WO2020168952A1 - 修饰核苷及其合成方法 - Google Patents

修饰核苷及其合成方法 Download PDF

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WO2020168952A1
WO2020168952A1 PCT/CN2020/074825 CN2020074825W WO2020168952A1 WO 2020168952 A1 WO2020168952 A1 WO 2020168952A1 CN 2020074825 W CN2020074825 W CN 2020074825W WO 2020168952 A1 WO2020168952 A1 WO 2020168952A1
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group
modified
compound
substituted
nucleotides
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PCT/CN2020/074825
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English (en)
French (fr)
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蔡云松
李航文
刘连晓
李宁
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斯微(上海) 生物科技有限公司
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Priority to CN202080004025.2A priority Critical patent/CN114096550B/zh
Priority to EP20758779.1A priority patent/EP3929204A4/en
Priority to US17/431,524 priority patent/US20220135612A1/en
Publication of WO2020168952A1 publication Critical patent/WO2020168952A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H23/00—Compounds containing boron, silicon, or a metal, e.g. chelates, vitamin B12

Definitions

  • the present invention claims a PCT application, application number PCT/CN2019/075474, application date: priority on February 19, 2019, and the entire content of the PCT application is a part of the present invention.
  • the present invention relates to the field of nucleic acid, in particular to the modification and synthesis method of nucleic acid.
  • mRNA messenger RNA plays a vital role in human biology. Through a process called transcription, mRNA controls protein synthesis in the body. mRNA drugs can be used for genetic diseases, cancer and infectious diseases.
  • RNA Naturally occurring RNA is synthesized from the four basic ribonucleotides ATP, CTP, UTP and GTP, but may include modified nucleotides after transcription. Nearly 100 different nucleoside modifications have been identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). RNA Modification Database: 1999 update. Nucleic Acid Research 27: 196-197). However, when incorporated into mRNA, many modifications can cause an inhibitory immune response in the receptor and/or limit protein production, thus limiting the therapeutic effect of mRNA drugs. Therefore, new nucleosides, nucleotides and/or nucleic acid (for example mRNA) modifications are needed in the art to solve these problems.
  • This article discloses compounds, modified nucleosides, modified nucleotides, modified nucleic acids and methods for their synthesis.
  • R 1 , R 2 , R 4 and R 5 are each independently selected from the group included in the following set consisting of -H, -OH, -NH 2 , halogen group, substituted or unsubstituted C 1 -C 10 alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted C 1 -C 10 aralkyl, substituted or unsubstituted C 1 -C 10 cycloalkyl , Substituted or unsubstituted C 1 -C 10 heterocycles, substituted or unsubstituted acyl groups, -OR 6 , -C(O)R 6 , -C(O)-OR 6 , -C(O)-NH- R 6 and 1-N(R 6 ) 2 are composed;
  • R 3 is selected from one or several groups included in the following group consisting of -H, -OH, -NH 2 , halogen group, substituted or unsubstituted C 1 -C 10 alkyl, substituted or unsubstituted Aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted C 1 -C 10 aralkyl, substituted or unsubstituted C 1 -C 10 cycloalkyl, substituted or unsubstituted C 1 -C 10 Heterocyclic group, substituted or unsubstituted acyl group, -OR 6 , -C(O)R 6 , -C(O)-OR 6 , -C(O)-NH-R 6 , and -N(R 6 ) 2.
  • R 6 is H, a substituted or unsubstituted C 1 -C 10 alkyl group, and a substituted or unsubstituted acyl group.
  • R 1 , R 2 , R 4 and R 5 are each independently —H, —OH, or substituted or unsubstituted C 1 -C 10 alkyl.
  • R 3 is —H, —OH, substituted or unsubstituted C 1 -C 10 alkyl, phosphate, diphosphate, or triphosphate.
  • R 1 is —OH.
  • R 2 is —OH.
  • R 2 is -H.
  • R 3 is —OH.
  • R 4 is -H.
  • R 5 is -H.
  • the compound is a modified nucleoside, wherein R 1 , R 2 and R 3 are -OH, and R 4 and R 5 are -H.
  • the compound is a modified ribonucleoside.
  • the compound may have the structure of formula (Ia):
  • the compound is a modified nucleoside, wherein R 1 and R 3 are -OH, and R 2 , R 4 and R 5 are -H.
  • the compound is a modified deoxyribonucleoside.
  • the compound may have the structure of formula (Ib):
  • the compound is a modified nucleotide, wherein R 2 is -OH and R 3 is a phosphate group.
  • the compound may have the structure of formula (Ic):
  • the compound is a modified nucleotide, wherein R 2 is -H and R 3 is a phosphate group.
  • the compound may have the structure of formula (Id):
  • the compound is a modified nucleotide, such as a modified nucleoside triphosphate (NTP), where R 2 is -OH and R 3 is a triphosphate group.
  • NTP modified nucleoside triphosphate
  • the compound may have the structure of formula (Ie):
  • the compound is a modified nucleotide, such as a modified deoxynucleoside triphosphate (dNTP), where R 2 is -H and R 3 is a triphosphate group.
  • dNTP modified deoxynucleoside triphosphate
  • the compound may have the structure of formula (If):
  • the modified nucleoside triphosphate has the structure of formula (I-g):
  • the modified nucleoside triphosphate includes a modified cytidine triphosphate.
  • Y + is selected from the group consisting of Li + , Na + , K + , H + , NH 4 + and tetraalkylammonium ion.
  • the tetraalkylammonium is selected from the group consisting of tetraethylammonium, tetrapropylammonium, and tetrabutylammonium.
  • the modified nucleotide includes a modified deoxynucleoside triphosphate (dNTP).
  • dNTP deoxynucleoside triphosphate
  • the modified deoxynucleoside triphosphate has the structure of formula (I-h):
  • the modified deoxynucleoside triphosphate includes a modified deoxycytidine triphosphate.
  • Y + is selected from the group consisting of Li + , Na + , K + , H + , NH 4 + and tetraalkylammonium ion.
  • the tetraalkylammonium is selected from the group consisting of tetraethylammonium, tetrapropylammonium, and tetrabutylammonium.
  • a nucleic acid comprising two or more covalently linked nucleotides, wherein at least one of the two or more covalently linked nucleotides includes any of the nucleotides disclosed in this application Compound, modified nucleoside or modified nucleotide.
  • the nucleic acid is a ribonucleic acid (RNA).
  • the RNA is a messenger RNA (mRNA).
  • the RNA includes any compound, modified nucleoside, or modified nucleotide disclosed in this application.
  • the nucleic acid is a deoxyribonucleic acid (DNA).
  • the DNA includes any of the compounds, modified nucleosides, or modified nucleotides disclosed in this application.
  • a pharmaceutical composition including any compound disclosed in the present application, a modified nucleoside or a modified nucleotide, and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition includes any compound disclosed in the present application or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable excipient thereof.
  • the pharmaceutical composition includes any nucleic acid disclosed herein or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable excipient thereof.
  • the pharmaceutical composition includes any RNA disclosed herein or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable excipient thereof.
  • the pharmaceutical composition includes any mRNA disclosed herein or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable excipient thereof.
  • R 11 , R 12 and R 13 are each independently —H, —OH or —O—protecting group
  • R 14 and R 15 are each independently selected from -H, substituted or unsubstituted C 1 -C 10 alkyl, and substituted or unsubstituted acyl;
  • R 16 is selected from -NH 2 , halogen group and
  • R 11 , R 12 and R 13 are —O—protecting groups.
  • the protecting group is selected from the group consisting of acetyl, benzoyl, benzyl, ⁇ -methoxyethoxymethyl ether, dimethoxy Trityl [bis-(4-methoxyphenyl) phenylmethyl], methoxymethyl ether group, methoxytrityl [(4-methoxyphenyl) diphenyl Methyl], p-methoxybenzyl ether, methylthiomethyl ether, pivaloyl, tetrahydropyranyl, tetrahydrofuranyl, trityl (triphenylmethyl), silyl It is composed of ether group, methyl ether group and ethoxy ethyl ether group.
  • the protecting group is a silyl ether group selected from the group consisting of trimethylsilyl ether group (TMS), tert-butyldiphenylsilyl group Ether group (TBDPS), tert-butyl dimethyl silyl ether group (TBDMS) and triisopropyl silyl ether group (TIPS) are composed.
  • TMS trimethylsilyl ether group
  • TDPS tert-butyldiphenylsilyl group Ether group
  • TDMS tert-butyl dimethyl silyl ether group
  • TIPS triisopropyl silyl ether group
  • the protecting group is TBDMS.
  • R 14 and R 15 are -H.
  • R 16 is —NH 2 .
  • R 16 is: a halogen group, such as —F, —Cl, —Br, or —I.
  • R 16 has the following structure
  • the compound has the following structure:
  • a method for preparing a compound of formula (I-a) or formula (I-b) comprising: contacting a compound of formula (III) with a deprotecting agent,
  • R 31 and R 33 are each independently -O-protecting group; and R 32 is -H or -O-protecting group.
  • the deprotecting agent is selected from the group consisting of tetra-n-butylammonium fluoride (TBAF), tris(dimethylamino)sulfonium difluorotrimethylsilicate (TASF), hydrochloric acid (HCl), camphor sulfonic acid , Pyr ⁇ TsOH, Pyr ⁇ HF, BF 3 ⁇ OEt 2 , AcOH, LiBF 4 , Et 3 N ⁇ 3HF, Et 3 NBn + Cl - KF ⁇ 2H 2 O and any combination thereof.
  • the deprotection agent is TBAF.
  • the deprotection agent is Et 3 N ⁇ 3HF.
  • the contacting is performed in the presence of an organic solvent.
  • the organic solvent is selected from the group consisting of tetrahydrofuran (THF), methanol, ethanol, dichloromethane, dimethylformamide (DMF), acetonitrile, and any combination thereof.
  • the organic solvent is THF.
  • the method includes contacting a compound of formula (III-a) with guanidine hydrochloride to form a compound of formula (III),
  • X is a halogen group selected from the group consisting of -F, -Cl, -Br and -I; R 31 and R 33 are each independently -O-protecting group; and R 32 is -H or -O -Protecting group.
  • the contact is in dimethylformamide (DMF), acetonitrile, cryptand (e.g., triethylenediamine (DABCO)), crown ether (e.g., 15-crown-5, 18- Crown ether-6) or any combination thereof.
  • DMF and acetonitrile may have a ratio of about 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, or 1:5 Volume ratio.
  • the DMF and acetonitrile may have about 5:1 to 3:1, about 4:1 to 2:1, about 3:1 to 1:1, about 2:1 to 1:2, about 1:1 to 1:3, about 1:2 to 1:4, or about 1:3 to 1:5 in volume ratio.
  • X is -Cl.
  • the method includes contacting a compound of formula (III-b) with tetraethylammonium chloride (TEAC) to form a compound of formula (III-a),
  • TEAC tetraethylammonium chloride
  • X is -Cl
  • R 31 and R 33 are each independently -O-protecting group
  • R 32 is -H or -O-protecting group.
  • the contact is in a nitrite compound, such as amyl nitrite (e.g., isoamyl nitrite), n-butyl nitrite, tert-butyl nitrite, acetic acid, CH 2 Cl 2 , It is carried out in the presence of CCl 4 , NaHCO 3 or any combination thereof.
  • the method includes contacting cytidine or deoxycytidine with tert-butyldimethylchlorosilane to form a compound of formula (III-b).
  • the contacting is performed in the presence of a basic solvent, such as imidazole, CH 2 Cl 2 , pyridine, DMF, trimethylamine, DMSO, NaHCO 3 or any combination thereof.
  • the protecting group is selected from the group included in the following set, the set is composed of acetyl, benzoyl, benzyl, ⁇ -methoxyethoxymethyl ether, two Methoxytrityl [bis-(4-methoxyphenyl) phenylmethyl], methoxymethyl ether group, methoxytrityl [(4-methoxyphenyl) Diphenylmethyl], p-methoxybenzyl ether, methylthiomethyl ether, pivaloyl, tetrahydropyranyl, tetrahydrofuranyl, trityl (triphenylmethyl), Silyl ether group, methyl ether group, ethoxy ethyl ether group and any combination thereof.
  • the protecting group is a silyl ether group selected from the group included in the following set, the set is composed of trimethylsilyl ether group (TMS), tert-butyl diphenyl Silyl ether group (TBDPS), tert-butyldimethylsilyl ether group (TBDMS), triisopropylsilyl ether group (TIPS) and any combination thereof.
  • TMS trimethylsilyl ether group
  • TDPS tert-butyl diphenyl Silyl ether group
  • TDMS tert-butyldimethylsilyl ether group
  • TIPS triisopropylsilyl ether group
  • the protecting group is TBDMS.
  • Figure 1 shows a synthetic scheme for the preparation of modified cytidine (for example, 4-guanidinocytidine).
  • modified cytidine for example, 4-guanidinocytidine
  • Figure 2 shows a comparison experiment of the expression of several modified cytidines of the present invention and existing controls.
  • Figure 3 shows a comparison of cell expression experiments of the capped structure among several modified structures of the present invention.
  • Figure 4 shows the experimental results of cell expression in different modified forms of cytidine modified with different modification ratios of the present invention (specific structure of Invention 1).
  • Figure 5 shows the experimental results of cell expression in different modified forms of cytidine modified with different modification ratios of the present invention (specific structure of Invention 4).
  • Figure 6 shows the experimental results of cell expression in different modified forms of cytidine modified with different modification ratios of the present invention (specific structure of Invention 3).
  • Fig. 7 shows the experimental results of cell expression in different modified forms of cytidine modified with different modification ratios of the present invention (specific structure of Invention 3).
  • This article discloses compounds, modified nucleosides, modified nucleotides, modified nucleic acids and methods for their synthesis.
  • the disclosed may be a compound of formula (I):
  • R 1 , R 2 , R 4 and R 5 are each independently selected from the group included in the following group consisting of -H, -OH, -NH 2 , halogen group Group, substituted or unsubstituted C 1 -C 10 alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted C 1 -C 10 aralkyl, substituted or unsubstituted C 1 -C 10 cycloalkyl, substituted or unsubstituted C 1 -C 10 heterocyclic ring, substituted or unsubstituted acyl group, -OR 6 , -C(O)R 6 , -C(O)-OR 6 , -C(O)-NH-R 6 and -N(R 6 ) 2 composition;
  • R 3 is selected from groups included in the following set consisting of -H, -OH, -NH 2 , halogen groups, substituted or unsubstituted C 1 -C 10 alkyl groups, substituted or unsubstituted aromatic groups, Substituted or unsubstituted heteroaryl, substituted or unsubstituted C 1 -C 10 aralkyl, substituted or unsubstituted C 1 -C 10 cycloalkyl, substituted or unsubstituted C 1 -C 10 heterocyclic group , Substituted or unsubstituted acyl groups, -OR 6 , -C(O)R 6 , -C(O)-OR 6 , -C(O)-NH-R 6 , and -N(R 6 ) 2 , phosphoric acid And R 6 is H, a substituted or unsubstituted C 1 -C 10 alkyl group and a substituted or unsubsti
  • R 1 , R 2 and R 3 are -OH, and R 4 and R 5 are -H.
  • the compound may be a modified nucleoside, such as a modified cytidine (eg, 4-guanidinocytidine). As shown in Figure 1, modified cytidine can be prepared by the following synthetic scheme:
  • a compound of formula (IV) is also disclosed:
  • R 41 , R 42 and R 43 are each independently -H or -O-protecting group; and R 44 and R 45 are each independently selected from -H, substituted or unsubstituted C 1 -C 10 alkyl and substituted or unsubstituted acyl.
  • the compound of formula (IV) can be prepared by the following synthetic scheme of contacting cytidine or deoxycytidine with a protective agent, wherein R 41 and R 43 are each independently —O—protecting group, and R 42 is -H or -O-protecting group:
  • the protecting group is selected from the group included in the following set consisting of acetyl, benzoyl, benzyl, ⁇ -methoxyethoxymethyl ether, dimethoxy Trityl [bis-(4-methoxyphenyl) phenylmethyl], methoxymethyl ether group, methoxytrityl [(4-methoxyphenyl) diphenyl Methyl], p-methoxybenzyl ether, methylthiomethyl ether, pivaloyl, tetrahydropyranyl, tetrahydrofuranyl, trityl (triphenylmethyl), monosilane It is composed of base ether group, methyl ether group and ethoxy ethyl ether group.
  • the protecting group is a silyl ether group selected from the group consisting of trimethylsilyl ether group (TMS), tert-butyldiphenylsilyl group Ether group (TBDPS), tert-butyldimethylsilyl ether group (TBDMS), triisopropylsilyl ether group (TIPS) and any combination thereof.
  • TMS trimethylsilyl ether group
  • TDPS tert-butyldiphenylsilyl group Ether group
  • TDMS tert-butyldimethylsilyl ether group
  • TIPS triisopropylsilyl ether group
  • the protecting group is TBDMS.
  • the protective agent used to prepare the protecting group can be found in the Organic Synthesis Archive (https://www.synarchive.com/protecting-group).
  • the protective agent is tert-butyldimethylchlorosilane.
  • a compound of formula (IV-a) is also disclosed:
  • X is selected from a halogen group selected from the group consisting of -F, -Cl, -Br and -I; R 41 and R 43 are each independently -O-protecting group; R 42 is -H or -O-protecting group; and R 44 and R 45 are each independently selected from -H, substituted or unsubstituted C 1 -C 10 alkyl, and substituted or unsubstituted acyl.
  • the compound of formula (IV-a) can be prepared by the following synthetic scheme of contacting the compound of formula (IV) with a halide:
  • the halide includes a halogen salt, such as a fluoride salt, a chloride salt, a bromide salt, or an iodide salt.
  • the halide includes tetraethylammonium chloride (TEAC).
  • a compound of formula (IV-b) is also disclosed:
  • R 41 and R 43 are each independently —O—protecting group
  • R 42 is -H or -O-protecting group; and R 44 and R 45 are each independently selected from -H, substituted or unsubstituted C 1 -C 10 alkyl, and substituted or unsubstituted acyl.
  • the compound of formula (IV-b) can be prepared by the following synthetic scheme of contacting the compound of formula (IV-a) with a guanidine salt:
  • the guanidine salt is selected from the group consisting of agmatine sulfate, guanidine hydrochloride, aminoguanidine bicarbonate, tetramethylguanidine, guanidine thiocyanate, guanidine carbonate, 1,2,3-Triphenylguanidine, 1-methylguanidine hydrochloride, guanidine acetic acid, aminoguanidine sulfate, 1,3-diphenylguanidine, N-ethylguanidine hydrochloride, o-tolyl biguanide , Polyhexamethylene biguanide hydrochloride, N-(4-cyanophenyl)guanidine hydrochloride, N,N'-diphenylguanidine hydrobromide, di-o-toluidine, 4-hydroxyisoquine Guanidine, tetramethylammonium borohydride, N-acetylguanidine, 1-methyl-3-nitroguanidine, biguan
  • R 44 and R 45 are each independently selected from -H, substituted or unsubstituted C 1 -C 10 alkyl, and substituted or unsubstituted acyl.
  • the compound of formula (IV-c) or (IV-d) can be prepared by the following synthetic method of contacting a compound of formula (IV-b) with a deprotecting agent:
  • the deprotection agent is selected from the group consisting of tetra-n-tetra-n-butylammonium fluoride (TBAF), tris(dimethylamino)sulfonium difluorotrimethyl Silicate (TASF), hydrochloric acid (HCl), camphorsulfonic acid, Pyr ⁇ TsOH, Pyr ⁇ HF, BF 3 ⁇ OEt 2 , AcOH, LiBF 4 , Et 3 N ⁇ 3HF, Et 3 NBn + Cl - KF ⁇ 2H 2 O and any combination thereof.
  • the deprotection agent includes TBAF.
  • the deprotection agent includes Et 3 N ⁇ 3HF.
  • C 1 -C 10 alkyl (or interchangeably referred to as C 1-10 alkyl) is specifically used to separately disclose methyl, ethyl, C 3 alkyl, C 4 alkyl, C 5 alkyl, C 6 alkyl, C 7 alkyl, C 8 alkyl, C 9 alkyl and C 10 alkyl.
  • aromatic group means that the aromatic group may be substituted or unsubstituted, and the description includes substituted aromatic groups and unsubstituted aromatic groups.
  • substituted may refer to a group in which one or more hydrogen atoms are each independently substituted with the same or different substituents.
  • substituents include, but are not limited to, halogen groups, alkyl groups, aryl groups, aralkyl groups, cycloalkyl groups, or acyl groups.
  • the term "about” and its grammatical equivalents in relation to the reference values used herein and their grammatical equivalents can include a series of values, plus or minus 10% of the value, for example, the range of the value plus or minus 10 %, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%.
  • the amount of "about 10" includes the amount of 9-11.
  • the term "compound” in this application includes its solvates, esters and prodrugs.
  • the compounds disclosed herein may exist in different tautomeric forms, and all these forms are included within the scope of this application.
  • the compounds disclosed herein can contain one or more asymmetric centers, and therefore can produce enantiomers, diastereomers and other stereoisomeric forms. In terms of absolute stereochemistry, they can be defined as (R) or ( S). Unless otherwise stated, this application considers all stereoisomeric forms of the compounds disclosed herein.
  • this application is intended to include E and Z geometric isomers (e.g., cis or trans).
  • geometric isomer refers to the E or Z geometric isomer of the olefin double bond (for example, cis or trans).
  • positional isomers refers to structural isomers around the central ring, such as ortho-, meta-, and para-isomers around the benzene ring.
  • the compounds of the present application optionally contain unnatural proportions of atomic isotopes on one or more of the atoms constituting such compounds.
  • the compounds can be labeled with isotopes, such as deuterium ( 2 H), tritium ( 3 H), iodine-125 ( 125 I), or carbon-14 ( 14 C).
  • isotopes such as deuterium ( 2 H), tritium ( 3 H), iodine-125 ( 125 I), or carbon-14 ( 14 C).
  • 2 H, 11 C, 13 C, 14 C, 15 C, 12 N, 13 N, 15 N, 16 N, 16 O, 17 O, 14 F, 15 F, 16 F, 17 F, 18 F, 33 S Isotopic substitutions of, 34 S, 35 S, 36 S, 35 Cl, 37 Cl, 79 Br, 81 Br, and 125 I are all considered. All isotopic variants of the compounds of the present invention, whether radioactive or not, are included within the scope of the present invention.
  • the compounds disclosed herein have some or all of the 1 H atoms replaced by 2 H atoms.
  • the synthesis method of the deuterium-substituted heterocyclic derivative compound is known in the art, and only as a non-limiting example, includes the following synthesis method.
  • the structures described herein are intended to include compounds that differ only in the presence of one or more isotopically enriched atoms.
  • compounds with this structure are within the scope of this application except for replacing hydrogen with deuterium or tritium, or replacing carbon with 13 C- or 14 C-rich carbon.
  • solvate may include, but is not limited to, one or more solvates that retain the activity and/or properties of the compound, which is not undesirable.
  • solvates include, but are not limited to, compounds with water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, ethanolamine, or combinations thereof.
  • solvent may include, but is not limited to, non-polar, polar aprotic, polar protic solvents, and ionic liquids.
  • non-polar solvents include, but are not limited to, pentane, cyclopentane, hexane, cyclohexane, benzene, toluene, 1,4-dioxane, chloroform, diethyl ether, and dichloromethane (DCM).
  • polar aprotic solvents include but are not limited to tetrahydrofuran (THF), ethyl acetate, acetone, dimethylformamide (DMF), acetonitrile (MeCN), dimethyl sulfoxide (DMSO), nitromethane And propylene carbonate.
  • polar protic solvents include, but are not limited to, formic acid, n-butanol, isopropanol (IPA), n-propanol, ethanol, methanol, acetic acid, and water.
  • Illustrative examples of ionic liquids include, but are not limited to, 1-alkyl-3-methylimidazole cation, 1-alkylpyridine cation, N-methyl-N-alkylpyrrolidine cation, 1-butyl-3-methyl Imidazole tetrachloroferrate, 1-butyl-3-methylimidazole chloride and tetraalkylphosphonium iodide.
  • tautomer refers to a molecule in which a proton may be transferred from one atom of a molecule to another atom of the same molecule.
  • the compounds presented herein may exist as tautomers. Where tautomerization is possible, there will be a chemical equilibrium of tautomers. The exact ratio of tautomers depends on several factors, including physical state, temperature, solvent, and pH. Some examples of tautomeric equilibrium include:
  • esters refers to a compound that can be derived from an acid in which at least one -OH (hydroxy) group is replaced by an -O-alkyl (alkoxy) group.
  • prodrug means a compound that can be converted into a biologically active compound described herein under physiological conditions or by solvolysis. Therefore, the term “prodrug” refers to a precursor of a pharmaceutically acceptable biologically active compound. When administered to a subject, the prodrug may be inactive, but is converted to the active compound in the body, for example by hydrolysis.
  • Prodrug compounds generally provide advantages in solubility, tissue compatibility, or delayed release in mammalian organisms (see, for example, Bungard, H. (Bundgard, H.), Design of Prodrugs) (1985), pp. 7-9, 21-24 (Elsevier, Amsterdam).
  • protecting group refers to an atomic group that masks, reduces or prevents the reactivity of the functional group when it is attached to the reactive functional group in the molecule. Generally, the protecting group can be selectively removed as needed during the synthesis process. Examples of protecting groups can be found in: Woods (Wuts) and Greene (Greene), “Greene's Protective Groups in Organic Synthesis” 4th Edition ("Greene's Protective Groups in Organic Synthesis” 4th Ed.), Wei Wiley Interscience (2006), and Harrison, etc., Compendium of Synthetic Organic Methods, Volumes 1-8, 1971-1996, John Wiley & Sons, new York.
  • Functional groups that may have protecting groups include, but are not limited to, hydroxyl, amino, and carboxyl groups.
  • Representative amine protecting groups include, but are not limited to, formyl, acetyl (Ac), trifluoroacetyl, benzyl (Bn), benzoyl (Bz), carbamate, benzyloxycarbonyl ("CBZ "), p-methoxybenzylcarbonyl (Moz or MeOZ), tert-butoxycarbonyl (“Boc”), trimethylsilyl (“TMS”), 2-trimethylsilyl-ethanesulfonyl ( "SES”), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethoxycarbonyl (“FMOC”), nitro-veratryloxycarbonyl (“NVOC” ), p-methoxybenzyl (PMB), tosyl (Ts), etc.
  • salt is intended to include, but is not limited to, pharmaceutically acceptable salts.
  • pharmaceutically acceptable salt is intended to mean those salts that retain one or more of the biological activities and properties of free acids and bases and are not biologically or otherwise undesirable.
  • Illustrative examples of pharmaceutically acceptable salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate , Pyrophosphate, chloride, bromide, iodide, acetate, propionate, caprate, caprylate, acrylate, formate, isobutyrate, caproate, enanthate, Propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyl Alkyne 1,6-dioxate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, ortho Phthalate, sulfonate, xylene sulfonate, pheny
  • acid refers to a molecule or ion capable of providing hydrogen (proton or hydrogen ion H+), or capable of forming a covalent bond with an electron pair (for example, a Lewis acid). Acids may include, but are not limited to, inorganic acids, sulfonic acids, carboxylic acids, halogenated carboxylic acids, vinyl carboxylic acids, and nucleic acids.
  • inorganic acids include, but are not limited to, hydrogen halides and their solutions: hydrofluoric acid (HF), hydrochloric acid (HCl), hydrobromic acid (HBr), hydroiodic acid (HI); halogenated oxygen acids: hypochlorous Acid (HClO), chlorous acid (HClO2), chloric acid (HClO 3 ), perchloric acid (HClO 4 ), and corresponding bromine and iodine analogues, and hypofluoric acid (HFO), sulfuric acid (H 2 SO 4 ), fluorosulfuric acid (HSO 3 F), nitric acid (HNO 3 ), phosphoric acid (H 3 PO 4 ), fluoroantimonic acid (HSbF 6 ), fluoroboric acid (HBF 4 ), hexafluorophosphoric acid (HPF 6 ), chromic acid (H 2 CrO 4 ) and boric acid (H 3 BO 3 ).
  • HF hydrofluoric acid
  • HCl hydrochloric
  • sulfonic acids include but are not limited to methanesulfonic acid (or methanesulfonic acid, CH 3 SO 3 H), ethylsulfonic acid (or ethanesulfonic acid, CH 3 CH 2 SO 3 H), phenylsulfonic acid (Or benzenesulfonic acid, C 6 H 5 SO 3 H), p-toluenesulfonic acid (or toluenesulfonic acid, CH 3 C 6 H 4 SO 3 H), trifluoromethanesulfonic acid (or trifluoromethanesulfonic acid , CF 3 SO 3 H) and polystyrene sulfonic acid (sulfonated polystyrene, [CH 2 CH(C 6 H 4 )SO 3 H] n ).
  • methanesulfonic acid or methanesulfonic acid, CH 3 SO 3 H
  • ethylsulfonic acid or ethanesul
  • carboxylic acids include, but are not limited to, acetic acid (CH 3 COOH), citric acid (C 6 H 8 O 7 ), formic acid (HCOOH), gluconic acid (HOCH 2 -(CHOH) 4 -COOH), lactic acid (CH 3 -CHOH-COOH), oxalic acid (HOOC-COOH) and tartaric acid (HOOC-CHOH-CHOH-COOH).
  • halogenated carboxylic acids include, but are not limited to, fluoroacetic acid, trifluoroacetic acid, chloroacetic acid, dichloroacetic acid, and trichloroacetic acid.
  • vinyl carboxylic acids include, but are not limited to, ascorbic acid.
  • nucleic acids include, but are not limited to, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
  • bases refers to a molecule or ion capable of accepting protons from a proton donor and/or generating hydroxide ions (OH ⁇ ).
