WO2023207615A1 - Class of double-stranded rnai compounds containing overhang consisting of natural nucleotides - Google Patents

Class of double-stranded rnai compounds containing overhang consisting of natural nucleotides Download PDF

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WO2023207615A1
WO2023207615A1 PCT/CN2023/088123 CN2023088123W WO2023207615A1 WO 2023207615 A1 WO2023207615 A1 WO 2023207615A1 CN 2023088123 W CN2023088123 W CN 2023088123W WO 2023207615 A1 WO2023207615 A1 WO 2023207615A1
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seq
double
present
nucleotides
stranded
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PCT/CN2023/088123
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French (fr)
Chinese (zh)
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贺海鹰
陆剑宇
胡彦宾
陈实
吴孝琴
石璐
胡利红
丁照中
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南京明德新药研发有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the present invention relates to double-stranded RNAi compounds, in particular to a class of double-stranded RNAi compounds containing overhangs composed of natural nucleotides.
  • RNA interference The RNA interference (RNAi) mechanism has been closely watched since its discovery in 1998; this mechanism controls the function of the corresponding target by down-regulating the level of target mRNA to achieve the desired pharmacological effect.
  • RNAi drugs a number of RNAi drugs have been approved for marketing, and the feasibility verification of their application in human drugs has been completed. Drugs that achieve pharmacological effects through this mechanism are called RNAi drugs.
  • RNAi drugs usually consist of an RNA double strand containing 16-27 base pairs, and its double-stranded structure is an important factor in determining the properties of RNAi drugs.
  • Common double-stranded structures include: (1) symmetrical double-stranded structure with blunt ends, that is, a completely complementary double-stranded double strand in which neither the sense strand nor the antisense strand has overhangs; (2) symmetrical double-stranded structure with overhangs, that is, the sense strand The 3' end of the sense strand and the 5' end of the antisense strand have overhangs with the same number of bases, or the 5' end of the sense strand and the 3' end of the antisense strand have overhangs with the same number of bases; (3) One end It is a blunt-ended asymmetric double-stranded structure. This structure usually contains a varying number of unpaired bases at the 3’ end of the antisense strand.
  • the above three double-stranded structures have been widely reported and widely used
  • this patent provides a new RNA double-stranded structure that can be used in RNAi drugs. Compared with the known double-stranded structure, RNAi drugs using this new structure can significantly improve the effectiveness of the drug.
  • the invention provides a double-stranded RNAi compound, wherein the compound includes a sense strand and an antisense strand capable of forming a double-stranded region, the double-stranded region consisting of 14-29 nucleotide base pairs, and the antisense strand
  • the compound includes a sense strand and an antisense strand capable of forming a double-stranded region, the double-stranded region consisting of 14-29 nucleotide base pairs, and the antisense strand
  • the 5' end of the sense strand contains an overhang, and the overhang is composed of 1-10 natural nucleotides.
  • the 5' end of the sense strand or the 3' end of the antisense strand optionally contains an overhang, and the double end
  • the nucleotides in the strand region, the overhang at the 5' end of the sense strand, or the overhang at the 3' end of the antisense strand are optionally modified.
  • the above-mentioned double-stranded region consists of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 nucleotide bases
  • the basis pairs are composed, and other variables are as defined in the present invention.
  • the above-mentioned double-stranded region consists of 17, 18, 19, 20, 21, 22 or 23 nucleotides, and other variables are as defined in the present invention.
  • At least one modified nucleotide of the sense strand of the above-mentioned double-stranded region refers to a modified sugar containing cyclic nucleotides, and other variables are as defined herein.
  • nucleotides containing modified sugar rings are selected from glycerol nucleic acid (GNA), and other variables are as defined in the present invention.
  • GAA glycerol nucleic acid
  • At least one modified nucleotide of the sense strand of the above-mentioned double-stranded region refers to a nucleotide containing a modified nucleobase, and other variables are as defined in the present invention.
  • At least one modified nucleotide of the antisense strand of the double-stranded region refers to a nucleotide containing a modified nucleobase, and other variables are as defined in the present invention.
  • the above-mentioned modified nucleobase is selected from substituted or unsubstituted triazole, Other variables are as defined in the present invention.
  • the overhang of the 5' end of the above-mentioned sense strand contains 0, 1, 2, 3, 4, 5, 6, 7 or 8 nucleotides, and the nucleotides are optionally modified , other variables are as defined in the present invention.
  • the overhang of the 5' end of the above-mentioned sense strand contains 0, 1, 2 or 3 nucleotides, and the nucleotides are optionally modified, and other variables are as defined in the present invention.
  • nucleotides at the overhang of the 5' end of the sense strand are modified, and other variables are as defined in the present invention.
  • the overhang of the 3' end of the above-mentioned antisense strand contains 0, 1, 2, 3, 4, 5, 6, 7 or 8 nucleotides, and the nucleotides are optionally Modification, other variables are as defined in the present invention.
  • the overhang of the 3' end of the above-mentioned antisense strand contains 0, 1, 2, 3, 4, 5 or 6 nucleotides, and the nucleotides are optionally modified, and other variables As defined herein.
  • nucleotides of the overhang at the 3' end of the antisense strand are modified, and other variables are as defined in the present invention.
  • the overhang at the 5' end of the antisense strand is composed of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 natural nucleotides, and other variables are as in the present invention defined.
  • a ligand is conjugated to any position of the above-mentioned sense strand or antisense strand, and other variables are as defined in the present invention.
  • one end of the above-mentioned sense strand and one end of the antisense strand can also be connected through one or more nucleotides, and other variables are as defined in the present invention.
  • one end of the above-mentioned sense strand and one end of the antisense strand can also be connected through chemical groups, and other variables are as defined in the present invention.
  • a ligand is conjugated to any position of the above-mentioned sense strand and/or antisense strand, and other variables are as defined in the present invention.
  • the 3' end of the above-mentioned sense strand is conjugated with a ligand, and other variables are as defined in the present invention.
  • the 5' end of the above-mentioned sense strand is conjugated with a ligand, and other variables are as defined in the present invention.
  • the 3' end of the above-mentioned antisense chain is conjugated with a ligand, and other variables are as defined in the present invention.
  • the 5' end of the above-mentioned antisense strand is conjugated with a ligand, and other variables are as defined in the present invention.
  • the 3' end of the sense strand, the 5' end of the sense strand, the 3' end of the antisense strand and the 5' end conjugation ligand of the antisense strand refer to the 3' end or the 5' end of the antisense strand.
  • the hydroxyl group at the end is connected to the ligand through a phosphate bond and/or a phosphorothioate bond, and other variables are as defined in the present invention.
  • a ligand is conjugated to the nucleotide connecting the sense strand and the antisense strand, and other variables are as defined in the present invention.
  • a ligand is conjugated to the chemical group connecting the sense strand and the antisense strand, and other variables are as defined in the present invention.
  • the number of the above-mentioned conjugated ligands is 1, 2, 3, 4 or 5, and other variables are as defined in the present invention.
  • the above-mentioned ligand is one or more GalNAc derivatives attached using divalent or trivalent branched linkers, and other variables are as defined in the present invention.
  • the above-mentioned ligand is D01 or L96, and other variables are as defined in the present invention.
  • the above-mentioned sense strand is selected from SEQ ID NO: 1 or SEQ ID NO: 2, and other variables are as defined in the present invention.
  • the above-mentioned sense strand is selected from SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 13, and other variables are as defined in the present invention.
  • the above-mentioned antisense chain is selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 , SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12, and other variables are as defined in the present invention.
  • the above-mentioned antisense strand is selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 , SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23, and other variables are as defined in the present invention.
  • the above-mentioned sense strand is selected from SEQ ID NO: 1, the 3' end of the sense strand is conjugated with a ligand, and other variables are as defined in the present invention.
  • the above-mentioned sense strand is selected from SEQ ID NO: 1, the 3' end of the sense strand is conjugated to a ligand, the ligand is D01, and other variables are as defined in the present invention.
  • the above-mentioned sense strand is selected from SEQ ID NO: 2, and other variables are as defined in the present invention.
  • the above-mentioned conjugate is selected from S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S13, S14, S15, S16, S17, S18 , S19 and S20.
  • the above-mentioned conjugate is selected from S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S13, S14, S15, S16, S17, S18 , S19, S20, S21, S22, S23, S24, S25, S26, S27, S28, S29 and S30.
  • the above-mentioned double-stranded RNAi compounds are used to inhibit or block gene expression, and other variables are as defined in the present invention.
  • the above-mentioned genes include but are limited to estrogen receptor genes, AGT genes or KRAS genes, and other variables are as defined in the present invention.
  • the present invention also provides the following test methods:
  • the mouse model of high-pressure tail vein injection of pcDNA-CMV-AGT plasmid was used to evaluate the effect of targeting the target gene and inhibiting the target gene in the test sample.
  • mice Order BALB/c female mice aged 6-8 weeks, and the mice will adapt to quarantine for one week after arriving in the animal room.
  • mice On day 0, the mice were randomly divided into groups according to their body weight data, with 4 mice in each group. After grouping, all mice were given subcutaneous injection, a single dose, and the dosage volume was 10 mL/kg. The mice in the first group were given DPBS; Two groups of mice were given the conjugate of the present invention at 3 mg/kg.
  • mice in all groups were euthanized by CO2 inhalation, and 2 liver samples were collected from each mouse. Liver samples were treated with RNAlater overnight at 4°C, and then the RNAlater was removed and stored at -80°C for detection of AGT gene expression levels.
  • Transcriptome refers to the sum of all RNA transcribed from a specific tissue or cell at a certain time or in a certain state, mainly including mRNA and non-coding RNA.
  • Transcriptome sequencing is based on the Illumina sequencing platform and studies all the mRNA transcribed by a specific tissue or cell at a certain period. It is the basis for the study of gene function and structure and plays an important role in understanding the development of organisms and the occurrence of diseases. With the development of gene sequencing technology and the reduction of sequencing costs, RNA-seq has become the main method for transcriptome research due to its advantages of high throughput, high sensitivity, and wide application range.
  • the RNA-seq technology process mainly consists of two parts: library construction and sequencing and bioinformatics analysis.
  • RNA is extracted from tissues or cells using standard extraction methods, and then strict quality control is performed on the RNA samples.
  • the quality control method is mainly through the Agilent 2100 bioanalyzer: accurate detection of RNA integrity.
  • mRNA There are two main ways to obtain mRNA: one is to take advantage of the structural characteristics that most eukaryotic mRNAs have polyA tails, and use Oligo (dT) magnetic beads to enrich mRNAs with polyA tails. The second is to remove ribosomal RNA from total RNA to obtain mRNA. The obtained mRNA is then randomly fragmented with divalent cations in NEB Fragmentation Buffer, and the library is constructed according to the NEB general library construction method or the strand-specific library construction method.
  • Oligo Oligo
  • NEB ordinary library construction Using fragmented mRNA as a template and random oligonucleotides as primers, the first strand of cDNA is synthesized in the M-MuLV reverse transcriptase system, and then RNaseH is used to degrade the RNA strand, and the first strand of cDNA is synthesized in the DNA polymerase I system. Next, dNTPs are used as raw materials to synthesize the second strand of cDNA. The purified double-stranded cDNA is end-repaired, A-tailed, and connected to sequencing adapters.
  • AMPure XP beads are used to screen cDNA of about 250-300 bp, PCR amplification is performed, and AMPure XP beads are used again to purify the PCR products to finally obtain a library.
  • the kit for library construction is Ultra TM RNA Library Prep Kit for
  • Strand-specific library construction The method of reverse transcription to synthesize the first strand of cDNA is the same as the NEB general library construction method. The difference is that when synthesizing the second strand, dTTP in dNTPs is replaced by dUTP, and then the cDNA end repair and addition are also performed. A tail, ligation of sequencing adapters and length screening, then USER enzyme is used to degrade the second strand of U-containing cDNA and then PCR amplification is performed to obtain a library. Strand-specific libraries have many advantages, such as obtaining more effective information with the same amount of data; obtaining more precise gene quantification, positioning and annotation information; and providing antisense transcripts and the expression level of a single exon in each isoform.
  • the kit used for library construction is Ultra TM Directional RNA Library Prep Kit for
  • Sequencing adapter includes three parts: P5/P7, index and Rd1/Rd2 SP.
  • P5/P7 is the part where the PCR amplification primer and the primer on the flow cell are combined.
  • the index provides information to distinguish different libraries.
  • Rd1/Rd2 SP is the read1/read2 sequence primer, which is the binding area of the sequencing primer. The sequencing process is theoretically composed of Rd1/ Rd2 SP starts backward.
  • each sequencing cluster extends the complementary strand, each fluorescently labeled dNTP is added to release the corresponding fluorescence.
  • the sequencer captures the fluorescence signal and converts the light signal into a sequencing peak through computer software to obtain the sequence information of the fragment to be tested.
  • Method 3 Testing the agonistic activity of compounds on hTLR3, hTLR7, and hTLR8 cells
  • the main instruments used in this research are multifunctional microplate reader Flexstation III (Molecular Device) and Echo555 (Labcyte)
  • Compound activity detection Take 20 ⁇ L of cell supernatant from each well and add it to the experimental plate containing 180 ⁇ L of QUANTI-Blue TM reagent. After incubating at 37°C for 1 hour, use a multifunctional microplate reader Flexstation III to detect the absorbance value at 650 nm (OD650). .
  • Cell viability detection Operate according to Celltiter-Glo instructions, and the chemiluminescence signal (RLU) is detected with a multifunctional microplate reader Flexstation III.
  • Compound activity The OD650 value was analyzed using GraphPad Prism software, and the compound dose-effect curve was fitted to calculate the EC 50 value of the compound.
  • test sample was incubated with primary human hepatocytes (PHH) to evaluate the degree of down-regulation of AGT mRNA by the test sample.
  • PHL primary human hepatocytes
  • PSH Primary human hepatocytes
  • 96Kit (12) QIAGEN-74182
  • FastKing RT Kit With gDNase
  • TaqMan Gene Expression Assay TaqMan Gene Expression Assay
  • DPBS Dulbecco's Phosphate Buffer
  • the conjugate of the present invention is diluted to 10 times the concentration to be tested with DPBS solution. Transfer 10 ⁇ L of siRNA to a 96-well plate. PHH cells were thawed and transplanted into 96-well plates, 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 diluted 4 times.
  • oligonucleotide as used herein is a nucleotide sequence containing 14 to 80 nucleotides or nucleotide base pairs. In some embodiments of the 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 within the cell. The nucleotides may optionally be modified. In some embodiments of the invention, the oligonucleotide is capable of inhibiting or blocking expression of a gene in vitro or in vivo upon delivery to a cell expressing the gene.
  • 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 RNA shRNA, ribozymes, interfering RNA molecules, and Dicer enzyme substrates.
  • a “single-stranded compound” as used herein refers to a single-stranded oligonucleotide having a sequence that is at least partially complementary to the target mRNA, which is capable of operating under mammalian physiological conditions (or equivalent in vitro environment) through hydrogen bonding. hybridize with target mRNA.
  • the single-stranded oligonucleotide is a single-stranded antisense oligonucleotide.
  • double-stranded compound refers to a duplex structure containing two antiparallel and substantially complementary nucleotide strands, one of which is the sense strand and the other is the antisense strand.
  • antisense strand or “guide strand” used herein refers to the strand in an oligonucleotide compound that is substantially complementary to the corresponding region of the target sequence (e.g., AGT mRNA).
  • the "sense strand” or “passenger strand” used in the present invention refers to a strand capable of forming a substantially complementary region with the antisense strand.
  • the “substantially complementary” means that the corresponding positions of the two sequences can be completely complementary, or there can be one or more mismatches. When there is a mismatch, there are usually no more than 3, 2 or 1 mismatched bases. right.
  • the bases on one strand are paired with bases on the other strand in a complementary manner.
  • the purine base adenine (A) always pairs with the pyrimidine base uracil (U); the purine base guanine (C) always pairs with the pyrimidine base cytosine (G).
  • nucleotide optionally modified in the present invention means that the nucleotide may be an unmodified nucleotide or a modified nucleotide.
  • the "unmodified” “Nucleotide” refers to nucleotides composed of natural nucleobases, sugar rings and phosphates.
  • the “modified nucleotide” refers to a nucleotide composed of a modified nucleobase, and/or a modified sugar ring, and/or a modified phosphate ester.
  • modified nucleotides are composed of modified nucleobases, modified sugar rings and natural phosphates; in some embodiments of the invention, “modified nucleosides” “Acid” consists of modified nucleobases, modified phosphate esters and natural sugar rings; in some embodiments of the invention, “modified nucleotides” consist of natural nucleobases, modified sugar rings and modified Composed of phosphate esters; in some embodiments of the present invention, “modified nucleotides” are composed of modified nucleobases, natural sugar rings and natural phosphate esters; in some embodiments of the present invention, “modified nucleotides” “Modified nucleotides” are composed of natural nucleobases, modified sugar rings and natural phosphates; in some embodiments of the invention, “modified nucleotides” are composed of natural nucleobases, natural It consists of a sugar ring and a modified phosphate ester; in some embodiment
  • the “natural sugar ring” in the present invention is selected from the five-membered sugar ring of 2’-OH.
  • the "natural bases” mentioned in the present invention are selected from the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine.
  • the "modified nucleobase” mentioned in the present invention refers to a 5-12-membered saturated, partially unsaturated or aromatic heterocycle other than natural bases, including a single ring or a condensed ring.
  • examples include, but are not limited to, thiophene, thianthrene, furan, pyran, isobenzofuran, benzothiazine, pyrrole, imidazole, substituted or unsubstituted triazole, pyrazole, isothiazole, isoxazole, pyridazine , indolezine, indole, isoindole, isoquinoline, quinoline, naphthopyridine, quinazoline, carbazole, phenanthridine, piperidine, phenazine, phenanzine, phenothiazine, furane, Phenoxazine, pyrrolidine, pyrroline, imidazolidine,
  • the "modified sugar ring" described in 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 can be substituted or unsubstituted C 1 to C 10 alkyl group or C 2 to C 10 alkenyl and alkynyl groups.
  • 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 , where n and m are from 1 to 10.
  • modifications at the 2' position include, but are not limited to, one of the following: 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, heterocycle Alkaryl group, aminoalkylamino group, polyalkylamino group, substituted silyl group, RNA cleavage group, reporter group, intercalator, group for improving the pharmacokinetic properties of iRNA, or for improving iRNA groups with pharmacodynamic properties, and other substituents with similar properties.
  • the modification includes, but is not limited to, 2'-O-CH 2 CH 2 OCH 3 , also known as 2'-O-(2-methoxye
  • the "modified phosphate ester” in the present invention includes but is not limited to: phosphorothioate modification, the "phosphorothioate” “Acid ester” includes the (R)- and (S)-isomers and/or mixtures thereof.
  • modified nucleotides may comprise one or more locked nucleic acids (LNA).
  • Locked nucleic acids are nucleotides with a modified ribose moiety that contains an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose sugar in the 3'-endostructural conformation.
  • the modified nucleotides comprise 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, resulting in unlocked "sugar” residues.
  • UNA also encompasses monomers in which the bond between C1'-C4' has been removed (i.e., the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons).
  • the C2'-C3' bond of the sugar i.e., the covalent carbon-carbon bond between the C2' and C3' carbons
  • the modified nucleotides comprise one or more monomers of GNA (glycerol nucleic acid) nucleotides.
  • GNA includes GNA-A, GNA-T, GNA-C, GNA-G and GNA-U, all of which are in S configuration.
  • the structure of GNA-A is The structure of GNA-T is The structure of GNA-C is The structure of GNA-G is The structure of GNA-U is
  • modified nucleotides may also include one or more bicyclic sugar moieties.
  • Bicyclic sugars are furanosyl rings modified by a bridge of two atoms.
  • BNA Bicyclic nucleosides
  • BNA are nucleosides having a sugar moiety that contains a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system.
  • the bridge connects the 4'-carbon and 2'-carbon of the sugar ring.
  • conjugation in the present invention means that two or more chemical moieties each having a specific function are connected to each other in a covalent manner; accordingly, “conjugate” means that each A compound formed by covalent linkages between chemical parts.
  • ligand in the present invention refers to a targeting group, such as a cell or tissue targeting agent, such as a lectin, glycoprotein, lipid or protein, that binds to a specified cell type such as kidney cells, For example, antibodies.
  • a targeting group such as a cell or tissue targeting agent, such as a lectin, glycoprotein, lipid or protein, that binds to a specified cell type such as kidney cells, For example, antibodies.
  • Targeting groups can be thyroid stimulating hormone, melanocyte stimulating hormone, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine (GalNAc) , N-acetyl-glucosamine polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonates, polyglutamic acid, polyaspartic acid , lipids, cholesterol, steroids, cholic acid, folic acid, vitamin B12, vitamin A, biotin, or RGD peptide or RGD peptide mimetic.
