Conjugates of double-stranded RNAi analogues containing nucleotide analogues

Abstract

The invention relates to conjugates of double-stranded RNAi (Ribonucleic Acid Interference) analogues containing nucleotide analogues, and particularly discloses a sequence composition and a ligand structure of the conjugates of the double-stranded RNAi analogues.

Description

A class of conjugates of double-stranded RNAi analogs containing nucleotide analogs

[0001] This application claims priority to:

[0002] CN202210477929.8, April 29, 2022;

[0003] CN202211106626.1, September 9, 2022. Technical Field

[0004] The present invention relates to a class of conjugates of double-stranded RNAi analogs containing nucleotide analogs, and in particular to the conjugate or a pharmaceutically acceptable salt thereof and use thereof in medicines for treating related diseases. Background Art

[0005] The RNA interference (RNAi) mechanism has garnered significant attention since its discovery in 1998. This mechanism achieves its intended pharmacological effect by controlling the function of the corresponding target by downregulating target mRNA levels. RNAi drugs are typically double-stranded ribonucleic acids. After entering target cells via delivery methods, they bind to proteins such as Ago2 within the cell, forming an RNA-induced silencing complex (RISC). This complex binds to the target mRNA in the target cell and hydrolyzes and cleaves it, depriving the target mRNA of its ability to translate protein, thereby downregulating the target protein and inhibiting target function. In recent years, several RNAi drugs have been approved for marketing, completing feasibility studies for human drug application. Drugs that achieve their pharmacological effects through this mechanism are called RNAi drugs.

[0006] The renin-angiotensin-aldosterone (RAAS) system plays an important role in blood pressure regulation. Angiotensinogen (AGT) protein is the only known upstream substance of the RAAS system. AGT protein in the circulation system is mainly synthesized in the liver. Renin secreted by the kidney converts AGT in the circulation system into angiotensin I (Ang I), and angiotensin-converting enzyme (ACE) then converts the inactive Ang I into biologically active angiotensin II (Ang II). Ang II acts on its receptors, causing vasoconstriction and promoting aldosterone release, leading to increased blood pressure. The regulatory effect of the RAAS system on blood pressure has been deeply verified in clinical practice. Renin inhibitors (aliskiren), angiotensin-converting inhibitors (prils), and angiotensin II receptor antagonists (sartans) that target this pathway are already common medications for the clinical treatment of hypertension.

[0007] Hypertension is a clinical syndrome characterized by elevated systemic arterial pressure. Sustained elevated and fluctuating blood pressure are major risk factors for cardiovascular disease, leading to damage to target organs such as the heart, kidneys, brain, and blood vessels.

[0008] There are a huge number of people suffering from hypertension worldwide. The first comprehensive analysis report on global hypertension trends, hosted by Imperial College London and the World Health Organization and published in The Lancet on August 25, 2021, is the largest hypertension research report in the world to date. (Article source: 10.1016 / S0140-6736(21)01330-1.) The report shows that in the past 30 years, the number of adults aged 30-79 with hypertension has increased from 650 million to 1.28 billion. The study also found that more than half of hypertensive patients (53% women and 62% men, a total of 720 million people) do not receive necessary treatment. The harm caused by hypertension is enormous, and cardiovascular disease (CVD) caused by hypertension causes approximately 8.5 million deaths worldwide each year. Therefore, there is a serious unmet clinical need for effective blood pressure control.

[0009] Clinically, the main drugs used to treat hypertension are small molecules. Due to their rapid absorption and metabolism, short half-life, and insufficient duration of action, small molecules can easily cause nocturnal blood pressure fluctuations and poor 24-hour blood pressure control. Furthermore, small molecules carry a high medication burden and require daily administration, making missed doses common, leading to elevated blood pressure and target organ damage. Therefore, there is an urgent need for a long-acting antihypertensive drug to address this unmet clinical need.

[0010] Summary of the Invention

[0011] The first aspect of the present invention provides a conjugate of a double-stranded RNAi analog or a pharmaceutically acceptable salt thereof, wherein the double-stranded RNAi analog comprises a sense strand and an antisense strand capable of forming a double-stranded region,

[0012] The sense strand is 5'-as,us,Cf,Cf,a,Cf,Af,Af,u,g,a,g,a,g,u,a,c,a-3' (SEQ ID NO: 1),

[0013] The antisense strand is 3'-a,gs,cs,a,g,u,a,Gf,g,Uf,g,u,u,a,c,u,c,Z,c,a,u,Gfs,us,A,C,A,C,U,C-5' (SEQ ID NO: 2),

[0014] in,

[0015] Z is

[0016] The 5' end and / or 3' end of the sense strand and / or antisense strand is conjugated to a ligand, and the ligand is one or more GalNAc derivatives attached using a bivalent, trivalent or tetravalent branched linkage.

[0017] In some embodiments of the first aspect of the invention, the ligand is one or more GalNAc derivatives attached using a bivalent or trivalent branched linkage.

[0018] In some embodiments of the first aspect of the invention, the ligand is one or more GalNAc derivatives attached using a trivalent branched linkage.

[0019] In some versions of the first aspect of the invention, the ligand is a GalNAc derivative attached using a trivalent branched linkage.

[0020] The second aspect of the present invention provides a conjugate of a double-stranded RNAi analog or a pharmaceutically acceptable salt thereof, wherein the double-stranded RNAi analog comprises a sense strand and an antisense strand capable of forming a double-stranded region,

[0021] The sense strand is 5'-as,us,Cf,Cf,a,Cf,Af,Af,u,g,a,g,a,Gf,u,a,c,a-3' (SEQ ID NO: 3),

[0022] The antisense strand is 3'-a,gs,cs,a,g,u,a,Gf,g,Uf,g,u,u,a,c,u,c,Z,c,a,u,Gfs,us,A,C,A,C,U,C-5' (SEQ ID NO: 2),

[0023] in,

[0024] Z is

[0025] The 5' end and / or 3' end of the sense strand and / or antisense strand is conjugated to a ligand, and the ligand is one or more GalNAc derivatives attached using a bivalent, trivalent, or tetravalent branched linkage.

[0026] In some embodiments of the second aspect of the invention, the ligand is one or more GalNAc derivatives attached using a bivalent or trivalent branched linkage.

[0027] In some embodiments of the second aspect of the invention, the ligand is one or more GalNAc derivatives attached using a trivalent branched linkage.

[0028] In some versions of the second aspect of the invention, the ligand is a GalNAc derivative attached using a trivalent branched linkage.

[0029] The third aspect of the present invention provides a conjugate of a double-stranded RNAi analog or a pharmaceutically acceptable salt thereof, wherein the double-stranded RNAi analog comprises a sense strand and an antisense strand capable of forming a double-stranded region,

[0030] The sense strand is 5'-as,us,Cf,Cf,a,Cf,Af,Af,u,g,a,Gf,a,g,u,a,c,a-3' (SEQ ID NO: 4),

[0031] The antisense strand is 3'-a,gs,cs,a,g,u,a,Gf,g,Uf,g,u,u,a,c,u,c,Z,c,a,u,Gfs,us,A,C,A,C,U,C-5' (SEQ ID NO: 2),

[0032] in,

[0033] Z is

[0034] The 5' end and / or 3' end of the sense strand and / or antisense strand is conjugated to a ligand, and the ligand is one or more GalNAc derivatives attached using a bivalent, trivalent, or tetravalent branched linkage.

[0035] In some embodiments of the third aspect of the invention, the ligand is one or more GalNAc derivatives attached using a bivalent or trivalent branched linkage.

[0036] In some embodiments of the third aspect of the invention, the ligand is one or more GalNAc derivatives attached using a trivalent branched linkage.

[0037] In some versions of the third aspect of the invention, the ligand is a GalNAc derivative attached using a trivalent branched linkage.

[0038] In some technical solutions of the present invention, the ligand is conjugated to the 3' end of the sense strand, and other variables are as defined in the present invention.

[0039] In some technical solutions of the present invention, the ligand is conjugated to the 5' end of the sense strand, and other variables are as defined in the present invention.

[0040] In some technical embodiments of the present invention, the ligand is conjugated to the 3' end of the antisense strand, and other variables are as defined in the present invention.

[0041] In some embodiments of the present invention, the ligand is conjugated to the 5' end of the antisense strand, and the other variables are as defined in the present invention.

[0042] In some technical solutions of the first, second and / or third aspects of the present invention, the ligand is conjugated to the 3' end of the sense strand, and other variables are as defined in the present invention.

[0043] In some technical solutions of the first, second and / or third aspects of the present invention, the ligand is conjugated to the 5' end of the sense strand, and other variables are as defined in the present invention.

[0044] In some technical solutions of the first, second and / or third aspects of the present invention, the ligand is conjugated to the 3' end of the antisense strand, and other variables are as defined in the present invention.

