Marine Geology

Design, synthesis and cytotoxicity of novel glycosyl-2, 5-diketopiperazine derivatives

  • LIAO Shengrong , 1, 2 ,
  • XU Huayan 3 ,
  • LI Xiaolin 1, 2 ,
  • LIU Yonghong , 1, 2, 3
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  • 1. Key Laboratory of Tropical Marine Bio-resources and Ecology (South China Sea Institute of Oceanology, Chinese Academy of Sciences), Guangzhou 510301, China
  • 2. University of Chinese Academy of Sciences, Beijing 100049, China
  • 3. Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang 110016, China
LIAO Shengrong, email: ;
LIU Yonghong, email:

Copy editor: LIN Qiang

Received date: 2022-06-21

  Revised date: 2022-08-01

  Online published: 2022-08-18

Supported by

National Natural Science Foundation of China(82073762)

Guangdong Basic and Applied Basic Research Foundation(2020A1515011045)

Abstract

Exploring the cytotoxic compounds from marine natural products is one of the most important tasks for the discovery of anticancer agent in recent years. In this study, based on the marine natural product Piperafizine B, fourteen novel glycosyl-2, 5-diketopiperazine derivatives (1a-i, 2a-f) have been designed and synthesized by the glycosylation on the side phenol group at the 6-position of the 2, 5-diketopiperizine ring. The solubilities of the derivatives are obviously improved in both DMSO (dimethyl sulfoxide, 1.5~27.9mg·mL-1) and phosphate buffer (0.1~0.9mg·mL-1, pH=7.4). The cytotoxicity results show that some of the derivatives present good activities with the IC50 values ranging from 1.0~9.1μmol·L-1, and exhibit selective inhibitory activities on Huh-7 cell lines. Among the derivatives, compound 1c has the best inhibitory abilities on the cell lines K562, A549 and Huh-7 with an IC50 values at 1.0, 3.6 and 3.3μmol·L-1, respectively, while compounds 2a-f have no observable cytotoxicity.

Cite this article

LIAO Shengrong , XU Huayan , LI Xiaolin , LIU Yonghong . Design, synthesis and cytotoxicity of novel glycosyl-2, 5-diketopiperazine derivatives[J]. Journal of Tropical Oceanography, 2023 , 42(3) : 174 -185 . DOI: 10.11978/2022140

近年来, 从海洋天然产物中挖掘高细胞毒活性化合物成为抗肿瘤药物研究工作的一个热点(Matulja et al, 2020; Mondal et al, 2020; Nadar et al, 2022; )。然而多数情况下, 分离获得的海洋天然产物质量少, 结构多样性不足, 严重影响了活性分子的后续研究(包括构效关系、机制研究与成药性评价等), 为解决这些问题, 对活性分子进行合成或结构衍生已经成为海洋来源药物分子开发的一种重要手段(Popović-Djordjević et al, 2022; Rycek, 2022)。
由两分子氨基酸经胺基与羧基缩合形成的环二肽化合物称为2, 5-二酮哌嗪衍生物(图1)。许多陆地、海洋动植物及微生物均能产生这类天然产物(Huang et al, 2014; Song et al, 2021)。在这类天然产物中, 其3, 6-位上含一个或两个双键的衍生物, 由于双键能与2, 5-二酮哌嗪环形成一个近似平面的结构, 当环上的两个N原子上均无取代基时, 衍生物易形成分子间氢键而聚合(图2), 导致化合物的溶解度(水溶性与脂溶性)降低(Borthwick, 2012)。此类海洋来源的代表分子有Phenylahistin(来源于焦曲霉菌)(Kanoh et al, 1997), Piperafizine B(来源于灰锈赤链霉菌)(Kamei et al, 1990), XR334(来源于硫黄链霉菌)(Bryans et al, 1996)等(图1)。由于结构简单, 许多此类衍生物已被合成, 并且具有抗肿瘤(Dale et al, 1998; Liao et al, 2014; Liao et al, 2016a; Liao et al, 2016b; Chinh et al, 2021; Tham et al, 2021)、抗污损(Loughlin et al, 2000; Liao et al, 2015)、抑制纤溶酶原激活抑制物(Wang et al, 2002)等多样生物活性, 其中以Phenylahistin结构衍生的小分子Plinabulin(图1)( Singh et al, 2011; Blayney et al, 2022)已在国内进行上市申请, 并进入优先评审程序, 用于非小细胞肺癌与中性粒细胞减少症的治疗。
图1 2, 5-二酮哌嗪及其海洋来源的天然产物与结构衍生物Plinabulin

Fig. 1 2, 5-Diketopiperafizine, its marine originated natural products and the modified compound Plinabulin

图2 N-未取代的2, 5-二酮哌嗪衍生物易形成分子间氢键

Fig. 2 Inter-molecular hydrogen bonds formed between the N-nonsubstituted 2, 5-diketopiperafizines

在哌嗪环N原子上引入取代基, 破坏分子间氢键, 有利于增加衍生物的脂溶性, 如Piperafizine A(图1)的脂溶性要高于Piperafizine B。因此, 在我们前期的研究工作中(图3), 选用Piperafizine A进行结构修饰, 从考察侧链基团多样性、高脂溶性、芳环上引进F原子及N-取代基替换等四个方面设计合成了一系列Piperafizine A衍生物。细胞毒活性评价结果表明, 大部分衍生物的活性提高, 如含2, 3-氯苯叉衍生物4m(IC50为0.5μmol·L-1, U937细胞株)(Liao et al, 2014), 含戊叉基侧链衍生物3c(IC50为0.36μmol·L-1, U937细胞株)(Liao et al, 2016b), 与含萘叉衍生物2t(IC50为0.2μmol·L-1, U937细胞株)(汤勇 等, 2016)等的抗肿瘤活性均明显增强。同时发现, 将1-位N原子上的保护基替换为甲基, 活性最大提升接近10倍, 而3, 6-位为间甲氧基苄叉取代的化合物3h的细胞毒活性(IC50为0.011μmol·L-1, HL60细胞株)与Plinabulin的活性(IC50为0.015μmol·L-1, HL60细胞株)接近(Liao et al, 2016a)。上述研究表明, 不同的侧链取代基团与N上保护基均能影响衍生物的活性, 通过结构衍生途径有可能发现高活性的衍生物。在1-位N原子引入取代基破坏分子间氢键, 有利于改善化合物的脂溶性, 但更高脂溶性对衍生物活性的改善不明显。因此后续的结构修饰设计从改善化合物的脂溶性与水溶性两方面进行探索。
图3 前期对Piperafizine A进行结构修饰及获得的高活性代表衍生物

Fig. 3 Previous modifications on Piperafizine A and the representative compounds with high cytotoxicities

