5 '-terminal modified 2', 5 '-connected nucleotide and application thereof in RNAi (Ribonucleic Acid Interference)

Abstract

The invention provides a 5 '-modified 2', 5 '-connected nucleotide monomer and an application of the 5'-modified 2 ', 5'-connected nucleotide monomer at a siRNA antisense strand 5 '-terminal. The 5 '-modified nucleotide monomer provided by the invention is an unconventional 2', 5 '-connected nucleotide monomer. The oligonucleotide carrying the 5 '-modified 2', 5 '-connected nucleotide monomer is a double-stranded siRNA molecule, the 5'-modified 2 ', 5'-connected nucleotide monomer is introduced to the 1 # site of the 5 '-terminal of an antisense strand of the siRNA, so that the siRNA can silence a target gene mRNA with higher efficiency and longer timeliness, and the oligonucleotide can be used for treating related diseases caused by the target gene mRNA.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to a 5'-end modified 2',5'-linked nucleotide that can be used for covalent coupling to a nucleic acid interference (RNAi) drug molecule and its application. Background Art

[0002] RNA interference (RNAi) refers to a highly conserved phenomenon in the evolutionary process, which is induced by double-stranded RNA (dsRNA) and is characterized by efficient and specific degradation of homologous mRNA (sometimes also achieved through antisense oligonucleotide interference), which can downregulate the expression of the corresponding gene and lead to post-transcriptional silencing of the corresponding gene.

[0003] From a genetic level, RNAi can treat almost all diseases, including tumors, infectious diseases, hereditary diseases, etc. Therefore, RNAi has become the hottest field of biomedical research and the most promising direction for new drug development in the future. Currently, 9 antisense oligonucleotide (ASO) drugs and 6 siRNA drugs have entered clinical application as RNAi drugs. They play a unique role in anti-infection, metabolic and hereditary disease treatment.

[0004] The siRNA response mechanism is a type of RNA interference mediated by a nuclease complex. This nuclease complex is usually called the RNA-induced silencing complex (RISC). RISC binds to the antisense strand of the siRNA, recruits the target gene mRNA, and performs complementary pairing to degrade the target gene mRNA.

[0005] The advantage of siRNA is that it can target any gene in the pathogenic genome, has strong and lasting pharmacological effects and therapeutic effects, and can significantly reduce the frequency of dosing and improve patient compliance with medication, thereby achieving better therapeutic effects and better safety.

[0006] However, siRNA therapy has also encountered unique challenges in its development. In addition to how to deliver siRNA drug molecules to the cells they act on, it is also necessary to ensure that the delivered siRNA drug molecules can exert their efficacy safely, quickly, and permanently, that is, to improve the drugability of siRNA molecules, which has always been the core concern of drug developers.

[0007] It has been proven that small nucleic acid drugs can reduce immunogenicity and off-target effects in vivo by relying on unique chemical modifications; different modification sites have different effects on silencing efficiency and metabolic stability. Some known modified nucleotide monomers, when introduced into siRNA small nucleic acid sequences, will increase the safety window and / or activity of RNAi efficacy.

[0008] On the other hand, although the current GalNAc platform can efficiently deliver siRNA to hepatocytes, only a small part of siRNA molecules escape from endosomes and are then loaded into the RISC complex in the cytoplasm to exert their pharmacological effects. A large number of siRNA molecules are actually confined in endosomes. Therefore, chemical modification of nucleoside monomers to enhance the metabolic stability of small nucleic acid molecules or improve the RISC loading efficiency of siRNA molecules after escape will undoubtedly further improve the efficacy and drugability of small nucleic acid drug molecules.

[0009] Therefore, novel modifications of small nucleic acids, especially novel chemical modifications of nucleoside monomers, are of great value in this field. Summary of the invention

[0010] In order to further improve the drugability of siRNA, the present invention modifies unconventional nucleotide monomers through novel nucleic acid chemistry and introduces them into the siRNA sequence, thereby further optimizing the drugability of siRNA and improving the therapeutic efficacy of siRNA drug molecules.

[0011] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0012] the term:

[0013] As used in the present invention, the term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, n-hexyl, etc. Alkyl can be substituted or unsubstituted, and when substituted, the substituent can be substituted at any available point of attachment, and the substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, oxy, carboxyl or carboxylate.

[0014] As used in the present invention, the term "alkenyl" refers to an alkyl group as described above, which is composed of at least two carbon atoms and at least one carbon-carbon double bond, and the substitution site must be on one of the carbon atoms of the carbon-carbon double bond. For example, vinyl, 1-propenyl, 2-propenyl, 1-, 2- or 3-butenyl, etc. Alkenyl can be substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio.

[0015] As used in the present invention, "small nucleic acid" is an oligonucleotide sequence containing about 10-50 single-stranded nucleotides or double-stranded nucleotide base pairs. In some embodiments, the small nucleic acid has a core base sequence that is at least partially complementary to the core sequence of the target gene expressed in the cell. In some embodiments, the oligonucleotide can regulate the expression of the corresponding target gene after being delivered to the cell expressing the target gene. Target gene expression can be regulated in vitro or in vivo. "Small nucleic acid" includes but is not limited to: oligonucleotides, 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.

[0016] As used herein, the terms "oligonucleotide" and "polynucleotide" refer to a polymer of linked nucleosides, wherein each nucleoside may independently be modified or unmodified.

[0017] As used herein, "RNAi drug" refers to an oligonucleotide molecule containing RNA or RNA-like (eg, chemically modified RNA) that can reduce or inhibit the translation of messenger RNA (mRNA) in a sequence-specific manner.

[0018] As used in the present invention, RNAi drugs can act through RNA interference mechanisms (e.g., by inducing mRNA degradation through interaction with the mRNA interference pathway mechanism of mammalian cells (RNA-induced silencing complex RISC)), or other arbitrary mechanisms or pathways. Although it is believed that the term RNAi drug used in the present invention mainly acts through the RNA interference mechanism, the RNAi drug is not limited to or confined to any specific pathway or mechanism of action. RNAi drugs include, but are not limited to, single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA) and dicer enzyme substrates. The RNAi drug of the present invention is composed of an oligonucleotide chain that is at least partially complementary to the mRNA as a target. In some embodiments, the RNAi drug of the present invention is double-stranded and is composed of an antisense chain and a sense chain that is at least partially complementary to the antisense chain.

[0019] As used in the present invention, the terms "silencing", "reducing", "inhibiting", "down-regulating", or "knockdown" refer to the expression of a given gene, when the RNAi drug molecules of the present invention are used to treat cells, tissues, organs or animals for direct administration, compared with cells, tissues, organs or animals that have not been treated in this way, The level of expression of the gene is reduced or downregulated.

[0020] As used herein, the term "sequence" or "nucleotide sequence" refers to a sequence of nucleotide bases, or the order, or sequence of nucleotides, expressed in alphabetical order using standard nucleotide nomenclature.

[0021] As used herein, "nucleotide base" or "nucleobase" is a heterocyclic pyrimidine or purine compound, a standard component of conventional nucleic acids, including adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) conventional nucleotide bases. Nucleobases can be modified or replaced by novel unconventional nucleobases.

