WO2023155909A1 - Ribavirin analog and use thereof as embedding group - Google Patents

Ribavirin analog and use thereof as embedding group Download PDF

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Publication number
WO2023155909A1
WO2023155909A1 PCT/CN2023/077236 CN2023077236W WO2023155909A1 WO 2023155909 A1 WO2023155909 A1 WO 2023155909A1 CN 2023077236 W CN2023077236 W CN 2023077236W WO 2023155909 A1 WO2023155909 A1 WO 2023155909A1
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compound
seq
present
nucleotides
antisense strand
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PCT/CN2023/077236
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French (fr)
Chinese (zh)
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陆剑宇
安可
胡彦宾
江文
胡利红
丁照中
陈曙辉
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南京明德新药研发有限公司
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Publication of WO2023155909A1 publication Critical patent/WO2023155909A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/056—Triazole or tetrazole radicals
    • 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 invention relates to a class of nucleoside analogues, in particular to the ribavirin analogues and their use as intercalation groups in RNAi.
  • RNA interference The RNA interference (RNAi) mechanism has been paid close attention to since it was discovered in 1998; the mechanism achieves the desired pharmacological effect by down-regulating the target mRNA level to control the function of the corresponding target.
  • RNAi drugs a number of RNAi drugs have been approved for marketing, and the feasibility verification of human drugs has been completed. Drugs that achieve pharmacological effects through this mechanism are called RNAi drugs.
  • RNAi drugs have completed clinical verification, they still face some problems. The most notable one is the problem of selectivity: how to achieve specific inhibition of selected target mRNAs without affecting other non-target mRNAs.
  • selectivity the most notable one is the "miRNA effect" from RNAi drug molecules. Different from the RNAi mechanism, the miRNA mechanism does not require a perfect pairing with the corresponding target mRNA sequence, and the existence of a certain degree of mismatch does not hinder the generation of the miRNA effect.
  • a deeper mechanism study reveals that the new sequence of the "seed region" in the antisense strand of RNAi molecules is crucial for the generation of miRNA effects.
  • the "seed region” sequence refers to the 2-7 nucleotide sequence of the antisense strand of the RNAi molecule; after the RNAi molecule enters the cell to form a functional complex, the complex will first use the "seed region” sequence to scan intracellular mRNA, And the degree of adaptation to the seed region determines which mRNAs have pharmacological effects (RNAi effect or miRNA effect). Therefore, the regulation and optimization of the sequence of the seed region has become an important means to optimize the selectivity of RNAi molecules.
  • Alynlam uses the structure of chiral glycol nucleic acid (GNA) to optimize the structure of the "seed region" of the antisense strand of RNAi drugs to obtain better selectivity. How to obtain better activity on the basis of high selectivity is a key research point in the field.
  • GAA chiral glycol nucleic acid
  • the first aspect of the present invention provides a compound of formula (Z) or a pharmaceutically acceptable salt thereof,
  • the second aspect of the present invention provides the application of the compound of formula (Z) as an intercalating group of an oligonucleotide compound,
  • the oligonucleotide compound comprises 15-40 nucleotides, any one or more nucleosides of which are replaced by the compound of formula (Z), and the nucleotides are optionally modified;
  • connection mode of formula (Z) compound embedding oligonucleotide is
  • the third aspect of the present invention provides an oligonucleotide compound or a pharmaceutically acceptable salt thereof, the oligonucleotide comprises 15-40 nucleotides, any one or more nucleosides of which are Replaced by a compound of formula (Z), the nucleotides are optionally modified;
  • connection mode of formula (Z) compound embedding oligonucleotide is
  • the above-mentioned oligonucleotide compound is a single-chain compound, and other variables are as defined in the present invention.
  • the above-mentioned single-stranded compound is a single-stranded antisense oligonucleotide, and other variables are as defined in the present invention.
  • any one, two or three nucleosides in the above-mentioned single-chain compound are replaced by the compound of formula (Z), and other variables are as defined in the present invention.
  • any nucleoside in the above-mentioned single-chain compound is replaced by the compound of formula (Z), and other variables are as defined in the present invention.
  • the above-mentioned single-stranded compound is preferably composed of 18-29 nucleotides, more preferably 18-25, further preferably 18-23, most preferably 19-21, and other variables as defined herein.
  • the above-mentioned single-chain compound comprises 7, 6, 5, 4, 3, 2, 1 and 0 unmodified nucleotides, and other variables are as defined in the present invention.
  • nucleotides of the above-mentioned single-stranded compound are modified nucleotides, and other variables are as defined in the present invention.
  • the above-mentioned single-chain compound is conjugated with a ligand at any position, and other variables are as defined in the present invention.
  • the 3' end of the above-mentioned single-chain compound is conjugated with a ligand, and other variables are as defined in the present invention.
  • the 5' end of the above-mentioned single-chain compound is conjugated with a ligand, and other variables are as defined in the present invention.
  • the above-mentioned ligands are one or more GalNAc-derived ligands attached by multivalent branched linkages.
  • the other variables are as defined in the present invention.
  • the above-mentioned ligands are one or more GalNAc derivatives attached by bivalent, trivalent or tetravalent branched linkages, and other variables are as defined in the present invention.
  • the above-mentioned ligand is a GalNAc derivative attached by a bivalent, trivalent or tetravalent branched linkage, and other variables are as defined in the present invention.
  • the above-mentioned ligands are one or more GalNAc derivatives attached by bivalent or trivalent branched linkages, and other variables are as defined in the present invention.
  • the above-mentioned ligand is a GalNAc derivative attached by a tetravalent branched linkage, and other variables are as defined in the present invention.
  • the above-mentioned oligonucleotide compound is a double-stranded compound, and other variables are as defined in the present invention.
  • the above-mentioned oligonucleotide compound is a small interfering nucleotide (RNAi), and other variables are as defined in the present invention.
  • RNAi small interfering nucleotide
  • the above-mentioned double-stranded compound comprises a sense strand and an antisense strand capable of forming a double-stranded region, and other variables are as defined in the present invention.
  • any one, two or three nucleosides in the sense strand of the above-mentioned double-stranded compound are replaced by the compound of formula (Z), and other variables are as defined in the present invention.
  • any nucleoside in the sense strand of the above-mentioned double-stranded compound is replaced by the compound of formula (Z), and other variables are as defined in the present invention.
  • any one, two or three nucleosides in the antisense strand of the above-mentioned double-stranded compound are replaced by the compound of formula (Z), and other variables are as defined in the present invention.
  • any nucleoside in the antisense strand of the above-mentioned double-stranded compound is replaced by the compound of formula (Z), and other variables are as defined in the present invention.
  • the above-mentioned sense strand or antisense strand further comprises an overhang, 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 may also be connected by 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 may also be connected through a chemical group, and other variables are as defined in the present invention.
  • ligands are conjugated to any position of the aforementioned sense strand and/or antisense strand, and other variables are as defined in the present invention.
  • the 3' end of the sense strand is conjugated with a ligand, and other variables are as defined in the present invention.
  • the 5' end of the 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 strand is conjugated with a ligand, and other variables are as defined in the present invention.
  • the 5' end of the antisense strand is conjugated with a ligand, and other variables are as defined in the present invention.
  • ligands are conjugated to the above-mentioned nucleotides connecting the sense strand and the antisense strand, and other variables are as defined in the present invention.
  • ligands are conjugated to the above chemical groups 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 ligands are one or more GalNAc derivatives attached by divalent or trivalent branched linkages.
  • the above-mentioned ligand is D01 or L96, and other variables are as defined in the present invention.
  • the above-mentioned sense strand consists of 15-30 nucleotides, preferably 17-25 nucleotides, more preferably 18-23 nucleotides, and other variables are as in the present invention defined.
  • the above-mentioned antisense strand consists of 15-40 nucleotides, preferably 17-35 nucleotides, more preferably 19-30 nucleotides, most preferably 21 - Composition of 29 nucleotides, other variables are as defined herein.
  • the above-mentioned double-stranded region consists of 17-23 nucleotide base pairs, and other variables are as defined in the present invention.
  • the above sense strand comprises 7, 6, 5, 4, 3, 2, 1 and 0 unmodified nucleotides, and other variables are as defined in the present invention.
  • the above-mentioned antisense strand comprises 7, 6, 5, 4, 3, 2, 1 and 0 unmodified nucleotides, and other variables are as defined in the present invention.
  • the above-mentioned oligonucleotide compounds are used to inhibit or block the expression of genes, and the genes are AGT gene, ANGPTL3 gene, ApoA gene, Factor B gene, HBV related gene, HSD gene KRAS Related genes or complement 5 related genes.
  • the above-mentioned oligonucleotide compound is used to inhibit or block the expression of a gene, and the gene is the AGT gene.
  • the above-mentioned oligonucleotide compound is used to inhibit or block the expression of a gene, and the gene is ANGPTL3 gene.
  • the above-mentioned sense strand comprises the sequence shown in SEQ ID NO: 5, and other variables are as defined in the present invention.
  • the above-mentioned antisense strand comprises the sequence shown in SEQ ID NO: 6, and other variables are as defined in the present invention.
  • the above-mentioned sense strand comprises the sequence shown in SEQ ID NO:1
  • the above-mentioned antisense strand comprises the sequence shown in SEQ ID NO:4 or SEQ ID NO:8, on the sequence
  • the nucleotides are optionally modified, and other variables are as defined herein.
  • the above-mentioned sense strand comprises a sequence as shown in SEQ ID NO: 7 or SEQ ID NO: 9
  • the above-mentioned antisense strand comprises a sequence as shown in SEQ ID NO: 12, on the sequence
  • the nucleotides are optionally modified, and other variables are as defined herein.
  • the above-mentioned sense strand comprises the sequence shown in SEQ ID NO: 11, and other variables are as defined in the present invention.
  • the above-mentioned antisense strand comprises the sequence shown in SEQ ID NO: 13, and other variables are as defined in the present invention.
  • the above-mentioned sense strand comprises the sequence shown in SEQ ID NO:11
  • the above-mentioned antisense strand comprises the sequence shown in SEQ ID NO:13.
  • the present invention also has some technical proposals that come from the free combination of the above-mentioned variables.
  • the "oligonucleotide” in the present invention is a nucleotide sequence containing 10-50 nucleotides or nucleotide base pairs.
  • the oligonucleotide has a nucleobase sequence that is at least partially complementary to a coding sequence in a target nucleic acid expressed in a cell or a target gene.
  • the nucleotides are optionally modified.
  • the oligonucleotide is capable of inhibiting or blocking the expression of a gene in vitro or in vivo following delivery of the oligonucleotide 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 Clip RNA (shRNA), ribozymes, interfering RNA molecules, and Dicer enzyme substrates.
  • siRNA short interfering RNA
  • dsRNA double-stranded RNA
  • miRNA microRNA
  • shRNA short Clip RNA
  • ribozymes interfering RNA molecules
  • Dicer enzyme substrates Dicer enzyme substrates.
  • the "intercalation” mentioned in the present invention means that the intercalation group is connected to at least one nucleotide residue in the sequence, including the use of an intercalation group (such as r) to replace a nucleotide residue in the sequence base.
  • the "intercalation group” in the present invention is a residue of an analogue of a natural nucleotide base, which is different from any natural nucleotide base of a patent. After it is introduced into a nucleic acid sequence, it can be used The sequence has a certain function (such as bringing unexpected activity). As described in the present invention, Z, after being inserted into the oligonucleotide sequence as an intercalating group, can inhibit the expression of genes, and then produce unexpected activities.
  • oligonucleotide intercalation group in the present invention means that the intercalation group is connected to at least one nucleotide residue in the oligonucleotide, including the use of an intercalation group in the oligonucleotide (eg Z) replaces a nucleotide residue.
  • the "single-stranded compound" of the present invention refers to a single-stranded oligonucleotide having a sequence at least partially complementary to the target mRNA, which can undergo hydrogen bonding under mammalian physiological conditions (or equivalent in vitro environment) hybridize to the target mRNA.
  • the single stranded oligonucleotide is a single stranded antisense oligonucleotide.
  • double-stranded compound in the present invention refers to a duplex structure comprising two antiparallel and substantially complementary nucleotide strands, one of which is a sense strand and the other is an antisense strand.
  • siRNA short interfering RNA
  • siRNA short interfering RNA
  • the short interfering RNA (siRNA) of the present invention includes double-stranded siRNA (including sense strand and antisense strand) and single-stranded siRNA (eg only includes antisense strand).
  • the "inhibition" of the present invention when referring to the expression of a given gene, means that when compared with cells, cell groups or tissues that have not been so treated, when the oligonucleotides of the present invention are used Upon treatment of the cell, cell population or tissue, gene expression is reduced.
  • sequence or “nucleotide sequence” in the present invention means the order or sequence of nucleobases or nucleotides described by a sequence of letters using standard nucleotide nomenclature.
  • the "antisense strand” or “guide strand” in the present invention refers to the strand that is substantially complementary to the corresponding region of the target sequence (eg, AGT mRNA) in the oligonucleotide compound.
  • the "sense strand” or “passenger strand” in the present invention refers to the strand that can form a substantially complementary region with the antisense strand.
  • the "substantially complementary” means that the corresponding positions of the two sequences can be completely complementary, and there can also be one or more mismatches. When there are mismatches, there are usually no more than 3, 2 or 1 and no more than 1 mismatched bases. right.
  • bases on one strand pair 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 is optionally modified in the present invention means that each nucleotide can be an unmodified nucleotide independently or a modified nucleotide, each Modifications on modified nucleotides are also independently.
  • the "modification” includes, but not limited to, nucleobase modification, ribose modification and phosphate modification.
  • the "unmodified nucleotide” refers to a nucleotide composed of a naturally occurring nucleobase, a natural sugar ring and a natural phosphate.
  • modified nucleotide refers to a nucleotide comprising at least one of a modified nucleobase, a modified sugar ring and a modified phosphate.
  • modified nucleotide refers to a nucleotide composed of a modified nucleobase, and/or a modified sugar ring, and/or a modified phosphate.