  • the "overhang” in the present invention refers to at least one unpaired nucleotide protruding from the double-stranded region structure of the 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 comprise at least one nucleotide; or the overhang may comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five or more nucleosides acid.
  • the nucleotides of the nucleotide overhang are optionally modified nucleotides.
  • the overhang can be on the sense strand, the antisense strand, or any combination thereof. In addition, the overhang may be present at the 5'-end, the 3'-end, or both ends of the antisense or sense strand of the double-stranded compound.
  • the antisense strand has 1 to 10 nucleotides at the 3'-end and/or 5'-end (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides) overhang.
  • the sense strand has 1 to 10 nucleotides at the 3'-end and/or 5'-end (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides) overhang.
  • the antisense strand is at the 3'-end and the sense strand has 1 to 10 nucleotides at the 3'-end (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides) overhang. In some embodiments of the invention, the antisense strand is at the 5'-end and the sense strand has 1 to 10 nucleotides at the 5'-end (e.g., 1, 2, 3, 4, 5, 6, 7, 8 , 9 or 10 nucleotides) overhang.
  • one end of the sense strand and one end of the antisense strand of the present invention can also be connected through one or more nucleotides, which refers to the connection method described in the following formula (I): Where N represents an optionally modified nucleotide, n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; strand 1 and strand 2 are independently the sense strand or the antisense strand. .
  • multiple in the present invention refers to integers 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 conjugate of a double-stranded RNAi analog according to the present invention is a compound formed by connecting a double-stranded RNAi analog and a pharmaceutically acceptable conjugation group, and the double-stranded RNAi analogue and a pharmaceutically acceptable conjugation group are Groups are covalently connected.
  • linker refers to an organic moiety group that connects two parts of a compound, eg, covalently attaches two parts of a compound.
  • the bond usually contains a direct bond or an atom (such as oxygen or sulfur), an atomic group (such as: NRR, C(O), C(O)NH, SO, SO 2 , SO 2 NH), a substituted or unsubstituted Substituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted Substituted heterocycloalkyl, wherein optional one or more C atoms in the substituted or unsubstituted alkyl, substituted or substituted alkenyl, substituted or unsubstituted alkyny
  • the cleavable linker has sufficient stability outside the cell, but when it enters the target cell, it will be cleaved to release the two parts of the group that the linker is fixed together.
  • the compounds of the present invention may exist in specific geometric or stereoisomeric forms.
  • the present invention contemplates all such compounds, including the (R)- and (S)-enantiomers, diastereomers, and racemic and other mixtures thereof, such as enantiomers or diastereomers. body-enriched mixtures, all such mixtures are within the scope of the present invention.
  • Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are included within the scope of the present invention.
  • enantiomers or “optical isomers” refer to stereoisomers that are mirror images of each other.
  • diastereomer refers to stereoisomers whose molecules have two or more chiral centers and are in a non-mirror image relationship between the molecules.
  • use wedge-shaped solid line keys and wedge-shaped dotted keys Represents the absolute configuration of a three-dimensional center
  • using straight solid line keys and straight dotted keys Represent the relative configuration of the three-dimensional center with a wavy line
  • wedge-shaped solid line key or wedge-shaped dotted key or use tilde Represents a straight solid line key and/or straight dotted keys
  • the terms “enriched in an isomer,” “enantiomerically enriched,” “enriched in an enantiomer,” or “enantiomerically enriched” refer to one of the isomers or enantiomers.
  • the content of the 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%.
  • isomeric 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%, then the isomer or enantiomeric excess (ee value) is 80% .
  • optically active (R)- and (S)-isomers as well as the 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 invention is desired, it can be prepared by asymmetric synthesis or derivatization with chiral auxiliaries, in which the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide pure desired enantiomer.
  • a diastereomeric salt is formed with a suitable optically active acid or base, and then the salt is formed by conventional methods known in the art. Diastereomeric resolution is performed and the pure enantiomers are recovered. Furthermore, the separation of enantiomers and diastereomers is usually accomplished by the use of chromatography using chiral stationary phases, optionally combined with chemical derivatization methods (e.g., generation of amino groups from amines). formate).
  • the compounds of the present invention may contain unnatural proportions of atomic isotopes on one or more of the atoms that make up the compound.
  • compounds can be labeled with radioactive isotopes, such as tritium ( 3 H), iodine-125 ( 125 I), or C-14 ( 14 C).
  • deuterated drugs can be replaced by heavy hydrogen to form deuterated drugs.
  • the bond between deuterium and carbon is stronger than the bond between ordinary hydrogen and carbon.
  • deuterated drugs can reduce side effects and increase drug stability. , enhance efficacy, extend drug biological half-life and other advantages. All variations in the isotopic composition of the compounds of the invention, whether radioactive or not, are included within the scope of the invention.
  • salt refers to a salt of a compound of the present invention prepared from a compound having specific substituents discovered in the present invention and a relatively non-toxic acid or base.
  • the compound of the present invention contains a relatively acidic functional group, it can be prepared by using it in a pure solution or a suitable inert solvent.
  • Base addition salts are obtained by contacting a sufficient amount of base with such compounds.
  • Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts.
  • acid addition salts can be obtained by contacting such compounds with a sufficient amount of acid in neat solution or in a suitable inert solvent.
  • Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, Hydrogen sulfate, hydriodic acid, phosphorous acid, etc.; and organic acid salts, including acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, Similar acids such as fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid; 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 both basic and acidic functional
  • the salts of the present invention can be synthesized by conventional chemical methods from parent compounds containing acid groups or bases.
  • such salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of the two.
  • 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 synthesis methods, and methods well known to those skilled in the art. Equivalent alternatives and preferred embodiments include, but are not limited to, embodiments of the present invention.
  • the compounds of the present invention may contain unnatural proportions of atomic isotopes on one or more of the atoms that make up the compound.
  • compounds can be labeled with radioactive isotopes, such as tritium ( 3 H), iodine-125 ( 125 I), or C-14 ( 14 C).
  • deuterated drugs can be replaced by heavy hydrogen to form deuterated drugs. The bond between deuterium and carbon is stronger than the bond between ordinary hydrogen and carbon. Compared with non-deuterated drugs, deuterated drugs can reduce side effects and increase drug stability. , enhance efficacy, extend drug biological half-life and other advantages. All variations in the isotopic composition of the compounds of the invention, whether radioactive or not, are included within the scope of the invention.
  • the direction of connection is arbitrary, for example, The middle linking group L is -MW-.
  • -MW- can be connected to ring A and ring B in the same direction as the reading order from left to right. You can also connect ring A and ring B in the opposite direction to the reading order from left to right.
  • substituted means that any one or more hydrogen atoms on a specific atom are replaced by a substituent, which may include deuterium and hydrogen variants, as long as the valence state of the specific 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 or may not be substituted. Unless otherwise specified, the type and number of substituents may be arbitrary on the basis of chemical achievability.
  • any variable e.g., R
  • its definition in each instance is independent.
  • said group may optionally be substituted by up to two R's, with independent options for R in each case.
  • 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.
  • the substituent can be bonded through any atom thereof.
  • a pyridyl group as a substituent can be bonded through any one of the pyridine rings. The carbon atom is attached to the substituted group.
  • C 1-3 alkoxy means those alkyl groups containing 1 to 3 carbon atoms that are attached to the remainder of the molecule through an oxygen atom.
  • the C 1-3 alkoxy group includes C 1-2 , C 2-3 , C 3 and C 2 alkoxy groups, etc.
  • Examples of C 1-3 alkoxy include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), and the like.
  • C 2-4 alkenyl is used to mean a straight or branched hydrocarbon group consisting of 2 to 4 carbon atoms containing at least one carbon-carbon double bond. Can be located anywhere on the group.
  • the C 2-4 alkenyl group includes C 2-3 , C 4 , C 3 and C 2 alkenyl groups, etc.; the C 2-4 alkenyl group can be monovalent, divalent or multivalent. Examples of C 2-4 alkenyl groups include, but are not limited to, vinyl, propenyl, butenyl, butadienyl, and the like.
  • C 2-3 alkenyl is used to mean a linear or branched hydrocarbon group consisting of 2 to 3 carbon atoms containing at least one carbon-carbon double bond, carbon-carbon double bond Can be located anywhere on the group.
  • the C 2-3 alkenyl group includes C 3 and C 2 alkenyl groups; the C 2-3 alkenyl group can be monovalent, divalent or multivalent. Examples of C 2-3 alkenyl groups include, but are not limited to, vinyl, propenyl, and the like.
  • C 2-4 alkynyl is used to mean a straight-chain or branched hydrocarbon group consisting of 2 to 4 carbon atoms containing at least one carbon-carbon triple bond. Can be located anywhere on the group.
  • the C 2-4 alkynyl group includes C 2-3 , C 4 , C 3 and C 2 alkynyl groups, etc. It can be monovalent, bivalent or polyvalent. Examples of C 2-4 alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, and the like.
  • the number of atoms in a ring is usually defined as the number of ring members.
  • a "5- to 7-membered ring” refers to a “ring” with 5 to 7 atoms arranged around it.
  • C 3-6 cycloalkyl means a saturated cyclic hydrocarbon group composed of 3 to 6 carbon atoms, which is a monocyclic and bicyclic system, and the C 3-6 cycloalkyl group includes C 3-5 , C 4-5 and C 5-6 cycloalkyl, etc.; it can be monovalent, divalent or multivalent.
  • C 3-6 cycloalkyl Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.
  • C n-n+m or C n -C n+m includes any specific case of n to n+m carbons, for example, C 1-12 includes C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 , C 11 , and C 12 , also include any range from n to n+m, for example, C 1-12 includes C 1-3 , C 1-6 , C 1-9 , C 3-6 , C 3-9 , C 3-12 , C 6-9 , C 6-12 , and C 9-12 , etc.; similarly, n yuan to n The +m member indicates that the number of atoms in the ring is n to n+m.
  • a 3-12 membered ring includes a 3-membered ring, a 4-membered ring, a 5-membered ring, a 6-membered ring, a 7-membered ring, an 8-membered ring, and a 9-membered ring.
  • 3-membered ring includes 3-6-membered ring, 3-9-membered ring, 5-6-membered ring ring, 5-7 membered ring, 6-7 membered ring, 6-8 membered ring, and 6-10 membered ring, etc.
  • the term "4-8 membered heterocycloalkyl" by itself or in combination with other terms means a saturated cyclic group consisting of 4 to 8 ring atoms, with 1, 2, 3 or 4 ring atoms. are heteroatoms independently selected from O, S and N, and the remainder are carbon atoms, in which the nitrogen atoms are optionally quaternized, and the nitrogen and sulfur heteroatoms can be optionally oxidized (i.e., NO and S(O) p , p is 1 or 2). It includes single-ring and double-ring systems, where the double-ring system includes spiro rings, parallel rings and bridged rings.
  • a heteroatom may occupy the attachment position of the heterocycloalkyl to the rest of the molecule.
  • the 4-8-membered heterocycloalkyl group includes 4-5-membered, 4-6-membered, 5-6-membered, 4-membered, 5-membered and 6-membered heterocycloalkyl groups, etc.
  • 4-8 membered heterocycloalkyl examples include, but are not limited to, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrothienyl ( 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, 4-morpholinyl, etc.), Dioxanyl, dithianyl, isoxazolidinyl, isothiazolidinyl
  • C 6-10 aromatic ring and “C 6-10 aryl” in the present invention can be used interchangeably, and the term “C 6-10 aromatic ring” or “C 6-10 aryl” means A cyclic hydrocarbon group composed of 6 to 10 carbon atoms with a conjugated ⁇ electron system. It can be a monocyclic, fused bicyclic or fused tricyclic system, in which each ring is aromatic. It can be monovalent, divalent or multivalent, and C 6-10 aryl groups include C 6-9 , C 9 , C 10 and C 6 aryl groups, etc. Examples of C 6-10 aryl groups include, but are not limited to, phenyl, naphthyl (including 1-naphthyl, 2-naphthyl, etc.).
  • 5-10 membered heteroaromatic ring and “5-10 membered heteroaryl” in the present invention can be used interchangeably.
  • the term “5-10 membered heteroaryl” means a ring consisting of 5 to 10 rings.
  • the nitrogen atoms are optionally quaternized, and the nitrogen and sulfur heteroatoms are optionally oxidized (i.e., NO and S(O) p , p is 1 or 2).
  • a 5- to 10-membered heteroaryl group can be attached to the rest of the molecule through a heteroatom or a carbon atom.
  • the 5- to 10-membered heteroaryl groups include 5- to 8-membered, 5- to 7-membered, 5- to 6-membered, 5- to 6-membered heteroaryl groups, and the like.
  • Examples of the 5-10 membered heteroaryl include, but are not limited to, pyrrolyl (including N-pyrrolyl, 2-pyrrolyl and 3-pyrrolyl, etc.), pyrazolyl (including 2-pyrazolyl and 3-pyrrolyl).
  • azolyl group, etc. imidazolyl group (including N-imidazolyl, 2-imidazolyl, 4-imidazolyl and 5-imidazolyl, etc.), oxazolyl (including 2-oxazolyl, 4-oxazolyl and 5-oxazolyl) Oxazolyl, etc.), triazolyl (1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, 1H-1,2,4-triazolyl and 4H-1, 2,4-triazolyl, etc.), tetrazolyl, isoxazolyl (3-isoxazolyl, 4-iso Oxazolyl and 5-isoxazolyl, etc.), thiazolyl (including 2-thiazolyl, 4-thiazolyl and 5-thiazolyl, etc.), furyl (including 2-furyl and 3-furyl, etc.), Thienyl (including 2-thienyl and 3-thienyl, etc.), pyridyl
  • 5-6 membered heteroaromatic ring and “5-6 membered heteroaryl” may be used interchangeably in the present invention
  • the term “5-6 membered heteroaryl” means 5 to 6 ring atoms. It consists of a monocyclic group with a conjugated ⁇ electron system, in which 1, 2, 3 or 4 ring atoms are heteroatoms independently selected from O, S and N, and the rest are carbon atoms.
  • the nitrogen atoms are optionally quaternized, and the nitrogen and sulfur heteroatoms are optionally oxidized (i.e., NO and S(O)p, p is 1 or 2).
  • a 5-6 membered heteroaryl group can be attached to the rest of the molecule through a heteroatom or a carbon atom.
  • the 5-6 membered heteroaryl group includes 5-membered and 6-membered heteroaryl groups.
  • Examples of the 5-6 membered heteroaryl include but are not limited to pyrrolyl (including N-pyrrolyl, 2-pyrrolyl and 3-pyrrolyl, etc.), pyrazolyl (including 2-pyrazolyl and 3-pyrrolyl).
  • azolyl group, etc. imidazolyl group (including N-imidazolyl, 2-imidazolyl, 4-imidazolyl and 5-imidazolyl, etc.), oxazolyl (including 2-oxazolyl, 4-oxazolyl and 5-oxazolyl) Oxazolyl, etc.), triazolyl (1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, 1H-1,2,4-triazolyl and 4H-1, 2,4-triazolyl, etc.), tetrazolyl, isoxazolyl (3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, etc.), thiazolyl (including 2-thiazolyl , 4-thiazolyl and 5-thiazolyl, etc.), furyl (including 2-furyl and 3-furyl, etc.), thienyl (including 2-thienyl and 3-thienyl, etc.), pyrid
  • any one or more sites of the group can be connected to other groups through chemical bonds.
  • connection mode of the chemical bond is non-positioned and there are H atoms at the connectable site, when the chemical bond is connected, the number of H atoms at the site will be reduced correspondingly with the number of connected chemical bonds and become the corresponding valence. group.
  • the chemical bond connecting the site to other groups can be a straight solid line bond straight dashed key or wavy lines express.
  • the straight solid line bond in -OCH 3 means that it is connected to other groups through the oxygen atom in the group;
  • the straight dotted bond in means that it is connected to other groups through both ends of the nitrogen atoms in the group;
  • the wavy lines in indicate that the phenyl group is connected to other groups through the 1 and 2 carbon atoms in the phenyl group; Indicates that any connectable site on the benzene ring can be connected to it through a chemical bond connected to other groups, including at least These 4 connection methods.
  • 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 synthesis methods, and methods well known to those skilled in the art. Equivalent alternatives and preferred embodiments include, but are not limited to, embodiments of the present invention.
  • the structure of the compound of the present invention can be confirmed by conventional methods well known to those skilled in the art. If the present invention involves the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the art.
  • single crystal X-ray diffraction uses a Bruker D8 venture diffractometer to collect diffraction intensity data on the cultured single crystal.
  • the light source is CuK ⁇ radiation.
  • the scanning method is: After scanning and collecting relevant data, the direct method (Shelxs97) is further used to analyze the crystal structure, and the absolute configuration can be confirmed.
  • the solvent used in the present invention is commercially available.
  • the solvent ratios used for column chromatography and thin-layer silica gel chromatography in the present invention are all volume ratios.
  • aq represents water
  • HATU O-(7-azabenzotriazol-1-yl)-N, N, N', N'-tetramethylurea hexafluorophosphate
  • EDC represents N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride
  • m-CPBA 3-chloroperoxybenzoic acid
  • eq represents equivalent, equivalent
  • CDI represents Carbonyldiimidazole
  • DCM represents dichloromethane
  • PE represents petroleum ether
  • DIAD represents diisopropyl azodicarboxylate
  • DMF represents N, N-dimethylformamide
  • DMSO represents dimethyl sulfoxide
  • EtOAc represents ethyl acetate Ester
  • EtOH represents ethanol
  • MeOH represents methanol
  • CBz represents benzyloxycarbonyl, which is an amine protecting group
  • BOC represents tert-but
  • nucleotide monomers are used in the description of nucleic acid sequences, as shown in Table 3:
  • RNAi drugs containing the novel structure described in this patent can significantly reduce the level of target gene mRNA in both cell and mouse HDI models, indicating that they have good activity.
  • the novel structure of the RNAi drug described in this patent has no risk of immunogenicity in in vitro testing, indicating that it has good safety.
  • the synthesized sequences are all chemically synthesized oligonucleotides.
  • the oligonucleotides containing solid phase carriers and protective groups are synthesized through multi-step solid phase, and finally the ammonia is deprotected and purified.
  • the solid phase synthesis method is as follows:
  • nucleotide monomers are used in the description of nucleic acid sequences, as shown in Table 5:
  • D01 ligand is as follows:
  • the carrier containing the protective oligonucleotide sequence was obtained through solid-phase synthesis, and then cleaved and deprotected from the solid-phase carrier to obtain the crude oligonucleotide, which was then aminolyzed (aminolysis conditions: ammonia water, 55°C, 16h. HPLC purification was performed Afterwards, the single chain can be directly freeze-dried to obtain the pure product. The double chain needs to be annealed and freeze-dried to obtain conjugate S16.
  • conjugate S26 was prepared by referring to the method of conjugate S16.
  • the purpose of this study is to use Huh7 cells to evaluate the activity of the conjugate described in the patent in inhibiting target genes, such as angiotensinogen (AGT) gene, using the EC 50 value of the compound as an indicator to evaluate the effect of the compound on Inhibitory activity of the target gene AGT.
  • target genes such as angiotensinogen (AGT) gene
  • Huh7 cells were provided by Shanghai WuXi AppTec New Drug Development Co., Ltd. Huh7 cells were cultured in MEM medium containing 10% fetal calf serum and 1% penicillin-streptomycin.
  • the main reagents used in this experiment include Lipofectamine TM iRNAiMAX transfection reagent, FastStart Universal Probe Mast, RNA extraction kit, GAPDH gene expression kit, AGT gene expression kit, FastKing cDNA first strand synthesis kit, 96-well plate.
  • the main instruments used in this experiment include fluorescence qPCR instrument (Applied Biosystems, model QuantStudio 6 Flex) and cell counter (Vi-cellTM XR).
  • RNAiMAX Optim-MEM culture medium For each well of cells, add a certain amount of diluted compound to an equal amount of RNAiMAX Optim-MEM culture medium, mix well, and incubate for 15 minutes;
  • the cells were cultured in a 5% CO 2 and 37°C incubator for 24 hours.
  • Collect cells extract RNA according to the instructions of Qiagen-74182 RNA extraction kit, and reverse-transcribe RNA into cDNA according to the instructions of FastKing cDNA First Strand Synthesis Kit.
  • qPCR was used to detect the cDNA of the target gene, GAPDH was used as the internal reference gene, and qPCR was performed in a 384-well plate.
  • the qPCR reaction program is: heat at 95°C for 10 minutes, then enter the cycle mode, heat at 95°C for 15 seconds, then 60°C for 1 minute, a total of 40 cycles.
  • This method requires the introduction of the housekeeping gene GAPDH, because the housekeeping gene is expressed in all cells, and its product is necessary to maintain cell survival.
  • the expression level in the cell or the copy number of the genome is constant and is less affected by the environment.
  • the conjugate of the present invention can significantly inhibit the level of AGT gene in Huh7 cells.
  • the mouse model of high-pressure tail vein injection of pcDNA-CMV-AGT plasmid was used to evaluate the effect of targeting the target gene and inhibiting the target gene in the test sample.
  • pcDNA-CMV-AGT plasmid BALB/c female mouse, DPBS (Dulbecco's phosphate buffer saline), conjugate of the invention.
  • mice Order BALB/c female mice aged 6-8 weeks, and the mice will adapt to quarantine for one week after arriving in the animal room.
  • mice were randomly divided into groups according to their body weight data, with 4 mice in each group. After grouping, all mice were given subcutaneous injection, a single dose, and the dosage volume was 5 mL/kg.
  • the mice in the first group were given DPBS; Two groups of mice were given the conjugate of the present invention at 5 mg/kg.
  • mice in all groups were euthanized by CO2 inhalation, and 2 liver samples were collected from each mouse. Liver samples were treated with RNAlater overnight at 4°C, and then the RNAlater was removed and stored at -80°C for detection of AGT gene expression levels.
  • the conjugate of the present invention can significantly down-regulate the expression of AGT mRNA in mouse liver, and the magnitude of the down-regulation reaches 93%.
  • Test Example 3 Test of the agonistic activity of the conjugate of the present invention on hTLR3, hTLR7, and hTLR8 cells
  • the main instruments used in this research are multifunctional microplate reader Flexstation III (Molecular Device) and Echo555 (Labcyte)
  • Compound activity detection Take 20 ⁇ L of cell supernatant from each well and add it to the experimental plate containing 180 ⁇ L of QUANTI-Blue TM reagent. After incubating at 37°C for 1 hour, use a multifunctional microplate reader Flexstation III to detect the absorbance value at 650 nm (OD650). .
  • Cell viability detection Operate according to Celltiter-Glo instructions, and the chemiluminescence signal (RLU) is detected with a multifunctional microplate reader Flexstation III.
  • Compound activity The OD650 value was analyzed using GraphPad Prism software, and the compound dose-effect curve was fitted to calculate the EC 50 value of the compound.

