WO2024099316A1 - Groupe de conjugaison tétravalent contenant un cycle hétérocyclique à sept chaînons et son utilisation - Google Patents

Groupe de conjugaison tétravalent contenant un cycle hétérocyclique à sept chaînons et son utilisation Download PDF

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WO2024099316A1
WO2024099316A1 PCT/CN2023/130247 CN2023130247W WO2024099316A1 WO 2024099316 A1 WO2024099316 A1 WO 2024099316A1 CN 2023130247 W CN2023130247 W CN 2023130247W WO 2024099316 A1 WO2024099316 A1 WO 2024099316A1
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compound
mmol
present
solution
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Chinese (zh)
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陆剑宇
胡彦宾
宋雨晨
陈实
安可
胡利红
丁照中
贺海鹰
陈曙辉
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南京明德新药研发有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings

Definitions

  • the present invention relates to a tetravalent conjugated group containing a seven-membered heterocycle and applications thereof, and in particular to the structure of the conjugated group represented by formula (V) and applications thereof.
  • Tissue-specific delivery of nucleic acid molecules is one of the key technologies for developing nucleic acid drugs.
  • the technology of conjugating nucleic acid molecules with ligands and using ligands to deliver nucleic acid molecules to specific tissues has been widely used.
  • ligands containing terminal N-acetylgalactose (GalNAc) and its derivatives to nucleic acid molecules, and using the binding of N-acetylgalactose to asialoglycoprotein receptors (ASGPR)
  • GalNAc N-acetylgalactose
  • ASGPR asialoglycoprotein receptors
  • nucleic acid molecules are delivered to hepatocytes in a targeted manner, which is a more common delivery method.
  • Common GalNAc-based ligands usually contain three terminal GalNAc molecules, that is, trivalent GalNAc ligands.
  • the first aspect of the present invention provides a conjugated group represented by formula (V),
  • L 1 is selected from
  • L 2 is selected from
  • n is selected from 0 and 1;
  • t is selected from 2, 3, 4, 5, 6 and 7.
  • the present invention also provides a conjugated group represented by formula (I),
  • L 1 is selected from
  • L 2 is selected from
  • n is selected from 0 and 1;
  • t is selected from 2, 3, 4, 5, 6 and 7.
  • the above-mentioned conjugated group is selected from (D02),
  • the above-mentioned conjugated group is selected from (D02-1),
  • the conjugated group is selected from (D02-1-A), (D02-1-B), (D02-1-C), (D03), (D12) and (D13),
  • the second aspect of the present invention provides a conjugate or a pharmaceutically acceptable salt thereof, wherein the conjugate is selected from a compound formed by connecting a conjugated group defined in any one of the above technical solutions to an oligonucleotide via a phosphodiester bond or a thiophosphodiester bond.
  • the above-mentioned conjugated group is connected to the oligonucleotide via a phosphodiester bond or a thiophosphodiester bond, which refers to the following connection mode:
  • X is selected from S- or O- .
  • the above-mentioned conjugated group is connected to the oligonucleotide via a phosphodiester bond or a thiophosphodiester bond, which refers to the following connection mode:
  • X is selected from S- or O- .
  • the above-mentioned conjugated group is connected to the oligonucleotide via a phosphodiester bond or a thiophosphodiester bond, which refers to the following connection mode:
  • X is selected from S- or O- .
  • the above-mentioned oligonucleotide is selected from RNAi agents and ASO agents, and other variables are as defined in the present invention.
  • the RNAi agent is selected from single-stranded oligonucleotides and double-stranded oligonucleotides, and other variables are as defined in the present invention.
  • the above-mentioned single-stranded oligonucleotide is selected from single-stranded antisense oligonucleotide, and other variables are as defined in the present invention.
  • the double-stranded oligonucleotide is selected from double-stranded siRNA, and other variables are as defined in the present invention.
  • nucleotides of the above oligonucleotides are optionally modified, and other variables are as defined in the present invention.
  • the above-mentioned conjugate can inhibit or block the expression of a gene.
  • the third aspect of the present invention provides the use of the conjugated group defined in any of the above technical solutions as a delivery platform, wherein the delivery platform is used to enhance the binding of the therapeutic agent to a specific target location.
  • the present invention provides an intermediate compound for preparing a conjugated group represented by formula (V), the structure of which is shown in formula (IM), (V-M12) and (V-M13),
  • L 1 , L 2 , t and n are defined in the present invention.
  • the intermediate structure is as shown in formula (D02-M),
  • the intermediate structure is as shown in formula (D02-1-M),
  • * represents a chiral carbon atom.
  • the intermediate structures are as shown in formulas (D02-1-AM), (D02-1-BM), (D02-1-CM), (D03-M), (D12-M) and (D13-M).
  • the present invention also provides the conjugates shown in Table 1:
  • the present invention also provides the following test method
  • the conjugate of the present invention was incubated with freshly isolated primary mouse hepatocytes (PMH) at room temperature for 30 minutes, allowing the conjugate to enter the PMH cells in a free uptake manner. After 24 hours of cell culture, the cells were lysed, RNA was extracted and purified, and the down-regulation level of the target gene was detected by rt-PCR.
  • PMH primary mouse hepatocytes
  • mice were randomly divided into groups according to body weight data, with 4 mice in each group. After grouping, all mice were given subcutaneous injections. The single dose was given with a dosing volume of 10 mL/kg. Mice in group 1 were given PBS, and mice in group 2 were given the conjugate.
  • mice in all groups were euthanized by CO2 inhalation, and two liver samples were collected from each mouse.
  • the liver samples were treated with RNAlater at 4°C overnight, then RNAlater was removed and stored at -80°C for detection of AGT gene expression levels.
  • test samples were incubated with human primary hepatocytes to evaluate the degree of downregulation of AGT mRNA by the test samples.
  • the conjugate of the present invention was diluted to 10 times the concentration to be tested with PBS solution. 10uL siRNA was transferred to a 96-well plate. PHH cells were thawed and transplanted into a 96-well plate, and the final cell density was 5.4 ⁇ 10 5 cells/well. The conjugate of the present invention was tested at 10 concentration points, with the highest concentration being 500nM, and 4-fold dilution.
