Chemically modified siRNA molecule and application thereof

Abstract

The invention belongs to the field of nucleic acid medicines, and particularly relates to a chemically modified siRNA molecule and application thereof to disease treatment. The present disclosure improves the efficacy and / or activity duration of in vivo activity of siRNA molecules based on the design of specific chemical modification patterns of the siRNA molecules. The specific modification mode can be universally applied to siRNA molecules with different sequences and targets, and is used for inhibiting in-vivo target gene expression, so that the treatment purpose is achieved.

Description

Technical Field

[0001] This disclosure relates to the field of nucleic acid medicine, specifically to a chemically modified siRNA molecule and its applications. Background Technology

[0002] RNA interference (RNAi) therapy has proven to be a promising class of treatments, especially for targets that are considered untreatable by small molecule therapies.

[0003] To improve the stability and tolerability of siRNA molecules and reduce toxic side effects, existing technologies overcome the inherent problems of natural RNA through chemical modification, such as reducing the 2'-F content and adjusting the 2'-OMe. Although these adjustments to the 2'-OMe and 2'-F positions have shown enhanced in vivo efficacy and stability, antisense strand modification, which demands higher nuclease resistance, remains an important direction in siRNA modification research. In particular, factors that significantly influence nuclease resistance, such as antisense strand terminal modification and intra-chain phosphate thioester combination modifications, still require further optimization to achieve enhanced in vivo activity and long-term stability of siRNA. Summary of the Invention

[0004] This disclosure, based on the design of specific chemical modification patterns for siRNA molecules, improves the potency and / or duration of activity of siRNA molecules in vivo. The specific modification patterns described herein can be universally applied to siRNA molecules with different sequences and targets, and used to inhibit the expression of target genes in vivo, thereby achieving therapeutic purposes.

[0005] The objective of this disclosure is achieved through the following technical solution: In a first aspect, this disclosure provides an siRNA molecule containing specific chemical modifications, said siRNA comprising a sense strand and an antisense strand, said sense strand and said antisense strand being at least partially anticomplementary to form a double-stranded region having 17-23 base pairs; and said nucleotides in said antisense strand having a combination of the following chemical modification characteristics: (1) The antisense strand, starting from the 5' end, consists of nucleotides at positions 2, 6, 9, 14 and 16 that are 2′-fluorinated nucleotides, and nucleotides at the remaining positions that are 2′-nonfluorinated nucleotides; (2) The antisense strand contains five or more phosphate thioester nucleoside bonds between nucleotides, and: starting from the 5' end, the nucleoside bond between the 10th and 11th nucleotides is selected from phosphate thioester nucleoside bonds; as well as: (3) The two nucleotides at the 3' end of the antisense strand are selected from stable nucleotides, which are selected from [2'-R] a -2'-R b nucleotides modified with ]; where [2'-R a -2'-R b ] represents the disubstituted group at the 2'-position of the ribose ring, R a Selected from F and C 1-3 Alkyl or C 1-3 Alkoxy, R b Selected from C 1-3 Alkyl or C 1-3 Alkyl group.

[0006] In some specific embodiments of this disclosure, the first nucleotide of the antisense strand, starting from the 5' end, is selected from a nucleotide modified with a 5'-phosphate analogue, wherein the 5'-phosphate analogue-modified nucleotide is selected from any nucleotide with the following structure: , , , , , , , ; Wherein, Base represents a nucleoside base, which is selected from A, U, G, C or T; and R substituents are selected from -H, -OH, -CH3, -OCH3 or -F.

[0007] In some embodiments of this disclosure, the 5'-phosphate analog-modified nucleotide is selected from any of the following modified uridine nucleotides: , , , or .

[0008] In some embodiments of this disclosure, R a Selected from F or methyl, R b Selected from methyl or methoxy.

[0009] In some embodiments of this disclosure, R a Selected from F, R b Selected from methyl.

[0010] In some embodiments of this disclosure, the nucleotides that pair with the nucleotides at positions 10-13 of the sense strand (i.e., the para nucleotides in the double-stranded region) via base complementarity with the nucleotides at positions 10-13 of the antisense strand starting from the 5' end are 2′-fluorinated nucleotides, and the remaining positions are 2′-nonfluorinated nucleotides.

[0011] In some embodiments of this disclosure, the antisense strand contains five phosphate thionucleotide internucleotide bonds, and the antisense strand contains two consecutive phosphate thionucleotide internucleotide bonds between the terminal nucleotides at the 3' and 5' ends, respectively. The positive chain contains three thiophosphate nucleoside bonds, located between the first and second nucleotides starting from the 5' end, between the second and third nucleotides, and between the first and second nucleotides starting from the 3' end.

[0012] In some embodiments of this disclosure, the sense strand has 19 modified nucleotides and the antisense strand has 21 modified nucleotides; Furthermore, the justice chain and the antisense chain have the following combined modification patterns: Chain of Justice: 5'-NmsNmsNmNmNmNmNfNfNfNfNmNmNmNmNmNmNmNmNmsNm-3' antisense chain: 5'-PNmsNfsNmNmNmNfNmNmNfNmsNmNmNmNfNmNfNmNmNmsNdsNd-3'; or The sense strand has 21 modified nucleotides, and the antisense strand has 23 modified nucleotides; Furthermore, the justice chain and the antisense chain have the following combined modification patterns: Chain of Justice: 5'-NmsNmsNmNmNmNmNmNmNfNfNfNfNmNmNmNmNmNmNmNmsNm-3' antisense chain: 5'-PNmsNfsNmNmNmNfNmNmNfNmsNmNmNmNfNmNfNmNmNmNmNmsNdsNd-3'; Wherein, Nm represents any nucleotide modified with 2'-methoxy, Nf represents any nucleotide modified with 2'-fluoride, Nd represents any nucleotide modified with 2'-F-2'-CH3 disubstituent, the lowercase letter s indicates that the two nucleotides adjacent to the letter s are connected by a thiophosphate subunit, and P indicates that the nucleotide adjacent to the letter P to the right is any nucleotide modified with the 5'-phosphate analogue (i.e., represented by PN).

[0013] In some specific embodiments of this disclosure, the siRNA molecule further comprises a ligand covalently linked to the sense strand via a linker group; the ligand is targeted for delivery to hepatocytes or extrahepatic tissue cells.

[0014] In a second aspect of this disclosure, a pharmaceutical composition is provided comprising a siRNA molecule as described above, and a pharmaceutically acceptable carrier or excipient.

[0015] In a third aspect of this disclosure, the disclosure provides the use of the siRNA molecule as described above in the preparation of a medicament for inhibiting the expression of a target gene in a subject in need.

[0016] The effective amount of the double-stranded oligonucleotides or pharmaceutical compositions described in this disclosure may vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, such as four times a day, three times a day, twice a day, once a day, or every other day, or the number of daily doses may be proportionally reduced.

[0017] The drug may be administered to the subject via any suitable route known in the art, including but not limited to: oral or parenteral routes, including intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, airway administration (aerosol), pulmonary administration, nasal administration, rectal administration, and local administration (including oral administration and sublingual administration), with preferred routes of administration selected from intravenous injection.

[0018] This disclosure, based on the design of specific chemical modification patterns for siRNA molecules, improves the potency and / or duration of activity of siRNA molecules in vivo. The specific modification patterns described herein can be universally applied to siRNA molecules with different sequences and targets, and used to inhibit the expression of target genes in vivo, thereby achieving therapeutic purposes. Attached Figure Description

[0019] Figure 1 This refers to the relative expression level of the target gene ApoB in mouse liver tissue after administration of the siRNA described in this embodiment.

[0020] Figure 2 This refers to the relative expression level of the target gene in HuH1 cells after siRNA transfection with 0.5 nM as described in this embodiment.

[0021] Figure 3 This refers to the relative expression level of the target gene in HuH1 cells after siRNA transfection with 0.5 nM as described in this embodiment. Detailed Implementation

[0022] The technical solutions in the embodiments of this disclosure will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this disclosure, and not all of them. Based on the embodiments in this disclosure, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this disclosure.

[0023] Unless otherwise indicated, the practice of this disclosure will employ conventional techniques within the scope of the art, including molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology.

[0024] In the following embodiments, unless otherwise specified, the terms used are explained as follows: In this disclosure, unless otherwise specified, the terms “comprising” or “including” are open-ended expressions used to refer to the phrase “including but not limited to” and are used interchangeably with it, meaning that they include the contents specified in this disclosure but do not exclude other contents.

[0025] In this disclosure, unless otherwise specified, the terms "double-stranded oligonucleotide," "dsRNA," or "siRNA" refer to a ribonucleic acid molecule or ribonucleic acid molecule complex containing the double-stranded region defined above. The two parts forming the double-stranded region can be two different parts of a larger RNA molecule, or they can be separate RNA molecules. When the two parts are separate RNA molecules, the dsRNA described herein is referred to as small interfering RNA or short interfering RNA, or simply siRNA.

