CN117567543A - 一种白头翁皂苷b4衍生物及其制备方法与应用 - Google Patents
一种白头翁皂苷b4衍生物及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种白头翁皂苷B4衍生物及制备方法与应用，以化合物白头翁皂苷B4（AB4）为原料，针对C‑19环外双键或C‑28的羧基利用亲核取代、亲电加成、酯化反应或酰胺化反应制备衍生物，以AB4衍生物的活性成分制备具有抗炎、免疫调节作用的药物。本发明首次公开的多数AB4衍生物对巨噬细胞未显示出明显的细胞毒性，能够降低NF‑κB信号通路中P‑IκBa蛋白水平，抑制NLRP3信号通路活化，明显降低Pro‑IL‑1β水平（p<0.05），且效果优于AB4；这些结果提本发明的AB4衍生物有更好的抗炎活性。另外这些AB4衍生物对特应性皮炎治疗作用显著，耳肿胀水平降低、背部和耳朵皮肤溃烂明显改善、治疗效果优于阳性药***和AB4，且毒副作用显著小于***。
Description
技术领域
本发明属于生物医药技术领域，涉及一种白头翁皂苷B4衍生物及制备方法，及该类衍生物在制备抗炎，比如特应性皮炎药物中的应用。
背景技术
一般来说炎症是机体对于刺激的一种防御反应，常见症状是红热、肿胀疼痛。炎症根据不同原因而分类广泛，例急慢性炎症、局部和全身炎症、感染性与非感染性炎症等等。炎症与维持体内稳态平衡有着紧密联系，在炎症过程中，一方面损伤因子直接或间接造成组织和细胞的破坏，另一方面通过炎症充血和渗出反应，以稀释、杀伤和包围损伤因子；同时通过实质和***的再生使受损的组织得以修复和愈合。因此炎症在修复、重塑和更新不同组织这一过程中扮演重要角色。但当炎症反应在体内变得不可控时，会引发系列疾病，如关节炎、肺炎、胃炎等等。为了保证机体健康，恢复正常的生理机能，抗炎药是必须的。
众所周知，经典的抗炎药是非甾体抗炎药和类固醇，他们在不同类型的炎性疾病上都有着一定的治疗作用。但这两类药物都有一系列不良副作用，如胃肠紊乱、心脏变化、肾毒性、高血压、2型糖尿病、内脏肥胖和动脉粥样硬化等；随着时代的发展和人们对健康的更高需求，越来越多人致力于寻找开发有着疗效好、毒副作用低、被接受程度大等优势的抗炎药。而天然产物有着副作用小，安全性高、药效好的特点。
特应性皮炎（AD）是一种公共常见、免疫介导的炎症性皮肤病，其特点是复发性、瘙痒性、局部湿疹，常伴有季节性波动；特应性皮炎也被称为特应性湿疹、神经性皮炎、特应性皮肤炎，以及最常见的湿疹。目前对于湿疹的治疗药物包括各种局部皮质类固醇（TCS）、局部钙调神经磷酸酶抑制剂他克莫司、吡美莫司，还有磷酸二酯酶4（PDE4）抑制剂克立硼罗等。对于更严重的AD，除了使用紫外线外，目前的治疗指南建议环孢素A、甲氨蝶呤、硫唑嘌呤和霉酚酸酯，但存在副作用大或价格昂贵等问题，白头翁皂苷B4（AB4）对特应性皮炎有治疗作用，但目前存在AB4分子量和水溶性太大、生物利用度低等问题。
发明内容
现有抗炎药物具有明显的毒副作用，有些药物存在半衰期短的缺陷，为了解决此问题，本发明公开了一种白头翁皂苷B4衍生物及制备方法，及该类衍生物在制备抗炎（包括特应性皮炎）药物中的应用。
白头翁皂苷B4（AB4）对特应性皮炎有治疗作用，但目前存在AB4分子量和水溶性太大、生物利用度低等问题。对此，本发明采用化学方法对AB4进行结构修饰和改造，得到系列衍生物以期解决上述AB4成药性差的问题并得到活性优于AB4的化合物。
本发明采用如下技术方案：
一种白头翁皂苷B4衍生物，具有如下化学结构通式：
式中，R1为3-O-α-L-吡喃鼠李糖基-(1→2)-α-L-吡喃***糖基；R2为羟基或乙酰氧基；R3为2-烯丙基、2-丙基、3-羟基丙烯基、3-溴丙烯基、2-环氧乙烷甲基；R4为1-氧苯并三氮唑基、甲氧基、羟基、28-O-α-L-吡喃鼠李糖-(1→4)-β-D-吡喃葡萄糖-(1→6)-β-D-吡喃葡萄糖基、28-O-α-L-[2，3，4－三乙酰氧基－吡喃鼠李糖]-(1→4)-β-D-[2，3，6－三乙酰氧基－吡喃葡萄糖]-(1→6)-β-D-[2，3，4－三乙酰氧基－吡喃葡萄糖] 基、2-甲氧乙胺基、4-氨基丁酸甲酯基、环戊胺基、3-氯丙胺基、2-氟乙胺基、1-（3-氨基丙基）苯并三氮唑基、环己胺基、1-环丙基乙胺基、环丁基甲胺基、1-（3-氨基丙基）咪唑基、N-（2-氨乙基）吡咯烷基、4-氨基呋喃基、3-氨基环戊烷甲酸甲酯基、4-氨基环己基甲酸甲酯基、烯丙胺基、1-（2-氨乙基)哌啶基、2-噻唑乙胺基、四氢呋喃甲胺基、N-氨乙基吗啉基、1-甲基-4-哌啶甲胺基、1-甲基吡咯烷-3-甲胺基、4-氨基环己醇基、β-苯乙胺基、2-噻吩乙胺基、对羟基苯乙胺基、4-恶唑甲胺基、甘氨酸基、N-（2-氨基乙基）乙酰胺基、4-氨基丁酸基、5-氨基戊酸基、6-氨基己酸甲酯基、3-L-氨基环戊醇基、3-D-氨基环戊醇基。优选的，R3为2-烯丙基、2-丙基、3-羟基丙烯基中的一种；R4为1-氧苯并三氮唑基、1-（3-氨基丙基）苯并三氮唑基、N-氨乙基吗啉基、对羟基苯乙胺基中的一种；进一步优选的，白头翁皂苷B4衍生物为化合物B4-19、B4-33和B4-39等。
本发明公开了一种药物组合物，以上述白头翁皂苷B4衍生物为活性成分。
本发明公开了上述白头翁皂苷B4衍生物或者药物组合物在制备抗炎症药物中的应用。优选的，所述炎症包括体表炎症、体内炎症。进一步优选的，所述炎症包括皮肤炎症。作为示例，本发明公开了上述白头翁皂苷B4衍生物或者药物组合物在制备治疗特应性皮炎的药物中的应用。
本发明还公开了上述白头翁皂苷B4衍生物或者药物组合物在制备免疫调节药物中的应用。
本发明中，所述药物包括外用、口服、直肠或者肠胃外药物。所述药物被制成药学上允许的剂型，比如所述药物的剂型包括丸剂、片剂、粉剂、胶囊剂、颗粒剂（散剂）、膏剂、溶液剂、凝胶剂或者栓剂，溶液剂包括滴丸、滴剂、喷雾剂、注射剂、混悬液。
本发明公开了所述白头翁皂苷B4衍生物的制备方法，以化合物AB4为原料，采用亲核取代、亲电加成、酯化或酰胺化反应制备所述白头翁皂苷B4衍生物。
本发明首次公开的白头翁皂苷B4衍生物对THP-1巨噬细胞未显示出明显的细胞毒性，能够降低NF-κB信号通路中P-IκBa蛋白水平，抑制NLRP3信号通路活化，明显降低Pro-IL-1β水平（p<0.05），且效果优于AB4；这些结果说明本发明的AB4衍生物有更好的抗炎活性。另外在DNCB诱导的特应性皮炎小鼠中，AB4衍生物对特应性皮炎有较好的治疗作用，显著降低耳肿胀水平、有效改善小鼠背部和耳朵皮肤溃烂、水肿的状况，效果优于阳性药***和AB4，同时AB4衍生物具有一定的免疫调节作用，尤其是，本发明衍生物没有***的免疫抑制副作用。
附图说明
图1为本发明白头翁皂苷B4衍生物的制备方案（1）的反应示意图。
图2为本发明白头翁皂苷B4衍生物的制备方案（2）的反应示意图。
图3为本发明白头翁皂苷B4衍生物的制备方案（3）的反应示意图。
图4为50uM下AB4衍生物对HIEC和THP-1细胞毒性。
图5为LPS刺激下，无细胞毒的AB4衍生物（10uM）亚硝酸盐产生含量。
图6为AB4衍生物抗炎活性WB图。
图7为AB4衍生物抗炎活性WB统计结果。
图8为DNCB诱导的特应性皮炎小鼠背部皮肤情况。
图9为DNCB诱导的特应性皮炎小鼠耳部情况。
图10为小鼠体重变化和背部评分示意图。
图11为小鼠耳厚度差和耳重差示意图。
图12为小鼠脾指数示意图。
具体实施方式
本发明公开了所述白头翁皂苷B4衍生物的制备方法，以化合物AB4为原料，采用亲核取代、亲电加成、酯化或酰胺化反应制备所述白头翁皂苷B4衍生物。
采用以下方案对AB4进行结构修饰与改造：
（1）以AB4为原料，在氢氧化钠水溶液105℃下水解得到中间体A3，再对糖基部分进行乙酰基保护；通过氧化、亲核取代、还原、水解等反应对双键部分进行改造，得到部分白头翁皂苷B4衍生物，反应示意图参见图1，反应条件：（a）NaOH/H2O，105℃，10h；（b）AC2O，Py，DMAP，rt；（c）i m-CPBA,NaHCO3，DCM；ii m-CPBA,CHCl3，reflux，2days；iii NBS，CCl4，3days；（d）NaOH，THF/CH3OH/H2O，室温，过夜。
（2）根据方案（1）得到中间体A3后，以C28位羧基为改造基团，在TBTU（O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸酯）、DIEA（N,N-二异丙基乙胺）、DMF（N，N-二甲基甲酰胺）作用下进行酰胺缩合，得到系列酰胺类衍生物，为部分白头翁皂苷B4衍生物，反应示意图参见图2，反应条件：（a）NaOH/H2O，105℃，10h；（b）TBTU，DIEA，DMF，室温，过夜；（c）DIEA，Amine，室温，过夜；（d）NaOH，THF/CH3OH/H2O，室温，过夜。
（3）以AB4为原料，在保护整个糖基的情况下，通过氧化、还原、亲核取代等方法对双键部分进行改造，得到部分白头翁皂苷B4衍生物，反应示意图参见图3，反应条件：（a）AC2O，Py，DMAP，rt；（b）i NBS，CCl4，3days； ii H2，Pd/C；（c）i NaOH，THF/CH3OH/H2O或CHCl3-CH3OH，K2CO3，室温，过夜。
本领域技术人员根据本发明的原料以及反应条件，可根据常规技术得到本发明的产物（羽扇豆烷型五环三萜皂苷化合物），也可采用其他可得到本发明产物的方法。
本发明公开了上述白头翁皂苷B4衍生物在制备抗炎作用药物中的应用。尤其是，本发明公开了上述白头翁皂苷B4衍生物在制备治疗特应性皮炎的药物中的应用。
本发明公开的药物组合物，以上述白头翁皂苷B4衍生物为活性成分，还包括药学上可接受的载体。所述活性成分和药物组合物用于制备特应性皮炎疾病的药物。比如，所述药物含有治疗有效量的白头翁皂苷B4衍生物或其盐酸盐、高氯酸盐、甲磺酸盐、磷酸盐、柠檬酸盐或硫酸盐和药学上可接受的载体。
本发明中，药学上可接受的载体指一种或多种相容性固体或液体填料或凝胶物质，能够药用，有足够的纯度和低的毒性，而且药物组合物中各组份之间以及与本发明的活性成分之间相互掺和且不降低活性成分的药效。药学上可接受的载体包括稀释剂、增溶剂、潜溶剂、崩解剂、分散剂、润滑剂、矫味剂、抗氧剂、粘合剂、吸收剂、湿润剂、缓冲剂、交联剂。药学上可以接受的载体部分例子有纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、檄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、环糊精(如羟丙基β-环糊精)、乳化剂(如吐温)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。
本发明中，所述药物包括外用、口服、直肠或者肠胃外药物。所述药物被制成药学上允许的剂型，比如所述药物的剂型包括丸剂、片剂、粉剂、胶囊剂、颗粒剂（散剂）、膏剂、液剂、凝胶剂或者栓剂，液剂包括、滴丸、滴剂、喷雾剂、注射剂、混悬液。
本发明公开了一种白头翁皂苷B4衍生物在制备抗炎作用药物中的应用。本发明衍生物可以单独给药，或者与其他治疗药物联合给药。本发明的活性成分或药物组合物的施用方式没有特别限制，代表性的施用方式包括外用、口服、直肠、肠胃外(比如静脉内、肌肉内或皮下)等。用于口服给药的固体剂型包括胶囊剂、片剂、丸剂、散剂和颗粒剂；用于口服给药的液体剂型包括药学上可接受的乳液、溶液、悬浮液、糖浆或酊剂。除了活性成分外，液体剂型可包含本领域中常规采用的稀释剂，如水或其它溶剂、增溶剂和乳化剂，比如乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1,3-丁二醇、二甲基甲酰胺以及油，特别是棉籽油、花生油、玉米胚油、檄榄油、葩麻油和芝麻油或这些物质的混合物等。除了这些情性稀释剂外，组合物也可包含助剂，如润湿剂、乳化剂和悬浮剂、甜味剂、矫味剂和香料。除了活性成分外，悬浮液可包含悬浮剂，例如，乙氧基化异十八烷醇、聚氧乙烯山梨醇和脱水山梨醇酯、微晶纤维素、甲醇铝和琼脂或这些物质的混合物等。用于肠胃外注射的组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体,稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。
现有技术公开了白头翁皂苷B4（AB4）具有抗炎症的应用，但是白头翁皂苷B4水溶性极强，半衰期短，口服利用度低，因而限制了其在临床上的应用；本发明对AB4进行结构改造，获得抗炎活性更好、毒性很低的化合物。本发明白头翁皂苷B4衍生物的合成路线参见图1至图3，体内外活性结果见图4-12。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。除非另外说明，否则百分比和份数是重量百分比和重量份数。除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。下述制备实施例中，试剂为现有产品，主要由上海化学试剂公司提供；TLC薄层层析硅胶板由山东烟台江友硅胶开发公司，型号HSGF 254，化合物纯化使用的正相柱层析硅胶为北京伊诺凯科技有限公司生产，200-300目。NMR用Varian Mercury 400M核磁共振仪记录，化学位移以δ（ppm）表示；本文缩写所对应的中文如下：DMF：N，N-二甲基甲酰胺；DCM：二氯甲烷；THF：四氢呋喃；TBTU：O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸酯；DIPEA：N,N-二异丙基乙胺；PE：石油醚；EA：乙酸乙酯；K2CO3：碳酸钾；amine：胺。
