as an alternative". If any numerical range is given, the range includes any value in the range (including the upper and lower values), and includes any sub-range in the range. For example, a range from 1 to 3 may include any value in 1, 2, and 3, and sub-ranges from 1 to 2 and from 2 to 3.

[0033] As used herein, the terms "oligonucleotide," "polynucleotide," or "oligomer" are used interchangeably and refer to a polymer of nucleotides, specifically single-stranded nucleic acid molecules of DNA, RNA, or DNA / RNA hybrids, oligonucleotide chains containing regular and irregular alternating deoxyribose and ribose moieties, as well as modified and naturally or non-naturally occurring frameworks for such oligonucleotides, for example, phosphorodiamidate morpholino oligonucleotides (PMOs).

[0034] As used herein, the term "complementary" refers to the ability to form base pairs between two oligonucleotide chains. Base pairs are generally formed by hydrogen bonds between the nucleotides in antiparallel oligonucleotide chains. The bases of complementary oligonucleotide chains can be paired in Watson-Crick mode (e.g., A to T, A to U and C to G) or in any other mode (e.g., RNA-RNA or DNA-RNA duplexes by Hoogsteen or reverse Hoogsteen base pairing) to allow formation of homologous or heteroduplexes.

[0035] As used herein, the term "SMN2FL mRNA" or "SMN2Δ7 mRNA" refers to the messenger RNA (mRNA) produced by the expression of the SMN2 gene or the transcription of the SMN2 gene.

[0036] As used herein, capitalized "SCN9A" or "SCN9A gene" refers to the human gene; and capitalized "Scn9a" or "Scn9a gene" refers to the mouse gene.

[0037] As used herein, the term "SCN9A mRNA" refers to messenger RNA (mRNA) produced by the expression of the SCN9A gene or the transcription of the SCN9A gene.

[0038] As used herein, capitalized "DMD" or "DMD gene" refers to the human gene.

[0039] The present disclosure also relates, at least in part, to an antisense oligonucleotide (ASO) comprising an oligonucleotide sequence ranging in length from 12 to 30 consecutive nucleotides, wherein the oligonucleotide sequence of the SMN2 gene is selected from SEQ ID NO: 1 and their oligonucleotide delivery agents SEQ ID NO: 2-21, wherein the ASO targets the pre-mRNA of the SMN2 gene, regulates its splicing by including exon 7 into the mature mRNA, thereby "converting" SMN2Δ7 (i.e., SMN2 with exon 7 deleted) in the cell into SMN2FL (i.e., full-length SMN2 including exon 7) mRNA, thereby increasing the level of SMN protein encoded by the SMN2FL mRNA.

[0040] The present disclosure also relates, at least in part, to an antisense oligonucleotide (ASO) comprising an oligonucleotide sequence ranging in length from 12 to 30 contiguous nucleotides, wherein the oligonucleotide sequence comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% complementary to an equal length portion of SEQ ID NO: 22, wherein the ASO inhibits at least 50% of the mRNA transcript of the SCN9A gene compared to a baseline of SCN9A mRNA levels.

[0041] The inventors also found that the optimal antisense oligonucleotide (ASO) sequence comprising the 3′-UTR region in the SCN9A mRNA transcript includes a sequence having: (1) 40% to 70% GC content; (2) less than 5 consecutive identical nucleotides; (3) less than 3 dinucleotide or trinucleotide repeats; and (4) not in the 5′UTR region of the SCN9A mRNA. As a beneficial result, the target sequence (e.g., an isolated nucleic acid sequence comprising the target sequence) inhibits at least 50% of the mRNA transcript of the SCN9A gene compared to the baseline of the SCN9A mRNA level.

[0042] Certain embodiments of the present application relate to an ASO target sequence comprising a 3′-UTR region in an SCN9A mRNA transcript, wherein the target sequence in the mRNA transcript has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or at least 100% homology with a nucleotide sequence selected from SEQ ID NOs: 23-141.

[0043] Certain embodiments of the present application also relate to an ASO comprising a single-stranded oligonucleotide sequence, which has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% homology or is 100% identical to a nucleotide sequence selected from chemically modified SEQ ID NOs: 261-379, SEQ ID NOs: 388 and SEQ ID NOs: 390, and their unmodified naked sequences SEQ ID NOs: 142-260, wherein the ASO inhibits the mRNA transcript of the SCN9A gene by at least 50% compared to the baseline of the SCN9A mRNA level.

[0044] Certain embodiments of the present application also relate to an ASO comprising a single-stranded oligonucleotide sequence, which has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% homology or is 100% identical to a nucleotide sequence selected from the oligonucleotide delivery agents SEQ ID NO: 389 and SEQ ID NO: 391, wherein the ASO inhibits the mRNA transcript of the SCN9A gene by at least 50% compared to the baseline of SCN9A mRNA levels.

[0045] The present disclosure also relates, at least in part, to antisense oligonucleotides (ASOs) comprising an oligonucleotide sequence ranging in length from 12 to 30 contiguous nucleotides, wherein the oligonucleotide sequence comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, 95% or 100% complementary to an equal length portion of SEQ ID NO: 392, wherein the ASO enhances exon 51 skipping levels of the DMD gene by at least 30% compared to a baseline of DMD exon 51 skipping levels.

[0046] The inventors also found that the optimal antisense oligonucleotide (ASO) sequence in the DMD exon 51 region includes a sequence having: (1) a GC content of 40% to 70%; (2) less than 5 consecutive identical nucleotides; (3) less than 3 dinucleotide or trinucleotide repeats. As a beneficial result, the target sequence (e.g., an isolated nucleic acid sequence comprising the target sequence) enhances the exon 51 skipping level of the DMD gene by at least 30% compared to the baseline of the DMD exon 51 skipping level.

[0047] Certain embodiments of the present application relate to an ASO target sequence in the exon 51 region of DMD, wherein the target sequence in the exon 51 region has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or at least 100% homology with a nucleotide sequence selected from SEQ ID NOs: 393-442.

[0048] Certain embodiments of the present application also relate to an ASO comprising a single-stranded oligonucleotide sequence, which has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% homology or is 100% identical to a nucleotide sequence selected from the oligonucleotide delivery agent SEQ ID NOs: 493-542 and their unmodified naked sequences SEQ ID NOs: 443-492, wherein the ASO enhances the exon 51 skipping level of the DMD gene by at least 30% compared to the baseline of the DMD exon 51 skipping level.

[0049] Also provided herein are vectors and cells comprising the oligonucleotide delivery agents of the present disclosure. In some embodiments, the cell is a mammalian cell, optionally a human cell. In some embodiments, the cell is a host cell. In some embodiments, the cell is in vitro. In some embodiments, the cell is present in a mammal.

[0050] In various embodiments, the oligonucleotide delivery agent comprises a single stranded oligonucleotide of 15 to 35 nucleotides in length, e.g., a single stranded oligonucleotide of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in length.

[0051] In various embodiments, the oligonucleotide delivery agent comprises a lipid portion, which is a C12, C13, C14, C15, C16, C17, C18, C19 or C20 straight chain saturated or unsaturated hydrocarbon group, for example, a straight chain alkyl or straight chain alkenyl. The straight chain alkenyl may contain one to four ethylenically unsaturated groups, for example, one or two ethylenically unsaturated groups. In an exemplary embodiment, the lipid portion comprises 2-dodecenyl, 3-dodecenyl, 4-dodecenyl, 5-dodecenyl, 6-dodecenyl, 7-dodecenyl, 8-dodecenyl, 9-dodecenyl, 10-dodecenyl, 2-hexadecenyl, 3-hexadecenyl, 4-hexadecenyl, 5-hexadecenyl, 6-hexadecenyl, 7-hexadecenyl, 8-hexadecenyl, 9-hexadecenyl, 10-hexadecenyl, 11-hexadecenyl, 12- octadecenyl, 2-octadecenyl, 3-octadecenyl, 4-octadecenyl, 5-octadecenyl, 6-octadecenyl, 7-octadecenyl, 8-octadecenyl, 9-octadecenyl, 10-octadecenyl, 11-octadecenyl, 12-octadecenyl, 4,7-hexadecadienyl, 5,8-hexadecadienyl, 6,9-hexadecadienyl, 7,10-hexadecadienyl, 5,8-octadecadienyl, 6,9-octadecadienyl or 7,10-octadecadienyl.

[0052] In certain embodiments, the unsaturated lipid moiety comprises at least one cis-unsaturated double bond in the alkenyl group. In certain embodiments, the unsaturated lipid moiety comprises at least one trans-unsaturated double bond in the alkenyl group.

[0053] In various embodiments, the oligonucleotide delivery agent comprises one or more lipid moieties coupled directly or indirectly to the oligonucleotide. As used herein, the term "lipid moiety coupled indirectly to the oligonucleotide" refers to embodiments in which the lipid and the oligonucleotide are both coupled to the same DEC moiety, wherein the connection between the DEC moiety and the oligonucleotide can be a covalent bond or a linking moiety, such as those listed below.

[0054] On the other hand, the term "lipid moiety directly coupled to the oligonucleotide" refers to embodiments in which the oligonucleotide delivery agent does not include a DEC moiety, and thus the lipid and oligonucleotide are linked to each other via a covalent bond or a linking moiety such as those listed below.

[0055] In some embodiments, the oligonucleotide delivery agent has a structure represented by formula A, wherein the lipid portion is coupled to the oligonucleotide via a DEC portion, wherein M refers to the number of lipid portions, which is an integer of 1, 2, 3, 4, or 5, and N refers to the number of DEC compounds coupled to the oligonucleotide, which is an integer of 1, 2, or 3. In an exemplary embodiment, the delivery enhancing compound portion is a divalent to hexavalent portion comprising at least one nitrogen-containing heterocycle, and the lipid portion and the oligonucleotide are each coupled to the delivery enhancing compound portion. In another embodiment, the delivery enhancing compound portion is a divalent, trivalent, tetravalent, pentavalent, or hexavalent portion. Preferably, the valence of the delivery enhancing compound portion is equal to M+1. More preferably, the delivery enhancing compound portion is a divalent, trivalent, or tetravalent portion.

[0056] As used herein, the terms "delivery enhancing compound" and "DEC" are used interchangeably and refer to a compound from which a delivery enhancing compound moiety can be derived.

[0057] In an exemplary embodiment, the nitrogen-containing heterocycle included in DEC may have a core structure shown in any one of the following formulae, wherein all chemical bonds, atoms, and substituents connected to the ring atoms of the core structure are omitted for simplicity:

[0058]

[0059] The core structure shown above comprises different ring atoms, and the condensed ring core structure can be electrically neutral or locally charged in the form of, for example, sulfonium or quaternary ammonium. It can be seen that, due to the delocalized π electron conjugation effect, one or more unsaturated double bonds in the condensed ring core structure may move, and all substituents, including any substituents that exist / do not exist, can change according to the category and valence of each ring atom connected to the substituent. For example, other substituents can be connected to sulfonium or quaternary ammonium ions that exist as the ring atoms of the condensed ring core structure. All these changes are within the conception of the present disclosure.

[0060] In one embodiment, the linkage of the at least one lipid moiety to the DEC moiety (ie, the combination of the lipid moiety and the delivery enhancing compound moiety, LDEC) may form a structure represented by Formula A'

[0061]

[0062] The lipid portion comprises at least one C6-C 22 Straight chain alkyl or alkenyl, for example at least one C8-C 18 A straight chain alkyl or alkenyl group, or at least one (e.g., one, two, or three) C 10 -C 12 The straight chain alkyl or alkenyl, DEC moiety and M are as defined above.

[0063] Exemplary compounds from which the structure represented by Formula A' is derived are specifically defined below, and the definition of the DEC moiety can be clearly and unambiguously determined by subtracting the at least one lipid moiety from the structure represented by Formula A'.

[0064] In one embodiment, the structure represented by formula A' is derived from the structure represented by formula I or formula II, for example, by subtracting at least one hydrogen atom or at least one terminal group from the structure represented by formula I or formula II. According to an embodiment, the terminal group can be selected from hydroxyl, carboxyl, ester, ether, alkyl, alkoxy, phosphate, phosphonate, sulfate, sulfonate, amide, acyl, and combinations thereof.

[0065]

[0066] Among them, each independently represent a covalent single or double bond; each occurrence of X is an atom selected from carbon, nitrogen, oxygen and sulfur; and F, G, H and I are each independently selected from carbon, nitrogen, oxygen and sulfur.

[0067] In one embodiment, m is an integer of 1, 2 or 3, n is an integer of 1, 2 or 3, and m+n=4. In some exemplary embodiments, m=1 and n=3; or m=2 and n=2; or m=3 and n=1.

[0068] In one embodiment, C is either absent or selected from the group consisting of hydrogen, a halogen atom such as fluorine, chlorine, bromine or iodine, a hydroxyl group, (C1-C 20 )alkyl, such as -(C2-C 19 )alkyl, or -(C3-C 18 )alkyl, or -(C4-C 16 )alkyl, or -(C6-C 12 )alkyl, or -(C8-C 10 ) alkyl; (C1-C 20 ) alkoxy, such as -(C2-C 19 )alkoxy, or -(C3-C 18 )alkoxy, or -(C4-C 16 )alkoxy, or -(C6-C 12 ) alkoxy; halo(C1-C 20 )alkyl, such as halo(C2-C 19 ) alkyl, or halogenated (C3-C 18 ) alkyl, or halogenated (C4-C 16 ) alkyl, or halogenated (C6-C 12 ) alkyl, or halogenated (C8-C 10 ) alkyl; and halo (C1-C 20 ) alkoxy, such as halo(C1-C 20 ) alkoxy, such as halo(C2-C 19 ) alkoxy, or halogenated (C3-C 18 ) alkoxy, or halogenated (C4-C 16 ) alkoxy, or halogenated (C6-C 12 ) alkoxy, or halogenated (C8-C 10 )alkoxy; wherein "halo" can be fluorinated, chloro, bromo, iodo or a combination thereof. In one embodiment, in the structure represented by Formula I, each B is connected to any one of F, G, H and I, and C is connected to the remaining ones of F, G, H and I. In one embodiment, B is connected to H and three C's are connected to each of F, G and I, respectively. In another embodiment, B is connected to G and three C's are connected to each of F, H and I, respectively. In another embodiment, B is connected to F and three C's are connected to each of G, H and I, respectively. In another embodiment, B is connected to I and three C's are connected to each of F, G and H, respectively. In another embodiment, two B's are connected to G and H, respectively, and two C's are connected to each of F and I, respectively.

[0069] In one embodiment, B is independently selected at each occurrence from the group consisting of hydroxyl; -C(O)OH; -P(O)2-OH; -P(O)-OH; -P(O)(S)-OH; -CN; -(C1-C 22 )alkyl, such as -(C2-C 20 )alkyl, or -(C3-C 16 )alkyl, or -(C6-C 12 )alkyl; -O-(C1-C 22 )alkyl, such as -O-(C2-C 20 )alkyl, or -O-(C3-C 16 )alkyl, or -O-(C6-C 12 )alkyl; -(C1-C 22 )alkenyl, such as -(C2-C 20 )alkenyl, or -(C3-C 16 )alkenyl, or -(C6-C 12 )alkenyl; -(C1-C 22 ) alkylene-OH, for example -(C2-C 20 ) alkylene-OH, or -(C3-C 16 ) alkylene-OH, or -(C6-C 12 )alkylene-OH; -(C3-C 22 )cycloalkylene-OH, for example -(C5-C 16 )cycloalkylene-OH, or -(C6-C 12 )cycloalkylene-OH, or -(C6-C 10 )cycloalkylene-OH; -(C6-C 22 )arylene-OH; -(C6-C 22 )heteroarylene-OH; -(C1-C 22 )alkylene-C(O)OH, for example -(C2-C 18 )alkylene-C(O)OH, or -(C3-C 16 )alkylene-C(O)OH, or -(C6-C 12 )alkylene-C(O)OH; -(C3-C 22 )cycloalkylene-C(O)OH; -(C6-C 22 )arylene-C(O)OH; -(C5-C 22 )heteroarylene-C(O)OH; -OC(O)-(C1-C 22 )alkylene-C(O)NH2, for example -OC(O)-(C2-C 18 )alkylene-C(O)NH2, or -OC(O)-(C3-C 16)alkylene-C(O)NH2, or -OC(O)-(C6-C 12 )alkylene-C(O)NH2; -(C1-C 22 )alkylene-OC(O)-(C1-C 22 )alkylene-C(O)NH2, for example -(C2-C 18 )alkylene-OC(O)-(C2-C 18 )alkylene-C(O)NH2, or -(C3-C 16 )alkylene-OC(O)-(C3-C 16 )alkylene-C(O)NH2, or -(C6-C 12 )alkylene-OC(O)-(C6-C 12 )alkylene-C(O)NH2; -OC(O)-(C1-C 22 )alkylene-C(O)OH; -(C1-C 22 )alkylene-OC(O)-(C1-C 22 )alkylene-C(O)OH; -C(O)-(C1-C 22 )alkylene-C(O)NH2; -(C1-C 22 )alkylene-C(O)-(C1-C 22 )alkylene-C(O)NH2; -C(O)-NH-(C1-C 22 )alkylene-OH; -C(O)-NH-(C1-C 22 )alkylene-C(O)OH; -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-C(O)OH; -(C1-C 30 )alkylene-P(O)2-OH; -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-CN, for example -(C2-C 18 )alkylene-OP(-N(C2-C 18 Alkyl)2)-O-(C2-C 18 )alkylene-CN, or -(C3-C 16 )alkylene-OP(-N(C3-C 16 Alkyl)2)-O-(C3-C 16 )alkylene-CN, or -(C6-C 12 )alkylene-OP(-N(C6-C 12 Alkyl)2)-O-(C6-C 12)alkylene-CN; -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-OH; -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-NH2; -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-C(O)OH; -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-CN; -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-OH; -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 30 )alkylene-C(O)OH; -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-NH2; -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-CN; -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-OH; -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-C(O)OH; -(C1-C 22)alkylene-C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-NH2; -(C1-C 22 )alkylene-P(O)-OH; -(C1-C 22 )alkylene-P(O)(S)-OH and -(C1-C 22 ) alkylene-CN. B contains (C1-C 22 )alkyl or (C1-C 22 ) The alkylene groups can each be an alkyl or alkylene group containing 1 to 22 carbon atoms, or 2 to 20 carbon atoms, or 3 to 16 carbon atoms, or 4 to 12 carbon atoms, or 6 to 12 carbon atoms, or 8 to 10 carbon atoms.

[0070] In one embodiment, each of A1, A2 and A3 is either absent or a substituent independently selected from the group consisting of: -H, -OH, -OR 1 ,-SH,-(C1-C 22 )alkyl, halo-(C1-C 22 )alkyl, -(C2-C 22 )alkenyl, -(C1-C 22 ) alkylene-OH, -(C1-C 22 )Alkenylene-OH, -(C3-C 22 )cycloalkyl, -(C3-C 22 )cycloalkenyl, -(C1-C 22 )alkylene-(C3-C 22 )cycloalkyl, -(C1-C 22 )alkylene-R 1 、-(C1-C 22 )Alkenylene-R 1 、-(C1-C 22 )alkylene-OR 1 、-(C1-C 22 )Alkenylene-OR 1 、-(C1-C 22 )Alkylene-COOR 1 、-(C1-C 22 )Alkenylene-COOR 1 、-C(O)OR 1 、-O-(C1-C 22 )alkyl, -S-(C1-C 22 )alkyl, -C(O)-R 1 、-C(O)-(C1-C 22)alkyl, -OC(O)-(C1-C 22 )alkyl, -OC(O)-R 1 、-(C1-C 22 )alkylene-OC(O)-R 1 、-C(O)-(C1-C 22 )alkylene-OH, -C(O)-(C1-C 22 )alkylene-R 1 、-C(O)-(C1-C 22 )alkylene-NH-R 1 、-C(O)-(C1-C 22 )Alkylene-NR 2 -R 1 、-OC(O)-(C1-C 22 )alkylene-OH, -OC(O)-(C1-C 22 )alkylene-R 1 , -adamantyl, -(C1-C 22 )alkylene-adamantyl, -O-adamantyl, -C(O)-(C1-C 22 ) alkylene-adamantyl, -(C1-C 22 )alkylene-C(O)-(C1-C 22 ) alkylene-adamantyl, -NH-C(O)-(C1-C 22 ) alkylene-adamantyl, -(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkyl, -(C1-C 22 )alkylene-NH-C(O)-halogeno(C1-C 22 )alkyl, -CH(NH-CO-(C1-C 22 )alkyl)-(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkyl, -CH(NH-CO-halogenated(C1-C 22 )alkyl)-(C1-C 22 )alkylene-NH-C(O)-halogeno(C1-C 22 )alkyl, -(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 ) alkylene-adamantyl, -(C1-C 22 )Alkylene-NR 2 -C(O)-(C1-C 22 ) alkylene-adamantyl, -(C1-C 22 )alkylene-(C1-C6 epoxyalkyl)(1-20) -NH-C(O)-(C1-C 22 ) alkylene-adamantyl, -C(O)NH-(C1-C 22 )alkyl, -C(O)NH-R 1 、-C(O)NR 2 -R 1 、-C(O)NH-(C1-C 22 )alkylene-OH, -C(O)NH-(C1-C 22 )alkylene-COOH, -NH-C(O)-(C1-C 22 )alkyl, -NH-C(O)-R 1 、-NR 2 -C(O)-R 1 、-OP(O)2-OR 1 、-OP(O)(S)-OR 1 、-OP(O)-OR 1 、-NH-R 1 、-NR 2 -R 1 、-(C1-C 22 )alkylene-NH-R 1 、-(C1-C 22 )Alkylene-NR 2 -R 1 、-C(O)-(C1-C 22 )alkylene-C(O)-R 1 、-C(O)-(C1-C 22 )alkylene-C(O)OR 1 、-C(O)-(C1-C 22 )alkylene-NH-C(O)-R 1 、-C(O)-(C1-C 22 )Alkylene-NR 2 -C(O)-R 1 、-(C1-C 22 )alkylene-C(O)-R 1 、-(C1-C 22 )alkylene-NH-C(O)-R 1 、-(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkylene-R 1 、-(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkylene-R 1、-(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-R 1 、-(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-R 1 、-(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkylene-R 1 、-(C1-C 22 )Alkylene-NR 2 -C(O)-(C1-C 22 )alkylene-R 1 、-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-CN, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-OH, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-NH2, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-C(O)OH, -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-CN, -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-OH, -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 30 )alkylene-C(O)OH, -C(O)-NH-(C1-C 22)alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-NH2, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-CN, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-OH, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-C(O)OH, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-NH2, -(C1-C 22 )alkylene-P(O)-OH, -(C1-C 22 )alkylene-P(O)(S)-OH, -(C1-C 22 )alkylene-CN, substituted or unsubstituted pyrrole, substituted or unsubstituted pyrroline, substituted or unsubstituted pyrrolidine, substituted or unsubstituted pyrazole, substituted or unsubstituted pyrazoline, substituted or unsubstituted pyrazolidine, substituted or unsubstituted imidazole, substituted or unsubstituted oxazole, substituted or unsubstituted thiazole, substituted or unsubstituted benzopyrrole, substituted or unsubstituted benzopyrroline, substituted or unsubstituted benzopyrrolidine, substituted or unsubstituted benzopyrazole, substituted or unsubstituted benzopyrazoline, substituted or unsubstituted benzopyrazolidine, substituted or unsubstituted benzimidazole, substituted or unsubstituted benzoxazole, substituted or unsubstituted benzothiazole, wherein when the above ring is substituted, the substituent may include hydroxyl, halogen atom (such as fluorine, chlorine, bromine or iodine), (C1-C 16 )alkyl, (C1-C 12 ) alkoxy; and a substituent represented by formula III,

[0071]

[0072] In one embodiment, Y is selected from carbon, nitrogen, oxygen, and sulfur; and each of P, Q, S, and T is independently selected from carbon, nitrogen, oxygen, and sulfur; and the asterisk refers to the site of attachment of the substituent represented by Formula III to the structure represented by Formula I or Formula II;

[0073] In one embodiment, R 3 , R 4 and R 5 Each of the above is either absent or is a substituent independently selected from the group consisting of: -H, -OH, -OR 1 ,-SH,-(C1-C 22 )alkyl, halo-(C1-C 22 )alkyl, -(C2-C 22 )alkenyl, -(C1-C 22 ) alkylene-OH, -(C3-C 22 )cycloalkyl, -(C3-C 22 )cycloalkenyl, -(C1-C 22 )alkylene-(C3-C 22 )cycloalkyl, -(C1-C 22 )alkylene-R 1 、-(C1-C 22 )alkylene-OR 1 、-(C1-C 22 )Alkylene-COOR 1 、-C(O)OR 1 、-O-(C1-C 22 )alkyl, -S-(C1-C 22 )alkyl, -C(O)-R 1 、-C(O)-(C1-C 22 )alkyl, -OC(O)-(C1-C 22 )alkyl, -OC(O)-R 1 、-(C1-C 22 )alkylene-OC(O)-R 1 、-C(O)-(C1-C 22 )alkylene-OH, -C(O)-(C1-C 22 )alkylene-R 1 、-C(O)-(C1-C 22 )alkylene-NH-R 1 、-C(O)-(C1-C 22 )Alkylene-NR 2 -R 1 、-OC(O)-(C1-C 22 )alkylene-OH, -OC(O)-(C1-C 22 )alkylene-R1 , -adamantyl, -(C1-C 22 )alkylene-adamantyl, -O-adamantyl, -C(O)-(C1-C 22 ) alkylene-adamantyl, -(C1-C 22 )alkylene-C(O)-(C1-C 22 ) alkylene-adamantyl, -NH-C(O)-(C1-C 22 ) alkylene-adamantyl, -(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkyl, -(C1-C 22 )alkylene-NH-C(O)-halogeno(C1-C 22 )alkyl, -CH(NH-CO-(C1-C 22 )alkyl)-(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkyl, -CH(NH-CO-halogenated(C1-C 22 )alkyl)-(C1-C 22 )alkylene-NH-C(O)-halogeno(C1-C 22 )alkyl, -(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 ) alkylene-adamantyl, -(C1-C 22 )Alkylene-NR 2 -C(O)-(C1-C 22 ) alkylene-adamantyl, -(C1-C 22 )alkylene-(C1-C6 epoxyalkyl) (1-20) -NH-C(O)-(C1-C 22 ) alkylene-adamantyl, -C(O)NH-(C1-C 22 )alkyl, -C(O)NH-R 1 、-C(O)NR 2 -R 1 、-C(O)NH-(C1-C 22 )alkylene-OH, -C(O)NH-(C1-C 22 )alkylene-COOH, -NH-C(O)-(C1-C 22 )alkyl, -NH-C(O)-R 1 、-NR 2 -C(O)-R 1 、-OP(O)2-OR 1 、-OP(O)(S)-OR 1、-OP(O)-OR 1 、-NH-R 1 、-NR 2 -R 1 、-(C1-C 22 )alkylene-NH-R 1 、-(C1-C 22 )Alkylene-NR 2 -R 1 、-C(O)-(C1-C 22 )alkylene-C(O)-R 1 、-C(O)-(C1-C 22 )alkylene-C(O)OR 1 、-C(O)-(C1-C 22 )alkylene-NH-C(O)-R 1 、-C(O)-(C1-C 22 )Alkylene-NR 2 -C(O)-R 1 、-(C1-C 22 )alkylene-C(O)-R 1 、-(C1-C 22 )alkylene-NH-C(O)-R 1 、-(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkylene-R 1 、-(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkylene-R 1 、-(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-R 1 、-(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-R 1 、-(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkylene-R 1 、-(C1-C 22 )Alkylene-NR 2 -C(O)-(C1-C 22 )alkylene-R1 、-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-CN, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-OH, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-NH2, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-C(O)OH, -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-CN, -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-OH, -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 30 )alkylene-C(O)OH, -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-NH2, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-CN, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-OH, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22)alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-C(O)OH, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-NH2, -(C1-C 22 )alkylene-P(O)-OH, -(C1-C 22 )alkylene-P(O)(S)-OH, -(C1-C 22 )alkylene-CN, substituted or unsubstituted pyrrole, substituted or unsubstituted pyrroline, substituted or unsubstituted pyrrolidine, substituted or unsubstituted pyrazole, substituted or unsubstituted pyrazoline, substituted or unsubstituted pyrazolidine, substituted or unsubstituted imidazole, substituted or unsubstituted oxazole, substituted or unsubstituted thiazole, substituted or unsubstituted benzopyrrole, substituted or unsubstituted benzopyrroline, substituted or unsubstituted benzopyrrolidine, substituted or unsubstituted benzopyrazole, substituted or unsubstituted benzopyrazoline, substituted or unsubstituted benzopyrazolidine, substituted or unsubstituted benzimidazole, substituted or unsubstituted benzoxazole and substituted or unsubstituted benzothiazole, wherein when the above ring is substituted, the substituent may include hydroxyl, halogen atoms (such as fluorine, chlorine, bromine or iodine), (C1-C 16 )alkyl, (C1-C 12 )alkoxy.

