Conjugates of duplex RNAi analogues containing nucleotide analogues

Abstract

Conjugates of nucleotide analog-containing double-stranded RNAi analogs are disclosed, in particular the sequence compositions and ligand structures of the conjugates of double-stranded RNAi analogs.

Description

[Technical field]

[0001] This patent application claims priority to: CN202210477929.8, April 29, 2022; CN202211106626.1, September 9, 2022.

[0002] Technology background The present invention relates to conjugates of nucleotide analogue-containing double-stranded RNAi analogues, in particular the conjugates or pharma- ceutically acceptable salts thereof, and their use in medicaments for the treatment of associated diseases. [Background technology]

[0003] background The RNA interference (RNAi) mechanism has been attracting attention since its discovery in 1998; this mechanism achieves the control of the corresponding target function by down-regulating the level of the target mRNA to achieve the expected pharmacological effect. RNAi drugs are generally ribonucleic acids having a double-stranded structure, which, after entering the target cell by delivery, bind to Ago2 protein or the like in the target cell to form an RNA-induced silencing complex (RISC). This complex binds to the target mRNA in the target cell, hydrolyzes and cleaves the target mRNA, thus losing the protein translation function of the target mRNA, thereby down-regulating the amount of the target protein and achieving the inhibition of the target function. In recent years, several RNAi drugs have been approved on the market, and drug feasibility verification for application to the human body has been completed. Drugs that achieve pharmacological effects by this mechanism are known as RNAi drugs.

[0004] The renin-angiotensin-aldosterone (RAAS) system plays an important role in blood pressure regulation. Angiotensinogen (AGT) protein is the only known upstream substance in the RAAS system. AGT protein in the circulation is mainly from liver synthesis. Renin secreted from the kidney converts AGT to angiotensin I (Ang I) in the circulation, and then angiotensin-converting enzyme (ACE) converts inactive Ang I to biologically active angiotensin II (Ang II). Ang II acts on receptors to produce effects such as vasoconstriction and promotion of aldosterone release, thereby increasing blood pressure. The regulatory effect of the RAAS system on blood pressure has been deeply verified in clinical trials, and renin inhibitors (aliskiren), angiotensin-converting inhibitors (drugs with the suffix -pril in their names) and angiotensin II receptor antagonists (drugs with the suffix -sartan in their names) that target this pathway have become commonly used drugs in the clinical treatment of hypertension.

[0005] Hypertension is a clinical syndrome characterized primarily by elevated systemic arterial pressure. Continuously elevated and volatile blood pressure is a major risk factor for cardiovascular disease and can lead to damage to target organs such as the heart, kidneys, brain, and blood vessels.

[0006] There are many people with high blood pressure worldwide. The first comprehensive analysis of global hypertension trends, written by Imperial College London and the World Health Organization and published in The Lancet on August 25, 2021, is the world's largest study on hypertension to date. (Article source: 10.1016 / S0140-6736(21)01330-1.). The report showed that over the past 30 years, the number of adults aged 30-79 with high blood pressure has increased from 650 million to 1.28 billion. The study also found that more than half of people with high blood pressure (53% women and 62% men, a total of 720 million) do not receive the treatment they need. High blood pressure can cause significant harm, and cardiovascular disease (CVD) caused by hypertension is responsible for approximately 8.5 million deaths worldwide each year. Thus, there is a significant clinical unmet need for effective blood pressure control.

[0007] Drugs for clinical treatment of hypertension are mainly small molecule drugs. Small molecule drugs are absorbed and metabolized quickly, have short half-life, and insufficient duration of action, which is prone to cause nocturnal hypertension fluctuations and poor 24-hour blood pressure control. In addition, small molecule drugs have high drug load and need to be taken every day. In clinical practice, it is easy to forget to take the drug, which leads to increased blood pressure and damage to target organs. Therefore, there is an urgent need for long-acting antihypertensive drugs in clinical practice to meet clinical unmet needs. Summary of the Invention

[0008] overview A first aspect of the present invention provides a conjugate of a double-stranded RNAi analog or a pharma- ceutically 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 is TIFF2025514400000001.tif10160, The antisense strand is TIFF2025514400000002.tif10156, Where: Z is TIFF2025514400000003.tif21128, The 5' and / or 3' ends of the sense and / or antisense strands are conjugated to a ligand, which is one or more GalNAc derivatives attached via a bivalent, trivalent or tetravalent branched linker.

[0009] In some embodiments of the first aspect of the invention, the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker.

[0010] In some embodiments of the first aspect of the invention, the ligand is one or more GalNAc derivatives attached via a trivalent branched linker.

[0011] In some embodiments of the first aspect of the invention, the ligand is a GalNAc derivative attached via a trivalent branched linker.

[0012] A second aspect of the present invention provides a conjugate of a double-stranded RNAi analogue or a pharma- ceutically acceptable salt thereof, wherein the double-stranded RNAi analogue comprises a sense strand and an antisense strand capable of forming a double-stranded region; The sense strand is TIFF2025514400000004.tif10156, The antisense strand is TIFF2025514400000005.tif10156, Where: Z is TIFF2025514400000006.tif21128, The 5' and / or 3' ends of the sense and / or antisense strands are conjugated to a ligand, which is one or more GalNAc derivatives attached via a bivalent, trivalent or tetravalent branched linker.

[0013] In some embodiments of the second aspect of the invention, the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker.

[0014] In some embodiments of the second aspect of the invention, the ligand is one or more GalNAc derivatives attached via a trivalent branched linker.

[0015] In some embodiments of the second aspect of the invention, the ligand is a GalNAc derivative attached via a trivalent branched linker.

[0016] A third aspect of the present invention provides a conjugate of a double-stranded RNAi analogue or a pharma- ceutically acceptable salt thereof, wherein the double-stranded RNAi analogue comprises a sense strand and an antisense strand capable of forming a double-stranded region; The sense strand is TIFF2025514400000007.tif10156, The antisense strand is TIFF2025514400000008.tif10156, Where: Z is TIFF2025514400000009.tif21128, The 5' and / or 3' ends of the sense and / or antisense strands are conjugated to a ligand, which is one or more GalNAc derivatives attached via a bivalent, trivalent or tetravalent branched linker.

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

[0018] In some embodiments of the third aspect of the invention, the ligand is one or more GalNAc derivatives attached via a trivalent branched linker.

[0019] In some embodiments of the third aspect of the invention, the ligand is a GalNAc derivative attached via a trivalent branched linker.

[0020] In some technical embodiments of the invention, the ligand is conjugated to the 3' end of the sense strand, with the other variables being as defined in the invention.

[0021] In some technical embodiments of the invention, the ligand is conjugated to the 5' end of the sense strand, with the other variables being as defined in the invention.

[0022] In some technical embodiments of the invention, the ligand is conjugated to the 3' end of the antisense strand, with the other variables being as defined in the invention.

[0023] In some technical embodiments of the invention, the ligand is conjugated to the 5' end of the antisense strand, with the other variables being as defined in the invention.

[0024] In some technical embodiments of the first, second and / or third aspect of the invention, the ligand is conjugated to the 3' end of the sense strand, and the other variables are as defined herein.

[0025] In some technical embodiments of the first, second and / or third aspect of the invention, the ligand is conjugated to the 5' end of the sense strand, and the other variables are as defined herein.

[0026] In some technical embodiments of the first, second and / or third aspect of the invention, the ligand is conjugated to the 3' end of the antisense strand, and the other variables are as defined herein.

[0027] In some technical embodiments of the first, second and / or third aspect of the invention, the ligand is conjugated to the 5' end of the antisense strand, and the other variables are as defined herein.

[0028] A fourth aspect of the present invention provides the following conjugate, or a pharma- ceutically acceptable salt thereof: Conjugate 1: Sense strand: TIFF2025514400000010.tif10160Antisense strand: TIFF2025514400000011.tif10156 Conjugate 2: Sense strand: TIFF2025514400000012.tif10156Antisense strand: TIFF2025514400000013.tif10156 Conjugate 3: Sense strand: TIFF2025514400000014.tif10156Antisense strand: TIFF2025514400000015.tif10156, where the structure of D01 is as follows: TIFF2025514400000016.tif51165, D01 is linked to a via a phosphodiester bond.

