CN115286523B - 用于递送活性成分的脂质分子及其组合物的制备和应用 - Google Patents
用于递送活性成分的脂质分子及其组合物的制备和应用 Download PDFInfo
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- CN115286523B CN115286523B CN202210829701.0A CN202210829701A CN115286523B CN 115286523 B CN115286523 B CN 115286523B CN 202210829701 A CN202210829701 A CN 202210829701A CN 115286523 B CN115286523 B CN 115286523B
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C219/00—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton
- C07C219/02—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
- C07C219/04—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
- C07C219/14—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having at least one of the hydroxy groups esterified by a carboxylic acid having the esterifying carboxyl group bound to a carbon atom of a six-membered aromatic ring
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
- A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
- A61K48/0033—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being non-polymeric
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/5123—Organic compounds, e.g. fats, sugars
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
- C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
- C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
- C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
- C07C229/12—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of acyclic carbon skeletons
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
- C07C235/42—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton
- C07C235/44—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton with carbon atoms of carboxamide groups and singly-bound oxygen atoms bound to carbon atoms of the same non-condensed six-membered aromatic ring
- C07C235/50—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton with carbon atoms of carboxamide groups and singly-bound oxygen atoms bound to carbon atoms of the same non-condensed six-membered aromatic ring having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by nitrogen atoms not being part of nitro or nitroso groups
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Abstract
本发明公开了用于递送活性成分的脂质分子及其组合物的制备和应用,涉及生物医药领域。该新型脂质分子包含通式(Ⅰ)、(Ⅱ)或(Ⅲ):可用于将活性成分(如核酸、多肽、蛋白)递送至细胞和/或器官。本发明的实施方案提供了多种新脂质分子的核酸‑脂质纳米粒子组合物,由其组成的脂质纳米递送系统用于递送mRNA;在细胞水平和动物水平上,均表现出优于目前上市的产品DLin‑MC3‑DMA递送效率,可以作为核酸药物的递送新的方法,促进核酸药物的发展。
Description
技术领域
本发明属于生物医药领域,具体涉及用于递送活性成分的脂质分子及其组合物的制备和应用。
背景技术
继小分子药物和抗体药物后,核酸药物已成为现代制药的第三次浪潮。核酸药物通常是将特定的核酸分子导入靶细胞或组织,替代、补偿、阻断、修正特定基因,以达到预防和治疗疾病的目的。虽然核酸药物优势明显,但是血清中核酸酶的存在会导致核酸的快速降解,核酸分子的负电性也使得其很难跨过细胞膜,从而导致核酸半衰期极短,无法进入细胞内,达不到治疗效果。因此,需要开发特定的化合物及递送系统来改善这一现状,以促进核酸药物可以作为疾病预防和治疗的重要手段。
目前经验证,含脂质分子的纳米粒子组合物可作为一种递送活性成分安全高效的载体。此类含脂质分子的组合物可以阻断血清中RNA的降解并促进寡核苷酸的细胞摄取,除递送核酸分子,还能有效地将小分子药物、多肽药物和蛋白质药物递送至靶细胞和/或器官。尽管已经公开了多种含脂质分子的组合物,但其安全性、功效和特异性有待进一步改良。因此,仍需设计和筛选新的脂质分子及其纳米组合物用于各种特定核酸分子的递送。
发明内容
本发明的目的在于提供了一种新型脂质分子以及包含所述脂质分子的纳米粒子组合物,此类纳米粒子组合物能将活性成分按照本发明的相关方法递送至细胞和/或器官。
本发明为实现上述目的所采取的技术方案为:
用于递送活性成分的脂质分子,包含:通式(Ⅰ)、(Ⅱ)、(Ⅲ)代表的脂质化合物,或其药物可用的盐、立体异构体、互变异构体、溶剂化物、螯合物、非共价复合物;
其中,
M选自苯环、环丁烷、环戊基、环己基、吡咯环、吡啶、哌嗪、咪唑、联苯、萘环、蒽环、嘧啶环或4-8元杂环;
G1、G’1各独立地选自-(CH2)x-O(C=O)-、-(CH2)x-(C=O)O-、-(CH2)x-(C=O)S-、-(CH2)x-(C=O)NH-、-(CH2)x-O-、-(CH2)x-O(C=O)NH-、-(CH2)x-O(C=O)O-、-(CH2)xNH(C=O)-中的一种,其中x为0至4之间的整数;
L1、L’1各独立地选自未取代的C1-6烷基中的一种;
G2选自-(CH2)0-3-、-O-(CH2)y-(C=O)O-、-(CH2)y-(C=O)O-、-(CH2)y-(C=O)NH-、-S-(CH2)y-(C=O)O-、-(CH2)y-(C=O)S-、-S-、-O-中的一种,其中y为0至4之间的整数;
G1、G’1、G2各自独立地与M中的任意位点连接,上述位点为碳或者氮原子;
X选自碳或者氮原子;n选自0至6之间的整数;
L2选自H、OH、C1-3烷基、C2-3烯基中的一种;
L3、L4各独立地选自C0-25烷基、C2-25烯基、C3-25炔基中的一种;
G3、G4各独立地选自-CH2-、-O(C=O)-、-(C=O)O-、-O(C=O)O-、-(C=O)NH-、-NH(C=O)-、-S(C=O)-、-(C=O)S-、-S-S-中的一种;
L5、L6各独立地选自C1-25烷基、C2-25烯基、C3-25炔基中的一种;
R1、R2和R’1、R’2各独立地选自任意取代或未取代的C1-6烷基、C2-6烯基、C2-6炔基、C3-8环烷基、C3-8环烯基、C3-8环炔基、苯基、-(C=O)C1-3烷基、中的一种,其中取代基团为1或2或3或4或5个独立的OH、SH、硝基、氰基、氨基、C1-3羟基、C1-3烷氧基、-(C=O)OC1-3烷基、C1-3烷基;X1、X2各独立地选自C1-3烷基;
或者
R1和R2、R’1和R’2结合起来形成任意取代或未取代的4-8元杂环、嘧啶环、嘌呤环;其中取代基团为1或2或3或4或5个独立的OH、SH、硝基、氰基、氨基、C1-3羟基、C1-3烷氧基、-(C=O)OC1-3烷基、C1-3烷基;
式(Ⅱ)中的Z选自H、F、-OH、-SH-、-NH2、-CF3、-NH-(CH2)rCH3、-N(CH3)-(CH2)rCH3中的一种,其中r为0至4之间的整数;
式(Ⅲ)中的Z’选自任意取代或未取代的H、C1-6烷基、C2-6烯基、C2-6炔基、C3-8环烷基、C3-8环烯基、C3-8环炔基、苯基、4-8元杂环中的一种,其中取代基团为1或2个独立的OH、SH、C1-3羟基、C1-3烷氧基、氨基、硝基、氰基、-(C=O)OC1-3烷基。
在某些实施方案中,上述通式(Ⅰ)的脂质化合物包含式(Ia)、(Ib)、(Ic)或(Id)所示的结构,或其药物可用的盐、立体异构体、互变异构体、溶剂化物、螯合物、非共价复合物;
在某些实施方案中,上述通式(Ⅱ)的脂质化合物包含式(Ⅱa)、(Ⅱb)、(Ⅱc)、(Ⅱd)、(Ⅱe)、(Ⅱf)、(Ⅱg)或(Ⅱh)所示结构,或其药物可用的盐、立体异构体、互变异构体、溶剂化物、螯合物、非共价复合物;
在某些实施方案中,上述通式(Ⅲ)的脂质化合物包含式(Ⅲa)、(Ⅲb)或(Ⅲc)所示结构,或其药物可用的盐、立体异构体、互变异构体、溶剂化物、螯合物、非共价复合物;
其中,G1、G’1各独立地选自-(CH2)x-O(C=O)-、-(CH2)x-(C=O)O-、-(CH2)x-(C=O)S-、-(CH2)x-(C=O)NH-、-(CH2)x-O-、-(CH2)x-O(C=O)NH-、-(CH2)x-O(C=O)O-、-(CH2)xNH(C=O)-中的一种,其中x为0至4之间的整数;
L1、L’1各独立地选自未取代的C1-6烷基中的一种;
G1、G’1各自独立地与苯环中的任意位点连接;
Z’选自任意取代或未取代的H、C1-6烷基、C2-6烯基、C2-6炔基、C3-8环烷基、C3-8环烯基、C3-8环炔基、苯基、4-8元杂环中的一种;其中取代基团为1或2个独立的OH、SH、C1-3羟基、C1-3烷氧基、氨基、硝基、氰基、-(C=O)OC1-3烷基;
R1、R2和R’1、R’2各独立地选自任意取代或未取代的C1-6烷基、C2-6烯基、C2-6炔基、C3-8环烷基、C3-8环烯基、C3-8环炔基、苯基、-(C=O)C1-3烷基、中的一种,其中取代基团为1或2或3或4或5个独立的OH、SH、硝基、氰基、氨基、C1-3羟基、C1-3烷氧基、-(C=O)OC1-3烷基、C1-3烷基;X1、X2各独立地选自C1-3烷基;
或者
R1和R2、R’1和R’2结合起来形成任意取代或未取代的4-8元杂环、嘧啶环、嘌呤环;其中取代基团为1或2或3或4或5个独立的OH、SH、硝基、氰基、氨基、C1-3羟基、C1-3烷氧基、-(C=O)OC1-3烷基、C1-3烷基;
E选自氧或者硫原子;
m为0至4之间的整数;
T包含式(1)-式(18)所示结构中的一种:
在某些实施方案中,上述脂质化合物,其中各独立地选自式Y01-Y30所示结构中的一种:
在某些实施方案中,脂质分子选自如下化合物的一种或多种:
本发明还提供了一种纳米粒子组合物,包含上述的脂质化合物中的一种或多种。
在某些实施方案中,上述纳米粒子组合物还包含治疗剂和/或预防剂。
在某些实施方案中,上述治疗剂和/或预防剂被包封在纳米粒子内或与纳米粒子缔合。
在某些实施方案中,上述治疗剂和/或预防剂包含核酸、小分子化合物、多肽或蛋白质;上述核酸包含单链DNA、双链DNA、短异构体、agomir、antagomir、反义分子、小干扰RNA(siRNA)、不对称干扰RNA(aiRNA)、microRNA(miRNA)、Dicer-substrate RNA(dsRNA)、小发夹RNA(shRNA)、转运RNA(tRNA)、信使RNA(mRNA)、环状RNA(circRNA)、核酸适体(aptamer)中的至少一种。
在某些实施方案中,上述核酸还包含mRNA。
在某些实施方案中,上述纳米粒子组合物还包含一种或多种中性脂质、一种或多种甾族化合物、一种或多种聚合物缀合的脂质;其中,上述用于递送活性成分的脂质分子摩尔百分比为20-100%;上述甾族化合物摩尔百分比为0-80%;上述中性脂质摩尔百分比为0-40%;上述聚合物缀合的脂质摩尔百分比为0-20%。
优选地,上述用于递送活性成分的脂质分子摩尔百分比为20-95%;上述甾族化合物摩尔百分比为5-80%;上述中性脂质摩尔百分比为5-40%;上述聚合物缀合的脂质摩尔百分比为10-20%。
在某些实施方案中,上述中性脂质包含1,2-二硬脂酰基-sn-甘油-3-磷酸胆碱(DSPC)、1,2-二棕榈酰基-sn-甘油-3-磷酸胆碱(DPPC)、1,2-二肉豆蔻酰基-sn-甘油-3-磷酸胆碱(DMPC)、1-棕榈酰基-2-油酰基-sn-甘油-3-磷酸胆碱(POPC)、1,2-二油酰基-sn-甘油-3-磷酸胆碱(DOPC)、1,2-二油酰基-sn-甘油-3-磷酸乙醇胺(DOPE)、棕榈酰基油酰基磷脂酰乙醇胺(POPE)、二硬脂酰基-磷脂酰-乙醇胺(DSPE)、二棕榈酰基磷脂酰乙醇胺(DPPE)、二肉豆蔻酰基磷酸乙醇胺(DMPE)、1-硬脂酰基-2-油酰基-硬脂酰乙醇胺(SOPE)、1-硬脂酰基-2-油酰基-磷脂酰胆碱(SOPC)、鞘磷脂(SM)、神经酰胺、甾醇及其衍生物中的至少一种。