[0074] In one embodiment, R 7 is connected to any one of P, Q, S and T at each occurrence and is either absent or selected from hydrogen, a halogen atom, a hydroxyl group, (C1-C 20 )alkyl, (C1-C 20 ) alkoxy, halogenated (C1-C 20 ) alkyl and halogenated (C1-C 20 )alkoxy.

[0075] In one embodiment, p is an integer of 0, 1, 2 or 3.

[0076] In one embodiment, R 6 is connected to any one of P, Q, S and T, and is selected from a direct bond, -O-, -C(O)O-, -OC(O)-, -P(O)2-O-, -OP(O)2-O-, -P(O)(S)-O-, -OP(O)(S)-O-, -OP(O)-O-, -(C1-C 22 )alkylene-, -(C1-C 22)alkylene-O-, -O-(C1-C 22 )alkylene-, -(C1-C 22 ) alkylene-NH-, -NH-(C1-C 22 )alkylene-, -C(O)-(C1-C 22 )alkylene-, -(C1-C 22 )alkylene-C(O)-, -C(O)-O-(C1-C 22 )alkylene-, -(C1-C 30 )alkylene-C(O)-O-, -C(O)-NH-(C1-C 22 )alkylene-, -C(O)-NH-(C1-C 22 )alkylene-C(O)-O-, -C(O)-NH-(C1-C 22 )alkylene-OC(O)-, -C(O)-NH-(C1-C 22 )alkylene-OC(O)-O-, -C(O)-NH-(C1-C 22 )alkylene-O-, -C(O)-N((C1-C 22 )alkyl)-(C1-C 22 )alkylene-, -C(O)-N((C1-C 22 )alkyl)-(C1-C 22 )alkylene-O-, -C(O)-NH-(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-, -C(O)-NH-(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkylene-, -C(O)-NH-(C1-C 22 )alkylene-C(O)-NH-, -C(O)-N((C1-C 22 )alkyl)-(C1-C 22 )alkylene-C(O)-N((C1-C 22 )alkyl)-(C1-C 22 )alkylene-, -C(O)-N((C1-C 22 )alkyl)-(C1-C 22 )alkylene-C(O)-N((C1-C 22 )alkyl)-, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-, -(C1-C 22)alkylene-C(O)-NH-(C1-C 22 )alkylene-C(O)-NH-, -NH-C(O)-(C1-C 22 )alkylene-, -NH-C(O)-(C1-C 22 )alkylene-C(O)-O-, -NH-C(O)-(C1-C 22 )alkylene-C(O)-, -NH-C(O)-(C1-C 22 )alkylene-O-, -N((C1-C 22 )alkyl)-C(O)-(C1-C 22 )alkylene-, -N((C1-C 22 )alkyl)-C(O)-(C1-C 22 )alkylene-O-, -NH-C(O)-(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkylene-, -NH-C(O)-(C1-C 22 )alkylene-NH-C(O)-, -N((C1-C 22 )alkyl)-C(O)-(C1-C 22 )alkylene-N((C1-C 22 )alkyl)-C(O)-(C1-C 22 )alkylene-, -N((C1-C 22 )alkyl)-C(O)-(C1-C 22 )alkylene-N((C1-C 22 )alkyl)-C(O)-, -(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkylene-, -(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkylene-NH-C(O)-, -(C1-C 22 )alkylene-P(O)2-O-, -(C1-C 22 )alkylene-OP(O)2-O-, -(C3-C 22 )cycloalkylene-, -(C3-C 22 )cycloalkylene-O-, -O-(C3-C 22 )cycloalkylene-, -(C6-C 22 )arylene-, -(C6-C 22 )arylene-O-, -O-(C6-C 22 )arylene-, -(C6-C 22)arylene-NH-, -NH-(C6-C 22 )arylene-, -C(O)-(C6-C 22 )arylene-, -(C6-C 22 )arylene-C(O)-, -C(O)-O-(C6-C 22 )arylene-, -(C6-C 22 )arylene-C(O)-O-, -C(O)-NH-(C6-C 22 )arylene- and -C(O)-NH-(C6-C 22 )arylene-C(O)-O-.

[0077] In one embodiment, R 1 is independently selected at each occurrence from hydrogen, hydroxy, -(C1-C 22 )alkyl, -(C3-C 22 )cycloalkyl, -(C6-C 22 )aryl, -(C1-C 22 ) alkoxy, -(C3-C 22 )cycloalkoxy, -(C6-C 22 ) aryloxy, -C(O)-(C1-C 22 )alkyl, -OC(O)(C1-C 22 )alkyl, -C(O)-O-(C1-C 22 )alkyl, -C(O)-(C3-C 22 )cycloalkyl, -OC(O)-(C3-C 22 )cycloalkyl, -C(O)-O-(C3-C 22 )cycloalkyl, -C(O)-(C6-C 22 )aryloxy, -OC(O)-(C6-C 22 ) aryloxy, -C(O)-O-(C6-C 22 ) aryloxy, -C(O)-phosphate group, phosphodiester group, phosphoramidite group, saturated fatty acid group, unsaturated fatty acid group, lipid, PEG, steroid, lipophilic substance, carbohydrate, cholesterol, adamantane, amino acid, peptide, chloroquine and alkaloid.

[0078] In one embodiment, R 2 Each occurrence is independently selected from the group consisting of: halogen atoms, (C1-C 12 )alkyl, (C1-C 12 ) alkoxy, (C1-C 12 ) alkoxycarbonyl, (C6-C 16 )aryl or (C6-C 16 ) aryloxycarbonyl.

[0079] In one embodiment, A1, A2, A3, B, C, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 One or more hydroxyl, carboxyl, amino, nitrile and phosphate groups contained in the P Protection, the terminal protecting group R P Selected from the following group: (C1-C 22 )alkyl, (C1-C 22 ) alkoxy, (C1-C 22 )alkylcarbonyl, (C1-C 22 ) alkoxycarbonyl, (C6-C 22 ) aryl, (C6-C 22 ) aryloxy, (C6-C 22 ) arylcarbonyl, (C6-C 22 ) aryloxycarbonyl, tri((C1-C 22 )alkyl)silyl and tri((C1-C 22 )alkoxy)silyl, wherein the protecting group R p Included in (C1-C 22 ) The alkyl group may be an alkyl group containing 1 to 22 carbon atoms, for example, 2 to 20 carbon atoms, or 3 to 18 carbon atoms, or 4 to 16 carbon atoms, or 6 to 12 carbon atoms; R p Included in (C1-C 22 ) The alkoxy group may be an alkoxy group containing 1 to 22 carbon atoms, for example, 2 to 20 carbon atoms, or 3 to 18 carbon atoms, or 4 to 16 carbon atoms, or 6 to 12 carbon atoms; R p Included in (C6-C 22 ) The aryl group may be an aryl group containing 6 to 22 carbon atoms, for example 6 to 20 carbon atoms, or 6 to 18 carbon atoms, or 6 to 16 carbon atoms, or 6 to 12 carbon atoms, or 8-10 carbon atoms; R p Included in (C6-C 22 ) The aryloxy group may be an aryloxy group containing 6 to 22 carbon atoms, for example 6 to 20 carbon atoms, or 6 to 18 carbon atoms, or 6 to 16 carbon atoms, or 6 to 12 carbon atoms, or 8-10 carbon atoms.

[0080] In one embodiment, A1, A2 and A3 are not simultaneously hydrogen and R 3 , R 4 and R 5In one exemplary embodiment, X is carbon, and A1, A2 and A3 are all present. In another embodiment, X is nitrogen, A1 and A2 are all present and A3 is not present. In another embodiment, X is positively charged nitrogen (i.e., quaternary ammonium), and A1, A2 and A3 are all present. In another embodiment, X is sulfur or oxygen, A1 and A3 are all absent and A2 is present. In another embodiment, X is positively charged sulfur (i.e., sulfonium), A2 and A3 are all present and A1 is not present.

[0081] In one embodiment, A1, A2, A3, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 Included in (C1-C 22 )alkyl or (C1-C 22 ) The alkylene groups can each be an alkyl or alkylene group containing 1 to 22 carbon atoms, or 2 to 20 carbon atoms, or 3 to 16 carbon atoms, or 4 to 12 carbon atoms, or 6 to 12 carbon atoms, or 8 to 10 carbon atoms.

[0082] In one embodiment, A1, A2, A3, B, C, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 One or more hydroxyl, carboxyl, amino, nitrile and phosphate groups contained therein may be connected to a support material selected from the group consisting of silica, silica gel, glass, ceramic, polymer, cellulose and combinations thereof. In some embodiments, the solid material is in the form of beads. The beads may be made of any material, including but not limited to magnetic beads, paramagnetic beads, silica beads, agarose beads, and the like.

[0083] In one embodiment, A1, A2, A3, R 3 , R 4 and R 5 At least one of A1, A2, A3, R 3 and R 4 One, two or three of the above correspond to the lipid moieties described above. In another embodiment, the structure of formula A' does not include R 3 , R 4 and R 5 And at least one of A1, A2 and A3 corresponds to a lipid moiety.

[0084] In another embodiment, the compound of the present disclosure may have a structure represented by any one of Formula IV to Formula XIII:

[0085]

[0086] wherein A1, A2, A3, B, C, F, G, H, I, m and n are as defined above.

[0087] In some embodiments, the oligonucleotide delivery agent is a compound comprising at least one nitrogen-containing linear group, at least one nitrogen-containing linear-carbocyclic group, or a combination thereof. In some exemplary embodiments, the delivery enhancing compound portion may include a structure selected from the group consisting of: -(C1-C 16 )alkylene-NH-C(O)-, -(C1-C 16 )alkylene-C(O)-NH-, -(C1-C 16 )alkylene-O-(C1-C 16 )alkylene-NH-C(O)-, -(C1-C 16 )alkylene-O-(C1-C 16 )alkylene-O-(C1-C 16 )alkylene-NH-C(O)-, -(C3-C 12 )cycloalkylene-NH-C(O)-, -(C1-C 16 )alkylene-(C3-C 12 )cycloalkylene-NH-C(O)-, -(C1-C 16 )alkylene-(C3-C 12 )cycloalkylene-(C1-C 16 )alkylene-NH-C(O)-, -(C1-C 16 )alkylene-O-(C1-C 16 )alkylene-C(O)-NH-, -(C1-C 16 )alkylene-O-(C1-C 16 )alkylene-O-(C1-C 16 )alkylene-C(O)-NH-, -(C3-C 12 )cycloalkylene-C(O)-NH-, -(C1-C 16 )alkylene-(C3-C 12 )cycloalkylene-C(O)-NH-, -(C1-C 16 )alkylene-(C3-C 12 )cycloalkylene-(C1-C 16 )alkylene-C(O)-NH-; for example -(C2-C 12 )alkylene-NH-C(O)-, -(C2-C12 )alkylene-C(O)-NH-, -(C2-C 12 )alkylene-O-(C2-C 12 )alkylene-NH-C(O)-, -(C2-C 12 )alkylene-O-(C2-C 12 )alkylene-O-(C2-C 12 )alkylene-NH-C(O)-, -(C3-C8)cycloalkylene-NH-C(O)-, -(C1-C 12 )alkylene-(C3-C8)cycloalkylene-NH-C(O)-, -(C1-C 12 )alkylene-(C3-C8)cycloalkylene-(C1-C 12 )alkylene-NH-C(O)-, -(C2-C 12 )alkylene-O-(C2-C 12 )alkylene-C(O)-NH-, -(C2-C 12 )alkylene-O-(C1-C 12 )alkylene-O-(C1-C 12 )alkylene-C(O)-NH-, -(C3-C8)cycloalkylene-C(O)-NH-, -(C2-C 12 )alkylene-(C3-C8)cycloalkylene-C(O)-NH-, -(C2-C 12 )alkylene-(C3-C8)cycloalkylene-(C2-C 12 )alkylene-C(O)-NH-; or -(C1-C6)alkylene-NH-C(O)-, -(C1-C6)alkylene-C(O)-NH-, -(C1-C6)alkylene-O-(C1-C6)alkylene-NH-C(O)-, -(C1-C6)alkylene-O-(C1-C6)alkylene-O-(C1-C6)alkylene-NH-C(O)-, -(C3-C6)cycloalkylene-NH-C(O)-, -(C1-C6)alkylene-(C3-C6)cycloalkylene-NH-C(O)-, -(C1-C6)alkylene-(C3 -C6) cycloalkylene-(C1-C6) alkylene-NH-C(O)-, -(C1-C6) alkylene-O-(C1-C6) alkylene-C(O)-NH-, -(C1-C6) alkylene-O-(C1-C6) alkylene-C(O)-NH-, -(C3-C6) cycloalkylene-C(O)-NH-, -(C1-C6) alkylene-(C3-C6) cycloalkylene-C(O)-NH-, -(C1-C6) alkylene-(C3-C6) cycloalkylene-(C1-C6) alkylene-C(O)-NH-.

[0088] In another embodiment, when any one or more of the above-mentioned substituents contain at least one double bond, the double bond and the atom or moiety connected to the double bond can each be in the E form or the Z form.

[0089] In some embodiments, at least one hydrogen atom (i.e., H) contained in the lipid portion and / or the delivery enhancing compound portion is substituted with a deuterium atom (i.e., D). In an exemplary embodiment, the lipid portion and / or the delivery enhancing compound portion contains 1 to 20 deuterium atoms, e.g., 1 to 15, or 1 to 12, or 1 to 10, or 1 to 8, or 1 to 6, or 1 to 3, or 1 to 2 deuterium atoms. In an exemplary embodiment, 1 mol% to 100 mol%, or 2 mol% to 90 mol%, or 5 mol% to 80 mol%, or 10 mol% to 70 mol%, or 20 mol% to 60 mol%, or 30 mol% to 50 mol%, or 40 mol% to 45 mol% of the hydrogen atoms contained in the lipid portion and / or the delivery enhancing compound portion are substituted with deuterium atoms. In another exemplary embodiment, the deuterium substitution rate may be within the range of values ​​obtained by combining any two of the endpoint values ​​described above.

[0090] In an exemplary embodiment, the combination of the lipid moiety and the delivery enhancing compound moiety represented by Formula A' can be obtained, for example, by subtracting at least one hydrogen atom or terminal group from any of the following compounds C1 to C71:

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098] in means a supporting material as defined above.

[0099] In one embodiment, the oligonucleotides included in the oligonucleotide delivery agent of the present disclosure can be selected from antisense oligonucleotides (ASO), antisense RNA, short interfering RNA (siRNA), microRNA (miRNA), small initiating RNA (saRNA), dsRNA, small guide RNA (sgRNA) and phosphorodiamidate morpholino oligonucleotides (PMO). Preferably, the oligonucleotide is an antisense oligonucleotide (ASO).

[0100] According to other embodiments of the present disclosure, the structure represented by formula A' is connected to the oligonucleotide by a covalent bond or by at least one linking portion to form an oligonucleotide delivery agent. According to other embodiments of the present disclosure, the at least one lipid (i.e., the straight chain alkyl or alkenyl) is connected to the oligonucleotide by a covalent bond or by at least one linking portion to form an oligonucleotide delivery agent. In one embodiment, when present, the linking portion may be selected from the group consisting of: -O-, -S-, -C(O)-, -NH-, -N((C1-C 12 )alkyl)-, -N((C1-C 12 )Alkyl)-C(O)-O-, -OC(O)-, -C(O)-O-, -OC(O)-O-, -C(O)-NH-, -OP(O)2O-, -P(O)(O-)O- , -OP(O)O-, -OP(O)(S)O-, -OP(O)(S-)O-, -OS(O)2-O-, -S(O)2-O-, -S(O)-O-, -(C1-C 22 )alkylene-, -(C1-C 22 ) alkylene-NH-, -NH-(C1-C 22 )alkylene-, -(C1-C 22 )alkylene-NH-C(O)-, -(C1-C 22 )alkylene-C(O)-, -(C1-C 22 )alkylene-C(O)-O-, -C(O)-(C1-C 22 )alkylene-, -NH-C(O)-(C1-C 22 )alkylene-, -C(O)-NH-(C1-C 22 )alkylene-, -C(O)-(C1-C 22 ) alkylene-NH-, -NH-(C1-C 22 )alkylene-C(O)-, -C(O)-(C1-C 22 )alkylene-C(O)-, -NH-(C1-C 22 )alkylene-NH-, -C(O)-(C1-C 22 )alkylene-C(O)O-, -OC(O)-(C1-C22 )alkylene-C(O)-O-, -C(O)-O-(C1-C 22 )alkylene-OC(O)-, -C(O)-(C1-C 22 ) alkylene-NH-C(O)-, -NH-C(O)-(C1-C 22 )alkylene-C(O)-, -NH-C(O)-(C1-C 22 )alkylene-C(O)-NH-, -C(O)-NH-(C1-C 22 )alkylene-NH-C(O)-, -(C1-C 22 )alkylene-OP(O)2O-, -(C1-C 22 )alkylene-OP(O)(O-)O-, -(C1-C 22 )alkylene-OP(O)(O-)O-(C1-C 22 )alkylene-, -(C1-C 22 )alkylene-OP(O)O-, -(C1-C 22 )alkylene-OP(O)(S)O-, -(C1-C 22 )alkylene-OS(O)2-O-, -(C1-C 22 )alkylene-S(O)2-O-, -(C1-C 22 )alkylene-S(O)-O-, -OP(O)2-O-(C1-C 22 )alkylene-OP(O)2O-, -OP(O)-O-(C1-C 22 )alkylene-OP(O)O-, -OP(O)(S)O-(C1-C 22 )alkylene-OP(O)(S)O-, -OS(O)2-O-(C1-C 22 )alkylene-OS(O)2-O-, -S(O)2-O-(C1-C 22 )alkylene-S(O)2-O- and -OS(O)-(C1-C 22 )alkylene-S(O)-O-; wherein the connecting part contains -(C1-C 22 )Alkylene- may be an alkylene containing 1 to 22 carbon atoms, for example 2 to 20 carbon atoms, or 3 to 18 carbon atoms, or 4 to 16 carbon atoms, or 5 to 12 carbon atoms, or 6 to 10 carbon atoms.

[0101] In one embodiment, the oligonucleotide delivery agent may include one, two or three (ie, N = 1, 2 or 3) structures of formula A', each of which is connected to the oligonucleotide via one, two or three linking moieties as described above. For example, the oligonucleotide delivery agent may have a structure represented by any one of the following formulas AI to AVII:

[0102]

[0103] Wherein, L represents the connecting part, represents a combination of a lipid moiety and a delivery enhancing compound moiety represented by formula A', represents a single-stranded oligonucleotide, and a, b and c are each independently an integer from 1 to 50, such as an integer from 2 to 45, or an integer from 3 to 40, or an integer from 4 to 35, or an integer from 5 to 30, or an integer from 10 to 20. Each linking portion, as well as a combination of a lipid portion represented by formula A' and a delivery enhancing compound portion can be linked to the 3' end, the 5' end or any intermediate position of the single-stranded oligonucleotide, such as the nth nucleotide.

[0104] In one embodiment, one or more of the substituents A1, A2, A3, B and C of the delivery enhancing compound can be linked to a linking moiety or to an oligonucleotide (when the linking moiety is a direct bond). For example, any one of A1, A2, A3, B, preferably A2 or B, is linked to a linking moiety or an oligonucleotide. Without being limited to any theory, one or more of the terminal groups (e.g., DMTrO-C1-C 22 Alkylene-or-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-CN) can be cleaved or hydrolyzed to expose the active site (e.g., -(C1-C 22 )alkylene-OH group), which is then linked to the oligonucleotide via a linking moiety (e.g., -P(O)(O-)O- or -OP(O)(S-)O-). For example, exemplary structures of oligonucleotide delivery agents include O1 to O20, which are referred to as RD-14644, RD-14645, RD-14646, RD-15865, RD-16070, RD-16071, RD-16072, RD-16073, RD-16074, RD-16075, RD-16076, RD-16077, RD-16078, RD-16079, RD-16080, RD-16081, RD-16098, RD-16935, RD-16936, and RD-16937, respectively, in the subsequent examples and figures as shown below, wherein Y represents an O or S atom.

[0105]

[0106]

[0107]

[0108]

[0109]

[0110] It can be seen that in the structures of oligonucleotide delivery agents O1, O2 and O3, the delivery enhancing compounds are each connected by a linker, such as OP(O)2O-, (-P(O)(O - )-O-) or (-P(O)(S - )-O-, linked to a single-stranded RNA (including but not limited to ASO) at one or both of the 3' and 5' ends.

[0111] Another embodiment of the present disclosure provides a pharmaceutical composition comprising an oligonucleotide delivery agent. In another embodiment, the pharmaceutical composition may comprise one or more additional ingredients, such as a pharmaceutically acceptable carrier, excipient, solvent, diluent, stabilizer, dispersant, buffer, extender, preservative, and combinations thereof.

[0112] Another embodiment of the present disclosure provides a method for regulating the expression of a target gene in vitro or in vivo, the method comprising administering a pharmaceutical composition to a subject, or contacting the subject's cells with a pharmaceutical composition. For example, it is estimated that the oligonucleotide delivery agent and the pharmaceutical composition can be applied to various organs, tissues and cells, such as the central nervous system (e.g., brain and spinal cord), heart, eye, ear, liver, lung, kidney, spleen, adipose tissue, intestine, pancreas, gallbladder, muscle (e.g., smooth muscle, skeletal muscle and myocardium), diaphragm, thoracic aorta and bone, and regulate the expression of one or more target genes in its cells. In some embodiments, the oligonucleotides contained in the oligonucleotide delivery agent are targeted to one or more genes selected from SMN2, SCN9A, Scn9a and DMD. In some specific embodiments, the oligonucleotides contained in the oligonucleotide delivery agent targeting SMN2 include at least a portion of the oligonucleotide sequence selected from SEQ ID NO: 1. In some specific embodiments, the oligonucleotide included in the oligonucleotide delivery agent targeting SCN9A comprises at least a portion of an oligonucleotide sequence selected from SEQ ID NOs: 142-260, SEQ ID NOs: 261-379, SEQ ID NOs: 388, and SEQ ID NOs: 390. In some specific embodiments, the oligonucleotide included in the oligonucleotide delivery agent targeting DMD comprises at least a portion of an oligonucleotide sequence selected from SEQ ID NOs: 443-492.

[0113] Another embodiment of the present disclosure provides a pharmaceutical composition comprising an oligonucleotide delivery agent that can treat muscle system diseases. In another embodiment, muscle system diseases include primary muscle system diseases, such as polymyositis, dermatomyositis, muscular dystrophy, for example, Duchenne muscular dystrophy, Becker muscular dystrophy, congenital muscular dystrophy, myotonic dystrophy, limb-girdle muscular dystrophy, Emery-Dreifuss muscular dystrophy, telemuscular dystrophy and oculopharyngeal muscular dystrophy, myasthenia gravis, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), rhabdomyolysis, cardiomyopathy and sarcopenia. In another embodiment, muscle system diseases include secondary muscle system diseases, such as muscle diseases caused by infectious diseases, endocrine disorders or glandular diseases, metabolic diseases such as diabetes, immune diseases such as HIV / AIDS and vascular diseases.

[0114] Another embodiment of the present disclosure provides the use of a pharmaceutical composition comprising an oligonucleotide delivery agent in the preparation of a medicament for treating muscle diseases, including muscle cancers such as sarcomas, neuromuscular diseases such as amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), autoimmune diseases such as myasthenia gravis (MG), and various types of myopathies, inflammatory myasthenia caused by polymyositis, cardiovascular diseases such as coronary artery disease, chronic pain diseases such as fibromyalgia, genetic diseases such as muscular dystrophy (e.g., Duchenne muscular dystrophy, Becker muscular dystrophy, congenital muscular dystrophy, myotonic dystrophy, limb-girdle muscular dystrophy, Emery-Dreifuss muscular dystrophy, telemuscular dystrophy and oculopharyngeal muscular dystrophy, infectious injuries to muscle fibers such as Lyme disease, malaria and Rocky Mountain spotted fever, muscle strains and paralysis caused by injuries, and muscle pain caused by drugs or severe allergic reactions to drugs or toxic substances. DETAILED DESCRIPTION

[0116] One specific embodiment includes an oligonucleotide delivery agent comprising one or more lipid moieties coupled directly or indirectly to an oligonucleotide, wherein at least one lipid moiety comprises at least one C6-C 22 Straight chain alkyl, at least one C6-C 22 A linear alkenyl or a combination thereof, and the oligonucleotide is a single-stranded oligonucleotide.

[0117] In another specific embodiment, the lipid moiety comprises C 12 -C 20 Straight chain alkyl, C 12 -C 20 alkenyl or a combination thereof, and C 12 -C 20 An alkenyl group contains one or more ethylenically unsaturated groups.

[0118] In another specific embodiment, C6-C 22 The straight-chain alkyl group is selected from the group consisting of a straight-chain dodecyl group, a straight-chain tetradecyl group, a straight-chain pentadecyl group, a straight-chain hexadecyl group, a straight-chain octadecyl group, a straight-chain eicosyl group, a straight-chain dodecenyl group, a straight-chain tetradecenyl group, a straight-chain pentadecenyl group, a straight-chain hexadecenyl group, a straight-chain octadecenyl group, a straight-chain eicosenyl group, a straight-chain dodecadienyl group, a straight-chain tetradecadienyl group, a straight-chain pentadecadienyl group, a straight-chain hexadecadienyl group, a straight-chain octadecadienyl group, and a straight-chain eicosadienyl group.

[0119] In another specific embodiment, the oligonucleotide is a single-stranded oligonucleotide.

[0120] In another specific embodiment, the single stranded oligonucleotide is 15 to 35 oligonucleotides in length.

[0121] In another specific embodiment, the lipid moiety is coupled to the single-stranded oligonucleotide directly via a covalent bond or is linked to the single-stranded oligonucleotide via a linker moiety having the following structure:

[0122]

[0123] where Y 1 is oxygen or sulfur, an asterisk (*) refers to the position where the linking moiety is attached to the single-stranded oligonucleotide, and a double asterisk (**) refers to the position where the linking moiety is attached to the lipid moiety.

[0124] In another specific embodiment, the oligonucleotide delivery agent has a structure represented by Formula A:

[0125]

[0126] The lipid portion comprises at least one C6-C 22 Straight chain alkyl, at least one C6-C 22 A linear alkenyl group or a combination thereof, and M is an integer from 1 to 5, the oligonucleotide is a single-stranded oligonucleotide, and N is an integer from 1, 2 or 3; and

[0127] The delivery enhancing compound portion is a divalent to hexavalent portion comprising at least one nitrogen-containing heterocyclic ring, at least one nitrogen-containing linear group, at least one nitrogen-containing linear-carbocyclic group, or a combination thereof, and the lipid portion and the oligonucleotide are each coupled directly or indirectly to the delivery enhancing compound portion.

[0128] In another specific embodiment, the lipid moiety is a C12, C13, C14, C15, C16, C17, C18, C19 or C20 straight chain saturated or unsaturated hydrocarbon group, and M is an integer of 1, 2, 3, 4 or 5.

[0129] In another specific embodiment, the unsaturated lipid moiety has at least one cis or trans unsaturated double bond in the hydrocarbon group.

[0130] In another specific embodiment, the oligonucleotide is at least one single-stranded oligonucleotide selected from antisense oligonucleotides (ASOs), antisense RNAs, short interfering RNAs (siRNAs), micro RNAs (miRNAs), cytoplasmic small RNAs (scRNAs), small guide RNAs (sgRNAs), and phosphorodiamidate morpholino oligonucleotides (PMOs).

[0131] In another specific embodiment, the combination of the lipid portion and the delivery enhancing compound portion represented by Formula A' has a structure obtained by subtracting at least one hydrogen atom or terminal group from the structure represented by Formula I or Formula II, wherein the terminal group is selected from hydroxyl, carboxyl, ester, ether, phosphate, phosphonate, sulfate, sulfonate, amide, acyl, and combinations thereof, and provided that A1, A2, A3, R 3 , R 4 and R 5 At least one of them is C6-C 22 A straight chain alkyl group, preferably a straight chain hexadecyl group.

