海洋科学  2019, Vol. 43 Issue (8): 43-49   PDF    
http://dx.doi.org/10.11759/hykx20190411002

文章信息

李冬艳, 王晶, 陈科良, 朱虎成, 陈春梅, 张锦文. 2019.
LI Dong-yan, WANG Jing, CHEN Ke-liang, ZHU Hu-cheng, CHEN Chun-mei, ZHANG Jin-wen. 2019.
来源于印度洋深海区的烟曲霉次生代谢产物的研究
Study of the secondary metabolites of Aspergillus fumigatus from the deepsea zone of the Indian Ocean
海洋科学, 43(8): 43-49
Marina Sciences, 43(8): 43-49.
http://dx.doi.org/10.11759/hykx20190411002

文章历史

收稿日期：2019-04-11
修回日期：2019-05-25
引用本文
李冬艳, 王晶, 陈科良, 朱虎成, 陈春梅, 张锦文. 2019. 来源于印度洋深海区的烟曲霉次生代谢产物的研究[J]. 海洋科学, 43(8): 43-49.
LI Dong-yan, WANG Jing, CHEN Ke-liang, ZHU Hu-cheng, CHEN Chun-mei, ZHANG Jin-wen. 2019. Study of the secondary metabolites of Aspergillus fumigatus from the deepsea zone of the Indian Ocean[J]. Marina Sciences, 43(8): 43-49.
来源于印度洋深海区的烟曲霉次生代谢产物的研究
李冬艳1, 王晶1, 陈科良2, 朱虎成2, 陈春梅2, 张锦文1     
1. 华中科技大学同济医学院附属同济医院药学部, 湖北 武汉 430030;
2. 华中科技大学同济医学院药学院, 湖北 武汉 430030
收稿日期：2019-04-11；修回日期：2019-05-25
基金项目：国家自然科学基金青年项目（31600266）
作者简介：李冬艳(1984-), 女, 讲师, 博士, 从事天然药物活性成分研究, 电话:027-83663558, E-mail:lidongyan2016@sina.com.
通信作者：张锦文, 通信作者, 电话:027-83663643, E-mail:tjzhangjinwen@163.com.
摘要：为了研究烟曲霉次生代谢产物，我们采用硅胶柱层析、sephadex LH-20凝胶柱层析、半制备液相色谱等分离方法对来源于印度洋深海区的烟曲霉（Aspergillus fumigatus）的乙醇提取液进行系统的次生代谢产物分离，根据所分离得到的化合物的理化性质和波谱数据确定化合物的结构。共分离鉴定了15个化合物，其中11个二酮哌嗪类生物碱（diketopiperazine），4个喹唑啉类衍生物（fumiquinazoline），分别为：fumitremorgin B（1），13-oxofumitremorgin B（2），fumitremorgin C（3），12-hydroxyfumitremorgin C（4），cyclotyprostatins B（5），cyclotyprostatins C（6），cyclotyprostatins E（7），verruculogen（8），spirotryprostatin C（9），spirotryprostatin D（10），spirotryprostatin F（11），fumiquinazoline C（12），fumiquinazoline D（13），3-hydroxyfumiquinazoline A（14）和fumiquinazoline E（15）。分离得到的15个化合物，丰富了烟曲霉次生代谢产物的类型，为烟曲霉的开发利用提供了化学研究基础。
关键词：烟曲霉(Aspergillusfumigatus)    曲霉属(Aspergillus)    次生代谢产物    化学成分    波谱数据    
Study of the secondary metabolites of Aspergillus fumigatus from the deepsea zone of the Indian Ocean
LI Dong-yan1, WANG Jing1, CHEN Ke-liang2, ZHU Hu-cheng2, CHEN Chun-mei2, ZHANG Jin-wen1     
1. Department of Pharmacy, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China;
2. Pharmaceutical college, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
Received: Apr. 11, 2019
Foundation: Project supported by the National Science Foundation for Young Sciencists of China, No. 31600266
Abstract: This study was conducted to analyze the chemical constituents of the secondary metabolites of Aspergillus fumigatus for which the chemical constituents of the ethanol extract of A. fumigatus from the deep sea zone of the Indian Ocean were separated systematically using silica gel column chromatography, Sephadex LH-20 column chromatography, and RP-HPLC. Physicochemical properties and spectral data were used to determine the structures of the isolated compounds. A total of 15 compounds were isolated that were identified as fumitremorgin B (1), 13-oxofumitremorgin B (2), fumitremorgin C (3), 12-hydroxyfumitremorgin C (4), cyclotyprostatins B (5), cyclotyprostatins C (6), cyclotyprostatins E (7), verruculogen (8), spirotryprostatin C (9), spirotryprostatin D (10), spirotryprostatin F (11), fumiquinazoline C (12), fumiquinazoline D (13), 3-hydroxyfumiquinazoline A (14), and fumiquinazoline E (15). These 15 compounds isolated from A. fumigatus enriched the type of the secondary metabolites of the fungus and provided a basis for exploring its development and conducting chemical research.
Key words: Aspergillus fumigatus    Aspergillus    the secondary metabolites    chemical constituents    spectral data    