Abstract

Provided is a double-stranded RNAi compound, particularly a class of double-stranded RNAi compounds containing an overhang consisting of natural nucleotides.

Description

一类含由天然核苷酸组成突出端的双链RNAi化合物A class of double-stranded RNAi compounds containing overhangs composed of natural nucleotides
本发明主张如下的优先权：The present invention claims the following priority:
申请号：CN202210452045.7，申请日：2022年4月26日。Application number: CN202210452045.7, application date: April 26, 2022.
技术领域Technical field
本发明涉及双链RNAi化合物，具体涉及一类含由天然核苷酸组成突出端的双链RNAi化合物。The present invention relates to double-stranded RNAi compounds, in particular to a class of double-stranded RNAi compounds containing overhangs composed of natural nucleotides.
背景技术Background technique
RNA干扰(RNAi)机制在1998年被发现以来一直被密切关注；该机制通过下调靶标mRNA水平来实现对相应靶点功能的控制从而达到预期的药理效果。近年来，有多个RNAi药物获批上市，完成了应用于人体药物的可行性验证。通过该机制实现药理效果的药物被称为RNAi药物。The RNA interference (RNAi) mechanism has been closely watched since its discovery in 1998; this mechanism controls the function of the corresponding target by down-regulating the level of target mRNA to achieve the desired pharmacological effect. In recent years, a number of RNAi drugs have been approved for marketing, and the feasibility verification of their application in human drugs has been completed. Drugs that achieve pharmacological effects through this mechanism are called RNAi drugs.
RNAi药物通常由一条含有16-27个碱基对的RNA双链组成，其双链结构是决定RNAi药物性质的重要因素。常见的双链结构包括：(1)平末端的对称双链结构，即正义链和反义链均无突出端的完全互补配对的双链；(2)带突出端的对称双链结构，即正义链的3’端和反义链的5’带有相同碱基数的突出端，或正义链的5’端和反义链的3’端带有相同碱基数的突出端；(3)一端为平末端的非对称双链结构。该结构中，通常是在反义链的3’端含有不同数量的非配对碱基。上述三种双链结构被大量报道，且广泛应用于已经上市或正在开发中的RNAi药物。RNAi drugs usually consist of an RNA double strand containing 16-27 base pairs, and its double-stranded structure is an important factor in determining the properties of RNAi drugs. Common double-stranded structures include: (1) symmetrical double-stranded structure with blunt ends, that is, a completely complementary double-stranded double strand in which neither the sense strand nor the antisense strand has overhangs; (2) symmetrical double-stranded structure with overhangs, that is, the sense strand The 3' end of the sense strand and the 5' end of the antisense strand have overhangs with the same number of bases, or the 5' end of the sense strand and the 3' end of the antisense strand have overhangs with the same number of bases; (3) One end It is a blunt-ended asymmetric double-stranded structure. This structure usually contains a varying number of unpaired bases at the 3’ end of the antisense strand. The above three double-stranded structures have been widely reported and widely used in RNAi drugs that are already on the market or under development.
尽管如此，开发新的RNA双链结构依然是提高RNAi药物有效性和长效性的重要手段。因此，本专利提供了一种可用于RNAi药物新的RNA双链结构。与已知的双链结构相比，使用该新型结构的RNAi药物可以显著提高药物的有效性。Despite this, developing new RNA double-stranded structures is still an important means to improve the effectiveness and long-term effects of RNAi drugs. Therefore, this patent provides a new RNA double-stranded structure that can be used in RNAi drugs. Compared with the known double-stranded structure, RNAi drugs using this new structure can significantly improve the effectiveness of the drug.
发明内容Contents of the invention
本发明提供了一种双链RNAi化合物，其中所述化合物包含能够形成双链区的正义链和反义链，所述双链区由14-29个核苷酸碱基对组成，所述反义链的5’端含有突出端，所述突出端由1-10个天然核苷酸组成，所述正义链的5’端或反义链的3’端任选含有突出端，所述双链区、正义链5’端的突出端或反义链3’端的突出端的核苷酸任选经修饰。The invention provides a double-stranded RNAi compound, wherein the compound includes a sense strand and an antisense strand capable of forming a double-stranded region, the double-stranded region consisting of 14-29 nucleotide base pairs, and the antisense strand The 5' end of the sense strand contains an overhang, and the overhang is composed of 1-10 natural nucleotides. The 5' end of the sense strand or the 3' end of the antisense strand optionally contains an overhang, and the double end The nucleotides in the strand region, the overhang at the 5' end of the sense strand, or the overhang at the 3' end of the antisense strand are optionally modified.
在本发明的一些技术方案中，上述双链区由14、15、16、17、18、19、20、21、22、23、24、25、26、27、28或29个核苷酸碱基对组成，其他变量如本发明所定义。In some technical solutions of the present invention, the above-mentioned double-stranded region consists of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 nucleotide bases The basis pairs are composed, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述双链区由17、18、19、20、21、22或23个核苷酸组成，其他变量如本发明所定义。In some technical solutions of the present invention, the above-mentioned double-stranded region consists of 17, 18, 19, 20, 21, 22 or 23 nucleotides, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述双链区的核苷酸全部经修饰，其他变量如本发明所定义。In some technical solutions of the present invention, all the nucleotides in the above-mentioned double-stranded region are modified, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述双链区的正义链的至少一个经修饰的核苷酸是指包含修饰的糖 环的核苷酸，其他变量如本发明所定义。In some technical solutions of the present invention, at least one modified nucleotide of the sense strand of the above-mentioned double-stranded region refers to a modified sugar containing cyclic nucleotides, and other variables are as defined herein.
在本发明的一些技术方案中，上述包含修饰的糖环的核苷酸选自甘油核酸(GNA)，其他变量如本发明所定义。In some technical solutions of the present invention, the above-mentioned nucleotides containing modified sugar rings are selected from glycerol nucleic acid (GNA), and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述双链区的正义链的至少一个经修饰的核苷酸是指包含修饰的核碱基的核苷酸，其他变量如本发明所定义。In some technical solutions of the present invention, at least one modified nucleotide of the sense strand of the above-mentioned double-stranded region refers to a nucleotide containing a modified nucleobase, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述双链区的反义链的至少一个经修饰的核苷酸是指包含修饰的核碱基的核苷酸，其他变量如本发明所定义。In some technical solutions of the present invention, at least one modified nucleotide of the antisense strand of the double-stranded region refers to a nucleotide containing a modified nucleobase, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述修饰的核碱基选自取代或未取代的三氮唑、 其他变量如本发明所定义。In some technical solutions of the present invention, the above-mentioned modified nucleobase is selected from substituted or unsubstituted triazole, Other variables are as defined in the present invention.
在本发明的一些技术方案中，上述正义链的5’端的突出端含有0、1、2、3、4、5、6、7或8个核苷酸，所述核苷酸任选经修饰，其他变量如本发明所定义。In some technical solutions of the present invention, the overhang of the 5' end of the above-mentioned sense strand contains 0, 1, 2, 3, 4, 5, 6, 7 or 8 nucleotides, and the nucleotides are optionally modified , other variables are as defined in the present invention.
在本发明的一些技术方案中，上述正义链的5’端的突出端含有0、1、2或3个核苷酸，所述核苷酸任选经修饰，其他变量如本发明所定义。In some technical solutions of the present invention, the overhang of the 5' end of the above-mentioned sense strand contains 0, 1, 2 or 3 nucleotides, and the nucleotides 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，上述正义链5’端的突出端的核苷酸全部经修饰，其他变量如本发明所定义。In some technical solutions of the present invention, all the nucleotides at the overhang of the 5' end of the sense strand are modified, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述反义链的3’端的突出端含有0、1、2、3、4、5、6、7或8个核苷酸，所述核苷酸任选经修饰，其他变量如本发明所定义。In some technical solutions of the present invention, the overhang of the 3' end of the above-mentioned antisense strand contains 0, 1, 2, 3, 4, 5, 6, 7 or 8 nucleotides, and the nucleotides are optionally Modification, other variables are as defined in the present invention.
在本发明的一些技术方案中，上述反义链的3’端的突出端含有0、1、2、3、4、5或6个核苷酸，所述核苷酸任选经修饰，其他变量如本发明所定义。In some technical solutions of the present invention, the overhang of the 3' end of the above-mentioned antisense strand contains 0, 1, 2, 3, 4, 5 or 6 nucleotides, and the nucleotides are optionally modified, and other variables As defined herein.
在本发明的一些技术方案中，上述反义链3’端的突出端的核苷酸全部经修饰，其他变量如本发明所定义。In some technical solutions of the present invention, all the nucleotides of the overhang at the 3' end of the antisense strand are modified, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述反义链5’端的突出端由1、2、3、4、5、6、7、8、9或10个天然核苷酸组成，其他变量如本发明所定义。In some technical solutions of the present invention, the overhang at the 5' end of the antisense strand is composed of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 natural nucleotides, and other variables are as in the present invention defined.
在本发明的一些技术方案中，上述正义链或反义链的任意位置缀合配体，其他变量如本发明所定义。 In some technical solutions of the present invention, a ligand is conjugated to any position of the above-mentioned sense strand or antisense strand, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述正义链和反义链之间除了碱基之间的氢键外，不存在其他的共价连接，其他变量如本发明所定义。In some technical solutions of the present invention, except for hydrogen bonds between bases, there are no other covalent connections between the above-mentioned sense strand and antisense strand, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述正义链的一端与反义链的一端还可以通过一个或多个核苷酸连接，其他变量如本发明所定义。In some technical solutions of the present invention, one end of the above-mentioned sense strand and one end of the antisense strand can also be connected through one or more nucleotides, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述正义链的一端与反义链的一端还可以通过化学基团连接，其他变量如本发明所定义。In some technical solutions of the present invention, one end of the above-mentioned sense strand and one end of the antisense strand can also be connected through chemical groups, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述正义链和/或反义链的任意位置缀合配体，其他变量如本发明所定义。In some technical solutions of the present invention, a ligand is conjugated to any position of the above-mentioned sense strand and/or antisense strand, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述正义链的3’端缀合配体，其他变量如本发明所定义。In some technical solutions of the present invention, the 3' end of the above-mentioned sense strand is conjugated with a ligand, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述正义链的5’端缀合配体，其他变量如本发明所定义。In some technical solutions of the present invention, the 5' end of the above-mentioned sense strand is conjugated with a ligand, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述反义链的3’端缀合配体，其他变量如本发明所定义。In some technical solutions of the present invention, the 3' end of the above-mentioned antisense chain is conjugated with a ligand, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述反义链的5’端缀合配体，其他变量如本发明所定义。In some technical solutions of the present invention, the 5' end of the above-mentioned antisense strand is conjugated with a ligand, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述正义链的3’端、正义链的5’端、反义链的3’端和反义链的5’端缀合配体是指3’端或5’端的羟基通过磷酸酯键和/或硫代磷酸酯键与配体连接，其他变量如本发明所定义。In some technical solutions of the present invention, the 3' end of the sense strand, the 5' end of the sense strand, the 3' end of the antisense strand and the 5' end conjugation ligand of the antisense strand refer to the 3' end or the 5' end of the antisense strand. The hydroxyl group at the end is connected to the ligand through a phosphate bond and/or a phosphorothioate bond, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述连接正义链与反义链之间的核苷酸上缀合配体，其他变量如本发明所定义。In some technical solutions of the present invention, a ligand is conjugated to the nucleotide connecting the sense strand and the antisense strand, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述连接正义链与反义链之间的化学基团上缀合配体，其他变量如本发明所定义。In some technical solutions of the present invention, a ligand is conjugated to the chemical group connecting the sense strand and the antisense strand, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述缀合配体的数量为1、2、3、4或5个，其他变量如本发明所定义。In some technical solutions of the present invention, the number of the above-mentioned conjugated ligands is 1, 2, 3, 4 or 5, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述配体为一种或多种利用二价或三价的分支键联体附接的GalNAc衍生物，其他变量如本发明所定义。In some technical solutions of the present invention, the above-mentioned ligand is one or more GalNAc derivatives attached using divalent or trivalent branched linkers, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述配体为D01或L96，其他变量如本发明所定义。
In some technical solutions of the present invention, the above-mentioned ligand is D01 or L96, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述正义链选自SEQ ID NO:1或SEQ ID NO:2，其他变量如本发明所定义。In some technical solutions of the present invention, the above-mentioned sense strand is selected from SEQ ID NO: 1 or SEQ ID NO: 2, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述正义链选自SEQ ID NO:1、SEQ ID NO:2或SEQ ID NO:13，其他变量如本发明所定义。In some technical solutions of the present invention, the above-mentioned sense strand is selected from SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 13, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述反义链选自SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11或SEQ ID NO:12，其他变量如本发明所定义。In some technical solutions of the present invention, the above-mentioned antisense chain is selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 , SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述反义链选自SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ  ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、或SEQ ID NO:23，其他变量如本发明所定义。In some technical solutions of the present invention, the above-mentioned antisense strand is selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 , SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述正义链选自SEQ ID NO:1，所述正义链的3’端缀合配体，其他变量如本发明所定义。In some technical solutions of the present invention, the above-mentioned sense strand is selected from SEQ ID NO: 1, the 3' end of the sense strand is conjugated with a ligand, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述正义链选自SEQ ID NO:1，所述正义链的3’端缀合配体，所述配体为D01，其他变量如本发明所定义。In some technical solutions of the present invention, the above-mentioned sense strand is selected from SEQ ID NO: 1, the 3' end of the sense strand is conjugated to a ligand, the ligand is D01, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述正义链选自SEQ ID NO:2，其他变量如本发明所定义。In some technical solutions of the present invention, the above-mentioned sense strand is selected from SEQ ID NO: 2, and other variables are as defined in the present invention.
本发明还有一些方案由上述各变量任意组合而来。There are also some solutions of the present invention that are derived from any combination of the above variables.
在本发明的一些技术方案中，上述缀合物选自S1、S2、S3、S4、S5、S6、S7、S8、S9、S10、S11、S12、S13、S14、S15、S16、S17、S18、S19和S20。In some technical solutions of the present invention, the above-mentioned conjugate is selected from S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S13, S14, S15, S16, S17, S18 , S19 and S20.
在本发明的一些技术方案中，上述缀合物选自S1、S2、S3、S4、S5、S6、S7、S8、S9、S10、S11、S12、S13、S14、S15、S16、S17、S18、S19、S20、S21、S22、S23、S24、S25、S26、S27、S28、S29和S30。In some technical solutions of the present invention, the above-mentioned conjugate is selected from S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S13, S14, S15, S16, S17, S18 , S19, S20, S21, S22, S23, S24, S25, S26, S27, S28, S29 and S30.
本发明相关序列和缀合物如表1所示，Relevant sequences and conjugates of the present invention are shown in Table 1.
表1本发明相关序列和缀合物