[0029] A fifth aspect of the present invention provides a pharmaceutical composition comprising an effective amount of a conjugate as defined in any of the above technical embodiments, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier.

[0030] In some embodiments of the fifth aspect of the invention, the pharmaceutical composition comprises an effective amount of a conjugate of any one, two, three, four, or five of the double-stranded RNAi analogs described above, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier.

[0031] In some embodiments of the fifth aspect of the present invention, the pharmaceutical composition comprises an effective amount of a conjugate of any one of the double-stranded RNAi analogs described above, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier.

[0032] A sixth aspect of the present invention provides the use of a conjugate of any of the above double-stranded RNAi analogues, or a pharma- ceutically acceptable salt thereof, in the preparation of a medicament for treating an AGT-associated disease.

[0033] In some embodiments of the sixth aspect of the present invention, the AGT-related disease is hypertension.

[0034] In some embodiments of the sixth aspect of the present invention, the AGT-related disease is a cardiovascular disease.

[0035] A seventh aspect of the present invention provides a method for treating an AGT-associated disease in a subject in need thereof, comprising administering to the subject an effective amount of a conjugate as defined in any of the above technical embodiments, or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0036] In the event that information in the instant sequence listing conflicts with the present specification, the present specification shall control.

[0037] The present invention also provides the following test method.

[0038] Test method 1: Primary human hepatocyte (PHH) free uptake experiment 1. Experimental principle: The sample to be tested is incubated with primary human hepatocytes. The degree of downregulation of AGT mRNA by the sample to be tested is evaluated.

[0039] 2. Experimental materials: Primary human hepatocytes (PHH), RNeasy® 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).

[0040] 3. Experimental Method: The conjugate of the present invention is diluted in PBS solution so that the resulting concentration is 10 times the concentration to be tested. 10 μL of siRNA is transferred to a 96-well plate. PHH cells are thawed and seeded into a 96-well plate to a final cell density of 5.4 × 10 5 cells / well. The conjugates of the invention are tested over 10 concentration points, with the highest concentration being 500 nM at 4-fold dilutions.

[0041] The cells are incubated at 37° C. and 5% CO 2 for 48 hours, and the state of the cells is observed using a microscope.

[0042] After the incubation is completed, the cells are lysed, and total RNA is extracted from the resulting lysate using RNeasy® 96 kit (QIAGEN-74182), and reverse transcribed to obtain cDNA using FastKing RT kit (with gDNase) (Tiangen-KR116-02). AGT cDNA is detected by qPCR.

[0043] 4. Experimental Results: The conjugate of the present invention can significantly down-regulate the level of AGT mRNA in PHH cells.

[0044] Test method 2: Mouse hAGT HDI model 1. Experimental principle: The targeting of the target gene in the tested sample and the inhibitory effect on the target gene are evaluated by a mouse model injected with pcDNA-CMV-AGT plasmid via the tail vein at high pressure.

[0045] 2. Experimental materials: pcDNA-CMV-AGT plasmid, BALB / c female mice, DPBS (Dulbecco's phosphate buffered saline), and the conjugate of the present invention or a pharma- ceutically acceptable salt thereof.

[0046] 3. Experimental Method: Purchase 6-8 week old BALB / c female mice and allow the mice to adapt to quarantine for 1 week after arrival in the animal room.

[0047] On day 0, mice are randomly divided into groups according to body weight data, each group has 4 mice, and after grouping, all mice are subcutaneously administered with a single dose of 10mL / kg. Group 1 mice are administered with DPBS; Group 2 mice are administered with the conjugate of the present invention or its pharmacologic acceptable salt at 3mg / kg.

[0048] On the third day after administration, inject all mice with the pcDNA-CMV-AGT plasmid via the tail vein within 5 seconds in a volume of 8% of the body weight (injection volume (mL) = mouse body weight (g) × 8%). The mass of plasmid injected into each mouse is 10 µg.

[0049] Four days after administration, all groups of mice are euthanized by CO2 inhalation, and two liver samples are collected from each mouse. The liver samples are treated with RNAlater overnight at 4°C, then RNAlater is removed, and the RNAlater-free samples are then stored at -80°C to detect the expression level of AGT gene.

[0050] 4. Experimental conclusion: The conjugate of the present invention has an excellent down-regulating effect on the level of AGT mRNA in mouse liver.

[0051] Test method 3: In vivo PD experiments in non-human primates (cynomolgus monkeys) 1. Experimental principle: The degree of downregulation of cynomolgus monkey plasma AGT by the samples being tested is observed through a non-human primate cynomolgus monkey model in which the AGT gene is highly homologous to that in humans.

[0052] 2. Experimental materials: cynomolgus monkeys, 1x PBS (phosphate buffer solution), AD-85481 (US2021095290), and the conjugate of the present invention.

[0053] 3. Experimental Method: Purchase 10 cynomolgus monkeys weighing 2-3 kg and allow the animals to adapt to quarantine for one week after their arrival in the animal room.

[0054] On day -6, the cynomolgus monkeys were randomly divided into groups according to body weight data, with each group containing two cynomolgus monkeys. After grouping, the AGT protein concentration in the plasma of all cynomolgus monkeys was detected, which became the experimental database line for each group.

[0055] Administration is performed on day 1. Blood is collected on days 1, 2, 4, 8, 15, 22, 29, 32, 35, 43, 57, and 71 after administration to detect the AGT protein concentration in the plasma of each cynomolgus monkey. The AGT protein concentration in the plasma after administration is compared with the AGT protein concentration on day -6 to obtain the relative downregulation rate of AGT protein in each animal group.

[0056] 4. Experimental Results: The conjugate of the present invention can significantly down-regulate AGT protein concentration in the plasma of cynomolgus monkeys.

[0057] Test method 4: In vitro RNA sequencing study 1. Experimental Introduction: Transcriptome refers to the sum of all RNA transcribed from a particular tissue or cell at a particular time or state, mainly including mRNA and non-coding RNA. Transcriptome sequencing is the study of all mRNA transcribed from a particular tissue or cell in a particular period based on the Illumina sequencing platform, which is the basis of gene function and structure research and plays an important role in understanding organism development and disease occurrence. With the development of gene sequencing technology and the reduction of sequencing costs, RNA-seq has become the main method for transcriptome research, with the advantages of high throughput, high sensitivity, and wide range of applications. The technical process of RNA-seq mainly includes two parts: library construction and sequencing, and biological information analysis.

[0058] 2. RNA Extraction and Detection: RNA is extracted from tissues or cells using standard extraction methods, and then stringent quality control is performed on the RNA samples, which is primarily accomplished by an Agilent 2100 bioanalyzer, which accurately detects the integrity of the RNA.

[0059] 3. Library construction and quality inspection: mRNA is obtained mainly in two ways: one is to enrich for mRNAs containing polyA tails using oligo(dT) magnetic beads, taking advantage of the structural feature that most eukaryotic mRNAs have polyA tails; the other is to obtain mRNAs by removing ribosomal RNA from total RNA. The obtained mRNA is then randomly fragmented using divalent cations in NEB fragmentation buffer, and library construction is performed using either the normal library construction method or the strand-specific library construction method of NEB.

[0060] NEB's regular library construction: The first strand of cDNA is synthesized with M-MuLV reverse transcriptase system using fragmented mRNA as template and random oligonucleotides as primers. Then, the RNA strand is digested using RNaseH, and the second strand of cDNA is synthesized with DNA polymerase I system using dNTPs as raw materials. The purified double-stranded cDNA is subjected to end repair, A-tail addition, and sequencing linker ligation. Approximately 250-300 bp of cDNA is screened using AMPure XP beads and subjected to PCR amplification, and the PCR product is purified again using AMPure XP beads to finally obtain the library. The kit for library construction is NEBNext® Ultra® RNA Library Prep Kit for Illumina®.