所有数据的统计分析使用GraphPad Prism 8软件进行，统计学显著性平均值之间的差异由GraphPad Prism 8进行比较，全部数据采用单因素方差分析或双因素方差分析进行，通常用P值表示两者间的差别。如果P≤0.05，则表明两者存在统计学差异，通常用“#”或“*”表示，“#”表示正常组与模型组相比，“*”表示给药组与模型组相比（在细胞毒实验中“*”表示给药组与正常组相比）。如果P≤0.01，则表明两者存在显著统计学差异，用“##”或“**”表示。如果P≤0.001，则表明两者存在高度显著统计学差异，用“###”或“***”表示；如果P≤0.0001，则表明两者存在极其显著统计学差异，用“####”或“****”表示。
以下实施例各白头翁皂苷B4衍生物具体结构如下：
实施例一
B4-1：将白头翁皂苷B4（1g，0.819mmol）溶于20ml吡啶，加入醋酸酐2.5ml和DMAP（14mg，0.082mmol）常温搅拌28小时后往反应液中加入50ml乙酸乙酯再加入100ml水萃取得有机相，再水洗两遍后干燥有机相，旋干得全乙酰化B4。将全乙酰化B4（300mg，0.2456mmol）溶于15ml四氯化碳，加入NBS（48mg，0.2702mmol），室温搅拌24小时得中间体3-O-α-L-[3，4－二乙酰氧基－吡喃鼠李糖基]-(1→2)-α-L-[2，3，4－三乙酰氧基－吡喃***糖基]-3β,23-二羟基羽扇豆烷-Δ20(29)烯-30-溴-28-O-α-L-[2，3，4－三乙酰氧基－吡喃鼠李糖]-(1→4)-β-D-[2，3，6－三乙酰氧基－吡喃葡萄糖]-(1→6)-β-D-[2，3，4－三乙酰氧基－吡喃葡萄糖]。将上述中间体（100mg，0.052mmol）溶于4ml二氯甲烷:甲醇=（2:1）混合溶液中，加入碳酸钾（161mg，1.165mmol），室温搅拌16小时，反应结束后减压浓缩，经制备液相（60%甲醇-水）纯化得白色固体12mg，产率22%。1H NMR (400 MHz, Methanol-d 4) δ 5.51 (1H,d,J= 8.1 Hz, 1-H of glc), 5.20 (1H, brs, 1-H of rha ), 5.00 (1H, brs, 1´-H ofrha ), 4.98 (1H, brs, H1-29 ), 4.60 (1H, d,J= 4.7 Hz, 1-H of ara ), 4.42 (1H,d,J= 7.8 Hz, 1´-H of glc), 4.16 (1H, d,J= 11.9 Hz, H1-23), 3.95 (2H, s, H-30), 1.30 (3H, d,J= 6.3 Hz, 6-H3 of rha), 1.28 ( 3H, d,J= 6.2 Hz, 6´-H3 ofrha ), 1.07(3H, s, H-27), 1.00 (3H, s, H-26 ), 0.93 (3H, s, H-25 ), 0.72 (3H,s, H-24).13C NMR (101 MHz, MeOD) δ 176.33, 152.63, 110.16, 104.60, 104.28,102.91, 101.86, 95.28, 82.30, 79.56, 78.26, 77.98, 76.88, 76.71, 76.63,76.12, 75.28, 73.94, 73.74, 73.66, 72.43, 72.21, 72.14, 72.02, 70.98, 70.67,70.16, 69.62, 69.11, 64.73, 64.59, 61.93, 58.55, 57.98, 51.92, 51.10, 49.50,44.44, 44.04, 43.61, 41.99, 39.91, 39.37, 37.82, 37.42, 34.93, 33.00, 32.77,30.87, 27.99, 26.69, 22.20, 18.78, 17.96, 17.84, 17.28, 16.79, 15.11, 13.54。
实施例二
B4-4：将白头翁皂苷B4（1g，0.82mmol）溶于15ml水中，加入氢氧化钠（65.6mg，1.64mmol）后105℃条件下搅拌12小时后将反应液过滤并水洗沉淀两次得白头翁皂苷A3。再将白头翁皂苷A3（1g，1.33mmol）溶于20ml吡啶，加入DMAP（20mg，0.164mmol）室温搅拌18小时后往反应液中加入50ml乙酸乙酯再加入100ml水萃取得有机相，再水洗两遍后干燥有机相，旋干后得中间体3-O-α-L-[3，4－二乙酰氧基－吡喃鼠李糖基]-(1→2)-α-L-[2，3，4－三乙酰氧基－吡喃***糖基]-3β,23-二羟基羽扇豆烷-Δ20(29)烯-28-酸。将上述中间体（1g，0.998mmol）溶于DCM、甲醇混合溶液中（2:1），加入Pd/C（50mg），氢气条件下室温反应18小时，HPLC监测反应，反应结束后过滤，减压浓缩除去溶剂，经C18制备分离（75%甲醇水），得白色固体523mg，产率52.3%。1H NMR (400 MHz, Chloroform-d) δ 5.04 (1H, brs, 1-Hof rha), 4.42 (1H, d,J= 6.4 Hz, 1-H of ara ),4.11 (1H, d,J= 11.5 Hz, H1-23),3.58 (1H, d,J= 11.5 Hz, H2-23 ), 2.13 (3H, s, H3 of -OAc ), 2.10 (3H, s, H3of -OAc), 2.09 (3H, s, H3 of -OAc), 2.05 (3H, s, H3 of -OAc), 2.03 (3H, s, H3of -OAc), 1.96 (3H, s, H3 of -OAc), 1.21 (3H, d,J= 6.2 Hz, 6-H3 of rha ),0.93 (3H, s, H-27), 0.91 (3H, s, H-26 ), 0.86 (3H, s, H-25 ), 0.85 (3H, d,J=7.2 Hz, H-29 ), 0.77 (3H, s, H-24), 0.75 (3H, d,J= 6.7 Hz, H-30).13C NMR (101MHz, CDCl3) δ 181.66, 170.58, 170.52, 170.44, 170.29, 170.19, 169.78, 103.69,98.31, 82.11, 77.36, 74.47, 72.01, 71.15, 69.69, 68.73, 67.98, 67.27, 65.25,62.86, 56.89, 50.63, 48.85, 48.13, 44.25, 42.64, 42.11, 40.83, 38.74, 38.36,37.53, 36.87, 34.24, 32.13, 29.86, 29.71, 27.04, 25.84, 23.10, 22.85, 21.17,21.11, 21.07, 20.95, 20.91, 20.80, 18.10, 17.47, 16.71, 16.13, 14.80, 14.58,12.62。
B4-5：将B4-4（200 mg, 0.20 mmol）溶于4 mL甲醇/四氢呋喃/水（2:1:1）混合溶液中，加入氢氧化钠（72 mg, 1.8 mmol），室温搅拌12h，反应结束，减压除去溶剂，用50 mL水洗除盐，干燥得白色固体120 mg，产率80.1%。1H NMR (400 MHz, DMSO-d 6) δ 4.96 (1H,brs, 1-H of rha),4.22 (1H, d,J= 4.6 Hz, 1-H of ara) , 4.04 (1H, d,J= 11.1 Hz,H1-23), 1.06 (3H, d,J= 5.8 Hz, 6-H3 of rha ), 0.89 (3H, s, H-27 ), 0.86 (3H,s, H-26), 0.82 (3H, s, H-25), 0.80 (3H, d,J= 6.1 Hz, H-29), 0.72 (3H, d,J=6.2 Hz, H-30), 0.69 (3H, s, H-24).13C NMR (101 MHz, DMSO) δ 170.16, 103.36,100.28, 80.72, 74.67, 72.23, 72.03, 70.63, 70.42, 68.60, 67.53, 64.70, 64.04,56.11, 50.13, 48.38, 47.81, 43.95, 42.26, 41.69, 40.48, 38.38, 37.55, 36.51,33.91, 29.62, 29.36, 29.24, 26.83, 25.42, 23.23, 22.73, 20.90, 20.75, 17.98,16.53, 16.04, 14.81, 14.27, 12.55。
实施例三
B4-6：将中间体3-O-α-L-[3，4－二乙酰氧基－吡喃鼠李糖基]-(1→2)-α-L-[2，3，4－三乙酰氧基－吡喃***糖基]-3β,23-二羟基羽扇豆烷-Δ20(29)烯-28-酸（1g，0.998mmol），溶于10ml氯仿，低温下加入间氯过氧苯甲酸（207mg，1.20mmol），完全溶解后升温至65℃，回流反应16小时，反应结束后减压浓缩出去溶剂，硅胶柱层析（二氯甲烷:甲醇=80:1→70:1）得白色固体305mg，产率30%。1H NMR (400 MHz, Chloroform-d) δ 5.22 (1H,brs, 1-H of rha), 5.04 (1H, brs, H1-29), 4.97 (1H, s, H1 of -OH on C-30),4.92 (1H, brs, H2-29), 4.41 (1H, d,J= 6.4 Hz, 1-H of ara ), 4.12 (2H, s, H2of H-30 ), 2.13 (3H, s, H3 of -OAc), 2.10 (3H, s, H3 of -OAc), 2.10 (3H, s,H3 of -OAc), 2.05 (3H, s, H3 of -OAc), 2.03 (3H, s, H3 of -OAc), 1.96 (3H, s,H3 of -OAc), 1.21 (3H, d,J= 6.2 Hz, 6-H3 of rha ), 0.96 (3H, s, H-27), 0.91(3H, s, H-26), 0.85 (3H, s, H-25), 0.77 (3H, s, H-24).13C NMR (101 MHz, CDCl3)δ 181.05, 170.90, 170.84, 170.76, 170.61, 170.51, 170.10, 155.14, 107.35,104.01, 98.63, 82.36, 77.68, 74.80, 72.34, 71.48, 70.01, 69.05, 68.30, 67.58,65.71, 65.57, 56.72, 51.14, 50.40, 48.47, 43.00, 42.76, 42.42, 41.15, 39.03,38.79, 37.21, 34.51, 32.81, 32.40, 30.16, 27.21, 21.49, 21.43, 21.39, 21.27,21.23, 21.12, 17.79, 17.08, 16.44, 14.98, 12.93。
B4-7：将B4-6（100mg，0.098mmol）溶于4 mL甲醇/四氢呋喃/水（2:1:1）混合溶液中，加入氢氧化钠（35.3 mg, 0.882 mmol），室温搅拌12h，反应结束，减压浓缩除去溶剂，硅胶柱层析（二氯甲烷:甲醇=10:1）得15mg白色固体，产率20%。1H NMR (400 MHz, DMSO-d 6) δ12.03 (1H, s, H1 of -COOH ),4.95 (1H, brs, 1-H of rha ), 4.85 (1H, s, H1 of -OH on C-30), 4.76 (1H, brs, H1-29), 4.66 (1H, brs, H2-29),4.21 (1H, d,J= 4.7Hz, 1-H of ara), 4.03 (1H, d,J= 11.3 Hz, H1-23), 3.69 (1H, d,J= 11.8 Hz, H2-23), 3.87 (2H, s, H2of H-30 ), 1.05 (3H, d,J= 6.1 Hz, 6-H3 of rha ), 0.90(3H, s, H-27), 0.84 (3H, s, H-26), 0.79 (3H, s, H-25), 0.67 (3H, s, H-24 ).13CNMR (101 MHz, DMSO) δ 170.49, 155.94, 106.07, 103.68, 100.58, 81.06, 74.97,72.54, 72.33, 70.95, 70.72, 68.92, 67.85, 63.40, 55.94, 50.66, 49.42, 48.14,42.85, 42.71, 42.38, 42.01, 40.78, 38.63, 38.07, 36.85, 34.12, 32.44, 29.56,29.11, 27.09, 25.75, 22.63, 21.22, 18.30, 18.01, 16.88, 16.28, 14.70, 14.49,12.86。
实施例四
