CN118878604B

CN118878604B - Phosphoester backbone modified nucleotides and oligonucleotides

Info

Publication number: CN118878604B
Application number: CN202411356089.5A
Authority: CN
Inventors: 李海涛; 黄渊余; 高永鑫
Original assignee: Beijing Xuanjingrui Pharmaceutical Technology Co ltd
Current assignee: Beijing Xuanjingrui Pharmaceutical Technology Co ltd
Priority date: 2024-09-27
Filing date: 2024-09-27
Publication date: 2025-01-24
Anticipated expiration: 2044-09-27
Also published as: CN118878604A

Abstract

The present disclosure provides a phosphoester skeleton modified nucleotide and an oligonucleotide, which belong to the technical field of small nucleic acid drug delivery. The present disclosure provides a double-stranded oligonucleotide and a conjugate thereof, wherein the double-stranded oligonucleotide contains at least one nucleotide modified by a phosphate backbone, and the nucleotide modified by the phosphate backbone has the efficacy of enhancing the activity of the double-stranded oligonucleotide and can improve the metabolic stability of the double-stranded oligonucleotide in vivo.

Description

Phosphoester skeleton modified nucleotide and oligonucleotide

Technical Field

The present disclosure relates to the technical field of small nucleic acid drug delivery, and in particular to a phosphorus ester skeleton modified nucleotide and an oligonucleotide.

Background

Currently, one of the commonly used modifications for improving the metabolic stability or long-acting activity of RNAi is Phosphorothioate (PS) modification. However, oligonucleotides (including siRNAs) that rely solely on phosphorothioate modifications remain degraded by endogenous nucleases, resulting in rapid decrease in vivo activity.

Thus, there remains a need in the field of RNAi therapy for new modifications, such as the formation of new phosphate backbone structures at the 5 'or 3' end of oligonucleotides (including siRNA, miRNA, and antisense oligonucleotides, etc.), to provide longer lasting efficacy of activity or higher metabolic stability in the oligonucleotide.

Disclosure of Invention

The present disclosure provides a double-stranded oligonucleotide and conjugates thereof, the double-stranded oligonucleotide containing a phosphate backbone modified nucleotide capable of enhancing the efficacy of the oligonucleotide, enhancing activity and/or longevity, and enhancing metabolic stability in vivo.

In a first aspect of the present disclosure, the present disclosure provides a double-stranded oligonucleotide comprising a sense strand and an antisense strand, each of the sense strand and the antisense strand independently having a length of 15-35 nucleotides, each of the nucleotides in the double-stranded oligonucleotide independently selected from a modified nucleotide or an unmodified nucleotide.

Wherein the double-stranded oligonucleotide contains at least one nucleotide modified with a phosphate backbone, and the nucleotide modified with a phosphate backbone has a structure represented by formula (IA):

wherein X is selected from methylene, O or S;

Z is selected from hydroxyl or mercapto;

Base is selected from nucleotide bases or analogues thereof;

Each R ₁、R₂、R₃、R₄ is independently selected from H, halogen, substituted or unsubstituted C ₁-C₆ alkyl, substituted or unsubstituted C ₁-C₆ alkoxy, and at least one of R ₁、R₂ is selected from substituted or unsubstituted C ₁-C₆ alkyl, substituted or unsubstituted C ₁-C₆ alkoxy;

R ₅ is selected from halogen, substituted or unsubstituted C ₁-C₆ alkyl, substituted or unsubstituted C ₁-C₆ alkoxy;

R ₆ is selected from H, halogen, substituted or unsubstituted C ₁-C₆ alkyl.

In some alternative embodiments, one of R ₁、R₂、R₃、R₄ is selected from halogen, substituted or unsubstituted C ₁-C₆ alkyl, substituted or unsubstituted C ₁-C₆ alkoxy.

In some alternative embodiments, one of R ₁、R₂ is selected from halogen, substituted or unsubstituted C ₁-C₆ alkyl, substituted or unsubstituted C ₁-C₆ alkoxy, and R ₁、R₂ is different.

In some alternative embodiments, R ₁ is selected from halogen, substituted or unsubstituted C ₁-C₆ alkyl, substituted or unsubstituted C ₁-C₆ alkoxy, and R ₂、R₃、R₄ is selected from H. Preferably, R ₁ is selected from halogen, C ₁-C₃ alkyl.

In some alternative embodiments, R ₁ is selected from F, methyl, ethyl, methoxy, or ethoxy, and R ₂、R₃、R₄ is selected from H.

In some alternative embodiments, R ₁ is selected from methyl or ethyl and R ₂、R₃、R₄ is selected from H.

In some embodiments, R ₁ is selected from methyl and R ₂、R₃、R₄ is selected from H.

In some alternative embodiments, the phosphoester backbone modified nucleotide has a structure represented by formula (IIA), formula (IIA-1), or formula (IIA-2), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

、、。

in some embodiments, X is selected from O.

In some alternative embodiments, the Base is selected from nucleobases A, U, G, C, T.

In some alternative embodiments, R ₅ is selected from F, methyl, ethyl, methoxy, or ethoxy.

In some alternative embodiments, R ₅ is selected from methoxy or ethoxy.

In some embodiments, R ₅ is selected from methoxy.

In some alternative embodiments, R ₆ is selected from H, F, methyl, or ethyl.

In some embodiments, R ₆ is selected from H.

In particular embodiments of the present disclosure, the nucleotide modified with a phosphate backbone is selected from any one of the following structures, or stereoisomers thereof, or tautomers thereof, or pharmaceutically acceptable salts thereof:

、、。

In some alternative embodiments, the antisense strand of the double-stranded oligonucleotide comprises at least one of the phosphate backbone modified nucleotides.

In some alternative embodiments, one of the phosphate backbone modified nucleotides is located at position 1 of the antisense strand of the double stranded oligonucleotide starting at the 5 'end or at position 1 starting at the 3' end.

In some alternative embodiments, at least one of the phosphate backbone modified nucleotides is each located independently from position 2-8 of the antisense strand of the double stranded oligonucleotide starting at the 5 'end or from position 1-6 of the antisense strand starting at the 3' end.

In some alternative embodiments, at least one of the phosphorus-backbone-modified nucleotides is each located independently from 1-6, starting at the 3' -terminus. For example, 1-5 positions from the 3 '-end, 1-4 positions from the 3' -end, 1-3 positions from the 3 '-end, or 1-2 positions from the 3' -end, etc.

In some embodiments, the antisense strand of the double-stranded oligonucleotide comprises two of the phosphate backbone modified nucleotides.

In some alternative embodiments, the two phosphate backbone modified nucleotides are each located independently from the antisense strand of the double stranded oligonucleotide at positions 2-8 starting at the 5 'end or 1-6 starting at the 3' end.

In some alternative embodiments, the two phosphoester backbone modified nucleotides are each located independently from 1-6 of the antisense strand of the double-stranded oligonucleotide starting at the 3' terminus. For example, 1-5 positions from the 3 '-end, 1-4 positions from the 3' -end, 1-3 positions from the 3 '-end, or 1-2 positions from the 3' -end, etc.

In some embodiments, the two phosphate backbone modified nucleotides are each located at positions 1 and 2 of the antisense strand of the double stranded oligonucleotide, starting at the 3' end.

In some alternative embodiments, the double-stranded oligonucleotide further comprises other modifications;

Wherein the other modifications include, but are not limited to, 2' -halo-modified nucleotides, 2' -deoxy-modified nucleotides, 2' -O-substituted or unsubstituted C ₁-C₆ alkyl-modified nucleotides, 2' -O- (CH ₂)_n -O-R modified nucleotides, 2' -amino-modified nucleotides, abasic nucleotides or nucleotide-like.