  • modified nucleotides consist of modified nucleobases, natural sugar rings and natural phosphates; in some embodiments of the present invention, “modified nucleosides Acids” consist of natural nucleobases, modified sugar rings, and natural phosphates; in some embodiments of the invention, "modified nucleotides” Consists of a natural nucleobase, a natural sugar ring, and a modified phosphate; in some embodiments of the invention, a "modified nucleotide” consists of a natural nucleobase, a modified sugar ring, and a modified phosphate ester composition; in some embodiments of the invention, "modified nucleotides” consist of modified nucleobases, natural sugar
  • the "natural sugar ring" in the present invention is selected from 2'-OH five-membered sugar ring and 2'-deoxy five-membered sugar ring.
  • the "natural base” of the present invention is selected from the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • the "modified nucleobase” in the present invention refers to 5-12 membered saturated, partially unsaturated or aromatic heterocyclic rings other than natural bases, including monocyclic or fused rings, specifically Examples include, but are not limited to, thiophene, thianthrene, furan, pyran, isobenzofuran, benzothiazine, pyrrole, imidazole, substituted or unsubstituted triazole, pyrazole, isothiazole, isoxazole, pyridazine , indoxazine, indole, isoindole, isoquinoline, quinoline, naphthopyridine, quinazoline, carbazole, phenanthridine, piperidine, phenazine, phenanthazine, phenothiazine, furane, Phenoxazine, pyrrolidine, pyrroline, imidazolidine, imidazoline, pyrazolidine
  • the "modified sugar ring” in the present invention can include but not limited to one of the following modifications at the 2' position: H; F; O -, S- or N-alkyl; O-, S- or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl, alkenyl and Alkynyl may be substituted or unsubstituted C 1 to C 10 alkyl or C 2 to C 10 alkenyl and alkynyl.
  • Exemplary suitable modifications include O[( CH2 ) nO ] mCH3 , O( CH2 ) nOCH3 , O (CH2) nNH2 , O( CH2 ) nCH3 , 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.
  • the modification at the 2' position includes, but is 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, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, group for improving the pharmacokinetic properties of iRNA, or for improving the iRNA Groups with pharmacodynamic characteristics, and other substituents with similar properties.
  • the modification includes , but is not limited to, 2'-methoxyethoxy (2' - O- CH2CH2OCH3 , also known as 2'-O-(2-methoxyethyl ) or 2'- MOE).
  • the "modified phosphate” in the present invention includes, but is not limited to: phosphorothioate modification, and the “phosphorothioate” includes (R)- and (S)-isomers and/or or a mixture thereof.
  • the modified nucleotides may comprise one or more locked nucleic acids (LNAs).
  • Locked nucleic acids are nucleotides that have a modified ribose moiety that includes an additional bridge connecting the 2' carbon to the 4' carbon. This structure effectively "locks" the ribose in the 3'-endo structural 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 linkages have been removed, resulting in unlocked "sugar” residues.
  • the UNA also covers 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 i.e., the covalent carbon-carbon bond between the C2' and C3' carbons
  • modified nucleotides may also include one or more bicyclic sugar moieties.
  • a "bicyclic sugar” is a furanosyl ring modified by a bridge of two atoms.
  • a "bicyclic nucleoside” (“BNA”) is a nucleoside having a sugar moiety comprising a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system.
  • the bridge connects the 4'-carbon and the 2'-carbon of the sugar ring.
  • sequences described in the present invention may include those listed in “further modified sequences" in Table 2 below.
  • 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; correspondingly, “conjugate” means that each Compounds formed by covalent linkages between chemical moieties, such as through “phosphoester linkages”, “phosphodiester linkages”, “phosphorothioate linkages”, etc.
  • the "ligand" in the present invention refers to a targeting group, such as a cell or tissue targeting agent that binds to a specified cell type such as a kidney cell, such as a lectin, glycoprotein, lipid or protein, For example antibodies.
  • a targeting group such as a cell or tissue targeting agent that binds to a specified cell type such as a kidney cell, such as a lectin, glycoprotein, lipid or protein, For example antibodies.
  • Targeting groups can be thyrotropin, melanin, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine (GalNAc) , N-acetyl-glucosamine polyvalent mannose, polyvalent fucose, glycosylated polyamino acid, polyvalent galactose, transferrin, bisphosphonate, polyglutamic acid, polyaspartic acid , lipids, cholesterol, steroids, cholic acid, folic acid, vitamin B12, vitamin A, biotin, or RGD peptides or RGD peptidomimetics.
  • 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 strand extends beyond the 5'-end of the other strand, or the 5'-end of one strand extends beyond the 3'-end of the other strand.
  • 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 overhangs are optionally modified.
  • the overhang can be on the sense strand, the antisense strand, or any combination thereof.
  • overhangs may be present at the 5'-end, 3'-end, or both of the antisense or sense strands of the double-stranded compound.
  • the antisense strand has 1 to 10 nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides) overhang.
  • the sense strand has 1 to 10 nucleotides (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) overhangs.
  • 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) overhangs.
  • said SEQ ID NO: 2 may include a modified or unmodified GA overhang in the 5' and/or 3' stretch.
  • one end of the sense strand and one end of the antisense strand described in the present invention can also be connected by one or more nucleotides, referring to the connection mode shown in the following formula (I): Wherein N represents optionally modified nucleotides, n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; strand 1 and strand 2 are independently sense strand or antisense strand .
  • one end of the sense strand and one end of the antisense strand described in the present invention can also be connected through a chemical group, which refers to the connection method shown in the following formula (II):
  • L is selected from C 1-100 alkylene
  • multiple in the terms “multiple” and “multivalent” in the present invention refers to an integer greater than or equal to 2, including but not limited to 2, 3, 4, 5, 6, 7, 8, 9, 10 .
  • the double-stranded siRNA analog conjugate of the present invention is a compound formed by linking a double-stranded siRNA analog and a pharmaceutically acceptable conjugating group, and the double-stranded siRNA analog and a pharmaceutically acceptable conjugating group group covalently linked.
  • linker refers to an organic moiety group that connects two parts of a compound, eg, covalently attaches two parts of a compound.
  • the linkage usually contains a direct bond or atom (such as: oxygen or sulfur), atomic group (such as: NRR, C(O), C(O)NH, SO, SO 2 , SO 2 NH), substituted or un 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 one or more optional C atoms in the substituted or unsubstituted alkyl, substituted or substituted alkenyl, substituted or unsubstituted alkynyl can be substituted or un
  • a cleavable linker is sufficiently stable outside the cell, but upon entry into the target cell, it is cleaved to release the two moieties to which the linker is co-immobilized.
  • the compounds of the invention may exist in particular geometric or stereoisomeric forms.
  • the present invention contemplates all such compounds, including (R)- and (S)-enantiomers, diastereomers, and racemic and other mixtures thereof, such as enantiomers or diastereomers Body-enriched mixtures, all of which are within the scope of the present invention.
  • Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are included within the scope of the present invention.
  • enantiomer or “optical isomer” refer to stereoisomers that are mirror images of each other.
  • diastereoisomer refers to stereoisomers whose molecules have two or more chiral centers and which are not mirror images of the molecules.
  • keys with wedge-shaped solid lines and dotted wedge keys Indicates the absolute configuration of a stereocenter, with a straight solid-line bond and straight dashed keys Indicates the relative configuration of the stereocenter, with a wavy line Indicates wedge-shaped solid-line bond or dotted wedge key or with tilde Indicates a straight solid line key and/or straight dotted key
  • the terms “enriched in an isomer”, “enriched in an isomer”, “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%.
  • the terms “isomer excess” or “enantiomeric excess” refer 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 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 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 auxiliary agents, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide pure desired enantiomer.
  • a diastereoisomeric salt is formed with an appropriate optically active acid or base, and then a diastereomeric salt is formed by a conventional method 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 in combination with chemical derivatization methods (e.g. amines to amino groups formate).
  • the compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compounds.
  • compounds may be labeled with radioactive isotopes such as tritium ( 3 H), iodine-125 ( 125 I) or C-14 ( 14 C).
  • radioactive isotopes such as tritium ( 3 H), iodine-125 ( 125 I) or C-14 ( 14 C).
  • heavy hydrogen can be used to replace hydrogen to form deuterated drugs.
  • the bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon.
  • deuterated drugs can reduce toxic side effects and increase drug stability. , enhance the efficacy, prolong the biological half-life of drugs and other advantages. All changes in 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 a specific substituent found 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 obtained by using enough in a pure solution or a suitable inert solvent Base addition salts are obtained by contacting such compounds with an appropriate amount of base.
  • 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 the acid, either neat solution or in a suitable inert solvent.
  • Examples of pharmaceutically acceptable acid addition salts include salts of inorganic acids including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogenphosphate, dihydrogenphosphate, sulfuric acid, Hydrogen sulfate, hydriodic acid, phosphorous acid, etc.; and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, Fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid and similar acids; also salts of amino acids such as arginine and the like , and salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain basic and acidic functional groups and can thus be converted into
  • the salts of the present invention can be synthesized from the parent compound containing acid groups or bases by conventional chemical methods.
  • 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 both.
  • the compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by combining them with other chemical synthesis methods, and the methods well known to those skilled in the art Equivalent alternatives, preferred embodiments include but are not limited to the examples of the present invention.
  • the compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compounds.
  • compounds may be labeled with radioactive isotopes such as tritium ( 3 H), iodine-125 ( 125 I) or C-14 ( 14 C).
  • radioactive isotopes such as tritium ( 3 H), iodine-125 ( 125 I) or C-14 ( 14 C).
  • heavy hydrogen can be used to replace hydrogen to form deuterated drugs.
  • the bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon.
  • deuterated drugs can reduce toxic side effects and increase drug stability. , enhance the efficacy, prolong the biological half-life of drugs and other advantages. All changes in isotopic composition of the compounds of the invention, whether radioactive or not, are included within the scope of the invention.
  • linking group listed does not indicate its linking direction
  • its linking direction is arbitrary, for example,
  • the connecting group L in the middle is -MW-, at this time -MW- can connect ring A and ring B in the same direction as the reading order from left to right to form It can also be formed by connecting loop A and loop B in the opposite direction to the reading order from left to right
  • substituted means that any one or more hydrogen atoms on a specified atom are replaced by a substituent, which may include deuterium and hydrogen variants, as long as the valence of the specified atom is normal and the substituted compound is stable.
  • Oxygen substitution does not occur on aromatic groups.
  • optionally substituted means that it may or may not be substituted, and unless otherwise specified, the type and number of substituents may be arbitrary on a chemically realizable basis.
  • any variable such as R
  • its definition in each case is unique standing.
  • said group may optionally be substituted with up to two R, with independent options for each occurrence of R.
  • substituents and/or variations thereof are permissible only if such combinations result in stable compounds.
  • linking group When the number of a linking group is 0, such as -(CRR) 0 -, it means that the linking group is a single bond.
  • substituent When a substituent is vacant, it means that the substituent does not exist. For example, when X in A-X is vacant, it means that the structure is actually A. When the enumerated substituent does not indicate which atom it is connected to the substituted group, this substituent can be bonded through any atom, for example, pyridyl as a substituent can be connected to any atom on the pyridine ring. The carbon atom is attached to the group being substituted.
  • the compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by combining them with other chemical synthesis methods, and the methods well known to those skilled in the art Equivalent alternatives, preferred embodiments include but are not limited to the examples of the present invention.
  • the structure of the compounds of the present invention can be confirmed by conventional methods 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, in single crystal X-ray diffraction (SXRD), the cultured single crystal is collected with a Bruker D8 venture diffractometer to collect diffraction intensity data, the light source is CuK ⁇ radiation, and the scanning method is: After scanning and collecting relevant data, the absolute configuration can be confirmed by further analyzing the crystal structure by direct method (Shelxs97).
  • SXRD single crystal X-ray diffraction
  • the solvent used in the present invention is commercially available.
  • the ratios of solvents used in column chromatography and preparative thin-layer silica gel chromatography of the present invention are volume ratios.
  • aq stands for water
  • HATU O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate
  • EDC represents N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride
  • m-CPBA 3-chloroperoxybenzoic acid
  • eq represents equivalent, equivalent
  • CDI represents Carbonyldiimidazole
  • DCM stands for dichloromethane
  • PE stands for petroleum ether
  • DIAD stands for diisopropyl azodicarboxylate
  • DMF stands for N,N-dimethylformamide
  • DMSO stands for dimethylsulfoxide
  • EtOAc stands for ethyl acetate EtOH stands for ethanol
  • MeOH stands for methanol
  • CBz stands for benzyloxycarbonyl, an amine protecting group
  • nucleotide monomer specifically as shown in Table 2:
  • the compound of the present invention and its application can produce better curative effect and safety.
  • Figure 1 shows the expression results of AGT in mouse liver.
  • Figure 2 and Figure 3 are the results of in vitro RNA sequence studies.
  • Fig. 4 is the test result of the agonistic activity of the compound on hTLR3, hTLR7, hTLR8 cells.
  • Fig. 5 is a 3D structure diagram of AU base pairing in EL86.
  • Figure 6 is a 3D structure diagram of ZU pairing in EL86.
  • Fig. 7 is a 3D structure diagram of UA base pairing in EL86.
  • Figure 8 is a 3D structure diagram of ZA pairing in EL86.
  • Figure 9 is a 3D structure diagram of CG base pairing in EL86.
  • Figure 10 is a 3D structure diagram of ZG pairing in EL86.
  • Figure 11 is a 3D structure diagram of GC base pairing in EL86.
  • Figure 12 is a 3D structure diagram of ZC pairing in EL86.
  • Figure 13 is a crystal structure diagram of EL86.
  • Figure 14 is the result of AD05488 and negative control.
  • Figure 15 shows the results of ANG001 and negative controls.
  • Step 2 Use Maestro software (Maestro V12.4, New York, NY, 2020), respectively constructing the structure of Z replacing A4/U6/C9/G16, the specific operation is the first step: delete the base part of A4, and the remaining hydrogen atom is used as the connection site of the triazole, Step 2: Use the Enumeration tool in Maesro to splice the triazole structure onto the above connection point to obtain the structure in which Z replaces A, and obtain the sequence shown in Table 4 (SS 5 ⁇ 3 of a, SEQ ID NO:16 SS 5 ⁇ 3 of b, SEQ ID NO:17; SS 5 ⁇ 3 of c, SEQ ID NO:18; SS 5 ⁇ 3 of d, SEQ ID NO:19; as of a, b, c and d 3 ⁇ 5, SEQ ID NO: 15):
  • Step A 1-1 (10 g, 19.82 mmol) was dissolved in acetonitrile (120 ml) and 1,2-dichloroethane (80 ml), and 1-2 (6.02 g, 42.62 mg mol) and trimethylsilyl trifluoromethanesulfonate (11.01 g, 49.56 mmol, 8.95 ml), and the mixture was stirred at 35°C for 12 hours. The reaction solution was slowly added saturated aqueous sodium bicarbonate (100 ml L) quenched and extracted with dichloromethane (100 mL ⁇ 2).