在Plinabulin分子中, 因其二酮哌嗪环上咪唑一侧的酰胺H能与咪唑N原子形成分子内氢键(Yamazaki et al, 2012), 其溶解度(脂溶性与水溶性)增加。同时由于N上两个H原子的存在, 有利于衍生物与靶蛋白之间的氢键作用。借鉴Plinabulin结构的这些特点, 保留二酮哌嗪环上裸露的N原子(两个N原子无取代基), 以增加衍生物分子与靶蛋白、水分子的氢键作用, 达到既改善衍生物的脂溶性与水溶性, 又提高其生物活性目的。但类似Piperafizine B结构的分子, 因其易形成分子间氢键, 溶解度低而导致其生物利用度和活性降低(Lipinski, 2000; van de Waterbeemd et al, 2001)。为克服这个缺点, 除前期研究中在N上引入取代基外, 在分子中引入大体积基团增加分子间位阻, 也是一种有效手段。因此我们在设计上选用葡萄糖分子(Chen et al, 2020)对Piperafizine B进行结构修饰, 一是葡萄糖骨架较大, 有可能阻止两分子靠近; 二是葡萄糖具有较好的水溶性, 有利于改善分子的水溶性, 从而衍生物的脂溶性与水溶性均得到改善。根据这种设计思路, 本研究以Piperafizine B为衍生骨架, 在2, 5-二酮哌嗪环6-位苄叉的邻位采用四乙酰基葡萄糖或葡萄糖进行结构修饰, 因此设计合成了两个系列衍生物(图4, 目标物12)。
图4 本研究的设计思路

Fig. 4 The designing in this work

1 材料与方法

1.1 仪器与试剂

采用SGW-X4熔点仪测定化合物的熔点, IR Affinity-1傅里叶变换红外光谱仪测定化合物的红外光谱与MCP 500旋光仪测定化合物的旋光。采用AV-500或700型超导核磁共振波谱仪(四甲基硅烷为内标)与德国Bruker maXis型高分辨飞行时间质谱仪(Q-TOF)与电喷雾(ESI)离子源对中间体或产物进行结构表征。细胞毒活性评价中阳性对照样品使用生物纯试剂taxol(紫杉醇), 其他试剂或中间体如2, 5-二酮哌嗪、芳香醛衍生物、α-溴代乙酰-D-葡萄糖等购置于常规化学试剂公司, 所有化学中间体、试剂与洗脱溶剂均为市售分析纯, 反应用加热盘、加热油浴锅等购置于常规仪器设备代理公司。反应跟踪与产物提纯分别采用薄层板与色谱硅胶(200~300目)。溶解度测定采用Waters 1525高效液相仪及Waters 2996 紫外检测仪(photodiode array detector), 检测波长为254nm, 分析柱采用Agilent eclipse extra-dense bonding-C18, 5μm, 4.6×250mm, 流速为1.0mL·min-1, 流动相的比例为甲醇/水=65/35(体积/体积)。

1.2 化学合成

1.2.1 (2R, 3R, 4S, 5R, 6S)-2-(乙酰氧甲基)-6-(2-甲醛基苯酚基)四氢-2H-吡喃-3, 4, 5-三醋酸酯(中间体I)的合成

在50mL反应瓶中, 依次加入乙酰溴-α-D-葡萄糖(3.5g, 8.5mmol, 4.2equiv)、水杨醛(216μL, 2mmol, 1.0equiv)与二氯甲烷(DCM)(12mL), 然后再加入四丁基溴化铵(TBAB)(644mg, 2mmol, 1.0equiv)与5%氢氧化钠(NaOH)溶液(4.0mmol, 6.4mL, 4.0equiv), 混合溶液室温搅拌(薄层色谱跟踪)。待反应完成后, 反应体系中加入二氯甲烷(50mL), 然后加水(每次20mL)萃洗三次, 有机层无水硫酸镁(MgSO4)干燥, 减压旋去溶剂后, 残余物经硅胶柱层析纯化得到中间体I, 收率95%。熔点: 118~120℃; [ α ] D 2 5-45.1 (c 0.1, dimethyl sulfoxide, DMSO); 1H NMR (500MHz, CDCl3) δ 10.32 (s, 1H), 7.83 (dd, J = 7.7, 1.1 Hz, 1H), 7.61~7.48 (m, 1H), 7.17 (t, J = 7.5 Hz, 1H), 7.10 (d, J = 8.4 Hz, 1H), 5.34 (dt, J = 18.1, 9.4 Hz, 2H), 5.22~5.11 (m, 2H), 4.28 (dd, J = 12.4, 5.3 Hz, 1H), 4.16 (dd, J = 12.3, 2.3 Hz, 1H), 3.90 (ddd, J = 10.0, 5.3, 2.4 Hz, 1H), 2.05 (s, 3H), 2.04 (s, 6H), 2.03 (s, 3H). 13C NMR (126MHz, CDCl3) δ 189.1, 170.4, 170.1, 169.3, 169.1, 158.7, 135.7, 128.2, 126.1, 123.5, 115.9, 98.9, 72.3, 72.1, 70.8, 68.1, 61.7; FT-IR (neat): 3089, 2882, 1744, 1693, 1600, 1483, 1367, 1209, 1033, 906, 762, 735, 598cm-1; HRMS (ESI) m/z: [M+Na]+ Calcd for C21H24NaO11 475.1211; found 475.1210.

1.2.2 (2R, 3R, 4S, 5R, 6S)-2-(乙酰氧甲基)-6-(2-(Z)-(4-乙酰基-3, 6-二酮哌嗪-2-苄叉)苯酚基)四氢-2H-吡喃-3, 4, 5-三醋酸酯(中间体II)的合成

在50mL反应瓶中, 依次加入中间体I(1.14g, 2.52mmol, 1.0equiv), 双乙酰-2, 5-二酮哌嗪(750mg, 3.78mmol, 1.5equiv)与N, N-dimethylformamide(DMF) (15mL), 然后加入碳酸铯(Cs2CO3)(1.23g, 3.78mmol, 1.5equiv), 室温搅拌。待反应完成后, 将反应液倾入50mL水中, 10%稀盐酸(HCl)调节pH至4~5, 然后用乙酸乙酯(每次25mL)萃取三次, 有机层水洗一次, MgSO4干燥。减压除去溶剂后, 残余物经硅胶柱层析纯化得到中间体II, 收率50%。熔点: 157~160℃; [ α ] D 2 5-133.0 (c 0.1, DMSO); 1H NMR (500MHz, CDCl3) δ 7.88 (s, 1H), 7.39~7.32 (m, 1H), 7.27 (d, J = 6.9 Hz, 1H), 7.15 (dd, J = 16.1, 8.2 Hz, 2H), 6.98 (s, 1H), 5.24 (p, J = 9.4 Hz, 2H), 5.14 (t, J = 9.5 Hz, 1H), 5.01 (d, J = 7.5 Hz, 1H), 4.56 (d, J = 18.5 Hz, 1H), 4.46 (d, J = 18.5 Hz, 1H), 4.27 (dd, J = 12.3, 5.4 Hz, 1H), 4.20 (dd, J = 12.3, 2.5 Hz, 1H), 3.88 (ddd, J = 10.0, 5.3, 2.5 Hz, 1H), 2.63 (s, 3H), 2.08 (s, 3H), 2.02 (s, 3H), 1.99 (s, 3H), 1.96 (s, 3H). 13C NMR (126MHz, CDCl3) δ 172.5, 170.5, 170.1, 169.2, 162.8, 159.9, 153.7, 130.6, 127.4, 124.0, 122.8, 116.2, 114.1, 99.6, 72.2, 72.1, 70.5, 68.2, 61.8, 46.3, 27.1, 20.6, 20.5, 20.4; FT-IR (neat): 3076, 2877, 1747, 1697, 1637, 1491, 1367, 1219, 1309, 908, 762, 734, 598cm-1; HRMS (ESI) m/z: [M+Na]+ Calcd for C27H30N2NaO13 613.1640; found 613.1642.

