CN115381844B - 一种细菌荚膜寡糖衍生物及其制备方法、药物组合物和用途 - Google Patents
一种细菌荚膜寡糖衍生物及其制备方法、药物组合物和用途 Download PDFInfo
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- CN115381844B CN115381844B CN202211201644.8A CN202211201644A CN115381844B CN 115381844 B CN115381844 B CN 115381844B CN 202211201644 A CN202211201644 A CN 202211201644A CN 115381844 B CN115381844 B CN 115381844B
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- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/02—Acyclic radicals, not substituted by cyclic structures
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
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Abstract
本申请公开了一种细菌荚膜寡糖衍生物及其制备方法、药物组合物和用途。所述的衍生物如式(I)所示,取代基详见说明书;该衍生物具有抗炎活性,可用于治疗脓毒症。
Description
技术领域
本文涉及生物医药技术,尤指一种细菌荚膜寡糖衍生物及其制备方法、药物组合物和用途。
背景技术
Colwellia psychrerythraea 34H细菌是一种从北极海洋沉积物中分离得到的嗜冷菌,其荚膜多糖能够通过模拟抗冻蛋白,具有良好的抗冻活性,从而适应低温环境。2015年,Colwellia psychrerythraea 34H细菌的荚膜多糖结构被报道,它是一种带有氨基酸修饰的糖胺聚糖,主链由→4)-β-D-GlcA-(1→3)-β-D-GlcNAc-(1→2)-α-D-GalA-(1→3)-β-D-GalNAc-(1→四糖重复单元组成,并在半乳糖醛酸的羧基处通过酰胺键连有一个L-苏氨酸(Sara Carillo,et al.,A Unique Capsular Polysaccharide Structure from thePsychrophilic Marine Bacterium Colwellia psychrerythraea 34H That MimicsAntifreeze(Glyco)proteins.J.Am.Chem.Soc.2015,137,179)。该四糖的非还原端二糖为透明质酸二糖,而还原端的二糖则是以前从未见报道的新结构。而该聚糖除了抗冻活性外,未见其它活性的报道。
2019年,发现该聚糖结构的课题组报道了其四糖苏氨酸单元的合成工作。在其报道的合成策略中,从四种单糖砌块出发,通过保护基操作和糖基化进行寡糖的组装,合成荚膜多糖寡糖片段,从单糖砌块算起共超过40步反应,总收率低于0.028%(Giulia Vessellaet al.,Synthesis of the tetrasaccharide repeating unit of the cryoprotectantcapsular polysaccharide from Colwellia psychrerythraea 34H.Org.Biomol.Chem.,2019,17,3129)。因此,开发新的荚膜多糖寡糖片段的化学合成方法对促进其药物化学研究具有重要意义。
发明内容
以下是对本文详细描述的主题的概述。本概述并非是为了限制权利要求的保护范围。
一方面,本申请提供了一种细菌荚膜寡糖衍生物或其药学上可接受的盐、溶剂化物、前药作为抗炎药物的用途,所述的衍生物如式I所示:
其中,式(I)中R1为OH、未取代或取代的C1-C6烷氧基、未取代得或取代的C2-C6烯烃氧基、未取代得或取代的C2-C6炔氧基、未取代或取代的C1-C6烷硫基、未取代的或取代的C1-C6烷酰氧基、或者未取代的或取代的芳烃氧基;
R2为OH、-N(H)-R15、N(R16)-R17、未取代或取代的C1-C6烷氧基、或者未取代的或取代的芳烃氧基,这里,R15为不包括脯氨酸的氨基酸残基;R16和R17一起形成脯氨酸残基;
R3和R4各自独立地为未取代的或取代的C1-C6烷酰基;
R5、R6、R7、R8、R9、R10、R11、R12和R13各自独立地为氢、或者未取代的或取代的C1-C6烷酰基、或者未取代的或取代的C1-C6烷基;
R14为OH、未取代或取代的C1-C6烷氧基、或者未取代的或取代的芳烃氧基。
第二方面,本申请提供了一种预防或治疗炎症的方法,所述的方法包括对需要的个体施用治疗上有效量的细菌荚膜寡糖衍生物或其药学上可接受的盐、溶剂化物、前药,所述的衍生物如式I所示:
其中,式(I)中R1为OH、未取代或取代的C1-C6烷氧基、未取代得或取代的C2-C6烯烃氧基、未取代得或取代的C2-C6炔氧基、未取代或取代的C1-C6烷硫基、未取代的或取代的C1-C6烷酰氧基、或者未取代的或取代的芳烃氧基;
R2为OH、-N(H)-R15、N(R16)-R17、未取代或取代的C1-C6烷氧基、或者未取代的或取代的芳烃氧基,这里,R15为不包括脯氨酸的氨基酸残基;R16和R17一起形成脯氨酸残基;
R3和R4各自独立地为未取代的或取代的C1-C6烷酰基;
R5、R6、R7、R8、R9、R10、R11、R12和R13各自独立地为氢、或者未取代的或取代的C1-C6烷酰基、或者未取代的或取代的C1-C6烷基;
R14为OH、未取代或取代的C1-C6烷氧基、或者未取代的或取代的芳烃氧基。
第三方面,本申请提供了上述细菌荚膜寡糖衍生物的制备方法,所述的制备方法包括如下步骤:
式(I-13)化合物与式(I-18)化合物反应,得到化合物(I-19)
这里,化合物式(I-13)、式(I-18)或(I-19)中Ac为乙酰基,Ph为苯基,Bn为苄基,Me为甲基,TFA为三氟乙酰基,Lev为乙酰丙酰基。
第四方面,本申请提供了一种新型的细菌荚膜寡糖衍生物,所述的衍生物如式(I’)所示,或其药学上可接受的盐、溶剂化物、前药:
其中,式(I’)中R1’为OH、未取代或取代的C1-C6烷氧基、未取代得或取代的C2-C6烯烃氧基、未取代得或取代的C2-C6炔氧基、未取代或取代的C1-C6烷硫基、未取代的或取代的C1-C6烷酰氧基、或者未取代的或取代的芳烃氧基;
R2’为OH、-N(H)-R15、N(R16)-R17、未取代或取代的C1-C6烷氧基、或者未取代的或取代的芳烃氧基,这里,R15为不包括脯氨酸的氨基酸残基;R16和R17一起形成脯氨酸残基;
R3’和R4’各自独立地为未取代的或取代的C1-C6烷酰基;
R5’、R6’、R7’、R8’、R9’、R10’、R11’、R12’和R13’各自独立地为氢、未取代的或其取代的C1-C6烷酰基、或者未取代的或取代的C1-C6烷基;
R14’为OH、未取代或取代的C1-C6烷氧基、或者未取代的或取代的芳烃氧基;
并规定:式(I’)中R1’不是正丙氧基或烯丙氧基。
第五方面,本申请提供了包含上述新型细菌荚膜寡糖衍生物的药物组合物。
第六方面,本申请提供了上述新型细菌荚膜寡糖衍生物或其药物组合物的抗炎活性用途。
第七方面,本申请提供了上述新型细菌荚膜寡糖衍生物或其药物组合物,用于抗炎药物。
第八方面,对个体施用本申请提供了上述新型细菌荚膜寡糖衍生物或其药物组合物来预防或治疗炎症的方法。
在第一方面或第二方面的实施方案中,本申请提供了一种细菌荚膜寡糖衍生物或其药学上可接受的盐、溶剂化物、前药作为抗炎药物的用途,或者预防或治疗炎症的方法,所述的衍生物如式I所示:
其中,式(I)中R1为OH、未取代或取代的C1-C6烷氧基、未取代得或取代的C2-C6烯烃氧基、未取代得或取代的C2-C6炔氧基、未取代或取代的C1-C6烷硫基、未取代的或取代的C1-C6烷酰氧基、或者未取代的或取代的芳烃氧基;这里,所述取代的C1-C6烷氧基、取代的C2-C6烯烃氧基、取代的C2-C6炔氧基、取代的C1-C6烷硫基、取代的C1-C6烷酰氧基和取代的芳烃氧基是指C1-C6烷氧基、C2-C6烯烃氧基、C2-C6炔氧基、C1-C6烷硫基、C1-C6烷酰氧基或芳烃氧基中一个或多个氢被选自如下基团所取代:羟基、C1-C6烷氧基、卤素、硝基、氰基、乙酰基、丙酰基和苯基;
R2为OH、-N(H)-R15、N(R16)-R17、未取代或取代的C1-C6烷氧基、或者未取代的或取代的芳烃氧基,这里,R15为不包括脯氨酸的氨基酸残基;R16和R17一起形成脯氨酸残基;所述取代的C1-C6烷氧基和取代的芳烃氧基是指C1-C6烷氧基、或芳烃氧基中一个或多个氢被选自如下基团所取代:羟基、C1-C6烷氧基、卤素、硝基、氰基和苯基;
R3和R4各自独立地为未取代的或取代的C1-C6烷酰基;这里,所述取代的C1-C6烷酰基是指C1-C6烷酰基中一个或多个氢被选自如下基团所取代:羟基、C1-C6烷氧基、卤素、硝基、氰基、乙酰基、丙酰基和苯基;
R5、R6、R7、R8、R9、R10、R11、R12和R13各自独立地为氢、未取代的或其取代的C1-C6烷酰基、或者未取代的或取代的C1-C6烷基;这里,所述取代的C1-C6烷酰基或是取代的C1-C6烷基指C1-C6烷酰基或C1-C6烷基中一个或多个氢被选自如下基团所取代:羟基、C1-C6烷氧基、卤素、硝基、氰基、乙酰基、丙酰基和苯基;任选地,所述苯基可以被选自C1-C4烷氧基和硝基中一个或多个所取代;
R14为OH、未取代或取代的C1-C6烷氧基、或者未取代的或取代的芳烃氧基;这里,所述取代的C1-C6烷氧基和取代的芳烃氧基是指C1-C6烷氧基、或芳烃氧基中一个或多个氢被选自如下基团所取代:羟基、C1-C6烷氧基、卤素、硝基、氰基和苯基。
在第一方面或第二方面的一些实施方案中,所述式(I)中R1为OH、未取代C1-C6烷氧基、苯取代的C1-C6烷氧基、或未取代得C2-C6烯烃氧基。
在第一方面或第二方面的一些实施方案中,所述式(I)中R1为OH、甲氧基、乙氧基、正丙氧基、异丙氧基、烯丙氧基、或苄氧基。
在第一方面或第二方面的一些实施方案中,所述式(I)中R2为OH、-N(H)-R15、N(R16)-R17、未取代C1-C6烷氧基、或苯取代的C1-C6烷氧基,这里,R15为不包括脯氨酸的氨基酸残基;R16和R17一起形成脯氨酸残基。
在第一方面或第二方面的一些实施方案中,所述式(I)中R2为OH、甲氧基、-N(H)-R15或N(R16)-R17,这里,R15为甘氨酸残基、丙氨酸残基、缬氨酸残基、亮氨酸残基、异亮氨酸残基、甲硫氨酸残基、色氨酸残基、丝氨酸残基、酪氨酸残基、半胱氨酸残基、苯丙氨酸残基、天门冬酰胺残基、谷氨酰胺残基、苏氨酸残基、天冬氨酸残基、谷氨酸残基、赖氨酸残基、精氨酸残基或组氨酸残基;R16和R17一起形成脯氨酸残基。
在第一方面或第二方面的一些实施方案中,所述式(I)中R2为OH或甲氧基。
在第一方面或第二方面的一些实施方案中,所述式(I)中R2为-N(H)-R15,这里,R15为甘氨酸残基、丙氨酸残基、缬氨酸残基、亮氨酸残基、异亮氨酸残基、甲硫氨酸残基、色氨酸残基、丝氨酸残基、酪氨酸残基、半胱氨酸残基、苯丙氨酸残基、天门冬酰胺残基、谷氨酰胺残基、苏氨酸残基、天冬氨酸残基、谷氨酸残基、赖氨酸残基、精氨酸残基或组氨酸残基。
在第一方面或第二方面的一些实施方案中,所述式(I)中R2为-N(H)-R15,这里,R15为苏氨酸残基。
在第一方面或第二方面的一些实施方案中,所述氨基酸可以是L构型或D构型。
在第一方面或第二方面的一些实施方案中,所述式(I)中R2为-N(H)-R15,这里,R15为L-苏氨酸残基。
在第一方面或第二方面的一些实施方案中,所述式(I)中R3和R4各自独立地为未取代的或取代的C1-C6烷酰基;这里,所述取代的C1-C6烷酰基是指C1-C6烷酰基中一个或多个氢被选自如下基团所取代:卤素、硝基、氰基、乙酰基、丙酰基和苯基。
在第一方面或第二方面的一些实施方案中,所述式(I)中R3和R4各自独立地为乙酰基或三氟乙酰基。
在第一方面或第二方面的一些实施方案中,所述式(I)中R5、R6、R7、R8、R9、R10、R11、R12和R13各自独立地为氢、未取代的C1-C6烷酰基、苯取代的C1-C6烷酰基、或者取代苯基甲基。
在第一方面或第二方面的一些实施方案中,所述式(I)中R5、R6、R7、R8、R9、R10、R11、R12和R13各自独立地为氢、乙酰基、苄基、或4-甲氧苄基。
在第一方面或第二方面的一些实施方案中,所述式(I)中R14为OH、或者未取代或取代的C1-C6烷氧基。
在第一方面或第二方面的一些实施方案中,所述式(I)中R14为OH或甲氧基。
在第一方面或第二方面的一些实施方案中,所述式(I)中R1为OH、甲氧基、乙氧基、正丙氧基、异丙氧基、烯丙氧基、或苄氧基;
R2为OH、甲氧基或-N(H)-R15这里,R15为苏氨酸残基;
R3和R4各自独立地为乙酰基或三氟乙酰基;
R5、R6、R7、R8、R9、R10、R11、R12和R13各自独立地为氢、乙酰基、苄基、或4-甲氧苄基;和
R14为OH或甲氧基。
在第一方面或第二方面的一种实施方案中,所述式(I)中R1为正丙氧基或烯丙氧基、或苄氧基;
R2为OH或-N(H)-R15这里,R15为苏氨酸残基;
R3和R4各自独立地为乙酰基;
R5、R6、R7、R8、R9、R10、R11、R12和R13各自独立地为氢;和R14为OH。
在第一方面或第二方面的一种实施方案中,所述式(I)中R1为OH;
R2为OH或-N(H)-R15这里,R15为苏氨酸残基;
R3和R4各自独立地为乙酰基;
R5、R6、R7、R8、R9、R10、R11、R12和R13各自独立地为氢;和R14为OH。
在第一方面或第二方面的一种实施方案中,所述式(I)中R1为甲氧基;
R2为OH或-N(H)-R15这里,R15为苏氨酸残基;
R3和R4各自独立地为乙酰基;
R5、R6、R7、R8、R9、R10、R11、R12和R13各自独立地为氢;和R14为OH。
在第一方面或第二方面的一种实施方案中,所述式(I)中R1为乙氧基;
R2为OH或-N(H)-R15这里,R15为苏氨酸残基;
R3和R4各自独立地为乙酰基;
R5、R6、R7、R8、R9、R10、R11、R12和R13各自独立地为氢;和R14为OH。
在第一方面或第二方面的一种实施方案中,所述式(I)中R1为异丙氧基;
R2为OH或-N(H)-R15这里,R15为苏氨酸残基;
R3和R4各自独立地为乙酰基;
R5、R6、R7、R8、R9、R10、R11、R12和R13各自独立地为氢;和R14为OH。
在第一方面或第二方面的一些实施方案中,本申请提供了上述细菌荚膜寡糖衍生物的用途为抗炎活性,或者预防或治疗炎症的方法,可以抑制一氧化氮和前列腺素E2的生成;和/或抑制一氧化氮合酶和环氧化酶2的蛋白表达;和/或降低白介素1β、白介素6和肿瘤坏死因子α的释放。
在第一方面或第二方面的一些实施方案中,本申请提供了上述细菌荚膜寡糖衍生物的用途或者预防或治疗炎症的方法为治疗脓毒症;这里,所述脓毒症包括脓毒血症。
脓毒血症是一种危及生命的临床综合征,表现为机体对感染的过度反应而导致的器官功能障碍;临床常见于手术、创伤及免疫力低下等状况下,细菌进入血液循环,并在其中生长繁殖、产生毒素而引起的全身性严重感染,表现为发热、严重毒血症状、皮疹瘀点、肝脾肿大和白细胞数增高等。
在第三方面的一些实施方案中,本申请提供了上述细菌荚膜寡糖衍生物的制备方法,所述的制备方法包括如下步骤:
式(I-13)化合物与式(I-18)化合物反应,得到化合物(I-19);式(I-13-Me)化合物与式(I-18)化合物反应,得到化合物(I-19-Me);式(I-13-Et)化合物与式(I-18)化合物反应,得到化合物(I-19-Et);式(I-13-Pr)化合物与式(I-18)化合物反应,得到化合物(I-19-Pr)。
这里,化合物式(I-13)、式(I-13-Me)、式(I-13-Et)、式(I-13-Pr)、式(I-18)、式(I-19)、式(I-19-Me)、式(I-19-Et)或式(I-19-Pr)中Ac为乙酰基,Ph为苯基,Bn为苄基,Me为甲基,Et为乙基,Pr为异丙基,TFA为三氟乙酰基,Lev为乙酰丙酰基。
在第三方面的一些实施方案中,所述化合物式(I-13)、式(I-13-Me)、式(I-13-Et)和式(I-13-Pr)可以采用如下路线来制备:
上述路线中,取代基PMB为对甲氧苄基,Tol为对甲苯基。
在第三方面的一些实施方案中,所述化合物式(I-18)可以以透明质酸钠为原料,采用如下路线来制备:
在第三方面的一些实施方案中,所述细菌荚膜寡糖衍生物的制备方法在得到化合物式(I-19)、式(I-19-Me)、式(I-19-Et)或式(I-19-Pr)后还包括选择性处理Lev基团,然后与保护的氨基酸进行酰胺化反应,最终完全地脱除保护基从而得到不含保护基的式(I)化合物;或者,在得到化合物式(I-19)、式(I-19-Me)、式(I-19-Et)或式(I-19-Pr)后还包括完全地脱除保护基从而得到不含保护基的式(I)化合物:
在第四方面的一些实施方案中,本申请提供了一种新型的细菌荚膜寡糖衍生物,所述的衍生物如式(I’)所示,或其药学上可接受的盐、溶剂化物、前药:
其中,式(I’)中R1’为氢、未取代或取代的C1-C6烷氧基、未取代得或取代的C2-C6烯烃氧基、未取代得或取代的C2-C6炔氧基、未取代或取代的C1-C6烷硫基、未取代的或取代的C1-C6烷酰氧基、或者未取代的或取代的芳烃氧基:这里,所述取代的C1-C6烷氧基、取代的C2-C6烯烃氧基、取代的C2-C6炔氧基、取代的C1-C6烷硫基、取代的C1-C6烷酰氧基和取代的芳烃氧基是指C1-C6烷氧基、C2-C6烯烃氧基、C2-C6炔氧基、C1-C6烷硫基、C1-C6烷酰氧基或芳烃氧基中一个或多个氢被选自如下基团所取代:羟基、C1-C6烷氧基、卤素、硝基、氰基、乙酰基、丙酰基和苯基;
R2’为OH、-N(H)-R15、N(R16)-R17、未取代或取代的C1-C6烷氧基、或者未取代的或取代的芳烃氧基,这里,R15为不包括脯氨酸的氨基酸残基;R16和R17一起形成脯氨酸残基;所述取代的C1-C6烷氧基和取代的芳烃氧基是指C1-C6烷氧基、或芳烃氧基中一个或多个氢被选自如下基团所取代:羟基、C1-C6烷氧基、卤素、硝基、氰基和苯基;
R3’和R4’各自独立地为未取代的或取代的C1-C6烷酰基;这里,所述取代的C1-C6烷酰基是指C1-C6烷酰基中一个或多个氢被选自如下基团所取代:羟基、C1-C6烷氧基、卤素、硝基、氰基、乙酰基、丙酰基和苯基;
R5’、R6’、R7’、R8’、R9’、R10’、R11’、R12’和R13’各自独立地为氢或未取代的或其取代的C1-C6烷酰基;这里,所述取代的C1-C6烷酰基是指C1-C6烷酰基中一个或多个氢被选自如下基团所取代:羟基、C1-C6烷氧基、卤素、硝基、氰基、乙酰基、丙酰基和苯基;