烟曲霉(Aspergillus fumigatus)属于丝孢菌纲(Hyphomycetes)、丛梗孢目(Hyphomycetales)、曲霉属(Aspergillus)真菌[1], 为曲霉属中最常见的菌种之一。烟曲霉来源很广泛, 如药用植物, 植物共生菌和动物器官等, 其产生的某些代谢成分能够引起机体的严重损害, 导致侵袭性曲霉病感染的形成和扩散, 严重危及生命健康。比如, 烟曲霉的主要次生代谢产物烟曲霉毒素(Fumitremorgin)严重危害畜禽健康及食品安全, 是对亚洲地区家禽类和农作物类致病力最强、危害最大的霉菌毒素[2]。

不同于传统的陆生真菌, 海洋真菌因其特有的生存条件, 使得其具有特殊的代谢体系以及耐碱和耐盐的生理特性, 从而能够产生许多结构特异、药理作用优良的次生代谢产物[3]。自20世纪50年代Newton[2]等学者从海洋来源的头孢霉菌(Cephalosporium acremonium)的发酵物中发现抗菌化合物Cephalosporin C以来, 海洋微生物的抗菌活性研究日益成为新药研究领域的热门。已经进入治疗非小细胞肺癌II期临床实验阶段的Plinabulin (NPI-2358)[4], 就是从曲霉属海洋真菌Aspergillus sp. CNC-139中获得的次生代谢产物为模板合成的。鉴于海洋来源的真菌次生代谢产物丰富的化学结构和多样的生物活性, 我们对来源于海拔下2 434 m印度洋深海热液区深海沉积物的烟曲霉, 采用大米固体发酵, 对其发酵物的乙醇提取液进行了系统的次生代谢产物化学成分研究, 分离鉴定了11个二酮哌嗪类生物碱(diketopiperazine)和4个喹唑啉类衍生物(fumiquinazoline), 分别为: fumitremorgin B (1), 13-oxofumitremorgin B (2), fumitremorgin C (3), 12-hydroxyfumitremorgin C (4), cyclotyprostatins B (5), cyclotyprostatins C (6), cyclotyprostatins E (7), verruculogen (8), spirotryprostatin C (9), spirotryprostatin D (10), spirotryprostatin F (11), fumiquinazoline C (12), fumiquinazoline D (13), 3-hydroxyfumiquinazoline A (14)和fumiquinazoline E (15)(图 1)。化合物13和15为首次从该菌种发现, 丰富了烟曲霉次生代谢产物类型, 为烟曲霉的开发利用提供了化学基础。

图 1 烟曲霉分离纯化得到的化合物结构 Fig. 1 Chemical structures of compounds 1-15 isolated from Aspergillus fumigatus
图选项
1 仪器与试剂

旋光仪: Perkin-Elmer 341 polarimeter; 红外光谱仪: Bruker Vertex 70;紫外全波长扫描仪: Varian Cary 50;高分辨质谱仪: Thermo Fisher LC-LTQ- Orbitrap XL spectrometer; 圆二色光谱: JASCO-810 CD spectrometer; 核磁共振仪: Bruker AM-400;高效液相色谱仪:美国Dionex公司和美国Agilent公司; C18反相填料:德国Merck公司; 凝胶填料:瑞典GE Healthcare Bio-Sciences AB公司; 柱层析、薄层层析硅胶:青岛海洋化工厂。其他所用试剂均为分析纯或色谱纯。

2 菌株来源

烟曲霉菌株于2014年购买于中国海洋微生物菌种保藏管理中心(Marine Culture Collection of China, MCCC 3A00161), 来源于印度洋深海热液区深海沉积物, 海拔下2 434 m。菌株固体斜面样品保存于华中科技大学同济医学院药学院天然药物化学教研室, 由汪建平副教授通过提取基因组, 基因测序比对鉴定为烟曲霉。

3 菌株发酵及提取分离 3.1 菌株发酵培养

烟曲霉保藏菌株转移接种到PDA培养基上, 放置于28℃恒温箱。该菌生长较快, 约1周后观察菌落表面孢子呈墨绿色块状, 菌落背面呈黄褐色, 可见绒毛状分生孢子。之后将种子培养基在无菌条件下按照10%的接种量转接到灭过菌的混合培养基(0.5%蛋白胨混合固体大米)中, 1 L锥形瓶中含200 g大米和200 mL水, 123℃灭菌0.5 h, 搅拌均匀, 之后静置于28℃培养4周。

3.2 提取分离

室温下, 30 kg烟曲霉大米发酵物加95%乙醇浸泡10 h, 提取6次, 浸提液减压浓缩后得总浸膏(2 kg)。总浸膏用水混悬后, 加等体积的乙酸乙酯溶剂进行萃取, 共得到287 g乙酸乙酯部位萃取物。乙酸乙酯部位进行硅胶柱层析(80~100目), 二氯甲烷-甲醇(100︰0→0︰100)梯度洗脱, 用TLC检测并合并相同流分, 得到6个组分(A—F)。

组分B经Sephadex LH-20凝胶柱(二氯甲烷︰甲醇, 1︰1)和正相硅胶柱层析(石油醚︰丙酮, 15︰1)划段得到B1和B2两个主要部位; B2经高效液相色谱(乙腈/水40/60, 220 nm)得化合物12(28.23 mg)。B1经反相中压色谱柱(甲醇-水, 20︰80→100︰0)得到3个组分B1-1, B1-2和B1-3, B1-1和B1-2分别经半制备高效液相色谱柱纯化, 得到化合物1(24.12 mg)和8(5.78 mg)。

