CN111848712A - 末端加帽的核酸分子 - Google Patents
末端加帽的核酸分子 Download PDFInfo
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- CN111848712A CN111848712A CN202010737580.8A CN202010737580A CN111848712A CN 111848712 A CN111848712 A CN 111848712A CN 202010737580 A CN202010737580 A CN 202010737580A CN 111848712 A CN111848712 A CN 111848712A
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- 125000002347 octyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 229940049964 oleate Drugs 0.000 description 1
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
- 239000013110 organic ligand Substances 0.000 description 1
- 125000001181 organosilyl group Chemical group [SiH3]* 0.000 description 1
- 125000001715 oxadiazolyl group Chemical group 0.000 description 1
- 125000000160 oxazolidinyl group Chemical group 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- KDLHZDBZIXYQEI-UHFFFAOYSA-N palladium Substances [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 1
- MUJIDPITZJWBSW-UHFFFAOYSA-N palladium(2+) Chemical compound [Pd+2] MUJIDPITZJWBSW-UHFFFAOYSA-N 0.000 description 1
- 229940014662 pantothenate Drugs 0.000 description 1
- 235000019161 pantothenic acid Nutrition 0.000 description 1
- 239000011713 pantothenic acid Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 125000001147 pentyl group Chemical group C(CCCC)* 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- JRKICGRDRMAZLK-UHFFFAOYSA-L peroxydisulfate Chemical compound [O-]S(=O)(=O)OOS([O-])(=O)=O JRKICGRDRMAZLK-UHFFFAOYSA-L 0.000 description 1
- 125000004934 phenanthridinyl group Chemical group C1(=CC=CC2=NC=C3C=CC=CC3=C12)* 0.000 description 1
- 125000004625 phenanthrolinyl group Chemical group N1=C(C=CC2=CC=C3C=CC=NC3=C12)* 0.000 description 1
- 125000005561 phenanthryl group Chemical group 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 125000005499 phosphonyl group Chemical group 0.000 description 1
- 150000008298 phosphoramidates Chemical class 0.000 description 1
- 150000003014 phosphoric acid esters Chemical class 0.000 description 1
- 238000001394 phosphorus-31 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 229940075930 picrate Drugs 0.000 description 1
- OXNIZHLAWKMVMX-UHFFFAOYSA-M picrate anion Chemical compound [O-]C1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O OXNIZHLAWKMVMX-UHFFFAOYSA-M 0.000 description 1
- 125000004193 piperazinyl group Chemical group 0.000 description 1
- 125000003386 piperidinyl group Chemical group 0.000 description 1
- 229950010765 pivalate Drugs 0.000 description 1
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- 229920000083 poly(allylamine) Polymers 0.000 description 1
- 229920000333 poly(propyleneimine) Polymers 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 230000023603 positive regulation of transcription initiation, DNA-dependent Effects 0.000 description 1
- 159000000001 potassium salts Chemical class 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- PSHHQIGKVLIVBD-UHFFFAOYSA-N purine-2,4-diamine Chemical compound C1=NC(N)=NC2(N)N=CN=C21 PSHHQIGKVLIVBD-UHFFFAOYSA-N 0.000 description 1
- 125000004309 pyranyl group Chemical group O1C(C=CC=C1)* 0.000 description 1
- 125000003373 pyrazinyl group Chemical group 0.000 description 1
- 125000003226 pyrazolyl group Chemical group 0.000 description 1
- 125000002098 pyridazinyl group Chemical group 0.000 description 1
- 125000000714 pyrimidinyl group Chemical group 0.000 description 1
- 125000000719 pyrrolidinyl group Chemical group 0.000 description 1
- 125000001422 pyrrolinyl group Chemical group 0.000 description 1
- 125000000168 pyrrolyl group Chemical group 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 125000001453 quaternary ammonium group Chemical group 0.000 description 1
- QQXQGKSPIMGUIZ-AEZJAUAXSA-N queuosine Chemical compound C1=2C(=O)NC(N)=NC=2N([C@H]2[C@@H]([C@H](O)[C@@H](CO)O2)O)C=C1CN[C@H]1C=C[C@H](O)[C@@H]1O QQXQGKSPIMGUIZ-AEZJAUAXSA-N 0.000 description 1
- 125000002294 quinazolinyl group Chemical group N1=C(N=CC2=CC=CC=C12)* 0.000 description 1
- 125000005493 quinolyl group Chemical group 0.000 description 1
- 125000001567 quinoxalinyl group Chemical group N1=C(C=NC2=CC=CC=C12)* 0.000 description 1
- 239000012857 radioactive material Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000006722 reduction reaction Methods 0.000 description 1
- 238000006268 reductive amination reaction Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 239000003161 ribonuclease inhibitor Substances 0.000 description 1
- DWRXFEITVBNRMK-JXOAFFINSA-N ribothymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 DWRXFEITVBNRMK-JXOAFFINSA-N 0.000 description 1
- 238000006798 ring closing metathesis reaction Methods 0.000 description 1
- 238000007363 ring formation reaction Methods 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 239000012898 sample dilution Substances 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 229930195734 saturated hydrocarbon Natural products 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 125000002914 sec-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 125000000467 secondary amino group Chemical group [H]N([*:1])[*:2] 0.000 description 1
- JRPHGDYSKGJTKZ-UHFFFAOYSA-N selenophosphoric acid Chemical class OP(O)([SeH])=O JRPHGDYSKGJTKZ-UHFFFAOYSA-N 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- AWUCVROLDVIAJX-GSVOUGTGSA-N sn-glycerol 3-phosphate Chemical compound OC[C@@H](O)COP(O)(O)=O AWUCVROLDVIAJX-GSVOUGTGSA-N 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- CDBYLPFSWZWCQE-UHFFFAOYSA-L sodium carbonate Substances [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- RMLUKZWYIKEASN-UHFFFAOYSA-M sodium;2-amino-9-(2-hydroxyethoxymethyl)purin-6-olate Chemical compound [Na+].O=C1[N-]C(N)=NC2=C1N=CN2COCCO RMLUKZWYIKEASN-UHFFFAOYSA-M 0.000 description 1
- ZHZRSXKCIPFRLW-UHFFFAOYSA-N sodium;[5-(2-amino-6-oxo-3h-purin-9-yl)-3,4-dihydroxyoxolan-2-yl]methyl dihydrogen phosphate Chemical compound [Na+].C1=2NC(N)=NC(=O)C=2N=CN1C1OC(COP(O)(O)=O)C(O)C1O ZHZRSXKCIPFRLW-UHFFFAOYSA-N 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- WGRULTCAYDOGQK-UHFFFAOYSA-M sodium;sodium;hydroxide Chemical compound [OH-].[Na].[Na+] WGRULTCAYDOGQK-UHFFFAOYSA-M 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 229940063673 spermidine Drugs 0.000 description 1
- 229940013618 stevioside Drugs 0.000 description 1
- 229910052682 stishovite Inorganic materials 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 1
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 150000003892 tartrate salts Chemical class 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 125000001302 tertiary amino group Chemical group 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- FAGUFWYHJQFNRV-UHFFFAOYSA-N tetraethylenepentamine Chemical compound NCCNCCNCCNCCN FAGUFWYHJQFNRV-UHFFFAOYSA-N 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- WHRNULOCNSKMGB-UHFFFAOYSA-N tetrahydrofuran thf Chemical compound C1CCOC1.C1CCOC1 WHRNULOCNSKMGB-UHFFFAOYSA-N 0.000 description 1
- 125000003718 tetrahydrofuranyl group Chemical group 0.000 description 1
- 125000001412 tetrahydropyranyl group Chemical group 0.000 description 1
- PHCBRBWANGJMHS-UHFFFAOYSA-J tetrasodium;disulfate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O PHCBRBWANGJMHS-UHFFFAOYSA-J 0.000 description 1
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 125000001113 thiadiazolyl group Chemical group 0.000 description 1
- 125000001984 thiazolidinyl group Chemical group 0.000 description 1
- 125000000335 thiazolyl group Chemical group 0.000 description 1
- 125000001544 thienyl group Chemical group 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- 125000004568 thiomorpholinyl group Chemical group 0.000 description 1
- 229930192474 thiophene Natural products 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 229960003087 tioguanine Drugs 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 125000005490 tosylate group Chemical group 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 125000001425 triazolyl group Chemical group 0.000 description 1
- IMFACGCPASFAPR-UHFFFAOYSA-O tributylazanium Chemical compound CCCC[NH+](CCCC)CCCC IMFACGCPASFAPR-UHFFFAOYSA-O 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 229910052905 tridymite Inorganic materials 0.000 description 1
- 150000008648 triflates Chemical class 0.000 description 1
- WJKHJLXJJJATHN-UHFFFAOYSA-N triflic anhydride Chemical compound FC(F)(F)S(=O)(=O)OS(=O)(=O)C(F)(F)F WJKHJLXJJJATHN-UHFFFAOYSA-N 0.000 description 1
- 125000002264 triphosphate group Chemical group [H]OP(=O)(O[H])OP(=O)(O[H])OP(=O)(O[H])O* 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- ZDPHROOEEOARMN-UHFFFAOYSA-N undecanoic acid Chemical class CCCCCCCCCCC(O)=O ZDPHROOEEOARMN-UHFFFAOYSA-N 0.000 description 1
- RVCNQQGZJWVLIP-VPCXQMTMSA-N uridin-5-yloxyacetic acid Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(OCC(O)=O)=C1 RVCNQQGZJWVLIP-VPCXQMTMSA-N 0.000 description 1
- YIZYCHKPHCPKHZ-UHFFFAOYSA-N uridine-5-acetic acid methyl ester Natural products COC(=O)Cc1cn(C2OC(CO)C(O)C2O)c(=O)[nH]c1=O YIZYCHKPHCPKHZ-UHFFFAOYSA-N 0.000 description 1
- NQPDZGIKBAWPEJ-UHFFFAOYSA-N valeric acid Chemical class CCCCC(O)=O NQPDZGIKBAWPEJ-UHFFFAOYSA-N 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- 239000003039 volatile agent Substances 0.000 description 1
- QAOHCFGKCWTBGC-QHOAOGIMSA-N wybutosine Chemical compound C1=NC=2C(=O)N3C(CC[C@H](NC(=O)OC)C(=O)OC)=C(C)N=C3N(C)C=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O QAOHCFGKCWTBGC-QHOAOGIMSA-N 0.000 description 1
- QAOHCFGKCWTBGC-UHFFFAOYSA-N wybutosine Natural products C1=NC=2C(=O)N3C(CCC(NC(=O)OC)C(=O)OC)=C(C)N=C3N(C)C=2N1C1OC(CO)C(O)C1O QAOHCFGKCWTBGC-UHFFFAOYSA-N 0.000 description 1