R14’为OH、未取代或取代的C1-C6烷氧基、或者未取代的或取代的芳烃氧基:这里,所述取代的C1-C6烷氧基和取代的芳烃氧基是指C1-C6烷氧基、或芳烃氧基中一个或多个氢被选自如下基团所取代:羟基、C1-C6烷氧基、卤素、硝基、氰基和苯基;
并规定:式(I’)中R1’不是正丙氧基或烯丙氧基。
在第四方面的一些实施方案中,所述式(I’)中R1’为OH、甲氧基、乙氧基、异丙氧基、正丁氧基、异丁氧基、叔丁氧基、或苄氧基。
在第四方面的一些实施方案中,所述式(I’)中R1’为OH。
在第四方面的一些实施方案中,所述式(I’)中R1’为甲氧基。
在第四方面的一些实施方案中,所述式(I’)中R1’为乙氧基。
在第四方面的一些实施方案中,所述式(I’)中R1’为异丙氧基。
在第四方面的一些实施方案中,所述式(I’)中R2’为OH、-N(H)-R15、N(R16)-R17、未取代C1-C6烷氧基、或苯取代的C1-C6烷氧基,这里,R15为不包括脯氨酸的氨基酸残基;R16和R17一起形成脯氨酸残基。
在第四方面的一些实施方案中,所述式(I’)中R2’为OH、甲氧基、-N(H)-R15或N(R16)-R17这里,R15为甘氨酸残基、丙氨酸残基、缬氨酸残基、亮氨酸残基、异亮氨酸残基、甲硫氨酸残基、色氨酸残基、丝氨酸残基、酪氨酸残基、半胱氨酸残基、苯丙氨酸残基、天门冬酰胺残基、谷氨酰胺残基、苏氨酸残基、天冬氨酸残基、谷氨酸残基、赖氨酸残基、精氨酸残基或组氨酸残基;R16和R17一起形成脯氨酸残基。
在第四方面的一些实施方案中,所述式(I’)中R2’为OH或甲氧基。
在第四方面的一些实施方案中,所述式(I’)中R2’为-N(H)-R15这里,R15为甘氨酸残基、丙氨酸残基、缬氨酸残基、亮氨酸残基、异亮氨酸残基、甲硫氨酸残基、色氨酸残基、丝氨酸残基、酪氨酸残基、半胱氨酸残基、苯丙氨酸残基、天门冬酰胺残基、谷氨酰胺残基、苏氨酸残基、天冬氨酸残基、谷氨酸残基、赖氨酸残基、精氨酸残基或组氨酸残基。
在第四方面的一些实施方案中,所述式(I’)中R2’为-N(H)-R15这里,R15为苏氨酸残基。
在第四方面的一些实施方案中,所述氨基酸可以是L构型或D构型。
在第四方面的一些实施方案中,所述式(I’)中R2’为-N(H)-R15,这里,R15为L-苏氨酸残基。
在第四方面的一些实施方案中,所述式(I’)中R3’和R4’各自独立地为未取代的或取代的C1-C6烷酰基;这里,所述取代的C1-C6烷酰基是指C1-C6烷酰基中一个或多个氢被选自如下基团所取代:卤素、硝基、氰基、乙酰基、丙酰基和苯基。
在第四方面的一些实施方案中,所述式(I’)中R3’和R4’各自独立地为乙酰基或三氟乙酰基。
在第四方面的一些实施方案中,所述式(I’)中R5’、R6’、R7’、R8’、R9’、R10’、R11’、R12’和R13’各自独立地为氢、未取代的C1-C6烷酰基、苯取代的C1-C6烷酰基、或者取代苯基甲基。
在第四方面的一些实施方案中,所述式(I’)中R5’、R6’、R7’、R8’、R9’、R10’、R11’、R12’和R13’各自独立地为氢、乙酰基、苄基、或4-甲氧苄基。
在第四方面的一些实施方案中,所述式(I’)中R14’为OH、或者未取代或取代的C1-C6烷氧基。
在第四方面的一些实施方案中,所述式(I’)中R14’为OH或甲氧基。
在第四方面的一些实施方案中,所述式(I’)中R1’为OH、甲氧基、乙氧基或异丙氧基;
R2’为OH、甲氧基或-N(H)-R15这里,R15为苏氨酸残基;
R3’和R4’各自独立地为乙酰基或三氟乙酰基;
R5’、R6’、R7’、R8’、R9’、R10’、R11’、R12’和R13’各自独立地为氢、乙酰基、苄基、或4-甲氧苄基;和
R14’为OH或甲氧基。
在第四方面的一种实施方案中,所述式(I’)中R1’为OH;
R2’为OH;
R3’和R4’各自独立地为乙酰基;
R5’、R6’、R7’、R8’、R9’、R10’、R11’、R12’和R13’各自独立地为氢;和
R14’为OH;
即式(I’)为化合物CP-1:
在第四方面的一种实施方案中,所述式(I’)中R1’为OH;
R2’为-N(H)-R15,这里,R15为苏氨酸残基;
R3’和R4’各自独立地为乙酰基;
R5’、R6’、R7’、R8’、R9’、R10’、R11’、R12’和R13’各自独立地为氢;和R14’为OH;
即式(I’)为化合物CP-2:
在第四方面的一种实施方案中,所述式(I’)中R1’为甲氧基;
R2’为OH;
R3’和R4’各自独立地为乙酰基;
R5’、R6’、R7’、R8’、R9’、R10’、R11’、R12’和R13’各自独立地为氢;和R14’为OH;
即式(I’)为化合物CP-Me:
在第四方面的一种实施方案中,所述式(I’)中R1’为乙氧基;
R2’为OH;
R3’和R4’各自独立地为乙酰基;
R5’、R6’、R7’、R8’、R9’、R10’、R11’、R12’和R13’各自独立地为氢;和R14’为OH;
即式(I’)为化合物CP-Et:
在第四方面的一种实施方案中,所述式(I’)中R1’为异丙氧基;
R2’为OH;
R3’和R4’各自独立地为乙酰基;
R5’、R6’、R7’、R8’、R9’、R10’、R11’、R12’和R13’各自独立地为氢;和
R14’为OH;
即式(I’)为化合物CP-Pr:
在第五方面的一些实施方案中,本申请提供了包含上述新型细菌荚膜寡糖衍生物的药物组合物。本申请提供的上述药物组合物,可以是口服或非胃肠给药的制剂形式,剂量可以是0.1-5000mg/次/日。
在第一方面、第二方面、第六方面、第七方面或第八方面的一些实施方案中,本申请提供了上述新型细菌荚膜寡糖衍生物或其药物组合物的用途为抗炎活性,或者预防或治疗炎症的方法,可以抑制一氧化氮和前列腺素E2的生成;和/或抑制一氧化氮合酶和环氧化酶2的蛋白表达;和/或降低白介素1β、白介素6和肿瘤坏死因子α的释放。
在第一方面、第二方面、第六方面、第七方面或第八方面的一些实施方案中,本申请提供了上述新型细菌荚膜寡糖衍生物或其药物组合物的用途为抗炎活性,或者预防或治疗炎症的方法,可以抑制一氧化氮和前列腺素E2的生成;和/或抑制一氧化氮合酶和环氧化酶2的蛋白表达;和/或降低白介素1β、白介素6和肿瘤坏死因子α的释放。
在第一方面、第二方面、第六方面、第七方面或第八方面的一些实施方案中,本申请提供了上述新型细菌荚膜寡糖衍生物或其药物组合物的用途为治疗脓毒症或者预防或治疗炎症的方法为治疗脓毒症;这里,所述脓毒症包括脓毒血症。
本申请的其它特征和优点将在随后的说明书中阐述,并且,部分地从说明书中变得显而易见,或者通过实施本申请而了解。本申请的其他优点可通过在说明书以及附图中所描述的方案来实现和获得。
附图说明
附图用来提供对本申请技术方案的理解,并且构成说明书的一部分,与本申请的实施例一起用于解释本申请的技术方案,并不构成对本申请技术方案的限制。
图1表示的是本发明化合物CP-1/CP-2对LPS诱导的RAW264.7细胞中NO生成的影响;其中,NC表示空白对照组;与LPS组相比,****p<0.0001,n=3;
图2表示的是本发明化合物CP-1/CP-2对LPS诱导的RAW264.7细胞中PGE2生成的影响;其中,NC表示空白对照组;与LPS组相比,****p<0.0001,n=3;
图3表示的是本发明化合物CP-1/CP-2对LPS诱导的RAW264.7细胞中IL-1β、IL-6、TNF-α释放的影响;其中,NC表示空白对照组;与LPS组相比,****p<0.0001,n=3;
图4表示的是本发明化合物CP-1/CP-2对LPS诱导的RAW264.7细胞中iNOS和COX-2蛋白表达的影响;注:NC表示空白对照组;与LPS组相比,*p<0.05,**p<0.001,n=3;
图5表示的是本发明化合物CP-1对LPS诱导的小鼠脓毒症模型存活率的影响;
图6表示的是本发明化合物CP-1对LPS诱导的小鼠脓毒症模型血清中炎性因子的抑制作用;
图7表示的是本发明化合物CP-Me对LPS诱导的小鼠脓毒症模型血清中炎性因子的抑制作用;
图8表示的是雄性C57BL/6小鼠被分成对照组(a/e)、LPS组(b/f)、CP-1组(c/g)、地塞米松组(d/h);腹腔注射给与LPS 24小时后,通过HE染色研究小鼠肺的组织病理(A);炎症打分分别由三位实验者独立打分得到(B,n=6)。
具体实施方式
为使本申请的目的、技术方案和优点更加清楚明白,下文中将对本发明的实施例进行详细说明。需要说明的是,在不冲突的情况下,本申请中的实施例及实施例中的特征可以相互任意组合。
缩写
Ac为乙酰基;
PE为石油醚;
EtOAc为乙酸乙酯
CDCl3为氘代氯仿
DCM为二氯甲烷
MeOH为甲醇
TLC为薄层色谱
IL-1β为白介素-1β
IL-6为白介素-6
TNF-α为肿瘤坏死因子-α
LPS为脂多糖
PEG2为前列腺素E2
NO为一氧化氮
iNOS为人一氧化氮合酶
COX-2为环氧化酶2
Dex为地塞米松
实施例1
对甲苯基2,3,4,6-四-O-乙酰基-1-硫-β-D-吡喃半乳糖的合成
将全乙酰半乳糖(5.0g,12.8mmol)和对甲苯硫酚(1.75g,14.1mmol,1.1equiv.)溶于二氯甲烷(50.0mL),冷却至0℃,加入三氟化硼乙醚(4.0mL,32.0mmol,2.5equiv.),室温反应过夜。反应液用二氯甲烷稀释,依次用饱和碳酸氢钠和饱和食盐水洗涤,无水硫酸钠干燥,过滤,减压浓缩,柱层析纯化(石油醚/EtOAc 2:1)得到白色固体(5.76g,99%)。Rf=0.22(石油醚/EtOAc 2:1);1H NMR(400MHz,CDCl3,TMS)δ7.41(2H,d,aromatic,J=8.1Hz),7.13(2H,d,aromatic,J=7.9Hz),5.41(1H,d,H-4,J=2.9Hz),5.22(1H,t,H-2,J=10.0Hz),5.04(1H,dd,H-3,J=3.3Hz,J=10.0Hz),4.65(1H,d,H-1,J=9.9Hz),4.19(1H,dd,H-6a,J=6.9Hz,J=11.3Hz),4.11(1H,dd,H-6b,J=6.4Hz,J=11.4Hz),3.91(1H,t,H-5,J=6.9Hz),2.35(3H,s,CH3 of STol),2.12(3H,s,COCH3),2.10(3H,s,COCH3),2.04(3H,s,COCH3),1.97(3H,s,COCH3);13C NMR(100MHz,CDCl3,TMS)δ170.4,170.2,170.0,169.4,138.5,133.2,129.6,128.6,87.0,74.4,72.0,67.3,67.2,61.6,21.2,20.9,20.7,20.6,20.5.
对甲苯基4,6-O-苯亚甲基-1-硫-β-D-吡喃半乳糖的合成
将对甲苯基2,3,4,6-四-O-乙酰基-1-硫-β-D-吡喃半乳糖(5.0g,11.0mmol)溶于甲醇(50.0mL),加入甲醇钠调节反应液pH至9~10,室温反应1小时。IR-120阳离子交换树脂中和反应液pH=7,过滤,甲醇洗涤树脂,浓缩滤液。将浓缩后的粗产物和(+)-樟脑磺酸(1.28g,5.5mmol,0.5equiv.)溶于无水乙腈(70.0mL),加入苯甲醛二甲基缩醛(2.48mL,16.5mmol,1.5equiv.),室温反应过夜。TLC监测反应完全后,滴加适量三乙胺淬灭反应,反应液减压浓缩,柱层析纯化(DCM/MeOH 30:1)得到白色固体(3.87g,两步收率94%)。Rf=0.40(DCM/MeOH 20:1);1H NMR(400MHz,CDCl3,TMS)δ7.55(2H,d,aromatic,J=8.1Hz),7.40-7.34(5H,m,aromatic),7.08(2H,d,aromatic,J=8.1Hz),5.44(1H,s),4.39(1H,d,J=9.1Hz),4.31(1H,dd,J=1.0Hz,J=12.4Hz),4.07(1H,d,J=1.6Hz),3.93(1H,dd,J=1.4Hz,J=12.4Hz),3.60(2H,d,J=6.6Hz),3.39(1H,s),3.09(1H,d,J=7.6Hz),3.07(1H,s),2.34(3H,s,CH3 of STol);13C NMR(100MHz,CDCl3,TMS)δ138.3,137.8,134.2,129.7,129.3,128.2,127.0,126.7,101.3,87.0,75.5,73.6,69.9,69.3,21.3.
对甲苯基3-O-苄基-4,6-O-苯亚甲基-1-硫-β-D-吡喃半乳糖的合成
将对甲苯基4,6-O-苯亚甲基-1-硫-β-D-吡喃半乳糖(3.50g,9.35mmol)和二丁基氧化锡(2.79g,11.2mmol,1.2equiv.)溶于无水甲苯(50.0mL),120℃反应8小时,冷却至室温,减压浓缩除去溶剂,所得中间体真空干燥1小时。将中间体和四丁基溴化铵(4.52g,14.0mmol,1.5equiv.)溶于无水甲苯(30.0mL),加入溴化苄(1.66mL,14.0mmol,1.5equiv.),60℃反应12小时。TLC监测反应结束后,反应液用乙酸乙酯稀释,依次用1M盐酸和饱和食盐水洗涤,无水硫酸钠干燥,过滤,减压浓缩,柱层析纯化(Toluene/EtOAc 9:1)得到白色固体(4.08g,94%)。Rf=0.30(石油醚/EtOAc 2:1);1H NMR(400MHz,CDCl3,TMS)δ7.57(2H,d,aromatic,J=8.1Hz),7.26-7.41(10H,m,aromatic),7.05(2H,d,aromatic,J=8.1Hz),5.41(1H,s,PhCH),4.71(2H,d,PhCH2,J=2.0Hz),4.46(1H,d,H-1,J=9.4Hz),4.34(1H,dd,H-6a,J=1.6Hz,J=12.3Hz),4.12(1H,d,H-4,J=3.3Hz),3.96(1H,dd,H-6b,J=1.7Hz,J=12.3Hz),3.87(1H,t,H-2,J=9.4Hz),3.50(1H,dd,H-3,J=3.3Hz,J=9.3Hz),3.43(1H,d,H-5,J=0.9Hz),2.45(1H,s,OH),2.33(3H,s,CH3 of STol);13C NMR(100MHz,CDCl3,TMS)δ138.4,138.0,137.9,134.4,129.7,129.0,128.5,128.1,128.0,126.6,126.5,101.2,87.1,80.2,73.3,71.7,70.0,69.4,67.1,21.2.ESI-Q-TOF(positive mode)计算值C27H32NO5S+[M+NH4]+m/z 482.2001,实测值482.1999.
对甲苯基2-O-对甲氧苄基-3-O-苄基-4,6-O-苯亚甲基-1-硫-β-D-吡喃半乳糖的合成
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将对甲苯基3-O-苄基-4,6-O-苯亚甲基-1-硫-β-D-吡喃半乳糖(2.60g,5.60mmol)溶于无水N,N-二甲基甲酰胺(50.0mL),冰浴条件下分批加入氢化钠(60%,448mg,11.2mmol,2.0equiv.),加入对甲氧基氯化苄(1.52mL,11.2mmol,2.0equiv.),随后移入室温搅拌反应2小时。TLC监测反应结束后,滴加适量甲醇淬灭,反应液用乙酸乙酯稀释,有机相依次经水和饱和食盐水洗涤,无水硫酸钠干燥,过滤,减压浓缩,柱层析纯化(石油醚/EtOAc 3:1)得到白色固体(3.18g,97%)。Rf=0.38(石油醚/EtOAc 2:1);1H NMR(400MHz,CDCl3,TMS)δ7.61-7.59,7.51-7.50,7.35-7.25,6.98-6.96,6.86-6.84(18H,m,aromatic),5.43(1H,s,PhCH),4.67(2H,s,2PhCH2),4.63(2H,s,2PhCH2),4.52(1H,d,H-1,J=9.4Hz),4.28(1H,d,H-6a,J=12.1Hz),4.07(1H,d,H-4,J=2.9Hz),3.89(1H,s,H-6b),3.82(1H,t,H-2,J=9.2Hz),3.74(3H,s,OCH3),3.56(1H,dd,H-3,J=2.4Hz,J=8.8Hz),3.26(1H,s,H-5),2.26(3H,s,CH3 of STol);13C NMR(100MHz,CDCl3,TMS)δ159.4,138.4,138.2,137.7,133.4,131.0,129.9,129.8,129.1,129.0,128.5,128.2,127.9,126.8,113.9,101.3,86.7,81.6,75.3,75.2,73.7,69.8,69.5,55.4,21.3.ESI-Q-TOF(positive mode)计算值C35H40NO6S+[M+NH4]+m/z 602.2576,实测值602.2583.
对甲苯基2-O-对甲氧苄基-3,4-二-O-苄基-1-硫-β-D-吡喃半乳糖的合成
氩气保护下,将对甲苯基2-O-对甲氧苄基-3-O-苄基-4,6-O-苯亚甲基-1-硫-β-D-吡喃半乳糖(2.0g,3.42mmol)溶于无水二氯甲烷(30.0mL),冰浴条件下加入1M硼烷四氢呋喃溶液(17.1mL,17.1mmol,5.0equiv.),搅拌10分钟后滴加三氟甲磺酸三甲基硅酯(93μL,0.51mmol,0.15equiv.),恢复室温反应2小时。TLC监测反应结束后,加入适量三乙胺淬灭反应,滴加甲醇至无氢气释放,减压浓缩,柱层析纯化(石油醚/EtOAc 4:1)得到白色固体(1.81g,90%)。Rf=0.26(石油醚/EtOAc 2:1);1H NMR(400MHz,CDCl3,TMS)δ7.46-7.44,7.34-7.29,7.01-6.99,6.85-6.83(18H,m,aromatic),4.94(1H,d,J=11.7Hz),4.73(3H,m),4.66(1H,d,J=10.0Hz),4.61(1H,d,J=11.5Hz),4.56(1H,d,J=9.4Hz),3.89(1H,t,J=9.4Hz),3.83-3.78(2H,m),3.76(3H,s),3.57-3.50(2H,m),3.39(1H,t,J=6.0Hz),2.26(3H,s);13C NMR(100MHz,CDCl3,TMS)δ159.4,138.5,138.3,137.4,132.1,130.6,130.2,130.1,129.7,128.6,128.4,128.2,127.8,127.6,113.8,88.0,84.3,78.9,77.2,75.3,74.3,73.5,73.0,62.2,55.4,21.2;ESI-Q-TOF(positive mode)计算值C35H42NO6S+[M+NH4]+m/z 604.2733,实测值604.2737.