  • the obtained organic phase was washed with saturated aqueous sodium chloride (200 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a crude product.
  • Step B Dissolve 1-3 (6.7 g, 13.05 mmol) in methanolic ammonia solution (7 mol/L, 50 mL), and stir the mixture at 40°C for 12 hours.
  • Step C Dissolve 1-4 (2.3 g, 11.43 mmol) in anhydrous pyridine (25 mL), add 1,3 dichloro-1,1,3,3-tetraisopropyldisila at 0 °C Oxane (3.64 g, 11.55 mmol, 3.69 mL), the mixture was stirred at 20°C for 12 hours.
  • the reaction solution was quenched by adding water (30 mL), and extracted by adding ethyl acetate (30 mL ⁇ 2).
  • the obtained organic phase was washed successively with hydrochloric acid (1 mol/L, 30 mL ⁇ 3) and saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a crude product.
  • Step D 1-5 (4 g, 9.02 mmol) was dissolved in acetonitrile (40 ml), silver oxide (8.36 g, 36.06 mmol) was added successively, Molecular sieves (3 g), anhydrous pyridine (1.78 g, 22.54 mmol, 1.82 mL) and iodomethane (6.4 g, 45.08 mmol, 2.81 mL), the mixture was stirred at 25°C for 12 hours.
  • the reaction solution was added ethyl acetate (50 mL) and stirred at 20°C for 1 hour, filtered through a Buchner funnel pad with celite and collected the filtrate, which was extracted by adding water (100 mL) and ethyl acetate (100 mL ⁇ 2).
  • the obtained organic phase was washed with saturated brine (200 ml), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a crude product.
  • Step F Dissolve 1-7 (1 g, 4.65 mmol) in anhydrous pyridine (10 mL), add 4,4-bismethoxytrityl chloride (1.57 g, 4.65 mmol), and the mixture Stir for 12 hours at 20°C.
  • the reaction solution was quenched by adding water (20 mL) and extracted with ethyl acetate (20 mL ⁇ 2).
  • the obtained organic phase was washed with saturated brine (40 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a crude product.
  • Step G Compounds 1-8 (3 g, 5.80 mmol) and 1-9 (1.92 g, 6.38 mmol) were dissolved in anhydrous dichloromethane (30 mL), and 4,5-bis Cyanoimidazole (342.26 mg, 2.90 mmol). The mixture was stirred at 25°C for 2 hours. 15 mL of saturated aqueous sodium bicarbonate solution and 15 mL of water were added to the reaction solution and extracted with dichloromethane (30 mL ⁇ 2). The combined organic phases were washed with saturated brine (30 mL ⁇ 2), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product.
  • the synthesized sequences are all chemically synthesized oligonucleotides, through multi-step solid-phase synthesis of oligonucleotides containing solid-phase carriers and protective groups, and finally ammonia deprotection and purification.
  • the solid-phase synthesis method is as follows:
  • nucleic acid sequence descriptions use abbreviations for nucleotide monomers, as follows:
  • D01 represents a conjugated group, and the specific structure is as follows:
  • the carrier containing the protective group oligonucleotide sequence was obtained by solid-phase synthesis, and then cut and deprotected from the solid-phase carrier to obtain the crude oligonucleotide, followed by ammonolysis (ammonolysis conditions: ammonia water, 55°C, 16h), and HPLC After purification, the single chain can be directly lyophilized to obtain a pure product.
  • the purity of the sense strand (SS) of AG001 is: 98.3%, and the molecular weight is 8768.3.
  • the purity of the antisense strand (AS) is 97.8%, and the molecular weight is 7631.4; the double strand needs to be annealed and freeze-dried to obtain AG001.
  • the purity of the sense strand (SS) of ANG001 is: 95.8%, and the molecular weight is 9084.3.
  • the purity of the antisense strand (AS) is: 96.8%.
  • the subweight is: 6879.5; the double strand needs to be annealed and freeze-dried to obtain ANG001.
  • the mouse model of pcDNA-CMV-AGT plasmid was injected into the high-pressure tail vein to evaluate the target gene and the inhibitory effect of the target gene in vivo.
  • mice On the 0th day, mice were randomly divided into groups according to body weight data, with 4 mice in each group. After grouping, all mice were subcutaneously injected with a single dose, and the volume of administration was 10 mL/kg.
  • the mice in the first group were given DPBS;
  • the mice in group 2 were given AD-85481, 3mg/kg;
  • the mice in group 3 were given AG001, 3mg/kg.
  • mice in all groups were euthanized by CO2 inhalation, and 2 liver samples were collected from each mouse. Liver samples were treated overnight at 4°C with RNAlater, and then RNAlater was removed and stored at -80°C for detection of ANGPTL3 gene expression levels.
  • Transcriptome refers to the sum of all RNAs 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 to study all mRNAs transcribed from 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 cost, RNA-seq has become the main method of transcriptome research due to its advantages of high throughput, high sensitivity and wide application range.
  • the RNA-seq technical process mainly includes two parts: library construction and sequencing and bioinformatics analysis.
  • Standard extraction methods are used to extract RNA from tissues or cells, followed by strict quality control of RNA samples, mainly through Agilent 2100bioanalyzer: accurate detection of RNA integrity.
  • mRNA There are two main ways to obtain mRNA: one is to use the structural characteristics of polyA tails in most mRNAs of eukaryotes, and enrich mRNAs with polyA tails through Oligo(dT) magnetic beads. The second is to remove ribosomal RNA from total RNA to obtain mRNA. Then, the obtained mRNA was randomly fragmented with divalent cations in NEB Fragmentation Buffer, and the library was constructed according to the NEB common library construction method or chain-specific library construction method.
  • NEB general library construction using fragmented mRNA as a template and random oligonucleotides as primers, synthesize the first strand of cDNA in the M-MuLV reverse transcriptase system, then use RNaseH to degrade the RNA strand, and use it in the DNA polymerase I system Next, the second strand of cDNA is synthesized from dNTPs. After the purified double-stranded cDNA is end-repaired, A-tailed and connected to a sequencing adapter, the cDNA of about 250-300bp is screened with AMPure XP beads, PCR is amplified, and the PCR product is purified again using AMPure XP beads to finally obtain a library. Kits for library construction are 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 that of NEB general library construction, the difference is that when synthesizing the second strand, the dTTP in dNTPs is replaced by dUTP, and then the cDNA ends are also repaired and added. A tail, ligation sequencing adapter and length screening, and then use USER enzyme to degrade the second strand of cDNA containing U, and then perform PCR amplification to obtain the library. Strand-specific libraries have many advantages, such as more effective information can be obtained with the same amount of data; more accurate gene quantification, positioning and annotation information can be obtained; antisense transcripts and the expression level of a single exon in each isoform can be provided.
  • the kits used for library construction are Ultra TM Directional RNA Library Prep Kit for
  • Sequencing linker including P5/P7, index and Rd1/Rd2 SP three parts.
  • P5/P7 is the PCR amplification primer and the primer binding part on the flow cell
  • the index provides information to distinguish different libraries
  • Rd1/Rd2 SP is the read1/read2 sequence primer, which is the binding region of the sequencing primer, and the sequencing process is theoretically controlled by Rd1/ Rd2 SP proceeds backwards.
  • the basic principle of sequencing is Sequencing by Synthesis. Add four kinds of fluorescently labeled dNTPs, DNA polymerase and adapter primers to the sequenced flow cell for amplification. When each sequencing cluster extends the complementary strand, each added fluorescently labeled dNTP can release the corresponding fluorescence , the sequencer obtains the sequence information of the fragment to be tested by capturing the fluorescent signal and converting the optical signal into a sequencing peak through computer software.
  • AD85481 (US2021095290) affects 1161 genes in total, up-regulates 608 genes, and down-regulates 553 genes; AG001 affects a total of 763 genes, up-regulates 345 genes, and down-regulates 418 genes Gene. Judging from the ratio of the effect, AD-85481 and AG001 have a relatively small effect on the gene. At the same time, the number of genes affected by AG001 was lower than that of AD-85481.
  • AD05488 (WO2019055633) has an effect on 1633 genes, up-regulates 490 genes, and down-regulates 1143 genes;
  • ANG001 affects a total of 826 genes, up-regulates 284 genes, and down-regulates 542 genes. gene. Judging from the proportion of the effect, the proportion of AG001's influence on the gene is relatively small.
  • the main instruments used in this research are multifunctional microplate reader Flexstation III (Molecular Device) and Echo555 (Labcyte)
  • Detection of compound activity Take 20 ⁇ l of cell supernatant from each well, add it to the experimental plate containing 180 ⁇ l of QUANTI-Blue TM reagent, incubate at 37°C for 1 hour, and use a multifunctional microplate reader Flexstation III to detect the absorbance value at 650 nm (OD650) .
  • Detection of cell viability operate according to the instructions of Celltiter-Glo, and detect the chemiluminescent signal (RLU) with a multifunctional microplate reader Flexstation III.
  • the OD650 value was analyzed by GraphPad Prism software, and the compound dose-effect curve was fitted to calculate the EC 50 value of the compound.
  • Cell Viability Detection The calculation formula of cell viability % is as follows. The cell activity % value was analyzed with GraphPad Prism software, and the dose-effect curve of the compound was fitted to calculate the CC 50 value of the compound on the cells.

Abstract

Disclosed are a ribavirin analog and use thereof as an embedding group in RNAi, and particularly disclosed is a compound as shown in formula (Z).

Description

三氮唑核苷类似物及其作为嵌入基团的应用Ribavirin analogues and their use as intercalation groups
本申请主张如下优先权：This application claims the following priority:
CN202210153288.0，申请日2022年02月18日；CN202210153288.0, the application date is February 18, 2022;
CN202210452061.6，申请日2022年04月26日。CN202210452061.6, the application date is April 26, 2022.
技术领域technical field
本发明涉及一类核苷类似物，具体涉及该三氮唑核苷类似物及其作为嵌入基团在RNAi中的应用。The invention relates to a class of nucleoside analogues, in particular to the ribavirin analogues and their use as intercalation groups in RNAi.
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 paid close attention to since it was discovered in 1998; the mechanism achieves the desired pharmacological effect by down-regulating the target mRNA level to control the function of the corresponding target. In recent years, a number of RNAi drugs have been approved for marketing, and the feasibility verification of human drugs has been completed. Drugs that achieve pharmacological effects through this mechanism are called RNAi drugs.
尽管RNAi药物完成了临床验证，但是其本身还是面临一些问题，比较显著的一个就是选择性问题：如何实现对选定靶点mRNA的专一性抑制而不影响其他非靶mRNA。研究认为，RNAi选择性不佳的主要原因之一是来自于RNAi药物分子的“miRNA效应”。与RNAi机制不同，miRNA机制不要求与对应靶mRNA的序列完美配对，存在一定程度的错配并不妨碍miRNA效应的产生。更深层次的机制研究揭示，RNAi分子反义链中“种子区”新序列对于miRNA效应的产生至关重要。“种子区”序列指的是RNAi分子反义链第2-7号核苷酸序列；在RNAi分子进入细胞形成功能复合物后，该复合物会先使用“种子区”序列扫描胞内mRNA，并通过与种子区的适配程度决定对哪些mRNA产生药理作用(RNAi效应或者miRNA效应)。因此，对于种子区序列的调节优化成为优化RNAi分子选择性的重要手段。Although RNAi drugs have completed clinical verification, they still face some problems. The most notable one is the problem of selectivity: how to achieve specific inhibition of selected target mRNAs without affecting other non-target mRNAs. Studies suggest that one of the main reasons for the poor selectivity of RNAi is the "miRNA effect" from RNAi drug molecules. Different from the RNAi mechanism, the miRNA mechanism does not require a perfect pairing with the corresponding target mRNA sequence, and the existence of a certain degree of mismatch does not hinder the generation of the miRNA effect. A deeper mechanism study reveals that the new sequence of the "seed region" in the antisense strand of RNAi molecules is crucial for the generation of miRNA effects. The "seed region" sequence refers to the 2-7 nucleotide sequence of the antisense strand of the RNAi molecule; after the RNAi molecule enters the cell to form a functional complex, the complex will first use the "seed region" sequence to scan intracellular mRNA, And the degree of adaptation to the seed region determines which mRNAs have pharmacological effects (RNAi effect or miRNA effect). Therefore, the regulation and optimization of the sequence of the seed region has become an important means to optimize the selectivity of RNAi molecules.
Alynlam公司使用手性乙二醇核酸(GNA)结构来优化RNAi药物反义链“种子区”结构从而获得更优的选择性。如何在高选择性的基础上获得更好的活性是领域内的一个关键研究点。
Alynlam uses the structure of chiral glycol nucleic acid (GNA) to optimize the structure of the "seed region" of the antisense strand of RNAi drugs to obtain better selectivity. How to obtain better activity on the basis of high selectivity is a key research point in the field.
参考文献：J.Am.Chem.Soc.2017,139,25,8537–8546.References: J.Am.Chem.Soc.2017,139,25,8537–8546.