B4-8:将中间体3-O-α-L-[3，4－二乙酰氧基－吡喃鼠李糖基]-(1→2)-α-L-[2，3，4－三乙酰氧基－吡喃***糖基]-3β,23-二羟基羽扇豆烷-Δ20(29)烯-28-酸（500mg，0.499mmol）,溶于5ml二氯甲烷中，并加入碳酸氢钠（46.1mg，0.55mmol）,室温搅拌8小时，反应结束后过滤，减压浓缩，经硅胶柱层析（二氯甲烷:甲醇=70:1）得白色固体211mg，产率41.5%。1H NMR (400 MHz, DMSO-d 6) δ 12.09 (1H, s, H1 of -COOH), 5.02 (1H, brs,1-H of rha), 4.50 (1H, d,J= 6.9 Hz, 1-H of ara), 3.99 (1H, d,J= 11.4 Hz, H1-23), 2.56 (2H, dd,J= 3.4 Hz, H-29), 2.10 (3H, s, H3 of -OAc), 2.07 (3H, s, H3of -OAc), 2.06 (3H, s, H3 of -OAc), 2.02 (3H, s, H3 of -OAc), 1.95 (3H, s, H3of -OAc), 1.93 (3H, s, H3 of -OAc), 1.16 (3H, s, H-30), 1.10 (3H, d,J= 6.2Hz, 6-H3 of rha), 0.92 (3H, s, H-27), 0.86 (3H, s, H-26), 0.83 (3H, s, H-25),0.73 (3H, s, H-24).13C NMR (101 MHz, DMSO) δ 177.14, 170.10, 169.90, 169.88,169.64, 169.61, 169.48, 102.35, 97.34, 80.44, 73.92, 71.88, 70.03, 68.83,68.03, 67.83, 66.38, 64.42, 62.63, 59.57, 58.48, 55.77, 55.52, 49.89, 49.08,47.57, 45.10, 41.91, 41.30, 40.22, 38.12, 36.86, 36.28, 36.09, 33.55, 31.56,28.94, 26.96, 26.22, 25.43, 20.74, 20.71, 20.66, 20.52, 20.45, 20.40, 18.05,17.44, 17.10, 16.30, 15.68, 14.05, 12.20。
实施例五
B4-9:将中间体3-O-α-L-[3，4－二乙酰氧基－吡喃鼠李糖基]-(1→2)-α-L-[2，3，4－三乙酰氧基－吡喃***糖基]-3β,23-二羟基羽扇豆烷-Δ20(29)烯-28-酸（500mg，0.499mmol）溶于10ml四氯化碳中，加入NBS（90mg，0.499mmol），室温搅拌20小时，反应结束后过滤、减压浓缩、经硅胶柱层析（二氯甲烷:甲醇=80:1)得类白色固体324mg，产率60%。1HNMR (400 MHz, Chloroform-d) δ 5.21 (1H, brs, 1-H of rha), 5.14 (1H, brs, H1-29), 5.04 (1H, brs, H2-29), 4.41 (1H, d,J= 6.3 Hz, 1-H of ara), 4.11 (1H, d,J= 11.4 Hz, H1-23 ), 3.99 (2H, s, H-30 ), 3.88 (1H, d,J= 10.9 Hz, H2-23), 2.13(3H, s, H3 of -OAc), 2.10 (3H, s, H3 of -OAc), 2.09 (3H, s, H3 of -OAc), 2.05(3H, s, H3 of -OAc), 2.03 (3H, s, H3 of -OAc), 1.96 (3H, s, H3 of -OAc), 1.21(3H, d,J= 6.1 Hz, 6-H3 of rha), 0.97 (3H, s, H-27 ), 0.92 (3H, s,H-26 ), 0.86(3H, s, H-25 ), 0.77 (3H, s, H-24).13C NMR (101 MHz, CDCl3) δ 170.56, 170.50,170.42, 170.28, 170.17, 169.77, 151.40, 113.62, 103.65, 98.32, 82.02, 77.36,74.50, 71.98, 71.19, 69.71, 68.75, 67.97, 67.28, 65.27, 62.82, 56.52, 50.86,48.18, 43.19, 42.50, 42.12, 40.87, 38.76, 38.54, 36.92, 34.23, 33.18, 29.84,29.46, 27.35, 26.96, 25.84, 22.83, 21.15, 21.10, 21.06, 20.94, 20.91, 20.79,18.10, 17.47, 16.77, 16.17, 14.69, 14.25, 12.62。
B4-10:将B4-9（200mg，0.185mmol）溶于4 mL甲醇/四氢呋喃/水（2:1:1）混合溶液中，加入氢氧化钠（66.6 mg, 1.67mmol），室温搅拌12h，反应结束，减压浓缩除去溶剂，硅胶柱层析（二氯甲烷:甲醇=10:1）得100mg白色固体，产率66.7%。1H NMR (400 MHz,Methanol-d 4) δ 5.19 (1H, brs, 1-H of rha), 5.09 (1H, brs, H1-29), 4.96 (1H,brs, H2-29), 4.59 (1H, d,J= 4.7 Hz, 1-H of ara), 4.09 (1H, s, H1 of -OH on C-23), 3.93 (2H, s, H-30), 1.27 (3H, d,J= 6.3 Hz, 6-H3 of rha), 1.07 (3H, s, H-27), 1.00 (3H, s, H-26), 0.92 (3H, s, H-25 ), 0.71 (3H, s, H-24 ).13C NMR (101MHz, MeOD) δ 153.40, 113.70, 104.28, 101.88, 82.26, 76.66, 76.19, 73.93,73.64, 72.14, 72.02, 70.16, 69.11, 66.65, 64.72, 64.57, 58.54, 57.53, 51.91,51.03, 44.39, 44.04, 43.63, 41.89, 39.91, 39.68, 37.81, 35.03, 34.27, 33.21,30.85, 28.23, 26.69, 22.23, 18.77, 17.95, 17.20, 16.69, 15.14, 13.51。
实施例六
B4-11:将中间体3-O-α-L-[3，4－二乙酰氧基－吡喃鼠李糖基]-(1→2)-α-L-[2，3，4－三乙酰氧基－吡喃***糖基]-3β,23-二羟基羽扇豆烷-Δ20(29)烯-28-酸（500mg，0.499mmol）溶于12ml DMF中，加入TBTU（240.3mg，0.7485mmol），DIEA（726mg，2.495mmol），室温搅拌10小时，反应结束后加60ml水至反应液中，析出白色固体，过滤，经硅胶柱层析（石油醚:乙酸乙酯=2:1）得白色固体452mg，产率81%。1H NMR (400 MHz, Chloroform-d) δ8.08 (1H, d,J= 8.4 Hz, H1 of benzene), 7.52–7.56 (1H, m, H1 of benzene ),7.40–7.44(1H, m, H1 of benzene), 7.36 (1H, d,J= 8.3 Hz, H1 of benzene ), 5.04(1H, brs, 1-H of rha), 4.73 (1H, brs, H1-29), 4.64 (1H, brs, H2-29), 4.42 (1H,d,J= 6.4 Hz,1-H of ara), 4.12 ( 1H, d,J= 12.1 Hz, H1-23), 3.57 (1H, d,J= 11.2Hz, H2-23), 2.13 (3H, s, H3 of -OAc), 2.11 (3H, s, H3 of -OAc), 2.10 (3H, s,H3 of -OAc), 2.05 (3H, s, H3 of -OAc), 2.03 (3H, s, H3 of -OAc), 1.97 (3H, s,H3 of -OAc), 1.71 (3H, s, H-30), 1.21 (3H, d,J= 6.2 Hz, 6-H3 of rha), 1.03(3H, s, H-27 ), 0.98 (3H, s, H-26), 0.85 (3H, s, H-25), 0.78 (3H, s, H-24).13CNMR (101 MHz, CDCl3) δ 171.97, 170.54, 170.50, 170.42, 170.28, 170.17,169.77, 149.36, 143.76, 129.01, 128.79, 124.84, 120.78, 110.53, 108.04,103.67, 98.24, 81.97, 77.36, 74.34, 72.05, 71.16, 69.70, 68.72, 67.99, 67.24,65.28, 62.88, 57.13, 50.89, 50.08, 48.21, 46.68, 42.54, 42.11, 40.93, 38.76,38.61, 36.92, 34.21, 30.44, 30.17, 29.83, 25.84, 25.55, 21.18, 21.11, 21.07,20.95, 20.91, 20.80, 19.55, 18.08, 17.46, 16.78, 16.25, 14.80, 12.64。
实施例七
B4-13：将中间体3-O-α-L-[3，4－二乙酰氧基－吡喃鼠李糖基]-(1→2)-α-L-[2，3，4－三乙酰氧基－吡喃***糖基]-3β,23-二羟基羽扇豆烷-Δ20(29)烯-28-酸（100mg，0.0998mmol）溶于5ml DMF中，加入碳酸钾（13.8mg，0.0998mmol）和碘甲烷（14.2mg，0.0998mmol），室温搅拌8小时，反应结束后过滤、减压浓缩除去溶剂，经硅胶柱层析（石油醚:乙酸乙酯=2:1）得白色固体89.4mg，产率88%。1H NMR (400 MHz, Chloroform-d) δ5.04(1H, brs, 1-H of rha )4.73 (1H, brs, H1-29), 4.60 (1H, brs, H2-29), 4.41(1H, d,J= 6.4 Hz,1-H of ara), 3.66 (3H, s, H3 of -COOCH3), 2.13 (3H, s, H3 of-OAc), 2.10 (3H, s, H3 of -OAc), 2.09 (3H, s, H3 of -OAc), 2.05 (3H, s, H3 of-OAc), 2.02 (3H, s, H3 of -OAc), 1.96 (3H, s, H3 of -OAc), 1.68 (3H, s, H-30), 1.21 (3H, d,J= 6.2 Hz, 6-H3 of rha), 0.94 (3H, s, H-27 ), 0.90 (3H, s,H-26), 0.85 (3H, s, H-25), 0.77 (3H, s, H-24).13C NMR (101 MHz, CDCl3) δ176.76, 170.55, 170.49, 170.41, 170.26, 170.18, 169.74, 150.72, 109.72,103.67, 98.27, 82.04, 74.42, 72.04, 71.18, 69.71, 68.74, 68.00, 67.25, 65.27,62.87, 56.68, 51.40, 50.89, 49.61, 48.18, 47.09, 42.45, 42.11, 40.81, 38.75,38.38, 37.07, 36.91, 34.17, 32.26, 30.75, 29.83, 29.74, 25.86, 25.64, 21.15,21.09, 21.05, 20.93, 20.89, 20.78, 19.52, 18.09, 17.45, 16.73, 16.11, 14.66,12.64。
实施例八
B4-14:将化合物A3（3-O-α-L-吡喃鼠李糖基-(1→2)-α-L-吡喃***糖基-3β,23-二羟基羽扇豆烷-Δ20(29)烯-28-酸）(100mg，0.1333mmol）溶于4ml DMF中，加入DIEA（70ul，0.3999mmol），室温搅拌8小时，反应结束完全生成中间体后加入2-甲氧乙胺（15mg，0.2mmol）和DIEA（116ul，0.6665mmol），室温搅拌16小时，反应结束后加入50ml水至反应液中，析出固体，过滤、经硅胶柱层析（二氯甲烷:甲醇=10:1→8:1）得类白色固体72.6mg，产率66%。1H NMR (400 MHz, DMSO-d 6) δ 7.58 (1H, s, H1 of -CONH), 5.05 (1H, brs, 1-Hof rha), 4.65 (1H, brs, H1-29), 4.53 (1H, brs, H1-29), 4.33 (1H, d,J= 5.9 Hz,1-H of ara), 3.22 (3H, s, H3 of -OCH3), 1.62 (3H, s, H-30), 1.07 (3H, d,J=6.2 Hz, 6-H3 of rha), 0.90 (3H, s, H-27 ), 0.84 (3H, s, H-26 ), 0.78 (3H, s,H-25 ), 0.54 (3H, s, H-24).13C NMR (101 MHz, DMSO) δ 175.63, 150.93, 109.24,102.91, 99.90, 79.37, 74.19, 72.82, 72.03, 70.70, 70.43, 70.36, 68.13, 67.78,64.31, 62.44, 57.87, 54.89, 50.08, 49.69, 46.47, 46.23, 42.34, 41.93, 40.21,38.40, 38.07, 37.65, 36.69, 36.15, 33.53, 32.35, 30.35, 28.81, 25.49, 25.30,20.57, 19.08, 17.78, 17.07, 16.40, 15.80, 14.27, 12.81。
以下化合物的制备方法类似于B4-14，更换2-甲氧乙胺，可得到对应产物。