[0026] "Double-stranded oligonucleotides" or "siRNAs" are interchangeable terms. They are a class of double-stranded RNA molecules containing a sense strand and an antisense strand, which mediate the silencing of a target RNA (e.g., mRNA) that is complementary or substantially complementary to the antisense strand. siRNAs are typically double-stranded, consisting of an antisense strand complementary to the target RNA and a sense strand complementary or substantially complementary to that antisense strand. Such genes are also called target genes.

[0027] In this disclosure, unless otherwise specified, the term "siRNA" is a double-stranded RNA of 17 to 25 nucleotides in length, comprising a sense strand and an antisense strand. siRNA mediates targeted cleavage of RNA transcripts via the RISC pathway by forming an RNA-induced silencing complex (RISC). Specifically, siRNA directs the specific degradation of mRNA sequences through a known RNA interference (RNAi) process, inhibiting the translation of mRNA into amino acids and its conversion into proteins.

[0028] In this disclosure, unless otherwise specified, the terms "sequence" and "nucleotide sequence" refer to a sequence of nucleobases or nucleotides. As used herein, "base," "nucleotide base," or "nucleobase" is a pyrimidine or purine compound that is a component of a nucleotide and includes purine bases adenine and guanine, and pyrimidine bases cytosine, thymine, and uracil. Nucleobases may be further modified. The synthesis of modified nucleobases (including phosphorous amide compounds of modified nucleobases) is known in the art.

[0029] In this disclosure, unless otherwise specified, the terms "double-stranded oligonucleotide" and "siRNA" are used interchangeably, referring to a double-stranded structure formed by partial or complete base pairing of two oligonucleotides. The two oligonucleotides include a sense strand and an antisense strand, which may or may not be of the same length. As long as at least some base-pairing regions exist to form a double-stranded region, the oligonucleotide with a double-stranded structure is considered a double-stranded oligonucleotide as described in this disclosure. The nucleotides constituting the double-stranded oligonucleotide in this disclosure can be modified or unmodified nucleotides. When referring to modified nucleotides, unless otherwise specified, the modification does not specifically refer to the modified site. In addition to the modification of the nucleotides, the linking bonds between the nucleotides in the double-stranded oligonucleotide in this disclosure can also be modified. Double-stranded oligonucleotides containing modified linking bonds between nucleotides also belong to the double-stranded oligonucleotides described in this invention. Besides the nucleotide portion, the double-stranded oligonucleotide in this disclosure may also contain compound molecules or modifiers acceptable in the art to improve the properties of the double-stranded oligonucleotide, such as linking ligands to form conjugates.

[0030] In this disclosure, unless otherwise specified, the term "antisense strand (or guide strand)" includes a region substantially complementary to a target sequence. The term "sense strand (or follower strand)" refers to an iRNA strand containing an iRNA strand substantially complementary to the antisense strand. The term "substantially complementary" means completely complementary or at least partially complementary, for example, the antisense strand being completely complementary or at least partially complementary to the target sequence. In the case of partial complementarity, mismatches may be present in the internal or terminal regions of the molecule, wherein the most tolerant mismatches are present in the terminal regions, for example, within 5, 4, 3, or 2 nucleotides at the 5'- and / or 3' ends of the iRNA. It should be noted that "at least partially substantially complementary" of the antisense strand to the mRNA means that the antisense strand has a polynucleotide substantially complementary to a continuous portion of the mRNA of interest.

[0031] In this disclosure, the terms “complementary,” “fully complementary,” “partially complementary,” and “substantially complementary” are used to refer to nucleobase or nucleotide matching between the sense and antisense strands of an RNAi agent, or between the antisense strand of an RNAi agent and the sequence of the target mRNA.

[0032] Unless otherwise specified, the term "complementarity" refers to the ability of an oligonucleotide in the first sequence to hybridize with an oligonucleotide in the second sequence under certain conditions and form a double-stranded structure. "At least partially complementary" means that the two sequences can be completely complementary, or have no more than 5, 4, 3, or 2 mismatched base pairs in total, while retaining the ability to hybridize under the relevant conditions. Furthermore, in cases where two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs should not be considered mismatches for determining complementarity. Correspondingly, in this invention, unless otherwise specified, "mismatch" means that in the siRNA double-stranded molecule, the bases at corresponding positions are not paired in a complementary manner.

[0033] In this disclosure, unless otherwise specified, the term "complete complementarity" means that in a hybridized nucleobase or nucleotide sequence pair, all (100%) bases of the sequential sequence of the first oligonucleotide will hybridize with the same number of bases of the sequential sequence of the second oligonucleotide. The sequential sequence may comprise all or part of the first or second nucleotide sequence. "Partial complementarity" means that in a hybridized nucleobase or nucleotide sequence pair, a majority, such as at least 70% (but not all), of the bases of the sequential sequence of the first oligonucleotide will hybridize with the same number of bases of the sequential sequence of the second oligonucleotide. The sequential sequence may comprise all or part of the first or second nucleotide sequence.

[0034] In this disclosure, unless otherwise specified, the terms "ligand" or "conjugation group" refer to an atom or group of atoms that binds to an oligonucleotide or other oligomer. Generally, a conjugation group modifies one or more properties of the compound to which it is linked, including but not limited to pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge, and / or clearance properties. When referring to a link between two molecules, the term "link" as used herein means that the two molecules are directly or indirectly connected by a covalent bond, or that the two molecules are associated by a non-covalent bond (e.g., a hydrogen bond or an ionic bond).

[0035] In this disclosure, unless otherwise specified, the terms “link” or “association” when referring to a link between two compounds or molecules mean that the two molecules are linked by a covalent bond or associated via a non-covalent bond (e.g., a hydrogen bond or an ionic bond). Unless otherwise specified, the terms “link” and “association” as used in this disclosure may refer to a link between a first compound and a second compound, with or without any inserted atoms or groups of atoms.

[0036] In this disclosure, a linking group is one or more atoms that connect one molecule or a portion of a molecule to another second molecule or a second portion of a molecule. A linking group can contain any number of atoms or functional groups. In some embodiments, the linking group is used solely to link two bioactive molecules.

[0037] In this disclosure, the term "unsubstituted" means that the specified group does not contain substituents.

[0038] In this disclosure, the terms “substituted,” “replaced,” and “substituted” are used interchangeably to indicate that any one or more hydrogen atoms in the given structure are replaced by a specific substituent (e.g., a C1-3 alkyl, C1-3 alkoxy, or halogen) provided that the substitution does not exceed the normal valence of the specified atom and yields a stable compound. Unless otherwise indicated, a substituted group may have one substituent at each substituted position of the group. When more than one position in the given structural formula can be replaced by one or more substituents selected from a specific group, the substituents may be substituted at the substituted positions in the same or different manner.

[0039] In this disclosure, "phosphate diester nucleoside inter-bond" and "phosphate diester bond" can be used interchangeably, and their structural formula is as follows.

[0040] In this disclosure, "thiophosphate nucleoside internucleotide bond" and "thiophosphate bond" can be used interchangeably, and their structural formula is as follows: The structural formula of the "dithiophosphate nucleoside internucleotide bond" is: .

[0041] In this disclosure, the structural formula of the nucleotide modified with 2'-O-methyl is as follows: In this context, Base represents a nucleoside base, such as A, U, G, C, or T; Z is selected from -OH, -O-, -SH, or -S-.

[0042] In this disclosure, the structural formula of the nucleotide modified with 2'-O-methoxyethyl is as follows: In this context, Base represents a nucleoside base, such as A, U, G, C, or T; Z is selected from -OH, -O-, -SH, or -S-.

[0043] In this disclosure, the structural formula of the 2'-fluorinated nucleotide is as follows: , Where Base represents a nucleoside base, such as A, U, G, C or T; Z is selected from -OH, -O-, -SH or -S-.

[0044] In this disclosure, [2'-R a -2'-R b [] represents a disubstituted group at the 2'-position of the ribose ring, for example: the structural formula of a nucleotide modified with 2',2'-[F,CH3] disubstituted group is... Wherein, Base represents a nucleoside base, which is selected from A, U, G, C, or T.

[0045] In this disclosure, the term "pharmaceutical composition" or "composition" can refer to something used for the treatment of a disease or for use in in vitro cell culture experiments. When used for the treatment of a disease, the term "pharmaceutical composition" generally refers to a unit dose form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with excipients constituting one or more adjunct components. Typically, compositions are prepared by uniformly and adequately combining active siRNA with liquid excipients, finely pulverized solid excipients, or both.

[0046] In this disclosure, the term "pharmaceutical acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components of the formulation and / or the mammals to which it is treated. Preferably, "pharmaceutical acceptable" as used in this disclosure means approved by a federal regulatory agency or national government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals, particularly in humans.

