CN111836822A - Lna-二元羧酸衍生物以及它们的制备方法 - Google Patents
Lna-二元羧酸衍生物以及它们的制备方法 Download PDFInfo
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- CN111836822A CN111836822A CN201980017513.4A CN201980017513A CN111836822A CN 111836822 A CN111836822 A CN 111836822A CN 201980017513 A CN201980017513 A CN 201980017513A CN 111836822 A CN111836822 A CN 111836822A
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- dicarboxylic acid
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- 238000000034 method Methods 0.000 title claims description 18
- 238000002360 preparation method Methods 0.000 title claims description 12
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- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 claims description 22
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 claims description 20
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 17
- 125000006239 protecting group Chemical group 0.000 claims description 17
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 claims description 17
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- 125000003545 alkoxy group Chemical group 0.000 claims description 3
- OFOBLEOULBTSOW-UHFFFAOYSA-N Malonic acid Chemical compound OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 claims description 2
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- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 3
- 150000001340 alkali metals Chemical class 0.000 description 3
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- 125000004191 (C1-C6) alkoxy group Chemical group 0.000 description 2
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- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 2
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- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
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- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 2
- 229910052783 alkali metal Inorganic materials 0.000 description 2
- 229910052784 alkaline earth metal Inorganic materials 0.000 description 2
- 125000000217 alkyl group Chemical group 0.000 description 2