Table 1 Related sequences and conjugates of the present invention

在本发明的一些技术方案中，上述双链RNAi化合物用于抑制或阻断基因的表达，其他变量如本发明所定义。In some technical solutions of the present invention, the above-mentioned double-stranded RNAi compounds are used to inhibit or block gene expression, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述基因包括但限于雌激素受体基因、AGT基因或KRAS基因，其他变量如本发明所定义。 In some technical solutions of the present invention, the above-mentioned genes include but are limited to estrogen receptor genes, AGT genes or KRAS genes, and other variables are as defined in the present invention.
本发明还提供如下测试方法：The present invention also provides the following test methods:
方法1：小鼠HDI AGT模型Method 1: Mouse HDI AGT model
1.实验原理：1. Experimental principle:
通过高压尾静脉注射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。The mouse model of high-pressure tail vein injection of pcDNA-CMV-AGT plasmid was used to evaluate the effect of targeting the target gene and inhibiting the target gene in the test sample.
2.实验材料：pcDNA-CMV-AGT质粒，BALB/c雌性小鼠，DPBS(Dulbecco's磷酸缓冲液)，本发2. Experimental materials: pcDNA-CMV-AGT plasmid, BALB/c female mice, DPBS (Dulbecco's phosphate buffer), the present invention
明缀合物。Ming conjugates.
3.实验方法：3.Experimental method:
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 BALB/c female mice aged 6-8 weeks, and the mice will adapt to quarantine for one week after arriving in the animal room.
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DPBS；第2组小鼠给本发明缀合物，3mg/kg。On day 0, the mice were randomly divided into groups according to their body weight data, with 4 mice in each group. After grouping, all mice were given subcutaneous injection, a single dose, and the dosage volume was 10 mL/kg. The mice in the first group were given DPBS; Two groups of mice were given the conjugate of the present invention at 3 mg/kg.
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 a volume of 8% of their body weight of pcDNA-CMV-AGT plasmid via the tail vein within 5 seconds (injection volume (mL) = mouse body weight (g) × 8%), each mouse The mass of plasmid injected into mice 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 4th day after administration, mice in all groups were euthanized by CO2 inhalation, and 2 liver samples were collected from each mouse. Liver samples were treated with RNAlater overnight at 4°C, and then the RNAlater was removed and stored at -80°C for detection of AGT gene expression levels.
方法2：体外RNA sequence研究Method 2: In vitro RNA sequence study
1.实验介绍：1. Experiment introduction:
转录组是指特定组织或细胞在某个时间或某个状态下转录出来的所有RNA的总和，主要包括mRNA和非编码RNA。转录组测序是基于Illumina测序平台，研究特定组织或细胞在某个时期转录出来的所有mRNA，是基因功能与结构研究的基础，对理解生物体的发育和疾病的发生具有重要作用。随着基因测序技术的发展以及测序成本的降低，RNA-seq凭借高通量、高灵敏度、应用范围广等优势，已成为转录组研究的主要方法。RNA-seq技术流程主要包含两个部分：建库测序和生物信息分析。Transcriptome refers to the sum of all RNA transcribed from a specific tissue or cell at a certain time or in a certain state, mainly including mRNA and non-coding RNA. Transcriptome sequencing is based on the Illumina sequencing platform and studies all the mRNA transcribed by a specific tissue or cell at a certain period. It is the basis for the study of gene function and structure and plays an important role in understanding the development of organisms and the occurrence of diseases. With the development of gene sequencing technology and the reduction of sequencing costs, RNA-seq has become the main method for transcriptome research due to its advantages of high throughput, high sensitivity, and wide application range. The RNA-seq technology process mainly consists of two parts: library construction and sequencing and bioinformatics analysis.
2.RNA提取与检测：2.RNA extraction and detection:
采用标准提取方法从组织或细胞中提取RNA，随后对RNA样品进行严格质控，质控方式主要是通过Agilent 2100 bioanalyzer：精确检测RNA完整性。RNA is extracted from tissues or cells using standard extraction methods, and then strict quality control is performed on the RNA samples. The quality control method is mainly through the Agilent 2100 bioanalyzer: accurate detection of RNA integrity.
3.文库构建与质检：3. Library construction and quality inspection:
mRNA的获取主要有两种方式：一是利用真核生物大部分mRNA都带有polyA尾的结构特征，通过Oligo(dT)磁珠富集带有polyA尾的mRNA。二是从总RNA中去除核糖体RNA，从而得到mRNA。 随后在NEB Fragmentation Buffer中用二价阳离子将得到的mRNA随机打断，按照NEB普通建库方式或链特异性建库方式进行建库。There are two main ways to obtain mRNA: one is to take advantage of the structural characteristics that most eukaryotic mRNAs have polyA tails, and use Oligo (dT) magnetic beads to enrich mRNAs with polyA tails. The second is to remove ribosomal RNA from total RNA to obtain mRNA. The obtained mRNA is then randomly fragmented with divalent cations in NEB Fragmentation Buffer, and the library is constructed according to the NEB general library construction method or the strand-specific library construction method.
NEB普通建库：以片段化的mRNA为模版，随机寡核苷酸为引物，在M-MuLV逆转录酶体系中合成cDNA第一条链，随后用RNaseH降解RNA链，并在DNA polymerase I体系下，以dNTPs为原料合成cDNA第二条链。纯化后的双链cDNA经过末端修复、加A尾并连接测序接头，用AMPure XP beads筛选250-300bp左右的cDNA，进行PCR扩增并再次使用AMPure XP beads纯化PCR产物，最终获得文库。建库用试剂盒为UltraTM RNA Library Prep Kit for NEB ordinary library construction: Using fragmented mRNA as a template and random oligonucleotides as primers, the first strand of cDNA is synthesized in the M-MuLV reverse transcriptase system, and then RNaseH is used to degrade the RNA strand, and the first strand of cDNA is synthesized in the DNA polymerase I system. Next, dNTPs are used as raw materials to synthesize the second strand of cDNA. The purified double-stranded cDNA is end-repaired, A-tailed, and connected to sequencing adapters. AMPure XP beads are used to screen cDNA of about 250-300 bp, PCR amplification is performed, and AMPure XP beads are used again to purify the PCR products to finally obtain a library. The kit for library construction is Ultra TM RNA Library Prep Kit for
链特异性建库：逆转录合成cDNA第一条链方法与NEB普通建库方法相同，不同之处在于合成第二条链时，dNTPs中的dTTP由dUTP取代，之后同样进行cDNA末端修复、加A尾、连接测序接头和长度筛选，然后先使用USER酶降解含U的cDNA第二链再进行PCR扩增并获得文库。链特异性文库具有诸多优势，如相同数据量下可获取更多有效信息；能获得更精准的基因定量、定位与注释信息；能提供反义转录本及每一isoform中单一exon的表达水平。建库所用试剂盒为UltraTM Directional RNA Library Prep Kit for Strand-specific library construction: The method of reverse transcription to synthesize the first strand of cDNA is the same as the NEB general library construction method. The difference is that when synthesizing the second strand, dTTP in dNTPs is replaced by dUTP, and then the cDNA end repair and addition are also performed. A tail, ligation of sequencing adapters and length screening, then USER enzyme is used to degrade the second strand of U-containing cDNA and then PCR amplification is performed to obtain a library. Strand-specific libraries have many advantages, such as obtaining more effective information with the same amount of data; obtaining more precise gene quantification, positioning and annotation information; and providing antisense transcripts and the expression level of a single exon in each isoform. The kit used for library construction is Ultra TM Directional RNA Library Prep Kit for
注：测序接头：包括P5/P7，index和Rd1/Rd2 SP三个部分。其中P5/P7是PCR扩增引物及flow cell上引物结合的部分，index提供区分不同文库的信息，Rd1/Rd2 SP即read1/read2 sequence primer，是测序引物结合区域，测序过程理论上由Rd1/Rd2 SP向后开始进行。Note: Sequencing adapter: includes three parts: P5/P7, index and Rd1/Rd2 SP. Among them, P5/P7 is the part where the PCR amplification primer and the primer on the flow cell are combined. The index provides information to distinguish different libraries. Rd1/Rd2 SP is the read1/read2 sequence primer, which is the binding area of the sequencing primer. The sequencing process is theoretically composed of Rd1/ Rd2 SP starts backward.
文库构建完成后，先使用Qubit2.0 Fluorometer进行初步定量，稀释文库至1.5ng/ul，随后使用Agilent 2100 bioanalyzer对文库的insert size进行检测，insert size符合预期后，qRT-PCR对文库有效浓度进行准确定量(文库有效浓度高于2nM)，以保证文库质量。After the library construction is completed, first use Qubit2.0 Fluorometer for preliminary quantification, dilute the library to 1.5ng/ul, and then use Agilent 2100 bioanalyzer to detect the insert size of the library. After the insert size meets the expectations, qRT-PCR is performed on the effective concentration of the library. Accurate quantification (effective library concentration higher than 2nM) to ensure library quality.
4.上机测序：4. Sequencing on computer:
库检合格后，把不同文库按照有效浓度及目标下机数据量的需求pooling后进行Illumina测序。测序的基本原理是边合成边测序(Sequencing by Synthesis)。在测序的flow cell中加入四种荧光标记的dNTP、DNA聚合酶以及接头引物进行扩增，在每一个测序簇延伸互补链时，每加入一个被荧光标记的dNTP就能释放出相对应的荧光，测序仪通过捕获荧光信号，并通过计算机软件将光信号转化为测序峰，从而获得待测片段的序列信息。After the library passes the inspection, different libraries are pooled according to the effective concentration and target off-machine data volume requirements, and then Illumina sequencing is performed. The basic principle of sequencing is Sequencing by Synthesis. Four fluorescently labeled dNTPs, DNA polymerase and adapter primers are added to the sequencing flow cell for amplification. When each sequencing cluster extends the complementary strand, each fluorescently labeled dNTP is added to release the corresponding fluorescence. , the sequencer captures the fluorescence signal and converts the light signal into a sequencing peak through computer software to obtain the sequence information of the fragment to be tested.
方法3：化合物对hTLR3、hTLR7、hTLR8细胞的激动活性测试Method 3: Testing the agonistic activity of compounds on hTLR3, hTLR7, and hTLR8 cells
1.实验材料1. Experimental materials
表2实验材料表