[0047] In this disclosure, the term "pharmaceutically acceptable carrier or excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for a specific target dosage form. The use of any conventional excipients that are incompatible with the siRNA of this disclosure, such as those that produce any adverse biological effects or interactions with any other component of the pharmaceutically acceptable composition in a harmful manner, is also within the scope of this disclosure.

[0048] In this disclosure, the term "subject" refers to any animal being examined, studied, or treated, and is not intended to limit this disclosure to any particular type of subject. In some embodiments, humans are preferred subjects. In other embodiments, non-human animals are preferred subjects, including but not limited to mice, monkeys, ferrets, cattle, sheep, goats, pigs, chickens, turkeys, dogs, cats, horses, and reptiles. In still other embodiments, cells are preferred subjects.

[0049] In this disclosure, the terms “treatment,” “relief,” or “improvement” are used interchangeably. These terms refer to methods of achieving beneficial or desired outcomes, including, but not limited to, treatment benefits. A “treatment benefit” means the eradication or improvement of the underlying disorder being treated. Here, a treatment benefit is achieved by eradicating or improving one or more physical symptoms associated with the underlying disorder, thereby observing improvement in the subject, although the subject may still be suffering from the underlying disorder.

[0050] In this disclosure, the terms “prevention” and “avoidance” are used interchangeably to refer to methods for obtaining beneficial or desired results, including but not limited to preventive benefits. To obtain a “preventive benefit,” the conjugate, RNAi reagent, or composition may be given to a subject at risk of developing a specific disease, or to a subject who reports one or more physiological symptoms of a disease, even if a diagnosis of the disease may not have been made.

[0051] In this disclosure, the term "administration" generally refers to the introduction of a pharmaceutical preparation of this disclosure into the body of a subject by any route of introduction or delivery. Any method known to those skilled in the art for contacting cells, organs, or tissues with the drug may be employed. Administration may include, but is not limited to, intravenous, intra-arterial, intranasal, intraperitoneal, intramuscular, subcutaneous, or oral administration. A daily dose may be divided into one, two, or more doses in suitable forms to be administered at one, two, or more times during a period of time.

[0052] As in this disclosure, the term "regulation of gene expression" means that the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, is upregulated or downregulated such that the expression, level, or activity is greater or less than that observed in the absence of a regulator. For example, the term "regulation" may mean "inhibition," but the use of the word "regulation" is not limited to this definition.

[0053] In this disclosure, unless otherwise specified, the term "suppression" refers to a downregulation of target gene expression due to siRNA-mediated mRNA degradation. "Downregulation" means a decrease in target gene expression level of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more relative to the absence of siRNA treatment.

[0054] In this disclosure, the term "effective amount" means an amount sufficient to treat a disease when administered to a subject for the purpose of treating that disease.

[0055] In addition to any conventional excipients, the use of any range of siRNAs incompatible with the present disclosure, such as any adverse biological effects produced or interactions with any other component of a pharmaceutically acceptable composition in a harmful manner, is also within the scope of this disclosure.

[0056] The present disclosure is further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present disclosure in any way.

[0057] Unless otherwise stated, the siRNA sequences used in this disclosure were all synthesized by Suzhou Xuanjing Biotechnology Co., Ltd. and Suzhou Beixin Biotechnology Co., Ltd.; the PCR primers used in this disclosure were all synthesized by Beijing Qingke Biotechnology Co., Ltd.; the experimental animals C57BL / 6J mice used in this disclosure were all purchased from Spiford (Beijing) Biotechnology Co., Ltd., and the HuH1 cells were purchased from Wuhan Pronosai Life Science Technology Co., Ltd.

[0058] Unless otherwise stated, the CPG vector (loading capacity 80 μmol / g) used in this disclosure was purchased from Beijing Coupling Technology Co., Ltd. The CPG vector is denoted as […]. ; This refers to a glass sphere with a controllable aperture (CPG).

[0059] Unless otherwise stated, all reagents, reagent consumables, and instruments used in this disclosure are commercially available. The main reagents are shown in Table 1, the main reagent consumables in Table 2, and the main instruments in Table 3.

[0060] Table 1. Main Reagents Table 2. Main Reagents and Consumables Table 3. Main Instruments and Equipment Some of the compounds disclosed herein can be prepared by referring to the preparation methods in the patent applications shown in Table 4.

[0061] Table 4. Compound structures, names, and related patent application publication numbers Preparation Example 1: Preparation of Compound NM151 In this preparation example, the synthetic route of compound NM151 is as follows: (1-1) Synthesis of compound NM151-2 Compound 1 (4 g, 10.75 mmol, 1.0 eq) was dissolved in tetrahydrofuran (10 mL, THF), and NaH (1.2 g, 32.2 mmol, 3.0 eq) was added. The mixture was purged with nitrogen three times and stirred at 0 °C for 1 hour. Then, diethyl p-toluenesulfonyloxymethylphosphonate (8 g, 26.875 mmol, 2.5 eq) was added. After the reaction was complete, the mixture was brought to room temperature, and extracted with saturated ammonium chloride aqueous solution (50 mL). The extract was then washed with saturated sodium chloride aqueous solution (20 mL × 3), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reversed-phase column chromatography (elution gradient, acetonitrile:water = 1:1, v / v) to give compound NM151-2 (3.3 g, yield 58.9%). MS ESI (m / z) = 523.2 [M + H] + .

[0062] (1-2) Synthesis of compound NM151-3 Compound NM151-2 (1.6 g, 3.06 mmol, 1.0 eq) was dissolved in THF (16 mL), and tetrabutylammonium fluoride (0.7 mL, 2 M, 4.0 eq, abbreviated as TBAF) (0.7 mL, 2 M, 4.0 eq) was added. The mixture was stirred at 25 °C for 2–3 hours. After the reaction was complete, the mixture was purified by reverse-phase column chromatography (elution gradient, acetonitrile:water = 1:2, v / v) to give 670 mg of compound NM151-3 (yield 55.8%). MS ESI (m / z) = 409.1 [M + H] + .

[0063] (1-3) Synthesis of compound NM151 Compound NM151-3 (0.67 g, 1 eq) was dissolved in 10 mL of anhydrous dichloromethane. 4,5-Dicyanimidazole (130 mg, 0.8 eq, abbreviated as DCI) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (630 mg, 1.5 eq, CAS No. 102691-36-1) were added separately. The mixture was purged with nitrogen three times, stirred at room temperature for 2 hours, and then 10 mL of saturated sodium bicarbonate solution was added. The mixture was extracted three times with dichloromethane (20 mL × 3) to separate the organic phase. The organic phase was dried, concentrated, and purified by reverse mixing (acetonitrile:water = 13:20 v / v, i.e., 65% ACN). The purified phase was then dried under vacuum for 12 hours to obtain 510 mg of compound NM151. MS ESI (m / z) = 631 [M + Na] + .

[0064] 1 H NMR (400 MHz, DMSO-d 6) δ 11.38 (s, 1H), 7.88 (dd, J = 8.1, 2.7 Hz, 1H), 5.90 (t, J = 5.5 Hz, 1H), 5.65 (d, J = 8.1 Hz, 1H), 4.40 (ddt, J = 25.7, 9.9,4.5 Hz, 1H), 4.20 (q, J = 3.0 Hz, 1H), 4.09 (ddt, J = 10.7, 7.8, 4.8 Hz, 6H), 4.02 – 3.90 (m, 3H), 3.92 – 3.81 (m, 1H), 3.82 (d, J = 3.3 Hz, 1H), 3.83 – 3.76(m, 1H), 3.79 – 3.68 (m, 1H), 3.63 (tdd, J = 16.1, 8.2, 4.5 Hz, 2H), 3.41 (s,2H), 3.35 (s, 1H), 2.82 (t, J = 5.9 Hz, 2H), 1.28 (dd, J = 7.1, 2.3 Hz, 6H), 1.18(td, J = 6.2, 3.2 Hz, 12H).

[0065] Preparation Example 2: Preparation of Compound NM152 In this preparation example, the synthetic route of compound NM152 is shown below: (2-1) Synthesis of compound NM152-2 Compound 1 (20 g, 10.75 mmol, 1.0 eq) was dissolved in ACN (10 mL), and IBX (22.6 g, 32.2 mol, 1.5 eq) was added. The mixture was stirred at 80 °C for 2 hours. After the reaction was complete, the mixture was cooled to room temperature, filtered, and concentrated to obtain 20 g of crude compound NM152-2, which was used directly in the next step.

[0066] (2-2) Synthesis of compound NM152-3 Trimethylphenylphosphine bromide (30 g, 3 eq) was dissolved in 50 mL of THF. Potassium tert-butoxide (13 g, 3 eq) was added under ice bath conditions. The mixture was stirred at 0 °C for 30 minutes, followed by the addition of a THF solution of compound NM152-2 (15 g, 1.0 eq). The reaction was allowed to proceed overnight at room temperature. The mixture was extracted three times with saturated ammonium chloride and ethyl acetate, concentrated under reduced pressure, dried, filtered, and purified by normal chromatography (PE / EA = 2:1, v / v) to give compound NM152-3 (9.8 g white solid, 65.7% yield). MS ESI (m / z) = 369 [M + H] + .

