WO2024099316A1 - Tetravalent conjugation group containing seven-membered heterocyclic ring and use thereof - Google Patents

Tetravalent conjugation group containing seven-membered heterocyclic ring and use thereof 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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present
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PCT/CN2023/130247
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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

Disclosed are a tetravalent conjugation group containing a seven-membered heterocyclic ring and a use thereof, and specifically disclosed are the structure of a conjugation group represented by formula (V) and a use thereof.

Description

含七元杂环的四价缀合基团及其应用Quaternary conjugated groups containing seven-membered heterocycles and their applications
本发明主张如下的优先权：The present invention claims the following priority:
申请号：CN202211389523.0，申请日：2022年11月8日；Application number: CN202211389523.0, application date: November 8, 2022;
申请号：CN202310166298.2，申请日：2023年2月24日；Application number: CN202310166298.2, application date: February 24, 2023;
申请号：CN202310900468.5，申请日：2023年7月20日；Application number: CN202310900468.5, application date: July 20, 2023;
申请号：CN202311425671.8，申请日：2023年10月30日。Application number: CN202311425671.8, application date: October 30, 2023.
技术领域Technical Field
本发明涉及一类含七元杂环的四价缀合基团及其应用，具体涉及式(V)所示缀合基团的结构及其应用。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.
背景技术Background technique
核酸分子的组织特异性递送是开发核酸类药物的关键技术之一。将核酸分子与配体缀合，利用配体将核酸分子递送到特定组织的技术已经被广泛应用。其中，通过将包含末端N-乙酰基半乳糖(GalNAc)及其衍生物的配体缀合至核酸分子，利用N-乙酰基半乳糖与脱唾液酸糖蛋白受体(ASGPR)的结合，将核酸分子靶向地递送到肝细胞，是较为常见的一种递送手段。常见的基于GalNAc的配体通常包含三个末端GalNAc分子，即三价的GalNAc配体。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. Among them, by conjugating ligands containing terminal N-acetylgalactose (GalNAc) and its derivatives to nucleic acid molecules, and using the binding of N-acetylgalactose to asialoglycoprotein receptors (ASGPR), 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.
公开报道和文献表明，与三价的GalNAc配体分子相比，包含四个末端GalNAc分子，即四价的GalNAc配体，具有更好的递送效率和组织特异性。但四个GalNAc分子的构象不易控制，使得包含四个末端GalNAc基团的配体与ASGPR蛋白的结合效率降低，进而影响配体的递送效率。因此，本发明专利提供了一种新颖的控制四个GalNAc分子构象的方式，并得到递送效率更高的四价GalNAc缀合基团。Public reports and literature show that compared with trivalent GalNAc ligand molecules, tetravalent GalNAc ligands containing four terminal GalNAc molecules have better delivery efficiency and tissue specificity. However, the conformation of the four GalNAc molecules is not easy to control, which reduces the binding efficiency of the ligand containing four terminal GalNAc groups with the ASGPR protein, thereby affecting the delivery efficiency of the ligand. Therefore, the patent of the present invention provides a novel way to control the conformation of four GalNAc molecules and obtain a tetravalent GalNAc conjugated group with higher delivery efficiency.
发明内容Summary of the invention
本发明第一方面提供式(V)所示的缀合基团，
The first aspect of the present invention provides a conjugated group represented by formula (V),
其中，in,
L1选自 L 1 is selected from
L2选自 L 2 is selected from
选自 Selected from
n选自0和1；n is selected from 0 and 1;
t选自2、3、4、5、6和7。t is selected from 2, 3, 4, 5, 6 and 7.
本发明还提供式(I)所示的缀合基团，
The present invention also provides a conjugated group represented by formula (I),
其中，in,
L1选自 L 1 is selected from
L2选自 L 2 is selected from
n选自0和1；n is selected from 0 and 1;
t选自2、3、4、5、6和7。t is selected from 2, 3, 4, 5, 6 and 7.
在本发明的一些技术方案中，上述缀合基团选自(D02)，
In some technical solutions of the present invention, the above-mentioned conjugated group is selected from (D02),
在本发明的一些技术方案中，上述缀合基团选自(D02-1)，
In some technical solutions of the present invention, the above-mentioned conjugated group is selected from (D02-1),
其中*表示手性碳原子。Where * represents a chiral carbon atom.
在本发明的一些技术方案中，上述缀合基团选自(D02-1-A)、(D02-1-B)、(D02-1-C)、(D03)、(D12)和(D13)，


In some technical solutions of the present invention, the conjugated group is selected from (D02-1-A), (D02-1-B), (D02-1-C), (D03), (D12) and (D13),


本发明第二方面提供一种缀合物或其药学上可接受的盐，其中，所述缀合物选自上述任意一项技术方案所限定的缀合基团通过磷酸二酯键或硫代磷酸二酯键与寡聚核苷酸连接形成的化合物。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.
在本发明的一些技术方案中，上述缀合基团通过磷酸二酯键或硫代磷酸二酯键与寡聚核苷酸连接，是指如下连接方式：
In some technical solutions of the present invention, 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:
其中X选自S-或O-。wherein X is selected from S- or O- .
在本发明的一些技术方案中，上述缀合基团通过磷酸二酯键或硫代磷酸二酯键与寡聚核苷酸连接，是指如下连接方式：
In some technical solutions of the present invention, 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:
其中X选自S-或O-。wherein X is selected from S- or O- .
在本发明的一些技术方案中，上述缀合基团通过磷酸二酯键或硫代磷酸二酯键与寡聚核苷酸连接，是指如下连接方式：
In some technical solutions of the present invention, 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:
其中X选自S-或O-。wherein X is selected from S- or O- .
在本发明的一些技术方案中，上述寡聚核苷酸选自RNAi试剂和ASO试剂，其他变量如本发明所定义。In some technical solutions of the present invention, the above-mentioned oligonucleotide is selected from RNAi agents and ASO agents, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述RNAi试剂选自单链寡聚核苷酸和双链寡聚核苷酸，其他变量如本发明所定义。In some technical solutions of 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.
在本发明的一些技术方案中，上述单链寡聚核苷酸选自单链反义寡聚核苷酸，其他变量如本发明所定义。In some technical solutions of 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.
在本发明的一些技术方案中，上述双链寡聚核苷酸选自双链siRNA，其他变量如本发明所定义。In some technical solutions of the present invention, the double-stranded oligonucleotide is selected from double-stranded siRNA, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述寡聚核苷酸的核苷酸任选被修饰，其他变量如本发明所定义。In some technical embodiments of the present invention, the nucleotides of the above oligonucleotides are optionally modified, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述缀合物能够抑制或阻断基因的表达。In some technical embodiments of the present invention, the above-mentioned conjugate can inhibit or block the expression of a gene.
本发明第三方面提供上述任意技术方案所限定的缀合基团作为递送平台的应用，其中，所述递送平台是用于增强治疗剂与特定靶标位置的结合。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.
本发明第四方面提一种用于制备式(V)所示缀合基团的中间体化合物，其结构如式(I-M)、(V-M12)和(V-M13)所示，
In a fourth aspect, 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),
L1、L2、t、n本发明所定义。 L 1 , L 2 , t and n are defined in the present invention.
在本发明的一些技术方案中，上述中间体结构如式(D02-M)所示，
In some technical solutions of the present invention, the intermediate structure is as shown in formula (D02-M),
在本发明的一些技术方案中，上述中间体结构如式(D02-1-M)所示，
In some technical solutions of the present invention, the intermediate structure is as shown in formula (D02-1-M),
其中，*表示手性碳原子。Here, * represents a chiral carbon atom.
在本发明的一些技术方案中，上述中间体结构如式(D02-1-A-M)、(D02-1-B-M)、(D02-1-C-M)、(D03-M)、(D12-M)和(D13-M)所示，


In some technical solutions of the present invention, the intermediate structures are as shown in formulas (D02-1-AM), (D02-1-BM), (D02-1-CM), (D03-M), (D12-M) and (D13-M).


本发明还提供表1所示的缀合物：The present invention also provides the conjugates shown in Table 1:
表1本发明缀合物列表

