CN116284179B - 淫羊藿次苷ii衍生物的制备及应用 - Google Patents
淫羊藿次苷ii衍生物的制备及应用 Download PDFInfo
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- CN116284179B CN116284179B CN202211093682.6A CN202211093682A CN116284179B CN 116284179 B CN116284179 B CN 116284179B CN 202211093682 A CN202211093682 A CN 202211093682A CN 116284179 B CN116284179 B CN 116284179B
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- icariside
- liver cancer
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Abstract
淫羊藿次苷II衍生物的制备及应用，属于药物化学领域，本发明制备了式(I)、式(II)、式(III)或式(IV)所示的一系列具有较高抗肝癌活性的淫羊藿次苷II衍生物或其药学上可接受的盐，并进一步提供了包含所述衍生物或其药学上可接受的盐的药物组合物，以及所述衍生物或其药学上可接受的盐的制备方法。此外，还提供了所述衍生物或其药学上可接受的盐或者其药物组合物在制备抗原发性肝癌感染或治疗原发性肝癌的药物中的用途。
Description
技术领域
本发明属于药物化学领域，涉及黄酮衍生物及其制备方法和应用，具体涉及异戊烯基黄酮衍生物在抗肝癌方面的应用。
背景技术
原发性肝癌(Hepatocellular carcinoma,HCC)是发生于肝脏上皮的恶性肿瘤，其发生率和死亡率位列全球癌症相关疾病的第三位和第六位(CA-A Cancer Journal forClinicians,2021,71:209-249)。发病人群为35岁以上的青壮年。其死亡率居高不下，且发病率以每年3％-9％的速度稳步增长。临床上主要表现为乏力、消瘦、食欲减退、腹胀等症状。病患在患病早期常无明显阳性体征或肝硬化体征。中晚期通常出现肝脏肿大、黄疸、腹水等体征。
到目前为止，HCC的病因及确切分子机制尚不完全清楚。目前认为其发病是受多因素、多步骤的复杂过程影响，受环境以及饮食双重因素影响造成的。乙肝病毒(HBV)和丙肝病毒(HCV)感染、酗酒、黄曲霉素、肝硬化、亚硝胺类物质等都是造成肝癌发生的重要因素。临床上常以手术切除、肝移植、化疗等方式对患者进行有效治疗。但手术或肝移植常受到配体有限、手术风险高等问题的制约。而与手术相比，利用抗肝癌药物进行化疗具有减轻患者痛苦、降低治疗风险、提高患者生存质量等优点。且中晚期患者难以进行手术治疗，常以化疗来延续生命，发现及研制新型抗肝癌药物则至关重要。市面上，分子靶向药物索拉菲尼(sorafenib)和仑伐替尼(Lenvatinib)，虽是抗肝癌一线药物，但只能延长患者1年左右的中位生存期，且对于原发性肝癌不敏感(Liver cancer,2021,10:52-62)。因此，发现有效治疗HCC的药物便迫在眉睫。
异戊烯基黄酮作为一类广泛存在于植物中的天然产物，具有良好的抗肿瘤活性。但其水溶性差、口服吸收率低、生物利用度低以及代谢不稳定等，限制了天然黄酮类化合物的药效及成药性(Cancer Cell International,2016,16:1-11)。黄酮结构的酚羟基上引入含氮亲水基团，可以很大程度地提高其水溶性和口服吸收，从而促进并大大提高了生物利用度(Journal of Medicinal Chemistry,2017,60:6152-6165)。但是天然来源的含氮黄酮类化合物的含量十分有限，这限制了其在药物研发中的应用。一方面为了改善化合物的水溶性、提高化合物的生物利用度；另一方面为了进一步提高其药理活性。通过调研文献，以淫羊藿次苷II为母核，基于活性片段组合以及生物电子等排的原理，设计合成了一系列具有显著抑制肝细胞癌活性的衍生物，它不同于目前已知的肝癌抑制剂。