[0067] (2-3) Synthesis of compound NM152-4 Compound NM152-3 (9.8 g, 1.0 eq) was dissolved in 50 mL of THF. 9-BBN (150 mL, 6 eq) was slowly added under ice bath conditions. The mixture was stirred at room temperature for 6 hours. Methanol (30 mL) was added at low temperature, followed by water (50 mL), and the mixture was stirred for 10 minutes. Sodium perborate tetrahydrate (6.9 g, 5 eq) was then added, and the mixture was reacted overnight at room temperature. The mixture was extracted three times with saturated ammonium chloride and ethyl acetate, dried, filtered, and concentrated under reduced pressure. The resulting solution was then dissolved in a 1:1 THF:water solution. Sodium perborate tetrahydrate (6.9 g, 5 eq) was added again, and the mixture was reacted overnight at room temperature. The mixture was extracted three times with saturated ammonium chloride and ethyl acetate, dried, filtered, and concentrated under reduced pressure. The solution was purified by normal chromatography (DCM / MeOH = 10:1, v / v) to give compound NM152-4 (5.4 g white solid, yield 65.7%). MS ESI (m / z) = 387 [M + H] + .

[0068] (2-4) Synthesis of compound NM152-5 Compound NM152-4 (5.4 g, 13.9 mmol, 1.0 eq) was dissolved in ACN (10 mL), and IBX (5.87 g, 20.98 mmol, 1.5 eq) was added. The mixture was stirred at 80 °C for 2 hours. After the reaction was complete, the mixture was cooled to room temperature, filtered, and concentrated to obtain 5 g of crude compound NM152-5, which was used directly in the next step.

[0069] (2-5) Synthesis of compound NM152-6 NaH (884 mg, 1.0 eq) was dissolved in 50 mL THF and stirred in an ice bath for 30 min. The reaction system was then cooled to -78 °C. Tetramethylmethylene diphosphate (2.65 g, 1.3 eq) was dissolved in THF and added dropwise to the reaction system. The reaction was carried out at low temperature for 30 min. Then, compound NM152-5 (3.4 g, 1.0 eq) was dissolved in THF and added dropwise to the reaction system. The reaction was carried out at -78 °C for 1 hour. The mixture was extracted three times with saturated ammonium chloride and ethyl acetate, dried, filtered, concentrated under reduced pressure, and purified by normal chromatography (DCM / MeOH = 10:1, v / v) to give compound NM152-6 (4 g of yellow oil, yield 32%). MS ESI (m / z) = 491 [M + H] + .

[0070] (2-6) Synthesis of compound NM152-7 Compound NM152-6 (4.3 g, 1.0 eq) was dissolved in 10 mL of methanol, Pd / C (400 mg) was added, and the mixture was purged with hydrogen three times. The reaction was carried out at room temperature for 4 hours. After the reaction was completed, the mixture was filtered and concentrated to obtain 3.5 g of crude compound NM152-7, which was used directly in the next step. MS ESI (m / z) = 493 [M + H] + .

[0071] (2-7) Synthesis of compound NM152-8 Compound NM152-7 (3.5 g, 1.0 eq) was dissolved in 15 mL of THF, and TBAF (3.5 g, 1.5 eq) was added. The mixture was reacted at room temperature for 2 hours. Purification was then performed by reverse chromatography (elution buffer: acetonitrile / water = 1:4, v / v) to give compound NM152-8 (1.45 g white solid, yield 55.5%). MS ESI (m / z) = 379 [M + H] + .

[0072] (2-8) Synthesis of compound NM152 Compound NM152-8 (1.45 g, 1.0 eq) was dissolved in 20 mL of DCM, and DCI (360 mg, 0.8 eq) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.5 g, 1.2 eq, CAS No. 102691-36-1) were added. The mixture was reacted at room temperature for 3 hours, and purified by reverse chromatography (ACN / H2O = 1:1, v / v) to give compound NM152 (950 mg white solid, yield 43.1%). MSESI (m / z) = 601 [M + Na] + .

[0073] 1 H NMR (400 MHz, DMSO- d 6) δ 11.37 (s, 1H), 7.64 (dd, J = 8.1, 3.0 Hz,1H), 5.83 – 5.75 (m, 1H), 5.68 (d, J = 8.0 Hz, 1H), 4.19 (ddt, J = 24.5, 10.3,4.9 Hz, 1H), 4.05 (q, J = 5.1 Hz, 1H), 3.98 – 3.87 (m, 1H), 3.87 – 3.75 (m,2H), 3.63 (dd, J = 10.7, 3.1 Hz, 8H), 3.40 (s, 2H), 3.36 (s, 1H), 2.82 (q, J =5.3, 3.9 Hz, 2H), 1.93 – 1.46 (m, 6H), 1.18 (dd, J = 6.8, 4.3 Hz, 12H).

[0074] Preparation Example 3: Preparation of Compound NM153 The synthetic route for compound NM153 is as follows: (3-1) Synthesis of compound NM153-2 Compound NM153-1 ((2'R)-2'-deoxy-3'-O-(tert-butyl-dimethylsilyl)-2'-fluoro-2'-methyl-uridine, 4 g, 10.75 mmol, 1.0 eq) was dissolved in THF (10 mL), and NaH (320 mg, 32.2 mmol, 3 eq) was added under ice bath conditions. The mixture was stirred for 1 hour, and then diethyl p-toluenesulfonyloxymethylphosphonate (5 g, 10.75 mmol, 2.5 eq) was added and reacted for 2 hours. After the reaction was completed, the mixture was extracted three times with saturated ammonium chloride and ethyl acetate, and concentrated under reduced pressure to obtain 2 g of crude compound NM153-2, which was directly used in the next step.

[0075] (3-2) Synthesis of compound NM153-3 Compound NM153-2 (2 g, 1.0 eq) was dissolved in 15 mL of THF, and TBAF (750 mg, 1.5 eq) was added. The mixture was reacted at room temperature for 2 hours. Purification by reverse chromatography (elution buffer: acetonitrile / water = 1:4, v / v) yielded compound NM153-3 (720 mg white solid, 48% yield). MS ESI (m / z) = 412 [M + H] + .

[0076] (3-3) Synthesis of compound NM153 Compound NM153-3 (720 mg, 1.0 eq) was dissolved in 10 mL of DCM, and DCI (160 mg, 0.8 eq) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (317 mg, 1.2 eq) were added. The mixture was reacted at room temperature for 2 hours, and purified by reverse chromatography (elution buffer: acetonitrile / water = 2:1, v / v) to give compound 4 (400 mg white solid, yield 37.3%). MS ESI (m / z) = 633 [M + Na] + .

[0077] 1 H NMR (400 MHz, DMSO- d 6) δ 11.51 (s, 1H), 7.87 (t, J = 8.1 Hz, 1H), 6.07 (s, 1H), 5.69 (dd, J = 8.1, 3.0 Hz, 1H), 4.09 (dtd, J = 9.1, 7.0, 5.1 Hz,5H), 4.05 – 3.85 (m, 3H), 3.89 – 3.71 (m, 2H), 3.75 – 3.62 (m, 1H), 3.66 –3.56 (m, 1H), 2.82 (dt, J = 13.4, 5.9 Hz, 2H), 1.36 (dd, J = 22.6, 17.8 Hz, 3H), 1.31 – 1.15 (m, 18H).

[0078] Preparation Example 4: Preparation of Compound NM156 The synthetic route for compound NM156 is as follows: (4-1) Synthesis of compound NM156-5 The synthesis of compound NM152-4 was described in Example 2. Compound NM152-4 (3.2 g, 8.29 mmol, 1.0 eq) was dissolved in THF (10 mL). NaH (1.32 g, 24.8 mmol, 3 eq) was added under ice bath conditions, and the mixture was stirred for 1 hour. Then, diethyl p-toluenesulfonyloxymethylphosphonate (3.19 g, 9.95 mmol, 1.2 eq) was added, and the reaction was carried out for 2 hours. After the reaction was complete, the mixture was extracted three times with saturated ammonium chloride and ethyl acetate. The extracts were separated, and the organic phase was concentrated under reduced pressure. The solution was purified by normal chromatography (DCM / MeOH = 10:1, v / v) to give compound NM156-5 (830 mg of yellow oil). MS ESI (m / z) = 537 [M + H] + .

[0079] (4-2) Synthesis of compound NM156-6 Compound NM156-5 (1.7 g, 1.0 eq) was dissolved in 15 mL of THF, and TBAF (1 g, 1.3 eq) was added. The mixture was reacted at room temperature for 2 hours, and purified by reverse chromatography (ACN / H2O = 1:4, v / v) to give compound NM156-6 (830 mg white solid, yield 55.5%). MS ESI ( m / z = 423 [M + H] + .

