WO2024104386A1 - 一类含七元杂环的三齿缀合基团 - Google Patents

一类含七元杂环的三齿缀合基团 Download PDF

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WO2024104386A1
WO2024104386A1 PCT/CN2023/131818 CN2023131818W WO2024104386A1 WO 2024104386 A1 WO2024104386 A1 WO 2024104386A1 CN 2023131818 W CN2023131818 W CN 2023131818W WO 2024104386 A1 WO2024104386 A1 WO 2024104386A1
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compound
present
mmol
solution
oligonucleotide
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French (fr)
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陆剑宇
胡彦宾
宋雨晨
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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical

Definitions

  • the present invention relates to the field of oligonucleotides, and in particular to a tridentate conjugated group containing a seven-membered heterocycle 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 patent of the present invention provides a three-ligand GalNAc molecule with a rigid seven-membered ring bridging point, which greatly reduces the difficulty of synthesis and reduces the cost while maintaining activity.
  • the first aspect of the present invention provides a conjugated group represented by formula (IV),
  • L 1 is selected from
  • L 2 is selected from wherein Ring A is selected from C 5-6 cycloalkyl and 5-6 membered heterocycloalkyl;
  • n is selected from 0 and 1;
  • n 0, 1, 2 and 3;
  • t is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6 and 7.
  • the above L2 is selected from Other variables are as defined in the present invention.
  • n is selected from 0, and other variables are as defined in the present invention.
  • n is selected from 1, and other variables are as defined in the present invention.
  • the above t is selected from 0, and other variables are as defined in the present invention.
  • the above t is selected from 1, and other variables are as defined in the present invention.
  • the above t is selected from 2, and other variables are as defined in the present invention.
  • the above t is selected from 3, and other variables are as defined in the present invention.
  • the above t is selected from 4, and other variables are as defined in the present invention.
  • the above t is selected from 5, and other variables are as defined in the present invention.
  • the above t is selected from 6, and other variables are as defined in the present invention.
  • the above t is selected from 7, and other variables are as defined in the present invention.
  • the above m is selected from 0, and other variables are as defined in the present invention.
  • the above m is selected from 1, and other variables are as defined in the present invention.
  • the above m is selected from 2, and other variables are as defined in the present invention.
  • the above m is selected from 3, and other variables are as defined in the present invention.
  • the present invention also provides a conjugated group represented by formula (I),
  • L 1 is selected from
  • L 2 is selected from wherein Ring A is selected from C 5-6 cycloalkyl and 5-6 membered heterocycloalkyl;
  • n is selected from 0 and 1;
  • n 0, 1, 2 and 3;
  • t is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6 and 7.
  • the above L2 is selected from Other variables are as defined in the present invention.
  • n is selected from 0, and other variables are as defined in the present invention.
  • n is selected from 1, and other variables are as defined in the present invention.
  • the above t is selected from 0, and other variables are as defined in the present invention.
  • the above t is selected from 1, and other variables are as defined in the present invention.
  • the above t is selected from 2, and other variables are as defined in the present invention.
  • the above t is selected from 3, and other variables are as defined in the present invention.
  • the above t is selected from 4, and other variables are as defined in the present invention.
  • the above t is selected from 5, and other variables are as defined in the present invention.
  • the above t is selected from 6, and other variables are as defined in the present invention.
  • the above t is selected from 7, and other variables are as defined in the present invention.
  • the above m is selected from 0, and other variables are as defined in the present invention.
  • the above m is selected from 1, and other variables are as defined in the present invention.
  • the above m is selected from 2, and other variables are as defined in the present invention.
  • the above m is selected from 3, and other variables are as defined in the present invention.
  • the conjugated group is selected from (D08), (D05), (D06), (D07), (D10) and (D11),
  • the second aspect of the present invention provides a conjugate or a pharmaceutically acceptable salt thereof, wherein the conjugate is a compound formed by connecting the conjugation group defined in any 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 O- and S- ;
  • L 1 , L 2 , n, m and t are as defined in the present invention.
  • the above-mentioned conjugated group is connected to the oligonucleotide via a phosphodiester bond or a thiophosphodiester bond.
  • X is selected from O- and S- ;
  • L 1 , L 2 , n, m and t are as defined in 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:
  • X is selected from O- and S- ;
  • L 1 , L 2 , n, m and t are as defined in the present invention.
  • the above-mentioned conjugated group is connected to the 3' end of the sense strand of the oligonucleotide via a phosphodiester bond or a phosphorothioate diester bond.
  • the above-mentioned oligonucleotide is selected from RNAi 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. righteous.
  • the double-stranded oligonucleotide is selected from double-stranded siRNA, and other variables are as defined in the present invention.
  • nucleotides of the above-mentioned oligonucleotide are optionally modified, and other variables are as defined in the present invention.
  • the above conjugate is capable of inhibiting or blocking gene expression, and other variables are as defined in the present invention.
  • the above-mentioned genes are selected from AGT, ANGPTL3, PCSK9, XDH, HSD, HAO, APP, TTR, complement proteins, etc., and other variables are as defined in the present invention.
  • 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 refers to enhancing the binding of the therapeutic agent to a specific target location.
  • the fourth aspect of the present invention provides an intermediate compound for preparing the conjugated group defined in any of the above technical solutions, whose structure is shown in formula (IM),
  • R 1 is selected from H or a hydroxyl protecting group, preferably Ac;
  • R2 is selected from hydroxyl protecting groups, preferably DMTr.
  • the intermediate structures are as shown in formulas (D08-M), (D05-M), (D06-M), (D07-M), (D10-M) and (D11-M).
  • the present invention also provides the conjugates shown in Table 1:
  • the present invention also provides the following testing 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 primary human 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. 10 ⁇ L of siRNA was transferred to a 96-well plate. PHH cells were thawed and transplanted into a 96-well plate, with a final cell density of 5.4 ⁇ 10 5 cells/well. The conjugate of the present invention was tested at 10 concentration points, with the highest concentration being 500 nM, and 4-fold dilution.
  • the cells were incubated at 37 °C, 5% CO2 for 48 h, and the cell status was examined under a microscope.
  • the present invention also provides a method for preparing the conjugate:
  • Oligonucleotides were synthesized using the phosphoramidite solid phase synthesis technique. or ) and the intermediate compound corresponding to the conjugated group are synthesized on a solid support made by covalently linking. All phosphoramidite monomers and auxiliary reagents of 2'-modified nucleotides are commercially available reagents. All phosphoramidite monomers are dissolved in anhydrous acetonitrile in sequence from 3' to 5' and molecular sieves are added.
  • ETT 5-ethylthio-1H-tetrazole
  • Synthesis of single-stranded oligonucleotides without conjugated groups Synthesize oligoribonucleotides using phosphoramidite solid phase synthesis technology. All 2'-modified nucleotide phosphoramidite monomers and auxiliary reagents are commercially available reagents. All phosphoramidite monomers are dissolved in anhydrous acetonitrile in sequence from 3' to 5' and molecular sieves are added.
  • ETT 5-ethylthio-1H-tetrazole
  • Oligos 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 oligonucleotides to produce siRNA anneal the single-stranded oligonucleotides with sterile RNase Free H 2 O (RNase-free water). Prepared to 200 ⁇ M. Set up the annealing reaction system as follows: place 10 nmol of the total volume of 100 ⁇ L of the mixture in a 95°C water bath for 10 minutes ( ⁇ 100 nmol requires high temperature for 20 minutes) ⁇ quickly place in a 60°C water bath, cool naturally ⁇ the solution after annealing cannot be stored at high temperature. Complementary chains are formed by combining equimolar single-stranded oligonucleotide solutions.
  • the conjugated group described in 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 cells more efficiently, and has excellent effectiveness and long-term effect on the inhibition of C5 protein.
  • the molecular structure of the GalNAc delivery platform that has been approved or is in the clinical trial stage has problems such as complicated synthesis and complex impurities, and the CMC difficulty is relatively high.
  • the molecule in the patent of the present invention is a three-ligand GalNAc molecule connected by a seven-membered ring, and the molecular rigidity is further enhanced, and it can be easily obtained from commercial raw materials. At the same time, the molecular structure is simple and the structural unit is single. During the research and development process, it was unexpectedly found that the molecule in the present invention has ideal activity, and the synthesis is simpler and the CMC difficulty is reduced.
  • 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.
  • 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 at least partially binds to a coding sequence in a target nucleic acid or target gene expressed in a cell. Complementary. 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 the underlying 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 can include modified nucleotides and/or one or more non-phosphodiester linkages.In some embodiments, the RNAi agents described herein are 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 14 to 30 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.
  • modified nucleotides consist of modified nucleobases, modified sugar rings and natural phosphates; in some embodiments of the present invention, “modified nucleotides” consist of modified nucleobases, modified phosphates and natural sugar rings; in some embodiments of the present invention, “modified nucleotides” consist of natural nucleobases, modified sugar rings and modified phosphates; in some embodiments of the present invention, “modified nucleotides” consist of modified nucleobases, natural sugar rings and natural phosphates; in some embodiments of the present invention, “modified nucleotides” consist of natural nucleobases, modified sugar rings and natural phosphates; in some embodiments of the present invention, “modified nucleotides” consist of natural nucleobases, natural sugar rings and modified phosphates; in some embodiments of the present invention, “modified nucleotides” consist of natural nucleobases, natural sugar rings and modified phosphates; in some embodiments of the present
  • 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 alkyl, alkenyl and alkynyl may be substituted or Unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl.
  • Exemplary suitable modifications include O[( CH2 ) nO ] mCH3 , O( CH2 ) nOCH3 , O( CH2 ) nNH2 , O( CH2 ) nCH3 , O( CH2 ) nONH2 , and O( CH2 ) nON [( CH2 ) nCH3 ) ] 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 is The structure of GNA-C is The structure of GNA-G is GNA- The U structure 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 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 mixtures enriched in enantiomers or diastereomers, 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.
  • all tautomeric forms are also included. For example, tautomers of all phosphate and thiophosphate groups are included.
  • 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., ).
  • 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.
  • 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.
  • 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 on each occurrence is independent.
  • the group may be optionally substituted with up to two R, with each occurrence of R being an independent choice.
  • substituents and/or variations thereof are permissible 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.
  • C 5-6 cycloalkyl means a saturated cyclic hydrocarbon group consisting of 5 to 6 carbon atoms, which is a monocyclic and bicyclic system, and the C 5-6 cycloalkyl includes C 5 and C 6 cycloalkyl, etc.; it can be monovalent, divalent or polyvalent. Examples of C 5-6 cycloalkyl include cyclopentyl and cyclohexyl, etc.
  • the term "5-6 membered heterocycloalkyl" by itself or in combination with other terms refers to a saturated cyclic group consisting of 5 to 6 ring atoms, 1, 2, 3 or 4 of which are heteroatoms independently selected from O, S and N, and the rest are carbon atoms, wherein the nitrogen atom is optionally quaternized, and the nitrogen and sulfur heteroatoms may be optionally oxidized (i.e., NO and S(O) p , p is 1 or 2). It includes monocyclic and bicyclic ring systems, wherein the bicyclic ring system includes spirocyclic, cyclic and bridged rings.
  • heteroatoms may occupy the position where the heterocycloalkyl is connected to the rest of the molecule.
  • the 5-6 membered heterocycloalkyl includes 5-membered and 6-membered heterocycloalkyl.
  • 5-6 membered heterocycloalkyl groups include, but are not limited to, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrothiophenyl (including tetrahydrothiophen-2-yl and tetrahydrothiophen-3-yl, etc.), tetrahydrofuranyl (including tetrahydrofuran-2-yl, etc.), tetrahydropyranyl, piperidinyl (including 1-piperidinyl, 2-piperidinyl and 3-piperidinyl, etc.), piperazinyl (including 1-piperazinyl and 2-piperazinyl, etc.), morpholinyl (including 3-morpholinyl and 4-morpholinyl, etc.), dioxanyl, dithianyl, isoxazolidinyl, isothiazolidinyl, 1,2-oxazinyl, 1,2-thiazinyl, hexahydro
  • hydroxy protecting group refers to a protecting group suitable for preventing side reactions of the hydroxy group.
  • Representative hydroxy protecting groups include, but are not limited to, alkyl groups such as methyl, ethyl and tert-butyl; acyl groups such as alkanoyl (such as acetyl); arylmethyl groups such as benzyl (Bn), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm) and diphenylmethyl (diphenylmethyl, DPM); silyl groups such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS); 4,4'-dimethoxytriphenyl (DMTr) and the like.
  • alkyl groups such as methyl, ethyl and tert-butyl
  • acyl groups such as alkanoyl (such as acetyl)
  • arylmethyl groups such as benzyl (Bn),
  • 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 used in the description of nucleic acid sequences are 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 adverse 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 thereof 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.
  • 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 2-1, which was directly used in the next step.
  • reaction solution was stirred at 25°C for half an hour. After the reaction, 150 ml of water was added and extracted with dichloromethane (100 ml*2). The organic phases were combined and washed with saturated brine (100 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated and purified by silica gel column chromatography to give compound 2-5 (gradient elution, dichloromethane/methanol, 1/0 to 10/1).
  • Triethylamine 53.58 mg, 529.52 ⁇ mol
  • 4-dimethylaminopyridine 32.35 mg, 264.76 ⁇ mol
  • compound 1-16 158.97 mg, 1.59 mmol
  • the reaction solution was stirred at 25°C 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]; gradient: 23%-53% acetonitrile, 10 minutes) to obtain compound D05-M.
  • Triethylamine 46.87 mg, 463.22 ⁇ mol
  • 4-dimethylaminopyridine 28.30 mg, 231.61 ⁇ mol
  • compound 1-16 139.07 mg, 1.39 mmol
  • the reaction solution was stirred at 25°C for 16 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]; gradient: 27%-57% acetonitrile, 10 minutes) to obtain compound D06-M.
  • the crude product was purified by preparative HPLC (column: Waters xbridge 150*25 mm*10 ⁇ m; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; gradient: 25%-55% acetonitrile, 10 minutes) to obtain compound 4-5.
  • Triethylamine (101.67 mg, 1 mmol), 4-dimethylaminopyridine (30.69 mg, 251.18 ⁇ mol), and compound 1-16 (100.55 mg, 1 mmol) were added to a dichloromethane solution (6 ml) of compound 4-7 (600 mg, 251.18 ⁇ mol).
  • the reaction solution was stirred at 25°C for 12 hours.
  • concentration under reduced pressure the mixture was purified by preparative HPLC (column: Waters Xbridge 150*25 mm*5 ⁇ m; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; gradient: 30%-60% acetonitrile, 10 minutes) to obtain compound D07-M.
  • 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.
  • 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°C 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.
  • the conjugates of the present invention showed 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.
  • 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 gradient, with a total of 10 concentration points, and then 10 ⁇ L was taken to the collagen-coated 96-well cell plate.
  • One human primary hepatocyte was transferred to the preheated InvitroGRO CP Medium complete culture medium, and inoculated into the 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°C incubator for 48 hours.
  • the target gene AGT was efficiently expressed in the mouse liver, thereby evaluating the knockdown effect of the test conjugate on the target gene after entering the mouse body.
  • 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 the 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.

