Double-stranded siRNA analogs containing R and E and their conjugates

Abstract

The present invention discloses a series of double-stranded siRNA analogs containing R and / or E, conjugates thereof, salts of the conjugates, and uses thereof.

Description

Detailed Description of the Invention

[0001] This invention claims priority from: CN2023101774460, filing date: February 24, 2023; CN2023101914409, filing date: February 28, 2023; CN2023104534435, filing date: April 23, 2023; CN202311511833X, filing date: November 13, 2023; CN2024100843576, filing date: January 19, 2024.

[0002] [Technical field] The present invention relates to the field of biomedicine, and specifically to a double-stranded siRNA analog containing E and R, a conjugate thereof, a salt of the conjugate, and use thereof.

[0003] [Background technology] Viral hepatitis B (HBV) is a disease caused by infection with the hepatitis B virus (HBV). The HBV is a hepadnavirus that primarily resides within liver cells and damages them, causing inflammation, necrosis, and fibrosis. Viral hepatitis B is classified into acute and chronic forms. Acute hepatitis B in adults is usually self-limited by the immune system. However, chronic hepatitis B (CHB) has become a major global health challenge and is a leading cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma (HCC). It is estimated that 2 billion people worldwide are infected with chronic hepatitis B virus, more than 350 million develop the disease, and approximately 600,000 die annually from complications of chronic hepatitis B. China is a region with a high incidence of hepatitis B, with a large cumulative number of hepatitis B patients and severe harm. According to data, there are currently approximately 93 million people infected with hepatitis B virus in China, of which approximately 20 million patients are diagnosed with chronic hepatitis B. Of these, 10% to 20% may progress to cirrhosis, and 1% to 5% may develop hepatocellular carcinoma.

[0004] The key to functional cure of hepatitis B is the removal of HBsAg (hepatitis B virus surface antigen) and the production of surface antibodies. In chronically infected patients, HBsAg reduction and seroconversion are rarely observed. Currently approved anti-HBV drugs mainly consist of immunomodulators (interferon-α and polyethylene glycolated interferon-α-2α) and antiviral drugs (lamivudine, adefovirboxil, entecavir, telbivudine, tenofovir, clalvudine, etc.). Among these, antiviral drugs belong to the nucleotide class, whose mechanism of action is to inhibit HBV DNA synthesis and cannot directly reduce HBsAg levels. Clinically, there are treatments that can reduce HBsAg levels, but their therapeutic efficacy is insufficient. Therefore, suppressing viral gene expression at the genetic level and inhibiting HBV production and replication, especially the production of HBsAg and HBeAg (hepatitis B S and E antigens), could fundamentally reduce viral metabolism and hepatocyte infection.

[0005] Small interfering RNA (siRNA) is a therapeutic approach based on the mechanism of RNA interference (RNAi), which inhibits or blocks target gene expression in a sequence-specific manner, exerting its inhibitory effect at the stage of mRNA-to-protein translation. This ideal approach for treating hepatitis B requires stabilizing modifications of siRNA and a corresponding delivery system to target organs and cells, improving metabolic stability. However, current siRNAs are still unable to effectively reduce the levels of hepatitis B virus S and E antigens. At the same time, siRNAs regulate the expression of genes corresponding to specific mRNAs by partially complementarily pairing with them. In particular, the 5'-terminal seed region of the siRNA antisense strand complementarily pairs with non-target genes, resulting in partial or complete suppression of the gene expression. This phenomenon is the main cause of off-target effects caused by siRNA in vivo and in vitro. This drawback has been identified in siRNAs for hepatitis B treatment in both clinical and preclinical stages. Although nucleotide modifications can reduce off-target risks, they also reduce the silencing effect, necessitating improvements in the therapeutic safety margin.

[0006] [Summary of the Invention] The present invention provides a double-stranded siRNA analog, a conjugate thereof, a salt thereof, or a salt of the conjugate, which comprises a sense strand and an antisense strand capable of forming a double-stranded region, wherein the double-stranded siRNA analog is selected from any one of the double strands shown in Table 1, and each nucleotide in the double strand is independently and selectively modified.

[0007] In some embodiments of the present invention, the double-stranded siRNA analog is selected from S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S13, S14, S15, S16, S17, S18, S19, S20, S21, S22, S23, S24, S25, S26, S27, and S28.

[0008] In some embodiments of the invention, the double-stranded siRNA analog is selected from S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, and S11. The present invention provides a double-stranded siRNA analog selected from S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S13, S14, S15, S16, S17, S18, S19, S20, S21, S22, S23, S24, S25 and S26, a conjugate thereof, a salt thereof, or a salt of the conjugate.

[0009] The unmodified double-stranded siRNA analogs provided by the present invention are as shown in Table 1.

[0010] [Table 1] TIFF2026507087000002.tif63167

[0011] The R and E of the present invention are each

[0012] [ka] is.

[0013] In some embodiments of the present invention, when the R or E is located at the 5' end of the AS or SS of the siRNA sequence, the 5' position of the R or E may be a hydroxyl group, a phosphate ester bond or a thiophosphate ester bond, and the 2' position of the R or E may be modified.

[0014] In some embodiments of the present invention, the sense strand of the double-stranded siRNA analog comprises 0, 1, 2, 3, 4, 5, or 6 unmodified nucleotides. In some embodiments of the present invention, the antisense strand of the double-stranded siRNA analog comprises 0, 1, 2, 3, 4, 5, or 6 unmodified nucleotides.

[0015] In some embodiments of the present invention, the sense strand of the double-stranded siRNA analogue comprises 0, 1, 2, or 3 nucleosides substituted with R.

[0016] In some embodiments of the present invention, the antisense strand of the double-stranded siRNA analogue comprises 0, 1, 2, or 3 nucleosides substituted with R.

[0017] In some embodiments of the present invention, the sense strand of the double-stranded siRNA analogue comprises nucleotides in which 0, 1, 2, or 3 nucleosides are substituted with E.

[0018] In some embodiments of the present invention, the antisense strand of the double-stranded siRNA analogue comprises nucleotides in which 0, 1, 2, or 3 nucleosides are substituted with E.

[0019] In some embodiments of the invention, the double-stranded siRNA analog is selected from any one of the duplexes shown in Table 2.

[0020] [Table 2] TIFF2026507087000005.tif215167

[0021] In some embodiments of the invention, the mR is

[0022] [ka] and the intermediate for synthesizing mR is shown in Compound 2:

[0023] [ka] .

[0024] In some embodiments of the present invention, MOE R is

[0025] [ka] and MOE The intermediate for synthesizing R is shown in compound 1:

[0026] [ka] . In some embodiments of the present invention, E is

[0027] [ka] and the intermediate for synthesizing E is shown in Compound 3:

[0028] [ka] .

[0029] In some embodiments of the present invention, the conjugate of the double-stranded siRNA analog or the salt of the conjugate is formed by binding the double-stranded siRNA analog with a pharmaceutically acceptable conjugate group.

[0030] In some embodiments of the present invention, the pharmaceutically acceptable conjugate group in the conjugate of the double-stranded siRNA analogue or the conjugate salt thereof contains 1 to 5 GalNAc groups.

[0031] In some embodiments of the present invention, the pharmaceutically acceptable conjugate group is attached to any position of the double-stranded siRNA analog. In some embodiments of the present invention, the pharmaceutically acceptable conjugate group is attached to the 3'-end of the sense strand of the double-stranded siRNA analogue.

[0032] In some embodiments of the present invention, the pharmaceutically acceptable conjugate groups in the double-stranded siRNA analog conjugate or salt thereof are D1, D2, D3 and L96,

[0033] [ka] Selected from TIFF2026507087000013.tif67170.

[0034] In some embodiments of the present invention, in the conjugate group

[0035] [ka] indicates that the conjugate group and the double-stranded siRNA analogue are linked at the relevant site via a phosphodiester bond or a thiophosphate bond.

[0036] Other embodiments of the present invention include any combination of the above embodiments. The present invention provides a conjugate of a double-stranded siRNA analog selected from Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16, or a salt thereof.

[0037] The double-stranded siRNA analog conjugates provided by the present invention are as shown in Table 3.

[0038] [Table 3] TIFF2026507087000016.tif232167TIFF2026507087000017.tif75167

[0039] The present invention further provides use of the above-mentioned double-stranded siRNA analog, a conjugate thereof, a salt thereof, or a salt of the conjugate in the manufacture of a therapeutic agent for hepatitis B.

[0040] Technical Effects The double-stranded siRNA analogs, their conjugates, their salts, or salts of the conjugates of the present invention have excellent anti-HBV biological activity. The compounds of the present invention exhibit excellent anti-HBsAg activity both in vitro and in vivo, and particularly exhibit excellent in vivo anti-HBV DNA activity in an AAV-HBV mouse model. At the same time, the compounds of the present invention have good in vitro liver S9 stability, low immunogenicity risk and off-target risk, and preliminary safety evaluations have confirmed that the compounds have good safety.

[0041] Definitions and Explanations Unless otherwise specified, the following terms and phrases used herein have the following meanings: If a particular term or phrase is not specifically defined, it should not be considered indefinite or unclear, but should be interpreted according to the meaning understood by a person of ordinary skill in the art. When a trade name is mentioned herein, it is intended to refer to the corresponding product or its active ingredient.

[0042] Unless otherwise specified, the terms "comprise," "include," and "contain" as used herein are open-ended and are meant to encompass the stated elements, components, or steps as well as other elements, components, or steps not specifically stated.

[0043] The terms "optionally" or "optionally" mean that the subsequently described event or condition may, but need not, occur, and that the description includes both cases where the event or condition occurs and cases where it does not occur.

[0044] Unless otherwise specified, the term "nucleic acid" as used herein refers to a polymer containing at least two nucleotides (i.e., deoxyribonucleotides or ribonucleotides) in single- or double-stranded form, including DNA and RNA. A "nucleotide" contains a sugar, deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked to each other by the phosphate group. "Base" includes purines and pyrimidines, including the naturally occurring compounds adenine, thymine, guanine, cytosine, uracil, inosine, and naturally occurring analogs.

[0045] Unless otherwise specified, the term "oligonucleotide" or "oligomeric nucleotide" as used herein refers to a polymer or oligomer of nucleotide or nucleoside monomers composed of naturally occurring bases, sugars, and intersugar (backbone) linkages, and further includes polymers or oligomers containing non-naturally occurring monomers or portions thereof that function similarly.

[0046] Unless otherwise specified, the term "oligo" or "oligonucleotide" as used herein refers to a nucleotide sequence containing 10 to 50 nucleotides or nucleotide base pairs. In some embodiments of the present invention, the oligonucleotide has a base sequence that is at least partially complementary to the coding sequence of a target nucleic acid or target gene expressed in a cell. The nucleotides may be optionally modified. In some embodiments of the present invention, after delivery of the oligonucleotide to a cell expressing the gene, the oligonucleotide can suppress or inhibit gene expression in vitro or in vivo. "Oligonucleotide" includes, but is not limited to, single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), ribozymes, interfering RNA molecules, and Dicer enzyme substrates.

[0047] Unless otherwise specified, the term "short interfering RNA (siRNA)" as used herein refers to a type of RNA molecule having a length of 14 to 30 base pairs, similar to miRNA, which acts within the RNA interference (RNAi) pathway, interfering with the translation of mRNA of a specific gene with a complementary nucleotide sequence, resulting in mRNA degradation. The short interfering RNA (siRNA) of the present invention includes double-stranded siRNA (containing both a sense strand and an antisense strand) and single-stranded siRNA (containing only an antisense strand, for example).

[0048] Unless otherwise specified, the term "inhibition" as used herein, when referring to the expression of a particular gene, means that gene expression is reduced when a cell, group of cells or tissue is treated with an oligonucleotide as described herein, compared to a cell, group of cells or tissue that is not treated with an oligonucleotide as described herein.

[0049] Unless otherwise specified, the term "double-stranded siRNA analog" in the present invention refers to a complex of ribonucleic acid molecules with a double-stranded structure, comprising two nucleotide strands that are antiparallel and substantially complementary to each other, and have "sense" and "antisense" orientations with respect to target RNA.In the present invention, "complementary" has the meaning well known to those skilled in the art, that is, in a double-stranded nucleic acid molecule, the bases of one strand are complementary paired with the bases of the other strand.The purine base adenine (A) always pairs with the pyrimidine base uracil (U), and the purine base guanine (C) always pairs with the pyrimidine base cytosine (G).Each base pair contains one purine and one pyrimidine.If adenine on one strand always pairs with uracil on the other strand, and guanine always pairs with cytosine, the two strands are considered to be complementary to each other, and the sequence of the strand can be deduced from the sequence of the complementary strand.

[0050] Unless otherwise specified, the term "substantially complementary" as used herein means that corresponding positions in two sequences may be completely complementary, or one or more mismatches may exist, and if a mismatch exists, typically there is a mismatch of less than 5, 4, 3, 2, or 1 base pair.

[0051] Unless otherwise specified, a "sequence" or "nucleotide sequence" of the present invention refers to the order or arrangement of nucleic acid bases or nucleotides represented by a series of letters written using standard nucleotide nomenclature.

[0052] Unless otherwise specified, the terms "antisense strand" (AS), template strand, or "guide strand" of the present invention refer to the strand of an oligonucleotide compound that is substantially complementary to the corresponding region of a target sequence (e.g., AGT mRNA).

[0053] Unless otherwise specified, the terms "sense strand," "useful strand," "significant strand" (SS), coding strand, or "passenger strand" of the present invention refer to a strand that can form a region substantially complementary to an antisense strand. The term "substantially complementary" means that the corresponding positions of the two sequences may be completely complementary, or one or more mismatches may exist, and if a mismatch exists, there is usually a mismatch of 3, 2, or less than 1 base pair. In a double-stranded nucleic acid molecule, bases on one strand complementarily pair with bases on the other strand. The purine base adenine (A) always pairs with the pyrimidine base uracil (U), and the purine base guanine (C) always pairs with the pyrimidine base cytosine (G).

[0054] The nucleotide monomers "A," "U," "G," and "C" described in the present invention represent adenosine-3'-phosphate, uridine-3'-phosphate, guanosine-3'-phosphate, and cytidine-3'-phosphate, respectively.

[0055] Unless otherwise specified, as used herein, the term "conjugation" refers to two or more chemical moieties, each having a specific function, being linked together by a covalent bond, and correspondingly, "conjugate" refers to a compound formed by covalent bonds between said chemical moieties.

[0056] Unless otherwise specified, the term "conjugate of double-stranded siRNA analog" in the present invention refers to a compound formed by linking a double-stranded siRNA analog with a pharmaceutically acceptable conjugate group, and the double-stranded siRNA analog and the pharmaceutically acceptable conjugate group are covalently bonded.

[0057] Unless otherwise specified, the "pharmaceutically acceptable conjugate groups" of the present invention are advantageous for the internal delivery of nucleic acids and compositions suitable for internal therapeutic applications. In some embodiments, the "pharmaceutically acceptable conjugate groups" of the present invention have the effect of enhancing the affinity of nucleic acids and compositions suitable for internal therapeutic applications to their targets (target tissues / cells). Exemplary conjugate groups include, but are not limited to, L96, compound group D1, compound group D2, compound group D3, and the like.

[0058] Unless otherwise specified, the "modification" of the present invention refers to a modification to the base on the nucleotide or substituted nucleotide group (e.g., R, E, etc.), the sugar ring, the bond between the nucleotide and / or substituted nucleotide group (e.g., R, E, etc.). Illustratively, the "modification" of the present invention includes an MOE modification, a methoxy modification, a fluorination modification, ( <tg2725> E< / tg2725> Modifications include, but are not limited to, 3'-vinyl phosphate modifications, phosphorothioate ester groups, or replacing nucleotides with GNA (glycerol nucleic acids). Illustratively, the "modifications" of the present invention may include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety, where the ribose moiety includes an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose into a 3'-endo structure.

