CN114763367A - Compounds, conjugates and uses thereof - Google Patents

Abstract

The invention relates to the technical field of small nucleic acid drug delivery, and particularly discloses a compound, a conjugate and application thereof. The invention provides a nucleic acid drug targeted delivery monomer and a conjugate, and a preparation method thereof, wherein the monomer and the conjugate with a specific design structure formed by the monomer can specifically target and deliver an active drug (or small nucleic acid) to a cell receptor (or surface) and are combined with the receptor. The conjugate connected with the active drug (or the small nucleic acid) can improve the cell penetration capacity of the nucleic acid drug by utilizing the structural characteristics, enhance the stability of the nucleic acid drug in cells and effectively solve the problem of drug targeted delivery. The delivery monomer and the conjugate have the advantages of simple preparation process, wide application and the like, and are suitable for delivery of various targeted drugs.

Description

Compounds, conjugates and uses thereof

technical field

The present invention relates to the technical field of small nucleic acid drug delivery, in particular to compounds, conjugates and uses thereof.

Background technique

The delivery system is one of the core key technologies in the development of small nucleic acid drugs. At present, the most widely studied type of delivery system for small nucleic acid delivery systems in the world is the targeted conjugation delivery technology. Although tissue-specific active agents represented by specific oligonucleotides and oligonucleotide analogs have partially progressed in their application as therapeutic agents, there is still a need for improvements in their pharmacological properties, such as targeting The tropism is delivered to the lesion to improve the selectivity of the therapeutic agent, improve its biological activity and efficacy. Recently targeted conjugation delivery technology has emerged as the most widely studied class of delivery systems, especially focusing on liver-targeted delivery.

SUMMARY OF THE INVENTION

The purpose of the present invention is to solve the problems existing in the prior art, and the present invention provides a compound, a conjugate and a preparation method and use thereof.

In order to achieve the above object, the first aspect of the present invention provides a compound M, the compound M has the structure shown in formula (V) or (V'):

In formula (V) or (V'),

A is an aptamer, which has a structure derived from a carbohydrate or a polypeptide;

L1 is a straight-chain group having an ether bond and/or an amide bond with a length of 1-50 carbon atoms;

Z has a structure derived from a first phosphorus compound selected from one of phosphoramidites, H-phosphates, and phosphoric triesters, and capable of covalently covalently passing through a phosphate bond or a phosphoramide diester bond Bonds are attached to adjacent groups.

The second aspect of the present invention provides a conjugate N, the conjugate N has the structure shown in formula (VI):

(M)m-R” (VI)

In formula (VI), m is an integer of 1-4, M is provided by a compound having the structure shown in formula (V) or (V') in claim 1, and when m is greater than 1, multiple Ms are the same or different;

R" has an active site capable of linking with Nu.

The third aspect of the present invention also provides a conjugate T, the conjugate T has the structure shown in formula (VIII):

In formula (VIII), at least one of M1, M2 and M3 has a structure derived from compound M, wherein the structure of compound M is the same as defined in the aforementioned first aspect; M1, M2 and M3 are the same or different.

The fourth aspect of the present invention provides the use of the aforementioned compound M, conjugates N and T in the preparation of small nucleic acid drugs.

The present invention provides a nucleic acid drug targeted delivery monomer and conjugate and a preparation method thereof. The monomer and the conjugate with a specific designed structure formed by the monomer can specifically target and deliver active drugs (or small nucleic acids) to cellular receptors (or surfaces) and bind to the receptors. Conjugates linked to active drugs (or small nucleic acids) can improve the cell penetration ability of nucleic acid drugs by utilizing their structural properties, and enhance their intracellular stability, which can effectively solve the problem of targeted drug delivery. The delivery monomer and conjugate of the present invention have the advantages of simple preparation process, suitable for delivery of various targeted drugs, and wide range of uses.

Description of drawings

Fig. 1 is a schematic diagram of the preparation process of the siRNA conjugate of a specific embodiment of the present invention;

Figure 2 shows the results of siRNA (SNK-17) reducing liver TTR mRNA expression in C57BL/6 mice;

Figure 3 shows the results of siRNA (SNK-18) reducing liver TTR mRNA expression in C57BL/6 mice.

Detailed ways

The endpoints of ranges and any values disclosed herein are not limited to the precise ranges or values, which are to be understood to encompass values proximate to those ranges or values. For ranges of values, the endpoints of each range, the endpoints of each range and the individual point values, and the individual point values can be combined with each other to yield one or more new ranges of values that Ranges should be considered as specifically disclosed herein.

In the present invention, a C1-C10 group refers to a group having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, and the same applies to C6-C10 or C5-C10.

In the present invention, the integer of 1-10 includes 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and the integer of 1-4 or the integer of 1-6 is also the same.

In the present invention, Ac is an acetyl group.

In the present invention, Nu is an active drug, preferably functional oligoribonucleic acid and/or oligodeoxyribonucleic acid. In the present invention, the active drug Nu is preferably siRNA, such as the siRNA described in the examples, and the conjugate of the present invention can be connected to the 5' end or 3' end of the sense strand of the siRNA through a phosphodiester bond , can also be linked to the 5' or 3' end of the antisense strand of siRNA through a phosphodiester bond.

A first aspect of the present invention provides a compound L2, L2 has the structure shown in formula (I), (II), (III) or (IV):

Wherein, in formula (I), (II), (III) or (IV), Ra is independently H or a hydroxyl protecting group; the hydroxyl protecting group can be selected from DMT(4,4'-dimethoxytri benzyl), MMT (4-methoxytrityl), trityl, TBDMS (tert-butyldimethylsilyl), 4-oxopentanoyl, 2-cyanoethyl, One of PNT (4-pentenoyl) and acyloxyalkyl;

R ¹ and R ² in formula (I) and R ⁴ in formula (II) all have a covalent bonding site;

In formula (III) or (IV), n is an integer of 1-10;

表示通过活性位点连接活性基团后得到的结构；其中，所述活性位点为可以通过共价键连接的位点；所述活性基团选自亚磷酰胺基、H-磷酸酯基、磷酸酯基和多羟基烷基中的一种。In formula (II), (III) or (IV),

Represents a structure obtained by connecting an active group through an active site; wherein, the active site is a site that can be connected by covalent bonds; the active group is selected from phosphoramidite, H-phosphate, One of phosphate ester group and polyhydroxyalkyl group.

R ¹ , R ² in formula (I) and R ⁴ in formula (II) are each independently a substituted or unsubstituted straight-chain group with a length of 1-70 carbon atoms, wherein one or more carbon atoms are optional is replaced by one or more selected from the group consisting of C(O), NH, O, and S; and wherein R ¹ may optionally have in the group consisting of Any one or more of the substituents: C1-C10 alkyl, C6-C10 aryl, C5-C10 heteroaryl, C1-C10 haloalkyl, -O-C1-C10 alkyl, - O-C1-C10 alkylphenyl, -C1-C10 alkyl-OH, -O-C1-C10 haloalkyl, -S-C1-C10 alkyl, -SC1-C10 alkylphenyl , -C1-C10 alkyl-SH, -S-C1-C10 halogenated alkyl, halogen substituent, -OH, -SH, -NH ₂ , -C1-C10 alkyl-NH ₂ , -N-( C1-C10 alkyl) (C1-C10 alkyl), -NH- (C1-C10 alkyl), cyano, nitro, -CO ₂ H, -C(O)-O-(C1- C10 alkyl), -CON- (C1-C10 alkyl) (C1-C10 alkyl), -CONH (C1-C10 alkyl), -CONH ₂ , -NHC(O) (C1-C10 alkyl), -NHC(O)(phenyl), -N(C1-C10 alkyl)C(O)(C1-C10 alkyl), -N(C1-C10 alkyl)C(O) (Phenyl), -C(O)C1-C10 alkyl, -C(O)C1-C10 alkylphenyl, -C(O)C1-C10 haloalkyl, -OC(O)C1-C10 Alkyl, -SO ₂ (C1-C10 alkyl), -SO ₂ (phenyl), -SO ₂ (C1-C10 haloalkyl), -SO ₂ NH ₂ , -SO ₂ NH (C1-C10 -SO ₂ NH (phenyl), -NHSO ₂ (C1-C10 alkyl), -NHSO ₂ (phenyl) and -NHSO ₂ (C1-C10 halogenated alkyl);

In formula (I), R ² can also be H;

In formula (II), R ³ is a substituted or unsubstituted C1-C20 alkyl group; and wherein, R ³ may optionally have any one or more substituents from the group consisting of: C1 -C10 alkyl group, C6-C10 aryl group, C5-C10 heteroaryl group, C1-C10 halogenated alkyl group, -OC1-C10 alkyl group.

According to some embodiments of the present invention, each R ¹ is independently selected from one or more of the following groups, which are connected in any number and in any manner;

-(CH ₂ )n-, -O- and -CONH-; wherein n is an integer of 1-10.

According to some embodiments of the present invention, R ² and R ⁴ are each independently selected from one or more of the following groups, which are connected in any number and in any manner;

-(CH ₂ )n-, -O-, -CONH- and -(OCH ₂ CH ₂ )n-; wherein n is an integer of 1-10.

According to some embodiments of the present invention, R ³ is a C1-C6 alkyl group.

According to some embodiments of the present invention, the compound L2 has one selected from the structures represented by formulae (A1)-(A24):

可以表示通过活性位点连接活性基团后得到的结构；其中，所述活性位点为可以通过共价键连接的位点；所述活性基团可以选自亚磷酰胺基、H-磷酸酯基、磷酸酯基和多羟基烷基中的一种。Wherein, m or n in formula (A2)-(A24) are each independently an integer of 1-10;

It can represent the structure obtained by connecting active groups through active sites; wherein, the active sites are sites that can be connected by covalent bonds; the active groups can be selected from phosphoramidite, H-phosphate ester one of a phosphate group, a phosphate group and a polyhydroxyalkyl group.

According to some embodiments of the present invention, in formulas (A2)-(A24), -OH represents a hydroxyl group or a hydroxyl group protected by a hydroxyl protecting group. Wherein, the hydroxyl protecting group can be selected from DMT (4,4'-dimethoxytrityl), MMT (4-methoxytrityl), trityl, TBDMS (tert-butyl group) One of dimethylsilyl), (4-oxopentanoyl), 2-cyanoethyl and PNT (4-pentenoyl) and acyloxyalkyl.

A second aspect of the present invention provides a compound M, the compound M has a structure represented by formula (V) or (V'):

In formula (V) or (V'),

A is an aptamer, which is a structure that can promote the high-affinity and strong-specific binding of the delivered substance to the corresponding ligand (such as asialoglycoprotein receptor), and has a structure derived from sugar or polypeptide;

L1 is a straight-chain group having an ether bond and/or an amide bond with a length of 1-50 carbon atoms;

The structure of L2 is as described above (the attachment position of L2 to A, L1 and Z can be on oxygen, sulfur, nitrogen, carbon atoms);

Z is a structure derived from a first phosphorus compound, which mainly acts as a linking and coupling function, and the first phosphorus compound may be selected from one of phosphoramidite, H-phosphate and phosphoric triester, and may pass through a phosphate bond Or phosphoramide diester bonds are covalently linked to adjacent groups.

According to some embodiments of the present invention, L1 can be selected from the group obtained by connecting one or more of the following groups in any number and in any manner:

_-CH2- , -O-, _-OCH2- and -CONH-.

According to some embodiments of the present invention, L1 may be selected from one of the following groups:

-O-[ _CH2CH2O ] _-n , -[ _CH2 ] _m -CONH-[ _CH2 ] _nO- and -O-[ _CH2CH2O ] _{m - CONH-} [ _CH2 ] _nO -; wherein m and n are each independently an integer of 1-10.

According to some embodiments of the present invention, the sugar may be selected from one of monosaccharides, disaccharides, trisaccharides or polysaccharides; preferably, the sugar may be a modified sugar.