对甲苯基2-O-对甲氧苄基-3,4-二-O-苄基-6-乙酰丙酰基-1-硫-β-D-吡喃半乳糖的合成
将对甲苯基2-O-对甲氧苄基-3,4-二-O-苄基-1-硫-β-D-吡喃半乳糖(1.80g,3.07mmol)溶于无水二氯甲烷(30.0mL),依次加入乙酰丙酸(713mg,6.14mmol,2.0equiv.),1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(1.18g,6.14mmol,2.0equiv.)和催化量4-二甲氨基吡啶,室温反应3小时。TLC监测反应结束后,反应液用二氯甲烷稀释,有机相依次经饱和碳酸氢钠溶液和饱和食盐水洗涤,无水硫酸钠干燥,过滤,减压浓缩,柱层析纯化(石油醚/EtOAc3:1)得到白色固体(12.3g,92%)。Rf=0.30(石油醚/EtOAc 2:1);1H NMR(400MHz,CDCl3,TMS)δ7.47-7.45,7.38-7.29,7.02-7.00,6.86-6.84(18H,m,aromatic),4.98(1H,d,J=11.4Hz,PhCH2),4.75(3H,m,PhCH2),4.67(1H,d,J=9.8Hz,PhCH2),4.63(1H,d,J=11.4Hz,PhCH2),4.55(1H,d,J=9.6Hz,H-1),4.27(1H,m,H-6a),4.15(1H,m,H-6b),3.89(2H,m,H-2,H-4),3.78(3H,s,OCH3),3.59-3.55(2H,m,H-3,H-5),2.71-2.68,2.51-2.47(4H,m,CH2 of Lev),2.29(3H,s,PhCH3),2.14(3H,s,CH3CO);13C NMR(100MHz,CDCl3,TMS)δ206.5,159.3,138.5,138.3,137.3,132.2,130.6,130.2,130.0,129.6,128.5,128.3,128.1,127.8,127.6,113.8,88.1,84.2,77.0,75.9,75.3,74.3,73.4,73.0,63.4,55.3,37.9,29.8,27.9,21.1;HRMS(ESI-MS)计算值C40H44NaO8S+[M+Na]+m/z 707.2649,实测值707.2635.
2-去氧-2-三氟乙酰氨基-1,3,4,6-四-O-乙酰基-β-D-吡喃半乳糖的合成
将全乙酰氨基半乳糖(5.0g,12.8mmol)溶于无水吡啶(100.0mL)中,加入三氟乙酸酐(9.02mL,64.0mmol,5.0equiv.),升温至135℃反应1小时。TLC监测反应结束后,加入适量甲醇淬灭反应,反应液用二氯甲烷稀释,有机相依次经1M盐酸溶液和饱和食盐水洗涤,无水硫酸钠干燥,过滤,减压浓缩,柱层析纯化(DCM/MeOH 60:1)得到白色固体(5.0g,88%)。Rf=0.34(DCM/MeOH 30:1);1H NMR(400MHz,CDCl3,TMS)δ7.19(1H,d,J=9.4Hz,NHAc),5.80(1H,d,H-1,J=8.9Hz),5.42(1H,d,H-4,J=3.0Hz),5.20(1H,d,H-3,J=3.2Hz,J=11.3Hz),4.50(1H,m,H-2),4.21-4.07(3H,m,H-5,H-6a,H-6b),2.20(3H,s,CH3CO),2.14(3H,s,CH3CO),2.06(3H,s,CH3CO),2.03(3H,s,CH3CO);13C NMR(100MHz,CDCl3,TMS)δ170.8,170.7,170.2,169.6,157.7(q,J=37.4Hz,COCF3),117.0(q,J=285.1Hz,COCF3),92.3,72.0,69.9,66.2,61.4,50.2,20.6,20.5,20.4.ESI-Q-TOF(positive mode)计算值C16H24F3N2O10 +[M+NH4]+m/z 461.1383,实测值461.1381.
对甲苯基2-去氧-2-三氟乙酰氨基-3,4,6-三-O-乙酰基-1-硫-β-D-吡喃半乳糖的合成
将2-去氧-2-三氟乙酰氨基-1,3,4,6-四-O-乙酰基-β-D-吡喃半乳糖(5.0g,11.3mmol)和对甲苯硫酚(2.1g,17.0mmol,1.5equiv.)溶于无水二氯甲烷(100.0mL)中,冰浴下滴加三氟化硼乙醚络合物(4.28mL,33.9mmol,3.0equiv.),恢复室温反应过夜。TLC监测反应结束后,加入适量三乙胺淬灭,反应液用二氯甲烷稀释,有机相依次经饱和碳酸氢钠溶液和饱和食盐水洗涤,无水硫酸钠干燥,过滤,减压浓缩,得到黄色糖浆(5.68g,99%),Rf=0.24(石油醚/EtOAc 2:1),直接用于下一步反应。
苄基2-去氧-2-三氟乙酰氨基-3,4,6-三-O-乙酰基-β-D-吡喃半乳糖的合成
氩气保护下,将对甲苯基2-去氧-2-三氟乙酰氨基-3,4,6-三-O-乙酰基-1-硫-β-D-吡喃半乳糖(5.68g,11.2mmol)、分子筛溶于无水二氯甲烷(100.0mL)中,加入苄醇(2.33mL,22.4mmol,2.0equiv.),室温搅拌2小时,随后降温至-40℃,加入N-碘代丁二酰亚胺(3.53g,15.7mmol,1.4equiv.),搅拌15分钟后,滴加三氟甲磺酸(300μL,3.36mmol,0.3equiv.),继续搅拌反应3小时。TLC监测反应结束后,加入适量三乙胺淬灭,硅藻土滤除分子筛,二氯甲烷洗涤滤饼数次,合并滤液,减压浓缩,柱层析纯化(石油醚/EtOAc 2:1)得到白色固体(5.06g,92%)。Rf=0.19(石油醚/EtOAc 2:1);1H NMR(400MHz,CDCl3,TMS)δ7.35-7.26(5H,m,aromatic),7.01(1H,br s,NHAc),5.36(1H,d,J=3.0Hz),5.20(1H,dd,J=11.5Hz,J=3.3Hz),4.89(1H,d,J=12.1Hz),4.63(1H,d,J=8.4Hz),4.61(1H,d,J=12.3Hz),4.29-4.13(3H,m),3.93(1H,t,J=6.8Hz),2.15(3H,s),2.06(3H,s),1.92(3H,s);13C NMR(100MHz,CDCl3,TMS)δ170.7,170.6,170.4,157.5(q,J=37.1Hz,COCF3),136.4,128.5,128.2,127.9,115.6(q,J=286.2Hz,COCF3),99.0,70.8,70.7,69.7,66.6,61.7,51.5,20.6,20.5,20.4;ESI-Q-TOF(positive mode)计算值C21H28F3N2O9 +[M+NH4]+m/z509.1747,实测值509.1747.
苄基2-去氧-2-三氟乙酰氨基-β-D-吡喃半乳糖的合成
将苄基2-去氧-2-三氟乙酰氨基-3,4,6-三-O-乙酰基-β-D-吡喃半乳糖(2.50g,5.09mmol)溶于甲醇(50.0mL),加入适量甲醇钠调节pH至9~10,室温下搅拌反应2小时。TLC监测反应结束后,向反应液中加入阳离子树脂中和至pH=7,过滤,滤液减压浓缩至干,得到黄色糖浆。Rf=0.45(DCM/MeOH30:1),直接用于下一步反应。
苄基2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖的合成
氩气保护下,将苄基2-去氧-2-三氟乙酰氨基-β-D-吡喃半乳糖(1.80g,4.93mmol)和(+)-樟脑磺酸(572mg,2.46mmol,0.5equiv.)溶于无水乙腈(50.0mL)中,加入苯甲醛二甲缩醛(1.11mL,7.40mmol,1.5equiv.),升温至40℃反应过夜。TLC监测反应完全后,滴加适量三乙胺淬灭反应,反应液减压浓缩,柱层析(石油醚/EtOAc 2:1)纯化得到化合物白色固体(2.03g,91%)。Rf=0.30(石油醚/EtOAc 1:1);1H NMR(400MHz,DMSO-d6)δ9.26(1H,d,NH,J=9.0Hz),7.52-7.49,7.42-7.26(10H,m,aromatic),5.63(1H,s,PhCH),5.25(1H,d,OH,J=6.4Hz),4.82(1H,d,PhCH2,J=12.5Hz),4.58(1H,d,H-1,J=8.3Hz),4.55(1H,d,PhCH2,J=12.3Hz),4.17-4.09(3H,m,H-4,H-6a,H-6b),3.95(1H,m,H-2),3.82(1H,m,H-3),3.57(1H,s,H-5);13C NMR(100MHz,DMSO-d6)δ157.0(q,J=35.6Hz,COCF3),138.9,138.3,129.2,128.6,128.4,128.0,127.6,126.8,116.5(q,J=287.0Hz,COCF3),100.6,100.3,75.5,70.4,69.0,66.7,53.2;HRMS(ESI-MS)计算值C22H22NNaO6F3 +[M+Na]+m/z 476.1291,实测值476.1292.
苄基2-O-对甲氧苄基-3,4-二-O-苄基-6-乙酰丙酰基-α-D-吡喃半乳糖-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖
氩气保护下,将对甲苯基2-O-对甲氧苄基-3,4-二-O-苄基-6-乙酰丙酰基-1-硫-β-D-吡喃半乳糖(680mg,0.99mmol,1.5equiv.)、苄基2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖(300mg,0.66mmol)溶于无水二氯甲烷/N,N-二甲基甲酰胺(10.0mL/2.0mL),加入分子筛(1.0g),室温搅拌2小时后,冷却至0℃,依次加入N-碘代丁二酰亚胺(297mg,1.32mmol,2.0equiv.)和三氟甲磺酸银(51mg,0.20mmol,0.3equiv.),0℃搅拌反应2小时后,缓慢升至室温反应过夜。TLC监测反应结束后,滴加适量三乙胺淬灭反应,硅藻土过滤除去分子筛,滤液减压浓缩,柱层析分离(DCM/MeOH100:1)纯化得到化合物I-12(555mg,83%),白色固体。Rf=0.28(DCM/MeOH30:1);1H NMR(600MHz,CDCl3,TMS)δ7.55,7.44,7.34-7.25,6.92,6.63(25H,m,aromatic,NH),5.56(1H,s,PhCH),5.23(1H,d,H-1Gal,J=3.5Hz),4.94(2H,m,PhCH2),4.87(1H,d,H-1GalNAc,J=8.3Hz),4.80(1H,d,PhCH2,J=11.8Hz),4.63(2H,m,PhCH2),4.54(1H,d,PhCH2,J=11.5Hz),4.45-4.38(5H,m,H-2GalNAc,H-4GalNAc,H-6aGalNAc,PhCH2),4.17(1H,dd,H-3GalNAc,J=3.5Hz,J=10.9Hz),4.16-4.12(2H,m,H-6aGal,H-6bGalNAc),4.07(1H,dd,H-6bGal,J=7.8Hz,J=11.7Hz),4.03(1H,dd,H-2Gal,J=3.5Hz,J=9.9Hz),3.84(1H,dd,H-3Gal,J=2.7Hz,J=9.9Hz),3.80(1H,m,H-4Gal),3.75(3H,s,OCH3),3.73(1H,m,H-5Gal),3.51(1H,s,H-5GalNAc),2.83-2.78,2.63-2.58,2.52-2.49,2.40-2.34(4H,m,CH2 of Lev),2.04(3H,s,CH3CO);13C NMR(150MHz,CDCl3,TMS)δ208.9,172.4,158.8,157.1(q,J=24.2Hz,COCF3),138.6,138.2,137.4,137.3,130.5,129.3,129.1,128.3,128.2(2C),127.9,127.7(2C),127.6,127.5,126.4,115.9(q,J=191.2Hz,COCF3),113.4,101.2,98.8,92.7,78.1,75.4,74.9,74.5,73.7,71.4,71.3,70.6,70.1,69.4,66.5,64.5,55.2,52.4,37.9,29.7,27.8;HRMS(ESI-MS)计算值C55H58NNaO14F3 +[M+Na]+m/z 1036.3702,实测值1036.3699.
苄基3,4-二-O-苄基-6-乙酰丙酰基-α-D-吡喃半乳糖-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖
将苄基2-O-对甲氧苄基-3,4-二-O-苄基-6-乙酰丙酰基-β-D-吡喃半乳糖-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖(340mg,0.34mmol)溶于二氯甲烷/水(6.0mL/0.6mL),分批加入2,3-二氯-5,6-二氰对苯醌(154mg,0.68mmol,2.0equiv.),室温反应1小时。TLC监测反应结束后,反应液用二氯甲烷稀释,有机相依次经饱和碳酸氢钠溶液、饱和硫代硫酸钠溶液以及饱和食盐水洗涤,无水硫酸钠干燥,过滤,减压浓缩,柱层析纯化(DCM/MeOH 60:1)得到白色固体(258mg,85%)。Rf=0.36(DCM/MeOH30:1);1H NMR(600MHz,CDCl3+CD3OD,TMS)δ7.59,7.40-7.26(20H,m,aromatic),5.64(1H,s,PhCH),5.14(1H,d,H-1Gal,J=3.5Hz),4.93(2H,m,PhCH2),4.83(1H,d,PhCH2,J=11.8Hz),4.76(1H,d,H-1GalNAc,J=8.6Hz),4.62(2H,m,PhCH2),4.50(1H,d,PhCH2,J=11.4Hz),4.42(2H,m,H-4GalNAc,H-6aGalNAc),4.35(1H,t,H-2GalNAc,J=10.0Hz),4.15(1H,d,H-3GalNAc,J=12.2Hz),4.09(3H,m,H-6aGal,H-6bGalNAc),4.03(1H,dd,H-6bGal,J=2.9Hz,J=10.8Hz),3.73(2H,m,H-3Gal,),3.60(1H,dd,H-4Gal,J=2.0Hz,J=10.0Hz),3.52(1H,s,),2.82-2.76,2.69-2.64,2.52-2.47,2.43-2.39(4H,m,CH2 of Lev),2.07(3H,s,CH3CO);13C NMR(150MHz,CDCl3+CD3OD,TMS)δ209.5,172.6,158.2(q,J=24.5Hz,COCF3),138.5,138.2,137.3,137.2,129.2,128.5,128.4,128.3,127.9,128.4,128.3,127.9(2C),127.7,126.4,116.0(q,J=190.9Hz,COCF3),101.1,99.0,95.2,79.2,75.1,74.8,73.8,73.0,70.6,70.4,70.1,69.3,68.9,66.6,63.9,51.5,38.0,29.8,27.7;HRMS(ESI-MS)计算值C47H50NNaO13F3 +[M+Na]+m/z 916.3126,实测值916.3132.
2,3,4-三-O-乙酰基-β-D-吡喃葡萄糖醛酸甲酯-(1→3)-1,4,6-三-O-乙酰基-2-去氧-2-乙酰氨基-α,β-D-吡喃葡萄糖的合成
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将5.0g透明质酸(Mw=500kDa)溶于300.0mL去离子水中,溶胀过夜,缓慢滴加浓盐酸(12M,13.0mL,终浓度为0.5M),80℃加热反应两天。TLC监测反应结束后,加入碳酸氢钠固体调节pH至7.0,减压浓缩反应液体积至约20.0mL,缓慢滴入1000.0mL乙醇中,得到浅棕色絮状沉淀,减压过滤,沉淀于红外烘箱中烘干,得到黄色粉末状固体。将粗产物溶于盐酸甲醇溶液(0.02M,200.0mL)中,4℃反应4天,TLC监测反应完全后,加入三乙胺调节pH至7.0,减压浓缩,用甲苯带3次,得到棕色粉末状固体。将棕色粉末状固体溶于吡啶(100.0mL),冰浴条件下缓慢滴加乙酸酐(50.0mL),自然升至室温,反应过夜。TLC监测反应结束后,冰浴条件下滴加适量甲醇淬灭反应,反应液减压浓缩,二氯甲烷稀释,1M盐酸溶液洗涤,二氯甲烷反萃三次,合并有机相后分别经饱和碳酸氢钠溶液和饱和食盐水洗涤,无水硫酸钠干燥,过滤,减压浓缩。柱层析纯化(DCM/MeOH 60:1,0.1%三乙胺)得到浅黄色固体(2.60g)(α/β=2.5/1)。Rf=0.40(DCM/MeOH 20:1)。混合物NMR谱图见附录,端基异构体比例由1H NMR确认。ESI-Q-TOF(positive mode)计算值C27H41N2O18 +[M+NH4]+m/z 681.2354,实测值681.2360.
2,3,4-三-O-乙酰基-β-D-吡喃葡萄糖醛酸甲酯-(1→3)-1,4,6-三-O-乙酰基-2-去氧-2-三氟乙酰氨基-α-D-吡喃葡萄糖的合成
将2,3,4-三-O-乙酰基-β-D-吡喃葡萄糖醛酸甲酯-(1→3)-1,4,6-三-O-乙酰基-2-去氧-2-乙酰氨基-α,β-D-吡喃葡萄糖(500mg,0.75mmol)溶于无水吡啶(3.0mL),加入三氟乙酸酐(425μL,3.01mmol,4.0equiv.),135℃回流反应30分钟。TLC监测反应完全后,冰浴条件下加入适量甲醇淬灭反应,反应液减压浓缩后用二氯甲烷稀释,1M盐酸溶液洗涤,二氯甲烷反萃三次,合并有机相后经饱和食盐水洗涤,无水硫酸钠干燥,过滤,减压浓缩。柱层析纯化(PE/丙酮4:1)得到棕色固体(468mg,87%)。Rf=0.36(PE/丙酮1:1)。1HNMR(400MHz,CDCl3)δ6.95(1H,s,NH),6.08(1H,d,H-1GlcNAc,J=3.3Hz),5.16-5.03(3H,m,H-3GlcA,H-5GlcA,H-4GlcNAc),4.84(1H,t,H-2GlcA,J=8.0Hz),4.66(1H,d,H-1GlcA,J=7.8Hz),4.43(1H,m,H-2GlcNAc),4.18(1H,dd,H-6aGlcNAc,J=3.7Hz,J=12.5Hz),4.11-3.98(4H,m,H-6bGlcNAc,H-3GlcNAc,H-5GlcNAc,H-4GlcA),3.70(3H,s,OCH3),2.15(3H,s,CH3CO),2.09(3H,s,CH3CO),2.05(3H,s,CH3CO),1.97(6H,s,2CH3CO),1.94(3H,s,CH3CO);13C NMR(100MHz,CDCl3)δ171.0,170.1,169.7,169.6,169.3,168.6,166.9,157.2(q,J=39.6Hz,COCF3),115.7(q,J=286.3Hz,COCF3),100.1,90.3,75.5,72.4,72.0,71.0,70.0,69.4,67.5,61.7,52.8,52.2,20.8,20.7,20.5(2C),20.3,20.2;ESI-Q-TOF(positive mode)计算值C27H38F3N2O18 +[M+NH4]+m/z 735.2072,实测值735.2081.
