GalNAc compound containing triazole structure and oligonucleotide conjugate thereof

Abstract

The present invention provides a GalNAc compound containing a triazole structure and an oligonucleotide conjugate thereof, specifically disclosing a GalNAc compound as shown in formula (I) or a pharmaceutically acceptable salt thereof. The GalNAc compound in the present invention can form a conjugate with an oligonucleotide, and the conjugate can achieve efficient liver-targeted delivery of the oligonucleotide, regulate gene expression, and can be used to prevent and / or treat diseases caused by the expression of specific genes in liver cells. #imgabs0#

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a GalNAc compound having a triazole ring structure and a GalNAc oligonucleotide conjugate prepared therefrom. Background Art

[0002] In recent years, following small molecule and antibody drugs, oligonucleotide drugs are gradually becoming the third wave of new drug research and development. Oligonucleotide drugs mainly bind to the upstream mRNA of pathogenic proteins and can regulate at the gene level. Therefore, they can provide treatment possibilities for some urgent clinical needs that are not available for drugs, and have shown great potential in the treatment of neuropathy and cardiovascular diseases.

[0003] Small nucleic acid drugs (oligonucleotides) are gradually developing into alternatives to traditional small molecule drugs due to their unique properties, and are used to regulate the functions of disease-related proteins. Oligonucleotides can be used to silence or activate the gene expression of specific diseases, thereby preventing or promoting the formation of specific proteins and playing a role in treating diseases. Oligonucleotides include but are not limited to antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), small activating RNAs (saRNAs), and microRNAs (microRNAs, miRNAs). Due to the good efficacy of oligonucleotide drugs and the breakthroughs in technology, they have become the most popular type of technology at present, and several drugs have been approved for marketing worldwide.

[0004] Currently, the delivery systems used for oligonucleotide drugs on the market include: lipid nanoparticle (LNP) delivery system and N-acetylgalactosamine (GalNAc) delivery system, among which the GalNAc delivery system is the most widely used. GalNAc can combine with ASGPR (asialoglycoprotein receptor), an endocytic receptor specifically expressed on the surface of hepatocytes, to achieve liver-targeted delivery of oligonucleotide drugs. Therefore, improvements in GalNAc can improve the delivery efficiency of oligonucleotide drugs.

[0005] Galactose (Gal) and N-acetylgalactosamine (GalNAc) are ligands that can bind to ASGPR on the liver surface. The affinity of GalNAc for binding to ASGPR is about 50 times higher than that of Gal. Studies have found that the order of affinity of GalNAc with different antennae is: tetra-antennae>tri-antennae>>bi-antennae>>mono-antennae galactosamine. In recent years, some progress has been made in the liver-targeted delivery of nucleic acid drugs using N-acetylgalactosamine (GalNAc), a high-affinity ligand of ASGPR, as a targeting molecule. Various GalNAc can be conjugated with siRNA and ASO, and this technology has been used to develop drugs for diseases such as amyloid lesions, hemophilia, hypercholesterolemia, and hepatitis B.

[0006] Different GalNAc structures have very different nucleic acid delivery efficiencies. In order to improve the delivery efficiency of liver-targeted drugs, such as complement inhibitors, there is a need in the art to develop new GalNAc compounds. Summary of the invention

[0007] The present invention provides a novel GalNAc compound containing a triazole structure, and the triazole is introduced into the linker of the GalNAc compound. With the advantage of click chemistry, a series of novel GalNAc compounds containing a triazole structure can be constructed modularly, simply and quickly. By using the phosphoramidite solid phase synthesis method, the GalNAc ligand is coupled to the 3' end of the oligonucleotide. Compared with the prior art, the efficiency of the GalNAc compound of the present invention in delivering the oligonucleotide to the liver is significantly improved, and it can be used to develop drugs for treating liver tissue-related diseases.

[0008] The present invention provides a GalNAc compound represented by formula (I) or a pharmaceutically acceptable salt thereof:

[0009]

[0010] Wherein, L1 is -HNC(O)- or -C(O)NH-;

[0011] L2 is -(CH2)n1- or -(CH2CH2O)n2CH2-, wherein n1 and n2 are integers of 1-7;

[0012] Z1 is -(CH2)n3-, wherein n3 is an integer from 1 to 7;

[0013] L3 is -HNC(O)- or -C(O)NH-;

[0014] n is an integer from 0 to 10;

[0015] L4 is -(CH2)n4-, wherein n4 is an integer from 0 to 7;

[0016] A is , R1 is hydrogen, C 1-6 Alkyl, C 1-6 Alkoxy or halogen; R2 is 4, 4'-dimethoxytrityl, monomethoxytrityl, trityl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triisopropylsilyl or isopropyldimethylsilyl; R3 is hydrogen, hydroxyl protecting group, phosphorus-containing reactive group, -CO(CH2) x CONH-E or -CO(CH2) x COOH, wherein x is an integer of 1 to 10, and E is controlled pore glass (CPG) or polystyrene;

[0017] B is oxygen or sulfur;

[0018] G is ;

[0019] in,

[0020] X1 is -(CH2) a -or-(CH2CH2O) a CH2-, a is an integer from 1 to 5;

[0021] X2 is -HNC(O)- or -C(O)NH-;

[0022] X3 is -(CH2) b -, b is an integer from 1 to 6;

[0023] X4 , where c, d are integers from 0 to 5;

[0024] Y1 is 0 or 1;

[0025] Y2 is 0 or 1;

[0026] Y3 is 1, 2 or 3;

[0027] When Y3 is 1, K is CH2; when Y3 is 2, K is CH; when Y3 is 3, K is carbon.

[0028] In some embodiments of the present invention, L1 is -C(O)NH-, and the * terminus is connected to G.

[0029] In some embodiments of the present invention, L2 is -(CH2)3-, -(CH2)4-, -(CH2)5-, -(CH2)6-, -(CH2)7- or -(CH2CH2O)3CH2-.

[0030] In some embodiments of the present invention, L2 is -(CH2)5- or -(CH2CH2O)3CH2-.

[0031] In some embodiments of the present invention, n is zero.

[0032] In some embodiments of the present invention, L4 is absent, -(CH2)3-, -(CH2)4-, -(CH2)5-, -(CH2)6-, or -(CH2)7-.

[0033] In some embodiments of the present invention, L4 is absent, -(CH2)3-, or -(CH2)5-.

[0034] In some embodiments of the present invention, R1 is hydrogen or methoxy.

[0035] In some embodiments of the present invention, R2 is 4,4'-dimethoxytrityl (DMTr).

[0036] In some embodiments of the present invention, R3 is -CO(CH2)2CONH-E, and E is a controlled pore glass.

[0037] In some embodiments of the present invention, B is oxygen.

[0038] In some embodiments of the present invention, X1 is -(CH2)3-.

[0039] In some embodiments of the present invention, X2 is -HNC(O)-**, and the ** end is connected to X3.

[0040] In some embodiments of the present invention, X3 is -(CH2)2-, -(CH2)4-, or -(CH2)5-.

[0041] In some embodiments of the present invention, X4 is , the *** end is connected to X3.

[0042] In some embodiments of the present invention, Y1 is 1.

[0043] In some embodiments of the present invention, Y2 is 1.

[0044] In some embodiments of the present invention, Y3 is 3 and K is carbon.

[0045] In some embodiments of the present invention, A is or , E is a controlled pore glass.

[0046] In some embodiments of the invention, G is:

[0047] ,

[0048] or

[0049] .

[0050] In some embodiments of the present invention, the compound of formula (I) has a structure as shown in formula (IA):

[0051] ,

[0052] Wherein, L1, L2, L4, B, A, X1, X2, X3, X4, Y1 and Y2 are defined as above.

[0053] In some embodiments of the present invention, the compound of formula (I) or a pharmaceutically acceptable salt thereof is capable of binding to asialoglycoprotein receptor (ASGPR).

[0054] In some embodiments of the present invention, the compound represented by formula (I) is any of the following compounds:

[0055] ,

[0056] , , , or .

[0057] The present invention also provides a conjugate as shown in formula (IIA) or (IIB) or a pharmaceutically acceptable salt thereof,

[0058] or

[0059] Wherein, Oligo represents oligonucleotide, X represents hydroxyl or thiol, G1 represents ;

[0060] L1, L2, Z1, L3, L4, n, R1, R2, R3, X1, X2, X3, X4, Y1, Y2, Y3 and K are defined as above.

[0061] It will be understood by those skilled in the art that in the conjugates shown in formula (IIA) or (IIB), the structural unit The oligonucleotide is synthesized in solid phase by reacting a monomer with a five-membered sugar ring (i.e. or ) is formed by connecting the hydroxyl groups in

[0062] In the conjugate represented by formula (IIA), R3 is preferably hydrogen.

[0063] In the conjugate represented by formula (IIB), R2 is preferably hydrogen.

[0064] In some embodiments of the present invention, the oligonucleotide is selected from any one of the group consisting of small interfering nucleotides (siRNA), DNA, micro RNA (miRNA), small activating RNA (saRNA), small guide RNA (sgRNA), transfer RNA (tRNA), antisense oligonucleotides (ASO) and aptamers, or a combination of at least two of them, preferably antisense oligonucleotides (ASO) or small interfering nucleotides (siRNA).

[0065] In some embodiments of the present invention, each nucleotide in the antisense oligonucleotide (ASO) or small interfering nucleotide (siRNA) is independently a modified or unmodified nucleotide.

[0066] In some embodiments of the invention, the oligonucleotide modulates expression of a target gene.

[0067] In some embodiments of the invention, the oligonucleotide is a double-stranded RNAi agent.

[0068] In some embodiments of the present invention, the double-stranded RNAi agent is a double-stranded RNAi agent that inhibits the expression of the PCSK9 gene.

[0069] In some embodiments of the invention, at least one strand of the double-stranded RNAi agent comprises a 3' overhang of at least 1 nucleotide.

[0070] In some embodiments of the invention, at least one strand of the double-stranded RNAi agent comprises a 3' overhang of at least 2 nucleotides.

[0071] In some embodiments of the invention, the double-stranded region of the double-stranded RNAi agent is 15-30 nucleotide pairs in length.

[0072] In some embodiments of the invention, the double-stranded region of the double-stranded RNAi agent is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotide pairs in length.

[0073] In some embodiments of the invention, the double-stranded region of the double-stranded RNAi agent is 17-25 nucleotide pairs in length.

[0074] In some embodiments of the invention, the double-stranded region of the double-stranded RNAi agent is 19-23 nucleotide pairs in length.

[0075] In some embodiments of the invention, the double-stranded region of the double-stranded RNAi agent is 21 nucleotide pairs in length.

[0076] In some embodiments of the invention, each strand of the double-stranded RNAi agent has 15-30 nucleotides.

[0077] In some embodiments of the present invention, the number of nucleotides of the sense strand and / or antisense strand of the double-stranded RNAi agent is each selected from any one of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30.

[0078] In some embodiments of the invention, each strand of the double-stranded RNAi agent has 19-25 nucleotides.

[0079] In some embodiments of the invention, the double-stranded RNAi agent has a sense strand of 21 nucleotides and an antisense strand of 23 nucleotides.

[0080] In some embodiments of the present invention, the double-stranded RNAi agent comprises any one or a combination of at least two selected from the group consisting of an oligonucleotide duplex formed by pairing an unmodified sense strand and an unmodified antisense strand as listed in the following table (the sequence is from Table 8 of the specification of CN117210468A):

[0081]

[0082] In some embodiments of the present invention, the sense strand of the double-stranded RNAi agent has 1 to 3 nucleotides substituted, added or deleted compared to any one of SEQ ID NOs. 1-12.

[0083] In some embodiments of the present invention, the antisense strand of the double-stranded RNAi agent has 1 to 3 nucleotides substituted, added or deleted compared to any one of SEQ ID NOs. 13-24.

[0084] In some embodiments of the invention, the double-stranded RNAi agent comprises at least one modified nucleotide selected from the group consisting of deoxy-nucleotides, 3'-terminal deoxy-thymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally restricted nucleotides, restricted ethyl nucleotides, abasic nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, The invention also provides any one of the following: modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing non-natural bases, tetrahydropyran-modified nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing thiophosphate groups, nucleotides containing methylphosphate groups, nucleotides containing 5'-phosphates, and nucleotides containing 5'-phosphate mimetics, or a combination of at least two of the following:

[0085] In some embodiments of the present invention, the nucleotide modification of the sense strand and the antisense strand of the double-stranded RNAi agent is chemical modification of the 2' position of the ribose of the nucleotide.

[0086] In some embodiments of the present invention, the chemical modification of the 2' position of the nucleotide ribose of the double-stranded RNAi agent is selected from any one of the group consisting of 2'-methoxy modification, 2'-O-methoxyethyl modification, 2'-fluoro modification, 2'-benzyloxy modification, 2'-methylcarbonylamino modification and 2'-pyridylmethoxy modification, or a combination of at least two of them.

[0087] In some embodiments of the present invention, the chemical modification of the 2' position of each ribose nucleotide of the double-stranded RNAi agent is selected from a combination of 2'-methoxy and 2'-fluoro.

[0088] In some embodiments of the present invention, the chemical modification of the 2' position of each nucleotide ribose of the double-stranded RNAi agent is as follows: the odd-numbered positions of the sense chain are all 2'-fluoro modified, and the even-numbered positions are all 2'-methoxy modified; the odd-numbered positions of the antisense chain are all 2'-methoxy modified, and the even-numbered positions are all 2'-fluoro modified.

[0089] In some embodiments of the present invention, the double-stranded RNAi agent nucleotide monomers are connected by 3',5'-phosphodiester bonds.

[0090] In some embodiments of the present invention, the double-stranded RNAi agent nucleotide monomers are connected by thio-modified 3',5'-phosphodiester bonds.

[0091] In some embodiments of the invention, the double-stranded RNAi agent has the following modifications:

[0092] The antisense strand has any of the following A, B, or C modifications:

[0093]

[0094] The positive strand has any of the following a and b modifications:

[0095]

[0096] In the table, 2'-OMe is 2'-methoxy; 2'-F is 2'-fluoro; and PS is a thiophosphate skeleton.