组分C先用反相中压色谱仪(MPLC)以MeOH- H2O系统梯度洗脱(30%甲醇→100%甲醇)得到4个极性部分C1—C4。C1再次经MPLC(40/60→80/20甲醇/水)得到C1-1→C1-44个组分, 其中C1-3经正相硅胶色谱及反复的HPLC纯化得到化合物3(16.67 mg), 11(6.54 mg), 13(26.45 mg), 14(6.99 mg)和15 (3.40 mg)。C4经Sephadex LH-20凝胶、MPLC、正相硅胶柱、半制备HPLC等色谱分离方法进行分离纯化, 最终得到化合物2(17.0 mg), 4(6.35 mg)和9(12.3 mg)。

组分E先经Sephadex LH-20凝胶柱(二氯甲烷︰甲醇1︰1)划段得到3个亚组分E1—E3。YQ3-3-1, YQ3-3-2和YQ3-3-3。E2经MPLC(甲醇/水)反复梯度洗脱、正相硅胶柱层析及半制备HPLC(乙腈/水85/15)纯化得单体化合物7(3.56 mg)和化合物10 (6.89 mg)。E3经正相硅胶柱层析(二氯甲烷︰甲醇100︰1→0︰100)划段, 然后经MPLC(MeOH/H2O 30︰70→100︰0甲醇)及半制备液相色谱纯化得到化合物5(10.90 mg)和化合物6(10.35 mg)。

4 结构鉴定

化合物1:白色无定型粉末, [α]D20–13.7 (c = 0.22, CHCl3); ESI-MS: m/z 502.2280 [M + Na]+, 分子式为C27H33N3O5; 1H-NMR (DMSO-d6, 400 MHz) δH : 5.87 (1H, d, J=9.97 Hz, H-3), 4.51 (1H, dd, J=4.63, 9.21 Hz, H-9), 5.52 (1H, d, J=1.95 Hz, H-13), 7.67 (1H, d, J=8.66 Hz, H-16), 6.66 (1H, dd, J=2.16, 8.67 Hz, H-17), 6.74 (1H, d, J=2.04 Hz, H-19), 4.48 (2H, d, J=6.1 Hz, H-21), 4.94 (1H, t, J=6.03 Hz, H-22), 1.84 (3H, s, H-24), 1.66 (3H, s, H-25), 4.73 (1H, d, J=10.0 Hz, H-26), 1.58 (3H, s, H-28), 1.91 (3H, s, H-29), 3.73 (3H, s, H-30). 13C-NMR (DMSO-d6, 100 MHz) δC : 130.5 (C-2), 48.2 (C-3), 170.6 (C-5), 58.6 (C-6), 29.1 (C-7), 22.4 (C-8), 45.2 (C-9), 166.3 (C-11), 83.4 (C-12), 68.3 (C-13), 106.5 (C-14), 120.7 (C-15), 121.3 (C-16), 108.9(C-17), 155.6 (C-18), 93.7 (C-19), 137.6 (C-20), 41.3 (C-21), 120.6 (C-22), 133.9 (C-23), 18.4 (C-24), 25.6 (C-25), 123.8 (C-26), 134.3 (C-27), 18.2 (C-28), 25.6 (C-29), 55.9 (C-30)。通过与文献[5]报道中的数据对比, 确定此化合物为吲哚二酮哌嗪类生物碱fumitremorgin B。

化合物2:无色固体结晶, [α]D20+21.5 (c = 0.41, CHCl3); δH : 6.02 (1H, d, J= 9.78 Hz, H-3), 2.35 (1H, m, H-7a), 2.11 (1H, m, H-7b), 2.09 (1H, m, H-8a), 1.93 (1H, m, H-8b), 3.61 (2H, dd, J=9.02, 16.71 Hz, H-9), 8.11 (1H, d, J=8.63 Hz, H-16), 6.88 (1H, dd, J=8.62, 1.99 Hz, H-17), 6.62 (1H, d, J=1.94 Hz, H-19), 4.47 (2H, m, H-21), 4.95 (1H, t, J=5.56 Hz, H-22), 1.82 (3H, s, H-24), 1.71 (3H, s, H-25), 1.94 (3H, s, H-28), 1.63 (3H, s, H-29), 3.82(3H, s, H-18-OCH3). 13C-NMR (CDCl3, 100 MHz) δC : 147.5 (C-2), 48.7 (C-3), 165.2 (C-5), 60.2 (C-6), 28.6 (C-7), 23.2 (C-8), 45.5 (C-9), 173.1 (C-11), 81.9 (C-12), 181.5 (C-13), 108.2 (C-14), 118.5 (C-15), 123.0 (C-16), 111.7 (C-17), 55.6 (C-18), 94.9 (C-19), 139.0 (C-20), 42.7 (C-21), 118.3 (C-22), 136.4 (C-23), 18.3 (C-24), 25.5 (C-25), 122.1 (C-26), 138.2 (C-27), 18.7 (C-28), 25.9 (C-29), 55.8 (C-18- OCH 3)。以上数据与文献[6]对照, 确定该化合物为二酮哌嗪类化合物13-oxofumitremorgin B。