[0022] As used in the present invention, the term "heterocyclic base moiety" is a novel nitrogen-containing heterocyclic base or modified nucleobase as defined in the present invention that can replace a nucleobase. In some embodiments, the heterocyclic base moiety is a pyrimidine, a substituted pyrimidine, a purine or a substituted purine, and a nitrogen-containing heterocycle. In some embodiments, the heterocyclic base moiety is an unconventional purine or a substituted purine. In some embodiments, the heterocyclic base moiety is an unconventional pyrimidine or a substituted pyrimidine. In some embodiments, the heterocyclic base moiety contains a non-natural five-membered nitrogen-containing heterocycle. In some embodiments, especially when the 5'-modified nucleotides described in the present invention are phosphoramidite compounds, the heterocyclic base moiety may include one or more protecting groups.

[0023] As used in the present invention, "ribosyl substitution group" refers to a structure that can replace the five-membered furanose ring of naturally occurring ribonucleotides, such as morpholinyl, cyclohexenyl, cyclohexanehexol, or an open ring structure.

[0024] As used in the present invention, the term "complementary" is used to describe the relationship between a first nucleotide sequence (e.g., RNAi drug sense strand or target mRNA) and a second nucleotide sequence (e.g., single-stranded antisense oligonucleotide or double-stranded RNAi drug antisense strand), and refers to the ability of an oligonucleotide or poly-oligonucleotide comprising a first nucleotide sequence to form base pairing and form a duplex or double helix structure with an oligonucleotide or poly-oligonucleotide comprising a second nucleotide sequence under certain conditions (under mammalian physiological conditions or similar in vitro conditions). Complementary sequences include Watson-Crick base pairing or non-Watson-Crick base pairing, and include natural or modified nucleotides or nucleotide analogs to at least the extent that the above pairing requirements are met. For example, for the purpose of determining identity or complementarity, monomers a and Af are complementary to U (or T) and are equivalent to A.

[0025] When the compounds described herein contain double bonds (e.g., in olefins or imines), it is intended that the compounds may contain both Z and E geometric isomers (also known as cis and trans isomers). Likewise, all tautomeric forms are intended to be included. The configuration illustrations of any bonds appearing in the present invention are selected for convenience only and are not intended to limit a particular configuration unless otherwise indicated herein.

[0026] As used in the present invention, "protecting group" refers to an unstable chemical moiety known in the art for preventing reactive groups (e.g., hydroxyl, amino, carboxyl and sulfhydryl) from reacting undesirably during synthesis. Protecting groups are typically selectively and / or orthogonally used to protect sites at other reactive sites in the reaction, and are subsequently removed to release the unprotected group so that it can be used for further reactions. In some embodiments, a "substituted" group or substituent comprises a protecting group.

[0027] The compounds and compositions described herein may have certain atoms (e.g., N, O, or S atoms) in a protonated or deprotonated state, depending on the environment in which the compound or composition is located. Thus, as used herein, the structures described herein take into account that certain functional groups, such as OH, SH, or NH, may be protonated or deprotonated. The disclosure of the present invention is intended to encompass the above-described compounds and compositions, regardless of their protonation state based on the pH of the environment, as will be readily appreciated by those of ordinary skill in the art.

[0028] As used in the present invention, the term "sense strand" refers to the strand on the RNA molecule that carries the nucleotide sequence encoding protein amino acid information, which is called the sense strand, also known as the coding strand, sense strand, or positive strand, and the other nucleotide sequence complementary to it is the antisense strand.

[0029] As used in the present invention, the term "alkoxy" refers to-O-(alkyl) or-O-(cycloalkyl), wherein the definition of alkyl is as described above. The non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy. Alkoxy can be optionally substituted or unsubstituted, and when substituted, substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate.

[0030] As used herein, the term "haloalkyl" refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.

[0031] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (ie, rings which share adjacent pairs of carbon atoms) group having a conjugated π electron system, preferably 6- to 10-membered, such as phenyl and naphthyl.

[0032] The aryl group may be substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate.

[0033] The term "hydroxy" refers to an -OH group.

[0034] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0035] The term "amino" refers to -NH2.

[0036] The term "cyano" refers to -CN.

[0037] The term "nitro" refers to -NO2.

[0038] The term "carbonyl" refers to =0.

[0039] The term "carboxy" refers to -C(O)OH.

[0040] The term "thiol" refers to -SH.

[0041] The term "ester group" refers to -C(O)O(alkyl) or -C(O)O(cycloalkyl), wherein alkyl and cycloalkyl are as defined above.

[0042] The term "acyl" refers to a compound containing a -C(O)R group, wherein R is alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl.

[0043] The term "optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs or does not occur. For example, "a heterocyclic group optionally substituted with an alkyl group" means that the alkyl group may but need not be present, and the description includes instances where the heterocyclic group is substituted with an alkyl group and instances where the heterocyclic group is not substituted with an alkyl group.

[0044] The term "substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms in a group are replaced independently of each other by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, which the skilled person can determine by means of the prior art and experimental conditions (by experiment or theory) as possible or impossible substitutions. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom with an unsaturated (e.g. olefinic) bond.

[0045] The term "pharmaceutical composition" refers to a mixture containing one or more compounds described herein or their pharmaceutically acceptable salts or prodrugs under physiological conditions and other chemical components, as well as other components such as pharmaceutically acceptable carriers and excipients under physiological conditions. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredients, and thus exert biological activity.

[0046] In one aspect, the present invention provides a compound having a structure of Formula I:

[0047]

[0048] in:

[0049] X 1 =O or S;

[0050] Sug is a ribosyl group or a ribosyl substituted group;

[0051] PG is an alkyl group and an alkyl group derived therefrom, or an aryl group and an aryl group derived therefrom, etc.;

[0052] PA is a phosphate moiety, a phosphorothioate moiety, or a phosphoramidite moiety derived from an RNAi drug;

[0053] R 1 , R 2 may be independently selected to be H, F, or methyl;

[0054] Bx is a heterocyclic base, preferably an unconventional base.

[0055] Based on the general formula I, the compound has the structure of the general formula II or the general formula III:

[0056]

[0057] or its meso, racemate, enantiomer, diastereoisomer or the like;

[0058] in:

[0059] X 1 =O or S;

[0060] X 2 =O or S or chemical bond or C 1-4 Alkylene;

[0061] PG is an alkyl group and an alkyl group derived therefrom, or an aryl group and an aryl group derived therefrom, etc.;

[0062] Y1 is optionally H, OH, F, OCH3, OCH2OCH2CH3, OCH2SCH3, OCH2CH2CONHR (R is H, or C1-C3 alkyl), etc., preferably H;

[0063] Y2 is optionally H, OH, F, OCH3, OCH2OCH2CH3, OCH2SCH3, OCH2CH2CONHR (R is H, or C1-C3 alkyl), etc., preferably H;

[0064] PA is a phosphate moiety, a phosphorothioate moiety, or a phosphoramidite moiety derived from an RNAi drug;

[0065] R 1 , R 2 can be independently selected as H, F, or methyl, preferably H;

[0066] Bx is a heterocyclic base.

[0067] Preferably, the heterocyclic base is modified or unmodified uracil (U), thymine (T), adenine (A), guanine (G) and cytosine (C), more preferably uracil (U).

[0068] Preferably, PA in the general formula is phosphoramidite, the compound is a phosphoramidite nucleotide monomer, and the compound has one of the following structures:

[0069]

[0070] In the compounds, Y1 and / or Y2 are as defined above and below.