B4-15：制备方法类似于B4-14，产率58% ；除酰胺基团外新增标志氢甲酯上的甲基（3.57ppm）。1H NMR (400 MHz, DMSO-d 6) δ 7.60 (1H, s, H1 of -CONH), 5.05 (1H,brs, 1-H of rha), 4.64 (1H, brs, H1-29), 4.59 (1H, brs, H2-29), 4.53 (1H, s,H1 of -OH on C-23), 4.44 (1H, d,J= 5.9 Hz, 1-H of ara), 3.57 (3H, s, H3 of -COOCH3), 2.28 (2H, t,J= 7.5 Hz, H2 of -CH2-COOCH3), 1.62 (3H, s, H-30), 1.07(3H, d,J= 6.2 Hz, 6-H3 of rha), 0.90 (3H, s, H-27 ), 0.82 (3H, s, H-26), 0.78(3H, s, H-25), 0.54 (3H, s, H-24).13C NMR (101 MHz, DMSO) δ 176.18, 173.79,151.55, 109.88, 103.54, 100.53, 80.00, 74.83, 73.44, 72.66, 71.06, 70.99,68.76, 68.42, 64.93, 63.07, 55.49, 51.87, 50.72, 50.30, 47.10, 46.80, 42.97,42.56, 40.83, 39.03, 38.35, 38.23, 37.27, 36.78, 34.16, 33.04, 31.33, 30.97,29.47, 26.12, 25.93, 25.28, 21.22, 19.69, 18.41, 17.69, 17.04, 16.42, 14.88,13.45。
B4-16：制备方法类似于B4-14，产率54%；除酰胺基团外新增标志氢为环戊烷上单基氢（3.97ppm）。1H NMR (400 MHz, DMSO-d 6) δ 7.28 (1H, s, H1 of -CONH), 5.06 (1H,brs, 1-H of rha), 4.65 (1H, brs, H1-29), 4.53 (1H, brs, H2-29), 4.43 (1H, d,J=5.7 Hz, 1-H of ara), 3.97 (1H, p,J= 6.7 Hz, H1 of -CH on cyclopentane), 1.63(3H, s, H-30), 1.07 (3H, d,J= 6.2 Hz, 6-H3 of rha), 0.91 (3H, s, H-27 ), 0.84(3H, s, H-26), 0.79 (3H, s, H-25), 0.55 (3H, s, H-24).13C NMR (101 MHz, DMSO)δ 175.14, 150.99, 109.17, 102.89, 99.90, 79.37, 74.21, 72.79, 72.03, 70.43,70.36, 68.12, 67.76, 64.28, 62.44, 54.64, 50.23, 50.10, 49.79, 46.48, 46.17,42.34, 41.88, 40.22, 38.39, 37.66, 36.58, 36.15, 33.51, 32.43, 32.32, 31.46,30.41, 28.77, 25.49, 25.30, 23.57, 23.53, 20.59, 19.07, 17.77, 17.05, 16.39,15.81, 14.24, 12.82。
B4-17：制备方法类似于B4-14，产率43%；除酰胺基团外新增标志氢为连接氯原子的亚甲基（3.60ppm）。1H NMR (400 MHz, DMSO-d 6) δ 7.66 (1H, s, H1 of -CONH), 5.06(1H, brs, 1-H of rha), 4.65 (1H, brs, H1-29), 4.54 (1H, brs, H2-29), 4.33 (1H,d,J= 5.8 Hz, 1-H of ara), 3.60 (2H, t,J= 6.7 Hz, H2 of -CH2Cl), 1.63 (3H, s,H-30), 1.07 (3H, d,J= 6.2 Hz, 6-H3 of rha), 0.91 (3H, s, H-27), 0.83 (3H, s,H-26), 0.78 (3H, s, H-25), 0.54 (3H, s, H-24).13C NMR (101 MHz, DMSO) δ175.91, 151.11, 109.47, 103.12, 100.10, 79.57, 74.39, 73.05, 72.24, 70.65,70.57, 68.33, 68.01, 64.53, 62.64, 55.09, 50.30, 49.86, 48.81, 46.68, 46.38,43.34, 42.55, 42.14, 40.43, 38.61, 37.89, 36.88, 36.36, 36.11, 33.73, 32.70,30.56, 29.07, 25.70, 25.51, 20.80, 19.28, 17.99, 17.26, 16.63, 16.04, 14.46,13.03。
B4-18：制备方法类似于B4-14，产率38%；除酰胺基团外新增标志为氟原子（19.44ppm）。1H NMR (400 MHz, DMSO-d 6) δ 7.76 (1H, s, H1 of -CONH), 5.04 (1H,brs, 1-H of rha ), 4.57 (1H, brs, H1-29), 4.56 ((1H, brs, H2-29)) ,4.53 (1H,s, H1 of -OH on C-23), 4.42 (1H，d,J= 5.9 Hz, 1-H of ara), 1.62 (3H, s, H-30),1.06 (3H, d,J= 6.2 Hz, 6-H3 of rha), 0.90 (3H, s, H-27), 0.82 (3H, s, H-26),0.77 (3H, s, H-25), 0.53 (3H, s, H-24).13C NMR (101 MHz, DMSO) δ 175.99,150.88, 109.27, 102.89, 99.91, 83.03, 81.39, 79.37, 74.23, 72.78, 72.04,70.45, 70.37, 68.14, 67.76, 64.27, 62.45, 54.96, 50.07, 49.69, 46.47, 46.23,42.35, 41.92, 40.21, 38.39, 37.57, 36.72, 36.15, 33.49, 32.28, 30.33, 29.84,25.48, 25.29, 20.56, 19.07, 17.78, 17.05, 16.39, 15.72, 14.27, 12.82.19F NMR(377 MHz, DMSO) δ 19.44。
B4-19：制备方法类似于B4-14，产率33%；除酰胺基团外新增标志氢为苯环上的四个氢（7-8ppm）。1H NMR (400 MHz, DMSO-d 6) δ 8.06 (1H, d,J= 8.4 Hz, H1 of benzene ),7.92 (1H, d,J= 8.4 Hz, H1 of benzene ), 7.73 (1H, s, H1 of -CONH), 7.65 –7.59 (1H, m, H1 of benzene), 7.50 – 7.44 (1H, m, H1 of benzene), 5.06 (1H,brs, 1-H of rha), 4.64 (1H, brs, H1-29), 4.57 (1H, brs, H2-29), 4.43 (1H, d,J=5.9 Hz, 1-H of ara), 1.62 (3H, s, H-30), 1.08 (3H, d,J= 6.2 Hz, 6-H3 of rha),0.88 (3H, s, H-27), 0.71 (3H, s, H-26), 0.57 (3H, s, H-25), 0.54 (3H, s, H-24).13C NMR (101 MHz, DMSO) δ 175.79, 150.86, 142.75, 128.28, 126.97, 124.92,119.64, 109.41, 109.24, 102.88, 99.89, 79.35, 79.00, 74.18, 72.79, 72.02,70.43, 70.36, 68.13, 67.76, 64.27, 62.43, 54.88, 50.02, 49.61, 46.42, 46.16,42.32, 41.86, 40.10, 38.37, 37.68, 36.65, 36.09, 34.66, 33.39, 32.31, 30.31,28.84, 28.40, 25.46, 25.26, 20.51, 19.05, 17.77, 17.01, 16.35, 15.54, 14.18,12.79。
B4-20：制备方法类似于B4-14，产率47%；除酰胺基团外新增标志为环己烷上的碳（20-40ppm）。1H NMR (400 MHz, DMSO-d 6) δ 7.17 (1H, s, H1 of -CONH), 5.05 (1H,brs, 1-H of rha), 4.58 (1H, brs, H1-29), 4.52 (1H, brs, H2-29), 4.32 (1H, d,J=5.9 Hz, 1-H of ara), 1.62 (3H, s, H-30), 1.06 (3H, d,J= 6.2 Hz, 6-H3 of rha),0.90 (3H, s, H-27), 0.82 (3H, s, H-26), 0.77 (3H, s, H-25), 0.54 (3H, s, H-24).13C NMR (101 MHz, DMSO) δ 174.51, 151.00, 109.18, 102.91, 99.90, 79.37,74.20, 72.83, 72.03, 70.43, 70.36, 68.12, 67.79, 64.32, 62.43, 54.68, 50.09,49.76, 48.60, 47.24, 46.48, 46.23, 42.34, 41.88, 40.23, 38.40, 37.79, 36.62,36.14, 33.50, 32.53, 32.36, 32.06, 30.40, 28.80, 25.49, 25.33, 24.97, 24.91,20.59, 19.07, 17.78, 17.03, 16.39, 15.87, 14.25, 12.83。
B4-21：制备方法类似于B4-14，产率59%；除酰胺基团外新增标志氢为环丙基乙烷上的甲基（1.08ppm）。1H NMR (400 MHz, DMSO-d 6) δ 7.31 (1H, s, H1 of -CONH), 5.06(1H, brs, 1-H of rha), 4.66 (1H, brs, H1-29), 4.54 (1H, brs, H2-29), 4.33 (1H,d,J= 5.9 Hz, 1-H of ara), 1.63 (3H, s, H-30), 1.07 (3H, d,J= 6.2 Hz, 6-H3 ofrha) 1.08 (3H, s, H3 of -CH3 on 1-cyclopropyletan), 0.91 (3H, s, H-27), 0.86(1H, s, H1 of -CH to CONH ), 0.83 (3H, s, H-26 ), 0.78 (3H, s, H-25), 0.55(3H, s, H-24), 0.42–0.35 (1H, m, H1 of cyclopropyl), 0.31–0.25 (1H, m, H1 ofcyclopropyl), 0.22 – 0.15 (1H, m, H1 of cyclopropyl), 0.14 – 0.06 (1H, m, H1of cyclopropyl).13C NMR (101 MHz, DMSO) δ 174.75, 151.20, 109.38, 103.12,100.11, 79.58, 74.41, 73.03, 72.24, 70.64, 70.57, 68.33, 68.00, 64.52, 62.64,55.00, 50.30, 49.96, 47.90, 46.69, 46.38, 42.55, 42.09, 40.41, 38.60, 38.10,36.78, 36.36, 33.72, 32.50, 30.63, 28.99, 25.70, 25.51, 20.74, 19.33, 17.99,17.48, 17.24, 17.06, 16.60, 16.00, 14.46, 13.04, 3.14, 2.72。
B4-22：制备方法类似于B4-14，产率51%；除酰胺基团外新增标志氢为环丁基上的单基氢（2.34-2.45ppm）。1H NMR (400 MHz, DMSO-d 6) δ 7.53 (1H, s, H1 of -CONH),5.05 (1H, brs, 1-H of rha), 4.64 (1H, brs, H1-29), 4.52 (1H, brs, H2-29), 4.32(1H, d,J= 5.9 Hz, 1-H of ara ), 2.45 – 2.34 (1H, m, H1 of -CH on cyclobutyl ),1.62 (3H, s, H-30), 1.06 (3H, d,J= 6.2 Hz, 6-H3 of rha ), 0.90 (3H, s, H-27),0.83 (3H, s, H-26), 0.78 (3H, s, H-25), 0.53 (3H, s, H-24).13C NMR (101 MHz,DMSO) δ 175.23, 150.75, 108.98, 102.68, 99.67, 79.15, 73.96, 72.61, 71.82,70.22, 70.15, 67.90, 67.56, 64.09, 62.22, 54.69, 49.90, 49.48, 46.26, 45.93,43.14, 42.12, 41.72, 40.00, 38.19, 37.63, 36.41, 35.93, 34.82, 33.34, 32.30,30.14, 28.61, 25.27, 25.11, 24.95, 24.87, 20.39, 18.86, 17.56, 17.52, 16.83,16.20, 15.64, 14.04, 12.60。