1.2.3 目标产物1的合成通用方法

在10mL 反应瓶中, 依次加入中间体II(70mg, 0.14mmol, 1.0equiv), 苯甲醛衍生物(0.21mmol, 1.5equiv)和Cs2CO3 (68mg, 0.21mmol, 1.5equiv), 然后加入DMF (2mL), 混合物溶液于80℃搅拌(TLC跟踪)。反应完成后加水20mL, 10%HCl溶液调pH至5~6。用乙酸乙酯( 3×5mL) 萃取水层, 有机层无水硫酸镁干燥, 减压除去溶剂后残余物经硅胶柱纯化得目标产物1

1.2.3.1 (2R, 3R, 4S, 5R, 6S)-2-(乙酰氧甲基)-6-(2-((Z)-5-((Z)-苄叉)-3, 6-二酮哌嗪-2-苄叉)苯酚基)四氢-2H-吡喃-3, 4, 5-三醋酸酯(1a)的合成

采用通用合成方法合成, 加入的芳香醛为苯甲醛(21μL), 收率为22%。熔点: 211~214℃; [ α ] D 2 5-58.7 (c 0.1, DMSO); 1H NMR (500MHz, CDCl3) δ 8.23 (s, 1H), 8.16 (s, 1H), 7.46 (t, J = 7.5 Hz, 2H), 7.41 (d, J = 7.3 Hz, 1H), 7.39~7.30 (m, 4H), 7.22 (d, J = 8.2 Hz, 1H), 7.17 (t, J = 7.5 Hz, 1H), 7.04 (s, 1H), 6.87 (s, 1H), 5.33~5.23 (m, 2H), 5.17 (t, J = 9.3 Hz, 1H), 5.02 (d, J = 7.3 Hz, 1H), 4.30 (dd, J = 12.3, 5.3 Hz, 1H), 4.23 (dd, J = 12.3, 2.3 Hz, 1H), 3.88 (ddd, J = 9.8, 5.2, 2.4 Hz, 1H), 2.11 (s, 3H), 2.04 (s, 3H), 2.01 (s, 3H), 2.00 (s, 3H). 13C NMR (126MHz, CDCl3) δ 170.6, 170.2, 169.3, 169.2, 156.9, 154.0, 132.9, 130.5, 130.4, 129.4, 128.8, 128.5, 126.8, 125.8, 124.2, 123.3, 116.9, 116.5, 111.6, 100.1, 72.3, 72.2, 70.5, 68.2, 61.8, 20.7, 20.5, 20.4; FT-IR (neat): 3186, 2916, 1747, 1680, 1627, 1491, 1389, 1356, 1250, 1037, 752, 690cm-1; HRMS (ESI) m/z: [M+H]+ Calcd for C32H33N2O12 637.2028; found 637.2044.

1.2.3.2 (2R, 3R, 4S, 5R, 6S)-2-(乙酰氧甲基)-6-(2-((Z)-5-((Z)-2, 3-二氯苄叉)-3, 6-二酮哌嗪-2-苄叉)苯酚基)四氢-2H-吡喃-3, 4, 5-三醋酸酯(1b)的合成

采用通用合成方法合成, 加入的芳香醛为2, 3-二氯苯甲醛(37mg), 收率为44%。熔点: > 250℃; [ α ] D 2 5-50.3 (c 0.1, DMSO); 1H NMR (500MHz, CDCl3) δ 8.29 (s, 1H), 8.24 (s, 1H), 7.49~7.45 (m, 1H), 7.34 (dd, J = 10.5, 4.5 Hz, 2H), 7.30 (t, J = 7.6 Hz, 2H), 7.21 (d, J = 8.2 Hz, 1H), 7.16 (dd, J = 10.0, 4.5 Hz, 2H), 7.04 (s, 1H), 6.85 (s, 1H), 5.33~5.23 (m, 2H), 5.16 (t, J = 9.5 Hz, 1H), 5.03 (d, J = 7.3 Hz, 1H), 4.29 (dd, J = 12.2, 5.2 Hz, 1H), 4.22 (dd, J = 12.4, 2.3 Hz, 1H), 3.88 (ddd, J = 9.7, 5.2, 2.4 Hz, 1H), 2.09 (s, 3H), 2.03 (s, 3H), 1.99 (s, 3H), 1.99 (s, 3H). 13C NMR (126MHz, CDCl3) δ 170.6, 170.2, 169.3, 169.1, 157.0, 156.3, 154.0, 134.4, 133.5, 132.7, 130.5, 130.5, 127.8, 127.6, 127.5, 126.5, 124.2, 123.0, 116.7, 113.0, 112.3, 99.9, 72.3, 72.2, 70.5, 68.2, 61.8, 20.6, 20.5, 20.4; FT-IR (neat): 3021, 3057, 2957, 2926, 1747, 1686, 1647, 1491, 1389, 1346, 1226, 1041, 754, 704cm-1; HRMS (ESI) m/z: [M+H]+ Calcd for C32H31Cl2N2O12 705.1249; found 705.1256.