发明内容
为了解决上述技术问题，本发明制备了一系列具有较高抗肝癌活性的淫羊藿次苷II衍生物或其药学上可接受的盐，并进一步提供了包含所述衍生物或其药学上可接受的盐的药物组合物，以及所述衍生物或其药学上可接受的盐的制备方法。此外，还提供了所述衍生物或其药学上可接受的盐或者其药物组合物在制备抗原发性肝癌感染或治疗原发性肝癌的药物中的用途。
具体地，通过以下几个方面的技术方案实现了本发明：
在第一个方面中，本发明提供了具有抗原发性肝癌活性的淫羊藿次苷II衍生物或其药学上可接受的盐，所述衍生物具有式(I)、式(II)、式(III)或式(IV)所示的化学结构。
其中，式(I)中n为1-10，式(II)中n为1-5，R1和R2各自独立地选自H、C1-C9烷基，或者R1、R2同所连接的氮原子共同形成，取代或未取代的含氮杂环、含氮生物碱或其盐；
式(III)中，羟基为R或S构型，R1和R2各自独立地选自H、C1-C9烷基，或者R1、R2同所连接的氮原子共同形成，取代或未取代的含氮杂环、含氮生物碱或其盐；
式(IV)中，n为1-10。
优选地，所述R1和R2各自独立地选自：H、
或者，R1、R2同所连接的氮原子共同形成：
进一步优选地，式(I)、(IV)中，n为1-10；式(II)中，n为1-5；R1、R2同所连接的氮原子共同形成：NH2、N(CH3)2、N(CH3)CH2CH3、N(CH2CH3)2、N(CH2CH2CH3)2、N(CH2CH2CH2CH3)2、
最优地，所述具有抗原发性肝癌活性的淫羊藿次苷II衍生物选自以下化合物中任一种：
在第二个方面中，本发明提供一种药物组合物，所述药物组合物包含所述淫羊藿次苷II衍生物或其药学上可接受的盐，以及药学上可接受的载体。
所述药物组合物的剂型为口服剂型或注射剂型。其中，口服剂型为胶囊剂、片剂、颗粒剂、口服液、缓释制剂或控释制剂。
在第三个方面中，本发明提供所述的淫羊藿次苷II衍生物或其药学上可接受的盐制备方法，所述制备方法包括如下步骤：
淫羊藿次苷II为起始原料，先制得中间体1-11，然后中间体1-4与有机胺类化合物反应，生成11a-14f；中间体5-7与有机胺化合物反应，生成15c-17f；中间体8与有机胺化合物反应，生成18c-18f；中间体9-11在0℃下在氢氧化钠水溶液中与盐酸羟胺反应，生成19-21。
具体的，所述制备方法包括下述步骤：
(1)中间体1-4的合成：将淫羊藿次苷II溶于溶剂中，在搅拌下加入碱，加入相应的α,ω-二溴烷烃，回流反应，除去溶剂，萃取，洗涤，干燥，抽滤，纯化；
(2)中间体5-7的合成：将淫羊藿次苷II溶于溶剂中，在搅拌下加入碱，加入相应的α,ω-聚乙二醇二溴烷烃，回流反应，后处理方式同(1)；
(3)中间体8的合成：将淫羊藿次苷II溶于溶剂中，在搅拌下加入碱，加入R/S-环氧氯丙烷，回流反应，后处理方式同(1)；
(4)中间体9-11的合成：将淫羊藿次苷II溶于溶剂中，在搅拌下加入碱，加入溴酸甲酯，回流反应，后处理方式同(1)；
(5)11a-14f的合成：将中间体1-4溶于溶剂中，搅拌下加入碱，催化量的碘化钾或碘化钠，以及不同的胺，反应完全，后处理方式同(1)；
(6)15c-17f的合成：将中间体1-4溶于溶剂中，搅拌下加入碱，催化量的碘化钾或碘化钠，以及不同的胺，加热或回流反应，后处理方式同(1)；
(7)18c-18f的合成：将中间体8溶于溶剂中，搅拌下加入不同的胺，加热或回流反应，后处理方式同(1)；
(8)19-21的合成：将中间体9-11溶于强碱，搅拌下加入盐酸羟胺，0℃反应后转为室温反应，去除溶剂，萃取，洗涤，干燥，抽滤，纯化。
上述制备方法，其中：
所述溶剂为无水乙腈、无水丙酮、N,N-二甲基甲酰胺、乙酸乙酯或二甲基亚砜。
所述碱为无水碳酸钾、无水碳酸钠、无水碳酸钾、氢氧化钠或无水碳酸铯。
所述不同的胺为有机胺或苄胺。
所述强碱为氢氧化钠、氢氧化钾、氢氧化锂、乙醇钠、甲醇钠、乙醇钾或甲醇钾。
在第四个方面中，本发明提供所述淫羊藿次苷II衍生物或其药学上可接受的盐，或者所述药物组合物在制备抗肝癌药物中的用途。