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Abstract

本发明公开了一类含七元杂环的三齿缀合基团，并具体公开了式(IV)所示缀合基团及其应用。

Description

一类含七元杂环的三齿缀合基团
本发明主张如下的优先权：
申请号：CN202211438284.3，申请日：2022年11月16日；
申请号：CN202310179731.6，申请日：2023年2月28日；
申请号：CN202310899385.9，申请日：2023年7月20日。
技术领域
本发明涉及寡聚核苷酸领域，具体涉及一类含七元杂环的三齿缀合基团及其应用。
背景技术
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。
公开报道和文献表明，GalNAc长链之间受刚性和柔性平衡的影响，过刚或者过柔都会造成分子不适配ASGPR的立体构象，导致活性或者递送效率上产生损失。同时，获批上市药物中的GalNAc分子在合成上存在技术较为复杂，合成路线较长，反应要求条件苛刻，杂质较难控制等问题。这也是该领域内存在主要问题之一。
本发明专利提供了一种具备刚性七元环桥接点的三配体GalNAc分子，在保持活性的同时，大大降低了合成上难度，降低了成本。
发明内容
本发明第一方面提供式(IV)所示的缀合基团，
其中，
L1选自
L2选自其中环A选自C5-6环烷基和5-6元杂环烷基；
选自
n选自0和1；
m选自0、1、2和3；
t选自0、1、2、3、4、5、6和7。
在本发明的一些技术方案中，上述L2选自其他变量如本发明所定义。
在本发明的一些技术方案中，上述n选自0，其他变量如本发明所定义。
在本发明的一些技术方案中，上述n选自1，其他变量如本发明所定义。
在本发明的一些技术方案中，上述t选自0，其他变量如本发明所定义。
在本发明的一些技术方案中，上述t选自1，其他变量如本发明所定义。
在本发明的一些技术方案中，上述t选自2，其他变量如本发明所定义。
在本发明的一些技术方案中，上述t选自3，其他变量如本发明所定义。
在本发明的一些技术方案中，上述t选自4，其他变量如本发明所定义。
在本发明的一些技术方案中，上述t选自5，其他变量如本发明所定义。
在本发明的一些技术方案中，上述t选自6，其他变量如本发明所定义。
在本发明的一些技术方案中，上述t选自7，其他变量如本发明所定义。
在本发明的一些技术方案中，上述m选自0，其他变量如本发明所定义。
在本发明的一些技术方案中，上述m选自1，其他变量如本发明所定义。
在本发明的一些技术方案中，上述m选自2，其他变量如本发明所定义。
在本发明的一些技术方案中，上述m选自3，其他变量如本发明所定义。
在本发明的一些技术方案中，上述选自 其他变量如本发明所定义。
在本发明的一些技术方案中，上述选自 其他变量如本发明所定义。
本发明还提供式(I)所示的缀合基团，
其中，
L1选自
L2选自其中环A选自C5-6环烷基和5-6元杂环烷基；
n选自0和1；
m选自0、1、2和3；
t选自0、1、2、3、4、5、6和7。
在本发明的一些技术方案中，上述L2选自其他变量如本发明所定义。
在本发明的一些技术方案中，上述n选自0，其他变量如本发明所定义。
在本发明的一些技术方案中，上述n选自1，其他变量如本发明所定义。
在本发明的一些技术方案中，上述t选自0，其他变量如本发明所定义。
在本发明的一些技术方案中，上述t选自1，其他变量如本发明所定义。
在本发明的一些技术方案中，上述t选自2，其他变量如本发明所定义。
在本发明的一些技术方案中，上述t选自3，其他变量如本发明所定义。
在本发明的一些技术方案中，上述t选自4，其他变量如本发明所定义。
在本发明的一些技术方案中，上述t选自5，其他变量如本发明所定义。
在本发明的一些技术方案中，上述t选自6，其他变量如本发明所定义。
在本发明的一些技术方案中，上述t选自7，其他变量如本发明所定义。
在本发明的一些技术方案中，上述m选自0，其他变量如本发明所定义。
在本发明的一些技术方案中，上述m选自1，其他变量如本发明所定义。
在本发明的一些技术方案中，上述m选自2，其他变量如本发明所定义。
在本发明的一些技术方案中，上述m选自3，其他变量如本发明所定义。
在本发明的一些技术方案中，上述选自 其他变量如本发明所定义。
在本发明的一些技术方案中，上述选自 其他变量如本发明所定义。
在本发明的一些技术方案中，上述缀合基团选自(D08)、(D05)、(D06)、(D07)、(D10)和(D11)，

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，所述缀合物为上述任意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。
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：
其中，X选自O-和S-；
L1、L2、n、m和t如本发明所定义。
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：
其中，X选自O-和S-；
L1、L2、n、m和t如本发明所定义。
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：
其中，X选自O-和S-；
L1、L2、n、m和t如本发明所定义。
在本发明的一些实施方案中，上述缀合基团通过磷酸二酯键或硫代磷酸二酯键连接在寡聚核苷酸正义链的3’端。
在本发明的一些技术方案中，上述寡聚核苷酸选自RNAi试剂，其他变量如本发明所定义。
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。
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。
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。
在本发明的一些技术方案中，上述的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。
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，其他变量如本发明所定义。
在本发明的一些技术方案中，上述基因选自AGT、ANGPTL3、PCSK9、XDH、HSD、HAO、APP、TTR、补体蛋白等，其他变量如本发明所定义。
本发明第三方面提供上述任意技术方案限定的缀合基团作为递送平台的应用，其中，所述递送是指用于增强治疗剂与特定靶标位置的结合。
本发明第四方面提供一种用于制备上述任意技术方案限定的缀合基团的中间体化合物，其结构如式(I-M)所示，

其中，
R1选自H或羟基保护基，优选Ac；
R2选自羟基保护基，优选为DMTr。
在本发明的一些技术方案中，上述中间体结构如式(D08-M)、(D05-M)、(D06-M)、(D07-M)、(D10-M)和(D11-M)所示，