B4-23：制备方法类似于B4-14，产率55%；除酰胺基团外新增标志氢为咪唑基上的氢（6.5-8ppm）和碳（115-140ppm）。1H NMR (400 MHz, DMSO-d 6) δ 7.65 (1H, s, H1 of -CONH), 7.61 (1H, s, H1 of imidazole ), 7.16 (1H, s, H1 of imidazole ), 6.88(1H, s, H1 of imidazole ), 5.05 (1H, brs, 1-H of rha), 4.59 (1H, brs, H1-29),4.54 (1H, brs, H2-29), 4.33 (1H, d,J= 5.8 Hz, 6-H3 of rha ), 3.92 (2H, t,J=7.5 Hz, H2 of -CH2 to imidazole ), 1.63 ( 3H, s, H-30), 1.07 (3H, d,J= 6.2 Hz,6-H3 of rha ), 0.91 (3H, s, H-27), 0.82 (3H, s, H-26), 0.77 (3H, s, H-25),0.54 (3H, s, H-24).13C NMR (101 MHz, DMSO) δ 176.25, 151.36, 137.74, 128.85,119.82, 109.75, 103.37, 100.37, 79.84, 74.68, 73.27, 72.51, 70.91, 70.84,68.60, 68.24, 64.75, 62.92, 55.37, 50.57, 50.13, 49.07, 46.95, 46.62, 44.15,42.82, 42.42, 40.71, 38.87, 38.21, 37.13, 36.62, 36.09, 33.99, 32.90, 31.52,30.84, 29.38, 25.96, 25.77, 21.06, 19.53, 18.25, 17.53, 16.89, 16.35, 14.72,13.28。
B4-24：制备方法类似于B4-14，产率44%；除酰胺基团外新增标志氢为四氢吡咯烷上的碳(20-50ppm)。1H NMR (400 MHz, DMSO-d 6) δ 7.51 (1H, s, H1 of -CONH), 5.06(1H, brs, 1-H of rha), 4.65 (1H, brs, H1-29), 4.54 (1H, brs, H2-29), 4.33 (1H,d,J= 5.8 Hz, 1-H of ara ), 1.63 (3H, s, H-30), 1.07 (3H, d,J= 6.2 Hz, 6-H3 ofrha), 0.91 (3H, s, H-27), 0.84 (3H, s, H-26), 0.78 (3H, s, H-25), 0.54 (3H,s, H-24).13C NMR (101 MHz, DMSO) δ 175.43, 150.90, 109.23, 102.88, 99.90,79.36, 74.21, 72.78, 72.03, 70.43, 70.35, 68.13, 67.76, 64.27, 62.44, 54.87,54.83, 53.58, 50.07, 49.64, 46.46, 46.23, 42.34, 41.94, 40.20, 38.38, 37.67,37.52, 36.69, 36.14, 33.52, 32.41, 30.33, 28.82, 25.47, 25.29, 23.11, 20.57,19.05, 17.77, 17.07, 16.39, 15.82, 14.25, 12.79。
B4-25：制备方法类似于B4-14，产率50%;除酰胺基团外新增标志氢为四氢吡喃环上的碳(20-70ppm)。1H NMR (400 MHz, DMSO-d 6) δ 7.33 (1H, s, H1 of -CONH), 5.05(1H, brs, 1-H of rha), 4.64 (1H, brs, H1-29), 4.52 (1H, brs, H1-29), 4.32 (1H,d,J= 5.9 Hz, 1-H of ara), 1.61 (3H, s, H-30), 1.05 (3H, d,J= 6.2 Hz, 6-H3 ofrha), 0.90 (3H, s, H-27), 0.81 (3H, s, H-26), 0.76 (3H, s, H-25), 0.53 (3H,s, H-24).13C NMR (101 MHz, DMSO) δ 174.85, 150.95, 109.23, 102.92, 99.91,79.38, 74.21, 72.84, 72.03, 70.44, 70.37, 68.13, 67.80, 66.24, 66.15, 64.33,62.44, 54.73, 50.09, 49.73, 46.48, 46.20, 44.71, 42.35, 41.89, 40.22, 38.40,37.72, 36.61, 36.15, 33.50, 32.56, 32.32, 32.09, 30.38, 28.80, 25.49, 25.30,20.59, 19.07, 17.78, 17.04, 16.40, 15.86, 14.25, 12.83。
B4-26：制备方法类似于B4-14，产率53%;除酰胺基团外新增标志氢为甲酯上的甲基（3.60ppm）。1H NMR (400 MHz, DMSO-d 6) δ 7.38 (1H, s, H1 of -CONH), 5.06 (1H,brs, 1-H of rha), 4.66 (1H, brs, H1-29), 4.54 (1H, brs, H2-29), 4.34 (1H, d,J=5.9 Hz, 1-H of ara), 3.60 (3H, s, H3 of -COOCH3 ), 1.63 (3H, s, H-30), 1.08(3H, d,J= 6.2 Hz, 6-H3 of rha), 0.91 (3H, s, H-27), 0.83 (3H, s, H-26), 0.79(3H, s, H-25), 0.55 (3H, s, H-24).13C NMR (101 MHz, DMSO) δ 176.02, 175.24,150.94, 109.20, 102.89, 99.89, 79.36, 74.20, 72.80, 72.03, 70.43, 70.36,68.12, 67.76, 64.28, 62.44, 54.71, 51.50, 50.02, 49.83, 49.73, 46.47, 42.33,41.88, 41.26, 41.21, 38.38, 37.58, 36.63, 36.14, 35.03, 33.50, 31.88, 31.62,30.37, 28.79, 27.59, 27.04, 25.48, 25.28, 20.58, 19.06, 17.77, 17.03, 16.39,15.80, 14.24, 12.81。
B4-27：制备方法类似于B4-14，产率47%;除酰胺基团外新增标志氢为甲酯上的甲基（3.58ppm）。1H NMR (400 MHz, DMSO-d 6) δ 7.26 (1H, s, H1 of -CONH), 5.05 (1H,brs, 1-H of rha), 4.64 (1H, brs, H1-29), 4.52 (1H, brs, H2-29), 4.43 (1H, d,J=5.9 Hz, 1-H of ara), 3.58 (3H, s, H3 of -COOCH3), 1.62 (3H, s, H-30), 1.06(3H, d,J= 6.2 Hz, 6-H3 of rha), 0.90 (3H, s, H-27), 0.83 (3H, s, H-26), 0.77(3H, s, H-25), 0.54 (3H, s, H-24).13C NMR (101 MHz, DMSO) δ 175.21, 174.70,150.96, 109.18, 102.89, 99.90, 79.37, 74.21, 72.80, 72.03, 70.43, 70.35,68.12, 67.76, 64.27, 62.44, 54.70, 51.31, 50.08, 49.73, 46.72, 46.47, 46.21,42.34, 41.88, 41.69, 40.22, 38.38, 37.75, 36.63, 36.14, 33.49, 32.28, 31.25,30.83, 30.38, 28.80, 27.77, 27.69, 25.48, 25.29, 20.58, 19.05, 17.77, 17.03,16.38, 15.90, 14.24, 12.82。
B4-28：制备方法类似于B4-14，产率41%;除酰胺基团外新增标志氢为丙烯基上的氢（5-6ppm）。1H NMR (400 MHz, DMSO-d 6) δ 7.74 (1H, s, H1 of -CONH), 5.81– 5.72(1H, m, H1 of -CH on CH=CH2), 5.07 (1H, d,J= 13.5 Hz, H1 of -CH2on CH=CH2),5.05 (1H, brs, 1-H of rha), 5.00 (1H, d,J= 10.2 Hz, H2 of -CH2on CH=CH2), 4.65(1H, brs, H1-29), 4.53 (1H, brs, H2-29), 4.41 (1H, d,J= 5.9 Hz, 1-H of ara ),1.63 (3H, s, H-30 ), 1.07 (3H, d,J= 6.1 Hz, 6-H3 of rha ), 0.91 (3H, s, H-27), 0.82 (3H, s, H-26), 0.78 (3H, s, H-25), 0.54 (3H, s, H-24).13C NMR (101MHz, DMSO) δ 175.27, 150.90, 136.21, 114.28, 109.22, 102.87, 99.88, 79.36,74.19, 72.78, 72.02, 70.42, 70.35, 68.12, 67.75, 64.26, 62.43, 54.90, 50.08,49.70, 46.46, 46.13, 42.33, 41.92, 40.60, 40.22, 38.38, 37.72, 36.61, 36.14,33.51, 32.34, 30.32, 28.86, 25.46, 25.28, 20.57, 19.06, 17.76, 17.04, 16.39,15.85, 14.23, 12.80。
B4-30：制备方法类似于B4-14，产率41%;除酰胺基团外新增标志氢为哌啶环上的碳（20-70ppm）。1H NMR (400 MHz, DMSO-d 6) δ 7.39 (1H, s, H1 of -CONH), 5.05 (1H,brs, 1-H of rha), 4.62 (1H, brs, H1-29), 4.56 (1H, brs, H2-29), 4.33 (1H, d,J=6.2 Hz, 1-H of ara ), 1.62 (3H, s, H-30), 1.06 (3H, d,J= 6.2 Hz, 6-H3 of rha ),0.90 (3H, s, H-27), 0.83 (3H, s, H-26), 0.78 (3H, s, H-25), 0.54 (3H, s, H-24).13C NMR (101 MHz, DMSO) δ 175.34, 150.88, 109.23, 102.86, 99.89, 79.36,74.21, 72.75, 72.02, 70.42, 70.34, 68.12, 67.73, 64.24, 62.44, 57.51, 54.88,53.92, 50.05, 49.58, 46.45, 46.24, 42.33, 41.95, 40.20,40.20, 38.37, 37.65,36.70, 36.13, 35.91, 33.51, 32.46, 30.32, 28.84, 25.47, 25.27, 23.91, 20.55,19.03, 17.76, 17.05, 16.38, 15.85, 14.24, 12.77。
B4-31：制备方法类似于B4-14，产率52%;除酰胺基团外新增标志氢为噻唑上的氢（7-8ppm）。1H NMR (400 MHz, DMSO-d 6) δ 7.72 (1H, s, H1 of -CONH), 7.70 (1H, d,J=3.3 Hz, H1 of thiazole ), 7.58 (1H, d,J= 3.3 Hz, H1´ of thiazole), 5.05 (1H,brs, 1-H of rha), 4.65 (1H, brs, H1-29), 4.54 (1H, brs, H2-29), 4.34 (1H, d,J=5.7 Hz, 1-H of ara ), 1.63 (3H, s, H-30), 1.07 (3H, d,J= 6.1 Hz, 6-H3 ofrha), 0.90 (3H, s, H-27), 0.82 (3H, s, H-26), 0.78 (3H, s, H-25), 0.55 (3H,s, H-24).13C NMR (101 MHz, DMSO) δ 175.68, 167.51, 150.87, 142.21, 119.43,109.24, 102.86, 99.89, 79.36, 74.21, 72.75, 72.02, 70.43, 70.34, 68.12,67.73, 64.24, 62.45, 54.86, 50.07, 49.61, 46.45, 46.14, 42.33, 41.90, 40.21,38.68, 38.37, 37.56, 36.60, 36.13, 33.47, 32.52, 32.34, 30.27, 28.89, 25.47,25.27, 20.56, 19.03, 17.76, 17.07, 16.39, 15.85, 14.22, 12.79。