[0059] Unless otherwise specified, the term "selectively modified nucleotides" used herein means that each nucleotide may be independently unmodified or modified, and the modifications on each modified nucleotide are also independent. The "modifications" include, but are not limited to, modifications of the nucleobase, ribose, and phosphate. The term "unmodified nucleotide" refers to a nucleotide composed of a naturally occurring nucleobase, a natural sugar ring, and a natural phosphate. The term "modified nucleotide" refers to a nucleotide containing at least one of a modified nucleobase, a modified sugar ring, and a modified phosphate. In some embodiments of the present invention, the term "modified nucleotide" refers to a nucleotide composed of a modified nucleobase, a modified sugar ring, and a modified phosphate. In some embodiments of the present invention, a "modified nucleotide" is composed of a modified nucleobase, a natural sugar ring, and a natural phosphate ester; in some embodiments of the present invention, a "modified nucleotide" is composed of a natural nucleobase, a natural sugar ring, and a modified phosphate ester; in some embodiments of the present invention, a "modified nucleotide" is composed of a natural nucleobase, a natural sugar ring, and a modified phosphate ester; in some embodiments of the present invention, a "modified nucleotide" is composed of a natural nucleobase, a modified sugar ring, and a modified phosphate ester; in some embodiments of the present invention, a "modified nucleotide" is composed of a modified nucleobase, a natural sugar ring, and a modified phosphate ester; in some embodiments of the present invention, a "modified nucleotide" is composed of a modified nucleobase, a modified sugar ring, and a natural phosphate ester; in some embodiments of the present invention, a "modified nucleotide" is composed of a modified nucleobase, a modified sugar ring, and a modified phosphate ester. Unless otherwise specified, in the present invention, a "natural sugar ring" is selected from a 2'-OH five-membered sugar ring or a 2'-deoxy five-membered sugar ring.

[0060] Unless otherwise specified, the "natural bases" of the present invention are selected from the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0061] Unless otherwise specified, the "modified nucleobase" of the present invention refers to a 5- to 12-membered saturated, partially unsaturated, or aromatic heterocycle other than a natural base, and includes a single ring or a fused ring. Specific examples include thiophene, thianthrene, furan, pyran, isobenzofuran, benzothiazine, pyrrole, imidazole, substituted or unsubstituted triazole, pyrazole, isothiazole, isoxazole, pyridazine, indolizine, indole, isoindole, isoquinoline, quinoline, naphthopyridine, quinazoline, carbazole, phenanthridine, piperidine, phenazine, phenazine, phenothiazine, furanan, phenoxazine, pyrrolidine, pyrroline, imidazolidine, imidazoline, pyrazolidine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine ... adenine, 2-aminoguanine, 2-propyladenine and guanine and other alkyl derivatives, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl 5-hydroxymethyl and other 8-substituted adenines and guanines; 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines; 7-methylguanine and adenine; 8-azaguanine and adenine; 7-deazaguanine and adenine; and 3-deazaguanine and adenine.

[0062] Unless otherwise specified, the "modified sugar ring" of the present invention can include, but is not limited to, any of the following modifications at the 2' position: H, F, O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C 10 Alkyl or C2-C 10 It may be alkenyl or alkynyl. Examples of suitable modifications include O[(CH2) n O] m CH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2, and O(CH2) n ON[(CH2) n CH3)]2, where n and m are 1 to 10. In other embodiments, the 2'-position includes a substituted or unsubstituted C1 to C 10 Modifications include, but are not limited to, lower alkyl, alkylaryl, arylalkyl, O-alkylaryl or O-arylalkyl, SH, SCH, OCN, Cl, Br, CN, CF, OCF, SOCH, SOCH, ONO, NO, N, NH, heterocyclic alkyl, heterocyclic alkylaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, intercalator, group for improving the pharmacokinetic properties of iRNA, group for improving the therapeutic properties of iRNA, and other substituent modifications with similar properties. In some embodiments, such modifications include, but are not limited to, 2'-methoxyethoxy (2'-O-CHCHOCH, also known as 2'-O-(2-methoxyethyl) or 2'-MOE).

[0063] Unless otherwise specified, the "modified phosphate esters" of the present invention include, but are not limited to, phosphorothioate ester modifications, which "phosphorothioate esters" include (R)- and (S)-isomers and / or mixtures thereof.

[0064] In some embodiments of the present invention, the modified nucleotide may comprise one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety, where the ribose moiety includes an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in a 3'-endo conformation.

[0065] In some embodiments of the present invention, the modified nucleotide comprises one or more UNAs (unlocked nucleic acids). A UNA is an unlocked acyclic nucleic acid in which any sugar linkage has been removed to form an unlocked "sugar" residue. In one example, the UNA further comprises a monomer in which the C1'-C4' bond has been removed (i.e., a covalent carbon-oxygen-carbon bond between the C1' and C4' carbons). In another example, the C2'-C3' bond of the sugar has been removed (i.e., a covalent carbon-carbon bond between the C2' and C3' carbons).

[0066] In some embodiments of the present invention, the modified nucleotide comprises one or more GNAs (glycerol nucleic acids). GNAs include GNA-A, GNA-T, GNA-C, GNA-G, and GNA-U. The structure of GNA-A is

[0067] [ka] and the structure of GNA-T is

[0068] [ka] and the structure of GNA-C is

[0069] [ka] and the structure of GNA-G is

[0070] [ka] and the structure of GNA-U is

[0071] [ka] is.

[0072] In some embodiments of the present invention, modified nucleotides may contain one or more bicyclic sugar moieties. A "bicyclic sugar" refers to a furanosyl ring modified with a two-atom bridge. A "bicyclonucleoside" ("BNA") is a nucleoside containing a sugar moiety bridging two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4' and 2' carbons of the sugar ring.

[0073] Unless otherwise specified, the term "covalent bond" as used herein includes, but is not limited to, bonds such as "phosphate ester bond" (i.e., "phosphodiester bond"), "thiophosphate bond", and the like.

[0074] In the present invention, the "modification" of a nucleotide includes a modification of the nucleic acid base, a modification of the sugar ring, a modification of the internucleotide bond, etc. Exemplary "modification" of a nucleotide in the present invention includes, but is not limited to, a methoxy modification, a fluorine modification, an (E)-vinyl phosphate ester modification, a phosphorothioate ester bond, or substitution of a nucleotide with an (S)-glycerol nucleic acid, etc.

[0075] Unless otherwise specified, when "R," "E," "A," "U," "G," and "C" are preceded by "m," it indicates that the nucleotide is 2'-O-methyl modified, and when "f" is preceded by "R," "E," "A," "U," "G," and "C," it indicates that the nucleotide is 2'-O-methyl modified.

[0076] Unless otherwise specified, the "R", "E", "A", "U", "G" and "C" of the present invention are linked by phosphate esters. Illustratively, the chemical structure of 5'-fAmRmC-3' is as follows:

[0077] [ka]

[0078] When "s" is added between "R," "E," "A," "U," "G," and "C" in the present invention, it indicates that the nucleotides are linked by a phosphorothioate ester group. In this invention, "R", "E", "A", "U", "C" and "G" are preceded by " MOE " indicates that the nucleotide or substituted nucleotide group is modified with 2'-O-methoxyethyl.

[0079] In some embodiments of the present invention, when "R", "E", "A", "U", "C" and "G" are preceded by "m", it indicates that the nucleotide or substituted nucleotide group is 2'-O-methyl modified (which belongs to methoxy modification), when "f" is preceded by "R", "E", "A", "U", "C" and "G", it indicates that the nucleotide or substituted nucleotide group is 2'-O-methyl modified (which belongs to methoxy modification), when "f" is preceded by "R", "E", "A", "U", "C" and "G", it indicates that the nucleotide or substituted nucleotide group is 2'-fluoronucleotide modified (which belongs to fluorination modification), when "f" is preceded by "f ... MOE " indicates that the nucleotide or substituted nucleotide group is 2'-O-methoxyethyl modified (belonging to MOE modification). In some embodiments of the present invention, when "s" is added between the "R", "E", "A", "U", "C" and "G", it indicates that the nucleotide is bonded via a phosphorothioate ester group. For example, 5'-mRs MOE The chemical structure of GsmCmAfC-3' is as follows:

[0080] [ka]

[0081] Unless otherwise specified, the term "multiple" and "multivalent" as used herein refers to an integer of 2 or greater, including, but not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, up to the theoretically possible maximum number of GalNAc derivatives attached to the siRNA analog or branched conjugate.

[0082] The sense strand or antisense strand of the double-stranded siRNA analogue described in the present invention may comprise " overhang ", for example, asymmetric protruding nucleotides that are not directly involved in the RNA double helix structure formed by the pair of " sense strand " and " antisense strand " as defined herein. Illustratively, such overhang may comprise one or more modified or unmodified U, T and A.

[0083] The compounds of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including (R)- and (S)-enantiomers, diastereomers, and racemic and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, and all such mixtures are within the scope of the present invention. Substituents such as alkyl groups may also have additional asymmetric carbon atoms. All such isomers and mixtures thereof are within the scope of the present invention.

[0084] Unless otherwise specified, the term "enantiomer" or "optical isomer" refers to mirror-image stereoisomers. Unless otherwise specified, the term "diastereomer" refers to stereoisomers in which the molecules have two or more centers of chirality and there is no mirror-image relationship between the molecules.

[0085] Unless otherwise specified, solid wedge bonds (

[0086] [ka] ) and wedge-shaped dashed bond (

[0087] [ka] ) represents the absolute configuration of the stereocenter, and the straight solid bond (

[0088] [ka] ) and straight dashed bond (

[0089] [ka] ) indicates the relative configuration of the stereocenter.

[0090] [ka] ) is a solid wedge bond (

[0091] [ka] ) or dashed wedge bond (

[0092] [ka] ), or a wavy line (

[0093] [ka] ) is a straight solid line bond (

[0094] [ka] ) and / or straight dashed bond (

[0095] [ka] )

[0096] Unless otherwise specified, if a group has one or more bondable positions, any one or more of the positions on the group can be linked to other groups by a chemical bond. The chemical bond connecting the positions to other groups is represented by a straight solid bond (

[0097] [ka] ), straight dashed bond (

[0098] [ka] ), or a wavy line (

[0099] [ka] ) can be expressed as

[0100] Unless otherwise specified, the terms "enriched in one isomer," "isomer-enriched," "enriched in one enantiomer," or "enantiomerically enriched" refer to less than 100% isomer or enantiomer content, and the content of that 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.

[0101] Unless otherwise specified, the terms "isomeric excess" or "enantiomeric excess" refer to the relative percentage difference between two isomers or two enantiomers. For example, if one isomer or enantiomer is 90% and the other isomer or enantiomer is 10%, the isomeric or enantiomeric excess (ee) is 80%.

[0102] Optically active (R)- and (S)-isomers, as well as D- and L-isomers, can be prepared by chiral synthesis, chiral reagents, and other conventional techniques. If a single enantiomer of a compound of the present invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, in which case the resulting diastereomeric mixture can be separated and the desired enantiomer obtained purely by cleavage of the auxiliary. Alternatively, if the molecule contains a basic (e.g., amino) or acidic (e.g., carboxyl) functional group, non-diastereomeric salt formation with an appropriate optically active acid or base can be used for diastereomeric resolution by conventional methods well known in the art, followed by recovery of the pure enantiomer. Furthermore, separation of enantiomers and diastereomers is typically accomplished by chromatography using chiral stationary phases, optionally combined with chemical derivatization (e.g., carbamate formation from amines). The compounds of the present invention may contain unnatural proportions of isotopes at one or more of their constituent atoms. For example, tritium ( 3 H), iodine-125( 125 I) or carbon-14 ( 14 Deuterium compounds can be labeled with radioactive isotopes such as CI, ...

[0103] The term "salt" refers to a salt of a compound of the present invention, which is prepared from a compound having a specific substituent discovered in this invention and a relatively non-toxic acid or base. When a compound of the present invention contains a relatively acidic functional group, a base addition salt can be obtained by contacting the compound with a sufficient amount of base in a pure solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts, or similar salts. When a compound of the present invention contains a relatively basic functional group, an acid addition salt can be obtained by contacting the compound with a sufficient amount of acid in a pure solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic and organic acid salts, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogenphosphate, dihydrogenphosphate, sulfuric acid, hydrogen sulfate, hydroiodide, and phosphorous acid, and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid, as well as salts of amino acids such as arginine and organic acids such as glucuronic acid. Certain compounds of the present invention contain basic and acidic functional groups, which allow them to be converted into either base or acid addition salts.

[0104] The salts of the present invention can be synthesized from parent compounds containing acidic or basic groups by conventional chemical methods. Generally, the preparation of such salts is carried out by reacting the free acid or base form of these compounds with the stoichiometrically appropriate base or acid in water or an organic solvent, or a mixture thereof.

[0105] The compounds of the present invention can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combination with other chemical synthetic methods, and equivalent alternative 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 compounds of the present invention can be confirmed by conventional methods known to those skilled in the art, and when the present invention relates to the absolute configuration of a compound, the absolute configuration can be verified by conventional technical means in the art. For example, in single crystal X-ray diffraction (SXRD), the grown single crystal is used to collect diffraction intensity data using a Bruker D8 venture diffractometer, with a CuKα radiation source and a φ / ω scanning method. After collecting relevant data, the crystal structure can be analyzed by a direct method (Shelxs97) to verify the absolute configuration.

[0107] The solvents used in the present invention are commercially available. Unless otherwise specified, the mixing ratios of solvents used in column chromatography and preparative thin-layer silica gel chromatography in the present invention are all volume ratios.

[0108] [Table 4] TIFF2026507087000039.tif20167

[0109] Compounds are named according to conventional naming conventions in the art or using ChemDraw® software, adopting supplier catalog names for commercially available compounds. DETAILED DESCRIPTION OF THE INVENTION

[0110] The present invention will be described in detail below through examples, but these are not intended to limit the present invention in any way. 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 alternative 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 obvious to those skilled in the art that various modifications and improvements can be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

[0111] Example 1: Synthesis of Compound 1

[0112] [ka]

[0113] Step A: Compound 1-1 (30.00 g, 94.26 mmol) and Compound A (11.98 g, 94.26 mmol) were dissolved in methyl acetate (220 mL) and concentrated to near dryness at 90 °C under 1 bar pressure. A solution of trifluoromethanesulfonic acid (141.46 mg, 0.94 mmol) in methyl acetate (2 mL) was then added. After the addition, the reaction mixture was stirred at 125 °C under 30 mbar pressure for 4 hours. The reaction mixture was cooled to 70 °C, ethanol (70 mL) was added, and the mixture was stirred at 70 °C until a homogeneous solution was formed. The stirring was then stopped and the mixture was cooled to 50 °C. The mixture was allowed to cool to 25 °C and then allowed to stand at 0 °C for 16 hours. The mixture was filtered and the filter cake was washed with 180 mL of ethanol (60 mL × 3). The filter cake was collected and dried under reduced pressure to obtain Compound 1-2. 1 H NMR (400 MHz, CDCl3): δ=8.40 (s, 1H), 6.04 (d, J=3.42 Hz, 1H), 5.81 - 5.69 (m, 1H), 5.54 (t, J=5.38 Hz, 1H), 4.51 - 4.42 (m, 2H), 4.30 - 4.16 (m, 1H), 3.98 (s, 3H), 2.18 - 2.05 (m, 9H).

[0114] Step B: Compound 1-2 (15.00 g, 38.93 mmol) and triethylamine (4.14 g, 40.87 mmol) were dissolved in methanol (100 mL), and the mixture was stirred under nitrogen gas protection at 50° C. for 17 hours. The reaction mixture was concentrated under reduced pressure to give compound 1-3. 1 H NMR (400 MHz, CD3OD): δ=8.87 (s, 1H), 5.93 (d, J=3.42 Hz, 1H), 4.48 (dd, J=3.48, 4.83 Hz, 1H), 4.33 (t, J=5.26 Hz, 1H), 4.16 - 4.10 (m, 1H), 3.95 (s, 3H), 3.84 (dd, J=3.24, 12.29 Hz, 1H), 3.70 (dd, J=4.46, 12.29 Hz, 1H).

[0115] Step C: Compound 1-3 (10.00 g, 38.58 mmol) was dissolved in pyridine (250 mL) and cooled to 0 °C. Then, 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (12.29 g, 38.97 mmol) was added dropwise. After the addition, the reaction mixture was allowed to warm to 25 °C and stirred for 16 h. The reaction mixture was concentrated under reduced pressure, and the concentrate was completely suspended in ethyl acetate (250 mL). After filtration, the filtrate was washed three times with 3 mol / L hydrochloric acid (250 mL × 3), followed by one wash with 250 mL of saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered under reduced pressure, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: V / V / V petroleum ether / dichloromethane / ethyl acetate = 3 / 1 / 1) to obtain compound 1-4. 1 H NMR (400MHz, CDCl3): δ=8.43 (s, 1H), 5.95 (s, 1H), 4.73 (dd, J=4.75, 8.00 Hz, 1H), 4.41 (d, J=4.75 Hz, 1H), 4.19 - 4.09 (m, 2H), 4.03 - 3.94 (m, 4H), 3.34 - 2.71 (m, 1H), 1.15 - 1.01 (m, 28H).