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- 239000012043 crude product Substances 0.000 description 2
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- 239000006260 foam Substances 0.000 description 2
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- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical class CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
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- 229910052708 sodium Inorganic materials 0.000 description 2
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- 238000004809 thin layer chromatography Methods 0.000 description 2
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 2
- 125000004169 (C1-C6) alkyl group Chemical group 0.000 description 1
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- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 239000007832 Na2SO4 Substances 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
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- 238000010533 azeotropic distillation Methods 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
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- 159000000007 calcium salts Chemical class 0.000 description 1
- 125000003917 carbamoyl group Chemical group [H]N([H])C(*)=O 0.000 description 1
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- 239000012876 carrier material Substances 0.000 description 1
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- 238000010511 deprotection reaction Methods 0.000 description 1
- ZBCBWPMODOFKDW-UHFFFAOYSA-N diethanolamine Chemical compound OCCNCCO ZBCBWPMODOFKDW-UHFFFAOYSA-N 0.000 description 1
- KPUWHANPEXNPJT-UHFFFAOYSA-N disiloxane Chemical compound [SiH3]O[SiH3] KPUWHANPEXNPJT-UHFFFAOYSA-N 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 125000001301 ethoxy group Chemical group [H]C([H])([H])C([H])([H])O* 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 150000002430 hydrocarbons Chemical group 0.000 description 1
- 125000000959 isobutyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])* 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- UBLQIESZTDNNAO-UHFFFAOYSA-N n,n-diethylethanamine;phosphoric acid Chemical compound [O-]P([O-])([O-])=O.CC[NH+](CC)CC.CC[NH+](CC)CC.CC[NH+](CC)CC UBLQIESZTDNNAO-UHFFFAOYSA-N 0.000 description 1