[0080] (4-3) Synthesis of compound NM156 Compound NM156-6 (800 mg, 1.0 eq) was dissolved in 20 mL of DCM, and DCI (180 mg, 0.8 eq) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (750 mg, 1.2 eq) were added. The reaction was carried out at room temperature for 3 hours, and purified by reverse chromatography (ACN / H2O = 1:1, v / v) to give compound NM156 (780 mg white solid, yield 66.1%). MS ESI ( m / z ) = 645[M + Na] + .

[0081] 1 H NMR (400 MHz, DMSO- d 6) δ 11.40 (s, 1H), 7.68 (dd, J = 8.1, 2.7 Hz,1H), 5.84 – 5.75 (m, 1H), 5.68 (dd, J = 8.1, 1.3 Hz, 1H), 4.25 (ddt, J= 22.7,10.2, 4.9 Hz, 1H), 4.12 – 3.97 (m, 6H), 3.79 (dq, J = 8.6, 4.3, 3.3 Hz, 4H), 3.76 – 3.56 (m, 4H), 3.37 (d, J = 16.7 Hz, 3H), 2.81 (dd, J = 6.8, 5.1 Hz, 2H),2.09 – 1.82 (m, 2H), 1.25 (td, J = 7.1, 3.3 Hz, 6H), 1.22 – 1.14 (m, 12H).

[0082] Preparation Example 5: Preparation of Compound NM157 The synthetic route for compound NM157 is as follows: (5-1) Synthesis of compound NM157-2 3-Deoxyuridine (10.0 g, 43.86 mmol, 1.0 eq) was dissolved in pyridine (100 mL), and the solution was cooled to 0 °C. 4,4'-Dimethoxytriphenylmethylchloro (15.56 g, 46.05 mmol, 1.05 eq, DMTrCl, CAS No. 40615-36-9) was added in portions. The mixture was purged with nitrogen three times, and stirred at 25 °C for 3 hours. After the reaction was complete, methanol (10 mL) was added to quench the reaction, and the solution was concentrated and purified by column chromatography (elution gradient, ethyl acetate:petroleum ether = 7:3, v / v) to give 16.3 g of a yellow solid compound NM157-2 (yield 70.0%). MS ESI (m / z) = 531 [M + H] + .

[0083] (5-2) Synthesis of compound NM157-3 Compound NM157-2 (15.8 g, 29.75 mmol, 1.0 eq) was dissolved in dichloromethane (158 mL), and imidazole (4.05 g, 59.51 mmol, 2.0 eq) was added. Then, tert-butyldimethylchlorosilane (TBSCl, 12.27 g, 44.63 mmol, 1.5 eq) was added. The mixture was purged with nitrogen three times, and stirred at 25 °C for 16 hours. After the reaction was complete, the solution was concentrated and subjected to column chromatography (elution gradient, ethyl acetate:petroleum ether = 10:90, v / v) to give 17.26 g of off-white solid NM157-3 (yield 90.1%). MS ESI (m / z) = 645 [M + H] + .

[0084] (5-3) Synthesis of compound NM157-4 Compound NM157-3 (17.26 g, 7.09 mmol, 1.0 eq) was added to a reaction vessel, followed by 170 mL of a 5 wt% dichloromethane / methanol solution of p-toluenesulfonyl chloride (dichloromethane:methanol = 7:3, v / v). The mixture was stirred for 30 minutes, washed with 50 mL of saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (elution gradient, ethyl acetate:petroleum ether = 70:30, v / v) to give 7.3 g of a yellow solid, compound NM157-4 (yield 79.6%). MS ESI (m / z) = 343 [M + H] + .

[0085] (5-4) Synthesis of compound NM157-5 Compound NM157-4 (3.5 g, 10.2 mmol, 1.0 eq) was dissolved in tetrahydrofuran (35 mL). Sodium tert-butoxide (3.93 g, 40.9 mmol, 4.0 eq) was added at 0 °C, and the mixture was slowly heated to 25 °C and stirred for 1 hour. Diethyl p-toluenesulfonyloxymethylphosphonate (8.23 g, 25.6 mmol, 2.5 eq) was added, and the mixture was stirred at 25 °C for 2 hours. After the reaction was complete, saturated ammonium chloride aqueous solution (50 mL) was added, followed by extraction with ethyl acetate (20 mL × 3), washing with saturated sodium chloride aqueous solution (10 mL × 3), drying with anhydrous sodium sulfate, filtration, and concentration. The solution was purified by column chromatography (elution gradient, petroleum ether:ethyl acetate = 1:4, v / v) to give 1.78 g of compound NM157-5 (yield 35.4%). MS ESI (m / z) = 493.3 [M + H] + .

[0086] (5-5) Synthesis of compound NM157-6 The crude compound NM157-5 (1.78 g, 3.61 mmol, 1.0 eq) was dissolved in THF (18 mL), and a THF solution of TBAF (5.4 mL, 1 M, 1.5 eq) was added. The mixture was stirred at 25 °C for 16 hours. After the reaction was complete, the solvent was removed by rotary evaporation, and the product was purified by normal column chromatography (elution gradient, dichloromethane:methanol = 10:1, v / v) to give 1.35 g of compound NM157-6 (yield 98.8%). MS ESI (m / z) = 379.2 [M+H] + .

[0087] (5-6) Synthesis of compound NM157 Compound NM157-6 (1.15 g, 3.04 mmol, 1.0 eq) was repeatedly dried with acetonitrile (3 × 15 mL), dissolved in dichloromethane (15 mL), and a solution of bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.37 g, 4.52 mmol, 1.5 eq) in dichloromethane (15 mL) was added after drying with acetonitrile (3 × 10 mL). 4,5-Dicyanimidazole (286 mg, 2.41 mmol, 0.8 eq) was added. The mixture was purged three times with nitrogen, and the reaction solution was stirred at 25 °C for 1 hour under a nitrogen atmosphere. After the reaction was complete, the mixture was diluted with 30 mL of saturated sodium bicarbonate aqueous solution, extracted with dichloromethane (3 × 20 mL), the organic phases were combined, washed with 2 × 30 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (elution gradient, petroleum ether:ethyl acetate = 9:1, v / v) to give 650 mg of white solid NM157 (yield 37.0%). MS ESI (m / z) = 579.1 [M + H] + .

[0088] 1 H NMR (400 MHz, DMSO) δ 11.26 (s, 1H), 7.86 (dd, J = 10.6, 8.1 Hz, 1H), 5.79 (dd, J = 15.4, 2.4 Hz, 1H), 5.59 (d, J = 8.0 Hz, 1H), 4.49 (s, 1H), 4.39 (s, 1H), 4.05 (tt, J = 13.7, 6.2 Hz, 6H), 3.81 – 3.65 (m, 3H), 3.65 – 3.52(m, 2H), 2.76 (q, J = 5.6 Hz, 2H), 2.07 (q, J = 5.7 Hz, 1H), 1.99 (s, 2H), 1.24(td, J = 7.1, 1.8 Hz, 6H), 1.13 (h, J = 4.7 Hz, 12H).

[0089] 31 P NMR (162 MHz, DMSO) δ 148.68 (d, J = 10.4 Hz).

[0090] Preparation Example 6: Preparation of siRNA In this preparation example, the synthesis process of the siRNA conjugate is as follows: (1) Composition of the justice chain (SS) and the antisense chain (AS): The phosphoramidol nucleic acid solid-phase synthesis method utilizes the aforementioned compounds linked to a solid-phase support (e.g., compound CR01008Z, with nucleoside monomers linked sequentially from the 3' to the 5' end of the nucleotide sequence). During the synthesis, compounds CR01008, NM054, NM151, NM152, NM153, NM156, and NM157 are each considered as a nucleoside monomer.

[0091] Each connection of a nucleoside monomer involves four steps: deprotection, coupling, capping, and oxidation or sulfidation. The synthetic conditions are as follows: The nucleoside monomer was prepared into an acetonitrile solution with a concentration of 0.1 M.

[0092] The deprotection reaction conditions were the same for each step. The deprotection reaction conditions were: temperature 25℃, reaction time 70 seconds, deprotection reagent was a dichloromethane solution of dichloroacetic acid (3% (v / v)), and the molar ratio of dichloroacetic acid to the 4,4'-dimethoxytriphenylmethyl protecting group on the solid support was 5:1.

[0093] The conditions for each coupling reaction were identical. The coupling reaction conditions were as follows: temperature 25℃, molar ratio of nucleic acid sequence to nucleoside monomer on the solid-phase support 1:10, molar ratio of nucleic acid sequence to coupling reagent on the solid-phase support 1:65, reaction time 600 seconds, coupling reagent 0.5M acetonitrile solution of 5-ethylthio-1H-tetrazole, and thioreagent 0.2mol / L acetonitrile / pyridine mixed solution of hydrogenated xanthanin (acetonitrile and pyridine volume ratio 1:1).

