CN116559458A - 基于protac蛋白靶向降解技术的活性天然产物靶点鉴定方法及protac化合物 - Google Patents
基于protac蛋白靶向降解技术的活性天然产物靶点鉴定方法及protac化合物 Download PDFInfo
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Abstract
本发明的基于PROTAC蛋白靶向降解技术的活性天然产物靶点鉴定方法及PROTAC化合物，属于医药技术领域，涉及以PROTAC技术为支撑，以活性天然产物为POI配体，合成Linker长度不同的靶蛋白水解靶向嵌合分子，通过蛋白质组学、分子生物学、结构生物学等技术确定活性天然产物的靶点，进一步的确定千金二萜烷类化合物的抗炎靶点为MAFF蛋白。本发明涉及的PROTAC蛋白靶向降解技术为活性天然产物的靶点鉴定提供了新的策略方法和新的技术补充。同时，为千金烷二萜类化合物介导的炎症、自身免疫性疾病和免疫损伤的治疗，提供了新的机制基础和治疗策略。
Description
技术领域：
本发明属于医药技术领域，具体涉及一种基于PROTAC蛋白靶向降解技术的活性天然 产物靶点鉴定方法及PROTAC化合物。
背景技术：
活性天然产物因其独特的生物相容性、新颖的结构和广泛的药理活性，已成为发现先导 化合物和新治疗药物的重要来源。然而，天然产物的生物学活性不仅仅来自于单个蛋白质或 通路的调节，而是通过与大量细胞成分的相互作用来实现的。这种作用机制的复杂性为天然 产物的精确靶点鉴定带来了很大困难。而且，通常情况下，天然产物与靶蛋白的结合亲和力 不是很强，这也为其靶点鉴定工作带来挑战。蛋白质靶向嵌合体(PROTAC)技术利用蛋白酶体 具有特异性降解蛋白底物的功能，依靠化学合成方法，将靶蛋白结合配体和泛素连接酶E3的 配体通过连接臂(Linker)连接起来，形成可以自发介导靶蛋白降解的双功能分子化合物，具有 广阔的研究和应用价值。目前PROTAC被广泛用于合成活性更好的双功能小分子抑制剂，由 于PROTAC降解靶蛋白不需较强的结合力，因此我们推测该技术很适合用于鉴定中药及天然 来源小分子化合物的确切靶点。(参见：Lai A.C.,etal.Nat.Rev.Drug Discov.2017,16,101-104； Schneekloth J.S.,etal.J.Am.Chem.Soc.2004,126,3748-3754；Deshaies R.J.,Nat.Chem.Biol. 2015,11,634-635；Zeng S.,et al.Eur.J.Med.Chem.2021,210,112981；Burslem G.M.,et al.Cell.2020,181,102-114.)
千金二萜烷类化合物是中药千金子的主要活性成分，具有广泛的生物活性，包括抗炎、抗 肿瘤、抗肿瘤多药耐药等。近年来，对从千金子中分离得到的千金二萜烷类化合物的抗炎活 性研究越来越多，申请人课题组前期研究发现新化合物 2S,3S,4S,5R,9S,11R,15R)-15-acetoxy-3-cinnamoyloxy-5-hydroxy-14-oxolathyra-6(17),12E-diene 在脂多糖(LPS)刺激的小鼠巨噬细胞RAW264.7细胞中，表现出最强的抑制一氧化氮(NO)生成 的活性，其IC50值为3.0±1.1μM，然而千金二萜烷类化合物的确切靶点和抗炎机制仍不清楚。 鉴于千金二萜烷类化合物的良好抗炎活性，其确切作用靶点的问题亟待阐明，以便更好地了解其抗炎治疗的分子机制，为后续优化寻找抗炎活性更好的先导化合物奠定基础。(参见：Zhang C.Y.,et al.J.Nat.Prod.2019,82,756-764；Wang J.X.,et al.Chem.Pharm.Bull.(Tokyo), 2018,66,674-677)
目前本领域没有以PROTAC技术作为天然来源小分子化合物靶点鉴定方法的有关报道， 也没有千金二萜烷类化合物确切作用靶点研究和基于靶点的相关用途研究。
发明内容：
本发明的目的是克服上述现有技术存在的不足，提供一种基于PROTAC蛋白靶向降解 技术的活性天然产物靶点鉴定方法及PROTAC化合物，具体以活性天然产物为靶蛋白(POI) 配体靶向细胞内靶蛋白的PROTAC分子，来鉴定细胞中活性天然产物的靶点，为中药及天然 药物来源的小分子化合物的靶点鉴定提供了新的方法，同时在体内外水平为千金二萜烷类化 合物治疗炎症相关疾病提供治疗策略。在此基础上，发现了千金二萜烷类化合物的潜在作用 靶点，包括MAFF、TRIR(C19orf43)、CHTOP、CDV3、MTDH等，并发现千金二萜烷类化 合物可以靶向MAFF在体内外发挥抗炎作用，用于治疗自身免疫性疾病，以及用于制备治疗 自身免疫性疾病和免疫损伤疾病药物的应用。
一种基于PROTAC蛋白靶向降解技术的活性天然产物靶点鉴定方法，包括以下步骤：
(1)以活性天然产物为POI配体，以不同E3连接酶配体，合成的一系列基于活性天然产 物的双功能PROTAC化合物；
(2)通过活性筛选，确定活性最佳的PROTAC化合物，将活性天然产物和PROTAC化合物分别处理的细胞裂解液进行差异蛋白质组学分析，鉴定蛋白裂解液中的差异表达蛋白，并 进行KEGG分析，确定PROTAC化合物和活性天然产物作用的潜在靶蛋白；
(3)将潜在的靶点蛋白质进行western blot验证其经PROTAC化合物降解的活性以及降解 特性，并经体外靶点验证方法MST、CETSA和DARTS等验证活性天然产物与靶蛋白的直接 结合力，经细胞生物学技术手段RNAi等和体内靶点相关通路验证等确定活性天然产物对靶 蛋白功能及靶蛋白所影响的下游蛋白的作用，进一步判断活性天然产物是否作用于该靶点， 从而验证靶蛋白的真实性，最终确证活性天然产物的靶点蛋白质。
所述的基于PROTAC蛋白靶向降解技术的活性天然产物靶点鉴定方法基于 POI-PROTAC-E3连接酶三元复合物的形成，不需活性天然产物与POI的强亲和力，能够有效 地进行较低亲和力靶蛋白的降解，适用于中药及天然药物活性小分子化合物的靶点鉴定。
所述的步骤(1)中，活性天然产物为具有千金二萜烷母核类物质，包括化合物和天然物， 所述的天然物包括但不限于续随子醇Lathyrol和ZCY020，所述Lathyrol和ZCY020结构式分 别如下通式I-1和通式I-2所示：
所述的步骤(1)中，PROTAC化合物为基于千金二萜烷类化合物不同结构、不同E3连接 酶配体、不同连接臂的诱导靶蛋白降解的双功能(靶向化合物靶蛋白和E3连接酶)化合物，或 其药学上可接受的盐、水合物或前药，所述的PROTAC化合物结构式如下通式I-3所示：
所述的通式I-3中：
所述的R1，R2为H，肉桂酰Cinnamoyl，对羟基肉桂酰P-hydroxycinnamoyl，乙酰Acetyl， 丁酰基Butyryl，苯甲酰Benzoyl或烟碱酰Nicotinoyl；
所述的B是E3泛素连接酶复合体的小分子配体，其选自Cereblon蛋白配体(沙利度胺、 来那度胺、泊马度胺及其衍生物)；VHL配体；CIAP配体；MDM2配体；UBR7配体；RNF114 配体；CBLB配体；KEAP1配体等。
所述的L是连接臂，通过共价键与羟基和B相连，共同构成双功能分子化合物。
所述的B为如下所示的任意结构之一：
其中：W选自CH2、C＝O、SO2、NH、N-C1-C4烷基；X选自O、S；Z选自氢、C1-C4 烷基、C3-C6环烷基、卤素；G、G′选自H、C1-C4烷基、-OH、C1-C4烷基取代的5-10元 杂环基，所述杂环基含有1-3个N、O或S的杂原子；R3自H、D、卤素、硝基、氨基、氰 基、羟基、C1-C4烷基、卤代C1-C4烷基、氘代C1-C4烷基。
其中，所述的L通过共价键与羟基和B相连，所述L优选为如下所示的任意结构之一：
其中：n选自1-10之间的整数。
本发明优选如通式II所示的双功能分子化合物或其药学上可接受的盐、水合物或前药：
L为如下结构中的任意一种：
n选自1-10之间的整数。
本发明优选化合物包括，但不限于：
所述的步骤(1)中，药学上可接受的盐包括与下列酸形成的加成盐：盐酸、氢溴酸、硫酸、 磷酸、甲磺酸、乙磺酸、对甲苯磺酸、苯磺酸、萘二磺酸、乙酸、丙酸、乳酸、三氟乙酸、马来酸、柠檬酸、富马酸、草酸、酒石酸、丙酮酸、琥珀酸、苯甲酸等。此外，本发明还包 括本发明衍生物的前药。它们自身可能具有较弱的活性甚至没有活性，但是在给药后，在生 理条件下(如通过代谢、溶剂分解或另外的方式)被转化成相应的生物活性形式。
所述的步骤(2)中，所述的活性筛选方法包括但不限于炎症刺激模型中抗炎活性筛选、肿 瘤细胞中抗肿瘤活性筛选、氧化应激模型中抗氧化活性筛选等，检测方法视具体模型而变； 所述炎症刺激模型中抗炎活性筛选方法具体步骤如下：
(1)将上述合成的一系列基于活性天然产物的双功能PROTAC化合物加入培养的处于对 数生长期的小鼠巨噬细胞RAW264.7中处理3小时后，以脂多糖LPS刺激细胞24小时，建立小鼠巨噬细胞炎症模型；
(2)用Griss试剂检测细胞上清中总一氧化氮(NO)含量，依据PROTAC小分子处理后对NO释放的抑制作用，进行分析，判定所合成的PROTAC化合物的抗炎活性，筛选抗炎活性最佳的PROTAC化合物，即ZCY-PROTAC。
所述的ZCY-PROTAC结构式为：
所述的步骤(2)中，进行差异蛋白质组学分析的蛋白裂解液可以是细胞全裂解液，也可以 是细胞的细胞器组分的裂解液。
所述的步骤(1)中，PROTAC化合物的制备方法，包括以下步骤：
(1)用无水DMF溶解续随子醇Lathyrol，加入氢化钠与溴丙炔，室温反应后；经稀释，清 洗与纯化，得到中间体1；
(2)在中间体1和中间体2的四氢呋喃和水的混合体系中加入维生素C钠(23mg,0.36 mmol,3.0eq)和无水硫酸铜；
(3)反应体系在室温条件下反应20-30分钟，待反应结束后，过滤除去固体，滤液减压蒸 干后经柱层析分离得到对应的终产物；其中：
所述的中间体2为中间体2-1、中间体2-2、中间体2-3、中间体2-4、或中间体2-5中的 一种；
所述的中间体2-1制备过程如下：
取沙利度胺衍生物，加入溶剂，搅拌下依次加入叠氮基-PEG-胺和DIPEA，经加热反应 后，萃取，干燥，浓缩，纯化，洗脱，得黄色油状物，即中间体2-1；
所述的中间体2-2制备过程如下：
称取M7，溶解后，加入叠氮基-PEG-胺，EDCI，HOBt和DIPEA，室温反应后，稀释， 洗涤，干燥，纯化，得到中间体2-2产物；
所述的中间体2-3制备过程如下：
称取B5，溶解后，加入叠氮基-PEG-胺，EDCI，HOBt和DIPEA，室温反应后，经稀释，洗涤，干燥与纯化，制得中间体2-3；
所述的中间体2-4制备过程如下：
称取B4盐酸盐，溶解后，加入叠氮羧酸，EDCI，HOBt和DIPEA，室温反应后，经稀 释，洗涤，干燥与纯化，制得中间体2-4；
所述的中间体2-5制备过程如下：