The nucleotide-like is selected from one or more of peptide nucleic acid (peptide nucleic acid, PNA), morpholine (Morpholino, MNA), bridge nucleic acid (bridged nucleic acid, BNA), locked nucleic acid (locked nucleic acid, LNA), ethylene glycol nucleic acid/glycerol nucleic acid (glycol nucleic acid, GNA), threose nucleic acid (threose nucleic acid, TNA) or unlocked nucleic acid (unlocked nucleic acid, UNA);

Wherein n is selected from 1 or 2, R is selected from substituted or unsubstituted C ₁-C₆ alkyl, substituted or unsubstituted C ₁-C₆ alkoxy, and if R contains a substituent, the substituent is selected from halogen, C ₁-C₆ alkoxy, hydroxy or amino.

In some embodiments, the 2 '-halo-modified nucleotide is selected from the group consisting of 2' -fluoro-modified nucleotides;

In some embodiments, the 2 '-O-substituted or unsubstituted C ₁-C₆ alkyl-modified nucleotide is selected from 2' -O-methyl-modified nucleotides.

In some alternative embodiments, the 2'-O- (CH ₂)_n -R modified nucleotide is selected from the group consisting of a 2' -O-methoxyethyl modified nucleotide, a2 '-O-ethoxymethyl modified nucleotide, and a 2' -O-2, 2-trifluoroethoxymethyl modified nucleotide.

In some embodiments, the 2'-O- (CH ₂)_n -R) modified nucleotide is selected from 2' -O-methoxyethyl modified nucleotides.

In some embodiments, the additional modification is selected from a 2' -O-methyl modified nucleotide, a 2' -fluoro modified nucleotide, or a 2' -O-methoxyethyl modified nucleotide.

In some alternative embodiments, the sense strand or the antisense strand comprises a 3' -overhang of at least 1 nucleotide.

In some alternative embodiments, the antisense strand comprises a 3' overhang of at least 1 nucleotide.

In some embodiments, the antisense strand comprises a 3' overhang of 2 nucleotides.

In some alternative embodiments, the sense strand and/or the antisense strand independently comprise one or more phosphorothioate linkages.

In some embodiments, the sense strand comprises two consecutive phosphorothioate linkages between the terminal nucleotides starting at the 5' terminus.

In some embodiments, the antisense strand comprises two consecutive phosphorothioate linkages between the 3 'terminal-initiated and 5' terminal-initiated terminal nucleotides, respectively.

In some embodiments, the sense strand has a length of 19 nucleotides and the antisense strand has a length of 21 nucleotides.

In some alternative embodiments, at least four nucleotides at positions 2, 6, 9-10, 14, 16 in the nucleotide sequence of the antisense strand are selected from the group consisting of 2' -fluoro modified nucleotides, at least one nucleotide at positions 16-21 is selected from the group consisting of the phosphoester backbone modified nucleotides, and the remaining position nucleotides are each independently selected from the group consisting of 2' -O-methyl modified nucleotides or 2' -O-methoxyethyl modified nucleotides, in a 5' end to 3' end orientation;

And/or, at least three nucleotides at positions 7-10 in the sense strand nucleotide sequence are selected from 2' -fluoro modified nucleotides, and the nucleotides at the remaining positions are each independently selected from 2' -O-methyl modified nucleotides or 2' -O-methoxyethyl modified nucleotides, in a 5' end to 3' end direction.

In some alternative embodiments, the nucleotides at positions 2,6, 9, 14, 16 in the nucleotide sequence of the antisense strand are selected from 2' -fluoro modified nucleotides, the 15 th nucleotide is selected from 2' -O-methoxyethyl modified nucleotides, any two of the nucleotides at positions 16-21 are selected from the phosphoester backbone modified nucleotides, and the remaining position nucleotides are selected from 2' -O-methyl modified nucleotides, in a 5' to 3' end orientation;

In some embodiments, the nucleotides at positions 2, 6,9, 14, 16 in the nucleotide sequence of the antisense strand are selected from 2' -fluoro modified nucleotides, the 15 th nucleotide is selected from 2' -O-methoxyethyl modified nucleotides, the 20 th to 21 th nucleotides are selected from the phosphate backbone modified nucleotides, and the remaining position nucleotides are selected from 2' -O-methyl modified nucleotides in a 5' to 3' end orientation;

and/or, according to the direction from the 5 'end to the 3' end, the 7 th to 10 th nucleotides in the sense strand nucleotide sequence are selected from 2 '-fluoro modified nucleotides, and the rest nucleotides are each independently selected from 2' -O-methyl modified nucleotides.

In some alternative embodiments, the nucleotide sequence of the antisense strand is selected from UmsAfsGmUmUmCfUmUmGfGmUmGmCmUfC (moe) UfUmGmGms (NM 101) s (NM 101) and the nucleotide sequence of the sense strand is selected from CmsCmsAmAmGmAmGfCfAfCfCmAmAmGmAmAmCmUmAm in the 5 'to 3' end direction.

Wherein, (NM 101) has the structural formula ofA represents adenine, U represents uracil, G represents guanine, C represents cytosine, m represents that the nucleotide adjacent to the left side is a 2' -O-methyl modified nucleotide, f represents that the nucleotide adjacent to the left side is a 2' -fluoro modified nucleotide, (moe) represents that the nucleotide adjacent to the left side is a 2' -O-methoxyethyl modified nucleotide, and s represents that phosphorothioate bond connection is formed between the two nucleotides adjacent to the left side and the right side.

In some alternative embodiments, the antisense strand is substantially reverse complementary, or fully reverse complementary to a nucleotide sequence in an mRNA expressed by the target gene.

In some embodiments, the double stranded oligonucleotide is selected from siRNA.

In a second aspect of the present disclosure, the present disclosure provides a conjugate comprising a double stranded oligonucleotide according to the first aspect of the present disclosure and one or more ligands capable of binding to a cell surface receptor.

In some embodiments, the cell is selected from a liver cell.

In some embodiments, the ligand is selected from the group consisting of asialoglycoprotein receptor ligands (ASGPR ligands).

In some embodiments, the asialoglycoprotein receptor ligand comprises galactose and/or derivatives thereof.

In some embodiments, the galactose derivative is selected from the group consisting of galactose derivatives having an affinity for the asialoglycoprotein receptor equal to or exceeding galactose, such as N-acetylgalactosamine (N-acetylgalactosamine, galNAc).

In some embodiments, the galactose derivative is selected from the group consisting of N-acetylgalactosamine. Wherein the structural formula of the N-acetylgalactosamine is。

In some embodiments, the ligand is selected from the following structures, or stereoisomers thereof, or tautomers thereof, or pharmaceutically acceptable salts thereof:

wherein Z' represents a hydroxyl group or a mercapto group.

In some alternative embodiments, the number of ligands is selected from 1,2, 3, or 4. The ligand is independently selected from the group consisting of conjugation linked to the 3 'end of the sense strand, the 5' end of the sense strand, the 3 'end of the antisense strand, or the 5' end of the antisense strand, and the conjugation sites of the ligand on the double-stranded oligonucleotide are different.

In some alternative embodiments, the number of ligands is selected from 1, wherein one of the ligands may be conjugated to the 3 'end of the sense strand, or one of the ligands may be conjugated to the 5' end of the sense strand, or one of the ligands may be conjugated to the 3 'end of the antisense strand, or one of the ligands may be conjugated to the 5' end of the antisense strand.

In some embodiments, the number of ligands is selected from one, the ligand conjugate is attached to the 3' end of the sense strand of the double-stranded oligonucleotide.

In some embodiments, the conjugate has the structural formula:

wherein Z' represents a hydroxyl group or a mercapto group;

nu represents the double stranded oligonucleotide of the first aspect of the disclosure.