[0061] Strand-specific library construction: The method for synthesizing the first strand of cDNA by reverse transcription is the same as NEB's normal library construction method, except that when the second strand is synthesized, dTTP in dNTP is replaced with dUTP, and then end repair, A-tail addition, sequencing linker ligation, and cDNA length screening are performed in the same way. Then, the second strand of cDNA containing U is digested using USER enzyme, followed by PCR amplification to obtain a library. Strand-specific library has many advantages, such as more effective information can be obtained with the same amount of data; more accurate gene quantification, localization, and annotation information can be obtained; and antisense transcripts and the expression level of a single exon in each isoform can be provided. The kit used for library construction is NEBNext® Ultra™ Directional RNA Prep Kit for Illumina®.

[0062] NOTE: The sequencing linker is composed of three components, namely P5 / P7, index, and Rd1 / Rd2 SP. Among these, P5 / P7 is the part where the PCR amplification primer and the primer on the flow cell bind, the index provides information to distinguish different libraries, and Rd1 / Rd2SP, i.e. the read1 / read2 sequence primers, is the region where the sequencing primer binds. The sequencing process theoretically starts from Rd1 / Rd2 SP in the reverse direction.

[0063] Once library construction is complete, perform preliminary quantification using a Qubit2.0 fluorometer. Dilute the library to 1.5ng / ul, then use an Agilent 2100 bioanalyzer to detect the insert size of the library. After the insert size is as expected, accurately quantify the effective concentration of the library by qRT-PCR (the effective concentration of the library is higher than 2nM) to ensure the quality of the library.

[0064] 4. On-machine sequencing: After the library test is qualified, the different libraries are subjected to Illumina sequencing after pooling according to the requirements of effective concentration and target offline data amount. The basic principle of sequencing is sequencing by synthesis. Four fluorescently labeled dNTPs and DNA polymerase, as well as a linker primer, are added to the sequencing flow cell for amplification. When the complementary strand is extended in each sequencing cluster, the addition of fluorescently labeled dNTPs will release the corresponding fluorescence, and the sequencer captures the fluorescent signal and converts the light signal into a sequencing peak through computer software to obtain the sequence information of the fragment being tested.

[0065] 5. Experimental Results: The conjugates of the present invention significantly down-regulate the AGT gene in RNA sequencing studies.

[0066] Test method 5: Agonist activity test of conjugates in hTLR3, hTLR7, and hTLR8 cells 1. Experimental Materials [Table 1]

[0067] 2. Experimental Apparatus The main equipment used in this study was the multifunctional microplate reader Flexstation III (Molecular Device) and Echo555 (Labcyte).

[0068] 3. Experimental Steps 1) Dilute the compound to a concentration 20 times the final concentration, mix the diluted compound with RNAiMAX-OPTI MEM (RNAiMAX:OPTI MEM=3:47) in a 1:1 ratio, and incubate at room temperature for 15 minutes. ("Compound" refers to the conjugate of the present invention for testing.) 2) 10 μL of incubated compound is added to the corresponding cell plate, for a total of 10 concentrations, with duplicate wells for each concentration. 3) Add 10 μL of PBS to each of the negative control wells, 10 μL of 20 μg / mL poly(I:C) HMW to each of the hTLR3 positive control wells, and 10 μL of 10 μg / mL R848 to each of the hTLR7 and hTLR8 positive control wells. Add 10 μL of 0.5 μM BLOCK-iT™ Alexa Fluor® red fluorescent control to the two transfection positive control wells. 4) Seed 90 μL of cells into compound-containing 96-well plates at a density of 50,000 cells / well. 5) The compounds and cells are co-incubated for 24 hours in an incubator at 37° C. and 5% CO 2 . 6) Observe the transfection effect of the transfection positive control wells using a fluorescent microscope and take pictures. 7) Compound activity detection: 20 μL of cell supernatant from each well is taken and added to an experimental plate containing 180 μL of QUANTI-Blue™ reagent, then incubated at 37° C. for 1 hour, followed by detecting the absorbance value at 650 nm (OD650) using a multifunctional microplate reader FlexstationIII. 8) Cell activity detection: Operate according to the Celltiter-Glo specification method and detect the chemiluminescence signal (RLU) using the multifunctional microplate reader FlexstationIII.

[0069] 3. Data Analysis Compound activity: OD650 values ​​were analyzed using GraphPad Prism software and dose-effect curves of compounds were fitted to determine the EC 50 Cellular activity detection: The formula for calculating % cellular activity is as follows: GraphPadPrism software was used to analyze the % cellular activity values, fit the dose-effect curves of the compounds, and calculate the CC of the compounds on the cells. 50 Calculate the value. Cellular activity %=RLU compound / RLU DMSO control * 100%

[0070] 4. Experimental results: The conjugates of the present invention show good efficacy in this test.

[0071] Technical effects The conjugates of the present invention have low off-target risk, low immunogenicity risk, and can significantly down-regulate AGT mRNA levels in PHH cells, mouse liver AGT mRNA levels in a mouse hAGT HDI model, and AGT protein concentrations in non-human primate cynomolgus monkey plasma.

[0072] Definitions and Examples Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings: Certain terms or phrases should not be considered unclear or unclear without a specific definition, but should be understood according to the meaning understood by those of ordinary skill in the art. When a trade name appears herein, it is intended to refer to the corresponding product or its active ingredients.

[0073] Unless otherwise specified, the terms "comprising," "including," and "containing" or their equivalents are open-ended and mean that in addition to the recited components, components, or steps, other unrecited components, components, or steps may be encompassed.

[0074] A double-stranded RNAi analog refers to a ribonucleic acid molecule complex that has a double-stranded structure, contains two parallel, substantially complementary nucleic acid strands in opposite directions, and has a "sense" and "antisense" orientation with respect to the target RNA. In this disclosure, "complementary" has the meaning well known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, bases on 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 contains one purine and one pyrimidine. If the adenine of one strand always pairs with the uracil of the other strand and the guanine always pairs with the cytosine, the two strands are considered to be complementary to each other, and the sequence of the strands can be inferred from the sequence of its complementary strand.

[0075] "Modifications" of the nucleotides described in this disclosure include, but are not limited to, methoxy modifications, fluoro modifications, (E)-vinyl phosphate modifications, thiophosphate linkages, or replacement of the nucleotide with GNA (glycerol nucleic acid) nucleotide monomers, where GNA includes GNA-A, GNA-T, GNA-C, GNA-G, and GNA-U. The sequences described in this disclosure may include the "further modified sequences" in Table 2 below.

[0076] In this disclosure, a fluoro-modified nucleotide refers to a nucleotide formed by replacing the 2'-hydroxyl of the ribosyl group of a nucleotide with fluorine, and a methoxy-modified nucleotide refers to a nucleotide formed by replacing the 2'-hydroxyl of a ribosyl with methoxy.

[0077] All thiophosphate linkage methods described in this disclosure refer to a method reading from the 5' end, where the previous and next nucleotides are linked by a thiophosphate bond; e.g., 5'-as,us,Cf...-3' of the sense strand, where "as,us" represents the linkage between nucleotide residues a and u by a thiophosphate bond, and "us,Cf" represents the linkage between nucleotide residues u and Cf by a thiophosphate bond; e.g., 3'-a of the antisense strand, where "as,us" represents the linkage between nucleotide residues a and u by a thiophosphate bond, and "us,Cf" represents the linkage between nucleotide residues u and Cf by a thiophosphate bond; ,gs,cs,a...u,Gfs,us,A...-5', where "Gfs,us" represents the linkage between nucleotide residue u and nucleotide residue Gf by a thiophosphate bond, "u,Gfs" represents the linkage between nucleotide residue Gf and nucleotide residue u by a thiophosphate bond, "gs,cs" represents the linkage between nucleotide residue c and nucleotide residue g by a thiophosphate bond, and "a,gs" represents the linkage between nucleotide residue g and nucleotide residue a by a thiophosphate bond.

[0078] GNA (glycerol nucleic acid) nucleotide monomers of the present disclosure (GNA-A, GNA-T, GNA-C, GNA-G, and GNA-U): The structure of GNA-A is TIFF2025514400000018.tif38128; the structure of GNA-T is TIFF2025514400000019.tif37128; the structure of GNA-C is TIFF2025514400000020.tif37128; the structure of GNA-G is TIFF2025514400000021.tif40128; the structure of GNA-U is The file is TIFF2025514400000022.tif37128.