[0071] The compound can be selected from the following structures:

[0072]

[0073] where Base is as defined by the context.

[0074] In yet another aspect, the present invention provides an RNAi molecule having the aforementioned modified nucleotide monomer at the 5'-end.

[0075] Preferably, the modified nucleotide monomer is a 5'-modified nucleotide.

[0076] Furthermore, the modification point of the 5'-modified nucleotide is located at the group portion on the 5'-carbon of the nucleotide sugar group (or at the corresponding position of the ribose replacement group).

[0077] Preferably, the modified nucleotide monomer can be connected to the antisense strand of the double-stranded RNAi molecule; preferably, connected to the 1st position at the 5'-end of the antisense strand of the double-stranded RNAi molecule to improve the drugability of siRNA.

[0078] Preferably, the RNAi molecule is selected from the following general formula III:

[0079]

[0080] siRNA is a double-stranded small interfering RNA molecule with the following structure:

[0081] Sense strand: 5′-NNNNNNNN…NNNNNNNN-3′;

[0082] Antisense strand: 3′-NNNNNNNN…NNNNNNNN1-5′;

[0083] N in the double-stranded structure of siRNA represents a nucleotide monomer; the 5'-modified nucleotide monomer involved in the present invention is covalently coupled to the N1 position at the 5'-end of the antisense strand; that is, the new nucleotide monomer is connected to the 5'-end of the siRNA antisense strand;

[0084] siRNA structure diagram is shown in Figure 4 , the yellow end position is the 5'-end of the siRNA antisense strand, that is, the location of the 5'-modified nucleotide monomer described in the present invention.

[0085] X 1 =O or S;

[0086] X 2 =O or S or chemical bond or C 1-4 Alkylene;

[0087] Y1 is optionally H, OH, F, OCH3, OCH2OCH2CH3, OCH2SCH3, OCH2CH2CONHR (R is H, or C1-C3 alkyl), etc., preferably H;

[0088] Y2 is optionally H, OH, F, OCH3, OCH2OCH2CH3, OCH2SCH3, OCH2CH2CONHR (R is H, or C1-C3 alkyl), etc., preferably H;

[0089] The H atoms of sp3 CH or sp2 CH in the general formula can be arbitrarily replaced by D or F atoms.

[0090] The 5'-modified nucleotide monomers of the present invention can enhance the resistance of the 5'-end of the antisense chain in the siRNA molecule to nuclease degradation. In addition, since the phosphorylation state of the 5'-end of the antisense chain of the RNAi drug directly affects its RISC loading efficiency, directly introducing a stable phosphonic acid group on the nucleotide at the 5'-end of the antisense chain can significantly improve the RISC loading efficiency of the antisense chain, enhance the silencing efficiency of the target gene and the timeliness of interfering with the RNAi molecule.

[0091] In another aspect, the present invention provides use of the aforementioned compound or RNAi molecule in the preparation of RNAi drugs.

[0092] The RNAi drug includes the aforementioned RNAi molecule or is prepared by using the aforementioned compound.

[0093] In some expressions, the RNAi drug of the present invention is also referred to as an RNAi initiator.

[0094] The compound is prepared as RNAi drug by modifying siRNA.

[0095] The siRNA may be known or unknown in the art.

[0096] The RNAi drug molecules comprising 5'-modified nucleotides of the present invention can also be conjugated with a GalNAc carrier or a peptide ligand.

[0097] The RNAi drug molecules comprising 5'-modified nucleotides of the present invention can also be covalently coupled with polyethylene glycol (PEG) or lipids (such as cholesterol).

[0098] The RNAi drug molecules comprising 5'-modified nucleotides of the present invention can also be delivered to the effected cells via lipid nanoparticles (LNP) or polypeptide nanoparticles (PNP) to produce corresponding pharmacological effects.

[0099] The present invention also provides a conjugate, a pharmaceutical composition, a cell or a kit comprising the aforementioned RNAi molecule.

[0100] The present invention also provides RNAi drugs comprising the aforementioned RNAi molecules or prepared by the aforementioned compounds.

[0101] In some embodiments, at least one of the RNAi drugs comprising 5'-modified nucleotides is used to prepare therapeutic pharmaceutical compositions (i.e., drugs) for subjects that would benefit from reduction or inhibition of gene expression. These pharmaceutical compositions are used to inhibit gene expression in cells, tissues, or organisms.

[0102] In the case of disease treatment or drug formulation or a composition for treating a disease, an expression inhibitory oligonucleic acid such as an RNAi drug containing one or more 5'-modified nucleotides.

[0103] As discussed elsewhere herein, for example, ribosyl substituted moieties can be used in conjunction with phosphonic acid modifications of 5'-modified nucleotides to change the five-membered furanose ring to a different structure that can replace the five-membered furanose ring, such as morpholinyl, cyclohexenyl, cyclohexane hexol, or an open ring structure, etc. Such changes fall within the scope of the invention described herein.

[0104] RNAi drugs, targeting ligands, and oligonucleotide delivery

[0105] Oligonucleic acids (eg, RNAi drugs) having a sequence at least partially complementary to a target nucleic acid have been shown to alter the function and activity of the target nucleic acid both in vitro and in vivo.

[0106] In some embodiments, the RNAi drug comprising the 5'-modified nucleotides of the present invention is double-stranded. In the case of double-stranded RNAi drugs, the length of the sense and antisense strands of the RNAi drug is independently 16-30 nucleotides. In some embodiments, the double-stranded RNAi drug comprises a sense strand and an antisense strand that are at least partially complementary to each other (at least 70% complementary). The antisense strand comprises a region having a sequence that is perfectly complementary (100% complementary) or at least partially complementary (at least 85% complementary) to a sequence in the target mRNA. The sense and antisense strands can be the same length or different lengths.

[0107] In some embodiments, the length of the sense strand is about 19 nucleotides, and the length of the antisense strand is about 21 nucleotides. In some embodiments, the length of the sense strand is about 21 nucleotides, and the length of the antisense strand is about 23 nucleotides. The perfect or partially complementary region between the sense strand and the antisense strand is generally a length of 15-25 nucleotides, and is located at the 5'-end of the antisense strand or near (such as 1,2,3 or 4 nucleotides from the 5'-end of the antisense strand in this region, which is imperfect or partially complementary). In some embodiments, the 5'-modified nucleotides are the terminal nucleotides on the 5'-end of the double-stranded RNAi drug antisense strand.

[0108] In some embodiments, the 5'-modified nucleotide is connected to the end of the RNAi drug and improves the RISC loading efficiency, thereby improving the drugability of the RNAi drug. In some embodiments, the RNAi drug comprising a 5'-modified nucleotide at the end of the present invention can inhibit or knock down the expression of the target gene through the biological process of RNA interference (RNAi) in vitro or in vivo by being delivered to cells.

[0109] For double-stranded RNAi drugs, if there are other sense strand nucleotides, they can be identical or different from the corresponding sequence in the target mRNA. If there are other antisense strand nucleotides, they can be complementary or non-complementary to other nucleotides (if present) of the corresponding sense strand.

[0110] In some embodiments, the sense strand and antisense strand of the double-stranded RNAi drug comprising terminal 5'-modified nucleotides of the present invention contain the same number of nucleotides. In some embodiments, the sense strand and antisense strand of the RNAi drug of the present invention contain different numbers of nucleotides.