Further, ligand conjugation is attached to the 3' -end of the sense strand of the double-stranded oligonucleotide.

In some alternative embodiments, the number of ligands is selected from 2. Wherein two of said ligands are conjugated to the 3 'end of said sense strand and the 5' end of said sense strand, respectively, or two of said ligands are conjugated to the 3 'end of said sense strand and the 3' end of said antisense strand, respectively, or two of said ligands are conjugated to the 3 'end of said sense strand and the 5' end of said antisense strand, respectively, or two of said ligands are conjugated to the 5 'end of said sense strand and the 3' end of said antisense strand, respectively, or two of said ligands are conjugated to the 5 'end of said sense strand and the 5' end of said antisense strand, respectively. In some alternative embodiments of the disclosure, the number of ligands is selected from 2, and two of the ligands are conjugated to the 3 'end of the sense strand and the 5' end of the sense strand, respectively.

In some alternative embodiments, the number of ligands is selected from 3. Wherein three of said ligands are conjugated to the 3 'end of said sense strand, the 5' end of said sense strand and the 3 'end of said antisense strand, respectively, or three of said ligands are conjugated to the 3' end of said sense strand, the 5 'end of said sense strand and the 5' end of said antisense strand, respectively, or three of said ligands are conjugated to the 3 'end of said sense strand, the 3' end of said antisense strand and the 5 'end of said antisense strand, respectively, or three of said ligands are conjugated to the 5' end of said sense strand, the 3 'end of said antisense strand and the 5' end of said antisense strand, respectively.

In some alternative embodiments, the number of ligands is selected from 4. Wherein four of the ligands are conjugated to the 3 'end of the sense strand, the 5' end of the sense strand, the 3 'end of the antisense strand, and the 5' end of the antisense strand, respectively.

In a specific embodiment of the present disclosure, the antisense strand of the conjugate is selected from UmsAfsGmUmUmCfUmUmGfGmUmGmCmUfC (moe) UfUmGmGms (NM 101) s (NM 101) and the sense strand of the conjugate is selected from CmsCmsAmAmGmAmGfCfAfCfCmAmAmGmAmAmCmUmAm _L96, in a 5 'end to 3' end direction.

Wherein "_L96" means that the L96 vector is linked to the 3' end of the sense strand as shown above by phosphodiester bond conjugation.

In a third aspect of the present disclosure, the present disclosure provides a use of any of the following in the manufacture of a medicament for the treatment and/or prevention of a disease or disorder associated with deregulation of mRNA levels of target gene expression:

(I) A double-stranded oligonucleotide according to the first aspect of the present disclosure, and/or

(II) conjugates of the second aspect of the disclosure.

In a fourth aspect of the present disclosure, the present disclosure provides a pharmaceutical composition comprising any of the following together with pharmaceutically acceptable excipients or adjuvants:

(II) conjugates of the second aspect of the disclosure.

In a fifth aspect of the present disclosure, the present disclosure provides a method of modulating a dysregulation of a target gene expression mRNA in a cell, the method comprising contacting any of the following with the cell:

(II), conjugates of the second aspect of the disclosure, and/or

(III) pharmaceutical compositions according to the fourth aspect of the present disclosure.

In a sixth aspect of the present disclosure, the present disclosure provides a modified nucleoside monomer having a structure represented by formula (IB), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

Wherein X, base and R ₁、R₂、R₃、R₄、R₅、R₆ are as defined in the first aspect of the disclosure, wherein, if an amino group is contained in Base, the amino group is protected by an amino protecting group.

In some alternative embodiments, the modified nucleoside monomer has a structure represented by formula (IIB), formula (IIB-1), or formula (IIB-2), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

、、。

R ₇ is selected from H or a hydroxyl protecting group;

In some alternative embodiments, the hydroxy protecting group is selected from the group consisting of trityl, 4-methoxytrityl, 4 '-dimethoxytrityl, or 4,4',4 "-trimethoxytrityl.

In some embodiments, the hydroxy protecting group is selected from 4,4' -dimethoxytrityl.

In some embodiments, R ₇ is selected from H or 4,4' -dimethoxytrityl.

In some embodiments, R ₇ is selected from 4,4' -dimethoxytrityl.

R ₈ is selected from H orWherein each R _8a is independently selected fromOr C _1-6 alkoxy containing cyano substituent, and at least one R _8a is selected fromEach R _8a' is independently selected from optionally substituted C _1-6 alkyl.

In some embodiments, each R _8a is independently selected fromOr (b)And at least one R _8a is selected from。

In some of the embodiments of the present invention in the alternative,Selected from the group consisting ofOr (b)。

In some embodiments of the present invention, in some embodiments,Selected from the group consisting of。

In some alternative embodiments, R ₈ is selected from H or。

In some embodiments, R ₈ is selected from。

In particular embodiments of the present disclosure, the modified nucleoside monomer is selected from any one of the following structures, or stereoisomers thereof, or tautomers thereof, or pharmaceutically acceptable salts thereof:

、、。

Experiments prove that the nucleotide modified by the phosphate skeleton has the effect of enhancing the activity of the double-stranded oligonucleotide and can improve the in-vivo metabolic stability of the double-stranded oligonucleotide.

Drawings

FIG. 1 relative expression levels of target genes of interest in mice following administration of siRNA conjugates.

Detailed Description

The present disclosure discloses a double-stranded oligonucleotide comprising a nucleotide modified by a phosphate backbone and conjugates thereof, and those skilled in the art can, in light of the disclosure herein, suitably modify the process parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present disclosure. While the methods and applications of the present disclosure have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and appropriate changes and combinations of the methods and applications described herein can be made to practice and use the disclosed technology without departing from the spirit and scope of the disclosure.

Interpretation of the terms

In this disclosure, the terms "comprises" and "comprising" are open-ended terms that include what is indicated in the disclosure, but do not exclude other aspects.

In this disclosure, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or condition may, but need not, occur, and that the description includes instances in which the event or condition occurs, as well as instances in which the event or condition does not.

In the present disclosure, the term "small interfering RNA (SMALL INTERFERING RNA, SIRNA)" is a double-stranded RNA of 17 to 25 nucleotides in length, comprising a sense strand and an antisense strand. siRNA mediates targeted cleavage of RNA transcripts of the RISC pathway by forming silencing complexes (RNA-induced silencing complex, RISC). Specifically, siRNA directs the specific degradation of mRNA sequences through known RNA interference (RNAi) processes, inhibiting translation of mRNA into amino acids and conversion to proteins.

In the present disclosure, the term "antisense strand (or guide strand)" includes a region substantially complementary to a target sequence, such as the mRNA of AGT. "sense strand (or" follower strand) "means that it contains an iRNA strand that is substantially complementary to the antisense strand. The term "substantially complementary" refers to complete complementarity or at least partial complementarity, e.g., the antisense strand is complete complementarity or at least partial complementarity to a target sequence. In the case of partial complementarity, the mismatch may be present within the interior or terminal region of the molecule, wherein the most tolerated mismatch is present within the terminal region, e.g., within 5, 4, 3, or 2 nucleotides of the 5 'end and/or 3' end of the iRNA.

It is noted that "at least partially substantially complementary" of the antisense strand to the mRNA means that the antisense strand has a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest. Or if a polynucleotide is substantially non-intermittently complementary to a portion of the mRNA encoding the target gene, the antisense strand is complementary to at least a portion of the mRNA of the target gene.

In the present disclosure, the term "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription encoding a gene of interest, including mRNA that is the RNA processing product of the primary transcript.

In the present disclosure, the term "complementary" refers to the ability of an oligonucleotide of a first sequence to hybridize under certain conditions to an oligonucleotide of a second sequence and form a double-stranded structure.