在某些实施方案中,上述甾族化合物包含胆固醇、粪固醇、非甾醇、谷固醇、麦角固醇、菜油固醇、豆固醇、菜籽固醇、芸苔甾醇、番茄碱、番茄碱、熊果酸、α-生育酚、皮质类固醇及其衍生物中的至少一种。
在某些实施方案中,上述聚合物缀合的脂质包含聚乙二醇修饰的磷脂酰乙醇胺、聚乙二醇修饰的磷脂酸、聚乙二醇修饰的神经酰胺、聚乙二醇修饰的二烷基胺、聚乙二醇修饰的二酰基甘油、聚乙二醇修饰的二烷基甘油中的至少一种。
在某些实施方案中,上述聚合物缀合的脂质包含DMG-PEG2000或者DMPE-PEG2000。
本发明还公开了一种含上述脂质化合物的纳米粒子组合物将核酸递送至细胞和/或器官的方法。
在某些实施方案中,上述纳米粒子组合物给药方式包括但不限于静脉、肌肉、皮内、皮下或滴鼻施用。
本发明还公开了上述纳米粒子组合物在药物制备中的应用。
需要说明的是,上述药物包括核酸药物、核酸疫苗、小分子药物、多肽药物、蛋白质药物等。
本发明开发了一种新型的脂质分子,该新型脂质分子可用于将活性成分(如核酸、多肽、蛋白)递送至细胞和/或器官。本发明的实施方案提供了多种包含本发明描述的新脂质分子的核酸-脂质纳米粒子组合物,由其组成的脂质纳米递送系统用于递送mRNA。在细胞水平和动物水平上,都表现出优于目前上市的产品DLin-MC3-DMA递送效率,可以作为核酸药物递送的新方法,促进核酸药物的发展。
本发明制备的脂质化合物能够递送核酸分子、小分子化合物、多肽或蛋白质等,使用本发明的脂质化合物制备的载体对核酸分子的包封效率高,可将核酸分子成功转运至细胞和/或器官中,并且高效进行表达;且本发明制备的纳米粒子组合物可以在动物体内有效递送mRNA,并且高水平地表达相关蛋白。
附图说明
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图2为IBMC-002的核磁共振谱图;
图3为IBMC-003的核磁共振谱图;
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图5为IBMC-005的核磁共振谱图;
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图11为IBMC-018的核磁共振谱图;
图12为IBMC-019的核磁共振谱图;
图13为IBMC-020的核磁共振谱图;
图14为IBMC-023的核磁共振谱图;
图15为IBMC-024的核磁共振谱图;
图16为IBMC-025的核磁共振谱图;
图17为IBMC-028的核磁共振谱图;
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图21为IBMC-034的核磁共振谱图;
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图28为IBMC-043的核磁共振谱图;
图29为IBMC-044的核磁共振谱图;
图30为IBMC-048的核磁共振谱图;
图31为IBMC-051的核磁共振谱图;
图32为IBMC-052的核磁共振谱图;
图33为IBMC-053的核磁共振谱图;
图34为IBMC-054的核磁共振谱图;
图35为IBMC-055的核磁共振谱图;
图36为IBMC-056的核磁共振谱图;
图37为IBMC-057的核磁共振谱图;
图38为IBMC-058的核磁共振谱图;
图39为IBMC-059的核磁共振谱图;
图40为IBMC-060的核磁共振谱图;
图41为IBMC-061的核磁共振谱图;
图42为IBMC-062的核磁共振谱图;
图43为IBMC-066的核磁共振谱图;
图44为不同N/P下脂质纳米粒子组合物细胞转染EGFP mRNA的效果图;
图45为脂质纳米粒子组合物Hela细胞转染效果;
图46为脂质纳米粒子组合物293T细胞转染效果;
图47为不同注射方式脂质纳米粒子组合物(LNP制剂)体内递送水平测试结果;
图48为不同时间点脂质纳米粒子组合物(LNP制剂)体内递送水平测试结果;
图49为脂质纳米粒子组合物(LNP制剂)体内递送SARS-CoV2 Spike诱导肌肉中S蛋白的表达水平;
图50为脂质纳米粒子组合物(LNP制剂)体内递送SARS-CoV2 Spike诱导肝脏中S蛋白的表达水平;
图51为脂质纳米粒子组合物(LNP制剂)体内递送SARS-CoV2 Spike诱导血液中S蛋白的表达水平。
具体实施方式
以下结合具体实施方式和附图对本发明的技术方案作进一步详细描述:
合成根据通式(I)、(Ia)、(Ib)、(Ic)、(Ⅱ)、(Ⅱa)、(Ⅱb)、(Ⅱc)、(Ⅱd)、(Ⅱe)、(Ⅱf)、(Ⅱg)、(Ⅱh)、(Ⅲ)、(Ⅲa)、(Ⅲb)、(Ⅲc)所代表的脂质分子;上述脂质分子均可采用模块化方式合成。本发明具体实施例中,除非特殊说明,否则使用的所有原料和试剂均为商购获得,并且无需进一步纯化。
实施例1:IBMC-001的合成路线,如下图所示:
IBMC-001的具体合成步骤如下:步骤1:Bi-12的合成:将79mmol Bi-01(5-羟基间苯二甲酸)、160mmol卞溴和240mmol碳酸氢钠溶于100mL的DMF中,40℃下搅拌8h,将反应物用二氯甲烷稀释并用饱和碳酸氢钠洗涤,氯化钠洗涤,无水硫酸钠干燥后旋干,用硅胶柱纯化,得到Bi-12(14.2g,49.58%)。1H NMR(400MHz,Chloroform-d)δ8.31(s,1H),7.79(d,J=1.5Hz,2H),7.51–7.29(m,10H),6.41(s,1H),5.37(s,4H)。
步骤2:Bi-13的合成:将10mmol Bi-12、20mmol溴乙酸叔丁酯和30mmol碳酸钾溶于100mL乙腈,70℃下搅拌8h,用乙酸乙酯稀释反应液,饱和碳酸氢钠洗涤,饱和氯化钠洗涤,无水硫酸钠干燥后旋干,用硅胶柱纯化,得到Bi-13(4.35g,90.52%)。1H NMR(400MHz,Chloroform-d)δ8.39(t,J=1.4Hz,1H),7.77(d,J=1.4Hz,2H),7.47–7.32(m,10H),5.37(s,4H),4.58(s,2H),1.47(s,9H)。
步骤3:Bi-14的合成:将6mmol Bi-13溶于50mL的三氟乙酸/二氯甲烷=4/1混合溶剂中,室温下搅拌12h,旋干溶剂,乙酸乙酯萃取,饱和氯化钠洗涤,无水硫酸钠干燥后旋干,用硅胶柱纯化,得到Bi-14(1.92g,77.05%)。1H NMR(400MHz,Chloroform-d)δ8.40(t,J=1.4Hz,1H),7.80(d,J=1.4Hz,2H),7.48–7.30(m,11H),5.37(s,4H),4.75(s,2H)。
步骤4:Bi-140013的合成:将1.52mmol Bi-14和3.04mmol T-13溶于50mL二氯甲烷,加入4-二甲氨基吡啶3.04mmol,1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐4.56mmol,室温下搅拌8h,二氯甲烷萃取,饱和氯化钠洗涤,无水硫酸钠干燥后旋干,用硅胶柱纯化,得到Bi-140013(1.56g,83.78%)。1H NMR(400MHz,Chloroform-d)δ8.39(s,1H),7.80(d,J=1.4Hz,2H),7.46–7.34(m,10H),5.37(s,4H),5.04–4.97(m,1H),4.69(s,2H),4.05(dt,J=13.4,6.6Hz,4H),2.30(td,J=9.0,4.4Hz,2H),1.68–1.19(m,64H),0.87(tt,J=7.1,2.2Hz,12H)。
步骤5:Bi-150013的合成:将1.11mmol Bi-140013、100mg Pd/C溶于50mL甲醇,通上氢气后室温下搅拌8h,抽滤后旋干滤液,得到Bi-150013(682mg,79.95%)。1H NMR(400MHz,Chloroform-d)δ8.45(s,1H),7.80(s,2H),5.01(s,1H),4.74(s,2H),4.05(d,J=7.1Hz,4H),2.31(s,2H),1.67–1.16(m,64H),0.90-0.83(m,12H)。
步骤6:IBMC-001的合成:将0.067mmol Bi-150013和0.266mmol N-甲基二乙醇胺溶于5mL二氯甲烷,加入4-二甲氨基吡啶0.1995mmol,1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐0.3325mmol,室温下搅拌8h,二氯甲烷萃取,饱和氯化钠洗涤,无水硫酸钠干燥后旋干,用硅胶柱纯化,得到IBMC-001(41mg,55.79%)。1H NMR(400MHz,Chloroform-d)δ8.33(s,1H),7.78(d,J=1.4Hz,2H),5.01(t,J=6.3Hz,1H),4.72(s,2H),4.44(t,J=5.6Hz,4H),4.04(t,J=6.6Hz,4H),3.61(t,J=5.3Hz,4H),2.86(t,J=5.6Hz,4H),2.65(t,J=5.3Hz,4H),2.38(s,6H),2.30(s,2H),1.66–1.19(m,64H),0.87(t,J=6.7Hz,12H),核磁氢谱如图1所示。
T-13合成路线,如下图所示:
T-13的具体合成步骤如下:
步骤1:T-3-3的合成:在三口烧瓶中,称取48.7mmol TosMIC溶于150mL的DMSO,冰浴下将166mmol NaH、107.2mmol T-3-1(5-溴代戊基乙酸酯)和9.7mmol TBAI加入反应瓶,加完后室温搅拌过夜;反应完成后,向反应液中加冰水,再用二氯甲烷萃取,用饱和碳酸氢钠溶液洗,饱和盐水洗,无水硫酸钠干燥,浓缩后得到中间体T-3-2(20.9g,95%),直接进行下一步。将20.9g的T-3-2溶于250mL的二氯甲烷中,滴加50mL饱和浓盐酸,搅拌1h后,向反应液中加水,用二氯甲烷萃取,用饱和碳酸氢钠溶液洗,饱和盐水洗,无水硫酸钠干燥,浓缩后,柱层析分离得到T-3-3(7.5g,53.9%)。1H NMR(400MHz,CDCl3)δ4.05(t,J=6.76Hz,4H),2.42(t,J=7.44,4H),2.04(s,6H),1.56-1.67(m,8H),1.30-1.39(m,4H)。
步骤2:T-3-4的合成:将24.4mmol T-3-3溶于100mL的甲醇/水(4:1)的混合液中,加入73mmol氢氧化钠,40℃下搅拌4h,旋蒸除去甲醇,乙酸乙酯萃取,浓缩后柱层析分离得到T-3-4(3.5g,71%)。1H NMR(400MHz,CDCl3)δ3.65(t,J=6.50Hz,4H),2.42(t,J=7.31,4H),1.54-1.64(m,8H),1.30-1.39(m,4H)。
步骤3:T-3-5的合成:将17.3mmol T-3-4、51.98mmol 2-己基癸酸溶于100mL的二氯甲烷中,再加入4-二甲氨基吡啶34.6mmol,1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐34.6mmol,室温搅拌过夜,用饱和氯化钠洗涤,无水硫酸钠干燥,旋干用硅胶柱纯化得到T-3-5(9.5g,80.9%)。1H NMR(400MHz,CDCl3)δ(ppm)4.06(t,J=6.63Hz,4H),2.40(t,J=7.42Hz,4H),2.27-2.34(m,2H),1.55-1.66(m,16H),1.25-1.28(m,44H),0.87(t,J=13.38Hz,12H)。
步骤4:T-13的合成:将14mmol T-3-5溶于150mL甲醇,加入硼氢化钠56mmol,室温下搅拌3h后,加入50mL冰水淬灭,二氯甲烷萃取,饱和盐水洗,无水硫酸钠干燥,蒸发浓缩得到T-13(9.5g,99%)。1H NMR(400MHz,CDCl3)δ4.00(t,J=6.61Hz,4H),3.51-3.52(m,1H),2.20-2.27(m,2H),1.48-1.60(m,8H),1.26-1.42(m,12H),1.18-1.25(s,44H),0.80(t,J=13.31,12H)。
实施例2:IBMC-002的合成路线,如下图所示:
IBMC-002的具体合成步骤与实施例1的区别:
1)Bi-140011的合成步骤与Bi-140013的区别:采用T-11替代T-13;
2)Bi-150011的合成步骤与Bi-150013的区别:采用Bi-140011替代Bi-140013。
IBMC-002的氢谱:1H NMR(400MHz,Chloroform-d)δ8.31(s,1H),7.78(s,2H),5.00(p,J=6.0Hz,1H),4.71(s,2H),4.46(t,J=5.6Hz,4H),4.03(td,J=6.5,4.4Hz,4H),3.64(t,J=5.3Hz,4H),2.92(t,J=5.6Hz,4H),2.70(t,J=5.3Hz,4H),2.42(s,6H),2.28(q,J=7.5,6.8Hz,3H),1.64–1.17(m,52H),0.86(t,J=6.6Hz,9H),核磁氢谱如图2所示。
T-11合成路线,如下图所示:
T-11的具体合成步骤如下:
步骤1:T-3-7的合成:将17.3mmol T-3-4、4.32mmol 2-己基癸酸溶于50mL的二氯甲烷中,再加入4-二甲氨基吡啶8.64mmol,1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐8.64mmol,室温搅拌过夜,用饱和氯化钠洗涤,无水硫酸钠干燥后旋干,用硅胶柱纯化得到T-3-7(1.7g,90%)。1H NMR(400MHz,CDCl3)δ4.06(t,J=6.63Hz,2H),3.65(t,J=6.51,2H),2.39-2.43(m,4H),2.27-2.34(m,1H),1.54-1.66(m,10H),1.25-1.44(m,26H),0.87(t,J=6.67,6H)。