  • the cells were incubated at 37°C, 5% CO2 for 48 hours and the cell status was examined under a microscope.
  • the conjugate of the present invention can significantly downregulate the level of AGT mRNA in PHH cells.
  • the present invention also provides a method for preparing the conjugate:
  • Oligoribonucleotides were synthesized using phosphoramidite solid phase synthesis technology. or ) and the intermediate compound corresponding to the conjugated group are synthesized on a solid support prepared by covalent bonding. All 2'-modified RNA phosphoramidites and auxiliary reagents are commercially available reagents. All amides are dissolved in anhydrous acetonitrile and molecular sieves are added.
  • the coupling time was 5 min using 5-ethylthio-1H-tetrazole (ETT) as an activator, followed by 3 min reaction in 50 mM I 2 -water/pyridine (volume ratio 1/9) solution to generate phosphate bonds, or in 50 mM 3-((dimethyl
  • Oligoribonucleotides were synthesized using phosphoramidite solid phase synthesis technology. or ) were synthesized. All 2'-modified RNA phosphoramidites and auxiliary reagents were commercially available reagents. All amides were dissolved in anhydrous acetonitrile and molecular sieves were added.
  • ETT 5-ethylthio-1H-tetrazole
  • Oligomers were purified by HPLC using NanoQ anion exchange.
  • Buffer A was 10 mM sodium perchlorate solution, 20 mM Tris, 1 mM EDTA, pH 7.4 and contained 20% acetonitrile
  • buffer B was 500 mM sodium perchlorate, 20 mM Tris, 1 mM EDTA, pH 7.4 and contained 20% acetonitrile.
  • the desired product was isolated and desalted using a reverse phase C18 column.
  • Annealing of single-stranded oligoribonucleotides to produce siRNA The single-stranded oligoribonucleotides to be annealed are prepared into 200 ⁇ M with sterile RNase Free H2O (no RNA hydrolase). The annealing reaction system is set up as follows: a total volume of 100 ⁇ L of the mixture, 10nmol, is placed in a 95°C water bath for 10 minutes ( ⁇ 100nmol requires high temperature for 20 minutes) ⁇ quickly placed in a 60°C water bath, cooled naturally ⁇ the solution after annealing is completed cannot be stored at high temperature. Complementary chains are formed by combining equimolar single-stranded oligoribonucleotide solutions.
  • the conjugated group of the present invention after being conjugated to the nucleic acid molecule, can efficiently and specifically deliver the nucleic acid molecule to the liver tissue.
  • the oligonucleotide using the conjugated group can better bind to the ASGPR protein, thereby allowing the oligonucleotide to enter the liver cell more efficiently.
  • the conjugated group makes the nucleic acid molecule conjugated thereto have good tissue specificity, that is, reduces the enrichment degree of the nucleic acid molecule in the extrahepatic tissue.
  • the conjugated group has low toxicity in vivo, so that when it is used as a delivery platform for the oligonucleotide, the toxicity of the oligonucleotide is also lower.
  • the siRNA obtained by connecting the double-stranded RNA sequence of different target genes using the conjugated group shows excellent effectiveness and long-term effect in the in vivo model.
  • the route is concise, the synthesis operation is simple, and it is easy to post-processing operation.
  • the "therapeutic agent” mentioned in the present invention refers to an agent used to treat a disease or improve symptoms, and the agent includes but is not limited to chemotherapeutic agents and biological therapeutic agents.
  • the conjugated groups of the present invention can enhance the delivery of therapeutic agents to specific target locations (e.g., specific organs or tissues) in objects such as humans or animals. In some embodiments of the present invention, the conjugated groups can enhance the targeted delivery of expression inhibitory oligonucleotides. In some embodiments of the present invention, the conjugated groups can enhance the delivery of expression inhibitory oligonucleotides to the liver.
  • the conjugated groups of the present invention can be directly or indirectly connected to a compound, such as a therapeutic agent, for example, an expression inhibitory oligonucleotide, for example, the 3' or 5' end of an expression inhibitory oligonucleotide.
  • a therapeutic agent for example, an expression inhibitory oligonucleotide, for example, the 3' or 5' end of an expression inhibitory oligonucleotide.
  • the expression inhibitory oligonucleotide comprises one or more modified nucleotides.
  • the expression inhibitory oligonucleotide is an RNAi agent, such as a double-stranded RNAi agent comprising a sense strand and an antisense strand.
  • the conjugated groups disclosed herein are connected to the 5' end of the sense strand of the double-stranded RNAi agent. In some embodiments, the conjugated groups disclosed herein are connected to the expression inhibitory oligonucleotide agent at the 5' end of the sense strand of the double-stranded RNAi agent via a phosphate, a thiophosphate or a phosphonate group.
  • linked when referring to the connection between two molecules, means that the two molecules are connected by a covalent bond or the two molecules are associated via a non-covalent bond (eg, a hydrogen bond or an ionic bond).
  • the "oligonucleotide” of the present invention is a nucleotide sequence containing 10 to 80 nucleotides or nucleotide base pairs.
  • the oligonucleotide has a nucleobase sequence that is at least partially complementary to a coding sequence in a target nucleic acid or target gene expressed in a cell.
  • the nucleotides may be optionally modified.
  • the oligonucleotide after the oligonucleotide is delivered to a cell expressing a gene, the oligonucleotide is able to inhibit the expression of a potential gene, and is referred to as an "expression inhibitory oligonucleotide" in the present invention, which can inhibit gene expression in vitro or in vivo.
  • Oligonucleotides include, but are not limited to: single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), ribozymes, interfering RNA molecules, and Dicer enzyme substrates.
  • siRNA short interfering RNA
  • dsRNA double-stranded RNA
  • miRNA microRNA
  • shRNA short hairpin RNA
  • ribozymes interfering RNA molecules
  • Dicer enzyme substrates Dicer enzyme substrates.