1.2.3.3 (2R, 3R, 4S, 5R, 6S)-2-(乙酰氧甲基)-6-(2-((Z)-5-((Z)-1-萘叉)-3, 6-二酮哌嗪-2-苄叉)苯酚基)四氢-2H-吡喃-3, 4, 5-三醋酸酯(1c)的合成

采用通用合成方法合成, 加入的芳香醛为1-萘醛(28μL), 收率为42%。熔点: > 250℃; [ α ] D 2 5-47.9 (c 0.1, DMSO); 1H NMR (500MHz, CDCl3) δ 8.15 (s, 1H), 8.06~8.00 (m, 1H), 7.95 (s, 1H), 7.90 (dd, J = 7.2, 3.3 Hz, 2H), 7.60~7.49 (m, 5H), 7.37 (t, J = 7.8 Hz, 1H), 7.33 (d, J = 7.6 Hz, 1H), 7.25 (d, J = 9.6 Hz, 1H), 7.19 (t, J = 7.5 Hz, 1H), 6.87 (s, 1H), 5.34 (t, J = 8.6 Hz, 1H), 5.28 (t, J = 9.3 Hz, 1H), 5.20 (t, J = 9.5 Hz, 1H), 5.02 (t, J = 10.1 Hz, 1H), 4.32 (dd, J = 11.8, 4.8 Hz, 1H), 4.25 (d, J = 12.2 Hz, 1H), 3.95~3.86 (m, 1H), 2.12 (s, 3H), 2.05 (s, 3H), 2.01 (s, 6H). 13C NMR (126MHz, CDCl3) δ 170.6, 170.2, 169.3, 169.2, 156.7, 156.7, 154.0, 133.9, 131.4, 130.5, 130.5, 129.7, 129.5, 128.7, 127.4, 127.0, 126.9, 126.7, 126.1, 125.5, 124.7, 124.3, 123.3, 117.0, 114.8, 111.6, 100.2, 72.3, 72.3, 70.5, 68.2, 61.9, 20.7, 20.6, 20.4-; FT-IR (neat): 3186, 3051, 3916, 2945, 2878, 1751, 1683, 1636, 1491, 1398, 1228, 1041, 773cm-1; HRMS (ESI) m/z: [M+H]+ Calcd for C36H35N2O12 687.2185; found 687.2192.

1.2.3.4 (2R, 3R, 4S, 5R, 6S)-2-(乙酰氧甲基)-6-(2-((Z)-5-((Z)-3-甲氧基苄叉)-3, 6-二酮哌嗪-2-苄叉)苯酚基)四氢-2H-吡喃-3, 4, 5-三醋酸酯(1d)的合成

采用通用合成方法合成, 加入的芳香醛为3-甲氧基苯甲醛(26μL), 收率为59%。熔点: 188~191℃; [ α ] D 2 5-44.0 (c 0.02, DMSO); 1H NMR (500MHz, CDCl3) δ 8.19 (s, 1H), 8.08 (s, 1H), 7.37 (d, J = 7.8 Hz, 2H), 7.35~7.30 (m, 1H), 7.23 (d, J = 8.2 Hz, 1H), 7.18 (t, J = 7.7 Hz, 1H), 7.00 (d, J = 7.1 Hz, 2H), 6.91 (d, J = 6.4 Hz, 2H), 6.87 (s, 1H), 5.33~5.24 (m, 2H), 5.18 (t, J = 9.4 Hz, 1H), 5.02 (d, J = 7.4 Hz, 1H), 4.30 (dd, J = 12.3, 5.3 Hz, 1H), 4.24 (dd, J = 12.2, 2.3 Hz, 1H), 3.92~3.86 (m, 1H), 3.84 (s, 3H), 2.12 (s, 3H), 2.04 (s, 3H), 2.02 (s, 3H), 2.00 (s, 3H). 13C NMR (126MHz, CDCl3) δ 170.6, 170.2, 169.3, 169.2, 160.3, 156.8, 154.0, 134.2, 130.6, 130.5, 130.5, 126.8, 126.1, 124.3, 123.3, 120.4, 116.9, 116.3, 114.3, 114.2, 111.5, 100.1, 100.0, 72.3, 72.3, 70.5, 68.2, 61.9, 55.3, 20.7, 20.6, 20.4; FT-IR (neat): 3196, 3061, 2957, 2926, 1746, 1682, 1627, 1491, 1377, 1213, 1035, 748, 688cm-1; HRMS (ESI) m/z: [M+H]+ Calcd for C33H35N2O13 667.2134; found 667.2142.

1.2.3.5 (2R, 3R, 4S, 5R, 6S)-2-(乙酰氧甲基)-6-(2-((Z)-5-((Z)-4-甲氧基苄叉)-3, 6-二酮哌嗪-2-苄叉)苯酚基)四氢-2H-吡喃-3, 4, 5-三醋酸酯(1e)的合成

采用通用合成方法合成, 加入的芳香醛为4-甲氧基苯甲醛(26μL), 收率为77%。熔点: 178~181℃; [ α ] D 2 5-64.5 (c 0.1, DMSO); 1H NMR (500MHz, CDCl3) δ 8.15 (s, 1H), 8.07 (s, 1H), 7.36 (t, J = 7.7 Hz, 3H), 7.32 (d, J = 8.0 Hz, 1H), 7.23 (d, J = 8.2 Hz, 1H), 7.17 (t, J = 7.4 Hz, 1H), 6.99 (d, J = 4.8 Hz, 2H), 6.97 (s, 1H), 6.86 (s, 1H), 5.29 (dt, J = 18.7, 9.5 Hz, 2H), 5.18 (t, J = 9.4 Hz, 1H), 5.02 (d, J = 7.4 Hz, 1H), 4.30 (dd, J = 12.3, 5.3 Hz, 1H), 4.24 (dd, J = 12.3, 2.3 Hz, 1H), 3.92~3.86 (m, 1H), 3.85 (s, 3H), 2.12 (s, 3H), 2.04 (s, 3H), 2.02 (s, 3H), 2.00 (s, 3H). 13C NMR (126MHz, CDCl3) δ 170.6, 170.2, 169.3, 169.2, 159.9, 157.1, 156.9, 154.0, 130.4, 130.4, 130.1, 127.0, 124.5, 124.3, 123.4, 117.0, 116.6, 114.9, 111.2, 100.1, 72.4, 72.3, 70.6, 68.2, 61.9, 55.4, 20.7, 20.6, 20.4; FT-IR (neat): 3232, 3070, 2958, 2930, 1747, 1682, 1643, 1602, 1491, 1377, 1219, 1034, 760, 743cm-1; HRMS (ESI) m/z: [M+H]+ Calcd for C33H35N2O13 667.2134; found 667.2134.