本发明还提供表1所示的缀合物：
表1本发明缀合物列表

本发明还提供如下测试方法：
一、体外活性测试
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。
二、体内活性测试
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。
实验过程：
1.实验材料：2-pcDNA-CMV-AGT质粒，BALB/c小鼠，PBS(磷酸缓冲液)，缀合物。
2.实验方法：
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。
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。
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。
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。
三：人原代肝细胞自由摄取实验
1.实验原理：
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。
2.实验材料：
人原代肝细胞(PHH)，96Kit(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：
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PHH细胞解冻并移植到96孔板中，最终的细胞密度为5.4×105细胞/孔。本发明缀合物测试10个浓度点，最高浓度为500nM，4倍稀释。
细胞在37℃，5％CO2中孵育48小时，用显微镜检查细胞状态。
孵育完成后，将细胞裂解，使用获得裂解液，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 cDNA。
本发明还提供缀合物的制备方法：
含缀合基团的单链寡核苷酸的合成：按照亚磷酰胺固相合成技术合成寡核苷酸。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’-修饰的核苷酸的亚磷酰胺(phosphoramidite)单体和辅助试剂均为商品化可得试剂。所有的亚磷酰胺单体按序列从3’到5’的顺序依次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。所得序列在最后脱除DMTr基团后即合成。
不含缀合基团的单链寡核苷酸的合成：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上进行合成。所有的2’-修饰的核苷酸亚磷酰胺(phosphoramidite)单体和辅助试剂均为商品化可得试剂。所有的亚磷酰胺单体按序列从3’到5’的顺序依次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。所得序列在最后脱除DMT基团后即合成。
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℃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。
单链寡核苷酸的纯化：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。
单链寡核苷酸的退火产生siRNA：把待退火的单链寡核苷酸用无菌RNase Free H2O(无RNA酶水) 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苷酸溶液形成互补链。
技术效果
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，对C5蛋白抑制具有优秀的有效性和长效性。目前已经用于获批或者正在临床试验阶段的GalNAc递送平台分子结构具有合成繁复，杂质复杂等问题，CMC难度较高。本发明专利中的分子为七元环连接的三配体GalNAc分子，分子刚性进一步增强，且可从商品化原料中简单获得，同时分子结构简单，结构单元单一。在研发过程中，意外的发现本发明中的分子具备理想的活性，同时合成更加简洁，CMC难度降低。
定义和说明
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。
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。
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。
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。
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。
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。
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。
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。
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。
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。
本发明所述“短干扰RNA(siRNA)”是一类RNA分子，双链区长度为14～30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)。
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。
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。
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。
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。
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)。
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、
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)。
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。
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。
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。
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结构为GNA-C结构为GNA-G结构为GNA- U结构为
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
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。
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。
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。
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。
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。
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。
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。
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。
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。
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。在发明的化合物中，还包括所有互变异构体形式。例如，包括所有磷酸酯和硫代磷酸酯基团的互变异构体。
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。
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，)。
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。
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。
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
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。
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％。
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％。
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)。
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。
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。
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。
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。
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。
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。
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。
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。
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。
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。
除非另有规定，“C5-6环烷基”表示由5至6个碳原子组成的饱和环状碳氢基团，其为单环和双环体系，所述C5-6环烷基包括C5和C6环烷基等；其可以是一价、二价或者多价。C5-6环烷基的实例包括环戊基和环己基等。
除非另有规定，术语“5-6元杂环烷基”本身或者与其他术语联合分别表示由5至6个环原子组成的饱和环状基团，其1、2、3或4个环原子为独立选自O、S和N的杂原子，其余为碳原子，其中氮原子任选地被季铵化，氮和硫杂原子可任选被氧化(即NO和S(O)p，p是1或2)。其包括单环和双环体系，其中双环体系包括螺环、并环和桥环。此外，就该“5-6元杂环烷基”而言，杂原子可以占据杂环烷基与分子其余部分的连接位置。所述5-6元杂环烷基包括5元和6元杂环烷基。5-6元杂环烷基的实例包括但不限于吡咯烷基、吡唑烷基、咪唑烷基、四氢噻吩基(包括四氢噻吩-2-基和四氢噻吩-3-基等)、四氢呋喃基(包括四氢呋喃-2-基等)、四氢吡喃基、哌啶基(包括1-哌啶基、2-哌啶基和3-哌啶基等)、哌嗪基(包括1-哌嗪基和2-哌嗪基等)、吗啉基(包括3-吗啉基和4-吗啉基等)、二噁烷基、二噻烷基、异噁唑烷基、异噻唑烷基、1,2-噁嗪基、1,2-噻嗪基、六氢哒嗪基等。
除非另有规定，术语“羟基保护基”是指适合用于阻止羟基副反应的保护基。代表性羟基保护基包括但不限于：烷基，如甲基、乙基和叔丁基；酰基，例如链烷酰基(如乙酰基)；芳基甲基，如苄基(Bn)，对甲氧基苄基(PMB)、9-芴基甲基(Fm)和二苯基甲基(二苯甲基，DPM)；甲硅烷基，如三甲基甲硅烷基(TMS)和叔丁基二甲基甲硅烷基(TBS)；4,4'-二甲氧基三苯基(DMTr)等等。
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。
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。
本发明所使用的溶剂可经市售获得。
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。
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。
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：
表2核苷酸单体的缩写

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。
具体实施方式
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。
实施例1

