WO2019105404A1 - 一种核酸、含有该核酸的组合物与缀合物及制备方法和用途 - Google Patents

一种核酸、含有该核酸的组合物与缀合物及制备方法和用途 Download PDF

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WO2019105404A1
WO2019105404A1 PCT/CN2018/118107 CN2018118107W WO2019105404A1 WO 2019105404 A1 WO2019105404 A1 WO 2019105404A1 CN 2018118107 W CN2018118107 W CN 2018118107W WO 2019105404 A1 WO2019105404 A1 WO 2019105404A1
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nucleotide
sirna
group
nucleotide sequence
alkyl
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PCT/CN2018/118107
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French (fr)
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张鸿雁
高山
康代武
陈庚容
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苏州瑞博生物技术有限公司
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Priority to CN201880049190.2A priority Critical patent/CN111050807A/zh
Publication of WO2019105404A1 publication Critical patent/WO2019105404A1/zh

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses

Definitions

  • Hepatitis B (also known as hepatitis B or hepatitis B) is a type of infectious disease that poses a serious threat to the world, especially China.
  • two major types of hepatitis B prevention drugs are recognized as interferons and nucleoside analogues.
  • interferon which may cause adverse reactions, drug resistance of nucleoside drugs, and recurrence after drug withdrawal. Therefore, if the gene expression of the virus can be silenced from the gene level, blocking the production and replication of HBV, thereby fundamentally reducing viral metabolism and infection of liver cells, it will undoubtedly be the most ideal treatment for hepatitis B.
  • Small interfering RNA can inhibit or block any gene of interest (such as a gene that causes diseases such as cancer) in a sequence-specific manner based on the mechanism of RNA interference (RNAi). The expression to achieve the purpose of treating the disease.
  • siRNA stabilization modification and its delivery system are two key technologies in the development of small RNA drugs.
  • the disclosure provides an siRNA capable of inhibiting expression of an HBV gene, the siRNA comprising a sense strand and an anti-sense strand, each of the nucleotides of the siRNA being independently a modified or unmodified nucleus Glycosyl acid, wherein the sense strand contains a nucleotide sequence I, the antisense strand contains a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially inverted Complementarily forming a double-stranded region, wherein the nucleotide sequence I comprises a nucleotide sequence A which is equal in length to the nucleotide sequence shown in SEQ ID NO: 1, and no more than 3 Nucleotide difference, and the nucleotide sequence II contains a nucleotide sequence B which is equal in length to the nucleotide sequence shown in SEQ ID NO: 2 and no more than 3 Nucleotide differences:
  • nucleotide sequence A contains a nucleotide Z A corresponding to a position Z
  • nucleotide sequence B contains a nucleotide Z' B whose position corresponds to Z', which is Z' B The first nucleotide of the 5' end of the antisense strand.
  • the present disclosure provides a pharmaceutical composition comprising a siRNA of the present disclosure and a pharmaceutically acceptable carrier.
  • the disclosure provides an siRNA conjugate comprising an siRNA provided herein and a conjugation group conjugated to the siRNA.
  • the disclosure provides for the preparation of a siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure for the treatment and/or prevention of a pathological condition or disease caused by an infection with hepatitis B virus. Use in medicine.
  • the present disclosure provides a method of treating and/or preventing a pathological condition or disease caused by an infection with hepatitis B virus, the method comprising combining an effective amount of a siRNA and/or a pharmaceutical combination of the present disclosure
  • the siRNA and/or siRNA conjugate is administered to a patient in need thereof.
  • the present disclosure provides a kit comprising the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure.
  • Figure 1 shows the results of semi-quantitative detection of stability of the tested siRNA conjugates in vitro Tritosome.
  • Figure 2 shows the results of semi-quantitative detection of the stability of the tested siRNA conjugates in human plasma in vitro.
  • Figure 3 shows the results of semi-quantitative detection of the stability of the tested siRNA conjugates in monkey plasma in vitro.
  • Figure 4 shows the results of inhibitory activity of conjugate 20 in vitro.
  • Figure 5 shows the results of detection of IC50 and off-target effects of conjugate 4 in an in vitro psiCHECK system.
  • Figure 6 shows the results of inhibition of HBV mRNA expression by conjugate 4 in mice.
  • Figure 7 shows the results of detection of inhibition of HBsAg expression by a single administration of conjugate 4 on the M-Tg model.
  • the HBV gene refers to a DNA sequence such as the gene shown in Genbank Accession No. NC_003977.1.
  • the capital letters C, G, U, A, T represent the base composition of the nucleotide; the lowercase letter d indicates that the nucleotide adjacent to the right side of the letter d is deoxygenated.
  • Ribonucleotide indicates that one nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide; the lowercase letter f indicates that one nucleotide adjacent to the left side of the letter f is fluorinated Modified nucleotide; lowercase s indicates that the two nucleotides adjacent to the letter s are phosphorothioate-linked; P1 indicates that one nucleotide adjacent to the right of P1 is 5'- A nucleotide modified with a phosphate nucleotide or a 5'-phosphate analog, especially a vinyl phosphate modified nucleotide (indicated by VP in the following examples), 5'-phosphate nucleotide (in the following examples) The nucleotide represented by P or 5'-phosphorothioate (indicated by Ps in the following examples).
  • the fluoro-modified nucleotide refers to a nucleotide formed by replacing a hydroxyl group at the 2'-position of a ribose group of a nucleotide with a fluorine
  • the non-fluorinated modified nucleotide refers to a nucleotide
  • a nucleotide or nucleotide analog formed by substitution of a hydroxyl group at the 2' position of a ribose group with a non-fluoro group which means that the nucleotide can be substituted in the nucleic acid, but the structure is different from the adenine ribonucleotide a group of guanine ribonucleotides, cytosine ribonucleotides, uridine ribonucleotides or thymine deoxyribonucleotides.
  • the methoxy-modified nucleotide refers to a nucleotide formed by substituting a 2'-hydroxyl group of a ribose group with a methoxy group.
  • the terms "complementary” or “reverse complementation” are used interchangeably and have the meanings well known to those skilled in the art that in a double stranded nucleic acid molecule, the base of one strand is linked to another strand.
  • the bases on are paired in a complementary manner.
  • the purine base adenine (A) is always paired with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) is always with the pyrimidine base.
  • Cytosine (G) is paired. Each base pair includes a purine and a pyrimidine.
  • mismatch in the art means that in a double-stranded nucleic acid, the bases at corresponding positions are not paired in a complementary form.
  • substantially reverse complementation means that there are no more than three base mismatches between the two nucleotide sequences involved; substantially reverse complementation means two There is no more than one base mismatch between the segment nucleotide sequences; complete complementation means that there is no base mismatch between the two nucleotide sequences.
  • nucleotide difference between one nucleotide sequence and another nucleotide sequence means that the base type of the nucleotide at the same position is changed in the former compared with the latter.
  • one nucleotide base is A
  • the corresponding nucleotide base at the same position in the former is U, C, G or T
  • a nucleotide at the home position is replaced with an abasic nucleotide or an equivalent thereof, a nucleotide difference is also considered to occur at that position.
  • the nucleoside monomer means, according to the preparation to be prepared
  • the type and sequence of nucleotides in siRNA or siRNA conjugates, modified or unmodified nucleoside phosphoramidites used in solid phase synthesis of phosphoramidite unmodified or modified RNA phosphoramidites, sometimes RNA phosphoramidites also known as Nucleoside phosphoramidites ).
  • the phosphoramidite solid phase synthesis is a method used in RNA synthesis well known to those skilled in the art.
  • the nucleoside monomers used in the present disclosure are commercially available.
  • a short cross that is not between two letters or between two symbols is the position used to indicate the attachment point of the substituent.
  • -C 1 -C 10 alkyl-NH 2 is attached via a C 1 -C 10 alkyl group.
  • optionally substituted alkyl includes “alkyl” and “substituted alkyl” as defined below.
  • alkyl refers to straight and branched chains having the indicated number of carbon atoms, typically from 1 to 20 carbon atoms, such as from 1 to 10 carbon atoms, such as from 1 to 8 Or 1 to 6 carbon atoms.
  • a C 1 -C 6 alkyl group contains a straight chain and a branched alkyl group of 1 to 6 carbon atoms.
  • alkyl residue having a specific number of carbons it is intended to cover all branched and straight-chain forms having this amount of carbon; thus, for example, "butyl” means including n-butyl, sec-butyl Base, isobutyl and tert-butyl; “propyl” includes n-propyl and isopropyl.
  • An alkylene group is a subset of an alkyl group and refers to a residue that is the same as the alkyl group but has two points of attachment.
  • alkenyl refers to an unsaturated branched or straight-chain alkyl group having at least one carbon-carbon double bond, which is derived from adjacent carbon atoms of the parent alkyl group. Obtained by removing one hydrogen molecule. The group can be in the cis or trans configuration of the double bond.
  • Typical alkenyl groups include, but are not limited to, ethenyl; propenyl, such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl Base), prop-2-en-2-yl; butenyl group, for example, but-1-en-1-yl, but-1-en-2-yl, 2-methylprop-1-ene-1- Base, but-2-en-1-yl, but-2-en-2-yl, butan-1,3-dien-1-yl, butan-1,3-dien-2-yl and the like.
  • an alkenyl group has 2 to 20 carbon atoms, and in other embodiments, 2 to 10, 2 to 8, or 2 to 6 carbon atoms.
  • An alkenylene group is a subset of an alkenyl group and refers to a residue that is the same as an alkenyl group but has two points of attachment.
  • alkynyl refers to an unsaturated branched or straight-chain alkyl group having at least one carbon-carbon triple bond which is derived from adjacent carbon atoms of the parent alkyl group. Obtained by removing two hydrogen molecules.
  • Typical alkynyl groups include, but are not limited to, ethynyl; propynyl, such as prop-1-yn-1-yl, prop-2-yn-1-yl; butynyl, such as but-1-yne- 1-yl, but-1-yn-3-yl, but-3-yn-1-yl and the like.
  • an alkynyl group has 2 to 20 carbon atoms, and in other embodiments, 2 to 10, 2 to 8, or 2 to 6 carbon atoms.
  • An alkynylene group is a subset of an alkynyl group and refers to a residue that is the same as an alkynyl group but has two points of attachment.
  • alkoxy refers to an alkyl group of the indicated number of carbon atoms attached through an oxygen bridge, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, Sec-butoxy, tert-butoxy, pentyloxy, 2-pentyloxy, isopentyloxy, neopentyloxy, hexyloxy, 2-hexyloxy, 3-hexyloxy, 3-methyl Pentyloxy and the like.
  • the alkoxy group usually has 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms attached through an oxygen bridge.
  • aryl refers to a radical derived from an aromatic monocyclic or polycyclic hydrocarbon ring system formed by the removal of a hydrogen atom from a ring carbon atom.
  • the aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and carbon of 6 to 18 carbon atoms, wherein at least one of the rings is completely unsaturated, ie it comprises a ring according to the Hückel theory. Delocalized (4n+2) ⁇ -electron system.
  • the aryl group includes, but is not limited to, a group such as a phenyl group, a fluorenyl group, and a naphthyl group.
  • An arylene group is a subset of an aryl group and refers to a residue that is the same as the aryl group but has two points of attachment.
  • cycloalkyl refers to a non-aromatic carbocyclic ring, typically having from 3 to 7 cyclic carbon atoms. The ring can be saturated or have one or more carbon-carbon double bonds.
  • cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl and cyclohexenyl, as well as bridged and caged ring groups such as norbornane.
  • halogen substituent or “halo” refers to fluoro, chloro, bromo and iodo, and the term “halogen” includes fluoro, chloro, bromo and iodo.
  • haloalkyl refers to an alkyl group as defined above, wherein a specified number of carbon atoms are replaced by one or more, up to a maximum allowable number of halogen atoms.
  • haloalkyl groups include, but are not limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl, and pentafluoroethyl.
  • Heterocyclyl means a stable 3- to 18-membered non-aromatic cyclic group containing from 2 to 12 carbon atoms and from 1 to 6 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. Unless otherwise stated in the specification, a heterocyclic group is a monocyclic, bicyclic, tricyclic or tetracyclic system and may include a fused ring or bridged ring system. The heteroatoms in the heterocyclic radical may optionally be oxidized. One or more nitrogen atoms, if present, are optionally quaternized. Heterocyclyl groups are partially saturated or fully saturated. A heterocyclic group can be attached to the remainder of the molecule through any atom of the ring.
  • heterocyclic groups include, but are not limited to, dioxoalkyl, thienyl [1,3]disulfonyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, heterophilic Azolidinyl, morpholinyl, octahydroindenyl, octahydroisoindolyl, 2-oxapiperazinyl, 2-oxapiperidinyl, 2-oxapyrimidinyl, oxazolidinyl, piperidine Pyridyl, piperazinyl, 4-piperidinone, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuranyl, trisulfonyl, tetrahydropyranyl, thiomorpholinyl, Thiomorpholinyl, 1-oxoal
  • Heteroaryl refers to a radical derived from a 3- to 18-membered aromatic ring radical comprising from 2 to 17 carbon atoms and from 1 to 6 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur.
  • a heteroaryl group can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system in which at least one ring in the ring system is completely unsaturated, ie, according to Hückel theory, comprising a circular delocalization (4n) +2) ⁇ -electron system.
  • Heteroaryl groups include fused ring or bridged ring systems. The heteroatoms in the heteroaryl are optionally oxidized.
  • heteroaryl group is attached to the remainder of the molecule through any atom in the ring.
  • heteroaryl groups include, but are not limited to, azepandinyl, acridinyl, benzimidazolyl, benzindenyl, 1,3-benzobisoxazolyl, benzofuranyl, benzene And oxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]bisoxazolyl, benzo[b][1,4]oxazolyl, 1 , 4-benzobisoxazolyl, benzonaphthofuranyl, benzodiazolyl, benzodioxanyl, benzopyranyl, benzopyranone, benzofuranyl, benzene And furanone, benzothienyl, benzothiophene [3,2-d]pyrimidin
  • hydroxy protecting groups can be used in the present disclosure.
  • a protecting group renders a chemical functionality insensitive to a particular reaction condition and can be added and removed in that functionality in the molecule without substantially damaging the remainder of the molecule.
  • Representative hydroxy protecting groups are disclosed in Beaucage et al, Tetrahedron 1992, 48, 2223-2311, and Greeneand Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d ed, John Wiley & Sons, New York, 1991, cited The above documents are incorporated herein in their entirety.
  • the protecting group is stable under basic conditions, but can be removed under acidic conditions.
  • non-exclusive examples of hydroxy protecting groups that may be used herein include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthene-9-yl ( Pixyl) and 9-(p-methoxyphenyl)xanthene-9-yl (Mox).
  • non-exclusive examples of hydroxy protecting groups that may be used herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxy). Trityl) and TMTr (4,4',4"-trimethoxytrityl).
  • subject refers to any animal, such as a mammal or marsupial.
  • Subject matter of the present disclosure includes, but is not limited to, humans, non-human primates (eg, rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cows, sheep, rats, and poultry of any kind.
  • therapeutic methods As used herein, “therapeutic methods,” “treatment,” “alleviation,” or “improvement” are used interchangeably herein. These terms refer to methods of obtaining beneficial or desired results, including but not limited to therapeutic benefits.
  • “Therapeutic benefit” means eradication or improvement of a potential disorder to be treated. In addition, the therapeutic benefit is obtained by eradicating or ameliorating one or more physiological symptoms associated with the underlying disorder, thereby observing an improvement in the patient, although the patient may still be afflicted with a potential disorder.
  • prevention and “prevention” are used interchangeably. These terms refer to methods of obtaining beneficial or desired results including, but not limited to, prophylactic benefits.
  • prophylactic benefit the conjugate or composition can be administered to a patient at risk for a particular disease, or to a patient who reports one or more pathological symptoms of the disease, even though the diagnosis of the disease may not have been made.
  • the present disclosure provides an siRNA capable of inhibiting the expression of a hepatitis B virus gene.
  • the siRNA of the present disclosure contains a nucleotide group as a basic structural unit, which is well known to those skilled in the art, and the nucleotide group contains a phosphate group, a ribose group, and a base, and will not be described herein.
  • CN102140458B discloses an siRNA that specifically inhibits the HBV gene, and various chemical modification strategies of the siRNA have been studied. The study found that different modification strategies have a very different impact on siRNA stability, biological activity and cytotoxicity. In this study, seven effective modifications were demonstrated. Compared to unmodified siRNA, one of the modified siRNAs improved blood stability while remaining substantially equivalent to unmodified siRNA. Inhibitory activity.
  • the siRNA of the present disclosure contains a sense strand and an antisense strand, each of the nucleotides of the siRNA being independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleotide sequence I,
  • the antisense strand comprises a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially reversely complementary to form a double-stranded region, wherein the nucleotide sequence I contains a nucleoside Acid sequence A, the nucleotide sequence A is equal in length to the nucleotide sequence shown in SEQ ID NO: 1, and is not more than 3 nucleotide differences, and the nucleotide sequence II contains nucleotides Sequence B, the nucleotide sequence B is equal in length to the nucleotide sequence shown in SEQ ID NO: 2, and no more than 3 nucleotide differences:
  • nucleotide sequence A contains a nucleotide Z A corresponding to a position Z
  • nucleotide sequence B contains a nucleotide Z' B whose position corresponds to Z', which is Z' B The first nucleotide of the 5' end of the antisense strand.
  • positional correspondence means the same position in the nucleotide sequence from the same end of the nucleotide sequence.
  • the first nucleotide at the 3' end of nucleotide sequence A is a nucleotide that corresponds to the 1st nucleotide of the 3' end of SEQ ID NO: 1.
  • the sense strand comprises only nucleotide sequence I
  • the antisense strand comprises only nucleotide sequence II.
  • the sense strand comprises nucleotide sequence I
  • the antisense strand comprises only nucleotide sequence II.
  • the nucleotide sequence A differs from the nucleotide sequence set forth in SEQ ID NO: 1 by no more than 1 nucleotide difference, and/or the nucleotide sequence B and SEQ There is no more than one nucleotide difference between the nucleotide sequences shown by ID NO:2.
  • the nucleotide difference between the nucleotide sequence B and the nucleotide sequence set forth in SEQ ID NO: 2 comprises a difference at the Z' B position, and Z' B is selected from A, C or G.
  • the nucleotide difference is a difference at the Z' B position, Z' B is selected from A, C or G, and Z A is a nucleotide complementary to Z' B.
  • the nucleotide sequence A and the nucleotide sequence B are substantially reverse complementary, substantially reverse complementary or fully reverse complementary; the substantially reverse complement refers to two cores There are no more than 3 base mismatches between the nucleotide sequences; the substantially reverse complement means that there are no more than one base mismatch between the two nucleotide sequences; complete reverse complementation It means that there is no mismatch between the two nucleotide sequences.
  • nucleotide sequence A is the nucleotide sequence set forth in SEQ ID NO: 3
  • nucleotide sequence B is the nucleotide sequence set forth in SEQ ID NO: 4:
  • Z' B is the first nucleotide at the 5' end of the antisense strand
  • Z A is selected from A, U, G or C
  • Z' B is a nucleotide complementary to Z A
  • Z A is A
  • Z′ B is U
  • the sense strand and the antisense strand are the same or different in length, the sense strand is 19-23 nucleotides in length, and the antisense strand is 20-26 nucleotides in length.
  • the length ratio of the sense strand and the antisense strand of the siRNA provided by the present disclosure may be 19/20, 19/21, 19/22, 19/23, 19/24, 19/25, 19/26, 20/20, 20/21, 20/22, 20/23, 20/24, 20/25, 20/26, 21/20, 21/21, 21/22, 21/23, 21/24, 21/25, 21/ 26, 22/20, 22/21, 22/22, 22/23, 22/24, 22/25, 22/26, 23/20, 23/21, 23/22, 23/23, 23/24, 23/25 or 23/26.
  • the length ratio of the sense strand and the antisense strand of the siRNA is 19/21, 21/23 or 23/25.
  • the sense strand and the antisense strand are of the same length
  • the nucleotide sequence I further comprises a nucleotide sequence III
  • the nucleotide sequence II further comprises a nucleotide sequence IV, a nucleotide
  • the sequence III and nucleotide sequence IV lengths are each independently 1-4 nucleotides; the nucleotide sequence III is ligated at the 5' end of nucleotide sequence A, which is ligated to the nucleus
  • the 3' end of the nucleotide sequence B, the nucleotide sequence III and the nucleotide sequence IV are equal in length.
  • nucleotide sequence III and the nucleotide sequence IV may or may not be complementary.
  • the nucleotide sequence III and the nucleotide sequence IV are at least partially complementary; in some implementations
  • nucleotide sequence III and nucleotide sequence IV are more than 80% base complementary, or more than 90% base complementary; in some embodiments, nucleotide sequence III and nucleotide sequence IV are substantially Reverse complementary or fully reverse complementary; said substantially reverse complement refers to the presence of no more than one base mismatch between two nucleotide sequences; complete reverse complementation refers to two nucleotide sequences There is no mismatch between them; in some embodiments, nucleotide sequence III and nucleotide sequence IV are completely reverse complementary.
  • the siRNA sense strand and the antisense strand are of equal length and have a length ratio of 20/20, 21/21, 22/22 or 23/23. In some embodiments, the length ratio of the sense strand and the antisense strand of the siRNA is 21/21 or 23/23.
  • the nucleotide sequence III and the nucleotide sequence IV are each 1 nucleotide in length, the nucleotide sequence III has a base G, and the nucleotide sequence IV has a base C.
  • the length ratio of the sense strand and the antisense strand is 20/20; or, the nucleotide sequences III and IV are both 2 nucleotides in length, and the nucleoside is in the direction from the 5' end to the 3' end.
  • the base composition of the acid sequence III is CG
  • the base composition of the nucleotide sequence IV is GC; at this time, the length ratio of the sense strand to the antisense strand is 21/21; or the length of the nucleotide sequences III and IV All are 3 nucleotides.
  • the base composition of nucleotide sequence III is CCG
  • the base composition of nucleotide sequence IV is CGG
  • the sense strand and the opposite The length ratio of the sense strand is 22/22
  • the lengths of the nucleotide sequences III and IV are 4 nucleotides
  • the base composition of the nucleotide sequence III is in the direction from the 5' end to the 3' end.
  • the nucleotide composition of nucleotide sequence IV is CGGG; at this time, the length ratio of the sense strand to the antisense strand is 23/23.
  • the nucleotide sequence III and the nucleotide sequence IV are 2 nucleotides in length, and the nucleotide composition of the nucleotide sequence III is CG according to the direction from the 5' end to the 3' end.
  • the nucleotide composition of nucleotide sequence IV is GC; at this time, the length ratio of the sense strand to the antisense strand is 21/21.
  • nucleotide sequence III and nucleotide sequence IV are the same length and are completely reverse-complementary, thus giving the nucleotide sequence III base, and the nucleotide sequence IV base is also It is determined.
  • the sense strand and the antisense strand are different in length
  • the nucleotide sequence II further comprises a nucleotide sequence V
  • the nucleotide sequence V is 1 to 3 nucleotides in length, and is ligated
  • the 3' end of the antisense strand constitutes the 3' overhanging end of the antisense strand.
  • the length ratio of the sense strand and the antisense strand of the siRNA provided by the present disclosure may be 19/20, 19/21, 19/22, 20/21, 20/22, 20/23, 21/22, 21/23. , 21/24, 22/23, 22/24, 22/25, 23/24, 23/25 or 23/26.
  • the nucleotide sequence V is 2 nucleotides in length, whereby the length ratio of the siRNA sense strand and the antisense strand provided by the present disclosure may be 19/21, 21/23 or 23 /25.
  • Each of the nucleotide sequences V may be any nucleotide, and the nucleotide sequence V is two consecutive thymidine deoxyribonucleotides for ease of synthesis and to save on synthesis cost ( TT) or two consecutive uracil ribonucleotides (UU); in order to increase the affinity of the siRNA antisense strand to the target mRNA, the nucleotide sequence V is complementary to the nucleotide at the corresponding position of the target mRNA.
  • the ratio of the sense strand and the antisense strand of the siRNA of the present disclosure is 19/21 or 21/23, in which case the siRNA of the present disclosure has better mRNA silencing activity.
  • the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 3, the anti-sense strand of the siRNA comprising the nucleotide sequence set forth in SEQ ID NO: 4:
  • Z' B is the first nucleotide at the 5' end of the antisense strand and Z A is selected from A, U, G or C, and Z' B is a nucleotide complementary to Z A .
  • the siRNA of the present disclosure is siHBVX1
  • Antisense strand 5'-Z'CAGAUGAGAAGGCACAGACG-3' (SEQ ID NO: 5),
  • the nucleotides in the siRNAs of the present disclosure are each independently a modified or unmodified nucleotide.
  • the nucleotide in the siRNA of the present disclosure is an unmodified nucleotide; in some embodiments, some or all of the nucleotides in the siRNA of the present disclosure are modified nucleotides, a nucleus These modifications on the glucoside group do not result in a significant impairment or loss of the ability of the siRNA conjugates of the present disclosure to inhibit hepatitis B virus gene expression.
  • the siRNA of the present disclosure contains at least one modified nucleotide.
  • modified nucleotide refers to a nucleotide or nucleotide analog formed by the substitution of a hydroxyl group at the 2'-position of a ribose group of a nucleotide with another group, or a nucleoside.
  • the base on the acid is the nucleotide of the modified base.
  • the modified nucleotide does not result in a significant attenuation or loss of the function of the siRNA to inhibit gene expression.
  • modified nucleotides disclosed in J. K. Watts, G. F. Deleavey, and M. J. Damha Chemically modified siRNA: tools and applications. Drug Discov Today, 2008, 13 (19-20): 842-55 can be selected.
  • the sense strand of the siRNA provided by the disclosure or at least one nucleotide of the antisense strand is a modified nucleotide
  • at least one phosphate group is a phosphate having a modifying group
  • at least a portion of the phosphate group and/or ribose group in the sense strand and at least one single-stranded phosphate-sugar backbone of the sense strand is a phosphate group having a modifying group and/or Or a ribosyl group having a modifying group.
  • the sense strand and/or all of the nucleotides in the antisense strand are modified nucleotides.
  • each of the sense strands of the siRNA provided by the disclosure and the antisense strand are independently fluoro-modified nucleotides or non-fluoro-modified nucleotides.
  • the inventors of the present disclosure have surprisingly found that the siRNAs described in the present disclosure achieve a high balance of plasma stability and gene silencing efficiency in animal experiments.
  • the fluoro-modified nucleotide is located in nucleotide sequence A and nucleotide sequence B, and, in the direction from the 5' end to the 3' end, the nucleotide sequence A
  • the nucleotides at positions 7, 8, and 9 are fluoro-modified nucleotides
  • the nucleus at positions 2, 6, 14, and 16 of the nucleotide sequence B in the direction from the 5' end to the 3' end Glycosylates are fluoro-modified nucleotides.
  • the fluoro-modified nucleotide is located in nucleotide sequence A and nucleotide sequence B, wherein the nucleotide sequence A has no more than five fluoro-modified nucleotides, And, in the direction from the 5' end to the 3' end, the nucleotides at positions 7, 8, and 9 of the nucleotide sequence A are fluoro-modified nucleotides; the nucleotide sequence B is fluorine The number of modified nucleotides is not more than 7, and the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence B are fluoro-modified nucleotides.
  • the nucleotides at positions 7, 8, 9 or 5, 7, 8, and 9 of the nucleotide sequence A are in the sense strand in the direction from the 5' end to the 3' end.
  • the nucleotide is a fluoro-modified nucleotide, and the nucleotides at the rest of the sense strand are non-fluoro-modified nucleotides; in the antisense strand in the direction from the 5' end to the 3' end
  • the nucleotide at position 2, 6, 14, 16 or 2, 6, 8, 9, 14, 16 of the nucleotide sequence B is a fluoro-modified nucleotide in the antisense strand
  • the nucleotides at the remaining positions are non-fluoro modified nucleotides.
  • fluoro-modified nucleotide refers to a nucleotide formed by substituting a hydroxyl group at the 2'-position of a ribose group of a nucleotide with fluorine, which has a structure represented by the following formula (101).
  • Non-fluorinated modified nucleotide refers to a nucleotide or nucleotide analog formed by the substitution of a hydroxyl group at the 2' position of the ribose group of a nucleotide with a non-fluoro group.
  • each non-fluoro modified nucleotide is independently selected from a nucleotide or nucleotide analog formed by the substitution of a hydroxyl group at the 2' position of the ribose group of a nucleotide with a non-fluoro group.
  • Nucleotides formed by substitution of a hydroxyl group at the 2' position of the ribose group with a non-fluoro group are well known to those skilled in the art, and these nucleotides may be selected from 2'-alkoxy-modified nucleotides, 2'- Substituted alkoxy-modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'- One of a substituted amino-modified nucleotide, 2'-deoxynucleotide.
  • the 2'-alkoxy-modified nucleotide is a methoxy-modified nucleotide (2'-OMe) as shown in formula (102).
  • the 2'-substituted alkoxy-modified nucleotide may, for example, be a 2'-O-methoxyethyl modified nucleotide (2'-MOE) as shown in formula (103).
  • the 2'-amino modified nucleotide (2'-NH2) is represented by the formula (104).
  • the 2'-deoxynucleotide (DNA) is represented by the formula (105).
  • a nucleotide analog refers to the ability to replace a nucleotide in a nucleic acid, but differs in structure from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uridine ribonucleotides or thymine deoxygenation.
  • the nucleotide analog can be, for example, an isonucleotide, a bridged nucleic acid (BNA) or an acyclic nucleotide.
  • the BNA refers to a restricted or inaccessible nucleotide.
  • the BNA may contain a five-membered ring, a six-membered ring, or a seven-membered ring with a bridge structure of "fixed" C3'-endo-glycans.
  • the bridge is typically incorporated into the 2'-, 4'-position of the ribose to provide a 2',4'-BNA nucleotide, such as LNA, ENA, cET BNA, etc., wherein the LNA is as in formula (106).
  • the ENA is as shown in the formula (107)
  • the cET BNA is as shown in the formula (108).
  • An acyclic nucleotide is a type of nucleotide formed by the opening of a sugar ring of a nucleotide, such as an unlocked nucleic acid (UNA) or a glycerol nucleic acid (GNA), wherein UNA is as shown in formula (109), and GNA is as in the formula ( 110) shown.
  • UNA unlocked nucleic acid
  • GNA glycerol nucleic acid
  • R is selected from H, OH or alkoxy group (O-alkyl group).
  • An isonucleotide refers to a compound formed by a change in the position of a base in a nucleotide on a ribose ring, for example, a base is moved from a 1'-position to a 2'-position or a 3'-position of a ribose ring.
  • the compound is represented by the formula (111) or (112).
  • Base represents a base such as A, U, G, C or T; and R is selected from H, OH, F or a non-fluorine group as described above.
  • the nucleotide analog is selected from the group consisting of an isonucleotide, LNA, ENA, cET, UNA, and GNA.
  • each non-fluoro-modified nucleotide is a methoxy-modified nucleotide, and above and below, the methoxy-modified nucleotide refers to a 2' of a ribose group. a nucleotide formed by substituting a hydroxyl group with a methoxy group.
  • fluoro-modified nucleotide has the same meaning, and both refer to a compound in which the 2'-hydroxyl group of the nucleotide is substituted by fluorine, and has a structure represented by the formula (207);
  • methoxy-modified nucleotide has a structure represented by the formula (208) formed by substituting a 2'-hydroxy group of an acid ribose group with a methoxy group.
  • the siRNA of the present disclosure is an siRNA having a modification in the direction of the 5' end to the 3' end, in the sense strand, positions 7, 8, and 9 of the nucleotide sequence A.
  • the nucleotides at positions 5, 7, 8, and 9 are fluoro-modified nucleotides, and the nucleotides at the remaining positions in the sense strand are methoxy-modified nucleotides;
  • the nucleotides at positions 2, 6, 14, 16 or 2, 6, 8, 9, 14, 16 of the nucleotide sequence B are fluoro-modified nucleotides, the antisense The nucleotides remaining in the chain are methoxy modified nucleotides.
  • the siRNA of the present disclosure is an siRNA having a modification of the 5th, 7th, 8th, and 9th positions of the nucleotide sequence A in the sense strand of the siRNA in the direction from the 5' end to the 3' end.
  • the nucleotide is a fluoro-modified nucleotide
  • the nucleotide at the remaining position of the sense strand of the siRNA is a methoxy-modified nucleotide, and, in the direction from the 5' end to the 3' end, the siRNA
  • the nucleotides at positions 2, 6, 8, 9, 14 and 16 of the nucleotide sequence B in the antisense strand are fluoro-modified nucleotides
  • the nucleotides at the rest of the antisense strand of the siRNA are methoxy groups.
  • Modified nucleotide is a fluoro-modified nucleotide
  • nucleotides at positions 5, 7, 8, and 9 of the nucleotide sequence A in the sense strand of the siRNA are fluoro-modified nucleotides in the direction from the 5' end to the 3' end, and the siRNA is sensed.
  • the nucleotides at the rest of the chain are methoxy-modified nucleotides, and, in the direction from the 5' end to the 3' end, nucleotides B, 2, 6, and 14 of the antisense strand of the siRNA And the nucleotide at position 16 is a fluoro-modified nucleotide, and the nucleotide at the remaining position of the antisense strand of the siRNA is a methoxy-modified nucleotide;
  • the nucleotides at positions 7, 8 and 9 of the nucleotide sequence A in the sense strand of the siRNA are -fluoro-modified nucleotides, and the sense strand of siRNA
  • the nucleotides at the remaining positions are methoxy-modified nucleotides, and, in the direction from the 5' end to the 3' end, the second, sixth, and fourth nucleotide sequences of the antisense strand of the siRNA are
  • the nucleotide at position 16 is a fluoro-modified nucleotide
  • the nucleotide at the rest of the antisense strand of the siRNA is a methoxy-modified nucleotide.
  • the ribose group in the phosphate-sugar backbone of the siRNA has a modifying group, respectively: in the direction from the 5' end to the 3' end, the nucleotide sequence A of the siRNA is in the fifth, seventh,
  • the glycosyl group at positions 8 and 9 is a 2'-fluororibosyl group, and the sugar group at the remaining position of the sense strand of the siRNA is a 2'-methoxyribosyl group, and, according to the 5' end to the 3' end
  • the glycosyl group at positions 2, 6, 8, 9, 14 and 16 of the nucleotide sequence B in the antisense strand of the siRNA is a 2'-fluororibosyl group, and the nucleotides at the remaining positions of the antisense strand of the siRNA
  • the glycosyl group is a 2'-methoxyribosyl group;
  • the glycosyl group at positions 5, 7, 8 and 9 of the nucleotide sequence A in the sense strand of the siRNA is 2'-fluororibosyl, in the direction from the 5' end to the 3' end, the sense strand of siRNA
  • the remaining position of the nucleotide has a 2'-methoxyribosyl group, and, in the direction from the 5' end to the 3' end, the nucleotide sequence B of the antisense strand of the siRNA is 2, 6,
  • the glycosyl group at positions 14 and 16 is a 2'-fluororibose group, and the glycosyl group at the remaining position of the antisense strand of the siRNA is a 2'-methoxyribose group;
  • the glycosyl group at positions 7, 8 and 9 of the nucleotide sequence A in the sense strand of the siRNA is 2'-fluororibosyl, in the direction from the 5' end to the 3' end, and the rest of the sense strand of the siRNA
  • the glycosyl group at the position nucleotide is a 2'-methoxyribosyl group
  • the second, sixth, and fourth nucleotide sequences B in the antisense strand of the siRNA are in the direction from the 5' end to the 3' end.
  • the glycosyl group at position 16 is a 2'-fluororibose group
  • the sugar group at the rest of the antisense strand of the siRNA is a 2'-methoxyribosyl group.
  • the siRNA provided by the present disclosure is siHBVX2 or siHBVX3:
  • the capital letters C, G, U, and A represent the base composition of the nucleotide;
  • the lowercase letter m indicates that the nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide;
  • the lowercase letter f indicates One nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide.
  • the siRNA having the above modification is not only low in cost, but also makes it difficult for the ribonuclease in the blood to cleave the nucleic acid, thereby increasing the stability of the nucleic acid and making the nucleic acid more resistant to nuclease hydrolysis.
  • the phosphate groups in the phosphate-sugar backbone of at least one single strand of the sense strand and the antisense strand of the siRNA provided by the disclosure are phosphate groups having a modifying group.
  • the phosphate group having a modifying group is a phosphorothioate group formed by substitution of at least one oxygen atom of a phosphodiester bond in a phosphate group with a sulfur atom; in some embodiments, The phosphate group having a modifying group is a phosphorothioate group having a structure represented by the formula (1):
  • This modification stabilizes the double-stranded structure of the siRNA, maintaining high specificity and high affinity for base pairing.
  • the phosphorothioate linkage is present at at least one of the following positions: between the first and second nucleotides at either end of the sense strand or the antisense strand ; between the second and third nucleotides at either end of the sense strand or the antisense strand; or any combination of the above. In some embodiments, the phosphorothioate linkage is present at all of the above positions except for the 5' end of the sense strand. In some embodiments, the phosphorothioate linkage is present at all of the above positions except for the 3' end of the sense strand. In some embodiments, the phosphorothioate linkage is present at at least one of the following locations:
  • the 2' end of the 3' end of the antisense strand is between the 2nd nucleotide and the 3rd nucleotide.
  • the siRNA provided by the present disclosure is siHBVX4 or siHBVX5:
  • the capital letters C, G, U, and A represent the base composition of the nucleotide;
  • the lowercase letter m indicates that the nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide;
  • the lowercase letter f indicates One nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide;
  • the lower-case letter s indicates a phosphorothioate-based linkage between the two nucleotides to the left and right of the letter.
  • the 5' terminal nucleotide of the siRNA antisense strand is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide.
  • 5'-phosphate nucleotides may have the following structure:
  • R is selected from H, OH, methoxy, and fluorine; and Base represents a base selected from A, U, C, G or T.
  • the 5'-phosphate nucleotide is a 5'-phosphate modified nucleotide represented by formula (2), and the 5'-phosphate analog modified nucleotide comprises a vinyl phosphate ( 5'-(E)-vinylphosphonate, E-VP) modified nucleotide, as shown in formula (3), or a phosphorothioate-modified nucleotide, as shown in formula (5).
  • the 5'-phosphate analog modified nucleotide comprises a vinyl phosphate ( 5'-(E)-vinylphosphonate, E-VP) modified nucleotide, as shown in formula (3), or a phosphorothioate-modified nucleotide, as shown in formula (5).
  • the siRNA provided by the present disclosure is siHBVX6, siHBVX7, siHBVX8 or siHBVX9:
  • the capital letters C, G, U, and A represent the base composition of the nucleotide;
  • the lowercase letter m indicates that the nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide;
  • the lowercase letter f indicates The nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide;
  • the lowercase letter s indicates a phosphorothioate linkage between the two nucleotides of the letter;
  • the uppercase letter P1 indicates the letter to the right.
  • One nucleotide adjacent to the side is a nucleotide modified by a 5'-phosphate nucleotide or a 5'-phosphate analog.
  • the inventors of the present disclosure have unexpectedly discovered that the siRNA provided by the present disclosure not only has significantly enhanced plasma and lysosomal stability, but also retains high gene inhibitory activity.
  • the siRNA provided by the present disclosure can be obtained by a conventional siRNA preparation method (for example, a method of solid phase synthesis and liquid phase synthesis) in the art. Among them, solid phase synthesis already has commercial customized services.
  • a method of preparing a nucleoside monomer having a corresponding modification and introducing a modified nucleotide group can be carried out by introducing a modified nucleotide group having a corresponding modified nucleoside monomer into the siRNA described in the present disclosure. Methods of siRNA are also well known to those skilled in the art.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the siRNA as described above as an active ingredient and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may be a carrier conventionally used in the field of siRNA administration, such as but not limited to magnetic nanoparticles (such as Fe3O4 or Fe2O3-based nanoparticles), carbon nanotubes, mesopores.
  • PEO polyethyleni
  • the pharmaceutical composition has no particular requirement for the amount of siRNA and pharmaceutically acceptable carrier, and in some embodiments, the weight ratio of siRNA to pharmaceutically acceptable carrier can be 1: ( 1-500), in some embodiments, the above weight ratio is 1: (1-50).
  • the pharmaceutical composition may further comprise other excipients that are pharmaceutically acceptable, and the excipients may be one or more of various formulations or compounds conventionally employed in the art.
  • the pharmaceutically acceptable other excipient may include at least one of a pH buffer, a protective agent, and an osmotic pressure adjusting agent.
  • the pH buffer may be a trishydroxymethylaminomethane hydrochloride buffer having a pH of 7.5-8.5 and/or a phosphate buffer having a pH of 5.5-8.5, for example, a phosphoric acid having a pH of 5.5-8.5. Salt buffer.
  • the protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose.
  • the protective agent may be included in an amount of from 0.01 to 30% by weight based on the total weight of the pharmaceutical composition.
  • the osmotic pressure adjusting agent may be sodium chloride and/or potassium chloride.
  • the osmotic pressure adjusting agent is present in an amount such that the osmotic pressure of the pharmaceutical composition is from 200 to 700 milliosmoles per kilogram.
  • the content of the osmotic pressure adjusting agent can be easily determined by those skilled in the art depending on the desired osmotic pressure.
  • the pharmaceutical composition may be a liquid preparation, such as an injection solution; or a lyophilized powder injection, which is mixed with a liquid adjuvant when administered, and formulated into a liquid preparation.
  • the liquid formulation can be, but is not limited to, for subcutaneous, intramuscular or intravenous administration, and can be, but is not limited to, administered to the lungs by spraying, or administered to other organ tissues (such as the liver) via the lungs by spraying.
  • the pharmaceutical composition is for intravenous administration.
  • the pharmaceutical composition can be in the form of a liposomal formulation.
  • the pharmaceutically acceptable carrier used in the liposome formulation comprises an amine-containing transfection compound (which may also be referred to hereinafter as an organic amine), helper lipid, and/or PEGylation. Lipid.
  • the organic amine, helper lipid, and pegylated lipid are each selected from the group consisting of amine-containing transfection compounds described in CN103380113A (incorporated herein by reference in its entirety) One or more of the accepted salts or derivatives, helper lipids, and pegylated lipids.
  • the organic amine can be a compound of formula (201) or a pharmaceutically acceptable salt thereof as described in CN103380113A:
  • X 101 and X 102 are each independently O, S, NA or CA, wherein A is hydrogen or a C 1 - C 20 hydrocarbon chain;
  • R 101 , R 102 , R 103 , R 104 , R 105 , R 106 and R 107 are each independently hydrogen, cyclic or acyclic, substituted or unsubstituted, branched or straight-chain aliphatic radical a cyclic, acyclic or acyclic, substituted or unsubstituted, branched or straight chain heteroaliphatic group, substituted or unsubstituted, branched or straight chain acyl group, substituted or not Substituted, branched or straight-chain aryl, substituted or unsubstituted, branched or straight-chain heteroaryl;
  • x is an integer from 1 to 10;
  • n is an integer from 1 to 3
  • m is an integer from 0 to 20
  • R 103 and the nitrogen in the formula (201) form a structure as shown in the formula (202) or the formula (203):
  • HCC represents a hydrocarbon chain
  • N represents a nitrogen atom in the formula (201).
  • R 103 is a polyamine. In other embodiments, R 103 is a ketal. In some embodiments, each of R 101 and R 102 in formula (201) is independently any substituted or unsubstituted, branched or straight chain alkyl or alkenyl group, said alkane
  • the base or alkenyl group has from 3 to about 20 carbon atoms, such as from 8 to about 18 carbon atoms, and from 0 to 4 double bonds, such as from 0 to 2 double bonds.
  • R 103 can be any of the following formulas (204) - (213):
  • g, e and f are each independently an integer of 1-6
  • each "HCC” represents a hydrocarbon chain
  • each * shows R 103 and is in the formula (201)
  • a possible point of attachment of a nitrogen atom in which each H at any * position can be replaced to effect attachment to the nitrogen atom in formula (201).
  • the compound of the formula (201) can be produced according to the description in CN103380113A.
  • the organic amine is an organic amine as shown in formula (214) and/or an organic amine as shown in formula (215):
  • the helper lipid is a cholesterol, an analog of cholesterol, and/or a derivative of cholesterol;
  • the PEGylated lipid is 1,2-dipalmitamide-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)]-2000.
  • the molar ratio between the organic amine, the helper lipid, and the PEGylated lipid is (19.7-80): (19.7-80) in the pharmaceutical composition. ): (0.3-50), for example, may be (50-70): (20-40): (3-20).
  • the pharmaceutical composition particles formed from the siRNA of the present disclosure and the amine-containing transfection reagent described above have an average diameter of from about 30 nm to about 200 nm, typically from about 40 nm to about 135 nm, and more typically, the liposome
  • the average diameter of the particles is from about 50 nm to about 120 nm, from about 50 nm to about 100 nm, from about 60 nm to about 90 nm, or from about 70 nm to about 90 nm, for example, the average diameter of the liposome particles is about 30, 40, 50, 60, 70. , 75, 80, 85, 90, 100, 110, 120, 130, 140, 150 or 160 nm.
  • the weight of the siRNA and all lipids in a pharmaceutical composition formed from the siRNAs of the present disclosure and the amine-containing transfection reagents described above
  • the ratio is from about 1:1 to about 1:50, from about 1:1 to about 1:30, from about 1:3 to about 1:20, from about 1:4 to about 1: 18.
  • the weight ratio of the siRNA of the present disclosure to the total lipid is about 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1 : 11, 1: 12, 1: 13, 1:14, 1:15, 1:16, 1:17 or 1:18.
  • the components of the pharmaceutical composition may be present separately upon sale and may be in the form of a liquid formulation when used.
  • the pharmaceutical composition of the siRNA provided by the present disclosure and the above pharmaceutically acceptable carrier can be prepared according to various known methods, except that the siRNA provided by the present disclosure is substituted for the existing siRNA; In an embodiment, it can be prepared as follows:
  • the organic amine, the auxiliary lipid and the PEGylated lipid are suspended in the alcohol according to the above molar ratio and mixed to obtain a lipid solution; the amount of the alcohol is such that the total mass concentration of the obtained lipid solution is 2-25 mg/mL, For example, it can be 8-18 mg/mL.
  • the alcohol is selected from a pharmaceutically acceptable alcohol, such as an alcohol that is liquid near room temperature, for example, ethanol, propylene glycol, benzyl alcohol, glycerin, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400. One or more of them may be, for example, ethanol.
  • the siRNA provided in the present disclosure was dissolved in a buffered saline solution to obtain an aqueous siRNA solution.
  • the concentration of the buffer salt solution is 0.05-0.5M, for example, 0.1-0.2M, and the pH of the buffer salt solution is adjusted to 4.0-5.5, for example, 5.0-5.2, and the buffer solution is used in an amount such that the concentration of the siRNA does not exceed 0.6 mg. /mL, for example, may be 0.2-0.4 mg/mL.
  • the buffer salt is selected from one or more of soluble acetate and soluble citrate, and may be, for example, sodium acetate and/or potassium acetate.
  • the lipid solution and the siRNA aqueous solution are mixed, and the product obtained after the mixing is incubated at 40 to 60 ° C for at least 2 minutes, for example, 5 to 30 minutes, to obtain a liposome preparation after the incubation.
  • the volume ratio of the lipid solution to the aqueous siRNA solution is 1: (2-5), for example, 1:4.
  • the liposome preparation after the incubation is concentrated or diluted to remove impurities and sterilized, and the pharmaceutical composition provided by the present disclosure has the physical and chemical parameters of pH 6.5-8, encapsulation efficiency of not less than 80%, and particle size of 40-200nm, polydispersity index is not higher than 0.30, osmotic pressure is 250-400mOsm/kg; for example, physical and chemical parameters can be pH 7.2-7.6, encapsulation efficiency is not less than 90%, particle size is 60-100nm, more The dispersion index is not higher than 0.20, and the osmotic pressure is 300-400 mOsm/kg.
  • concentration or dilution can be carried out before, after or simultaneously with the removal of impurities.
  • the method of removing impurities can be carried out by various methods, for example, a phase-cut flow system, a hollow fiber column, ultrafiltration under a condition of 100 K Da, and an ultrafiltration exchange solution of phosphate buffer (PBS) having a pH of 7.4.
  • the sterilization method can be carried out by various methods, for example, it can be sterilized by filtration on a 0.22 ⁇ m filter.
  • the disclosure provides an siRNA conjugate comprising the siRNA described above and a conjugating group attached to the siRNA.
  • conjugation means that two or more chemical moieties each having a particular function are linked to each other in a covalently bonded manner; accordingly, the “conjugate” is A compound formed by covalent attachment between the various chemical moieties.
  • siRNA conjugate refers to a compound formed by covalent attachment of one or more chemical moieties having a particular function to an siRNA.
  • siRNA conjugate of the present disclosure is sometimes simply referred to as "conjugate”.
  • An siRNA conjugate is understood to be a generic term for an siRNA conjugate, a first siRNA conjugate or a second siRNA conjugate, depending on the context.
  • a "conjugated molecule” is understood to mean a compound that can be conjugated to a siRNA by reaction, ultimately forming an siRNA conjugate of the present disclosure.
  • the present disclosure provides a first siRNA conjugate comprising the siRNA described above and a conjugate group attached to the siRNA.
  • the conjugate group comprises a pharmaceutically acceptable at least one targeting group and an optional linker, and the siRNA, the linker and The targeting groups are linked in sequence.
  • the targeting group is from 1 to 6.
  • the targeting group is 2-4.
  • the siRNA molecule can be non-covalently or covalently conjugated to the conjugate group, for example, can be covalently conjugated to the conjugate group.
  • the conjugation site of the siRNA to the conjugate group may be at the 3' or 5' end of the siRNA sense strand, or at the 5' end of the antisense strand, or in the internal sequence of the siRNA. In some embodiments, the conjugation site of the siRNA to the conjugating group is at the 3' end of the siRNA sense strand.
  • the conjugate group can be attached to a phosphate group, a 2'-position hydroxy group or a base of a nucleotide. In some embodiments, the conjugated group can be attached to the 3'-position hydroxyl group, in which case the nucleotides are linked by a 2'-5' phosphodiester bond.
  • the conjugate group is typically attached to the phosphate group of the nucleotide; when the conjugate group is attached to the internal sequence of the siRNA, the conjugate group Usually attached to a ribose sugar ring or base.
  • linkages refer to: Muthiah Manoharan et. al. siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes. ACS Chemical biology, 2015, 10(5): 1181-7.
  • the siRNA and the conjugate group may be linked by an acid labile or reducible chemical bond, and in the acidic environment of the cell endosomes, these chemical bonds may degrade, thereby rendering the siRNA free.
  • the conjugate group can be attached to the sense strand of the siRNA to minimize the effect of conjugation on siRNA activity.
  • the pharmaceutically acceptable targeting group refers to a ligand that can be routinely used in the field of siRNA administration, such as the various ligands described in WO2009082607A2, all of which are incorporated by reference. Into this article.
  • the pharmaceutically acceptable targeting group can be selected from one or more of the following ligands or derivatives thereof: lipophilic molecules, such as cholesterol, bile acids, Vitamins (eg vitamin E), lipid molecules of different chain lengths; polymers such as polyethylene glycol; polypeptides such as transmembrane peptides; aptamers; antibodies; quantum dots; saccharides such as lactose, polylactose, mannose Sugar, galactose, N-acetylgalactosamine (GalNAc); folate; receptor ligands expressed by hepatocytes, such as asialoglycoprotein, asialoglycohol residues, lipoproteins (eg high density) Lipoprotein, low density lipoprotein, etc., glucagon, neurotransmitters (such as adrenaline), growth factors, transferrin and the like.
  • lipophilic molecules such as cholesterol, bile acids, Vitamins (eg vitamin E), lipid molecules of different chain lengths
  • polymers such as poly
  • each of said ligands is independently selected from a ligand capable of binding to a cell surface receptor.
  • at least one of the ligands is a ligand capable of binding to a hepatocyte surface receptor.
  • at least one of the ligands is a ligand capable of binding to a mammalian hepatocyte surface receptor.
  • at least one of the ligands is a ligand capable of binding to a human hepatocyte surface receptor.
  • at least one of the ligands is a ligand capable of binding to a hepatic surface asialoglycoprotein receptor (ASGPR).
  • ASGPR hepatic surface asialoglycoprotein receptor
  • the pharmaceutically acceptable targeting group can be any ligand that binds to an asialoglycoprotein receptor (ASGPR) on the surface of a mammalian liver cell.
  • each ligand is independently a asialoglycoprotein, such as asialouromusmucoid (ASOR) or asialofibrate (ASF).
  • ASOR asialouromusmucoid
  • ASF asialofibrate
  • the ligand is a derivative of a sugar or a sugar.
  • At least one of the ligands is a sugar. In some embodiments, each ligand is a sugar. In some embodiments, at least one of the ligands is a monosaccharide, a polysaccharide, a modified monosaccharide, a modified polysaccharide, or a sugar derivative. In some embodiments, at least one of the ligands can be a monosaccharide, a disaccharide or a trisaccharide. In some embodiments, at least one of the ligands is a modified sugar. In some embodiments, each ligand is a modified sugar.