称取VHL盐酸盐，溶解后，加入叠氮羧酸，EDCI，HOBt和DIPEA，室温反应后，经 稀释，洗涤，干燥，与纯化，制得中间体2-5。
所述的步骤(2)中，当中间体2为中间体2-1时，n＝2、3或4，获得的终产物为终产物1-3， 发生反应过程如下式III所示；当中间体2为中间体2-2时，n＝2、3或4，发生反应过程如下 式IV所示，获得的终产物为终产物4-6；当中间体2为中间体2-3时，n＝2、3或4，发生反应过程如下式V所示，获得的终产物为终产物7-9；当中间体2为中间体2-4时，n＝1、3或 5，发生反应过程如下式VI所示，获得的终产物为终产物10-12；当中间体2为中间体2-5时， n＝1、3或5，R1为H或CH3，发生反应过程如下式VII所示，获得的终产物为终产物13-18。
一种药物组合物，含有治疗有效量的上述任意一个基于PROTAC蛋白靶向降解技术的具 有千金二萜烷母核类物质或其立体异构体、互变异构体、药学上可接受的盐、水合物、前药 以及药学上可接受的载体、稀释剂、辅剂、媒介物或它们的组合。
所述的药物组合物的剂型为注射剂、片剂和胶囊剂中的任意一种。
所述的具有千金二萜烷母核类物质在制备治疗或预防炎症相关疾病的药物中的应用。
本发明在细胞水平和动物水平上确证了具有千金二萜烷母核类化合物为有效的MAFF-Nrf2激活剂，可用于治疗炎症、自身免疫性疾病和免疫性损伤，包括但不限于诸如系统性红斑狼疮、类风湿关节炎、系统性血管炎、硬皮病、皮肌炎、自身免疫性溶血性贫血、 溃疡性结肠炎、慢性淋巴细胞性甲状腺炎、甲状腺功能亢进、胰岛素依赖型糖尿病、重症肌 无力、溃疡性结肠炎、恶性贫血伴慢性萎缩性胃炎、肺出血肾炎综合征、寻常天疱疮、类天 疱疮、原发性胆汁性肝硬化、多发性脑脊髓硬化症、急性特发性多神经炎、感染所致的内毒 素血症、败血症等。
上述具有千金二萜烷母核类化合物包括千金二萜烷类天然小分子化合物、基于千金二萜 烷母核类化合物的靶向降解剂及其药学上可接受的盐或上述的组合物在制备治疗或预防炎症 相关疾病的药物中的应用。所述的炎症作用靶点为MAFF。
本发明的有益效果：
本发明以PROTAC技术为支撑，以中药及天然活性小分子化合物为原料，合成Linker 长度不同的靶蛋白水解靶向嵌合分子。本发明建立了基于双功能PROTAC蛋白靶向降解技术 的中药及天然药物小分子化合物的靶点鉴定方法。本发明通过合成一系列基于中药及天然药 物小分子化合物的双功能PROTAC分子，通过细胞活性筛选，确定活性最好的PROTAC，将 小分子化合物和PROTAC分别处理后的细胞裂解液进行差异蛋白质组学分析和KEGG通路分 析，对被降解的候选蛋白质进行降解机制验证，得到候选靶蛋白。通过体外靶点验证方法 MST、CETSA和DARTS等、细胞生物学技术RNAi等、体内靶点相关通路验证等进一步确 证中药及天然药物小分子的靶点蛋白质。
本发明在分子水平和细胞水平进一步验证了MAFF蛋白与ZCY-PROTAC的POI配体续随子醇Lathyrol和千金二萜烷类代表化合物ZCY020的直接结合力，明确MAFF是千金烷二萜类化合物的作用靶蛋白，为千金二萜烷类化合物介导的炎症和/或自身免疫性疾病的治疗， 提供了新的作用机制。
本发明在细胞水平和动物水平上进一步确证了千金二萜烷类化合物为有效的MAFF-Nrf2激活剂，可用于治疗炎症、自身免疫性疾病和免疫性损伤，包括但不限于诸如系统性红斑狼疮、类风湿关节炎、系统性血管炎、硬皮病、皮肌炎、自身免疫性溶血性贫血、 溃疡性结肠炎、慢性淋巴细胞性甲状腺炎、甲状腺功能亢进、胰岛素依赖型糖尿病、重症肌 无力、溃疡性结肠炎、恶性贫血伴慢性萎缩性胃炎、肺出血肾炎综合征、寻常天疱疮、类天 疱疮、原发性胆汁性肝硬化、多发性脑脊髓硬化症、急性特发性多神经炎、感染所致的内毒 素血症、败血症等。
附图说明：
图1为本发明实施例的ZCY-PROTAC介导MAFF蛋白的降解特性图，其中A为 ZCY-PROTAC的结构，B为续随子醇Lathyrol和ZCY-PROTAC的抗NO活性IC50，C为对照 组与ZCY-PROTAC给药细胞中差异蛋白质火山图，D炎症相关蛋白质KEGG通路分析为，E 为ZCY-PROTAC在RAW264.7和HEK293T细胞中降解MAFF的浓度和时间依赖性，F为 MG132预处理RAW264.7和HEK293T细胞后ZCY-PROTAC降解MAFF的活性；
图2为本发明实施例的千金二萜烷类母核Lathyrol和ZCY020的靶点验证图，其中A为Lathyrol和ZCY020的结构，B为Lathyrol和ZCY020与MAFF蛋白的MST结果图，C为Lathyrol和ZCY020与MAFF蛋白的CETSA结果图，D为Lathyrol和ZCY020与MAFF蛋 白的DARTS结果图；
图3为本发明实施例的ZCY020促进MAFF-Nrf2异源二聚体生成，其中A为LPS和不同浓度ZCY020处理后的COIP实验结果图，B为LPS和40μM的ZCY020处理后的COIP 实验结果图，C为不同浓度ZCY020处理RAW264.7细胞后的COIP实验结果图，D为HEK293T 细胞转染MAFF-GFP-HA质粒或MAFF-GFP-HA质粒加MAFG-HIS质粒或MAFF-GFP-HA 质粒加MAFK-GST质粒后的COIP实验结果图，E为HEK293T细胞转染MAFF-GFP-HA质 粒和Nrf2-FLAG质粒后的COIP实验结果图；
图4为本发明实施例的ZCY020激活MAFF-Nrf2/HO-1信号通路，其中A为不同浓度ZCY020处理的LPS刺激的RAW264.7细胞的RT-qPCR结果，B为Nrf2通路相关蛋白的表达， C为流式检测ROS水平，D为ML385干预后Nrf2和HO-1的表达，E为ML385干预后流式 检测ROS水平，F为shRNA处理RAW264.7细胞48h后MAFF的表达情况，G为shRNA处 理后HO-1的表达，H为shRNA处理后流式检测ROS水平；
图5为本发明实施例的ZCY020抑制NF-κB信号通路，其中A为IκBα的磷酸化水平， B为iNOS和COX-2的表达，C为ZCY020抑制NO生成的IC50图，D为细胞上清中炎症因 子的分泌水平，E为RT-qPCR结果，F为shRNA处理后ZCY020的抗NO活性，G为shRNA 处理后ZCY020对IκBα的磷酸化水平的影响；
图6为本发明实施例的ZCY020促进线粒体质量控制，其中A为p62,Keap1和Nrf2的表达，B为BNIP3和LC3B的表达，C为流式检测线粒体超氧化物mitoSOX水平，D为NAC 处理后ZCY020对p62,BNIP3和LC3B表达的影响，E为ML385干预后ZCY020对BNIP3 和LC3B表达的影响，F为ML385干预后ZCY020对mitoSOX水平的影响，G为shRNA处 理后ZCY020对p62,BNIP3和LC3B表达的影响；
图7为本发明实施例的ZCY020对LPS诱导的小鼠急性肺损伤模型的治疗作用，其中A 为高剂量LPS处理后小鼠的存活率，B为小鼠肺部和肝脏的病理组织学变化，C为小鼠血清中炎症因子水平，D为小鼠肺组织中RT-qPCR检测炎症因子mRNA水平，E为小鼠肺组织中 RT-qPCR检测Nrf2调控基因的mRNA水平，F为小鼠肺组织中相关通路蛋白的表达；
图8为本发明实施例的ZCY020对IMQ诱导的小鼠银屑病模型的治疗作用，其中A为实验过程中皮肤病变程度评分，B为实验最后一天小鼠背部皮肤图片，C为银屑病小鼠背部皮肤的病理组织学变化；
图9为本发明实施例的ZCY020对小鼠CIA关节炎模型的治疗作用，其中A为小鼠关节炎实验过程中关节肿胀程度评分，B为实验最后一天小鼠关节图片，C为关节炎小鼠的关节病理组织学变化，D为关节炎小鼠的脾脏系数。
具体实施方式：
下面结合实施例对本发明作进一步的详细说明。
实施例1中间体1的合成
用无水DMF溶解续随子醇(Lathyrol)，冰浴20分钟后加入1.0eq的氢化钠，继续冰浴10 分钟后加入1.2eq的溴丙炔，然后撤下冰浴，室温反应5小时。结束后，用乙酸乙酯稀释反 应体系，用饱和氯化钠溶液洗3次，有机相干燥旋干后柱色谱纯化，得到终产物为油状液体， 产率45％。
1H NMR(400MHz,CDCl3)δ7.61(s,1H),7.49(dd,J＝7.6,2.0Hz,1H),7.34–7.28(m,2H), 4.22(s,2H),3.70–3.57(m,4H),3.44(d,J＝2.4Hz,2H),2.22(t,J＝2.4Hz,1H),1.69–1.62(m, 4H),1.22(s,3H).
实施例2中间体2-1(n＝2)的合成
58mg沙利度胺衍生物(可购买获得)于茄形瓶中，加入3mL DMF，搅拌下依次加入50mg 叠氮基-PEG2-胺(1.2eq，可购买获得)和47μL DIPEA(2eq)，90℃反应3-4小时，加入30mL 水和30mL乙酸乙酯萃取，有机层用无水硫酸钠干燥，浓缩后得粗品，经硅胶柱层析纯化， 石油醚-乙酸乙酯1:2至1:4梯度洗脱，得黄色油状物40.9mg，收率41％。
1H NMR(400MHz,CDCl3)δ9.06(br s,1H),7.49(t,J＝7.8Hz,1H),7.09(dd,J＝7.2,2.0Hz, 1H),6.93(d,J＝8.4Hz,1H),6.50(t,J＝5.6Hz,1H),4.96–4.92(m,1H),3.74(t,J＝5.2Hz,2H), 3.70–3.67(m,6H),3.48(q,J＝5.6Hz,2H),3.38(t,J＝4.4Hz,2H),2.80–2.73(m,3H),2.13–2.10 (m,1H).
实施例3中间体2-1(n＝3)的合成
具体操作及配比参考中间体2-1(n＝2)的制备。产物为黄色油状物，收率40％。1HNMR(400 MHz,CDCl3)δ8.81(br s,1H),7.48(dd,J＝8.0,7.2Hz,1H),7.09(d,J＝7.2Hz,1H),6.92(d,J＝ 8.8Hz,1H),6.49(t,J＝5.6Hz,1H),4.95–4.90(m,1H),3.72(t,J＝5.2Hz,2H),3.68–3.66(m, 14H),3.48(q,J＝5.6Hz,2H),3.38(t,J＝5.0Hz,2H),2.88–2.72(m,3H),2.13–2.09(m,1H).
实施例4中间体2-1(n＝4)的合成
具体操作及配比参考中间体2-1(n＝2)的制备。
产物为黄色油状物，收率40％。1H NMR(400MHz,CDCl3)δ8.85(br s,1H),7.43(dd,J＝ 8.4,7.2Hz,1H),7.04(d,J＝7.2Hz,1H),6.88(d,J＝8.4Hz,1H),6.44(t,J＝5.6Hz,1H),4.92–4.87(m,1H),3.68(t,J＝5.4Hz,2H),3.64–3.61(m,10H),3.43(q,J＝5.6Hz,2H),3.33(t,J ＝5.0Hz,2H),2.83–2.68(m,3H),2.08–2.05(m,1H).