[0111] In some embodiments, both the sense and antisense strands contain 1 to 4 phosphorothioate linkages.

[0112] The RNAi drug of the present invention is synthesized by a method commonly used in the art. Double-stranded RNAi drugs can be formed by annealing the antisense strand and the sense strand together.

[0113] In some embodiments, the RNAi drug comprising 5'-modified nucleotides inhibits the expression of target mRNA in cells, tissues or in vivo. In some embodiments, a therapeutically effective dose of the RNAi drug comprising 5'-modified nucleotides of the present invention is administered into the body, thereby inhibiting the expression of target mRNA in the body.

[0114] In some embodiments, the RNAi drug is used to treat, prevent or control clinical symptoms associated with the expression of target mRNA. In some embodiments, a therapeutically or prophylactically effective dose of one or more RNAi drugs is administered to a subject in need of such treatment, prevention or control.

[0115] The RNAi drug comprising 5'-modified 2',5' linked nucleotides of the present invention and the composition comprising the RNAi drug of the present invention can be delivered to cells, tissues, or related organs using oligonucleotide delivery techniques known in the art. In general, any method suitable for delivering nucleic acid molecules (in vitro or in vivo) in the art can be applied to the RNAi drug comprising one or more 5'-modified 2',5' linked nucleotides of the present invention.

[0116] In some embodiments where the RNAi drug is double-stranded, a targeting ligand or targeting group, a linking group or a delivery vehicle is covalently linked to the sense strand. In some embodiments, a targeting ligand, a linking group and / or a delivery vehicle is covalently linked directly or indirectly to the 3'- or 5'-end of the sense strand via a linker.

[0117] In some embodiments, the targeting ligand or targeting group contains two to four terminal N-acetylgalactosamines (GalNAc) linked to an RNAi drug comprising one or more 5'-modified nucleotides. It is known that triantennary (GalNAc) binds to ASGPR with greater affinity than diantennary or monoantennary structures (Connolly et al., J. Biol. Chem., 1982, 257, 939-945).

[0118] The present invention relates to a class of modified novel nucleotides, which can provide RNAi drug molecules with improved RISC loading efficiency, increased metabolic stability and enhanced silencing efficacy on target gene mRNA, while also extending the timeliness of RNAi drug molecules, thereby significantly improving the drugability of RNAi drug molecules (such as siRNA). BRIEF DESCRIPTION OF THE DRAWINGS

[0119] Figure 1 The process flow for the synthesis of double-stranded nucleic acid sequences.

[0120] Figure 2 Schematic diagram of solid phase synthesis.

[0121] Figure 3 The relative changes in target gene expression between the siRNA in vivo silencing efficiency test sample and the control sample.

[0122] Figure 4 Schematic diagram of siRNA structure. The yellow end is the 5'-end of the siRNA antisense strand, which is the location of the modified nucleotide monomer described in the present invention. DETAILED DESCRIPTION

[0123] Further understand the compounds of the present invention and their preparation by examples, which illustrate some methods of preparing or using the compounds. However, it is to be understood that these examples are not limited to the present invention. Now known or further developed content of the present invention is considered to fall within the scope of the invention described herein and claimed.

[0124] The compounds of the present invention are prepared using convenient starting materials and general experimental methods and steps. The present invention provides typical or preferred reaction conditions, such as reaction temperature, time, solvent, pressure, and molar ratio of reactants. However, unless otherwise specified, other reaction conditions can also be adopted. The optimized conditions may change with the use of specific reactants or solvents, but in general, the reaction optimized steps and conditions can be determined.

[0125] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0126] The instruments, equipment and reagents used in the examples can be obtained through various channels, such as commercial purchase or laboratory preparation.

[0127] In addition, some protecting groups may be used in the present invention to protect certain functional groups from unnecessary reactions. Protecting groups suitable for various functional groups and their protection or deprotection conditions are widely known to those skilled in the art. For example, TW Greene and GM Wuts's "Protective Groups in Organic Preparations" (3rd edition, Wiley, New York, 1999 and references therein) describes in detail the protection or deprotection of a large number of protecting groups.

[0128] The separation and purification of compounds and intermediates can be carried out by appropriate methods and steps according to specific needs, such as filtration, extraction, distillation, crystallization, column chromatography, preparative thin layer plate chromatography, preparative high performance liquid chromatography, or a combination of the above methods. The specific use method can refer to the examples described in the present invention. Of course, other similar separation and purification means can also be adopted. Conventional methods (including physical constants and spectral data) can be used to characterize them.

[0129] The structures of the compounds were determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). -6 The unit of (ppm) is given. NMR measurements were performed using an Agent Pro Pulse 500 MHz nuclear magnetic resonance spectrometer, with deuterated dimethyl sulfoxide (DMSO-d6) and deuterated chloroform (CDCl3) as the measuring solvent and tetramethylsilane (TMS) as the internal standard.

[0130] Thermo Ultimate 3000-ISQ (manufacturer: Thermo) was used for MS measurement.

[0131] The high performance liquid chromatograph used was Thermo Ultimate 3000 (analytical column: DNApac RP, 4um 3.0×100mm) (manufacturer: Thermo)

[0132] The preparative liquid phase was prepared using LC-100 high performance liquid chromatography (preparative column: PS-15Q, 10×250 mm) (manufacturer: Jiangsu Zhirun Technology)

[0133] The nucleic acid synthesizer used was Dr.Oligo 48 (manufacturer: BIOLYTIC)

[0134] The thin layer chromatography silica gel plate used was Qingdao Ocean Chemical GF254 silica gel plate. The silica gel plate used in thin layer chromatography (TLC) had a specification of 0.15 mm to 0.2 mm, and the specification used for thin layer chromatography separation and purification products was 0.4 mm to 0.5 mm.

[0135] Column chromatography generally uses Qingdao Marine Silica Gel 100-200 mesh and 200-300 mesh silica gel as the carrier.

[0136] The known starting materials of the present invention can be synthesized by methods known in the art, or can be purchased from online shopping malls, Beijing Coupling, Sigma, J&K, Shanghai Aladdin, Shanghai Bid, and other companies.

[0137] Unless otherwise specified in the examples, the reactions can be carried out under an argon atmosphere or a nitrogen atmosphere.

[0138] Argon atmosphere or nitrogen atmosphere means that the reaction bottle is connected to an argon or nitrogen balloon with a capacity of about 1L.

[0139] Reaction solvent, organic solvent or inert solvent are each expressed as the solvent used that does not participate in the reaction under the described reaction conditions, including, for example, benzene, toluene, acetonitrile, tetrahydrofuran (THF), dimethylformamide (DMF), chloroform, dichloromethane, ether, methanol, N-methylpyrrolidone (NMP), pyridine, etc. Unless otherwise specified in the examples, the solution refers to an aqueous solution.

[0140] The chemical reactions described in the present invention are generally carried out under normal pressure. The reaction temperature is between -78°C and 200°C. The reaction time and conditions are, for example, between -78°C and 200°C at one atmosphere, and are completed within about 1 to 24 hours. If the reaction is left overnight, the reaction time is generally 16 hours. In the embodiments, unless otherwise specified, the reaction temperature is room temperature, which is 20°C to 30°C.