- 125000004108 n-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
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- 150000007524 organic acids Chemical class 0.000 description 1
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- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- UYWQUFXKFGHYNT-UHFFFAOYSA-N phenylmethyl ester of formic acid Natural products O=COCC1=CC=CC=C1 UYWQUFXKFGHYNT-UHFFFAOYSA-N 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 238000011176 pooling Methods 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 159000000001 potassium salts Chemical class 0.000 description 1
- 238000004237 preparative chromatography Methods 0.000 description 1
- 125000001501 propionyl group Chemical group O=C([*])C([H])([H])C([H])([H])[H] 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 150000003222 pyridines Chemical class 0.000 description 1
- 229930195734 saturated hydrocarbon Natural products 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
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
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- 238000003756 stirring Methods 0.000 description 1
- 150000003890 succinate salts Chemical class 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 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
- 125000000037 tert-butyldiphenylsilyl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1[Si]([H])([*]C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 125000001412 tetrahydropyranyl group Chemical group 0.000 description 1
- 125000004044 trifluoroacetyl group Chemical group FC(C(=O)*)(F)F 0.000 description 1
- 239000013638 trimer Substances 0.000 description 1
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- 229940035893 uracil Drugs 0.000 description 1
- 239000003039 volatile agent Substances 0.000 description 1
Classifications
-
- 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
- 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
- C07H19/16—Purine radicals