步骤A：中间体1-11-2的制备
氮气保护，0℃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℃下，滴加化合物1-11-1(17.86克,62.43毫摩尔)的四氢呋喃(50毫升)溶液。混合物在25℃下搅拌反应15.5小时。在10-25℃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1-11-2。
步骤B：中间体1-11-3的制备
氮气保护，60℃下，向化合物1-11-2(15克,41.06毫摩尔)在甲醇(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℃下搅拌反应1小时。在0-25℃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℃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1-11-3。
步骤C：中间体1-11-4的制备
将化合物1-11-3(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℃，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)纯化得化合物1-11-4。
步骤D：中间体1-11-5的制备
将化合物1-11-4(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℃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1-11-5。
步骤E：中间体1-11的制备
将化合物1-11-5(7.1克,10.93毫摩尔)和水合肼(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℃下反应16小时。35-40℃下，减压浓缩除去乙醇得中间体1-11。
步骤1：化合物1-3的制备
25℃下，向化合物1-2(13.24克，50.93毫摩尔)的甲苯溶液中(150毫升)加入三乙胺(10.10克，99.86毫摩尔，13.90毫升)和化合物1-1(12克，49.93毫摩尔，11.76毫升)的甲苯溶液(100毫升)。反应在100℃下搅拌16小时。反应液中加入饱和碳酸钠水溶液(200毫升)后分液。有机相用无水硫酸钠干燥，过滤，浓缩后得到粗产物，粗产物用制备HPLC(柱子:Agela Innoval ODS-2 250毫米*100毫米*10微米；流动相:[水(甲酸)-乙腈]；乙腈％:15％-35％，25分钟)分离得到化合物1-3。
步骤2：化合物1-4的制备
化合物1-3(12.6克，31.04毫摩尔)，钯碳(5g，10％含量)，碳酸酐二叔丁酯(14.90克，68.28毫摩尔，15.69毫升)的甲醇溶液(120毫升)氩气置换三次后，氢气置换三次，反应液在常压氢气氛围下25℃下搅拌16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1-4。
步骤3：化合物1-5的制备
向化合物1-4(7.5克，18.66毫摩尔)溶于盐酸乙酸乙酯溶液(4摩尔/升，18.66毫升)，氮气置换3次后，氮气保护下25℃搅拌1小时。减压浓缩后得到化合物1-5。
步骤4：化合物1-7的制备
将化合物1-6(2.2克，5.69毫摩尔)，化合物1-5(2.24克，9.68毫摩尔)，三乙胺(2.88克，28.47毫摩尔，3.96毫升)，1-丙基磷酸环酐(5.06g，7.95毫摩尔，4.73毫升，50％含量)的N，N-二甲基甲酰胺溶液(25毫升)氮气置换3次后，氮气保护下25℃搅拌1小时。反应液加入水(50毫升)后用乙酸乙酯 萃取(80毫升*2)。合并的有机相用饱和食盐水洗涤(100毫升*1)，无水硫酸钠干燥后，过滤并浓缩，得到的粗产品用制备级HPLC(柱子:Phenomenex luna C18 150*40毫米*15微米；流动相:[水(甲酸)-乙腈]；乙腈％:14％-44％，15分钟)纯化得到化合物1-7。
步骤5：化合物1-9的制备
将化合物1-7(2.2克，4.06毫摩尔)，化合物1-8(1.50克，6.09毫摩尔)，O-苯并三氮唑-N，N，N'，N'-四甲基脲四氟硼酸酯(2.22克，6.90毫摩尔)，三乙胺(1.23克，12.18毫摩尔，1.69毫升)的N，N-二甲基甲酰胺溶液(20毫升)氮气置换三次后，在氮气保护下25℃搅拌1小时。反应液加入水(80毫升)后用乙酸乙酯萃取(80毫升*2)。合并的有机相用饱和食盐水洗涤(100毫升*1)，无水硫酸钠干燥后，过滤浓缩得到粗产品，粗产品用硅胶柱色谱(洗脱剂：石油醚/乙酸乙酯，梯度淋洗)分离得到化合物1-9。
步骤6：化合物1-10的制备
向化合物1-9(2.5克，3.43毫摩尔)的甲醇(22毫升)和水(7毫升)混合溶液中加入一水合氢氧化锂(504.37毫克，12.02毫摩尔)。反应液在25℃下搅拌12小时。向反应液中加入水(50毫升)。水相用2M盐酸调节pH至4-5，用乙酸乙酯(100毫升*2)萃取水相。合并的有机相用饱和食盐水洗涤(50毫升)。用无水硫酸钠干燥后，过滤并浓缩滤液，得到化合物1-10。
步骤7：化合物1-12的制备
将化合物1-10(500毫克，710.49微摩尔)，化合物1-11(406.18毫克，781.54微摩尔)，苯并三氮唑-N，N，N'，N'-四甲基脲六氟磷酸酯(485.01毫克，1.28毫摩尔)，三乙胺(215.68毫克，2.13毫摩尔)的N，N-二甲基甲酰胺溶液(5毫升)氮气置换3次后，氮气氛围下25℃搅拌1小时。向反应液中加入水(80毫升)后用二氯甲烷萃取(80毫升*2)。合并的有机相用饱和食盐水洗涤(100毫升*1)，用无水硫酸钠干燥后，过滤并浓缩滤液，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％:57％-87％，10分钟)纯化得到化合物1-12。
步骤8：化合物1-13的制备
向化合物1-12(320毫克，203.93微摩尔)的甲醇溶液(10毫升)中加入钯碳(100毫克，10％含量)后氩气置换三次，后氢气置换三次，反应液在常压氢气氛围下，25℃搅拌16小时。反应液用硅藻土过滤后，滤液减压浓缩得到化合物1-13。
步骤9：化合物1-15的制备
将化合物1-13(178毫克，221.66微摩尔)，化合物1-14(413.14毫克，775.80微摩尔)，苯并三氮唑-N，N，N'，N'-四甲基脲六氟磷酸酯(336.25毫克，886.63微摩尔)，三乙胺(179.43毫克，1.77毫摩尔)的N.N-二甲基甲酰胺(4毫升)溶液氮气置换3次，反应液在氮气氛围下25℃下搅拌1小时。反应液加入水(100毫升)并用50毫升DCM/i-PrOH(3/1)萃取两次。合并的有机相用饱和食盐水洗涤(150毫升 *1)，并用无水硫酸钠干燥后过滤。滤液减压浓缩后，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％:36％-66％，10分钟)纯化得到化合物1-15。
步骤10：化合物D08-M的制备
将化合物1-15(240毫克，97.49微摩尔)，化合物1-16(34.15毫克，341.21微摩尔)，4-二甲氨基吡啶(11.91毫克，97.49微摩尔)，三乙胺(9.86毫克，97.49微摩尔)的二氯甲烷溶液(5毫升)氮气置换3次后，25℃氮气保护下搅拌16小时。反应液浓缩后用制备级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％:19％-49％，10分钟)纯化得到化合物D08-M。
1H NMR(400MHz，DMSO-d6)δppm 7.65-8.20(m，11H)7.28-7.39(m，4H)7.17-7.28(m，5H)6.78-7.03(m，4H)5.13-5.33(m，3H)4.89-5.10(m，3H)4.39-4.60(m，3H)3.94-4.15(m，15H)3.82-3.92(m，5H)3.64-3.81(m，12H)2.83-3.19(m，16H)2.08-2.19(m，14H)1.96-2.07(m，19H)1.86-1.92(m，9H)1.74-1.81(m，10H)1.54-1.65(m，7H)1.34-1.54(m，16H)1.03-1.33(m，16H)。
实施例2
步骤1：化合物2-1的制备
向化合物1-4(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℃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2-1，直接用于下一步。
步骤2：化合物2-2的制备
向化合物2-1(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℃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2-2。
步骤3：化合物2-3的制备
25℃下向化合物2-2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℃下搅拌0.5小时。减压浓缩得到化合物2-3，直接用于下一步。
步骤4：化合物2-5的制备