发明内容Contents of the invention
本发明第一方面提供了式(Z)化合物或其药学上可接受的盐，
The first aspect of the present invention provides a compound of formula (Z) or a pharmaceutically acceptable salt thereof,
本发明第二方面提供了式(Z)化合物作为寡聚核苷酸化合物的嵌入基团的应用，
The second aspect of the present invention provides the application of the compound of formula (Z) as an intercalating group of an oligonucleotide compound,
其中，所述寡聚核苷酸化合物包含15-40个核苷酸，其中的任意一个或多个核苷被式(Z)化合物替换，所述核苷酸任选被修饰；Wherein, the oligonucleotide compound comprises 15-40 nucleotides, any one or more nucleosides of which are replaced by the compound of formula (Z), and the nucleotides are optionally modified;
式(Z)化合物嵌入寡聚核苷酸的连接方式为 The connection mode of formula (Z) compound embedding oligonucleotide is
本发明第三方面提供了一种寡聚核苷酸化合物或其药学上可接受的盐，所述寡聚核苷酸包含15-40个核苷酸，其中的任意一个或多个核苷被式(Z)化合物替换，所述核苷酸任选被修饰；
The third aspect of the present invention provides an oligonucleotide compound or a pharmaceutically acceptable salt thereof, the oligonucleotide comprises 15-40 nucleotides, any one or more nucleosides of which are Replaced by a compound of formula (Z), the nucleotides are optionally modified;
式(Z)化合物嵌入寡聚核苷酸的连接方式为 The connection mode of formula (Z) compound embedding oligonucleotide is
在本发明的一些技术方案中，上述寡聚核苷酸化合物为单链化合物，其他变量如本发明所定义。In some technical solutions of the present invention, the above-mentioned oligonucleotide compound is a single-chain compound, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述单链化合物为单链反义寡核苷酸，其他变量如本发明所定义。In some technical solutions of the present invention, the above-mentioned single-stranded compound is a single-stranded antisense oligonucleotide, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述单链化合物中的任意一个、两个或三个核苷被式(Z)化合物替换，其他变量如本发明所定义。In some technical solutions of the present invention, any one, two or three nucleosides in the above-mentioned single-chain compound are replaced by the compound of formula (Z), and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述单链化合物中的任意一个核苷被式(Z)化合物替换，其他变量如本发明所定义。In some technical solutions of the present invention, any nucleoside in the above-mentioned single-chain compound is replaced by the compound of formula (Z), and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述单链化合物优选为由18-29个核苷酸组成，更优选为18-25，进一步优选为18-23个，最优选为19-21个，其他变量如本发明所定义。In some technical solutions of the present invention, the above-mentioned single-stranded compound is preferably composed of 18-29 nucleotides, more preferably 18-25, further preferably 18-23, most preferably 19-21, and other variables as defined herein.
在本发明的一些技术方案中，上述单链化合物包含7、6、5、4、3、2、1和0个未经修饰的核苷酸，其他变量如本发明所定义。In some technical solutions of the present invention, the above-mentioned single-chain compound comprises 7, 6, 5, 4, 3, 2, 1 and 0 unmodified nucleotides, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述单链化合物全部核苷酸是经修饰的核苷酸，其他变量如本发明所定义。In some technical solutions of the present invention, all nucleotides of the above-mentioned single-stranded compound are modified nucleotides, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述单链化合物在任意位置缀合配体，其他变量如本发明所定义。In some technical solutions of the present invention, the above-mentioned single-chain compound is conjugated with a ligand at any position, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述单链化合物的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 single-chain compound 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，上述单链化合物的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 single-chain compound 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，上述配体为一种或多种利用多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 ligands are one or more GalNAc-derived ligands attached by multivalent branched linkages. The 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、三价或四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 ligands are one or more GalNAc derivatives attached by bivalent, trivalent or tetravalent branched linkages, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述配体为一种利用二价、三价或四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 a GalNAc derivative attached by a bivalent, trivalent or tetravalent branched linkage, 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 ligands are one or more GalNAc derivatives attached by bivalent or trivalent branched linkages, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述配体为一种利用四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 a GalNAc derivative attached by a tetravalent branched linkage, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述寡聚核苷酸化合物为双链化合物，其他变量如本发明所定义。In some technical solutions of the present invention, the above-mentioned oligonucleotide compound is a double-stranded compound, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述寡聚核苷酸化合物为小干扰核苷酸(RNAi)，其他变量如本发明所定义。In some technical solutions of the present invention, the above-mentioned oligonucleotide compound is a small interfering nucleotide (RNAi), and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述双链化合物包含能够形成双链区的正义链和反义链，其他变量如本发明所定义。In some technical solutions of the present invention, the above-mentioned double-stranded compound comprises a sense strand and an antisense strand capable of forming a double-stranded region, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述双链化合物的正义链中的任意一个、两个或三个核苷被式(Z)化合物替换，其他变量如本发明所定义。In some technical solutions of the present invention, any one, two or three nucleosides in the sense strand of the above-mentioned double-stranded compound are replaced by the compound of formula (Z), and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述双链化合物的正义链中的任意一个核苷被式(Z)化合物替换，其他变量如本发明所定义。In some technical solutions of the present invention, any nucleoside in the sense strand of the above-mentioned double-stranded compound is replaced by the compound of formula (Z), and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述双链化合物的反义链中的任意一个、两个或三个核苷被式(Z)化合物替换，其他变量如本发明所定义。In some technical solutions of the present invention, any one, two or three nucleosides in the antisense strand of the above-mentioned double-stranded compound are replaced by the compound of formula (Z), and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述双链化合物的反义链中的任意一个核苷被式(Z)化合物替换，其他变量如本发明所定义。In some technical solutions of the present invention, any nucleoside in the antisense strand of the above-mentioned double-stranded compound is replaced by the compound of formula (Z), and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述正义链或反义链还进一步包含突出端，其他变量如本发明所定义。In some technical solutions of the present invention, the above-mentioned sense strand or antisense strand further comprises an overhang, 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, there is no other covalent connection between the sense strand and the antisense strand except for hydrogen bonds between bases, 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 may also be connected by 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 may also be connected through a chemical group, 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, ligands are conjugated to any position of the aforementioned 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 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 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 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 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, ligands are conjugated to the above-mentioned nucleotides 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, ligands are conjugated to the above chemical groups 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。In some technical solutions of the present invention, the above-mentioned ligands are one or more GalNAc derivatives attached by divalent or trivalent branched linkages.
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，上述正义链由15-30个核苷酸组成，优选由17-25个核苷酸组成，更优选由18-23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 sense strand consists of 15-30 nucleotides, preferably 17-25 nucleotides, more preferably 18-23 nucleotides, and other variables are as in the present invention defined.
在本发明的一些技术方案中，上述反义链由15-40个核苷酸组成，优选由17-35个核苷酸组成，更优选由19-30个核苷酸组成，最优选由21-29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 antisense strand consists of 15-40 nucleotides, preferably 17-35 nucleotides, more preferably 19-30 nucleotides, most preferably 21 - Composition of 29 nucleotides, other variables are as defined herein.
在本发明的一些技术方案中，上述双链区由17-23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 17-23 nucleotide base pairs, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述正义链包含7、6、5、4、3、2、1和0个未经修饰的核苷酸，其他变量如本发明所定义。In some technical solutions of the present invention, the above sense strand comprises 7, 6, 5, 4, 3, 2, 1 and 0 unmodified nucleotides, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述反义链包含7、6、5、4、3、2、1和0个未经修饰的核苷酸，其他变量如本发明所定义。In some technical solutions of the present invention, the above-mentioned antisense strand comprises 7, 6, 5, 4, 3, 2, 1 and 0 unmodified nucleotides, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述寡聚核苷酸化合物用于抑制或阻断基因的表达，所述基因为AGT基因、ANGPTL3基因、ApoA基因、Factor B基因、HBV相关基因、HSD基因KRAS相关基因或补体5相关基因。In some technical schemes of the present invention, the above-mentioned oligonucleotide compounds are used to inhibit or block the expression of genes, and the genes are AGT gene, ANGPTL3 gene, ApoA gene, Factor B gene, HBV related gene, HSD gene KRAS Related genes or complement 5 related genes.
在本发明的一些技术方案中，上述寡聚核苷酸化合物用于抑制或阻断基因的表达，所述基因为AGT基因。In some technical solutions of the present invention, the above-mentioned oligonucleotide compound is used to inhibit or block the expression of a gene, and the gene is the AGT gene.
在本发明的一些技术方案中，上述寡聚核苷酸化合物用于抑制或阻断基因的表达，所述基因为ANGPTL3基因。In some technical solutions of the present invention, the above-mentioned oligonucleotide compound is used to inhibit or block the expression of a gene, and the gene is ANGPTL3 gene.
在本发明的一些技术方案中，上述正义链包含如SEQ ID NO:5所示的序列，其他变量如本发明所定义。In some technical solutions of the present invention, the above-mentioned sense strand comprises the sequence shown in SEQ ID NO: 5, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述反义链包含如SEQ ID NO:6所示的序列，其他变量如本发明所定义。In some technical solutions of the present invention, the above-mentioned antisense strand comprises the sequence shown in SEQ ID NO: 6, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述正义链包含如SEQ ID NO:1所示的序列，上述反义链包含如SEQ ID NO:4或SEQ ID NO:8所示的序列，所述序列上的核苷酸任选被修饰，其他变量如本发明所定义。 In some technical solutions of the present invention, the above-mentioned sense strand comprises the sequence shown in SEQ ID NO:1, and the above-mentioned antisense strand comprises the sequence shown in SEQ ID NO:4 or SEQ ID NO:8, on the sequence The nucleotides are optionally modified, and other variables are as defined herein.
在本发明的一些技术方案中，上述正义链包含如SEQ ID NO:7或SEQ ID NO:9所示的序列，上述反义链包含如SEQ ID NO:12所示的序列，所述序列上的核苷酸任选被修饰，其他变量如本发明所定义。In some technical solutions of the present invention, the above-mentioned sense strand comprises a sequence as shown in SEQ ID NO: 7 or SEQ ID NO: 9, and the above-mentioned antisense strand comprises a sequence as shown in SEQ ID NO: 12, on the sequence The nucleotides are optionally modified, and other variables are as defined herein.
在本发明的一些技术方案中，上述正义链包含如SEQ ID NO:11所示的序列，其他变量如本发明所定义。In some technical solutions of the present invention, the above-mentioned sense strand comprises the sequence shown in SEQ ID NO: 11, and other variables are as defined in the present invention.
在本发明的一些技术方案中，上述反义链包含如SEQ ID NO:13所示的序列，其他变量如本发明所定义。In some technical solutions of the present invention, the above-mentioned antisense strand comprises the sequence shown in 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，上述正义链包含如SEQ ID NO:11所示的序列，上述反义链包含如SEQ ID NO:13所示的序列。In some technical solutions of the present invention, the above-mentioned sense strand comprises the sequence shown in SEQ ID NO:11, and the above-mentioned antisense strand comprises the sequence shown in SEQ ID NO:13.
在本发明的一些技术方案中，所述序列如表1-1和1-2所示。In some technical solutions of the present invention, the sequences are shown in Tables 1-1 and 1-2.
表1-1靶向AGT基因的双链siRNA类似物及相关序列
Table 1-1 Double-stranded siRNA analogs and related sequences targeting AGT gene
表1-2靶向ANGPTL3基因的双链siRNA类似物及相关序列
Table 1-2 Double-stranded siRNA analogs targeting ANGPTL3 gene and related sequences
本发明还有一些技术方案由上述变量自由组合而来。The present invention also has some technical proposals that come from the free combination of the above-mentioned variables.
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 specific term or phrase should not be regarded as indeterminate or unclear if there is no special definition, but should be interpreted according to the meaning understood by those of ordinary skill in the art. When 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 comprising" 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10～50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。所述核苷酸任选被修饰。在本发明一些实施方式中，在将寡聚核苷酸递送至表达基因的细胞后，寡聚核苷酸能够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, the "oligonucleotide" in the present invention is a nucleotide sequence containing 10-50 nucleotides or nucleotide base pairs. In some embodiments of the present invention, the oligonucleotide has a nucleobase sequence that is at least partially complementary to a coding sequence in a target nucleic acid expressed in a cell or a target gene. The nucleotides are optionally modified. In some embodiments of the invention, the oligonucleotide is capable of inhibiting or blocking the expression of a gene in vitro or in vivo following delivery of the oligonucleotide 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 Clip RNA (shRNA), ribozymes, interfering RNA molecules, and Dicer enzyme substrates.
除非另有规定，本发明所述所述“嵌入”表示嵌入基团与序列中至少一个核苷酸残基相连接，包括在序列中使用嵌入基团(如r)替换了一个核苷酸残基。Unless otherwise specified, the "intercalation" mentioned in the present invention means that the intercalation group is connected to at least one nucleotide residue in the sequence, including the use of an intercalation group (such as r) to replace a nucleotide residue in the sequence base.
除非另有规定，本发明所述“嵌入基团”为天然核苷酸碱基的类似物的残基，不同于任何公开专利的天然核苷酸碱基，将其引入核酸序列后，可使序列具备一定功能(如带来不可预料的活性)。如本发明所述Z，其作为嵌入基团嵌入寡聚核苷酸序列后，能够抑制基因的表达，进而产生不可预料的活性。Unless otherwise specified, the "intercalation group" in the present invention is a residue of an analogue of a natural nucleotide base, which is different from any natural nucleotide base of a patent. After it is introduced into a nucleic acid sequence, it can be used The sequence has a certain function (such as bringing unexpected activity). As described in the present invention, Z, after being inserted into the oligonucleotide sequence as an intercalating group, can inhibit the expression of genes, and then produce unexpected activities.
本发明所述“寡聚核苷酸嵌入基团”是指所述嵌入基团与寡聚核苷酸中至少一个核苷酸残基相连接，包括在寡聚核苷酸中使用嵌入基团(如Z)替换了一个核苷酸残基。The "oligonucleotide intercalation group" in the present invention means that the intercalation group is connected to at least one nucleotide residue in the oligonucleotide, including the use of an intercalation group in the oligonucleotide (eg Z) replaces a nucleotide residue.
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, the "single-stranded compound" of the present invention refers to a single-stranded oligonucleotide having a sequence at least partially complementary to the target mRNA, which can undergo hydrogen bonding under mammalian physiological conditions (or equivalent in vitro environment) hybridize to the 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, the "double-stranded compound" in the present invention refers to a duplex structure comprising two antiparallel and substantially complementary nucleotide strands, one of which is a sense strand and the other is an antisense strand.