In the present disclosure, the term "substantially complementary" means that the sense strand and the antisense strand are mismatched by no more than 3 nucleotides, no more than 2 nucleotides, or no more than 1 nucleotide in the duplex region, e.g., 3 nucleotides, 2 nucleotides, 1 nucleotide, or 0 nucleotide, while preserving the ability to hybridize under the relevant conditions. In addition, where two oligonucleotides are designed to hybridize to form one or more single stranded overhangs, such overhangs should not be considered mismatches in terms of determining complementarity. In the present disclosure, as long as the above requirements for hybridization capability are met, a "complementary" sequence may also include or be formed entirely from non-Watson (Watson) -Crick base pairs and/or base pairs formed from non-natural and modified nucleotides. Such non-Watson-Crick base pairs include, but are not limited to, G: U wobble base pairing or Hoogstein (Hu Gesi Teng) base pairing. Correspondingly, in the present disclosure, unless otherwise specified, "mismatch" means that bases at corresponding positions in the siRNA duplex molecule do not pair in a complementary form.

In the present disclosure, the term "complementary sequence with complementarity" refers to a mismatch of not more than 3 nucleotides, not more than 2 nucleotides or not more than 1 nucleotide, e.g. 3 nucleotides, 2 nucleotides, 1 nucleotide or 0 nucleotide, in the antisense strand of a double stranded oligonucleotide and the complementary sequence of an AGT mRNA.

In the present disclosure, the terms "nucleotide difference" and "nucleotide base difference" and the term "difference in nucleotide sequence" may be used interchangeably. Refers to a change in the base type of the nucleotide at the same or corresponding position as compared with the original nucleotide sequence. For example, when one nucleotide base in the original nucleotide sequence is A, in the case where the nucleotide base at the same or corresponding position is changed to U, C, G or dT, dC, dG, or the like, it is considered that there is a difference in nucleotide sequence at that position. Here, in the case where a nucleotide at the same or corresponding position differs from the original nucleotide sequence only in the presence or absence of modification or the type of modification, the difference in nucleotide sequence at that position is not considered. .

In the present disclosure, the term "overhang" refers to at least one unpaired nucleotide protruding from a double-stranded oligonucleotide duplex, as well as a nucleotide sequence in the siRNA structure other than the double-stranded region. For example, a nucleotide overhang is present when the 3 'end of one of the sense strand and/or the antisense strand extends beyond the 5' end of the other strand, or when the 5 'end of one of the sense strand and/or the antisense strand extends beyond the 3' end of the other strand. The overhang can comprise at least one nucleotide, at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. The nucleotide overhang may comprise or consist of nucleotide/nucleoside analogues, including deoxynucleotides/nucleosides. The overhang may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the overhanging nucleotides may occur at the 5 'end, the 3' end, or both ends of the antisense or sense strand.

In the present disclosure, the term "subject" refers to any animal being examined, studied, or treated and is not intended that the present disclosure be limited to any particular type of subject. In some embodiments of the present disclosure, humans are preferred subjects, while in other embodiments, non-human animals are preferred subjects, including but not limited to mice, monkeys, ferrets, cows, sheep, goats, pigs, chickens, turkeys, dogs, cats, horses, and reptiles.

In the present disclosure, "conjugate" refers to two or more chemical moieties linked to each other by covalent linkage, and "conjugate" refers to a compound formed by covalent linkage between the respective chemical moieties, and "conjugate molecule" is understood to be a specific compound that can be conjugated to an oligonucleotide by reaction, ultimately forming an oligonucleotide conjugate of the present disclosure.

In the present disclosure, "pharmaceutical composition" may refer to a composition for the treatment of a disease, as well as an in vitro culture experiment of cells. For the treatment of diseases, the term "pharmaceutical composition" generally refers to a unit dosage form and may be prepared by any of the methods well known in the pharmaceutical arts. All methods include the step of combining the active ingredient with adjuvants that constitute one or more adjunct ingredients. Generally, the compositions are prepared by uniformly and sufficiently combining the active siRNA with a liquid adjuvant, a finely divided solid adjuvant, or both.

In the present disclosure, the term "pharmaceutically acceptable" means that the substance or composition must be chemically and/or toxicologically compatible with the other ingredients comprising the formulation and/or the mammal being treated therewith. Preferably, the term "pharmaceutically acceptable" as used in the present disclosure refers to use in animals, particularly humans, approved by the federal regulatory agency or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia.

In the present disclosure, the term "pharmaceutically acceptable excipients or adjuvants" may include any solvents, solid excipients, diluents or other liquid excipients, etc., suitable for the particular target dosage form. In addition to the extent to which any conventional adjuvant is incompatible with the siRNA of the present disclosure, such as any adverse biological effect produced or interactions with any other component of the pharmaceutically acceptable composition that occur in a deleterious manner, their use is also contemplated by the present disclosure.

In the present disclosure, the term "treatment" refers to a treatment for obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or symptoms thereof, and/or may be therapeutic in terms of partially or completely curing the disease and/or adverse effects caused by the disease. "treating" as used herein encompasses diseases in mammals, particularly humans, including (a) preventing the occurrence of a disease or disorder in an individual susceptible to the disease but not yet diagnosed with the disease, (b) inhibiting the disease, e.g., arresting the development of the disease, or (c) alleviating the disease, e.g., alleviating symptoms associated with the disease. As used herein, "treating" or "treatment" encompasses any administration of a drug or siRNA to an individual to treat, cure, alleviate, ameliorate, reduce or inhibit a disease in the individual, including, but not limited to, administration of a drug comprising an siRNA or siRNA conjugate described herein to an individual in need thereof.

In addition to any conventional adjuvants, the scope of incompatibility with the siRNA of the present disclosure, e.g., any adverse biological effects produced or interactions with any other component of the pharmaceutically acceptable composition in a deleterious manner, is also contemplated by the present disclosure.

For the purposes of clarity, technical solutions and advantages of the present disclosure, embodiments of the present disclosure will be described in further detail below with reference to examples.

Unless otherwise indicated, reagent consumables and instrumentation used in the present disclosure are all derived from commercial products. The main reagent consumables and the sources thereof are shown in table 1, and the main instruments and the sources thereof are shown in table 2.

Table 1 major reagent consumables

TABLE 2 Main instrumentation

The disclosure is further illustrated below in conjunction with the examples:

Preparation of the Compounds

Preparation example 1 preparation of Compound 5

In this preparation, the synthetic route for compound 5 is shown below:

(1-1) Synthesis of Compound 2

Compound 1 (25 g, 1.0eq,2 '-methoxyuridine, CAS No. 2140-76-3) was dissolved in 250mL pyridine, 4' -dimethoxytrityl chloride (37 g, 1.3eq, abbreviated as DMTrCl) was added in an ice bath, reacted at room temperature for 3 hours, and quenched by addition of 150mL methanol. After the completion of the reaction, the reaction mixture was concentrated under reduced pressure, extracted with ethyl acetate 3 times, and concentrated by drying to give crude compound 2 (47 g) as a yellow oil, which was subjected to the next reaction without purification. MS ESI (M/z) =561 [ m+h ] ⁺.

(1-2) Synthesis of Compound 3

Compound 2 (47 g, 1 eq) was dissolved in 200mL dichloromethane. Imidazole (11.5 g, 2 eq) and t-butyldimethylchlorosilane (19 g, 1.2eq, CAS number 18162-48-6) were added and stirred overnight at room temperature. After the completion of the reaction, the reaction mixture was concentrated under reduced pressure, extracted with ethyl acetate for 3 times, and dried and concentrated to give crude compound 3 (60 g) as a yellow solid, and the compound 3 was directly subjected to the next reaction without purification. MS ESI (M/z) =675 [ m+h ] ⁺.