[0141] The reaction progress in the embodiment is monitored by thin layer chromatography (TLC), and the developing solvent systems used in the reaction are: A: dichloromethane and methanol system, B: n-hexane and ethyl acetate system, C: petroleum ether and ethyl acetate system, D: acetone, and the volume ratio of the solvent is adjusted according to the polarity of the compound.

[0142] The eluent system of column chromatography and the developing solvent system of thin layer chromatography used for purifying compounds include: A: dichloromethane and methanol system, B: n-hexane and ethyl acetate system, C: petroleum ether and ethyl acetate system. The volume ratio of the solvent is adjusted according to the polarity of the compound, and a small amount of alkaline or acidic reagents such as triethylamine and acetic acid can also be added for adjustment.

[0143] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equivalent to the described content can be used in the method of the present invention.

[0144] Abbreviations

[0145] NMR = Nuclear Magnetic Resonance

[0146] d = double peak

[0147] δ = chemical shift

[0148] ℃ = degrees Celsius

[0149] dd = double double peak

[0150] ddd=double double double peaks

[0151] DMAP=N,N-dimethylaminopyridine

[0152] DMP = Dess-Martin periodinane

[0153] DMSO = dimethyl sulfoxide

[0154] DMSO-d6 = deuterated dimethyl sulfoxide

[0155] DCM = dichloromethane

[0156] MeOH = methanol

[0157] EA = Ethyl acetate

[0158] EDCI = 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride

[0159] ETT = 5-ethylthiotetrazolyl

[0160] ESI = Electrospray Ionization

[0161] g = grams

[0162] HPLC = High Performance Liquid Chromatography

[0163] h = hours

[0164] Hz = Hertz

[0165] J = coupling constant (Hz)

[0166] LCMS = Liquid Chromatography-Mass Spectrometry

[0167] m = multiplet

[0168] [M+H] + = mass of the parent compound + one proton (positive charge)

[0169] mg = milligrams

[0170] min = minutes

[0171] mL = milliliters

[0172] mm = millimeters

[0173] mmol = millimole

[0174] MS = Mass Spectrometry

[0175] m / z = mass-to-charge ratio

[0176] nM = nanomoles per liter

[0177] PE = Petroleum Ether

[0178] ppm = parts per million

[0179] RP-HPLC = Reverse Phase High Performance Liquid Chromatography

[0180] s = single peak

[0181] t = triplet

[0182] TEA = triethylamine

[0183] TFA = trifluoroacetic acid

[0184] THF = Tetrahydrofuran

[0185] VP = Vinyl Phosphate

[0186] PN = phosphoramidite

[0187] Example 1: Preparation of [((((E)-2-((2S,4R,5R)-4-(((2-cyanoethoxy)(diisopropylamino)phosphinoyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1-(2H)-yl)tetrahydrofuran-2-yl)vinyl)phosphoryl)bis(oxy))bis(methylene)bis(2,2-dimethylpropionate)] (3'-deoxy-U-VP-PN, this monomer is referred to as d3UVP in the sequence table)

[0188]

[0189] The specific synthesis method is as follows:

[0190] Step 1: Preparation of 3'-deoxy-U2:

[0191] The starting material 3'-deoxy-U1 was synthesized according to the literature (Synthesis, 2000, 3, 399-402): 1 HNMR(500MHz,Chloroform-d)δ7.88(d,J=8.1Hz,1H),5.69(d,J=8.1Hz,1H),5.63(d,J=1.8Hz,1H),4.52(ddt,J=8.8,5.6,2.7Hz,1H),4.47(d t,J=4.6,2.2Hz,1H),4.10(d,J=12.1Hz,1H),3.75(d,J=12.0Hz,1H),2.11(ddd,J=12.9,9.7,5.2Hz,1H),1.83(ddd,J=13.0,5.8,2.4Hz,2H).

[0192] At 0°C, add 3'-deoxy-U1 (1 g, 2.9 mmol) and Dess-Martin periodinane (1.61 g, 3.9 mmol) to a reaction bottle containing 40 mL of anhydrous dichloromethane, seal, replace with argon three times, stir at 0°C for 1 hour; move to room temperature and stir for 1 hour. The reaction solution is quenched with saturated sodium bicarbonate solution and saturated sodium thiosulfate solution (V / V=1:1, 80 mL), and extracted with ethyl acetate (100 mL). The organic phase is washed with a mixed solution of saturated sodium bicarbonate solution and saturated sodium thiosulfate (V / V=1:1, 100 mL×3), dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated under reduced pressure to obtain a crude product 3'-deoxy-U2 (1 g, 2.9 mmol, white solid, yield: 100%), which is directly used in the next step.

[0193] LC-MS (ESI): m / z 341.16 [M+H] + .

[0194] Step 2: Preparation of 3'-deoxy-U3:

[0195] In an argon atmosphere at -30°C, sodium hydride (350 mg, 60% (content in mineral oil), 8.7 mmol) and tetrakis-(neopentyloxy)-methylmethylene diphosphate (4.5 g, 7.25 mmol) were added to a two-necked reaction bottle containing 10 mL of anhydrous tetrahydrofuran. Stir at -30°C for 15 minutes, and slowly drop 10 mL of anhydrous tetrahydrofuran solution containing 3'-deoxy-U2 (1 g, 2.9 mmol) into the above reaction solution. After the addition was completed (about 30 minutes), stir at -30°C for 1 hour, move to an ice-water bath and stir for 2 hours. Saturated ammonium chloride solution (50 mL) was added to the reaction solution to quench, and the resulting mixed solution was extracted with ethyl acetate (100 mL×3), and the organic phases were combined. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by flash silica gel column chromatography (EA / PE=1 / 3), and the target product eluate was collected and concentrated under reduced pressure to give 3'-deoxy-U3 (1.5 g, 2.3 mmol, white solid, two-step yield 80%, E isomer / Z isomer is about 9 / 1)

[0196] LC-MS (ESI): m / z 647.28 [M+H] + .

[0197] Step 3: Preparation of 3'-deoxy-U-VP:

[0198] At room temperature, 3'-deoxy-U3 (1.5 g, 2.3 mmol) was added to a reaction flask containing (50 mL) formic acid / water mixture (V / V = 1:1). Stir at room temperature for 24 hours. Dilute with water (50 mL) and extract with ethyl acetate (100 mL). The organic phase was washed with saturated sodium bicarbonate (50 mL × 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was separated into E / Z isomers by reverse phase preparative high pressure liquid chromatography (RP-HPLC, phase A: 10 mM ammonium acetate; phase B: acetonitrile), and the target product eluate was collected and concentrated to obtain the E-type isomer: 3'-deoxy-U-VP (850 mg, 1.6 mmol, white solid, yield: 69%).

[0199] LC-MS (ESI): m / z 533.20 [M+H] + .

[0200] Step 4: Preparation of 3'-deoxy-U-VP-PN:

[0201] Under argon protection, 3'-deoxy-U-VP (590 mg, 1.1 mmol) was co-rotated with acetonitrile three times. At 0°C, 10 mL of anhydrous dichloromethane, 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (1.0 mL, 3.3 mmol), and 5-ethylthiotetrazolyl (205 mg, 1.6 mmol) were added to the reaction bottle. The mixture was slowly heated to room temperature and stirred for 3 hours, and then directly spun dry. The crude product was purified by silica gel column chromatography (EA / PE=1 / 1), and the eluent of the target product was collected and concentrated under reduced pressure to obtain 3'-deoxy-U-VP-PN (650 mg, 0.89 mmol, colorless oily liquid, yield: 80%).

