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EP-4737469-A1 - NUCLEIC ACID CONJUGATES AS WELL AS PREPARATION METHOD THEREFOR AND USE THEREOF

EP4737469A1EP 4737469 A1EP4737469 A1EP 4737469A1EP-4737469-A1

Abstract

The present invention provides a compound for forming a conjugate with an oligonucleotide, the compound having a structure as shown in Formula (I). The present disclosure also provides a corresponding conjugate, and the use of said conjugate in the preparation of a medicament for preventing and/or treating related diseases. The conjugate of the present disclosure has the advantages of inexpensive raw materials, simple synthesis, and easy process development, while also having good oligonucleotide synthesis efficiency and in vivo biological activity in small animals.

Inventors

  • Shao, Hongxia
  • YANG, ZHIWEI
  • LIU, NAN

Assignees

  • Suzhou Siran Biotechnology Co., Ltd.

Dates

Publication Date
20260506
Application Date
20240627

Claims (20)

  1. A compound, wherein the compound has the structure shown in Formula (I): wherein, A 0 represents a ligand having affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells, or represents a group formed by replacing all or part of the hydroxyl groups in the ligand with a KCOO- group, wherein each K is independently selected from the group consisting of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl, and alkylphenyl; b is an integer in the range of 1-4; L 1 and L 2 independently represent a linking combination of one or more groups selected from Formula A1-A11: wherein, j1 is an integer from 1-20; j2 is an integer from 1-20; R' is a C 1 - C 10 alkyl; M represents a structural formula as shown in Formula (A12): wherein, m represents an integer from 0-6; * represents the bond connected to G 1 , Q represents a structural formula as shown in Formula (A13): wherein, connected to O represents the bond connected to G 1 ; R 2 and R 3 are the same or different, and are independently selected from H, C 1 - C 20 alkyl, C 1 - C 20 alkoxy, C 2 - C 20 alkenyl, or C 2 - C 20 alkynyl; Z is absent or represents a C 1 - C 10 alkylene; X represents a structural formula as shown in Formula (A14-1) or (A14-2): wherein R 4 and R 5 are the same or different, and are independently selected from H, fluoro, hydroxyl, C 1 - C 20 alkyl, C 1 - C 20 alkoxy, C 2 - C 20 alkenyl, or C 2 - C 20 alkynyl; p is an integer from 1-6; optionally, R 4 and R 5 are directly connected to form a three- to six-membered saturated carbocyclic ring; E represents a structural formula as shown in Formula (A15): wherein, represents a C 3 - C 18 cycloalkyl or a C 3 - C 18 heterocyclyl, and R 1 is selected from H, fluoro, hydroxyl, cyano, C 1 - C 20 alkyl, C 1 - C 20 alkoxy, C 2 - C 20 alkenyl, or C 2 - C 20 alkynyl; G 1 and G 2 each represent a phosphoramidite functional group having the structure shown in Formula (G-1) or a hydroxyl protecting group, provided that at least one of G 1 and G 2 has the structure shown in Formula (G-1), the hydroxyl protecting group is selected from any one of trityl, 4-methoxytrityl, 4,4'-dimethoxytrityl (DMTr), and 4,4',4''-trimethoxytrityl; wherein, B 1 is selected from substituted or unsubstituted C 1 - C 5 hydrocarbyl; represents the site of covalent bond attachment of the group.
  2. The compound according to claim 1, wherein L 1 and L 2 are independently selected from a linking combination of one or more of the following: A1, A2, A4, A6, A7, A8, A9, A10, and A11; the ligand is independently selected from any one of the following: D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, α -D-mannofuranose, β -D-mannofuranose, α -D-mannopyranose, β -D-mannopyranose, α -D-glucopyranose, β -D-glucopyranose, α -D-glucofuranose, β -D-glucofuranose, α -D-fructofuranose, α -D-fructopyranose, α -D-galactopyranose, β -D-galactopyranose, α -D-galactofuranose, β -D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy- β -D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-glycolyl- α -neuraminic acid, 5-thio- β -D-glucopyranose,methyl 2,3,4-tri-O-acetyl-1-thio-6-O-trityl- α -D-glucopyranoside, 4-thio- β -D-galactopyranose, ethyl 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio- α -D-glucoheptopyranoside, 2,5-anhydro-D-allononitrile, ribose, D-ribose, D-4-thioribose, L-ribose, and L-4-thioribose; b is 1, 2, or 3; m is 0, 1, or 2; R 2 and R 3 are independently selected from H, C 1 - C 6 alkyl, C 1 - C 6 alkoxy, C 2 - C 6 alkenyl, or C 2 - C 6 alkynyl; Z is absent or represents a C 1 - C 3 alkylene; R 4 and R 5 are independently selected from H, fluoro, hydroxyl, C 1 - C 6 alkyl, C 1 - C 6 alkoxy, C 2 - C 6 alkenyl, or C 2 - C 6 alkynyl; p is 1, 2, or 3; represents a C 4 - C 8 cycloalkyl or a C 3 - C 8 heterocyclyl; and R 1 is selected from H, fluoro, hydroxyl, cyano, C 1 - C 6 alkyl, C 1 - C 6 alkoxy, C 2 - C 6 alkenyl, or C 2 - C 6 alkynyl; B 1 is methyl, ethyl, or isopropyl; B 2 is selected from one of C 1 - C 5 alkyl, cyanoethyl, cyanopropyl, or cyanobutyl.
  3. The compound according to claim 2, wherein b is 1 or 3; represents a C 3 - C 6 heterocyclyl; A 0 is an N-acetylgalactosamine (GalNAc) group as shown below or a group formed by replacing all or part of the hydroxyl groups in the N-acetylgalactosamine group with a KCOO-group, wherein each K is independently selected from methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl, or alkylphenyl: the hydroxyl protecting group is 4,4'-dimethoxytrityl; the length of L 1 or L 2 is independently 1-25 atoms, wherein the length of L 1 or L 2 refers to the number of chain-forming atoms in the longest straight chain; each j1 is independently an integer from 1-10, and each j2 is independently an integer from 1-10.
  4. The compound according to claim 3, wherein represents a four- to six-membered nitrogen-containing saturated cycloalkyl; the length of L 1 or L 2 is independently 1-20 atoms; each j1 is independently an integer from 1-8, and each j2 is independently an integer from 1-8.
  5. The compound according to claim 2, wherein L 1 and L 2 are independently selected from a linking combination of at least 2 of A1, A2, A4, A8, A9, A10, and A11.
  6. The compound according to claim 2, wherein L 1 and L 2 are independently selected from a linking combination of at least 2 of A1, A4, A8, A10, and A11.
  7. The compound according to claim 1, wherein the compound has the structure (I-1) shown below: wherein, L is A10, wherein j1 is an integer from 2-8, or is A11, wherein j2 is an integer from 1-7; m is 0, 1, or 2; n is 0, 1, 2, or 3; Q represents a structural formula as shown in Formula (A13): wherein, represents the site of covalent bond attachment of the group; R 2 and R 3 are independently selected from H, C 1 - C 6 alkyl, C 1 - C 6 alkoxy, C 2 - C 6 alkenyl, or C 2 - C 6 alkynyl; X represents a structural formula as shown in Formula (A14-1) or (A14-2): wherein R 4 and R 5 are independently selected from H, fluoro, hydroxyl, C 1 - C 6 alkyl, C 1 - C 6 alkoxy, C 2 - C 6 alkenyl, or C 2 - C 6 alkynyl; p is 1, 2, or 3; optionally, R 4 and R 5 are directly connected to form a three- to six-membered saturated carbocyclic ring; represents a four- to eight-membered all-carbon or nitrogen-containing saturated ring, R 1 is selected from H, fluoro, hydroxyl, cyano, C 1 - C 6 alkyl, C 1 - C 6 alkoxy, C 2 - C 6 alkenyl, or C 2 - C 6 alkynyl; wherein, the hydroxyl groups in are all or partially replaced by a KCOO- group, wherein each K is independently selected from methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl, or alkylphenyl.
  8. The compound according to claim 1, wherein is a four-, five-, or six-membered saturated cycloalkyl containing one or two nitrogen atoms.
  9. The compound according to claim 1, wherein the compound has any one of the structures shown below:
  10. A nucleic acid conjugate, wherein the conjugate contains one or more structures as shown in Formula (II) connected to any position on an oligonucleotide sequence: wherein, R p and R q are each H or have the structure shown in Formula A16, provided that at least one of R p and R q has the structure shown in Formula A16; wherein, represents the site of covalent bond attachment of the group; E 1 is OH, SH, or BH 2 , A 0 represents a ligand having affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells, or represents a group formed by replacing all or part of the hydroxyl groups in the ligand with a KCOO- group, wherein each K is independently selected from the group consisting of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl, and alkylphenyl; b is an integer in the range of 1-4; L 1 and L 2 independently represent a linking combination of one or more groups selected from Formula A1-A11: wherein, j1 is an integer from 1-20; j2 is an integer from 1-20; R' is a C 1 - C 10 alkyl; M represents a structural formula as shown in Formula (A12): wherein, represents the site of covalent bond attachment of the group; m represents an integer from 0-6; * represents the bond connected to R p , Q represents a structural formula as shown in Formula (A13): wherein, represents the site of covalent bond attachment of the group, connected to O represents the bond connected to R p ; R 2 and R 3 are the same or different, and are independently selected from H, C 1 - C 20 alkyl, C 1 - C 20 alkoxy, C 2 - C 20 alkenyl, or C 2 - C 20 alkynyl, preferably H, C 1 - C 6 alkyl, C 1 - C 6 alkoxy, C 2 - C 6 alkenyl, or C 2 - C 6 alkynyl; Z is absent or represents a C 1 - C 10 alkylene; X represents a structural formula as shown in Formula (A14-1) or (A14-2): wherein R 4 and R 5 are the same or different, and are independently selected from H, fluoro, hydroxyl, C 1 - C 20 alkyl, C 1 - C 20 alkoxy, C 2 - C 20 alkenyl, or C 2 - C 20 alkynyl, preferably H, fluoro, hydroxyl, C 1 - C 6 alkyl, C 1 - C 6 alkoxy, C 2 - C 6 alkenyl, or C 2 - C 6 alkynyl; p is an integer from 1-6; optionally, R 4 and R 5 are directly connected to form a three- to six-membered saturated carbocyclic ring; E represents a structural formula as shown in Formula (A15): wherein, represents the site of covalent bond attachment of the group; represents a C 3 - C 18 cycloalkyl or a C 3 - C 18 heterocyclyl, and R 1 is selected from H, fluoro, hydroxyl, cyano, C 1 - C 20 alkyl, C 1 - C 20 alkoxy, C 2 - C 20 alkenyl, or C 2 - C 20 alkynyl.
  11. The nucleic acid conjugate according to claim 10, wherein the conjugate contains one or more structures as shown in Formula (II-A) connected to any position on an oligonucleotide sequence: wherein, a represents an integer from 0-7; the structure of W is as shown in the following formula: wherein, represents the site of covalent bond attachment of the group; E 1 is OH, SH, or BH 2 .
  12. The nucleic acid conjugate according to claim 11, wherein a represents an integer of 0, 1, 2, or 3.
  13. The nucleic acid conjugate according to claim 10 or 11, wherein L 1 and L 2 are independently selected from a linking combination of one or more of the following: A1, A2, A4, A6, A7, A8, A9, A10, and A11; the ligand is independently selected from any one of the following: D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, α -D-mannofuranose, β -D-mannofuranose, α -D-mannopyranose, β -D-mannopyranose, α -D-glucopyranose, β -D-glucopyranose, α -D-glucofuranose, β -D-glucofuranose, α -D-fructofuranose, α -D-fructopyranose, α -D-galactopyranose, β -D-galactopyranose, α -D-galactofuranose, β -D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy- β -D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-glycolyl- α -neuraminic acid, 5-thio- β -D-glucopyranose,methyl 2,3,4-tri-O-acetyl-1-thio-6-O-trityl- α -D-glucopyranoside, 4-thio- β -D-galactopyranose, ethyl 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio- α -D-glucoheptopyranoside, 2,5-anhydro-D-allononitrile, ribose, D-ribose, D-4-thioribose, L-ribose, and L-4-thioribose; b is 1, 2, or 3; m is 0, 1, or 2; R 2 , R 3 are independently selected from H, C 1 - C 6 alkyl, C 1 - C 6 alkoxy, C 2 - C 6 alkenyl, or C 2 - C 6 alkynyl; Z is absent or represents a C 1 - C 3 alkylene; R 4 and R 5 are independently selected from H, fluoro, hydroxyl, C 1 - C 6 alkyl, C 1 - C 6 alkoxy, C 2 - C 6 alkenyl, or C 2 - C 6 alkynyl; p is 1, 2, or 3; represents a C 4 - C 8 cycloalkyl or a C 3 - C 8 heterocyclyl; and R 1 is selected from H, fluoro, hydroxyl, cyano, C 1 - C 6 alkyl, C 1 - C 6 alkoxy, C 2 - C 6 alkenyl, or C 2 - C 6 alkynyl; B 1 is methyl, ethyl, or isopropyl; B 2 is selected from one of C 1 - C 5 alkyl, cyanoethyl, cyanopropyl, or cyanobutyl.