另外地，本发明提供所述淫羊藿次苷II衍生物或其药学上可接受的盐，或者所述药物组合物在制备治疗肝癌的药物中的用途，优选地，所述肝癌为原发性肝癌。
本发明所使用的，术语“药学上可接受的载体”选自稀释剂、润滑剂、粘合剂、崩解剂、稳定剂或溶剂中的一种或多种。
本发明所述稀释剂选自淀粉、微晶纤维素、蔗糖、糊精、乳糖、糖粉、葡萄糖；所述润滑剂选自硬脂酸镁、硬脂酸、氯化钠、油酸钠、月桂醇硫酸钠、泊洛沙姆；所述粘合剂选自水、乙醇、淀粉浆、糖浆、羟丙基甲基纤维素、羧甲基纤维素钠、海藻酸钠、聚乙烯吡咯烷酮；所述崩解剂选自淀粉泡腾混合物即碳酸氢钠和枸橼酸、酒石酸、低取代羟丙基纤维素；所述稳定剂选自多糖如金合欢胶、琼脂、藻酸、纤维素醚和羧甲基甲壳酯；所述溶剂选自水、平衡的盐溶液。
本发明相对于现有技术，具有以下有益效果：
本发明通过对淫羊藿次苷II进行必要的结构改造，设计并合成了一系列新型淫羊藿次苷II衍生物或其药学上可接受的盐。通过药理学实验对肝癌细胞增殖进行抗肿瘤药物筛选发现，本发明的淫羊藿次苷II衍生物或其药学上可接受的盐具有良好的肿瘤抑制活性，因此可以将其开发成新型抗原发性肝癌的药物，具有非常高的潜在研究价值和临床应用前景。
附图说明
图1 11c通过Hoechst33258染色观察到肝癌细胞凋亡；
图2 11c在高浓度下诱导HepG2和SMMC-7721细胞凋亡；
图3 A/B 11c诱导肝癌细胞在G0/G1期阻滞；C通过Western Blot实验验证11c诱导肝癌细胞在G0/G1期阻滞。
具体实施方式
下面参照具体的实施例对本发明做进一步说明。应当理解，此处所描述的具体实施例仅用于解释本发明，并不用于限定本发明的范围。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道购买得到的常规产品。
制备淫羊藿次苷II母核方法：
1、取淫羊藿苷用等量的纤维素酶在pH＝4.5磷酸二氢钾/磷酸缓冲盐溶液中水解，50℃下加热搅拌24h；
2、将水解液用正丁醇萃取，得淫羊藿次苷II粗品；
3、将所得淫羊藿次苷II粗品溶解后上硅胶，在二氯甲烷:甲醇＝4:1条件下进行洗脱，收集洗脱液，减压干燥得到淫羊藿次苷II干燥粉末，产率约为95％；
1H NMR(600MHz,DMSO-d6)δ12.52(s,1H),7.85(d,J＝8.9Hz,2H),7.11(d,J＝8.9Hz,2H),6.33(s,1H),5.26(d,J＝1.3Hz,1H),5.15(ddd,J＝7.0,5.8,1.3Hz,1H),3.99(dd,J＝3.1,1.6Hz,1H),3.85(s,3H),3.48(dd,J＝9.3,3.2Hz,1H),3.47–3.31(m,3H),3.16–3.05(m,2H),1.67(s,3H),1.62(s,3H),0.78(d,J＝6.1Hz,3H).
实施例1:中间体1的制备
25ml茄型瓶加入淫羊藿次苷II(ICA-II)(100.0mg,194.3μmol)，碳酸钾(33.0mg,237.5μmol)，二溴乙烷(325.0μmol)，无水丙酮10ml，加热回流下反应10h。反应完毕，依次用水、饱和食盐水萃取，分出有机层，干燥过滤浓缩得粉末，经硅胶柱色谱纯化(二氯甲烷:甲醇＝20:1)纯化得关键中间体1，结构鉴定数据如下：
1H NMR(600MHz,DMSO-d6):δ12.57(s,1H),7.88(d,J＝8.9Hz,2H),7.13(d,J＝8.9Hz,2H),6.53(s,1H),5.28(s,1H),5.20(t,J＝7.1Hz,1H),4.78(s,1H),4.46(d,J＝1.8Hz,1H),4.00(s,1H),3.86–3.83(m,4H),3.48(t,J＝7.8Hz,2H),3.41(dd,J＝14.4,7.1Hz,1H),3.35(s,3H,4'-OCH3),3.15(t,J＝9.4Hz,1H),3.10–3.05(m,1H),1.70(s,3H),1.62(s,3H),0.79(d,J＝6.1Hz,3H,CH3).