除非另有规定，本发明所述“短干扰RNA(siRNA)”是一类RNA分子，长度为14-30个碱基对，类似于miRNA，并且在RNA干扰(RNAi)途径内操作，它干扰了与核苷酸序列互补的特定基因的mRNA的翻译，导致mRNA降解。本发明所述短干扰RNA(siRNA)包括双链siRNA(包括正义链和反义链)和单链siRNA(如仅包括反义链)。Unless otherwise specified, "short interfering RNA (siRNA)" as used herein is a class of RNA molecules, 14-30 base pairs in length, similar to miRNA, and operating within the RNA interference (RNAi) pathway, which interferes with Inhibits the translation of the mRNA of a specific gene that is complementary to the nucleotide sequence, resulting in mRNA degradation. The short interfering RNA (siRNA) of the present invention includes double-stranded siRNA (including sense strand and antisense strand) and single-stranded siRNA (eg only includes antisense strand).
除非另有规定，本发明所述“抑制”，当指代表达给定基因时，表示与还没有经这样处理的细胞、细胞群或组织相比，当用本发明所述寡聚核苷酸处理该细胞、细胞群或组织时，基因表达降低。Unless otherwise specified, the "inhibition" of the present invention, when referring to the expression of a given gene, means that when compared with cells, cell groups or tissues that have not been so treated, when the oligonucleotides of the present invention are used Upon treatment of the cell, cell population or tissue, gene expression is reduced.
除非另有规定，本发明所述“序列”或“核苷酸序列”表示使用标准核苷酸命名的一序列字母描述的核碱基或核苷酸的次序或顺序物。Unless otherwise specified, the "sequence" or "nucleotide sequence" in the present invention means the order or sequence of nucleobases or nucleotides described by a sequence of letters using standard nucleotide nomenclature.
除非另有规定，本发明所述“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" in the present invention refers to the strand that is substantially complementary to the corresponding region of the target sequence (eg, AGT mRNA) in the oligonucleotide compound.
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" in the present invention refers to the strand that can form a substantially complementary region with the antisense strand. The "substantially complementary" means that the corresponding positions of the two sequences can be completely complementary, and there can also be one or more mismatches. When there are mismatches, there are usually no more than 3, 2 or 1 and no more than 1 mismatched bases. right. In a double-stranded nucleic acid molecule, bases on one strand pair 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“核苷酸任选被修饰”是指每一个核苷酸独立地可以是未经修饰的核苷酸，也可以是经修饰的核苷酸，每一个经修饰的核苷酸上的修饰也是独立地。所述“修饰”包括但不限于对核碱基的修饰、对核糖的修饰以及对磷酸酯的修饰。所述“未经修饰的核苷酸”是指由天然存在的核碱基、天然的糖环及天然的磷酸酯组成的核苷酸。所述“经修饰的核苷酸”是指包含修饰的核碱基、修饰的糖环和修饰的磷酸酯中至少一种的核苷酸。在本发明的一些实施例中，“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、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、修饰的糖环和修饰的磷酸酯组成。Unless otherwise specified, "the nucleotide is optionally modified" in the present invention means that each nucleotide can be an unmodified nucleotide independently or a modified nucleotide, each Modifications on modified nucleotides are also independently. The "modification" includes, but not limited to, nucleobase modification, ribose modification and phosphate modification. The "unmodified nucleotide" refers to a nucleotide composed of a naturally occurring nucleobase, a natural sugar ring and a natural phosphate. The "modified nucleotide" refers to a nucleotide comprising at least one of a modified nucleobase, a modified sugar ring and a modified phosphate. In some embodiments of the present invention, "modified nucleotide" refers to a nucleotide composed of a modified nucleobase, and/or a modified sugar ring, and/or a modified phosphate. In some embodiments of the present invention, "modified nucleotides" consist of modified nucleobases, natural sugar rings and natural phosphates; in some embodiments of the present invention, "modified nucleosides Acids" consist of natural nucleobases, modified sugar rings, and natural phosphates; in some embodiments of the invention, "modified nucleotides" Consists of a natural nucleobase, a natural sugar ring, and a modified phosphate; in some embodiments of the invention, a "modified nucleotide" consists of a natural nucleobase, a modified sugar ring, and a modified phosphate ester composition; in some embodiments of the invention, "modified nucleotides" consist of modified nucleobases, natural sugar rings, and modified phosphates; in some embodiments of the invention, "modified Nucleotides" are composed of modified nucleobases, modified sugar rings and natural phosphates; in some embodiments of the present invention, "modified nucleotides" are composed of modified nucleobases, modified sugar rings ring and modified phosphate composition.
除非另有规定，本发明所述“天然的糖环”选自2’-OH的五元糖环和2’-脱氧的五元糖环。Unless otherwise specified, the "natural sugar ring" in the present invention is selected from 2'-OH five-membered sugar ring and 2'-deoxy five-membered sugar ring.
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 base" of the present invention is selected from the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
除非另有规定，本发明所述“修饰的核碱基”是指除天然碱基之外的5-12元的饱和、部分不饱和或芳香的杂环，包括单环或稠环，其具体例包括但不限于噻吩、噻蒽、呋喃、吡喃、异苯并呋喃、苯并噻嗪、吡咯、咪唑、取代或未取代的三氮唑、吡唑、异噻唑、异恶唑、哒嗪、吲哚嗪、吲哚、异吲哚、异喹啉、喹啉、萘并吡啶、喹唑啉、咔唑、菲啶、哌啶、吩嗪、菲那嗪、吩噻嗪、呋喃烷、吩恶嗪、吡咯烷、吡咯啉、咪唑烷、咪唑啉、吡唑烷、5-甲基胞嘧啶(5-me-C)、5-羟甲基胞嘧啶、黄嘌呤、次黄嘌呤、2-氨基腺嘌呤、2-氨基腺嘌呤、2-氨基鸟嘌呤、2-丙基的腺嘌呤和鸟嘌呤以及其他烷基衍生物、2-硫尿嘧啶、2-硫代胸腺嘧啶、2-硫代胞嘧啶、5-卤代尿嘧啶以及胞嘧啶、5-丙炔基尿嘧啶以及胞嘧啶、6-偶氮尿嘧啶、6-偶氮胞嘧啶、6-偶氮胸腺嘧啶、5-尿嘧啶(假尿嘧啶)、4-硫尿嘧啶、8-卤基，8-氨基，8-氢硫基，8-硫烷基，8-羟基以及其他8-取代腺嘌呤和鸟嘌呤、5-卤基尤其是5-溴，5-三氟甲基以及其他5-取代的尿嘧啶和胞嘧啶、7-甲基鸟嘌呤和7-甲基腺嘌呤、8-氮鸟嘌呤和8-氮腺嘌呤、7-脱氮鸟嘌呤和7-脱氮腺嘌呤、以及3-脱氮鸟嘌呤和3-脱氮腺嘌呤、 Unless otherwise specified, the "modified nucleobase" in the present invention refers to 5-12 membered saturated, partially unsaturated or aromatic heterocyclic rings other than natural bases, including monocyclic or fused rings, specifically Examples include, but are not limited to, thiophene, thianthrene, furan, pyran, isobenzofuran, benzothiazine, pyrrole, imidazole, substituted or unsubstituted triazole, pyrazole, isothiazole, isoxazole, pyridazine , indoxazine, indole, isoindole, isoquinoline, quinoline, naphthopyridine, quinazoline, carbazole, phenanthridine, piperidine, phenazine, phenanthazine, 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-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-mercapto, 8-sulfanyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halo groups especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine , 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine,
除非另有规定，本发明所述“修饰的糖环”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" in the present invention Unless otherwise specified, the "modified sugar ring" in the present invention can include but not limited to one of the following modifications at the 2' position: H; F; O -, S- or N-alkyl; O-, S- or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl, alkenyl and Alkynyl may be substituted or unsubstituted C 1 to C 10 alkyl or C 2 to C 10 alkenyl and alkynyl. Exemplary suitable modifications include O[( CH2 ) nO ] mCH3 , O( CH2 ) nOCH3 , O (CH2) nNH2 , O( CH2 ) nCH3 , 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, the modification at the 2' position includes, but is 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, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, group for improving the pharmacokinetic properties of iRNA, or for improving the iRNA Groups with pharmacodynamic characteristics, and other substituents with similar properties. In some embodiments, the modification includes , but is not limited to, 2'-methoxyethoxy (2' - O- CH2CH2OCH3 , also known as 2'-O-(2-methoxyethyl ) or 2'- MOE).
除非另有规定，本发明所述“修饰的磷酸酯“包括但不限于：硫代磷酸酯修饰，所述的“硫代磷酸酯”包括(R)-和(S)-异构体和/或其混合物。Unless otherwise specified, the "modified phosphate" in the present invention includes, but is not limited to: phosphorothioate modification, and the "phosphorothioate" includes (R)- and (S)-isomers and/or or a mixture thereof.
在本发明的一些实施例中，修饰的核苷酸可以包含一个或多个锁核酸(LNA)。锁核酸是具有修饰核糖部分的核苷酸，其中所述核糖部分包含连接2'碳和4'碳的额外桥。这个结构有效地将该核糖“锁”在3'-内切结构构象中。In some embodiments of the invention, the modified nucleotides may comprise one or more locked nucleic acids (LNAs). Locked nucleic acids are nucleotides that have a modified ribose moiety that includes an additional bridge connecting the 2' carbon to the 4' carbon. This structure effectively "locks" the ribose in the 3'-endo structural 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 linkages have been removed, resulting in unlocked "sugar" residues. In one example, the UNA also covers 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 (i.e., the covalent carbon-carbon bond between the C2' and C3' carbons) of the sugar has been removed.
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. A "bicyclic sugar" is a furanosyl ring modified by a bridge of two atoms. A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety comprising a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In a particular embodiment, the bridge connects the 4'-carbon and the 2'-carbon of the sugar ring.
本发明所述的序列可以包括如下表2中“进一步修饰的序列”所列。The sequences described in the present invention may include those listed in "further modified sequences" in Table 2 below.
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，如通过“磷酸酯键”、“磷酸二酯键”、“硫代磷酸酯键”等连接。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; correspondingly, "conjugate" means that each Compounds formed by covalent linkages between chemical moieties, such as through "phosphoester linkages", "phosphodiester linkages", "phosphorothioate linkages", etc.
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, the "ligand" in the present invention refers to a targeting group, such as a cell or tissue targeting agent that binds to a specified cell type such as a kidney cell, such as a lectin, glycoprotein, lipid or protein, For example antibodies. Targeting groups can be thyrotropin, melanin, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine (GalNAc) , N-acetyl-glucosamine polyvalent mannose, polyvalent fucose, glycosylated polyamino acid, polyvalent galactose, transferrin, bisphosphonate, polyglutamic acid, polyaspartic acid , lipids, cholesterol, steroids, cholic acid, folic acid, vitamin B12, vitamin A, biotin, or RGD peptides or RGD peptidomimetics.
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任选被修饰。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。例如，所述SEQ ID NO:2可以在5’和/或3’段包括修饰的或未修饰的GA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 strand extends beyond the 5'-end of the other strand, or the 5'-end of one strand extends beyond the 3'-end of the other strand. 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 overhangs are optionally modified. The overhang can be on the sense strand, the antisense strand, or any combination thereof. In addition, overhangs may be present at the 5'-end, 3'-end, or both of the antisense or sense strands of the double-stranded compound. In some embodiments of the invention, the antisense strand has 1 to 10 nucleotides (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 (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) overhangs. 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) overhangs. For example, said SEQ ID NO: 2 may include a modified or unmodified GA overhang in the 5' and/or 3' stretch.
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)所示的连接方式：其中N表示任选被修饰的核苷酸，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 described in the present invention can also be connected by one or more nucleotides, referring to the connection mode shown in the following formula (I): Wherein N represents optionally modified nucleotides, n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; strand 1 and strand 2 are independently sense strand or 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)所示的连接方式：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 described in the present invention can also be connected through a chemical group, which refers to the connection method shown in the following formula (II): Wherein L is selected from C 1-100 alkylene, and one or more CH 2 on C 1-100 alkylene 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 Atoms or group replacements of membered heterocycloalkyl groups, the C 1-100 alkylene group is optionally substituted by one or more R a ; L 2 and L 3 are independently selected from nonexistent and C 1-100 alkylene groups Alkyl, one or more CH 2 on the C 1-100 alkylene 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 membered heterocycloalkyl 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，本发明术语”多个”和“多价”中的“多”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类似物或者分支键联体附接的GalNAc衍生物的理论上限。Unless otherwise specified, "multiple" in the terms "multiple" and "multivalent" in the present invention refers to an integer greater than or equal to 2, including but not limited to 2, 3, 4, 5, 6, 7, 8, 9, 10 .
本发明所述的双链SiRNA类似物的缀合物是双链SiRNA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SiRNA类似物和药学上可接受的缀合基团共价连接。The double-stranded siRNA analog conjugate of the present invention is a compound formed by linking a double-stranded siRNA analog and a pharmaceutically acceptable conjugating group, and the double-stranded siRNA analog and a pharmaceutically acceptable conjugating group group covalently linked.
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 linkage usually contains a direct bond or atom (such as: oxygen or sulfur), atomic group (such as: NRR, C(O), C(O)NH, SO, SO 2 , SO 2 NH), substituted or un 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 one or more optional C atoms in the substituted or unsubstituted alkyl, substituted or substituted alkenyl, substituted or unsubstituted alkynyl can be substituted or unsubstituted Substituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl.
可裂解的键联体在细胞外具有充份稳定性，但当进入标靶细胞内时即会裂解而释出该键联体所共同固定的两个部分的基团。A cleavable linker is sufficiently stable outside the cell, but upon entry into the target cell, it is cleaved to release the two moieties to which the linker is co-immobilized.