将化合物2-4(1.11克,5.86毫摩尔)，三乙胺(1.19克,11.72毫摩尔,1.63毫升)和苯并三氮唑-N,N,N’,N’-四甲基脲六氟磷酸酯(2.44克,6.44毫摩尔)溶于N,N-二甲基甲酰胺(30毫升)中25℃下搅拌半小时。将该反应液缓慢滴加进化合物2-3(3.6克,11.72毫摩尔)，三乙胺(3.56克,35.15毫摩尔,4.89毫升)的N,N-二甲基甲酰胺(30毫升)溶液中。反应液在25℃下搅拌半小时。反应结束后加入150毫升水，并用二氯甲烷萃取(100毫升*2)。合并有机相后用饱和食盐水洗涤(100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2-5(梯度淋洗，二氯甲烷/甲醇，1/0至10/1)。
步骤5：化合物2-7的制备
将化合物2-6(1.81克,5.67毫摩尔)，N,N-二异丙基乙胺(2.20克,17.02毫摩尔,2.96毫升)和苯并三氮唑-N,N,N’,N’-四甲基脲六氟磷酸酯(3.23克,8.51毫摩尔)溶于N,N-二甲基甲酰胺(15毫升)，缓慢加入到化合物2-5(2.3克,5.67毫摩尔)的N,N-二甲基甲酰胺(15毫升)溶液中。反应液在25℃下搅拌半小时。反应结束后加入120毫升水，并用乙酸乙酯萃取(60毫升*2)。合并有机相后用依次用1M盐酸(120毫升)，饱和碳酸钠(120毫升)，饱和食盐水洗涤(120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2-7(梯度淋洗，石油醚/乙酸乙酯，1/0至3/1)。
步骤6：化合物2-8的制备
将化合物2-7(2.5克,3.54毫摩尔)溶于盐酸乙酸乙酯中(4摩尔/升,30.78毫升)。在25℃下搅拌3小时。减压浓缩得到化合物2-8，未经进一步纯化直接用于下一步。
步骤7：化合物2-9的制备
向化合物1-14(5.12克,9.61毫摩尔)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(4.52克,34.96毫摩尔,6.09毫升)，苯并三氮唑-N,N,N’,N’-四甲基脲六氟磷酸酯(3.98克,10.49毫摩尔)，在25℃下搅拌半小时，将化合物2-8(1.50克,2.91毫摩尔)的N,N-二甲基甲酰胺(5毫升)溶液滴加到反应液中。反应在25℃下搅拌12小时。加入100毫升水后用二氯甲烷/异丙醇＝3/1萃取水相(100毫升*3)，有机相合并，无水硫酸钠干燥后过滤，滤液浓缩得到粗产物。粗产物用制备级HPLC(柱子:Kromasil Eternity XT 250*80毫米*10微米；流动相:[水(碳酸氢铵)-乙腈]；梯度:22％-52％乙腈，20分钟)纯化得到化合物2-9。
步骤8：化合物2-10的制备
向化合物2-9(2克,1.03毫摩尔)的甲醇溶液(20毫升)加入钯碳(375毫克,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℃反应12小时。反应液硅藻土过滤后浓缩得到化合物2-10，直接用于下一步。
步骤9：化合物2-11的制备
向化合物2-10(2克,1.08毫摩尔)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(556.19毫克,4.30毫摩尔)，苯并三氮唑-N,N,N’,N’-四甲基脲六氟磷酸酯(612.03毫克,1.61毫摩尔)和化合物1-11(559.15毫克,1.08毫摩尔)。反应液在25℃下搅拌12小时。反应结束后加水120毫升，并用二氯甲烷萃取(120毫升*3)。合并后的有机相用120毫升饱和食盐水洗涤后，无水硫酸钠干燥。过滤后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2-11。
步骤10：化合物D05-M的制备
向化合物2-11(625毫克,264.76微摩尔)的二氯甲烷溶液(15毫升)加入三乙胺(53.58毫克,529.52微摩尔)，4-二甲氨基吡啶(32.35毫克,264.76微摩尔)，化合物1-16(158.97毫克,1.59毫摩尔)。反应液在25℃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]；梯度:23％-53％乙腈，10分钟)纯化得到化合物D05-M。
1H NMR(400MHz,DMSO-d6)δ＝8.44-7.96(m,2H),7.95-7.64(m,9H),7.40-7.26(m,4H),7.22(br d,J＝8.6Hz,5H),6.88(d,J＝8.8Hz,4H),5.21(d,J＝3.4Hz,3H),4.97(dd,J＝2.7,11.0Hz,3H),4.49(d,J＝8.2Hz,3H),4.16-3.80(m,18H),3.73(s,6H),2.55-2.49(m,54H),2.10(s,9H),1.89(s,9H),1.76(s,11H),1.62-1.41(m,19H),1.28-1.09(m,15H)。
实施例3
步骤1：化合物3-2的制备
将化合物3-1(1.19克,5.86毫摩尔)，三乙胺(1.19克,11.72毫摩尔,1.63毫升)和苯并三氮唑-N,N,N’,N’-四甲基脲六氟磷酸酯(2.44克,6.44毫摩尔)溶于N,N-二甲基甲酰胺(40毫升)中25℃下搅拌半小时。将该反应液缓慢滴加进化合物2-3(3.6克,11.72毫摩尔)，三乙胺(3.56克,35.15毫摩尔,4.89毫升)的N,N-二甲基甲酰胺(40毫升)溶液中。反应液在25℃下搅拌半小时。反应结束后加入100毫升水，并用二氯甲烷萃取(100毫升*3)。合并有机相后用饱和食盐水洗涤(80毫升*3)，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3-2(梯度淋洗，二氯甲烷/甲醇，1/0至10/1)。
步骤2：化合物3-3的制备
将化合物2-6(1.94克,6.08毫摩尔)，N,N-二异丙基乙胺(2.36克,18.24毫摩尔,3.18毫升)和苯并三氮唑-N,N,N’,N’-四甲基脲六氟磷酸酯(2.77克,7.29毫摩尔)溶于N,N-二甲基甲酰胺(52毫升)，加入化合物3-2(2.55克,6.08毫摩尔)。反应液在25℃下搅拌12小时。反应结束后加入300毫升水，并用乙酸乙酯萃取(300毫升*3)。合并有机相后用饱和食盐水洗涤(300毫升*3)，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3-3(柱子：Phenomenex luna C18(250*70毫米*10微米)；流动相:[水(甲酸)-乙腈]；梯度:45％-75％乙腈，20分钟)。
步骤3：化合物3-4的制备
将化合物3-3(2.44克,3.39毫摩尔)溶于盐酸乙酸乙酯中(4摩尔/升,47.20毫升)。在25℃下搅拌1小时。减压浓缩得到化合物3-4，未经进一步纯化直接用于下一步。
步骤4：化合物3-5的制备
向化合物1-14(5.98克,11.23毫摩尔)的N,N-二甲基甲酰胺(80毫升)溶液中加入N,N-二异丙基乙胺(5.28克,40.84毫摩尔,7.11毫升)，苯并三氮唑-N,N,N’,N’-四甲基脲六氟磷酸酯(4.65克,12.25毫摩尔)，在25℃下搅拌半小时，将化合物3-4(1.8克,3.40毫摩尔)加到反应液中。反应在25℃下搅拌12小时。加入500毫升水后用二氯甲烷/异丙醇＝5/1萃取水相(300毫升*4)，有机相合并，无水硫酸钠干燥后过滤，滤液浓缩得到粗产物。粗产物用制备级HPLC(柱子:Kromasil Eternity XT 250*80毫米*10微米；流动相:[水(碳酸氢铵)-乙腈]；梯度:22％-52％乙腈，20分钟)纯化得到化合物3-5。
步骤5：化合物3-6的制备
向化合物3-5(2克,1.02毫摩尔)的甲醇溶液(30毫升)加入钯碳(400毫克,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℃反应12小时。反应液硅藻土过滤后浓缩得到化合物3-6，直接用于下一步。
步骤6：化合物3-7的制备
向化合物3-6(1.39克,742.14微摩尔)的N,N-二甲基甲酰胺(28毫升)溶液中加入N,N-二异丙基乙胺(383.67毫克,2.97毫摩尔)，苯并三氮唑-N,N,N’,N’-四甲基脲六氟磷酸酯(422.18毫克,1.11毫摩尔)在25℃下搅拌半小时，加入化合物1-11(385.70毫克,742.14微摩尔)。反应液在25℃下搅拌12小时。反应结束后加水120毫升，并用二氯甲烷萃取(60毫升*3)。合并后的有机相用120毫升饱和食盐水洗涤后，无水硫酸钠干燥。过滤后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3-7。
步骤7：化合物D06-M的制备
向化合物3-7(550毫克,231.61微摩尔)的二氯甲烷溶液(11毫升)加入三乙胺(46.87毫克,463.22微摩尔)，4-二甲氨基吡啶(28.30毫克,231.61微摩尔)，化合物1-16(139.07毫克,1.39毫摩尔)。反应液在25℃下搅拌16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]；梯度:27％-57％乙腈，10分钟)纯化得到化合物D06-M。
1H NMR(400MHz,DMSO-d6)δ＝8.25-7.65(m,10H),7.39-7.28(m,4H),7.23(d,J＝8.4Hz,5H),6.89(d,J＝8.4Hz,4H),5.22(d,J＝3.6Hz,3H),4.97(dd,J＝3.2,11.2Hz,3H),4.49(d,J＝8.8Hz,3H),4.20-3.80(m,19H),3.80-3.61(m,12H),3.26-2.85(m,19H),2.43(s,4H),2.19-2.01(m,21H),2.00(s,9H),1.89(s,9H),1.78(s,10H),1.66-1.36(m,20H),1.33-0.96(m,15H)。