B4-32：制备方法类似于B4-14，产率53%;除酰胺基团外新增标志氢为呋喃环上的碳（20-70ppm）。1H NMR (400 MHz, DMSO-d 6) δ 7.58 (1H, s, H1 of -CONH), 5.05 (1H,brs, 1-H of rha), 4.64 (1H, brs, H1-29), 4.56 (1H, brs, H2-29), 4.53 (1H, s,H1 of -OH on C-23 ), 4.43 (1H, d,J= 5.8 Hz, 1-H of ara ), 1.62 (3H, s, H-30),1.07 (3H, d,J= 6.2 Hz, 6-H3 of rha), 0.90 (3H, s, H-27), 0.83 (3H, s, H-26),0.78 (3H, s, H-25), 0.54 (3H, s, H-24).13C NMR (101 MHz, DMSO) δ 175.55,150.93, 109.22, 102.88, 99.89, 79.36, 77.37, 74.18, 72.79, 72.02, 70.43,70.35, 68.12, 67.76, 67.02, 64.27, 62.44, 54.88, 50.10, 49.71, 46.47, 46.17,42.46, 42.33, 41.92, 40.23, 38.39, 37.67, 36.66, 36.14, 33.53, 32.34, 30.33,28.82, 28.57, 25.47, 25.30, 25.03, 20.58, 19.05, 17.76, 17.04, 16.40, 15.76,14.24, 12.79。
B4-33：制备方法类似于B4-14，产率66%;除酰胺基团外新增标志氢为吗啉环上的碳（40-75ppm）。1H NMR (400 MHz, DMSO-d 6) δ 7.44 (1H, s, H1 of -CONH), 5.05 (1H,brs, 1-H of rha), 4.63 (1H, brs, H1-29), 4.57 (1H, brs, H2-29), 4.41 (1H, d,J=5.9 Hz, 1-H of ara ), 1.63 (3H, s, H-30 ), 1.07 (3H, d,J= 6.2 Hz, 6-H3 ofrha), 0.91 (3H, s, H-27), 0.83 (3H, s, H-26), 0.78 (3H, s, H-25), 0.54 (3H,s, H-24).13C NMR (101 MHz, DMSO) δ 175.43, 150.91, 109.23, 102.88, 99.89,79.36, 74.21, 72.77, 72.02, 70.43, 70.35, 68.12, 67.74, 66.15, 64.25, 62.44,57.36, 54.87, 53.19, 53.15, 50.06, 49.61, 46.45, 46.21, 42.33, 41.94, 40.21,38.37, 37.67, 36.66, 36.14, 33.52, 32.44, 30.32, 28.85, 25.47, 25.26, 20.55,19.03, 17.76, 17.06, 16.38, 15.89, 14.23, 12.77。
B4-34：制备方法类似于B4-14，产率56%;除酰胺基团外新增标志氢为哌啶环上的甲基（2.11ppm）。1H NMR (400 MHz, DMSO-d 6) δ 7.58 (1H, s, H1 of -CONH), 5.05 (1H,brs, 1-H of rha), 4.65 (1H, brs, H1-29), 4.57 (1H, brs, H2-29), 4.53 (1H, s,H1 of -OH on C-23), 4.35 (1H, d,J= 5.1 Hz, 1-H of ara ), 2.11 (3H, s, H3 of -CH3on piperidine ), 1.62 (3H, s, H-30), 1.07 (3H, d,J= 6.1 Hz, 6-H3 of rha ),0.90 (3H, s, H-27), 0.82 (3H, s, H-26), 0.78 (3H, s, H-25), 0.54 (3H, s, H-24).13C NMR (101 MHz, DMSO) δ 175.41, 150.96, 109.20, 102.86, 99.90, 79.35,74.22, 72.75, 72.03, 70.44, 70.35, 68.13, 67.73, 64.24, 62.43, 55.16, 54.89,50.12, 49.70, 46.47, 46.20, 46.13, 44.06, 42.33, 41.94, 40.22, 38.39, 37.81,36.65, 36.14, 35.26, 33.59, 32.44, 30.37, 29.92, 29.85, 29.00, 28.86, 25.47,25.33, 20.62, 19.07, 17.76, 17.07, 16.43, 15.78, 14.24, 12.77。
B4-35：制备方法类似于B4-14，产率47%;除酰胺基团外新增标志氢为四氢吡咯环上的甲基（2.24ppm）。1H NMR (400 MHz, DMSO-d 6) δ 7.64 (1H, s, H1 of -CONH), 5.05(1H, brs, 1-H of rha), 4.65 (1H, brs, H1-29), 4.53 (1H, brs, H2-29), 4.33 (1H,d,J= 5.6 Hz, 1-H of ara), 2.24 (3H, s, H3 of -CH3on pyrrolidine ), 1.63 (3H,s, H-30 ), 1.07 (3H, d,J= 6.1 Hz, 6-H3 of rha ), 0.91 (3H, s, H-27 ), 0.83(3H, s, H-26 ), 0.78 (3H, s, H-25 ), 0.54 (3H, s, H-24 ).13C NMR (101 MHz,DMSO) δ 175.49, 150.93, 109.22, 102.88, 99.89, 79.35, 74.20, 72.79, 72.03,70.44, 70.35, 68.13, 67.76, 64.27, 62.44, 59.79, 59.62, 55.50, 54.87, 50.10,49.63, 46.47, 46.13, 42.33, 41.94, 41.81, 40.22, 38.39, 37.72, 37.50, 36.65,36.14, 33.55, 32.44, 30.35, 28.82, 28.31, 25.47, 25.31, 20.60, 19.06, 17.76,17.05, 16.41, 15.89, 14.24, 12.79。
B4-36：制备方法类似于B4-14，产率50%;除酰胺基团外新增标志氢为环己烷上的羟基（4.57ppm）。1H NMR (400 MHz, DMSO-d 6) δ 7.16 (1H, s, H1 of -CONH), 5.05 (1H,brs, 1-H of rha), 4.63 (1H, brs, H1-29), 4.57 (1H, s, H1 of -OH oncyclohexane ), 4.56 (1H, brs, H2-29), 4.53 (1H, s, H1 of -OH on C-23 ), 4.48(1H, d,J= 5.4 Hz, 1-H of ara ), 1.62 (3H, s, H-30 ), 1.07 (3H, d,J= 6.1 Hz,6-H3 of rha ), 0.90 (3H, s, H-27), 0.83 (3H, s, H-26), 0.78 (3H, s, H-25),0.54 (3H, s, H-24).13C NMR (101 MHz, DMSO) δ 174.75, 150.98, 109.17, 102.89,99.90, 79.37, 74.21, 72.79, 72.03, 70.43, 70.36, 68.40, 68.12, 67.75, 64.26,62.44, 54.70, 50.08, 49.74, 46.88, 46.47, 46.22, 42.34, 41.87, 40.22, 38.38,37.73, 36.63, 36.13, 34.28, 34.20, 33.50, 32.30, 30.35, 29.92, 28.79, 25.47,25.28, 20.58, 19.05, 17.76, 17.02, 16.37, 15.88, 14.23, 12.81。
B4-37：制备方法类似于B4-14，产率67%;除酰胺基团外新增标志氢为苯环上的氢（7-8ppm）。1H NMR (400 MHz, DMSO-d 6) δ 7.59 (1H, s, H1 of -CONH), 7.27 (1H, d,J=8.0 Hz, H1´ of benzene ), 7.26 (1H, d,J= 8.0 Hz, H1 of benzene ), 7.20 – 7.15(3H, m, each 1H of benzene), 5.05 (1H, brs, 1-H of rha), 4.64 (1H, brs, H1-29), 4.57 (1H, brs, H2-29), 4.42 (1H, d,J= 5.8 Hz, 1-H of ara ), 1.62 (3H, s,H-30 ), 1.07 (3H, d,J= 6.2 Hz, 6-H3 of rha ), 0.89 (3H, s, H-27 ), 0.79 (3H,s, H-26 ), 0.78 (3H, s, H-25 ), 0.54 (3H, s, H-24 ).13C NMR (101 MHz, DMSO) δ175.40, 150.93, 139.67, 128.56, 128.23, 125.92, 109.21, 102.87, 99.91, 79.38,74.23, 72.75, 72.03, 70.43, 70.35, 68.13, 67.73, 64.23, 62.46, 54.81, 50.07,49.68, 46.46, 46.15, 42.33, 41.89, 40.19, 38.37, 37.63, 36.57, 36.13, 35.26,33.46, 32.41, 30.27, 28.83, 25.47, 25.26, 20.55, 19.01, 17.76, 17.05, 16.37,15.84, 14.22, 12.79。
B4-38：制备方法类似于B4-14，产率52%;除酰胺基团外新增标志氢为噻吩环上的氢（6.5-7.5ppm）。1H NMR (400 MHz, DMSO-d 6) δ 7.69 (1H, s, H1 of -CONH), 7.32(1H, d,J=5.1 Hz, H1of thiophene ), 6.93 (1H, dd,J= 5.1, 3.4 Hz, H2of thiophene ),6.86 (1H, d,J=8.0 Hz, H3of thiophene ), 5.05 (1H, brs, 1-H of rha ), 4.63(1H, brs, H1-29), 4.57 (1H, brs, H2-29), 4.42 (1H, d,J= 5.9 Hz, 1-H of ara ),1.62 (3H, s, H-30), 1.07 (3H, d,J= 6.2 Hz, 6-H3 of rha ), 0.90 (3H, s, H-27),0.82 (3H, s, H-26), 0.78 (3H, s, H-25), 0.54 (3H, s, H-24).13C NMR (101 MHz,DMSO) δ 175.60, 150.92, 141.81, 126.87, 124.98, 123.87, 109.24, 102.87,99.90, 79.37, 74.22, 72.76, 72.03, 70.44, 70.35, 68.13, 67.74, 64.24, 62.45,54.84, 50.09, 49.66, 46.46, 46.17, 42.34, 41.91, 40.35, 40.22, 37.61, 36.61,36.13, 33.48, 32.37, 30.30, 29.37, 29.00, 28.89, 25.47, 25.27, 20.57, 19.03,17.76, 17.06, 16.39, 15.86, 14.23, 12.79。