[0094] The conditions for each capping reaction were identical. The conditions for the capping reaction were: temperature 25℃; reaction time 2 minutes; and the capping reagent solution was a mixture of Cap1 and Cap2. Cap1 was a 20% (v / v) N-methylimidazole pyridine / acetonitrile mixture, with a volume ratio of pyridine to acetonitrile of 3:5. Cap2 was a 20% (v / v) acetic anhydride acetonitrile solution. The molar ratio of N-methylimidazole in Cap1 and acetic anhydride in Cap2 to the nucleic acid sequence linked on the solid-phase support was 1:1:1.

[0095] The conditions for each oxidation reaction were identical. The oxidation reaction conditions were: temperature 25°C; reaction time 3 seconds; oxidizing agent concentration of 0.05M iodine solution, with a molar ratio of iodine to the nucleic acid sequence linked on the solid support in the coupling reaction of 30:1; the oxidation reaction was carried out in a water / pyridine mixed solvent (water to pyridine volume ratio 1:9). The sulfidation reaction conditions were: temperature 25°C; reaction time 360 ​​seconds; thioreagent concentration of 0.2M hydroflavin in pyridine solution, with a molar ratio of thioreagent to the nucleic acid sequence linked on the solid support in the coupling reaction of 4:1; the thioreagent reaction was carried out in a water / pyridine mixed solvent (water to pyridine volume ratio 1:9).

[0096] After the last nucleoside monomer is ligated, the nucleic acid sequence ligated on the solid-phase support is sequentially cut, deprotected, purified, and desalted, and then freeze-dried to obtain the sense or antisense strand, wherein: The cleavage and deprotection conditions were as follows: The synthesized nucleotide sequence linked to a solid-phase support was added to 25 wt% ammonia solution at a concentration of 0.5 mL / μmol. The reaction was carried out at 55 °C for 16 hours. The solvent was removed, and the solution was concentrated to dryness under vacuum. After ammonia treatment, the product was dissolved in 0.4 mL / μmol N-methylpyrrolidone relative to the amount of single-stranded nucleic acid. Subsequently, 0.3 mL / μmol triethylamine and 0.6 mL / μmol triethylamine trifluoride were added to deprotect the 2'-O-TBDMS protection on the ribose.

[0097] Purification and desalting conditions: Nucleic acid purification was performed using a preparative ion chromatography column (Source 15Q) with a NaCl gradient elution. Specifically: eluent 1 was 20 mM sodium phosphate (pH=8.1), and the solvent was a water / acetonitrile mixture (water to acetonitrile volume ratio 9:1); eluent 2 was 1.5 M sodium chloride and 20 mM sodium phosphate (pH=8.1), and the solvent was a water / acetonitrile mixture (water to acetonitrile volume ratio 9:1); the elution gradient was eluent 1: eluent 2 = (100:0) - (50:50). The product eluates were collected and combined, and desalting was performed using a reverse chromatographic purification column. Desalting conditions included using a dextran gel column (g25 packing material) and elution with deionized water.

[0098] After solid-phase synthesis, the dried solid support was ammonolyzed with 25% ammonia solution (treated at 55°C for 40 hours) and then purified. The 5' end nucleotide of the antisense strand was (NM152a).

[0099] After solid-phase synthesis, the dried solid support was first quenched with TMSI, then ammonolyzed with 25% ammonia solution (treated at 35°C for 20 hours), and then purified. The 5' end nucleotide of the antisense strand was (NM151), (NM152), (NM153), (NM156), or (NM157).

[0100] Detection: Purity was determined using ion exchange chromatography (IEX-HPLC); molecular weight was determined using liquid chromatography-mass spectrometry (LC-MS). The measured molecular weight was compared with the theoretical value. If the measured value and the theoretical value were consistent, it indicated that the target sense and antisense strands were obtained.

[0101] During the synthesis of the justice chains, the following justice chains, each linked to a target ligand (carrier), were synthesized: When the end of the justice chain 3' is joined with _(CR01008×3), its structural formula is: (6-2) Annealing of siRNA The sense and antisense strands synthesized in step (6-1) were mixed in an equimolar ratio, dissolved in water for injection, and heated to 95°C. The mixture was then slowly cooled to room temperature and kept at room temperature for 10 min to allow the sense and antisense strands to form a double-stranded structure through hydrogen bonds, thereby obtaining the siRNA of the delivery vector _(CR01008×3) at the 3' end of the sense strand.

[0102] Based on the above method, exemplary siRNA conjugates obtained in this disclosure are shown in Table 6, and their corresponding unmodified siRNA sequence information is shown in Table 5.

[0103] Table 5. Sequence information of unmodified siRNA Table 6. Sequence information of siRNA conjugates Unless otherwise stated, the base composition and modifications in this disclosure have the following meanings: uppercase letters A, U, G, C, and T represent the base composition of nucleotides; lowercase letter m indicates that the 2'-hydroxyl group on the ribose group of the nucleotide represented by the uppercase letter to its left is replaced by 2'-O-methyl; lowercase letter f indicates that the 2'-hydroxyl group on the ribose group of the nucleotide represented by the uppercase letter to its left is modified by 2'-fluorination; (moe) indicates that the 2'-hydroxyl group on the ribose group of the nucleotide represented by the uppercase letter to its left is replaced by 2'-O-MOE; lowercase letter s indicates that the two nucleotides represented by the two adjacent letters to its left and right are connected by a phosphate thioester bond; VP indicates that the 5' end of the antisense chain has a 5'-(E)-vinylphosphonate (5'-(E)-VP) modification; (NM151), (NM152), (NM152a), (NM153), (NM156), (NM157), and (NM054) each represent a 5'-phosphate analog modified nucleoside or nucleotide (non-limitingly, the corresponding base is U in this disclosure), wherein: The structural formula of (NM151) is: ; The structural formula of (NM152) is: ; The structural formula of (NM152a) is: ; The structural formula of (NM153) is: The structural formula of (NM156) is: The structural formula of (NM157) is: The structural formula of (NM054) is: or , For example, regarding the modification of 5'-(E)-vinylphosphonate (5'-(E)-VP), The structural formula of VPAm is or .

[0104] For example, the structural formula of a nucleotide modified with 2'-O-methyl is as follows: In this context, Base represents a nucleoside base, such as A, U, G, C, or T; Z is selected from -OH, -O-, -SH, or -S-.

[0105] For example, the structural formula of a 2'-fluorinated nucleotide is: , Where Base represents a nucleoside base, such as A, U, G, C or T; Z is selected from -OH, -O-, -SH or -S-.

[0106] For example, the structural formula of a nucleotide modified with 2'-O-methoxyethyl is as follows: In this context, Base represents a nucleoside base, such as A, U, G, C, or T; Z is selected from -OH, -O-, -SH, or -S-.

[0107] Example of bioassay: Biological detection experiments: mRNA expression level detection: RNA extraction: The tissue samples were taken from RNAlater and homogenized for 120 seconds in a Tissuelyser II fully automated tissue homogenizer. Total RNA was extracted from the liver tissue samples using a fully automated nucleic acid extractor and nucleic acid extraction kit from Yisheng Company, following the method described in the instruction manual.

[0108] Reverse transcription: Take 1 μg of total RNA and use a reverse transcription kit (Thermo Fisher Scientific, RevertAid First Strand cDNA Synthesis Kit, K1622) with Oligo (dT)18 reverse transcription primers. Prepare a 20 μL reverse transcription system according to the instructions of the reverse transcription kit and complete the reverse transcription reaction. After the reaction, add 80 μL of RNase-free water to the reverse transcription system to obtain a cDNA solution.

[0109] Real-time quantitative PCR: Next, the expression level of the target gene mRNA was detected using a real-time quantitative PCR kit (ABI, SYBR™ Select Master Mix, Catalog number: 4472908). A 20 µL real-time PCR reaction system was prepared for each PCR well according to the kit's instructions. Each reaction system contained 5 µL of cDNA solution obtained from the reverse transcription reaction, 10 µL of SYBR™ Select Master Mix, 0.5 µL of 10 µM upstream primer, 0.5 µL of 10 µM downstream primer, and 4 µL of RNase-free H2O. The prepared reaction systems were placed on a real-time quantitative PCR instrument (ABI, StepOnePlus™), and a three-step real-time PCR amplification was performed. The amplification program was: 95℃ pre-denaturation for 10 min, followed by 95℃ denaturation for 30 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s. This denaturation, annealing, and extension process was repeated for 40 cycles.

[0110] The expression level of the target gene mRNA was detected using the TaqMan™ PCR kit (ABI, TaqMan™ Fast Advanced Master Mix). Each reaction system contained 5 μL of cDNA solution obtained from the above reverse transcription reaction, 10 μL of TaqMan™ Fast Advanced Master Mix, 0.4 μL of 10 μM upstream primer, 0.4 μL of 10 μM downstream primer, 0.2 μL of 10 μM Probe primer, and 3 μL of RNase-Free H2O. The prepared reaction systems were placed on a real-time quantitative PCR instrument (ABI, StepOnePlus™) and amplified using a three-step method: 50℃ for 2 min, followed by 95℃ pre-denaturation for 20 s, 95℃ denaturation for 3 s, and 60℃ annealing for 30 s. The denaturation, annealing, and extension processes were repeated for 40 cycles.