实施例5中间体2-2(n＝2)的合成
称取M7，用二氯甲烷溶解后(每100毫克加入1毫升二氯甲烷)，加入1.05eq的linker， 1.1eq的EDCI和1.1eq的HOBt，室温反应3小时。反应结束后，反应液加入乙酸乙酯稀释体 系，有机相用饱和氯化铵溶液洗涤3次，有机相加入无水硫酸钠干燥，旋干，硅胶柱层析纯 化(二氯甲烷：甲醇40:1-30:1)，得到产物。产物为白色固体，产率85％。
1H NMR(400MHz,CDCl3)δ7.54(d,J＝8.5Hz,1H),7.03(d,J＝7.6Hz,4H),6.86(dd,J＝ 11.8,8.4Hz,4H),6.51(dd,J＝8.5,2.2Hz,1H),6.45(d,J＝2.1Hz,1H),6.27(t,J＝5.4Hz,1H), 5.59(d,J＝9.9Hz,1H),5.45(d,J＝9.9Hz,1H),4.59(dt,J＝12.0,6.0Hz,1H),3.94–3.78(m, 5H),3.75–3.68(m,2H),3.68–3.59(m,2H),3.57–3.39(m,6H),3.38–3.32(m,2H),3.31– 3.22(m,1H),3.10(t,J＝5.2Hz,2H),1.37(dd,J＝16.5,6.0Hz,6H).产率：85％
实施例6中间体2-2(n＝3)的合成
具体操作及配比参考中间体2-2(n＝2)的制备。产物为白色固体，产率90％。
1H NMR(400MHz,CDCl3)δ7.59(d,J＝8.5Hz,1H),7.06(dd,J＝20.9,8.5Hz,4H),6.91 (dd,J＝26.3,8.4Hz,4H),6.55(dd,J＝8.5,2.2Hz,1H),6.51–6.40(m,2H),5.57(d,J＝9.7Hz, 1H),5.48(d,J＝9.7Hz,1H),4.62(dt,J＝12.1,6.0Hz,1H),3.97–3.79(m,5H),3.78–3.57(m, 9H),3.57–3.46(m,3H),3.41(dt,J＝10.1,4.4Hz,4H),3.32–3.19(m,1H),3.11(t,J＝5.2Hz, 2H),1.36(dd,J＝14.8,6.0Hz,6H).产率：90％
实施例7中间体2-2(n＝4)的合成
具体操作及配比参考中间体2-2(n＝2)的制备。产物为白色固体，产率86％。
1H NMR(400MHz,CDCl3)δ7.62(d,J＝8.5Hz,1H),7.10(d,J＝8.5Hz,2H),7.04(d,J＝ 8.6Hz,2H),6.95(d,J＝8.4Hz,2H),6.88(d,J＝8.4Hz,2H),6.56(dd,J＝8.5,2.2Hz,1H),6.48 (d,J＝2.1Hz,1H),5.61(s,2H),4.69–4.52(m,1H),3.94(d,J＝15.7Hz,1H),3.86(s,3H),3.85– 3.75(m,2H),3.68–3.57(m,16H),3.51(dd,J＝12.7,7.7Hz,5H),3.43–3.34(m,4H),3.29(dd,J ＝13.1,6.2Hz,1H),3.13(s,2H),1.37(dd,J＝16.9,6.0Hz,6H).产率：86％
实施例8中间体2-3(n＝2)的合成
称取B5，用二氯甲烷溶解后(每100毫克加入1毫升二氯甲烷)，加入1.05eq的linker， 1.1eq的EDCI和1.1eq的HOBt，室温反应3小时。反应结束后，反应液加入乙酸乙酯稀释体 系，有机相用饱和氯化铵溶液洗涤3次，有机相加入无水硫酸钠干燥，旋干，硅胶柱层析纯 化(二氯甲烷：甲醇50:1-40:1)，得到产物。产物为白色固体，产率85％。
1H NMR(400MHz,CDCl3)δ7.33–7.27(m,2H),7.23(d,J＝7.5Hz,3H),6.57(s,1H),5.01 (d,J＝8.0Hz,1H),4.45(d,J＝4.7Hz,1H),4.13(s,1H),3.98(s,1H),3.67–3.60(m,2H),3.59– 3.52(m,2H),3.37(dd,J＝21.2,16.4Hz,3H),3.18(d,J＝13.9Hz,1H),3.03(s,1H),1.43–1.33 (m,8H),0.95–0.86(m,6H).产率：85％
实施例9中间体2-3(n＝3)的合成
具体操作及配比参考中间体2-3(n＝2)的制备。产物为白色固体，产率85％。
1H NMR(400MHz,CDCl3)δ7.32–7.27(m,2H),7.25–7.19(m,3H),6.67(s,1H),5.58(s, 1H),5.04(d,J＝8.2Hz,1H),4.45(dd,J＝11.6,6.5Hz,1H),4.09(dd,J＝26.7,24.2Hz,2H),3.71 –3.58(m,7H),3.55(dd,J＝9.4,5.0Hz,2H),3.51–3.43(m,2H),3.39(dd,J＝11.6,6.6Hz,3H), 3.07(d,J＝40.9Hz,2H),1.62(dt,J＝15.9,10.2Hz,3H),1.38(s,9H),0.91(dd,J＝12.5,6.1Hz, 6H).产率：85％
实施例10中间体2-3(n＝4)的合成
具体操作及配比参考中间体2-3(n＝2)的制备。产物为白色固体，产率90％。
1H NMR(400MHz,CDCl3)δ7.31–7.26(m,2H),7.22(dd,J＝8.6,4.5Hz,3H),6.88(s,1H), 5.04(s,1H),4.48(s,1H),4.13(s,2H),3.70–3.50(m,16H),3.40(dd,J＝18.6,13.7Hz,4H),3.02 (d,J＝16.1Hz,2H),1.71(dd,J＝11.1,6.4Hz,2H),1.64–1.51(m,2H),1.38(d,J＝11.6Hz,8H), 0.92(dd,J＝11.3,6.2Hz,6H).产率：90％
实施例11中间体2-4(n＝1)的合成
称取B4盐酸盐，用二氯甲烷溶解后(每100毫克加入1毫升二氯甲烷)，加入1.05eq的叠 氮羧酸，1.1eq的EDCI，1.1eq的HOBt和2.1eq的DIPEA，室温反应3小时。反应结束后，反应液加入乙酸乙酯稀释体系，有机相用饱和氯化铵溶液洗涤3次，有机相加入无水硫酸钠干燥，旋干，硅胶柱层析纯化(二氯甲烷：甲醇50:1-40:1)，得到产物。产物为白色固体，产率85％。
1H NMR(400MHz,CDCl3)δ7.31(dd,J＝9.3,5.3Hz,2H),7.23(d,J＝7.5Hz,3H),7.08(d, J＝8.3Hz,1H),6.88(d,J＝8.2Hz,1H),5.13(s,1H),4.64–4.49(m,1H),4.37(dd,J＝6.9,2.9 Hz,1H),4.22–4.15(m,1H),3.91(d,J＝16.6Hz,1H),3.82(d,J＝16.6Hz,1H),3.72(s,3H), 3.10(dd,J＝13.7,6.9Hz,1H),3.00(dd,J＝13.6,8.8Hz,1H),1.26(d,J＝1.6Hz,1H),0.93(dd,J ＝8.2,6.2Hz,6H).产率：85％
实施例12中间体2-4(n＝3)的合成
具体操作及配比参考中间体2-4(n＝3)的制备。产物为白色固体，产率88％。
1H NMR(400MHz,CDCl3)δ7.32–7.27(m,3H),7.25–7.18(m,3H),6.36(d,J＝8.1Hz,1H),5.78(s,1H),4.57(td,J＝8.9,5.0Hz,1H),4.29–4.21(m,1H),4.22–4.13(m,1H),3.73(s, 3H),3.18(t,J＝6.2Hz,2H),3.10(dd,J＝13.8,6.0Hz,1H),2.98(dd,J＝13.8,9.8Hz,1H),2.21– 2.06(m,2H),1.75(dt,J＝14.9,7.4Hz,3H),1.71–1.65(m,3H),1.60(ddd,J＝16.9,10.2,4.3Hz, 3H),0.93(dd,J＝8.0,6.2Hz,6H).
实施例13中间体2-4(n＝5)的合成
具体操作及配比参考中间体2-4(n＝3)的制备。产物为白色固体，产率92％。
1H NMR(400MHz,CDCl3)δ7.39(d,J＝8.6Hz,1H),7.27(dd,J＝8.1,6.1Hz,2H),7.21(t, J＝7.6Hz,3H),6.56(d,J＝8.1Hz,1H),4.57(td,J＝8.9,5.0Hz,1H),4.29–4.13(m,2H),3.72(s, 2H),3.28(t,J＝6.8Hz,1H),3.22(t,J＝6.9Hz,2H),3.11(dd,J＝13.8,5.8Hz,1H),2.96(dd,J＝ 13.8,9.9Hz,1H),2.35(t,J＝7.4Hz,1H),2.12–2.01(m,2H),1.71–1.39(m,9H),1.31–1.21(m, 3H),0.97–0.87(m,6H).产率：92％
实施例14中间体2-5(n＝1,R1＝H)的合成
称取VHL盐酸盐，用二氯甲烷溶解后(每100毫克加入1毫升二氯甲烷)，加入1.05eq的 叠氮乙酸，1.1eq的EDCI，1.1eq的HOBt和2.1eq的DIPEA，室温反应3小时。反应结束后，反应液加入乙酸乙酯稀释体系，有机相用饱和氯化铵溶液洗涤3次，有机相加入无水硫酸钠干燥，旋干，硅胶柱层析纯化(二氯甲烷：甲醇30:1-20:1)，得到产物。产物为白色固体，产率60％。
1H NMR(400MHz,CDCl3)δ8.69(s,1H),7.36(q,J＝8.3Hz,4H),6.93(d,J＝8.5Hz,1H), 4.72(t,J＝7.9Hz,1H),4.61–4.46(m,3H),4.34(dd,J＝14.9,5.2Hz,1H),4.03(dd,J＝17.9,9.7 Hz,2H),3.63(dd,J＝11.3,3.6Hz,1H),2.63–2.54(m,1H),2.52(d,J＝6.7Hz,3H),2.13(dd,J＝ 13.3,8.0Hz,1H),0,95(s,9H).产率：36％
实施例15中间体2-5(n＝3,R1＝H)的合成
具体操作及配比参考中间体2-5(n＝1,R＝H)的制备。产物为白色固体，产率68％。
1H NMR(400MHz,CDCl3)δ8.69(s,1H),7.35(q,J＝8.3Hz,5H),6.52–6.37(m,1H),4.69 (t,J＝7.9Hz,1H),4.53(dd,J＝12.3,7.7Hz,3H),4.35(dd,J＝15.0,5.4Hz,1H),4.03(d,J＝11.2 Hz,1H),3.65(dd,J＝11.2,3.6Hz,1H),3.31(td,J＝6.4,1.9Hz,2H),2.51(s,3H),2.48–2.40(m, 1H),2.29(dd,J＝14.3,7.2Hz,2H),2.13(s,1H),1.93–1.80(m,2H),0.95(s,9H).
实施例16中间体2-5(n＝5,R1＝H)的合成
具体操作及配比参考中间体2-5(n＝1,R＝H)的制备。产物为白色固体，产率64％。
1H NMR(400MHz,CDCl3)δ8.68(s,1H),7.36(q,J＝8.3Hz,4H),6.11(d,J＝8.8Hz,1H), 4.72(t,J＝8.0Hz,1H),4.64–4.47(m,3H),4.33(dd,J＝14.9,5.1Hz,1H),3.60(dd,J＝11.4,3.5 Hz,1H),3.26(t,J＝6.8Hz,2H),2.61–2.54(m,1H),2.53(d,J＝6.5Hz,3H),2.22(t,J＝7.5Hz, 2H),2.14(d,J＝8.2Hz,1H),1.60(ddd,J＝22.1,15.0,7.4Hz,4H),1.44–1.33(m,2H),0.93(s, 9H).产率：64％
实施例17中间体2-5(n＝1,R1＝CH3)的合成
具体操作及配比参考中间体2-5(n＝1,R＝H)的制备。产物为白色固体，产率74％。
1H NMR(400MHz,CDCl3)δ8.70(s,1H),7.38(q,J＝8.5Hz,4H),7.02(d,J＝8.6Hz,1H), 5.14–5.03(m,1H),4.73(t,J＝7.8Hz,1H),4.57(d,J＝8.7Hz,2H),4.10–4.04(m,1H),3.99(d, J＝16.7Hz,1H),3.63(dd,J＝11.3,3.7Hz,1H),2.60–2.50(m,4H),2.08(dd,J＝19.9,6.3Hz, 1H),1.48(d,J＝6.9Hz,3H),1.06(s,9H).产率：74％
实施例18中间体2-5(n＝3,R1＝CH3)的合成
具体操作及配比参考中间体2-5(n＝1,R＝H)的制备。产物为白色固体，产率70％。
1H NMR(400MHz,CDCl3)δ8.70(s,1H),7.46–7.33(m,5H),6.59(d,J＝8.9Hz,1H),5.15 –5.04(m,1H),4.68(t,J＝7.9Hz,1H),4.60(d,J＝8.9Hz,1H),4.51(s,1H),4.02(d,J＝11.3Hz, 1H),3.65(dd,J＝11.2,3.8Hz,1H),3.40–3.25(m,3H),2.52(s,3H),2.47–2.21(m,4H),2.06 (dd,J＝9.0,2.6Hz,1H),1.89(dt,J＝12.6,6.7Hz,3H),1.48(d,J＝6.9Hz,3H),1.04(s,9H).