Table 1 List of conjugates of the present invention

本发明还提供如下测试方法The present invention also provides the following test method
一、体外活性测试1. In vitro activity test
实验过程：将本发明缀合物与新鲜分离的原代小鼠肝细胞(PMH)在室温下孵育30分钟，让缀合物以自由摄取的方式进入到PMH细胞中。细胞培养24小时后，将细胞裂解，提取纯化RNA，用rt-PCR的方法检测靶基因的下调水平。Experimental process: 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.
实验结论：本发明缀合物在体外活性测试实验中表现良好。Experimental conclusion: The conjugate of the present invention performed well in the in vitro activity test experiment.
二、体内活性测试2. In vivo activity test
实验目的：通过高压尾静脉注射2-pcDNA-CMV-AGT质粒的小鼠模型来评价待测样品体内靶向目的基因并对目的基因的抑制效果。Experimental purpose: To evaluate the in vivo targeting and inhibitory effect of the target gene by using a mouse model of high-pressure tail vein injection of 2-pcDNA-CMV-AGT plasmid.
实验过程：experiment procedure:
1.实验材料：2-pcDNA-CMV-AGT质粒，BALB/c小鼠，PBS(磷酸缓冲液)，本发明缀合物。 1. Experimental materials: 2-pcDNA-CMV-AGT plasmid, BALB/c mice, PBS (phosphate buffered saline), and the conjugate of the present invention.
2.实验方法：2. Experimental methods:
订购6-8周龄的BALB/c雌性小鼠，小鼠到达动物房后适应检疫一周。Order 6-8 week old BALB/c female mice and acclimate to quarantine for one week after the mice arrive at the animal house.
第0天，按照体重数据将小鼠随机分组，每组4只，分组后所有小鼠皮下注射给药，单次给药，给药体积为10mL/kg，第1组小鼠给PBS；第2组小鼠给缀合物。On day 0, 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.
给药后第3天，所有小鼠在5秒内经尾静脉注射其体重8％体积的2-pcDNA-CMV-AGT质粒，(注射体积(mL)＝小鼠体重(g)×8％)，每只小鼠注射质粒的质量为10μg。On the third day after administration, all mice were injected with 2-pcDNA-CMV-AGT plasmid at a volume of 8% of their body weight through the tail vein within 5 seconds (injection volume (mL) = mouse body weight (g) × 8%), and the mass of the injected plasmid per mouse was 10 μg.
给药后第4天，所有组小鼠经CO2吸入安乐死，每只小鼠分别收集2份肝脏样品。肝脏样品经RNAlater4℃过夜处理，后移除RNAlater，保存于-80℃用于检测AGT基因表达水平。On the 4th day after administration, 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.
实验结论：本发明缀合物在体内活性测试实验中表现良好。Experimental conclusion: The conjugate of the present invention performed well in the in vivo activity test experiment.
三：人原代肝细胞自由摄取实验3: Free uptake experiment of human primary hepatocytes
1.实验原理：1. Experimental principle:
将待测样品与人原代肝细胞进行孵育，评估待测样品对AGT mRNA的下调程度。The test samples were incubated with human primary hepatocytes to evaluate the degree of downregulation of AGT mRNA by the test samples.
2.实验材料：2. Experimental Materials:
人原代肝细胞(PHH)，96 Kit(12)(QIAGEN-74182)，FastKing RT Kit(With gDNase)(Tiangen-KR116-02)，TaqMan Gene Expression Assay(GAPDH,Thermo,Assay ID-Hs02786624_g1)，TaqMan Gene Expression Assay(AGT,Thermo,Assay ID-Hs01586213_m1)。Primary human hepatocytes (PHH), 96 Kit (12) (QIAGEN-74182), FastKing RT Kit (With gDNase) (Tiangen-KR116-02), TaqMan Gene Expression Assay (GAPDH, Thermo, Assay ID-Hs02786624_g1), TaqMan Gene Expression Assay (AGT, Thermo, Assay ID-Hs01586213_m1).
3.实验方法：3. Experimental methods:
用PBS溶液将本发明缀合物稀释至待测浓度的10倍。转移10uL siRNA到96孔板中。将PHH细胞解冻并移植到96孔板中，最终的细胞密度为5.4×105cells/well。本发明缀合物测试10个浓度点，最高浓度为500nM，4倍稀释。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.
细胞在37摄氏度，5％CO2中孵育48小时，用显微镜检查细胞状态。The cells were incubated at 37°C, 5% CO2 for 48 hours and the cell status was examined under a microscope.
孵育完成后，将细胞裂解，使用获得裂解液，96 Kit(QIAGEN-74182)提取所有的RNA，并用FastKing RT Kit(With gDNase)(Tiangen-KR116-02)逆转录获得cDNA。用qPCR检测AGT cDNA。After the incubation is complete, the cells are lysed and the lysate is obtained. All RNA was extracted using QIAQUIN-96 Kit (QIAGEN-74182), and reverse transcribed using FastKing RT Kit (With gDNase) (Tiangen-KR116-02) to obtain cDNA. AGT cDNA was detected using qPCR.
4.实验结论：本发明缀合物能够显著下调AGT mRNA在PHH细胞中的水平。4. Experimental conclusion: The conjugate of the present invention can significantly downregulate the level of AGT mRNA in PHH cells.
本发明还提供缀合物的制备方法：The present invention also provides a method for preparing the conjugate:
含缀合基团的单链寡核糖核苷酸的合成：按照亚磷酰胺固相合成技术合成寡核糖核苷酸。在可控多孔玻璃(氨基CPG，或)与缀合基团对应的中间体化合物通过共价键连接制成的固体支持物上进行合成。所有的2’-修饰的RNA亚磷酰胺(phosphoramidite)和辅助试剂均为商品化可得试剂。所有的酰胺溶于无水乙腈中并且加入分子筛使用5-乙基硫-1H-四唑(ETT)作为活化剂的偶合时间为5分钟，然后在50mM I2-水/吡啶(体积比1/9)溶液中反应3分钟产生磷酸酯键，或者在50mM 3-((二甲 基氨基-亚甲基)氨基)-3H-1,2,4-二噻唑-3-硫酮(DDTT)的无水乙腈/吡啶(v/v＝1/1)溶液中反应3分钟产生硫代磷酸酯键。所有序列在最后脱除DMTr基团后即合成。Synthesis of single-stranded oligoribonucleotides containing conjugated groups: 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 The phosphorothioate bond was generated by reacting 3H-1,2,4-dithiazole-3-thione (DDTT) in anhydrous acetonitrile/pyridine (v/v=1/1) for 3 minutes. All sequences were synthesized after the DMTr group was removed at the end.
不含缀合基团的单链寡核糖核苷酸的合成：按照亚磷酰胺固相合成技术合成寡核糖核苷酸。在通用可控多孔玻璃CPG(或)上进行合成。所有的2’-修饰的RNA亚磷酰胺(phosphoramidite)和辅助试剂均为商品化可得试剂。所有的酰胺溶于无水乙腈中并且加入分子筛使用5-乙基硫-1H-四唑(ETT)作为活化剂的偶合时间为5分钟，然后在50mM I2-水/吡啶(体积比1/9)溶液中反应3分钟产生磷酸酯键，或者在50mM 3-((二甲基氨基-亚甲基)氨基)-3H-1,2,4-二噻唑-3-硫酮(DDTT)的无水乙腈/吡啶(v/v＝1/1)溶液中反应3分钟产生硫代磷酸酯键。所有序列在最后脱除DMT基团后即合成。Synthesis of single-stranded oligoribonucleotides without conjugated groups: 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. The coupling time was 5 minutes using 5-ethylthio-1H-tetrazole (ETT) as an activator, followed by 3 minutes of reaction in 50 mM I2 -water/pyridine (volume ratio 1/9) solution to generate phosphate bonds, or 3 minutes of reaction in 50 mM 3-((dimethylamino-methylene)amino)-3H-1,2,4-dithiazole-3-thione (DDTT) in anhydrous acetonitrile/pyridine (v/v=1/1) solution to generate phosphorothioate bonds. All sequences were synthesized after the DMT group was finally removed.
CPG上结合的低聚体的切割和去保护：在固相合成终止后，通过用含20％二乙胺的乙腈溶液处理30分钟去除保护基，而没有从CPG上切下寡核苷酸。随后，干燥的CPG在40摄氏度度下用浓氨水处理18小时。在离心之后，上清液被转移至新的管中并且用氨水洗涤CPG。浓缩合并的溶液得到固体混合物。Cleavage and deprotection of oligomers bound to CPG: After termination of solid phase synthesis, the protecting groups were removed by treatment with acetonitrile solution containing 20% diethylamine for 30 minutes without cutting the oligonucleotide from CPG. Subsequently, the dried CPG was treated with concentrated ammonia for 18 hours at 40 degrees Celsius. After centrifugation, the supernatant was transferred to a new tube and the CPG was washed with ammonia. The combined solution was concentrated to obtain a solid mixture.
单链寡核糖核苷酸的纯化：通过使用NanoQ阴离子交换经HPLC纯化的低聚体。缓冲液A是10mM高氯酸钠溶液，20mM Tris，1mM EDTA，pH 7.4和含有乙腈20％，以及缓冲液B，500mM高氯酸钠，20mM Tris，1mM EDTA，pH 7.4和含有乙腈20％。分离得到目标产物，并用反相C18柱脱盐。Purification of single-stranded oligoribonucleotides: 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, and 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.
单链寡核糖核苷酸的退火产生siRNA：把待退火的单链寡核糖核苷酸用无菌RNase Free H2O(无RNA水解酶)配制成200μM。如下设置退火反应体系，将总体积为100μL的混合液，10nmol放置95℃水浴锅10分钟(≥100nmol需求量需要高温20分钟)→迅速放入60℃水浴，自然降温→退火完成后的溶液不可放置在高温中储存。通过合并等摩尔的单链寡核糖核苷酸溶液形成互补链。Annealing of single-stranded oligoribonucleotides to produce siRNA: The single-stranded oligoribonucleotides to be annealed are prepared into 200μM with sterile RNase Free H2O (no RNA hydrolase). The annealing reaction system is set up as follows: a total volume of 100μL of the mixture, 10nmol, is placed in a 95℃ water bath for 10 minutes (≥100nmol requires high temperature for 20 minutes) → quickly placed in a 60℃ 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.
技术效果Technical Effects
本发明所述的缀合基团，在缀合到核酸分子后，可高效地特异性递送核酸分子到肝组织。使用该缀合基团的寡聚核苷酸，可以更好的与ASGPR蛋白结合，进而使寡聚核苷酸更高效地进入到肝细胞中。同时，该缀合基团使得与其缀合的核酸分子具有良好的组织特异性，即降低核酸分子在肝外组织中的富集程度。同时，该缀合基团在体内的毒性低，使得使用其作为寡聚核苷酸的递送平台时，寡聚核苷酸的毒性也更低。更重要的是，使用所述的缀合基团连接不同靶基因的双链RNA序列得到的siRNA在体内模型中展现出优秀的有效性和长效性。另外，对于本申请的缀合基团的合成，路线简练，合成操作简单，易于后处理操作。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. At the same time, 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. At the same time, 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. More importantly, 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. In addition, for the synthesis of the conjugated group of the present application, the route is concise, the synthesis operation is simple, and it is easy to post-processing operation.
定义和说明Definition and Description
除非另有说明，本文所用的下列术语和短语旨在具有下列含义。一个特定的术语或短语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照本领域普通技术人员所理解的含义去理解。当本文中出现商品名时，意在指代其对应的商品或其活性成分。 Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered to be indefinite or unclear in the absence of a special definition, but should be understood according to the meaning understood by a person of ordinary skill in the art. When a trade name appears in this article, it is intended to refer to the corresponding commercial product or its active ingredient.
除非另有说明，否则术语“包含、包括和含有”或等同物为开放式表述，意味着除所列出的要素、组分或步骤外，还可涵盖其他未指明的要素、组分或步骤。Unless otherwise specified, the terms "comprising, including and containing" or equivalents are open-ended expressions, meaning that in addition to the listed elements, components or steps, other unspecified elements, components or steps may also be included.
本发明所述“治疗剂”是指用于治疗疾病或改善症状的药剂，所述药剂包括但不限定于化学治疗剂和生物治疗剂。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.
本发明所述的缀合基团可以增强治疗剂向诸如人或动物的对象内特定靶位置(例如，特定器官或组织)的递送。在本发明的一些实施方式中，所述缀合基团可以增强表达抑制性寡聚核苷酸的靶向递送。在本发明的一些实施方式中，缀合基团可以增强表达抑制性寡聚核苷酸向肝脏的递送。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.
本发明所述的缀合基团可以直接或间接地连接至化合物，诸如治疗剂，例如，表达抑制性寡聚核苷酸，例如，表达抑制性寡聚核苷酸的3’或5’末端。在本发明的一些实施方式中，表达抑制性寡聚核苷酸包括一个或多个修饰的核苷酸。在本发明的一些实施方式中，表达抑制性寡聚核苷酸是RNAi试剂，诸如包含正义链和反义链的双链RNAi试剂。在本发明的一些实施方式中，本文所公开的缀合基团连接至双链RNAi试剂的正义链的5’末端。在一些实施方式中，本文所公开的缀合基团经由磷酸酯、硫代磷酸酯或膦酸酯基团在双链RNAi试剂正义链5′末端与表达抑制性寡聚核苷酸试剂连接。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. In some embodiments of the present invention, the expression inhibitory oligonucleotide comprises one or more modified nucleotides. In some embodiments of the present invention, the expression inhibitory oligonucleotide is an RNAi agent, such as a double-stranded RNAi agent comprising a sense strand and an antisense strand. In some embodiments of the present invention, 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.
本发明所述术语“连接”，当表示两个分子之间的联系时，指两个分子通过共价键连接或者两个分子经由非共价键(例如，氢键或离子键)关联。The term "linked" as used herein, 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).
本发明所述“寡聚核苷酸”是含有10～80个核苷酸或核苷酸碱基对的核苷酸序列。在本发明的一些实施方式中，寡聚核苷酸具有这样的核碱基序列，其与细胞内表达的靶核酸或靶基因中的编码序列至少部分互补。所述核苷酸可以任选被修饰。在本发明一些实施方式中，在将寡聚核苷酸递送至表达基因的细胞后，寡聚核苷酸能够抑制潜在基因的表达，并且在本发明中被称为“表达抑制性寡聚核苷酸”，其可以体外或体内抑制基因表达。“寡聚核苷酸”包括但不限于：单链寡核苷酸，单链反义寡核苷酸，短干扰RNA(siRNA)，双链RNA(dsRNA)，微RNA(miRNA)，短发夹RNA(shRNA)，核糖酶，干扰RNA分子，和Dicer酶底物。The "oligonucleotide" of the present invention is a nucleotide sequence containing 10 to 80 nucleotides or nucleotide base pairs. In some embodiments of the present invention, 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. In some embodiments of the present invention, 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.
本发明所述“RNAi试剂”指含有能够以序列特异性方式降解或抑制靶mRNA的信使RNA(mRNA)转录本翻译的RNA或RNA样(如化学修饰的RNA)寡核苷酸分子的试剂。本发明所述RNAi试剂可以通过RNA干扰机制(即，通过与哺乳动物细胞的RNA干扰通路组成部分(RNA诱导的沉默复合物或RISC)相互作用诱导RNA干扰)操纵，或通过任何其它机制或途径起作用。尽管本发明所述RNAi试剂主要通过RNA干扰机制操纵，但是公开的RNAi试剂并不受限于或受约束于任何特定作用途径或机制。RNAi试剂包括但不限于：单链寡核苷酸，单链反义寡核苷酸，短干扰RNA(siRNA)，双链RNA(dsRNA)，微RNA(miRNA)，短发夹RNA(shRNA)和Dicer底物。本发明所述的RNAi试剂包括寡核苷酸，所述寡核苷酸具有与靶向的mRNA至少部分互补的链。在本发明的一些实施方式中，本文所述RNAi试剂是双链的，并且包括反义链 以及与反义链至少部分互补的正义链。RNAi试剂可以包括修饰的核苷酸和/或一个或多个非磷酸二酯连接。在一些实施方式中，本文所述的RNAi试剂是单链的。The "RNAi agent" of the present invention 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. Although 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. In some embodiments of the present invention, 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. In some embodiments, the RNAi agent described herein is single-stranded.
本发明所述术语“单链寡核苷酸”指具有与靶mRNA至少部分互补的序列的单链寡聚核苷酸，其能够通过氢键在哺乳动物生理条件(或相当的体外环境)下与靶mRNA杂交。在本发明的一些实施方式中，单链寡核苷酸是单链反义寡核苷酸。The term "single-stranded oligonucleotide" as used herein 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. In some embodiments of the present invention, the single-stranded oligonucleotide is a single-stranded antisense oligonucleotide.
本发明所述术语“双链寡核苷酸”指包含两个反向平行且基本互补的核苷酸链的双链体结构，其中一条链为正义链，另一条链为反义链，其中所述反义链是指与靶序列(例如，AGT mRNA)的相应区域基本上互补的链，其能够通过氢键在哺乳动物生理条件(或相当的体外环境)下与靶mRNA杂交。所述“基本上互补”是指两条序列的相应位置可以完全互补，也可以存在一个或多个错配，当存在错配式通常为存在不超过3、2或1个错配的碱基对。在双链核酸分子中，一条链的碱基与另一条链上的碱基以互补的方式相配对。嘌呤碱基腺嘌呤(A)始终与嘧啶碱基尿嘧啶(U)相配对；嘌呤碱基鸟嘌呤(C)始终与嘧啶碱基胞嘧啶(G)相配对。在本发明的一些实施方式中，双链寡核苷酸是双链siRNA。The term "double-stranded oligonucleotide" as used herein 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. The "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. In a double-stranded nucleic acid molecule, 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). In some embodiments of the present invention, the double-stranded oligonucleotide is a double-stranded siRNA.
本发明所述“短干扰RNA(siRNA)”是一类RNA分子，双链区长度为17～25个碱基对，类似于miRNA，并且在RNA干扰(RNAi)途径内操作，它干扰了与核苷酸序列互补的特定基因的mRNA的翻译，导致mRNA降解。本发明所述短干扰RNA(siRNA)包括双链siRNA(包括正义链和反义链)和单链siRNA(仅反义链)。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).
本发明所述“沉默”、“降低”、“抑制”、“下调”或“敲减”，当指代表达给定基因时，表示与还没有经这样处理的第二细胞、细胞群或组织相比，当用与本文所述缀合基团连接的寡聚核苷酸处理该细胞、细胞群、或组织时基因表达降低，如由从基因转录的RNA的水平或从转录基因的细胞、细胞群、组织或对象中mRNA翻译的多肽、蛋白质或蛋白质亚基的水平测量。As used herein, "silencing," "reducing," "inhibiting," "downregulating," or "knockdown," 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.
本发明所述“序列”或“核苷酸序列”表示使用标准核苷酸命名的一序列字母描述的核碱基或核苷酸的次序或顺序物。The "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.
除非另有规定，本发明所述的“核苷酸任选被修饰”是指核苷酸可以是未经修饰的核苷酸，也可以是经修饰的核苷酸，所述“未经修饰的核苷酸”是指由天然的核碱基、糖环及磷酸酯组成的核苷酸。所述“经修饰的核苷酸”是指由修饰的核碱基，和/或修饰的糖环，和/或修饰的磷酸酯组成的核苷酸。在本发明的一些实施例中，“经修饰的核苷酸”由修饰的核碱基、修饰的糖环和天然的磷酸酯组成；在本发明的一些实施例中，“经修饰的核苷酸”由修饰的核碱基、修饰的磷酸酯和天然的糖环组成；在本发明的一些实施例中，“经修饰的核苷酸”由天然的核碱基、修饰的糖环和修饰的磷酸酯组成；在本发明的一些实施例中，“经修饰的核苷酸”由修饰的核碱基、天然的糖环和天然的磷酸酯组成；在本发明的一些实施例中，“经修饰的核苷酸”由天然的核碱基、修饰的糖环和天然的磷酸酯组成；在本发明的一些实施例中，“经修饰的核苷酸”由天然的核 碱基、天然的糖环和修饰的磷酸酯组成；在本发明的一些实施例中，“经修饰的核苷酸”由修饰的核碱基、修饰的糖环和修饰的磷酸酯组成。Unless otherwise specified, the "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. The "modified nucleotides" refer to nucleotides composed of modified nucleobases, and/or modified sugar rings, and/or modified phosphates. In some embodiments of the present invention, 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, 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; base, a natural sugar ring and a modified phosphate; in some embodiments of the present invention, a "modified nucleotide" is composed of a modified nucleobase, a modified sugar ring and a modified phosphate.
除非另有规定，本发明所述“天然的糖环”选自2’-OH的五元糖环。Unless otherwise specified, the "natural sugar ring" described in the present invention is a five-membered sugar ring selected from 2'-OH.
除非另有规定，本发明所述“天然的碱基”选自嘌呤碱基腺嘌呤(A)和鸟嘌呤(G)，以及嘧啶碱基胸腺嘧啶(T)、胞嘧啶(C)和尿嘧啶(U)。Unless otherwise specified, 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).
除非另有规定，本发明所述“修饰的核碱基”是指除天然碱基之外的5-12元的饱和、部分不饱和或芳香的杂环，包括单环或稠环，其具体例包括但不限于噻吩、噻蒽、呋喃、吡喃、异苯并呋喃、苯并噻嗪、吡咯、咪唑、取代或未取代的三氮唑、吡唑、异噻唑、异恶唑、哒嗪、吲哚嗪、吲哚、异吲哚、异喹啉、喹啉、萘并吡啶、喹唑啉、咔唑、菲啶、哌啶、吩嗪、菲那嗪、吩噻嗪、呋喃烷、吩恶嗪、吡咯烷、吡咯啉、咪唑烷、咪唑啉、吡唑烷、5-甲基胞嘧啶(5-me-C)、5-羟甲基胞嘧啶、黄嘌呤、次黄嘌呤、2-氨基腺嘌呤、2-氨基腺嘌呤、2-氨基鸟嘌呤、2-丙基的腺嘌呤和鸟嘌呤以及其他烷基衍生物、2-硫尿嘧啶、2-硫代胸腺嘧啶、2-硫代胞嘧啶、5-卤代尿嘧啶以及胞嘧啶、5-丙炔基尿嘧啶以及胞嘧啶、6-偶氮尿嘧啶、6-偶氮胞嘧啶、6-偶氮胸腺嘧啶、5-尿嘧啶(假尿嘧啶)、4-硫尿嘧啶、8-卤基，8-氨基，8-氢硫基，8-硫烷基，8-羟基以及其他8-取代腺嘌呤和鸟嘌呤、5-卤基尤其是5-溴，5-三氟甲基以及其他5-取代的尿嘧啶和胞嘧啶、7-甲基鸟嘌呤和7-甲基腺嘌呤、8-氮鸟嘌呤和8-氮腺嘌呤、7-脱氮鸟嘌呤和7-脱氮腺嘌呤、以及3-脱氮鸟嘌呤和3-脱氮腺嘌呤、 Unless otherwise specified, 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, imidazoline, pyrazolidine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 2-aminoadenine, 2-aminoguanine, 2-propyl adenine and guanine and other alkyl derivatives, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-sulfhydryl, 8-sulfanyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine,
除非另有规定，本发明所述“修饰的糖环”可以在2'位置包含但不限于以下之一的修饰：H；F；O-、S-或N-烷基；O-、S-或N-烯基；O-、S-或N-炔基；或O-烷基-O-烷基，其中烷基、烯基和炔基可以是取代或未取代的C1至C10烷基或C2至C10烯基和炔基。示例性的适合的修饰包括O[(CH2)nO]mCH3、O(CH2)nOCH3、O(CH2)nNH2、O(CH2)nCH3、O(CH2)nONH2、和O(CH2)nON[(CH2)nCH3)]2，其中n和m是从1至10。在其他实施例中，在2'位置包含但不限于以下之一的修饰：取代或未取代的C1至C10低级烷基、烷芳基、芳烷基、O-烷芳基或O-芳烷基、SH、SCH3、OCN、Cl、Br、CN、CF3、OCF3、SOCH3、SO2CH3、ONO2、NO2、N3、NH2、杂环烷基、杂环烷芳基、氨烷基氨基、聚烷氨基、取代的甲硅烷基、RNA切割基团、报道基团、嵌入剂、用于改善iRNA的药物代谢动力学特性的基团、或用于改善iRNA的药效特征的基团、和具有相似特性的其他取代基。在一些实施例中，该修饰包括但不限于2'-甲氧基乙氧基(2'-O-CH2CH2OCH3，也称作2'-O-(2-甲氧基乙基)或2'-MOE)。 Unless otherwise specified, 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. In other embodiments, 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. In some embodiments, the modification includes but is not limited to 2'-methoxyethoxy (2'-O-CH 2 CH 2 OCH 3 , also known as 2'-O-(2-methoxyethyl) or 2'-MOE).
除非另有规定，本发明所述“修饰的磷酸酯“包括但不限于：硫代磷酸酯修饰，所述的“硫代磷酸酯”包括(R)-和(S)-异构体和/或其混合物。Unless otherwise specified, 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.
在本发明的一些实施例中，修饰的核苷酸可以包含一个或多个锁核酸(LNA)。锁核酸是具有修饰核糖部分的核苷酸，其中所述核糖部分包含连接2'碳和4'碳的额外桥。这个结构有效地将该核糖“锁”在3'-内切结构构象中。In some embodiments of the invention, 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.
在本发明的一些实施例中，修饰的核苷酸包含一个或多个是UNA(未锁核酸)核苷酸的单体。UNA是未锁的无环核酸，其中已经除去任何糖键，从而形成未锁的“糖”残基。在一个实例中，UNA还涵盖C1’-C4’之间的键已经除去的单体(即，C1’和C4’碳之间的共价碳-氧-碳键)。在另一个实例中，糖的C2’-C3’键(即，C2’和C3’碳之间的共价碳-碳键)已经除去。In some embodiments of the invention, 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. In one example, 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). In another example, the C2'-C3' bond of the sugar (i.e., a covalent carbon-carbon bond between C2' and C3' carbons) has been removed.
在本发明的一些实施例中，修饰的核苷酸包含一个或多个GNA(甘油核酸)核苷酸的单体。GNA包括GNA-A、GNA-T、GNA-C、GNA-G和GNA-U。GNA-A结构为GNA-T或者Tgn结构为GNA-C结构为GNA-G结构为GNA-U结构为 In some embodiments of the present invention, 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
在本发明的一些实施例序列中，修饰的核苷酸包含一个或多个dX(脱氧核苷酸)核苷酸的单体。dX包括dA、dT、dC、dG和dU。dA结构为dT结构为dC结构为dG结构为dU结构为 In some example sequences of the present invention, 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
在本发明的一些实施例中，修饰的核苷酸也可以包括一个或多个双环糖部分。“双环糖”是通过两个原子的桥接修饰的呋喃基(furanosyl)环。“双环核苷”(“BNA”)是具有糖部分的核苷，所述糖部分包含连接糖环的两个碳原子的桥，由此形成双环环系统。在特定实施方案中，桥连接糖环的4’-碳和2’-碳。In some embodiments of the present invention, 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. In a specific embodiment, the bridge connects 4 '-carbon and 2 '-carbon of a sugar ring.
本发明所述“多个”指大于等于2的整数，包括但不限于2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20个，至多可达所述siRNA类似物的理论上限。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.
除非另有规定，本发明所述“突出端”是指从双链化合物的双链区结构突出的至少一个未配对的核苷酸。例如一条链的3’-端延伸超出另一条链5’-端，或一条链的5’-端延伸超出另一条链3’-端。所述的突出端可包含含有至少一个核苷酸；或者该突出端可包含至少两个核苷酸、至少三个核苷酸、至少四个核苷酸、至少五个或更多个核苷酸。核苷酸突出端的核苷酸为任选被修饰的核苷酸。突出端可位于正义链、反义链或其任何组合上。此外，突出端可存在于双链化合物的反义或正义链的5’-端、3’-端或同时存在于两端。在本发明的一些实施例中，反义链在3’-端和/或5’-端具有1至10个核苷酸(例如1、2、3、4、5、6、7、8、9或10个核苷酸)的突出端。在本发明的一些实施例中，正义链在3’-端和/或5’-端具有1至10个核苷酸(例如1、2、3、4、5、6、7、8、9或10个核苷酸)的突出端。在本发明的一些实施例中，反义链在3’-端，正义链在3’-端具有1至10个核苷酸(例如1、2、3、4、5、6、7、8、9或10个核苷酸)的突出端。在本发明的一些实施例中，反义链在5’-端，正义链在5’-端具有1至10个核苷酸(例如1、2、3、4、5、6、7、8、9或10个核苷酸)的突出端。 Unless otherwise specified, 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. For example, 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. In addition, 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. In some embodiments of the present invention, 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. In some embodiments of the invention, 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. In some embodiments of the invention, 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. In some embodiments of the invention, 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.
本发明中，双链RNAi类似物的缀合物(也称“缀合物”)是双链RNAi类似物和药学上可接受的缀合基团连接形成的化合物，并且双链RNAi类似物和药学上可接受的缀合基团共价连接。In the present invention, the conjugate of the double-stranded RNAi analog (also referred to as "conjugate") 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.
本发明中，药学上可接受的缀合基团可连接至双链RNAi类似物的正义链和/或反义链的3’末端和/或5’末端。In the present invention, 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.
本发明中，药学上可接受的缀合基团的数量为1、2、3、4或5个，且所述药学上可接受的缀合基团分别独立地可连接至双链RNAi的正义链和/或反义链的3’末端和/或5’末端。In the present invention, 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.
在本发明的上下文中，除非另有说明，“缀合”是指两个或多个各自具有特定功能的化学部分之间以共价连接的方式彼此连接；相应地，“缀合物”是指该各个化学部分之间通过共价连接而形成的化合物。In the context of the present invention, unless otherwise specified, "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.
在本发明中，除非另有说明，“键联体”是指连接化合物的两个部分的有机部分基团，例如：共价附接化合物的两个部分。该键联体通常包含一个直接键联或原子(如：氧或硫)、原子团(如：NRR、C(O)、C(O)NH、SO、SO2、SO2NH)、取代或未取代的烷基、取代或未取代的烯基、取代或未取代的炔基、取代或未取代的芳基、取代或未取代的杂芳基、取代或未取代的环烷基、取代或未取代的杂环烷基，其中所述取代或未取代的烷基、取代或为取代的烯基、取代或未取代的炔基中的任选的一个或多个C原子能被取代或未取代的芳基、取代或未取代的杂芳基、取代或未取代的环烷基、取代或未取代的杂环烷基替换。In the present invention, unless otherwise specified, "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 unsubstituted alkynyl can be replaced by a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heterocycloalkyl.
可裂解的键联体在细胞外具有充份稳定性，但当进入标靶细胞内时即会裂解而释出该键联体所共同固定的两个部分的基团。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.
本发明的化合物可以存在特定的几何或立体异构体形式。本发明设想所有的这类化合物，包括(R)-和(S)-对映体、非对映异构体，及其外消旋混合物和其他混合物，例如对映异构体或非对映体富集的混合物，所有这些混合物都属于本发明的范围之内。烷基等取代基中可存在另外的不对称碳原子。所有这些异构体以及它们的混合物，均包括在本发明的范围之内。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.
除非另有说明，术语“对映异构体”或者“旋光异构体”是指互为镜像关系的立体异构体。Unless otherwise indicated, the term "enantiomer" or "optical isomer" refers to stereoisomers that are mirror images of one another.
除非另有说明，术语“非对映异构体”是指分子具有两个或多个手性中心，并且分子间为非镜像的关系的立体异构体。Unless otherwise indicated, the term "diastereomer" refers to stereoisomers that have two or more chiral centers and that are not mirror images of each other.
除非另有说明，用楔形实线键和楔形虚线键表示一个立体中心的绝对构型，用直形实线键和直形虚线键表示立体中心的相对构型，用波浪线表示楔形实线键或楔形虚线键或用波浪线表示直形实线键或直形虚线键 Unless otherwise specified, the key is a solid wedge. and dotted wedge key To indicate the absolute configuration of a stereocenter, use a straight solid bond. and straight dashed key To indicate the relative configuration of a stereocenter, use a wavy line Denotes a solid wedge bond or dotted wedge key Or use a wavy line Represents a straight solid bond or straight dashed key
本发明，结构片段中的直形实线键表示结构中所连的两个取代基是同向的。The present invention, structural fragment Straight solid bond in It means that the two substituents connected in the structure are in the same direction.
除非另有说明，术语“富含一种异构体”、“异构体富集”、“富含一种对映体”或者“对映体富集”指其中一种异构体或对映体的含量小于100％，并且，该异构体或对映体的含量大于等于60％，或者大于等于70％，或者大于等于80％，或者大于等于90％，或者大于等于95％，或者大于等于96％，或者大于等于97％，或者大于等于98％，或者大于等于99％，或者大于等于99.5％，或者大于等于99.6％，或者大于等于99.7％，或者大于等于99.8％，或者大于等于99.9％。Unless otherwise indicated, 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%.
除非另有说明，术语“异构体过量”或“对映体过量”指两种异构体或两种对映体相对百分数之间的差值。例如，其中一种异构体或对映体的含量为90％，另一种异构体或对映体的含量为10％，则异构体或对映体过量(ee值)为80％。Unless otherwise indicated, 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%.
可以通过的手性合成或手性试剂或者其他常规技术制备光学活性的(R)-和(S)-异构体以及D和L异构体。如果想得到本发明某化合物的一种对映体，可以通过不对称合成或者具有手性助剂的衍生作用来制备，其中将所得非对映体混合物分离，并且辅助基团裂开以提供纯的所需对映异构体。或者，当分子中含有碱性官能团(如氨基)或酸性官能团(如羧基)时，与适当的光学活性的酸或碱形成非对映异构体的盐，然后通过本领域所公知的常规方法进行非对映异构体拆分，然后回收得到纯的对映体。此外，对映异构体和非对映异构体的分离通常是通过使用色谱法完成的，所述色谱法采用手性固定相，并任选地与化学衍生法相结合(例如由胺生成氨基甲酸盐)。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. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), 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. In addition, 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).
本发明的化合物可以在一个或多个构成该化合物的原子上包含非天然比例的原子同位素。例如，可用放射性同位素标记化合物，比如氚(3H)，碘-125(125I)或C-14(14C)。又例如，可用重氢取代氢形成氘代药物，氘与碳构成的键比普通氢与碳构成的键更坚固，相比于未氘化药物，氘代药物有降低毒副作用、增加药物稳定性、增强疗效、延长药物生物半衰期等优势。本发明的化合物的所有同位素组成的变换，无论放射性与否，都包括在本发明的范围之内。The compounds of the present invention may contain non-natural proportions of atomic isotopes on one or more atoms constituting the compound. For example, the compound may be labeled with a radioactive isotope, such as tritium ( 3H ), iodine-125 ( 125I ) or C-14 ( 14C ). For another example, 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.
除非另有说明，术语“硫代磷酸酯”和“硫代磷酸酯(phosphothioate)”是指式其质子化形式(例如)及其互变异构体(例如)的化合物。 Unless otherwise indicated, the terms "phosphothioate" and "phosphothioate" refer to a thioester of the formula Its protonated form (e.g. ) and its tautomers (e.g. )compound of.
除非另有说明，术语“磷酸酯”以本领域技术人员理解的一般含义使用，并且包括其质子化形式(例如，)。Unless otherwise indicated, the term "phosphate" is used in its ordinary sense as understood by those skilled in the art and includes its protonated form (e.g., ).
术语“盐”是指本发明化合物的盐，由本发明发现的具有特定取代基的化合物与相对无毒的酸或碱制备。当本发明的化合物中含有相对酸性的功能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的碱与这类化合物接触的方式获得碱加成盐。药学上可接受的碱加成盐包括钠、钾、钙、铵、有机胺或镁盐或类似的盐。当本发明的化合物中含有相对碱性的官能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的酸与这类化合物接触的方式获得酸加成盐。药学上可接受的酸加成盐的实例包括无机酸盐，所述无机酸包括例如盐酸、氢溴酸、硝酸、碳酸、碳酸氢根、磷酸、磷酸一氢根、磷酸二氢根、硫酸、硫酸氢根、氢碘酸、亚磷酸等；以及有机酸盐，所述有机酸包括如乙酸、丙酸、异丁酸、马来酸、丙二酸、苯甲酸、琥珀酸、辛二酸、反丁烯二酸、乳酸、扁桃酸、邻苯二甲酸、苯磺酸、对甲苯磺酸、柠檬酸、酒石酸和甲磺酸等类似的酸；还包括氨基酸(如精氨酸等)的盐，以及如葡糖醛酸等有机酸的盐。本发明的某些特定的化合物含有碱性和酸性的官能团，从而可以被转换成任一碱或酸加成盐。The term "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. When the compound of the invention contains a relatively acidic functional group, 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. When the compound of the invention contains a relatively basic functional group, 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 base or acid addition salt.
本发明的盐可由含有酸根或碱基的母体化合物通过常规化学方法合成。一般情况下，这样的盐的制备方法是：在水或有机溶剂或两者的混合物中，经由游离酸或碱形式的这些化合物与化学计量的适当的碱或酸反应来制备。The salts of the present invention can be synthesized by conventional chemical methods from parent compounds containing acid radicals or bases. In general, 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.
本发明的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本发明的实施例。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.
本发明的化合物可以在一个或多个构成该化合物的原子上包含非天然比例的原子同位素。例如，可用放射性同位素标记化合物，比如氚(3H)，碘-125(125I)或C-14(14C)。又例如，可用重氢取代氢形成氘代药物，氘与碳构成的键比普通氢与碳构成的键更坚固，相比于未氘化药物，氘代药物有降低毒副作用、增加药物稳定性、增强疗效、延长药物生物半衰期等优势。本发明的化合物的所有同位素组成的变换，无论放射性与否，都包括在本发明的范围之内。The compounds of the present invention may contain non-natural proportions of atomic isotopes on one or more atoms constituting the compound. For example, the compound may be labeled with a radioactive isotope, such as tritium ( 3H ), iodine-125 ( 125I ) or C-14 ( 14C ). For another example, 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.
当所列举的连接基团没有指明其连接方向，其连接方向是任意的，例如，中连接基团L为-M-W-，此时-M-W-既可以按与从左往右的读取顺序相同的方向连接环A和环B构成也可以按照与从左往右的读取顺序相反的方向连接环A和环B构成 所述连接基团、取代基和/或其变体的组合只有在这样的组合会产生稳定的化合物的情况下才是被允许的。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.
术语“任选”或“任选地”指的是随后描述的事件或状况可能但不是必需出现的，并且该描述包括其中所述事件或状况发生的情况以及所述事件或状况不发生的情况。The terms "optional" or "optionally" mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.
术语“被取代的”是指特定原子上的任意一个或多个氢原子被取代基取代，取代基可以包括重氢和氢的变体，只要特定原子的价态是正常的并且取代后的化合物是稳定的。当取代基为氧(即＝O)时，意味着两个氢原子被取代。氧取代不会发生在芳香基上。术语“任选被取代的”是指可以被取代，也可以不被取代，除非另有规定，取代基的种类和数目在化学上可以实现的基础上可以是任意的。The term "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. When the substituent is oxygen (i.e., =O), it means that two hydrogen atoms are replaced. Oxygen substitution does not occur on aromatic groups. The term "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.
当任何变量(例如R)在化合物的组成或结构中出现一次以上时，其在每一种情况下的定义都是独立的。因此，例如，如果一个基团被0-2个R所取代，则所述基团可以任选地至多被两个R所取代，并且每种情况下的R都有独立的选项。此外，取代基和/或其变体的组合只有在这样的组合会产生稳定的化合物的情况下才是被允许的。When any variable (e.g., R) occurs more than once in a compound's composition or structure, its definition at each occurrence is independent. Thus, for example, if a group is substituted with 0-2 Rs, the group may be optionally substituted with up to two Rs, and each occurrence of R is an independent choice. In addition, combinations of substituents and/or variants thereof are permitted only if such combinations result in stable compounds.
当一个连接基团的数量为0时，比如-(CRR)0-，表示该连接基团为单键。When the number of a linking group is 0, such as -(CRR) 0 -, it means that the linking group is a single bond.
当一个取代基为空缺时，表示该取代基是不存在的，比如A-X中X为空缺时表示该结构实际上是A。当所列举的取代基中没有指明其通过哪一个原子连接到被取代的基团上时，这种取代基可以通过其任何原子相键合，例如，吡啶基作为取代基可以通过吡啶环上任意一个碳原子连接到被取代的基团上。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.
本发明的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本发明的实施例。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.
本发明的化合物可以通过本领域技术人员所熟知的常规方法来确认结构，如果本发明涉及化合物的绝对构型，则该绝对构型可以通过本领域常规技术手段予以确证。例如单晶X射线衍射法(SXRD)，把培养出的单晶用Bruker D8 venture衍射仪收集衍射强度数据，光源为CuKα辐射，扫描方式：扫描，收集相关数据后，进一步采用直接法(Shelxs97)解析晶体结构，便可以确证绝对构型。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. For example, 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 CuKα 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.
本发明所使用的溶剂可经市售获得。The solvent used in the present invention is commercially available.
如无特殊说明，本发明柱层析、制备薄层硅胶色谱所用溶剂配比均为体积比。Unless otherwise specified, the solvent ratios used in the column chromatography and preparative thin layer silica gel chromatography of the present invention are all volume ratios.
本发明采用下述缩略词：DMSO代表二甲亚砜；CBz代表苄氧羰基，是一种胺保护基团；Boc代表叔丁氧羰基是一种胺保护基团；Boc2O代表二-叔丁基二碳酸酯；DMTr代表二甲氧基三苯甲基；Fmoc代表9-芴基甲氧基羰基；ANGPTL3代表血管生成素样蛋白3(angiopoietin-like 3)；AGT代表血管紧张素原；补体C5代表补体成分5。 The present invention uses the following abbreviations: DMSO stands for 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; and complement C5 stands for complement component 5.
核酸序列描述中使用的是核苷酸单体的缩写，具体如表2所示：The abbreviations of nucleotide monomers are used in the description of nucleic acid sequences, as shown in Table 2:
表2核苷酸单体的缩写