  • each ligand is independently selected from the group consisting of a polysaccharide, a modified polysaccharide, a monosaccharide, a modified monosaccharide, a polysaccharide derivative, or a monosaccharide derivative.
  • each or at least one ligand is selected from the group consisting of glucose and its derivatives, mannan and its derivatives, galactose and its derivatives, xylose and its derivatives, ribose and its derivatives, A group consisting of fucose and its derivatives, lactose and its derivatives, maltose and its derivatives, arabinose and its derivatives, fructose and its derivatives, and sialic acid.
  • each of the ligands may be independently selected from the group consisting of D-mannopose, L-mannopose, D-arabinose, D-nitrofuran, L-nitroxylose, D- Glucose, L-glucose, D-galactose, L-galactose, ⁇ -D-furanmannose, ⁇ -D-furanmannose, ⁇ -D-mannopose, ⁇ -D-mannopyrose, ⁇ -D-glucopyranose, ⁇ -D-glucopyranose, ⁇ -D-glucofuranose, ⁇ -D-glucofuranose, ⁇ -D-fructofuranose, ⁇ -D-pyranose, ⁇ -D-pyridyl Galactose, ⁇ -D-galactopyranosyl, ⁇ -D-galactopyranosylose, ⁇ -D-galactofuranosamine, glucosamine, si
  • the pharmaceutically acceptable targeting group in the first siRNA conjugate can be galactose or N-acetylgalactosamine, wherein the galactose or N-acetylgalactosamine molecule can It is one price, two price, three price, and four price.
  • the monovalent, divalent, trivalent, and tetravalent terms described herein refer to the formation of siRNA conjugates of siRNA molecules and conjugated groups containing galactose or N-acetylgalactosamine molecules as targeting groups, respectively.
  • the molar ratio of the siRNA molecule to the galactose or N-acetylgalactosamine molecule in the siRNA conjugate is 1:1, 1:2, 1:3 or 1:4.
  • the pharmaceutically acceptable targeting group is N-acetylgalactosamine.
  • the siRNA of the present disclosure when the siRNA of the present disclosure is conjugated to a conjugating group comprising N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent.
  • the siRNA of the present disclosure is conjugated to a conjugating group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.
  • the conjugated molecule can be linked to the siRNA molecule via a suitable linker, and one skilled in the art can select a suitable linker depending on the particular type of targeting group.
  • suitable linker one skilled in the art can select a suitable linker depending on the particular type of targeting group.
  • the conjugated groups, the linkers, the types of targeting groups, and the manner of attachment to siRNAs can be found in the disclosure of WO 2015006740 A2, the entire contents of which is incorporated herein by reference.
  • a suitable linker can be a structure as shown in formula (301):
  • k is an integer from 1 to 3;
  • L A is a chain moiety comprising an amide bond having a structure represented by formula (302), each of said L A having an ether bond at its two ends with one of said targeting group and said L C moiety, respectively connection:
  • L B is a chain moiety comprising an N-acylpyrrolidine having a structure represented by the formula (303), the chain moiety having a carbonyl group at one end thereof and being bonded to the L C moiety via an amide bond at the other end Has an oxy group and is linked to the siRNA via a phosphate linkage:
  • L C is a 2-4 valent linking group based on hydroxymethylaminomethane, dimethylolaminomethane or trishydroxymethylaminomethane, and the L C is via an oxygen atom to each of the L A moieties via an ether linkage. It is linked and is linked to the L B moiety via an amide bond via a nitrogen atom.
  • L C is a trimethylolaminomethane-based tetravalent linking group, which is linked by -(L A ) 3 trishydroxymethylaminomethane-L B - as a linker
  • the first siRNA conjugate formed by the N-acetylgalactosamine molecule and the siRNA molecule has the structure shown in the following formula (304):
  • the double helix structure represents siRNA.
  • the conjugation site of the siRNA to the conjugated molecule can be at the 3' or 5' end of the siRNA sense strand, also at the 5' end of the antisense strand, and also within the internal sequence of the siRNA.
  • the 3' terminus of the sense strand of the siRNA of the present disclosure is covalently conjugated to three N-acetylgalactosamine (GalNAc) molecules via a linker-(LA)3 trishydroxymethylaminomethane-L B -
  • the first siRNA conjugate having a molar ratio of siRNA molecule to GalNAc molecule of 1:3 was obtained, which may also be referred to as (GalNAc)3-1-siRNA hereinafter, and its structure is as shown in the following formula (305):
  • double helix structure represents the siRNA and the linker is ligated to the 3' end of the sense strand of the siRNA.
  • a suitable linker can be a structure as shown in formula (306):
  • l is an integer from 0-3;
  • * indicates a site on the linker that is attached to the targeting group via an ether linkage
  • # indicates a site on the linker that is linked to the siRNA via a phosphate bond.
  • the first siRNA conjugate has a structure as shown in formula (307):
  • double helix structure represents the siRNA and the linker is ligated to the 3' end of the sense strand of the siRNA.
  • WO 2015006740 A2 describes in detail the preparation of various conjugates.
  • the first siRNA conjugate of the present disclosure is obtained by means well known to those skilled in the art.
  • the preparation method of the structure represented by the formula (305) is described in WO2014025805A1
  • the preparation method of the structure represented by the formula (307) is described by Rajeev et al. in ChemBioChem 2015, 16, 903-908.
  • the disclosure provides a second siRNA conjugate having the structure shown in Formula (308):
  • N1 is an integer selected from 1 to 3
  • n3 is an integer selected from 0 to 4;
  • M1, m2 and m3 are independently an integer selected from 2-10;
  • R 10 , R 11 , R 12 , R 13 , R 14 and R 15 are each independently H or selected from the group consisting of C 1 -C 10 alkyl, C 1 -C 10 haloalkane a group and a C 1 -C 10 alkoxy group;
  • R 3 is a group of the structure represented by the formula A59:
  • E 1 is OH, SH or BH 2 , and Nu is an siRNA disclosed;
  • L 1 may be selected from the group consisting of A1-A26 groups or any combination thereof, wherein the structures and definitions of A1-A26 are as follows:
  • j1 is an integer from 1 to 20;
  • j2 is an integer from 1 to 20;
  • R' is a C 1 -C 10 alkyl group
  • Ra is selected from one of the groups of the formula A27-A45:
  • Rb is a C 1 -C 10 alkyl group; Represents a site where a group is attached to the rest of the molecule.
  • L 1 is defined as a linear alkyl group, but it may not be a linear group or a different name, such as an amine or alkenyl group replaced the above and / or substitutions generated.
  • the length of L 1 is the number of atoms in the chain connecting the two attachment points.
  • a ring for example, a heterocyclylene group or a heteroarylene group obtained by substituting a carbon atom of the linear alkylene group is counted as one atom.
  • each M 1 represents a targeting group whose definition and selectable range are the same as those described above.
  • each M 1 is independently selected having one affinity ligand for the asialoglycoprotein receptor on the cell surface of mammalian liver.
  • n1 may be an integer from 1 to 3
  • n3 may be an integer from 0 to 4, Ensuring that the number of M 1 targeting groups in the conjugate is at least 2; in some embodiments, n1+n3 ⁇ 2 such that the number of M 1 targeting groups is at least 3, such that M 1
  • the targeting group binds more readily to the hepatic surface asialoglycoprotein receptor, thereby facilitating entry of the conjugate into the cell by endocytosis.
  • n1 is an integer of 1-2
  • n3 is an integer of 0-1
  • n1+n3 2-3.
  • the spatial position between the plurality of M 1 targeting groups can be adapted to the M 1 targeting group and the liver surface desialic acid
  • m1, m2 and m3 are each independently an integer from 2 to 5
  • R 10 , R 11 , R 12 , R 13 , R 14 and R 15 are each independently H, C 1 -C 10 alkyl, C 1 -C 10 haloalkyl, and C 1 when one kind -C 10 alkoxy group is, not change the nature of the conjugates disclosed herein are object of the present disclosure may be implemented.
  • R 10 , R 11 , R 12 , R 13 , R 14 , and R 15 are each independently selected from the group consisting of H, methyl, and ethyl.
  • R 10 , R 11 , R 12 , R 13 , R 14 and R 15 are both H.
  • R 3 is a group of the structure represented by formula A59, wherein E 1 is OH, SH or BH 2 , based on consideration of ease of preparation of the raw materials, in some embodiments In the case, E 1 is OH or SH.
  • R 2 is selected to achieve the A59 connection N on the backbone nitrogen.
  • the "nitrogen-containing skeleton” means a chain structure in which a carbon atom to which R 10 , R 11 , R 12 , R 13 , R 14 and R 15 are bonded to each other is bonded to N.
  • R 2 can be any linking group capable of attaching the A59 group to the N on the nitrogen-containing backbone in an appropriate manner.
  • the R 2 group needs to contain both a linkage to the N on the nitrogen-containing backbone and to the R 3 The P-phase is connected to the attachment site.
  • R 2 can be B5, B6, B5' or B6':
  • q 2 may range from an integer of 1-10, and in some embodiments, q 2 is an integer from 1 to 5.
  • L 1 is selected from the group consisting of one or more of the groups of formulas A1 to A26; in some embodiments, L 1 is selected from the group consisting of A1, A4, A5, A6, A8, A10, A11 and A13 connection of one or more thereof; in some embodiments, L 1 is selected from A1, A4, A8, A10 and A11 are connected to at least two compositions; in some embodiments, L 1 is selected from A combination of at least two of A1, A8, and A10.
  • L 1 can be from 3 to 25 atoms, from 3 to 20 atoms, from 4 to 15 atoms, or from 5 to 12 atoms. In some embodiments, the length of L 1 is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60 atom.
  • j1 is an integer from 2 to 10, and in some embodiments, j1 is an integer from 3-5.
  • J2 is an integer from 2 to 10, and in some embodiments, j2 is an integer from 3-5.
  • R' is a C 1 -C 4 alkyl group, and in some embodiments, R' is one of a methyl group, an ethyl group, and an isopropyl group.
  • Ra is one of A27, A28, A29, A30 and A31, and in some embodiments, Ra is A27 or A28.
  • Rb is a C 1 -C 5 alkyl group, and in some embodiments, Rb is one of a methyl group, an ethyl group, an isopropyl group, and a butyl group.
  • the respective formula A1-A26 of j1, j2, R ', Ra , Rb are selected in order to achieve targeting group M 1 on the N-linked nitrogen-containing backbone with targeting and M 1 The spatial position between the groups is more suitable for binding of the M 1 targeting group to the hepatic surface asialoglycoprotein receptor.
  • the second siRNA conjugate of the present disclosure has the formula (403), (404), (405), (406), (407), (408), (409), (410), ( Structures shown in 411), (412), (413), (414), (415), (416), (417), (418), (419), (420), (421), or (422) :
  • the P in the formula A59 can be linked to any possible position in the siRNA sequence represented by Nu, for example, P in the formula A59 can be linked to any one of the siRNA sense strands or the antisense strand of the siRNA represented by Nu Acid; in some embodiments, P in formula A59 is attached to any one of the nucleotides of the sense strand of siRNA represented by Nu. In some embodiments, P in Formula A59 is joined to the end of the sense or antisense strand of siRNA represented by Nu; in some embodiments, P in Formula A59 is joined to the end of the sense strand of siRNA represented by Nu.
  • the terminus of the siRNA represented by Nu refers to the siRNA sense strand represented by Nu or the first 4 nucleotides from the one end of the antisense strand.
  • P in Formula A59 is joined to the terminus of the sense or antisense strand of siRNA represented by Nu; in some embodiments, P in Formula A59 is joined to the 3' end of the sense strand of siRNA represented by Nu.
  • P in formula A59 can be attached to any possible position on the nucleotide in the siRNA represented by Nu, for example, the 5' position of the nucleotide, the 2' position of the nucleotide, the 3' position of the nucleotide or the nucleus On the base of the nucleotide.
  • P in formula A59 can be linked to the 2', 3' or 5' position of the nucleotide in the siRNA represented by Nu by forming a phosphodiester bond.
  • the P in the formula A59 is linked to an oxygen atom formed after dehydrogenation of the 3′ hydroxyl group of the 3′ terminal nucleotide of the siRNA sense strand represented by Nu, or the P represented by the substitution of Nu in the formula A59.
  • Hydrogen in the 2'-hydroxyl group of one nucleotide in the sense strand is linked to the nucleotide, or P in the formula A59 is substituted by Nu to represent the hydrogen in the 5' hydroxyl group of the 5' terminal nucleotide of the siRNA sense strand. Nucleotide linkage.
  • each adjacent nucleotide is linked by a phosphodiester bond or a phosphorothioate diester bond, and a non-bridging in a phosphodiester bond or a phosphorothioate diester bond
  • the oxygen atom or the sulfur atom has a negative charge, which may exist in the form of a hydroxyl group or a sulfhydryl group, and the hydrogen ion in the hydroxyl group or the thiol group may also be partially or completely substituted by a cation.
  • the cation may be any cation such as one of a metal cation, an ammonium ion NH 4 + , and an organic ammonium cation.
  • the cation is selected from one or more of an alkali metal ion, an ammonium cation formed by a tertiary amine, and a quaternary ammonium cation.
  • the alkali metal ion may be K + and/or Na +
  • the cation formed by the tertiary amine may be an ammonium ion formed by triethylamine and/or an ammonium ion formed by N,N-diisopropylethylamine.
  • the siRNA or the first or second siRNA conjugate of the present disclosure may exist at least partially in the form of a salt.
  • the non-bridging oxygen or sulfur atom in the phosphodiester bond or the phosphorothioate diester bond is at least partially bound to the sodium ion, the siRNA or the first or second siRNA conjugate of the present disclosure. It exists in the form of a sodium salt or a partial sodium salt.
  • modified nucleotide groups can be introduced into the siRNAs described in the present disclosure by using nucleoside monomers having corresponding modifications. Methods of preparing nucleoside monomers having corresponding modifications and methods of introducing modified nucleotide groups into siRNA are also well known to those skilled in the art. All modified nucleoside monomers are either commercially available or can be prepared by known methods.
  • the second siRNA conjugate of the present disclosure can be prepared using any reasonable synthetic route.
  • the second siRNA conjugate can be prepared by a method comprising, under the conditions of solid phase synthesis of phosphoramidite, according to the nucleotide type and order of the sense strand and the antisense strand of the siRNA, respectively.
  • the nucleoside monomers are sequentially linked in the direction of 3' to 5', and the linkage of each nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization; the sense strand and the antisense strand of the siRNA are isolated. , annealing, wherein the siRNA represented by Nu is the siRNA of the present disclosure described above;
  • the method further comprises contacting the compound represented by the formula (321) with a nucleoside monomer or a nucleotide sequence attached to the solid phase carrier in the presence of a coupling reaction condition and a coupling reagent, such that the formula (321) The compound shown is linked to the nucleotide sequence by a coupling reaction.
  • the compound represented by the formula (321) is also referred to as a conjugation molecule.
  • R 4 is a moiety capable of binding to the siRNA represented by Nu. In some embodiments, R 4 is a moiety capable of binding to a siRNA represented by Nu by a covalent bond. In some embodiments, R 4 is a moiety capable of being reacted to conjugate to any functional group of the siRNA represented by Nu by a phosphodiester bond;
  • Each S 1 is independently a group formed by substituting all of the active hydroxyl groups in M 1 with a YCOO- group, wherein each Y is independently selected from the group consisting of methyl, trifluoromethyl, difluoromethyl, and fluoro One of a group, a trichloromethyl group, a dichloromethyl group, a monochloromethyl group, an ethyl group, a n-propyl group, an isopropyl group, a phenyl group, a halogenated phenyl group, and an alkylphenyl group;
  • n1, n3, m1, m2, m3, R 10, R 11, R 12, R 13, R 14, R 15, L 1, M 1 are each as defined and selectable range as described above.
  • R 4 is selected to N in order to achieve a nitrogen-containing backbone connection, and to provide suitable reaction sites for the synthesis of siRNA conjugate of formula (308).
  • R 4 includes an R 2 linking group or a protected R 2 linking group, and a functional group capable of forming a structure represented by A59 with a siRNA by a reaction.
  • R 4 comprises a first functional group comprising a phosphite capable of forming a phosphite with a group represented by Nu or an nucleoside monomer, and a second functional group capable of reacting with a hydroxyl group or an amino group to form a covalent bond or containing A solid phase carrier joined by the covalent bond.
  • the first functional group is a phosphoramidite, a hydroxyl group, or a protected hydroxyl group.
  • the second functional group is a phosphoramidite, a carboxylic acid, or a carboxylate.
  • the second functional group is a solid support that is linked to other portions of the molecule via a covalent bond, the covalent bond being formed from a hydroxyl group or an amino group.
  • the solid support is linked via a phosphate bond, a carboxylate bond, or an amide bond.
  • the solid support is a resin.
  • the first functional group contains a hydroxyl group, -OR k or a group represented by formula (C3); and the second functional group contains formulas (C1), (C2), (C3), (C1' ) or the structure shown in (C3'):
  • q 1 is an integer of 1-4
  • X is O or NH
  • M + is a cation
  • R k is a hydroxy protecting group
  • SPS represents a solid phase carrier, indicating a site at which a group is covalently linked.
  • the first functional group contains a phosphoramidite functional group, as shown in formula (C3), and the phosphoramidite group may be a hydroxyl group at any position on the nucleotide, such as a 2' hydroxyl group or The hydroxy group undergoes a coupling reaction to form a phosphite, and is oxidized or vulcanized to form a phosphodiester bond or a phosphorothioate bond represented by the formula A59, and the conjugated molecule is conjugated to the siRNA.
  • the compound of the formula (321) can be conjugated to the nucleotide without affecting the acquisition of the siRNA conjugate represented by the formula (308).
  • the compound of the formula (321) is reacted with a hydroxyl group at the terminal nucleotide in the nucleotide sequence, and A phosphodiester bond or a phosphorothioate linkage is formed during oxidation or vulcanization, and the compound of formula (321) is conjugated to the siRNA.
  • the first functional group contains a protected hydroxyl group.
  • the second functional group comprises a group reactive with a solid support, the reaction providing a conjugation molecule comprising a solid support.
  • the second functional group contains a carboxyl group, a carboxylate or a phosphoramidite, as represented by formula (C1), (C2) or (C3), when the second functional group comprises a carboxyl group or a carboxylate.
  • the compound of the formula (321) is subjected to an esterification reaction or amidation reaction with a solid phase carrier such as a hydroxyl group or an amino group on the resin to form a conjugation molecule comprising a solid phase carrier which is bonded via a carboxylate bond or an amide bond.
  • a solid phase carrier such as a hydroxyl group or an amino group on the resin
  • the compound of the formula (321) is coupled with a general-purpose solid phase carrier, for example, a hydroxyl group on a resin, and is oxidized to form a phosphodiester-linked solid phase carrier. Conjugated molecules.
  • the nucleoside monomer is sequentially linked according to the phosphoramidite solid phase synthesis method, and the sense strand or the antisense strand of the siRNA to which the conjugated group is attached is obtained starting from the product after the above-mentioned solid phase carrier is attached.
  • the first functional group undergoes deprotection and subsequent coupling with a phosphoramidite group on the nucleoside monomer under coupling reaction conditions.
  • the first functional group contains a hydroxyl group or a protected hydroxyl group
  • the second functional group contains a solid phase carrier linked by a carboxylate bond or a solid phase carrier linked by an amide bond, or a phosphate bond.
  • the attached solid support is as shown in formula (C1') or (C3').
  • the nucleoside monomer is sequentially linked according to the phosphoramidite solid phase synthesis method to obtain a sense strand or an antisense strand of the siRNA to which the conjugated group is attached.
  • the carboxylate can be represented as -COO-M +, wherein, M + is a cation, for example selected from metal cation, ammonium cation NH +, organic ammonium cations of one 4.
  • M + is a cation, for example selected from metal cation, ammonium cation NH +, organic ammonium cations of one 4.
  • the metal ion is selected from one of the alkali metal ions, such as K + or Na + .
  • the organic ammonium ion is an ammonium cation or a quaternary ammonium cation formed by a tertiary amine, such as an ammonium ion formed by triethylamine or N, N-di Ammonium ion formed by isopropylethylamine.
  • the carboxylate is a triethylamine carboxylate or an N,N-diisopropylethylamine carboxylate.
  • R 4 comprises a structure of formula (B9), (B10), (B9'), (B10'), (B11), (B12), (B11'), or (B12'):
  • q 1 is an integer from 1 to 4
  • q 2 is an integer from 1 to 10
  • X is O or NH
  • M + is a cation
  • R k is a hydroxy protecting group
  • SPS is a solid phase carrier, indicating a group covalent The site to which the bond is connected.
  • q 1 is 1 or 2.
  • q 2 is an integer from 1 to 5.
  • R 4 contains a structure represented by formula (B9) or (B10).
  • R4 contains a structure represented by formula (B11) or (B12).
  • R k is Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-bismethoxytrityl), TMTr (4 One or more of 4', 4'-trimethoxybenzyl.
  • R k may be DMTr, i.e. methoxytrityl, 4,4'-bis (4,4'-dimethoxytrityl).
  • L 1 The definition of L 1 is as described above.
  • L 1 M 1 are used to connect the targeting group to the N atom a nitrogen-containing backbone to provide a liver function as a second targeting siRNA conjugate.
  • L 1 comprises any one of A1-A26 or a combination thereof.
  • the conjugated molecule can be linked by the above first functional group and optionally the second functional group as compared to the phosphoramidite solid phase synthesis method well known in the art.
  • a second siRNA conjugate to any possible position of the nucleotide sequence for example, the conjugate molecule is attached to the end of the nucleotide sequence and the conjugate molecule is ligated to the end of the nucleotide sequence.
  • each S 1 is independently M 1 . In some embodiments, each S 1 is independently a group formed by the protection of at least one reactive hydroxyl group of M 1 by a hydroxy protecting group. In some embodiments, each S 1 is independently a group formed by any of the active hydroxyl groups present in M 1 being protected by a hydroxy protecting group. In some embodiments, any known to those skilled hydroxy protecting groups may be used to protect the reactive hydroxyl groups of M 1.
  • the protected hydroxy group can be represented by the formula YCOO-, wherein each Y is independently selected from the group consisting of a C 1 -C 10 alkyl group and a C 6 -C 10 aryl group, the C The 1- C 10 alkyl group and the C 6 -C 10 aryl group are optionally substituted by one or more substituents selected from the group consisting of halogen and C 1 -C 6 alkyl groups.
  • each Y is independently selected from the group consisting of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl , monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl, and C 1 -C 6 alkylphenyl.
  • each S 1 is independently selected from the group consisting of Formulas A46-A54:
  • S 1 is Formula A49 or A50.
  • each Y is independently selected from the group consisting of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, and One of a propyl group, an isopropyl group, a phenyl group, a halogenated phenyl group, and an alkylphenyl group; for the purpose of simplifying the conjugation molecule of the present disclosure, in some embodiments, Y is a methyl group.
  • the preparation method of the second siRNA conjugate further comprises the steps of synthesizing another strand of the siRNA (for example, when the above step synthesizes the siRNA sense strand to which the conjugated group is attached, The phase synthesis method synthesizes the antisense strand of siRNA, and vice versa), separates the sense strand and the antisense strand, and anneals.
  • the solid phase carrier linked to the nucleotide sequence and/or the conjugated molecule is cleaved while the necessary protecting group is removed (in this case, each S in the compound of the formula (321) 1 group is converted to the corresponding M 1 targeting group), the siRNA sense strand (or antisense strand) linked to the conjugated group and the corresponding antisense strand (or sense strand), sense strand and antisense strand are obtained.
  • Annealing forms a double-stranded RNA structure to obtain a second siRNA conjugate.
  • the method of preparing the second siRNA conjugate comprises the steps of: reacting a compound of formula (321) with a sense strand or an antisense strand in the presence of coupling reaction conditions and a coupling reagent Contact with the first nucleoside monomer at the 3' end, such that the compound of formula (321) is attached to the first nucleotide of the sequence, under the conditions of solid phase synthesis of phosphoramidite, according to the desired sense strand or
  • the nucleotide sequence and sequence of the antisense strand are sequentially linked in the direction of 3' to 5' to synthesize the sense strand or the antisense strand of the siRNA; wherein the compound of (321) contains the first functional group in R4 and a second functional group, wherein the first functional group contains a protected hydroxyl group, and the second functional group has a compound represented by the formula (321) having a structure represented by the formula (C1') or (C3'), and is bonded to the first nucleoside
  • the compound of the formula (321) is deprotected; the linkage of each nucleoside monomer includes a four-step reaction of deprotection, coupling, capping, oxidation or vulcanization; and the sense strand or the antisense strand of the nucleic acid to which the conjugated molecule is attached is obtained.
  • nucleoside monomers are sequentially linked in the direction of 3' to 5' to synthesize the antisense strand or sense strand of the nucleic acid; the linkage of each nucleoside monomer includes deprotection, coupling, capping, oxidation or The four-step reaction is sulfided; the protecting group is removed and cleaved with the solid phase carrier, and the sense strand and the antisense strand of the nucleic acid are separated and purified, and annealed.
  • the method of making a second siRNA conjugate comprises the steps of: nucleating the 3' to 5' direction according to the nucleotide species and sequence of the sense strand or the antisense strand of the double stranded siRNA
  • the glycosides are sequentially linked to synthesize the sense strand and the antisense strand.
  • the linkage of each nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization to obtain a sense strand and linkage attached to the solid support.
  • An antisense strand on a solid support; in the presence of coupling reaction conditions and a coupling reagent, the compound of formula (321) is coupled to a sense strand attached to a solid support or to a solid support.
  • the compound of the formula (321) is attached to the sense strand or the antisense strand, wherein the compound of the formula (321) is a compound of the formula (321) wherein the first functional group is contained in R4 and the first functional group is a phosphoramidite group;
  • the protecting group is removed and cleaved with the solid phase vector, and purified, respectively, and the sense strand or the antisense strand of the siRNA represented by Nu is annealed, wherein a conjugate group is attached to the sense strand or the antisense strand of the siRNA.
  • the P in Formula A59 is linked to the 3' end of the sense strand in the siRNA, and the method of preparing the second siRNA conjugate comprises:
  • step (1) the method of removing the formula (321) R k protecting group in the compound comprising the deprotection conditions, the formula (321) contacting the compound with a deprotecting agent.
  • Deprotection conditions include a temperature of 0-50 ° C, in some embodiments 15-35 ° C, a reaction time of 30-300 seconds, in some embodiments 50-150 seconds, and a deprotection reagent may be selected from trifluoroacetic acid One or more of trichloroacetic acid, dichloroacetic acid, monochloroacetic acid, in some embodiments dichloroacetic acid.
  • the molar ratio of the deprotecting agent to the compound of formula (321) is from 10:1 to 1000:1, and in some embodiments from 50:1 to 500:1.
  • the coupling reaction conditions and the coupling reagent may use any conditions and reagents suitable for the above coupling reaction. In some embodiments, the same conditions and reagents as for the coupling reaction in the solid phase synthesis method employed can be used.
  • the conditions of the coupling reaction comprise a reaction temperature of from 0 to 50 ° C, and in some embodiments from 15 to 35 ° C.
  • the molar ratio of the compound of the formula (321) to the nucleoside monomer is from 1:1 to 1:50, in some embodiments from 1:2 to 1:5; the molar ratio of the compound of the formula (321) to the coupling reagent may be one. 1-1:50, in some embodiments 1:3-1:10, reaction time 200-3000 seconds, in some embodiments 500-1500 seconds.
  • the coupling reagent is selected from one or more of 1H-tetrazole, 5-ethylthio 1H-tetrazole, 5-benzylthio 1H-tetrazole, and in some embodiments 5-ethion Base 1H-tetrazole.
  • the coupling reaction can be carried out in an organic solvent selected from one or more of anhydrous acetonitrile, anhydrous DMF, anhydrous dichloromethane, and in some embodiments anhydrous acetonitrile.
  • the organic solvent is used in an amount of from 3 to 50 L/mol, and in some embodiments from 5 to 20 L/mol, relative to the compound of the formula (321).
  • step (2) the method of solid phase synthesis of phosphoramidite nucleic acid is used to synthesize the nucleoside monomer which is ligated to the solid phase carrier by the conjugation molecule, and is synthesized in the direction of 3′-5′.
  • the conjugate group is attached to the 3' end of the resulting sense strand.
  • conditions for solid phase synthesis described in steps (2) and (3) include nucleoside monomer deprotection conditions, type and amount of deprotection reagent, coupling reaction conditions, type and amount of coupling reagent, cap reaction
  • the conditions, the type and amount of the capping reagent, the oxidation reaction conditions, the type and amount of the oxidizing reagent, the vulcanization reaction conditions, the vulcanization reagent and the amount are various reagents, amounts and conditions conventionally used in the art.
  • the solid phase synthesis described in steps (2) and (3) can use the following conditions:
  • Nucleoside monomer deprotection conditions include a temperature of 0-50 ° C, in some embodiments 15-35 ° C, a reaction time of 30-300 seconds, in some embodiments 50-150 seconds, a deprotection reagent can be selected One or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, monochloroacetic acid, in some embodiments, dichloroacetic acid.
  • the molar ratio of the deprotecting agent to the 4,4'-dimethoxytrityl protecting group on the solid support is from 2:1 to 100:1, and in some embodiments from 3:1 to 50:1.
  • the coupling reaction conditions include a temperature of 0-50 ° C, in some embodiments 15-35 ° C, a molar ratio of the nucleic acid sequence attached to the nucleoside monomer on the solid support is 1:1 to 1:50, in some In the embodiment: 1:5-1:15; the molar ratio of the nucleic acid sequence and the coupling reagent attached to the solid phase support is from 1:1 to 1:100, and in some embodiments from 1:50 to 1:80,
  • the reaction time and the choice of coupling reagent are the same as described above.
  • the cap reaction conditions include a temperature of 0-50 ° C, in some embodiments 15-35 ° C, a reaction time of 5-500 seconds, in some embodiments 10-100 seconds, and the capping agent is selected in the same manner as previously described.
  • the molar ratio of the total amount of capping reagent to the nucleic acid sequence attached to the solid support is from 1:100 to 100:1, and in some embodiments from 1:10 to 10:1.
  • the capping reagent uses an equimolar amount of acetic anhydride and N-methylimidazole
  • the molar ratio of the nucleic acid sequence attached to the acetic anhydride, the N-methylimidazole, and the solid phase carrier is 1:1:10-10:10.
  • the oxidation reaction conditions include a temperature of 0-50 ° C, in some embodiments 15-35 ° C, a reaction time of 1-100 seconds, in some embodiments 5-50 seconds, and an oxidizing agent, in some embodiments, iodine. (In some embodiments, provided in the form of iodine water).
  • the molar ratio of the oxidizing reagent to the nucleic acid sequence attached to the solid support in the coupling step may range from 1:1 to 100:1, and in some embodiments from 5:1 to 50:1.
  • the vulcanization reaction conditions include a temperature of 0-50 ° C, in some embodiments 15-35 ° C, a reaction time of 50-2000 seconds, in some embodiments 100-1000 seconds, and a vulcanization reagent in some embodiments is hydrogenation. Xanthogen.
  • the molar ratio of the sulfurizing reagent to the nucleic acid sequence attached to the solid support in the coupling step may range from 10:1 to 1000:1, and in some embodiments from 10:1 to 500:1.
  • the method further comprises isolating the sense and antisense strands of the siRNA.
  • Methods for isolation are well known to those skilled in the art and generally involve cleavage of the resulting nucleotide sequence from a solid support, removal of protecting groups on bases, phosphates and ligands, purification and desalting. .
  • the cleavage of the synthesized nucleotide sequence from the solid phase support and removal of the protecting group on the base, on the phosphate group and on the ligand can be carried out according to conventional cleavage and deprotection methods in siRNA synthesis.
  • the obtained nucleotide sequence linked to the solid phase carrier is contacted with concentrated aqueous ammonia; during deprotection, the protecting group YCOO- of the A46-A54 group is converted into a hydroxyl group, and the S 1 group is converted into a corresponding one.
  • the M 1 group produces a conjugate of the formula (308).
  • the concentrated ammonia water may be 25-30% by weight of ammonia water, and the amount of concentrated ammonia water may be 0.2 ml/ ⁇ mol-0.8 ml/ ⁇ mol compared with the target siRNA sequence.
  • the method further comprises contacting the nucleotide sequence of the solid phase removal medium with triethylamine trihydrofluoride to remove the 2'-TBDMS protection.
  • the obtained target siRNA sequence has a corresponding nucleoside of a free 2'-hydroxy group.
  • the amount of the pure triethylamine trihydrofluorate is 0.4 ml/ ⁇ mol to 1.0 ml/ ⁇ mol as compared with the target siRNA sequence.
  • the siRNA conjugate of the formula (308) can be obtained.
  • the preparative ion chromatography purification column can be used to purify the nucleic acid by gradient elution with NaBr or NaCl; after product collection and combination, the reverse phase chromatography purification column can be used for desalting.
  • a non-bridging oxygen atom or a sulfur atom in a phosphodiester bond or a phosphorothioate diester bond between nucleotides substantially binds to a sodium ion
  • a second siRNA is conjugated.
  • the substance is basically present in the form of a sodium salt.
  • Other forms of the second siRNA conjugate can be obtained by replacing the sodium ion with a hydrogen ion and/or other cation using well known ion exchange methods. The cation is as previously described.
  • the purity and molecular weight of the nucleic acid sequence can be detected at any time during the synthesis to better control the quality of the synthesis. Such detection methods are well known to those skilled in the art. For example, nucleic acid purity can be detected by ion exchange chromatography and molecular weight can be determined by LC-MS chromatography.
  • the synthesized sense strand (S chain) and the antisense strand (AS chain) can be simply mixed in an equimolar ratio in water for injection to 70-95 ° C, followed by cooling at room temperature to form a double bond through hydrogen bonding. Chain structure. This gives a second siRNA conjugate.
  • the synthesized second siRNA conjugate can also be characterized by molecular weight detection or the like using a method such as LC/MS chromatography to determine the synthesis.
  • the siRNA conjugate is a second siRNA conjugate designed for the target, and the sequence of the synthesized siRNA matches the sequence of the siRNA to be synthesized, for example, one of the sequences listed in Table 2.
  • the compound of the formula (321) can be obtained by a production method comprising: a compound represented by the formula (313) and a cyclic group in an organic solvent under an esterification reaction condition and in the presence of a base and an ester-forming catalyst.
  • the acid anhydride is contacted, ion exchanged, and the compound represented by the formula (321) is obtained:
  • n1, n3, m1, m2, m3, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , L 1 , S 1 are as defined above;
  • R 6 is a group which provides R 4 in the formula (321). In some embodiments, R 6 has the structure shown by formula (A61):
  • R i is possible to achieve the N-linked nitrogen-containing backbone, and R k O connection has a connection to any free hydroxyl group, R k is a hydroxy protecting group.
  • R 4 contains a first functional group and a second functional group as a hydroxy protecting group, and the second functional group contains a compound of the formula (321) having a structure represented by the formula (C1) or (C2).
  • the esterification reaction conditions include a reaction temperature of 0-100 ° C, and a reaction time of 8-48 hours. In some embodiments, the esterification reaction conditions are a reaction temperature of 10-40 ° C, and a reaction time of 20-30. hour.
  • the organic solvent comprises an epoxy solvent, an ether solvent, a halogenated alkane solvent, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine.
  • the epoxy solvent is dioxane and/or tetrahydrofuran
  • the ether solvent is diethyl ether and/or methyl tert-butyl ether
  • the halogenated alkane solvent is dichloromethane, three One or more of methyl chloride and 1,2-dichloroethane.
  • the organic solvent is dichloromethane.
  • the organic solvent is used in an amount of from 3 to 50 L/mol, and in some embodiments from 5 to 20 L/mol, relative to the compound of the formula (313).
  • the cyclic anhydride is one of succinic anhydride, glutaric anhydride, adipic anhydride, or pimelic anhydride, and in some embodiments is succinic anhydride.
  • the molar ratio of the cyclic anhydride to the compound of formula (313) is from 1:1 to 10:1, and in some embodiments from 2:1 to 5:1.
  • the ester-forming catalyst may be any catalyst that catalyzes the esterification reaction, for example, the catalyst may be 4-dimethylaminopyridine.
  • the molar ratio of the catalyst to the compound of formula (313) is from 1:1 to 10:1, and in some embodiments from 2:1 to 5:1.
  • the base can be any inorganic base, organic base, or a combination thereof.
  • the base may be, for example, a tertiary amine organic base.
  • the tertiary amine organic base is triethylamine or N,N-diisopropylethylamine.
  • the molar ratio of the tertiary amine organic base to the compound of formula (313) is from 1:1 to 20:1, and in some embodiments from 3:1 to 10:1.
  • the ion exchange is the conversion of a compound of formula (321) to the desired carboxylic acid or carboxylate form, and methods of ion exchange are known to those skilled in the art, and suitable ion exchange solutions and exchange conditions can be used to obtain the foregoing.
  • the cation is a M + conjugation molecule and will not be described in detail herein.
  • the ion exchange reaction is carried out using a solution of triethylamine phosphate, the concentration of the triethylamine phosphate solution being from 0.2 to 0.8 M, and in some embodiments from 0.4 to 0.6 M, relative to the formula (313)
  • a compound, the triethylamine phosphate solution is used in an amount of from 3 to 6 L/mol, and in some embodiments from 4 to 5 L/mol.
  • the compound of formula (321) can be isolated from the reaction mixture using any suitable separation method.
  • the solvent can be removed directly to provide a crude product of the compound of formula (321) which can be used directly in the subsequent reaction.
  • the method for preparing the compound of the formula (321) further comprises the product obtained by the above ion exchange reaction in the presence of a condensing agent and a tertiary amine organic base in an organic solvent under condensation reaction conditions. Further contacting with a solid phase carrier containing an amino group or a hydroxyl group.
  • a compound of the formula (321) wherein R4 contains a first functional group and a second functional group, a first functional group contains a hydroxy protecting group, and a second functional group contains a structure represented by the formula (C1').
  • the solid phase support is one of the vectors used in solid phase synthesis of siRNA, some of which are well known to those skilled in the art.
  • the solid support may be selected from a solid support containing reactive hydroxyl or amino functional groups, and in some embodiments, the solid support is an amino resin or a hydroxy resin.
  • the amino or hydroxy resin has, in some embodiments, the following parameters: particle size 100-400 mesh, surface amino or hydroxyl loading of 0.2-0.5 mmol/g.
  • the ratio of the compound represented by the formula (321) to the solid phase carrier is from 10 to 400 ⁇ mol of the compound per gram of the solid phase carrier ( ⁇ mol/g). In some embodiments, the ratio of the compound represented by the formula (321) to the solid phase carrier is from 50 to 200 ⁇ mol/g.
  • the organic solvent may be any suitable solvent or mixed solvent known to those skilled in the art.
  • the organic solvent is acetonitrile, an epoxy solvent, an ether solvent, a halogenated alkane solvent, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropyl One or more of ethylamine.
  • the epoxy solvent is dioxane and/or tetrahydrofuran
  • the ether solvent is diethyl ether and/or methyl tert-butyl ether
  • the halogenated alkane solvent is dichloromethane, three One or more of methyl chloride and 1,2-dichloroethane.
  • the organic solvent is acetonitrile.
  • the organic solvent is used in an amount of from 20 to 200 L/mol, and in some embodiments from 50 to 100 L/mol, relative to the compound of the formula (321).
  • the condensing agent may be benzotriazol-1-yl-oxytripyrrolidinyl hexafluorophosphate, 3-diethoxyphosphoryl-1,2,3-oxazolyl 4(3H)-one and / or O-benzotriazole-tetramethylurea hexafluorophosphate, in some embodiments O-benzotriazole-tetramethylurea hexafluorophosphate.
  • the molar ratio of the condensing agent to the compound of formula (321) is from 1:1 to 20:1, and in some embodiments from 1:1 to 5:1.
  • the tertiary amine organic base is triethylamine and/or N,N-diisopropylethylamine, in some embodiments N,N-diisopropylethylamine;
  • the molar ratio of the tertiary amine organic base to the compound of formula (321) is from 1:1 to 20:1, and in some embodiments from 1:1 to 5:1.
  • the method for preparing the compound of the formula (321) may further comprise contacting the obtained condensation product with a capping reagent and an acylation catalyst in an organic solvent under a cap reaction condition, and separating the compound represented by the formula (321). Compound.
  • the effect of the cap reaction is to remove any reactive functional groups that have not been fully reacted to avoid the formation of unnecessary by-products in subsequent reactions.
  • the conditions for the cap reaction include a reaction temperature of 0-50 ° C, in some embodiments 15-35 ° C, a reaction time of 1-10 h, and in some embodiments 3-6 h.
  • the capping reagent can use a capping reagent used in siRNA solid phase synthesis, and capping reagents used in siRNA solid phase synthesis are well known to those skilled in the art.
  • the cap reagent consists of cap reagent 1 (cap1) and cap reagent 2 (cap2), wherein cap reagent A is N-methylimidazole, in some embodiments N-methylimidazole Provided as a mixed solution of pyridine/acetonitrile wherein the volume ratio of pyridine to acetonitrile is from 1:10 to 1:1, in some embodiments from 1:3 to 1:1, the total volume of pyridine and acetonitrile and N-A The volume of the imidazole is from 1:1 to 10:1, and in some embodiments from 3:1 to 7:1.
  • the capping agent B is acetic anhydride, and in some embodiments is provided as a solution of acetic anhydride in acetonitrile, wherein the volume of acetic anhydride and acetonitrile is from 1:1 to 1:10, and in some embodiments is 1:2- 1:6.
  • the ratio of the volume of the pyridine/acetonitrile mixed solution of the N-methylimidazole to the mass of the compound of the formula (321) is 5 ml/g to 50 ml/g, and in some embodiments, 15 ml/g- 30ml/g.
  • the ratio of the volume of the acetic anhydride in acetonitrile solution to the mass of the compound of formula (321) is from 0.5 ml/g to 10 ml/g, and in some embodiments from 1 ml/g to 5 ml/g.
  • the capping reagent uses an equimolar amount of acetic anhydride and N-methylimidazole.
  • the organic solvent is one of acetonitrile, an epoxy solvent, an ether solvent, a halogenated alkane solvent, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine. Or a variety.
  • the organic solvent is acetonitrile.
  • the organic solvent is used in an amount of 10 to 50 L/mol, and in some embodiments, 5 to 30 L/mol, relative to the compound of the formula (321).
  • the acylation catalyst may be selected from any catalyst useful for ester-forming condensation or amide condensation, such as a basic heterocyclic compound.
  • the acylation catalyst is 4-dimethylaminopyridine.
  • the mass ratio of the catalyst to the compound of formula (321) is from 0.001:1 to 1:1, and in some embodiments from 0.01:1 to 0.1:1.
  • the compound of formula (321) can be isolated from the reaction mixture using any suitable separation method.
  • the compound of formula (321) which is selected from the group consisting of acetonitrile, dichloromethane, can be obtained by extensive washing with an organic solvent and filtration to remove unreacted reactants, excess capping reagents, and other impurities.
  • Methanol in some embodiments acetonitrile.
  • the method for preparing the conjugated molecule of formula (321) comprises reacting the compound of formula (313) with phosphorousous acid in an organic solvent under coupling reaction conditions and in the presence of a coupling reagent.
  • the acyl diamine is contacted to obtain a compound of the formula (321).
  • a compound of the formula (321) wherein R4 contains a first functional group and a second functional group, a first functional group contains a hydroxy protecting group, and a second functional group contains a structure represented by the formula (C3).
  • the coupling reaction conditions include a temperature of 0 to 50 ° C, for example, 15 to 35 ° C, and a molar ratio of the compound of the formula (313) to the phosphorous diamine may be 1:1 to 1:50, for example,
  • the molar ratio of the compound of the formula (313) to the coupling reagent may be from 1:1 to 1:100, for example from 1:50 to 1:80; and the reaction time may be from 200 to 3000 seconds. For example, 500-1500 seconds.
  • the phosphoramidide for example, bis(diisopropylamino)(2-cyanoethoxy)phosphine, which is commercially available or can be obtained by a method known in the art, can be used.
  • the coupling reagent is selected from one or more of 1H-tetrazole, 5-ethylthio 1H-tetrazole, 5-benzylthio 1H-tetrazole, for example, 5-ethylthio 1H-tetra Azole.
  • the coupling reaction can be carried out in an organic solvent selected from one or more of anhydrous acetonitrile, anhydrous DMF, anhydrous dichloromethane, such as anhydrous acetonitrile.
  • the organic solvent is used in an amount of from 3 to 50 L/mol, for example, from 5 to 20 L/mol, relative to the compound of the formula (313).
  • a hydroxyl group in the compound of the formula (313) is reacted with a phosphorous diamine to form a phosphoramidite group.
  • the solvent can be removed directly to provide a crude product of the compound of formula (321), which can be used directly in the subsequent reaction.
  • the method of preparing the compound of the formula (321) further comprises the steps of further separating the isolated product from the hydroxyl group-containing product under coupling reaction conditions, in an organic solvent, and in the presence of a coupling reagent.
  • the solid support is contacted.
  • the compound of the formula (321) is isolated by a cap reaction and an oxidation reaction.
  • a compound of the formula (321) wherein R4 contains a first functional group and a second functional group, a first functional group contains a hydroxy protecting group, and a second functional group has a structure represented by the formula (C3').
  • the solid phase support is a solid phase support known in the art and useful for solid phase synthesis of nucleic acids, for example, may be a commercially available universal solid phase support after deprotection ( HL UnyLinker TM 300Oligonucleotide Synthesis Support, Kinovate Life Sciences Corporation, the structure as shown in Formula B80):
  • the deprotection conditions include a temperature of 0-50 ° C, such as 15-35 ° C; a reaction time of 30-300 seconds, such as 50-150 seconds.
  • the deprotecting agent can be selected from one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, monochloroacetic acid, and in some embodiments, the deprotecting reagent is dichloroacetic acid.
  • the molar ratio of the deprotecting agent to the -DMTr(4,4'-dimethoxytrityl) protecting group on the stationary phase is from 2:1 to 100:1, for example from 3:1 to 50:1.
  • the coupling reaction conditions and the choice of coupling reagent can be as described above.
  • the free hydroxyl group formed in the deprotection reaction reacts with the phosphoramidite group to form a phosphite linkage.
  • the cap reaction conditions comprise a temperature of 0-50 ° C, such as 15-35 ° C, a reaction time of 5-500 seconds, such as 10-100 seconds, and the cap reaction is carried out in the presence of a capping agent.
  • the selection and amount of cap reagent can be as described above.
  • the oxidation reaction conditions include a temperature of 0 to 50 ° C, for example, 15 to 35 ° C, a reaction time of 1 to 100 seconds, for example, 5 to 50 seconds, and an oxidizing agent such as iodine (in some embodiments, iodine) Provided in the form of water).
  • an oxidizing agent such as iodine (in some embodiments, iodine) Provided in the form of water).
  • the molar ratio of oxidizing agent to phosphite groups is from 1:1 to 100:1, such as from 5:1 to 50:1.
  • R 6 is one of the groups of formula B7 or B8,
  • the compound of the formula (313) can be obtained by the following production method: in an organic solvent, under the conditions of an amide formation reaction, and in the presence of an amide reaction condensing agent and a tertiary amine organic base, the formula (314) The compound shown is contacted with a compound of formula (A-1) or a compound of formula (A-2), followed by separation:
  • n1, n3, m1, m2, m3, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , L 1 , S 1 , q 2 and R k are each defined and selected as As mentioned before.
  • the amide forming reaction conditions may include a reaction temperature of 0-100 ° C and a reaction time of 1-48 hours. In some embodiments, the amide forming reaction conditions are a reaction temperature of 10-40 ° C and a reaction time of 2 16 hours.
  • the organic solvent is an alcohol solvent, an epoxy solvent, an ether solvent, a halogenated alkane solvent, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diiso One or more of propyl ethylamine.
  • the alcohol solvent is, in some embodiments, one or more of methanol, ethanol, propanol, and in some embodiments, ethanol.
  • the epoxy-based solvent is, in some embodiments, dioxane and/or tetrahydrofuran.
  • the ether solvent is, in some embodiments, diethyl ether and/or methyl tert-butyl ether.
  • the halogenated alkane solvent is, in some embodiments, one or more of dichloromethane, chloroform, and 1,2-dichloroethane.
  • the organic solvent is dichloromethane.
  • the organic solvent is used in an amount of from 3 to 50 L/mol, and in some embodiments from 3 to 20 L/mol, relative to the compound of the formula (314).
  • the amide-forming condensing agent is benzotriazol-1-yl-oxytripyrrolidinyl hexafluorophosphate, 3-diethoxyphosphoryl-1,2,3-benzene Azole 4(3H)-one, 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride, 2-ethoxy-1-ethoxycarbonyl- 1,2-Dihydroquinoline (EEDQ) or O-benzotriazole-tetramethylurea hexafluorophosphate, in a further embodiment 3-diethoxyphosphoryl-1,2,3 - benzoxazole 4(3H)-one.
  • the molar ratio of the amide-forming condensing agent to the compound of formula (314) may range from 1:1 to 10:1, and in some embodiments from 2.5:1 to 5:1.
  • the tertiary amine organic base is triethylamine or N,N-diisopropylethylamine, in some embodiments N,N-diisopropylethylamine.
  • the molar ratio of the tertiary amine organic base to the compound of formula (314) is from 3:1 to 20:1, and in some embodiments from 5:1 to 10:1.
  • Compounds of formula (A-1) and formula (A-2) can be prepared by any suitable means.
  • R k is a DMTr group
  • a compound of formula (A-1) can be prepared by reacting calcium glycerate with DMTrCl; similarly, 3-amino-1,2-propanediol can be first contacted with a cyclic anhydride, followed by The compound of formula (A-2) is then prepared by reaction with DMTrCl, which may be a cyclic anhydride having from 4 to 13, in some embodiments from 4 to 8.
  • the compound of formula (313) can also be prepared by reacting a compound of formula (314) with the cyclic anhydride, 3-amino-1,2-propanediol, and DMTrCl in that order.
  • a compound of formula (314) with the cyclic anhydride, 3-amino-1,2-propanediol, and DMTrCl in that order.
  • the compound of formula (313) can be isolated from the reaction mixture using any suitable separation method.
  • the solvent can be removed directly to provide a crude product of the compound of formula (313) which can be used directly in the subsequent reaction.
  • the compound of formula (314) can be obtained by the following preparation method: the method comprises contacting a compound of formula (315) with a haloacetic acid in an organic solvent under deprotection reaction conditions, followed by Separate:
  • R 7 is selected from the group represented by formula (330), (331), (332) or (333), and in some embodiments, the structure of R 7 is as shown in formula (330):
  • the haloacetic acid may be selected from one or more of the group consisting of dichloroacetic acid, trichloroacetic acid, monochloroacetic acid, and trifluoroacetic acid, and in some embodiments, dichloroacetic acid.
  • the deprotection reaction conditions include a reaction temperature of 0-100 ° C, a reaction time of 0.1-24 hours, in some embodiments a reaction temperature of 10-40 ° C, and a reaction time of 0.5-16 hours.
  • the organic solvent is an epoxy solvent, an ether solvent, a halogenated alkane solvent, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine.
  • the epoxy-based solvent is, in some embodiments, dioxane and/or tetrahydrofuran, and in some embodiments is diethyl ether and/or methyl tert-butyl ether
  • the halogenated alkane solvent is in some
  • the organic solvent is dichloromethane.
  • the organic solvent is used in an amount of from 3 to 50 L/mol, and in some embodiments from 5 to 20 L/mol, relative to the compound of the formula (315).
  • the molar ratio of the haloacetic acid to the compound of formula (315) is from 5:1 to 100:1, and in some embodiments from 10:1 to 50:1.
  • the compound of formula (314) can be isolated from the reaction mixture using any suitable separation method.
  • the solvent can be removed directly to provide a crude product of the compound of formula (314) which can be used directly in the subsequent reaction.
  • the compound of the formula (315) can be obtained by the following production method: the method comprises: in the presence of an amide reaction condensing agent and a tertiary amine organic base in an organic solvent, under the condensation reaction conditions, the formula (317) The compound is contacted with a compound of formula (316) and subsequently isolated:
  • n1, n3, m1, m2, m3, R 7 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , L 1 , S 1 are as defined above .
  • the compound of the formula (316) can be, for example, a compound disclosed in J. Am. Chem. Soc. 2014, 136, 16958-16961, or the compound of the formula (316) can be produced by a person skilled in the art by various methods, for example, Certain compounds of formula (316) are prepared by reference to the process disclosed in Example 1 of U.S. Patent No. 8,100,062, B2, the entire disclosure of which is incorporated herein in its entirety.
  • the condensation reaction conditions comprise a reaction temperature of 0-100 ° C, a reaction time of 0.1-24 hours, in some embodiments a reaction temperature of 10-40 ° C, and a reaction time of 0.5-16 hours.
  • the molar ratio of the compound of the formula (316) to the compound of the formula (317) may be from 2:1 to 10:1, and in some embodiments from 2.5:1 to 5:1.