  • bases include, but are not limited to, aluminum hydroxide (Al(OH) 3 ), ammonium hydroxide (NH 4 OH), arsenic hydroxide (As(OH) 3 ), barium hydroxide (Ba(OH) 2 ) , Beryllium hydroxide (Be(OH) 2 ), bismuth (III) hydroxide (Bi(OH) 3 ), boron hydroxide (B(OH) 3 ), cadmium hydroxide (Cd(OH) 2 ), hydroxide Calcium (Ca(OH) 2 ), cerium (III) hydroxide (Ce(OH) 3 ), cesium hydroxide (CsOH), chromium hydroxide (II) (Cr(OH) 2 ), chromium hydroxide (III) (Cr(OH) 3
  • alkyl refers to a straight or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, without unsaturation, and having 1 to 15 carbon atoms (for example, C 1-15 alkyl).
  • the alkyl group includes one to thirteen carbon atoms (e.g., C 1-13 alkyl group).
  • the alkyl group includes one to ten carbon atoms (e.g., a C 1-10 alkyl group).
  • the alkyl group includes one to eight carbon atoms (e.g., C 1-8 alkyl group).
  • the alkyl group includes one to five carbon atoms (e.g., C 1-5 alkyl).
  • the alkyl group includes one to four carbon atoms (e.g., C 1-4 alkyl group). In other embodiments, the alkyl group includes one to three carbon atoms (e.g., C 1-3 alkyl). In other embodiments, the alkyl group including one or two carbon atoms (e.g., C 1-2 alkyl group). In other embodiments, the alkyl group comprises a carbon atom (e.g., C 1 alkyl group). In other embodiments, the alkyl group includes five to fifteen carbon atoms (e.g., C 5-15 alkyl group). In other embodiments, the alkyl group includes five to ten carbon atoms (e.g., a C5-10 alkyl group).
  • the alkyl group includes five to eight carbon atoms (e.g., a C5-8 alkyl group). In other embodiments, the alkyl group includes two to five carbon atoms (e.g., C 2-5 alkyl group). In other embodiments, the alkyl group includes three to five carbon atoms (e.g., C 3-5 alkyl group).
  • the alkyl group is selected from methyl, ethyl, 1-propyl (n-propyl), 1-methylethyl (isopropyl), 1-butyl (n-butyl), 1 -Methylpropyl (sec-butyl), 2-methylpropyl (isobutyl), 1,1-dimethylethyl (tert-butyl), 1-pentyl (n-pentyl).
  • the alkyl group is connected to the rest of the molecule through a single bond.
  • alkyl groups are optionally substituted by one or more of the following substituents: halogen groups, cyano groups, nitro groups, oxo, thio, imino, oxime, trimethylsilanyl, -OR a, -SR a, -OC (O) -R a, -N (R a) 2, -C (O) R a, -C (O) OR a, -C ( O)N(R a ) 2 , -N(R a )C(O)OR a , -OC(O)-N(R a ) 2 , -N(R a )C(O)R a , -N (R a )S(O) t R a (where t is 1 or 2), -S(O) t OR a (where t is 1 or 2), -S(O) t R a (where t is 1 or 2), -S(O)
  • aromatic group refers to a group derived from an aromatic monocyclic or polycyclic hydrocarbon ring system by removing hydrogen atoms from ring carbon atoms.
  • the aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and 5 to 18 carbon atoms, wherein at least one ring in the ring system is completely unsaturated, that is, according to Hückel theory, it contains A ring-shaped delocalized (4n+2) ⁇ -electronic system.
  • the ring system derived from the aromatic group includes, but is not limited to, groups such as benzene, fluorene, indene, indene, tetralin, and naphthalene.
  • aryl or the prefix “ar-” (such as “aralkyl”) is meant to include an aryl group optionally substituted with one or more substituents Free radicals, the substituents are independently selected from alkyl, alkenyl, alkynyl, halogen, fluoroalkyl, cyano, nitro, optionally substituted aryl, optionally substituted aralkyl Group, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally substituted carbocyclyl, optionally substituted carbocyclylalkyl, optionally substituted heterocyclyl, any Optionally substituted heterocyclylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -R b -OR a , -R b -OC(O)-R a ,- R b -OC(O)-OR a , -R b -OR a , -R b
  • alkenyl refers to a straight or branched hydrocarbon chain radical group consisting only of carbon and hydrogen atoms, containing at least one carbon-carbon double bond, and having two to twelve carbon atoms. In certain embodiments, alkenyl groups include two to eight carbon atoms. In other embodiments, alkenyl groups include two to four carbon atoms. The alkenyl group is connected to the rest of the molecule through a single bond, for example, ethenyl (ie vinyl), propyl-1-enyl (ie allyl), butyl-1-alkenyl , Pentyl-1-alkenyl, pentyl-1,4-dienyl, etc.
  • alkenyl groups are optionally substituted with one or more of the following substituents: halogen groups, cyano groups, nitro groups, oxo, thio, imino, oxime groups , Trimethylsilyl, -OR a , -SR a , -OC(O)-R a , -N(R a ) 2 , -C(O)R a , -C(O)OR a , -C (O)N(R a ) 2 , -N(R a )C(O)OR a , -OC(O)-N(R a ) 2 , -N(R a )C(O)R a ,- N(R a )S(O) t R a (where t is 1 or 2), -S(O) t OR a (where t is 1 or 2), -S(O) t R a
  • alkynyl refers to a straight or branched chain hydrocarbyl radical group consisting only of carbon and hydrogen atoms, containing at least one carbon-carbon triple bond, and having two to twelve carbon atoms.
  • alkynyl groups include two to eight carbon atoms. In other embodiments, the alkynyl group has two to four carbon atoms.
  • the alkynyl group is connected to the rest of the molecule through a single bond, for example, ethynyl, propynyl, butynyl, pentynyl, hexynyl and the like.
  • an alkynyl group is optionally substituted with one or more of the following substituents: halogen group, cyano, nitro, oxo, thio, imino, oxime , Trimethylsilyl, -OR a , -SR a , -OC(O)-R a , -N(R a ) 2 , -C(O)R a , -C(O)OR a , -C (O)N(R a ) 2 , -N(R a )C(O)OR a , -OC(O)-N(R a ) 2 , -N(R a )C(O)R a ,- N(R a )S(O) t R a (where t is 1 or 2), -S(O) t OR a (where t is 1 or 2), -S(O) t R a (where t is 1 or 2),
  • alkylene or "alkylene chain” refers to a straight or branched divalent hydrocarbon chain that connects the rest of the molecule to a free radical group, which consists only of carbon and hydrogen and contains no unsaturation And has one to twelve carbon atoms, such as methylene, ethylene, propylene, butylene, etc.
  • the alkylene chain is connected to the rest of the molecule through a single bond and to the radical group through a single bond.
  • the point of attachment of the alkylene chain to the rest of the molecule and to the radical group can be through one carbon in the alkylene chain or any two carbons in the chain.
  • the alkylene group includes one to eight carbon atoms (e.g., C 1-8 alkylene group).
  • the alkylene group includes one to five carbon atoms (e.g., C 1-5 alkylene group). In other embodiments, the alkylene group includes one to four carbon atoms (e.g., C 1-4 alkylene group). In other embodiments, the alkylene group includes one to three carbon atoms (e.g., C 1-3 alkylene group). In other embodiments, the alkylene group includes one to two carbon atoms (e.g., C 1-2 alkylene group). In other embodiments, the alkylene group includes one carbon atom (e.g., C 1 alkylene group). In other embodiments, the alkylene group includes five to eight carbon atoms (e.g., C 5-8 alkylene group).
  • the alkylene group includes two to five carbon atoms (e.g., C 2-5 alkylene group). In other embodiments, the alkylene group includes three to five carbon atoms (e.g., C 3-5 alkylene group).
  • the alkylene chain is optionally substituted by one or more of the following substituents: halogen group, cyano group, nitro group, oxo, thio, imino, oxime group , Trimethylsilyl, -OR a , -SR a , -OC(O)-R a , -N(R a ) 2 , -C(O)R a , -C(O)OR a , -C (O)N(R a ) 2 , -N(R a )C(O)OR a , -OC(O)-N(R a ) 2 , -N(R a )C(O)R a ,- N(R a )S(O) t R a (where t is 1 or 2), -S(O) t OR a (where t is 1 or 2), -S(O) t R a
  • aralkyl refers to a group of formula -R c -aryl, where R c is an alkylene chain as defined above, such as methylene, ethylene and the like.
  • R c is an alkylene chain as defined above, such as methylene, ethylene and the like.
  • the alkylene chain portion of the aralkyl group is optionally substituted as described above for the alkylene chain.
  • the aryl portion of the aralkyl group is optionally substituted as described for the aryl group above.
  • aralkenyl refers to a group of formula -R d -aryl, where Rd is an alkenylene chain as defined above.
  • Rd is an alkenylene chain as defined above.
  • the aryl portion of the aromatic alkenyl group is optionally substituted as described for the aryl group above.
  • the alkenylene chain portion of the aralkenyl group is optionally substituted as defined above for the alkenylene group.
  • arylalkynyl group refers to a formula -R e - aromatic group, where R e is an alkynylene chain as defined above.
  • R e is an alkynylene chain as defined above.
  • the aryl portion of the aralkynyl group is optionally substituted as described above for aryl groups.
  • the alkynylene chain portion of the arylalkynyl group is optionally substituted as defined above for the alkynylene chain.
  • carbocyclyl refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon group composed only of carbon and hydrogen atoms, which includes fused or bridged ring systems having three to fifteen carbon atoms.
  • carbocyclyl groups include three to ten carbon atoms.
  • the carbocyclic group includes five to seven carbon atoms.
  • the carbocyclic group is connected to the rest of the molecule by a single bond.
  • the carbocyclic group can be saturated (that is, containing only a single CC bond) or unsaturated (that is, containing one or more double or triple bonds).
  • a fully saturated carbocyclic group is also called a "cycloalkyl".
  • Examples of monocyclic cycloalkyl groups include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
  • the unsaturated carbocyclic group is also called "cycloalkenyl".
  • Examples of monocyclic cycloalkenyl groups include, for example, cyclopentenyl, cyclohexenyl, cycloheptenyl, and cyclooctenyl.
  • Polycyclic carbocyclic groups include, for example, adamantyl, norbornyl (ie, bicyclo[2.2.1]heptyl), norbornenyl, decahydronaphthyl, 7,7-dimethylbicyclo[2.2.1 ] Heptyl et al.
  • carbocyclic group is meant to include carbocyclic groups optionally substituted with one or more substituents independently selected from alkyl, alkenyl, alkyne Group, halogen group, fluoroalkyl, oxo, thio, cyano, nitro, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, any Optionally substituted arylalkynyl, optionally substituted carbocyclyl, optionally substituted carbocyclylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally Ground-substituted heteroaryl, optionally substituted heteroarylalkyl, -R b -OR a , -R b -OC(O)-R a , -R b -OC(O)-OR a ,- R b -OC(O)-N(
  • fluoroalkyl refers to an alkyl group as defined above, which is substituted with one or more fluorinated groups as defined above, for example, trifluoromethyl, difluoromethyl, fluoromethyl, 2,2 , 2-Trifluoroethyl, 1-fluoromethyl-2-fluoroethyl.
  • the alkyl portion of the fluoroalkyl group may be optionally substituted as defined above for the alkyl group.
  • halo refers to a bromine, chlorine, fluorine or iodine substituent.
  • heterocyclyl refers to a stable three to eighteen membered non-aromatic ring group that includes two to twelve carbon atoms and one to six heteroatoms selected from nitrogen, oxygen, and sulfur.
  • a heterocyclic group is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems.
  • the heteroatoms in the heterocyclic group can be optionally oxidized. If present, one or more nitrogen atoms are optionally quaternized.
  • the heterocyclic group is partially or fully saturated.
  • the heterocyclic group can be connected to the rest of the molecule through any atom of the ring.
  • heterocyclic groups include, but are not limited to: dioxolane, thienyl[1,3]dithiazyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl , Isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazole Alkyl, piperidinyl, piperazinyl, 4-piperidinyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuranyl, trithiazyl, tetrahydropyranyl, sulfur Morpholinyl, thiamorpholinyl, 1-oxoxo
  • heterocyclyl is meant to include a heterocyclyl group as defined above, which is optionally selected from alkyl, alkenyl, alkynyl, halogen, fluoroalkane Group, oxo, thio, cyano, nitro, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, any Optionally substituted carbocyclyl, optionally substituted carbocyclylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heteroaryl, optionally Ground substituted heteroarylalkyl, -R b -OR a , -R b -OC(O)-R a , -R b -OC(O)-OR a , -R b -OC(O)-N (R a ) 2
  • heterocyclylalkyl refers to a formula -R c - heterocyclic group, wherein R c is alkylene as defined above. If the heterocyclic group is a nitrogen-containing heterocyclic group, the heterocyclic group is optionally attached to the alkyl group on the nitrogen atom.
  • the alkylene chain of the heterocyclylalkyl group is optionally substituted as defined above for the alkylene chain.
  • the heterocyclyl portion of the heterocyclylalkyl group is optionally substituted as defined above for the heterocyclyl group.
  • heteroaryl refers to a group derived from a three to eighteen membered aromatic ring group, which contains two to seventeen carbon atoms and one to six heteroatoms selected from nitrogen, oxygen, and sulfur.
  • a heteroaryl group can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, wherein at least one ring in the ring system is fully unsaturated, that is, according to Hückel theory, it includes A ring-shaped delocalized (4n+2) ⁇ -electronic system.
  • Heteroaryl groups include fused or bridged ring systems. The heteroatoms in the heteroaryl group are optionally oxidized.
  • heteroaryl group is connected to the rest of the molecule through any atom of the ring.
  • heteroaryl groups include, but are not limited to, azepanyl, acridinyl, benzimidazolyl, benzindolyl, 1,3-benzodioxolyl, benzofuranyl, Benzo[d]thiazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepenyl, benzo[b][1,4]oxa Azinyl, 1,4-benzodioxanyl, benzonaphthalenefuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzo Pyranone, benzofuranyl, benzofuranone, benzothienyl (benzothiophenyl (benzothiophenyl)
  • heteroaryl is meant to include heteroaryl groups as defined above, which are optionally selected from alkyl, alkenyl, alkynyl, halogen, and fluoro Alkyl, haloalkenyl, haloalkynyl, oxo, thio, cyano, nitro, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl , Optionally substituted arylalkynyl, optionally substituted carbocyclyl, optionally substituted carbocyclylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, Optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -R b -OR a , -R b -OC(O)-R a , -R b -OC(O)
  • nucleoside is defined as a compound including five-carbon sugars (ribose or deoxyribose) or derivatives thereof, and organic bases, purines or pyrimidines, or derivatives thereof.
  • the nucleosides described herein may be modified nucleosides.
  • nucleotide is defined as a nucleoside plus at least one phosphate group.
  • the nucleotide may include a phosphate group, a diphosphate group or a triphosphate group.
  • the nucleotides described herein may be modified nucleotides.
  • nucleic acid includes any compound and/or substance that is or can be incorporated into an oligonucleotide chain.
  • exemplary nucleic acids used in accordance with the present application include but are not limited to DNA, RNA includes messenger mRNA (mRNA), its hybrids, RNAi inducers, RNAi agents, siRNA, shRNA, miRNA, antisense RNA, ribozymes, catalytic DNA, induction
  • mRNA messenger mRNA
  • RNAi inducers RNAi agents
  • siRNA siRNA
  • shRNA miRNA
  • antisense RNA ribozymes
  • ribonucleic acid refers to a chain of at least two base-glycosyl-phosphate groups.
  • the term includes compounds comprising nucleotides, where the sugar moiety is ribose.
  • the term includes RNA and RNA derivatives in which the backbone is modified.
  • nucleotide refers to the monomer unit of a nucleic acid polymer.
  • the RNA may be tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), antisense RNA, small inhibitory RNA (siRNA), microRNA ( miRNA) and ribozymes.
  • siRNA and miRNA has been described (Caudy A (Caudy A) et al., Genes & Devel 16:2491-96 and cited references).
  • these forms of RNA can be single-stranded, double-stranded, triple-stranded, or four-stranded.
  • the term also includes artificial nucleic acids, which may contain other types of backbones but have the same bases.
  • the artificial nucleic acid is PNA (Peptide Nucleic Acid).
  • PNA contains a peptide backbone and nucleotide bases, and can bind to DNA and RNA molecules in another embodiment.
  • the nucleotides are oxetane modified.
  • the nucleotide is modified by replacing one or more phosphodiester bonds with phosphorothioate bonds.
  • the modified nucleic acid comprises any other variants of the phosphate backbone of natural nucleic acids known in the art.
  • nucleic acids are well known to those skilled in the art, and are described in, for example, Molecular Cloning (2001), Sambrook and Russell editing, and enzymatic methods: molecular cloning methods for eukaryotic cells (Methods in Enzymology: Methods for molecular cloning ineukaryotic cells) (2003), described in Purchio and GCFareed.
  • Each nucleic acid derivative represents a separate embodiment of the invention.
  • derivative can be used interchangeably with the term “analog.”
  • Compound A can be a derivative or analog of compound B, if 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 atoms of compound A are replaced by another atom or functional group (for example, amino , Halogen group, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, or substituted or unsubstituted cycloalkyl) to form compound B.
  • the terms “derivative” and “analog” can also be used interchangeably with the term “modified”, for example, if compound A is a derivative of compound B, compound A is also a modified compound B.
  • subject refers to a mammal that has been or will be the object of treatment, observation or experiment.
  • mammal is intended to have its standard meaning, and includes, for example, humans, dogs, cats, sheep, and cows.
  • the methods described herein can be used in human therapy and veterinary applications.
  • the subject is a human.
  • terapéuticaally effective amount refers to an amount effective to provide a therapeutic benefit when administered to a human or non-human subject, such as improving symptoms, slowing disease progression, or preventing disease.
  • treating includes the administration of at least one compound disclosed in this application or a pharmaceutically acceptable salt thereof to a mammalian subject in need thereof, especially a human subject, and This includes (i) preventing the development of clinical symptoms of the disease (e.g. cancer), (ii) regressing the clinical symptoms of the disease (e.g. cancer) and/or (iii) preventive treatment to prevent the onset of the disease (e.g. cancer).
  • a mammalian subject in need thereof, especially a human subject
  • This includes (i) preventing the development of clinical symptoms of the disease (e.g. cancer), (ii) regressing the clinical symptoms of the disease (e.g. cancer) and/or (iii) preventive treatment to prevent the onset of the disease (e.g. cancer).
  • the modified nucleoside may include a compound having the following structure:
  • R 4 and R 5 are each independently selected from H, -OH, -NH 2 , halogen group, substituted or unsubstituted C 1 -C 10 alkyl, substituted or unsubstituted Aryl group, substituted or unsubstituted C 1 -C 10 aralkyl group, substituted or unsubstituted C 1 -C 10 cycloalkyl group, substituted or unsubstituted acyl group, -OR 6 , -C(O)R 6 , And -NR 6 ; and wherein R 6 is H, a substituted or unsubstituted C 1 -C 10 alkyl group, and a substituted or unsubstituted acyl group.
  • R 4 is H.
  • R 5 is H.
  • the modified nucleoside may be a modified cytidine, such as 4-guanidinocytidine.
  • the modified nucleoside may be a compound of formula (Ia).
  • Modified nucleosides can also include m 1 A (1-methyladenosine), m 2 A (2-methyladenosine), Am (2'-O-methyladenosine), ms 2 m 6 A (2 -Methylthio-N 6 -methyladenosine), I 6 A (N 6 -prenyladenosine), ms 2 i 6 A (2-methylthio-N 6 -prenyladenosine) ), io 6 A (N 6 -(cis-hydroxyisopentenyl) adenosine), ms 2 io 6 A (2-methylthio-N 6 -(cis-hydroxyisopentenyl) adenosine), g 6 A (N 6 -glycylcarbamoyl adenosine), t 6 A (N 6 -threonylcarbamoyl adenosine), ms 2 t 6 A (2-methylthio
  • modified nucleosides can be found in Modomics (http://modomics.genesilico.pl/). See also US Patent No. 8,278,036 or WO2011012316 for discussion of modified nucleosides and their incorporation into mRNA.
  • modified nucleosides for example, compounds of formula (I-a)
  • nucleotides for example, compounds of formula (I-e) or (I-g)
  • process conditions ie, reaction temperature, time, molar ratio of reactants, solvent, pressure, etc.
  • optimal reaction conditions may vary with the specific reactants or solvents used, but these conditions can be determined by those skilled in the art through routine optimization procedures.
  • modified nucleosides and nucleotides can involve the protection and deprotection of various chemical groups. Those skilled in the art can easily determine the need for protection and deprotection and the choice of appropriate protecting groups. For example, the chemical properties of protecting groups can be found in Greene et al., Protective Groups in Organic Synthesis, Second Edition, Wiley & Sons, 1991, and the entire contents This application is incorporated by reference.
  • the reaction of the method described herein can be carried out in a suitable solvent, which can be easily selected by those skilled in the art of organic synthesis.
  • a suitable solvent may not substantially react with the starting materials (reactants), intermediates or products at the temperature at which the reaction proceeds, that is, the temperature ranges from the freezing and solidification temperature of the solvent to the boiling temperature of the solvent.
  • a given reaction can be carried out in one solvent or a mixture of more than one solvent.
  • a suitable solvent can be selected for the specific reaction step.
  • the resolution of the racemic mixture of modified nucleosides and nucleotides can be performed by any of many methods known in the art.
  • An exemplary method includes fractional recrystallization using "chiral resolving acids," which are optically active salt-forming organic acids.
  • Suitable resolving agents for the fractional recrystallization method are, for example, optically active acids such as D and L forms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid or various optically active acids.
  • Camphor sulfonic acid The resolution of the racemic mixture can also be performed by elution on a column packed with an optically active resolving agent (for example, dinitrobenzoylphenylglycine).
  • the suitable elution solvent composition can be determined by those skilled in the art.
  • modified nucleosides and nucleotides can be prepared according to the scheme provided below:
  • modified nucleosides and nucleotides can be prepared according to the scheme provided below:
  • Modified nucleosides and nucleotides can also be prepared according to the synthetic methods described therein, Ogata et al., Journal of Organic Chemistry 74: 2585-2588, 2009; Purmal et al., Nucleic Acids Nucleic Acids Research 22(1): 72-78, 1994; Fukuhara et al., Biochemistry 1(4): 563-568, 1962; and Xu et al., Tetrahedron ) 48(9): 1729-1740, 1992, each of which is fully incorporated into this application by reference.
  • RNA RNA
  • synthesis method a modified nucleic acid, such as mRNA
  • the nucleic acid used according to the present application can be based on any existing technology, including but not limited to chemical synthesis, enzymatic synthesis, usually in vitro transcription of the end of a longer precursor, enzymatic or chemical cleavage, etc.
  • the method of synthesizing RNA is well-known in the art (see, for example, Gait, MJ (Gait, MJ) (Editor) Oligonucleotide synthesis: a practical approach (Oligonucleotide synthesis: a practical approach), Oxford [Oxfordshire] ], Washington, District of Columbia: IRL Press, 1984; and Herdewijn, P.
  • RNA polymerase reaction buffer e.g., nucleotides such as ribonucleotides
  • RNA polymerase reaction buffers generally include salts/buffering agents such as Tris, HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, sodium phosphate, sodium chloride, and magnesium chloride.
  • the pH of the reaction mixture can be about 6 to 8.5, 6.5 to 8.0, 7.0 to 7.5, and in some embodiments, the pH is 7.5.
  • the reaction mixture includes NTP in the concentration range of 1-10 mM, DNA template in the concentration range of 0.01-0.5 mg/ml, and RNA polymerase in the concentration range of 0.01-0.1 mg/ml.
  • the reaction mixture includes the concentration It is 5mM NTP, 0.1mg/ml DNA template and 0.05mg/ml RNA polymerization.
  • modified nucleic acids such as modified mRNA
  • modified mRNA may include one or more natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); modified nucleosides (e.g., 2-aminoadenosine, 2-thiothymidine, inosine , Pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine , C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-d
  • natural nucleosides e.g., adenosine, guanosine, cy
  • RNA molecules can include at least two nucleotides. Nucleotides can be naturally occurring nucleotides or modified nucleotides. In some embodiments, the RNA molecule includes about 5 nucleotides to about 5,000 nucleotides. In some embodiments, the RNA molecule includes at least about 5 nucleotides. In some embodiments, RNA molecules include up to about 5,000 nucleotides.
  • the RNA molecule includes about 5 nucleotides to about 20 nucleotides, about 5 nucleotides to about 40 nucleotides, about 5 nucleotides to about 60 nucleotides , About 5 nucleotides to about 80 nucleotides, about 5 nucleotides to about 100 nucleotides, about 5 nucleotides to about 200 nucleotides, about 5 nucleotides to About 500 nucleotides, about 5 nucleotides to about 1,000 nucleotides, about 5 nucleotides to about 2,000 nucleotides, about 5 nucleotides to about 5,000 nucleotides Acid, about 20 nucleotides to about 40 nucleotides, about 20 nucleotides to about 60 nucleotides, about 20 nucleotides to about 80 nucleotides, about 20 nucleotides To about 100 nucleotides, about 20 nucleotides to about 200 nucleotides,
  • the RNA molecule includes about 5 nucleotides, about 20 nucleotides, about 40 nucleotides, about 60 nucleotides, about 80 nucleotides, about 100 nucleotides. , About 200 nucleotides, about 500 nucleotides, about 1,000 nucleotides, about 2000 nucleotides, or about 5000 nucleotides.
  • RNA molecules may include at least one modified nucleotide as described in this application. In some embodiments, the RNA molecule includes about 1 modified nucleotide to about 100 modified nucleotides. In some embodiments, the RNA molecule includes at least about 1 modified nucleotide. In some embodiments, RNA molecules include up to about 100 modified nucleotides.
  • the RNA molecule includes about 1 modified nucleotide to about 2 modified nucleotides, about 1 modified nucleotide to about 3 modified nucleotides, and about 1 modified nucleotide to about 4 kinds of modified nucleotides, about 1 kind of modified nucleotides to about 5 kinds of modified nucleotides, about 1 kind of modified nucleotides to about 10 kinds of modified nucleotides, about 1 kind of modified nucleotides to about 20 kinds Modified nucleotides, about 1 modified nucleotides to about 100 modified nucleotides, about 2 modified nucleotides to about 3 modified nucleotides, about 2 modified nucleotides to about 4 modified nuclei Nucleotides, about 2 modified nucleotides to about 5 modified nucleotides, about 2 modified nucleotides to about 10 modified nucleotides, about 2 modified nucleotides to about 20 modified nucleotides , About 2 modified nucleotides to about 2 modified nu
  • the RNA molecule includes about 1 modified nucleotide, about 2 modified nucleotides, about 3 modified nucleotides, about 4 modified nucleotides, about 5 modified nucleotides, about 10 kinds of modified nucleotides, about 20 kinds of modified nucleotides, or about 100 kinds of modified nucleotides.
  • RNA molecules can include at least 0.1% modified nucleotides.
  • the fraction of modified nucleotides can be calculated as: number of modified nucleotides/total number of nucleotides*100%.
  • the RNA molecule includes about 0.1% modified nucleotides to about 100% modified nucleotides.
  • the RNA molecule includes at least about 0.1% modified nucleotides.
  • RNA molecules include up to about 100% modified nucleotides.
  • the RNA molecule includes about 0.1% modified nucleotides to about 0.2% modified nucleotides, about 0.1% modified nucleotides to about 0.5% modified nucleotides, about 0.1% modified nucleotides to about About 1% modified nucleotides, about 0.1% modified nucleotides to about 2% modified nucleotides, about 0.1% modified nucleotides to about 5% modified nucleotides, about 0.1% modified nucleotides to about 10 % Modified nucleotides, about 0.1% modified nucleotides to about 20% modified nucleotides, about 0.1% modified nucleotides to about 50% modified nucleotides, about 0.1% modified nucleotides to about 100% modified Nucleotides, about 0.2% modified nucleotides to about 0.5% modified nucleotides, about 0.2% modified nucleotides to about 1% modified nucleotides, about 0.2% modified nucleotides to about 2% modified nucleosides Acid, about 0.2%
  • the RNA molecule includes about 0.1% modified nucleotides, about 0.2% modified nucleotides, about 0.5% modified nucleotides, about 1% modified nucleotides, about 2% modified nucleotides, about 5% modified nucleotides, about 10% modified nucleotides, about 20% modified nucleotides, about 50% modified nucleotides, or about 100% modified nucleotides.
  • a compound of formula (I) or (I-a) replaces about 1 cytosine in the modified RNA with about 10,000 cytosines in the modified RNA. In some embodiments, a compound of formula (I) or (I-a) replaces at least about 1 cytosine in the modified RNA. In some embodiments, a compound of formula (I) or (I-a) replaces up to about 10,000 cytosines in the modified RNA. In some embodiments, a compound of formula (I) or (Ia) replaces about 1 cytosine in the modified RNA with about 2 cytosines in the modified RNA, and about 1 cytosine in the modified RNA.
  • Cytosine is replaced with about 1,000 cytosines in modified RNA
  • about 10 cytosines in modified RNA is replaced with about 5,000 cytosines in modified RNA
  • about 10 cytosines in modified RNA are replaced with about 10,000 in modified RNA
  • About 50 cytosines in the modified RNA are replaced with about 100 cytosines in the modified RNA
  • about 50 cytosines in the modified RNA are replaced with about 500 cytosines in the modified RNA
  • about 500 cytosines in the modified RNA are replaced.
  • cytosines in modified RNA about 50 cytosines were replaced with about 1,000 cytosines in modified RNA, about 50 cytosines in modified RNA were replaced with about 5,000 cytosines in modified RNA, and about 50 cytosines in modified RNA were replaced with modifications About 10,000 cytosines in RNA, about 100 cytosines in modified RNA are replaced with about 500 cytosines in modified RNA, and about 100 cytosines in modified RNA are replaced with about 1,000 cytosines in modified RNA Pyrimidine, approximately 100 cytosines in modified RNA are replaced with approximately 5,000 cytosines in modified RNA, and approximately Replace 100 cytosines with about 10,000 cytosines in modified RNA, replace about 500 cytosines in modified RNA with about 1,000 cytosines in modified RNA, and replace about 500 cytosines in modified RNA with modified RNA about 5,000 cytosines in the modified RNA.