[0045] In some technical solutions of the first, second and / or third aspects of the present invention, the ligand is conjugated to the 5' end of the antisense strand, and other variables are as defined in the present invention.

[0046] The fourth aspect of the present invention provides the following conjugate or a pharmaceutically acceptable salt thereof,

[0047] Conjugate 1:

[0048] The sense strand is 5'-as,us,Cf,Cf,a,Cf,Af,Af,u,g,a,g,a,g,u,a,c,a,D01 (SEQ ID NO:5)

[0049] The antisense strand is 3'-a,gs,cs,a,g,u,a,Gf,g,Uf,g,u,u,a,c,u,c,Z,c,a,u,Gfs,us,A,C,A,C,U,C-5' (SEQ ID NO: 2);

[0050] Conjugate 2:

[0051] The sense strand is 5'-as,us,Cf,Cf,a,Cf,Af,Af,u,g,a,g,a,Gf,u,a,c,a,D01 (SEQ ID NO: 6)

[0052] The antisense strand is 3'-a,gs,cs,a,g,u,a,Gf,g,Uf,g,u,u,a,c,u,c,Z,c,a,u,Gfs,us,A,C,A,C,U,C-5' (SEQ ID NO: 2);

[0053] Conjugate 3:

[0054] The sense strand is 5'-as,us,Cf,Cf,a,Cf,Af,Af,u,g,a,Gf,a,g,u,a,c,a,D01 (SEQ ID NO:7)

[0055] The antisense strand is 3'-a,gs,cs,a,g,u,a,Gf,g,Uf,g,u,u,a,c,u,c,Z,c,a,u,Gfs,us,A,C,A,C,U,C-5' (SEQ ID NO: 2);

[0056] The structure of D01 is:

[0057] D01 and a are connected via a phosphodiester bond.

[0058] In a fifth aspect, the present invention provides a pharmaceutical composition comprising an effective dose of the conjugate defined in any of the above technical solutions or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0059] In some embodiments of the fifth aspect of the present invention, the pharmaceutical composition comprises an effective dose of a conjugate of any one, two, three, four or five double-stranded RNAi analogs described above or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0060] In some embodiments of the fifth aspect of the present invention, the pharmaceutical composition comprises an effective dose of any one of the above-mentioned double-stranded RNAi analog conjugates or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0061] In a sixth aspect, the present invention provides use of any of the above-mentioned conjugates of double-stranded RNAi analogs or pharmaceutically acceptable salts thereof in the preparation of a drug for treating AGT-related diseases.

[0062] In some embodiments of the sixth aspect of the present invention, the AGT-related disease is hypertension.

[0063] In some embodiments of the sixth aspect of the present invention, the AGT-related disease is a cardiovascular disease.

[0064] In a seventh aspect, the present invention provides a method for treating an AGT-related disease in a subject in need thereof, comprising providing the subject with an effective dose of the conjugate defined in any of the above technical solutions or a pharmaceutically acceptable salt or pharmaceutical composition thereof.

[0065] In the event of any discrepancy between the information in the sequence listing of the present invention and the description, the description shall prevail.

[0066] The present invention also provides the following testing method:

[0067] Test method 1: PHH cell free uptake experiment

[0068] 1. Experimental principle:

[0069] The test sample is incubated with human primary hepatocytes to evaluate the degree of downregulation of AGT mRNA by the test sample.

[0070] 2. Experimental Materials:

[0071] Primary human hepatocytes (PHH), 96 Kit(12)(QIAGEN-74182), FastKing RT Kit(With gDNase)(Tiangen-KR116-02), TaqMan Gene Expression Assay(GAPDH, Thermo, Assay ID-Hs02786624_g1), TaqMan Gene Expression Assay(AGT, Thermo, Assay ID-Hs01586213_m1).

[0072] 3. Experimental methods:

[0073] The conjugate of the present invention was diluted to 10 times the concentration to be tested with PBS solution. 10 μL of siRNA was transferred to a 96-well plate. PHH cells were thawed and transplanted into a 96-well plate to a final cell density of 5.4×10 5 The conjugate of the present invention was tested at 10 concentration points, with the highest concentration being 500 nM, and 4-fold dilution.

[0074] The cells were incubated at 37°C in 5% CO2 for 48 hours, and the cell status was examined under a microscope.

[0075] After the incubation is complete, the cells are lysed and the lysate is obtained. All RNA was extracted using the QIAGEN-96 Kit (QIAGEN-74182), and reverse transcribed using the FastKing RT Kit (with gDNase) (Tiangen-KR116-02) to obtain cDNA. AGT cDNA was detected by qPCR.

[0076] 4. Experimental results: The conjugate of the present invention can significantly downregulate the AGT mRNA level in PHH cells.

[0077] Test Method 2: Mouse hAGT HDI Model

[0078] 1. Experimental principle:

[0079] The mouse model of high-pressure tail vein injection of pcDNA-CMV-AGT plasmid was used to evaluate the in vivo targeting and inhibitory effect of the test sample on the target gene.

[0080] 2. Experimental materials: pcDNA-CMV-AGT plasmid, BALB / c female mice, DPBS (Dulbecco's phosphate buffered saline), the conjugate of the present invention or a pharmaceutically acceptable salt thereof.

[0081] 3. Experimental methods:

[0082] Order 6-8 week old BALB / c female mice and acclimate them to quarantine for one week after arrival at the animal house.

[0083] On day 0, mice were randomly divided into groups according to body weight data, with 4 mice in each group. After grouping, all mice were administered subcutaneously, with a single dose of 10 mL / kg. Group 1 mice were given DPBS; Group 2 mice were given the conjugate of the present invention or a pharmaceutically acceptable salt thereof, 3 mg / kg.

[0084] On the third day after administration, all mice were injected with pcDNA-CMV-AGT plasmid at a volume of 8% of their body weight (injection volume (mL) = mouse body weight (g) × 8%) via the tail vein within 5 seconds. The mass of the injected plasmid for each mouse was 10 μg.

[0085] On day 4 after administration, mice in all groups were euthanized by CO₂ inhalation, and two liver samples were collected from each mouse. The liver samples were treated with RNAlater overnight at 4°C, then the RNAlater was removed and stored at -80°C for analysis of AGT gene expression levels.

[0086] 4. Experimental conclusions:

[0087] The conjugate of the present invention has an excellent down-regulating effect on the level of AGT mRNA in mouse liver.

[0088] Test Method 3: In vivo PD test in non-human primates, cynomolgus monkeys

[0089] 1. Experimental principle:

[0090] The non-human primate cynomolgus macaque model, which is highly homologous to the human AGT gene, is used to observe the degree of downregulation of cynomolgus macaque plasma AGT protein by the test sample.

[0091] 2. Experimental materials: cynomolgus monkeys, 1x PBS (phosphate buffered saline), AD-85481 (US2021095290), and the conjugate of the present invention.

[0092] 3. Experimental methods:

[0093] Order 10 cynomolgus macaques weighing 2-3 kg and acclimate them to quarantine for one week upon arrival at the animal house.

[0094] On day -6, the macaques were randomly divided into groups according to their body weight, with 2 macaques in each group. After grouping, the plasma AGT protein concentration of all macaques was tested as the experimental data baseline for each group.

[0095] Dosing was performed on day 1, and blood was collected on days 1, 2, 4, 8, 15, 22, 29, 32, 35, 43, 57, and 71 after administration to measure plasma AGT protein concentration in each cynomolgus monkey. The plasma AGT protein concentration after administration was compared with the AGT protein concentration on day -6 to determine the relative reduction rate of AGT protein in each group of animals.

[0096] 4. Experimental results: The conjugate of the present invention can significantly downregulate the AGT protein concentration in cynomolgus monkey plasma.

[0097] Test Method 4: In vitro RNA sequencing studies

[0098] 1. Experimental Introduction:

[0099] The transcriptome refers to the sum of all RNA transcribed by a specific tissue or cell at a specific time or state, primarily including mRNA and non-coding RNA. Transcriptome sequencing, based on the Illumina sequencing platform, studies all mRNA transcribed by a specific tissue or cell at a specific time. It is fundamental to the study of gene function and structure and plays a crucial role in understanding biological development and disease. With the advancement of gene sequencing technology and the reduction in sequencing costs, RNA-seq has become a leading method for transcriptome research due to its high throughput, high sensitivity, and wide range of applications. The RNA-seq technical process primarily consists of two parts: library construction and sequencing, and bioinformatics analysis.

[0100] 2. RNA extraction and detection:

[0101] RNA is extracted from tissues or cells using standard extraction methods, and then the RNA samples are strictly quality controlled, mainly through the Agilent 2100 bioanalyzer: accurately detecting RNA integrity.