步骤2:T-3-8的合成:将3.8mmol T-3-7、4.6mmol壬酸溶于50mL的二氯甲烷中,再加入4-二甲氨基吡啶7.6mmol,1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐7.6mmol,室温搅拌过夜,用饱和氯化钠洗涤,无水硫酸钠干燥后旋干,用硅胶柱纯化得到T-3-8(0.9g,90%)。1H NMR(400MHz,CDCl3)δ4.06(td,J=6.6,2.9Hz,4H),2.40(t,J=7.4Hz,4H),2.30(q,J=8.4,7.5Hz,3H),1.67–1.52(m,12H),1.48–1.18(m,36H),0.87(t,J=6.6Hz,9H)。
步骤3:T-11的合成:将1.3mmol T-3-8溶于50mL甲醇,加入硼氢化钠5.2mmol,室温下搅拌3h后,加入50mL冰水淬灭,二氯甲烷萃取,饱和盐水洗,无水硫酸钠干燥,蒸发浓缩得到T-11(0.9g,99%)。1H NMR(400MHz,CDCl3)δ4.07(td,J=6.7,3.2Hz,4H),3.59(dd,J=7.5,4.0Hz,1H),2.38–2.21(m,3H),1.67–1.56(m,8H),1.48–1.22(m,44H),0.92–0.83(m,9H)。
T-3-4的具体合成步骤同实施例1。
实施例3:IBMC-003的合成路线,如下图所示:
IBMC-003的具体合成步骤与实施例2的区别:采用T-08替代T-11。
IBMC-003的氢谱:1H NMR(400MHz,Chloroform-d)δ8.33(t,J=1.5Hz,1H),7.79(d,J=1.5Hz,2H),5.00(p,J=6.2Hz,1H),4.72(s,2H),4.44(t,J=5.7Hz,4H),3.65–3.59(m,4H),2.87(t,J=5.6Hz,4H),2.70–2.63(m,4H),2.39(s,6H),1.18-1.25(m,68H),0.91–0.85(m,6H),核磁氢谱如图3所示。
T-08合成路线,如下图所示:
T-08的具体合成步骤如下:
步骤1:T-1-1的合成:冰水浴下,将17.8mmol T-1-0(亚油酸)溶于50mL的THF中缓慢滴加至50mL含17.8mmol LiAlH4的四氢呋喃中搅拌,滴完后室温搅拌2h,反应完成加冰水淬灭LiAlH4,抽滤,旋干,柱层析分离,得到T-1-1(4.5g,94.7%)。1H NMR(400MHz,CDCl3)δ5.29-5.42(m,4H),3.64(t,J=6.64Hz,2H),2.77(t,J=6.64Hz,2H),2.02-2.07(m,4H),1.53-1.60(m,2H),1.25-1.39(m,16H),0.89(t,J=6.89Hz,3H)。
步骤2:T-1-2的合成:冰水浴下,取16.9mmol T-1-1、17.7mmol CBr4溶于100mL的二氯甲烷中,再加入PPh3,反应2h,旋干溶剂,柱层析分离得到T-1-2(5.7g,90%)。1H NMR(400MHz,CDCl3)δ5.32-5.44(m,4H),3.43(t,J=6.87Hz,2H),2.79(t,J=6.49Hz,2H),2.07(dd,J=6.81Hz,4H),1.87(t,J=14.74Hz,2H),1.26-1.46(m,16H),0.91(t,J=6.86Hz,3H)。
步骤3:T-08的合成:在史莱克管中加入18.29mmol镁条并抽真空,加入超干的THF,最后将6mmol T-1-2溶于5mL的THF中,注入反应瓶中,室温下搅拌1h后加入甲酸乙酯搅拌过夜,加水淬灭反应,用二氯甲烷萃取,浓缩后柱层析分离得到T-08(0.9g,28%)。1H NMR(400MHz,CDCl3)δ5.29-5.42(m,8H),3.56-3.59(m,1H),2.77(t,J=6.41Hz,4H),2.02-2.07(m,8H),1.23-1.47(m,40H),0.89(t,J=6.89Hz,6H)。
实施例4:IBMC-004的合成路线,如下图所示:
IBMC-004的具体合成步骤如下:步骤1:Bi-02的合成:将55mmol Bi-01(5-羟基间苯二甲酸)、330mmol硼烷四氢呋喃溶于100mL超干THF,室温下搅拌24h,将反应物用饱和的氯化铵淬灭,用饱和的氯化钠洗涤后,用无水NaSO4干燥,过硅胶柱纯化得到Bi-02(5.25g,61%)。1H NMR(400MHz,Methanol-d4)δ6.77(q,J=1.1Hz,1H),6.67(d,J=1.5Hz,2H),4.49(s,4H)。
步骤2:Bi-04的合成:将64.6mmol Bi-02、77.2mmol溴乙酸甲酯溶于300mL乙腈,加入碳酸钾129.8mmol,80℃回流,反应4h,反应液冷却至室温后过滤,滤液旋干后硅胶柱纯化得到Bi-04(10.97g,75%)。1H NMR(400MHz,Chloroform-d)δ6.99(tt,J=1.3,0.7Hz,1H),6.85(d,J=1.4Hz,2H),4.67(d,J=0.7Hz,4H),4.66(s,2H),3.81(s,3H)。
步骤3:Bi-06的合成:将22.1mmol Bi-04、88.4mmol咪唑溶于100mL的无水二氯甲烷,将叔丁基二甲基氯硅烷溶于50mL无水二氯甲烷后滴加至反应液,室温下反应4h,旋干过柱纯化得到目标产物Bi-06(7.56g,70%)。1H NMR(400MHz,Chloroform-d)δ6.89(dt,J=1.9,0.9Hz,1H),6.84–6.72(m,2H),4.75–4.65(m,4H),4.64(s,2H),3.80(s,3H),0.94(s,18H),0.15(s,12H)。
步骤4:Bi-07的合成:将11mmol Bi-06溶于90mL甲醇;氢氧化钠22mmol溶于30mL去离子水中滴加至甲醇溶液中,反应2h,将溶液旋至三分之一后用乙酸乙酯萃取,无水硫酸钠干燥后,旋干过柱纯化得到目标产物Bi-07(2.9g,60%)。1H NMR(400MHz,Chloroform-d)δ6.94(s,1H),6.75(s,2H),4.67(d,J=12.8Hz,4H),3.48(d,J=1.1Hz,2H),0.93(d,J=7.2Hz,18H),0.08(d,J=7.1Hz,12H)。
步骤5:Bi-0713的合成:将0.227mmol Bi-07溶于50mL二氯甲烷中,依次加入T-130.114mmol,4-二甲氨基吡啶0.341mmol,1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐0.272mmol,室温搅拌8h,氯化钠溶液洗涤,无水硫酸钠干燥后,旋干用硅胶柱纯化,得到目标产物Bi-0713(2.29g,71%)。1H NMR(400MHz,Chloroform-d)δ6.83(d,J=6.7Hz,1H),6.73–6.66(m,2H),4.91(td,J=7.2,3.5Hz,1H),4.62(s,4H),4.54(d,J=5.8Hz,2H),3.97(t,J=6.7Hz,4H),2.23(tt,J=8.8,5.3Hz,2H),1.58–1.07(m,64H),0.87(s,18H),0.83–0.78(m,12H),0.02(s,12H)。
步骤6:Bi-070013的合成:将0.91mmol Bi-0713溶于50mL二氯甲烷,滴加5mL饱和浓盐酸,反应8h,旋干后过硅胶柱纯化得到目标产物Bi-070013(0.71g,81%)。1H NMR(400MHz,Chloroform-d)δ7.04–6.92(m,1H),6.85(d,J=1.3Hz,2H),5.09–4.93(m,1H),4.66(s,4H),4.64(s,2H),4.03(td,J=6.8,1.5Hz,4H),2.30(tt,J=8.9,5.4Hz,2H),1.69–1.16(m,64H),0.94–0.79(m,12H)。
步骤7:IBMC-004的合成:将0.08mmol Bi-070013溶于8mL二氯甲烷,依次加入3-(4-吗啉基)丙酸0.32mmol,4-二甲氨基吡啶0.48mmol,1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐0.38mmol,室温反应12h,饱和氯化钠溶液洗涤,无水硫酸钠干燥后旋干,用硅胶柱纯化,得到目标产物IBMC-004(42mg,45.38%)。1H NMR(400MHz,Chloroform-d)δ6.96(d,J=1.5Hz,1H),6.87(d,J=1.6Hz,2H),5.09(s,4H),5.01(q,J=6.0Hz,1H),4.61(s,2H),4.04(t,J=6.6Hz,4H),3.69(t,J=4.6Hz,8H),2.71(s,4H),2.57(s,4H),2.47(s,8H),2.33–2.26(m,2H),1.82–1.20(m,64H),0.91–0.83(m,12H),核磁氢谱如图4所示。
T-13的具体合成步骤同实施例1。
实施例5:IBMC-005的合成路线,如下图所示:
IBMC-005的具体合成步骤如下:步骤1:Bi-0711的合成:将0.227mmol Bi-07溶于5mL二氯甲烷中,依次加入0.114mmol T-11,4-二甲氨基吡啶0.342mmol,1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐0.227mmol,室温搅拌12h,氯化钠溶液洗,无水硫酸钠干燥后旋干,用硅胶柱纯化,得到目标产物Bi-0711(90mg,78.95%)。1H NMR(400MHz,Chloroform-d)δ6.81(d,J=6.4Hz,1H),6.72–6.63(m,2H),4.89(p,J=6.0Hz,1H),4.62–4.58(m,4H),4.54(s,1H),4.51(s,1H),3.94(td,J=6.7,3.6Hz,4H),3.71(s,1H),2.29–2.09(m,2H),1.59–1.06(m,52H),0.85(s,18H),0.82–0.75(m,9H),0.01(s,12H)。
步骤2:Bi-070011的合成:将0.045mmol Bi-0711溶于5mL二氯甲烷,滴加0.6mL的饱和浓盐酸,反应8h,旋干后过硅胶柱纯化,得到目标产物Bi-070011(53mg,75%)。1H NMR(400MHz,Chloroform-d)δ6.98(t,J=1.4Hz,1H),6.85(d,J=1.4Hz,2H),5.30(s,2H),5.05–4.94(m,1H),4.65(d,J=9.0Hz,4H),4.11–3.91(m,4H),2.35–2.19(m,3H),1.74–1.12(m,52H),0.93–0.81(m,9H)。
步骤3:IBMC-005的合成:将0.064mmol Bi-070011溶于5mL二氯甲烷,依次加入N,N-二甲基氨基丁酸盐酸盐0.032mmol,4-二甲氨基吡啶0.096mmol,1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐0.096mmol,室温反应12h,饱和氯化钠溶液洗涤,无水硫酸钠干燥后旋干,用硅胶柱纯化,得到目标产物IBMC-005(7mg,10.1%)。1H NMR(400MHz,Chloroform-d)δ7.01(s,1H),6.83(dt,J=13.1,2.3Hz,2H),5.08(s,2H),4.99(tt,J=7.2,3.5Hz,1H),4.64(d,J=15.2Hz,4H),4.03(td,J=6.7,4.1Hz,4H),2.45(dt,J=16.9,7.4Hz,6H),2.36(s,4H),2.28(t,J=7.6Hz,3H),1.93(p,J=7.2Hz,2H),1.70–1.16(m,52H),0.88(td,J=6.9,1.7Hz,9H),核磁氢谱如图5所示。
Bi-07的具体合成步骤同实施例4。
实施例6:IBMC-006的合成路线,如下图所示:
IBMC-006的具体合成步骤与实施例5的区别:采用T-08替代T-11。IBMC-006的氢谱:1H NMR(400MHz,Chloroform-d)δ6.96(d,J=1.6Hz,1H),6.75(dt,J=12.2,2.2Hz,2H),5.36–5.21(m,8H),5.02(s,2H),4.92(p,J=6.1Hz,1H),4.57(d,J=19.0Hz,4H),2.74–2.68(m,4H),2.61(t,J=7.9Hz,2H),2.45(s,6H),2.40(t,J=6.9Hz,2H),1.98(td,J=7.2,4.2Hz,8H),1.53–1.08(m,42H),0.85–0.75(m,6H),核磁氢谱如图6所示。
T-08的具体合成步骤同实施例3。
实施例7:IBMC-007的具体合成步骤:将0.067mmol Bi-150013和0.266mmol N,N-二甲基丙醇胺溶于5mL二氯甲烷,加入4-二甲氨基吡啶0.1995mmol,1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐0.3325mmol,室温下搅拌8h,二氯甲烷萃取,饱和氯化钠洗涤,无水硫酸钠干燥,旋干用硅胶柱纯化,得到IBMC-007(41mg,55.79%)。1H NMR(400MHz,Chloroform-d)δ8.30(t,J=1.4Hz,1H),7.76(d,J=1.4Hz,2H),5.04–4.97(m,1H),4.70(s,2H),4.38(t,J=6.6Hz,4H),4.04(t,J=6.6Hz,4H),2.42(dd,J=7.9,6.7Hz,4H),2.29(ddd,J=8.9,5.4,3.6Hz,2H),2.25(s,12H),1.99–1.90(m,4H),1.64–1.19(m,64H),0.86(td,J=6.8,1.2Hz,12H),核磁氢谱如图7所示。