[0202] LC-MS (ESI): m / z 733.32 [M+H] + .

[0203] Example 2: Preparation of [((((E)-2-((2S,4R,5R)-4-(((2-cyanoethoxy)(diisopropylamino)phosphinoyl)oxy)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1-(2H)-yl)tetrahydrofuran-2-yl)vinyl)phosphoryl)bis(oxy))bis(methylene)bis(2,2-dimethylpropionate)] (3'-deoxy-T-VP-PN, this monomer is referred to as d3TVP in the sequence table)

[0204]

[0205] The specific synthesis method is as follows:

[0206] Step 1: Preparation of 3'-deoxy-T2:

[0207] The starting material 3'-deoxy-T1 was synthesized according to the literature (Synthesis, 2000, 3, 399-402).

[0208] At 0°C, add 3'-deoxy-T1 (1 g, 2.8 mmol) and Dess-Martin periodinane (1.61 g, 3.9 mmol) to a reaction bottle containing 40 mL of anhydrous dichloromethane, seal, replace with argon three times, stir at 0°C for 1 hour; move to room temperature and stir for 1 hour. The reaction solution was quenched with saturated sodium bicarbonate solution and saturated sodium thiosulfate solution (V / V=1:1, 80 mL), and extracted with ethyl acetate (100 mL). The organic phase was washed with a mixed solution of saturated sodium bicarbonate solution and saturated sodium thiosulfate (V / V=1:1, 100 mL×3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product 3'-deoxy-T2 (0.9 g, 2.5 mmol, white solid, yield: 90%), which was directly used in the next step.

[0209] LC-MS (ESI): m / z 355.20 [M+H] + .

[0210] Step 2: Preparation of 3'-deoxy-T3:

[0211] In an argon atmosphere at -30°C, sodium hydride (350 mg, 60% (content in mineral oil), 8.7 mmol) and tetrakis-(neopentyloxy)-methylmethylene diphosphate (4.5 g, 7.25 mmol) were added to a two-necked reaction bottle containing 10 mL of anhydrous tetrahydrofuran. Stir at -30°C for 15 minutes, and slowly drop 10 mL of anhydrous tetrahydrofuran solution containing 3'-deoxy-T2 (1 g, 2.8 mmol) into the above reaction solution. After the addition was completed (about 30 minutes), stir at -30°C for 1 hour, move to an ice-water bath and stir for 2 hours. Saturated ammonium chloride solution (50 mL) was added to the reaction solution to quench, and the resulting mixed solution was extracted with ethyl acetate (100 mL×3), and the organic phases were combined. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by flash silica gel column chromatography (EA / PE=1 / 3), and the target product eluate was collected and concentrated under reduced pressure to give 3'-deoxy-T3 (1.5 g, 2.27 mmol, white solid, yield 81%, E isomer / Z isomer is about 9 / 1)

[0212] LC-MS (ESI): m / z 661.32 [M+H] + .

[0213] Step 3: Preparation of 3'-deoxy-T-VP:

[0214] At room temperature, 3'-deoxy-T3 (1.5 g, 2.27 mmol) was added to a reaction flask containing (40 mL) formic acid / water mixture (V / V = 1:1). Stir at room temperature for 24 hours. Water (50 mL) was added to dilute and extracted with ethyl acetate (100 mL). The organic phase was washed with saturated sodium bicarbonate (50 mL × 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (EA / PE = 1 / 2), concentrated, and separated by reverse phase preparative high pressure liquid chromatography (RP-HPLC, phase A: 10 mM ammonium acetate; phase B: acetonitrile) to separate the E / Z isomers. The target product eluate was collected and concentrated to obtain the E-type isomer: 3'-deoxy-T-VP (710 mg, 1.3 mmol, white solid, yield: 57%).

[0215] LC-MS (ESI): m / z 546.26 [M+H] + .

[0216] Step 4: Preparation of 3'-deoxy-T-VP-PN:

[0217] Under argon protection, 3'-deoxy-T-VP (370 mg, 0.7 mmol) was co-rotated with acetonitrile three times. At 0°C, 10 mL of anhydrous dichloromethane, 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (306 mg, 1.0 mmol), and 5-ethylthiotetrazolyl (90 mg, 0.7 mmol) were added to the reaction bottle. The mixture was slowly heated to room temperature and stirred for 3 hours, and then directly spun dry. The crude product was purified by silica gel column chromatography (EA / PE=1 / 1), and the eluent of the target product was collected and concentrated under reduced pressure to obtain 3'-deoxy-T-VP-PN (381 mg, 0.51 mmol, white solid, yield: 73%).

[0218] LC-MS (ESI): m / z 746.32 [M+H] + .

[0219] Example 3: Preparation of [((((E)-2-((2S,4R,5R)-4-(((2-cyanoethoxy)(diisopropylamino)phosphinoyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1-(2H)-yl)-3-methoxytetrahydrofuran-2-yl)vinyl)phosphoryl)bis(oxy))bis(methylene)bis(2,2-dimethylpropionate)] (3'-OMe-U-VP-PN, this monomer is referred to as R3MUVP in the sequence table)

[0220]

[0221] The specific synthesis method is as follows:

[0222] Step 1: Preparation of 3'-OMe-U2:

[0223] The starting material 3'-OMe-U1 was synthesized according to the literature (Heterocycles, 2009, 77, 217-225).

[0224] At room temperature, 3'-OMe-U1 (1.48 g, 4 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.86 g, 15 mmol) were added to the reaction flask, and the nitrogen was replaced after vacuuming for 1 hour. 25 mL of anhydrous dimethyl sulfoxide was added to the reaction flask to dissolve, and anhydrous pyridine (0.3 mL, 4 mmol) and trifluoroacetic acid (0.2 mL, 2.5 mmol) were added in sequence, and stirred overnight. After the reaction was completed by LCMS, a solution containing 3'-OMe-U2 was obtained, which was directly used for the next step reaction.

[0225] LC-MS (ESI): m / z 371.25 [M+H] + .

[0226] Step 2: Preparation of 3'-OMe-U3:

[0227] In an argon atmosphere at -30°C, sodium hydride (1.2 mg, 60% (content in mineral oil), 30 mmol) and tetrakis-(neopentyloxy)-methylmethylene diphosphate (7.8 g, 12.4 mmol) were added to a two-necked reaction bottle containing 70 mL of anhydrous tetrahydrofuran. Stir at -30°C for 15 minutes, and the solution containing 3'-deoxy-U2 (1.48 g, 4 mmol) was diluted with 30 mL of anhydrous tetrahydrofuran and slowly added dropwise to the above reaction solution. After the addition was completed (about 30 minutes), stir at -30°C for 1 hour, move to an ice-water bath and stir for 2 hours. Saturated ammonium chloride solution (50 mL) was added to the reaction solution to quench, and the resulting mixed solution was extracted with ethyl acetate (100 mL×3), and the organic phases were combined. The organic phase was washed with saturated ammonium chloride solution (100 mL×3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by flash silica gel column chromatography (EA / PE=1 / 2), and the target product eluate was collected and concentrated under reduced pressure to give 3'-OMe-U3 (1.08 g, 1.6 mmol, white solid, two-step yield 40%, E isomer / Z isomer is about 9 / 1)

[0228] LC-MS (ESI): m / z 677.28 [M+H] + .