According to some embodiments of the present invention, A may be selected from D-mannose, L-mannose, D-arabinose, D-xylfuranose, L-xylfuranose, D-glucose, L-glucose , D-galactose, L-galactose, α-D-mannose, β-D-mannose, α-D-mannose, β-D-mannose, α-D-pyridine Glucuronose, β-D-glucopyranose, α-D-glucofuranose, β-D-glucofuranose, α-D-fructose, α-D-fructose, α-D-galactopyranose, β -D-galactopyranosine, α-D-galactofuranos, β-D-galactofuranos, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine Lactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino-3-O-[(R)-1-carboxyethyl]-2 -Deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-carboxamido-2,3-di-O-methyl -D-mannanose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-glycolyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, 2,3 ,4-Tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside methyl ester, 4-thio-β-D-galactopyranoside, 3 ,4,6,7-Tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucopyranoside ethyl ester, 2,5-Anhydro-D-allosenitrile one of , ribose, D-ribose, D-4-ribose thio, L-ribose or L-4-ribose thio; preferably, the sugar is N-acetylgalactosamine (GalNAc).

According to some embodiments of the present invention, A can also be selected from one of proteins, monoclonal antibodies and nanobodies.

In the present invention, A can be selected from ligands that can bind to cell surface receptors; and, A has an active hydroxyl or amino group, and A can be connected to L1 by covalent bonding with the hydroxyl or amino group; the role of A is to utilize Its affinity with the receptor delivers the active drug Nu to the target site; wherein, when A is a sugar, the linking site usually includes the 1-position hydroxyl group.

In the present invention, the receptor can be a receptor on the surface of hepatocytes, or can be an asialoglycoprotein receptor on human hepatocytes.

In the present invention, the active drug Nu can be oligoribonucleic acid and/or oligodeoxyribonucleic acid.

According to some embodiments of the present invention, Z has a structure represented by formula (B1), (B2), (B3), (B4), (B5) or (B6):

表示可以通过共价键连接的位点。Wherein, M in formula (B3) is selected from the one in TEA (triethylamine), trimethylamine, triisopropylamine and tripropylamine. Wherein, R' in formula (B4)-(B6) can be selected from one of 2,2,2-trichloroethyl, phenyl, o-chlorophenyl, cyanoethyl, and Nu. (B1), (B2), (B3), (B4), (B5) or (B6)

Indicates sites that can be linked by covalent bonds.

According to some embodiments of the present invention, the compound M has a structure represented by formula (101), (102), (201), (202), (203), (301) or (302):

According to some embodiments of the present invention, the compound M can be linked with the active drug Nu; the active drug Nu can be linked to the active drug Nu through a phosphate bond (eg, a phosphodiester bond or a phosphorothioate bond) or a phosphoramide diester bond. The compound M is linked. The active drug Nu is as previously described.

The third aspect of the present invention provides a first conjugate N, the first conjugate N has a structure represented by formula (VI):

(M)m-R” (VI)

In formula (VI), m can be an integer of 1-4, when m is greater than 1, M can independently be the structure shown in formula (V) or (V') and multiple M can be the same or different; R" Has an active site capable of being linked to the active drug Nu; alternatively, R" can be a group to which the active drug Nu is linked through the active site.

According to some embodiments of the present invention, the first conjugate N has a structure represented by formula (a), (b), (c), (d) or (e):

Wherein, in formula (a), (b), (c), (d) or (e),

n, n ₁ or n ₂ are each independently an integer of 1-10, preferably an integer of 1-6; m is each independently an integer of 1-10, preferably an integer of 1-4; X is an oxygen atom or sulfur atom;

L1' is a straight-chain group having an ether bond and/or an amide bond with a length of 1-50 carbon atoms;

A' is an aptamer, which has a structure derived from a sugar or a polypeptide, and A is a structure that can promote the binding of the delivered substance with the corresponding ligand (such as asialoglycoprotein receptor) with high affinity and strong specificity;

In formula (a), R ⁵ is each independently selected from H, C1-C10 alkyl, C1-C10 haloalkyl or C1-C10 alkoxy;

In formula (c), R ⁶ is selected from H, C1-C10 alkyl, C1-C10 haloalkyl or C1-C10 alkoxy; R ⁷ is H or a hydroxyl protecting group, and the hydroxyl protecting group is selected from DMT (4 ,4'-dimethoxytrityl), MMT (4-methoxytrityl), trityl, TBDMS (tert-butyldimethylsilyl), 4-oxopentane One of acyl group, 2-cyanoethyl group, PNT (4-pentenoyl group) and acyloxyalkyl group, preferably, R ⁷ is 2-cyanoethyl group;

In formula (e), R ⁸ is H or a hydroxyl protecting group; the hydroxyl protecting group can be selected from DMT (4,4'-dimethoxytrityl), MMT (4-methoxytrityl) group), trityl, TBDMS (tert-butyldimethylsilyl), 4-oxopentanoyl, 2-cyanoethyl, PNT (4-pentenoyl) and acyloxyalkyl A kind of, preferably 2-cyanoethyl; Preferably, R ⁸ is 2-cyanoethyl;

可以表示通过共价键与活性药物Nu或其他基团连接的位点，或者，

可以表示通过共价键连接了活性药物Nu的基团。所述活性药物Nu如前所述。in,

Can represent the site to which the active drug Nu or other group is attached via a covalent bond, or,

Can represent a group to which the active drug Nu is attached by a covalent bond. The active drug Nu is as previously described.

According to some embodiments of the present invention, L1' can be selected from the group obtained by connecting one or more of the following groups in any number and in any manner:

_-CH2- , -O-, _-OCH2- and -CONH-.

According to some embodiments of the present invention, L1' can be selected from one of the following groups:

-O-[CH ₂ CH ₂ O] _n , -[CH ₂ ] _m -CONH-[CH ₂ ] _n O-, -O-[CH ₂ CH ₂ O] _m -CONH-[CH ₂ ] _n O- and -O-[ _CH2CH2O ] _m -CONH-[ _CH2 ]nO- _; wherein m and _n are each independently an integer of 1-10.

According to some embodiments of the present invention, A' may be selected from one of monosaccharides, disaccharides, trisaccharides or polysaccharides, and A' may be a modified sugar.

According to some embodiments of the present invention, A' may be selected from D-mannose, L-mannose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L- Glucose, D-Galactose, L-Galactose, α-D-Mannofuranosose, β-D-Mannofuranosose, α-D-Mannpyranose, β-D-Mannose, α-D- glucopyranose, β-D-glucopyranose, α-D-glucopyranose, β-D-glucofuranose, α-D-fructose, α-D-fructose, α-D-galactopyranose, β-D-galactopyranosine, α-D-galactofuranos, β-D-galactofuranos, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetyl Galactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino-3-O-[(R)-1-carboxyethyl]- 2-Deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-carboxamido-2,3-di-O-methyl yl-D-mannanose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-glycolyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, 2, 3,4-Tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside methyl ester, 4-thio-β-D-galactopyranoside, 3,4,6,7-Tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucopyranoside ethyl ester, 2,5-anhydro-D-allose One of nitrile, ribose, D-ribose, D-4-ribose thio, L-ribose or L-4-ribose thio; preferably, A' is N-acetylgalactosamine (GalNAc).

According to some embodiments of the present invention, A' can also be selected from one of proteins, monoclonal antibodies and nanobodies.

In the present invention, A' can be selected from ligands that can bind to cell surface receptors; and, A' has an active hydroxyl or amino group, and can be connected to L1 by forming a covalent bond with the hydroxyl or amino group; the role of A' In order to take advantage of its affinity with the receptor, the active drug Nu is delivered to the target site; wherein, when A' is a sugar, the attachment site usually includes the 1-position hydroxyl group.

According to some embodiments of the present invention, A' may be selected from one of proteins, monoclonal antibodies and nanobodies.

In the present invention, A' can be independently selected from ligands that can bind to cell surface receptors; and, A' has an active hydroxyl group or an amino group, and can be connected to L ₁ by forming a covalent bond with the hydroxyl group or amino group; A The role of ' includes using its affinity with the receptor to deliver the active drug Nu to the target site; wherein, when A' is a sugar, the linking site usually includes a 1-position hydroxyl group.

According to some embodiments of the present invention, the first conjugate N has a structure represented by formula (401), (402), (501), (502) or (503):

In formula (401), (402), (501), (502) or (503), X is an oxygen atom or a sulfur atom; Rx has a site group for connecting the solid support, and succinate on the solid support can be selected. amide group or hydroxyl group; Nu is a small nucleic acid of active drug (such as active functional oligonucleotide) linked by covalent bond; Rz is a hydroxyl protecting group, which can be selected from 2-cyanoethyl group.

According to some embodiments of the present invention, in formula (401), (402), (501), (502) or (503), Nu may be replaced by other groups having an active site.

The fourth aspect of the present invention provides a compound L3, the compound L3 has the structure shown in formula (VII):

Wherein, Y is a substituted or unsubstituted linear group with a length of 1-10 carbon atoms, wherein one or more carbon atoms are optionally replaced by one or more -CONH-;

In formula (VII), Ra' is independently H or a hydroxyl protecting group; the hydroxyl protecting group can be selected from DMT (4,4'-dimethoxytrityl), MMT (4-methoxytrityl) trityl), trityl, TBDMS (tert-butyldimethylsilyl), 4-oxopentanoyl, 2-cyanoethyl, PNT (4-pentenoyl) and acyloxy one of the alkyl groups;

R ⁹ and R ¹⁰ each independently have H or a straight chain group of 1-70 carbon atoms in length containing a hydroxy group and/or a hydroxy group protected at the end by a hydroxy protecting group, wherein one or more carbon atoms are optionally is replaced by one or more selected from the group consisting of C(O), NH, O, and S; and wherein R ⁹ and R ¹⁰ each independently optionally have the following groups Substituents of any one or more of the group consisting of: C1-C10 alkyl, C6-C10 aryl, C5-C10 heteroaryl, C1-C10 haloalkyl, -OC1-C10 alkyl , -OC1-C10 alkylphenyl, -C1-C10 alkyl-OH, -OC1-C10 halogenated alkyl, -SC1-C10 alkyl, -SC1-C10 alkylphenyl, -C1- C10 alkyl-SH, -SC1-C10 haloalkyl, halogen substituent, -OH, -SH, -NH ₂ , -C1-C10 alkyl-NH ₂ , -N (C1-C10 alkyl) (C1-C10 alkyl), -NH (C1-C10 alkyl), cyano, nitro, -CO ₂ H, -C(O)O (C1-C10 alkyl), -CON (C1 -C10 alkyl) (C1-C10 alkyl), -CONH (C1-C10 alkyl), -CONH ₂ , -NHC(O) (C1-C10 alkyl), -NHC(O)( Phenyl), -N(C1-C10 alkyl)C(O)(C1-C10 alkyl), -N(C1-C10 alkyl)C(O)(phenyl), -C(O)C1 -C10 alkyl, -C(O)C1-C10 alkylphenyl, -C(O)C1-C10 haloalkyl, -OC(O)C1-C10 alkyl, -SO ₂ (C1-C10 alkyl), -SO ₂ (phenyl), -SO ₂ (haloalkyl of C1-C10), -SO ₂ NH ₂ , -SO ₂ NH (alkyl of C1-C10), -SO ₂ NH (benzene base), _-NHSO2 (C1-C10 alkyl), _-NHSO2 (phenyl), and _-NHSO2 (C1-C10 haloalkyl). Among them, R ⁹ and R ¹⁰ can also represent groups having a site for linking the active drug Nu. R ⁹ and R ¹⁰ can also represent groups to which the active drug Nu is attached. The active drug Nu is as previously described.

According to some embodiments of the present invention, Y may be -CONH- or _CH2 .

According to some embodiments of the present invention, R ⁹ and R ¹⁰ are each independently selected from one or more of the following groups, which are obtained by connecting in any number and in any manner:

-NH ₂ -, -CH ₂ -, -O-, -CONH-, -(OCH ₂ CH ₂ )n-, Z',

可以由式(F1)、式(F2)、式(F3)、或式(F4)的基团衍生得到，In the present invention,

can be derived from groups of formula (F1), formula (F2), formula (F3), or formula (F4),

In formula (F1), formula (F2), formula (F3) or formula (F4), Rq is a hydroxyl protecting group, and Rq can be selected from trityl, 4-methoxytrityl, 4,4 One of '-dimethoxytrityl and 4,4',4"-trimethoxybenzyl. In formula (F3), M ⁺ can be a cation or a metal cation, preferably , M ⁺ is selected from ammonium cation, tertiary amine cation and quaternary ammonium cation. In formula (F4), X is O or NH, and SPS represents a solid phase carrier.