Bi-150013的具体合成步骤同实施例1。
实施例8:IBMC-011的具体合成步骤:将0.067mmol Bi-150013和0.266mmol吗啉基丙醇溶于5mL二氯甲烷,加入4-二甲氨基吡啶0.1995mmol,1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐0.3325mmol,室温下搅拌8h,二氯甲烷萃取饱和氯化钠洗涤,无水硫酸钠干燥,旋干用硅胶柱纯化,得到IBMC-011(41mg,55.79%)。1H NMR(400MHz,Chloroform-d)δ8.31–8.29(m,1H),7.77(d,J=1.5Hz,2H),5.05–4.99(m,1H),4.71(s,2H),4.41(t,J=6.6Hz,4H),4.05(t,J=6.6Hz,4H),3.74–3.68(m,8H),2.53–2.42(m,12H),2.30(tt,J=8.9,5.3Hz,2H),1.97(p,J=6.8Hz,4H),1.65–1.19(m,64H),0.90–0.85(m,12H),核磁氢谱如图8所示。
Bi-150013的具体合成步骤同实施例1。
实施例9:IBMC-012的具体合成步骤:同实施例8。IBMC-012的氢谱:1H NMR(400MHz,Chloroform-d)δ8.33–8.30(m,1H),7.79–7.73(m,2H),5.01(td,J=7.1,3.5Hz,1H),4.70(s,2H),4.39(q,J=7.0Hz,4H),4.04(t,J=6.6Hz,4H),3.71(t,J=4.6Hz,4H),2.48(dt,J=10.2,6.0Hz,4H),2.29(tt,J=8.9,5.3Hz,2H),1.97(p,J=6.8Hz,2H),1.68–1.16(m,64H),0.90–0.83(m,12H),核磁氢谱如图9所示。
实施例10:IBMC-015的具体合成步骤:IBMC-015的具体合成步骤:将0.022mmolBi-150013、0.067mmol EDCI和0.067mmol HOBT溶于5mL二氯甲烷,1h之后向其中加入吗啉基丙醇0.088mmol、DIEA 0.088mmol,室温下搅拌8h,二氯甲烷萃取,饱和氯化钠洗涤,无水硫酸钠干燥,旋干用硅胶柱纯化;加入0.089mmol苯乙醇溶于5mL二氯甲烷,加入4-二甲氨基吡啶0.067mmol,1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐0.11mmol,室温下搅拌8h,二氯甲烷萃取饱和氯化钠洗涤,无水硫酸钠干燥,旋干用硅胶柱纯化,得到IBMC-015(5mg,20.15%)。1H(400MHz,Chloroform-d)δ8.26(t,J=4.8Hz,1H),8.03(s,1H),7.76–7.68(m,2H),7.47–7.34(m,5H),5.38(s,2H),5.00(dq,J=12.4,6.6,5.9Hz,1H),4.70(s,2H),4.03(t,J=6.6Hz,4H),3.70(t,J=4.7Hz,4H),3.57(q,J=5.6Hz,2H),2.60–2.44(m,6H),2.29(tt,J=8.9,5.3Hz,2H),1.79(p,J=6.0Hz,2H),1.66–1.17(m,64H),0.90–0.84(m,12H),核磁氢谱如图10所示。
实施例11:IBMC-018的具体合成步骤:将0.075mmol Bi-150011和0.3mmol 1,4-双(2-羟乙基)哌嗪溶于5mL二氯甲烷,加入4-二甲氨基吡啶0.225mmol,1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐0.375mmol,室温下搅拌8h,二氯甲烷萃取,用饱和氯化钠洗涤,无水硫酸钠干燥,旋干用硅胶柱纯化,得到IBMC-018(21mg,25.3%)。1H NMR(400MHz,Chloroform-d)δ8.27(s,1H),7.76(s,2H),5.00(q,J=6.3Hz,1H),4.70(s,2H),4.46(t,J=5.8Hz,4H),4.03(q,J=6.2Hz,4H),3.70(t,J=5.2Hz,4H),2.89–2.57(m,24H),2.27(t,J=7.6Hz,3H),1.64–1.18(m,52H),0.86(t,J=6.6Hz,9H),核磁氢谱如图11所示。
Bi-150011的具体合成步骤同实施例2。
实施例12:IBMC-019的具体合成步骤:将0.075mmol Bi-150011和0.3mmol吗啉基丙醇溶于5mL二氯甲烷,加入4-二甲氨基吡啶0.225mmol,1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐0.375mmol,室温下搅拌8h,二氯甲烷萃取,用饱和氯化钠洗涤,无水硫酸钠干燥,旋干用硅胶柱纯化,得到IBMC-019(36mg,45.57%)。1H NMR(400MHz,Chloroform-d)δ8.28(t,J=1.5Hz,1H),7.75(d,J=1.4Hz,2H),5.01(dt,J=7.1,5.3Hz,1H),4.69(s,2H),4.39(t,J=6.6Hz,4H),4.03(td,J=6.6,4.6Hz,4H),3.70(t,J=4.7Hz,8H),2.47(dt,J=10.0,6.0Hz,12H),2.27(dd,J=9.2,6.1Hz,3H),1.96(q,J=6.9Hz,4H),1.65–1.18(m,52H),0.90–0.83(m,9H),核磁氢谱如图12所示。
Bi-150011的具体合成步骤同实施例2。
实施例13:IBMC-020的具体合成步骤:将0.075mmol Bi-150011和0.3mmol N,N-二甲基丙醇胺溶于5mL二氯甲烷,加入4-二甲氨基吡啶0.225mmol,1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐0.375mmol,室温下搅拌8h,二氯甲烷萃取,用饱和氯化钠洗涤,无水硫酸钠干燥,旋干用硅胶柱纯化,得到IBMC-020(25mg,34.39%)。1H NMR(400MHz,Chloroform-d)δ8.30(d,J=1.6Hz,1H),7.77(d,J=1.4Hz,2H),5.01(q,J=6.4Hz,1H),4.71(s,2H),4.40(t,J=6.5Hz,4H),4.04(q,J=6.4Hz,4H),2.52(t,J=7.4Hz,4H),2.32(s,12H),2.28(t,J=7.6Hz,3H),2.00(q,J=6.9Hz,4H),1.66–1.18(m,52H),0.91–0.84(m,9H),核磁氢谱如图13所示。
Bi-150011的具体合成步骤同实施例2。
实施例14:IBMC-023的具体合成步骤:将0.377mmol Bi-070013溶于10mL二氯甲烷,依次加入N,N-二甲基氨基丁酸盐酸盐0.189mmol,4-二甲氨基吡啶0.567mmol,1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐0.227mmol,室温反应12h,饱和氯化钠溶液洗涤,无水硫酸钠干燥后旋干,用硅胶柱纯化,得到目标产物IBMC-023(100mg,53.6%)。1H NMR(400MHz,Chloroform-d)δ6.95(s,1H),6.82–6.70(m,2H),5.01(s,2H),4.92(td,J=7.1,3.5Hz,1H),4.57(d,J=14.0Hz,4H),3.97(t,J=6.7Hz,4H),2.59(t,J=7.8Hz,2H),2.43(s,6H),2.39(d,J=6.9Hz,2H),2.23(tt,J=8.8,5.3Hz,2H),1.95(q,J=7.4Hz,2H),1.62–1.08(m,64H),0.80(t,J=6.7Hz,12H),核磁氢谱如图14所示。
Bi-070013的具体合成步骤同实施例4。
实施例15:IBMC-024的具体合成步骤:将0.235mmol Bi-070013溶于8mL二氯甲烷,依次加入3-(4-吗啉基)丙酸0.118mmol,4-二甲氨基吡啶0.353mmol,1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐0.142mmol,室温反应12h,氯化钠溶液洗涤,无水硫酸钠干燥后,旋干用硅胶柱纯化,得到目标产物IBMC-024(62.7mg,50.93%)。1H NMR(400MHz,Chloroform-d)δ6.90(s,1H),6.79(dt,J=7.8,2.3Hz,2H),5.02(s,2H),4.98–4.87(m,1H),4.58(d,J=16.4Hz,4H),3.96(t,J=6.6Hz,4H),3.67–3.55(m,4H),2.64(t,J=7.2Hz,2H),2.49(t,J=7.2Hz,2H),2.38(t,J=4.6Hz,4H),2.23(tt,J=8.9,5.3Hz,2H),1.66–1.08(m,64H),0.80(t,J=6.7Hz,12H),核磁氢谱如图15所示。
Bi-070013的具体合成步骤同实施例4。
实施例16:IBMC-025的合成路线,如下图所示:
IBMC-025的具体合成步骤:步骤1:1OH-02的合成:将65mmol 1OH-1(3,5-二羟基苯甲酸)、195mmol硼烷四氢呋喃溶于100mL超干THF,室温下搅拌24h;将反应物用饱和的氯化铵淬灭,用饱和的氯化钠洗涤后,用无水NaSO4干燥,过硅胶柱纯化得到1OH-02(5.9g,65%)。1H NMR(400MHz,DMSO-d6)δ9.05(s,2H),6.17(d,J=2.2Hz,2H),6.05(t,J=2.2Hz,1H),4.98(t,J=5.8Hz,1H),4.30(d,J=5.8Hz,2H)。
步骤2:1OH-03的合成:将71.4mmol 1OH-02、35.7mmol溴乙酸苄酯溶于200mL乙腈,50℃下,71.4mmol加入碳酸钾,反应4h,反应液冷却至室温后过滤,滤液旋干后硅胶柱纯化得到1OH-03(3.61g,59%)。1H NMR(400MHz,Chloroform-d)δ7.39–7.27(m,5H),7.13(s,1H),6.44–6.36(m,1H),6.32(dd,J=2.3,1.2Hz,1H),6.25(t,J=2.3Hz,1H),5.18(s,2H),4.52(s,2H),4.42(d,J=2.8Hz,2H)。
步骤3:1OH-04的合成:将11.1mmol 1OH-03、44.4mmol咪唑溶于100mL的无水二氯甲烷,将叔丁基二甲基氯硅烷溶于20mL无水二氯甲烷后滴加至反应液,室温下反应4h,旋干过柱纯化得到目标产物1OH-04(5.6g,97.7%)。1H NMR(400MHz,Chloroform-d)δ7.39–7.31(m,5H),6.48(tq,J=2.1,0.9Hz,2H),6.30(t,J=2.3Hz,1H),5.24(s,2H),4.63(d,J=1.7Hz,4H),0.97(s,9H),0.94(s,9H),0.18(s,6H),0.09(s,6H)。
步骤4:1OH-05的合成:将0.7g 1OH-04、0.07g Pd/C溶于20mL的甲醇,室温下搅拌1h。过滤旋干溶剂,硅胶柱纯化得到1OH-05(0.44g,76%)。1H NMR(400MHz,Chloroform-d)δ6.52–6.44(m,2H),6.31(s,1H),4.64(s,2H),4.60(s,2H),0.96(s,9H),0.93(s,9H),0.18(s,6H),0.08(s,6H)。
步骤5:1OH-06的合成:将3.3mmol 1OH-05溶于50mL二氯甲烷中,依次加入4.95mmol T-13、14.85mmol 4-二甲氨基吡啶、9.9mmol 1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐,室温搅拌8h,饱和氯化钠溶液洗涤,无水硫酸钠干燥后,旋干用硅胶柱纯化,得到目标产物1OH-06(3.07g,86%)。1H NMR(400MHz,Chloroform-d)δ6.47(ddt,J=3.4,2.2,1.2Hz,2H),6.28(t,J=2.3Hz,1H),4.99(qd,J=7.2,5.2Hz,1H),4.63(s,2H),4.55(s,2H),4.04(t,J=6.6Hz,4H),2.30(tt,J=8.9,5.3Hz,2H),1.59(dq,J=11.2,6.6Hz,12H),1.46–1.38(m,4H),1.33–1.21(m,48H),0.96(s,9H),0.93(s,9H),0.90–0.84(m,12H),0.18(s,6H),0.08(s,6H)。
步骤6:1OH-07的合成:将0.92mmol 1OH-06溶于50mL二氯甲烷,滴加1.2mL饱和浓盐酸,反应6h,旋干后过硅胶柱纯化得到目标产物1OH-07(0.68g,76%)。1H NMR(400MHz,Chloroform-d)δ6.52(dd,J=2.2,1.3Hz,1H),6.48(dd,J=2.2,1.3Hz,1H),6.33(t,J=2.3Hz,1H),4.99(qd,J=7.2,5.1Hz,1H),4.58(d,J=5.3Hz,4H),4.03(t,J=6.7Hz,4H),2.30(tt,J=8.9,5.4Hz,2H),1.62–1.52(m,12H),1.46–1.38(m,4H),1.33–1.20(m,48H),0.97(s,9H),0.90–0.84(m,12H),0.19(s,6H)。