HR-ESI-MS:621.1319[M+H]+,(calcd for C29H34BrO10,621.1330).
实施例2:中间体6的制备
25ml茄型瓶加入淫羊藿次苷II(ICA-II)(100.0mg,194.3μmol)，碳酸钾(33.0mg,237.5μmol)，二溴代二聚乙二醇(325.0μmol)，无水丙酮10ml，加热回流反应10h。反应完毕，依次用水、饱和食盐水萃取，分出有机层，干燥过滤浓缩得粉末，经硅胶柱层析纯化(二氯甲烷:甲醇＝20:1)得关键中间体6，结构鉴定数据如下：
1H NMR(600MHz,DMSO-d6):δ12.64(s,1H),7.87(d,J＝8.9Hz,2H),7.13(d,J＝9.0Hz,2H),6.58(s,1H),5.27(d,J＝1.4Hz,1H),5.16(t,J＝8.3Hz,1H),4.98(d,J＝4.4Hz,1H),4.72(d,J＝5.0Hz,1H),4.66(d,J＝5.8Hz,1H),4.24(dd,J＝5.7,3.1Hz,2H),3.99(t,J＝4.4Hz,1H),3.85(s,3H),3.81–3.79(m,2H),3.63–3.60(m,4H),3.56(t,J＝5.8Hz,4H),3.51–3.35(m,4H),3.16–3.07(m,2H),1.69(s,3H),1.62(s,3H),0.78(d,J＝6.1Hz,3H).
HR-ESI-MS:709.1854[M+H]+,(calcd for C33H42BrO12,709.1855).
实施例3:中间体8的制备
25ml茄型瓶加入淫羊藿次苷II(ICA-II)(100.0mg,194.3μmol)，碳酸钾(33.0mg,237.5μmol)，(R)-环氧氯丙烷(325.0μmol)，无水丙酮10ml，加热回流反应10h。反应完毕，依次用水、饱和食盐水萃取，分出有机层，干燥过滤浓缩得粉末，经硅胶柱层析(二氯甲烷:甲醇＝40:1-20:1)纯化得关键中间体8，结构鉴定数据如下：
1H NMR(600MHz,DMSO-d6):δ12.64(s,1H),7.88(d,J＝8.9Hz,2H),7.13(d,J＝9.0Hz,2H),6.57(s,1H),5.27(s,1H),5.15(t,J＝7.1Hz,1H),4.98(d,J＝4.5Hz,1H),4.73(d,J＝5.0Hz,1H),4.66(d,J＝5.9Hz,1H),4.51(ddd,J＝11.5,5.4,2.5Hz,1H),4.02–3.97(m,2H),3.85(s,3H),3.48(ddd,J＝16.3,8.1,5.2Hz,2H),3.42–3.36(m,2H),3.16–3.05(m,2H),2.88–2.86(m,1H),2.73(dd,J＝5.1,2.6Hz,1H),1.70(s,3H),1.62(s,3H),0.78(d,J＝6.1Hz,3H).
实施例4:氮杂环类衍生物的制备：
10ml茄型瓶加入中间体1(20.0mg,32.3μmol)，吡咯烷(4.6mg,65μmol),碳酸钾(6.6mg,47.5μmol)，碘化钾(1.2mg,7.2μmol),乙腈6ml，50℃下反应8h。反应完毕，依次用水、饱和食盐水萃取，分出有机层，干燥过滤浓缩得粉末，经反向高效液相色谱纯化(甲醇-水80:20,2.5ml/min)得到目标产物11c，结构鉴定数据如下：
1H NMR(600MHz,DMSO-d6):δ12.61(s,1H),7.86(d,J＝8.9Hz,2H),7.12(d,J＝8.9Hz,2H),6.56(s,1H),5.27(d,J＝1.2Hz,1H),5.14(t,J＝7.0Hz,1H),4.19(t,J＝5.6Hz,2H),3.99(s,1H),3.85(s,3H,4'-OCH3),3.48(dd,J＝9.2,3.2Hz,1H),3.44(dd,J＝15.1,7.5Hz,2H),3.28(d,J＝10.3Hz,2H),3.14(t,J＝9.4Hz,1H),3.07(dq,J＝12.3,6.1Hz,1H),2.82(t,J＝5.6Hz,2H),2.53(d,J＝9.2Hz,4H),1.75(s,1H),1.69(d,J＝3.2Hz,4H),1.67(s,3H,CH3),1.60(s,3H,CH3),0.78(d,J＝6.1Hz,3H,CH3).