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
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Abstract
本发明包括式I的LNA‑二元羧酸衍生物或其盐,其中R1是核碱基或经修饰的核碱基,R2是羟基保护基,且n是1至5的整数。式I的LNA‑二元羧酸衍生物可用于式III的预载固体载体,照此可用作适于寡核苷酸合成的起始材料。
Description
本发明涉及式I的LNA-二元羧酸衍生物或其盐、它们的制备方法、它们用于制备含有式I的LNA-二元羧酸衍生物的LNA-预载固体载体的用途、LNA-预载固体载体及其在寡核苷酸合成中的用途,
其中
R1是核碱基或经修饰的核碱基,
R2是羟基保护基,且
n是1至5的整数。
寡核苷酸通常在固体载体介质上制备。通常,第一合成子(例如单体,例如作为核苷)首先连接至固体载体介质,然后通过依次将单体偶联至固体载体结合的合成子而合成了寡核苷酸。这种反复的延伸最后产生了最终寡核苷酸化合物,其从载体上裂解,并且如果需要的话进一步后处理以产生最终的寡核苷酸化合物。
在进一步的研发中,已经引入了连接至固体载体的连接基分子用于更有效地进行寡核苷酸合成,特别是在当今使用的自动过程合成仪上。例如,国际专利公开WO 2005/049621或WO 2006/029023公开了这类连接基化合物。寡核苷酸延伸经常在连接基分子上由O-二甲氧基三苯甲基(O-DMT)开始。
但是,在LNA寡核苷酸的合成中发现,在一定程度上在连接基分子上第一次偶联失败,导致最终寡核苷酸含有大量(N-1)杂质,即,在3’-端缺少一个核苷的寡核苷酸。
因此,本发明的目的是在很大程度上减少3`-端(N-1)杂质的量,因为这类杂质难以通过现有技术的色谱纯化方法除去,引起产量的重大损失,例如通过在制备型色谱步骤中紧密的级分汇集和/或在分离的LNA寡核苷酸中令人不满意的杂质情况。
已经发现,采用可以连接至固体树脂(国际专利公开WO1992/006103)和作为预载树脂发挥作用的如上文所述的式I的LNA-二元羧酸衍生物,可以以很大程度降低N-1失败。
给出下文定义以解释和定义用于描述本发明的各种术语的含义和范围。
术语“C1-6-烷基”指具有1-6个碳原子、在更具体的实施方案中具有1-4个碳原子的单价直链或支链饱和烃基团。典型的实例包括甲基、乙基、丙基、异丙基、正丁基、异丁基、仲丁基或叔丁基,优选甲基或乙基。
术语“C1-6-烷氧基”指与氧原子连接的如上文所定义的C1-6-烷基基团、更优选C1-4-烷基基团。典型的实例包括甲氧基、乙氧基、丙氧基、异丙氧基、正丁氧基、异丁氧基、叔丁氧基,优选甲氧基或乙氧基。
术语“C1-6-烷酰基”指连接至羰基的如上文定义的C1-6-烷基基团、更优选C1-4-烷基基团。典型的实例包括乙酰基、乙烷酰基、丙酰基、异丙酰基、正丁酰基、异丁酰基或叔丁酰基,优选乙酰基。
用于R2的术语“羟基保护基”指意欲保护羟基的基团,包括形成酯的基团和形成醚的基团,特别是经修饰的三苯甲基基团、四氢吡喃基、酰基基团、氨甲酰基、苄基和硅烷基醚(例如TBS、TBDPS)基团。这些基团的其它实例见于T.W.Greene和P.G.M.Wuts,“ProtectiveGroups in Organic Synthesis”,第2版,John Wiley&Sons,Inc.,纽约,NY,1991,第2-3章;E.Haslam,“Protective Groups in Organic Chemistry”,J.G.W.McOmie编辑,PlenumPress,纽约,NY,1973,第5章和T.W.Greene,“Protective Groups in OrganicSynthesis”,John Wiley and Sons,纽约,NY,1981。
优选酸敏感性羟基基团,更优选烷氧基修饰的三苯甲基基团。此处的修饰指一个、两个或三个苯基环被一个或多个C1-6-烷氧基基团、优选甲氧基基团取代。
最优选的羟基保护基是4,4’-二甲氧基三苯甲基(DMT)。
术语“氨基保护基”指意欲保护氨基的基团,包括C1-6-烷酰基、苯甲酰基、苄基氧基羰基、苄氧羰基(CBZ或Z)、9-芴基甲氧羰基(FMOC)、对甲氧基苄氧羰基、对硝基苄氧羰基、叔丁氧羰基(BOC)和三氟乙酰基、二甲基甲脒(dmf)。这些基团的其它实例见于T.W.Greene和P.G.M.Wuts,“Protective Groups in Organic Synthesis”,第2版,John Wiley&Sons,Inc.,纽约,NY,1991,第7章;E.Haslam,“Protective Groups in Organic Chemistry”,J.G.W.McOmie编辑,Plenum Press,纽约,NY,1973,第5章,和T.W.Greene,“ProtectiveGroups in Organic Synthesis”,John Wiley and Sons,纽约,NY,1981。
优选的氨基保护基选自C1-6-烷酰基、苯甲酰基(bz)或二甲基甲脒(dmf)。