步骤7:1OH-08的合成:将0.21mmol 1OH-07溶于15mL二氯甲烷,依次加入0.41mmolN,N-二甲基氨基丁酸盐酸盐、75.13mmol 4-二甲氨基吡啶、94.32mmol 1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐,室温反应12h,饱和氯化钠溶液洗涤,无水硫酸钠干燥后旋干,用硅胶柱纯化,得到目标产物1OH-08(0.16g,71.7%)。1H NMR(400MHz,Chloroform-d)δ6.50(t,J=1.9Hz,1H),6.45(t,J=1.8Hz,1H),6.35(t,J=2.3Hz,1H),5.04–4.94(m,3H),4.56(s,2H),4.04(t,J=6.6Hz,4H),2.51(t,J=7.6Hz,2H),2.43(t,J=7.2Hz,2H),2.38(s,6H),2.29(ddd,J=8.9,5.4,3.6Hz,2H),1.91(p,J=7.3Hz,2H),1.63–1.50(m,12H),1.45–1.37(m,4H),1.24(s,48H),0.96(s,9H),0.90–0.83(m,12H),0.18(s,6H)。
步骤8:IBMC-025的合成:将0.046mmol 1OH-08溶于8mL的四氢呋喃,加入1mL的1MTBAF的四氢呋喃,室温反应2h,饱和氯化钠溶液洗涤,无水硫酸钠干燥后旋干,用硅胶柱纯化,得到目标产物IBMC-025(20mg,44.7%)。1H NMR(400MHz,Chloroform-d)δ6.39(d,J=1.8Hz,1H),6.34(t,J=1.8Hz,1H),6.26(t,J=2.3Hz,1H),4.96–4.88(m,3H),4.51(s,2H),3.99(tt,J=6.7,3.2Hz,4H),2.32(td,J=8.2,7.7,5.8Hz,4H),2.27–2.18(m,8H),1.79(q,J=7.5Hz,2H),1.58–1.43(m,12H),1.40–1.33(m,4H),1.18(d,J=3.5Hz,48H),0.80(t,J=6.7Hz,12H),核磁氢谱如图16所示。
实施例17:IBMC-028的具体合成步骤:将0.0558mmol Bi-150013和0.223mmol 1,4-双(2-羟乙基)哌嗪溶于5mL二氯甲烷,加入4-二甲氨基吡啶0.1674mmol,1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐0.279mmol,室温下搅拌8h,二氯甲烷萃取,饱和氯化钠洗涤,无水硫酸钠干燥后旋干,用硅胶柱纯化,得到IBMC-028(18mg,26.73%)。1H NMR(400MHz,Chloroform-d)δ8.28(d,J=1.6Hz,1H),7.76(d,J=1.4Hz,2H),5.00(td,J=7.0,3.5Hz,1H),4.70(s,2H),4.46(t,J=5.9Hz,4H),4.04(t,J=6.6Hz,4H),3.65–3.56(m,4H),2.78(t,J=5.9Hz,4H),2.65–2.52(m,16H),2.34–2.18(m,6H),1.65–1.17(m,64H),0.86(t,J=6.7Hz,12H),核磁氢谱如图17所示。
Bi-150013的具体合成步骤同实施例1。
实施例18:IBMC-029的具体合成步骤同实施例17。IBMC-029的氢谱:1H NMR(400MHz,Chloroform-d)δ8.30(d,J=1.6Hz,1H),7.76(dt,J=14.5,2.2Hz,2H),5.01(p,J=6.1Hz,1H),4.70(s,2H),4.46(t,J=6.0Hz,2H),4.04(t,J=6.6Hz,4H),3.93(s,2H),3.62(t,J=5.4Hz,2H),2.79(t,J=6.0Hz,2H),2.71–2.50(m,8H),2.31(dt,J=14.3,5.7Hz,2H),1.65–1.18(m,64H),0.86(t,J=6.7Hz,12H),核磁氢谱如图18所示。
实施例19:IBMC-030的具体合成步骤:将0.057mmol Bi-070013溶于5mL二氯甲烷,依次加入4-(4-甲基-1-哌嗪基)丁酸0.229mmol,4-二甲氨基吡啶0.342mmol,1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐0.229mmol,室温反应12h,饱和氯化钠溶液洗涤,无水硫酸钠干燥后旋干,用硅胶柱纯化,得到目标产物IBMC-030(40mg,57.7%)。1H NMR(400MHz,Chloroform-d)δ6.94(s,1H),6.85(s,2H),5.05(s,4H),4.98(q,J=6.3Hz,1H),4.60(s,2H),4.03(t,J=6.6Hz,4H),2.68–2.41(m,16H),2.39(t,J=7.3Hz,8H),2.33(s,6H),1.83(p,J=7.3Hz,4H),1.56(dd,J=13.4,7.0Hz,12H),1.45–1.18(m,54H),0.86(t,J=6.6Hz,12H),核磁氢谱如图19所示。
Bi-070013的具体合成步骤同实施例4。
实施例20:IBMC-031的具体合成步骤:将0.171mmol Bi-070013溶于8mL二氯甲烷,依次加入4-(4-甲基-1-哌嗪基)丁酸0.086mmol,4-二甲氨基吡啶0.258mmol,1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐0.103mmol,室温反应12h,饱和氯化钠溶液洗涤,无水硫酸钠干燥后,旋干用硅胶柱纯化,得到目标产物IBMC-031(89.68mg,50.3%)。1H NMR(400MHz,Chloroform-d)δ6.99(s,1H),6.91–6.77(m,2H),5.07(s,2H),5.06–4.95(m,1H),4.64(d,J=15.0Hz,4H),4.04(t,J=6.7Hz,4H),2.69–2.16(m,17H),1.84(p,J=7.2Hz,2H),1.71–1.12(m,64H),0.87(t,J=6.7Hz,12H),核磁氢谱如图20所示。
Bi-070013的具体合成步骤同实施例4。
实施例21:IBMC-034的合成路线,如下图所示:
IBMC-034的具体合成步骤:步骤1:m46-02的合成:将0.017mmol m46-01、0.0187mmol乙酸钾溶于30mL的DMF,室温下搅拌5h,将反应物用水淬灭,乙酸乙酯萃取,用饱和的氯化钠洗涤后,用无水NaSO4干燥,过硅胶柱纯化得到m46-02(1.81g,54.7%)。1H NMR(400MHz,Chloroform-d)δ11.04(d,J=0.5Hz,1H),9.90(d,J=0.6Hz,1H),7.60–7.57(m,1H),7.56–7.52(m,1H),7.00(d,J=8.5Hz,1H),5.08(s,2H),2.10(s,3H)。
步骤2:m46-03的合成:将3.09mmol m46-02溶于20mL的1M的NaOH溶液,室温下搅拌5h。将反应物用6M的盐酸调pH=3.5,乙酸乙酯萃取,用饱和的氯化钠洗涤后,用无水NaSO4干燥,过硅胶柱纯化得到m46-03(0.42g,89.4%)。1H NMR(400MHz,Chloroform-d)δ11.00(s,1H),9.90(s,1H),7.58(d,J=2.2Hz,1H),7.53(dd,J=8.5,2.3Hz,1H),6.99(d,J=8.6Hz,1H),4.69(s,2H)。
步骤3:m46-04的合成:将3.16mmol m46-04、3.79mmol溴乙酸甲酯溶于20mL乙腈,加入4.75mmol碳酸钾,80℃回流,反应4h。反应液冷却至室温后过滤,滤液旋干后,硅胶柱纯化得到m46-04(0.42g,59.4%)。1H NMR(400MHz,Chloroform-d)δ10.54(s,1H),7.84(d,J=2.4Hz,1H),7.58(dd,J=8.5,2.4Hz,1H),6.87(d,J=8.5Hz,1H),4.78(s,2H),4.67(s,2H),3.82(s,3H)。
步骤4:m46-05的合成:将1.16mmol m46-04、2.32mmol咪唑溶于20mL的无水二氯甲烷,将叔丁基二甲基氯硅烷溶于5mL无水二氯甲烷后滴加至反应液,室温下反应4h,旋干过柱纯化得到目标产物m46-05(0.28g,71.4%)。1H NMR(400MHz,Chloroform-d)δ10.55(s,1H),7.83–7.71(m,1H),7.55(ddt,J=8.5,2.2,0.7Hz,1H),6.84(d,J=8.6Hz,1H),4.76(s,2H),4.69(d,J=0.9Hz,2H),3.81(s,3H),0.93(s,9H),0.09(s,6H)。
步骤5:m46-06的合成:将0.83mmol m46-05溶于10mL甲醇,1.24mmol氢氧化钠溶于20mL去离子水中滴加至甲醇溶液中,反应2h,将溶液旋至三分之一后用乙酸乙酯萃取,无水硫酸钠干燥后,旋干过柱纯化得到目标产物m46-06(180g,67.2%)。1H NMR(400MHz,DMSO-d6)δ10.43(s,1H),7.61(d,J=2.3Hz,1H),7.47(dd,J=8.6,2.4Hz,1H),6.98(d,J=8.6Hz,1H),4.64(s,2H),4.41(d,J=5.2Hz,2H),0.89(s,9H),0.06(s,6H)。
步骤6:m46-07的合成:将1.39mmol m46-06溶于40mL二氯甲烷中,依次加入2.78mmol T-13、5.56mmol 4-二甲氨基吡啶、4.17mmol 1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐,室温搅拌8h,饱和氯化钠溶液洗涤,无水硫酸钠干燥后,旋干用硅胶柱纯化,得到目标产物m46-07(0.84g,61%)。1H NMR(400MHz,Chloroform-d)δ10.55(s,1H),7.78(d,J=2.3Hz,1H),7.57–7.50(m,1H),6.85(d,J=8.6Hz,1H),4.99(p,J=6.2Hz,1H),4.74(s,2H),4.69(s,2H),4.04(t,J=6.7Hz,4H),2.30(tt,J=8.9,5.3Hz,2H),1.61–1.54(m,12H),1.46–1.39(m,4H),1.25(d,J=3.6Hz,48H),0.92(s,9H),0.89–0.84(m,12H),0.09(s,6H)。
步骤7:m46-08的合成:将0.22mmol m46-07溶于20mL二氯甲烷中,滴加0.025mL饱和浓盐酸,室温搅拌8h,旋干用硅胶柱纯化,得到目标产物m46-08(0.13g,66.8%)。1H NMR(400MHz,Chloroform-d)δ10.55(s,1H),7.84(d,J=2.4Hz,1H),7.59(dd,J=8.5,2.4Hz,1H),6.87(d,J=8.6Hz,1H),5.03–4.96(m,1H),4.76(s,2H),4.67(s,2H),4.02(t,J=6.7Hz,4H),2.30(tt,J=8.9,5.3Hz,2H),1.60–1.53(m,12H),1.42(td,J=8.0,3.9Hz,4H),1.26(d,J=9.0Hz,48H),0.87(t,J=6.7Hz,12H)。
步骤8:m46-09的合成:将0.15mmol m46-08溶于10mL二氯甲烷中,依次加入0.3mmol N,N-二甲基氨基丁酸盐酸盐、0.75mmol 4-二甲氨基吡啶、0.36mmol 1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐,室温反应12h,饱和氯化钠溶液洗涤,无水硫酸钠干燥后旋干,用硅胶柱纯化,得到目标产物IBMC-004(0.12g,81.6%)。1H NMR(400MHz,Chloroform-d)δ10.54(s,1H),7.86(d,J=2.4Hz,1H),7.53(dd,J=8.6,2.4Hz,1H),6.85(d,J=8.5Hz,1H),5.06(s,2H),4.99(p,J=6.1Hz,1H),4.75(s,2H),4.04(t,J=6.6Hz,4H),2.38(t,J=7.4Hz,2H),2.33–2.26(m,4H),2.21(s,6H),1.80(p,J=7.4Hz,2H),1.63–1.52(m,12H),1.46–1.39(m,4H),1.32–1.20(m,48H),0.89–0.84(m,12H)。
步骤9:IBMC-034的合成:将0.05mmol m46-09溶于8mL甲醇中,加入0.056mmol硼氢化钠,室温反应2h,旋干溶剂,用硅胶柱纯化,得到目标产物IBMC-034(17.5g,35%)。1H NMR(400MHz,Chloroform-d)δ7.34(d,J=2.2Hz,1H),7.23(dd,J=8.3,2.3Hz,1H),6.76(d,J=8.3Hz,1H),5.05(s,2H),4.98(p,J=6.3Hz,1H),4.71(d,J=14.1Hz,4H),4.04(t,J=6.6Hz,4H),3.64(s,1H),2.52(t,J=7.7Hz,2H),2.41(d,J=7.7Hz,8H),2.30(tt,J=8.9,5.3Hz,2H),1.91(p,J=7.2Hz,2H),1.58(dt,J=13.4,6.5Hz,12H),1.42(t,J=6.9Hz,4H),1.25(d,J=7.3Hz,48H),0.87(t,J=6.7Hz,12H),核磁氢谱如图21所示。