13C NMR(150MHz,DMSO-d6):δ178.24,171.46,161.77,161.37,159.58,157.13,152.90,134.47,131.20,130.48,122.32,122.04,114.10,107.17,104.62,102.02,95.86,71.13,70.69,70.34,70.08,68.19,55.51,54.08,54.03,51.33,25.44,23.23,22.52,21.27,17.88,17.78,17.47.
HR-ESI-MS:612.2839[M+H]+,(calcd for C33H42NO10,612.2803).
实施例5:氮烷烃类衍生物的制备：
10ml茄型瓶加入中间体1(20.0mg,32.3μmol)，二乙胺(4.7mg,65μmol),碳酸钾(6.6mg,47.5μmol)，碘化钾(1.2mg,7.2μmol)，乙腈6ml，50℃下反应8h。反应完毕，依次用水、饱和食盐水萃取，分出有机层，干燥过滤浓缩得粉末，经反向高效液相色谱纯化(甲醇-水80:20,2.5ml/min)得到目标产物11f，结构鉴定数据如下：
1H NMR(600MHz,DMSO-d6):δ12.64(s,1H),7.86(d,J＝8.9Hz,2H),7.12(d,J＝9.0Hz,2H),6.58(s,1H),5.27(d,J＝1.3Hz,1H),5.14(t,J＝7.1Hz,1H),4.18(t,J＝5.6Hz,2H),3.99(s,1H),3.85(s,3H,4'-OCH3),3.48(dd,J＝9.2,3.2Hz,1H),3.45–3.40(m,1H),3.36(dd,J＝15.2,7.2Hz,4H),3.14(t,J＝9.4Hz,1H),3.09–3.04(m,1H),2.66(t,J＝5.6Hz,2H),2.50(s,3H,CH3),2.22(s,6H,2CH3),1.68(s,3H,CH3),1.61(s,3H,CH3),0.78(d,J＝6.1Hz,3H,CH3).
13C NMR(150MHz,DMSO-d6):δ178.24,161.77,161.37,159.58,157.13,152.89,134.47,131.19,130.48,122.32,122.00,114.10,107.17,104.62,102.02,95.90,71.12,70.69,70.31,70.08,67.19,57.60,55.50,45.56,39.80,39.66,39.38,39.24,25.45,21.26,17.78,17.47.
HR-ESI-MS:614.2960[M+H]+,(calcd for C33H44NO10,614.2949).
实施例6:聚乙二醇氮杂环类衍生物的制备：
10ml茄型瓶加入中间体6(20.0mg,32.3μmol)，吡咯烷(4.7mg,65μmol),碳酸钾(6.6mg,47.5μmol)，碘化钾(1.2mg,7.2μmol)，乙腈6ml，60℃下反应8h。反应完毕，依次用水、饱和食盐水萃取，分出有机层，干燥过滤浓缩得粉末，经反向高效液相色谱纯化(甲醇-水85:15,2.5ml/min)得到目标产物16c，结构鉴定数据如下：
1H NMR(600MHz,DMSO-d6):δ12.63(s,1H),7.87(d,J＝8.9Hz,2H),7.12(d,J＝8.9Hz,2H),6.57(s,1H),5.26(d,J＝1.3Hz,1H),5.17–5.14(m,1H),4.99(s,1H),4.74(s,2H),4.23(dt,J＝9.6,4.8Hz,2H),3.99(s,1H),3.84(d,J＝6.8Hz,3H),3.77(d,J＝4.4Hz,2H),3.59(dd,J＝5.8,3.8Hz,2H),3.51(dd,J＝5.8,3.8Hz,2H),3.47(qd,J＝6.4,4.2Hz,6H),3.44(dd,J＝6.8,3.6Hz,1H),3.38(dd,J＝14.6,7.2Hz,2H),3.14(t,J＝9.3Hz,1H),3.09–3.05(m,1H),2.52(dd,J＝11.1,4.9Hz,2H),2.48–2.40(m,3H),2.38(s,4H),1.68(s,3H),1.65–1.62(m,3H),1.61(s,3H),1.60–1.59(m,2H),0.95–0.82(m,2H),0.78(d,J＝6.1Hz,3H).
13C NMR(150MHz,DMSO-d6):δ178.26,161.79,161.38,159.55,157.15,152.90,134.49,131.22,130.48,122.32,121.92,114.10,107.27,104.68,102.03,96.01,71.11,70.69,70.31,70.08,69.98,69.75,69.67,69.61,69.18,68.80,68.47,55.50,54.98,53.99,53.96,46.95,29.03,28.99,25.47,23.10,23.08,22.10,21.29,17.70,17.46.