实施例19中间体2-5(n＝5,R1＝CH3)的合成
具体操作及配比参考中间体2-5(n＝1,R＝H)的制备。产物为白色固体，产率60％。
1H NMR(400MHz,CDCl3)δ8.68(s,1H),7.45–7.33(m,4H),6.19(d,J＝8.8Hz,1H),5.15 –5.03(m,1H),4.72(t,J＝7.9Hz,1H),4.60–4.48(m,2H),4.17–4.05(m,1H),3.60(dd,J＝ 11.4,3.6Hz,1H),3.26(t,J＝6.8Hz,2H),2.58–2.49(m,3H),2.22(t,J＝7.5Hz,2H),2.12–2.02 (m,1H),1.70–1.54(m,4H),1.48(d,J＝6.9Hz,3H),1.44–1.33(m,2H),1.05(s,8H).产率： 60％
实施例20终产物1的合成
在中间体1(0.12mmol,1.0eq)和中间体2-1(n＝2)(0.14mmol,1.2eq)的四氢呋喃(THF，1 mL)和水(0.3mL)的混合体系中加入维生素C钠(0.36mmol,3.0eq)和无水硫酸铜(19mg, 0.12mmol,1.0eq)。反应体系在室温条件下反应20-30分钟。待反应结束后，过滤除去固体， 滤液减压蒸干后经柱层析分离得到终产物1。产物为淡黄色固体，产率60％。
1H NMR(600MHz,DMSO)δ11.11(s,1H),8.04(s,1H),7.98(s,1H),7.58(t,J＝7.8Hz, 1H),7.13(dd,J＝8.6,3.9Hz,1H),7.04(d,J＝7.0Hz,1H),6.60(s,1H),5.76(s,2H),5.06(dd,J＝ 12.8,5.4Hz,1H),4.84(s,1H),4.73(d,J＝12.0Hz,1H),4.68–4.62(m,1H),4.54–4.43(m,3H), 4.35(d,J＝9.5Hz,1H),3.86–3.76(m,2H),3.61–3.56(m,2H),3.54(s,4H),3.47–3.41(m,2H), 2.95–2.77(m,2H),2.62–2.52(m,2H),2.17(dd,J＝9.5,3.5Hz,1H),2.08–1.78(m,5H),1.56 (s,2H),1.49–1.37(m,2H),1.21–1.14(m,1H),1.10(d,J＝22.9Hz,4H),1.04(s,2H),1.01(d,J ＝6.7Hz,2H),0.94(d,J＝6.7Hz,2H).13C NMR(101MHz,DMSO)δ202.16,173.24,170.53, 169.43,167.74,150.20,146.86,145.91,145.33,144.86,136.69,133.91,132.55,124.29,117.89, 113.05,111.16,110.24,109.72,88.32,70.11,70.05,69.31,69.25,65.67,65.51,61.30,56.28,55.37, 50.10,49.85,49.03,42.14,38.04,35.93,31.45,28.99,28.46,25.35,22.61,22.12,16.65,15.10, 12.93.
实施例21终产物2的合成
具体操作及配比参考终产物1的制备。产物为淡黄色固体，产率64％。
1H NMR(600MHz,DMSO)δ11.11(s,1H),8.04(s,1H),7.98(s,1H),7.63–7.51(m,1H), 7.14(d,J＝8.6Hz,1H),7.04(d,J＝7.0Hz,1H),6.60(t,J＝5.4Hz,1H),5.76(s,1H),5.06(dd,J ＝12.9,5.4Hz,1H),4.74(d,J＝11.9Hz,1H),4.66(d,J＝8.1Hz,1H),4.53–4.43(m,3H),4.35 (d,J＝9.5Hz,1H),4.15(s,1H),3.83–3.75(m,2H),3.61(dd,J＝8.7,5.0Hz,2H),3.56–3.53(m, 2H),3.52–3.43(m,9H),2.86(ddd,J＝38.7,20.1,8.6Hz,2H),2.57(dd,J＝27.0,15.4Hz,2H), 2.17(dd,J＝9.3,2.8Hz,1H),2.07–1.79(m,4H),1.56(s,2H),1.48(dd,J＝28.2,15.7Hz,2H), 1.43–1.37(m,1H),1.20–1.14(m,1H),1.13(s,3H),1.10(s,1H),1.05(s,2H),1.01(d,J＝6.7 Hz,1H),0.95(d,J＝6.6Hz,2H).13C NMR(101MHz,DMSO)δ202.16,173.26,170.52,169.40, 167.75,150.20,146.86,145.94,145.31,144.85,136.68,133.91,132.55,124.31,117.89,113.05, 111.14,110.24,109.70,88.32,70.23,70.18,70.03,69.34,69.22,65.67,61.34,56.28,55.37,50.10, 49.81,49.02,42.16,38.04,35.93,31.45,28.99,28.47,25.36,22.62,22.12,16.66,15.10,15.07, 12.93.
实施例22终产物3的合成
具体操作及配比参考终产物1的制备。产物为淡黄色固体，产率55％。
1H NMR(600MHz,DMSO)δ11.11(s,1H),8.05(s,1H),7.98(s,1H),7.58(dd,J＝8.3,7.3 Hz,1H),7.14(d,J＝8.6Hz,1H),7.04(d,J＝7.0Hz,1H),6.60(t,J＝5.7Hz,1H),5.06(dd,J＝ 12.9,5.4Hz,1H),4.82(d,J＝8.0Hz,1H),4.77–4.71(m,1H),4.69–4.63(m,1H),4.50(dt,J＝ 15.3,5.2Hz,2H),4.46(s,1H),4.35(s,1H),4.16(t,J＝3.3Hz,1H),3.83–3.75(m,2H),3.61(t,J ＝5.3Hz,2H),3.57–3.54(m,2H),3.54–3.40(m,12H),2.96–2.75(m,2H),2.63–2.52(m,2H), 2.18(dd,J＝9.5,3.5Hz,1H),2.01(dd,J＝25.0,18.1Hz,4H),1.56(s,2H),1.53–1.37(m,3H), 1.17(d,J＝4.0Hz,2H),1.12(d,J＝13.9Hz,4H),1.05(s,2H),1.01(d,J＝6.7Hz,2H),0.95(d,J ＝6.7Hz,2H).13C NMR(151MHz,DMSO)δ202.15,173.26,170.52,169.39,167.75,150.20, 146.86,145.94,145.31,144.85,136.68,133.89,132.55,124.33,117.90,113.04,111.13,110.23, 109.69,88.30,70.28,70.23,70.09,69.99,69.34,69.23,69.19,65.66,65.48,61.33,56.29,50.11, 49.79,49.01,42.15,38.04,35.93,35.47,31.44,28.99,28.46,25.36,22.60,22.12,16.66,15.10, 15.07,12.93.
实施例23终产物4的合成
具体操作及配比参考终产物1的制备。产物为白色固体，产率50％。
1H NMR(600MHz,DMSO)δ8.01(d,J＝36.9Hz,1H),7.92(dt,J＝11.0,5.5Hz,1H),7.53 (d,J＝8.8Hz,1H),7.13(dd,J＝21.6,8.2Hz,4H),7.01(dd,J＝38.9,8.1Hz,4H),6.61(d,J＝7.2 Hz,2H),5.66(d,J＝9.8Hz,1H),5.58(d,J＝9.8Hz,1H),4.89–4.78(m,1H),4.77–4.63(m, 3H),4.53–4.44(m,3H),4.35(t,J＝8.7Hz,1H),4.16(s,1H),3.83(s,4H),3.77(dd,J＝10.0,5.1 Hz,2H),3.72(d,J＝15.9Hz,2H),3.59(d,J＝17.1Hz,1H),3.42–3.35(m,3H),3.17(dd,J＝6.8, 4.3Hz,4H),2.99(s,2H),2.81(dd,J＝12.8,9.1Hz,1H),2.56(dd,J＝12.7,6.7Hz,1H),2.24(s, 1H),2.18(dd,J＝9.4,3.3Hz,1H),1.97–1.90(m,1H),1.84(s,1H),1.56(s,2H),1.53–1.38(m, 3H),1.27(d,J＝5.9Hz,3H),1.22(t,J＝6.4Hz,5H),1.12(d,J＝14.8Hz,5H),1.05(s,2H),1.01 (d,J＝6.5Hz,2H),0.95(d,J＝6.6Hz,2H).13C NMR(151MHz,DMSO)δ202.18,167.81, 164.84,162.83,160.39,156.97,154.65,150.20,145.93,145.38,144.91,137.82,136.88,133.90, 132.42,131.71,131.58,130.15,129.18,127.93,127.89,124.32,113.80,113.06,110.25,105.46, 99.78,88.30,71.52,70.32,69.08,68.92,68.30,65.64,61.29,56.27,55.89,50.10,49.69,49.44, 49.00,46.93,42.48,38.77,38.05,35.92,28.99,28.46,26.80,25.35,22.19,22.12,16.66,15.09, 14.54,14.41,12.93.
实施例24终产物5的合成
具体操作及配比参考终产物1的制备。产物为白色固体，产率56％。
1H NMR(600MHz,DMSO)δ7.98(s,1H),7.94(t,J＝5.6Hz,1H),7.53(d,J＝8.9Hz,1H), 7.15(d,J＝8.5Hz,2H),7.12(d,J＝8.7Hz,2H),7.04(d,J＝8.5Hz,2H),6.98(d,J＝8.2Hz,2H), 6.62(s,2H),5.65(d,J＝9.7Hz,1H),5.58(d,J＝9.7Hz,1H),4.77–4.70(m,2H),4.68–4.64(m, 1H),4.50(dt,J＝15.6,5.3Hz,3H),4.38–4.33(m,1H),4.16(t,J＝3.4Hz,1H),3.83(s,3H),3.80 (dt,J＝10.3,5.3Hz,2H),3.75–3.66(m,2H),3.62–3.56(m,1H),3.53–3.49(m,2H),3.46(d,J ＝2.6Hz,6H),3.37(td,J＝5.9,1.9Hz,3H),3.25–3.14(m,3H),2.99(s,2H),2.81(dd,J＝13.0, 8.9Hz,1H),2.57(dd,J＝13.1,6.7Hz,1H),2.24(s,1H),2.18(dd,J＝9.5,3.6Hz,0H),1.97–1.90 (m,1H),1.89–1.81(m,1H),1.56(s,2H),1.53–1.46(m,1H),1.46–1.38(m,2H),1.27(d,J＝ 6.0Hz,3H),1.22(d,J＝6.0Hz,3H),1.13(s,3H),1.10(d,J＝8.9Hz,1H),1.05(s,1H),1.01(d,J ＝6.8Hz,2H),0.97–0.93(m,1H).13CNMR(151MHz,DMSO)δ167.79,164.82,162.83,160.41, 156.98,154.68,150.22,145.97,145.33,144.87,137.84,136.90,132.42,131.71,131.58,130.16, 129.19,127.94,127.90,124.32,113.83,113.04,110.25,105.48,99.78,88.30,71.55,70.32,70.15,70.10,70.08,70.02,69.39,69.24,69.20,68.31,65.66,61.36,56.29,55.90,50.11,49.81,49.77, 49.47,48.97,46.91,42.48,40.52,38.99,38.04,35.94,29.00,28.47,26.81,25.37,22.19,22.12, 16.67,15.10,15.07,12.94.