[0132]

[0133] Among them, each independently represents a covalent single or double bond; each occurrence of X is selected from carbon, nitrogen, oxygen and sulfur; F, G, H and I are each independently selected from carbon, nitrogen, oxygen and sulfur;

[0134] wherein m is an integer of 1, 2 or 3, n is an integer of 1, 2 or 3, and m+n=4;

[0135] wherein C is either absent or selected from the group consisting of hydrogen, halogen, hydroxyl, (C1-C 20 )alkyl, (C1-C 20 ) alkoxy, halogenated (C1-C 20 ) alkyl and halogenated (C1-C 20 ) alkoxy;

[0136] wherein B is independently selected at each occurrence from the group consisting of hydroxy, -C(O)OH, -P(O)2-OH, -P(O)-OH, -P(O)(S)-OH, -CN, -(C1-C 22 )alkyl, -(C1-C 22 )alkenyl, -(C1-C 22 ) alkylene-OH, -(C3-C 22 )cycloalkylene-OH, -(C6-C 22 )arylene-OH, -(C6-C 22 )heteroarylene-OH, -(C1-C 22 )alkylene-C(O)OH, -(C3-C 22 )cycloalkylene-C(O)OH, -(C6-C 22 )arylene-C(O)OH, -(C5-C 22 )heteroarylene-C(O)OH, -OC(O)-(C1-C 22 )alkylene-C(O)NH2, -(C1-C 22)alkylene-OC(O)-(C1-C 22 )alkylene-C(O)NH2, -OC(O)-(C1-C 22 )alkylene-C(O)OH, -(C1-C 22 )alkylene-OC(O)-(C1-C 22 )alkylene-C(O)OH, -C(O)-(C1-C 22 )alkylene-C(O)NH2, -(C1-C 22 )alkylene-C(O)-(C1-C 22 )alkylene-C(O)NH2, -C(O)-NH-(C1-C 22 ) alkylene-OH, -C(O)-NH-(C1-C 22 )alkylene-C(O)OH, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-C(O)OH, -(C1-C 30 )alkylene-P(O)2-OH, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-CN, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-OH, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-NH2, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-C(O)OH, -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-CN, -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-OH, -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 30)alkylene-C(O)OH, -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-NH2, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-CN, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-OH, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-C(O)OH, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-NH2, -(C1-C 22 )alkylene-P(O)-OH, -(C1-C 22 )alkylene-P(O)(S)-OH, -(C1-C 22 ) alkylene-CN,

[0137] Wherein, each of A1, A2 and A3 is either absent or a substituent independently selected from the following groups: -H, -OH, -OR 1 ,-SH,-(C1-C 22 )alkyl, halo-(C1-C 22 )alkyl, -(C2-C 22 )alkenyl, -(C1-C 22 ) alkylene-OH, -(C3-C 22 )cycloalkyl, -(C3-C 22 )cycloalkenyl, -(C1-C 22 )alkylene-(C3-C 22 )cycloalkyl, -(C1-C 22 )alkylene-R 1 、-(C1-C 22 )Alkenylene-R1 、-(C1-C 22 )alkylene-OR 1 、-(C1-C 22 )Alkenylene-OR 1 、-(C1-C 22 )Alkylene-COOR 1 、-(C1-C 22 )Alkenylene-COOR 1 、-C(O)OR 1 、-O-(C1-C 22 )alkyl, -S-(C1-C 22 )alkyl, -C(O)-R 1 、-C(O)-(C1-C 22 )alkyl, -OC(O)-(C1-C 22 )alkyl, -OC(O)-R 1 、-(C1-C 22 )alkylene-OC(O)-R 1 、-C(O)-(C1-C 22 )alkylene-OH, -C(O)-(C1-C 22 )alkylene-R 1 、-C(O)-(C1-C 22 )alkylene-NH-R 1 、-C(O)-(C1-C 22 )Alkylene-NR 2 -R 1 、-OC(O)-(C1-C 22 )alkylene-OH, -OC(O)-(C1-C 22 )alkylene-R 1 , -adamantyl, -(C1-C 22 )alkylene-adamantyl, -O-adamantyl, -C(O)-(C1-C 22 ) alkylene-adamantyl, -(C1-C 22 )alkylene-C(O)-(C1-C 22 ) alkylene-adamantyl, -NH-C(O)-(C1-C 22 ) alkylene-adamantyl, -(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkyl, -(C1-C 22 )alkylene-NH-C(O)-halogeno(C1-C 22 )alkyl, -CH(NH-CO-(C1-C 22 )alkyl)-(C1-C 22)alkylene-NH-C(O)-(C1-C 22 )alkyl, -CH(NH-CO-halogenated(C1-C 22 )alkyl)-(C1-C 22 )alkylene-NH-C(O)-halogeno(C1-C 22 )alkyl, -(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 ) alkylene-adamantyl, -(C1-C 22 )Alkylene-NR 2 -C(O)-(C1-C 22 ) alkylene-adamantyl, -(C1-C 22 )alkylene-(C1-C6 epoxyalkyl) (1-20) -NH-C(O)-(C1-C 22 ) alkylene-adamantyl, -C(O)NH-(C1-C 22 )alkyl, -C(O)NH-R 1 、-C(O)NR 2 -R 1 、-C(O)NH-(C1-C 22 )alkylene-OH, -C(O)NH-(C1-C 22 )alkylene-COOH, -NH-C(O)-(C1-C 22 )alkyl, -NH-C(O)-R 1 、-NR 2 -C(O)-R 1 、-OP(O)2-OR 1 、-OP(O)(S)-OR 1 、-OP(O)-OR 1 、-NH-R 1 、-NR 2 -R 1 、-(C1-C 22 )alkylene-NH-R 1 、-(C1-C 22 )Alkylene-NR 2 -R 1 、-C(O)-(C1-C 22 )alkylene-C(O)-R 1 、-C(O)-(C1-C 22 )alkylene-C(O)OR 1 、-C(O)-(C1-C 22 )alkylene-NH-C(O)-R 1 、-C(O)-(C1-C 22)Alkylene-NR 2 -C(O)-R 1 、-(C1-C 22 )alkylene-C(O)-R 1 、-(C1-C 22 )alkylene-NH-C(O)-R 1 、-(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkylene-R 1 、-(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkylene-R 1 、-(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-R 1 、-(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-R 1 、-(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkylene-R 1 、-(C1-C 22 )Alkylene-NR 2 -C(O)-(C1-C 22 )alkylene-R 1 、-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-CN, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-OH, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-NH2, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22)alkylene-C(O)OH, -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-CN, -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-OH, -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 30 )alkylene-C(O)OH, -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-NH2, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-CN, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-OH, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-C(O)OH, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-NH2, -(C1-C 22 )alkylene-P(O)-OH, -(C1-C 22 )alkylene-P(O)(S)-OH, -(C1-C 22) alkylene-CN, substituted or unsubstituted pyrrole, substituted or unsubstituted pyrroline, substituted or unsubstituted pyrrolidine, substituted or unsubstituted pyrazole, substituted or unsubstituted pyrazoline, substituted or unsubstituted pyrazolidine, substituted or unsubstituted imidazole, substituted or unsubstituted oxazole, substituted or unsubstituted thiazole, substituted or unsubstituted benzopyrrole, substituted or unsubstituted benzopyrroline, substituted or unsubstituted benzopyrrolidine, substituted or unsubstituted benzopyrazole, substituted or unsubstituted benzopyrazoline, substituted or unsubstituted benzopyrazolidine, substituted or unsubstituted benzimidazole, substituted or unsubstituted benzoxazole, substituted or unsubstituted benzothiazole, and the structure represented by formula III:

[0138]

[0139] wherein Y is selected from carbon, nitrogen, oxygen, and sulfur; and each of P, Q, S, and T is independently selected from carbon, nitrogen, oxygen, and sulfur; and the asterisk refers to the site of attachment of the substituent represented by Formula III to the structure represented by Formula I or Formula II;

[0140] Among them, R 3 , R 4 and R 5 Each of the above is either absent or is a substituent independently selected from the group consisting of: -H, -OH, -OR 1 ,-SH,-(C1-C 22 )alkyl, halo-(C1-C 22 )alkyl, -(C2-C 22 )alkenyl, -(C1-C 22 ) alkylene-OH, -(C3-C 22 )cycloalkyl, -(C3-C 22 )cycloalkenyl, -(C1-C 22 )alkylene-(C3-C 22 )cycloalkyl, -(C1-C 22 )alkylene-R 1 、-(C1-C 22 )Alkenylene-R 1 、-(C1-C 22 )alkylene-OR 1 、-(C1-C 22 )Alkenylene-OR 1 、-(C1-C 22 )Alkylene-COOR 1 、-(C1-C 22 )Alkenylene-COOR 1 、-C(O)OR 1 、-O-(C1-C 22 )alkyl, -S-(C1-C 22)alkyl, -C(O)-R 1 、-C(O)-(C1-C 22 )alkyl, -OC(O)-(C1-C 22 )alkyl, -OC(O)-R 1 、-(C1-C 22 )alkylene-OC(O)-R 1 、-C(O)-(C1-C 22 )alkylene-OH, -C(O)-(C1-C 22 )alkylene-R 1 、-C(O)-(C1-C 22 )alkylene-NH-R 1 、-C(O)-(C1-C 22 )Alkylene-NR 2 -R 1 、-OC(O)-(C1-C 22 )alkylene-OH, -OC(O)-(C1-C 22 )alkylene-R 1 , -adamantyl, -(C1-C 22 )alkylene-adamantyl, -O-adamantyl, -C(O)-(C1-C 22 ) alkylene-adamantyl, -(C1-C 22 )alkylene-C(O)-(C1-C 22 ) alkylene-adamantyl, -NH-C(O)-(C1-C 22 ) alkylene-adamantyl, -(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkyl, -(C1-C 22 )alkylene-NH-C(O)-halogeno(C1-C 22 )alkyl, -CH(NH-CO-(C1-C 22 )alkyl)-(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkyl, -CH(NH-CO-halogenated(C1-C 22 )alkyl)-(C1-C 22 )alkylene-NH-C(O)-halogeno(C1-C 22 )alkyl, -(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 ) alkylene-adamantyl, -(C1-C 22 )Alkylene-NR 2 -C(O)-(C1-C 22) alkylene-adamantyl, -(C1-C 22 )alkylene-(C1-C6 epoxyalkyl) (1-20) -NH-C(O)-(C1-C 22 ) alkylene-adamantyl, -C(O)NH-(C1-C 22 )alkyl, -C(O)NH-R 1 、-C(O)NR 2 -R 1 、-C(O)NH-(C1-C 22 )alkylene-OH, -C(O)NH-(C1-C 22 )alkylene-COOH, -NH-C(O)-(C1-C 22 )alkyl, -NH-C(O)-R 1 、-NR 2 -C(O)-R 1 、-OP(O)2-OR 1 、-OP(O)(S)-OR 1 、-OP(O)-OR 1 、-NH-R 1 、-NR 2 -R 1 、-(C1-C 22 )alkylene-NH-R 1 、-(C1-C 22 )Alkylene-NR 2 -R 1 、-C(O)-(C1-C 22 )alkylene-C(O)-R 1 、-C(O)-(C1-C 22 )alkylene-C(O)OR 1 、-C(O)-(C1-C 22 )alkylene-NH-C(O)-R 1 、-C(O)-(C1-C 22 )Alkylene-NR 2 -C(O)-R 1 、-(C1-C 22 )alkylene-C(O)-R 1 、-(C1-C 22 )alkylene-NH-C(O)-R 1 、-(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkylene-R 1 、-(C1-C 22 )alkylene-NH-C(O)-(C1-C 22)alkylene-NH-C(O)-(C1-C 22 )alkylene-R 1 、-(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-R 1 、-(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-R 1 、-(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkylene-R 1 、-(C1-C 22 )Alkylene-NR 2 -C(O)-(C1-C 22 )alkylene-R 1 、-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-CN, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-OH, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-NH2, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-C(O)OH, -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-CN, -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-OH, -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 30)alkylene-C(O)OH, -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-NH2, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-CN, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-OH, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-C(O)OH, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-NH2, -(C1-C 22 )alkylene-P(O)-OH, -(C1-C 22 )alkylene-P(O)(S)-OH, -(C1-C 22 )alkylene-CN, substituted or unsubstituted pyrrole, substituted or unsubstituted pyrroline, substituted or unsubstituted pyrrolidine, substituted or unsubstituted pyrazole, substituted or unsubstituted pyrazoline, substituted or unsubstituted pyrazolidine, substituted or unsubstituted imidazole, substituted or unsubstituted oxazole, substituted or unsubstituted thiazole, substituted or unsubstituted benzopyrrole, substituted or unsubstituted benzopyrroline, substituted or unsubstituted benzopyrrolidine, substituted or unsubstituted benzopyrazole, substituted or unsubstituted benzopyrazoline, substituted or unsubstituted benzopyrazolidine, substituted or unsubstituted benzimidazole, substituted or unsubstituted benzoxazole and substituted or unsubstituted benzothiazole,

[0141] Among them, R 7 is connected to any one of P, Q, S and T at each occurrence and is either absent or selected from hydrogen, a halogen atom, a hydroxyl group, (C1-C 20 )alkyl, (C1-C 20 ) alkoxy, halogenated (C1-C20 ) alkyl and halogenated (C1-C 20 ) alkoxy;

[0142] Wherein, p is an integer of 0, 1, 2 or 3;

[0143] Among them, R 6 is connected to any one of P, Q, S and T, and is selected from a direct bond, -O-, -C(O)O-, -OC(O)-, -P(O)2-O-, -OP(O)2-O-, -P(O)(S)-O-, -OP(O)(S)-O-, -OP(O)-O-, -(C1-C 22 )alkylene-, -(C1-C 22 )alkylene-O-, -O-(C1-C 22 )alkylene-, -(C1-C 22 ) alkylene-NH-, -NH-(C1-C 22 )alkylene-, -C(O)-(C1-C 22 )alkylene-, -(C1-C 22 )alkylene-C(O)-, -C(O)-O-(C1-C 22 )alkylene-, -(C1-C 30 )alkylene-C(O)-O-, -C(O)-NH-(C1-C 22 )alkylene-, -C(O)-NH-(C1-C 22 )alkylene-C(O)-O-, -C(O)-NH-(C1-C 22 )alkylene-OC(O)-, -C(O)-NH-(C1-C 22 )alkylene-OC(O)-O-, -C(O)-NH-(C1-C 22 )alkylene-O-, -C(O)-N((C1-C 22 )alkyl)-(C1-C 22 )alkylene-, -C(O)-N((C1-C 22 )alkyl)-(C1-C 22 )alkylene-O-, -C(O)-NH-(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-, -C(O)-NH-(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkylene-, -C(O)-NH-(C1-C 22 )alkylene-C(O)-NH-, -C(O)-N((C1-C 22 )alkyl)-(C1-C22 )alkylene-C(O)-N((C1-C 22 )alkyl)-(C1-C 22 )alkylene-, -C(O)-N((C1-C 22 )alkyl)-(C1-C 22 )alkylene-C(O)-N((C1-C 22 )alkyl)-, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-C(O)-NH-, -NH-C(O)-(C1-C 22 )alkylene-, -NH-C(O)-(C1-C 22 )alkylene-C(O)-O-, -NH-C(O)-(C1-C 22 )alkylene-C(O)-, -NH-C(O)-(C1-C 22 )alkylene-O-, -N((C1-C 22 )alkyl)-C(O)-(C1-C 22 )alkylene-, -N((C1-C 22 )alkyl)-C(O)-(C1-C 22 )alkylene-O-, -NH-C(O)-(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkylene-, -NH-C(O)-(C1-C 22 )alkylene-NH-C(O)-, -N((C1-C 22 )alkyl)-C(O)-(C1-C 22 )alkylene-N((C1-C 22 )alkyl)-C(O)-(C1-C 22 )alkylene-, -N((C1-C 22 )alkyl)-C(O)-(C1-C 22 )alkylene-N((C1-C 22 )alkyl)-C(O)-, -(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkylene-, -(C1-C 22 )alkylene-NH-C(O)-(C1-C 22)alkylene-NH-C(O)-, -(C1-C 22 )alkylene-P(O)2-O-, -(C1-C 22 )alkylene-OP(O)2-O-, -(C3-C 22 )cycloalkylene-, -(C3-C 22 )cycloalkylene-O-, -O-(C3-C 22 )cycloalkylene-, -(C6-C 22 )arylene-, -(C6-C 22 )arylene-O-, -O-(C6-C 22 )arylene-, -(C6-C 22 )arylene-NH-, -NH-(C6-C 22 )arylene-, -C(O)-(C6-C 22 )arylene-, -(C6-C 22 )arylene-C(O)-, -C(O)-O-(C6-C 22 )arylene-, -(C6-C 22 )arylene-C(O)-O-, -C(O)-NH-(C6-C 22 )arylene-and-C(O)-NH-(C6-C 22 )arylene-C(O)-O-;

[0144] Among them, R 1 is independently selected at each occurrence from hydrogen, hydroxy, -(C1-C 22 )alkyl, -(C3-C 22 )cycloalkyl, -(C6-C 22 )aryl, -(C1-C 22 ) alkoxy, -(C3-C 22 )cycloalkoxy, -(C6-C 22 ) aryloxy, -C(O)-(C1-C 22 )alkyl, -OC(O)(C1-C 22 )alkyl, -C(O)-O-(C1-C 22 )alkyl, -C(O)-(C3-C 22 )cycloalkyl, -OC(O)-(C3-C 22 )cycloalkyl, -C(O)-O-(C3-C 22 )cycloalkyl, -C(O)-(C6-C 22 )aryloxy, -OC(O)-(C6-C 22 ) aryloxy, -C(O)-O-(C6-C 22) aryloxy, -C(O)-phosphate group, phosphodiester group, phosphoramidite group, saturated fatty acid group, unsaturated fatty acid group, lipid, PEG, steroid, lipophilic substance, carbohydrate, cholesterol, adamantane, amino acid, peptide, chloroquine and alkaloid,

[0145] Among them, R 2 Each occurrence is independently selected from the group consisting of: halogen atoms, (C1-C 12 )alkyl, (C1-C 12 ) alkoxy, (C1-C 12 ) alkoxycarbonyl, (C6-C 16 )aryl or (C6-C 16 ) aryloxycarbonyl; and

[0146] Among them, A1, A2, A3, B, C, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 One or more hydroxyl, carboxyl, amino, nitrile and phosphate groups contained therein are optionally attached to a supporting material or protected by a terminal protecting group.

[0147] In another specific embodiment, A1, A2, A3, B, C, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 One or more hydroxyl, carboxyl, amino, nitrile and phosphate groups contained in each are optionally protected by a terminal protecting group R P Protection, the terminal protection group R P Selected from the following group: (C1-C 22 )alkyl, (C1-C 22 ) alkoxy, (C1-C 22 )alkylcarbonyl, (C1-C 22 ) alkoxycarbonyl, (C6-C 22 ) aryl, (C6-C 22 ) aryloxy, (C6-C 22 ) arylcarbonyl, (C6-C 22 ) aryloxycarbonyl, tri((C1-C 22 )alkyl)silyl and tri((C1-C 22 )alkoxy)silyl; and

[0148] Wherein, the supporting material is selected from silicon dioxide, silica gel, glass, ceramic, polymer, cellulose and a combination thereof.

[0149] In another specific embodiment, the combination of the lipid portion and the delivery enhancing compound portion represented by Formula A' has a structure that can be obtained by subtracting at least one hydrogen atom or terminal group from the structure represented by Formula IV to Formula XIII:

[0150]

[0151]

[0152] In another specific embodiment, each of F, G, H and I is carbon, m is 1 and n is 3, B is attached to G or each of H, P, Q, S and T is carbon, R 6 Connect to either Q or S;

[0153] Among them, the protecting group R P Selected from the group consisting of benzyloxycarbonyl (Cbz), tert-butyldimethylsilyl (TBS), 4,4'-dimethoxytrityl (DMTr), tert-butyloxycarbonyl (Boc), benzyl (Bn) and benzyloxy (BnO);

[0154] Wherein, each occurrence of C is selected from the group consisting of hydrogen, halogen atoms, hydroxyl groups, (C1-C 12 )alkyl, (C1-C 12 ) alkoxy, halogenated (C1-C 12 ) alkyl and halogenated (C1-C 12 ) alkoxy;

[0155] Among them, each occurrence of B is -(C1-C 22 )alkylene-OH, -OC(O)-(C1-C 16 )alkylene-C(O)NH2, -(C1-C 16 )alkylene-OC(O)-(C1-C 16 )alkylene-C(O)NH2, -OC(O)-(C1-C 16 )alkylene-C(O)OH, -(C1-C 16 )alkylene-OC(O)-(C1-C 16 )alkylene-C(O)OH, -C(O)-(C1-C 16 )alkylene-C(O)NH2, -(C1-C 16 )alkylene-C(O)-(C1-C 16 )alkylene-C(O)NH2, -C(O)-NH-(C1-C 16) alkylene-OH, -C(O)-NH-(C1-C 16 )alkylene-C(O)OH, -(C1-C 16 )alkylene-C(O)-NH-(C1-C 16 )alkylene-C(O)OH, -(C1-C 16 )alkylene-OP(-N(C1-C 16 Alkyl)2)-O-(C1-C 16 )alkylene-CN, -(C1-C 16 )alkylene-OP(-N(C1-C 16 Alkyl)2)-O-(C1-C 16 ) alkylene-OH, -(C1-C 16 )alkylene-OP(-N(C1-C 16 Alkyl)2)-O-(C1-C 16 ) alkylene-NH2, -(C1-C 16 )alkylene-OP(-N(C1-C 16 Alkyl)2)-O-(C1-C 16 )alkylene-C(O)OH, -C(O)-NH-(C1-C 16 )alkylene-OP(-N(C1-C 16 Alkyl)2)-O-(C1-C 16 ) alkylene-CN,

[0156] wherein each of A1, A2 and A3 is either absent or a substituent independently selected from the following groups: -H, -OH, a linear or branched -(C6-C 22 ) alkyl, linear or branched-(C2-C 22 )alkenyl, -(C1-C 22 ) alkylene-OH, -(C1-C 22 )Alkenylene-OH, -(C3-C 22 )cycloalkyl, -(C3-C 22 )cycloalkenyl, -(C1-C 22 )alkylene-(C3-C 22 )cycloalkyl, -(C1-C 22 )alkylene-R 1 、-(C1-C 22 )Alkenylene-R 1 、-(C1-C 22 )alkylene-OR 1 、-(C1-C 22 )Alkenylene-OR 1 、-(C1-C 22 )Alkylene-COOR1 、-(C1-C 22 )Alkenylene-COOR 1 、-O-(C1-C 22 )alkyl, -(C6-C 22 ) alkylene-adamantyl, -(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkyl, -(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 ) alkylene-adamantyl, -(C1-C 22 )Alkylene-NR 2 -C(O)-(C1-C 22 ) alkylene-adamantyl, -(C1-C 22 )alkylene-(C1-C6 epoxyalkyl) (1-20) -NH-C(O)-(C1-C 22 )alkylene-adamantyl, -C(O)NH-R 1 、-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-CN, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-OH, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-NH2, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-C(O)OH, -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22)alkylene-CN, substituted or unsubstituted pyrrole, substituted or unsubstituted pyrroline, substituted or unsubstituted pyrrolidine, substituted or unsubstituted pyrazole, substituted or unsubstituted pyrazoline, substituted or unsubstituted pyrazolidine, substituted or unsubstituted imidazole, substituted or unsubstituted oxazole, substituted or unsubstituted thiazole, substituted or unsubstituted benzopyrrole, substituted or unsubstituted benzopyrroline, substituted or unsubstituted benzopyrrolidine, substituted or unsubstituted benzopyrazole, substituted or unsubstituted benzopyrazoline, substituted or unsubstituted benzopyrazolidine, substituted or unsubstituted benzimidazole, substituted or unsubstituted benzoxazole, substituted or unsubstituted benzothiazole, and a substituent represented by formula III,

[0157] wherein Y is selected from carbon, nitrogen, oxygen and sulfur; and each of P, Q, S and T is carbon;

[0158] Among them, R 3 , R 4 and R 5 Each of the above is either absent or is a substituent independently selected from the following groups: -H, -OH, a linear or branched -(C6-C 22 ) alkyl, linear or branched-(C2-C 22 )alkenyl, -(C1-C 22 ) alkylene-OH, -(C3-C 22 )cycloalkyl, -(C3-C 22 )cycloalkenyl, -(C1-C 22 )alkylene-(C3-C 22 )cycloalkyl, -(C1-C 22 )alkylene-R 1 、-(C1-C 22 )alkylene-OR 1 、-(C1-C 22 )Alkylene-COOR 1 、-O-(C1-C 22 )alkyl, -(C6-C 22 ) alkylene-adamantyl, -(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkyl, -(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 ) alkylene-adamantyl, -(C1-C 22 )Alkylene-NR 2 -C(O)-(C1-C 22 ) alkylene-adamantyl, -(C1-C 22 )alkylene-(C1-C6 epoxyalkyl)( 1-20) -NH-C(O)-(C1-C22 )alkylene-adamantyl, -C(O)NH-R 1 、-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-CN, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-OH, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-NH2, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-C(O)OH, -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-CN, substituted or unsubstituted pyrrole, substituted or unsubstituted pyrroline, substituted or unsubstituted pyrrolidine, substituted or unsubstituted pyrazole, substituted or unsubstituted pyrazoline, substituted or unsubstituted pyrazolidine, substituted or unsubstituted imidazole, substituted or unsubstituted oxazole, substituted or unsubstituted thiazole, substituted or unsubstituted benzopyrrole, substituted or unsubstituted benzopyrroline, substituted or unsubstituted benzopyrrolidine, substituted or unsubstituted benzopyrazole, substituted or unsubstituted benzopyrazoline, substituted or unsubstituted benzopyrazolidine, substituted or unsubstituted benzimidazole, substituted or unsubstituted benzoxazole and substituted or unsubstituted benzothiazole,

[0159] Among them, R 7 is connected to any one of P, Q, S and T at each occurrence, and is either absent or selected from hydrogen, a halogen atom, a hydroxyl group, (C1-C 20 )alkyl, (C1-C 20 ) alkoxy, halogenated (C1-C 20 ) alkyl and halogenated (C1-C 20 ) alkoxy;

[0160] Where p is an integer of 0, 1, 2 or 3;

[0161] Where R 6 is connected to any one of P, Q, S and T, and is selected from: -(C1-C 16 )alkylene-, -(C1-C 16)alkylene-O-, -O-(C1-C 16 )alkylene-, -(C1-C 16 ) alkylene-NH-, -NH-(C1-C 16 )alkylene-, -C(O)-(C1-C 16 )alkylene-, -(C1-C 16 )alkylene-C(O)-, -C(O)-O-(C1-C 16 )alkylene-, -(C1-C16)alkylene-C(O)-O-, -C(O)-NH-(C1-C 16 )alkylene-, -C(O)-NH-(C1-C 16 )alkylene-C(O)-O-, -C(O)-NH-(C1-C 16 )alkylene-OC(O)-, -C(O)-NH-(C1-C 16 )alkylene-OC(O)-O-, -C(O)-NH-(C1-C 16 )alkylene-O-, -C(O)-N((C1-C 16 )alkyl)-(C1-C 16 )alkylene-, -C(O)-N((C1-C 16 )alkyl)-(C1-C 16 )alkylene-O-, -C(O)-NH-(C1-C 16 )alkylene-C(O)-NH-(C1-C 16 )alkylene-, -C(O)-NH-(C1-C 16 )alkylene-NH-C(O)-(C1-C 16 )alkylene-, -C(O)-NH-(C1-C 16 )alkylene-C(O)-NH-, -C(O)-N((C1-C 16 )alkyl)-(C1-C 16 )alkylene-C(O)-N((C1-C 16 )alkyl)-(C1-C 16 )alkylene-, -C(O)-N((C1-C 16 )alkyl)-(C1-C 16 )alkylene-C(O)-N((C1-C 16 )alkyl)-, -(C1-C 16 )alkylene-C(O)-NH-(C1-C 16 )alkylene-C(O)-NH-(C1-C 16 )alkylene-, -(C1-C 16 )alkylene-C(O)-NH-(C1-C16 )alkylene-C(O)-NH-, -NH-C(O)-(C1-C 16 )alkylene-, -NH-C(O)-(C1-C 16 )alkylene-C(O)-O-, -NH-C(O)-(C1-C 16 )alkylene-C(O)-, -NH-C(O)-(C1-C 16 )alkylene-O-, -N((C1-C 16 )alkyl)-C(O)-(C1-C 16 )alkylene-, -N((C1-C 16 )alkyl)-C(O)-(C1-C 16 )alkylene-O-, -NH-C(O)-(C1-C 16 )alkylene-NH-C(O)-(C1-C 16 )alkylene-, -NH-C(O)-(C1-C 16 )alkylene-NH-C(O)-, -N((C1-C 16 )alkyl)-C(O)-(C1-C 16 )alkylene-N((C1-C 16 )alkyl)-C(O)-(C1-C 16 )alkylene-, -N((C1-C 16 )alkyl)-C(O)-(C1-C 16 )alkylene-N((C1-C 16 )alkyl)-C(O)-, -(C1-C 16 )alkylene-NH-C(O)-(C1-C 16 )alkylene-NH-C(O)-(C1-C 16 )alkylene-, -(C1-C 16 )alkylene-NH-C(O)-(C1-C 16 )alkylene-NH-C(O)-; and

[0162] The conditions are A1, A2, A3, R3, R 4 and R 5 At least one of them is C6-C 22 A straight chain alkyl group, preferably a straight chain hexadecyl group.