  • RNAi agent refers to an agent containing RNA or RNA-like (such as chemically modified RNA) oligonucleotide molecules that can degrade or inhibit the translation of messenger RNA (mRNA) transcripts of target mRNA in a sequence-specific manner.
  • the RNAi agent of the present invention can be manipulated by an RNA interference mechanism (i.e., by inducing RNA interference by interacting with components of the RNA interference pathway of mammalian cells (RNA-induced silencing complex or RISC)), or by any other mechanism or pathway.
  • RNA interference mechanism i.e., by inducing RNA interference by interacting with components of the RNA interference pathway of mammalian cells (RNA-induced silencing complex or RISC)
  • RISC RNA-induced silencing complex
  • the RNAi agent of the present invention is mainly manipulated by an RNA interference mechanism, the disclosed RNAi agent is not limited to or constrained by any specific pathway or mechanism of action.
  • RNAi agents include, but are not limited to, single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA) and Dicer substrates.
  • the RNAi agent of the present invention includes an oligonucleotide having a strand that is at least partially complementary to the targeted mRNA.
  • the RNAi agent described herein is double-stranded and includes an antisense strand. and a sense strand that is at least partially complementary to the antisense strand.
  • the RNAi agent may include modified nucleotides and/or one or more non-phosphodiester linkages.
  • the RNAi agent described herein is single-stranded.
  • single-stranded oligonucleotide refers to a single-stranded oligonucleotide having a sequence that is at least partially complementary to a target mRNA, which can hybridize to the target mRNA under mammalian physiological conditions (or an equivalent in vitro environment) through hydrogen bonds.
  • the single-stranded oligonucleotide is a single-stranded antisense oligonucleotide.
  • double-stranded oligonucleotide refers to a duplex structure comprising two reverse parallel and substantially complementary nucleotide chains, wherein one chain is a sense chain and the other chain is an antisense chain, wherein the antisense chain refers to a chain substantially complementary to the corresponding region of the target sequence (e.g., AGT mRNA), which can hybridize with the target mRNA under mammalian physiological conditions (or an equivalent in vitro environment) through hydrogen bonds.
  • target sequence e.g., AGT mRNA
  • substantially complementary means that the corresponding positions of the two sequences can be completely complementary, or there can be one or more mismatches, and when there are mismatches, there are usually no more than 3, 2 or 1 mismatched base pairs.
  • the bases of one chain are paired with the bases on the other chain in a complementary manner.
  • the purine base adenine (A) is always paired with the pyrimidine base uracil (U); the purine base guanine (C) is always paired with the pyrimidine base cytosine (G).
  • the double-stranded oligonucleotide is a double-stranded siRNA.
  • the "short interfering RNA (siRNA)" of the present invention is a type of RNA molecule with a double-stranded region length of 17 to 25 base pairs, similar to miRNA, and operates within the RNA interference (RNAi) pathway, which interferes with the translation of mRNA of a specific gene complementary to the nucleotide sequence, resulting in mRNA degradation.
  • the short interfering RNA (siRNA) of the present invention includes double-stranded siRNA (including sense strand and antisense strand) and single-stranded siRNA (antisense strand only).
  • silencing when referring to the expression of a given gene, means that the expression of the gene is reduced when the cell, cell population, or tissue is treated with an oligonucleotide linked to a conjugated group as described herein, as measured by the level of RNA transcribed from the gene or the level of a polypeptide, protein, or protein subunit translated from mRNA in a cell, cell population, tissue, or subject in which the gene is transcribed, compared to a second cell, cell population, or tissue that has not been so treated.
  • sequence or “nucleotide sequence” of the present invention refers to the order or sequence of nucleobases or nucleotides described by a sequence of letters using standard nucleotide nomenclature.
  • nucleotides are optionally modified” described in the present invention means that the nucleotides can be unmodified nucleotides or modified nucleotides, and the "unmodified nucleotides” refer to nucleotides composed of natural nucleobases, sugar rings and phosphates.
  • modified nucleotides refer to nucleotides composed of modified nucleobases, and/or modified sugar rings, and/or modified phosphates.
  • the "modified nucleotides” are composed of modified nucleobases, modified sugar rings and natural phosphates; in some embodiments of the present invention, the "modified nucleotides” are composed of modified nucleobases, modified phosphates and natural sugar rings; in some embodiments of the present invention, the "modified nucleotides” are composed of natural nucleobases, modified sugar rings and modified phosphates; in some embodiments of the present invention, the "modified nucleotides” are composed of modified nucleobases, natural sugar rings and natural phosphates; in some embodiments of the present invention, the "modified nucleotides” are composed of natural nucleobases, modified sugar rings and natural phosphates; in some embodiments of the present invention, the "modified nucleotides” are composed of natural nucleobases, modified sugar rings and natural phosphates; in some embodiments of the present invention, the "modified nucleotides” are composed of natural nucleobases,
  • the "natural sugar ring" described in the present invention is a five-membered sugar ring selected from 2'-OH.
  • the "natural bases" of the present invention are selected from the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • the "modified nucleobase” of the present invention refers to a 5-12 membered saturated, partially unsaturated or aromatic heterocycle other than a natural base, including a monocyclic or condensed ring, and specific examples thereof include but are not limited to thiophene, thianthrene, furan, pyran, isobenzofuran, benzothiazine, pyrrole, imidazole, substituted or unsubstituted triazole, pyrazole, isothiazole, isoxazole, pyridazine, indolizine, indole, isoindole, isoquinoline, quinoline, naphthopyridine, quinazoline, carbazole, phenanthridine, piperidine, phenazine, phenazine, phenothiazine, furanane, phenoxazine, pyrrolidine, pyrroline, imidazolidine, imidazo
  • the "modified sugar ring" of the present invention may include, but is not limited to, one of the following modifications at the 2' position: H; F; O-, S- or N-alkyl; O-, S- or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C 1 to C 10 alkyl or C 2 to C 10 alkenyl and alkynyl.
  • Exemplary suitable modifications include O[(CH 2 ) n O] m CH 3 , O(CH 2 ) n OCH 3 , O(CH 2 ) n NH 2 , O(CH 2 ) n CH 3 , O(CH 2 ) n ONH 2 , and O(CH 2 ) n ON[(CH 2 ) n CH 3 )] 2 , wherein n and m are from 1 to 10.