实施例25终产物6的合成
具体操作及配比参考终产物1的制备。产物为白色固体，产率53％。
1H NMR(600MHz,DMSO)δ7.96(d,J＝4.7Hz,2H),7.54(d,J＝6.8Hz,1H),7.15(d,J＝ 8.3Hz,2H),7.12(d,J＝8.5Hz,2H),7.03(d,J＝7.8Hz,2H),6.97(d,J＝7.8Hz,2H),6.62(s, 2H),5.67(d,J＝9.4Hz,1H),5.60(s,1H),5.16(s,1H),5.01(s,1H),4.84(d,J＝4.4Hz,1H),4.79 (s,1H),4.76–4.68(m,2H),4.49(t,J＝5.3Hz,2H),4.45(d,J＝10.3Hz,1H),4.39(s,1H),4.32(s, 1H),4.18(d,J＝10.8Hz,1H),4.11(s,1H),3.83(s,4H),3.80(t,J＝5.2Hz,3H),3.71(s,2H),3.59 (d,J＝17.2Hz,1H),3.51(dd,J＝5.6,3.0Hz,2H),3.49–3.44(m,12H),3.37(t,J＝5.9Hz,3H), 3.24(d,J＝10.7Hz,1H),3.21–3.15(m,3H),2.99(s,2H),2.19(dd,J＝11.1,6.4Hz,1H),2.12– 2.05(m,1H),2.02(d,J＝13.4Hz,1H),1.80(s,3H),1.56(dd,J＝15.9,11.0Hz,1H),1.49(dd,J＝ 15.4,8.8Hz,1H),1.34–1.28(m,1H),1.27(d,J＝5.9Hz,3H),1.22(d,J＝5.9Hz,4H),1.04(s, 4H),0.91(s,2H),0.90(d,J＝6.9Hz,3H),0.84(d,J＝6.6Hz,1H).13C NMR(151MHz,DMSO)δ 167.78,164.79,162.87,156.95,154.64,148.10,137.75,136.81,135.99,132.37,131.87,131.73, 131.61,130.14,129.18,127.94,124.43,116.86,113.70,105.48,99.78,81.70,79.89,72.45,70.34, 70.21,70.16,70.07,70.02,69.98,69.37,69.16,60.79,55.90,55.72,49.75,49.45,48.95,46.89, 42.46,41.43,38.98,35.23,33.63,29.17,26.59,26.24,24.65,23.85,23.76,22.19,22.11,18.00, 16.16,12.52.
实施例26终产物7的合成
具体操作及配比参考终产物1的制备。产物为白色固体，产率64％。
1H NMR(600MHz,DMSO)δ8.04(s,1H),7.98(s,1H),7.60(d,J＝8.9Hz,1H),7.26(t,J＝ 7.4Hz,2H),7.19(dd,J＝16.9,7.2Hz,4H),6.16(dd,J＝9.3,4.2Hz,1H),5.97(d,J＝5.7Hz,1H), 4.73(s,1H),4.67(d,J＝11.0Hz,1H),4.52–4.43(m,3H),4.34(d,J＝8.5Hz,2H),4.16(s,1H), 3.95(d,J＝7.3Hz,1H),3.81(s,1H),3.77(dt,J＝10.2,5.1Hz,2H),3.44–3.36(m,2H),3.17(d,J ＝4.9Hz,2H),2.77(d,J＝7.3Hz,1H),2.67(s,1H),2.24(s,1H),2.20–2.15(m,1H),2.00–1.92 (m,1H),1.85(s,1H),1.56(s,2H),1.54–1.51(m,1H),1.46(dd,J＝19.5,10.2Hz,3H),1.40(dd, J＝11.9,8.2Hz,1H),1.33(s,1H),1.28(s,8H),1.24(d,J＝8.1Hz,3H),1.15(s,2H),1.13(s,3H), 1.10(d,J＝9.3Hz,2H),1.05(s,1H),1.01(d,J＝6.6Hz,2H),0.95(d,J＝6.7Hz,1H),0.85(d,J＝ 6.4Hz,3H),0.81(d,J＝6.3Hz,4H).13C NMR(151MHz,DMSO)δ202.17,172.31,171.98, 155.33,150.19,145.95,145.39,144.91,139.18,133.89,129.85,129.68,128.59,126.47,124.29, 113.05,110.25,88.34,78.04,71.87,69.08,68.95,65.65,61.32,56.29,54.93,50.75,50.11,49.70, 42.08,38.80,38.02,35.93,29.44,29.00,28.57,28.46,28.16,25.36,24.44,23.59,22.17,21.52, 16.67,15.10,12.93.
实施例27终产物8的合成
具体操作及配比参考终产物1的制备。产物为白色固体，产率56％。
1H NMR(400MHz,DMSO)δ8.04(s,2H),7.98(s,1H),7.59(d,J＝9.0Hz,1H),7.26(d,J＝ 7.3Hz,3H),7.21(d,J＝7.1Hz,4H),6.16(d,J＝9.4Hz,1H),5.97(d,J＝6.2Hz,1H),4.86(s, 1H),4.81(d,J＝8.0Hz,1H),4.73(d,J＝6.5Hz,1H),4.70–4.63(m,1H),4.49(d,J＝5.0Hz, 4H),4.39–4.29(m,2H),4.16(d,J＝3.2Hz,1H),3.95(q,J＝7.4Hz,1H),3.79(d,J＝5.4Hz, 4H),3.56–3.42(m,5H),3.17(d,J＝5.2Hz,3H),2.85–2.75(m,2H),2.66(dd,J＝13.3,7.7Hz, 1H),2.29–2.14(m,1H),2.04–1.79(m,4H),1.48(d,J＝66.2Hz,11H),1.29(s,10H),1.13(t,J＝ 7.5Hz,9H),1.05(s,2H),1.01(d,J＝6.7Hz,3H),0.95(d,J＝6.7Hz,2H),0.84(dd,J＝16.3,6.4 Hz,6H).13C NMR(101MHz,DMSO)δ202.14,172.28,171.95,155.33,150.21,145.97,145.34, 144.88,139.19,133.92,129.68,128.59,126.47,124.31,113.03,110.24,88.37,88.31,78.05,71.89, 69.93,69.90,69.87,69.31,69.24,69.20,65.67,61.37,56.29,54.94,50.77,50.10,49.77,42.17, 39.01,38.03,35.94,29.01,28.92,28.58,28.47,28.17,26.81,25.35,24.44,23.60,22.20,16.67, 15.10,12.93.
实施例28终产物9的合成
具体操作及配比参考终产物1的制备。产物为白色固体，产率50％。
1H NMR(400MHz,DMSO)δ8.05(s,1H),7.59(d,J＝9.0Hz,1H),7.26(d,J＝7.3Hz,2H), 7.21(d,J＝8.2Hz,4H),6.15(d,J＝9.4Hz,1H),5.95(t,J＝9.2Hz,1H),4.77(dd,J＝12.5,10.0 Hz,1H),4.71–4.63(m,2H),4.50(d,J＝5.2Hz,4H),4.39–4.30(m,2H),4.18–4.12(m,1H), 3.95(dt,J＝9.7,5.0Hz,1H),3.81(d,J＝5.7Hz,3H),3.54–3.50(m,2H),3.47(s,11H),3.38(d,J ＝5.8Hz,3H),3.18(dd,J＝10.2,5.4Hz,3H),2.84–2.74(m,2H),2.66(dd,J＝13.3,7.7Hz,1H), 2.24(d,J＝6.9Hz,1H),2.18(dd,J＝9.4,3.5Hz,1H),1.96(dd,J＝15.5,7.3Hz,2H),1.87(d,J＝ 18.8Hz,2H),1.68–1.60(m,2H),1.56(s,2H),1.51(dd,J＝13.1,7.0Hz,2H),1.48(s,3H),1.40 (s,3H),1.29(s,9H),1.24(s,2H),1.15(s,2H),1.13(s,3H),1.11(s,2H),1.05(s,2H),1.01(d,J＝ 6.6Hz,2H),0.96(d,J＝6.7Hz,2H),0.86(d,J＝6.5Hz,3H),0.82(d,J＝6.4Hz,4H).13C NMR (101MHz,DMSO)δ202.13,172.28,171.94,155.34,150.22,146.00,145.33,144.87,139.20, 133.93,129.69,129.13,128.60,126.48,124.32,113.02,110.24,88.39,88.31,78.05,71.89,70.23, 70.18,70.12,70.09,70.04,69.32,69.25,69.21,65.68,65.49,61.39,56.30,54.94,50.78,50.10, 49.82,42.21,39.04,38.03,35.95,29.01,28.59,28.48,28.17,26.81,25.35,24.45,23.60,22.23, 16.67,15.09,12.93.
实施例29终产物10的合成
具体操作及配比参考终产物1的制备。产物为白色固体，产率44％。
1H NMR(400MHz,DMSO)δ8.22–8.14(m,1H),7.87–7.82(m,1H),7.78(s,1H),7.26(s, 2H),7.23(s,3H),6.30(d,J＝5.8Hz,1H),5.06(dd,J＝16.1,13.7Hz,1H),4.93(dd,J＝16.2,8.8 Hz,1H),4.86(s,1H),4.81–4.73(m,1H),4.67(d,J＝6.0Hz,1H),4.37(dt,J＝12.9,8.6Hz,2H), 4.26–4.19(m,1H),4.16(s,1H),3.94–3.87(m,1H),3.61(s,3H),2.85(dt,J＝19.2,9.5Hz,2H), 2.71–2.62(m,1H),2.25(d,J＝8.6Hz,1H),2.18(dd,J＝9.5,3.5Hz,1H),1.97(dd,J＝16.2,6.8 Hz,2H),1.87(d,J＝10.9Hz,2H),1.71–1.58(m,3H),1.56(s,2H),1.55–1.38(m,5H),1.27– 1.15(m,4H),1.13(s,5H),1.05(s,1H),1.02(d,J＝6.8Hz,3H),0.89(d,J＝6.3Hz,3H),0.83(d,J ＝6.3Hz,3H).13C NMR(101MHz,DMSO)δ202.11,173.14,172.15,165.22,150.26,145.99, 145.18,144.77,138.90,133.92,129.64,128.74,126.69,125.14,113.02,110.22,88.42,71.09, 65.74,61.38,61.08,56.32,54.15,52.39,51.91,50.16,38.04,37.33,35.97,29.02,28.50,25.35, 24.64,23.23,21.69,16.68,15.10,12.93.
实施例30终产物11的合成
具体操作及配比参考终产物1的制备。产物为白色固体，产率43％。
1H NMR(600MHz,DMSO)δ8.03(s,1H),7.85(d,J＝8.4Hz,1H),7.62(s,1H),7.26(t,J＝ 7.5Hz,2H),7.22(d,J＝7.3Hz,2H),7.17(s,1H),6.19(d,J＝6.1Hz,1H),4.81(d,J＝8.1Hz, 1H),4.73(s,1H),4.72(s,1H),4.66(s,1H),4.45(s,1H),4.34(d,J＝8.7Hz,2H),4.21(dd,J＝ 11.7,6.8Hz,3H),4.15(s,1H),3.87(d,J＝2.4Hz,1H),3.60(s,3H),3.17(d,J＝5.2Hz,1H),2.80 (d,J＝7.4Hz,2H),2.66(dd,J＝13.2,8.2Hz,1H),2.57(dd,J＝13.1,6.9Hz,1H),2.17(dd,J＝ 9.5,3.4Hz,1H),2.11–2.04(m,1H),1.99(s,2H),1.93(s,2H),1.91(d,J＝7.2Hz,3H),1.67– 1.58(m,2H),1.56(s,2H),1.52(s,1H),1.48(d,J＝13.3Hz,2H),1.45(d,J＝3.8Hz,1H),1.40 (dd,J＝11.8,8.5Hz,1H),1.23(s,2H),1.17(d,J＝7.4Hz,2H),1.13(s,3H),1.04(s,3H),1.01(d, J＝6.1Hz,1H),0.95(d,J＝6.7Hz,2H),0.85(d,J＝5.9Hz,1H),0.82(d,J＝6.5Hz,3H),0.76(d, J＝6.4Hz,3H).13C NMR(151MHz,DMSO)δ202.15,173.18,172.41,171.08,150.20,145.41, 139.23,133.86,129.66,128.61,126.54,123.83,110.24,88.36,71.65,65.71,56.31,53.62,52.37, 50.13,50.01,49.26,49.05,38.05,37.36,35.93,32.39,28.99,28.47,26.53,25.38,24.47,23.26, 22.13,21.57,16.68,15.08,12.93.