  • About 5,000 cytosines in the modified RNA about 500 cytosines in the modified RNA are replaced
  • the compound of formula (I) or (Ia) replaces about 1 cytosine in modified RNA, about 2 cytosines in modified RNA, about 10 cytosines in modified RNA, and about 10 cytosines in modified RNA.
  • 50 cytosines about 100 cytosines in modified RNA, about 500 cytosines in modified RNA, about 1,000 cytosines in modified RNA, about 5,000 cytosines in modified RNA, or about 10,000 cytosines in modified RNA.
  • a compound of formula (I) or (I-a) replaces about 0.01% of the cytosine in the modified RNA with about 100% of the cytosine in the modified RNA. In some embodiments, a compound of formula (I) or (I-a) replaces at least about 0.01% of cytosine in the modified RNA. In some embodiments, a compound of formula (I) or (I-a) replaces up to about 100% of nucleosides in the modified RNA. In some embodiments, a compound of formula (I) or (Ia) replaces about 0.01% of cytosine in modified RNA with about 0.1% of cytosine in modified RNA, and replaces about 0.01% of cytosine in modified RNA.
  • Pyrimidine is replaced with about 0.5% of cytosine in modified RNA
  • about 0.01% of cytosine in modified RNA is replaced with about 1% of cytosine in modified RNA
  • about 0.01% of cytosine in modified RNA is replaced with modified RNA
  • about 5% of cytosine about 0.01% of cytosine in modified RNA is replaced with about 10% of cytosine in modified RNA
  • about 0.01% of cytosine in modified RNA is replaced with about 50% of cytosine in modified RNA
  • replace about 0.1% of cytosine in modified RNA with about 0.5% of cytosine in modified RNA replace about 0.5% of cytosine in modified RNA.
  • a compound of formula (I) or (Ia) replaces about 0.01% of cytosine in modified RNA, about 0.1% of cytosine in modified RNA, about 0.5% of cytosine in modified RNA, Modify about 1% of cytosine in RNA, modify about 5% of cytosine in RNA, modify about 10% of cytosine in RNA, modify about 50% of cytosine in RNA, or modify about 100% of cytosine in RNA .
  • the concentration of each nucleotide, such as ribonucleotides (for example, ATP, UTP, GTP, and CTP) in the reaction mixture may be about 0.1 mM to about 100 mM. In some embodiments, the concentration of each nucleotide is at least about 0.1 mM. In some embodiments, the concentration of each nucleotide is at most about 100 mM.
  • ribonucleotides for example, ATP, UTP, GTP, and CTP
  • the concentration of each nucleotide is about 0.1 mM to about 0.5 mM, about 0.1 mM to about 1 mM, about 0.1 mM to about 5 mM, about 0.1 mM to about 10 mM, about 0.1 mM to about 20 mM, About 0.1mM to about 50mM, about 0.1mM to about 75mM, about 0.1mM to about 100mM, about 0.5mM to about 1mM, about 0.5mM to about 5mM, about 0.5mM to about 10mM, about 0.5mM to about 20mM, about 0.5mM to about 50mM, about 0.5mM to about 75mM, about 0.5mM to about 100mM, about 1mM to about 5mM, about 1mM to about 10mM, about 1mM to about 20mM, about 1mM to about 50mM, about 1mM to about 75mM, About 1mM to about 100mM, about 5mM, about 1m
  • the concentration of each nucleotide is about 0.1 mM, about 0.5 mM, about 1 mM, about 5 mM, about 10 mM, about 20 mM, about 50 mM, about 75 mM, or about 100 mM.
  • the total concentration of nucleotides used in the reaction is between 0.5 mM and about 500 mM. In some embodiments, the total concentration of nucleotides is about 0.5 mM to about 500 mM. In some embodiments, the total concentration of nucleotides is at least about 0.5 mM. In some embodiments, the total concentration of nucleotides is at most about 500 mM.
  • nucleotides used in the reaction such as ribonucleotides (such as combined ATP, GTP, CTP, and UTP) is between 0.5 mM and about 500 mM. In some embodiments, the total concentration of nucleotides is about 0.5 mM to about 500 mM. In some embodiments, the total concentration of nucleotides is at least about 0.5 mM. In some embodiments, the total concentration of nucleotides is at most about 500 mM.
  • the total concentration of nucleotides is about 0.5mM to about 1mM, about 0.5mM to about 5mM, about 0.5mM to about 10mM, about 0.5mM to about 50mM, about 0.5mM to about 100mM, about 0.5 mM to about 200mM, about 0.5mM to about 300mM, about 0.5mM to about 500mM, about 1mM to about 5mM, about 1mM to about 10mM, about 1mM to about 50mM, about 1mM to about 100mM, about 1mM to about 200mM, about 1mM to about 300mM, about 1mM to about 500mM, about 5mM to about 10mM, about 5mM to about 50mM, about 5mM to about 100mM, about 5mM to about 200mM, about 5mM to about 300mM, about 5mM to about 500mM, about 10mM to About 50mM, about 10mM to about 100mM, about 5mM to
  • 5'caps and/or 3'tails can be added.
  • the presence of the cap can provide resistance to nucleases found in most eukaryotic cells.
  • the presence of “tails” can be used to protect mRNA from exonuclease degradation and/or to regulate protein expression levels.
  • RNA terminal phosphatase removes one terminal phosphate group from the 5'nucleotide, leaving two terminal phosphate groups; then guanosine triphosphate (GTP) It is added to the terminal phosphate to generate a 5'5'5 triphosphate bond; then the 7-nitrogen of guanine is methylated with a methyltransferase.
  • GTP guanosine triphosphate
  • Examples of cap structures include, but are not limited to, m7G(5')ppp(5'(A, G(5')ppp(5')A and G(5')ppp(5')G. More cap structures are available in The published U.S. application No.
  • the tail structure may include poly(A) and/or poly(C) tails.
  • the poly-A tail on the 3'end of the mRNA (for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides at the 3'end) may include at least 50%, 55% , 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% adenosine nucleotides.
  • the poly-A tail on the 3'end of the mRNA may include at least 50%, 55% , 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% cytosine nucleotides.
  • 5'caps and/or 3'tails can help to detect invalid transcripts produced during in vitro synthesis, because without capping and/or tailing, the size of those mRNA transcripts that are terminated prematurely It may be too small to be detected. Therefore, in some embodiments, 5'caps and/or 3'tails are added to the synthetic mRNA before testing for mRNA purity (eg, the level of invalid transcripts present in the mRNA). In some embodiments, 5'caps and/or 3'tails are added to the synthesized mRNA before purifying the mRNA as described in this application. In other embodiments, after purifying the mRNA as described in this application, 5'caps and/or 3'tails are added to the synthesized mRNA.
  • mRNA synthesized according to the present invention can be used without further purification.
  • mRNA synthesized according to the present invention can be used without a step of removing short polymers.
  • mRNA synthesized according to the present invention can be further purified.
  • various methods can be used to purify synthesized mRNA. For example, centrifugation, filtration, and/or chromatography can be used to purify mRNA.
  • the synthesized mRNA is purified by ethanol precipitation or filtration or chromatography, or gel purification, or any other suitable method.
  • mRNA is purified by HPLC.
  • mRNA is extracted in a standard phenol:chloroform:isoamyl alcohol solution, which is well known to those skilled in the art.
  • tangential flow filtration is used to purify mRNA.
  • Suitable purification methods include US 2016/0040154, US 2015/0376220, PCT application PCT/US18/19954 filed on February 27, 2018, titled “Method for Purifying Messenger RNA", and in February 2018 The method described in the PCT application PCT/US18/19978 entitled “Method for Purifying Messenger RNA” filed on the 27th, all of which are incorporated into this application by reference and can be used to implement the present invention.
  • the mRNA is purified before capping and tailing. In some embodiments, mRNA is purified after capping and tailing. In some embodiments, mRNA is purified both before and after capping and tailing. In some embodiments, the mRNA is purified by centrifugation before or after capping and tailing, or both. In some embodiments, the mRNA is purified by filtration before or after capping and tailing, or both. In some embodiments, mRNA is purified by tangential flow filtration (TFF) before or after capping and tailing, or both. In some embodiments, the mRNA is purified by chromatography before or after capping and tailing, or both.
  • TMF tangential flow filtration
  • any method available in the art can be used to detect and quantify full-length or null transcripts of mRNA.
  • the synthetic mRNA molecules are detected using blotting, capillary electrophoresis, chromatography, fluorescence, gel electrophoresis, HPLC, silver staining, spectroscopy, ultraviolet (UV) or UPLC or a combination thereof.
  • UV absorption spectroscopy is used to detect synthesized mRNA molecules by capillary electrophoresis separation.
  • prior to gel electrophoresis (“glyoxal gel electrophoresis"), mRNA is denatured with glyoxal dye.
  • the synthesized mRNA is characterized before capping or tailing.
  • synthetic mRNA is characterized after capping and tail sealing.
  • the mRNA produced by the methods disclosed herein includes less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% , Less than 1%, less than 0.5%, less than 0.1% of impurities other than full-length mRNA.
  • Impurities include IVT contaminants, such as proteins, enzymes, free nucleotides and/or short polymers.
  • the mRNA prepared according to the invention is substantially free of short polymers or null transcripts.
  • the mRNA prepared according to the present invention includes undetectable levels of short polymers or invalid transcripts.
  • the term "short polymer” or “null transcript” refers to any transcript that is less than full length.
  • the length of the "short polymer” or “null transcript” is less than 100 nucleotides, less than 90, less than 80, less than 70, less than 60, less than 50, less than 40, less than 30, less than 20, or length Less than 10 nucleotides.
  • short polymers are detected or quantified after the addition of 5'-caps and/or 3'-poly A tails.
  • compositions provided in the present application, including compounds, modified nucleosides, modified nucleotides, or modified nucleic acids.
  • the pharmaceutical composition of the present invention can be administered to a subject by any method known to those skilled in the art, such as parenteral, para-cancerous, transmucosal, transdermal, intramuscular, intravenous, intradermal, Subcutaneous, intraperitoneal, intraventricular, intracranial, intravaginal, or tumor.
  • the pharmaceutical composition can be administered by intravenous, intraarterial or intramuscular injection of liquid formulations.
  • suitable liquid preparations include solutions, suspensions, dispersions, emulsions, oils and the like.
  • the pharmaceutical composition is administered intravenously and therefore is formulated in a form suitable for intravenous administration.
  • the pharmaceutical composition is administered intraarterially and therefore is formulated in a form suitable for intraarterial administration.
  • the pharmaceutical composition is administered intramuscularly and therefore is formulated in a form suitable for intramuscular administration.
  • the pharmaceutical composition can be administered using vesicles, for example, liposomes (see Langer, Science 249:1527-1533 (1990); Treat et al., liposomes are effective in infectious diseases and Role in Cancer Treatment (Liposomes in the Therapy of Infectious Disease and Cancer), Lopez-Berestein and Fidler (Editors), Liss, New York, pages 353-365 (1989); Lopez-Berestein, ibid., pages 317-327; see ibid.).
  • liposomes see Langer, Science 249:1527-1533 (1990); Treat et al., liposomes are effective in infectious diseases and Role in Cancer Treatment (Liposomes in the Therapy of Infectious Disease and Cancer), Lopez-Berestein and Fidler (Editors), Liss, New York, pages 353-365 (1989); Lopez-Berestein, ibid., pages 317-327; see ibid.).
  • the pharmaceutical composition can be administered orally, and therefore can be formulated into a form suitable for oral administration, that is, a solid or liquid preparation.
  • Suitable solid oral preparations may include tablets, capsules, pills, granules, pellets and the like.
  • Suitable liquid oral preparations may include solutions, suspensions, dispersions, emulsions, oils and the like.
  • the pharmaceutical composition can be administered topically to the body surface and therefore can be formulated into a form suitable for topical administration.
  • Suitable topical preparations may include gels, ointments, creams, lotions, drops and the like.
  • the composition or a physiologically tolerable derivative thereof can be prepared and applied to a physiologically acceptable diluent as a solution, suspension or emulsion with or without a pharmaceutical carrier.
  • the pharmaceutical composition can be administered as a suppository, such as a rectal suppository or a urethral suppository.
  • the pharmaceutical composition is subcutaneously implanted with microparticles.
  • the pellets provide controlled release of the agent over a period of time.
  • the pharmaceutical composition may additionally include pharmaceutically acceptable excipients, as used in this application, including any and all solvents, dispersion media, diluents or other liquid carriers, dispersion or suspension aids, surfactants, isotonic agents, Thickeners or emulsifiers, preservatives, solid binders, lubricants, etc. are suitable for the specific dosage form required.
  • pharmaceutically acceptable excipients including any and all solvents, dispersion media, diluents or other liquid carriers, dispersion or suspension aids, surfactants, isotonic agents, Thickeners or emulsifiers, preservatives, solid binders, lubricants, etc. are suitable for the specific dosage form required.
  • the purity of the pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.
  • excipients are approved for human and veterinary use.
  • the excipient is approved by the United States Food and Drug Administration (United States Food and Drug Administration).
  • the excipient is pharmaceutical grade.
  • the excipient meets the standards of the United States Pharmacopoeia (USP), European Pharmacopoeia (EP), British Pharmacopoeia and/or International Pharmacopoeia.
  • the pharmaceutically acceptable carrier for liquid formulations may be an aqueous or non-aqueous solution, suspension, emulsion or oil.
  • non-aqueous solvents may be propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers can include water, alcohol/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • oils may be oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and cod liver oil.
  • Carriers for parenteral administration may include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution and fixed oil .
  • Intravenous vehicles include fluid and nutritional supplements, electrolyte supplements, such as Ringer's dextrose-based electrolyte supplements, and the like.
  • Examples may be sterile liquids, such as water and oil, with or without addition of surfactants, and other pharmaceutically acceptable adjuvants.
  • water, saline, aqueous glucose and related sugar solutions, and glycols such as propylene glycol or polyethylene glycol are the preferred liquid carriers, especially for injectable solutions.
  • oils may be oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and cod liver oil.
  • the pharmaceutical composition may further include a binder (e.g. gum arabic, corn starch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone) , Disintegrants (such as corn starch, potato starch, alginic acid, silicon dioxide, croscarmellose sodium, crospovidone, guar gum, sodium starch glycolate), various pH and ionic strength Buffer (e.g. Tris-HCl, acetate, phosphate), albumin or gelatin and other additives to prevent absorption on the surface, detergent (e.g.
  • a binder e.g. gum arabic, corn starch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone
  • Disintegrants such as corn starch, potato starch, alginic acid, silicon dioxide, croscar
  • Tween 20, Tween 80, pluronic F68, bile salt) Protease inhibitors, surfactants (such as sodium lauryl sulfate), penetration enhancers, solubilizers (such as glycerin, polyethylene glycol glycerin), antioxidants (such as ascorbic acid, sodium metabisulfite, butylated Hydroxyanisole), stabilizers (e.g. hydroxypropyl cellulose, hydroxypropyl methyl cellulose), thickeners (e.g. carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum), sweeteners Flavoring agents (e.g. aspartame, citric acid), preservatives (e.g.
  • lubricants e.g. stearic acid, magnesium stearate, polyethylene glycol, twelve Sodium alkyl sulfate
  • flow aids such as colloidal silicon dioxide
  • plasticizers such as diethyl phthalate, triethyl citrate
  • emulsifiers such as carbomer, hydroxypropyl cellulose) , Sodium lauryl sulfate
  • polymer coatings e.g. poloxamer or poloxamine
  • coatings and film formers e.g. ethyl cellulose, acrylate, polymethacrylate
  • Adjuvant e.g. ethyl cellulose, acrylate, polymethacrylate
  • the pharmaceutical composition provided in the application may be a controlled release composition, that is, a composition in which the compound is released within a period of time after administration.
  • Controlled release or sustained release compositions may include formulations in lipophilic depots (e.g. fatty acids, waxes, oils).
  • the pharmaceutical composition may be an immediate release composition, that is, a composition in which the entire compound is released immediately after administration.
  • Suitable devices for delivering the intradermal pharmaceutical compositions described in this application may include short needle devices, such as those described in U.S. Patent Nos. 4,886,499, 5,190,521, 5,328,483, 5,527,288, 4,270,537, 5,015,235, 5,141,496, and 5,417,662.
  • the intradermal composition can be applied through a device that limits the effective penetration length of the needle into the skin, such as those described in PCT Publication WO 99/34850 and their functional equivalents.
  • a jet injection device that delivers the liquid composition to the dermis through a liquid jet syringe and/or through a needle that pierces the stratum corneum and generates a jet that reaches the dermis may be suitable.
  • Jet injection equipment is described in, for example, U.S. Patent Nos. 5,480,381, 5,599,302, 5,334,144, 5,993,412, 5,649,912, 5,569,189, 5,704,911, 5,383,851, 5,893,397, 5,466,220, 5,339,163, 5,312,335, 5,503,639,790, PCT4 /37705 and WO 97/13537.
  • Ballistic powder/particle delivery devices that use compressed gas to accelerate vaccine in powder form through the outer layer of the skin to the dermis may be suitable.
  • conventional syringes can be used in the classic tuberculin intradermal method of intradermal administration.
  • the mRNA is encapsulated in a transfer carrier, such as a nanoparticle.
  • a transfer carrier such as a nanoparticle.
  • one purpose of such encapsulation is usually to protect nucleic acids from the environment that may contain enzymes or chemicals that may degrade nucleic acids and/or cause rapid excretion of nucleic acids or receptors. Therefore, in some embodiments, a suitable delivery vehicle can enhance the stability of the mRNA contained therein and/or facilitate the delivery of the mRNA to the target cell or tissue.
  • the nanoparticles may be lipid-based nanoparticles, for example including liposomes or polymer-based nanoparticles.
  • the nanoparticles may have a diameter of less than about 40-100 nm.
  • the nanoparticle may include at least 1 ⁇ g, 10 ⁇ g, 100 ⁇ g, 1 mg, 10 mg, 100 mg, 1 g or more mRNA.
  • the delivery vehicle is a liposomal vesicle, or other means to facilitate the transfer of nucleic acid to target cells and tissues.
  • Suitable transport carriers can include, but are not limited to, liposomes, nanoliposomes, ceramide-containing nanoliposomes, proteoliposomes, nanoparticles, phosphosilicate nanoparticles, calcium phosphate nanoparticles, silica Nanoparticles, nanocrystalline particles, semiconductor nanoparticles, poly(D-arginine), nanodendrimers, starch-based delivery systems, micelles, emulsions, liposomes, plasmids, viruses, calcium phosphate nucleotides , Aptamers, peptides and other carrier tags. It is also considered to use bio-nanocapsules and other viral capsid protein assemblies as suitable transfer vectors. (Human Gene Ther. (Hum. Gene Ther.) September 2008; 19(9): 887-95).
  • Liposomes may include one or more cationic lipids, one or more non-cationic lipids, one or more sterol-based lipids, and/or one or more PEG-modified lipids. Liposomes may include three or more different lipid components, one of which is a sterol-based cationic lipid.
  • the sterol-based cationic lipid is cholesteryl imidazole or "ICE" lipid (see WO2011/068810, which is incorporated herein by reference in its entirety).
  • sterol-based cationic lipids may constitute no more than 70% (eg, no more than 65% and 60%) of the total lipids in lipid nanoparticles (e.g., liposomes).
  • lipids may include, for example, phosphatidyl compounds (eg, phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides).
  • phosphatidyl compounds eg, phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides.
  • Non-limiting examples of cationic lipids may include C12-200, MC3, DLinDMA, DLinkC2DMA, cKK-E12, ICE (imidazolyl), HGT5000, HGT5001, OF-02, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DODMA and DMDMA, DODAC, DLenDMA, DMRIE, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA, and HGT4003 or a combination thereof.
  • Non-limiting examples of non-cationic lipids can include ceramide, cephalin, cerebroside, diacylglycerol, 1,2-dipalmitoyl-sn-glyceryl-3-glyceryl-3-phosphate sodium salt (DPPG), 1, 2-Distearoyl-sn-glyceryl-3-phosphoethanolamine (DSPE), 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC), 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleyl-sn-glycerol-3-phosphatidylcholine Alkali (DOPC), 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), and 1, 2-Dio
  • the PEG-modified lipid may be a poly(ethylene) glycol chain up to 5 kDa in length, which is covalently attached to a lipid having an alkyl chain of C6-C20 length.
  • PEG-modified lipids can include DMG-PEG, DMG-PEG2K, C8-PEG, DOGPEG, ceramide PEG, and DSPE-PEG, or combinations thereof.
  • polymers as transfer vehicles, either alone or in combination with other transfer vehicles.
  • Suitable polymers may include, for example, polyacrylate, polyalkylcyanoacrylate, polylactide, polylactide-polyglycolide copolymer, polycaprolactone, dextran, albumin, gelatin, Alginate, collagen, chitosan, cyclodextrin and polyethyleneimine.
  • the polymer-based nanoparticles may include polyethyleneimine (PEI), such as branched PEI.
  • cytosine nucleoside (2.43g, 10mmol), imidazole (6.13g, 90mmol), tert-butyldimethylchlorosilane and tert-triethylchlorosilane (9.04g, 6mmol) were mixed with a basic solvent ( 60 mL) were mixed together and stirred at room temperature for 2 days.
  • the reaction mixture was then poured into ice water (250 mL), and extracted and washed with dichloromethane (2 ⁇ 300 mL).
  • dichloromethane 2 ⁇ 300 mL
  • the organic phase was concentrated and dissolved in 50 mL of 80% acetic acid aqueous solution, reacted at room temperature for 30 minutes, and then extracted with dichloromethane (2 ⁇ 300 mL).
  • Example 2 1-((2R,3R,4R,5R)-3,4-bis((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl) (Yl)oxy)methyl)tetrahydrofuran-2-yl)-4-chloropyrimidin-2(1H)-one).
  • Et 4 NCl (3 g, 18 mmol) was mixed into a solution of compound 1 (2.34 g, 4 mmol) in dry dichloromethane (60 mL). Then the nitrous acid compound (40 mmol) was added dropwise to the reaction mixture. After the addition was completed, the reaction was continued at room temperature for 10 hours, transferred to a separatory funnel, saturated sodium bicarbonate solution and dichloromethane were added, and then extracted. The extracted dichloromethane phase was washed with concentrated brine, collected and dried over anhydrous sodium sulfate.
  • Example 3 2-(1-((2R,3R,4R,5R)-3,4-bis((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethyl Synthesis of ylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)guanidine.
  • Guanidine hydrochloride (3.3 g, 34 mmol) and 90% sodium hydride (0.91 g, 34 mmol) were added to the reaction tube. Then, 14 mL of anhydrous dimethylformamide (DMF) and acetonitrile were added and stirred at room temperature for 12 hours to activate the guanidine hydrochloride. The stirring was stopped, and the reaction mixture was allowed to stand for 1 hour. The supernatant was extracted with the long needle of a disposable syringe and injected into a Schlenk reaction tube containing compound 2 (1.04 g, 1.7 mmol) and cryptether or crown ether (1.7 mmol). The reaction tube was covered with a rubber stopper and nitrogen was added.
  • DMF dimethylformamide
  • the reaction was continued for 2 hours at 50°C and then for 12 hours at room temperature.
  • the reaction was terminated by adding 1 mL of acetic acid.
  • the reaction solution was concentrated, 40 mL of water was added, and extraction was performed with dichloromethane (3 ⁇ 50 mL).
  • the organic phase was concentrated, and then separated by column chromatography on silica gel (the elution solvent from 2% ethanol-ethyl acetate to 10% ethanol-ethyl acetate) to obtain pure compound 3 (0.15g, 14% yield), white solid.
  • the NMR spectrum was measured using a Bruker 400MHz NMR spectrometer.
  • the mass spectrum (ESI) was measured using a Thermo q-exactive mass spectrometer.
  • the reaction is carried out under the protection of N 2 . All reagents were purchased from Sigma-Aldrich and SCRC and can be used without further purification.
  • the reaction solvent is an anhydrous reagent.
  • the 4-guanidinocytidine-5'-triphosphate or 4-guanidinodeoxycytidine-5'-triphosphate disclosed herein can be synthesized by the following reaction:
  • TEA salt and sodium perchlorate (5.0g) were ion-exchanged twice in acetone (100.0mL) to obtain 4-guanidinocytidine-5'-triphosphate or 4-guanidinodeoxycytidine-5 '-Sodium salt of triphosphate.
  • Example 7 Experiments using modified luciferase to report mRNA expression in dendritic cells.
  • FLuc luciferase reporter
  • modified luciferase mRNA From the luciferase DNA sequence, it can be transcribed into mRNA in vitro using transcriptase and common reagent conditions. During the transcription process, it can be obtained according to modified C (cytidine) and no modified C. To obtain different ratios of modified mRNA, the modified mRNA may contain different ratios of modified U mRNA.
  • the sequence is synthesized in vitro by mRMA modified as follows to form a new modified luciferase. In the above sequence (SEQ NO:1), replace cytidine with the modified C* of the present invention.
  • modified C* is the expression of 5 kinds of modified single cytidine modified mRNA, and the modification ratio 100% (that is, all Cs are replaced with the following 5 different C* modifications, m 4 C (N4-methylcytidine), m 4 Cm (N 4 ,2'-O-dimethylcytidine) ,
  • the details are as follows in Table 1. ⁇
  • the replacement ratio can be 100%, of course, it can also be a different ratio, and the replacement method can also be a mixed replacement of different modified forms instead of a single replacement.
  • the modification of cytidine may be that the cytidine at certain positions is replaced by one or more of the specific compounds of 1, 2, 3 and 4 of the present invention. This method of producing modified mRNA is described in detail in the Chinese invention patent CN102947450B. Each method in the patent specification is a specific embodiment of the present invention.
  • DOPE was purchased from Avanti
  • mPEG2000-DSPE was purchased from cordenpharma
  • PBS was purchased from Invitrogen.
  • 1.2.:3 Preparation of phospholipid/mRNA: Use a BD syringe to draw 3mL mRNA and 3mL phospholipid solution (concentration of 12mg/mL) into the microfluidic chip (the microfluidic here should be a small package that can produce nanoparticles Equipment, please tell the company or manufacturer you purchased it), set the mixing number as: volume: 9.0mL; flow rate ratio: 3:1, total flow rate: 1mL/min, temperature: 37.0°C, starting dosage is 0.35mL, the final dosage is 0.10mL to obtain a phospholipid/mRNA solution, that is, a mixed solution of phospholipid-encapsulated mRNA particles and phospholipids is obtained.
  • Centrifugal ultrafiltration add the phospholipid/mRNA solution to the ultrafiltration tube for centrifugal ultrafiltration, the sample volume is 12mL, the volume of the ultrafiltration medium phosphate buffer is 12mL, the ultrafiltration doping number is set to: centrifugal force 3400g, The centrifugation time is 60min, the temperature is 4°C, and the number of cycles is 3 times. Thus, the wrapped mRNA vector for each treatment is obtained.
  • the package prevention in this specific example is the LPP method.
  • any other method can be used to package the mRNA, or the naked mRNA can be used to infect cells, tissues or any living tissues without packaging.
  • gene gun or transgenic methods can be used to transfer mRNA into cells to express the target protein.
  • the amount of expression can directly indicate the amount of mRNA expression.
  • 1.3.6 Take an equal volume of cell lysate (100 ⁇ l) into the centrifuge tube in step 5, mix quickly, and read the absorbance value on a luminometer. Note: The luminescence reaction will decay rapidly. The absorbance value must be read within 5 seconds after adding the cell lysate to the reaction solution.
  • the mRNA Poly(A) tail is not encoded by DNA, but the transcribed pre-mRNA uses ATP as the precursor, and is polymerized to the 3'end by the RNA terminal adenylate transferase, namely Ploy(A) polymerase. It is known that the function of the mRNA Poly(A) tail is: 1It may help the transfer of mRNA from the nucleus to the cytoplasm; 2Avoid ribozyme degradation in the cell and enhance the stability of mRNA; 3As a recognition signal for ribosomes. The structure of increasing Ploy(A) can also be realized in vitro.
  • mRNA molecules generally have regions of different sequences located before the translation start codon and after the untranslated translation stop codon. These regions (referred to as the 5'untranslated region (5'UTR) and 3'untranslated region (3'UTR), respectively) can affect mRNA stability, mRNA localization, and translation efficiency of the mRNA linked to them. It is known that certain 5'and 3'UTRs, such as the 5'and 3'UTRs of ⁇ and ⁇ globin, improve mRNA stability and mRNA expression. therefore.
  • mRNA encoding a reprogramming factor is displayed in the cell to 5'UTR and/or 3'UTR (e.g., iPSC inducer) that leads to higher mRNA stability and higher mRNA expression , Alpha globin or beta globin 5'UTR and/or 3'UTR; for example, Xenopus laevis or human alpha globin or beta globin 5'UTR and/or 3'UTR, or for example tobacco etch virus (TEV )5'UTR).
  • a reprogramming factor e.g., iPSC inducing factor
  • the core function (mRNA) has stronger stability and the superiority of other inventions, which can be achieved by the technology disclosed in the following patent applications.
  • the method described in the specification of Chinese invention patent CN102947450B is a part of the present invention.
  • Example 8 Effect on modified mRNA expression (dendritic cells) in the case of in vitro capping structure of mRNA (Ploy(A)).
  • Example 9 The effect of different modification ratios on mRNA expression.
  • the modified cytidine compound of the present invention replaces part of the cytidine in proportions of 0.5%, 5%, 10%, 20%, 30% , 40%, 50%, 70%, 80% and 90%
  • the specific replacement method is through the in vitro luciferase DNA under the action of transcriptase, the supply of raw materials with AUCG, and the conventional method for transcription.
  • the synthetic method is controlled and replaced, that is, part of the cytidine in the mRNA is replaced according to the above ratio.
  • Investigate the effect of different ratios of substitution on mRNA expression The detailed investigation refers to the method of Example 1, and the results are as follows.
  • the modified cytidine according to Invention 1 is substituted for the unmodified cytidine.
  • the expression of the target mRNA is higher.
  • the expression level of 80%-100% modification is significantly different from other modification ratios. It is indicated that if it is desired to use the cytidine structure of Invention 1 to modify mRNA, the modification ratio is greater than 80%.