Table 2 Abbreviations of nucleotide monomers

化合物依据本领域常规命名原则或者使用软件命名，市售化合物采用供应商目录名称。Compounds are named according to the conventional nomenclature in the art or using The software names were used, and commercially available compounds were named using the supplier's catalog names.
具体实施方式Detailed ways
下面通过实施例对本发明进行详细描述，但并不意味着对本发明任何不利限制。本发明的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本发明的实施例。对本领域的技术人员而言，在不脱离本发明精神和范围的情况下针对本发明具体实施方式进行各种变化和改进将是显而易见的。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.
实施例1


Example 1


步骤A:Step A:
氮气保护，0摄氏度下，向丙二酸二乙酯(10克,62.43毫摩尔,9.43毫升)的四氢呋喃(100毫升)溶液中逐次加入钠氢(2.75克,68.68毫摩尔,60％纯度)。加毕，搅拌反应0.5小时，然后在0摄氏度下，滴加化合物1-1(17.86克,62.43毫摩尔)的四氢呋喃(50毫升)溶液。混合物在25摄氏度下搅拌反应15.5小时。在10-25摄氏度下，加入饱和氯化铵(20毫升)淬灭反应，加水(20毫升)稀释，乙酸乙酯(50毫升*2)萃取，饱和食盐水(30毫升*2)洗涤，无水硫酸钠干燥，过滤，减压浓缩得粗品。粗品通过硅胶柱过柱(石油醚/乙酸乙酯＝1/0到9/1)纯化得化合物1-2。Under nitrogen protection, sodium hydrogen (2.75 g, 68.68 mmol, 60% purity) was added successively to a solution of diethyl malonate (10 g, 62.43 mmol, 9.43 ml) in tetrahydrofuran (100 ml) at 0 degrees Celsius. After the addition, the reaction was stirred for 0.5 hours, and then a solution of compound 1-1 (17.86 g, 62.43 mmol) in tetrahydrofuran (50 ml) was added dropwise at 0 degrees Celsius. The mixture was stirred and reacted at 25 degrees Celsius for 15.5 hours. At 10-25 degrees Celsius, saturated ammonium chloride (20 ml) was added to quench the reaction, diluted with water (20 ml), extracted with ethyl acetate (50 ml * 2), 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 silica gel column (petroleum ether/ethyl acetate = 1/0 to 9/1) to obtain compound 1-2.
步骤B: Step B:
氮气保护，60摄氏度下，向化合物1-2(15克,41.06毫摩尔)在甲醇(150毫升)的甲醇(150毫升)溶液中逐次加入硼氢化钠(15.2克,401.77毫摩尔)，0.5小时加毕，搅拌反应0.5小时，混合物在65摄氏度下搅拌反应1小时。在0-25摄氏度下，加水淬灭反应，搅拌0.5小时，加水(50毫升)稀释，反应液减压30-35摄氏度下浓缩除去甲醇，乙酸乙酯(100毫升*2)萃取，饱和食盐水(50毫升*2)洗涤，无水硫酸钠干燥，过滤，减压浓缩得化合物1-3。Under nitrogen protection, sodium borohydride (15.2 g, 401.77 mmol) was added to a solution of compound 1-2 (15 g, 41.06 mmol) in methanol (150 ml) at 60 degrees Celsius in a methanol (150 ml) solution. The addition was completed in 0.5 hours, and the mixture was stirred for 0.5 hours. The mixture was stirred for 1 hour at 65 degrees Celsius. Water was added to quench the reaction at 0-25 degrees Celsius, stirred for 0.5 hours, and diluted with water (50 ml). The 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.
步骤C:Step C:
将化合物1-3(8.5克,30.22毫摩尔)和邻苯二甲酰亚胺钾盐(14.00克,75.56毫摩尔)混合在N,N-二甲基甲酰胺(90毫升)溶液中，氮气置换3次，反应液在100摄氏度，氮气保护下反应16小时。反应液加水(300毫升)稀释，有白色固体析出，过滤收集滤饼，滤饼通过硅胶柱过柱(石油醚/乙酸乙酯＝1/0到1/1)纯化的化合物1-4。Compound 1-3 (8.5 g, 30.22 mmol) and potassium phthalimide (14.00 g, 75.56 mmol) were mixed in N,N-dimethylformamide (90 ml) solution, replaced with nitrogen three times, and the reaction solution was reacted at 100 degrees Celsius for 16 hours under nitrogen protection. The reaction solution was diluted with water (300 ml), and a white solid precipitated. The filter cake was collected by filtration, and the filter cake was purified by passing through a silica gel column (petroleum ether/ethyl acetate = 1/0 to 1/1) to obtain compound 1-4.
步骤D:Step D:
将化合物1-4(6.1克,17.56毫摩尔)，4,4-二甲氧基三苯甲基氯(7.14克,21.07毫摩尔)和三乙烯二胺(2.95克,26.33毫摩尔,2.90毫升)混合在二氯甲烷(60毫升)溶液中，氮气置换3次，氮气保护下，反应液25摄氏度反应16小时。反应液加水(30毫升)稀释，二氯甲烷(50毫升)萃取，有机相用饱和食盐水(30毫升*2)洗涤，无水硫酸钠干燥，过滤，减压浓缩得粗品。粗品通过硅胶柱过柱(石油醚/乙酸乙酯＝1/0到4/1，添加0.1％三乙胺)纯化得化合物1-5。Compound 1-4 (6.1 g, 17.56 mmol), 4,4-dimethoxytrityl chloride (7.14 g, 21.07 mmol) and triethylenediamine (2.95 g, 26.33 mmol, 2.90 ml) were mixed in a dichloromethane (60 ml) solution, replaced with nitrogen three times, and the reaction solution was reacted at 25 degrees Celsius for 16 hours under nitrogen protection. The reaction solution was diluted with water (30 ml), extracted with dichloromethane (50 ml), and 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 passing through a silica gel column (petroleum ether/ethyl acetate = 1/0 to 4/1, with 0.1% triethylamine added) to obtain compound 1-5.
步骤E:Step E:
将化合物1-5(7.1克,10.93毫摩尔)和一水合肼(1.52克,29.76毫摩尔,1.48毫升,98％纯度)混合在乙醇(80毫升)溶液中，氮气置换3次。在氮气保护下，反应液在70摄氏度下反应16小时。35-40摄氏度下，减压浓缩除去乙醇得化合物1-6。Compound 1-5 (7.1 g, 10.93 mmol) and hydrazine monohydrate (1.52 g, 29.76 mmol, 1.48 ml, 98% purity) were mixed in an ethanol (80 ml) solution and replaced with nitrogen three times. Under nitrogen protection, the reaction solution was reacted at 70 degrees Celsius for 16 hours. At 35-40 degrees Celsius, the ethanol was removed by vacuum concentration to obtain compound 1-6.
步骤F:Step F:
将化合物1-7A(15克,62.41毫摩尔,14.71毫升)，化合物1-7B(15.35克,62.41毫摩尔)和三乙胺(20.21克,199.71毫摩尔,27.80毫升)混合在甲苯(150毫升)和甲醇(15毫升)溶液中，氮气置换3次。氮气保护下，反应液在70摄氏度下反应16小时。减压浓缩除去甲苯得粗品。粗品加0.5摩尔每升的盐酸(50毫升)稀释，乙酸乙酯(50毫升*2)萃取，保留水相，水相用碳酸钠调pH为13，乙酸乙酯(50毫升*2)萃取，保留水相。水相减压浓缩得粗品，用甲醇比乙酸乙酯1比4(50毫升)打浆，过滤，收集母液，减压浓缩得粗品。粗品再通过制备色谱纯化(甲酸条件；色谱柱:Phenomenex luna C18 250*80毫米*10微米；流动相:[水(盐酸)-乙腈]；乙腈％:5％-35％,20分钟)得化合物1-7。 Compound 1-7A (15 g, 62.41 mmol, 14.71 ml), compound 1-7B (15.35 g, 62.41 mmol) and triethylamine (20.21 g, 199.71 mmol, 27.80 ml) were mixed in a toluene (150 ml) and methanol (15 ml) solution and replaced with nitrogen three times. Under nitrogen protection, the reaction solution was reacted at 70 degrees Celsius for 16 hours. The crude product was concentrated under reduced pressure to remove toluene. The crude product was diluted with 0.5 mol hydrochloric acid (50 ml), extracted with ethyl acetate (50 ml * 2), and the aqueous phase was retained. 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 crude product was then purified by preparative chromatography (formic acid conditions; chromatographic column: Phenomenex luna C18 250*80 mm*10 μm; mobile phase: [water (hydrochloric acid)-acetonitrile]; acetonitrile%: 5%-35%, 20 minutes) to obtain compound 1-7.
步骤G:Step G:
将化合物1-6(5.68克,10.93毫摩尔)，化合物1-7(5.13克,14.21毫摩尔,HCl)，三乙胺(4.42克,43.72毫摩尔,6.08毫升)和三正丙基环磷酸酐(14.47克,22.74毫摩尔,13.52毫升,50％纯度)混合在二氯甲烷(50毫升)溶液中，氮气置换3次，氮气保护下，反应液在25摄氏度下反应16小时。反应液加水(30毫升)稀释，二氯甲烷(100毫升*2)萃取。有机相用饱和食盐水(30毫升*2)洗涤，无水硫酸钠干燥，过滤，减压浓缩得粗品。粗品通过制备色谱纯化(中性条件；色谱柱:Kromasil Eternity XT 250*80毫米*10微米；流动相:[水(碳酸氢铵)-乙腈]；乙腈％:70％-100％,20分钟)得化合物1-8。Compound 1-6 (5.68 g, 10.93 mmol), compound 1-7 (5.13 g, 14.21 mmol, HCl), triethylamine (4.42 g, 43.72 mmol, 6.08 ml) and tri-n-propyl cyclophosphoric anhydride (14.47 g, 22.74 mmol, 13.52 ml, 50% purity) were mixed in a dichloromethane (50 ml) solution, replaced with nitrogen three times, and the reaction solution was reacted at 25 degrees Celsius for 16 hours under nitrogen protection. The reaction solution was diluted with water (30 ml) and extracted with dichloromethane (100 ml*2). 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 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; acetonitrile%: 70%-100%, 20 minutes) to obtain compound 1-8.
步骤H:Step H:
将化合物1-8(4.9克,5.93毫摩尔)和钯碳(10％)混合在乙酸乙酯(100毫升)溶液中，氢气置换三次，在氢气(15psi)压力下，反应液在25摄氏度搅拌反应100小时。过滤，收集滤液，减压浓缩得化合物1-9。Compound 1-8 (4.9 g, 5.93 mmol) and palladium carbon (10%) were mixed in ethyl acetate (100 ml) solution, and hydrogen was replaced three times. The reaction solution was stirred at 25 degrees Celsius under hydrogen pressure (15 psi) for 100 hours. The filtrate was collected by filtration and concentrated under reduced pressure to obtain compound 1-9.
步骤I:Step I:
向盐酸乙酸乙酯(4摩尔/升,50.00毫升)溶液中加入化合物1-10(5克,16.43毫摩尔)，在25摄氏度下反应1小时。反应液减压浓缩得化合物1-11。Compound 1-10 (5 g, 16.43 mmol) was added to a solution of hydrochloric acid and ethyl acetate (4 mol/L, 50.00 ml) and reacted at 25 degrees Celsius for 1 hour. The reaction solution was concentrated under reduced pressure to obtain compound 1-11.
步骤J:Step J:
向化合物1-11(2.91克,16.44毫摩尔)的氢氧化钠(2M,24.66毫升)溶液中，0摄氏度下，2分钟内加入氯甲酸苄酯(6.17克,36.16毫摩尔,5.14毫升)。加毕，在0摄氏度下加入氢氧化钠(2M,18.08毫升)溶液。反应液在25摄氏度下反应0.5小时。反应液加水(50毫升)稀释，乙酸乙酯(100毫升*2)萃取，保留水相，水相加2摩尔每升的盐酸调pH至4-5，乙酸乙酯(100毫升*2)萃取。有机相加饱和食盐水(50毫升)洗涤，无水硫酸钠干燥，过滤。减压浓缩得化合物1-12。To a solution of compound 1-11 (2.91 g, 16.44 mmol) in sodium hydroxide (2M, 24.66 ml), add benzyl chloroformate (6.17 g, 36.16 mmol, 5.14 ml) at 0 degrees Celsius within 2 minutes. After the addition, add sodium hydroxide (2M, 18.08 ml) solution at 0 degrees Celsius. The reaction solution is reacted at 25 degrees Celsius for 0.5 hours. The 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.
步骤K:Step K:
将化合物1-12(1.2克,1.86毫摩尔)，化合物1-9(1.52克,4.09毫摩尔)，三乙胺(752.02毫克,7.43毫摩尔,1.03毫升)和O-(7-氮杂苯并三氮唑-1-基)-N,N,N,N-四甲基脲六氟膦盐(1.55克,4.09毫摩尔)混合在N,N-二甲基甲酰胺(10毫升)溶液中，氮气置换3次。氮气保护下，反应液在25摄氏度下反应2小时。反应液加水(50毫升)稀释，乙酸乙酯(50毫升*2)萃取，无水硫酸钠干燥，过滤，减压浓缩得粗品。粗品通过制备色谱纯化(中性条件；色谱柱:Waters Xbridge C18 150*50毫米*10微米；流动相:[水(碳酸氢铵)-乙腈]；乙腈％:64％-94％,10分钟)得化合物1-13。Compound 1-12 (1.2 g, 1.86 mmol), compound 1-9 (1.52 g, 4.09 mmol), triethylamine (752.02 mg, 7.43 mmol, 1.03 ml) and O-(7-azabenzotriazole-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphonate (1.55 g, 4.09 mmol) were mixed in N,N-dimethylformamide (10 ml) solution and replaced with nitrogen three times. Under nitrogen protection, the reaction solution was reacted at 25 degrees Celsius for 2 hours. 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 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; acetonitrile%: 64%-94%, 10 minutes) to obtain compound 1-13.
步骤L： Step L:
将化合物1-13(1.5克,1.11毫摩尔)和钯碳(200毫克,10％纯度)混合在甲醇(10毫升)和四氢呋喃(5毫升)的溶液中，氢气置换3次。氢气(15psi)压力下，25摄氏度反应40小时。过滤，收集母液，减压浓缩得化合物1-14。Compound 1-13 (1.5 g, 1.11 mmol) and palladium carbon (200 mg, 10% purity) were mixed in a solution of methanol (10 ml) and tetrahydrofuran (5 ml), and hydrogen was replaced 3 times. The mixture was reacted at 25 degrees Celsius for 40 hours under hydrogen pressure (15 psi). The mother liquor was collected by filtration and concentrated under reduced pressure to obtain compound 1-14.
步骤M:Step M:
将化合物1-14(465毫克,568.42微摩尔)，化合物1-15(1.51克,2.84毫摩尔)，三乙胺(575.19毫克,5.68毫摩尔,791.18微升)和苯并三氮唑-N,N,N,N-四甲基脲六氟磷酸酯(1.51克,3.98毫摩尔)混合在N,N-二甲基甲酰胺(5毫升)溶液中，氮气置换3次。氮气保护下，25摄氏度反应16小时。反应液过滤，收集母液，通过制备色谱纯化(中性条件；色谱柱:Waters Xbridge C18 150*50毫米*10微米；流动相:[水(碳酸氢铵)-乙腈]；乙腈％:48％-78％,10分钟)得化合物1-16。Compound 1-14 (465 mg, 568.42 μmol), compound 1-15 (1.51 g, 2.84 mmol), triethylamine (575.19 mg, 5.68 mmol, 791.18 μL) and benzotriazole-N,N,N,N-tetramethyluronium hexafluorophosphate (1.51 g, 3.98 mmol) were mixed in N,N-dimethylformamide (5 ml) solution and replaced with nitrogen three times. Under nitrogen protection, the reaction was carried out at 25 degrees Celsius for 16 hours. The 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 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; acetonitrile%: 48%-78%, 10 minutes).
步骤N:Step N:
将化合物1-16(402毫克,139.77微摩尔),化合物1-15(1.51克,2.84毫摩尔)，丁二酸酐(47.84毫克,419.31微摩尔)，4-二甲氨基吡啶(3.42毫克,27.95微摩尔)和三乙胺(42.43毫克,419.31微摩尔,58.36微升)混合在二氯甲烷(10毫升)溶液中，氮气置换3次。氮气保护，反应液25摄氏度下搅拌反应16小时。减压浓缩除去二氯甲烷得粗品。粗品通过制备色谱纯化(中性条件；色谱柱:Waters Xbridge 150*25毫米*5微米；流动相:[水(碳酸氢铵)-乙腈]；乙腈％:28％-58％,8分钟)得化合物D02-M。Compound 1-16 (402 mg, 139.77 μmol), compound 1-15 (1.51 g, 2.84 mmol), succinic anhydride (47.84 mg, 419.31 μmol), 4-dimethylaminopyridine (3.42 mg, 27.95 μmol) and triethylamine (42.43 mg, 419.31 μmol, 58.36 μl) were mixed in a dichloromethane (10 ml) solution and replaced with nitrogen three times. Under nitrogen protection, the reaction solution was stirred at 25 degrees Celsius for 16 hours. The dichloromethane was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by preparative chromatography (neutral conditions; chromatographic column: Waters Xbridge 150*25 mm*5 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; acetonitrile%: 28%-58%, 8 minutes) to obtain compound D02-M.
1H NMR(400MHz,DMSO-d6)δ＝8.55-7.59(m,10H),7.40-7.27(m,4H),7.23(br d,J＝8.3Hz,5H),6.89(br d,J＝8.6Hz,4H),5.21(d,J＝2.8Hz,3H),4.97(br dd,J＝2.9,11.1Hz,4H),4.87-4.62(m,1H),4.49(br d,J＝8.4Hz,3H),4.24-3.96(m,13H),3.93-3.81(m,5H),3.79-3.60(m,12H),3.56-3.35(m,13H),3.13-2.87(m,15H),2.47-2.34(m,6H),2.16-2.01(m,27H),2.00(s,12H),1.89(s,11H),1.77(s,14H),1.68-1.33(m,28H),1.32-0.98(m,16H)。 1 H NMR (400 MHz, DMSO-d6) δ = 8.55-7.59 (m, 10H), 7.40-7.27 (m, 4H), 7.23 (br d, J = 8.3 Hz, 5H), 6.89 (br d, J = 8.6 Hz, 4H), 5.21 (d, J = 2.8 Hz, 3H), 4.97 (br dd, J = 2.9, 11.1 Hz, 4H), 4.87-4.62 (m, 1H), 4.49 (br d, J = 8.4 Hz, 3H), 4.24-3.96 (m, 13H), 3.93-3.81 (m, 5H), 3.79-3.60 (m, 12H), 3.56-3.35 (m, 13H), 3.13-2.87 (m, 15H), 2.47-2.34 (m, 6H), 2.16-2.01 (m, 27H), 2.00 (s, 12H), 1.89 (s, 11H), 1.77 (s, 14H), 1.68-1.33 (m, 28H), 1.32-0.98 (m, 16H).
实施例2