1.2.3.6 (2R, 3R, 4S, 5R, 6S)-2-(乙酰氧甲基)-6-(2-((Z)-5-((Z)-4-羟基苄叉)-3, 6-二酮哌嗪-2-苄叉)苯酚基)四氢-2H-吡喃-3, 4, 5-三醋酸酯(1f)的合成

采用通用合成方法合成, 加入的芳香醛为4-羟基苯甲醛(21μL), 收率为33%。熔点: 224~226℃; [ α ] D 2 5-23.7 (c 0.06, DMSO); 1H NMR (500MHz, CDCl3) δ 8.33 (s, 1H), 8.15 (s, 1H), 7.34 (dd, J = 15.4, 7.3 Hz, 2H), 7.27 (d, J = 8.7 Hz, 2H), 7.21 (d, J = 8.3 Hz, 1H), 7.17 (t, J = 7.6 Hz, 1H), 7.11 (s, 1H), 6.97 (s, 1H), 6.90 (d, J = 8.7 Hz, 2H), 6.87 (s, 1H), 5.34~5.24 (m, 2H), 5.17 (t, J = 9.3 Hz, 1H), 5.04 (d, J = 7.4 Hz, 1H), 4.30 (dd, J = 12.3, 5.3 Hz, 1H), 4.23 (dd, J = 12.3, 2.3 Hz, 1H), 3.89 (ddd, J = 9.8, 5.3, 2.4 Hz, 1H), 2.10 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H), 1.99 (s, 3H). 13C NMR (126MHz, CDCl3) δ 170.7, 170.3, 169.5, 169.4, 157.3, 157.2, 157.0, 153.9, 130.5, 130.4, 130.3, 126.9, 124.6, 124.2, 124.0, 123.3, 117.4, 116.7, 116.5, 111.4, 99.9, 72.3, 72.2, 70.6, 68.3, 61.9, 20.7, 20.6, 20.4; FT-IR (neat): 3178, 2956, 2926, 1746, 1682, 1625, 1490, 1387, 1352, 1221, 1032, 742, 682cm-1; HRMS (ESI) m/z: [M+H]+ Calcd for C32H33N2O13 653.1991; found 653.1997.

1.2.3.7 (2R, 3R, 4S, 5R, 6S)-2-(乙酰氧甲基)-6-(2-((Z)-5-((Z)-2-氟苄叉)-3, 6-二酮哌嗪-2-苄叉)苯酚基)四氢-2H-吡喃-3, 4, 5-三醋酸酯(1g)的合成

采用通用合成方法合成, 加入的芳香醛为2-氟苯甲醛(26μL), 收率为60%。熔点: 205~208℃; [ α ] D 2 5-65.9 (c 0.1, DMSO); 1H NMR (500MHz, CDCl3) δ 8.13 (s, 1H), 8.07 (s, 1H), 7.42 (t, J = 7.4 Hz, 1H), 7.40~7.34 (m, 2H), 7.32 (d, J = 7.0 Hz, 1H), 7.24 (dd, J = 7.6, 5.7 Hz, 2H), 7.17 (dd, J = 12.7, 5.6 Hz, 2H), 7.00 (s, 1H), 6.89 (s, 1H), 5.33~5.24 (m, 2H), 5.18 (t, J = 9.4 Hz, 1H), 5.03 (d, J = 7.5 Hz, 1H), 4.30 (dd, J = 12.3, 5.3 Hz, 1H), 4.24 (dd, J = 12.3, 2.4 Hz, 1H), 3.88 (ddd, J = 9.9, 5.2, 2.5 Hz, 1H), 2.12 (s, 4H), 2.04 (s, 4H), 2.02 (s, 3H), 2.00 (s, 3H). 13C NMR (126MHz, CDCl3) δ 170.6, 170.2, 169.3, 169.2, 159.8 (d, J = 249.6 Hz), 156.7, 156.5, 154.0, 130.8 (d, JC-F = 8.4 Hz), 130.5, 130.2 (d, JC-F = 3.3 Hz), 127.0, 126.8, 124.9 (d, JC-F = 3.2 Hz), 124.3, 123.2, 120.6 (d, JC-F = 15.0 Hz), 117.0, 116.6 (d, JC-F = 22.0 Hz), 111.9, 109.7, 100.1, 72.3, 72.3, 70.5, 68.2, 61.8, 20.7, 20.6, 20.4; FT-IR (neat): 3192, 3045, 2877, 1746, 1682, 1633, 1489, 1400, 1217, 1036, 750, 598cm-1; HRMS (ESI) m/z: [M+H]+ Calcd for C32H32FN2O12 655.1934; found 655.1943.

1.2.3.8 (2R, 3R, 4S, 5R, 6S)-2-(乙酰氧甲基)-6-(2-((Z)-5-((Z)- 4-三氟甲基苄叉)-3, 6-二酮哌嗪-2-苄叉)苯酚基)四氢-2H-吡喃-3, 4, 5-三醋酸酯(1h)的合成

采用通用合成方法合成, 加入的芳香醛为4-三氟甲基苯甲醛(29μL), 收率为93%。熔点: 218~221℃; [ α ] D 2 5-53.0 (c 0.03, DMSO); 1H NMR (500MHz, CDCl3) δ 8.28 (s, 1H), 8.22 (s, 1H), 7.70 (d, J = 8.1 Hz, 2H), 7.54 (d, J = 8.1 Hz, 2H), 7.39~7.33 (m, 1H), 7.31 (d, J = 7.5 Hz, 1H), 7.22 (d, J = 8.2 Hz, 1H), 7.17 (t, J = 7.5 Hz, 1H), 7.01 (s, 1H), 6.85 (s, 1H), 5.31~5.24 (m, 2H), 5.20~5.12 (m, 1H), 5.02 (dd, J = 4.6, 2.9 Hz, 1H), 4.30 (dd, J = 12.3, 5.3 Hz, 1H), 4.23 (dd, J = 12.3, 2.4 Hz, 1H), 3.89 (ddd, J = 9.9, 5.3, 2.5 Hz, 1H), 2.10 (s, 3H), 2.03 (s, 3H), 1.99 (s, 3H), 1.99 (s, 3H). 13C NMR (126MHz, CDCl3) δ 170.6, 170.1, 169.3, 169.2, 157.0, 156.4, 153.9, 136.6, 130.6 (d, JC-F = 15.5 Hz), 130.1 (q, JC-F = 33.2 Hz), 128.8, 127.3, 126.6, 126.3 (q, JC-F = 3.7 Hz), 125.9 (q, JC-F = 273.4 Hz), 124.8, 124.2, 123.0, 122.6, 116.7, 114.2, 112.2, 100.0, 72.3, 72.2, 70.5, 68.2, 61.8, 20.7, 20.5, 20.4; FT-IR (neat): 3232, 3072, 2959, 1755, 1686, 1643, 1491, 1379, 1325, 1227, 1124, 1067, 1041, 762cm-1; HRMS (ESI) m/z: [M+H]+ Calcd for C33H32F3N2O12 705.1902; found 705.1903.