  • the organic solvent is acetonitrile, an epoxy solvent, an ether solvent, a halogenated alkane solvent, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropyl
  • ethylamines which in some embodiments are dioxane and/or tetrahydrofuran, and in some embodiments are diethyl ether and/or methyl tert-butyl.
  • the alkyl ether which in some embodiments is one or more of dichloromethane, chloroform, and 1,2-dichloroethane, in some embodiments, the organic solvent is acetonitrile .
  • the organic solvent is used in an amount of from 3 to 50 L/mol, and in some embodiments from 5 to 20 L/mol, relative to the compound of the formula (317).
  • the amide-forming reaction condensing agent is benzotriazol-1-yl-oxytripyrrolidinylphosphorus hexafluorophosphate, 3-diethoxyphosphoryl-1,2,3-oxazolyl 4(3H)- Ketone (DEPBT), O-benzotriazole-tetramethylurea hexafluorophosphate or 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride In some embodiments is 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride.
  • the molar ratio of the amide-forming condensing agent to the compound of formula (317) is from 2:1 to 10:1, and in some embodiments from 2.5:1 to 5:1.
  • the tertiary amine organic base is N-methylmorpholine, triethylamine or N,N-diisopropylethylamine, in some embodiments N-methylmorpholine; the tertiary amine
  • the molar ratio of the organic base to the compound of formula (317) is from 3:1 to 20:1, and in some embodiments from 5:1 to 10:1.
  • the compound of formula (315) can be isolated from the reaction mixture using any suitable separation method.
  • the solvent can be removed by evaporation, followed by chromatographic separation of the compound of formula (315).
  • the solvent can be removed directly to provide a crude product of formula (315) which can be used directly in the subsequent reaction.
  • a compound of formula (317) is reacted in a single reaction with a sufficient amount of a compound of formula (316) to form the desired compound of formula (315), in which case the individual S 1 -L 1 moieties are identical to each other.
  • the formula (317) with a different batch of the compound of formula (316) compound i.e. compound L 1 and / or S 1 different from formula (316) reacts so that the resulting formula (315)
  • the compound contains two or more kinds of S 1 and/or L 1 .
  • a compound of formula (317) may be first contacted with 2 eq of the first compound of formula (316), and the first S 1 -L may be attached to the two terminal primary amine groups of the compound of formula (317). Part 1 , followed by contacting it with a compound of the second formula (316) of (n3+n1-1) eq (the definitions and ranges of values for n3 and n1 are as described above), in the compound of formula (317) The second S 1 -L 1 moiety is attached to the (n3+n1-1) secondary amine group.
  • the compound of formula (317) can be obtained by a process comprising: contacting a compound of formula (318) with an aqueous solution of methylamine in the presence of an organic solvent under deprotection conditions, Subsequent separation:
  • n1, n3, m1, m2 , m3, R 7, R 10, R 11, R 12, R 13, R 14, R 15 are each as defined and selectable range as described above.
  • the deprotection reaction conditions include a reaction temperature of 0 to 150 ° C, a reaction time of 5 to 72 hours, and in some embodiments, a reaction temperature of 20 to 80 ° C and a reaction time of 10 to 30 hours.
  • the organic solvent is selected from the group consisting of alcohols, in some embodiments one of methanol, ethanol, and isopropanol, and in some embodiments methanol; the organic solvent is used in an amount of 1 relative to the compound of formula (318) -20 L/mol, in some embodiments from 1.5 to 10 L/mol.
  • the concentration of the aqueous solution of the methylamine may be from 30 to 40% by mass, and the molar ratio of the methylamine to the compound of the formula (318) may be from 10:1 to 500:1, and in some embodiments from 50:1 to 200: 1.
  • the compound of formula (317) can be isolated from the reaction mixture using any suitable separation method.
  • the solvent can be removed by evaporation, followed by chromatographic separation of the compound of formula (317).
  • the solvent can be removed directly to provide a crude product of the compound of formula (317), which can be used directly in the subsequent reaction.
  • the compound of formula (318) can be obtained by the following preparation method: the method comprises the compound of formula (319) and triphenylchloromethane (in the presence of an organic solvent under the substitution reaction conditions). TrCl), diphenylethylphenylchloromethane, phenyldiethylphenylchloromethane or triethylphenylchloromethane, in some embodiments triphenylchloromethane (TrCl) contact, followed by separation:
  • n1, n3, m1, m2 , m3, R 10, R 11, R 12, R 13, R 14, R 15 are each as defined and selectable range as described above.
  • the substitution reaction conditions may include a reaction temperature of 0-100 ° C and a reaction time of 5 to 72 hours. In some embodiments, the reaction conditions include a reaction temperature of 10 to 40 ° C and a reaction time of 10 to 30 hours.
  • Triphenylchloromethane (TrCl), diphenylethylphenylchloromethane, phenyldiethylphenylchloromethane or triethylphenylchloromethane are commercially available, triphenylchloromethane (TrCl), diphenyl
  • the molar ratio of ethylphenylchloromethane, phenyldiethylphenylchloromethane or triethylphenylchloromethane to the compound of formula (319) may range from 1:1 to 10:1, and in some embodiments, 1: 1-3:1.
  • the organic solvent may be one of an epoxy solvent, an ether solvent, a halogenated alkane solvent, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine or A variety.
  • the epoxy-based solvent is, in some embodiments, dioxane and/or tetrahydrofuran, and in some embodiments is diethyl ether and/or methyl tert-butyl ether
  • the halogenated alkane solvent is in some
  • the organic solvent may be used in an amount of from 3 to 50 L/mol, and in some embodiments from 5 to 20 L/mol, relative to the compound of the formula (319).
  • the compound of formula (318) can be isolated from the reaction mixture using any suitable separation method.
  • the solvent can be removed by evaporation, followed by chromatographic separation of the compound of formula (318).
  • the compound of formula (319) can be obtained by the following preparation method: the method comprises contacting a compound of the formula (320) with ethyl trifluoroacetate under an alternative reaction condition in an organic solvent. Subsequent separation:
  • n1, n3, m1, m2 , m3, R 10, R 11, R 12, R 13, R 14, R 15 are each as defined and selectable range as described above.
  • the organic solvent is acetonitrile, an epoxy solvent, an ether solvent, a halogenated alkane solvent, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropyl One or more of ethylamine.
  • the epoxy-based solvent is dioxane and/or tetrahydrofuran
  • the ether solvent is diethyl ether and/or methyl tert-butyl ether
  • the halogenated alkane solvent is one or more of dichloromethane, chloroform and 1,2-dichloroethane, and in some embodiments, the organic solvent is acetonitrile.
  • the organic solvent is used in an amount of from 1 to 50 L/mol, and in some embodiments from 1 to 20 L/mol, relative to the compound of the formula (320).
  • the substitution reaction conditions may include a reaction temperature of 0-100 ° C and a reaction time of 5 to 72 hours. In some embodiments, the substitution reaction conditions include a reaction temperature of 10 to 40 ° C and a reaction time of 10 to 30. hour.
  • the molar ratio of ethyl trifluoroacetate to the compound of formula (320) is from 2:1 to 10:1, and in some embodiments from 3:1 to 5:1.
  • the compound of formula (319) can be isolated from the reaction mixture using any suitable separation method.
  • the solvent can be removed by evaporation, followed by chromatographic separation of the compound of formula (319).
  • the solvent can be removed directly to provide a crude product of the compound of formula (319) which can be used directly in the subsequent reaction.
  • the first or second siRNA conjugate of the present disclosure may also be combined with other pharmaceutically acceptable excipients, which may be one or more of various formulations or compounds conventionally employed in the art, for details. See above for a description of the pharmaceutical compositions of the present disclosure.
  • siRNA of the present disclosure pharmaceutical composition containing the same, first siRNA conjugate, and second siRNA conjugate
  • the disclosure provides siRNA, a pharmaceutical composition comprising the siRNA, a first siRNA conjugate, and a second siRNA conjugate prepared for treatment and/or prevention by the hepatitis B
  • siRNA a pharmaceutical composition
  • a pharmaceutical composition comprising the siRNA, a first siRNA conjugate, and a second siRNA conjugate prepared for treatment and/or prevention by the hepatitis B
  • the present disclosure provides a method of treating a pathological condition or disease caused by an infection of hepatitis B virus, the method comprising administering to a patient an siRNA, a pharmaceutical composition, a first one provided by the present disclosure siRNA conjugate and a second siRNA conjugate.
  • the present disclosure provides a method for inhibiting expression of a hepatitis B virus gene in a hepatitis cell infected with chronic hepatitis B virus, the method comprising the siRNA, a pharmaceutical composition, and the An siRNA conjugate and a second siRNA conjugate are contacted with the hepatitis B virus infected with chronic hepatitis B virus.
  • the pathological condition or disease caused by the infection of hepatitis B virus is selected from the group consisting of chronic liver disease, hepatitis, liver fibrosis disease, and liver proliferative disease.
  • the siRNA and/or pharmaceutical composition of the present disclosure By administering the siRNA and/or pharmaceutical composition of the present disclosure, the first siRNA conjugate, and the second siRNA conjugate to a patient in need thereof, the purpose of treating hepatitis B can be achieved by a mechanism of RNA interference.
  • the siRNA and/or pharmaceutical compositions of the present disclosure, the first siRNA conjugate, and the second siRNA conjugate can be used to prevent and/or treat hepatitis B, or to prepare for the prevention and/or treatment of hepatitis B. drug.
  • administering/administering refers to the desired effect by at least partially positioning the siRNA or pharmaceutical composition, the first siRNA conjugate, and the second siRNA conjugate at a desired site.
  • Routes of administration suitable for the methods of the present disclosure include topical administration and systemic administration. In general, topical administration results in delivery of more siRNA or pharmaceutical composition, first siRNA conjugate, and second siRNA conjugate to a particular site than the entire body of the patient; The siRNA or pharmaceutical composition, the first siRNA conjugate, and the second siRNA conjugate are delivered to the patient's substantially entire body.
  • a mode of administration capable of delivering a drug to the liver is preferred.
  • Administration can be administered to a patient by any suitable route known in the art including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, Airway administration (aerosol), pulmonary administration, nasal administration, rectal administration, and topical administration (including buccal administration and sublingual administration).
  • the frequency of administration can be one or more times per day, every week, every two weeks, every three weeks, every month or every year.
  • the siRNA or pharmaceutical composition, the first siRNA conjugate, and the second siRNA conjugate of the present disclosure may be administered at a dosage conventional in the art, which may be based on various parameters, particularly a patient's Determined by age, weight and gender. Toxicity and efficacy can be determined by standard pharmaceutical procedures in cell culture or laboratory animals, such as determining LD50 (a dose that kills 50% of the population) and ED50 (in a quantitative response, a dose that causes 50% of the maximum response intensity, in a qualitative response) The middle finger causes a dose of 50% of the subjects to have a positive reaction). The dose ratio between toxicity and efficacy is the therapeutic index and can be expressed as the ratio of LD50/ED50. Preferred are siRNA or pharmaceutical compositions that exhibit a high therapeutic index, a first siRNA conjugate, and a second siRNA conjugate. The range of human doses can be derived based on data obtained from cell culture assays and animal studies.
  • the first siRNA conjugate, and the second siRNA conjugate for example, for male or female, 6-12 weeks old, body weight 18-25 g C57BL/6J or C3H/HeNCrlVr mice, based on the amount of siRNA in the pharmaceutical composition or siRNA conjugate: (i) a pharmaceutical composition for siRNA and a pharmaceutically acceptable carrier, the siRNA may be used in an amount of 0.001-50 mg /kg body weight, in a further embodiment 0.01 to 10 mg/kg body weight, in still further embodiments 0.05 to 5 mg/kg body weight, and in still further embodiments 0.1 to 3 mg/kg body weight; (ii) For the first and/or second siRNA conjugate formed by the siRNA and the pharmaceutically acceptable conjugated molecule, the amount of siRNA may range from 0.001 to 100 mg/kg body weight, and in further embodiments from 0.01 to 50 mg /kg body weight, in still further embodiments, 0.05-20 mg/kg body weight, and in still further embodiments 0.1
  • the siRNA and/or pharmaceutical composition of the present disclosure by introducing the siRNA and/or pharmaceutical composition of the present disclosure, the first siRNA conjugate, and the second siRNA conjugate into hepatitis cells infected with chronic HBV, inhibition of infection with chronic HBV can also be achieved by the mechanism of RNA interference.
  • the cell is a HepG2.2.15 cell.
  • the amount of siRNA is generally such an amount:
  • the expression of the target gene is reduced and results in an extracellular concentration of 1 pM to 1 ⁇ M, or 0.01 nM to 100 nM, or 0.05 nM to 50 nM or to about 5 nM at the surface of the target cell.
  • the amount required to achieve this local concentration will vary with a variety of factors including the method of delivery, the site of delivery, the number of cell layers between the delivery site and the target cell or tissue, whether the delivery is local or systemic, and the like.
  • the concentration at the delivery site can be significantly higher than the concentration at the surface of the target cells or tissue.
  • the present disclosure provides a kit comprising an effective amount of at least one of an siRNA of the present disclosure, a pharmaceutical composition, a first siRNA conjugate, and a second siRNA conjugate.
  • kits described herein can provide modified siRNA in one container.
  • the kits described herein can comprise a container that provides a pharmaceutically acceptable excipient.
  • other components such as stabilizers or preservatives and the like may also be included in the kit.
  • the kits described herein can comprise at least one additional therapeutic agent in a container other than a container that provides the modified siRNA described herein.
  • the kit can include instructions for mixing the modified siRNA with a pharmaceutically acceptable carrier and/or adjuvant or other ingredients, if any.
  • the modified siRNA and a pharmaceutically acceptable carrier and/or adjuvant and the modified siRNA, pharmaceutical composition, first siRNA conjugate, and/or second siRNA conjugate may be provided in any form, such as a liquid form, a dry form, or a lyophilized form.
  • the modified siRNA and the pharmaceutically acceptable carrier and/or adjuvant and the pharmaceutical composition and/or conjugate and optionally the pharmaceutically acceptable excipient are substantially pure and/or Sterile.
  • sterile water can be provided in a kit of the present disclosure.
  • the siRNA, composition or siRNA conjugate provided by the present disclosure may have greater stability, lower toxicity, and/or higher activity in vivo. In some embodiments, the siRNA, siRNA composition or siRNA conjugate provided by the present disclosure exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95 in vivo. % inhibition of HBV gene expression. In some embodiments, the siRNA, siRNA composition or siRNA conjugate provided by the present disclosure exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95 in vivo. % inhibition of HBV gene expression in the liver.
  • the siRNA, siRNA composition or siRNA conjugate provided by the present disclosure exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95 in vivo. Inhibition rate of HBV gene expression in the liver in % of animal models. In some embodiments, the siRNA, siRNA composition or siRNA conjugate provided by the present disclosure exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95 in vivo. % HBV surface antigen expression inhibition rate. In some embodiments, the siRNA, composition or siRNA conjugates provided by the present disclosure do not exhibit significant off-target effects. The off-target effect can be, for example, inhibition of normal expression of a gene of a non-target gene. It is believed that the off-target effect is insignificant if the binding/inhibition of off-target gene expression is less than 50%, 40%, 30%, 20% or 10% compared to the target gene effect.
  • the siRNA conjugates provided by the present disclosure have better in vitro inhibitory activity with an inhibition rate of up to 99% at 0.1 nM.
  • the siRNA conjugates provided by the present disclosure have better in vivo inhibitory activity with an inhibition rate of up to 93.8% at 1 mg/kg.
  • the siRNA conjugates provided by the present disclosure also exhibit low off-target effects while having excellent target mRNA inhibitory effects.
  • the siRNA conjugates provided by the present disclosure maintain long-term non-degradation in Tritosome, showing good stability.
  • the siRNA conjugates provided by the present disclosure are still not degraded in human plasma up to 72 h, exhibiting excellent stability in human plasma.
  • the siRNA conjugates provided by the present disclosure are still not degraded in cynomolgus plasma until 72 h, showing excellent stability in monkey plasma.
  • a single administration of 3 mg/kg of conjugate 4 has a maximum inhibition of HBsAg of greater than 90% and is maintained for at least 21 days.
  • conjugate 4 (hereinafter, also referred to as L10-siHB1M1SVP conjugate) was synthesized, and it is planned to synthesize conjugate 18 (hereinafter, also referred to as L10-siHB1M1SP) and conjugate 19 (hereinafter, also Called L10-siHB1M1SPs).
  • the conjugate is a conjugate formed by conjugation of L-9 conjugated molecules to siRNAs numbered siHB1M1SVP, siHB1M1SP or siHB1M1SPs, respectively. See Table 2 for the sequence of the siRNA conjugated in this conjugate.
  • the L-10 compound was synthesized as follows:
  • GAL-1 N-acetyl-D-galactosamine hydrochloride, CAS No.: 1772-03-8, purchased from Ningbo Hongxiang Biochemical Co., Ltd., 463.8 mmol
  • acetic anhydride purchased from Enox Corporation, 5565.6 mmol
  • GAL-2 (35.1 g, 90.0 mmol) obtained in the step (1-1-1a) was dissolved in 213 ml of anhydrous 1,2-dichloroethane, and 24.0 g of TMSOTf was added in an ice water bath under nitrogen atmosphere. (CAS No.: 27607-77-8, purchased from Macleans, 108.0 mmol), reacted overnight at room temperature.
  • the reaction solution was diluted with 400 ml of dichloromethane, and filtered with celite, and then 1 L of saturated aqueous sodium hydrogen carbonate solution was added, and the mixture was stirred, and the organic phase was separated.
  • the aqueous phase was extracted twice with dichloroethane, 300 ml each time.
  • the organic phase was washed with 300 ml of a saturated aqueous sodium hydrogen carbonate solution and 300 ml of brine, and the organic phase was evaporated, dried over anhydrous sodium sulfate, and evaporated.
  • GAL-3 (26.9 g, 81.7 mmol) obtained in the step (1-1-1b) was dissolved in 136 ml of anhydrous 1,2-dichloroethane, and dried. 30 g of molecular sieve powder, and then added 9.0 g of 5-hexen-1-ol (CAS No.: 821-41-0, available from Adamas-beta, 89.9 mmol), stirred at room temperature for 30 minutes, added under ice bath and nitrogen protection. 9.08 g of TMSOTf (40.9 mmol) was stirred at room temperature overnight.
  • GAL-4 (14.9 g, 34.7 mmol) obtained according to the method described in the step (1-1-1c) was dissolved in a mixed solvent of 77 ml of dichloromethane and 77 ml of acetonitrile, and respectively, 103 ml of deionized water and 29.7 g of high were added.
  • Sodium iodate (CAS No.: 7790-28-5, purchased from Aladdin, 138.8 mmol), stirred for 10 minutes in an ice water bath, and added with antimony trichloride (CAS No.: 14988-67-0, purchased from Anji Company, 238 mg, 1.145 mmol), reacted overnight at room temperature.
  • the reaction solution was diluted with 300 ml of water and stirred, and adjusted to pH 7.5 with saturated sodium hydrogencarbonate.
  • the organic phase was separated and discarded, and the aqueous phase was extracted three times with dichloromethane (200 ml), and the organic phase was discarded.
  • the aqueous phase was adjusted to pH 3 with citric acid solids, and extracted twice with dichloromethane (200 ml).
  • the crude M-11-T3-Tr obtained in the step (1-1-3) (7.763 g, 10 mmol) was dissolved in 100 ml of methanol, and then 100 ml of aqueous methylamine solution (40% by mass) was added, and the reaction was stirred at 50 ° C for 23 h. The insoluble granules were removed by filtration, and the solvent was evaporated to dryness.
  • the solvent was evaporated to drynessjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjj
  • the solvent was evaporated to dryness overnight, purified by 200-300 mesh normal phase silica gel column, petroleum ether packed column, 1 wt%
  • the product eluate was collected, and the solvent was evaporated under reduced pressure. The bubble was dried to obtain a pure product M-18-Tr 2.887 g.
  • the reaction mixture was diluted with 200 ml of dichloromethane, and the organic phase was washed with 100 ml of saturated sodium hydrogen carbonate, and the organic phase was washed with anhydrous sodium sulfate.
  • the L-5-Tr (5.94 g, 3.456 mmol) obtained in the step (1-1-5) was dissolved in 69 ml of dichloromethane, then dichloroacetic acid (13.367 g, 103.67 mmol) was added, and reacted at room temperature for 2 h, added
  • DMTrCl (4,4'-bismethoxytrityl chloride, 38.12 g, 112.5 mmol) was dissolved in 450 ml of anhydrous pyridine, and DL-calcium glycerate hydrate (12.88 g, 45.0 mmol) was added at 45 After reacting for 22 h at ° C, the reaction solution was filtered, and the filter cake was rinsed with 200 ml of DCM, and the filtrate was concentrated to dryness under reduced pressure.
  • an L-10 compound was prepared by attaching an L-9 conjugating molecule to a solid support.
  • the L-9 conjugated molecule obtained in the step (1-1-8) (0.233 g, 0.1126 mmol), O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, 0.064 g, 0.1689 mmol)
  • HBTU O-benzotriazole-tetramethylurea hexafluorophosphate
  • DIEA diisopropylethylamine
  • CapA and CapB are capping reagent solutions
  • CapA is a pyridine/acetonitrile mixed solution of 20% by volume of N-methylimidazole, and the volume ratio of pyridine to acetonitrile is 3:5
  • CapB is a solution of 20% by volume of acetic anhydride in acetonitrile.
  • the nucleoside monomer was ligated one by one from the 3'-5' direction by the solid phase phosphoramidite method using the L-10 compound prepared in the above procedure.
  • Each nucleoside monomer is linked to a four-step reaction of deprotection, coupling, capping, oxidation or sulfurization.
  • a phosphate linkage is used between two nucleotides
  • a nucleoside monomer is attached, a four-step reaction including deprotection, coupling, capping, and oxidation is included.
  • a phosphorothioate is used between two nucleotides, when a nucleoside monomer is attached, a four-step reaction of protection, coupling, capping, and sulfurization is included.
  • the synthesis conditions are given as follows:
  • the nucleoside monomer is provided in a 0.1 M acetonitrile solution.
  • the deprotection reaction conditions are the same for each step, that is, the temperature is 25 ° C, the reaction time is 70 seconds, and the deprotecting reagent is dichloroacetic acid in dichloromethane (3%). v/v), the molar ratio of dichloroacetic acid to the 4,4'-dimethoxytrityl protecting group on the solid support is 5:1.
  • the coupling reaction conditions are the same in each step, including the temperature of 25 ° C, the molar ratio of the nucleic acid sequence and the nucleoside monomer attached to the solid phase carrier is 1:10, and the nucleic acid sequence and the coupling reagent are attached to the solid phase carrier.
  • the ratio was 1:65, the reaction time was 600 seconds, and the coupling reagent was a solution of 5-ethylthio-1H-tetrazole in 0.5 M acetonitrile.
  • the cap conditions were the same for each step, including a temperature of 25 ° C and a reaction time of 15 seconds.
  • the oxidation reaction conditions were the same for each step, including a temperature of 25 ° C, a reaction time of 15 seconds, and an oxidizing reagent of iodine water having a concentration of 0.05 M.
  • the molar ratio of iodine to the nucleic acid sequence attached to the solid support in the coupling step was 30:1.
  • the conditions of the vulcanization reaction were the same for each step, including a temperature of 25 ° C, a reaction time of 300 seconds, and a sulfurization reagent of hydrogenated xanthogen.
  • the molar ratio of the sulfurizing reagent to the nucleic acid sequence attached to the solid phase support in the coupling step was 120:1.
  • the cleavage and deprotection conditions were as follows: The synthesized nucleotide sequence linked to the carrier was added to a 25 wt% aqueous ammonia solution, and the amount of ammonia water was 0.5 ml/ ⁇ mol, which was reacted at 55 ° C for 16 h, the liquid was removed, and concentrated to dryness in vacuo.
  • the product eluate was collected and combined, and subjected to desalting using a reverse phase chromatography purification column.
  • the specific conditions include desalination using a Sephadex column, and the filler was a glucan gel G25, which was eluted with deionized water.
  • Detection Purity was measured using ion exchange chromatography (IEX-HPLC), and molecular weight was analyzed using liquid chromatography-mass spectrometry (LC-MS).
  • the theoretical value is 7423.22, and the measured value is 7422.6.
  • the measured values are in agreement with the theoretical values, indicating that the sense strand S is conjugated to the L-9 conjugated molecule at the 3' end.
  • Detection Purity was determined by ion exchange chromatography (IEX-HPLC); molecular weight was analyzed by LC-MS. The theoretical value was 7207.78, and the measured value was 7207.2. The measured values are in agreement with the theoretical values, indicating that the antisense strand AS having the target sequence is synthesized.
  • VP-Um vinyl phosphate modified 2'-methoxy modified uridine monomer
  • the VP-U-2 molecule was synthesized as follows:
  • the aqueous phase was extracted with dichloromethane (DCM) three times, 300 ml each time, and the organic phase was combined and washed with 5% oxalic acid to pH. ⁇ 5.
  • DCM dichloromethane
  • the crude VP-U-1 was obtained after evaporation of the solvent to dryness and used directly for the subsequent synthesis of VP-U-2.
  • the VP-U-1 crude product was dissolved in 100 ml of dichloromethane, and then stirred in an ice bath for 10 minutes, and then 450 ml of 2% p-toluenesulfonic acid solution (solvent as a solvent of 3:7 by volume) which was previously stored in a refrigerator at 4 ° C was added. - dichloromethane mixed solvent), reacted for 10 minutes. The reaction was quenched by the addition of EtOAc (EtOAc) (EtOAc) The combined aqueous phases were extracted twice with dichloromethane (200 ml), and the organic phase was combined and washed twice with 200 ml of brine and evaporated to dryness.
  • EtOAc EtOAc
  • VP-U-2 (19.84 g, 40.0 mmol), dicyclohexylcarbodiimide (DCC, 16.48 g, 80.0 mmol), pyridine (4.20 g, 53.2 mmol), trifluoroacetic acid (6.61 g, 53.2 mmol)
  • DMSO dimethyl sulfoxide
  • tetraethyl methylene diphosphate 21.44 g, 74.4 mmol
  • t-BuOK 11.36 g, 101.2 mmol
  • the reaction was quenched with water and the aqueous phase was extracted with dichloromethane three times, 200 ml each time.
  • VP-U-4 14.00 g, 22.29 mmol was dissolved in 100 ml of tetrahydrofuran, and triethylamine trihydrofluoric acid (17.96 g, 111.45 mmol) was added, and the mixture was stirred at room temperature for 20 h. The solvent was directly evaporated to dryness, then taken up in dichloromethane and then evaporated to dryness twice twice with 50 ml of dichloromethane. Purified by a 200-300 mesh normal phase silica gel column, packed with petroleum ether and eluted with a gradient of petroleum ether:ethyl acetate:dichloromethane:methanol:1:1:1:0.05 to 1:1:1:0.25.
  • VP-U-5 (391 mg, 1.0 mmol), trifluoroacetic acid pyridinium salt (0.232 g, 1.2 mmol), N-methylimidazole (0.099 g, 1.2) was added to 10 ml of anhydrous dichloromethane under argon.
  • Methyl) bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.452 g, 1.5 mmol) was stirred at room temperature for 5 h.
  • the antisense strand of conjugate 18 differs from the antisense strand of conjugate 4 only in that the first nucleotide modification at the 5'-end is different.
  • the last nucleoside monomer to be linked is a 2'-methoxy modified uridine monomer (Um), which is then deprotected, coupled, capped, and oxidized.
  • Um 2'-methoxy modified uridine monomer
  • the step reaction ligated the CPR-I monomer (Suzhou Jima, Cat. No. 13#-2601-XX) to the 5' end of the antisense strand to form a 5'-phosphate modification.
  • the universal solid phase carrier used deprotection, coupling, capping, oxidation or sulfurization reaction conditions, cutting and deprotection, purification and desalting conditions are the same as the synthetic sense strand.
  • conjugate 18 antisense strand The same synthetic procedure as the conjugate 18 antisense strand is used, except that when the CPR-I monomer is attached, the oxidation reaction conditions are replaced by the sulfurization reaction conditions, and it is expected that a 5'-phosphorothioate modified conjugation can be obtained. 19 antisense strand.
  • conjugate 4 the S chain and the AS chain were respectively dissolved in water for injection to obtain a solution of 40 mg/mL, mixed in an equimolar ratio, heated at 50 ° C for 15 min, and cooled at room temperature to form a double-stranded structure by hydrogen bonding. .
  • the conjugate was diluted to a concentration of 0.2 mg/mL using ultrapure water (manufactured by Milli-Q ultrapure water meter, resistivity 18.2 M ⁇ *cm (25 ° C)), using a LC-MS, LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, Model: LCT Premier) for molecular weight testing.
  • the measured values are in agreement with the theoretical values, indicating that the synthesized conjugate 4 is a double-stranded nucleic acid sequence with a L-9-conjugated molecule of interest.
  • conjugates 18 and 19 annealing was carried out as described above, and it is expected that conjugates 18 and 19 can be synthesized.
  • the structures of the above conjugates 4, 18 and 19 are as shown in the formula (403).
  • the title conjugate can be prepared, except that: 1) the siRNAs are respectively shown in Table 2 corresponding to conjugates 1-3, 5-9 and comparative Sequence of conjugate 1; 2) When the target sequence contains unmodified nucleotides, in the conditions of cleavage and deprotection, after ammonia treatment, with respect to the amount of single-stranded nucleic acid, 0.4 ml/ ⁇ mol of N-A is used.
  • the P-10 compound was synthesized as follows:
  • the crude GAL5-C4-1 obtained in the step (3-1-1) (30.3 g, 30 mmol) was dissolved in 180 ml of formic acid, and the mixture was stirred at room temperature for 16 hours. The solvent was evaporated to dryness and purified by column chromatography (200-300 m. -C4-2 total 14.84g.
  • the organic phase was washed with 10 ml of saturated sodium bicarbonate, and the aqueous phase was extracted with dichloromethane (3 ml), 10 ml each time, and the organic phase was washed with 10 ml of brine, and the aqueous phase was extracted twice with 10 ml each time, and the organic phase was combined.
  • P-10 was prepared by the same method as the step (1-1-9) in Preparation Example 1. The difference is that the P-9 conjugated molecule is substituted for the L-9 conjugated molecule to obtain a P-9 conjugated molecule attached to the solid support.
  • the conjugate 10 was prepared by the same method as the steps (1-2), (1-3A), (1-4) in Preparation Example 1, except that the P-10 compound was substituted for the L-10 compound to initiate justice. Chain synthesis. It is expected that a P10-siHB1M1SVP conjugate can be obtained, which has the structure shown by the formula (404).
  • the R-5 compound was synthesized as follows:
  • GAL-3 (26.4 g, 80.2 mmol) obtained according to the method described in the step (1-1-1b) was dissolved in 134 ml of anhydrous 1,2-dichloroethane, and added. 60 g of molecular sieve powder, further adding 7-octene-1-ol (11.3 g, 88.2 mmol), stirring for 10 minutes at room temperature, adding trimethylsilyl trifluoromethanesulfonate (8.9 g) under ice bath and nitrogen protection. , 40.1 mmol), and the reaction was stirred at room temperature for 24 hours.
  • GAL-C7-1 (33.3 g, 72.8 mmol) obtained in the step (4-1-1) was dissolved in a mixed solvent of 160 ml of dichloromethane and 160 ml of acetonitrile, and 216 ml of water and sodium periodate solid were respectively added ( 62.3 g, 291.2 mmol), stirred for 10 minutes in an ice water bath, and the catalyst was added to ruthenium trichloride (498 mg, 2.4 mmol). The reaction solution was diluted with 200 ml of water and stirred, and saturated sodium hydrogencarbonate was added to adjust the pH to 7.5.
  • R-2 (2.391 g, 1.532 mmol) and A-1 (2.342 g, 4.596 mmol) were mixed and dissolved in 16 ml of dichloromethane, and 3-diethoxyphosphoryl-1,2,3-oxazole 4 was added.
  • 3H)-ketone (DEPBT) (1.375 g, 4.596 mmol)
  • diisopropylethylamine (1.188 g, 9.191 mmol) was added, and the reaction was stirred at 25 ° C for 2 h.
  • the organic phase was washed with 10 ml of saturated sodium bicarbonate, and the aqueous phase was extracted three times with dichloromethane (10 ml), and the organic phase was washed with 10 ml of saturated brine, and the aqueous phase was extracted twice with 10 ml of dichloromethane.
  • the organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness.
  • R-3 (795 mg, 0.4074 mmol), succinic anhydride (82 mg, 0.8148 mmol) and 4-dimethylaminopyridine (DMAP, 100 mg, 0.8148 mmol) were mixed and dissolved in 4 ml of dichloromethane, then diisopropyl B was added.
  • the amine (DIPEA, 100 mg, 0.8148 mmol) was stirred at 25 ° C for 18 h.
  • R-5 was prepared by the same method as the step (1-1-9) in Preparation Example 1. The difference is that the R-4 conjugated molecule replaces the L-9 conjugated molecule to give an R-4 conjugated molecule attached to a solid support.
  • the conjugate 11 was prepared by the same method as the steps (1-2), (1-3A), (1-4) in Preparation Example 1, except that the R-5 compound was substituted for the L-10 compound to initiate justice. Chain synthesis. It is expected that an R5-siHB1M1SVP conjugate can be obtained, which has the structure shown in formula (407).
  • LA-5 compound can be synthesized:
  • the conjugate 12 was prepared by the same method as the steps (1-2), (1-3A), (1-4) in Preparation Example 1, except that the LA-5 compound was substituted for the L-10 compound to initiate justice. Chain synthesis. It is expected that a LA5-siHB1M1SVP conjugate can be obtained, which has the structure shown by the formula (412).
  • the LB-5 compound was synthesized as follows:
  • LB-3 (822 mg, 0.415 mmol), succinic anhydride (83 g, 0.83 mmol) and 4-dimethylaminopyridine (DMAP, 102 mg, 0.83 mmol) were dissolved in 4 ml of dichloromethane, then DIPEA (270 mg, 2.075) Methyl), the reaction was stirred at 25 ° C overnight. The reaction solution was washed three times with 0.5 M triethylamine phosphate, and the aqueous phase was extracted three times with dichloromethane (2 ml).
  • DMAP 4-dimethylaminopyridine
  • LB-5 was prepared by the same method as the step (1-1-9) in Preparation Example 1. The difference is that the LB-4 conjugated molecule replaces the L-9 conjugated molecule to obtain an LB-4 conjugated molecule linked to a solid support.
  • the conjugate 13 was prepared by the same method as the steps (1-2), (1-3A), (1-4) in Preparation Example 1, except that the LB-5 compound was substituted for the L-10 compound to initiate justice. Chain synthesis. It is expected that an LB5-siHB1M1SVP conjugate can be obtained, which has the structure shown by the formula (413).
  • the conjugate 14 was prepared by the same method as the steps (1-2), (1-3A), (1-4) in Preparation Example 1, except that the V-8 compound was substituted for the L-10 compound to initiate justice. Chain synthesis. It is expected that a V8-siHB1M1SVP conjugate can be obtained, which has the structure shown in formula (414).
  • the W-8 compound was synthesized as follows:
  • the crude W-2 (8.012 g, 10 mmol) was dissolved in 100 ml of methanol, then 100 ml of aqueous methylamine solution (40 wt%) was added, and the reaction was stirred at 50 ° C for 23 h. The insoluble granules were removed by filtration, and the solvent was evaporated to dryness. EtOAc was evaporated. EtOAcjjjjjjjjjjjjjjj The organic phase was combined and dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated to dryness under reduced pressure.
  • the solvent was evaporated to dryness overnight on a vacuum oil pump, purified on a 200-300 mesh normal phase silica gel column, packed with petroleum ether, and neutralized with 1 wt% triethylamine.
  • the product eluate was collected, and the solvent was evaporated under reduced pressure.
  • Product W-3 3.062g.
  • W-3 (0.675 g, 1.517 mmol) was mixed with GAL-C7-2 (2.60 g, 5.46 mmol) in 47 ml of acetonitrile, then diisopropylethylamine (1.57 g, 12.14 mmol), and finally 3- Diethoxyphosphoryl-1,2,3-oxazolyl 4(3H)-one (DEPBT, 1.816 g, 6.04 mmol) was stirred at room temperature for 2.5 h.
  • DEPBT 3- Diethoxyphosphoryl-1,2,3-oxazolyl 4(3H)-one
  • the reaction mixture was diluted with 100 ml of methylene chloride, and the organic phase was washed with 80 ml of saturated sodium hydrogen carbonate solution, and the organic phase was washed with 80 ml of brine, and the organic phase was combined and dried over anhydrous sodium sulfate.
  • Purification of 300 mesh normal phase silica gel column, petroleum ether packed column, neutralized silica gel with 1 wt% triethylamine, eluted with dichloromethane:methanol 100:5-100:7 gradient, collected product eluent, decompressed Evaporated to give a pure product W-4 1.610 g.
  • W-8 was prepared by the same method as the step (1-1-9) in Preparation Example 1. The difference is that the W-9 conjugated molecule replaces the L-9 conjugated molecule to obtain a W-7 conjugated molecule attached to a solid support.
  • the conjugate 15 was prepared by the same method as the steps (1-2), (1-3A), (1-4) in Preparation Example 1, except that the W-8 compound was substituted for the L-10 compound to initiate justice. Chain synthesis. It is expected that a W8-siHB1M1SVP conjugate can be obtained, which has the structure shown in formula (415).
  • the conjugate 16 was prepared by the same method as the steps (1-2), (1-3A), (1-4) in Preparation Example 1, except that the X-8 compound was substituted for the L-10 compound to initiate justice. Chain synthesis. It is expected that an X8-siHB1M1SVP conjugate can be obtained, which has the structure shown by the formula (421).
  • the Z-5 compound was synthesized as follows:
  • the reaction mixture was diluted with 100 ml of methylene chloride, and the organic phase was washed with 80 ml of saturated sodium hydrogen carbonate solution, and the organic phase was washed with 80 ml of brine, and the organic phase was combined and dried over anhydrous sodium sulfate.
  • Purified by a 300-mesh normal phase silica gel column, packed with petroleum ether, neutralized silica gel with 1 wt% triethylamine, and eluted with a gradient of dichloromethane:methanol 30:1 to 15:1. Evaporation to give pure product Z-1 3.97 g. MS m/z: C 98 H 143 N 10 O 33 , [M+H] + , Theory: 1987.98, Found: 1987.90.
  • the organic phase was washed twice with saturated sodium bicarbonate twice, 30 ml each time, and the aqueous phase was extracted with 10 methylene chloride.
  • the organic phase was combined and washed with 50 ml of saturated brine. After drying, the solvent was evaporated to dryness under reduced pressure, and then evaporated to dryness with vacuo.
  • Z-5 was prepared by the same method as the step (1-1-9) in Preparation Example 1. The difference is that the Z-4 conjugated molecule replaces the L-9 conjugated molecule to obtain a Z-4 conjugated molecule attached to a solid support.
  • the conjugate 17 was prepared by the same method as the steps (1-2), (1-3A), (1-4) in Preparation Example 1, except that the Z-5 compound was substituted for the L-10 compound to initiate justice. Chain synthesis. It is expected that a Z5-siHB1M1SVP conjugate can be obtained, which has the structure shown by the formula (422).
  • conjugate 20 (hereinafter also referred to as FIN-siHB1M1SVP conjugate) was synthesized. See Table 2 for the sequence of the siRNA conjugated in this conjugate.
  • the FIN-2 conjugated molecule was synthesized according to the preparation method described in Rajeev et al., ChemBioChem 2015, 16, 903-908 according to the following route:
  • PRO-6 L-hydroxyproline, CAS No.: 51-35-4, available from Ange, 22.4 mmol
  • 1,4-dioxane 1,4-dioxane
  • Ring, CAS No.: 123-91-1 add 34 ml of 10% (w/w) aqueous solution of Na2CO3 in a suspension state, and 6.95 g of Fmoc-Cl (-9-mercaptomethyl chloroformate, CAS) No.: 28920-43-6, purchased from Angyi Company, 26.8 mmol) dissolved in 34 ml of 1,4-dioxane, added to the above suspension under ice bath, and naturally raised to room temperature overnight.
  • Fmoc-Cl -9-mercaptomethyl chloroformate
  • reaction solution was poured into 150 ml of ice water, extracted with methyl t-butyl ether three times, 100 ml each time, the organic phase was discarded, the aqueous phase was adjusted to pH ⁇ 5 with concentrated HCl, and extracted twice with 100 ml of ethyl acetate. The organic layer was dried over anhydrous sodium sulfate (MgSO4).
  • the silica gel column was basified with pyridine and then dissolved in DCM to dissolve the crude product, first containing 1% (v) /v) dimethyl pyridine was eluted with EtOAc (EtOAc) eluted eluted eluted eluted eluted eluted eluted eluted eluted eluted eluted eluted eluted eluted eluted eluted eluted eluted eluted eluted eluted
  • EtOAc EtOAc
  • GAL-5 (4.5 g, 10 mmol) obtained according to the method described in (1-1-1) was dissolved in 40 ml of DMF, and 3.9 g of DIPEA (N,N-diisopropylethylamine, CAS number: 7087-68-5, purchased from Aladdin, 30mmol) and 3.8g HBTU (benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, CAS No.:94790-37 -2, commercially available from Aladdin, 11 mmol), stirred at room temperature for 10 minutes, and PRO-10 (4.2 g, 10 mmol) obtained in the step (11-1-1d) was dissolved in 40 ml of DMF, and then added to the above reaction.
  • DIPEA N,N-diisopropylethylamine, CAS number: 7087-68-5, purchased from Aladdin, 30mmol
  • HBTU benzotriazole-N,N
  • FIN-1 (3.0 g, 3.53 mmol) obtained in the step (11-1-2) was azeotropically dehydrated with acetonitrile, dried under reduced pressure, dissolved in 10 ml of DMF, and 2.
  • Isopropylamino)(2-cyanoethoxy)phosphine available from Adamas Company, trade number 11356B, 7.06 mmol
  • 346 mg tetrazolium (CAS number: 288-94-8, purchased from Aladdin, 4.94 mmol)
  • the reaction was stirred at room temperature, and 10 ml of DMF was added thereto, and the reaction was further stirred for 1 hour.
  • the solvent was evaporated under reduced pressure and purified by silica gel column chromatography.
  • the FIN-2 conjugated molecule obtained in the step (11-1-3) is connected to the universal solid phase carrier (UnyLinkerTM loaded ) through three cycles. HL Solid Supports) to achieve attachment of the conjugate group (FIN_FIN_FIN) to the 3' end of the RNA sense strand.
  • the above-described ligation is carried out in accordance with the preparation method described in Rajeev et al., ChemBioChem 2015, 16, 903-908. Specifically, first, starting from the above-mentioned universal solid phase carrier, the hydroxyl protecting group on the solid phase carrier is removed, and Coupling conditions and coupling reagents are coupled to the FIN-2 conjugated molecule in the presence of a coupling reagent, and after the cap reaction and the oxidation reaction, a FIN conjugated molecule attached to the solid phase carrier is obtained; and the attachment to the solid phase carrier is removed.
  • the hydroxy protecting group DMTr on the FIN conjugated molecule is coupled to the FIN-2 conjugated molecule for capping reaction and oxidation reaction, and the above-mentioned deprotection-coupling-cap-oxidation step is repeated once again.
  • One FIN-2 conjugated molecule, the conjugated group (FIN_FIN_FIN) attached to the solid support was obtained.
  • the title conjugate was prepared by the same method as the steps (1-2), (1-3A), (1-4) in Preparation Example 1, except that: 1) obtained by the step (11-2) The compound initiates sense strand synthesis; 2) the conjugated siRNA has the sequence corresponding to conjugate 20 shown in Table 2.
  • the preparation of the conjugate of the present disclosure described above is completed, it is lyophilized as a solid powder for storage by standard means. In use, it can be used, for example, by reconstituting it with water for injection to a solution of the desired concentration.
  • Test sample preparation by lysosomal lysate preparation Conjugate 4 (provided as a 0.9% sodium chloride aqueous solution having a siRNA concentration of 20 ⁇ M in an amount of 6 ⁇ l per group) and 27.2 ⁇ L of an aqueous solution of sodium citrate (pH 5.0), respectively. 4.08 ⁇ L of deionized water and 2.72 ⁇ L of Tritosomes (commercially available from Xenotech, Cat. No. R0610LT, Lot No. 1610069) were mixed. Incubate at 37 ° C.
  • Reference sample preparation without lysosomal lysate preparation Take 1.5 ⁇ l of equimolar amount of conjugate 4 (20 ⁇ M) and 7.5 ⁇ L of sodium citrate aqueous solution (pH 5.0), mix with 1 ⁇ L of deionized water, add 30 ⁇ L The 9 M urea solution was denatured, then 8 ⁇ L of 6 ⁇ loading buffer was added and mixed, and immediately frozen in a -80 ° C refrigerator to terminate the reaction.
  • the reference sample is labeled Con in the electropherogram.
  • a 16% by weight non-denaturing polyacrylamide gel was prepared. 20 ⁇ l of each of the above test sample and reference sample was applied to the gel, and after electrophoresis for 10 min under a constant current of 20 mA, electrophoresis was continued for 30 min under a constant current of 40 mA. After the end of the electrophoresis, the gel was placed on a shaker and stained with Gelred dye (BioTium, Cat. No. 13G1203) for 10 min. The gel was observed and photographed, and the results are shown in Fig. 1.
  • Figure 1 shows the results of semi-quantitative detection of the stability of the tested siRNA conjugates in vitro lysosomes. The results show that the conjugate of the present disclosure can be maintained in lysosomes for a long time without degradation, showing good stability.
  • Conjugate 4 (provided as a 0.9% sodium chloride aqueous solution having a siRNA concentration of 20 ⁇ M, 12 ⁇ l) and Comparative Sequence 1 (20 ⁇ M, 12 ⁇ l) were mixed with 108 ⁇ L of 90% human plasma (Human plasma, PBS diluted), respectively. Incubate at 37 ° C. 10 ⁇ L of the sample was taken at 0, 2, 4, 6, 8, 24, 48, and 72 hours, and immediately frozen in liquid nitrogen, and frozen in a refrigerator at -80 °C. After sampling at each time point, the above frozen samples were diluted 5 times with 1 ⁇ PBS (pH 7.4), and 10 ⁇ L of each sample was taken for use.
  • 1 ⁇ PBS pH 7.4
  • siRNA 2 ⁇ M, 2 ⁇ l
  • siRNA conjugate siRNA concentration of 2 ⁇ M, 2 ⁇ l
  • a 20% by weight non-denaturing polyacrylamide gel was prepared, and all samples of each of the above-mentioned spare samples were mixed with 4 ⁇ L of loading buffer (20 mM EDTA, 36% by weight glycerol, 0.06% by weight aqueous solution of bromophenol blue). Then, the sample was applied to the above gel, and electrophoresed under a constant current of 80 mA for 60 minutes. After completion of electrophoresis, the cells were stained with 1 ⁇ Sybr Gold dye (Invitrogen, Cat. 11494) for 15 minutes, and the results were as shown in Fig. 2 .
  • Antisense strand UUUGAAGUAUGCCUCAAGGUU (SEQ ID NO: 30)
  • Figure 2 shows the results of semi-quantitative detection of the stability of the tested conjugates in human plasma in vitro.
  • Figure 3 shows the results of semi-quantitative detection of the stability of the tested conjugates in monkey plasma in vitro.
  • siRNA conjugates of the present disclosure did not degrade in cynomolgus monkey plasma until 72 h, showing excellent stability in monkey plasma.
  • Experimental Example 2 This Experimental Example demonstrates the inhibitory activity of the siRNA conjugate of the present disclosure in vitro (in vitro)
  • the HEK293A cells used in this experimental example were provided by the Nucleic Acid Technology Laboratory of the Institute of Molecular Medicine, Peking University, using 20% fetal bovine serum (FBS, Hyclone) and 0.2% by volume of scleromycin double antibody (Penicillin- The cells were cultured in DMEM complete medium (Hyclone) of Streptomycin, Gibco, Invitrogen, and cultured at 37 ° C in an incubator containing 5% CO 2 /95% air.
  • FBS fetal bovine serum
  • scleromycin double antibody Penicillin- The cells were cultured in DMEM complete medium (Hyclone) of Streptomycin, Gibco, Invitrogen, and cultured at 37 ° C in an incubator containing 5% CO 2 /95% air.
  • conjugate 20 targeted complete match target sequence was determined (the nucleotide sequence thereof and the conjugate) The full length nucleotide sequence of the antisense strand is fully complementary).
  • modified siRNA with a DNA seed arm is a powerful tool for mammalian gene silencing with significantly reduced off-target effect.
  • Nucleic Acids Research, 2008.36 (7 a method described in 2136-2151, constructing a detection plasmid, co-transfected with the siRNA conjugate to be evaluated into HEK293A cells, and reacting the expression level of the double luciferase reporter gene to reflect the target of the siRNA conjugate Activity and off-target effects. Specific steps are as follows:
  • the target plasmid was constructed using a psiCHECKTM-2 (PromegaTM) plasmid containing a target sequence that completely complements all 21 nucleotide sequences of the antisense strand in the conjugate to be tested.
  • the target sequence was cloned into the Xho I/Not I site of the psiCHECKTM-2 plasmid.
  • NC is a universal negative control B01001 with no homology between the gene and the gene sequence of the gene.
  • the dual luciferase reporter gene assay kit (Promega, cat. E2940) was used to lyse HEK293A cells according to the instructions for use to detect the double luciferase reporter gene.
  • the Renilla luciferase protein level was normalized to the firefly luciferase protein level. The result is shown in Figure 4.
  • modified siRNA with a DNA seed arm is a powerful tool for mammalian gene silencing with significantly reduced off-target effect.
  • Nucleic Acids Research, 2008.36 (7 , the method described in 2136-2151, constructs a detection plasmid, co-transfected with the conjugate to be tested into HEK293A cells, and reacts the target activity and off-target of the conjugate by the expression level of the double luciferase reporter gene. effect. Specific steps are as follows:
  • plasmids Four recombinant plasmids were constructed using psiCHECKTM-2 (PromegaTM) plasmid, in which GSCM was expressed in the target plasmid, and PSCM, GSSM, and PSSM were expressed as off-target plasmids:
  • GSCM comprising a target sequence which is fully complementary to all 21 nucleotide sequences of the antisense strand in conjugate 4;
  • PSCM which contains a target sequence which is identical to all 21 nucleotide sequences of the antisense strand in conjugate 4;
  • GSSM comprising a target sequence which is fully complementary to the 1-8 nucleotide sequence of the 5' end of the antisense strand of the siRNA to be tested, and the remaining part of the target sequence is antisense to the siRNA to be tested.
  • the 5' end of the strand corresponds to the 9-21 nucleotide sequence, and the sequence is completely non-complementary, that is, the nucleotide at any position of the 9-21 position of the 5' end of the antisense strand in the siRNA to be tested is G.
  • C, A or U the nucleotides at the corresponding positions of the target sequence are T, A, C or G, respectively.
  • PSSM which contains a target sequence which is fully complementary to the nucleotide sequence of 1-8 from the 5' end of the sense strand in the siRNA to be tested, and the remainder of the target sequence and the sense strand 5 in the siRNA to be tested.
  • the nucleotide sequences corresponding to positions 9-19 are corresponding, and the sequences are completely non-complementary, that is, the nucleotides at any position from the 5' position of the sense strand at the 5' end of the sense siRNA are G, C, A. Or U, the nucleotides corresponding to the position of the target sequence are T, A, C or G, respectively.
  • nucleotides T and A are sequentially added to the 3' end of the target sequence.
  • the target sequence was cloned into the Xho I/Not I site of the psiCHECKTM-2 plasmid.
  • HEK293A cells were cultured for 24 hours, the cells were lysed according to the instruction manual using a Dual luciferase reporter gene assay kit (Promega, cat. E2940) to detect the expression level of the double fluorescent reporter gene.
  • a Dual luciferase reporter gene assay kit Promega, cat. E2940
  • Each specific concentration of the conjugate test group was treated with the conjugate-free treatment group as a control.
  • the Renilla luciferase protein level (Ren) was normalized to the firefly luciferase protein level (Fir).
  • the Graphpad 5.0 software log (inhibitor) vs. response-Variable slope function was used to fit the dose-response curve, and the IC50 value of the siRNA-targeted GSCM was calculated according to the dose-response curve.
  • the calculation method is as follows:
  • Y is the expression level of residual mRNA
  • X is the logarithm of the concentration of transfected siRNA
  • Bot is the Y value at the bottom of the steady state period.
  • Top is the Y value at the top of the steady state period.
  • the conjugate 4 exhibited a low off-target effect while having an excellent target mRNA inhibitory effect.
  • Experimental Example 3 This experimental example demonstrates inhibition of HBV mRNA expression by conjugates of the present disclosure in mice
  • mice C57BL/6J-Tg(Alb1HBV)44Bri/J mice were purchased from the Department of Laboratory Animal Science of Peking University Medical School, and the serum HBsAg was detected using the hepatitis B virus surface antigen diagnostic kit (ELISA) (Shanghai Kehua Bio).