- JCZSFCLRSONYLH-QYVSTXNMSA-N wyosin Chemical compound N=1C(C)=CN(C(C=2N=C3)=O)C=1N(C)C=2N3[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O JCZSFCLRSONYLH-QYVSTXNMSA-N 0.000 description 1
- 229940075420 xanthine Drugs 0.000 description 1
Classifications
-
- 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
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
- C07F9/65616—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings containing the ring system having three or more than three double bonds between ring members or between ring members and non-ring members, e.g. purine or analogs
-
- 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
- C07H19/16—Purine radicals
- C07H19/167—Purine radicals with ribosyl as the saccharide radical
-
- 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
- C07H19/16—Purine radicals
- C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
- C07H19/207—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine adenine dinucleotide or nicotinamide-adenine dinucleotide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/5759—Products of obesity genes, e.g. leptin, obese (OB), tub, fat
Abstract
本公开内容涉及末端加帽的核酸分子,尤其涉及在5’‑末端、3’‑末端或5’‑末端和3’‑末端含有修饰的核酸,并且公开了可用于制备经修饰的核酸的化合物。与未修饰的核酸相比,经修饰的核酸具有改善的表达、较低的免疫原性和改善的稳定性。
Description
本申请为2015年12月16日提交的、发明名称为“末端加帽的核酸分子”的PCT申请PCT/IB2015/059697的分案申请,所述PCT申请进入中国国家阶段的日期为2017年8月15日,申请号为201580076159.4。
背景技术
分离的核酸,特别是编码蛋白质的那些,是各种临床应用的有吸引力的候选者。特别是编码蛋白质的RNA(例如mRNA)为临床应用提供了许多潜在的优点。例如,RNA可以体内、体外或离体转染到细胞中以诱导用于治疗或诊断疾病的期望的治疗或诊断蛋白质的表达。然而,RNA治疗的潜力受RNA的稳定性和半衰期以及编码的蛋白质的表达水平的限制。
天然存在的mRNA含有5’帽结构,其有助于稳定RNA并且是真核基因表达的基础(Shuman,S.等人Mol.Microbiol.1995,17,405-410)。这些mRNA还含有3’poly A尾。两种修饰都能稳定和改善编码的蛋白质的翻译。在真核信使RNA(mRNA)和许多病毒RNA的5’末端发现的5’-帽结构由通过5’-5’三磷酸连接与前mRNA的5’-末端核苷连接的N7-甲基鸟苷核苷组成(Shatkin,A.J.Cell 1976,2,645-53;Shuman,S.Prog.Nucleic AcidRes.Mol.Biol.2001,66,1-40;Decroly,E.等人PLoS Pathog.2011,7,e1002059)。这种帽结构实现最终导致mRNA翻译的许多作用。RNA加帽对于其他过程也是重要的,例如RNA剪接和从细胞核导出和避免通过细胞先天免疫机制的mRNA识别(Daffis,S.等人Nature 2010,468,452-6;Züst,R.等人Nat.Immunol.2011,12,137-43)。已经描述了许多合成的帽类似物。参见例如WO2009/149253、WO2011/015347。
合成的mRNA通常通过使用RNA聚合酶和DNA模板,然后通过酶促加入5’-帽和3’-末端的酶促合成进行制备(Peyrane,F.等人Nucleic Acids Res.2007,35,e26)。然而,该方法昂贵且难以控制,因此对于商业规模生产是不合需要的。
因此,需要在5’-末端、3’-末端或者5’-末端和3’-末端被修饰的RNA,其在可以被有效产生,并且具有改善的由RNA编码的产物(例如蛋白质)的表达、降低免疫原性和/或提高稳定性。
发明内容
本公开内容涉及在5’-末端、3’-末端或5’-末端和3-末端两者都含有修饰的核酸分子(例如RNA和DNA分子)。核酸分子优选为编码产物如多肽或核酸的RNA分子,更优选为mRNA,包括用于CRISPR基因组编辑技术的编码Cas9蛋白的mRNA和/或用于CRISPR基因组编辑技术的sgRNA(或crRNA和tracrRNA)的3’-末端。
一方面,本公开涉及式I化合物及其盐(优选药学上可接受的盐):
其中
Z1是
Z2是不存在或是连接部分(linking moiety),所述连接部分选自–O–、–S–、任选取代的低级烷基、任选取代的氨基烷基、任选取代的芳基、任选取代的杂芳基、任选取代的环烷基、任选取代的环烯基、任选取代的杂环基、
A1和A3独立地选自不存在、NH、S和O;
A2是不存在或选自>CR6R7、>NR6、>NNR6R7、>NOR6、>S和>O;
Y是不存在或是连接部分,所述连接部分选自任选取代的低级烷基、任选取代的烯基、任选取代的炔基、–(CH2)nOR15、–(CH2)nCOOR15和–(CH2)nC(O)NR12;
R1选自H、任选取代的环烷基、任选取代的环烯基、任选取代的芳基、任选取代的杂芳基和任选取代的杂环基;
R2选自H、–OH、任选取代的烷基、–C(O)NR12R13和–NR12R13;
R5、R6和R8独立地选自H、任选取代的烷基、聚胺、PEGs、–(CH2)n1NR12R13、–(CH2)n1NR14C(O)R15、–(CH2)n1OR15、–(CH2)n1C(O)OR15、–(CH2)n1C(O)R15、–(CH2)n1C(O)NR12R13、–O–(CH2)n3–C(O)–(NR12)2–C(O)–X2、–O–(CH2)n3–C(O)–[NR12–C(O)–(CH2)n3]1-3–X2,或R6和R8一起形成环,该环任选被取代且含有10-80个环原子,其中10-40个环原子可以是杂原子,或者R6和R7一起形成3-8元环,其任选被取代且其中1-6个环原子可以是杂原子;
R7、R11、R12、R13、R14、R15和R16独立地选自H、任选取代的低级烷基和任选取代的酰基;
R9选自H和任选取代的低级烷基;
R10选自H、–NR12R13和–OR16;
n是1-4;
n1是0-10;
n2是1-12;
n3是1-8;
X选自O、S、NH和任选取代的烷二基;
R3和R4独立地选自H和–OR16,或R3和R4一起形成O-Q-O;
Q选自–CH2–和–C(Me)2–;
X2选自亲和部分和检测部分,且
Xn是核碱基。
在一些方面,式I化合物在5’-末端和3’-末端被修饰并且是式II、III、IV化合物或其盐(优选药学上可接受的盐),
式II、III和IV中的变量如式I中所定义,前提是-A1–R5和-A3–R8不都是–OH,或A2不是不存在。在一些实施方案中,所述化合物是式II、III或IV化合物,前提是-A1–R5和-A3–R8不都是–OH,或A2不是不存在。
在其它方面中,式I化合物在5’-末端被修饰并且在3’-末端未被修饰,且是式VI、式VII、式VIII化合物或其盐(优选药学上可接受的盐),
式VI、VII和VIII中的变量如式I中所定义,前提是-A1–R5和-A3–R8都是–OH且A2是不存在。
在某些优选的实施方案中,所述化合物是式I–IV和VI–VIII中任何一式的化合物,其中Y是烷基连接部分,优选低级烷基连接部分例如(-CH2-);且R1是取代的芳基或取代的杂芳基,例如苯基取代的芳基或苯基取代的杂芳基。在特别优选的实施方案中,
在其它方面中,式I化合物在3’-末端被修饰且在5’-末端未被修饰,且是式V化合物或其盐(优选药学上可接受的盐)
式I–VIII化合物含有核碱基Xn,其可以是任何期望的核碱基例如腺嘌呤、鸟嘌呤、胞嘧啶、尿嘧啶,或修饰的核碱基例如假尿嘧啶。优选的核碱基是腺嘌呤。
式I–VIII化合物中的RNA可以是任何期望的RNA分子,但其优选编码产物例如蛋白质。
在其它方面中,本公开涉及鸟苷或嘌呤衍生物,其可以用于制备本文所公开的5’-末端修饰的核酸(例如RNA)。在特别方面,本公开涉及式IX、X、XI化合物或其盐(药学上可接受的盐):
在式IX、X和XI的每一式中,
n4是0-2;并且其它变量如式I中所定义。在该方面的某些优选的实施方案中,Y是烷基连接部分,优选低级烷基连接部分例如(-CH2-);且R1是取代的芳基或取代的杂芳基,例如苯基取代的芳基或苯基取代的杂芳基。在特别优选的实施方案中,-Y-R1可以是
本发明还涉及使用和制备本文公开的化合物的方法。
发明详述
尽管下面详细描述了本发明,但是应当理解,本发明不限于本文所述的特定方法、方案和试剂,因为它们可以变化。还应当理解,本文使用的术语仅用于描述特定实施方案的目的,并不意图限制本发明的范围,本发明的范围将仅由所附权利要求限制。除非另有定义,本文使用的所有技术和科学术语具有与本领域普通技术人员通常理解的相同的含义。
本公开内容涉及在5’-末端、3’-末端或者5’-末端和3-末端含有修饰的核酸分子(例如RNA和DNA分子)。核酸分子优选是编码产物如多肽或核酸的RNA分子,更优选是mRNA。如本文公开的修饰的RNA或mRNA可以使用任何期望的方法如酶促合成或化学合成来制备。5’-末端修饰包括基于鸟苷或嘌呤的帽结构,其增加由RNA编码的产物(例如蛋白质)的表达,降低免疫原性和/或提高RNA的稳定性。3’-末端修饰包括在RNA的末端核糖的3’和2'位上的顺式二醇的修饰,例如通过在这些位置之间插入环原子或用其它取代基替换一个或两个羟基,以增加由RNA编码的产物(例如蛋白质)的表达和/或提高RNA的稳定性(例如,通过降低降解速率)。如果需要,3’-末端修饰可以包括多个功能部分,例如亲和部分或检测部分。含有这些部分的化合物特别可用于例如成像、生物化学分析和生物缀合。
如本文所述和所示例,已经制备了含有本文公开的基于鸟苷或嘌呤的5’-末端帽结构的RNA。这些修饰的RNA显示结合eIF4E并在细胞中翻译。还制备了3’-末端RNA,与未修饰的RNA相比,其半衰期和表达增加。
一方面,本公开涉及含有5’-末端帽结构、3’-末端修饰或者5’-末端帽结构和3’-末端修饰的RNA分子。这样的RNA分子是式I化合物和其盐(优选药学上可接受的盐):
其中
Z1是
A1和A3独立地选自不存在、NH、S和O;
A2是不存在或选自>CR6R7、>NR6、>NNR6R7、>NOR6、>S和>O;
Y是不存在或是连接部分,所述连接部分选自任选取代的低级烷基、任选取代的烯基、任选取代的炔基、–(CH2)nOR15、–(CH2)nCOOR15和–(CH2)nC(O)NR12;
R1选自H、任选取代的环烷基、任选取代的环烯基、任选取代的芳基、任选取代的杂芳基和任选取代的杂环基;
R2选自H、–OH、任选取代的烷基、–C(O)NR12R13和–NR12R13;
R5、R6和R8独立地选自H、任选取代的烷基、聚胺、PEGs、–(CH2)n1NR12R13、–(CH2)n1NR14C(O)R15、–(CH2)n1OR15、–(CH2)n1C(O)OR15、–(CH2)n1C(O)R15、–(CH2)n1C(O)NR12R13、–O–(CH2)n3–C(O)–(NR12)2–C(O)–X2、–O–(CH2)n3–C(O)–[NR12–C(O)–(CH2)n3]1-3–X2,或R6和R8一起形成环,该环任选被取代且含有10-80个环原子,其中10-40个环原子可以是杂原子,或者R6和R7一起形成3-8元环,其任选被取代且其中1-6个环原子可以是杂原子;
R7、R11、R12、R13、R14、R15和R16独立地选自H、任选取代的低级烷基和任选取代的酰基;
R9选自H和任选取代的低级烷基;
R10选自H、–NR12R13和–OR16;
n是1-4;
n1是0-10;
n2是1-12;
n3是1-8;
X选自O、S、NH和任选取代的烷二基;
R3和R4独立地选自H和–OR16,或R3和R4一起形成O-Q-O;
Q选自–CH2–和–C(Me)2–;
X2选自亲和部分和检测部分,且
Xn是核碱基。
在某些优选的实施方案中,所述化合物是式I化合物,其中
Z1是
Y是烷基连接部分,优选低级烷基连接部分例如(-CH2-);且
在其它优选的实施方案中,所述化合物是式I化合物,其中-A1–R5和-A3–R8不都是–OH,或A2不是不存在。在另一优选的实施方案中,-A1–R5和-A3–R8不都是–OH,且A2不是不存在。
在一些方面,式I化合物在5’-末端和3’-末端都被修饰且是式II、III或IV化合物或其盐,
式II、III和IV中的变量如式I中所定义,前提是-A1–R5和-A3–R8不都是–OH,或A2不是不存在。在一些实施方案中,所述化合物是式II、III或IV化合物,前提是-A1–R5和-A3–R8不都是–OH,或A2不是不存在。
在该方面的某些优选的实施方案中,Y是烷基连接部分,优选低级烷基连接部分例如(-CH2-);且R1是取代的芳基或取代的杂芳基,例如苯基取代的芳基或苯基取代的杂芳基。在特别优选的实施方案中,-Y-R1可以是
在其它方面中,式I化合物在5’-末端被修饰且在3’-末端未被修饰,且是式VI、式VII、式VIII化合物或其盐,
式VI、VII和VIII中的变量如式I中所定义,前提是-A1–R5和-A3–R8都是–OH且A2是不存在。在该方面的某些优选的实施方案中,Y是烷基连接部分,优选低级烷基连接部分例如(-CH2-);且R1是取代的芳基或取代的杂芳基,例如苯基取代的芳基或苯基取代的杂芳基。在特别优选的实施方案中,-Y-R1可以是
在其它方面中,式I化合物在3’-末端被修饰且在5’-末端未被修饰,且是式V化合物或其盐
式I–VIII化合物包含核碱基Xn,其可以是任何期望的核碱基,例如腺嘌呤、鸟嘌呤、胞嘧啶、尿嘧啶,或修饰的核碱基例如假尿嘧啶。优选的核碱基是腺嘌呤。
式I-VIII化合物中的RNA可以是任何期望的RNA分子,但其优选编码产物例如蛋白质。合适的蛋白质包括治疗性蛋白质,例如抗体或其抗原结合片段、受体、细胞因子和生长因子;抗原,例如来自病原体或肿瘤细胞的抗原。RNA例如mRNA可以含有核糖-磷酸骨架,其具有与核糖环的1”位结合的常见核碱基腺嘌呤、胞嘧啶、鸟嘌呤和尿嘧啶。如果需要,RNA中可以以需要的程度包含一个或多个修饰的碱基。例如,0.1%至100%的核碱基可以是修饰的碱基,例如假尿嘧啶。如果需要,RNA分子可以含有一个或多个氨基磷酸酯、硫代磷酸酯、甲基膦酸酯、硒代磷酸酯(phosphoroselenoate)或其它合适的连接(linkages)。
在其它方面,本公开涉及可用于制备本文公开的5’-末端修饰的核酸(例如RNA)的鸟苷或嘌呤衍生物。
在具体方面,本公开涉及式IX、X、XI的化合物或其盐(药学上可接受的盐):
在各式IX、X和XI中,
n4是0-2;且其它变量如式I中所定义。在该方面的某些优选的实施方案中,Y是烷基连接部分,优选低级烷基连接部分例如(-CH2-);且R1是取代的芳基或取代的杂芳基,例如苯基取代的芳基或苯基取代的杂芳基。在特别优选的实施方案中,-Y-R1可以是
应用末端加帽的核酸的方法
本文公开的末端加帽的核酸可用于多种目的,包括诱导细胞产生所需蛋白质,用于诊断或治疗目的。例如,编码蛋白质的末端加帽的mRNA可以在体内或体外转染到细胞中。用于转染mRNA的合适方法是本领域公知的,并且包括例如使用阳离子聚合物、磷酸钙或阳离子脂质以促进转染的方法以及直接注射、生物射弹(biolistic)粒子递送、电穿孔、激光照射、超声和磁性纳米颗粒法。参见例如Kim等人,Anal Bioanal Chem.397:3173-3178(2010)。
待转染的细胞优选是动物细胞,例如来自哺乳动物、鱼、鸟,更优选人。合适的动物受试者包括例如牛、猪、马、鹿、绵羊、山羊、野牛(bison)、兔、猫、狗、鸡、鸭、火鸡等。
本发明的末端加帽的RNA分子也可以递送到离体细胞,例如从个体患者外植的细胞(例如,淋巴细胞、骨髓抽吸物、组织活检),随后通常在对已经用末端加帽的RNA分子转染的细胞进行选择后将细胞重新植入到患者中。递送给患者的适当量的细胞将随着患者状况和期望的效果而变化,这可由本领域技术人员来确定。优选地,使用的细胞是自体的,即从被治疗的患者获得的细胞。
末端加帽的核酸(例如,mRNA)可用于被称为CRISPR(规律成簇间隔短回文重复序列)的基因组编辑技术,其是细菌抗病毒防御系统的一部分,并且可用于诱导真核细胞中的靶向基因组编辑。当用作基因组编辑工具时,II型CRISPR系统通常由2或3种组分组成:是RNA依赖性DNA核酸内切酶的Cas9蛋白,每个DNA链切割位点有一个或两个RNA(天然2RNA系统包括CRISPR-RNA(crRNA)和反式激活(tracrRNA),而在单RNA系统中,2个crRNA和tracrRNA被重新设计成单功能转录物,称为单向导RNA(sgRNA))。CRISPR编辑的标准系统可以在慢病毒载体中编码sgRNA或多个RNA和Cas9蛋白。然而,病毒载体的挑战在于对其可以编码的另外的基因的大小是有限制的,并且长度为~1000-2000个氨基酸的大小的Cas9蛋白质可能难以在病毒载体中编码。使用与体外转录的sgRNA(或cr RNA+tracrRNA)共同配制的体外转录的Cas9 mRNA的所有RNA CRISPR系统可共同配制用于在体内、体外或离体转染至细胞以特异性编辑转染的细胞的基因组。本文公开的末端加帽的mRNA非常适合于该应用。
末端加帽的核酸也可以用于各种生化和分析目的。例如,末端加帽的核酸可用于成像研究,研究RNA的代谢和研究RNA与其他生物分子的相互作用。
合成流程
本发明的化合物可以通过以下流程或实施例中描述的途径制备。
流程1:式VI所定义的通式结构I的化学加帽的mRNA可以如下获得:使mRNA-5’-单磷酸与通式结构II的咪唑活化的帽结构在适当缓冲的盐水溶液(缓冲液:HEPES、TRIS、MES、PBS)在范围为5.5-7.5的合适的pH的条件下、在存在或不存在有机溶剂如DMF和DMSO的情况下、在合适的路易斯酸性活化剂如MnCl2、NiCl2、ZnCl2存在下反应。
流程2:其中Y和R1如式I中定义的通式结构II的化合物可以如下制备:用咪唑或N-甲基-咪唑、合适的叔膦例如三苯基膦、合适的氧化剂如2,2'-二吡啶基二硫化物以及合适的碱(例如三乙胺)在合适的溶剂如DMF、DMSO、NMP、磷酸三甲酯中,在合适的温度例如室温下处理磷酸酯III(三丁基铵盐),(a)M.Lewdorowicz,Y.Yoffe,J.Zuberek,J.Jemielity,J.Stepinski,R.Kierzek,R.Stolarski,M.Shapira,E.Darzynkiewicz,RNA 2004,10,1469;b)R.Worch,J.Stepinski,A.Niedzwiecka,M.Jankowska-Anyszka,C.Mazza,S.Cusack,R.Stolarski,E.Darzynkiewicz,Nucleosides Nucleotides and Nucleic Acids 2005,24,1131;c)M.Warminski,J.Kowalska,J.Buck,J.Zuberek,M.Lukaszewicz,C.Nicola,A.N.Kuhn,U.Sahin,E.Darzynkiewicz,J.Jemielity,Bioorg.Med.Chem.,2013,23,3753)。
流程3:其中Y和R1如式I中定义的通式结构III的化合物可以如下制备:使活化的磷酸酯IV与磷酸三乙铵或类似的磷酸盐在合适的溶剂如DMF、DMSO或水中,在诸如氯化锌、氯化镁或氯化锰的路易斯酸存在下,在合适的温度如室温下反应,(a)M.Lewdorowicz,Y.Yoffe,J.Zuberek,J.Jemielity,J.Stepinski,R.Kierzek,R.Stolarski,M.Shapira,E.Darzynkiewicz,RNA 2004,10,1469;b)R.Worch,J.Stepinski,A.Niedzwiecka,M.Jankowska-Anyszka,C.Mazza,S.Cusack,R.Stolarski,E.Darzynkiewicz,NucleosidesNucleotides and Nucleic Acids 2005,24,1131;c)M.Warminski,J.Kowalska,J.Buck,J.Zuberek,M.Lukaszewicz,C.Nicola,A.N.Kuhn,U.Sahin,E.Darzynkiewicz,J.Jemielity,Bioorg.Med.Chem.,2013,23,3753)。
其中Y和R1如式I中定义的通式结构III的化合物可以通过使用合适的烷基化试剂如烷基卤化物、三氟甲磺酸酯或甲磺酸酯在合适的溶剂如DMF、DMSO或NMP中,在范围为室温至60℃的适宜反应温度下烷基化V来制备。
当X=O时,其中Y和R1如式I中定义的通式结构III的化合物也可以通过使VI与二磷酸四氯化物(diphosphoric acid tetrachloride)在合适的溶剂如磷酸三甲酯中、在0℃至室温的适当的温度下反应来制备(J.Emsley,J.Moore,P.B.Udy,J.Chem.Soc.(A)Inorg.Phys.Theor.1971,2863)。
当X=NH时,其中Y和R1如式I中定义的通式结构III的化合物也可以如下制备:使IV与亚氨基-双(磷酰二氯)在合适的溶剂如磷酸三甲酯中在0℃至室温的适当的温度反应(A.M.Rydzik,M.Kulis,M.Lukaszewicz,J.Kowalska,J.Zuberek,Z.M.Darzynkiewicz,E.Darzynkiewicz,J.Jemielity,Bioorganic&Med.Chem.2012,20,1699)。