1.2.3.9 (2R, 3R, 4S, 5R, 6S)-2-(乙酰氧甲基)-6-(2-((Z)-5-((Z)-6-溴-2-吡啶苄叉)-3, 6-二酮哌嗪-2-苄叉)苯酚基)四氢-2H-吡喃-3, 4, 5-三醋酸酯(1i)的合成

采用通用合成方法合成, 加入的芳香醛为6-溴吡啶-2-甲醛(39mg), 收率为58%。熔点: 116~119℃; [ α ] D 2 5-35.2 (c 0.04, DMSO); 1H NMR (500MHz, CDCl3) δ 11.87 (s, 1H), 8.16 (s, 1H), 7.57 (t, J = 7.8 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.35~7.31 (m, 2H), 7.29 (d, J = 7.7 Hz, 1H), 7.26 (s, 1H), 7.22 (d, J = 8.3 Hz, 1H), 7.16 (t, J = 7.6 Hz, 1H), 6.93 (s, 1H), 6.71 (s, 1H), 5.32~5.22 (m, 3H), 5.16 (dd, J = 11.6, 6.4 Hz, 1H), 5.01 (d, J = 7.3 Hz, 1H), 4.30 (dd, J = 12.3, 5.3 Hz, 1H), 4.23 (d, J = 12.1 Hz, 1H), 3.88 (ddd, J = 9.8, 5.2, 2.4 Hz, 1H), 2.11 (s, 2H), 2.02 (s, 3H), 2.01 (s, 3H), 1.97 (s, 3H). 13C NMR (126MHz, CDCl3) δ 170.6, 170.1, 169.3, 169.3, 156.4, 156.4, 155.6, 153.8, 140.6, 139.1, 131.3, 130.9, 130.4, 126.8, 126.4, 124.5, 124.2, 123.2, 116.8, 112.1, 107.7, 100.2, 72.2, 70.4, 68.2, 61.8, 20.7, 20.5, 20.5, 20.4; FT-IR (neat): 3285, 3120, 2960, 2939, 1755, 1693, 1645, 1578, 1454, 1367, 1344, 1225, 1122, 1040, 1041, 734cm-1; HRMS (ESI) m/z: [M+H]+ Calcd for C31H31BrN3O12 716.1086; found 716.1094, 718.1083.

1.2.4 目标产物2的合成通用方法

将目标产物1(0.1mmol, 1.0equiv)溶解在甲醇(1mL)中, 然后加入氢氧化锂 (LiOH)(34mg, 0.8mmol, 8.0equiv), 并置于室温搅拌, 待反应完成后将反应液缓慢加入到冰水混合物中, 10%稀盐酸溶液调节pH至3~4。所得固体抽滤, 水洗, 真空干燥得到目标化合物2

1.2.4.1 (Z)-3-(苄叉基)-6-((Z)-2-(((2S, 3R, 4S, 5S, 6R)-3, 4, 5-三羟基-6-(羟甲基)四氢-2H-吡喃-2-基)氧)苄叉)哌嗪-2, 5-二酮(2a)的合成

采用通用合成方法合成, 加入1a的质量为63.6mg, 收率为56%。熔点: 214~217℃; [ α ] D 2 5-46.7 (c 0.1, DMSO); 1H NMR (500MHz, DMSO) δ 10.25 (s, 1H), 9.86 (s, 1H), 7.55 (d, J = 7.5 Hz, 2H), 7.48 (d, J = 7.4 Hz, 1H), 7.43 (t, J = 7.5 Hz, 2H), 7.33 (t, J = 7.3 Hz, 2H), 7.24 (d, J = 8.5 Hz, 1H), 7.07 (t, J = 7.3 Hz, 1H), 6.90 (s, 1H), 6.78 (s, 1H), 5.39 (d, J = 4.2 Hz, 1H), 5.12 (d, J = 6.0 Hz, 2H), 5.08 (d, J = 5.3 Hz, 1H), 4.56 (t, J = 5.7 Hz, 1H), 3.70 (dd, J = 10.8, 4.8 Hz, 1H), 3.49 (dt, J = 11.7, 6.0 Hz, 2H), 3.31 (s, 3H), 3.21 (d, J = 5.1 Hz, 1H). 13C NMR (126MHz, DMSO) δ 157.8, 157.6, 154.4, 133.1, 130.7, 130.2, 129.9, 129.4, 128.8, 128.2, 127.6, 126.4, 126.3, 122.6, 122.1, 114.9, 111.5, 99.8, 77.3, 76.7, 73.1, 69.6, 60.7-; FT-IR (neat): 3302, 2876, 1666, 1622, 1489, 1352, 1244, 1072, 1032, 939, 746, 687cm-1; HRMS (ESI) m/z: [M+H]+ Calcd for C24H25N2O8 439.1605; found 469.1598.

1.2.4.2 (Z)-3-(萘叉基)-6-((Z)-2-(((2S, 3R, 4S, 5S, 6R)-3, 4, 5-三羟基-6-(羟甲基)四氢-2H-吡喃-2-基)氧)苄叉)哌嗪-2, 5-二酮(2b)的合成

采用通用合成方法合成, 加入1b的质量为69mg, 收率为39%。熔点: > 250℃; [ α ] D 2 5-38.8 (c 0.05, DMSO); 1H NMR (500MHz, DMSO) δ 10.22 (s, 1H), 9.95 (s, 1H), 8.03~7.97 (m, 1H), 7.94 (d, J = 8.3 Hz, 2H), 7.66 (d, J = 7.0 Hz, 1H), 7.63~7.55 (m, 3H), 7.51 (d, J = 7.8 Hz, 1H), 7.36 (t, J = 7.5 Hz, 1H), 7.27 (d, J = 8.5 Hz, 1H), 7.24 (s, 1H), 7.10 (t, J = 7.4 Hz, 1H), 6.91 (s, 1H), 5.41 (d, J = 4.7 Hz, 1H), 5.14 (t, J = 5.7 Hz, 2H), 5.10 (d, J = 5.2 Hz, 1H), 4.58 (t, J = 5.7 Hz, 1H), 3.76~3.68 (m, 1H), 3.51 (dt, J = 11.7, 5.9 Hz, 1H), 3.45~3.36 (m, 3H), 3.27~3.20 (m, 1H). 13C NMR (126MHz, DMSO) δ 157.5, 157.2, 154.4, 133.4, 131.3, 130.8, 130.0, 129.9, 128.6, 128.5, 128.4, 127.2, 126.6, 126.3, 126.2, 125.8, 124.3, 122.6, 122.1, 114.8, 112.3, 111.5, 99.8, 99.6, 77.3, 76.7, 73.1, 69.6, 60.7; FT-IR (neat): 3391, 3196, 2922, 2853, 1670, 1635, 1541, 1394, 1246, 1076, 1043, 759, 669cm-1; HRMS (ESI) m/z: [M+Na]+ Calcd for C28H26N2NaO8 541.1581; found 541.1585.