实施例4
步骤1：化合物4-1的制备
将化合物2-6(2.1克,6.60毫摩尔)，N,N-二异丙基乙胺(8.53克,65.96毫摩尔,11.49毫升)和苯并三氮唑-N,N,N’,N’-四甲基脲六氟磷酸酯(2.75克,7.26毫摩尔)溶于N,N-二甲基甲酰胺(20毫升)中25℃下搅拌半小时。将该反应液缓慢滴加进化合物2-3(4.05克,13.19毫摩尔)的N,N-二甲基甲酰胺(20毫升)溶液中。反应液在25℃下搅拌1小时。反应结束后加入150毫升水，并用乙酸乙酯萃取(100毫升*2)。合并有机相后用饱和食盐水洗涤(100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4-1(梯度淋洗，二氯甲烷/甲醇，1/0至10/1)。
步骤2：化合物4-3的制备
将化合物4-2(931.39毫克,4.29毫摩尔)，N,N-二异丙基乙胺(1.39克,10.72毫摩尔,1.87毫升)和苯并三氮唑-N,N,N’,N’-四甲基脲六氟磷酸酯(2.03克,5.36毫摩尔)溶于N,N-二甲基甲酰胺(15毫升)，加入化合物4-1(1.91克,3.57毫摩尔)。反应液在25℃下搅拌12小时。反应结束后加入50毫升水，并用乙酸乙酯萃取(50毫升*2)。合并有机相后用饱和食盐水洗涤(100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4-3(梯度淋洗，石油醚/乙酸乙酯，1/0至0/1)。
步骤3：化合物4-4的制备
将化合物4-3(2.27克,3.09毫摩尔)溶于盐酸乙酸乙酯中(4摩尔/升,20毫升)。在25℃下搅拌12小时。减压浓缩得到化合物4-4，未经进一步纯化直接用于下一步。
步骤4：化合物4-5的制备
向化合物1-14(5.37克,10.09毫摩尔)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(3.56克,27.52毫摩尔,4.79毫升)，2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(4.65克,12.23毫摩尔)，在25℃下搅拌半小时，将化合物4-4(1.66克,3.06毫摩尔)加到反应液中。反应在25℃下搅拌12小时。加入50毫升水后用二氯甲烷/异丙醇＝5/1萃取水相(50毫升*3)，有机相合并，150mL饱和食盐水洗涤，有机相无水硫酸钠干燥后过滤，滤液浓缩得到粗产物。粗产物用制备级HPLC(柱子:Waters xbridge 150*25毫米*10微米；流动相:[水(碳酸氢铵)-乙腈]；梯度:25％-55％乙腈，10分钟)纯化得到化合物4-5。
步骤5：化合物4-6的制备
向化合物4-5(2.83克,1.43毫摩尔)的甲醇溶液(30毫升)加入钯碳(1.52克,10％钯含量)。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℃反应12小时。反应液硅藻土过滤后浓缩得到化合物4-6，直接用于下一步。
步骤6：化合物4-7的制备
向化合物4-6(2.55克,1.35毫摩尔)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(698.62毫克,5.41毫摩尔)，苯并三氮唑-N,N,N’,N’-四甲基脲六氟磷酸酯(768.74毫克,2.03毫摩尔)，化合物1-11(632.09毫克,1.22毫摩尔)。反应液在25℃下搅拌12小时。反应结束后加水100毫升，并用二氯甲烷萃取(50毫升*3)。合并后的有机相用150毫升饱和食盐水洗涤后，无水硫酸钠干燥。过滤后所得滤液浓缩并用制备级HPLC(柱子:Kromasil Eternity XT 250*80毫米*10微米；流动相:[水(碳酸氢铵)-乙腈]；梯度:40％-70％乙腈，20分钟)纯化得到化合物4-7。
步骤7：化合物D07-M的制备
向化合物4-7(600毫克,251.18微摩尔)的二氯甲烷溶液(6毫升)加入三乙胺(101.67毫克,1毫摩尔)，4-二甲氨基吡啶(30.69毫克,251.18微摩尔)，化合物1-16(100.55毫克,1毫摩尔)。反应液在25℃下搅拌12小时。减压浓缩后，用制备级HPLC(柱子:Waters Xbridge 150*25毫米*5微米；流动相:[水(碳酸氢铵)-乙腈]；梯度:30％-60％乙腈，10分钟)纯化得到化合物D07-M。
1H NMR(400MHz,DMSO-d6)δ＝8.24-7.64(m,11H),7.47-7.13(m,10H),6.88(d,J＝8.7Hz,4H),5.21(d,J＝3.3Hz,3H),4.97(dd,J＝3.4,11.2Hz,3H),4.48(d,J＝8.4Hz,3H),4.13-3.97(m,12H),3.93-3.78(m,6H),3.77-3.62(m,10H),3.47-3.35(m,5H),3.23-2.87(m,19H), 2.85-2.62(m,2H),2.10(s,11H),2.03(br s,10H),1.99(s,9H),1.89(s,9H),1.77(s,9H),1.68-1.31(m,26H),1.30-0.99(m,16H)。
实施例5
步骤1：化合物5-2的制备
将化合物5-1(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)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。反应液中加入盐酸水溶液(200毫升，1M)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，室温打浆12小时，得到化合物5-2。
步骤2：化合物5-4的制备
将化合物5-2(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(4.08毫升,23.44毫摩尔)，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5-3(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(淋洗剂：石油醚/乙酸乙酯＝1/1)纯化得化合物5-4。
步骤3：化合物5-5的制备
将化合物5-4(3.8克,4.69毫摩尔)，二乙胺(19.31毫升,187.42毫摩尔)溶于二氯甲烷(30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5-5。
步骤4：化合物5-8的制备
将化合物5-6(37.7克，68.72毫摩尔，草酸盐)和化合物5-7(22.45克，82.47毫摩尔)，三乙胺(33.48毫升，240.53毫摩尔)溶解于乙腈(400毫升)中，使用氮气置换三次。将混合液在25℃搅拌3.5小时。将混合液用甲基叔丁基醚400毫升稀释，分别使用饱和碳酸氢钠水溶液(400毫升*2)，食盐水(400毫升)，饱和氯化铵(400毫升*2)和食盐水(400毫升)清洗，有机相用无水硫酸钠干燥，过滤，真空中浓缩得化合物5-8。
步骤5：化合物5-9的制备
将化合物5-8(42.31克，68.72毫摩尔)溶解于甲醇(200毫升)和水(50毫升)中，之后，在5℃下，加入氢氧化钠(6.87克，171.79毫摩尔)。将混合液在25℃搅拌2小时。将混合液浓缩除去大部分甲醇，用水(100毫升)稀释，在5℃下，使用1M盐酸调节溶液pH＝4-5，使用二氯甲烷(300毫升*2)萃取。有机相用无水硫酸钠干燥，过滤，真空中浓缩得化合物5-9。
步骤6：化合物5-10的制备
将化合物5-9(5克,8.31毫摩尔)溶于二氯甲烷(50毫升)，依次加入1,8-二氮杂二环[5.4.0]十一烷-7-烯(1.52克,9.97毫摩尔)和溴化苄(1.71克,9.97毫摩尔)。反应液在25℃下反应12小时。反应液减压浓缩除去二氯甲烷，加水溶液(100毫升)，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。粗品通过制备级HPLC纯化(柱子:Kromasil Eternity XT 250*80毫米*10微米；流动相:[水(碳酸氢铵)-乙腈]；梯度:43％-73％乙腈，20分钟)得到化合物5-10。
步骤7：化合物5-11的制备
化合物5-10(3.7克,5.35毫摩尔)加入盐酸乙酸乙酯溶液(2M,92.50毫升)。反应液在25℃下反应12小时。反应液减压浓缩得化合物5-11。
步骤8：化合物5-12的制备
将化合物1-14(9.47克,17.79毫摩尔)溶于二氯甲烷(60毫升)溶液，依次加入N,N-二异丙基乙胺 (8.45毫升,48.52毫摩尔)，O-(7-氮杂苯并三氮唑-1-基)-N,N,N,N-四甲基脲六氟膦盐(8.20克,21.56毫摩尔)和化合物5-11(2.7克,5.39毫摩尔)。反应液在25℃下反应12小时。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(碳酸氢铵)-乙腈]；梯度:20％-50％乙腈，20分钟)得到化合物5-12。
步骤9：化合物5-13的制备