[0229] Step 3: Preparation of 3'-OMe-U-VP:

[0230] At room temperature, 3'-OMe-U3 (1.0 g, 1.6 mmol) was added to a reaction flask containing (60 mL) formic acid / water mixture (V / V = 1:1). Stir at room temperature for 24 hours. Add water (50 mL) to dilute, and extract with ethyl acetate (100 mL). The organic phase was washed with saturated sodium bicarbonate (50 mL × 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (MeOH in DCM = 2%), concentrated, and separated by reverse phase preparative high pressure liquid chromatography (RP-HPLC, phase A: 10 mM ammonium acetate; phase B: acetonitrile) to separate the E / Z isomers. The target product eluate was collected and concentrated to obtain the E-type isomer: 3'-OMe-U-VP (600 mg, 1.07 mmol, white solid, yield: 67%).

[0231] LC-MS (ESI): m / z 563.20 [M+H] + .

[0232] Step 4: Preparation of 3'-OMe-U-VP-PN:

[0233] Under argon protection, 3'-OMe-U-VP (200 mg, 0.37 mmol) was co-rotated with acetonitrile three times. At 0°C, 10 mL of anhydrous dichloromethane, 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (0.12 mL, 1.1 mmol), and 5-ethylthiotetrazolyl (50 mg, 0.37 mmol) were added to the reaction bottle. The mixture was slowly heated to room temperature and stirred for 3 hours, and then directly spun dry. The crude product was purified by silica gel column chromatography (EA / PE=1 / 1), and the eluent of the target product was collected and concentrated under reduced pressure to obtain 3'-OMe-U-VP-PN (160 mg, 0.21 mmol, colorless oily liquid, yield: 56%).

[0234] LC-MS (ESI): m / z 763.30 [M+H] + .

[0235] Example 4: Synthesis of double-stranded nucleic acid sequences

[0236] See the process flow Figure 1 The process is described as follows:

[0237] Oligo synthesis starts with Universal-CPG, and uses 3'-O-(2-cyanoethyl)phosphoramidite / 4,4'dimethoxytrityl (DMT) group protection method to assemble oligonucleotide chains on a solid support (controlled pore glass (CPG). According to the sequence information, the corresponding crude oligonucleotide single-stranded CPG-Oligo is obtained by cyclic synthesis. The crude oligonucleotide single-stranded is cleaved from the solid support and the relevant protecting groups are removed. Then, the final product is obtained through preparative chromatography purification, ultrafiltration desalting, annealing, and freeze-drying steps. See Figure 2 .

[0238] (1) Synthesis procedure

[0239] The following units are included:

[0240] 1) De-blocking: The 5'-OH end of the ribonucleotide is protected by a DMT group (dimethoxytrityl). In the first step of the synthesis, trichloroacetic acid (TCA) is used to remove the DMT protecting group from the solid phase support and the ribonucleotide, so that the 5'-OH of the naked ribonucleotide can be coupled with a new base.

[0241] 2) Coupling: Nucleotide monomers are mixed with activation reagents and react with CPG-Oligo in the synthesis column. The activation reagent provides a proton to the N atom of diisopropylamide on the 3'-phosphoric acid to form a phosphoramidite tetrazole active intermediate. When phosphoramidite tetrazole comes into contact with CPG-Oligo, it undergoes a nucleophilic reaction with its 5'-hydroxyl group, coupling occurs and tetrazole is removed, extending a nucleotide.

[0242] 3) Capping: Since the coupling efficiency cannot reach 100%, in order to prevent the uncoupled CPG-Oligo from continuing to the next step of coupling, the CPG-Oligo 5'-hydroxyl group is capped with a Cap reagent.

[0243] 4) Oxidation or thiolation: After the coupling reaction, the nucleotide is connected to the oligonucleotide on the CPG through a phosphite bond (trivalent phosphorus). This phosphite bond is unstable and easily hydrolyzed by acid and alkali. The trivalent phosphorus here is oxidized to pentavalent phosphorus by an oxidizing agent. Thiolation refers to the reaction of the trivalent phosphorus of the phosphite bond to form a phosphorus-sulfur bond under weak alkaline conditions by a thiolation agent.

[0244] 5) A series of syntheses were performed according to standard oligo solid phase synthesis. The synthesis sequence is as follows:

[0245]

[0246] The expression of nucleoside monomer modification is as follows:

[0247] a represents 2'-O-methyladenine, u represents 2'-O-methyluridine, g represents 2'-O-methylguanosine, c represents 2'-O-methylcytosine, Af represents 2'-fluoroadenine, Uf represents 2'-fluorouridine, Gf represents 2'-fluoroguanosine, Cf represents 2'-fluorocytosine, UVP represents 2'-O-methyluridine-5'-olefin phosphate (reference patents WO2011139702A2 and WO2013033230A1), d3UVP and d3TVP are as defined above. s represents connection with a phosphorothioate bond, and there is no symbol between directly adjacent nucleotides.

[0248] The raw materials (phosphoramidite monomers) and auxiliary materials required for the synthesis of the above nucleic acid chains were purchased from the following companies:

[0249]

[0250] L96 is a commonly used carrier for liver-targeted delivery of small nucleic acid drugs. For details, please refer to the relevant patents of Alnylam or the following documents: JAm Chem Soc. 2014, 136, 16958-16961.

[0251] (2) VP protecting group removal and aminolysis

[0252] 1) Removal of protective groups of antisense strands 177, 195, and 148

[0253] The synthesized CPG-oligo was transferred from the synthesis column to a 3 mL centrifuge tube, and 3% ethylenediamine / ammonia solution was added at 1 mL / 4 μmol, and aminolysis was performed at 60° C. for 5 hours. After the aminolysis was completed, the ammonia solution was removed by vacuum centrifugation and purified.

[0254] 2) Removal of the protective group of the sense chain in the above table

[0255] The synthesized CPG-oligo was transferred from the synthesis column to a 3 mL centrifuge tube, and 28% ammonia water was added at 1 mL / 4 μmol, and aminolysis was performed at 60° C. for 5 hours. After the aminolysis was completed, the ammonia water was removed by vacuum centrifugation and purification was performed.

[0256] (3) Purification

[0257] The sample diluted by ammonolysis was purified by anion column exchange chromatography, and the liquid phase system adopted high performance liquid chromatography. The chromatographic conditions were as follows.

[0258] Chromatographic column: PS-15Q 10×250mm

[0259] Flow rate: 4mL / min

[0260] Detection wavelength: 260nm

[0261] Ion column preparation purification gradient program:

[0262] Mobile phase: Phase A: 10 mM NaOH solution (pH = 10) Phase B: 10 mM NaOH solution (pH = 10) + 2 M NaCl

[0263]

[0264] After preparation, the samples were confirmed by mass spectrometry and purity.

[0265] The above sequences were analyzed by mass spectrometry, and the summary data (calculated and experimentally detected molecular weights) are as follows:

[0266]

[0267] (4) Ultrafiltration

[0268] The purified sample was dissolved in 4 mL of PBS, transferred to a 1K (pore size with a molecular weight of 1000) ultrafiltration tube, centrifuged at 5000 r / min for 45 min, and ultrafiltered three times, each time for 45 min.