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 invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including (R)- and (S)-enantiomers, diastereomers, and racemic and other mixtures thereof, such as enantiomers or diastereomers Body-enriched mixtures, all of which are within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl 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 "enantiomer" or "optical isomer" 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 indicated, the term "diastereoisomer" refers to stereoisomers whose molecules have two or more chiral centers and which are not mirror images of 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 noted, keys with wedge-shaped solid lines and dotted wedge keys Indicates the absolute configuration of a stereocenter, with a straight solid-line bond and straight dashed keys Indicates the relative configuration of the stereocenter, with a wavy line Indicates wedge-shaped solid-line bond or dotted wedge key or with tilde Indicates a straight solid line key and/or straight dotted key
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", "enriched in an isomer", "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 terms "isomer excess" or "enantiomeric excess" refer 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 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。Optically active (R)- and (S)-isomers as well as 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 auxiliary agents, wherein 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 diastereoisomeric salt is formed with an appropriate optically active acid or base, and then a diastereomeric salt is formed by a conventional method 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 in combination with chemical derivatization methods (e.g. amines to amino groups formate). The compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compounds. For example, compounds may be labeled with radioactive isotopes such as tritium ( 3 H), iodine-125 ( 125 I) or C-14 ( 14 C). For another example, heavy hydrogen can be used to replace hydrogen to form deuterated drugs. The bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon. Compared with non-deuterated drugs, deuterated drugs can reduce toxic side effects and increase drug stability. , enhance the efficacy, prolong the biological half-life of drugs and other advantages. All changes in 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 a specific substituent found 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 obtained by using enough in a pure solution or a suitable inert solvent Base addition salts are obtained by contacting such compounds with an appropriate amount of base. 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 functionalities, acid addition salts can be obtained by contacting such compounds with a sufficient amount of the acid, either neat solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include salts of inorganic acids including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogenphosphate, dihydrogenphosphate, sulfuric acid, Hydrogen sulfate, hydriodic acid, phosphorous acid, etc.; and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, Fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid and similar acids; also salts of amino acids such as arginine and the like , and salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain basic and acidic functional groups and can thus 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 from the parent compound containing acid groups or bases by conventional chemical methods. 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 both.
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, the embodiments formed by combining them with other chemical synthesis methods, and the methods well known to those skilled in the art Equivalent alternatives, preferred embodiments include but are not limited to the examples of the present invention.
本发明的化合物可以在一个或多个构成该化合物的原子上包含非天然比例的原子同位素。例如，可用放射性同位素标记化合物，比如氚(3H)，碘-125(125I)或C-14(14C)。又例如，可用重氢取代氢形成氘代药物，氘与碳构成的键比普通氢与碳构成的键更坚固，相比于未氘化药物，氘代药物有降低毒副作用、增加药物稳定性、增强疗效、延长药物生物半衰期等优势。本发明的化合物的所有同位素组成的变换，无论放射性与否，都包括在本发明的范围之内。The compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compounds. For example, compounds may be labeled with radioactive isotopes such as tritium ( 3 H), iodine-125 ( 125 I) or C-14 ( 14 C). For another example, heavy hydrogen can be used to replace hydrogen to form deuterated drugs. The bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon. Compared with non-deuterated drugs, deuterated drugs can reduce toxic side effects and increase drug stability. , enhance the efficacy, prolong the biological half-life of drugs and other advantages. All changes in 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 linking group listed does not indicate its linking direction, its linking direction is arbitrary, for example, The connecting group L in the middle is -MW-, at this time -MW- can connect ring A and ring B in the same direction as the reading order from left to right to form It can also be formed by connecting loop A and loop B in the opposite direction to the reading order from left to right Combinations of the described linking groups, substituents and/or variations thereof are permissible only if such combinations result in stable compounds.
术语“任选”或“任选地”指的是随后描述的事件或状况可能但不是必需出现的，并且该描述包括其中所述事件或状况发生的情况以及所述事件或状况不发生的情况。The term "optional" or "optionally" means that the subsequently described event or circumstance can but need not occur, and that the description includes instances where said event or circumstance occurs and instances where said event or circumstance 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 specified atom are replaced by a substituent, which may include deuterium and hydrogen variants, as long as the valence of the specified atom is normal and the substituted compound is stable. When a substituent is oxygen (ie =0), 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, and unless otherwise specified, the type and number of substituents may be arbitrary on a chemically realizable basis.
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 (such as R) occurs more than once in the composition or structure of a compound, its definition in each case is unique standing. Thus, for example, if a group is substituted with 0-2 R, said group may optionally be substituted with up to two R, with independent options for each occurrence of R. Also, combinations of substituents and/or variations thereof are permissible 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 enumerated substituent does not indicate which atom it is connected to the substituted group, this substituent can be bonded through any atom, for example, pyridyl as a substituent can be connected to any atom on the pyridine ring. The carbon atom is attached to the group being substituted.
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, the embodiments formed by combining them with other chemical synthesis methods, and the methods well known to those skilled in the art Equivalent alternatives, preferred embodiments include but are not limited to the examples of the present invention.
本发明的化合物可以通过本领域技术人员所熟知的常规方法来确认结构，如果本发明涉及化合物的绝对构型，则该绝对构型可以通过本领域常规技术手段予以确证。例如单晶X射线衍射法(SXRD)，把培养出的单晶用Bruker D8 venture衍射仪收集衍射强度数据，光源为CuKα辐射，扫描方式：扫描，收集相关数据后，进一步采用直接法(Shelxs97)解析晶体结构，便可以确证绝对构型。The structure of the compounds of the present invention can be confirmed by conventional methods 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, in single crystal X-ray diffraction (SXRD), the cultured single crystal is collected with a Bruker D8 venture diffractometer to collect diffraction intensity data, the light source is CuKα radiation, and the scanning method is: After scanning and collecting relevant data, the absolute configuration can be confirmed by further analyzing the crystal structure by direct method (Shelxs97).
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 ratios of solvents used in column chromatography and preparative thin-layer silica gel chromatography of the present invention are 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 following abbreviations are used in the present invention: aq stands for water; HATU stands for O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate ; EDC represents N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride; m-CPBA represents 3-chloroperoxybenzoic acid; eq represents equivalent, equivalent; CDI represents Carbonyldiimidazole; DCM stands for dichloromethane; PE stands for petroleum ether; DIAD stands for diisopropyl azodicarboxylate; DMF stands for N,N-dimethylformamide; DMSO stands for dimethylsulfoxide; EtOAc stands for ethyl acetate EtOH stands for ethanol; MeOH stands for methanol; CBz stands for benzyloxycarbonyl, an amine protecting group; BOC stands for tert-butoxycarbonyl, an amine protecting group; HOAc stands for acetic acid; NaCNBH3 stands for sodium cyanoborohydride ; rt stands for room temperature; O /N stands for overnight; THF stands for tetrahydrofuran; Boc2O stands for di-tert-butyldicarbonate; TFA stands for trifluoroacetic acid; DIPEA stands for diisopropylethylamine; Sulfone; CS 2 represents carbon disulfide; TsOH represents p-toluenesulfonic acid; NFSI represents N-fluoro-N-(benzenesulfonyl)benzenesulfonamide; NCS represents 1-chloropyrrolidine-2,5-dione; n-Bu 4 NF stands for tetrabutylammonium fluoride; iPrOH stands for 2-propanol; mp stands for melting point; LDA stands for lithium diisopropylamide.
本发明的核酸序列描述中使用的是核苷酸单体的缩写，具体如表2所示：What is used in the nucleic acid sequence description of the present invention is the abbreviation of nucleotide monomer, specifically as shown in Table 2:
表2核苷酸单体的缩写

Table 2 Abbreviations for Nucleotide Monomers

(invAb)s：
(invAb)s:
化合物依据本领域常规命名原则或者使用软件命名，市售化合物采用供应商目录名称。Compounds are named according to the conventional naming principles in this field or using The software is named, and the commercially available compounds adopt the supplier catalog name.
技术效果technical effect
本发明化合物及其应用能够产生较好的疗效及安全性。The compound of the present invention and its application can produce better curative effect and safety.
附图说明Description of drawings
图1为小鼠肝脏中AGT的表达结果。Figure 1 shows the expression results of AGT in mouse liver.
图2和图3为体外RNA sequence研究结果。Figure 2 and Figure 3 are the results of in vitro RNA sequence studies.
图4为化合物对hTLR3、hTLR7、hTLR8细胞的激动活性测试结果。Fig. 4 is the test result of the agonistic activity of the compound on hTLR3, hTLR7, hTLR8 cells.
图5为EL86中AU碱基配对3D结构图。Fig. 5 is a 3D structure diagram of AU base pairing in EL86.
图6为EL86中ZU配对3D结构图。Figure 6 is a 3D structure diagram of ZU pairing in EL86.
图7为EL86中UA碱基配对3D结构图。Fig. 7 is a 3D structure diagram of UA base pairing in EL86.
图8为EL86中ZA配对3D结构图。Figure 8 is a 3D structure diagram of ZA pairing in EL86.
图9为EL86中CG碱基配对3D结构图。 Figure 9 is a 3D structure diagram of CG base pairing in EL86.
图10为EL86中ZG配对3D结构图。Figure 10 is a 3D structure diagram of ZG pairing in EL86.
图11为EL86中GC碱基配对3D结构图。Figure 11 is a 3D structure diagram of GC base pairing in EL86.
图12为EL86中ZC配对3D结构图。Figure 12 is a 3D structure diagram of ZC pairing in EL86.
图13为EL86晶体结构图。Figure 13 is a crystal structure diagram of EL86.
图14为AD05488和阴性的对照结果。Figure 14 is the result of AD05488 and negative control.
图15为ANG001和阴性的对照结果。Figure 15 shows the results of ANG001 and negative controls.
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 will be described in detail through examples below, but it does not imply any unfavorable limitation 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, the embodiments formed by combining them with other chemical synthesis methods, and the methods well known to those skilled in the art Equivalent alternatives, preferred embodiments include but are not limited to the examples of the present invention. Various changes and modifications to the specific embodiments of the invention will be apparent to those skilled in the art without departing from the spirit and scope of the invention.
计算例1Calculation example 1
实验目的：Purpose:
探究式(Z)化合物上的三氮唑与天然碱基A、U、G和C之间的氢键配对关系。Explore the hydrogen bond pairing relationship between the triazole on the compound of formula (Z) and the natural bases A, U, G and C.
实验操作：Experimental operation:
1)从pdb数据库下载得到代表性的SiRNA EL86的晶体结构，其中EL86的序列(SS 5→3，SEQ ID NO:14；as 3→5，SEQ ID NO:15)如表3所示，晶体结构如附图13所示：1) Download the crystal structure of representative SiRNA EL86 from the pdb database, wherein the sequence of EL86 (SS 5→3, SEQ ID NO:14; as 3→5, SEQ ID NO:15) is shown in Table 3, the crystal The structure is shown in Figure 13:
表3 EL86序列
Table 3 EL86 sequence
2)使用Maestro软件(Maestro V12.4,New York,NY,2020)，分别构建了Z替代A4/U6/C9/G16的结构，具体操作为第一步：删除A4的碱基部分，留下的氢原子作为三唑的连接位点，第二步：应用Maesro中的Enumeration工具将三唑结构拼接到上述的连接点上，即可得到Z替换A的结构，得到如表4的序列(a的SS 5→3，SEQ ID NO:16；b的SS 5→3，SEQ ID NO:17；c的的SS 5→3，SEQ ID NO:18；d的SS 5→3，SEQ ID NO:19；a、b、c和d的as 3→5，SEQ ID NO:15)：2) Use Maestro software (Maestro V12.4, New York, NY, 2020), respectively constructing the structure of Z replacing A4/U6/C9/G16, the specific operation is the first step: delete the base part of A4, and the remaining hydrogen atom is used as the connection site of the triazole, Step 2: Use the Enumeration tool in Maesro to splice the triazole structure onto the above connection point to obtain the structure in which Z replaces A, and obtain the sequence shown in Table 4 (SS 5→3 of a, SEQ ID NO:16 SS 5→3 of b, SEQ ID NO:17; SS 5→3 of c, SEQ ID NO:18; SS 5→3 of d, SEQ ID NO:19; as of a, b, c and d 3→5, SEQ ID NO: 15):
表4 Z替代A4/U6/C9/G16后得到的序列
Table 4 Sequence obtained after Z replaces A4/U6/C9/G16
3)使用Pymol软件(Pymol,New York,NY,2020)对得到的结构进行了碱基间距离分析和结构可视化，实验结果如附图5、6、7和8所示。3) Use Pymol software (Pymol, New York, NY, 2020) performed interbase distance analysis and structure visualization on the obtained structure, and the experimental results are shown in Figures 5, 6, 7 and 8.
实验结论：Z上的三氮唑与天然碱基A、U、G和C均无法形成氢键，不能形成配对关系。Experimental conclusion: the triazole on Z cannot form hydrogen bonds with the natural bases A, U, G and C, and cannot form a pairing relationship.
实施例1
Example 1
步骤A：将1-1(10克，19.82毫摩尔)溶于乙腈(120毫升)和1,2-二氯乙烷(80毫升)，于0摄氏度下加入1-2(6.02克，42.62毫摩尔)和三甲硅基三氟甲磺酸酯(11.01克，49.56毫摩尔，8.95毫升)，混合液在35摄氏度下搅拌12小时。反应液在0摄氏度下缓慢加入饱和碳酸氢钠水溶液(100毫 升)淬灭和二氯甲烷(100毫升×2)萃取。所得有机相用饱和氯化钠水溶液(200毫升)洗涤，无水硫酸钠干燥，过滤减压浓缩得到粗品。粗品经硅胶柱纯化(洗脱剂：石油醚/乙酸乙酯＝20：1)得到1-3。Step A: 1-1 (10 g, 19.82 mmol) was dissolved in acetonitrile (120 ml) and 1,2-dichloroethane (80 ml), and 1-2 (6.02 g, 42.62 mg mol) and trimethylsilyl trifluoromethanesulfonate (11.01 g, 49.56 mmol, 8.95 ml), and the mixture was stirred at 35°C for 12 hours. The reaction solution was slowly added saturated aqueous sodium bicarbonate (100 ml L) quenched and extracted with dichloromethane (100 mL×2). The obtained organic phase was washed with saturated aqueous sodium chloride (200 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column (eluent: petroleum ether/ethyl acetate=20:1) to obtain 1-3.
步骤B：将1-3(6.7克，13.05毫摩尔)溶于氨的甲醇溶液(7摩尔/升，50毫升)，混合液于40摄氏度下搅拌12小时。反应液经减压浓缩得到粗品，经硅胶柱纯化(洗脱剂：乙酸乙酯/甲醇＝50/1至10/1)得到1-4。Step B: Dissolve 1-3 (6.7 g, 13.05 mmol) in methanolic ammonia solution (7 mol/L, 50 mL), and stir the mixture at 40°C for 12 hours. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was purified by silica gel column (eluent: ethyl acetate/methanol=50/1 to 10/1) to obtain 1-4.