[0079] The term "multiple" as described in this disclosure refers to an integer greater than or equal to 2, i.e., up to the theoretical upper limit of the siRNA analog, 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.

[0080] Unless otherwise specified, the "overhang" described in the present invention refers to at least one unpaired nucleotide that protrudes from the double-stranded region structure of a double-stranded compound. For example, the 3' end of one strand extends beyond the 5' end of the other strand, or the 5' end of one strand extends beyond the 3' end of the other strand. The overhang may comprise at least one nucleotide, or the overhang may comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more nucleotides. The nucleotides of the nucleotide overhang are optionally modified nucleotides. The overhang may be located on the sense strand, the antisense strand, or any combination thereof. Furthermore, the overhang may be present at the 5' end or the 3' end of the antisense strand or the sense strand of the double-stranded compound, or may be present at both ends simultaneously. In some embodiments of the 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 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 5' end. In some embodiments of the invention, the antisense strand and the sense strand have 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 invention, the antisense strand and the sense strand have 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.

[0081] In the present disclosure, a conjugate of a double-stranded RNAi analog (also known as a "conjugate") is a compound formed by linking a double-stranded RNAi analog with a pharma- ceutically acceptable conjugation group, where the double-stranded RNAi analog is covalently linked to the pharma- ceutically acceptable conjugation group.

[0082] In the present disclosure, a pharma- ceutically acceptable conjugation group can be linked to the 3'-end and / or the 5'-end of the sense strand and / or the antisense strand of the double-stranded RNAi analog.

[0083] In some embodiments of the present disclosure, the pharma- ceutically acceptable conjugation group is D01.

[0084] In the present disclosure, the number of pharma- ceutically acceptable conjugation groups is 1, 2, 3, 4, or 5, and each pharma-ceutically acceptable conjugation group is independently linked to the 3' end and / or the 5' end of the sense strand and / or the antisense strand of the double-stranded RNAi.

[0085] In the context of this disclosure, unless otherwise specified, "conjugation" refers to the interconnection between two or more chemical moieties, each having a specific function, and accordingly, "conjugate" refers to a compound formed by a covalent bond between chemical moieties.

[0086] In this disclosure, unless otherwise specified, "linker" refers to an organic moiety that connects two parts of a compound, for example, covalently bonds two parts of a compound. A linker generally includes one direct link or atom (such as oxygen or sulfur), a group of atoms (such as NRR, C(O), C(O)NH, SO, SO2, SO2NH), a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkynyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted cycloalkyl group, and a substituted or unsubstituted heterocycloalkyl group, where any one or more C atoms in the substituted or unsubstituted alkyl group, the substituted or unsubstituted alkenyl group, and the substituted or unsubstituted alkynyl group can be replaced with a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted cycloalkyl group, and a substituted or unsubstituted heterocycloalkyl group.

[0087] The cleavable linker is sufficiently stable outside the cell but is cleaved upon entry into the target cell, separating the groups of the two moieties that are fixed together by the linker.

[0088] The compounds of the present disclosure may have specific geometric or stereoisomeric forms. The present disclosure contemplates all such compounds, including (R)- and (S)-enantiomers, diastereomers, and racemic mixtures thereof, as well as other mixtures, such as enantiomerically or diastereomerically enriched mixtures. All of these mixtures are included within the scope of the present disclosure. Other asymmetric carbon atoms may be present in substituents, such as alkyl groups. All of these isomers and mixtures thereof are included within the scope of the present disclosure.

[0089] Unless otherwise specified, the terms "enantiomers" or "optical isomers" refer to stereoisomers that are mirror images of each other.

[0090] Unless otherwise specified, the term "diastereomer" refers to stereoisomers with two or more chiral centers and which are not mirror-image related.

[0091] Solid wedge connections unless otherwise stated TIFF2025514400000023.tif5128 and wedge-shaped dashed line combination TIFF2025514400000024.tif5128 is used to represent the absolute configuration of stereocenters; straight solid bonds TIFF2025514400000025.tif5128 and straight line dashed line combination TIFF2025514400000026.tif5128 is used to represent the relative arrangement of stereocenters; wavy lines TIFF2025514400000027.tif5128 is a solid wedge combination TIFF2025514400000028.tif5128 or wedge-shaped dashed line combination TIFF2025514400000029.tif5128 is used to represent a wavy line TIFF2025514400000030.tif5128 is a straight line combination TIFF2025514400000031.tif5128 or straight line dashed line combination It is used to represent TIFF2025514400000032.tif5128.

[0092] Unless otherwise specified, the terms "enriched in one isomer," "isomer-enriched," "enriched in one enantiomer," or "enantiomer-enriched" refer to a situation in which the amount of one of the isomers or enantiomers is less than 100%, and the amount of the isomer or enantiomer is 60% or more, or 70% or more, or 80% or more, or 90% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more, or 99.5% or more, or 99.6% or more, or 99.7% or more, or 99.8% or more, or 99.9% or more.

[0093] Unless otherwise specified, the term "isomer excess" or "enantiomeric excess" refers to the difference between the relative percentages of two isomers or two enantiomers. For example, if one of the isomers or enantiomers is present at 90% and the other isomer or enantiomer at 10%, then the isomeric or enantiomeric excess (ee value) is 80%.

[0094] Optically active (R)- and (S)-isomers, as well as D- and L-isomers, can be prepared by chiral synthesis, chiral reagents, or other conventional techniques. If one enantiomer of a compound disclosed herein is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral adjuvant, in which case the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to obtain the pure desired enantiomer. Alternatively, if the molecule contains an alkaline functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), the molecule can be reacted with an appropriate optically active acid or base to form a diastereomeric salt, and then separation of the diastereomers can be carried out using conventional methods known in the art, followed by recovery to obtain the pure enantiomers. Furthermore, separation of enantiomers and diastereomers is typically achieved by chromatography, which employs a chiral stationary phase, optionally combined with chemical derivatization methods (e.g., generating carbamates from amines). The compounds of the present disclosure can contain unnatural proportions of atomic isotopes on one or more of the atoms that constitute the compound. For example, the compound is tritium ( 3 H), iodine-125( 125 I), or C-14( 14 As another example, hydrogen can be replaced with deuterium to form a deuterated drug. The bond formed by deuterium and carbon is stronger than the bond formed by normal hydrogen and carbon. Compared to non-deuterated drugs, deuterated drugs have the advantages of reducing toxic side effects, increasing drug stability, increasing efficacy, and extending the drug half-life. All isotopic composition variations of the compounds of the present disclosure, whether radioactive or not, are included within the scope of the present disclosure.

[0095] The term "salt" refers to a salt of a compound of the present disclosure prepared from a compound having certain substituents found in the present disclosure and a relatively non-toxic acid or base. When a compound of the present disclosure contains a relatively acidic functional group, a base addition salt can be obtained by contacting such a compound with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium salts, potassium salts, calcium salts, ammonium salts, organic amine salts, or magnesium salts, or similar salts. When a compound of the present disclosure contains a relatively basic functional group, an acid addition salt can be obtained by contacting such a compound with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharma- ceutically acceptable acid addition salts include salts of inorganic acids, including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, phosphate monohydrate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, and the like; salts of organic acids, including acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, octanedioic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and other similar acids; salts of amino acids, such as arginine, and salts of organic acids, such as glucuronic acid. Certain compounds of the present disclosure contain basic and acidic functional groups and can therefore be converted into any base or acid addition salt.

[0096] The salts of the present disclosure can be synthesized by conventional chemical methods using parent compounds that contain acidic or basic groups. In general, the preparation of such salts is as follows: these compounds in their free acid or base form can be prepared by reacting them with the appropriate stoichiometric base or acid in water or an organic solvent or a mixture thereof.

[0097] The compounds of the present disclosure can be prepared by a number of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining with other chemical synthetic methods, and equivalent substitution methods known to those skilled in the art. Preferred embodiments include, but are not limited to, the examples of the present disclosure.