将化合物5-12(5.2克,2.69毫摩尔)溶于甲醇(50毫升)，在氮气氛围保护下，向反应液中加入Pd/C(1克,939.67微摩尔,10％钯含量)，氢气置换三次，在氢气(15Psi)，25℃条件下反应12小时。过滤出去钯碳，过滤液减压浓缩得化合物5-13。
步骤10：化合物5-14的制备
将化合物5-13(1克,542.03微摩尔)溶于二氯甲烷(10毫升)溶液，依次加入N,N-二异丙基乙胺(283.24微升,1.63毫摩尔)，1-羟基-7-氮杂苯并三唑(110.67毫克,813.05微摩尔)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(155.86毫克,813.05微摩尔)和化合物5-5(462.52毫克,542.03微摩尔)。反应液在25℃下反应12小时。减压浓缩得粗品，粗品通过制备级HPLC(柱子:Waters Xbridge C18 150*50毫米*10微米；流动相:[水(碳酸氢铵)-乙腈]；梯度:31％-61％乙腈10分钟)纯化得到化合物5-14。
步骤11：化合物D10-M的制备
将化合物5-14(0.47克,194.56微摩尔)溶于二氯甲烷(5毫升)溶液，依次加入三乙胺(148.94微升,1.07毫摩尔)，4-二甲基氨基吡啶(23.77毫克,194.56微摩尔)和丁二酸酐(97.35毫克,972.82微摩尔)。反应液在25℃下反应12小时，检测反应完成。减压浓缩得粗品，粗品通过制备级HPLC(柱子:Waters Xbridge C18 150*50毫米*10微米；流动相:[水(碳酸氢铵)-乙腈]；梯度:22％-52％乙腈10分钟)得到化合物D10-M。
1H NMR(400MHz,DMSO-d6)δ＝8.46-7.60(m,9H),7.38-7.09(m,9H),6.93-6.80(m,4H),5.21(d,J＝3.3Hz,3H),4.97(dd,J＝3.4,11.1Hz,3H),4.48(d,J＝8.4Hz,3H),4.26-4.15(m,1H),4.08-3.93(m,10H),3.92-3.78(m,5H),3.78-3.63(m,11H),3.57-3.39(m,6H),3.16-2.86(m,18H),2.40-2.19(m,6H),2.17-2.08(m,13H),2.07-2.01(m,8H),1.99(s,10H),1.89(s,9H),1.77(s,9H),1.65-1.33(m,22H),1.33-1.08(m,11H).
实施例6
步骤1：化合物6-3的制备
将化合物5-2(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(1.53毫升,8.79毫摩尔)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6-3。
步骤2：化合物6-5的制备
将化合物6-3(2.86克,5.65毫摩尔)溶于二氯甲烷(30毫升)溶液，加入化合物6-4(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6-5。
步骤3：化合物6-6的制备
将化合物6-5(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(479.25微升,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6-6。
步骤4：化合物6-7的制备
将化合物6-6(1.78克,2.09毫摩尔)，二乙胺(11.13毫升,108.00毫摩尔)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6-7。
步骤5：化合物6-8的制备
将化合物5-13(1.5克,813.05微摩尔)溶于二氯甲烷(15毫升)溶液，依次加入N,N-二异丙基乙胺(424.86微升,2.44毫摩尔)，1-羟基-7-氮杂苯并三唑(166.00毫克,1.22毫摩尔)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(233.79毫克,1.22毫摩尔)。搅拌均匀后加入化合物6-7(854.86毫克,813.05微摩尔)。反应液在25℃下反应12小时，检测反应完成。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:34％-64％乙腈，10分钟)得到化合物6-8。
步骤6：化合物D11-M的制备
将化合物6-8(450毫克,183.09微摩尔)溶于二氯甲烷(3毫升)溶液，依次加入三乙胺(229.36微升,1.65毫摩尔)，4-二甲基氨基吡啶(22.37毫克,183.09微摩尔)和丁二酸酐(146.58毫克,1.46毫摩尔)。氮气保护下，反应液在35℃下反应24小时。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:23％-53％乙腈，10分钟)得到化合物D11-M。
1H NMR(400MHz,DMSO-d6)δ＝8.47-7.70(m,10H),7.41-7.15(m,9H),6.87(br d,J＝7.1Hz,4H),5.21(d,J＝3.2Hz,3H),4.97(br dd,J＝2.6,11.2Hz,3H),4.49(br d,J＝8.3Hz,3H),4.18-3.91(m,12H),3.91-3.79(m,5H),3.76-3.61(m,14H),3.22-2.82(m,27H),2.10(s,11H),1.99(s,18H),1.88(s,9H),1.77(s,8H),1.66-1.30(m,22H),1.23(br d,J＝6.2Hz,10H),1.11-1.02(m,3H),0.97(br d,J＝2.8Hz,3H)。
生物测试
实验例1：人原代肝细胞自由摄取实验-靶基因AGT
1.实验原理：
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。
2.实验材料：
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)。
3.实验方法：
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。
细胞在37℃，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。
4.实验结果见表3
表3本发明缀合物在人原代肝细胞中自由摄取实验结果
结论：本发明缀合物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递送系统对siRNA序列具有良好的肝靶向递送能力。
实验例2：人原代肝细胞自由摄取实验靶基因ANGPTL3
1.实验原理：
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。
2.实验材料：
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)。
3.实验方法：
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℃孵箱中培养48小时。
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。
4.实验结果见表4
表4本发明缀合物在人原代肝细胞中自由摄取实验结果
结论：本发明缀合物在人原代肝细胞中均展现出对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。
实验例3：小鼠体内活性测试靶基因AGT
1.实验原理：
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。
2.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。
3.实验方法：
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。
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。
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。
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。
4.实验结果表5
表5本发明缀合物体内活性测试实验结果(每组4只小鼠)
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。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。
实验例4：小鼠体内活性测试靶基因补体C5
1.实验原理：
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。
2.实验材料：
C57 BL/6小鼠，PBS(磷酸缓冲液)，本发明缀合物。
3.实验方法：
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。
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％。
结论：本实验展现出本发明缀合物对C5蛋白抑制具有优秀的有效性和长效性，由于补体C5 mRNA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。