  • the cytidine modification of the present invention can be used to replace cytidine in mRNA to significantly increase the expression level in vivo.
  • the specific time method of the present invention is experimentally verified against luciferase, it is understandable that for other mRNAs, such as mRNA for a certain line of cancer treatment, mRNA for infectious disease vaccines or therapeutic vaccines, or any other mRNA.
  • the cytidine modification of the present invention can find an appropriate ratio through reasonable experiments, and significantly increase the expression of target mRNA in vivo. This is easily understood by those skilled in the art, luciferase is an expressed reporter gene, and an increase in its expression level also indicates an increase in target mRNA.
  • luciferase is only a commonly used tool for verification.
  • it can be used for the modification of meaningful nucleic acids, such as messenger RNA, such as many cancer or tumor-related mRNA genes.
  • the modification of infectious disease mRNA or any other related mRNA also has effects and effects.
  • it also includes any plant, animal, bacterial, and algae-related mRNA modification. Modification of the mRNA by the modified cytidine compound of the present invention can significantly increase the expression and translation of the target mRNA in the cell.

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Abstract

一种修饰的胞苷化合物，即在胞苷的4位增加了胍基而产生新的衍生胞苷以及含有该修饰化合物的核酸，例如RNA。含有修饰胞苷的核酸，特别是mRNA可以显著提高mRNA在体内的表达量。

Description

修饰核苷及其合成方法
本发明主张PCT申请，申请号PCT/CN2019/075474，申请日：2019年2月19日的优先权，该PCT申请的全部内容作为本发明的一部分。
技术领域
本发明涉及核酸领域，具体涉及核酸的修饰以及合成方法。
背景技术
信使RNA(mRNA)在人类生物学中起着至关重要的作用。通过一个被称为转录的过程，mRNA控制人体内的蛋白质合成。mRNA药物可用于遗传疾病、癌症和传染病。
天然存在的RNA由四种基本核糖核苷酸ATP、CTP、UTP以及GTP合成，但可能包括转录后的修饰核苷酸。在RNA(Rozenski,J,Crain,P,以及McCloskey,J.(1999)。RNA修饰数据库：1999更新。核酸研究27：196-197)中已经鉴定出近100种不同的核苷修饰。然而，当掺入mRNA中时，许多修饰会在受体中引起抑制性免疫反应和/或限制蛋白质的产生，因此限制了mRNA药物的治疗效果。因此，本领域需要新的核苷、核苷酸和/或核酸(例如mRNA)修饰来解决这些问题。
发明内容
本文披露了化合物、修饰核苷、修饰核苷酸、修饰核酸及其合成方法。
在一个方面，披露了一种具有化学式(I)的化合物：
Figure PCTCN2020074825-appb-000001
其中,R 1、R 2、R 4和R 5各自独立地选自以下集合所包括的基团，该集合由–H、–OH、–NH 2、卤素基团、取代或未取代的C 1-C 10烷基、取代或未取代的芳香基、取代或未取代的杂芳基、取代或未取代的C 1-C 10芳烷基、取代或未取代的C 1-C 10环烷基、取代或未取代的C 1-C 10杂环、取代或未取代的酰基、–OR 6、–C(O)R 6、–C(O)-O-R 6、–C(O)-NH-R 6以及1–N(R 6) 2组成；
R 3选自以下集合所包括的基团一个或者几个，该集合由–H、–OH、–NH 2、卤素基团、取代或未取代的C 1-C 10烷基、取代或未取代的芳香基、取代或未取代的杂芳基、取代或未取代的C 1-C 10芳烷基、取代或未取代的C 1-C 10环烷基、取代或未取代的C 1-C 10杂环基、取代或未取代的酰基、–OR 6、–C(O)R 6、–C(O)-O-R 6、–C(O)-NH-R 6、和–N(R 6) 2、磷酸基、二磷酸基以及三磷酸基组成。其中，R 6为H、取代或未取代的C 1-C 10烷基以及取代或未取代的酰基。
在一些实施例中，R 1、R 2、R 4和R 5各自独立为–H、–OH或者取代或未取代的C 1-C 10烷基。在一些实施例中，R 3为–H、–OH、取代或未取代的C 1-C 10烷基、磷酸基、二磷酸基或者三磷酸基。在一些实施例中，R 1为–OH。在一些实施例中，R 2为–OH。在一些实施例中，R 2为–H。在一些实施例中，R 3为–OH。在一些实施例中，R 4为–H。在一些实施例R 5为–H。
在一些实施例中，该化合物是一种修饰核苷，其中R 1、R 2和R 3为–OH，并且R 4和R 5为–H。在一些实施例中，该化合物是一种修饰核糖核苷。例如，该化合物可以具有化学式(I-a)的结构：
Figure PCTCN2020074825-appb-000002
在一些实施例中，该化合物是一种修饰核苷，其中R 1和R 3为–OH，并且R 2、R 4和R 5为–H。在一些实施例中，该化合物是一种修饰脱氧核糖核苷。例如，该化合物可以具有化学式(I-b)的结构：
Figure PCTCN2020074825-appb-000003
在一些实施例中，该化合物是一种修饰核苷酸，其中R 2为–OH且R 3为磷酸基。例如，该化合物可以具有化学式(I-c)的结构：
Figure PCTCN2020074825-appb-000004
在一些实施例中，该化合物是一种修饰核苷酸，其中R 2为–H且R 3为磷酸基。例如，该化合物可以具有化学式(I-d)的结构：
Figure PCTCN2020074825-appb-000005
在一些实施例中，该化合物是一种修饰核苷酸，例如修饰核苷三磷酸(NTP)，其中R 2为–OH且R 3为三磷酸基。例如，该化合物可以具有化学式(I-e) 的结构：
Figure PCTCN2020074825-appb-000006
在一些实施例中，该化合物是一种修饰核苷酸，例如修饰脱氧核苷三磷酸(dNTP)，其中R 2为–H且R 3为三磷酸基。例如，该化合物可以具有化学式(I-f)的结构：
Figure PCTCN2020074825-appb-000007
在一些实施例中，修饰核苷三磷酸具有化学式(I-g)的结构：
Figure PCTCN2020074825-appb-000008
Figure PCTCN2020074825-appb-000009
其中Y +是阳离子。
在一些实施例中，修饰核苷三磷酸包括一种修饰胞苷三磷酸。在一些实施例中，Y +选自由Li +、Na +、K +、H +、NH 4 +以及四烷基铵离子组成的组。在一些实施例中，该四烷基铵选自由四乙基铵、四丙基铵以及四丁基铵组成的组。
在一些实施例中，修饰核苷酸包括一种修饰脱氧核苷三磷酸(dNTP)。在一些实施例中，该修饰脱氧核苷三磷酸具有化学式(I-h)的结构：
Figure PCTCN2020074825-appb-000010
其中Y +是阳离子。
在一些实施例中，修饰脱氧核苷三磷酸包括一种修饰脱氧胞苷三磷酸。在一些实施例中，Y +选自由Li +、Na +、K +、H +、NH 4 +以及四烷基铵离子组成的组。在一些实施例中，该四烷基铵选自由四乙基铵、四丙基铵以及四丁基铵组成的组。
在另一个方面，披露了一种包括两个或以上共价键连接的核苷酸的核酸，其中所述两个或以上共价键连接的核苷酸中的至少一个包括本申请披露的任何化合物、修饰核苷或者修饰核苷酸。在一些实施例中，核酸为一种核糖核酸(RNA)。在一些实施例中，该RNA为一种信使RNA(mRNA)。在一些实施例中，该RNA包括本申请披露的任何化合物、修饰核苷或者修饰核苷酸。在一些实施例中，核酸为一种脱氧核糖核酸(DNA)。在一些实施例中，该DNA包括本申请披露的任何化合物、修饰核苷或者修饰核苷酸。
在另一个方面，披露了一种药物组合物，包括本申请披露的任何化合物、修饰核苷或者修饰核苷酸，及其药学上可接受的赋形剂。在一些实施例中，药物组合物包括本申请披露的任何化合物或其药学上可接受的盐；及其药学上可接受的赋形剂。在一些实施例中，药物组合物包括本文披露的任何核酸或其药学上可接受的盐；及其药学上可接受的赋形剂。在一些实施例中，药物组合物包括本文披露的任何RNA或其药学上可接受的盐；及其药学上可接受的赋形剂。在一些实施例中，药物组合物包括本文披露的任何mRNA或其药学上可接受的盐；及其药学上可接受的赋形剂。
在另一个方面，披露了一种具有化学式(II)的化合物：
Figure PCTCN2020074825-appb-000011
或其药学上可接受的盐，其中：
R 11、R 12和R 13各自独立为–H、–OH或者–O–保护基团；
R 14和R 15各自独立选自–H、取代或未取代的C 1-C 10烷基以及取代或未取代的酰基；以及
R 16是选自–NH 2、卤素基团以及
Figure PCTCN2020074825-appb-000012
。
在一些实施例中，R 11、R 12和R 13为–O–保护基团。在一些实施例中，保护基团选自以下集合所包括的基团，该集合由乙酰基、苯甲酰基、苯甲基、β-甲氧基乙氧基甲基醚基、二甲氧基三苯甲基[双-(4-甲氧基苯基)苯基甲基]、甲氧基甲基醚基、甲氧基三苯甲基[(4-甲氧基苯基)二苯基甲基]、对-甲氧基苄基醚基、甲硫基甲基醚基、新戊酰基、四氢吡喃基、四氢呋喃基、三苯甲基(三苯基甲基)、甲硅烷基醚基、甲醚基以及乙氧基乙基醚基组成。在一些实施例中，保护基团是选自以下集合所包括的基团中的甲硅烷基醚基，该集合由三甲基硅烷基醚基(TMS)、叔丁基二苯基甲硅烷基醚基(TBDPS)、叔丁基二甲基甲硅烷基醚基(TBDMS)以及三异丙基甲硅烷基醚基(TIPS)组成。在一些实施例中，保护基团是TBDMS。
在一些实施例中，R 14和R 15为–H。在一些实施例中，R 16为–NH 2。在一些实施例中，R 16为：卤素基团，例如–F、–Cl、–Br或者–I。在一些实施例中，R 16为以下结构
Figure PCTCN2020074825-appb-000013
在一些实施例中，该化合物具有以下结构：
Figure PCTCN2020074825-appb-000014
在另一个方面，披露了一种制备具有化学式(I-a)或化学式(I-b)的化合物的方法，包括：使具有化学式(III)的化合物与脱保护剂接触，
Figure PCTCN2020074825-appb-000015
其中：R 31和R 33各自独立为–O–保护基团；以及R 32为–H或者–O–保护基团。
在一些实施例中，脱保护剂选自由四正丁基氟化铵(TBAF)、三(二甲基氨基)锍二氟三甲基硅酸盐(TASF)、盐酸(HCl)、樟脑磺酸，Pyr·TsOH、Pyr·HF、BF 3·OEt 2、AcOH、LiBF 4、Et 3N·3HF、Et 3NBn +Cl -KF·2H 2O及其任意组合组成的组。在一些实施例中，所述脱保护剂为TBAF。在一些实施例中，所述脱保护剂为Et 3N·3HF。在一些实施例中，所述接触是在有机溶剂存在下进行的。在一些实施例中，所述有机溶剂选自由四氢呋喃(THF)、甲醇、乙醇、 二氯甲烷、二甲基甲酰胺(DMF)、乙腈及其任意组合组成的组。在一些实施例中，所述有机溶剂为THF。
在一些实施例中，该方法包括使具有化学式(III-a)的化合物与盐酸胍接触，以形成具有化学式(III)的化合物，
Figure PCTCN2020074825-appb-000016
其中：X是选自由–F、–Cl、–Br以及–I组成的组的卤素基团；R 31和R 33各自独立地为–O–保护基团；以及R 32为–H或者–O–保护基团。在一些实施例中，所述接触是在二甲基甲酰胺(DMF)、乙腈、穴醚(例如，三乙二胺(DABCO))、冠醚(例如，15-冠醚-5，18-冠醚-6)或者其任意组合存在下进行的。在一些实施例中，所述DMF和乙腈可以具有约5:1、4:1、3:1、2:1、1:1、1:2、1:3、1:4或者1:5的体积比。在一些实施例中，所述DMF和乙腈可以具有约5:1至3:1、约4:1至2:1、约3:1至1:1、约2:1至1:2、约1:1至1:3、约1:2至1:4、或者约1:3至1:5的体积比。在一些实施例中，X是–Cl。
在一些实施例中，该方法包括使具有化学式(III-b)的化合物与四乙基氯化铵(TEAC)接触，以形成具有化学式(III-a)的化合物，
Figure PCTCN2020074825-appb-000017
其中：X是–Cl；R 31和R 33各自独立地为–O–保护基团；以及R 32为–H或者–O–保护基团。在一些实施例中，所述接触是在亚硝酸盐化合物，例如亚硝酸戊酯(例如，亚硝酸异戊酯)、亚硝酸正丁酯、亚硝酸叔丁酯、乙酸、CH 2Cl 2、CCl 4、NaHCO 3或其任意组合存在下进行的。
在一些实施例中，该方法包括使胞苷或脱氧胞苷与叔丁基二甲基氯硅烷接触，以形成具有化学式(III-b)的化合物。在一些实施例中，所述接触是在碱性溶剂，例如咪唑、CH 2Cl 2、吡啶、DMF、三甲胺、DMSO、NaHCO 3或其任意组合存在下进行的。在一些实施例中，所述保护基团选自以下集合所包括的基团，所述集合由乙酰基、苯甲酰基、苯甲基、β-甲氧基乙氧基甲基醚基、二甲氧基三苯甲基[双-(4-甲氧基苯基)苯基甲基]、甲氧基甲基醚基、甲氧基三苯甲基[(4-甲氧基苯基)二苯基甲基]、对-甲氧基苄基醚基、甲硫基甲基醚基、新戊酰基、四氢吡喃基、四氢呋喃基、三苯甲基(三苯基甲基)、甲硅烷基醚基、甲醚基、乙氧基乙基醚基及其任意组合组成。在一些实施例中，所述保护基团是选自以下集合所包括的基团中的甲硅烷基醚基，所述集合由三甲基硅烷基醚基(TMS)、叔丁基二苯基甲硅烷基醚基(TBDPS)、叔丁基二甲基甲硅烷基醚基(TBDMS)、三异丙基甲硅烷基醚基(TIPS)及其任意组合组成。在一些实施例中，所述保护基团是TBDMS。
在另一个方面，披露了一种制备具有化学式(I-a)的化合物的方法，包括
(a)使胞苷与叔丁基二甲基氯硅烷接触，以形成具有化学式(II-a)的化合物：
Figure PCTCN2020074825-appb-000018
(b)使具有化学式(II-a)的化合物与四乙基氯化铵(TEAC)接触，以形成具有化学式(II-b)的化合物
Figure PCTCN2020074825-appb-000019
(c)使具有化学式(II-b)的化合物与盐酸胍接触，以形成具有化学式(II-c)的化合物：
Figure PCTCN2020074825-appb-000020
以及(d)使具有化学式(II-c)的化合物与四正丁基氟化铵(TBAF)接触 ，以形成具有化学式(I-a)的化合物：
Figure PCTCN2020074825-appb-000021
附图说明
图1显示了用于制备修饰胞苷(例如，4-胍基胞苷)的合成方案。
图2显示了本发明的几种修饰的胞苷与现有对照的几种胞苷的表达的比较实验。
图3显示了本发明的几个修饰结构中加帽届结构的细胞表达实验比较。
图4显示了本发明的不同修饰形式的胞苷修饰不同修饰比例下细胞表达实验结果图(发明1的具体结构)。
图5显示了本发明的不同修饰形式的胞苷修饰不同修饰比例下细胞表达实验结果图(发明4的具体结构)。
图6显示了本发明的不同修饰形式的胞苷修饰不同修饰比例下细胞表达实验结果图(发明3的具体结构)。
图7显示了本发明的不同修饰形式的胞苷修饰不同修饰比例下细胞表达实验结果图(发明3的具体结构)。
本发明的新颖特征在所附的权利要求中进行了具体阐述。通过参考以下具体实施方式及其附图，将更好地理解本发明的特征和优点，其中具体实施方式详尽介绍了利用本发明原理的说明性实施例，所述附图包括：
具体实施方式
本文披露了化合物、修饰核苷、修饰核苷酸、修饰核酸及其合成方法。
所披露的可以是一种具有化学式(I)的化合物:
Figure PCTCN2020074825-appb-000022
或其药学上可接受的盐，其中：R 1、R 2、R 4和R 5各自独立地选自以下集合所包括的基团，该集合由–H、–OH、–NH 2、卤素基团、取代或未取代的C 1-C 10烷基、取代或未取代的芳香基、取代或未取代的杂芳基、取代或未取代的C 1-C 10芳烷基、取代或未取代的C 1-C 10环烷基、取代或未取代的C 1-C 10杂环、取代或未取代的酰基、–OR 6、–C(O)R 6、–C(O)-O-R 6、–C(O)-NH-R 6以及–N(R 6) 2组成；
R 3选自以下集合所包括的基团，该集合由–H、–OH、–NH 2、卤素基团、取代或未取代的C 1-C 10烷基、取代或未取代的芳香基、取代或未取代的杂芳基、取代或未取代的C 1-C 10芳烷基、取代或未取代的C 1-C 10环烷基、取代或未取代的C 1-C 10杂环基、取代或未取代的酰基、–OR 6、–C(O)R 6、–C(O)-O-R 6、–C(O)-NH-R 6、和–N(R 6) 2、磷酸基、二磷酸基以及三磷酸基组成；以及R 6为H、取代或未取代的C 1-C 10烷基以及取代或未取代的酰基。所述具有化学式(I)的化合物可以以不同的互变异构形式存在，并且所有这些形式都包括在本申请的范围内。
在一些实施例中，R 1、R 2和R 3为–OH，并且R 4和R 5为–H。所述化合物可以 是修饰核苷，例如修饰胞苷(例如，4-胍基胞苷)。如图1所示，修饰胞苷可以通过以下合成方案制备：
Figure PCTCN2020074825-appb-000023
还披露了一种具有化学式(IV)的化合物：
Figure PCTCN2020074825-appb-000024
或其药学上可接受的盐，其中：R 41、R 42和R 43各自独立为–H或–O–保护基 团；以及R 44和R 45各自独立选自–H、取代或未取代的C 1-C 10烷基以及取代或未取代的酰基。
在一些实施例中，具有化学式(IV)的化合物可以通过以下使胞苷或者脱氧胞苷与保护剂接触的合成方案来制备，其中R 41和R 43各自独立为–O–保护基团，并且R 42为–H或–O–保护基团：
Figure PCTCN2020074825-appb-000025
在一些实施例中，保护基团选自以下集合所包括的基团，该集合由乙酰·基、苯甲酰基、苯甲基、β-甲氧基乙氧基甲基醚基、二甲氧基三苯甲基[双-(4-甲氧基苯基)苯基甲基]、甲氧基甲基醚基、甲氧基三苯甲基[(4-甲氧基苯基)二苯基甲基]、对-甲氧基苄基醚基、甲硫基甲基醚基、新戊酰基、四氢吡喃基、四氢呋喃基、三苯甲基(三苯基甲基)、甲硅烷基醚基、甲醚基以及乙氧基乙基醚基组成。在一些实施例中，保护基团是选自以下集合所包括的基团中的甲硅烷基醚基，该集合由三甲基硅烷基醚基(TMS)、叔丁基二苯基甲硅烷基醚基(TBDPS)、叔丁基二甲基甲硅烷基醚基(TBDMS)以及三异丙基甲硅烷基醚基(TIPS)及其任意组合组成。在一些实施例中，所述保护基团是TBDMS。用于制备保护基团的保护剂可以在有机合成档案( https://www.synarchive.com/protecting-group)中找到。在一些实施例中，所述保护剂为叔丁基二甲基氯硅烷。
还披露了一种具有化学式(IV-a)的化合物：
Figure PCTCN2020074825-appb-000026
或其药学上可接受的盐，其中：X选自由–F、–Cl、–Br以及–I组成的集合的卤素基团；R 41和R 43各自独立地为–O–保护基团；R 42为–H或者–O–保护基团；以及R 44和R 45各自独立选自–H、取代或未取代的C 1-C 10烷基以及取代或未取代的酰基。
在一些实施例中，具有化学式(IV-a)的化合物可以通过以下使具有化学式(IV)的化合物与卤化物接触的合成方案来制备：
Figure PCTCN2020074825-appb-000027
在一些实施例中，卤化物包括卤素盐，例如氟化物盐，氯化物盐，溴化物盐或碘化物盐。在一些实施例中，卤化物包括四乙基氯化铵(TEAC)。
还披露了一种具有化学式(IV-b)的化合物：
Figure PCTCN2020074825-appb-000028
或其药学上可接受的盐，其中：R 41和R 43各自独立地为–O–保护基团；
R 42为–H或者–O–保护基团；以及R 44和R 45各自独立选自–H、取代或未取代的C 1-C 10烷基以及取代或未取代的酰基。
在一些实施例中，具有化学式(IV-b)的化合物可以通过以下使具有化学式(IV-a)的化合物与胍盐接触的合成方案来制备：
Figure PCTCN2020074825-appb-000029
在一些实施例中，胍盐选自选自以下集合所包括的基团，该集合由硫酸胍丁胺、盐酸胍、氨基胍碳酸氢盐、四甲基胍、硫氰酸胍、碳酸胍、1,2,3-三苯基胍、1-甲基胍盐酸盐、胍乙酸、氨基胍硫酸盐、1,3-二苯基胍、N-乙基胍盐酸盐、邻甲苯基双胍、聚六亚甲基双胍盐酸盐、N-(4-氰基苯基)胍盐酸盐、N，N'-二苯基胍氢溴酸盐、二邻甲苯胍、4-羟基异喹胍、四甲基硼氢化铵、N-乙酰基胍、1-甲基-3-硝基胍、双胍盐酸盐、甲基胍盐酸盐、异喹胍、十二烷基胍、1-苯基胍、4-胍基苄胺、1-邻甲苯基双胍、N-(3,4-二氯苯基)胍、N-[4-(二甲基氨基)苯基]胍及其任意组合组成。在一些实施例中，胍盐是盐酸胍。
还披露了一种具有化学式(IV-c)或化学式(IV-d)的化合物：
Figure PCTCN2020074825-appb-000030
或其药学上可接受的盐，其中：
R 44和R 45各自独立选自–H、取代或未取代的C 1-C 10烷基以及取代或未取代的酰基。
在一些实施例中，所述具有化学式(IV-c)或化学式(IV-d)的化合物可以通过以下使具有化学式(IV-b)的化合物与脱保护剂接触的合成方法来制备：
Figure PCTCN2020074825-appb-000031
在一些实施例中，脱保护剂选自以下集合所包括的基团，该集合由tetra-n-四正丁基氟化铵(TBAF)、三(二甲基氨基)锍二氟三甲基硅酸盐(TASF)、盐酸(HCl)、樟脑磺酸、Pyr·TsOH、Pyr·HF、BF 3·OEt 2、AcOH、LiBF 4、Et 3N·3HF、Et 3NBn +Cl -KF·2H 2O及其任意组合组成。在一些实施例中，脱保护剂包括TBAF。在一些实施例中，脱保护剂包括Et 3N·3HF。
定义
除非另有定义，这里使用的所有技术和科学术语与本发明所属领域的普通技术人员通常理解的含义相同。尽管与本文描述的那些类似或等同的任何方法和材料可用于本文的制剂或单位剂量的实践或测试，但现在描述一些方法和材料。除非另有说明，否则本文采用或考虑的技术是标准方法，材料、方法以及实施例仅是说明性的而非限制性。
如本文和所附权利要求中所使用的，单数形式“一(个)”、“和”、“ 以及”、“该”以及“所述”包括复数指示物，除非上下文另有明确说明。因此，例如，提及“化合物”包括多个这样的试剂，对“盐”的提及包括提及本领域普通技术人员所知的一种或以上盐(或多种盐)及其等同物，等等。
除非另有说明，本文的一些实施例考虑了数值范围。当提供数值范围时，除非另有说明，否则该范围包括范围端点。除非另有说明，否则数值范围包括其中的所有值和子范围，如同明确写出的那样。例如，术语“C 1-C 10烷基”(或可互换的称为C 1-10烷基)专门用于单独披露了甲基、乙基、C 3烷基、C 4烷基、C 5烷基、C 6烷基、C 7烷基、C 8烷基、C 9烷基和C 10烷基。
术语“任选的”或“任选地”是指随后描述的事件或情况可能发生或可能不发生，并且该描述包括事件或情况发生时的情况和不发生事件或情况的情况。例如，“任选地取代的芳香基”是指芳香基基团可以被取代或不被取代，并且该描述包括取代的芳香基团和没有取代的芳香基团。
术语“取代的”可以指基团，其中一个或以上氢原子各自独立地被相同或不同的取代基取代。典型的取代基包括但不限于卤素基团、烷基、芳香基、芳烷基、环烷基或酰基。
与本文所用的参考数值及其语法等价物相关的术语“约”及其语法等同物可以包含一系列值，加上或减去该值的10％，例如，该值的范围加上或减去10％、9％、8％、7％、6％、5％、4％、3％、2％或1％。例如，“约10”的量包括9至11的量。
术语“包括”(和诸如“包括”或“包含”或“具有”或“包括”的相关术语)并不旨在排除在其他某些实施例中，例如，本文所述的任何物质的组成、组合物、方法或过程等的实施例可以“由......组成”或“基本上由......组成”所描述的特征。
本申请的术语“化合物”包括其溶剂化物、酯和前药。本文披露的化合物可以以不同的互变异构形式存在，并且所有这些形式都包括在本申请的范围内。本文披露的化合物可以含有一个或以上不对称中心，因此可以产生对映异构体、非对映异构体和其他立体异构形式，就绝对立体化学而言，可以定义为( R)或(S)。除非另有说明，否则本申请考虑本文披露的化合物的所有立体异构形式。当本文所述的化合物含有烯烃双键时，除非另有说明，本申请意图包括E和Z几何异构体(例如顺式或反式)。同样地，所有可能的异构体，以及它们的外消旋和光学纯形式，以及所有互变异构形式也都应包括在内。术语“几何异构体”是指烯烃双键的E或Z几何异构体(例如，顺式或反式)。术语“位置异构体”是指中心环周围的结构异构体，例如苯环周围的邻-、间-和对-异构体。本申请的化合物任选地在构成这种化合物的一个或以上原子上含有非天然比例的原子同位素。例如，化合物可以用同位素标记，例如氘( 2H)、氚( 3H)、碘-125( 125I)或碳-14( 14C)。 2H、 11C、 13C、 14C、 15C、 12N、 13N、 15N、 16N、 16O、 17O、 14F、 15F、 16F、 17F、 18F、 33S、 34S、 35S、 36S、 35Cl、 37Cl、 79Br、 81Br、 125I的同位素取代都是被考虑的。本发明的化合物的所有同位素变体，无论是否具有放射性，都包括在本发明的范围内。在某些实施例中，本文披露的化合物具有部分或全部 1H原子被 2H原子取代。含氘取代的杂环衍生物化合物的合成方法是本领域已知的，并且仅作为非限制性实例，包括以下合成方法。除非另有说明，否则本文描述的结构旨在包括仅在一个或多个同位素富集的原子存在下不同的化合物。例如，具有该结构的化合物除了用氘或氚代替氢，或用富含 13C-或 14C-的碳代替碳以外，都在本申请的范围内。
术语“溶剂化物”可包括但不限于保留化合物的活性和/或性质的一种或以上的溶剂化物，这并非是不希望的。溶剂化物的实例包括但不限于与水、异丙醇、乙醇、甲醇、DMSO、乙酸乙酯、乙酸、乙醇胺或其组合的化合物。
术语“溶剂”可包括但不限于非极性、极性非质子、极性质子溶剂以及离子液体。非极性溶剂的说明性实例包括但不限于戊烷、环戊烷、己烷、环己烷、苯、甲苯、1,4-二恶烷、氯仿、乙醚和二氯甲烷(DCM)。极性非质子溶剂的说明性实例包括但不限于四氢呋喃(THF)、乙酸乙酯、丙酮、二甲基甲酰胺(DMF)、乙腈(MeCN)、二甲基亚砜(DMSO)、硝基甲烷以及碳酸亚丙酯。极性质子溶剂的说明性实例包括但不限于甲酸、正丁醇、异丙醇(IPA)、正丙醇、乙醇、甲醇、乙酸以及水。离子液体的说明性实例包括但不限于1-烷基-3-甲基咪唑阳离子、1-烷基吡啶阳离子、N-甲基-N-烷基吡咯烷阳离子、1-丁基-3-甲基咪唑四氯高铁酸盐、1-丁基-3-甲基咪唑氯化物和四烷基碘化鏻。
术语“互变异构体”是指一种分子，其中质子可能从一个分子的一个原子转移到同一分子的另一个原子。在某些实施例中，本文提出的化合物可以互变异构体形式存在。在可能发生互变异构化的情况下，将存在互变异构体的化学平衡。互变异构体的确切比例取决于几个因素，包括物理状态、温度、溶剂和pH。互变异构平衡的一些例子包括：