2,3,4-三-O-乙酰基-β-D-吡喃葡萄糖醛酸甲酯-(1→3)-4,6-二-O-乙酰基-2-去氧-2-三氟乙酰氨基-α,β-D-吡喃葡萄糖的合成
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将2,3,4-三-O-乙酰基-β-D-吡喃葡萄糖醛酸甲酯-(1→3)-1,4,6-三-O-乙酰基-2-去氧-2-三氟乙酰氨基-α-D-吡喃葡萄糖(400mg,0.56mmol)溶于四氢呋喃(6.0mL)中,滴加3-二甲氨基丙胺(348μL,2.79mmol,5.0equiv.),室温反应1小时。TLC监测反应完全后,反应液用二氯甲烷稀释,1M盐酸溶液洗涤,二氯甲烷反萃三次,合并有机相后经饱和食盐水洗涤,无水硫酸钠干燥,过滤,减压浓缩,得到的粗产物不经进一步纯化直接用于下一步反应。
2,3,4-三-O-乙酰基-β-D-吡喃葡萄糖醛酸甲酯-(1→3)-4,6-二-O-乙酰基-2-去氧-2-三氟乙酰氨基-D-吡喃葡萄糖[2,1,-d]2-噁唑啉的合成
氩气保护下,将2,3,4-三-O-乙酰基-β-D-吡喃葡萄糖醛酸甲酯-(1→3)-4,6-二-O-乙酰基-2-去氧-2-三氟乙酰氨基-α,β-D-吡喃葡萄糖溶于无水乙腈(10.0mL)中,加入甲磺酸酐(293mg,1.68mmol),室温反应25分钟后,加入三乙胺(1.55mL,11.2mmol),室温反应2小时。TLC监测反应完全后,反应液用二氯甲烷稀释,饱和碳酸氢钠溶液洗涤,二氯甲烷反萃三次,合并有机相后经饱和食盐水洗涤,无水硫酸钠干燥,过滤,减压浓缩。柱层析纯化(PE/丙酮3:1)得到白色固体(284mg,两步收率77%)。Rf=0.49(PE/丙酮1:1)。1H NMR(400MHz,CDCl3)δ6.26(1H,d,H-1GlcNAc,J=7.6Hz),5.27-5.16(3H,m,H-3GlcA,H-4GlcA,H-4GlcNAc),4.97(1H,t,H-2GlcA,J=7.9Hz),4.87(1H,d,H-1GlcA,J=7.9Hz),4.26(1H,d,H-2GlcNAc,J=7.3Hz),4.18(3H,m,H-3GlcNAc,H-6aGlcNAc,H-6bGlcNAc),4.11(1H,d,H-5GlcA,J=9.6Hz),3.72(3H,s,OCH3),3.65(1H,m,H-5GlcNAc),2.07(3H,s,CH3CO),2.04(3H,s,CH3CO),2.02(3H,s,CH3CO),2.00(3H,s,CH3CO),1.99(3H,s,CH3CO);13C NMR(100MHz,CDCl3)δ170.7,170.1,169.8,169.4,169.2,166.9,156.3(q,J=40.9Hz,COCF3),116.0(q,J=273.1Hz,COCF3),102.9,100.9,76.7,72.4,72.1,71.2,69.1,68.8,67.1,64.8,63.4,53.0,20.8,20.7(2C),20.6(2C);HRMS(ESI-MS)计算值C25H30NNaO16F3 +[M+Na]+m/z 680.1409,实测值680.1391.
苄基2,3,4-三-O-乙酰基-β-D-吡喃葡萄糖醛酸甲酯-(1→3)-4,6-二-O-乙酰基-2-去氧-2-三氟乙酰氨基-β-D-吡喃葡萄糖-(1→2)-3,4-二-O-苄基-6-乙酰丙酰基-α-D-吡喃半乳糖-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖的合成
氩气保护下,将2,3,4-三-O-乙酰基-β-D-吡喃葡萄糖醛酸甲酯-(1→3)-4,6-二-O-乙酰基-2-去氧-2-三氟乙酰氨基-D-吡喃葡萄糖[2,1,-d]2-噁唑啉(140mg,0.21mmol,1.5equiv.)和苄基3,4-二-O-苄基-6-乙酰丙酰基-α-D-吡喃半乳糖-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖(127mg,0.14mmol)溶于无水二氯甲烷中(2.0mL),加入分子筛(200mg),室温搅拌2小时后,冷却至-20℃,加入三氟甲磺酸三甲基硅酯(7.6μL,0.042mmol,0.3equiv.),-20℃搅拌反应2小时后,缓慢升至室温反应过夜。TLC监测反应结束后,滴加适量三乙胺淬灭反应,硅藻土过滤除去分子筛,滤液减压浓缩,柱层析分离(PE/丙酮2:1)纯化得到白色固体(195mg,90%)。Rf=0.39(PE/丙酮1:1);1H NMR(600MHz,CDCl3,TMS)δ8.08(1H,s,NH),7.88(1H,d,NH,J=9.4Hz),7.62,7.46-7.41,7.36-7.25,7.15(20H,m,aromatic),5.62(1H,s,PhCH),5.16(1H,t,H-4GlcA,J=9.6Hz),5.10(1H,d,H-1Gal,J=3.6Hz),5.07(1H,t,H-3GlcA,J=9.4Hz),4.98-4.94(3H,m,H-2GlcA,H-1GlcNAc,PhCH2),4.80(1H,d,PhCH2,J=11.6Hz),4.72(1H,d,H-1GalNAc,J=8.3Hz),4.67(1H,d,PhCH2,J=12.0Hz),4.62(3H,m,H-3GlcNAc,H-4GlcNAc,PhCH2),4.54(1H,q,H-2GalNAc,J=9.4Hz),4.45(1H,d,H-4GalNAc,J=3.2Hz),4.40-4.36(3H,m,H-6aGalNAc,H-6aGlcNAc,H-1GlcA),4.30(1H,d,PhCH2,J=12.0Hz),4.27(1H,d,PhCH2,J=11.7Hz),4.16(1H,m,H-6bGalNAc),4.13(1H,dd,H-6aGal,J=2.1Hz,J=11.9Hz),4.08(1H,dd,H-2Gal,J=3.6Hz,J=10.2Hz),3.93(2H,m,H-5GlcA,H-3GalNAc),3.88(1H,dd,H-6bGlcNAc,J=2.1Hz,J=12.2Hz),3.83(1H,m,H-6bGal),3.71(3H,s,OCH3),3.70(1H,dd,H-3Gal,J=2.8Hz,J=10.5Hz),3.63(1H,d,H-5Gal,J=8.3Hz),3.58(1H,m,H-5GlcNAc),3.49(1H,s,H-5GalNAc),3.45(1H,s,H-4Gal),2.94-2.86(1H,m,CH2 ofLev),2.56-2.51(3H,m,CH2 of Lev,H-2GlcNAc),2.31-2.28(1H,m,CH2 of Lev),2.10(3H,s,CH3CO),2.04(3H,s,CH3CO),2.00(12H,m,CH3CO);13CNMR(150MHz,CDCl3,TMS)δ210.4,172.3,170.7,169.9,169.8,169.4(2C),166.9,157.9(q,J=24.5Hz,COCF3),157.7(q,J=24.3Hz,COCF3),138.1,137.9,137.3,137.2,130.3,128.6(2C),128.3,128.2,128.0,127.9,127.8,127.7,126.4,116.0(q,J=191.1Hz,COCF3),115.4(q,J=191.2Hz,COCF3),101.7,100.3,99.1,98.7,93.8,77.6,76.1,75.8,74.7,74.0,73.6,72.6(2C),72.4,71.9,70.9,70.1,70.0,69.9,69.4,69.3,68.6,66.5,64.8,62.3,58.4,52.6,51.3,37.9,29.7,27.7,20.8,20.6,20.4;ESI-Q-TOF(positive mode)计算值C72H84F6N3O29 +[M+NH4]+m/z1568.5095,实测值1568.5099.
苄基2,3,4-三-O-乙酰基-β-D-吡喃葡萄糖醛酸甲酯-(1→3)-4,6-二-O-乙酰基-2-去氧-2-三氟乙酰氨基-β-D-吡喃葡萄糖-(1→2)-3,4-二-O-苄基-α-D-吡喃半乳糖-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖的合成
将苄基2,3,4-三-O-乙酰基-β-D-吡喃葡萄糖醛酸甲酯-(1→3)-4,6-二-O-乙酰基-2-去氧-2-三氟乙酰氨基-β-D-吡喃葡萄糖-(1→2)-3,4-二-O-苄基-6-乙酰丙酰基-α-D-吡喃半乳糖-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖(224mg,0.14mmol)溶于二氯甲烷(2.0mL),冰浴条件下滴加0.5M醋酸肼(0.87mL,水合肼溶于吡啶/乙酸=3:2的混合溶液中),缓慢升至室温反应1小时。TLC监测反应结束后,反应液用二氯甲烷稀释,有机相依次经1M盐酸溶液、饱和碳酸氢钠溶液和饱和食盐水洗涤,无水硫酸钠干燥,减压浓缩,柱层析纯化(DCM/MeOH=50:1)纯化得到白色固体(178mg,85%)。Rf=0.21(DCM/MeOH 30:1);1H NMR(600MHz,CDCl3,TMS)δ7.56,7.39,7.32-7.19,7.01(23H,m,aromatic,NH,OH),5.61(1H,s,PhCH),5.14(1H,t,H-4GlcA,J=9.4Hz),5.10(1H,t,H-3GlcA,J=8.9Hz),5.06(1H,d,H-1Gal,J=3.4Hz),4.90(2H,m,H-2GlcA,PhCH2),4.81(1H,d,H-1GlcNAc,J=8.4Hz),4.73(2H,m,H-4GlcNAc,PhCH2),4.65-4.59(3H,m,H-1GalNAc,PhCH2),4.41(1H,d,H-1GlcA,J=8.0Hz),4.39-4.29(5H,m,H-2GalNAc,H-4GalNAc,H-6aGalNAc,PhCH2),4.26(1H,dd,H-6aGlcNAc,J=5.6Hz,J=12.6Hz),4.17(2H,m,H-3GlcNAc,H-6bGalNAc),4.06(1H,m,H-6bGlcNAc),4.00(1H,dd,H-2Gal,J=3.5Hz,J=10.2Hz),3.93(2H,m,H-3GalNAc,H-5GlcA),3.76(1H,dd,H-3Gal,J=2.6Hz,J=10.1Hz),3.72(3H,s,OCH3),3.70(1H,m,H-5Gal),3.61(2H,m,H-6aGal,H-5GlcNAc),3.50(1H,d,H-4Gal,J=1.4Hz),3.43(2H,m,H-5GalNAc,H-2GlcNAc),3.32(1H,m,H-6bGal),2.11(3H,s,CH3CO),2.00(9H,m,CH3CO),1.92(3H,s,CH3CO);13C NMR(150MHz,CDCl3,TMS)δ171.2,169.9,169.6,169.4,169.3,166.8,157.8(q,J=24.5Hz,COCF3),157.5(q,J=24.6Hz,COCF3),138.0,137.8,137.7,137.0,129.4,128.6,128.4(2C),128.2,128.0(2C),127.9(2C),127.7,126.3,115.8(q,J=191.1Hz,COCF3),115.5(q,J=191.1Hz,COCF3),101.0,100.6,100.2,98.2,97.5,77.3,76.7,75.8,74.7,72.9,72.4(2C),72.3,71.7,70.9,69.9,69.3,69.1,68.1,66.8,62.5,62.1,56.7,52.8,52.7,20.8,20.6,20.5,20.4(2C);ESI-Q-TOF(positive mode)计算值C67H78F6N3O27 +[M+NH4]+m/z 1470.4727,实测值1470.4736.
苄基2,3,4-三-O-乙酰基-β-D-吡喃葡萄糖醛酸甲酯-(1→3)-4,6-二-O-乙酰基-2-去氧-2-三氟乙酰氨基-β-D-吡喃葡萄糖-(1→2)-3,4-二-O-苄基-α-D-吡喃半乳糖醛酸-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖的合成
将苄基2,3,4-三-O-乙酰基-β-D-吡喃葡萄糖醛酸甲酯-(1→3)-4,6-二-O-乙酰基-2-去氧-2-三氟乙酰氨基-β-D-吡喃葡萄糖-(1→2)-3,4-二-O-苄基-α-D-吡喃半乳糖-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖的合成(155mg,0.11mmol)溶于二氯甲烷/水(0.8mL/0.4mL),冰浴条件下依次加入2,2,6,6-四甲基哌啶-氮-氧化物(7mg,0.044mmol,0.4equiv.)、二乙酰氧基碘苯(71mg,0.22mmol,2.0equiv.),自然升至室温反映过夜。TLC监测反应结束后,反应液用二氯甲烷稀释,有机相经饱和硫代硫酸钠溶液洗涤,无水硫酸钠干燥,减压浓缩,柱层析(DCM/MeOH=60:1,0.1%CH3COOH)纯化得到淡黄色固体(135mg,86%)。Rf=0.18(DCM/MeOH 30:1);1H NMR(600MHz,CDCl3+CD3COOD,TMS)δ7.58,7.39,7.31-7.24,7.18,7.15(22H,m,aromatic,NH),5.69(1H,s,PhCH),5.24(1H,s,H-1GalA),5.15(2H,m,H-3GlcA,H-4GlcA),4.95(1H,t,H-2GlcA,J=8.3Hz),4.91(1H,d,PhCH2,J=12.2Hz),4.78(1H,t,H-4GlcNAc,J=9.5Hz),4.71-4.64(5H,m,H-1GlcNAc,H-1GalNAc,3PhCH2),4.52(1H,d,H-1GlcA,J=8.1Hz),4.46(1H,s,H-4GalNAc),4.36(2H,m,H-6aGalNAc,PhCH2),4.30-4.23(5H,m,H-2GalNAc,H-5GalA,H-6aGlcNAc,H-6bGalNAc,PhCH2),4.13(1H,m,H-3GlcNAc),4.05(2H,m,H-3GalNAc,H-6bGlcNAc),3.99(2H,m,H-2GalA,H-5GlcA),3.86(1H,dd,H-3GalA,J=2.2Hz,J=10.0Hz),3.80(1H,s,H-4GalA),3.73(3H,s,OCH3),3.71-3.60(2H,m,H-2GlcNAc,H-5GlcNAc),3.47(1H,s,H-5GalNAc),2.13(3H,s,CH3CO),2.04(3H,s,CH3CO),2.00(6H,s,2CH3CO),1.93(3H,s,CH3CO);13C NMR(150MHz,CDCl3+CD3COOD,TMS)δ172.8,171.6,170.3,170.0,169.9,169.5,167.2,157.7(q,J=24.7Hz,2COCF3),137.7,137.0,136.9,129.5,128.8,128.5,128.4,128.2,128.1(2C),128.0,127.8,126.5,115.8(q,J=191.1Hz,2COCF3),101.4,101.2,100.5,98.5,77.7,75.8,75.6,73.8,72.5(3C),71.0,70.8,70.1,69.5,69.1,68.2,66.7,62.3,56.1,52.9,52.5,20.8,20.6(2C),20.4;ESI-Q-TOF(positivemode)计算值C67H76F6N3O28 +[M+NH4]+m/z 1484.4520,实测值1484.4539.
苄基2,3,4-三-O-乙酰基-β-D-吡喃葡萄糖醛酸甲酯-(1→3)-4,6-二-O-乙酰基-2-去氧-2-三氟乙酰氨基-β-D-吡喃葡萄糖-(1→2)-N-L-苏氨酸甲酯-3,4-二-O-苄基-α-D-吡喃半乳糖醛酸-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖的合成
将苄基2,3,4-三-O-乙酰基-β-D-吡喃葡萄糖醛酸甲酯-(1→3)-4,6-二-O-乙酰基-2-去氧-2-三氟乙酰氨基-β-D-吡喃葡萄糖-(1→2)-3,4-二-O-苄基-α-D-吡喃半乳糖醛酸-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖(70.0mg,0.048mmol)溶于无水二氯甲烷(0.6mL),依次加入N-羟基琥珀酰亚胺(11.0mg,0.096mmol,2.0equiv.)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(18.4mg,0.096mmol,2.0equiv.),室温反应过夜。TLC监测反应结束后,反应液用二氯甲烷稀释,有机相依次经1M盐酸溶液、饱和碳酸氢钠溶液和饱和食盐水洗涤,无水硫酸钠干燥,减压浓缩,得到粗产物直接用于下一步反应。将粗产物与L-苏氨酸甲酯(40.7mg,0.24mmol,5.0equiv.)溶于N,N-二甲基甲酰胺/三乙胺(2.0mL/0.1mL),升温至40℃反应过夜。TLC监测反应结束后,直接将反应液减压浓缩,柱层析(DCM/MeOH=60:1)纯化得到淡黄色固体(59.6mg,两步收率79%)。Rf=0.26(DCM/MeOH 30:1);1H NMR(600MHz,CDCl3,TMS)δ8.00,7.84,7.54,7.39,7.33-7.17,7.04(23H,m,aromatic,NH),5.68(1H,s,PhCH),5.23(1H,d,H-1GalA,J=2.9Hz),5.15-5.10(2H,m,H-3GlcA,H-4GlcA),4.92(2H,m,H-2GlcA,PhCH2),4.79(2H,m,H-1GlcNAc,H-4GlcNAc),4.70(1H,d,PhCH2,J=10.9Hz),4.65(2H,m,PhCH2,H-1GalNAc),4.59(1H,d,PhCH2,J=12.3Hz),4.50(1H,d,H-1GlcA,J=8.0Hz),4.48(1H,d,H-4GalNAc,J=2.8Hz),4.45(1H,dd,CH,J=2.2Hz,J=8.9Hz),4.41(1H,d,PhCH2,J=11.0Hz),4.37(2H,m,PhCH2,H-6aGalNAc),4.32-4.23(6H,m,H-5GalA,H-6aGlcNAc,H-2GalNAc,H-6bGalNAc,H-3GlcNAc,CH),4.12(2H,m,H-4GalA,H-6bGlcNAc),4.02(2H,m,H-2GalA,H-3GalNAc),3.94(1H,m,H-5GlcA),3.81(1H,d,H-3GalA,J=2.2Hz,J=9.9Hz),3.71(6H,s,2OCH3),3.62(1H,m,H-5GlcNAc),3.49(1H,s,H-5GalNAc),3.44(1H,m,H-2GlcNAc),2.12(3H,s,CH3CO),2.02(3H,s,CH3CO),1.99(6H,s,2CH3CO),1.89(3H,s,CH3CO),0.96(3H,d,CH3,J=6.4Hz);13CNMR(150MHz,CDCl3,TMS)δ171.4(2C),170.0,169.7,169.6,169.4,168.8,167.0,162.8,157.8(q,J=24.6Hz,2COCF3),157.5(q,J=24.6Hz,2COCF3),138.4,137.7,137.5,137.1,129.4,128.6,128.5,128.4,128.1,128.0,127.9,127.8,127.7,127.6,126.3,115.9(q,J=191.1Hz,2COCF3),115.7(q,J=191.1Hz,2COCF3),101.0,100.9,100.2,98.4,97.7,77.5,76.5,76.3,76.2,75.3,75.1,72.9,72.6,72.5,72.4(2C),72.1,70.9,70.1,69.4,69.0,68.0,67.4,66.8,62.1,57.0,56.6,52.8,52.7,52.6,20.7,20.6,20.5,20.4(2C),19.6;ESI-Q-TOF(positive mode)计算值C72H85F6N4O30 +[M+NH4]+m/z1599.5153,实测值1599.5133.