Claims (13)

  1. 式(IV)所示的缀合基团，
    其中，
    L1选自
    L2选自其中环A选自C5-6环烷基和5-6元杂环烷基；
    选自
    n选自0和1；
    m选自0、1、2和3；
    t选自0、1、2、3、4、5、6和7。
  2. 根据权利要求1所述的缀合基团，其中，所述选自
  3. 根据权利要求1所述的缀合基团，其中，所述选自
  4. 根据权利要求1所述的缀合基团，其选自(D08)、(D05)、(D06)、(D07)、(D10)和(D11)，

  5. 一种缀合物或其药学上可接受的盐，其中，所述缀合物为权利要求1-4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。
  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，所述寡聚核苷酸选自RNAi试剂。
  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RNAi试剂选自单链寡聚核苷酸和双链寡聚核苷酸。
  8. 根据权利要求7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。
  9. 根据权利要求7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。
  10. 根据权利要求5-9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。
  11. 权利要求1-4任意一项所述的缀合基团作为递送平台的应用，其中，所述递送是指用于增强治疗剂与特定靶标位置的结合。
  12. 一种用于制备权利要求1-3任意一项所述的缀合基团的中间体化合物，其结构如式(I-M)、(IV-M10)和(IV-M11)所示，

    其中，
    R1选自H或羟基保护基，优选Ac；
    R2选自羟基保护基，优选为DMTr。
  13. 一种用于制备权利要求4所述的缀合基团的中间体化合物，其结构如式(D08-M)、(D05-M)、(D06-M)、(D07-M)、(D10-M)和(D11-M)所示，

PCT/CN2023/131818 2022-11-16 2023-11-15 一类含七元杂环的三齿缀合基团 WO2024104386A1 (zh)

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WO2020191183A1 (en) * 2019-03-21 2020-09-24 Mitotherapeutix Llc Multivalent ligand clusters for targeted delivery of therapeutic agents
WO2021046260A1 (en) * 2019-09-03 2021-03-11 Arcturus Therapeutics, Inc. Asialoglycoprotein receptor mediated delivery of therapeutically active conjugates
WO2021249484A1 (zh) * 2020-06-10 2021-12-16 南京明德新药研发有限公司 缀合基团及其缀合物
CN115028670A (zh) * 2022-06-24 2022-09-09 四川大学华西医院 一种n-乙酰基-d-半乳糖胺三聚体前体的制备方法
US20230100220A1 (en) * 2021-08-30 2023-03-30 Hongene Biotech Corporation Functionalized n-acetylgalactosamine analogs
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Publication number Priority date Publication date Assignee Title
WO2020191183A1 (en) * 2019-03-21 2020-09-24 Mitotherapeutix Llc Multivalent ligand clusters for targeted delivery of therapeutic agents
WO2021046260A1 (en) * 2019-09-03 2021-03-11 Arcturus Therapeutics, Inc. Asialoglycoprotein receptor mediated delivery of therapeutically active conjugates
WO2021249484A1 (zh) * 2020-06-10 2021-12-16 南京明德新药研发有限公司 缀合基团及其缀合物
US20230100220A1 (en) * 2021-08-30 2023-03-30 Hongene Biotech Corporation Functionalized n-acetylgalactosamine analogs
US20230279041A1 (en) * 2021-12-15 2023-09-07 Hongene Biotech Corporation Functionalized n-acetylgalactosamine analogs
CN115028670A (zh) * 2022-06-24 2022-09-09 四川大学华西医院 一种n-乙酰基-d-半乳糖胺三聚体前体的制备方法

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