Example 2

步骤A:Step A:
25摄氏度下向化合物2-2(11.03克,42.44毫摩尔)的甲苯溶液(100毫升)逐滴加入化合物2-1(10克,41.61毫摩尔,9.80毫升)和三乙胺(8.42克,83.21毫摩尔,11.58毫升)的甲苯溶液(100毫升)。反应在100摄氏度搅拌12小时。反应溶液加入饱和碳酸钠水溶液(100毫升)后用乙酸乙酯萃取(100毫升*2)。合并有机相用饱和食盐水洗涤(100毫升*2)，无水硫酸钠干燥后过滤，滤液浓缩后硅胶柱色谱分离得到化合物2-3。 Compound 2-1 (10 g, 41.61 mmol, 9.80 ml) and triethylamine (8.42 g, 83.21 mmol, 11.58 ml) in toluene (100 ml) were added dropwise to a toluene solution (100 ml) of compound 2-2 (11.03 g, 42.44 mmol) at 25 degrees Celsius. The reaction was stirred at 100 degrees Celsius for 12 hours. Saturated aqueous sodium carbonate solution (100 ml) was added to the reaction solution and then extracted with ethyl acetate (100 ml*2). The combined organic phases were washed with saturated brine (100 ml*2), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated and separated by silica gel column chromatography to obtain compound 2-3.
步骤B:Step B:
化合物2-3(9.5克,26.21毫摩尔)的甲醇溶液(95毫升)中加入钯碳(5克,26.21毫摩尔,10％含量)和Boc2O(12.59克,57.67毫摩尔,13.25毫升)。氩气置换三次后，氢气置换三次，常压氢气氛围下，25摄氏度下反应3小时。反应液用硅藻土过滤后减压浓缩。粗产物硅胶柱色谱纯化得到化合物2-4，产物未经纯化直接在下一步使用。Palladium carbon (5 g, 26.21 mmol, 10% content) and Boc 2 O (12.59 g, 57.67 mmol, 13.25 ml) were added to a methanol solution (95 ml) of compound 2-3 (9.5 g, 26.21 mmol). After argon replacement three times, hydrogen replacement three times, under normal pressure hydrogen atmosphere, at 25 degrees Celsius for 3 hours. The reaction solution was filtered with diatomaceous earth and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain compound 2-4, which was used directly in the next step without purification.
步骤C:Step C:
向化合物2-4(7.3克,20.37毫摩尔)的四氢呋喃溶液(25毫升)加入氢氧化钠(3.24克,81.06毫摩尔)的甲醇(25毫升)和水(25毫升)混合溶液。反应液在25摄氏度搅拌2小时。反应液浓缩除去甲醇后，加入100毫升水并用乙酸乙酯洗涤水相(100毫升*2)，乙酸乙酯相丢弃。用1摩尔/升盐酸调节水相pH＝2-3，用二氯甲烷萃取水相(100毫升*2)。合并的二氯甲烷相用饱和食盐水洗涤(100毫升)后用无水硫酸钠干燥，过滤后滤液浓缩得到化合物2-5，直接用于下一步。To a tetrahydrofuran solution (25 ml) of compound 2-4 (7.3 g, 20.37 mmol) was added a mixed solution of methanol (25 ml) and water (25 ml) of sodium hydroxide (3.24 g, 81.06 mmol). The reaction solution was stirred at 25 degrees Celsius for 2 hours. After the reaction solution was concentrated to remove methanol, 100 ml of water was added and the aqueous phase was washed with ethyl acetate (100 ml * 2), and the ethyl acetate phase was discarded. 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.
步骤D:Step D:
向化合物2-5(5.1克,14.81毫摩尔)的N,N-二甲基甲酰胺(50毫升)溶液中加入碳酸钾(4.09克,29.62毫摩尔)和溴化苄(3.04克,17.77毫摩尔,2.11毫升)。反应液在25摄氏度搅拌12小时。反应液加入200毫升水后，用乙酸乙酯萃取(100毫升*2)。合并有机相用100毫升饱和食盐水洗涤后，无水硫酸钠干燥。过滤后的滤液浓缩，经硅胶柱色谱分离得到化合物2-6。Potassium carbonate (4.09 g, 29.62 mmol) and benzyl bromide (3.04 g, 17.77 mmol, 2.11 ml) were added to a solution of compound 2-5 (5.1 g, 14.81 mmol) in N,N-dimethylformamide (50 ml). The reaction solution was stirred at 25 degrees Celsius for 12 hours. After adding 200 ml of water to the reaction solution, it was extracted with ethyl acetate (100 ml*2). The combined organic phases were washed with 100 ml of saturated brine and dried over anhydrous sodium sulfate. The filtrate after filtration was concentrated and separated by silica gel column chromatography to obtain compound 2-6.
步骤E:Step E:
25摄氏度下向化合物2-6的乙酸乙酯(10毫升)溶液中加入盐酸乙酸乙酯溶液(4M,48毫升)。反应液在25摄氏度下搅拌0.5小时。减压浓缩得到化合物2-7，直接用于下一步。To a solution of compound 2-6 in ethyl acetate (10 ml) was added a hydrochloric acid ethyl acetate solution (4M, 48 ml) at 25 degrees Celsius. The reaction mixture was stirred at 25 degrees Celsius for 0.5 hours and concentrated under reduced pressure to obtain compound 2-7, which was used directly in the next step.
步骤F:Step F:
向化合物2-8(3.07克,10.10毫摩尔)的N,N-二甲基甲酰胺溶液(25毫升)中分别加入N,N-二异丙基乙胺(4.75克,36.72毫摩尔,6.40毫升)和苯并三氮唑-N,N,N’,N’-四甲基脲六氟磷酸酯(4.35克,11.47毫摩尔)，化合物2-7(1.41克,4.59毫摩尔)。反应在25摄氏度下搅拌12小时。反应液用水(150毫升)稀释后用乙酸乙酯萃取(100毫升*2)，合并的有机相用饱和食盐水洗涤(100毫升)，无水硫酸钠干燥后，减压过滤，滤液浓缩后用制备级HPLC(柱子:Phenomenex luna C18 250*80毫米*10微米；流动相:[水(甲酸)-乙腈]；乙腈％:65％-95％,20分钟)分离得到化合物2-9。To a solution of compound 2-8 (3.07 g, 10.10 mmol) in N,N-dimethylformamide (25 ml), N,N-diisopropylethylamine (4.75 g, 36.72 mmol, 6.40 ml), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (4.35 g, 11.47 mmol), and compound 2-7 (1.41 g, 4.59 mmol) were added respectively. The reaction was stirred at 25 degrees Celsius for 12 hours. The reaction solution was diluted with water (150 ml) and extracted with ethyl acetate (100 ml * 2). The combined organic phase was washed with saturated brine (100 ml), dried over anhydrous sodium sulfate, and filtered under reduced pressure. The filtrate was concentrated and separated by preparative HPLC (column: Phenomenex luna C18 250*80 mm*10 μm; mobile phase: [water (formic acid)-acetonitrile]; acetonitrile%: 65%-95%, 20 minutes) to obtain compound 2-9.
步骤G:Step G:
将化合物2-9(3.4克,4.21毫摩尔)溶于盐酸乙酸乙酯溶液(4M,50毫升)中，反应在25摄氏度搅拌2小时。反应液浓缩后真空干燥，得到化合物2-10。 Compound 2-9 (3.4 g, 4.21 mmol) was dissolved in a hydrochloric acid ethyl acetate solution (4M, 50 ml), and the reaction was stirred at 25 degrees Celsius for 2 hours. The reaction solution was concentrated and dried in vacuo to obtain compound 2-10.
步骤H:Step H:
向化合物2-11的N,N-二甲基甲酰胺(30毫升)溶液中分别加入苯并三氮唑-N,N,N’,N’-四甲基脲六氟磷酸酯(2.92克,7.69毫摩尔)，N,N-二异丙基乙胺(3.18克,24.62毫摩尔,4.29毫升)，化合物2-10(0.85克,1.54毫摩尔)。反应液在25摄氏度下搅拌12小时。反应液加入水(150毫升)后用二氯甲烷/异丙醇＝3/1(200毫升*3)萃取。合并的有机相用无水硫酸钠干燥后，过滤并减压浓缩滤液，得到的粗产品用制备级HPLC(柱子:Phenomenex luna C18(250*70毫米,10微米)；流动相:[水(三氟乙酸)-乙腈]；乙腈％:25％-50％,19分钟)纯化得到化合物2-12。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. The reaction solution was stirred at 25 degrees Celsius for 12 hours. Water (150 ml) was added to the reaction solution, and then extracted with dichloromethane/isopropanol = 3/1 (200 ml * 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure. The crude product was purified by preparative HPLC (column: Phenomenex luna C18 (250*70 mm, 10 μm); mobile phase: [water (trifluoroacetic acid)-acetonitrile]; acetonitrile%: 25%-50%, 19 minutes) to obtain compound 2-12.
步骤I:Step I:
向化合物2-12(1.85克,750.64微摩尔)的甲醇溶液(30毫升)中加入钯碳(0.4克,750.64微摩尔,10％含量)氮气置换三次后，氢气置换三次。混合物在氢气常压氛围下25摄氏度搅拌12小时。反应液硅藻土过滤后，滤液浓缩得到化合物2-13。Palladium carbon (0.4 g, 750.64 μmol, 10% content) was added to a methanol solution (30 ml) of compound 2-12 (1.85 g, 750.64 μmol). The nitrogen atmosphere was replaced three times, and then the hydrogen atmosphere was replaced three times. The mixture was stirred at 25 degrees Celsius for 12 hours under a hydrogen atmosphere at normal pressure. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated to obtain compound 2-13.
步骤J:Step J:
向化合物2-13(0.8克,336.92微摩尔)的N,N-二甲基甲酰胺(15毫升)溶液中加入苯并三氮唑-N,N,N’,N’-四甲基脲六氟磷酸酯(191.66毫克,505.38微摩尔)，N,N-二异丙基乙胺(174.18毫克,1.35毫摩尔)和化合物2-14(175.10毫克,336.92微摩尔)。反应液在25摄氏度下搅拌12小时。向反应液中加入水(100毫升)后用二氯甲烷萃取(100毫升*2)，合并的有机相用无水硫酸钠干燥后，制备级HPLC纯化得到化合物2-15(柱子:Waters XbridgeC18 150*50毫米*10微米；流动相:[水(碳酸氢铵)-乙腈]；乙腈％:36％-66％,10分钟)。To a solution of compound 2-13 (0.8 g, 336.92 μmol) in N,N-dimethylformamide (15 ml) were added benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (191.66 mg, 505.38 μmol), N,N-diisopropylethylamine (174.18 mg, 1.35 mmol) and compound 2-14 (175.10 mg, 336.92 μmol). The reaction solution was stirred at 25 degrees Celsius for 12 hours. Water (100 ml) was added to the reaction solution, followed by extraction with dichloromethane (100 ml * 2). The combined organic phase was dried over anhydrous sodium sulfate and purified by preparative HPLC to obtain compound 2-15 (column: Waters Xbridge C18 150*50 mm*10 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; acetonitrile%: 36%-66%, 10 minutes).
步骤K:Step K:
向化合物2-15(600毫克,208.61微摩尔)的二氯甲烷溶液(15毫升)中加入4-二甲氨基吡啶(25.49毫克,208.61微摩尔)，三乙胺(42.22毫克,417.23微摩尔)，化合物2-16(125.26毫克,1.25毫摩尔)。反应液在25摄氏度下搅拌12小时后，减压浓缩后制备级HPLC(柱子:Waters Xbridge C18 150*50毫米*10微米；流动相:[水(碳酸氢铵)-乙腈]；乙腈％:24％-54％,10分钟)纯化得到化合物D02-1-A-M。LCMS([(M-2)/2],m/z:1487)，1H NMR(400MHz,DMSO-d6)δppm 0.99-1.33(m,19H)1.35-1.67(m,28H)1.77(s,12H)1.89(s,12H)1.99(s,12H)2.01-2.14(m,28H)2.42(s,4H)2.89-3.13(m,14H)3.35-3.44(m,6H)3.62-3.79(m,12H)3.82-3.93(m,5H)3.94-4.23(m,16H)4.48(d,J＝8.44Hz,4H)4.97(dd,J＝11.19,3.24Hz,4H)5.21(d,J＝3.30Hz,4H)6.88(d,J＝8.68Hz,4H)7.18-7.26(m,5H)7.27-7.39(m,4H)7.72-7.89(m,9H)7.91-8.25(m,3H)。4-Dimethylaminopyridine (25.49 mg, 208.61 μmol), triethylamine (42.22 mg, 417.23 μmol), and compound 2-16 (125.26 mg, 1.25 mmol) were added to a dichloromethane solution (15 ml) of compound 2-15 (600 mg, 208.61 μmol). After the reaction solution was stirred at 25 degrees Celsius for 12 hours, it was concentrated under reduced pressure and purified by preparative HPLC (column: Waters Xbridge C18 150*50 mm*10 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; acetonitrile%: 24%-54%, 10 minutes) to obtain compound D02-1-AM. LCMS ([(M-2)/2], m/z: 1487), 1 H NMR (400 MHz, DMSO-d6) δppm 0.99-1.33 (m, 19H) 1.35-1.67 (m, 28H) 1.77 (s, 12H) 1.89 (s, 12H) 1.99 (s, 12H) 2.01-2.14 (m, 28H) 2.42 (s, 4H) 2.89-3.13 (m, 14H) 3.35-3.44 (m, 6H) 3.62-3.79 (m, 12H) 3.82-3.93 (m, 5H) 3.9 4-4.23 (m, 16H) 4.48 (d, J = 8.44 Hz, 4H) 4.97 (dd, J = 11.19, 3.24 Hz, 4H) 5.21 (d, J = 3.30 Hz, 4H) 6.88 (d, J = 8.68 Hz, 4H) 7.18-7.26 (m, 5H) 7.27-7.39 (m, 4H) 7.72-7.89 (m, 9H) 7.91-8.25 (m, 3H).
实施例3
Example 3
步骤A:Step A:
向化合物3-1(3.07克,10.10毫摩尔)，N,N-二异丙基乙胺(1.19克,9.18毫摩尔,1.60毫升)和苯并三氮唑-N,N,N’,N’-四甲基脲六氟磷酸酯(4.35克,11.47毫摩尔)的N,N-二甲基甲酰胺(20毫升)溶液中加入加入化合物2-7(1.41克,4.59毫摩尔)和N,N-二异丙基乙胺(3.56克,27.54毫摩尔,4.80毫升)。反应液在25摄氏度下搅拌12小时。反应结束后加入80毫升水，并用乙酸乙酯萃取(80毫升*2)。合并有机相后用饱和食盐水洗涤(80毫升*2)，无水硫酸钠干燥后过滤，得到的滤液浓缩并用制备级HPLC纯化得到化合 物3-2(柱子:Phenomenex luna C18(250*70毫米,10微米)；流动相:[水(甲酸)-乙腈]；乙腈％:50％-80％,26分钟)。Compound 2-7 (1.41 g, 4.59 mmol) and N,N-diisopropylethylamine (3.56 g, 27.54 mmol, 4.80 ml) were added to a solution of compound 3-1 (3.07 g, 10.10 mmol), N,N-diisopropylethylamine (1.19 g, 9.18 mmol, 1.60 ml) and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (4.35 g, 11.47 mmol) in N,N-dimethylformamide (20 ml). The reaction solution was stirred at 25 degrees Celsius for 12 hours. After the reaction was completed, 80 ml of water was added and extracted with ethyl acetate (80 ml*2). The organic phases were combined and washed with saturated brine (80 ml*2), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated and purified by preparative HPLC to obtain compound Compound 3-2 (column: Phenomenex luna C18 (250*70 mm, 10 μm); mobile phase: [water (formic acid)-acetonitrile]; acetonitrile%: 50%-80%, 26 minutes).
步骤B:Step B:
将化合物3-2(3.79克,4.52毫摩尔)溶于盐酸乙酸乙酯中(4M,50毫升)。在25摄氏度下搅拌2小时。减压浓缩得到化合物3-3，未经进一步纯化直接用于下一步。Compound 3-2 (3.79 g, 4.52 mmol) was dissolved in ethyl acetate (4M, 50 ml) and stirred at 25 degrees Celsius for 2 hours. The mixture was concentrated under reduced pressure to obtain compound 3-3, which was used directly in the next step without further purification.
步骤C:Step C:
向化合物2-11(5.43克,10.20毫摩尔)的N,N-二甲基甲酰胺(60毫升)溶液中加入N,N-二异丙基乙胺(4.90克,37.95毫摩尔,6.61毫升)，苯并三氮唑-N,N,N’,N’-四甲基脲六氟磷酸酯(4.50克,11.86毫摩尔)和化合物3-3(1.31克,2.37毫摩尔)。反应在25摄氏度下搅拌12小时。加入200毫升水后用二氯甲烷/异丙醇＝3/1萃取水相(100毫升*4)，有机相合并后用饱和食盐水(200毫升*2)洗涤，干燥后过滤，滤液浓缩得到粗产物。粗产物用制备级HPLC(柱子:Phenomenex Synergi Max-RP 250*50毫米*10微米；流动相:[水(三氟乙酸)-乙腈]；乙腈％:25％-50％,17分钟)纯化得到化合物3-4。To a solution of compound 2-11 (5.43 g, 10.20 mmol) in N,N-dimethylformamide (60 ml) were added N,N-diisopropylethylamine (4.90 g, 37.95 mmol, 6.61 ml), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (4.50 g, 11.86 mmol) and compound 3-3 (1.31 g, 2.37 mmol). The reaction was stirred at 25 degrees Celsius for 12 hours. After adding 200 ml of water, the aqueous phase was extracted with dichloromethane/isopropanol = 3/1 (100 ml * 4), the organic phases were combined and washed with saturated brine (200 ml * 2), dried and filtered, and the filtrate was concentrated to obtain a crude product. The crude product was purified by preparative HPLC (column: Phenomenex Synergi Max-RP 250*50 mm*10 μm; mobile phase: [water (trifluoroacetic acid)-acetonitrile]; acetonitrile%: 25%-50%, 17 minutes) to obtain compound 3-4.
步骤D:Step D:
向化合物3-4(380毫克,151.95微摩尔)的甲醇溶液(10毫升)加入钯碳(50毫克,151.95微摩尔,10％含量)。反应经三次氩气置换和三次氢气置换后，在常压氢气氛围下25摄氏度反应12小时。反应液硅藻土过滤后浓缩得到化合物3-5，直接用于下一步。Palladium carbon (50 mg, 151.95 μmol, 10% content) was added to a methanol solution (10 ml) of compound 3-4 (380 mg, 151.95 μmol). The reaction was replaced by argon three times and hydrogen three times, and then reacted at 25 degrees Celsius under normal pressure hydrogen atmosphere for 12 hours. The reaction solution was filtered through diatomaceous earth and concentrated to obtain compound 3-5, which was directly used in the next step.
步骤E:Step E:
向化合物3-5(310毫克,130.56微摩尔)的N,N-二甲基甲酰胺(10毫升)溶液中加入N,N-二异丙基乙胺(67.49毫克,522.23微摩尔)，苯并三氮唑-N,N,N’,N’-四甲基脲六氟磷酸酯(74.27毫克,195.84微摩尔)和化合物2-14(61.07毫克,117.50微摩尔)。反应液在25摄氏度下搅拌12小时。反应结束后加入200毫升水，用乙酸乙酯萃取(100毫升*2)。有机相用100毫升饱和食盐水洗涤后，无水硫酸钠干燥，过滤。所得滤液浓缩并用制备级HPLC(柱子:Waters Xbridge C18 150*50毫米*10微米；流动相:[水(碳酸氢铵)-乙腈]；乙腈％:37％-67％,10分钟)纯化得到化合物3-6。To a solution of compound 3-5 (310 mg, 130.56 μmol) in N,N-dimethylformamide (10 ml), N,N-diisopropylethylamine (67.49 mg, 522.23 μmol), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (74.27 mg, 195.84 μmol) and compound 2-14 (61.07 mg, 117.50 μmol) were added. The reaction solution was stirred at 25 degrees Celsius for 12 hours. After the reaction was completed, 200 ml of water was added and extracted with ethyl acetate (100 ml*2). The organic phase was washed with 100 ml of saturated brine, dried over anhydrous sodium sulfate, and filtered. The obtained filtrate was concentrated and purified by preparative HPLC (column: Waters Xbridge C18 150*50 mm*10 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; acetonitrile%: 37%-67%, 10 minutes) to obtain compound 3-6.
步骤F:Step F:
向化合物3-6(142毫克,46.38微摩尔)的二氯甲烷溶液(5毫升)加入三乙胺(4.69毫克,46.38微摩尔)，4-二甲氨基吡啶(5.67毫克,46.38微摩尔)，化合物2-16(11.60毫克,115.94微摩尔)。反应液在25摄氏度下搅拌12小时。减压浓缩后，用制备级HPLC(柱子:Waters Xbridge C18 150*50毫米*10毫米；流动相:[水(碳酸氢铵-乙腈)]；乙腈％:25％-55％,10分钟)纯化得到化合物D02-1-B-M。LCMS([(M-2)/2], m/z:1487)，1H NMR(400MHz,DMSO-d6)δ＝7.95-7.68(m,9H),7.40-7.28(m,4H),7.23(d,J＝8.6Hz,5H),6.89(d,J＝8.4Hz,4H),5.22(d,J＝3.3Hz,4H),4.98(dd,J＝3.1,11.3Hz,4H),4.90-4.76(m,1H),4.49(d,J＝8.4Hz,4H),4.15-3.97(m,15H),3.94-3.83(m,5H),3.77-3.66(m,11H),3.40(br s,7H),3.13-2.89(m,14H),2.69-2.66(m,1H),2.44-2.30(m,6H),2.10(s,14H),2.08-2.01(m,13H),2.01-1.98(m,12H),1.89(s,12H),1.78(s,12H),1.58(br d,J＝2.9Hz,8H),1.46(br s,18H),1.29-1.02(m,15H)。Triethylamine (4.69 mg, 46.38 μmol), 4-dimethylaminopyridine (5.67 mg, 46.38 μmol), and compound 2-16 (11.60 mg, 115.94 μmol) were added to a dichloromethane solution (5 ml) of compound 3-6 (142 mg, 46.38 μmol). The reaction solution was stirred at 25 degrees Celsius for 12 hours. After 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. LCMS ([(M-2)/2], m/z: 1487), 1 H NMR (400 MHz, DMSO-d6) δ = 7.95-7.68 (m, 9H), 7.40-7.28 (m, 4H), 7.23 (d, J = 8.6 Hz, 5H), 6.89 (d, J = 8.4 Hz, 4H), 5.22 (d, J = 3.3 Hz, 4H), 4.98 (dd, J = 3.1, 11.3 Hz, 4H), 4.90-4.76 (m, 1H), 4.49 (d, J = 8.4 Hz, 4H), 4.15-3.97 (m, 15H), 3.94-3.83 (m, 5H), 3.77-3.66 (m, 11H), 3.40 (br s, 7H), 3.13-2.89(m, 14H), 2.69-2.66(m, 1H), 2.44-2.30(m, 6H), 2.10(s, 14H), 2.08-2.01(m, 13H), 2.01-1.98(m, 12H), 1.89(s, 12H), 1.78(s, 12H), 1.58(br d, J＝2.9Hz, 8H), 1.46(br s, 18H), 1.29-1.02(m, 15H).
实施例4