Figure PCTCN2020074825-appb-000032
术语“酯”是指可以衍生自酸的化合物，其中至少一个-OH(羟基)基团被-O-烷基(烷氧基)基团代替。
术语“前药”意指可以在生理条件下或通过溶剂分解转化为本文所述的生物活性化合物的化合物。因此，术语“前药”是指药学上可接受的生物活性化合物的前体。当施用于受试者时，前药可以是无活性的，但是在体内转化为活性化合物，例如通过水解。前药化合物通常在哺乳动物生物体中提供溶解性、组织相容性或延迟释放的优点(参见，例如，邦加德，H.(Bundgard，H.)，前药的设计(Design of Prodrugs)(1985)，pp.7-9,21-24(爱思唯尔，阿姆斯特丹(Elsevier，Amsterdam))。
术语“保护基团”是指当连接到分子中的反应性官能基团时，掩蔽、降低或防止功能基团的反应性的原子团。通常，可以在合成过程中根据需要选择性地除去保护基团。保护基团的实例可以在以下中找到：伍兹(Wuts)和格林(Greene )，“有机合成中的格林的保护基团”第4版("Greene's Protective Groups in Organic Synthesis"4th Ed.)，威利跨科学(Wiley Interscience)(2006)，以及哈里森(Harrison)等，合成有机方法纲要(Compendium of Synthetic Organic Methods)，第1-8卷，1971-1996，约翰威利父子(John Wiley&Sons)，纽约。可具有保护基团的功能基团包括但不限于羟基、氨基和羧基。代表性胺保护基团包括但不限于甲酰基、乙酰基(Ac)、三氟乙酰基、苯甲基(Bn)、苯甲酰基(Bz)、氨基甲酸酯、苄氧基羰基(“CBZ”)、对甲氧基苄基羰基(Moz或MeOZ)、叔丁氧基羰基(“Boc”)、三甲基硅烷基(“TMS”)、2-三甲基硅烷基-乙磺酰基(“SES”)、三苯甲基和取代的三苯甲基基团、烯丙氧基羰基、9-芴基甲氧基羰基(“FMOC”)、硝基-藜芦氧羰基(“NVOC”)、对-甲氧基苄基(PMB)、甲苯磺酰基(Ts)等。
术语“盐”旨在包括但不限于药学上可接受的盐。并且术语“药学上可接受的盐”旨在表示保留游离酸和碱的一种或以上生物活性和性质并且在生物学上或其它方面不是不合需要的那些盐。药学上可接受的盐的说明性实例包括但不限于硫酸盐、焦硫酸盐、硫酸氢盐、亚硫酸盐、亚硫酸氢盐、磷酸盐、磷酸一氢盐、磷酸二氢盐、偏磷酸盐、焦磷酸盐、氯化物、溴化物、碘化物、乙酸盐、丙酸盐、癸酸盐、辛酸盐、丙烯酸盐、甲酸盐、异丁酸酯、己酸盐、庚酸酯、丙炔酸酯、草酸盐、丙二酸盐、琥珀酸盐、辛二酸盐、癸二酸酯、富马酸酯、马来酸酯、丁炔-1,4-二酸盐、己炔1,6-二酸盐、苯甲酸酯、氯苯甲酸盐、甲基苯甲酸盐、二硝基苯甲酸盐、羟基苯甲酸酯、甲氧基苯甲酸盐、邻苯二甲酸盐、磺酸盐、二甲苯磺酸盐、乙酸苯酯、丙酸苯酯、丁酸苯酯、柠檬酸盐、乳酸盐、y-羟基丁酸酯、乙醇酸酯、酒石酸盐、甲磺酸盐、丙磺酸盐、萘-1-磺酸盐、萘-2-磺酸盐和扁桃酸盐。
术语“酸”是指能够提供氢(质子或氢离子H+)，或者，能够与电子对(例如路易斯酸)形成共价键的分子或离子。酸可包括但不限于无机酸、磺酸、羧酸、卤代羧酸、乙烯基羧酸以及核酸。无机酸的说明性实例包括但不限于卤化氢及其溶液：氢氟酸(HF)、盐酸(HCl)、氢溴酸(HBr)、氢碘酸(HI)；卤代含氧酸：次氯酸(HClO)、亚氯酸(HClO2)、氯酸(HClO 3)、高氯酸(HClO 4)、以及相应的溴和碘类似物、以及次氟酸(HFO)、硫酸(H 2SO 4 )、氟代硫酸(HSO 3F)、硝酸(HNO 3)、磷酸(H 3PO 4)、氟锑酸(HSbF 6)、氟硼酸(HBF 4)、六氟磷酸(HPF 6)、铬酸(H 2CrO 4)以及硼酸(H 3BO 3)。磺酸的说明性实例包括但不限于甲基磺酸(或甲磺酸，CH 3SO 3H)、乙基磺酸(或乙磺酸，CH 3CH 2SO 3H)、苯基磺酸(或苯磺酸，C 6H 5SO 3H)、对甲苯基磺酸(或甲苯磺酸，CH 3C 6H 4SO 3H)、三氟甲基磺酸(或三氟甲磺酸，CF 3SO 3H)以及聚苯乙烯基磺酸(磺化聚苯乙烯，[CH 2CH(C 6H 4)SO 3H] n)。羧酸的说明性实例包括但不限于乙酸(CH 3COOH)、柠檬酸(C 6H 8O 7)、甲酸(HCOOH)、葡萄糖酸(HOCH 2-(CHOH) 4-COOH)、乳酸(CH 3-CHOH-COOH)、草酸(HOOC-COOH)以及酒石酸(HOOC-CHOH-CHOH-COOH)。卤代羧酸的说明性实例包括但不限于氟乙酸、三氟乙酸、氯乙酸、二氯乙酸以及三氯乙酸。乙烯基羧酸的说明性实例包括但不限于抗坏血酸。核酸的说明性实例包括但不限于脱氧核糖核酸(DNA)和核糖核酸(RNA)。
术语“碱”是指能够从质子供体接受质子和/或产生氢氧根离子(OH -)的分子或离子。碱的说明性实例包括但不限于氢氧化铝(Al(OH) 3)、氢氧化铵(NH 4OH)、氢氧化砷(As(OH) 3)、氢氧化钡(Ba(OH) 2)、氢氧化铍(Be(OH) 2)、氢氧化铋(III)(Bi(OH) 3)、氢氧化硼(B(OH) 3)、氢氧化镉(Cd(OH) 2)、氢氧化钙(Ca(OH) 2)、氢氧化铈(III)(Ce(OH) 3)、氢氧化铯(CsOH)、氢氧化铬(II)(Cr(OH) 2)、氢氧化铬(III)(Cr(OH) 3)、氢氧化铬(V)(Cr(OH) 5)、氢氧化铬(VI)(Cr(OH) 6)、氢氧化钴(II)(Co(OH) 2)、氢氧化钴(III)(Co(OH) 3)、氢氧化铜(I)(CuOH)、氢氧化铜(II)(Cu(OH) 2)、氢氧化镓(II)(Ga(OH) 2)、氢氧化镓(III)(Ga(OH) 3)、氢氧化金(I)(AuOH)、氢氧化(III)金(Au(OH) 3)、氢氧化铟(I)(InOH)、氢氧化铟(II)(In(OH) 2)、氢氧化铟(III)(In(OH) 3)、氢氧化铱(III)(Ir(OH) 3)、氢氧化铁(II)(Fe(OH) 2)、氢氧化铁(III)(Fe(OH) 3)、氢氧化镧(La(OH))、氢氧化铅(II)(Pb(OH) 2)、氢氧化铅(IV)(Pb(OH) 4)、氢氧化锂(LiOH)、氢氧化镁(Mg(OH) 2)、氢氧化锰(II)(Mn(OH) 2)、氢氧化锰(III)(Mn(OH) 3)、氢氧化锰(IV)(Mn(OH) 4)、氢氧化锰(VII)(Mn(OH) 7)、氢氧化汞(I)(Hg 2(OH) 2)、氢氧化汞(II)(Hg(OH) 2)、氢氧化钼(Mo(OH) 3)、氢氧化钕(Nd(OH) 3)、氧代氢氧化镍(NiOOH)、氢氧 化镍(II)(Ni(OH) 2)、氢氧化镍(III)(Ni(OH) 3)、氢氧化铌(Nb(OH) 3)、氢氧化锇(IV)(Os(OH) 4)、氢氧化钯(II)(Pd(OH) 2)、氢氧化钯(IV)(Pd(OH) 4)、氢氧化铂(II)(Pt(OH) 2)、氢氧化铂(IV)(Pt(OH) 4)、氢氧化钚(IV)(Pu(OH) 4)、氢氧化钾(KOH)、氢氧化镭(Ra(OH) 2)、氢氧化铷(RbOH)、氢氧化钌(III)(Ru(OH) 3)、氢氧化钪(Sc(OH) 3)、氢氧化硅(Si(OH) 4)、氢氧化银(AgOH)、氢氧化钠(NaOH)、氢氧化锶(Sr(OH) 2)、氢氧化钽(V)(Ta(OH) 5)、氢氧化锝(II)(Tc(OH) 2)、四甲基氢氧化铵(C 4H 12NOH)、氢氧化铊(I)(TlOH)、氢氧化铊(III)(Tl(OH) 3)、氢氧化钍(Th(OH) 4)、氢氧化锡(II)(Sn(OH) 2)、氢氧化锡(IV)(Sn(OH) 4)、氢氧化钛(II)(Ti(OH) 2)、氢氧化钛(III)(Ti(OH) 3)、氢氧化钛(IV)(Ti(OH) 4)、氢氧化钨(II)(W(OH) 2)、氢氧化铀酰((UO 2) 2(OH) 4)、氢氧化钒(II)(V(OH) 2)、氢氧化钒(III)(V(OH) 3)、氢氧化钒(V)(V(OH) 5)、氢氧化镱(Yb(OH) 3)、氢氧化钇(Y(OH) 3)、氢氧化锌(Zn(OH) 2)以及氢氧化锆(Zr(OH) 4)。
术语“烷基”是指仅由碳和氢原子组成的直链或支链烃链基团，不含不饱和度，具有1至15个碳原子(例如，C 1-15烷基)。在某些实施例中，烷基包括一至十三个碳原子(例如，C 1-13烷基)。在某些实施例中，烷基包括一至十个碳原子(例如，C 1-10烷基)。在某些实施例中，烷基包括一至八个碳原子(例如，C 1-8烷基)。在其他实施例中，烷基包括一至五个碳原子(例如，C 1-5烷基)。在其他实施例中，烷基包括一至四个碳原子(例如，C 1-4烷基)。在其他实施例中，烷基包括一至三个碳原子(例如，C 1-3烷基)。在其他实施例中，烷基包括一至两个碳原子(例如，C 1-2烷基)。在其他实施例中，烷基包括一个碳原子(例如，C 1烷基)。在其他实施例中，烷基包括五至十五个碳原子(例如，C 5-15烷基)。在其他实施例中，烷基包括五至十个碳原子(例如，C 5-10烷基)。在其他实施例中，烷基包括五至八个碳原子(例如，C 5-8烷基)。在其他实施例中，烷基包括二至五个碳原子(例如，C 2-5烷基)。在其他实施例中，烷基包括三至五个碳原子(例如，C 3-5烷基)。在其他实施例中，该烷基选自甲基、乙基、1-丙基(正丙基)、1-甲基乙基(异丙基)、1-丁基(正丁基)、1-甲基丙基(仲丁基)、2-甲基丙基(异丁基)、1,1-二甲基乙基(叔丁 基)、1-戊基(正戊基)。烷基通过单键与分子的其余部分连接。除非在本说明书中另外特别说明，否则烷基基团任选地被一个或以上的下述取代基取代：卤素基团、氰基、硝基、氧代、硫代、亚氨基、肟基、三甲基硅烷基、-OR a、-SR a、-OC(O)-R a、-N(R a) 2、-C(O)R a、-C(O)OR a、-C(O)N(R a) 2、-N(R a)C(O)OR a、-OC(O)-N(R a) 2、-N(R a)C(O)R a、-N(R a)S(O) tR a(其中t为1或2)、-S(O) tOR a(其中t为1或2)、-S(O) tR a(其中t为1或2)以及-S(O) tN(R a) 2(其中t为1或2)，其中每个R a独立地为氢、烷基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、氟代烷基、碳环基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、碳环基烷基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、芳香基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、芳烷基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、杂环基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、杂环基烷基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、杂芳基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)，或杂芳基烷基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)。
术语“芳香基”是指通过从环碳原子上除去氢原子而衍生自芳族单环或多环烃环系的基团。芳香族单环或多环烃环系统仅含有氢和5至18个碳原子的碳，其中环系统中的至少一个环是完全不饱和的，即，根据休克尔理论(Hückel theory)，它包含一个环状离域(4n+2)π–电子系统。芳香基基团衍生的环系包括但不限于基团，如苯、芴、二氢化茚、茚、四氢化萘和萘。除非在说明书中另有特别说明，术语“芳香基”或前缀“芳(ar-)”(例如“芳烷基(aralkyl)”)意指包括任选地被一个或以上取代基取代的芳香基自由基，所述取代基独立地选自烷基、烯基、炔基、卤素基团、氟代烷基、氰基、硝基、任选地取代的芳香基、任选地取代的芳烷基、任选地取代的芳烯基、任选地取代的芳炔基、任选地取代的碳环基、任选地取代的碳环基烷基、任选地取代的杂环基、任选地取代的杂环基烷基、任选地取代的杂芳基、任选地取代的杂芳基烷基、-R b-OR a、-R b-OC(O)-R a、-R b-OC(O)-OR a、-R b-OC(O)-N(R a) 2、-R b-N(R a) 2、-R b-C(O)R a、-R b-C(O)OR a、-R b-C(O)N(R a) 2、-R b-O-R c-C(O)N(R a) 2、-R b-N(R a)C(O)OR a、-R b-N(R a)C(O)R a、-R b-N(R a)S(O) tR a(其中t为1或2)、 -R b-S(O) tOR a(其中t为1或2)以及-R b-S(O) tN(R a) 2(其中t为1或2)，其中每个R a独立地为氢、烷基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、氟烷基、环烷基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、环烷基烷基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、芳香基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、芳烷基(任选地被卤素、羟基、甲氧基或三氟甲基取代)、杂环基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、杂环基烷基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、杂芳基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)，或杂芳基烷基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)，每个R b独立地为直接键或直链或支链亚烷基或亚烯基链，以及R c是直链或支链亚烷基或亚烯基链，并且除非另有说明，否则上述取代基中的每一个是未取代的。
术语“烯基”是指直链或支链烃链自由基基团，其仅由碳和氢原子组成，含有至少一个碳-碳双键，并具有二至十二个碳原子。在某些实施例中，烯基包括二至八个碳原子。在其他实施例中，烯基包括二至四个碳原子。链烯基通过单键与分子的其余部分连接，例如，乙烯基(ethenyl)(即乙烯基(vinyl))、丙基-1-烯基(即烯丙基)、丁基-1-烯基、戊基-1-烯基、戊基-1,4-二烯基等。除非在本说明书中另有特别说明，否则烯基基团任选地被一个或以上的下述取代基取代：卤素基团、氰基、硝基、氧代、硫代、亚氨基、肟基、三甲基硅烷基、-OR a、-SR a、-OC(O)-R a、-N(R a) 2、-C(O)R a、-C(O)OR a、-C(O)N(R a) 2、-N(R a)C(O)OR a、-OC(O)-N(R a) 2、-N(R a)C(O)R a、-N(R a)S(O) tR a(其中t为1或2)、-S(O) tOR a(其中t为1或2)、-S(O) tR a(其中t为1或2)以及-S(O) tN(R a) 2(其中t为1或2)，其中每个R a独立地为氢、烷基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、氟烷基、碳环基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、碳环基烷基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、芳香基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、芳烷基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、杂环基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、杂环基烷基(任选被卤素基团、羟基、甲氧基或三氟甲基取代)、杂芳基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)，或杂芳基烷基(任选地被卤素基团、羟基、甲氧 基或三氟甲基取代)。
术语“炔基”是指直链或支链烃基自由基基团，其仅由碳和氢原子组成，含有至少一个碳-碳三键，具有二至十二个碳原子。在某些实施例中，炔基包括二至八个碳原子。在其他实施例中，炔基具有二至四个碳原子。炔基通过单键与分子的其余部分连接，例如，乙炔基、丙炔基、丁炔基、戊炔基、己炔基等。除非在本说明书中另有特别说明，否则炔基基团任选地被一个或以上的下述取代基取代：卤素基团、氰基、硝基、氧代、硫代、亚氨基、肟基、三甲基硅烷基、-OR a、-SR a、-OC(O)-R a、-N(R a) 2、-C(O)R a、-C(O)OR a、-C(O)N(R a) 2、-N(R a)C(O)OR a、-OC(O)-N(R a) 2、-N(R a)C(O)R a、-N(R a)S(O) tR a(其中t为1或2)、-S(O) tOR a(其中t为1或2)、-S(O) tR a(其中t为1或2)以及-S(O) tN(R a) 2(其中t为1或2)，其中每个R a独立地为氢、烷基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、氟代烷基、碳环基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、碳环基烷基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、芳香基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、芳烷基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、杂环基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、杂环基烷基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、杂芳基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)，或杂芳基烷基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)。
术语“亚烷基”或“亚烷基链”是指将分子的其余部分连接至自由基基团的直链或支链二价烃链，其仅由碳和氢组成，不含不饱和度且具有一至十二个碳原子，例如亚甲基、亚乙基、亚丙基、亚丁基等。亚烷基链通过单键与分子的其余部分连接，并通过单键与自由基基团连接。亚烷基链与分子的其余部分和与自由基基团的连接点可以通过亚烷基链中的一个碳或链内的任意两个碳。在某些实施例中，亚烷基包括一至八个碳原子(例如，C 1-8亚烷基)。在其他实施例中，亚烷基包括一至五个碳原子(例如，C 1-5亚烷基)。在其他实施例中，亚烷基包括一至四个碳原子(例如，C 1-4亚烷基)。在其他实施例中，亚烷基包括一至三个碳原子(例如，C 1-3亚烷基)。在其他实施例中，亚烷基包括一至两个碳原子(例如，C 1-2亚烷基)。在其他实施例中，亚烷基包括一个碳原子(例如 ，C 1亚烷基)。在其他实施例中，亚烷基包括五至八个碳原子(例如，C 5-8亚烷基)。在其他实施例中，亚烷基包括二至五个碳原子(例如，C 2-5亚烷基)。在其他实施例中，亚烷基包括三至五个碳原子(例如，C 3-5亚烷基)。除非在本说明书中另有特别说明，否则亚烷基链任选地被一个或以上的下述取代基取代：卤素基团、氰基、硝基、氧代、硫代、亚氨基、肟基、三甲基硅烷基、-OR a、-SR a、-OC(O)-R a、-N(R a) 2、-C(O)R a、-C(O)OR a、-C(O)N(R a) 2、-N(R a)C(O)OR a、-OC(O)-N(R a) 2、-N(R a)C(O)R a、-N(R a)S(O) tR a(其中t为1或2)、-S(O) tOR a(其中t为1或2)、-S(O) tR a(其中t为1或2)以及-S(O) tN(R a) 2(其中t为1或2)，其中每个R a独立地为氢、烷基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、氟代烷基、碳环基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、碳环基烷基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、芳香基(任选被卤素基团、羟基、甲氧基或三氟甲基取代)、芳烷基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、杂环基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、杂环基烷基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、杂芳基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)，或杂芳基烷基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)。
术语“芳烷基”是指式–R c–芳香基的基团，其中R c是如上定义的亚烷基链，例如亚甲基、亚乙基等。芳烷基的亚烷基链部分如上文对亚烷基链所述任选地被取代。芳烷基的芳香基部分如上述芳香基基团所述任选地被取代。
术语“芳烯基”是指式–R d–芳香基的基团，其中R d是如上定义的亚烯基链。芳香烯基的芳香基部分任选地如上述芳香基基团所述被取代。芳烯基的亚烯基链部分任选地如上文对亚烯基基团所定义的那样被取代。
术语“芳炔基”是指式–R e–芳香基的基团，其中R e是如上定义的亚炔基链。芳炔基的芳香基部分任选地如上文对芳香基基团所述被取代。芳炔基的亚炔基链部分任选地如上文对亚炔基链所定义的那样被取代。
术语“碳环基”是指仅由碳和氢原子组成的稳定的非芳族单环或多环烃基，其包括具有三至十五个碳原子的稠合或桥环系统。在某些实施例中，碳环基包 括三至十个碳原子。在其他实施例中，碳环基包括五至七个碳原子。碳环基通过单键与分子的其余部分连接。碳环基可以是饱和的(即仅含有单个C-C键)或不饱和的(即含有一个或以上双键或三键)。完全饱和的碳环基也称为“环烷基”。单环环烷基的实例包括例如环丙基、环丁基、环戊基、环己基、环庚基以及环辛基。不饱和碳环基也称为“环烯基”。单环环烯基的实例包括例如环戊烯基、环己烯基、环庚烯基和环辛烯基。多环碳环基包括，例如，金刚烷基、降冰片基(即双环[2.2.1]庚基)、降冰片烯基、十氢萘基、7,7-二甲基双环[2.2.1]庚基等。除非在本说明书中另有特别说明，否则术语“碳环基”意指包括任选地被一个或以上取代基取代的碳环基，所述取代基独立地选自烷基、烯基、炔基、卤素基团、氟代烷基、氧代、硫代、氰基、硝基、任选地取代的芳香基、任选地取代的芳烷基、任选地取代的芳烯基、任选地取代的芳炔基、任选地取代的碳环基、任选地取代的碳环基烷基、任选地取代的杂环基、任选地取代的杂环基烷基、任选地取代的杂芳基、任选地取代的杂芳基烷基、-R b-OR a、-R b-OC(O)-R a、-R b-OC(O)-OR a、-R b-OC(O)-N(R a) 2、-R b-N(R a) 2、-R b-C(O)R a、-R b-C(O)OR a、-R b-C(O)N(R a) 2、-R b-O-R c-C(O)N(R a) 2、-R b-N(R a)C(O)OR a、-R b-N(R a)C(O)R a、-R b-N(R a)S(O) tR a(其中t为1或2)、-R b-S(O) tR a(其中t为1或2)、-R b-S(O) tOR a(其中t为1或2)以及-R b-S(O) tN(R a) 2(其中t为1或2)，其中每个R a独立地为氢、烷基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、氟代烷基、环烷基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、环烷基烷基(任选地被卤素基团取代、羟基、甲氧基或三氟甲基)、芳香基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、芳烷基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、杂环基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、杂环基烷基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、杂芳基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)，或杂芳基烷基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)，每个R b独立为直接键或直链或支链亚烷基或亚烯基链，R c是直链或支链亚烷基或亚烯基链，并且除非另有说明，否则上述取代基中的每一个是未取代。
术语“氟代烷基”是指如上定义的烷基基团，其被如上定义的一个或以 上氟代基团取代，例如，三氟甲基、二氟甲基、氟甲基、2,2,2-三氟乙基、1-氟甲基-2-氟乙基。氟烷基的烷基部分可以如上文对于烷基基团所定义的那样任选地被取代。
术语“卤代”、“卤素”或“卤素基团”是指溴、氯、氟或碘取代基。
术语“杂环基”是指稳定的三至十八元非芳族环基，其包括二至十二个碳原子和一至六个选自氮、氧和硫的杂原子。除非在本说明书中另有特别说明，否则杂环基团是单环、双环、三环或四环环系，其可包括稠合或桥环系统。杂环基团中的杂原子可任选地被氧化。如果存在，一个或以上氮原子任选地被季铵化。杂环基团是部分或完全饱和的。杂环基可以通过环的任何原子与分子的其余部分连接。这种杂环基团的实例包括但不限于：二氧戊环基、噻吩基[1,3]二噻烷基、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-氧代哌嗪基、2-氧代哌啶基、2-氧代吡咯烷基、恶唑烷基、哌啶基、哌嗪基、4-哌啶基、吡咯烷基、吡唑烷基、奎宁环基、噻唑烷基、四氢呋喃基、三噻烷基、四氢吡喃基、硫代吗啉基、噻吗啉基、1-氧代硫代吗啉基以及1,1-二氧代硫代吗啉基。除非在说明书中另有特别说明，术语“杂环基”意指包括如上定义的杂环基基团，其任选地被选自烷基、烯基、炔基、卤素基团、氟代烷基、氧代、硫代、氰基、硝基、任选地取代的芳香基、任选地取代的芳烷基、任选地取代的芳烯基、任选地取代的芳炔基、任选地取代的碳环基、任选地取代的碳环基烷基、任选地取代的杂环基、任选地取代的杂环基烷基、任选地取代的杂芳基、任选地取代的杂芳基烷基、-R b-OR a、-R b-OC(O)-R a、-R b-OC(O)-OR a、-R b-OC(O)-N(R a) 2、-R b-N(R a) 2、-R b-C(O)R a、-R b-C(O)OR a、-R b-C(O)N(R a) 2、-R b-O-R c-C(O)N(R a) 2、-R b-N(R a)C(O)OR a、-R b-N(R a)C(O)R a、-R b-N(R a)S(O) tR a(其中t为1或2)、-R b-S(O) tR a(其中t为1或2)、-R b-S(O) tOR a(其中t为1或2)以及-R b-S(O) tN(R a) 2(其中t为1或2)的一个或以上取代基取代，其中每个R a独立地为氢、烷基(任选被卤素基团、羟基、甲氧基或三氟甲基取代)、氟代烷基、环烷基(任选被卤素基团、羟基、甲氧基或三氟甲基取代)、环烷基烷基(任选被卤素基团、羟基、甲氧基或三氟甲基取代)、芳香基(任选被卤素基团、羟基、甲氧基或三氟甲基取代)、芳烷基(任选被卤素基团、羟基、甲氧 基或三氟甲基取代)、杂环基(任选被卤素基团、羟基、甲氧基或三氟甲基取代)、杂环基烷基(任选被卤素基团、羟基、甲氧基或三氟甲基取代)、杂芳基(任选被卤素基团、羟基、甲氧基或三氟甲基取代)，或杂芳基烷基(任选被卤素基团、羟基、甲氧基或三氟甲基取代)，每个R b独立地是直接键或直链或支链亚烷基或亚烯基链，R c是直链或支链亚烷基或亚烯基链，并且除非另有说明，否则上述取代基中的每一个是未取代的。
术语“杂环基烷基”是指式–R c–杂环基的基团，其中R c是如上定义的亚烷基。如果杂环基是含氮杂环基，则杂环基任选地在氮原子上连接至烷基基团。杂环基烷基的亚烷基链如上文对亚烷基链的定义任选地被取代。杂环基烷基的杂环基部分如上文对杂环基基团所定义的那样任选地被取代。