当X=CH2时,其中Y和R1如式I中定义的通式结构III的化合物也可以如下制备:使IV与亚甲基双(膦酰二氯)在合适的溶剂如磷酸三甲酯中在适当的温度例如0℃或室温下反应(a)M.Honcharenko,M.Zytek,B.Bestas,P.Moreno,J.Jemielity,E.Darzynkiewicz,C.I.E.Smith,R.Stroemberg,Bioorg.Med.Chem.,2013,21,7921;b)M.Kalek,J.Jemielity,Z.M.Darzynkiewicz,E.Bojarska,J.Stepinski,R.Stolarski,R.E.Davis,E.Darzynkiewic,Bioorg.Med.Chem.2006,14,3223;c)M.Kalek,J.Jemielity,J.Stepinski,R.Stolarski,E.Darzynkiewics,Tetrahedron Lett.2005,46,2417)。
流程4:其中Y和R1如式I中定义的通式结构IV的化合物可以如下制备:用咪唑、合适的叔膦如三苯基膦、合适的氧化剂例如2,2'-二吡啶基二硫化物和合适的碱如三乙胺在合适的溶剂如DMF或DMSO中,在合适的温度例如室温下处理磷酸酯VII(三丁基铵盐)(P.C.Joshi,M.F.Aldersley,D.V.Zagorevskii,J.P.Ferris,Nucleosides,Nucleotidesand Nucleic Acids2012,31,7,536)。
流程5:其中Y和R1如式I中定义的通式结构VII的化合物可以如下制备:使用合适的烷基化试剂如烷基卤化物、三氟甲磺酸酯或甲磺酸酯在合适的溶剂如DMF或DMSO中,在范围为室温至50℃的适宜的反应温度下烷基化VIII。
其中Y和R1如式I所定义的通式结构VIII的化合物可以如下获得:在合适的溶剂如磷酸三甲酯中,在范围为0℃或室温的合适的温度,使X与合适的磷酰化试剂例如磷酰氯反应。
其中Y和R1如式I中定义的通式结构VII的化合物也可以如下制备:在合适的溶剂如磷酸三甲酯中,在范围为0℃至室温的合适的温度,使IX与合适的磷酰化试剂例如磷酰氯反应。
其中Y和R1如式I中定义的通式结构IX的化合物可以如下制备:使用合适的烷基化试剂如烷基卤化物、三氟甲磺酸酯或甲磺酸酯在合适的溶剂如DMF或DMSO中在范围为室温至50℃的适宜的反应温度烷基化X。
流程6:其中Y和R1如式I中定义的通式结构VI的化合物可以如下制备:使用合适的烷基化试剂如烷基卤化物、三氟甲磺酸酯或甲磺酸酯在合适的溶剂如DMF或DMSO中,在范围为室温至50℃的适宜的反应温度烷基化X。
流程7:当X=Br、Cl、I且A=CH3时,通式结构XI和XII的化合物可以使用合适的自由基引发剂如偶氮二异丁腈和卤化物供体如N-溴代琥珀酰亚胺、N-溴代乙酰胺、N-氯代琥珀酰亚胺、N-碘代琥珀酰亚胺、溴化物、氯化物或碘化物,在合适的溶剂如四氯化碳中,在合适的反应温度如85℃分别由XIII和XIV通过自由基卤化来制备(a)K.Ziegler,A.Spath,E.Schaaf,W.Schumann,E.Winkelmann,Ann.1942,551,80;b)A.Nechvatal,Advances inFree-Radical Chemistry(London)1972,4,175)。
当X=Br、Cl、I且A=CH2OH时,通式结构XI和XII的化合物可以分别由XIII和XIV通过与合适的膦如三苯基膦和合适的氧化剂如四溴化碳、四氯化碳、N-碘代琥珀酰亚胺在合适的溶剂如二氯甲烷中,在合适的反应温度如室温反应来制备(a)R.Appel,Angew.Chem.Int.Ed.1979,14,801;b)Cadogan,J,ed.(1979).Organophosphorus Reagentsin Organic Synthesis.London:Academic Press)。
当X=Br、Cl、I且A=CH2OH时,通式结构XI和XII的化合物可以分别由XIII和XIV通过用合适的酸如HBr(48%水溶液)、HCl(12M aq.)或HI(aq.)处理来制备(a)M.Uchida,F.Tabusa,M.Komatsu,S.Morita,T.Kanbe,K.Nakagawa,Chem.Pharm.Bull.1985,33,3775;b)V.Boekelheide,G.K.Vick,J.Am.Chem.Soc.1956,78,653;c)K.M.Doxsee,M.Feigel,K.D.Stewart,J.W.Canary,C.B.Knobler,D.J.Cram,J.Am.Chem.Soc.1987,109,3098.)。
当X=OMs、OTf且A=CH2OH时,通式结构XI和XII的化合物可以分别由XIII和XIV通过与合适的试剂如MsCl、(Ms)2O、(Tf)2O在合适的碱例如叔胺例如三乙胺存在下,在合适的溶剂如二氯甲烷中反应来制备。
当A=CH2OH时,通式结构XIII的化合物可以如下制备:由相应的酸(A=COOH)与合适的还原剂如硼烷二甲硫醚加合物或氢化铝锂在合适的溶剂如四氢呋喃中在合适的反应温度例如室温下反应来制备(a)N.G.Gaylord,Reduction with Complex Metal Hydrides,Wiley,NY,.1956,322;b)H.C.Brown,W.Korytnyk,J.Am.Chem.Soc.1960,82,3866)。
当A=CH3、CH2OH时,通式结构XIII和XIV的化合物可以使用合适的芳基底物XV-XVII作为起始原料,通过Suzuki交联反应来制备(N.Miyaura,A.Suzuki,Chem.Rev.1995,95,2457)。
如果B=Br、I、OTf,则交联反应包括使用合适的催化剂如Sphos Palladacycle G2(氯(2-二环己基膦基-2′,6′-二甲氧基-1,1′-联苯基)[2-(2′-氨基-1,1′-联苯基)]钯(II))和合适的碱例如K2CO3在合适的溶剂混合物例如DMF/H2O中,在合适的温度例如室温、50℃或80℃使XV或XVI与硼试剂XVII(C=B(OH)2,Bpin)反应(T.E.Barder,S.D.Walker,J.R.Martinelli,S.L.Buchwald,S.L.J.Am.Chem.Soc.2005,127 4685;b)R.A.Altman,S.L.Buchwald,Nature Protocols 2007,2,3115–3121)。
如果B=B(OH)2、Bpin,交叉偶联反应包括使用合适的催化剂如SphosPalladacycle G2(氯(2-二环己基膦基-2′,6′-二甲氧基-1,1′-联苯基)[2-(2′-氨基-1,1′-联苯基)]钯(II))和合适的碱例如K2CO3在合适的溶剂混合物如DMF/H2O在合适的温度如室温、50℃或80℃使XV或XVI与试剂XVII(C=Br、I、OTf)反应。化合物XVI-XVII是市售的。
流程8:如式V所定义的通式结构XVIII的化学3’-修饰的mRNA可以如下获得:使RNA与通式结构XX和NaIO4在适当的缓冲水溶液(缓冲剂:NaOAc、TRIS、PBS、MES、HEPES),在范围为5.0-7.5的适宜的pH,范围为0℃至室温的温度反应,然后用合适的亲核试剂例如肼、酰腙、羟胺、1,2-氨基硫醇、胺处理。
如式V中定义的通式结构XVIII的化学3’-修饰的mRNA可以如下获得:使RNA与通式结构XX和NaIO4在适当的缓冲水溶液(缓冲剂:NaOAc、TRIS、PBS、MES、HEPES),在范围为5.0-7.5的适宜的pH值,在范围为0℃至室温的温度反应,然后用合适的胺亲核试剂例如肼、酰腙、羟胺、胺处理,随后用合适的还原剂例如NaCNBH3在范围为室温至37℃的温度处理。
如式I中定义的通式结构XIX的RNA可以如下获得:使RNA与通式结构XX和NaIO4在适当的缓冲水溶液(缓冲剂:NaOAc、TRIS、PBS、MES、HEPES)中在范围为5.0-7.5的合适的pH下,范围为0℃至室温的温度反应,随后用合适的亲核试剂如Meldrum酸处理。
流程9:如下定义的通式结构XXI和XXII的5’3’-双修饰的RNA可通过在流程1中描述的条件下与鸟苷衍生物II反应分别由结构XXIII和XXIV的3’-修饰的RNA获得。
流程10:通式结构XXV的化学加帽的mRNA可以如下获得:使mRNA-5’-单磷酸与通式结构XXVI的咪唑活化的帽结构在合适的缓冲盐水溶液(缓冲液:HEPES、TRIS、MES、PBS)在范围为5.5-7.5的合适的pH,在存在或不存在有机溶剂如DMF和DMSO的情况下,在合适的路易斯酸性活化剂如例如MnCl2、NiCl2、ZnCl2存在下反应。
可以制备的示例性5’帽结构包括
流程11:其中Y和R1如式I所定义的通式结构XXVI的化合物可以如下制备:用咪唑或N-甲基-咪唑、合适的叔膦例如三苯基膦、合适的氧化剂如2,2'-二吡啶基二硫化物以及合适的碱(例如三乙胺)在合适的溶剂如DMF、DMSO、NMP、磷酸三甲酯中,在合适的温度例如室温下处理磷酸酯XXVII(三丁基铵盐)(a)M.Lewdorowicz,Y.Yoffe,J.Zuberek,J.Jemielity,J.Stepinski,R.Kierzek,R.Stolarski,M.Shapira,E.Darzynkiewicz,RNA2004,10,1469;b)R.Worch,J.Stepinski,A.Niedzwiecka,M.Jankowska-Anyszka,C.Mazza,S.Cusack,R.Stolarski,E.Darzynkiewicz,Nucleosides Nucleotides and NucleicAcids 2005,24,1131;c)M.Warminski,J.Kowalska,J.Buck,J.Zuberek,M.Lukaszewicz,C.Nicola,A.N.Kuhn,U.Sahin,E.Darzynkiewicz,J.Jemielity,Bioorg.Med.Chem.,2013,23,3753)。
流程12:其中Y、R1和Z2如式I中所定义的通式结构XXVII的化合物可以如下制备:在合适的溶剂例如DMF、DMSO或水中,在诸如氯化锌、氯化镁或氯化锰的路易斯酸存在下,在合适的温度例如室温下,使活化的磷酸酯XXIX与磷酸三乙铵或类似的磷酸盐反应(a)M.Lewdorowicz,Y.Yoffe,J.Zuberek,J.Jemielity,J.Stepinski,R.Kierzek,R.Stolarski,M.Shapira,E.Darzynkiewicz,RNA 2004,10,1469;b)R.Worch,J.Stepinski,A.Niedzwiecka,M.Jankowska-Anyszka,C.Mazza,S.Cusack,R.Stolarski,E.Darzynkiewicz,Nucleosides Nucleotides and Nucleic Acids 2005,24,1131;c)M.Warminski,J.Kowalska,J.Buck,J.Zuberek,M.Lukaszewicz,C.Nicola,A.N.Kuhn,U.Sahin,E.Darzynkiewicz,J.Jemielity,Bioorg.Med.Chem.,2013,23,3753)。
当X=O时,其中Y、R1和Z2如式I中定义的通式结构XXVII化合物也可以通过在合适的溶剂如磷酸三甲酯中,在0℃至室温的适当的温度,使XXVIII与二磷酸四氯化物反应来制备(J.Emsley,J.Moore,P.B.Udy,J.Chem.Soc.(A)Inorg.Phys.Theor.1971,2863)。
当X=NH时,其中Y、R1和Z2如式I中定义的通式结构XXVII的化合物也可以通过使XXVIII与亚氨基-双(磷酰二氯)在合适的溶剂如磷酸三甲酯中以范围为0℃至室温的合适的温度反应来制备(A.M.Rydzik,M.Kulis,M.Lukaszewicz,J.Kowalska,J.Zuberek,Z.M.Darzynkiewicz,E.Darzynkiewicz,J.Jemielity,Bioorganic&Med.Chem.2012,20,1699)。
当X=CH2时,其中Y、R1和Z2如式I中定义的通式结构XXVII的化合物也可以通过在合适的溶剂如磷酸三甲酯中在合适的温度例如0℃或室温下使XXVIII与亚甲基双(膦酰二氯)反应来制备(a)M.Honcharenko,M.Zytek,B.Bestas,P.Moreno,J.Jemielity,E.Darzynkiewicz,C.I.E.Smith,R.Stroemberg,Bioorg.Med.Chem.,2013,21,7921;b)M.Kalek,J.Jemielity,Z.M.Darzynkiewicz,E.Bojarska,J.Stepinski,R.Stolarski,R.E.Davis,E.Darzynkiewic,Bioorg.Med.Chem.2006,14,3223;c)M.Kalek,J.Jemielity,J.Stepinski,R.Stolarski,E.Darzynkiewics,Tetrahedron Lett.2005,46,2417)。
其中Y、R1和Z2如式I中所定义的通式结构XXIX(Z=咪唑、N-甲基咪唑)的化合物可以通过用咪唑或N-甲基-咪唑、合适的叔膦如三苯基膦、合适的氧化剂如2,2'-联吡啶二硫化物以及合适的碱如三乙胺,在合适的溶剂如DMF、DMSO、NMP、磷酸三甲酯中的适宜的温度例如室温处理磷酸酯XXIX(Z=OH,三丁基铵盐)来制备(a)M.Lewdorowicz,Y.Yoffe,J.Zuberek,J.Jemielity,J.Stepinski,R.Kierzek,R.Stolarski,M.Shapira,E.Darzynkiewicz,RNA 2004,10,1469;b)R.Worch,J.Stepinski,A.Niedzwiecka,M.Jankowska-Anyszka,C.Mazza,S.Cusack,R.Stolarski,E.Darzynkiewicz,NucleosidesNucleotides and Nucleic Acids 2005,24,1131;c)M.Warminski,J.Kowalska,J.Buck,J.Zuberek,M.Lukaszewicz,C.Nicola,A.N.Kuhn,U.Sahin,E.Darzynkiewicz,J.Jemielity,Bioorg.Med.Chem.,2013,23,3753)。
其中Y、R1和Z2如式I所定义的通式结构XXIX(Z=OH)的化合物也可以通过在合适的溶剂如磷酸三甲酯中在范围为0℃至室温的合适的温度使XXVIII与合适的磷酰化试剂例如磷酰氯反应来制备。
其中Y、R1和Z2如实施方案式I所定义的通式结构XXVII的化合物也可以通过使用合适的芳基底物和化合物XXX作为起始原料的交联反应来制备(N.Miyaura,A.Suzuki,Chem.Rev.1995,95,2457)。
化合物XXX可以通过使用合适的烷基化试剂如烷基卤化物、三氟甲磺酸酯或甲磺酸酯,在合适的溶剂如DMF、DMSO或NMP中,在范围为室温至60℃的温度下烷基化市售的8-溴-3-甲基-1H-嘌呤-2,6(3H,7H)-二酮(A=H)或8-溴-1,3-二甲基-1H-嘌呤-2,6(3H,7H)-二酮(A=Me)来制备。
流程13:其中Y、R1和Z2如式I所定义的通式结构XXVIII的化合物可以使用适当的碱例如叔丁醇钠、叔丁醇钾或异丙醇钠,在合适的溶剂如乙醇、异丙醇或THF中,在范围为50-100℃的温度下通过环化由通式结构XXXII的酰胺得到。
其中Y、R1和Z2如式I所定义的通式结构XXIX的化合物可以通过使用合适的路易斯酸例如三甲基甲硅烷基溴、三溴化硼或三氯化铝在溶剂如DMF、DMSO、NMP或THF中,在范围为0℃至30℃的温度下通过水解通式结构XXXI的单-或二烷基膦和磷酸酯获得。
其中Y、R1和Z2如实施方案式I中所定义的通式结构XXXI的化合物可以由通式结构XXXII的酰胺通过使用适当的碱例如叔丁醇钠、叔丁醇钾或异丙醇钠在合适的溶剂如乙醇、异丙醇或THF中,在50-100℃的温度下进行环化而获得。
流程14:其中Y、R1和Z2如实施方案XXX中所定义的通式结构XXXII的化合物可以通过使用合适的活化剂例如HATU、HBTU、O-(苯并三唑-1-基)-N,N,N′,N′-四甲基脲四氟硼酸盐或(苯并三唑-1-基氧基)三吡咯烷子基六氟磷酸盐和合适的碱例如三乙胺或Hunig碱在合适溶剂例如DMF、DMSO或NMP中,在范围为室温至50℃的温度下酰化XXXIII来获得。
其中Y、R1如实施方案式I中所定义的通式结构XXXIII的化合物可以通过使用还原剂如氢化铝锂或Red-Al在合适的溶剂如THF、乙醚或二烷中,范围为室温至120℃的温度下来还原XXXIV(其中Y是取代基R1的相应羧酸)来获得。
通式结构XXXIV的化合物可以如下获得:使用合适的活化剂例如HATU、HBTU、O-(苯并三唑-1-基)-N,N,N′,N′-四甲基脲四氟硼酸盐或(苯并三唑-1-基氧基)三吡咯烷子基六氟磷酸盐和合适的碱例如三乙胺或Hunig碱在合适溶剂例如DMF、DMSO或NMP中、在范围为室温至50℃的温度下用合适的羧酸酰化市售的5,6-二氨基嘧啶-4(3H)-酮(XXXV)以引入取代基R1。
流程15:如上所定义的通式结构XXXVI和XXXVII的5’3’-双修饰的RNA可分别由结构XL和XLI的3’修饰的RNA通过与鸟苷衍生物XXVI在流程1中描述的条件下反应得到。流程10描述了XXVI的合成。
定义
术语“酰基”是指任选取代的烷基羰基、任选取代的芳基羰基。这些酰基的实例包括乙酰基、苯甲酰基等。
术语“亲和部分”是指特异性结合目的分子例如蛋白质或核酸或其他分子的分子。亲和部分的实例包括但不限于生物素和地高辛(digoxigenin)。
术语“烷二基”是指衍生自脂族烃的通式CnH2n的二价基团。除非另有说明,此类烷二基包括取代的烷二基。合适的实例包括亚甲基(-CH2-),乙二基(-CH2-CH2-)等。
本文单独或组合使用的术语“烯基”和“炔基”是指含有1至20个碳原子并包括一个或多个不饱和单元的脂族直链或支链烃链。烯基含有至少一个碳-碳双键,但不含碳-碳叁键。炔基含有至少一个碳-碳叁键。优选的烯基和炔基可以包含2至10个碳原子或2至6个碳原子。合适的烯基包括例如乙烯基、烯丙基、1-丙烯基、2-丙烯基、1-丁烯基、2-丁烯基、3-丁烯基、1-戊烯基、2-戊烯基、3-戊烯基、4-戊烯基、1-己烯基、2-己烯基、3-己烯基、4-己烯基、5-己烯基、1-庚烯基、2-庚烯基、3-庚烯基、4-庚烯基、5-庚烯基、6-庚烯基、1-辛烯基、2-辛烯基、3-辛烯基、4-辛烯基、5-辛烯基、6-辛烯基、7-辛烯基、1-壬烯基、2-壬烯基、3-壬烯基、4-壬烯基、5-壬烯基、6-壬烯基、7-壬烯基、8-壬烯基、1-癸烯基、2-癸烯基、3-癸烯基、4-癸烯基、5-癸烯基、6-癸烯基、7-癸烯基、8-癸烯基或9-癸烯基。合适的炔基包括例如乙炔基、1-丙炔基、2-丙炔基、1-丁炔基、2-丁炔基、3-丁炔基、1-戊炔基、2-戊炔基、3-戊炔基、4-戊炔基、1-己炔基、2-己炔基、3-己炔基、4-己炔基、5-己炔基、1-庚炔基、2-庚炔基、3-庚炔基、4-庚炔基、5-庚炔基、6-庚炔基、1-辛炔基、2-辛炔基、3-辛炔基、4-辛炔基、5-辛炔基、6-辛炔基、7-辛炔基、1-壬炔基、2-壬炔基、3-壬炔基、4-壬炔基、5-壬炔基、6-壬炔基、7-壬炔基、8-壬炔基、1-癸炔基、2-癸炔基、3-癸炔基、4-癸炔基、5-癸炔基、6-癸炔基、7-癸炔基、8-癸炔基或9-癸炔基。烯基和炔基可以如本文所述任选地被取代。
本文单独使用或组合使用的术语“烷基”是指含有1至20个碳原子的脂肪族直链或支链饱和烃链。在某些实施方案中,烷基可以包含1至10个碳原子。在另外的实施方案中,烷基可以包含1至6个碳原子。烷基可以如本文所述任选地被取代。烷基的实例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、戊基、异戊基、己基、辛基、壬基(noyl)等。
术语“氨基”是指基团–NH2。
术语“氨基烷基”是指被伯、仲或叔氨基取代的烷基。优选的氨基烷基包括具有一个或多个伯、仲和/或叔胺基团以及1至约12个碳原子、更优选1至约8个碳原子、还更优选1、2、3、4、5或6个碳原子的那些氨基烷基。此类氨基烷基的实例包括氨基甲基、氨基乙基等。
本文单独或组合使用的术语“芳基”表示含有至少一个芳环的碳环芳烃环体系。芳环体系可以是包括芳族或非芳族烃环的稠环体系或包括非芳族烃环的芳环体系。术语“芳基”包括芳族基团如苯基、萘基、蒽基和菲基。芳基可以如本文所述任选地被取代。
术语“帽0”是指其中仅有的甲基化在m7G中的帽。术语“帽1”是指在N1中具有另外的甲基化的帽。概括的帽结构表示为m7G(5’)ppp(5’)Nl mpN2 mpN3p....其中N是任何核苷酸,优选嘌呤或嘧啶,p是磷酸酯基团,且m是甲基。在鸟苷的N7位含有甲基的m7G位于mRNA的最5’-末端。另一方面,在N1和N2位置的甲基化是核糖部分的2'-OH基团处的取代。各种帽结构根据其含有的甲基数量进行分类(Banerjee,A.K.Microbiological Rev.,1980,44,175)。
除非另有说明,本文单独或与其它术语组合使用的术语“环烯基”表示含有至少一个碳-碳双键的环状形式的“烯基”,具有优选3至10个碳原子,即3、4、5、6、7、8、9或10个碳原子,优选3至6个碳原子形成环。术语“环烯基”也意在包括其双环形式。如果形成双环,则优选各个环在两个相邻的碳原子处彼此连接,然而,或者两个环通过相同的碳原子连接,即它们形成螺环体系或它们形成桥环体系。环烯基的实例包括环丙烯基、环丁烯基、环戊烯基、环戊二烯基、环己烯基、1,3-环己烯基、环庚烯基、环庚三烯基、环辛烯基、环壬烯基、环癸烯基、二环[2.2.1]-2-庚烯基、二环[2.2.1]-2-辛烯基或二环[4.4.0]-2-癸烯基。环烯基可以如本文所述任选地被取代。
除非另有说明,本文单独或与其它术语组合使用的术语“环烷基”表示包括双环和多环烷基的环状形式的“烷基”。双环或多环体系的环可以被稠合通过单个共享原子连接,即它们形成螺环体系或桥环体系。环烷基可优选含有3至10个碳原子,即3、4、5、6、7、8、9或10个碳原子,优选3至6个碳原子,形成环。合适的环烷基的实例包括环丙基、环丁基、环戊基、环己基、环庚基、环辛基、环壬基或环癸基。合适的环烷基的实例包括环丙基、环丁基、环戊基、环己基、环庚基、环辛基、螺[3,3]庚基、螺[3,4]辛基、螺[4,3]辛基、二环[4.1.0]庚基、二环[3.2.0]庚基、二环[2.2.1]庚基、二环[2.2.2]辛基、二环[5.1.0]辛基、二环[4.2.0]辛基等。环烷基可以如本文所述任选地被取代。