(1-3) Synthesis of Compound 4

Compound 3 (25 g, 1.0 eq) was dissolved in a mixed solution of dichloromethane (70 ml) and methanol (30 ml), p-toluenesulfonic acid (14 g, 2eq, CAS number 104-15-4) was added, and stirred at room temperature for 3 hours. After the completion of the reaction, the reaction mixture was extracted 3 times with ethyl acetate and saturated aqueous sodium bicarbonate, backwashed 3 times with saturated aqueous sodium chloride, dried and concentrated, and subjected to normal phase purification (eluent: petroleum ether PE/ethyl acetate ea=1/1, volume ratio v/v) to give compound 4 (20 g) as an off-white solid. MS ESI (m/z) =373 [ m+h ] ⁺.

(1-4) Synthesis of Compound 5

Compound 4 (20 g, 1.0 eq) and 2-iodoxybenzoic acid (9.4 g, 2.5eq, abbreviated as IBX, CAS number 61717-82-6) were dissolved in 150ml acetonitrile and reacted for 3 hours at 85 ℃. After the reaction was completed, the reaction system was cooled to room temperature, the reaction solution was filtered and washed 3 times with ethyl acetate, and the obtained filtrate was concentrated by drying to obtain crude compound 5 (20 g) as a pink-yellow solid, and compound 5 was directly involved in the subsequent reaction without purification. MS ESI (M/z) =371 [ m+h ] ⁺.

PREPARATION EXAMPLE 2 Synthesis of Compound NM100

In this preparation, the synthetic route for compound NM100 is shown below:

(2-1) Synthesis of Compound NM100-1

Triphenylphosphine (15 g,2.5eq, CAS No. 1779-49-3) was dissolved in tetrahydrofuran (50 ml, abbreviated as THF), potassium tert-butoxide (4.8 g,2.5eq, CAS No. 865-47-4) was added under ice-bath, stirred at 0deg.C for 30 min, and then a solution of Compound 5 (7.2 g,1.0 eq) in tetrahydrofuran was added and reacted overnight at room temperature. After the completion of the reaction, the reaction mixture was extracted 3 times with saturated ammonium chloride and ethyl acetate, concentrated under reduced pressure, dried and filtered, and purified in normal phase (eluent: PE/ea=2/1, v/v) to give compound NM100-1 (3.2 g, yield 44.7%) as a white solid. MS ESI (M/z) =369 [ m+h ] ⁺.

(2-2) Synthesis of Compound NM100-2

Compound NM100-1 (3.2 g, 1.0 eq) was dissolved in 50ml THF, 9-borabicyclo [3.3.1] nonane (100 ml, 6eq, abbreviated as 9-BBN, CAS number 280-64-8) was slowly added under ice bath, stirred at room temperature for 6 hours, methanol (30 ml), water (50 ml) and stirring for 10 minutes were sequentially added at low temperature, sodium perborate tetrahydrate (6.9 g, 5 eq) was added, and reacted overnight at room temperature. After the completion of the reaction, the reaction mixture was extracted 3 times with saturated ammonium chloride and ethyl acetate, dried, filtered and concentrated under reduced pressure to give crude compound NM100-2 (7 g) as a white solid. MS ESI (M/z) =387 [ m+h ] ⁺.

(2-3) Synthesis of Compound NM100-3

Compound NM100-2 (7 g, 1.0 eq) was dissolved in 45mL pyridine, DMTrCl (3.2 g, 1.3 eq) was added under ice bath, reacted at room temperature for 3 hours, quenched by adding 15mL methanol. After the completion of the reaction, the reaction mixture was concentrated under reduced pressure and purified in the normal phase (eluent: PE/ea=1/1, v/v) to give compound NM100-3 (370 mg) as a yellow solid. MS ESI (M/z) =689 [ m+h ] ⁺.

(2-4) Synthesis of Compound NM100-4

Compound NM100-3 (730 mg, 1.0 eq) was dissolved in 15ml THF, tetrabutylammonium fluoride (1.25 ml, 1.2eq, TBAF for short, CAS number 429-41-4) was added, and reacted at room temperature for 1 hour. After the completion of the reaction, the reaction mixture was directly purified in reverse phase to give compound NM100-4 (400 mg, yield 65.5%) as a white solid. MS ESI (M/z) =575 [ m+h ] ⁺.

(2-5) Synthesis of Compound NM100

Compound NM100-4 (400 mg, 1.0 eq) was dissolved in 20ml dichloromethane (DCM for short), 4, 5-dicyanoimidazole (64 mg, 0.8eq, DCI for short, CAS No. 1122-28-7) and bis (diisopropylamino) (2-cyanoethoxy) phosphine (247 mg, 1.2eq, CAS No. 102691-36-1) were added and reacted at room temperature for 3 hours. After the completion of the reaction, the reaction mixture was directly purified in reverse phase to give compound NM100 (480 mg, yield 74.6%) as a white solid. MS ESI (M/z) =775 [ m+h ] ⁺.

¹H NMR (400 MHz, DMSO-d6) δ 11.45 – 11.40 (s, 1H), 7.43 – 7.23 (m, 10H), 6.92 – 6.83 (m, 4H), 5.85 – 5.78 (dd, J = 6.9, 5.2 Hz, 1H), 5.62 – 5.54 (dd, J = 8.1, 3.7 Hz, 1H), 4.53 – 4.46 (ddd, J = 10.4, 4.9, 2.6 Hz, 1H), 4.14 – 4.00 (ddd, J = 19.5, 7.0, 4.9 Hz, 1H), 3.91 – 3.78 (m, 2H), 3.76 – 3.71 (t, J = 1.8 Hz, 6H), 3.71 – 3.57 (m, 1H), 3.40 – 3.32 (d, J = 7.5 Hz, 4H), 3.13 – 2.86 (m, 2H), 2.84 – 2.76 (dt, J = 12.0, 5.9 Hz, 2H), 2.12 – 2.07 (s, 1H), 1.31 – 1.14 (ddd, J = 8.1, 6.4, 2.7 Hz, 12H).

PREPARATION EXAMPLE 3 Synthesis of Compound NM101

In this preparation, the synthetic route for compound NM101 is shown below:

(3-1) Synthesis of Compound NM101-1

Compound 5 (20 g, 1 eq) was dissolved in 200ml THF, a solution of 3M methyl magnesium bromide in THF (50 ml) was added at-20℃and replaced 3 times with nitrogen, the reaction system was stirred under nitrogen atmosphere at-20℃for 1 hour and quenched by addition of 20ml saturated aqueous ammonium chloride. After the completion of the reaction, the reaction mixture was extracted 3 times with ethyl acetate, the organic phase was separated, dried and concentrated, and purified in the normal phase (eluent: PE/ea=1/1, v/v) to give compound NM101-1 (7 g, yield 33.6%) as a white solid. MS ESI (M/z) =409 [ m+na ] ⁺.

(3-2) Synthesis of Compound NM101-2

Compound NM101-1 (7 g,1.0 eq) and 2-iodoxybenzoic acid (13.3 g, 2.5eq, abbreviated as IBX, CAS No. 61717-82-6) were dissolved in 70ml acetonitrile and reacted for 3 hours at 85 ℃. After the completion of the reaction, the reaction system was cooled to room temperature, the reaction solution was filtered and washed 3 times with ethyl acetate, and the obtained filtrate was concentrated by drying to give crude compound NM101-2 (20 g) as a pink-yellow solid. MS ESI (M/z) =385 [ m+h ] ⁺.