Example 4

步骤A:Step A:
化合物4-1(20克,76.95毫摩尔)溶于200毫升氯仿，控制温度在0-5摄氏度之间。搅拌下15分钟内逐滴加入溴化苄(20.61克,192.36毫摩尔,20.97毫升,2.5eq)并保持温度在0-5摄氏度。N,N-二异丙基乙胺(19.89克,153.89毫摩尔,26.81毫升,2eq)在25摄氏度下缓慢加入，并升温至70摄氏度反应两小时。反应自然降温至25摄氏度后，有机相依次用水(2*200毫升)，饱和食盐水(2*200毫升)洗涤后，无水硫酸钠干燥。过滤后的滤液浓缩，并用硅胶柱色谱(石油醚/乙酸乙酯，梯度淋洗)纯化得到化合物4-2。步骤B:Compound 4-1 (20 g, 76.95 mmol) was dissolved in 200 ml of chloroform and the temperature was controlled between 0-5 degrees Celsius. Benzyl bromide (20.61 g, 192.36 mmol, 20.97 ml, 2.5 eq) was added dropwise within 15 minutes under stirring and the temperature was kept at 0-5 degrees Celsius. N,N-diisopropylethylamine (19.89 g, 153.89 mmol, 26.81 ml, 2 eq) was slowly added at 25 degrees Celsius and the temperature was raised to 70 degrees Celsius for two hours. After the reaction was cooled to 25 degrees Celsius naturally, the organic phase was washed with water (2*200 ml) and saturated brine (2*200 ml) in turn and dried over anhydrous sodium sulfate. The filtrate after filtration was concentrated and purified by silica gel column chromatography (petroleum ether/ethyl acetate, gradient elution) to obtain compound 4-2. Step B:
向化合物4-2(13克,41.61毫摩尔)的甲醇(80毫升)和醋酸(160毫升)混合溶液中加入钯碳(2克,10％含量)。氩气置换三次，氢气置换三次后，反应在50PSI压力的氢气氛围下38摄氏度反应15小时。反应液冷却后过滤，滤液减压浓缩。加入200毫升乙腈后重新浓缩，重复两次后，将得到的产物溶于甲醇(125毫升)并降温至15摄氏度,缓慢的加入23毫升浓盐酸，保持温度不高于26摄氏度。浓缩溶液后，再用同体积的甲醇和浓盐酸重复一遍。得到的产物用甲醇(25毫升)和甲基叔丁基醚(500毫升)在25摄氏度打浆20分钟。过滤后得到化合物4-3。产物直接投下一步。Palladium carbon (2 g, 10% content) was added to a mixed solution of compound 4-2 (13 g, 41.61 mmol) in methanol (80 ml) and acetic acid (160 ml). After argon replacement three times and hydrogen replacement three times, the reaction was carried out at 38 degrees Celsius for 15 hours under a hydrogen atmosphere of 50 PSI pressure. The reaction solution was cooled and filtered, and the filtrate was concentrated under reduced pressure. After adding 200 ml of acetonitrile, it was re-concentrated. After repeating twice, the obtained product was dissolved in methanol (125 ml) and cooled to 15 degrees Celsius. 23 ml of concentrated hydrochloric acid was slowly added, and the temperature was kept below 26 degrees Celsius. After concentrating the solution, it was repeated with the same volume of methanol and concentrated hydrochloric acid. The obtained product was slurried with methanol (25 ml) and methyl tert-butyl ether (500 ml) at 25 degrees Celsius for 20 minutes. Compound 4-3 was obtained after filtration. The product was directly put into the next step.
步骤C:Step C:
向化合物4-3(1.3克,6.34毫摩尔)的乙醇(13毫升)悬浊液加入碳酸氢钠(1.60克,19.02毫摩尔)和Boc2O(2.84克,12.99毫摩尔,2.99毫升)。反应液在45摄氏度搅拌3小时。减压浓缩后加入50毫升水，并用乙酸乙酯进行萃取(50毫升*2)。合并的有机相用100毫升饱和食盐水洗涤，并用无水硫酸钠干燥。过滤后的滤液减压浓缩，硅胶柱色谱纯化(石油醚/乙酸乙酯，梯度淋洗)得到化合物4-4。Sodium bicarbonate (1.60 g, 19.02 mmol) and Boc 2 O (2.84 g, 12.99 mmol, 2.99 ml) were added to a suspension of compound 4-3 (1.3 g, 6.34 mmol) in ethanol (13 ml). The reaction solution was stirred at 45 degrees Celsius for 3 hours. After concentration under reduced pressure, 50 ml of water was added and extracted with ethyl acetate (50 ml * 2). The combined organic phase was washed with 100 ml of saturated brine and dried over anhydrous sodium sulfate. The filtrate after filtration was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether/ethyl acetate, gradient elution) to obtain compound 4-4.
步骤D: Step D:
向化合物4-4(1.95克,5.87毫摩尔)的四氢呋喃(6毫升)，甲醇(6毫升)和水(6毫升)的混合溶液中加入氢氧化钠(469.29毫克,11.73毫摩尔)。反应液在25摄氏度搅拌2小时。减压浓缩后加入20毫升的水，并用饱和柠檬酸溶液调节pH至3-4溶液用乙酸乙酯萃取后(100毫升)，有机相用100毫升饱和食盐水洗涤，并减压浓缩得到化合物4-5。To a mixed solution of compound 4-4 (1.95 g, 5.87 mmol) in tetrahydrofuran (6 ml), methanol (6 ml) and water (6 ml) was added sodium hydroxide (469.29 mg, 11.73 mmol). The reaction solution was stirred at 25 degrees Celsius for 2 hours. After concentration under reduced pressure, 20 ml of water was added, and the pH was adjusted to 3-4 with saturated citric acid solution. The solution was extracted with ethyl acetate (100 ml), and the organic phase was washed with 100 ml of saturated brine and concentrated under reduced pressure to obtain compound 4-5.
步骤E:Step E:
向化合物4-5(1.20克,3.76毫摩尔)的DMF溶液中(6毫升)加入N,N-二异丙基乙胺(1.85克,14.32毫摩尔,2.49毫升)，2-(7-氮杂苯并三氮唑)-N,N,N’,N’-四甲基脲六氟磷酸酯(1.70克,4.48毫摩尔)和化合物4-6(550毫克,1.79毫摩尔)。反应液在25摄氏度反应3小时。反应液加入乙酸乙酯(100毫升)和水(50毫升)。分液后有机相用无水硫酸钠干燥，过滤后得到的滤液减压浓缩，硅胶色谱柱纯化(石油醚/乙酸乙酯，梯度淋洗)得到化合物4-7。To a DMF solution (6 ml) of compound 4-5 (1.20 g, 3.76 mmol), N,N-diisopropylethylamine (1.85 g, 14.32 mmol, 2.49 ml), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.70 g, 4.48 mmol) and compound 4-6 (550 mg, 1.79 mmol) were added. The reaction solution was reacted at 25 degrees Celsius for 3 hours. Ethyl acetate (100 ml) and water (50 ml) were added to the reaction solution. After separation, the organic phase was dried over anhydrous sodium sulfate, and the filtrate obtained after filtration was concentrated under reduced pressure, and purified by silica gel chromatography (petroleum ether/ethyl acetate, gradient elution) to obtain compound 4-7.
步骤F:Step F:
将化合物4-7(1.5克,1.80毫摩尔)溶于盐酸乙酸乙酯(4M，20毫升)。反应液在25摄氏度下搅拌5小时。反应液减压浓缩后得到化合物4-8的粗产品。粗产物未经纯化直接投下一步。Compound 4-7 (1.5 g, 1.80 mmol) was dissolved in ethyl acetate hydrochloride (4 M, 20 ml). The reaction solution was stirred at 25 degrees Celsius for 5 hours. The reaction solution was concentrated under reduced pressure to obtain a crude product of compound 4-8. The crude product was directly used in the next step without purification.
步骤G:Step G:
向化合物2-11(3.63克,6.82毫摩尔)的N,N-二甲基甲酰胺(30毫升)溶液加入N,N-二异丙基乙胺(3.28克,25.36毫摩尔,4.42毫升)，苯并三氮唑-N,N,N’,N’-四甲基脲六氟磷酸酯(2.71克,7.13毫摩尔)和化合物4-8(920毫克,1.59毫摩尔)。反应在25摄氏度下搅拌12小时。减压浓缩后粗产品用制备级HPLC分离得到化合物4-9(柱子:Kromasil Eternity XT 250*80毫米*10微米；流动相:[水(碳酸氢铵)-乙腈]；乙腈％:20％-50％,20分钟)。To a solution of compound 2-11 (3.63 g, 6.82 mmol) in N,N-dimethylformamide (30 ml) were added N,N-diisopropylethylamine (3.28 g, 25.36 mmol, 4.42 ml), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (2.71 g, 7.13 mmol) and compound 4-8 (920 mg, 1.59 mmol). The reaction was stirred at 25 degrees Celsius for 12 hours. After concentration under reduced pressure, the crude product was separated by preparative HPLC to obtain compound 4-9 (column: Kromasil Eternity XT 250*80 mm*10 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; acetonitrile%: 20%-50%, 20 minutes).
步骤H:Step H:
化合物4-9(1.92克,770.27微摩尔)溶于20毫升甲醇中，加入钯碳(300毫克,10％含量)。反应氩气置换三次后再氢气置换三次，在常压氢气氛围下25摄氏度搅拌12小时。反应结束后用硅藻土过滤，滤液浓缩后得到化合物4-10。Compound 4-9 (1.92 g, 770.27 μmol) was dissolved in 20 ml methanol, and palladium carbon (300 mg, 10% content) was added. The reaction was replaced with argon three times and then replaced with hydrogen three times, and stirred at 25 degrees Celsius for 12 hours under normal pressure hydrogen atmosphere. After the reaction was completed, it was filtered with diatomaceous earth, and the filtrate was concentrated to obtain compound 4-10.
步骤I:Step I:
化合物4-10(1.7克,707.60微摩尔)的N,N-二甲基甲酰胺溶液(10毫升)加入N,N-二异丙基乙胺(365.81毫克,2.83毫摩尔)，苯并三氮唑-N,N,N’,N’-四甲基脲六氟磷酸酯(402.53毫克,1.06毫摩尔)，化合物2-14(330.97毫克,636.84微摩尔)。反应在25摄氏度下搅拌12小时。反应液过滤后，滤液旋干并用 制备级HPLC(柱子:Waters Xbridge C18 150*50毫米*10微米；流动相:[水(碳酸氢铵)-乙腈]；乙腈％:46％-76％,10分钟)纯化得到化合物4-11。Compound 4-10 (1.7 g, 707.60 μmol) in N,N-dimethylformamide solution (10 ml) was added with N,N-diisopropylethylamine (365.81 mg, 2.83 mmol), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (402.53 mg, 1.06 mmol), and compound 2-14 (330.97 mg, 636.84 μmol). The reaction was stirred at 25 degrees Celsius for 12 hours. After the reaction solution was filtered, the filtrate was dried and then Compound 4-11 was purified by preparative HPLC (column: Waters Xbridge C18 150*50 mm*10 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; acetonitrile%: 46%-76%, 10 minutes).
步骤J:Step J:
向化合物4-11(804毫克,276.84微摩尔)的二氯甲烷溶液(8毫升)中加入三乙胺(112.05毫克,1.11毫摩尔)，4-二甲氨基吡啶(33.82毫克,276.84微摩尔)和化合物2-16(110.82毫克,1.11毫摩尔)。反应液在25摄氏度下搅拌12小时。反应液过滤后减压浓缩，通过制备级HPLC(柱子:Waters Xbridge C18 150*50毫米*10微米；流动相:[水(碳酸氢铵)-乙腈]；乙腈％:24％-54％,10分钟)纯化得到化合物D03-M。LCMS([(M-2)/2],m/z:1501)，1H NMR(DMSO-d6)δ＝9.94-9.65(m,1H),8.72(br s,1H),8.22-8.19(m,1H),8.15(s,2H),8.05(br d,J＝8.0Hz,2H),7.66(s,1H),7.58(s,2H),5.09-4.92(m,1H),4.11-3.88(m,3H),3.53(br s,3H),3.48-3.32(m,2H),3.23(br d,J＝2.1Hz,3H),2.25-2.06(m,3H),1.91-1.65(m,3H),1.64-1.55(m,1H),1.34(s,6H)。Triethylamine (112.05 mg, 1.11 mmol), 4-dimethylaminopyridine (33.82 mg, 276.84 μmol) 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 μmol). 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 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; acetonitrile%: 24%-54%, 10 minutes) to obtain compound D03-M. LCMS ([(M-2)/2], m/z: 1501), 1 H NMR (DMSO-d6) δ = 9.94-9.65 (m, 1H), 8.72 (br s, 1H), 8.22-8.19 (m, 1H), 8.15 (s, 2H), 8.05 (br d, J = 8.0 Hz, 2H), 7.66 (s, 1H), 7.58 (s, 2H), 5.09-4.92 (m, 1H), 4.11-3.88 (m, 3H), 3.53 (br s, 3H), 3.48-3.32 (m, 2H), 3.23 (br d, J = 2.1 Hz, 3H), 2.25-2.06 (m, 3H), 1.91-1.65 (m, 3H), 1.64-1.55 (m, 1H), 1.34 (s, 6H).
实施例5