术语“检测部分”是指可以使用合适的方法容易地检测的化学部分。检测部分的实例包括但不限于荧光材料、发光材料、生物发光材料和放射性材料。
术语“半衰期”(T1/2)涉及消除分子的活性、量或数量的一半所需要的时间。在本发明的上下文中,RNA的半衰期表示所述RNA的稳定性。
术语“杂原子”是指氮、氧和硫。
本文所用的术语“杂芳基”是其中一个或多个碳环原子独立地被O、S和N代替的芳基。在某些实施方案中,杂芳基可以包含5至7个碳原子。该术语还包括其中杂环与芳环稠合、其中杂芳基环与其它杂芳基环稠合、其中杂芳基环与杂环烷基环稠合或其中杂芳基环与环烷基环稠合的稠合多环基团。杂芳基的实例包括吡咯基、吡咯啉基、咪唑基、吡唑基、吡啶基、嘧啶基、吡嗪基、哒嗪基、三唑基、吡喃基、呋喃基、噻吩基、唑基、异唑基、二唑基、噻唑基、噻二唑基、异噻唑基、吲哚基、异吲哚基、吲嗪基、苯并咪唑基、喹啉基、异喹啉基、喹喔啉基、喹唑啉基、吲唑基、苯并三唑基、苯并间二氧杂环戊烯基、苯并吡喃基、苯并唑基、苯并二唑基、苯并噻唑基、苯并噻二唑基、苯并呋喃基、苯并噻吩基、色酮基(chromonyl)、香豆素基、苯并吡喃基、四氢喹啉基、四唑并哒嗪基、四氢异喹啉基、噻吩并吡啶基、呋喃并吡啶基、吡咯并吡啶基等。示例性的三环杂环基包括咔唑基、苯并吲哚基、菲咯啉基、二苯并呋喃基、吖啶基、菲啶基、吨基等。杂芳基可以如本文所述任选地被取代。
术语“杂环基”是指如上定义的环烷基,其中环中的1至3个碳原子独立地被O、S或N代替。这种杂环基的实例是吡咯烷基、咪唑烷基、唑烷基、噻唑烷基、四氢呋喃基、哌啶基、哌嗪基、吗啉基、硫代吗啉基、四氢吡喃基、二烷基、二氢吲哚基、异二氢吲哚基、二氢苯并呋喃基、二氢异苯并呋喃基、四氢喹啉基、四氢异喹啉基、四氢喹喔啉基、色满基、异色满基、二氢苯并嗪基、二氢苯并噻嗪基或二氢苯并二氧芑基。杂环基可以如本文所述任选地被取代。
术语“免疫原性”是指诱导免疫反应的能力。
术语“连接基团”是指连接两个其它部分的二价基团。连接基团通常将含有1至40个原子,并且可以是例如烷基、烯基、炔基、环烷基、环烯基、芳基、杂芳基或杂环连接基团,其各自可以如本文所述任选地被取代。例如,合适的连接基团包括亚甲基(-CH2-)、乙二基(-CH2-CH2-)、乙烯二基(-CH=CH-)、乙炔二基(-C≡C-)、-CH2CH2CH2-、-CH2CH(CH3)-、-C(CH3)2-、-(C6H4)-、-(C6H10)-等。合适的连接基团的另外的实例包括但不限于
本文单独或组合使用的术语“低级”,当没有另外具体定义时,意味着含有1至6(包括6)个碳原子。
术语“核碱基”是指为核苷酸成分的含氮碱基。核碱基包括例如初级核碱基胞嘧啶、鸟嘌呤、腺嘌呤、胸腺嘧啶、尿嘧啶;假尿嘧啶;以及修饰的核碱基,例如m5C(5-甲基胞苷)、m5U(5-甲基尿苷)、m6A(N6-甲基腺苷)、s2U(2-硫尿苷)、Um(2'-0-甲基尿苷)、m1A(l-甲基腺苷);m2A(2-甲基腺苷);Am(2-1-O-甲基腺苷);ms2m6A(2-甲硫基-N6-甲基腺苷);i6A(N6-异戊烯基腺苷);ms2i6A(2-甲硫基-N6异戊烯基腺苷);io6A(N6-(顺-羟基异戊烯基)腺苷);ms2io6A(2-甲硫基-N6-(顺-羟基异戊烯基)腺苷);g6A(N6-甘氨酰氨基甲酰基腺苷);t6A(N6-苏氨酰氨基甲酰基腺苷);ms2t6A(2-甲硫基-N6-苏氨酰氨基甲酰基腺苷);m6t6A(N6-甲基-N6-苏氨酰氨基甲酰基腺苷);hn6A(N6-羟基正缬氨酰氨基甲酰基腺苷);ms2hn6A(2-甲硫基-N6-羟基正缬氨酰氨基甲酰基腺苷);Ar(p)(2'-O-核糖基腺苷(磷酸酯(phosphate)));I(肌苷);m11(1-甲基肌苷);m'Im(1,2'-O-二甲基肌苷);m3C(3-甲基胞苷);Cm(2T-O-甲基胞苷);s2C(2-硫胞苷);ac4C(N4-乙酰基胞苷);f5C(5-fonnyl胞苷);m5Cm(5,2-O-二甲基胞苷);ac4Cm(N4乙酰基2TO甲基胞苷);k2C(赖胞苷);m1G(1-甲基鸟苷);m2G(N2-甲基鸟苷);m7G(7-甲基鸟苷);Gm(2'-O-甲基鸟苷);m22G(N2,N2-二甲基鸟苷);m2Gm(N2,2'-O-二甲基鸟苷);m22Gm(N2,N2,2'-O-三甲基鸟苷);Gr(p)(2'-O-核糖基鸟苷(磷酸酯(phosphate)));yW(怀丁苷(wybutosine));o2yW(过氧怀丁苷);OHyW(羟基怀丁苷);OHyW*(修饰下的羟基怀丁苷);imG(怀俄苷(wyosine));mimG(甲基鸟苷);Q(辫苷(queuosine));oQ(环氧辫苷);galQ(半乳糖基-辫苷);manQ(甘露糖基-辫苷);preQo(7-氰基-7-去氮杂鸟苷);preQi(7-氨基甲基-7-去氮杂鸟苷);G(古嘌苷);D(二氢尿苷);m5Um(5,2'-O-二甲基尿苷);s4U(4-硫尿苷);m5s2U(5-甲基-2-硫尿苷);s2Um(2-硫-2'-O-甲基尿苷);acp3U(3-(3-氨基-3-羧基丙基)尿苷);ho5U(5-羟基尿苷);mo5U(5-甲氧基尿苷);cmo5U(尿苷5-氧基乙酸);mcmo5U(尿苷5-氧基乙酸甲基酯);chm5U(5-(羧基羟基甲基)尿苷));mchm5U(5-(羧基羟基甲基)尿苷甲基酯);mcm5U(5-甲氧基羰基甲基尿苷);mcm5Um(S-甲氧基羰基甲基-2-O-甲基uricjine);mcm5s2U(5-甲氧基羰基甲基-2-硫尿苷);nm5s2U(5-氨基甲基-2-硫尿苷);mnm5U(5-甲基氨基甲基尿苷);mnm5s2U(5-甲基氨基甲基-2-硫尿苷);mnm5se2U(5-甲基氨基甲基-2-硒尿苷);ncm5U(5-氨基甲酰基甲基尿苷);ncm5Um(5-氨基甲酰基甲基-2'-O-甲基尿苷);cmnm5U(5-羧基甲基氨基甲基尿苷);cnmm5Um(5-羧基甲基氨基甲基-2-L-O甲基尿苷);cmnm5s2U(5-羧基甲基氨基甲基-2-硫尿苷);m62A(N6,N6-二甲基腺苷);Tm(2'-O-甲基肌苷);m4C(N4-甲基胞苷);m4Cm(N4,2-O-二甲基胞苷);hm5C(5-羟基甲基胞苷);m3U(3-甲基尿苷);cm5U(5-羧基甲基尿苷);m6Am(N6,T-O-二甲基腺苷);rn62Am(N6,N6,O-2-三甲基腺苷);m2'7G(N2,7-二甲基鸟苷);m2'2'7G(N2,N2,7-三甲基鸟苷);m3Um(3,2T-O-二甲基尿苷);m5D(5-甲基二氢尿苷);f5Cm(5-甲酰基-2'-O-甲基胞苷);m1Gm(1,2'-O-二甲基鸟苷);m'Am(1,2-0-二甲基腺苷)irino甲基尿苷);tm5s2U(S-taurino甲基-2-硫尿苷));imG-14(4-去甲基鸟苷);imG2(异鸟苷);或ac6A(N6-乙酰基腺苷)、次黄嘌呤、肌苷、8-氧代-腺嘌呤、其7-取代的衍生物、二氢尿嘧啶、假尿嘧啶、2-硫尿嘧啶、4-硫尿嘧啶、5-氨基尿嘧啶、5-(C1-C6)-烷基尿嘧啶、5-甲基尿嘧啶、5-(C2-C6)-烯基尿嘧啶、5-(C2-C6)-炔基尿嘧啶、5-(羟基甲基)尿嘧啶、5-氯尿嘧啶、5-氟尿嘧啶、5-溴尿嘧啶、5-羟基胞嘧啶、5-(C1-C6)-烷基胞嘧啶、5-甲基胞嘧啶、5-(C2-C6)-烯基胞嘧啶、5-(C2-C6)-炔基胞嘧啶、5-氯胞嘧啶、5-氟胞嘧啶、5-溴胞嘧啶、N2-二甲基鸟嘌呤、7-去氮杂鸟嘌呤、8-氮杂鸟嘌呤、7-去氮杂-7-(C2-C6)炔基鸟嘌呤、8-羟基鸟嘌呤、6-硫鸟嘌呤、8-氧代鸟嘌呤、2-氨基嘌呤、2-氨基-6-氯嘌呤、2,4-二氨基嘌呤、2,6-二氨基嘌呤、8-氮杂嘌呤等。
当基团被定义为“不存在(null)”时,意思是该基团不存在。
术语“任选取代的”表示所涉基团可以是取代的或未被取代的。当被取代时,“任选取代的”基团的取代基可以包括但不限于一个或多个独立地选自单独或组合的以下基团或特别指定的一组基团的取代基:低级烷基、低级烯基、低级炔基、低级烷酰基、低级杂烷基、低级杂环烷基、低级卤代烷基、低级卤代烯基、低级卤代炔基、低级全卤代烷基、低级全卤代烷氧基、低级环烷基、苯基、芳基、芳氧基、低级烷氧基、低级卤代烷氧基、氧代、低级酰氧基、羰基、羧基、低级烷基羰基、低级羧基酯、低级甲酰氨基、氰基、卤素、羟基、氨基、低级烷基氨基、芳基氨基、酰氨基、硝基、硫醇、低级烷硫基、低级卤代烷硫基、低级全卤代烷硫基、芳硫基、磺酸酯、磺酸、三取代甲硅烷基、N3、SH、SCH3、C(O)CH3、CO2CH3、CO2H、腈、CF3、环烷基、吡啶基、噻吩、呋喃基、低级氨基甲酸酯、卤代苯基、羟基苯基、卤代烷基和羟烷基。两个取代基可以连接在一起以形成含有一至三个杂原子的五、六或七元芳族或非芳族碳环或杂环,例如形成亚甲二氧基或亚乙二氧基。任选取代的基团可以是未完全取代的(例如-CH2CH3),完全取代的(例如-CF2CF3),单取代的(-CH2CH2F)或在完全取代和单取代之间的任何水平取代(例如-CH2CF3)。当取代基被列出而没有关于取代的资格时,包括取代和未取代的形式。当取代基被认定为“取代的”时,取代的形式是特别意图的。另外,可以根据需要限定特定部分的不同组的任选取代基;在这些情况下,任选的取代被定义,通常紧随短语“任选被...取代”之后。
根据本发明的化合物可以提供在“药学上可接受的制剂”中。这样的组合物可以含有盐、缓冲剂、防腐剂、载体和任选的其它治疗剂。“药学上可接受的盐”包括生理上相容并优选无毒的盐。例如,可以例如通过将化合物溶液与药学上可接受的酸如盐酸、硫酸、富马酸、马来酸、琥珀酸、乙酸、苯甲酸、柠檬酸、酒石酸、碳酸或磷酸的溶液混合而形成酸加成盐。此外,当化合物携带酸性部分时,其合适的药学上可接受的盐可包括碱金属盐(例如钠盐或钾盐);碱土金属盐(如钙盐或镁盐);与合适的有机配体形成的盐(例如,使用抗衡阴离子如卤化物、氢氧化物、羧酸盐、硫酸盐、磷酸盐、硝酸盐、烷基磺酸盐和芳基磺酸盐形成的铵、季铵和胺阳离子)。药学上可接受的盐的说明性实例包括但不限于乙酸盐、己二酸盐、藻酸盐、抗坏血酸盐、天冬氨酸盐、苯磺酸盐、苯甲酸盐、碳酸氢盐、硫酸氢盐、酒石酸氢盐、硼酸盐、溴化物、丁酸盐、依地酸钙、樟脑酸盐(camphorate)、樟脑磺酸盐、右旋樟脑磺酸盐(camsylate)、碳酸盐、氯化物、柠檬酸盐、克拉维酸盐、环戊烷丙酸盐、二葡糖酸盐、二盐酸盐、十二烷基硫酸盐、依地酸盐、乙二磺酸盐、丙酸酯月桂硫酸盐(estolate)、乙磺酸盐(esylate)、乙烷磺酸盐(ethanesulfonate)、甲酸盐、富马酸盐、葡庚糖酸盐(gluceptate)、葡庚糖酸盐(glucoheptonate)、葡糖酸盐、谷氨酸盐、甘油磷酸盐、glycolylarsanilate、半硫酸盐、庚酸盐、己酸盐、己基resorcinate、海巴明(hydrabamine)、氢溴酸盐、盐酸盐、氢碘酸盐、2-羟基-乙磺酸盐、羟基萘甲酸盐、碘化物、异硫化羟酸盐(isothionate)、乳酸盐、乳糖酸盐、月桂酸盐、月桂基硫酸盐、苹果酸盐、马来酸盐、丙二酸盐、扁桃酸盐、甲磺酸盐、甲烷磺酸盐、甲基硫酸盐、粘酸盐、2-萘磺酸盐、萘磺酸盐、烟酸盐、硝酸盐、N-甲基葡糖胺铵盐、油酸盐、草酸盐、扑酸盐(双羟萘酸盐)、棕榈酸盐、泛酸盐、果胶酸盐、过硫酸盐、3-苯基丙酸盐、磷酸盐/二磷酸盐(diphosphate)、苦味酸盐、新戊酸盐、聚半乳糖醛酸盐、丙酸盐、水杨酸盐、硬脂酸盐、硫酸盐、次乙酸盐、琥珀酸盐、鞣酸盐、酒石酸盐、teoclate、甲苯磺酸盐、三乙基碘(triethiodide)、十一酸盐、戊酸盐等(参见例如Berge,S.M.等人J.Pharm.Sci.1977,66,1-19)。
术语“PEG”是指具有重复单元的聚合物,其中n2是2至12。直链或支链聚乙二醇聚合物包括在此术语中,并且包括聚乙二醇的单甲基醚(mPEG)。术语“PEG”还包括Newkome型树突状分子,例如PEG以多种配方(例如CarbowaxTM(Dow Chemical,Midland,MI)、Poly-(ArchChemicals,Norwalk CT)和Solbase)是商业可得的。
术语“聚胺”是指含有具有至少一个氨基氢原子的至少两个氨基的胺化合物。聚胺的实例包括但不限于聚乙烯亚胺、聚丙烯-亚胺、聚乙烯胺、聚烯丙胺、乙二胺、六亚甲基二胺、二亚乙基三胺、三亚乙基四胺、四亚乙基五胺和双六亚甲基三胺。
具体实施方式
实施例
材料和方法:除非另有说明,所有试剂均购自商业来源,不经处理便使用。在TopSpin程序控制下,使用ICON-NMR在Bruker AVANCE 400MHz或500MHz NMR波谱仪上进行NMR波谱测定。记录13C NMR和31P NMR波谱。在298K下测量波谱,除非另有说明,并且参考相对于溶剂共振。化学位移(δ)以ppm表示,信号描述为s(单峰)、d(双峰)、t(三重峰)、q(四重峰)和m(多重峰)。由以下配置的各种仪器使用电喷雾电离方法在LC-MS系统上获得质谱。[M+H+]+是指化学物质的质子化分子离子。[M-H+]-指化学物质的去质子化分子离子。
LCMS方法1 RXNMON_ACIDC
仪器 Agilent 1100HPLC;Waters Micromass ZQ质谱仪
柱 Sunfire C18 3.5μm 30x3.0 mm,3.5μm
柱温 40℃
洗脱剂 A:H2O+0.05%TFA,B:乙腈
流速 2ml/min
梯度 5%-95%B,在1.7min内
LCMS方法2 RXNMON Acidic_Polar_=RXNMON
Acidic_Polar_PosNeg
仪器 Agilent 1100HPLC;Waters Micromass ZQ质谱仪
柱 Sunfire C18 3.5μm 30x3.0 mm,3.5μm
柱温 40℃
洗脱剂 A:H2O+0.05%TFA,B:乙腈
流速 2ml/min
梯度 在1.20min内1%至30%B,在0.65min内30%至95%B,
LCMS方法4RXNMON_Basic_Polar
仪器 Agilent 1100HPLC;Waters Micromass ZQ质谱仪
柱 Xbridge C18 3.5μm 30x3.0 mm,3.5μm
柱温 40℃
洗脱剂 A:H2O+5mM氢氧化铵,B:乙腈
流速 2ml/min
梯度 在1.20min内1%至30%B,在0.65min内30%至95%B,
LCMS方法5SQ4mRNAcap_FIA Acidic
仪器 Waters Acquity UPLC,具有SQ检测器
柱 Acquity BEH C18 1.7μm 2.1x50mm
柱温 50℃
洗脱剂 A:H2O+0.1%甲酸,B:乙腈+0.1%甲酸
流速 1ml/min
梯度 50%B等度,2.16min
LCMS方法6 Acquity LCTp2 Tof_产物分析酸性的
仪器 Waters Acquity UPLC,具有Waters LCT Premier XE检测器
柱 Acquity BEH C18 1.7μm 2.1x50mm
柱温 50℃
洗脱剂 A:H2O+0.1%甲酸,B:乙腈+0.1%甲酸
流速 1ml/min
梯度 2%-98%B,在7.5min内
LCMS方法7 SQ4酸性极性的
仪器 Waters Acquity UPLC,具有SQ检测器
柱 Acquity BEH C18 1.7μm 2.1x50mm
柱温 50℃
洗脱剂 A:H2O+0.1%甲酸,B:乙腈+0.1%甲酸
流速 1ml/min
梯度 在1.20min内1%-30%B;在0.95min内30%-98%B
LCMS方法8 SQ4酸性的
仪器 Waters Acquity UPLC,具有SQ检测器
柱 Acquity BEH C18 1.7μm 2.1x50mm
柱温 50℃
洗脱剂 A:H2O+0.1%甲酸,B:乙腈+0.1%甲酸
流速 1ml/min
梯度 2%-98%B,在1.76min内
LCMS方法9IPC
仪器 Micromass Quattro micro API,Agilent 1100Series泵
柱 Xbridge BEH C18 2.5μm 3.0x100mm
柱温 80℃
洗脱剂 A:H2O+200mM HFIP+8mM TEA,B:甲醇
流速 1ml/min
梯度 30%B直到3.65min;在3.75min至99%B;99%B,直到3.85min
LCMS方法10监控(scout)碱性肽
仪器 Agilent 1100HPLC;Waters Micromass ZQ质谱仪
柱 Xbridge C18 3.5μm,30x3.0 mm,3.5μm
柱温 40℃
洗脱剂 A:H2O+5mM氢氧化铵,B:乙腈
流速 2ml/min
梯度 在4.30min内5%至80%B,在5.0min至95%B
缩写:
ATP 腺苷5’-三磷酸
Atm 大气
AcOH 乙酸
Aq 含水的
Ar 芳基
br 宽的
br.s.,bs 宽单峰
BSA 牛血清白蛋白
℃ 摄氏度
CDCl3 氘代氯仿
CH2Cl2,DCM 二氯甲烷
CH3CN,MeCN 乙腈
d 双重峰
dd 双二重峰
ddd 双重峰的双二重峰
DIPEA N-乙基二异丙基胺
DMF N,N-二甲基甲酰胺
DMAP 二甲基氨基吡啶
DMSO 二甲基亚砜
dt 双三重峰
ESI 电喷雾电离
EtOAc 乙酸乙酯
EtOH 乙醇
FCC 快速柱色谱法
G 号(gauge)
GMP 鸟苷5’-单磷酸
GDP 鸟苷5’-二磷酸
GTP 鸟苷5’-三磷酸
h 小时
HCl 盐酸
HEPES 2-[4-(2-羟基乙基)哌嗪-1-基]乙烷磺酸
HFIP 六氟异丙醇
HOBt 羟基苯并三唑
HPLC 高压液相色谱法
i-PrOH 异丙醇
H2O 水
K 开尔文
KOH 氢氧化钾
LC 液相色谱法
M 摩尔的
m 多重峰,质量
MeOH 甲醇
MES 2-(N-吗啉代)乙烷磺酸
MgSO4 硫酸镁
MHz 兆赫
mL 毫升
mm 毫米
mmol 毫摩尔
min. 分钟
mRNA 信使核糖核酸
MS 质谱
mw 微波
m/z 质荷比
NaOH 氢氧化钠
Na2SO4 硫酸钠
NEt3 三乙胺
NH3 氨
NMR 核磁共振
PBS 磷酸盐缓冲液
ppt 沉淀
ppm 百万分之率
rbf 圆底烧瓶
Rf 阻滞因子
RP 反相
RT,rt 室温
Rt 保留时间
s 单峰
sat. 饱和的
SM 原料
t 三重峰
TBA 三丁胺
TEA 三乙胺
TEAB 三乙基碳酸氢铵
TFA 三氟乙酸
THF 四氢呋喃
TLC 薄层色谱法
TRIS 2-氨基-2-羟基甲基-丙烷-1,3-二醇
UPLC 超高效液相色谱法
Tris·HCl 氨基三(羟基甲基)甲烷盐酸盐
wt 重量
Xphos Pd G2 第2代Xphos Precatalyst,氯(2-二环己基膦基-2′,4′,6′-三异丙基-1,1′-联苯基)[2-(2′-氨基-1,1′-联苯基)]钯(II)
μg 微克
μL 微升
核苷酸三烷基铵盐根据文献方法(Y.Thillier,E.Decroly,F.Morvan,B.Canard,J.-J.Vasseur,F.Debart RNA2012,18,856-868;GMP Na-盐:Sigma-Aldrich No.G8377;GDPNa-盐:Sigma-Aldrich No.G7127;2’-脱氧GMP Na-盐:Sigma-Aldrich No.D9500;肌苷5’-单磷酸Na-盐:Sigma-Aldrich No.I4625;肌苷5’-二磷酸Na-盐:Sigma-Aldrich No.I4375)或在类似于下述那些条件从相应的市售钠盐获得。
GMP三乙基铵盐:
将GMP钠盐(2.0g,4.91mmol)溶解于无RNase水(40mL)中并通过DOWEX树脂(50WX8,40g,用H2O彻底淋洗)进入冰冷的三乙胺(10g,98mmol)的EtOH(60mL)溶液中。洗脱,随后测量洗脱液的UV吸收,并将树脂用水淋洗,直到没有进一步的GMP洗脱出。将得到的溶液在真空中浓缩至约500mL,然后冻干,获得标题产物,为白色固体(2.2g,GMP/TEA 1/2,通过1H-NMR判断).
GDP三丁基铵盐:
将GDP-二钠盐(1.17g,2.40mmol)溶解于水(100mL)中并通过DOWEX树脂(50WX8,10mL,>1.7meq/mL,用H2O彻底淋洗)进入冰冷的三丁基胺(1.34g,7.20mmol)的EtOH(20mL)溶液中。将树脂用水(1000mL)淋洗,并将得到的溶液在真空中浓缩至约500mL),然后冻干,获得标题产物,为无色固体(1.88g,GDP/TBA 1/2.5,通过1H-NMR判断)。
GMP N7烷基化的通用方法
方法A:在2-打兰(dram)小瓶中,将鸟苷单磷酸三丁基铵盐(50mg,68umol)溶解于DMSO(680uL)中,并用商业可获得的烷基溴或烷基氯试剂(4equiv.)处理。当使用氯化物烷基化试剂时并且在(2-溴乙氧基)-苯底物情况下,加入NaI(5mg,0.5eq.)。将获得的溶液在40℃振摇或搅拌18h。将获得的溶液直接通过HPLC(反相,H2O+0.1%TFA至MeCN+0.1%TFA0-100%)纯化。将含有需要的产物的级分合并,并通过冻干除去溶剂,获得纯产物,为无色固体或泡沫状物。
方法B:在2-打兰小瓶中,将0.1M GMP或GDP三乙基-或三丁基铵盐的DMSO溶液用溴化物烷基化试剂(4equiv.)处理。将溶液在室温或55℃搅拌18h,然后直接通过反相柱色谱法(ISCO Teledyne C18aq.洗脱液:0.1M三乙基碳酸氢铵(pH=8.0)至MeCN 0-100%)纯化。将含有需要的产物的级分合并,并通过冻干除去溶剂,获得为三丁基铵盐的纯产物,为无色固体或泡沫状物。
实施例1.N7-([1,1'-联苯]-4-基甲基)-5’-GMP TEA盐
将鸟苷单磷酸三乙基铵盐(100mg,0.177mmol)和4-联苯基甲基溴(175mg,0.710mmol)的DMSO(1mL)溶液在55℃搅拌过夜。将溶液直接通过反相柱色谱法(ISCOTeledyne C18 30g Gold柱,洗脱液A:0.1M TEAB;B:20%MeCN的0.1M TEAB溶液;梯度:0-100%B/A)纯化。合并产物级分并冻干,得到为白色粉末的标题化合物(38mg,28%).1H NMR(400MHz,D2O)δppm:7.58-7.67(4H,m),7.46-7.53(4H,m),7.38-7.46(1H,m),5.97-6.03(1H,m),5.63-5.76(2H,m),4.62-4.69(1H,m),4.46-4.52(1H,m),4.32-4.40(1H,m),3.97-4.18(2H,m),3.11-3.25(8.6H,q),1.20-1.34(13H,t).31P NMR(162MHz,D2O):δppm:3.73(1P).LCMS方法2Rt:1.39min,MS[M+H]+观测值:529.8.计算值:530.1.