  • ELISA hepatitis B virus surface antigen diagnostic kit
  • mice with S/COV>10 were randomly divided into groups (all females), and 4 mice in each group were numbered respectively, and the saline NS control group was added. All animals were dosed according to body weight, administered in a single dose by subcutaneous injection, and conjugate 4 was administered at different doses of 1 mg/kg and 0.1 ml/kg, respectively (0.2 mg/ml and 0.9% chlorine at 0.02 mg/ml).
  • the administration volume is 5 ml/kg.
  • the animals were sacrificed on the 7th day after the administration, and the liver was collected and stored with RNA later (Sigma Aldrich); the liver tissue was homogenized with a tissue homogenizer, and total RNA was extracted by Trizol according to the standard procedure of total RNA extraction.
  • Real-time quantitative PCR was used to detect the expression of HBV mRNA in liver tissue. Specifically, the extracted total RNA was reverse-transcribed into cDNA using the ImpProm-IITM reverse transcription kit (Promega) according to the instructions, followed by fluorescent quantitative PCR reagent. Box (Beijing Kangwei Century Biotechnology Co., Ltd.) tested the inhibition efficiency of siRNA on HBV mRNA expression in liver tissue. In the real-time PCR method, HBV and ⁇ -actin were detected using a ⁇ -actin gene as an internal reference gene, and a primer against HBV and a primer against ⁇ -actin.
  • the expression level of HBV mRNA is expressed by the remaining amount of HBV X gene expression, and is calculated as follows:
  • Remaining amount of HBV X gene expression (copy number of HBV X gene in test group / copy number of ⁇ -actin in test group) / (copy number of HBV X gene in control group / copy number of ⁇ -actin in control group) ⁇ 100%, The figure indicates the amount of HBV X/ ⁇ -actin mRNA expression.
  • the inhibition rate of the conjugate to mRNA (the remaining amount of 1-HBV X gene expression) ⁇ 100%
  • control group was a control group of mice administered with NS in the experiment, and each test group was a group of mice administered with different siRNA conjugates.
  • the results are shown in Figure 6.
  • the above-described conjugate of the present disclosure showed a good inhibitory effect against the target mRNA at a dose of 1 mg/kg of 93.8%.
  • Experimental Example 4 This experiment demonstrates the inhibitory effect of the siRNA conjugate of the present disclosure on HBsAg in a single administration on the M-Tg model.
  • HBV transgenic mice purchased from the Animal Department of Shanghai Public Health Center
  • M-TgHBV mice purchased from the Animal Department of Shanghai Public Health Center
  • serum HBsAg content (6 in each group, all male), which were respectively saline (NS) control group.
  • Blood was taken from the orbital venous plexus of mice before administration (denoted as D0) and on days 7, 14, 21, 28, 42, 56, 70, and 85 after administration, and serum HBsAg levels were measured at each time point.
  • the eyelids take about 0.5 ml of blood each time, and the serum after centrifugation is not less than 200 ⁇ l. Serum HBsAg levels were measured using the HBsAg CLIA kit (Antu Bio, CL0310).
  • Standardized serum HBsAg levels (HssAg content in the test group after administration / HBsAg content in the test group before administration) x 100%.
  • HBsAg inhibition rate (1 - HBsAg content after administration / HBsAg content before administration) ⁇ 100%.
  • the HBsAg content is expressed by how many equivalents (UI) of HBsAg are contained per milliliter (ml) of serum.
  • Figure 7 shows the results of the detection of HBsAg expression by a single administration of the above test siRNA conjugate on the M-Tg model.
  • the experimental data were expressed by X ⁇ SEM, and the data analysis was performed using Graphpad prism 5.0 statistical analysis software.
  • the data is normally distributed and tested for homogeneity of variance. Consistent with normal distribution (p>0.20) and variance (p>0.10): multiple comparisons were performed using LSS method with one-way analysis of variance for multiple comparisons, p ⁇ 0.05 considered statistically significant; non-conforming to normal distribution or variance

Abstract

提供了一种抑制乙型肝炎病毒基因表达的siRNA，及含有该siRNA的药物组合物和缀合物。该siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸。该siRNA含有正义链和反义链，该正义链含有核苷酸序列A，该核苷酸序列A与SEQ ID NO：1所示的核苷酸序列长度相等，且不多于3个核苷酸差异；该反义链含有核苷酸序列B，该核苷酸序列B与SEQ ID NO：2所示的核苷酸序列长度相等，且不多于3个核苷酸差异。

Description

一种核酸、含有该核酸的组合物与缀合物及制备方法和用途 背景技术
乙型病毒性肝炎(又称乙型肝炎或乙肝)是严重威胁全球、特别是中国的一类传染病，目前全球公认的两大类乙肝防止药物为干扰素和核苷类似物，但是这两类药物存在使用后容易产生耐药性或使用受限等多种弊端，如干扰素容易产生不良反应、核苷类药物存在耐药性和停药后复发问题。因此，若能从基因水平沉默病毒的基因表达，阻断HBV的生成和复制，由此从根本上降低病毒代谢和对肝细胞的侵染，无疑将是最为理想的乙肝治疗手段。小干扰RNA(small interfering RNA，siRNA)可基于RNA干扰(RNA interference，RNAi)这一机制，以序列特异性的方式抑制或阻断任何感兴趣的目的基因(例如引发如癌症等疾病的基因)的表达，从而达到治疗疾病的目的。
siRNA稳定化修饰及其递送系统是小RNA药物开发中的两个关键技术。
发明内容
在一些实施方式中，本公开提供了一种能够抑制HBV基因表达的siRNA，该siRNA含有正义链和反义链，所述siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述正义链含有一段核苷酸序列I，所述反义链含有一段核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II至少部分地反向互补形成双链区，其中，所述核苷酸序列I含有核苷酸序列A，所述核苷酸序列A与SEQ ID NO：1所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II含有核苷酸序列B，所述核苷酸序列B与SEQ ID NO：2所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
5’-UCUGUGCCUUCUCAUCUGZ-3’(SEQ ID NO：1)；
5’-Z′CAGAUGAGAAGGCACAGA-3′(SEQ ID NO：2)，
其中，Z为A，Z′为U；
并且，所述核苷酸序列A中包含位置对应于Z的核苷酸Z A，所述核苷酸序列B中包含位置对应于Z′的核苷酸Z′ B，所述Z′ B是所述反义链5′末端的第一个核苷酸。
在一些实施方式中，本公开提供了一种药物组合物，所述药物组合物含有本公开的siRNA和药学上可接受的载体。
在一些实施方式中，本公开提供了一种siRNA缀合物，所述siRNA缀合物 含有本公开提供的siRNA以及缀合连接至该siRNA的缀合基团。
在一些实施方式中，本公开提供了本公开的siRNA和/或药物组合物和/或siRNA缀合物在制备用于治疗和/或预防由乙型肝炎病毒的感染引起的病理状况或疾病的药物中的用途。
在一些实施方式中，本公开提供了一种治疗和/或预防由乙型肝炎病毒的感染引起的病理状况或疾病的方法，所述方法包括将有效量的本公开的siRNA和/或药物组合物和/或siRNA缀合物给予有需要的患者。
在一些实施方式中，本公开提供了一种试剂盒，所述试剂盒含有本公开的siRNA和/或药物组合物和/或siRNA缀合物。
附图说明
图1表示所所测试siRNA缀合物在体外Tritosome中的稳定性半定量检测结果。
图2表示所测试siRNA缀合物在体外人血浆中的稳定性半定量检测结果。
图3表示所测试siRNA缀合物在体外猴血浆中的稳定性半定量检测结果。
图4表示缀合物20在体外的抑制活性结果。
图5表示缀合物4在体外psiCHECK系统中IC50及脱靶效应的检测结果。
图6表示缀合物4在小鼠中对HBV mRNA表达的抑制结果。
图7表示缀合物4在M-Tg模型上单次给药对HBsAg表达抑制作用的检测结果。
具体实施方式
以下对本公开的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本公开，并不用于限制本公开。
定义
在本公开中，HBV基因是指DNA序列如Genbank注册号NC_003977.1所示的基因。
在上文及下文中，如无特别说明，大写字母C、G、U、A、T表示核苷酸的碱基组成；小写字母d表示该字母d右侧相邻的一个核苷酸为脱氧核糖核苷酸；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸；小写字母s表示与该字母s左右相邻的两个核苷酸之间为硫代磷酸酯基连接；P1表示该 P1右侧相邻的一个核苷酸为5′-磷酸核苷酸或5′-磷酸类似物修饰的核苷酸，尤指乙烯基磷酸酯修饰的核苷酸(以下实施例中以VP表示)、5′-磷酸核苷酸(以下实施例中以P表示)或5′-硫代磷酸酯修饰的核苷酸(以下实施例中以Ps表示)。
在上文及下文中，所述氟代修饰的核苷酸指核苷酸的核糖基2′位的羟基被氟取代形成的核苷酸，非氟代修饰的核苷酸指核苷酸的核糖基2′位的羟基被非氟基团取代形成的核苷酸或核苷酸类似物，核苷酸类似物指能够在核酸中代替核苷酸，但结构不同于腺嘌呤核糖核苷酸、鸟嘌呤核糖核苷酸、胞嘧啶核糖核苷酸、尿嘧啶核糖核苷酸或胸腺嘧啶脱氧核糖核苷酸的基团。如异核苷酸、桥联的核苷酸(bridged nucleic acid，简称BNA)或无环核苷酸。所述甲氧基修饰的核苷酸指核糖基的2′-羟基被甲氧基取代而形成的核苷酸。
在本文的上下文中，“互补”或“反向互补”一词可互相替代使用，并具有本领域技术人员周知的含义，即，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。在DNA中，嘌呤碱基腺嘌呤(A)始终与嘧啶碱基胸腺嘧啶(T)(或者在RNA中为尿嘧啶(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。每个碱基对都包括一个嘌呤和一个嘧啶。当一条链上的腺嘌呤始终与另一条链上的胸腺嘧啶(或尿嘧啶)配对，以及鸟嘌呤始终与胞嘧啶配对时，两条链被认为是彼此相互补的，以及从其互补链的序列中可以推断出该链的序列。与此相应地，“错配”在本领域中意指在双链核酸中，对应位置上的碱基并未以互补的形式配对存在。
在上文及下文中，如无特别说明，基本上反向互补是指所涉及的两段核苷酸序列之间存在不多于3个的碱基错配；实质上反向互补是指两段核苷酸序列之间存在不多于1个的碱基错配；完全互补是指两段核苷酸序列之间不存在碱基错配。
在上文及下文中，一个核苷酸序列与另外一个核苷酸序列存在“核苷酸差异”，是指前者与后者相比，相同位置的核苷酸的碱基种类发生了改变，例如，在后者中一个核苷酸碱基为A时，在前者的相同位置处的对应核苷酸碱基为U、C、G或者T的情况下，认定为两个核苷酸序列之间在该位置处存在核苷酸差异。在一些实施方式中，以无碱基核苷酸或其等同物代替原位置的核苷酸时，也可认为在该位置处产生了核苷酸差异。
在上文及下文中，特别是在描述本公开的缀合分子的制备方法或siRNA缀合物的制备方法时，除非特别说明，所述核苷单体(nucleoside monomer)指， 根据欲制备的siRNA或siRNA缀合物中核苷酸的种类和顺序，亚磷酰胺固相合成中使用的修饰或未修饰的核苷亚磷酰胺单体(unmodified or modified RNA phosphoramidites，有时RNA phosphoramidites也称为Nucleoside phosphoramidites)。亚磷酰胺固相合成为本领域技术人员所公知的RNA合成中所用的方法。本公开所用的核苷单体均可商购得到。
如本文所使用的，不介于两个字母之间或两个符号之间的短横(“-”)是用于指示取代基附着点的位置。例如：-C 1-C 10烷基-NH 2通过C 1-C 10烷基而附着。
如本文所使用的，“任选的”或“任选地”是指其后描述的事件或状况可以发生或不发生，并且所述描述包括事件或状况发生的情况和其中不发生的情况。例如，“任选的取代的烷基”包括下面定义的“烷基”和“取代烷基”。本领域技术人员将理解的是，对于包含一个或多个取代基的任何基团，这些基团不打算引入空间上不切实际、合成上不可行和/或内在不稳定的任何取代或取代型式。
如本文所使用的，“烷基”是指具有指定数量的碳原子的直链和支链，所述数量通常为1到20个碳原子，例如1至10个碳原子，如1至8个或1至6个碳原子。例如，C 1-C 6烷基包含1至6个碳原子的直链和支链烷基。当对具有特定数量的碳的烷基残基进行命名时，旨在涵盖所有具有该数量的碳的支链和直链形式；因此，例如，“丁基”意味着包括正丁基、仲丁基、异丁基和叔丁基；“丙基”包括正丙基和异丙基。亚烷基是烷基的子集，指与烷基相同、但具有两个附着点的残基。
如本文所使用的，“烯基”是指具有至少一个碳-碳双键的不饱和支链或直链烷基，所述碳-碳双键是通过从母体烷基的相邻碳原子中除去一个氢分子而获得的。该基团可以处于双键的顺式或反式构型。典型的烯基基团包括但不限于：乙烯基；丙烯基，如丙-1-烯-1-基、丙-1-烯-2-基、丙-2-烯-1-基(烯丙基)、丙-2-烯-2-基；丁烯基，例如丁-1-烯-1-基、丁-1-烯-2-基、2-甲基丙-1-烯-1-基、丁-2-烯-1-基、丁-2-烯-2-基、丁-1，3-二烯-1-基、丁-1，3-二烯-2-基等等。在某些实施方式中，烯基基团具有2到20个碳原子，而在其他实施方式中，具有2至10个、2至8个或2至6个碳原子。亚烯基是烯基的一个子集，指与烯基相同、但具有两个附着点的残基。
如本文所使用的，“炔基”是指具有至少一个碳-碳三键的不饱和支链或直链烷基，所述碳-碳三键是通过从母体烷基的相邻碳原子中除去两个氢分子而获得的。典型的炔基基团包括但不限于：乙炔基；丙炔基，如丙-1-炔-1-基，丙-2-炔-1-基；丁炔基，例如丁-1-炔-1-基，丁-1-炔-3-基，丁-3-炔-1-基等。在某些实 施方式中，炔基具有2到20个碳原子，而在其他实施方式中，具有2至10、2至8或2至6个碳原子。亚炔基是炔基的一个子集，指的是与炔基相同、但有两个附着点的残基。
如本文所使用的，“烷氧基”是指通过氧桥附着的指定数量碳原子的烷基，例如，甲氧基、乙氧基、丙氧基、异丙氧基、正丁氧基、仲丁氧基、叔丁氧基、戊氧基、2-戊氧基、异戊氧基、新戊氧基、己氧基、2-己氧基、3-己氧基、3-甲基戊氧基等。烷氧基通常具有1至10个、1至8个、1至6个，或1至4个通过氧桥附着的碳原子。
如本文所使用的，“芳基”是指衍生自芳香族单环或多环烃环系统、通过从环碳原子中除去氢原子而形成的基团。所述芳香族单环或多环烃环系统仅含有氢和6至18个碳原子的碳，其中所述环系统中的至少一个环是完全不饱和的，即，其根据Hückel理论包含环状、离域的(4n+2)π-电子系统。芳基包括但不限于苯基、芴基和萘基等基团。亚芳基是芳基的一个子集，指与芳基相同、但具有两个附着点的残基。
如本文所使用的，“环烷基”是指非芳香碳环，通常具有3至7个环状碳原子。环可以是饱和的，或具有一个或多个碳-碳双键。环烷基的实例包括环丙基、环丁基、环戊基、环戊烯基、环己基和环己烯基，以及桥联和笼状环基团，如降冰片烷(norbornane)。
如本文所使用的，“卤素取代基”或“卤”指氟代、氯代、溴代和碘代，术语“卤素”包括氟、氯、溴和碘。
如本文所使用的，“卤代烷基”是指指定数量的碳原子被一个或多个、直至最大允许数量的卤素原子取代的如上述所定义的烷基。卤代烷基的实例包括但不限于三氟甲基、二氟甲基、2-氟乙基和五氟乙基。
“杂环基”是指一个稳定的3-到18-元非芳香环基，包含2-12个碳原子和1-6个杂原子，所述杂原子选自氮、氧和硫。除非说明书中另有说明，杂环基是单环、双环、三环或四环系统，可包括稠环或桥环系统。杂环自由基中的杂原子可以任选地被氧化。一个或多个氮原子(如果存在的话)任选地被季铵化。杂环基是部分饱和或完全饱和的。杂环基可以通过环的任何原子附着至分子的其余部分。此类杂环基的实例包括但不限于：二噁烷基、噻吩基[1，3]二硫酰基、十氢异喹啉基、咪唑啉基、咪唑烷基、异噻唑烷基、异恶唑烷基、吗啉基、八氢吲哚基、八氢异吲哚基、2-氧杂哌嗪基、2-氧杂哌啶基、2-氧杂嘧啶基、恶唑烷基、哌啶基、哌嗪基、4-哌啶酮基、吡咯烷基、吡唑烷基、奎宁环基、噻唑 烷基、四氢呋喃基、三硫酰基、四氢吡喃基、硫吗啉基、硫杂吗啉基、1-氧杂硫吗啉基和1，1-二氧杂硫吗啉基。
“杂芳基”指由3-至18-元芳香环自由基衍生而成的基团，其包含2个至17个碳原子和选自氮、氧和硫的1至6个杂原子。如本文所使用的，杂芳基可以是单环、双环、三环或四环系统，其中环系统中的至少一个环是完全不饱和的，即，根据Hückel理论，包含环状离域(4n+2)π-电子体系。杂芳基包括稠环或桥环系统。杂芳基中的杂原子被任选地氧化。一个或多个氮原子(如果存在的话)任选地被季铵化。杂芳基通过环中的任何原子附着至分子的其余部分。杂芳基的实例包括但不限于：氮杂环庚三烯基、吖啶基、苯并咪唑基、苯并吲哚基、1，3-苯并二恶唑基、苯并呋喃基、苯并恶唑基、苯并[d]噻唑基、苯并噻二唑基、苯并[b][1，4]二恶唑基、苯并[b][1，4]恶唑基、1，4-苯并二恶唑基、苯并萘并呋喃基、苯并二唑基、苯并二氧杂苯基、苯并吡喃基、苯并吡喃酮基、苯并呋喃基、苯并呋喃酮基、苯并噻吩基、苯并噻吩[3，2-d]嘧啶基、苯并三唑基、苯并[4，6]咪唑[1，2-a]吡啶基、咔唑基、噌啉基、环戊基[d]嘧啶基、6，7-二氢-5H-环戊基[4，5]噻吩[2，3-d]嘧啶基、5，6-二氢苯并[h]喹唑啉基、5，6-二氢苯并[h]辛诺林基、6，7-二氢-5H-苯并[6，7]环庚[1，2-c]哒嗪基、二苯并呋喃基、二苯并噻吩基、呋喃基、呋喃酮基、呋喃[3，2-c]吡啶基、5，6，7，8，9，10-六氢环庚烷[d]嘧啶基、5，6，7，8，9，10-六氢环辛酸[d]哒嗪基、5，6，7，8，9，10-六氢环辛酸[d]吡啶基、异噻唑基、吲唑基、咪唑基、吲哚基、异吲哚基、二氢吲哚基、异二氢氮茚基、异喹啉基、吲哚嗪基、异恶唑基、5，8-甲基-5，6，7，8-四氢喹唑啉基、萘啶酮基、1，6-萘啶酮基、恶二唑基、2-氧氮杂庚基、恶唑基、恶草酰基、5，6，6a，7，8，9，10，10a-八氢苯并[H]喹唑啉基、1-苯基-1H-吡咯基、吩嗪基、吩噻嗪基、吩恶嗪基、邻苯二甲酰基、蝶啶基、嘌呤基、吡咯基、吡唑基、吡唑并[3，4-d]嘧啶基、吡啶基、吡啶并[3，2-d]嘧啶基、吡啶并[3，4-d]嘧啶基、吡嗪基、嘧啶基、哒嗪基、吡咯基、喹唑啉基、喹喔啉基、喹啉基、异喹啉基、四氢喹啉基、5，6，7，8-四氢喹唑啉基、5，6，7，8-四氢苯并[4，5]噻吩[2，3-d]嘧啶基、6，7，8，9四氢-5H-环庚烷[4，5]噻吩[2，3-d]嘧啶基、5，6，7，8-四氢吡啶并[4，5-c]哒嗪基、噻唑基、噻二唑基、三唑基、四唑基、三嗪基、噻吩[2，3-d]嘧啶基、噻吩[3，2-d]嘧啶基、噻吩[2，3-c]普萘基和噻吩基。
在本公开中可以使用各种羟基保护基团。一般来说，保护基团使化学官能度对特定的反应条件不敏感，并且可以在分子中的该官能度上附加以及去除，而不实质上损害分子的其余部分。代表性的羟基保护基团公开于Beaucage等人，Tetrahedron 1992，48，2223-2311，以及Greeneand Wuts，Protective Groups in  Organic Synthesis，Chapter 2，2d ed，John Wiley&Sons，New York，1991中，以引用的方式将上述文献整体并入本文。在一些实施方式中，保护基团在碱性条件下稳定，但可以在酸性条件下脱除。在一些实施方式中，本文可使用的羟基保护基的非排他性实例包括二甲氧基三苯甲基(DMT)、单甲氧基三苯甲基、9-苯基黄嘌呤-9-基(Pixyl)和9-(对甲氧基苯基)黄嘌呤-9-基(Mox)。在一些实施方式中，本文可使用的羟基保护基的非排他性实例包括Tr(三苯甲基)、MMTr(4-甲氧基三苯甲基)、DMTr(4，4’-二甲氧基三苯甲基)和TMTr(4，4’，4”-三甲氧基三苯甲基)。
“受试者”一词，如本文所使用的，指任何动物，例如哺乳动物或有袋动物。本公开的主题包括但不限于人类、非人灵长类(例如，恒河猴或其他类型的猕猴)、小鼠、猪、马、驴、牛、绵羊、大鼠和任何种类的家禽。
如本文所使用的，“治疗方法”、“治疗”、“减轻”或“改善”可在此处互换使用。这些术语指的是获得有益的或期望的结果的方法，包括但不限于治疗益处。“治疗益处”意味着根除或改善被治疗的潜在障碍。此外，治疗益处通过根除或改善与潜在障碍相关的一个或多个生理症状，从而在患者中观察到改善而获得，尽管患者可能仍然受到潜在障碍的折磨。
如本文所使用的，“防止”和“预防”可互换使用。这些术语指获得有益或期望的结果的方法，包括但不限于预防性益处。为了获得“预防性益处”，可将缀合物或组合物给予有罹患特定疾病风险的患者，或给予报告疾病的一种或多种病理症状的患者，即便可能该疾病的诊断尚未作出。
siRNA
本公开提供了一种能够抑制乙型肝炎病毒基因表达的siRNA。
本公开的siRNA含有核苷酸基团作为基本结构单元，本领域技术人员公知，所述核苷酸基团含有磷酸基团、核糖基团和碱基，在此不再赘述。
CN102140458B公开了一种特异性抑制HBV基因的siRNA，并对该siRNA的多种化学修饰策略进行了研究。该研究发现，不同修饰策略会对siRNA的稳定性、生物活性及细胞毒性等指标产生截然不同的影响。在该研究中，证实了7种有效的修饰方式，与未经修饰的siRNA相比，其中一种修饰方式所得的siRNA在提高血液稳定性的同时，还保持了与未经修饰的siRNA基本相当的抑制活性。
本公开的siRNA含有正义链和反义链，所述siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述正义链含有一段核苷酸序列I，所述 反义链含有一段核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II至少部分地反向互补形成双链区，其中，所述核苷酸序列I含有核苷酸序列A，所述核苷酸序列A与SEQ ID NO：1所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II含有核苷酸序列B，所述核苷酸序列B与SEQ ID NO：2所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
5’-UCUGUGCCUUCUCAUCUGZ-3’(SEQ ID NO：1)；
5’-Z′CAGAUGAGAAGGCACAGA-3’(SEQ ID NO：2)，
其中，Z为A，Z′为U；
并且，所述核苷酸序列A中包含位置对应于Z的核苷酸Z A，所述核苷酸序列B中包含位置对应于Z′的核苷酸Z′ B，所述Z′ B是所述反义链5′末端的第一个核苷酸。
在上文与下文中，“位置对应”是指从核苷酸序列相同端起算，处于核苷酸序列中相同的位置。例如，核苷酸序列A的3′端第1个核苷酸是位置对应于SEQ ID NO：1的3′端第1个核苷酸的核苷酸。
在一些实施方式中，所述正义链仅包含核苷酸序列I，所述反义链仅包含核苷酸序列II。
在一些实施方式中，所述正义链包含核苷酸序列I，所述反义链仅包含核苷酸序列II。在一些实施方式中，所述核苷酸序列A与SEQ ID NO：1所示的核苷酸序列之间不多于1个核苷酸差异，和/或所述核苷酸序列B与SEQ ID NO：2所示的核苷酸序列之间不多于1个核苷酸差异。
在一些实施方式中，所述核苷酸序列B与SEQ ID NO：2所示的核苷酸序列之间的核苷酸差异包括Z′ B位置处的差异，且Z′ B选自A、C或G。在一些实施方式中，所述核苷酸差异为Z′ B位置处的差异，Z′ B选自A、C或G，并且Z A是与Z′ B互补的核苷酸。这些核苷酸差异并不会显著降低siRNA缀合物的靶基因抑制能力，而这些包含核苷酸差异的siRNA缀合物也在本公开的保护范围之内。
在一些实施方式中，所述核苷酸序列A和所述核苷酸序列B基本上反向互补、实质上反向互补或完全反向互补；所述基本上反向互补是指两个核苷酸序列之间存在不多于3个的碱基错配；所述实质上反向互补是指两个核苷酸序列之间存在不多于1个的碱基错配；完全反向互补是指两个核苷酸序列之间没有错配。
在一些实施方式中，核苷酸序列A是SEQ ID NO：3所示的核苷酸序列， 核苷酸序列B是SEQ ID NO：4所示的核苷酸序列：
5′-UCUGUGCCUUCUCAUCUGZ A-3′(SEQ ID NO：3)；
5′-Z′ BCAGAUGAGAAGGCACAGA-3’(SEQ ID NO：4)，
其中，所述Z′ B是反义链5′末端的第一个核苷酸，Z A选自A、U、G或C，并且Z′ B是与Z A互补的核苷酸；在一些实施方式中，Z A为A，Z′ B为U；
并且，所述正义链和反义链长度相同或不同，所述正义链的长度为19-23个核苷酸，反义链的长度为20-26个核苷酸。这样，本公开提供的siRNA正义链和反义链的长度比可以是19/20、19/21、19/22、19/23、19/24、19/25、19/26、20/20、20/21、20/22、20/23、20/24、20/25、20/26、21/20、21/21、21/22、21/23、21/24、21/25、21/26、22/20、22/21、22/22、22/23、22/24、22/25、22/26、23/20、23/21、23/22、23/23、23/24、23/25或23/26。在一些实施方式中，所述siRNA正义链和反义链的长度比为19/21、21/23或23/25。
在一些实施方式中，所述正义链和反义链长度相同，所述核苷酸序列I还含有核苷酸序列III，所述核苷酸序列II还含有核苷酸序列IV，核苷酸序列III和核苷酸序列IV长度各自独立地为1-4个核苷酸；所述核苷酸序列III连接在核苷酸序列A的5’末端，所述核苷酸序列IV连接在核苷酸序列B的3’末端，所述核苷酸序列III和所述核苷酸序列IV长度相等。
所述核苷酸序列III和核苷酸序列IV可以互补或不互补，为了增加siRNA的稳定性，在一些实施方式中，核苷酸序列III和核苷酸序列IV至少部分互补；在一些实施方式中，核苷酸序列III和核苷酸序列IV 80％以上的碱基互补，或者90％以上的碱基互补；在一些实施方式中，核苷酸序列III和核苷酸序列IV实质上反向互补或完全反向互补；所述实质上反向互补是指两个核苷酸序列之间存在不多于1个的碱基错配；完全反向互补是指两个核苷酸序列之间没有错配；在一些实施方式中，核苷酸序列III和核苷酸序列IV完全反向互补。由此，所述siRNA正义链和反义链等长，其长度比为20/20、21/21、22/22或23/23。在一些实施方式中，所述siRNA正义链和反义链的长度比为21/21或23/23。
在一些实施方式中，所述核苷酸序列III和核苷酸序列IV的长度均为1个核苷酸，核苷酸序列III的碱基为G，核苷酸序列IV的碱基为C；此时，正义链和反义链的长度比为20/20；或者，核苷酸序列III和IV的长度均为2个核苷酸，按照5’末端到3’末端的方向，核苷酸序列III的碱基组成为CG，核苷酸序列IV的碱基组成为GC；此时，正义链和反义链的长度比为21/21；或者，核苷酸序列III和IV的长度均为3个核苷酸，按照5’末端到3’末端的方向，核 苷酸序列III的碱基组成为CCG，核苷酸序列IV的碱基组成为CGG；此时，正义链和反义链的长度比为22/22；或者，核苷酸序列III和IV的长度均为4个核苷酸，按照5’末端到3’末端的方向，核苷酸序列III的碱基组成为CCCG，核苷酸序列IV的碱基组成为CGGG；此时，正义链和反义链的长度比为23/23。在一些实施方式中，所述核苷酸序列III和核苷酸序列IV的长度为2个核苷酸，按照5’末端到3’末端的方向，核苷酸序列III的碱基组成为CG，核苷酸序列IV的碱基组成为GC；此时，正义链和反义链的长度比为21/21。
在一些实施方式中，核苷酸序列III和核苷酸序列IV的长度相同，并且完全反向互补，因此，给出了核苷酸序列III的碱基，核苷酸序列IV的碱基也就确定了。
在一些实施方式中，所述正义链和反义链长度不同，所述核苷酸序列II还含有核苷酸序列V，核苷酸序列V的长度为1至3个核苷酸，连接在所述反义链的3’末端，构成反义链的3’悬垂末端。由此，本公开提供的siRNA正义链和反义链的长度比可以是19/20、19/21、19/22、20/21、20/22、20/23、21/22、21/23、21/24、22/23、22/24、22/25、23/24、23/25或23/26。在一些实施方式中，所述核苷酸序列V的长度为2个核苷酸，由此，本公开提供的siRNA正义链和反义链的长度比可以是19/21、21/23或23/25。
所述核苷酸序列V中的每一个核苷酸可以是任意的核苷酸，为了便于合成并节约合成成本，所述核苷酸序列V为连续的2个胸腺嘧啶脱氧核糖核苷酸(TT)或连续的2个尿嘧啶核糖核苷酸(UU)；为了提高siRNA反义链与靶mRNA的亲和力，核苷酸序列V与靶mRNA的相应位置的核苷酸互补。因此，在一些实施方式中，本公开的siRNA的正义链和反义链的长度之比为19/21或21/23，此时，本公开的siRNA具有更好的mRNA沉默活性。
在一些实施方式中，所述siRNA的正义链含有如SEQ ID NO：3所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO：4所示的核苷酸序列：
5’-UCUGUGCCUUCUCAUCUGZ A-3’(SEQ ID NO：3)；
5’-Z′ BCAGAUGAGAAGGCACAGACG-3’(SEQ ID NO：4)；其中，所述Z′ B是反义链5′末端的第一个核苷酸，Z A选自A、U、G或C，并且Z′ B是与Z A互补的核苷酸。
根据本公开一些具体的实施例，本公开所述siRNA为siHBVX1
正义链：5’-UCUGUGCCUUCUCAUCUGZ-3’(SEQ ID NO：1)，
反义链：5’-Z′CAGAUGAGAAGGCACAGACG-3’(SEQ ID NO：5)，
其中，Z为A，Z′为U。
如前所述，本公开的siRNA中的核苷酸各自独立地为修饰或未修饰的核苷酸。在一些实施方式中，本公开的siRNA中的核苷酸为未经修饰的核苷酸；在一些实施方式中，本公开的siRNA中的部分或全部核苷酸为修饰的核苷酸，核苷酸基团上的这些修饰不会导致本公开的siRNA缀合物抑制乙肝病毒基因表达的功能明显削弱或丧失。
在一些实施方式中，本公开的siRNA至少含有1个修饰的核苷酸。在本公开的上下文中，所使用的术语“修饰的核苷酸”是指核苷酸的核糖基2′位羟基被其他基团取代形成的核苷酸或核苷酸类似物，或者核苷酸上的碱基是经修饰的碱基的核苷酸。所述修饰的核苷酸不会导致siRNA抑制基因表达的功能明显削弱或丧失。例如，可以选择J.K.Watts，G.F.Deleavey，and M.J.Damha，Chemically modified siRNA：tools and applications.Drug Discov Today，2008，13(19-20)：842-55中公开的修饰的核苷酸。
在一些实施方式中，本公开提供的siRNA的正义链或所述反义链中的至少一个核苷酸为修饰的核苷酸，和/或至少一个磷酸酯基为具有修饰基团的磷酸酯基；换句话说，所述正义链和所述反义链中至少一条单链的磷酸-糖骨架中的磷酸酯基和/或核糖基的至少一部分为具有修饰基团的磷酸酯基和/或具有修饰基团的核糖基。
在一些实施方式中，所述正义链和/或所述反义链中的全部核苷酸均为修饰的核苷酸。在一些实施方式中，本公开提供的siRNA的正义链和所述反义链中的每一个核苷酸独立地为氟代修饰的核苷酸或非氟代修饰的核苷酸。
本公开的发明人惊奇地发现，本公开所述的siRNA在动物实验中获得了血浆中稳定性和基因沉默效率的高度平衡。
在一些实施方式中，所述氟代修饰的核苷酸位于核苷酸序列A和核苷酸序列B中，并且，按照5′末端到3′末端的方向，所述核苷酸序列A的第7、8、9位的核苷酸为氟代修饰的核苷酸；按照5′末端到3′末端的方向，所述核苷酸序列B的第2、6、14、16位的核苷酸为氟代修饰的核苷酸。
在一些实施方式中，所述氟代修饰的核苷酸位于核苷酸序列A和核苷酸序列B中，所述核苷酸序列A中氟代修饰的核苷酸不多于5个，并且，按照5′末端到3′末端的方向，所述核苷酸序列A的第7、8、9位的核苷酸为氟代修饰的核苷酸；所述核苷酸序列B中氟代修饰的核苷酸不多于7个，并且，所述核苷酸序列B的第2、6、14、16位的核苷酸为氟代修饰的核苷酸。
在一些实施方式中，按照5′末端到3′末端的方向，在所述正义链中，所述核苷酸序列A的第7、8、9位或者5、7、8、9位的核苷酸为氟代修饰的核苷酸，所述正义链中其余位置的核苷酸为非氟代修饰的核苷酸；按照5′末端到3′末端的方向，在所述反义链中，所述核苷酸序列B的第2、6、14、16位或者2、6、8、9、14、16位的核苷酸为氟代修饰的核苷酸，所述反义链中其余位置的核苷酸为非氟代修饰的核苷酸。
在本公开的上下文中，“氟代修饰的核苷酸”指核苷酸的核糖基2′位的羟基被氟取代形成的核苷酸，其具有以下式(101)所示的结构。“非氟代修饰的核苷酸”指核苷酸的核糖基2′位的羟基被非氟基团取代形成的核苷酸或核苷酸类似物。在一些实施方式中，每一个非氟代修饰的核苷酸独立地选自核苷酸的核糖基2′位的羟基被非氟基团取代形成的核苷酸或核苷酸类似物中的一种。
这些核糖基2′位的羟基被非氟基团取代形成的核苷酸是本领域技术人员所公知的，这些核苷酸可以选自2′-烷氧基修饰的核苷酸、2′-经取代的烷氧基修饰的核苷酸、2′-烷基修饰的核苷酸、2′-经取代的烷基修饰的核苷酸、2′-氨基修饰的核苷酸、2′-经取代的氨基修饰的核苷酸、2′-脱氧核苷酸中的一种。
在一些实施方式中，2′-烷氧基修饰的核苷酸为甲氧基修饰的核苷酸(2′-OMe)，如式(102)所示。2′-经取代的烷氧基修饰的核苷酸，例如可以是2′-O-甲氧基乙基修饰的核苷酸(2′-MOE)，如式(103)所示。2′-氨基修饰的核苷酸(2′-NH2)如式(104)所示。2′-脱氧核苷酸(DNA)如式(105)所示。
Figure PCTCN2018118107-appb-000001
核苷酸类似物指能够在核酸中代替核苷酸，但结构不同于腺嘌呤核糖核苷酸、鸟嘌呤核糖核苷酸、胞嘧啶核糖核苷酸、尿嘧啶核糖核苷酸或胸腺嘧啶脱氧核糖核苷酸的基团。在一些实施方式中，核苷酸类似物可以是如异核苷酸、桥联的核苷酸(bridged nucleic acid，简称BNA)或无环核苷酸。
BNA是指受约束的或不能接近的核苷酸。BNA可以含有五元环、六元环、或七元环的具有“固定的”C3′-内切糖缩拢的桥联结构。通常将该桥掺入到该核糖的2′-、4′-位处以提供一个2′，4′-BNA核苷酸，如LNA、ENA、cET BNA等，其中，LNA如式(106)所示，ENA如式(107)所示，cET BNA如式(108)所示。
Figure PCTCN2018118107-appb-000002
无环核苷酸是核苷酸的糖环被打开形成的一类核苷酸，如解锁核酸(UNA)或甘油核酸(GNA)，其中，UNA如式(109)所示，GNA如式(110)所示。
Figure PCTCN2018118107-appb-000003
上述式(109)和式(110)中，R选自H、OH或烷氧基(O-烷基)。
异核苷酸是指核苷酸中碱基在核糖环上的位置发生改变而形成的化合物，例如，碱基从核糖环的1′-位移动至2′-位或3′-位而形成的化合物，如式(111)或(112)所示。
Figure PCTCN2018118107-appb-000004
上述式(111)-式(112)化合物中，Base表示碱基，例如A、U、G、C或T；R选自H、OH、F或者如上所述的非氟基团。
在一些实施方式中，核苷酸类似物选自异核苷酸、LNA、ENA、cET、UNA和GNA中的一种。在一些实施方式中，每一个非氟代修饰的核苷酸均为甲氧基修饰的核苷酸，在上文和下文中，所述甲氧基修饰的核苷酸指核糖基的2′-羟基被甲氧基取代而形成的核苷酸。
在上文及下文中，“氟代修饰的核苷酸”、“2′-氟修饰的核苷酸”、“核糖基团的2′-羟基被氟取代的核苷酸”和“2′-氟代核糖基”意义相同，均指核苷酸的2′-羟基被氟取代，而形成的具有如式(207)所示结构的化合物；“甲氧基修饰的核苷酸”、“2′-甲氧基修饰的核苷酸”、“核糖基团的2′-羟基被甲氧基取代的核苷酸”和“2′-甲氧基核糖基”意义相同，均指核苷酸核糖基团的2′-羟基被甲氧基取代而形成的具有如式(208)所示结构的化合物。
在一些实施方式中，本公开的siRNA是具有以下修饰的siRNA：按照5′末 端到3′末端的方向，在所述正义链中，所述核苷酸序列A的第7、8、9位或者第5、7、8、9位的核苷酸为氟代修饰的核苷酸，所述正义链中其余位置的核苷酸为甲氧基修饰的核苷酸；在所述反义链中，所述核苷酸序列B的第2、6、14、16位或者第2、6、8、9、14、16位的核苷酸为氟代修饰的核苷酸，所述反义链中其余位置的核苷酸为甲氧基修饰的核苷酸。
在一些实施方式中，本公开的siRNA是具有以下修饰的siRNA：按照5’末端到3’末端的方向，所述siRNA的正义链中核苷酸序列A的第5、7、8和9位的核苷酸为氟代修饰的核苷酸，siRNA的正义链的其余位置的核苷酸为甲氧基修饰的核苷酸，并且，按照5’末端到3’末端的方向，所述siRNA的反义链中核苷酸序列B的第2、6、8、9、14和16位的核苷酸为氟代修饰的核苷酸，siRNA的反义链其余位置的核苷酸为甲氧基修饰的核苷酸；
或者，按照5’末端到3’末端的方向，所述siRNA的正义链中核苷酸序列A的第5、7、8和9位的核苷酸为氟代修饰的核苷酸，siRNA的正义链的其余位置的核苷酸为甲氧基修饰的核苷酸，并且，按照5’末端到3’末端的方向，所述siRNA的反义链中核苷酸序列B的第2、6、14和16位的核苷酸为氟代修饰的核苷酸，siRNA的反义链其余位置的核苷酸为甲氧基修饰的核苷酸；
或者，按照5’末端到3’末端的方向，所述siRNA的正义链中核苷酸序列A的第7、8和9位的核苷酸为-氟代修饰的核苷酸，siRNA的正义链的其余位置的核苷酸为甲氧基修饰的核苷酸，并且，按照5’末端到3’末端的方向，所述siRNA的反义链中核苷酸序列B的第2、6、14和16位的核苷酸为氟代修饰的核苷酸，siRNA的反义链其余位置的核苷酸为甲氧基修饰的核苷酸。
换句话说，该siRNA的磷酸-糖骨架中的核糖基分别具有如下修饰基团：按照5’末端到3’末端的方向，所述siRNA的正义链中核苷酸序列A的第5、7、8和9位的糖基为2’-氟代核糖基，siRNA的正义链的其余位置核苷酸的糖基为2’-甲氧基核糖基，并且，按照5’末端到3’末端的方向，所述siRNA的反义链中核苷酸序列B的第2、6、8、9、14和16位的糖基为2’-氟代核糖基，siRNA的反义链其余位置核苷酸的糖基为2’-甲氧基核糖基；
或者，按照5’末端到3’末端的方向，所述siRNA的正义链中核苷酸序列A的第5、7、8和9位的糖基为2’-氟代核糖基，siRNA的正义链的其余位置核苷酸的糖基为2’-甲氧基核糖基，并且，按照5’末端到3’末端的方向，所述siRNA的反义链中核苷酸序列B的第2、6、14和16位的糖基为2’-氟代核糖基，siRNA的反义链其余位置核苷酸的糖基为2’-甲氧基核糖基；
或者，按照5’末端到3’末端的方向，所述siRNA的正义链中核苷酸序列A的第7、8和9位的糖基为2’-氟代核糖基，siRNA的正义链的其余位置核苷酸的糖基为2’-甲氧基核糖基，并且，按照5’末端到3’末端的方向，所述siRNA的反义链中核苷酸序列B的第2、6、14和16位的糖基为2’-氟代核糖基，siRNA的反义链其余位置核苷酸的糖基为2’-甲氧基核糖基。
在一些实施方式中，本公开提供的siRNA为siHBVX2或siHBVX3：
siHBVX2
正义链：
5’-UmCmUmGmUmGmCfCfUfUmCmUmCmAmUmCmUmGmAm-3’(SEQ ID NO：6)，
反义链：
5’-UmCfAmGmAmUfGmAmGmAmAmGmGmCfAmCfAmGmAmCmGm-3′(SEQ ID NO：7)，
siHBVX3
正义链：
5’-UmCmUmGmUfGmCfCfUfUmCmUmCmAmUmCmUmGmAm-3’(SEQ ID NO：8)，
反义链：
5’-UmCfAmGmAmUfGmAfGfAmAmGmGmCfAmCfAmGmAmCmGm-3’(SEQ ID NO：9)，
其中，大写字母C、G、U、A表示核苷酸的碱基组成；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸。
具有上述修饰的siRNA不仅成本低，而且可使血液中的核糖核酸酶不易切割核酸，由此增加核酸的稳定性，使核酸具有更强的抵抗核酸酶水解的性能。
在一些实施方式中，本公开提供的siRNA的正义链和反义链中至少一条单链的磷酸-糖骨架中的磷酸酯基中的至少一部分为具有修饰基团的磷酸酯基。在一些实施方式中，具有修饰基团的磷酸酯基为磷酸酯基中的磷酸二酯键中的至少一个氧原子被硫原子取代而形成的硫代磷酸酯基；在一些实施方式中，所述具有修饰基团的磷酸酯基为具有如式(1)所示结构的硫代磷酸酯基：
Figure PCTCN2018118107-appb-000005
这种修饰能稳定siRNA的双链结构，保持碱基配对的高特异性和高亲和力。
在一些实施方式中，本公开提供的siRNA中，硫代磷酸酯基连接存在于以下位置中的至少一处：正义链或反义链任意一端的第一个和第二个核苷酸之间；正义链或反义链任意一端的第二个和第三个核苷酸之间；或上述的任意组合。在一些实施方式中，硫代磷酸酯基连接存在于除正义链5′末端以外的全部上述位置处。在一些实施方式中，硫代磷酸酯基连接存在于除正义链3′末端以外的全部上述位置处。在一些实施方式中，硫代磷酸酯基连接存在于以下位置中的至少一处：
所述正义链的5′末端端部第1个核苷酸和第2个核苷酸之间；
所述正义链的5′末端端部第2个核苷酸和第3个核苷酸之间；
所述正义链的3′末端端部第1个核苷酸和第2个核苷酸之间；
所述正义链的3′末端端部第2个核苷酸和第3个核苷酸之间；
所述反义链的5′末端端部第1个核苷酸和第2个核苷酸之间；
所述反义链的5′末端端部第2个核苷酸和第3个核苷酸之间；
所述反义链的3′末端端部第1个核苷酸和第2个核苷酸之间；以及
所述反义链的3′末端端部第2个核苷酸和第3个核苷酸之间。
在一些实施方式中，本公开提供的siRNA为siHBVX4或siHBVX5：
siHBVX4
正义链：
5’-UmsCmsUmGmUmGmCfCfUfUmCmUmCmAmUmCmUmGmAm-3’(SEQ ID NO：10)，
反义链：
5’-UmsCfsAmGmAmUfGmAmGmAmAmGmGmCfAmCfAmGmAmsCmsGm-3′(SEQ ID NO：11)，
siHBVX5
正义链：
5’-UmsCmsUmGmUfGmCfCfUfUmCmUmCmAmUmCmUmGmAm-3’(SEQ ID NO：12)，
反义链：
5’-UmsCfsAmGmAmUfGmAfGfAmAmGmGmCfAmCfAmGmAmsCmsGm-3’(SEQ ID NO：13)，
其中，大写字母C、G、U、A表示核苷酸的碱基组成；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸；小写字母s表示该字母左右两个核苷酸之间为硫代磷酸酯基连接。
在一些实施方式中，所述siRNA反义链的5’末端核苷酸为5’-磷酸核苷酸或5’-磷酸类似物修饰的核苷酸。
常用的所述5’-磷酸核苷酸或5’-磷酸类似物修饰的核苷酸是本领域技术人员所公知的，如5’-磷酸核苷酸可具有如下结构：
Figure PCTCN2018118107-appb-000006
再如，Anastasia Khvorova and Jonathan K.Watts，The chemical evolution of oligonucleotide therapies of clinical utility.Nature Biotechnology，2017，35(3)：238-48中公开了如下4种5’-磷酸类似物修饰的核苷酸：
Figure PCTCN2018118107-appb-000007
其中，R选自H、OH、甲氧基、氟；Base表示碱基，选自A、U、C、G或T。
在一些实施方式中，5′-磷酸核苷酸为式(2)所示的含有5′-磷酸修饰的核苷酸，5’-磷酸类似物修饰的核苷酸为含有乙烯基磷酸酯(5’-(E)-vinylphosphonate，E-VP)修饰的核苷酸，如式(3)所示，或者为硫代磷酸酯修饰的核苷酸，如式(5)所示。
在一些实施方式中，本公开提供的siRNA为siHBVX6、siHBVX7、siHBVX8或siHBVX9：
siHBVX6
正义链：
5’-UmCmUmGmUmGmCfCfUfUmCmUmCmAmUmCmUmGmAm-3’(SEQ ID NO：6)，
反义链：
5’-P1-UmCfAmGmAmUfGmAmGmAmAmGmGmCfAmCfAmGmAmCmGm-3′(SEQ ID NO：14)，
siHBVX7
正义链：
5’-UmCmUmGmUfGmCfCfUfUmCmUmCmAmUmCmUmGmAm-3’(SEQ ID NO：8)，
反义链：
5’-P1-UmCfAmGmAmUfGmAfGfAmAmGmGmCfAmCfAmGmAmCmGm-3’(SEQ ID NO：15)，
siHBVX8
正义链：
5’-UmsCmsUmGmUmGmCfCfUfUmCmUmCmAmUmCmUmGmAm-3’(SEQ ID NO：10)，
反义链：
5’-P1-UmsCfsAmGmAmUfGmAmGmAmAmGmGmCfAmCfAmGmAmsCmsGm-3′(SEQ ID NO：16)，
siHBVX9
正义链：
5’-UmsCmsUmGmUfGmCfCfUfUmCmUmCmAmUmCmUmGmAm-3’(SEQ ID NO：12)，
反义链：
5’-P1-UmsCfsAmGmAmUfGmAfGfAmAmGmGmCfAmCfAmGmAmsCmsGm-3’(SEQ ID NO：17)，
其中，大写字母C、G、U、A表示核苷酸的碱基组成；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸；小写字母s表示该字母左右两个核苷酸之间为硫代磷酸酯基连接；大写字母P1表示该字母右侧相邻的一个核苷酸为5’-磷酸核苷酸或5’-磷酸类似物修饰的核苷酸。
本公开的发明人意外发现，本公开提供的siRNA不仅具有显著增强的血浆 和溶酶体稳定性，还保留很高的基因抑制活性。
本公开提供的siRNA可以通过本领域常规的siRNA制备方法(例如固相合成和液相合成的方法)得到。其中，固相合成已经有商业化订制服务。可以通过使用具有相应修饰的核苷单体来将修饰的核苷酸基团引入本公开所述的siRNA中，制备具有相应修饰的核苷单体的方法及将修饰的核苷酸基团引入siRNA的方法也是本领域技术人员所熟知的。
药物组合物
本公开提供了一种药物组合物，所述药物组合物含有如上所述的siRNA作为活性成分和药学上可接受的载体。
所述药学上可接受的载体可以是siRNA给药领域常规使用的载体，例如但不限于磁性纳米粒(magnetic nanoparticles，如基于Fe3O4或Fe2O3的纳米粒)、碳纳米管(carbon nanotubes)、介孔硅(mesoporous silicon)、磷酸钙纳米粒(calcium phosphate nanoparticles)、聚乙烯亚胺(polyethylenimine，PEI)、聚酰胺型树形高分子(polyamidoamine(PAMAM)dendrimer)、聚赖氨酸(poly(L-lysine)，PLL)、壳聚糖(chitosan)、1，2-二油酰基-3-三甲铵丙烷(1，2-dioleoyl-3-trimethylammonium-propane，DOTAP)、聚D型或L型乳酸/羟基乙酸共聚物(poly(D&L-lactic/glycolic acid)copolymer，PLGA)、聚(氨乙基乙撑磷酸酯)(poly(2-aminoethyl ethylene phosphate)，PPEEA)和聚(甲基丙烯酸-N，N-二甲氨基乙酯)(poly(2-dimethylaminoethyl methacrylate)，PDMAEMA)以及它们的衍生物中的一种或多种。
在一些实施方式中，所述药物组合物中，对siRNA和药学上可接受的载体的含量没有特别要求，在一些实施方式中，siRNA与药学上可接受的载体的重量比可以为1∶(1-500)，在一些的实施方式中，上述重量比为1∶(1-50)。
在一些实施方式中，所述药物组合物中，还可以包含药学上可接受的其它辅料，该辅料可以为本领域常规采用的各种制剂或化合物的一种或多种。例如，所述药学上可接受的其它辅料可以包括pH值缓冲液、保护剂和渗透压调节剂中的至少一种。
所述pH值缓冲液可以为pH值7.5-8.5的三羟甲基胺基甲烷盐酸盐缓冲液和/或pH值5.5-8.5的磷酸盐缓冲液，例如可以为pH值5.5-8.5的磷酸盐缓冲液。
所述保护剂可以为肌醇、山梨醇、蔗糖、海藻糖、甘露糖、麦芽糖、乳糖和葡萄糖中的至少一种。以所述药物组合物的总重量为基准，所述保护剂的含量可以为0.01-30重量％。
所述渗透压调节剂可以为氯化钠和/或氯化钾。所述渗透压调节剂的含量使所述药物组合物的渗透压为200-700毫渗摩尔/千克。根据所需渗透压，本领域技术人员可以容易地确定所述渗透压调节剂的含量。
在一些实施方式中，所述药物组合物可以为液体制剂，例如注射液；也可以为冻干粉针剂，实施给药时与液体辅料混合，配制成液体制剂。所述液体制剂可以但不限于用于皮下、肌肉或静脉注射给药，也可以但不限于通过喷雾给药到肺脏、或通过喷雾经肺脏给药到其它脏器组织(如肝脏)。在一些实施方式中，所述药物组合物用于静脉注射给药。
在一些实施方式中，所述药物组合物可以为脂质体制剂的形式。在一些实施方式中，所述脂质体制剂中使用的药学上可接受的载体包含含胺的转染化合物(下文也可将其称为有机胺)、辅助脂质和/或聚乙二醇化脂质。其中，所述有机胺、辅助脂质和聚乙二醇化脂质可分别选自于CN103380113A(通过引用的方式将其整体并入本文)中所描述的含胺的转染化合物或其药学上可接受的盐或衍生物、辅助脂质和聚乙二醇化脂质中的一种或多种。
在一些实施方式中，所述有机胺可为CN103380113A中描述的如式(201)所示的化合物或其药学上可接受的盐：
Figure PCTCN2018118107-appb-000008
其中：
X 101和X 102各自独立地是O、S、N-A或C-A，其中A是氢或C 1-C 20烃链；
Y和Z各自独立地是C＝O、C＝S、S＝O、CH-OH或SO 2；
R 101、R 102、R 103、R 104、R 105、R 106和R 107各自独立地是氢，环状或无环的、被取代的或未被取代的、支链或直链脂族基团，环状或无环的、被取代的或未被取代的、支链或直链杂脂族基团，被取代的或未被取代的、支链或直链酰基，被取代的或未被取代的、支链或直链芳基，被取代的或未被取代的、支链或直链杂芳基；
x是1-10的整数；
n是1-3的整数，m是0-20的整数，p是0或1；其中，如果m＝p＝0，则R 102是氢；
并且，如果n或m中的至少一个是2，那么R 103和在式(201)中的氮形成如式(202)或式(203)所示的结构：
Figure PCTCN2018118107-appb-000009
其中，g、e和f各自独立地是1-6的整数，“HCC”代表烃链，且每个＊N代表式(201)中的氮原子。
在一些实施方式中，R 103是多胺。在其它实施方式中，R 103是缩酮。在一些实施方式中，在式(201)中的R 101和R 102中的每一个独立地是任意的被取代的或未被取代的、支链或直链烷基或烯基，所述烷基或烯基具有3至约20个碳原子，诸如8至约18个碳原子，和0至4个双键，诸如0至2个双键。
在一些实施方式中，如果n和m中的每一个独立地具有1或3的值，那么R 103可以是下述式(204)-式(213)中的任一个：
Figure PCTCN2018118107-appb-000010
Figure PCTCN2018118107-appb-000011
其中，式(204)-式(213)中，g、e和f各自独立地是1-6的整数，每个“HCC”代表烃链，且每个＊显示R 103与在式(201)中的氮原子的可能连接点，其中在任意＊位置上的每个H可以被替换以实现与在式(201)中的氮原子的连接。
其中，式(201)所示化合物可以根据CN103380113A中的描述制备。
在一些实施方式中，所述有机胺为如式(214)所示的有机胺和/或如式(215)所示的有机胺：
Figure PCTCN2018118107-appb-000012
所述辅助脂质为胆固醇、胆固醇的类似物和/或胆固醇的衍生物；
所述聚乙二醇化脂质为1，2-二棕榈酰胺-sn-甘油-3-磷脂酰乙醇胺-N-[甲氧基(聚乙二醇)]-2000。
在一些实施方式中，所述药物组合物中，所述有机胺、所述辅助脂质和所述聚乙二醇化脂质三者之间的摩尔比为(19.7-80)∶(19.7-80)∶(0.3-50)，例如 可以为(50-70)∶(20-40)∶(3-20)。
在一些实施方式中，由本公开的siRNA与上述含胺的转染试剂形成的药物组合物颗粒具有约30nm至约200nm的平均直径，通常为约40nm至约135nm，更通常地，该脂质体颗粒的平均直径是约50nm至约120nm、约50nm至约100nm、约60nm至约90nm或约70nm至约90nm，例如，该脂质体颗粒的平均直径是约30、40、50、60、70、75、80、85、90、100、110、120、130、140、150或160nm。
在一些实施方式中，由本公开的siRNA与上述含胺的转染试剂形成的药物组合物中，siRNA与全部脂质(例如有机胺、辅助脂质和/或聚乙二醇化脂质)的重量比(重量/重量比)在从约1∶1至约1∶50、从约1∶1至约1∶30、从约1∶3至约1∶20、从约1∶4至约1∶18、从约1∶5至约1∶17、从约1∶5至约1∶15、从约1∶5至约1∶12、从约1∶6至约1∶12或从约1∶6至约1∶10的范围内，例如，本公开的siRNA与全部脂质的重量比为约1∶5、1∶6、1∶7、1∶8、1∶9、1∶10、1∶11、1∶12、1∶13、1∶14、1∶15、1∶16、1∶17或1∶18。
在一些实施方式中，所述药物组合物在销售时各组分可以独立存在，在使用时可以液体制剂的形式存在。在一些实施方式中，本公开提供的siRNA与上述药学上可接受的载体形成的药物组合物可以按照已知的各种方法制备，只是用本公开提供的siRNA替代现有siRNA即可；在一些实施方式中，可以按照如下方法制备：
将有机胺、辅助脂质和聚乙二醇化脂质按照上述摩尔比悬浮于醇中并混匀得到脂质溶液；醇的用量使得到的脂质溶液的总质量浓度为2-25mg/mL，例如可以为8-18mg/mL。所述醇选自药学上可接受的醇，诸如在室温附近为液体的醇，例如，乙醇、丙二醇、苯甲醇、甘油、聚乙二醇200，聚乙二醇300，聚乙二醇400中的一种或多种，例如可以为乙醇。
将本公开提供的siRNA溶解于缓冲盐溶液中，得到siRNA水溶液。缓冲盐溶液的浓度为0.05-0.5M，例如可以为0.1-0.2M，调节缓冲盐溶液的pH至4.0-5.5，例如可以为5.0-5.2，缓冲盐溶液的用量使siRNA的浓度不超过0.6mg/mL，例如可以为0.2-0.4mg/mL。所述缓冲盐选自可溶性醋酸盐、可溶性柠檬酸盐中的一种或多种，例如可以为醋酸钠和/或醋酸钾。
将脂质溶液和siRNA水溶液混合，将混合后得到的产物在40-60℃孵育至少2分钟，例如可以为5-30分钟，得到孵育后的脂质体制剂。脂质溶液和siRNA水溶液的体积比为1∶(2-5)，例如可以为1∶4。
将孵育后的脂质体制剂浓缩或稀释，去除杂质，除菌，得到本公开提供的药物组合物，其理化参数为pH值为6.5-8，包封率不低于80％，粒径为40-200nm，多分散指数不高于0.30，渗透压为250-400mOsm/kg；例如理化参数可以为pH值为7.2-7.6，包封率不低于90％，粒径为60-100nm，多分散指数不高于0.20，渗透压为300-400mOsm/kg。
其中，浓缩或稀释可以在去除杂质之前、之后或同时进行。去除杂质的方法可以采用现有各种方法，例如可以使用切相流系统、中空纤维柱，在100K Da条件下超滤，超滤交换溶液为pH7.4的磷酸盐缓冲液(PBS)。除菌的方法可以采用现有各种方法，例如可以在0.22μm滤器上过滤除菌。
第一种siRNA缀合物
在一个方面，本公开提供了一种siRNA缀合物，所述siRNA缀合物包含上述的siRNA以及连接至该siRNA的缀合基团。
在本公开的上下文中，除非另有说明，“缀合”是指两个或多个各自具有特定功能的化学部分之间以共价连接的方式彼此连接；相应地，“缀合物”是指该各个化学部分之间通过共价连接而形成的化合物。进一步地，“siRNA缀合物”表示一个或多个具有特定功能的化学部分共价连接至siRNA上而形成的化合物。在下文中，有时也将本公开的siRNA缀合物简称为“缀合物”。siRNA缀合物应根据上下文，理解为siRNA缀合物的总称，第一种siRNA缀合物或第二种siRNA缀合物。在本公开的上下文中，“缀合分子”应当理解为可通过反应缀合至siRNA、最终形成本公开的siRNA缀合物的化合物。
本公开提供了第一种siRNA缀合物，所述siRNA缀合物包含上述的siRNA以及连接至该siRNA的缀合基团。一般来说，对于第一种siRNA缀合物，所述缀合基团包含药学上可接受的至少一个靶向基团和任选的接头(linker)，并且，所述siRNA、所述接头和所述靶向基团依次连接。在一种实施方式中，所述靶向基团为1-6个。在一种实施方式中，所述靶向基团为2-4个。所述siRNA分子可以非共价或共价缀合至所述缀合基团，例如可以共价缀合至所述缀合基团。siRNA与缀合基团的缀合位点可以在siRNA正义链的3’端或5’端，也可在反义链的5’端，还可以在siRNA的内部序列中。在一些实施方式中，所述siRNA与缀合基团的缀合位点在siRNA正义链的3’端。
在一些实施方式中，所述缀合基团可以连接在核苷酸的磷酸基团、2’-位羟基或者碱基上。在一些实施方式中，所述缀合基团可以连接在3’-位羟基上，此时核苷酸之间采用2’-5’磷酸二酯键连接。当缀合基团连接在siRNA链的末端时， 所述缀合基团通常连接在核苷酸的磷酸基团上；当缀合基团连接在siRNA的内部序列时，所述缀合基团通常连接在核糖糖环或者碱基上。各种连接方式可参考：Muthiah Manoharan et.al.siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes.ACS Chemical biology，2015，10(5)：1181-7.