Example 5

步骤A:Step A:
将化合物2-8(3.68克,12.08毫摩尔)，三乙胺(6.11克,60.38毫摩尔,8.40毫升)和苯并三氮唑-N,N,N’,N’-四甲基脲六氟磷酸酯(10.08克,26.57毫摩尔)溶于N,N-二甲基甲酰胺(50毫升)中25摄氏度下搅拌半小时。将该反应液缓慢滴加进化合物2-7(7.42克,24.15毫摩尔)的N,N-二甲基甲酰胺(50毫升)溶液中。反应液在25摄氏度下搅拌半小时。反应结束后加入500毫升水，并用二氯甲烷萃取(500毫升*2)。合并有机相后用饱和食盐水洗涤(500毫升)，无水硫酸钠干燥后过滤，得到的滤液浓缩并用硅胶柱色谱纯化得到化合物5-1(梯度淋洗，石油醚/乙酸乙酯依次为1/1，1/2，1/3，0/1，均含0.1％三乙胺)。步骤B:Compound 2-8 (3.68 g, 12.08 mmol), triethylamine (6.11 g, 60.38 mmol, 8.40 ml) and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (10.08 g, 26.57 mmol) were dissolved in N,N-dimethylformamide (50 ml) and stirred at 25 degrees Celsius for half an hour. The reaction solution was slowly added dropwise to a solution of compound 2-7 (7.42 g, 24.15 mmol) in N,N-dimethylformamide (50 ml). The reaction solution was stirred at 25 degrees Celsius for half an hour. After the reaction was completed, 500 ml of water was added and extracted with dichloromethane (500 ml*2). The organic phases were combined and washed with saturated brine (500 ml), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated and purified by silica gel column chromatography to obtain compound 5-1 (gradient elution, petroleum ether/ethyl acetate was 1/1, 1/2, 1/3, 0/1, all containing 0.1% triethylamine). Step B:
将化合物3-1(2.86克,9.41毫摩尔)，N,N-二异丙基乙胺(3.65克,28.24毫摩尔,4.92毫升)和苯并三氮唑-N,N,N’,N’-四甲基脲六氟磷酸酯(5.35克,14.12毫摩尔)溶于N,N-二甲基甲酰胺(30毫升)，缓慢加入化合物5-1(4.9克,9.41毫摩尔)的N,N-二甲基甲酰胺(30毫升)溶液中。反应液在25摄氏度下搅拌12小时。反应结束后加入400毫升水，并用乙酸乙酯萃取(400毫升*2)。合并有机相后用饱和食盐水洗涤(500毫升)，无水硫酸钠干燥后过滤，得到的滤液浓缩并用制备级高效液相色谱纯化得到化合物5-2(柱子:Waters Xbridge C18 150*50毫米*10微米；流动相:[水(碳酸氢铵)-乙腈]；乙腈％:24％-54％,10min)。 Compound 3-1 (2.86 g, 9.41 mmol), N,N-diisopropylethylamine (3.65 g, 28.24 mmol, 4.92 ml) and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.35 g, 14.12 mmol) were dissolved in N,N-dimethylformamide (30 ml), and slowly added to a solution of compound 5-1 (4.9 g, 9.41 mmol) in N,N-dimethylformamide (30 ml). The reaction solution was stirred at 25 degrees Celsius for 12 hours. After the reaction was completed, 400 ml of water was added and extracted with ethyl acetate (400 ml*2). The organic phases were combined and washed with saturated brine (500 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated and purified by preparative HPLC to obtain compound 5-2 (column: Waters Xbridge C18 150*50 mm*10 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; acetonitrile%: 24%-54%, 10 min).
步骤C:Step C:
将化合物5-2(6克,7.44毫摩尔)溶于盐酸乙酸乙酯中(4M,64.62毫升)。在25摄氏度下搅拌3小时。减压浓缩得到化合物5-3，未经进一步纯化直接用于下一步。Compound 5-2 (6 g, 7.44 mmol) was dissolved in ethyl acetate hydrochloride (4 M, 64.62 ml). The mixture was stirred at 25 degrees Celsius for 3 hours. The mixture was concentrated under reduced pressure to obtain compound 5-3, which was used directly in the next step without further purification.
步骤D:Step D:
向化合物2-11(6.51克,12.22毫摩尔)的N,N-二甲基甲酰胺(60毫升)溶液中加入N,N-二异丙基乙胺(5.62克,43.45毫摩尔,7.57毫升)，苯并三氮唑-N,N,N’,N’-四甲基脲六氟磷酸酯(5.15克,13.58毫摩尔)和化合物5-3(1.50克,2.72毫摩尔)。反应在25摄氏度下搅拌12小时。加入400毫升饱和碳酸氢钠溶液后用二氯甲烷/异丙醇＝3/1萃取水相(300毫升*3)，有机相合并，干燥后过滤，滤液浓缩得到粗产物。粗产物用制备级HPLC(柱子:Phenomenex luna C18 150*40毫米*15微米；流动相:[水(三氟乙酸)-乙腈]；梯度:30％-60％乙腈，10分钟)纯化得到化合物5-4。To a solution of compound 2-11 (6.51 g, 12.22 mmol) in N,N-dimethylformamide (60 ml) were added N,N-diisopropylethylamine (5.62 g, 43.45 mmol, 7.57 ml), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.15 g, 13.58 mmol) and compound 5-3 (1.50 g, 2.72 mmol). The reaction was stirred at 25 degrees Celsius for 12 hours. After adding 400 ml of saturated sodium bicarbonate solution, the aqueous phase was extracted with dichloromethane/isopropanol = 3/1 (300 ml * 3), the organic phases were combined, dried and filtered, and the filtrate was concentrated to obtain a crude product. The crude product was purified by preparative HPLC (column: Phenomenex luna C18 150*40 mm*15 μm; mobile phase: [water (trifluoroacetic acid)-acetonitrile]; gradient: 30%-60% acetonitrile, 10 minutes) to obtain compound 5-4.
步骤E:Step E:
向化合物5-4(2.6克,1.05毫摩尔)的甲醇溶液(30毫升)加入钯碳(200毫克,1.05毫摩尔,10％含量)。反应经三次氩气置换和三次氢气置换后，在常压氢气氛围下25摄氏度反应16小时。反应液硅藻土过滤后浓缩得到化合物5-5，直接用于下一步。Palladium carbon (200 mg, 1.05 mmol, 10% content) was added to a methanol solution (30 ml) of compound 5-4 (2.6 g, 1.05 mmol). The reaction was replaced with argon three times and hydrogen three times, and then reacted at 25 degrees Celsius under normal pressure hydrogen atmosphere for 16 hours. The reaction solution was filtered through diatomaceous earth and concentrated to obtain compound 5-5, which was directly used in the next step.
步骤F:Step F:
向化合物5-5(2.2克,926.53微摩尔)的N,N-二甲基甲酰胺(20毫升)溶液中加入N,N-二异丙基乙胺(478.99毫克,3.71毫摩尔)，苯并三氮唑-N,N,N’,N’-四甲基脲六氟磷酸酯(478.99毫克,3.71毫摩尔)和化合物2-14(433.99毫克,833.88微摩尔)。反应液在25摄氏度下搅拌4小时。反应结束后过滤。所得滤液浓缩并用制备级HPLC(柱子:Waters Xbridge C18 150*50毫米*10微米；流动相:[水(碳酸氢铵)-乙腈]；梯度:35％-65％乙腈，10分钟)纯化得到化合物5-6。To a solution of compound 5-5 (2.2 g, 926.53 μmol) in N,N-dimethylformamide (20 ml) were added N,N-diisopropylethylamine (478.99 mg, 3.71 mmol), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (478.99 mg, 3.71 mmol) and compound 2-14 (433.99 mg, 833.88 μmol). The reaction solution was stirred at 25 degrees Celsius for 4 hours. After the reaction was completed, the mixture was filtered. The filtrate was concentrated and purified by preparative HPLC (column: Waters Xbridge C18 150*50 mm*10 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; gradient: 35%-65% acetonitrile, 10 minutes) to obtain compound 5-6.
步骤G:Step G:
向化合物5-6(500毫克,46.38微摩尔)的二氯甲烷溶液(5毫升)加入三乙胺(70.36毫克,695.38微摩尔)，4-二甲氨基吡啶(21.24毫克,173.84微摩尔)，化合物2-16(69.59毫克,695.38微摩尔)。反应液在25摄氏度下搅拌12小时。减压浓缩后，用制备级HPLC(柱子:Waters Xbridge C18 150*50毫米*10毫米；流动相:[水(碳酸氢铵-乙腈)]；乙腈％:25％-55％,10分钟)纯化得到化合物D02-1-C-M。LCMS([(M-2)/2],m/z:1487)，1H NMR(400MHz,DMSO-d6)δ＝8.33-7.70(m,12H),7.38-7.16(m,9H),6.88(d,J＝8.7Hz,4H),5.21(d,J＝3.3Hz,4H),4.97(dd,J＝3.5,11.2Hz,4H),4.48(d,J＝8.4Hz,4H),4.09-3.97(m,14H),3.92-3.81(m,5H),3.76-3.66(m,11H),3.47-3.36(m,5H),3.16-2.88(m,14H),2.68-2.65(m,4H),2.42(s,7H), 2.34-2.31(m,4H),2.10(s,15H),2.04(br d,J＝3.7Hz,12H),1.99(s,13H),1.89(s,12H),1.77(s,12H),1.63-1.37(m,27H),1.30-1.11(m,14H)。Triethylamine (70.36 mg, 695.38 μmol), 4-dimethylaminopyridine (21.24 mg, 173.84 μmol), and compound 2-16 (69.59 mg, 695.38 μmol) were added to a dichloromethane solution (5 ml) of compound 5-6 (500 mg, 46.38 μmol). The reaction solution was stirred at 25 degrees Celsius for 12 hours. After 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. LCMS ([(M-2)/2], m/z: 1487), 1 H NMR (400 MHz, DMSO-d6) δ = 8.33-7.70 (m, 12H), 7.38-7.16 (m, 9H), 6.88 (d, J = 8.7 Hz, 4H), 5.21 (d, J = 3.3 Hz, 4H), 4.97 (dd, J = 3.5, 11.2 Hz, 4H), 4.48 (d, J = 8.4 Hz, 4H), 4.09-3.97 (m, 14H), 3.92-3.81 (m, 5H), 3.76-3.66 (m, 11H), 3.47-3.36 (m, 5H), 3.16-2.88 (m, 14H), 2.68-2.65 (m, 4H), 2.42 (s, 7H), 2.34-2.31 (m, 4H), 2.10 (s, 15H), 2.04 (br d, J=3.7 Hz, 12H), 1.99 (s, 13H), 1.89 (s, 12H), 1.77 (s, 12H), 1.63-1.37 (m, 27H), 1.30-1.11 (m, 14H).
实施例6
Example 6
步骤A:Step A:
将化合物6-1(2克,10.68毫摩尔)溶于乙腈(20毫升)和水(20毫升)的混合溶液中，依次加入碳酸氢钠(1.79克,21.36毫摩尔,831.06微升)和N-(9-芴甲氧羰基氧基)琥珀酰亚胺(4.32克,12.82毫摩尔)。反应液在25℃下反应12小时。反应液加1摩尔/升的盐酸水溶液(200毫升)稀释，乙酸乙酯(100毫升*2)萃取，无水硫酸钠干燥，过滤，减压浓缩得粗品。粗品加入石油醚(10毫升)和乙酸乙酯(10毫升)的混合溶液，打浆12小时，得到化合物6-2。Compound 6-1 (2 g, 10.68 mmol) was dissolved in a mixed solution of acetonitrile (20 ml) and water (20 ml), and sodium bicarbonate (1.79 g, 21.36 mmol, 831.06 μL) and N-(9-fluorenylmethoxycarbonyloxy)succinimide (4.32 g, 12.82 mmol) were added in sequence. The reaction solution was reacted at 25°C for 12 hours. The 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.
步骤B:Step B:
将化合物6-2(3.2克,7.81毫摩尔)溶于二氯甲烷(50毫升)溶液，依次加入N,N-二异丙基乙胺(23.44毫摩尔,4.08毫升)，1-羟基-7-氮杂苯并三唑(1.60克,11.72毫摩尔)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(2.25克,11.72毫摩尔)和化合物6-3(3.11克,7.42毫摩尔)。反应液在25℃下反应12小时。 反应液加水(200毫升)稀释，二氯甲烷(100毫升*2)萃取，无水硫酸钠干燥，过滤，减压浓缩得粗品。粗品通过硅胶柱过柱纯化得化合物6-4。Compound 6-2 (3.2 g, 7.81 mmol) was dissolved in dichloromethane (50 ml) solution, and N,N-diisopropylethylamine (23.44 mmol, 4.08 ml), 1-hydroxy-7-azabenzotriazole (1.60 g, 11.72 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.25 g, 11.72 mmol) and compound 6-3 (3.11 g, 7.42 mmol) were added in sequence. The reaction solution was reacted at 25°C for 12 hours. The 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.
步骤C:Step C:
将化合物6-4(3.8克,4.69毫摩尔)，二乙胺(187.42毫摩尔,19.31毫升)溶于二氯甲烷(30毫升)溶液，反应液在25℃下反应12小时。减压浓缩得化合物6-5。Compound 6-4 (3.8 g, 4.69 mmol) and diethylamine (187.42 mmol, 19.31 ml) were dissolved in dichloromethane (30 ml) solution, and the reaction solution was reacted at 25° C. for 12 hours and concentrated under reduced pressure to obtain compound 6-5.
步骤D:Step D:
氮气保护，将化合物4-10(1.7克,707.60微摩尔)溶在二氯甲烷溶液(17毫升)中，25-30℃下，依次加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(203.47毫克，1.06毫摩尔)，1-羟基-7-氮杂苯并三唑(144.47毫克，1.06毫摩尔)，N,N-二异丙基乙胺(2.12毫摩尔，369.75微升)，混合溶液在25-30℃下搅拌半小时，再加入化合物6-5(603.79毫克，707.60微摩尔)，然后在25-30℃继续搅拌12小时，反应结束后，在40-45℃下浓缩除掉二氯甲烷，通过制备级HPLC纯化(柱子:Waters Xbridge C18 150*50毫米*10微米；流动相:[水(碳酸氢铵)-乙腈]；梯度:30％-60％乙腈，10分钟)得化合物6-6。Under nitrogen protection, compound 4-10 (1.7 g, 707.60 μmol) 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 μl) 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 μmol) was added, and then stirring was continued at 25-30°C for 12 hours. After the reaction was completed, dichloromethane was removed by concentration at 40-45°C, and compound 6-6 was obtained by purification by preparative HPLC (column: Waters Xbridge C18 150*50 mm*10 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; gradient: 30%-60% acetonitrile, 10 minutes).
步骤E:Step E:
氮气保护，将化合物6-6(350毫克,105.94微摩尔)，丁二酸酐(53.01毫克,529.69微摩尔)，4-二甲氨基吡啶(12.94毫克,105.94微摩尔)和三乙胺(635.63微摩尔,88.47微升)混合在二氯甲烷(3毫升)溶液中，氮气置换3次。氮气保护，反应液25℃下搅拌反应16小时。检测反应完成。减压浓缩除去二氯甲烷得粗品。粗品通过制备级HPLC纯化(柱子:Waters Xbridge C18 150*50毫米*10微米；流动相:[水(碳酸氢铵)-乙腈]；梯度:30％-60％乙腈，10分钟)得化合物D12-M。1H NMR(400MHz,DMSO-d6)δppm:7.55-8.38(m,13H)7.11-7.40(m,9H)6.78-6.97(m,4H)5.21(d,J＝3.18Hz,4H)4.97(dd,J＝11.25,3.18Hz,4H)4.49(d,J＝8.44Hz,4H)4.02(s,12H)3.81-3.93(m,7H)3.65-3.77(m,12H)2.84-3.25(m,33H)2.49-2.50(m,3H)2.44(br d,J＝19.20Hz,6H)2.17-2.30(m,3H)2.10(s,12H)2.01-2.08(m,14H)1.99(s,12H)1.89(s,12H)1.77(s,12H)1.54-1.66(m,8H)1.46(br s,20H)1.25(br s,10H)。Under nitrogen protection, compound 6-6 (350 mg, 105.94 μmol), succinic anhydride (53.01 mg, 529.69 μmol), 4-dimethylaminopyridine (12.94 mg, 105.94 μmol) and triethylamine (635.63 μmol, 88.47 μl) were mixed in a dichloromethane (3 ml) solution and replaced with nitrogen three times. Under nitrogen protection, the reaction solution was stirred at 25°C for 16 hours. The reaction was detected to be complete. The dichloromethane was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by preparative HPLC (column: Waters Xbridge C18 150*50 mm*10 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; gradient: 30%-60% acetonitrile, 10 minutes) to obtain compound D12-M. 1 H NMR (400 MHz, DMSO-d6) δppm: 7.55-8.38 (m, 13H) 7.11-7.40 (m, 9H) 6.78-6.97 (m, 4H) 5.21 (d, J = 3.18 Hz, 4H) 4.97 (dd, J = 11.25, 3.18 Hz, 4H) 4.49 (d, J = 8.44 Hz, 4H) 4.02 (s, 12H) 3.81-3.93 (m, 7H) 3.65-3.77 (m, 12H) 2.84-3.25 (m, 33H) 2.49-2.50 (m, 3H) 2.44 (br d, J = 19.20 Hz, 6H) 2.17-2.30 (m, 3H) 2.10 (s, 12H) 2.01-2.08 (m, 14H) 1.99 (s, 12H) 1.89 (s, 12H) 1.77 (s, 12H) 1.54-1.66 (m, 8H) 1.46 (br s, 20H) 1.25 (br s, 10H).
实施例7
Example 7
步骤A:Step A:
将化合物6-2(3克,7.33毫摩尔)溶于四氢呋喃(30毫升)溶液，依次加入N,N-二异丙基乙胺(1.14克,8.79毫摩尔,1.53毫升)和2-琥珀酰亚胺基-1,1,3,3-四甲基脲四氟硼酸酯(2.43克,8.06毫摩尔)。反应液在25℃下反应12小时。反应液加水(100毫升)稀释，过滤得到粗品。粗品通过硅胶柱过柱纯化(淋洗剂：石油醚：乙酸乙酯＝2:1)得化合物7-1。Compound 6-2 (3 g, 7.33 mmol) was dissolved in tetrahydrofuran (30 ml) solution, and N,N-diisopropylethylamine (1.14 g, 8.79 mmol, 1.53 ml) and 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (2.43 g, 8.06 mmol) were added in sequence. The reaction solution was reacted at 25°C for 12 hours. The reaction solution was diluted with water (100 ml) and filtered to obtain a crude product. The crude product was purified by silica gel column (eluent: petroleum ether: ethyl acetate = 2:1) to obtain compound 7-1.
步骤B:Step B:
将化合物7-1(2.86克,5.65毫摩尔)溶于二氯甲烷(30毫升)溶液，加入化合物7-2(1.44克,9.03毫摩尔)。反应液在25℃下反应12小时。反应液加水(200毫升)稀释，二氯甲烷(100毫升*2)萃取，无水硫酸钠干燥，过滤，减压浓缩得粗品。粗品通过制备级HPLC纯化(柱子:Waters Xbridge C18 150*50毫米*10微米；流动相:[水(碳酸氢铵)-乙腈]；梯度:40％-70％乙腈，10分钟)得化合物7-3。Compound 7-1 (2.86 g, 5.65 mmol) was dissolved in dichloromethane (30 ml) solution, and compound 7-2 (1.44 g, 9.03 mmol) was added. The reaction solution was reacted at 25°C for 12 hours. The reaction solution was diluted with water (200 ml), extracted with dichloromethane (100 ml*2), dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by preparative HPLC (column: Waters Xbridge C18 150*50 mm*10 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; gradient: 40%-70% acetonitrile, 10 minutes) to obtain compound 7-3.
步骤C:Step C:
将化合物7-3(2克,3.63毫摩尔)溶于二氯甲烷(20毫升)溶液，依次加入4A分子筛(2克)，三乙烯二胺(488.84毫克,4.36毫摩尔)和4,4-二甲氧基三苯甲基氯(1.35克,3.99毫摩尔)。反应液在25℃下 反应16小时。反应液加水(200毫升)稀释，二氯甲烷(100毫升*2)萃取，无水硫酸钠干燥，过滤，减压浓缩得粗品，粗品通过制备级HPLC纯化(柱子:Kromasil Eternity XT 250*80毫米*10微米；流动相:[水(氨水)-乙腈]；梯度:60％-90％乙腈，25分钟)得到化合物7-4。Compound 7-3 (2 g, 3.63 mmol) was dissolved in dichloromethane (20 ml) solution, and 4A molecular sieves (2 g), triethylenediamine (488.84 mg, 4.36 mmol) and 4,4-dimethoxytrityl chloride (1.35 g, 3.99 mmol) were added in sequence. The reaction solution was heated at 25 °C. The 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 μm; mobile phase: [water (ammonia water)-acetonitrile]; gradient: 60%-90% acetonitrile, 25 minutes) to obtain compound 7-4.
步骤D:Step D:
将化合物7-4(1.78克,2.09毫摩尔)，二乙胺(7.90克,108.00毫摩尔,11.13毫升)溶于二氯甲烷(15毫升)溶液，反应液在25℃下反应12小时，检测反应完成。减压浓缩得化合物7-5。Compound 7-4 (1.78 g, 2.09 mmol) and diethylamine (7.90 g, 108.00 mmol, 11.13 ml) were dissolved in dichloromethane (15 ml) solution, and the reaction solution was reacted at 25°C for 12 hours, and the reaction was detected to be complete. The solution was concentrated under reduced pressure to obtain compound 7-5.
步骤E:Step E:
氮气保护，将化合物4-10(1.5克,624.35微摩尔,1当量)溶在二氯甲烷溶液(17毫升)中，25-30摄氏度下，依次加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(179.53毫克，936.53微摩尔)，1-羟基-7-氮杂苯并三唑(127.47毫克，936.53微摩尔)，N,N-二异丙基乙胺(1.87毫摩尔，326.25微升)，混合溶液在25-30摄氏度下搅拌半小时，再加入化合物7-5(472.65毫克，749.22微摩尔)，然后在25-30摄氏度继续搅拌16小时，反应结束后，在40-45摄氏度下浓缩除掉二氯甲烷，通过制备级HPLC纯化(柱子:Waters Xbridge C18 150*50毫米*10微米；流动相:[水(碳酸氢铵)-乙腈]；梯度:34％-64％乙腈，10分钟)得化合物7-6。Under nitrogen protection, compound 4-10 (1.5 g, 624.35 μmol, 1 equivalent) was dissolved in dichloromethane solution (17 ml), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (179.53 mg, 936.53 μmol), 1-hydroxy-7-azabenzotriazole (127.47 mg, 936.53 μmol), N,N-diisopropylethylamine (1.87 mmol, 326.25 μl) were added in sequence at 25-30 degrees Celsius. The mixed solution was stirred at 25 The mixture was stirred at -30 degrees Celsius for half an hour, and compound 7-5 (472.65 mg, 749.22 μmol) was added, and then stirring was continued at 25-30 degrees Celsius for 16 hours. After the reaction was completed, dichloromethane was removed by concentration at 40-45 degrees Celsius, and compound 7-6 was obtained by purification by preparative HPLC (column: Waters Xbridge C18 150*50 mm*10 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; gradient: 34%-64% acetonitrile, 10 minutes).