步骤C：将1-4(2.3克，11.43毫摩尔)溶于无水吡啶(25毫升)，于0摄氏度下加入1,3二氯-1,1,3,3-四异丙基二硅氧烷(3.64克，11.55毫摩尔，3.69毫升)，混合液在20摄氏度下搅拌12小时。反应液加入水(30毫升)淬灭，并加入乙酸乙酯(30毫升×2)萃取。所得有机相依次用盐酸(1摩尔/升，30毫升×3)和饱和食盐水(30毫升)洗涤，无水硫酸钠干燥，过滤减压浓缩得到粗品。粗品经硅胶柱纯化(洗脱剂，石油醚/乙酸乙酯＝7/1至5/1)得到1-5。Step C: Dissolve 1-4 (2.3 g, 11.43 mmol) in anhydrous pyridine (25 mL), add 1,3 dichloro-1,1,3,3-tetraisopropyldisila at 0 °C Oxane (3.64 g, 11.55 mmol, 3.69 mL), the mixture was stirred at 20°C for 12 hours. The reaction solution was quenched by adding water (30 mL), and extracted by adding ethyl acetate (30 mL×2). The obtained organic phase was washed successively with hydrochloric acid (1 mol/L, 30 mL×3) and saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column (eluent, petroleum ether/ethyl acetate=7/1 to 5/1) to obtain 1-5.
步骤D：将1-5(4克，9.02毫摩尔)溶于乙腈(40毫升)，依次加入氧化银(8.36克，36.06毫摩尔)，分子筛(3克)，无水吡啶(1.78克，22.54毫摩尔，1.82毫升)和碘甲烷(6.4克，45.08毫摩尔，2.81毫升)，混合液在25摄氏度下搅拌12小时。反应液加入乙酸乙酯(50毫升)在20摄氏度搅拌1小时，用布氏漏斗垫硅藻土过滤并收集滤液，滤液加入水(100毫升)和乙酸乙酯(100毫升×2)萃取。所得有机相用饱和食盐水(200毫升)洗涤，无水硫酸钠干燥，过滤减压浓缩得到粗品。粗品经硅胶柱纯化(洗脱剂，石油醚/乙酸乙酯＝15/1至10/1至8/1)得到1-6。Step D: 1-5 (4 g, 9.02 mmol) was dissolved in acetonitrile (40 ml), silver oxide (8.36 g, 36.06 mmol) was added successively, Molecular sieves (3 g), anhydrous pyridine (1.78 g, 22.54 mmol, 1.82 mL) and iodomethane (6.4 g, 45.08 mmol, 2.81 mL), the mixture was stirred at 25°C for 12 hours. The reaction solution was added ethyl acetate (50 mL) and stirred at 20°C for 1 hour, filtered through a Buchner funnel pad with celite and collected the filtrate, which was extracted by adding water (100 mL) and ethyl acetate (100 mL×2). The obtained organic phase was washed with saturated brine (200 ml), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column (eluent, petroleum ether/ethyl acetate=15/1 to 10/1 to 8/1) to obtain 1-6.
步骤E：将1-6(2克，4.37毫摩尔)溶于无水四氢呋喃(20毫升)，加入三乙胺三氟化氢盐(1.55克，9.61毫摩尔，1.57毫升)，混合液在20摄氏度下搅拌12小时。反应液经减压浓缩得到粗品，经硅胶柱纯化(洗脱剂：乙酸乙酯/甲醇＝100/1至50/1)得到1-7。Step E: 1-6 (2 g, 4.37 mmol) was dissolved in anhydrous tetrahydrofuran (20 ml), triethylamine trihydrogen trifluoride (1.55 g, 9.61 mmol, 1.57 ml) was added, and the mixture was heated at 20 degrees Celsius Stir for 12 hours. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was purified by silica gel column (eluent: ethyl acetate/methanol=100/1 to 50/1) to obtain 1-7.
步骤F：将1-7(1克，4.65毫摩尔)溶于无水吡啶(10毫升)，加入4,4-双甲氧基三苯甲基氯(1.57克，4.65毫摩尔)，混合液在20摄氏度搅拌12小时。反应液加入水(20毫升)淬灭和乙酸乙酯(20毫升×2)萃取。所得有机相用饱和食盐水(40毫升)洗涤，无水硫酸钠干燥，过滤减压浓缩得到粗品。粗品经硅胶柱纯化(洗脱剂：石油醚/乙酸乙酯＝3/1至1/1)得到1-8。1H NMR(400MHz,DMSO-d6):δ＝8.78(s,1H),8.14-8.03(m,1H),7.41-7.31(m,2H),7.29-7.16(m,7H),6.92-6.76(m,4H),6.08(d,J＝3.2Hz,1H),5.25(d,J＝6.5Hz,1H),4.48-4.34(m,1H),4.13-4.01(m,2H),3.73(s,6H),3.38(s,3H),3.18-3.03(m,2H)。Step F: Dissolve 1-7 (1 g, 4.65 mmol) in anhydrous pyridine (10 mL), add 4,4-bismethoxytrityl chloride (1.57 g, 4.65 mmol), and the mixture Stir for 12 hours at 20°C. The reaction solution was quenched by adding water (20 mL) and extracted with ethyl acetate (20 mL×2). The obtained organic phase was washed with saturated brine (40 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column (eluent: petroleum ether/ethyl acetate=3/1 to 1/1) to obtain 1-8. 1 H NMR (400MHz, DMSO-d 6 ): δ=8.78(s,1H),8.14-8.03(m,1H),7.41-7.31(m,2H),7.29-7.16(m,7H),6.92- 6.76(m,4H),6.08(d,J=3.2Hz,1H),5.25(d,J=6.5Hz,1H),4.48-4.34(m,1H),4.13-4.01(m,2H),3.73 (s,6H), 3.38(s,3H), 3.18-3.03(m,2H).
步骤G：将化合物1-8(3克，5.80毫摩尔)和1-9(1.92克，6.38毫摩尔)溶于无水二氯甲烷(30毫升)，在氮气氛围下加入4，5-二氰基咪唑(342.26毫克，2.90毫摩尔)。混合液在25摄氏度搅拌2小时。向反应液加入15毫升饱和碳酸氢钠水溶液和15毫升水并用二氯甲烷萃取(30毫升×2)。合并的有机相用饱和食盐水洗涤(30毫升×2)，无水硫酸钠干燥、过滤、浓缩得粗品。经硅胶柱层析纯化(洗脱剂：石油醚/乙酸乙酯(0.1％三乙胺)＝1/0至2/1)得到实施例1。1H NMR(400MHz,DMSO-d6):δ＝8.83(s,1H),8.11(d,J＝6.2Hz,1H),7.38(br t,J＝6.9Hz,2H),7.32-7.15(m,7H),6.92-6.76(m,4H),6.26-6.05(m,1H),4.78-4.56(m,1H),4.31(br d,J＝3.8Hz,1H),4.25-4.12(m,1H),3.85-3.77(m,1H),3.73(br s,6H),3.66-3.46(m,3H),3.39(br d,J＝15.7Hz,3H),3.32-3.19(m,1H),3.10(br dd,J＝5.4,10.0Hz,1H),2.78(br t,J＝5.7Hz,1H),2.60(br t,J＝5.5Hz,1H),1.22-1.04(m,9H),0.98(br d,J＝6.5Hz,3H)。31P NMR(162MHz,DMSO-d6):δ＝149.33(br d,J＝11.7Hz,1P)。LCMS(ESI)m/z：718,719[M+H]+。 Step G: Compounds 1-8 (3 g, 5.80 mmol) and 1-9 (1.92 g, 6.38 mmol) were dissolved in anhydrous dichloromethane (30 mL), and 4,5-bis Cyanoimidazole (342.26 mg, 2.90 mmol). The mixture was stirred at 25°C for 2 hours. 15 mL of saturated aqueous sodium bicarbonate solution and 15 mL of water were added to the reaction solution and extracted with dichloromethane (30 mL×2). The combined organic phases were washed with saturated brine (30 mL×2), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Purification by silica gel column chromatography (eluent: petroleum ether/ethyl acetate (0.1% triethylamine) = 1/0 to 2/1) gave Example 1. 1 H NMR (400MHz, DMSO-d 6 ): δ=8.83(s, 1H), 8.11(d, J=6.2Hz, 1H), 7.38(br t, J=6.9Hz, 2H), 7.32-7.15( m,7H),6.92-6.76(m,4H),6.26-6.05(m,1H),4.78-4.56(m,1H),4.31(br d,J＝3.8Hz,1H),4.25-4.12(m ,1H),3.85-3.77(m,1H),3.73(br s,6H),3.66-3.46(m,3H),3.39(br d,J＝15.7Hz,3H),3.32-3.19(m,1H ),3.10(br dd,J=5.4,10.0Hz,1H),2.78(br t,J=5.7Hz,1H),2.60(br t,J=5.5Hz,1H),1.22-1.04(m,9H ), 0.98 (br d, J = 6.5Hz, 3H). 31 P NMR (162 MHz, DMSO-d 6 ): δ=149.33 (br d, J=11.7 Hz, 1P). LCMS (ESI) m/z: 718,719 [M+H] + .
实施例2：AG001及ANG001的合成Example 2: Synthesis of AG001 and ANG001
1.合成原理1. Synthesis 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, through multi-step solid-phase synthesis of oligonucleotides containing solid-phase carriers and protective groups, and finally ammonia deprotection and purification. The solid-phase synthesis method is as follows:
2.实验设计2. Experimental design
AG001的实验设计如下：The experimental design of AG001 is as follows:
表5 AG001及ANG001序列
Table 5 AG001 and ANG001 sequences
核酸序列描述中使用的是核苷酸单体的缩写，具体如下：Nucleic acid sequence descriptions use abbreviations for nucleotide monomers, as follows:
表5 AG001和ANG001中核苷酸单体的缩写

Table 5 Abbreviations of Nucleotide Monomers in AG001 and ANG001

另外，D01代表缀合基团，具体结构如下：
In addition, D01 represents a conjugated group, and the specific structure is as follows:
注：PS为树脂。Note: PS is resin.
3.实验结果3. Experimental results
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。The carrier containing the protective group oligonucleotide sequence was obtained by solid-phase synthesis, and then cut and deprotected from the solid-phase carrier to obtain the crude oligonucleotide, followed by ammonolysis (ammonolysis conditions: ammonia water, 55°C, 16h), and HPLC After purification, the single chain can be directly lyophilized to obtain a pure product.
AG001正义链(SS)的纯度为：98.3％，分子量为8768.3。反义链(AS)的纯度为：97.8％，分子量为：7631.4；双链需要进行退火、冻干得到AG001。The purity of the sense strand (SS) of AG001 is: 98.3%, and the molecular weight is 8768.3. The purity of the antisense strand (AS) is 97.8%, and the molecular weight is 7631.4; the double strand needs to be annealed and freeze-dried to obtain AG001.
ANG001正义链(SS)的纯度为：95.8％，分子量为9084.3。反义链(AS)的纯度为：96.8％，分 子量为：6879.5；双链需要进行退火、冻干得到ANG001。The purity of the sense strand (SS) of ANG001 is: 95.8%, and the molecular weight is 9084.3. The purity of the antisense strand (AS) is: 96.8%. The subweight is: 6879.5; the double strand needs to be annealed and freeze-dried to obtain ANG001.
实验例1：体内AGT HDI模型Experimental example 1: In vivo AGT 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 pcDNA-CMV-AGT plasmid was injected into the high-pressure tail vein to evaluate the target gene and the inhibitory effect of the target gene in vivo.
2.实验材料：pcDNA-CMV-AGT质粒，BALB/c雌性小鼠，DPBS(Dulbecco's磷酸缓冲液)，AD-85481(US2021095290)，AG0012. Experimental materials: pcDNA-CMV-AGT plasmid, BALB/c female mice, DPBS (Dulbecco's phosphate buffer), AD-85481 (US2021095290), AG001
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 adapt to quarantine for one week after the mice arrive at 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AD-85481，3mg/kg；第3组小鼠给AG001，3mg/kg。On the 0th day, mice were randomly divided into groups according to body weight data, with 4 mice in each group. After grouping, all mice were subcutaneously injected with a single dose, and the volume of administration was 10 mL/kg. The mice in the first group were given DPBS; The mice in group 2 were given AD-85481, 3mg/kg; the mice in group 3 were given AG001, 3mg/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 3rd day after administration, all mice were injected with pcDNA-CMV-AGT plasmid of 8% volume of their body weight via tail vein within 5 seconds, (injection volume (mL)=mouse body weight (g)×8%), each The quality 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ANGPTL3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 overnight at 4°C with RNAlater, and then RNAlater was removed and stored at -80°C for detection of ANGPTL3 gene expression levels.
4.实验结论：4. Experimental conclusion:
如图1所示，与Vehicle组相比，AD-85481和AG001对小鼠肝脏中AGT的表达均有优秀的抑制作用。且AG001的抑制效果优于AD-85481。As shown in Figure 1, compared with the Vehicle group, both AD-85481 and AG001 had excellent inhibitory effects on the expression of AGT in the mouse liver. And the inhibitory effect of AG001 is better than that of AD-85481.