Table 2 Experimental materials list

2.仪器2.Instruments
本研究所使用主要仪器为多功能酶标仪Flexstation III(Molecular Device)和Echo555(Labcyte)The main instruments used in this research are multifunctional microplate reader Flexstation III (Molecular Device) and Echo555 (Labcyte)
3.实验步骤3. Experimental steps
1)将化合物稀释到终浓度的20倍，与RNAiMAX-OPTI MEM(RNAiMAX:OPTI MEM＝3:47)1:1混合，室温孵育15分钟。1) Dilute the compound to 20 times the final concentration, mix 1:1 with RNAiMAX-OPTI MEM (RNAiMAX:OPTI MEM=3:47), and incubate at room temperature for 15 minutes.
2)取10μL孵育的化合物加入到相应的细胞板中，共10个浓度，每个浓度双复孔。2) Add 10 μL of the incubated compound to the corresponding cell plate for a total of 10 concentrations, with duplicate wells for each concentration.
3)阴性对照孔每孔加入10μL PBS，hTLR3的阳性对照孔每孔加入10μL 20μg/mL的Poly(I:C)HMW，hTLR7和hTLR8的阳性对照孔每孔加入10μL 10μg/mL R848。2个转染阳性对照孔加入10μL 0.5μM BLOCK-iTTM AlexaRed Fluorescent Control。3) Add 10 μL PBS to each well of the negative control well, add 10 μL 20 μg/mL Poly(I:C) HMW to each well of the positive control well of hTLR3, add 10 μL 10 μg/mL R848 to each well of the positive control wells of hTLR7 and hTLR8. 2 pcs. Add 10 μL of 0.5 μM BLOCK-iT TM Alexa to the transfection positive control well. Red Fluorescent Control.
4)将90μL细胞种于已经加好化合物的96孔板中，50,000细胞/孔。4) Seed 90 μL of cells into a 96-well plate with compound added, 50,000 cells/well.
5)将化合物和细胞在37℃、5％CO2培养箱共孵育24小时。5) Incubate the compound and cells in a 37°C, 5% CO2 incubator for 24 hours.
6)用荧光显微镜观察转染阳性对照孔的转染效果，并拍照。6) Use a fluorescence microscope to observe the transfection effect of the positive control wells and take photos.
7)化合物活性检测：每孔取20μL细胞上清，加入含有180μL QUANTI-BlueTM试剂的实验板中，37℃孵育1小时之后，用多功能酶标仪Flexstation III检测650nm的吸光度值(OD650)。7) Compound activity detection: Take 20 μL of cell supernatant from each well and add it to the experimental plate containing 180 μL of QUANTI-Blue TM reagent. After incubating at 37°C for 1 hour, use a multifunctional microplate reader Flexstation III to detect the absorbance value at 650 nm (OD650). .
8)细胞活性检测：按照Celltiter-Glo说明书方法操作，化学发光信号(RLU)用多功能酶标仪Flexstation III检测。8) Cell viability detection: Operate according to Celltiter-Glo instructions, and the chemiluminescence signal (RLU) is detected with a multifunctional microplate reader Flexstation III.
3.数据分析3.Data analysis
化合物活性：OD650值用GraphPad Prism软件分析，并拟合化合物剂量效应曲线，计算化合物的EC50值。细胞活性检测：细胞活性％计算公式如下。细胞活性％值用GraphPad Prism软件分析，并 拟合化合物剂量效应曲线，计算化合物对细胞的CC50值。
细胞活性％＝RLU化合物/RLUDMSO控制*100％Compound activity: The OD650 value was analyzed using GraphPad Prism software, and the compound dose-effect curve was fitted to calculate the EC 50 value of the compound. Cell activity detection: The calculation formula of cell activity % is as follows. Cell viability % values were analyzed using GraphPad Prism software, and Fit the compound dose-effect curve and calculate the CC 50 value of the compound on cells.
Cell activity %=RLU compound/RLUDMSO control*100%
方法4：Huh7细胞系靶基因下调Method 4: Downregulation of target genes in Huh7 cell line
1.实验材料：Huh7，Opti-MEM，Lipofectamine RNAiMax，96孔板1. Experimental materials: Huh7, Opti-MEM, Lipofectamine RNAiMax, 96-well plate
2.实验方案:96孔板，每孔添加各个浓度下siRNA双链体(10μL)，添加9.7μL含有Huh7细胞Opti-MEM和0.3μL Lipofectamine RNAiMax(Invitrogen-13778-150)；然后将混合物在室温下孵育15分钟。在转染期间，洗涤细胞并以2×105个细胞/mL重新悬浮。然后将含有适当细胞数的100μL完全生长培养基添加到20μL siRNA混合物中。在细胞培养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。2. Experimental protocol: 96-well plate, add siRNA duplexes (10 μL) at various concentrations to each well, add 9.7 μL containing Huh7 cell Opti-MEM and 0.3 μL Lipofectamine RNAiMax (Invitrogen-13778-150); then incubate the mixture at room temperature Incubate for 15 minutes. During transfection, cells were washed and resuspended at 2 × 10 cells/mL. Then add 100 μL of complete growth medium containing the appropriate number of cells to the 20 μL siRNA mixture. 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.
方法5：人原代肝细胞中靶基因下调Method 5: Downregulation of target genes in human primary hepatocytes
1.实验原理：1. Experimental principle:
将待测样品与人原代肝细胞(PHH)进行孵育，评估待测样品对AGT mRNA的下调程度。The test sample was incubated with primary human hepatocytes (PHH) to evaluate the degree of down-regulation of AGT mRNA by the test sample.
2.实验材料：2. Experimental materials:
人原代肝细胞(PHH)，96Kit(12)(QIAGEN-74182)，FastKing RT Kit(With gDNase)，TaqMan Gene Expression Assay，TaqMan Gene Expression Assay，DPBS(Dulbecco's磷酸缓冲液)，本发明缀合物。Primary human hepatocytes (PHH), 96Kit (12) (QIAGEN-74182), FastKing RT Kit (With gDNase), TaqMan Gene Expression Assay, TaqMan Gene Expression Assay, DPBS (Dulbecco's Phosphate Buffer), conjugate of the present invention.
3.实验方法：3.Experimental method:
用DPBS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5.4×105cells/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 is diluted to 10 times the concentration to be tested with DPBS solution. Transfer 10 μL of siRNA to a 96-well plate. PHH cells were thawed and transplanted into 96-well plates, 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 diluted 4 times.
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。Cells were incubated at 37 °C, 5% CO for 48 h, and cell status was examined microscopically.
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)逆转录获得cDNA。用qPCR检测AGT cDNA。After the incubation is completed, the cells are lysed and the lysate obtained is used. All RNA was extracted with 96Kit (QIAGEN-74182) and reverse transcribed using FastKing RT Kit (With gDNase) to obtain cDNA. Detection of AGT cDNA using qPCR.
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 stated, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered uncertain or unclear in the absence of a specific definition, but should be interpreted in accordance with its meaning as understood by one of ordinary skill in the art. Where a trade name appears herein, it is intended to refer to its corresponding trade name or its active ingredient.
本发明中，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。 In the present invention, unless otherwise stated, 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.
除非另有规定，本发明所述“寡聚核苷酸”是含有14～80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经修饰。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，寡聚核苷酸能够在体外或体内抑制或阻断基因的表达。“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。Unless otherwise specified, "oligonucleotide" as used herein is a nucleotide sequence containing 14 to 80 nucleotides or nucleotide base pairs. In some embodiments of the 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 within the cell. The nucleotides may optionally be modified. In some embodiments of the invention, the oligonucleotide is capable of inhibiting or blocking expression of a gene in vitro or in vivo upon delivery to a cell expressing the gene. "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 RNA shRNA, ribozymes, interfering RNA molecules, and Dicer enzyme substrates.
除非另有规定，本发明所述“单链化合物”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。Unless otherwise specified, a "single-stranded compound" as used herein refers to a single-stranded oligonucleotide having a sequence that is at least partially complementary to the target mRNA, which is capable of operating under mammalian physiological conditions (or equivalent in vitro environment) through hydrogen bonding. hybridize with target mRNA. In some embodiments of the invention, the single-stranded oligonucleotide is a single-stranded antisense oligonucleotide.
除非另有规定，本发明所述“双链化合物”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。Unless otherwise specified, a "double-stranded compound" as used herein refers to a duplex structure containing two antiparallel and substantially complementary nucleotide strands, one of which is the sense strand and the other is the antisense strand.
除非另有规定，本发明所述“反义链”或“指导链”指寡聚核苷酸化合物中与靶序列(例如，AGT mRNA)的相应区域基本上互补的链。Unless otherwise specified, the "antisense strand" or "guide strand" used herein refers to the strand in an oligonucleotide compound that is substantially complementary to the corresponding region of the target sequence (e.g., AGT mRNA).
除非另有规定，本发明所述“正义链”或“过客链”是指能够与反义链形成基本上互补区的链。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和错配的碱基对。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。Unless otherwise specified, the "sense strand" or "passenger strand" used in the present invention refers to a strand capable of forming a substantially complementary region with the antisense strand. The "substantially complementary" means that the corresponding positions of the two sequences can be completely complementary, or there can be one or more mismatches. When there is a mismatch, there are usually no more than 3, 2 or 1 mismatched bases. right. In a double-stranded nucleic acid molecule, the bases on one strand are paired with bases on the other strand in a complementary manner. The purine base adenine (A) always pairs with the pyrimidine base uracil (U); the purine base guanine (C) always pairs with the pyrimidine base cytosine (G).
除非另有规定，本发明所述的“核苷酸任选经修饰”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 "nucleotide optionally modified" in the present invention means that the nucleotide may be an unmodified nucleotide or a modified nucleotide. The "unmodified" "Nucleotide" refers to nucleotides composed of natural nucleobases, sugar rings and phosphates. The "modified nucleotide" refers to a nucleotide composed of a modified nucleobase, and/or a modified sugar ring, and/or a modified phosphate ester. In some embodiments of the invention, "modified nucleotides" are composed of modified nucleobases, modified sugar rings and natural phosphates; in some embodiments of the invention, "modified nucleosides" "Acid" consists of modified nucleobases, modified phosphate esters and natural sugar rings; in some embodiments of the invention, "modified nucleotides" consist of natural nucleobases, modified sugar rings and modified Composed of phosphate esters; in some embodiments of the present invention, "modified nucleotides" are composed of modified nucleobases, natural sugar rings and natural phosphate esters; in some embodiments of the present invention, "modified nucleotides" "Modified nucleotides" are composed of natural nucleobases, modified sugar rings and natural phosphates; in some embodiments of the invention, "modified nucleotides" are composed of natural nucleobases, natural It consists of a sugar ring and a modified phosphate ester; in some embodiments of the invention, the "modified nucleotide" consists of a modified nucleobase, a modified sugar ring and a modified phosphate ester.
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” in the present invention is selected from the five-membered sugar ring of 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" mentioned in the present invention are selected from the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine. Adenine (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" mentioned in the present invention refers to a 5-12-membered saturated, partially unsaturated or aromatic heterocycle other than natural bases, including a single ring or a condensed ring. Specifically, Examples include, but are not limited to, thiophene, thianthrene, furan, pyran, isobenzofuran, benzothiazine, pyrrole, imidazole, substituted or unsubstituted triazole, pyrazole, isothiazole, isoxazole, pyridazine , indolezine, indole, isoindole, isoquinoline, quinoline, naphthopyridine, quinazoline, carbazole, phenanthridine, piperidine, phenazine, phenanzine, phenothiazine, furane, 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-thio Cytosine, 5-halogenated uracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-hydroxysulfanyl, 8-sulfanyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halo In particular, 5-bromo, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azadenine , 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" described in 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 can be substituted or unsubstituted C 1 to C 10 alkyl group or C 2 to C 10 alkenyl and alkynyl groups. 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 , where n and m are from 1 to 10. In other embodiments, modifications at the 2' position include, but are not limited to, one of the following: 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, heterocycle Alkaryl group, aminoalkylamino group, polyalkylamino group, substituted silyl group, RNA cleavage group, reporter group, intercalator, group for improving the pharmacokinetic properties of iRNA, or for improving iRNA groups with pharmacodynamic properties, and other substituents with similar properties. In some embodiments, the modification includes, but is not limited to, 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 ester" in the present invention includes but is not limited to: phosphorothioate modification, the "phosphorothioate" "Acid ester" includes the (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, modified nucleotides may comprise one or more locked nucleic acids (LNA). Locked nucleic acids are nucleotides with a modified ribose moiety that contains an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose sugar in the 3'-endostructural 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 comprise 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, resulting in unlocked "sugar" residues. In one example, UNA also encompasses monomers in which the bond between C1'-C4' has been removed (i.e., the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons). In another example, the C2'-C3' bond of the sugar (i.e., the covalent carbon-carbon bond between the 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，其均为S构型。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 In some embodiments of the invention, the modified nucleotides comprise one or more monomers of GNA (glycerol nucleic acid) nucleotides. GNA includes GNA-A, GNA-T, GNA-C, GNA-G and GNA-U, all of which are in S configuration. The structure of GNA-A is The structure of GNA-T 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 invention, modified nucleotides may also include one or more bicyclic sugar moieties. "Bicyclic sugars" are furanosyl rings modified by a bridge of two atoms. "Bicyclic nucleosides" ("BNA") are nucleosides having a sugar moiety that contains a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In a specific embodiment, the bridge connects the 4'-carbon and 2'-carbon of the sugar ring.
除非另有规定，本发明所述“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。 Unless otherwise specified, "conjugation" in the present invention means that two or more chemical moieties each having a specific function are connected to each other in a covalent manner; accordingly, "conjugate" means that each A compound formed by covalent linkages between chemical parts.
除非另有规定，本发明所述“配体”是指靶向基团，例如与指定的细胞类型如肾细胞结合的细胞或组织靶向剂，例如凝集素、糖蛋白、脂质或蛋白质，例如抗体。靶向基团可以是促甲状腺激素、促黑素、凝集素、糖蛋白、表面活性蛋白质A、黏蛋白碳水化合物、多价乳糖、多价半乳糖、N-乙酰基-半乳糖胺(GalNAc)、N-乙酰基-葡糖胺多价甘露糖、多价岩藻糖、糖基化聚氨基酸、多价半乳糖、转铁蛋白、双膦酸盐、聚谷氨酸、聚天冬氨酸、脂质、胆固醇、类固醇、胆酸、叶酸、维生素B12、维生素A、生物素、或RGD肽或RGD肽模拟物。Unless otherwise specified, "ligand" in the present invention refers to a targeting group, such as a cell or tissue targeting agent, such as a lectin, glycoprotein, lipid or protein, that binds to a specified cell type such as kidney cells, For example, antibodies. Targeting groups can be thyroid stimulating hormone, melanocyte stimulating hormone, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine (GalNAc) , N-acetyl-glucosamine polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonates, polyglutamic acid, polyaspartic acid , lipids, cholesterol, steroids, cholic acid, folic acid, vitamin B12, vitamin A, biotin, or RGD peptide or RGD peptide mimetic.
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经修饰的核苷酸。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" in the present invention refers to at least one unpaired nucleotide protruding from the double-stranded region structure of the 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 comprise at least one nucleotide; or the overhang may comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five or more nucleosides acid. The nucleotides of the nucleotide overhang are optionally modified nucleotides. The overhang can be on the sense strand, the antisense strand, or any combination thereof. In addition, the overhang may be present at the 5'-end, the 3'-end, or both ends of the antisense or sense strand of the double-stranded compound. In some embodiments of the invention, the antisense strand has 1 to 10 nucleotides at the 3'-end and/or 5'-end (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides) overhang. In some embodiments of the invention, the sense strand has 1 to 10 nucleotides at the 3'-end and/or 5'-end (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides) overhang. In some embodiments of the invention, the antisense strand is at the 3'-end and the sense strand has 1 to 10 nucleotides at the 3'-end (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides) overhang. In some embodiments of the invention, the antisense strand is at the 5'-end and the sense strand has 1 to 10 nucleotides at the 5'-end (e.g., 1, 2, 3, 4, 5, 6, 7, 8 , 9 or 10 nucleotides) overhang.
除非另有规定，本发明所述的正义链的一端与反义链的一端还可以通过一个或多个核苷酸连接是指如下式(I)所述的连接方式：其中N表示任选经修饰的核苷酸，n选自1、2、3、4、5、6、7、8、9或10；strand 1、strand 2分别独立地为正义链或反义链。Unless otherwise specified, one end of the sense strand and one end of the antisense strand of the present invention can also be connected through one or more nucleotides, which refers to the connection method described in the following formula (I): Where N represents an optionally modified nucleotide, n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; strand 1 and strand 2 are independently the sense strand or the antisense strand. .
除非另有规定，本发明所述的正义链的一端与反义链的一端还可以通过化学基团连接是指通过下式(II) 所述的连接方式：其中L1选自C1-100亚烷基，C1-100亚烷基上的一个或多个CH2各自独立地任选被选自-NH-、＝N-、-O-、-S-、-C(＝O)-、-C(＝O)O-、-NHC(＝O)-、-NHC(＝O)O-、-NHC(＝O)NH-、-S(＝O)-、-S(＝O)2-、-S(＝O)2NH-、＝NO-、-P(＝O)(OH)-、-P(＝O)(NRR)-、-P(＝O)(R)NH-、C2-4烯基、C2-4炔基、C6-10芳基、5-10元杂芳基、C3-6环烷基和4-8元杂环烷基的原子或基团置换，所述C1-100亚烷基任选被一个或多个Ra取代；L2和L3分别独立地选自不存在和C1-100亚烷基，所述C1-100亚烷基上的一个或多个CH2各自独立地任选被选自-NH-、＝N-、-O-、-S-、-C(＝O)-、-C(＝O)O-、-NHC(＝O)-、-NHC(＝O)O-、-NHC(＝O)NH-、-S(＝O)-、-S(＝O)2-、-S(＝O)2NH-、＝NO-、-P(＝O)(OH)-、-P(＝O)(NRR)-、-P(＝O)(R)NH-、C2-4烯基、C2-4炔基、C6-10芳基、5-10元杂芳基、C3-6环烷基和4-8元杂环烷基的原子或基团置换，所述C1-100亚烷基任选被一个或多个Ra取代；R和Ra分别独立地选自F、Cl、Br、I、OH、NH2、CN、C1-3烷基、C6-10芳基和5-10元杂芳基；strand 1、strand 2分别独立地为正义链或反义链。Unless otherwise specified, one end of the sense strand and one end of the antisense strand of the present invention can also be connected through a chemical group, which means through the following formula (II) Described connection method: Wherein L 1 is selected from C 1-100 alkylene group, and one or more CH 2 on the C 1-100 alkylene group are each independently selected from -NH-, =N-, -O-, -S -, -C(=O)-, -C(=O)O-, -NHC(=O)-, -NHC(=O)O-, -NHC(=O)NH-, -S(=O )-, -S(=O) 2 -, -S(=O) 2 NH-, =NO-, -P(=O)(OH)-, -P(=O)(NRR)-, -P (=O)(R)NH-, C 2-4 alkenyl, C 2-4 alkynyl, C 6-10 aryl, 5-10 membered heteroaryl, C 3-6 cycloalkyl and 4-8 The atoms or groups of the heterocyclic alkyl group are replaced, and the C 1-100 alkylene group is optionally substituted by one or more Ra ; L 2 and L 3 are independently selected from the group consisting of absence and C 1-100 substituents. Alkyl group, one or more CH 2 on the C 1-100 alkylene group are each independently optionally selected from -NH-, =N-, -O-, -S-, -C(=O) -, -C(=O)O-, -NHC(=O)-, -NHC(=O)O-, -NHC(=O)NH-, -S(=O)-, -S(=O ) 2 -, -S(＝O) 2 NH-, =NO-, -P(＝O)(OH)-, -P(＝O)(NRR)-, -P(＝O)(R)NH -, C 2-4 alkenyl, C 2-4 alkynyl, C 6-10 aryl, 5-10 membered heteroaryl, C 3-6 cycloalkyl and 4-8 membered heterocycloalkyl atoms or Group replacement, the C 1-100 alkylene group is optionally substituted by one or more R a ; R and R a are independently selected from F, Cl, Br, I, OH, NH 2 , CN, C 1 -3 alkyl, C 6-10 aryl and 5-10 membered heteroaryl; strand 1 and strand 2 are independently sense strand or antisense strand.
除非另有规定，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。Unless otherwise specified, "multiple" in the present invention refers to integers 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.
本发明所述的双链RNAi类似物的缀合物是双链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。The conjugate of a double-stranded RNAi analog according to the present invention is a compound formed by connecting a double-stranded RNAi analog and a pharmaceutically acceptable conjugation group, and the double-stranded RNAi analogue and a pharmaceutically acceptable conjugation group are Groups are covalently connected.
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。Unless otherwise stated, "linker" refers to an organic moiety group that connects two parts of a compound, eg, covalently attaches two parts of a compound. The bond usually contains a direct bond or an atom (such as oxygen or sulfur), an atomic group (such as: NRR, C(O), C(O)NH, SO, SO 2 , SO 2 NH), a substituted or unsubstituted Substituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted Substituted heterocycloalkyl, wherein optional one or more C atoms in the substituted or unsubstituted alkyl, substituted or substituted alkenyl, substituted or unsubstituted alkynyl can be substituted or unsubstituted Aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl substitution.
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 has sufficient stability outside the cell, but when it enters the target cell, it will be cleaved to release the two parts of the group that the linker is fixed together.
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 the (R)- and (S)-enantiomers, diastereomers, and racemic and other mixtures thereof, such as enantiomers or diastereomers. body-enriched mixtures, all such mixtures are within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, as well as mixtures thereof, 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 stated, the terms "enantiomers" or "optical isomers" refer to stereoisomers that are mirror images of each other.
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 stated, the term "diastereomer" refers to stereoisomers whose molecules have two or more chiral centers and are in a non-mirror image relationship between the molecules.
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和/或直形虚线键 Unless otherwise stated, use wedge-shaped solid line keys and wedge-shaped dotted keys Represents the absolute configuration of a three-dimensional center, using straight solid line keys and straight dotted keys Represent the relative configuration of the three-dimensional center with a wavy line Represents wedge-shaped solid line key or wedge-shaped dotted key or use tilde Represents a straight solid line key and/or straight dotted keys