According to some embodiments of the present invention, R ⁹ and R ¹⁰ each independently have a structure represented by formula (D1), (D2) or (D3):

Wherein, m is an integer of 0-6 (such as 0, 1, 2, 3, 4, 5, 6); M ⁺ is triethylamine cation;

Rk ^' is each independently selected from H, hydroxyl protecting group or Nu; the hydroxyl protecting group is selected from 4,4'-dimethoxytrityl, 4-methoxytrityl, trityl , one of tert-butyldimethylsilyl, 4-oxopentanoyl, 2-cyanoethyl, 4-pentenoyl and acyloxyalkyl; Nu is an active drug. R _k' can also represent a site with a linking active drug Nu. R _k' can also represent the group to which the active drug Nu is attached through the active site.

Wherein, n is an integer of 1-10; Z' is the second phosphorus compound, and can be connected with other groups in a covalent manner through a phosphate bond or a phosphoramide diester bond.

According to some embodiments of the present invention, Z' has a structure represented by formula (C1) or (C2):

wherein, Q and Q' are each independently O or S; R ¹¹ and R ¹² are each independently selected from H, C1-C10 alkyl, C1-C10 haloalkyl, C1-C10 alkoxy; and wherein R ¹¹ and R ¹² each independently contain groups as described below: a nitrile group, an amino group, a hydroxyl group, a carboxyl group and an amide group.

According to some embodiments of the present invention, the compound L3 has a structure represented by formulae (E1)-(E12):

Wherein, in formula (E1)-(E12), m and n are integers of 1-10; R _b -R _k' are each independently selected from H, hydroxyl protecting group or Nu; the hydroxyl protecting group is selected from DMT ( 4,4'-dimethoxytrityl), MMT (4-methoxytrityl), trityl, TBDMS (tert-butyldimethylsilyl), 4-oxopentane One of alkanoyl, 2-cyanoethyl, PNT (4-pentenoyl) and acyloxyalkyl.

The fifth aspect of the present invention provides a second conjugate T, the second conjugate T has a structure represented by formula (VIII):

In formula (VIII), at least one of M1, M2 and M3 has a structure derived from the aforementioned compound M, wherein the structure of the compound M is as described above; A, L1, L2 or Z are obtained by connecting any number and any way. Wherein, the compound represented by formula (VIII) can also represent that the active drug Nu is connected; the active drug Nu is as described above.

According to some embodiments of the invention, M1, M2 and M3 are the same or different.

According to some embodiments of the invention, M1, M2 and M3 all have the structure from compound M previously described, the same or different.

According to some embodiments of the present invention, the second conjugate T has the structure represented by formula (IX) or formula (X):

In formula (IX), the structure of A, L1, L2, L3 or Z' is as described above; wherein, Z' can be the structure formed after Z and L3 are connected. In formula (X), the structures of A and L1 are as described above. Among them, the compounds represented by formula (IX) and formula (X) can also represent that the active drug Nu is connected. Among them, Nu can also represent a protective group or a solid phase carrier. The protecting groups are as previously described. Wherein, the type of the solid phase carrier is not particularly limited, and can be a conventional choice in the field.

According to some embodiments of the present invention, the conjugate T has a structure represented by formula (IX-1) or formula (X-1):

In formula (IX-1) or formula (X-1), m and n are each independently an integer of 0-6; Rt and Rt' are each independently selected from H, hydroxyl protecting group, active drug Nu, solid phase carrier , C2-C6 carboxyl group, carboxylate or amide group; the hydroxyl protecting group is selected from 4,4'-dimethoxytrityl, 4-methoxytrityl, trityl, One of tert-butyldimethylsilyl, 4-oxopentanoyl, 2-cyanoethyl, 4-pentenoyl and acyloxyalkyl.

According to some embodiments of the present invention, the second conjugate T has a structure represented by formula (601), (602), (603), (604), (605), (606) or (607):

Wherein, in formula (601), (602), (603), (604), (605), (606) or (607), X is an oxygen atom or a sulfur atom; Nu is an active drug small nucleic acid.

In the present invention, the conjugate of the structure represented by the formula (603) can be contacted by the formula (603A) with an acid anhydride (such as succinic anhydride) to obtain the corresponding carboxylate (603B), and (603B) is further connected to the solid phase to obtain ( 603C), (603C) is then connected to the active group Nu (such as functional small nucleic acid),

In the present invention, the conjugate of the structure represented by the formula (606) can be prepared from the compound represented by the formula (101) by solid-phase synthesis. The method of solid-phase synthesis is not particularly limited, and can be a conventional choice in the field.

According to some embodiments of the present invention, in formula (601), (602), (603), (604), (605), (606) or (607), Nu may be replaced by other groups having an active site .

In the present invention, the preparation of compound M can be prepared according to the following method (taking the preparation of formula (101) as an example):

(a) under nitrogen protection, in the first solvent, compound 3 is contacted with N,N,N'N'-tetraisopropyl chlorophosphite and diisopropylethylamine to react; wherein, the contacted The condition can be room temperature;

(b) in the presence of the first solvent, add 1-O-dimethoxytrityl-1,3-propanediol and ethylthiotetrazole into the solution after the above reaction, and continue to carry out under the protection of nitrogen at room temperature reaction. Among them, compound 3, N,N,N'N'-tetraisopropyl chlorophosphite, diisopropylethylamine, 1-O-dimethoxytrityl-1,3-propanediol and ethyl acetate The molar ratio of thiotetrazole may be 1:(1.2-2):(2.5-3.5):(1.2-2):(0.2-0.6). Wherein, the type and amount of the first solvent are not particularly limited, as long as the requirements of the present invention can be met. For example, the first solvent may be dichloromethane.

The present invention has no particular limitation on the post-treatment for the preparation of compound M, which can be carried out with reference to the conventional manner in the art. For example, the post-treatment step may include: washing the reacted system with saturated brine, and extracting it with an extraction solvent (such as dichloromethane), combining the organic phases and concentrating, and then separating by column chromatography to obtain compound (101) .

In the present invention, the preparation of compound 3 is well known to those skilled in the art, and the preparation of compound 3 is not particularly limited in the present invention, and can be carried out with reference to conventional methods in the art.

In the present invention, the preparation of the second conjugate T is prepared according to the following method (taking the preparation of formula (603A) as an example):

(a) At room temperature, in the presence of a second solvent, compound 22 is mixed with part of N,N-diisopropylethylamine to obtain a mixture; wherein, the moles of compound 22 and N,N-diisopropylethylamine The ratio can be 1:(3-5);

(b) at -5°C to 5°C, combine the above mixture with 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate and N,N-diisopropylethylamine and compound 14 were mixed and reacted with stirring for 0.5-1 hour; and continued to add part of N,N-diisopropylethylamine to the reaction solution at -5°C to 5°C Amine, continue to react for 1-2 hours; wherein, compound 22, compound 14, N,N-diisopropylethylamine and 2-(7-azabenzotriazole)-N,N,N',N The molar ratio of '-tetramethylurea hexafluorophosphate can be 1:(1-1.2):(5-10):(1-1.2); preferably, part of N,N-diisopropylethylamine, And the mol ratio of the added N,N-diisopropylethylamine can be 1:(0.5-1);

Wherein, the type and amount of the second solvent are not particularly limited, as long as it can meet the requirements of the present invention. For example, the second solvent may be dichloromethane.

The present invention has no particular limitation on the post-processing of the preparation of the second conjugate T, which can be carried out in a conventional manner in the art. For example, the post-treatment step may include: gradually increasing the temperature of the reacted system from 0° C. to room temperature, and then continuing to stir for 2-5 hours. The reacted system is washed with saturated brine, extracted with an extraction solvent (such as dichloromethane), the combined organic phases are concentrated, and then separated by column chromatography.

In the present invention, the preparation of compound 22 and compound 14 is well known to those skilled in the art, and the preparation of compound 22 and compound 14 is not particularly limited in the present invention, and can be carried out with reference to conventional methods in the art.

According to the first and second conjugates of the present invention, wherein the active functional oligonucleotide can be selected from the following nucleic acid substances: small interfering RNA, microRNA, single-stranded RNA, antisense nucleic acid, inducing oligonucleotide , stem-loop RNA, etc. Wherein, the functional oligonucleotide is composed of single-stranded oligonucleotide or double-stranded oligonucleotide. The small interfering RNAs in the present invention are selected from double-stranded oligonucleotides, which include a sense strand and an antisense strand, and the sense and antisense strands are complementary, that is, in a double-stranded nucleic acid molecule, the bases of one strand and the other Bases on one strand pair in a complementary fashion through hydrogen bonding interactions.

In the present invention, both the aforementioned first conjugate and second conjugate may be nucleic acid conjugates. Preferably, the nucleic acid conjugate is a siRNA conjugate comprising the following sequence:

Sequence of SNK-17

Chain of Justice (S):

5'-AfsmAsCfmAGfmUGfmUUfCfUfmUGfmCUfmCUfmAUfmAAf-EgGaGaGaC3 (base sequence shown in SQE ID NO: 1)

Antisense Strand (AS):

5'-mUsUfsmAUfmAGfmAGfmCAfmAmGmAAfmCAfmCUfmGUfmUsmUsmU (base sequence shown in SQEID NO: 2)

Sequence of SNK-18:

Chain of Justice (S):

5'-AfsmAsCfmAGfmUGfmUUfCfUfmUGfmCUfmCUfmAUfmAAf-TriGalNac (base sequence shown in SQE ID NO: 1)

Antisense Strand (AS):

5'-mUsUfsmAUfmAGfmAGfmCAfmAmGmAAfmCAfmCUfmGUfmUsmUsmU (the base sequence is shown in SQEID NO: 2).

In the present invention, the modified nucleotides in the above sequences can be obtained commercially, or can be modified by themselves, and the modification methods of nucleotides are well known to those skilled in the art, and can be carried out with reference to conventional methods in the art. The present invention does not make special restrictions.

In the present invention, the oligonucleotide conjugate can be prepared by the following method. The nucleoside monomers are sequentially connected in the direction of 5', and the connection of each nucleoside monomer includes a four-step reaction of deprotection, coupling, capping, oxidation or sulfurization; in some embodiments, the method further comprises: In the presence of coupling reaction conditions and coupling reagents, the compound represented by formula (101) is contacted with a nucleoside monomer or a nucleotide sequence linked to a solid support, so that the compound represented by formula (101) is The coupling reaction connects to the nucleotide sequence. Wherein, the method further comprises a step of removing the protecting group and cleaving with the solid phase carrier, a separation and purification step, and an optional annealing step.

In the present invention, after connecting all nucleoside monomers and before annealing, the method further includes separating the sense strand and antisense strand of the siRNA. The separation method is well known to those skilled in the art, and generally includes cutting the synthesized nucleotide sequence from the solid support, removing the protective groups on the base, the phosphate group and the ligand, purification and desalting. .

In the present invention, the methods and specific conditions involved in the preparation process of the above-mentioned siRNA conjugates are well known to those skilled in the art, and can be carried out with reference to conventional methods in the art.

The present invention also provides the use of the aforementioned compound M and the conjugate in the preparation of small nucleic acid drugs;

Preferably, the specific target gene of the small nucleic acid in the small nucleic acid drug can be selected from PCSK9, HBV, TTR, AGT and the like.

The present invention will be described in detail below by means of examples.

In the following examples, unless otherwise specified, all commercially available raw materials and reagents were used directly without further processing. The organic solvent was concentrated under reduced pressure by rotary evaporator.