B4-39：制备方法类似于B4-14，产率70%;除酰胺基团外新增标志氢为苯环上的氢（6-8ppm）。1H NMR (400 MHz, DMSO-d 6) δ 9.11 (1H, s, H1 of -OH on benzene), 7.53(1H, s, H1 of -CONH), 6.96 (1H, d,J= 8.5 Hz, H1of benzene ), 6.96 (1H, d,J=8.5 Hz, H2of benzene ), 6.65 (1H, d,J= 8.4 Hz, H3of benzene), 6.65 (1H, d,J=8.4 Hz, H4of benzene), 5.05 (1H, brs, 1-H of rha), 4.63 (1H, brs, H1-29), 4.57(1H, brs, H2-29), 4.53 (1H, s, H1 of -OH on C-23 ), 4.42 (1H, d,J= 5.9 Hz, 1-H of ara ), 1.62 (3H, s, H-30 ), 1.07 (3H, d,J= 6.1 Hz, 6-H3 of rha), 0.89(3H, s, H-27), 0.80 (3H, s, H-26), 0.78 (3H, s, H-25), 0.54 (3H, s, H-24).13CNMR (101 MHz, DMSO) δ 175.33, 155.55, 150.95, 129.65, 129.36, 115.02, 109.21,102.87, 99.90, 79.38, 74.23, 72.76, 72.03, 70.44, 70.35, 68.13, 67.74, 64.24,62.46, 54.81, 50.07, 49.69, 46.46, 46.18, 42.34, 41.90, 40.36, 40.19, 38.38,37.65, 36.59, 36.13, 34.47, 33.44, 32.43, 30.30, 28.85, 25.47, 25.26, 20.55,19.02, 17.77, 17.07, 16.38, 15.82, 14.22, 12.80。
B4-40：制备方法类似于B4-14，产率33%;除酰胺基团外新增标志氢为恶唑环上的氢(7-8ppm)。1H NMR (400 MHz, DMSO-d 6) δ 8.27 ( 1H, s, H1of oxazole), 8.02 (1H,s, H1 of -CONH), 7.76 (1H, s, H2of oxazole), 5.05 (1H, brs, 1-H of rha), 4.63(1H, brs, H1-29), 4.57 (1H, brs, H2-29), 4.41 (1H, d,J= 5.9 Hz, 1-H of ara ),1.62 (3H, s, H-30 ), 1.07 (3H, d,J= 6.2 Hz, 6-H3 of rha ), 0.89 (3H, s, H-27), 0.77 (3H, s, H-26), 0.73 (3H, s, H-25), 0.54 (3H, s, H-24).13C NMR (101MHz, DMSO) δ 175.53, 151.70, 150.89, 138.39, 135.55, 109.23, 102.87, 99.89,79.36, 74.20, 72.77, 72.03, 70.43, 70.35, 68.13, 67.75, 64.26, 62.44, 54.86,50.08, 49.70, 48.58, 46.45, 46.16, 42.33, 41.90, 40.18, 38.39, 37.54, 36.65,36.13, 33.48, 32.22, 30.33, 28.79, 25.47, 25.29, 20.56, 19.06, 17.76, 17.05,16.39, 15.64, 14.23, 12.78。
B4-41：制备方法类似于B4-14，产率34%;除酰胺基团外新增标志为羧基碳（174ppm）。1H NMR (400 MHz, DMSO-d 6) δ 6.82 (1H, s, H1 of -CONH), 5.05 (1H, brs,1-H of rha), 4.65 (1H, brs, H1-29), 4.54 (1H, brs, H2-29), 4.33 (1H, d,J= 5.7Hz, 1-H of ara ), 1.63 (3H, s, H-30), 1.07 (3H, d,J= 6.1 Hz, 6-H3 of rha ),0.91 (3H, s, H-27), 0.82 (3H, s, H-26), 0.78 (3H, s, H-25), 0.54 (3H, s, H-24).13C NMR (101 MHz, DMSO) δ 174.25, 171.56, 150.86, 109.28, 102.88, 99.88,79.37, 74.22, 72.78, 72.07, 70.44, 70.36, 68.12, 67.71, 64.25, 62.46, 54.76,50.03, 49.51, 48.59, 46.46, 43.93, 42.34, 42.03, 40.20, 38.38, 37.74, 36.90,36.14, 33.46, 32.94, 30.40, 28.95, 25.47, 25.26, 20.52, 19.01, 17.78, 17.06,16.36, 15.87, 14.26, 12.79。
B4-42：制备方法类似于B4-14，产率54%;除酰胺基团外新增标志氢为和酰胺键相连的甲基（1.79ppm）。1H NMR (400 MHz, DMSO-d 6) δ 7.83 (1H, s, H1 of -CONH), 7.57(1H, s, H1´ of -CONH), 5.06 (1H, brs, 1-H of rha), 4.66 (1H, brs, H1-29),4.54 (1H, brs, H2-29), 4.34 (1H, d,J= 5.8 Hz, 1-H of ara ), 1.79 (3H, s, H3of -CH3to CONH), 1.63 (3H, s, H-30), 1.07 (3H, d,J= 6.2 Hz, 6-H3 of rha ),0.91 (3H, s, H-27), 0.83 (3H, s, H-26), 0.78 (3H, s, H-25), 0.55 (3H, s, H-24).13C NMR (101 MHz, DMSO) δ 175.54, 169.14, 150.68, 109.02, 102.68, 99.68,79.15, 74.00, 72.57, 71.82, 70.22, 70.14, 67.91, 67.54, 64.06, 62.23, 54.66,49.84, 49.43, 46.25, 45.99, 42.13, 41.71, 39.99, 38.34, 38.14, 37.41, 36.46,35.93, 33.26, 32.16, 30.12, 28.65, 25.26, 25.06, 22.41, 20.35, 18.83, 17.56,16.84, 16.18, 15.63, 14.04, 12.59。
B4-43：将B4-15（100mg，0.118mmol）溶于4ml四氢呋喃:甲醇:水（2:1:1）混合溶液中，加入氢氧化钠（42.4mg，1.06mmol），室温搅拌12小时，反应结束后加入5ml水，过滤，经硅胶柱层析（二氯甲烷:甲醇=8:1→6:1）得类白色固体30mg，产率30.5%。1H NMR (400 MHz,DMSO-d 6) δ 7.78 (1H, s, H1 of -CONH), 5.05 (1H, brs, 1-H of rha), 4.65 (1H,brs, H1-29), 4.53 (1H, brs, H2-29), 4.33 (1H, d,J= 5.7 Hz, 1-H of ara ), 2.04(2H, t,J= 7.4 Hz, H2 of -CH2 to -COOH ), 1.62 (3H, s, H-30), 1.07 (3H, d,J=6.1 Hz, 6-H3 of rha ), 0.90 (3H, s, H-27), 0.83 (3H, s, H-26), 0.78 (3H, s,H-25), 0.54 (3H, s, H-24).13C NMR (101 MHz, DMSO) δ 176.17, 175.38, 151.01,109.18, 102.87, 99.87, 79.36, 74.22, 72.78, 72.07, 70.47, 70.35, 68.12,67.71, 64.23, 62.46, 54.82, 50.11, 49.72, 46.49, 46.19, 42.34, 41.92, 40.21,38.70, 38.40, 37.77, 36.65, 36.15, 33.80, 33.51, 32.40, 30.39, 28.87, 25.41,25.32, 20.59, 19.07, 17.78, 17.06, 16.40, 15.87, 14.26, 12.80。
B4-44：制备方法类似于B4-14，产率42%;除酰胺基团外新增标志氢为与羧基相连的亚甲基（2.19ppm）和羧基碳（175ppm）。1H NMR (400 MHz, DMSO-d 6) δ 7.58 (1H, s, H1of -CONH), 5.06 (1H, brs, 1-H of rha), 4.68 (1H, brs, H1-29), 4.53 (1H, brs,H2-29), 4.39 (1H, s, H1 of -OH on C-23), 4.34 (1H, d,J= 5.7 Hz, 1-H of ara ),2.19 (2H, t,J= 7.1 Hz, H2 of -CH2to -COOH), 1.63 (3H, s, H-30 ), 1.07 (3H, d,J= 6.2 Hz, 6-H3 of rha), 0.91 (3H, s, H-27), 0.83 (3H, s, H-26), 0.78 (3H, s,H-25), 0.54 (3H, s, H-24).13C NMR (101 MHz, DMSO) δ 175.33, 174.66, 150.97,109.19, 102.86, 99.88, 79.34, 74.24, 72.77, 72.06, 70.49, 70.34, 68.11,67.73, 64.24, 62.43, 54.84, 50.10, 49.69, 46.47, 46.17, 42.34, 41.93, 40.21,38.39, 37.81, 36.64, 36.14, 33.48, 32.43, 30.36, 28.78, 26.55, 25.48, 25.31,22.08, 21.93, 20.59, 19.06, 17.78, 17.08, 16.41, 15.82, 14.24, 12.80。
B4-46：制备方法类似于B4-14，产率58%;除酰胺基团外新增标志氢为甲酯上的甲基（3.57ppm）。1H NMR (400 MHz, DMSO-d 6) δ 7.52 (1H, s, H1 of -CONH ), 4.64 (1H,brs, H1-29), 4.53 (1H, brs, H2-29), 4.35 (1H, s, H1 of -OH on -C3), 4.12 (1H,s, H1 of -OH on -C23), 3.57 (3H, s, H3 of -COOCH3), 3.41 (1H, m, H-19), 2.27(2H, t,J= 7.4 Hz, H2 of -CH2to -COOCH3), 1.62 (3H, s, H-30), 0.91 (3H, s, H-27), 0.83 (3H, s, H-26), 0.78 (3H, s, H-25), 0.51 (3H, s, H-24).13C NMR (101MHz, DMSO) δ 175.31, 173.25, 150.96, 109.15, 70.30, 64.43, 54.80, 51.13,50.10, 49.66, 48.58, 46.69, 46.15, 41.92, 40.20, 38.13, 37.94, 37.71, 36.61,36.42, 33.57, 33.26, 32.44, 30.33, 28.91, 28.83, 26.75, 25.82, 25.24, 24.14,20.59, 19.03, 17.44, 16.29, 15.82, 14.30, 12.39。
B4-47：制备方法类似于B4-14，产率31%;除酰胺基团外新增标志氢为环戊烷环上的羟基（4.30ppm）。1H NMR (400 MHz, Methanol-d 4 ) δ 5.16 (1H, brs, 1-H of rha),4.72 (1H, brs, H1-29), 4.59 ((1H, brs, H2-29)), 4.56 (1H, d,J= 4.8 Hz, 1-H ofara ), 4.30 (1H, s, H1 of -OH on cyclopentane ), 4.20 (1H, s, H1 of -OH on C-23), 1.70 ( 3H, s, H-30), 1.25 (3H, d,J= 6.2 Hz, 6-H3 of rha ), 1.02 (3H, s,H-27), 0.98 (3H, s, H-26), 0.90 (3H, s, H-25), 0.69 (3H, s, H-24).13C NMR (101MHz, Methanol-d 4 ) δ 178.21, 152.32, 109.98, 104.30, 101.89, 82.27, 76.66,73.93, 73.65, 73.36, 72.13, 72.02, 70.17, 69.12, 64.74, 64.57, 56.83, 52.02,51.31, 50.77, 48.22, 44.04, 43.62, 42.05, 42.00, 39.93, 39.28, 39.09, 37.82,35.01, 34.69, 34.16, 32.10, 31.97, 30.50, 27.01, 26.69, 22.16, 19.63, 18.77,17.95, 17.22, 16.78, 15.08, 13.51。