HR-ESI-MS:700.3360[M+H]+,(calcd for C37H50NO12,700.3328).
实施例7:聚乙二醇氮烷烃类衍生物的制备：
10ml茄型瓶加入中间体6(20.0mg,32.3μmol)，二乙胺(4.9mg,65μmol),碳酸钾(6.6mg,47.5μmol)，碘化钾(1.2mg,7.2μmol)，乙腈6ml，60℃下反应8h。反应完毕，依次用水、饱和食盐水萃取，分出有机层，干燥过滤浓缩得粉末，经反向高效液相色谱纯化(甲醇-水85:15,2.5ml/min)得到目标产物16f，结构鉴定数据如下：
1H NMR(600MHz,DMSO-d6):δ7.87(d,J＝8.9Hz,2H),7.13(d,J＝9.0Hz,2H),6.57(s,1H),5.16(t,J＝7.2Hz,1H),5.01(s,1H),4.76(s,2H),4.24(dd,J＝5.5,2.9Hz,2H),3.99(s,1H),3.85(d,J＝3.3Hz,3H),3.80–3.76(m,2H),3.59(dd,J＝5.8,3.7Hz,2H),3.51(dd,J＝5.8,3.7Hz,2H),3.49–3.44(m,2H),3.42(t,J＝6.5Hz,2H),3.38(dd,J＝14.4,7.2Hz,2H),3.14(t,J＝9.4Hz,1H),3.07(dd,J＝9.4,6.1Hz,1H),2.48(s,1H),2.42(q,J＝7.1Hz,3H),1.69(s,3H),1.61(s,3H),0.89(t,J＝7.1Hz,6H),0.78(d,J＝6.1Hz,3H).
13C NMR(150MHz,DMSO-d6):δ178.27,161.80,161.38,159.56,157.16,152.90,134.48,131.22,130.48,129.65,122.32,121.92,114.11,107.26,104.69,102.03,96.00,71.11,70.69,70.31,70.08,69.99,69.83,69.21,68.81,68.48,55.51,51.91,46.94,29.03,28.99,25.47,21.29,17.70,17.46,11.84.
HR-ESI-MS:702.3485[M+H]+,(calcd for C37H52NO12,702.3484).
实施例8:胺基醇衍生物的制备：
10ml茄型瓶加入中间体8(24.0mg,42μmol)，吡咯烷(30mg，42μmol),乙醇6ml，加热回流反应24h。反应完毕，依次用水、饱和食盐水萃取，分出有机层，干燥过滤浓缩得黄色粉末，经PTLC(二氯甲烷-甲醇，10：1)纯化得到目标产物18c。结构鉴定数据如下：
1H NMR(600MHz,DMSO-d6):δ12.60(s,1H),7.88(d,J＝8.4Hz,2H),7.14(d,J＝8.5Hz,2H),6.55(s,1H),5.28(s,1H),5.16(t,J＝7.0Hz,1H),5.12–4.67(m,4H),4.10(s,1H),4.04(s,1H),3.98(d,J＝36.9Hz,2H),3.86(s,3H),3.63–3.46(m,2H),3.43–3.20(m,3H),3.20–2.93(m,2H),2.66(dd,J＝11.9,6.1Hz,1H),2.51(s,4H),1.77–1.56(m,10H),0.79(d,J＝5.8Hz,3H).
13C NMR(150MHz,DMSO-d6):δ178.21,162.03,161.36,159.63,157.09,152.91,134.46,131.07,130.48,122.32,122.23,114.10,107.23,104.62,101.96,95.88,71.72,71.16,70.66,70.40,70.05,67.65,58.72,55.52,54.28,25.47,23.19,21.24,17.87,17.47.
HR-ESI-MS:642.2924[M+H]+,(calcd for C34H44NO11,642.2909).