  14. The nucleic acid conjugate according to claim 13, wherein b is 1 or 3; represents a C 3 - C 6 heterocyclyl; A 0 is an N-acetylgalactosamine (GalNAc) group as shown below or a group formed by replacing all or part of the hydroxyl groups in the N-acetylgalactosamine group with a KCOO-group, wherein each K is independently selected from methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl, or alkylphenyl: the hydroxyl protecting group is 4,4'-dimethoxytrityl; the length of L 1 or L 2 is independently 1-25 atoms, wherein the length of L 1 or L 2 refers to the number of chain-forming atoms in the longest straight chain; each j1 is independently an integer from 1-10, and each j2 is independently an integer from 1-10.
  15. The nucleic acid conjugate according to claim 14, wherein represents a four- to six-membered nitrogen-containing saturated cycloalkyl; the length of L 1 or L 2 is independently 1-20 atoms; each j1 is independently an integer from 1-8, and each j2 is independently an integer from 1-8.
  16. The nucleic acid conjugate according to claim 10 or 11, wherein L 1 and L 2 are independently selected from a linking combination of at least 2 of A1, A2, A4, A8, A9, and A10.
  17. The nucleic acid conjugate according to claim 10 or 11, wherein L 1 and L 2 are independently selected from a linking combination of at least 2 of A1, A4, A8, and A10.
  18. The nucleic acid conjugate according to claim 10 or 11, which has any one of the structures shown below: wherein Y is O or S, is an oligonucleotide.
  19. The nucleic acid conjugate according to claim 10 or 11, wherein the oligonucleotide is selected from one of the following: small interfering RNA, microRNA, anti-microRNA, microRNA antagonist, microRNA mimetic, decoy oligonucleotide, immunostimulant, G-quadruplex, splice variant, single-stranded RNA, antisense nucleic acid, nucleic acid aptamer, stem-loop RNA, mRNA fragment, and activating RNA; optionally, the oligonucleotide is a single-stranded oligonucleotide or a double-stranded oligonucleotide; optionally, the oligonucleotide is a single-stranded oligonucleotide, the P atom in Formula (A16) is connected to an end of the single-stranded oligonucleotide, the end of the single-stranded oligonucleotide refers to the first 4 nucleotides from one end of the single-stranded oligonucleotide; optionally, the P atom in Formula (A16) is connected to a terminus of the single-stranded oligonucleotide; optionally, the P atom in Formula (A16) is connected to the 3' terminus of the single-stranded oligonucleotide; optionally, the oligonucleotide is a double-stranded oligonucleotide, the double-stranded oligonucleotide comprises a sense strand and an antisense strand, the P atom in the Formula (A16) is connected to an end of the double-stranded oligonucleotide, the end of the double-stranded oligonucleotide refers to the first 4 nucleotides from one end of the sense strand or the antisense strand; optionally, the P atom in Formula (A16) is connected to a terminus of the sense strand or the antisense strand; optionally, the P atom in Formula (A16) is connected to the 5' terminus of the antisense strand; optionally, the P atom in Formula (A16) is connected to the 2', 3', or 5' position of a nucleotide in the nucleic acid conjugate by forming a phosphodiester bond.
  20. The nucleic acid conjugate according to claim 19, wherein the double-stranded oligonucleotide is an siRNA.