术语“杂芳基”是指衍生自三至十八元芳环基团的基团，其包含二至十七个碳原子和一至六个选自氮、氧和硫的杂原子。如本文所用，杂芳基基团可以是单环、双环、三环或四环环系，其中环系统中的至少一个环是完全不饱和的，即根据休克尔理论(Hückel theory)，它包括一个环状离域(4n+2)π–电子系统。杂芳基包括稠合或桥环系统。杂芳基基团中的杂原子任选地被氧化。如果存在，一个或以上氮原子任选地被季铵化。杂芳基通过环的任何原子与分子的其余部分连接。杂芳基的实例包括但不限于氮杂环庚烯基、吖啶基、苯并咪唑基、苯并吲哚基、1,3-苯并二氧杂环戊烯基、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]二氧杂环庚烯基、苯并[b][1,4]恶嗪基、1,4-苯并二恶烷基、苯并萘呋喃基、苯并恶唑基、苯并二氧杂环戊烯基、苯并二恶英基、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(苯并苯硫基)、苯并噻吩并[3,2-d]嘧啶基、苯并三唑基、苯并[4,6]咪唑并[1,2-a]吡啶基、咔唑基、喹啉基、环戊二烯并[d]嘧啶基、6,7-二氢-5H-环戊二烯并[4,5]噻吩并[2,3-d]嘧啶基、5,6-二氢苯并[h]喹唑啉基、5,6-二氢苯并[h]喹啉基、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、呋喃并[3,2-c]吡啶基、5,6,7,8,9,10-六氢环辛烷并[d]嘧啶基、5,6,7,8,9,10-六氢环辛烷并[d]哒嗪基、5,6,7,8,9,10-六氢环辛烷并[d]吡啶基、异噻唑基、咪唑基、吲唑基、吲哚基、异吲哚基、吲哚啉、异吲哚啉、异喹啉基、吲哚嗪基、异恶唑基、5,8-甲醇-5,6,7,8-四氢喹唑啉基、萘啶基、1,6-萘啶酮基、 恶二唑基、2-氧代氮杂环庚烯基、恶唑基、环氧乙烷基、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、酞嗪基、哌啶基、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]噻吩并[2,3-d]嘧啶基、6,7,8,9-四氢-5H-环庚[4,5]噻吩并[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、噻吩并[2,3-d]嘧啶基、噻吩并[3,2-d]嘧啶基、噻吩并[2,3-c]脯氨酰基以及噻吩基(thiophenyl)(即噻吩基(thienyl))。除非在说明书中另有特别说明，否则术语“杂芳基”意指包括如上定义的杂芳基基团，其任选地被选自烷基、烯基、炔基、卤素基团、氟代烷基、卤代烯基、卤代炔基、氧代、硫代、氰基、硝基、任选地取代的芳香基、任选地取代的芳烷基、任选地取代的芳烯基、任选地取代的芳炔基、任选地取代的碳环基、任选地取代的碳环基烷基、任选地取代的杂环基、任选地取代的杂环基烷基、任选地取代的杂芳基、任选地取代的杂芳基烷基、-R b-OR a、-R b-OC(O)-R a、-R b-OC(O)-OR a、-R b-OC(O)-N(R a) 2、-R b-N(R a) 2、-R b-C(O)R a、-R b-C(O)OR a、-R b-C(O)N(R a) 2、-R b-O-R c-C(O)N(R a) 2、-R b-N(R a)C(O)OR a、-R b-N(R a)C(O)R a、-R b-N(R a)S(O) tR a(其中t为1或2)、-R b-S(O) tR a(其中t为1或2)、-R b-S(O) tOR a(其中t为1或2)以及-R b-S(O) tN(R a) 2(其中t为1或2)的一个或以上取代基取代，其中每个R a独立地为氢、烷基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、氟代烷基、环烷基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、环烷基烷基(任选地被卤素基团、羟基取代、甲氧基、或三氟甲基取代)、芳香基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、芳烷基(任选地被卤素基团取代、羟基、甲氧基或三氟甲基取代)、杂环基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、杂环基烷基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)、杂芳基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)，或杂芳基烷基(任选地被卤素基团、羟基、甲氧基或三氟甲基取代)，每个R b独立地是直接键或直链或支链亚烷基或亚烯基链，R c是直链或支链亚烷基或亚烯基链，并且除非另有说明，否则上述取代基中的每一个是未取代的。
术语“核苷”定义为包括五碳糖(核糖或脱氧核糖)或其衍生物，和有机碱、嘌呤或嘧啶、或其衍生物的化合物。本文描述的核苷可以是修饰核苷。
术语“核苷酸”定义为核苷加至少一个磷酸基基团。核苷酸可包括磷酸基基团、二磷酸基基团或三磷酸基基团。本文所述的核苷酸可以为修饰核苷酸。
术语“核酸”包括寡核苷酸链中或可以掺入寡核苷酸链中的任何化合物和/或物质。根据本申请使用的示例性核酸包括但不限于DNA，RNA包括信使mRNA(mRNA)、其杂交体、RNAi诱导剂、RNAi剂、siRNA、shRNA、miRNA、反义RNA、核酶、催化DNA、诱导三螺旋形成的RNA、适体、载体等，在本申请中详细描述。
术语“核糖核酸”、“RNA”或“RNA分子”是指至少2个碱基-糖基-磷酸基组合串成的链。在一个实施例中，术语包括包含核苷酸的化合物，其中糖部分是核糖。在另一个实施例中，该术语包括其中主链被修饰的RNA和RNA衍生物。在另一个实施例中，“核苷酸”是指核酸聚合物的单体单元。在一个实施例中，RNA可能是以tRNA(转移RNA)、snRNA(小核RNA)、rRNA(核糖体RNA)、mRNA(信使RNA)、反义RNA、小抑制RNA(siRNA)、微小RNA(miRNA)以及核酶的形式存在。siRNA和miRNA的用途已有描述(考迪A A(Caudy A A)等人，基因与发育(Genes&Devel)16:2491-96及其引用的参考文献)。另外，这些形式的RNA可以是单链、双链、三链或四链的绞合。在另一实施例中，该术语也包括人造核酸，其可以包含其他类型的主链但具有相同的碱基。在另一实施例中，人造核酸为PNA(肽核酸)。PNA含有肽主链和核苷酸碱基，并且能够在另一实施例中与DNA和RNA分子结合。在另一实施例中，核苷酸为氧杂环丁烷修饰的。在另一实施例中，通过用硫代磷酸酯键取代一个或以上磷酸二酯键来修饰核苷酸。在另一实施例中，修饰核酸包含本领域已知的天然核酸的磷酸基主链的任何其他变体。本领域普通技术人员熟悉硫代磷酸酯核酸和PNA的用途，其描述，例如，尼尔森P E(Neilsen P E)，结构生物学新见(Curr Opin Struct Biol)9：353-57；[0280]，以及拉兹N K(Raz N K)等，生物化学与生物物理研究通讯(Biochem Biophys Res Commun.)。核酸的生产和使用是本领域技术人员熟知的，并且在例如分子克 隆(Molecular Cloning)(2001)，桑布鲁克(Sambrook)和罗素(Russell)编辑，以及酶学方法：真核细胞的分子克隆方法(Methods in Enzymology:Methods for molecular cloning ineukaryotic cells)(2003)，普基奥(Purchio)和G.C.法里德(G.C.Fareed)中有描述。每个核酸衍生物代表本发明的单独实施例。
术语“衍生物”可与术语“类似物”互换使用。化合物A可以是化合物B的衍生物或类似物，如果化合物A的1、2、3、4、5、6、7、8、9或10个原子被另一个原子或官能基团(例如，氨基、卤素基团、取代或未取代的烷基、取代或未取代的芳香基、取代或未取代的芳烷基，或取代或未取代的环烷基)取代，形成化合物B。术语“衍生物”和“类似物”也可与术语“修饰(的)”互换使用，例如，如果化合物A是化合物B的衍生物，则化合物A也是经修饰的化合物B。
术语“受试者”是指已经或将要成为治疗、观察或实验对象的哺乳动物。术语“哺乳动物”旨在具有其标准含义，并且包括例如人、狗、猫、绵羊以及奶牛。本文描述的方法可用于人类治疗和兽医应用。在一些实施例中，受试者为人。
本文所述的化学实体的术语“治疗有效量”是指当施用于人或非人受试者时有效提供治疗益处，例如改善症状、减缓疾病进展或预防疾病的量。
术语“治疗(treating)”或“治疗(treatment)”包括对需要的哺乳动物受试者，特别是人类受试者，施用本申请披露的至少一种化合物或其药学上可接受的盐，并包括(i)阻止疾病(例如癌症)临床症状的发展，(ii)使疾病(例如癌症)的临床症状退化和/或(iii)预防疾病(例如癌症)发作的预防性治疗。
修饰核苷
修饰核苷可以包括一种具有以下结构的化合物：
Figure PCTCN2020074825-appb-000033
或其药学上可接受的盐，其中：R 4和R 5各自独立选自H、-OH、-NH 2、卤素基团、取代或未取代的C 1-C 10烷基、取代或未取代的芳香基、取代或未取代的C 1-C 10芳烷基、取代或未取代的C 1-C 10环烷基、取代或未取代的酰基、–OR 6、–C(O)R 6、以及–NR 6；以及其中，R 6为H、取代或未取代的C 1-C 10烷基，以及取代或未取代的酰基。在一些实施例中，R 4为H。在一些实施例中，R 5为H。修饰核苷可以是修饰胞苷，例如4-胍基胞苷。修饰核苷可以是具有化学式(I-a)的化合物。
修饰核苷还可以包括m 1A(1-甲基腺苷)、m 2A(2-甲基腺苷)、Am(2'-O-甲基腺苷)、ms 2m 6A(2-甲硫基-N 6-甲基腺苷)、I 6A(N 6-异戊烯基腺苷)、ms 2i 6A(2-甲硫基-N 6-异戊烯基腺苷)、io 6A(N 6-(顺式羟基异戊烯基)腺苷)、ms 2io 6A(2-甲硫基-N 6-(顺式羟基异戊烯基)腺苷)、g 6A(N 6-甘氨酰氨基甲酰基腺苷)、t 6A(N 6-苏氨酰氨基甲酰基腺苷)、ms 2t 6A(2-甲硫基-N 6-苏氨酰氨基甲酰基腺苷)、m 6t 6A(N 6-甲基-N 6-苏氨酰氨基甲酰基腺苷)、hn 6A(N 6-羟基正缬氨酰基氨基甲酰腺苷)、ms 2hn 6A(2-甲硫基-N 6-羟基正缬氨酰基氨基甲酰腺苷)、Ar(p)(2'-O-核糖基腺苷(磷酸基))、I(肌苷)、m 1I(1-甲肌苷)、m 1Im(1,2'-O-二甲肌苷)、m 3C(3-甲基胞苷)、Cm(2'-O-甲基胞苷)、s 2C(2-硫代胞苷)、ac 4C(N 4-乙酰基胞苷)、f 5C(5-甲酰基胞苷)、m 5Cm(5,2'-O-二甲基胞苷)、ac 4Cm(N 4-乙酰基-2'-O-甲基胞苷)、k 2C(赖西丁)、m 1G(1-甲基鸟苷)、m 2G(N 2-甲基鸟苷)、m 7G(7-甲基鸟苷)、Gm(2'-O-甲基鸟苷)、m 2  2G(N 2,N 2-二甲基鸟苷)、m 2Gm(N 2,2'-O-二甲基鸟苷)、m 2  2Gm(N 2,N 2,2'-O-三甲基鸟苷)、Gr(p)(2'-O-核糖基鸟苷(磷酸基))、yW(怀丁苷)、o 2yW(过氧基怀丁苷)、OHyW(羟基怀丁苷)、OHyW*(未修饰羟基怀丁苷)、imG(怀俄苷)、mimG(甲基怀俄苷)、Q(辫苷)、oQ(环氧基辫苷)、galQ(半乳糖基辫苷)、manQ(甘露糖基-辫苷)、preQ 0(7-氰基-7-脱氮鸟苷)、preQ1(7-氨基甲基-7-脱氮鸟苷)、G+(古嘌苷)、D(二氢尿苷)、m 5Um(5,2'-O-二甲基尿苷)、s 4U(4-硫代尿苷)、m 5s 2U(5-甲基-2-硫代尿苷)、s 2Um(2- 硫代-2'-O-甲基尿苷)、acp 3U(3-(3-氨基-3-羧丙基)尿苷)、ho 5U(5-羟基尿苷)、mo 5U(5-甲氧基尿苷)、cmo 5U(尿苷5-氧基乙酸)、mcmo 5U(尿苷5-氧基乙酸甲酯)、chm 5U(5-(羧基羟基甲基)尿苷)、mchm 5U(5-(羧基羟基甲基)尿苷甲酯)、mcm 5U(5-甲氧基羰基甲基尿苷)、mcm 5Um(5-甲氧羰基甲基-2'-O-甲基尿苷)、mcm 5s 2U(5-甲氧基羰基甲基-2-硫代尿苷)、nm 5S 2U(5-氨基甲基-2-硫代尿苷)、mnm 5U(5-甲基氨基甲基尿苷)、mnm 5s 2U(5-甲基氨基甲基-2-硫代尿苷)、mnm 5se 2U(5-甲基氨基甲基-2-硒基尿苷)、ncm 5U(5-氨基甲酰基甲基尿苷)、ncm 5Um(5-氨基甲酰基甲基-2'-O-甲基尿苷)、cmnm 5U(5-羧基甲基氨基甲基尿苷)、cmnm 5Um(5-羧基甲基氨基甲基-2'-O-甲基尿苷)、cmnm 5s 2U(5-羧基甲基氨基甲基-2-硫代尿苷)、m 6  2A(N 6,N 6-二甲基腺苷)、Im(2'-O-甲基肌苷)、m 4C(N4-甲基胞苷)、m 4Cm(N 4,2'-O-二甲基胞苷)、hm 5C(5-羟基甲基胞苷)、m 3U(3-甲基尿苷)、cm 5U(5-羧基甲基尿苷)、m 6Am(N 6,2'-O-二甲基腺苷)、m 6  2Am(N 6,N 6,O-2'-三甲基腺苷)、m 2 , 7G(N 2,7-二甲基鸟苷)、m2'2'7G(N 2,N 2,O-2'-三甲基鸟苷)、m 3Um(3,2'-O-二甲基尿苷)、m 5D(5-甲基二氢尿苷)、f 5Cm(5-甲酰基-2'-O-甲基胞苷)、m 1Gm(1,2'-O-二甲基鸟苷)、m 1Am(1,2'-O-二甲基腺苷)、τm 5U(5-牛磺酰甲基尿苷)、τm 5s 2U(5-牛磺酰甲基-2-硫代尿苷)、imG-14(4-去甲基怀俄苷)、imG 2(异怀俄苷)、ac 6A(N 6-乙酰基腺苷)或其任意组合。其他修饰核苷可以从Modomics中找到(http://modomics.genesilico.pl/)。关于修饰核苷及其掺入mRNA的讨论另见美国专利号8,278,036或WO2011012316。
修饰核苷酸
本文披露的修饰核苷(例如，具有化学式(I-a)的化合物)和核苷酸(例如，具有化学式(I-e)或(I-g)的化合物)可以通过以下一般方法和程序从易获得的原材料制备。应理解，给出了典型或优选的工艺条件(即反应温度、时间、反应物的摩尔比、溶剂、压力等)；除非另有说明，否则也可以使用其他工艺条件。最佳反应条件可随所用的特定反应物或溶剂而变化，但这些条件可由本领域技术人员通过常规优化程序确定。
修饰核苷和核苷酸的制备可涉及各种化学基团的保护和去保护。本领域技术人员可以容易地确定保护和去保护的需要以及适当的保护基团的选择。例如，保护基团的化学性质可以在格林(Greene)等，有机合成中的保护基团(Protective Groups in Organic Synthesis)，第二版，约翰威利父子(Wiley&Sons )，1991中找到，其全部内容通过引用并入本申请。
本文所述方法的反应可在合适的溶剂中进行，所述溶剂可由有机合成技术领域的人员容易地选择。合适的溶剂可以与原料(反应物)、中间体或产物在反应进行的温度下基本上不反应，即，温度范围从溶剂的冷冻结固温度到溶剂的沸腾温度。给定的反应可以在一种溶剂或一种以上溶剂的混合物中进行。取决于具体的反应步骤，可以选择用于特定反应步骤的合适溶剂。修饰核苷和核苷酸的外消旋混合物的拆分可以通过本领域已知的许多方法中的任何一种进行。一种示例性方法包括使用“手性拆分酸”的分级重结晶，所述“手性拆分酸”是光学活性的成盐有机酸。用于分级重结晶方法的合适的拆分剂是，例如，光学活性酸，如D和L形式的酒石酸、二乙酰酒石酸、二苯甲酰基酒石酸、扁桃酸、苹果酸、乳酸或各种光学活性樟脑磺酸。外消旋混合物的拆分也可以通过在填充有光学活性拆分剂(例如，二硝基苯甲酰基苯基甘氨酸)的柱上洗脱来进行。合适的洗脱溶剂组合物可由本领域技术人员确定。
可根据下面提供的方案制备修饰核苷和核苷酸：
Figure PCTCN2020074825-appb-000034
可根据下面提供的方案制备修饰核苷和核苷酸：
Figure PCTCN2020074825-appb-000035
修饰核苷和核苷酸也可以根据其中描述的合成方法制备，绪方(Ogata)等，有机化学杂志(Journal of Organic Chemistry)74：2585-2588，2009；帕玛尔(Purmal)等，核酸研究(Nucleic Acids Research)22(1)：72-78，1994；福原(Fukuhara)等，生物化学(Biochemistry)1(4)：563-568，1962；以及徐(Xu)等，四面体(Tetrahedron)48(9)：1729-1740，1992，每一篇都是通过引用完整地并入本申请。
修饰核酸
本文披露了一种修饰核酸，例如mRNA，及其合成方法。
根据本申请使用的核酸可根据任何现有技术，包括但不限于化学合成、酶促合成，通常是较长前体的末端体外转录、酶促或化学切割等。合成RNA的方法是本领域熟知的(参见，例如，盖特,M.J.(Gait,M.J.)(编辑)寡核苷酸合成：一种实用的方法(Oligonucleotide synthesis：a practical approach)，牛津[牛津郡]，华盛顿，哥伦比亚特区：IRL出版社，1984；以及赫德威金，P.(Herdewijn,P.)(编辑)寡核苷酸合成：方法和应用(Oligonucleotide synthesis：methods and applications)，分子生物学方法(Methods in Molecular Biology)，v.288(克利夫顿，新泽西州)托托华，新泽西州：胡玛娜出版社(Humana Press)，2005；两篇都是通过引用完整的并入本申请)。mRNA可以用包括RNA聚合酶、线 性DNA模板以及RNA聚合酶反应缓冲液(例如，核苷酸如核糖核苷酸)的反应混合物产生。美国专利公布US20120195936和国际公布WO2011012316披露了RNA的用途，这两篇文献的全部内容通过引用并入本申请。
RNA聚合酶反应缓冲液通常包括盐/缓冲剂，例如Tris、HEPES、硫酸铵、碳酸氢钠、柠檬酸钠、乙酸钠、磷酸钾、磷酸钠、氯化钠以及氯化镁。反应混合物的pH可以为约6至8.5、6.5至8.0、7.0至7.5，并且在一些实施例中，pH为7.5。
在一个实例中，反应混合物包括浓度范围为1-10mM的NTP、浓度范围为0.01-0.5mg/ml的DNA模板以及浓度范围为0.01-0.1mg/ml的RNA聚合酶，例如，反应混合物包括浓度为5mM的NTP、浓度为0.1mg/ml的DNA模板和浓度为0.05mg/ml的RNA聚合。
根据本发明，天然存在的或修饰核苷和/或核苷酸可以用于制备修饰核酸，例如修饰mRNA。例如，修饰mRNA可包括一种或以上天然核苷(例如，腺苷、鸟苷、胞苷、尿苷)；修饰核苷(例如，2-氨基腺苷、2-硫代胸苷、肌苷、吡咯并嘧啶、3-甲基腺苷、5-甲基胞苷、C-5丙炔基-胞苷、C-5丙炔基-尿苷、2-氨基腺苷、C5-溴尿苷、C5-氟尿苷、C5-碘尿苷、C5-丙炔基-尿苷、C5-丙炔基-胞苷、C5-甲基胞苷、2-氨基腺苷、7-脱氮腺苷、7-脱氮鸟苷、8-氧代腺苷、8-氧代鸟苷、O(6)-甲基鸟嘌呤、假尿苷、(例如，N-1-甲基-假尿苷)、2-硫代尿苷以及2-硫代胞苷)；化学修饰碱基；生物修饰碱基(例如，甲基化碱基)；插入碱基；修饰糖(例如，2'-氟代核糖、核糖、2'-脱氧核糖、阿拉伯糖以及己糖)；修饰磷酸基基团(例如硫代磷酸基和5'-N-亚磷酰胺键)，或其任意组合。
RNA分子(例如，mRNA)可以包括至少两种核苷酸。核苷酸可以是天然存在的核苷酸或修饰核苷酸。在一些实施例中，RNA分子包括约5种核苷酸至约5,000种核苷酸。在一些实施例中，RNA分子包括至少约5种核苷酸。在一些实施例中，RNA分子包括至多约5,000种核苷酸。在一些实施例中，RNA分子包括约5种核苷酸至约20种核苷酸、约5种核苷酸至约40种核苷酸、约5种核苷酸至约60种核苷酸、约5种核苷酸至约80种核苷酸、约5种核苷酸至约100种核苷酸、约 5种核苷酸至约200种核苷酸、约5种核苷酸至约500种核苷酸、约5种核苷酸至约1,000种核苷酸核苷酸、约5种核苷酸至约2,000种核苷酸、约5种核苷酸至约5,000种核苷酸、约20种核苷酸至约40种核苷酸、约20种核苷酸至约60种核苷酸、约20种核苷酸至约80种核苷酸、约20种核苷酸至约100种核苷酸、约20种核苷酸至约200种核苷酸、约20种核苷酸至约500种核苷酸、约20种核苷酸至约1,000种核苷酸、约20种核苷酸至约2,000种核苷酸、约20种核苷酸至约5,000种核苷酸、约40种核苷酸至约60种核苷酸、约40种核苷酸至约80种核苷酸、约40种核苷酸至约100种核苷酸、约40种核苷酸至约200种核苷酸、约40种核苷酸约500种核苷酸、约40种核苷酸至约1,000种核苷酸、约40种核苷酸至约2000种核苷酸、约40种核苷酸至约5,000种核苷酸、约60种核苷酸至约80种核苷酸、约60种核苷酸至约100种核苷酸、约60种核苷酸至约200种核苷酸、约60种核苷酸至约500种核苷酸、约60种核苷酸至约1,000种核苷酸、约60种核苷酸至约2,000种核苷酸、约60种核苷酸至约5,000种核苷酸、约80种核苷酸至约100种核苷酸、约80种核苷酸至约200种核苷酸、约80种核苷酸至约500种核苷酸、约80种核苷酸至约1,000种核苷酸、约80种核苷酸至约2,000种核苷酸、约80种核苷酸至约5,000种核苷酸、约100种核苷酸至约200种核苷酸、约100种核苷酸至约500种核苷酸、约100种核苷酸至约1,000种核苷酸、约100种核苷酸至约2000种核苷酸、约100种核苷酸至约5,000种核苷酸、约200种核苷酸至约500种核苷酸、约200种核苷酸至约1,000种核苷酸、约200种核苷酸至约2000种核苷酸、约200种核苷酸约5000种核苷酸、约500种核苷酸至约1,000种核苷酸、约500种核苷酸至约2000种核苷酸、约500种核苷酸至约5,000种核苷酸、约1,000种核苷酸以约2000种核苷酸、约1,000种核苷酸至约5,000种核苷酸或约2000种核苷酸至约5,000种核苷酸。在一些实施例中，RNA分子包括约5种核苷酸、约20种核苷酸、约40种核苷酸、约60种核苷酸、约80种核苷酸、约100种核苷酸、约200种核苷酸、约500种核苷酸、约1,000种核苷酸、约2000种核苷酸或约5000种核苷酸。
RNA分子(例如，mRNA)可以包括至少一种本申请所述的修饰核苷酸。在一些实施例中，RNA分子包括约1种修饰核苷酸至约100种修饰核苷酸。在一些实施例中，RNA分子包括至少约1种修饰核苷酸。在一些实施例中，RNA分子包括至多约100种修饰核苷酸。在一些实施例中，RNA分子包括约1种修饰核苷 酸至约2种修饰核苷酸、约1种修饰核苷酸至约3种修饰核苷酸、约1种修饰核苷酸至约4种修饰核苷酸、约1种修饰核苷酸至约5种修饰核苷酸、约1种修饰核苷酸至约10种修饰核苷酸、约1种修饰核苷酸至约20种修饰核苷酸、约1种修饰核苷酸至约100种修饰核苷酸、约2种修饰核苷酸至约3种修饰核苷酸、约2种修饰核苷酸至约4种修饰核苷酸、约2种修饰核苷酸至约5种修饰核苷酸、约2种修饰核苷酸至约10种修饰核苷酸、约2种修饰核苷酸至约20种修饰核苷酸、约2种修饰核苷酸至约100种修饰核苷酸、约3种修饰核苷酸至约4种修饰核苷酸、约3种修饰核苷酸至约5种修饰核苷酸、约3种修饰核苷酸至约10种修饰核苷酸、约3种修饰核苷酸至约20种修饰核苷酸、约3种修饰核苷酸至约100种修饰核苷酸、约4种修饰核苷酸至约5种修饰核苷酸、约4种修饰核苷酸至约10种修饰核苷酸、约4种修饰核苷酸至约20种修饰核苷酸、约4种修饰核苷酸至约100种修饰核苷酸、约5种修饰核苷酸至约10种修饰核苷酸、约5种修饰核苷酸至约20种修饰核苷酸、约5种修饰核苷酸至约100种修饰核苷酸、约10种修饰核苷酸至约20种修饰核苷酸、约10种修饰核苷酸至约100种修饰核苷酸或者约20种修饰核苷酸至约100种修饰核苷酸。在一些实施例中，RNA分子包括约1种修饰核苷酸、约2种修饰核苷酸、约3种修饰核苷酸、约4种修饰核苷酸、约5种修饰核苷酸、约10种修饰核苷酸、约20种修饰核苷酸或者约100种修饰核苷酸。
RNA分子(例如，mRNA)可以包括至少0.1％的修饰核苷酸。修饰核苷酸的分数可以计算为：修饰核苷酸的数量/核苷酸的总数*100％。在一些实施例中，RNA分子包括约0.1％修饰核苷酸至约100％修饰核苷酸。在一些实施例中，RNA分子包括至少约0.1％的修饰核苷酸。在一些实施例中，RNA分子包括至多约100％修饰核苷酸。在一些实施例中，RNA分子包括约0.1％修饰核苷酸至约0.2％修饰核苷酸、约0.1％修饰核苷酸至约0.5％改性核苷酸、约0.1％修饰核苷酸至约1％修饰核苷酸、约0.1％修饰核苷酸至约2％修饰核苷酸、约0.1％修饰核苷酸至约5％修饰核苷酸、约0.1％修饰核苷酸至约10％修饰核苷酸、约0.1％修饰核苷酸至约20％修饰核苷酸、约0.1％修饰核苷酸至约50％修饰核苷酸、约0.1％修饰核苷酸至约100％修饰核苷酸、约0.2％修饰核苷酸至约0.5％修饰核苷酸、约0.2％修饰核苷酸至约1％修饰核苷酸、约0.2％修饰核苷酸至约2％修饰核苷酸、约0.2％修饰核苷酸至约5％修饰核苷酸、约0.2％修饰核苷酸至约10％修饰核苷酸、约 0.2％修饰核苷酸至约20％修饰核苷酸、约0.2％修饰核苷酸至约50％修饰核苷酸、约0.2％修饰核苷酸至约100％修饰核苷酸、约0.5％修饰核苷酸至约1％修饰核苷酸、约0.5％修饰核苷酸至约2％修饰核苷酸、约0.5％修饰核苷酸至约5％修饰核苷酸、约0.5％修饰核苷酸至约10％修饰核苷酸、约0.5％修饰核苷酸至约20％修饰核苷酸、约0.5％修饰核苷酸至约50％修饰核苷酸、约0.5％修饰核苷酸至约100％修饰核苷酸、约1％修饰核苷酸至约2％修饰核苷酸、约1％修饰核苷酸至约5％修饰核苷酸、约1％修饰核苷酸至约10％修饰核苷酸、约1％修饰核苷酸至约20％修饰核苷酸、约1％修饰核苷酸至约50％修饰核苷酸、约1％修饰核苷酸至约100％修饰核苷酸、约2％修饰核苷酸至约5％修饰核苷酸、约2％修饰核苷酸至约10％修饰核苷酸、约2％修饰核苷酸至约20％修饰核苷酸、约2％修饰核苷酸至约50％修饰核苷酸、约2％修饰核苷酸至约100％修饰核苷酸、约5％修饰核苷酸至约10％修饰核苷酸、约5％修饰核苷酸至约20％修饰核苷酸、约5％修饰核苷酸至约50％修饰核苷酸、约5％修饰核苷酸至约100％修饰核苷酸、约10％修饰核苷酸至约20％修饰核苷酸、约10％修饰核苷酸至约50％修饰核苷酸、约10％改性核苷酸至约100％修饰核苷酸、约20％修饰核苷酸至约50％修饰核苷酸、约20％修饰核苷酸至约100％修饰核苷酸或者约50％修饰核苷酸至约100％修饰核苷酸。在一些实施例中，RNA分子包括约0.1％修饰核苷酸、约0.2％修饰核苷酸、约0.5％修饰核苷酸、约1％修饰核苷酸、约2％修饰核苷酸、约5％修饰核苷酸、约10％修饰核苷酸、约20％修饰核苷酸、约50％修饰核苷酸、或约100％修饰核苷酸。