[0102] 3. Library construction and quality control:

[0103] There are two main methods for obtaining mRNA: 1. Taking advantage of the structural characteristic of most eukaryotic mRNAs with polyA tails, mRNA with polyA tails can be enriched using Oligo(dT) magnetic beads. 2. mRNA can be obtained by removing ribosomal RNA from total RNA. The resulting mRNA is then randomly fragmented with divalent cations in NEB Fragmentation Buffer and constructed using either the NEB standard library construction method or the strand-specific library construction method.

[0104] NEB general library construction: Using fragmented mRNA as a template and random oligonucleotides as primers, the first cDNA chain is synthesized in the M-MuLV reverse transcriptase system, followed by RNaseH degradation of the RNA chain, and the second cDNA chain is synthesized using dNTPs as raw materials in the DNA polymerase I system. The purified double-stranded cDNA is end-repaired, A-tailed, and ligated to sequencing adapters. AMPure XP beads are used to screen cDNA of about 250-300bp, PCR amplification is performed, and the PCR product is purified again using AMPure XP beads to finally obtain the library. The kit used for library construction is Ultra TM RNA Library Prep Kit for

[0105] Chain-specific library construction: The method for reverse transcription synthesis of the first chain of cDNA is the same as the NEB general library construction method. The difference is that when synthesizing the second chain, dTTP in dNTPs is replaced by dUTP. After that, the cDNA end repair, A tail addition, sequencing adapter connection and length screening are also performed. Then, the second chain of cDNA containing U is degraded with USER enzyme and PCR amplification is performed to obtain the library. Chain-specific libraries have many advantages, such as obtaining more effective information with the same amount of data; obtaining more accurate gene quantification, positioning and annotation information; and providing the expression level of antisense transcripts and a single exon in each isoform. The kit used for library construction is Ultra TM Directional RNA Library Prep Kit for

[0106] Note: Sequencing adapters consist of three parts: P5 / P7, index, and Rd1 / Rd2SP. P5 / P7 are the PCR amplification primers and the primer binding sites on the flow cell. The index provides information to distinguish different libraries. Rd1 / Rd2SP, or read1 / read2sequence primers, is the sequencing primer binding region. Theoretically, sequencing proceeds backward from Rd1 / Rd2SP.

[0107] After the library is constructed, a Qubit 2.0 Fluorometer is used for preliminary quantification, and the library is diluted to 1.5 ng / ul. The insert size of the library is then detected using an Agilent 2100 bioanalyzer. Once the insert size meets expectations, qRT-PCR is used to accurately quantify the effective concentration of the library (the effective concentration of the library is greater than 2 nM) to ensure library quality.

[0108] 4. Sequencing:

[0109] After passing the library inspection, different libraries are pooled according to effective concentration and target data volume requirements before Illumina sequencing. The basic principle of sequencing is sequencing by synthesis. Four fluorescently labeled dNTPs, DNA polymerase, and adapter primers are added to the sequencing flow cell for amplification. As each sequencing cluster extends the complementary chain, each added fluorescently labeled dNTP releases corresponding fluorescence. The sequencer captures the fluorescent signal and converts it into sequencing peaks through computer software, thereby obtaining the sequence information of the fragment to be tested.

[0110] 5. Experimental results: The conjugate of the present invention significantly down-regulated the AGT gene in RNA-Sequecing studies.

[0111] Test method 5: Agonistic activity test of the conjugate on hTLR3, hTLR7, and hTLR8 cells

[0112] 1. Experimental Materials

[0113] Table 1 Experimental materials

[0114] 2. Instruments

[0115] The main instruments used in this study are the multifunctional microplate reader Flexstation III (Molecular Device) and Echo555 (Labcyte).

[0116] 3. Experimental Procedure

[0117] 1) Dilute the compound to 20 times the final concentration and mix with RNAiMAX-OPTI MEM (RNAiMAX:OPTI MEM=3:47)

[0118] Mix 1:1 and incubate at room temperature for 15 minutes. (“Compound” refers to the conjugate of the present invention used in the test.)

[0119] 2) Take 10 μL of the incubated compound and add it to the corresponding cell plate, for a total of 10 concentrations, with duplicate wells for each concentration.

[0120] 3) Add 10 μL of PBS to each negative control well, 10 μL of 20 μg / mL Poly(I:C)HMW to each hTLR3 positive control well, and 10 μL of 10 μg / mL R848 to each hTLR7 and hTLR8 positive control well. Add 10 μL of 0.5 μM BLOCK-iT to each of the two transfection positive control wells.TM Alexa Red Fluorescent Control.

[0121] 4) 90 μL of cells were seeded into a 96-well plate to which compounds had been added, at a density of 50,000 cells per well.

[0122] 5) The compound and cells were incubated in a 37°C, 5% CO2 incubator for 24 hours.

[0123] 6) Observe the transfection effect of the positive control wells using a fluorescence microscope and take photos.

[0124] 7) Compound activity detection: Take 20 μL of cell supernatant from each well and add 180 μL of QUANTI-Blue TM After incubation at 37° C. for 1 hour in the test plate containing the reagent, the absorbance at 650 nm (OD650) was measured using a multifunctional microplate reader Flexstation III.

[0125] 8) Cell viability assay: The assay was performed according to the Celltiter-Glo instructions, and the chemiluminescence signal (RLU) was detected using a multifunctional microplate reader Flexstation III.

[0126] 3. Data Analysis

[0127] Compound activity: OD650 values ​​were analyzed using GraphPad Prism software, and the compound dose-effect curve was fitted to calculate the EC of the compound. 50 Cell viability assay: The formula for calculating cell viability % is as follows. Cell viability % values ​​were analyzed using GraphPad Prism software, and the compound dose-effect curve was fitted to calculate the CC of the compound on the cells. 50 value.

[0128] Cell activity % = RLU compound / RLU DMSO control * 100%

[0129] 4. Experimental results:

[0130] The conjugate of the present invention showed better results in this test.

[0131] Technical Effects

[0132] The conjugate of the present invention has low off-target risk and low immunogenicity risk, and can significantly downregulate the AGT mRNA level in PHH cells, significantly downregulate the AGT mRNA level in mouse liver in the mouse hAGT HDI model, and significantly downregulate the AGT protein concentration in the plasma of non-human primates, crab-eating macaques.

[0133] Definition and Description

[0134] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. The absence of a specific definition for a particular term or phrase should not be construed as undefined or ambiguous, but rather should be understood as meaning understood by one of ordinary skill in the art. When a trade name appears herein, it is intended to refer to the corresponding commercial product or its active ingredient.

[0135] Unless otherwise stated, the terms "comprises, includes and contains" or equivalents are open-ended expressions and mean that in addition to the listed elements, components or steps, other unspecified elements, components or steps may also be included.

[0136] Double-stranded RNAi analogs refer to complexes of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands having "sense" and "antisense" orientations relative to the target RNA. In the present disclosure, "complementary" has the meaning known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand are paired with bases on the other strand in a complementary manner. The purine base adenine (A) always pairs with the pyrimidine base uracil (U); the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair includes a purine and a pyrimidine. When adenine on one strand always pairs with uracil on the other strand, and guanine always pairs with cytosine, the two strands are considered to be complementary to each other, and the sequence of that strand can be inferred from the sequence of its complementary strand.

[0137] The "modification" of the nucleotides described in the present disclosure includes, but is not limited to, methoxy modification, fluoro modification, (E)-vinyl phosphate modification, phosphorothioate linkage, or replacement of the nucleotide with a monomer of a GNA (glycerol nucleic acid) nucleotide, wherein the GNA includes GNA-A, GNA-T, GNA-C, GNA-G, and GNA-U. The sequences described in the present disclosure may include those listed in the "further modified sequences" in Table 2 below.

[0138] The fluorinated modified nucleotides disclosed herein refer to nucleotides in which the hydroxyl group at the 2' position of the ribose group is replaced by fluorine, and the methoxy-modified nucleotides refer to nucleotides in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.

[0139] The phosphorothioate linkage methods described in the present disclosure all indicate that reading starts from the 5' end, and the previous nucleotide and the next nucleotide are connected by a phosphorothioate bond: such as 5'-as,us,Cf...-3' of the sense chain, wherein "as,us" indicates that the nucleotide residue a and the nucleotide residue u are connected by a phosphorothioate bond, and "us,Cf" indicates that the nucleotide residue u and the nucleotide residue Cf are connected by a phosphorothioate bond; such as 3'-a,gs,cs,a...u,Gfs,us,A...-5' of the antisense chain, wherein "Gfs,us" indicates that the nucleotide residue u and the nucleotide residue Gf are connected by a phosphorothioate bond, "u,Gfs" indicates that the nucleotide residue Gf and the nucleotide residue u are connected by a phosphate bond, "gs,cs" indicates that the nucleotide residue c and the nucleotide residue g are connected by a phosphorothioate bond, and "a,gs" indicates that the nucleotide residue g and the nucleotide residue a are connected by a phosphorothioate bond.