  • the 2' position includes but is not limited to one of the following modifications: substituted or unsubstituted C 1 to C 10 lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkylaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, group for improving the pharmacokinetic properties of iRNA, or group for improving the pharmacodynamic characteristics of iRNA, and other substituents with similar properties.
  • the modification includes but is not limited to 2'-methoxyethoxy (2'-O-CH 2 CH 2 OCH 3 , also known as 2'-O-(2-methoxyethoxy (2'-
  • the "modified phosphate” of the present invention includes but is not limited to: thiophosphate modification, and the “thiophosphate” includes (R)- and (S)-isomers and/or mixtures thereof.
  • the modified nucleotides may comprise one or more locked nucleic acids (LNA).
  • Locked nucleic acids are nucleotides with a modified ribose moiety, wherein the ribose moiety comprises an additional bridge connecting the 2' carbon and the 4' carbon. This structure effectively "locks" the ribose in a 3'-endo conformation.
  • the modified nucleotides include one or more monomers that are UNA (unlocked nucleic acid) nucleotides.
  • UNA is an unlocked acyclic nucleic acid in which any sugar bonds have been removed to form unlocked "sugar" residues.
  • UNA also encompasses monomers in which the bond between C1'-C4' has been removed (i.e., a covalent carbon-oxygen-carbon bond between C1' and C4' carbons).
  • the C2'-C3' bond of the sugar i.e., a covalent carbon-carbon bond between C2' and C3' carbons
  • the modified nucleotide comprises one or more monomers of GNA (glycerol nucleic acid) nucleotides.
  • GNA includes GNA-A, GNA-T, GNA-C, GNA-G and GNA-U.
  • the structure of GNA-A is The structure of GNA-T or Tgn is The structure of GNA-C is The structure of GNA-G is The structure of GNA-U is
  • the modified nucleotides contain one or more dX (deoxynucleotide) nucleotide monomers.
  • dX includes dA, dT, dC, dG and dU.
  • the structure of dA is The structure of dT is The structure of dC is The structure of dG is The structure of dU is
  • the modified nucleotides may also include one or more bicyclic sugar moieties.
  • bicyclic sugar is a furanyl (furanosyl) ring modified by the bridging of two atoms.”
  • bicyclic nucleoside (“ BNA ”) is a nucleoside with a sugar moiety, and the sugar moiety comprises a bridge connecting two carbon atoms of a sugar ring, thus forming a bicyclic ring system.
  • the bridge connects 4 '-carbon and 2 '-carbon of a sugar ring.
  • the “multiple” mentioned in the present invention refers to an integer greater than or equal to 2, including but not limited to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, up to the theoretical upper limit of the siRNA analogs.
  • the "overhang" of the present invention refers to at least one unpaired nucleotide protruding from the double-stranded region structure of a double-stranded compound.
  • the 3'-end of one chain extends beyond the 5'-end of the other chain, or the 5'-end of one chain extends beyond the 3'-end of the other chain.
  • the overhang may contain at least one nucleotide; or the overhang may contain at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five or more nucleotides.
  • the nucleotides at the nucleotide overhang are optionally modified nucleotides.
  • the overhang may be located on the sense strand, the antisense strand, or any combination thereof.
  • the overhang may be present at the 5'-end, 3'-end, or both ends of the antisense or sense strand of the double-stranded compound.
  • the antisense strand has an overhang of 1 to 10 nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides) at the 3'-end and/or 5'-end.
  • the sense strand has an overhang of 1 to 10 nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides) at the 3'-end and/or the 5'-end.
  • the antisense strand is at the 3'-end, and the sense strand has an overhang of 1 to 10 nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides) at the 3'-end.
  • the antisense strand is at the 5'-end, and the sense strand has an overhang of 1 to 10 nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides) at the 5'-end.
  • the conjugate of the double-stranded RNAi analog is a compound formed by linking the double-stranded RNAi analog and a pharmaceutically acceptable conjugating group, and the double-stranded RNAi analog and the pharmaceutically acceptable conjugating group are covalently linked.
  • the pharmaceutically acceptable conjugated group can be linked to the 3' end and/or 5' end of the sense strand and/or antisense strand of the double-stranded RNAi analog.
  • the number of pharmaceutically acceptable conjugated groups is 1, 2, 3, 4 or 5, and the pharmaceutically acceptable conjugated groups can be independently connected to the 3' end and/or 5' end of the sense strand and/or antisense strand of the double-stranded RNAi.
  • conjugation refers to the covalent linkage of two or more chemical moieties, each having a specific function, to each other; accordingly, “conjugate” refers to a compound formed by covalent linkage of the chemical moieties.
  • linker refers to an organic moiety group that connects two parts of a compound, for example, covalently attaches two parts of a compound.
  • the linker usually contains a direct bond or an atom (such as oxygen or sulfur), an atom group (such as NRR, C(O), C(O)NH, SO, SO 2 , SO 2 NH), a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heterocycloalkyl, wherein one or more C atoms in the substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or un
  • the cleavable linker is sufficiently stable outside the cell, but will be cleaved once inside the target cell to release the two moieties to which the linker co-fixes.
  • the compounds of the present invention may exist in specific geometric or stereoisomeric forms.
  • the present invention contemplates all such compounds, including (R)- and (S)-enantiomers, diastereomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present invention.
  • Additional asymmetric carbon atoms may be present in substituents such as alkyl. All of these isomers and their mixtures are included within the scope of the present invention.
  • enantiomer or “optical isomer” refers to stereoisomers that are mirror images of one another.
  • diastereomer refers to stereoisomers that have two or more chiral centers and that are not mirror images of each other.
  • the key is a solid wedge. and dotted wedge key
  • a straight solid bond To indicate the absolute configuration of a stereocenter, use a straight solid bond. and straight dashed key
  • a wavy line Denotes a solid wedge bond or dotted wedge key
  • use a wavy line Represents a straight solid bond or straight dashed key
  • the terms “enriched in one isomer”, “isomerically enriched”, “enriched in one enantiomer” or “enantiomerically enriched” mean that the content of one isomer or enantiomer is less than 100%, and the content of the isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.