1.2.4.3 3-((Z)-3-甲氧基苄叉)-6-((Z)-2-(((2S, 3R, 4S, 5S, 6R)-3, 4, 5-三羟基-6-(羟甲基)四氢-2H-吡喃-2-基)氧)苄叉)哌嗪-2, 5-二酮(2c)的合成

采用通用合成方法合成, 加入1c的量为67mg, 收率为25%。熔点: 217~220℃; [ α ] D 2 5-141.1 (c 0.1, DMSO); 1H NMR (500MHz, DMSO) δ 10.26 (s, 1H), 9.86 (s, 1H), 7.54~7.42 (m, 1H), 7.34 (t, J = 7.9 Hz, 2H), 7.26~7.21 (m, 1H), 7.13~7.03 (m, 3H), 6.93~6.89 (m, 2H), 6.75 (s, 1H), 5.38 (d, J = 4.5 Hz, 1H), 5.14 (s, 1H), 5.10 (dd, J = 10.7, 6.3 Hz, 2H), 4.56 (t, J = 5.7 Hz, 1H), 3.80 (s, 3H), 3.69 (dd, J = 11.1, 4.4 Hz, 1H), 3.53~3.44 (m, 2H), 3.31 (m, 2H), 3.25~3.16 (m, 1H). 13C NMR (126MHz, DMSO) δ 159.3, 157.8, 157.5, 154.4, 134.4, 130.7, 129.9, 129.8, 126.6, 126.3, 122.6, 122.0, 121.7, 114.8, 114.8, 114.6, 114.1, 111.5, 99.8, 77.3, 76.6, 73.1, 69.5, 60.7, 55.1; FT-IR (neat): 3366, 3182, 2926, 2851, 1670, 1633, 1490, 1402, 1357, 1244, 1078, 1045, 763, 687cm-1; HRMS (ESI) m/z: [M+H]+ Calcd for C25H27N2O9 499.1711; found 499.1718.

1.2.4.4 3-((Z)-4-甲氧基苄叉)-6-((Z)-2-(((2S, 3R, 4S, 5S, 6R)-3, 4, 5-三羟基-6-(羟甲基)四氢-2H-吡喃-2-基)氧)苄叉)哌嗪-2, 5-二酮(2d)的合成

采用通用合成方法合成, 加入1d的量为60mg, 收率为21%。熔点: > 250℃; [ α ] D 2 5-125.5 (c 0.1, DMSO); 1H NMR (500MHz, DMSO) δ 10.18 (s, 1H), 9.78 (s, 1H), 7.52 (d, J = 8.7 Hz, 2H), 7.48 (d, J = 7.4 Hz, 1H), 7.33 (t, J = 7.8 Hz, 1H), 7.24 (d, J = 8.3 Hz, 1H), 7.07 (t, J = 7.4 Hz, 1H), 7.00 (d, J = 8.7 Hz, 2H), 6.88 (s, 1H), 6.75 (s, 1H), 5.38 (d, J = 4.8 Hz, 1H), 5.12 (d, J = 3.7 Hz, 2H), 5.07 (d, J = 5.2 Hz, 1H), 4.55 (t, J = 5.8 Hz, 1H), 3.80 (s, 3H), 3.69 (dd, J = 10.4, 5.0 Hz, 1H), 3.49 (dt, J = 11.9, 6.1 Hz, 1H), 3.42~3.35 (m, 1H), 3.29 (m, 2H), 3.22 (dd, J = 9.0, 5.3 Hz, 1H). 13C NMR (126MHz, DMSO) δ 159.3, 157.8, 157.8, 154.3, 131.1, 130.7, 129.8, 126.4, 125.5, 124.7, 122.6, 122.0, 115.2, 114.8, 114.2, 111.1, 99.7, 77.9, 76.6, 73.1, 69.5, 60.7, 55.3; FT-IR (neat): 3391, 2939, 2833, 1668, 1624, 1508, 1396, 1250, 1078, 1035, 752, 667cm-1; HRMS (ESI) m/z: [M+H]+ Calcd for C25H27N2O9 499.1711; found 499.1712.

1.2.4.5 3-((Z)-6-溴2-吡啶苄叉基)-6-((Z)-2-(((2S, 3R, 4S, 5S, 6R)-3, 4, 5-三羟基-6-(羟甲基)四氢-2H-吡喃-2-基)氧)苄叉)哌嗪-2, 5-二酮(2e)的合成

采用通用合成方法合成, 加入1e的量为71.5mg, 收率为42%。熔点: > 250℃; [ α ] D 2 5-41.0 (c 0.01, DMSO); 1H NMR (500MHz, DMSO) δ 11.68 (s, 1H), 10.07 (s, 1H), 7.86 (t, J = 7.8 Hz, 1H), 7.73 (d, J = 7.6 Hz, 1H), 7.63 (d, J = 7.9 Hz, 1H), 7.50 (d, J = 7.4 Hz, 1H), 7.36 (dd, J = 11.5, 4.2 Hz, 1H), 7.26 (d, J = 8.3 Hz, 1H), 7.09 (t, J = 7.5 Hz, 1H), 6.98 (s, 1H), 6.70 (s, 1H), 5.39 (d, J = 4.9 Hz, 1H), 5.14 (d, J = 4.4 Hz, 1H), 5.10 (d, J = 7.2 Hz, 1H), 5.08 (d, J = 5.3 Hz, 1H), 4.56 (t, J = 5.8 Hz, 1H), 3.71 (dd, J = 10.2, 5.2 Hz, 1H), 3.50 (dt, J = 11.9, 6.1 Hz, 1H), 3.39 (dd, J = 7.7, 6.0 Hz, 1H), 3.33~3.27 (m, 2H), 3.25~3.15 (m, 1H). 13C NMR (126MHz, DMSO) δ 156.7, 156.1, 155.7, 154.5, 140.7, 139.6, 131.8, 130.9, 130.2, 126.4, 125.7, 125.4, 122.3, 122.1, 114.9, 112.8, 105.9, 99.9, 77.3, 76.6, 73.1, 69.5, 60.7; FT-IR (neat): 3398, 3296, 2908, 2866, 1688, 1653, 1460, 1404, 1359, 1230, 1119, 1080, 1018, 950, 765cm-1; HRMS (ESI) m/z: [M+Na]+ Calcd for C23H22BrN3NaO8 570.0482; found 570.0486, 572.0466.