实施例31终产物12的合成
具体操作及配比参考终产物1的制备。产物为白色固体，产率52％。
1H NMR(600MHz,DMSO)δ7.99(s,1H),7.81(t,J＝12.8Hz,1H),7.50(d,J＝7.5Hz,1H), 7.30–7.15(m,5H),6.23(s,1H),4.80(dd,J＝29.2,21.2Hz,1H),4.68(dd,J＝37.6,9.7Hz,2H), 4.27(s,3H),4.22(s,3H),3.85(s,1H),3.61(s,3H),2.79(dd,J＝12.8,7.1Hz,1H),2.70–2.60(m, 1H),2.19(dd,J＝24.8,8.4Hz,1H),2.03(dt,J＝15.1,7.7Hz,1H),1.95(d,J＝7.4Hz,3H),1.85 (s,1H),1.73(s,2H),1.63(d,J＝7.8Hz,2H),1.56(s,3H),1.44(dd,J＝21.6,8.7Hz,3H),1.41– 1.34(m,3H),1.23(s,2H),1.11(d,J＝16.1Hz,6H),1.05–0.99(m,3H),0.92(dd,J＝12.5,7.0 Hz,3H),0.83(d,J＝6.1Hz,3H),0.78(d,J＝6.2Hz,3H).13C NMR(151MHz,DMSO)δ173.19, 172.05,167.42,150.19,145.95,145.40,144.90,139.23,133.90,132.16,131.98,129.64,128.61, 126.52,123.75,113.01,110.24,88.31,71.60,65.67,65.48,61.34,61.04,56.31,53.62,52.37,50.94, 49.59,38.00,37.30,35.37,28.99,28.43,26.80,25.86,25.05,24.48,23.29,21.64,16.66,15.10, 12.92.
实施例32终产物13的合成
具体操作及配比参考终产物1的制备。产物为白色固体，产率30％。
1H NMR(600MHz,DMSO)δ8.99(s,1H),8.63(s,1H),8.58–8.52(m,1H),7.41(t,J＝11.9 Hz,5H),5.26–5.19(m,1H),5.14(d,J＝3.5Hz,1H),4.89–4.71(m,2H),4.67(dd,J＝14.8,8.8 Hz,1H),4.54(dd,J＝17.6,8.3Hz,2H),4.46(s,3H),4.39–4.30(m,2H),4.29–4.14(m,3H), 3.72–3.54(m,2H),2.82(dd,J＝12.8,8.9Hz,1H),2.56(s,1H),2.47–2.42(m,4H),2.30(d,J＝ 53.5Hz,1H),2.18(dd,J＝9.5,3.5Hz,1H),2.03(dd,J＝22.4,14.7Hz,1H),1.93(s,4H),1.56(s, 3H),1.51–1.37(m,2H),1.22(d,J＝20.2Hz,1H),1.13(s,4H),1.06(d,J＝2.4Hz,2H),1.02– 0.90(m,14H).13C NMR(151MHz,DMSO)δ172.34,169.48,165.81,151.94,150.24,148.18, 145.16,139.96,131.63,130.11,129.10,127.86,125.55,113.05,110.23,100.00,88.33,69.33, 65.75,59.23,57.31,57.01,56.31,51.74,50.10,42.11,38.38,38.09,36.04,29.01,28.47,26.73, 25.37,16.72,16.42,15.09,12.93.
实施例33终产物14的合成
具体操作及配比参考终产物1的制备。产物为白色固体，产率45％。
1H NMR(600MHz,DMSO)δ8.99(s,1H),8.54–8.40(m,2H),7.44(d,J＝8.2Hz,2H),7.39(d,J＝8.0Hz,2H),5.27–5.16(m,2H),5.12(d,J＝3.4Hz,1H),4.93(s,1H),4.83(d,J＝8.0 Hz,1H),4.75(s,1H),4.67(s,1H),4.52(s,2H),4.49–4.41(m,2H),4.31–4.22(m,1H),4.16(t,J ＝3.3Hz,1H),3.62(d,J＝7.4Hz,1H),3.54(dd,J＝19.6,9.3Hz,1H),2.82(dd,J＝13.0,9.0Hz, 0H),2.46(s,4H),2.25(s,0H),2.19(dd,J＝9.4,3.5Hz,1H),2.07–2.00(m,1H),1.95(d,J＝2.4 Hz,1H),1.88(d,J＝8.1Hz,1H),1.78(ddd,J＝24.2,12.2,7.8Hz,1H),1.57(s,3H),1.52–1.42 (m,2H),1.39(d,J＝7.1Hz,4H),1.23(s,1H),1.15–1.09(m,4H),1.06(s,2H),1.04–0.92(m, 14H).13C NMR(151MHz,DMSO)δ202.20,171.01,169.38,165.77,151.95,150.24,148.22, 145.95,145.19,145.10,144.72,133.88,131.58,130.17,129.30,126.85,125.55,113.05,110.24, 88.36,69.24,67.43,65.76,65.49,61.34,60.22,59.11,57.38,56.90,56.29,51.77,50.10,48.20, 38.17,36.02,36.01,35.98,35.95,29.02,28.49,26.81,25.37,22.89,22.14,16.72,16.45,15.13, 15.09,14.55,12.94.
实施例34终产物15的合成
具体操作及配比参考终产物1的制备。产物为白色固体，产率50％。
1H NMR(600MHz,DMSO)δ8.99(s,1H),8.57(t,J＝6.0Hz,1H),8.02(s,1H),7.40(dd,J＝ 22.7,8.2Hz,5H),5.16–5.11(m,1H),4.81(d,J＝8.0Hz,1H),4.73(s,1H),4.66(s,1H),4.55(dd, J＝9.3,3.8Hz,1H),4.43(dd,J＝15.5,6.8Hz,3H),4.34(dd,J＝15.2,7.7Hz,4H),4.21(dd,J＝ 15.9,5.4Hz,1H),4.15(t,J＝3.3Hz,1H),3.66(q,J＝10.6Hz,2H),2.81(dd,J＝12.9,9.0Hz, 1H),2.45(d,J＝2.9Hz,3H),2.32–2.14(m,4H),2.03(d,J＝7.6Hz,3H),1.97–1.82(m,4H), 1.56(s,2H),1.46(dd,J＝23.4,10.8Hz,2H),1.40(dd,J＝11.8,8.4Hz,1H),1.24(s,2H),1.13(s, 3H),1.10(d,J＝7.1Hz,1H),1.05(s,2H),1.01(d,J＝6.8Hz,2H),0.97–0.91(m,12H).13C NMR (101MHz,DMSO)δ202.14,172.38,171.55,170.08,151.89,150.23,148.19,145.98,145.47, 144.97,139.97,133.91,131.63,130.12,129.10,127.90,123.84,113.02,110.23,88.38,88.30, 69.35,65.73,61.36,59.17,56.96,56.83,56.29,55.36,50.08,49.42,42.13,38.41,38.03,35.94, 35.68,32.11,29.01,28.47,26.85,26.66,25.35,22.13,16.40,15.09,14.55,12.93.
实施例35终产物16的合成
具体操作及配比参考终产物1的制备。产物为白色固体，产率54％。
1H NMR(600MHz,DMSO)δ8.99(s,1H),8.37(s,1H),8.09(s,1H),7.95(dd,J＝9.1,4.1 Hz,1H),7.43(d,J＝8.1Hz,3H),7.38(d,J＝8.1Hz,2H),5.11(d,J＝2.3Hz,1H),4.92(dd,J＝ 14.3,7.1Hz,1H),4.80(t,J＝11.6Hz,1H),4.73(s,1H),4.66(s,1H),4.55–4.48(m,2H),4.44(d, J＝19.1Hz,2H),4.37–4.31(m,3H),4.28(s,1H),4.16(s,1H),3.61(s,2H),2.81(dd,J＝13.0, 9.1Hz,1H),2.46(s,3H),2.25(ddd,J＝21.2,14.2,6.8Hz,2H),2.18(s,2H),2.06–1.98(m,4H), 1.98–1.90(m,2H),1.88(d,J＝7.3Hz,1H),1.79(ddd,J＝12.8,8.4,4.7Hz,1H),1.56(s,2H), 1.48(s,2H),1.37(d,J＝7.0Hz,3H),1.23(s,2H),1.12(d,J＝10.3Hz,5H),1.05(s,2H),1.02(d, J＝6.8Hz,2H),0.97–0.91(m,13H).13C NMR(101MHz,DMSO)δ202.15,171.51,171.07, 169.99,151.92,150.23,148.23,145.98,145.48,145.10,144.98,133.91,131.58,130.17,129.28, 126.85,123.84,113.02,110.24,88.38,69.24,65.73,65.51,61.36,59.02,57.03,56.72,56.29,50.08, 49.42,48.16,38.17,38.04,35.95,35.64,32.12,29.01,28.48,26.91,26.66,25.35,22.87,22.12, 16.68,16.44,15.09,12.93.
实施例36终产物17的合成
具体操作及配比参考终产物1的制备。产物为白色固体，产率50％。
1H NMR(600MHz,DMSO)δ8.98(s,1H),8.56(t,J＝6.0Hz,1H),8.07(s,1H),8.00(s,1H), 7.86(dd,J＝9.3,3.7Hz,1H),7.40(dd,J＝22.6,8.2Hz,6H),5.13(d,J＝2.8Hz,1H),4.80(t,J＝ 8.6Hz,1H),4.76–4.63(m,3H),4.53(dt,J＝14.7,7.3Hz,2H),4.44(d,J＝17.1Hz,4H),4.33(s, 5H),4.21(dd,J＝15.8,5.6Hz,2H),4.18–4.12(m,1H),3.66(dt,J＝17.4,6.8Hz,2H),2.81(dd,J ＝13.0,8.9Hz,1H),2.44(s,4H),2.30–2.21(m,2H),2.19–2.08(m,2H),1.99(s,10H),1.56(s, 10H),1.24(s,6H),1.13(s,5H),1.05(s,2H),1.01(d,J＝6.7Hz,2H),0.96–0.89(m,13H).13C NMR(151MHz,DMSO)δ202.14,172.41,172.38,170.16,151.92,150.21,148.18,145.97,145.39, 144.89,139.98,131.63,130.10,129.10,127.88,123.81,113.03,110.24,88.31,69.33,65.69,61.37, 60.22,59.15,56.83,56.75,56.30,50.11,49.64,42.11,40.53,38.42,38.02,35.94,35.67,35.09, 29.95,29.00,28.47,26.84,25.97,25.37,25.27,22.13,16.68,16.41,15.12,15.09,12.94.
实施例37终产物18的合成
具体操作及配比参考终产物1的制备。产物为白色固体，产率47％。
1H NMR(600MHz,DMSO)δ8.98(s,1H),8.37(s,1H),8.08(s,1H),8.02–8.00(m,1H),7.81(dd,J＝9.2,2.9Hz,1H),7.43(d,J＝8.2Hz,3H),7.38(d,J＝8.2Hz,3H),5.11(d,J＝3.2Hz, 2H),4.95–4.89(m,2H),4.83–4.78(m,1H),4.77–4.64(m,3H),4.51(t,J＝9.6Hz,2H),4.42(s, 3H),4.33(s,5H),4.16(t,J＝3.3Hz,1H),3.60(s,3H),2.82(dd,J＝13.0,8.9Hz,1H),2.46(s,4H), 2.28–2.20(m,2H),2.18(dd,J＝9.5,3.5Hz,1H),2.15–2.08(m,2H),2.04–2.00(m,1H),1.84– 1.76(m,5H),1.56(s,8H),1.38(d,J＝7.0Hz,5H),1.23(s,4H),1.15–1.08(m,5H),1.06(s,2H), 1.01(s,2H),0.94(dd,J＝13.9,4.5Hz,15H).13C NMR(151MHz,DMSO)δ202.15,172.34, 171.09,170.05,151.94,150.21,148.22,145.97,145.40,145.12,144.89,133.90,131.58,130.16, 129.29,126.85,123.81,113.03,110.25,88.32,69.23,65.70,61.38,59.01,56.82,56.73,50.11, 49.65,48.16,40.52,38.19,38.03,35.94,35.64,35.10,29.94,29.00,28.48,26.90,25.97,25.37, 25.26,22.89,22.14,16.45,15.12,15.10,14.55,12.93.