[0140] The monomers of the GNA (glycerol nucleic acid) nucleotides disclosed herein (GNA-A, GNA-T, GNA-C, GNA-G and GNA-U): GNA-A structure is The structure of GNA-T is The structure of GNA-C is The structure of GNA-G is The structure of GNA-U is

[0141] The "multiple" mentioned in the present disclosure refers to an integer greater than or equal to 2, including but not limited to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, up to the theoretical upper limit of the siRNA analog.

[0142] Unless otherwise specified, the "overhang" of the present invention refers to at least one unpaired nucleotide protruding from the double-stranded region structure of a double-stranded compound. For example, the 3'-end of one chain extends beyond the 5'-end of the other chain, or the 5'-end of one chain extends beyond the 3'-end of the other chain. The overhang may contain at least one nucleotide; or the overhang may contain at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five or more nucleotides. The nucleotides at the nucleotide overhang are optionally modified nucleotides. The overhang may be located on the sense strand, the antisense strand, or any combination thereof. In addition, the overhang may be present at the 5'-end, 3'-end, or both ends of the antisense or sense strand of the double-stranded compound. In some embodiments of the present invention, the antisense strand has an overhang of 1 to 10 nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides) at the 3'-end and / or 5'-end. In some embodiments of the present invention, the sense strand has an overhang of 1 to 10 nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides) at the 3'-end and / or the 5'-end. In some embodiments of the present invention, the antisense strand is at the 3'-end and the sense strand has an overhang of 1 to 10 nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides) at the 3'-end. In some embodiments of the present invention, the antisense strand is at the 5'-end and the sense strand has an overhang of 1 to 10 nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides) at the 5'-end.

[0143] In the present disclosure, the conjugate of a double-stranded RNAi analog (also referred to as "conjugate") is a compound formed by linking a double-stranded RNAi analog and a pharmaceutically acceptable conjugating group, and the double-stranded RNAi analog and the pharmaceutically acceptable conjugating group are covalently linked.

[0144] In the present disclosure, the pharmaceutically acceptable conjugated group can be linked to the 3' end and / or 5' end of the sense strand and / or antisense strand of the double-stranded RNAi analog.

[0145] In some embodiments of the present disclosure, the pharmaceutically acceptable conjugated group is D01.

[0146] In the present disclosure, the number of pharmaceutically acceptable conjugated groups is 1, 2, 3, 4 or 5, and the pharmaceutically acceptable conjugated groups can be independently linked to the 3' end and / or 5' end of the sense strand and / or antisense strand of the double-stranded RNAi.

[0147] In the context of the present disclosure, unless otherwise specified, "conjugation" refers to the covalent linkage of two or more chemical moieties, each of which has a specific function, to each other; accordingly, "conjugate" refers to a compound formed by covalent linkage of the chemical moieties.

[0148] In this disclosure, unless otherwise indicated, "linker" refers to an organic moiety group that connects two parts of a compound, e.g., covalently attaches two parts of a compound. The linker typically comprises a direct bond or an atom (e.g., oxygen or sulfur), an atom group (e.g., NRR, C(O), C(O)NH, SO, SO2, SO2NH), a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted cycloalkyl, or a substituted or unsubstituted heterocycloalkyl, wherein one or more carbon atoms in the substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl group can be replaced with a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted cycloalkyl, or a substituted or unsubstituted heterocycloalkyl group.

[0149] The cleavable linker is sufficiently stable outside the cell, but upon entering the target cell, it is cleaved to release the two moieties to which the linker co-immobilizes.

[0150] The compounds of the present disclosure may exist in specific geometric or stereoisomeric forms. This disclosure contemplates all such compounds, including (R)- and (S)-enantiomers, diastereomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of this disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and their mixtures are included within the scope of this disclosure.

[0151] Unless otherwise indicated, the term "enantiomer" or "optical isomer" refers to stereoisomers that are mirror images of one another.

[0152] Unless otherwise indicated, the term "diastereomer" refers to stereoisomers that have two or more chiral centers and that are not mirror images of each other.

[0153] Unless otherwise specified, use a solid wedge key. and dotted wedge key To indicate the absolute configuration of a stereocenter, use a straight solid bond and straight dashed key Indicate the relative configuration of stereocenters with a wavy line Indicates a wedge-shaped solid key or dotted wedge key Or use a wavy line Indicates a straight solid bond or straight dashed key

[0154] Unless otherwise indicated, the terms "enriched in one isomer", "isomerically enriched", "enriched in one enantiomer" or "enantiomerically enriched" mean that the content of one isomer or enantiomer is less than 100%, and the content of that isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.

[0155] Unless otherwise indicated, the term "isomer excess" or "enantiomeric excess" refers to the difference between the relative percentages of two isomers or two enantiomers. For example, if the content of one isomer or enantiomer is 90% and the content of the other isomer or enantiomer is 10%, the isomer or enantiomeric excess (ee value) is 80%.

[0156] Optically active (R)- and (S)-isomers and D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present disclosure is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a diastereomeric salt is formed with an appropriate optically active acid or base, and then the diastereoisomers are split by conventional methods known in the art, and then the pure enantiomer is recovered. In addition, the separation of enantiomers and diastereoisomers is typically accomplished using chromatography, which employs a chiral stationary phase and is optionally combined with a chemical derivatization method (e.g., carbamate is generated from an amine). The compounds of the present disclosure may contain unnatural ratios of atomic isotopes on one or more of the atoms constituting the compound. For example, compounds can be labeled with radioactive isotopes, such as tritium ( 3 H), iodine-125( 125 I) or C-14( 14 C). For another example, deuterated drugs can be formed by replacing hydrogen with heavy hydrogen. The bond between deuterium and carbon is stronger than the bond between ordinary hydrogen and carbon. Compared with non-deuterated drugs, deuterated drugs have advantages such as reduced toxic side effects, increased drug stability, enhanced efficacy, and prolonged drug biological half-life. All isotopic variations of the compounds disclosed herein, whether radioactive or not, are included within the scope of this disclosure.

[0157] The term "salt" refers to salts of the compounds of the present disclosure, which are prepared by reacting the compounds of the present disclosure with relatively nontoxic acids or bases. When the compounds of the present disclosure contain relatively acidic functional groups, base addition salts can be obtained by contacting the compounds with a sufficient amount of base in a neat solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts. When the compounds of the present disclosure contain relatively basic functional groups, acid addition salts can be obtained by contacting the compounds with a sufficient amount of acid in a neat solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, bisulfate, hydroiodic acid, phosphorous acid, and the like; and organic acid salts such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid; and salts of amino acids such as arginine, and salts of organic acids such as glucuronic acid. Certain specific compounds of the present disclosure contain basic and acidic functional groups and can be converted into either base or acid addition salts.

[0158] The salts of the present disclosure can be synthesized from parent compounds containing acid radicals or bases by conventional chemical methods. Generally, such salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or an organic solvent or a mixture of the two.

[0159] The compounds disclosed herein can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include, but are not limited to, the examples disclosed herein.

[0160] The compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compound. For example, the compounds may be labeled with radioactive isotopes, such as tritium ( 3 H), iodine-125( 125 I) or C-14( 14 C). For example, deuterated drugs can be formed by replacing hydrogen with heavy hydrogen. The bond between deuterium and carbon is stronger than the bond between ordinary hydrogen and carbon. Compared with non-deuterated drugs, deuterated drugs have advantages such as reduced toxic side effects, increased drug stability, enhanced efficacy, and prolonged drug biological half-life. All isotopic variations of the compounds of the present invention, whether radioactive or not, are included within the scope of this invention.

[0161] When the listed linking groups do not specify their linking direction, their linking direction is arbitrary, for example, The connecting group L is -MW-, in which case -MW- can connect ring A and ring B in the same direction as the reading order from left to right to form You can also connect ring A and ring B in the opposite direction of reading from left to right to form Combinations of linkers, substituents, and / or variations thereof are permissible only if such combinations result in stable compounds.

[0162] The terms "optional" or "optionally" mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0163] The term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, which may include deuterium and hydrogen variants, as long as the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is oxygen (i.e., =O), it means that two hydrogen atoms are replaced. Oxygen substitution does not occur on aromatic groups. The term "optionally substituted" means that it may be substituted or unsubstituted, and unless otherwise specified, the type and number of substituents can be any on the basis of chemical achievable.

[0164] When any variable (e.g., R) occurs more than once in a compound's composition or structure, its definition at each occurrence is independent. Thus, for example, if a group is substituted with 0-2 Rs, the group may be optionally substituted with up to two Rs, and each occurrence of R is an independent choice. Furthermore, combinations of substituents and / or their variants are permissible only if such combinations result in stable compounds.