苄基β-D-吡喃葡萄糖醛酸-(1→3)-2-去氧-2-乙酰氨基-β-D-吡喃葡萄糖-(1→2)-3,4-二-O-苄基-α-D-吡喃半乳糖醛酸-(1→3)-2-去氧-2-乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖的合成
将苄基2,3,4-三-O-乙酰基-β-D-吡喃葡萄糖醛酸甲酯-(1→3)-4,6-二-O-乙酰基-2-去氧-2-三氟乙酰氨基-β-D-吡喃葡萄糖-(1→2)-3,4-二-O-苄基-α-D-吡喃半乳糖醛酸-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖(30.0mg,0.02mmol)溶于甲醇(1.0mL),滴加饱和氢氧化锂溶液(1.0mL),升温至35℃反应48小时。TLC监测反应结束后,IR-120阳离子交换树脂中和反应液至pH=7,过滤,用甲醇洗涤树脂,滤液减压浓缩,浓缩得到的粗产物溶于甲醇/水(1.0mL/1.0mL)混合溶液,加入碳酸钾固体调节pH=11~12,滴加乙酸酐(0.3mL),再加入碳酸钾固体调节pH=11~12,室温反应过夜。TLC监测反应结束后,IR-120阳离子交换树脂中和反应液至pH=7,过滤,用甲醇洗涤树脂,滤液减压浓缩,以CH2Cl2/MeOH 1:1作为洗脱液,使用Sephadex LH-20纯化得到白色固体(18.6mg,两步收率82%)。Rf=0.58(CHCl3/MeOH/H2O=1:1:0.3);1H NMR(600MHz,MeOD-d6,TMS)δ7.64,7.40,7.36-7.30,7.27-7.19(20H,m,aromatic),5.61(1H,s,PhCH),5.41(1H,s,H-1GalA),4.89(1H,m,PhCH2),4.77(3H,m,2PhCH2,H-4GalNAc),4.68-4.57(5H,m,3PhCH2,H-1GlcNAc,H-1GalNAc),4.29-4.19(6H,m,H-4GalA,H-5GalA,H-1GlcA,H-2GalNAc,H-6aGalNAc,H-6bGalNAc),4.03(1H,d,H-2GalA,J=7.4Hz),3.93-3.81(5H,m,H-3GalA,H-2GlcNAc,H-6aGlcNAc,H-6bGlcNAc,H-3GalNAc),3.66(1H,m,H-3GlcNAc),3.59(1H,m,H-5GlcA),3.54(1H,s,H-5GalNAc),3.47(2H,m,H-4GlcA,H-4GlcNAc),3.38(1H,m,H-3GlcA),3.35(1H,s,H-5GlcNAc),3.26(1H,t,H-2GlcA,J=8.0Hz);13CNMR(150MHz,MeOD-d6,TMS)δ175.9,174.9,174.1,173.9,140.2,139.8,139.1,129.7,129.4,129.3,129.1,129.0,128.9,128.6,128.4,128.3,127.8,105.1,104.8,102.0,101.2,86.5,80.7,77.4,77.3,76.1,75.4,74.6,74.2,73.8,73.3,72.9,71.5,70.4,70.1,68.3,62.3,56.6,52.3,49.6,23.3,23.2;ESI-Q-TOF(negative mode)计算值C56H65N2O23 -[M-H]-m/z 1133.3984,实测值1133.3983.
β-D-吡喃葡萄糖醛酸-(1→3)-2-去氧-2-乙酰氨基-β-D-吡喃葡萄糖-(1→2)-α-D-吡喃半乳糖醛酸-(1→3)-2-去氧-2-乙酰氨基-α,β-D-吡喃半乳糖(化合物CP-1)的合成
将苄基β-D-吡喃葡萄糖醛酸-(1→3)-2-去氧-2-乙酰氨基-β-D-吡喃葡萄糖-(1→2)-3,4-二-O-苄基-α-D-吡喃半乳糖醛酸-(1→3)-2-去氧-2-乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖(18.6mg,16.4μmol)溶于甲醇/水(1.0mL/1.0mL)混合溶液中,加入10%氢氧化钯碳(37.0mg),于40Pa的氢气压力下,室温反应48小时。TLC监测反应结束后,过滤,减压浓缩,以纯水作为洗脱液,使用Sephadex LH-20纯化得到白色固体(10.8mg,85%)。Rf=0.11(CHCl3/MeOH/H2O=1:1:0.3);1H NMR(600MHz,D2O)δ5.31(0.97H,s),5.19(0.53H,d,J=3.3Hz),4.68(1.69H,m),4.49(1.02H,d,J=7.7Hz),4.37(0.58H,s),4.31(0.49H,s),4.24(1.52H,m),4.18(0.63H,s),4.11(0.52H,m),3.98-3.75(11.26H,m),3.70(0.53H,m),3.51(3.46H,m),3.35(0.92H,m),2.02(3H,s),1.98(2.88H,d,J=4.4Hz);13C NMR(150MHz,D2O)δ175.5,103.9,103.8,103.7,97.8,97.5,95.8,92.0,83.2,78.7,78.6,78.4,76.6,76.2(3C),76.0,75.4,73.5,72.7,72.4,71.7,70.7,69.4(2C),69.1,69.0,66.3,65.4,61.9,61.7,61.6,61.5,55.4,52.8,49.2,23.0,22.9,22.7;ESI-Q-TOF(positive mode)计算值C28H42N2O23 2-[M-2H]2-m/z 387.1095,实测值387.1090.
苄基β-D-吡喃葡萄糖醛酸-(1→3)-2-去氧-2-乙酰氨基-β-D-吡喃葡萄糖-(1→2)-N-L-苏氨酸-3,4-二-O-苄基-α-D-吡喃半乳糖醛酸-(1→3)-2-去氧-2-乙酰氨基-β-D-吡喃半乳糖的合成
将苄基2,3,4-三-O-乙酰基-β-D-吡喃葡萄糖醛酸甲酯-(1→3)-4,6-二-O-乙酰基-2-去氧-2-三氟乙酰氨基-β-D-吡喃葡萄糖-(1→2)-N-L-苏氨酸甲酯-3,4-二-O-苄基-α-D-吡喃半乳糖醛酸-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖(30mg,19.0μmol)溶于80%AcOH水溶液(2.0mL)中,加热至60℃反应2小时,反应结束恢复室温后,用甲苯带干溶剂得到粗产物,随后直接将粗产物溶于甲醇(1.0mL),滴加饱和氢氧化锂溶液(1.0mL),升温至35℃反应48小时。TLC监测反应结束后,IR-120阳离子交换树脂中和反应液至pH=7,过滤,用甲醇洗涤树脂,滤液减压浓缩,浓缩得到的粗产物溶于甲醇/水(1.0mL/1.0mL)混合溶液,加入碳酸钾固体调节pH=11~12,滴加乙酸酐(0.3mL),再加入碳酸钾固体调节pH=11~12,室温反应过夜。TLC监测反应结束后,IR-120阳离子交换树脂中和反应液至pH=7,过滤,用甲醇洗涤树脂,滤液减压浓缩,以CH2Cl2/MeOH 1:1作为洗脱液,使用Sephadex LH-20纯化得到白色固体(15.1mg,三步收率69%)。1H NMR(600MHz,CD3OD+D2O)δ7.42-7.25(15H,m,aromatic),5.52(1H,d,H-1GalA,J=3.7Hz),4.92-4.64(6H,m,5PhCH2,H-1GlcNAc),4.49(2H,m,H-1GalNAc,PhCH2),4.42(1H,d,H-1GlcA,J=7.8Hz),4.37(1H,d,H-4GalNAc,J=2.8Hz),4.32(1H,m,H-4GalA),4.29(1H,s,H-5GalA),4.21-4.16(3H,m,CHOH,CHNH,H-2GalNAc),4.12(1H,dd,H-2GalA,J=3.6Hz,J=10.1Hz),3.98(1H,dd,H-3GalA,J=2.9Hz,J=10.3Hz),3.94-3.77(7H,m,H-6aGalNAc,H-6bGalNAc,H-3GalNAc,H-2GlcNAc,H-3GlcNAc,H-6aGlcNAc,H-6bGlcNAc),3.68(1H,d,H-5GlcA,J=9.6Hz),3.65(1H,t,H-5GalNAc,J=6.7Hz),3.53-3.45(4H,m,H-4GlcA,H-4GlcNAc,H-3GlcA,H-5GlcNAc),3.32(1H,m,H-2GlcA),1.84(3H,s,CH3CO),1.75(3H,s,CH3CO),0.98(3H,d,CH3,J=6.2Hz);13C NMR(150MHz,CD3OD+D2O)δ175.6,173.7,173.3,169.2,138.2,137.8,137.4,128.4,128.3,128.1,127.8,127.7,127.4,103.3,103.1,100.9,96.9,83.2,78.0,77.9,76.6,76.1,76.0,75.7,75.2,74.8,74.6,73.0,72.9,71.9,71.1,70.8,69.0,67.7,64.4,60.9(2C),59.0,55.0,50.5,22.0,21.8,19.1;ESI-Q-TOF(negative mode)计算值C53H67N3O25 2-[M-2H]2-m/z 572.7037,实测值572.7036.
实施例2
β-D-吡喃葡萄糖醛酸-(1→3)-2-去氧-2-乙酰氨基-β-D-吡喃葡萄糖-(1→2)-N-L-苏氨酸-α-D-吡喃半乳糖醛酸-(1→3)-2-去氧-2-乙酰氨基-α,β-D-吡喃半乳糖(化合物CP-2)的合成
将苄基β-D-吡喃葡萄糖醛酸-(1→3)-2-去氧-2-乙酰氨基-β-D-吡喃葡萄糖-(1→2)-N-L-苏氨酸-3,4-二-O-苄基-α-D-吡喃半乳糖醛酸-(1→3)-2-去氧-2-乙酰氨基-β-D-吡喃半乳糖(19.0mg,16.6μmol)溶于甲醇/水(1.0mL/1.0mL)混合溶液中,加入10%氢氧化钯碳(38.0mg),于40Pa的氢气压力下,室温反应48小时。TLC监测反应结束后,过滤,减压浓缩,以纯水作为洗脱液,使用Sephadex LH-20纯化得到白色固体(12.5mg,86%)。1H NMR(600MHz,D2O)δ5.46(0.86H,m),5.18(0.48H,d,J=3.6Hz),4.81(0.56H,m),4.66(0.95H,m),4.48(0.98H,d,J=7.9Hz),4.39(0.47H,d,J=2.4Hz),4.32-4.23(5.18H,m),4.14-4.06(1.13H,m),3.99-3.68(11.95H,m),3.55-3.47(4.46H,m),3.34(1.26H,m);13C NMR(150MHz,D2O)δ175.69,175.03,170.93,170.76,103.9,103.8,103.7,98.0,97.7,96.0,92.0,83.1,79.0,78.3,78.2,76.6,76.4,76.2(2C),76.0,75.4,73.5,72.5,72.2,72.1,70.7,70.6(2C),69.4,69.3,69.1,69.0,68.7(2C),66.4,65.5,61.9,61.6,61.5,60.4,55.4,52.6,49.2,23.0,22.8,22.5,20.0;ESI-Q-TOF(negative mode)计算值C32H49N3O25 2-[M-2H]2-m/z437.6333,实测值437.6323.
实施例3
甲基2-去氧-2-三氟乙酰氨基-3,4,6-三-O-乙酰基-β-D-吡喃半乳糖的合成
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氩气保护下,将对甲苯基2-去氧-2-三氟乙酰氨基-3,4,6-三-O-乙酰基-1-硫-β-D-吡喃半乳糖(101mg,0.20mmol)、N-碘代丁二酰亚胺(70mg,0.31mmol,1.5equiv.)、分子筛溶于无水二氯甲烷(1.7mL)中,加入甲醇(50μL,1.24mmol,6.0equiv.),室温搅拌2小时,随后降温至-20℃,滴加三氟甲磺酸(5.0μL,62.5μmol,0.30equiv.),继续搅拌反应3小时,移至室温反应过夜。TLC监测反应结束后,加入适量三乙胺淬灭,硅藻土滤除分子筛,二氯甲烷洗涤滤饼数次,合并滤液,减压浓缩,柱层析纯化(石油醚/EtOAc 3:1)得到白色固体(73mg,88%)。Rf=0.39(石油醚/EtOAc 1:1);1H NMR(400MHz,CCl3,TMS)δ7.29(1H,d,J=8.9Hz,NH),5.37(1H,d,J=3.2Hz),5.28(1H,dd,J=11.2,3.4Hz),4.57(1H,d,J=8.4Hz)4.25-4.06(3H,m),3.98(1H,t,J=6.5),3.49(3H,s),2.15(3H,s),2.03(3H,s),1.96(3H,s);13C NMR(100MHz,CDCl3,TMS)δ170.5,170.5,170.2,157.5,114.2,101.2,70.7,69.5,66.5,61.3,57.0,51.8,20.6,20.6,20.3.MS(ESI-MS)计算值C15H21F3NO9 +[M+H]+m/z 415.3,实测值416.3.
甲基2-去氧-2-三氟乙酰氨基-β-D-吡喃半乳糖的合成
将甲基2-去氧-2-三氟乙酰氨基-3,4,6-三-O-乙酰基-β-D-吡喃半乳糖(3.10g,7.48mmol)溶于甲醇(50.0mL),加入适量甲醇钠调节pH至9~10,室温下搅拌反应2小时。TLC监测反应结束后,向反应液中加入阳离子树脂中和至pH=7,过滤,滤液减压浓缩至干,得到黄色糖浆。Rf=0.75(DCM/MeOH5:1),直接用于下一步反应。
甲基2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖的合成
氩气保护下,将甲基2-去氧-2-三氟乙酰氨基-β-D-吡喃半乳糖(2.16g,7.48mmol)和(+)-樟脑磺酸(3.46g,14.9mmol,2.0equiv.)溶于无水乙腈(60.0mL)中,加入苯甲醛二甲缩醛(4.36mL,29.0mmol,4.0equiv.),升温至40℃反应过夜。TLC监测反应完全后,滴加适量三乙胺淬灭反应,反应液减压浓缩,柱层析(石油醚/EtOAc 2:1)纯化得到化合物白色固体(2.24g,80%)。Rf=0.72(石油醚/丙酮1:2).MS(ESI-MS)计算值C16H19F3NO6 +[M+H]+m/z378.3,实测值378.3.
甲基2-O-对甲氧苄基-3,4-二-O-苄基-6-乙酰丙酰基-α-D-吡喃半乳糖-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖
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氩气保护下,将对甲苯基2-O-对甲氧苄基-3,4-二-O-苄基-6-乙酰丙酰基-1-硫-β-D-吡喃半乳糖(5.80g,8.48mmol,1.5equiv.)、甲基2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖(2.15g,5.70mmol)溶于无水二氯甲烷/N,N-二甲基甲酰胺(70.0mL/14.0mL),加入分子筛(8.0g),室温搅拌2小时后,冷却至0℃,依次加入N-碘代丁二酰亚胺(2.50g,11.1mmol,2.0equiv.)和三氟甲磺酸银(731mg,2.85mmol,0.5equiv.),0℃搅拌反应2小时后,缓慢升至室温反应过夜。TLC监测反应结束后,滴加适量三乙胺淬灭反应,硅藻土过滤除去分子筛,滤液减压浓缩,柱层析分离(石油醚/EtOAc 1:1)纯化得到化合物I-12(4.66g,87%),白色固体。Rf=0.58(DCM/MeOH 25:1);1H NMR(400MHz,CDCl3,TMS)δ7.75(1H,b),7.61-7.48(2H,m),7.39-7.17(13H,m),6.95(2H,d,J=8.5Hz),6.62(2H,d,J=8.6Hz),5.54(1H,s,),5.20(1H,d,J=3.6Hz),4.94(1H,d,J=11.4Hz),4.85-4.73(2H,m),4.65(1H,d,J=11.8Hz),4.54(1H,d,J=11.4Hz),4.46(2H,s),4.42(1H,s),4.32(1H,d,J=12.4),4.20(3H,q,J=5.3,4.0Hz),4.12-4.02(4H,m),3.92-3.87(1H,m),3.85-3.81(1H,m),3.79-3.74(1H,m),3.73(3H,s)3.50(3H,s),2.90-2.79(1H,m),2.75-2.38(3H,m),2.18(3H,s);13C NMR(100MHz,CDCl3,TMS)δ208.9,172.5,158.9,157.2(COCF3),138.6,138.3,137.6,130.5,129.5,129.0,128.4,128.3(2C),128.2,127.8,127.7,127.5,126.4,116.0(COCF3),113.5,101.1,100.6,92.8,78.2,75.4,74.7,74.6,73.5,71.4,71.1,70.5,70.1,69.4,66.5,64.2,56.5,55.2,52.5,38.0,29.8,27.8;ESI-Q-TOF(positive mode)计算值C49H54F3NO14 +[M+NH4]+m/z 955.3840,实测值955.3956.
甲基3,4-二-O-苄基-6-乙酰丙酰基-α-D-吡喃半乳糖-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖
将甲基2-O-对甲氧苄基-3,4-二-O-苄基-6-乙酰丙酰基-β-D-吡喃半乳糖-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖(4.66g,4.97mmol)溶于二氯甲烷/水(100mL/10.0mL),分批加入2,3-二氯-5,6-二氰对苯醌(2.17g,9.56mmol,1.9equiv.),室温反应1小时。TLC监测反应结束后,反应液用二氯甲烷稀释,有机相依次经饱和碳酸氢钠溶液、饱和硫代硫酸钠溶液以及饱和食盐水洗涤,无水硫酸钠干燥,过滤,减压浓缩,柱层析纯化(DCM/MeOH 40:1)得到白色固体(3.32mg,82%)。Rf=0.79(石油醚/丙酮1:1);1H NMR(400MHz,CDCl3,TMS)δ7.71(1H,d,J=9.3Hz),7.55(2H,dd,J=7.8,1.8Hz),7.43-7.22(13H,m),5.60(1H,s),5.19(1H,d,J=3.9Hz),4.94(1H,d,J=11.4Hz),4.86(1H,d,J=11.8Hz),4.67-4.57(2H,m),4.50(1H,d,J=11.5Hz),4.47-4.35(3H,m),4.20(1H,dd,J=11.8,2.7Hz),4.15-4.02(4H,m),3.83-3.76(1H,m),3.72(1H,b),3.58(1H,dd,J=10.0,2.7Hz),3.52-3.46(4H,m),2.93(1H,ddd,J=19.1,10.1,3.5Hz),2.74-2.55(2H,m),2.42(1H,m),2.21(3H,s);13C NMR(100MHz,CDCl3,TMS)δ210.3,172.2,157.3(COCF3),138.5,138.2,137.2,129.1,128.4,128.3,128.3(2C),127.8(2C),127.6,126.2,115.9(COCF3),101.0(2C),94.5,79.6,75.5,74.6,74.0,73.0,70.6,70.3,69.2,69.1,66.5,64.7,56.3,50.8,38.1,30.0,27.7.ESI-Q-TOF(positive mode)计算值C41H50F3NO13 +[M+NH4]+m/z835.3840,实测值835.3956.