将上述合成的一系列基于活性天然产物的双功能PROTAC化合物加入培养的处于对数生 长期的小鼠巨噬细胞RAW264.7中处理3小时后，以脂多糖LPS刺激细胞24小时，建立小 鼠巨噬细胞炎症模型；用Griss试剂检测细胞上清中总一氧化氮(NO)含量，依据PROTACs小分子处理后对NO释放的抑制作用，进行分析，判定所合成的PROTAC化合物的抗炎活性，筛选抗炎活性最佳的PROTAC化合物，即ZCY-PROTAC；
将活性天然产物和PROTAC化合物分别处理的细胞裂解液进行差异蛋白质组学分析，鉴 定蛋白裂解液中的差异表达蛋白，并进行KEGG分析，确定PROTAC化合物和活性天然产 物作用的潜在靶蛋白；将潜在的靶点蛋白质进行western blot验证其经PROTAC化合物降解 的活性以及降解特性，并经体外靶点验证方法MST、CETSA和DARTS等验证活性天然产物 与靶蛋白的直接结合力，经细胞生物学技术手段RNAi等和体内实验等确定活性天然产物对 靶蛋白功能及靶蛋白所影响的下游蛋白的作用，进一步判断活性天然产物是否作用于该靶点， 从而验证靶蛋白的真实性，最终确证活性天然产物的靶点蛋白质，具体过程如下：
实施例38 Griess法检测一氧化氮(NO)生成
对数生长期的小鼠巨噬细胞RAW264.7细胞以每孔2.5×104个接种于96孔板后，培养24 h，弃上清液，换成含浓度梯度化合物(续随子醇Lathyrol和合成的PROTACs)的培养液。培养 3h后，每孔加入LPS使其终浓度为1μg/mL。继续培养24h后，取细胞上清，按照一氧化 氮试剂盒说明书，加入Griess试剂，在540nm处检测OD值，计算下列公式计算化合物的NO抑制率：NO抑制率＝(OD模型-OD化合物)/(OD模型-OD对照)×100％。结果表明，千金二萜烷类母 核化合物续随子醇Lathyrol对LPS刺激的NO生成有良好的抑制作用，IC50值为11.10±1.14 μM，合成的PROTACs中终产物2，即ZCY-PROTAC(图1A)具有最佳的抗NO活性，IC50值 为8.34±1.09μM(图1B)。
表1.续随子醇PROTAC终产物抑制RAW264.7细胞NO生成IC50值表
实施例39细胞蛋白质组质谱分析
RAW264.7细胞与ZCY-PROTAC(8μM)、Lathyrol(11μM)或DMSO孵育48小时后离心收集，分别加入4倍体积的裂解缓冲液(8M尿素，1％蛋白酶抑制剂)，超声破碎取上清，用BCA试剂盒进行蛋白定量。每个样品取等量的蛋白质样品进行酶解，溶解，用6-plex串联质量标签(TMT)标记试剂盒进行肽段标记，取1μg进行质谱检测。
定量蛋白质组学质谱分析结果显示，与DMSO组相比，ZCY-PROTAC处理后，有548 个蛋白的水平上调，有284个蛋白的水平下调。在所有蛋白中，潜在的降解靶蛋白有MAFF、TRIR(C19orf43)、CHTOP、CDV3、MTDH等，其中MAFF蛋白的降解倍数最高，ZCY-PROTAC 处理后其蛋白水平下调了7倍以上(图1C)。同时，我们采用KEGG通路富集分析，对这些炎 症相关蛋白进行了分析。KEGG分析结果表明，具有差异表达的蛋白质主要与溶酶体、铁死 亡、胆固醇代谢和线粒体自噬有关，其中MAFF蛋白也与炎症密切相关(图1D)。
实施例40细胞中ZCY-PROTAC介导的MAFF蛋白的降解
收取不同浓度ZCY-PROTAC处理不同时间的细胞样品，裂解出蛋白质后进行蛋白质免疫 印迹(Weatern blot)分析。
结果表明，ZCY-PROTAC以浓度依赖性和时间依赖性的方式显著降低RAW264.7细胞和 HEK293T细胞内MAFF的蛋白水平，此外蛋白酶体抑制剂MG132可以完全阻断这一过程，说明ZCY-PROTAC具有通过蛋白酶体途径特异性地诱导MAFF蛋白降解的作用(图1E-1F)。
实施例41微量热泳动实验
将MAFF蛋白用蛋白缓冲液(25mM Hepes pH 7.5；200mM NaCl)配制成10μM，加入2.5 μL MST荧光染料，室温避光孵育30min。用小分子筛分离出已荧光标记的蛋白质溶液。用 蛋白缓冲液梯度稀释小分子化合物(续随子醇Lathyrol和ZCY020)，配成12个浓度梯度，在 与荧光标记蛋白等体积混匀。通过标准毛细管吸取样品置于微量热泳动仪上检测。结果表明， 续随子醇Lathyrol和ZCY020与MAFF蛋白在体外的平衡解离常数KD值分别为20.90±2.34 μM和19.50±1.70μM，均与MAFF蛋白具有较强的体外结合能力(图2B)。
实施例42 CETSA实验
RAW264.7细胞给予DMSO或40μM小分子化合物(续随子醇Lathyrol和ZCY020)处理4h后，离心收集细胞，用PBS重悬细胞，并等分为6份，分别于64、66、68、70或72℃加 热10min，随后细胞于液氮中反复冻融三次进行裂解，4℃12000g离心10min，取上清，加 入SDS-PAGE上样缓冲液，95℃加热变性5min后，进行免疫印迹检测。结果表明，续随子 醇Lathyrol和ZCY020能够与MAFF直接结合，提高了MAFF蛋白的热稳定性(图2C)。
实施例43 DARTS实验
将RAW264.7细胞裂解液均分为5等份，分为对照组、酶解组、给药组(10、20和40μM的续随子醇Lathyrol或ZCY020)，先于室温使化合物与裂解液孵育1h，然后酶解组与给药组中加入1:1000稀释的蛋白酶Pronase，对照组给予等体积的PBS，混合均匀后，于37℃水浴锅中孵育15min。立即取出，加入SDS-PAGE上样缓冲液，95℃加热变性5min后，进行免 疫印迹检测。结果表明，续随子醇Lathyrol和ZCY020处理后，MAFF蛋白更加稳定，Pronase 蛋白酶对MAFF的降解程度显著降低(图2D)。
以上结果证明，ZCY020能够与MAFF蛋白直接结合，因此MAFF蛋白是ZCY020的作 用靶点。
实施例44 ZCY020促进MAFF-Nrf2异二聚体的形成
细胞离心收集后，用含有1％PMSF的非变性裂解液冰上裂解，取上清进行BCA定量。每 个样品取0.2-1mg，加入0.2-2μg用于免疫沉淀的蛋白一抗，4℃缓慢摇动过夜。再加入20μL 充分重悬的Protein A+G Agarose，4℃缓慢摇动2h。13000rpm瞬时离心，小心吸除上清，用 PBS洗涤沉淀5次。加入40μL的1×SDS-PAGE上样缓冲液涡旋重悬沉淀，95℃加热变性5min 后，用于免疫印迹分析。
结果表明，在LPS的诱导下，MAFF在巨噬细胞中过表达，而ZCY020则在此基础上下调了过量表达的MAFF水平，同时促进MAFF-Nrf2异二聚体的形成(图3A)。同时ZCY020 抑制了MAFF与SUMO-2/3的结合(图3B)，同时在RAW264.7细胞和HEK293T细胞中均抑 制了MAFF同源二聚体的形成，促进了MAFF-Nrf2异源二聚体的形成(图3C-3E)。
实施例45 ZCY020激活MAFF-Nrf2/HO-1信号通路
1、ZCY020促进Nrf2调控基因的转录
RAW264.7细胞离心收集后，加入TRIZOL试剂室温裂解10min，提取RNA进行反转录得到样品CDNA。以得到的CDNA为模板，进行PCR扩增反应，以β-Atin基因作为阳性内 参。根据软件分析得到每个样品反应的Ct值，计算得到△Ct，△Ct＝待测基因Ct值-内参基 因Ct值，然后计算2-△△Ct值进行数据统计，计算各组样品各基因的相对mRNA转录水平。
结果表明，在LPS诱导的RAW264.7细胞中，外源性内毒素LPS轻度增加了HMOX1、NQO1和GCLM的基因转录水平。而在此基础上，ZCY020浓度依赖性地增强了HMOX1、 NQO1和GCLM的转录。此外，应对LPS刺激，GCLC的转录下调，而ZCY020则恢复了受 损的GCLC基因的转录。因此，ZCY020能够通过激活MAFF-Nrf2通路中重要调控基因的转 录(图4A)。
2、ZCY020促进Nrf2入核及其调控蛋白HO-1的表达
RAW264.7细胞贴壁后加入不同浓度的ZCY020，处理2小时后，加入LPS使其终浓度为1 μg/mL，24h后收集细胞进行免疫印迹分析，结果表明，在LPS诱导的RAW264.7细胞中，ZCY020显著上调Nrf2的蛋白水平，并减弱了LPS诱导的MAFF蛋白的过度表达。同时，与Nrf2的变化一致，HO-1蛋白表达也呈剂量依赖性的增加(图4B)。
3、ZCY020抑制活性氧的生成
将处于对数期的RAW264.7细胞以2×105/mL的细胞悬液加入6孔板中，贴壁后加入不同 浓度的ZCY020处理2小时，加入LPS孵育12h。弃掉上清，细胞用PBS清洗两次，加入1 mL10μM的DCFH-DA，37℃孵育20min。PBS洗涤细胞三次，重悬，用流式细胞仪检测 ROS的水平。
结果表明，ZCY020治疗可以剂量依赖性地抑制LPS刺激的ROS产生。由此可知，ZCY020 可以通过激活Nrf2/HO-1通路发挥抗氧化活性(图4C)。
4、Nrf2抑制剂ML385和MAFF shRNA抑制ZCY020的活性
RAW264.7细胞贴壁后加入不同浓度的ZCY020和Nrf2抑制剂ML385(5μM)，处理2小时后，加入LPS，24h后收集细胞进行免疫印迹实验和活性氧检测实验，结果表明Nrf2抑制剂ML385抑制了ZCY020增强的Nrf2和HO-1的表达，并逆转了ZCY020对ROS的抑制作 用(图4D-4E)。
RAW264.7细胞贴壁后，转染MAFF敲低质粒shMAFF，24h后加入不同浓度的ZCY020处理2小时，加入LPS，24h后收集细胞进行免疫印迹实验和活性氧检测实验，结果表明shMAFF处理的RAW264.7细胞中，ZCY020对HO-1表达的增强作用、以及对ROS产生的 抑制作用也明显减弱(图4F-4H)。综合以上结果表明，ZCY020通过促进MAFF-Nrf2异二聚 体形成，激活Nrf2/HO-1通路来发挥抗氧化作用。
实施例46ZCY020抑制NF-κB信号通路
1、ZCY020抑制LPS诱导的NF-κB通路蛋白的表达
制备不同浓度ZCY020处理2h后，加入LPS处理24h的RAW264.7细胞样品，进行免 疫印迹检测，结果表明，化合物ZCY020能够抑制LPS诱导的IκBα的磷酸化，浓度依赖性 地降低了iNOS和COX-2的表达水平的表达水平，并抑制了炎症介质NO的释放(图5A-5C)。 ELISA分析结果表明，在RAW264.7细胞中，ZCY020显著下调了LPS诱导的IL-6、IL-1β 和TNF-α的分泌(图6D)。同时，RT-qPCR分析表明，LPS刺激后IL-1β、IL-6和TNF-α的转 录水平显著上调，而在ZCY020处理之后，它们的转录水平都明显降低(图6E)。
2、MAFF shRNA抑制ZCY020的抗NF-κB通路活性
用MAFF shRNA质粒敲低MAFF的表达后，再检测化合物ZCY020对LPS诱导的NF-κB通路蛋白的抑制活性。结果表明，MAFF shRNA(shMAFF)处理后，ZCY020对LPS诱导的IκBα 的磷酸化和NO生成的抑制作用明显减弱(图5F和5G)。结果证明，ZCY020通过靶向MAFF 抑制NF-κB通路的激活。
实施例47ZCY020促进线粒体质量控
1、ZCY020抑制过量mtROS的产生
处于对数生长期的RAW264.7细胞按5×105个每孔接种于6孔板中，培养24h，给予化 合物处理3h后，加入LPS 1μg/mL孵育12h。将线粒体超氧化物(mitoSOX)检测试剂用DMSO配置成5mM的储备液。用HBSS缓冲液将储备液1:1000稀释成5μM的工作液。弃上清， 取1mL工作液加入孔中，37℃避光孵育10min。用预热的HBSS缓冲液清洗细胞3次。用 适量HBSS重悬细胞，用流式细胞仪检测MitoSOX的水平。