[0163] In another specific embodiment, the delivery enhancing compound portion comprises a structure selected from the group consisting of: -(C1-C 16 )alkylene-NH-C(O)-, -(C1-C 16 )alkylene-C(O)-NH-, -(C1-C 16 )alkylene-O-(C1-C16 )alkylene-NH-C(O)-, -(C1-C 16 )alkylene-O-(C1-C 16 )alkylene-O-(C1-C 16 )alkylene-NH-C(O)-, -(C3-C 12 )cycloalkylene-NH-C(O)-, -(C1-C 16 )alkylene-(C3-C 12 )cycloalkylene-NH-C(O)-, -(C1-C 16 )alkylene-(C3-C 12 )cycloalkylene-(C1-C 16 )alkylene-NH-C(O)-, -(C1-C 16 )alkylene-O-(C1-C 16 )alkylene-C(O)-NH-, -(C1-C 16 )alkylene-O-(C1-C 16 )alkylene-O-(C1-C 16 )alkylene-C(O)-NH-, -(C3-C 12 )cycloalkylene-C(O)-NH-, -(C1-C 16 )alkylene-(C3-C 12 )cycloalkylene-C(O)-NH-, -(C1-C 16 )alkylene-(C3-C 12 )cycloalkylene-(C1-C 16 )alkylene-C(O)-NH-, and / or

[0164] Wherein, the delivery enhancing compound portion is directly coupled to the single-stranded oligonucleotide via a covalent bond or is connected to the single-stranded oligonucleotide via a linker portion having the following structure:

[0165]

[0166] where Y 1 is oxygen or sulfur, an asterisk (*) refers to the position where the linking moiety is attached to the single-stranded oligonucleotide, and a double asterisk (**) refers to the position where the linking moiety is attached to the delivery enhancing compound moiety.

[0167] In another specific embodiment, at least one hydrogen atom contained in the lipid moiety is replaced with a deuterium atom.

[0168] In another specific embodiment, at least one hydrogen atom contained in the lipid moiety and / or the delivery enhancing compound moiety is replaced with a deuterium atom.

[0169] In another specific embodiment, the combination of the lipid portion and the delivery enhancing compound portion represented by Formula A' has a structure that can be obtained by subtracting at least one hydrogen atom or terminal group from any of Compounds C1 to C71:

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177] in Indicates supporting material.

[0178] In another specific embodiment, the oligonucleotide is linked to the delivery enhancing compound moiety via at least one linking moiety selected from the group consisting of: -O-, -S-, -C(O)-, -NH-, -N((C1-C 12 )alkyl)-, -N((C1-C 12 )Alkyl)-C(O)-O-, -OC(O)-, -C(O)-O-, -OC(O)-O-, -C(O)-NH-, -OP(O)2O-, -P(O)(O-)O- , -OP(O)O-, -OP(O)(S)O-, -OP(O)(S-)O-, -OS(O)2-O-, -S(O)2-O-, -S(O)-O-, -(C1-C 22 )alkylene-, -(C1-C 22 ) alkylene-NH-, -NH-(C1-C 22 )alkylene-, -(C1-C 22 )alkylene-NH-C(O)-, -(C1-C 22 )alkylene-C(O)-, -(C1-C 22 )alkylene-C(O)-O-, -C(O)-(C1-C 22 )alkylene-, -NH-C(O)-(C1-C 22 )alkylene-, -C(O)-NH-(C1-C 22 )alkylene-, -C(O)-(C1-C 22 ) alkylene-NH-, -NH-(C1-C22 )alkylene-C(O)-, -C(O)-(C1-C 22 )alkylene-C(O)-, -NH-(C1-C 22 )alkylene-NH-, -C(O)-(C1-C 22 )alkylene-C(O)O-, -OC(O)-(C1-C 22 )alkylene-C(O)-O-, -C(O)-O-(C1-C 22 )alkylene-OC(O)-, -C(O)-(C1-C 22 ) alkylene-NH-C(O)-, -NH-C(O)-(C1-C 22 )alkylene-C(O)-, -NH-C(O)-(C1-C 22 )alkylene-C(O)-NH-, -C(O)-NH-(C1-C 22 )alkylene-NH-C(O)-, -(C1-C 22 )alkylene-OP(O)2O-, -(C1-C 22 )alkylene-OP(O)(O-)O-, -(C1-C 22 )alkylene-OP(O)(O-)O-(C1-C 22 )alkylene-, -(C1-C 22 )alkylene-OP(O)O-, -(C1-C 22 )alkylene-OP(O)(S)O-, -(C1-C 22 )alkylene-OS(O)2-O-, -(C1-C 22 )alkylene-S(O)2-O-, -(C1-C 22 )alkylene-S(O)-O-, -OP(O)2-O-(C1-C 22 )alkylene-OP(O)2O-, -OP(O)-O-(C1-C 22 )alkylene-OP(O)O-, -OP(O)(S)O-(C1-C 22 )alkylene-OP(O)(S)O-, -OS(O)2-O-(C1-C 22 )alkylene-OS(O)2-O-, -S(O)2-O-(C1-C 22 )alkylene-S(O)2-O- and -OS(O)-(C1-C 22 )alkylene-S(O)-O-.

[0179] In another specific embodiment, the oligonucleotide delivery agent has a structure represented by any one of Formula AI to AVII:

[0180]

[0181] Wherein, L represents the connecting part, represents a combination of a lipid moiety and a delivery enhancing compound moiety represented by formula A', represents a single-stranded oligonucleotide in the 5′ to 3′ direction or in the 3′ to 5′ direction, and a, b and c are each independently an integer from 1 to 50.

[0182] In another specific embodiment, the oligonucleotide delivery agent has a structure represented by any one of Formulas O1 to O20:

[0183]

[0184]

[0185]

[0186]

[0187]

[0188] wherein Y represents oxygen or sulfur.

[0189] In another specific embodiment, the oligonucleotide targets a gene comprising at least a portion of an oligonucleotide sequence selected from the group consisting of SMN2, SCN9A, Scn9a, and DMD.

[0190] In another specific embodiment, the oligonucleotide comprises at least a portion of an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 142-260 and SEQ ID NOs: 443-492. In another specific embodiment, the oligonucleotide comprises at least a portion of an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 1, SEQ ID NOs: 261-379, SEQ ID NOs: 388, and SEQ ID NOs: 390. In another specific embodiment, the oligonucleotide is complementary to a contiguous target sequence comprising at least a portion of an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 23-141 and SEQ ID NOs: 393-442. In another specific embodiment, the oligonucleotide comprises at least a portion of an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 2-21, SEQ ID NOs: 389, SEQ ID NOs: 391, and SEQ ID NOs: 493-542.

[0191] In another specific embodiment, the present disclosure provides a pharmaceutical composition comprising: a) an oligonucleotide delivery agent as described herein; and b) optionally, one or more ingredients selected from the following group: a pharmaceutically acceptable carrier, an excipient, a solvent, a diluent, a stabilizer, a dispersant, a buffer, a volume extender, a preservative, and a combination thereof.

[0192] In another specific embodiment, the present disclosure provides a method for regulating the expression of a target gene in a subject, the method comprising the step of administering the pharmaceutical composition as described herein.

[0193] In another specific embodiment, the pharmaceutical composition is administered to at least one of the cardiac muscle, skeletal muscle, smooth muscle, and liver of the subject.

[0194] In another specific embodiment, the pharmaceutical composition increases the expression of a target gene.

[0195] In another specific embodiment, the pharmaceutical composition reduces the expression of a target gene.

[0196] In another specific embodiment, the subject is a mammal.

[0197] In another specific embodiment, the mammal is a rodent.

[0198] In another specific embodiment, the rodent is a mouse or a rat.

[0199] In another specific embodiment, the mammal is a non-human primate.

[0200] In another specific embodiment, the mammal is a human.

[0201] In another specific embodiment, the target gene is associated with a disease or disorder.

[0202] In another specific embodiment, the target gene is associated with a disease or disorder of the central nervous system, eye, spleen, muscle, heart, kidney, or liver.

[0203] In another specific embodiment, the oligonucleotide targets a gene comprising at least a portion of an oligonucleotide sequence selected from the group consisting of SMN2, SCN9A, Scn9a, and DMD.

[0204] In another specific embodiment, the oligonucleotide comprises at least a portion of an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 142-260 and SEQ ID NOs: 443-492. In another specific embodiment, the oligonucleotide comprises at least a portion of an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 1, SEQ ID NOs: 261-379, SEQ ID NOs: 388, and SEQ ID NOs: 390. In another specific embodiment, the oligonucleotide is complementary to a contiguous target sequence comprising at least a portion of an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 23-141 and SEQ ID NOs: 393-442. In another specific embodiment, the oligonucleotide comprises at least a portion of an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 2-21, SEQ ID NOs: 389, SEQ ID NOs: 391, and SEQ ID NOs: 493-542.

[0205] In another specific embodiment, the method comprises contacting a cell with a pharmaceutical composition described herein.

[0206] In another specific embodiment, the cell is from skeletal muscle, cardiac muscle, smooth muscle or liver.

[0207] In another specific embodiment, the pharmaceutical composition increases the expression of a target gene.

[0208] In another specific embodiment, the pharmaceutical composition reduces the expression of a target gene.

[0209] In another specific embodiment, said cell is a mammalian cell.

[0210] In another specific embodiment, the mammalian cell is a mouse cell or a rat cell.

[0211] In another specific embodiment, the mammalian cell is a non-human primate cell.

[0212] In another specific embodiment, said mammalian cell is a human cell.

[0213] In another specific embodiment, the target gene is associated with a disease or disorder.

[0214] In another specific embodiment, the target gene is associated with a disease or disorder of the central nervous system, eye, spleen, muscle, heart, kidney, or liver.

[0215] In another specific embodiment, the oligonucleotide targets a gene comprising at least a portion of an oligonucleotide sequence selected from the group consisting of SMN2, SCN9A, Scn9a, and DMD.

[0216] In another specific embodiment, wherein the oligonucleotide comprises at least a portion of an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 142-260 and SEQ ID NOs: 443-492. In another specific embodiment, wherein the oligonucleotide comprises at least a portion of an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 1, SEQ ID NOs: 261-379, SEQ ID NOs: 388, and SEQ ID NOs: 390. In another specific embodiment, wherein the oligonucleotide is complementary to a contiguous target sequence comprising at least a portion of an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 23-141 and SEQ ID NOs: 393-442. In another specific embodiment, the oligonucleotide comprises at least a portion of an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 2-21, SEQ ID NOs: 389, SEQ ID NOs: 391, and SEQ ID NOs: 493-542.

[0217] Example

[0218] Some embodiments of the present invention will now be described in the following examples, wherein, unless otherwise specified, all parts and percentages are by weight. However, the scope of the present disclosure is certainly not limited to the formulations described in these examples. More specifically, the examples are merely inventive embodiments of the present disclosure.

[0219] The following examples are provided for the purpose of fully disclosing and describing to those of ordinary skill in the art how to make and use the present invention, and these examples are not intended to limit the scope of the invention as the inventors believe, nor do they represent that the experiments described below are all or the only experiments performed. Efforts have been made to ensure the accuracy of the values ​​used (such as amounts, temperatures, etc.), but some experimental errors and deviations should be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is degrees Celsius, and pressure is atmospheric pressure or near atmospheric pressure. Standard abbreviations may be used, for example, bp: base pair; kb: kilobase; nM: nanomole; s or sec: second; min: minute; h or hr: hour; aa: amino acid; nt: nucleotide; im: intramuscular; ip: intraperitoneal; sc: subcutaneous; ivt or IVT: intravitreal; iv or IV: tail vein, intravenous; icv or icv or ICV: intracerebroventricular, etc.

[0220] Unless otherwise noted, all starting materials, reagents and solvents used below were obtained from commercial sources and used as is. Purification of the reaction products was performed using silica gel column chromatography, including silica gel (200-300 mesh) and hexane / ethyl acetate, DCM / MeOH eluents. Thin layer chromatography (TLC) was performed using pre-coated silica gel GF plates and visualized using KMnO4 staining. CDCl3 and TMS were used and recorded at 400 or 500 MHz (Varian / Varian Company). 1 H-NMR spectroscopy. High-resolution mass spectra (HRMS) were recorded on LC / MS (Agilent Technologies 1260 Infinity II / 6120 quadrupole) and time-of-flight mass spectrometers by ESI or matrix-assisted laser desorption / ionization (MALDI).

[0221] Example 1: Preparation of Compound C1

[0222] In this example, compound C1 was prepared using the following procedure:

[0223]

[0224] (1) Preparation of Compound 5

[0225]

[0226] Under nitrogen atmosphere, compound 3 (19.56 g, 81 mmol, 1.0 eq.) was added to a solution of 2-(4-fluoro-3-nitrophenyl)acetic acid methyl ester 4 (17.3 g, 81 mmol, 1.0 eq.) and KCO (11.2 g, 81 mmol) in anhydrous DMF (200 mL). The reaction mixture was stirred at 55 °C for 6 h, and then cold water (100 mL) was added. The mixture was extracted three times with ethyl acetate, and then the organic phase was washed three times with saturated LiCl solution and once with brine. The organic phase was then dried over anhydrous NaSO and concentrated under reduced pressure to form compound 5 as a yellow oil, which was used directly in the next step without further purification.

[0227] (2) Preparation of Compound 6

[0228]

[0229] To a solution of compound 5 (35.18 g, 81 mmol, 1.0 eq.) in THF / H2O (9:1, 280 mL) was added HCOONH4 (30.67 g, 486 mmol, 6.0 eq.) and Zn powder (31.78 g, 486 mmol, 6.0 eq.) under an ice bath. After stirring for 10 minutes, the reaction mixture was removed from the ice bath and stirred at room temperature overnight. Then, the reaction mixture was filtered and concentrated under reduced pressure. After this, water (200 mL) was added to the mixture, and then the mixture was extracted three times with ethyl acetate, and the organic phase was washed once with brine. After drying over Na2SO4 and concentrating under reduced pressure, product 6 was formed, which was then used directly in the next step without further purification. Compound 6 was characterized by mass spectrometry. MW calculated: 404.34; MW found: 405.3 [M+H] + .

[0230] (3) Preparation of Compound 8

[0231]

[0232] Under nitrogen atmosphere, 3-((tert-butyldimethylsilyl)oxy)propanal 7 (9.0 g, 48 mmol, 1.0 eq.) and AcOH (11 mL, 192 mmol, 4.0 eq.) were added to a solution of compound 6 (19.36 g, 48 mmol, 1.0 eq.) in EtOH (200 mL). The reaction mixture was stirred at 80 ° C overnight and then concentrated under reduced pressure. Saturated NaHCO solution (100 mL) was then added, and the mixture was extracted three times with ethyl acetate, and the organic phases were combined and washed with brine, dried over Na2SO4 and concentrated. The resulting residual compound 8 was used directly in the next step without further purification.

[0233] (4) Preparation of Compound 9

[0234]

[0235] Under nitrogen atmosphere, 1 M TBAF THF solution (26.3 mL, 26.3 mmol, 1.5 equiv.) was added to a solution of compound 8 (10 g, 17.5 mmol, 1.0 equiv.) in anhydrous THF (50 mL). The reaction mixture was stirred at room temperature for 1 h and then concentrated under reduced pressure. Water (100 mL) was then added, the mixture was extracted three times with DCM, the organic phases were then combined and washed with brine, dried over Na2SO4 and concentrated. The resulting residue was dissolved in 50 mL of pyridine, and DMTrCl (7.12 g, 21 mmol, 1.2 equiv.) was added thereto. The reaction mixture was stirred at room temperature for 6 h and then concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-3% MeOH / DCM) to provide compound 9 (8.1 g, 61% yield) as a yellow solid. The product was purified by mass spectrometry and 1 H NMR characterization. MW calculated: 760.48; MW found: 761.8 [M+H] + . 1 H NMR (400MHz, CDCl3) δ7.59-7.53 (m, 1H), 7.38-7.31 (m, 2H), 7.23-7.19 (m, 6H), 7.18-7.14 (m, 3H), 6.76 (dd, J=7.8, 5.6Hz, 4H), 4.17-4. 02 (m, 2H), 3.76 (s, 6H), 3.73 (s, 2H), 3.67 (s, 3H), 3.59 (t, J = 7.0Hz, 2H), 3.19-3.05 (m, 2H), 1.29-1.25 (m, 28H), 0.88 (t, J = 6.5Hz, 3H).

[0236] (5) Preparation of Compound C1

[0237]

[0238] To a solution of compound 9 (2.7 g, 3.55 mmol, 1.0 eq.) in anhydrous THF (20 mL) was added LiAlH4 (202 mg, 5.33 mmol, 1.5 eq.) under a nitrogen atmosphere and an ice bath. After 10 minutes, the mixture was moved to room temperature and stirred for 1 h. The reaction was then moved to an ice bath and saturated potassium sodium tartrate solution (20 mL) was slowly added to the mixture. After 30 minutes, the reaction was extracted three times with Et2O, and the organic phases were combined and washed with brine, dried over Na2SO4 and concentrated. The crude product (300 mg, 0.41 mmol, 1.0 eq.) was dissolved in anhydrous DCM (5 mL), and then DIPEA (204 μL, 1.23 mmol, 3.0 eq.) and 3-((chloro(diisopropylamino)phosphine)oxy)propionitrile (274 μL, 1.23 mmol, 3.0 eq.) were added at 25°C under a nitrogen atmosphere. The reaction mixture was stirred for 1 h. The mixture was extracted twice with DCM, then washed with brine and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure and the resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-5% MeOH / DCM, 1% Et3N) to provide compound C1 (299 mg, 78% yield) as a colorless oil. The product was purified by mass spectrometry and 1 H NMR characterization. 1 HNMR (400MHz, CDCl3) δ7.62 (s, 1H), 7.32 (dd, J=7.6, 4.1Hz, 2H), 7.27-7.11 (m, 9H), 6 .73(dd, J=7.9, 5.6Hz, 4H), 4.34-4.31(m, 2H), 4.10-4.06(m, 2H), 3.75(s, 6H), 3.64-3 .61 (m, 2H), 3.58 (dd, J=11.8, 5.1Hz, 2H), 3.24-3.18 (m, 2H), 3.09-2.88 (m, 4H), 2.65- 2.55 (m, 4H), 1.37-1.28 (m, 28H), 1.22 (dd, J=6.8, 3.2Hz, 12H), 0.88 (t, J=6.5Hz, 3H).

[0239] Example 2: Preparation of Compound C2

[0240] In this example, compound C2 was prepared using the following procedure:

[0241]

[0242] (1) Preparation of Compound 11

[0243]

[0244] Under nitrogen atmosphere, compound 3 (18.18 g, 75.3 mmol, 1.0 equivalent) was added to a solution of 4-fluoro-3-nitrobenzoic acid methyl ester 10 (15 g, 75.3 mmol, 1.0 equivalent) and KCO (10.4 g, 75.3 mmol, 1.0 equivalent) in anhydrous DMF (240 mL). The reaction mixture was stirred at 25 ° C for 6 h, and then cold water (100 mL) was added. The mixture was extracted three times with ethyl acetate, and then the organic phase was washed three times with saturated LiCl solution and once with brine. It was then dried over anhydrous NaSO and concentrated under reduced pressure to form compound 11, which was a brown oil that was directly used in the next step without further purification.

[0245] (2) Preparation of Compound 12

[0246]

[0247] Under ice bath, to the solution of compound 11 (31.65 g, 75.3 mmol, 1.0 equiv) in THF / H2O (9:1, 300 mL) was added HCOONH4 (28.5 g, 451.8 mmol, 6.0 equiv) and Zn powder (29.5 g, 451.8 mmol, 6.0 equiv). After 10 minutes, the reaction mixture was moved to room temperature and stirred overnight. Then, the reaction mixture was filtered and concentrated under reduced pressure. After this, water (100 mL) was added to the mixture, which was then extracted three times with ethyl acetate and the organic phase was washed once with brine. After drying over Na2SO4 and concentrating under reduced pressure, a brown solid compound 12 was formed, which was then used directly in the next step without further purification.

[0248] (3) Preparation of Compound 14

[0249]

[0250] To a solution of compound 12 (6.0 g, 15.4 mmol, 1.0 equiv) in EtOH (90 mL) was added compound 13 (1.57 g, 15.4 mmol, 1.0 equiv) and AcOH (3.54 mL, 61.6 mmol, 4.0 equiv) under nitrogen atmosphere. The reaction mixture was stirred at 80 °C overnight and then concentrated under reduced pressure. Saturated NaHCO3 solution (80 mL) was then added and the mixture was extracted three times with ethyl acetate, then the organic phases were combined and washed with brine, dried over Na2SO4 and concentrated. The residue was used directly in the next step without further purification. The residue was characterized by mass spectrometry. MW calculated: 472.37; MW found: 473.4 [M+H] + .

[0251] The obtained residue (1.0 g, 2.12 mol, 1.0 eq) was dissolved in 10 mL DCM, and Et3N (0.44 mL, 3.17 mmol, 1.5 eq) and DMTrCl (860 mg, 2.54 mmol, 1.2 eq) were added thereto. The reaction mixture was stirred at room temperature for 6 h, after which it was concentrated under reduced pressure. The obtained residue was purified by flash chromatography (silica gel, gradient eluent: 1-5% MeOH / DCM) to provide compound 14 (1.27 g, 77% yield) as a yellow solid. The product was purified by mass spectrometry and 1 H NMR characterization. MW calculated: 774.50; MW found: 303.2 [DMT] - ,473.4[DMT off+H] + . 1 H NMR (400MHz, CDCl3) δ8.42 (d, J=0.9Hz, 1H), 7.97 (dd, J=8.5, 1.4Hz, 1H), 7.42 (d, J=7.4Hz, 2H), 7.33-7.28 (m, 5H), 7.25 (d, J=6.8Hz, 2H), 7.19 (t, J=7.3Hz, 1H), 6.80 (dd, J=9.4, 2.4Hz, 4H), 4.05 (t, J=7.5Hz, 2H), 3.93 (s, 3H), 3.78 (s, 6H), 3.12 (t, J=6.3Hz, 2H), 2.92 2.80(m,2H),2.07 1.97 (m, 2H), 1.82-1.71 (m, 4H), 1.28-1.24 (m, 26H), 0.88 (t, J=6.8Hz, 3H).

[0252] (4) Preparation of Compound 15

[0253]

[0254] Under nitrogen atmosphere and ice bath, to a solution of compound 14 (1.27 g, 1.64 mmol, 1.0 equiv) in anhydrous THF (10 mL) was added LiAlH4 (93 mg, 2.46 mmol, 1.5 equiv). After 10 minutes, the mixture was moved to room temperature and stirred for 1 h. The reaction was then moved to an ice bath and saturated potassium sodium tartrate solution (10 mL) was slowly added to the mixture. After 30 minutes, the reaction was extracted three times with Et2O, and then the organic phases were combined and washed with brine, dried over Na2SO4 and concentrated. The crude product (500 mg, 0.67 mmol, 1.0 equiv) was dissolved in DCM (10 mL), and then DMAP (286 mg, 2.34 mmol, 3.5 equiv) and succinic anhydride (134 mg, 1.34 mmol, 2.0 equiv) were added under nitrogen atmosphere. The reaction mixture was stirred at room temperature overnight, then H2O (10 mL) was added to the reaction, the mixture was extracted with DCM (3*10 mL) and the organic phases were combined, dried over Na2SO4 and concentrated. The resulting residue was purified by flash chromatography. The resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-10% MeOH / DCM) to provide compound 15 (425 mg, 62% yield) as a colorless oil. The product was purified by mass spectrometry and 1 H NMR characterization. MW calculated: 846.52; MW found: 303.2 [DMT] - , 545.4[DMT off+H] + . 1 H NMR (400MHz, CDCl3) δ7.81 (s, 1H), 7.42 (d, J=7.5Hz, 2H), 7.30 (d, J=8.8Hz, 4H), 7. 25 (d, J=7.6Hz, 3H), 7.19 (dd, J=14.9, 7.9Hz, 2H), 6.80 (d, J=8.8Hz, 4H), 5.26 (s, 2H ), 4.03 (t, J = 7.4Hz, 2H), 3.77 (s, 6H), 3.11 (t, J = 6.1Hz, 2H), 2.90 (t, J = 7.7Hz, 2H), 2.73-2.66 (m, 4H), 1.99 (dt, J=15.3, 7.7Hz, 2H), 1.74 (dd, J=13.5, 6.9Hz, 4H), 1.29 1.25 (m, 26H), 0.87 (t, J=6.7Hz, 3H).

[0255] (5) Preparation of Compound C2

[0256]

[0257] Under nitrogen atmosphere, HBTU (102 mg, 0.268 mmol, 2.0 equiv) was added to a solution of compound 15 (114 mg, 0.134 mmol, 1.0 equiv), controlled pore glass (CPG) (2.36 g), DIPEA (67 μL, 0.4 mmol, 3.0 equiv) in acetonitrile (18 mL). The mixture was stirred overnight at 25 ° C, then washed with DCM and ether to produce a crude solid support. Under nitrogen atmosphere, a solution of acetic anhydride (8.04 mL), pyridine (15.5 mL), NEt3 (241 μL) in acetonitrile (10 mL) was added to a crude solid support. The reaction mixture was stirred overnight at 25 ° C, then washed with DCM and ether to produce compound C2 (2.2 g) of the present disclosure.

[0258] Example 3: Preparation of Compound C55 of the Present Disclosure

[0259] In this example, compound C55 was prepared using the following procedure:

[0260]

[0261] (1) Preparation of Compound 5

[0262]

[0263] Under nitrogen atmosphere, compound 3 (11.2 g, 46.4 mmol, 1.0 eq.) was added to a solution of 2-(4-fluoro-3-nitrophenyl)acetic acid methyl ester 4 (9.9 g, 46.4 mmol, 1.0 eq.) and KCO (6.4 g, 46.4 mmol, 1.0 eq.) in anhydrous DMF (80 mL). The reaction mixture was stirred at 55 °C for 8 h, and then cold water (100 mL) was added. The mixture was extracted three times with ethyl acetate, and then the organic phase was washed three times with saturated LiCl solution and once with brine. The organic phase was then dried over anhydrous NaSO and concentrated under reduced pressure to form compound 5 as a yellow oil, which was used directly in the next step without further purification.

[0264] (2) Preparation of Compound 16

[0265]

[0266] Under nitrogen atmosphere and ice bath, LiAlH4 (1.36 g, 35.87 mmol, 1.0 equiv) was slowly added to a solution of compound 5 (15.58 g, 35.87 mmol, 1.0 equiv) in anhydrous THF (160 mL). After 10 minutes, the mixture was moved to room temperature and stirred for 1 h. The reaction was then moved to an ice bath, and saturated potassium sodium tartrate solution (30 mL) was slowly added to the mixture. After 30 minutes, the reaction was extracted three times with EtOAc, and then the organic phases were combined and washed with brine, dried over Na2SO4 and concentrated. The crude residue was then dissolved in anhydrous DMF (140 mL). Next, imidazole (3.66 g, 53.8 mmol, 1.5 equiv) and TBSCl (6.5 g, 43.04 mmol, 1.2 equiv) were added under nitrogen atmosphere. The reaction mixture was stirred at room temperature for 2 h, and then H2O (100 mL) was added. The mixture was extracted three times with ethyl acetate, and then the organic phase was washed three times with saturated LiCl solution and once with brine, then dried over anhydrous Na2SO4 and concentrated under reduced pressure. The resulting residue 16 was used directly in the next step without further purification. The product was characterized by mass spectrometry. MW calculated: 520.41; MW found: 521.4 [M+H] + .

[0267] (3) Preparation of Compound 17

[0268]

[0269] To a solution of compound 16 (35.87 mmol, 1.0 equiv) in THF / H2O (9:1, 178 mL) was added HCOONH4 (13.58 g, 215.2 mmol, 6.0 equiv) and Zn powder (14.1 g, 215.2 mmol, 6.0 equiv) under an ice bath. After stirring for 10 minutes, the reaction mixture was removed from the ice bath and stirred at room temperature overnight. Then, the reaction mixture was filtered and concentrated under reduced pressure. After this, water (200 mL) was added to the mixture, and then the mixture was extracted three times with ethyl acetate, and the organic phase was washed once with brine. After drying over Na2SO4 and concentrating under reduced pressure, product 17 was formed, which was then used directly in the next step without further purification. Compound 17 was characterized by mass spectrometry. MW calculated: 490.43; MW found: 491.40 [M+H] + .