(3-3) Synthesis of Compound NM101-3

Triphenylphosphine methyl bromide (15 g, 2.5 eq) was dissolved in 50ml THF, potassium tert-butoxide (4.8 g, 2.5 eq) was added under ice-bath, stirred at 0 ℃ for 30min, and then a THF solution of compound NM101-2 (7.2 g,1.0 eq) was added and reacted overnight at room temperature. After the completion of the reaction, the reaction mixture was extracted 3 times with saturated aqueous ammonium chloride and ethyl acetate, concentrated under reduced pressure, dried and filtered, and purified in normal phase (eluent: PE/ea=2/1, v/v) to give compound NM101-3 (3.2 g, yield 44.7%) as a white solid. MS ESI (M/z) =383 [ m+h ] ⁺.

(3-4) Synthesis of Compound NM101-4

Compound NM101-3 (3.2 g, 1.0 eq) was dissolved in 50ml THF, 9-BBN (100 ml, 6 eq) was slowly added under ice bath, stirred at room temperature for 6 hours, methanol (30 ml), water (50 ml) and stirring for 10 minutes were sequentially added at low temperature, sodium perborate tetrahydrate (6.9 g, 5 eq) was added, and the reaction was carried out at room temperature overnight. After the completion of the reaction, the reaction mixture was extracted 3 times with a saturated aqueous ammonium chloride solution and ethyl acetate, dried, filtered, and concentrated under reduced pressure to give crude compound NM101-4 (7 g) as a white solid. MS ESI (M/z) =401 [ m+h ] ⁺.

(3-5) Synthesis of Compound NM101-5

Compound NM101-4 (7 g, 1.0 eq) was dissolved in 45mL pyridine, DMTrCl (3.2 g, 1.3 eq) was added under ice bath, reacted at room temperature for 3 hours, quenched by adding 15mL methanol. After the completion of the reaction, the reaction mixture was concentrated under reduced pressure and purified in the normal phase (eluent: PE/ea=1/1, v/v) to give compound NM101-5 (370 mg) as a yellow solid. MS ESI (M/z) =703 [ m+h ] ⁺.

(3-6) Synthesis of Compound NM101-6

Compound NM101-5 (730 mg, 1.0 eq) was dissolved in 15ml THF, TBAF (1.25 ml, 1.2 eq) was added and reacted at room temperature for 1 hour. After the completion of the reaction, the reaction mixture was directly purified in reverse phase to give compound NM101-6 (400 mg, yield 65.5%) as a white solid. MS ESI (M/z) =589 [ m+h ] ⁺.

(3-7) Synthesis of Compound NM101

Compound NM101-6 (400 mg, 1.0 eq) was dissolved in 20ml DCM, DCI (64 mg, 0.8 eq) and bis (diisopropylamino) (2-cyanoethoxy) phosphine (247 mg, 1.2 eq) were added and reacted at room temperature for 3 hours. After the completion of the reaction, the reaction mixture was directly purified in reverse phase to give compound NM101 (400 mg, yield 74.6%) as a white solid. MS ESI (M/z) =789 [ m+h ] ⁺.

¹H NMR (400 MHz, DMSO-d6) δ 11.45 – 11.40 (s, 1H), 7.43 – 7.23 (m, 10H), 6.92 – 6.83 (m, 4H), 5.85 – 5.78 (dd, J = 6.9, 5.2 Hz, 1H), 5.62 – 5.54 (dd, J = 8.1, 3.7 Hz, 1H), 4.53 – 4.46 (ddd, J = 10.4, 4.9, 2.6 Hz, 1H), 4.14 – 4.00 (ddd, J = 19.5, 7.0, 4.9 Hz, 1H), 3.91 – 3.78 (m, 2H), 3.76 – 3.71 (t, J = 1.8 Hz, 6H), 3.71 – 3.57 (m, 1H), 3.40 – 3.32 (d, J = 7.5 Hz, 4H), 3.13 – 2.86 (m, 2H), 2.84 – 2.76 (dt, J = 12.0, 5.9 Hz, 2H), 2.12 – 2.07 (s, 1H), 1.31 – 1.14 (ddd, J = 8.1, 6.4, 2.7 Hz, 12H), 1.08 – 1.00 (dd, J = 13.4, 6.7 Hz, 3H).

PREPARATION EXAMPLE 4 preparation of siRNA conjugates

Wherein, the compound L96-PS is purchased from Kaileying medical group (Tianjin) Co., ltd, and the loading is 120+/-12 mu mol/g (the detection method is UV/HPLC). The structural formula of the compound L96-PS is as follows:

Wherein PS stands for polystyrene (Polystyrene) resin solid phase carrier.

(4-1) Synthesis of Sense Strand (SS)

By the method of phosphoramidite nucleic acid solid phase synthesis, nucleoside monomers are linked one by one according to the nucleotide sequence in the 3'-5' direction using the compound L96-PS initiation cycle. Each nucleoside monomer attached includes four steps of deprotection, coupling, capping, oxidation or vulcanization. The synthesis conditions were given as follows:

Nucleoside monomers were formulated as an acetonitrile solution of nucleoside monomers at a concentration of 0.1M.

The deprotection conditions are the same for each step. The deprotection reaction was carried out at 25℃for 70 seconds with a molar ratio of dichloroacetic acid to 4,4' -dimethoxytrityl protecting group on the solid support of 5:1 in the presence of a dichloromethane solution (3% by volume) of dichloroacetic acid as the deprotection reagent.

The conditions for each coupling reaction were identical. The conditions of the coupling reaction are that the temperature is 25 ℃, the mole ratio of the nucleic acid sequence connected on the solid carrier to the nucleoside monomer is 1:10, the mole ratio of the nucleic acid sequence connected on the solid carrier to the coupling reagent is 1:65, the reaction time is 600 seconds, the coupling reagent is acetonitrile solution of 5-ethylthio-1H-tetrazole with the concentration of 0.5M, and the thio reagent is acetonitrile/pyridine mixed solution of hydrogenated Huang Yuansu with the concentration of 0.2mol/L (the volume ratio of acetonitrile to pyridine is 1:1).

The conditions for the capping reaction were the same for each step. The Cap reaction conditions are that the temperature is 25 ℃, the reaction time is 2 minutes, the Cap reagent solution is a mixed solution of Cap1 and Cap2 with the molar ratio of 1:1, cap1 is a pyridine/acetonitrile mixed solution of N-methylimidazole with the concentration of 20 volume percent, the volume ratio of pyridine to acetonitrile is 3:5, cap2 is an acetonitrile solution of acetic anhydride with the annual attack rate of 20 volume percent, and the molar ratio of N-methylimidazole in the Cap1 Cap reagent, acetic anhydride in the Cap2 Cap reagent and a nucleic acid sequence connected to a solid phase carrier is 1:1:1.

The conditions for each oxidation reaction are the same. The conditions of the oxidation reaction were 25℃for 3 seconds, 0.05M iodine water as the oxidizing agent, a molar ratio of iodine to nucleic acid sequence attached to the solid support in the coupling reaction of 30:1, and a water/pyridine mixed solvent (volume ratio of water to pyridine of 1:9). The conditions of the sulfidation reaction were 25℃for 360 seconds, 0.2M solution of pyridine hydrogenated Huang Yuansu in concentration of the thio reagent, 4:1 molar ratio of thio reagent to nucleic acid sequence attached to the solid support in the coupling reaction, and the thio reaction was carried out in a water/pyridine mixed solvent (volume ratio of water to pyridine: 1:9).

After the last nucleoside monomer is connected, the nucleic acid sequence connected on the solid phase carrier is sequentially cut, deprotected, purified and desalted, and then freeze-dried to obtain the sense strand, wherein:

The cleavage and deprotection conditions were such that the synthesized nucleotide sequence to which the solid support was attached was added to aqueous ammonia having a concentration of 25% by mass, the amount of aqueous ammonia was 0.5 ml/. Mu.mol, reacted at 55℃for 16 hours, the solvent was removed, and concentrated to dryness in vacuo. After the ammonia treatment, the product was dissolved with 0.4 ml/. Mu.mol of N-methylpyrrolidone, followed by the addition of 0.3 ml/. Mu.mol of triethylamine and 0.6 ml/. Mu.mol of triethylamine-tricofluoride, relative to the amount of single-stranded nucleic acid, and the 2' -O-TBDMS protection on ribose was removed.