化合物3:白色粉末, [α]D20–7.9 (c = 0.13, CHCl3); 1H-NMR (DMSO-d6, 400 MHz) δH : 5.85 (1H, d, J = 9.78 Hz, H-3), 4.27 (2H, m, H-6, 12), 2.20 (1H, m, H-7a), 2.01(2H, m, H-8a), 1.85 (2H, m, H-7b, 8b), 3.44 (1H, dd, J=5.86, 7.71 Hz, H-9), 3.31 (1H, dd, J=15.79, 5.21 Hz, H-13a), 2.86 (1H, dd, J=15.82, 10.96 Hz, H-13b), 7.42 (1H, d, J=8.26 Hz, H-16), 6.66(1H, dd, J=8.32, 2.36 Hz, H-17), 6.84 (1H, d, J=2.42 Hz, H-19), 4.84 (1H, d, J=9.24 Hz, H-21), 1.59(3H, s, H-23), 1.94 (3H, s, H-24), 3.81 (3H, s, H-25). 13C-NMR (DMSO- d6, 100 MHz) δC: 130.8 (C-2), 48.3 (C-3), 167.5 (C-5), 56.8 (C-6), 26.4 (C-7), 21.1 (C-8), 43.2 (C-9), 163.8 (C-11), 54.4 (C-12), 19.3 (C-13), 103.2 (C-14), 116.8 (C-15), 118.7 (C-16), 106.8 (C-17), 153.8(C-18), 93.3 (C-19), 135.2 (C-20), 123.2 (C-21), 131.2 (C-22), 23.8 (C-23), 16.3 (C-24), 53.7 (C-25)。以上数据与文献[7]对照, 确定该化合物为二酮哌嗪类化合物fumitremorgin C。

化合物4:无色针晶固体, [α]D20+15.8 (c = 0.15, CHCl3); 1H-NMR (DMSO-d6, 400 MHz) δH : 6.02 (1H, d, J=9.52 Hz, H-3), 4.38 (2H, dd, J=9.47 Hz, H-6), 3.65 (1H, d, J=15.97 Hz, H-13a), 3.27 (1H, d, J=16.24 Hz, H-13b), 7.53 (1H, d, J=8.68 Hz, H-16), 6.87 (1H, dd, J=2.14, 8.63 Hz, H-17), 6.85 (1H, d, J=2.12 Hz, H-19), 4.76 (2H, d, J=9.37 Hz, H-21), 1.88 (3H, s, H-23), 1.61(3H, s, H-24), 3.78 (3H, s, H-25). 13C-NMR (DMSO-d6, 100 MHz) δC : 131.8 (C-2), 50.2(C-3), 170.9 (C-5), 59.2 (C-6), 29.2 (C-7), 22.8 (C-8), 45.6 (C-9), 164.3 (C-11), 84.3 (C-12), 30.4 (C-13), 103.5 (C-14), 121.8 (C-15), 119.2 (C-16), 109.9 (C-17), 157.4 (C-18), 95.8 (C-19), 137.6 (C-20), 123.1 (C-21), 134.5 (C-22), 25.8 (C-23), 18.4 (C-24), 55.7 (C-25)。以上数据与文献[8]对照, 确定该化合物为二酮哌嗪类化合物烟曲霉毒素12-hydroxyfumitremorgin C。

化合物5:淡黄色结晶, [α]D20+19.9 (c = 0.37, CHCl3); 1 H-NMR (CDCl3, 400 MHz) δH : 6.65 (1H, d, J=9.70 Hz, H-3), 4.38 (2H, m, H-6), 2.44 (1H, m, H-7a), 1.96 (1H, m, H-7b), 2.13 (1H, m, H-8a), 1.98 (1H, m, H-8b), 3.72 (2H, m, H-9), 7.45 (1H, d, J= 8.63 Hz, H-16), 6.81 (1H, dd, J=8.62, 2.23 Hz, H-17), 6.88 (1H, d, J=2.12 Hz, H-19), 5.56 (1H, d, J=9.72 Hz, H-21), 1.78 (3H, d, J=0.93 Hz, H-23), 2.04 (3H, d, J=1.08 Hz, H-24), 3.37 (3H, s, H-13-OCH3), 3.85 (3H, s, H-18-OCH3). 13C-NMR (CDCl3, 100 MHz) δC: 133.7 (C-2), 49.2 (C-3), 167.1 (C-5), 60.1 (C-6), 29.8 (C-7), 22.2 (C-8), 45.9 (C-9), 166.0 (C-11), 84.9 (C-12), 76.7 (C-13), 105.4 (C-14), 122.6 (C-15), 118.5 (C-16), 110.2 (C-17), 156.7 (C-18), 95.4 (C-19), 136.8 (C-20), 123.6 (C-21), 138.0 (C-22), 26.2 (C-23), 18.3 (C-24), 56.8 (C-13-OCH3), 55.7 (C-18-OCH3)。以上数据与文献[9]对照, 确定该化合物为二酮哌嗪类化合物cyclotyprostatins B。