在一些实施方式中，所述siRNA与缀合基团间可以通过酸不稳定的、或可还原的化学键相连，在细胞内涵体的酸性环境下，这些化学键可降解，从而使siRNA成为自由状态。对于不可降解的缀合方式，缀合基团可连接在siRNA的正义链，从而尽量降低缀合对siRNA活性的影响。
在一些实施方式中，所述药学上可接受的靶向基团指可以是siRNA给药领域常规使用的配体，例如WO2009082607A2中描述的各种配体，以引用的方式将其全部公开内容并入本文。
在一些实施方式中，所述药学上可接受的靶向基团可以选自以下靶向分子或其衍生物形成的配体中的一种或多种：亲脂分子，例如胆固醇、胆汁酸、维生素(例如维生素E)、不同链长的脂质分子；聚合物，例如聚乙二醇；多肽，例如透膜肽；适配体；抗体；量子点；糖类，例如乳糖、聚乳糖、甘露糖、半乳糖、N-乙酰半乳糖胺(GalNAc)；叶酸(folate)；肝实质细胞表达的受体配体，例如去唾液酸糖蛋白、去唾液酸糖残基、脂蛋白(如高密度脂蛋白、低密度脂蛋白等)、胰高血糖素、神经递质(如肾上腺素)、生长因子、转铁蛋白等。
在一些实施方式中，所述的每个配体独立地选自一个能够与细胞表面受体结合的配体。在一些实施方式中，至少一个配体是能够与肝细胞表面受体结合的配体。在一些实施方式中，至少一个配体是能够与哺乳动物肝细胞表面受体结合的配体。在一些实施方式中，至少一个配体是能够与人肝细胞表面受体结合的配体。在一些实施方式中，至少一个配体是能够与肝表面去唾液酸糖蛋白受体(ASGPR)结合的配体。这些配体的种类为本领域技术人员所公知，其作用一般是与靶细胞表面的特异性受体相结合，介导与配体连接的siRNA递送至靶细胞。
在一些实施方式中，所述药学上可接受的靶向基团可以是与哺乳动物肝细胞表面上的去唾液酸糖蛋白受体(ASGPR)结合的任意一种配体。在一种实施方式中，每个配体独立地为去唾液酸糖蛋白，例如去唾液酸血清类粘蛋白(asialoorosomucoid，ASOR)或去唾液酸胎球蛋白(asialofetuin，ASF)。在一种实施方式所述配体为糖或糖的衍生物。
在一些实施方式中，至少一个配体是糖。在一些实施方式中，每个配体均是糖。在一些实施方式中，至少一个配体是单糖、多糖、修饰的单糖、修饰的多糖或糖衍生物。在一些实施方式中，至少一个所述配体可以是单糖，双糖或三糖。在一些实施方式中，至少有一个配体是修饰的糖。在一些实施方式中，每一个配体均为修饰的糖。在一些实施方式中，每个配体均独立地选自多糖、修饰的多糖、单糖、修饰的单糖、多糖衍生物或单糖衍生物。在一些实施方式中，每一个或至少一个配体选自由葡萄糖及其衍生物组、甘露聚糖及其衍生物、半乳糖及其衍生物、木糖及其衍生物、核糖及其衍生物、岩藻糖及其衍生物、乳糖及其衍生物、麦芽糖及其衍生物，阿拉伯糖及其衍生物、果糖及其衍生物和唾液酸组成的组。
在一些实施方式中，每个所述配体可独立地选自D-吡喃甘露糖、L-吡喃甘露糖、D-阿拉伯糖、D-呋喃木糖、L-呋喃木糖、D-葡萄糖、L-葡萄糖、D-半乳糖、L-半乳糖、α-D-呋喃甘露糖、β-D-呋喃甘露糖、α-D-吡喃甘露糖、β-D-吡喃甘露糖、α-D-吡喃葡萄糖、β-D-吡喃葡萄糖、α-D-呋喃葡萄糖、β-D-呋喃葡萄糖、α-D-呋喃果糖、α-D-吡喃果糖、α-D-吡喃半乳糖、β-D-吡喃半乳糖、α-D-呋喃半乳糖、β-D-呋喃半乳糖、葡糖胺、唾液酸、半乳糖胺、N-乙酰基半乳糖胺、N-三氟乙酰基半乳糖胺、N-丙酰基半乳糖胺、N-正丁酰基半乳糖胺、N-异丁酰基半乳糖胺、2-氨基-3-O-[(R)-1-羧乙基]-2-脱氧-β-D-吡喃葡萄糖、2-脱氧-2-甲基氨基-L-吡喃葡萄糖、4，6-二脱氧-4-甲酰胺基-2，3-二-O-甲基-D-吡喃甘露糖、2-脱氧-2-磺氨基-D-吡喃葡萄糖、N-乙醇酰基-α-神经氨酸、5-硫代-β-D-吡喃葡萄糖、2，3，4-三-O-乙酰基-1-硫代-6-O-三苯甲基-α-D-吡喃葡萄糖苷甲酯、4-硫代-β-D-吡喃半乳糖、3，4，6，7-四-O-乙酰基-2-脱氧-1，5-二硫代-α-D-吡喃葡庚糖苷乙酯、2，5-脱水-D-阿洛糖腈、核糖、D-核糖、D-4-硫代核糖、L-核糖或L-4-硫代核糖。所述配体的其它选择可参见例如CN105378082A的记载，以引用的方式将其全部公开内容并入本文。
在一些实施方式中，所述第一种siRNA缀合物中药学上可接受的靶向基团可以是半乳糖或N-乙酰半乳糖胺，其中，半乳糖或N-乙酰半乳糖胺分子可以是一价、二价、三价、四价。应当理解的是，这里所述的一价、二价、三价、四价分别指siRNA分子与含有作为靶向基团的半乳糖或N-乙酰半乳糖胺分子的缀合基团形成siRNA缀合物后，该siRNA缀合物中siRNA分子与半乳糖或N-乙酰半乳糖胺分子的摩尔比为1∶1、1∶2、1∶3或1∶4。在一些实施方式中，所述药学上可接受的靶向基团是N-乙酰半乳糖胺。在一些实施方式中，当本公开所述 的siRNA与含有N-乙酰半乳糖胺的缀合基团缀合时，N-乙酰半乳糖胺分子是三价或四价。在一些实施方式中，当本公开所述的siRNA与含有N-乙酰半乳糖胺的缀合基团缀合时，N-乙酰半乳糖胺分子是三价。
当本公开所述的siRNA与缀合分子缀合时，缀合分子可经由合适的接头与siRNA分子相连，本领域技术人员可以根据靶向基团的具体类型选择合适的接头。这些缀合基团、接头、靶向基团的种类以及与siRNA的连接方式，可参见WO2015006740A2的公开内容，通过引用的方式将其整体内容并入本文。
在一些实施方式中，当所述靶向基团为N-乙酰半乳糖胺时，合适的接头可以为如式(301)所示的结构：
Figure PCTCN2018118107-appb-000013
其中，
k为1-3的整数；
L A为具有如式(302)所示结构的包含酰胺键的链状部分，每个所述L A在其两端分别与一个所述靶向基团和所述L C部分通过醚键相连接：
Figure PCTCN2018118107-appb-000014
L B为具有如式(303)所示结构的包含N-酰基吡咯烷的链状部分，所述链状部分在其一端具有羰基并与所述L C部分通过酰胺键相连接，在另一端具有氧基并与所述siRNA通过磷酸酯键相连接：
Figure PCTCN2018118107-appb-000015
L C为基于羟甲基氨基甲烷、二羟甲基氨基甲烷或三羟甲基氨基甲烷的2-4价连接基团，所述L C经由氧原子与各个所述L A部分通过醚键相连接，并且经由氮原子与所述L B部分通过酰胺键相连接。
在一些实施方式中，当n＝3，L C为基于三羟甲基氨基甲烷的4价连接基团时，由作为接头的-(L A)3三羟甲基氨基甲烷-L B-连接N-乙酰半乳糖胺分子和siRNA分子所形成的第一种siRNA缀合物，其结构如下式(304)所示：
Figure PCTCN2018118107-appb-000016
式中，双螺旋结构表示siRNA。
同样，siRNA与缀合分子的缀合位点可以在siRNA正义链的3′端或5′端，也可在反义链的5′端，还可以在siRNA的内部序列中。
在一些实施方式中，本公开所述siRNA的正义链3′末端通过接头-(LA)3三羟甲基氨基甲烷-L B-与三个N-乙酰半乳糖胺(GalNAc)分子共价缀合，得到siRNA分子与GalNAc分子的摩尔比为1∶3的第一种siRNA缀合物，下文也可将其称为(GalNAc)3-1-siRNA，其结构如下式(305)所示：
Figure PCTCN2018118107-appb-000017
其中，双螺旋结构表示所述siRNA，并且所述接头连接至所述siRNA的正义链3′末端。
在一些实施方式中，当所述靶向基团为N-乙酰半乳糖胺时，合适的接头可以为如式(306)所示的结构：
Figure PCTCN2018118107-appb-000018
其中，
l为0-3的整数；
＊表示接头上通过醚键与靶向基团连接的位点；
#表示接头上通过磷酸酯键与siRNA连接的位点。
在一些实施方式中，当l＝2时，所述第一种siRNA缀合物具有如式(307)所示的结构：
Figure PCTCN2018118107-appb-000019
其中，双螺旋结构表示所述siRNA，并且所述接头连接至所述siRNA的正义链3′末端。
上述缀合物可以通过现有技术中已经详细描述的方法进行合成。例如，WO2015006740A2中详细描述了多种缀合物的制备方法。通过本领域技术人员熟知的方式，获得本公开的第一种siRNA缀合物。如WO2014025805A1中记载了式(305)所示结构的制备方法，Rajeev等人在ChemBioChem 2015，16，903-908中描述了式(307)所示结构的制备方法。
第二种siRNA缀合物
在一些实施方式中，本公开提供了第二种siRNA缀合物，该第二种siRNA缀合物具有如式(308)所示的结构：
Figure PCTCN2018118107-appb-000020
其中：
n1为选自1-3的整数，n3为选自0-4的整数；
m1、m2和m3独立地为选自2-10的整数；
R 10、R 11、R 12、R 13、R 14和R 15各自独立地为H，或选自于由以下基团所组成的组：C 1-C 10烷基、C 1-C 10卤代烷基以及C 1-C 10烷氧基；
R 3为式A59所示结构的基团：
Figure PCTCN2018118107-appb-000021
其中，E 1为OH、SH或BH 2，Nu为本公开的siRNA；
R 2是长度为1-20个碳原子的直链亚烷基，其中一个或多个碳原子任选地被选自于以下基团所组成的组中的一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O) 2、C 2-C 10亚烯基、C 2-C 10亚炔基、C 6-C 10亚芳基、C 3-C 18亚杂环基和C 5-C 10亚杂芳基；并且其中，R 2可任选地具有由以下基团所组成的组中的任何一个或多个的取代基：C 1-C 10烷基、C 6-C 10芳基、C 5-C 10杂芳基、C 1-C 10卤代烷基、-OC 1-C 10烷基、-OC 1-C 10烷基苯基、-C 1-C 10烷基-OH、-OC 1-C 10卤代烷基、-SC 1-C 10烷基、-SC 1-C 10烷基苯基、-C 1-C 10烷基-SH、-SC 1-C 10卤代烷基、卤素取代基、-OH、-SH、-NH 2、-C 1-C 10烷基-NH 2、-N(C 1-C 10烷基)(C 1-C 10烷基)、-NH(C 1-C 10烷基)、氰基、硝基、-CO 2H、-C(O)O(C 1-C 10烷基)、-CON(C 1-C 10烷基)(C 1-C 10烷基)、-CONH(C 1-C 10烷基)、-CONH 2、-NHC(O)(C 1-C 10烷基)、-NHC(O)(苯基)、-N(C 1-C 10烷基)C(O)(C 1-C 10烷基)、-N(C 1-C 10烷基)C(O)(苯基)、-C(O)C 1-C 10烷基、-C(O)C 1-C 10烷基苯基、-C(O)C 1-C 10卤烷基、-OC(O)C 1-C 10烷基、-SO 2(C 1-C 10烷基)、-SO 2(苯基)、-SO 2(C 1-C 10卤代烷基)、-SO 2NH 2、 -SO 2NH(C 1-C 10烷基)、-SO 2NH(苯基)、-NHSO 2(C 1-C 10烷基)、-NHSO 2(苯基)和-NHSO 2(C 1-C 10卤代烷基)。
每个L 1是长度为1-70个碳原子的直链亚烷基，其中一个或多个碳原子任选地被选自于以下基团所组成的组中的一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O) 2、C 2-C 10亚烯基、C 2-C 10亚炔基、C 6-C 10亚芳基、C 3-C 18亚杂环基和C 5-C 10亚杂芳基；并且其中，L 1可任选地具有由以下基团所组成的组中的任何一个或多个的取代基：C 1-C 10烷基、C 6-C 10芳基、C 5-C 10杂芳基、C 1-C 10卤代烷基、-OC 1-C 10烷基、-OC 1-C 10烷基苯基、-C 1-C 10烷基-OH、-OC 1-C 10卤代烷基、-SC 1-C 10烷基、-SC 1-C 10烷基苯基、-C 1-C 10烷基-SH、-SC 1-C 10卤代烷基、卤素取代基、-OH、-SH、-NH 2、-C 1-C 10烷基-NH 2、-N(C 1-C 10烷基)(C 1-C 10烷基)、-NH(C 1-C 10烷基)、氰基、硝基、-CO 2H、-C(O)O(C 1-C 10烷基)、-CON(C 1-C 10烷基)(C 1-C 10烷基)、-CONH(C 1-C 10烷基)、-CONH 2、-NHC(O)(C 1-C 10烷基)、-NHC(O)(苯基)、-N(C 1-C 10烷基)C(O)(C 1-C 10烷基)、-N(C 1-C 10烷基)C(O)(苯基)、-C(O)C 1-C 10烷基、-C(O)C 1-C 10烷基苯基、-C(O)C 1-C 10卤烷基、-OC(O)C 1-C 10烷基、-SO 2(C 1-C 10烷基)、-SO 2(苯基)、-SO 2(C1-C 10卤代烷基)、-SO 2NH 2、-SO 2NH(C 1-C 10烷基)、-SO 2NH(苯基)、-NHSO 2(C 1-C 10烷基)、-NHSO 2(苯基)和-NHSO 2(C 1-C 10卤代烷基)。
并且，在一些实施方式中，L 1可选自于由A1-A26基团或其任意组合所组成的组，其中A1-A26的结构和定义如下所示：
Figure PCTCN2018118107-appb-000022
Figure PCTCN2018118107-appb-000023
其中，j1为1-20的整数；j2为1-20的整数；
R’为C 1-C 10的烷基；
Ra选自式A27-A45基团中的一种：
Figure PCTCN2018118107-appb-000024
Figure PCTCN2018118107-appb-000025
Rb为C 1-C 10的烷基；
Figure PCTCN2018118107-appb-000026
表示基团连接至分子其余部分的位点。
技术人员会理解的是，尽管为了方便起见，L 1被定义为线性烷基，但是它可能不是线性基团或者名称不同，例如由于上述替换和/或置换而产生的胺或烯基。为了本公开内容的目的，L 1的长度是连接两个附着点的链中的原子数。为此目的，将替换所述直链亚烷基的碳原子而得到的环(如亚杂环基或亚杂芳基)计为一个原子。
M 1表示靶向基团，其定义和可选择的范围与上述靶向基团相同。在一些实施方式中，每个M 1独立地选自对哺乳动物肝脏细胞表面上的去唾液酸糖蛋白受体具有亲合力的配体中的一种。
当M 1为对哺乳动物肝脏细胞表面上的去唾液酸糖蛋白受体具有亲和力的配体时，在一些实施方式中，n1可以是1-3的整数，n3可以是0-4的整数，保证所述缀合物中M 1靶向基团的个数至少为2；在一些实施方式中，n1+n3≥2，这样可以使得M 1靶向基团的个数至少为3，使得M 1靶向基团与肝表面去唾液酸糖蛋白受体更容易结合，进而促进所述缀合物通过内吞作用进入细胞。实验表明，当M 1靶向基团的个数大于3个时，M 1靶向基团与肝表面去唾液酸糖蛋白受体结合的容易程度增加并不明显，因此，从合成容易程度、结构/工艺成本和递送效率等多方面综合考虑，在一些实施方式中，n1为1-2的整数，n3为0-1的整数，且n1+n3＝2-3。
在一些实施方式中，m1、m2和m3独立地选自2-10的整数时，可以使多个M 1靶向基团之间的空间位置适合M 1靶向基团与肝表面去唾液酸糖蛋白受体的结合，为了使本公开提供的缀合物更为简单，更容易合成和/或降低成本，在一些实施方式中，m1、m2和m3各自独立地为2-5的整数，在一些实施方式中，m1＝m2＝m3。
本领域技术人员可以理解，当R 10、R 11、R 12、R 13、R 14和R 15各自独立地为H、C 1-C 10烷基、C 1-C 10卤代烷基、以及C 1-C 10烷氧基中的一种时，不会改变本文公开的缀合物的性质，均可以实现本公开的目的。在一些实施方式中，R 10、R 11、R 12、R 13、R 14和R 15各自独立地选自H、甲基和乙基。在一些实施方式中，R 10、R 11、R 12、R 13、R 14和R 15均为H。
根据本公开提供的第二种siRNA缀合物，R 3为式A59所示结构的基团，其中，E 1为OH、SH或BH 2，基于制备原料易获取性的考虑，在一些实施方式中，E 1为OH或SH。
在一些实施方式中，R 2的选择是为了实现与含氮骨架上的N与A59的连接。在本公开的上下文中，“含氮骨架”是指连接有R 10、R 11、R 12、R 13、R 14和R 15的碳原子与N互相连接的链状结构。因此，R 2可以是任何能够以适当方式将A59基团连接至含氮骨架上的N的连接基团。在一些实施方式中，在通过固相合成的工艺制备第二种siRNA缀合物的情况下，R 2基团中需要同时含有与含氮骨架上的N连接的连接位点和与R 3中的P相连接的连接位点。在一些实施方式中，R 2中所述与含氮骨架上的N连接的位点与N形成酰胺键，所述与R 3上的P连接的位点与P形成磷酸酯键。在一些实施方式中，R 2可以是B5、B6、B5’或B6’：
Figure PCTCN2018118107-appb-000027
其中，表示基团共价键连接的位点。
q 2的取值范围可以是1-10的整数，在一些实施方式中，q 2为1-5的整数。
L 1的作用是将M 1靶向基团与含氮骨架上的N连接，为本公开的第二种siRNA缀合物提供肝靶向功能。在一些实施方式中，L 1选自式A1-A26基团中的一种或多种的连接组合；在一些实施方式中，L 1选自A1、A4、A5、A6、A8、A10、A11和A13中的一种或多种的连接组合；在一些实施方式中，L 1选自A1、A4、A8、A10和A11中至少2个的连接组合；在一些实施方式中，L 1选自A1、A8、A10中至少2个的连接组合。
在一些实施方式中，L 1的长度可以为3-25个原子，3-20个原子、4-15个原子或5-12个原子。在一些实施方式中，L 1的长度为3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个、25个、30个、35个、40个、45个、50个、55个、60个原子。
在一些实施方式中，j1为2-10的整数，在一些实施方式中，j1为3-5的整数。j2为2-10的整数，在一些实施方式中，j2为3-5的整数。R’为C 1-C 4的烷基，在一些实施方式中，R’为甲基、乙基和异丙基中的一种。Ra为A27、A28、A29、A30和A31中的一种，在一些实施方式中，Ra为A27或A28。Rb为C 1-C 5的烷基，在一些实施方式中，Rb为甲基、乙基、异丙基和丁基中的一种。在一些实施方式中，在式A1-A26中各自对j1、j2、R’、Ra、Rb进行选择，以实现M 1靶向基团与含氮骨架上的N连接，并使M 1靶向基团之间的空间位置更适合M 1靶向基团与肝表面去唾液酸糖蛋白受体结合。
在一些实施方式中，本公开的第二siRNA缀合物具有式(403)、(404)、(405)、(406)、(407)、(408)、(409)、(410)、(411)、(412)、(413)、(414)、(415)、(416)、(417)、(418)、(419)、(420)、(421)或(422)所示的结构：
Figure PCTCN2018118107-appb-000028
Figure PCTCN2018118107-appb-000029
Figure PCTCN2018118107-appb-000030
Figure PCTCN2018118107-appb-000031
Figure PCTCN2018118107-appb-000032
Figure PCTCN2018118107-appb-000033
在一些实施方式中，式A59中的P可以连接到Nu代表的siRNA序列中任何可能的位置，例如，式A59中的P可以连接到Nu代表的siRNA正义链或反义链的任何一个核苷酸上；在一些实施方式中，式A59中的P连接到Nu代表的siRNA正义链的任何一个核苷酸上。在一些实施方式中，式A59中的P连接到Nu代表的siRNA正义链或反义链的端部；在一些实施方式中，式A59中的P连接到Nu代表的siRNA正义链的端部。Nu代表的siRNA的端部指Nu代表的siRNA正义链或所述反义链中从其一端起算的前4个核苷酸。在一些实施方式中，式A59中的P连接到Nu代表的siRNA正义链或反义链的末端；在一些实施方式中，式A59中的P连接到Nu代表的siRNA正义链的3′末端。在连接至Nu代表的siRNA的正义链的上述位置的情况下，第二种siRNA缀合物进入细胞后，在解旋时，可以释放出单独的siRNA反义链，以阻断HBV的mRNA翻译蛋白质的过程，抑制乙型肝炎病毒(hepatitis B virus，HBV)基因表达。
式A59中的P可以连接到Nu代表的siRNA中的核苷酸上任何可能的位置，例如，核苷酸的5′位、核苷酸的2′位、核苷酸的3′位或核苷酸的碱基上。在一些实施方式中，式A59中的P可通过形成磷酸二酯键连接至Nu代表的siRNA中的核苷酸的2′位、3′位或5′位。在一些实施方式中，式A59中的P连接在Nu代表的siRNA正义链3′末端核苷酸的3′羟基脱氢后形成的氧原子上，或者式A59中的P通过取代Nu代表的siRNA正义链中的一个核苷酸的2′-羟基中的氢与核苷酸连接，或者式A59中的P通过取代Nu代表的siRNA正义链5′末端核苷酸的5′羟基中的氢与核苷酸连接。
本公开所述siRNA或siRNA缀合物中，每个相邻核苷酸之间由磷酸二酯键或硫代磷酸二酯键连接，磷酸二酯键或硫代磷酸二酯键中的非桥接氧原子或硫原子带有负电荷，它可以以羟基或巯基的形式存在，羟基或巯基中的氢离子也可以部分或全部被阳离子取代。所述阳离子可以是任意的阳离子，如金属阳离子，铵离子NH 4 +，有机铵阳离子中的一种。出于提高溶解性考虑，在一种实施方式中，所述阳离子选自碱金属离子、三级胺形成的铵阳离子和季铵阳离子中的一种或多种。碱金属离子可以是K +和/或Na +，三级胺形成的阳离子可以是三乙胺形成的铵离子和/或N，N-二异丙基乙胺形成的铵离子。因此，本公开所述siRNA或第一种或第二种siRNA缀合物可以至少部分以盐的形式存在。在一种方式中，磷酸二酯键或硫代磷酸二酯键中的非桥接氧原子或硫原子至少部分与钠离子结合，本公开所述siRNA或第一种或第二种siRNA缀合物以钠盐或部分 钠盐的形式存在。
本领域技术人员清楚知晓的是，可以通过使用具有相应修饰的核苷单体来将修饰的核苷酸基团引入本公开所述的siRNA中。制备具有相应修饰的核苷单体的方法及将修饰的核苷酸基团引入siRNA的方法也是本领域技术人员所熟知的。所有修饰的核苷单体均可以商购得到或者采用已知方法制备得到。
第二种siRNA缀合物的制备
可以采用任意合理的合成路线制备本公开的第二种siRNA缀合物。
在一些实施方式中，第二种siRNA缀合物可以采用如下方法制备，该方法包括在亚磷酰胺固相合成的条件下，分别按照siRNA正义链和反义链的核苷酸种类和顺序，按照3′到5′的方向将核苷单体依次连接，每个核苷单体的连接包括脱保护、偶联、盖帽、氧化或硫化四步反应；分离出siRNA的正义链和反义链，退火，其中，Nu代表的siRNA为上述本公开的siRNA；
并且，该方法还包括在偶联反应条件和偶联试剂存在下，将式(321)所示的化合物与核苷单体或连接在固相载体上的核苷酸序列接触，使式(321)所示的化合物经偶联反应连接至核苷酸序列。下文中，式(321)所示的化合物也称作缀合分子。
Figure PCTCN2018118107-appb-000034
其中：
R 4为能够结合至Nu代表的siRNA的部分。在一些实施方式中，R 4为能够通过共价键结合至Nu代表的siRNA的部分。在一些实施方式中，R 4为能够经反应而通过磷酸二酯键缀合至Nu代表的siRNA的任意官能团的部分；
每个S 1独立地是M 1中全部活性羟基被YCOO-基团取代而形成的基团，其中，每个Y独立地选自甲基、三氟甲基、二氟甲基、一氟甲基、三氯甲基、二氯甲基、一氯甲基、乙基、正丙基、异丙基、苯基、卤代苯基以及烷基苯基中的一种；
n1、n3、m1、m2、m3、R 10、R 11、R 12、R 13、R 14、R 15、L 1、M 1各自的定义和可选择的范围如前所述。
R 4的选择是为了实现与含氮骨架上的N的连接，并且为合成式(308)的 siRNA缀合物提供合适的反应位点。在一些实施方式中，R 4中包括R 2连接基团或经保护的R 2连接基团，以及可通过反应与siRNA形成A59所示结构的官能团。
在一些实施方式中，R 4含包含可与Nu代表的siRNA或核苷单体上的基团形成亚磷酸酯的第1官能团以及可与羟基或氨基反应形成共价键的第2官能团或者含有由所述共价键连接的固相载体。在一些实施方式中，所述第1官能团为亚磷酰胺、羟基或被保护的羟基。在一些实施方式中，所述第2官能团为亚磷酰胺、羧酸或羧酸盐。在一些实施方式中，所述第2官能团为经由共价键连接至分子其他部分的固相载体，所述共价键由羟基或氨基形成。在一些实施方式中，所述固相载体经由磷酸酯键、羧酸酯键或酰胺键连接。在一些实施方式中，所述固相载体为树脂。
在一些实施方式中，所述第1官能团含有羟基、-OR k或式(C3)所示的基团；所述第2官能团含有式(C1)、(C2)、(C3)、(C1’)或(C3’)所示的结构：
Figure PCTCN2018118107-appb-000035
式中，q 1为1-4的整数，X为O或NH，M +为阳离子，R k为羟基保护基团，SPS表示固相载体，表示基团共价连接的位点。
在一些实施方式中，所述第1官能团含有亚磷酰胺官能团，如式(C3)所示，该亚磷酰胺基团可以与核苷酸上的任意位置的羟基，如2′位羟基或3′位羟基发生偶联反应形成亚磷酸酯，并经氧化或硫化形成式A59所示的磷酸二酯键或硫代磷酸酯键，将缀合分子缀合至siRNA。此时，即使所述第2官能团并不存在，式(321)化合物也能够缀合至核苷酸，不影响式(308)所示siRNA缀合物的获得。在此情况下，在经由亚磷酰胺固相合成等方法获得siRNA的正义链或反义链后，使式(321)化合物与核苷酸序列中末端核苷酸上的羟基反应，并在后续的氧化或硫化过程中形成磷酸二酯键连接或硫代磷酸酯连接，将式 (321)化合物缀合至siRNA。
在一些实施方式中，所述第1官能团含有被保护的羟基。在一些实施方式中，所述第2官能团包含可与固相载体反应的基团，所述反应提供包含固相载体的缀合分子。在一些实施方式中，所述第2官能团含有羧基、羧酸盐或亚磷酰胺，如式(C1)、(C2)或(C3)所示，当所述第2官能团包含羧基或羧酸盐时，式(321)化合物与固相载体，例如树脂上的羟基或氨基进行酯化反应或酰胺化反应，形成经羧酸酯键连接或经酰胺键连接的包含固相载体的缀合分子。当所述第2官能团包含亚磷酰胺官能团时，式(321)化合物与通用固相载体，例如树脂上的羟基发生偶联反应，并经氧化形成经磷酸二酯键连接的包含固相载体的缀合分子。随后，以上述连接固相载体后的产物作为起始，按照亚磷酰胺固相合成方法依次连接核苷单体，获得连接有缀合基团的siRNA的正义链或反义链。在亚磷酰胺固相合成过程中，所述第1官能团发生脱保护，随后在偶联反应条件下与核苷单体上的亚磷酰胺基团发生偶联。
在一些实施方式中，所述第1官能团含有羟基或被保护的羟基；所述第2官能团含有经羧酸酯键连接的固相载体或经酰胺键连接的固相载体、或者经磷酸酯键连接的固相载体，如式(C1’)或(C3’)所示。此时，由式(321)化合物代替固相载体作为起始，按照亚磷酰胺固相合成方法依次连接核苷单体，获得连接有缀合基团的siRNA的正义链或反义链。
在一些实施方式中，羧酸盐可以表示为-COO-M +，其中，M +是阳离子，例如选自金属阳离子，铵阳离子NH 4 +，有机铵阳离子中的一种。在一种实施方式中，所述金属离子选自碱金属离子中的一种，如K +或Na +。出于提高溶解性、使反应顺利进行的考虑，在一些实施方式中，有机铵离子为三级胺形成的铵阳离子或季铵阳离子，如，三乙胺形成的铵离子或N，N-二异丙基乙胺形成的铵离子。在一些实施方式中，羧酸盐是三乙胺羧酸盐或N，N-二异丙基乙胺羧酸盐。
在一些实施方式中，R 4含有式(B9)、(B10)、(B9’)、(B10’)、(B11)、(B12)、(B11’)或(B12’)所示的结构：
Figure PCTCN2018118107-appb-000036
Figure PCTCN2018118107-appb-000037
其中，q 1为1-4的整数，q 2为1-10的整数，X为O或NH，M +为阳离子，R k为羟基保护基团，SPS表示固相载体，表示基团共价键连接的位点。在一些实施方式中，q 1为1或2。在一些实施方式中，q 2为1-5的整数。在一些实施方式中，R 4含有式(B9)或(B10)所示的结构。在一些实施方式中，R4含有式(B11)或(B12)所示的结构。
在一些实施方式中，R k是Tr(三苯甲基)、MMTr(4-甲氧基三苯甲基)、DMTr(4，4’-双甲氧基三苯甲基)、TMTr(4，4’，4’-三甲氧基苯甲基)中的一种或多种。在一些实施方式中，R k可以是DMTr，即4，4’-双甲氧基三苯甲基(4，4’-dimethoxytrityl)。
L 1的定义如前所述。
在一些实施方式中，L 1被用于将M 1靶向基团连接至含氮骨架上的N原子，从而为第二种siRNA缀合物提供肝靶向功能。在一些实施方式中，L 1包含A1-A26中的任一个或其组合。
根据上述描述，本领域技术人员容易理解的是，相较于本领域公知的亚磷酰胺固相合成方法而言，可通过上述第1官能团以及任选的第2官能团，获得 将缀合分子连接至核苷酸序列的任意可能的位置的第二种siRNA缀合物，例如，缀合分子连接至核苷酸序列的端部，缀合分子连接至核苷酸序列的末端。相应地，除非另有说明，以下涉及缀合制备的描述中，当提及“脱保护”、“偶联”、“盖帽”、“氧化”、“硫化”等反应时，应当理解为本领域公知的亚磷酰胺核酸固相合成方法中所涉及的反应条件和试剂也同样适用于这些反应。示例性的反应条件和试剂将在后文详细描述。
在一些实施方式中，每个S 1独立地是M 1。在一些实施方式中，每个S 1独立地是M 1中至少一个活性羟基被羟基保护基团保护而形成的基团。在一些实施方式中，每个S 1独立地是M 1中任何存在的活性羟基全部被羟基保护基团保护而形成的基团。在一些实施方式中，任何本领域技术人员已知的羟基保护基团均可被用于保护M 1中的活性羟基。在一些实施方式中，被保护的羟基可以式YCOO-表示，其中，每个Y独立地选自于由C 1-C 10烷基和C 6-C 10芳基所组成的组，所述C 1-C 10烷基和C 6-C 10芳基任选地被一个或多个取代基取代，所述取代基选自于由卤素和C 1-C 6烷基所组成的组。在一些实施方式中，每个Y独立地选自于由以下基团所组成的组：甲基、三氟甲基、二氟甲基、单氟甲基、三氯甲基、二氯甲基、一氯甲基、乙基、正丙基、异丙基、苯基、卤苯基，以及C 1-C 6烷基苯基。
在一些实施方式中，每个S 1各自独立地选自于由式A46-A54所组成的组：
Figure PCTCN2018118107-appb-000038
Figure PCTCN2018118107-appb-000039
在一些实施方式中，S 1为式A49或A50。
在一些实施方式中，每个Y独立地选自甲基、三氟甲基、二氟甲基、一氟甲基、三氯甲基、二氯甲基、一氯甲基、乙基、正丙基、异丙基、苯基、卤代苯基以及烷基苯基中的一种；出于简化本公开的缀合分子的目的，在一些实施方式中，Y为甲基。
如前所述，第二种siRNA缀合物的制备方法还包括以下步骤：合成siRNA的另一链(例如，当上述步骤合成了连接有缀合基团的siRNA正义链时，还包括按照固相合成方法合成siRNA的反义链，反之亦然)，分离正义链和反义链，以及退火。具体地，在分离步骤中，连接至核苷酸序列和/或缀合分子的固相载体被切割下来，同时必要的保护基团被脱除(此时，式(321)化合物中的各S 1基团转化为对应的M 1靶向基团)，获得连接有缀合基团的siRNA正义链(或反义链)以及对应的反义链(或正义链)，正义链与反义链退火形成双链RNA结构，获得第二种siRNA缀合物。
在一些实施方式中，所述第二种siRNA缀合物的制备方法包含以下步骤：在偶联反应条件和偶联试剂存在下，将式(321)所示的化合物与正义链或反义链的3′端的第一个核苷单体接触，使式(321)所示的化合物连接上序列中第一个核苷酸，在亚磷酰胺固相合成的条件下，按照期望的正义链或反义链核苷酸种类和顺序，按照3′到5′的方向将核苷单体依次连接，合成siRNA的正义链或反义链；其中，(321)化合物为R4中含有第1官能团和第2官能团，第1官能团含有被保护的羟基，第2官能团具有如式(C1’)或(C3’)所示结构的式(321)所示的化合物，与第一个核苷单体连接前，式(321)化合物经过脱保护；每个核苷单体的连接包括脱保护、偶联、盖帽、氧化或硫化四步反应；得到连接有缀合分子的核酸的正义链或反义链；在亚磷酰胺固相合成的条件下，按照反义链或正义链核苷酸种类和顺序，按照3′到5′的方向将核苷单体依次连接，合成核酸的反义链或正义链；每个核苷单体的连接包括脱保护、偶联、盖帽、氧化或硫化四步反应；脱除保护基并与固相载体切割，分离纯化获得核酸的正义链 和反义链，退火。
在一些实施方式中，第二种siRNA缀合物的制备方法包含以下步骤：按照该双链siRNA中正义链或反义链的核苷酸种类和顺序，按照3′到5′的方向将核苷单体依次连接，合成正义链和反义链，每个核苷单体的连接包括脱保护、偶联、盖帽、氧化或硫化四步反应，得到连接在固相载体上的正义链和连接在固相载体上的反义链；在偶联反应条件和偶联试剂存在下，将式(321)所示的化合物与连接在固相载体上的正义链或连接在固相载体上的反义链接触，将式(321)化合物连接至正义链或反义链，其中，式(321)化合物是R4中含有第1官能团，第1官能团为亚磷酰胺基团的式(321)化合物；脱除保护基并与固相载体切割，分别分离纯化，Nu代表的siRNA的正义链或反义链，退火，其中，所述siRNA的正义链或反义链上连接有缀合基团。
在一些实施方式中，式A59中的P连接至siRNA中的正义链的3′末端，第二种siRNA缀合物的制备方法包括：
(1)脱除式(321)化合物(其中，式(321)化合物为R4中含有第1官能团和第2官能团，第1官能团含有被保护的羟基OR k，第2官能团具有如式(C1’)或(C3’)所示结构的化合物)中的羟基保护基团R k；在偶联反应条件和偶联试剂存在下，将脱保护得到的产物与核苷单体接触，得到通过缀合分子连接至固相载体的核苷单体；
(2)以该通过缀合分子连接至固相载体的核苷单体起始，按照3′-5′的方向通过亚磷酰胺固相合成方法合成siRNA的正义链；
(3)通过亚磷酰胺固相合成方法，合成siRNA的反义链；
(4)分离出siRNA的正义链和反义链并退火，获得第二种siRNA缀合物。
其中，在步骤(1)中，脱除式(321)化合物中的保护基团R k的方法包括在脱保护条件下，将式(321)化合物与脱保护试剂接触。脱保护条件包括温度为0-50℃，在一些实施方式中为15-35℃，反应时间为30-300秒，在一些实施方式中为50-150秒，脱保护试剂可以选自三氟乙酸、三氯乙酸、二氯乙酸、一氯乙酸中的一种或多种，在一些实施方式中为二氯乙酸。脱保护试剂与式(321)化合物的摩尔比为10∶1-1000∶1，在一些实施方式中为50∶1-500∶1。
所述偶联反应条件和偶联试剂可使用任何适合于上述偶联反应的条件和试剂。在一些实施方式中，可使用与所采用的固相合成方法中的偶联反应相同的条件与试剂。
在一些实施方式中，所述偶联反应的条件包括反应温度为0-50℃，在一些 实施方式中为15-35℃。式(321)化合物与核苷单体的摩尔比为1∶1-1∶50，在一些实施方式中为1∶2-1∶5；式(321)化合物和偶联试剂的摩尔比可以1∶1-1∶50，在一些实施方式中为1∶3-1∶10，反应时间为200-3000秒，在一些实施方式中为500-1500秒。偶联试剂选自1H-四氮唑、5-乙硫基1H-四氮唑、5-苄硫基1H-四氮唑中的一种或多种，在一些实施方式中为5-乙硫基1H-四氮唑。所述偶联反应可在有机溶剂中进行，所述有机溶剂选自无水乙腈、无水DMF、无水二氯甲烷中的一种或多种，在一些实施方式中为无水乙腈。相对于式(321)化合物，所述有机溶剂的用量为3-50L/mol，在一些实施方式中为5-20L/mol。
在步骤(2)中，通过亚磷酰胺核酸固相合成的方法，利用上述步骤制备的通过缀合分子连接至固相载体的核苷单体起始，按照3′-5′的方向合成第二种siRNA缀合物的正义链S。此时，缀合基团连接至所得到的正义链的3′末端。
步骤(2)和(3)中所述固相合成的其它条件，包括核苷单体脱保护条件，脱保护试剂种类和用量，偶联反应条件，偶联试剂的种类和用量，盖帽反应的条件，盖帽试剂的种类和用量，氧化反应条件，氧化试剂种类和用量，硫化反应条件，硫化试剂和用量采用本领域中常规使用的各种试剂、用量和条件。
例如，在一些实施方式中，步骤(2)和(3)中所述固相合成可使用如下条件：
核苷单体脱保护条件包括温度为0-50℃，在一些实施方式中为15-35℃，反应时间为30-300秒，在一些实施方式中为50-150秒，脱保护试剂可以选自三氟乙酸、三氯乙酸、二氯乙酸、一氯乙酸、中的一种或多种，在一些实施方式中为二氯乙酸。脱保护试剂与固相载体上4，4′-二甲氧基三苯甲基保护基的的摩尔比为2∶1-100∶1，在一些实施方式中为3∶1-50∶1。
偶联反应条件包括温度为0-50℃，在一些实施方式中为15-35℃，固相载体上连接的核酸序列与核苷单体的摩尔比为1∶1-1∶50，在一些实施方式中为1∶5-1∶15；固相载体上连接的核酸序列和偶联试剂的摩尔比为1∶1-1∶100，在一些实施方式中为1∶50-1∶80，反应时间和偶联试剂的选择与前述相同。
盖帽反应条件包括温度为0-50℃，在一些实施方式中为15-35℃，反应时间为5-500秒，在一些实施方式中为10-100秒，盖帽试剂的选择与前述相同。盖帽试剂的总量与固相载体上连接的核酸序列的摩尔比为1∶100-100∶1，在一些实施方式中为1∶10-10∶1。在盖帽试剂使用等摩尔量的乙酸酐与N-甲基咪唑的情况下，乙酸酐、N-甲基咪唑以及固相载体上连接的核酸序列的摩尔比为1∶1∶10-10∶10∶1，在一些实施方式中为1∶1∶2-2∶2∶1。
氧化反应条件包括温度为0-50℃，在一些实施方式中为15-35℃，反应时间为1-100秒，在一些实施方式中为5-50秒，氧化试剂在一些实施方式中为碘(在一些实施方式中，以碘水的形式提供)。氧化试剂与偶联步骤中固相载体上连接的核酸序列的摩尔比可以为1∶1-100∶1，在一些实施方式中为5∶1-50∶1。在一些实施方式中，所述氧化反应在四氢呋喃∶水∶吡啶＝3∶1∶1-1∶1∶3的混合溶剂中进行。硫化反应条件包括温度为0-50℃，在一些实施方式中为15-35℃，反应时间为50-2000秒，在一些实施方式中为100-1000秒，硫化试剂在一些实施方式中为氢化黄原素。硫化试剂与偶联步骤中固相载体上连接的核酸序列的摩尔比可以为10∶1-1000∶1，在一些实施方式中为10∶1-500∶1。在一些实施方式中，所述硫化反应在乙腈∶吡啶＝1∶3-3∶1的混合溶剂中进行。
在将所有核苷单体连接之后，退火之前，该方法还包括分离出siRNA的正义链和反义链。分离的方法为本领域技术人员所公知，一般包括将合成得到的核苷酸序列从固相载体上切割下来，脱除碱基上、磷酸基上和配体上的保护基团，纯化和脱盐。
将合成得到的核苷酸序列从固相载体上切割下来，并脱除碱基上、磷酸基上和配体上的保护基团可按照siRNA合成中常规的切割和脱保护方法进行。例如，将得到的连接有固相载体的核苷酸序列与浓氨水接触；在脱保护的过程中，A46-A54基团的保护基团YCOO-转化为羟基，S 1基团转化为相应的M 1基团，生成式(308)所示的缀合物。其中，所述浓氨水可以是25-30重量％的氨水，浓氨水的用量与目标siRNA序列相比可以为0.2ml/μmol-0.8ml/μmol。
在所合成的核苷酸序列上存在至少一个2′-TBDMS保护时，所述方法还包括将脱除了固相载体的核苷酸序列与三乙胺三氢氟酸盐接触，以脱除该2′-TBDMS保护。此时，所得到的目标siRNA序列中具有游离的2′-羟基的相应核苷。三乙胺三氢氟酸盐纯品的用量与目标siRNA序列相比为0.4ml/μmol-1.0ml/μmol。这样即可得到式(308)的siRNA缀合物。
纯化和脱盐的方法是本领域技术人员熟知的。例如，可利用制备型离子色谱纯化柱，通过NaBr或NaCl的梯度洗脱，完成核酸的纯化；产品收集合并后，可采用反相色谱纯化柱进行脱盐。
这样得到的第二种siRNA缀合物中，核苷酸之间的磷酸二酯键或硫代磷酸二酯键中的非桥接氧原子或硫原子基本与钠离子结合，第二种siRNA缀合物基本以钠盐形式存在。可以采用熟知的离子交换方法，用氢离子和/或其他阳离子取代所述钠离子，得到其他形式的第二种siRNA缀合物。所述阳离子如前所述。
在合成过程中，可随时对核酸序列的纯度和分子量进行检测，更好地把控合成质量，此类检测方法为本领域技术人员所公知。例如，可通过离子交换色谱检测核酸纯度，并通过液质联用色谱测定分子量。
退火的方法也是本领域技术人员熟知的。例如，可简单地将所合成的正义链(S链)与反义链(AS链)以等摩尔比混合在注射用水中加热至70-95℃，随后室温冷却，使其通过氢键形成双链结构。这样即可得到第二种siRNA缀合物。
在获得本公开的缀合物后，在一些实施方式中，还可利用例如液质联用色谱等方法，通过分子量检测等方式对所合成的第二种siRNA缀合物进行表征，确定所合成的siRNA缀合物为目标设计的第二种siRNA缀合物，且所合成的siRNA的序列与欲合成的siRNA的序列相符，例如为表2中所列的序列之一。
式(321)所示化合物可以通过以下制备方法得到：该方法包括在有机溶剂中，在酯化反应条件下，以及在碱和成酯催化剂存在下，将式(313)所示化合物与环状酸酐接触，离子交换，分离得到式(321)所示化合物：
Figure PCTCN2018118107-appb-000040
其中，n1、n3、m1、m2、m3、R 10、R 11、R 12、R 13、R 14、R 15、L 1、S 1各自的定义和可选择的范围如前所述；