结果表明，LPS明显增加线粒体超氧化物的产生，ZCY020预处理剂量依赖性地逆转了 这种情况(图6C)。
2、ZCY020促进线粒体自噬
免疫印迹检测结果表明，在Nrf2蛋白水平上调，而Keap1水平下降的同时，ZCY020给药 处理后P62的表达也明显上调，同时化合物ZCY020浓度依赖性地升高了BNIP3的蛋白水平， 同时促使了LC3B-II的富集，由此可知ZCY020促进线粒体自噬以维持线粒体稳态(图6A-6B)。
3、活性氧清除剂NAC、ML385和MAFF shRNA抑制ZCY020的活性
RAW264.7细胞贴壁后，①加入不同浓度的ZCY020和活性氧清除剂NAC(5mM)或ML385，处理2小时后加入LPS 24h后收集细胞；②转染MAFF敲低质粒shMAFF后加入不同浓度的ZCY020处理2小时，后加入LPS 24h后收集细胞。细胞进行免疫印迹分析和流式分析。
结果表明，NAC和ML385预处理显著减弱了ZCY020对p62、BNIP3和LC3B-II的作用(图 6D-6E)；ZCY020对线粒体超氧化物产生的抑制作用被ML385阻断(图6F)；MAFF敲低后，ZCY020对ZCY020对p62、BNIP3和LC3B-II的作用均被阻断(图6G)。
实施例48 ZCY020对LPS诱导的小鼠急性肺损伤模型的治疗作用
BALB/C小鼠40只，7-8周龄，雌雄各半，随机分组。实验开始时，第一天灌胃给药或生理盐水，2h后腹腔注射LPS(15mg/kg)，并于12h、48h、72h和96h时灌胃给予药物或 生理盐水，观察每组小鼠一周生存率情况。第八天解剖取肝、肺、脾，进行HE染色、免疫 印迹和RT-qPCR分析。结果表明，而在口服ZCY020后，LPS引起的死亡率显著下降，且呈 现剂量依赖性(图7A)。血清ELISA分析表明，化合物ZCY020能够显著降低LPS内毒素引 起的炎症细胞因子的产生(图7C)。病理组织分析表明，化合物ZCY020治疗明显改善了小鼠 肺部和肝脏的病理损伤：炎症细胞浸润、肺泡壁增厚和肝细胞损伤(图7B)。组织免疫印迹和 RT-qPCR分析表明，ZCY020激活了Nrf2通路调控基因的转录和蛋白的表达，并抑制NF-κB 通路的激活，促进线粒体的保护性自噬(图7D-7F)。
实施例49 ZCY020对IMQ诱导的小鼠银屑病模型的治疗作用
雄性BALB/C小鼠40只，7-8周龄，进行背部除毛，除毛面积为2.5cm×2.5cm。将小鼠随机分为4组，将适量的咪喹莫特(IMQ)乳膏连续涂抹于小鼠背部已除毛皮肤6天。对照组给予同等剂量的凡士林软膏。在IMQ涂抹的同时进行灌胃给药或生理盐水。实验期间，每天测体重且进行临床症状评分PASI(Psoriasis Area and Severity Index，发病面积大小和严重程度 指数)。结果表明，在IMQ暴露期间，ZCY020治疗明显降低了红斑、磷屑和皮肤病变增厚的 严重程度，并改善IMQ诱导的银屑病样皮肤炎症(图8A-8C)。
实施例50 ZCY020对小鼠CIA关节炎模型的治疗作用
雄性C57BL/6J小鼠40只，20-25g，随机分为4组。将II型胶原溶液和同体积的完全弗 氏佐剂(CFA，5mg/mL)在冰浴中完全乳化，将CII/CFA乳化剂0.1mL皮内注射于模型组与给 药组每只小鼠尾根部，进行初次免疫。第21天，以同样的方式进行二次免疫。与此同时，每 组小鼠每天进行灌胃给药或生理盐水直至初次免疫后的第37天。期间，每天进行关节炎指数 评分。结果表明，ZCY020治疗剂量依赖性地降低了CIA小鼠的足肿胀严重程度，并显著改 善了软骨和骨破坏以及炎症浸润程度(图9A-9C)。同时用药后，因关节炎而引起的脾脏指数的 升高也有明显降低(图9D)。
Claims (10)
1.基于PROTAC蛋白靶向降解技术的活性天然产物靶点鉴定方法，其特征在于，包括以下步骤：
(1)以活性天然产物为POI配体，以不同E3连接酶配体，合成一系列基于活性天然产物的双功能PROTAC化合物；
(2)通过活性筛选，确定活性最佳的PROTAC化合物，将活性天然产物和PROTAC化合物分别处理的细胞裂解液进行差异蛋白质组学分析，鉴定蛋白裂解液中的差异表达蛋白，并进行KEGG分析，确定PROTAC化合物和活性天然产物作用的潜在靶蛋白；
(3)将潜在的靶点蛋白质进行western blot验证其经PROTAC化合物降解的活性以及降解特性，并经体外靶点验证方法MST、CETSA和DARTS验证活性天然产物与靶蛋白的直接结合力，经细胞生物学技术手段RNAi和体内靶点相关通路验证确定活性天然产物对靶蛋白功能及靶蛋白所影响的下游蛋白的作用，进一步判断活性天然产物是否作用于该靶点，从而验证靶蛋白的真实性，最终确证活性天然产物的靶点蛋白质。
2.根据权利要求1所述的基于PROTAC蛋白靶向降解技术的活性天然产物靶点鉴定方法，其特征在于，所述的步骤(1)中：
活性天然产物为具有千金二萜烷母核类物质，包括化合物和天然产物，所述的天然产物包括但不限于续随子醇Lathyrol和ZCY020，所述Lathyrol和ZCY020结构式分别如下通式I-1和通式I-2所示：
PROTAC化合物为基于千金二萜烷类化合物不同结构、不同E3连接酶配体、不同连接臂的诱导靶蛋白降解的靶向化合物靶蛋白和E3连接酶双功能化合物，或其药学上可接受的盐、水合物或前药，所述的PROTAC化合物结构式如下通式I-3所示：
所述的通式I-3中：
所述的R1，R2为H，肉桂酰Cinnamoyl，对羟基肉桂酰P-hydroxycinnamoyl，乙酰Acetyl，丁酰基Butyryl，苯甲酰Benzoyl或烟碱酰Nicotinoyl；
所述的B是E3泛素连接酶复合体的小分子配体，其选自Cereblon蛋白配体、VHL配体、CIAP配体、MDM2配体、UBR7配体、RNF114配体、CBLB配体或KEAP1配体；
所述的L是连接臂，通过共价键与羟基和B相连，共同构成双功能分子化合物；
所述的B为如下所示的任意结构之一：
其中：
W选自CH2、C＝O、SO2、NH、N-C1-C4烷基；
X选自O、S；
Z选自氢、C1-C4烷基、C3-C6环烷基、卤素；
G、G′选自H、C1-C4烷基、-OH、C1-C4烷基取代的5-10元杂环基，所述杂环基含有1-3个N、O或S的杂原子；
R3自H、D、卤素、硝基、氨基、氰基、羟基、C1-C4烷基、卤代C1-C4烷基、氘代C1-C4烷基；
其中，所述的L通过共价键与羟基和B相连。
3.根据权利要求2所述的基于PROTAC降解的活性天然产物靶点鉴定方法，其特征在于，所述的步骤(1)中，L为如下所示的任意结构之一：
其中：n选自1-10之间的整数。
4.根据权利要求1所述的基于PROTAC降解的活性天然产物靶点鉴定方法，其特征在于，所述的步骤(1)中，PROTAC化合物包括：
5.根据权利要求1所述的基于PROTAC降解的活性天然产物靶点鉴定方法，其特征在于，所述的步骤(1)中，PROTAC化合物的制备方法，包括以下步骤：
(1)用无水DMF溶解续随子醇Lathyrol，加入氢化钠与溴丙炔，室温反应后；经稀释，清洗与纯化，得到中间体1；
(2)在中间体1和中间体2的四氢呋喃和水的混合体系中加入维生素C钠和无水硫酸铜；
(3)反应体系在室温条件下反应20-30分钟，待反应结束后，过滤除去固体，滤液减压蒸干后经柱层析分离得到对应的终产物；其中：
所述的中间体2为中间体2-1、中间体2-2、中间体2-3、中间体2-4、或中间体2-5中的一种；
所述的中间体2-1制备过程如下：
取沙利度胺衍生物，加入溶剂，搅拌下依次加入叠氮基-PEG-胺和DIPEA，经加热反应后，萃取，干燥，浓缩，纯化，洗脱，得黄色油状物，即中间体2-1；
所述的中间体2-2制备过程如下：
称取M7，溶解后，加入叠氮基-PEG-胺，EDCI，HOBt和DIPEA，室温反应后，稀释，洗涤，干燥，纯化，得到中间体2-2产物；
所述的中间体2-3制备过程如下：
称取B5，溶解后，加入叠氮基-PEG-胺，EDCI，HOBt和DIPEA，室温反应后，经稀释，洗涤，干燥与纯化，制得中间体2-3；
所述的中间体2-4制备过程如下：
称取B4盐酸盐，溶解后，加入叠氮羧酸，EDCI，HOBt和DIPEA，室温反应后，经稀释，洗涤，干燥与纯化，制得中间体2-4；
所述的中间体2-5制备过程如下：
称取VHL盐酸盐，溶解后，加入叠氮羧酸，EDCI，HOBt和DIPEA，室温反应后，经稀释，洗涤，干燥，与纯化，制得中间体2-5。
6.根据权利要求5所述的基于PROTAC降解的活性天然产物靶点鉴定方法，其特征在于，所述的步骤(2)中，当中间体2为中间体2-1时，n＝2、3或4，获得的终产物为终产物1-3，发生反应过程如下式III所示；当中间体2为中间体2-2时，n＝2、3或4，发生反应过程如下式IV所示，获得的终产物为终产物4-6；当中间体2为中间体2-3时，n＝2、3或4，发生反应过程如下式V所示，获得的终产物为终产物7-9；当中间体2为中间体2-4时，n＝1、3或5，发生反应过程如下式VI所示，获得的终产物为终产物10-12；当中间体2为中间体2-5时，n＝1、3或5，R1为H或CH3，发生反应过程如下式VII所示，获得的终产物为终产物13-18；
7.根据权利要求1所述的基于PROTAC蛋白靶向降解技术的活性天然产物靶点鉴定方法，其特征在于，所述的步骤(2)中，所述的活性筛选方法包括但不限于炎症刺激模型中抗炎活性筛选、肿瘤细胞中抗肿瘤活性筛选、氧化应激模型中抗氧化活性筛选，检测方法视具体模型而变；所述炎症刺激模型中抗炎活性筛选方法具体步骤如下：
(1)将合成的一系列基于活性天然产物的双功能PROTAC化合物加入培养的处于对数生长期的小鼠巨噬细胞RAW264.7中处理3小时后，以脂多糖LPS刺激细胞24小时，建立小鼠巨噬细胞炎症模型；
(2)用Griss试剂检测细胞上清中总一氧化氮含量，依据PROTAC小分子处理后对NO释放的抑制作用，进行分析，判定所合成的PROTAC化合物的抗炎活性，筛选抗炎活性最佳的PROTAC化合物，即ZCY-PROTAC，所述的ZCY-PROTAC结构式为：
8.一种药物组合物，其特征在于，含有权利要求2所述的具有千金二萜烷母核类物质或其立体异构体、互变异构体、药学上可接受的盐、水合物、前药以及药学上可接受的载体、稀释剂、辅剂、媒介物或它们的组合；所述的药物组合物的剂型为注射剂、片剂和胶囊剂中的任意一种。
9.权利要求2所述的具有千金二萜烷母核类物质在制备治疗或预防炎症相关疾病的药物中的应用，具体作为MAFF-Nrf2激活剂的应用，用于治疗炎症、自身免疫性疾病和免疫性损伤，包括诸如系统性红斑狼疮、类风湿关节炎、系统性血管炎、硬皮病、皮肌炎、自身免疫性溶血性贫血、溃疡性结肠炎、慢性淋巴细胞性甲状腺炎、甲状腺功能亢进、胰岛素依赖型糖尿病、重症肌无力、溃疡性结肠炎、恶性贫血伴慢性萎缩性胃炎、肺出血肾炎综合征、寻常天疱疮、类天疱疮、原发性胆汁性肝硬化、多发性脑脊髓硬化症、急性特发性多神经炎、感染所致的内毒素血症或败血症。
10.基于权利要求2所述的具有千金二萜烷母核类物质的靶向降解剂及其药学上可接受的盐在制备治疗或预防炎症相关疾病的药物中的应用，所述的炎症作用靶点为MAFF。
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