化合物6:白色针晶固体, [α]D20+27.8 (c = 0.22, CHCl3); 1H-NMR (CDCl3, 400 MHz) δH: 7.93(1H, brs, H-1), 5.94 (1H, d, J=9.70 Hz, H-3), 4.47 (2H, dd, J=1.02, 7.15 Hz, H-6), 2.48 (1H, m, H-7a), 2.08 (1H, m, H-7b), 2.13 (1H, m, H-8a), 1.98 (1H, m, H-8b), 3.62 (2H, m, H-9), 5.77 (1H, d, J=2.88 Hz, H-13), 7.84 (1H, d, J=7.63 Hz, H-16), 7.15 (1H, t, J=7.62 Hz, H-17), 7.19 (1H, t, J=7.82, 1.03 Hz, H-18), 7.33 (1H, d, J=7.78 Hz, H-19), 4.86 (1H, m, J=9.72 Hz, H-21), 1.68 (3H, d, J=0.93 Hz, H-23), 2.04 (3H, d, J=1.02 Hz, H-24). 13C-NMR (CDCl3, 100 MHz) δC: 131.5 (C-2), 49.9 (C-3), 171.0 (C-5), 59.1 (C-6), 29.2 (C-7), 22.5 (C-8), 45.3 (C-9), 166.0 (C-11), 84.9 (C-12), 68.7 (C-13), 105.3 (C-14), 125.9 (C-15), 119.7 (C-16), 120.7 (C-17), 122.5 (C-18), 111.1 (C-19), 136.6 (C-20), 123.8 (C-21), 135.0 (C-22), 21.4 (C-23), 18.2 (C-24)。以上数据与文献[9]对照, 确定该化合物为二酮哌嗪类化合物cyclotyprostatins C。

化合物7:浅黄色无定型粉末, [α]D20+24.2 (c = 0.22, CHCl3); 1H-NMR (DMSO-d6, 400 MHz) δH : 5.97 (1H, dd, J=5.96, 6.02 Hz, H-3), 4.29 (1H, dd, J=6.02, 11.03 Hz, H-6), 2.51 (1H, m, H-7a), 1.97 (1H, m, H-7b), 2.13 (1H, m, H-8a), 2.02 (1H, m, H-8b), 2.23 (1H, d, J=6.31 Hz, H-9a), 2.15 (1H, d, J=6.26 Hz, H-9b), 4.83 (1H, s, H-13), 7.48 (1H, d, J=8.96 Hz, H-16), 6.76 (1H, dd, J=2.46, 8.98 Hz, H-17), 6.97 (1H, d, J=2.52 Hz, H-19), 3.69 (2H, m, H-21), 1.37 (1H, s, H-23), 1.43 (3H, s, H-24), 3.37 (3H, s, H-13-OCH3), 4.83 (3H, s, H-18-OCH3). 13C-NMR (DMSO-d6, 100 MHz) δC : 136.3 (C-2), 49.2 (C-3), 1167.8 (C-5), 60.7 (C-6), 31.0 (C-7), 22.7 (C-8), 46.2 (C-9), 169.2 (C-11), 87.4 (C-12), 77.7 (C-13), 105.2 (C-14), 138.2 (C-15), 119.4 (C-16), 110.8 (C-17), 157.4 (C-18), 96.1 (C-19), 123.7 (C-20), 50.6 (C-21), 71.4 (C-22), 31.5 (C-23), 29.1 (C-24), 57.4 (C-13-OCH3), 56.2 (C-18-OCH3)。以上数据与文献[10]对照, 确定此化合物为二酮哌嗪类化合物cyclotryprostatin E。

化合物8:淡黄色晶体, [α]D20+4.7 (c = 0.20, CHCl3); 1H-NMR (CDCl3, 400 MHz) δH : 6.04 (1H, d, J=10.07 Hz, H-3), 4.49 (1H, dd, J=9.45, 7.13 Hz, H-6), 3.64 (2H, dd, J=8.73, 4.33 Hz, H-9), 4.07 (1H, s, H-12-OH), 5.68 (1H, s, H-13), 4.76 (1H, s, H-13-OH), 7.90 (1H, d, J=8.73 Hz, H-16), 6.83 (1H, dd, J=8.73, 2.12 Hz, H-17), 6.59 (1H, d, J=2.02 Hz, H-19), 6.64 (1H, d, J=8.08 Hz, H-21), 5.04 (1H, d, J=8.13 Hz, H-22), 1.74 (3H, s, H-24), 2.00 (3H, s, H-25), 1.02 (3H, s, H-28), 1.72 (3H, s, H-29), 3.83 (3H, s, H-18-OCH3). 13C-NMR (CDCl3, 100 MHz) δC : 131.7 (C-2), 48.9 (C-3), 166.2 (C-5), 58.7 (C-6), 29.0 (C-7), 22.6 (C-8), 51.1 (C-9), 170.7 (C-11), 82.5 (C-12), 68.6 (C-13), 105.5 (C-14), 121.0 (C-15), 121.6 (C-16), 109.3 (C-17), 156.3 (C-18), 93.9 (C-19), 136.2 (C-20), 85.8 (C-21), 118.5 (C-22), 143.1 (C-23), 18.8 (C-24), 24.3 (C-25), 55.4 (C-18-OCH3)。以上数据与参考文献[11]对照, 确定该化合物为二酮哌嗪类化合物verruculogen。