1.2.5 化合物溶解度的测定

吡啶标准溶液的配置: 称量30.0mg吡啶溶解于70μL DMSO中, 然后用甲醇稀释至4.52×10-3μmol·μL-1。样品饱和溶液的配置: 室温(25℃)条件下, 将衍生物溶解于DMSO或磷酸钾缓冲溶液中(pH=7.4)形成饱和溶液, 室温储存备用。
溶解度的测定: 在20μL的离心管中, 依次加入5.0μL MeOH, 3.0μL吡啶标准溶液及1.0uL的饱和样品溶液, 并混合均匀。从混合后的溶液中取2.5uL采用高效液相进行分析, 然后根据吡啶与样品的峰面积比, 求出样品的溶解度(Mota et al, 2009)。

1.2.6 化合物细胞毒活性测定

癌细胞株K562, A549与Huh-7购于中国科学院上海癌细胞库。细胞以400~800个/孔的密度置于384孔的培养皿中, 在含有10%胎牛血清(FBS)与1%青霉素-链霉素双抗(penicillin/streptomycin)的Roswell park memorial institute或Dulbeccos modified eagle medium培养基, 置于培养箱(5%CO2, 37℃)中进行培养。癌细胞分别用不同浓度(30.0、10.0、3.33、1.11、0.37、0.12、0.04、0.01、0.004、0.001μmol·L-1)的待测化合物、紫杉醇(阳性对照)或Dulbeccos磷酸盐缓冲液(DPBS溶液, 空白对照)处理72h, 每个处理三个重复。细胞成活率采用cell counting kit-8 (DOjinDo, Japan)方法测定, 溶液吸光度(波长为450nm)用紫外-可见光谱仪envision 2104 multi-label reader (Perkin elmer, USA)进行测定。利用Prism 5.0 (GraphPad software Inc., USA)软件对数据进行处理并计算化合物的IC50值(Liao et al, 2014)。

2 结果与讨论

2.1 化合物的合成

目标化合物1采用三步合成(图5): 首先将装配了四乙酰基葡萄糖的水杨醛中间体I与2, 5-二酮哌嗪在Cs2CO3存在的条件下, 室温反应得到中间体II。随后该中间体与另一分子苯甲醛同系物在Cs2CO3存在的条件下, 加热(80℃)反应得到目标物1。目标物2的合成利用目标物1在LiOH存在的条件下, 于甲醇中室温水解制备。需要注意的是在目标物2的后处理过程中, 最好将反应液缓慢滴入到冰水混合物中, 放置至产物形成良好的固体颗粒, 利于抽滤, 并防止产物稀键构型的改变而出现异构体。然后将固体抽滤, 真空干燥, 最终得到目标物2
图5 衍生物12的合成

Fig. 5 The synthesis of derivatives 1

2.2 化合物的溶解度

将化合物于室温条件下, 分别溶解于DMSO或缓冲溶液中, 然后以吡啶为对照样, 采用高效液相分析方法对衍生物的溶解度进行测定, 结果如表1所示。从表中的数据可以看出, 化合物在DMSO中均具有良好的溶解性(1.5~27.9mg·mL-1), 但溶解度2稍低于1。在缓冲溶液中, 化合物12的溶解性均低于在DMSO中的溶解度, 但均明显高于Pierafizine B的溶解度。这些结果说明通过糖基修饰, 衍生物的溶解性得到了较大改善。同时, 从脂溶性结果来分析, 葡萄糖上四个羟基均被乙酰化后, 乙酰糖基部分的体积足够大, 有效阻止了分子靠近形成牢固氢键而聚合, 脂溶性提高。脱除了乙酰基的葡萄糖基团体积可能还不够大, 虽然未能提供有效空间位阻, 但可能减弱了分子间氢键的键能, 使其结构不稳定, 聚合物易破裂。因此相对于Piperafizine B, 衍生物2的溶解度也相应增加, 从而溶解性得到改善。
表1 化合物的溶解度

Tab. 1 The solubilities of the synthesized derivatives

化合物 溶解度/(mg·mL-1)
CDMSO C缓冲液
1a 21.5 0.9
1b 11.8 0.4
1c 13.9 0.2
1d 17.0 0.9
1e 12.2 0.5
1f 18.0 0.6
1g 21.2 0.8
1h 17.6 0.5
1i 27.9 0.2
2a 18.3 0.6
2b 12.6 0.1
2c 5.70 0.3
2d 7.20 0.8
2e 1.50 0.2
Piperafizine B 0.10 0.05

2.3 化合物的细胞毒活性

化合物的细胞毒活性如表2所示, 化合物系列1中, 1a1c对三种癌细胞株(K562、A549与Huh-7)均具有较好的活性, 其中化合物1c对三种癌细胞株的抑制活性最强(分别为1、3.3、3.6μmol·mL-1)。其它化合物如1f(IC50=4.2μmol·mL-1, Huh-7), 1g(IC50= 2.9μmol·mL-1, A549; IC50=3.6μmol·mL-1, Huh-7), 1i (IC50=9.1μmol·mL-1, K562; IC50 =4.5μmol·mL-1, Huh-7)均显示出一定的细胞毒活性。综合分析这些结果表明, 半数化合物对Huh-7细胞具有生长抑制活性, 并对该癌细胞的选择性略好于K562与A549。从2, 5-二酮哌嗪环3-位取代芳香基团来分析, 只有含萘基的衍生物(1c)仍具有好的活性, 与前期的研究一致, 苯基取代的衍生物(1a)活性也得到保留, 但含有其它基团衍生物如含2, 3-二氯苯基衍生物(1b), 3-甲氧基苯基衍生物(1d)等无明显活性, 说明萘结构可能更适合后续结构修饰。另外, 如含4-甲氧基苯基衍生物(1e), 4-羟基苯基衍生物(1f), 4-三氟甲基苯基衍生物(1h)也无明显活性。然而让人意外的是, 脱乙酰基的化合物2a-e与Piperafizine B一样, 均无明显活性。这些结果说明, 尽管衍生物的溶解度得到了改善, 但因细胞毒活性的强弱受取代基团的种类、电性、体积大小及取代位置等多种因素的影响, 导致其活性强弱与溶解度高低规律的不一致性。
表2 化合物的细胞毒活性

Tab. 2 The cytotoxicities of the synthesized derivatives

化合物 IC50/(μmol·mL-1)
K562 A549 Huh-7
1a 4.5±0.8 5.2±0.6 3.9±0.9
1b >10 >10 >10
1c 1.0±0.08 3.6±0.2 3.3±0.4
1d >10 >10 >10
1e >10 >10 >10
1f >10 >10 4.2±0.6
1g >10 2.9±0.2 3.6±0.6
1h >10 >10 >10
1i 9.1±1.0 >10 4.5±0.5
2a-e >10 >10 >10
Piperafizine B >10 >10 >10
Taxol 0.003±0.0002 0.002±0.0008 0.007±0.0005

3 结论

以Piperafizine B 为衍生物骨架, 共合成了14个葡萄糖基修饰的衍生物。相较于Piperafizine B, 糖基修饰的衍生物的溶解度均增加。糖基上的乙酰基未水解的衍生物1的脂溶性普遍优于乙酰基水解后的衍生物2, 但两者在缓冲溶液中的溶性差别不大。化合物系列1的部分衍生物具有中等程度的细胞毒活性, 而衍生物系列2无明显活性。由于糖基修饰后衍生物的水溶性仍较低, 单糖修饰可能未能有效提供空间位阻, 同时衍生物的活性并未明显提高, 引入较大糖基团尽管有利于改善化合物的溶解度, 但其是否会对衍生物的细胞毒活性带来负面影响, 值得进一步研究。因此后续研究可以对Piperafizine B采用二糖甚至多糖的方式进行衍生, 以详细考察糖基如何影响衍生物的溶解度及其生物活性。
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