[0098] The compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compounds. For example, the compounds may contain tritium ( 3 H), iodine-125( 125 I), or C14( 14 As another example, hydrogen can be replaced with deuterium to form a deuterated drug. The bond formed by deuterium and carbon is stronger than the bond formed by normal hydrogen and carbon. Compared to non-deuterated drugs, deuterated drugs have the advantages of reducing toxic side effects, increasing drug stability, increasing efficacy, and extending drug half-life. All isotopic composition variations of the compounds of the present invention, whether radioactive or not, are included within the scope of the present invention.

[0099] When the direction of the linkage of the listed linking groups is not indicated, the direction of the linkage is arbitrary, for example: The linking group L in TIFF2025514400000033.tif11128 is -MW-, and at this stage, -MW- is linked to ring A and ring B with the same reading direction from left to right. TIFF2025514400000034.tif13128 can be constructed, or -MW- can be linked to ring A and ring B with the sequence reading in the opposite direction from left to right. TIFF2025514400000035.tif13128. Combinations of linking groups, substituents, and / or variants thereof are permissible only if such combinations result in stable compounds.

[0100] The term "optional" or "optionally" refers to the fact that the subsequent event or circumstance described may, but does not necessarily, occur, and the description includes circumstances under which the event or circumstance occurs as well as circumstances under which the event or circumstance does not occur.

[0101] The term "substituted" refers to the replacement of any one or more hydrogen atoms on a particular atom with a substituent. Substituents can 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 replacement does not occur in aromatic groups. The term "optionally substituted" refers to the situation where a group is substituted or unsubstituted, unless otherwise specified, and the type and number of substituents can be optional based on chemical feasibility.

[0102] When any variable (e.g., R) occurs more than one time in a composition or structure of a compound, its definition is independent at each occurrence. Thus, for example, if a group is substituted with 0-2 R, then that group may be optionally substituted with up to 2 R, with R having an independent option at each occurrence. Further, substituents and / or variants thereof are permissible only if such combinations result in stable compounds.

[0103] -(CRR) 0- When the number of linking groups is 0, it indicates that the linking group is a single bond.

[0104] If one substituent is empty, it indicates that the substituent is not present. For example, if X in AX is empty, it indicates that the structure is actually A. If the atom to which the listed substituents are attached is not shown, such a substituent can be attached through any atom. For example, a pyridyl group as a substituent can be attached to the substituent through any carbon atom on the pyridine ring.

[0105] The compounds of the present invention can be prepared by various synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining with other chemical synthetic methods, and equivalent substitution methods known to those skilled in the art. Preferred embodiments include, but are not limited to, the examples of the present invention.

[0106] The structure of the compound of the present invention can be confirmed by conventional methods well known to those skilled in the art. When the present invention involves the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the art. For example, in single crystal X-ray diffraction (SXRD) method, the diffraction intensity data of the cultured single crystal is collected using a Bruker D8 venture diffractometer; the light source is CuKα radiation; and the scanning method is φ / ω scanning; after collecting relevant data, the crystal structure can be further divided using direct method (Shelxs97) to confirm the absolute configuration.

[0107] The solvents used in this disclosure are commercially available.

[0108] Unless otherwise specified, all solvent ratios used for column chromatography and preparative thin layer silica gel chromatography in this disclosure are by volume.

[0109] The present invention employs the following abbreviations: aq stands for aqueous; HATU stands for O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate; EDC stands for N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride; m-CPBA stands for 3-chloroperoxybenzoic acid; eq stands for equivalent; CDI stands for carbonyldiimidazole; DCM stands for dichloromethane; PE stands for petroleum ether. DIAD represents diisopropyl azodicarboxylate; DMF represents N,N-dimethylformamide; DMSO represents dimethylsulfoxide; EtOAc represents ethyl acetate; EtOH represents ethanol; MeOH represents methanol; CBz represents the amine protecting group benzyloxycarbonyl; BOC represents the amine protecting group tert-butoxycarbonyl; HOAc represents acetic acid; NaCNBH3 represents sodium cyanoborohydride; cyanoborocyanide; rt represents room temperature; O / N represents overnight; THF represents tetrahydropyran; Boc2O represents di-tert-butyl dicarbonate; TFA represents trifluoroacetic acid; DLPEA represents diisopropylethylamine; SOCl2 represents thionyl chloride; CS2 represents carbon disulfide; TsOH represents p-toluenesulfonic acid; NFSI represents N-fluoro-N-(benzenesulfonyl)benzenesulfonamide; NCS represents 1-chloropyrrolidine-2,5-dione; n-Bu4NF represents tetrabutylamine fluoride; iPrOH represents 2-propanol; mp represents melting point; LDA represents lithium diisopropylamide.

[0110] Nucleotide monomer abbreviations are used in the description of the nucleic acid sequences, specifically as set forth in Table 2. [Table 2]

[0111] Compounds are named according to conventional naming conventions in the art or using ChemDraw® software, and names provided by the Suppliers Directory are adopted for commercially available compounds. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0112] The present invention will now be described in detail through examples, but no adverse limitations are implied for the present invention. The compounds of the present invention can be prepared by various synthesis methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining with other chemical synthesis methods, and equivalent replacement methods known to those skilled in the art. Preferred embodiments include, but are not limited to, the examples of the present invention. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention. EXAMPLES

[0113] Synthesis Example 1: Synthesis of Z1 and Z2 TIFF2025514400000037.tif75165 Step A: Compound 1-1 (10 g, 19.82 mmol) was dissolved in acetonitrile (120 mL) and 1,2-dichloroethane (80 mL), and compound 1-2 (6.02 g, 42.62 mmol) and trimethylsilyl trifluoromethanesulfonate (11.01 g, 49.56 mmol, 8.95 mL) were added to the above mixture at 0° C. The above system was stirred at 35° C. for 12 h. The stirred system was quenched by slowly adding saturated aqueous sodium bicarbonate solution (100 mL) and extracted with dichloromethane (100 mL×2) at 0° C., and the organic phase was washed with saturated aqueous sodium chloride solution (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column (eluent: petroleum ether / ethyl acetate=20:1) to give compound 1-3.

[0114] Step B: Compound 1-3 (6.7 g, 13.05 mmol) was dissolved in ammonia methanol solution (7 mol / L, 50 mL). The above system was stirred at 40° C. for 12 hours, and the reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column (eluent: ethyl acetate / methanol=50 / 1 to 10 / 1) to obtain compound 1-4.

[0115] Step C: Compound 1-4 (2.3 g, 11.43 mmol) was dissolved in anhydrous pyridine (25 mL), and 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (3.64 g, 11.55 mmol, 3.69 mL) was added at 0° C., and then the above system was stirred at 20° C. for 12 h. The stirred system was quenched with water (30 mL) and extracted with ethyl acetate (30 mL×2). The organic phase was washed successively with hydrochloric acid (1 mol / L, 30 mL×3) and saturated saline (30 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=7 / 1 to 5 / 1) to obtain compound 1-5.

[0116] Step D: Compound 1-5 (4 g, 9.02 mmol) was dissolved in acetonitrile (40 mL), and 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 above system was stirred at 25° C. for 12 hours. Ethyl acetate (50 mL) was added to the system, and the above material was stirred at 20° C. for 1 hour, filtered using a Buchner funnel containing diatomaceous earth, and the filtrate was collected. The filtrate was extracted with water (100 mL) and ethyl acetate (100 mL×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 by silica gel column (eluent, petroleum ether / ethyl acetate=15 / 1-10 / 1-8 / 1) to obtain compound 1-6.

[0117] Step E: Compound 1-6 (2 g, 4.37 mmol) was dissolved in anhydrous tetrahydrofuran (20 mL), and then triethylamine trihydrofluoride (1.55 g, 9.61 mmol, 1.57 mL) was added. The above system was stirred at 20° C. for 12 h, and the reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column (eluent: ethyl acetate / methanol=100 / 1 to 50 / 1) to obtain compound 1-7.