[0165] When the number of a linking group is 0, such as -(CRR)0-, it means that the linking group is a single bond.

[0166] When a substituent is vacant, it means that the substituent does not exist. For example, when X in AX is vacant, it means that the structure is actually A. When the substituent is listed without specifying which atom it is connected to the substituted group, the substituent can be bonded through any atom of the substituent. For example, a pyridyl substituent can be connected to the substituted group through any carbon atom on the pyridine ring.

[0167] The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present invention.

[0168] The structures of the compounds of the present invention can be confirmed by conventional methods well known to those skilled in the art. If the present invention relates to the absolute configuration of the compounds, the absolute configuration can be confirmed by conventional techniques in the art. For example, single crystal X-ray diffraction (SXRD) is used to collect diffraction intensity data on the cultured single crystal using a Bruker D8 venture diffractometer, using CuKα radiation as the light source and scanning mode: After scanning and collecting relevant data, the crystal structure is further analyzed using the direct method (Shelxs97) to confirm the absolute configuration.

[0169] The solvents used in the present disclosure are commercially available.

[0170] Unless otherwise specified, the solvent ratios used in the column chromatography and preparative thin-layer silica gel chromatography disclosed herein are all volume ratios.

[0171] The present invention uses the following abbreviations: aq represents water; HATU represents O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; EDC represents N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride; m-CPBA represents 3-chloroperoxybenzoic acid; eq represents equivalent; CDI represents carbonyldiimidazole; DCM represents dichloromethane; PE represents petroleum ether; DIAD represents diisopropyl azodicarboxylate; DMF represents N,N-dimethylformamide; DMSO represents dimethyl sulfoxide; EtOAc represents ethyl acetate; EtOH represents ethanol; MeOH represents methanol; CBz represents benzyloxycarbonyl, which is an amine protection group; BOC stands for tert-butyloxycarbonyl, which is an amine protecting group; HOAc stands for acetic acid; NaCNBH3 stands for sodium cyanoborohydride; rt stands for room temperature; O / N stands for overnight; THF stands for tetrahydrofuran; Boc2O stands for di-tert-butyl dicarbonate; TFA stands for trifluoroacetic acid; DIPEA stands for diisopropylethylamine; SOCl2 stands for thionyl chloride; CS2 stands for carbon disulfide; TsOH stands for p-toluenesulfonic acid; NFSI stands for N-fluoro-N-(phenylsulfonyl)benzenesulfonamide; NCS stands for 1-chloropyrrolidine-2,5-dione; n-Bu4NF stands for tetrabutylammonium fluoride; iPrOH stands for 2-propanol; mp stands for melting point; LDA stands for lithium diisopropylamide.

[0172] The abbreviations of nucleotide monomers used in the description of nucleic acid sequences are shown in Table 2:

[0173] Table 2 Abbreviations of nucleotide monomers

[0174] Compounds are named according to the conventional nomenclature in the art or using Software naming, commercially available compounds use supplier catalog names. DETAILED DESCRIPTION

[0175] The present invention is described in detail below by examples, but it is not intended to limit the present invention in any way. The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by combining them with other chemical synthesis methods, and equivalent replacement modes well known to those skilled in the art. Preferred embodiments include but are not limited to the embodiments of the present invention. It will be apparent to those skilled in the art that various changes and modifications will be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

[0176] Synthesis Example 1: Synthesis of Z1 and Z2

[0177] Step A: Dissolve 1-1 (10 g, 19.82 mmol) in acetonitrile (120 mL) and 1,2-dichloroethane (80 mL). Add 1-2 (6.02 g, 42.62 mmol) and trimethylsilyl trifluoromethanesulfonate (11.01 g, 49.56 mmol, 8.95 mL) at 0°C. Stir at 35°C for 12 hours. The mixture is quenched by slowly adding saturated aqueous sodium bicarbonate (100 mL) at 0°C and extracted with dichloromethane (100 mL x 2). The organic phase is washed with saturated aqueous sodium chloride (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford the crude product. The crude product is purified on a silica gel column (eluent: petroleum ether / ethyl acetate = 20:1) to afford 1-3.

[0178] Step B: Dissolve 1-3 (6.7 g, 13.05 mmol) in methanolic ammonia (7 mol / L, 50 mL). Stir at 40°C for 12 hours. The reaction mixture is concentrated under reduced pressure to give a crude product. The crude product is purified on a silica gel column (eluent: ethyl acetate / methanol = 50 / 1 to 10 / 1) to give 1-4.

[0179] Step C: Dissolve 1-4 (2.3 g, 11.43 mmol) in anhydrous pyridine (25 mL). Add 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (3.64 g, 11.55 mmol, 3.69 mL) at 0°C. Stir the mixture at 20°C for 12 hours. Quench the mixture with water (30 mL) and extract with ethyl acetate (30 mL x 2). The organic phase is washed sequentially with hydrochloric acid (1 mol / L, 30 mL x 3) and saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to yield the crude product. The crude product is purified on a silica gel column (eluent: petroleum ether / ethyl acetate = 7 / 1 to 5 / 1) to yield 1-5.

[0180] Step D: 1-5 (4 g, 9.02 mmol) was dissolved in acetonitrile (40 mL). Silver oxide (8.36 g, 36.06 mmol), 4A molecular sieves (3 g), anhydrous pyridine (1.78 g, 22.54 mmol, 1.82 mL), and iodomethane (6.4 g, 45.08 mmol, 2.81 mL) were added sequentially. The mixture was stirred at 25°C for 12 hours. Ethyl acetate (50 mL) was added and stirred at 20°C for 1 hour. The mixture was filtered through a Buchner funnel with a pad of celite and the filtrate was collected. The filtrate was extracted with water (100 mL) and ethyl acetate (100 mL x 2). The organic phase was washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified on a silica gel column (eluent: petroleum ether / ethyl acetate = 15 / 1 to 10 / 1 to 8 / 1) to obtain 1-6.

[0181] Step E: Dissolve 1-6 (2 g, 4.37 mmol) in anhydrous tetrahydrofuran (20 mL) and add triethylamine trihydrofluoride (1.55 g, 9.61 mmol, 1.57 mL). Stir at 20°C for 12 hours. The reaction mixture is concentrated under reduced pressure to give a crude product. The crude product is purified on a silica gel column (eluent: ethyl acetate / methanol = 100 / 1 to 50 / 1) to give 1-7.

[0182] Step F: Dissolve 1-7 (1 g, 4.65 mmol) in anhydrous pyridine (10 mL), add 4,4-bismethoxytrityl chloride (1.57 g, 4.65 mmol), and stir at 20°C for 12 hours. The mixture is quenched by the addition of water (20 mL) and extracted with ethyl acetate (20 mL x 2). The organic phase is washed with saturated brine (40 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product is purified on a silica gel column (eluent: petroleum ether / ethyl acetate = 3 / 1 to 1 / 1) to obtain Z1. 1H NMR (400MHz, DMSO-d6): δ = 8.78 (s, 1H), 8.14-8.03 (m, 1H), 7.41-7.31 (m, 2H), 7.29-7.16 (m, 7H), 6.92-6.76 (m, 4H), 6.08 ( d,J=3.2Hz,1H),5.25(d,J=6.5Hz,1H),4.48-4.34(m,1H),4.13-4.01(m,2H),3.73(s,6H),3.38(s,3H),3.18-3.03(m,2H).

[0183] Step G: 5 g of starting material Z1 (9.66 mmol, 1.0 eq) was dissolved in 50 ml of dichloromethane. 3.2 g of bis(diisopropylamino)(2-cyanoethoxy)phosphine (10.63 mmol, 1.1 eq) and 0.57 g of 4,5-dicyanoimidazole (4.83 mmol, 0.5 eq) were added and reacted at 25°C for 2 hours. After completion, the reaction was quenched by the addition of 20 ml of saturated sodium bicarbonate at 20-25°C. The mixture was diluted with 20 ml of water and extracted with 50 ml of dichloromethane. The combined organic phases were washed with 30 ml of saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and the mother liquor was concentrated under reduced pressure to obtain the crude product. The crude product was purified on a silica gel column (eluent: petroleum ether / ethyl acetate (0.1% triethylamine) = 1 / 0 to 1 / 1) to yield 6.8 g of Z2 as a white solid powder. NMR (400MHz, DMSO-d6) δ = 8.82 (s, 1H), 8.11 (d, J = 6.6Hz, 1H), 7.42-7.32 (m, 2H ),7.30-7.16(m,7H),6.90-6.76(m,4H),6.20-6.08(m,1H),4.76-4.51(m,1H) ,4.29(q,J=4.2Hz,1H),4.23-4.09(m,1H),3.83-3.75(m,1H),3.72(d,J=2.4H z,6H),3.61-3.45(m,3H),3.38(d,J=15.2Hz,3H),3.30-3.16(m,1H),3.07(br dd,J=5.6,10.4Hz,1H),2.77(t,J=5.9Hz,1H),2.63-2.55(m,1H),1.16-1.07(m,9H),0.96(d,J=6.6Hz,3H)

[0184] Method for preparing conjugate

[0185] Preparation method: oligoribonucleotides were synthesized by phosphoramidite solid phase synthesis technique. ) was synthesized on a solid support prepared by covalently linking compound 2. All RNA phosphoramidites in the sequence were dissolved in anhydrous acetonitrile and molecular sieves were added. The coupling time was 5 minutes using 5-ethylthio-1H-tetrazole (ETT) as an activator. A 50 mM I2-water / pyridine (1 / 9 by volume) solution was used to generate the phosphate bond, with a reaction time of 3 minutes. A 50 mM 3-((dimethylamino-methylene)amino)-3H-1,2,4-dithiazole-3-thione (DDTT) solution in anhydrous acetonitrile / pyridine (1 / 1 by volume) was used to generate the phosphorothioate bond, with a reaction time of 3 minutes. The resulting sequence was synthesized after the final removal of the DMT group.