在一些实施例中，一种具有化学式(I)或(I-a)的化合物将修饰RNA中的约1种胞嘧啶替换为修饰RNA中的约10,000种胞嘧啶。在一些实施例中，一种具有化学式(I)或(I-a)的化合物替换修饰RNA中的至少约1种胞嘧啶。在一些实施例中，一种具有化学式(I)或(I-a)的化合物替换修饰RNA中的至多约10,000种胞嘧啶。在一些实施例中，一种具有化学式(I)或(I-a)的化合物将修饰RNA中的约1种胞嘧啶替换为修饰RNA中的约2种胞嘧啶、将修饰RNA中约1种胞嘧啶替换为修饰RNA中的约10种胞嘧啶、将修饰RNA中约1种胞嘧啶替换为修饰RNA中约50种胞嘧啶、将修饰RNA中约1种胞嘧啶替换为修饰RNA中约100种胞嘧啶、将修饰RNA中约1种胞嘧啶替换为修饰RNA中约500种胞嘧啶、将修饰RNA中约1种胞嘧啶替换为修饰RNA中约1,000种胞嘧啶、将修饰RNA中约1种胞 嘧啶替换为修饰RNA中约5,000种胞嘧啶、将修饰RNA中约1种胞嘧啶替换为修饰RNA中的约10,000种胞嘧啶、将修饰RNA中约2种胞嘧啶替换为修饰RNA中的约10种胞嘧啶、将修饰RNA中约2种胞嘧啶替换为修饰RNA中的约50种胞嘧啶、将修饰RNA中约2种胞嘧啶替换为修饰RNA中约100种胞嘧啶、将修饰RNA中约2种胞嘧啶替换为修饰RNA中约500种胞嘧啶、将修饰RNA中约2种胞嘧啶替换为修饰RNA中的约1,000种胞嘧啶、将修饰RNA中的约2种胞嘧啶替换为修饰RNA中的约5,000种胞嘧啶、将改性RNA中的约2种胞嘧啶替换为修饰RNA中的约10,000种胞嘧啶、将修饰RNA中约10种胞嘧啶替换为修饰RNA中的约50种胞嘧啶、将修饰RNA中约10种胞嘧啶替换为修饰RNA中的约100种胞嘧啶、将修饰RNA中约10种胞嘧啶替换为修饰RNA中约500种胞嘧啶、将修饰RNA中约10种胞嘧啶替换为改性RNA中约1,000种胞嘧啶、将修饰RNA中约10种胞嘧啶替换为修饰RNA中约5,000种胞嘧啶、将修饰RNA中约10种胞嘧啶替换为修饰RNA中约10,000种胞嘧啶、将修饰RNA中约50种胞嘧啶替换为修饰RNA中的约100种胞嘧啶、将修饰RNA中约50种胞嘧啶替换为修饰RNA中的约500种胞嘧啶、将修饰RNA中约50种胞嘧啶替换为修饰RNA中的约1,000种胞嘧啶、将修饰RNA中约50种胞嘧啶替换为修饰RNA中的约5,000种胞嘧啶、将修饰RNA中约50种胞嘧啶替换为修饰RNA中的约10,000种胞嘧啶、将修饰RNA中约100种胞嘧啶替换为修饰RNA中的约500种胞嘧啶、将修饰RNA中的约100种胞嘧啶替换为修饰RNA中的约1,000种胞嘧啶、将修饰RNA中约100种胞嘧啶替换为修饰RNA中的约5,000种胞嘧啶、将修饰RNA中约100种胞嘧啶替换为修饰RNA中的约10,000种胞嘧啶、将修饰RNA中约500种胞嘧啶替换为修饰RNA中的约1,000种胞嘧啶、将修饰RNA中约500种胞嘧啶替换为修饰RNA中的约5,000种胞嘧啶、将修饰RNA中约500种胞嘧啶替换为修饰RNA中的约10,000种胞嘧啶、将修饰RNA中约1,000种胞嘧啶替换为修饰RNA中的约5,000种胞嘧啶、将修饰RNA中的约1,000种胞嘧啶替换为修饰RNA中的约10,000种胞嘧啶或者将修饰RNA中的约5,000种胞嘧啶替换为修饰RNA中的约10,000种胞嘧啶。在一些实施例中，化学式(I)或(I-a)的化合物取代修饰RNA中的约1种胞嘧啶、修饰RNA中约2种胞嘧啶、修饰RNA中约10种胞嘧啶、改性RNA中约50种胞嘧啶、修饰RNA中约100种胞嘧啶、修饰RNA中约500种胞嘧啶、修饰RNA中约1,000种胞嘧啶、修饰RNA 中约5,000种胞嘧啶或者修饰RNA中约10,000种胞嘧啶。
在一些实施例中，一种具有化学式(I)或(I-a)的化合物将修饰RNA中约0.01％的胞嘧啶替换为修饰RNA中约100％的胞嘧啶。在一些实施例中，一种具有化学式(I)或(I-a)的化合物替换修饰RNA中至少约0.01％的胞嘧啶。在一些实施例中，一种具有化学式(I)或(I-a)的化合物替换修饰RNA中至多约100％的核苷。在一些实施例中，一种具有化学式(I)或(I-a)的化合物将修饰RNA中约0.01％的胞嘧啶替换为修饰RNA中约0.1％的胞嘧啶、将修饰RNA中约0.01％的胞嘧啶替换为修饰RNA中约0.5％的胞嘧啶、将修饰RNA中约0.01％的胞嘧啶替换为修饰RNA中约1％的胞嘧啶、将修饰RNA中约0.01％的胞嘧啶替换为修饰RNA中约5％的胞嘧啶、将修饰RNA中约0.01％的胞嘧啶替换为修饰RNA中约10％的胞嘧啶、将修饰RNA中约0.01％的胞嘧啶替换为修饰RNA中约50％的胞嘧啶、将修饰RNA中约0.01％的胞嘧啶替换为修饰RNA中约100％的胞嘧啶、将修饰RNA中约0.1％的胞嘧啶替换为修饰RNA中约0.5％的胞嘧啶、将修饰RNA中约0.1％的胞嘧啶替换为修饰RNA中约1％的胞嘧啶、将修饰RNA中约0.1％的胞嘧啶替换为修饰RNA中约5％的胞嘧啶、将修饰RNA中约0.1％的胞嘧啶替换为修饰RNA中约10％的胞嘧啶、将修饰RNA中约0.1％的胞嘧啶替换为修饰RNA中约50％的胞嘧啶、将修饰RNA中约0.1％的胞嘧啶替换为修饰RNA中约100％的胞嘧啶、将修饰RNA中约0.5％的胞嘧啶替换为修饰RNA中约1％的胞嘧啶、将修饰RNA中约0.5％的胞嘧啶替换为修饰RNA中约5％的核苷、将修饰RNA中约0.5％的胞嘧啶替换为修饰RNA中约10％的胞嘧啶、将修饰RNA中约0.5％的胞嘧啶替换为修饰RNA中约50％的胞嘧啶、将修饰RNA中约0.5％的胞嘧啶替换为修饰RNA中约100％的胞嘧啶、将修饰RNA中约1％的胞嘧啶替换为修饰RNA中约5％的胞嘧啶、将修饰RNA中约1％的胞嘧啶替换为修饰RNA中约10％的胞嘧啶、将修饰RNA中约1％的胞嘧啶替换为修饰RNA中约50％的胞嘧啶、将修饰RNA中约1％的胞嘧啶替换为修饰RNA中约100％的胞嘧啶、将修饰RNA中约5％的胞嘧啶替换为修饰RNA中约10％的胞嘧啶、将修饰RNA中约5％的胞嘧啶替换为修饰RNA中约50％的胞嘧啶、将修饰RNA中约5％的胞嘧啶替换为修饰RNA中约100％的胞嘧啶、将修饰RNA中约10％的胞嘧啶替换为修饰RNA中约50％的胞嘧啶、将修饰RNA中约10％的胞嘧啶替换为修饰RNA中约100％的胞嘧啶、或修饰 RNA中约50％的胞嘧啶替换为修饰RNA中约100％的胞嘧啶。在一些实施例中，一种具有化学式(I)或(I-a)的化合物替换修饰RNA中约0.01％的胞嘧啶、修饰RNA中约0.1％的胞嘧啶、修饰RNA中约0.5％的胞嘧啶、修饰RNA中约1％的胞嘧啶、修饰RNA中约5％的胞嘧啶、修饰RNA中约10％的胞嘧啶、修饰RNA中约50％的胞嘧啶、或修饰RNA中约100％的胞嘧啶。
反应混合物中每种核苷酸，例如核糖核苷酸(例如，ATP、UTP、GTP以及CTP))的浓度可以为约0.1mM至约100mM。在一些实施例中，每种核苷酸的浓度至少约为0.1mM。在一些实施例中，每种核苷酸的浓度至多约为100mM。在一些实施例中，每种核苷酸的浓度为约0.1mM至约0.5mM、约0.1mM至约1mM、约0.1mM至约5mM、约0.1mM至约10mM、约0.1mM至约20mM、约0.1mM至约50mM、约0.1mM至约75mM、约0.1mM至约100mM、约0.5mM至约1mM、约0.5mM至约5mM、约0.5mM至约10mM、约0.5mM至约20mM、约0.5mM至约50mM、约0.5mM至约75mM、约0.5mM至约100mM、约1mM至约5mM、约1mM至约10mM、约1mM至约20mM、约1mM至约50mM、约1mM至约75mM、约1mM至约100mM、约5mM至约10mM、约5mM至约20mM、约5mM至约50mM、约5mM至约75mM、约5mM至约100mM、约10mM至约20mM、约10mM至约50mM、约10mM至约75mM、约10mM至约100mM、约20mM至约50mM、约20mM至约75mM、约20mM至约100mM、约50mM至约75mM、约50mM至约100mM或者约75mM至约100mM。在一些实施例中，每种核苷酸的浓度为约0.1mM、约0.5mM、约1mM、约5mM、约10mM、约20mM、约50mM、约75mM或约100mM。
在反应中使用的核苷酸，例如核糖核苷酸(例如组合的ATP、GTP、CTP以及UTP)的总浓度在0.5mM至约500mM之间。在一些实施例中，核苷酸的总浓度为约0.5mM至约500mM。在一些实施例中，核苷酸的总浓度为至少约0.5mM。在一些实施例中，核苷酸的总浓度为至多约500mM。在一些实施例中，核苷酸的总浓度为约0.5mM至约1mM、约0.5mM至约5mM、约0.5mM至约10mM、约0.5mM至约50mM、约0.5mM至约100mM、约0.5mM至约200mM、约0.5mM至约300mM、约0.5mM至约500mM、约1mM至约5mM、约1mM至约10mM、约1mM至约50mM、约1mM至约100mM、约1mM至约200mM、约1mM至约300mM 、约1mM至约500mM、约5mM至约10mM、约5mM至约50mM、约5mM至约100mM、约5mM至约200mM、约5mM至约300mM、约5mM至约500mM、约10mM至约50mM、约10mM至约100mM、约10mM至约200mM、约10mM至约300mM、约10mM至约500mM、约50mM至约100mM、约50mM至约200mM、约50mM至约300mM、约50mM至约500mM、约100mM至约200mM、约100mM至约300mM、约100mM至约500mM、约200mM至约300mM、约200mM至约500mM或者约300mM至约500mM。在一些实施例中，核苷酸的总浓度为约0.5mM、约1mM、约5mM、约10mM、约50mM、约100mM、约200mM、约300mM或者约500mM。
合成后处理
合成后可加入5'帽和/或3'尾。帽的存在可以提供对大多数真核细胞中发现的核酸酶的抗性。“尾”的存在可以用于保护mRNA免于核酸外切酶降解和/或调节蛋白质表达水平。
可以如下添加5'帽：第一，RNA末端磷酸酶从5'核苷酸中去除一个末端磷酸基，留下两个末端磷酸基；然后通过鸟苷酰转移酶将鸟苷三磷酸(GTP)加入到末端磷酸基中，产生5'5'5三磷酸键；然后用甲基转移酶将鸟嘌呤的7-氮甲基化。帽结构的实例包括但不限于m7G(5')ppp(5'(A，G(5')ppp(5')A和G(5')ppp(5')G。更多帽结构在已公布的美国申请No.US 2016/0032356中进行了描述，阿什奎尔海克(Ashiqul Haque)等，“化学修饰的hCFTR mRNA在囊性纤维化小鼠模型中恢复肺功能”(Chemicallymodified hCFTR mRNAs recuperate lung function in a mouse model of cysticfibrosis)，科学报告(Scientific Reports)(2018)8：16776，以及科尔(Kore)等，“5'-端帽类似物的最新进展：合成和生物分支”(Recent Developments in 5’-Terminal Cap Analogs：Synthesis and Biological Ramifications)有机化学迷你读物(Mini-Reviews in Organic Chemistry)2008，5，179-192，其通过引用并入本申请。
尾结构可以包括聚(A)和/或聚(C)尾。mRNA的3'末端(例如，3'末端的10、20、30、40、50、60、70、80、90或100种核苷酸)上的聚-A尾可以包括至少50％、55％、65％、70％、75％、80％、85％、90％、92％、94％、95％、96％ 、97％、98％或99％的腺苷核苷酸。mRNA的3'末端(例如，3'末端的10、20、30、40、50、60、70、80、90或100种核苷酸)上的聚-A尾可以包括至少50％、55％、65％、70％、75％、80％、85％、90％、92％、94％、95％、96％、97％、98％或99％胞嘧啶核苷酸。
如本申请所述，添加5'帽和/或3'尾可以有助于检测体外合成期间产生的无效转录物，因为没有加帽和/或加尾，那些过早中止的mRNA转录物的大小可能太小而无法被检测到。因此，在一些实施例中，在测试mRNA纯度(例如，mRNA中存在的无效转录物的水平)之前，将5'帽和/或3'尾加入合成的mRNA中。在一些实施例中，在如本申请所述纯化mRNA之前，将5'帽和/或3'尾加入到合成的mRNA中。在其他实施例中，在如本申请所述纯化mRNA后，将5'帽和/或3'尾加入到合成的mRNA中。
根据本发明合成的mRNA可以不需进一步纯化而使用。特别地，可以使用根据本发明合成的mRNA而无需去除短聚物的步骤。在一些实施例中，根据本发明合成的mRNA可以进一步纯化。根据本发明可以使用各种方法纯化合成的mRNA。例如，可以使用离心、过滤和/或色谱方法进行mRNA的纯化。在一些实施例中，合成的mRNA通过乙醇沉淀或过滤或色谱法、或凝胶纯化或任何其它合适的方法纯化。在一些实施例中，通过HPLC纯化mRNA。在一些实施例中，在标准酚：氯仿：异戊醇溶液中提取mRNA，这是本领域技术人员所熟知的。在一些实施例中，使用切向流过滤纯化mRNA。合适的纯化方法包括在US 2016/0040154、US 2015/0376220、于2018年2月27日提交的PCT申请PCT/US18/19954，名称为“用于纯化信使RNA的方法”以及于2018年2月27日提交的题为“纯化信使RNA的方法”的PCT申请PCT/US18/19978中所描述的方法，所有这些都通过引用并入本申请并且可以用于实施本发明。
在一些实施例中，mRNA在加帽和封尾之前被纯化。在一些实施例中，在加帽和封尾之后纯化mRNA。在一些实施例中，在加帽和封尾之前和之后均纯化mRNA。在一些实施例中，通过离心在加帽和封尾之前或之后或之前和之后均纯化mRNA。在一些实施例中，通过过滤在加帽和封尾之前或之后或之前和之后均纯化mRNA。在一些实施例中，通过切向流过滤(TFF)在加帽和封尾 之前或之后或之前和之后均纯化mRNA。在一些实施例中，通过色谱法在加帽和封尾之前或之后或之前和之后均纯化mRNA。
可以使用本领域可用的任何方法来检测和定量mRNA的全长或无效转录物。在一些实施例中，使用印迹、毛细管电泳、色谱、荧光、凝胶电泳、HPLC、银染色、光谱、紫外(UV)或UPLC或其组合检测合成的mRNA分子。本领域已知的其他检测方法包括在本发明中。在一些实施例中，使用UV吸收光谱法通过毛细管电泳分离来检测合成的mRNA分子。在一些实施例中，在凝胶电泳(“乙二醛凝胶电泳”)之前，mRNA被乙二醛染料变性。在一些实施例中，合成的mRNA在加帽或封尾前表征。在一些实施例中，合成的mRNA在加帽和封尾后表征。
在一些实施例中，通过本文披露的方法产生的mRNA包括小于10％、小于9％、小于8％、小于7％、小于6％、小于5％、小于4％、小于3％、小于2％、小于1％、小于0.5％、小于0.1％的除全长mRNA之外的杂质。杂质包括IVT污染物，例如蛋白质、酶、游离核苷酸和/或短聚物。
在一些实施例中，根据本发明制备的mRNA基本上没有短聚物或无效转录物。特别地，通过毛细管电泳或乙二醛凝胶电泳，根据本发明制备的mRNA包括不可检测水平的短聚物或无效转录物。如本文所用，术语“短聚物”或“无效转录物”是指任何小于全长的转录物。在一些实施例中，“短聚物”或“无效转录物”的长度小于100核苷酸、小于90、小于80、小于70、小于60、小于50、小于40、小于30、小于20或者长度小于10核苷酸。在一些实施例中，在添加5'-帽和/或3'-聚A尾后检测或量化短聚物。
药物组合物
还披露了本申请提供的包括化合物、修饰核苷、修饰核苷酸或修饰核酸的药物组合物。
在一些实施例中，本发明的药物组合物可以通过本领域技术人员已知的任何方法给予受试者，例如肠胃外、癌旁、经粘膜、经皮、肌肉内、静脉内、皮内、皮下、腹膜内、心室内、颅内、阴道内或肿瘤内。
药物组合物可以通过静脉内、动脉内或肌内注射液体制剂来施用。合适的液体制剂包括溶液、悬浮液、分散液、乳液、油等。在一些实施例中，药物组合物是静脉内给药的，因此配制成适于静脉内给药的形式。在一些实施例中，药物组合物是动脉内给药的，因此配制成适于动脉内给药的形式。在一些实施例中，药物组合物是肌肉内给药的，因此配制成适于肌内给药的形式。
药物组合物可以使用囊泡给药，例如，脂质体(参见朗格(Langer)，科学(Science)249：1527-1533(1990)；崔特(Treat)等，脂质体在传染病和癌症治疗中的作用(Liposomes in the Therapy of Infectious Disease and Cancer)，洛佩兹-贝雷斯坦(Lopez-Berestein)和菲德勒(编辑)，利斯(Liss)，纽约，页数353-365(1989)；洛佩兹-贝雷斯坦(Lopez-Berestein)，同上，页数317-327；见同上)。
药物组合物可以口服给药，因此可以配制成适于口服给药的形式，即固体或液体制剂。合适的固体口服制剂可以包括片剂、胶囊、药丸、颗粒、微丸等。合适的液体口服制剂可以包括溶液、悬浮液、分散液、乳液、油等。
药物组合物可以局部给予体表，因此可以配制成适于局部给药的形式。合适的局部制剂可包括凝胶、软膏、乳膏、洗剂、滴剂等。对于局部给药，可以制备组合物或其生理学上可耐受的衍生物，并在有或没有药物载体的情况下作为溶液、悬浮液或乳液施用于生理学上可接受的稀释剂中。
药物组合物可以作为栓剂给药，例如直肠栓剂或尿道栓剂。在一些实施例中，药物组合物通过皮下植入微粒药。在一些实施例中，微丸在一段时间内提供药剂的受控释放。
药物组合物可另外包括药学上可接受的赋形剂，如本申请所用，包括任何和所有溶剂、分散介质、稀释剂或其他液体载体、分散或悬浮助剂、表面活性剂、等渗剂、增稠剂或乳化剂、防腐剂、固体粘合剂、润滑剂等，适合于所需的特定剂型。雷明顿(Remington)的《药学的科学与实践》(The Science and Practice of Pharmacy)，第21版，A.R.格纳罗(A.R.Gennaro)(利平科特，威廉斯与威尔金斯(Lippincott,Williams&Wilkins,Baltimore)，马里兰州，2006 ；通过引用并入本文)披露了用于配制药物组合物的各种赋形剂和用于其制备的已知技术。
在一些实施例中，药学上可接受的赋形剂的纯度为至少95％、至少96％、至少97％、至少98％、至少99％或100％。在一些实施例中，赋形剂被批准用于人类和兽医用途。在一些实施例中，赋形剂经美国食品和药物管理局(United States Food and Drug Administration)批准。在一些实施例中，赋形剂是药用级的。在一些实施例中，赋形剂符合美国药典(United States Pharmacopoeia，USP)、欧洲药典(European Pharmacopoeia，EP)、英国药典和/或国际药典的标准。
用于液体制剂的药学上可接受的载体可以是水性或非水性溶液、悬浮液、乳液或油。非水溶剂的实例可以是丙二醇、聚乙二醇和可注射的有机酯，例如油酸乙酯。含水载体可包括水、醇/水溶液、乳液或悬浮液、包括盐水和缓冲介质。油的实例可以是石油、动物、植物或合成来源的油，例如花生油、大豆油、矿物油、橄榄油、向日葵油和鱼肝油。
非消化道给药的载体(用于皮下、静脉内、动脉内或肌内注射)可包括氯化钠溶液、林格氏葡萄糖、右旋糖和氯化钠、乳酸林格氏液和固定油。静脉内载体包括液体和营养补充剂、电解质补充剂，例如基于林格氏葡萄糖的电解质补充剂等。实例可以是无菌液体，例如水和油、添加或不添加表面活性剂以及其他药学上可接受的佐剂。通常，水、盐水、葡萄糖水溶液和相关糖溶液、以及二醇如丙二醇或聚乙二醇是优选的液体载体，特别是对于可注射溶液。油的实例可以是石油、动物、植物或合成来源的油，例如花生油、大豆油、矿物油、橄榄油、向日葵油和鱼肝油。
药物组合物可进一步包括粘合剂(例如阿拉伯胶、玉米淀粉、明胶、卡波姆、乙基纤维素、瓜尔胶、羟丙基纤维素、羟丙基甲基纤维素、聚维酮)、崩解剂(例如玉米淀粉、马铃薯淀粉、海藻酸、二氧化硅、交联羧甲基纤维素钠、交聚维酮、瓜尔胶、羟基乙酸淀粉钠)、各种pH和离子强度的缓冲剂(例如Tris-HCl、乙酸盐、磷酸盐)、白蛋白或明胶等添加剂、以防止吸收到表面、洗涤剂(例如吐温20、吐温80、普朗尼克F68、胆汁酸盐)、蛋白酶抑制剂、表面活性剂(例如十二烷基硫酸钠)、渗透增强剂、增溶剂(例如甘油、聚乙 二醇甘油)、抗氧化剂(例如抗坏血酸、偏亚硫酸氢钠、丁基化羟基苯甲醚)、稳定剂(例如羟丙基纤维素、羟丙基甲基纤维素)、增粘剂(例如卡波姆、胶体二氧化硅、乙基纤维素、瓜尔胶)、甜味剂(例如阿斯巴甜、柠檬酸)、防腐剂(如硫柳汞、苯甲醇、对羟基苯甲酸酯)、润滑剂(如硬脂酸、硬脂酸镁、聚乙二醇、十二烷基硫酸钠)、流动助剂(如胶体二氧化硅)、增塑剂(如邻苯二甲酸二乙酯、柠檬酸三乙酯)、乳化剂(例如卡波姆、羟丙基纤维素、十二烷基硫酸钠)、聚合物涂层(例如泊洛沙姆或泊洛沙胺)、涂料和成膜剂(例如乙基纤维素、丙烯酸酯、聚甲基丙烯酸酯)和/或佐剂。
本申请提供的药物组合物可以是控释组合物，即其中化合物在给药后的一段时间内释放的组合物。控释或缓释组合物可包括在亲脂性贮库(例如脂肪酸、蜡、油)中的制剂。在一些实施例中，药物组合物可以是速释组合物，即其中整个化合物在给药后立即释放的组合物。
用于递送本申请所述的皮内药物组合物的合适设备可包括短针设备，例如在美国专利4,886,499、5,190,521、5,328,483、5,527,288、4,270,537、5,015,235、5,141,496以及5,417,662中描述的那些。皮内组合物可以通过限制针进入皮肤的有效穿透长度的设备施用，例如PCT公布WO 99/34850中描述的那些及其功能等同物。通过液体喷射注射器和/或通过刺穿角质层并产生到达真皮的射流的针将液体组合物输送到真皮的喷射注射设备可能是合适的。喷射注射设备描述于例如美国专利5,480,381、5,599,302、5,334,144、5,993,412、5,649,912、5,569,189、5,704,911、5,383,851、5,893,397、5,466,220、5,339,163、5,312,335、5,503,627、5,064,413、5,520,639、4,596,556、4,790,824、4,941,880、4,940,460以及PCT公布WO 97/37705和WO 97/13537。使用压缩气体将粉末形式的疫苗加速通过皮肤外层到达真皮的弹道粉末/颗粒递送设备可能是合适的。作为另外一种选择或除此之外，常规注射器可用于皮内施用的经典结核菌素皮内方法中。
可以使用任何方法配制和递送根据本发明合成的mRNA用于体内蛋白质生产。在一些实施例中，将mRNA封装到转移载体中，例如纳米颗粒。除此之外，这种封装的一个目的通常是保护核酸免受可能含有降解核酸和/或引起核酸快 速排泄的系统或受体的酶或化学物质的环境的影响。因此，在一些实施例中，合适的递送载体能够增强其中所含mRNA的稳定性和/或促进mRNA递送至靶细胞或组织。在一些实施例中，纳米颗粒可以是基于脂质的纳米颗粒，例如包括脂质体或基于聚合物的纳米颗粒。在一些实施例中，纳米颗粒可以具有小于约40-100nm的直径。纳米颗粒可包括至少1μg、10μg、100μg、1mg、10mg、100mg、1g或更多mRNA。
在一些实施例中，转运载体是脂质体囊泡、或促进核酸转移至靶细胞和组织的其他手段。合适的转运载体可以包括但不限于脂质体、纳米脂质体、含神经酰胺的纳米脂质体、蛋白脂质体、纳米颗粒、磷硅酸盐纳米颗粒、磷酸钙纳米颗粒、二氧化硅纳米颗粒、纳米晶颗粒、半导体纳米颗粒、聚(D-精氨酸)、纳米树枝状聚合物、淀粉基递送系统、胶束、乳剂、脂质体、质粒、病毒、钙磷酸基核苷酸、适体、肽以及其他载体标签。还考虑使用生物纳米胶囊和其他病毒衣壳蛋白组装体作为合适的转移载体。(人类基因治疗(Hum.Gene Ther.)2008 9月；19(9)：887-95)。
脂质体可以包括一种或以上阳离子脂质、一种或以上非阳离子脂质、一种或以上基于甾醇的脂质、和/或一种或以上PEG-修饰脂质。脂质体可包括三种或更多种不同的脂质组分，脂质的一种不同组分是基于甾醇的阳离子脂质。在一些实施例中，基于甾醇的阳离子脂质是咪唑胆固醇酯或“ICE”脂质(参见WO2011/068810，其通过引用整体并入本申请)。在一些实施例中，基于甾醇的阳离子脂质可构成脂质纳米颗粒(例如，脂质体)中总脂质的不超过70％(例如，不超过65％和60％)。
合适的脂质的实例可包括，例如，磷脂酰化合物(例如，磷脂酰甘油、磷脂酰胆碱、磷脂酰丝氨酸、磷脂酰乙醇胺、鞘脂、脑苷脂和神经节苷脂)。
阳离子脂质的非限制性实例可包括C12-200、MC3、DLinDMA、DLinkC2DMA、cKK-E12、ICE(咪唑基)、HGT5000、HGT5001、OF-02、DODAC、DDAB、DMRIE、DOSPA、DOGS、DODAP、DODMA和DMDMA、DODAC、DLenDMA、DMRIE、CLinDMA、CpLinDMA、DMOBA、DOcarbDAP、DLinDAP、DLincarbDAP、DLinCDAP、KLin-K-DMA、DLin-K-XTC2-DMA和HGT4003 或者其组合。
非阳离子脂质的非限制性实例可以包括神经酰胺、脑磷脂、脑苷脂、二酰基甘油、1,2-二棕榈酰基-sn-甘油基-3-磷酸甘油钠盐(DPPG)、1,2-二硬脂酰-sn-甘油基-3-磷酸乙醇胺(DSPE)、1,2-二硬脂酰-sn-甘油-3-磷酸胆碱(DSPC)、1,2-二棕榈酰-sn-甘油-3-磷酸胆碱(DPPC)、1,2-二油基-sn-甘油-3-磷酸乙醇胺(DOPE)、1,2-二油基-sn-甘油-3-磷脂酰胆碱(DOPC)、1,2-二棕榈酰-sn-甘油基-3-磷酸乙醇胺(DPPE)、1,2-二肉豆蔻酰-sn-甘油-3-磷酸乙醇胺(DMPE)、和1,2-二油酰基-sn-甘油-3-磷酸-(1'-rac-甘油)(DOPG)，1-棕榈酰-2-油酰基-磷脂酰乙醇胺(POPE)、1-棕榈酰-2-油酰基-sn-甘油-3-磷酸胆碱(POPC)、1-硬脂酰基-2-油酰基-磷脂酰乙醇胺(SOPE)、鞘磷脂或者其组合。
在一些实施例中，PEG修饰脂质可以是长度高达5kDa的聚(乙烯)二醇链，其共价连接到具有C6-C20长度的烷基链的脂质上。PEG修饰脂质的非限制性实例可包括DMG-PEG、DMG-PEG2K、C8-PEG、DOGPEG、神经酰胺PEG，和DSPE-PEG、或其组合。
还考虑使用聚合物作为转移载体，无论是单独使用还是与其他转移载体组合使用。合适的聚合物可包括，例如，聚丙烯酸酯、聚烷基氰基丙烯酸酯、聚丙交酯、聚丙交酯-聚乙交酯共聚物、聚己内酯、葡聚糖、白蛋白、明胶、藻酸盐、胶原、壳聚糖、环糊精以及聚乙烯亚胺。基于聚合物的纳米颗粒可包括聚乙烯亚胺(PEI)，例如支链的PEI。
尽管与本文描述的那些类似或等同的方法和材料可用于本发明的实践或测试中，但下文描述了合适的方法和材料。本文提及的所有出版物、专利申请、专利以及其他参考文献都通过引用整体并入。本文引用的参考文献不被认为是要求保护的发明的现有技术。另外，材料、方法和以及实例仅是说明性的，而并非旨在进行限制。
具体实施方式
实例1：(4-氨基-1-((2R，3R，4R，5R)-3，4-双((叔丁基二甲基甲硅烷基)氧基)-5-(((叔丁基二甲基甲硅烷基)氧基)甲基)四氢呋喃基 -2-基)嘧啶-2(1H)-酮)的合成
通过以下反应合成题述化合物：
Figure PCTCN2020074825-appb-000036
将胞嘧啶核苷(2.43g，10mmol)、咪唑(6.13g，90mmol)、叔丁基二甲基氯硅烷以及叔三乙基氯硅烷(9.04g，6mmol)在反应烧瓶中与碱性溶剂(60mL)混合在一起，并在室温下搅拌2天。然后将反应混合物倒入冰水(250mL)中，并用二氯甲烷(2×300mL)萃取洗涤。浓缩有机相，并溶解在50mL 80％乙酸水溶液，在室温下反应30分钟，然后用二氯甲烷(2×300mL)萃取。将碳酸氢钠NaHCO 3颗粒加入有机相，搅拌20分钟，然后水洗。浓缩有机相以得到粗产物，将其通过硅胶柱色谱纯化(从100％正己烷到5％乙酸乙酯-正己烷的洗脱溶剂)以得到纯化合物1(4.68g，80％得率)，白色粉末。
1H NMR(DMSO-d 6)：δ＝7.78-7.73(d，1H，CH)，7.16-7.09(br，2H，NH 2)，5.81-5.75(d，1H，CH)，5.70-5.64(d，1H，CH)，4.09-4.03(m，1H，CH 2)，4.03-3.98(m，1H，CH)，3.91-3.81(m，2H，CH+CH 2a)，3.71-3.64(d，1H，CH 2b)，0.93-0.73(m，27H，CH 3)，0.11-0.00(m，18H，CH 3)。ESI m/z计算值C 27H 55N 3O 5Si 3(M+H)：586.34，实际值：586.71。
实例2：1-((2R，3R，4R，5R)-3，4-双((叔丁基二甲基甲硅烷基)氧基)-5-(((叔丁基二甲基甲硅烷基)氧基)甲基)四氢呋喃基-2-基)-4-氯嘧啶-2(1H)-酮)的合成。
通过以下反应合成题述化合物：
Figure PCTCN2020074825-appb-000037
将Et 4NCl(3g，18mmol)混合到化合物1(2.34g，4mmol)的无水二氯甲烷(60mL)溶液中。然后将亚硝酸化合物(40mmol)滴加到反应混合物中。滴加完成后，反应在室温下继续10小时，转移至分液漏斗，加入饱和碳酸氢钠溶液和二氯甲烷，然后萃取。萃取的二氯甲烷相用浓盐水洗涤，收集并用无水硫酸钠干燥。浓缩有机相，然后在硅胶上进行柱色谱分离(用5％乙酸乙酯-正己烷和20％乙酸乙酯-正己烷的洗脱溶剂)，得到纯化合物2(1.04g，43％得率)，黄色粘稠液体。
1H NMR(DMSO-d 6)：δ＝8.03-7.94(m，1H，CH)，5.89-5.80(d，1H，CH)，5.70-5.62(d，1H，CH 2)，4.07-4.02(br，3H，CH+CH 2a)，4.00-3.93(d，1H，CH)，3.76-3.70(d，1H，CH 2b)，0.98-0.79(m，27H，CH 3)，0.20-0.01(m，18H，CH 3).ESI m/z计算值C 27H 53ClN 2O 5Si 3(M+K)：644.30，实际值：644.81。
实例3：2-(1-((2R，3R，4R，5R)-3，4-双((叔丁基二甲基甲硅烷基)氧基)-5-(((叔丁基二甲基甲硅烷基)氧基)甲基)四氢呋喃基-2-基)-2-氧代-1，2-二氢嘧啶-4-基)胍的合成。
通过以下反应合成题述化合物：
Figure PCTCN2020074825-appb-000038