  • the term “isomer excess” or “enantiomeric excess” refers to the difference between the relative percentages of two isomers or two enantiomers. For example, if the content of one isomer or enantiomer is 90% and the content of the other isomer or enantiomer is 10%, the isomer or enantiomeric excess (ee value) is 80%.
  • Optically active (R)- and (S)-isomers and D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer.
  • a diastereomeric salt is formed with an appropriate optically active acid or base, and then the diastereoisomers are separated by conventional methods known in the art, and then the pure enantiomer is recovered.
  • the separation of enantiomers and diastereomers is usually accomplished by using chromatography, which uses a chiral stationary phase and is optionally combined with a chemical derivatization method (for example, a carbamate is generated from an amine).
  • the compounds of the present invention may contain non-natural proportions of atomic isotopes on one or more atoms constituting the compound.
  • the compound may be labeled with a radioactive isotope, such as tritium ( 3H ), iodine-125 ( 125I ) or C-14 ( 14C ).
  • deuterated drugs may be formed by replacing hydrogen with heavy hydrogen. The bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon. Compared with undeuterated drugs, deuterated drugs have the advantages of reducing toxic side effects, increasing drug stability, enhancing therapeutic effects, and extending the biological half-life of drugs. All isotopic composition changes of the compounds of the present invention, whether radioactive or not, are included in the scope of the present invention.
  • phosphothioate and “phosphothioate” refer to a thioester of the formula Its protonated form (e.g. ) and its tautomers (e.g. )compound of.
  • phosphate is used in its ordinary sense as understood by those skilled in the art and includes its protonated form (e.g., ).
  • salt refers to a salt of a compound of the invention, prepared from a compound having a specific substituent discovered by the invention and a relatively nontoxic acid or base.
  • a base addition salt can be obtained by contacting such a compound with a sufficient amount of a base in a pure solution or a suitable inert solvent.
  • Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts.
  • an acid addition salt can be obtained by contacting such a compound with a sufficient amount of an acid in a pure solution or a suitable inert solvent.
  • Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid, and also include salts of amino acids (such as arginine, etc.), and salts of organic acids such as glucuronic acid.
  • Certain specific compounds of the present invention contain basic and acidic functional groups, and thus can be converted into any
  • the salts of the present invention can be synthesized by conventional chemical methods from parent compounds containing acid radicals or bases.
  • the preparation method of such salts is: in water or an organic solvent or a mixture of the two, via the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid to prepare.
  • the compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present invention.
  • the compounds of the present invention may contain non-natural proportions of atomic isotopes on one or more atoms constituting the compound.
  • the compound may be labeled with a radioactive isotope, such as tritium ( 3H ), iodine-125 ( 125I ) or C-14 ( 14C ).
  • deuterated drugs may be formed by replacing hydrogen with heavy hydrogen. The bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon. Compared with undeuterated drugs, deuterated drugs have the advantages of reducing toxic side effects, increasing drug stability, enhancing therapeutic effects, and extending the biological half-life of drugs. All isotopic composition changes of the compounds of the present invention, whether radioactive or not, are included in the scope of the present invention.
  • linking group L When the linking group is listed without specifying its linking direction, its linking direction is arbitrary, for example,
  • the connecting group L is -MW-, in which case -MW- can connect ring A and ring B in the same direction as the reading order from left to right to form You can also connect ring A and ring B in the opposite direction of the reading order from left to right to form Combinations of linkers, substituents, and/or variations thereof are permissible only if such combinations result in stable compounds.
  • substituted means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, which may include a variant of deuterium and hydrogen, as long as the valence state of the particular atom is normal and the substituted compound is stable.
  • oxygen it means that two hydrogen atoms are replaced.
  • Oxygen substitution does not occur on aromatic groups.
  • optionally substituted means that it may be substituted or not substituted, and unless otherwise specified, the type and number of the substituents may be arbitrary on the basis of chemical achievable.
  • any variable e.g., R
  • its definition at each occurrence is independent.
  • the group may be optionally substituted with up to two Rs, and each occurrence of R is an independent choice.
  • substituents and/or variants thereof are permitted only if such combinations result in stable compounds.
  • linking group When the number of a linking group is 0, such as -(CRR) 0 -, it means that the linking group is a single bond.
  • substituent When a substituent is vacant, it means that the substituent does not exist. For example, when X in A-X is vacant, it means that the structure is actually A. When the listed substituent does not specify which atom it is connected to the substituted group through, the substituent can be bonded through any atom of it. For example, pyridyl as a substituent can be connected to the substituted group through any carbon atom on the pyridine ring.
  • the compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present invention.
  • the structure of the compound of the present invention can be confirmed by conventional methods known to those skilled in the art. If the present invention relates to the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the art.
  • single crystal X-ray diffraction (SXRD) is used to collect diffraction intensity data of the cultured single crystal using a Bruker D8 venture diffractometer, the light source is CuK ⁇ radiation, and the scanning mode is: After scanning and collecting relevant data, the crystal structure is further analyzed using the direct method (Shelxs97) to confirm the absolute configuration.
  • SXRD single crystal X-ray diffraction
  • the solvent used in the present invention is commercially available.
  • the solvent ratios used in the column chromatography and preparative thin layer silica gel chromatography of the present invention are all volume ratios.
  • DMSO dimethyl sulfoxide
  • CBz stands for benzyloxycarbonyl, which is an amine protecting group
  • Boc stands for tert-butyloxycarbonyl, which is an amine protecting group
  • Boc2O stands for di-tert-butyl dicarbonate
  • DMTr stands for dimethoxytrityl
  • Fmoc stands for 9-fluorenylmethoxycarbonyl
  • ANGPTL3 stands for angiopoietin-like 3
  • AGT stands for angiotensinogen
  • complement C5 stands for complement component 5.
  • nucleotide monomers are used in the description of nucleic acid sequences, as shown in Table 2:
  • the present invention is described in detail below by examples, but it is not intended to limit the present invention in any way.