Compound (101) was prepared according to the following route

Synthesis of Compound 2

N-acetylgalactosamine tetraacetate 1 (10 g, 25.68 mmol) was dissolved in dichloroethane (60 mL) at room temperature, and trimethylsilyl trifluoromethanesulfonate (8.6 g, 38.66 mmol) was stirred Add to the above solution, continue stirring and heat this solution to 50°C. After reacting at 50°C for 2 hours, heating was stopped, and stirring was continued at room temperature for 12 hours. The reacted solution was poured into ice water containing saturated sodium bicarbonate, extracted with dichloromethane, and the organic phase was washed with water. The organic phase was separated, dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness under reduced pressure to obtain Compound 2 in the form of a brown-yellow foam candy. This compound was used directly in the next reaction without purification.

Synthesis of Compound 3

Compound 2 (5 g, 15.18 mmol) was mixed with tetravinyl alcohol (29.49 g, 151.8 mmol) and pyridinium p-toluenesulfonate (3.43 g, 13.66 mmol), and heated to 80° C. with stirring for 18 hours. The reaction solution was poured into saturated brine and saturated sodium bicarbonate solution in a volume ratio of 1:1, and extracted with 300 mL of dichloromethane. The organic phase was separated, dried over anhydrous sodium sulfate, and evaporated to semi-dryness under reduced pressure. Purify by silica gel chromatography using a gradient elution. Rinse with dichloromethane first, then with a mixed solvent (dichloromethane/methanol, 92:8, v/v), and dry the solvent under reduced pressure to obtain nearly white compound 3 (3.61 g, 45.4%). ¹ H NMR (CDCl ₃ ): δ, 6.79-6.77(1H,m), 5.31-5.29(1H,m), 5.11-5.08(1H,m), 4.81-4.79(1H,m), 4.22-4.10( 7H,m), 3.93-3.89(2H,m), 3.86-3.81(1H,m), 3.74-3.61(8H,m), 2.17(3H,s), 2.17-2.15(1H,s), 2.12( 4H,s), 2.10(7H,m).

Synthesis of 1-O-Dimethoxytrityl-1,3-propanediol (A-1)

4,4'-Dimethoxytrityl chloride (18 g, 0.053 mol) was dissolved in a mixed solvent (40 mL of dichloromethane, 8.7 mL of triethylamine, 0.1 mL of 4-(dimethylamino) ) pyridine). 1,3-Propanediol (21 g, 0.265 mol) was added dropwise to the above solution with stirring, and stirring was continued at room temperature for 12 hours. The reaction solution was poured into 100 mL of saturated brine, extracted with 300 mL of dichloromethane, the organic phase was separated, dried over anhydrous sodium sulfate, and evaporated to semi-dryness under reduced pressure. Purified by silica gel column chromatography, using a gradient elution, first with mixed solvent (n-hexane/ethyl acetate, 3:1, v/v), then with another mixed solvent (n-hexane/ethyl acetate, 1 : 1, v/v) rinsed, and the solvent was dried under reduced pressure to obtain an orange compound 1-O-dimethoxytrityl-1,3-propanediol (16 g, 80%). ¹ H NMR (CDCl ₃ ): δ, 7.43-7.41(2H,m), 7.33-7.27(6H,m), 7.29-7.27(1H,m), 6.85-6.82(4H,m), 3.79-3.75( 7H,m), 3.29-3.27(2H,m), 2.20-2.18(1H,br), 1.88-1.83(2H,br), 1.57(1H,s).

Synthesis of Compound (101)

Compound 3 (687 mg, 1.31 mmol) was dissolved in 10 mL of dry dichloromethane, and then N,N,N'N'-tetraisopropyl chlorophosphite (570 mg, 2.1 mmol) and diisopropylethyl acetate were rapidly dissolved. Amine (550 mg, 4.2 mmol) was added to the above solution. The above reaction solution was stirred at room temperature for 20 minutes under nitrogen protection. 1-O-Dimethoxytrityl-1,3-propanediol (800 mg, 2.1 mmol) was dissolved in 5 mL of dry dichloromethane, and then poured into the above solution, and at the same time, ethylthiotetrazole (70 mg, 0.525 mmol) was added. mmol) and continued to stir the reaction for 2 hours at room temperature under nitrogen protection. The reacted solution was poured into saturated brine, extracted with 2×60 mL of dichloromethane, the organic phase was separated, dried over anhydrous sodium sulfate, and evaporated to semi-dryness under reduced pressure. Purified by silica gel chromatography using a gradient of first with a mixed solvent (n-hexane/ethyl acetate/triethylamine, 48:48; 4, v/v/v) and then with another mixed solvent (acetic acid Ethyl ester/triethylamine, 96:4, v/v) rinsed, and the solvent was sucked dry under reduced pressure to obtain compound 101 as a transparent foam and nearly white. ¹ H NMR (CD ₃ CN): δ, 7.44-7.42(3H,m), 7.32-7.30(6H,m), 7.29-7.21(1H,m), 6.87-6.84(4H,m), 6.50(1H) ,m),5.28-5.27(1H,m),5.00-4.97(1H,m),4.65-4.63(1H,m),4.11-4.09(3H,m),4.07-4.06(3H,m),3.97 -3.95(1H,m),3.76(6H,s),3.69-3.57(6H,m),3.56-3.50(8H,m),3.11-3.10(2H,m),2.21(4H,s),1.98 (3H,s),1.97(3H,s),1.94(3H,s),1.85(4H,s),1.37-1.34(1H,m),1.28-1.27(1H,m),1.20(4H,m) ), 1.14(4H,m), 1.12(1H,m). ³¹ P NMR (CD ₃ CN): δ, 147.95, 147.41 ppm. HRMS(ESI) m/z, C ₅₂ H ₇₅ N ₂ O ₁₇ P(M+Na ⁺ +H ⁺ )/2 Theoretical value: 527.56, found value: 527.20.

Synthesis of Conjugate (603A)

1. Compound 7 was prepared according to the following route

Synthesis of Compound Oxazoline 5

N-acetylgalactosamine tetraacetate 4 (10 g, 25.68 mmol) was dissolved in dichloroethane (60 mL) at room temperature, and trimethylsilyl trifluoromethanesulfonate (8.6 g, 38.66 mmol) was stirred Add to the above solution, continue stirring and heat this solution to 50°C. After reacting at 50°C for 2 hours, heating was stopped and stirring was continued at room temperature for 12 hours. The solution was poured into ice water containing saturated sodium bicarbonate, extracted with dichloromethane, and the organic phase was washed with water. The organic phase was separated, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure to obtain Compound 5 in the form of a brown-yellow foam sugar. This compound 5 was directly used in the next reaction.

Synthesis of compound 6

Compound oxazoline 5 (4.26 g, 12.9 mmol) was dissolved in dichloromethane (20 mL) at room temperature, and was dissolved with azide-tripolyethylene glycol (3.4 g, 19 mmol) in dry dichloromethane at 0 °C. (20 mL) of the solution was mixed and stirred. Trisilyl trifluoromethanesulfonate (TMSOTf, 1.4 g, 6.45 mmol) was slowly added to the above solution at 0°C and stirring was continued for one hour. The mixed solution was continued to stir at room temperature for 14 hours, then the solution was poured into ice water containing saturated sodium bicarbonate, extracted with dichloromethane (2 x 50 mL), and the organic phase was washed with water. The organic phase was separated, dried over anhydrous sodium sulfate, and concentrated to semi-dryness by rotary evaporation under reduced pressure. Continue to purify with silica gel column, use a gradient elution, first elute with mixed solvent (ethyl acetate/methanol, 10:1, v/v), collect product components, and dry the solvent under reduced pressure to obtain a nearly white compound 6 (5.3g, 81%) ¹ H NMR (CDCl ₃ ): δ, 6.15 (d, 1H, NH), 5.32 (d, 1H, sugar-H-4'), 5.07 (dd, 1H, J=11.2 Hz,J=3.3Hz,sugar-H-3'),4.76(d,1H,J=8.6Hz,sugar-H-1'),4.17(m,3H,sugar-H-2',sugar-H -6'), 3.91(m, 2H, -CH ₂ O), 3.89(m, 1H, suager-H-5'), 3.76-3.61(m, 8H, -CH ₂ O), 3.47(m, 2H ,-CH ₂ N ₃ ), 2.16(s,3H,-CH ₃ ,NHAc),1.99,2.00,2.05(3xs,9H,-CH ₃ ,Ac).HRMS(ESI)m/z,C ₂₀ H ₃₂ N ₄ O ₁₁ (M+H ⁺ ) theoretical value: 505.49, measured value: 505.20.

Synthesis of compound 7

Azide 6 (522 mg, 1.04 mmol) was dissolved in 10 mL of ethyl acetate, Pd/C (80 mg), and added to 30 mL of ethyl acetate under nitrogen protection. The reaction flask was connected to a hydrogen balloon, and after repeated hydrogen replacement, the reaction flask was connected to the hydrogen balloon at room temperature, and the reaction solution was continuously stirred for 3 hours. After the Pd/C was filtered through Celite, 0.5 mL of hydrochloric acid (2M) was slowly added dropwise to the reaction flask, and the solution was reacted under continuous stirring for 30 minutes at a reaction temperature of 0°C. 10 mL of acetonitrile was added to the reaction solution, and the mixture was azeotropically concentrated under reduced pressure twice. The concentrated solution was mixed with dichloromethane (10 mL) and concentrated again under reduced pressure twice to give crude product 7 (500 mg) as an oily foam, which was used directly in the next reaction without further purification. ¹ H NMR (CDCl ₃ ): δ, 8.25 (m, 2H, -NH ₂ ), 5.34 (d, 1H, sugar-H-4'), 5.21 (dd, 1H, J=11.2 Hz, J=3.3 Hz ,sugar-H-3'),4.91(d,1H,J=8.5Hz,sugar-H-1'),4.12(m,3H,sugar-H-6',sugar-H-2'),4.07 (m,2H,sugar-H-5',-NH),3.76(m,2H, _-CH2O ),3.68(m,2H, _-CH2O ),3.61(m,2H, _-CH2O ), 3.58(m, 4H, 2x-CH ₂ O), 3.20(m, 2H, NH ₂ ), 2.09(s, 3H, -NHCO ₂ CH ₃ ), 2.04, 1.96, 1.89(3x s, 9H, - CO ₂ CH ₃ ).HRMS (ESI) m/z, C ₂₀ H ₃₄ N ₂ O ₁₁ (M+H ⁺ ) Theoretical value: 479.49, found value: 479.20.

2. Compound 12 was prepared according to the following route

Synthesis of compound 9

Tris (10 g, 82.6 mmol) was dissolved in 15 mL of dioxane, 1.26 mL of 40 wt% potassium hydroxide aqueous solution was added dropwise to the reaction solution, and 20 mL of dioxane was added at room temperature. . At 0°C, acrylonitrile (18 mL, 272 mmol) was slowly added dropwise to the reaction flask for about 1 hour. The reaction solution was continuously stirred at room temperature for 24 hours. The reaction solution was poured into saturated aqueous sodium chloride solution, extracted with dichloromethane (2 x 50 mL), and the organic phase was washed with water. The organic phase was separated, dried over anhydrous sodium sulfate, and concentrated to semi-dryness by rotary evaporation under reduced pressure. Purification was continued with a silica gel column eluting first with dichloromethane and then a mixed solvent (dichloromethane/methanol, 10:1, v/v) to collect the product fractions. Concentration under reduced pressure by rotary evaporation gave 9 (20 g, 86%) as a pale yellow oil. ¹ H NMR (CDCl ₃ ): δ, 3.68 (t, 6H, J=7.1 Hz, 3x-CH ₂ O), 3.44 (s, 6H, 3x-CH ₂ CNH ₂ ), 2.61 (t, 6H, J= 6.2Hz,3x–CH ₂ CN),1.70(s,2H,-NH ₂ ).HRMS(ESI)m/z,C ₁₃ H ₂₀ N ₄ O ₃ (M+H ⁺ )Theoretical: 281.32, found :281.20.