实施例2.N7-([1,1'-联苯]-4-基甲基)-5’-GDP TEA盐
将鸟苷二磷酸三乙基铵盐(400mg,0.538mmol)和4-联苯基甲基溴(400mg,1.619mmol)的DMSO(2mL)溶液在室温搅拌过夜。加入1M NaClO4丙酮溶液(3.23mL),然后用丙酮稀释。通过离心分离沉淀,用丙酮洗涤并在真空下干燥。将如此获得的固体溶解于0.1MTEAB(5mL)中。将得到的溶液通过反相柱色谱法纯化(ISCO,Teledyne C18,30g Gold柱,洗脱液A:0.1M TEAB;B:90%MeCN的0.1M TEAB溶液;梯度:0-100%B/A)。合并产物级分并冻干,得到标题化合物,为白色粉末(90mg,18%).1H NMR(400MHz,D2O)δppm:7.57-7.67(4H,m),7.39-7.53(5H,m),5.92-6.00(1H,d),5.64-5.75(2H,m),4.67-4.72(2H,m),4.56-4.64(1H,m),4.34-4.40(1H,m),4.26-4.34(2H,m),3.12-3.24(12H,q),1.27(18H,t).31P NMR(162MHz,D2O):δppm:7.16(1P),10.93(1P).LCMS方法2Rt:1.43min,MS[M-H]+观测值:608.2,计算值:608.1.
实施例3.N7-(4-氯苯氧基乙基)-5’-GDP TEA盐
将鸟苷二磷酸三乙基铵盐(500mg,0.672mmol)和4-氯苯基2-溴乙基醚(633mg,2.69mmol)的DMSO(4mL)溶液在55℃搅拌过夜。加入1M NaClO4丙酮溶液(4mL),然后用丙酮稀释。通过离心分离沉淀,用丙酮洗涤并在真空下干燥。将如此获得的固体溶解于0.1MTEAB(5mL)中。将得到的溶液通过离子交换色谱法纯化(TOSOH,TSKgel DEAE-5PW,21.5mm x15cm,13μm,洗脱液A:H2O;B:1M TEAB的水溶液;梯度:0-100%B/A)。合并产物级分并冻干,得到标题化合物,为白色粉末(25mg,4%).1H NMR(400MHz,D2O)δppm:7.22-7.31(2H,d),6.86-6.94(2H,d),5.96-6.03(1H,m),4.46-4.52(1H,m),4.37-4.44(1H,m),4.29-4.37(1H,m),4.19-4.29(2H,m),3.12-3.26(9H,q),1.20-1.34(16H,t).31P NMR(162MHz,D2O):δppm:9.91(1P),11.28(1P).LCMS方法2Rt:1.20min,MS[M-H]+观测值:596.8,计算值:596.0.
实施例4.N7-(4-氯苯氧基乙基)-5’-GMP TEA盐
如先前所述制备(A.R.Kore等人Bioorg.Med.Chem.2013,21,4570;Chen等人J.Med.Chem.2012,55,3837)。获得产物,为白色粉末(84mg,12%).1H NMR(400MHz,D2O)δppm:7.18-7.27(2H,d),6.83-6.93(2H,d),5.92-6.02(1H,m),4.41-4.48(1H,m),4.33-4.41(1H,m),4.11-4.20(1H,m),3.97-4.09(1H,m),3.13-3.26(10H,q),1.20-1.34(15H,t).31PNMR(162MHz,D2O):δppm:3.39(1P).LCMS方法2Rt:1.20min,MS[M+H]+观测值:517.8,计算值:518.1.
实施例5.N7-苄基-5’-GMP TEA盐
如先前所述制备(Brown等人J.Mol Biol.2007,372,7-15).获得产物,为白色粉末(55mg,38%).1H NMR(400MHz,D2O)δppm:7.33-7.45(5H,m),6.02-6.11(1H,m),5.64-5.73(2H,m),4.44-4.53(1H,m),4.33-4.42(1H,m),3.96-4.17(2H,m),3.19(10H,q),1.27(14H,t).31P NMR(162MHz,D2O):δppm:3.78(1P).LCMS方法2Rt:0.84min,MS[M+H]+观测值:454.2,计算值:454.1.
实施例6.N7-(6-苯基吡啶-3-基)甲基)-5’-GMP TEA盐
步骤1.5-(溴甲基)-2-苯基吡啶
将(6-苯基吡啶-3-基)甲醇(300mg,1.620mmol)在33%HBr的乙酸溶液(2.93mL,16.20mmol)中的溶液在40℃搅拌过夜。蒸发挥发物。将残留物在DCM间分配,用碳酸氢钠溶液稀释,并分离有机层。将有机层用水洗涤,干燥并在真空下浓缩,得到固体(277mg,69%).LCMS方法3Rt:1.36min,MS[M+H]+观测值:249.9,计算值:250.0.
步骤2.N7-(6-苯基吡啶-3-基)甲基)-5’-GMP TEA盐
通过实施例5中所述的方法由130mg GMP TEA盐制备标题化合物(40mg,23%)。1HNMR(400MHz,D2O)δppm:8.57-8.66(1H,m),7.85-7.93(1H,m),7.66-7.82(3H,m),7.39-7.52(3H,m),5.91-6.02(1H,m),5.58-5.77(2H,m),4.42-4.50(1H,m),4.28-4.37(1H,m),4.06-4.17(1H,m),3.95-4.02(1H,m),3.06-3.23(10H,m),1.11-1.30(15H,m).31P NMR(162MHz,D2O):δppm:3.54(1P).LCMS方法2Rt:0.91min,MS[M+H]+观测值:531.0,计算值:531.1.
实施例7.N7-([1,1'-联苯]-4-基甲基)-2’,3’-亚异丙基-5’-GMP TEA盐
步骤1:N7-([1,1'-联苯]-4-基甲基)-2’,3’-亚异丙基鸟苷
将2’,3’-亚异丙基鸟苷(500mg,1.547mmol)和4-联苯基甲基溴(145mg,0.849mmol)的DMSO(2mL)溶液在室温搅拌过夜。将溶液通过反相HPLC直接纯化(X-Bridge50x50mmm,5μm柱,洗脱液A:含有5mM NH4OH的水;B含有5mM NH4OH的MeCN;梯度:15-40%B/A)。合并产物级分并冻干,得到标题化合物,为白色粉末(260mg,34%).LCMS方法1Rt:1.02min,MS[M+H]+观测值:490.1,计算值:490.2.
步骤2:N7-([1,1'-联苯]-4-基甲基)-2’,3’-亚异丙基-5’-GMP TEA盐
在0℃和在N2搅拌下,将三氯氧磷(95μL,1.021mmol)缓慢加入N7-([1,1'-联苯]-4-基甲基)-2’,3’-亚异丙基鸟苷(100mg,0.204mmol)的磷酸三甲酯(1mL)混合物中,持续3hrs。在0℃将反应混合物滴加至1M TEAB溶液(3mL)中,将得到的混合物离心。将如此获得的溶液通过反相柱色谱法纯化(ISCO Teledyne C18 30g Gold柱,洗脱液A:0.1M TEAB;B:30%MeCN的0.1M TEAB溶液;梯度:0-100%B/A)。合并产物级分并冻干,得到标题化合物,为白色粉末(38mg,24%).1H NMR(400MHz,D2O)δppm:9.70-9.90(1H,br),7.57-7.69(4H,m),7.29-7.57(5H,m),6.01-6.13(1H,m),5.61-5.78(2H,m),5.34-5.41(1H,m),5.09-5.15(1H,m),4.52-4.60(1H,m),3.98-4.09(1H,m),3.80-3.92(1H,m),2.60(8H,q),1.50(3H,s),1.34(4H,s),1.00(14H,t).31C(162MHz,D2O):δppm:0.224(1P).LCMS方法4Rt:0.94min,MS[M+H]+观测值:570.1,计算值:570.2.
无环鸟苷(Sigma-Aldrich No.A4669)的烷基化按照通用方法B进行。LC-MS方法1Rt=0.95mins;MS m/z[M+H]+392.2.
将步骤1中获得的醇(73mg,0.19mmol)混悬在PO(OMe)3(1.9mL)中,并加入POCl3(86mg,0.56mmol)。将溶液在rt搅拌3h,然后通过加入TEAB(0.1M,1mL)淬灭。将获得的溶液直接通过柱色谱法纯化(ISOC RP C18Aq用TEAB 0.1M和MeCN洗脱;0-100%MeCN)。将获得的级分冻干后,获得产物,为无色泡沫(84mg,0.14mmol,75%,磷酸酯与NEt3比例1/1.4,通过1H-NMR测定).LC-MS方法6Rt=1.79mins;MS m/z[M+H]+472.1168;计算值:472.1145,1H NMR(400MHz,D2O)δ7.45 7.44(3H,m),7.54(2H,d,J=8.14Hz),7.50(2H,d,J=7.80Hz),5.53(2H,s),5.50(2H,s),3.78–3.74(2H,m),3.65–3.62(2H,m),3.05(8.4H,q,J=7.34Hz,NEt3),1.13(12.7H,t,J=7.56Hz,NEt3).
步骤1:将无环鸟苷磷酸酯三乙基铵盐(如上所述获得的,77mg,0.13mmol)混悬在DMF(无水的,6.7mL)和咪唑(101mg,1.48mmol)中,加入2,2’-二吡啶基二硫醚(151mg,0.685mmol)和NEt3(34mg,0.24mmol)。将混悬液在rt搅拌10min,然后加入三苯基膦(183mg,0.698mmol)。反应混合物变为浅黄色,并在rt搅拌5h。加入NaClO4的丙酮溶液(1M,2.1mL)和丙酮(15ml),并将混悬液在冰上保持10min。将得到的溶液离心(3min,2000x g),并将沉淀物用10mL冰冷的丙酮洗涤两次。将沉淀物在真空下干燥获得咪唑活化的磷酸酯(50mg,0.096mmol,71%),其没有进一步纯化直接使用。
步骤2:将步骤1中获得的活化的磷酸酯混悬在DMF(0.5mL)中,并加入磷酸三丁基铵盐(1.0M的DMF溶液,0.5mL,0.479mmol)和ZnCl2(13mg,0.096mmol)。将溶液在rt剧烈搅拌5h,然后直接通过柱色谱法在ISCO RPAq18上纯化,用0.1M TEAB和MeCN洗脱,0至100%MeCN。冻干含有产物的级分,得到标题产物,为无色固体(52mg,0.065mmol,68%,磷酸酯与NEt3的比例1/2.45,通过1H NMR测定).LC-MS:Rt=0.99mins;MS m/z[M+H]+552.1LCMS方法1;1H NMR(400MHz,D2O)δ7.59–7.54(4H,m),7.40–7.36(4H,m),7.32–7.28(1H,m),5.58(2H,s),5.55(2H,s),3.98–3.94(2H,m),3.73–3.71(2H,m),3.08(q,J=7.22Hz,NEt3),1.13(12.7H,t,J=7.56Hz,NEt3).
实施例10. 7-((2-氯-[1,1'-联苯]-4-基)甲基)-5’-GDP TEA盐
步骤1:2-氯-4-甲基-1,1'-联苯
在2打兰小瓶中,将苯基碘(200mg,0.98mmol)和(2-氯-4-甲基苯基)硼酸(167mg,0.98mmol)溶解在DMF(4.9mL)中。加入K2CO3水溶液(542mg在400μL H2O中)和SphosPalladacycle G2(7.1mg,9.8μmol),并将得到的混悬液在80℃剧烈搅拌3h。冷却至室温后,加入DCM(5mL),并将有机层用水(10mL),10%LiCl(aq.,3x 10mL)洗涤,并用Na2SO4干燥。将在真空中除去溶剂后获得的残留物通过快速色谱法纯化(SiO2,庚烷至20%EtOAc的庚烷溶液)。获得2-氯-4-甲基-1,1'-联苯,为无色油状物(147mg,0.725mmol,74%).1H NMR(400MHz,CDCl3)δ7.45 7.44(3H,m),7.41–7.36(1H,m),7.32(1H,br s),7.25(1H,d,J=7.58Hz),7.14(1H,d,J=7.58Hz),2.40(3H,s).
步骤2:4-(溴甲基)-2-氯-1,1'-联苯
将2-氯-4-甲基-1,1'-联苯(140mg,0.691mmol)、N-溴琥珀酰亚胺(135mg,0.760mmol)和偶氮二异丁腈(11mg,0.069mmol)溶解在CCl4(6.9mL)中,并将溶液在85℃搅拌1h。在真空下除去溶剂,并将残留物通过快速色谱法纯化(SiO2,庚烷至20%EtOAc的庚烷溶液),获得标题化合物,为含有原料的混合物(3:1,,通过1H NMR判断),为无色固体(173mg,0.406mmol,66%)。将如此的混合物用于随后的烷基化反应。
步骤3:7-((2-氯-[1,1'-联苯]-4-基)甲基)-5’-GDP TEA盐
按照通用方法B,将GDP三丁基铵盐(70mg,0.086mmol)用步骤2的产物(73mg,0.26mmol)烷基化。获得标题产物,为无色固体(30mg,0.036mmol,42%).LCMS方法1:Rt=1.01mins;MS m/z[M+H]+644.0;1H NMR(400MHz,DMSO-d6)δ10.42(1H,s),7.93(1H,s),7.78(1H,d,J=8.12Hz),7.72-7.63(1H,m),7.47-7.35(9H,m),5.81(1H,s),5.72(1H,br s),4.48(1H,br s),4.40(1H,br s),4.14-4.04(3H,m),3.34(br,NEt3),1.03(12.7H,t,J=7.56Hz,NEt3).
实施例11. 7-((3-甲氧基-[1,1'-联苯]-4-基)甲基)-5’-GDP TEA盐
步骤1:3-甲氧基-4-甲基-1,1'-联苯
在2打兰小瓶中,将苯基碘(200mg,0.98mmol)和(3-甲氧基-4-甲基苯基)硼酸(163mg,0.98mmol)溶解在DMF(4.9mL)中。加入K2CO3水溶液(542mg在400μL H2O中),并加入Sphos Palladacycle G2(7.1mg,9.8μmol),并将得到的混悬液在80℃剧烈搅拌12h。冷却至室温后,加入DCM(5mL),并将有机层用水(10mL),10%LiCl(aq.,3x 10mL)洗涤,并用Na2SO4干燥。将在真空中除去溶剂后获得的残留物通过快速色谱法纯化(SiO2,庚烷至20%EtOAc的庚烷溶液)。获得3-甲氧基-4-甲基-1,1'-联苯,为无色油状物(149mg,0.752mmol,77%).1H NMR(400MHz,DMSO-d6)δ7.70-7.66(2H,m),7.53–7.49(2H,m),7.44–7.39(1H,m),7.28(1H,d,J=7.04Hz),7.19(1H,dd,J=7.60,1.77Hz),7.13(1H,d,J=1.50Hz),3.97(3H,s),2.37(3H,s).
步骤2:4-(溴甲基)-3-甲氧基-1,1'-联苯
将3-甲氧基-4-甲基-1,1'-联苯(147mg,0.741mmol)、N-溴琥珀酰亚胺(145mg,0.816mmol)和偶氮二异丁腈(12mg,0.074mmol)溶解在CCl4(7.4mL)中,并将溶液在85℃搅拌1h。在真空下除去溶剂,并将残留物通过快速色谱法纯化(SiO2,庚烷至20%EtOAc的庚烷溶液),获得标题化合物,为无色固体(163mg,0.588mmol,79%).1H NMR(400MHz,CDCl3)δ7.62-7.59(2H,m),7.49–7.45(2H,m),7.43–7.39(2H,m),7.18(1H,dd,J=7.75,1.66Hz),7.11(1H,d,J=1.59Hz),4.65(2H,s),3.99(3H,s).
步骤3:7-((3-甲氧基-[1,1'-联苯]-4-基)甲基)-5’-GDP TEA盐
按照通用方法B,将GDP三丁基铵盐(100mg,0.123mmol)用步骤2的产物(102mg,0.369mmol)烷基化。获得标题产物,为无色固体(46mg,0.056mmol,45%).LCMS方法1Rt=0.99mins;MS m/z[M+H]+640.0;1H NMR(400MHz,DMSO-d6)δ9.80(1H,s),7.66(2H,d,J=7.49Hz),7.47-7.43(3H,m),7.38–7.34(1H,m),7.21(1H,br s),7.15(1H,d,J=7.59Hz),5.83(1H,s),5.56(2H,s),4.48(2H,s),4.14(1H,br s),4.06(1H,br s),3.95(3H,s),2.68(br,NEt3),1.04(t,J=6.81Hz,NEt3).
实施例12. 7-(6-苯基吡啶-2(1H)-酮-3-基)-5’-GDP TEA盐
步骤1:3-(羟基甲基)-6-苯基吡啶-2(1H)-酮
向2-氧代-6-苯基-1,2-二氢吡啶-3-甲酸(533mg,2.48mmol)的THF(24mL)溶液中,加入硼烷二甲基硫醚络合物(1.0M的THF溶液,991μL,9.91mmol),并将混悬液在室温搅拌18h。缓慢加入MeOH,直到气体产生停止。将得到的溶液在EtOAc和盐水间分配,并将有机层用NaSO4干燥。在真空下除去溶剂,并通过快速色谱法(SiO2,DCM至5%MeOH的DCM溶液)纯化残留物,得到标题化合物,为淡黄色固体(331mg,1.65mmol,66%).LCMS方法1Rt=0.89mins;MS m/z[M-OH-]+183.6.
步骤2:3-(溴甲基)-6-苯基吡啶-2(1H)-酮
将3-(羟基甲基)-6-苯基吡啶-2(1H)-酮(331mg,1.65mmol)用HBr(aq.48%,16.5mL)处理,并将混悬液在室温搅拌1h,然后在60℃搅拌1h。在此期间,混悬液首先澄清,然后形成沉淀。过滤沉淀,用水充分洗涤,并在真空中干燥,得到3-(溴甲基)-6-苯基吡啶-2(1H)-酮,为无色粉末(388mg,1.469mmol,89%).1H NMR(400MHz,DMSO-d6)δ7.08-7.74(4H,m),7.55-7.51(3H,m),7.50-7.46(1H,m),5.28(2H,s).
步骤3:7-(6-苯基吡啶-2(1H)-酮-3-基)-5’-GDP TEA盐
按照通用方法B将GDP三丁基铵盐(100mg,0.123mmol)用步骤2的产物(102mg,0.369mmol)烷基化。获得标题产物,为无色固体(46mg,0.056mmol,45%).LCMS方法1Rt=0.73mins;MS m/z[M+H]+627.1;1H NMR(400MHz,DMSO-d6)δ9.7(1H,s),7.78–7.76(2H,m),7.63(1H,d,J=7.33Hz),7.47-7.55(5H,m),6.69(1H,d,J=7.33Hz),5.82(1H,d,J=2.04Hz),5.45(2H,s),4.47(1H,br),4.40(1H,dd,J=5.48Hz),4.13(1H,br),4.08(1H,br),4.03(1H,br),3.35(br,NEt3),1.05(t,J=6.49Hz,NEt3).
实施例13. 7-(3,5-二甲基苄基)-5’-GDP TEA盐
按照通用方法B将GDP三丁基铵盐(150mg,0.184mmol)用3,5-二甲基苄基溴(110mg,0.553mmol)烷基化。获得标题产物,为无色固体(66mg,0.090mmol,49%).LCMS方法1Rt=0.80mins;MS m/z[M+H]+561.9;1H NMR(400MHz,DMSO-d6)δ10.38(1H,s),8.20(1H,d,J=7.48Hz),7.95(1H,d,J=8.73Hz),7.65–7.58(1H,m),5.81(2H,br s),4.44(1H,br s),4.38(1H,br s),4.13–4.04(2H,m),2.69(br,NEt3),1.05(t,J=6.49Hz,NEt3).
实施例14. 7-(3-氟苄基)-鸟苷-5’-GDP TEA盐
按照通用方法B将GDP三丁基铵盐(150mg,0.184mmol)用3-氟苄基溴(105mg,0.553mmol)烷基化。获得标题产物,为无色固体(40mg,0.055mmol,30%).LCMS方法1:Rt=0.59mins;MS m/z[M+H]+551.9;1H NMR(400MHz,DMSO-d6)δ10.38(1H,s),7.61(1H,d,J=9.96Hz),7.54(1H,d,J=7.87Hz),7.38(1H,dd,J=14.50,7.44Hz),5.79(1H,d,J=1.82Hz),5.66(1H,br s),4.45(1H,br s),4.39(1H,br s),4.11–4.05(2H,m),2.73(br,NEt3),1.05(t,J=6.49Hz,NEt3).