  • the compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by the combination of the specific embodiments with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art, and preferred embodiments include but are not limited to the embodiments of the present invention. It will be apparent to those skilled in the art that various changes and improvements are made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.
  • reaction solution was concentrated under reduced pressure at 30-35 degrees Celsius to remove methanol, extracted with ethyl acetate (100 ml*2), washed with saturated brine (50 ml*2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 1-3.
  • the aqueous phase was adjusted to pH 13 with sodium carbonate, extracted with ethyl acetate (50 ml * 2), and the aqueous phase was retained.
  • the aqueous phase was concentrated under reduced pressure to obtain a crude product, slurried with methanol to ethyl acetate 1:4 (50 ml), filtered, the mother liquor was collected, and concentrated under reduced pressure to obtain a crude product.
  • the organic phase was washed with saturated brine (30 ml*2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product.
  • the crude product was purified by preparative chromatography (neutral conditions; chromatographic column: Kromasil Eternity XT 250*80 mm*10 ⁇ m; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; acetonitrile%: 70%-100%, 20 minutes) to obtain compound 1-8.
  • reaction solution is diluted with water (50 ml), extracted with ethyl acetate (100 ml*2), the aqueous phase is retained, 2 mol/L hydrochloric acid is added to the aqueous phase to adjust the pH to 4-5, and extracted with ethyl acetate (100 ml*2).
  • the organic phase is washed with saturated brine (50 ml), dried over anhydrous sodium sulfate, and filtered. Concentrated under reduced pressure to obtain compound 1-12.
  • the reaction solution was diluted with water (50 ml), extracted with ethyl acetate (50 ml*2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product.
  • the crude product was purified by preparative chromatography (neutral conditions; chromatographic column: Waters Xbridge C18 150*50 mm*10 ⁇ m; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; acetonitrile%: 64%-94%, 10 minutes) to obtain compound 1-13.
  • reaction solution was filtered, the mother liquor was collected, and the compound 1-16 was obtained by preparative chromatography (neutral conditions; chromatographic column: Waters Xbridge C18 150*50 mm*10 ⁇ m; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; acetonitrile%: 48%-78%, 10 minutes).
  • the pH of the aqueous phase was adjusted to 2-3 with 1 mol/L hydrochloric acid, and the aqueous phase was extracted with dichloromethane (100 ml * 2).
  • the combined dichloromethane phase was washed with saturated brine (100 ml) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to obtain compound 2-5, which was directly used in the next step.
  • Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (2.92 g, 7.69 mmol), N,N-diisopropylethylamine (3.18 g, 24.62 mmol, 4.29 ml), and compound 2-10 (0.85 g, 1.54 mmol) were added to a solution of compound 2-11 in N,N-dimethylformamide (30 ml), respectively.
  • Triethylamine (4.69 mg, 46.38 ⁇ mol), 4-dimethylaminopyridine (5.67 mg, 46.38 ⁇ mol), and compound 2-16 (11.60 mg, 115.94 ⁇ mol) were added to a dichloromethane solution (5 ml) of compound 3-6 (142 mg, 46.38 ⁇ mol).
  • the reaction solution was stirred at 25 degrees Celsius for 12 hours.
  • concentration under reduced pressure the product was purified by preparative HPLC (column: Waters Xbridge C18 150*50 mm*10 mm; mobile phase: [water (ammonium bicarbonate-acetonitrile)]; acetonitrile%: 25%-55%, 10 minutes) to obtain compound D02-1-BM.
  • Triethylamine (112.05 mg, 1.11 mmol), 4-dimethylaminopyridine (33.82 mg, 276.84 ⁇ mol) and compound 2-16 (110.82 mg, 1.11 mmol) were added to a dichloromethane solution (8 ml) of compound 4-11 (804 mg, 276.84 ⁇ mol).
  • the reaction solution was stirred at 25 degrees Celsius for 12 hours.
  • the reaction solution was filtered and concentrated under reduced pressure, and purified by preparative HPLC (column: Waters Xbridge C18 150*50 mm*10 ⁇ m; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; acetonitrile%: 24%-54%, 10 minutes) to obtain compound D03-M.
  • Triethylamine 70.36 mg, 695.38 ⁇ mol
  • 4-dimethylaminopyridine 21.24 mg, 173.84 ⁇ mol
  • compound 2-16 69.59 mg, 695.38 ⁇ mol
  • the reaction solution was stirred at 25 degrees Celsius for 12 hours.
  • concentration under reduced pressure the product was purified by preparative HPLC (column: Waters Xbridge C18 150*50 mm*10 mm; mobile phase: [water (ammonium bicarbonate-acetonitrile)]; acetonitrile%: 25%-55%, 10 minutes) to obtain compound D02-1-CM.
  • reaction solution was diluted with 1 mol/L aqueous hydrochloric acid solution (200 ml), extracted with ethyl acetate (100 ml*2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product.
  • the crude product was added to a mixed solution of petroleum ether (10 ml) and ethyl acetate (10 ml), and slurried for 12 hours to obtain compound 6-2.
  • reaction solution was diluted with water (200 ml), extracted with dichloromethane (100 ml*2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product.
  • the crude product was purified by silica gel column to obtain compound 6-4.
  • compound 4-10 (1.7 g, 707.60 ⁇ mol) was dissolved in dichloromethane solution (17 ml), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (203.47 mg, 1.06 mmol), 1-hydroxy-7-azabenzotriazole (144.47 mg, 1.06 mmol), N,N-diisopropylethylamine (2.12 mmol, 369.75 ⁇ l) were added in sequence at 25-30°C. The mixed solution was stirred at 25-3 After stirring at 0°C for half an hour, compound 6-5 (603.79 mg, 707.60 ⁇ mol) was added, and then stirring was continued at 25-30°C for 12 hours.
  • reaction mixture was diluted with water (200 ml), extracted with dichloromethane (100 ml * 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product, which was purified by preparative HPLC (column: Kromasil Eternity XT 250*80 mm*10 ⁇ m; mobile phase: [water (ammonia water)-acetonitrile]; gradient: 60%-90% acetonitrile, 25 minutes) to obtain compound 7-4.