Purification and desalting conditions purification of nucleic acid was accomplished by gradient elution with NaCl using a preparative ion chromatography purification column (Source 15Q). Specifically, the eluent 1 is 20mM sodium phosphate (pH=8.1), the solvent is a water/acetonitrile mixed solution (the volume ratio of water to acetonitrile is 9:1), the eluent 2 is 1.5M sodium chloride, the solvent is 20mM sodium phosphate (pH=8.1), the solvent is a water/acetonitrile mixed solution (the volume ratio of water to acetonitrile is 9:1), and the elution gradient is eluent 1, eluent 2= (100:0) - (50:50). Collecting and combining product eluents, desalting by using a reverse chromatography purification column, wherein the desalting conditions comprise desalting by using a sephadex column, eluting with deionized water, wherein the filler is sephadex G25.

Detecting, namely detecting the purity by using ion exchange chromatography (IEX-HPLC), detecting the molecular weight by using liquid chromatography-mass spectrometry (LC-MS), and comparing the measured value and the theoretical value of the molecular weight, wherein if the measured value and the theoretical value are consistent, the obtained compound is conjugated at the 3' -end of the siRNA sense strand.

(4-2) Synthesis of Antisense Strand (AS)

Antisense strands were synthesized using a universal solid support. The conditions of deprotection, coupling, capping, oxidation or sulfidation reaction conditions, cleavage and deprotection conditions, purification and desalting in the solid phase synthesis method of antisense strand are the same as those of step (4-1) for synthesizing sense strand.

Detecting, namely detecting the purity by using ion exchange chromatography (IEX-HPLC), detecting the molecular weight by using liquid chromatography-mass spectrometry (LC-MS), and comparing the measured value and the theoretical value of the molecular weight, wherein if the measured value and the theoretical value are consistent, the siRNA antisense strand is obtained.

(4-3) Synthesis of siRNA conjugates

The sense strand synthesized in step (4-1) and the antisense strand synthesized in step (4-2) were mixed in an equimolar ratio, dissolved in water for injection and heated to 95 ℃, slowly cooled to room temperature and maintained at room temperature for 10 minutes, and the sense strand and the antisense strand formed a double-stranded structure through hydrogen bonding, thereby obtaining siRNA conjugates having the sense strand and the antisense strand shown in table 3.

Wherein, the structural formula of the siRNA conjugate is as follows:

wherein, Representing siRNA. L96 is linked to the 3' end of the sense strand of siRNA by phosphodiester linkage conjugation.

The siRNA conjugates described in table 3 were modified from double stranded oligonucleotides as follows:

CCAAGAGCACCAAGAACUA (SEQ ID NO. 1) sense strand (5 '-3')

Antisense strand (5 '-3') UAGUUCUUGGUGCUCUUGGUU (SEQ ID NO. 2)

In the process of preparing an RNA sequence by sequence table preparation software, U is required to be represented by T, so that the siRNA sequence disclosed by the disclosure cannot be correctly represented in the sequence table, and all sequences are subject to the description.

TABLE 3 siRNA conjugate sequence information

Illustratively, "_l96" means that the L96 vector is linked to the 3' end of the sense strand as shown in RZ597114 by phosphodiester linkage conjugation.

Unless otherwise indicated, the base compositions and modifications described in the various embodiments of the present disclosure are defined by uppercase letters A, U, G, C, T representing the base composition of the nucleotides, lowercase letter m representing the nucleotide to the left of the letter m that is 2' -O-methyl modified (also referred to as 2' -methoxy modified), lowercase letter f representing the nucleotide to the left of the letter f that is 2' -fluoro modified, lowercase letter d representing the nucleotide to the left of the letter d that is 2' -deoxy modified ribonucleic acid (also referred to as deoxyribonucleic acid), and (moe) representing the nucleotide to the left of the combination identifier (moe) that is 2' -O-methoxyethyl modified, lowercase letter s representing the phosphorothioate linkage between the two nucleotides adjacent to the left and right of the letter s.

(NM 100) represents a nucleotide of the formula: It is obtained from the nucleoside monomer NM100 involved in the synthesis of siRNA conjugates.

(NM 101) represents a nucleotide of the formula: It is obtained from the nucleoside monomer NM101 involved in the synthesis of siRNA conjugates.

The structural formula of the 2' -methoxy modified nucleotide is。

The structural formula of the 2' -fluorine modified nucleotide is。

Wherein Z represents a hydroxyl group or a sulfhydryl group, and Base represents a Base of a nucleotide, such as A, U, G, C, T.

Table 4 siRNA molecular weight information for conjugates

Biological assay

Unless otherwise indicated, the siRNA sequences used in the present disclosure were all assigned to the Soviet Bei Xin Biotechnology Inc., PCR primer synthesis was all assigned to the Beijing engine Biotechnology Inc., and laboratory animals C57BL/6J mice were all purchased from the St Bei Fu (Beijing) Biotechnology Inc.

Method for evaluating inhibition activity of siRNA conjugate in vivo target gene of mouse

The 6-8 week old C57BL/6J mice were randomly grouped by body weight (females). The mice in each group were dosed on a single basis by abdominal subcutaneous injection, with each siRNA conjugate being formulated with PBS solution as a corresponding concentration (calculated as siRNA) solution for dosing, at a volume of 5ml (calculated as siRNA)/kg (calculated as mice). The PBS control group was given 5ml/kg (in mice) of PBS solution (without drug conjugate). Administration when the day of administration was recorded as day 0 (recorded as D0), 5 mice were sacrificed for each group at a preset time after administration. The sacrificed mice were each subjected to gross dissection and liver tissue of each sacrificed mouse was collected, and the liver tissue was cut into approximately 2mm ³ pieces and stored with RNA Later.

Taking liver tissue samples at different time points from the RNA later, crushing the liver tissue samples in Tissuelyser II type full-automatic tissue homogenizer for 60s, and extracting total RNA by using a full-automatic nucleic acid extractor (purchased from Zhejiang Han Wei technology Co., ltd.) and a nucleic acid extraction kit (purchased from Zhejiang Han Wei technology Co., ltd.) according to standard operation steps of total RNA extraction.

MRNA expression level detection:

Using the above 1. Mu.g of total RNA, a reverse transcription kit (Promega, reverse Transcription System, A3500) was used and Oligo (dT) 15 reverse transcription primer was selected, and a 20. Mu.L reverse transcription system was prepared according to the method described in the specification of the reverse transcription kit to complete the reverse transcription reaction. After completion of the reaction, 80. Mu.L of RNase-Free water was added to the reverse transcription system to obtain a cDNA solution. The amount of mRNA expression of the target gene in liver tissue was then detected using a real-time fluorescent quantitative PCR kit (ABI Co., SYBRTM SELECT MASTER Mix, catalyst number: 4472908). In the real-time fluorescent quantitative PCR method, a primer for a target gene and a primer for an internal reference gene are used to detect the target gene and the internal reference gene, respectively. 20. Mu.L Real-time PCR reaction systems were prepared for each PCR detection well according to the method described in the Real-time fluorescent quantitative PCR kit, each reaction system containing 5. Mu.L of the cDNA solution obtained by the above-mentioned reverse transcription reaction, 10. Mu. L SYBRTM SELECT MASTER Mix, 0.5. Mu.L of 10. Mu.M upstream primer, 0.5. Mu.L of 10. Mu.M downstream primer, and 4. Mu.L of RNase-Free H ₂ O. The prepared reaction system is placed on a Real-time fluorescence quantitative PCR instrument (ABI company, stepOnePlusTM) and Real-time PCR amplification is carried out by using a three-step method, wherein the amplification procedure is that the reaction is carried out for 10min at 95 ℃, then the reaction is carried out for 30s at 95 ℃, the reaction is carried out for 30s at 60 ℃ and the reaction is carried out for 30s at 72 ℃, and the denaturation, annealing and extension processes are repeated for 40 cycles. In the real-time fluorescence quantitative PCR method, the delta Ct method is adopted to perform relative quantitative calculation on the expression level and the inhibition rate of the target gene mRNA in each test group, and the calculation method is as follows:

Delta Ct (test group) =ct (test group target gene) -Ct (test group reference gene)

Delta Ct (control) =ct (control target gene) -Ct (control reference gene)

ΔΔct (test group) =Δct (test group) - Δct (control group average)

ΔΔct (control) =Δct (control) - Δct (control average)

Wherein, Δct (control group average) is the arithmetic average of Δct (control group) of each of 5 mice sacrificed at the same time point in the control group. Thus, each sample of the test and control groups corresponds to one ΔΔct value.

Test group target gene mRNA relative expression level = 2 ^-ΔΔCt( test group) ×100%

And normalizing the mRNA expression level of the target gene of the test group by taking the control group as a reference, and defining the mRNA expression level of the target gene of the control group as 100%.

Test group target gene mRNA expression inhibition rate (%) =1-test group target gene mRNA relative expression level

Unless otherwise indicated, in vivo activity assay data are all expressed as X.+ -. STDEV, and the assay data are plotted and analyzed using GRAPHPAD PRISM 8.0.0 software.

EXAMPLE 1 siRNA evaluation of the Activity of the conjugate for inhibition of the target Gene angiopoietin-like 3 (Angiopoietin-like 3, ANGPTL 3) in mice

This example uses a mouse in vivo target gene inhibition activity assessment method to evaluate the inhibition activity of ANGPTL3 target siRNA comprising antisense strand 20, conjugate RZ597151 comprising NM101 group at position 21 and control conjugate RZ597150 comprising NM100 group at positions 20, 21, and conjugate RZ597114 comprising no surrogate group on target gene ANGPTL3 in mice.

The 6-8 week old C57BL/6j mice were randomly grouped into 4 groups of 15 mice each by weight, and each group of mice was given the above siRNA conjugates by subcutaneous abdominal administration, wherein each mouse in the PBS control group was given a dose of 5 mL/kg, and the siRNA conjugate experimental group was given a dose of 3 mg/kg (in terms of siRNA) per dose of 5 mL/kg. Administration when the diary was day 0 (D0), 5 mice were sacrificed on each of the 7 th (D7), 28 th (D28) and 56 th (D56) groups after administration, animals were subjected to general dissection, liver tissue was collected, and a number of 2mm ³ pieces were cut and stored in RNA later. RNA extraction and Real-time PCR detection were as described above, the gene expression differences were calculated by the delta Ct method.

TABLE 5 primer sequence listing

The results are shown in the following table:

TABLE 6 inhibitory Activity of target genes of interest in mice following administration of siRNA conjugates described in this example

The results of example 1 show that conjugate RZ597150 comprising the NM100 group and conjugate RZ597151 comprising the NM101 group have higher inhibitory activity at D7, D28 and D56, respectively, than the control conjugate RZ597114, the antisense strand of which does not comprise any surrogate groups, while conjugate RZ597151 comprising the NM101 group has higher inhibitory activity at D56 than the conjugate RZ597150 comprising the NM100 group. (FIG. 1, table 6)

The above detailed description is illustrative of the present invention and is not meant to be limiting. Various modifications and improvements may be made by those skilled in the art without departing from the spirit and substance of the disclosure, and are also considered to be within the scope of the disclosure.

Claims

1. A double-stranded oligonucleotide comprising a sense strand and an antisense strand, each of the sense strand and the antisense strand independently having a length of 15-35 nucleotides, each of the double-stranded oligonucleotides independently selected from a modified nucleotide or an unmodified nucleotide;

Wherein X is selected from O;

Z is selected from hydroxyl or mercapto;

base is selected from nucleotide bases;

R ₁ is selected from unsubstituted C ₁-C₆ alkyl, R ₂、R₃、R₄ is selected from H;

r ₅ is selected from halogen, unsubstituted C ₁-C₆ alkyl, unsubstituted C ₁-C₆ alkoxy;

r ₆ is selected from H, halogen, unsubstituted C ₁-C₆ alkyl.

2. The double-stranded oligonucleotide of claim 1, wherein R ₁ is selected from methyl and R ₂、R₃、R₄ is selected from H;

and, the nucleotide modified by the phosphate backbone has a structure represented by formula (IIA), formula (IIA-1) or formula (IIA-2), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

、、;

Wherein X is selected from O;

base is selected from nucleobases A, U, G, C, T;

r ₅ is selected from F, methyl, ethyl, methoxy or ethoxy;

R ₆ is selected from H, F, methyl or ethyl.

3. The double-stranded oligonucleotide of claim 2, wherein the phosphoester backbone modified nucleotide is selected from any one of the following structures, or stereoisomers thereof, or tautomers thereof, or pharmaceutically acceptable salts thereof:

、、;

z is selected from hydroxyl or mercapto.

4. A double-stranded oligonucleotide according to claim 3, wherein the antisense strand of the double-stranded oligonucleotide comprises two of said phosphate backbone modified nucleotides, each of said two phosphate backbone modified nucleotides being located independently at positions 1 and 2 of the antisense strand of the double-stranded oligonucleotide, starting at the 3' end.

5. A double stranded oligonucleotide according to claim 3, further comprising a further modification selected from a 2' -O-methyl modified nucleotide, a 2' -fluoro modified nucleotide or a 2' -O-methoxyethyl modified nucleotide.

6. The double-stranded oligonucleotide of claim 3, wherein the antisense strand of the double-stranded oligonucleotide comprises a3 'overhang of 2 nucleotides, wherein the sense strand comprises two consecutive phosphorothioate linkages between the 5' end-initiated terminal nucleotides, and wherein the antisense strand comprises two consecutive phosphorothioate linkages between the 3 'end-initiated and 5' end-initiated terminal nucleotides, respectively.

7. The double-stranded oligonucleotide of any one of claims 1-6, wherein the sense strand has a length of 19 nucleotides, the antisense strand has a length of 21 nucleotides, at least four nucleotides at positions 2, 6, 9-10, 14, 16 in the nucleotide sequence of the antisense strand are selected from 2' -fluoro modified nucleotides, at least one nucleotide at positions 16-21 is selected from the phospho-backbone modified nucleotides, and the remaining position nucleotides are each independently selected from 2' -O-methyl modified nucleotides or 2' -O-methoxyethyl modified nucleotides;

8. A conjugate comprising the double stranded oligonucleotide of any one of claims 1-7 and one or more ligands capable of binding to a cell surface receptor, said ligand selected from the group consisting of asialoglycoprotein receptor ligands.

9. Use of any of the following for the manufacture of a medicament for the treatment and/or prevention of a disease or disorder associated with deregulation of the mRNA levels of target gene expression:

(I) The double-stranded oligonucleotide of any one of claims 1-7, and/or

(II) the conjugate of claim 8.

10. A pharmaceutical composition comprising any of the following pharmaceutically acceptable excipients or adjuvants:

(I) The double-stranded oligonucleotide of any one of claims 1-7, and/or

(II) the conjugate of claim 8.

11. A modified nucleoside monomer selected from any one of the following structures, or stereoisomers thereof, or tautomers thereof, or pharmaceutically acceptable salts thereof:

、、。