实施例15. 7-((3-氟-[1,1'-联苯]-4-基)甲基)-5’-GDP TEA盐
步骤1:3-氟-4-甲基-1,1'-联苯
在2打兰小瓶中,将苯基碘(800mg,3.92mmol)和(3-氟-4-甲基苯基)硼酸(604mg,3.92mmol)溶解在DMF(19.6mL)中。加入K2CO3水溶液(2.17g在1.9mL H2O中)和SphosPalladacycle G2(28.3mg,39.0μmol),并将得到的混悬液在80℃剧烈搅拌16h。冷却至室温后,加入DCM(15mL),并将有机层用水(10mL)、10%LiCl(aq.,3x 10mL)洗涤,并用Na2SO4干燥。将在真空中除去溶剂后获得的残留物通过快速色谱法纯化(SiO2,庚烷至20%EtOAc的庚烷溶液)。获得3-氟-4-甲基-1,1'-联苯,为无色油状物(746mg,3.61mmol,91%,90%纯度).1H NMR(400MHz,DMSO-d6)δ7.69–7.66(2H,m),7.48–7.36(6H,m),2.27(3H,d,J=1.68Hz).
步骤2:4-(溴甲基)-3-氟-1,1'-联苯
将3-氟-4-甲基-1,1'-联苯(300mg,1.611mmol)、N-溴琥珀酰亚胺(315mg,1.77mmol)和偶氮二异丁腈(26.5mg,0.161mmol)溶解在CCl4(16mL)中,并将溶液在85℃搅拌1h。在真空下除去溶剂,并将残留物通过快速色谱法纯化(SiO2,庚烷至20%EtOAc的庚烷溶液),获得标题化合物,为淡黄色固体(311mg,1.17mmol,72%).1H NMR(400MHz,DMSO-d6)δ7.74–7.71(2H,m),7.62(1H,d,J=7.92Hz),7.57(1H,dd,J=11.47,1.70Hz),7.53(1H,dd,J=7.92,1.60Hz),7.51–7.46(2H,m),7.43–7.38(1H,m).
步骤3:7-((3-氟-[1,1'-联苯]-4-基)甲基)-5’-GDP TEA盐
按照通用方法B将GDP三丁基铵盐(150mg,0.184mmol)用步骤2中获得的溴化物(147mg,0.553mmol)烷基化。获得标题产物,为无色固体(45mg,0.056mmol,30%).LCMS方法1Rt=0.96mins;MS m/z[M+H]+627.9;1H NMR(400MHz,DMSO-d6)δ10.13(1H,s),7.69–7.67(2H,m),7.55–7.50(2H,m),7.48–7.44(3H,m),7.41–7.38(1H,m),5.84–5.78(2H,m),4.46–4.43(2H,m),4.11–4.06(2H,m),2.75(br,NEt3),1.05(t,J=6.49Hz,NEt3).
实施例16. 7-(二苯甲基)-鸟苷-5’-GDP TEA盐
按照通用方法B将GDP三丁基铵盐(150mg,0.184mmol)用二苯甲基溴(91mg,0.369mmol)烷基化。获得标题产物,为无色固体(20mg,0.021mmol,30%).LCMS方法1Rt=0.90mins;MS m/z[M+H]+610.1;1H NMR(400MHz,DMSO-d6)δ7.53–7.47(2H,m),7.44–7.29(8H,m),5.86(1H,d,J=4.62Hz),5.82(1H,d,J=4.32),4.72(1H,br s),4.48–4.40(1H,brs),4.20–4.02(3H,m),3.98–3.92(2H,m),2.62(br,NEt3),1.00(t,J=6.49Hz,NEt3).
N7-烷基化GMP衍生物的分析数据提供在表1中。下面的类似物通过与通用方法A或B或实施例1-17中所述的方法类似的方法制备。
表1:N7-烷基化GMP衍生物的分析数据.
N7-烷基化GDP衍生物的分析数据提供在表2中。下面的类似物通过与通用方法A或B或实施例1-17中所述的方法类似的方法制备。
表2:N7-烷基化GDP衍生物的分析数据
N7-烷基化肌苷衍生物的分析数据提供在3表中。下面的类似物通过与通用方法A或B或实施例1-17中所述的方法类似的方法制备。
表3:N7-烷基化肌苷衍生物的分析数据.
C8取代的黄嘌呤的合成方法
实施例18. 7-([1,1'-联苯]-4-基甲基)-8-溴-3-甲基-1H-嘌呤-2,6(3H,7H)-二酮
将8-溴-3-甲基-1H-嘌呤-2,6(3H,7H)-二酮(245mg,1.000mmol)、4-(溴甲基)-1,1'-联苯(247mg,1.000mmol)和K2CO3(38.5mg,1.000mmol)的DMF(5mL)混合物在RT搅拌过夜。将水加入反应混合物。将得到的固体通过过滤收集,用水洗涤并干燥。ISCO纯化(Silica80g,0-5%MeOH的DCM溶液)提供标题产物。
实施例19:(4-(7-([1,1'-联苯]-4-基甲基)-3-甲基-2,6-二氧代-2,3,6,7-四氢-1H-嘌呤-8-基)苯基)膦酸
将实施例18(60mg,0.146mmol)、(4-(4,4,5,5-四甲基-1,3,2-二氧硼杂环戊-2-基)苯基)膦酸二甲酯(45.5mg,0.146mmol)、Na2CO3(30.9mg,0.292mmol)、四(三苯基膦)钯(16.9mg,0.015mmol)和水(0.2mL)在二氧六环(1mL)中的反应混合物用N2冲洗2分钟,然后在密封小瓶中在100℃搅拌过夜。Prep-HPLC纯化(Waters X-Bridge C18 30x50mm 5um柱,ACN/H2O w/5mM NH4OH@75ml/min,15-40%ACN,历经3.5min梯度)分离单-甲基酯和二甲基酯。将含有这两种产物的级分合并,并在真空下浓缩。将残留物溶解于DMF(1mL)中,加入溴三甲基硅烷(112mg,0.729mmol)。将反应混合物在RT搅拌过夜。加入水和MeOH,淬灭反应,并在真空下浓缩得到的溶液。Prep-HPLC纯化(Waters X-Bridge C18 30x50mm 5um柱ACN/H2Ow/5mM NH4OH@75ml/min,5-20%ACN,历经3.2min梯度)提供需要的产物。1H NMR(400MHz,DMSO-d6):δppm 7.64-7.77(2H,m),7.54-7.64(6H,m),7.27-7.46(3H,m),7.03-7.15(2H,m),5.56-5.71(2H,m),3.42(3H,s).LCMS(XBridge C18 3.5μm 3.0x30mm ACN/H2O w/5mMNH4OH@75ml/min,5-95,历经2min梯度)Rt=0.51min,MS[M+H]+观测值:489.3,计算值:489.4.
实施例20:7-([1,1'-联苯]-4-基甲基)-8-溴-1,3-二甲基-1H-嘌呤-2,6(3H,7H)-二酮
根据上面所述的方法类似地制备标题产物。1H NMR(400MHz,甲醇-d4):δppm7.88-8.00(2H,m),7.68-7.78(2H,m),7.49-7.59(4H,m),7.27-7.44(3H,m),7.03-7.15(2H,m),5.72-5.81(2H,m),3.63(3H,s),3.38(3H,s).LCMS(XBridge C18 3.5μm 3.0x30mm ACN/H2O w/5mM NH4OH@75ml/min,5-95,历经2min梯度)Rt:0.63min,MS[M+H]+观测值:503.3,计算值:503.4.
C8取代的嘌呤的通用合成:
步骤1:6-氨基-5-((2-(4-氯苯氧基)乙基)氨基)嘧啶-4(3H)-酮
将2-(4-氯苯氧基)乙酸(13.3g,71.4mmol)溶解于DMF(90mL,0.8M)中,并加入三乙胺(14.4g,143mmol,2equiv.)和HATU(27.1g,71.4mmol)。将反应在室温搅拌30min,然后加入吡啶酮(9.0g,71.4mmol)。将混合物在室温搅拌过夜后,将反应用水淬灭,并通过加入5NHCl将pH调至1。收集白色沉淀,用1N HCl洗涤并在真空下干燥2天(12.7g,43.1mmol,60%)。将获得的酰胺直接用于下一步骤。
将上面获得的酰胺(10g,33.9mmol)溶解于THF(250mL)中并冷却至0℃。缓慢加入LiAlH4(7.73g,204mmol),并将混合物在室温搅拌30min,在50℃搅拌5小时。然后将混合物冷却至0℃,并通过加入1N HCl淬灭。收集白色沉淀,用1N HCl洗涤并在真空下干燥2天。获得产物,为白色固体,其没有进一步纯化即使用(3.0g,10.7mmol,32%).
步骤2:用于酰化和闭环的通用方法:
将羧酸溶取在DMF(0.1M)中,并用HATU(2equiv.)和三乙胺(4.5equiv.)处理。将溶液在室温搅拌15min,然后加入步骤1中获得的嘧啶。将得到的溶液在室温搅拌16h,然后用EtOAc稀释并用缓冲溶液(pH=7.0)洗涤。将含水层用EtOAc萃取2x,并将合并的有机层用Na2SO4干燥。在真空下浓缩溶液,得到棕色油状物,将其通过RP-HPLC纯化。
对于R=OH:将上面获得的酰胺溶取在异丙醇(0.1M)中,加入叔丁醇钠(5equiv.),并将混合物在80℃搅拌4h。加入乙酸,直到溶液成酸性,并且形成沉淀。过滤沉淀,用水洗涤并在真空中干燥。将得到的醇通过用POCl3的磷酸三甲酯溶液在0℃处理2h磷酸化。将反应用TEAB(1M)淬灭,并直接通过RP-HPLC纯化。
对于R=PO(OEt)2:将上面获得的磷酸酯通过用TMSBr(5.5eq.)处理0.7M在DMF中的溶液来水解。在室温搅拌16h后,通过加入MeOH/H2O 1/1和1M TEAB将反应淬灭。过滤混悬液并直接通过RP-HPLC纯化。
C8-取代的嘌呤的分析数据提供在表4中。通过上面所述方法制备下面的类似物。
表4:C8取代的嘌呤的分析数据
咪唑活化的mRNA帽的制备
用于GMP和GDP衍生物的咪唑活化的通用方法:
向N7-烷基化的-GMP/GDP衍生物的三乙基铵盐的DMF溶液中加入咪唑(10eq.)、2,2'-二吡啶基二硫醚(4eq.)和TEA(2eq.)。将反应在RT搅拌5min,然后加入三苯基膦(4eq.),反应变为黄色。在RT搅拌16-18h后,将1M NaClO4的丙酮溶液(10eq.)加入反应中,产生白色沉淀。将另外的丙酮加入反应中,并将得到的混悬液在4℃冷却20min并离心。将上清液倾出,并用丙酮洗涤沉淀,在4℃冷却10min并离心。将该洗涤操作重复一次或两次。将得到的沉淀在真空下干燥,得到咪唑活化的N7-烷基化GMP/GDP衍生物的钠盐。
实施例21:P2-咪唑阴离子(imidazolide)N7-([1,1'-联苯]-4-基甲基)-5’-GDP Na盐
将N7-([1,1'-联苯]-4-基甲基)-5’GDP的三乙基铵盐(45mg,0.049mmol)、咪唑(50mg,0.742mmol)、2,2’-二硫二吡啶(65mg,0.297mmol),三乙胺(207μL,1.484mmol)和三苯基膦(78mg,0.297mmol)的二甲基甲酰胺(1.0mL)混合物在室温搅拌3h。加入高氯酸钠的丙酮溶液(1M,1mL),然后用丙酮(40mL)稀释。通过离心分离沉淀,用丙酮洗涤三次,并在真空下干燥,得到为钠盐的标题化合物(34mg,93%).1H NMR(400MHz,D2O)δppm:7.80-7.90(1H,s),7.63-7.72(4H,m),7.38-7.56(5H,m),7.18-7.27(1H,s),6.86-6.95(1H,s),5.97-6.07(1H,m),5.59-5.75(2H,m),4.58-4.64(2H,m),4.33-4.40(3H,m),4.16-4.29(1H,m),4.06-4.16(1H,m).13C NMR(162MHz,D2O):δppm:11.712(1P),19.863(1P).LCMS方法2Rt:1.35min,MS[M-H]+观测值:659.1,计算值:659.1.
实施例22:P2-咪唑阴离子N7-([1,1'-联苯]-4-基甲基)-5’-GMP Na盐
用与实施例21中所述的方法类似的方法合成标题化合物。1H NMR(400MHz,D2O)δppm:7.78-7.83(1H,s),7.43-7.49(4H,m),7.16-7.40(5H,m),7.01-7.08(1H,s),6.83-6.90(1H,s),5.79-5.87(1H,m),5.51-5.76(2H,m),4.50-4.57(1H,m),4.20-4.35(2H,m),4.00-4.16(2H,m).31P NMR(162MHz,D2O):δppm:8.134(1P).LCMS方法2Rt:1.32min,MS[M-H]+观测值:577.9,计算值:578.2.
按照上面所述的二磷酸酯的咪唑活化的通用方法合成标题化合物。LCMS方法5MS[M-H]+观测值:602.3,计算值:602.1.
实施例24:P2-咪唑阴离子-N7-(2-(4-氯苯氧基)乙基)-2’-O-甲基-5’-GDP Na盐
步骤1:N7-(2-(4-氯苯氧基)乙基)-2’-O-甲基-鸟苷
将2'OMe-鸟苷(0.673mmol)和1-(2-溴乙氧基)-4-氯苯(2.70mmol)溶取在4ml无水DMSO中,并在50℃搅拌20h。将反应冷却至RT,用DMSO稀释,并在制备HPLC上纯化[ShimadzuPrep HPLC(Sunfire Prep C18 5μm,100mm x 30mm柱;0-20min:5至30%0.1%TFA的ACN溶液/0.1%TFA的H2O溶液;42mL/min)]。合并产物级分并浓缩,同时与甲苯共沸,得到标题化合物的三氟乙酸盐,为白色固体(99mg,0.17mmol,26%).LCMS方法2Rt=1.38mins;MS m/z[M+H]+452.2.
步骤2:N7-(2-(4-氯苯氧基)乙基)-2’-O-甲基-5’-GMP TEA盐
向冷却至0℃的N7-(2-(4-氯苯氧基)乙基)-2’-O-甲基-鸟苷(0.085mmol)的1.0ml磷酸三甲酯溶液中缓慢滴加加入POCl3(0.536mmol)。将反应在0℃搅拌6.5h,然后冷却至-20℃反应16h。然后将反应升温至0℃,持续2h。将另外的POCl3(0.536mmol)加至反应中,并将反应在0℃搅拌3h。在0℃用1M TEAB(aq.)(3.5ml)将反应缓慢淬灭。一旦TEAB加入完成,将反应升温至RT,以确保淬灭过量的POCl3。将淬灭的反应物通过制备离子交换色谱法直接纯化[(Tosoh TSKgel DEAE-5PW,13μm,21.5x150mm,0-30min:0-100%1M TEAB,5ml/min)]。冻干产物级分得到标题化合物的三乙基铵盐,为白色固体,将其直接用于下一步骤中。LCMS方法2Rt=1.34mins;MS m/z[M+H]+532.9;1H NMR(D2O)δ:7.16-7.22(m,2H),6.80-6.86(m,2H),6.03(d,J=3.4Hz,1H),4.78-4.84(m,2H),4.48-4.54(m,2H),4.46(t,J=5.3Hz,1H),4.25-4.30(m,1H),4.18-4.22(m,1H),4.09-4.17(m,1H),3.96-4.03(m,1H),3.51(s,3H),3.14(q,J=7.4Hz,NEt3),1.21(t,J=7.3Hz,NEt3).
步骤3:P1-咪唑阴离子N7-(2-(4-氯苯氧基)乙基)-2’-O-甲基-5’-GMP Na盐
向N7-(2-(4-氯苯氧基)乙基)-2’-O-甲基-5’-GMP(0.05mmol)的1.0ml无水的DMF混悬液中加入咪唑(0.514mmol)、2,2'-二吡啶基二硫醚(0.227mol)和TEA(0.072mmol)。然后将PPh3(0.229mmol)加至反应物中,使其变为黄色。在RT搅拌18h后,将1M NaClO4的丙酮溶液(0.250mmol)加入反应物中,随后加入4ml丙酮。将得到的混悬液在4℃冷却20min并离心。倾出上清液并将得到的沉淀用5ml丙酮洗涤,在4℃冷却20min并再离心。将该洗涤操作再重复两次。将得到的灰白色固体在真空下干燥,得到标题化合物的钠盐(30mg)。将该化合物直接用于步骤4中。LCMS方法5MS(ES-):m/z=580.4(M-H+).
步骤4:N7-(2-(4-氯苯氧基)乙基)-2’-O-甲基-5’-GDP TEA盐
向P1-咪唑阴离子N7-(2-(4-氯苯氧基)乙基)-2’-O-甲基-5’-GMP Na盐(0.05mmol)的1ml无水DMF溶液中滴加加入正磷酸三丁基铵盐(如:A.R.Kore,G.Parmar,Syn.Comm.2006,36,3393-3399中所述制备;1M的DMF溶液,0.250mmol)中,随后加入ZnCl2(0.051mmol)。将反应在RT搅拌4.5h,然后用水(1ml)淬灭并浓缩至油性固体。将该固体溶取在DMF(1ml)中,并加入1M NaClO4的丙酮溶液(0.500mmol),随后加入7ml丙酮。将得到的混悬液冷却至0℃,保持~10min,离心并倾倒上清液。将固体用丙酮洗涤一次,离心并倾倒上清液。然后将得到的固体在真空下干燥,以除去任何残留物有机物并溶取在H2O中,采用制备离子交换色谱法纯化[(Tosoh TSKgel DEAD-5PW,13μm,21.5x150 mm,0-100%1M TEAB,5ml/min)].合并产物级分,浓缩,并与乙醇共沸,得到标题化合物的三乙基铵盐,为白色固体(5.7mg,6.6μmol,13%).LCMS方法5MS(ES+):m/z=612.0(M+H+);1H NMR(D2O)δ:9.25(s,1H),7.13(d,J=8.9Hz,2H),6.79(d,J=8.9Hz,2H),5.99(d,J=3.1Hz,1H),4.76-4.87(m,2H),4.40-4.54(m,3H),4.24-4.35(m,2H),4.13-4.23(m,2H),3.50(s,3H),3.13(q,J=7.3Hz,NEt3),1.17-1.23(m,J=7.3,7.3Hz,NEt3).
步骤5:P2-咪唑阴离子-N7-(2-(4-氯苯氧基)乙基)-2’-O-甲基-5’-GDP Na盐
向N7(2-(4-氯苯氧基)乙基)-2’-O-甲基-5’-GDP TEA盐(6.55μmol)的0.5ml无水DMF混悬液中加入咪唑(88μmol)、2,2'-二吡啶基二硫醚(27.2μmol)和TEA(14.3μmol)。然后将PPh3(26.7μmol)加至反应物中,使其变为黄色。在RT搅拌18h后,将1M NaClO4的丙酮(0.100mmol)加入反应物中,随后加入丙酮(2ml),产生白色沉淀。将该混悬液在4℃冷却20min并离心。倾出上清液并将得到的沉淀用丙酮(3ml)洗涤,在4℃冷却10min并离心。将该丙酮洗涤操作再重复一次。将得到的白色固体在真空下干燥,得到标题化合物的钠盐(4.6mg,6.4μmol,97%)。将该化合物直接用于mRNA加帽反应。LCMS方法5MS(ES-):m/z=660.2(M-H+).
实施例25:P2-咪唑阴离子-N7-(2-(4-氯苯氧基)乙基)-3’-O-甲基-5’-GDP Na盐
步骤1:N7-(2-(4-氯苯氧基)乙基)-3’-O-甲基-5’-鸟苷
将3’OMe-鸟苷(0.673mmol)和1-(2-溴乙氧基)-4-氯苯(2.70mmol)溶取在4ml无水DMSO中,并在50℃搅拌2 0h。将反应冷却至RT,用DMSO稀释并在制备HPLC上纯化[ShimadzuPrep HPLC(Sunfire Prep C18 5μm,100mm x 30mm柱;0-20min:5至30%0.1%TFA的ACN溶液/0.1%TFA的H2O溶液;42mL/min)]。合并产物级分并浓缩,同时与甲苯共沸,得到标题化合物的三氟乙酸盐,为白色固体(124mg,0.223mmol,33%).LCMS方法2Rt=1.39mins;MS m/z[M+H]+452.9.
步骤2:N7-(2-(4-氯苯氧基)乙基)-3’-O-甲基-5’-GMP TEA盐
向冷却至0℃的N7-(2-(4-氯苯氧基)乙基)-3’-O-甲基-5’-鸟苷(0.115mmol)的1.0ml磷酸三甲酯溶液缓慢滴加加入POCl3(0.536mmol)。将反应在0℃搅拌6.5h,然后冷却至-20℃,保持16h(置于冰箱中过夜)。然后将反应升温至0℃持续2h。将另外的POCl3(0.536mmol)加入反应中,并将反应在0℃搅拌3h。将反应在0℃用1M TEAB(aq.)(3.5ml)缓慢淬灭。一旦TEAB加入完成,将反应升温至RT,以确保淬灭过量的POCl3。将淬灭的反应物通过制备离子交换色谱法直接纯化[(Tosoh TSKgel DEAE-5PW,13μm,21.5x150mm,0-30min:0-100%1M TEAB,5ml/min)]。冻干产物级分得到标题化合物的三乙基铵盐,为白色固体,将其如获得那样用于下一步骤。LCMS方法2Rt=1.34mins;MS m/z[M+H]+532.8;1H NMR(D2O)δ:7.16-7.23(m,2H),6.79-6.87(m,2H),5.94(d,J=4.0Hz,1H),4.78-4.86(m,2H),4.47-4.52(m,J=5.0,5.0Hz,2H),4.36-4.43(m,1H),4.09-4.17(m,1H),4.07(t,J=5.0Hz,1H),3.93-4.01(m,1H),3.43(s,3H),3.13(q,J=7.4Hz,NEt3),1.21(t,J=7.3Hz,NEt3).