实施例22:IBMC-035的具体合成步骤:将0.022mmol Bi-150013、0.067mmol EDCI和0.067mmol HOBT溶于5mL二氯甲烷,1h后加入N,N-二甲基丙醇胺0.088mmol、DIEA0.088mmol,室温下搅拌8h,二氯甲烷萃取,饱和氯化钠洗涤,无水硫酸钠干燥,旋干用硅胶柱纯化,得到IBMC-035(8mg,32%)。1H NMR(400MHz,Chloroform-d)δ8.74(t,J=4.8Hz,2H),7.96(s,1H),7.52(s,2H),4.99(t,J=6.2Hz,1H),4.70(s,2H),4.04(t,J=6.6Hz,4H),3.56(q,J=5.6Hz,4H),2.59(t,J=6.1Hz,4H),2.40(s,12H),2.30(ddd,J=8.8,5.3,3.5Hz,2H),1.83(p,J=6.1Hz,4H),1.64–1.19(m,64H),0.87(t,J=6.7Hz,12H),核磁氢谱如图22所示。
Bi-150013的具体合成步骤同实施例1。
实施例23:IBMC-036的具体合成步骤:将0.022mmol Bi-150013、0.067mmol EDCI和0.067mmol HOBT溶于5mL二氯甲烷,1h之后向其中加入吗啉基丙醇0.088mmol、DIEA0.088mmol,室温下搅拌8h,二氯甲烷萃取,饱和氯化钠洗涤,无水硫酸钠干燥,旋干用硅胶柱纯化,得到IBMC-036(14mg,54.73%)。1H NMR(400MHz,Chloroform-d)δ8.06(t,J=4.8Hz,2H),7.87(d,J=1.6Hz,1H),7.52(d,J=1.4Hz,2H),5.04–4.96(m,1H),4.72(s,2H),4.06(dt,J=10.4,6.6Hz,4H),3.74(t,J=4.6Hz,8H),3.57(q,J=5.8Hz,4H),2.53(dt,J=18.6,5.3Hz,12H),2.30(tt,J=8.8,5.4Hz,2H),1.81(q,J=6.1Hz,4H),1.65–1.20(m,64H),0.87(td,J=6.8,1.5Hz,12H),核磁氢谱如图23所示。
Bi-150013的具体合成步骤同实施例1。
实施例24:IBMC-037的具体合成步骤:将0.022mmol Bi-150013、0.067mmol EDCI和0.067mmol HOBT溶于5mL二氯甲烷,1h之后向其中加入0.088mmol DIEA和吗啉基乙醇0.088mmol,室温下搅拌8h,二氯甲烷萃取,饱和氯化钠洗涤,无水硫酸钠干燥,旋干用硅胶柱纯化,得到IBMC-037(14mg,56%)。1H NMR(400MHz,Chloroform-d)δ8.10–8.02(m,2H),7.88(s,1H),7.53(d,J=1.4Hz,2H),5.00(p,J=6.5Hz,1H),4.71(s,2H),4.04(t,J=6.6Hz,4H),3.74(t,J=4.7Hz,8H),3.57(q,J=5.8Hz,4H),2.62–2.43(m,10H),2.30(tt,J=8.8,5.4Hz,2H),1.80(p,J=6.0Hz,4H),1.66–1.20(m,62H),0.87(td,J=6.8,1.5Hz,12H),核磁氢谱如图24所示。
Bi-150013的具体合成步骤同实施例1。
实施例25:IBMC-040的合成路线,如下图所示:
IBMC-040的具体合成步骤:步骤1:Si-H02的合成:将36.2mmol Si-H01(对羟基苯甲酸)、38.0mmol溴化苄和43.4mmol碳酸钠溶于150mL的DMF,在60℃下搅拌5h。二氯甲烷稀释,水洗,饱和氯化钠洗,无水硫酸钠干燥,蒸发浓缩,柱层析分离,得到Si-H02(7.1g,85.9%)。1H NMR(400MHz,Chloroform-d)δ8.00(d,J=8.8Hz,2H),7.48–7.30(m,5H),6.85(d,J=8.8Hz,2H),5.34(s,1H),5.18(s,1H)。
步骤2:Si-H0303的合成:将3.0mmol Si-H02、6.0mmol碳酸铯和3.3mmol T-13-Br溶于100mL的DMF,在80℃下搅拌5h,二氯甲烷萃取,饱和氯化钠洗涤,无水硫酸钠干燥,蒸发浓缩,柱层析分离得到Si-H0303(1.2g,44.6%)。1H NMR(400MHz,Chloroform-d)δ8.01(d,J=8.9Hz,2H),7.46–7.27(m,5H),6.87(d,J=8.9Hz,2H),5.33(s,2H),4.31(p,J=5.8Hz,1H),4.05(t,J=6.6Hz,4H),2.30(tt,J=9.0,5.3Hz,2H),1.76–1.50(m,11H),1.50–1.33(m,9H),1.24(br,42H),0.94–0.77(m,12)。
步骤3:Si-H030003的合成:将2.5mmol Si-H0303、80mg钯碳溶于20mL的四氢呋喃,通入氢气,在25℃下搅拌12h,将反应液用硅藻土过滤,蒸发浓缩得到Si-H030003(450.0mg,67.0%)。1H NMR(400MHz,Chloroform-d)δ8.02(d,J=8.6Hz,2H),6.88(d,J=8.6Hz,2H),4.33(p,J=5.9Hz,1H),4.05(t,J=6.6Hz,4H),2.29(tt,J=8.9,5.3Hz,2H),1.79–1.51(m,8H),1.51–1.32(m,12H),1.25(m,44H),0.94–0.81(m,12H)。
步骤4:IBMC-040的合成:将124.8μmol Si-H030003、282.6μmol N,N-二甲基丙醇胺和374.4μmol DMAP溶于15mL无水DCM中,在25℃下搅拌24h,饱和盐水洗,无水硫酸钠干燥,蒸发浓缩,柱层析分离得到IBMC-040(18mg,10.3%)。1H NMR(400MHz,Chloroform-d)δ8.03–7.89(m,2H),6.92–6.81(m,2H),4.33(dt,J=14.9,6.0Hz,3H),4.05(t,J=6.6Hz,4H),2.60(t,J=7.6Hz,2H),2.39(s,6H),2.32–2.24(m,2H),2.09–1.98(m,2H),1.62(ddq,J=22.2,14.7,8.4,6.9Hz,12H),1.48–1.33(m,12H),1.25(d,J=3.8Hz,40H),0.87(td,J=6.9,2.3Hz,12H),核磁氢谱如图25所示。
T-13-Br的具体合成步骤如下:将3.7mmol四溴化碳和3.7mmol三苯基膦溶于20mL的无水四氢呋喃中,室温下搅拌,加入1.2mmol T-13,升温至45℃反应1h,蒸发浓缩,柱层析分离得到T-13-Br(1.0g,92.9%)。1H NMR(400MHz,Chloroform-d)δ4.07(t,J=6.6Hz,4H),4.00(tt,J=8.0,5.0Hz,1H),2.31(tt,J=8.9,5.3Hz,2H),1.93–1.73(m,4H),1.71–1.52(m,9H),1.51–1.33(m,8H),1.32–1.08(m,42H),0.87(t,J=6.7Hz,12H)。
实施例26:IBMC-041的具体合成步骤与实施例24的区别:采用吗啉基丙醇替代N,N-二甲基丙醇胺。IBMC-041的氢谱:1H NMR(400MHz,Chloroform-d)δ8.00–7.89(m,2H),6.94–6.80(m,2H),4.33(dt,J=14.0,6.1Hz,3H),4.05(t,J=6.6Hz,4H),3.74–3.70(m,4H),2.50(dd,J=13.3,5.9Hz,4H),2.29(tt,J=8.9,5.4Hz,2H),2.00–1.89(m,2H),1.70–1.52(m,12H),1.48–1.33(m,12H),1.30–1.19(m,42H),0.87(td,J=6.9,2.3Hz,12H),核磁氢谱如图26所示。
实施例27:IBMC-042的具体合成步骤与实施例24的区别:采用3-(4-甲基-1-哌嗪基)-1-丙醇替代N,N-二甲基丙醇胺。IBMC-042的氢谱:1H NMR(400MHz,Chloroform-d)δ8.01–7.88(m,2H),6.95–6.78(m,2H),4.32(dt,J=8.8,6.0Hz,3H),4.05(t,J=6.6Hz,4H),2.66–2.44(m,8H),2.33(s,3H),2.32–2.25(m,2H),1.99–1.91(m,2H),1.71–1.52(m,12H),1.48–1.33(m,12H),1.25(d,J=3.8Hz,42H),0.87(td,J=6.9,2.3Hz,12H),核磁氢谱如图27所示。
实施例28:IBMC-043的合成路线,如下图所示:
IBMC-043的具体合成步骤:步骤1:Si-H03的合成:将8.8mmol Si-H02、17.5mmol碳酸铯和9.6mmol N,N-二甲基-3-氯丙胺溶于100mL的DMF,在80℃下搅拌5h,二氯甲烷稀释,水洗,饱和氯化钠洗,无水硫酸钠干燥,蒸发浓缩,柱层析分离,得到Si-H03(1.8g,65.6%)。1HNMR(400MHz,Chloroform-d)δ8.01(d,J=9.0Hz,2H),7.47–7.30(m,5H),6.91(d,J=8.9Hz,2H),5.33(s,2H),4.07(t,J=6.4Hz,2H),2.46(t,J=7.2Hz,2H),2.26(s,4H),1.98(p,2H)。
步骤2:Si-H04的合成:将2.55mmol Si-H03、80mg钯碳溶于20mL的甲醇,通入氢气,在25℃下搅拌12h,蒸发浓缩得到Si-H04(550mg,96.5%)。1H NMR(400MHz,Methanol-d4)δ7.87(d,J=8.8Hz,2H),6.89(d,J=8.8Hz,2H),4.13(t,J=5.9Hz,2H),3.14(t,J=7.7Hz,2H),2.77(s,6H),2.16(p,2H)。
步骤3:IBMC-043的合成:将223.9μmol Si-H04、268.7μmol T-13、335.9μmol EDCI和671.8μmol DMAP溶于15mL的DCM中,在30℃下搅拌24h,将反应液采用饱和氯化钠洗涤,无水硫酸钠干燥,蒸发浓缩,柱层析得到IBMC-043(40mg,23.1%)。1H NMR(400MHz,Chloroform-d)δ7.97(d,J=8.9Hz,2H),6.91(d,J=8.9Hz,2H),5.10(dq,J=9.8,3.7,2.4Hz,1H),4.08(t,J=6.3Hz,2H),4.04(t,J=6.6Hz,4H),3.75(t,J=4.7Hz,2H),2.53(br,6H),2.29(tt,J=9.0,5.4Hz,2H),2.02(br,2H),1.73–1.47(m,8H),1.48–1.31(m,12H),1.24(s,44H),0.90–0.83(m,12H),核磁氢谱如图28所示。
实施例29:
IBMC-044的具体合成步骤与实施例28的区别:采用T-11替代T-13。IBMC-044的氢谱:1H NMR(400MHz,Chloroform-d)δ2.13(s,6H),2.29(t,2H),1.85(m,2H),3.97-4.05(t,6H),7.05-7.93(dd,4H),4.42(m,1H),2.15(m,1H),2.29(t,2H),1.29-1.63(m,52H),0.93(t,9H),核磁氢谱如图29所示。
实施例30:IBMC-048的合成路线,如下图所示:
IBMC-048的合成步骤同实施例29的区别:
1)Si-H05的合成方法与Si-H03的区别:采用吗啉基丙醇替代N,N-二甲基-3-氯丙胺;
2)Si-H06的合成方法与Si-H04的区别:采用Si-H05替代Si-H03。
IBMC-048的氢谱:1H NMR(400MHz,Chloroform-d)δ7.97(d,J=8.9Hz,2H),6.91(d,J=8.9Hz,2H),5.19–4.98(m,1H),4.08(t,J=6.3Hz,2H),4.04(t,J=6.6Hz,4H),3.84–3.59(m,4H),2.53(br,6H),2.02(br,2H),1.72–1.50(m,12H),1.47–1.32(m,12H),1.24(m,40H),0.92–0.81(m,12H),核磁氢谱如图30所示。
实施例31:IBMC-051的合成路线,如下图所示:
IBMC-051的具体合成步骤:
步骤1:Bi-070813的合成:将0.277mmol Bi-070013溶于10mL二氯甲烷,依次加入0.138mmol C3Y09,4-二甲氨基吡啶0.415mmol,1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐0.166mmol,室温反应12h,饱和氯化钠溶液洗涤,无水硫酸钠干燥后旋干,用硅胶柱纯化,得到目标产物Bi-070813(71mg,45.5%)。1H NMR(400MHz,Chloroform-d)δ6.93(s,1H),6.80(dt,J=13.8,2.1Hz,2H),5.02(s,2H),5.01–4.91(m,1H),4.59(d,J=14.8Hz,4H),3.98(t,J=6.7Hz,4H),3.64(t,J=6.5Hz,2H),2.45(t,J=6.5Hz,2H),2.39–2.21(m,6H),2.20(s,3H),1.75(p,J=7.4Hz,2H),1.61–1.11(m,64H),0.83(m,18H)。
步骤2:IBMC-051的合成:将0.075mmol Bi-070813溶于4mL二氯甲烷中,滴加1.52mL的饱和浓盐酸,反应12h,旋干用硅胶柱纯化,得到目标产物IBMC-051(23mg,30.1%)。1H NMR(400MHz,Chloroform-d)δ6.94(s,1H),6.77(dt,J=10.1,2.2Hz,2H),5.01(s,2H),4.92(td,J=7.2,3.6Hz,1H),4.57(d,J=10.8Hz,4H),3.97(t,J=6.7Hz,4H),3.60–3.44(m,2H),2.56–2.47(m,2H),2.45(t,J=7.1Hz,2H),2.35(t,J=7.0Hz,2H),2.29–2.19(m,5H),1.81(p,J=7.0Hz,2H),1.58–1.10(m,64H),0.80(t,J=6.7Hz,12H),核磁氢谱如图31所示。