Description

Technical Field The present disclosure belongs to the field of nucleic acid delivery, and specifically relates to a nucleic acid conjugate, a preparation method and a use thereof. Background Art SiRNA is a negatively charged macromolecule composed of two oligonucleotide chains, which cannot effectively target tissues in the body on its own, nor can it enter cells autonomously. SiRNA requires special delivery vehicles to achieve enrichment in target tissues and enable it to enter cells to exert its therapeutic effect. Lipid Nanoparticles (LNPs) can encapsulate siRNA and effectively achieve liver-targeted delivery. The drug Onpattro, which uses LNP technology, has been marketed. However, the clinical application of LNP technology still has many shortcomings, for example: LNP formulations have a short validity period; immune responses are generated during application, often requiring the synergistic use of immunosuppressants such as dexamethasone, which complicates the clinical application of LNPs. Targeted delivery of drugs into cells through endocytosis mediated by cell surface receptors is an effective strategy. The asialoglycoprotein receptor (ASGPR) is a receptor that is highly specifically expressed on the surface of hepatocytes, characterized by high abundance and high receptor recycling efficiency. By covalently conjugating ligands that can specifically recognize ASGPR, such as monosaccharides and polysaccharides like galactose, galactosamine, and N-acetylgalactosamine (GalNAc), with siRNA, the siRNA can be targeted for delivery into hepatocytes, allowing the siRNA to exert its gene-silencing effect on the target gene. Therefore, the research and development of targeting ligands suitable for siRNA delivery is of great significance for the clinical application of siRNA. Currently, some progress has been made globally in research on achieving effective liver-targeted delivery of siRNA by targeting ASGPR. A representative delivery molecule is L96 from Alnylam Pharmaceuticals (its structural formula is shown below), which has been used in four marketed siRNA drugs and has shown good delivery activity and selectivity in both in vitro and in vivo experiments. However, there are still problems such as the in vivo biological activity and efficacy of siRNA can be further improved, and the CMC (Chemistry, Manufacturing, and Controls) process and technology for delivery molecules are complex and production costs are high. Summary of the Invention The delivery compounds and corresponding nucleic acid conjugates of the present disclosure use natural amino acids such as serine, threonine, etc., as the core linking backbone, resulting in a new class of GalNAc conjugates with completely new chemical structures. Compared with existing delivery technologies, they have the advantages of simple molecular structure, inexpensive and readily available raw materials, and easy-to-develop CMC processes, as well as good oligonucleotide synthesis efficiency and in vivo biological activity. In a first aspect, the present disclosure provides a compound having the structure shown in Formula (I): wherein, A0 represents a ligand having affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells, or represents a group formed by replacing all or part of the hydroxyl groups in said ligand with a KCOO- group, wherein each K is independently selected from the group consisting of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl, and alkylphenyl;wherein said ligand is preferably independently selected from any one of the following: D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-mannofuranose, β-D-mannofuranose, α-D-mannopyranose, β-D-mannopyranose, α-D-glucopyranose, β-D-glucopyranose, α-D-glucofuranose, β-D-glucofuranose, α-D-fructofuranose, α-D-fructopyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactofuranose, β-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-glycolyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, methyl 2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside, 4-thio-β-D-galactopyranose, ethyl 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucoheptopyranoside, 2,5-anhydro-D-allononitrile, ribose, D-ribose, D-4-thioribose, L-ribose, L-4-thioribose;b is an integer ranging from 1 to 4, preferably 1, 2, or 3, more preferably 1 or 3;L1 and L2 independently represent a linking combination of one or more groups selected from Formula A1-A11: wherein, j1 is an integer f