[0116] Step D: Compound 1-4 (20.00 g, 39.86 mmol) was dissolved in anhydrous toluene (200 mL), and silver oxide (92.38 g, 398.63 mmol) and 2-iodoethyl methyl ether (22.24 g, 119.56 mmol) were added sequentially. The reaction mixture was stirred at 130 °C for 24 h. After cooling to room temperature, the reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain crude compound 1-5, which was used directly in the next reaction. LC-MS (ESI) m / z: 604.3 [M+H] + .

[0117] Step E: Compound 1-5 (20.00 g, 33.12 mmol) was dissolved in anhydrous methanol (200 mL) and triethylamine (3.38 g, 33.45 mmol) was added. The reaction mixture was stirred at 25 °C for 12 h. After cooling to room temperature, the mixture was concentrated under reduced pressure to give crude compound 1-6, which was used directly in the next reaction. LC-MS (ESI) m / z: 560.3 [M+H] + .

[0118] Step F: At 0 °C, compound 1-6 (18.50 g, 33.12 mmol) was dissolved in anhydrous tetrahydrofuran (185 mL), and then triethylamine trihydrofluoroacid (11.72 g, 72.70 mmol) was added. The reaction mixture was stirred at 25 °C for 12 h. After cooling to room temperature, it was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: V / V dichloromethane / methanol = 40 / 1 to 10 / 1) to give compound 1-7. 1 H NMR (400 MHz, CD3OD) δ=8.87 (s, 1H), 6.04 (d, J=3.3 Hz, 1H), 4.45 - 4.36 (m, 2H), 4.15 - 4.09 (m, 1H), 3.96 (s, 3H), 3.87 - 3.67 (m, 4H), 3.59 - 3.54 (m, 2H), 3.34 (s, 3H).

[0119] Step G: Compound 1-7 (6.00 g, 18.91 mmol) was dissolved in anhydrous pyridine (20 mL) at 0 °C, and DMTr-Cl (7.69 g, 22.69 mmol) was added. The reaction mixture was allowed to warm naturally and stirred at 20–25 °C for 12 h. After concentration under reduced pressure, the residue was purified by silica gel column chromatography (eluent: V / V petroleum ether / ethyl acetate = 30 / 1 to 0 / 1, containing 0.2% triethylamine) to give compound 1-8. 1 H NMR (400 MHz, DMSO-d6) δ=8.95 (s, 1H), 7.33 (d, J=7.4 Hz, 2H), 7.26 - 7.17 (m, 7H), 6.82 (t, J=7.9 Hz, 4H), 6.12 (d, J=1.8 Hz, 1H), 5.23 (d, J=6.1Hz, 1H), 4.43 - 4.29 (m, 2H), 4.12 - 4.03 (m, 1H), 3.83 (s, 3H), 3.79 - 3.75 (m, 1H), 3.72 (s, 6H), 3.67 (brs, 1H), 3.48 - 3.42 (m, 2H), 3.20 (s, 3H), 3.16 - 3.08 (m, 2H).

[0120] Step H: At 0°C, compound 1-8 (1.10 g, 1.77 mmol) was dissolved in anhydrous dichloromethane (8 mL), followed by the addition of compound B (0.62 g, 2.61 mmol) and 4,5-dicyanoimidazole (0.10 g, 0.89 mmol). After the addition was complete, the reaction mixture was stirred at 20°C for 0.5 h. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: V / V petroleum ether / ethyl acetate = 50 / 1 to 1 / 2) to give compound 1. LCMS (ESI) m / z: 820.3 [M+H] + .

[0121] Example 2: Synthesis of Compound 2

[0122] [ka]

[0123] Step A: 1-4 (8.23 g, 16.40 mmol), potassium carbonate (11.34 g, 82.02 mmol), and silver oxide (19.01 g, 82.02 mmol) were added sequentially to N,N-dimethylformamide (50 mL) at room temperature, followed by the addition of methyl iodide (11.64 g, 82.02 mmol). The reaction mixture was stirred at room temperature for 3 h, then diluted with ethyl acetate (300 mL) and filtered. The filtrate was washed once each with saturated aqueous sodium thiosulfate (250 mL), water (250 mL), and saturated brine (250 mL). After drying over anhydrous sodium sulfate, the filtrate was filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: V / V petroleum ether / ethyl acetate = 5 / 1) to give 1-9. 1 H NMR (400 MHz, CDCl3): δ=8.58 (s, 1H), 5.91 (s, 1H), 4.46 (dd, J=4.22, 9.35 Hz, 1H), 4.28 - 4.17 (m, 2H), 4.06 - 3.96 (m, 5H), 3.68 (s, 3H), 1.13 - 0.99 (m, 28H).

[0124] Step B: At 0 °C, 1-9 (3.27 g, 6.34 mmol) was added to tetrahydrofuran (50 mL) and stirred to dissolve. Triethylamine trihydrofluoride (2.25 g, 13.95 mmol) was then added dropwise. After the addition, the reaction mixture was allowed to warm to room temperature and stirred for 16 h. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: V / V dichloromethane / methanol = 20 / 1) to give 1-10. 1H NMR (400 MHz, CD3OD): δ=8.88 (s, 1H), 6.04 (d, J=3.26 Hz, 1H), 4.44 (t, J=5.33 Hz, 1H), 4.20 (dd, J=3.33, 4.83 Hz, 1H), 4.14 - 4.07 (m, 1H), 3.96 (s, 3H), 3.84 (dd, J=3.20, 12.36 Hz, 1H), 3.69 (dd, J=4.39, 12.30 Hz, 1H), 3.52 (s, 3H).

[0125] Step C: At 0 °C, 1-10 (1.30 g, 4.76 mmol) was added to anhydrous pyridine (20 mL). After stirring, DMTr-Cl (2.42 g, 7.14 mmol) was added. The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was diluted with ethyl acetate (70 mL), saturated aqueous sodium bicarbonate (20 mL) and water (40 mL) were added, and the mixture was stirred uniformly and then allowed to stand for separation. The organic phase was washed once with water (60 mL) and saturated brine (60 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC (separation column: Phenomenex Luna C18 (specifications: 250 mm × 50 mm, particle size: 10 μm); mobile phase: Phase A: 10 mM aqueous ammonium bicarbonate; Phase B: acetonitrile; elution gradient: 35%–65%, 20 min) to give 1-11. 1H NMR (400 MHz, CDCl3): δ=8.44 (s, 1H), 7.45 - 7.38 (m, 2H), 7.34 - 7.28 (m, 5H), 7.27 - 7.18 (m, 2H), 6.92 - 6.70 (m, 4H), 5.97 (d, J=2.88 Hz, 1H), 4.43 - 4.37 (m, 1H), 4.33 (dd, J=2.88, 5.00 Hz, 1H), 4.25 - 4.19 (m, 1H), 3.98 (s, 3H), 3.80 (s, 6H), 3.58 (s, 3H), 3.49 - 3.43 (m, 1H), 3.40 - 3.33 (m, 1H), 2.55 (d, J=6.88 Hz, 1H). LCMS (ESI) m / z: 574.2 [MH] - .

[0126] Step D: At 0 °C, 1-11 (1.10 g, 1.91 mmol) was added to anhydrous dichloromethane (8 mL), followed by the addition of compound B (678.45 mg, 2.87 mmol) and 4,5-dicyanoimidazole (0.11 g, 0.96 mmol). After the addition, the reaction mixture was stirred at room temperature under a nitrogen atmosphere for 0.5 h. After concentration under reduced pressure, the residue was purified by silica gel column chromatography (eluent: V / V petroleum ether / ethyl acetate = 50 / 1 to 1 / 2) to give compound 2. LCMS (ESI) m / z: 776.3 [M+H] + .

[0127] Example 3: Synthesis of Compound 3

[0128] [ka]

[0129] Step A: 1-12 (10 g, 36.06 mmol) was dissolved in anhydrous pyridine (200 mL) and trimethylchlorosilane (48.98 g, 450.81 mmol) was added. The reaction mixture was stirred at 25 °C for 2 hours, after which isobutyric anhydride (71.32 g, 450.81 mmol) was added and stirring was continued at 25 °C for an additional 12 hours. The reaction mixture was cooled and 28% aqueous ammonia (109.37 g, 873.85 mmol) was slowly added dropwise at 0 °C. After the addition was complete, stirring was continued at 0 °C for an additional 15 minutes. The mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (eluent: V / V dichloromethane / methanol = 1 / 0 to 30 / 1, containing 0.2% triethylamine) and preparative HPLC (column: Kromasil Eternity XT 250 × 80 mm × 10 μm; mobile phase: Phase A: 0.05% aqueous ammonia; Phase B: 5% to 35% acetonitrile; 20 min) to give 1-13. 1 H NMR (400 MHz, DMSO-d6) δ=7.97 (s, 1H), 5.47 (dd, J=7.9, 10.3 Hz, 1H), 5.13 (br s, 1H), 4.58 (t, J=2.1 Hz, 1H), 4.33 - 4.21 (m, 1H), 3.55 (d, J=6.6 Hz, 2H), 2.84 - 2.69 (m, 1H), 2.58 - 2.52 (m, 1H), 2.43 - 2.33 (m, 1H), 2.28 (br s, 1H), 2.07 (s, 1H), 1.11 (d, J=6.8 Hz, 6H).LC-MS (ESI) m / z: 348.1 [M+H] + .

[0130] Step B: 1-13 (11.9 g, 34.26 mmol) was dissolved in anhydrous pyridine (120 mL) and DMTr-Cl (13.93 g, 41.11 mmol) was added at 0 °C. The reaction mixture was stirred at 20 °C for 12 h. After filtration, the filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by silica gel column chromatography (eluent: V / V dichloromethane / methanol = 1 / 0 to 30 / 1, containing 0.2% triethylamine) and preparative HPLC (column: Kromasil Eternity XT 250 × 80 mm × 10 μm; mobile phase: Phase A: 0.05% aqueous ammonia; Phase B: 35% to 65% acetonitrile; 21 min) to give compound 1-14. 1 H NMR (400 MHz, DMSO-d6) δ=12.27 - 11.39 (m, 2H), 7.74 (s, 1H), 7.43 - 7.22 (m, 9H), 6.90 (d, J=8.6 Hz, 4H), 5.46 (t, J=8.8 Hz, 1H), 5.11 - 4.89 (m, 2H), 4.56 (br s, 1H), 4.22 (br s, 1H), 3.74 (s, 6H), 3.22 (d, J=6.4 Hz, 2H), 2.74 (s, 1H), 2.63 (br s, 1H), 2.24 - 2.05 (m, 2H), 1.11 (d, J=6.8Hz, 6H). LC-MS (ESI) m / z:650.3 [M+H] + .

[0131] Step C: Compound 1-14 was pre-prepared by azeotropic dehydration three times with anhydrous acetonitrile. Compound 1-14 (5.10 g, 7.85 mmol) was dissolved in anhydrous dichloromethane (50 mL) at 0 °C under a nitrogen atmosphere, followed by the addition of compound B (3.55 g, 11.8 mmol, 3.74 mL) and 4,5-dicyanoimidazole (1.21 g, 10.2 mmol). After the addition, the reaction mixture was stirred at 20 °C for 2 h, then diluted with dichloromethane (20 mL), followed by the addition of saturated aqueous sodium bicarbonate (20 mL). The organic phase was extracted and separated. The organic phase was washed again with saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated, and the residue was purified by silica gel column chromatography (eluent: V / V petroleum ether / ethyl acetate = 3 / 1 to 1 / 1) to obtain compound 3. 1 H NMR (400 MHz, CD3CN) δ=7.55 (d, J=12.0 Hz, 1H), 7.51 - 7.44 (m, 2H), 7.39 - 7.19 (m, 7H), 6.91 - 6.80 (m, 4H), 5.43 - 5.37 (m, 1H), 5.05 (m, 1H), 4.68 (m, 1H), 4.53 (m, 1H), 3.78 (m, 1H), 3.76 (s, 6H), 3.75 - 3.43 (m, 3H), 3.37 - 3.26 (m, 2H), 2.87 (m, 1H), 2.68 - 2.55 (m, 2H), 2.54 - 2.25 (m, 3H), 1.21 - 1.09 (m, 18H). 31 P NMR (162 MHz, CD3CN) δ=-147.46 (s, 1P), -146.79 (s, 1P).

[0132] Example 4: Synthesis of D01

[0133] [ka] TIFF2026507087000044.tif48167

[0134] Step A: 11-Dodecyn-1-ol (2-1, 25 g, 137.14 mmol) and triethylamine (16.65 g, 164.56 mmol) were dissolved in dichloromethane (250 mL), and methanesulfonyl chloride (18.85 g, 164.56 mmol) was added at 0 °C. The mixture was stirred at 0 °C for 2 h. The reaction mixture was diluted with water (400 mL) and then extracted with 800 mL of dichloromethane (400 mL × 2). The combined organic phase was washed with 400 mL of water (200 mL × 2) and saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 2-2.

[0135] Step B: 2-3 (20 g, 67.26 mmol) was dissolved in N,N-dimethylformamide (200 mL) and added with sodium hydride (w / w = 60%, 4.04 g, 100.89 mmol) at 0 °C, followed by 2-2 (19.27 g, 73.99 mmol). The reaction mixture was stirred at 25 °C for 16 h, then quenched with water (1 L) and extracted with 1.6 L (800 mL × 2) of dichloromethane. The combined organic layer was washed once with 800 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 2-4. 1 H NMR (400 MHz, DMSO-d6): δ=7.63 - 6.89 (m, 10H), 5.64 - 5.52 (m, 2H), 4.27 - 4.01 (m, 2H), 3.98 - 3.77 (m, 2H), 3.72 - 3.18 (m, 4H), 2.23 - 2.14 (m, 2H), 1.98 - 1.92 (m, 1H), 1.54 - 1.23 (m, 16H).

[0136] Step C: 2-4 (48 g, 103.98 mmol) was dissolved in methanol (870 mL), followed by the addition of a 4 mol / L solution of hydrogen chloride in methanol (400 mL). The reaction mixture was stirred at 30°C for 2 hours, after which an additional 350 mL of 4 mol / L solution of hydrogen chloride in methanol was added. Stirring was continued at 30°C for an additional 16 hours. The reaction mixture was concentrated under reduced pressure, followed by the addition of 200 mL of chloroform, and further concentration under reduced pressure until a white solid precipitated. Toluene (130 mL) and petroleum ether (130 mL) were added to the residue, and stirring was continued at 15°C for 16 hours. The mixture was then filtered through a Buchner funnel, and the filter cake was collected and dried under reduced pressure to obtain a white solid. The white solid was dissolved in dichloromethane (50 mL), and a solution of sodium hydroxide (6.59 g, 164.66 mmol) in water (50 mL) was added. The mixture was stirred at 20°C for 1 hour, then diluted with water (500 mL), and extracted with 1 L of dichloromethane (500 mL x 2). The combined organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 2-5.

[0137] Step D: 2-5 (23 g, 80.58 mmol) and sodium hydroxide (322.31 mg, 8.06 mmol) were added to a mixture of dimethyl sulfoxide (70 mL) and water (6 mL), followed by the addition of tert-butyl acrylate (22.72 g, 177.28 mmol). The reaction mixture was stirred under a nitrogen atmosphere at 25 °C for 16 h, then diluted with water (500 mL) and extracted with 1 L of ethyl acetate (500 mL × 2). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: V / V / V petroleum ether / ethyl acetate / ethanol (containing 0.1% aqueous ammonia) = 36 / 3 / 1 to 16 / 3 / 1) to give 2-6. 1 H NMR (400 MHz, DMSO-d6): δ=3.60 - 3.54 (m, 4H), 3.32 (br s, 5H), 3.15 (s, 5H), 2.74 - 2.66 (m, 1H), 2.40 (t, J=6.0 Hz, 4H), 2.18 - 2.11 (m, 2H), 1.58 - 1.38 (m, 22H), 1.34 - 1.23 (m, 12H).