Synthesis of Compound 10

Tris[(cyanoethoxy)methyl]aminomethane 9 (1.2 g, 4.28 mmol) was dissolved in 10 mL of absolute ethanol, and 2 mL of concentrated sulfuric acid and 10 mL of concentrated sulfuric acid were slowly added dropwise to the solution in the reaction flask at room temperature. of anhydrous ethanol. The reaction solution was heated to 80°C, and the reaction solution was kept at reflux for about 36 hours. After cooling the reaction solution to room temperature, 25 mL of saturated sodium bicarbonate ice solution was added. Rotary evaporated under reduced pressure, and the ethanol was evaporated. The aqueous solution was extracted with ethyl acetate (2×50 mL), the obtained organic phase was dried over anhydrous sodium sulfate, and then concentrated by rotary evaporation under reduced pressure to obtain light yellow oil 10 (0.8 g, 46%), the crude product Used directly in the next reaction without further purification.

Synthesis of Compound 11

The crude compound 10 (0.8 g, 1.9 mmol) was dissolved in 20 mL of dichloromethane. To the reaction solution were added di-tert-butyl dicarbonate (2 mL, 8.8 mmol) and 5 mL of triethylamine. The reaction solution was stirred for 14 hours at room temperature. The reaction solution was poured into an aqueous solution containing saturated sodium bicarbonate, extracted with dichloromethane (2 x 50 mL), and the organic phase was washed with water. The organic phase was separated, dried over anhydrous sodium sulfate, concentrated to semi-dryness by rotary evaporation under reduced pressure, continued to purify with a silica gel chromatographic column, and eluted with a gradient, first washed with dichloromethane solvent, and then with (dichloromethane/methanol) , 96:4, v/v) rinsed, the product components were collected, and the solvent was evaporated under reduced pressure to obtain a nearly white oily substance 11 (0.5 g, 51%), ¹ H NMR (CDCl ₃ ): δ, 4.92 (b ,1H,-CONH-),4.14(m,3x2H,-CO ₂ CH ₂ -),3.69(m,3x2H,-OCH ₂ -),3.63(s,3x2H,-OCH ₂ -),2.53(m, 3x2H, -COCH ₂ -), 1.45(s, 3x3H, -CH ₃ ), 1.26(t, 3x3H, -CH ₂ CH ₃ ).HRMS (ESI) m/z, C ₂₄ H ₄₃ NO ₁₁ (M+H ⁺ ) Theoretical value: 522.60, measured value: 522.40.

Synthesis of Compound 12

The Boc-protected compound 11 (0.6 g, 1.43 mmol) was dissolved in 20 mL of absolute ethanol, 4 mL of sodium hydroxide (4M) was slowly added dropwise to the reaction solution, the reaction solution was kept at 0°C, and stirred for 14 hours. The reaction progress was monitored by liquid quality from time to time. When the peaks of the reactants disappeared, rotary-evaporated under reduced pressure. After the ethanol was evaporated, 10 mL of potassium hydrogen sulfate (1M) was added to the reaction solution and stirred for 15 minutes at 0°C. The above reaction solution was extracted with ethyl acetate (2×50 mL), and the obtained organic phase was dried over anhydrous sodium sulfate, and then concentrated by rotary evaporation under reduced pressure to obtain a viscous substance 12 (0.5 g, 81%). The crude product was used directly in the next reaction without further purification. 1H NMR (CDCl3): d, 9.40 (b, 3H, -CO ₂ H), 5.0 (b, 1H, -CONH-), 3.70 (m, m, 3x2H, -OCH ₂ -), 3.65 (s, 3x2H ,-OCH ₂ -),2.60(m,3x2H,-COCH ₂ -),1.42(s,3x3H,-CH ₃ ).HRMS(ESI)m/z,C ₁₈ H ₃₁ NO ₁₁ (M+H ⁺ ) Theoretical value: 438.32, measured value: 438.20.

Compound 14 was prepared according to the following route

Synthesis of Compound 13

Tricarboxylic acid 12 (0.5 g, 1.14 mmol) was dissolved in 20 mL of dichloromethane, and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea was added Hexafluorophosphate (1.3 g, 3.42 mmol) and N,N-diisopropylethylamine (0.2 g, 3.95 mmol) were added simultaneously with 8 mL of dimethylformamide. Compound amine 7 (2.19 g, 4.08 mmol) was dissolved in 5 mL of dimethylformamide, and 1 mL of N,N-diisopropylethylamine was added. The two solutions were mixed and stirred at room temperature, and the stirring was continued for 14 hours. After chromatographic detection, it was determined that the reactant completely disappeared. 20 mL of saturated aqueous sodium bicarbonate solution was added to the reaction solution, and extracted with 2 × 50 mL of dichloromethane. The organic phase was concentrated to semi-dryness by rotary evaporation, and purified by silica gel column. Washed with chloromethane solvent, then rinsed with (dichloromethane/methanol, 85:15, v/v), the product components were collected, and the solvent was vacuumed to dryness to obtain a near-yellow oily crude product 13 (2 g, 86%). The crude product 13 was further purified by reversed-phase chromatography, the elution solvent was (H ₂ O/MeOH, 1:1, v/v), the product components were collected, and concentrated to complete dryness through rotary evaporation to obtain compound 13 (1.24 g , 60%). ¹ H NMR (CDCl ₃ ): δ, 5.32 (d, 3H, J=3.0 Hz, sugar-H-4'), 5.18 (dd, 3H, sugar-H-3'), 4.78 (d, 3H, sugar -H-1'),4.18-4.06(m,24H,-OCH ₂ ,sugar-H-5'),3.93(m,9H,sugar-2xH-6',sugar-H-2'),3.77, 3.64, 3.46(m, 6H, -CH ₂ NH-), 2.44, 2.20(m, 6H, -COCH ₂ -), 2.15(s, 9H, -NHCOCH ₃ ), 2.09(s, 2H, -CH ₂ - ), 2.05, 1.99, 1.95(3xs, 27H, -OCOCH ₃ ), 1.81(s, 9H, CH ₃ , Boc).HRMS(ESI) m/z, C ₇₈ H ₁₂₇ N ₇ O ₄₁ (M+2H ⁺ )/2, theoretical value: 910.1, measured value: 910.0.

Synthesis of compound 14

The purified compound 13 (2 g, 1.1 mmol) was dissolved in 30 mL of dichloromethane, and 1 mL of hydrochloric acid solution (4M) and 1 mL of dioxane were slowly added to the above-mentioned dichloromethane reaction solution at a temperature of 0 °C middle. The above reaction solution was continuously stirred at a temperature of 0°C for 30 minutes and then at room temperature for 30 minutes. It was concentrated to complete dryness by rotary evaporation to obtain a white foamy crude product 14 (1.7 g, 90%), which was directly used in the next reaction without further purification. ¹ H NMR (CDCl ₃ ): δ, 8.20 (b, 2H, -NH ₂ ), 5.35 (d, 3H, J=3.0 Hz, sugar-H-4'), 5.22 (dd, 3H, sugar-H- 3'), 4.80(d, 3H, sugar-H-1'), 4.13(m, 9H, sugar-2xH-6', sugar-H-2'), 3.94-3.44(m, 24H, -OCH ₂ ,sugar-H-5'),3.77,3.64,3.46(m,6H,-CH ₂ NH-),2.55,2.43(m,6H,-COCH ₂ -),2.15(s,9H,-NHCOCH ₃ ) ,2.09(s,2H,-CH ₂ -),2.05,1.98,1.96(3xs,27H,-OCOCH ₃ ).HRMS(ESI)m/z,C ₇₃ H ₁₁₉ N ₇ O ₃₉ (M+H ⁺ ) /2, theoretical value: 860.4, measured value: 860.0.

Compound 21 was prepared according to the following route

Synthesis of compound 16

4,4'-Dimethoxytrityl chloride (1.8 g, 5.3 mmol) was dissolved in 5 mL of dichloromethane, and this solution was slowly added dropwise to the solution containing 3-hydroxy-2-hydroxyl group at room temperature. Methyl-2-methyl-propionic acid 15 (0.8 g, 5.97 mmol) in dry pyridine (10 mL). The above solution was continuously stirred for 14 hours at room temperature. 20 mL of water was added to the reaction mixture and extracted with 2 x 50 mL of ethyl acetate. The organic phase was concentrated to semi-dryness by rotary evaporation, and was further purified by silica gel chromatography, using a gradient elution, first with n-hexane solvent, and then with (n-hexane/ethyl acetate, 1:1, v/v) , the product components were collected, and the solvent was drained under reduced pressure to obtain a yellow solid 16 (1.5 g, 58%). The product 16 was directly used in the next reaction.

Synthesis of compound 19

Monomethyl adipate 17 (0.16 g, 1 mmol) and N-(tert-butoxycarbonyl)-1,3-diaminopropane tert-butyl N-(3-aminopropyl)carbamate 18 (0.174 g, 1 mmol) was dissolved in 5 mL of anhydrous tetrahydrofuran at room temperature. The above solution was mixed with 0.892 mL of 1-propylphosphoric acid cyclic anhydride (0.892 mL, 1.5 mmol, in 50% (volume ratio 1:1) ethyl acetate) and 0.522 mL of N,N-diisopropylethylamine ( DIPEA, 0.522 mL, 3 mmol) were mixed. The mixed reaction solution was stirred at room temperature for 30 minutes, then 20 mL of ethyl acetate was added to the reaction solution to dilute, 20 mL of saturated brine was added, and extraction was performed with 2×20 mL of ethyl acetate. The organic phase was separated, dried over anhydrous sodium sulfate, and concentrated to dryness by rotary evaporation to obtain a pale yellow foamy crude product 19 (0.29 g, 91%). The crude product 19 was directly used in the next reaction without further purification. ¹ H NMR (CDCl ₃ ), δ, 5.89 (b, 1H, -CONH-), 4.97 (b, 1H, -NHCO-), 3.75 (s, 3H, -CH ₃ ), 3.27 (m, 2H), 3.15(m,2H),2.34(m,2H),2.19(m,2H),1.67(m 2x2H),1.64(m,2H),1.4(s,9H).HRMS(ESI)m/z,C ₁₅ H ₂₈ N ₂ O ₅ , theoretical value: 316.39, measured value: 316.40.

Synthesis of Compound 20

Compound 19 (0.8 g, 2.5 mmol) was dissolved in 5 mL of ethyl acetate, the reaction solution was mixed with 3.2 mL of aqueous hydrochloric acid (4 M), and 5 mL of dioxane was added. The mixed reaction solution was continuously stirred for 30 minutes at room temperature. After rotary evaporation and concentration, a viscous crude product 20 (0.5 g, 92%) was obtained, which was directly used in the next reaction.

Synthesis of Compound 21

The above crude product compound 20 (0.252 g, 1 mmol) was dissolved in 5 mL of dimethylformamide, and the above reaction solution was mixed with 3-O-4,4'-dimethoxytrityl at 0 °C. -2-Hydroxy-2-methylpropionic acid 16 (0.45 g, 0.9 mmol) was mixed, and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyl was added Urea hexafluorophosphate (0.46 g, 1.2 mmol) and N,N-diisopropylethylamine (0.52 mL) and stirred for 20 minutes. The temperature of the above reaction solution was slowly raised to room temperature, and stirring was continued for 14 hours. The reaction solution was mixed with 20 mL of saturated aqueous sodium chloride solution, and extracted with 2×50 mL of ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, and then concentrated by rotary evaporation under reduced pressure to obtain crude product 21. Continue to purify with silica gel column, use a gradient elution, first wash with ethyl acetate solvent, then with (ethyl acetate/methanol, 90:10, v/v), collect product fractions, and dry under reduced pressure The solvent gave compound 21 (0.38 g, 61%). ¹ H NMR (CDCl ₃ ): δ, 8.01 (b, 1H, -CONH-), 7.26 (m, 4H, Trityl), 7.05 (m, 4H, Trityl), 6.67 (m, 4H, Trityl), 6.48 ( b, 1H, -NHCO-), 5.25(s, 3H, -OCH ₃ ), 3.78(s, 6H, 2x-OCH ₃ ), 3.64(s, 4H, 2X-CH ₂ -), 3.26(m, 2H ,-NHCH ₂ -), 3.17(m, 2H, -CH ₂ NH-), 2.79(m, 3H, -OCH ₃ ), 2.31(m, 2H), 2.18(m, 2H), 1.65-1.56(m ,6H),1.25(s,3H).HRMS(ESI)m/z,C ₃₆ H ₄₆ N ₂ O ₈ ,(M+Na ⁺ )Theoretical value: 657.34, Found value: 657.40.