化合物9:淡黄色粉末, [α]D20+43.2 (c = 0.12, CHCl3); 1H-NMR (DMSO-d6, 400 MHz) δH : 7.15 (1H, d, J=8.28 Hz, H-4), 6.58 (1H, dd, J=2.11, 8.35 Hz, H-5), 6.45 (1H, d, J=2.11 Hz, H-7), 4.72 (1H, s, H-8), 2.57 (1H, s, H-8-OH), 5.72 (1H, s, H-9-OH), 4.51 (1H, t, J=8.03 Hz, H-12), 2.19 (1H, m, H-14a), 1.88 (1H, m, H-14b), 4.59 (1H, d, J=9.05 Hz, H-18), 5.04 (1H, d, J=8.95 Hz, H-19), 0.97 (3H, s, H-21), 1.57 (3H, s, H-22), 4.29 (2H, qd, J=6.88, 15.46 Hz, H-23), 5.11 (1H, t, J=6.22 Hz, H-24), 1.76 (3H, s, H-26), 1.67 (3H, s, H-27), 3.74 (3H, s, H-6-OCH3). 13C-NMR (DMSO-d6, 100 MHz) δC : 179.7 (C-2), 61.5 (C-3), 118.3 (C-3a), 127.6 (C-4), 107.2 (C-5), 160.2 (C-6), 96.7 (C-7), 144.7 (C-7a), 74.5 (C-8), 87.0 (C-9), 169.1 (C-11), 60.5 (C-12), 27.9 (C-13), 23.0 (C-14), 44.9 (C-15), 164.7 (C-17), 56.5 (C-18), 123.1 (C-19), 137.0 (C-20), 17.7 (C-21), 25.1 (C-22), 38.1 (C-23), 118.2 (C-24), 136.4 (C-25), 18.3 (C-26), 25.7 (C-27), 55.7 (C-6-OCH3)。以上数据与文献[12]对照, 确定该化合物为具有独特的螺环结构骨架的二酮哌嗪类化合物spirotryprostatin C。

化合物10:淡黄色粉末, [α]D20+47.7 (c = 0.22, CHCl3); 1H-NMR (DMSO-d6, 400 MHz) δH : 7.05 (1H, d, J=8.32 Hz, H-4), 6.59 (1H, dd, J=2.32, 8.25 Hz, H-5), 6.46 (1H, d, J=2.31 Hz, H-7), 4.74 (1H, s, H-8), 2.32 (1H, m, H-13a), 2.41 (1H, m, H-13b), 2.34 (1H, m, H-14a), 2.13 (1H, m, H-14b), 4.62 (1H, d, J=8.95 Hz, H-18), 5.14 (1H, d, J=9.12 Hz, H-19), 1.09 (3H, s, H-21), 1.59 (3H, s, H-22), 4.31 (2H, dd, J=6.78, 15.46 Hz, H-23), 5.14 (1H, t, J=6.25 Hz, H-24), 1.74 (3H, s, H-26), 1.65 (3H, s, H-27), 3.82 (3H, s, H-6-OCH3). 13C-NMR (DMSO-d6, 100 MHz) δC : 178.9 (C-2), 61.5 (C-3), 118.3 (C-3a), 127.6 (C-4), 107.2 (C-5), 160.2 (C-6), 96.7 (C-7), 144.7 (C-7a), 74.5 (C-8), 87.0 (C-9), 169.3 (C-11), 90.4 (C-12), 33.9 (C-13), 21.5 (C-14), 45.1 (C-15), 164.7 (C-17), 56.5 (C-18), 121.2 (C-19), 139.2 (C-20), 17.9 (C-21), 25.4 (C-22), 38.0 (C-23), 118.1 (C-24), 136.1 (C-25), 18.3 (C-26), 25.4 (C-27), 55.7 (C-6-OCH3)。以上数据归属情况与参考文献[12]对照, 确定此化合物为具有独特螺环结构骨架的二酮哌嗪类化合物spirotryprostatin D。

化合物11:淡黄色粉末, [α]D20+35.1 (c = 0.25, CHCl3); 1H-NMR (DMSO-d6, 400 MHz) δH : 6.96 (1H, d, J=8.31 Hz, H-4), 6.54 (1H, dd, J=2.21, 8.46 Hz, H-5), 6.42 (1H, d, J=2.27 Hz, H-7), 4.77 (1H, s, H-8), 4.56 (1H, t, J=7.25, 9.43 Hz, H-12), 2.23 (1H, m, H-13a), 2.03 (1H, m, H-13b), 2.37 (1H, m, H-14a), 1.85 (1H, m, H-14b), 3.69 (2H, m, H-15), 4.69 (1H, m, H-18), 5.34 (1H, d, J=8.45 Hz, H-19), 1.97 (3H, s, H-21), 1.27 (3H, s, H-22), 3.76 (3H, s, H-6-OCH3). 13C-NMR (DMSO-d6, 100 MHz) δC : 179.6 (C-2), 61.3 (C-3), 114.2 (C-3a), 127.3 (C-4), 107.4 (C-5), 160.4 (C-6), 97.0 (C-7), 142.3 (C-7a), 75.3 (C-8), 87.2 (C-9), 169.5 (C-11), 60.3 (C-12), 27.8 (C-13), 23.2 (C-14), 45.0 (C-15), 165.3 (C-17), 57.5 (C-18), 121.5 (C-19), 139.0 (C-20), 25.3 (C-21), 20.1 (C-22)。以上数据与文献[13]对照, 确定该化合物为具有独特的螺环结构骨架的二酮哌嗪类化合物spirotryprostatin F。