[0097] In some embodiments of the present invention, the antisense strand of the double-stranded RNAi agent has any one of the following modifications (I) to (X):

[0098] (I) From the 5'-3' direction, the 2nd, 3rd, 4th, 6th, 14th, and 16th positions are all 2'-fluorinated, the 7th position is GNA-modified, and the other positions are all 2'-methoxy-modified; the 1st and 2nd nucleotides, the 2nd and 3rd nucleotides, the 1st and 2nd last nucleotides, and the 2nd and 3rd last nucleotides are all connected by phosphorothioate bonds;

[0099] (II) From the 5'-3' direction, the 2nd, 3rd, 4th, 6th, 14th, and 16th positions are all 2'-fluorinated, and the other positions are all 2'-methoxy modified; the 1st and 2nd nucleotides, the 2nd and 3rd nucleotides, the 1st and 2nd last nucleotides, and the 2nd and 3rd last nucleotides are all connected by phosphorothioate bonds;

[0100] (III) From the 5'-3' direction, the 2nd, 3rd, 4th, 6th, 14th and 16th positions are all 2'-fluorinated, and the other positions are all 2'-methoxy modified; the 1st and 2nd nucleotides, the 2nd and 3rd nucleotides, the 1st and 2nd last nucleotides, and the 2nd and 3rd last nucleotides are all connected by phosphorothioate bonds; the 1st position is a 5'-trans vinyl phosphate nucleotide;

[0101] (IV) From the 5'-3' direction, the 2nd, 4th, 5th, 6th, 14th and 16th positions are all 2'-fluorinated, the 7th position is GNA-modified, and the other positions are all 2'-methoxy-modified; the 1st and 2nd nucleotides, the 2nd and 3rd nucleotides, the 1st and 2nd last nucleotides, and the 2nd and 3rd last nucleotides are all connected by phosphorothioate bonds; the 1st position is a 5'-trans-vinyl phosphate nucleotide;

[0102] (V) From the 5'-3' direction, the 2nd, 4th, 5th, 6th, 14th, and 16th positions are all 2'-fluorinated, and the other positions are all 2'-methoxy modified; the 1st and 2nd nucleotides, the 2nd and 3rd nucleotides, the 1st and 2nd last nucleotides, and the 2nd and 3rd last nucleotides are all connected by phosphorothioate bonds;

[0103] (VI) From the 5'-3' direction, the 2nd, 4th, 5th, 6th, 14th and 16th positions are all 2'-fluorinated, and the other positions are all 2'-methoxy modified; the 1st and 2nd nucleotides, the 2nd and 3rd nucleotides, the 1st and 2nd last nucleotides, and the 2nd and 3rd last nucleotides are all connected by phosphorothioate bonds; the 1st position is a 5'-trans vinyl phosphate nucleotide;

[0104] (VII) From the 5'-3' direction, the 2nd, 6th, 14th and 16th positions are all 2'-fluorinated, the 7th position is GNA modified, and the other positions are all 2'-methoxy modified; the 1st and 2nd nucleotides, the 2nd and 3rd nucleotides, the 1st and 2nd last nucleotides, and the 2nd and 3rd last nucleotides are all connected by phosphorothioate bonds;

[0105] (VIII) From the 5'-3' direction, the 2nd, 6th, 14th and 16th positions are all 2'-fluorinated, and the other positions are all 2'-methoxy modified; the 1st and 2nd nucleotides, the 2nd and 3rd nucleotides, the 1st and 2nd last nucleotides, and the 2nd and 3rd last nucleotides are all connected by phosphorothioate bonds;

[0106] (IX) From the 5'-3' direction, the 2nd, 6th, 14th and 16th positions are all 2'-fluorinated, and the other positions are all 2'-methoxy modified; the 1st and 2nd nucleotides, the 2nd and 3rd nucleotides, the 1st and 2nd last nucleotides, and the 2nd and 3rd last nucleotides are all connected by phosphorothioate bonds; the 1st position is a 5'-trans vinyl phosphate nucleotide;

[0107] (X) From the 5'-3' direction, the 2nd, 4th, 5th, 6th, 8th, 10th, 12th, 14th, 16th, and 18th positions are all 2'-fluorinated, and the other positions are all 2'-methoxy modified; the 1st and 2nd nucleotides, the 2nd and 3rd nucleotides, the 1st and 2nd to last nucleotides, and the 2nd and 3rd to last nucleotides are all connected by phosphorothioate bonds.

[0108] In some embodiments of the present invention, the sense strand of the double-stranded RNAi agent has any one of the following modifications (1) to (3):

[0109] (1) From the 5'-3' direction, positions 7, 9, 10, 11, and 17 are all 2'-fluorinated, and the other positions are all 2'-methoxy modified; the 1st and 2nd nucleotides, and the 2nd and 3rd nucleotides are all connected by phosphorothioate bonds;

[0110] (2) From the 5'-3' direction, positions 7, 9, 11, and 17 are all 2'-fluorinated, and the other positions are all 2'-methoxy modified; the 1st and 2nd nucleotides, and the 2nd and 3rd nucleotides are all connected by phosphorothioate bonds;

[0111] (3) From the 5'-3' direction, the 7th and 9th positions are both 2'-fluorinated, and the other positions are all 2'-methoxy modified; the 1st and 2nd nucleotides, and the 2nd and 3rd nucleotides are all connected by phosphorothioate bonds, and the 11th position is thymidine.

[0112] In some embodiments of the invention, the double-stranded RNAi agent has any of the following modifications:

[0113] The antisense strand has the above-mentioned modification method A, and the sense strand has the above-mentioned modification method a;

[0114] The antisense strand has the above-mentioned modification method B, and the sense strand has the above-mentioned modification method a;

[0115] The antisense strand has the above-mentioned modification method C, and the sense strand has the above-mentioned modification method a;

[0116] The antisense strand has the above-mentioned modification method B, and the sense strand has the above-mentioned modification method b;

[0117] The antisense strand has the above-mentioned modification pattern c, and the sense strand has the above-mentioned modification pattern b.

[0118] In some embodiments of the present invention, the antisense strand of the double-stranded RNAi agent has a modification group in the second to eighth positions from the 5' end, wherein the modification group is UNA, GNA or DNA, wherein the structures of UNA and GNA are as follows:

[0119] ; The base is adenine, guanine, cytosine, thymine or uracil.

[0120] In some embodiments of the present invention, the phosphorylation of the 5'-carbon atom of the 5'-terminal nucleotide glycoside of the antisense strand modified by the double-stranded RNAi agent includes but is not limited to the following 5'-phosphorylation groups: 5'-vinyl phosphonate group (5'-E-VP); 5'-methyl phosphonate group (5'-MP); 5'-C-methyl phosphate group; 5'-thiophosphate group (5'-PS); 5'-phosphate group (5'-P), the structure of which is shown as follows:

[0121] ;

[0122] Wherein, R is hydrogen, hydroxyl, amine, C 1-4 Alkyl, aromatic, C 1-4 Alkoxy, C 1-4 Alkylcarbonylamino or halogen;

[0123] The bases are adenine, guanine, cytosine, thymine or uracil.

[0124] In some embodiments of the present invention, the double-stranded RNAi agent is any oligonucleotide duplex consisting of a pair of a modified sense strand and a modified antisense strand as listed in the following table (the sequence is from Table 38 of the CN117210468A specification):

[0125]

[0126]

[0127]

[0128] In some embodiments of the present invention, the conjugate represented by formula (IIA) or (IIB) is conjugated with a double-stranded RNAi agent and YK-GAL-401, YK-GAL-402, YK-GAL-403, YK-GAL-404, YK-GAL-405 or YK-GAL-406 as described above, and the double-stranded RNAi agent comprises any oligonucleotide duplex consisting of the following sense strand and antisense strand pairing:

[0129] (1) the sense strand has the sequence shown in SEQ ID NO. 29; and the antisense strand has the sequence shown in SEQ ID NO. 41;

[0130] (2) the sense strand has the sequence shown in SEQ ID NO. 30; and the antisense strand has the sequence shown in SEQ ID NO. 42; and

[0131] (3) The sense strand has the sequence shown in SEQ ID NO. 30; and the antisense strand has the sequence shown in SEQ ID NO. 41.

[0132] In some embodiments of the present invention, the conjugate is conjugated with a double-stranded RNAi agent and YK-GAL-401, YK-GAL-402, YK-GAL-403, YK-GAL-404, YK-GAL-405 or YK-GAL-406 as described above, and the double-stranded RNAi agent comprises an oligonucleotide duplex consisting of a pair of a sense strand shown in SEQ ID NO. 30 and an antisense strand shown in SEQ ID NO. 41.

[0133] In some embodiments of the present invention, the conjugate represented by formula (IIA) or (IIB) is conjugated with a double-stranded RNAi agent and YK-GAL-401, YK-GAL-402, YK-GAL-403, YK-GAL-404, YK-GAL-405 or YK-GAL-406 as described above, and the sequence of the double-stranded RNAi agent is as follows:

[0134] Sense strand: 5'-Cms-Ums-Am-Gm-Am-Cm-Cf-Um-Gf-Um-dT-Um-Um-Gm-Cm-Um-Um-Um-Um-Gm-Um-3' (SEQ ID NO. 46);

[0135] Antisense strand: 5'-Ams-Cfs-Am-Af-Af-Af-Gm-Cf-Am-Af-Am-Af-Cm-Af-Gm-Gf-Um-Cf-Um-Am-Gms-Ams-Am-3' (SEQ ID NO. 47).

[0136] In some embodiments of the present invention, the conjugate represented by formula (IIA) is any of the following conjugates:

[0137] ,

[0138] ,

[0139] ,

[0140] ,

[0141] or

[0142] ;

[0143] Among them, the positive strand of siRNA has the sequence shown as SEQ ID NO. 46; the antisense strand of siRNA has the sequence shown as SEQ ID NO. 47.

[0144] The present invention also provides a pharmaceutical composition comprising the conjugate represented by formula (IIA) or (IIB) as described above and at least one pharmaceutically acceptable excipient.

[0145] The present invention also provides a use of the conjugate represented by formula (IIA) or (IIB) as described above or the pharmaceutical composition as described above in the preparation of a drug for treating and / or preventing a pathological condition or disease caused by the expression of a specific gene in hepatocytes.

[0146] In some embodiments of the present invention, the disease caused by the expression of specific genes in hepatocytes is selected from chronic liver disease, hepatitis, liver fibrosis, liver proliferative disease, dyslipidemia and complement-related disease.

[0147] The present invention also provides a method for inhibiting the expression of a specific gene in a hepatocyte, wherein the method comprises contacting an effective amount of the conjugate represented by formula (IIA) or (IIB) described above with the hepatocyte.

[0148] The present invention provides a kit, comprising the conjugate represented by formula (IIA) or (IIB) as described above.

[0149] All publications and patents mentioned in the present invention are hereby incorporated into the present invention by reference in their entirety. If the purposes or terms used in any publications and patents incorporated by reference conflict with the purposes or terms used in the present invention, then the purposes and terms of the present invention shall prevail.

[0150] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0151] Unless otherwise specified, all technical and scientific terms used herein have the common meaning in the field to which the claimed subject matter belongs. If there are multiple definitions for a term, the definition herein shall prevail.

[0152] Except in the working examples or otherwise indicated, all numbers of quantitative properties such as dosage stated in the specification and claims should be understood to be modified by the term "about" in all cases. It should also be understood that any numerical range recited in the present invention is intended to include all subranges within the range and any combination of the respective endpoints of the range or subrange.

[0153] The words "include", "contain" or "comprises" and the like used in the present invention mean that the elements preceding the word include the elements listed after the word and their equivalents, without excluding unrecorded elements. The terms "contain" or "includes (comprising)" used herein may be open, semi-closed and closed. In other words, the term also includes "essentially consisting of" or "consisting of".

[0154] In the present invention, "hydroxyl protecting group" refers to a protecting group commonly used to protect the hydroxyl group of the ribose structure in the synthesis of RNA or its derivatives, such as acetyl, phenoxyacetyl, pivaloyl, benzyl, 4-methoxybenzyl, benzoyl, triphenylmethyl, 4, 4'-dimethoxytrityl (DMr, 4, 4'-dimethoxytrityl), monomethoxytrityl (MMTr, monomethoxytrityl), 9-phenyl-xanthene-9-yl (9-phenylxanthen-9-yl), 9-p-tolyl-xanthene-9-yl (9-(p-tolyl)-xanthen-9-yl), trimethylsilyl, tert-butyldimethylsilyl (TBDMS), cyanomethoxymethyl, 2-(cyanoethoxy)ethyl, cyanoethoxymethyl, etc., preferably 4, 4'-dimethoxytrityl.

[0155] In the present invention, "phosphorus-containing reactive groups" refer to phosphorus-containing groups that can react with a hydroxyl or amine group contained in another molecule, especially in another nucleotide unit or another nucleotide analog, through a nucleophilic attack reaction. Typically, such reactions produce ester-type nucleoside bonds connecting a nucleotide unit or a nucleotide analog unit to another nucleotide unit or a nucleotide analog unit. These phosphorus-containing reactive groups are known in the art and include P III or P V The phosphorus atom in a valence state, the phosphorus-containing reactive groups include but are not limited to phosphoramidites, H-phosphonates, phosphotriesters and phosphorus-containing chiral auxiliary agents, such as .

[0156] In the present invention, controlled pore glass (CPG) and polystyrene (highly cross-linked polystyrene microbeads) are solid supports for oligonucleotide synthesis, are insoluble particles bound to oligonucleotides during the synthesis process, and are commercially available.

[0157] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0158] The term "alkyl" refers to a straight or branched chain alkyl group with a specified number of carbon atoms (e.g., C1 to C6). Alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, etc.

[0159] The term "alkoxy" refers to a group R X -O-, where R X is an alkyl group as defined above.

[0160] The term "pharmaceutically acceptable salt" refers to a salt obtained by reacting a compound with a pharmaceutically acceptable (relatively non-toxic, safe, and suitable for use by patients) acid or base. When the compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the free form of the compound with a sufficient amount of a pharmaceutically acceptable base in a suitable inert solvent. Pharmaceutically acceptable base addition salts include, but are not limited to, sodium salts, potassium salts, calcium salts, aluminum salts, magnesium salts, bismuth salts, ammonium salts, and the like. When the compound contains a relatively basic functional group, an acid addition salt can be obtained by contacting the free form of the compound with a sufficient amount of a pharmaceutically acceptable acid in a suitable inert solvent. Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochlorides, sulfates, methanesulfonates, and the like.

[0161] The term "pharmaceutically acceptable excipient" in the present invention refers to all substances contained in the pharmaceutical preparation except the active ingredient.

[0162] The term "treat" refers to any of the following: (1) ameliorating one or more biological manifestations of a disease; (2) interfering with one or more points in the biological cascade that leads to a disease; or (3) slowing the progression of one or more biological manifestations of a disease.

[0163] The term "prevention" refers to reducing the risk of developing a disease.

[0164] The oligonucleotides in the present invention include single-stranded oligonucleotides (such as antisense oligonucleotides, referred to as ASOs) and double-stranded oligonucleotides (such as small interfering nucleotides, referred to as siRNAs).

[0165] The oligonucleotides in the present invention include natural oligonucleotides and chemically modified oligonucleotides. Chemical modifications herein include nucleoside modifications (including sugar moiety modifications and nucleobase modifications) and internucleoside linkage modifications. Chemical modifications of oligonucleotides do not include situations where there are differences only in the nucleobase sequence. Natural herein refers to situations corresponding to naturally occurring RNA or DNA.

[0166] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0167] The reagents and raw materials used in the present invention are commercially available.

[0168] The positive and progressive effects of the present invention are: the present invention designs a series of novel GalNAc compounds, which have completely different chemical structures compared with the prior art GalNAc compounds. The GalNAc compounds designed by the present invention introduce a triazole structure in the connecting arm, and with the advantages of click chemistry, a series of novel GalNAc compounds containing triazole structures can be quickly constructed. The GalNAc oligonucleotide conjugates prepared from the GalNAc compounds of the present invention can achieve efficient liver-targeted delivery, and the activity is significantly improved compared with the representative GalNAc compounds of the prior art.