甲基2,3,4-三-O-乙酰基-β-D-吡喃葡萄糖醛酸甲酯-(1→3)-4,6-二-O-乙酰基-2-去氧-2-三氟乙酰氨基-β-D-吡喃葡萄糖-(1→2)-3,4-二-O-苄基-6-乙酰丙酰基-α-D-吡喃半乳糖-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖的合成
氩气保护下,将2,3,4-三-O-乙酰基-β-D-吡喃葡萄糖醛酸甲酯-(1→3)-4,6-二-O-乙酰基-2-去氧-2-三氟乙酰氨基-D-吡喃葡萄糖[2,1,-d]2-噁唑啉(1.50g,2.28mmol,1.4equiv.)和甲基3,4-二-O-苄基-6-乙酰丙酰基-α-D-吡喃半乳糖-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖(1.35g,1.65mmol)溶于无水二氯甲烷中(30.0mL),加入分子筛(3.00g),室温搅拌2小时后,冷却至-20℃,加入三氟甲磺酸三甲基硅酯(81μL,0.47mmol,0.3equiv.),-20℃搅拌反应2小时后,缓慢升至室温反应过夜。TLC监测反应结束后,滴加适量三乙胺淬灭反应,硅藻土过滤除去分子筛,滤液减压浓缩,柱层析分离(石油醚/丙酮2:1)纯化得到白色固体(2.11g,87%)。Rf=0.43(石油醚/丙酮1:1);1HNMR(400MHz,CDCl3,TMS)δ8.47(1H,d,J=6.6Hz),7.98(1H,d,J=9.4Hz),7.66-7.55(2H,m),7.51-7.39(3H,m),7.38-7.23(8H,m),7.19-7.06(2H,m),5.60(1H,s),5.16(1H,t,J=9.6Hz),5.12-5.03(2H,m),5.01-4.91(2H,m),4.79(1H,d,J=11.6Hz),4.72-4.60(3H,m),4.58(1H,d,J=8.5Hz),4.48-4.33(5H,m),4.27(2H,dd,J=11.9,6.4Hz),4.20(1H,dd,J=11.9,2.1Hz),4.17-4.06(2H,m),3.95(1H,d,J=9.9Hz),3.92-3.86(2H,m),3.83(1H,dd,J=8.1,3.8Hz),3.71(3H,s),3.67(1H,d,J=2.7Hz),3.65-3.56(2H,m),3.51(3H,s),3.48(2H,b),3.01(1H,ddd,J=19.0,11.2,3.3Hz),2.62-2.55(2H,m),2.46(1H,q,J=8.6,8.1Hz),2.33(1H,ddd,J=17.0,5.5,3.3Hz),2.22(3H,s),2.10(3H,s),2.02(3H,s),2.02(3H,s),2.00(6H,s);13C NMR(100MHz,CDCl3,TMS)δ210.9,172.4,170.8,169.9(2C),169.5,169.4,167.0,157.9(COCF3),157.8(COCF3),138.1,137.9,137.1,130.5,128.7,128.6,128.3,128.2,128.0,127.8,127.8,126.4,117.4(COCF3),115.4(COCF3),101.8,100.9,100.3,98.5,93.5,77.8,76.2,75.8,74.8,73.7,73.5,72.7,72.4,71.9,70.9,70.0,69.8,69.4,69.3,68.7,66.5,64.9,62.4,58.5,56.3,52.6,51.0,38.0,29.9,27.7,20.6(2C),20.4(2C);ESI-Q-TOF(positive mode)计算值C66H76F6N2O29 +[M+NH4]+m/z 1492.4782,实测值1492.4733.
甲基2,3,4-三-O-乙酰基-β-D-吡喃葡萄糖醛酸甲酯-(1→3)-4,6-二-O-乙酰基-2-去氧-2-三氟乙酰氨基-β-D-吡喃葡萄糖-(1→2)-3,4-二-O-苄基-α-D-吡喃半乳糖-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖的合成
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将甲基2,3,4-三-O-乙酰基-β-D-吡喃葡萄糖醛酸甲酯-(1→3)-4,6-二-O-乙酰基-2-去氧-2-三氟乙酰氨基-β-D-吡喃葡萄糖-(1→2)-3,4-二-O-苄基-6-乙酰丙酰基-α-D-吡喃半乳糖-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖(1.51g,1.02mmol)溶于二氯甲烷/甲醇(10.0mL/2.0mL),冰浴条件下加入醋酸肼(182mg,2.02mmol,2.0equiv.),缓慢升至室温反应过夜。TLC监测反应结束后,反应液用二氯甲烷稀释,有机相依次经1M盐酸溶液、饱和碳酸氢钠溶液和饱和食盐水洗涤,无水硫酸钠干燥,减压浓缩,柱层析纯化(DCM/MeOH=50:1)纯化得到白色固体(1.20g,86%)。Rf=0.46(DCM/MeOH 20:1);1H NMR(400MHz,CDCl3,TMS)δ8.59(1H,b),8.16(1H,d,J=9.7Hz),7.58-7.49(2H,m),7.45-7.37(3H,m),7.35-7.22(9H,m),7.19-7.11(2H,m),5.50(1H,s),5.21-4.99(3H,m),4.97-4.86(2H,m),4.77(1H,d,J=11.1Hz),4.66(1H,d,J=12.1Hz),4.59-4.46(2H,m),4.45-4.25(5H,m),4.26-4.14(2H,m),4.11(1H,s),4.05(1H,dd,J=10.1,3.2Hz),3.94(1H,d,J=9.7Hz),3.92-3.82(3H,m),3.78-3.66(6H,m),3.56(2H,s),3.52-3.46(5H,m),3.24(1H,s),2.08(3H,s),2.04(3H,s),1.99(6H,s),1.93(3H,s);13C NMR(100MHz,CDCl3,TMS)δ171.1,169.9,169.8,169.7,169.4,167.0,158.1(COCF3),157.8(COCF3)138.0,137.9,137.4,130.0,128.7,128.6,128.4,128.0,128.0,126.2,117.0(COCF3),114.3(COCF3),100.9,100.7,100.3(2C),99.5,77.9,74.8,73.5,72.5,72.0,70.8,69.4,68.5,66.3,62.4,57.7,56.6,52.7,20.8,20.6,20.5,20.4,20.2.
甲基2,3,4-三-O-乙酰基-β-D-吡喃葡萄糖醛酸甲酯-(1→3)-4,6-二-O-乙酰基-2-去氧-2-三氟乙酰氨基-β-D-吡喃葡萄糖-(1→2)-3,4-二-O-苄基-α-D-吡喃半乳糖醛酸-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖的合成
将甲基2,3,4-三-O-乙酰基-β-D-吡喃葡萄糖醛酸甲酯-(1→3)-4,6-二-O-乙酰基-2-去氧-2-三氟乙酰氨基-β-D-吡喃葡萄糖-(1→2)-3,4-二-O-苄基-α-D-吡喃半乳糖-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖的合成(950mg,0.69mmol)溶于二氯甲烷/水(16.0mL/8.0mL),冰浴条件下依次加入2,2,6,6-四甲基哌啶-氮-氧化物(44mg,0.28mmol,0.4equiv.)、二乙酰氧基碘苯(440mg,1.36mmol,2.0equiv.),自然升至室温反映过夜。TLC监测反应结束后,反应液用二氯甲烷稀释,有机相经饱和硫代硫酸钠溶液洗涤,无水硫酸钠干燥,减压浓缩,柱层析(DCM/MeOH=50:1)纯化得到淡黄色固体(902mg,94%)。Rf=0.18(DCM/MeOH 30:1);1H NMR(400MHz,DMSO-d6,TMS)δ7.56(2H,d,J=7.4Hz),7.48-7.09(15H,m),5.72(1H,s),5.29(1H,s),5.19(1H,t,J=9.3Hz),4.92(1H,t,J=9.7Hz),4.82-4.61(6H,m),4.61-4.43(3H,m),4.41-4.29(4H,m),4.20(1H,d,J=12.1Hz),4.16-4.07(3H,m),4.00(2H,d,J=11.7Hz),3.90(5H,s),3.80-3.70(1H,m),3.66(3H,s),3.58(1H,s),3.38(3H,s),2.13-1.92(12H,m),1.88(3H,s);13C NMR(100MHz,DMSO-d6,TMS)δ170.6,169.9,169.8,169.7,169.6,167.6,156.9(COCF3),156.6(COCF3)139.0,138.7,138.6,129.0,128.7,128.5,128.4,128.1,128.0,127.9,126.4,117.6(COCF3),114.8(COCF3),101.7,101.1(2C),100.0,75.6,75.4,73.1,72.0,71.8,71.3,70.6,69.8,69.0,66.5,62.4,56.5,53.1,21.1,20.9,20.7,20.6,20.4;ESI-Q-TOF(positive mode)计算值C61H68F6N2O28 +[M+NH4]+m/z 1408.4207,实测值1408.4362.
甲基β-D-吡喃葡萄糖醛酸-(1→3)-2-去氧-2-乙酰氨基-β-D-吡喃葡萄糖-(1→2)-3,4-二-O-苄基-α-D-吡喃半乳糖醛酸-(1→3)-2-去氧-2-乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖的合成
将甲基2,3,4-三-O-乙酰基-β-D-吡喃葡萄糖醛酸甲酯-(1→3)-4,6-二-O-乙酰基-2-去氧-2-三氟乙酰氨基-β-D-吡喃葡萄糖-(1→2)-3,4-二-O-苄基-α-D-吡喃半乳糖醛酸-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖(468mg,0.34mmol)溶于甲醇(8.0mL),滴加饱和氢氧化锂溶液(8.0mL),升温至35℃反应48小时。TLC监测反应结束后,IR-120阳离子交换树脂中和反应液至pH=7,过滤,用甲醇洗涤树脂,滤液减压浓缩,浓缩得到的粗产物溶于甲醇/水(8.0mL/8.0mL)混合溶液,加入碳酸钾固体调节pH=11~12,滴加乙酸酐(2.4mL),再加入碳酸钾固体调节pH=11~12,室温反应过夜。TLC监测反应结束后,IR-120阳离子交换树脂中和反应液至pH=7,过滤,用甲醇洗涤树脂,滤液减压浓缩,以CH2Cl2/MeOH 1:1作为洗脱液,使用Sephadex LH-20纯化得到白色固体(含盐,直接投入下一步)。Rf=0.70(CHCl3/MeOH/H2O/丙酮=4:3:1:2);1H NMR(600MHz,CD3OD,TMS)δ7.67(2H,d,J=7.7Hz),7.47(2H,t,J=7.6Hz),7.40(1H,t,J=7.5Hz),7.36-7.16(10H,m),5.84(1H,s),5.48(1H,b),4.78-4.75(1H,m),4.73-4.68(1H,m),4.69-4.60(2H,m),4.56(1H,d,J=12.1Hz),4.53-4.44(2H,m),4.44-4.34(2H,m),4.24(2H,s),4.10-4.00(3H,m),3.96-3.82(2H,m),3.72-3.65(3H,m),3.64-3.55(3H,m),3.53-3.40(4H,m),3.31(4H,s),1.95(3H,s),1.64(3H,s);13C NMR(100MHz,CD3OD,TMS)δ172.9,172.8(2C),138.6,137.7,129.4,128.7,128.1,127.8,127.7,127.2,127.1,126.9,126.3,104.4,103.7,101.8,101.1,77.3,76.6,76.2,75.4,74.8,74.2,73.5,72.0(2C),71.5,69.0,68.9,66.6,55.8,54.5,50.1,22.8,21.8;ESI-Q-TOF(positive mode)计算值C50H62N2O23 +[M+NH4]+m/z 1076.4087,实测值1076.4073.
甲基β-D-吡喃葡萄糖醛酸-(1→3)-2-去氧-2-乙酰氨基-β-D-吡喃葡萄糖-(1→2)-α-D-吡喃半乳糖醛酸-(1→3)-2-去氧-2-乙酰氨基-α,β-D-吡喃半乳糖的合成
将苄基β-D-吡喃葡萄糖醛酸-(1→3)-2-去氧-2-乙酰氨基-β-D-吡喃葡萄糖-(1→2)-3,4-二-O-苄基-α-D-吡喃半乳糖醛酸-(1→3)-2-去氧-2-乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖(上一步全部,预为0.34mmol)溶于甲醇/水(5.0mL/5.0mL)混合溶液中,加入20%氢氧化钯碳(480mg),于40Pa的氢气压力下,室温反应48小时。TLC监测反应结束后,过滤,减压浓缩,以纯水作为洗脱液,使用Sephadex LH-20纯化得到白色固体(95mg,两步合计52%)。Rf=0.11(CHCl3/MeOH/H2O=1:1:0.3);1H NMR(400MHz,D2O)δ5.29(1H,s),4.64(1H,d,J=8.5Hz),4.48(1H,d,J=8.0Hz),4.41(1H,d,J=8.7Hz),4.34-4.11(3H,m),3.98-3.69(11H,m),3.69-3.60(1H,m),3.60-3.42(7H,m),3.39-3.22(1H,m),1.98(3H,s),1.94(3H,s);13C NMR(100MHz,D2O)δ176.2,103.1,103.0,102.2,96.7,82.6,77.8,75.5,75.3,74.7,72.8,72.0,71.7,71.1,68.7,68.4,64.7,61.0,60.8,57.0,54.7,50.7,22.3,22.1;ESI-Q-TOF(negative mode)计算值C29H46N2O23 -[M-H+]-m/z 789.2413,实测值789.2446.
实施例4
乙基2-去氧-2-三氟乙酰氨基-3,4,6-三-O-乙酰基-β-D-吡喃半乳糖的合成
氩气保护下,将对甲苯基2-去氧-2-三氟乙酰氨基-3,4,6-三-O-乙酰基-1-硫-β-D-吡喃半乳糖(2.0g,3.94mmol)、N-碘代丁二酰亚胺(1.24g,5.51mmol,1.4equiv.)、分子筛溶于无水二氯甲烷(30mL)中,加入乙醇(0.69mL,11.8mmol,3.0equiv.),室温搅拌2小时,随后降温至-30℃,滴加三氟甲磺酸三甲基硅脂(0.27mL,1.55mmol,0.40equiv.),继续搅拌反应3小时,移至室温反应过夜。TLC监测反应结束后,加入适量三乙胺淬灭,硅藻土滤除分子筛,二氯甲烷洗涤滤饼数次,合并滤液,减压浓缩,柱层析纯化(石油醚/EtOAc 3:1)得到白色固体(1.51g,89%)。Rf=0.45(石油醚/EtOAc 1:1);1H NMR(400MHz,CDCl3,TMS)δ7.21(1H,d,J=9.0Hz),5.37(1H,d,J=3.4),5.33–5.18(2H,m),4.66(1H,d,J=8.3Hz,),4.23-4.07(2H,m),4.00-3.94(1H,m),3.94-3.86(1H,m),3.62-3.52(1H,m),2.14(3H,s),2.03(3H,s),1.97(3H,s),1.18(3H,t,J=7.1Hz);13C NMR(100MHz,CDCl3,TMS)δ170.7,170.6,170.3,157.8(COCF3),115.7(COCF3),100.2,70.7,69.6,66.7,65.6,61.6,51.8,20.6,20.6,20.4,14.8.
乙基2-去氧-2-三氟乙酰氨基-β-D-吡喃半乳糖的合成
将乙基2-去氧-2-三氟乙酰氨基-3,4,6-三-O-乙酰基-β-D-吡喃半乳糖(1.51g,3.52mmol)溶于甲醇(50.0mL),加入适量甲醇钠调节pH至9~10,室温下搅拌反应2小时。TLC监测反应结束后,向反应液中加入阳离子树脂中和至pH=7,过滤,滤液减压浓缩至干,得到黄色糖浆。Rf=0.57(DCM/MeOH5:1),直接用于下一步反应。
乙基2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖的合成
氩气保护下,将乙基2-去氧-2-三氟乙酰氨基-β-D-吡喃半乳糖(1.07g,3.52mmol)和(+)-樟脑磺酸(1.90g,8.18mmol,2.3equiv.)溶于无水乙腈(30.0mL)中,加入苯甲醛二甲缩醛(2.50mL,16.6mmol,4.6equiv.),升温至40℃反应过夜。TLC监测反应完全后,滴加适量三乙胺淬灭反应,反应液减压浓缩,柱层析(石油醚/EtOAc=2:1)纯化得到化合物白色固体(1.01g,74%)。Rf=0.68(石油醚/丙酮=1:2)。
乙基2-O-对甲氧苄基-3,4-二-O-苄基-6-乙酰丙酰基-α-D-吡喃半乳糖-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖
氩气保护下,将对甲苯基2-O-对甲氧苄基-3,4-二-O-苄基-6-乙酰丙酰基-1-硫-β-D-吡喃半乳糖(2.21g,3.23mmol,1.4equiv.)、乙基2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖(0.90g,2.30mmol)溶于无水二氯甲烷/N,N-二甲基甲酰胺(30.0mL/6.0mL),加入分子筛(3.6g),室温搅拌2小时后,冷却至0℃,依次加入N-碘代丁二酰亚胺(0.90g,4.00mmol,1.7equiv.)和三氟甲磺酸银(193mg,0.75mmol,0.3equiv.),0℃搅拌反应2小时后,缓慢升至室温反应过夜。TLC监测反应结束后,滴加适量三乙胺淬灭反应,硅藻土过滤除去分子筛,滤液减压浓缩,柱层析分离(石油醚/EtOAc 1:1)纯化得到化合物I-12(1.96g,89%),白色固体。Rf=0.74(DCM/MeOH 20:1);1H NMR(400MHz,CDCl3,TMS)δ7.58-7.47(3H,m),7.28(13H,m),6.92(2H,d,J=8.6Hz),6.62(2H,d,J=8.6Hz),5.53(1H,s),5.23(1H,d,J=3.5Hz),4.96(1H,d,J=11.5Hz),4.84-4.76(2H,m),4.65(1H,d,J=11.7Hz),4.54(1H,d,J=11.5Hz),4.48-4.38(3H,m),4.36–4.27(2H,m),4.22(1H,dd,J=11.7,3.2Hz),4.15(1H,dd,J=10.9,3.4Hz),4.11-4.00(3H,m),3.94(1H,dd,J=9.7,7.1Hz),3.87(1H,dd,J=10.0,2.7Hz),3.85-3.80(1H,b),3.79-3.71(4H,m),3.61-3.51(1H,m),3.46(1H,s),2.94-2.82(1H,m),2.72-2.51(2H,m),2.46-2.37(1H,m),2.17(3H,s),1.17(3H,t,J=7.0Hz);13C NMR(100MHz,CDCl3,TMS)δ209.2,172.5,158.9,157.2(COCF3),138.7,138.3,137.6,130.6,129.3,129.1,128.4,128.3(2C),128.2,127.8,127.7,127.5,126.5,116.0(COCF3),113.5,101.2,99.6,92.7,78.2,75.6,74.9,74.6,73.7,71.5,71.3,70.5,70.2,69.5,66.5,64.8,64.6,55.3,52.4,38.0,29.8,27.8,15.0;ESI-Q-TOF(positive mode)计算值C50H56F3NO14 +[M+NH4]+m/z 969.3997,实测值969.4011.
乙基3,4-二-O-苄基-6-乙酰丙酰基-α-D-吡喃半乳糖-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖
将乙基2-O-对甲氧苄基-3,4-二-O-苄基-6-乙酰丙酰基-β-D-吡喃半乳糖-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖(140mg,0.15mmol)溶于二氯甲烷/水(2.90mL/0.29mL),分批加入2,3-二氯-5,6-二氰对苯醌(44mg,0.22mmol,1.5equiv.),室温反应1小时。TLC监测反应结束后,反应液用二氯甲烷稀释,有机相依次经饱和碳酸氢钠溶液、饱和硫代硫酸钠溶液以及饱和食盐水洗涤,无水硫酸钠干燥,过滤,减压浓缩,柱层析纯化(DCM/MeOH 40:1)得到白色固体(88mg,72%)。Rf=0.69(石油醚/丙酮1:1);1H NMR(400MHz,CDCl3,TMS)δ7.67(1H,d,J=9.4Hz),7.58-7.50(2H,m),7.44-7.21(13H,m),5.58(1H,s),5.18(1H,d,J=3.9Hz),4.93(1H,d,J=11.4Hz),4.86(1H,d,J=11.8Hz),4.69-4.58(2H,m),4.50(1H,d,J=11.4Hz),4.48-4.31(3H,m),4.19(1H,dd,J=11.8,2.9Hz),4.17-4.00(4H,m),3.92(1H,dd,J=9.7,7.0Hz),3.83-3.77(1H,m),3.75-3.70(1H,m),3.59(1H,dd,J=10.0,2.8Hz),3.53(1H,dd,J=9.7,7.0Hz),3.44(1H,s),2.96-2.85(1H,m),2.76-2.35(3H,m),2.20(3H,s),1.17(3H,t,J=7.0Hz);13C NMR(100MHz,CDCl3,TMS)δ210.2,172.2,157.3(COCF3),138.5,138.2,137.3,129.0,128.4,128.3(2C),127.8,127.6,126.2,115.9(COCF3),100.9,100.0,94.5,79.7,75.4,74.6,73.9,73.1,70.6,70.4,69.3,69.1,66.4,64.7,64.6,51.1,38.1,29.9,27.7,15.0.