R 6为提供式(321)中R 4的基团。在一些实施方式中，R 6具有式(A61)所示的结构：
Figure PCTCN2018118107-appb-000041
其中，R i为能够实现与含氮骨架上的N连接、与R kO连接并且连接有一个游离羟基的任意基团，R k为羟基保护基团。此时，所获得的是R 4中含有作为羟基保护基团的第1官能团和第2官能团，所述第2官能团含有如式(C1)或(C2)所示结构的式(321)化合物。
所述酯化反应条件包括反应温度为0-100℃，反应时间为8-48小时，在一些实施方式中，所述酯化反应条件为反应温度为10-40℃，反应时间为20-30小时。
在一些实施方式中，所述有机溶剂包含环氧类溶剂、醚类溶剂、卤代烷类溶剂、二甲基亚砜、N，N-二甲基甲酰胺和N，N-二异丙基乙胺中的一种或多种。在一些实施方式中，所述环氧类溶剂为二氧六环和/或四氢呋喃，所述醚类溶剂为乙醚和/或甲基叔丁基醚，所述卤代烷类溶剂为二氯甲烷、三氯甲烷和1，2-二氯乙烷中的一种或多种。在一些实施方式中，所述有机溶剂为二氯甲烷。相对于所述式(313)所示化合物，所述有机溶剂的用量为3-50L/mol，在一些实施方式中为5-20L/mol。
在一些实施方式中，所述环状酸酐为丁二酸酐、戊二酸酐、己二酸酐或庚二酸酐中的一种，在一些实施方式中为丁二酸酐。所述环状酸酐与所述式(313)所示化合物的摩尔比为1∶1-10∶1，在一些实施方式中为2∶1-5∶1。
所述成酯催化剂可以是任何对该酯化反应起到催化作用的催化剂，例如该催化剂可以是4-二甲氨基吡啶。所述催化剂与式(313)所示化合物的摩尔比为1∶1-10∶1，在一些实施方式中为2∶1-5∶1。
在一些实施方式中，所述碱可以是任意的无机碱，有机碱或者它们的结合。考虑溶解性和产物稳定性，所述碱可以是例如三级胺类有机碱。在一些实施方式中，所述三级胺类有机碱为三乙胺或N，N-二异丙基乙胺。所述三级胺类有机碱与式(313)所示化合物的摩尔比为1∶1-20∶1，在一些实施方式中为3∶1-10∶1。
所述离子交换作用是将式(321)化合物转化为期望的羧酸或羧酸盐的形式，离子交换的方法为本领域技术人员所公知，可以使用合适的离子交换溶液和交换条件，得到前述阳离子为M +的缀合分子，在此不做详述。在一些实施方式中，所述离子交换反应使用三乙胺磷酸盐溶液进行，所述三乙胺磷酸盐溶液的浓度为0.2-0.8M，在一些实施方式中为0.4-0.6M，相对于式(313)化合物，所述三乙胺磷酸盐溶液的用量为3-6L/mol，在一些实施方式中为4-5L/mol。
可使用任何合适的分离方法从反应混合物中分离式(321)化合物。在一些实施方式中，可通过蒸发除去溶剂、随后通过色谱方法分离式(321)化合物，例如，可使用如下色谱条件进行分离：(1)正相纯化硅胶：200-300目硅胶填料，使用含1wt‰三乙胺的二氯甲烷∶甲醇＝100∶18-100∶20梯度洗脱；或者(2)反相纯化：C18、C8反相填料，使用甲醇∶乙腈＝0.1∶1-1∶0.1梯度洗脱。在一些实施方式中，可以直接除去溶剂得到式(321)化合物粗产品，该粗产品可以直接用 于后续反应。
在一些实施方式中，式(321)化合物的制备方法还进一步包括在缩合反应条件下，在有机溶剂中，在缩合剂和三级胺类有机碱的存在下，将上述离子交换反应得到的产物进一步与含有氨基或羟基的固相载体进行接触。此时，所获得的是R4中含有第1官能团和第2官能团，第1官能团含有羟基保护基团，第2官能团含有如式(C1’)所示结构的式(321)化合物。
所述固相载体为固相合成siRNA中所用的载体中的一种，其中的一些为本领域技术人员所公知。例如，所述固相载体可以选自含有活性羟基或氨基官能团的固相载体，在一些实施方式中，所述固相载体为氨基树脂或羟基树脂。为了便于后续进行核酸固相合成，所述氨基或羟基树脂在一些实施方式中具有如下参数：粒径100-400目(mesh)，表面氨基或羟基载量为0.2-0.5mmol/g。所述式(321)所示化合物与固相载体的用量比为10-400μmol化合物/每克固相载体(μmol/g)。在一些实施方式中，所述式(321)所示化合物与固相载体的用量比为50-200μmol/g。
所述有机溶剂可以是本领域技术人员已知的任何合适的溶剂或混合溶剂。在一些实施方式中，所述有机溶剂为乙腈、环氧类溶剂、醚类溶剂、卤代烷类溶剂、二甲基亚砜、N，N-二甲基甲酰胺和N，N-二异丙基乙胺中的一种或多种。在一些实施方式中，所述环氧类溶剂为二氧六环和/或四氢呋喃，所述醚类溶剂为乙醚和/或甲基叔丁基醚，所述卤代烷类溶剂为二氯甲烷、三氯甲烷和1，2-二氯乙烷中的一种或多种。在一些实施方式中，所述有机溶剂为乙腈。相对于式(321)化合物，所述有机溶剂的用量为20-200L/mol，在一些实施方式中为50-100L/mol。
所述缩合剂可以是六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷、3-二乙氧基磷酰基-1，2，3-苯唑4(3H)-酮和/或O-苯并三氮唑-四甲基脲六氟磷酸酯，在一些实施方式中为O-苯并三氮唑-四甲基脲六氟磷酸酯。所述缩合剂与式(321)所示化合物的摩尔比为1∶1-20∶1，在一些实施方式中为1∶1-5∶1。
在一些实施方式中，所述三级胺类有机碱为三乙胺和/或N，N-二异丙基乙胺，在一些实施方式中为N，N-二异丙基乙胺；所述三级胺类有机碱与式(321)所示化合物的摩尔比为1∶1-20∶1，在一些实施方式中为1∶1-5∶1。
在一些实施方式中，式(321)化合物的制备方法还可以包括将得到的缩合产物在盖帽反应条件下，在有机溶剂中，与盖帽试剂和酰化催化剂接触，分离得到式(321)所示化合物。所述盖帽反应的作用在于除去任何尚未反应完全的 活性反应官能团，以避免在后续反应中产生不必要的副产物。所述盖帽反应的条件包括反应温度为0-50℃，在一些实施方式中为15-35℃，反应的时间为1-10h，在一些实施方式中为3-6h。盖帽试剂可以使用siRNA固相合成中所使用的盖帽试剂，siRNA固相合成中所使用的盖帽试剂为本领域技术人员所公知。
在一些实施方式中，所述盖帽试剂由盖帽试剂1(cap1)和盖帽试剂2(cap2)组成，其中，盖帽试剂A为N-基甲基咪唑，在一些实施方式中以N-甲基咪唑的吡啶/乙腈混合溶液形式提供，其中，吡啶与乙腈的体积比为1∶10-1∶1，在一些实施方式中为1∶3-1∶1，吡啶与乙腈的总体积与N-甲基咪唑的体积为1∶1-10∶1，在一些实施方式中为3∶1-7∶1。所述盖帽试剂B为乙酸酐，在一些实施方式中以乙酸酐的乙腈溶液形式提供，其中，乙酸酐和乙腈的体积为1∶1-1∶10，在一些实施方式中为1∶2-1∶6。
在一些实施方式中，所述N-甲基咪唑的吡啶/乙腈混合溶液的体积与式(321)化合物的质量之比为5ml/g-50ml/g，在一些实施方式中为15ml/g-30ml/g。所述乙酸酐的乙腈溶液的体积与式(321)化合物的质量之比为0.5ml/g-10ml/g，在一些实施方式中为1ml/g-5ml/g。
在一些实施方式中，盖帽试剂使用等摩尔量的乙酸酐与N-甲基咪唑。所述有机溶剂为乙腈、环氧类溶剂、醚类溶剂、卤代烷类溶剂、二甲基亚砜、N，N-二甲基甲酰胺和N，N-二异丙基乙胺中的一种或多种。在一些实施方式中，所述有机溶剂为乙腈。相对于式(321)化合物，所述有机溶剂的用量为10-50L/mol，在一些实施方式中为5-30L/mol。
所述酰化催化剂可以选自任何可用于成酯缩合或成酰胺缩合的催化剂，例如碱性杂环化合物。在一些实施方式中，所述酰化催化剂为4-二甲氨基吡啶。所述催化剂与式(321)所示化合物的质量之比为0.001∶1-1∶1，在一些实施方式中为0.01∶1-0.1∶1。
可使用任何合适的分离方法从反应混合物中分离式(321)化合物。在一些实施方式中，可通过以有机溶剂充分洗涤，并过滤，去除未反应的反应物、过量的盖帽试剂及其它杂质，得到式(321)化合物，所述有机溶剂选自乙腈、二氯甲烷、甲醇，在一些实施方式中为乙腈。
在一些实施方式中，式(321)所示缀合分子的制备方法包括在有机溶剂中，在偶联反应条件下，以及在偶联试剂存在下，将式(313)所示化合物与亚磷酰二胺接触，分离得到式(321)所示化合物。此时，所获得的是R4中含有第1官能团和第2官能团，第1官能团含有羟基保护基团，第2官能团含有如式(C3) 所示结构的式(321)化合物。
在一些实施方式中，偶联反应条件包括温度为0-50℃，例如为15-35℃，式(313)化合物与亚磷酰二胺的摩尔比可以为1∶1-1∶50，例如为1∶5-1∶15；式(313)化合物和偶联试剂的摩尔比可以为1∶1-1∶100，例如为1∶50-1∶80；反应时间可以为200-3000秒，例如为500-1500秒。所述亚磷酰二胺例如可使用双(二异丙基氨基)(2-氰基乙氧基)膦，其可商购获得或按照本领域中公知的方法合成获得。偶联试剂选自1H-四氮唑、5-乙硫基1H-四氮唑、5-苄硫基1H-四氮唑中的一种或多种，例如为5-乙硫基1H-四氮唑。所述偶联反应可在有机溶剂中进行，所述有机溶剂选自无水乙腈、无水DMF、无水二氯甲烷中的一种或多种，例如为无水乙腈。在一些实施方式中，相对于式(313)化合物，所述有机溶剂的用量为3-50L/mol，例如可以为5-20L/mol。通过进行该偶联反应，式(313)化合物中的羟基与亚磷酰二胺反应形成亚磷酰胺基团。在一些实施方式中，可以直接除去溶剂得到式(321)化合物粗产品，该粗产品可以直接用于后续反应。
在一些实施方式中，式(321)化合物的制备方法还进一步包括以下步骤：在偶联反应条件下，在有机溶剂中，以及在偶联试剂存在下，将分离得到的产物进一步与含有羟基的固相载体进行接触。随后，经盖帽反应、氧化反应，分离得到式(321)化合物。此时，所获得的是R4中含有第1官能团和第2官能团，第1官能团含有羟基保护基团，第2官能团具有如式(C3’)所示结构的式(321)化合物。
在一些实施方式中，所述固相载体为本领域中公知的可用于核酸固相合成的固相载体，例如，可以是经脱保护反应后的市售的通用固相载体(
Figure PCTCN2018118107-appb-000042
HL UnyLinker TM 300Oligonucleotide Synthesis Support，Kinovate Life Sciences公司，结构如式B80所示)：
Figure PCTCN2018118107-appb-000043
脱保护反应为本领域技术人员所公知。在一些实施方式中，脱保护条件包括温度为0-50℃，例如为15-35℃；反应时间为30-300秒，例如为50-150秒。 脱保护试剂可以选自三氟乙酸、三氯乙酸、二氯乙酸、一氯乙酸中的一种或多种，在一些实施方式中，脱保护试剂为二氯乙酸。脱保护试剂与固定相上的-DMTr(4，4′-二甲氧基三苯甲基)保护基的摩尔比为2∶1-100∶1，例如为3∶1-50∶1。通过进行所述脱保护，在所述固相载体表面上获得具有反应活性的游离羟基，便于进行下一步的偶联反应。
偶联反应条件以及偶联试剂的选择可如上所述。通过进行该偶联反应，脱保护反应中形成的游离羟基与亚磷酰胺基团反应形成亚磷酸酯连接。
在一些实施方式中，盖帽反应条件包括温度为0-50℃，例如为15-35℃，反应时间为5-500秒，例如为10-100秒，所述盖帽反应在盖帽试剂存在下进行。盖帽试剂的选择和用量可如上所述。
氧化反应条件包括温度为0-50℃，例如可以为15-35℃，反应时间为1-100秒，例如可以为5-50秒，氧化试剂例如可以为碘(在一些实施方式中，以碘水的形式提供)。在一些实施方式中，氧化试剂与亚磷酸酯基团的摩尔比为1∶1-100∶1，例如可以为5∶1-50∶1。在一些实施方式中，所述氧化反应在四氢呋喃∶水∶吡啶＝3∶1∶1-1∶1∶3的混合溶剂中进行。
在一些实施方式中，R 6为式B7或B8基团中的一种，
Figure PCTCN2018118107-appb-000044
其中q 2的定义如前所述，
此时，式(313)所示化合物可以通过以下制备方法得到：在有机溶剂中，在成酰胺反应条件下，以及在成酰胺反应缩合剂和三级胺类有机碱存在下，将式(314)所示化合物与式(A-1)所示化合物或式(A-2)化合物接触，随后进行分离：
Figure PCTCN2018118107-appb-000045
Figure PCTCN2018118107-appb-000046
其中，n1、n3、m1、m2、m3、R 10、R 11、R 12、R 13、R 14、R 15、L 1、S 1、q 2和R k各自的定义和可选择的范围如前所述。
所述成酰胺反应条件可包括反应温度为0-100℃，反应时间为1-48小时，在一些实施方式中，所述成酰胺反应条件为反应温度为10-40℃，反应时间为2-16小时。
在一些实施方式中，所述有机溶剂为醇类溶剂、环氧类溶剂、醚类溶剂、卤代烷类溶剂、二甲基亚砜、N，N-二甲基甲酰胺和N，N-二异丙基乙胺中的一种或多种。所述醇类溶剂在一些实施方式中为甲醇、乙醇、丙醇中的一种或多种，在一些实施方式中为乙醇。所述环氧类溶剂在一些实施方式中为为二氧六环和/或四氢呋喃。所述醚类溶剂在一些实施方式中为为乙醚和/或甲基叔丁基醚。所述卤代烷类溶剂在一些实施方式中为为二氯甲烷、三氯甲烷和1，2-二氯乙烷中的一种或多种。在一些实施方式中，所述有机溶剂为二氯甲烷。相对于式(314)化合物，有机溶剂用量为3-50L/mol，在一些实施方式中为3-20L/mol。
在一些实施方式中，所述成酰胺反应缩合剂为六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷、3-二乙氧基磷酰基-1，2，3-苯唑4(3H)-酮、4-(4，6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐、2-乙氧基-1-乙氧碳酰基-1，2-二氢喹啉(EEDQ)或O-苯并三氮唑-四甲基脲六氟磷酸酯，在进一步的实施方式中为3-二乙氧基磷酰基-1，2，3-苯唑4(3H)-酮。所述成酰胺反应缩合剂与式(314)所示化合物的摩尔比可以为1∶1-10∶1，在一些实施方式中为2.5∶1-5∶1。
在一些实施方式中，所述三级胺类有机碱为三乙胺或N，N-二异丙基乙胺，在一些实施方式中为N，N-二异丙基乙胺。所述三级胺类有机碱与式(314)所示化合物的摩尔比为3∶1-20∶1，在一些实施方式中为5∶1-10∶1。
式(A-1)和式(A-2)化合物可通过任何适当的方式制备。例如，当R k为DMTr基团时，可通过甘油酸钙与DMTrCl反应制备式(A-1)化合物；类似地，可先将3-氨基-1，2-丙二醇与环状酸酐接触，随后再与DMTrCl反应制备式(A-2)化合物，所述环状酸酐可以是碳原子数为4-13、在一些实施方式中为4-8的环状酸酐。本领域技术人员容易理解的是，所述环状酸酐的选择对应于(A-2)化合物中q 2的不同值，例如，当所述环状酸酐为丁二酸酐时，q 2＝1， 当所述环状酸酐为戊二酸酐时，q 2＝2，以此类推。
在一些变型中，也可通过使式(314)所示化合物依次与所述环状酸酐、3-氨基-1，2-丙二醇和DMTrCl反应，制备式(313)化合物。本领域技术人员容易理解的是，这些变型不会影响式(313)化合物的结构与功能，并且这些变型是本领域技术人员在上述方法的基础上容易实现的。
与上述类似地，可使用任何合适的分离方法从反应混合物中分离式(313)化合物。在一些实施方式中，可通过蒸发除去溶剂、随后通过色谱方法分离式(313)化合物，例如，可使用如下两种色谱条件进行分离：(1)正相纯化硅胶：200-300目硅胶填料，使用石油醚∶乙酸乙酯∶二氯甲烷∶N，N-二甲基甲酰胺＝1∶1∶1∶0.5-1∶1∶1∶0.6梯度洗脱；以及(2)反相纯化：C18、C8反相填料，使用甲醇∶乙腈＝0.1∶1-1∶0.1梯度洗脱。在一些实施方式中，可以直接除去溶剂得到式(313)化合物粗产品，该粗产品可以直接用于后续反应。
在一些实施方式中，式(314)所示化合物可以通过以下制备方法得到：该方法包括在有机溶剂中，在脱保护反应条件下，将式(315)所示化合物与卤代乙酸接触，随后进行分离：
Figure PCTCN2018118107-appb-000047
其中，R 7选自式(330)、(331)、(332)或(333)所示的基团，在一些实施方式中，R 7的结构如式(330)所示：
Figure PCTCN2018118107-appb-000048
n1、n3、m1、m2、m3、R 10、R 11、R 12、R 13、R 14、R 15、L 1、S 1各自的定义和可选择的范围如前所述。
所述卤代乙酸可选自二氯乙酸、三氯乙酸、一氯乙酸和三氟乙酸中的一种 或多种，在一些实施方式中为二氯乙酸。
所述脱保护反应条件包括反应温度为0-100℃，反应时间为0.1-24小时，在一些实施方式中为反应温度为10-40℃，反应时间为0.5-16小时。
在一些实施方式中，所述有机溶剂为环氧类溶剂、醚类溶剂、卤代烷类溶剂、二甲基亚砜、N，N-二甲基甲酰胺和N，N-二异丙基乙胺中的一种或多种。所述环氧类溶剂在一些实施方式中为二氧六环和/或四氢呋喃，所述醚类溶剂在一些实施方式中为乙醚和/或甲基叔丁基醚，所述卤代烷类溶剂在一些实施方式中为二氯甲烷、三氯甲烷和1，2-二氯乙烷中的一种或多种，在一些实施方式中，所述有机溶剂为二氯甲烷。相对于式(315)化合物，有机溶剂用量为3-50L/mol，在一些实施方式中为5-20L/mol。
所述卤代乙酸与所述式(315)所示化合物的摩尔比为5∶1-100∶1，在一些实施方式中为10∶1-50∶1。
与上述类似地，可使用任何合适的分离方法从反应混合物中分离式(314)化合物。在一些实施方式中，可通过蒸发除去溶剂、随后通过色谱方法分离式(314)化合物，例如，可使用如下两种色谱条件进行分离：(1)正相纯化硅胶：200-300目硅胶填料，使用二氯甲烷∶甲醇＝100∶30-100∶40梯度洗脱；以及(2)反相纯化：C18、C8反相填料，使用甲醇∶乙腈＝0.1∶1-1∶0.1梯度洗脱。在一些实施方式中，可以直接除去溶剂得到式(314)化合物粗产品，该粗产品可以直接用于后续反应。
式(315)所示化合物可以通过以下制备方法得到：该方法包括在有机溶剂中，在成酰胺反应缩合剂和三级胺类有机碱存在下，在缩合反应条件下，将式(317)所示化合物与式(316)所示化合物接触，随后进行分离：
Figure PCTCN2018118107-appb-000049
其中，n1、n3、m1、m2、m3、R 7、R 10、R 11、R 12、R 13、R 14、R 15、L 1、S 1各自的定义和可选择的范围如前所述。
式(316)化合物可使用例如J.Am.Chem.Soc.2014，136，16958-16961中所公开的化合物，或者，式(316)化合物可由本领域技术人员通过各种方法制 备，例如，可参照美国专利US8106022B2实施例1中所公开的方法制备某些式(316)化合物，以引用的方式将以上文献的全部内容整体并入本文。
在一些实施方式中，所述缩合反应条件包括反应温度为0-100℃，反应时间为0.1-24小时，在一些实施方式中为反应温度为10-40℃，反应时间为0.5-16小时。
所述式(316)所示化合物与所述式(317)所示化合物的摩尔比可以为2∶1-10∶1，在一些实施方式中为2.5∶1-5∶1。
在一些实施方式中，所述有机溶剂为乙腈、环氧类溶剂、醚类溶剂、卤代烷类溶剂、二甲基亚砜、N，N-二甲基甲酰胺和N，N-二异丙基乙胺中的一种或多种，所述环氧类溶剂在一些实施方式中为二氧六环和/或四氢呋喃，所述醚类溶剂在一些实施方式中为乙醚和/或甲基叔丁基醚，所述卤代烷类溶剂在一些实施方式中为二氯甲烷、三氯甲烷和1，2-二氯乙烷中的一种或多种，在一些实施方式中，所述有机溶剂为乙腈。相对于式(317)化合物，所述有机溶剂的用量为3-50L/mol，在一些实施方式中为5-20L/mol。
所述成酰胺反应缩合剂为六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷、3-二乙氧基磷酰基-1，2，3-苯唑4(3H)-酮(DEPBT)、O-苯并三氮唑-四甲基脲六氟磷酸酯或4-(4，6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐，在一些实施方式中为4-(4，6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐。所述成酰胺反应缩合剂与式(317)所示化合物的摩尔比为2∶1-10∶1，在一些实施方式中为2.5∶1-5∶1。
所述三级胺类有机碱为N-甲基吗啉、三乙胺或N，N-二异丙基乙胺，在一些实施方式中为N-甲基吗啉；所述三级胺类有机碱与式(317)所示化合物的摩尔比为3∶1-20∶1，在一些实施方式中为5∶1-10∶1。
与上述类似地，可使用任何合适的分离方法从反应混合物中分离式(315)化合物。在一些实施方式中，可通过蒸发除去溶剂、随后通过色谱方法分离式(315)化合物例如，可使用如下两种色谱条件进行分离：(1)正相纯化硅胶：200-300目硅胶填料，使用二氯甲烷∶甲醇＝100∶5-100∶7梯度洗脱；以及(2)反相纯化：C18、C8反相填料，使用甲醇∶乙腈＝0.1∶1-1∶0.1梯度洗脱。在一些实施方式中，可以直接除去溶剂得到式(315)化合物粗产品，该粗产品可以直接用于后续反应。
在一些实施方式中，式(317)化合物与足量的一种式(316)化合物一次反应即生成期望的式(315)化合物，此时，各个S 1-L 1部分彼此相同。在一些实施方式中，可根据需要，通过使式(317)化合物分批与不同的式(316)化 合物，即L 1和/或S 1不同的式(316)化合物发生反应，使得生成的式(315)化合物中含有两种以上的S 1和/或L 1。例如，对于1eq的式(317)化合物，可先使其与2eq的第一式(316)化合物接触，在式(317)化合物中的两个末端伯胺基团上连接第一S 1-L 1部分，随后，使其继续与(n3+n1-1)eq的第二式(316)化合物接触(n3和n1的定义和取值范围如前所述)，在式(317)化合物中的(n3+n1-1)个仲胺基团上连接第二S 1-L 1部分。
在一些实施方式中，式(317)所示化合物可以通过以下制备方法得到：该方法包括在有机溶剂存在下，在脱保护反应条件下，将式(318)所示化合物与甲胺水溶液接触，随后进行分离：
Figure PCTCN2018118107-appb-000050
其中，n1、n3、m1、m2、m3、R 7、R 10、R 11、R 12、R 13、R 14、R 15各自的定义和可选择的范围如前所述。
所述脱保护反应条件包括反应温度为0-150℃，反应时间为5-72小时，在一些实施方式中为反应温度为20-80℃，反应时间为10-30小时。
所述有机溶剂选自醇，在一些实施方式中为甲醇、乙醇和异丙醇中的一种，在一些实施方式中为甲醇；相对于式(318)化合物，所述有机溶剂的用量为1-20L/mol，在一些实施方式中为1.5-10L/mol。
所述甲胺水溶液的浓度可以为30-40质量％，甲胺与式(318)所示化合物的摩尔比可以为10∶1-500∶1，在一些实施方式中为50∶1-200∶1。
与上述类似地，可使用任何合适的分离方法从反应混合物中分离式(317)化合物。在一些实施方式中，可通过蒸发除去溶剂、随后通过色谱方法分离式(317)化合物例如，可使用如下两种色谱条件进行分离：正相纯化硅胶：(1)200-300目硅胶填料，使用二氯甲烷∶甲醇∶氨水(25wt％)＝1∶1∶0.05-1∶1∶0.25梯度洗脱；以及(2)反相纯化：C18、C8反相填料，使用甲醇∶乙腈＝0.1∶1-1∶0.1梯度洗脱。在一些实施方式中，可以直接除去溶剂得到式(317)化合物粗产品，该粗产品可以直接用于后续反应。
在一些实施方式中，式(318)所示化合物可以通过以下制备方法得到：该方法包括在有机溶剂存在下，在取代反应条件下，将式(319)所示化合物与三苯基氯甲烷(TrCl)、二苯基乙苯基氯甲烷、苯基二乙苯基氯甲烷或三乙苯基氯 甲烷、在一些实施方式中为三苯基氯甲烷(TrCl)接触，随后进行分离：
Figure PCTCN2018118107-appb-000051
其中，n1、n3、m1、m2、m3、R 10、R 11、R 12、R 13、R 14、R 15各自的定义和可选择的范围如前所述。
所述取代反应条件可包含反应温度为0-100℃，反应时间为5-72小时，在一些实施方式中，反应条件包含反应温度为10-40℃，反应时间为10-30小时。
三苯基氯甲烷(TrCl)、二苯基乙苯基氯甲烷、苯基二乙苯基氯甲烷或三乙苯基氯甲烷可商购得到，三苯基氯甲烷(TrCl)、二苯基乙苯基氯甲烷、苯基二乙苯基氯甲烷或三乙苯基氯甲烷与式(319)所示化合物的摩尔比可以为1∶1-10∶1，在一些实施方式中为1∶1-3∶1。
所述有机溶剂可以为环氧类溶剂、醚类溶剂、卤代烷类溶剂、二甲基亚砜、N，N-二甲基甲酰胺和N，N-二异丙基乙胺中的一种或多种。所述环氧类溶剂在一些实施方式中为二氧六环和/或四氢呋喃，所述醚类溶剂在一些实施方式中为乙醚和/或甲基叔丁基醚，所述卤代烷类溶剂在一些实施方式中为二氯甲烷、三氯甲烷和1，2-二氯乙烷中的一种或多种；在一些实施方式中，所述有机溶剂为二氯甲烷。相对于式(319)化合物，所述有机溶剂的用量可以为3-50L/mol，在一些实施方式中为5-20L/mol。
与上述类似地，可使用任何合适的分离方法从反应混合物中分离式(318)化合物。在一些实施方式中，可通过蒸发除去溶剂、随后通过色谱方法分离式(318)化合物例如，可使用如下两种色谱条件进行分离：(1)正相纯化硅胶：200-300目硅胶填料，使用甲醇∶二氯甲烷＝0.01∶1-0.5∶1梯度洗脱；或者使用甲醇∶二氯甲烷∶乙酸乙酯∶石油醚＝0.1∶1∶1∶1-1∶1∶1∶1梯度洗脱。以及(2)反相纯化：C18、C8反相填料，使用甲醇∶乙腈＝0.1∶1-1∶0.1梯度洗脱。在一些实施方式中，可以直接除去溶剂得到式(318)化合物粗产品，该粗产品可以直接用于后续反应。
在一些实施方式中，式(319)所示化合物可以通过以下制备方法得到：该方法包括在有机溶剂中，在取代反应条件下，将式(320)所示化合物与三氟乙酸乙酯接触，随后进行分离：
Figure PCTCN2018118107-appb-000052
其中，n1、n3、m1、m2、m3、R 10、R 11、R 12、R 13、R 14、R 15各自的定义和可选择的范围如前所述。
在一些实施方式中，所述有机溶剂为乙腈、环氧类溶剂、醚类溶剂、卤代烷类溶剂、二甲基亚砜、N，N-二甲基甲酰胺和N，N-二异丙基乙胺中的一种或多种。在一些实施方式中，所述环氧类溶剂为二氧六环和/或四氢呋喃，在一些实施方式中，所述醚类溶剂为乙醚和/或甲基叔丁基醚，在一些实施方式中，所述卤代烷类溶剂为二氯甲烷、三氯甲烷和1，2-二氯乙烷中的一种或多种，在一些实施方式中，所述有机溶剂为乙腈。相对于式(320)化合物，所述有机溶剂的用量为1-50L/mol，在一些实施方式中为1-20L/mol。
所述取代反应条件可包括反应温度为0-100℃，反应时间为5-72小时，在一些实施方式中，所述取代反应条件包括为反应温度为10-40℃，反应时间为10-30小时。
式(320)化合物可商购获得，或者由本领域技术人员使用已知的方法获得。例如，当m1＝m2＝m3＝3，n1＝1，n3＝2，且R 10、R 11、R 12、R 13、R 14、R 15均为H时，式(320)化合物可自阿法埃莎公司商购获得。
所述三氟乙酸乙酯与式(320)所示化合物的摩尔比为2∶1-10∶1，在一些实施方式中为3∶1-5∶1。
与上述类似地，可使用任何合适的分离方法从反应混合物中分离式(319)化合物。在一些实施方式中，可通过蒸发除去溶剂、随后通过色谱方法分离式(319)化合物例如，可使用如下两种色谱条件进行分离：(1)正相纯化硅胶：200-300目硅胶填料，使用甲醇∶二氯甲烷＝0.01∶1-0.5∶1梯度洗脱；或者使用甲醇∶二氯甲烷∶乙酸乙酯∶石油醚＝0.1∶1∶1∶1-1∶1∶1∶1梯度洗脱，以及(2)反相纯化：C18、C8反相填料，使用甲醇∶乙腈＝0.1∶1-1∶0.1梯度洗脱。在一些实施方式中，可以直接除去溶剂得到式(319)化合物粗产品，该粗产品可以直接用于后续反应。
本公开的第一种或第二种siRNA缀合物也可以与药学上可接受的其它辅料联用，该辅料可以为本领域常规采用的各种制剂或化合物的一种或多种，详情可参见上文关于本公开的药物组合物的描述。
本公开的siRNA、含该siRNA的药物组合物、第一种siRNA缀合物以及第二种siRNA缀合物的应用
在一些实施方式中，本公开提供了siRNA、含该siRNA的药物组合物、第一种siRNA缀合物以及第二种siRNA缀合物在制备用于治疗和/或预防由所述乙型肝炎病毒的感染引起的病理状况或疾病的药物中的用途。
按照本公开的一种实施方式，本公开提供了一种治疗乙型肝炎病毒的感染引起的病理状况或疾病的方法，该方法包括向患者给予本公开提供的siRNA、药物组合物、第一种siRNA缀合物以及第二种siRNA缀合物。
按照本公开另外一种实施方式，本公开提供了一种抑制感染慢性乙型肝炎病毒的肝炎细胞中乙型肝炎病毒基因表达的方法，该方法包括将本公开提供的siRNA、药物组合物、第一种siRNA缀合物以及第二种siRNA缀合物与所述感染慢性乙型肝炎病毒的肝炎细胞接触。
所述由乙型肝炎病毒的感染引起的病理状况或疾病选自慢性肝病、肝炎、肝纤维化疾病和肝增生性疾病。
通过将本公开的siRNA和/或药物组合物、第一种siRNA缀合物以及第二种siRNA缀合物给予有需要的患者，可以通过RNA干扰的机制达到治疗乙肝的目的。因此，本公开的siRNA和/或药物组合物、第一种siRNA缀合物以及第二种siRNA缀合物可用于预防和/或治疗乙肝、或用于制备用于预防和/或治疗乙肝的药物。
本文所使用的术语“给药/给予”是指通过使得至少部分地将siRNA或药物组合物、第一种siRNA缀合物以及第二种siRNA缀合物定位于期望的位点以产生期望效果的方法或途径，将siRNA或药物组合物、第一种siRNA缀合物以及第二种siRNA缀合物放置入患者体内。适于本公开方法的给药途径包括局部给药和全身给药。一般而言，局部给药导致与患者整个身体相比将更多siRNA或药物组合物、第一种siRNA缀合物以及第二种siRNA缀合物递送至特定位点；而全身给药导致将所述siRNA或药物组合物、第一种siRNA缀合物以及第二种siRNA缀合物递送至患者的基本整个身体。考虑到本公开旨在提供预防和/或治疗乙肝的手段，优选能够将药物递送至肝脏的给药方式。
可通过本领域已知的任何合适途径向患者给药，所述途径包括但不仅限于：口服或胃肠外途径，包括静脉内给药、肌肉内给药、皮下给药、经皮给药、气道给药(气雾剂)、肺部给药、鼻部给药、直肠给药和局部给药(包括口腔含化给药和舌下给药)。给药频率可以是每天、每周、每两周、每三周、每个月或每 年1次或多次。
本公开所述的siRNA或药物组合物、第一种siRNA缀合物以及第二种siRNA缀合物的使用剂量可为本领域常规的剂量，所述剂量可以根据各种参数、尤其是患者的年龄、体重和性别来确定。可在细胞培养或实验动物中通过标准药学程序测定毒性和疗效，例如测定LD50(使50％的群体致死的剂量)和ED50(在量反应中指能引起50％最大反应强度的剂量，在质反应中指引起50％实验对象出现阳性反应时的剂量)。毒性和疗效之间的剂量比为治疗指数，可以用LD50/ED50的比值来表示。优选显示出高治疗指数的siRNA或药物组合物、第一种siRNA缀合物以及第二种siRNA缀合物。可基于由细胞培养分析和动物研究得到的数据得出人用剂量的范围。
在给予本公开所述的药物组合物、第一种siRNA缀合物以及第二种siRNA缀合物时，例如，对于雄性或雌性、6-12周龄、体重18-25g的C57BL/6J或C3H/HeNCrlVr小鼠，以所述药物组合物或siRNA缀合物中的siRNA的量计：(i)对于siRNA与药学上可接受的载体形成的药物组合物，其siRNA用量可以为0.001-50mg/kg体重，在进一步的实施方式中为0.01-10mg/kg体重，在更进一步的实施方式中为0.05-5mg/kg体重，在又进一步的实施方式中为0.1-3mg/kg体重；(ii)对于siRNA与药学上可接受的缀合分子形成的第一种和/或第二种siRNA缀合物，其siRNA用量可以为0.001-100mg/kg体重，在进一步的实施方式中为0.01-50mg/kg体重，在更进一步的实施方式中为0.05-20mg/kg体重，在又进一步的实施方式中为0.1-10mg/kg体重。在给予本公开所述的siRNA时，可优选上述用量。
另外，通过将本公开的siRNA和/或药物组合物、第一种siRNA缀合物以及第二种siRNA缀合物导入感染慢性HBV的肝炎细胞，还可以通过RNA干扰的机制达到抑制感染慢性HBV的肝炎细胞中HBV基因的表达这一目的。在一些优选的实施方式中，所述细胞为HepG2.2.15细胞。
采用本公开提供的方法抑制HBV基因在细胞中表达，无论使用提供的siRNA还是药物组合物还是第一种siRNA缀合物以及第二种siRNA缀合物，siRNA用量一般是这样的量：其足以减少靶基因的表达，并导致在靶细胞表面处1pM至1μM、或0.01nM至100nM、或0.05nM至50nM或至约5nM的细胞外浓度。达到该局部浓度所需的量将随各种因素而变化，所述因素包括递送方法、递送部位、在递送部位和靶细胞或组织之间的细胞层的数目、递送是局部还是全身等。在递送部位处的浓度可以显著高于在靶细胞或组织的表面处的浓 度。
试剂盒
本公开提供了一种试剂盒，该试剂盒含有有效量的本公开的siRNA、药物组合物、第一种siRNA缀合物和第二种siRNA缀合物中的至少一种。
在一些实施方式中，本文所述的试剂盒可在一个容器中提供修饰的siRNA。在一些实施方式中，本文所述的试剂盒可包含一个提供药学上可接受的赋形剂的容器。在一些实施方式中，所述试剂盒中还可包含其它成分，如稳定剂或防腐剂等。在一些实施方式中，本文所述的试剂盒可在不同于提供本文所述修饰的siRNA的容器以外的其它容器中包含至少一种其它治疗剂。在一些实施方式中，所述试剂盒可包含用于将修饰的siRNA与药学上可接受的载体和/或辅料或其它成分(若有的话)进行混合的说明书。
在本公开的试剂盒中，所述修饰的siRNA和药学上可接受的载体和/或辅料以及所述修饰的siRNA、药物组合物、第一种siRNA缀合物和/或第二种siRNA缀合物和/或缀合物，和/或药学上可接受的辅料可以任何形式提供，例如液体形式、干燥形式或冻干形式。在一些实施方式中，所述修饰的siRNA和药学上可接受的载体和/或辅料以及所述药物组合物和/或缀合物和任选的药学上可接受的辅料基本上纯净和/或无菌。在一些实施方式中，可在本公开的试剂盒中提供无菌水。
下面将通过实施例来进一步说明本公开，但是本公开并不因此而受到任何限制。
有益效果
在一些实施方式中，本公开提供的siRNA、组合物或siRNA缀合物可在体内具有更高的稳定性、更低的毒性和/或更高的活性。在一些实施方式中，本公开提供的siRNA、siRNA组合物或siRNA缀合物在体内显示出至少20％，30％，40％，50％，60％，70％，80％，90％或95％的HBV基因表达抑制率。在一些实施方式中，本公开提供的siRNA、siRNA组合物或siRNA缀合物在体内显示出至少20％，30％，40％，50％，60％，70％，80％，90％或95％的肝内HBV基因表达抑制率。在一些实施方式中，本公开提供的siRNA、siRNA组合物或siRNA缀合物在体内显示出至少20％，30％，40％，50％，60％，70％，80％，90％或95％的动物模型中肝内HBV基因表达抑制率。在一些实施方式中，本公开提供的siRNA、siRNA组合物或siRNA缀合物在体内显示出至少20％，30％，40％，50％，60％，70％，80％，90％或95％的HBV表面抗原表达抑制 率。在一些实施方式中，本公开提供的siRNA、组合物或siRNA缀合物未显示出明显脱靶效应。脱靶效应可以是例如抑制非靶基因的基因正常表达。据认为，如果脱靶基因表达的结合/抑制与在靶基因效果相比低于50％、40％、30％、20％或10％时，该脱靶效应就是不显著的。
在一些实施方式中，本公开提供的siRNA缀合物具有较好的体外抑制活性，0.1nM下抑制率高达99％。
在一些实施方式中，本公开提供的siRNA缀合物具有较好的体内抑制活性，1mg/kg下抑制率高达93.8％。
在一些实施方式中，本公开提供的siRNA缀合物在具有优异的靶mRNA抑制效果的同时，还显示出低的脱靶效应。
在一些实施方式中，本公开提供的siRNA缀合物在Tritosome中可维持长时间不降解，显示出很好的稳定性。
在一些实施方式中，本公开提供的siRNA缀合物在人血浆中直至72h时仍未降解，显示出优异的在人血浆中的稳定性。
在一些实施方式中，本公开提供的siRNA缀合物在食蟹猴血浆中直至72h仍未降解，显示出优异的在猴血浆中的稳定性。
在一些实施方式中，单次给药3mg/kg的缀合物4对HBsAg的最大抑制率在90％以上，且至少维持21天。
本公开的其他特征和优点将在随后的具体实施方式部分予以详细说明
实施例
以下将通过实施例对本公开进行详细描述。除非特别说明，以下实施例中所用到的试剂、培养基均为市售商品，所用到的核酸电泳、real-time PCR等操作均参照Molecular Cloning(Cold Spring Harbor Laboratory Press(1989))所记载的进行。
若无其它说明，以下提供的试剂比例均按体积比(v/v)计算。
制备例1：缀合物4、18和19的制备
本制备例中，合成了缀合物4(以下，也称为L10-siHB1M1SVP缀合物)，计划合成缀合物18(以下，也称为L10-siHB1M1SP)和缀合物19(以下，也称为L10-siHB1M1SPs)。所述缀合物为L-9缀合分子分别与编号为siHB1M1SVP、siHB1M1SP或siHB1M1SPs的siRNA缀合后形成的缀合物。该缀合物中所缀合的siRNA的序列参见表2。
(1-1)L-10化合物的合成
按照以下方法，合成了L-10化合物：
Figure PCTCN2018118107-appb-000053
(1-1-1)缀合末端段GAL-5的合成
Figure PCTCN2018118107-appb-000054
(1-1-1a)GAL-2的合成
将100.0g GAL-1(N-乙酰-D-半乳糖胺盐酸盐，CAS号：1772-03-8，购自宁波弘翔生化公司，463.8mmol)溶于1000ml无水吡啶，冰水浴下加入540ml乙酸酐(购自Enox公司，5565.6mmol)，室温搅拌反应1.5小时。将反应液倒入10L冰水中，减压抽滤，滤饼用2L冰水洗涤后，加乙腈/甲苯混合溶剂(体积比乙腈∶甲苯＝1∶1)至完全溶解，蒸干溶剂，得到白色固体产品GAL-2 130.0g。
(1-1-1b)GAL-3的合成
将步骤(1-1-1a)中获得的GAL-2(35.1g，90.0mmol)溶解于213ml无水1，2-二氯乙烷中，在冰水浴且氮气保护条件下，加入24.0g TMSOTf(CAS号：27607-77-8，购自麦克林公司，108.0mmol)，室温反应过夜。
在反应液中加入400ml二氯甲烷稀释，以硅藻土过滤，再加入1L饱和碳酸氢钠水溶液，搅拌均匀，分出有机相，水相用二氯乙烷萃取两次，每次300ml，合并有机相，分别用300ml饱和碳酸氢钠水溶液和300ml饱和食盐水洗涤，分出有机相，无水硫酸钠干燥，减压蒸干溶剂，得到浅黄色粘稠糖稀状产品GAL-3 26.9g。
(1-1-1c)GAL-4的合成
将步骤(1-1-1b)中获得的GAL-3(26.9g，81.7mmol)溶于136ml无水1，2-二氯乙烷中，加入干燥的
Figure PCTCN2018118107-appb-000055
分子筛粉末30g，再加入9.0g 5-己烯-1-醇(CAS号：821-41-0，购自Adamas-beta公司，89.9mmol)，室温下搅拌30分钟，冰浴和氮气保护下加入9.08g TMSOTf(40.9mmol)，室温下搅拌反应过夜。过滤除去
Figure PCTCN2018118107-appb-000056
分子筛粉末，滤液中加入300ml二氯甲烷稀释，以硅藻土过滤，再加入 500ml饱和碳酸氢钠水溶液搅拌10分钟洗涤，分出有机相，水相用300ml二氯乙烷萃取一次，合并有机相并分别用300ml饱和碳酸氢钠水溶液和300ml饱和食盐水洗涤，分出有机相，无水硫酸钠干燥，减压蒸干溶剂，得到黄色糖稀状产品GAL-4 41.3g，不进行纯化直接进行下一步氧化反应。
(1-1-1d)GAL-5的合成
将按照步骤(1-1-1c)中描述的方法得到的GAL-4(14.9g，34.7mmol)溶于77ml二氯甲烷和77ml乙腈的混合溶剂中，分别加入103ml去离子水和29.7g高碘酸钠(CAS号：7790-28-5，购自阿拉丁公司，138.8mmol)，冰水浴下搅拌10分钟，加入三氯化钌(CAS号：14898-67-0，购自安耐吉公司，238mg，1.145mmol)，室温反应过夜。反应液加入300ml水稀释搅拌，加饱和碳酸氢钠调pH约为7.5，分出并弃去有机相，水相用二氯甲烷萃取三次，每次200ml，弃去有机相。水相用柠檬酸固体调节pH约为3，用二氯甲烷萃取三次，每次200ml，合并有机相，无水硫酸钠干燥，减压蒸干溶剂，得到白色泡沫状固体产品GAL-56.85g。 1H NMR(400MHz，DMSO)δ12.01(br，1H)，7.83(d，J＝9.2Hz，1H)，5.21(d，J＝3.2Hz，1H)，4.96(dd，J＝11.2，3.2Hz，1H)，4.49(d，J＝8.4Hz，1H)，4.07-3.95(m，3H)，3.92-3.85(m，1H)，3.74-3.67(m，1H)，3.48-3.39(m，1H)，2.20(t，J＝6.8Hz，2H)，2.11(s，3H)，2.00(s，3H)，1.90(s，3H)，1.77(s，3H)，1.55-1.45(m，4H).