步骤F:Step F:
氮气保护，将化合物7-6(570毫克,184.84微摩尔)，丁二酸酐(83.24毫克,831.77微摩尔)，4-二甲氨基吡啶(22.58毫克,184.84微摩尔)和三乙胺(924.19微摩尔,128.63微升)混合在二氯甲烷(5毫升)溶液中，氮气置换3次。氮气保护，反应液25摄氏度下搅拌反应16小时。检测反应完成。减压浓缩除去二氯甲烷得粗品。粗品通过制备级HPLC纯化(柱子:Waters Xbridge C18 150*50毫米*10微米；流动相:[水(碳酸氢铵)-乙腈]；梯度:23％-53％乙腈，10分钟)得化合物D13-M。1H NMR(400MHz,DMSO-d6)δppm7.65-8.35(m,13H)7.15-7.42(m,9H)6.82-6.94(m,4H)5.21(d,J＝3.18Hz,4H)4.97(dd,J＝11.25,3.30Hz,4H)4.49(d,J＝8.44Hz,4H)4.02(s,12H)3.81-3.95(m,7H)3.67-3.77(m,12H)2.89-3.25(m,33H)2.49-2.50(m,4H)2.29-2.44(m,6H)2.10(s,12H)2.06(br d,J＝13.20Hz,14H)1.99(s,12H)1.89(s,12H)1.77(s,12H)1.54-1.69(m,9H)1.46(br s,21H)1.24(br d,J＝6.60Hz,10H)1.09(br d,J＝10.15Hz,3H)0.97(br d,J＝2.93Hz,3H)。Under nitrogen protection, compound 7-6 (570 mg, 184.84 μmol), succinic anhydride (83.24 mg, 831.77 μmol), 4-dimethylaminopyridine (22.58 mg, 184.84 μmol) and triethylamine (924.19 μmol, 128.63 μl) were mixed in a dichloromethane (5 ml) solution and replaced with nitrogen three times. Under nitrogen protection, the reaction solution was stirred at 25 degrees Celsius for 16 hours. The reaction was detected to be complete. The dichloromethane was removed by vacuum concentration to obtain a crude product. The crude product was purified by preparative HPLC (column: Waters Xbridge C18 150*50 mm*10 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; gradient: 23%-53% acetonitrile, 10 minutes) to obtain compound D13-M. 1 H NMR (400 MHz, DMSO-d6) δppm7.65-8.35 (m, 13H)7.15-7.42 (m, 9H)6.82-6.94 (m, 4H)5.21 (d, J＝3.18 Hz, 4H)4.97 (dd, J＝11.25,3.30 Hz, 4H)4.49 (d, J＝8.44 Hz, 4H)4.02 (s, 12H)3.81-3.95 (m, 7H)3.67-3.77 (m, 12H)2.89-3.25 (m, 33H)2.49-2.50 (m, 4H)2.29-2.44 (m, 6H)2.10 (s, 12H)2.06 (br d, J = 13.20 Hz, 14H) 1.99 (s, 12H) 1.89 (s, 12H) 1.77 (s, 12H) 1.54-1.69 (m, 9H) 1.46 (br s, 21H) 1.24 (br d, J = 6.60 Hz, 10H) 1.09 (br d, J = 10.15 Hz, 3H) 0.97 (br d, J = 2.93 Hz, 3H).
实验例1：人原代肝细胞自由摄取实验-靶基因AGTExperimental Example 1: Free uptake experiment of human primary hepatocytes - target gene AGT
1.实验原理：1. Experimental principle:
GalNAc可以被肝细胞表面特异性ASGPR识别，并通过胞吞作用将与其缀合的siRNA摄取进入肝细胞中，从而实现GalNAc-siRNA对靶基因mRNA水平的下调。 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.
2.实验材料：2. Experimental Materials:
人原代肝细胞(BioIVT)，96Kit(12)(QIAGEN-74182)，FastKing RT Kit(With gDNase)(Tiangen-KR116-02)，AceQ Universal U+Probe Master Mix V2(Vazyme-Q513-03)，TaqMan Gene Expression Assay(GAPDH,Thermo,Assay ID-Hs02786624_g1)，TaqMan Gene Expression Assay(AGT,Thermo,Assay ID-Hs01586213_m1)。Human primary hepatocytes (BioIVT), 96Kit (12) (QIAGEN-74182), FastKing RT Kit (With gDNase) (Tiangen-KR116-02), AceQ Universal U+Probe Master Mix V2 (Vazyme-Q513-03), TaqMan Gene Expression Assay (GAPDH, Thermo, Assay ID-Hs02786624_g1), TaqMan Gene Expression Assay (AGT, Thermo, Assay ID-Hs01586213_m1).
3.实验方法：3. Experimental methods:
用PBS溶液将本发明缀合物稀释至待测浓度的10倍。转移10μL siRNA到96孔板中。将人原代肝细胞解冻并转移到胶原包被的96孔板中，最终的细胞密度为5.4×105细胞/100μL/孔。本发明缀合物测试10个浓度点，最高浓度为500nM，4倍稀释，2复孔。The conjugate of the present invention was diluted to 10 times the concentration to be tested with PBS solution. 10 μL 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 μL/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.
细胞在37摄氏度，5％CO2条件下与本发明缀合物孵育48小时，孵育完成后，将细胞裂解，用96Kit(QIAGEN-74182)提取所有的RNA，并用FastKing RT Kit(With gDNase)(Tiangen-KR116-02)逆转录获得cDNA。用qPCR检测AGT mRNA表达水平。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.
4.实验结果：实验结果见表3，表4和表54. Experimental results: The experimental results are shown in Table 3, Table 4 and Table 5
表3本发明缀合物在人原代肝细胞中自由摄取实验结果
Table 3 Results of the free uptake experiment of the conjugates of the present invention in human primary hepatocytes
表4本发明缀合物在人原代肝细胞中自由摄取实验结果
Table 4 Results of the free uptake experiment of the conjugates of the present invention in human primary hepatocytes
表5本发明缀合物在人原代肝细胞中自由摄取实验结果
Table 5 Results of the free uptake experiment of the conjugates of the present invention in human primary hepatocytes
注：表3表4表5为不同批次测试，人原代肝细胞的来源不同，活性受此影响较大。 Note: 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.
结论：本发明缀合物在在人原代肝细胞中均展现出对AGT mRNA较高的抑制活性，证明本发明的GalNAc递送系统均对siRNA序列具有良好的肝靶向递送能力。Conclusion: 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.
实验例2：人原代肝细胞自由摄取实验靶基因补体C5Experimental Example 2: Free uptake of target gene complement C5 by human primary hepatocytes
1.实验原理：1. Experimental principle:
GalNAc可以被肝细胞表面特异性ASGPR识别，并通过胞吞作用将与其缀合的siRNA摄取进入肝细胞中，从而实现GalNAc-siRNA对靶基因mRNA水平的下调。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.
2.实验材料：2. Experimental Materials:
人原代肝细胞(BioIVT)，本实验使用的主要试剂包括HiScript III RT SuperMix for qPCR(Vazyme，货号R323-01)，RNA提取试剂盒(Qiagen，货号74182)，AceQ Universal U+Probe Master Mix V2(Vazyme，货号Q513-03)，TaqMan Gene Expression Assay(GAPDH,Thermo,Assay ID-Hs02786624_g1)和TaqMan Gene Expression Assay(C5,Thermo,Assay ID-Hs00156197_m1)。Human primary hepatocytes (BioIVT). The main reagents used in this experiment included HiScript III RT SuperMix for qPCR (Vazyme, Catalog No. R323-01), RNA extraction kit (Qiagen, Catalog No. 74182), AceQ Universal U+Probe Master Mix V2 (Vazyme, Catalog No. Q513-03), TaqMan Gene Expression Assay (GAPDH, Thermo, Assay ID-Hs02786624_g1) and TaqMan Gene Expression Assay (C5, Thermo, Assay ID-Hs00156197_m1).
3.实验方法：3. Experimental methods:
第一天，用Nuclease-free water稀释siRNA到5000nM为起始点，4倍梯度稀释，共10个浓度点，然后取10μL到胶原包被过的96孔细胞板。取1支人原代肝细胞转移至预热好的InvitroGRO CP Medium完全培养基中，以每孔54000个细胞的密度(90μL/孔)接种到96孔细胞板中，每孔最终的培养液为100μL。本发明缀合物测试10个浓度点，最高浓度为500nM，4倍稀释，2复孔。细胞置于5％CO2、37摄氏度孵箱中培养48小时。孵育完成后，将细胞裂解，使用获得裂解液，参照RNA提取试剂盒(Qiagen，74182)说明书提取RNA，参照HiScript III RT SuperMix for qPCR(Vazyme，货号R323-01)说明书将RNA反转录为cDNA后进行qPCR检测C5mRNA的表达水平。On the first day, 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 10μL 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 (90μL/well), and the final culture medium per well was 100μL. 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. After the incubation, the cells were lysed, and the lysate was used to extract RNA according to the instructions of the 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.
4.实验结果：实验结果见表64. Experimental results: The experimental results are shown in Table 6
表6本发明缀合物在人原代肝细胞中细胞自由摄取实验结果
Table 6 Results of the free cell uptake experiment of the conjugates of the present invention in human primary hepatocytes
结论：本发明缀合物在人原代肝细胞中均展现出对C5mRNA较高的抑制活性，证明递送系统均对siRNA序列具有良好的肝靶向递送能力。Conclusion: The 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.
实验例3：人原代肝细胞自由摄取实验靶基因ANGPTL3Experimental Example 3: Human primary hepatocytes free uptake experiment target gene ANGPTL3
1.实验原理： 1. Experimental principle:
GalNAc可以被肝细胞表面特异性ASGPR识别，并通过胞吞作用将与其缀合的siRNA摄取进入肝细胞中，从而实现GalNAc-siRNA对靶基因mRNA水平的下调。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.
2.实验材料：2. Experimental Materials:
本实验使用的主要试剂包括FastQuant RT Kit(with gDNase)(TianGen，货号KR106-02)，RNA提取试剂盒(Qiagen，货号74182)，FastStart Universal Probe Master(Rox)(Roche，货号04914058001)，TaqMan Gene Expression Assay(GAPDH,Thermo,Assay ID-Hs02786624_g1)和TaqMan Gene Expression Assay(ANGPTL3,Thermo,Assay ID-Hs00205581_m1)。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).
3.实验方法：3. Experimental methods:
第一天，用Nuclease-free water稀释siRNA到5000nM为起始点，4倍梯度稀释，共10个浓度点，然后取10μL到胶原包被过的96孔细胞板。取1支人原代肝细胞转移至预热好的InvitroGRO CP Medium完全培养基中，以每孔54000个细胞的密度(90μL/孔)接种到96孔板中，每孔最终的培养液为100μL。本发明缀合物测试10个浓度点，最高浓度为500nM，4倍稀释，2复孔。细胞置于5％CO2、37摄氏度孵箱中培养48小时。On the first day, 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 10μL 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 (90μL/well), and the final culture medium per well was 100μL. 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.
参照RNA提取试剂盒(Qiagen，74182)说明书提取RNA，参照FastQuant RT Kit(with gDNase)(TianGen，货号KR106-02)说明书将RNA反转录为cDNA后使用qPCR检测ANGPTL3mRNA的水平。RNA was extracted according to the instructions of the RNA extraction kit (Qiagen, 74182), and RNA was reverse transcribed into cDNA according to the instructions of the FastQuant RT Kit (with gDNase) (TianGen, catalog number KR106-02), and the level of ANGPTL3 mRNA was detected by qPCR.
4.实验结果：实验结果见表74. Experimental results: The experimental results are shown in Table 7
表7本发明缀合物在人原代肝细胞中自由摄取实验结果
Table 7 Results of the free uptake experiment of the conjugates of the present invention in human primary hepatocytes
结论：本发明缀合物在人原代肝细胞中中均展现出对ANGPTL3mRNA较高的抑制活性，证明本发明的GalNAc递送系统均对siRNA序列具有良好的肝靶向递送能力。Conclusion: The conjugates of the present invention all exhibited high inhibitory activity against ANGPTL3 mRNA in human primary hepatocytes, demonstrating that the GalNAc delivery system of the present invention has good liver-targeted delivery capability for siRNA sequences.
实验例4：小鼠体内活性测试靶基因AGTExperimental Example 4: Activity test target gene AGT in mice
1.实验原理：1. Experimental principle:
通过高压尾静脉快速注射含目的基因重组质粒2-pcDNA-CMV-AGT的生理盐水，实现目的基因AGT在小鼠肝脏中的高效表达，从而评估待测缀合物进入小鼠体内后对目的基因的敲低效果。By rapidly injecting physiological saline containing the target gene recombinant plasmid 2-pcDNA-CMV-AGT through high-pressure tail vein, efficient expression of the target gene AGT in the mouse liver is achieved, thereby evaluating the knockdown effect of the test conjugate on the target gene after entering the mouse body.
2.实验材料：2. Experimental Materials:
2-pcDNA-CMV-AGT质粒，BALB/c小鼠，PBS(磷酸缓冲液)，本发明缀合物。2-pcDNA-CMV-AGT plasmid, BALB/c mice, PBS (phosphate buffered saline), conjugate of the present invention.
3.实验方法：3. Experimental methods:
订购6-8周龄的BALB/c雌性小鼠，小鼠到达动物房后适应检疫一周。 Order 6-8 week old BALB/c female mice and acclimate to quarantine for one week after the mice arrive at the animal house.
第0天，按照体重数据将小鼠随机分组，分组后所有小鼠皮下注射给药，单次给药，给药体积为10mL/kg，第1组小鼠给PBS；其他组小鼠给缀合物。On day 0, 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.
给药后第3天，所有小鼠在5秒内经尾静脉注射含2-pcDNA-CMV-AGT质粒的生理盐水，注射体积(mL)＝小鼠体重(g)×8％，每只小鼠注射质粒的质量为10μg。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 μg.
给药后第4天，所有组小鼠经CO2吸入安乐死，每只小鼠分别收集2份肝脏样品。肝脏样品经RNAlater4℃过夜处理，后移除RNAlater，保存于-80℃用于检测AGT基因表达水平。On the 4th day after administration, 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.
4.实验结果：实验结果见表8和94. Experimental results: The experimental results are shown in Tables 8 and 9
表8本发明缀合物体内活性测试实验结果(每组4只小鼠)
Table 8 In vivo activity test results of the conjugates of the present invention (4 mice per group)
注：“AGT mRNA下调百分比”是指给药组小鼠肝脏中AGT-mRNA相对PBS空白组下调百分比Note: "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
表9本发明缀合物体内活性测试实验结果(每组3只小鼠)
Table 9 Results of in vivo activity test of the conjugates of the present invention (3 mice per group)
注：“AGT mRNA下调百分比”是指给药组小鼠肝脏中AGT-mRNA相对PBS空白组下调百分比Note: "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
结论：本发明缀合物在AGT-HDI小鼠模型中均可以产生对肝脏中AGT-mRNA的下调，且下调活性展现出了剂量依赖性。由于本此测试为肝脏内的mRNA水平，因此，可以证明本发明的GalNAc递送系统可以有效的进行序列的肝靶向递送。Conclusion: 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.
实验例5：小鼠体内活性测试靶基因AGTExperimental Example 5: Activity test target gene AGT in mice
1.实验原理： 1. Experimental principle:
通过高压尾静脉快速注射含目的基因重组质粒2-pcDNA-CMV-AGT的生理盐水，实现目的基因AGT在小鼠肝脏中的高效表达，从而评估待测缀合物进入小鼠体内后对目的基因的敲低效果。By rapidly injecting physiological saline containing the target gene recombinant plasmid 2-pcDNA-CMV-AGT through high-pressure tail vein, efficient expression of the target gene AGT in the mouse liver is achieved, thereby evaluating the knockdown effect of the test conjugate on the target gene after entering the mouse body.
2.实验材料：2. Experimental Materials:
2-pcDNA-CMV-AGT质粒，BALB/c小鼠，PBS(磷酸缓冲液)，本发明缀合物。2-pcDNA-CMV-AGT plasmid, BALB/c mice, PBS (phosphate buffered saline), conjugate of the present invention.
3.实验方法：3. Experimental methods:
订购6-8周龄的BALB/c雌性小鼠，小鼠到达动物房后适应检疫一周。以小鼠高压尾静脉当天定义为实验第0天，往前一天为第-1天，往后一天为第1天，以此类推。Order 6-8 week old BALB/c female mice, and acclimate them to quarantine for one week after they arrive at the animal room. The day of high pressure tail vein injection is defined as day 0 of the experiment, the day before is day -1, the day after is day 1, and so on.
第-14天，按照体重数据将小鼠随机分组，每组4只，分组后3组小鼠皮下注射给药，单次给药，给药体积为10mL/kg，第1组小鼠给PBS；其他2组小鼠给缀合物，剩余组小鼠常规饲养，不进行给药操作。On day -14, the 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.
第-3天，将剩余组小鼠给缀合物。On day -3, the remaining groups of mice were given the conjugate.
第0天，所有小鼠在5秒内经尾静脉注射含2-pcDNA-CMV-AGT质粒的生理盐水，(注射体积(mL)＝小鼠体重(g)×8％)，每只小鼠注射质粒的质量为10μg。On day 0, all mice were injected with physiological saline containing 2-pcDNA-CMV-AGT plasmid via tail vein within 5 seconds (injection volume (mL) = mouse body weight (g) × 8%), and the mass of the injected plasmid for each mouse was 10 μg.
第1天，所有组小鼠经CO2吸入安乐死，每只小鼠分别收集2份肝脏样品。肝脏样品经RNAlater 4℃过夜处理，后移除RNAlater，保存于-80℃用于检测AGT基因表达水平。On the first day, 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.
4.实验结果：实验结果见表104. Experimental results: The experimental results are shown in Table 10
表10本发明缀合物体内活性测试实验结果
Table 10 Results of in vivo activity test of the conjugates of the present invention
注：“AGT mRNA下调百分比”是指给药组小鼠肝脏AGT-mRNA相对PBS空白组下调百分比Note: "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
结论：在测试时间内，缀合物均在AGT-HDI小鼠模型中均对AGT mRNA产生下调。由于本测试为肝脏内的mRNA水平，因此可以证明本发明的GalNAc递送系统可以有效的进行序列的肝靶向递送。Conclusion: Within the test time, the conjugates all down-regulated AGT mRNA in the AGT-HDI mouse model. Since this test is for 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.
实验例6：小鼠体内活性测试靶基因补体C5Experimental Example 6: In vivo activity test of target gene complement C5 in mice
1.实验原理： 1. Experimental principle:
C57BL/6小鼠表达补体C5，待测样本经皮下注射进入小鼠体内可达到肝脏，进而抑制肝细胞中靶基因C5的表达。通过测定给药后不同时间点小鼠血浆中靶蛋白C5的浓度，可评估待测样品的体内活性和长效性。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. 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.
2.实验材料：2. Experimental Materials:
C57BL/6小鼠，PBS(磷酸缓冲液)，本发明缀合物。C57BL/6 mice, PBS (phosphate buffered saline), conjugate of the present invention.
3.实验方法：3. Experimental methods:
在siRNA给药前2天(Day-2)，采集C57BL/6(雌性，7周龄)小鼠血浆样本，通过ELISA方法(Abcam)测定小鼠血浆中C5蛋白水平，根据测试结果挑选入组小鼠后进行随机分组，每组5只。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.
所有动物根据体积计算给药量，在第0天(Day 0)采用皮下注射方式单次给药，siRNA给药体积为10mL/kg。在给药后第7天，14天，21天和28天收集小鼠血浆，通过ELISA方法测定小鼠血浆中C5蛋白的浓度，以各组小鼠血浆中C5蛋白的相对表达水平评估不同siRNA的体内有效性。C5蛋白的相对表达水平按照如下公式计算：C5相对表达水平＝不同时间点给药组C5浓度/Day-2给药组血浆中C5浓度×100％。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. The relative expression level of C5 protein was calculated according to the following formula: C5 relative expression level = C5 concentration in the administration group at different time points/C5 concentration in the plasma of the administration group on Day-2 × 100%.
4.实验结果：实验结果见表11和表12。4. Experimental results: The experimental results are shown in Tables 11 and 12.
表11本发明缀合物体内活性测试实验结果
Table 11 Results of in vivo activity test of the conjugates of the present invention
注：“C5蛋白下调百分比”是指各给药组不同时间点小鼠血浆中C5蛋白浓度相对于各组小鼠给药前(Day-2)血浆C5蛋白下降的百分比Note: "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)
表12本发明缀合物体内活性测试实验结果