[0186] Cleavage and deprotection of oligomers bound to CPG: After termination of solid-phase synthesis, the protecting groups are removed by treatment with 10% diethylamine in acetonitrile for 30 minutes, without cleaving the nucleotides from the CPG. Subsequently, the dried CPG is treated with concentrated ammonia for 18 hours at 40°C. After centrifugation, the supernatant is transferred to a new tube, and the CPG is washed with water. The combined solution is concentrated to yield a solid mixture. The solid mixture is dissolved in DMSO (10V), followed by the addition of triethylammonium hydrofluoride (2V), and stirred at 40°C for 16 hours. Purification is performed after completion of the reaction.

[0187] Purification of Oligoribonucleotides: Oligomers were purified using NanoQ anion exchange HPLC. Buffer A consisted of 10 mM sodium perchlorate, 20 mM Tris, 1 mM EDTA, pH 7.4, and 20% acetonitrile, and buffer B consisted of 500 mM sodium perchlorate, 20 mM Tris, 1 mM EDTA, pH 7.4, and 20% acetonitrile. The desired product was isolated and desalted using a reversed-phase C18 column.

[0188] Annealing of oligoribonucleotides to produce siRNA: Prepare the RNA oligomers to be annealed at a concentration of 200 μM using sterile RNase-free water (without RNA hydrolases). Complementary strands are formed by combining equimolar RNA solutions. The annealing reaction is set up as follows: Place 10 nmol of the 100 μL mixture in a 95°C water bath for 10 minutes, then quickly place it in a 60°C water bath and allow to cool naturally. Do not store the annealed solution at high temperatures.

[0189] Examples 1-3 can all be prepared according to the above method by adjusting the order of the raw materials according to the order of nucleotides and Z shown in the sequence.

[0190] Example 1: Preparation of Conjugate 1

[0191] The sense strand is 5'-as,us,Cf,Cf,a,Cf,Af,Af,u,g,a,g,a,g,u,a,c,a,D01 (SEQ ID NO:5)

[0192] The antisense strand is 3'-a,gs,cs,a,g,u,a,Gf,g,Uf,g,u,u,a,c,u,c,Z,c,a,u,Gfs,us,A,C,A,C,U,C-5' (SEQ ID NO: 2);

[0193] Z is

[0194] The structure of D01 is:

[0195] D01 and a are connected via a phosphodiester bond.

[0196] Example 2: Preparation of Conjugate 3

[0197] The sense strand is 5'-as,us,Cf,Cf,a,Cf,Af,Af,u,g,a,Gf,a,g,u,a,c,a,D01 (SEQ ID NO:7)

[0198] The antisense strand is 3'-a,gs,cs,a,g,u,a,Gf,g,Uf,g,u,u,a,c,u,c,Z,c,a,u,Gfs,us,A,C,A,C,U,C-5' (SEQ ID NO: 2);

[0199] Z is

[0200] The structure of D01 is:

[0201] D01 and a are connected via a phosphodiester bond.

[0202] Example 3: Preparation of Conjugate 2

[0203] The sense strand is 5'-as,us,Cf,Cf,a,Cf,Af,Af,u,g,a,g,a,Gf,u,a,c,a,D01 (SEQ ID NO: 6)

[0204] The antisense strand is 3'-a,gs,cs,a,g,u,a,Gf,g,Uf,g,u,u,a,c,u,c,Z,c,a,u,Gfs,us,A,C,A,C,U,C-5' (SEQ ID NO: 2);

[0205] Z is

[0206] The structure of D01 is:

[0207] D01 and a are connected via a phosphodiester bond.

[0208] Biological testing

[0209] Test Example 1: PHH cell free uptake experiment

[0210] 1. Experimental principle:

[0211] The test sample is incubated with human primary hepatocytes to evaluate the degree of downregulation of AGT mRNA by the test sample.

[0212] 2. Experimental Materials:

[0213] Primary human hepatocytes (PHH), 96 Kit(12)(QIAGEN-74182), FastKing RT Kit(With gDNase)(Tiangen-KR116-02), TaqMan Gene Expression Assay(GAPDH, Thermo, Assay ID-Hs02786624_g1), TaqMan Gene Expression Assay(AGT, Thermo, Assay ID-Hs01586213_m1).

[0214] 3. Experimental methods:

[0215] The conjugate of the present invention was diluted to 10 times the concentration to be tested with PBS solution. 10 uL of siRNA was transferred to a 96-well plate. PHH cells were thawed and transplanted into a 96-well plate. The final cell density was 5.4×10 5 The conjugate of the present invention was tested at 10 concentration points, with the highest concentration being 500 nM, and 4-fold dilution.

[0216] The cells were incubated at 37°C in 5% CO2 for 48 hours, and the cell status was examined under a microscope.

[0217] After the incubation is complete, the cells are lysed and the lysate is obtained. All RNA was extracted using the QIAGEN-96 Kit (QIAGEN-74182) and reverse transcribed using the FastKing RT Kit (with gDNase) (Tiangen-KR116-02) to obtain cDNA. AGT cDNA was detected by qPCR. 4. Experimental Results: The experimental results are shown in Table 3.

[0218] Table 3 Results of the PHH cell free uptake experiment of the conjugates of the present invention

[0219] 5. Experimental conclusion: The conjugate of the present invention can significantly downregulate the level of AGT mRNA in PHH cells and has a good effect in reducing the level of AGT mRNA.

[0220] Test Example 2: Mouse hAGT HDI Model

[0221] 1. Experimental principle:

[0222] The mouse model of high-pressure tail vein injection of pcDNA-CMV-AGT plasmid was used to evaluate the in vivo targeting and inhibitory effect of the test sample on the target gene.

[0223] 2. Experimental Materials:

[0224] pcDNA-CMV-AGT plasmid, BALB / c female mice, DPBS (Dulbecco's phosphate buffered saline), and the conjugate of the present invention.

[0225] 3. Experimental methods:

[0226] Order 6-8 week old BALB / c female mice and acclimate them to quarantine for one week after arrival at the animal house.

[0227] On day 0, mice were randomly divided into groups according to body weight data, with 4 mice in each group. After grouping, all mice were administered subcutaneously with a single dose of 10 mL / kg. Group 1 mice were given DPBS; Group 2 mice were given AD-85481, 5 mg / kg; Group 3 mice were given conjugate 3 of the present invention, 5 mg / kg; Group 4 mice were given conjugate 2 of the present invention, 5 mg / kg.

[0228] On the third day after administration, all mice were injected with pcDNA-CMV-AGT plasmid at a volume of 8% of their body weight (injection volume (mL) = mouse body weight (g) × 8%) via the tail vein within 5 seconds. The mass of the injected plasmid for each mouse was 10 μg.

[0229] On day 4 after administration, mice in all groups were euthanized by CO2 inhalation, and two liver samples were collected from each mouse. The liver samples were treated with RNAlater overnight at 4°C, then the RNAlater was removed and stored at -80°C for analysis of AGT gene expression levels.

[0230] 4. Experimental results: The results are shown in Table 4. The data in the table represent the AGT mRNA expression level in the liver of mice after administration / the AGT mRNA expression level in the liver of mice before administration.

[0231] Table 4. Down-regulation of liver AGT mRNA levels by the conjugates of the present invention in mice

[0232] 5. Experimental conclusion: The conjugate of the present invention can significantly downregulate the expression of AGT mRNA in mouse liver, with the downregulation reaching 97%.

[0233] Test Example 3: In vivo PD experiment in non-human primates, cynomolgus monkeys

[0234] 1. Experimental principle:

[0235] The non-human primate cynomolgus macaque model, which is highly homologous to the human AGT gene, is used to observe the degree of downregulation of cynomolgus macaque plasma AGT protein by the test sample.