实施例9：中间体10的制备：
25ml茄型瓶加入淫羊藿次苷II(ICA-II)(100.0mg,194.3μmol)，碳酸钾(33.0mg,237.5μmol)，溴戊酸甲酯(325.0μmol)，无水丙酮10ml，加热回流反应10h。反应完毕，依次用水、饱和食盐水萃取，分出有机层，干燥过滤浓缩得粉末，经硅胶柱层析纯化(二氯甲烷:甲醇＝20:1)得关键中间体10，结构鉴定数据如下：
1H NMR(600MHz,DMSO-d6):δ7.86(d,J＝8.9Hz,2H),7.12(d,J＝8.9Hz,2H),6.51(s,1H),5.27(s,1H),5.11(t,J＝7.0Hz,1H),4.10(t,J＝6.0Hz,2H),4.00(s,1H),3.85(s,3H),3.61(s,3H),3.49(dd,J＝9.2,3.1Hz,1H),3.42(dd,J＝14.4,6.9Hz,1H),3.34(m,1H),3.18–3.03(m,2H),2.33(t,J＝7.2Hz,2H),2.02–1.79(m,2H),1.67(s,3H),1.61(s,3H),0.78(d,J＝6.1Hz,3H).
实施例10:异羟肟酸衍生物的制备：
在5ml甲醇盐酸羟胺(100mg，144μmol)溶液中加入0℃氢氧化钠(120mg，216μmol)。将混合物在0℃下搅拌0.5h，形成白色固体。然后过滤残渣，在甲醇溶液中得到羟胺。将中间体10(88mg，144μmol)在0℃下溶于含羟胺的甲醇溶液2ml，并在室温下搅拌0.5h。经PTLC(二氯甲烷-甲醇，10：1)纯化得到目标产物20。结构鉴定数据如下：
1H NMR(600MHz,DMSO-d6):δ7.86(d,J＝8.9Hz,2H),7.12(d,J＝8.9Hz,2H),6.51(s,1H),5.27(s,1H),5.11(t,J＝7.0Hz,1H),4.10(t,J＝6.0Hz,2H),4.00(s,1H),3.85(s,3H),3.49(dd,J＝9.2,3.1Hz,1H),3.42(dd,J＝14.4,6.9Hz,1H),3.34(dd,J＝14.4,7.0Hz,1H),3.18–3.03(m,2H),2.33(t,J＝7.2Hz,2H),2.02–1.79(m,2H),1.67(s,3H),1.61(s,3H),0.78(d,J＝6.1Hz,3H).
13C NMR(150MHz,DMSO-d6):δ178.21,174.68,161.80,161.35,159.62,157.02,152.84,134.46,131.22,130.47,122.34,122.02,114.09,107.07,104.54,102.02,95.69,71.16,70.71,70.32,70.09,68.11,55.49,30.99,25.43,24.62,21.23,17.73,17.48.
HR-ESI-MS:630.2513[M+H]+,(calcd for C32H40NO11,630.2572).
实施例11:药学筛选
为了证明本发明的淫羊藿次苷II衍生物具有潜在治疗肝癌的效果，对制备的化合物进行了肝癌药效学筛选。
具体的抗肝癌化合物筛选方法及结果，如下所示：
1)利用MTT法进行化合物初筛，以索拉非尼(sorafenib)、淫羊藿素(ICT)作为阳性对照。选择生长状态良好的肝癌细胞或正常肝细胞，胰蛋白酶消化，用细胞完全培养基分散成单细胞悬液，进行细胞计数，调整细胞浓度5×104个/ml，轻轻吹打细胞使其均匀分散，均匀铺于96孔板中(100μl/孔)，CO2培养箱中培养过夜。第二天弃掉培养基，分别加入指定浓度的药物，并设置空白组以扣除背景的影响，每组设3个复孔。药物于CO2培养箱中作用48h后，取出96孔板，以提前配制好的MTT溶液，20μl/孔，于培养箱中继续孵育4h后。随后弃掉MTT溶液，每孔加入150μl DMSO溶液溶解甲瓒，室温振荡5min使结晶充分溶解。酶标仪调至OD 490nm处，检测各孔的吸光度值，实验重复3次。细胞生长抑制率按照如下公式计算：抑制率(％)＝(A对照-A加药)/(A对照-A空白)×100％。计算不同浓度下的抑制率，并绘制量-效曲线。计算生长抑制率50％时的浓度，即为IC50值。结果表明大部分化合物具有相当程度抑制肝癌细胞增殖的活性，如表1所示。
表1化合物的抗肝癌细胞增殖活性
aN.T.代表未检测；b.48h后测定最大半抑制浓度