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 stated, the terms "enriched in an isomer," "enantiomerically enriched," "enriched in an enantiomer," or "enantiomerically enriched" refer to one of the isomers or enantiomers. The content of the 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 stated, the term "isomeric 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%, then 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)。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 optically active (R)- and (S)-isomers as well as the 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 invention is desired, it can be prepared by asymmetric synthesis or derivatization with chiral auxiliaries, in which the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide 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 a suitable optically active acid or base, and then the salt is formed by conventional methods known in the art. Diastereomeric resolution is performed and the pure enantiomers are recovered. Furthermore, the separation of enantiomers and diastereomers is usually accomplished by the use of chromatography using chiral stationary phases, optionally combined with chemical derivatization methods (e.g., generation of amino groups from amines). formate). The compounds of the present invention may contain unnatural proportions of atomic isotopes on one or more of the atoms that make up the compound. For example, compounds can be labeled with radioactive isotopes, such as tritium ( 3 H), iodine-125 ( 125 I), or C-14 ( 14 C). For another example, deuterated drugs can be replaced by heavy hydrogen to form deuterated drugs. The bond between deuterium and carbon is stronger than the bond between ordinary hydrogen and carbon. Compared with non-deuterated drugs, deuterated drugs can reduce side effects and increase drug stability. , enhance efficacy, extend drug biological half-life and other advantages. All variations in the isotopic composition of the compounds of the invention, whether radioactive or not, are included within the scope of the invention.
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 present invention prepared from a compound having specific substituents discovered in the present invention and a relatively non-toxic acid or base. When the compound of the present invention contains a relatively acidic functional group, it can be prepared by using it in a pure solution or a suitable inert solvent. Base addition salts are obtained by contacting a sufficient amount of base with such compounds. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts. When compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting such compounds with a sufficient amount of acid in neat solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, Hydrogen sulfate, hydriodic acid, phosphorous acid, etc.; and organic acid salts, including acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, Similar acids such as fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid; 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 both basic and acidic functional groups and thus can be converted into either base or acid addition salts.
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 groups or bases. In general, such salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of the two.
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 synthesis methods, and methods well known to those skilled in the art. Equivalent alternatives and preferred embodiments include, but are not limited to, embodiments 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 unnatural proportions of atomic isotopes on one or more of the atoms that make up the compound. For example, compounds can be labeled with radioactive isotopes, such as tritium ( 3 H), iodine-125 ( 125 I), or C-14 ( 14 C). For another example, deuterated drugs can be replaced by heavy hydrogen to form deuterated drugs. The bond between deuterium and carbon is stronger than the bond between ordinary hydrogen and carbon. Compared with non-deuterated drugs, deuterated drugs can reduce side effects and increase drug stability. , enhance efficacy, extend drug biological half-life and other advantages. All variations in the isotopic composition of the compounds of the invention, whether radioactive or not, are included within the scope of the 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 listed linking groups do not specify the direction of connection, the direction of connection is arbitrary, for example, The middle linking group L is -MW-. At this time, -MW- can be connected to ring A and ring B in the same direction as the reading order from left to right. You can also connect ring A and ring B in the opposite direction to the reading order from left to right. Combinations of the linking groups, substituents and/or variants 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 "optionally" or "optionally" mean that the subsequently described event or condition may, but need not, occur, and that the description includes its circumstances in which the event or condition described occurs and circumstances in which the event or condition does not occur.
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 specific atom are replaced by a substituent, which may include deuterium and hydrogen variants, as long as the valence state of the specific 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 or may not be substituted. Unless otherwise specified, the type and number of substituents may be arbitrary on the basis of chemical achievability.
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 the composition or structure of a compound, its definition in each instance is independent. Thus, for example, if a group is substituted by 0-2 R, then said group may optionally be substituted by up to two R's, with independent options for R in each case. Furthermore, 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 thereof. For example, a pyridyl group as a substituent can be bonded through any one of the pyridine rings. The carbon atom is attached to the substituted group.
除非另有规定，术语“C1-3烷氧基”表示通过一个氧原子连接到分子的其余部分的那些包含1至3个碳原子的烷基基团。所述C1-3烷氧基包括C1-2、C2-3、C3和C2烷氧基等。C1-3烷氧基的实例包括但不限于甲氧基、乙氧基、丙氧基(包括正丙氧基和异丙氧基)等。Unless otherwise specified, the term "C 1-3 alkoxy" means those alkyl groups containing 1 to 3 carbon atoms that are attached to the remainder of the molecule through an oxygen atom. The C 1-3 alkoxy group includes C 1-2 , C 2-3 , C 3 and C 2 alkoxy groups, etc. Examples of C 1-3 alkoxy include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), and the like.
除非另有规定，“C2-4烯基”用于表示直链或支链的包含至少一个碳-碳双键的由2至4个碳原子组成的碳氢基团，碳-碳双键可以位于该基团的任何位置上。所述C2-4烯基包括C2-3、C4、C3和C2烯基等；所述C2-4烯基可以是一价、二价或者多价。C2-4烯基的实例包括但不限于乙烯基、丙烯基、丁烯基、丁间二烯基等。除非另有规定，“C2-3烯基”用于表示直链或支链的包含至少一个碳-碳双键的由2至3个碳原子组成的碳氢基团，碳-碳双键可以位于该基团的任何位置上。所述C2-3烯基包括C3和C2烯基；所述C2-3烯基可以是一价、二价或者多价。C2-3烯基的实例包括但不限于乙烯基、丙烯基等。Unless otherwise specified, "C 2-4 alkenyl" is used to mean a straight or branched hydrocarbon group consisting of 2 to 4 carbon atoms containing at least one carbon-carbon double bond. Can be located anywhere on the group. The C 2-4 alkenyl group includes C 2-3 , C 4 , C 3 and C 2 alkenyl groups, etc.; the C 2-4 alkenyl group can be monovalent, divalent or multivalent. Examples of C 2-4 alkenyl groups include, but are not limited to, vinyl, propenyl, butenyl, butadienyl, and the like. Unless otherwise specified, "C 2-3 alkenyl" is used to mean a linear or branched hydrocarbon group consisting of 2 to 3 carbon atoms containing at least one carbon-carbon double bond, carbon-carbon double bond Can be located anywhere on the group. The C 2-3 alkenyl group includes C 3 and C 2 alkenyl groups; the C 2-3 alkenyl group can be monovalent, divalent or multivalent. Examples of C 2-3 alkenyl groups include, but are not limited to, vinyl, propenyl, and the like.
除非另有规定，“C2-4炔基”用于表示直链或支链的包含至少一个碳-碳三键的由2至4个碳原子组成的碳氢基团，碳-碳三键可以位于该基团的任何位置上。所述C2-4炔基包括C2-3、C4、C3和C2炔基等。其可以是一价、二价或者多价。C2-4炔基的实例包括但不限于乙炔基、丙炔基、丁炔基等。Unless otherwise specified, "C 2-4 alkynyl" is used to mean a straight-chain or branched hydrocarbon group consisting of 2 to 4 carbon atoms containing at least one carbon-carbon triple bond. Can be located anywhere on the group. The C 2-4 alkynyl group includes C 2-3 , C 4 , C 3 and C 2 alkynyl groups, etc. It can be monovalent, bivalent or polyvalent. Examples of C 2-4 alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, and the like.
除非另有规定，环上原子的数目通常被定义为环的元数，例如，“5-7元环”是指环绕排列5-7个原子的“环”。Unless otherwise specified, the number of atoms in a ring is usually defined as the number of ring members. For example, a "5- to 7-membered ring" refers to a "ring" with 5 to 7 atoms arranged around it.
除非另有规定，“C3-6环烷基”表示由3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C3-6环烷基包括C3-5、C4-5和C5-6环烷基等；其可以是一价、二价或者多价。C3-6环烷基的 实例包括，但不限于，环丙基、环丁基、环戊基、环己基等。Unless otherwise specified, "C 3-6 cycloalkyl" means a saturated cyclic hydrocarbon group composed of 3 to 6 carbon atoms, which is a monocyclic and bicyclic system, and the C 3-6 cycloalkyl group includes C 3-5 , C 4-5 and C 5-6 cycloalkyl, etc.; it can be monovalent, divalent or multivalent. C 3-6 cycloalkyl Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.
除非另有规定，Cn-n+m或Cn-Cn+m包括n至n+m个碳的任何一种具体情况，例如C1-12包括C1、C2、C3、C4、C5、C6、C7、C8、C9、C10、C11、和C12，也包括n至n+m中的任何一个范围，例如C1- 12包括C1-3、C1-6、C1-9、C3-6、C3-9、C3-12、C6-9、C6-12、和C9-12等；同理，n元至n+m元表示环上原子数为n至n+m个，例如3-12元环包括3元环、4元环、5元环、6元环、7元环、8元环、9元环、10元环、11元环、和12元环，也包括n至n+m中的任何一个范围，例如3-12元环包括3-6元环、3-9元环、5-6元环、5-7元环、6-7元环、6-8元环、和6-10元环等。Unless otherwise specified, C n-n+m or C n -C n+m includes any specific case of n to n+m carbons, for example, C 1-12 includes C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 , C 11 , and C 12 , also include any range from n to n+m, for example, C 1-12 includes C 1-3 , C 1-6 , C 1-9 , C 3-6 , C 3-9 , C 3-12 , C 6-9 , C 6-12 , and C 9-12 , etc.; similarly, n yuan to n The +m member indicates that the number of atoms in the ring is n to n+m. For example, a 3-12 membered ring includes a 3-membered ring, a 4-membered ring, a 5-membered ring, a 6-membered ring, a 7-membered ring, an 8-membered ring, and a 9-membered ring. , 10-membered ring, 11-membered ring, and 12-membered ring, also including any range from n to n+m, for example, 3-12-membered ring includes 3-6-membered ring, 3-9-membered ring, 5-6-membered ring ring, 5-7 membered ring, 6-7 membered ring, 6-8 membered ring, and 6-10 membered ring, etc.
除非另有规定，术语“4-8元杂环烷基”本身或者与其他术语联合分别表示由4至8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“4-8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4-8元杂环烷基包括4-5元、4-6元、5-6元、4元、5元和6元杂环烷基等。4-8元杂环烷基的实例包括但不限于氮杂环丁基、氧杂环丁基、硫杂环丁基、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、高哌嗪基、高哌啶基或二氧杂环庚烷基等。Unless otherwise specified, the term "4-8 membered heterocycloalkyl" by itself or in combination with other terms means a saturated cyclic group consisting of 4 to 8 ring atoms, with 1, 2, 3 or 4 ring atoms. are heteroatoms independently selected from O, S and N, and the remainder are carbon atoms, in which the nitrogen atoms are optionally quaternized, and the nitrogen and sulfur heteroatoms can be optionally oxidized (i.e., NO and S(O) p , p is 1 or 2). It includes single-ring and double-ring systems, where the double-ring system includes spiro rings, parallel rings and bridged rings. Furthermore, in the case of the "4- to 8-membered heterocycloalkyl", a heteroatom may occupy the attachment position of the heterocycloalkyl to the rest of the molecule. The 4-8-membered heterocycloalkyl group includes 4-5-membered, 4-6-membered, 5-6-membered, 4-membered, 5-membered and 6-membered heterocycloalkyl groups, etc. Examples of 4-8 membered heterocycloalkyl include, but are not limited to, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrothienyl ( 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, 4-morpholinyl, etc.), Dioxanyl, dithianyl, isoxazolidinyl, isothiazolidinyl, 1,2-oxazinyl, 1,2-thiazinyl, hexahydropyridazinyl, homopiperazinyl, homopiperazinyl Aldyl or dioxepanyl, etc.
除非另有规定，本发明术语“C6-10芳环”和“C6-10芳基”可以互换使用，术语“C6-10芳环”或“C6-10芳基”表示由6至10个碳原子组成的具有共轭π电子体系的环状碳氢基团，它可以是单环、稠合双环或稠合三环体系，其中各个环均为芳香性的。其可以是一价、二价或者多价，C6-10芳基包括C6-9、C9、C10和C6芳基等。C6-10芳基的实例包括但不限于苯基、萘基(包括1-萘基和2-萘基等)。Unless otherwise specified, the terms "C 6-10 aromatic ring" and "C 6-10 aryl" in the present invention can be used interchangeably, and the term "C 6-10 aromatic ring" or "C 6-10 aryl" means A cyclic hydrocarbon group composed of 6 to 10 carbon atoms with a conjugated π electron system. It can be a monocyclic, fused bicyclic or fused tricyclic system, in which each ring is aromatic. It can be monovalent, divalent or multivalent, and C 6-10 aryl groups include C 6-9 , C 9 , C 10 and C 6 aryl groups, etc. Examples of C 6-10 aryl groups include, but are not limited to, phenyl, naphthyl (including 1-naphthyl, 2-naphthyl, etc.).
除非另有规定，本发明术语“5-10元杂芳环”和“5-10元杂芳基”可以互换使用，术语“5-10元杂芳基”是表示由5至10个环原子组成的具有共轭π电子体系的环状基团，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。其可以是单环、稠合双环或稠合三环体系，其中各个环均为芳香性的。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)。5-10元杂芳基可通过杂原子或碳原子连接到分子的其余部分。所述5-10元杂芳基包括5-8元、5-7元、5-6元、5元和6元杂芳基等。所述5-10元杂芳基的实例包括但不限于吡咯基(包括N-吡咯基、2-吡咯基和3-吡咯基等)、吡唑基(包括2-吡唑基和3-吡唑基等)、咪唑基(包括N-咪唑基、2-咪唑基、4-咪唑基和5-咪唑基等)、噁唑基(包括2-噁唑基、4-噁唑基和5-噁唑基等)、三唑基(1H-1,2,3-三唑基、2H-1,2,3-三唑基、1H-1,2,4-三唑基和4H-1,2,4-三唑基等)、四唑基、异噁唑基(3-异噁唑基、4-异 噁唑基和5-异噁唑基等)、噻唑基(包括2-噻唑基、4-噻唑基和5-噻唑基等)、呋喃基(包括2-呋喃基和3-呋喃基等)、噻吩基(包括2-噻吩基和3-噻吩基等)、吡啶基(包括2-吡啶基、3-吡啶基和4-吡啶基等)、吡嗪基、嘧啶基(包括2-嘧啶基和4-嘧啶基等)、苯并噻唑基(包括5-苯并噻唑基等)、嘌呤基、苯并咪唑基(包括2-苯并咪唑基等)、苯并噁唑基、吲哚基(包括5-吲哚基等)、异喹啉基(包括1-异喹啉基和5-异喹啉基等)、喹喔啉基(包括2-喹喔啉基和5-喹喔啉基等)或喹啉基(包括3-喹啉基和6-喹啉基等)。Unless otherwise specified, the terms "5-10 membered heteroaromatic ring" and "5-10 membered heteroaryl" in the present invention can be used interchangeably. The term "5-10 membered heteroaryl" means a ring consisting of 5 to 10 rings. A cyclic group composed of atoms with a conjugated π electron system, of which 1, 2, 3 or 4 ring atoms are heteroatoms independently selected from O, S and N, and the rest are carbon atoms. It can be a monocyclic, fused bicyclic or fused tricyclic system, in which each ring is aromatic. The nitrogen atoms are optionally quaternized, and the nitrogen and sulfur heteroatoms are optionally oxidized (i.e., NO and S(O) p , p is 1 or 2). A 5- to 10-membered heteroaryl group can be attached to the rest of the molecule through a heteroatom or a carbon atom. The 5- to 10-membered heteroaryl groups include 5- to 8-membered, 5- to 7-membered, 5- to 6-membered, 5- to 6-membered heteroaryl groups, and the like. Examples of the 5-10 membered heteroaryl include, but are not limited to, pyrrolyl (including N-pyrrolyl, 2-pyrrolyl and 3-pyrrolyl, etc.), pyrazolyl (including 2-pyrazolyl and 3-pyrrolyl). azolyl group, etc.), imidazolyl group (including N-imidazolyl, 2-imidazolyl, 4-imidazolyl and 5-imidazolyl, etc.), oxazolyl (including 2-oxazolyl, 4-oxazolyl and 5-oxazolyl) Oxazolyl, etc.), triazolyl (1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, 1H-1,2,4-triazolyl and 4H-1, 2,4-triazolyl, etc.), tetrazolyl, isoxazolyl (3-isoxazolyl, 4-iso Oxazolyl and 5-isoxazolyl, etc.), thiazolyl (including 2-thiazolyl, 4-thiazolyl and 5-thiazolyl, etc.), furyl (including 2-furyl and 3-furyl, etc.), Thienyl (including 2-thienyl and 3-thienyl, etc.), pyridyl (including 2-pyridyl, 3-pyridyl and 4-pyridyl, etc.), pyrazinyl, pyrimidinyl (including 2-pyrimidinyl and 4-pyrimidinyl, etc.), benzothiazolyl (including 5-benzothiazolyl, etc.), purinyl, benzimidazolyl (including 2-benzimidazolyl, etc.), benzoxazolyl, indole ( Including 5-indolyl, etc.), isoquinolinyl (including 1-isoquinolinyl and 5-isoquinolinyl, etc.), quinoxalinyl (including 2-quinoxalinyl and 5-quinoxalinyl) etc.) or quinolyl (including 3-quinolyl and 6-quinolyl, etc.).
除非另有规定，本发明术语“5-6元杂芳环”和“5-6元杂芳基”可以互换使用，术语“5-6元杂芳基”表示由5至6个环原子组成的具有共轭π电子体系的单环基团，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)。5-6元杂芳基可通过杂原子或碳原子连接到分子的其余部分。所述5-6元杂芳基包括5元和6元杂芳基。所述5-6元杂芳基的实例包括但不限于吡咯基(包括N-吡咯基、2-吡咯基和3-吡咯基等)、吡唑基(包括2-吡唑基和3-吡唑基等)、咪唑基(包括N-咪唑基、2-咪唑基、4-咪唑基和5-咪唑基等)、噁唑基(包括2-噁唑基、4-噁唑基和5-噁唑基等)、三唑基(1H-1,2,3-三唑基、2H-1,2,3-三唑基、1H-1,2,4-三唑基和4H-1,2,4-三唑基等)、四唑基、异噁唑基(3-异噁唑基、4-异噁唑基和5-异噁唑基等)、噻唑基(包括2-噻唑基、4-噻唑基和5-噻唑基等)、呋喃基(包括2-呋喃基和3-呋喃基等)、噻吩基(包括2-噻吩基和3-噻吩基等)、吡啶基(包括2-吡啶基、3-吡啶基和4-吡啶基等)、吡嗪基、哒嗪基或嘧啶基(包括2-嘧啶基和4-嘧啶基等)。Unless otherwise specified, the terms "5-6 membered heteroaromatic ring" and "5-6 membered heteroaryl" may be used interchangeably in the present invention, and the term "5-6 membered heteroaryl" means 5 to 6 ring atoms. It consists of a monocyclic group with a conjugated π electron system, in which 1, 2, 3 or 4 ring atoms are heteroatoms independently selected from O, S and N, and the rest are carbon atoms. The nitrogen atoms are optionally quaternized, and the nitrogen and sulfur heteroatoms are optionally oxidized (i.e., NO and S(O)p, p is 1 or 2). A 5-6 membered heteroaryl group can be attached to the rest of the molecule through a heteroatom or a carbon atom. The 5-6 membered heteroaryl group includes 5-membered and 6-membered heteroaryl groups. Examples of the 5-6 membered heteroaryl include but are not limited to pyrrolyl (including N-pyrrolyl, 2-pyrrolyl and 3-pyrrolyl, etc.), pyrazolyl (including 2-pyrazolyl and 3-pyrrolyl). azolyl group, etc.), imidazolyl group (including N-imidazolyl, 2-imidazolyl, 4-imidazolyl and 5-imidazolyl, etc.), oxazolyl (including 2-oxazolyl, 4-oxazolyl and 5-oxazolyl) Oxazolyl, etc.), triazolyl (1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, 1H-1,2,4-triazolyl and 4H-1, 2,4-triazolyl, etc.), tetrazolyl, isoxazolyl (3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, etc.), thiazolyl (including 2-thiazolyl , 4-thiazolyl and 5-thiazolyl, etc.), furyl (including 2-furyl and 3-furyl, etc.), thienyl (including 2-thienyl and 3-thienyl, etc.), pyridyl (including 2-thienyl, etc.) -pyridyl, 3-pyridyl and 4-pyridyl, etc.), pyrazinyl, pyridazinyl or pyrimidinyl (including 2-pyrimidinyl, 4-pyrimidinyl, etc.).
除非另有规定，当某一基团具有一个或多个可连接位点时，该基团的任意一个或多个位点可以通过化学键与其他基团相连。当该化学键的连接方式是不定位的，且可连接位点存在H原子时，则连接化学键时，该位点的H原子的个数会随所连接化学键的个数而对应减少变成相应价数的基团。所述位点与其他基团连接的化学键可以用直形实线键直形虚线键或波浪线表示。例如-OCH3中的直形实线键表示通过该基团中的氧原子与其他基团相连；中的直形虚线键表示通过该基团中的氮原子的两端与其他基团相连；中的波浪线表示通过该苯基基团中的1和2位碳原子与其他基团相连；表示苯环上的任意可连接位点可以通过1个化学键与其 他基团相连，至少包括这4种连接方式。Unless otherwise specified, when a certain group has one or more attachable sites, any one or more sites of the group can be connected to other groups through chemical bonds. When the connection mode of the chemical bond is non-positioned and there are H atoms at the connectable site, when the chemical bond is connected, the number of H atoms at the site will be reduced correspondingly with the number of connected chemical bonds and become the corresponding valence. group. The chemical bond connecting the site to other groups can be a straight solid line bond straight dashed key or wavy lines express. For example, the straight solid line bond in -OCH 3 means that it is connected to other groups through the oxygen atom in the group; The straight dotted bond in means that it is connected to other groups through both ends of the nitrogen atoms in the group; The wavy lines in indicate that the phenyl group is connected to other groups through the 1 and 2 carbon atoms in the phenyl group; Indicates that any connectable site on the benzene ring can be connected to it through a chemical bond connected to other groups, including at least These 4 connection methods.
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 synthesis methods, and methods well known to those skilled in the art. Equivalent alternatives and preferred embodiments include, but are not limited to, embodiments 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 well known to those skilled in the art. If the present invention involves 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) uses a Bruker D8 venture diffractometer to collect diffraction intensity data on the cultured single crystal. The light source is CuKα radiation. The scanning method is: After scanning and collecting relevant data, the direct method (Shelxs97) is further used to analyze the crystal structure, and the absolute configuration can be confirmed.
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 for column chromatography and thin-layer silica gel chromatography in the present invention are all volume ratios.
本发明采用下述缩略词：aq代表水；HATU代表O-(7-氮杂苯并三唑-1-基)-N，N，N'，N'-四甲基脲六氟磷酸盐；EDC代表N-(3-二甲基氨基丙基)-N'-乙基碳二亚胺盐酸盐；m-CPBA代表3-氯过氧苯甲酸；eq代表当量、等量；CDI代表羰基二咪唑；DCM代表二氯甲烷；PE代表石油醚；DIAD代表偶氮二羧酸二异丙酯；DMF代表N，N-二甲基甲酰胺；DMSO代表二甲亚砜；EtOAc代表乙酸乙酯；EtOH代表乙醇；MeOH代表甲醇；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；HOAc代表乙酸；NaCNBH3代表氰基硼氢化钠；r.t.代表室温；O/N代表过夜；THF代表四氢呋喃；Boc2O代表二-叔丁基二碳酸酯；TFA代表三氟乙酸；DIPEA代表二异丙基乙基胺；SOCl2代表氯化亚砜；CS2代表二硫化碳；TsOH代表对甲苯磺酸；NFSI代表N-氟-N-(苯磺酰基)苯磺酰胺；NCS代表1-氯吡咯烷-2,5-二酮；n-Bu4NF代表氟化四丁基铵；iPrOH代表2-丙醇；mp代表熔点；LDA代表二异丙基胺基锂。The present invention adopts the following abbreviations: aq represents water; HATU represents O-(7-azabenzotriazol-1-yl)-N, N, N', N'-tetramethylurea hexafluorophosphate ; EDC represents N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride; m-CPBA represents 3-chloroperoxybenzoic acid; eq represents equivalent, equivalent; CDI represents Carbonyldiimidazole; DCM represents dichloromethane; PE represents petroleum ether; DIAD represents diisopropyl azodicarboxylate; DMF represents N, N-dimethylformamide; DMSO represents dimethyl sulfoxide; EtOAc represents ethyl acetate Ester; EtOH represents ethanol; MeOH represents methanol; CBz represents benzyloxycarbonyl, which is an amine protecting group; BOC represents tert-butoxycarbonyl, which is an amine protecting group; HOAc represents acetic acid; NaCNBH 3 represents sodium cyanoborohydride ; rt represents room temperature; O/N represents overnight; THF represents tetrahydrofuran; Boc 2 O represents di-tert-butyl dicarbonate; TFA represents trifluoroacetic acid; DIPEA represents diisopropylethylamine; SOCl 2 represents methylene chloride Sulfone; CS 2 represents carbon disulfide; TsOH represents p-toluenesulfonic acid; NFSI represents N-fluoro-N-(phenylsulfonyl)benzenesulfonamide; NCS represents 1-chloropyrrolidine-2,5-dione; n-Bu 4 NF represents tetrabutylammonium fluoride; iPrOH represents 2-propanol; mp represents melting point; LDA represents lithium diisopropylamide.
核酸序列描述中使用的是核苷酸单体的缩写，具体如表3所示：The abbreviations of nucleotide monomers are used in the description of nucleic acid sequences, as shown in Table 3:
表3核苷酸单体的缩写