实验例2体外RNA sequence研究Experimental example 2 RNA sequence research in vitro
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 RNAs 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 to study all mRNAs transcribed from 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 cost, RNA-seq has become the main method of transcriptome research due to its advantages of high throughput, high sensitivity and wide application range. The RNA-seq technical process mainly includes 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 2100bioanalyzer：精确检测RNA完整性。Standard extraction methods are used to extract RNA from tissues or cells, followed by strict quality control of RNA samples, mainly through Agilent 2100bioanalyzer: 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 use the structural characteristics of polyA tails in most mRNAs of eukaryotes, and enrich mRNAs with polyA tails through Oligo(dT) magnetic beads. The second is to remove ribosomal RNA from total RNA to obtain mRNA. Then, the obtained mRNA was randomly fragmented with divalent cations in NEB Fragmentation Buffer, and the library was constructed according to the NEB common library construction method or chain-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UltraTMRNA Library Prep Kit for NEB general library construction: using fragmented mRNA as a template and random oligonucleotides as primers, synthesize the first strand of cDNA in the M-MuLV reverse transcriptase system, then use RNaseH to degrade the RNA strand, and use it in the DNA polymerase I system Next, the second strand of cDNA is synthesized from dNTPs. After the purified double-stranded cDNA is end-repaired, A-tailed and connected to a sequencing adapter, the cDNA of about 250-300bp is screened with AMPure XP beads, PCR is amplified, and the PCR product is purified again using AMPure XP beads to finally obtain a library. Kits for library construction are 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UltraTMDirectional 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 that of NEB general library construction, the difference is that when synthesizing the second strand, the dTTP in dNTPs is replaced by dUTP, and then the cDNA ends are also repaired and added. A tail, ligation sequencing adapter and length screening, and then use USER enzyme to degrade the second strand of cDNA containing U, and then perform PCR amplification to obtain the library. Strand-specific libraries have many advantages, such as more effective information can be obtained with the same amount of data; more accurate gene quantification, positioning and annotation information can be obtained; antisense transcripts and the expression level of a single exon in each isoform can be provided. The kits used for library construction are 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 linker: including P5/P7, index and Rd1/Rd2 SP three parts. Among them, P5/P7 is the PCR amplification primer and the primer binding part on the flow cell, the index provides information to distinguish different libraries, Rd1/Rd2 SP is the read1/read2 sequence primer, which is the binding region of the sequencing primer, and the sequencing process is theoretically controlled by Rd1/ Rd2 SP proceeds backwards.
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Agilent2100 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, use Qubit2.0 Fluorometer for preliminary quantification, dilute the library to 1.5ng/ul, and then use Agilent2100 bioanalyzer to detect the insert size of the library. After the insert size meets the expectation, qRT-PCR will accurately measure the effective concentration of the library. Quantification (the effective concentration of the library is higher than 2nM) to ensure the quality of the library.
4.上机测序：4. On-machine sequencing:
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 inspection is qualified, different libraries are pooled according to the requirements of the effective concentration and the target off-machine data volume, and then Illumina sequencing is performed. The basic principle of sequencing is Sequencing by Synthesis. Add four kinds of fluorescently labeled dNTPs, DNA polymerase and adapter primers to the sequenced flow cell for amplification. When each sequencing cluster extends the complementary strand, each added fluorescently labeled dNTP can release the corresponding fluorescence , the sequencer obtains the sequence information of the fragment to be tested by capturing the fluorescent signal and converting the optical signal into a sequencing peak through computer software.
5.实验结果：5. Experimental results:
从上图2和图3可以看出，AD85481(US2021095290)共对1161个基因产生影响，上调608个基因，下调553个基因；AG001共对763个基因产生影响，上调345个基因，下调418个基因。从产生影响的比例看，AD-85481和AG001对基因的影响比例较小。同时AG001影响基因的数量低于AD-85481影响的数量。From Figure 2 and Figure 3 above, it can be seen that AD85481 (US2021095290) affects 1161 genes in total, up-regulates 608 genes, and down-regulates 553 genes; AG001 affects a total of 763 genes, up-regulates 345 genes, and down-regulates 418 genes Gene. Judging from the ratio of the effect, AD-85481 and AG001 have a relatively small effect on the gene. At the same time, the number of genes affected by AG001 was lower than that of AD-85481.
从图14和图15可以看出，AD05488(WO2019055633)共对1633个基因产生影响，上调490个基因，下调了1143个基因；ANG001共对826个基因产生影响，上调284个基因，下调了542个基因。从产生影响的比例看，AG001对基因的影响比例较小。It can be seen from Figure 14 and Figure 15 that AD05488 (WO2019055633) has an effect on 1633 genes, up-regulates 490 genes, and down-regulates 1143 genes; ANG001 affects a total of 826 genes, up-regulates 284 genes, and down-regulates 542 genes. gene. Judging from the proportion of the effect, the proportion of AG001's influence on the gene is relatively small.
结论：从上述实验结果可知，引入Z能够显著调节siRNA分子的选择性，且选择性优于使用GNA的siRNA的分子。Conclusion: From the above experimental results, it can be seen that the introduction of Z can significantly regulate the selectivity of siRNA molecules, and the selectivity is better than that of siRNA molecules using GNA.
实验例3化合物对hTLR3、hTLR7、hTLR8细胞的激动活性测试Test of the agonistic activity of the compound of Experimental Example 3 on hTLR3, hTLR7, hTLR8 cells
1.实验材料 1. Experimental materials
表6实验材料表
Table 6 Experimental material list
2.仪器2. Instrument
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 it with RNAiMAX-OPTI MEM (RNAiMAX:OPTI MEM=3:47) 1:1, 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) Take 10 μl of the incubated compound and add it to the corresponding cell plate, a total of 10 concentrations, and 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-iTTMAlexaRed Fluorescent Control。3) Add 10 μl of PBS to each well of the negative control well, add 10 μl of 20 μg/ml Poly(I:C) HMW to each well of the positive control well of hTLR3, add 10 μl of 10 μg/ml R848 to each well of the positive control well of hTLR7 and hTLR8. 2 Add 10μl 0.5μM BLOCK-iT TM Alexa to the positive control wells of transfection 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 in a 96-well plate to which the compound has been 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) Co-incubate the compound and the cells in a 37°C, 5% CO 2 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) Observe the transfection effect of the transfection positive control wells with a fluorescence microscope, and take pictures.
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) Detection of compound activity: Take 20 μl of cell supernatant from each well, add it to the experimental plate containing 180 μl of QUANTI-Blue TM reagent, incubate at 37°C for 1 hour, and 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) Detection of cell viability: operate according to the instructions of Celltiter-Glo, and detect the chemiluminescent signal (RLU) 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。Compound activity: The OD650 value was analyzed by GraphPad Prism software, and the compound dose-effect curve was fitted to calculate the EC 50 value of the compound. Cell Viability Detection: The calculation formula of cell viability % is as follows. The cell activity % value was analyzed with GraphPad Prism software, and the dose-effect curve of the compound was fitted to calculate the CC 50 value of the compound on the cells.
细胞活性％＝RLU化合物/RLUDMSO控制*100％Cell viability % = RLU compound/RLUDMSO control * 100%
5.实验结果：5. Experimental results:
如上图4所示，从EC50曲线看，对于TLR3的激动活性研究，在阳性对照Poly(I:C)HMW具有剂量依赖的激动活性的情况下，参比AD85481和AG001均没有激动作用；对于TLR7的激动活性研究，在阳性对照R848具有剂量依赖的激动活性的情况下，参比AD85481和AG001均没有激动作用；对于 TLR8的激动活性研究，在阳性对照R848具有剂量依赖的激动活性的情况下，参比AD85481和AG001均没有激动作用。同时，从CC50的曲线看，阳性对照、参比、AG001对相应的细胞均没有细胞毒性。 As shown in Figure 4 above, from the EC 50 curve, for the agonistic activity of TLR3, when the positive control Poly(I:C)HMW has a dose-dependent agonistic activity, neither the reference AD85481 nor AG001 has an agonistic effect; The agonistic activity of TLR7, in the case of the positive control R848 has a dose-dependent agonistic activity, the reference AD85481 and AG001 have no agonistic effect; for In the study of the agonistic activity of TLR8, in the case of the positive control R848 having a dose-dependent agonistic activity, neither the reference AD85481 nor AG001 had an agonistic effect. At the same time, from the curve of CC50 , the positive control, reference, and AG001 have no cytotoxicity to the corresponding cells.

Claims (16)

  1. 式(Z)化合物或其药学上可接受的盐，
    A compound of formula (Z) or a pharmaceutically acceptable salt thereof,
  2. 式(Z)化合物作为寡聚核苷酸化合物的嵌入基团的应用，
    The application of formula (Z) compound as the intercalation group of oligonucleotide compound,
    其中，所述寡聚核苷酸化合物包含15-40个核苷酸，其中的任意一个或多个核苷被式(Z)化合物替换，所述核苷酸任选被修饰；Wherein, the oligonucleotide compound comprises 15-40 nucleotides, any one or more nucleosides of which are replaced by the compound of formula (Z), and the nucleotides are optionally modified;
    式(Z)化合物嵌入寡聚核苷酸化合物的连接方式为 The connection mode of formula (Z) compound embedding oligonucleotide compound is
  3. 根据权利要求2所述的应用，其中所述寡聚核苷酸化合物为双链化合物。The application according to claim 2, wherein the oligonucleotide compound is a double-stranded compound.
  4. 根据权利要求3所述的应用，其中所述双链化合物为RNAi。The use according to claim 3, wherein the double-stranded compound is RNAi.
  5. 根据权利要求3所述的应用，其中所述双链化合物包含能够形成双链区的正义链和反义链，所述正义链由15-40个核苷酸组成，优选由17-25个核苷酸组成，更优选由18-23个核苷酸组成；所述反义链由15-40个核苷酸组成，优选由17-35个核苷酸组成，更优选由19-30个核苷酸组成，最优选由21-29个核苷酸组成；所述双链区由17-23个核苷酸碱基对组成。The application according to claim 3, wherein the double-stranded compound comprises a sense strand and an antisense strand capable of forming a double-stranded region, and the sense strand is composed of 15-40 nucleotides, preferably 17-25 nuclei Nucleotide composition, more preferably composed of 18-23 nucleotides; the antisense strand is composed of 15-40 nucleotides, preferably composed of 17-35 nucleotides, more preferably composed of 19-30 cores The nucleotide composition is most preferably composed of 21-29 nucleotides; the double-stranded region is composed of 17-23 nucleotide base pairs.
  6. 根据权利要求5所述的应用，其中所述正义链和/或反义链包含7、6、5、4、3、2、1和0个未经修饰的核苷酸。The use according to claim 5, wherein the sense strand and/or the antisense strand comprise 7, 6, 5, 4, 3, 2, 1 and 0 unmodified nucleotides.
  7. 根据权利要求2-6任意一项所述的应用，其中所述的寡聚核苷酸化合物进一步包含配体。The use according to any one of claims 2-6, wherein the oligonucleotide compound further comprises a ligand.
  8. 根据权利要求7所述的应用，其中所述配体缀合在正义链或反义链的任意位置。The use according to claim 7, wherein the ligand is conjugated at any position of the sense strand or the antisense strand.
  9. 根据权利要求8所述的应用，其中所述配体缀合在正义链的3’端或5’端。The use according to claim 8, wherein the ligand is conjugated at the 3' end or the 5' end of the sense strand.
  10. 根据权利要求8所述的应用，其中所述配体缀合在反义链的3’端或5’端。The use according to claim 8, wherein the ligand is conjugated at the 3' end or the 5' end of the antisense strand.
  11. 根据权利要求8-10任意一项所述的应用，其中所述配体为一种或多种利用多价的分支键联体附接的GalNAc衍生物；The use according to any one of claims 8-10, wherein the ligand is one or more GalNAc derivatives attached by multivalent branched linkages;
    或者，所述配体为一种或多种利用二价、三价或四价的分支键联体附接的GalNAc衍生物；Alternatively, the ligand is one or more GalNAc derivatives attached using bivalent, trivalent or tetravalent branched linkages;
    或者，所述配体为一种利用二价、三价或四价的分支键联体附接的GalNAc衍生物。Alternatively, the ligand is a GalNAc derivative attached using a bivalent, trivalent or tetravalent branched linkage.
  12. 根据权利要求2-11所述的应用，其中所述寡聚核苷酸化合物用于抑制或阻断基因的表达，所述基因为AGT基因、ANGPTL3基因、ApoA基因、Factor B基因、HBV相关基因、HSD基因KRAS相关基 因或补体5相关基因。The application according to claims 2-11, wherein the oligonucleotide compound is used to inhibit or block the expression of genes, and the genes are AGT gene, ANGPTL3 gene, ApoA gene, Factor B gene, HBV related gene , HSD gene KRAS related gene cause or complement 5-related genes.
  13. 一种寡聚核苷酸化合物，所述寡聚核苷酸化合物为双链化合物，其中所述双链包含能够形成双链区的正义链和反义链，所述正义链包含如SEQ ID NO:1所示的序列，所述反义链包含如SEQ ID NO:4或SEQ ID NO:8所示的序列，或者，所述正义链包含如SEQ ID NO:7或SEQ ID NO:9所示的序列，所述反义链包含如SEQ ID NO:12所示的序列，所述序列上的核苷酸任选被修饰。An oligonucleotide compound, the oligonucleotide compound is a double-stranded compound, wherein the double strand comprises a sense strand and an antisense strand capable of forming a double-stranded region, and the sense strand comprises such as SEQ ID NO The sequence shown in: 1, the antisense strand comprises the sequence shown in SEQ ID NO: 4 or SEQ ID NO: 8, or, the sense strand comprises the sequence shown in SEQ ID NO: 7 or SEQ ID NO: 9 The sequence shown, the antisense strand comprises the sequence shown in SEQ ID NO: 12, and the nucleotides on the sequence are optionally modified.
  14. 根据权利要求13所示的寡聚核苷酸化合物，所述正义链包含如SEQ ID NO:5所示的序列，所述反义链包含如SEQ ID NO:6所示的序列，或者，所述正义链包含如SEQ ID NO:11所示的序列，所述反义链包含如SEQ ID NO:13所示的序列。According to the oligonucleotide compound shown in claim 13, the sense strand comprises the sequence shown in SEQ ID NO:5, and the antisense strand comprises the sequence shown in SEQ ID NO:6, or, the The sense strand comprises the sequence shown in SEQ ID NO:11, and the antisense strand comprises the sequence shown in SEQ ID NO:13.
  15. 根据权利要求13或14所述的寡聚核苷酸化合物，其中所述如SEQ ID NO:1、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9或SEQ ID NO:11所示序列的3’端缀合配体。The oligonucleotide compound according to claim 13 or 14, wherein said as described in SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:11 The 3' end of the indicated sequence was conjugated with the ligand.
  16. 根据权利要求15所述的寡聚核苷酸化合物，所述配体为一种或多种利用多价的分支键联体附接的GalNAc衍生物。 The oligonucleotide compound according to claim 15, wherein the ligand is one or more GalNAc derivatives attached using multivalent branched linkages.
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