[0270] (4) Preparation of Compound 18

[0271]

[0272] Under nitrogen atmosphere, 5-hydroxypentanal (1.04 g, 10.2 mmol, 1.0 eq.) and AcOH (2.35 mL, 40.8 mmol, 4.0 eq.) were added to a solution of compound 17 (5 g, 10.2 mmol, 1.0 eq.) in EtOH (25 mL). The reaction mixture was stirred at 80 ° C overnight and then concentrated under reduced pressure. Saturated NaHCO3 solution (25 mL) was then added, and the mixture was extracted three times with ethyl acetate, and the organic phases were then combined and washed with brine, dried over Na2SO4 and concentrated. The resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-5% MeOH / DCM) to provide compound 18 (1.9 g, 33% yield). The product was purified by mass spectrometry and 1 H NMR characterization. MW calculated: 572.47; MW found: 573.61 [M+H] + . 1 H NMR (400MHz, CDCl3) δ7.53 (s, 1H), 7.21 (d, J=8.2Hz, 1H), 7.11 (dd, J=8.2, 1.4Hz, 1H), 4.07 (t, J=7.5Hz, 2H), 3.84 (dd, J=9.3, 5.6Hz, 2H), 3. 69(t, J=6.1Hz, 2H), 2.93(dt, J=19.8, 7.3Hz, 4H), 2.13-2.03(m, 2H), 1 .84-1.70(m, 4H), 1.401.21(m, 26H), 0.93-0.85(m, 12H), 0.03(s, 6H).

[0273] (5) Preparation of Compound 19

[0274]

[0275] Under nitrogen atmosphere, Dess-Martin periodinane (DMP) (1.69 g, 3.98 mmol, 1.2 eq.) was added to a solution of compound 18 (1.9 g, 3.32 mmol, 1.0 eq.) in DCM (10 mL). The reaction mixture was stirred for 6 h. After the reaction, NaHCO solution (20 mL) was added to the mixture and the mixture was stirred for 30 min. The mixture was extracted three times with DCM, and then the organic phase was washed once with brine. After drying over anhydrous Na2SO4 and concentrating under reduced pressure, the resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-5% MeOH / DCM) to provide compound 19 (619 mg, 33% yield) as a brown oil. The product was purified by mass spectrometry and 1 H NMR characterization. MW calculated: 570.46; MW found: 571.61 [M+H] + .1 H NMR (400MHz, CDCl3) δ7.98 (s, 1H), 7.35 (d, J=8.2Hz, 2H), 7.30 (s, 1H), 4.25 (t, J=7.5Hz, 2H), 3.89 (t, J=6.9Hz, 2H), 3.18-3.12 (m, 2H), 3.01 (t, J=6.8Hz, 2H), 2.55-2.50 (m, 2H), 2.32-2.24 (m, 2H), 1.85 (dd, J=14.3, 7.2Hz, 2H), 1.36-1.28 (m, 26H), 0.93-0.86 (m, 12H), 0.03 (s, 6H).

[0276] (6) Preparation of Compound 20

[0277]

[0278] To a solution of compound 8 (733 mg, 1.16 mmol, 1.2 equiv) in anhydrous THF (5 mL) was added NaHH (60% dispersion in mineral oil, 77 mg, 1.93 mmol, 2.0 equiv) under a nitrogen atmosphere and an ice bath. The reaction mixture was stirred for 15 min under an ice bath, and then compound 19 (550 mg, 0.964 mmol, 1.0 equiv) was added to the reaction mixture. The mixture was stirred at room temperature for 2 h, and then the reaction was moved to an ice bath, and saturated ammonium chloride (5 mL) was slowly added to the mixture. After 5 minutes, the reaction was extracted twice with ethyl acetate, and the organic phase was washed once with brine. It was then dried over anhydrous Na2SO4 and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-5% MeOH / DCM) to provide compound 20 (699 mg, 81% yield). The product was purified by mass spectrometry and 1 H NMR characterization. MW calculated: 876.58; MW found: 877.96 [M+H] + . 1 HNMR (400MHz, CDCl3) δ7.54 (s, 1H), 7.22 (d, J=8.2Hz, 1H), 7.15-7.10 (m, 1H), 6.97-6.84 (m, 1H), 5.73-5.65 (m, 5H), 4.07 (t, J=7.5Hz, 2H), 3.85 (t , J=7.4Hz, 2H), 2.96 (t, J=7.4Hz, 2H), 2.88 (t, J=7.5Hz, 2H), 2.18-2.06 ( m, 2H), 1.85-1.70 (m, 3H), 1.33-1.18 (m, 48H), 0.90 (s, 9H), 0.03 (s, 6H).

[0279] (7) Preparation of Compound 21

[0280]

[0281] To a solution of compound 20 (690 mg, 0.77 mmol, 1.0 equiv.) in THF (2 mL) was added 2 M HCl (2 mL). The reaction mixture was stirred at room temperature for 4 h. 5 mL of H2O was added to the reaction. The mixture was then extracted twice with ethyl acetate, washed with brine and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure and the resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-5% MeOH / DCM) to provide compound 21 (488 mg, 82% yield). The product was purified by mass spectrometry and 1 HNMR characterization. MW calculated: 762.49; MW found: 763.76 [M+H] + . 1 H NMR (400MHz, CDCl3) δ7.72 (s, 1H), 7.33 (d, J=8.3Hz, 1H), 7.24 (d, J=8.0Hz, 1H), 6.87-6.74 (m, 1H), 5.75

[0282] 5.59 (m, 5H), 4.14 (t, J = 7.4Hz, 2H), 3.91 (t, J = 6.4Hz, 2H), 3.09-2.95 (m, 4H), 2.46 (d, J = 6.8Hz, 2H), 2.17 (dt, J=14.4, 7.3Hz, 2H), 1.87-1.75 (m, 3H), 1.31-1.17 (m, 44H), 0.88 (t, J=6.8Hz, 3H).

[0283] (8) Preparation of Compound C55

[0284]

[0285] To a solution of compound 21 (480 mg, 0.62 mmol, 1.0 equiv) and diisopropylammonium tetrazolium salt (212 mg, 1.24 mmol, 2.0 equiv) in anhydrous DCM (5 mL) was added 3-((bis(diisopropylamino)phosphino)oxy)propionitrile (374 mg, 1.24 mmol, 2.0 equiv) under a nitrogen atmosphere at room temperature. The reaction mixture was stirred for 4 h. The mixture was extracted twice with DCM, then washed with brine and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-3% MeOH / DCM, 1% Et3N) to provide compound C55 (514 mg, 84% yield) as a yellow oil. The product was purified by mass spectrometry and 1H NMR characterization. MW calculated: 962.60; MW found: 880.82 [M-83+H] + . 1 H NMR (400MHz, CDCl3) δ7.53 (s, 1H), 7.22 (d, J=8.2Hz, 1H), 7.11 (d, J=8.2, 1H), 6.95-6.82 (m, 1H ), 5.83-5.59 (m, 5H), 4.05 (t, J=7.5Hz, 2H), 3.83-3.73 (m, 2H), 3.51-3.46 (m, 2H), 3.04 (t, J=7 .3Hz, 2H), 2.86 (t, J=7.5Hz, 2H), 2.76 (td, J=6.2, 1.9Hz, 5H), 2.10 (dd, J=15.1, 7.6Hz, 2H), 1. 81-1.72 (m, 3H), 1.26-1.24 (m, 26H), 1.21 (s, 18H), 1.18-1.34 (m, 12H), 0.88 (t, J=6.8Hz, 3H).

[0286] Example 4: Preparation of Compound C5 of the Present Disclosure

[0287] In this example, compound C56 was prepared using the following procedure:

[0288]

[0289] (1) Preparation of Compound C56

[0290]

[0291] To a solution of compound 22 (1.00 g, 5.37 mmol, 1.0 equiv) and diisopropylammonium tetrazolium (1.83 g, 10.73 mmol, 2.0 equiv) in anhydrous DCM (27 mL) was added 3-((bis(diisopropylamino)phosphino)oxy)propionitrile (3.4 mL, 10.73 mmol, 2.0 equiv) under a nitrogen atmosphere and room temperature. The reaction mixture was stirred for 4 h. The mixture was extracted twice with DCM, then washed with brine and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-2% ethyl acetate / petroleum ether, 1% Et3N) to provide compound C56 (1.3 g, 63% yield) as a colorless oil. The product was purified by mass spectrometry and 1 H NMR characterization. MW calculated: 386.13; MW found: 387.56 [M+H] + . 1HNMR (400MHz, CDCl3) δ3.90-3.78 (m, 2H), 3.69-3.51 (m, 4H), 2.64 (t, J=6.6Hz , 2H), 1.65-1.57(m, 2H), 1.27-1.24(m, 18H), 1.22-1.15(m, 12H), 0.88(t, 3H).

[0292] Example 5: Preparation of Compound C57 of the Present Disclosure

[0293] In this example, compound C57 was prepared using the following procedure:

[0294]

[0295] (1) Preparation of Compound C57

[0296]

[0297] To a solution of compound 23 (1.0 g, 4.7 mmol, 1.0 equiv) and diisopropylammonium tetrazolium salt (1.6 g, 9.4 mmol, 2.0 equiv) in anhydrous DCM (10 mL) was added 3-((bis(diisopropylamino)phosphino)oxy)propionitrile (2.8 g, 9.4 mmol, 2.0 equiv) under a nitrogen atmosphere at room temperature. The reaction mixture was stirred for 4 h. The mixture was extracted twice with DCM, then washed with brine and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, gradient eluent: 3-5% ethyl acetate / petroleum ether, 1% Et3N) to provide compound C57 (1.8 g, 93% yield). The product was obtained by 1 H NMR characterization. 1 H NMR (400MHz, CDCl3) δ3.90-3.76 (m, 2H), 3.70-3.53 (m, 4H), 2.64 (t, J=6.6Hz, 2H), 1.64-1.57 (m, 2H), 1.29-1.25 (m, 22H), 1.21-1.16 (m, 12H), 0.88 (t, J=6.8Hz, 3H).

[0298] Example 6: Preparation of Compound C58 of the Present Disclosure

[0299] In this example, compound C58 was prepared using the following procedure:

[0300]

[0301] (1) Preparation of Compound C58

[0302]

[0303] To a solution of compound 24 (1.00 g, 4.12 mmol, 1.0 equiv) and diisopropylammonium tetrazolium (1.41 g, 8.25 mmol, 2.0 equiv) in anhydrous DCM (21 mL) was added 3-((bis(diisopropylamino)phosphino)oxy)propionitrile (2.61 mL, 8.25 mmol, 2.0 equiv) under a nitrogen atmosphere at room temperature. The reaction mixture was stirred for 4 h. The mixture was extracted twice with DCM, then washed with brine and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-2% ethyl acetate / petroleum ether, 1% Et3N) to provide compound C58 (1.7 g, 94% yield) as a colorless oil. The product was obtained by 1 H NMR characterization. 1 H NMR (400MHz, CDCl3) δ3.89-3.77(m, 2H), 3.70-3.52(m, 4H), 2.64(t, J=6.6Hz, 2H), 1.65-1.56(m, 2H), 1.33-1.23(m, 26H), 1.22-1.14(m, 12H), 0.87(t, 3H).

[0304] Example 7: Preparation of Compound C59 of the Present Disclosure

[0305] In this example, compound C59 was prepared using the following procedure:

[0306]

[0307] (1) Preparation of Compound C59

[0308]

[0309] To a solution of compound 25 (1.0 g, 4.2 mmol, 1.0 equiv) and diisopropylammonium tetrazolium salt (1.4 g, 8.4 mmol, 2.0 equiv) in anhydrous DCM (10 mL) was added 3-((bis(diisopropylamino)phosphino)oxy)propionitrile (2.5 g, 8.4 mmol, 2.0 equiv) under nitrogen atmosphere and room temperature. The reaction mixture was stirred for 4 h. The mixture was extracted twice with DCM, then washed with brine and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, gradient eluent: 2-5% ethyl acetate / petroleum ether, 1% Et3N) to provide compound C59 (1.4 g, 77% yield). The product was obtained by 1 H NMR characterization. 1H NMR (400MHz, CDCl3) 65.37-5.32 (m, 2H), 3.87-3.77 (m, 2H), 3.67-3.51 (m, 4H), 2.68-2.60 (m, 2H), 2 .06-1.99 (m, 4H), 1.63-1.56 (m, 2H), 1.30-1.25 (m, 18H), 1.21-1.15 (m, 12H), 0.89 (t, J=7.0Hz, 3H).

[0310] Example 8: Preparation of Compound C60 of the Present Disclosure

[0311] In this example, compound C60 was prepared using the following procedure:

[0312]

[0313] (1) Preparation of Compound C60

[0314]

[0315] To a solution of compound 26 (1.00 g, 4.20 mmol, 1.0 equiv) and diisopropylammonium tetrazolium (1.43 g, 8.40 mmol, 2.0 equiv) in anhydrous DCM (18 mL) was added 3-((bis(diisopropylamino)phosphino)oxy)propionitrile (2.66 mL, 8.40 mmol, 2.0 equiv) under a nitrogen atmosphere at room temperature. The reaction mixture was stirred for 4 h. The mixture was extracted twice with DCM, then washed with brine and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-2% ethyl acetate / petroleum ether, 1% Et3N) to provide compound C60 (1.6 g, 88% yield) as a colorless oil. The product was obtained by 1 H NMR characterization. 1 HNMR (400MHz, CDCl3) δ6.28-6.15 (m, 1H), 6.13-5.98 (m, 1H), 3.87-3.77 (m, 2H), 3.69-3.44 (m, 4H), 2.70-2.6 2 (m, 2H), 1.65-1.57 (m, 2H), 1.39-1.36 (m, 6H), 1.26-1.25 (m, 24H), 1.21-1.15 (m, 6H), 0.89 (t, J=6.5Hz, 3H).

[0316] Example 9: Preparation of Compound C61 of the Present Disclosure

[0317] In this example, compound C61 was prepared using the following procedure:

[0318]

[0319] (1) Preparation of Compound C61

[0320]

[0321] To a solution of compound 25 (1.0 g, 3.7 mmol, 1.0 equiv) and diisopropylammonium tetrazolium salt (1.3 g, 7.4 mmol, 2.0 equiv) in anhydrous DCM (10 mL) was added 3-((bis(diisopropylamino)phosphino)oxy)propionitrile (2.2 g, 7.4 mmol, 2.0 equiv) under nitrogen atmosphere and room temperature. The reaction mixture was stirred for 4 h. The mixture was extracted twice with DCM, then washed with brine and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, gradient eluent: 2-5% ethyl acetate / petroleum ether, 1% Et3N) to provide compound C61 (860 mg, 49% yield). The product was obtained by 1 H NMR characterization. 1 H NMR (400MHz, CDCl3) δ3.94-3.73(m, 2H), 3.72-3.48(m, 4H), 2.64(t, J=6.6Hz, 2H), 1. 60 (t, J=13.7Hz, 3H), 1.39-1.22 (m, 30H), 1.21-1.15 (m, 12H), 0.88 (t, J=6.8Hz, 1H).

[0322] Example 10: Preparation of Compound C62 of the Present Disclosure

[0323] In this example, compound C62 was prepared using the following procedure:

[0324]

[0325] (1) Preparation of Compound C62

[0326]

[0327] To a solution of compound 28 (1.00 g, 3.73 mmol, 1.0 equiv) and diisopropylammonium tetrazolium (1.27 g, 7.45 mmol, 2.0 equiv) in anhydrous DCM (19 mL) was added 3-((bis(diisopropylamino)phosphino)oxy)propionitrile (2.36 mL, 7.45 mmol, 2.0 equiv) under a nitrogen atmosphere at room temperature. The reaction mixture was stirred for 8 h. The mixture was extracted twice with DCM, then washed with brine and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-2% ethyl acetate / petroleum ether, 1% Et3N) to provide compound C62 (1.6 g, 91% yield) as a colorless oil. The product was obtained by 1 H NMR characterization. 1 H NMR (400MHz, CDCl3) δ6.28-6.15 (m, 1H), 6.13-5.98 (m, 1H), 3.87-3.77 (m, 2H), 3.69-3.44 (m, 4H), 2.70-2.62 (m, 2H), 1.65-1.57 (m, 2H), 1.39-1.36 (m, 6H), 1.26-1.25 (m, 24H), 1.21-1.15 (m, 6H), 0.89 (t, J=6.5Hz, 3H).

[0328] Example 11: Preparation of Compound C63 of the Present Disclosure

[0329] In this example, compound C63 was prepared using the following procedure:

[0330]

[0331] (1) Preparation of Compound C63

[0332]

[0333] To a solution of compound 29 (1.00 g, 3.35 mmol, 1.0 equivalent) and diisopropylammonium tetrazolium (1.15 g, 6.70 mmol, 2.0 equivalent) in anhydrous DCM (17 mL) was added 3-((bis(diisopropylamino)phosphino)oxy)propionitrile (2.12 mL, 6.70 mmol, 2.0 equivalent) under a nitrogen atmosphere and room temperature. The reaction mixture was stirred for 4 h. The mixture was extracted twice with DCM, then washed with brine and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-2% ethyl acetate / petroleum ether, 1% Et3N) to provide compound C63 (1.3 g, 78% yield) as a colorless oil. The product was purified by mass spectrometry and 1H NMR characterization. MW calculated: 498.78; MW found: 613.55 [M-diisopropyl + H] + . 1 H NMR (400MHz, CDCl3) δ3.84-3.74 (m, 2H), 3.61-3.51 (m, 4H), 2.64 (t, J=6.5Hz, 2H) , 1.631.57 (m, 2H), 1.38-1.22 (m, 34H), 1.20-1.17 (m, 12H), 0.88 (t, J=6.7Hz, 3H).

[0334] Example 12: Preparation of Compound C64 of the Present Disclosure

[0335] In this example, compound C64 was prepared using the following procedure:

[0336]

[0337] (1) Preparation of compound 31

[0338]

[0339] Under nitrogen atmosphere, HBTU (11 g, 29.1 mmol, 1.5 eq.) was added to a solution of compound 40 (2 g, 19.4 mmol, 1.0 eq.), compound 30 (5.97 g, 23.28 mmol, 1.2 eq.) and DIPEA (4.82 mL, 29.1 mmol, 1.5 eq.) in DCM (40 mL). The reaction mixture was stirred overnight. The mixture was extracted twice with DCM, then washed with brine and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-5% MeOH / DCM) to provide compound 31 (4.63 g, 70% yield) as a white solid. The product was purified by mass spectrometry and 1 HNMR characterization. MW calculated: 341.33; MW found: 342.39 [M+H] + . 1 H NMR (400MHz, CDCl3) δ3.64 (t, J=6.4Hz, 2H), 3.25 (dd, J=13.0, 6.9Hz, 2H), 2.19-2.10 (m, 2H), 1.59 (dd, J=14 .3, 7.3Hz, 4H), 1.52 (dd, J=15.0, 7.9Hz, 2H), 1.44-1.38 (m, 2H), 1.27-1.21 (m, 26H), 0.87 (t, J=6.8Hz, 3H).

[0340] (2) Preparation of Compound C64

[0341]

[0342] To a solution of compound 31 (1 g, 2.9 mmol, 1.0 equiv) and diisopropylammonium tetrazolium salt (745 mg, 4.35 mmol, 1.5 equiv) in anhydrous DCM (10 mL) was added 3-((bis(diisopropylamino)phosphino)oxy)propionitrile (1.31 g, 4.35 mmol, 1.5 equiv) under nitrogen atmosphere and room temperature. The reaction mixture was stirred for 2 h. The mixture was extracted twice with DCM, then washed with brine and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-5% MeOH / DCM, 1% Et3N) to provide compound C64 (1.25 g, 79% yield). The product was purified by mass spectrometry and 1 H NMR characterization. MW calculated: 541.44; MW found: 459.41 [M-83+H] + . 1 H NMR (400MHz, CDCl3) δ3.55-3.51 (m, 2H), 3.19-3.16 (m, 2H), 2.69 (td, J=6.3, 1.7Hz, 1H), 2.652.45 (m, 3H), 2.14-2.02 (m, 2H), 1. 54 (dt, J=11.8, 4.8Hz, 4H), 1.49-1.39 (m, 2H), 1.38-1.30 (m, 2H), 1.22-1.15 (m, 26H), 1.12-1.09 (m, 12H), 0.81 (t, J=6.8Hz, 3H).

[0343] Example 13: Preparation of Compound C65 of the Present Disclosure

[0344] In this example, compound C65 was prepared using the following procedure:

[0345]

[0346] (1) Preparation of compound 33

[0347]

[0348] Under nitrogen atmosphere, HBTU (10.82 g, 28.53 mmol, 1.5 eq.) was added to a solution of compound 32 (5.85 g, 22.83 mmol, 1.2 eq.), compound 41 (2 g, 19.02 mmol, 1.0 eq.) and DIPEA (4.73 mL, 28.53 mmol, 1.5 eq.) in DCM (50 mL). The reaction mixture was stirred overnight. The mixture was extracted twice with DCM, then washed with brine and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-8% MeOH / DCM) to provide compound 33 (2.3 g, 35% yield) as a white solid. The product was purified by mass spectrometry and 1 HNMR characterization. MW calculated: 343.31; MW found: 344.37 [M+H] + . 1 H NMR (400MHz, CDCl3) δ5.85 (s, 1H), 3.78-3.73 (m, 2H), 3.62-3.54 (m, 4H), 3.47 (dd, J=10.5, 5 .2Hz, 2H), 2.21-2.15(m, 2H), 1.62-1.60(m, 2H), 1.33-1.21(m, 24H), 0.88(t, J=6.8Hz, 3H).

[0349] (2) Preparation of Compound C65

[0350]

[0351] To a solution of compound 33 (1.5 g, 4.37 mmol, 1.0 equiv) and diisopropylammonium tetrazolium salt (1.5 g, 8.74 mmol, 2.0 equiv) in anhydrous DCM (28 mL) was added 3-((bis(diisopropylamino)phosphino)oxy)propionitrile (2.63 g, 8.74 mmol, 2.0 equiv) under nitrogen atmosphere at room temperature. The reaction mixture was stirred for 2 h. The mixture was extracted twice with DCM, then washed with brine and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-5% MeOH / DCM, 1% Et3N) to provide compound C65 (2.02 g, 85% yield). The product was purified by mass spectrometry and 1 H NMR characterization. MW calculated: 543.42; MW found: 485.35 [M-81+Na] + . 1H NMR (400MHz, CDCl3) δ3.64 (d, J=7.1Hz, 2H), 3.57-3.53 (m, 4H), 3.50-3.43 (m, 4H), 2.78-2.74 (m, 4H), 2.66 -2.64 (d, J=6.4Hz, 2H), 2.19-2.15 (m, 2H), 1.26-1.25 (m, 24H), 1.20-1.17 (m, 12H), 0.88 (t, J=6.8Hz, 3H).

[0352] Example 14: Preparation of Compound C66 of the Present Disclosure

[0353] In this example, compound C66 was prepared using the following procedure:

[0354]

[0355] (1) Preparation of Compound 35

[0356]

[0357] Under nitrogen atmosphere, HBTU (3.81 g, 10.05 mmol, 1.5 eq.) was added to a solution of compound 34 (2.06 g, 8.04 mmol, 1.2 eq.), compound 42 (1 g, 6.7 mmol, 1.0 eq.), DIPEA (1.67 mL, 10.05 mmol, 1.5 eq.) in DCM (15 mL). The reaction mixture was stirred overnight. The mixture was extracted twice with DCM, then washed with brine and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-8% MeOH / DCM) to provide compound 35 (2.2 g, 85% yield) as a white solid. The product was purified by mass spectrometry and 1 HNMR characterization. MW calculated: 387.33; MW found: 388.93 [M+H] + . 1 H NMR (400MHz, CDCl3) δ6.05 (s, 1H), 3.77-3.73 (m, 2H), 3.70-3.66 (m, 2H), 3.66-3.61 (m, 4H), 3.59-3.55 (m, 2H), 3. 46 (dd, J=10.4, 5.2Hz, 2H), 2.21-2.14 (m, 2H), 1.66-1.58 (m, 2H), 1.27 (d, J=11.8Hz, 24H), 0.88 (t, J=6.8Hz, 3H).

[0358] (2) Preparation of Compound C66

[0359]

[0360] To a solution of compound 35 (2.0 g, 5.17 mmol, 1.0 equiv) and diisopropylammonium tetrazolium salt (1.77 g, 10.34 mmol, 2.0 equiv) in anhydrous DCM (40 mL) was added 3-((bis(diisopropylamino)phosphino)oxy)propionitrile (3.12 g, 10.34 mmol, 2.0 equiv) under nitrogen atmosphere at room temperature. The reaction mixture was stirred for 2 h. The mixture was extracted twice with DCM, then washed with brine and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-5% MeOH / DCM, 1% Et3N) to provide compound C66 (2.43 g, 80% yield). The product was purified by mass spectrometry and 1 H NMR characterization. MW calculated: 587.44; MW found: 505.32 [M-83+H] + . 1 H NMR (400MHz, CDCl3) δ3.68-3.65 (m, 4H), 3.64-3.58 (m, 4H), 3.55 (dd, J=9.6, 3.9Hz, 4H), 3.44 (dd, J=9.7, 5.3Hz, 2H), 2.77 (td, J=6.3, 1.5H z, 2H), 2.68-2.63 (m, 2H), 2.20-2.14 (m, 2H), 1.61 (dd, J=14.5, 7.1Hz, 2H), 1.27-1.24 (m, 24H), 1.19-1.15 (m, 12H), 0.88 (t, J=6.8Hz, 3H).

[0361] Example 15: Preparation of Compound C67 of the Present Disclosure

[0362] In this example, compound C67 was prepared using the following procedure:

[0363]

[0364] (1) Preparation of compound 37

[0365]

[0366] Under nitrogen atmosphere, HBTU (1.98 g, 5.23 mmol, 1.5 eq.) was added to a solution of compound 36 (1.07 g, 4.19 mmol, 1.2 eq.), compound 43 (500 mg, 3.49 mmol, 1.0 eq.) and DIPEA (866 μL, 5.23 mmol, 1.5 eq.) in DCM (10 mL). The reaction mixture was stirred overnight. The mixture was extracted twice with DCM, then washed with brine and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-5% MeOH / DCM) to provide compound 37 (998 mg, 75% yield) as a white solid. The product was purified by mass spectrometry and 1 H NMR characterization. MW calculated: 381.36; MW found: 405.10 [M+Na] + . 1 H NMR (400MHz, CDCl3) 65.48 (s, 1H), 3.46 (d, J = 6.3Hz, 2H), 3.11 (t, J = 6.4Hz, 2H), 2.24-2.09 (m, 2H), 1.88-1.74 (m, 4H) , 1.65-1.57 (m, 2H), 1.45 (dd, J=11.3, 7.8Hz, 2H), 1.33-1.20 (m, 24H), 0.96 (t, J=10.9Hz, 4H), 0.88 (t, J=6.8Hz, 3H).

[0367] (2) Preparation of Compound C67

[0368]

[0369] To a solution of compound 37 (900 mg, 2.36 mmol, 1.0 equiv) and diisopropylammonium tetrazolium salt (606 mg, 3.54 mmol, 1.5 equiv) in anhydrous DCM (10 mL) was added 3-((bis(diisopropylamino)phosphino)oxy)propionitrile (1.07 g, 3.54 mmol, 1.5 equiv) under a nitrogen atmosphere at room temperature. The reaction mixture was stirred for 2 h. The mixture was extracted twice with DCM, then washed with brine and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-5% MeOH / DCM, 1% Et3N) to provide compound C67 (1.09 g, 80% yield). The product was obtained by 1 H NMR characterization. 1H NMR (400MHz, CDCl3) δ3.88-3.76 (m, 2H), 3.58-3.45 (m, 4H), 3.10 (t, J=6.4Hz, 2H), 2.76 (tt, J=6.1, 3.1Hz, 2H), 2.64 (t, J=6.5Hz, 2H), 2.19 -2.13 (m, 2H), 1.861.73 (m, 4H), 1.69-1.60 (m, 2H), 1.26-1.23 (m, 24H), 1.20-1.15 (m, 12H), 0.96 (t, J=10.5Hz, 4H), 0.88 (t, J=6.8Hz, 3H).

[0370] Example 16: Preparation of Compound C68 of the Present Disclosure

[0371] In this example, compound C68 was prepared using the following procedure:

[0372]

[0373] (1) Preparation of Compound 39

[0374]

[0375] Under nitrogen atmosphere, HBTU (4.85 g, 12.8 mmol, 1.5 eq.) was added to a solution of compound 38 (2.19 g, 8.53 mmol, 1.0 eq.), compound 44 (1 g, 8.53 mmol, 1.0 eq.), DIPEA (2.12 mL, 12.8 mmol, 1.5 eq.) in DCM (50 mL). The reaction mixture was stirred overnight. The mixture was extracted twice with DCM, then washed with brine and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure to provide compound 39 (2.44 g, 80% yield) as a white solid. The product was purified by mass spectrometry and 1 H NMR characterization. MW calculated: 355.35; MW found: 356.16 [M+H] + . 1 H NMR (400MHz, CDCl3) δ5.43 (s, lH), 3.64 (t, J=6.5Hz, 2H), 3.25 (dd, J=13.1, 7.0Hz, 2H), 2.18- 2.11 (m, 2H), 1.67-1.48 (m, 6H), 1.46-1.34 (m, 4H), 1.33-1.16 (m, 24H), 0.88 (t, J=6.8Hz, 3H).