[0169] 1. The oligonucleotide conjugates prepared from the GalNAc compounds YK-Gal-401 to YK-Gal-403 of the present invention significantly improve the inhibition rate of PCSK9 protein expression in mouse serum compared with the prior art GalNAc. For example, the inhibition rate of inc-YK-Gal-401 on day 7 was 21.5%, 14.5% and 12.7% higher than that of inc-YK-Gal-301, inc-GalNAc 1b and inc-NAG0052, and the inhibition rates on day 14 were increased by 17.3%, 10.4% and 10.8%, respectively.

[0170] 1) The siRNA conjugated with the GalNAc compound of the present invention has significant differences in the inhibition rate of PCSK9 protein expression in mouse serum, and YK-Gal-401~YK-Gal-403 are significantly higher than YK-Gal-404~406. For example, inc-YK-Gal-401 increased by 20.7% and 16.0% on the 7th day and 14th day respectively compared with inc-YK-Gal-404.

[0171] 2) Compared with the GalNAc compounds of the prior art, the conjugated siRNA of the present invention, YK-Gal-401 to YK-Gal-403, significantly improves the inhibition rate of PCSK9 protein expression in mouse serum. For example, inc-YK-Gal-401 increased the PCSK9 protein expression by 21.5% and 17.3% on the 7th day and 14th day respectively compared with inc-YK-Gal-301.

[0172] 3) The conjugates of GalNAc compounds with similar structures and siRNA have significant differences in the inhibition rate of PCSK9 protein expression in mouse serum. Therefore, it is not necessarily the case that GalNAc compounds with similar chemical structures have similar oligonucleotide delivery efficiencies. The inhibition rates of PCSK9 protein expression in mouse serum by the prepared GalNAc oligonucleotide conjugates are not consistent and may have very large differences.

[0173] 2. The oligonucleotide conjugates prepared from the GalNAc compounds YK-Gal-401 to YK-Gal-403 of the present invention significantly improve the inhibition rate of PCSK9 gene expression in mouse liver compared with the prior art GalNAc. For example, the inhibition rate of inc-YK-Gal-401 was significantly improved by 21.1% and 9.8% compared with inc-YK-Gal-301 and inc-GalNAc 1b, respectively.

[0174] 1) The siRNA conjugated with the GalNAc compound of the present invention has significant differences in the inhibition rate of PCSK9 gene expression in mouse liver, and YK-Gal-401~YK-Gal-403 are significantly higher than YK-Gal-404~YK-Gal-406. For example, inc-YK-Gal-401 is 19.4% higher than inc-YK-Gal-404.

[0175] 2) Compared with the prior art GalNAc compounds, the conjugated siRNA of the present invention YK-Gal-401 to YK-Gal-403 significantly improves the inhibition rate of PCSK9 gene expression in mouse liver. For example, inc-YK-Gal-401 is 21.1% higher than inc-YK-Gal-301.

[0176] 3) The conjugates of GalNAc compounds with similar structures and siRNA have significant differences in the inhibition rate of PCSK9 gene expression in mouse liver. Therefore, it is not necessarily the case that GalNAc compounds with similar chemical structures have similar oligonucleotide delivery efficiencies. The inhibition rates of the prepared GalNAc oligonucleotide conjugates on the PCSK9 gene in mouse liver are not consistent and may have very large differences.

[0177] 3. The oligonucleotide conjugates prepared by the GalNAc compounds YK-Gal-401 to YK-Gal-403 of the present invention significantly improve the level of LDL-C reduction in mouse serum compared with the prior art GalNAc. For example, the level of LDL-C reduction in mice in the inc-YK-Gal-401 group on day 14 was significantly improved by 36.4%, 20.6% and 17.8% compared with the inc-YK-Gal-301, inc-GalNAc 1b and NAG0052 groups, respectively.

[0178] 1) The siRNA conjugated with the GalNAc compound of the present invention has a significant difference in reducing the LDL-C level in mouse serum, and the reduction level of YK-Gal-401~YK-Gal-403 is significantly higher than that of YK-Gal-404~406. For example, inc-YK-Gal-401 increased by 33.0% and 36.0% on the 7th day and 14th day respectively compared with inc-YK-Gal-404, which is a significant improvement.

[0179] 2) Compared with the prior art GalNAc compounds, the conjugated siRNA of the present invention YK-Gal-401 to YK-Gal-403 significantly improved the level of LDL-C reduction in mouse serum. For example, inc-YK-Gal-401 increased LDL-C by 33.4% and 36.4% on the 7th day and 14th day respectively compared with inc-YK-Gal-301.

[0180] 3) The conjugates of GalNAc compounds with similar structures and siRNA have significant differences in the reduction of LDL-C in mouse serum. Therefore, it is not necessarily the case that GalNAc compounds with similar chemical structures have similar oligonucleotide delivery efficiencies. The inhibition rates of PCSK9 protein expression in mouse serum by the prepared GalNAc oligonucleotide conjugates are not consistent and may have very large differences.

[0181] 4. The designed compounds of the present invention can efficiently deliver oligonucleotides to the liver.

[0182] For example, the liver fluorescence intensities of inc-YK-Gal-401, inc-YK-Gal-402 and inc-YK-Gal-403 groups can reach 9.03E+09, 8.79E+09 and 8.92E+09 respectively at 4 hours, and can reach 9.11E+09, 8.95E+09 and 9.01E+09 respectively at 8 hours, indicating that the GalNAc compounds designed in the present invention can efficiently deliver oligonucleotides to animal livers. BRIEF DESCRIPTION OF THE DRAWINGS

[0183] Figure 1 This is the inhibition rate of PCSK9 protein expression in mouse serum 7 days and 14 days after administration of each test drug group (inc-YK-Gal-401, inc-YK-Gal-402, inc-YK-Gal-403, inc-YK-Gal-404, inc-YK-Gal-405, inc-YK-Gal-406, inc-YK-Gal-301, inc-GalNAc 1b and inc-NAG0052).

[0184] Figure 2This is the inhibition rate of PCSK9 gene expression in mouse liver 14 days after administration of each test drug group (inc-YK-Gal-401, inc-YK-Gal-402, inc-YK-Gal-403, inc-YK-Gal-404, inc-YK-Gal-405, inc-YK-Gal-406, inc-YK-Gal-301, inc-GalNAc 1b and inc-NAG0052).

[0185] Figure 3 The reduction levels of LDL-C in mouse serum were measured 7 days after administration and 14 days after administration of each test drug group (inc-YK-Gal-401, inc-YK-Gal-402, inc-YK-Gal-403, inc-YK-Gal-404, inc-YK-Gal-405, inc-YK-Gal-406, inc-YK-Gal-301, inc-GalNAc 1b and inc-NAG0052).

[0186] Figure 4 The fluorescence intensity in the liver and kidney of mice at 4 hours and 8 hours after administration of each test drug group (inc-YK-Gal-401, inc-YK-Gal-402, inc-YK-Gal-403, inc-YK-Gal-404, inc-YK-Gal-405, inc-YK-Gal-406, inc-YK-Gal-301, inc-GalNAc 1b and inc-NAG0052). DETAILED DESCRIPTION

[0187] In order to make the purpose, technical solution and advantages of the embodiment of the present invention clearer, the technical solution of the embodiment of the present invention will be clearly and completely described below. Obviously, the described embodiment is a part of the embodiment of the present invention, not all of the embodiments. Based on the described embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0188] The present invention may be implemented in other specific forms without departing from the basic attributes of the present invention. It should be understood that, without conflict, any and all embodiments of the present invention may be combined with the technical features in any other embodiment or multiple other embodiments to obtain other embodiments. The present invention includes other embodiments obtained by such combination.

[0189] The present invention is further described below in conjunction with the examples. However, the present invention is not limited to the following examples. The implementation conditions used in the examples can be further adjusted according to the different requirements of specific use, and the implementation conditions not specified are conventional conditions in the industry. In the specific examples of the present invention, the raw materials used can be obtained commercially. Unless otherwise specified, all temperatures are given in degrees Celsius. The technical features involved in each embodiment of the present invention can be combined with each other as long as they do not conflict with each other.

[0190] The following abbreviations represent the following reagents: DCM: dichloromethane; PE: petroleum ether; EA: ethyl acetate; THF: tetrahydrofuran; DMF: N, N-dimethylformamide; ACN: acetonitrile; KOH: potassium hydroxide; DCE: 1,2-dichloroethane; TsOH·H2O: p-toluenesulfonic acid monohydrate; EDCI: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; HOBt: 1-hydroxybenzotriazole; THPTA: tris(3-hydroxypropyltriazolylmethyl)amine; VC Na: sodium ascorbate; TFA: trifluoroacetic acid; TEAB: triethylammonium bicarbonate aqueous solution; HBTU: O-benzotriazole-tetramethyluronium hexafluorophosphate; DIPEA: N, N-Diisopropylethylamine; DMAP: 4-dimethylaminopyridine; TMSOTf: trimethylsilyl trifluoromethanesulfonate; Py: pyridine; Ac2O: acetic anhydride; CPG-NH2: amino-modified controlled pore glass (from WuXi AppTec).

[0191] Example 1: Synthesis of GalNAc compounds

[0192] 1. Synthesis of YK-GAL-401

[0193] The synthetic route is as follows:

[0194]

[0195]

[0196] Step 1: Synthesis of G1-2

[0197] Compound G1-1 (5.0 g, 22.6 mmol, 1.0 eq) was dissolved in anhydrous DMF (60.0 mL) and stirred at 0 °C. Then propargyl bromide (40.3 g, 271 mmol, 29.2 mL, 12.0 eq) was added, and finely ground KOH (15.2 g, 271 mmol, 12.0 eq) was added to the reaction solution in batches within 15 minutes. The temperature was raised to 35 °C and stirred for 24 h. The sample was taken and tested by LC / MS, which showed that the starting material disappeared. Ethyl acetate (300 mL) was added to the reaction solution and washed with water (50 mL × 3). The organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure to remove the solvent. The crude product was purified by silica gel column (petroleum ether / ethyl acetate) to obtain yellow oil G1-2 (5.30 g, 15.8 mmol, 69.9 % yield). MS (ESI) m / z [M - H] - : 334.2; 1 H NMR: δ ppm 4.90 (s, 1 H), 4.14 (d, J = 2.4, 6 H), 3.77 (s, 6 H), 2.43-2.41 (m, 3H), 1.41 (s, 9 H).

[0198] Step 2: Synthesis of G1-4

[0199] Compound G1-3 (22.3 g, 57.3 mmol, 1.2 eq) was dissolved in DCE (200 mL), 3Å molecular sieves (20.0 g) were added, and the temperature was lowered to 0 °C. Then, TMSOTf (21.2 g, 95.6 mmol, 17.3 mL, 2.0 eq) and 3-(benzyloxycarbonylamino)-1-propanol (10.0 g, 47.8 mmol, 1.0 eq) were added at 0 °C. The temperature was raised to 15 °C and stirred for 10 min. The sample was taken and tested by LC / MS, which showed that the starting material disappeared. Saturated sodium bicarbonate solution (200 mL) was added to the reaction solution, and extracted with dichloromethane (200 mL × 3). The organic phase was dried over Na2SO4, filtered, and then concentrated under reduced pressure to remove the solvent. The crude product was purified by silica gel column (petroleum ether / ethyl acetate) to obtain a white solid G1-4 (13.4 g, 24.9 mmol, 52.1% yield). MS (ESI) m / z [M + H] + : 539.1; 1H NMR (400 MHz, CDCl3) δ ppm 7.38 - 7.29 (m, 5 H), 6.31 (d, J = 8.7 Hz, 1 H), 5.33 - 5.28 (m, 1 H), 5.14 - 5.02 (m, 3 H), 4.98 (dd, J = 11.1, 3.3 Hz, 1 H), 4.32 (d, J = 8.5 Hz, 1 H), 4.16 - 4.09 (m, 3 H), 3.98 - 3.93 (m, 1 H), 3.76 (t, J = 6.7 Hz, 1 H), 3.57 - 3.48 (m, 1 H), 3.39(td, J = 9.9, 3.1 Hz, 1H), 3.12 - 3.05 (m, 1 H), 2.13 (s, 3 H), 2.04 (s, 3H), 1.99 (s, 3 H), 1.93 (s, 3 H), 1.83 - 1.76 (m, 1 H), 1.65 - 1.57 (m, 1 H).

[0200] Step 3: Synthesis of G1-5

[0201] Compound G1-4 (13.0 g, 24.1 mmol, 1.0 eq) and TsOH·H2O (4.59 g, 24.1 mmol, 1.0 eq) were added to a solution of palladium carbon (6.50 g, 26.4 mmol, 1.09 eq) in tetrahydrofuran (130 mL). The reaction solution was stirred at 25 °C for 2 h under hydrogen (15 psi). The sample was taken and tested by LC / MS, which showed that the starting material disappeared. The reaction solution was filtered and concentrated under reduced pressure to remove the solvent to obtain a white solid crude product G1-5 (15.0 g). MS (ESI) m / z [M - H] - : 403.2.

[0202] Step 4: Synthesis of G1-6

[0203] Add EDCI (7.47 g, 38.9 mmol, 1.05 eq) and HOBt (5.51 g, 40.8 mmol, 1.1 eq) to a dichloromethane solution (150 mL) of 5-azidopentanoic acid (5.31 g, 37.1 mmol, 1.0 eq) and cool to 0 °C. Then, add DIPEA (9.59 g, 74.2 mmol, 12.9 mL, 2.0 eq) and compound G1-5 (15.0 g, 37.1 mmol, 1.0 eq) to the above solution. Heat to 20 °C and continue stirring for 1 h. Samples were taken and tested by LC / MS, showing that the starting material disappeared. The reaction solution was diluted with dichloromethane (150 mL) and washed with saturated NaHCO3 (150 mL × 3). The organic phase was dried over Na2SO4, filtered and concentrated under reduced pressure to remove the solvent, yielding a colorless liquid crude product G1-6 (18.0 g). MS (ESI) m / z [M -H] - : 528.3; 1 H NMR (400 MHz, CDCl3) δ ppm 6.30 (d, J = 8.8 Hz, 1 H), 6.17 (dd,J = 7.1, 4.6 Hz, 1 H), 5.34 (dd, J = 3.4, 1.1 Hz, 1 H), 5.03 (dd, J = 11.2,3.4 Hz, 1 H), 4.44 (d, J = 8.4 Hz, 1 H), 4.29 - 4.08 (m, 3 H), 4.03 -3.98 (m,1 H), 3.88 (td, J = 6.7, 1.2 Hz, 1 H), 3.71 -3.57 (m, 1 H), 3.45 (td, J =9.2, 3.5 Hz, 1 H), 3.31 (t, J = 6.7 Hz, 2 H), 3.14 -3.02 (m, 1 H), 2.27 (t, J= 7.2 Hz, 2 H), 2.16 (s, 3 H), 2.04 (s, 3 H), 2.00 (s, 3 H), 1.96 (s, 3 H),1.84 - 1.58 (m, 6 H).