用于R1的术语“核碱基”或“经修饰的核碱基”表示五个核碱基腺嘌呤(A)、胞嘧啶(C)、鸟嘌呤(G)、胸腺嘧啶(T)和尿嘧啶(U)和它们的修饰形式。
在本发明式I的本发明的LNA-二元羧酸衍生物的上下文中的术语“盐”表示由无机碱或有机碱形成的盐。
无机盐通常是来自无机的碱金属或碱土金属氢氧化物碱或来自有机的碱金属或碱土金属醇化物的碱金属或碱土金属盐,例如钠盐、钾盐、镁盐或钙盐,优选钠盐或钾盐。
有机盐通常是来自有机胺、通常来自脂族胺或芳香胺、优选来自叔胺、优选三-C1-4-烷基胺或吡啶、更优选三乙胺或吡啶的铵盐。
有机盐相比于无机盐是优选的。
式I的LNA-二元羧酸衍生物可以以游离酸和来自无机或有机碱的盐的任意混合物的形式存在。
典型的经修饰的核碱基是5-甲基胞嘧啶(5-MeC)。
在优选的实施方案中,R1是任选经修饰的和/或氨基被保护的腺嘌呤(A)、胞嘧啶(C)、5-甲基胞嘧啶(5-MeC)、鸟嘌呤(G)或胸腺嘧啶(T),优选氨基被保护的腺嘌呤(A)、氨基被保护的胞嘧啶(C)、氨基被保护的5-甲基胞嘧啶(5-MeC)、氨基被保护的鸟嘌呤(G)或胸腺嘧啶(T)。
上文已经定义了典型的氨基保护基,对腺嘌呤(A)、胞嘧啶(C)和5-甲基胞嘧啶(5-MeC)而言优选的氨基保护基是苯甲酰基、对鸟嘌呤(G)而言优选的氨基保护基是异丁酰基或二甲基甲脒(dmf)。
在优选的实施方案中,本发明的LNA-二元羧酸衍生物具有式I或其来自有机胺的铵盐,
其中
R1是任选经修饰的和/或氨基被保护的腺嘌呤(A)、胞嘧啶(C)、5-甲基胞嘧啶(5-MeC)、鸟嘌呤(G)或胸腺嘧啶(T),优选氨基被保护的腺嘌呤、氨基被保护的胞嘧啶(C)、氨基被保护的5-甲基胞嘧啶(5-MeC)、氨基被保护的鸟嘌呤(G)或胸腺嘧啶(T);
R2是酸敏感性羟基保护基,选自烷氧基修饰的三苯甲基基团;
n是1至5的整数。
在进一步优选的实施方案中,本发明的LNA-二元羧酸衍生物具有式I或其来自叔胺、选自三-C1-4-烷基胺、更优选三乙胺或来自吡啶的铵盐;
其中:
R1是苯甲酰基保护的腺嘌呤(A)、苯甲酰基保护的胞嘧啶(C)、苯甲酰基保护的5-甲基胞嘧啶(5-MeC)、异丁酰基或二甲基甲脒保护的鸟嘌呤(G)或胸腺嘧啶(T);
R2是4,4’-二甲氧基三苯甲基(DMT);
n是1。
本发明的最优选的LNA-二元羧酸衍生物具有式I,并且通过如下定义表征:
·R1=苯甲酰基保护的腺嘌呤(A);R2=4,4’-二甲氧基三苯甲基(DMT);n=1;为三乙铵盐或吡啶鎓盐的形式
·R1=苯甲酰基保护的胞嘧啶(C);R2=4,4’-二甲氧基三苯甲基(DMT);n=1;为三乙铵盐或吡啶鎓盐的形式
·R1=苯甲酰基保护的5-甲基胞嘧啶(5-MeC);R2=4,4’-二甲氧基三苯甲基(DMT);n=1;为三乙铵盐或吡啶鎓盐的形式
·R1=异丁酰基保护的鸟嘌呤(G);R2=4,4’-二甲氧基三苯甲基(DMT);n=1;为三乙铵盐或吡啶鎓盐的形式
·R1=二甲基甲脒保护的鸟嘌呤(G);R2=4,4’-二甲氧基三苯甲基(DMT);n=1;为三乙铵盐或吡啶鎓盐的形式
·R1=胸腺嘧啶(T);R2=4,4’-二甲氧基三苯甲基(DMT);n=1;为三乙铵盐或吡啶鎓盐的形式。
如上文概括的优选的本发明的LNA-二元羧酸衍生物可以作为游离酸或如上文所述的铵盐形式或还可以作为铵盐和游离酸的任意混合物存在。
制备式I的LNA-二元羧酸衍生物的方法包括使式II的LNA醇与C2-6-二元羧酸酸酐在碱和有机溶剂的存在下反应,
其中R1和R2如上所定义,形成式I的LNA-二元羧酸衍生物。
式II的LNA醇通常可购买获得或者可以按照Wengel等人,Tetrahedron 54(1998)3607-3630制得。
同样,C2-6-二元羧酸酸酐是可购买获得的化合物。典型的C2-6-二元羧酸酸酐是琥珀酸酐。
通常,在初始步骤,起始材料在与适宜有机溶剂如甲苯的共沸条件下除去残余水。
上文已经记载了适宜的碱,但是优选使用有机胺。
优选叔胺、更优选三-C1-4-烷基胺或吡啶、甚至更优选三乙胺或吡啶是常用的碱。
适宜的有机溶剂是卤化烃,例如二氯甲烷。
反应常规地在惰性气氛下在10℃至40℃的反应温度下进行。
反应完全后,通常是约2小时后,经由提取步骤、适宜地应用反应所用的溶剂可以获得铵盐。
从合并的有机相中除去溶剂和任选地通过色谱法进一步纯化,可以以94-99.9%产率获得式I的LNA-二元羧酸衍生物的各个铵盐,HPLC纯度为97-99.5面积-%(不包括残余甲苯)。
式I的LNA-二元羧酸衍生物还可以以游离酸的形式获得。这可以通过将本发明的式I的LNA-二元羧酸衍生物的盐用盐酸或有机酸如柠檬酸在适宜的有机溶剂如二氯甲烷或乙酸乙酯中转化来完成。
在本发明的进一步实施方案中,式I的LNA-二元羧酸衍生物可用于制备式III的预载固体载体,
其中R1、R2和n如上所定义,SOLID SUPPORT是适于寡核苷酸合成的固体载体材料。