[0138] Step E: To a solution of 2-6 (24.5 g, 45.22 mmol) in dichloromethane (250 mL), triethylamine (9.15 g, 90.45 mmol) and succinic anhydride (6.79 g, 67.83 mmol) were added and stirred for 16 hours at 20° C. Dichloromethane (1 L) and hydrochloric acid (1 mol / L, 1 L) were added to the reaction mixture, which was stirred and then allowed to stand for separation. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to give 2-7. 1 H NMR (400 MHz, CDCl3): δ=6.49 - 6.37 (m, 1H), 3.72 (s, 2H), 3.70 - 3.57 (m, 8H), 3.37 (t, J=6.7 Hz, 2H), 2.69 - 2.51 (m, 4H), 2.50 - 2.36 (m, 4H), 2.22 - 2.13 (m, 2H), 1.96 - 1.90 (m, 1H), 1.57 - 1.47 (m, 4H), 1.46 - 1.40 (m, 18H), 1.40 - 1.31 (m, 2H), 1.30 - 1.21 (m, 10H).

[0139] Step F: 2-7 (27.4 g, 42.69 mmol) was dissolved in formic acid (140 mL). The solution was stirred under a nitrogen atmosphere at 20° C. for 16 hours. After concentration under reduced pressure, toluene (150 mL) was added to the residue, which was then concentrated to dryness under reduced pressure. Toluene (150 mL) was added again, and the mixture was concentrated to dryness under reduced pressure to give 2-8. 1 H NMR (400 MHz, CDCl3): δ=9.79 - 9.22 (m, 3H), 6.44 - 6.23 (m, 1H), 3.88 - 3.43 (m, 10H), 3.39 - 3.20 (m, 2H), 2.77 - 2.31 (m, 8H), 2.15 - 2.06 (m, 2H), 1.87 (t, J=2.6 Hz, 1H), 1.48 - 1.28 (m, 6H), 1.26 - 1.12 (m, 10H).

[0140] Step G: 2-8 (22.6 g, 42.67 mmol), N,N-diisopropylethylamine (33.09 g, 256.03 mmol), and HATU (51.92 g, 136.55 mmol) were dissolved in N,N-dimethylformamide (250 mL) and tert-butyl N-(3-aminopropyl)aminocarboxylate (29.74 g, 170.69 mmol) was added. The reaction mixture was stirred at 20 °C for 16 h, after which dichloromethane (1 L) and hydrochloric acid (1 mol / L, 1 L) were added. The mixture was stirred and allowed to stand for 10 min before separation. The organic phase was washed successively with water (1 L), saturated aqueous sodium bicarbonate (1 L), and saturated brine (1 L). The mixture was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: V / V / V petroleum ether / ethyl acetate / ethanol) = (40 / 3 / 1 to 10 / 3 / 1) to give 2-9. 1 H NMR (400 MHz, CDCl3): δ=7.22 - 6.79 (m, 3H), 6.77 - 6.44 (m, 1H), 5.45 - 5.00 (m, 3H), 3.86 - 3.73 (m, 2H), 3.72 - 3.63 (m, 4H), 3.62 - 3.45 (m, 4H), 3.41 - 3.32 (m, 2H), 3.32 - 3.20 (m, 6H), 3.19 - 3.03 (m, 6H), 2.56 - 2.47 (m, 4H), 2.47 - 2.39 (m, 4H), 2.21 - 2.12 (m, 2H), 1.95 - 1.90 (m, 1H), 1.70 - 1.57 (m, 6H), 1.56 - 1.47 (m, 4H), 1.46 - 1.38 (m, 29H), 1.30 - 1.25 (m, 10H).

[0141] Step H: 2-9 (15 g, 15.03 mmol) was dissolved in dichloromethane (114 mL) and trifluoroacetic acid (38 mL) was added. The reaction mixture was stirred at 20°C for 16 hours. After concentration under reduced pressure, a mixture of toluene / acetonitrile (V / V = 3 / 1, 200 mL) was added and the mixture was concentrated to dryness under reduced pressure. The above concentration procedure was repeated three times to obtain 2-10.

[0142] Step I: 2-11 (22.15 g, 49.50 mmol), N,N-diisopropylethylamine (7.75 g, 60.00 mmol), HOAt (6.12 g, 45.00 mmol), and HATU (20.53 g, 54.00 mmol) were dissolved in N,N-dimethylformamide (90 mL), followed by the addition of a solution of 2-10 (15.6 g, 15.00 mmol) and N,N-diisopropylethylamine (21.32 g, 165.00 mmol) in N,N-dimethylformamide (120 mL). The mixture was stirred at 20 °C for 16 h, followed by the addition of dichloromethane (1.2 L) and hydrochloric acid (1 mol / L, 1 L). After stirring, the layers were separated, and the organic phase was washed successively with 1 L of water, 1 L of aqueous sodium bicarbonate solution, and 1 L of saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: V / V / V dichloromethane / methanol = 100 / 1, 10 / 1, dichloromethane / ethanol = 1 / 1) to give 2-12. 1 H NMR (400 MHz, DMSO-d6): δ=7.87 - 7.66 (m, 9H), 7.09 (s, 1H), 5.21 (d, J=3.4 Hz, 3H), 4.96 (dd, J=3.4, 11.3 Hz, 3H), 4.48 (d, J=8.5 Hz, 3H), 4.06 - 3.98 (m, 9H), 3.91 - 3.82 (m, 3H), 3.74 - 3.66 (m, 3H), 3.58 - 3.46 (m, 12H), 3.31 (br s, 3H), 3.07 - 2.98 (m, 12H), 2.71 (t, J=2.6 Hz, 1H), 2.33 - 2.22 (m, 8H), 2.16 - 2.12 (m, 2H), 2.10 (s, 9H), 2.04 (br t, J=7.1 Hz, 6H), 1.99 (s, 9H), 1.89 (s, 9H), 1.81 - 1.74 (m, 9H), 1.54 - 1.39 (m, 22H), 1.32 (dd, J=4.5, 6.7 Hz, 2H), 1.24 (s, 10H).

[0143] Step J: 2-12 (1.00 g, 0.50 mmol) and N-methyl-N,N,N-trinortylammonium chloride (20.35 mg, 50.35 μmol) were dissolved in a mixture of acetic acid (2.7 mL) and n-pentane (6.3 mL). To this mixture was added a solution of potassium permanganate (0.40 g, 2.52 mmol) in water (9 mL) dropwise at 0 °C. The mixture was stirred at 0–15 °C for 2 h. The reaction was quenched with sodium bisulfite (1.27 g), hydrochloric acid (2 mol / L, 5 mL) and water (30 mL) were added, and the mixture was extracted with 120 mL of a chloroform / isopropanol mixture (V / V = 3 / 1, 40 mL × 3). The combined organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. 30 mL of a mixture of toluene and acetonitrile was added to the residue, and the mixture was concentrated under reduced pressure again. The above concentration procedure was repeated six times to obtain 2-13. 1 H NMR (400 MHz, CD3OD): δ=5.34 (d, J=2.9Hz, 3H), 5.06 (dd, J=3.3, 11.2 Hz, 3H), 4.56 (d, J=8.4 Hz, 3H), 4.19 - 4.06 (m, 9H), 4.04 - 3.98 (m, 3H), 3.87 (td, J=5.7, 9.9 Hz, 4H), 3.72 - 3.64 (m, 9H), 3.57 - 3.50 (m, 3H), 3.39 (br t, J=6.4 Hz, 2H), 3.22 (q, J=6.4 Hz, 12H), 2.51 - 2.40 (m, 9H), 2.21 (br t, J=7.3 Hz, 6H), 2.14 (s, 9H), 2.03 (s, 9H), 1.94 (d, J=7.9 Hz, 18H), 1.72 - 1.57 (m, 22H), 1.39 (br s, 12H).

[0144] Step K: To a stirred solution of 2-13 (1.00 g, 0.50 mmol) in N,N-dimethylformamide (10 mL) was added N,N-diisopropylethylamine (0.26 g, 1.99 mmol) and HATU (0.23 g, 0.60 mmol), followed by 2-14 (0.23 g, 0.55 mmol). The reaction mixture was stirred at 15 °C for 16 h, and then dichloromethane (50 mL) and water (50 mL) were added. After stirring, the mixture was separated. The organic phase was washed sequentially with saturated aqueous sodium bicarbonate (50 mL), water (50 mL), and saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: V / V dichloromethane / methanol = 20 / 1 to 10 / 1) to give 2-15. 1 H NMR (400 MHz, DMSO-d6): δ=7.90 - 7.82 (m, 6H), 7.78 (d, J=4.8 Hz, 3H), 7.40 - 7.26 (m, 10H), 6.91 (dd, J=3.1, 9.0 Hz, 4H), 5.26 (d, J=3.4 Hz, 3H), 5.03 - 4.99 (m, 3H), 4.53 (d, J=8.4 Hz, 3H), 4.43 (d, J=3.8 Hz, 1H), 4.23 - 4.14 (m, 1H), 4.12 - 4.02 (m, 9H), 3.92 (td, J=9.0, 11.0 Hz, 3H), 3.78 (s, 6H), 3.77 - 3.71 (m, 3H), 3.66 - 3.51 (m, 13H), 3.49 - 3.41 (m, 4H), 3.11 - 3.01 (m, 16H), 2.38 - 2.37 (m, 1H), 2.32 (br s, 9H), 2.14 (s, 9H), 2.08 (br t, J=6.9 Hz, 7H), 2.04 (s, 9H), 1.93 (s, 9H), 1.82 (s, 9H), 1.57 - 1.46 (m, 22H), 1.31 - 1.26 (m, 12H).

[0145] Step L: To a solution of 2-15 (0.80 g, 0.33 mmol) in dichloromethane (8 mL), triethylamine (67.24 mg, 0.64 mmol), 4-N,N-dimethylaminopyridine (0.12 g, 1.00 mmol), and succinic anhydride (83.13 mg, 0.83 mmol) were added sequentially. The mixture was stirred at 10 °C for 16 h, followed by the addition of dichloromethane (50 mL), water (30 mL), and saturated brine (30 mL). The separated organic phase was washed sequentially with water (30 mL) and saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC (preparative column: Waters Xbridge C18 (specifications: 150 mm × 50 mm, particle size: 10 μm); mobile phase: [water (10 mM ammonium bicarbonate)-acetonitrile]; elution gradient: 27% to 57%, 11 minutes) to obtain compound D01. 1 H NMR (400 MHz, DMSO-d6): δ=7.96 - 7.69 (m, 9H), 7.33 - 7.09 (m, 10H), 6.90 - 6.78 (m, 4H), 5.21 (d, J=3.3 Hz, 3H), 4.97 (dd, J=3.3, 11.2 Hz, 3H), 4.49 (d, J=8.4 Hz, 3H), 4.06 - 3.97 (m, 9H), 3.91 - 3.83 (m, 3H), 3.79 - 3.66 (m, 11H), 3.63 - 3.45 (m, 18H), 3.02 (br d, J=4.6Hz, 14H), 2.46 - 2.37 (m, 4H), 2.35 - 2.14 (m, 12H), 2.10 (s, 9H), 2.04 (t, J=7.0 Hz, 6H), 1.99 (s, 9H), 1.88 (s, 9H), 1.77 (s, 9H), 1.57 - 1.37 (m, 22H), 1.22 (br s, 12H).

[0146] Example 5: Synthesis of D02

[0147] [ka] TIFF2026507087000046.tif158167

[0148] Step A: Compound 3-1 (50 g, 271.27 mmol), trimethylamine hydrochloride (2.59 g, 27.13 mmol), and p-toluenesulfonyl chloride (77.98 g, 406.91 mmol) were added sequentially to dichloromethane (500 mL), and triethylamine (32.94 g, 325.53 mmol) was added dropwise at 0 °C. After the addition, the reaction mixture was allowed to warm to room temperature and stirred for 12 h. The reaction mixture was diluted with dichloromethane (500 mL) and washed sequentially with sodium hydroxide solution (1 mol / L, 500 mL), dilute hydrochloric acid (1 mol / L, 500 mL), water (500 mL), and saturated brine (500 mL). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to give compound 3-2. 1 H NMR (400 MHz, DMSO-d6) δ= 7.77 (d, J=8.2 Hz, 2 H), 7.48 (d, J=8.2 Hz, 2H), 5.79 - 5.77 (m, 1 H), 5.04 - 4.90 (m, 2 H), 3.99 (t, J=6.2 Hz, 2 H), 2.42 (s, 3H), 2.00 (q, J=6.75 Hz, 2 H), 1.59 - 1.47 (m, 2 H), 1.35 - 1.13 (m, 14 H).

[0149] Step B: To a solution of compound 3-2 (50 g, 147.71 mmol) in tetrahydrofuran (500 mL), potassium hydroxide (41.14 g, 738.54 mmol) and 2-3 (46.12 g, 155.09 mmol) were added. The mixture was stirred at 80 °C for 12 h. After completion of the reaction, the reaction mixture was quenched with ice water (1000 mL) and then washed with 2000 mL of methyl tert-butyl ether (1000 mL per wash, twice). The organic phase was washed sequentially with water (1000 mL) and saturated brine (1000 mL), and then dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to give 3-3, which was used directly in the next step.

[0150] Step C: Compound 3-3 (65.53 g, 141.34 mmol) was dissolved in a mixture of methanol (230 mL) and water (77 mL), followed by the addition of concentrated hydrochloric acid (12 mol / L, 40 mL). The reaction mixture was stirred at 50 °C for 12 h and then concentrated under reduced pressure until approximately 10 mL of liquid remained. Dilute hydrochloric acid (2 mol / L, 500 mL), tert-butyl methyl ether (500 mL), and petroleum ether (500 mL) were added to the residue, mixed uniformly, and allowed to stand for an extended period of time for layer separation (three layers). The liquid interlayer was collected and adjusted to pH > 11 with sodium hydroxide solution (1 mol / L), followed by two extractions with dichloromethane (500 mL × 2). The combined organic phase was washed once with saturated brine (200 mL), dried over anhydrous sodium sulfate, filtered under reduced pressure, and the filtrate was concentrated under reduced pressure. Acetonitrile (90 mL) was added to the residue, and the mixture was heated to 60°C and stirred to dissolve. The mixture was then gradually cooled to 0°C and stirred for another 30 minutes to precipitate a solid. The filter cake was collected by low-temperature filtration and dried under reduced pressure to obtain compound 3-4. 1 H NMR (400 MHz, DMSO-d6) δ=5.80 - 5.76 (m, 1H), 5.07 - 4.83 (m, 2H), 4.34 (t, J=5.44 Hz, 2H), 3.21 (d, J=5.26 Hz, 4H), 3.16 (s, 2H), 2.03 - 1.97 (m, 2H), 1.46 (t, J=6.36 Hz, 2H), 1.32 (d, J=6.72 Hz, 2H), 1.24 (br s, 12H).

[0151] Step D: To a solution of compound 3-4 (17.91 g, 62.31 mmol) in dimethyl sulfoxide (100 mL), sodium hydroxide (224.3 mg, 5.61 mmol) and water (10 mL) were added, and tert-butyl acrylate (17.57 g, 137.08 mmol) was added under a nitrogen atmosphere. After the addition was complete, the reaction mixture was stirred at room temperature for 12 h. The reaction mixture was diluted with 0.2% aqueous sodium chloride (250 mL) and extracted twice with 400 mL of dichloromethane (250 mL × 2). The combined organic layers were dried over anhydrous sodium sulfate and filtered under reduced pressure. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: V / V / V petroleum ether / ethyl acetate / ethanol) = 80 / 3 / 1 to 32 / 3 / 1) to give 3-5. 1 H NMR (400 MHz, CDCl3) δ=5.83 - 5.77 (m, 1H), 5.10 - 4.83 (m, 2H), 3.64 (t, J=6.42 Hz, 5H), 3.40 - 3.24 (m, 8H), 2.44 (t, J=6.36 Hz, 5H), 2.02 (q, J=7.01 Hz, 2H), 1.54 - 1.48 (m, 2H), 1.43 (s, 18H), 1.38 - 1.33 (m, 2H), 1.25 (br s, 12H). LC-MS (ESI) m / z: 544.4 [M+H] + .