[0204] Step 5: Synthesis of G1-7

[0205] G1-2 (400 mg, 1.19 mmol, 1.0 eq) was added to a DMSO solution (50.0 mL) of G1-6 (2.53 g, 4.77 mmol, 4.0 eq). Then, CuSO4 (1.71 g, 10.7 mmol, 1.65 mL, 9.0 eq) and THPTA (3.11 g, 7.16 mmol, 6.0 eq) were added to water (15.0 mL), followed by VC Na (3.54 g, 17.89 mmol, 15.0 eq) to prepare an aqueous solution, which was added to the above DMSO solution and stirred at 25 °C for 2 h. Samples were taken and tested by LC / MS, which showed that the starting material disappeared. The reaction solution was diluted with dichloromethane (100 mL) and washed with water (100 mL ×2). The organic phase was dried over Na2SO4, filtered and concentrated under reduced pressure to remove the solvent. The crude product was purified by silica gel column (petroleum ether / ethyl acetate, dichloromethane / methanol) to obtain a white solid G1-7 (1.90 g, 987.5 μmol, 82.8% yield). MS (ESI) m / z [M – 2 H] 2- : 960.8; 1 H NMR (400 MHz, CDCl3) δ ppm 7.62 (s, 3 H), 6.99 (d, J= 9.0 Hz, 3 H), 6.73 (t, J = 6.2 Hz, 3 H), 5.32 (d, J = 3.3 Hz, 3 H), 5.28 (s, 3 H), 5.05 (dd, J = 11.2, 3.4 Hz, 3 H), 4.55- 4.49 (m, 10 H), 4.37 (t, J= 7.0 Hz, 6 H), 4.27- 4.06 (m, 10 H), 4.00- 3.95 (m, 3 H), 3.92- 3.85 (m, 3H), 3.67 (s, 6H), 3.54- 3.42 (m, 6 H), 3.13 - 3.08 (m, 3 H), 2.32 - 2.23 (m, 7 H), 2.13 (s, 10 H), 2.01 (s, 10 H), 1.96 (s, 10 H), 1.88 (s, 9 H), 1.70-1.61 (m, 9 H), 1.38 (s, 10 H).

[0206] Step 6: Synthesis of G1-8

[0207] Trifluoroacetic acid (9.21 g, 80.7 mmol, 6.00 mL, 81.7 eq) was added to a dichloromethane solution (18.0 mL) of G1-7 (1.90 g, 988 μmol, 1.0 eq) and stirred at 25 °C for 0.5 h. The sample was detected by TLC (dichloromethane / methanol), which showed that the starting material disappeared. The reaction solution was directly concentrated under reduced pressure to remove the solvent, and the crude product was co-rotated with toluene (50.0 mL) three times to obtain a yellow oil G1-8 (1.80 g, 967 μmol, 97.8% yield, 98.0% purity). LC / MS(ESI) m / z [M – 2 H] 2- : 910.7.

[0208] Step 7: Synthesis of G1-10

[0209] HBTU (174 mg, 460 μmol, 1.5 eq) and DIPEA (79.4 mg, 614 μmol, 107 μL, 2.0 eq) were added to a DMF solution (3.0 mL) of G1-9 (preparation method reference CN116854754B) (200 mg, 307 μmol, 1.0 eq). Then, the crude product G1-8 (955 mg, 491 μmol, 1.6 eq, TFA) (pH ~7 adjusted with DIPEA) was added to the above reaction solution and stirred at 25 °C for 6 h. The sample was taken and detected by LC / MS, which showed that the starting material disappeared. The reaction solution was used directly in the next step. MS (ESI) m / z [M – 2 H] 2- : 1227.0.

[0210] Step 8: Synthesis of G1-11

[0211] G1-10 (750 mg, 305 μmol, 1.0 eq) was added to DMF (4.0 mL). DIPEA (118 mg, 915 μmol, 159 μL, 3.0 eq), succinic anhydride (244 mg, 2.44 mmol, 8.00 eq) and DMAP (37.3 mg, 305 μmol, 1.0 eq) were then added, and the reaction solution was stirred at 25 °C for 40 h. The sample was taken and detected by LC / MS, and the main peak was the target product. The reaction solution was filtered and the filtrate was purified by preparative chromatography to obtain a white solid G1-11 (120.0 mg, 46.0 μmol). MS (ESI) m / z [M – 2 H] 2- : 1276.8; 11H NMR (400 MHz, CDCl3) δ 7.55 (s, 2H), 7.43 - 7.36 (m, 2H), 7.26 (dd, J = 9.1, 2.8 Hz, 3H), 7.21 - 7.17 (m, 6H), 7.11 (dd, J = 8.3, 6.2 Hz, 1H), 6.88 (d, J = 9.0 Hz, 2H), 6.76 - 6.70 (m, 3H), 6.68 (t, J = 6.0 Hz, 2H), 5.28 (d, J = 3.3 Hz, 2H), 5.09 (t, J = 5.2 Hz, 1H), 5.00 (dd, J = 11.2, 3.4 Hz, 2H), 4.93 (d, J = 2.3 Hz, 1H), 4.57 - 4.41 (m, 7H), 4.32 (t, J = 6.9 Hz, 4H), 4.24 - 4.00 (m, 8H), 3.95 - 3.90 (m, 2H), 3.86 - 3.82 (m, 3H), 3.71 - 3.61 (m, 10H), 3.48 - 3.37 (m, 5H), 3.34 - 3.29 (m, 3H), 3.18 - 3.02 (m, 5H), 2.73 (q, J = 7.3 Hz, 7H), 2.64 - 2.57 (m, 2H), 2.47 (t, J = 6.9 Hz, 2H), 2.32 - 1.75 (m, 54H), 1.72 - 1.58 (m, 11H), 1.50 - 1.41 (m, 4H), 1.19 - 1.09 (m, 20H).

[0212] Step 9: Synthesis of YK-GAL-401

[0213] G1-11 (160 mg, 62.5 μmol, 1.0 eq) was added to DMF (10.0 mL). DIPEA (64.7 mg, 500 μmol, 87.20 μL, 8.0 eq), HBTU (118 mg, 312 μmol, 5.0 eq), DMAP (37.3 mg, 305 μmol, 1.0 eq) and CPG-NH2 (1.1 g) were then added, and the reaction solution was stirred at 40 °C for 16 h. Samples were taken and tested by LC / MS, which showed that the starting material disappeared. The reaction solution was filtered, and the filter cake was washed with MeOH (20 mL × 4) and DCM (20 mL × 4) successively, and dried under nitrogen to obtain a light yellow solid, which was directly used in the next step.

[0214] The crude product (1.2 g) was dissolved in pyridine (10.0 mL). Acetic anhydride (2.17 g, 21.3 mmol, 2.0 mL, 45.3 eq) was then added, and the reaction solution was stirred at 40 °C for 1 h. The reaction solution was then filtered, and the filter cake was washed with MeOH (20 mL × 4) and DCM (20 mL × 4) successively, and dried under nitrogen to obtain a light yellow solid YK-GAL-401 (1.0 g, loading: 37.1 μmol / g).

[0215] General determination method for carrier loading: Weigh YK-GAL-401 in a 1.5 mL centrifuge tube, then add 1.0 mL PTSA (CAS: 104-15-4) (0.1 M in ACN) to the centrifuge tube. Seal the test tube and mix well. The solution is orange and let stand for 1 hour. Take 100 uL of the solution and add it to a 4.0 mL centrifuge tube. Add 3.0 mL PTSA (0.1 M in ACN) to the centrifuge tube. Seal the test tube and mix well, and fill the reference cuvette with PTSA (0.1 M in ACN). Measure the absorbance of the blank solution at 411 nm. Fill the cuvette with the sample solution, measure the absorbance of the sample solution at 411 nm, and calculate the loading capacity.

[0216] 2. Synthesis of YK-GAL-402

[0217] The synthetic route is as follows:

[0218]

[0219]

[0220] Step 1: Synthesis of G2-3

[0221] Compounds G2-1 (1.30 g, 11.3 mmol, 1.04 eq) and G2-2 (2.00 g, 10.9 mmol, 1.0eq) were dissolved in anhydrous ethyl acetate (20.0 mL) and cooled to 0 °C. EDCI (2.17 g, 11.3mmol, 1.04 eq) was then added in batches and stirred at 0 °C for 1 h. The temperature was then slowly raised to 25 °C and stirred for 4 h. The sample was taken and tested by LC / MS, which showed that the starting material disappeared. The reaction solution was washed with water (20 mL × 3), 10% NaHCO3 aqueous solution (20 mL × 3), water (20mL × 2), 5% KHSO4 aqueous solution (20 mL × 2) and water (20 mL × 3). The organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure to remove the solvent to obtain a brown oil G2-3 (2.50 g, 8.89 mmol, 81.8% yield). 1 H NMR (400 MHz, CDCl3) δ ppm 3.72 (t, J = 6.4 Hz, 2 H), 2.94 (t, J = 6.4 Hz, 2 H); 19 F NMR (376 MHz, CDCl3) δ -152.54 - -152.63 (m, 2 F), -157.46 (t, J = 21.8Hz, 1 F), -161.98 - -162.13 (m, 2 F).

[0222] Step 2: Synthesis of G2-4

[0223] DIPEA (1.54 g, 11.9 mmol, 2.08 mL, 2.0 eq) was added to a dichloromethane solution (30.0 mL) of compound G1-5 (3.55 g, 5.97 mmol, 1.0 eq) and compound G2-3 (1.68 g, 5.97 mmol, 1.0 eq). Stir at 25 °C for 2 h. Samples were taken and tested by LC / MS, showing that the starting material disappeared. The reaction solution was washed with NaHCO3 aqueous solution (30.0 mL × 1) and water (30 mL × 3) in turn. The organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure to remove the solvent. The crude product was purified by preparative chromatography to obtain a white solid G2-4 (710 mg, 1.42 mmol, 23.7% yield). MS (ESI) m / z [M - H] - : 500.2; 1H NMR (400 MHz, CDCl3) δ ppm 6.37 - 6.28 (m, 1 H), 6.14 (d, J = 8.8 Hz, 1 H), 5.35 (d, J = 3.3 Hz, 1 H), 5.02 (dd, J = 11.2, 3.4 Hz,1 H), 4.43 (d, J = 8.4 Hz, 1 H), 4.30 - 4.23 (m, 1 H), 4.20 - 4.10 (m, 2 H), 4.06 - 4.01 (m, 1 H), 3.92 - 3.84 (m, 1 H), 3.71 - 3.57 (m, 3 H), 3.46 (td, J= 9.1, 3.3 Hz, 1H), 3.21 - 3.09 (m, 1 H), 2.62 - 2.44 (m, 2 H), 2.17 (s, 3H), 2.04 (s, 3 H), 2.01 (s, 3 H), 1.98 (s, 3 H), 1.71 - 1.66 (m, 2 H).

[0224] Step 3: Synthesis of G2-5

[0225] G1-2 (110 mg, 328 μmol, 1.0 eq) was added to a DMSO solution (20.0 mL) of G2-4 (575 mg, 1.15 mmol, 3.5 eq). Then, CuSO4 (471 mg, 2.95 mmol, 453 μL, 9.0 eq) and THPTA (855 mg, 1.97 mmol, 6.0 eq) were added to water (4.0 mL), followed by VC Na (974 mg, 4.92mmol, 15.0 eq) to prepare an aqueous solution, which was added to the above DMSO solution and stirred at 25 °C for 2 h. Samples were taken and tested by LC / MS, which showed that the starting material disappeared. Water (2.0 mL) was added to the reaction solution, extracted with dichloromethane (3 mL × 3), and washed with water (2 mL × 3). The organic phase was dried over Na2SO4, filtered and concentrated under reduced pressure to remove the solvent. The crude product was purified by preparative chromatography to obtain a white solid G2-5 (350 mg, 190 μmol, 58.0% yield). MS (ESI) m / z [M – 2 H] 2- :918.6; 1H NMR (400 MHz, CDCl3) δ ppm 7.69 (s, 3 H), 7.09 (t, J = 5.7 Hz, 3 H), 6.97 (d, J = 9.0 Hz, 3 H), 5.35 (d, J = 3.2 Hz, 3 H), 5.08 (dd, J = 11.3, 3.4Hz, 3 H), 4.77 - 4.66 (m, 6 H), 4.52 (s, 6 H), 4.47 (d, J = 8.5 Hz, 3 H), 4.26 - 4.11 (m, 9 H), 3.96 - 3.91 (m, 6 H), 3.60 (s, 6 H), 3.52 - 3.47 (m, 2H), 3.42 - 3.36 (m, 3 H), 3.16 - 3.11 (m, 3 H), 3.04 - 2.97 (m, 3 H), 2.86 -2.80 (m, 3 H), 2.15 (s, 9 H), 2.04 (s, 9 H), 2.00 (s, 9 H), 1.85 (s, 9 H), 1.78 (s, 8 H), 1.42 (s, 9 H).

[0226] Step 4: Synthesis of G2-6

[0227] Trifluoroacetic acid (1.07 g, 9.42 mmol, 700 μL, 49.5 eq) was added to a dichloromethane solution (2.8 mL) of G2-5 (350 mg, 190μmol, 1.0 eq) and stirred at 25 °C for 0.5 h. The sample was tested by LC / MS, which showed that the starting material disappeared. The reaction solution was directly concentrated under reduced pressure to remove the solvent, and the crude product was co-evaporated with toluene (10.0 mL) three times to obtain a red oil G2-6 (523 mg). MS (ESI) m / z [M – 2 H] 2- : 868.6.

[0228] Step 5: Synthesis of G2-7

[0229] HBTU (65.6 mg, 173 μmol, 1.5 eq) and DIPEA (29.8 mg, 230 μmol, 40.2 μL, 2.0 eq) were added to a DMF solution (1.5 mL) of G1-9 (75.0 mg, 115 μmol, 1.0 eq). Then, the crude product G2-6 (200 mg, 115 μmol, 1.0 eq, TFA) (pH ~ 7 adjusted with DIPEA) was added to the above reaction solution and stirred at 25 °C for 2 h. The sample was taken and detected by LC / MS, which showed that the starting material disappeared. The reaction solution was used directly in the next step. MS (ESI) m / z [M –2 H] 2- : 1184.8.