乙基2,3,4-三-O-乙酰基-β-D-吡喃葡萄糖醛酸甲酯-(1→3)-4,6-二-O-乙酰基-2-去氧-2-三氟乙酰氨基-β-D-吡喃葡萄糖-(1→2)-3,4-二-O-苄基-6-乙酰丙酰基-α-D-吡喃半乳糖-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖的合成
氩气保护下,将2,3,4-三-O-乙酰基-β-D-吡喃葡萄糖醛酸甲酯-(1→3)-4,6-二-O-乙酰基-2-去氧-2-三氟乙酰氨基-D-吡喃葡萄糖[2,1,-d]2-噁唑啉(400mg,0.61mmol,1.4equiv.)和乙基3,4-二-O-苄基-6-乙酰丙酰基-α-D-吡喃半乳糖-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖(360mg,0.43mmol)溶于无水二氯甲烷中(8.0mL),加入分子筛(800mg),室温搅拌2小时后,冷却至-20℃,加入三氟甲磺酸三甲基硅酯(21.6μL,0.13mmol,0.3equiv.),-20℃搅拌反应2小时后,缓慢升至室温反应过夜。TLC监测反应结束后,滴加适量三乙胺淬灭反应,硅藻土过滤除去分子筛,滤液减压浓缩,柱层析分离(石油醚/丙酮2:1)纯化得到白色固体(664mg,94%)。Rf=0.45(石油醚/丙酮1:1);1H NMR(400MHz,CDCl3,TMS)δ8.52(1H,d,J=6.6Hz),8.00(1H,d,J=9.3Hz),7.67-7.50(2H,m),7.53-7.39(3H,m),7.39-7.21(8H,m),7.14(2H,dd,J=7.7,1.8Hz),5.59(1H,s),5.16(1H,t,J=9.6Hz),5.11-5.02(2H,m),5.00-4.93(2H,m),4.79(1H,d,J=11.6Hz),4.74-4.57(4H,m),4.44(1H,d,J=3.6Hz),4.43-4.36(3H,m),4.34(1H,d,J=12.0Hz),4.27(2H,dd,J=11.8,6.1Hz),4.20(1H,d,J=11.9),4.16-4.08(2H,m),3.99-3.92(2H,m),3.89(2H,dt,J=12.3,2.7Hz),3.81(1H,dd,J=11.8,8.5Hz),3.71(3H,s),3.68(1H,d,J=2.8Hz),3.65-3.56(2H,m),3.53(1H,dd,J=9.6,7.1Hz),3.46(2H,b),3.07-2.95(1H,m),2.69-2.55(2H,m),2.46(1H,dd,J=17.2,8.5Hz),2.33(1H,dt,J=16.9,4.1Hz),2.21(3H,s),2.10(3H,s),2.02(3H,s),2.01(3H,s),1.99(6H,s),1.20(3H,t,J=7.0Hz);13C NMR(100MHz,CDCl3,TMS)δ210.8,172.4,170.7,169.9(2C),169.5,169.4,167.0,157.9(COCF3),157.6(COCF3),138.1,137.9,137.2,130.4,128.7,128.6,128.3,128.2,128.0,127.8,127.8,126.4,117.4(COCF3),116.9(COCF3),101.8,100.3,99.9,98.5,93.5,77.9,76.2,75.8,74.7,73.7,73.4,72.7,72.5,72.4,71.9,70.9,70.1,69.8,69.4,69.3,68.7,66.5,64.8,62.4,58.5,52.6,51.3,38.0,29.8,27.6,20.8,20.6,20.5,20.4(2C),15.0;ESI-Q-TOF(positive mode)计算值C67H78F6N2O29 +[M+NH4]+m/z 1506.4938,实测值1506.4886.
乙基2,3,4-三-O-乙酰基-β-D-吡喃葡萄糖醛酸甲酯-(1→3)-4,6-二-O-乙酰基-2-去氧-2-三氟乙酰氨基-β-D-吡喃葡萄糖-(1→2)-3,4-二-O-苄基-α-D-吡喃半乳糖-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖的合成
将乙基2,3,4-三-O-乙酰基-β-D-吡喃葡萄糖醛酸甲酯-(1→3)-4,6-二-O-乙酰基-2-去氧-2-三氟乙酰氨基-β-D-吡喃葡萄糖-(1→2)-3,4-二-O-苄基-6-乙酰丙酰基-α-D-吡喃半乳糖-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖(660mg,0.44mmol)溶于二氯甲烷/甲醇(5.0mL/1.0mL),冰浴条件下加入醋酸肼(200mg,2.22mmol,5.0equiv.),缓慢升至室温反应过夜。TLC监测反应结束后,反应液用二氯甲烷稀释,有机相依次经1M盐酸溶液、饱和碳酸氢钠溶液和饱和食盐水洗涤,无水硫酸钠干燥,减压浓缩,柱层析纯化(DCM/MeOH=50:1)纯化得到白色固体(450mg,73%)。Rf=0.52(DCM/MeOH=20:1);1H NMR(400MHz,CDCl3,TMS)δ8.33(1H,d,J=7.5Hz),8.04(1H,d,J=9.1Hz),7.56(2H,dd,J=7.5,2.1Hz),7.46-7.38(3H,m),7.34-7.24(9H,m),7.17(2H,dd,J=7.3,2.3Hz),5.54(1H,s),5.23-5.00(3H,m),4.93(1H,t,J=8.5Hz),4.88(1H,d,J=8.3Hz),4.75(1H,d,J=11.3Hz),4.66(1H,s),4.63(1H,d,J=4.7Hz),4.58(1H,t,J=9.5Hz),4.50-4.29(5H,m),4.26-4.14(3H,m),4.06(1H,dd,J=10.2,3.4Hz),4.00(1H,d,J=11.9Hz),3.97-3.85(4H,m),3.76(1H,d,J=2.7Hz),3.71(3H,s),3.67(2H,s),3.62-3.53(2H,m),3.50(1H,d,J=2.8Hz),3.45-3.37(1H,m),3.36(1H,s),3.02-2.91(1H,m),2.10(3H,s),2.04(3H,s),1.99(6H,d,J=0.9Hz),1.96(3H,s),1.16(3H,t,J=7.0Hz);13C NMR(100MHz,CDCl3,TMS)δ171.1,170.0,169.9,169.6,169.4,167.0,158.2(COCF3),157.8(COCF3),138.0,137.8,137.4,130.0,128.7,128.6,128.4,128.0,127.9,126.3,117.2(COCF3),114.3(COCF3),101.2,100.3,99.6,99.4,95.3,77.7,76.2,76.1,74.8,73.4,72.5,72.4,72.1,71.3,71.0,70.9,69.4,69.3,68.5,66.4,64.7,62.7,62.4,57.5,52.7,52.1,20.9,20.6,20.5,20.4,20.3,14.8.
乙基2,3,4-三-O-乙酰基-β-D-吡喃葡萄糖醛酸甲酯-(1→3)-4,6-二-O-乙酰基-2-去氧-2-三氟乙酰氨基-β-D-吡喃葡萄糖-(1→2)-3,4-二-O-苄基-α-D-吡喃半乳糖醛酸-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖的合成
将乙基2,3,4-三-O-乙酰基-β-D-吡喃葡萄糖醛酸甲酯-(1→3)-4,6-二-O-乙酰基-2-去氧-2-三氟乙酰氨基-β-D-吡喃葡萄糖-(1→2)-3,4-二-O-苄基-α-D-吡喃半乳糖-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖的合成(430mg,0.31mmol)溶于二氯甲烷/水(8.0mL/4.0mL),冰浴条件下依次加入2,2,6,6-四甲基哌啶-氮-氧化物(20mg,0.13mmol,0.4equiv.)、二乙酰氧基碘苯(200mg,0.62mmol,2.0equiv.),自然升至室温反映过夜。TLC监测反应结束后,反应液用二氯甲烷稀释,有机相经饱和硫代硫酸钠溶液洗涤,无水硫酸钠干燥,减压浓缩,柱层析(DCM/MeOH=50:1)纯化得到淡黄色固体(320mg,74%)。Rf=0.19(DCM/MeOH=30:1);1H NMR(400MHz,DMSO-d6,TMS)δ7.56(2H,d,J=7.4Hz),7.45-7.10(15H,m),5.70(1H,s),5.28(1H,s),5.19(1H,t,J=9.3Hz),4.91(1H,t,J=9.7Hz),4.83-4.49(7H,m),4.49-4.26(6H,m),4.25-3.69(13H,m),3.65(3H,s),3.57(2H,s),2.01-1.95(9H,m),1.94(3H,s),1.88(3H,s),1.07(3H,b);13C NMR(100MHz,DMSO-d6,TMS)δ170.6,169.9,169.8,169.7(2C),167.6,157.0(COCF3),156.7(COCF3),139.5,138.9(2C),138.8(2C),128.7,128.4,128.1,128.0,127.9,126.4,117.7(COCF3),114.8(COCF3),100.1(2C),100.0(2C),75.4,72.0,71.8,71.2,70.7,69.8,69.1(2C),68.0,66.4,64.7,62.4,53.1,20.9,20.8,20.7,20.6,20.5,15.4;ESI-Q-TOF(positive mode)计算值C66H76F6N2O29 +[M+NH4]+m/z 1422.4363,实测值1422.4349.
乙基β-D-吡喃葡萄糖醛酸-(1→3)-2-去氧-2-乙酰氨基-β-D-吡喃葡萄糖-(1→2)-α-D-吡喃半乳糖醛酸-(1→3)-2-去氧-2-乙酰氨基-α,β-D-吡喃半乳糖的合成
采用同实施例3相同的方法,从I-21-Et经两步得到CP-Et
实施例5
异丙基2-去氧-2-三氟乙酰氨基-3,4,6-三-O-乙酰基-β-D-吡喃半乳糖的合成
氩气保护下,将对甲苯基2-去氧-2-三氟乙酰氨基-3,4,6-三-O-乙酰基-1-硫-β-D-吡喃半乳糖(2.0g,mmol)、N-碘代丁二酰亚胺(1.24g,mmol,equiv.)、分子筛溶于无水二氯甲烷(30mL)中,加入异丙醇(1.0mL,mmol,equiv.),室温搅拌2小时,随后降温至-20℃,滴加三氟甲磺酸(0.11mL,mmol,equiv.),继续搅拌反应3小时,移至室温反应过夜。TLC监测反应结束后,加入适量三乙胺淬灭,硅藻土滤除分子筛,二氯甲烷洗涤滤饼数次,合并滤液,减压浓缩,柱层析纯化(石油醚/EtOAc=3:1)得到白色固体(1.72g,98%)。Rf=0.49(石油醚/EtOAc=1:1);1H NMR(400MHz,CDCl3,TMS)δ6.60(1H,d,J=8.7Hz,NH),5.32(1H,d,J=2.1Hz),5.28(1H,dd,J=11.2,3.4Hz),4.69(1H,d,J=8.3Hz),4.16-4.03(2H,m),4.02-3.93(1H,m),3.92-3.80(2H,m),2.09(3H,s),1.98(3H,s),1.93(3H,s),1.18(d,J=6.1Hz,3H),1.06(d,J=6.1Hz,3H);13C NMR(100MHz,CDCl3,TMS)δ170.61,170.53,170.28,157.5,117.2,99.19,73.18,70.70,69.42,66.58,61.50,52.40,23.24,21.72,20.66,20.47.
异丙基2-去氧-2-三氟乙酰氨基-β-D-吡喃半乳糖的合成
将异丙基2-去氧-2-三氟乙酰氨基-3,4,6-三-O-乙酰基-β-D-吡喃半乳糖(1.71g,3.86mmol)溶于甲醇(50.0mL),加入适量甲醇钠调节pH至9~10,室温下搅拌反应2小时。TLC监测反应结束后,向反应液中加入阳离子树脂中和至pH=7,过滤,滤液减压浓缩至干,得到黄色糖浆。Rf=0.57(DCM/MeOH=5:1),直接用于下一步反应。
异丙基2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖的合成
氩气保护下,将异丙基2-去氧-2-三氟乙酰氨基-β-D-吡喃半乳糖(1.22g,3.86mmol)和(+)-樟脑磺酸(1.60g,6.89mmol,1.8equiv.)溶于无水乙腈(40.0mL)中,加入苯甲醛二甲缩醛(3.87mL,25.7mmol,6.6equiv.),升温至40℃反应过夜。TLC监测反应完全后,滴加适量三乙胺淬灭反应,反应液减压浓缩,柱层析(石油醚/EtOAc=2:1)纯化得到化合物白色固体(1.12g,71%)。Rf=0.55(石油醚/丙酮=1:2)。
异丙基2-O-对甲氧苄基-3,4-二-O-苄基-6-乙酰丙酰基-α-D-吡喃半乳糖-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖
氩气保护下,将对甲苯基2-O-对甲氧苄基-3,4-二-O-苄基-6-乙酰丙酰基-1-硫-β-D-吡喃半乳糖(227mg,0.33mmol,1.3equiv.)、异丙基2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖(103mg,0.25mmol)溶于无水二氯甲烷/N,N-二甲基甲酰胺(5.0mL/1.0mL),加入分子筛(400mg),室温搅拌2小时后,冷却至0℃,依次加入N-碘代丁二酰亚胺(100mg,0.44mmol,1.7equiv.)和三氟甲磺酸银(30mg,0.12mmol,0.5equiv.),0℃搅拌反应2小时后,缓慢升至室温反应过夜。TLC监测反应结束后,滴加适量三乙胺淬灭反应,硅藻土过滤除去分子筛,滤液减压浓缩,柱层析分离(石油醚/EtOAc=1:1)纯化得到化合物I-12(210mg,86%),白色固体。Rf=0.61(DCM/MeOH=20:1);1H NMR(400MHz,CDCl3,TMS)δ7.61-7.50(3H,m),7.41-7.22(13H,m),6.96(2H,d,J=8.5Hz),6.64(2H,d,J=8.6Hz),5.54(1H,s),5.21(1H,d,J=3.6Hz),5.00-4.92(2H,m),4.78(1H,d,J=11.7Hz),4.66(1H,d,J=11.8Hz),4.55(1H,d,J=11.4Hz),4.47(2H,dd,J=11.8,2.3Hz),4.42(1H,d,J=3.4Hz),4.35-4.24(2H,m),4.19(1H,dd,J=11.6,4.0Hz),4.13–4.03(4H,m),3.96(1H,p,J=6.3Hz),3.89(1H,dd,J=10.0,2.7Hz),3.84(1H,b),3.79-3.71(4H,m),3.48(1H,s),2.91-2.80,2.75-2.42(4H,m),2.19(3H,s),1.23(3H,d,J=6.1Hz),1.10(3H,d,J=6.0Hz);13CNMR(100MHz,CDCl3,TMS)δ208.5,172.5,158.9,157.3(COCF3),138.6,138.3,137.7,130.4,129.5,129.0,128.4,128.3(2C),128.2,127.7(2C),127.5,126.4,115.9(COCF3),113.5,101.0,98.2,92.8,78.3,75.2,74.7,74.6,73.4,72.0,71.5,70.9,70.7,70.0,69.5,66.4,64.1,55.2,53.3,38.0,29.8,27.8,23.4,21.8;ESI-Q-TOF(positive mode)计算值C51H58F3NO14 +[M+NH4]+m/z 983.3433,实测值983.4160.
异丙基3,4-二-O-苄基-6-乙酰丙酰基-α-D-吡喃半乳糖-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖
将异丙基2-O-对甲氧苄基-3,4-二-O-苄基-6-乙酰丙酰基-β-D-吡喃半乳糖-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖(714mg,0.86mmol)溶于二氯甲烷/水(14.6mL/1.46mL),分批加入2,3-二氯-5,6-二氰对苯醌(204mg,0.90mmol,1.0equiv.),室温反应1小时。TLC监测反应结束后,反应液用二氯甲烷稀释,有机相依次经饱和碳酸氢钠溶液、饱和硫代硫酸钠溶液以及饱和食盐水洗涤,无水硫酸钠干燥,过滤,减压浓缩,柱层析纯化(DCM/MeOH=40:1)得到白色固体(490mg,80%)。Rf=0.63(石油醚/丙酮=1:1);1H NMR(400MHz,CDCl3,TMS)δ7.64(1H,d,J=9.3Hz),7.60-7.47(2H,m),7.42-7.10(13H,m),5.59(1H,s),5.18(1H,d,J=3.9Hz),4.94(1H,d,J=11.5Hz),4.86(1H,d,J=11.9Hz),4.70(1H,d,J=8.3Hz),4.62(1H,d,J=11.9Hz),4.51(1H,d,J=11.5Hz),4.42-4.24(3H,m),4.20-4.02(6H,m),3.95(1H,p,J=6.2Hz),3.78(1H,d,J=7.7Hz),3.72(1H,s),3.59(1H,dd,J=10.0,2.8Hz),3.44(1H,s),2.92(1H,ddd,J=18.9,10.0,3.5Hz),2.71-2.55(2H,m),2.48-2.40(1H,m),2.20(3H,s),1.23(3H,d,J=6.2Hz),1.11(3H,d,J=6.1Hz);13C NMR(100MHz,CDCl3,TMS)δ210.0,172.2,157.3(COCF3),138.5,138.2,137.3,129.0,128.4,128.3(2C),127.8(2C),127.6,126.2,116.1(COCF3),100.9,98.9,94.6,79.6,75.4,74.6,73.9,73.1,71.7,70.6,70.4,69.4,69.1,66.3,64.6,51.6,38.1,29.9,27.6,23.4,21.8;ESI-Q-TOF(positive mode)计算值C43H50F3NO13 +[M+NH4]+m/z 863.3578,实测值863.3588.
异丙基2,3,4-三-O-乙酰基-β-D-吡喃葡萄糖醛酸甲酯-(1→3)-4,6-二-O-乙酰基-2-去氧-2-三氟乙酰氨基-β-D-吡喃葡萄糖-(1→2)-3,4-二-O-苄基-6-乙酰丙酰基-α-D-吡喃半乳糖-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖的合成
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氩气保护下,将2,3,4-三-O-乙酰基-β-D-吡喃葡萄糖醛酸甲酯-(1→3)-4,6-二-O-乙酰基-2-去氧-2-三氟乙酰氨基-D-吡喃葡萄糖[2,1,-d]2-噁唑啉(488mg,0.74mmol,1.4equiv.)和异丙基3,4-二-O-苄基-6-乙酰丙酰基-α-D-吡喃半乳糖-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖(438mg,0.52mmol)溶于无水二氯甲烷中(7.0mL),加入分子筛(700mg),室温搅拌2小时后,冷却至-20℃,加入三氟甲磺酸三甲基硅酯(26.5μL,0.16mmol,0.3equiv.),-20℃搅拌反应2小时后,缓慢升至室温反应过夜。TLC监测反应结束后,滴加适量三乙胺淬灭反应,硅藻土过滤除去分子筛,滤液减压浓缩,柱层析分离(石油醚/丙酮2:1)纯化得到白色固体(635mg,82%)。Rf=0.49(石油醚/丙酮1:1);1H NMR(400MHz,CDCl3,TMS)δ7.76(2H,t,J=9.5Hz),7.66-7.58(2H,m),7.50–7.23(11H,m),7.19-7.14(2H,m),5.59(1H,s),5.16(1H,t,J=9.6Hz),5.13-5.02(2H,m),4.96(2H,dd,J=9.4,8.1Hz),4.81(1H,d,J=11.6Hz),4.74-4.51(4H,m),4.42(1H,d,J=3.5Hz),4.40-4.24(6H,m),4.20-4.10(2H,m),4.07(1H,dd,J=10.2,3.5Hz),4.03-3.78(5H,m),3.74(1H,d,J=2.9Hz),3.72(3H,s),3.65(1H,d,J=8.0Hz),3.59(1H,td,J=6.3,2.9Hz),3.45(2H,b),3.07-2.92(1H,m),2.69-2.55(3H,m),2.35(1H,dt,J=16.4,4.2Hz),2.20(3H,s),2.10(3H,s),2.05(3H,s),2.02(3H,s),2.00(6H,s),1.24(3H,d,J=6.2Hz),1.11(3H,d,J=6.1Hz).13C NMR(100MHz,CDCl3,TMS)δ210.2,172.5,170.9,170.1,169.9,169.5(2C),167.1,157.9(COCF3),157.8(COCF3),138.3,138.1,137.6,130.3,128.7,128.5,128.2,128.1,128.0,127.9,126.6,117.0(COCF3),115.7(COCF3),101.6,100.4,99.1,98.9,94.2,77.8,77.4,76.3,76.0,74.9,73.7,72.9,72.7,72.6,72.1,71.8,71.1,70.4,70.0,69.5,68.7,66.5,64.9,62.5,58.5,52.8,52.0,38.1,29.9,27.8,23.6,21.9,20.9,20.7,20.6;ESI-Q-TOF(positive mode)计算值C68H80F6N2O29 +[M+NH4]+m/z 1520.5095,实测值1520.5051.