[0152] Step E: Compound 3C (10.47 g, 22.07 mmol) was dissolved in N,N-dimethylformamide (140 mL), followed by the sequential addition of HATU (6.70 g, 17.65 mmol) and triethylamine (4.10 mL). The mixture was stirred at 35 °C for 5 min, followed by the addition of compound 3-5 (8.00 g, 14.71 mmol). After the addition was complete, stirring was continued for an additional 12 h at 35 °C. The reaction mixture was diluted with water (100 mL) and extracted with ethyl acetate (300 mL). The organic phase was washed twice with purified water (100 mL) and once with saturated brine (50 mL). After concentration under reduced pressure, the residue was purified by preparative HPLC (separation column: Agela Innoval ODS-2 250 mm × 100 mm × 10 μm; mobile phase: Phase A: 10 mM trifluoroacetic acid aqueous solution, Phase B: acetonitrile; elution gradient: 90% to 100%, 20 min) to obtain compound 3-6. 1 H NMR (400 MHz, CDCl3) δ=9.08 (s, 1H), 8.34 (s, 2H), 8.29 (s, 1H), 7.73 (d, J=7.60 Hz, 2H), 7.57 (d, J=7.60 Hz, 2H), 7.36 (t, J=7.20 Hz, 2H), 7.28 - 7.24 (m, 1H), 6.76 (s, 1H), 5.94 - 5.93 (m, 1H), 5.86 - 5.76 (m, 1H), 5.01 - 4.92 (m, 2H), 4.38 (d, J=7.20 Hz, 2H), 4.19 - 4.16 (m, 1H), 3.91 - 3.83 (m, 6H), 3.75 - 3.72 (m, 4H), 3.63 - 3.59 (m, 2H), 3.46 (t, J=6.80 Hz, 2H), 2.74 - 2.71 (m, 2H), 2.51 (t, J=6.00 Hz, 4H), 2.04 - 2.01 (m, 2H), 1.56 - 1.54 (m, 2H), 1.42 (s, 18H), 1.30 - 1.25 (m, 16H). LC-MS (ESI) m / z: 1014.6 [M+H] + .

[0153] Step F: Compound 3-6 (2.55 g, 2.55 mmol) was dissolved in N,N-dimethylformamide (50 mL), followed by the sequential addition of HATU (1.07 g, 2.80 mmol) and triethylamine (0.35 mL). The reaction mixture was stirred at 25–35°C for 5 min, after which compound 3D (0.81 g, 2.55 mmol) and triethylamine (0.35 mL) were added. Stirring was continued for an additional 3 h after the addition was complete. The reaction mixture was diluted with water (50 mL) and then extracted with ethyl acetate (150 mL). The organic phase was washed once with purified water (50 mL) and once with saturated brine (50 mL). After concentration under reduced pressure, compound 3-7 was obtained. 1 H NMR (400 MHz, CDCl3) δ=8.40 (s, 1H), 8.20 (s, 1H), 8.04 (s, 2H), 8.00 (s, 1H), 7.83 (s, 1H), 7.75 - 7.73 (m, 4H), 7.59 - 7.57 (m, 4H), 7.47 (brs, 1H), 7.39 - 7.35 (m, 4H), 7.26 - 7.24 (m, 2H), 6.69 (s, 1H), 5.83 - 5.79 (m, 2H), 5.70 (brs, 1H), 5.02 - 4.93 (m, 2H), 4.40 - 4.36 (m, 3H), 4.21 - 4.17 (m, 2H), 3.90 - 3.88 (m, 2H), 3.80 - 3.78 (m, 4H), 3.73 - 3.70 (m, 4H), 3.62 - 3.54 (m, 3H), 3.47 - 3.40 (m, 4H), 2.62 (brs, 2H), 2.48 (t, J=6.00 Hz, 4H), 2.05 - 2.01 (m, 2H), 1.55 - 1.52 (m, 2H), 1.38 (s, 18H), 1.30 - 1.26 (m, 16H). LC-MS (ESI) m / z: 1265.0 [M+H] + .

[0154] Step G: Compound 3-7 (9 g, 7.12 mmol) was dissolved in anhydrous dioxane (15 mL), and a solution of hydrochloric acid in dioxane (4 M, 71.17 mL) was added. The reaction mixture was stirred at room temperature for 1 hour and then concentrated under reduced pressure to give compound 3-8. 1 H NMR (400 MHz, DMSO-d6) δ=10.22 (s, 1H), 8.56 (t, J=5.3 Hz, 1H), 8.24 (s, 1H), 8.06 (s, 1H), 7.95 - 7.81 (m, 5H), 7.69 (d, J=7.4 Hz, 4H), 7.47 - 7.24 (m, 10H), 5.76 (tdd, J=6.6, 10.3, 17.0 Hz, 1H), 5.05 - 4.82 (m, 2H), 4.42 - 4.27 (m, 4H), 4.22 (d, J=6.4 Hz, 2H), 3.71 (s, 3H), 3.68 - 3.60 (m, 6H), 3.36 (dd, J=6.8, 13.4 Hz, 6H), 3.22 - 3.16 (m, 2H), 2.57 - 2.53 (m, 2H), 2.45 (t, J=6.3 Hz, 3H), 2.10 - 1.86 (m, 2H), 1.58 - 1.01 (m, 18H). LC-MS (ESI) m / z: 1152.6 [M+H] + .

[0155] Step H: Compound 3-8 (9 g, 7.81 mmol) was dissolved in N,N-dimethylformamide (100 mL), followed by the addition of HATU (6.83 g, 17.96 mmol), N,N-diisopropylethylamine (1.01 g, 7.81 mmol, 1.36 mL), and tert-butyl (3-aminopropyl)aminocarboxylate (2.99 g, 17.18 mmol). The reaction mixture was stirred at 25 °C for 4 h and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 30 / 1 to 0 / 1, v / v) to give compound 3-9.

[0156] Step I: Compound 3-9 (15 g, 10.24 mmol) was dissolved in anhydrous dichloromethane (110 mL), followed by the addition of tetrahydropyrrole (1.82 g, 25.60 mmol, 2.14 mL). The reaction mixture was stirred at room temperature for 3 hours and then concentrated under reduced pressure. The residue was purified by preparative HPLC (preparative column: Waters Xbridge C18 (specifications: 250 mm × 100 mm, particle size: 10 μm); mobile phase: Phase A: 10 mM aqueous trifluoroacetic acid; Phase B: acetonitrile; elution gradient: 35% to 55%, 25 min) to obtain a colorless oil. This colorless oil (5 g) was dissolved in anhydrous dioxane (35 mL), and a hydrochloric acid / dioxane solution (4 mol / L, 38 mL) was added. The mixture was stirred at 25 °C for 12 hours. The mixture was concentrated under reduced pressure to obtain compound 3-10. 1 H NMR (400 MHz, DMSO-d6) δ=10.66 (s, 1H), 9.04 (t, J=5.1 Hz, 1H), 8.35 (s, 1H), 8.24 - 8.18 (m, 3H), 7.42 (s, 1H), 5.92 - 5.82 (m, 1H), 5.04 - 4.94 (m, 2H), 3.83 - 3.57 (m, 13H), 3.48 - 3.39 (m, 2H), 3.20 - 3.02 (m, 8H), 2.91 - 2.76 (m, 6H), 2.06 (q, J=6.8 Hz, 2H), 1.73 (q, J=6.9 Hz, 4H), 1.58 - 1.47 (m, 2H), 1.45 - 1.18 (m, 16H).LC-MS (ESI) m / z: 821.0 [M+H] + .

[0157] Step J: Compound 3-10 (9.37 g, 20.94 mmol), N,N-diisopropylethylamine (2.71 g, 20.94 mmol), and HATU (9.27 g, 20.94 mmol) were added sequentially to N,N-dimethylformamide (50 mL), and the reaction mixture was stirred at 10-15 °C for 2 h. Next, compound 2-11 (4.6 g, 4.76 mmol) and N,N-diisopropylethylamine (2.46 g, 19.04 mmol) were slowly added to the reaction mixture. After the addition, the reaction mixture was stirred at 10-15 °C for 12 h. The reaction mixture was gradually poured into water (500 mL) and extracted with dichloromethane (500 mL). The organic phase was washed once with saturated brine (200 mL), dried over anhydrous sodium sulfate, filtered under reduced pressure, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Welch Ultimate XB-CN, 250 mm x 70 mm x 10 μm; mobile phase: Phase A: n-hexane; Phase B: ethanol; 15 min) to give compound 3-11. 1 H NMR (400 MHz, CDCl3) δ=8.62 (br s, 1H), 8.20 (s, 1H), 8.07 (s, 1H), 7.96 - 7.88 (m, 2H), 7.82 (br d, J=9.0 Hz, 6H), 7.71 (t, J=5.3 Hz, 1H), 7.27 (br s, 1H), 5.22 (d, J=3.3 Hz, 4H), 5.01 - 4.94 (m, 4H), 4.48 (br d, J=8.5 Hz, 4H), 4.03 (s, 12H), 3.95 - 3.83 (m, 4H), 3.78 - 3.58 (m, 14H), 3.38 - 3.27 (m, 19H), 3.00 (d, J=5.6 Hz, 7H), 2.38 - 2.27 (m, 4H), 2.11 (s, 12H), 2.04 (d, J=6.8 Hz, 8H), 2.00 (s, 11H), 1.89 (s, 12H), 1.77 (d, J=1.5 Hz, 12H), 1.57 - 1.42 (m, 20H), 1.27 - 1.15 (m, 16H).

[0158] Step K: Compound 3-11 (2.0 g, 788.10 μmol) and ruthenium trichloride trihydrate (4.12 mg, 15.76 μmol) were added to a mixture of dichloromethane (6 mL), acetonitrile (6 mL), and water (9 mL) at room temperature. Sodium periodate (1.26 g, 5.9 mmol) and sodium bicarbonate (132.41 mg, 1.58 mmol) were then added. The reaction mixture was stirred at room temperature for 16 hours, then slowly poured into water (50 mL), and extracted again with dichloromethane (50 mL). The organic phase was washed once with saturated sodium sulfite solution (20 mL), once with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered under reduced pressure, and the filtrate was concentrated under reduced pressure to give compound 3-12.

[0159] Step L: Compound 3-12 (1.3 g, 508.66 μmol) was dissolved in dichloromethane (15 mL), and HATU (232.09 mg, 610.40 μmol) and N,N-diisopropylethylamine (65.74 mg, 508.66 μmol) were added. The reaction mixture was stirred at 10-15°C for 15 min, after which compound 3E (234.73 mg, 559.53 μmol) was added to the reaction mixture, and stirring was continued for an additional 1 h at 10-15°C. The reaction mixture was added to 5% aqueous sodium bicarbonate solution (50 mL) and extracted again with dichloromethane (50 mL). The organic phase was washed once with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered under reduced pressure, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Welch Ultimate XB-SiOH, 250 mm×70 mm×10 μm; mobile phase: Phase A: n-hexane; Phase B: ethanol; 15 min) to give compound 3-13. 1H NMR (400 MHz, CDCl3) δ=8.69 - 8.57 (m, 1H), 8.20 (s, 1H), 8.07 (s, 1H), 7.91 (q, J=5.5 Hz, 2H), 7.83 (d, J=9.2 Hz, 6H), 7.72 (t, J=5.3 Hz, 2H), 7.37 - 7.26 (m, 5H), 7.22 - 7.15 (m, 4H), 6.92 - 6.83 (m, 4H), 5.21 (d, J=3.3 Hz, 4H), 4.99 - 4.95 (m, 4H), 4.48 (d, J=8.4 Hz, 4H), 4.02 (s, 12H), 3.93 - 3.82 (m, 4H), 3.76 - 3.55 (m, 21H), 3.42 - 3.34 (m, 22H), 3.25 - 3.14 (m, 3H), 3.04 - 2.91 (m, 9H), 2.33 - 2.27 (m, 4H), 2.10 (s, 12H), 2.05 - 1.99 (m, 21H), 1.89 (s, 12H), 1.77 (d, J=1.7 Hz, 12H), 1.51 - 1.42 (m, 20H), 1.28 - 1.16 (m, 16H).

[0160] Step M: Compound 3-13 (1 g, 338.16 μmol), triethylamine (119.76 mg, 1.18 mmol), and 4-dimethylaminopyridine (20.66 mg, 169.08 μmol) were added sequentially to dichloromethane (10 mL), followed by succinic anhydride (101.52 mg, 1.01 mmol). The reaction mixture was stirred at room temperature for 48 hours. The reaction mixture was diluted with water (20 mL) and extracted with dichloromethane (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered under reduced pressure. The filtrate was concentrated under reduced pressure. The residue was subjected to preparative HPLC (Waters Xbridge C18, 150 mm × 50 mm × 10 μm column; mobile phase: Phase A: 10 mM ammonium bicarbonate aqueous solution; Phase B: acetonitrile; 10 min) to give compound D02. 1H NMR (400 MHz, CD3CN) δ=9.68 (br s, 1H), 8.29 (br s, 1H), 8.15 (d, J=2.5 Hz, 2H), 7.87 (s, 1H), 7.43 - 7.19 (m, 13H), 7.15 - 6.97 (m, 7H), 6.93 - 6.80 (m, 4H), 5.47 - 5.26 (m, 5H), 5.04 (dd, J=2.7, 11.2 Hz, 4H), 4.62 - 4.48 (m, 4H), 4.25 - 3.92 (m, 18H), 3.87 - 3.65 (m, 22H), 3.54 - 3.34 (m, 13H), 3.10 - 3.01 (m, 8H), 2.44 - 2.38 (m, 6H), 2.32 - 2.28 (m, 2H), 2.20 - 2.09 (m, 22H), 2.00 (br s, 12H), 1.97 (d, J=2.4 Hz, 4H), 1.96 - 1.92 (m, 12H), 1.87 (s, 12H), 1.64 - 1.41 (m, 24H), 1.34 - 1.13 (m, 12H). LC-MS (ESI) m / z:1529.3 [M+2H] 2+ .

[0161] Modulation of intermediate 3C:

[0162]

change

[0163] Compound 3B (42.97 g, 138.01 mmol) was dissolved in N,N-dimethylformamide (500 mL) and cooled to 0-10 °C. HATU (57.72 g, 151.81 mmol) and N,N-diisopropylethylamine (21.40 g, 165.61 mmol) were added. The reaction mixture was stirred at room temperature for 0.5 h, followed by the addition of compound 3A (25.00 g, 138.01 mmol). After the addition was complete, stirring was continued for an additional 1 h at room temperature. The reaction mixture was poured into a dilute aqueous hydrochloric acid solution (23 mL of concentrated hydrochloric acid and 2.5 L of water) and stirred for 0.5 h. After filtration, the filter cake was added to ethanol (650 mL) and stirred at 60 °C for 1 h. After cooling to room temperature, the mixture was filtered under reduced pressure, and the filter cake was collected and dried under reduced pressure to obtain compound 3C. 1 H NMR (400 MHz, DMSO-d6) δ=10.34 (s, 1H), 8.46 (s, 2H), 8.15 (s, 1H), 7.95 (s, 1H), 7.87 (d, J=7.50 Hz, 2H), 7.67 (br d, J=7.38 Hz, 2H), 7.45 (t, J=5.50 Hz, 1H), 7.39 (t, J=7.44 Hz, 2H), 7.26 - 7.33 (m, 2H), 4.26 - 4.30 (m, 2H), 4.18 - 4.24 (m, 1H), 4.04 - 4.04 (m, 1H), 2.54 (t, J=5.57 Hz, 2H).

[0164] Example 6: Synthesis of D03

[0165] [ka] TIFF2026507087000049.tif52168

[0166] Step A: At 25 °C, a toluene solution (150 mL) of compound 4-2 (13.24 g, 50.93 mmol) was added to a toluene solution (100 mL) of compound 4-1 (12 g, 49.93 mmol, 11.76 mL). The reaction was stirred at 100 °C for 16 h. Saturated aqueous sodium carbonate (200 mL) was added to the reaction mixture, and the layers were separated. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product. The crude product was separated by preparative HPLC (column: Agela Innoval ODS-2 250 mm x 100 mm x 10 μm; mobile phase: [water (0.225% formic acid)-acetonitrile]; acetonitrile %: 15% to 35%, 25 min) to give compound 4-3.

[0167] Step B: A methanol solution (120 mL) of compound 4-3 (12.6 g, 31.04 mmol), palladium carbon (5 g, 10% content), and di-tert-butyl dicarbonate (14.90 g, 68.28 mmol, 15.69 mL) was purged with argon three times and then with hydrogen three times. The reaction mixture was stirred at 25°C under a hydrogen atmosphere at atmospheric pressure for 16 hours. The reaction mixture was filtered through diatomaceous earth and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain compound 4-4.