2)取处于对数生长期的肝癌细胞制成细胞悬液，以每孔1×104个的密度均匀地接种于24孔板中，在5％CO2，37℃培养箱中培养过夜后加药组加入不同浓度的11c(HepG2和SMMC-7721细胞处理浓度分别为2、4、8μM)，阴性对照组加入等体积的空白培养基继续培养48h后将孔中培养基弃去。向每个孔中加4％的多聚甲醛100μl室温固定15min后，加入Hoechst 33258(5μg/mL)染料没过24孔板孔，室温避光染色30min。染色结束后，吸去染料并用PBS轻洗两次。使用倒置荧光显微镜，紫色激发光激发下观察荧光以及核断裂情况并拍照。在倒置荧光显微镜下可看到，对照组的细胞核染色均匀，无明显的细胞核断裂等现象。但随着11c给药浓度的增加，胞质浓染加深，核断裂加深，证明11c可诱导HCC细胞大浓度凋亡(图1)。
3)取对数生长期的人肝癌细胞，消化后制成密度为2×105细胞/孔的细胞悬液，均匀接种于6孔板中，置于培养箱培养过夜。实验组每孔加入不同浓度的化合物11c(HepG2细胞处理浓度分别2、4、8μM；SMMC-7721细胞处理浓度分别为2、4、8μM)，对照组加等体积的培养基。培养48h后，去除培养基，用PBS洗三次，加入不含EDTA的胰酶消化并收集细胞，每组样品中各加入400μl Binding Buffer重悬细胞，再依次加入Annexin V-FITC、PI各4μl染色液并混匀，室温避光孵育15min即可用流式细胞仪检测凋亡，使用Flow JO 7.6及Graph PadPrism 7.0软件分析并统计实验结果。
结果如图2所示，加入11c处理后的两种肝癌细胞AV+PI-与AV+PI+细胞比例随着浓度的升高而增加，表明11c在大浓度下能够显著地诱导HepG2和SMMC-7721细胞产生凋亡。
4)使用不同浓度的11c(2、4和8μM)处理HepG2细胞以及SMMC-7721细胞72h，然后用碘化丙啶(PI)染色，并通过流式细胞术进行细胞周期分析。如图3A和3B所示，在HepG2细胞中，随着化合物11c浓度的提升，处于G0/G1期的细胞从阴性对照的64.11％分别增加到91.15％，且呈浓度依赖性；在SMMC-7721细胞中，随着化合物11c浓度的提升，处于G0/G1期的细胞从阴性对照的59.57％分别增加到78.65％，且呈浓度依赖性。
HepG2细胞或SMMC-7721细胞与化合物作用后，收集细胞，PBS清洗两次，加入适量的细胞裂解液及蛋白酶抑制剂(RIPA裂解液：PMSF＝100:1)，12,000×g离心10min后，加入5×上样缓冲液混匀，100℃煮沸10min，再应用Bio-Rad蛋白分析仪检测上清液中蛋白的含量，通过10％十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)进行分离，然后转移至PVDF膜上，薄膜浸入5％脱脂奶中室温下孵育2h，加入不同种类的一抗4℃孵育过夜后，再同方法加入辣根过氧化酶(HRP)标记的二抗的封闭液，于暗室用化学发光液显影，扫描胶片保存结果。最后利用Image J计算各蛋白条的相对密度。
结果显示，HepG2和SMMC-7721细胞经11c处理48h后,G1期相关蛋白P21的蛋白水平明显升高，并且呈一定的浓度依赖性(图3C)。结果表明11c能够通过上调P21，下调Cdc2p34和CDK4蛋白的表达，诱导肝癌细胞G0/G1期细胞周期阻滞。
综上所述，通过应用上述药理学实验对肝癌细胞增殖进行抗肿瘤药物筛选发现，本发明的淫羊藿次苷II衍生物具有良好的抗肝癌细胞增殖活性，可以进一步用于开发治疗肝癌的药物。
Claims (6)
1.一种淫羊藿次苷II衍生物或其药学上可接受的盐，其特征在于，所述的淫羊藿次苷II衍生物选自以下化合物中任一种：
2.一种药物组合物，其特征在于，包含权利要求1所述的淫羊藿次苷II衍生物或其药学上可接受的盐，以及药学上可接受的载体。
3.根据权利要求2所述的药物组合物，其特征在于，所述药物组合物剂型为口服剂型或注射剂型；其中，口服剂型为胶囊剂、片剂、颗粒剂、口服液、缓释制剂或控释制剂。
4.权利要求1所述淫羊藿次苷II衍生物或其药学上可接受的盐或权利要求2或3所述药物组合物在制备抗肝癌药物中的应用。
5.权利要求1所述淫羊藿次苷II衍生物或其药学上可接受的盐或权利要求2或3所述药物组合物在制备治疗肝癌的药物中的应用。
6.根据权利要求5所述的应用，其特征在于，所述肝癌为原发性肝癌。
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