[0118] Step F: Compound 1-7 (1 g, 4.65 mmol) was dissolved in anhydrous pyridine (10 mL), and then 4,4-dimethoxytriphenylmethyl chloride (1.57 g, 4.65 mmol) was added. The above system was stirred at 20 °C for 12 h. The stirred system was quenched with water (20 mL) and extracted with ethyl acetate (20 mL × 2). The organic phase was 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 was purified by silica gel column (eluent: petroleum ether / ethyl acetate = 3 / 1 to 1 / 1) to obtain Z1. 1 H NMR (400 MHz, 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 = 6.5 Hz, 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).

[0119] Step G: 5 g of raw material Z1 (9.66 mmol, 1.0 equiv.) was dissolved in 50 mL of dichloromethane, 3.2 g of bis(diisopropylamino)(2-cyanoethoxy)phosphine (10.63 mmol, 1.1 equiv.) and 0.57 g of 4,5-dicyanoimidazole (4.83 mmol, 0.5 equiv.) were added, and the above materials were reacted at 25 ° C for 2 h. After the reaction was completed, the reaction was quenched with 20 mL of saturated sodium bicarbonate at 20-25 ° C, diluted with 20 mL of water, extracted with dichloromethane (50 mL × 2), the organic phases were combined, washed with saturated sodium chloride (30 mL × 2), dried over anhydrous sodium sulfate, filtered, and then the mother liquor was concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column (eluent, petroleum ether / ethyl acetate (0.1% triethylamine) = 1 / 0 to 1 / 1) to obtain 6.8 g of white solid powder Z2. 1H 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 = 4.2 Hz, 1H), 4.23-4.09 (m, 1H), 3.83-3.75 (m, 1H), 3.72 (d, J = 2.4 Hz, 6H), 3.61 - 3.45 (m, 3H), 3.38 (d,J = 15.2 Hz, 3H), 3.30-3.16 (m, 1H), 3.07 (br dd, J = 5.6, 10.4 Hz, 1H), 2.77 (t, J = 5.9 Hz, 1H), 2.63-2.55 (m,1H), 1.16-1.07 (m, 9H), 0.96 (d, J = 6.6 Hz, 3H).

[0120] Methods for preparing conjugates Preparation method: Oligoribonucleotides were synthesized using phosphoramidite solid-phase synthesis technology. Synthesis was performed on a solid support made by covalently linking controllable pore glass (CPG, 500 Å) with compound 2. All RNA phosphoramidites on the sequence were dissolved sequentially in anhydrous acetonitrile and molecular sieves (3 Å) were added. 5-Ethylthio-1H-tetrazole (ETT) was used as the activator, and the coupling time was 5 min. Phosphate bonds were generated using a 50 mM I2-water / pyridine (volume ratio 1 / 9) solution, and the reaction time was 3 min. Thiophosphate bonds were generated using a solution of 50 mM 3-((dimethylamino)methylene)amino)-3H-1,2,4-dithiazole-3-thioketone (DDT) in anhydrous acetonitrile / pyridine (volume ratio 1 / 1), and the reaction time was 3 min. After final removal of the DMT groups of the resulting sequence, the conjugates were synthesized.

[0121] Cleavage and deprotection of oligomers bound to CPG: After the solid-phase synthesis was completed, the CPG was treated with acetonitrile solution containing 10% diethylamine for 30 minutes to remove the protecting groups without cleaving the nucleotides from the CPG. The dried CPG was then treated with concentrated ammonia solution at 40°C for 18 hours. After centrifugation, the supernatant was transferred to a new tube and the CPG was washed with water. The combined solution was concentrated to obtain a solid mixture. The solid mixture was dissolved in DMSO (10V), then triethylamine hydrofluride (2V) was added, and the above materials were stirred at 40°C for 16 hours. After the reaction was completed, the reaction product was purified.

[0122] Purification of oligoribonucleotides: Oligomers were purified using NanoQ anion exchange HPLC. Buffer A was 10 mM sodium perchlorate solution, 20 mM Tris, 1 mM EDTA, pH 7.4, and 20% acetonitrile, and buffer B was 500 mM sodium perchlorate, 20 mM Tris, 1 mM EDTA, pH 7.4, and 20% acetonitrile. Target products were obtained by separation and desalting using a reversed-phase C18 column.

[0123] Oligoribonucleotides were annealed to generate siRNA: RNA oligomers to be annealed were prepared in 20 μM RNA oligomer solution using sterile RNase-free water (containing no RNA hydrolase). Equimolar RNA solutions were combined to form complementary strands. The annealing reaction system was set up as follows: 100 μL of 10 nmol mixed solution was placed in a 95°C water bath for 10 minutes, then immediately placed in a 60°C water bath, and then naturally cooled. The solution after annealing cannot be stored at high temperatures.

[0124] Examples 1 to 3 were carried out according to the above method, and the order of the raw materials was adjusted based on the order of nucleotides and Z shown in the sequence to prepare the target product.

[0125] Example 1: Preparation of Conjugate 1 Sense strand: TIFF2025514400000038.tif10160Antisense strand: TIFF2025514400000039.tif10156Z is TIFF2025514400000040.tif21128; wherein the structure of D01 is as follows: TIFF2025514400000041.tif52165, D01 was linked to a via a phosphodiester bond.

[0126] Example 2: Preparation of Conjugate 3 Sense strand: TIFF2025514400000042.tif10156Antisense strand: TIFF2025514400000043.tif10156Z is TIFF2025514400000044.tif21128; wherein the structure of D01 is as follows: TIFF2025514400000045.tif51165, D01 was linked to a via a phosphodiester bond.

[0127] Example 3: Preparation of Conjugate 2 Sense strand: TIFF2025514400000046.tif10156Antisense strand: TIFF2025514400000047.tif10156Z is TIFF2025514400000048.tif21128; wherein the structure of D01 is as follows: TIFF2025514400000049.tif54165, D01 was linked to a via a phosphodiester bond.

[0128] Bioassay Test Example 1: Free uptake experiment of PHH cells 1. Experimental principle: The samples to be tested were incubated with primary human hepatocytes. The degree of downregulation of AGT mRNA by the samples to be tested was evaluated.

[0129] 2. Experimental materials: Primary human hepatocytes (PHH), RNeasy® 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).

[0130] 3. Experimental Method: The complexes of the present invention were diluted with PBS solution so that the resulting concentration was 10 times that of the concentration to be tested. 10 uL of siRNA was transferred to a 96-well plate. PHH cells were thawed and seeded into a 96-well plate, with a final cell density of 5.4 x 10 5cells / well. The conjugates of the present invention were tested over 10 concentration points, with the highest concentration being 500 nM at 4-fold dilutions.

[0131] The cells were incubated at 37° C. and 5% CO 2 for 48 hours, and the state of the cells was observed using a microscope.

[0132] After the incubation was completed, the cells were lysed, and total RNA was extracted from the resulting lysate using RNeasy® 96 kit (QIAGEN-74182), and reverse transcribed to obtain cDNA using FastKing RT kit (with gDNase) (Tiangen-KR116-02). AGT cDNA was detected by qPCR.

[0133] 4. Experimental Results: The experimental results can be seen in Table 3. [Table 3]

[0134] 5. Experimental Conclusion: The conjugate of the present invention can significantly down-regulate the level of AGT mRNA in PHH cells, and has an excellent effect of reducing the level of AGT mRNA.

[0135] Test Example 2: Mouse hAGT HDI model 1. Experimental principle: The targeting of the target gene in the tested sample and the inhibitory effect on the target gene were evaluated by a mouse model in which the pcDNA-CMV-AGT plasmid was injected via the tail vein at high pressure.

[0136] 2. Experimental materials: pCDNA-CMV-AGT plasmid, BALB / c female mice, DPBS (Dulbecco's phosphate buffered saline), and the conjugate of the present invention.

[0137] 3. Experimental Method: Purchase 6-8 week old BALB / c female mice and allow the mice to adapt to quarantine for 1 week after arrival in the animal room.