Compound (603A) was prepared according to the following route

Synthesis of Compound 22

The purified compound 21 (0.7 g, 1.1 mmol) was dissolved in 5 mL of anhydrous methanol, and then 1.5 mL of lithium chloride in methanol (2M) was slowly added to the reaction solution at 0°C. After stirring at 0°C for 30 minutes, the reaction solution was warmed to room temperature, and stirring was continued at room temperature for 2 hours. At room temperature, the reaction solution was mixed with 2 mL of water, concentrated to semi-dryness by rotary evaporation, methanol was removed, and then separated by preparative reversed-phase high pressure liquid chromatography, and the mobile phase solvent was methanol and water (MeOH:H ₂ O, 1:1, v/v). The product fractions were collected, and the solvent was evaporated under reduced pressure to obtain compound 22 as a yellow solid (0.66 g, 93%). ¹ H NMR (CDCl ₃ ): δ, 7.38 (m, 4H, Trityl), 7.29 (b, 1H, -CONH-), 7.28 (m, 5H, Trityl), 7.16 (b, 1H, -NHCO-), 6.79(m, 4H, Trityl), 3.76(s, 10H, 2x-OCH _3, 2x-CH ₂ ), 3.71(m, 2H, -CH ₂ -), 3.21(m, 2H, -NHCH ₂ -), 2.07(m,2H,-CH ₂ NH-),1.87(m,2H),1.50(m,2H),1.27(m,2H),1.21(s,3H).HRMS(ESI)m/z,C ₃₅ H ₄₃ N ₂ O ₈ , (M+H ⁺ +Na ⁺ ) Theoretical value: 643.72 Measured value: 643.20.

Synthesis of Compound (603A)

The purified compound 22 (0.65 g, 1.04 mmol) was dissolved in 15 mL of dichloromethane, then the above solution was mixed with 0.723 mL of N,N-diisopropylethylamine (4.16 mmol) at room temperature. At 0°C, this mixed solution was mixed with purified compound 14 (1.83 g, 1.04 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyl Urea hexafluorophosphate (0.435 g, 1.1 mmol) and N,N-diisopropylethylamine (0.723 mL, 4.16 mmol) were mixed and stirred for 30 minutes. Continuing at 0°C, 1 mL of N,N-diisopropylethylamine was added to the reaction solution, and the reaction solution was continuously stirred for 1 hour. The reaction temperature was gradually increased from 0°C to room temperature, and then stirring was continued for 2 hours. The reaction solution was mixed with 5 mL of saturated aqueous sodium chloride solution, and extracted with 2×50 mL of dichloromethane. The organic phase was dried over anhydrous sodium sulfate, and then concentrated by rotary evaporation under reduced pressure to obtain crude product 603A. Purification was continued with a silica gel column using a gradient elution, first with dichloromethane solvent, then with (dichloromethane/methanol/triethylamine, 94:5:1, v/v/v), and the collected The product components were dried under reduced pressure to obtain yellow solid compound 603A (1.56 g, 65%). ¹ H NMR (CDCl ₃ ): δ, 7.38 (m, 3H, -NH-), 7.28 (m, 4H, trityl), 7.26 (m, 1H, -NH-), 7.18 (m, 1H, -NH-) ), 6.84 (m, 5H, trityl), 6.37 (m, 4H, trityl), 5.33 (m, 3H, sugar-H-4'), 5.16 (dd, J=3.4Hz, J=11.3Hz, 3H, sugar-H-3'), 4.77(d, J=8.4Hz, 3H, sugar-H-1'), 4.18-4.07(m, 3x2H, 3x1H, sugar-H-5', sugar-H-6' ), 3.94(m, 3H, sugar-H-2'), 3.77-3.53(m, 14H), 3.42(m, 2H, -NHCH ₂ -), 3.30-3.19(m, 2H, -CH ₂ NH- ), 2.42(m, 2H), 2.19(m, 4H), 2.15(s, 9H), 2.07(m, 2H), 2.05(s, 9H, 2.01(s, 9H), 1.96(s, 9H), 1.20(s, 3H).HRMS(ESI) m/z, C ₈₇ H ₁₄₃ N ₉ O ₄₄ , (M-trityl+H ⁺ )/2, theoretical value: 1010.55, observed value: 1010.4.

The conjugate (603B) triethylamine carboxylate was prepared according to the following route

Compound (603A) (1.5 g, 0.646 mmol) was dissolved in 30 mL of dry dichloromethane, and then 5 mL of triethylamine was added. 4-Dimethylaminopyridine (0.159 g, 1.3 mmol) was stirred and dissolved in the reaction solution, and succinic anhydride (0.13 g, 1.3 mmol) was stirred and dissolved in the reaction solution at room temperature, and the reaction was stirred for 8 hours. Succinic anhydride (32 mg, 0.32 mmol) was continued to be added and stirring was continued for 14 hours at room temperature. The above-reacted solution was poured into saturated brine, extracted with 2×50 mL of dichloromethane, the organic phase was separated, dried over anhydrous sodium sulfate, and evaporated to semi-dryness under reduced pressure. Purified by silica gel chromatography, using a gradient elution, first with mixed solvent (dichloromethane/methanol/triethylamine, 100:2; 1, v/v/v), followed by mixed solvent (dichloromethane/ Methanol/triethylamine, 100:5:1, v/v/v) rinse, and finally with mixed solvent (dichloromethane/methanol/triethylamine, 100:5:1, v/v/v) rinse The final product was obtained, and the solvent was evaporated under reduced pressure to obtain a white compound (603B) (1.56 g, 65%). ¹ H NMR(CDCl ₃ )δ, ¹ H NMR(CDCl ₃ ):δ, 7.39(m,3H,-NH-),7.28(m,5H,trityl),7.22(m,1H,-NH-), 7.10(m,1H,-NH-),6.80(m,4H,trityl),6.37(m,4H,trityl),5.32(m,3H,sugar-H-4'),5.29(s,2H), 5.15(dd,J=3.4Hz,J=11.3Hz,3H,sugar-H-3'),4.77(d,J=8.4Hz,3H,sugar-H-1'),4.18-4.07(m,3x2H ,3x1H,sugar-H-5',sugar-H-6'),3.94(m,3H,sugar-H-2'),3.67(m,9H),3.61-3.53(m,42H),3.42( m, 2H, -NHCH ₂ -), 3.30-3.19(m, 2H, -CH ₂ NH-), 2.42(m, 2H), 2.19(m, 4H), 2.15(s, 9H), 2.07(m, 2H), 2.05(s, 9H, 2.01(s, 9H), 1.96(s, 9H), 1.23(s, 3H).HRMS(ESI) m/z, C ₁₁₂ H ₁₆₅ N ₉ O ₄₉ , (MH ⁺ )/2, theoretical value: 1209.27 Measured value: 1209.83.

The solid phase support of the (603C) conjugated molecule was prepared by attaching the (603B) conjugated molecule to the solid support according to the following procedure

The conjugate hemisuccinate (603B) (50 mg, 0.021 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate The salt (10 mg, 0.026 mmol) was dissolved in 1.25 mL of anhydrous acetonitrile at room temperature. N,N-diisopropylethylamine (10 μL) was added to the reaction solution. After all the reagents were dissolved, 125 mg of long-chain aminoalkane glass sand (500°A, native lcaa-CPG, manufacturer Chemgenes, USA) was added. added to the above reaction solution. At room temperature, the solid and liquid phases were rotated and stirred at 300 rpm. After the reaction continued for 2 hours, the residue was filtered off, and the long-chain aminoalkane glass sand solid support was washed three times with acetonitrile (3×1 mL). Mix 0.5 mL of capping reagent A (acetic anhydride) in tetrahydrofuran with a concentration of (10%, v/v) and 0.5 mL of capping reagent B (N-methylimidazole in a mixed solvent of pyridine and acetonitrile with a concentration of (15%) : 10:75, v/v/v)) with long-chain aminoalkane glass sand for 1 hour at room temperature with rotary stirring. The reaction solution was filtered off, the long-chain aminoalkane glass sand solid phase carrier was rinsed three times with acetonitrile, and the long-chain aminoalkane glass sand solid phase carrier was dried under reduced pressure for 2 hours with a vacuum oil pump. A glass sand solid phase carrier (603C, 130 mg) was obtained. Weigh the long-chain aminoalkane glass sand 603C solid phase carrier (8.3 mg), add it to 100 mL of a 3% trichloroacetic acid solution in dichloromethane, rotate and stir for 30 seconds, and let stand for 1 minute. The visible light absorption was measured at 498 nm from the supernatant, and the absorbance was 0.309. The calculated loading of the conjugate 603C (that is, the loading of (603B) on CPG) was 53.25 μmol/g.

Preparation of TTR siRNA Sense and Antisense Sequences (Preparation of siRNA Conjugates)

The small interfering RNAs in the present invention are selected from double-stranded oligonucleotides, which include a sense strand and an antisense strand, and the sense and antisense strands are complementary, that is, in a double-stranded nucleic acid molecule, the bases of one strand and the other Bases on one strand pair in a complementary fashion through hydrogen bonding interactions. In double-stranded oligonucleotides, the purine base adenine (A) is paired with the pyrimidine bases thymine (T) or uracil (U); the purine base guanine (G) is always paired with a pyrimidine base Cytosine (C) is paired. The sequence of the two complementary strands is from 5'- to 3'-, and the other is from 3'- to 5'-ordered. Each nucleotide in the small interfering RNA is independently a modified or unmodified nucleotide, and modification means that the 2'-hydroxyl group is replaced by other groups. The modification at the 2'-position of the nucleotide can be selected from 2'-methoxy, 2'-fluoro, 2'-methoxyethyl, 2'-2,4-dinitrophenol, 2'-amino, 2'-4'-ring-locked ethyl and other groups. Each nucleoside in the sequence is linked by a phosphodiester bond, in which an oxygen atom in the phosphodiester bond is replaced by a sulfur atom to form a phosphorothioate bond. The sense strand and one antisense strand usually consist of 19 (or 21) nucleotides in length and form a double-stranded pair in a complementary manner. The sense strand nucleoside sequence is a stretch of nucleotides in the target mRNA. The antisense strand is usually linked with two consecutive deoxythymidine nucleotides or two consecutive uracil nucleotides. Target mRNAs generally refer to mRNAs in genes whose proteins are abnormally expressed in cells.

The preparation of functional oligonucleotide conjugates can be prepared according to the process shown in Figure 1:

According to the method of phosphoramidite solid-phase synthesis, the nucleoside monomers are connected one by one in the direction from 3' to 5' according to the above sequence sequence. Each linking of a nucleoside monomer includes four steps of deprotection, coupling, capping, and oxidation.

Preparation of solid-phase synthesis reagents :

The deprotection reagent is trichloroacetic acid or dichloroacetic acid in dichloromethane solution (3%, v/v). The nucleoside monomer is dissolved in anhydrous acetonitrile, the concentration is (0.05M-0.1M), an appropriate amount of molecular sieve 3A is added °Do anhydrous treatment. The coupling activator is anhydrous acetonitrile of 5-Ethylthio-1H-Tetrazole (5-Ethylthio-1H-Tetrazole), the concentration is (0.25M, or 0.45M), and the activator can also be 1H-tetrazolium 5-Benzylthio-1H-Tetrazole; 4,5-Dicyanoimidazole. The capping reagent A is a solution of acetic anhydride in tetrahydrofuran with a concentration of (10%, v/v). The capping reagent B is a mixed solvent of N-methylimidazole in pyridine and acetonitrile with a concentration of (15:10:75, v/v/v). The oxidizing reagent was iodine in water and pyridine (0.05M, 95 wt% pyridine in water). The sulfurizing reagent is N-Dimethylaminomethylidene)amino]-3H-1,2,4-dithiazoline-3-thione((dimethylamino-methylene)amino-3H-1-2,4-thiazole-3-thione ) at a concentration of (0.05M, pyridine/acetonitrile, 2:3, v/v). The cleavage and deprotection reagent is 28wt% concentrated ammonia water.