(1-1-2)M-11-T3的合成：
Figure PCTCN2018118107-appb-000057
将J-0(1.883g，10mmol，商购自阿法埃莎公司)溶于25ml乙腈中，加入三乙胺(4.048g，40mmol)冰水浴冷却至0℃，加入三氟乙酸乙酯(5.683g，40mmol)，室温下反应22h，减压蒸干溶剂，真空油泵发泡干燥18h，得到5.342g粗品固体M-11-T3，不经进一步纯化地直接用于后续反应。MS m/z：C 15H 22F 9N 4O 3，[M+H] +，理论：477.35，实测：477.65。
(1-1-3)M-11-T3-Tr的合成：
Figure PCTCN2018118107-appb-000058
将M-11-T3粗品(5.342g，10mmol)溶于50ml二氯甲烷，向反应液中加入TrCl(3.345g，12mmol)和三乙胺(1.518g，15mmol)，室温下搅拌反应20h，用饱和碳酸氢钠洗涤反应液2次，每次20ml，20ml饱和食盐水洗涤1次，有机相用无水硫酸钠干燥，过滤后减压蒸干有机溶剂，真空油泵发泡干燥过夜，得到粗品固体M-11-T3-Tr 7.763g。MS m/z：C 34H 36F 9N 4O 3，[M+Na] +，理论：741.25，实测：741.53。粗品固体M-11-T3-Tr不经纯化地继续用于下一步M-18-Tr的合成。
(1-1-4)M-18-Tr的合成：
Figure PCTCN2018118107-appb-000059
将步骤(1-1-3)中获得的M-11-T3-Tr粗品(7.763g，10mmol)溶于100ml甲醇，再加入100ml甲胺水溶液(40质量％)，在50℃搅拌反应23h，过滤除去不溶颗粒物，减压蒸干溶剂，加入200ml体积比为1∶1的二氯甲烷∶甲醇混合溶剂，用50ml饱和碳酸氢钠洗涤，水相再用二氯甲烷(DCM)萃取3次，每次50ml，合并有机相，用无水硫酸钠干燥，过滤后减压蒸干溶剂，真空油泵发泡干燥过夜，用200-300目正相硅胶柱纯化，石油醚装柱，以1wt％三乙胺中和硅胶酸性，以二氯甲烷∶甲醇∶氨水(25wt％)＝1∶1∶0.05-1∶1∶0.25梯度洗脱，收集产物洗脱液，减压蒸干溶剂，真空油泵发泡干燥得到纯品M-18-Tr 2.887g。 1H NMR(400MHz，DMSO)δ7.47-7.39(m，6H)，7.32-7.24(m，6H)，7.19-7.12(m，3H)，2.60-2.47(m，4H)，2.46-2.19(m，13H)，1.70-1.55(m，4H)，1.40(p，J＝6.8Hz，2H).MS m/z：C 28H 39N 4，[M+H] +，理论：431.65，实测：432.61。
(1-1-5)L-5-Tr的合成：
Figure PCTCN2018118107-appb-000060
将步骤(1-1-4)中获得的M-18-Tr(2.02g，4.69mmol)与步骤(1-1-1)中获得的GAL-5(6.93g，15.48mmol)混合溶于47ml乙腈，加入N-甲基吗啉(3.13g， 30.96mmol)和4-(4，6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐(DMTMM，4.28g，15.48mmol)，室温搅拌反应2h。以200ml二氯甲烷稀释反应液，100ml饱和碳酸氢钠溶液洗涤有机相，100ml饱和食盐水洗涤有机相，有机相用无水硫酸钠干燥，过滤后减压蒸干溶剂得粗品。200-300目正相硅胶柱纯化，石油醚装柱，以1wt％三乙胺中和硅胶酸性，以二氯甲烷∶甲醇＝100∶5-100∶7梯度洗脱，收集产物洗脱液，减压蒸干得到纯品L-5-Tr 7.49g。 1H NMR(400MHz，DMSO)δ7.83-7.10(m，4H)，7.67-7.60(m，1H)，7.44-7.34(m，6H)，7.33-7.24(m，6H)，7.20-7.15(m，3H)，5.22(s，3H)，4.97(d，J＝11.3Hz，3H)，4.49(d，J＝8.4Hz，3H)，4.06-3.07(m，9H)，3.95-3.83(m，3H)，3.77-3.64(m，3H)，3.45-3.35(m，3H)，3.12-2.87(m，8H)，2.30-2.15(m，3H)，2.11-1.98(m，22H)，1.95-1.84(m，11H)，1.81-1.61(m，14H)，1.54-1.36(m，14H).MS m/z：C 85H 119N 7O 30，[M+H] +，理论：1718.81，实测：1718.03。
(1-1-6)L-8的合成：
Figure PCTCN2018118107-appb-000061
将步骤(1-1-5)中得到的L-5-Tr(5.94g，3.456mmol)溶于69ml二氯甲烷，再加入二氯乙酸(13.367g，103.67mmol)，室温下反应2h，加入100ml二氯甲烷稀释反应液，再加饱和碳酸氢钠溶液洗涤调节pH＝7-8之间，水相以二氯甲烷萃取6次，每次30ml，合并有机相，用无水硫酸钠干燥，过滤后减压蒸干溶剂得粗品。纯化使用200-300目正相硅胶，以10wt％三乙胺中和硅胶酸性，1wt‰三乙胺平衡柱子，以二氯甲烷∶甲醇＝100∶30-100∶40梯度洗脱，收集产物洗脱液，减压蒸干溶剂得到纯品L-84.26g。 1H NMR(400MHz，DMSO)δ7.84(d，J＝9.0Hz，3H)，7.27-7.23(m，1H)，7.13-7.18(m，1H)，5.22(d，J＝3.1Hz，3H)，4.97(dd，J＝11.3，3.1Hz，3H)，4.48(d，J＝8.4Hz，3H)，4.09-3.98(m，9H)，3.88(dd，J＝19.3，9.3Hz，3H)，3.75-3.66(m，3H)，3.44-3.38(m，3H)，3.17-3.30(m，4H)， 3.10-2.97(m，4H)，2.35-2.20(m，6H)，2.15-2.08(m，9H)，2.07-1.98(m，13H)，1.94-1.87(m，9H)，1.81-1.74(m，9H)，1.65-1.42(m，18H).MS m/z：C 85H 119N 7O 30，[M+H] +，理论：1477.59，实测：1477.23。
(1-1-7a)A-1的合成
Figure PCTCN2018118107-appb-000062
将DMTrCl(4，4′-双甲氧基三苯甲基氯，38.12g，112.5mmol)溶于450ml无水吡啶中，加入DL-甘油酸钙水合物(12.88g，45.0mmol)，在45℃反应22h，将反应液过滤，滤饼用200ml DCM淋洗，滤液减压浓缩至干，剩余物用500ml二氯甲烷重新溶解，0.5M三乙胺磷酸盐(pH＝7-8)洗涤2次，每次200ml，水相以二氯甲烷萃取2次，每次200ml，合并有机相，用无水硫酸钠干燥，过滤，减压蒸干溶剂，200-300目正相硅胶柱纯化，以石油醚∶乙酸乙酯∶二氯甲烷∶甲醇＝1∶1∶1∶0.35-1∶1∶1∶0.55梯度洗脱，收集产物洗脱液，减压蒸干溶剂，500ml二氯甲烷重新溶解，以200ml 0.5M三乙胺磷酸盐洗涤1次，水相用二氯甲烷萃取2次，每次200ml，合并有机相，无水硫酸钠干燥，过滤，减压蒸干溶剂，真空油泵减压下过夜，得到白色固体产品A-120.7g。 1H NMR(400MHz，DMSO-d 6)δ7.46(ddd，J＝6.5，2.3，1.1Hz，1H)，7.40-7.28(m，7H)，6.89-6.81(m，4H)，4.84(d，J＝5.0Hz，1H)，4.36-4.24(m，1H)，4.29(s，6H)，3.92(dd，J＝12.4，7.0Hz，1H)，3.67(dd，J＝12.3，7.0Hz，1H)，2.52(q，J＝6.3Hz，6H)，1.03(t，J＝6.3Hz，9H).MS m/z：C 24H 23O 6，[M-H] -，理论：407.15，实测：406.92。
(1-1-7b)L-7的合成：
Figure PCTCN2018118107-appb-000063
将步骤(1-1-6)中获得的L-8(2.262g，1.532mmol)和步骤(1-1-7a)中 获得的A-1(2.342g，4.596mmol)混合，溶于16ml二氯甲烷，加入3-二乙氧基磷酰基-1，2，3-苯唑4(3H)-酮(DEPBT)(1.375g，4.596mmol)，再加入二异丙基乙胺(1.188g，9.191mmol)，25℃下搅拌反应2h，用10ml饱和碳酸氢钠洗涤有机相，水相以二氯甲烷萃取3次，每次10ml，以10ml饱和食盐水洗涤有机相，水相以二氯甲烷萃取2次，每次10ml，合并有机相并以无水硫酸钠干燥，过滤后减压蒸干溶剂，真空油泵发泡干燥过夜，得到粗品4.900g。柱纯化使用120g200-300目正相硅胶，以20ml三乙胺中和硅胶酸性，以含1wt％三乙胺的石油醚平衡柱子，以石油醚∶乙酸乙酯∶二氯甲烷∶N，N-二甲基甲酰胺＝1∶1∶1∶0.5-1∶1∶1∶0.6梯度洗脱，收集产物洗脱液，减压蒸干溶剂得到纯品L-72.336g。 1H NMR(400MHz，DMSO)δ7.90-7.78(m，4H)，7.75-7.64(m，1H)，7.38-7.18(m，9H)，6.91-6.83(m，4H)，5.25-5.10(m，4H)，4.97(dd，J＝11.2，3.2Hz，3H)，4.48-4.30(m，4H)，4.02(s，9H)，3.93-3.84(m，3H)，3.76-3.66(m，9H)，3.45-3.35(m，3H)，3.24-2.98(m，10H)，2.30-2.20(m，2H)，2.11-1.88(m，31H)，1.80-1.40(m，28H).MS m/z：C 90H 128N 7O 35，[M-DMTr] +，理论：1564.65，实测：1564.88。
(1-1-8)L-9缀合分子的合成：
Figure PCTCN2018118107-appb-000064
将步骤(1-1-7b)中获得的L-7(2.300g，1.26mmol)、丁二酸酐(0.378g，3.78mmol)和4-二甲氨基吡啶(DMAP，0.462g，3.78mmol)混合溶于13ml二氯甲烷，再加入二异丙基乙胺(DIEA，0.814g，6.30mmol)，25℃下搅拌24h，5ml 0.5M三乙胺磷酸盐洗涤反应液，水相以二氯甲烷萃取3次，每次5ml，合并有机相减压蒸干得到2.774g粗品。柱纯化使用60g 200-300目正相硅胶，以1wt％三乙胺中和硅胶酸性，以二氯甲烷平衡柱子，以含1wt‰三乙胺的二氯甲烷∶甲醇＝100∶18-100∶20梯度洗脱，收集产物洗脱液，减压蒸干溶剂得到纯品L-9缀合分子1.874g。 1H NMR(400MHz，DMSO)δ8.58(d，J＝4.2Hz，1H)，7.94- 7.82(m，3H)，7.41-7.29(m，5H)，7.22(d，J＝8.1Hz，5H)，6.89(d，J＝8.3Hz，4H)，5.49-5.37(m，1H)，5.21(d，J＝3.0Hz，3H)，4.97(d，J＝11.1Hz，3H)，4.49(d，J＝8.2Hz，3H)，4.02(s，9H)，3.88(dd，J＝19.4，9.4Hz，3H)，3.77-3.65(m，9H)，3.50-3.39(m，6H)，3.11-2.90(m，5H)，2.61-2.54(m，4H)，2.47-2.41(m，2H)，2.26-2.17(m，2H)，2.15-1.95(m，22H)，1.92-1.84(m，9H)，1.80-1.70(m，10H)，1.65-1.35(m，17H)，1.31-1.19(m，4H)，0.96(t，J＝7.1Hz，9H).MS m/z：C 94H 132N 7O 38，[M-DMTr] +，理论：1664.72，实测：1665.03。
(1-1-9)L-10化合物的合成：
Figure PCTCN2018118107-appb-000065
此步骤中，通过将L-9缀合分子连接至固相载体，制备了L-10化合物。
将步骤(1-1-8)中获得的L-9缀合分子(0.233g，0.1126mmol)、O-苯并三氮唑-四甲基脲六氟磷酸酯(HBTU，0.064g，0.1689mmol)和二异丙基乙胺(DIEA，0.029g，0.2252mmol)混合，溶于19ml乙腈，室温搅拌5分钟，向反应液中加入氨甲基树脂(0.901g，100-200目，氨基载量400μmol/g，购自南开和成公司)，25℃下进行摇床反应，转速220转/分钟，反应15h后过滤，滤饼以DCM淋洗2次，每次30ml，乙腈淋洗3次，每次30ml，30ml乙醚淋洗1次，真空油泵干燥2h，随后再按照表1中示出的投料配比加入原料(CapA、CapB、4-二甲氨基吡啶(DMAP)和乙腈)进行盖帽反应。25℃下置于摇床上，转速200转/分钟，反应5h，反应液过滤，滤饼用乙腈淋洗3次，每次30ml，抽滤至干，真空油泵减压下干燥过夜，得到L-10化合物(即，连接固相载体的L-9缀合分子)1.100g，载量90.8μmol/g。
表1 盖帽反应投料配比
Figure PCTCN2018118107-appb-000066
Figure PCTCN2018118107-appb-000067
其中，CapA和CapB为盖帽试剂溶液，CapA为20体积％N-甲基咪唑的吡啶/乙腈混合溶液，吡啶与乙腈的体积比为3∶5；CapB为20体积％乙酸酐的乙腈溶液。
(1-2)合成缀合物4、18和19的正义链
缀合物4、18和19的正义链序列相同，故其制备方法也相同。
通过固相亚磷酰胺法，利用上述步骤制备的L-10化合物起始循环，按照正义链核苷酸排布顺序自3′-5′方向逐一连接核苷单体。每连接一个核苷单体都包括脱保护、偶联、盖帽、氧化或硫化四步反应。其中，两个核苷酸之间采用磷酸酯连接时，连接后一个核苷单体时，包括脱保护、偶联、盖帽、氧化四步反应。两个核苷酸之间采用硫代磷酸酯连接时，连接后一个核苷单体时，包括保护、偶联、盖帽、硫化四步反应。合成条件给定如下：
核苷单体以0.1M浓度的乙腈溶液提供，每一步的脱保护反应的条件相同，即温度为25℃，反应时间为70秒，脱保护试剂为二氯乙酸的二氯甲烷溶液(3％v/v)，二氯乙酸与固相载体上4，4′-二甲氧基三苯甲基保护基的摩尔比为5∶1。
每一步偶联反应条件均相同，包括温度为25℃，固相载体上连接的核酸序列与核苷单体的摩尔比为1∶10，固相载体上连接的核酸序列和偶联试剂的摩尔比为1∶65，反应时间为600秒，偶联试剂为5-乙硫基-1H-四氮唑的0.5M乙腈溶液。
每一步盖帽条件均相同，包括温度为25℃，反应时间为15秒。盖帽试剂溶液为摩尔比为1∶1的CapA和CapB的混合溶液，盖帽试剂与固相载体上连接的核酸序列的摩尔比为乙酸酐∶N-甲基咪唑∶固相载体上连接的核酸序列＝1∶1∶1。
每一步氧化反应条件相同，包括温度为25℃，反应时间为15秒，氧化试剂为浓度为0.05M的碘水。碘与偶联步骤中固相载体上连接的核酸序列的摩尔比为30∶1。反应在四氢呋喃∶水∶吡啶＝3∶1∶1的混合溶剂中进行。
每一步硫化反应的条件相同，包括温度为25℃，反应时间为300秒，硫化试剂为氢化黄原素。硫化试剂与偶联步骤中固相载体上连接的核酸序列的摩尔 比为120∶1。反应在乙腈∶吡啶＝1∶1的混合溶剂中进行。
切割和脱保护条件如下：将合成的连接有载体的核苷酸序列加入浓度为25wt％的氨水中，氨水用量为0.5ml/μmol，在55℃反应16h，除去液体，真空浓缩至干。
纯化与脱盐：利用制备型离子色谱纯化柱(Source 15Q)，通过NaCl的梯度洗脱，完成核酸的纯化。具体而言为：洗脱剂A：20mM磷酸钠(pH 8.1)，溶剂为水/乙腈＝9∶1(体积比)；洗脱剂B：1.5M氯化钠，20mM磷酸钠(pH 8.1)，溶剂为水/乙腈＝9∶1(体积比)；洗脱梯度：洗脱剂A∶洗脱剂B＝100∶0-50∶50梯度洗脱。收集产品洗脱液后合并，采用反相色谱纯化柱进行脱盐，具体条件包括采用葡聚糖凝胶柱进行脱盐，填料为葡聚糖凝胶G25，以去离子水洗脱。
检测：使用离子交换色谱(IEX-HPLC)检测纯度，使用液质联用(LC-MS)分析分子量。
对于缀合物4的正义链分子量，理论值为7423.22，实测值为7422.6。实测值与理论值相符，表明所合成的是3′末端缀合了L-9缀合分子的正义链S。
(1-3)合成缀合物4、18和19的反义链
(1-3A)缀合物4反义链的制备
通过固相亚磷酰胺法，利用通用固相载体(UnyLinker TM loaded 
Figure PCTCN2018118107-appb-000068
HL Solid Supports，Kinovate Life Sciences公司)起始循环，合成缀合物4的反义链AS。固相合成方法中的脱保护、偶联、盖帽、氧化或硫化反应条件，切割和脱保护，纯化与脱盐条件与合成正义链相同。
检测：纯度采用离子交换色谱(IEX-HPLC)进行检测；分子量采用液质联用(LC-MS)进行分析，理论值为7207.78，实测值为7207.2。实测值与理论值相符，表明所合成的是具有目标序列的反义链AS。
其中，乙烯基磷酸酯修饰的2′-甲氧基修饰尿嘧啶核苷单体(VP-Um)按照以下方法合成：
Figure PCTCN2018118107-appb-000069
(1-3-1)VP-U-2的合成
按照以下方法，合成了VP-U-2分子：
Figure PCTCN2018118107-appb-000070
将2′-甲氧基修饰的尿嘧啶核苷(2′-OMe-U，51.30g，91.6mmol)，叔丁基二苯基氯硅烷(TBDPSCl，50.35g，183.2mmol)，咪唑(12.47g，183.2mmol)混合溶于450ml N，N-二甲基甲酰胺(DMF)，室温下搅拌反应20h。蒸除DMF，用600ml二氯甲烷溶解后加300ml饱和碳酸氢钠洗涤，水相再用二氯甲烷(DCM)萃取3次，每次300ml，合并有机相，用5％草酸洗涤至水相pH＜5，蒸发溶剂至干后获得VP-U-1粗品直接用于随后VP-U-2的合成。
将VP-U-1粗品用100ml二氯甲烷溶解后，外加冰浴搅拌10分钟，再加入预先在4℃冰箱冷藏好的450ml 2％对甲苯磺酸溶液(溶剂为体积比3∶7的甲醇-二氯甲烷混合溶剂)，反应10分钟。再加入200ml饱和碳酸氢钠淬灭反应，有机相加入饱和碳酸氢钠水溶液洗涤至pH＝8。合并水相，用二氯甲烷萃取2次，每次200ml，合并有机相，再用200ml饱和食盐水洗涤一次，蒸发溶剂至干。200-300目正相硅胶柱纯化，石油醚装柱，以石油醚∶乙酸乙酯∶二氯甲烷∶甲醇＝1∶1∶1∶0.05-1∶1∶1∶0.25梯度洗脱，收集产物洗脱液，减压蒸干溶剂，真空油泵发泡干燥得到纯品VP-U-2共40.00g。 1H NMR(400MHz，DMSO-d 6)δ7.96(d，J＝7.8Hz，1H)，7.64(dtd，J＝5.1，4.0，2.2Hz，4H)，7.41-7.30(m，6H)，6.79(d，J＝4.7Hz，1H)，5.73(d，J＝7.6Hz，1H)，4.94(t，J＝7.0Hz，1H)，4.12(td，J＝4.6，3.9Hz， 1H)，4.05(dd，J＝4.8，4.0Hz，1H)，3.96(t，J＝4.7Hz，1H)，3.68(ddd，J＝11.8，7.0，4.6Hz，1H)，3.57-3.46(m，1H)，3.39(s，3H)，1.05(s，8H).MS m/z：C 26H 33N 2O 6Si，[M+H] +，理论：497.21，实测：497.45。
(1-3-2)VP-U-4的合成：
Figure PCTCN2018118107-appb-000071
将VP-U-2(19.84g，40.0mmol)，二环己基碳二亚胺(DCC，16.48g，80.0mmol)，吡啶(4.20g，53.2mmol)，三氟乙酸(6.61g，53.2mmol)混合溶于200ml二甲基亚砜(DMSO)，室温下搅拌反应20h。另取亚甲基二磷酸四乙酯(21.44g，74.4mmol)溶于120ml THF，冰浴降温，在冰浴温度下加入t-BuOK(11.36g，101.2mmol)，先在冰浴温度下反应10min，再升至室温反应0.5h，然后加入至前述反应液中，约1h加完，冰浴温度下反应1h，再升至室温反应18h。加水淬灭反应，水相以二氯甲烷提取3次，每次200ml。合并有机相，用200ml饱和食盐水水洗一次后蒸发溶剂至干。用200-300目正相硅胶柱纯化，石油醚装柱，以石油醚∶乙酸乙酯＝1∶1-1∶4梯度洗脱，收集产物洗脱液，减压蒸干溶剂，真空油泵发泡干燥得到纯品VP-U-4共14.00g。 1H NMR(400MHz，DMSO-d 6)δ7.96(d，J＝7.8Hz，1H)，7.64(dtd，J＝5.1，4.0，2.2Hz，4H)，7.41-7.30(m，6H)，6.82-6.71(m，2H)，5.90(ddd，J＝25.9，15.0，1.0Hz，1H)，5.73(d，J＝7.6Hz，1H)，4.36-4.21(m，3H)，4.18(t，J＝4.9Hz，1H)，4.05(ddq，J＝9.7，8.5，6.9Hz，2H)，3.87(t，J＝4.8Hz，1H)，3.39(s，3H)，1.32(td，J＝6.9，0.7Hz，6H)，1.05(s，8H).MS m/z：C 31H 42N 2O 8PSi，[M+H] +，理论：629.24，实测：629.51。
(1-3-3)VP-U-5的合成：
Figure PCTCN2018118107-appb-000072
将VP-U-4(14.00g，22.29mmol)溶于100ml四氢呋喃，加入三乙胺三氢氟酸(17.96g，111.45mmol)，室温搅拌20h反应完全。直接蒸发溶剂至干，再用二氯甲烷溶解随后蒸干2次，每次使用50ml二氯甲烷，得到粗品。用200-300 目正相硅胶柱纯化，石油醚装柱，以石油醚∶乙酸乙酯∶二氯甲烷∶甲醇＝1∶1∶1∶0.05-1∶1∶1∶0.25梯度洗脱，收集产物洗脱液，减压蒸干溶剂，真空油泵发泡干燥得到纯品VP-U-5共6.70g。 1H NMR(400MHz，DMSO-d 6)δ7.96(d，J＝7.8Hz，1H)，6.77(dd，J＝15.0，6.2Hz，1H)，5.99-5.82(m，2H)，5.73(d，J＝7.6Hz，1H)，5.27(d，J＝5.1Hz，1H)，5.10(dd，J＝5.3，4.7Hz，1H)，4.29(ddq，J＝9.8，8.6，7.0Hz，2H)，4.17(ddd，J＝6.2，5.2，1.0Hz，1H)，4.12-3.98(m，3H)，3.39(s，2H)，1.32(td，J＝6.9，0.6Hz，6H).MS m/z：C 15H 24N 2O 8P，[M+H] +，理论：391.13，实测：391.38。
(1-3-4)VP-U-6的合成：
Figure PCTCN2018118107-appb-000073
在氩气保护条件下向10ml无水二氯甲烷中加入VP-U-5(391mg，1.0mmol)、三氟乙酸吡啶盐(0.232g，1.2mmol)、N-甲基咪唑(0.099g，1.2mmol)，双(二异丙基氨基)(2-氰基乙氧基)膦(0.452g，1.5mmol)，室温搅拌反应5小时。蒸除溶剂至干，柱层析纯化(200-300目正相硅胶，二氯甲烷∶乙腈(含0.5wt％三乙胺)＝3∶1-1∶3梯度洗脱)，收集产物洗脱液，浓缩除去溶剂，得到目标产物VP-U-6共508mg。 31P NMR(161MHz，DMSO-d 6)δ150.34，150.29，17.07，15.50.MS m/z：C 24H 41N 4O 9P 2，[M+H] +，理论：591.23，实测：591.55。表明VP-U-6是目标产物VP-Um，作为核苷单体参与RNA链合成。
(1-3B)缀合物18的反义链的制备
缀合物18的反义链与缀合物4的反义链的区别仅在于5′-末端第一个核苷酸修饰不同。按照固相亚磷酰胺法制备反义链时，最后连接的核苷单体为2′-甲氧基修饰尿嘧啶核苷单体(Um)，再经脱保护、偶联、盖帽、氧化四步反应将CPR-I单体(苏州吉玛，货号Cat#13-2601-XX)连接至反义链5′末端，形成5′-磷酸酯修饰。
Figure PCTCN2018118107-appb-000074
合成中，使用的通用固相载体，脱保护、偶联、盖帽、氧化或硫化反应条件，切割和脱保护，纯化与脱盐条件与合成正义链相同。
(1-3C)缀合物19的反义链的制备
采用与缀合物18反义链相同的合成工艺，区别在于连接CPR-I单体时，以硫化反应条件代替上述氧化反应条件，预期能够制得具有5′-硫代磷酸酯修饰的缀合物19反义链。
(1-4)合成缀合物4、18、19
对于缀合物4，将S链与AS链分别溶于注射用水中，得到40mg/mL的溶液，以等摩尔比混合，50℃加热15min，室温冷却后，使它们通过氢键形成双链结构。使用超纯水(Milli-Q超纯水仪自制，电阻率18.2MΩ＊cm(25℃))将缀合物稀释至浓度为0.2mg/mL后，利用液质联用仪(LC-MS，Liquid Chromatography-Mass Spectrometry，购于Waters公司，型号：LCT Premier)进行分子量检测。实测值与理论值一致，说明所合成的缀合物4是目标设计的带有L-9缀合分子的双链核酸序列。
对于缀合物18和19，按照上述方法进行退火，预期能够合成缀合物18和19。上述缀合物4、18和19的结构均如式(403)所示。
制备例2：缀合物1-3、5-9与对比缀合物1的制备
采用与制备例1相同的方法，预期能够制得题述缀合物，不同的是：1)所述siRNA分别为表2中所示的对应于缀合物1-3、5-9及对比缀合物1的序列；2)当目标序列中含有未修饰的核苷酸时，切割与脱保护条件中，在氨水处理后，相对于单链核酸的量，用0.4ml/μmol N-甲基吡咯烷酮溶解产品，随后加入0.3ml/μmol三乙胺和0.6ml/μmol三乙胺三氢氟酸盐，以脱除核糖上的2′-TBDMS保护。
题述缀合物中所缀合的siRNA的序列参见表2。
表2 siRNA缀合物
Figure PCTCN2018118107-appb-000075
Figure PCTCN2018118107-appb-000076
Figure PCTCN2018118107-appb-000077
制备例3：P10-siHB1M1SVP(缀合物10)的制备
(3-1)P-10化合物的合成
按照以下方法，合成了P-10化合物：
Figure PCTCN2018118107-appb-000078
(3-1-1)GAL5-C4-1的合成
向40ml N，N-二甲基甲酰胺中加入按照上述(1-1-1)中描述的方法得到的GAL-5(13.43g，30.0mmol)、4-氨基酸叔丁酯盐酸盐(5.87g，30.0mmol)、O-苯并三氮唑-四甲基脲六氟磷酸酯(13.65g，36.0mmol)和二异丙基乙胺(11.63g，90.0mmol)，溶解均一后室温搅拌反应5小时。向反应液中加入300ml饱和碳酸 氢钠水溶液，用乙酸乙酯萃取3次，每次200ml，合并有机相，用200ml饱和食盐水洗涤一次，分出有机相，再用无水硫酸钠干燥，减压蒸除溶剂至干得到30.3g油状物粗品GAL5-C4-1，直接进行下一步反应。
(3-1-2)GAL5-C4-2的合成
将步骤(3-1-1)中获得的GAL5-C4-1粗品(30.3g，30mmol)溶于180ml甲酸中，室温搅拌反应16小时。蒸发溶剂至干，柱层析纯化(200-300目正相硅胶，二氯甲烷∶甲醇＝100∶18-100∶20梯度洗脱)，收集反应洗脱液，浓缩除去溶剂，得到目标产物GAL5-C4-2共14.84g。
(3-1-3)P-6的合成：
将按照步骤(1-1-4)中描述的方法得到的M-18-Tr(2.02g，4.69mmol)与将步骤(3-1-2)中获得的GAL5-C4-2(8.24g，15.48mmol，由两批产物合并获得)混合溶于47ml乙腈，再加入N-甲基吗啉(3.13g，30.96mmol)，最后加入4-(4，6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐(DMTMM，4.28g，15.48mmol)，室温搅拌反应2h。以20ml二氯甲烷稀释反应液，10ml饱和碳酸氢钠溶液洗涤有机相，10ml饱和食盐水洗涤有机相，合并有机相并以无水硫酸钠干燥，过滤后减压蒸干溶剂得粗品，200-300目正相硅胶柱纯化，石油醚装柱，以1wt％三乙胺中和硅胶酸性，以二氯甲烷∶甲醇＝100∶5-100∶7梯度洗脱，收集产物洗脱液，减压蒸干得到纯品P-6共8.27g。
(3-1-4)P-7的合成：
将按照上述(3-1-3)中得到的P-6(6.82g，3.456mmol)溶于69ml二氯甲烷，再加入二氯乙酸(13.367g，103.67mmol)，室温下反应2h。加入100ml二氯甲烷稀释反应液，再加入饱和碳酸氢钠溶液洗涤调节pH＝7-8之间，水相以二氯甲烷萃取6次，每次30ml，合并有机相，无水硫酸钠干燥，过滤后减压蒸干溶剂得粗品。用200-300目正相硅胶纯化，以10wt％三乙胺中和硅胶酸性，以1wt‰三乙胺平衡柱子，二氯甲烷∶甲醇＝100∶30-100∶40梯度洗脱，收集产物洗脱液，减压蒸干溶剂得到P-7共4.82g。MS m/z：C 78H 127N 10O 33，[M+H] +，理论：1732.91，实测：1735.73。
(3-1-5)P-8的合成：
Figure PCTCN2018118107-appb-000079
将P-7(2.653g，1.532mmol)和A-1(2.342g，4.596mmol)混合溶于16ml 二氯甲烷，加入3-二乙氧基磷酰基-1，2，3-苯唑4(3H)-酮(DEPBT)(1.375g，4.596mmol)，再加入二异丙基乙胺(1.188g，9.191mmol)，25℃下搅拌反应2h。用10ml饱和碳酸氢钠洗涤有机相，水相以二氯甲烷萃取3次，每次10ml，10ml饱和食盐水洗涤有机相，水相以二氯甲烷萃取2次，每次10ml，合并有机相，用无水硫酸钠干燥，过滤后减压蒸干溶剂，真空油泵发泡干燥过夜得到粗品。柱纯化使用120g 200-300目正相硅胶，以20ml三乙胺中和硅胶酸性，以含1wt％三乙胺的石油醚平衡柱子，以石油醚∶乙酸乙酯∶二氯甲烷∶N，N-二甲基甲酰胺＝1∶1∶1∶0.5-1∶1∶1∶0.6梯度洗脱，收集产物洗脱液，减压蒸干溶剂得到纯品P-8共2.793g。
(3-1-6)P-9的合成：
将P-8(490mg，0.231mmol)、丁二酸酐(69mg，0.693mmol)和4-二甲氨基吡啶(DMAP，68mg，0.554mmol)混合溶于2.3ml二氯甲烷，再加入二异丙基乙胺(DIPEA，149mg，1.155mmol)，25℃下搅拌反应21h。50ml二氯甲烷稀释反应液，再加入100ml 0.5M三乙胺磷酸盐洗涤反应液，水相以二氯甲烷萃取3次，每次10ml，合并有机相，减压蒸干得到粗品。柱纯化使用80g 200-300目正相硅胶，以1wt％三乙胺中和硅胶酸性，以二氯甲烷平衡柱子，以含1wt‰三乙胺的二氯甲烷∶甲醇＝100∶18-100∶20梯度洗脱，收集产物洗脱液，减压蒸干溶剂得到纯品P-9缀合分子共200mg。MS m/z：C 106H 153N 10O 41，[M-DMTr] +，理论：1921.05，实测：1920.97。
(3-1-7)P-10的合成
通过与制备例1中步骤(1-1-9)相同的方法，制备P-10。不同的是以P-9缀合分子代替L-9缀合分子，得到连接固相载体的P-9缀合分子。
(3-2)合成P10-siHB1M1SVP缀合物
通过与制备例1中步骤(1-2)、(1-3A)、(1-4)相同的方法，制备缀合物10，不同的是以P-10化合物代替L-10化合物起始正义链合成。预期可以得到P10-siHB1M1SVP缀合物，其结构如式(404)所示。
制备例4：R5-siHB1M1SVP缀合物(缀合物11)的制备
(4-1)R-5化合物的合成
按照以下方法，合成了R-5化合物：
Figure PCTCN2018118107-appb-000080
(4-1-1)GAL-C7-1的合成
将按照步骤(1-1-1b)中描述的方法得到的GAL-3(26.4g，80.2mmol)溶 于134ml无水1，2-二氯乙烷中，加入
Figure PCTCN2018118107-appb-000081
分子筛粉末60g，再加入7-辛烯-1-醇(11.3g，88.2mmol)，室温下搅拌反应10分钟，冰浴和氮气保护下加入三氟甲基磺酸三甲基硅酯(8.9g，40.1mmol)，室温搅拌反应24小时。过滤除去
Figure PCTCN2018118107-appb-000082
分子筛粉末，滤液中加入500ml饱和碳酸氢钠水溶液洗涤，分出有机相，水相用100ml二氯甲烷萃取一次，合并有机相并用250ml饱和食盐水洗涤一次，分出有机相，用无水硫酸钠干燥，减压蒸除溶剂至干得到黄色糖稀状产品GAL-C7-133.3g，不进行纯化直接进行下一步氧化反应。
(4-1-2)GAL-C7-2的合成
将按照步骤(4-1-1)中得到的GAL-C7-1(33.3g，72.8mmol)溶于160ml二氯甲烷和160ml乙腈的混合溶剂中，分别加入216ml水和高碘酸钠固体(62.3g，291.2mmol)，冰水浴下搅拌10分钟，加入催化剂三氯化钌(498mg，2.4mmol)自然升至室温搅拌反应23小时。反应液加入200ml水稀释搅拌，加饱和碳酸氢钠调节pH值为7.5，分掉有机相，水相再用二氯甲烷萃取三次，弃去有机相，水相用柠檬酸固体调节pH约为3，用二氯甲烷萃取三次，每次200ml，合并有机相，无水硫酸钠干燥，减压蒸除溶剂后柱层析(200-300目正相硅胶，二氯甲烷∶甲醇＝100∶18-100∶20梯度洗脱)纯化得到白色泡沫状固体产品GAL-C7-222.4g。MS m/z：C 21H 32NO 11，[M+H] +，理论：476.50，实测：475.94。
(4-1-3)R-1的合成：
将按照步骤(1-1-4)中描述的方法得到的M-18-Tr(2.02g，4.69mmol)与GAL-C7-2(7.36g，15.48mmol)混合溶于47ml乙腈，再加入N-甲基吗啉(3.13g，30.96mmol)，最后加入4-(4，6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐(DMTMM，4.28g，15.48mmol)，室温搅拌反应2h。以200ml二氯甲烷稀释反应液，100ml饱和碳酸氢钠溶液洗涤有机相，100ml饱和食盐水洗涤有机相，合并有机相并以无水硫酸钠干燥，过滤后减压蒸干溶剂得粗品，200-300目正相硅胶柱纯化，石油醚装柱，以1wt％三乙胺中和硅胶酸性，二氯甲烷∶甲醇＝100∶5-100∶7梯度洗脱，收集产物洗脱液，减压蒸干得到纯品R-17.82g。
(4-1-4)R-2的合成：
将R-1(6.23g，3.456mmol)溶于69ml二氯甲烷，再加入二氯乙酸(13.367g，103.67mmol)，室温下反应2h。加入100ml二氯甲烷稀释反应液，再加饱和碳酸氢钠溶液洗涤调节pH＝7-8之间，水相以二氯甲烷萃取6次，每次30ml，合并有机相并以无水硫酸钠干燥，过滤后减压蒸干溶剂得粗品。200-300目正相硅胶，以10wt％三乙胺中和硅胶酸性，以1wt‰三乙胺平衡柱子，二氯甲烷∶甲醇 ＝100∶30-100∶40梯度洗脱，减压蒸干溶剂得到纯品R-24.49g。
(4-1-5)R-3的合成：
将R-2(2.391g，1.532mmol)和A-1(2.342g，4.596mmol)混合溶于16ml二氯甲烷，加入3-二乙氧基磷酰基-1，2，3-苯唑4(3H)-酮(DEPBT)(1.375g，4.596mmol)，再加入二异丙基乙胺(1.188g，9.191mmol)，25℃下搅拌反应2h。用10ml饱和碳酸氢钠洗涤有机相，水相以二氯甲烷萃取3次，每次10ml，以10ml饱和食盐水洗涤有机相，水相以二氯甲烷萃取2次，每次10ml，合并有机相并以无水硫酸钠干燥，过滤后减压蒸干溶剂，真空油泵发泡干燥过夜得到粗品。柱纯化使用120g 200-300目正相硅胶，以20ml三乙胺中和硅胶酸性，以含1wt％三乙胺的石油醚平衡柱子，石油醚∶乙酸乙酯∶二氯甲烷∶N，N-二甲基甲酰胺＝1∶1∶1∶0.5-1∶1∶1∶0.6梯度洗脱，减压蒸干溶剂得到纯品R-32.642g。
(4-1-6)R-4的合成：
将R-3(795mg，0.4074mmol)、丁二酸酐(82mg，0.8148mmol)和4-二甲氨基吡啶(DMAP，100mg，0.8148mmol)混合溶于4ml二氯甲烷，再加入二异丙基乙胺(DIPEA，100mg，0.8148mmol)，25℃下搅拌反应18h。5ml 0.5M三乙胺磷酸盐洗涤反应液，水相以二氯甲烷萃取3次，每次5ml，合并有机相减压蒸干得到粗品。柱纯化使用30g 200-300目正相硅胶，以1wt％三乙胺中和硅胶酸性，以二氯甲烷平衡柱子，含1wt‰三乙胺的二氯甲烷∶甲醇＝100∶18-100∶20梯度洗脱，收集产物洗脱液，减压蒸干溶剂得到纯品R-4缀合分子505mg。
(4-1-7)R-5的合成：
通过与制备例1中步骤(1-1-9)相同的方法，制备R-5。不同的是以R-4缀合分子代替L-9缀合分子，得到连接固相载体的R-4缀合分子。
(4-2)合成R5-siHB1M1SVP缀合物
通过与制备例1中步骤(1-2)、(1-3A)、(1-4)相同的方法，制备缀合物11，不同的是以R-5化合物代替L-10化合物起始正义链合成。预期可以得到R5-siHB1M1SVP缀合物，其结构如式(407)所示。
制备例5：LA5-siHB1M1SVP缀合物(缀合物12)的制备
按照以下工艺路线，预期能够合成LA-5化合物：
Figure PCTCN2018118107-appb-000083
通过与制备例1中步骤(1-2)、(1-3A)、(1-4)相同的方法，制备缀合物12，不同的是以LA-5化合物代替L-10化合物起始正义链合成。预期可以得到LA5-siHB1M1SVP缀合物，其结构如式(412)所示。
制备例6：LB5-siHB1M1SVP缀合物(缀合物13)的制备
(6-1)LB-5化合物的合成
按照以下方法，合成了LB-5化合物：
Figure PCTCN2018118107-appb-000084
(6-1-1)LB-1的合成：
将按照步骤(1-1-6)中描述的方法得到的L-8(5.0g，3.386mmol)、己二酸酐(870mg，6.772mmol)和4-二甲氨基吡啶(DMAP，827mg，6.772mmol)混合溶于130ml二氯甲烷，再加入二异丙基乙胺(DIPEA，2.2g，16.931mmol)，25℃下搅拌反应4h。加入70ml二氯甲烷稀释反应液，以0.5M三乙胺磷酸盐洗涤反应液，水相以二氯甲烷萃取4次，每次10ml，合并有机相减压蒸干得到粗品。柱纯化使用120g 200-300目正相硅胶，以1wt％三乙胺中和硅胶酸性，以二氯甲烷平衡柱子，石油醚∶乙酸乙酯∶二氯甲烷∶甲醇＝1∶1∶1∶0.2-1∶1∶1∶1梯度洗脱，减压蒸干溶剂得到纯品LB-1 4.267g。
(6-1-2)LB-2的合成：
将按照步骤(6-1-1)中描述的方法得到的LB-1(4.697g，2.753mmol，由两批次产物合并而得)、3-氨基-1，2-丙二醇(313mg，3.442mmol)、4-(4，6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐(DMTMM，953mg，3.442mmol)和N-甲基 吗啉(700mg，6.884mmol)先后加入30ml乙腈和3ml甲醇的混合液中，室温搅拌反应过夜。蒸发溶剂至干，柱层析(200-300目正相硅胶，二氯甲烷∶甲醇＝1∶0.07-1∶0.5梯度洗脱)纯化，收集产物洗脱液，浓缩除去溶剂，得到目标产物LB-2 3.27g。
(6-1-3)LB-3的合成：
将LB-2(2.27g，1.353mmol)用14ml无水吡啶溶解。再加入4，4′-双甲氧基三苯甲基氯(688mg，2.03mmol)室温下搅拌反应过夜。加150ml甲醇淬灭，蒸发溶剂至干。柱层析(200-300目正相硅胶，二氯甲烷∶甲醇＝1∶0.05-1∶0.2梯度洗脱)纯化，收集产物洗脱液，浓缩除去溶剂，得到目标产物LB-3 1.647g。
(6-1-4)LB-4的合成：
将LB-3(822mg，0.415mmol)、丁二酸酐(83g，0.83mmol)和4-二甲氨基吡啶(DMAP，102mg，0.83mmol)混合溶于4ml二氯甲烷，再加入DIPEA(270mg，2.075mmol)，25℃下搅拌反应过夜。0.5M三乙胺磷酸盐洗涤反应液3次，水相以二氯甲烷萃取3次，每次2ml，合并有机相减压蒸干得到粗品。柱纯化使用200-300目正相硅胶，以5wt％三乙胺中和硅胶酸性，以石油醚平衡柱子，用含1wt‰三乙胺的二氯甲烷∶甲醇＝100∶5-100∶20梯度洗脱，减压蒸干溶剂得到纯品LB-4缀合分子787mg。
(6-1-5)LB-5的合成：
通过与制备例1中步骤(1-1-9)相同的方法，制备LB-5。不同的是以LB-4缀合分子代替L-9缀合分子，得到连接固相载体的LB-4缀合分子。
(6-2)合成LB5-siHB1M1SVP缀合物
通过与制备例1中步骤(1-2)、(1-3A)、(1-4)相同的方法，制备缀合物13，不同的是以LB-5化合物代替L-10化合物起始正义链合成。预期可以得到LB5-siHB1M1SVP缀合物，其结构如式(413)所示。
制备例7：V8-siHB1M1SVP缀合物(缀合物14)的合成
按照以下工艺路线，预期能够合成V-8化合物：
Figure PCTCN2018118107-appb-000085
通过与制备例1中步骤(1-2)、(1-3A)、(1-4)相同的方法，制备缀合物14，不同的是以V-8化合物代替L-10化合物起始正义链合成。预期可以得到V8-siHB1M1SVP缀合物，其结构如式(414)所示。
制备例8：W8-siHB1M1SVP缀合物(缀合物15)的制备
(8-1)W-8化合物的合成
按照以下方法，合成了W-8化合物：
Figure PCTCN2018118107-appb-000086
(8-1-1)W-1的合成：
将W-0(2.024g，10mmol)溶于25ml乙腈中，再加三乙胺(4.048g，40mmol)，冰水浴冷却至0℃左右，加入三氟乙酸乙酯(5.683g，40mmol)，室温下反应22h。减压蒸干溶剂，真空油泵发泡干燥18h，得到5.835g粗品固体W-1。
(8-1-2)W-2的合成：
将W-1粗品(5.835g，10mmol)溶于50ml二氯甲烷，向反应液中加入TrCl(3.345g，12mmol)和三乙胺(1.518g，15mmol)，室温下搅拌反应20h。用20ml饱和碳酸氢钠洗涤反应液2次，用20ml饱和食盐水洗涤1次，合并有机相并以无水硫酸钠干燥，过滤后减压蒸干有机溶剂，真空油泵发泡干燥过夜，得到粗品固体W-2 8.012g。不经处理，进行下一步脱保护反应。
(8-1-3)W-3的合成：
将W-2粗品(8.012g，10mmol)溶于100ml甲醇，再加入100ml甲胺水溶液(40wt％)，在50℃下搅拌反应23h。过滤除去不溶颗粒物，减压蒸干溶剂，加入200ml体积比为1∶1的DCM-甲醇混合溶剂，以50ml饱和碳酸氢钠洗涤有机相，水相再用二氯甲烷萃取3次，每次50ml，合并有机相并以无水硫酸钠干燥，过滤后减压蒸干溶剂，真空油泵发泡干燥过夜，200-300目正相硅胶柱纯化，石油醚装柱，以1wt％三乙胺中和硅胶酸性，以二氯甲烷∶甲醇∶氨水(25wt％)＝1∶1∶0.05-1∶1∶0.25梯度洗脱，收集产物洗脱液，减压蒸干溶剂，真空油泵发泡干燥得到纯品W-3 3.062g。
(8-1-4)W-4的合成：
将W-3(0.675g，1.517mmol)与GAL-C7-2(2.60g，5.46mmol)混合溶于47ml乙腈，再加二异丙基乙胺(1.57g，12.14mmol)，最后加入3-二乙氧基磷酰基-1，2，3-苯唑4(3H)-酮(DEPBT，1.816g，6.04mmol)，室温搅拌反应2.5h。以100ml二氯甲烷稀释反应液，80ml饱和碳酸氢钠溶液洗涤有机相，80ml饱和食盐水洗涤有机相，合并有机相并以无水硫酸钠干燥，过滤后减压蒸干溶剂得粗品，200-300目正相硅胶柱纯化，石油醚装柱，以1wt％三乙胺中和硅胶酸性，以二氯甲烷∶甲醇＝100∶5-100∶7梯度洗脱，收集产物洗脱液，减压蒸干得到纯品W-4 1.610g。
(8-1-5)W-5的合成：
将W-4(1.61g，0.886mmol)溶于125ml二氯甲烷，再加入二氯乙酸(3.5ml，42.43mmol)，室温下反应1h。加入150ml吡啶中和反应液，减压蒸干溶剂得粗品。200-300目正相硅胶，10wt％三乙胺中和硅胶酸性，1wt‰三乙胺平衡柱子，二氯甲烷∶甲醇＝100∶30-100∶40梯度洗脱，收集产物洗脱液，减压蒸干溶剂得到纯品W-5 1.26g。
(8-1-6)W-6的合成：
将W-5(1.25g，0.793mmol)和按照步骤(1-1-7a)中描述的方法得到的A-1(1.21g，2.38mmol)混合溶于12ml二氯甲烷，加入3-二乙氧基磷酰基-1，2，3- 苯唑4(3H)-酮(DEPBT，0.712g，2.38mmol)，再加入二异丙基乙胺(0.615g，4.76mmol)，25℃下搅拌反应3h。用80ml饱和碳酸氢钠洗涤有机相，水相以二氯甲烷萃取3次，每次10ml，合并有机相并以10ml饱和食盐水洗涤，合并有机相并以无水硫酸钠干燥，过滤后减压蒸干溶剂，真空油泵发泡干燥过夜得到粗品。柱纯化使用185g 200-300目正相硅胶，20ml三乙胺中和硅胶酸性，以含1wt％三乙胺的石油醚平衡柱子，以石油醚∶乙酸乙酯∶二氯甲烷∶N，N-二甲基甲酰胺＝1∶1∶1∶0.1-1∶1∶0.7梯度洗脱，收集产物洗脱液，减压蒸干溶剂得到纯品W-6 1.57g。
(8-1-7)W-7的合成：
将W-6(1.238g，0.63mmol)、丁二酸酐(0.189g，1.89mmol)和4-二甲氨基吡啶(DMAP，0.231g，1.89mmol)混合溶于7ml二氯甲烷，再加入DIEA(0.407g，3.15mmol)，25℃下搅拌反应24h。以5ml 0.5M三乙胺磷酸盐洗涤反应液，水相以二氯甲烷萃取3次，每次5ml，合并有机相减压蒸干得到粗品。柱纯化使用30g 200-300目正相硅胶，以1wt％三乙胺中和硅胶酸性，二氯甲烷平衡柱子，以含1wt‰三乙胺的二氯甲烷∶甲醇＝100∶18-100∶20梯度洗脱，收集产物洗脱液，减压蒸干溶剂得到纯品W-7缀合分子1.033g。MS m/z：C 101H 146N 7O 38，[M-DMTr] +，理论：1763.92，实测：1763.21。
(8-1-8)W-8的合成：
通过与制备例1中步骤(1-1-9)相同的方法，制备W-8。不同的是以W-7缀合分子代替L-9缀合分子，得到连接固相载体的W-7缀合分子。
(8-2)合成W8-siHB1M1SVP缀合物
通过与制备例1中步骤(1-2)、(1-3A)、(1-4)相同的方法，制备缀合物15，不同的是以W-8化合物代替L-10化合物起始正义链合成。预期可以得到W8-siHB1M1SVP缀合物，其结构如式(415)所示。
制备例9：X8-siHB1M1SVP缀合物(缀合物16)的制备
按照以下工艺路线，预期能够合成X-8化合物：
Figure PCTCN2018118107-appb-000087
通过与制备例1中步骤(1-2)、(1-3A)、(1-4)相同的方法，制备缀合物16，不同的是以X-8化合物代替L-10化合物起始正义链合成。预期可以得到X8-siHB1M1SVP缀合物，其结构如式(421)所示。
制备例10：Z5-siHB1M1SVP缀合物(缀合物17)的制备
(10-1)Z-5化合物的合成
按照以下方法，合成了Z-5化合物：
Figure PCTCN2018118107-appb-000088
(10-1-1)Z-1的合成：
将按照步骤(8-1-3)中描述的方法得到的W-3(1.50g，3.37mmol)与按照步骤(3-1-2)中描述的方法得到的GAL5-C4-2(7.18g，13.48mmol)混合溶于34ml二氯甲烷，再加入二异丙基乙胺(3.48g，26.96mmol)，最后加入3-二乙氧基磷酰基-1，2，3-苯唑4(3H)-酮(DEPBT，4.04g，13.48mmol)，室温搅拌反应4.5h。以100ml二氯甲烷稀释反应液，80ml饱和碳酸氢钠溶液洗涤有机相，80ml饱和食盐水洗涤有机相，合并有机相并以无水硫酸钠干燥，过滤后减压蒸干溶剂得粗品，200-300目正相硅胶柱纯化，石油醚装柱，以1wt％三乙胺中和硅胶酸性，以二氯甲烷∶甲醇＝30∶1-15∶1梯度洗脱，收集产物洗脱液，减压蒸干得到纯品Z-1 3.97g。MS m/z：C 98H 143N 10O 33，[M+H] +，理论：1987.98，实测：1987.90。
(10-1-2)Z-2的合成：
将Z-1(3.97g，2.00mmol)溶于250ml二氯甲烷，再加入二氯乙酸(10.941g，84.85mmol)，室温下反应1h。加入吡啶中和反应液至中性，减压蒸干溶剂得粗品。220g 200-300目正相硅胶装柱，10％吡啶中和硅胶酸性，1‰吡啶平衡柱子，二氯甲烷∶甲醇＝10∶1-2∶1梯度洗脱，收集产物洗脱液，减压蒸干溶剂得到纯品 Z-23.49g。MS m/z：C 79H 129N 10O 33，[M+H] +，理论：1746.94，实测：1746.90。
(10-1-3)Z-3的合成：
将Z-2(3.49g，2.0mmol)和按照步骤(1-1-7a)中描述的方法得到的A-1(3.06g，6.0mmol)混合溶于30ml二氯甲烷，加入3-二乙氧基磷酰基-1，2，3-苯唑4(3H)-酮(DEPBT，1.80g，6.0mmol)，再加入二异丙基乙胺(1.55g，12.0mmol)，25℃下搅拌反应3h。100ml二氯甲烷稀释反应液，用饱和碳酸氢钠洗涤有机相2次，每次30ml，水相以10二氯甲烷萃取，合并有机相并以50ml饱和食盐水洗涤，合并有机相无水硫酸钠干燥，过滤后减压蒸干溶剂，真空油泵发泡干燥过夜得到粗品。柱纯化使用200g 200-300目正相硅胶，20ml三乙胺中和硅胶酸性，以含1wt％三乙胺的石油醚平衡柱子，二氯甲烷∶甲醇＝25∶1-15∶1梯度洗脱，收集产物洗脱液，减压蒸干溶剂得到纯品Z-3 2.2g。MS m/z：C 103H 151N 10O 38，[M+H] +，理论：2136.02，实测：2136.20。
(10-1-4)Z-4的合成：
将Z-3(2.10g，0.983mmol)溶解在含有DIEA(0.635g，4.915mmol)的14.8ml二氯甲烷中，加入4-二甲氨基吡啶(DMAP，240mg，1.966mmol)搅拌澄清后，加入丁二酸酐(197mg，1.966mmol)，25℃下搅拌反应18h。加入50ml二氯甲烷稀释反应液，以80ml 0.5M三乙胺磷酸盐洗涤有机相，水相以二氯甲烷萃取2次，每次50ml，合并有机相减压蒸干得到粗品。柱纯化使用188g 200-300目正相硅胶，以1wt％三乙胺中和硅胶酸性，二氯甲烷平衡柱子，以含1wt‰三乙胺的二氯甲烷∶甲醇＝10∶1-3∶1梯度洗脱，收集产物洗脱液，减压蒸干溶剂得到纯品Z-4缀合分子1.95g。MS m/z：C 107H 155N 10O 41，[M+H] +，理论：1935.07，实测：1935.29。
(10-1-5)Z-5的合成
通过与制备例1中步骤(1-1-9)相同的方法，制备Z-5。不同的是以Z-4缀合分子代替L-9缀合分子，得到连接固相载体的Z-4缀合分子。
(10-2)合成Z5-siHB1M1SVP缀合物
通过与制备例1中步骤(1-2)、(1-3A)、(1-4)相同的方法，制备缀合物17，不同的是以Z-5化合物代替L-10化合物起始正义链合成。预期可以得到Z5-siHB1M1SVP缀合物，其结构如式(422)所示。
制备例11：缀合物20的制备
本制备例合成了缀合物20(以下，也称为FIN-siHB1M1SVP缀合物)。该缀合物中所缀合的siRNA的序列参见表2。
(11-1)FIN-2缀合分子的合成
参照Rajeev等人，ChemBioChem 2015，16，903-908中描述的制备方法，按照以下工艺路线，合成了FIN-2缀合分子：
(11-1-1)PRO-10的合成
Figure PCTCN2018118107-appb-000089
(11-1-1a)PRO-7的合成
将2.93g PRO-6(L-羟基脯氨酸，CAS号：51-35-4，购自安耐吉公司，22.4mmol)溶于22.5ml 1，4-dioxane(1，4-二氧六环，CAS号：123-91-1)中，加入34ml 10％(w/w)Na2CO3的水溶液，呈悬浊液状态，将6.95g Fmoc-Cl(氯甲酸-9-芴基甲酯，CAS号：28920-43-6，购自安耐吉公司，26.8mmol)溶于34ml 1，4-dioxane，冰浴下加入到上述悬浊液中，自然升至室温反应过夜。将反应液倒入150ml冰水中，用甲基叔丁基醚萃取三次，每次100ml，弃去有机相，水相用浓HCl调节至pH≤5，用100ml乙酸乙酯萃取两次，合并有机相，无水硫酸钠干燥，减压蒸干溶剂得到白色泡沫状固体产品PRO-77.83g。 1H NMR(400MHz，DMSO-d 6)δ7.91(t，J＝7.2Hz，2H)，7.67(d，J＝7.5Hz，2H)，7.48-7.39(m，2H)，7.38-7.27(m，2H)，5.17(s，1H)，4.27(s，2H)，4.23-4.11(m，2H)，3.55-3.41(m，3H)，2.31-2.10(m，1H)，2.08-1.88(m，1H).HRMS(ESI)m/z理论C 20H 19NO 5[M-H] -352.1190，实测352.1033.
(11-1-1b)PRO-8的合成
将7.83g PRO-7(22.2mmol)溶于80ml THF(CAS号：109-99-9)中，油浴加热到65℃，回流状态下加入36.6ml 2mol/L的BH3-Me2S的THF溶液(CAS号13292-87-0，购自百灵威公司，73.2mmol)，继续回流反应3小时。倒出反应液，用甲醇溶解剩余固体，搅拌下加入甲醇至反应液无气体放出并继续搅拌30 分钟，减压蒸除溶剂后用石油醚提纯三次后得白色固体产物PRO-8 7.1g。 1H NMR(400MHz，DMSO-d 6)δ7.91(t，J＝6.7Hz，2H)，7.67(d，J＝7.2Hz，2H)，7.49-7.39(m，2H)，7.38-7.26(m，2H)，5.18(dd，J＝6.1，3.8Hz，1H)，4.28(s，2H)，4.23-4.13(m，2H)，3.55-3.38(m，2H)，2.32-2.11(m，1H)，2.08-1.89(m，1H).HRMS(ESI)m/z理论C 20H 21NO 4[M+Na] +362.1368，实测362.1012.