步骤3:P1-咪唑阴离子-N7-(2-(4-氯苯氧基)乙基)-3’-O-甲基-5’-GMP Na盐
向N7-(2-(4-氯苯氧基)乙基)-3’-O-甲基-5’-GMP TEA盐(0.075mmol)的1.0ml无水DMF混悬液中加入咪唑(0.734mol)、2,2'-二吡啶基二硫醚(0.340mmol)和TEA(0.108mmol)。然后将PPh3(0.343mmol)加至反应物中,使其变为黄色。在RT搅拌18h后,加入1M NaClO4的丙酮溶液(0.375mmol),随后加入4ml丙酮。将得到的混悬液在4℃冷却20min并离心。倾出上清液并将得到的沉淀用丙酮(5ml)洗涤,在4℃冷却20min并离心。将该洗涤操作再重复两次。将得到的灰白色固体在真空下干燥,得到标题化合物的钠盐(45mg)。将该化合物直接用于步骤4。LCMS方法5MS(ES-):m/z=580.4(M-H+).
步骤4:N7-(2-(4-氯苯氧基)乙基)-3’-O-甲基-5’-GDP TEA盐
向P1-咪唑阴离子-N7-(2-(4-氯苯氧基)乙基)-3’-O-甲基-5’-GMP Na盐(0.075mmol)的1ml无水DMF溶液中滴加加入正磷酸三丁基铵盐(如:A.R.Kore,G.Parmar,Syn.Comm.2006,36,3393-3399中所述制备;1M的DMF溶液,0.375mmol),随后加入ZnCl2(0.076mmol)。将rxn在RT搅拌4.5h,然后用水(1ml)淬灭并浓缩至油性固体。将该固体溶取在DMF(1ml)中,并加入1M NaClO4的丙酮溶液(1.00mmol),随后加入丙酮(7ml)。将得到的混悬液冷却至0℃持续10min,离心并倾倒上清液。将固体用丙酮洗涤一次,离心并倾倒上清液。然后将得到的固体在真空下干燥,以除去任何残留物有机物,并采用制备离子交换色谱法纯化[(Tosoh TSKgel DEAD-5PW,13μm,21.5x150mm,0-100%1M TEAB,5ml/min)]。合并产物级分,浓缩,并与乙醇共沸,得到标题化合物的三乙基铵盐,为白色固体(14.3mg,4.21μmol,8%).LCMS方法5MS(ES-):m/z=610.2(M-H+);1H NMR(D2O)δ:9.30(s,1H),7.15(d,J=9.0Hz,2H),6.80(d,J=9.0Hz,2H),5.92(d,J=4.0Hz,1H),4.77-4.87(m,2H),4.71-4.75(m,1H),4.39-4.49(m,3H),4.23-4.32(m,1H),4.06-4.17(m,2H),3.43(s,3H),3.13(q,J=7.4Hz,NEt3),1.21(t,J=7.3Hz,NEt3).
步骤5:P2-咪唑阴离子-N7-(2-(4-氯苯氧基)乙基)-3’-O-甲基-5’-GDP Na盐
向2-氨基-7-(2-(4-氯苯氧基)乙基)-9-((2R,3R,4S,5R)-3-羟基-5-(((羟基(膦酰基氧基)磷酰基)氧基)甲基)-4-甲氧基四氢呋喃-2-基)-6-氧代-6,9-二氢-1H-嘌呤-7-(4.20μmol)的0.5ml无水的DMF混悬液中加入咪唑(88μmol)、2,2'-二吡啶基二硫醚(27.2μmol)和TEA(14.3μmol)。然后将PPh3(26.7μmol)加至反应物中,使其变为黄色。在RT搅拌18h后,将1M NaClO4的丙酮溶液(0.100mmol)加入反应物中,随后加入丙酮(2ml),产生白色沉淀。将该混悬液在4℃冷却20min并离心。倾出上清液并将得到的沉淀用丙酮(3ml)洗涤,在4℃冷却10min。并离心。将该丙酮洗涤操作再重复一次。将得到的白色固体在真空下干燥,得到标题化合物的钠盐(7.2mg).将该化合物直接用于mRNA加帽反应。LCMS方法5MS(ES-):m/z=660.2(M-H+).
实施例26:7-([1,1'-联苯]-4-基甲基)-2-氨基-9-((2R,3R,4S,5R)-3,4-二羟基-5-(((羟基((羟基(1H-咪唑-1-基)磷酰基)甲基)磷酰基)氧基)甲基)四氢呋喃-2-基)-6-氧代-6,9-二氢-1H-嘌呤-7-
步骤1:N7-([1,1'-联苯]-4-基甲基)-鸟苷
将鸟苷(2.65mmol)和4-溴甲基联苯(6.07mmol)溶取在无水的DMSO(7.5ml)中,并在RT搅拌60h。将反应物用DMSO稀释并通过制备RP-HPLC纯化。将合并的产物级分浓缩,同时与甲苯共沸,溶取在1:1H2O/乙腈中,冷冻并冻干,得到标题化合物的三氟乙酸盐,为白色固体。LCMS方法1Rt=0.94mins;MS m/z[M+H]+450.0.
步骤2:7-([1,1'-联苯]-4-基甲基)-2-氨基-9-((2R,3R,4S,5R)-3,4-二羟基-5-(((羟基(膦酰基甲基)磷酰基)氧基)甲基)四氢呋喃-2-基)-6-氧代-6,9-二氢-1H-嘌呤-7-
向在-10℃的N7-([1,1'-联苯]-4-基甲基)-鸟苷(0.266mmol)的1.0ml磷酸三甲酯溶液中缓慢加入到冷却至-10℃的亚甲基二膦酰二氯(1.00mmol)的1.0ml磷酸三甲酯溶液中。将反应在-10℃-0℃搅拌6.5h,然后置于-20℃保持60h。通过将其在0℃加入1M TEAB(9ml)中,缓慢淬灭反应。一旦加入完成,将反应升温至RT,以确保淬灭过量的亚甲基二膦酰二氯。将得到的溶液直接在制备HPLC上纯化[(Phenomenex Gemini-NX 5μm C18 100mm x30mm柱;0-15min:10至40%ACN/0.1M TEAB的H2O溶液,25ml/min)]。合并产物级分,冷冻并冻干,得到标题化合物的三乙基铵盐,为白色固体(16mg,0.02mmol,7%).LCMS方法2Rt=1.46mins;MS m/z[M+H]+608.1;1H NMR(D2O)δ:9.51(s,1H),7.25-7.35(m,4H),7.10-7.25(m,5H),5.70(d,J=3.4Hz,1H),5.42-5.58(m,2H),4.50(t,J=3.9Hz,1H),4.40(t,J=5.1Hz,1H),4.28-4.33(m,1H),4.20-4.28(m,1H),4.06-4.17(m,1H),3.12(q,J=7.3Hz,NEt3),2.17(t,J=19.7Hz,2H),1.20(t,J=7.3Hz,NEt3).
步骤3:7-([1,1'-联苯]-4-基甲基)-2-氨基-9-((2R,3R,4S,5R)-3,4-二羟基-5-(((羟基((羟基(1H-咪唑-1-基)磷酰基)甲基)磷酰基)氧基)甲基)四氢呋喃-2-基)-6-氧代-6,9-二氢-1H-嘌呤-7-
向7-([1,1'-联苯]-4-基甲基)-2-氨基-9-((2R,3R,4S,5R)-3,4-二羟基-5-(((羟基(膦酰基甲基)磷酰基)氧基)甲基)四氢呋喃-2-基)-6-氧代-6,9-二氢-1H-嘌呤-7-(0.016mmol)的1.0ml无水的DMF溶液中加入咪唑(0.176mmol)、2,2’-二吡啶基二硫醚(0.082mmol)和TEA(0.029mmol)。将反应在RT搅拌10min,然后加入三苯基膦(0.084mmol),使反应物变为黄色。在RT搅拌18h后,将1M NaClO4的丙酮溶液(0.250mmol)加入反应物中,随后加入丙酮(5ml),产生白色沉淀。将得到的混悬液在4℃冷却20min并离心。倾出上清液并将沉淀用丙酮(5ml)洗涤,在4℃冷却10min并离心。将该洗涤操作再重复一次。将得到的沉淀在真空下干燥,得到标题化合物的钠盐,为白色固体(10.8mg,0.013mmol,81%)。将该化合物直接用于mRNA加帽反应。LCMS方法5MS(ES-):m/z=657.5(M-H+).
实施例27:7-([1,1'-联苯]-4-基甲基)-2-氨基-9-((2R,3R,4S,5R)-3,4-二羟基-5-(((羟基((羟基(1H-咪唑-1-基)磷酰基)氨基)磷酰基)氧基)甲基)四氢呋喃-2-基)-6-氧代-6,9-二氢-1H-嘌呤-7-
步骤1:7-([1,1'-联苯]-4-基甲基)-2-氨基-9-((2R,3R,4S,5R)-3,4-二羟基-5-(((羟基(膦酰基氨基)磷酰基)氧基)甲基)四氢呋喃-2-基)-6-氧代-6,9-二氢-1H-嘌呤-7-
在0℃将N7-([1,1'-联苯]-4-基甲基)-鸟苷(0.177mmol)加至二氯氧膦基亚氨代磷酰三氯(dichlorophosphinylphosphorimidic trichloride)(J.Emsley,J.Moore,P.B.Udy,J.Chem.Soc.A 1971,2863-2864;0.891mmol)的磷酸三甲酯(2.0ml)溶液中。将反应在0℃搅拌1.5h,然后冷却至-20℃并放置16h。将反应升温至0℃,将另外的二氯氧膦基亚氨代磷酰三氯(0.891mmol)加入反应物中。将反应在0℃搅拌5h,直到通过LCMS(方法2RXNMON_Acidic_Polar_PosNeg.olp-ZQ1)监测,仅剩余痕量的起始原料。通过在0℃缓慢地将反应物加入1M TEAB(10ml)中,将反应淬灭。一旦加入完成,将反应升温至RT,以确保淬灭过量的二氯氧膦基亚氨代磷酰三氯。将得到的混悬液过滤并将滤液提供至制备HPLC[(Phenomenex Gemini-NX 5μm C18 100mm x 30mm柱;0-15min:10至50%ACN/0.1M TEAB的H2O溶液,25ml/min)]。合并产物级分,冷冻并冻干,得到标题化合物的三乙基铵盐,为白色固体(13.5mg,0.018mmol,10%).LCMS方法6计算值:608.1186;实测值:609.1290[M+H+];1HNMR(D2O)δ:9.51(s,1H),7.49-7.57(m,4H),7.38-7.44(m,4H),7.31-7.38(m,1H),5.90(d,J=3.6Hz,1H),5.55-5.68(m,2H),4.63(t,J=4.3Hz,1H),4.48(t,J=5.1Hz,1H),4.29-4.38(m,1H),4.17-4.26(m,1H),4.04-4.17(m,1H),3.13(q,J=7.3Hz,NEt3),1.21(t,J=7.3Hz,NEt3).
步骤2:7-([1,1'-联苯]-4-基甲基)-2-氨基-9-((2R,3R,4S,5R)-3,4-二羟基-5-(((羟基((羟基(1H-咪唑-1-基)磷酰基)氨基)磷酰基)氧基)甲基)四氢呋喃-2-基)-6-氧代-6,9-二氢-1H-嘌呤-7-
将7-([1,1'-联苯]-4-基甲基)-2-氨基-9-((2R,3R,4S,5R)-3,4-二羟基-5-(((羟基(膦酰基氨基)磷酰基)氧基)甲基)四氢呋喃-2-基)-6-氧代-6,9-二氢-1H-嘌呤-7-(0.007mmol)溶取在1.0ml无水DMF中,并加入咪唑(0.073mmol)、2,2'-二吡啶基二硫醚(0.036mmol)和TEA(0.014mmol)。将反应在RT搅拌5min,然后加入三苯基膦(0.038mmol),使反应物变为黄色。在RT搅拌16h后,将1M NaClO4的丙酮溶液(0.100mmol)加入反应物中,随后加入丙酮(5ml),产生白色沉淀。将得到的混悬液在4℃冷却20min并离心。倾出清液并将沉淀用丙酮(5ml)洗涤,在4℃冷却10min并离心。将该洗涤操作再重复一次。将得到的沉淀在真空下干燥,得到标题化合物的钠盐,为白色固体(2.3mg,3.4μmol,33%).将该化合物直接用于mRNA加帽反应。LCMS方法5MS(ES-):m/z=658.3(M-H+).
咪唑活化的GDP衍生物的分析数据提供在表5中。根据上面所述的用于咪唑活化的通过方法或类似于实施例21-27中所述的方法制备下面的实施例。
表5.咪唑活化的GDP衍生物的分析数据。
咪唑活化的GMP衍生物的分析数据提供在表6中。根据上面所述的用于咪唑活化的通过方法或类似于实施例21-27中所述的方法制备下面的实施例。
表6.咪唑活化的GMP衍生物的分析数据。
对于寡核苷酸和RNA的加帽,将如上面所示的实施例中获得的咪唑活化的单-和二磷酸酯溶解在无RNAse的水中,得到20mM咪唑活化的GMP/GDP衍生物的H2O溶液(基于样品中存在的活化的咪唑的量,通过1H-NMR测定)。
寡核苷酸的3’-末端修饰的通用方法:
寡5-/5Phos/rGrArAr ArArA rArArAr ArA-3(寡核苷酸1)和5-/5Phos/rGrU/iFluorT/rUrCrG rCrCrA rUrU/i6-TAMN/rArArA rArArA rArArA rA-3(寡核苷酸2)购自IDT。
方法A(缩合反应):将含有在PBS缓冲液中的10μM寡核苷酸的溶液用50x NaIO4(5mM贮备液,500μM终浓度)处理并在0℃保持1h。将获得的溶液用100x Na2SO3(10mM贮备液,1mM终浓度)处理,并升到RT。10min后,加入100x亲核试剂的水溶液(10mM贮备液,1mM终浓度),并将溶液保持在RT过夜。使用Princeton Separation CS100spin柱将溶液脱盐。
方法B(还原胺化反应):将含有在PBS缓冲液中的10μM寡核苷酸的溶液用50xNaIO4(5mM贮备液,500μM终浓度)处理并在0℃保持1h。将获得的溶液用200x Na2SO3(10mM贮备液,2mM终浓度)处理,并升到RT。10min后,加入100x亲核试剂(10mM在水中的贮备液,1mM终浓度)和NaBH3CN(100mM在水中的贮备液,10mM终浓度)。将溶液在37℃振摇过夜,并使用Princeton Separation CS 100spin柱脱盐。
3’-修饰的寡核苷酸的分析数据提供在表7中。根据上面所述的通用方法制备下面的实施例。
表7:3’-修饰的寡核苷酸的分析数据。
a显示的结构代表可能的区域异构的缩合产物之一。
b如果无亲核试剂加入获得的化合物。
c LCMS方法9
mRNA的3’-末端修饰的通用方法:
将mRNA的水溶液(0.5-1.5mg/mL,90μL)用NaIIO4(0.1M在3M NaOAc缓冲液中,pH5.2,终浓度10mM)处理,并将溶液在冰上在暗处保持1h。采用用PBS缓冲液平衡的PrincetonSeparations Centrispin柱使样品脱盐。将溶液用适当的亲核试剂(在H2O或DMSO中,终浓度5mM)处理,用H2O稀释0.75x,并在500rpm在室温振摇2h。将得到的溶液采用PrincetonSeparations CentriSpin 10柱(PBS平衡,然后H2O平衡的)脱盐两次。将获得的RNA溶液通过LiCl沉淀进一步纯化。
生物学数据
结合生物素化和表面固定的hEIF4E的帽类似物的分析:
将双重组氨酸和Avi标记的人EIF4E(His6-3C-avi-eIF4E)表达在8L TB培养基中。在2.0OD600发生通过0.4mM IPTG的诱导,在15OD600收获细胞。将225克沉淀物在缓冲液(50mM Tris,500mM NaCl,2mM MgCl2,1mM TCEP,pH 7.5,含有10%甘油,蛋白酶抑制剂和DNase)中稀释至体积为600mL,并在微流化器上通过一次。在5mL HisTrap HP IMAC上以4.0mL/min IMAC运行样品,对于一柱体积25mM至500mM咪唑梯度。将GST-PreScission蛋白酶(2mg,内部制备)加入到样品中,并在4℃下反应过夜。使切割的集合物在重力柱中通过0.5mL GST和0.5IMAC树脂。使用50mM Tris pH 7.5,1mM TCEP使样品体积增加至800mL,并以4.0mL/min通过5mL hiTrap SP FF,采用历经20柱体积的0至1M NaCl梯度。然后将样品以20mg/mL注射到124mL S75凝胶过滤柱上。将最终的Avi-eIF4E(26931Da)在1x N-二(羟乙基)甘氨酸(Bicine)缓冲液中稀释至1mg/mL,至2.5mg的体积,并与ATP/生物素混合物(10mMATP,10mM Mg(OAc)2,50μM d-生物素最终)混合。将生物素连接酶(25μg内部制备的BirA)加入到反应中。在Eppendorf ThermoMixer R上在30℃下混合(500rpm)反应60分钟,并使用LC-MS检查其完全性。向样品中加入100μl固定化的谷胱甘肽(1:1,与缓冲液),并在4℃下混合15分钟,以结合C3和Bir3,并通过离心除去。使用用20mM HEPES,100mM KCl,1mM DTT,pH7.5平衡的两个连续的PD-10柱缓冲交换样品。
由于低的eIF4E稳定性,制备了链霉亲和素包被的芯片,并在Biacore T200上在10℃下运行。将eIF4E(0.04mg/mL,150μl)与S系列Sensor Chip SA(GE Life Sciences,BR-1005-31)的样品通道结合,至5000至7000RU(响应单位)的表面密度。使用1X PBS、50mMNaCl、0.1%甘油、0.1%CHAPS和1%DMSO,缓冲液以30μL/min流过芯片。将各种帽类似物的样品稀释至在100μM至小于1nM的范围内的各浓度。将样品以两分钟的缔合时间和5分钟的解离时间注入到Biacore芯片中。对于每个样品进行几次缓冲液注入,用于空白扣除。
使用Biacore T200评价软件对所有组进行分析。所有化合物的结合分析报告为1微摩尔化合物的响应单位,其中RU的更高值被解释为与表面固定的eIF4E蛋白质结合更高的配体。一亚组化合物被进一步表征,以使用动力学结合和热力学(稳态亲和力)结合分析中的一种或两者确定解离常数。使用默认设置(注射停止前4秒,具有5秒窗口)完成稳态亲和力拟合。动力学拟合通常用1:1结合模型进行,常数RI=0,所有其他变量设置为全局拟合。
使用上述方法的帽类似物的Biacore结合数据示于表8中。
表8.帽类似物的Biacore结合数据
N/D=未测定
帽类似物与EIF4E的竞争性结合的FRET测定
材料:His标记的eIF4E蛋白购自Fitzgerald Industries International(80R-125;Acton,MA),tRNA购自SIGMA(R5636;St.Louis,MO)。生物素化的20个核苷酸的RNA寡(5’Phos/rGrGrA rCrCrC rCrUrC rUrCrC rCrUrC rCrCrC rCrC生物素-3’)购自IntegratedDNA Technologies(Coralville,IA)。铕标记的抗his抗体购自Perkin Elmer(AD0110;Waltham,MA)和链霉亲和素缀合的alexa fluor 647购自Life Technologies(S32357;Grand Island,NY)。
用于eIF4E结合的TR-FRET测定:
使用四个单独的加入步骤,在PBS(pH 7.4)、0.02%吐温和0.1%BSA的测定缓冲液中进行TR-FRET测定。最初,将5μl His-eIF4E(10nM)和tRNA(0.1mg/ml)加入到黑色有围墙的384孔小体积板中。接下来,使用Beckman Coulter Biomek FX液体分配器加入1μl/孔新鲜稀释的化合物,最终DMSO浓度为1%。使化合物和eIF4E在室温下结合15分钟。第三个添加由以下组成:向每个孔中加入4μl化学加帽的7mGDP咪唑mRNA-生物素(10nM),除了仅含有缓冲液的一列用作背景对照。将结合反应物在室温下孵育1小时。最后,将5μl的铕标记的抗His抗体(10nM)和链霉亲和素缀合的alexa fluor 647(10nM)TR-FRET检测试剂加入每个孔中,并使其在室温下避光1小时。在Envision板读数器(Perkin-Elmer)上读取板以测量来自Alexa fluor(665nm)和铕(615nm)的信号。计算作为帽类似物浓度的函数的alexa fluor和铕荧光的比例用于数据分析,并且在Graph Pad中将数据拟合至标准EC50抑制曲线。
使用上述方法的N7-烷基化GMP衍生物的FRET测定数据提供在表9中。
表9:N7-烷基化GMP衍生物的FRET测定数据。
*报告单次测量的值,除非另外指出.