Bi-070013的具体合成步骤同实施例4。
C3Y09的合成路线,如下图所示:
C3Y09的具体合成步骤:步骤1:C3-2的合成:将6.52mmol C3-1(4-(甲基氨基)丁酸甲酯盐酸盐)和19.5mmol碳酸钾溶于40mL的乙腈,升温至50℃,加入(2-溴乙氧基)-叔丁基二甲基硅烷8.5mmol后,升至80℃回流5h,反应完全后,旋干溶剂,过柱纯化得C3-2(1.25g,66.4%)。1H NMR(400MHz,Chloroform-d)δ3.68(td,J=6.6,1.3Hz,2H),3.65(d,J=1.4Hz,3H),2.50(td,J=6.6,1.4Hz,2H),2.40(dd,J=7.9,6.5Hz,2H),2.33(td,J=7.5,1.4Hz,2H),2.25(d,J=1.4Hz,3H),1.77(pd,J=7.4,1.3Hz,2H),0.88(d,J=1.4Hz,9H),0.06(d,J=1.2Hz,6H)。
步骤2:C3Y09的合成:将1.72mmol C3-2和5.2mmol氢氧化钠溶于10mL的甲醇和5mL的水,室温搅拌3h,反应完全后,旋蒸除掉甲醇,乙酸乙酯洗涤,水相用稀盐酸酸化,乙酸乙酯萃取,无水硫酸钠干燥,旋干得C3Y09(0.35g,74%)。1H NMR(400MHz,Chloroform-d)δ3.93–3.82(m,2H),2.83(qd,J=5.4,3.1Hz,4H),2.64–2.56(m,2H),2.53(s,3H),1.87–1.77(m,2H),0.89(s,9H),0.07(d,J=2.7Hz,6H)。
实施例32:IBMC-052的具体合成步骤:将0.125mmol Bi-070013溶于10mL二氯甲烷,依次加入N,N-二甲基氨基丁酸盐酸盐0.498mmol,4-二甲氨基吡啶0.747mmol,1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐0.597mmol,室温反应12h,饱和氯化钠溶液洗涤,无水硫酸钠干燥后旋干,用硅胶柱纯化,得到目标产物IBMC-052(40mg,33.8%)。1H NMR(400MHz,Chloroform-d)δ6.89(d,J=1.7Hz,1H),6.79(d,J=1.4Hz,2H),5.00(s,4H),4.93(td,J=7.0,3.5Hz,1H),4.54(s,2H),3.98(t,J=6.6Hz,4H),2.34(t,J=7.4Hz,4H),2.30–2.25(m,4H),2.24–2.21(m,2H),2.18(s,12H),1.78(h,J=7.4,6.9Hz,4H),1.59–1.08(m,64H),0.83–0.77(m,12H),核磁氢谱如图32所示。
Bi-070013的具体合成步骤同实施例4。
实施例33:IBMC-053的合成路线,如下图所示:
IBMC-053的具体合成步骤:步骤1:JY-02的合成:将2.43mmol JY-01(2-(4-甲氧基苯基)丙二酸-1,3-二甲酯)溶解在10mL无水DCM中,-20℃条件下加入三溴化硼2.92mmol并搅拌过夜,将反应物用冰水混合物淬灭后用DCM洗涤萃取三次,合并有机相,用无水Na2SO4干燥,柱层析得到JY-02(0.84g,90.0%)。1H NMR(400MHz,CDCl3)δ(ppm)7.27(d,J=8.6Hz,2H),6.81(d,J=8.6Hz,2H),4.58(s,1H),3.75(s,6H)。
步骤2:JY-03的合成:将2.85mmol JY-02溶解在15mL的乙腈中,加入4.28mmolCs2CO3、3.43mmol(3-溴丙基)二甲基胺后,于60℃搅拌反应12h,反应结束后,过滤,旋干,柱层析得到JY-03(0.31g,35.0%)。1H NMR(400MHz,CDCl3)δ(ppm)7.16(d,J=8.6Hz,2H),6.87(d,J=8.6Hz,2H),4.12(t,J=6.1Hz,2H),4.02-3.99(m,4H),3.48(s,1H),3.33(br,12H)。
步骤3:IBMC-053的合成:将1.21mmol JY-03溶解在5mL的EtOH中,冰浴下加入7.26mmol NaBH4,室温搅拌反应6h,反应结束后,加饱和NH4Cl水溶液淬灭反应,用DCM萃取3次,旋干后用二氯甲烷溶解,依次加入3.02mmol ROH、0.84mmol EDC、0.84mmol DMAP室温搅拌反应24h,反应结束后,过滤,旋干,柱层析得到IBMC-053(367.0mg,39.0%)。1H NMR(400MHz,CDCl3)δ(ppm)7.16-7.12(m,2H),6.87-6.82(m,2H),5.42-5.28(m,8H),3.81-3.78(m,7H),2.77(t,J=6.4Hz,4H),2.12-2.02(m,8H),1.58(br,4H),1.37-1.23(m,42H),0.89(t,J=6.8Hz,6H),核磁氢谱如图33所示。
实施例34:IBMC-054的具体合成步骤:将223.9μmol Si-H04、268.7μmol T-08、335.9μmol EDCI和671.8μmol DMAP溶于15mL的DCM中,在30℃下搅拌24h,饱和NaCl洗涤,无水硫酸钠干燥,蒸发浓缩,柱层析分离,得到IBMC-054(30mg,18.3%)。1H NMR(400MHz,Chloroform-d)δ7.98(d,J=8.5Hz,2H),6.90(d,J=8.5Hz,2H),5.47–5.24(m,8H),5.08(q,J=6.2Hz,1H),4.08(t,J=6.2Hz,2H),2.76(t,J=6.5Hz,2H),2.62(t,J=7.4Hz,2H),2.37(s,6H),2.12–1.95(m,8H),1.76–1.50(m,4H),1.45–1.15(m,40H),0.95–0.76(m,6H),核磁氢谱如图34所示。
Si-H04的具体合成步骤同实施例28。
T-08的具体合成步骤同实施例3。
实施例35:IBMC-055的具体合成步骤与实施例34的区别:采用吗啉基丙醇替代N,N-二甲基-3-氯丙胺。IBMC-055的氢谱:1H NMR(400MHz,Chloroform-d)δ7.98(d,J=8.5Hz,2H),6.90(d,J=8.7Hz,2H),5.34(tq,J=10.9,6.7,5.8Hz,8H),5.08(p,J=6.0Hz,1H),4.08(t,J=6.2Hz,2H),3.82–3.62(m,4H),2.76(t,J=6.4Hz,2H),2.62–2.47(m,4H),2.16–1.90(m,8H),1.74–1.52(m,4H),1.41–1.16(m,40H),0.88(t,J=6.7Hz,6H),核磁氢谱如图35所示。
实施例36:IBMC-056的具体合成步骤与实施例3的区别:采用吗啉基丙醇替代N-甲基二乙醇胺。IBMC-056的氢谱:1H NMR(400MHz,Chloroform-d)δ8.29(t,J=1.4Hz,1H),7.76(d,J=1.4Hz,2H),5.00(p,J=6.2Hz,1H),4.70(s,2H),4.40(t,J=6.6Hz,4H),3.75–3.68(m,8H),2.55–2.36(m,12H),1.96(p,J=6.8Hz,4H),1.24(q,J=3.5,2.8Hz,68H),0.90–0.85(m,6H),核磁氢谱如图36所示。
实施例37:IBMC-057的具体合成步骤与实施例3的区别:采用N,N-二甲基丙醇胺替代N-甲基二乙醇胺。IBMC-057的氢谱:1H NMR(400MHz,Chloroform-d)δ8.31(d,J=1.5Hz,1H),7.77(d,J=1.4Hz,2H),5.01(q,J=6.1Hz,1H),4.70(s,2H),4.39(t,J=6.6Hz,4H),2.44(t,J=7.3Hz,4H),2.27(s,12H),1.97(h,J=6.8,6.0Hz,4H),1.54(t,J=6.1Hz,4H),1.24(d,J=5.4Hz,64H),0.88(t,J=6.8Hz,6H),核磁氢谱如图37所示。
实施例38:IBMC-058的具体合成步骤与实施例3的区别:采用1,4-双(2-羟乙基)哌嗪替代N-甲基二乙醇胺。IBMC-058的氢谱:1H NMR(400MHz,Chloroform-d)δ8.29(t,J=1.5Hz,1H),7.77(d,J=1.5Hz,2H),5.00(p,J=6.2Hz,1H),4.70(s,2H),4.47(t,J=5.9Hz,4H),3.66–3.58(m,4H),2.78(q,J=7.0,6.5Hz,4H),2.54(d,J=5.3Hz,20H),1.25(d,J=4.9Hz,68H),0.90–0.85(m,6H),核磁氢谱如图38所示。
实施例39:IBMC-059的具体合成步骤:将0.23mmol Bi-070013溶于10mL二氯甲烷,依次加入0.11mmol N,N-二甲基氨基丙酸盐酸盐、0.34mmol 4-二甲氨基吡啶、0.14mmol 1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐,室温反应12h,饱和氯化钠溶液洗涤,无水硫酸钠干燥后旋干,用硅胶柱纯化,得到目标产物IBMC-059(60mg,54.1%)。1H NMR(400MHz,Chloroform-d)δ6.97(s,1H),6.86(dt,J=13.6,2.2Hz,2H),5.09(s,2H),5.02–4.97(m,1H),4.64(d,J=16.0Hz,4H),4.03(t,J=6.7Hz,4H),2.69(t,J=7.1Hz,2H),2.57(t,J=6.9Hz,2H),2.34–2.24(m,8H),1.62–1.52(m,12H),1.42(ddd,J=13.6,7.2,3.4Hz,4H),1.25(s,48H),0.89–0.85(m,12H),核磁氢谱如图39所示。
Bi-070013的具体合成步骤同实施例4。
实施例40:IBMC-060的具体合成步骤:将0.23mmol Bi-070013溶于10mL二氯甲烷,依次加入0.11mmol N,N-二甲基氨基戊酸盐酸盐、0.34mmol 4-二甲氨基吡啶、0.14mmol 1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐,室温反应12h,饱和氯化钠溶液洗涤,无水硫酸钠干燥后旋干,用硅胶柱纯化,得到目标产物IBMC-060(100mg,53.6%)。1H NMR(400MHz,Chloroform-d)δ7.02(s,1H),6.80(dt,J=13.9,2.1Hz,2H),5.06(s,2H),4.98(td,J=7.2,3.6Hz,1H),4.62(d,J=12.7Hz,4H),4.02(t,J=6.7Hz,4H),2.79–2.73(m,2H),2.57(s,6H),2.41(t,J=6.3Hz,2H),2.29(ddd,J=8.9,7.1,4.5Hz,2H),1.72–1.68(m,2H),1.61–1.51(m,12H),1.44–1.38(m,4H),1.24(d,J=7.9Hz,48H),0.85(t,J=6.7Hz,12H),核磁氢谱如图40所示。
Bi-070013的具体合成步骤同实施例4。
实施例41:IBMC-061的具体合成步骤:将0.23mmol Bi-070013溶于10mL二氯甲烷,依次加入0.11mmol 1-哌啶丙酸、0.34mmol 4-二甲氨基吡啶、0.14mmo 1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐,室温反应12h,饱和氯化钠溶液洗涤,无水硫酸钠干燥后旋干,用硅胶柱纯化,得到目标产物IBMC-061(100mg,53.6%)。1H NMR(400MHz,Chloroform-d)δ6.97(s,1H),6.85(d,J=18.1Hz,2H),5.07(s,2H),5.00(p,J=6.1Hz,1H),4.64(d,J=14.3Hz,4H),4.04(t,J=6.7Hz,4H),2.72(t,J=7.4Hz,2H),2.59(t,J=7.4Hz,2H),2.49–2.37(m,4H),2.30(tt,J=9.2,5.3Hz,2H),1.64–1.52(m,J=5.5,5.0Hz,16H),1.43(p,J=6.8Hz,6H),1.33–1.20(m,48H),0.87(t,J=6.5Hz,12H),核磁氢谱如图41所示。
Bi-070013的具体合成步骤同实施例4。
实施例42:IBMC-062的具体合成步骤:将0.23mmol Bi-070013溶于10mL二氯甲烷,依次加入0.11mmol 3-吡咯烷-1-基丙酸、0.34mmol 4-二甲氨基吡啶、0.14mmol 1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐,室温反应12h,饱和氯化钠溶液洗涤,无水硫酸钠干燥后旋干,用硅胶柱纯化,得到目标产物IBMC-062(100mg,53.6%)。1H NMR(400MHz,Chloroform-d)δ6.98(s,1H),6.85(dt,J=16.9,2.1Hz,2H),5.09(s,2H),5.00(tt,J=7.2,3.6Hz,1H),4.65(d,J=15.6Hz,4H),4.03(t,J=6.7Hz,4H),2.96(t,J=7.5Hz,2H),2.78–2.63(m,6H),2.30(td,J=8.9,4.5Hz,2H),1.91–1.82(m,4H),1.63–1.52(m,12H),1.47–1.39(m,4H),1.33–1.21(m,48H),0.87(t,J=6.7Hz,12H),核磁氢谱如图42所示。
Bi-070013的具体合成步骤同实施例4。