Table 12 Results of in vivo activity test of the conjugates of the present invention

注：“C5蛋白相对表达水平”是指各组小鼠给药后不同时间点血浆C5蛋白浓度相对于每组给药前第2天(Day-2)的百分比Note: "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.
结论：本实验展现出本发明缀合物对C5蛋白的持续抑制和抑制效果的剂量相关性，具有优秀的有效性和长效性，同时展示出对C5蛋白良好的持续抑制和抑制效果的剂量相关性，由于补体C5mRNA具有肝源性的特征，因此本实验结果展示出GalNAc递送系统良好的肝靶向递送能力。 Conclusion: 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.

Claims (13)

  1. 式(V)所示的缀合基团，
    The conjugated group represented by formula (V)
    其中，in,
    L1选自 L 1 is selected from
    L2选自 L 2 is selected from
    选自 Selected from
    n选自0和1；n is selected from 0 and 1;
    t选自2、3、4、5、6和7。t is selected from 2, 3, 4, 5, 6 and 7.
  2. 根据权利要求1所述的缀合基团，其中，缀合基团选自(D02)，
    The conjugated group according to claim 1, wherein the conjugated group is selected from (D02),
  3. 根据权利要求1所述的缀合基团，其中，缀合基团选自(D02-1-A)、(D02-1-B)、(D02-1-C)、(D03)、(D12)和(D13)，


    The conjugated group according to claim 1, wherein the conjugated group is selected from (D02-1-A), (D02-1-B), (D02-1-C), (D03), (D12) and (D13),


  4. 一种缀合物或其药学上可接受的盐，其中，所述缀合物选自如权利要求1-3任意一项所述的缀合基团通过磷酸二酯键或硫代磷酸二酯键与寡聚核苷酸连接形成的化合物。A conjugate or a pharmaceutically acceptable salt thereof, wherein the conjugate is selected from a compound formed by connecting the conjugated group as described in any one of claims 1 to 3 to an oligonucleotide through a phosphodiester bond or a thiophosphodiester bond.
  5. 根据权利要求4所述的缀合物或其药学上可接受的盐，其中，所述寡聚核苷酸选自RNAi试剂和ASO试剂。The conjugate according to claim 4 or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide is selected from an RNAi agent and an ASO agent.
  6. 根据权利要求5所述的缀合物或其药学上可接受的盐，其中，所述RNAi试剂选自单链寡聚核苷酸和双链寡聚核苷酸。The conjugate according to claim 5 or a pharmaceutically acceptable salt thereof, wherein the RNAi agent is selected from a single-stranded oligonucleotide and a double-stranded oligonucleotide.
  7. 根据权利要求6所述的缀合物或其药学上可接受的盐，其中，所述单链寡聚核苷酸选自单链反义寡聚核苷酸。The conjugate according to claim 6 or a pharmaceutically acceptable salt thereof, wherein the single-stranded oligonucleotide is selected from a single-stranded antisense oligonucleotide.
  8. 根据权利要求6所述的缀合物或其药学上可接受的盐，其中，所述双链寡聚核苷酸选自双链siRNA。The conjugate according to claim 6 or a pharmaceutically acceptable salt thereof, wherein the double-stranded oligonucleotide is selected from double-stranded siRNA.
  9. 根据权利要求4-8任意一项所述的缀合物或其药学上可接受的盐，其中，所述的寡聚核苷酸的核苷酸任选被修饰。The conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 4 to 8, wherein the nucleotides of the oligonucleotide are optionally modified.
  10. 根据权利要求1-3任意一项所述的缀合基团作为递送平台的应用，其中，所述递送平台是用于增强治疗剂与特定靶标位置的结合。Use of the conjugated group according to any one of claims 1 to 3 as a delivery platform, wherein the delivery platform is used to enhance the binding of a therapeutic agent to a specific target location.
  11. 一种用于制备权利要求1所述缀合基团的中间体化合物，其结构如式(I-M)、(V-M12)和(V-M13)所示，
    An intermediate compound for preparing the conjugated group according to claim 1, whose structure is shown in formula (IM), (V-M12) and (V-M13),
    L1、L2、t、n如权利要求1所定义。 L 1 , L 2 , t, and n are as defined in claim 1 .
  12. 一种用于制备权利要求2所述缀合基团的中间体化合物，其结构如式(D02-M)所示，
    An intermediate compound for preparing the conjugated group according to claim 2, whose structure is shown in formula (D02-M),
  13. 一种用于制备权利要求3所述缀合基团的中间体化合物，其结构分别如式(D02-1-A-M)、(D02-1-B-M)、(D02-1-C-M)、(D03-M)、(D12-M)和(D13-M)所示，


    An intermediate compound for preparing the conjugated group according to claim 3, whose structures are shown in formulas (D02-1-AM), (D02-1-BM), (D02-1-CM), (D03-M), (D12-M) and (D13-M), respectively.


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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RO104721B1 (en) * 1989-07-10 1994-12-12 Inst Medicina Farmacie Hydroxi-alkyl-homo-piperazidine of ruthozide derivatives and preparation method therefore
WO2013166155A1 (en) * 2012-05-02 2013-11-07 Merck Sharp & Dohme Corp. Novel tetragalnac and peptide containing conjugates and methods for delivery of oligonucleotides
WO2021037205A1 (en) * 2019-08-29 2021-03-04 苏州瑞博生物技术股份有限公司 Compound and drug conjugate, and preparation method and use thereof
WO2022098990A1 (en) * 2020-11-06 2022-05-12 Arbutus Biopharma Corporation Targeted conjugates comprising modified sirna
CN114805113A (en) * 2022-01-22 2022-07-29 苏州天澜生物材料科技有限公司 Safe and efficient degradable lipid nanoparticle as well as preparation method and application thereof
WO2023143374A1 (en) * 2022-01-30 2023-08-03 成都凌泰氪生物技术有限公司 Ligand, method for preparing same, and use thereof
CN116655715A (en) * 2023-07-27 2023-08-29 北京炫景瑞医药科技有限公司 GalNAc derivative, conjugate, composition and application thereof

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RO104721B1 (en) * 1989-07-10 1994-12-12 Inst Medicina Farmacie Hydroxi-alkyl-homo-piperazidine of ruthozide derivatives and preparation method therefore
WO2013166155A1 (en) * 2012-05-02 2013-11-07 Merck Sharp & Dohme Corp. Novel tetragalnac and peptide containing conjugates and methods for delivery of oligonucleotides
WO2021037205A1 (en) * 2019-08-29 2021-03-04 苏州瑞博生物技术股份有限公司 Compound and drug conjugate, and preparation method and use thereof
WO2022098990A1 (en) * 2020-11-06 2022-05-12 Arbutus Biopharma Corporation Targeted conjugates comprising modified sirna
CN114805113A (en) * 2022-01-22 2022-07-29 苏州天澜生物材料科技有限公司 Safe and efficient degradable lipid nanoparticle as well as preparation method and application thereof
WO2023143374A1 (en) * 2022-01-30 2023-08-03 成都凌泰氪生物技术有限公司 Ligand, method for preparing same, and use thereof
CN116655715A (en) * 2023-07-27 2023-08-29 北京炫景瑞医药科技有限公司 GalNAc derivative, conjugate, composition and application thereof

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