[0230] Step 6: Synthesis of G2-8

[0231] G2-7 (273 mg, 115 μmol, 1.0 eq) was added to DMF (3.0 mL). DIPEA (44.7 mg, 345 μmol, 60.2 μL, 3.0 eq), succinic anhydride (104 mg, 1.04 mmol, 9.0 eq) and DMAP (56.3 mg, 461 μmol, 4.0 eq) were then added, and the reaction solution was stirred at 30 °C for 24 h. The sample was taken and tested by LC / MS, which showed that the starting material disappeared. The reaction solution was filtered and the filtrate was purified by preparative chromatography to obtain a white solid G2-8 (110 mg, 44.5 μmol, 38.6% yield). MS (ESI) m / z [M – 2 H] 2- : 1234.9; 11H NMR (400 MHz, CDCl3) δ ppm 7.66 (s, 3 H), 7.45 (d, J = 7.7 Hz, 2 H), 7.33 (d, J = 8.8 Hz, 4 H), 7.28 (s, 1 H), 7.24 (s, 1 H), 7.20 - 7.13 (m, 4 H), 7.02 (d, J = 9.0 Hz, 2 H), 6.81 (d, J = 8.7 Hz, 3 H), 5.35 (d, J = 3.3 Hz, 3 H), 5.08 (dd, J = 11.2, 3.4 Hz, 3 H), 4.73 - 4.69 (m, 5 H), 4.53 - 4.47 (m, 8 H), 4.26 - 4.10 (m, 11 H), 3.97 - 3.89 (m, 7 H), 3.78 (s, 6 H), 3.65 (s, 5 H), 3.56 - 3.44 (m, 4 H), 3.40 - 3.37 (m, 5 H), 3.24 - 3.21 (m, 2 H), 3.16 - 3.09 (m, 4 H), 3.01 - 2.94 (m, 4 H), 2.82 - 2.78 (m, 2 H), 2.69 - 2.64 (m, 2 H), 2.56 - 2.53 (m, 2 H), 2.15 (s, 9 H), 2.04 (s, 9 H), 2.00 (s, 9 H), 1.93 - 1.76 (m, 32 H), 1.27 (br, 10 H).

[0232] Step 7: Synthesis of YK-GAL-402

[0233] G2-8 (110 mg, 42.74 μmol, 1.0 eq, TEA) was added to DMF (8.0 mL), followed by DIPEA (44.2 mg, 342 μmol, 59.5 μL, 8.0 eq), HBTU (81.0 mg, 213 μmol, 5.0 eq), DMAP (5.22 mg, 42.7 μmol, 1.0 eq) and CPG-NH2 (713 mg, 1.21 mmol, 28.4 eq). The reaction solution was stirred at 40 °C for 16 h. Samples were taken and tested by LC / MS, showing that the starting material disappeared. The reaction solution was filtered, and the filter cake was washed with MeOH (10 mL × 4) and DCM (10 mL × 4) successively, and dried under nitrogen to obtain a light yellow solid, which was directly used in the next step.

[0234] The crude product was dissolved in pyridine and acetic anhydride solution (Py / Ac2O = 5 / 1, 9.0 mL), the reaction solution was stirred at 40 °C for 40 min, and then the reaction solution was filtered, the filter cake was washed with DCM (5.0 mL × 4) and MeOH (5.0 mL × 4) successively, and dried under nitrogen to obtain a light yellow solid YK-GAL-402 (726 mg, loading: 33.15 μmol / g). The carrier loading was determined according to the general method for determining the carrier loading.

[0235] 3. Synthesis of YK-GAL-403

[0236] The synthetic route is as follows:

[0237]

[0238]

[0239] Step 1: Synthesis of G3-1

[0240] EDCI (1.92 g, 10.0 mmol, 1.05 eq) and HOBt (1.42 g, 10.50 mmol, 1.1 eq) were added to a dichloromethane solution (40.0 mL) of 6-azidohexanoic acid (1.50 g, 9.54 mmol, 1.0 eq) and the temperature was lowered to 0 °C. Then, DIPEA (2.47 g, 19.1 mmol, 3.32 mL, 2.0 eq) and compound G1-5 (3.86 g, 9.54mmol, 1.0 eq) were added to the above solution. The temperature was raised to 25 °C and stirred for 2 h. The sample was taken and tested by LC / MS, which showed that the starting material disappeared. The reaction solution was diluted with dichloromethane (20.0 mL), washed with saturated NaHCO3 (40.0 mL) and then washed with water (40.0 mL). The organic phase was dried over Na2SO4, filtered, and then concentrated under reduced pressure to remove the solvent to obtain a yellow oily crude product G3-1 (1.42 g, 2.61 mmol, 32.7% yield). MS (ESI) m / z [M – H] - : 542.2; 1 H NMR (400 MHz, CDCl3) δ ppm 6.32 (d, J = 8.8 Hz, 1 H), 6.09 - 6.01 (m, 1 H), 5.34 (d, J =2.7 Hz, 1 H), 5.04 (dd, J = 11.2, 3.3 Hz, 1 H), 4.45 (d, J = 8.4 Hz, 1 H),4.28 - 4.09 (m, 3 H), 4.03 - 3.98 (m, 1 H), 3.88 (td, J = 6.7, 1.2 Hz, 1 H),3.71 - 3.62 (m, 1 H), 3.44 (td, J = 9.3, 3.4 Hz, 1 H), 3.28 (t, J = 6.8 Hz, 2H), 3.10 - 3.03 (m, 1 H), 2.24 (t, J = 7.5 Hz, 2 H), 2.16 (s, 3 H), 2.04 (s,3 H), 2.01 (s, 3 H), 1.96 (s, 3 H), 1.76 - 1.63 (m, 6 H), 1.46 - 1.38 (m, 2H).

[0241] Step 2: Synthesis of G3-2

[0242] G1-2 (230 mg, 686 μmol, 1.0 eq) was added to a DMSO solution (50.0 mL) of G3-1 (1.30 g, 2.40 mmol, 3.5 eq). Then, CuSO4 (985 mg, 6.17 mmol, 947 μL, 9.0 eq) and THPTA (1.79 g, 4.11 mmol, 6.0 eq) were added to water (6.0 mL), followed by the addition of VC Na (2.04 g, 10.3mmol, 15.0 eq) to prepare an aqueous solution, which was added to the above DMSO solution and stirred at 25 °C for 2 h. Samples were taken and tested by LC / MS, which showed that the starting material disappeared. The reaction solution was diluted with water (30.0 mL), extracted with dichloromethane (30.0 mL × 3), and then washed with water (30.0 mL × 3). The organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure to remove the solvent. The crude product was purified by preparative chromatography to obtain a white solid G3-2 (1.0 g, 508 μmol, 74.2% yield). MS (ESI) m / z [M - 2H] 2- :981.8; 1 H NMR (400 MHz, CDCl3) δ ppm 7.60 (s, 3 H), 6.84 (d, J = 8.9 Hz, 3 H), 6.62 - 6.54 (m, 3 H), 5.34 (d, J = 3.3 Hz, 3 H), 5.06 (dd, J = 11.2, 3.4 Hz,3 H), 4.57 (s, 6 H), 4.50 (d, J = 8.5 Hz, 3 H), 4.36 (t, J = 6.9 Hz, 6 H), 4.28 - 4.07 (m, 10 H), 4.00 - 3.95 (dt, J = 9.9, 5.0 Hz, 3 H), 3.90 (t, J =6.7 Hz, 3 H), 3.69 (s, 6 H), 3.59 - 3.55 (m, 3 H), 3.51 - 3.41 (m, 3 H), 3.11- 3.06 (m, 3 H), 2.21 (t, J = 7.4 Hz, 6 H), 2.15 (s, 9 H), 2.03 (s, 9 H), 1.99 (s, 9 H), 1.93 (s, 9 H), 1.82 (s, 9 H), 1.72 - 1.61 (m, 9 H), 1.40 (s, 9H), 1.37 - 1.29 (m, 6 H).

[0243] Step 3: Synthesis of G3-3

[0244] Trifluoroacetic acid (3.07 g, 26.9 mmol, 2.0 mL, 52.9 eq) was added to a dichloromethane solution (10.0 mL) of G3-2 (1.00 g, 508 μmol, 1.00 eq) and stirred at 25 °C for 1 h. The sample was detected by TLC (dichloromethane / methanol), which showed that the starting material disappeared. The reaction solution was directly concentrated under reduced pressure to remove the solvent, and the crude product was co-rotated with toluene (20.0 × 3 mL) three times to obtain a yellow oil G3-3 (950 mg, 458.21 μmol, 90.09% yield, 90% purity). MS (ESI) m / z [M - 2H] 2- : 931.7.

[0245] Step 4: Synthesis of G3-4

[0246] HBTU (174 mg, 460 μmol, 1.5 eq) and DIPEA (79.4 mg, 614 μmol, 107 μL, 2.0 eq) were added to a DMF solution (3.0 mL) of G1-9 (200 mg, 307 μmol, 1.0 eq). Then, the crude product G3-3 (837 mg, 430 μmol, 1.4 eq, TFA) (pH adjusted to 7 with DIPEA) was added to the above reaction solution and stirred at 25 °C for 4 h. The sample was taken and detected by LC / MS, which showed that the starting material disappeared. The reaction solution was used directly in the next step. MS (ESI) m / z [M -2H] 2- : 1247.8.

[0247] Step 5: Synthesis of G3-5

[0248] G3-4 (760 mg, 304 μmol, 1.0 eq) was added to DMF (4.0 mL). DIPEA (112 mg, 912 μmol, 158 μL, 3.0 eq), succinic anhydride (243 mg, 2.43 mmol, 8.0 eq) and DMAP (37.1 mg, 304 μmol, 1.0 eq) were then added, and the reaction solution was stirred at 35 °C for 24 h. The sample was taken and tested by LC / MS, which showed that the starting material disappeared and the target product was detected. The reaction solution was filtered and the filtrate was purified by preparative chromatography to obtain a white solid G3-5 (400 mg, 142 μmol, 46.7% yield, 96.0% purity, TEA). MS (ESI) m / z [M - 2H] 2- : 1298.2; 1HNMR (400 MHz, CDCl3) δ ppm 7.53 (s, 3 H), 7.40 - 7.36 (m, 2 H), 7.31 - 7.24(m, 4 H), 7.22 - 7.07 (m, 6 H), 6.84 (d, J = 8.9 Hz, 2 H), 6.77 - 6.70 (m, 4H), 6.59 (t, J = 6.1 Hz, 3 H), 6.08 (s, 1 H), 5.28 (d, J = 3.3 Hz, 3 H), 5.09(t, J = 5.1 Hz, 1 H), 5.04 - 4.90 (m, 4 H), 4.50 (s, 6 H), 4.45 (d, J = 8.4Hz, 3 H), 4.30 (t, J = 6.9 Hz, 6 H), 4.21 - 4.02 (m, 10 H), 3.93 - 3.88 (m, 3H), 3.87 - 3.81 (m, 4 H), 3.70 (d, J = 9.3 Hz, 12 H), 3.50 (d, J = 7.8 Hz, 3H), 3.39 (td, J = 9.2, 3.7 Hz, 3 H), 3.32 (s, 3 H), 3.16 (dd, J = 9.8, 4.3Hz, 2 H), 3.08 - 2.99 (m, 4 H), 2.76 (q, J = 7.3 Hz, 9 H), 2.63 - 2.57 (m, 2H), 2.47 - 2.44 (m, 2 H), 2.15 (t, J = 7.3 Hz, 6 H), 2.09 (s, 9 H), 1.97 (s,9 H), 1.92 (s, 9 H), 1.87 (s, 9 H), 1.71 - 1.67 (m, 3 H), 1.66 - 1.56 (m, 9H), 1.51 - 1.40 (m, 4 H), 1.29 - 1.24 (m, 6 H), 1.10 (t, J = 7.2 Hz, 11 H).

[0249] Step 6: Synthesis of YK-GAL-403

[0250] G3-5 (400 mg, 154 μmol, 1.0 eq) was added to DMF (10.0 mL). DIPEA (159 mg, 1.23 mmol, 214 μL, 8.0 eq), HBTU (291 mg, 769 μmol, 5.0 eq), DMAP (18.8 mg, 154 μmol, 1.00 eq) and CPG-NH2 (2.25 g) were then added, and the reaction solution was stirred at 40 °C for 16 h. Samples were taken and tested by LC / MS, which showed that the starting material disappeared. The reaction solution was filtered, and the filter cake was washed with MeOH (20 mL × 4) and DCM (20 mL × 4) successively, and dried under nitrogen to obtain a light yellow solid, which was directly used in the next step.

[0251] The crude product (2.4 g) was dissolved in pyridine (15.0 mL). Acetic anhydride (3.26 g, 31.9 mmol, 3.0 mL, 34.5 eq) was then added, and the reaction solution was stirred at 40 °C for 1 h. The reaction solution was then filtered, and the filter cake was washed with MeOH (30 mL × 4) and DCM (30 mL × 4) successively, and dried under nitrogen to obtain a light yellow solid YK-GAL-403 (2.2 g, loading: 32.8 μmol / g). The carrier loading was determined according to the general method for determining the carrier loading.

[0252] 4. Synthesis of YK-GAL-404

[0253] The synthetic route is as follows:

[0254]

[0255] Step 1: Synthesis of G4-1

[0256] Using crude product G1-8 (537 mg, 280 μmol, 1.4 eq, TFA) and G1-9A (preparation method reference CN116854754B) (124 mg, 200 μmol, 1.0 eq) as raw materials, the reaction was carried out according to the method for synthesizing G1-10. The sample was taken and tested by LC / MS, which showed that the raw material disappeared. The reaction solution was directly used in the next step. MS (ESI) m / z [M – 2 H] 2- :1212.0.

[0257] Step 2: Synthesis of G4-2

[0258] Using G4-1 (485 mg, 200 umol, 1.0 eq) as the starting material, the reaction was carried out according to the method for synthesizing G1-11 to obtain white solid G4-2 (187 mg, 74.2 umol, 37.1%). MS (ESI) m / z [M – 2 H] 2- : 1262.1; 1 H NMR (400 MHz, CDCl3) δ 7.63 (s, 2H), 7.53 - 7.45 (m, 2H), 7.36 - 7.33 (m, 3H), 7.29 - 7.11 (m, 7H) , 6.92 (d, J = 9.1 Hz, 2H), 6.79 - 6.68 (m, 5H), 5.12 -5.05 (m, 3H), 5.06 (d, J = 2.3 Hz, 1H), 5.01 - 4.97 (m, 2H), 4.79 - 4.61 (m,8H), 4.53 (t, J = 6.0 Hz, 4H), 4.34 - 4.20 (m, 3H), 4.14 - 4.09 (m, 4H), 3.98- 3.86 (m, 5H), 3.80 (s, 6H), 3.77 - 3.65 (m, 2H), 3.47 - 3.32 (m, 10H), 3.25- 3.12 (m, 3H), 2.85 - 2.61 (s, 9H), 2.53 - 2.44 (m, 2H), 2.39 - 1.89 (m,52H), 1.82 - 1.49 (m, 19H), 1.19 - 1.09 (m, 18H).

[0259] Step 3: Synthesis of YK-GAL-404

[0260] G4-2 (126 mg, 50.0 umol, 1.0 eq) was used as the raw material, and the reaction was carried out according to the method for synthesizing YK-GAL-401 to obtain a light yellow solid YK-GAL-404 (783 mg, loading: 35.9 μmol / g). The carrier loading was determined according to the general method for determining the carrier loading.

[0261] 5. Synthesis of YK-GAL-405

[0262] The synthetic route is as follows:

[0263]

[0264] Step 1: Synthesis of G5-1

[0265] Using crude product G1-8 (537 mg, 280 μmol, 1.4 eq, TFA) and G1-9B (preparation method reference CN116854754B) (125 mg, 200 μmol, 1.0 eq) as raw materials, the reaction was carried out according to the method for synthesizing G1-10. The sample was taken and tested by LC / MS, which showed that the raw material disappeared. The reaction solution was directly used in the next step. MS (ESI) m / z [M – 2 H] 2- :1215.2.