[0236] 2. Experimental materials: cynomolgus monkeys, 1x PBS (phosphate buffered saline), AD-85481 (US2021095290), and the conjugate of the present invention.

[0237] 3. Experimental methods:

[0238] Order 10 cynomolgus macaques weighing 2-3 kg and acclimate them to quarantine for one week upon arrival at the animal house.

[0239] On day -6, the macaques were randomly divided into groups according to their body weight, with 2 macaques in each group. After grouping, the plasma AGT protein concentration of all macaques was tested as the experimental data baseline for each group.

[0240] On day 1, the first group received 1x PBS; the second group received a subcutaneous injection of AD-85481 at a dose of 0.3 mpk; the third group received a subcutaneous injection of AD-85481 at a dose of 3 mpk; the fourth group received a subcutaneous injection of Conjugate 2 of the present invention at a dose of 0.3 mpk; and the fifth group received a subcutaneous injection of Conjugate 2 of the present invention at a dose of 3 mpk. Blood was collected on days 1, 2, 4, 8, 15, 22, 29, 32, 35, 43, 57, and 71 after dosing to measure plasma AGT protein concentration in each cynomolgus monkey. Plasma AGT protein concentrations after dosing were compared with AGT protein concentrations on day -6 to determine the relative reduction in AGT protein in each group.

[0241] 4. Experimental results: The results are shown in Table 5. The data in the table represent the AGT gene expression level after administration / AGT gene expression level before administration.

[0242] Table 5. Down-regulation of plasma AGT protein levels in cynomolgus monkeys after a single subcutaneous injection of the conjugate of the present invention

[0243] 5. Experimental conclusion: The conjugate of the present invention can significantly downregulate the AGT protein concentration in the plasma of crab-eating macaques. Compared with the plasma AGT protein concentration on day -6, the AGT protein concentration of the 3mpk group downregulated by 97.53% on the 22nd day after a single dose, and the AGT protein concentration downregulated by 90.12% on the 71st day.

[0244] Test Example 4: In vitro RNA sequencing study

[0245] 1. Experimental introduction:

[0246] The transcriptome refers to the sum of all RNA transcribed by a specific tissue or cell at a specific time or state, primarily including mRNA and non-coding RNA. Transcriptome sequencing, based on the Illumina sequencing platform, studies all mRNA transcribed by a specific tissue or cell at a specific time. It is fundamental to the study of gene function and structure and plays a crucial role in understanding biological development and disease. With the advancement of gene sequencing technology and the reduction in sequencing costs, RNA-seq has become a leading method for transcriptome research due to its high throughput, high sensitivity, and wide range of applications. The RNA-seq technical process primarily consists of two parts: library construction and sequencing, and bioinformatics analysis.

[0247] 2. RNA extraction and detection:

[0248] RNA is extracted from tissues or cells using standard extraction methods, and then the RNA samples are strictly quality controlled, mainly through the Agilent 2100 bioanalyzer: accurately detecting RNA integrity.

[0249] 3. Library construction and quality control:

[0250] There are two main methods for obtaining mRNA: 1. Taking advantage of the structural characteristic of most eukaryotic mRNAs with polyA tails, mRNA with polyA tails can be enriched using Oligo(dT) magnetic beads. 2. mRNA can be obtained by removing ribosomal RNA from total RNA. The resulting mRNA is then randomly fragmented with divalent cations in NEB Fragmentation Buffer and constructed using either the NEB standard library construction method or the strand-specific library construction method.

[0251] NEB general library construction: Using fragmented mRNA as a template and random oligonucleotides as primers, the first cDNA chain is synthesized in the M-MuLV reverse transcriptase system, followed by RNaseH degradation of the RNA chain, and the second cDNA chain is synthesized using dNTPs as raw materials in the DNA polymerase I system. The purified double-stranded cDNA is end-repaired, A-tailed, and ligated to sequencing adapters. AMPure XP beads are used to screen cDNA of about 250-300bp, PCR amplification is performed, and the PCR product is purified again using AMPure XP beads to finally obtain the library. The kit used for library construction is Ultra TM RNA Library Prep Kit for

[0252] Chain-specific library construction: The method for reverse transcription synthesis of the first chain of cDNA is the same as the NEB general library construction method. The difference is that when synthesizing the second chain, dTTP in dNTPs is replaced by dUTP. After that, the cDNA end repair, A tail addition, sequencing adapter connection and length screening are also performed. Then, the second chain of cDNA containing U is degraded with USER enzyme and PCR amplification is performed to obtain the library. Chain-specific libraries have many advantages, such as obtaining more effective information with the same amount of data; obtaining more accurate gene quantification, positioning and annotation information; and providing the expression level of antisense transcripts and a single exon in each isoform. The kit used for library construction is Ultra TM Directional RNA Library Prep Kit for

[0253] Note: Sequencing adapters consist of three parts: P5 / P7, index, and Rd1 / Rd2SP. P5 / P7 are the PCR amplification primers and the primer binding sites on the flow cell. The index provides information to distinguish different libraries. Rd1 / Rd2SP, or read1 / read2 sequence primers, is the sequencing primer binding region. Theoretically, sequencing proceeds backward from Rd1 / Rd2SP.

[0254] After library construction, a Qubit2.0 Fluorometer was used for preliminary quantification, and the library was diluted to 1.5 ng / uL. The insert size of the library was then detected using an Agilent 2100 bioanalyzer. Once the insert size was in line with expectations, qRT-PCR was used to accurately quantify the effective concentration of the library (the effective concentration of the library was greater than 2 nM) to ensure library quality.

[0255] 4. Sequencing:

[0256] After passing the library inspection, different libraries are pooled according to effective concentration and target data volume requirements before Illumina sequencing. The basic principle of sequencing is sequencing by synthesis. Four fluorescently labeled dNTPs, DNA polymerase, and adapter primers are added to the sequencing flow cell for amplification. As each sequencing cluster extends the complementary chain, each added fluorescently labeled dNTP releases corresponding fluorescence. The sequencer captures the fluorescent signal and converts it into sequencing peaks through computer software, thereby obtaining the sequence information of the fragment to be tested.

[0257] 5. Experimental results: The experimental results are shown in Table 6

[0258] Table 6. In vitro RNA sequencing results of the conjugates of the present invention

[0259] 6. Experimental conclusion: The conjugate of the present invention has a low off-target risk.

[0260] Test Example 5: Test of the agonist activity of the conjugate of the present invention on hTLR3, hTLR7, and hTLR8 cells

[0261] 1. Experimental Materials

[0262] Table 7 Experimental materials

[0263] 2. Experimental Instruments

[0264] The main instruments used in this study are the multifunctional microplate reader Flexstation III (Molecular Device) and Echo555 (Labcyte).

[0265] 3. Experimental Procedure

[0266] 1) The conjugate of the present invention was diluted to 20 times the final concentration, mixed with RNAiMAX-OPTI MEM (RNAiMAX:OPTI MEM=3:47) in a 1:1 ratio, and incubated at room temperature for 15 minutes.

[0267] 2) Take 10 μL of the incubated conjugate of the present invention and add it to the corresponding cell plate, with a total of 10 concentrations, the maximum concentration being 200 nM, and each

[0268] Concentrations were measured in duplicate.

[0269] 3) Add 10 μL of PBS to each negative control well, 10 μL of 20 μg / mL Poly(I:C)HMW to each hTLR3 positive control well, and 10 μL of 10 μg / mL R848 to each hTLR7 and hTLR8 positive control well. Add 10 μL of 0.5 μM BLOCK-iT to each of the two transfection positive control wells. TM Alexa Red Fluorescent Control.

[0270] 4) 90 μL of cells were seeded into a 96-well plate to which the conjugate had been added, at a density of 50,000 cells per well.

[0271] 5) The conjugate of the present invention and cells were co-incubated in a 37° C., 5% CO 2 incubator for 24 hours.

[0272] 6) Observe the transfection effect of the positive control wells using a fluorescence microscope and take photos.

[0273] 7) Compound activity detection: Take 20 μL of cell supernatant from each well and add 180 μL of QUANTI-Blue TM After incubation at 37° C. for 1 hour in the test plate containing the reagent, the absorbance at 650 nm (OD650) was measured using a multifunctional microplate reader Flexstation III.

[0274] 8) Cell viability assay: The assay was performed according to the Celltiter-Glo instructions, and the chemiluminescence signal (RLU) was detected using a multifunctional microplate reader Flexstation III.

[0275] 4. Data Analysis

[0276] Compound activity: OD650 values ​​were analyzed using GraphPad Prism software, and the compound dose-effect curve was fitted to calculate the EC of the compound. 50 Cell viability assay: The formula for calculating cell viability % is as follows. Cell viability % values ​​were analyzed using GraphPad Prism software, and the compound dose-effect curve was fitted to calculate the CC of the compound on the cells. 50 value.