Table 3 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 conventional naming principles in the field or use For software naming, commercially available compounds adopt supplier catalog names.
技术效果Technical effect
本发明化合物具有良好的活性和安全性。含有本专利所述的新型结构的RNAi药物在细胞和小鼠HDI模型中均可显著降低靶基因mRNA的水平，表明其具有良好的活性。同时本专利所述的新型结构的RNAi药物在体外测试中没有免疫原性的风险，表明其具有较好的安全性。The compound of the present invention has good activity and safety. RNAi drugs containing the novel structure described in this patent can significantly reduce the level of target gene mRNA in both cell and mouse HDI models, indicating that they have good activity. At the same time, the novel structure of the RNAi drug described in this patent has no risk of immunogenicity in in vitro testing, indicating that it has good safety.
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 through examples, which do not mean any adverse limitations to the present invention. 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 synthesis methods, and methods well known to those skilled in the art. Equivalent alternatives and preferred embodiments include, but are not limited to, embodiments of the present invention. It will be apparent to those skilled in the art that various changes and modifications can be made in the specific embodiments of the invention without departing from the spirit and scope of the invention.
实施例1：缀合物S16的合成Example 1: Synthesis of conjugate S16
1.合成原理1.Synthetic principle
合成的序列均为化学合成的寡核苷酸，通过多步固相合成含固相载体和保护基的寡核苷酸，最后氨解脱保护和纯化。固相合成方法如下：
The synthesized sequences are all chemically synthesized oligonucleotides. The oligonucleotides containing solid phase carriers and protective groups are synthesized through multi-step solid phase, and finally the ammonia is deprotected and purified. The solid phase synthesis method is as follows:
2.实验设计2. Experimental design
S16的实验设计如表4所示：The experimental design of S16 is shown in Table 4:
表4 S16序列
Table 4 S16 sequence
核酸序列描述中使用的是核苷酸单体的缩写，具体如表5所示：The abbreviations of nucleotide monomers are used in the description of nucleic acid sequences, as shown in Table 5:
表5 S16中核苷酸单体的缩写
Table 5 Abbreviations of nucleotide monomers in S16
另外，D01配体，具体结构如下：
In addition, the specific structure of D01 ligand is as follows:
3.实验过程3. Experimental process
通过固相合成得到含保护基寡核苷酸序列的载体，然后从固相载体上切割脱保护得到寡核苷酸粗品，随后氨解(氨解条件：氨水，55℃，16h。进行HPLC纯化后，单链可直接冻干得到纯品。双链需要进行退火、冻干得到缀合物S16。The carrier containing the protective oligonucleotide sequence was obtained through solid-phase synthesis, and then cleaved and deprotected from the solid-phase carrier to obtain the crude oligonucleotide, which was then aminolyzed (aminolysis conditions: ammonia water, 55°C, 16h. HPLC purification was performed Afterwards, the single chain can be directly freeze-dried to obtain the pure product. The double chain needs to be annealed and freeze-dried to obtain conjugate S16.
实施例2缀合物S26的合成Example 2 Synthesis of conjugate S26
表6 S26序列
Table 6 S26 sequence
缀合物S26的合成参考缀合物S16的方法制备得到。The synthesis of conjugate S26 was prepared by referring to the method of conjugate S16.
表7 S26中核苷酸单体的缩写