[0266] Step 2: Synthesis of G5-2

[0267] Using G5-1 (481 mg, 198 umol, 1.0 eq) as the starting material, the reaction was carried out according to the method for synthesizing G1-11 to obtain a white solid G5-2 (173 mg, 68.4 umol). MS (ESI) m / z [M – 2 H] 2- : 1265.2; 1 HNMR (400MHz, CDCl3) δ ppm 7.53 (s, 1H), 7.50 (s, 1H), 7.44 (d, J = 8.0 Hz, 2H), 7.25- 7.16 (m, 4H), 7.05 - 6.91 (m, 4H), 6.85 - 6.72 (m, 6H), 6.85 - 6.72 (m,2H), 5.48 - 5.45 (m, 4H), 5.39 (d, J = 4.0 Hz, 2H), 5.12 - 4.99 (m, 3H), 4.57- 4.32 (m, 11H), 4.19 - 4.06 (m, 6H), 3.96 - 3.87 (m, 4H), 3.85 - 3.79 (m,14H), 3.68 (s, 6H), 3.63 - 3.56 (m, 12H), 3.37 - 3.25 (m, 11H), 3.15 (t, J =5.9 Hz, 5H), 3.11 - 2.89 (m, 4H), 2.48 - 2.34 (m, 12H), 2.21 - 1.98 (m, 25H), 1.82 - 1.61 (m, 14H), 1.28 - 1.08 (m, 13H).

[0268] Step 3: Synthesis of YK-GAL-405

[0269] G5-2 (171 mg, 67.5 umol, 1.0 eq) was used as the raw material, and the reaction was carried out according to the method for synthesizing YK-GAL-401 to obtain a light yellow solid YK-GAL-405 (1.1 g, loading: 35.9 μmol / g). The carrier loading was determined according to the general method for determining the carrier loading.

[0270] 6. Synthesis of YK-GAL-406

[0271] The synthetic route is as follows:

[0272]

[0273] Step 1: Synthesis of G6-1

[0274] Using crude product G1-8 (402 mg, 210 μmol, 1.4 eq, TFA) and G1-9C (preparation method reference CN116854754B) (88 mg, 150 μmol, 1.0 eq) as raw materials, the reaction was carried out according to the method for synthesizing G1-10. The sample was taken and tested by LC / MS, which showed that the raw material disappeared. The reaction solution was directly used in the next step. MS (ESI) m / z [M – 2 H] 2- :1198.0.

[0275] Step 2: Synthesis of G6-2

[0276] Using G6-1 (479 mg, 150 umol, 1.0 eq) as the starting material, the reaction was carried out according to the method for synthesizing G1-11 to obtain white solid G6-2 (146 mg, 58.8 umol). MS (ESI) m / z [M – 2 H] 2- : 1247.8; 1HNMR (400MHz, CDCl3) δ ppm 7.47 (d, J = 6.4 Hz, 2H), 7.36 - 7.31 (m, 3H), 7.27 - 7.23(m, 4H), 7.20 (d, J = 6.0 Hz, 1H), 7.15 - 7.08 (m, 2H), 7.04 - 6.97 (m, 4H), 6.90 - 6.83 (m, 3H), 5.54 - 5.51 (m, 2H), 5.42 (d, J = 3.9 Hz, 2H), 5.36 (t,J = 4.0 Hz, 2H), 4.89 - 4.81 (m, 1H), 4.55 - 4.47 (m, 5H), 4.38 - 4.24 (m,7H), 4.16 - 4.04 (m, 4H), 3.80 (s, 6H), 3.74 - 3.65 (m, 11H), 3.59 - 3.52 (m,4H), 3.46 - 3.28 (m, 15H), 2.83 - 2.78 (m, 7H), 2.64 (t, J = 6.5 Hz, 4H), 2.59 - 2.50 (m, 4H), 2.47 - 2.41 (m, 4H), 2.33 - 1.97 (m, 33H), 1.86 - 1.62(m, 20H), 1.37 - 1.29 (m, 5H), 1.16 - 1.13 (m, 13H).

[0277] Step 3: Synthesis of YK-GAL-406

[0278] G6-2 (146 mg, 58.8 umol, 1.0 eq) was used as the raw material, and the reaction was carried out according to the method for synthesizing YK-GAL-401 to obtain a light yellow solid YK-GAL-406 (832 mg, loading: 34.1 μmol / g). The carrier loading was determined according to the general method for determining the carrier loading.

[0279] 7. Synthesis of reference substance YK-GAL-301

[0280]

[0281] According to the synthesis method of YK-GAL-301 on page 35 of the CN116854754B specification, the target product was obtained.

[0282] 8. Synthesis of reference substance GalNAc 1b

[0283]

[0284] According to the synthesis method of GalNAc 1b on page 145 of WO2023014938A1, 377.2 mg of product was obtained with a total yield of 0.58%, MS (ESI) m / z [M + H] + = 1137.9.

[0285] 9. Synthesis of reference substance NAG0052

[0286]

[0287] According to the synthesis method of NAG0052 on page 100 of the specification of WO2023109938A1, 288 mg of product was obtained with a total yield of 1.26% and a loading of 31.5 μmol / g.

[0288] Example 2: Coupling of GalNAc compounds to oligonucleotides

[0289] In this example, the same siRNA sequence was used for synthesis, and the conjugated siRNA sequence was a sequence numbered inc, and the inc sequence was as follows:

[0290] Sense strand (inc-SS): 5'-Cms-Ums-Am-Gm-Am-Cm-Cf-Um-Gf-Um-dT-Um-Um-Gm-Cm-Um-Um-Um-Um-Gm-Um-3' (SEQ ID NO. 46)

[0291] Antisense strand (inc-AS): 5'-Ams-Cfs-Am-Af-Af-Af-Gm-Cf-Am-Af-Am-Af-Cm-Af-Gm-Gf-Um-Cf-Um-Am-Gms-Ams-Am-3' (SEQ ID NO. 47)

[0292] Among them, A, U, C, and G represent the base composition of the nucleotide; dT represents deoxythymidine nucleotide; m represents that the nucleotide adjacent to the left of m is 2'-OMe modified; f represents that the nucleotide adjacent to the left of f is 2'-F modified; s represents that the two adjacent nucleotides on the left and right of s are connected by thiophosphate groups.

[0293] The sequence comes from a public sequence and is the oligonucleotide sequence of the siRNA drug inclisiran that has been marketed overseas.

[0294] The inclisiran sequence is a synthetic, chemically modified double-stranded small interfering RNA (siRNA) that targets and binds to the mRNA encoding the PCSK9 protein, inhibiting the production of the PCSK9 protein through the RNA interference mechanism, thereby regulating the recycling and reuse of the LDL receptor, enhancing its binding with LDL, and achieving the purpose of lowering the LDL level in the blood.

[0295] 1. Preparation of GalNAc-conjugated siRNA sense strand

[0296] Oligonucleotide GalNAc conjugates were synthesized on a solid support according to the phosphoramidite chemistry. When synthesizing inc-YK-Gal-401, inc-YK-Gal-402, inc-YK-Gal-403, inc-YK-Gal-404, inc-YK-Gal-405, inc-YK-Gal-406, inc-YK-Gal-301 and inc-NAG0052 conjugates, the GalNAc-CPG compound synthesized in Example 1 was used as a solid support. When synthesizing inc-GalNAc 1b conjugates, universal CPG was used as a solid support, and the reference substance inc-GalNAc 1b monomer was coupled as the first monomer. Among them, GalNAc was conjugated to the 3' end of the siRNA sense strand, and the specific structure of the GalNAc compound used is shown in Table 1.

[0297] Table 1 GalNAc compounds conjugated to the positive strand of siRNA

[0298]

[0299]

[0300] Experimental process:

[0301] (1) Preparation of reagents and monomers

[0302] By using an acetonitrile solution of the monomer (1 / 20, w / v), a 0.25 M 5-benzylmercaptotetrazole solution in acetonitrile as an activating agent, a 0.2 M solution of hydroxanthin in acetonitrile / pyridine (1 / 4, v / v) as a thiolation reagent, a 0.05 M solution of iodine in water / pyridine (1 / 9, v / v) as an oxidizing reagent, 20% acetic anhydride in acetonitrile (v / v) as a capping agent A, 20 / 30 / 50 (1-methylimidazole / pyridine / acetonitrile, v / v / v) as a capping agent B, 20% diethylamine in acetonitrile (v / v) as a decyanoethylation reagent, and 3% dichloroacetic acid in toluene (v / v) as a de-DMT reagent, the corresponding CPG carrier was selected and loaded into the designated reagent position of a 192 P model DNA / RNA automatic synthesizer.

[0303] (2) Crude product synthesis

[0304] Enter the designated oligonucleotide sequence and set the synthesis program. After checking that everything is correct, start the cyclic oligonucleotide synthesis. The monomer coupling time is about 1 minute, of which the oxygen generation time is about 30-45 seconds and the thio generation time is about 2 minutes. After the cycle is completed, the solid phase synthesis of the oligonucleotide is completed.

[0305] (3) Deprotection

[0306] After the synthesis is completed, the solid phase carrier is transferred to a reactor, and the oligonucleotide is cleaved from the solid phase carrier with concentrated ammonia (25-28%) at 50-60 ° C for 16-24 hours. The system is cooled to room temperature and then filtered, rinsed with a mixed solution of purified water and ethanol, the filtrate is combined, and the filtrate is concentrated at low temperature to obtain a crude residue.

[0307] (4) Purification

[0308] The crude residue after deprotection was dissolved in purified water and purified by HPLC. The product peak solution was collected and the content was measured by an enzyme marker, and the molecular weight was confirmed by ESI MS.

[0309] 2. Preparation of siRNA antisense strand without GalNAc conjugation

[0310] The siRNA antisense chain was synthesized according to the method for synthesizing the siRNA sense chain, wherein a universal CPG solid phase carrier was used, and the synthesis scale of each antisense chain complementary to the sense chain was 1 μmol.

[0311] 3. Preparation of conjugated double-stranded siRNA

[0312] The siRNA sense strand and the complementary antisense strand were mixed in a ratio of 1:1 according to the UV absorption content, heated to 95°C, and cooled to room temperature after 3 minutes to form a double strand. The obtained double-stranded solution was characterized by HPLC, and the content was determined by an ELISA instrument after the product purity was qualified, and lyophilized to obtain a solid powder for storage.

[0313] The GalNAc compounds in Table 1 were conjugated with siRNA numbered inc, and the resulting conjugates were named inc-YK-Gal-401, inc-YK-Gal-402, inc-YK-Gal-403, inc-YK-Gal-404, inc-YK-Gal-405, inc-YK-Gal-406, inc-YK-Gal-301, inc-GalNAc 1b and inc-NAG0052.

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323] Example 3: Inhibitory effect of GalNAc-conjugated siRNA on PCSK9 gene in mouse serum and liver and its effect on LDL-C level

[0324] After the GalNAc-conjugated inclisiran siRNA sequence enters the blood, it binds to the ASGPR on the liver cell membrane through GalNAc and enters the liver cell. After the inclisiran siRNA sequence enters the liver cell, it binds to the RNA-induced silencing complex (RISC) and binds to the mRNA encoding the PCSK9 protein under the mediation of the antisense strand, which inhibits the production of the PCSK9 protein. The reduction of PCSK9 protein in the liver promotes the circulation of LDL-R, increases the number of LDL-R receptors on the surface of liver cells, and further reduces the plasma LDL-C level. Therefore, the higher the amount of Inclisiran siRNA sequence delivered to the liver, that is, the higher the efficiency of GalNAc delivery, the lower the amount of PCSK9 protein in the liver and serum, and the lower the level of LDL-C in the liver.

[0325] The results showed that compared with the existing GalNAc oligonucleotide conjugates, the PCSK9 gene expression inhibition rate and LDL-C reduction levels in mouse serum and liver in the inc-YK-Gal-401, inc-YK-Gal-402 and inc-YK-Gal-403 experimental groups were significantly improved. For example, compared with inc-YK-Gal-301, inc-GalNAc 1b, and inc-NAG0052, the inhibition rate of PCSK9 protein in serum by inc-YK-Gal-401 was increased by 21.5%, 14.5%, and 12.7% on the 7th day, and by 17.3%, 10.4%, and 10.8% on the 14th day, respectively; the inhibition rate of PCSK9 gene in mouse liver was increased by 21.1%, 9.8%, and 5.1% on the 14th day, respectively; the LDL-C reduction level was significantly increased by 33.4%, 19.1%, and 16.7% on the 7th day, and by 36.4%, 20.6%, and 17.8% on the 14th day, respectively.

[0326] Animal preparation:

[0327] First, hPCSK9 transgenic mice (SPF grade, purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.) were adaptively raised and then randomly divided into a negative control group (no medication) and a siRNA test drug group according to the serum PSCK9 protein content, with 5 mice in each group, all of which were male.

[0328] Administration and dosage:

[0329] The drug was administered by a single subcutaneous injection at a dose of 6 mg / kg, a volume of 1 mL / kg, and a concentration of 6 mg / mL. The day of administration was designated as day 0.

[0330] 1. Inhibitory effects of different GalNAc-conjugated siRNAs on PCSK9 protein expression in mouse serum

[0331] Compared with the prior art GalNAc, the oligonucleotide conjugate prepared by the GalNAc compound of the present invention significantly improves the inhibition rate of PCSK9 protein expression in mouse serum. For example, the inhibition rate of inc-YK-Gal-401 on the 7th day was 21.5%, 14.5% and 12.7% higher than that of inc-YK-Gal-301, inc-GalNAc 1b and inc-NAG0052, and the inhibition rate on the 14th day was 17.3%, 10.4% and 10.8% higher, respectively.

[0332] Experimental process:

[0333] Before administration (D0), 7 days after administration (D7), and 14 days after administration (D14), about 200 μL of blood (without anticoagulation) was collected from the orbital venous plexus of the experimental animals. The whole blood samples were temporarily stored in an ice box before centrifugation, and the serum was separated by centrifugation at 4000 r / min for 10 min at 4°C. The PCSK9 protein level in the serum was detected using an ELISA kit (Sino Biological Company).

[0334] Experimental results:

[0335] The inhibition rates of PCSK9 protein in serum of the negative control group and each test drug group 7 days and 14 days after administration are shown in Table 3 and Figure 1 GraphPad Prism 9 software was used for data statistics and analysis.

[0336] 1. Structural differences of GalNAc compounds

[0337] Table 2 Comparison of GalNAc compound structures

[0338]

[0339] 1) The chemical structures of the GalNAc compounds YK-GAL-401 to YK-Gal-406 designed in the present invention are very similar.

[0340] It can be seen from Table 1 and Table 2 that the chemical structures of the GalNAc compounds YK-GAL-401, YK-GAL-402, YK-GAL-403, YK-GAL-404, YK-GAL-405 and YK-GAL-406 designed in the present invention are very similar, for example, the 1' position of ribose is oxygen, the number of antennae is 3, and the antennae contain triazole structures.