HLUnyLinker^TM 300，或500°A,native lcaa-CPG,厂商Chemgenes,USA)。Wherein, the solid-phase carrier for nucleotide solid-phase synthesis is a commercially available general-purpose solid-phase carrier (

HLUnyLinker ^™ 300, or 500°A, native lcaa-CPG, manufacturer Chemgenes, USA).

Steps of solid phase synthesis :

A solution of the 4,4'-dimethoxytrityl protecting group on the solid-phase support or the nucleoside monomer linked to the support and trichloroacetic acid in dichloromethane (3%, v/ v), the molar ratio is (1:30). The solid-phase reaction was performed at room temperature for 1.5 minutes, and the operation was repeated three times. When the color of the eluent of the solid-phase carrier changed from red to colorless, stop adding the deprotection solution dropwise. After repeated washing with anhydrous acetonitrile, add nucleoside monomer and activator (coupling activator, 5-ethylthiotetrazolium) (the ratio of nucleoside monomer and activator (molar ratio is 1:20) , the molar ratio of solid phase carrier and nucleoside monomer is 1:(5-6). The reaction time of room temperature reagent and solid phase is 3-4 minutes for one cycle, after two cycles, the reaction is stopped. Wash with anhydrous acetonitrile Then, add the oxidizing reagent solution, and the molar ratio of the solid phase carrier and the oxidizing agent is 1:6. The reaction time of the oxidizing reagent and the solid phase carrier at room temperature is about 2 minutes, and the operation is repeated twice. After the coupling reaction, if the vulcanization reaction step is required , add sulfidation reagent solution, the molar ratio of solid phase carrier and sulfidation agent is 1:6. The reaction time of oxidizing reagent and solid phase carrier at room temperature is about 4-5 minutes, repeat the operation twice. Cap protection reaction, add cap reaction reagent , the molar ratio of the solid phase carrier and the capping reagent is 1:80. The reaction time of the capping reagent and the solid phase carrier at room temperature is about 1-2 minutes, and the operation is repeated twice. The above steps of deprotection, coupling, oxidation, and capping are cycled until To complete the coupling of the last nucleotide. Transfer the solid phase carrier carrying the sense or antisense strand of the nucleic acid sequence to a small glass vial, add 28% aqueous ammonia solution, and tightly seal the glass cover at 55°C Under the temperature of , the base protecting group in the sense strand or antisense strand is hydrolyzed off, and the sense strand or antisense strand is hydrolyzed and separated from the solid phase carrier at the same time. The reaction lasted for 16 hours. The obtained small nucleic acid sequence chain was separated by hydrolysis. The solution is separated from the solid phase carrier by filtration. After concentration, the crude product of the small nucleic acid sequence chain is obtained.

Purification separation and desalting by preparative high pressure liquid chromatography

Small nucleic acids were purified by gradient elution of NaBr using a preparative anion exchange chromatography column (Source 15Q). Mobile phase A: 20 mM sodium phosphate (pH 8.0), mobile phase B: 20 mM sodium phosphate (pH 8.0), 1 M sodium bromide in 10% acetonitrile in water. The column temperature was 65°C. The flow rate was 10 mL/min. The elution gradient was started with mobile phase A followed by an increase in mobile phase B from 0% to 20% in 12 minutes. After 15 minutes, increase mobile phase B from 20% to 50%. The product eluates were collected and subjected to fractional analysis and fractional pooling. Desalting is carried out using reversed-phase chromatographic purification columns, or dialysis desalting. After concentration and lyophilization, purified small nucleotides were obtained. For the above-synthesized sense and antisense strands, anion exchange liquid chromatography (AEX-HPLC) was used to detect the purity, and reversed-phase liquid chromatography-mass spectrometry (LC-MS) was used to identify and analyze the molecular weight of the whole sequence. The theoretical values are in agreement, confirming the synthesized nucleic acid sequence.

annealing

The synthesized sense strand (S strand) and antisense strand (AS strand) were mixed in an equimolar ratio in physiological saline for injection, heated at 90°C for 5 minutes, then slowly cooled to room temperature, and stored at 4°C The siRNA conjugates (sequences of SNK-17 and SNK-18) can be obtained by placing them in a refrigerator for 12 hours to form a double-stranded structure through hydrogen bonds.

The siRNA targeting murine TTR is the sequence numbered SNK-17:

Chain of Justice (S):

5'-AfsmAsCfmAGfmUGfmUUfCfUfmUGfmCUfmCUfmAUfmAAf-EgGaGaGaC3 (base sequence shown in SQE ID NO: 1)

Antisense Strand (AS):

5'-mUsUfsmAUfmAGfmAGfmCAfmAmGmAAfmCAfmCUfmGUfmUsmUsmU (base sequence shown in SQEID NO: 2)

The siRNA targeting murine TTR is the sequence numbered SNK-18:

Chain of Justice (S):

5'-AfsmAsCfmAGfmUGfmUUfCfUfmUGfmCUfmCUfmAUfmAAf-TriGalNac (base sequence shown in SQE ID NO: 1)

Antisense Strand (AS):

5'-mUsUfsmAUfmAGfmAGfmCAfmAmGmAAfmCAfmCUfmGUfmUsmUsmU (base sequence shown in SQEID NO: 2)

S: sense strand; AS: antisense strand

Among them, capital letters C, G, U, A represent the base composition of nucleotides; lowercase letter m represents that the adjacent nucleotide to the right of the letter m is a 2'-methoxy-modified nucleotide (ie The pentose sugar 2'-OH of the nucleotide is replaced by a methoxy group); the lowercase letter f indicates that the adjacent nucleotide to the left of the letter f is a 2'-fluorine modified nucleotide (that is, the pentane of the nucleotide is The sugar 2'-OH is replaced by fluorine); the lowercase letter s indicates that the two nucleotides adjacent to the letter s are connected by a phosphorothioate bond (that is, the non-bridging oxygen atom in the phosphodiester bond is Sulfur atom substitution); there are no other letters between two nucleotides adjacent to the left and right are indicated as phosphodiester linkages. TriGalNac represents the moiety other than Nu in formula (603), Eg is ethylene glycol, and three Ga represent a single N-acetylgalactosamine (formula (101)) (606) prepared by solid-phase synthesis ), C3 is 1,3-propanediol.

The liquid mass analysis data confirmed that the sense strand (S) in the sequence of SNK-17, the theoretical value of HRMS (ESI) m/z: 8704.23; the measured value: 8704.0; HRMS(ESI) m/z theoretical value: 7595.43, measured value: 7595.0. Sense strand (S) in the sequence of SNK-18, HRMS (ESI) m/z theoretical value: 8503.94; Measured value: 8503.1; antisense strand (A) in the sequence of SNK-18, HRMS (ESI) m/z The theoretical value of z is: 7595.43, and the measured value is: 7595.0.

Activity test (bioactivity test targeting murine TTR)

Female C57BL/6 mice (4-6 weeks old) were randomly divided into groups by body weight, injected with 5 mg/kg of the siRNA conjugate (SNK-17) obtained above on day 0, and the livers were harvested after killing the mice on day 21 Tissue, RNA was extracted according to the instructions of the RNAeasy Mini Kit (QIAGEN, Cat. No. 74104). The control group was administered by injecting normal saline without drug. RT-PCR was performed according to the recommendations of High Capacity cDNA Reverse Transcription Kits (Thermo Fisher, Cat. No. 4368814), and each reaction contained 1 μg of RNA. Gene expression was quantified by real-time fluorescent PCR. The TaqMan probe for mouse TTR was Mm00443267_m1, and the probe for the internal reference gene (mouse HPRT) was Mm03024075_m1 (Thermo Fisher Scientific, Waltham, MA, USA). PCR conditions were 1 cycle of 95°C for 20 seconds, 40 cycles of 95°C for 1 second and 60°C for 20 seconds, and the real-time fluorescence PCR instrument was StepOne Plus (Thermo Fisher). The TTR gene expression was calculated as 2^-ΔΔCt, and the mouse HPRT gene expression was used as an internal reference. The expression of TTR gene was compared with the expression of liver TTR gene of control mice (see Figure 2).

Female C57BL/6 mice (4-6 weeks old) were randomly divided into groups according to body weight, injected with 5 mg/kg siRNA (SNK-18) on days 0, 2, and 4, and liver tissues were collected after killing the mice on day 21. RNAeasy Mini kit (QIAGEN, Cat. No. 74104) was instructed to extract RNA. The control group was administered by injecting normal saline without drug. RT-PCR was performed according to the recommendations of HighCapacity cDNA Reverse Transcription Kits (Thermo Fisher, Cat. No. 4368814), and each reaction contained 1 μg of RNA. Gene expression was quantified by real-time fluorescent PCR. The TaqMan probe for mouse TTR was Mm00443267_m1, and the probe for the internal reference gene (mouse HPRT) was Mm03024075_m1 (Thermo Fisher Scientific, Waltham, MA, USA). PCR conditions were 1 cycle of 95°C for 20 seconds, 40 cycles of 95°C for 1 second and 60°C for 20 seconds, and the real-time fluorescence PCR instrument was StepOne Plus (Thermo Fisher). The TTR gene expression was calculated as 2^-ΔΔCt, and the mouse HPRT gene expression was used as an internal reference. The expression of TTR gene was compared with the expression of liver TTR gene of control mice (see Figure 3).

It can be seen from Fig. 2 and Fig. 3 that the conjugate of the present invention has the performance of directional delivery of small nucleic acid, and has strong in vivo stability. Further experiments show that the double-stranded oligonucleotides or oligonucleotide conjugates provided by the present invention have higher stability, lower toxicity and/or higher inhibitory activity of liver TTR mRNA expression in vivo.

The siRNA-100 with the base sequence of the sense strand being 5'-CUAGGAGGCUGUAGGCAUAAA-3' (SEQ ID NO: 3) and the base sequence of the antisense strand being 5'-UUUAUGCCUACAGCCUCCUAG-3' (SEQ ID NO: 4) was used as the active drug Nu can obtain better effect of inhibiting the replication of hepatitis B virus. Further, siRNA-100 was modified in the following manner so that the modified siRNA had a more stable nucleic acid structure, higher specificity and affinity base pairing; and the siRNA with the following modifications showed more excellent in vivo inhibitory effect , can further reduce the immunogenicity of the siRNA of the present invention in vivo (see the description of the invention titled "Nucleic acid targeting hepatitis B virus and its use"):

5'- C s U s AGGA Gf G Cf U Gf UAGGCAUAAA -3'

5'- U sUfs U Af U Gf CCUACAG Cf C Uf CCUAG s U s U -3'

Among them, the lowercase letter f indicates that a nucleotide adjacent to the left of the letter f is a 2'-fluorine modified nucleotide (that is, the 2'-OH of the pentose sugar of the nucleotide is replaced by fluorine); the lowercase letter s indicates that The two nucleotides adjacent to the letter s are connected by a phosphorothioate bond (that is, the non-bridging oxygen atom in the phosphodiester bond is replaced by a sulfur atom); the underlined nucleotide represents the nucleus The 2' hydroxyl group of the glucoside is replaced by a methoxy group.

The preferred embodiments of the present invention have been described above in detail, however, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, a variety of simple modifications can be made to the technical solutions of the present invention, including combining various technical features in any other suitable manner. These simple modifications and combinations should also be regarded as the content disclosed in the present invention. All belong to the protection scope of the present invention.