  • compound 4-10 (1.5 g, 624.35 ⁇ mol, 1 equivalent) was dissolved in dichloromethane solution (17 ml), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (179.53 mg, 936.53 ⁇ mol), 1-hydroxy-7-azabenzotriazole (127.47 mg, 936.53 ⁇ mol), N,N-diisopropylethylamine (1.87 mmol, 326.25 ⁇ l) were added in sequence at 25-30 degrees Celsius.
  • GalNAc can be recognized by ASGPR on the surface of hepatocytes, and the siRNA conjugated to it can be taken up into hepatocytes through endocytosis, thereby achieving downregulation of target gene mRNA levels by GalNAc-siRNA.
  • the conjugate of the present invention was diluted to 10 times the concentration to be tested with PBS solution. 10 ⁇ L of siRNA was transferred to a 96-well plate. Human primary hepatocytes were thawed and transferred to a collagen-coated 96-well plate, with a final cell density of 5.4 ⁇ 10 5 cells/100 ⁇ L/well. The conjugate of the present invention was tested at 10 concentration points, with the highest concentration being 500 nM, 4-fold dilution, and 2 replicates.
  • the cells were incubated with the conjugate of the present invention at 37 degrees Celsius and 5% CO2 for 48 hours. After the incubation, the cells were lysed and All RNA was extracted using QIAGEN-74182 and reverse transcribed using FastKing RT Kit (With gDNase) (Tiangen-KR116-02) to obtain cDNA. The expression level of AGT mRNA was detected using qPCR.
  • Tables 3, 4 and 5 are from different batches of tests. The sources of human primary hepatocytes are different, and their activity is greatly affected by this.
  • the conjugates of the present invention exhibited high inhibitory activity against AGT mRNA in primary human hepatocytes, demonstrating that the GalNAc delivery system of the present invention has good liver-targeted delivery capability for siRNA sequences.
  • GalNAc can be recognized by ASGPR on the surface of hepatocytes, and the siRNA conjugated to it can be taken up into hepatocytes through endocytosis, thereby achieving downregulation of target gene mRNA levels by GalNAc-siRNA.
  • siRNA was diluted to 5000nM with Nuclease-free water as the starting point, and then diluted 4-fold gradiently for a total of 10 concentration points, and then 10 ⁇ L was taken to a 96-well cell plate coated with collagen.
  • One human primary hepatocyte was transferred to the preheated InvitroGRO CP Medium complete culture medium and inoculated into a 96-well cell plate at a density of 54,000 cells per well (90 ⁇ L/well), and the final culture medium per well was 100 ⁇ L.
  • the conjugate of the present invention was tested at 10 concentration points, with the highest concentration of 500nM, 4-fold dilution, and 2 replicates. The cells were cultured in a 5% CO 2 , 37 degrees Celsius incubator for 48 hours.
  • RNA extraction kit Qiagen, 74182
  • the RNA was reverse transcribed into cDNA according to the instructions of HiScript III RT SuperMix for qPCR (Vazyme, catalog number R323-01), and then qPCR was performed to detect the expression level of C5 mRNA.
  • conjugates of the present invention all exhibited high inhibitory activity against C5 mRNA in human primary hepatocytes, demonstrating that the delivery systems have good liver-targeted delivery capabilities for siRNA sequences.
  • GalNAc can be recognized by ASGPR on the surface of hepatocytes, and the siRNA conjugated to it can be taken up into hepatocytes through endocytosis, thereby achieving downregulation of target gene mRNA levels by GalNAc-siRNA.
  • the main reagents used in this experiment include FastQuant RT Kit (with gDNase) (TianGen, Catalog No. KR106-02), RNA extraction kit (Qiagen, Catalog No. 74182), FastStart Universal Probe Master (Rox) (Roche, Catalog No. 04914058001), TaqMan Gene Expression Assay (GAPDH, Thermo, Assay ID-Hs02786624_g1) and TaqMan Gene Expression Assay (ANGPTL3, Thermo, Assay ID-Hs00205581_m1).
  • siRNA was diluted to 5000nM with Nuclease-free water as the starting point, and then diluted 4-fold to a total of 10 concentration points, and then 10 ⁇ L was taken to a 96-well cell plate coated with collagen.
  • One human primary hepatocyte was transferred to the preheated InvitroGRO CP Medium complete culture medium and inoculated into a 96-well plate at a density of 54,000 cells per well (90 ⁇ L/well), and the final culture medium per well was 100 ⁇ L.
  • the conjugate of the present invention was tested at 10 concentration points, with the highest concentration of 500nM, 4-fold dilution, and 2 replicates. The cells were cultured in a 5% CO 2 , 37 degrees Celsius incubator for 48 hours.
  • 2-pcDNA-CMV-AGT plasmid BALB/c mice, PBS (phosphate buffered saline), conjugate of the present invention.
  • mice were randomly divided into groups according to body weight data. After grouping, all mice were given subcutaneous injections. The single dose was given with a dosing volume of 10 mL/kg. Mice in group 1 were given PBS; mice in other groups were given the conjugate.
  • mice On the third day after administration, all mice were injected with physiological saline containing 2-pcDNA-CMV-AGT plasmid via the tail vein within 5 seconds.
  • the injection volume (mL) mouse body weight (g) ⁇ 8%, and the mass of the injected plasmid for each mouse was 10 ⁇ g.
  • mice in all groups were euthanized by CO2 inhalation, and two liver samples were collected from each mouse.
  • the liver samples were treated with RNAlater at 4°C overnight, then RNAlater was removed and stored at -80°C for detection of AGT gene expression levels.
  • AGT mRNA downregulation percentage refers to the downregulation percentage of AGT-mRNA in the liver of mice in the drug-treated group relative to the PBS blank group
  • AGT mRNA downregulation percentage refers to the downregulation percentage of AGT-mRNA in the liver of mice in the drug-treated group relative to the PBS blank group
  • the conjugates of the present invention can down-regulate AGT-mRNA in the liver in the AGT-HDI mouse model, and the down-regulation activity shows a dose-dependency. Since this test is the mRNA level in the liver, it can be proved that the GalNAc delivery system of the present invention can effectively carry out liver-targeted delivery of sequences.