Table 7 Abbreviations of nucleotide monomers in S26

生物测试biological testing
测试例1.Huh7细胞转染实验Test example 1. Huh7 cell transfection experiment
1.实验目的：本研究的目的是应用Huh7细胞评价该专利所述缀合物抑制靶基因，如血管紧张素原(AGT)基因的活性，以化合物的EC50值为指标，来评价化合物对靶基因AGT的抑制活性。1. Experimental purpose: The purpose of this study is to use Huh7 cells to evaluate the activity of the conjugate described in the patent in inhibiting target genes, such as angiotensinogen (AGT) gene, using the EC 50 value of the compound as an indicator to evaluate the effect of the compound on Inhibitory activity of the target gene AGT.
2.实验材料:2.Experimental materials:
2.1细胞系：Huh7细胞，本发明缀合物2.1 Cell line: Huh7 cells, conjugate of the present invention
Huh7细胞由上海药明康德新药开发有限公司提供。Huh7细胞在含10％胎牛血清、1％青霉素-链霉素的MEM培养基中培养。Huh7 cells were provided by Shanghai WuXi AppTec New Drug Development Co., Ltd. Huh7 cells were cultured in MEM medium containing 10% fetal calf serum and 1% penicillin-streptomycin.
2.2试剂：2.2 Reagents:
本实验使用的主要试剂包括LipofectamineTMiRNAiMAX转染试剂，FastStart Universal Probe Mast，RNA提取试剂盒，GAPDH基因表达试剂盒，AGT基因表达试剂盒，FastKing cDNA第一链合成试剂盒，96孔板。The main reagents used in this experiment include Lipofectamine TM iRNAiMAX transfection reagent, FastStart Universal Probe Mast, RNA extraction kit, GAPDH gene expression kit, AGT gene expression kit, FastKing cDNA first strand synthesis kit, 96-well plate.
2.3耗材与仪器：2.3 Consumables and instruments:
本实验使用的主要仪器包括荧光qPCR仪(Applied Biosystems，型号QuantStudio 6 Flex)、细胞计数仪(Vi-cellTM XR)。The main instruments used in this experiment include fluorescence qPCR instrument (Applied Biosystems, model QuantStudio 6 Flex) and cell counter (Vi-cellTM XR).
3.实验步骤和方法：3. Experimental procedures and methods:
3.1转染3.1 Transfection
1)第一天，根据(RNAiMAX转染试剂：Opti-MEM＝1.5:48.5)按需配置两者混合液，室温孵 育15分钟；1) On the first day, prepare the mixture of the two according to (RNAiMAX transfection reagent: Opti-MEM=1.5:48.5) as needed, and incubate at room temperature. Incubate for 15 minutes;
2)对于每孔细胞，取一定量的稀释好的化合物加入到等量的RNAiMAX Optim-MEM培养液中，混匀，孵育15分钟；2) For each well of cells, add a certain amount of diluted compound to an equal amount of RNAiMAX Optim-MEM culture medium, mix well, and incubate for 15 minutes;
3)取Huh7细胞，先用DPBS洗涤后，加入胰蛋白酶进行消化，调整细胞到合适的密度；3) Take Huh7 cells, wash them with DPBS first, add trypsin for digestion, and adjust the cells to a suitable density;
4)种细胞同时取20μL Opti-MEM RNAiMax和化合物的混合物加入到细胞培养板中，以每孔2,0000个细胞的密度接种到96孔板中，每孔最终的培养液为120μL。4) While seeding cells, add 20 μL of the mixture of Opti-MEM RNAiMax and compounds into the cell culture plate, and inoculate it into a 96-well plate at a density of 20,000 cells per well. The final culture medium in each well is 120 μL.
5)细胞置于5％CO2、37℃孵箱中培养24小时。5) The cells were cultured in a 5% CO 2 and 37°C incubator for 24 hours.
3.2 RNA提取及反转录3.2 RNA extraction and reverse transcription
收集细胞，参照Qiagen-74182 RNA提取试剂盒说明书提取RNA，参照FastKing cDNA第一链合成试剂盒说明书将RNA反转录为cDNA。Collect cells, extract RNA according to the instructions of Qiagen-74182 RNA extraction kit, and reverse-transcribe RNA into cDNA according to the instructions of FastKing cDNA First Strand Synthesis Kit.
3.3 QPCR检测3.3 QPCR detection
利用qPCR检测目的基因cDNA，GAPDH作为内参基因，qPCR在384孔板中进行。qPCR反应程序为：95℃加热10分钟，然后进入循环模式，95℃加热15秒，随后60℃1分钟，共40个循环。qPCR was used to detect the cDNA of the target gene, GAPDH was used as the internal reference gene, and qPCR was performed in a 384-well plate. The qPCR reaction program is: heat at 95°C for 10 minutes, then enter the cycle mode, heat at 95°C for 15 seconds, then 60°C for 1 minute, a total of 40 cycles.
3.4数据分析：ΔΔCt法(Ct差值比较法)3.4 Data analysis: ΔΔCt method (Ct difference comparison method)
此方法里面需要引进看家基因GAPDH，因为看家基因在所有细胞中均表达，其产物是维持细胞生存所必须，且在细胞中的表达量或基因组的拷贝数恒定，受环境影响小。qRT-PCR后，内参的CT值同时会被记录，被称为Ct(GAPDH)，样品的CT值，被称为Ct(样品)。
ΔCt(样品)＝Ct(样品)-Ct(GAPDH)
ΔCt(对照)＝Ct(对照)-Ct(GAPDH)
ΔΔCt＝ΔCt(样品)-ΔCt(对照)
基因的相对表达量＝2^-ΔΔCt
抑制率％＝(1-样品的相对表达量/对照组平均的相对表达量)×100This method requires the introduction of the housekeeping gene GAPDH, because the housekeeping gene is expressed in all cells, and its product is necessary to maintain cell survival. The expression level in the cell or the copy number of the genome is constant and is less affected by the environment. After qRT-PCR, the CT value of the internal control will be recorded at the same time, which is called Ct(GAPDH), and the CT value of the sample is called Ct(sample).
ΔCt(sample)=Ct(sample)-Ct(GAPDH)
ΔCt(control)=Ct(control)-Ct(GAPDH)
ΔΔCt=ΔCt(sample)-ΔCt(control)
Relative expression level of gene=2^-ΔΔCt
Inhibition rate % = (1 - relative expression level of sample/average relative expression level of control group) × 100
4.实验结果：如表8所示。4. Experimental results: As shown in Table 8.
表8本发明缀合物S26在Huh7细胞转染实验中结果
Table 8 Results of the Huh7 cell transfection experiment of the conjugate S26 of the present invention
结论：本发明缀合物可显著抑制AGT基因在Huh7细胞中的水平。Conclusion: The conjugate of the present invention can significantly inhibit the level of AGT gene in Huh7 cells.
测试例2：小鼠hAGT HDI模型Test Example 2: Mouse hAGT HDI Model
1.实验原理： 1. Experimental principle:
通过高压尾静脉注射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。The mouse model of high-pressure tail vein injection of pcDNA-CMV-AGT plasmid was used to evaluate the effect of targeting the target gene and inhibiting the target gene in the test sample.
2.实验材料：2. Experimental materials:
pcDNA-CMV-AGT质粒，BALB/c雌性小鼠，DPBS(Dulbecco's磷酸缓冲液)，本发明缀合物。pcDNA-CMV-AGT plasmid, BALB/c female mouse, DPBS (Dulbecco's phosphate buffer saline), conjugate of the invention.
3.实验方法：3.Experimental method:
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 BALB/c female mice aged 6-8 weeks, and the mice will adapt to quarantine for one week after arriving in the animal room.
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5mL/kg，第1组小鼠给DPBS；第2组小鼠给本发明缀合物，5mg/kg。On day 0, the mice were randomly divided into groups according to their body weight data, with 4 mice in each group. After grouping, all mice were given subcutaneous injection, a single dose, and the dosage volume was 5 mL/kg. The mice in the first group were given DPBS; Two groups of mice were given the conjugate of the present invention at 5 mg/kg.
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 a volume of 8% of their body weight of pcDNA-CMV-AGT plasmid via the tail vein within 5 seconds (injection volume (mL) = mouse body weight (g) × 8%), each mouse The mass of plasmid injected into mice 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 4th day after administration, mice in all groups were euthanized by CO2 inhalation, and 2 liver samples were collected from each mouse. Liver samples were treated with RNAlater overnight at 4°C, and then the RNAlater was removed and stored at -80°C for detection of AGT gene expression levels.
4.实验结果：结果如表9所示，表格中数据表示的是给药后小鼠肝脏AGT mRNA表达量/给药前小鼠肝脏AGT mRNA表达量。4. Experimental results: The results are shown in Table 9. The data in the table represent the expression amount of AGT mRNA in mouse liver after administration/the expression amount of AGT mRNA in mouse liver before administration.
表9.本发明缀合物在小鼠中对肝脏AGT mRNA水平的下调
Table 9. Down-regulation of liver AGT mRNA levels by conjugates of the present invention in mice
实验结论：本发明缀合物能够显著下调AGT mRNA在小鼠肝脏中的表达，下调的幅度达到93％。Experimental conclusion: The conjugate of the present invention can significantly down-regulate the expression of AGT mRNA in mouse liver, and the magnitude of the down-regulation reaches 93%.
测试例3本发明缀合物对hTLR3、hTLR7、hTLR8细胞的激动活性测试Test Example 3 Test of the agonistic activity of the conjugate of the present invention on hTLR3, hTLR7, and hTLR8 cells
1.实验材料1. Experimental materials
表10实验材料表

Table 10 Experimental materials list

2.实验仪器2. Experimental instruments
本研究所使用主要仪器为多功能酶标仪Flexstation III(Molecular Device)和Echo555(Labcyte)The main instruments used in this research are multifunctional microplate reader Flexstation III (Molecular Device) and Echo555 (Labcyte)
3.实验步骤3. Experimental steps
1)将缀合物稀释到终浓度的20倍，与RNAiMAX-OPTI MEM(RNAiMAX:OPTI MEM＝3:47)1:1混合，室温孵育15分钟。1) Dilute the conjugate to 20 times the final concentration, mix 1:1 with RNAiMAX-OPTI MEM (RNAiMAX:OPTI MEM=3:47), and incubate at room temperature for 15 minutes.
2)取10μL孵育的缀合物加入到相应的细胞板中，共10个浓度，最大浓度为200nM，每个浓度双复孔。2) Add 10 μL of the incubated conjugate to the corresponding cell plate for a total of 10 concentrations. The maximum concentration is 200 nM. Each concentration is in duplicate wells.
3)阴性对照孔每孔加入10μL PBS，hTLR3的阳性对照孔每孔加入10μL 20μg/mL的Poly(I:C)HMW，hTLR7和hTLR8的阳性对照孔每孔加入10μL 10μg/mL R848。2个转染阳性对照孔加入10μL 0.5μM BLOCK-iTTM AlexaRed Fluorescent Control。3) Add 10 μL PBS to each well of the negative control well, add 10 μL 20 μg/mL Poly(I:C) HMW to each well of the positive control well of hTLR3, add 10 μL 10 μg/mL R848 to each well of the positive control wells of hTLR7 and hTLR8. 2 pcs. Add 10 μL of 0.5 μM BLOCK-iT TM Alexa to the transfection positive control well. Red Fluorescent Control.
4)将90μL细胞种于已经加好缀合物的96孔板中，50,000细胞/孔。4) Seed 90 μL of cells into a 96-well plate with the conjugate added, 50,000 cells/well.
5)将化合物和细胞在37℃、5％CO2培养箱共孵育24小时。5) Incubate the compound and cells in a 37°C, 5% CO2 incubator for 24 hours.
6)用荧光显微镜观察转染阳性对照孔的转染效果，并拍照。6) Use a fluorescence microscope to observe the transfection effect of the positive control wells and take photos.
7)化合物活性检测：每孔取20μL细胞上清，加入含有180μL QUANTI-BlueTM试剂的实验板中，37℃孵育1小时之后，用多功能酶标仪Flexstation III检测650nm的吸光度值(OD650)。7) Compound activity detection: Take 20 μL of cell supernatant from each well and add it to the experimental plate containing 180 μL of QUANTI-Blue TM reagent. After incubating at 37°C for 1 hour, use a multifunctional microplate reader Flexstation III to detect the absorbance value at 650 nm (OD650). .
8)细胞活性检测：按照Celltiter-Glo说明书方法操作，化学发光信号(RLU)用多功能酶标仪Flexstation III检测。8) Cell viability detection: Operate according to Celltiter-Glo instructions, and the chemiluminescence signal (RLU) is detected with a multifunctional microplate reader Flexstation III.
4.数据分析4.Data analysis
化合物活性：OD650值用GraphPad Prism软件分析，并拟合化合物剂量效应曲线，计算化合物的EC50值。细胞活性检测：细胞活性％计算公式如下。细胞活性％值用GraphPad Prism软件分析，并 拟合化合物剂量效应曲线，计算化合物对细胞的CC50值。
细胞活性％＝RLU化合物/RLUDMSO控制*100％Compound activity: The OD650 value was analyzed using GraphPad Prism software, and the compound dose-effect curve was fitted to calculate the EC 50 value of the compound. Cell activity detection: The calculation formula of cell activity % is as follows. Cell viability % values were analyzed using GraphPad Prism software, and Fit the compound dose-effect curve and calculate the CC 50 value of the compound on cells.
Cell activity %=RLU compound/RLUDMSO control*100%
5.实验结果：实验结果如表11-13所示。5. Experimental results: The experimental results are shown in Table 11-13.
表11.缀合物对TLR3细胞的激动活性
Table 11. Agonistic activity of conjugates on TLR3 cells
表12.缀合物对TLR7细胞的激动活性
Table 12. Agonistic activity of conjugates on TLR7 cells
表13.缀合物对TLR8细胞的激动活性
Table 13. Agonistic activity of conjugates on TLR8 cells
实验结论：本发明中的缀合物在所述实验条件下均未激起TLR3、hTLR7和hTLR8响应，免疫原性风险低。 Experimental conclusion: The conjugates of the present invention did not stimulate TLR3, hTLR7 and hTLR8 responses under the experimental conditions, and the risk of immunogenicity is low.

Claims (14)

  1. 一种双链RNAi化合物，其中所述化合物包含能够形成双链区的正义链和反义链，其中，所述双链区由14-29个核苷酸碱基对组成，所述反义链的5’端含有突出端，所述突出端由1-10个天然核苷酸组成，所述正义链的5’端或反义链的3’端任选含有突出端，所述双链区、正义链5’端的突出端或反义链3’端的突出端的核苷酸任选经修饰。A double-stranded RNAi compound, wherein the compound comprises a sense strand and an antisense strand capable of forming a double-stranded region, wherein the double-stranded region consists of 14-29 nucleotide base pairs, and the antisense strand The 5' end contains an overhang, the overhang is composed of 1-10 natural nucleotides, the 5' end of the sense strand or the 3' end of the antisense strand optionally contains an overhang, and the double-stranded region , the overhang at the 5' end of the sense strand or the overhang at the 3' end of the antisense strand is optionally modified.
  2. 根据权利要求1所述的双链RNAi化合物，其中所述双链区由14、15、16、17、18、19、20、21、22、23、24、25、26、27、28或29个核苷酸碱基对组成。The double-stranded RNAi compound according to claim 1, wherein the double-stranded region consists of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 Composed of nucleotide base pairs.
  3. 根据权利要求2所述的双链RNAi化合物，其中所述双链区由17、18、19、20、21、22或23个核苷酸碱基对组成。The double-stranded RNAi compound of claim 2, wherein the double-stranded region consists of 17, 18, 19, 20, 21, 22 or 23 nucleotide base pairs.
  4. 根据权利要求1-3任意一项所述的双链RNAi化合物，其中所述双链区的核苷酸全部经修饰。The double-stranded RNAi compound according to any one of claims 1-3, wherein all nucleotides in the double-stranded region are modified.
  5. 根据权利要求1所述的双链RNAi化合物，其中所述正义链的5’端的突出端含有0、1、2或3个任选经修饰的核苷酸。The double-stranded RNAi compound of claim 1, wherein the overhang of the 5' end of the sense strand contains 0, 1, 2 or 3 optionally modified nucleotides.
  6. 根据权利要求1所示的双链RNAi化合物，其中所述反义链的3’端的突出端含有0、1、2、3、4、5或6个任选经修饰的核苷酸。The double-stranded RNAi compound according to claim 1, wherein the overhang of the 3' end of the antisense strand contains 0, 1, 2, 3, 4, 5 or 6 optionally modified nucleotides.
  7. 根据权利要求5或6所述的双链RNAi化合物，其中正义链5’端的突出端或反义链3’端的突出端的核苷酸全部经修饰。The double-stranded RNAi compound according to claim 5 or 6, wherein all the nucleotides of the overhang at the 5' end of the sense strand or the overhang at the 3' end of the antisense strand are modified.
  8. 根据权利要求1所述的双链RNAi化合物，所述反义链5’端的突出端由1、2、3、4、5、6、7、8、9或10个天然核苷酸组成。According to the double-stranded RNAi compound of claim 1, the overhang at the 5' end of the antisense strand consists of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 natural nucleotides.
  9. 根据权利要求1所述的双链RNAi化合物，所述正义链的3’端缀合配体。The double-stranded RNAi compound according to claim 1, the 3' end of the sense strand is conjugated with a ligand.
  10. 根据权利要求9所述的双链RNAi化合物，所述配体为一种或多种利用二价或三价或四价的分支键联体附接的GalNAc衍生物。The double-stranded RNAi compound according to claim 9, wherein the ligand is one or more GalNAc derivatives attached using bivalent, trivalent or tetravalent branched linkages.
  11. 根据权利要求10所述的双链RNAi化合物，所述配体选自D01，
    The double-stranded RNAi compound according to claim 10, said ligand is selected from D01,
  12. 根据权利要求1所述的双链RNAi化合物，所述正义链选自SEQ ID NO:1、SEQ ID NO:2和SEQ  ID NO:13。The double-stranded RNAi compound according to claim 1, the sense strand is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO:13.
  13. 根据权利要求1所述的双链RNAi化合物，所述反义链选自SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22和SEQ ID NO:23。The double-stranded RNAi compound according to claim 1, the antisense strand is selected from SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO :17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23.
  14. 缀合物选自S1、S2、S3、S4、S5、S6、S7、S8、S9、S10、S11、S12、S13、S14、S15、S16、S17、S18、S19、S20、S21、S22、S23、S24、S25、S26、S27、S28、S29和S30。 The conjugate is selected from S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S13, S14, S15, S16, S17, S18, S19, S20, S21, S22, S23 , S24, S25, S26, S27, S28, S29 and S30.
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