化合物12:淡黄色粉末, [α]D20–99.7 (c = 0.15, CHCl3); ESI-MS: m/z 466.1487 [M +Na]+, 分子式C24H21N5O4; 1H-NMR (DMSO-d6, 400 MHz) δH : 7.78 (1H, d, J=8.05 Hz, H-7), 7.86 (1H, t, J=7.65 Hz, H-8), 7.63 (1H, t, J=7.54 Hz, H-9), 8.16 (1H, d, J=7.87 Hz, H-10), 5.36 (1H, d, J=6.92 Hz, H-14), 2.95 (1H, dd, J=7.26, 15.22 Hz, H-15a), 2.04 (1H, d, J=15.01 Hz, H-15b), 1.87 (3H, s, H-16), 5.08 (1H, d, J=9.18 Hz, H-18), 2.30 (1H, t, J=9.42 Hz, H-20), 7.31 (3H, m, H-24, 25, 27), 7.21 (1H, t, J=7.45 Hz, H-26). 13C- NMR (DMSO-d6, 100 MHz) δC : 172.5 (C-1), 85.9 (C-3), 150.7 (C-4), 146.5 (C-6), 128.3 (C-7), 134.8 (C-8), 128.4 (C-9), 126.3 (C-10), 121.5 (C-11), 159.6 (C-12), 51.2 (C-14), 31.5 (C-15), 24.3 (C-16), 87.4 (C-17), 85.9 (C-18), 58.4 (C-20), 170.3 (C-21), 136.4 (C-23), 114.8 (C-24), 130.1 (C-25), 125.8 (C-26), 125.4 (C-27), 138.7 (C-28), 18.1 (C-29)。以上数据与文献[14]对照, 确定此化合物确为喹唑啉类衍生物fumiquinazoline C。

化合物13:淡黄色粉末, [α]D20–24.7 (c = 0.09, CHCl3); 1H-NMR (DMSO-d6, 400 MHz) δH : 7.64 (1H, d, J=7.89 Hz, H-7), 7.82 (1H, m, H-8), 7.53 (1H, m, H-9), 8.12 (1H, dd, J=7.97, 1.16 Hz, H-10), 5.56 (1H, dd, J=1.32 Hz, H-14), 3.06 (1H, dd, J=10.32, 14.92 Hz, H-15a), 2.08 (1H, dd, J=14.96, 2.13 Hz, H-15b), 1.95 (3H, s, H-16), 5.21 (1H, d, J=10.02 Hz, H-18), 4.20 (1H, m, H-20), 7.48 (1H, d, J=7.42 Hz, H-24), 7.29 (1H, ddd, J=7.87, 7.69, 1.12 Hz, H-25), 7015 (1H, ddd, J=7.48, 7.46, 1.08 Hz, H-26), 7.35 (1H, d, J=7.36 Hz, H-27). 13C-NMR (DMSO-d6, 100 MHz) δC : 172.4 (C-1), 70.7 (C-3), 153.4 (C-4), 146.2 (C-6), 127.6 (C-7), 134.9 (C-8), 127.6 (C-9), 126.5 (C-10), 120.3 (C-11), 160.2 (C-12), 53.2 (C-14), 43.0 (C-15), 18.8 (C-16), 82.8 (C-17), 85.3 (C-18), 58.4 (C-20), 168.7 (C-21), 137.7 (C-23), 114.7 (C-24), 129.4 (C-25), 125.2 (C-26), 124.6 (C-27), 139.2 (C-28), 17.3 (C-29)。以上数据与文献[14]对照, 确定该化合物为fumiquinazoline D。

化合物14:淡黄色粉末, [α]D20–48.7 (c = 0.07, MeOH); 1H-NMR (DMSO-d6, 400 MHz) δH : 7.68 (1H, d, J=8.26 Hz, H-7), 7.84 (1H, t, J=7.62 Hz, H-8), 7.41 (1H, d, J=7.54 Hz, H-9), 8.06 (1H, dd, J=7.87, 1.23 Hz, H-10), 5.65 (1H, m, H-14), 2.53 (1H, m, H-15a), 2.27 (1H, m, H-15b), 1.86 (3H, s, H-16), 5.28 (1H, dd, J=5.94, 1.43 Hz, H-18), 4.14 (1H, d, J=5.21 Hz, H-20), 7.52 (1H, m, H-24), 7.35 (1H, d, J=6.51 Hz, H-25), 7.17 (1H, t, J=7.36 Hz, H-26), 7.64 (1H, d, J=7.58 Hz, H-27), 1.11 (1H, d, J=6.62 Hz, H-29). 13C-NMR (DMSO-d6, 100 MHz) δ C : 171.7 (C-1), 80.3 (C-3), 152.4 (C-4), 146.7 (C-6), 127.6 (C-7), 134.9 (C-8), 127.2 (C-9), 126.5 (C-10), 120.3 (C-11), 160.4 (C-12), 53.0 (C-14), 38.2 (C-15), 26.1 (C-16), 79.9 (C-17), 86.0 (C-18), 58.5 (C-20), 171.3 (C-21), 136.5 (C-23), 114.3 (C-24), 129.1 (C-25), 125.8 (C-26), 124.6 (C-27), 139.6 (C-28), 18.2 (C-29)。以上数据与文献[15]对照, 确定该化合物为3-hydroxyfumiquinazoline A。