[0168] Step C: Compound 4-4 (7.5 g, 18.66 mmol) was dissolved in a solution of hydrochloric acid and ethyl acetate (4 mol / L, 18.66 mL). The mixture was purged with nitrogen three times and stirred under a nitrogen atmosphere at 25° C. for 1 hour. After concentration under reduced pressure, compound 4-5 was obtained.

[0169] Step D: A solution of compound 4-5 (2.24 g, 9.68 mmol), compound 4-6 (2.2 g, 5.69 mmol), triethylamine (2.88 g, 28.47 mmol, 3.96 mL), and 1-propylphosphonic acid cyclic anhydride (5.06 g, 7.95 mmol, 4.73 mL, 50% content) in N,N-dimethylformamide (25 mL) was purged with nitrogen three times and stirred at 25 °C under a nitrogen atmosphere for 1 hour. Water (50 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (80 mL × 2). The combined organic layer was washed with saturated brine (100 mL × 1), dried over anhydrous sodium sulfate, filtered, and concentrated. The obtained crude product was separated by preparative HPLC (column: Phenomenexluna C18 150 × 40 mm × 15 μm; mobile phase: [water (0.225% formic acid)-acetonitrile]; acetonitrile%: 14% to 44%, 15 min) to obtain compounds 4-7.

[0170] Step E: A solution of compound 4-7 (2.2 g, 4.06 mmol), compound 4-8 (1.50 g, 6.09 mmol), O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (2.22 g, 6.90 mmol), and triethylamine (1.23 g, 12.18 mmol, 1.69 mL) in N,N-dimethylformamide (20 mL) was purged with nitrogen three times and stirred at 25 °C under a nitrogen atmosphere for 1 h. Water (80 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (80 mL × 2). The combined organic layer was washed with saturated brine (100 mL × 1), dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product. The crude product was separated by silica gel column chromatography (eluent: petroleum ether / ethyl acetate, gradient elution) to give compound 4-9.

[0171] Step F: To a solution of compound 4-9 (2.5 g, 3.43 mmol) in a mixture of methanol (22 mL) and water (7 mL) was added lithium hydroxide monohydrate (504.37 mg, 12.02 mmol). The reaction was stirred at 25 °C for 12 hours. Water (50 mL) was added to the reaction mixture. The aqueous phase was adjusted to pH 4-5 with 2 M hydrochloric acid and extracted with ethyl acetate (100 mL × 2). The combined organic phase was washed with saturated brine (50 mL). After drying over anhydrous sodium sulfate, the mixture was filtered, and the filtrate was concentrated to give compound 4-10.

[0172] Step G: A solution of compound 4-10 (500 mg, 710.49 μmol), compound 4-11 (406.18 mg, 781.54 μmol), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (485.01 mg, 1.28 mmol), and triethylamine (215.68 mg, 2.13 mmol) in N,N-dimethylformamide (5 mL) was purged with nitrogen three times and stirred at 25°C under a nitrogen atmosphere for 1 hour. Water (80 mL) was added to the reaction solution, which was then extracted with dichloromethane (80 mL × 2). The combined organic layer was washed with saturated brine (100 mL × 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by preparative HPLC (column: Waters Xbridge C18 150 × 50 mm × 10 μm; mobile phase: [water (10 mM ammonium bicarbonate)-acetonitrile]; acetonitrile %: 57% to 87%, 10 min) to obtain compound 4-12.

[0173] Step H: Palladium carbon (100 mg, 10% content) was added to a methanol solution (10 mL) of compound 4-12 (320 mg, 203.93 μmol), and the atmosphere was replaced with argon three times and then replaced with hydrogen three times. The reaction mixture was stirred under atmospheric pressure at 25° C. for 16 hours. The reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give compound 4-13.

[0174] Step I: A solution of compound 4-13 (178 mg, 221.66 μmol), compound 4-14 (413.14 mg, 775.80 μmol), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (336.25 mg, 886.63 μmol), and triethylamine (179.43 mg, 1.77 mmol) in N,N-dimethylformamide (4 mL) was purged with nitrogen three times, and the reaction mixture was stirred at 25 °C under a nitrogen atmosphere for 1 h. Water (100 mL) was added to the reaction mixture, and the mixture was extracted twice with 50 mL of DCM / i-PrOH (3 / 1). The combined organic phase was washed with saturated brine (150 mL × 1), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and then purified by preparative HPLC (column: Waters Xbridge C18 150×50 mm×10 μm; mobile phase: [water (10 mM ammonium bicarbonate)-acetonitrile]; acetonitrile %: 36% to 66%, 10 minutes) to obtain compound 4-15.

[0175] Step J: A dichloromethane solution (5 mL) of compound 4-15 (240 mg, 97.49 μmol), compound 4-16 (34.15 mg, 341.21 μmol), 4-dimethylaminopyridine (11.91 mg, 97.49 μmol), and triethylamine (9.86 mg, 97.49 μmol) was purged with nitrogen three times and then stirred at 25°C under a nitrogen atmosphere for 16 hours. The reaction mixture was concentrated and purified by preparative HPLC (Waters Xbridge C18 150 x 50 mm x 10 μm column; mobile phase: [water (10 mM ammonium bicarbonate)-acetonitrile]; acetonitrile %: 19% to 49%, 10 min) to give compound D03. LC-MS (ESI) m / z: 1172 [M-2H] 2- . 1H NMR (400 MHz, DMSO-d6)δ=7.65 - 8.20(m, 11 H)7.28 - 7.39(m, 4 H)7.17 - 7.28(m, 5 H)6.78 - 7.03(m, 4 H)5.13 ​​- 5.33(m, 3 H)4.89 - 5.10(m, 3) H)4.39 - 4.60(m, 3 H)3.94 - 4.15(m, 15 H)3.82 - 3.92(m, 5 H)3.64 - 3.81(m, 12 H)2.83 - 3.19(m, 16 H)2.08 - 2.19(m, 14 H)1.96 - 2.07(m, 19) H)1.86 - 1.92(m, 9 H)1.74 - 1.81(m, 10 H)1.54 - 1.65(m, 7 H)1.34 - 1.54(m, 16 H)1.03 - 1.33(m, 16 H).

[0176] Example 7: Synthesis of double-stranded siRNA analogs or their conjugates Synthesis of single-stranded oligoribonucleotides containing D1, D2, or D3: Oligoribonucleotides were synthesized using phosphoramidite solid-phase synthesis. Synthesis was performed on a solid support in which D01, D02, or D03 was covalently immobilized on controllable pore glass (amino CPG, 500 Å). All 2'-modified RNA phosphoramidites and auxiliary reagents were commercially available. All amides were dissolved in anhydrous acetonitrile with the addition of molecular sieves (3 Å). 5-ethylthio-1H-tetrazole (ETT) was used as the activator, with a coupling time of 5 min. Alternatively, phosphorothioate ester bonds were generated using a solution of 50 mM 3-((dimethylamino-methylene)amino)-3H-1,2,4-dithiazole-3-thione (DDTT) in anhydrous acetonitrile / pyridine (V / V = 1 / 1). The reaction time was 3 min. The synthesis of all sequences was completed after the final removal of the DMTr group.

[0177] Synthesis of single-stranded oligoribonucleotides without D1, D2, or D3: Oligoribonucleotides were synthesized according to the sequence design using phosphoramidite solid-phase synthesis technology. Synthesis was performed on universal controllable pore glass (CPG) (500 Å). All 2'-modified RNA phosphoramidites and auxiliary reagents were commercially available. Modified R and E were obtained as in Examples 1, 2, and 3. All phosphoramidites were sequentially dissolved in anhydrous acetonitrile, and molecular sieves (3 Å) were added. The coupling time was 5 minutes using 5-ethylthio-1H-tetrazole (ETT) as an activator. Alternatively, phosphorothioate ester bonds were generated using a solution of 50 mM 3-((dimethylamino-methylene)amino)-3H-1,2,4-dithiazole-3-thione (DDTT) in anhydrous acetonitrile / pyridine (V / V = 1 / 1). The reaction time was 3 minutes. The synthesis of all sequences was completed after the DMTr group was finally removed.

[0178] Cleavage and deprotection of CPG-bound oligomers: After solid-phase synthesis, the protecting groups were removed by treatment with 20% diethylamine in acetonitrile for 30 minutes, without cleaving the oligonucleotides from the CPG. The dried CPG was then treated with concentrated aqueous ammonia at 40°C for 18 hours. After centrifugation, the supernatant was transferred to a new tube, and the CPG was washed with aqueous ammonia. The solutions were combined and concentrated to obtain a solid mixture.

[0179] Purification of single-stranded oligoribonucleotides: Oligomers were purified by HPLC using a NanoQ anion-exchange column. Buffer A contained 10 mM sodium perchlorate, 20 mM Tris, 1 mM EDTA, pH 7.4, and 20% acetonitrile. Buffer B contained 500 mM sodium perchlorate, 20 mM Tris, 1 mM EDTA, pH 7.4, and 20% acetonitrile. The target product was isolated and desalted on a reverse-phase C18 column.

[0180] Annealing of single-stranded oligoribonucleotides to generate double-stranded siRNA: The single-stranded oligoribonucleotides to be annealed were prepared at 200 μM in sterile RNase-free HO (containing no RNases). Equimolar amounts of the single-stranded oligoribonucleotide solutions were mixed to form complementary strands. The annealing reaction system was set up as follows: a total of 100 μL of the mixture (10 nmol) was placed in a 95°C water bath for 10 minutes (20 minutes of high-temperature treatment is required for amounts greater than 100 nmol), then rapidly transferred to a 60°C water bath and allowed to cool naturally to complete the annealing. The purity of the resulting double-stranded siRNA was confirmed by HPLC, and the molecular weights of the sense and antisense strands were measured by HRMS to confirm the molecular weight of the double-stranded siRNA and identify it as the target product.

[0181] Experimental Example 1: The inhibitory activity of test compounds against HBV was evaluated using HepG2.2.15 cells. 1. Experimental Objective: The purpose of this study was to evaluate the inhibitory activity of test compounds against HBV in HepG2.2.15 cells.

[0182] 2. Experimental materials: 1) Cells and major reagents HepG2.2.15 cells were provided by Shanghai Yaoming Kangde New Drug Development Co., Ltd. Transfection reagent Lipofectamine RNAiMAX (Invitrogen -13778-150).

[0183] Hepatitis B virus surface antigen quantitative detection kit (Antu Bio-CL-0310). Hepatitis B virus e antigen quantitative detection kit (Anzu Bio-CL-0312). Fluorescent probe FastStart Universal Probe Master (Roche, 04914058001).

[0184] 2)Main equipment 79001 Real Time PCR System (Applied Biosystems), Synergy 2 (BioTek), and cell counter (Vi-cell™ XR).

[0185] 3. Experimental Procedures and Methods: siRNA was introduced into HepG2.2.15 cells by transfection, as follows: On day 0, siRNA was diluted to eight concentrations in PBS. HepG2.2.15 cells were harvested, washed with DPBS, digested with trypsin, and adjusted to the appropriate density. At the same time as cell seeding, siRNA was transfected into HepG2.2.15 cells using Lipofectamine RNAiMax. Cells were seeded at a density of 15,000 cells / well in a 96-well plate, with each well containing 150 μL of culture medium. The cells were cultured for three days in a 5% CO2, 37°C incubator. Test siRNAs were prepared at eight concentrations in a 3-fold dilution series, in duplicate.

[0186] On the third day, the medium was replaced with fresh medium, and the culture was continued for another 3 days. The cell culture supernatant was collected on day 6. The supernatant samples were assayed for HBsAg and HBeAg according to the protocol of the Hepatitis B virus surface antigen and e antigen quantitative assay kit, and HBV DNA was detected by qPCR.

[0187] The procedure for detecting HBsAg or HBeAg is briefly described below: 50 μL of sample and standard were added to a reaction plate, 50 μL of enzyme conjugate was added to each well, and after shaking and mixing, the plate was incubated at 37°C for 60 minutes. After washing five times with washing solution, 50 μL of luminescent substrate was added to each well, mixed evenly, and reacted at room temperature for 10 minutes in the dark. Finally, the chemiluminescence intensity was measured using a microplate reader.

[0188] The HBV DNA detection procedure was as follows: a qPCR reaction system was prepared and a solution containing either the supernatant sample or the HBV full-length plasmid standard was added. The PCR reaction program was 95°C for 10 minutes, followed by a cycle of 95°C for 15 seconds, followed by 60°C for 1 minute, for a total of 40 cycles. The amount of HBV DNA in each sample was calculated based on the Ct value.

[0189] Data Analysis: HBsAg or HBeAg or HBV DNA suppression rate = (1 - sample HBsAg or HBeAg or HBV DNA content / RNAiMAX transfection reagent control HBsAg or HBeAg or HBV DNA average value) x 100% The 50% inhibitory concentration (EC) of compounds against HBV was calculated using GraphPad Prism software. 50 ) values ​​were calculated.

[0190] 4. Experimental results: See Table 5.

[0191] [Table 5]

[0192] Experimental conclusion: The compounds of the present invention had the biological activity of significantly reducing HBV HBsAg, HBeAg and HBV DNA in HepG2.2.15 cells.

[0193] Experimental Example 2: Evaluation of the inhibitory activity of test compounds against HBV using primary human hepatocytes (PHH) 1. Experimental Objective: The amount of HBV DNA in the supernatant of human primary hepatocyte (PHH) cultures was measured using real-time quantitative PCR, and the amounts of hepatitis B surface antigen and e antigen were detected using ELISA. The total RNA amount was measured, and the EC 50 The inhibitory effect of the compound on HBV was evaluated using this value as an index.

[0194] 2. Experimental materials: 1) Cells and reagents: Cryopreserved primary human hepatocytes (PHH) Hepatitis B virus surface antigen quantitative detection kit (Antu Bio-CL-0310).

[0195] Hepatitis B virus e antigen quantitative detection kit (Anzu Bio-CL-0312). RNA extraction kit (Qiagen). FastKing cDNA first-strand synthesis kit (TianGen).

[0196] Fluorescent probe FastStart Universal Probe Master (Roche, 04914058001). 2) Main equipment: 79001 PCR system (Applied Biosystems), Synergy 2 (BioTek), and cell counter (Vi-cell™ XR).

[0197] 3. Experimental Procedures and Methods: On day 0, compounds were diluted to 7 concentrations with PBS and added to the cell plates (22 μL / well). Cryopreserved PHHs were thawed and plated at a density of 6.67 × 10 5 After adjusting the concentration to cells / mL, the mixture was seeded into a 48-well plate (198 μL / well). The total volume of each well was 220 μL, and after mixing, the well was placed in an incubator and cultured overnight at 37°C and 5% CO2. Seven 3-fold dilution series of each compound were prepared and tested in duplicate.

[0198] On day 1, the culture medium was removed from the culture plate, and PHHs were infected with type D HBV (concentrated from the HepG2.2.15 cell culture supernatant) (100GE / cell). The final concentration of DMSO in the culture medium was 2%.

[0199] Fresh medium (without compounds) was replaced on days 2, 4 and 6. On day 8, cell culture supernatants were collected and HBsAg and HBeAg were measured according to the protocol of the Hepatitis B virus surface antigen and e antigen quantitative assay kit, and HBV DNA was detected by qPCR. Finally, cells were collected and HBV RNA was detected.

[0200] The steps for detecting HBsAg or HBeAg were as follows: 50 μL of sample and standard were added to a reaction plate, 50 μL of enzyme conjugate was added to each well, and after shaking and mixing, the plate was incubated at 37°C for 60 minutes. The reaction plate was washed five times with washing solution, 50 μL of luminescent substrate was added to each well, and after mixing, the plate was reacted at room temperature in the dark for 10 minutes. Finally, the chemiluminescence intensity was measured using a microplate reader.

[0201] HBV DNA detection was performed as follows: qPCR reactions were prepared and either supernatant samples or a solution containing a full-length HBV plasmid standard was added. The PCR reaction program consisted of heating at 95°C for 30 seconds, followed by 40 cycles of denaturation at 95°C for 5 seconds and extension at 60°C for 34 seconds. A final cycle of 95°C for 15 seconds and 60°C for 1 minute was performed. The amount of HBV DNA in each sample was calculated based on the Ct value.