[0376] (2) Preparation of Compound C68

[0377]

[0378] To a solution of compound 39 (1 g, 2.82 mmol, 1.0 eq.) and diisopropylammonium tetrazolium salt (724 mg, 4.23 mmol, 1.5 eq.) in anhydrous DCM (10 mL) was added 3-((bis(diisopropylamino)phosphino)oxy)propionitrile (1.27 g, 4.23 mmol, 1.5 eq.) under a nitrogen atmosphere and room temperature. The reaction mixture was stirred for 2 h. The mixture was extracted twice with DCM, then washed with brine and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-5% MeOH / DCM, 1% Et3N) to provide compound C68 (1.29 g, 82% yield) as a white solid. The product was obtained by 1 H NMR characterization. 1 H NMR (400MHz, CDCl3) δ3.90-3.75 (m, 2H), 3.63-3.57 (m, 2H), 3.23 (dd, J=13.1, 6.9Hz, 2H), 2.65 (t, J=6.5Hz, 2H), 2.19-2.10 (m , 2H), 1.67-1.57(m, 4H), 1.54-1.45(m, 2H), 1.39-1.34(m, 2H), 1.33-1.22(m, 28H), 1.20-1.15(m, 12H), 0.88(t, J=6.8Hz, 3H).

[0379] Example 17: Preparation of Compound C69

[0380] In this example, compound C69 was prepared using the following procedure:

[0381]

[0382] (1) Preparation of Compound 45

[0383]

[0384] Under nitrogen atmosphere, to a solution of methyl 2-(4-fluoro-3-nitrophenyl)acetate 4 (5 g, 23.5 mmol, 1.0 eq.) and KCO (4.9 g, 35.3 mmol, 1.5 eq.) in anhydrous DMF (80 mL) was added compound 51 (6.9 g, 25.8 mmol, 1.1 eq.). The reaction mixture was stirred at 55 °C for 6 h, and then cold water (200 mL) was added. The mixture was extracted three times with ethyl acetate, and then the organic phase was washed three times with saturated LiCl solution and once with brine. The organic phase was then dried over anhydrous NaSO and concentrated under reduced pressure to form compound 45 as a yellow oil which was used directly in the next step without further purification.

[0385] (2) Preparation of Compound 46

[0386]

[0387] To a solution of compound 45 (10.8 g, 23.5 mmol, 1.0 eq.) in THF / H2O (9:1, 130 mL) was added HCOONH4 (8.9 g, 141 mmol, 6.0 eq.) and Zn powder (9.2 g, 141 mmol, 6.0 eq.) under an ice bath. After stirring for 10 minutes, the reaction mixture was removed from the ice bath and stirred at room temperature overnight. Then, the reaction mixture was filtered and concentrated under reduced pressure. After this, water (200 mL) was added to the mixture, and then the mixture was extracted three times with ethyl acetate and the organic phase was washed once with brine. After drying over Na2SO4 and concentrating under reduced pressure, product 46 was formed, which was then used directly in the next step without further purification.

[0388] (3) Preparation of Compound 47

[0389]

[0390] Under nitrogen atmosphere, 3-((tert-butyldimethylsilyl)oxy)propanal 7 (3.3 g, 17.4 mmol, 1.5 eq.) and AcOH (2.7 mL, 46.4 mmol, 4.0 eq.) were added to a solution of compound 46 (5 g, 11.6 mmol, 1.0 eq.) in EtOH (60 mL). The reaction mixture was stirred at 80 ° C overnight and then concentrated under reduced pressure. Saturated NaHCO3 solution (100 mL) was then added, and the mixture was extracted three times with ethyl acetate, and the organic phases were then combined and washed with brine, dried over Na2SO4 and concentrated. The resulting residual compound was purified by flash chromatography (silica gel, gradient eluent: 10-40% ethyl acetate / petroleum ether) to provide compound 47 (1.5 g, 22% yield) as a light yellow oil. The product was purified by mass spectrometry and 1 H NMR characterization. MW calculated: 598.99; MW found: 599.72 [M+H] + . 1H NMR (400MHz, CDCl3) δ7.59 (s, 1H), 7.24 (d, J = 8.3Hz, 1H), 7.16 (d, J = 8.3Hz, 1H), 5.40-5.31 (m, 2H), 4.17-4.09 (m, 4H), 3.74 (s, 2H), 3.68 ( s, 3H), 3.09 (t, J=6.9Hz, 2H), 2.06-1.92 (m, 4H), 1.81-1.72 (m, 2H), 1.39-1.19 (m, 22H), 0.88 (t, J=6.5Hz, 3H), 0.86 (s, 9H), 0.01 (s, 6H).

[0391] (4) Preparation of Compound 48

[0392]

[0393] Under nitrogen atmosphere, 2MHCl solution (10mL) was added to a solution of compound 47 (1.5g, 2.5mmol, 1.0 equivalent) in THF (10mL). The reaction mixture was stirred at room temperature for 2h. 20mL saturated NaHCO3 solution was added to the reaction. The mixture was then extracted twice with ethyl acetate, washed with brine and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure. The resulting residue was dissolved in DCM (10mL), and triethylamine (0.4g, 3.8mmol, 1.5 equivalents) and DMTrCl (1.1g, 3.3mmol, 1.3 equivalents) were added thereto. The reaction mixture was stirred at room temperature for 6h, after which it was concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, gradient eluent: 20-50% ethyl acetate / petroleum ether) to provide compound 48 (1.5g, 76% yield), which was a light yellow oil. The product was purified by mass spectrometry and 1 H NMR characterization. MW calculated: 787.1; MW found: 788.1 [M+H] + . 1 H NMR (400MHz, CDCl3) δ7.56 (s, 1H), 7.34 (d, J=7.1Hz, 2H), 7.25-7.13 (m, 9H), 6.75 (d, J=8.8Hz, 4H), 5.41-5.30 (m, 2H), 4.13-4 .07 (m, 2H), 3.76 (s, 6H), 3.73 (s, 2H), 3.67 (s, 3H), 3.60 (t, J = 7.0Hz, 2H), 3.11 (t, J = 6.9Hz, 2H), 1.99 (t, J = 15.9Hz, 4H), 1.77

[0394] 1.65(m, 2H), 1.33-1.23(m, 22H), 0.87(t, J=6.8Hz, 3H).

[0395] (5) Preparation of Compound C69

[0396]

[0397] Under nitrogen atmosphere and ice bath, LiAlH4 (73 mg, 1.9 mmol, 1.0 equiv) was added to a solution of compound 48 (1.5 g, 1.9 mmol, 1.0 equiv) in anhydrous THF (20 mL). After 10 minutes, the mixture was moved to room temperature and stirred for 1 h. The reaction was then moved to an ice bath, and saturated potassium sodium tartrate solution (10 mL) was slowly added to the mixture. After 30 minutes, the reaction was extracted three times with ethyl acetate, and then the organic phases were combined and washed with brine, dried over Na2SO4 and concentrated. Under nitrogen atmosphere and room temperature, the crude product (1.3 g, 1.7 mmol, 1.0 equiv) and diisopropylammonium tetrazole (587 mg, 3.4 mmol, 2 equiv) were dissolved in anhydrous DCM (10 mL), and 3-((bis(diisopropylamino)phosphino)oxy)propionitrile (1.0 g, 3.4 mmol, 2 equiv) was added. The reaction mixture was stirred for 6 h. The mixture was extracted twice with DCM, then washed with brine and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure and the resulting residue was purified by flash chromatography (silica gel, gradient eluent: 10-30% ethyl acetate / petroleum ether, 1% Et3N) to provide compound C69 (1.2 g, 78% yield) as a light yellow oil. The product was purified by mass spectrometry and 1 H NMR characterization. MW calculated: 959.31; MW found: 876.75 [M-diisopropyl + H] + . 1 H NMR (400MHz, CDCl3) δ7.50 (s, 1H), 7.34 (d, J = 7.2Hz, 2H), 7.25-7.14 (m, 8H), 7.09 (d, J = 8.2Hz, 1 H), 6.75 (d, J=8.8Hz, 4H), 5.41-5.31 (m, 2H), 4.13-4.05 (m, 2H), 3.93-3.83 (m, 1H), 3.81-3.70 ( m, 9H), 3.64-3.54 (m, 4H), 3.11 (t, J=7.0Hz, 2H), 3.03 (t, J=7.3Hz, 2H), 2.57-2.47 (m, 2H), 2.07 -1.92 (m, 4H), 1.77-1.66 (m, 2H), 1.34-1.24 (m, 22H), 1.18-1.12 (m, 12H), 0.87 (t, J=6.7Hz, 3H).

[0398] Example 18: Preparation of Compound C70 of the Present Disclosure

[0399] In this example, compound C70 was prepared using the following procedure:

[0400]

[0401] (1) Preparation of Compound C70

[0402]

[0403] To a solution of compound 49 (1.00 g, 3.73 mmol, 1.0 equiv) and diisopropylammonium tetrazolium (1.27 g, 7.45 mmol, 2.0 equiv) in anhydrous DCM (19 mL) was added 3-((bis(diisopropylamino)phosphino)oxy)propionitrile (2.36 mL, 7.45 mmol, 2.0 equiv) under a nitrogen atmosphere and room temperature. The reaction mixture was stirred for 8 h. The mixture was extracted twice with DCM, then washed with brine and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-2% ethyl acetate / petroleum ether, 1% Et3N) to provide compound C70 (1.65 g, 94% yield) as a colorless oil. The product was obtained by 1 H NMR characterization. 1 H NMR (400MHz, CDCl3) δ5.40-5.36 (m, 2H), 3.86-3.76 (m, 2H), 3.62-3.52 (m, 4H), 2.65 (t, 2H), 1.99 -1.91 (m, 4H), 1.66-1.59 (m, 2H), 1.35-1.25 (m, 22H), 1.20-1.18 (m, 12H), 0.88 (t, J=6.8Hz, 3H).

[0404] Example 19: Preparation of Compound C71 of the Present Disclosure

[0405] In this example, compound C71 was prepared using the following procedure:

[0406]

[0407] (1) Preparation of Compound C71

[0408]

[0409] To a solution of compound 50 (1.00 g, 3.75 mmol, 1.0 equiv) and diisopropylammonium tetrazolium (1.28 g, 7.51 mmol, 2.0 equiv) in anhydrous DCM (20 mL) was added 3-((bis(diisopropylamino)phosphino)oxy)propionitrile (2.38 mL, 7.51 mmol, 2.0 equiv) under a nitrogen atmosphere at room temperature. The reaction mixture was stirred for 6 h. The mixture was extracted twice with DCM, then washed with brine and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-2% ethyl acetate / petroleum ether, 1% Et3N) to provide compound C71 (1.5 g, 90% yield) as a colorless oil. The product was obtained by 1 H NMR characterization. 1 H NMR (400MHz, CDCl3) δ5.45-5.28(m, 4H), 3.85-3.75(m, 2H), 3.64-3.53(m, 4H), 2.65(t, J=6.6Hz, 2H), 2.09-2.00 (m, 4H), 1.64-1.58 (m, 2H), 1.41-1.24 (m, 18H), 1.21-1.17 (m, 12H), 0.89 (t, J=6.7Hz, 3H).

[0410] Example 20: Synthesis and design of LDEC-coupled ASO

[0411] RD-10004 (ASO-10-27, SEQ ID NO: 1) is an ASO that targets the pre-mRNA of the SMN2 gene, modulating its splicing by including exon 7 into the mature mRNA and thus "converting" SMN2Δ7 (SMN2 lacking exon 7) in cells to SMN2FL (i.e., full-length SMN2 containing exon 7) mRNA, thereby increasing the level of SMN protein encoded by SMN2FL mRNA. It is synthesized by medicinal chemistry (i.e., 2'MOE and PS modification) and is not lipid-coupled.

[0412] When the exemplary LDEC conjugates are synthesized by covalently linking LDEC to the 5' end, 3' end, or 3' and 5' ends of RD-10004, respectively, the sequence and chemical modifications of RD-10004 remain unchanged. Table 1 below shows the oligonucleotide sequences and conjugates.

[0413]

[0414]

[0415] Example 21: In vivo activity of LDEC-ASO in exon 7 inclusion and in type III SMA mice by SC administration Induction of SMN2FL mRNA in murine skeletal muscle

[0416] To compare the activity of LDEC-ASO (i.e., RD-14644) and unconjugated RD-10004 in regulating SMN2 splicing in skeletal muscle tissue, female mice with type III SMA were treated with 10 mg / kg, 30 mg / kg, or 100 mg / kg doses of RD-14644 or RD-10004 by SC injection at PND 40-50. Treatment with saline alone was used as a vehicle control to establish baseline expression levels of SMN2FL and SMN2Δ7 mRNA. Mice were sacrificed on day 14 after treatment, and biceps ( Figure 1A )、Semitendinosus( Figure 1B )、Platinum( Figure 1C ) and gluteal muscles ( Figure 1D ) muscle tissues. At all doses, RD-14644 measured SMN2FL mRNA levels higher than SMN2Δ7 mRNA levels in each muscle tissue compared to RD-10004 ( Figure 1A Table 2 summarizes the mean fold changes in SMN2FL and SMN2Δ7 transcript levels following SC treatment relative to saline controls. Overall, LDEC-conjugated ASOs (e.g., RD-14644) have improved efficacy on exon 7 inclusion and SMN2FL transcript induction in muscle tissue compared to unconjugated ASOs (i.e., RD-10004).

[0417] Table 2: SMN2FL and SMN2Δ7 mRNA levels in skeletal muscle after LDEC-ASO treatment in vivo

[0418]

[0419] Note: "SEM" means standard error of the mean. * / # means p<0.05, ** / ## means p<0.01, *** / ### means p<0.001, **** / #### means p<0.0001, and "ns" means no statistical significance (compared to the saline group).

[0420] Example 22: SC administration of LDEC-ASOs to reduce the expression of LDECs in peripheral tissues (i.e., heart and liver) of type III SMA mice SMN2Δ7 is converted into SMN2FL transcripts

[0421] To compare the activity of LDEC-ASOs (i.e., RD-14644, RD-14645, and RD-14646) and unconjugated RD-10004 in regulating SMN2 splicing in the liver, SMN2FL and SMN2Δ7 expression in tissues of female mice with type III SMA was quantified by RT-qPCR 14 days after administration via SC injection. Figure 2AAs shown, each LDEC-ASO (i.e., RD-14644, RD-14645, and RD-14646) had a dose-dependent response in increasing SMN2FL production compared to RD-10004, and for RD-10004, statistical differences in splicing activity were only measured at the highest dose (i.e., 100 mg / kg).

[0422] In the heart, SMN2FL mRNA levels were also measured to be higher than SMN2Δ7 mRNA levels with LDEC-ASO (ie, RD-14644) treatment compared to RD-10004 ( Figure 2B In fact, only the LDEC conjugate (ie, RD-14644) demonstrated dose-dependent splicing activity, while RD-10004 had no or little activity at the doses tested (ie, 10 mg / kg, 30 mg / kg, and 100 mg / kg).

[0423] Table 3 summarizes the results of SMN2FL and SMN2Δ7 expression in liver and heart following treatment with the indicated LDEC-ASOs relative to saline controls. Similar to the results in muscle tissue, the data suggest that LDEC conjugation also enhances the delivery and subsequent splicing activity of RD-10004 in peripheral tissues (i.e., heart and liver).

[0424] Table 3: SMN2FL and SMN2Δ7 mRNA levels in liver or heart after LDEC-ASO treatment in vivo

[0425]

[0426] Note: "SEM" means standard error of the mean. * / # means p<0.05, ** / ## means p<0.01, *** / ### means p<0.001, **** / #### means p<0.0001, and "ns" means no statistical significance (compared to the saline group).

[0427] Example 23: SMN2 expression in peripheral tissues and skeletal muscle of type III SMA mice was reduced by SC administration of LDEC-ASO. Δ7 is converted into SMN2FL transcripts

[0428] To further compare the activity of LDEC-ASO (ie, RD-14644) and unconjugated RD-10004 in regulating SMN2 splicing in peripheral tissues and skeletal muscle, 30 mg / kg of LDEC-ASO (ie, RD-14644) or unconjugated control (ie, RD-10004) was administered to female mice with type III SMA by SC injection at PND 40-50. Treatment with aCSF alone was used as a vehicle control to establish baseline expression levels of SMN2FL and SMN2Δ7 mRNA. SMN2FL and SMN2Δ7 expression in tissues of female mice with type III SMA was quantified by RT-qPCRC at 14 days after SC injection. The average expression levels of SMN2FL or SMN2Δ7 in peripheral tissues (i.e., heart, liver, adipose tissue, diaphragm, thoracic aorta with vein) and skeletal muscles (i.e., biceps, semitendinosus, platysma, and gluteal muscles) of treated animals are shown in Figure 3A-3B , which showed that LDEC conjugation also enhanced the delivery and subsequent splicing activity of RD-10004 in peripheral tissues and skeletal muscle.

[0429] Example 24: IV administration of LDEC-ASO converts SMN2Δ7 in peripheral tissues of type III SMA mice SMN2FL transcript

[0430] To compare the activity of LDEC-ASOs (i.e., RD-14644 and RD-14645) and unconjugated RD-10004 in regulating SMN2 splicing, SMN2FL and SMN2Δ7 expression was quantified by RT-qPCR using tissues from type III SMA female mice at day 14 after femoral vein (IV) injection. Treatment with saline alone was used as a vehicle control to establish baseline expression levels of SMN2FL and SMN2Δ7 mRNA. Figures 4A-4F As shown, each LDEC-ASO (i.e., RD-14644 and RD-14645) increased SMN2FL production compared to RD-10004 in peripheral tissues (i.e., liver, adipose tissue, thoracic aorta, heart, diaphragm, and lung). Table 4 shows the results of SMN2FL and SMN2Δ7 expression after treatment with the indicated LDEC-ASOs relative to saline controls.

[0431] Table 4: SMN2FL and SMN2Δ7 mRNA levels after LDEC-ASO treatment in vivo

[0432]

[0433]

[0434] Note: “SEM” stands for standard error of the mean.

[0435] Example 25: Conversion of SMN2Δ7 in GM03813 and GM09677 cells to SMN2FL transcript

[0436] To test the in vitro activity of various LDEC-ASOs, the indicated LDEC-ASOs (i.e., RD-15865, RD-16070, RD-16071, RD-16072, RD-16073, RD-16074, RD-16075, RD-16076, RD-16077, RD-16078, RD-16079, RD-16080, RD-16081, and RD-16098) were transfected into GM03813 and GM09677 cells at 0.1 nM, 1 nM, and 10 nM, respectively, for 3 days. Mock treatment was transfection in the absence of oligonucleotides. dsCon2 served as a nonspecific duplex control. The mRNA levels of SMN2FL and SMN2Δ7 were determined by RT-qPCR. Figure 5A-5B The mRNA expression of SMN2FL and SMN2Δ7 at 1 nM and 10 nM is shown. Table 5 summarizes the expression results of SMN2FL and SMN2Δ7 in GM03813 cells.

[0437] Table 5: SMN2FL and SMN2Δ7 mRNA levels in GM03813 cells after LDEC-ASo treatment (fold change)

[0438]

[0439]

[0440] Note: "SEM" means standard error of the mean.

[0441] Further, Figure 6A-6B The mRNA expression of SMN2FL and SMN2217 at 0.1 nM and 1 nM in GM09677 and GM03813 cells is shown and the results are in Table 6.

[0442] Table 6: SMN2FL and SMN2Δ7 mRNA levels in GM09677 and GM03813 cells after LDEC-ASO treatment (Fold change)

[0443]

[0444]

[0445] Note: “SEM” stands for standard error of the mean.

[0446] Example 26: Conversion of SMN2Δ7 to SMN2FL transcripts in GM03813 cells by LDEC-ASO treatment

[0447] To test the in vitro activity of LDEC-ASOs in which the lipids contained 18 carbon atoms, the indicated LDEC-ASOs (i.e., RD-16935, RD-16936, and RD-16937) were transfected into GM03813 cells at 1 nM and 10 nM for 3 days. RD-10004 was transfected and used as a non-conjugated control. Fig. 7A As shown, at 1 nM, all LDEC-ASOs exhibited splicing activity similar to or superior to that of the unconjugated RD-10004. Figure 7B As shown, when the concentration increased to 10 nM, all LDEC-ASOs presented similar splicing activities compared to the unconjugated RD-10004, suggesting that the splicing activity of the ASOs may reach a plateau at 10 nM. The mRNA expression of SMN2FL and SMN2Δ7 at 1 nM and 10 nM in GM03813 cells relative to the mock control is summarized in Table 7.

[0448] Table 7: SMN2FL and SMN2Δ7 mRNA levels in GM03813 cells after LDEC-ASO treatment

[0449]

[0450]

[0451] Note: "SEM" means standard error of the mean.

[0452] Example 27: Design and synthesis of ASOs targeting human SCN9A transcripts

[0453] A 20-nucleotide (nt) Gapmer ASO with a 10 nt DNA gap and two 5 nt wings was designed using the PFRD program for the SCN9A mRNA sequence (ENST00000409672, SEQ ID NO: 22, see Table 8) (Sciabola et al., PFRED: A computational platform for siRNA and antisense oligonucleotides design. PLoS One. 2021; 16(1): e0238753. Published January 22, 2021. doi: 10.1371 / joumal.pone.0238753).

[0454] Table 8: Human SCN9A cDNA sequences used for ASO design

[0455]

[0456]

[0457]

[0458] The obtained ASOs were first screened using the following criteria: (i) 40% to 70% GC content; (ii) less than 5 consecutive identical nucleotides; (iii) less than 3 dinucleotide or trinucleotide repeats; and (iv) not in the 5'UTR region of SCN9A mRNA. The remaining ASOs were then ranked from largest to smallest according to the PLSpred_optimized score of each ASO provided by the program, and the top 119 ASOs were selected as candidates for in vitro screening. The ASO sequences were synthesized by chemical modification and are listed in Table 9 together with their cognate target sequences.

[0459]

[0460]

[0461]

[0462]

[0463]

[0464]

[0465]

[0466] Example 28: Screening of ASOs targeting human SCN9A transcripts

[0467] To evaluate the knockdown activity of SCN9A ASO, SK-N-AS cells were transfected with each of the aforementioned ASOs at 25 nM and 100 nM for 24 hours. Mock treatment was transfected without oligonucleotides (not shown). RD-10750 was used as a nonspecific control (not shown). RD-12746 was transfected to serve as a silencing dsRNA control (not shown). SCN9A mRNA levels were quantified by one-step RT-qPCR using a gene-specific primer set in each PCR reaction.

[0468] The relative changes in SCN9A expression caused by ASO treatment are summarized in Table 8 and plotted on Fig. 8A middle. Figure 8B The E of ASO to SCN9A mRNA is shown between 25 nM and 100 nM. 最大 Knockdown activity. The solid rectangular boxes mark the 32 best performing ASOs. The results showed that 4 (3.4%) and 34 (28.6%) ASOs had high (≥70%) and moderate (50-70%) knockdown activities on SCN9A expression and are summarized in Table 10.

[0469] Table 10: Summary of the knockdown activity of ASOs screened on SCN9A mRNA in SK-N-AS cells.

[0470]

[0471] Example 29: Knockdown activity of ASO on SCN9A mRNA in SK-N-AS cells

[0472] To confirm the screening results for SCN9A knockdown activity, 32 functional SCN9A ASOs were transfected into SK-N-AS cells at a concentration gradient (i.e., 0.02, 0.1, 0.39, 1.56, 6.25, 25, 100, and 400 nM) for 24 h to generate a dose-response curve by RT-qPCR, as shown in Figures 9A-9D As shown. Extrapolated IC 50 Value and E 最大 Knockdown levels were determined to define the potency of maximal activity of each tested ASO that demonstrated dose-dependent knockdown activity of SCN9A. Table 11 summarizes the EC 50 Value and E 最大 Knock down the level.

[0473] Table 11: IC in SK-N-AS cells after ASO treatment 50 Value and E 最大 Knock down the level.

[0474]

[0475]

[0476] Example 30: Thermal Sensitivity Functional Test of LDEC-coupled ASO in SD Rats

[0477] To test the thermosensitivity of SCN9A ASOs, 0.5 and 1 mg of the indicated LDEC-ASOs (i.e., RD-13767 and RD-15069) and unconjugated ASOs (i.e., RD-12835 and RD-15064) were administered to adult female SD rats by IT injection. RD-15873 was treated at 1 mg and used as a positive control. Table 12 lists the aforementioned ASO sequences. Treatment with aCSF alone was used as a vehicle control to establish a baseline for tail-flick latency. Fig.10 Tail-flick reaction latency was tested by hot water tail-flick test on day 27 to evaluate thermal sensitivity and the mean values ​​are summarized in Table 13.

[0478] Table 12: Human SCN9A ASO sequences

[0479]

[0480] Note: Capital letters, RNA; lowercase letters, DNA; d, DNA; *, phosphorothioate (PS) backbone modification; f, 2′-fluoro; me, 2′-O-methoxyethyl (2'MOE); E, cET (constrained ethyl); C, 5-methylcytosine; U, 5-methyluracil.

[0481] Table 13: Tail-flick latency values ​​on day 27 after IT injection in SD rats

[0482]

[0483] Note: “SEM” stands for standard error of the mean.

[0484] Example 31: LDEC-coupled ASo-mediated knockdown activity of rat Scn9a mRNA in SD rats

[0485] To evaluate the knockdown activity of LDEC-ASO on rat Scn9a mRNA, 0.5 and 1 mg of the indicated LDEC-ASO (i.e., RD-13767 and RD-15069) and uncoupled ASO (i.e., RD-12835 and RD-15064) were administered by IT injection. RD-15873 was treated with 1 mg and used as a positive control. Treatment with aCSF alone was used as a vehicle control to establish baseline expression of Scn9a mRNA. Rats were sacrificed on day 27 after administration, and Scn9a mRNA levels were quantified by RT-qPCR. Figures 11A-11B The remaining Scn9a mRNA quantified in tissues of the brain (ie, cerebrum), spinal cord (ie, cervical, thoracic and lumbar vertebrae) and dorsal root ganglia (DRG) are shown. Table 14 summarizes the average knockdown activity on Scn9a mRNA at day 27 after ASO treatment in SD rats.

[0486] Table 14: Average knockdown activity of Scn9a mRNA on day 27 after ASO treatment

[0487]

[0488] Note: “ / ” means not tested.

[0489] Example 32: Design and synthesis of ASO targeting human DMD exon 51 transcript

[0490] To evaluate the knockdown activity of ASOs, the coding strand sequence of human DMD exon 51 transcript (SEQ ID NO: 392, as shown in Table 15) was retrieved from the UCSC genome database.

[0491] Table 15: Human DMD cDNA sequences used for ASO design

[0492]

[0493] Fifty ASOs of 20 nucleotides (nt) in length were designed based on the following criteria: (1) 40% to 70% GC content; (2) less than 5 consecutive identical nucleotides; (3) less than 3 dinucleotide or trinucleotide repeats. The ASOs were synthesized by chemical modification, and their sequences (including homologous targets in human DMD exon 51 transcripts) are listed in Table 16.

[0494]

[0495]

[0496]

[0497]

[0498]

[0499]

[0500] Example 33: Levels of human DMD mRNA skipping mediated by LDEC-ASO in RD cells.

[0501] To evaluate the level of exon 51 skipping of human DMD mRNA mediated by LDEC-ASO, 50 LDEC-ASOs were transfected into RD cells individually at 25 nM for 48 h. RD-16271 and RD-16272 were transfected and served as nonspecific controls. RD-15946 was transfected and served as a positive control for LDEC-ASO. The level of exon 51 skipping was quantified by nested PCR using two gene-specific primer sets and plotted on Fig.12 and are summarized in Table 16. Fig.12 As shown, seven LDEC-ASOs (i.e., RD-15905, RD-15935, RD-15936, RD-15941, RD-15942, RD-15943, and RD-15944) exhibited high levels of exon 51 skipping compared to the LDEC-ASO positive control (RD-15946).

[0502] Example 34: Dose-dependent exon 51 skipping of human DMD mRNA mediated by LDEC-ASO in RD cells flat.

[0503] To further verify the level of exon 51 skipping, the best performing 7 LDEC-ASOs (i.e., RD-15905, RD-15935, RD-15936, RD-15941, RD-15942, RD-15943, and RD-15944) were transfected into RD cells at 10 nM, 25 nM, and 100 nM for 48 hours. RD-16271 was used as a nonspecific control. RD-15945 was transfected and used as a non-conjugated ASO positive control. RD-15946 was transfected and used as a LDEC-ASO positive control. Exon 51 skipping levels were quantified by nested PCR using two gene-specific primer sets. Fig.13 As shown, nearly all LDEC-ASOs showed a dose-dependent increase in exon 51 skipping.

[0504] Materials and methods

[0505] General methods for synthesizing oligonucleotides

[0506] Single-strand synthesis method

[0507] Single-stranded oligonucleotides were synthesized by solid phase synthesis on a K&A DNA synthesizer (K&A Laborgeraete, Schaffheim, Germany).