适宜的固体载体材料例如在Guzaev,A.P.Solid-phase supports foroligonucleotide synthesis.In:Current protocols in nucleic acid chemistry.(John Wiley&Sons,Inc.)(2013),第3章,第3.1单元.,第3.1.1-3.1.60页中有充分记载。典型的商业固体载体是来自GE Healthcare的Primer Support 5G系列或来自Nitto Denko的HL固体载体。
上文对R1、R2和n提供的定义和优选也同样应用于式III的预载固体载体。
在另一个实施方案中,本发明涉及式III的预载固体载体
其中R1、R2、n和SOLID SUPPORT如上所定义。上文对R1、R2、n和SOLID SUPPORT提供的优选同样应用于式III的预载固体载体。
在本发明的另一个实施方案,式III的预载固体载体可用作用于寡核苷酸合成的起始材料,
其中R1、R2、n和SOLID SUPPORT如上所定义。上文对R1、R2、n和SOLID SUPPORT提供的定义和优选同样可用于式III的预载固体载体。
式I的LNA-二元羧酸衍生物可以通过本领域技术人员已知的方法和例如如国际专利公开WO 1992/006103中所述连接至固体载体。
寡核苷酸合成的原则是本领域熟知的,在文献和公开论坛如Wikipedia(参见例如Oligonucleotide synthesis;Wikipedia,the free encyclopedia;https://en.wikipedia.org/wiki/Oligonucleotide_synthesis,2016年3月15日)中有充分的记载。
当今,较大规模的寡核苷酸合成采用计算机控制合成仪自动地进行。
通常,寡核苷酸合成是固相合成,其中被装配的寡核苷酸经由其3'-端羟基共价结合至固体载体材料并在链装配的整个过程中保持与固体载体材料连接。适宜的固体载体如上文所述。
优选地,寡核苷酸由任选修饰的DNA或LNA核苷单体或其组合组成,其长度为10至25个核苷酸。
鉴于式III的预载固体载体包含LNA核苷的事实,所产生的寡核苷酸链的3’端核苷始终是LNA核苷。
原则上,寡核苷酸合成是向生长链的5'-端逐步添加核苷酸残基,直至装配了预期序列。
通常,每个添加被称为合成周期,原则上由化学反应组成
a1)式III的预载固体载体上的被保护的羟基的去阻断,
a2)将具有游离羟基的作为活化亚磷酰胺的第一核苷偶联在固体载体上,
a3)使各自的P-连接的核苷氧化或硫化,形成各自的磷酸三酯(P=O)或各自的硫代磷酸酯(P=S);
a4)任选地,给固体载体上任何未反应的羟基加帽;
a5)使连接至固体载体的第二核苷的5’羟基去阻断;
a6)使作为活化亚磷酰胺的第三核苷偶联,形成各自的P-连接的三聚物;
a7)使各自的P-连接的二核苷氧化或硫化,形成各自的磷酸三酯(P=O)或各自的硫代磷酸酯(P=S);
a8)任选地,给任何未反应的5’羟基加帽;
a9)重复前述步骤a5至a8,直至装配了预期序列;
a10)全面脱保护,获得寡核苷酸粗品,其可以通过典型的寡核苷酸纯化技术如色谱法和/或超滤和/或冻干法进行进一步纯化。
以下实施例应当解释而非限制本发明。
实施例
缩写
EtOAc =乙酸乙酯
MeCN =乙腈
MeOH =甲醇
DCM =二氯甲烷
DMAP =4-二甲基氨基吡啶
TEA =三乙胺
THF =四氢呋喃
TLC =薄层色谱法
rt =室温
eq =当量
方法流程
通用操作
向圆底烧瓶装入核苷2(1eq)和琥珀酸酐(1.5eq)。对于残余水的共沸除去,将混合物于室温混悬于绝对甲苯(每1g核苷2而言20mL)。接下来,真空蒸除挥发物。重复该操作一次,然后蒸除甲苯,产生固体,但不是完全干燥的残余物。以下操作全部在氩气气氛下进行。向以上形成的固体中加入绝对DCM(每1g核苷2而言18mL)。然后一次性加入TEA(相对于2a而言3.0eq,相对于2b而言5.0eq),将混合物于室温搅拌,30分钟后获得澄清的反应混合物。通过TLC或LC-MS测定转化(对于1a.TEA参见实施例1表1,对于1b.TEA参见实施例2表2)。
通常,转化在2小时后完全,但是在所有情况中混合物另外于室温搅拌另外1小时。然后将反应混合物倒入三乙铵磷酸盐缓冲溶液中(对于每1g核苷2而言15mL缓冲液。缓冲溶液制备:将85%H3PO4水溶液(8.5mL,1eq)、TEA(26mL,1.5eq)和水(465.5mL)混合)。分离所形成的各层,将水层另外用DCM萃取(对于每1g核苷2而言2x10mL)。将合并的有机层经无水Na2SO4垫过滤,真空浓缩。获得粗产物,为白色泡沫,将其通过硅胶柱过滤进一步纯化(对于每1g粗产物而言20g硅胶;洗脱剂:EtOH在DCM中的2.5%至10%溶液,混合物进一步补充有3vol-%TEA)。将目标级分浓缩至干,立即混悬于绝对甲苯中,在真空中共蒸发。再重复一次共蒸发。
实施例1:1a.TEA的合成