[0202] The procedure for HBV RNA detection is briefly described below: Cells were harvested, RNA was extracted according to the Qiagen-74182 RNA Extraction Kit protocol, and the RNA was reverse transcribed into cDNA according to the FastKing cDNA First-Strand Synthesis Kit protocol. Target gene cDNA was detected by qPCR, with GAPDH used as an internal control gene.

[0203] Based on the Ct value of each sample, the ΔΔCt relative quantification method was used to calculate the RNA expression level of the target gene in the sample. The relative expression level of the target gene was calculated as 2 -ΔΔCT It is expressed as:

[0204] The formula is as follows: ΔCT = average Ct value of the gene of interest - average Ct value of the internal control gene; ΔΔCT = ΔCT (medication group) - ΔCT (control group); relative expression level of target gene mRNA = 2 -ΔΔCT

[0205] 4. Data Analysis: HBsAg or HBeAg or HBV DNA suppression rate = (1 - HBsAg or HBeAg or HBV DNA content of sample / average HBsAg or HBeAg or HBV DNA content of culture medium control group) x 100% The 50% inhibitory concentration (EC) of compounds against HBsAg or HBeAg or HBV DNA was calculated using GraphPad Prism software. 50 ) was calculated.

[0206] 5. Experimental results: See Table 6.

[0207] [Table 6]

[0208] Experimental conclusion: The compounds of the present invention had the biological activity of significantly reducing HBV HBsAg, HBeAg, HBV DNA and RNA in PHH. Experimental Example 3. Evaluation of the competitive efficiency of non-fluorescently labeled siRNA with GalNac3-Cy5 in PHH

[0209] 1. Research purpose The purpose of this study was to evaluate the competitive efficiency of unlabeled compounds with GalNac3-Cy5 in PHH using flow cytometry.

[0210] 2. Control compound GalNac3 and competitor compounds: The control compound was GalNac3 (EE(ahGalNAc)3 described in Kornilova AY, Algayer B, Breslin M, Uebele V. Anal Biochem. 2012;425(1):43-46.), and the competitor compound was GalNac3 labeled with Cy5 fluorescent label. 3. Main Reagents and Cells

[0211] [Table 7]

[0212] 4. Experimental Protocol On day 0, the resuscitated PHH suspension was adjusted to the appropriate density and the cells were seeded into 48-well plates.

[0213] On the first day, pre-mixed diluted test samples and Cy5 fluorescently labeled GalNac3 (DMSO final concentration 2%) were added, and the test samples were diluted three-fold to 11 concentration points in a single well starting from 20 μM.

[0214] After incubating the test products with the cells for 4 hours, the cells were digested and fixed, and the corresponding fluorescence intensity of PHH was measured by flow cytometry. The control compound was unlabeled GalNac3, and the test and control compounds were set at 11 concentration points and tested in a single well. The competitor compound was Cy5 fluorescently labeled GalNac3, with a final concentration of 0.05 μM.

[0215] Analysis was performed using Graphpad Prism software (four parameter logistic equations) to obtain EC 50 The value was calculated.

[0216] [Table 8]

[0217] Experimental results: The compounds of the present invention showed good competitive efficiency against GalNac3-Cy5 in PHH. Experimental Example 4: Study of anti-hepatitis B virus activity and safety in a recombinant adeno-associated virus type 8 vector-mediated hepatitis B virus mouse model (AAV-HBV)

[0218] 1. Experimental Objective: The AAV vector-mediated HBV transfection mouse model is a rapid and efficient HBV model. Taking advantage of the high hepatotropism of the AAV8 vector, recombinant adeno-associated virus type 8 (rAAV8-1.3HBV) carrying 1.3 copies of the HBV genome was administered via the tail vein of mice, resulting in efficient delivery of 1.3 copies of the HBV genome into hepatocytes. Due to the properties of the AAV viral vector, the vector mediated by the vector was expressed persistently for a long period of time. Using the AAV / HBV model, sustained HBV DNA replication and expression of HBsAg and HBeAg were achieved in the mouse liver.

[0219] Using an AAV-HBV mouse model, the in vivo anti-HBV effect and safety of the test compound were evaluated by measuring HBsAg, HBeAg, and DNA in the mouse serum and mouse body weight after administration.

[0220] 2. Experimental materials: C57BL / 6 mice were incubated in PBS (RNase-free), and the test compound and recombinant virus rAAV8-1.3HBV were used. The main reagents used in this project included the QIAamp96 DNA kit (Qiagen, 51162), FastStart Universal Probe Master (Rox) (Roche, 04914058001), Hepatitis B virus surface antigen detection kit (Antu Bio, CL0310), Hepatitis B virus e antigen detection kit (Antu Bio, CL0918), and PureLink. TM The kit includes the Pro 96 Viral RNA / DNA Kit (Invitrogen, 12280-096A), the FastQuant RT Kit (with gDNase) (TIANGEN, KR106-02), a centrifuge (Beckman Allegra X-15R), a multifunction plate reader (BioTek, Synergy 2), a real-time PCR instrument (Applied Biosystems, 7900HT Fast Real-time PCR system), and a plate reader (Molecular Devices, SpectraMax 340PC384).

[0221] 3. Experimental Method: a) Subcutaneous administration to mice was initiated on day 34 after virus injection, with the dose being 3 or 30 mg / kg, and this day was designated as day 0. Prior to administration, submandibular blood was collected from all mice to obtain plasma.

[0222] b) Blood samples were collected from the submandibular vein on days 0, 14, and 21 after administration to the mice, and plasma was collected. The collected blood samples were anticoagulated with K2-EDTA and then stored for 4 hours. o C, Plasma was collected after centrifugation at 7000 g / min for 10 min.

[0223] c) Plasma was collected from all mice by submandibular vein bleeding, after which the mice were euthanized by CO2 inhalation, plasma samples were collected by cardiac bleeding, and liver samples were harvested.

[0224] d) The sample was sent for detection. 4. Sample analysis: Measurement of HBsAg and HBeAg contents in mouse serum by ELISA: The experimental procedure was carried out according to the instructions for the HBsAg ELISA (Anzu Bioscience, CL0310) and HBeAg ELISA (Anzu Bioscience, CL0918) kits.

[0225] Measurement of HBV DNA content in mouse plasma by qPCR: HBV DNA was extracted from plasma, and the HBV DNA content in mouse plasma was measured by qPCR according to the QIAamp 96 DNA Blood Kit instructions.

[0226] Samples from each mouse group are expressed as mean ± standard error, and unless otherwise specified, n = 5. Statistical analysis was performed using Student's t-test. 5. Experimental results: a) The anti-HBV activity of the test compound in the AAV-HBV mouse model was evaluated based on the HBsAg content in serum. The HBsAg content in mouse plasma was measured by ELISA. The results are shown in Table 9.

[0227] [Table 9]

[0228] b) The anti-HBV activity of the test compound in the AAV-HBV mouse model was evaluated based on the serum HBeAg content. The HBeAg content in mouse plasma was measured by ELISA. The results are shown in Table 10.

[0229] [Table 10]

[0230] c) The anti-HBV activity of the test compounds in the AAV-HBV mouse model was evaluated based on serum DNA content. HBV DNA content in mouse plasma was measured by quantitative PCR. The results are shown in Table 11.

[0231] [Table 11]

[0232] d) Changes in body weight. Weight was compared to the baseline weight on day 0. According to IACUC regulations, a 20% weight loss was defined as a humane endpoint, and mice showing weight loss of more than 20% were excluded from the experiment. No mice were excluded from the experiment due to weight loss.

[0233] Testing Conclusion: In this study, the test compound significantly reduced HBsAg, HBeAg, and HBV DNA in the AAV-HBV mouse model, and the mice tolerated the treatment well.

[0234] Experimental Example 5: Immunogenicity evaluation of compounds using hPBMC Study objective: To evaluate the immunogenicity of test compounds using hPBMCs 1. Experimental materials and equipment: hPMBC cells: freshly isolated peripheral blood mononuclear cells (PBMCs) purchased from Shanghai Sega Biotechnology Co., Ltd.

[0235] Compound:

[0236] [Table 12]

[0237] reagent:

[0238] [Table 13]

[0239] Experimental equipment:

[0240] [Table 14]

[0241] Testing Procedure: Day 1: hPBMC cells were cultured at high density overnight. Prepare 10% FBS-containing RPMI-1640 cell culture medium at the following ratio: RPMI 1640 media (1x) contains 10% FBS and 1% penicillin-streptomycin solution.

[0242] Two newly purchased lots of hPBMC were centrifuged together in a 50 mL centrifuge tube at 400 xg and 25°C for 5 minutes. The supernatant was discarded and the hPBMCs were resuspended in 10 mL of medium and then counted.

[0243] Cell density was increased to 10 7 Adjust to 25cm 2 The cells were transferred to a cell culture flask and cultured overnight in an incubator at 37°C under 5% CO2.

[0244] Day 2: hPBMCs were plated and compound treatment was performed. Compound dilution Test compounds were diluted 3-fold starting at 15 μg / mL to set four concentration points. 10x gradient dilutions of compounds were prepared in a 96-well V-bottom plate according to the following dilution scheme: The reference compound GS9688 had a stock solution concentration of 20 mM and was diluted 2-fold starting at 0.5 μM to set two concentration points. 10x gradient dilutions of compounds were prepared in a 96-well V-bottom plate according to the following dilution scheme: The reference compound LPS_TLR4 activator, standardized at 25 mg / tube, was prepared as a 20 mg / mL stock solution by adding 1.25 mL of endotoxin-free physiological water. Starting at 1 μg / mL, the compound was diluted 4-fold starting at 1 μg / mL to set two concentration points. 10x gradient dilutions of compounds were prepared in a 96-well V-bottom plate according to the following dilution scheme:

[0245] hPBMCs cultured overnight the previous day were collected in a 50 mL centrifuge tube and centrifuged at 400 × g for 5 minutes. The supernatant was removed, and 15 mL of RPMI-1640 medium containing 10% FBS was added to the cell pellet to resuspend the cells.

[0246] Count the resuspended cells using a cell counter and measure the cell density to 2.2 x 10 6 Adjusted to cells / mL. Transfer 10 μL of the compound from step 1 to a 96-well cell plate, and add 90 μL of the cell suspension with the density adjusted in step 3 to each well. The total volume in each well is 100 μL, and the cell number is 2.0 × 10 6 There were 100 pieces.

[0247] The cell plate was cultured in a 5% CO2, 37°C incubator for 1 day. Day 3: hPBMC cytokine measurement The contents of IFNα, IFNβ, IL-6, and TNFα in hPBMC supernatants were measured using the Human ProcartaPlex Mix&Match 4-plex kit. The specific procedures were performed according to the instructions of the reagent kit, roughly as follows: 1. The cytokine detection plate was marked based on the hPBMC plate map.

[0248] 2. The magnetic beads were vortexed for 1 minute and then dispensed in 50 μL portions into each well of the detection plate using a multi-pipette. 3. The detection plate was placed on a magnetic plate for 2 minutes to collect the beads. After removing the liquid, 150 μL of washing solution was added, and the plate was left for 30 seconds before removing the liquid to wash the beads in the detection plate. This washing process was repeated twice.

[0249] 4. 50 μL of the standard and measurement sample were added to each well of the detection plate. 5. The assay plate was sealed with the sealing film provided in the kit, a black microplate cover was attached, and the plate was incubated overnight at 4°C on a shaker at 500 rpm.

[0250] Day 4: Plate reading for hPBMC cytokine measurements The measurement plate, which had been incubated overnight the previous day, was transferred to room temperature, shaken at 500 rpm for 30 minutes, washed using a magnetic plate as in the previous procedure, and all subsequent operations were performed in the dark: 25 μL of detection antibody mixture was added to each well of the assay plate, and after attaching a sealing film, the plate was covered with a black microplate cover and incubated on a shaker at room temperature at 500 rpm for 30 minutes.

[0251] The previous washing step was repeated. 50 μL of SAPE solution was added to each well, and the wells were sealed with a plate sealer. A black microplate cover was then attached, and the wells were incubated on a shaker at room temperature at 500 rpm for 30 minutes. The previous washing step was repeated.

[0252] 120 μL of reading buffer was added to the detection wells, a seal was attached, a black detection plate cover was attached, and then the plate was incubated at room temperature at 500 rpm for 5 minutes using a shaker.

[0253] The plate seal was removed from the detection plate, and the plate was set in the Luminex instrument for measurement and reading. Luminex experiments were set up and concentration data were measured using Bio-plex™200 System's Bio-plex Manager 5.0 software. A five-parameter (5-PL) regression model was used to fit the standard curves of target factors (Luminex auto-fitting), and the target factor contents in samples were analyzed based on the fluorescence values ​​measured by the instrument.

[0254] Result analysis: The contents of IL-6, TNF-α, IFN-α and IFN-β in the supernatants of hPBMCs treated with the compounds were measured using Luminex, and the immunogenicity of the compounds to hPBMCs was evaluated.

[0255] hPBMCs from donor 1 and donor 2 were treated with different doses of test compound, and the IFN-α, IFN-beta, IL-6, and TNF-α contents in the cell supernatant were measured. hPBMCs from donor 3 were treated with different doses of test compound, and the IL-6 and TNF-α contents in the cell supernatant were measured.

[0256] Test Results:

[0257] [Table 15]

[0258] [Table 16]

[0259] [Table 17]

[0260] Experimental conclusion: The compounds of the present invention have low immunogenicity risk to hPBMC. Experimental Example 6: Study of anti-hepatitis B virus activity and safety in a recombinant adeno-associated virus type 8 vector-mediated hepatitis B virus mouse model (AAV-HBV) 1. Experimental Objective: The AAV vector-mediated HBV transfection mouse model is a rapid and efficient HBV model. Taking advantage of the high hepatotropism of the AAV8 vector, recombinant adeno-associated virus type 8 (rAAV8-1.3HBV) carrying 1.3 copies of the HBV genome was administered via the tail vein of mice, resulting in efficient delivery of 1.3 copies of the HBV genome into hepatocytes. Due to the properties of the AAV viral vector, the vector mediated by the vector was expressed persistently for a long period of time. Using the AAV / HBV model, sustained HBV DNA replication and expression of HBsAg and HBeAg were achieved in the mouse liver.

[0261] Using an AAV-HBV mouse model, the in vivo anti-HBV effect and safety of the test compound were evaluated by measuring HBsAg, HBeAg, and DNA in the mouse serum and mouse body weight after administration.

[0262] 2. Experimental materials: C57BL / 6 mice were incubated in PBS (RNase-free), and the test compound and recombinant virus rAAV8-1.3HBV were used. The main reagents used in this project included the QIAamp96 DNA kit (Qiagen, 51162), FastStart Universal Probe Master (Rox) (Roche, 04914058001), Hepatitis B virus surface antigen detection kit (Antu Bio, CL0310), Hepatitis B virus e antigen detection kit (Antu Bio, CL0918), and PureLink. TMThe kit includes the Pro 96 Viral RNA / DNA Kit (Invitrogen, 12280-096A), the FastQuant RT Kit (with gDNase) (TIANGEN, KR106-02), a centrifuge (Beckman Allegra X-15R), a multifunction plate reader (BioTek, Synergy 2), a real-time PCR instrument (Applied Biosystems, 7900HT Fast Real-time PCR system), and a plate reader (Molecular Devices, SpectraMax 340PC384).

[0263] 3. Experimental Method: a) Mice were administered subcutaneously starting on day 34 after virus injection, at a dose of 2, 6, or 12 mg / kg, with this day designated as day 0. Prior to administration, blood was collected from the submandibular region of all mice to obtain plasma.

[0264] b) Blood samples were collected from the submandibular vein on days 0, 14, 21, and 28 after administration to the mice, and plasma was collected. The collected blood samples were anticoagulated with K2-EDTA and centrifuged at 4°C and 7000 g / min for 10 minutes, after which the plasma was collected.

[0265] c) Plasma was collected from all mice by submandibular vein bleeding, after which the mice were euthanized by CO2 inhalation, plasma samples were collected by cardiac bleeding, and liver samples were harvested.

[0266] d) The sample was sent for detection. 4. Sample analysis: Measurement of HBsAg and HBeAg contents in mouse serum by ELISA: The experimental procedure was carried out according to the instructions for the HBsAg ELISA (Anzu Bioscience, CL0310) and HBeAg ELISA (Anzu Bioscience, CL0918) kits.