[0508] The starting material is a general solid support or a special solid support, which is commercially available or synthesized as disclosed in the previous context. Generally, phosphoramidite monomers (0.1M in acetonitrile or dichloromethane) including various joints and conjugates are sequentially added to the solid support in the DNA synthesizer to produce the required full-length oligonucleotide. Each amidite addition cycle consists of four chemical reactions, including detritylation, coupling, oxidation / thiolation and end-capping (capping). In the first step, detritylation is carried out using a DCM solution of 3% dichloroacetic acid (TCA) for 45 seconds. In the second step, all amidates were subjected to phosphoramidite coupling at 12 equivalents for 6 minutes; in the third step, oxidation was performed using 0.02M iodine in THF:pyridine:water (70:20:10, v / v / v) for 1 minute; if phosphorothioate modification was required, oxidation was replaced by thiolation, which was performed using 0.1M hydroxanthin in pyridine:ACN (50:50, v / v) for 3 minutes. In the fourth step, end-capping was performed using THF:acetic anhydride:pyridine (80:10:10, v / v / v) (CAPA) and N-methylimidazole:THF (10:90, v / v), (CAPB) for 20 seconds. The number of cycles of the four chemical reactions will depend on the length of the individual oligonucleotides.

[0509] Deprotection I (Nucleobase Deprotection): After the synthesis is completed, the solid support is transferred to a screw cap microcentrifuge tube. For a 1 μmol synthesis scale, 1 ml of a mixture of methylamine and ammonium hydroxide is added. The tube containing the solid support is then heated in an oven at 60°C to 65°C for 15 minutes and then cooled to room temperature. The cleavage solution is collected and evaporated to dryness in a speedvac to provide a crude single-stranded oligonucleotide.

[0510] Deprotection II (removal of 2'-TBDMS group): The crude RNA oligonucleotide still carrying the 2'-TBDMS group is dissolved in 0.1 ml of DMSO. After adding 1 ml of triethylamine trihydrofluoride, cover the test tube, shake the mixture vigorously to ensure complete dissolution, and then heat it in an oven at 65°C for 15 minutes. Remove the test tube from the oven and cool to room temperature. Cool the solution containing the completely desilylated oligonucleotide on dry ice. Carefully add 2 ml of ice-cold n-butanol (-20°C) in 0.5 ml portions to precipitate the oligonucleotide. Filter the precipitate and wash with 1 ml of ice-cold n-butanol, then dissolve the precipitate in 0.01 M tris (hydroxymethyl) aminomethanol hydrochloric acid buffer.

[0511] (2) Single-strand purification

[0512] Purification of oligonucleotides was performed on an AKTA explorer 10 equipped with a Source 15Q 4.6 / 100PE column using the following conditions: Buffer A: (10 mM Tris-HCl, 1 mM EDTA, pH 7.5), B: (10 mM Tris-HCl, 1 mM EDTA, 2 M NaCl, pH 7.5), gradient: 10% B to 60% B in 25 min, flow rate: 1 ml / min. Pure oligonucleotides were collected and desalted by a HiPrep 26 / 10 desalting column and then lyophilized to powder.

[0513] Cell culture and treatment

[0514] SMA patient-derived fibroblasts were obtained from the Coriell Research Institute (Camden, NJ, USA), including GM03813 (SMA type II, with 3 copies of the SMN2 gene) and GM09677 (SMAI type, with 3 copies of the SMN2 gene), which were cultured in modified MEM medium (Gibco, Thermo Fisher Scientific, Carlsbad, CA) supplemented with 15% calf serum (Sigma-Aldrich), 1% NEAA (Gibco), and 1% penicillin / streptomycin (Gibco) at 5% CO2 and 37°C. SK-N-AS (Procell, Wuhan, China, catalog number CL-0621) cells and RD cells (TCHu 45, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences) were cultured in modified DMEM medium (Gibco, Thermo Fisher Scientific, Carlsbad, CA) supplemented with 10% fetal calf serum (Sigma-Aldrich) and 1% penicillin / streptomycin (Gibco) at 37°C, 5% CO2. Transfections were performed using Lipofectamine RNAiMax (Thermo Fisher Scientific, Waltham, MA, USA) in growth medium without antibiotics according to the manufacturer's instructions.

[0515] Animal procedures

[0516] All animal procedures were performed by certified laboratory personnel using protocols that complied with local and state regulations and were approved by the Institutional Animal Care and Use Committee. SMA-like mice were derived by homozygous knockout of Smn exon 7 in mice with a human SMN2 transgene (Smn - / - SMN2 + / - ), as previously described by Hsieh-Li et al. (Hsieh-Li, HM et al., 2000), the SMA-like mice were obtained from Jackson Laboratories. Generation of type III SMA female mice [Smnl - / - , SMN2 + / + (For genotyping, see WO2022022617A1 patent)] and administered with test compounds. Sprague-Dawley (SD) female rats (A102, SPF, China) were purchased from SPF (Suzhou) Biotechnology Co., Ltd. The animals were randomly assigned to the study groups.

[0517] Femoral vein (IV)

[0518] Mice were anesthetized using 5% isoflurane in a custom-made chamber. Anesthetized mice were placed on a heating pad at 37°C and maintained anesthesia by inhalation of 2% isoflurane through a mask. The hind limbs of the mice were fixed with tape in a slight abduction, the inguinal area of ​​the calf was shaved, disinfected with 70% ethanol, and exposed for surgery. The femoral vein was exposed through a 3 to 4 mm skin incision parallel to the inguinal ligament and then separated from the surrounding tissue. 200 μL of the test compound solution was slowly infused intravenously in the femoral vein over 2 minutes. At the end of the infusion, the femoral vein injection site was pressed firmly with a cotton swab to prevent reflux of the administration solution and / or blood. The wound was rinsed with 1 mL of 0.9% saline and the incision was closed with 6-0 sutures.

[0519] Intrathecal (IT) injection

[0520] Anesthesia was maintained for 10 min using 3.0% isoflurane in an induction chamber. The hair around the injection site at the base of the tail was shaved and cleaned with 75% ethanol. The space between the L5-L6 spinous processes was identified, and a 30-gauge needle connected to a microliter syringe containing the appropriate drug preparation was slowly inserted into the intradural space until a tail flick was observed. The needle position was then fixed and a total volume of 30 μL of solution was injected over 1 min.

[0521] Hot water tail flick test

[0522] The tail-flick test measures the pain response (tail-flick latency) to 55°C hot water. Pick up the rat and expose its tail. Place the rat above a water bath and immerse 1 cm of the tail remotely in the water bath. Record the time required for the rat to respond (e.g., tail flick). If there is no response within 10 seconds, the test is terminated. For each animal, the average response time of 3 independent measurements is calculated.

[0523] Tissue harvesting

[0524] Treated animals were euthanized by carbon dioxide inhalation and placed on a support board. The hair on the forelimbs, hind limbs, neck and buttocks of the animals was shaved. The skin of the animals was disinfected with 75% ethanol. Skeletal muscles (i.e., biceps, semitendinosus, platysma and gluteal muscles) were isolated according to the anatomical diagram and immediately immersed in RNAlater TM (Rol18-500mL, Beyotime (Biyuntian Company, China). The indicated brain, spinal cord and peripheral tissues were isolated and stored in RNAlater TM middle.

[0525] RNA Isolation

[0526] Prior to sample processing, animal tissues were immersed and stored in RNAlater TMAnimal tissues were cut into 2-3 slices less than 0.5 square in thickness, and the resulting fragments were transferred to a 2 mL microtube containing 1 mL RNAVzol (Bisharp (White Shark), BS259A, China) and 6-8 ceramic beads (ALLSHENG (Ao Sheng) ceramic beads, China). The tissue was homogenized with a Bioprep-24 homogenizer (ALLSHENG Bioprep-24, China). 200 μL of chloroform was pipetted into the homogenate, then vortexed and incubated for 5 minutes, and then centrifuged at 12000g for 15 minutes. 200 μL of the top aqueous phase was pipetted into a new test tube. RNA was then isolated in an Auto-pure96 machine (ALLSHENG, Zhejiang, China) using the MagPure Total RNA Micro LQ kit (Magen, R6621, Guangdong, China) according to the instructions provided by the manufacturer. The RNA concentration was determined by an ELISA system (Infinite M2000Pro, Tecan).

[0527] Reverse transcription quantitative polymerase chain reaction (RT-qPCR)

[0528] The obtained RNA (~1 μg) was reverse transcribed into cDNA using the PrimeScript RT reagent kit (Takara, Shiga, Japan) containing gDNA Eraser. The obtained cDNA was amplified in a Roche LightCycler 480 multiwell plate 384 (Roche, reference number: 4729749001, USA) using SYBR Premix Ex Taq II (Takara, RR820A, Shiga, Japan) reagent and primers (which specifically amplify the target gene of interest). The experimental conditions are as follows: reverse transcription reaction (stage 1): 42°C for 5 minutes, 95°C for 10 seconds; PCR reaction (stage 2): 95°C for 5 seconds, 60°C for 30 seconds, 72°C for 10 seconds; 40 amplification cycles; and, melting curve (stage 3). The PCR reaction conditions are shown in Tables 17 and 18. The primer sequences are listed in Table 19.

[0529] Table 17: RT reaction

[0530]

[0531]

[0532] Table 18: RT-qPgCR reaction

[0533]

[0534] Table 19: Primer sequences used for RT-qPCR assay

[0535]

[0536]

[0537] Single reference gene

[0538] To calculate the expression level of target mRNA in dsRNA or ASO treated samples relative to control treatment (E rel ), substitute the Ct values ​​of the target gene and the reference gene into Formula BI:

[0539] E rel =2 (CtTm-CtTs) / 2 (CtRm-CtRs)

[0540] (formula BI)

[0541] Among them, CtT m is the Ct value of the target gene from the control treated sample; CtT s is the Ct value of the target gene from the dsRNA or ASO treated sample; CtR m is the Ct value of the reference gene from the control treated sample; CtR s is the Ct value of the reference gene from the dsRNA or ASO treated samples.

[0542] Dual reference genes

[0543] To calculate the expression level of target gene mRNA in dsRNA-treated samples relative to that in control samples (E rel ), substitute the Ct values ​​of the target gene and the two internal reference genes into the following formula BII:

[0544] E rel =2 (CtTm-CtTs) / ((2 (CtR1m-CtR1s) *2 (CtR2m-CtR2s) ) (1 / 2) )

[0545] (Formula BII)

[0546] Among them, CtT m is the Ct value of the target gene from the control treated sample; CtT s is the Ct value of the target gene from the dsRNA treated sample; CtR1 m is the Ct value of the reference gene 1 from the control treated sample; CtR1 s is the Ct value of the internal reference gene 1 from the dsRNA treated sample; CtR2 m is the Ct value of the reference gene 2 from the control treated sample; and CtR2 s is the Ct value of internal reference gene 2 from dsRNA treated samples.

[0547] Nested PCR and gel electrophoresis analysis

[0548] Total cellular RNA was isolated from cell cultures using the RNeasy Plus Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The resulting RNA (500 ng) was reverse transcribed into cDNA using the PrimeScript RT kit (Takara, Shiga, Japan) containing gDNA Eraser as shown in Table 17. Primer-specific, exon-specific, optimized nested PCR amplifications were then performed in Roche Light Cycler 480 multiwell plates 384 (Roche, Reference No. 4729749001, USA) using SYBR Premix Ex Taq II (Takara, Shiga, Japan) reagents and primer pair sets as shown in Table 20 below. The indicated primer pairs are shown in Table 21, which correspond to the forward or reverse (F / R) and outer or inner primer pairs (O / I) for primary or secondary amplification, respectively. The secondary amplification products were then added to 10× loading buffer (Takara Bio, Shiga, Japan), separated by 2% agarose gel at 180 V for 30 min, and Nucleic acid gel stain (Biotium, Fremont, USA) was used for visualization. Images were captured by a ChemiDoc XRS+ system (Biorad, Hercules, USA) and quantified by Image J.

[0549] Table 20: Nested PCR reaction

[0550]

[0551] 95℃ for 30sec; 95℃ for 5sec, 60℃ for 60sec, 15 cycles; dilution

[0552] 40 times

[0553]

[0554] Table 21: Primer sequences used for nested PCR assay

[0555]

[0556] Statistical analysis

[0557] One-way analysis of variance (ANOVA) and Dunnett's multiple comparisons were used to compare the differences between the groups of continuous variables. P values ​​less than 0.05 were considered statistically significant between the two groups. * / # represents p < 0.05; ** / ## represents p < 0.01; ** / ### represents p < 0.001; and **** / #### represents p < 0.0001.

[0558] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternative embodiments, it will be understood by those skilled in the relevant art that various changes in form and details may be made without departing from the spirit and scope of the invention.

[0559] References

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[0561] 2. IONIS PHARMACEUTICALS, 1NC: Modulation of smnexpression; April 10, 2015.

[0562] 3. Stein, C.A. and D. Castanotto. FDA-Approved Oligonucleotide Therapies in 2017. Mol Thera, 2017 May 3;25(5):1069-1075.

[0563] 4. P Duda, GA Laforet, C DiJohnson, H Eliopoulos, A Narayana, DE Frank, SRuff, JR Mendell. Development of Phosphorodiamidate Morpholino Oligomers (PMOs) for the Treatment of Duchenne Muscular Dystrophy (DMD) (S42.008). Neurology, 2017 Apr 18;88(16 Suppl).

[0564] 5. HM Hsieh-Li 1, JG Chang, YJ Jong, MH Wu, NM Wang, CH Tsai, H Li. A mouse model for spinal muscular atrophy. Nature Genet, 2000 Jan;24(1):66-70.

Claims

1. An oligonucleotide delivery agent comprising one or more lipid moieties coupled directly or indirectly to an oligonucleotide, wherein: At least one of the lipid moieties comprises at least one C6-C 22 Straight chain alkyl, at least one C6-C 22 A linear alkenyl or a combination thereof, and the oligonucleotide is a single-stranded oligonucleotide.

2. The oligonucleotide delivery agent according to claim 1, wherein The lipid portion contains C 12 -C 20 Straight chain alkyl, C 12 -C 20 A straight chain alkenyl group or a combination thereof, and C 12 -C 20 A straight chain alkenyl group contains one or two ethylenically unsaturated groups.

3. The oligonucleotide delivery agent according to claim 1, wherein C6-C 22 The straight-chain alkyl group is selected from the group consisting of a straight-chain dodecyl group, a straight-chain tetradecyl group, a straight-chain pentadecyl group, a straight-chain hexadecyl group, a straight-chain octadecyl group, a straight-chain eicosyl group, a straight-chain dodecenyl group, a straight-chain tetradecenyl group, a straight-chain pentadecenyl group, a straight-chain hexadecenyl group, a straight-chain octadecenyl group, a straight-chain eicosenyl group, a straight-chain dodecadienyl group, a straight-chain tetradecadienyl group, a straight-chain pentadecadienyl group, a straight-chain hexadecadienyl group, a straight-chain octadecadienyl group, and a straight-chain eicosadienyl group.

4. The oligonucleotide delivery agent according to any one of claims 1 to 3, wherein Oligonucleotides are single-stranded oligonucleotides.

5. The oligonucleotide delivery agent according to claim 4, wherein the single-stranded oligonucleotide has a length of 15 to 35 nucleotides.

6. The oligonucleotide delivery agent according to claim 1, wherein The lipid moiety is coupled to the single-stranded oligonucleotide directly via a covalent bond or via a linker moiety having the following structure: where Y 1 is oxygen or sulfur, an asterisk (*) refers to the position where the linking moiety is attached to the single-stranded oligonucleotide, and a double asterisk (**) refers to the position where the linking moiety is attached to the lipid moiety.

7. The oligonucleotide delivery agent according to claim 1, which has a structure represented by formula A: The lipid portion comprises at least one C6-C 22 Straight chain alkyl, at least one C6-C 22 A linear alkenyl group or a combination thereof, and M is an integer from 1 to 5, the oligonucleotide is a single-stranded oligonucleotide, and N is an integer from 1, 2 or 3; and The delivery enhancing compound portion is a divalent to hexavalent portion comprising at least one nitrogen-containing heterocyclic ring, at least one nitrogen-containing linear group, at least one nitrogen-containing linear-carbocyclic group, or a combination thereof, and the lipid portion and the oligonucleotide are each coupled directly or indirectly to the delivery enhancing compound portion.

8. The oligonucleotide delivery agent according to claim 7, wherein The lipid portion is a C12, C13, C14, C15, C16, C17, C18, C19 or C20 straight chain saturated or unsaturated hydrocarbon group, and M is an integer of 1, 2, 3, 4 or 5.

9. The oligonucleotide delivery agent according to claim 8, wherein The unsaturated lipid moiety has at least one cis or trans unsaturated double bond in the hydrocarbon group.

10. The oligonucleotide delivery agent according to any one of claims 1 to 3 and 7, wherein The oligonucleotide is at least one single-stranded oligonucleotide selected from antisense oligonucleotide (ASO), antisense RNA, short interfering RNA (siRNA), micro RNA (miRNA), small promoter RNA (saran), cytoplasmic small RNA (scRNA), small guide RNA (sgRNA) and phosphorodiamidate morpholino oligonucleotide (PMO).

11. The oligonucleotide delivery agent according to claim 7, wherein The combination of the lipid portion and the delivery enhancing compound portion represented by Formula A' has a structure that can be obtained by subtracting at least one hydrogen atom or terminal group from the structure represented by Formula I or Formula II, wherein the terminal group is selected from hydroxyl, carboxyl, ester, ether, phosphate, phosphonate, sulfate, sulfonate, amide, acyl, and combinations thereof, and provided that A1, A2, A3, R 3 , R 4 and R 5 At least one of them is C6-C 22 Straight chain alkyl, preferably straight chain hexadecyl, Among them, each independently represents a covalent single or double bond; each occurrence of X is selected from carbon, nitrogen, oxygen and sulfur; F, G, H and I are each independently selected from carbon, nitrogen, oxygen and sulfur; wherein m is an integer of 1, 2 or 3, n is an integer of 1, 2 or 3, and m+n=4; wherein C is either absent or selected from the group consisting of hydrogen, halogen, hydroxyl, (C1-C 20 )alkyl, (C1-C 20 ) alkoxy, halogenated (C1-C 20 ) alkyl and halogenated (C1-C 20 ) alkoxy; wherein B is independently selected at each occurrence from the group consisting of hydroxy, -C(O)OH, -P(O)2-OH, -P(O)-OH, -P(O)(S)-OH, -CN, -(C1-C 22 )alkyl, -(C1-C 22 )alkenyl, -(C1-C 22 ) alkylene-OH, -(C3-C 22 )cycloalkylene-OH, -(C6-C 22 )arylene-OH, -(C6-C 22 )heteroarylene-OH, -(C1-C 22 )alkylene-C(O)OH, -(C3-C 22 )cycloalkylene-C(O)OH, -(C6-C 22 )arylene-C(O)OH, -(C5-C 22 )heteroarylene-C(O)OH, -OC(O)-(C1-C 22 )alkylene-C(O)NH2, -(C1-C 22 )alkylene-OC(O)-(C1-C 22 )alkylene-C(O)NH2, -OC(O)-(C1-C 22 )alkylene-C(O)OH, -(C1-C 22 )alkylene-OC(O)-(C1-C 22 )alkylene-C(O)OH, -C(O)-(C1-C 22 )alkylene-C(O)NH2, -(C1-C 22 )alkylene-C(O)-(C1-C 22 )alkylene-C(O)NH2, -C(O)-NH-(C1-C 22 ) alkylene-OH, -C(O)-NH-(C1-C 22 )alkylene-C(O)OH, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-C(O)OH, -(C1-C 30 )alkylene-P(O)2-OH, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-CN, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-OH, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-NH2, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-C(O)OH, -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-CN, -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-OH, -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 30 )alkylene-C(O)OH, -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-NH2, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-CN, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-OH, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-C(O)OH, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-NH2, -(C1-C 22 )alkylene-P(O)-OH, -(C1-C 22 )alkylene-P(O)(S)-OH, -(C1-C 22 ) alkylene-CN, wherein each of A1, A2 and A3 is either absent or a substituent independently selected from the following group: -H, -OH, -OR 1 ,-SH,-(C1-C 22 )alkyl, halo-(C1-C 22 )alkyl, -(C2-C 22 )alkenyl, -(C1-C 22 ) alkylene-OH, -(C3-C 22 )cycloalkyl, -(C3-C 22 )cycloalkenyl, -(C1-C 22 )alkylene-(C3-C 22 )cycloalkyl, -(C1-C 22 )alkylene-R 1 、-(C1-C 22 )Alkenylene-R 1 、-(C1-C 22 )alkylene-OR 1 、-(C1-C 22 )Alkenylene-OR 1 、-(C1-C 22 )Alkylene-COOR 1 、-(C1-C 22 )Alkenylene-COOR 1 、-C(O)OR 1 、-O-(C1-C 22 )alkyl, -S-(C1-C 22 )alkyl, -C(O)-R 1 、-C(O)-(C1-C 22 )alkyl, -OC(O)-(C1-C 22 )alkyl, -OC(O)-R 1 、-(C1-C 22 )alkylene-OC(O)-R 1 、-C(O)-(C1-C 22 )alkylene-OH, -C(O)-(C1-C 22 )alkylene-R 1 、-C(O)-(C1-C 22 )alkylene-NH-R 1 、-C(O)-(C1-C 22 )Alkylene-NR 2 -R 1 、-OC(O)-(C1-C 22 )alkylene-OH, -OC(O)-(C1-C 22 )alkylene-R 1 , -adamantyl, -(C1-C 22 )alkylene-adamantyl, -O-adamantyl, -C(O)-(C1-C 22 ) alkylene-adamantyl, -(C1-C 22 )alkylene-C(O)-(C1-C 22 ) alkylene-adamantyl, -NH-C(O)-(C1-C 22 ) alkylene-adamantyl, -(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkyl, -(C1-C 22 )alkylene-NH-C(O)-halogeno(C1-C 22 )alkyl, -CH(NH-CO-(C1-C 22 )alkyl)-(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkyl, -CH(NH-CO-halogenated(C1-C 22 )alkyl)-(C1-C 22 )alkylene-NH-C(O)-halogeno(C1-C 22 )alkyl, -(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 ) alkylene-adamantyl, -(C1-C 22 )Alkylene-NR 2 -C(O)-(C1-C 22 ) alkylene-adamantyl, -(C1-C 22 )alkylene-(C1-C6 epoxyalkyl) (1-20) -NH-C(O)-(C1-C 22 ) alkylene-adamantyl, -C(O)NH-(C1-C 22 )alkyl, -C(O)NH-R 1 、-C(O)NR 2 -R 1 、-C(O)NH-(C1-C 22 )alkylene-OH, -C(O)NH-(C1-C 22 )alkylene-COOH, -NH-C(O)-(C1-C 22 )alkyl, -NH-C(O)-R 1 、-NR 2 -C(O)-R 1 、-OP(O)2-OR 1 、-OP(O)(S)-OR 1 、-OP(O)-OR 1 、-NH-R 1 、-NR 2 -R 1 、-(C1-C 22 )alkylene-NH-R 1 、-(C1-C 22 )Alkylene-NR 2 -R 1 、-C(O)-(C1-C 22 )alkylene-C(O)-R 1 、-C(O)-(C1-C 22 )alkylene-C(O)OR 1 、-C(O)-(C1-C 22 )alkylene-NH-C(O)-R 1 、-C(O)-(C1-C 22 )Alkylene-NR 2 -C(O)-R 1 、-(C1-C 22 )alkylene-C(O)-R 1 、-(C1-C 22 )alkylene-NH-C(O)-R 1 、-(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkylene-R 1 、-(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkylene-R 1 、-(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-R 1 、-(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-R 1 、-(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkylene-R 1 、-(C1-C 22 )Alkylene-NR 2 -C(O)-(C1-C 22 )alkylene-R 1 、-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-CN, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-OH, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-NH2, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-C(O)OH, -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-CN, -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-OH, -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 30 )alkylene-C(O)OH, -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-NH2, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-CN, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-OH, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-C(O)OH, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-NH2, -(C1-C 22 )alkylene-P(O)-OH, -(C1-C 22 )alkylene-P(O)(S)-OH, -(C1-C 22 )alkylene-CN, substituted or unsubstituted pyrrole, substituted or unsubstituted pyrroline, substituted or unsubstituted pyrrolidine, substituted or unsubstituted pyrazole, substituted or unsubstituted pyrazoline, substituted or unsubstituted pyrazolidine, substituted or unsubstituted imidazole, substituted or unsubstituted oxazole, substituted or unsubstituted thiazole, substituted or unsubstituted benzopyrrole, substituted or unsubstituted benzopyrroline, substituted or unsubstituted benzopyrrolidine, substituted or unsubstituted benzopyrazole, substituted or unsubstituted benzopyrazoline, substituted or unsubstituted benzopyrazolidine, substituted or unsubstituted benzimidazole, substituted or unsubstituted benzoxazole, substituted or unsubstituted benzothiazole, and substituents represented by formula III: wherein Y is selected from carbon, nitrogen, oxygen, and sulfur; and each of P, Q, S, and T is independently selected from carbon, nitrogen, oxygen, and sulfur; and the asterisk refers to the site of attachment of the substituent represented by Formula III to the structure represented by Formula I or Formula II; Where R 3 , R 4 and R 5 Each of the above is either absent or is a substituent independently selected from the group consisting of: -H, -OH, -OR 1 ,-SH,-(C1-C 22 )alkyl, halo-(C1-C 22 )alkyl, -(C2-C 22 )alkenyl, -(C1-C 22 ) alkylene-OH, -(C3-C 22 )cycloalkyl, -(C3-C 22 )cycloalkenyl, -(C1-C 22 )alkylene-(C3-C 22 )cycloalkyl, -(C1-C 22 )alkylene-R 1 、-(C1-C 22 )Alkenylene-R 1 、-(C1-C 22 )alkylene-OR 1 、-(C1-C 22 )Alkenylene-OR 1 、-(C1-C 22 )Alkylene-COOR 1 、-(C1-C 22 )Alkenylene-COOR 1 、-C(O)OR 1 、-O-(C1-C 22 )alkyl, -S-(C1-C 22 )alkyl, -C(O)-R 1 、-C(O)-(C1-C 22 )alkyl, -OC(O)-(C1-C 22 )alkyl, -OC(O)-R 1 、-(C1-C 22 )alkylene-OC(O)-R 1 、-C(O)-(C1-C 22 )alkylene-OH, -C(O)-(C1-C 22 )alkylene-R 1 、-C(O)-(C1-C 22 )alkylene-NH-R 1 、-C(O)-(C1-C 22 )Alkylene-NR 2 -R 1 、-OC(O)-(C1-C 22 )alkylene-OH, -OC(O)-(C1-C 22 )alkylene-R 1 , -adamantyl, -(C1-C 22 )alkylene-adamantyl, -O-adamantyl, -C(O)-(C1-C 22 ) alkylene-adamantyl, -(C1-C 22 )alkylene-C(O)-(C1-C 22 ) alkylene-adamantyl, -NH-C(O)-(C1-C 22 ) alkylene-adamantyl, -(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkyl, -(C1-C 22 )alkylene-NH-C(O)-halogeno(C1-C 22 )alkyl, -CH(NH-CO-(C1-C 22 )alkyl)-(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkyl, -CH(NH-CO-halogenated(C1-C 22 )alkyl)-(C1-C 22 )alkylene-NH-C(O)-halogeno(C1-C 22 )alkyl, -(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 ) alkylene-adamantyl, -(C1-C 22 )Alkylene-NR 2 -C(O)-(C1-C 22 ) alkylene-adamantyl, -(C1-C 22 )alkylene-(C1-C6 epoxyalkyl) (1-20) -NH-C(O)-(C1-C 22 ) alkylene-adamantyl, -C(O)NH-(C1-C 22 )alkyl, -C(O)NH-R 1 、-C(O)NR 2 -R 1 、-C(O)NH-(C1-C 22 )alkylene-OH, -C(O)NH-(C1-C 22 )alkylene-COOH, -NH-C(O)-(C1-C 22 )alkyl, -NH-C(O)-R 1 、-NR 2 -C(O)-R 1 、-OP(O)2-OR 1 、-OP(O)(S)-OR 1 、-OP(O)-OR 1 、-NH-R 1 、-NR 2 -R 1 、-(C1-C 22 )alkylene-NH-R 1 、-(C1-C 22 )Alkylene-NR 2 -R 1 、-C(O)-(C1-C 22 )alkylene-C(O)-R 1 、-C(O)-(C1-C 22 )alkylene-C(O)OR 1 、-C(O)-(C1-C 22 )alkylene-NH-C(O)-R 1 、-C(O)-(C1-C 22 )Alkylene-NR 2 -C(O)-R 1 、-(C1-C 22 )alkylene-C(O)-R 1 、-(C1-C 22 )alkylene-NH-C(O)-R 1 、-(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkylene-R 1 、-(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkylene-R 1 、-(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-R 1 、-(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-R 1 、-(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkylene-R 1 、-(C1-C 22 )Alkylene-NR 2 -C(O)-(C1-C 22 )alkylene-R 1 、-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-CN, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-OH, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-NH2, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-C(O)OH, -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-CN, -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-OH, -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 30 )alkylene-C(O)OH, -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-NH2, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-CN, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-OH, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-C(O)OH, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-NH2, -(C1-C 22 )alkylene-P(O)-OH, -(C1-C 22 )alkylene-P(O)(S)-OH, -(C1-C 22 )alkylene-CN, substituted or unsubstituted pyrrole, substituted or unsubstituted pyrroline, substituted or unsubstituted pyrrolidine, substituted or unsubstituted pyrazole, substituted or unsubstituted pyrazoline, substituted or unsubstituted pyrazolidine, substituted or unsubstituted imidazole, substituted or unsubstituted oxazole, substituted or unsubstituted thiazole, substituted or unsubstituted benzopyrrole, substituted or unsubstituted benzopyrroline, substituted or unsubstituted benzopyrrolidine, substituted or unsubstituted benzopyrazole, substituted or unsubstituted benzopyrazoline, substituted or unsubstituted benzopyrazolidine, substituted or unsubstituted benzimidazole, substituted or unsubstituted benzoxazole and substituted or unsubstituted benzothiazole, Where R 7 is connected to any one of P, Q, S and T at each occurrence and is either absent or selected from hydrogen, a halogen atom, a hydroxyl group, (C1-C 20 )alkyl, (C1-C 20 ) alkoxy, halogenated (C1-C 20 ) alkyl and halogenated (C1-C 20 ) alkoxy; Where p is an integer of 0, 1, 2 or 3; Where R 6 is connected to any one of P, Q, S and T, and is selected from a direct bond, -O-, -C(O)O-, -OC(O)-, -P(O)2-O-, -OP(O)2-O-, -P(O)(S)-O-, -OP(O)(S)-O-, -OP(O)-O-, -(C1-C 22 )alkylene-, -(C1-C 22 )alkylene-O-, -O-(C1-C 22 )alkylene-, -(C1-C 22 ) alkylene-NH-, -NH-(C1-C 22 )alkylene-, -C(O)-(C1-C 22 )alkylene-, -(C1-C 22 )alkylene-C(O)-, -C(O)-O-(C1-C 22 )alkylene-, -(C1-C 30 )alkylene-C(O)-O-, -C(O)-NH-(C1-C 22 )alkylene-, -C(O)-NH-(C1-C 22 )alkylene-C(O)-O-, -C(O)-NH-(C1-C 22 )alkylene-OC(O)-, -C(O)-NH-(C1-C 22 )alkylene-OC(O)-O-, -C(O)-NH-(C1-C 22 )alkylene-O-, -C(O)-N((C1-C 22 )alkyl)-(C1-C 22 )alkylene-, -C(O)-N((C1-C 22 )alkyl)-(C1-C 22 )alkylene-O-, -C(O)-NH-(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-, -C(O)-NH-(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkylene-, -C(O)-NH-(C1-C 22 )alkylene-C(O)-NH-, -C(O)-N((C1-C 22 )alkyl)-(C1-C 22 )alkylene-C(O)-N((C1-C 22 )alkyl)-(C1-C 22 )alkylene-, -C(O)-N((C1-C 22 )alkyl)-(C1-C 22 )alkylene-C(O)-N((C1-C 22 )alkyl)-, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-, -(C1-C 22 )alkylene-C(O)-NH-(C1-C 22 )alkylene-C(O)-NH-, -NH-C(O)-(C1-C 22 )alkylene-, -NH-C(O)-(C1-C 22 )alkylene-C(O)-O-, -NH-C(O)-(C1-C 22 )alkylene-C(O)-, -NH-C(O)-(C1-C 22 )alkylene-O-, -N((C1-C 22 )alkyl)-C(O)-(C1-C 22 )alkylene-, -N((C1-C 22 )alkyl)-C(O)-(C1-C 22 )alkylene-O-, -NH-C(O)-(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkylene-, -NH-C(O)-(C1-C 22 )alkylene-NH-C(O)-, -N((C1-C 22 )alkyl)-C(O)-(C1-C 22 )alkylene-N((C1-C 22 )alkyl)-C(O)-(C1-C 22 )alkylene-, -N((C1-C 22 )alkyl)-C(O)-(C1-C 22 )alkylene-N((C1-C 22 )alkyl)-C(O)-, -(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkylene-, -(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkylene-NH-C(O)-, -(C1-C 22 )alkylene-P(O)2-O-, -(C1-C 22 )alkylene-OP(O)2-O-, -(C3-C 22 )cycloalkylene-, -(C3-C 22 )cycloalkylene-O-, -O-(C3-C 22 )cycloalkylene-, -(C6-C 22 )arylene-, -(C6-C 22 )arylene-O-, -O-(C6-C 22 )arylene-, -(C6-C 22 )arylene-NH-, -NH-(C6-C 22 )arylene-, -C(O)-(C6-C 22 )arylene-, -(C6-C 22 )arylene-C(O)-, -C(O)-O-(C6-C 22 )arylene-, -(C6-C 22 )arylene-C(O)-O-, -C(O)-NH-(C6-C 22 )arylene- and -C(O)-NH-(C6-C 22 )arylene-C(O)-O-; Where R 1 is independently selected at each occurrence from hydrogen, hydroxy, -(C1-C 22 )alkyl, -(C3-C 22 )cycloalkyl, -(C6-C 22 )aryl, -(C1-C 22 ) alkoxy, -(C3-C 22 )cycloalkoxy, -(C6-C 22 ) aryloxy, -C(O)-(C1-C 22 )alkyl, -OC(O)(C1-C 22 )alkyl, -C(O)-O-(C1-C 22 )alkyl, -C(O)-(C3-C 22 )cycloalkyl, -OC(O)-(C3-C 22 )cycloalkyl, -C(O)-O-(C3-C 22 )cycloalkyl, -C(O)-(C6-C 22 )aryloxy, -OC(O)-(C6-C 22 ) aryloxy, -C(O)-O-(C6-C 22 ) aryloxy, -C(O)-phosphate group, phosphodiester group, phosphoramidite group, saturated fatty acid group, unsaturated fatty acid group, lipid, PEG, steroid, lipophilic substance, carbohydrate, cholesterol, adamantane, amino acid, peptide, chloroquine and alkaloid, Where R 2 Each occurrence is independently selected from the group consisting of: halogen atoms, (C1-C 12 )alkyl, (C1-C 12 ) alkoxy, (C1-C 12 ) alkoxycarbonyl, (C6-C 16 )aryl or (C6-C 16 ) aryloxycarbonyl; and Among them, A1, A2, A3, B, C, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 One or more hydroxyl, carboxyl, amino, nitrile and phosphate groups contained therein are optionally attached to a supporting material or protected by a terminal protecting group.