[0138] On day 0, mice were randomly divided into groups according to body weight data, each group had 4 mice, and all mice were subcutaneously administered with a single dose of 10mL / kg after grouping. Group 1 mice were administered with DPBS; Group 2 mice were administered with AD-85481 at a dose of 5mg / kg; Group 3 mice were administered with the conjugate 3 of the present invention at a dose of 5mg / kg. Group 4 mice were administered with the conjugate 2 of the present invention at a dose of 5mg / kg.

[0139] On the third day after administration, all mice were injected with pcDNA-CMV-AGT plasmid via the tail vein within 5 seconds in a volume of 8% of the body weight (injection volume (mL) = mouse body weight (g) × 8%). The mass of plasmid injected into each mouse was 10 μg.

[0140] On the fourth day after administration, all groups of mice 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 the samples without RNAlater were then stored at -80°C to detect the expression level of the AGT gene.

[0141] 4. Experimental results: The results are shown in Table 4. In the table, the data represents the AGT mRNA expression in mouse liver after administration / AGT mRNA expression in mouse liver before administration. [Table 4]

[0142] 5. Experimental conclusion: The conjugate of the present invention can significantly down-regulate the expression of AGT mRNA in mouse liver, with the range of down-regulation being up to 97%.

[0143] Test Example 3: In vivo PD experiments in non-human primates (cynomolgus monkeys) 1. Experimental principle: The degree of downregulation of cynomolgus monkey plasma AGT by the samples tested was observed through a non-human primate cynomolgus monkey model in which the AGT gene is highly homologous to that in humans.

[0144] 2. Experimental materials: cynomolgus monkeys, 1x PBS (phosphate buffer solution), AD-85481 (US2021095290), and the conjugate of the present invention.

[0145] 3. Experimental Method: Ten cynomolgus monkeys weighing 2-3 kg were purchased and the animals were adapted to quarantine for 1 week after arrival in the animal room.

[0146] On day -6, the cynomolgus monkeys were randomly divided into groups according to body weight data, with each group containing two cynomolgus monkeys. After group division, the AGT protein concentrations in the plasma of all cynomolgus monkeys were detected and used as the experimental database line for each group.

[0147] Administration was carried out on day 1. Group 1 was administered 1xPBS; Group 2 was subcutaneously injected with AD-85481 at a dose of 0.3mpk. Group 3 was subcutaneously injected with AD-85481 at a dose of 3mpk. Group 4 was subcutaneously injected with conjugate 2 of the present invention at a dose of 0.3mpk. Group 5 was subcutaneously injected with conjugate 2 of the present invention at a dose of 3mpk. Blood was collected on days 1, 2, 4, 8, 15, 22, 29, 32, 35, 43, 57, and 71 after administration, and the AGT protein concentration in the plasma of each cynomolgus monkey was detected. The AGT protein concentration in the plasma after administration was compared with the AGT protein concentration on day -6 to obtain the relative downregulation rate of AGT protein in each animal group.

[0148] 4. Experimental results: The results are shown in Table 5. In the table, the data represents the AGT gene expression level after administration / the AGT gene expression level before administration. [Table 5]

[0149] 5. Experimental conclusion: The conjugate of the present invention can significantly down-regulate the concentration of AGT protein in the plasma of cynomolgus monkeys. Compared with the plasma AGT protein concentration on day -6, the AGT protein concentration on day 22 after single administration of 3mpk group was down-regulated by 97.53%, and the AGT protein concentration on day 71 was down-regulated by 90.12%.

[0150] Test Example 4: In vitro RNA sequencing studies 1. Experimental Introduction: Transcriptome refers to the sum of all RNA transcribed from a particular tissue or cell at a particular time or state, mainly including mRNA and non-coding RNA. Transcriptome sequencing is the study of all mRNA transcribed from a particular tissue or cell in a particular period based on the Illumina sequencing platform, which is the basis of gene function and structure research and plays an important role in understanding organism development and disease occurrence. With the development of gene sequencing technology and the reduction of sequencing costs, RNA-seq has become the main method for transcriptome research, with the advantages of high throughput, high sensitivity, and wide range of applications. The technical process of RNA-seq mainly includes two parts: library construction and sequencing, and biological information analysis.

[0151] 2. RNA Extraction and Detection: RNA was extracted from tissues or cells using standard extraction methods, and then stringent quality control was performed on the RNA samples, which was mainly achieved by Agilent 2100 Bioanalyzer, which accurately detected the integrity of the RNA.

[0152] 3. Library construction and quality inspection: The mRNAs were obtained mainly by the following two methods: one was to enrich for mRNAs containing polyA tails using oligo(dT) magnetic beads, taking advantage of the structural feature that most eukaryotic mRNAs have polyA tails; the other was to obtain mRNAs by removing ribosomal RNA from total RNA. The obtained mRNAs were then randomly fragmented using divalent cations in NEB fragmentation buffer, and library construction was performed using either the normal library construction method or the strand-specific library construction method of NEB.

[0153] NEB's regular library construction: The first strand of cDNA was synthesized with M-MuLV reverse transcriptase system using fragmented mRNA as template and random oligonucleotides as primers. Then, the RNA strand was digested using RNaseH, and the second strand of cDNA was synthesized with DNA polymerase I system using dNTPs as raw materials. The purified double-stranded cDNA was subjected to end repair, A-tail addition, and sequencing linker ligation. Approximately 250-300 bp of cDNA was screened using AMPure XP beads and subjected to PCR amplification, and the PCR products were purified again using AMPure XP beads to finally obtain the library. The kit for library construction was NEBNext® Ultra® RNA Library Prep Kit for Illumina®.

[0154] Strand-specific library construction: The method for synthesizing the first strand of cDNA was the same as NEB's normal library construction method, except that when the second strand was synthesized, dTTP in dNTP was replaced with dUTP, and then end repair, A-tail addition, sequencing linker ligation, and cDNA length screening were performed in the same way. Then, the second strand of cDNA containing U was digested using USER enzyme, followed by PCR amplification to obtain a library. Strand-specific library construction has many advantages, such as more effective information can be obtained with the same amount of data; more accurate gene quantification, localization, and annotation information can be obtained; and antisense transcripts and the expression levels of single exons in each isoform can be provided. The kit used for library construction was NEB Next® Ultra™ Directional RNA Prep Kit for Illumina®.

[0155] NOTE: The sequencing linker is composed of three components, namely P5 / P7, index, and Rd1 / Rd2 SP. Among these, P5 / P7 is the part where the PCR amplification primer and the primer on the flow cell bind, the index provides information to distinguish different libraries, and Rd1 / Rd2SP, i.e. the read1 / read2 sequence primers, is the region where the sequencing primer binds. The sequencing process theoretically starts from Rd1 / Rd2 SP in the reverse direction.

[0156] Once library construction was completed, preliminary quantification was performed using a Qubit2.0 fluorometer. The library was diluted to 1.5ng / ul, and then an Agilent 2100 bioanalyzer was used to detect the insert size of the library. After the insert size was as expected, the effective concentration of the library was accurately quantified by QRT-PCR (the effective concentration of the library was higher than 2nM) to ensure the quality of the library.

[0157] 4. On-machine sequencing: After the library test was qualified, the different libraries were subjected to Illumina sequencing after pooling according to the requirements of effective concentration and target offline data amount. The basic principle of sequencing was sequencing by synthesis. Four fluorescently labeled dNTPs and DNA polymerase, as well as a linker primer, were added to the sequencing flow cell for amplification. When the complementary strand was extended in each sequencing cluster, the corresponding fluorescence was released when the fluorescently labeled dNTPs were added, and the sequencer captured the fluorescent signal and converted the light signal into a sequencing peak through computer software to obtain the sequence information of the fragment being tested.

[0158] 5. Experimental Results: The results are shown in Table 6. [Table 6]

[0159] 6. Experimental Conclusion: The conjugate of the present invention has low off-target risk.

[0160] Test Example 5 Agonistic activity of the conjugate of the present invention against hTLR3, hTLR7, and hTLR8 cells 1. Experimental Materials [Table 7]

[0161] 2. Experimental Apparatus The main equipment used in this study was the multifunctional microplate reader Flexstation III (Molecular Device) and Echo555 (Labcyte).