[0111] The relative quantification of target gene mRNA in each test group was performed using the ΔΔCt method. The calculation method is summarized below: ΔCt(test group) = Ct(target gene in test group) – Ct(internal reference gene in test group) ΔCt(control group) = Ct(target gene in control group) – Ct(internal reference gene in control group) ΔCt(test group) = ΔCt(test group) - ΔCt(control group average) ΔCt(control group) = ΔCt(control group) - ΔCt(control group average) The relative residual expression level of the target gene mRNA in the test group was 2. -ΔΔCt(测试组) ×100% Using the control group as a baseline, the mRNA expression level of the target gene in the test group was normalized, and the residual mRNA expression level of the target gene in the control group was defined as 100%. The inhibition rate of target gene mRNA in the test group = 100% - the relative expression level of target gene mRNA in the test group All activity experimental data are based on X±STDEV indicates that all experimental data were plotted and analyzed using GraphPad Prism 8.0 software.

[0112] Example 1: Evaluation of the activity of the modified sequence against apolipoprotein B (ApoB) in mice. In this embodiment, the inhibitory effects of sequences R598014, R598015, R598016, R598017, R598018, and R598020 with the new general formula modification pattern and the control sequence R5980019 on the target gene ApoB were evaluated using an in vivo mouse activity assessment method. The difference is that the former has an added VP analog modification at the 5' end of the antisense strand, and the 18th and 19th positions of the sense strand and the 10th and 11th positions of the antisense strand are phosphate thiophosphate substitution links.

[0113] siRNA sequence subcutaneous administration activity evaluation assay in mice: Six- to eight-week-old C57BL / 6j mice were randomly divided into groups according to body weight. Each group of mice was administered siRNA subcutaneously at a dose of 3 mg / kg, with a volume of 5 mL / kg. The dosage for each animal was calculated based on its most recent body weight, accurate to the smallest readable graduation on the syringe. The PBS control group received the same volume of siRNA-free PBS solution. The day of administration was designated as day 0 (D0), and mice were sacrificed on day 14. Liver tissue was harvested and cut into several 2 mm sections. 3 Small pieces are stored separately in RNAlater.

[0114] Example of bioassay: Biological detection experiments: mRNA expression level detection: RNA extraction: The tissue samples were taken from RNAlater and homogenized for 120 seconds in a Tissuelyser II fully automated tissue homogenizer. Total RNA was extracted from the liver tissue samples using a fully automated nucleic acid extractor and nucleic acid extraction kit from Yisheng Company, following the method described in the instruction manual.

[0115] Reverse transcription: Take 1 μg of total RNA and use a reverse transcription kit (Thermo Fisher Scientific, RevertAid First Strand cDNA Synthesis Kit, K1622) with Oligo (dT)18 reverse transcription primers. Prepare a 20 μL reverse transcription system according to the instructions of the reverse transcription kit and complete the reverse transcription reaction. After the reaction, add 80 μL of RNase-free water to the reverse transcription system to obtain a cDNA solution.

[0116] Real-time quantitative PCR: Next, the expression level of the target gene mRNA was detected using a real-time quantitative PCR kit (ABI, SYBR™ Select Master Mix, Catalog number: 4472908). A 20 µL real-time PCR reaction system was prepared for each PCR well according to the kit's instructions. Each reaction system contained 5 µL of cDNA solution obtained from the reverse transcription reaction, 10 µL of SYBR™ Select Master Mix, 0.5 µL of 10 µM upstream primer, 0.5 µL of 10 µM downstream primer, and 4 µL of RNase-free H2O. The prepared reaction systems were placed on a real-time quantitative PCR instrument (ABI, StepOnePlus™), and a three-step real-time PCR amplification was performed. The amplification program was: 95℃ pre-denaturation for 10 min, followed by 95℃ denaturation for 30 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s. This denaturation, annealing, and extension process was repeated for 40 cycles.

[0117] The expression level of the target gene mRNA was detected using the TaqMan™ PCR kit (ABI, TaqMan™ Fast Advanced Master Mix). Each reaction system contained 5 μL of cDNA solution obtained from the above reverse transcription reaction, 10 μL of TaqMan™ Fast Advanced Master Mix, 0.4 μL of 10 μM upstream primer, 0.4 μL of 10 μM downstream primer, 0.2 μL of 10 μM Probe primer, and 3 μL of RNase-Free H2O. The prepared reaction systems were placed on a real-time quantitative PCR instrument (ABI, StepOnePlus™) and amplified using a three-step method: 50℃ for 2 min, followed by 95℃ pre-denaturation for 20 s, 95℃ denaturation for 3 s, and 60℃ annealing for 30 s. The denaturation, annealing, and extension processes were repeated for 40 cycles.

[0118] The relative quantification of target gene mRNA in each test group was performed using the ΔΔCt method. The calculation method is summarized below: ΔCt(test group) = Ct(target gene in test group) – Ct(internal reference gene in test group) ΔCt(control group) = Ct(target gene in control group) – Ct(internal reference gene in control group) ΔCt(test group) = ΔCt(test group) - ΔCt(control group average) ΔCt(control group) = ΔCt(control group) - ΔCt(control group average) The relative residual expression level of the target gene mRNA in the test group was 2. -ΔΔCt(测试组) ×100% Using the control group as a baseline, the mRNA expression level of the target gene in the test group was normalized, and the residual mRNA expression level of the target gene in the control group was defined as 100%. The inhibition rate of target gene mRNA in the test group = 100% - the relative expression level of target gene mRNA in the test group All activity experimental data are based on X±STDEV indicates that all experimental data were plotted and analyzed using GraphPad Prism 8.0 software.

[0119] Example 1: Evaluation of the activity of the modified sequence against apolipoprotein B (ApoB) in mice. In this embodiment, the inhibitory effects of sequences R598014, R598015, R598016, R598017, R598018, and R598020 with the new general formula modification pattern and the control sequence R5980019 on the target gene ApoB were evaluated using an in vivo mouse activity assessment method. The difference is that the former has an added VP analog modification at the 5' end of the antisense strand, and the 18th and 19th positions of the sense strand and the 10th and 11th positions of the antisense strand are phosphate thiophosphate substitution links.

[0120] siRNA sequence subcutaneous administration activity evaluation assay in mice: Six- to eight-week-old C57BL / 6j mice were randomly divided into groups according to body weight. Each group of mice was administered siRNA subcutaneously at a dose of 3 mg / kg, with a volume of 5 mL / kg. The dosage for each animal was calculated based on its most recent body weight, accurate to the smallest readable graduation on the syringe. The PBS control group received the same volume of siRNA-free PBS solution. The day of administration was designated as day 0 (D0), and mice were sacrificed on day 14. Liver tissue was harvested and cut into several 2 mm sections. 3 Small pieces are stored separately in RNAlater.

[0121] Table 7. Primer sequence listing The results of Example 1 showed that the activity of the new general formula pattern modification sequence was comparable to that of the control sequence R598019 ( Figure 1 (Table 8).

[0122] Table 8. Inhibitory activity level of the target gene ApoB in mouse liver tissue after administration of the siRNA described in this example. Example 2: Modified sequence in Huh1 cells for solute carrier family 22 member 12. Activity assessment of 22 member 12 (SLC22A12 / URAT1) In this embodiment, an in vitro cell transfection method was used to evaluate the inhibitory activity of the sequence R214090 with the new general formula modification pattern and the control sequence R291023 on URAT1 targeting in Huh1 cells. The difference is that R214090 has a 5'-phosphate analog modification added to the 5' end of the antisense strand, and the 20th and 21st bases of the 3' end are replaced by NM054 monomer; at the same time, there are phosphate thiophosphate substitution links between positions 18 and 19 of the sense strand and between positions 10 and 11 of the antisense strand.

[0123] HuH1 cells (Wuhan Pronosai Biotechnology Co., Ltd.) were cultured in complete medium (DMEM medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibiotics) at 5% CO2 and 37°C. After reaching 80% confluence, the cells were digested with trypsin and resuspended. The resuspended cells were then adjusted to a density of 0.6 × 10⁻⁶ cells / mL using complete medium. 5 / wells were inoculated into 24-well plates and transfected after 24 hours of incubation.