[0267] Measurement of HBV DNA content in mouse plasma by qPCR: HBV DNA was extracted from plasma, and the HBV DNA content in mouse plasma was measured by qPCR according to the QIAamp 96 DNA Blood Kit instructions.

[0268] Samples from each mouse group are expressed as mean ± standard error, and unless otherwise specified, n = 5. Statistical analysis was performed using Student's t-test. 5. Experimental results: a) The anti-HBV activity of the test compound in the AAV-HBV mouse model was evaluated based on the HBsAg content in serum. The HBsAg content in mouse plasma was measured by ELISA. The results are shown in Table 18.

[0269] [Table 18]

[0270] b) The anti-HBV activity of the test compound in the AAV-HBV mouse model was evaluated based on the serum HBeAg content. The HBeAg content in mouse plasma was measured by ELISA. The results are shown in Table 19.

[0271] [Table 19]

[0272] c) The anti-HBV activity of the test compounds in the AAV-HBV mouse model was evaluated based on serum DNA content. HBV DNA content in mouse plasma was measured by quantitative PCR. The results are shown in Table 20.

[0273] [Table 20]

[0274] d) Changes in body weight. Weight was compared to the baseline weight on day 0. According to IACUC regulations, a 20% weight loss was defined as a humane endpoint, and mice showing weight loss of more than 20% were excluded from the experiment. No mice were excluded from the experiment due to weight loss.

[0275] Testing Conclusion: In this experiment, the compound of the present invention significantly reduced HBsAg, HBeAg and HBV DNA in the AAV-HBV mouse model, showing a clear dose-dependent relationship. The mice showed good tolerance during the experiment.

[0276] Experimental Example 7 In vitro plasma stability test 1. Experimental Materials a) CD-1 mouse plasma, SD rat plasma, human plasma, b) Test compound: 50 μL of test compound (1 mg / mL, solvent: nuclease-free water) was diluted with 450 μL of nuclease-free water to a concentration of 100 μL / mL.

[0277] c) Control compound: Patisiran, supplier: MedChemExpress 2. Experimental Procedure a) Frozen plasma was thawed under cold water for 10 to 20 minutes and centrifuged at 3220 × g for 5 minutes.

[0278] b) Working solutions of test and control compounds were prepared and at each time point, 2 μL of working solution was mixed with 98 μL of blank plasma in duplicate. c) For TO samples, blank plasma and working solutions of test and control compounds were added, followed immediately by the addition of stop solution.

[0279] d) Samples at each time point (0.5, 1, 2, 4, 6, 8, 24 hours) except for TO were incubated in a 37°C water bath. e) At the end of each time point, 100 μL of an aqueous solution containing 100 mM ammonium acetate (pH 10.0), 2 mM tris(2-carboxyethyl)phosphine hydrochloride, 1 mM ethylenediaminetetraacetic acid, and 750 ng / mL internal standard was added and vortex mixed for 60 seconds.

[0280] f) 100 μL of PCL (phenol / chloroform / isoamyl alcohol=25:24:1) reagent and 200 μL of dichloromethane were added to each sample well, mixed thoroughly, and then centrifuged at 3220×g for 20 minutes.

[0281] g) The aqueous layer was transferred to a new 96-well plate and stored at 4°C before LC-MS analysis. 3. Data Analysis The residual rate of the test compound after incubation in plasma was calculated using the following formula: residual rate (%) = 100 * (PAR at incubation time / PAR at TO), where PAR is the peak area ratio of the analyte to the internal standard.

[0282] The incubation time points were T0 (0 min) and Tn (n = 0, 0.5, 1, 2, 4, 6, 8, 24 h). 4. Experimental results: See Table 21

[0283] [Table 21]

[0284] Experimental results: The compound of the present invention was rapidly metabolized in the plasma of three species: rats, mice and humans. Experimental Example 8 In vitro liver S9 stability test 1. Experimental Materials a) Liver S9: Human and animal S9 were purchased from certified suppliers such as BioIVT and stored in a -80°C freezer.

[0285] b) Reference compound: Patisiran, supplier: MedChemExpress, lot number: 155113 2. Experimental Procedure 2.1 Preparation of buffers and working solutions Buffer solution: A buffer solution containing 100 mM tris(hydroxymethyl)aminomethane hydrochloride, 1 mM magnesium chloride, and 1x penicillin-streptomycin mixed solution was prepared using nuclease-free ultrapure water. The pH of the solution was adjusted to 6.00 ± 0.10 using hydrochloric acid.

[0286] S9 working solution: Liver S9 solution was diluted to 1.05 mg / mL with buffer. Working solution of test or control compound: 1.00 mg / mL of test or control compound was diluted to 2.00 μg / mL using nuclease-free water.

[0287] 2.2 Experimental Procedure a) Seven 96-well incubation plates were prepared and designated T0, T1, T4, T8, T24, T48, and Blank48. The reaction time points corresponding to the first six incubation plates were 0, 1, 4, 8, 24, and 48 hours, respectively. No test or control compound was added to the Blank48 plate, and the reaction was stopped after 48 hours of incubation. All time-point samples were performed in duplicate.

[0288] b) 190 μL of S9 working solution (protein concentration 1.05 mg / mL) was added to each of the T0, T1, T4, T8, T24, T48, and Blank48 plates, and the incubation plates except for T0 were pre-incubated in a 37°C water bath for approximately 10 minutes.

[0289] c) After the preincubation, 10 μL of test compound or control stock solution was added to each of the TO, T1, T4, T8, T24, and T48 plates, and 10 μL of water was added to the Blank 48 plate. The incubation plates (except TO, T1, T4, T8, T24, T48, and Blank 48) were then placed in a 37°C water bath to initiate the reaction. The final reaction volume was 200 μL, and the liver S9 protein concentration in the sample wells containing liver S9 was 1.00 mg / mL, while the final reaction concentration in the sample wells containing the test compound or control stock solution was 2.00 μg / mL.

[0290] d) 200 μL of stop solution (2.00 mM tris(2-carboxyethyl)phosphine hydrochloride, 100 mM ammonium acetate, 1.00 mM ethylenediaminetetraacetic acid, and an aqueous solution containing 750 ng / mL of internal standard) was added to the TO sample and thoroughly stirred. After that, 200 μL of a mixed solution (phenol:chloroform:isoamyl alcohol=25:24:1) was added and thoroughly shaken for 10 minutes. After that, 400 μL of dichloromethane was added, and the mixture was thoroughly shaken again and centrifuged at 4°C and 3220 × g for 20 minutes.

[0291] e) At the end of each incubation time point (1, 4, 8, 24, and 48 hours), the corresponding incubation plate was removed from the water bath and processed in the same manner as the TO sample. 100 μL of supernatant was collected from each sample and subjected to LC-MS / MS analysis.

[0292] 3. Sample analysis: The antisense strands of the test compounds were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS). The retention times of the analytes and internal standards, chromatogram collection, and chromatogram integration were performed using the software Analyst (Sciex, Framingham, MA, USA).

[0293] 4. Data Analysis The peak area ratio of the compound and the internal standard substance was converted into a residual rate using the following formula, and the in vitro elimination rate constant k of the test compound and the control compound was calculated. e asked for:

[0294]

number

[0295] k e From the data, the in vitro hepatic S9 specific clearance (CL int(S9) ) and hepatic intrinsic clearance (CL int(肝臓) ) was calculated. CL int(S9) =0.693 / T 1 / 2 / S9 protein content (mg / mL S9 concentration at incubation) CL int(肝臓) =CL int(S9) × S9 protein amount in liver (mg / g) × liver weight to body weight ratio The parameters used in the formula are shown in Table 22.

[0296] [Table 22]

[0297] Experimental results: See Table 23.

[0298] [Table 23]

[0299] Experimental conclusion: The antisense strand of the compound of the present invention showed good stability in liver S9 of three species: mouse, rat, and human. Experimental Example 9 RNA sequence off-target study of the compounds of the present invention

[0300] 1. Experimental Overview: The transcriptome is the sum of all RNA transcribed in a specific tissue or cell at a given time or state, primarily consisting of mRNA and non-coding RNA. Transcriptome sequencing, based on the Illumina sequencing platform, studies all mRNA transcribed by a specific tissue or cell over a given period. This is the basis for studying gene function and structure and plays an important role in understanding the mechanisms of organismal development and disease. With the development of gene sequencing technology and the decline in sequencing costs, RNA-seq has become the primary method for transcriptome research due to its advantages of high throughput, high sensitivity, and wide applicability. The RNA-seq technology process mainly consists of two parts: library construction and sequencing, and bioinformatics analysis.

[0301] 2. Research method: After incubating HepG2.2.15 cells with siRNA or PBS for 24 hours, the cells were lysed and RNA was extracted. RNA-seq was used to analyze differentially enriched genes between each siRNA group and the PBS group. Different databases were compared to identify differentially enriched genes. HepG2.2.15 cells were transfected with 100 nM siRNA using Lipofectamine RNAiMax and incubated in a 5% CO2, 37°C incubator for 24 hours. Cells were then harvested and RNA extracted. Strict quality control was then performed on the RNA samples, primarily using an Agilent 2100 bioanalyzer for precise RNA integrity detection.

[0302] 3. Library construction and quality check: There are two main methods for obtaining mRNA. One is to utilize the structural feature that most eukaryotic mRNAs have polyA tails and enrich for polyA-tailed mRNAs using Oligo(dT) magnetic beads. The other method is to remove ribosomal RNA from total RNA to obtain mRNA. The obtained mRNA was then randomly fragmented using divalent cations in NEB Fragmentation Buffer, and libraries were constructed according to the NEB standard library preparation method or strand-specific library preparation method.

[0303] NEB standard library preparation: Using fragmented mRNA as a template and random oligonucleotides as primers, the first strand of cDNA was synthesized using the M-MuLV reverse transcriptase system. The RNA strand was then degraded with RNase H, and the second strand of cDNA was synthesized using dNTPs as raw materials with the DNA polymerase I system. The purified double-stranded cDNA underwent end repair and A-tailing, followed by ligation of a sequence adapter. Approximately 250-300 bp of cDNA was selected using AMPure XP beads. After PCR amplification, the PCR product was purified again using AMPure XP beads, and the final library was obtained. The library construction kit was NEBNext® Ultra TM The RNA Library Prep Kit for Illumina® was used.

[0304] Strand-specific library construction: First-strand cDNA synthesis was performed using the same NEB standard library construction method, except that dTTP in the dNTPs was replaced with dUTP during second-strand synthesis. This was followed by cDNA end repair, A-tailing, sequencing adapter ligation, and size selection. U-containing second-strand cDNA was digested with USER enzyme, followed by PCR amplification to obtain the library. Strand-specific libraries offer many advantages, including the ability to obtain more effective information from the same amount of data, more accurate gene quantification, localization, and annotation information, and the ability to provide information on antisense transcripts and single-exon expression levels within each isoform. NEBNext® Ultra was used for library construction. TM The Directional RNA Library Prep Kit for Illumina® was used.

[0305] NOTE: The sequencing adapter consists of three parts: P5 / P7, index, and Rd1 / Rd2 SP. P5 / P7 are PCR amplification primers and primer binding sites on the flow cell. The index provides information to distinguish between different libraries. Rd1 / Rd2 SP (read1 / read2 sequence primers) are sequencing primer binding regions, and the sequencing process theoretically starts from Rd1 / Rd2 SP.

[0306] After completing the library construction, we first performed initial quantification using a Qubit2.0 Fluorometer and diluted the library to 1.5 ng / μL. We then used an Agilent 2100 bioanalyzer to detect the insert size of the library. After confirming that the insert size was as expected, we accurately quantified the effective concentration of the library using qRT-PCR (the effective concentration of the library was 2 nM or higher) to ensure the quality of the library.

[0307] 4. Sequencing: After library testing, the different libraries were pooled based on the effective concentration and target sequence data volume, and then subjected to Illumina sequencing. The basic principle of sequencing is sequencing by synthesis. Four types of fluorescently labeled dNTPs, DNA polymerase, and adapter primers are added to the sequencing flow cell to perform amplification. When the complementary strand is extended in each sequencing cluster, a corresponding fluorescent light is emitted with each addition of a fluorescently labeled dNTP. The sequencer detects this fluorescent signal, and computer software converts the optical signal into a sequencing peak, thereby obtaining the base sequence information of the target fragment.

[0308] 5. Experimental results: For differential gene analysis, the threshold was DESeq2 padj≦0.05|log2FoldChange|≧1.0, and the statistical results are shown in Table 24 below.

[0309] [Table 24]

[0310] Experimental results: The off-target risk of the compounds of the present invention is relatively low. Experimental Example 10: Preliminary safety study of the compounds of the present invention in rats 1. Experimental Materials a) Test compound: a compound of the present invention b) Male rats (3 rats), SPF grade, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0311] 2. Experimental Procedure a) Preparation of dosage formulation: Enzyme-free phosphate buffered saline (pH 7.4, 1X) was used as the solvent. The test compound was prepared into a 15 mg / mL clear solution.

[0312] b) The body weight of the animals was measured before administration, and the dose was calculated based on the body weight (based on a 150 mpk dose). On the day of administration, the animals' overall health and body surface were observed. c) On day 1, a single subcutaneous injection of the test compound solution was administered, and cage-side observations were carried out daily.

[0313] d) After 336 hours, blood (approximately 1.5 mL) was collected by puncturing the jugular vein and dispensed into blood collection tubes containing dipotassium ethylenediaminetetraacetic acid anticoagulant, sodium citrate anticoagulant, and silicon dioxide coagulation accelerator, then stored on wet ice and used for general blood tests, blood coagulation tests, and blood biochemistry tests.

[0314] 3. Experimental Results The experimental results for compound Z13 were as follows: a) Weight and behavior monitoring: No abnormalities b) General blood tests: No abnormalities were found in any of the indices, including white blood cells (WBC), neutrophils (NEUT), lymphocytes (LYMPH), monocytes (MONO), eosinophils (EOS), basophils (BASO), red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width (RDW), platelet count (PLT), and mean platelet volume (MPV). However, reticulocytes (RET) were slightly elevated in one rat.

[0315] c) Blood coagulation: There were no abnormalities in prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen (FIB). d) Blood biochemistry: Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were normal.

[0316] 4. Experimental conclusion: The preliminary safety of the compound of the present invention was good.

Claims

1. A double-stranded siRNA analog, a conjugate thereof, a salt thereof, or a salt of the conjugate thereof, comprising a sense strand and an antisense strand capable of forming a double-stranded region, selected from any one of the double strands shown in Table 1, wherein each nucleotide in the double strand is independently and selectively modified.

2. The double-stranded siRNA analog, its conjugate, its salt, or the salt of the conjugate according to claim 1, wherein the double-stranded siRNA analog is selected from S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S13, S14, S15, S16, S17, S18, S19, S20, S21, S22, S23, S24, S25, S26, S27 and S28.

3. The double-stranded siRNA analog, its conjugate, its salt, or the salt of the conjugate according to claim 2, wherein the double-stranded siRNA analog is selected from S1, S2, S3, S4, S5, S6, S7, S8, S9, S10 and S11.

4. The double-stranded siRNA analog, its conjugate, its salt, or the salt of the conjugate according to claim 1 , wherein the double-stranded siRNA analog is selected from any one of the double strands shown in Table 2.

5. The double-stranded siRNA analog, its conjugate, its salt, or the salt of the conjugate according to claim 1, which is formed by binding a pharmaceutically acceptable conjugate group to the double-stranded siRNA analog according to any one of claims 1 to 4.

6. The double-stranded siRNA analogue, a conjugate thereof, a salt thereof, or a salt of the conjugate according to claim 5, wherein the pharmaceutically acceptable conjugate group contains 1 to 5 GalNAc groups.

7. The double-stranded siRNA analog, its conjugate, its salt, or the salt of the conjugate according to claim 6, wherein the pharmaceutically acceptable conjugate group is linked to the 3' end of the sense strand of the double-stranded siRNA analog.

8. The pharmaceutically acceptable conjugate group is 【Chemistry 1】 【change】 The double-stranded siRNA analogue according to claim 5 , a conjugate thereof, a salt thereof, or a salt of the conjugate thereof, selected from the group consisting of:

9. A conjugate of a double-stranded siRNA analog selected from Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16, or a salt thereof.

10. Use of the double-stranded siRNA analogue according to any one of claims 1 to 9, a conjugate thereof, a salt thereof, or a salt of the conjugate thereof in the manufacture of a therapeutic agent for hepatitis B.

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