实施例43:IBMC-066的具体合成步骤:将0.23mmol Bi-070013溶于10mL二氯甲烷,依次加入0.11mmol 4-(氮杂环庚烷-1-基)丁酸、0.34mmol 4-二甲氨基吡啶、0.14mmol 1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐,室温反应12h,饱和氯化钠溶液洗涤,无水硫酸钠干燥后旋干,用硅胶柱纯化,得到目标产物IBMC-066(100mg,53.6%)。1H NMR(400MHz,Chloroform-d)δ7.04(s,1H),6.82(dt,J=16.6,2.2Hz,2H),5.08(s,2H),5.01–4.95(m,1H),4.64(d,J=19.0Hz,4H),4.03(t,J=6.6Hz,4H),3.11(s,4H),2.97–2.89(m,2H),2.48(t,J=6.6Hz,2H),2.30(tt,J=8.9,5.3Hz,2H),2.16(dq,J=14.1,6.7Hz,2H),1.92(s,4H),1.70(s,4H),1.57(dq,J=14.3,6.6Hz,12H),1.42(td,J=8.4,7.8,4.6Hz,4H),1.26(d,J=14.1Hz,48H),0.86(t,J=6.7Hz,12H),核磁氢谱如图43所示。
Bi-070013的具体合成步骤同实施例4。
实施例44:脂质纳米粒子组合物(LNP制剂)的制备和测试
为了制备将治疗剂和/或预防剂递送至细胞的纳米粒子组合物,制备测试了一系列纳米粒子配制物,并且对纳米粒子组合物各脂质组分进行了优化。纳米粒子可以通过手动和微流控的方法制备,这些方法都不是穷举,只要是能制备本发明配方的纳米粒子组合物,都在本发明的保护范围内。
将通式(I)、(Ia)、(Ib)、(Ic)、(Ⅱ)、(Ⅱa)、(Ⅱb)、(Ⅱc)、(Ⅱd)、(Ⅱe)、(Ⅱf)、(Ⅱg)、(Ⅱh)、(Ⅲ)、(Ⅲa)、(Ⅲb)、(Ⅲc)所代表的脂质分子、中性脂质(如DSPC,艾伟拓(上海)医药科技有限公司)、甾族化合物(如胆固醇,艾伟拓(上海)医药科技有限公司)和聚合物缀合的脂质(如DMG-PEG2000,艾伟拓(上海)医药科技有限公司)按照摩尔百分比(脂质分子:DSPC:胆固醇:DMG-PEG2000=20%-100%:0%-40%:0%-80%:0%-20%)配制脂质组合物。这个摩尔百分比范围的配方都可以制备纳米粒子组合物,只要是通过这样的配方得到的LNP,均在本发明的保护范围内。
本实施例中,上述脂质组分按50:10:38.5:1.5的摩尔百分比制备总浓度为50mM的乙醇相溶液备用。脂质DLin-MC3-DMA(MC3)是本领域当前的标准品,因此,使用MC3:DSPC:胆固醇:DMG-PEG2000=50:10:38.5:1.5的摩尔百分比制备标准MC3纳米粒子组合物作为本研究的对照。
将活性成分(如mRNA,EGFP、Luciferase或SARS-CoV2 Spike)加到10-50mM缓冲液(柠檬酸盐、醋酸盐,pH=3~6)中制备mRNA水相溶液备用。通过使用手动或微流控装置将乙醇相脂质溶液和水相mRNA溶液混合,制备出纳米粒子组合物。其中水相和乙醇相的体积比在1:1至5:1之间,本实施例设定体积比为3:1;其中总脂质与mRNA的质量比在5~65:1之间(或按脂质分子与mRNA的N/P在4~12:1),本实施例分别设定三个N/P,为8:1、6:1、4:1,得到三组实验数据;其中手动操作为快速注入后涡旋30-60s,本实施例设定涡旋30s;其中微流控系统总液体流速10~25mL/min之间,本实施例设定总液体流速为20mL/min。
纳米粒子组合物通过透析纯化,将纳米粒子组合物溶液通过多次DPBS透析除去乙醇和游离分子,再用0.2μm无菌过滤器过滤,获得包封mRNA的脂质纳米粒子组合物(LNP制剂)。
纳米粒子组合物的测试,使用Malvern Zetasizer Nano ZS ZEN3600(MalvernUK)通过动态光散射测定脂质纳米粒子组合物的粒径大小、多分散指数(PDI)和电位,使用RNA定量试剂盒Quant-iTTM RiboGreenTM RNA Assay Kit(Thermo Fisher Scientific)评价纳米粒子组合物对mRNA的包封率。
如表1A-1C中所示,比较根据通式(I)、(Ia)、(Ib)、(Ic)、(Ⅱ)、(Ⅱa)、(Ⅱb)、(Ⅱc)、(Ⅱd)、(Ⅱe)、(Ⅱf)、(Ⅱg)、(Ⅱh)、(Ⅲ)、(Ⅲa)、(Ⅲb)、(Ⅲc)代表的脂质分子的纳米粒子组合物包载mRNA(EGFP和/或Luciferase、SARS-CoV2 Spike)的粒径、PDI、电位和包封率。
表1A包载供细胞转染的纳米粒子组合物理化性质参数,其中mRNA为EGFP和Luciferase等质量比例混合
/>
#=脂质分子与mRNA的N/P为6
表1B包载供动物施用的纳米粒子组合物理化性质参数,其中mRNA为Luciferase
脂质分子 | 粒径(nm) | PDI | 电位(mV) | 包封率% |
MC3#N/P=6 | 161.13 | 0.18 | -4.35 | 94.3 |
MC3*N/P=6 | 121.97 | 0.20 | -7.53 | 96.4 |
SM-102#N/P=6 | 168.90 | 0.20 | -5.29 | 95.8 |
SM-102*N/P=6 | 91.80 | 0.15 | -2.61 | 97.2 |
IBMC-023#N/P=6 | 147.23 | 0.23 | -3.61 | 93.2 |
IBMC-023*N/P=6 | 97.33 | 0.20 | -1.41 | 96.7 |
IBMC-023#N/P=8 | 157.23 | 0.22 | -2.01 | 95.4 |
IBMC-023#N/P=4 | 157.43 | 0.24 | -2.52 | 95.3 |
#=手动操作制备纳米粒子组合物;*=微流控制备纳米粒子混合物
表1C包载供动物施用的纳米粒子组合物理化性质参数,其中mRNA为SARS-CoV2Spike
#=手动操作制备纳米粒子组合物;*=微流控制备纳米粒子混合物
测试结果如表1A、1B、1C所示,由表1A、1B、1C可知,纳米粒子组合物对mRNA的包封率与上市产品的相差不大,说明本产品制备的纳米粒子组合物达到上市要求。
实施例45:脂质纳米粒子组合物(LNP制剂)细胞转染效果评估
在96孔板中,每孔铺2×104个293T或者Hela细胞,培养24h后,待细胞汇合度为70-90%,以每孔0.1μg EGFP和0.1μg Luciferase的混合mRNA剂量的脂质纳米粒子组合物孵育细胞,24h后,通过奥林巴斯CKX53荧光显微镜拍摄EGFP的20X荧光图像。
测试结果如图44、45、46所示,由图44、45、46可知,在细胞水平上,新脂质分子可以有效将mRNA递送至细胞并且表达,并且筛选出的部分脂材优于已上市的脂质分子MC3。
实施例46:脂质纳米粒子组合物(LNP制剂)体内递送水平评估
以0.1mg/kg的剂量对6-8周龄雌性Babl/c小鼠通过肌肉或静脉注射的方式施用包载Luciferase mRNA的纳米粒子配制物,并在给药后特定的时间节点通过IVIS Lumina III(PE公司)进行小动物荧光成像(3h、6h和24h)。
测试结果如图47、48所示,由图47、48可知,脂质分子IBMC-023的纳米粒子组合物可以在动物体内有效递送mRNA,并且高水平地表达相关蛋白;同时,相比MC3,IBMC-023没有观察到明显的肝脏富集现象。
实施例47:脂质纳米粒子组合物(LNP制剂)体内递送SARS-CoV2 Spike诱导S蛋白表达水平评估
以0.5mg/kg的剂量对6-8周龄雌性Babl/c小鼠通过肌肉注射的方式施用包载SARS-CoV2 Spike mRNA的纳米粒子配制物,并在给药后6h根据商品化的SARS-CoV-2(2019-nCoV)SpikeELISA试剂盒(义翘神州,KIT40591)的步骤分析小鼠血液、肌肉、肝脏中S蛋白的表达量。
测试结果如图49、50、51所示,由图49、50、51可知,脂质分子IBMC-023的纳米粒子组合物可以在动物体内有效递送mRNA,并且高水平地表达相关蛋白。
实验证明,本发明的阳离子脂质化合物能够递送核酸分子、小分子化合物、多肽或蛋白质等,使用本发明的阳离子脂质化合物制备的载体对核酸分子的包封效率高,可将核酸分子成功转运至细胞和/或器官中,并且高效进行表达。
上述实施例中的常规技术为本领域技术人员所知晓的现有技术,故在此不再详细赘述。以上所述,仅为本发明的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到变化或替换,都应涵盖在本发明的保护范围之内。因此,本发明的保护范围应以所述权利要求的保护范围为准。
Claims (8)
1.一种脂质分子在制备药物中的用途,所述脂质分子用于递送活性成分,所述脂质分子包含:通式(Ⅱ)代表的脂质化合物,或其药物可用的盐中的至少一种;
其中,
M选自苯环;
G1各独立地选自-(CH2)x-O(C=O)-、-(CH2)x-(C=O)O-、-(CH2)x-(C=O)S-、-(CH2)x-(C=O)NH-、-(CH2)x-O-、-(CH2)x-O(C=O)NH-、-(CH2)x-O(C=O)O-、-(CH2)xNH(C=O)-中的一种,其中x为0至4之间的整数;
L1各独立地选自未取代的C1-6烷基中的一种;
G2选自-(CH2)0-3-、-O-(CH2)y-(C=O)O-、-(CH2)y-(C=O)O-、-(CH2)y-(C=O)NH-、-S-
(CH2)y-(C=O)O-、-(CH2)y-(C=O)S-、-S-、-O-中的一种,其中y为0至4之间的整数;
G1、G2各自独立地与M中的任意位点连接,所述位点为碳或者氮原子;
X选自碳或者氮原子;n选自0至6之间的整数;
L2选自H、OH、C1-3烷基、C2-3烯基中的一种;
L3、L4各独立地选自C0-25烷基、C2-25烯基、C3-25炔基中的一种;
G3、G4各独立地选自-CH2-、-O(C=O)-、-(C=O)O-、-O(C=O)O-、-(C=O)NH-、-NH(C=O)-、-S(C=O)-、-(C=O)S-、-S-S-中的一种;
L5、L6各独立地选自C1-25烷基、C2-25烯基、C3-25炔基中的一种;
R1、R2各独立地选自任意取代或未取代的C1-6烷基、C2-6烯基、C2-6炔基、C3-8环烷基、C3-8环烯基、C3-8环炔基、苯基、-(C=O)C1-3烷基、中的一种,其中取代基团为1或2或3或4或5个独立的OH、SH、硝基、氰基、氨基、C1-3羟基、C1-3烷氧基、-(C=O)OC1-3烷基、C1-3烷基;X1、X2各独立地选自C1-3烷基;
或者
R1和R2结合起来形成任意取代或未取代的4-8元杂环、嘧啶环、嘌呤环;其中取代基团为1或2或3或4或5个独立的OH、SH、硝基、氰基、氨基、C1-3羟基、C1-3烷氧基、-(C=O)OC1-3烷基、C1-3烷基;
式(Ⅱ)中的Z选自H、F、-OH、-SH-、-NH2、-CF3、-NH-(CH2)rCH3、-N(CH3)-(CH2)rCH3中的一种,其中r为0至4之间的整数。
2.根据权利要求1所述的一种脂质分子在制备药物中的用途,其特征在于:所述通式(Ⅱ)的脂质化合物包含式(Ⅱa)、(Ⅱb)、(Ⅱc)、(Ⅱd)、(Ⅱe)、(Ⅱf)、(Ⅱg)或(Ⅱh)所示结构,或其药物可用的盐;
其中,
G1选自-(CH2)x-O(C=O)-、-(CH2)x-(C=O)O-、-(CH2)x-(C=O)S-、-(CH2)x-(C=O)NH-、-
(CH2)x-O-、-(CH2)x-O(C=O)NH-、-(CH2)x-O(C=O)O-、-(CH2)xNH(C=O)-中的一种,其中x为0至4之间的整数;
L1选自未取代的C1-6烷基中的一种;G1与苯环中的任意位点连接;
R1、R2各独立地选自任意取代或未取代的C1-6烷基、C2-6烯基、C2-6炔基、C3-8环烷基、C3-8环烯基、C3-8环炔基、苯基、-(C=O)C1-3烷基、中的一种,其中取代基团为1或2或3或4或5个独立的OH、SH、硝基、氰基、氨基、C1-3羟基、C1-3烷氧基、-(C=O)OC1-3烷基、C1-3烷基;X1、X2各独立地选自C1-3烷基;
或者
R1和R2结合起来形成任意取代或未取代的4-8元杂环、嘧啶环、嘌呤环;其中取代基团为1或2或3或4或5个独立的OH、SH、硝基、氰基、氨基、C1-3羟基、C1-3烷氧基、-(C=O)OC1-3烷基、C1-3烷基;
E选自氧或者硫原子;
m为0至4之间的整数;
T包含式(5)或式(7)-(18)所示结构中的一种:
3.根据权利要求1所述的一种脂质分子在制备药物中的用途,其特征在于:所述和/>各独立地选自式Y01-Y30所示结构中的一种:
4.根据权利要求1所述的一种脂质分子在制备药物中的用途,其特征在于:所述脂质化合物在制备纳米粒子组合物中的用途。
5.根据权利要求4所述的一种脂质分子在制备药物中的用途,其特征在于:所述纳米粒子组合物还包含治疗剂和/或预防剂。
6.根据权利要求5所述的一种脂质分子在制备药物中的用途,其特征在于:所述治疗剂和/或预防剂包含核酸、小分子化合物、多肽或蛋白质;所述核酸包含单链DNA、双链DNA、短异构体、agomir、antagomir、反义分子、小干扰RNA、不对称干扰RNA、microRNA、Dicer-substrate RNA、小发夹RNA、转运RNA、信使RNA、环状RNA、核酸适体中的至少一种。
7.根据权利要求4所述的一种脂质分子在制备药物中的用途,其特征在于:所述纳米粒子组合物还包含一种或多种中性脂质、一种或多种甾族化合物、一种或多种聚合物缀合的脂质;其中,权利要求1所述用于递送活性成分的脂质分子摩尔百分比为20-100%;所述甾族化合物摩尔百分比为0-80%;所述中性脂质摩尔百分比为0-40%;所述聚合物缀合的脂质摩尔百分比为0-20%。
8.根据权利要求4所述的一种脂质分子在制备药物中的用途,其特征在于:所述纳米粒子组合物在药物制备中的应用。
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