如通用操作中所述将8.6g 2a转化为1a.TEA,得到11.3g的1a.TEA,产率为96%,纯度为99.1HPLC面积-%(不包括0.67mol eq残余甲苯)(柱:Apollo C-18(10μm)(4.6x250mm)10.65min;流动相:梯度40%的MeCN+60%的0.1%H3PO4水溶液;流速1.0mL/min;柱温40℃;检测:210nm,254nm;样品浓度:1mg/mL,在MeCN中;进样体积:0.002mL)。
LC-MS ESI(m/z):671.2[M-H]
1H-NMR(D4-MeOH,400MHz):δ7.77-7.73(m,1H),7.50-7.43(m,2H),7.38-7.28(m,6H),7.26-7.21(m,1H),6.91-6.85(m,4H),5.62(s,1H),5.21(s,1H),4.56(s,1H),3.89-3.79(m,2H),3.78(s,6H),3.57(d,J=11.2Hz,1H),3.47(d,J=11.2Hz,1H),2.94(q,J=7.3Hz,6H),2.64-2.46(m,2H),2.46-2.36(m,2H),1.61(d,J=1.2Hz,3H),1.29(t,J=7.3Hz,9H)。
表1
实施例2:1b.TEA的制备
如通用操作中所述将8.9g 2b转化为1b.TEA,得到11.0g的1b.TEA,产率为94%,纯度为97.2HPLC面积-%(不包括0.75mol eq残余甲苯)(柱:Apollo C-18(10μm)(4.6x250mm)9.18min;流动相:梯度40%的MeCN+60%的0.1%H3PO4水溶液;流速1.0mL/min;柱温40℃;检测:210nm,254nm;样品浓度:1mg/mL,在MeCN中;进样体积:0.002mL).LC-MS ESI(m/z):753.3[M+H]+
1H-NMR(D4-MeOH,400MHz):δ8.70(s,1H),7.98(s,1H),7.44-7.39(m,2H),7.32-7.15(m,7H),6.87-6.81(m,4H),6.00(s,1H),5.85(s,1H),4.80(s,1H),4.01-3.89(m,2H),3.76(s,6H),3.53-3.74(m,2H),3.15(q,J=7.3Hz,6H),3.09(s,3H),3.07(s,3H),2.63-2.46(m,2H),2.45-2.33(m,2H),1.26(t,J=7.3Hz,9H)。
表2
实施例3:1c.TEA的制备
如通用操作中所述将10g 2c转化为1c.TEA,得到13.6g 1c.TEA,产率为98%,纯度为98.6HPLC面积-%(不包括1.20mol eq残余甲苯)(柱:Apollo C-18(10μm)(4.6x250mm)11.00min;流动相:梯度40%的MeCN+60%的0.1%H3PO4水溶液;流速1.0mL/min;柱温40℃;检测:210nm,254nm;样品浓度:1mg/mL,在MeCN中;进样体积:0.002mL)。
LC-MS ESI(m/z):768.5[M-TEA+H]+
1H-NMR(D4-MeOH,400MHz):δ8.11(s,1H),7.46–7.40(m,2H),7.34–7.26(m,4H),7.25–7.06(m,5H),6.90–6.82(m,4H),5.99(s,1H),5.35(s,1H),4.85(s,1H),3.96(q,J=8.3Hz,2H),3.76(s,6H),3.56–3.47(m,2H),3.16(q,J=7.3Hz,6H),2.71(hept,J=6.8Hz,1H),2.61–2.38(m,4H),1.27(t,J=7.3Hz,9H),1.21(d,J=6.9Hz,6H)。
实施例4:1d.TEA的制备
如通用操作中所述将10g 2d转化为1d.TEA,以>99%产率和99HPLC面积-%纯度得到14.0g 1d.TEA(不包括0.50mol eq残余甲苯)(柱:Apollo C-18(10μm)(4.6x250mm)12.89min;流动相:梯度40%的MeCN+60%的0.1%H3PO4水溶液;流速1.0mL/min;柱温40℃;检测:210nm,254nm;样品浓度:1mg/mL,在MeCN中;进样体积:0.002mL)。
LC-MS ESI(m/z):775.5[M-TEA+H]+
1H-NMR(D4-MeOH,400MHz):δ8.22(s,1H),8.19(s,1H),7.98(s,1H),7.59–7.52(m,1H),7.51–7.42(m,4H),7.40–7.32(m,4H),7.28–7.09(m,5H),6.95–6.84(m,4H),5.69(s,1H),5.23(s,1H),4.64(s,1H),3.87(dd,J=15.6Hz,J=8.2Hz,2H),3.79(s,6H),3.55(dd,J=34.1Hz,J=11.3Hz,2H),3.13(q,J=7.3Hz,6H),2.67–2.37(m,4H),1.83(s,3H),1.27(t,J=7.3Hz,9H)。
实施例5:1e.TEA的制备