[0341] These compounds differ only slightly in individual groups. For example, the 2' substituent of ribose in YK-GAL-401, YK-GAL-402 and YK-GAL-403 is methoxy, and YK-GAL-404, YK-GAL-405 and YK-GAL-406 is hydrogen. The main chain structures of YK-GAL-401~YK-Gal-404 are all -(CH2) 10 C(O)NH-, YK-GAL-405 is -(CH2CH2O)3CH2C(O)NH-, and YK-GAL-406 is -(CH2)8C(O)NH-. In addition, the length of the chain in the antennae is slightly different, but the other structures are exactly the same.

[0342] 2) The chemical structures of the GalNAc compounds YK-GAL-401 to YK-GAL-406 designed in the present invention are very similar to those of the GalNAc compounds in the prior art, while others are very different.

[0343] The GalNAc compounds YK-GAL-401, YK-GAL-402, YK-GAL-403, YK-GAL-404, YK-GAL-405 and YK-GAL-406 designed in the present invention have very similar chemical structures compared to the prior art GalNAc compounds YK-GAL-301 and NAG0052. For example, YK-GAL-301 and NAG0052 also contain three antennae, and the main chain structure contains a fatty chain and an amide bond, but the antennae of YK-GAL-301 and NAG0052 do not contain a triazole structure.

[0344] The GalNAc compound designed in the present invention has a great difference in chemical structure from GalNAc 1b. For example, GalNAc 1b has only one antenna, a very short chain, and does not contain a triazole structure in the chain.

[0345] 2. Inhibition rate of PCSK9 protein expression in mouse serum by different GalNAc-conjugated siRNAs

[0346] Table 3 PCSK9 protein expression inhibition rate in mouse serum

[0347]

[0348] 1) The siRNA conjugated with the GalNAc compound of the present invention has significant differences in the inhibition rate of PCSK9 protein expression in mouse serum, and YK-GAL-401~YK-Gal-403 are significantly higher than YK-GAL-404~406. For example, inc-YK-Gal-401 increased by 20.7% and 16.0% on the 7th day and 14th day respectively compared with inc-YK-Gal-404.

[0349] As can be seen from Table 3, the siRNA conjugated with the GalNAc compound of the present invention can inhibit the expression of PCSK9 protein in mouse serum. Different GalNAc conjugates have significant differences in the inhibition rate of PCSK9 protein expression in mouse serum. The inhibition rates of inc-YK-Gal-401, inc-YK-Gal-402, inc-YK-Gal-403, inc-YK-Gal-404, inc-YK-Gal-405 and inc-YK-Gal-406 on the 7th day were 94.2%, 92.4%, 93.1%, 73.5%, 84.3% and 85.0%, respectively, and the inhibition rates on the 14th day were 95.4%, 92.8%, 94.3%, 79.4%, 88.7% and 90.2%, respectively.

[0350] The inhibition rates of inc-YK-Gal-401, inc-YK-Gal-402, and inc-YK-Gal-403 all exceeded 90%, while that of inc-YK-Gal-404 was between 70% and 80%. The inhibition rate of inc-YK-Gal-401, which had the highest inhibition rate, was significantly higher by 20.7% and 16.0% on the 7th and 14th days than that of inc-YK-Gal-404, which had the lowest inhibition rate.

[0351] 2) Compared with the GalNAc compounds of the prior art, the conjugated siRNA of the present invention, YK-GAL-401 to YK-GAL-403, significantly improves the inhibition rate of PCSK9 protein expression in mouse serum. For example, inc-YK-Gal-401 increased the PCSK9 protein expression by 21.5% and 17.3% on the 7th day and 14th day respectively compared with inc-YK-Gal-301.

[0352] The inhibition rates of siRNA conjugates inc-YK-Gal-301, inc-GalNAc 1b and inc-NAG0052 prepared by the prior art GalNAc compound were 72.7%, 79.7% and 81.5% on day 7, and 78.1%, 85.0% and 84.6% on day 14, respectively.

[0353] Compared with the GalNAc compounds in the prior art, the GalNAc compound of the present invention significantly improves the inhibition rate of PCSK9 protein expression in mouse serum by the conjugated siRNA. For example, the inhibition rates of inc-YK-Gal-401, inc-YK-Gal-402 and inc-YK-Gal-403 on the 7th day were significantly increased by 21.5%, 19.7% and 20.4% compared with inc-YK-Gal-301, by 14.5%, 11.9% and 13.4% compared with inc-GalNAc 1b, and by 12.7%, 10.9% and 11.6% compared with inc-NAG0052, respectively; the inhibition rates on the 14th day were significantly increased by 17.3%, 14.7% and 16.2% compared with inc-YK-Gal-301, by 10.4%, 7.8% and 9.3% compared with inc-GalNAc 1b, and by 10.8%, 8.2% and 9.7% compared with inc-NAG0052, respectively.

[0354] 3) The conjugates of GalNAc compounds with similar structures and siRNA have significantly different inhibition rates on PCSK9 protein expression in mouse serum.

[0355] The GalNAc compounds designed by the present invention have very similar structures, but the activities of the siRNA conjugates are very different. For example, YK-GAL-401 and YK-GAL-404 are different only in the 2' position of the ribose ring, YK-GAL-401 is methoxy, and YK-GAL-404 is hydrogen, and the other structures are exactly the same, but the inhibition rate of PCSK9 protein expression in mouse serum by inc-YK-Gal-401 can be increased by more than 20% compared with inc-YK-Gal-404, which is significantly improved.

[0356] Compared with YK-GAL-301, YK-GAL-406 only differs in the main chain and antennae. The main chain of YK-GAL-406 has 2 fewer methylene groups than that of YK-GAL-301. The antennae of YK-GAL-406 have a triazole structure, while YK-GAL-301 does not. However, the inhibition rate of inc-YK-Gal-406 on the 7th day was significantly increased by 12.3% compared with inc-YK-Gal-301.

[0357] Compared with inc-NAG0052, YK-GAL-401 has only slight differences in individual groups, but the inhibition rate of inc-YK-Gal-401 on the 7th day was 12.7% higher than that of inc-NAG0052, which is a significant improvement.

[0358] Therefore, GalNAc compounds with similar chemical structures do not necessarily have similar oligonucleotide delivery efficiencies. The inhibition rates of PCSK9 protein expression in mouse serum by the prepared GalNAc oligonucleotide conjugates are not consistent and may have very large differences.

[0359] 3. Inhibitory effects of different GalNAc-conjugated siRNAs on PCSK9 in mouse liver

[0360] Compared with the prior art GalNAc, the oligonucleotide conjugate prepared by the GalNAc compound of the present invention significantly improves the inhibition rate of PCSK9 gene expression in mouse liver. For example, the inhibition rate of inc-YK-Gal-401 is 21.1% and 9.8% higher than that of inc-YK-Gal-301 and inc-GalNAc 1b, respectively, which is significantly improved.

[0361] Experimental process:

[0362] On the 14th day (D14) after administration, the experimental animals were anesthetized and killed, and then perfused to collect the liver.

[0363] According to the tissue weight: RNA lysis solution (trizol, Ambion) = 100 mg: 1 mL, quickly put it into a 1.5 mL RNase-free EP tube containing 1 mL RNA lysis solution (trizol), add 3 3 mm steel beads (RNA-free treated) to the tube, put it into a tissue homogenizer, run at 50 Hz for 30 seconds, stop for 10 seconds, run 3 times, and tissue homogenate can be prepared.

[0364] Centrifuge at 4°C, 12,000 × g for 3 minutes, transfer 400 μL of homogenate supernatant to a 1.5 mL RNase-free EP tube and place on ice. Add 80 μL of chloroform to each tube, shake vigorously for 15 seconds, and let stand at room temperature for 5 minutes. Centrifuge at 4°C, 12,000 × g for 15 minutes, and take 150 μL of supernatant to a new EP tube.

[0365] Add an equal volume of isopropanol, gently mix the liquid in the tube by inverting it upside down, let it stand at -20°C for 10 minutes, centrifuge at 4°C, 12,000 × g for 15 minutes, and discard the supernatant.

[0366] Add 1 mL of 75% ethanol, gently wash the RNA precipitate, centrifuge at 7,500 × g at 4°C for 5 minutes, and remove the supernatant. Repeat the rinse once, centrifuge at 7,500 × g at 4°C for 5 minutes, and remove any remaining ethanol with a micropipette.

[0367] The residual ethanol was dried at room temperature for 10 minutes, and 150 μL RNase-free ddH2O was added to dissolve. The RNA concentration was detected using a micro-volume UV spectrophotometer. The PCSK9 gene expression was detected by qPCR.

[0368] Experimental results:

[0369] The qPCR results (Mean ± SD) were statistically analyzed using GraphPad Prism 9 software for plotting and data analysis. The specific results are shown in Tables 4 and Figure 2 .

[0370] Table 4 PCSK9 gene expression inhibition rate in mouse liver

[0371]

[0372] 1) The siRNA conjugated with the GalNAc compound of the present invention has significant differences in the inhibition rate of PCSK9 gene expression in mouse liver, and YK-GAL-401~YK-GAL-403 are significantly higher than YK-GAL-404~YK-GAL-406. For example, inc-YK-Gal-401 is 19.4% higher than inc-YK-Gal-404.

[0373] As can be seen from Table 4, the siRNA conjugated with the GalNAc compound of the present invention can inhibit the expression of PCSK9 gene in mouse liver. Different GalNAc conjugates have significant differences in the inhibition rate of PCSK9 gene expression in mouse liver. The inhibition rates of inc-YK-Gal-401, inc-YK-Gal-402, inc-YK-Gal-403, inc-YK-Gal-404, inc-YK-Gal-405 and inc-YK-Gal-406 are 90.9%, 89.3%, 90.1%, 71.5%, 83.2% and 81.6%, respectively.

[0374] The inhibition rates of inc-YK-Gal-401 and inc-YK-Gal-403 both exceeded 90%, and that of inc-YK-Gal-402 also reached 89.3%, while that of inc-YK-Gal-404 was only 71.5%. The inhibition rate of inc-YK-Gal-401 with the highest inhibition rate was significantly higher than that of inc-YK-Gal-404 with the lowest inhibition rate, which was 19.4%.

[0375] 2) Compared with the prior art GalNAc compounds, the conjugated siRNA of the present invention, YK-GAL-401 to YK-GAL-403, significantly increased the inhibition rate of PCSK9 gene expression in mouse liver. For example, inc-YK-Gal-401 increased by 21.1% compared with inc-YK-Gal-301.

[0376] The inhibition rates of siRNA conjugates inc-YK-Gal-301, inc-GalNAc 1b and inc-NAG0052 prepared from the prior art GalNAc compounds were 69.8%, 81.1% and 85.8%, respectively.

[0377] Compared with the GalNAc compounds of the prior art, the GalNAc compounds of the present invention significantly improve the inhibition rate of PCSK9 protein expression in mouse serum by the conjugated siRNA. For example, the inhibition rates of inc-YK-Gal-401, inc-YK-Gal-402 and inc-YK-Gal-403 are 21.1%, 19.5% and 20.3% higher than those of inc-YK-Gal-301, and 9.8%, 8.2% and 9.0% higher than those of inc-GalNAc 1b, respectively, which are significantly improved.

[0378] 3) The conjugates of GalNAc compounds with similar structures and siRNA have significantly different inhibition rates on PCSK9 gene expression in mouse liver.

[0379] The GalNAc compounds designed by the present invention have very similar structures, but the activities of the siRNA conjugates are very different. For example, YK-GAL-401 and YK-GAL-404 are different only in the 2' position of the ribose ring, YK-GAL-401 is methoxy, and YK-GAL-404 is hydrogen. The other structures are exactly the same, but the inhibition rate of PCSK9 gene expression in mouse liver by inc-YK-Gal-401 can be increased by 19.4% compared with inc-YK-Gal-404, which is significantly improved.

[0380] Compared with YK-GAL-301, YK-GAL-406 only differs in the main chain and antennae. The main chain of YK-GAL-406 has two fewer methylene groups than that of YK-GAL-301. The antennae of YK-GAL-406 have a triazole structure, while YK-GAL-301 does not. However, the inhibition rate of inc-YK-Gal-406 is significantly increased by 11.8% compared with inc-YK-Gal-301.

[0381] Therefore, GalNAc compounds with similar chemical structures do not necessarily have similar oligonucleotide delivery efficiencies. The inhibition rates of the PCSK9 gene in mouse livers by the prepared GalNAc oligonucleotide conjugates are not consistent and may vary greatly.

[0382] 4. Effects of different GalNAc-conjugated siRNAs on LDL-C levels in mouse serum

[0383] Compared with the prior art GalNAc, the oligonucleotide conjugate prepared by the GalNAc compound of the present invention significantly improves the level of LDL-C reduction in mouse serum. For example, the level of LDL-C reduction in the inc-YK-Gal-401 group of mice on day 14 was significantly improved by 36.4%, 20.6% and 17.8% compared with the inc-YK-Gal-301, inc-GalNAc 1b and NAG0052 groups, respectively.

[0384] Experimental process:

[0385] Before administration (D0), 7 days after administration (D7), and 14 days after administration (D14), about 200 μL of blood (without anticoagulation) was collected from the orbital venous plexus of the experimental animals. The whole blood samples were temporarily stored in an ice box before centrifugation, and the serum was separated by centrifugation at 4000 r / min for 10 minutes at 4 ℃, and the serum LDL-C level was detected.

[0386] Experimental results:

[0387] Serum LDL-C (Mean±SD) was statistically analyzed using GraphPad Prism 9 software for plotting and data analysis. The specific results are shown in Tables 5 and Figure 3 .

[0388] Table 5 Reduction level of LDL-C in mouse serum

[0389]

[0390] 1) The siRNA conjugated with the GalNAc compound of the present invention has a significant difference in reducing the LDL-C level in mouse serum, and the reduction level of YK-GAL-401~YK-GAL-403 is significantly higher than that of YK-GAL-404~406. For example, inc-YK-Gal-401 increased by 33.0% and 36.0% on the 7th day and 14th day respectively compared with inc-YK-Gal-404, which is a significant improvement.

[0391] As can be seen from Table 5, the siRNA conjugated with the GalNAc compound of the present invention can reduce the LDL-C level in mouse serum. Different GalNAc conjugates have significant differences in the reduction level of LDL-C in mouse serum. The reduction levels of inc-YK-Gal-401, inc-YK-Gal-402, inc-YK-Gal-403, inc-YK-Gal-404, inc-YK-Gal-405 and inc-YK-Gal-406 on the 7th day were 49.2%, 39.6%, 44.5%, 16.2%, 33.7% and 30.3%, respectively, and the reduction levels on the 14th day were 53.5%, 41.3%, 50.4%, 17.5%, 35.0% and 32.2%, respectively.

[0392] The reduction levels of inc-YK-Gal-401 and inc-YK-Gal-403 on the 7th day were both over 40%, and on the 14th day the reduction levels were both over 50%. The reduction levels of inc-YK-Gal-402 on the 7th day were also 39.6%, and on the 14th day the reduction levels were 41.3%. However, the reduction levels of inc-YK-Gal-404 on the 7th day were only 16.2%, and on the 14th day the reduction levels were only 17.5%. The reduction levels of inc-YK-Gal-401, inc-YK-Gal-402 and inc-YK-Gal-403 on the 7th day were 33.0%, 23.4% and 28.3% higher than those of inc-YK-Gal-404, respectively, and on the 14th day the reduction levels were 36.0%, 23.8% and 32.9% higher, respectively, which were significantly higher.