[0269] (5) Annealing

[0270] After ultrafiltration of the sense and antisense strands, dilute the samples at a concentration of 3 mg / mL, and mix the sense and antisense strands at a molar ratio of 1:1. Heat the water bath to 90°C, place the sense and antisense strands mixed in proportion in the water bath, and anneal for 30 minutes. Turn off the water bath and let it cool to room temperature for 16 hours. Take 10 μL of the sample for HPLC detection.

[0271] Example 5: siRNA in vitro silencing efficiency test

[0272] In vitro cell model Hep 3B (ATCC) cells were cultured in DMEM medium containing 10% FBS at 37°C and 5% CO 2 On the day of transfection, cells were plated at 8 x 10 4 Cells / well were inoculated in 24-well plates. The transfection process was based on the standard procedure in the instruction manual of Lipofectemine RNAiMAX (Thermofisher). The transfection system included 1 μL Lipofectemine RNAiMAX / well and the selected dose of siRNA test substance. Two parallel wells were set for each transfection condition. The negative control group was composed of only transfection reagent without adding siRNA test substance.

[0273] After 24 hours of transfection, RNA from tissues or cells was extracted using Trizol. A high-throughput QPCR instrument (Analytikjena qTOWER 384G) and a one-step SYBR green method (HiScript IIOne Step qRT-PCRSYBR Green Kit, Norwegian) were used to quantify the expression level of the target gene. Experimental results: The ΔΔCT method was used to analyze the relative changes in the expression of the target gene between the test sample and the control sample. The PCR primer information for human ANGPTL3 is as follows: upstream primer 5'-ATTTTAGCCAATGGCCTCCTTC-3', downstream primer 5'-CTGGTTTGCAGCGATAGATCATA-3'.

[0274] Experimental results:

[0275]

[0276] The results of cell experiments showed that compared with the control sequence (146), the siRNA molecule antisense strand modified with a novel nucleotide d3UVP (177) at the 5'-end showed higher silencing efficiency against the target gene ANGPTL3 at different doses. Furthermore, compared with the sequence 195 with d3TVP, the sequence 177 with d3UVP mediated higher silencing efficiency at all tested doses.

[0277] Example 6: siRNA in vivo silencing efficiency test

[0278] The experimental animals C57BL / 6J were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. and were SPF grade animals. The mice were weighed and observed before administration, and animals with uniform weight and normal condition were selected for subsequent experiments. The experimental animals were randomly divided into groups, with 7 mice in each group, and the test substance was administered subcutaneously at the selected dose of 1 mg / kg. On the 7th, 21st and 35th days after administration, the animals were sacrificed by cervical dislocation and liver tissue was obtained.

[0279] RNA was extracted from tissues or cells using Trizol. According to the measured RNA concentration, 1 μg of RNA was taken for subsequent experiments. The cDNA samples were prepared using a reverse transcription kit (HiScriptRII 1st Strand cDNA Synthesis Kit R211-01 / 02, Novazon) according to standard methods. After reverse transcription, the cDNA samples were diluted 20 times and the expression level of the target gene was quantified using a high-throughput QPCR instrument (Analytikjena qTOWER 384G) and SYBR green method (ChamQ Universal SYBR qPCR Master MixQ711-02 / 03, Novazon) QPCR. Experimental results: The relative changes in the expression of the target gene in the test sample and the control sample were analyzed using the ΔΔCT method.

[0280]

[0281] In vivo experimental results showed that the use of a novel nucleotide (177) at the 5'-end of the antisense strand of the siRNA molecule had a better silencing effect on the target gene ANGPTL3 compared with the control sequence (146 and 148).

Claims

1. A compound, characterized in that Having the structure of general formula I: in: X 1 =O or S; Sug is a ribosyl group or a ribosyl substituted group; PG is an alkyl group and an alkyl group derived therefrom, or an aryl group and an aryl group derived therefrom; PA is a phosphate moiety, a phosphorothioate moiety, or a phosphoramidite moiety derived from an RNAi drug; R 1 , R 2 are independently selected to be H, F, or methyl; Bx is a heterocyclic base, preferably an unconventional base.

2. The compound according to claim 1, characterized in that It has the structure of general formula II: or its meso, racemate, enantiomer, diastereomer; in: X 1 =O or S; X 2 =O or S or chemical bond or C 1-4 Alkylene; PG is an alkyl group and an alkyl group derived therefrom, or an aryl group and an aryl group derived therefrom; Y1 is optionally H, OH, F, OCH3, OCH2OCH2CH3, OCH2SCH3, OCH2CH2CONHR, R is H, or a C1-C3 alkyl group, preferably H; Y2 is optionally H, OH, F, OCH3, OCH2OCH2CH3, OCH2SCH3, OCH2CH2CONHR, R is H, or a C1-C3 alkyl group, preferably H; PA is a phosphate moiety, a phosphorothioate moiety, or a phosphoramidite moiety derived from an RNAi drug; R 1 , R 2 Can be independently selected as H, F, or methyl, preferably H; Bx is a heterocyclic base.

3. The compound according to claim 2, characterized in that The Bx is a heterocyclic base, preferably modified or unmodified uracil, thymine, adenine, guanine and cytosine, more preferably uracil.

4. The compound according to claim 2, characterized in that The PA is a phosphoramidite, the compound is a phosphoramidite nucleotide monomer, and the compound has one of the following structures: In the compound, Y1 and / or Y2 are as defined in claim 2, and Base is as defined in claim 3.

5. The compound according to claim 2, characterized in that The compound is selected from: Wherein Base is as defined in claim 3.

6. An RNAi molecule having a modified nucleotide monomer according to any one of claims 1 to 5 at its end.

7. The RNAi molecule according to claim 6, characterized in that The RNAi molecule is a double-stranded RNAi molecule.

8. The RNAi molecule according to claim 7, characterized in that The modified nucleotide monomer is connected to the 1st position of the 5'-end of the antisense strand.

9. The RNAi molecule according to claim 8, characterized in that The RNAi molecule is selected from the following general formula III: Among them, siRNA is double-stranded and has the following structure: Sense strand: 5′-NNNNNNNN…NNNNNNNN-3′; Antisense strand: 3′-NNNNNNNN…NNNNNNNN1-5′; The N in the double-stranded structure of siRNA represents a nucleotide monomer; the 5'-modified nucleotide monomer involved in the present invention is connected to the N1 position of the 5'-terminus of the antisense strand; X 1 =O or S; X 2 =O or S or chemical bond or C 1-4 Alkylene; Y1 is optionally H, OH, F, OCH3, OCH2OCH2CH3, OCH2SCH3, OCH2CH2CONHR, R is H, or a C1-C3 alkyl group, preferably H; Y2 is optionally H, OH, F, OCH3, OCH2OCH2CH3, OCH2SCH3, OCH2CH2CONHR, R is H, or a C1-C3 alkyl group, preferably H.

10. The RNAi molecule according to claim 9, characterized in that The H atom of sp3 CH or sp2 CH in the general formula may be replaced by a deuterium or fluorine atom.

11. Use of the compound according to any one of claims 1 to 5 or the RNAi molecule according to any one of claims 8 to 10 in the preparation of RNAi drugs.

12. A conjugate, a pharmaceutical composition, a cell or a kit comprising the RNAi molecule of any one of claims 8 to 10.

13. An RNAi drug, which is prepared by using the compound according to any one of claims 1 to 5.

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