[0124] Preparation of siRNA transfection mixture: Prepare a 0.2 µM stock solution of siRNA using PBS. Then, add 1.5 µL of the siRNA stock solution to each well and dilute with 48.5 µL of Opti-MEM medium to prepare a 50 µL siRNA working solution. Dilute 3 µL of Lipofectamine® RNAiMAX transfection reagent to each well and add to 47 µL of Opti-MEM medium. Incubate at room temperature for 5 minutes to prepare a 50 µL transfection reagent working solution. Mix the 50 µL siRNA working solution with the 50 µL transfection reagent working solution to prepare the siRNA transfection mixture. For the MOCK group, mix 50 µL of Opti-MEM medium with 50 µL of transfection reagent working solution. On the day of transfection, replace 500 µL of Opti-MEM medium per well in the 24-well plate and add 100 µL of the corresponding siRNA transfection mixture or MOCK group transfection mixture to each well, with a final siRNA concentration of 0.5 nM per well.

[0125] Table 9. Primer sequence information The results of Example 2 showed that the new general formula pattern modification sequence R2141090 exhibited superior inhibitory activity against the target gene in HuH1 compared to the control sequence R291023. Figure 2 (Table 10).

[0126] Table 10. Inhibitory activity of target genes in HuH1 cells after siRNA transfection with 0.5 nM as described in this example. Example 3: Evaluation of the activity of the modified sequence against the URAT1 target gene in Huh1 cells In this embodiment, an in vitro cell transfection method was used to evaluate the inhibitory activity of the sequence R214119 with the new general formula modification pattern and the control sequence R291058 on URAT1 targeting in Huh1 cells. The difference is that R214119 has a 5'-phosphate analog modification added to the 5' end of the antisense strand, and the 20th and 21st bases of the 3' end are replaced by NM054 monomer; at the same time, there are phosphate thiophosphate substitution links between positions 18 and 19 of the sense strand and between positions 10 and 11 of the antisense strand.

[0127] Cell culture and transfection were performed as described in Example 2. HuH1 cells were cultured at 0.6 × 10⁻⁶ cells per cell line. 5 / wells were inoculated into 24-well plates and transfected after 24 hours of incubation.

[0128] Preparation of siRNA transfection mixture: Prepare a 0.2 µM stock solution of siRNA using PBS. Then, dilute 1.5 µL of the siRNA stock solution in 48.5 µL of Opti-MEM medium per well to obtain a 50 µL siRNA working solution. Dilute 3 µL of Lipofectamine® RNAiMAX transfection reagent in 47 µL of Opti-MEM medium per well and incubate at room temperature for 5 minutes to obtain a 50 µL transfection reagent working solution. Mix the 50 µL siRNA working solution with the 50 µL transfection reagent working solution to prepare the siRNA transfection mixture. For the MOCK group, mix 50 µL of Opti-MEM medium with 50 µL of transfection reagent working solution. On the day of transfection, replace 500 µL of Opti-MEM medium per well in the 24-well plate and add 100 µL of the corresponding siRNA transfection mixture or MOCK group transfection mixture to each well, with a final siRNA concentration of 0.5 nM per well.

[0129] The results of Example 3 showed that the new general formula pattern modification sequence R214119 exhibited superior inhibitory activity against target genes in HuH1 compared to the control sequence R291058. Figure 3 (Table 11).

[0130] Table 11. Inhibitory activity of target genes in HuH1 cells after siRNA transfection with 0.5 nM as described in this example. Other embodiments of this application will readily occur to those skilled in the art upon consideration of the specification and practice of the disclosure herein. This application is intended to cover any variations, uses, or adaptations of this application that follow the general principles of this application and include common knowledge or customary techniques in the art not disclosed herein. The specification and examples are to be considered exemplary only, and the true scope and spirit of this application are indicated by the claims.

[0131] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. A chemically modified siRNA molecule, characterized in that, The siRNA comprises a sense strand and an antisense strand, the sense strand and the antisense strand being at least partially anticomplementary to form a double-stranded region having 17-23 base pairs; and the nucleotides in the antisense strand having a combination of the following chemical modifications: (1) The antisense strand, starting from the 5' end, consists of nucleotides at positions 2, 6, 9, 14 and 16 that are 2′-fluorinated nucleotides, and nucleotides at the remaining positions that are 2′-nonfluorinated nucleotides; (2) The antisense strand contains five or more phosphate thioester nucleoside bonds between nucleotides, and: starting from the 5' end, the nucleoside bond between the 10th and 11th nucleotides is selected from phosphate thioester nucleoside bonds; as well as: (3) The two nucleotides at the 3' end of the antisense strand are selected from stable nucleotides, which are selected from [2'-R] a -2'-R b nucleotides modified with ]; where [2'-R a -2'-R b ] represents the disubstituted group at the 2'-position of the ribose ring, R a Selected from F and C 1-3 Alkyl or C 1-3 Alkoxy, R b Selected from C 1-3 Alkyl or C 1-3 Alkyl group.

2. The siRNA molecule according to claim 1, characterized in that, The first nucleotide of the antisense strand, starting from the 5' end, is selected from a nucleotide modified with a 5'-phosphate analogue, wherein the 5'-phosphate analogue is selected from any nucleotide with the following structure: 、 、 、 、 、 、 、 ； Wherein, Base represents a nucleoside base, which is selected from A, U, G, C or T; and R substituents are selected from -H, -OH, -CH3, -OCH3 or -F.

3. The siRNA molecule according to claim 1 or 2, characterized in that, in, R a Selected from F or methyl, R b Selected from methyl or methoxy; Preferably, R a Selected from F, R b Selected from methyl.

4. The siRNA molecule according to any one of claims 1-3, characterized in that, In the sense strand, the nucleotides at positions 10-13 (starting from the 5' end) that are paired with the nucleotides in the antisense strand through base complementarity are 2′-fluorinated nucleotides, while the remaining positions are 2′-nonfluorinated nucleotides.

5. The siRNA molecule according to any one of claims 1-4, characterized in that, The antisense strand contains five thiophosphate nucleotide inter-bonds, and the antisense strand contains two consecutive thiophosphate nucleotide inter-bonds between the terminal nucleotides at the 3' and 5' ends, respectively. The positive chain contains three thiophosphate nucleoside bonds, located between the first and second nucleotides starting from the 5' end, between the second and third nucleotides, and between the first and second nucleotides starting from the 3' end.

6. The siRNA molecule according to any one of claims 1-5, characterized in that, The 2'-nonfluorinated nucleotide is selected from 2'-O-methyl modified nucleotides, 2'-deoxynucleotides, locked nucleotides (LNA), open-ring nucleotides (UNA), ethylene glycol nucleotides (GNA), bicyclic nucleotides (BNA), 2'-F-arabinonucleotides, 2'-O-methoxyethyl nucleotides, baseless nucleotides, inverse baseless nucleotides (Invab), any nucleotide modified with 2'-F-2'-CH3 disubstituents, or nucleotides containing non-natural bases; Preferably, the 2'-nonfluorinated nucleotide is selected from 2'-O-methyl modified nucleotides or 2'-F-2'-CH3 disubstituted nucleotides; More preferably, when the 2'-nonfluorinated modified nucleotide is selected from nucleotides with 2'-F-2'-CH3 disubstituted modification, it is located only in the overhanging region at the 3' end of the antisense strand.

7. The siRNA molecule according to any one of claims 1-6, characterized in that, The sense strand has 19 modified nucleotides, and the antisense strand has 21 modified nucleotides; Furthermore, the justice chain and the antisense chain have the following combined modification patterns: Chain of Justice: 5'-NmsNmsNmNmNmNmNfNfNfNfNmNmNmNmNmNmNmNmNmsNm-3' antisense chain: 5'-PNmsNfsNmNmNmNfNmNmNfNmsNmNmNmNfNmNfNmNmNmsNdsNd-3'; or The sense strand has 21 modified nucleotides, and the antisense strand has 23 modified nucleotides; Furthermore, the justice chain and the antisense chain have the following combined modification patterns: Chain of Justice: 5'-NmsNmsNmNmNmNmNmNmNfNfNfNfNmNmNmNmNmNmNmNmsNm-3' antisense chain: 5'-PNmsNfsNmNmNmNfNmNmNfNmsNmNmNmNfNmNfNmNmNmNmNmsNdsNd-3'; Wherein, Nm represents any nucleotide modified with 2'-methoxy, Nf represents any nucleotide modified with 2'-fluoride, Nd represents any nucleotide modified with 2'-F-2'-CH3 disubstituent, the lowercase letter s indicates that the two nucleotides adjacent to the letter s on the left and right are connected by a thiophosphate subunit, and P indicates that the nucleotide adjacent to the letter P on the right is any nucleotide modified with the 5'-phosphate analogue.

8. The siRNA molecule according to any one of claims 1-7, characterized in that, The siRNA molecule further includes a ligand covalently linked to the sense strand via a linker group; the ligand is targeted for delivery to hepatocytes or extrahepatic tissue cells.

9. A pharmaceutical composition comprising an siRNA molecule as described in any one of claims 1-8, and a pharmaceutically acceptable carrier or excipient.

10. Use of the siRNA molecule as described in any one of claims 1-8 in the preparation of a medicament for inhibiting the expression of a target gene in a subject in need.

Images