异丙基2,3,4-三-O-乙酰基-β-D-吡喃葡萄糖醛酸甲酯-(1→3)-4,6-二-O-乙酰基-2-去氧-2-三氟乙酰氨基-β-D-吡喃葡萄糖-(1→2)-3,4-二-O-苄基-α-D-吡喃半乳糖-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖的合成
将异丙基2,3,4-三-O-乙酰基-β-D-吡喃葡萄糖醛酸甲酯-(1→3)-4,6-二-O-乙酰基-2-去氧-2-三氟乙酰氨基-β-D-吡喃葡萄糖-(1→2)-3,4-二-O-苄基-6-乙酰丙酰基-α-D-吡喃半乳糖-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖(490mg,0.33mmol)溶于二氯甲烷/乙醇(17.0mL/2.0mL),冰浴条件下加入醋酸肼(177mg,1.96mmol,6.0equiv.),缓慢升至室温反应过夜。TLC监测反应结束后,反应液用二氯甲烷稀释,有机相依次经1M盐酸溶液、饱和碳酸氢钠溶液和饱和食盐水洗涤,无水硫酸钠干燥,减压浓缩,柱层析纯化(DCM/MeOH=50:1)纯化得到白色固体(436mg,97%)。Rf=0.44(DCM/MeOH=20:1);1H NMR(400MHz,CDCl3,TMS)δ7.56(2H,dd,J=7.3,2.4Hz),7.42-7.35(3H,m),7.33-7.18(12H,m),7.07(1H,d,J=8.3Hz),5.61(1H,s),5.17-5.07(3H,m),4.92(1H,t,J=8.5Hz),4.86(1H,d,J=8.4Hz),4.82-4.72(3H,m),4.61(1H,d,J=12.2Hz),4.44-4.28(6H,m),4.22(1H,d,J=9.6Hz),4.16(2H,d,J=12.5Hz),4.08-3.99(3H,m),3.96(1H,t,J=6.2Hz),3.92(1H,d,J=9.6Hz),3.78(1H,d,J=2.8Hz),3.73(3H,s),3.69-3.58(3H,m),3.51(1H,d,J=2.8Hz),3.46(1H,s),3.45-3.38(1H,m),3.34(1H,dd,J=10.6,3.7Hz),2.12(3H,s),2.05(3H,s),2.01(6H,s),2.00(3H,s),1.23(3H,d,J=6.2Hz),1.10(3H,d,J=6.1Hz);13C NMR(100MHz,CDCl3,TMS)δ171.1,170.0,169.7,169.4,169.3,166.9,157.9(COCF3),157.8(COCF3),138.1,137.8,137.8,128.6,128.4(2C),128.1,128.0(2C),127.7,126.3,117.0(COCF3),115.7(COCF3),101.0,100.5,100.2,98.1,75.6,74.7,72.9,72.4,72.2,71.9,71.0,69.3,68.2,66.6,62.4,56.9,52.8,23.3,21.7,21.0,20.6,20.5,20.4;ESI-Q-TOF(positive mode)计算值C63H74F6N2O27 +[M+NH4]+m/z 1422.4727,实测值1422.4666.
异丙基2,3,4-三-O-乙酰基-β-D-吡喃葡萄糖醛酸甲酯-(1→3)-4,6-二-O-乙酰基-2-去氧-2-三氟乙酰氨基-β-D-吡喃葡萄糖-(1→2)-3,4-二-O-苄基-α-D-吡喃半乳糖醛酸-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖的合成
将异丙基2,3,4-三-O-乙酰基-β-D-吡喃葡萄糖醛酸甲酯-(1→3)-4,6-二-O-乙酰基-2-去氧-2-三氟乙酰氨基-β-D-吡喃葡萄糖-(1→2)-3,4-二-O-苄基-α-D-吡喃半乳糖-(1→3)-2-去氧-2-三氟乙酰氨基-4,6-O-苯亚甲基-β-D-吡喃半乳糖的合成(413mg,0.29mmol)溶于二氯甲烷/水(8.0mL/4.0mL),冰浴条件下依次加入2,2,6,6-四甲基哌啶-氮-氧化物(23mg,0.15mmol,0.5equiv.)、二乙酰氧基碘苯(189mg,0.59mmol,2.0equiv.),自然升至室温反映过夜。TLC监测反应结束后,反应液用二氯甲烷稀释,有机相经饱和硫代硫酸钠溶液洗涤,无水硫酸钠干燥,减压浓缩,柱层析(DCM/MeOH=50:1)纯化得到淡黄色固体(384mg,92%)。Rf=0.19(DCM/MeOH=30:1);1H NMR(400MHz,CDCl3,TMS)δ7.62-7.47(4H,m),7.42-7.35(3H,m),7.31-7.16(10H,m),5.62(1H,s),5.20(1H,s),5.12(2H,p,J=9.3Hz),4.92(1H,t,J=8.2Hz),4.82(1H,d,J=8.3Hz),4.79-4.67(3H,m),4.63(1H,d,J=12.1Hz),4.44(1H,d,J=7.9Hz),4.41-4.21(7H,m),4.18-4.09(2H,m),4.06(2H,d,J=9.0Hz),4.01-3.90(4H,m),3.83(1H,d,J=10.3Hz),3.71(3H,s),3.65-3.56(1H,m),3.33(2H,b),2.11(3H,s),2.07-1.94(12H,m),1.21(3H,d,J=6.1Hz),1.07(3H,d,J=6.0Hz);13CNMR(100MHz,CDCl3,TMS)δ171.4,170.4,170.0,169.9,169.6,169.4,167.0,157.9(COCF3),157.5(COCF3),137.9,137.7,137.4,129.7,128.6,128.5,128.3,128.2,127.8(2C),126.4,117.1(COCF3),114.3(COCF3),101.1,100.3,98.1,76.4,75.8,75.4,73.1,72.4(2C),72.0,70.9,69.4,69.2,68.3,66.4,62.4,57.0,53.1,52.8,23.3,21.7,20.8,20.6,20.5,20.4(2C);ESI-Q-TOF(positive mode)计算值C63H72F6N2O28 +[M+NH4]+m/z 1436.4520,实测值1436.4529.
异丙基β-D-吡喃葡萄糖醛酸-(1→3)-2-去氧-2-乙酰氨基-β-D-吡喃葡萄糖-(1→2)-α-D-吡喃半乳糖醛酸-(1→3)-2-去氧-2-乙酰氨基-α,β-D-吡喃半乳糖的合成
采用同实施例3相同的方法,从I-21-Pr经两步得到CP-Pr。
试验例1
体外抗炎活性测试
化合物CP-1/CP-2对LPS诱导的RAW 264.7细胞中NO生成的影响
将细胞分为空白对照组、LPS组、不同浓度的化合物CP-1组和CP-2组(化合物浓度为0.01、0.03、0.1、0.3、1、3、10μM),每组设3个复孔,每孔100μL。按每孔1×105个细胞的密度,将细胞接种于96孔细胞培养板中,于37℃下孵育过夜。次日分别加入不同浓度的化合物处理2小时后,加入浓度为100ng/mL的脂多糖(LPS)溶液,37℃继续培养24小时。培养结束后,参照一氧化氮试剂盒说明书对细胞中的一氧化氮水平进行检测。如图1所示,与空白对照组相比,100ng/mL的脂多糖处理细胞24小时后能够显著促进一氧化氮的释放;与LPS模型组相比,CP-1和CP-2均能剂量依赖地抑制一氧化氮的释放,差异具有统计学意义。
化合物CP-1/CP-2对LPS诱导的RAW 264.7细胞中PGE2生成的影响
将细胞分为空白对照组、LPS组、不同浓度的化合物CP-1组和CP-2组(化合物浓度为0.01、0.03、0.1、0.3、1、3、10μM),每组设3个复孔,每孔100μL。按每孔1×105个细胞的密度,将细胞接种于96孔细胞培养板中,于37℃下孵育过夜。次日分别加入不同浓度的化合物处理2小时后,加入浓度为100ng/mL的脂多糖(LPS)溶液,37℃继续培养24小时。培养结束后,4℃,300g离心5分钟,收集细胞上清液,参照前列腺素E2试剂盒说明书对上清液中的前列腺素E2水平进行检测。如图2所示,与空白对照组相比,100ng/mL的脂多糖处理细胞24小时后能够显著促进前列腺素E2的释放;与LPS模型组相比,CP-1和CP-2均能剂量依赖地抑制前列腺素E2的释放,差异具有统计学意义。
化合物CP-1/CP-2对LPS诱导的RAW 264.7细胞中IL-1β、IL-6、TNF-α释放的影响
将细胞分为空白对照组、LPS组、不同浓度的化合物CP-1组和CP-2组(化合物浓度为0.01、0.03、0.1、0.3、1、3、10μM),每组设3个复孔,每孔100μL。按每孔1×105个细胞的密度,将细胞接种于96孔细胞培养板中,于37℃下孵育过夜。次日分别加入不同浓度的化合物处理2小时后,加入浓度为100ng/mL的脂多糖(LPS)溶液,37℃继续培养24小时。培养结束后,4℃,300g离心5分钟,收集细胞上清液,参照相应的Elisa试剂盒说明书对上清液中IL-1β、IL-6、IL-10、TNF-α的浓度进行检测。如图3所示,与空白对照组相比,100ng/mL的脂多糖处理细胞24小时后能够显著促进IL-1β、IL-6、TNF-α的释放;与LPS模型组相比,CP-1和CP-2均能剂量依赖地抑制IL-1β、IL-6、TNF-α的释放,差异具有统计学意义。
试验例2
化合物CP-1/CP-2对LPS诱导的RAW 264.7细胞中iNOS和COX-2蛋白表达的影响
将细胞分为空白对照组、LPS组、不同浓度的化合物CP-1组和CP-2组(化合物浓度为1、3、10、30、100μM),每组设3个复孔,每孔100μL。按每孔4×105个细胞的密度,将细胞接种于6孔细胞培养板中,于37℃下孵育过夜。次日分别加入不同浓度的化合物处理2小时后,加入浓度为100ng/mL的脂多糖(LPS)溶液,37℃继续培养24小时。培养结束后,采用Westernblot法检测细胞中iNOS和COX-2蛋白表达情况。弃去细胞上清液,加入预冷的PBS润洗3次,加入含有PMSF的RIPA细胞裂解液,置于冰上15分钟使细胞充分裂解,4℃,300g离心5分钟,收集细胞上清液,按照BCA蛋白定量试剂盒说明书测定蛋白浓度后,置于100℃金属浴煮沸5分钟使蛋白变性。蛋白经电泳分离后,转膜,室温封闭1小时。4℃一抗孵育过夜,次日用TBST洗膜3次后,置于室温二抗孵育1小时。TBST洗膜3次后,加入化学发光液,置于化学发光凝胶成像系统中成像。Western blot结果如图4所示,与空白对照组相比,100ng/mL的脂多糖处理细胞24小时后能够显著促进细胞内iNOS和COX-2蛋白表达;与LPS模型组相比,CP-1和CP-2均能剂量依赖地抑制iNOS和COX-2蛋白表达,差异具有统计学意义。
试验例3体内抗炎活性测试
化合物CP-1对LPS诱导的小鼠脓毒症模型存活率的影响
选取6-8周的雄性C57BL/6小鼠共计60只,体重20±2g。小鼠安置于温度为20-24℃、湿度为50%-60%的饲养室内,自由饮水进食,实验前适应性饲养7天。
小鼠随机分为六组,分别为对照组、模型组、CP-1高剂量组(30mg/kg)、中剂量组(10mg/kg)、低剂量组(3mg/kg)、地塞米松组,每组10只。CP-1高剂量组按照30mg/kg的浓度腹腔注射CP-1溶液200μL,中剂量组按照10mg/kg的浓度腹腔注射等体积CP-1溶液,低剂量组按照3mg/kg的浓度腹腔注射等体积CP-1溶液,地塞米松组腹腔注射等体积30mg/kg的地塞米松溶液,对照组腹腔注射等体积0.9%生理盐水。除对照组外,各组均腹腔注射45mg/kg的LPS溶液200μL造模。造模前48小时、24小时以及造模后30分钟,各组分别给药一次,共计三次。给药后观察记录小鼠状态与存活率,每6小时观察记录一次,连续观察记录72小时。
实验结果如图所示,给药72小时后,模型组小鼠存活率为40%,对照组小鼠存活率为100%,CP-1高剂量组和低剂量组小鼠存活率为70%,CP-1中剂量组小鼠存活率为80%,阳性药物地塞米松组小鼠存活率为60%。
化合物CP-1对LPS诱导的脓毒症小鼠模型血清中细胞因子的影响
选取6-8周的雄性C57BL/6小鼠共计24只,体重20±2g。小鼠安置于温度为20-24℃、湿度为50%-60%的饲养室内,自由饮水进食,实验前适应性饲养7天。小鼠随机分为四组,分别为对照组、CP-1组、LPS组、LPS+CP-1组,每组6只。CP-1组和LPS+CP-1组按照10mg/kg的浓度腹腔注射CP-1溶液200μL,对照组和LPS组腹腔注射等体积0.9%生理盐水。LPS组和LPS+CP-1组腹腔注射25mg/kg的LPS溶液200μL造模。造模前24小时以及造模后30分钟,各组分别给药一次,共计两次。给药12小时后,麻醉小鼠并心脏取血,血液静置2小时后,5000rpm离心10分钟收集血清,依据Elisa试剂盒说明书,使用多功能酶标仪于450nm的波长下检测每孔吸光度,根据标准曲线计算细胞因子IL-1β、IL-6、IL-18、TNF-α、Gal-3、INF-γ、HMGB1含量。
化合物CP-Me对LPS诱导的脓毒症小鼠模型血清中细胞因子的影响
选取6-8周的雄性C57BL/6小鼠共计24只,体重20±2g。小鼠安置于温度为20-24℃、湿度为50%-60%的饲养室内,自由饮水进食,实验前适应性饲养7天。小鼠随机分为四组,分别为对照组、CP-Me组、LPS组、LPS+CP-Me组,每组6只。CP-Me 10mg/kg组和CP-Me 3mg/kg组分别按照10mg/kg和3mg/kg的浓度腹腔注射CP-Me溶液200μL,对照组和LPS组腹腔注射等体积0.9%生理盐水。LPS组和给药组腹腔注射25mg/kg的LPS溶液200μL造模。造模前12小时以及造模后30分钟,各组分别给药一次,共计两次。给药12小时后,麻醉小鼠并心脏取血,血液静置2小时后,5000rpm离心10分钟收集血清,依据Elisa试剂盒说明书,使用多功能酶标仪于450nm的波长下检测每孔吸光度,根据标准曲线计算细胞因子IL-1β、IL-6、TNF-α含量。
HE染色和炎症指数分析
选取6-8周的雄性C57BL/6小鼠共计60只,体重20±2g。小鼠安置于温度为20-24℃、湿度为50%-60%的饲养室内,自由饮水进食,实验前适应性饲养7天。小鼠随机分为六组,分别为对照组、LPS组、CP-1组(10mg/kg)、地塞米松组,每组6只。CP-1高剂量组按照30mg/kg的浓度腹腔注射CP-1溶液200μL,中剂量组按照10mg/kg的浓度腹腔注射等体积CP-1溶液,低剂量组按照3mg/kg的浓度腹腔注射等体积CP-1溶液,地塞米松组腹腔注射等体积30mg/kg的地塞米松溶液,对照组腹腔注射等体积0.9%生理盐水。除对照组外,各组均腹腔注射45mg/kg的LPS溶液200μL造模。造模前24小时以及造模后30分钟,各组分别给药一次,共计两次。
给药24h后,脱颈处死小鼠,取左肺放入福尔马林溶液中浸泡固定,固定后进行石蜡包埋,切片,通过伊红染色观察组织形态学上的变化;并通过不同的组织变化打分从0到4依次增加,例如:出血、中性粒细胞亲润肺泡、膜透明化增加、组织碎片增加以及不均称性中隔层增厚等。0分代表无损伤,1代表小于25%的损伤程度,2代表25%到50%的损伤程度,3代表50%到75%的损伤程度,4代表大于75%的损伤程度。炎症打分由三个不同的技术人员进行统计,通过计算三位所得的平均值作为最终结果。
结果显示LPS组相对空白组发生了非常显著的肺泡壁增厚、肺泡缩小、大量的肺间质炎性细胞浸润以及出血;而CP-1(10mg/kg)组可以显著的缓解LPS造成的肺损伤,保持肺组织的基本形态;地塞米松组同样可以达到类似的效果,但是形态学上不如CP-1效果好,而且可能早成更多出血的风险。
虽然本申请所揭露的实施方式如上,但所述的内容仅为便于理解本申请而采用的实施方式,并非用以限定本申请。任何本申请所属领域内的技术人员,在不脱离本申请所揭露的精神和范围的前提下,可以在实施的形式及细节上进行任何的修改与变化,但本申请的保护范围,仍须以所附的权利要求书所界定的范围为准。
Claims (16)
1.一种细菌荚膜寡糖衍生物或其药学上可接受的盐在制备抗炎药物中的用途,所述的衍生物如式(I)所示:
其中,式(I)中R1为OH、甲氧基、乙氧基、正丙氧基、异丙氧基、烯丙氧基、或苄氧基;
R2为OH或-N(H)-R15,这里,R15为苏氨酸残基;
R3和R4各自独立地为乙酰基;
R5、R6、R7、R8、R9、R10、R11、R12和R13各自独立地为氢;
R14为OH。
2.根据权利要求1所述的用途,其中,所述的衍生物为化合物CP-1:
3.根据权利要求1所述的用途,其中,所述的衍生物为化合物CP-2:
4.根据权利要求1所述的用途,其中,所述的衍生物为化合物CP-Me:
5.根据权利要求1所述的用途,其中,所述的衍生物为化合物CP-Et:
6.根据权利要求1所述的用途,其中,所述的衍生物为化合物CP-Pr:
7.权利要求1至6中任一项所述的用途,所述抗炎是指抑制一氧化氮和前列腺素E2的生成;和/或抑制一氧化氮合酶和环氧化酶2的蛋白表达;和/或降低白介素1β、白介素6和肿瘤坏死因子α的释放。
8.权利要求7所述的用途,所述抗炎为治疗脓毒症。
9.一种细菌荚膜寡糖衍生物,所述的衍生物为化合物CP-1:
10.一种细菌荚膜寡糖衍生物,所述的衍生物为化合物CP-Me:
11.一种细菌荚膜寡糖衍生物,所述的衍生物为化合物CP-Et:
12.一种细菌荚膜寡糖衍生物,所述的衍生物为化合物CP-Pr:
13.包含权利要求9至12中任一项所述的衍生物的药物组合物。
14.权利要求13所述的药物组合物在制备抗炎药物中的用途。
15.权利要求14所述的用途,所述抗炎是指抑制一氧化氮和前列腺素E2的生成;和/或抑制一氧化氮合酶和环氧化酶2的蛋白表达;和/或降低白介素1β、白介素6和肿瘤坏死因子α的释放。
16.权利要求15所述的用途,所述抗炎为治疗脓毒症。
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CN110724209A (zh) * | 2018-07-16 | 2020-01-24 | 北京大学 | 岩藻糖基化硫酸软骨素寡糖及其制备方法、组合物和用途 |
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