B4-48：制备方法类似于B4-14，产率32%;除酰胺基团外新增标志氢为环戊烷环上的羟基（4.30ppm）。1H NMR (400 MHz, Methanol-d 4 ) δ 5.16 (1H, brs, 1-H of rha),4.72 (1H, brs, H1-29), 4.60 (1H, brs, H2-29), 4.57 (1H, d,J= 5.0 Hz, 1-H ofara ), 4.30 (1H, s, H1 of -OH on cyclopentane), 4.21 (1H, s, H1 of -OH on C-23), 1.70 (3H, s, H-30), 1.25 (3H, d,J= 6.2 Hz, 6-H3 of rha), 1.02 (3H, s, H-27), 0.99 (3H, s, H-26), 0.90 (3H, s, H-25), 0.69 (3H, s, H-24).13C NMR (101MHz, D2O) δ 178.18, 152.27, 110.02, 104.30, 101.88, 82.27, 76.65, 73.93,73.66, 73.31, 72.13, 72.02, 70.16, 69.12, 64.74, 64.57, 56.88, 52.02, 51.18,50.66, 48.15, 44.04, 43.63, 42.42, 41.99, 39.92, 39.31, 39.06, 37.81, 34.99,34.68, 34.11, 31.93, 31.72, 30.53, 27.01, 26.69, 22.16, 19.62, 18.77, 17.96,17.22, 16.77, 15.08, 13.50。
以白头翁皂苷B4或衍生物为实验组药物，进行以下实验。
实施例八
细胞毒实验1: HIEC细胞以5000个/孔接种于96孔板，100uL/孔，常规培养，设定正常对照组、给药组。待细胞贴壁后除正常对照组外每孔加入除中间体以外的B4衍生物1 uL，使药物终浓度为50 uM，置于培养箱共孵育24 h。孵育结束后，每孔加入10 uL的CCK-8，避光孵育4h。用酶标仪在 450 nm 波长处测定各孔吸光度，计算每孔的细胞存活率，筛选出第一轮无明显细胞毒的化合物。
细胞毒实验2: THP-1细胞以1×104个/孔接种于96孔板，100 uL/孔，常规培养，待细胞达到80％后用100 ng/mL的佛波酯（phorbol-12-Myristate-13-Acetate，PMA）诱导12h后弃去原培养基，加入新的完全培养基100 uL。设置不加细胞的培养基调零孔以及不加药物的正常组；每孔加入第一轮中无明显细胞毒的B4衍生物1 uL，使药物终浓度为50 uM，置于培养箱共孵育24 h。孵育结束后，每孔加入10 uL的CCK-8，避光孵育4h。用酶标仪在 450nm 波长处测定各孔吸光度，计算每孔的细胞存活率，筛选出第二轮无明显细胞毒的化合物。
计算公式：细胞存活率（%）=[A(加药)-A(空白)]/[A(0加药)-A(空白)]×100
A(加药)：具有细胞、CCK-8、和药物溶液的孔的吸光度
A(空白)：具有培养基和CCK-8而没有细胞的孔的吸光度
A(0加药)：具有细胞、CCK-8而不加药物的孔的吸光度。
如图4所示，B4衍生物在50uM下的细胞毒情况，统计图中没有标注符号“*”的表明和正常对照相比没有显著性差异，即B4衍生物在50uM剂量下不存在明显的细胞毒性，由图可知存在较多无细胞毒的衍生物。
实施例九
亚硝酸盐含量检测: Raw 264.76细胞以6×104个/孔接种于96孔板，100 uL/孔，常规培养，设定空白对照组（N组）、造模组（M组）、阳性药***组（Y组）、给药组。待细胞贴壁后对应给药1uL，使其终浓度为10uM，1小时后给lps 1uL，使其终浓度为1ug/ml。置于培养箱共孵育24 h后从种的板中每孔取50ul培养基到新的96孔板中，避光条件下每孔加入Griess试剂A液50ul，再加入B液50uL，用酶标仪在 540 nm 波长处测定各孔吸光度。如图5所示，纵坐标B4衍生物在LPS刺激下所产生的亚硝酸盐含量，图中横坐标的数字表示不同白头翁皂苷B4衍生物，符号“#”表示模型与正常组相比存在显著性差异，即造模成立；符号“*”表示含有B4衍生物的给药组与模型组相比存在显著性差异，即疗效较好，有较可观的抗炎活性。
实施例十
Western blotting实验: THP-1细胞以2×106个/孔接种于6孔板，每孔2ml，常规培养，待细胞达到80％后用100 ng/mL的PMA诱导12 h后弃去原培养基，加入新的完全培养基。实验组分别加入AB4及其衍生物2 uL使其终浓度为10 uM，对照组为空白组；1 h后除去空白组其他组加入2uL 1 mg/mL LPS孵育2 h。然后在冰上进行以下操作，移除六孔板上清液，吸取4℃预冷的PBS沿边缘轻轻加入，洗涤两遍；加入1 mL PBS后用细胞刮刮下细胞，置于1.5 mL 离心管中，2000 g，4℃离心3 min，弃去上清，收集细胞沉淀。每管加入100 uLRIPA 裂解液（使用前现加入蛋白酶抑制剂和磷酸酶抑制剂），吹打混匀，冰上裂解10 min。超声破碎仪再次破碎细胞后，12000 g，4℃离心10 min，小心收集细胞上清液于新的EP管中，冰上保存。参考说明书，使用 BCA 蛋白定量试剂盒测量并计算蛋白总含量，蛋白样品用PBS稀释后，加入5×SDS-PAGE loading buffer（每mL已加入50 uL β-巯基乙醇），使蛋白终浓度为2 ug/uL；100℃煮沸 10 min 对其变性以防蛋白降解。根据所需蛋白分子量配置不同浓度的 SDS-PAGE 凝胶，并将蛋白样品以 20 mg/孔的含量装载在凝胶上进行电泳，使分离后的蛋白样品转印至聚偏二氟乙烯（PVDF）膜上，将PVDF膜以 Tris-HCl 缓冲盐溶液（TBST 缓冲液）洗涤3次，每次 10 min。室温下蛋白封闭液对其封闭 1 小时后，使用 TBST缓冲液多次洗涤至洗净封闭液，在 4℃冰箱中将 PVDF 膜与特定一抗按照说明书孵育过夜。次日，将PVDF膜用TBST缓冲液充分洗涤，并与相应的二抗室温结合1小时，再次用TBST缓冲液充分洗涤，最后参考特超敏 ECL 化学发光试剂盒说明书对蛋白曝光显色分析。
AB4及其衍生物可以抑制NF-κB/MAPK或NLRP3信号通路中关键蛋白的激活，如图6以及图7所示，图中横坐标的数字表示不同白头翁皂苷B4衍生物。通过第一轮Westernblotting显示THP-1巨噬细胞内P-IκBa蛋白水平，发现LPS刺激THP-1细胞2 h后，模型组P-IκBa蛋白水平明显上升（P<0.01）；而与LPS模型组相比，本发明化合物及其AB4能够降低P-IκBa蛋白水平，并且多个衍生物与AB4相比，P-IκBa蛋白水平明显降低（p<0.05），这些结果提示这些衍生物有更好的抗炎活性。将第一轮中活性较好的衍生物继续用于第二轮抗炎活性筛选，通过检测NLRP3通路相关指标进行比较判断，发现其中B4-19、B4-28、B4-33、B4-39、B4-40五个化合物活性较好。
实施例十一
白头翁皂苷B4衍生物对DNCB诱导的小鼠特应性皮炎的治疗作用:现有技术证明AB4对DNCB（2，4-二硝基氯苯）诱导的小鼠湿疹模型有改善作用；而通过先前体外实验表明有活性优于AB4的AB4衍生物，现通过体内实验进一步验证。设计如下实验。将56只Balb/c小鼠按体重随机分为正常对照组、模型组、***阳性药组（3mg/kg）、白头翁皂苷B4（AB4）对照组（6.6mg/kg）、B4-19给药组（6.6mg/kg），B4-33给药组（6.6mg/kg），B4-39给药组（6.6mg/kg），每组8只。实验前1天，用一次性备皮刀给小鼠背部皮肤刮毛，均选取约3cmx3cm范围备用。第1天，除正常对照组以外，其他组以5%DNCB 50uL外涂小鼠背部致敏:第2天同法强化涂抹1次,再次致敏:第3天在小鼠右耳壳内、外用移液枪外涂1%DNCB 50uL激发，第4天、第5天继续激发，连续激发三天，左耳壳内涂抹等量的丙酮基质。模型成功的标准为DNCB溶液反复刺激小鼠右耳部及背部皮肤后出现不同程度的潮红、丘疹、水疱、糜烂、渗出、结痂、脱屑的表现。Day 1-7天***组下午4点于背部和右耳壳内、外涂抹***乳膏共0.08g；AB4、B4-19、B4-33、B4-39 给药组上午10点分别背部和右耳壳内、外涂抹70%乙醇-水溶液(0.66mg/ml）共200uL，下午4点涂抹一次。模型组上午10点背部和右耳廓内、外共涂抹200ul纯水，下午6点涂抹一次。连续处理7d。第8天处死小鼠，取脾脏和小鼠耳朵，用以计算脾指数和耳重差（选用直径6mm打孔器打同一部位得耳圆片，称重）。
试验期间，每天观察每组小鼠背部皮肤的湿疹情况(皮肤上明显的红肿、斑疹、糜烂和渗出)并拍照，参考各组小鼠皮肤临床症状采用湿疹面积及严重指数(EASI)评分标准，从红斑、丘疹/脓疱、鳞屑、结痂4项指标进行评价，以0~3分进行记分:0分=无症状;1分=轻度;2分=中度;3分=重度，将各指标积分相加得到总积分。由两名观察者采取盲法的方式分别在干预第1、3、5和第7天进行评分，并采用数码照相的方法进行记录。末次给药后24h后用游标卡尺测量双耳厚度(取3个点测量后取均值)，计算厚度差:厚度差=右耳厚度-左耳厚度；每天称量体重并记录。
图8为DNCB诱导的特应性皮炎小鼠背部皮肤情况；图9为DNCB诱导的特应性皮炎小鼠耳部情况；图10为小鼠体重变化和背部评分示意图；图11为小鼠耳厚度差和耳重差示意图；图12为小鼠脾指数示图。
当局部组织受到DNCB刺激后,细胞的组织胺等介质被释放,通过H1和H2受体使耳部皮肤、粘膜毛细血管扩张及毛细血管壁通透性增高,导致水肿。实验结果表明，第三天首次涂抹DNCB后，与空白组相比，模型组耳廓皮肤微微发红，随着药物的持续作用，模型组从第 4天开始红肿日益严重，出现脱屑，第五天开始出现渗出，溃烂并开始结痂。对照组与模型组左右耳厚度差结果第7天（P<0.0001）有统计学意义，表明湿疹小鼠模型建立成功。AB4组及其衍生物组耳廓与模型组相比同期的耳肿胀症状明显减轻，耳朵几乎无溃烂；***阳性药组效果稍差，有部分渗出和结痂。其中AB4、B4-19、B4-33、B4-39组与模型组左右耳厚度差结果在第7天（P<0.0001）有统计学意义；阳性药组与模型组厚度差结果在第7天（P<0.0001）均有统计学意义。并且，实验结果表明背部皮肤涂抹DNCB后，与空白组相比，模型组皮肤出现渗出、糜烂及结痂，产生类似湿疹样皮损。随着药物的持续作用，模型组从第 2～3天开始皮损日益严重，出现明显的红斑，皮肤浸润、结痂明显，对照组与模型组EASI综合评分结果第3、5、7天（P<0.0001）均有统计学意义，表明湿疹小鼠模型建立成功。AB4和AB4衍生物给药组皮肤与模型组相比同期的皮损症状明显减轻，皮肤较光滑，渗出较少，结痂轻微或脱落最早；阳性药组皮肤结痂较严重，痂皮几乎无脱落，有明显的鳞屑和丘疹。其中AB4衍生物组与模型组EASI综合评分差异结果第3天（P<0.001）、5天（P<0.0001）、7天（P<0.001）均有统计学意义；阳性药组与模型组EASI综合评分差异在第3天（P<0.001）有统计学意义。AB4、B4-19、B4-33、B4-39组与模型组相比，背部评分均有统计学意义（P<0.0001），其中AB4效果不如衍生物。另外，实验结果表明给药7天后，模型组小鼠的脾指数明显升高，白头翁皂苷B4衍生物脾指数明显降低，***组相比较模型组脾指数明显低于正常组，说明***对小鼠产生了免疫抑制，而白头翁皂苷B4衍生物能够提高小鼠的免疫力。所有实验结果提示白头翁皂苷B4衍生物对DNCB引起的湿疹皮损改变具有保护作用，效果比白头翁皂苷B4和糖皮质激素效果更优。
Claims (10)
1.一种白头翁皂苷B4衍生物，具有如下化学结构通式：
式中，R1包括3-O-α-L-吡喃鼠李糖基-(1→2)-α-L-吡喃***糖基；R2包括羟基或乙酰氧基；R3包括2-烯丙基、2-丙基、3-羟基丙烯基、3-溴丙烯基、2-环氧乙烷甲基；R4包括1-氧苯并三氮唑基、甲氧基、羟基、28-O-α-L-吡喃鼠李糖-(1→4)-β-D-吡喃葡萄糖-(1→6)-β-D-吡喃葡萄糖基、28-O-α-L-[2，3，4－三乙酰氧基－吡喃鼠李糖]-(1→4)-β-D-[2，3，6－三乙酰氧基－吡喃葡萄糖]-(1→6)-β-D-[2，3，4－三乙酰氧基－吡喃葡萄糖] 基、2-甲氧乙胺基、4-氨基丁酸甲酯基、环戊胺基、3-氯丙胺基、2-氟乙胺基、1-（3-氨基丙基）苯并三氮唑基、环己胺基、1-环丙基乙胺基、环丁基甲胺基、1-（3-氨基丙基）咪唑基、N-（2-氨乙基）吡咯烷基、4-氨基呋喃基、3-氨基环戊烷甲酸甲酯基、4-氨基环己基甲酸甲酯基、烯丙胺基、1-（2-氨乙基)哌啶基、2-噻唑乙胺基、四氢呋喃甲胺基、N-氨乙基吗啉基、1-甲基-4-哌啶甲胺基、1-甲基吡咯烷-3-甲胺基、4-氨基环己醇基、β-苯乙胺基、2-噻吩乙胺基、对羟基苯乙胺基、4-恶唑甲胺基、甘氨酸基、N-（2-氨基乙基）乙酰胺基、4-氨基丁酸基、5-氨基戊酸基、6-氨基己酸甲酯基、3-L-氨基环戊醇基、3-D-氨基环戊醇基。
2.根据权利要求1所述白头翁皂苷B4衍生物，其特征在于，R3为2-烯丙基、2-丙基、3-羟基丙烯基中的一种；R4为1-（3-氨基丙基）苯并三氮唑基、N-氨乙基吗啉基、对羟基苯乙胺基中的一种。
3.权利要求1所述白头翁皂苷B4衍生物的制备方法，其特征在于，以化合物AB4为原料，采用亲核取代、亲电加成、酯化或酰胺化反应制备所述白头翁皂苷B4衍生物。
4.一种药物组合物，以权利要求1所述白头翁皂苷B4衍生物为活性成分。
5.权利要求1所述白头翁皂苷B4衍生物或者权利要求4所述药物组合物在制备抗炎症药物中的应用。
6.根据权利要求5所述的应用，其特征在于，所述炎症包括体表炎症、体内炎症。
7.根据权利要求5所述的应用，其特征在于，所述炎症包括皮肤炎症。
8.权利要求1所述白头翁皂苷B4衍生物或者权利要求4所述药物组合物在制备免疫调节药物中的应用。
9.根据权利要求5～8任意一项所述的应用，其特征在于，所述药物包括外用、口服、直肠或者肠胃外药物。
10.根据权利要求9所述的应用，其特征在于，所述药物的剂型包括丸剂、片剂、粉剂、胶囊剂、颗粒剂、膏剂、溶液剂、注射剂、凝胶剂或者栓剂。
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