[0393] 2) Compared with the prior art GalNAc compounds, the conjugated siRNA of the present invention YK-GAL-401 to YK-GAL-403 significantly improved the level of LDL-C reduction in mouse serum. For example, inc-YK-Gal-401 increased LDL-C by 33.4% and 36.4% on the 7th day and 14th day respectively compared with inc-YK-Gal-301.

[0394] The siRNA conjugates inc-YK-Gal-301, inc-GalNAc 1b and inc-NAG0052 prepared from the prior art GalNAc compounds reduced LDL-C by 15.8%, 30.1% and 32.5% on day 7, respectively, and by 17.1%, 32.9% and 35.7% on day 14, respectively.

[0395] Compared with the GalNAc compounds in the prior art, the GalNAc compounds of the present invention and the conjugated siRNA significantly improve the level of LDL-C reduction in mouse serum. For example, the reduction levels of inc-YK-Gal-401, inc-YK-Gal-402 and inc-YK-Gal-403 on the 7th day were 33.4%, 23.8% and 28.7% higher than those of inc-YK-Gal-301, 19.1%, 9.5% and 14.4% higher than those of inc-GalNAc 1b, and 16.7%, 7.1% and 12.0% higher than those of inc-NAG0052, respectively, which were significantly improved; the reduction levels on the 14th day were 36.4%, 24.2% and 33.3% higher than those of inc-YK-Gal-301, 20.6%, 8.4% and 17.5% higher than those of inc-GalNAc 1b, and 17.8%, 5.6% and 14.7% higher than those of inc-NAG0052, respectively, which were significantly improved.

[0396] 3) The conjugates of GalNAc compounds with similar structures and siRNA have significant differences in reducing the level of LDL-C in mouse serum.

[0397] The GalNAc compounds designed by the present invention have very similar structures, but the activities of the siRNA conjugates are very different. For example, YK-GAL-401 and YK-GAL-404 are different only in the 2' position of the ribose ring, YK-GAL-401 is methoxy, YK-GAL-404 is hydrogen, and the other structures are exactly the same, but inc-YK-Gal-401 can significantly improve the level of LDL-C reduction in mouse serum by more than 30% compared with inc-YK-Gal-404.

[0398] Compared with YK-GAL-301, YK-GAL-406 only differs in the main chain and antennae. The main chain of YK-GAL-406 has two fewer methylene groups than that of YK-GAL-301. The antennae of YK-GAL-406 have a triazole structure, while YK-GAL-301 does not. However, the LDL-C reduction level in the serum of inc-YK-Gal-406 mice can be increased by 15.1% compared with inc-YK-Gal-301, which is a significant improvement.

[0399] Compared with inc-NAG0052, YK-GAL-401 has only slight differences in individual groups, but the LDL-C reduction level in the serum of inc-YK-Gal-401 mice can be increased by 17.8% compared with inc-NAG0052, which is a significant improvement.

[0400] Therefore, GalNAc compounds with similar chemical structures do not necessarily have similar oligonucleotide delivery efficiencies. The inhibition rates of PCSK9 protein expression in mouse serum by the prepared GalNAc oligonucleotide conjugates are not consistent and may have very large differences.

[0401] Example 4: Distribution of different GalNAc-conjugated siRNAs in different tissues and organs of mice

[0402] The results show that the GalNAc compound designed by the present invention can efficiently deliver nucleic acids to animal livers.

[0403] Experimental process:

[0404] 6-8 week old wild-type C57BL / 6 mice were used, with 6 mice in each group. Each group was given siRNA conjugates with Cy5 fluorescent groups, inc-YK-GAL-401, inc-YK-GAL-402, inc-YK-GAL-403, inc-YK-GAL-404, inc-YK-GAL-405, inc-YK-GAL-406, inc-YK-GAL-301, inc-GalNAc 1b and inc-NAG0052. The negative control group was not given any drug. The mice were weighed and given a dose of 6 mg / kg, and the dose was given subcutaneously, and the injection volume did not exceed 0.1- 0.2 mL according to the administration volume.

[0405] After administration, the hair on the mouse abdomen was shaved clean, and the mouse was placed in a small animal living imaging system in a supine position after isoflurane anesthesia. The imaging of the mouse was observed under the Cy5 channel 4 hours and 8 hours after administration. The luminescence intensity of the mouse was counted using the living imaging software Living Image, and the differences between different test groups were compared.

[0406] Experimental results:

[0407] The results of the fluorescence intensity test in mouse liver and kidney (which can represent the relative content of oligonucleotides) are shown in Tables 6 and Figure 4 The results show that the GalNAc compound designed by the present invention can efficiently deliver nucleic acids to animal livers.

[0408] Table 6 Fluorescence intensity in mouse liver and kidney

[0409]

[0410] As can be seen from Table 6, oligonucleotides labeled with Cy5 fluorescence are distributed in the mouse liver and kidney, among which inc-YK-Gal-401 has the highest fluorescence intensity in the mouse liver (i.e., the highest relative content of oligonucleotides), and the liver fluorescence intensity can reach 9.03E+09 and 9.11E+09 at 4 hours and 8 hours, respectively. The liver fluorescence intensity of inc-YK-Gal-402 and inc-YK-Gal-403 is also very high, which is 8.79E+09 and 8.92E+09 at 4 hours, and 8.95E+09 and 9.01E+09 at 8 hours, respectively. This shows that the GalNAc compound designed by the present invention can efficiently deliver oligonucleotides to the liver.

[0411] In summary, the present invention designs a series of novel GalNAc compounds, which have completely different chemical structures compared with the prior art GalNAc compounds. The GalNAc compounds designed by the present invention introduce a triazole structure in the connecting arm, and by virtue of the advantages of click chemistry, a series of novel GalNAc compounds containing triazole structures can be quickly constructed. The GalNAc oligonucleotide conjugates prepared from the GalNAc compounds of the present invention can achieve efficient liver-targeted delivery, and the activity is significantly improved compared with the representative GalNAc compounds of the prior art.

[0412] 1. The oligonucleotide conjugates prepared from the GalNAc compounds YK-GAL-401 to YK-GAL-403 of the present invention significantly improve the inhibition rate of PCSK9 protein expression in mouse serum compared with the prior art GalNAc. For example, the inhibition rate of inc-YK-Gal-401 on day 7 was 21.5%, 14.5% and 12.7% higher than that of inc-YK-Gal-301, inc-GalNAc 1b and inc-NAG0052, and the inhibition rates on day 14 were increased by 17.3%, 10.4% and 10.8%, respectively.

[0413] 1) The siRNA conjugated with the GalNAc compound of the present invention has significant differences in the inhibition rate of PCSK9 protein expression in mouse serum, and YK-Gal-401~YK-Gal-403 are significantly higher than YK-Gal-404~406. For example, inc-YK-Gal-401 increased by 20.7% and 16.0% on the 7th day and 14th day respectively compared with inc-YK-Gal-404.

[0414] 2) Compared with the GalNAc compounds of the prior art, the conjugated siRNA of the present invention, YK-Gal-401 to YK-Gal-403, significantly improves the inhibition rate of PCSK9 protein expression in mouse serum. For example, inc-YK-Gal-401 increased the PCSK9 protein expression by 21.5% and 17.3% on the 7th day and 14th day respectively compared with inc-YK-Gal-301.

[0415] 3) The conjugates of GalNAc compounds with similar structures and siRNA have significant differences in the inhibition rate of PCSK9 protein expression in mouse serum. Therefore, it is not necessarily the case that GalNAc compounds with similar chemical structures have similar oligonucleotide delivery efficiencies. The inhibition rates of PCSK9 protein expression in mouse serum by the prepared GalNAc oligonucleotide conjugates are not consistent and may have very large differences.

[0416] 2. The oligonucleotide conjugates prepared from the GalNAc compounds YK-GAL-401 to YK-GAL-403 of the present invention significantly improve the inhibition rate of PCSK9 gene expression in mouse liver compared with the prior art GalNAc. For example, the inhibition rate of inc-YK-Gal-401 was significantly improved by 21.1% and 9.8% compared with inc-YK-Gal-301 and inc-GalNAc 1b, respectively.

[0417] 1) The siRNA conjugated with the GalNAc compound of the present invention has significant differences in the inhibition rate of PCSK9 gene expression in mouse liver, and YK-Gal-401~YK-Gal-403 are significantly higher than YK-Gal-404~406. For example, inc-YK-Gal-401 is 19.4% higher than inc-YK-Gal-404.

[0418] 2) Compared with the prior art GalNAc compounds, the conjugated siRNA of the present invention YK-Gal-401 to YK-Gal-403 significantly increased the inhibition rate of PCSK9 gene expression in mouse liver. For example, inc-YK-Gal-401 increased by 21.1% compared with inc-YK-Gal-301.

[0419] 3) The conjugates of GalNAc compounds with similar structures and siRNA have significant differences in the inhibition rate of PCSK9 gene expression in mouse liver. Therefore, it is not necessarily the case that GalNAc compounds with similar chemical structures have similar oligonucleotide delivery efficiencies. The inhibition rates of the prepared GalNAc oligonucleotide conjugates on the PCSK9 gene in mouse liver are not consistent and may have very large differences.

[0420] 3. The oligonucleotide conjugates prepared by the GalNAc compounds YK-GAL-401~YK-GAL-403 of the present invention significantly improve the level of LDL-C reduction in mouse serum compared with the prior art GalNAc. For example, the level of LDL-C reduction in mice in the inc-YK-Gal-401 group on day 14 was significantly improved by 36.4%, 20.6% and 17.8% compared with the inc-YK-Gal-301, inc-GalNAc 1b and NAG0052 groups, respectively.

[0421] 1) The siRNA conjugated with the GalNAc compound of the present invention has a significant difference in reducing the LDL-C level in mouse serum, and the reduction level of YK-Gal-401~YK-Gal-403 is significantly higher than that of YK-Gal-404~YK-Gal-406. For example, inc-YK-Gal-401 increased by 33.0% and 36.0% on the 7th day and 14th day respectively compared with inc-YK-Gal-404, which is a significant improvement.

[0422] 2) Compared with the prior art GalNAc compounds, the conjugated siRNA of the present invention YK-Gal-401 to YK-Gal-403 significantly improved the level of LDL-C reduction in mouse serum. For example, inc-YK-Gal-401 increased LDL-C by 33.4% and 36.4% on the 7th day and 14th day respectively compared with inc-YK-Gal-301.

[0423] 3) The conjugates of GalNAc compounds with similar structures and siRNA have significant differences in the reduction of LDL-C in mouse serum. Therefore, it is not necessarily the case that GalNAc compounds with similar chemical structures have similar oligonucleotide delivery efficiencies. The inhibition rates of PCSK9 protein expression in mouse serum by the prepared GalNAc oligonucleotide conjugates are not consistent and may have very large differences.

[0424] 4. The designed compounds of the present invention can efficiently deliver oligonucleotides to the liver.

[0425] For example, the liver fluorescence intensities of inc-YK-Gal-401, inc-YK-Gal-402 and inc-YK-Gal-403 groups can reach 9.03E+09, 8.79E+09 and 8.92E+09 respectively at 4 hours, and can reach 9.11E+09, 8.95E+09 and 9.01E+09 respectively at 8 hours, indicating that the GalNAc compounds designed in the present invention can efficiently deliver oligonucleotides to animal livers.

[0426] The applicant declares that the present invention illustrates the GalNAc compound and its application through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A GalNAc compound as represented by formula (I) or a pharmaceutically acceptable salt thereof, ， in, L1 is -C(O)NH-, with the * end connected to G; L2 is -(CH2)n1- or -(CH2CH2O)n2CH2-, wherein n1 and n2 are integers of 1-7; Z1 is -(CH2)n3-, wherein n3 is an integer from 1 to 7; L3 is -HNC(O)- or -C(O)NH-; n is 0; L4 is -(CH2)n4-, wherein n4 is an integer from 0 to 7; A is , R1 is hydrogen or C 1-6 Alkoxy; R2 is 4, 4'-dimethoxytrityl or monomethoxytrityl; R3 is -CO(CH2)2CONH-E, and E is controlled pore glass; B is oxygen or sulfur; G is ; in, X1 is -(CH2)3-; X2 is -HNC(O)-**, and the ** end is connected to X3; X3 is -(CH2)b-, where b is an integer from 1 to 6; X4 , the *** end is connected to X3; Y1 is 1; Y2 is 1; Y3 is 3 and K is carbon.

2. The GalNAc compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein the GalNAc compound of formula (I) satisfies one or more of the following conditions: (1) L2 is -(CH2)3-, -(CH2)4-, -(CH2)5-, -(CH2)6- or -(CH2)7-; (2) L4 is absent, -(CH2)3-, -(CH2)4-, -(CH2)5-, -(CH2)6- or -(CH2)7-; (3) R1 is hydrogen or methoxy; (4) R2 is 4, 4'-dimethoxytrityl; (5) B is oxygen.

3. The GalNAc compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein L2 is -(CH2CH2O)3CH2-.

4. The GalNAc compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein G is: , or .

5. The GalNAc compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, which is a compound YK-GAL-401, YK-GAL-402, YK-GAL-403, YK-GAL-404, YK-GAL-405 or YK-GAL-406 having the following structure, wherein E is a controlled pore glass; , , , , or .

6. A conjugate of formula (IIA) or (IIB) or a pharmaceutically acceptable salt thereof, or , Among them, Ol igo represents oligonucleotide, X represents hydroxyl or thiol, G1 is ; R2 is hydrogen; R3 is hydrogen; L1, L2, Z1, L3, L4, n, R1, X1, X2, X3, X4, Y1, Y2, Y3 and K are as defined in claim 1; The sense strand of the oligonucleotide has the sequence shown in SEQ ID NO. 46; the antisense strand has the sequence shown in SEQ ID NO.

47.

7. The conjugate according to claim 6 or a pharmaceutically acceptable salt thereof, wherein the conjugate is conjugated with YK-GAL-401, YK-GAL-402, YK-GAL-403, YK-GAL-404, YK-GAL-405 or YK-GAL-406 according to claim 5 by an oligonucleotide; the sense strand of the oligonucleotide has the sequence shown in SEQ ID NO. 46; the antisense strand of the oligonucleotide has the sequence shown in SEQ ID NO.

47.

8. The conjugate according to claim 6 or a pharmaceutically acceptable salt thereof, wherein the conjugate is any of the following conjugates: , , , , or ; in, The sense strand of the siRNA has the sequence shown in SEQ ID NO. 46; the antisense strand of the siRNA has the sequence shown in SEQ ID NO.

47.

9. A pharmaceutical composition comprising the conjugate according to any one of claims 6 to 8 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient.

10. A kit comprising the conjugate according to any one of claims 6 to 8 or a pharmaceutically acceptable salt thereof.

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