[0277] Cell activity % = RLU compound / RLU DMSO control * 100%

[0278] 5. Experimental results: The experimental results are shown in Tables 8-10.

[0279] Table 8. Agonist activity of the conjugates on hTLR3 cells

[0280] Table 9. Agonist activity of the conjugates on hTLR7 cells

[0281] Table 10. Agonistic activity of the conjugates on hTLR8 cells

[0282] 5. Experimental conclusion: The conjugates of the present invention did not elicit hTLR3, hTLR7, and hTLR8 responses under the experimental conditions described, indicating that the immunogenicity risk of the conjugates of the present invention is low.

[0283] Test Example 6. Compound Activity Experiment in Huh7 Cell Line

[0284] 1. Experimental purpose: The purpose of this study is to use Huh7 cells to evaluate the effect of the conjugate described in this patent on inhibiting target genes, using the EC 50 The value was used as an indicator to evaluate the inhibitory activity of the compound on the target gene AGT.

[0285] 2. Experimental Materials:

[0286] 2.1 Cell line: Huh7 cells, conjugate of the present invention

[0287] Huh7 cells were provided by Shanghai WuXi AppTec Pharmaceutical Development Co., Ltd. Huh7 cells were cultured in MEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin.

[0288] 2.2 Reagents:

[0289] The main reagents used in this experiment include Lipofectamine TM iRNAiMAX transfection reagent, FastStart Universal Probe Mast, RNA extraction kit, GAPDH gene expression kit, AGT gene expression kit, FastKing cDNA first-strand synthesis kit, 96-well plate.

[0290] 2.3 Consumables and instruments:

[0291] The main instruments used in this experiment include a fluorescence qPCR instrument (Applied Biosystems, model QuantStudio 6 Flex) and a cell counter (Vi-cellTM XR).

[0292] 3. Experimental steps and methods:

[0293] 3.1 Transfection

[0294] 1) On the first day, prepare a mixture of RNAiMAX transfection reagent and Opti-MEM (1.5:48.5) as needed and incubate at room temperature for 15 minutes.

[0295] 2) For each well of cells, add a certain amount of diluted compound to an equal amount of RNAiMAX Optim-MEM medium, mix well, and incubate for 15 minutes;

[0296] 3) Take Huh7 cells, wash them with DPBS, add trypsin for digestion, and adjust the cells to an appropriate density;

[0297] 4) 20 μL of the mixture of Opti-MEM RNAiMax and the compound was added to the cell culture plate at the same time, and the cells were seeded into a 96-well plate at a density of 2,000 cells per well. The final culture medium per well was 120 μL.

[0298] 5) The cells were cultured in a 5% CO2, 37°C incubator for 24 hours.

[0299] 3.2 RNA extraction and reverse transcription

[0300] The cells were collected, and RNA was extracted according to the instructions of the Qiagen-74182 RNA extraction kit. The RNA was reverse transcribed into cDNA according to the instructions of the FastKing cDNA first-strand synthesis kit.

[0301] 3.3 QPCR detection

[0302] Target gene cDNA was detected by qPCR, using GAPDH as an internal reference gene. qPCR was performed in a 384-well plate. The qPCR reaction program was as follows: heating at 95°C for 10 minutes, followed by cycling at 95°C for 15 seconds, followed by 60°C for 1 minute, for a total of 40 cycles.

[0303] 3.4 Data Analysis: ΔΔCt Method (Ct Difference Comparison Method)

[0304] This method requires the introduction of the internal reference gene GAPDH. This gene is expressed in all cells, its product is essential for cell survival, and its expression level or genomic copy number is constant, making it less susceptible to environmental influences. After qRT-PCR, the CT value of the internal reference is recorded, referred to as Ct(GAPDH), and the CT value of the sample is referred to as Ct(sample).

[0305] ΔCt(sample)=Ct(sample)-Ct(GAPDH)

[0306] ΔCt(control)=Ct(control)-Ct(GAPDH)

[0307] ΔΔCt = ΔCt(sample) - ΔCt(control)

[0308] Relative gene expression = 2^-ΔΔCt

[0309] 4. Experimental results: as shown in Table 11.

[0310] Table 11. Results of Huh7 cell transfection experiments with the conjugates of the present invention

[0311] 5. Experimental conclusion: The conjugate of the present invention can significantly downregulate the level of AGT mRNA in Huh7 cells and has a good effect in reducing AGT mRNA.

Claims

1. A conjugate of a double-stranded RNAi analog or a pharmaceutically acceptable salt thereof, wherein the double-stranded RNAi analog comprises a sense strand and an antisense strand capable of forming a double-stranded region, the sense strand being 5'-as,us,Cf,Cf,a,Cf,Af,Af,u,g,a,g,a,g,u,a,c,a-3' (SEQ ID NO: 1), and the antisense strand being 3'-a,gs,cs,a,g,u,a,Gf,g,Uf,g,u,u,a,c,u,c,Z,c,a,u,Gfs,us,A,C,A,C,U,C-5' (SEQ ID NO: 2), Where Z is The 5' end and / or 3' end of the sense strand and / or antisense strand is conjugated to a ligand, which is one or more GalNAc derivatives attached using a bivalent, trivalent or tetravalent branched linkage.

2. A conjugate of a double-stranded RNAi analog or a pharmaceutically acceptable salt thereof, wherein the double-stranded RNAi analog comprises a sense strand and an antisense strand capable of forming a double-stranded region, the sense strand being 5'-as,us,Cf,Cf,a,Cf,Af,Af,u,g,a,g,a,Gf,u,a,c,a-3' (SEQ ID NO: 3), and the antisense strand being 3'-a,gs,cs,a,g,u,a,Gf,g,Uf,g,u,u,a,c,u,c,Z,c,a,u,Gfs,us,A,C,A,C,U,C-5' (SEQ ID NO: 2), in, Z is The 5' end and / or 3' end of the sense strand and / or antisense strand is conjugated to a ligand, which is one or more GalNAc derivatives attached using a bivalent, trivalent or tetravalent branched linkage.

3. A conjugate of a double-stranded RNAi analog or a pharmaceutically acceptable salt thereof, wherein the double-stranded RNAi analog comprises a sense strand and an antisense strand capable of forming a double-stranded region, the sense strand being 5'-as,us,Cf,Cf,a,Cf,Af,Af,u,g,a,Gf,a,g,u,a,c,a-3' (SEQ ID NO:4), and the antisense strand being 3'-a,gs,cs,a,g,u,a,Gf,g,Uf,g,u,u,a,c,u,c,Z,c,a,u,Gfs,us,A,C,A,C,U,C-5' (SEQ ID NO:2), Where Z is The 5' end and / or 3' end of the sense strand and / or antisense strand is conjugated to a ligand, which is one or more GalNAc derivatives attached using a bivalent, trivalent or tetravalent branched linkage.

4. The conjugate according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, wherein: The ligand is conjugated to the 3' end of the sense strand.

5. The conjugate according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, wherein: The ligand is conjugated to the 5' end of the sense strand.

6. The conjugate according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, wherein: The ligand is conjugated to the 3' end of the antisense strand.

7. The conjugate according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, wherein: The ligand is conjugated to the 5' end of the antisense strand.

8. The conjugate according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, wherein: The conjugate is selected from: Conjugate 1: The sense strand is 5'-as,us,Cf,Cf,a,Cf,Af,Af,u,g,a,g,a,g,u,a,c,a,D01 (SEQ ID NO:5) The antisense strand is 3'-a,gs,cs,a,g,u,a,Gf,g,Uf,g,u,u,a,c,u,c,Z,c,a,u,Gfs,us,A,C,A,C,U,C-5' (SEQ ID NO: 2); Conjugate 2: The sense strand is 5'-as,us,Cf,Cf,a,Cf,Af,Af,u,g,a,g,a,Gf,u,a,c,a,D01 (SEQ ID NO: 6) The antisense strand is 3'-a,gs,cs,a,g,u,a,Gf,g,Uf,g,u,u,a,c,u,c,Z,c,a,u,Gfs,us,A,C,A,C,U,C-5' (SEQ ID NO: 2); Conjugate 3: The sense strand is 5'-as,us,Cf,Cf,a,Cf,Af,Af,u,g,a,Gf,a,g,u,a,c,a,D01 (SEQ ID NO:7) The antisense strand is 3'-a,gs,cs,a,g,u,a,Gf,g,Uf,g,u,u,a,c,u,c,Z,c,a,u,Gfs,us,A,C,A,C,U,C-5' (SEQ ID NO: 2); Wherein, the D01 structure is: D01 and a are connected via a phosphodiester bond.