如通用操作中所述将10g 2c转化为1c.TEA,以>99%产率和99.2HPLC面积-%纯度得到14.2g 1c.TEA(不包括1.00mol eq残余甲苯)(柱:Apollo C-18(10μm)(4.6x250mm)5.17min;流动相:梯度40%的MeCN+60%的0.1%H3PO4水溶液;流速1.0mL/min;柱温40℃;检测:210nm,254nm;样品浓度:1mg/mL,在MeCN中;进样体积:0.002mL)。
LC-MS ESI(m/z):786.5[M-TEA+H]+
1H-NMR(D4-MeOH,400MHz):δ8.73(s,1H),8.55(s,1H),8.11–8.05(m,2H),7.68–7.61(m,1H),7.59–7.52(m,2H),7.47–7.42(m,2H,Ar H),7.37–7.26(m,5H),7.25–7.07(m,3H,Ar H),6.91–6.82(m,4H),6.24(s,1H),5.44(s,1H),4.93(s,1H),4.02(dd,2H,J=16.5Hz,J=8.3Hz)、3.77(s,6H),3.57(br,2H),3.12(q,J=7.3Hz,6H),2.65–2.35(m,4H),1.25(t,J=7.3Hz,9H)。
实施例6:1e.TEA和1e(酸形式)的制备
将化合物2e(6.71g,9.80mmol)溶于干燥THF(70mL)中,于35℃真空浓缩,然后将固体于环境温度进一步真空干燥4小时,溶于干燥EtOAc(50mL)和TEA(1.60mL,11.7mmol)中,向核苷溶液中依次加入DMAP(0.30g,2.4mmol)和琥珀酸酐(1.37g,13.7mmol)。然后将反应混合物于50℃加热16小时。将反应混合物用EtOAc(100mL)和己烷(5mL)稀释,用10%柠檬酸溶液萃取(3x50mL)。将有机相用5%NaHCO3溶液萃取(4x 50mL)、用EtOAc稀释(150mL),进一步用10%柠檬酸溶液(2x75mL)和水(2x75mL)萃取。将合并的柠檬酸和水级分用EtOAc(2x50mL)反萃取。将合并的有机相干燥(Na2SO4),在减压下浓缩,得到琥珀酸盐1e(酸形式),为白色泡沫。
1H NMR(600MHz,DMSO-d6)δppm 12.27(br s,1H),11.28(s,1H),8.79(s,1H),8.51(s,1H),8.06(br d,J=7.5Hz,2H),7.64-7.70(m,1H),7.53-7.61(m,2H),7.39(br d,J=7.6Hz,2H),7.32(br t,J=7.7Hz,2H),7.23-7.28(m,4H),7.24(br s,1H),6.90(br d,J=8.6Hz,4H),6.23(s,1H),5.58(s,1H),4.93(s,1H),4.02-4.07(m,2H),3.89(br d,J=8.5Hz,1H),3.74(s,6H),3.53(br d,J=11.2Hz,1H),3.41(br d,J=11.2Hz,1H),2.40-2.45(m,2H)。
将固体重新溶于MeCN(70mL)中,加入TEA(6.8mL,49mmol)。搅拌10分钟后,将混合物于35℃真空浓缩10分钟,将剩余物于环境温度真空干燥过夜,得到1e.TEA盐(7.45g琥珀酸盐x 0.6TEA,90%产率,HPLC纯度>98%)。
1e.TEA的分析数据与实施例5中报道的那些一致。
Claims (17)
2.权利要求1的LNA-二元羧酸衍生物,其中R1是任选经修饰的和/或氨基被保护的腺嘌呤(A)、胞嘧啶(C)、鸟嘌呤(G)或胸腺嘧啶(T)。
3.权利要求1或2的LNA-二元羧酸衍生物,其中R1是氨基被保护的腺嘌呤(A)、氨基被保护的胞嘧啶(C)、氨基被保护的5-甲基胞嘧啶(5-MeC)、氨基被保护的鸟嘌呤(G)或者是胸腺嘧啶。
4.权利要求3的LNA-二元羧酸衍生物,其中氨基保护基选自C1-6-烷酰基、苯甲酰基(bz)或二甲基甲脒(dmf)。
5.权利要求1-4任一项的LNA-二元羧酸衍生物,其中R2是酸敏感性羟基保护基,其选自烷氧基修饰的三苯甲基基团,优选选自4,4`-二甲氧基三苯甲基。
6.权利要求1-5任一项的LNA-二元羧酸衍生物,其中n是1。
7.权利要求1-6任一项的LNA-二元羧酸衍生物,为来自无机或有机碱的盐的形式。
8.权利要求1-7任一项的LNA-二元羧酸衍生物,为有机胺的铵盐的形式。
9.权利要求8的LNA-二元羧酸衍生物,为叔胺、优选三-C1-4-烷基胺或吡啶、更优选三乙胺或吡啶的铵盐的形式。
10.权利要求1-9任一项的LNA-二元羧酸衍生物,为游离酸和来自无机或有机碱的盐的任意混合物的形式。
12.权利要求11的方法,其中C2-6-二元羧酸酸酐是琥珀酸酐。
13.权利要求11或12的方法,其中所述碱是有机胺,优选叔胺、更优选三-C1-4-烷基胺或吡啶,甚至更优选三乙胺或吡啶。
14.权利要求11-13任一项的方法,其中所述有机溶剂是卤化烃。
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