SEQUENCE LISTING

<110> Synerk Biotech Limited

<120> Compounds, conjugates and uses thereof

<130> I66088SNKB-CJ

<150> 202110049158.8

<151> 2021-01-14

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 21

<212> RNA

<213> Artificial Sequence

<220>

<223> The sequence is synthesized

<400> 1

aacaguguuc uugcucuaua a 21

<210> 2

<211> 23

<212> RNA

<213> Artificial Sequence

<220>

<223> The sequence is synthesized

<400> 2

uuauagagca agaacacugu uuu 23

<210> 3

<211> 21

<212> RNA

<213> Artificial Sequence

<220>

<223> The sequence is synthesized

<400> 3

cuaggaggcu guaggcauaa a 21

<210> 4

<211> 21

<212> RNA

<213> Artificial Sequence

<220>

<223> The sequence is synthesized

<400> 4

uuuaugccua cagccuccua g 21

Claims (10)

1. a compound M is characterized in that, this compound M has the structure shown in formula (V) or (V'):

In formula (V) or (V'), A is an aptamer, which has a structure derived from a carbohydrate or a polypeptide; L1 is a straight-chain group having an ether bond and/or an amide bond with a length of 1-50 carbon atoms; Z has a structure derived from a first phosphorous compound selected from one of phosphoramidites, H-phosphates, and phosphoric triesters, and capable of covalently covalently passing through a phosphoric acid ester bond or a phosphoramidite diester bond The way of the bond is connected with the adjacent group; L2 is provided by having a structure of formula (I), (II), (III) or (IV):

Wherein, R in formula (I), (II), (III) or (IV) is each independently H or hydroxyl protecting group; R ¹ and R ² in formula (I) and R ⁴ in formula (II) all have a covalent bonding site; In formula (III) or (IV), n is an integer of 1-10; In formula (II), (III) or (IV),

Represents the structure obtained by connecting active groups through active sites; wherein, the active sites are sites connected by covalent bonds; the active groups are selected from phosphoramidite, H-phosphate, phosphoric acid One of ester group and polyhydroxyalkyl group. 2. The compound according to claim 1, wherein L2 is provided by a structure having a structure selected from the structures represented by formula (A1)-(A24):

Wherein, m or n in formula (A2)-(A24) are each independently an integer of 1-10;

Represents the structure obtained by connecting active groups through active sites; wherein, the active sites are sites connected by covalent bonds; the active groups are selected from phosphoramidite, H-phosphate, phosphoric acid One of ester group and polyhydroxyalkyl group; In formula (A2)-(A24), -OH represents a hydroxyl group or a hydroxyl group protected by a hydroxyl protecting group, and the hydroxyl protecting group is selected from 4,4'-dimethoxytrityl, 4-methoxytrityl One of benzyl, trityl, tert-butyldimethylsilyl, 4-oxopentanoyl, 2-cyanoethyl, 4-pentenoyl and acyloxyalkyl; And/or, L1 is selected from one of the following groups: -O-[ _CH2CH2O ] _n- , -[ _CH2 ] _m -CONH-[ _CH2 ] _nO- and -O-[ _{CH 2} CH ₂ O] _m -CONH-[CH ₂ ] _n O-; wherein, m and n are each independently an integer of 1-10; And/or, the sugar is selected from one of monosaccharides, disaccharides, trisaccharides and polysaccharides; preferably, the sugar is N-acetylgalactosamine; And/or, Z has a structure represented by formula (B1), (B2), (B3), (B4), (B5) or (B6):

Wherein, M in formula (B3) is selected from a kind of in triethylamine, trimethylamine, triisopropylamine and tripropylamine; Wherein, R' in formula (B4)-(B6) is independently selected from 2, One of 2,2-trichloroethyl, phenyl, o-chlorophenyl, cyanoethyl and Nu; Nu is the active drug; in (B1), (B2), (B3) or (B4)

Indicates sites linked by covalent bonds. 3. The compound according to claim 1 or 2, wherein the compound M is represented by formula (101), (102), (201), (202), (203), (301) or (302) Structure:

4. A conjugate N, characterized in that, the conjugate N has the structure shown in formula (VI): (M)m-R” (VI) In formula (VI), m is an integer of 1-4, M is provided by a compound having the structure shown in formula (V) or (V') in claim 1, and when m is greater than 1, multiple Ms are the same or different; R" has an active site capable of being linked to Nu; preferably, Nu is an active drug; Preferably, the conjugate N has a structure represented by formula (a), (b), (c), (d) or (e):

Wherein, in formula (a), (b), (c), (d) or (e), n, n ₁ or n ₂ are each independently an integer of 1-10, preferably an integer of 1-6; m is each independently an integer of 1-10, preferably an integer of 1-4; X is an oxygen atom or sulfur atom; L1' is a straight-chain group having an ether bond and/or an amide bond with a length of 1-50 carbon atoms; A' is an aptamer with a structure derived from a sugar or a polypeptide; In formula (a), R ⁵ is each independently selected from H, C1-C10 alkyl, C1-C10 haloalkyl or C1-C10 alkoxy; In formula (c), R ⁶ is selected from H, C1-C10 alkyl, C1-C10 haloalkyl or C1-C10 alkoxy; R ⁷ is H or a hydroxyl protecting group, and the hydroxyl protecting group is selected from 4,4 '-Dimethoxytrityl, 4-methoxytrityl, trityl, tert-butyldimethylsilyl, 4-oxopentanoyl, 2-cyanoethyl, One of 4-pentenoyl and acyloxyalkyl, preferably, R ⁷ is 2-cyanoethyl; In formula (e), R ⁸ is H or a hydroxyl protecting group, and the hydroxyl protecting group is selected from 4,4'-dimethoxytrityl, 4-methoxytrityl, and trityl , one of tert-butyldimethylsilyl, 4-oxopentanoyl, 2-cyanoethyl, 4-pentenoyl and acyloxyalkyl, preferably 2-cyanoethyl; in,

represents the site of covalent linkage to Nu or an adjacent group, or

Indicates a group to which Nu is attached by covalent bonds. 5. A conjugate T, characterized in that, the conjugate T has the structure shown in formula (VIII):

In formula (VIII), at least one of M1, M2 and M3 has a structure derived from compound M, wherein the structure of said compound M is the same as defined in any one of claims 1-3; or, M1, M2 and M3 are each independently The ground is obtained by connecting A, L1, L2 or Z described in any one of claims 1-3 in any number and in any manner; M1, M2 and M3 are the same or different; Preferably, M1, M2 and M3 all have the structure from compound M according to any one of claims 1-3, the same or different; L3 is provided by the structure having the formula (VII):

Wherein, Y is a substituted or unsubstituted linear group with a length of 1-10 carbon atoms, wherein one or more carbon atoms are optionally replaced by one or more -CONH-; preferably, Y is - CONH- or -CH ₂ -; In formula (VII), Ra' is each independently H or a hydroxyl protecting group; the hydroxyl protecting group is selected from 4,4'-dimethoxytrityl, 4-methoxytrityl, trityl One of benzyl, tert-butyldimethylsilyl, 4-oxopentanoyl, 2-cyanoethyl, 4-pentenoyl and acyloxyalkyl; R ⁹ and R ¹⁰ are each independently H or a straight chain group of 1-70 carbon atoms in length containing a hydroxyl group and/or a hydroxyl group protected by a hydroxyl protecting group at the end group, wherein one or more carbon atoms are optionally is replaced by one or more selected from the group consisting of C(O), NH, O, and S; and wherein R ⁹ and R ¹⁰ each independently optionally have the following groups Substituents of any one or more of the group consisting of: C1-C10 alkyl, C6-C10 aryl, C5-C10 heteroaryl, C1-C10 haloalkyl, -OC1-C10 alkyl , -OC1-C10 alkylphenyl, -C1-C10 alkyl-OH, -OC1-C10 halogenated alkyl, -SC1-C10 alkyl, -SC1-C10 alkylphenyl, -C1- C10 alkyl-SH, -SC1-C10 haloalkyl, halogen substituent, -OH, -SH, -NH ₂ , -C1-C10 alkyl-NH ₂ , -N (C1-C10 alkyl) (C1-C10 alkyl), -NH (C1-C10 alkyl), cyano, nitro, -CO ₂ H, -C(O)O (C1-C10 alkyl), -CON (C1 -C10 alkyl) (C1-C10 alkyl), -CONH (C1-C10 alkyl), -CONH ₂ , -NHC(O) (C1-C10 alkyl), -NHC(O)( Phenyl), -N(C1-C10 alkyl)C(O)(C1-C10 alkyl), -N(C1-C10 alkyl)C(O)(phenyl), -C(O)C1 -C10 alkyl, -C(O)C1-C10 alkylphenyl, -C(O)C1-C10 haloalkyl, -OC(O)C1-C10 alkyl, -SO ₂ (C1-C10 alkyl), -SO ₂ (phenyl), -SO ₂ (haloalkyl of C1-C10), -SO ₂ NH ₂ , -SO ₂ NH (alkyl of C1-C10), -SO ₂ NH (benzene base), -NHSO ₂ (C1-C10 alkyl), -NHSO ₂ (phenyl) and -NHSO ₂ (C1-C10 haloalkyl); Preferably, R ⁹ and R ¹⁰ each independently have one or more groups selected from the following groups, which are obtained by connecting in any number and in any manner: -NH ₂ -, -CH ₂ -, -O-, -CONH-, -(OCH ₂ CH ₂ )n-, Z',

Preferably, Z' has a structure derived from the second phosphorus compound and is covalently linked to other groups through a phosphate bond or a phosphoramide diester bond; Preferably, Z' has a structure represented by formula (C1) or (C2):

Preferably, Q and Q' are each independently O or S; R ¹¹ and R ¹² are each independently selected from H, C1-C10 alkyl, C1-C10 haloalkyl, or C1-C10 alkoxy; and wherein , R ¹¹ and R ¹² each independently contain a group as described below: nitrile group, amino group, hydroxyl group, carboxyl group or amide group; More preferably, R ⁹ and R ¹⁰ each independently have a structure represented by formula (D1), (D2) or (D3):

Wherein, m is an integer of 0-6; M ⁺ is triethylamine cation; In formula (D1)-(D3), Rk' is independently selected from H, hydroxyl protecting group or Nu; the hydroxyl protecting group is selected from 4,4'-dimethoxytrityl, 4-methoxy one of trityl, trityl, tert-butyldimethylsilyl, 4-oxopentanoyl, 2-cyanoethyl, 4-pentenoyl and acyloxyalkyl ; Nu is the active drug. 6. The conjugate of claim 5, wherein the L3 is provided by a structure having the formula (E1)-(E12):

Wherein, in formula (E1)-(E12), m and n are integers of 1-10; R _b -R _k and Rk' are each independently selected from H, hydroxyl protecting group or Nu; the hydroxyl protecting group is selected from 4,4'-Dimethoxytrityl, 4-methoxytrityl, trityl, tert-butyldimethylsilyl, 4-oxopentanoyl, 2-cyano One of ethyl, 4-pentenoyl and acyloxyalkyl; Nu is the active drug. 7. The conjugate according to claim 5 or 6, wherein the conjugate T has a structure represented by formula (IX) or formula (X):

In formula (IX) or formula (X), the structure of A, L1, L2 or Z' is the same as that defined in any one of claims 1-3 and 5; Z' has a compound derived from the compound shown in claim 5 or 6 The structure of Z'. 8. The conjugate according to claim 5 or 6, wherein the conjugate T has a structure represented by formula (IX-1) or formula (X-1):

In formula (IX-1) or formula (X-1), m and n are each independently an integer of 0-6; Rt and Rt' are each independently selected from H, hydroxyl protecting group, active drug Nu, solid phase carrier , C2-C6 carboxyl group, carboxylate or amide group; the hydroxyl protecting group is preferably selected from 4,4'-dimethoxytrityl, 4-methoxytrityl, trityl , one of tert-butyldimethylsilyl, 4-oxopentanoyl, 2-cyanoethyl, 4-pentenoyl and acyloxyalkyl. 9. The conjugate of claim 5 or 6, wherein the conjugate T has formula (601), (602), (603), (604), (605), (606) or ( 607) shown in the structure:

In formula (601), (602), (603), (604), (605), (606) or (607), X is an oxygen atom or a sulfur atom. 10. Use of the compound M according to any one of claims 1-3 and the conjugate according to any one of claims 4 and 5-9 in the preparation of small nucleic acid drugs; Preferably, the specific target gene of the small nucleic acid in the small nucleic acid drug is selected from at least one of PCSK9, HBV, TTR and AGT.

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