  • 2-pcDNA-CMV-AGT plasmid BALB/c mice, PBS (phosphate buffered saline), conjugate of the present invention.
  • mice were randomly divided into groups according to body weight data, with 4 mice in each group. After grouping, the mice in the three groups were given subcutaneous injections, a single dose, and the administration volume was 10 mL/kg. The mice in the first group were given PBS; the other two groups of mice were given the conjugate, and the mice in the remaining groups were raised normally without any administration.
  • mice On day -3, the remaining groups of mice were given the conjugate.
  • mice in all groups were euthanized by CO2 inhalation, and two liver samples were collected from each mouse.
  • the liver samples were treated with RNAlater at 4°C overnight, then RNAlater was removed and stored at -80°C for detection of AGT gene expression levels.
  • AGT mRNA downregulation percentage refers to the downregulation percentage of AGT-mRNA in the liver of mice in the drug-treated group relative to the PBS blank group
  • C57BL/6 mice express complement C5.
  • the sample to be tested can reach the liver after subcutaneous injection into the mouse, thereby inhibiting the expression of the target gene C5 in hepatocytes.
  • concentration of target protein C5 in mouse plasma By measuring the concentration of target protein C5 in mouse plasma at different time points after administration, the in vivo activity and long-term effect of the sample to be tested can be evaluated.
  • mice Two days before siRNA administration (Day-2), plasma samples of C57BL/6 (female, 7 weeks old) mice were collected, and the C5 protein level in mouse plasma was measured by ELISA (Abcam). Mice were selected according to the test results and randomly divided into groups, with 5 mice in each group.
  • the dosage of all animals was calculated based on the volume.
  • a single subcutaneous injection was used for the administration of siRNA on Day 0, and the volume of siRNA administration was 10 mL/kg.
  • Mouse plasma was collected on days 7, 14, 21, and 28 after administration, and the concentration of C5 protein in mouse plasma was determined by ELISA.
  • the relative expression level of C5 protein in the plasma of each group of mice was used to evaluate the in vivo effectiveness of different siRNAs.
  • C5 protein downregulation percentage refers to the percentage of C5 protein concentration in the plasma of mice at different time points in each dosing group relative to the plasma C5 protein of each group of mice before dosing (Day-2)
  • Relative expression level of C5 protein refers to the percentage of plasma C5 protein concentration at different time points after administration of each group of mice relative to the 2nd day (Day-2) before administration of each group.
  • This experiment shows that the conjugate of the present invention has sustained inhibition on C5 protein and the dose-dependency of the inhibitory effect, and has excellent effectiveness and long-term effect. At the same time, it shows good sustained inhibition on C5 protein and the dose-dependency of the inhibitory effect. Since complement C5mRNA has the characteristics of hepatic origin, the results of this experiment show the good liver-targeted delivery ability of the GalNAc delivery system.

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Abstract

L'invention concerne un groupe de conjugaison tétravalent contenant un cycle hétérocyclique à sept chaînons et son utilisation, et concerne spécifiquement la structure d'un groupe de conjugaison représenté par la formule (V) et son utilisation.
PCT/CN2023/130247 2022-11-08 2023-11-07 Groupe de conjugaison tétravalent contenant un cycle hétérocyclique à sept chaînons et son utilisation WO2024099316A1 (fr)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RO104721B1 (ro) * 1989-07-10 1994-12-12 Inst Medicina Farmacie Derivați hidroxialchilhomopiperazinici ai rutozidului și procedeu de preparare a acestora
WO2013166155A1 (fr) * 2012-05-02 2013-11-07 Merck Sharp & Dohme Corp. Nouveaux conjugués contenant tétragalnac et peptide et procédés pour l'administration d'oligonucléotides
WO2021037205A1 (fr) * 2019-08-29 2021-03-04 苏州瑞博生物技术股份有限公司 Composé et conjugué de médicament, procédé de préparation associé et utilisation correspondante
WO2022098990A1 (fr) * 2020-11-06 2022-05-12 Arbutus Biopharma Corporation Conjugués ciblés conprenant un arnsi modifié
CN114805113A (zh) * 2022-01-22 2022-07-29 苏州天澜生物材料科技有限公司 一种安全高效的可降解脂质纳米颗粒及其制备方法和应用
WO2023143374A1 (fr) * 2022-01-30 2023-08-03 成都凌泰氪生物技术有限公司 Ligand, son procédé de préparation, et son utilisation
CN116655715A (zh) * 2023-07-27 2023-08-29 北京炫景瑞医药科技有限公司 一种GalNAc衍生物、缀合物、组合物以及它们的用途

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RO104721B1 (ro) * 1989-07-10 1994-12-12 Inst Medicina Farmacie Derivați hidroxialchilhomopiperazinici ai rutozidului și procedeu de preparare a acestora
WO2013166155A1 (fr) * 2012-05-02 2013-11-07 Merck Sharp & Dohme Corp. Nouveaux conjugués contenant tétragalnac et peptide et procédés pour l'administration d'oligonucléotides
WO2021037205A1 (fr) * 2019-08-29 2021-03-04 苏州瑞博生物技术股份有限公司 Composé et conjugué de médicament, procédé de préparation associé et utilisation correspondante
WO2022098990A1 (fr) * 2020-11-06 2022-05-12 Arbutus Biopharma Corporation Conjugués ciblés conprenant un arnsi modifié
CN114805113A (zh) * 2022-01-22 2022-07-29 苏州天澜生物材料科技有限公司 一种安全高效的可降解脂质纳米颗粒及其制备方法和应用
WO2023143374A1 (fr) * 2022-01-30 2023-08-03 成都凌泰氪生物技术有限公司 Ligand, son procédé de préparation, et son utilisation
CN116655715A (zh) * 2023-07-27 2023-08-29 北京炫景瑞医药科技有限公司 一种GalNAc衍生物、缀合物、组合物以及它们的用途

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