a超过22次测量的平均值
b超过2次测量的平均值
c超过33次测量的平均值
d超过3次测量的平均值
表10:N7-烷基化GDP衍生物的FRET测定数据
*报告单次测量的值,除非另外指出。
a超过2次测量的平均值
表11:N7-烷基化肌苷衍生物的FRET测定数据
编号 | n | AC50/μM* |
1 | 1 | 1.21 |
2 | 2 | 0.49<sup>a</sup> |
*报告单次测量的值,除非另外指出。
a超过2次测量的平均值
表12:C8-取代的嘌呤衍生物的FRET测定数据
5’-单磷酸mRNA的产生:
如果在最终产物中需要三磷酸结构,则标准T7转录反应导致5’-三磷酸mRNA,其与咪唑磷酸酯活化的帽类似物不相容。为了使用本文所述的咪唑活化的帽类似物产生标准的三磷酸帽结构,RNA底物必须具有用于化学加帽的5’-单磷酸mRNA(当使用咪唑二磷酸帽类似物时)或5’-二磷酸mRNA(当使用咪唑单磷酸帽类似物时)。为了产生单磷酸或二磷酸5’-末端mRNA,将向转录反应中加入10mM GMP或10mM GDP进行测试。虽然10mM GMP产生>95%的5’-单磷酸mRNA,10mM GDP完全抑制转录反应。因此,为了产生5’-三磷酸帽mRNA结构,我们专注于使用含有10mM GMP的转录反应和咪唑二磷酸活化的帽类似物。
mRNA(瘦素和荧光素酶)的体外转录:
为了产生用于体外转录的DNA模板,根据制造商的方案将质粒pGEM-oT7-TEV-oK-Gluc(NcoI)--2hBG-(NotI)-120A或pGEM-oT7-TEV-hLeptin-GAopt-2hBG-120A用限制酶BspQ1(New England Biolabs,Ipswich,MA)线性化。通过用三体积的100%乙醇和1/10体积的3M NaOAc,pH 5.1沉淀来纯化线性化的DNA载体。然后用70%乙醇洗涤,将DNA重新悬浮于水中。
体外转录在40mM Tris-HCl,pH 8、8mM MgCl2、1mM各NTP、10mM DTT、2mM亚精胺、0.004U/uL无机焦磷酸酶(New England Biolabs,Ipswich,MA)、1U/uL RNase抑制剂(NewEngland Biolabs,Ipswich,MA)、5U/μL T7RNA聚合酶(New England Biolabs,Ipswich,MA)和0.2μg/μL的线性化质粒DNA中进行。
如果mRNA制品用于5’-末端化学加帽,则将10mM GMP(Sigma-Aldrich,St.Louis,MO)掺入转录反应中。将反应物在37℃下孵育1.5小时。通过加入0.04U/μL TURBO DNase(Thermo Fisher,Waltham,MA)消化DNA模板,并在37℃下再温育30分钟。转录物用LiCl(终浓度2.81mM)沉淀,然后用70%乙醇洗涤。将沉淀重新悬浮于不含核酸酶的水中。
RNA的化学加帽:
使用的RNA是用5’-单磷酸(Integrated DNA Technologies:5’-P-rGrGrArCrCrCrCrUrCrUrCrCrCrUrCrCrCrCrCrC-3’)合成的20个核苷酸的合成RNA分子或如上所述体外转录的含有5’-单磷酸的mRNA(Gaussia Luciferase mRNA或人瘦素mRNA)。使用3’修饰和未修饰的RNA。
首先通过在65℃加热10分钟使RNA溶液变性。将混合物在冰上冷却5分钟,然后加入到加帽缓冲液(100mM MES缓冲液,pH 6.0,100mM NaCl和5mM MnCl2)中。然后将咪唑活化的帽类似物加入反应中至终浓度为5mM。使反应在Thermoshaker(Grant Instrument,Cambridge,UK)中在室温下进行过夜。通过加入EDTA终止反应,至终浓度为50mM。加帽的产物使用Amicon超离心过滤器(Millipore,Billerica,MA)脱盐。将产物用LiCl沉淀(最终2.81mM LiCl)进一步纯化,并用70%乙醇洗涤。将沉淀重新悬浮于不含核酸酶的水中。
在一些情况下,使用Waters XBridge Shield RP18 3.5um 2.1x100 mm柱(表13),采用反相HPLC进一步纯化化学加帽的mRNA。流动相A为在水中的0.1M乙酸三乙基铵(TEAA),流动相B为在75%水/25%乙腈中的0.1M TEAA。柱流速为0.8ml/分钟,柱温为65℃。手动收集级分。
表13:HPLC纯化梯度.
时间(min) | %流动相B |
0.1 | 44 |
3.0 | 44 |
13 | 64 |
14 | 90 |
15 | 90 |
15.1 | 44 |
20 | 44 |
20个核苷酸合成RNA寡聚体的加帽的LC/MS分析:
在Waters Acquity UPLC BEH C18,1.7μm,2.1x 75mm柱上分离加帽的和未加帽的寡核苷酸。含水流动相含有0.8μM EDTA,7.15mM三乙胺和192.3mM六氟异丙醇。有机流动相为甲醇。将柱保持在65℃,流速为0.35mL/min。典型的梯度在2分钟内从5%升至16%,随后在20分钟内从16%升至25%。使用Thermo LTQ-Orbitrap XL或ABSciex 6500QTrap进行寡核苷酸的Post HPLC MS分析。质谱仪以从735到1550m/z扫描的ESI-MS负模式运行。
表14:通过LC/MS测定的化学加帽的效率。
表15:通过LC/MS测定的化学加帽的效率。
表16:通过LC/MS测定的化学加帽的效率。
表17:通过LC/MS测定的化学加帽的效率。
荧光素酶mRNA的转染和发光读数
细胞培养:将HEK293以30,000个细胞/孔的密度接种在96孔聚D赖氨酸包被的平板中,并在37℃,5%CO2培养箱中孵育过夜。培养基是EMEM(ATCC,cat#30-2003),10%FBS(Invitrogen),无抗生素。
mRNA转染:使用0.4μl/孔的DharmaFECT制剂2(ThermoScientific T-2002-01)转染100ng/孔的mRNA。这是mRNA与转染试剂的1:4比例。将mRNA和转染试剂在OptiMEM中混合以获得10μl/孔的终体积。将混合物在室温下温育20分钟。通过轻击板从细胞中除去过夜的培养基。向每个孔中加入90μl新鲜培养基。向每个孔中加入10μl mRNA混合物。将板在37℃,5%CO2下孵育24小时。
培养基收集:Gaussia荧光素酶蛋白被分泌在培养基中。为了收集培养基,将90μl培养基从细胞转移到v-底的96孔板中。将板以1000rpm离心5分钟。将80μl上清液转移到新的v-底板中,并冷冻或直接用于荧光素酶表达测定。
荧光素酶表达测定:BioLux Gaussia荧光素酶测定试剂盒(New England BiolabsCat#E3300L)用于进行荧光素酶测定。将BioLux gLUC底物在测定缓冲液进行1:100稀释(10μl底物:1000μl测定缓冲液)。将20μl转染培养基加入到96孔白色透明底板(Greiner bio-one 655095)中。使用FlexStation 3微孔板读数器(Molecular Devices)读取板。在时间0,将50uL的底物混合物加入到培养基板中,收集数据持续60秒。相对发光单位(RLU)在30秒处达峰值,并使用该时间点计算结果。通过将每一修饰的mRNA的RLU针对酶促加帽的帽-0mRNA的RLU标准化并乘以100来计算%荧光素酶活性。
流程16:与HPLC纯化的化学加帽的mRNA相比,酶促加帽的HPLC纯化的mRNA(帽-1)的荧光素酶活性。
流程17:与化学加帽的mRNA相比的酶促加帽的mRNA(帽-0)的荧光素酶活性。
加帽的瘦素mRNA的转染和发光读数
细胞培养:将HEK293以30,000个细胞/孔的密度接种在96孔聚D赖氨酸包被的平板中,并在37℃,5%CO2培养箱中孵育过夜。培养基是EMEM(ATCC,cat#30-2003),10%FBS(Invitrogen),无抗生素。
mRNA转染:使用0.4μl/孔的DharmaFECT制剂2(ThermoScientific T-2002-01)转染100ng/孔的mRNA。这是mRNA与转染试剂的1:4比例。将mRNA和转染试剂在OptiMEM中混合以获得10μl/孔的终体积。将混合物在室温下温育20分钟。通过轻击板从细胞中除去过夜的培养基。向每个孔中加入90μl新鲜培养基。向每个孔中加入10μl mRNA混合物。将板在37℃,5%CO2下孵育24小时。
培养基收集:蛋白质被分泌在培养基中。为了收集培养基,将90μl培养基从细胞转移到v-底的96孔板中。将板以1000rpm离心5分钟。将80μl上清液转移到新的v-底板中。
人瘦素蛋白ELISA测定:通过ELISA测量小鼠血浆中的人瘦素(leptin)。从R&Dsystems duoset购买的抗体(Cat#DY398E,用于捕获抗体的部分#840279和用于检测抗体的部分#840280)用PBS重新配制,并再次用PBS滴定(titered)。在白色Maxisorp 384孔板(Cat#460372)上在30μl/孔中,以4μg/mL包被捕获抗体。在室温下孵育过夜后,吸取捕获抗体,室温下用90μL/孔的KPL乳封闭剂(Cat#50-82-00)封闭板2小时。一旦孵育完成,吸取板,并将重组标准品和样品在37℃下以30μL/孔加入到板中保持2小时,同时以600rpm振荡。使用酪蛋白样品稀释液(0.7%酪蛋白,1.7mM磷酸二氢钠,8.1mM磷酸氢二钠七水合物,0.15M NaCl,0.7%Triton X-100和0.1%叠氮化钠)制备样品/标准稀释液。然后使用Teknova板洗液(Cat#P1192),以100μl/孔洗涤/抽吸3次。接下来,使用酪蛋白检测抗体稀释剂(0.4%酪蛋白,1.7mM磷酸二氢钠,8.1mM磷酸氢二钠七水合物,0.15M NaCl和0.1%叠氮化钠)将检测抗体稀释至12.5ng/mL,并以30μl/孔室温加入,保持2小时。孵育后,再次洗涤平板,将在HRP稀释缓冲液(1.7mM磷酸二氢钠,8.1mM磷酸氢二钠七水合物,0.145M NaCl溶液,0.1%氯乙酰胺,1%无蛋白酶的BSA和0.05%吐温20)中以1:1250稀释的聚-链霉亲和素-HRP(Cat#21140)溶液加入到各孔(30μL/孔)中并在室温下温育30分钟。最后洗涤/抽吸除去HRP溶液,并以30μL/孔加入化学发光底物(Cat#1859678&1859679)。采用50ms积分时间,使用SpectramaxM5读板器快速读取该板。ELISA的动态范围是5-150pM的人瘦素。
流程18:采用化学加帽的HPLC纯化的mRNA的瘦素表达数据。
HEK293细胞S100提取物:S100提取物按照S100制备的标准方案制备。HEK293(Thermo Scientific SH30521.02)细胞在80%的FreeStyle 293(Invitrogen 12338)和20%的SFM4中、在8%的CO2中,在加湿的气氛下在以100rpm旋转的轨道摇床上生长。将2.5x109个细胞在临床离心机中以1500rpm离心5分钟。将沉淀物在1L冰冷的DPBS中洗涤,并在临床离心机中在4℃下以1,500rpm再次旋转10分钟。去除上清液,将细胞沉淀重新悬浮于250ml冰冷的PBS中,通过移液破碎,并在临床离心机中于4℃以1,700rpm离心6分钟。去除上清液,将沉淀物重新悬浮于5体积的缓冲液A(10mM HEPES-KOH,pH 7,9、1.5mM MgCl2、10mMKCl、新加入的0.5mM DTT)中。将细胞在冰上孵育10分钟,以2,000rpm离心10分钟,除去上清液。加入另外体积的缓冲液A,使用5冲程松散杵杜恩斯(dounce)均质器将沉淀物破碎。使用15冲程紧杵杜恩斯均质器裂解细胞,并采用显微镜确认裂解。将裂解的材料在临床离心机中以4,500rpm在4℃离心10分钟。移出上清液并用于制备S100提取物。将0.11体积的缓冲液B(0.3M HEPES-KOH,pH 7.9,1.4M KCl,30mM MgCl2)加入到S100中,通过倒置轻轻混合,并在超速离心机中在4℃下以102,000g旋转1小时。移出上清液并置于冷却的15mL锥形管中。从脂质层下面的底层移出清澈上清液,并在4℃用1L缓冲液D(20mM HEPES-KOH,pH 7.9,20%甘油,0.1M KCl,0.2mM EDTA,0.5mM DTT,0.5mM PMSF)透析>5小时。S100提取物在80℃下以小的等分试样储存,直到准备使用。
FRET RNA降解测定:按照Uhler等人J.Am.Chem.Soc.2003,125,14230-14231中的方案(进行了一些修改)进行RNA降解测定。寡5-/5Phos/rGrU/iFluorT/rUrCrG rCrCrArUrU/i6-TAMN/rArArA rArArA rArArA rA-3和5-/5Phos/rGrU/iFluorT/rUrCrG rCrCrArUrU/i6-TAMN/rArArA rArArA rArArA rA/3BIO-3购自IDT并用于RNA降解测定。测定缓冲液由130mM的谷氨酸K盐pH7.5,1mM MgCl2组成。在即将实验之前,将DTT加至终浓度为10mM。根据反应体积分别为25uL和50uL,使用100uM或200uM的寡核苷酸(oligo)。S100提取物从0.3%-20%v/v变化,对于所有样品,缓冲液D为总反应体积的20%。所有反应在37℃的384孔板中进行。在100秒时,将S100提取物加入到寡核苷酸缓冲液混合物中。荧光素在490nm处被激发,并且在520nm和585nm处测量荧光发射。每分钟收集数据,持续2小时。计算FRET比率Q Q(=F585/F520)并针对初始值Q0标准化。使用Prism 5.0,将数据拟合为单指数衰减函数y=yo–Plateau*exp(-K*X)+Plateau。为了提取Michaelis-Menten参数,使用Prism 5.0将速率常数kdec对%S100[X]的依赖性拟合至Michaelis-Menten方程Y=Vmax*X/(Km+X)。
表18:在1.2%HEK293S-100提取物中FRET RNA寡核苷酸的稳定性。
mRNA的3’-末端修饰
荧光素酶3’末端修饰的mRNA的转染和发光读数
细胞培养:将HEK293以30,000个细胞/孔的密度接种在96孔聚D赖氨酸包被的平板中,并在37℃,5%CO2培养箱中孵育过夜。培养基是EMEM(ATCC,cat#30-2003),10%FBS(Invitrogen),无抗生素。
mRNA转染:使用0.4μl/孔的DharmaFECT制剂2(ThermoScientific T-2002-01)转染30ng/孔的mRNA。将mRNA和转染试剂在OptiMEM中混合以获得10μl/孔的终体积。将混合物在室温下温育20分钟。通过轻击板从细胞中除去过夜的培养基。向每个孔中加入90μl新鲜培养基,并向每个孔中加入10μl mRNA混合物。将板在37℃,5%CO2下孵育24小时。
培养基收集:Gaussia荧光素酶蛋白被分泌在培养基中。为了收集培养基,转染后24小时,将90μl培养基从细胞转移到v-底的96孔板中,并以1000rpm离心5分钟。将80μl上清液转移到新的v-底板中,并冷冻或直接用于荧光素酶表达测定或瘦素ELISA。
荧光素酶表达测定:BioLux Gaussia荧光素酶测定试剂盒(New England BiolabsCat#E3300L)用于进行荧光素酶测定。将BioLux gLUC底物在测定缓冲液中进行1:100稀释(10μl底物:1000μl测定缓冲液)。将20μl转染培养基加入到96孔白色透明底板(Greinerbio-one 655095)中。使用FlexStation 3微孔板读数器(Molecular Devices)读取板。在时间0,将50uL的底物混合物加入到培养基板中,收集数据持续60秒。相对发光单位(RLU)在30秒处达峰值,并使用该时间点计算结果。通过将每一修饰的mRNA的RLU针对酶促加帽的帽1mRNA的RLU标准化并乘以100来计算%荧光素酶活性。
表19:HEK293细胞中的3’修饰的GLuc-mRNA表达数据。
*相对转染后24小未修饰的标准化
在本申请中,已经引用了各种出版物。这些出版物的全部内容通过引用并入本申请中,以便更充分地描述本发明所属领域的状态。
虽然已经参考所公开的实施方案描述了本发明,但是本领域技术人员将容易地理解,上面具体实施例和详细研究仅仅是对本发明的说明。应当理解,在不脱离本发明的精神的情况下,可以进行各种修改。因此,本发明仅由所附权利要求限制。
Claims (16)
1.式I化合物或其盐
其中
Z1是
A1和A3独立地选自不存在、NH、S和O;
A2是不存在或选自>CR6R7、>NR6、>NNR6R7、>NOR6、>S和>O;
Y是不存在或是连接部分,所述连接部分选自任选取代的低级烷基、任选取代的烯基、任选取代的炔基、–(CH2)nOR15、–(CH2)nCOOR15和–(CH2)nC(O)NR12;
R1选自H、任选取代的环烷基、任选取代的环烯基、任选取代的芳基、任选取代的杂芳基和任选取代的杂环基;
R2选自H、–OH、任选取代的烷基、–C(O)NR12R13和–NR12R13;
R5、R6和R8独立地选自H、任选取代的烷基、聚胺、PEGs、–(CH2)n1NR12R13、–(CH2)n1NR14C(O)R15、–(CH2)n1OR15、–(CH2)n1C(O)OR15、–(CH2)n1C(O)R15、–(CH2)n1C(O)NR12R13、–O–(CH2)n3–C(O)–(NR12)2–C(O)–X2、–O–(CH2)n3–C(O)–[NR12–C(O)–(CH2)n3]1-3–X2,或R6和R8一起形成环,该环任选被取代且含有10-80个环原子,其中10-40个环原子可以是杂原子,或者R6和R7一起形成3-8元环,其任选被取代且其中1-6个环原子可以是杂原子;
R7、R11、R12、R13、R14、R15和R16独立地选自H、任选取代的低级烷基和任选取代的酰基;
R9选自H和任选取代的低级烷基;
R10选自H、–NR12R13和–OR16;
n是1-4;
n1是0-10;
n2是1-12;
n3是1-8;
X选自O、S、NH和任选取代的烷二基;
R3和R4独立地选自H和–OR16,或R3和R4一起形成O-Q-O;
Q选自–CH2–和–C(Me)2–;
X2选自亲和部分和检测部分,且
Xn是核碱基。
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EP3233880A1 (en) * | 2014-12-16 | 2017-10-25 | Novartis AG | End capped nucleic acid molecules |
CN113584020A (zh) | 2015-09-21 | 2021-11-02 | 垂林克生物技术公司 | 用于合成5’-加帽rna的组合物和方法 |
WO2019087113A1 (en) | 2017-11-01 | 2019-05-09 | Novartis Ag | Synthetic rnas and methods of use |
US11439656B2 (en) * | 2018-08-30 | 2022-09-13 | Regents Of The University Of Minnesota | Pharmaceutical compounds and uses thereof |
KR20210089648A (ko) * | 2018-11-08 | 2021-07-16 | 트랜슬레이트 바이오 인코포레이티드 | 메신저 rna 정제를 위한 방법 및 조성물 |
WO2022140365A1 (en) * | 2020-12-22 | 2022-06-30 | Empirico Inc. | Galnac compositions for improving sirna bioavailability |
WO2023007019A1 (en) | 2021-07-30 | 2023-02-02 | CureVac SE | Cap analogs having an acyclic linker to the guanine derivative nucleobase |
WO2023178144A2 (en) | 2022-03-16 | 2023-09-21 | Empirico Inc. | Galnac compositions for improving sirna bioavailability |
CN115057903B (zh) * | 2022-06-22 | 2024-03-29 | 江苏申基生物科技有限公司 | 一种含吗啉环结构的起始加帽寡核苷酸引物及其制备方法和应用 |
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US20230183286A1 (en) | 2023-06-15 |
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JP6909157B2 (ja) | 2021-07-28 |
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