(11-1-1c)PRO-9的合成
将7.1g PRO-8(21mmol)溶于100ml吡啶中，加入14.2g DMTr-Cl(4，4′-双甲氧基三苯甲基氯，42mmol)，室温下搅拌反应5小时。减压蒸除溶剂，粗品用乙酸乙酯溶解后过滤除去盐类杂质，减压蒸除溶剂后硅胶柱纯化，硅胶柱预先用吡啶碱化后DCM溶解粗品上样，先用含1％(v/v)吡啶的DCM洗脱DMTr-Cl，随后用乙酸乙酯洗脱产物，收集产物洗脱液，减压蒸干溶剂，得白色固体产物PRO-9 8.2g；HRMS(ESI)m/z理论C 41H 39NO 6[M+Na] +664.2675，实测664.2348；C18 RP-HPLC(批号JJS160324-1)纯度94.20％。
(11-1-1d)PRO-10的合成
将8.2g PRO-9(12.8mmol)溶于64ml DMF(N，N-二甲基甲酰胺)中，加入40ml哌啶(384mmol)，室温下搅拌反应30分钟。反应液倒入300ml冰水中，乙酸乙酯萃取三次，每次150ml，合并有机相，用200ml饱和食盐水洗涤后，有机相以无水硫酸钠干燥，减压蒸除溶剂后硅胶柱纯化，硅胶柱预先用吡啶碱化后DCM溶解粗品上样，先用含1％(v/v)吡啶的DCM洗脱Fmoc，随后用乙酸乙酯洗脱产物，收集产物洗脱液，减压蒸干溶剂，得白色固体产物PRO-10 4.65g。 1H NMR(400MHz，DMSO-d 6)δ7.40(d，J＝7.2Hz，2H)，7.35-7.18(m，7H)，6.93-6.84(m，4H)，4.56(d，J＝3.9Hz，1H)，4.12(s，1H)，3.74(s，6H)，3.46-3.37(m，1H)，2.88(ddd，J＝18.5，10.0，5.5Hz，2H)，2.75(dd，J＝8.7，5.8Hz，1H)，2.62(dd，J＝11.0，2.7Hz，1H)，1.74-1.65(m，1H)，1.40(ddd，J＝12.9，8.5，5.9Hz，1H)；HRMS(ESI)m/z理论C 26H 29NO 4[M+Na] +442.1994，实测442.1999；C18 RP-HPLC(批号JJS160329-1)纯度97.07％。
(11-1-2)FIN-1的合成
Figure PCTCN2018118107-appb-000090
将按照(1-1-1)中描述的方法得到的GAL-5(4.5g，10mmol)溶于40ml DMF 中，依次加入3.9g DIPEA(N，N-二异丙基乙胺，CAS号：7087-68-5，购自阿拉丁公司，30mmol)和3.8g HBTU(苯并三氮唑-N，N，N′，N′-四甲基脲六氟磷酸盐，CAS号：94790-37-2，商购自阿拉丁公司，11mmol)，室温下搅拌10分钟，将步骤(11-1-1d)中获得的PRO-10(4.2g，10mmol)溶于40mlDMF中，随后加入到上述反应液中，反应液中加入无水硫酸钠干燥，室温搅拌2小时。将反应液倒入120ml冰水中，用乙酸乙酯萃取三次，每次60ml，合并有机相，分别用20ml水、20ml饱和食盐水洗涤，分出有机相并以无水硫酸钠干燥，减压蒸除溶剂，硅胶柱纯化，硅胶柱预先用吡啶碱化后上样，用含1体积％三乙胺和1体积％甲醇的二氯甲烷(DCM)溶液洗脱，收集产物洗脱液，减压蒸干溶剂，得到浅黄色泡沫状固体产品FIN-16.5g。 1H NMR(400MHz，DMSO-d 6)δ7.83(d，J＝9.2Hz，1H)，7.32(t，J＝6.6Hz，4H)，7.20(td，J＝8.9，3.5Hz，5H)，6.93-6.84(m，4H)，5.21(d，J＝3.2Hz，1H)，5.04-4.90(m，2H)，4.49(s，1H)，4.40(d，J＝4.4Hz，0.8H)，4.31(d，J＝5.0Hz，0.2H)，4.15(s，1H)，4.03(s，3H)，3.93(s，1H)，3.74(s，7H)，3.59(dt，J＝12.0，6.0Hz，1H)，3.50-3.40(m，1H)，3.39-3.25(m，3H)，3.13(dd，J＝8.9，5.2Hz，1H)，3.00(dq，J＝9.3，5.3，4.3Hz，1H)，2.22(s，2H)，2.07(s，3H)，1.99(s，3H)，1.90(s，4H)，1.74(s，3H)，1.50(s，3H)，1.36(s，1H)。C18 RP-HPLC(批号LJ160422)纯度95.45％。
(11-1-3)FIN-2的合成
Figure PCTCN2018118107-appb-000091
将步骤(11-1-2)中获得的FIN-1(3.0g，3.53mmol)与乙腈共沸除水，减压抽干，溶于10ml DMF，氮气保护下加入2.13g PA(双(二异丙基氨基)(2-氰基乙氧基)膦，购自Adamas公司，商品编号11356B，7.06mmol)、346mg四唑(CAS号：288-94-8，购自阿拉丁公司，4.94mmol)，室温下搅拌反应，补加10ml DMF，继续搅拌反应1小时。减压蒸除溶剂后以硅胶柱色谱纯化，硅胶柱预先用吡啶碱化后DCM溶解粗品上样，乙酸乙酯洗脱，收集产物洗脱液，减压蒸除溶剂，得无色糖浆状粗品4.5g。粗品用50体积％乙腈水溶液溶解至完全溶解，用C-18，330g，
Figure PCTCN2018118107-appb-000092
中压纯化柱纯化样品，柱子先用1体积％吡啶的乙腈溶液碱化，梯 度洗脱收集产品峰，减压蒸除溶剂得白色粉末产品FIN-2缀合分子2.2g。 31P NMR(162MHz，CDCl 3)δ148.04，147.94，147.62，147.19，磷谱纯度92％；C18 RP-HPLC纯度90.54％。
(11-2)FIN-2缀合分子连接到固相载体
采用核酸固相合成方法，将步骤(11-1-3)中得到的FIN-2缀合分子，通过三次循环，连接到通用固相载体(UnyLinker TM loaded
Figure PCTCN2018118107-appb-000093
HL Solid Supports)上，从而实现缀合基团(FIN_FIN_FIN)连接在RNA正义链的3′末端。
参照Rajeev等人，ChemBioChem 2015，16，903-908中描述的制备方法进行上述连接，具体而言，首先，由上述通用固相载体开始，脱除固相载体上的羟基保护基团，在偶联反应条件和偶联试剂存在下与FIN-2缀合分子接触发生偶联，经盖帽反应和氧化反应后，获得连接至固相载体的FIN缀合分子；脱除该连接至固相载体的FIN缀合分子上的羟基保护基团DMTr，与FIN-2缀合分子接触发生偶联，进行盖帽反应和氧化反应，并再重复一次上述脱保护-偶联-盖帽-氧化步骤，连接第三个FIN-2缀合分子，获得连接在固相载体上的的缀合基团(FIN_FIN_FIN)。
上述反应中，所述的脱保护、偶联、盖帽、氧化的反应条件、溶剂和试剂用量与前述步骤(1-2)中描述的核酸固相合成方法相同。
(11-3)缀合物20的合成
通过与制备例1中步骤(1-2)、(1-3A)、(1-4)相同的方法，制备题述缀合物，不同的是：1)以步骤(11-2)得到的化合物起始正义链合成；2)缀合的siRNA具有表2中所示的对应于缀合物20的序列。
利用液质联用仪(LC-MS，Liquid Chromatography-Mass Spectrometry，购于Waters公司，型号：LCT Premier)进行分子量检测。其结果，实测值与理论值相符，从而确定所合成的缀合物是目标设计的化合物，其结构如式(307)所示。
在上述本公开的缀合物制备完成后，使用标准手段冻干为固体粉末保存备用。在使用时，可使用例如注射用水将其重新溶解为所需浓度的溶液使用。
实验例1：本实验说明本公开的siRNA缀合物的稳定性
实验例1-1：siRNA缀合物在体外溶酶体裂解液中的稳定性
经溶酶体裂解液处理的测试样品制备：将缀合物4(以siRNA浓度为20μM的0.9％氯化钠水溶液形式提供，每组6μl)分别与27.2μL柠檬酸钠水溶液 (pH5.0)、4.08μL去离子水和2.72μL Tritosomes(商购自Xenotech公司，货号R0610LT，批号1610069)混匀。37℃恒温孵育。分别在0h、5min、15min、30min、1h、2h、4h、8h取出5μl样本，分别加入到15μL 9M的尿素中变性，随后加入4μl 6×上样缓冲液(索莱宝公司，货号20160830)，立即冷冻于-80℃冰箱终止反应。0小时表示，将待测样品与溶酶体裂解液混匀后，立即取出的时刻。
未经溶酶体裂解液处理的参比样品制备：取等摩尔量的缀合物4(20μM)1.5μl与7.5μL柠檬酸钠水溶液(pH5.0)、1μL去离子水混匀，加入30μL 9M的尿素溶液变性，随后加入8μL 6×上样缓冲液混匀，立即冷冻于-80℃冰箱终止反应。参比样品在电泳图中标记为Con。
配制16重量％的非变性聚丙烯酰胺凝胶，上述测试样品及参比样品各取20μl上样至凝胶，在20mA恒流条件下电泳10min后，继续在40mA恒流条件下电泳30min。电泳结束后，将凝胶置于摇床上，用Gelred染料(BioTium公司，货号13G1203)染色10min。凝胶成像观察并拍照，结果如图1所示。
图1显示了所测试siRNA缀合物在体外溶酶体中的稳定性半定量检测结果。结果显示，本公开的缀合物在溶酶体中可维持长时间不降解，显示出很好的稳定性。
实验例1-2：siRNA缀合物在人血浆中的稳定性
将缀合物4(以siRNA浓度为20μM的0.9％氯化钠水溶液形式提供，12μl)以及对比序列1(20μM，12μl)分别与108μL 90％人血浆(Human plasma，PBS稀释)混匀。37℃恒温孵育。分别在0、2、4、6、8、24、48、72小时取出10μL样本，立即进行液氮速冻，于-80℃冰箱中冻存。待各时间点取样完毕后，将上述冻存样品分别以1×PBS(pH7.4)稀释5倍后每一样品取10μL备用。同时，取等摩尔量的siRNA(2μM，2μl)或siRNA缀合物(siRNA浓度为2μM，2μl)，与8μl 1×PBS(pH7.4)混匀，制备成10μL未经人血浆处理的样品，记为Con。
配制20重量％的非变性聚丙烯酰胺凝胶，将上述备用样品中每一组的全部样品与4μL上样缓冲液(20mM EDTA，36重量％甘油，0.06重量％溴酚蓝的水溶液)混合，然后上样至前述凝胶，在80mA恒流条件下电泳60分钟。电泳结束后，用1×Sybr Gold染料(Invitrogen，Cat.11494)染色15分钟后成相，结果如图2所示。
对比序列1：
正义链：CCUUGAGGCAUACUUCAAA(SEQ ID NO：29)
反义链：UUUGAAGUAUGCCUCAAGGUU(SEQ ID NO：30)
图2示出了所测试缀合物在体外人血浆中的稳定性半定量检测结果。
由图2的结果可以看出，本公开的缀合物在人血浆中直至72h时仍未降解，显示出优异的在人血浆中的稳定性。
实验例1-3：siRNA缀合物在猴血浆中的稳定性
在另外的实验中，采用与实验例1-2相同的方法检测缀合物4在猴血浆(Monkey plasma，购自鸿泉生物，HQ70082，PBS稀释)中的稳定性，结果如图3所示。
图3出了所测试缀合物在体外猴血浆中的稳定性半定量检测结果。
由图3的结果可以看出，本公开的siRNA缀合物在食蟹猴血浆中直至72h仍未降解，显示出优异的在猴血浆中的稳定性。
实验例2：本实验例说明本公开的siRNA缀合物在体外(in vitro)的抑制活性
实验例2-1：体外psiCHECK系统中的在靶活性
本实验例中所使用的HEK293A细胞由北京大学分子医学研究所核酸技术实验室提供，用含有20％的胎牛血清(FBS，Hyclone公司)及0.2体积％的青链霉素双抗(Penicillin-Streptomycin，Gibco，Invitrogen公司)的DMEM完全培养基(Hyclone公司)培养细胞，于37℃在含5％CO2/95％空气的培养箱中培养。
本实验例考察了缀合物20在体外psiCHECK系统中的在靶活性(on-target activity)，即测定了缀合物20靶向完全匹配目标序列(其核苷酸序列与所述缀合物反义链的全长核苷酸序列完全互补)的活性。
根据Kumico Ui-Tei et.al.，Functional dissection of siRNA sequence by systematic DNA substitution：modified siRNA with a DNA seed arm is a powerful tool for mammalian gene silencing with significantly reduced off-target effect.Nucleic Acids Research，2008.36(7)，2136-2151描述的方法，构建检测质粒，与待评价的siRNA缀合物共转染至HEK293A细胞中，通过双萤光素酶报告基因的表达水平，来反应siRNA缀合物的在靶活性及脱靶效应。具体步骤如下：
[1]构建检测质粒
采用psiCHECKTM-2(PromegaTM)质粒构建在靶质粒，该质粒含有一个目标序列，该目标序列与待测缀合物中的反义链的所有21个核苷酸序列完全互 补。将目标序列克隆到psiCHECKTM-2质粒的Xho I/Not I位点。
[2]转染
在96孔板中，根据Lipofectamine TM 2000(Invitrogen公司)的使用说明，分别共转染siRNA缀合物和上述质粒，其中每孔转染质粒10ng，使用Lipofectamine TM 2000 0.2μL。缀合物的终浓度(以siRNA的浓度计算)依次为0.1nM、0.05nM和0.01nM。各组以无缀合物处理为对照。每组3个复孔。
NC为吉玛公司与目的基因序列无同源性的通用阴性对照B01001。
[3]检测
共转染24小时后，使用双萤光素酶报告基因检测试剂盒(Dual luciferase reporter gene assay kit，Promega公司，cat.E2940)，根据使用说明书裂解HEK293A细胞，检测双萤光素酶报告基因的表达水平。以海肾萤光素酶蛋白水平相对于萤火虫萤光素酶蛋白水平进行标准化。结果如图4所示。
结果表明，缀合物20具有较好的体外抑制活性。
实验例2-2：体外psiCHECK系统中IC 50的测定及脱靶检测
本实验例例考察了缀合物4在体外psiCHECK系统中的IC50及脱靶效应。
根据Kumico Ui-Tei et.al.，Functional dissection of siRNA sequence by systematic DNA substitution：modified siRNA with a DNA seed arm is a powerful tool for mammalian gene silencing with significantly reduced off-target effect.Nucleic Acids Research，2008.36(7)，2136-2151描述的方法，构建检测质粒，与待测缀合物共转染至HEK293A细胞中，通过双萤光素酶报告基因的表达水平，来反应缀合物的在靶活性及脱靶效应。具体步骤如下：
[1]构建检测质粒
采用psiCHECKTM-2(PromegaTM)质粒构建了4种重组质粒，其中GSCM表示在靶质粒，PSCM、GSSM、PSSM表示脱靶质粒：
(1)GSCM，含有一个目标序列，该目标序列与缀合物4中的反义链的所有21个核苷酸序列完全互补；
(2)PSCM，含有一个目标序列，该目标序列与缀合物4中的反义链的所有21个核苷酸序列完全一致；
(3)GSSM，含有一个目标序列，该目标序列与待测siRNA中反义链的5’端起1-8位核苷酸序列完全互补，该目标序列的剩余部分与待测siRNA中反义链5’端起9-21位的核苷酸序列相对应，其序列完全不互补，即待测siRNA中反义链5’端起9-21位中任一位置的核苷酸为G，C，A或U时，目标序列相应位 置的核苷酸分别为T，A，C或G。
(4)PSSM，含有一个目标序列，该目标序列与待测siRNA中正义链的5’端起1-8位核苷酸序列完全互补，该目标序列的剩余部分与待测siRNA中正义链5’端起9-19位的核苷酸序列相对应，其序列完全不互补，即待测siRNA中正义链5’端起9-19位中任一位置的核苷酸为G，C，A或U时，目标序列相应位置的核苷酸分别为T，A，C或G。为了与GSSM靶序列等长，目标序列的3’末端依次加入核苷酸T、A。
将目标序列克隆到psiCHECKTM-2质粒的Xho I/Not I位点。
[2]转染
在96孔板中，根据Lipofectamine TM 2000(Invitrogen公司)的使用说明，分别共转染缀合物和上述每一种质粒，其中每孔转染质粒10ng，使用Lipofectamine TM 2000 0.2μL，siRNA缀合物终浓度(以siRNA的量计)自0.1nM起始，倍比稀释至0.0001nM，一种质粒对应11组siRNA浓度，每组3个复孔。
[3]检测
将HEK293A细胞培养24小时后，使用双萤光报告基因检测试剂盒(Dual luciferase reporter gene assay kit，Promega公司，cat.E2940)，根据使用说明书裂解细胞，检测双萤光报告基因的表达水平。每一特定浓度的缀合物测试组以无缀合物处理组为对照。以海肾萤光素酶蛋白水平(Ren)相对于萤火虫萤光素酶蛋白水平(Fir)进行标准化。
根据采用不同siRNA浓度所测得的活性结果，利用Graphpad 5.0软件log(inhibitor)vs.response-Variable slope功能来拟合剂量-效应曲线，根据剂量-效应曲线计算待测siRNA靶向GSCM的IC50值，计算方法如下：
Figure PCTCN2018118107-appb-000094
式中：
Y是残留mRNA的表达水平，
X为转染siRNA浓度的对数值，
Bot是稳态期底部的Y值，
Top是稳态期顶部的Y值，
LogIC50是当Y在底部到顶部之间一半时的X值，而HillSlope则是曲线的斜率。结果如图5所示。
由图5可知，缀合物4在具有优异靶mRNA抑制效果的同时，还显示出低 的脱靶效应。
实验例3：本实验例说明本公开的缀合物在小鼠中对HBV mRNA表达的抑制
在本实验例中，对缀合物4在HBV转基因小鼠C57BL/6J-Tg(Alb1HBV)44Bri/J中对HBV mRNA表达量的抑制效率进行了考察。
C57BL/6J-Tg(Alb1HBV)44Bri/J小鼠购自北京大学医学部实验动物科学部，使用乙型肝炎病毒表面抗原诊断试剂盒(酶联免疫法)(上海科华生物)检测小鼠血清HBsAg含量，选取S/COV＞10的小鼠，随机分组(均为雌性)，每组4只小鼠，分别进行编号，并增加生理盐水NS对照组。所有动物根据体重计算药量，采用皮下注射方式单次给药，分别以1mg/kg以及0.1ml/kg的不同剂量给予缀合物4(以0.2mg/ml以及0.02mg/ml的0.9％氯化钠水溶液形式提供)，给药体积为5ml/kg。给药后第7天将动物处死，收集肝脏，用RNA later(Sigma Aldrich公司)保存；用组织匀浆仪匀浆肝组织，再用Trizol根据总RNA提取的标准操作步骤提取得到总RNA。
采用实时荧光定量PCR检测肝组织中HBV mRNA的表达量，具体地：使用ImProm-IITM反转录试剂盒(Promega公司)按其说明书将提取的总RNA逆转录为cDNA，接着用荧光定量PCR试剂盒(北京康为世纪生物科技有限公司)检测siRNA对肝组织中的HBV mRNA表达的抑制效率。在该荧光定量PCR法中，以β-肌动蛋白(β-actin)基因作为内参基因，使用针对HBV的引物和针对β-肌动蛋白的引物分别对HBV和β-肌动蛋白进行检测。
检测引物的序列参见表3。
表3检测引物的序列
Figure PCTCN2018118107-appb-000095
在该荧光定量PCR法中，HBV mRNA的表达量用HBV X基因表达剩余量表示，按如下等式计算：
HBV X基因表达剩余量＝(测试组HBV X基因的拷贝数/测试组β-actin的拷贝数)/(对照组HBV X基因的拷贝数/对照组β-actin的拷贝数)×100％，图中标 识为HBV X/β-actin mRNA表达量。
随后根据下式计算缀合物对mRNA抑制率：
缀合物对mRNA的抑制率＝(1-HBV X基因表达剩余量)×100％，
其中，对照组为本实验中施以NS的对照组小鼠，各测试组为分别施以不同siRNA缀合物的给药组小鼠。结果示于图6中。
由图6的结果可见，上述本公开的缀合物在1mg/kg的给药量下，对于靶mRNA的抑制率达93.8％，显示出良好的抑制效果。
实验例4：本实验说明本公开的siRNA缀合物在M-Tg模型上单次给药对HBsAg的抑制作用
将HBV转基因(M-TgHBV)小鼠(购自上海市公共卫生中心动物部)按血清HBsAg含量随机分成3组(每组6只，均为雄性)，分别为生理盐水(NS)对照组、缀合物41mg/kg和3mg/kg组。所有动物根据体重计算药量，给药体积为10ml/kg，皮下单次给药。在给药前(记为D0)与给药后第7、14、21、28、42、56、70、85天对小鼠眼眶静脉丛取血，在各时间点检测血清HBsAg水平。
眼眶取血每次约0.5ml，离心后血清不少于200μl。利用HBsAg CLIA试剂盒(安图生物，CL0310)检测血清中HBsAg的含量。
标准化的血清HBsAg水平＝(给药后测试组HBsAg含量/给药前测试组HBsAg含量)×100％。
HBsAg抑制率＝(1-给药后HBsAg含量/给药前HBsAg含量)×100％。
其中，HBsAg含量用每毫升(ml)血清含多少当量(UI)HBsAg表示。
以下图7示出了上述测试siRNA缀合物在M-Tg模型上单次给药对HBsAg表达抑制作用的检测结果。
从图7结果可以看出：单次给药3mg/kg的缀合物4对HBsAg的最大抑制率在90％以上，至少维持21天。
实验数据均以X±SEM表示，数据分析采用Graphpad prism5.0统计分析软件。首先对数据进行正态分布及方差齐性检验。符合正态分布(p＞0.20)及方差齐(p＞0.10)：多组间比较采用单因素方差分析的LSD法进行多重比较，p＜0.05认为有统计学意义；不符合正态分布或方差不齐：多组间比较采用非参数检验的Kruskal-Wallis H方法，如果Kruskal-wallis H检验结果显著(p＜0.05)，再将数据进行秩转换后，进行多组间两两比较，p＜0.05认为有统计学意义。图中“＊＊＊”表示p＜0.001，“＊＊”表示p＜0.01，“＊”表示p＜0.05。
以上详细描述了本公开的实施方式，但是，本公开并不限于上述实施方式中的具体细节，在本公开的技术构思范围内，可以对本公开的技术方案进行多种简单变型，这些简单变型均属于本公开的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合。为了避免不必要的重复，本公开对各种可能的组合方式不再另行说明。
此外，本公开的各种不同的实施方式之间也可以进行任意组合，只要其不违背本公开的思想，其同样应当视为本公开所公开的内容。
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Claims (74)

  1. 一种siRNA，所述siRNA含有正义链和反义链，所述siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述正义链含有一段核苷酸序列I，反义链含有一段核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II至少部分地反向互补形成双链区，其中，所述核苷酸序列I含有核苷酸序列A，所述核苷酸序列A与SEQ ID NO：1所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II含有核苷酸序列B，所述核苷酸序列B与SEQ ID NO：2所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
    5’-UCUGUGCCUUCUCAUCUGZ-3’(SEQ ID NO：1)；
    5’-Z′CAGAUGAGAAGGCACAGA-3’(SEQ ID NO：2)，
    其中，Z为A，Z′为U，
    所述核苷酸序列A中包含位置对应于Z的核苷酸Z A，所述核苷酸序列B中包含位置对应于Z′的核苷酸Z′ B，所述Z′ B是所述反义链5′末端的第一个核苷酸。
  2. 根据权利要求1所述的siRNA，其中，所述核苷酸序列A与SEQ ID NO：1所示的核苷酸序列之间不多于1个核苷酸差异，和/或所述核苷酸序列B与SEQ ID NO：2所示的核苷酸序列之间不多于1个核苷酸差异。
  3. 根据权利要求1或2所述的siRNA，其中，所述核苷酸序列B与SEQ ID NO：2所示的核苷酸序列之间的核苷酸差异包括Z′ B位置处的差异，且Z′ B选自A、C或G。
  4. 根据权利要求3所述的siRNA，其中Z A是与Z′ B互补的核苷酸。
  5. 根据权利要求1-4中任一项所述的siRNA，其中，所述核苷酸序列I和所述核苷酸序列II基本上反向互补、实质上反向互补或完全反向互补；所述基本上反向互补是指两个核苷酸序列之间存在不多于3个的碱基错配；所述实质上反向互补是指两个核苷酸序列之间存在不多于1个的碱基错配；完全反向互补是指两个核苷酸序列之间没有错配。
  6. 根据权利要求1-5中任一项所述的siRNA，其中，核苷酸序列A是SEQ ID NO：3所示的核苷酸序列，核苷酸序列B是SEQ ID NO：4所示的核苷酸序列：
    5′-UCUGUGCCUUCUCAUCUGZ A-3′(SEQ ID NO：3)；
    5′-Z′ BCAGAUGAGAAGGCACAGA-3’(SEQ ID NO：4)，
    其中，所述Z′ B是反义链5′末端的第一个核苷酸，Z A选自A、U、G或C，并且Z′ B是与Z A互补的核苷酸。
  7. 根据权利要求6所述的siRNA，其中Z A为A，Z′ B为U。
  8. 根据权利要求1-7中任一项所述的siRNA，其中，所述核苷酸序列I还含有核苷酸序列III，所述核苷酸序列II还含有核苷酸序列IV，核苷酸序列III和核苷酸序列IV的长度各自独立地为1-4个核苷酸，所述核苷酸序列III连接在核苷酸序列A的5’末端，核苷酸序列IV连接在核苷酸序列B的3’末端，所述核苷酸序列III和所述核苷酸序列IV长度相等并且实质上反向互补或完全反向互补；所述实质上反向互补是指两个核苷酸序列之间存在不多于1个的碱基错配；完全反向互补是指两个核苷酸序列之间没有错配。
  9. 根据权利要求8所述的siRNA，其中，所述核苷酸序列III和IV的长度均为1个核苷酸，所述核苷酸序列III的碱基为G；
    或者，所述核苷酸序列III和IV的长度均为2个核苷酸，按照5’末端到3’末端的方向，核苷酸序列III的碱基依次为CG；
    或者，所述核苷酸序列III和IV的长度均为3个核苷酸，按照5’末端到3’末端的方向，核苷酸序列III的碱基依次为CCG；
    或者，所述核苷酸序列III和IV的长度均为4个核苷酸，按照5’末端到3’末端的方向，核苷酸序列III的碱基依次为CCCG。
  10. 根据权利要求1-9所述的siRNA，其中，所述核苷酸序列II还含有核苷酸序列V，核苷酸序列V的长度为1至3个核苷酸，连接在所述反义链的3’末端，构成反义链的3’垂悬末端。
  11. 根据权利要求10所述的siRNA，其中，所述核苷酸序列V的长度为2个核苷酸。
  12. 根据权利要求10或11所述的siRNA，其中，所述核苷酸序列V为连续的两个胸腺嘧啶脱氧核糖核苷酸或连续的两个尿嘧啶核糖核苷酸，或者所述核苷酸序列V与靶mRNA相应位置的核苷酸互补。
  13. 根据权利要求1-12中任一项所述的siRNA，其中，所述siRNA的正义链含有如SEQ ID NO：3所示的核苷酸序列，所述反义链含有如SEQ ID NO：4所示的核苷酸序列：
    5’-UCUGUGCCUUCUCAUCUGZ A-3’(SEQ ID NO：3)；
    5’-Z′ BCAGAUGAGAAGGCACAGACG-3’(SEQ ID NO：4)；
    其中，所述Z′ B是反义链5′末端的第一个核苷酸，Z A选自A、U、G或C， 并且Z′ B是与Z A互补的核苷酸。
  14. 根据权利要求1-13中任一项所述的siRNA，其中，所述siRNA为siHBVX1：
    siHBVX1
    正义链：5’-UCUGUGCCUUCUCAUCUGZ-3’(SEQ ID NO：1)，
    反义链：5’-Z′CAGAUGAGAAGGCACAGACG-3’(SEQ ID NO：5)，
    其中，Z为A，Z′为U。
  15. 根据权利要求1-14中任一项所述的siRNA，其中，所述正义链或所述反义链中的至少一个核苷酸为修饰的核苷酸，和/或至少一个磷酸酯基为具有修饰基团的磷酸酯基。
  16. 根据权利要求1-15中任一项所述的siRNA，其中，所述正义链和所述反义链中的每一个核苷酸独立地为氟代修饰的核苷酸或非氟代修饰的核苷酸。
  17. 根据权利要求16所述的siRNA，其中，所述氟代修饰的核苷酸位于核苷酸序列A和核苷酸序列B中，并且，按照5′末端到3′末端的方向，所述核苷酸序列A的第7、8、9位的核苷酸为氟代修饰的核苷酸；按照5′末端到3′末端的方向，所述核苷酸序列B的第2、6、14、16位的核苷酸为氟代修饰的核苷酸。
  18. 据权利要求17所述的siRNA，其中，按照5′末端到3′末端的方向，在所述正义链中，所述核苷酸序列A的第7、8、9位或者5、7、8、9位的核苷酸为氟代修饰的核苷酸，所述正义链中其余位置的核苷酸为非氟代修饰的核苷酸；按照5′末端到3′末端的方向，在所述反义链中，所述核苷酸序列B的第2、6、14、16位或者2、6、8、9、14、16位的核苷酸为氟代修饰的核苷酸，所述反义链中其余位置的核苷酸为非氟代修饰的核苷酸。
  19. 根据权利要求16-18中任一项所述的siRNA，其中，每一个非氟代修饰的核苷酸独立地选自核苷酸的核糖基2′位的羟基被非氟基团取代形成的核苷酸或核苷酸类似物中的一种。
  20. 根据权利要求19所述的siRNA，其中，核苷酸的核糖基2′位的羟基被非氟基团取代形成的核苷酸选自2′-烷氧基修饰的核苷酸、2′-经取代的烷氧基修饰的核苷酸、2′-烷基修饰的核苷酸、2′-经取代的烷基修饰的核苷酸、2′-氨基修饰的核苷酸、2′-经取代的氨基修饰的核苷酸、2′-脱氧核苷酸中的一种；核苷酸类似物选自异核苷酸、LNA、ENA、cET、UNA和GNA中的一种。
  21. 根据权利要求20所述的siRNA，其中，每一个非氟代修饰的核苷酸均为甲氧基修饰的核苷酸，所述甲氧基修饰的核苷酸指核糖基的2′-羟基被甲氧基 取代而形成的核苷酸。
  22. 根据权利要求21所述的siRNA，其中，按照5’末端到3’末端的方向，所述siRNA的正义链中核苷酸序列A的第5、7、8和9位的核苷酸为氟代修饰的核苷酸，siRNA的正义链的其余位置的核苷酸为甲氧基修饰的核苷酸，并且，按照5’末端到3’末端的方向，所述siRNA的反义链中核苷酸序列B的第2、6、8、9、14和16位的核苷酸为氟代修饰的核苷酸，siRNA的反义链其余位置的核苷酸为甲氧基修饰的核苷酸；
    或者，按照5’末端到3’末端的方向，所述siRNA的正义链中核苷酸序列A的第5、7、8和9位的核苷酸为氟代修饰的核苷酸，siRNA的正义链的其余位置的核苷酸为甲氧基修饰的核苷酸，并且，按照5’末端到3’末端的方向，所述siRNA的反义链中核苷酸序列B的第2、6、14和16位的核苷酸为氟代修饰的核苷酸，siRNA的反义链其余位置的核苷酸为甲氧基修饰的核苷酸；
    或者，按照5’末端到3’末端的方向，所述siRNA的正义链中核苷酸序列A的第7、8和9位的核苷酸为氟代修饰的核苷酸，siRNA的正义链的其余位置的核苷酸为甲氧基修饰的核苷酸，并且，按照5’末端到3’末端的方向，所述siRNA的反义链中核苷酸序列B的第2、6、14和16位的核苷酸为氟代修饰的核苷酸，siRNA的反义链其余位置的核苷酸为甲氧基修饰的核苷酸。
  23. 根据权利要求22所述的siRNA，其中，所述siRNA为siHBVX2或siHBVX3：
    siHBVX2
    正义链：
    5’-UmCmUmGmUmGmCfCfUfUmCmUmCmAmUmCmUmGmAm-3’(SEQ ID NO：6)，
    反义链：
    5’-UmCfAmGmAmUfGmAmGmAmAmGmGmCfAmCfAmGmAmCmGm-3′(SEQ ID NO：7)，
    siHBVX3
    正义链：
    5’-UmCmUmGmUfGmCfCfUfUmCmUmCmAmUmCmUmGmAm-3’(SEQ ID NO：8)，
    反义链：
    5’-UmCfAmGmAmUfGmAfGfAmAmGmGmCfAmCfAmGmAmCmGm-3’(SEQ  ID NO：9)，
    其中，大写字母C、G、U、A表示核苷酸的碱基组成；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸。
  24. 根据权利要求15所述的siRNA，其中，所述具有修饰基团的磷酸酯基为磷酸酯基中的磷酸二酯键中的至少一个氧原子被硫原子取代而形成的硫代磷酸酯基。
  25. 根据权利要求15或24所述的siRNA，其中，所述具有修饰基团的磷酸酯基为具有如式(1)所示结构的硫代磷酸酯基：
    Figure PCTCN2018118107-appb-100001
  26. 根据权利要求25所述的siRNA，其中，所述siRNA中，硫代磷酸酯基连接存在于由以下位置所组成的组中的至少一处：
    所述正义链的5′末端端部第1个核苷酸和第2个核苷酸之间；
    所述正义链的5′末端端部第2个核苷酸和第3个核苷酸之间；
    所述正义链的3′末端端部第1个核苷酸和第2个核苷酸之间；
    所述正义链的3′末端端部第2个核苷酸和第3个核苷酸之间；
    所述反义链的5′末端端部第1个核苷酸和第2个核苷酸之间；
    所述反义链的5′末端端部第2个核苷酸和第3个核苷酸之间；
    所述反义链的3′末端端部第1个核苷酸和第2个核苷酸之间；以及
    所述反义链的3′末端端部第2个核苷酸和第3个核苷酸之间。
  27. 根据权利要求26所述的siRNA，其中，所述siRNA为siHBVX4或siHBVX5：
    siHBVX4
    正义链：
    5’-UmsCmsUmGmUmGmCfCfUfUmCmUmCmAmUmCmUmGmAm-3’(SEQ ID NO：10)，
    反义链：
    5’-UmsCfsAmGmAmUfGmAmGmAmAmGmGmCfAmCfAmGmAmsCmsGm-3′(SEQ ID NO：11)，
    siHBVX5
    正义链：
    5’-UmsCmsUmGmUfGmCfCfUfUmCmUmCmAmUmCmUmGmAm-3’(SEQ ID NO：12)，
    反义链：
    5’-UmsCfsAmGmAmUfGmAfGfAmAmGmGmCfAmCfAmGmAmsCmsGm-3’(SEQ ID NO：13)，
    其中，大写字母C、G、U、A表示核苷酸的碱基组成；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸；小写字母s表示该字母左右两个核苷酸之间为硫代磷酸酯基连接。
  28. 根据权利要求1-27中任一项所述的siRNA，其中，所述反义链的5’末端核苷酸为5’-磷酸核苷酸或5’-磷酸类似物修饰的核苷酸。
  29. 根据权利要求28所述的siRNA，其中，所述5’-磷酸核苷酸为具有如式(2)所示结构的核苷酸，所述5’-磷酸类似物修饰的核苷酸选自结构如式(3)-式(6)中任意一个所示的核苷酸：
    Figure PCTCN2018118107-appb-100002
    其中，R选自H、OH、甲氧基或氟；Base表示碱基，选自A、U、C、G或T。
  30. 根据权利要求28或29所述的siRNA，其中，所述siRNA为siHBVX6、siHBVX7、siHBVX8或siHBVX9：
    siHBVX6
    正义链：
    5’-UmCmUmGmUmGmCfCfUfUmCmUmCmAmUmCmUmGmAm-3’(SEQ ID NO：6)，
    反义链：
    5’-P1-UmCfAmGmAmUfGmAmGmAmAmGmGmCfAmCfAmGmAmCmGm-3′(SEQ ID NO：14)，
    siHRVX7
    正义链：
    5’-UmCmUmGmUfGmCfCfUfUmCmUmCmAmUmCmUmGmAm-3’(SEQ ID NO：8)，
    反义链：
    5’-P1-UmCfAmGmAmUfGmAfGfAmAmGmGmCfAmCfAmGmAmCmGm-3’
    (SEQ ID NO：15)，
    siHBVX8
    正义链：
    5’-UmsCmsUmGmUmGmCfCfUfUmCmUmCmAmUmCmUmGmAm-3’(SEQ ID NO：10)，
    反义链：
    5’-P1-UmsCfsAmGmAmUfGmAmGmAmAmGmGmCfAmCfAmGmAmsCmsGm-3′(SEQ ID NO：16)，
    siHBVX9
    正义链：
    5’-UmsCmsUmGmUfGmCfCfUfUmCmUmCmAmUmCmUmGmAm-3’(SEQ ID NO：12)，
    反义链：
    5’-P1-UmsCfsAmGmAmUfGmAfGfAmAmGmGmCfAmCfAmGmAmsCmsGm-3’(SEQ ID NO：17)，
    其中，大写字母C、G、U、A表示核苷酸的碱基组成；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸；小写字母s表示该字母左右两个核苷酸之间为硫代磷酸酯基连接；大写字母P1表示该字母右侧相邻的一个核苷酸为5’-磷酸核苷酸或5’-磷酸类似物修饰的核苷酸。
  31. 一种药物组合物，其特征在于，该药物组合物含有权利要求1-30中任意一项所述的siRNA和药学上可接受的载体。
  32. 根据权利要求31所述的药物组合物，其中，所述siRNA与药学上可接受的载体的重量比为1∶(1-500)。
  33. 根据权利要求31或32所述的siRNA，其中，所述siRNA与药学上可接受的载体的重量比为1∶(1-50)。
  34. 根据权利要求31-33中任一项所述的药物组合物，其中，所述药学上可 接受的载体含有有机胺、辅助脂质和聚乙二醇化脂质；其中，所述有机胺为如式(201)所示的化合物和/或其药学上可接受的盐：
    Figure PCTCN2018118107-appb-100003
    其中：
    X 101和X 102各自独立地是O、S、N-A或C-A，其中A是氢或C 1-C 20烃链；
    Y和Z各自独立地是C＝O、C＝S、S＝O、CH-OH或SO 2；
    R 101、R 102、R 103、R 104、R 105、R 106和R 107各自独立地是氢，环状或无环的、被取代的或未被取代的、支链或直链脂族基团，环状或无环的、被取代的或未被取代的、支链或直链杂脂族基团，被取代的或未被取代的、支链或直链酰基，被取代的或未被取代的、支链或直链芳基，被取代的或未被取代的、支链或直链杂芳基；
    x是1-10的整数；
    n是1-3的整数，m是0-20的整数，p是0或1；其中，如果m＝p＝0，则R102是氢；
    并且，如果n或m中的至少一个是2，那么R 103和在式(201)中的氮形成如式(202)或式(203)所示的结构：
    Figure PCTCN2018118107-appb-100004
    其中，g、e和f各自独立地是1-6的整数，“HCC”代表烃链，且每个＊N表示在式(201)中的氮原子。
  35. 根据权利要求34所述的药物组合物，其中，所述有机胺为如式(214) 所示的有机胺和/或如式(215)所示的有机胺：
    Figure PCTCN2018118107-appb-100005
    所述辅助脂质为胆固醇、胆固醇的类似物和/或胆固醇的衍生物；
    所述聚乙二醇化脂质为1，2-二棕榈酰胺-sn-甘油-3-磷脂酰乙醇胺-N-[甲氧基(聚乙二醇)]-2000。
  36. 根据权利要求34或35所述的药物组合物，其中，所述有机胺、所述辅助脂质和所述聚乙二醇化脂质三者之间的摩尔比为(19.7-80)∶(19.7-80)∶(0.3-50)。
  37. 根据权利要求36所述的药物组合物，其中，所述有机胺、所述辅助脂质和所述聚乙二醇化脂质三者之间的摩尔比为(50-70)∶(20-40)∶(3-20)。
  38. 一种siRNA缀合物，所述siRNA缀合物包含权利要求1-30中任意一项所述的siRNA以及缀合连接至该siRNA的缀合基团。
  39. 根据权利要求38所述的siRNA缀合物，其中，所述缀合基团包含药学上可接受的靶向基团和接头，并且，所述siRNA、所述接头和所述靶向基团依次共价或非共价连接。
  40. 根据权利要求39所述的siRNA缀合物，其中，所述接头具有如式(301)所示的结构：
    Figure PCTCN2018118107-appb-100006
    其中，k为1-3的整数；
    L A为具有如式(302)所示结构的包含酰胺键的链状部分，每个所述L A在其两端分别与一个所述靶向基团和所述L C部分通过醚键相连接：
    Figure PCTCN2018118107-appb-100007
    L B为具有如式(303)所示结构的包含N-酰基吡咯烷的链状部分，所述链状部分在其一端具有羰基并与所述L C部分通过酰胺键相连接，在另一端具有氧原子并与所述siRNA通过磷酸酯键相连接：
    Figure PCTCN2018118107-appb-100008
    L C为基于羟甲基氨基甲烷、二羟甲基氨基甲烷或三羟甲基氨基甲烷的2-4价连接基团，所述L C经由氧原子与各个所述L A部分通过醚键相连接，并且经由氮原子与所述L B部分通过酰胺键相连接。
  41. 根据权利要求38-40中任一项所述的siRNA缀合物，其中，所述siRNA缀合物具有如式(305)所示的结构：
    Figure PCTCN2018118107-appb-100009
    其中，双螺旋结构表示所述siRNA。
  42. 根据权利要求39所述的siRNA缀合物，其中，所述接头具有式(306)所示的结构：
    Figure PCTCN2018118107-appb-100010
    其中，l为0-3的整数；
    ＊表示所述接头上通过醚键与所述靶向基团连接的位点；
    #表示所述接头上通过磷酸酯键与所述siRNA连接的位点。
  43. 根据权利要求38、39和42中任一项所述的siRNA缀合物，其中，所述siRNA缀合物具有如式(307)所示的结构：
    Figure PCTCN2018118107-appb-100011
    其中，双螺旋结构表示所述siRNA。
  44. 根据权利要求39-43中任一项所述的siRNA缀合物，其中，所述接头连接至所述siRNA的正义链3’末端。
  45. 根据权利要求38所述的siRNA缀合物，其中，所述缀合物具有式(308)所示的结构：
    Figure PCTCN2018118107-appb-100012
    Figure PCTCN2018118107-appb-100013
    其中，
    n1为选自1-3的整数，n3为选自0-4的整数；
    m1、m2和m3独立地为选自2-10的整数；
    R 10、R 11、R 12、R 13、R 14和R 15各自独立地为H，或选自于由以下基团所组成的组：C 1-C 10烷基、C 1-C 10卤代烷基以及C 1-C 10烷氧基；
    R 3为式A59所示结构的基团：
    Figure PCTCN2018118107-appb-100014
    其中，E 1为OH、SH或BH 2，Nu为siRNA；
    R 2是长度为1-20个碳原子的直链亚烷基，其中一个或多个碳原子任选地被选自于以下基团所组成的组中的一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O) 2、C 2-C 10亚烯基、C 2-C 10亚炔基、C 6-C 10亚芳基、C 3-C 18亚杂环基和C 5-C 10亚杂芳基；并且其中，R 2可任选地具有由以下基团所组成中的任何一个或多个的取代基：C 1-C 10烷基、C 6-C 10芳基、C 5-C 10杂芳基、C 1-C 10卤代烷基、-OC 1-C 10烷基、-OC 1-C 10烷基苯基、-C 1-C 10烷基-OH、-OC 1-C 10卤代烷基、-SC 1-C 10烷基、-SC 1-C 10烷基苯基、-C 1-C 10烷基-SH、-SC 1-C 10卤代烷基、卤素取代基、-OH、-SH、-NH 2、-C 1-C 10烷基-NH 2、-N(C 1-C 10烷基)(C 1-C 10烷基)、-NH(C 1-C 10烷基)、氰基、硝基、-CO 2H、-C(O)O(C 1-C 10烷基)、-CON(C 1-C 10烷基)(C 1-C 10烷基)、-CONH(C 1-C 10烷基)、-CONH 2、-NHC(O)(C 1-C 10烷基)、-NHC(O)(苯基)、-N(C 1-C 10烷基)C(O)(C 1-C 10烷基)、-N(C 1-C 10烷基)C(O)(苯基)、-C(O)C 1-C 10烷基、-C(O)C1-C 10烷基苯基、-C(O)C 1-C 10卤烷基、-OC(O)C 1-C 10烷基、-SO 2(C 1-C 10烷基)、-SO 2(苯基)、-SO 2(C 1-C 10卤代烷基)、-SO 2NH 2、-SO 2NH(C 1-C 10烷基)、-SO 2NH(苯基)、-NHSO 2(C 1-C 10烷基)、-NHSO 2(苯基)和-NHSO 2(C 1-C 10卤代烷基)；
    每个L 1是长度为1-70个碳原子的直链亚烷基，其中一个或多个碳原子任选地被选自于以下基团所组成的组中的一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O) 2、C 2-C 10亚烯基、C 2-C 10亚炔基、C 6-C 10亚芳基、C 3-C 18亚杂环基和C 5-C 10亚杂芳基；并且其中，L 1可任选地具有由以下基团所组成中的任何一个或多个的取代基：C 1-C 10烷基、C 6-C 10芳基、C 5-C 10杂芳基、C 1-C 10卤代烷基、 -OC 1-C 10烷基、-OC 1-C 10烷基苯基、-C 1-C 10烷基-OH、-OC 1-C 10卤代烷基、-SC 1-C 10烷基、-SC 1-C 10烷基苯基、-C 1-C 10烷基-SH、-SC 1-C 10卤代烷基、卤素取代基、-OH、-SH、-NH 2、-C 1-C 10烷基-NH 2、-N(C 1-C 10烷基)(C 1-C 10烷基)、-NH(C 1-C 10烷基)、氰基、硝基、-CO 2H、-C(O)O(C 1-C 10烷基)、-CON(C 1-C 10烷基)(C 1-C 10烷基)、-CONH(C 1-C 10烷基)、-CONH 2、-NHC(O)(C 1-C 10烷基)、-NHC(O)(苯基)、-N(C 1-C 10烷基)C(O)(C 1-C 10烷基)、-N(C 1-C 10烷基)C(O)(苯基)、-C(O)C 1-C 10烷基、-C(O)C 1-C 10烷基苯基、-C(O)C 1-C 10卤烷基、-OC(O)C 1-C 10烷基、-SO 2(C 1-C 10烷基)、-SO 2(苯基)、-SO 2(C 1-C 10卤代烷基)、-SO 2NH 2、-SO 2NH(C 1-C 10烷基)、-SO 2NH(苯基)、-NHSO 2(C 1-C 10烷基)、-NHSO 2(苯基)和-NHSO 2(C 1-C 10卤代烷基)；
    Figure PCTCN2018118107-appb-100015
    表示基团连接至分子其余部分的位点；
    M 1表示靶向基团。
  46. 根据权利要求45所述的siRNA缀合物，其中，每个L 1独立地选自式A1-A26基团中的一种或多种的连接组合：
    Figure PCTCN2018118107-appb-100016
    Figure PCTCN2018118107-appb-100017
    其中，j1为1-20的整数；j2为1-20的整数；
    R’为C 1-C 10的烷基；
    Ra选自式A27-A45基团或其任意组合所组成的组：
    Figure PCTCN2018118107-appb-100018
    Figure PCTCN2018118107-appb-100019
    Rb为C 1-C 10的烷基。
  47. 根据权利要求46所述的siRNA缀合物，其中，L 1选自A1、A4、A5、A6、A8、A10、A11、A13中的一种或多种的连接组合。
  48. 根据权利要求47所述的siRNA缀合物，其中，L 1选自A1、A4、A8、A10和A11中至少2个的连接组合。
  49. 根据权利要求48所述的siRNA缀合物，其中，L 1选自A1、A8、A10中至少2个的连接组合。
  50. 根据权利要求45-49中任意一项所述的siRNA缀合物，其中，L 1的长度为3-25个原子。
  51. 根据权利要求50所述的siRNA缀合物，其中，L 1的长度为4-15个原子。
  52. 根据权利要求46-51中任意一项所述的siRNA缀合物，其中，j1为2-10的整数，j2为2-10的整数，R’为C 1-C 4的烷基，Ra为A27、A28、A29、A30和A31中的一种，Rb为C 1-C 5的烷基。
  53. 根据权利要求52所述的siRNA缀合物，其中，j1为3-5的整数，j2为3-5的整数，R’为甲基、乙基和异丙基中的一种，Ra为A27或A28，Rb为甲基、 乙基、异丙基和丁基中的一种。
  54. 根据权利要求45-53中任意一项所述的siRNA缀合物，其中，n1为1-2的整数，n3为0-1的整数，且n1+n3＝2-3。
  55. 根据权利要求45-54中任意一项所述的siRNA缀合物，其中，m1、m2和m3各自独立地为2-5的整数。
  56. 根据权利要求45-55中任一项所述的siRNA缀合物，其中，m1＝m2＝m3。
  57. 根据权利要求45-56中任意一项所述的siRNA缀合物，其中，R i0、R 11、R 12、R 13、R 14和R 15独立地为H、甲基或乙基。
  58. 根据权利要求45-57中任一项所述的siRNA缀合物，其中，R 2上同时含有与含氮骨架上的N连接的连接位点和与R3中的P连接的连接位点。
  59. 根据权利要求45-58中任一项所述的siRNA缀合物，其中，R 2上所述与含氮骨架上的N连接的位点与N形成酰胺键，所述与R 3上的P连接的位点与P形成磷酸酯键。
  60. 根据权利要求45-59中任一项所述的siRNA缀合物，其中，R 2选自B5、B6、B5’或B6’：
    Figure PCTCN2018118107-appb-100020
    其中，表示基团连接至分子其余部分的位点，q 2为1-10的整数。
  61. 根据权利要求60所述的siRNA缀合物，其中，q 2为1-5的整数。
  62. 根据权利要求39-61中任一项所述的siRNA缀合物，其中，每个所述靶向基团独立地为与哺乳动物肝细胞表面的去唾液酸糖蛋白受体亲和的配体。
  63. 根据权利要求中62所述的siRNA缀合物，其中，每个所述靶向基团独 立地为去唾液酸糖蛋白或糖。
  64. 根据权利要求63所述的siRNA缀合物，其中每个所述靶向基团独立地选自D-吡喃甘露糖、L-吡喃甘露糖、D-阿拉伯糖、D-呋喃木糖、L-呋喃木糖、D-葡萄糖、L-葡萄糖、D-半乳糖、L-半乳糖、α-D-呋喃甘露糖、β-D-呋喃甘露糖、α-D-吡喃甘露糖、β-D-吡喃甘露糖、α-D-吡喃葡萄糖、β-D-吡喃葡萄糖、α-D-呋喃葡萄糖、β-D-呋喃葡萄糖、α-D-呋喃果糖、α-D-吡喃果糖、α-D-吡喃半乳糖、β-D-吡喃半乳糖、α-D-呋喃半乳糖、β-D-呋喃半乳糖、葡糖胺、唾液酸、半乳糖胺、N-乙酰基半乳糖胺、N-三氟乙酰基半乳糖胺、N-丙酰基半乳糖胺、N-正丁酰基半乳糖胺、N-异丁酰基半乳糖胺、2-氨基-3-O-[(R)-1-羧乙基]-2-脱氧-β-D-吡喃葡萄糖、2-脱氧-2-甲基氨基-L-吡喃葡萄糖、4，6-二脱氧-4-甲酰胺基-2，3-二-O-甲基-D-吡喃甘露糖、2-脱氧-2-磺氨基-D-吡喃葡萄糖、N-乙醇酰基-α-神经氨酸、5-硫代-β-D-吡喃葡萄糖、2，3，4-三-O-乙酰基-1-硫代-6-O-三苯甲基-α-D-吡喃葡萄糖苷甲酯、4-硫代-β-D-吡喃半乳糖、3，4，6，7-四-O-乙酰基-2-脱氧-1，5-二硫代-α-D-吡喃葡庚糖苷乙酯、2，5-脱水-D-阿洛糖腈、核糖、D-核糖、D-4-硫代核糖、L-核糖、L-4-硫代核糖中的一种。
  65. 根据权利要求64所述的siRNA缀合物，其中，至少一个或每个所述靶向基团为半乳糖或N-乙酰基半乳糖胺。
  66. 根据权利要求45-65中任意一项所述的siRNA缀合物，其中，该缀合物具有式(403)、(404)、(405)、(406)、(407)、(408)、(409)、(410)、(411)、(412)、(413)、(414)、(415)、(416)、(417)、(418)、(419)、(420)、(421)或(422)所示的结构：
    Figure PCTCN2018118107-appb-100021
    Figure PCTCN2018118107-appb-100022
    Figure PCTCN2018118107-appb-100023
    Figure PCTCN2018118107-appb-100024
    Figure PCTCN2018118107-appb-100025
    Figure PCTCN2018118107-appb-100026
  67. 根据权利要求45-66中任意一项所述的siRNA缀合物，其中，式A59中的P连接到siRNA正义链或反义链的端部，所述端部指所述正义链或反义链中从其一端起算的前4个核苷酸。
  68. 根据权利要求67所述的siRNA缀合物，其中，式A59中的P连接到所述siRNA正义链或反义链的末端。
  69. 根据权利要求68所述的siRNA缀合物，其中，式A59中的P连接到 所述siRNA正义链的3′末端。
  70. 根据权利要求45-69中任意一项所述的siRNA缀合物，其中，式A59中的P通过形成磷酸二酯键连接至所述siRNA中的核苷酸的2′位、3′位或5′位。
  71. 权利要求1-30中任意一项所述的siRNA、权利要求31-37中任意一项所述的药物组合物和/或权利要求38-70中任意一项所述的siRNA缀合物在制备用于治疗和/或预防由乙型肝炎病毒的感染引起的病理状况或疾病的药物中的用途。
  72. 根据权利要求71所述的用途，其中，所述由乙型肝炎病毒的感染引起的病理状况或疾病选自慢性肝病、肝炎、肝纤维化疾病和肝增生性疾病。
  73. 一种治疗和/或预防由乙型肝炎病毒的感染引起的病理状况或疾病的方法，其中，所述方法包括将有效量的权利要求1-30中任意一项所述的siRNA、权利要求31-37中任意一项所述的药物组合物和/或权利要求38-70中任意一项所述的siRNA缀合物给予有需要的患者。
  74. 一种试剂盒，其中，该试剂盒含有权利要求1-30任意一项所述的siRNA、权利要求31-37中任意一项所述的药物组合物和/或权利要求38-70中任意一项所述的siRNA缀合物。
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