12. The oligonucleotide delivery agent according to claim 11, wherein A1, A2, A3, B, C, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 One or more hydroxyl, carboxyl, amino, nitrile and phosphate groups contained in each are optionally protected by a terminal protecting group R P Protection, the terminal protection group R P Selected from the following group: (C1-C 22 )alkyl, (C1-C 22 ) alkoxy, (C1-C 22 )alkylcarbonyl, (C1-C 22 ) alkoxycarbonyl, (C6-C 22 ) aryl, (C6-C 22 ) aryloxy, (C6-C 22 ) arylcarbonyl, (C6-C 22 ) aryloxycarbonyl, tri((C1-C 22 )alkyl)silyl and tri((C1-C 22 )alkoxy)silyl; and in, The support material is selected from the group consisting of silica, silica gel, glass, ceramic, polymer, cellulose and combinations thereof.

13. The oligonucleotide delivery agent according to claim 11, wherein The combination of the lipid portion and the delivery enhancing compound portion represented by Formula A' has a structure that can be obtained by subtracting at least one hydrogen atom or terminal group from the structure represented by Formula IV to Formula XIII:

14. The oligonucleotide delivery agent according to claim 11, wherein F, G, H and I are each carbon, m is 1 and n is 3, B is connected to G or H, each of P, Q, S and T is carbon, R 6 Connect to either Q or S; The protecting group R P Selected from benzyloxycarbonyl (Cbz), tert-butyldimethylsilyl (TBS), 4,4'-dimethoxytrityl (DMTr), tert-butyloxycarbonyl (Boc), benzyl (Bn) and benzyloxy (BnO); Each occurrence of C is selected from the group consisting of hydrogen, halogen atoms, hydroxyl groups, (C1-C 12 )alkyl, (C1-C 12 ) alkoxy, halogenated (C1-C 12 ) alkyl and halogenated (C1-C 12 ) alkoxy; Each occurrence of B is -(C1-C 22 )alkylene-OH, -OC(O)-(C1-C 16 )alkylene-C(O)NH2, -(C1-C 16 )alkylene-OC(O)-(C1-C 16 )alkylene-C(O)NH2, -OC(O)-(C1-C 16 )alkylene-C(O)OH, -(C1-C 16 )alkylene-OC(O)-(C1-C 16 )alkylene-C(O)OH, -C(O)-(C1-C 16 )alkylene-C(O)NH2, -(C1-C 16 )alkylene-C(O)-(C1-C 16 )alkylene-C(O)NH2, -C(O)-NH-(C1-C 16 ) alkylene-OH, -C(O)-NH-(C1-C 16 )alkylene-C(O)OH, -(C1-C 16 )alkylene-C(O)-NH-(C1-C 16 )alkylene-C(O)OH, -(C1-C 16 )alkylene-OP(-N(C1-C 16 Alkyl)2)-O-(C1-C 16 )alkylene-CN, -(C1-C 16 )alkylene-OP(-N(C1-C 16 Alkyl)2)-O-(C1-C 16 ) alkylene-OH, -(C1-C 16 )alkylene-OP(-N(C1-C 16 Alkyl)2)-O-(C1-C 16 ) alkylene-NH2, -(C1-C 16 )alkylene-OP(-N(C1-C 16 Alkyl)2)-O-(C1-C 16 )alkylene-C(O)OH, -C(O)-NH-(C1-C 16 )alkylene-OP(-N(C1-C 16 Alkyl)2)-O-(C1-C 16 ) alkylene-CN, wherein each of A1, A2 and A3 is either absent or a substituent independently selected from the following group: -H, -OH, a linear or branched -(C6-C 22 ) alkyl, linear or branched-(C2-C 22 )alkenyl, -(C1-C 22 ) alkylene-OH, -(C1-C 22 )Alkenylene-OH, -(C3-C 22 )cycloalkyl, -(C3-C 22 )cycloalkenyl, -(C1-C 22 )alkylene-(C3-C 22 )cycloalkyl, -(C1-C 22 )alkylene-R 1 、-(C1-C 22 )Alkenylene-R 1 、-(C1-C 22 )alkylene-OR 1 、-(C1-C 22 )Alkenylene-OR 1 、-(C1-C 22 )Alkylene-COOR 1 、-(C1-C 22 )Alkenylene-COOR 1 、-O-(C1-C 22 )alkyl, -(C6-C 22 ) alkylene-adamantyl, -(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkyl, -(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 ) alkylene-adamantyl, -(C1-C 22 )Alkylene-NR 2 -C(O)-(C1-C 22 ) alkylene-adamantyl, -(C1-C 22 )alkylene-(C1-C6 epoxyalkyl) (1-20) -NH-C(O)-(C1-C 22 )alkylene-adamantyl, -C(O)NH-R 1 、-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-CN, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-OH, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-NH2, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-C(O)OH, -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-CN, substituted or unsubstituted pyrrole, substituted or unsubstituted pyrroline, substituted or unsubstituted pyrrolidine, substituted or unsubstituted pyrazole, substituted or unsubstituted pyrazoline, substituted or unsubstituted pyrazolidine, substituted or unsubstituted imidazole, substituted or unsubstituted oxazole, substituted or unsubstituted thiazole, substituted or unsubstituted benzopyrrole, substituted or unsubstituted benzopyrroline, substituted or unsubstituted benzopyrrolidine, substituted or unsubstituted benzopyrazole, substituted or unsubstituted benzopyrazoline, substituted or unsubstituted benzopyrazolidine, substituted or unsubstituted benzimidazole, substituted or unsubstituted benzoxazole, substituted or unsubstituted benzothiazole, and a substituent represented by Formula III, wherein Y is selected from carbon, nitrogen, oxygen, and sulfur; and each of P, Q, S, and T is carbon; Where R 3 , R 4 and R 5 Each of the above is either absent or is a substituent independently selected from the following groups: -H, -OH, a linear or branched -(C6-C 22 ) alkyl, linear or branched-(C2-C 22 )alkenyl, -(C1-C 22 ) alkylene-OH, -(C3-C 22 )cycloalkyl, -(C3-C 22 )cycloalkenyl, -(C1-C 22 )alkylene-(C3-C 22 )cycloalkyl, -(C1-C 22 )alkylene-R 1 、-(C1-C 22 )alkylene-OR 1 、-(C1-C 22 )Alkylene-COOR 1 、-O-(C1-C 22 )alkyl, -(C6-C 22 ) alkylene-adamantyl, -(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 )alkyl, -(C1-C 22 )alkylene-NH-C(O)-(C1-C 22 ) alkylene-adamantyl, -(C1-C 22 )Alkylene-NR 2 -C(O)-(C1-C 22 ) alkylene-adamantyl, -(C1-C 22 )alkylene-(C1-C6 epoxyalkyl) (1-20) -NH-C(O)-(C1-C 22 )alkylene-adamantyl, -C(O)NH-R 1 、-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-CN, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-OH, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 ) alkylene-NH2, -(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-C(O)OH, -C(O)-NH-(C1-C 22 )alkylene-OP(-N(C1-C 22 Alkyl)2)-O-(C1-C 22 )alkylene-CN, substituted or unsubstituted pyrrole, substituted or unsubstituted pyrroline, substituted or unsubstituted pyrrolidine, substituted or unsubstituted pyrazole, substituted or unsubstituted pyrazoline, substituted or unsubstituted pyrazolidine, substituted or unsubstituted imidazole, substituted or unsubstituted oxazole, substituted or unsubstituted thiazole, substituted or unsubstituted benzopyrrole, substituted or unsubstituted benzopyrroline, substituted or unsubstituted benzopyrrolidine, substituted or unsubstituted benzopyrazole, substituted or unsubstituted benzopyrazoline, substituted or unsubstituted benzopyrazolidine, substituted or unsubstituted benzimidazole, substituted or unsubstituted benzoxazole and substituted or unsubstituted benzothiazole, Where R 7 is connected to any one of P, Q, S and T at each occurrence and is either absent or selected from hydrogen, a halogen atom, a hydroxyl group, (C1-C 20 )alkyl, (C1-C 20 ) alkoxy, halogenated (C1-C 20 ) alkyl and halogenated (C1-C 20 ) alkoxy; Where p is an integer of 0, 1, 2 or 3; Where R 6 Connected to any one of P, Q, S and T, and selected from -(C1-C 16 )alkylene-, -(C1-C 16 )alkylene-O-, -O-(C1-C 16 )alkylene-, -(C1-C 16 ) alkylene-NH-, -NH-(C1-C 16 )alkylene-, -C(O)-(C1-C 16 )alkylene-, -(C1-C 16 )alkylene-C(O)-, -C(O)-O-(C1-C 16 )alkylene-, -(C1-C16)alkylene-C(O)-O-, -C(O)-NH-(C1-C 16 )alkylene-, -C(O)-NH-(C1-C 16 )alkylene-C(O)-O-, -C(O)-NH-(C1-C 16 )alkylene-OC(O)-, -C(O)-NH-(C1-C 16 )alkylene-OC(O)-O-, -C(O)-NH-(C1-C 16 )alkylene-O-, -C(O)-N((C1-C 16 )alkyl)-(C1-C 16 )alkylene-, -C(O)-N((C1-C 16 )alkyl)-(C1-C 16 )alkylene-O-, -C(O)-NH-(C1-C 16 )alkylene-C(O)-NH-(C1-C 16 )alkylene-, -C(O)-NH-(C1-C 16 )alkylene-NH-C(O)-(C1-C 16 )alkylene-, -C(O)-NH-(C1-C 16 )alkylene-C(O)-NH-, -C(O)-N((C1-C 16 )alkyl)-(C1-C 16 )alkylene-C(O)-N((C1-C 16 )alkyl)-(C1-C 16 )alkylene-, -C(O)-N((C1-C 16 )alkyl)-(C1-C 16 )alkylene-C(O)-N((C1-C 16 )alkyl)-, -(C1-C 16 )alkylene-C(O)-NH-(C1-C 16 )alkylene-C(O)-NH-(C1-C 16 )alkylene-, -(C1-C 16 )alkylene-C(O)-NH-(C1-C 16 )alkylene-C(O)-NH-, -NH-C(O)-(C1-C 16 )alkylene-, -NH-C(O)-(C1-C 16 )alkylene-C(O)-O-, -NH-C(O)-(C1-C 16 )alkylene-C(O)-, -NH-C(O)-(C1-C 16 )alkylene-O-, -N((C1-C 16 )alkyl)-C(O)-(C1-C 16 )alkylene-, -N((C1-C 16 )alkyl)-C(O)-(C1-C 16 )alkylene-O-, -NH-C(O)-(C1-C 16 )alkylene-NH-C(O)-(C1-C 16 )alkylene-, -NH-C(O)-(C1-C 16 )alkylene-NH-C(O)-, -N((C1-C 16 )alkyl)-C(O)-(C1-C 16 )alkylene-N((C1-C 16 )alkyl)-C(O)-(C1-C 16 )alkylene-, -N((C1-C 16 )alkyl)-C(O)-(C1-C 16 )alkylene-N((C1-C 16 )alkyl)-C(O)-, -(C1-C 16 )alkylene-NH-C(O)-(C1-C 16 )alkylene-NH-C(O)-(C1-C 16 )alkylene-, -(C1-C 16 )alkylene-NH-C(O)-(C1-C 16 )alkylene-NH-C(O)-; and The conditions are: A1, A2, A3, R 3 , R 4 and R 5 At least one of them is C6-C 22 A straight chain alkyl group, preferably a straight chain hexadecyl group.

15. The oligonucleotide delivery agent according to claim 7, wherein the delivery enhancing compound portion comprises a structure selected from the group consisting of: -(C1-C 16 )alkylene-NH-C(O)-, -(C1-C 16 )alkylene-C(O)-NH-, -(C1-C 16 )alkylene-O-(C1-C 16 )alkylene-NH-C(O)-, -(C1-C 16 )alkylene-O-(C1-C 16 )alkylene-O-(C1-C 16 )alkylene-NH-C(O)-, -(C3-C 12 )cycloalkylene-NH-C(O)-, -(C1-C 16 )alkylene-(C3-C 12 ) cycloalkylene-NH-C(O)-, -(C1-C 16 )alkylene-(C3-C 12 )cycloalkylene-(C1-C 16 )alkylene-NH-C(O)-, -(C1-C 16 )alkylene-O-(C1-C 16 )alkylene-C(O)-NH-, -(C1-C 16 )alkylene-O-(C1-C 16 )alkylene-O-(C1-C 16 )alkylene-C(O)-NH-, -(C3-C 12 )cycloalkylene-C(O)-NH-, -(C1-C 16 )alkylene-(C3-C 12 )cycloalkylene-C(O)-NH-, -(C1-C 16 )alkylene-(C3-C 12 )cycloalkylene-(C1-C 16 )alkylene-C(O)-NH-, and / or in, The delivery enhancing compound moiety is coupled to the single stranded oligonucleotide directly via a covalent bond or is linked to the single stranded oligonucleotide via a linker moiety having the following structure: where Y 1 is oxygen or sulfur, an asterisk (*) refers to the position where the linking moiety is attached to the single-stranded oligonucleotide, and a double asterisk (**) refers to the position where the linking moiety is attached to the delivery enhancing compound moiety.

16. The oligonucleotide delivery agent according to any one of claims 1 to 6, wherein At least one hydrogen atom contained in the lipid moiety is substituted with a deuterium atom.

17. The oligonucleotide delivery agent according to any one of claims 7 to 15, wherein at least one hydrogen atom contained in the lipid portion, the delivery enhancing compound portion and / or the single-stranded oligonucleotide is substituted with a deuterium atom.

18. The oligonucleotide delivery agent according to claim 7, wherein The combination of the lipid portion and the delivery enhancing compound portion represented by Formula A' has a structure that can be obtained by subtracting at least one hydrogen atom or terminal group from any of Compounds C1 to C71: in Indicates supporting material.

19. The oligonucleotide delivery agent according to claim 7, wherein the oligonucleotide is linked to the delivery enhancing compound portion via at least one linking moiety selected from the group consisting of a direct bond, -O-, -S-, -C(O)-, -NH-, -N((C1-C 12 )alkyl)-, -N((C1-C 12 )Alkyl)-C(O)-O-, -OC(O)-, -C(O)-O-, -OC(O)-O-, -C(O)-NH-, -OP(O)2O-, -P(O)(O - )O-, -OP(O)O-, -OP(O)(S)O-, -OP(O)(S - )O-, -OS(O)2-O-, -S(O)2-O-, -S(O)-O-, -(C1-C 22 )alkylene-, -(C1-C 22 ) alkylene-NH-, -NH-(C1-C 22 )alkylene-, -(C1-C 22 )alkylene-NH-C(O)-, -(C1-C 22 )alkylene-C(O)-, -(C1-C 22 )alkylene-C(O)-O-, -C(O)-(C1-C 22 )alkylene-, -NH-C(O)-(C1-C 22 )alkylene-, -C(O)-NH-(C1-C 22 )alkylene-, -C(O)-(C1-C 22 ) alkylene-NH-, -NH-(C1-C 22 )alkylene-C(O)-, -C(O)-(C1-C 22 )alkylene-C(O)-, -NH-(C1-C 22 )alkylene-NH-, -C(O)-(C1-C 22 )alkylene-C(O)O-, -OC(O)-(C1-C 22 )alkylene-C(O)-O-, -C(O)-O-(C1-C 22 )alkylene-OC(O)-, -C(O)-(C1-C 22 ) alkylene-NH-C(O)-, -NH-C(O)-(C1-C 22 )alkylene-C(O)-, -NH-C(O)-(C1-C 22 )alkylene-C(O)-NH-, -C(O)-NH-(C1-C 22 )alkylene-NH-C(O)-, -(C1-C 22 )alkylene-OP(O)2O-, -(C1-C 22 )alkylene-OP(O)(O - )O-、-(C1-C 22 )alkylene-OP(O)(O - )O-(C1-C 22 )alkylene-, -(C1-C 22 )alkylene-OP(O)O-, -(C1-C 22 )alkylene-OP(O)(S)O-, -(C1-C 22 )alkylene-OS(O)2-O-, -(C1-C 22 )alkylene-S(O)2-O-, -(C1-C 22 )alkylene-S(O)-O-, -OP(O)2-O-(C1-C 22 )alkylene-OP(O)2O-, -OP(O)-O-(C1-C 22 )alkylene-OP(O)O-, -OP(O)(S)O-(C1-C 22 )alkylene-OP(O)(S)O-, -OS(O)2-O-(C1-C 22 )alkylene-OS(O)2-O-, -S(O)2-O-(C1-C 22 )alkylene-S(O)2-O-and-OS(O)-(C1-C 22 )alkylene-S(O)-O-.

20. The oligonucleotide delivery agent according to claim 19, which has a structure represented by any one of formulas AI to AVII: in, L represents the connecting part, represents a combination of a lipid moiety and a delivery enhancing compound moiety represented by formula A', represents a single-stranded oligonucleotide in the 5′ to 3′ direction or in the 3′ to 5′ direction, and a, b and c are each independently an integer from 1 to 50.

21. The oligonucleotide delivery agent according to claim 19, which has a structure represented by any one of formulas O1 to O20: wherein Y represents oxygen or sulfur.

22. The oligonucleotide delivery agent according to any one of claims 1 to 21, wherein The oligonucleotide targets a gene comprising at least a portion of an oligonucleotide sequence selected from the group consisting of SMN2, SCN9A, Scn9a, and DMD.

23. The oligonucleotide delivery agent according to any one of claims 1 to 22, wherein The oligonucleotide comprises at least a portion of an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 142-260 and SEQ ID NOs: 443-492; and / or wherein the oligonucleotide comprises at least a portion of an oligonucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 261-379, SEQ ID NO: 388 and SEQ ID NO: 390; and / or wherein the oligonucleotide is complementary to at least a portion of a continuous target sequence comprising an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 23-141 and SEQ ID NOs: 393-442.

24. The oligonucleotide delivery agent according to any one of claims 1 to 23, wherein The oligonucleotide comprises at least a portion of an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 2-21, SEQ ID NO: 389, SEQ ID NO: 391, and SEQ ID NOs: 493-542.

25. A pharmaceutical composition comprising: a) an oligonucleotide delivery agent according to any one of claims 1 to 24; and b) optionally, one or more ingredients selected from the group consisting of a pharmaceutically acceptable carrier, excipient, solvent, diluent, stabilizer, dispersant, buffer, extender, preservative, and combinations thereof.

26. A method for regulating the expression of a target gene in a subject, the method comprising the step of administering the pharmaceutical composition according to claim 25 to the subject.

27. The method according to claim 26, wherein: The pharmaceutical composition is administered to at least one of the cardiac muscle, skeletal muscle, smooth muscle, and liver of the subject.

28. The method according to claim 26, wherein: The pharmaceutical composition increases the expression of the target gene.

29. The method according to claim 26, wherein: The pharmaceutical composition reduces the expression of the target gene.

30. The method of any one of claims 26 to 29, wherein: The subject is a mammal.

31. The method according to claim 30, wherein: The mammal is a rodent.

32. The method according to claim 31, wherein: The rodent is a mouse or a rat.

33. The method of claim 32, wherein the mammal is a non-human primate.

34. The method of claim 30, wherein the mammal is a human.

35. The method according to any one of claims 26 to 29, wherein: The target gene is associated with a disease or disorder.

36. The method according to claim 35, wherein: The target gene is associated with a disease or disorder of the central nervous system, eye, spleen, muscle, heart, kidney, or liver.

37. The method according to any one of claims 26 to 36, wherein: The oligonucleotide targets a gene comprising at least a portion of an oligonucleotide sequence selected from the group consisting of SMN2, SCN9A, Scn9a, and DMD.

38. The method according to any one of claims 26 to 37, wherein: The oligonucleotide comprises at least a portion of an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 142-260 and SEQ ID NOs: 443-492; and / or wherein the oligonucleotide comprises at least a portion of an oligonucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 261-379, SEQ ID NO: 388 and SEQ ID NO: 390; and / or wherein the oligonucleotide is complementary to at least a portion of a continuous target sequence comprising an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 23-141 and SEQ ID NOs: 393-442.

39. The method according to any one of claims 26 to 38, wherein: The oligonucleotide comprises at least a portion of an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 2-21, SEQ ID NO: 389, SEQ ID NO: 391, and SEQ ID NOs: 493-542.

40. A method for regulating expression of a target gene, the method comprising contacting a cell with the pharmaceutical composition of claim 22.

41. The method according to claim 40, wherein: The cells were from skeletal muscle, cardiac muscle, smooth muscle or liver.

42. The method according to claim 40, wherein: The pharmaceutical composition increases the expression of the target gene.

43. The method according to claim 40, wherein: The pharmaceutical composition reduces the expression of the target gene.

44. The method of claim 41, wherein the cell is a mammalian cell.

45. The method according to claim 44, wherein: The mammalian cell is a mouse cell or a rat cell.

46. ​​The method of claim 44, wherein the mammal is a non-human primate.

47. The method of claim 44, wherein the mammalian cell is a human cell.

48. The method according to any one of claims 40 to 47, wherein: The target gene is associated with a disease or disorder.

49. The method according to claim 48, wherein: The target gene is associated with a disease or disorder of the central nervous system, eye, spleen, muscle, heart, kidney, or liver.

50. The method according to any one of claims 40 to 49, wherein: The oligonucleotide targets a gene comprising at least a portion of an oligonucleotide sequence selected from the group consisting of SMN2, SCN9A, Scn9a, and DMD.

51. The method according to any one of claims 40 to 50, wherein: The oligonucleotide comprises at least a portion of an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 142-260 and SEQ ID NOs: 443-492; and / or wherein the oligonucleotide comprises at least a portion of an oligonucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 261-379, SEQ ID NO: 388 and SEQ ID NO: 390; and / or wherein the oligonucleotide is complementary to at least a portion of a continuous target sequence comprising an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 23-141 and SEQ ID NOs: 393-442.

52. The method according to any one of claims 40 to 51, wherein: The oligonucleotide comprises at least a portion of an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 2-21, SEQ ID NO: 389, SEQ ID NO: 391, and SEQ ID NOs: 493-542.

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