[0162] 3. Experimental Steps 1) The conjugate of the present invention was diluted to a concentration 20 times the final concentration, and the diluted conjugate was mixed with RNAiMAX-OPTI MEM (RNAiMAX:OPTI MEM=3:47) in a 1:1 ratio and incubated at room temperature for 15 minutes. 2) To the corresponding cell plates, 10 μL of incubated conjugate of the invention was added for a total of 10 concentrations, with the highest concentration being 200 nM, with duplicate wells for each concentration. 3) 10 μL of PBS was added to each of the negative control wells, 10 μL of 20 μg / mL poly(I:C) HMW was added to each of the hTLR3 positive control wells, and 10 μL of 10 μg / mL R848 was added to each of the hTLR7 and hTLR8 positive control wells. 10 μL of 0.5 μM BLOCK-iT™ Alexa Fluor® red fluorescent control was added to the two transfection positive control wells. 4) 90 μL of cells were seeded into 96-well plates containing the conjugate at a density of 50,000 cells / well. 5) The conjugate of the present invention and the cells were co-incubated for 24 hours in an incubator under conditions of 37° C. and 5% CO2. 6) The transfection effect of the transfection positive control wells was observed using a fluorescent microscope and photographed. 7) Compound activity detection: 20 μL of cell supernatant from each well was taken and added to an experimental plate containing 180 μL of QUANTI-Blue™ reagent, then incubated at 37° C. for 1 hour, followed by detecting the absorbance value at 650 nm (OD650) using a multifunctional microplate reader FlexstationIII. 8) Cell activity detection: The procedure was performed according to the Celltiter-Glo specifications, and the chemiluminescence signal (RLU) was detected using the multifunctional microplate reader FlexstationIII.

[0163] 4. Data Analysis Compound activity: OD650 values ​​were analyzed using GraphPad Prism software and dose-effect curves of compounds were fitted to determine the EC 50The values ​​were calculated. Cellular activity detection: The calculation formula for % cellular activity was as follows: GraphPadPrism software was used to analyze the % cellular activity values, fit the dose-effect curves of the compounds, and calculate the CC of the compounds on the cells. 50 values ​​were calculated. Cellular activity %=RLU compound / RLU DMSO control * 100%

[0164] 5. Experimental results: The experimental results are shown in Tables 8 to 10.

[0165] [Table 8]

[0166] [Table 9]

[0167] [Table 10]

[0168] 5. Experimental Conclusion: The conjugates of the present invention do not induce hTLR3, hTLR7, and hTLR8 responses under the experimental conditions, indicating that the conjugates of the present invention have a low immunogenicity risk.

[0169] Test Example 6. Compound activity experiments in Huh7 cell line 1. Objective: This study aims to evaluate the inhibitory effect of the conjugate described in this patent on the target gene using Huh7 cell line. The inhibitory activity of the compound on the target gene AGT is evaluated using the EC 50 Evaluate based on value.

[0170] 2. Experimental materials: 2.1 Cell line: Huh7 cells and the conjugate of the invention Huh7 cells were provided by Shanghai WuXi AppTec New Drug Development Co., Ltd. Hub7 cells were cultured in MEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin.

[0171] 2.2 Reagents: The main reagents used in this experiment include Lipofectamine™ iRNAiMAX Transfection Reagent, FastStart Universal Probe Mast, RNA Extraction Kit, GAPDH Gene Expression Kit, AGT Gene Expression Kit, FastKing cDNA First Chain Synthesis Kit, and 96-well plates.

[0172] 2.3 Consumable materials and equipment: The main equipment used in this experiment included a fluorescent qPCR instrument (Applied Biosystems, model QuantStudio 6 Flex) and a cell counter (Vi-cell™ XR).

[0173] 3. Experimental steps and methods: 3.1 Transfection 1) On day 1, prepare a mixed solution of RNAiMAX transfection reagent and Opti-MEM as needed at a ratio of RNAiMAX transfection reagent:Opti-MEM=1.5:48.5 and incubate at room temperature for 15 minutes; 2) For each well of cells, a certain amount of diluted compound was taken and added to an equal amount of RNAiMAX Optim-MEM medium, mixed well, and then incubated for 15 minutes; 3) Huh7 cells were harvested, washed with DPBS, digested with trypsin, and the cell density was adjusted to an appropriate density; 4) Cells were seeded in 96-well plates at a density of 2,0000 cells / well while 20 μL of Opti-MEM RNAiMax and compound mixture was added to the cell culture plate, and the final medium volume in each well was 120 μL. 5) The cells were incubated in an incubator under conditions of 5% CO2 and 37°C for 24 hours.

[0174] 3.2 RNA extraction and reverse transcription The cells were harvested, and RNA was extracted according to the instructions of the Qiagen-74182 RNA extraction kit, and then cDNA was obtained according to the instructions of the FastKing cDNA First Chain synthesis kit.

[0175] 3.3 qPCR detection The cDNA of the target genes was detected by qPCR. qPCR was performed in 384-well plates using GAPDH as the internal reference gene. The qPCR reaction procedure was as follows: heating at 95°C for 10 min, then entering cycle mode, heating at 95°C for 15 s, heating at 60°C for 1 min, and performing a total of 40 cycles.

[0176] 3.4 Data analysis: ΔΔCt method (Ct difference comparison method) This method requires the introduction of an internal reference gene, GAPDH, because it is expressed in all cells and its product is necessary to maintain cell survival; the expression level or copy number of the genome in the cells remains constant and is less affected by environmental factors. After qRT-PCR, the CT value of the internal reference is also recorded and is called Ct(GAPDH), and the CT value of the sample is called Ct(sample). ΔCt(sample)=Ct(sample)-Ct(GAPDH) ΔCt(control)=Ct(control)-Ct(GAPDH) ΔΔCt(sample)=ΔCt(sample)-ΔCt(control) Relative expression level of a gene = 2^-ΔΔCt

[0177] 4. Experimental Results: The experimental results are shown in Table 11. [Table 11]

[0178] 5. Experimental conclusion: The conjugate of the present invention can significantly down-regulate the level of AGT mRNA in Huh7 cells, and has an excellent effect of lowering AGT mRNA.

Claims

1. A conjugate of a double-stranded RNAi analog or a pharma- ceutically acceptable salt thereof, wherein the double-stranded RNAi analog comprises a sense strand and an antisense strand capable of forming a double-stranded region, wherein the sense strand is and the antisense strand is and Here, Z is and the 5'-end and / or the 3'-end of the sense strand and / or the antisense strand are conjugated to a ligand, and the ligand is one or more GalNAc derivatives linked via a bivalent, trivalent or tetravalent branched linker; The conjugate or a pharma- ceutically acceptable salt thereof.

2. A conjugate of a double-stranded RNAi analog or a pharma- ceutically acceptable salt thereof, wherein the double-stranded RNAi analog comprises a sense strand and an antisense strand capable of forming a double-stranded region, wherein the sense strand is and the antisense strand is and Where: Z is, and the 5'-end and / or the 3'-end of the sense strand and / or the antisense strand are conjugated to a ligand, and the ligand is one or more GalNAc derivatives linked via a bivalent, trivalent or tetravalent branched linker; The conjugate or a pharma- ceutically acceptable salt thereof.

3. A conjugate of a double-stranded RNAi analog or a pharma- ceutically acceptable salt thereof, wherein the double-stranded RNAi analog comprises a sense strand and an antisense strand capable of forming a double-stranded region, wherein the sense strand is and the antisense strand is and Here, Z is and the 5'-end and / or the 3'-end of the sense strand and / or the antisense strand are conjugated to a ligand, and the ligand is one or more GalNAc derivatives linked via a bivalent, trivalent or tetravalent branched linker; The conjugate or a pharma- ceutically acceptable salt thereof.

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

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

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

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

8. 4. The conjugate according to claim 1, or a pharma- ceutically acceptable salt thereof, wherein the conjugate is Conjugate 1: Conjugate 2: Conjugate 3: is selected from wherein the structure of D01 is as follows: , wherein D01 is linked to a via a phosphodiester bond; The conjugate or a pharma- ceutically acceptable salt thereof.

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