将盐酸胍(3.3g，34mmol)和90％氢化钠(0.91g，34mmol)添加到反应管中。然后加入14mL无水二甲基甲酰胺(DMF)和乙腈，并在室温下搅拌12小时以活化盐酸胍。停止搅拌，并将反应混合物静置1小时。用一次性注射器的长针提取上清液，并注入含有化合物2(1.04g，1.7mmol)和穴醚或冠醚(1.7mmol)的施伦克(schlenk)反应管中。反应管用橡胶塞覆盖，并加入氮气。反应在50℃下继续进行2小时，然后在室温下进行12小时。通过添加1mL的乙酸来终止反应。浓缩反应溶液，加入40mL水，并用二氯甲烷(3×50mL)萃取。浓缩有机相，然后在硅胶上进行柱色谱分离(从2％乙醇-乙酸乙酯到10％乙醇-乙酸乙酯的洗脱溶剂)，得到纯化合物3(0.15g，14％得率)，白色固体。
1H NMR(DMSO-d 6)： 1H NMR(DMSO-d 6)：δ＝7.98-7.92(d，1H，CH)，7.54-6.47(m，4H，NH)，5.81-5.76(d，1H，CH)，5.75-5.70(d，1H，CH)，4.22-4.19(m，1H，CH)，4.14-4.10(m，1H，CH)，3.90-3.86(m，1H，CH 2a)，3.72-3.66(m，1H，CH)，3.55-3.52(m，1H，CH 2b)，0.89-0.82(m，27H，CH 3)，0.10-0.02(m，18H，CH 3).ESI m/z计算值C 28H 57N 5O 5Si 3(M)：628.05，实际值：628.38。
实例4：2-(1-((2R，3R，4S，5R)-3，4-二羟基-5-(羟基1-甲基)四氢呋喃基-2-基)-2-氧代-1，2-二氢嘧啶-4-基)胍(4-胍基胞苷)的合成。
通过以下反应合成题述化合物：
Figure PCTCN2020074825-appb-000039
将四氢呋喃溶液中的1M四丁基氟化铵(0.56mL，0.56mmol)加入化合物3(100mg，0.16mmol)的无水四氢呋喃溶液中。将反应混合物在室温搅拌3小时，加入3滴氨水，搅拌使反应碱化另外一个小时。浓缩反应混合物，并使用Dowex1×2(OH -)树脂进一步纯化。将产品完全溶于10mL乙醇中，然后置于-20℃的冰箱中重结晶，得到化合物4(30mg，65％得率)，白色固体。
1H NMR(DMSO-d 6)：δ＝7.86-7.84(d，1H，CH)，7.36-6.90(m，4H，NH)，6.00-5.74(m，2H，OH)，5.73-5.69(m，1H，CH)，5.68-5.62(m，1H，CH)，5.33-5.03(br，1H，OH)，3.94-3.87(m，2H，CH)，3.84-3.77(m，1H，CH2a)，3.66-3.61(m，1H，CH)，3.56-3.50(m，1H，CH 2b).ESI m/z计算值C 10H 15N 5O 5：285.11，实际值(M+H)：286.11。
实例5：材料与方法
使用Bruker 400MHz NMR光谱仪测量NMR光谱。使用Thermo q-exactive质谱仪测量质谱(ESI)。使用Merck TLC Silica Gel 60F2541荧光分析板生成薄层色谱。使用规格为200至300目的硅胶进行柱色谱分离。反应在N 2的保护下进行。所有试剂均购自Sigma-Aldrich和SCRC，无需进一步纯化即可使用。反应溶剂是无水试剂。
实例6：4-胍基胞苷-5′-三磷酸或4-胍基脱氧胞苷-5’-三磷酸的合成
本文披露的4-胍基胞苷-5’-三磷酸或4-胍基脱氧胞苷-5’-三磷酸可通过以下反应合成：
Figure PCTCN2020074825-appb-000040
在0℃下，向搅拌的4-胍基胞苷(R 41＝–OH)或4-胍基脱氧胞苷(R 41＝H)(1.11g，3.89mmol)的磷酸三甲酯(20mL)溶液中加入三氯氧磷(0.36mL，3.87mmol)并将混合物搅拌10分钟。将另一部分的三氯氧磷(0.36mL，3.87mmol)添加到反应混合物中，并进一步搅拌40分钟。将含有焦磷酸三丁基胺(5.29g，9.67mmol)、三丁胺(5.60mL，23.49mmol)和乙腈(15mL)的预冷混合物加入到反应物料中并在搅拌下保持10分钟。通过缓慢加入500mL水淬灭反应混合物，然后用二氯甲烷(3×100mL)萃取。将收集的水溶液调节至pH6.5并装载在DEAE琼脂柱上。使用0-1M TEAB的线性梯度洗脱所需产物，并将含有该产物的馏分合并、蒸发，并与水(3×100mL)共蒸发。将获得的TEA盐与高氯酸钠(5.0g)在丙酮(100.0mL)中进行离子交换两次，得到4-胍基胞苷-5’-三磷酸或4-胍基脱氧胞苷-5’-三磷酸的钠盐。
实施例7.利用修饰的荧光素酶报告mRNA在树突状细胞内的表达的实验。
1.1荧光素酶报告(FLuc)的mRNA序列如下(FLuc mRNA，来源：Trilink Biotechnologies)(自然的)：
Figure PCTCN2020074825-appb-000041
Figure PCTCN2020074825-appb-000042
修饰的荧光素酶的mRNA的获得：从荧光素酶的DNA序列，利用转录酶以及常用的试剂条件在体外可以转录为mRNA，在转录过程中，按照修饰C(胞苷)和没有修饰C得比例来获得不同比例的经过修饰的mRNA，其中修饰的mRNA中可以含有不同比例的修饰U的mRNA。序列通过体外合成如下修饰的mRMA,从而形成新的经过修饰的荧光素酶。在如上的序列(SEQ NO:1)中，把胞苷替换成本发明的修饰改性过的C*，如下的改性C*为5种改性的单胞苷修饰的mRNA的表达，修饰比例为100％(即所有的C都替换为下列5种不同的C*修饰，m 4C(N4-甲基胞苷)，m 4Cm(N 4,2'-O-二甲基胞苷)，具体如下表1。、
可以知道，合成经过修饰的mRNA的方法或者途径有很多种，现有的任何可以合成经过修饰的mRNA的方法都可以被运用到本发明的方法来实现。也可以购买商业的试剂盒来进行体外转录。这种实现可以达成100％的修饰，或者一定比例的修饰，例如90％，85％，80％，75％，60％，50％，40％，20％，10％，2％或者0.5％的修饰。例如在前述FLuc mRMA上，可以让所有的胞苷替换为修饰的胞苷，例如本发明的任何一种化学结构，也例如下面所举例的本发明1,2,3和4的具体修饰的胞苷所替换，这种替换的比例可以是100％，当然也可以是不同的比例，这种替换的方式也可以是不同的修饰形式混合替换，而非单一的替换。例如，对 于胞苷的修饰，可以是某些位置的胞苷被本发明1,2,3和4的具体化合物的一种或者几种替换。这种产生经过修饰的mRNA的方法，例如在中国发明专利CN102947450B中有具体的描述，该专利说明书中的每一种方法都是本发明的一个具体实施例子来运用。
表1：实施例子1实验处理数目。
对照 本发明1 本发明2 本发明3 m 4C m 4Cm 本发明4
0％ 100％ 100％ 100％ 100％ 100％ 100％
Figure PCTCN2020074825-appb-000043
(发明1，其中R4为H，R5为H，R2为-OH，R1为-OH，R3：既R3为-OH中H被三磷酸基取代，既：-O-三磷酸基。)
Figure PCTCN2020074825-appb-000044
(发明2，其中，R4为-CH 3，R5为-H，R2为-H，R1为-OH，R3:-OH，其中，H被为三磷酸基取代：-CH 2-O-三磷酸基)
Figure PCTCN2020074825-appb-000045
(发明3，其中R4为-OH，R5为-NH 2，R2为-OH，R1为-OH，R3：既R3为-CH 2-OH，其中，H被三磷酸基取代，既：-O-三磷酸基。)
Figure PCTCN2020074825-appb-000046
(发明4，其中R4为-OH，R5为－CH 3，R2为-OH，R1为-OH，R3：既R3为-OH，其中，H被三磷酸基取代，既：－O－三磷酸基)。
1.2LPP包裹过程按照如下的方法包裹：
1.2.1：磷脂混合液的制备：按比例将磷脂：DOPE：mPEG2000-DSPE＝49：49：2溶解于乙醇溶液。其中，DOPE采购于Avanti、mPEG2000-DSPE采购于cordenpharma，PBS采购于Invitrogen。
1.2.2：mRNA的制备：用BD注射器分别吸取各处理的1mL的mRNA(浓度为0.2mg/mL，总质量0.2mg的表格2中的mRNA)。
1.2.:3：磷脂/mRNA的制备：用BD注射器分别吸取3mL mRNA和3mL磷脂溶液(浓度为12mg/mL)插入微流控芯片(这里的微流控应该是可以产生纳米颗粒的包装的小型设备,请告诉购买的公司或者厂家就可以了)中，设置掺数为：体积 为:9.0mL；流速比为:3:1，总流速为：1mL/min，温度：37.0℃，开始用量为0.35mL，结束用量为0.10mL，得到磷脂/mRNA溶液,即得到磷脂包裹mRNA的颗粒以及磷脂的混合溶液。
1.2.4：离心超滤：将磷脂/mRNA溶液加入到超滤管中进行离心超滤，样品体积为12mL，超滤介质磷酸盐缓冲液体积为12mL，设置超滤掺数为：离心力3400g，离心时间为60min，温度4℃，循环次数为3次。从而获得包裹的各个处理的mRNA载体。
本具体实施例子中的包裹防范为LPP方法，当然可以采用任何其它的方法来包裹mRNA,或者不进行包裹直接用裸的mRNA来传染细胞，组织或者任何生命活体组织等等。当然，可以采用基因枪或者转基因的方法把mRNA转移到细胞中去进行目的蛋白的表达。这些都是现有技术中常规的方法。
1.3细胞传染实验
实验试剂：(1)harvest buffer(25ml)：1.25ml 1M Tris-HCl(pH7.5),25μl 1M DTT,250μl 10％Triton X-100,加水至25ml，4℃保存。(2)ATP buffer(10ml)：1.25ml 1M Tris-HCl(pH7.5),250μl 1M MgCl2,24mg ATP,加水至10ml,20℃保存。(3)luciferin buffer(36ml)：10mg luciferin,36ml 5mM KH2PO4(pH7.8),4℃保存。(4)PBS:20mMNaCl,2.68mMKCl,10mM Na2HPO4,1.76mM KH2PO4,4℃保存。
当然，也可以选择商品化的试剂盒来测试荧光素酶的表达量,表达量的多少直接可以说明mRNA的表达的数量多少。
分别用1.2获得的各个处理的包裹的含有mRNA的载体来传染树突状细胞的方法如下(每一个处理重复3次)：
1.3.1：实验第一天，消化并接种小鼠树突状细胞(购买自丰晖生物，D.2.4细胞)于35mm细胞培养皿，置于5％CO2、饱和湿度的37℃培养箱内培养过夜；
1.3.2：等细胞密度达到70％时用获得的各个处理来转染细胞
1.3.3：转染24小时后，吸去培养液，用冰冷的PBS洗涤细胞。注：荧光酶的酶促反应会被痕量的钙所抑制，故用磷酸钙转染的细胞在收集细胞前应充分洗涤除去含钙介质。
1.3.4：在每个培养皿中加入350μl预冷的harvest buffer,于4℃或冰上放置10min裂解细胞。
1.3.5：在细胞裂解期间，准备足量的1.5mL微量离心管，将ATP buffer与luciferin buffer以1：3.6比例混合成反应液后分装，每管100μl。
1.3.6：依次取等体积的细胞裂解液(100μl)至步骤5中的离心管中，迅速混匀，在发光仪(Luminometer)上读取吸光值。注：发光反应会迅速衰减，将细胞裂解液加入反应液后5秒内必须读取吸光值。
1.3.7：确保以相同操作手法读取全部样品吸光值后。
1.3.8：取剩余裂解液测定LacZ的活性，其读数作为内标用以矫正荧光素酶的读数。
1.3.9：用矫正后的读数作图，分析数据(见图2)。注：荧光素见光易氧化，已稀释未用的荧光素应丢弃。
1.4结果分析
从图2可以看出，荧光mRNA细胞中的表达蛋白(荧光蛋白)的含量与对照(无修饰的胞苷)随着不同的胞苷修饰形式具有明显的变化。本发明的具体的三种修饰方式的胞苷在细胞中的表达量都高于对照处理，经过方差显著性分析，本发明的1和发明2，发明3以及发明4分别与对照和m 4C，为极显著差异(P≤0.01)。m 4Cm(N 4,2'-O-二甲基胞苷)
当本发明与m 4Cm修饰的时候，本发明1和本发明4的方案与其程极显著差异，而本发明2和3与m 4Cm修饰的时候，与其不呈现显著差异，效果相当。
这说明,采用发明修饰的胞苷,在对mRNA表达具有显著的提高或者显著的影响。这种影响可能可以提高mRNA在包装载体中的稳定性，同时也可以能影响在包装载体中以后，载体在运输到细胞内，并且进入细胞核内，mRNA具有更高的稳定性和更好的翻译特性，并被表达出更稳定更具有活性的蛋白。这些多方面的影响最终导致荧光素酶的活性或者数量。从而，相对没有修饰的对照样品来讲， 具有更多的优越性(图2)。
为了让(mRNA)本身具有更强的稳定性，可以在起到核心功能的信使RNA的5‘端或者3’端增加一些结构，让其具有更强的稳定性和翻译蛋白的能力。这种附加的结构是现有技术可以容易实现的。例如，为了防止mRNA降解、增强其稳定性，通常在mRNA的3'端需要进行适当的加尾。因此，准确反映Ploy(A)尾长度对于mRNA在生产过程中的质量控制十分重要。大多数真核生物的mRNA 3’末端都有由100～200个A组成的Poly(A)尾巴，即mRNAPloy(A)尾。该mRNA Poly(A)尾不是由DNA编码的，而是转录后的前mRNA以ATP为前体，由RNA末端腺苷酸转移酶，即Ploy(A)聚合酶催化聚合到3’末端的。已知mRNA Poly(A)尾的功能是：①可能有助mRNA从核到细胞质转运；②避免在细胞中受到核酶降解，增强mRNA的稳定性；③担任核糖体的一个识别信号。这种增加Ploy(A)的结构也可以在体外实现。
在本领域还已知mRNA分子通常具有位于翻译起始密码子之前和未被翻译的翻译终止密码子之后的不同序列的区域。这些区域(分别称为5’端非翻译区(5′UTR)和3’端非翻译区(3′UTR))可影响mRNA稳定性、mRNA定位和与它们连接的mRNA的翻译效率。已知某些5’和3’UTR例如α和β珠蛋白的5’和3’UTR提高mRNA稳定性和mRNA的表达。因此。在某些优选实施方案中，编码重编程因子(例如，iPSC诱导因子)的mRNA展示在细胞中导致更高的mRNA稳定性和更高的mRNA表达的5’UTR和/或3’UTR(例如，α珠蛋白或β珠蛋白5’UTR和/或3’UTR；例如，非洲爪蟾或人α珠蛋白或β珠蛋白5’UTR和/或3’UTR，或例如烟草蚀纹病毒(TEV)5’UTR)。
具体对于核心功能的(mRNA)具有更强的稳定性和其它发明的优越性，可以通过如下专利申请公开的技术来实现。例如中国发明专利CN102947450B的说明书所描述的方法作为本发明的一部分内容。
实施例子8：在mRNA在体外进行体外加帽结构(Ploy(A)情况下对修饰的mRNA表达的影响(树突状细胞)。
为了考察在3‘端加Poly(A)对于翻译效果的影响，对对照和经过100％修饰的发明1-4修饰的荧光素酶表达的影响，在3’增加了120A作为尾巴。按照实施例子1 中的包装方法和传染细胞的方法测定对于酶表达的影响。在3‘端增加Ploy(A)结构后，无论对于修饰或者无修饰的mRNA的表达都有提高，通过图2比较可明显看出。但是，对于不同的修饰方式确显得有些不同，对于发明1的化合物修饰的mRNA提高比较明显，相对发明3修饰的mRNA提高并不十分明显。
同样的实验在传染HEK293细胞的时候，有表现出不同的结果，整体表达水平比在树突状细胞中要高2-3倍，但是趋势确表现一样(具体数据略)。
实施例子9：不同的修饰比例对mRNA表达的影响。
以实施例子1的荧光素酶的mRNA为例子，在该序列中，对于本发明的修饰胞苷的化合物替换其中部分的胞苷，比例为0.5％，5％，10％，20％，30％，40％，50％，70％，80％和90％，具体替换的方法通过体外荧光素酶DNA在转录酶的作用下，具有AUCG的原料供应下，以及常规的方法下进行转录，其中，合成的方法进行控制和替换，即按照以上的比例来替换mRNA中的部分胞苷。考察不同比例的替换对于mRNA表达的影响。具体考察参考实施例子1的方法进行，结果如下图。
从图4可以看出，对于按照发明1的修饰的胞苷来替换没有修饰的胞苷，随着修饰里比例提高，对于目的mRNA表达的也越高。80％-100％修饰的表达量与其它修饰比例为显著性差异。说明，如果希望采用发明1结构的胞苷来修饰mRNA，修饰的比例大于80％以上。
从图5可以看出，对于发明4的胞苷的修饰替换没有吸修饰的胞苷，随着比例的提高，表达水平也逐渐提高，但是在修饰比例在20％-40％处于相对高的表达量，在20％为最高。经过方差分析，20％比例修饰和其它修饰为显著性或者极显著性差异(具体分析过程略)。说明，如果希望采用发明4结构的胞苷来修饰mRNA，修饰的比例处于20％左右为最高。
从图6可以看出，对于发明2的胞苷的修饰替换没有修饰的胞苷，随着比例的提高，表达水平也逐渐提高，但是在修饰比例在20％-40％处于相对高的表达量，在40％为最高。经过方差分析，40％比例修饰和其它修饰为显著性或者极显著性差异(具体分析过程略)。说明，如果希望采用发明2结构的胞苷来修饰mRNA， 修饰的比例处于40％左右为最高。
从图7可以看出，对于发明3的胞苷的修饰替换没有吸修饰的胞苷，随着比例的提高，表达水平也逐渐提高，但是在修饰比例在5％处于相对高的表达量，在5％为最高。经过方差分析，5％比例修饰和其它修饰为显著性或者极显著性差异(具体分析过程略)。说明，如果希望采用发明3结构的胞苷来修饰mRNA，修饰的比例处于5％左右为最高。
另外，本发明这四种不同的修饰方案中，不同的修饰比例，都有最佳修饰比例来确定最高的表达量。这可能是不同位置的取代基不同而影响最终的表达量，但是，都是可能因为共同的在胞苷的4号位置具有共同的结构变化而体现出来的。
对于希望在体内获得高表达量的蛋白，可以通过使用本发明的胞苷的修饰替换mRNA中的胞苷的方式来显著提高在体内的表达量。虽然对于本发明的具体时候方式是针对荧光素酶来进行实验验证的，可以理解，对于其它mRNA，例如某系癌症治疗的mRNA，产染病疫苗或者治疗性疫苗的mRNA，或者其它任何mRNA，通过本发明的胞苷修饰，通过合理的实验，可以找出合适的比例，显著提高目的mRNA在体内的表达量。这是本领域的一般人员可以容易理解的，荧光素酶是表达的报告基因，它的表达量的增加，也表示目的mRNA的增加。
一般技术领域的人应当明白，本实施例子仅仅采用常用的荧光素酶来验证本发明的化合物可以用来对胞苷进行替换并达到修饰的效果，这仅仅是举例的方式来说明，而不能仅仅认为仅仅可以对于荧光素酶具有作用，相反，荧光素酶仅仅是一个常用的工具来验证，当然可以用于那些有意义的核酸，例如信使RNA的修饰，例如很多癌症或者肿瘤相关的mRNA基因、传染病mRNA、或者其它任何相关的mRNA的修饰也具有效果和作用。当然，也包括任何植物、动物、细菌、藻类相关的mRNA的修饰，通过本发明的修饰的胞苷化合物对mRNA进行修饰，可以显著提高目的mRNA在细胞中的表达和翻译。
本发明说明书中提到的所有专利和出版物都表示这些是本领域的公开技术，本发明可以使用。这里所引用的所有专利和出版物都被同样列在参考文献中，跟每一个出版物具体的单独被参考引用一样。这里所述的本发明可以在缺乏任何一种元素或多种元素，一种限制或多种限制的情况下实现，这里这种限制没有特别 说明。例如这里每一个实例中术语“包含”，“实质由……组成”和“由……组成”可以用两者之一的其余2个术语代替。这里的所谓的“一个”仅仅表示“一”的意思，而不排除仅仅只是包括一个，也可以表示包括2个以上。这里采用的术语和表达方式所为描述方式，而不受其限制，这里也没有任何意图来指明此书描述的这些术语和解释排除了任何等同的特征，但是可以知道，可以在本发明和权利要求的范围内做任何合适的改变或修改。可以理解，本发明所描述的实施例子都是一些优选的实施例子和特点，任何本领域的一般技术人员都可以根据本发明描述的精髓下做一些更改和变化，这些更改和变化也被认为属于本发明的范围和独立权利要求以及附属权利要求所限制的范围内。

Claims (49)

  1. 一种具有化学式(I)的化合物：
    Figure PCTCN2020074825-appb-100001
    或其药学上可接受的盐，其中：
    R 1、R 2、R 4和R 5各自独立地选自以下集合所包括的基团，该集合由–H、–OH、–NH 2、卤素基团、取代或未取代的C 1-C 10烷基、取代或未取代的芳香基、取代或未取代的杂芳基、取代或未取代的C 1-C 10芳烷基、取代或未取代的C 1-C 10环烷基、取代或未取代的C 1-C 10杂环、取代或未取代的酰基、–OR 6、–C(O)R 6、–C(O)-O-R 6、–C(O)-NH-R 6以及–N(R 6) 2组成；
    R 3选自以下集合所包括的基团，该集合由–H、–OH、–NH 2、卤素基团、取代或未取代的C 1-C 10烷基、取代或未取代的芳香基、取代或未取代的杂芳基、取代或未取代的C 1-C 10芳烷基、取代或未取代的C 1-C 10环烷基、取代或未取代的C 1-C 10杂环基、取代或未取代的酰基、–OR 6、–C(O)R 6、–C(O)-O-R 6、–C(O)-NH-R 6、和–N(R 6) 2、磷酸基、二磷酸基以及三磷酸基组成；以及其中，R 6为–H、取代或未取代的C 1-C 10烷基以及取代或未取代的酰基。
  2. 根据权利要求1所述的化合物，其中R 1、R 2、R 4和R 5各自独立为–H、–OH或取代或未取代的C 1-C 10烷基。
  3. 根据权利要求1或2所述的化合物，其中R 3为–H、–OH、取代或未取代的C 1-C 10烷基、磷酸基、二磷酸基或三磷酸基。
  4. 根据权利要求1-3中任一项所述的化合物，其中R 1为–OH。
  5. 根据权利要求1-4中任一项所述的化合物，其中R 2为–OH。
  6. 根据权利要求1-5中任一项所述的化合物，其中R 3为–OH。
  7. 根据权利要求1-6中任一项所述的化合物该化合物，其中R 4为–H。
  8. 根据权利要求1-7中任一项所述的化合物该化合物，其中R 5为–H。
  9. 根据权利要求8所述的化合物，具有化学式(I-a)的结构：
    Figure PCTCN2020074825-appb-100002
  10. 根据权利要求1-4中任一项所述的化合物，其中R 2为–H。
  11. 根据权利要求1所述的化合物，具有化学式(I-b)的结构：
    Figure PCTCN2020074825-appb-100003
  12. 根据权利要求5所述的化合物，其中R 2为–OH且R 3为磷酸基。
  13. 根据权利要求12所述的化合物，具有化学式(I-c)的结构：
    Figure PCTCN2020074825-appb-100004
  14. 根据权利要求10所述的化合物，其中R 2为–H且R 3为磷酸基。
  15. 根据权利要求14所述的化合物，具有化学式(I-d)的结构：
    Figure PCTCN2020074825-appb-100005
  16. 根据权利要求5所述的化合物，其中R 2为–OH且R 3为三磷酸基。
  17. 根据权利要求16所述的化合物，具有化学式(I-e)的结构：
    Figure PCTCN2020074825-appb-100006
  18. 根据权利要求10所述的化合物，其中R 2为–H且R 3为三磷酸基。
  19. 根据权利要求18所述的化合物，具有化学式(I-f)的结构：
    Figure PCTCN2020074825-appb-100007
  20. 一种修饰核苷三磷酸(NTP)具有化学式(I-g)的结构：
    Figure PCTCN2020074825-appb-100008
    其中，Y +是阳离子。
  21. 根据权利要求20所述的修饰核苷三磷酸，包括修饰胞苷三磷酸。
  22. 根据权利要求20或21所述的修饰核苷三磷酸，其中Y +选自由Li +、Na +、K +、H +、NH 4 +以及四烷基铵离子组成的组。
  23. 根据权利要求22所述的修饰核苷三磷酸，其中四烷基铵选自由四乙基铵、四丙基铵以及四丁基铵组成的组。
  24. 一种修饰脱氧核苷三磷酸(dNTP)具有化学式(I-h)的结构：
    Figure PCTCN2020074825-appb-100009
    其中，Y +是阳离子。
  25. 根据权利要求24所述的修饰脱氧核苷三磷酸，包括修饰脱氧胞苷三磷酸。
  26. 根据权利要求24或25所述的修饰脱氧核苷三磷酸，其中Y +选自由Li +、Na +、K +、H +、NH 4 +以及四烷基铵离子组成的组。
  27. 根据权利要求26所述的修饰脱氧核苷三磷酸，其中四烷基铵选自由四乙基铵、四丙基铵以及四丁基铵组成的组。
  28. 一种核酸，包括两个或以上共价键连接的核苷酸，其中所述两个或以上共价键连接的核苷酸中的至少一个包括如权利要求1-19中任一项所述的化合物。
  29. 根据权利要求28所述的核酸，其中核酸为核糖核酸(RNA)。
  30. 根据权利要求29所述的核酸，其中RNA包括如权利要求9或13所述的化合。
  31. 根据权利要求29或30所述的核酸，其中RNA为信使RNA(mRNA)。
  32. 根据权利要求31所述的核酸,其中所述的核苷酸中包括如下结构的修饰的胞苷:
    Figure PCTCN2020074825-appb-100010
    其中，R4为H，R5为H，R2为-OH，R1为-OH，R3为-OH，其中H被三磷酸基取代。
  33. 根据权利要求32所述的核酸,其中修饰的比例为30％-100％。
  34. 根据权利要求31所述的核酸,其中所述的核苷酸中包括如下结构的修饰的胞苷:
    Figure PCTCN2020074825-appb-100011
    其中，其中，R4为-CH 3，R5为-H，R2为-H，R1为-OH，R3:-OH，其中，H被为三磷酸基取代。
  35. 根据权利要求33所述的核酸,其中修饰的比例为10-40％或者70％-90％。
  36. 根据权利要求31所述的核酸,其中所述的核苷酸中包括如下结构的修饰的胞苷:
    Figure PCTCN2020074825-appb-100012
    其中，R4为-OH，R5为-NH 2，R2为-OH，R1为-OH，R3：既R3为-CH 2-OH，其中，H被三磷酸基取代。
  37. 根据权利要求36所述的核酸,其中修饰的比例为20-40％。
  38. 根据权利要求31所述的核酸,其中所述的核苷酸中包括如下结构的修饰的胞苷:
    Figure PCTCN2020074825-appb-100013
    其中，R4为-OH，R5为－CH 3，R2为-OH，R1为-OH，R3为-OH，其中，H被三磷酸基取代。
  39. 根据权利要求38所述的核酸,其中修饰的比例为10-40％或者70％-90％。
  40. 根据权利要求39所述的核酸，其中核酸为脱氧核糖核酸(DNA)。
  41. 根据权利要求39所述的核酸，其中DNA包括如权利要求11或15所述的化合。
  42. 一种药物组合物，包括如权利要求1-19中任一项所述的化合物，或其药学上可接受的盐；及其药学上可接受的赋形剂。
  43. 一种药物组合物，包括如权利要求20-33中任一项所述的核酸；及其药学上可接受的赋形剂。
  44. 一种具有化学式(II)的化合物：
    Figure PCTCN2020074825-appb-100014
    或其药学上可接受的盐，其中：
    R 11、R 12和R 13各自独立为–H、–OH或者–O–保护基团；R 14和R 15各自独立选自–H、取代或未取代的C 1-C 10烷基以及取代或未取代的酰基；以及R 16是选自–NH 2、卤素基团以及如下基团
    Figure PCTCN2020074825-appb-100015
  45. 根据权利要求44所述的化合物，其中R 11、R 12和R 13为–O–保护基团。
  46. 根据权利要求45所述的化合物，其中保护基团选自以下集合所包括的基团，该集合由乙酰基、苯甲酰基、苯甲基、β-甲氧基乙氧基甲基醚基、二甲氧基三苯甲基[双-(4-甲氧基苯基)苯基甲基]、甲氧基甲基醚基、甲氧基三苯甲基[(4-甲氧基苯基)二苯基甲基]、对-甲氧基苄基醚基、甲硫基甲基醚基、新戊酰基、四氢吡喃基、四氢呋喃基、三苯甲基(三苯基甲基)、甲硅烷基醚基、甲醚基以及乙氧基乙基醚基组成。
  47. 根据权利要求44或46所述的化合物，其中保护基团是选自以下集合所包括的基团中的甲硅烷基醚基，该集合由三甲基硅烷基醚基(TMS)、叔丁基二苯基甲 硅烷基醚基(TBDPS)、叔丁基二甲基甲硅烷基醚基(TBDMS)以及三异丙基甲硅烷基醚基(TIPS)组成。
  48. 根据权利要求44-47中任一项所述的化合物，其中所述保护基团是TBDMS。
  49. 根据权利要求44所述的化合物，具有结构：
    Figure PCTCN2020074825-appb-100016
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