WO2023155909A1 - Analogue de ribavirine et son utilisation comme groupe d'enrobage - Google Patents

Analogue de ribavirine et son utilisation comme groupe d'enrobage Download PDF

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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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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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    • 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.

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Abstract

La divulgation concerne un analogue de ribavirine et son utilisation en tant que groupe d'enrobage dans des ARNi, et en particulier un composé tel que représenté dans la formule (Z).
PCT/CN2023/077236 2022-02-18 2023-02-20 Analogue de ribavirine et son utilisation comme groupe d'enrobage WO2023155909A1 (fr)

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