化合物15:淡黄色粉末, [α]D20–53.4 (c = 0.10, MeOH); 1H-NMR (CDCl3, 400 MHz) δH : 7.73 (1H, m, H-7), 7.78 (1H, dd, J=6.92, 1.41 Hz, H-8), 7.36 (1H, m, H-9), 8.24 (1H, dd, J=8.12, 1.15 Hz, H-10), 5.92 (1H, dd, J=4.98, 9.01 Hz, H-14), 2.91 (1H, dd, J=9.04, 14.43 Hz, H-15a), 2.33 (1H, dd, J=4.99, 14.42 Hz, H-15b), 1.97 (3H, s, H-16), 5.48 (1H, d, J=4.46 Hz, H-18), 4.17 (1H, m, H-20), 7.56 (1H, m, H-24), 7.33 (1H, m, H-25), 7.16 (1H, ddd, J=0.95, 7.56, 7.54 Hz, H-26), 7.58 (1H, d, J=8.19 Hz, H-27), 1.33 (1H, d, J=6.66 Hz, H-29), 3.33 (3H, s, H-3-OCH3). 13C-NMR (CDCl3, 100 MHz) δC : 172.6 (C-1), 85.0 (C-3), 148.2 (C-4), 146.4 (C-6), 128.2 (C-7), 135.1 (C-8), 128.3 (C-9), 127.1 (C-10), 120.7 (C-11), 161.1 (C-12), 53.5 (C-14), 39.0 (C-15), 21.1 (C-16), 80.2 (C-17), 86.4 (C-18), 59.4 (C-20), 171.4 (C-21), 136.9 (C-23), 115.2 (C-24), 129.9 (C-25), 125.3 (C-26), 124.9 (C-27), 138.7 (C-28), 18.1 (C-29), 50.8 (C-3- CH3)。以上数据与文献[14]对照, 确定该化合物为fumiquinazoline E。

参考文献
[1]
Sun G Y, Tan Y J, Zhang R. The family Chaetomiaceae from China Ⅰ. species of the genus Chaetomium[J]. Mycosystema, 2004, 23(3): 333-337.
[2]
郝飞. 烟曲霉的致病因子及作用机制的研究进展[J]. 中华医院感染学杂志, 2004, 14(1): 119-120.
Hao Fei. Advances in research on Virulence and Pathogenesis of Aspergillus fumigatus[J]. Chinese Journal of Nosocomiology, 2004, 14(1): 119-120. DOI:10.3321/j.issn:1005-4529.2004.01.039
[3]
朱伟明, 王俊锋. 海洋真菌生物活性物质研究之管见[J]. 菌物学报, 2011, 30(2): 218-228.
Zhu Weiming, Wang Junfeng. A review on studies of secondary metabolites from marine fungi[J]. Mycosystema, 2011, 30(2): 218-228.
[4]
Mayer A M S, Glaser K B, Carmen C, et al. The odyssey of marine pharmaceuticals:a current pipeline perspective[J]. Trends Pharmacol Sci, 2010, 31(6): 255-265. DOI:10.1016/j.tips.2010.02.005
[5]
Liu J, Yang Z J, Meng Z H. The Isolation, Purification and Identification of Fumitremorgin B Produced by Aspergillus fumigatus[J]. Biom Environ Sci, 1996, 9(1): 1-11.
[6]
Fujimoto H, Fujimaki T, Okuyama E, et al. Immunosuppressive constituents from an Ascomycete, Sordaria gondaensis[J]. Jsm Mycotoxins, 2000, 50(2): 93-99. DOI:10.2520/myco1975.50.93
[7]
任虹, 曹学丽, 王巧娥, 等. 真菌萨氏曲霉D2-6抗肿瘤活性代谢产物[J]. 中国药学杂志, 2011, 46(8): 569-575.
Ren Hong, Cao Xueli, Wang Qiaoe, et al. Antitumor active metabolites from the fungus Aspergillus sydowi D2-6[J]. Chin J Hosp Pharm, 2011, 46(8): 569-575.
[8]
Wang Y, Lia Z, Baia J, et al. 2, 5-Diketopiperazines from the marine-derived fungus Aspergillus fumigatus YK-7[J]. Chem Biodivers, 2012, 9(2): 385-393. DOI:10.1002/cbdv.201100061
[9]
Cui C, Kakeya H, Osada H. Novel mammalian cell cycle inhibitors, Cyclotryprostatins A-D, produced by Aspergillus fumigatus, which inhibit mammalian cell cycle at G2/M phase[J]. Tetrahedron, 1996, 52(39): 12651-12666. DOI:10.1016/0040-4020(96)00737-5
[10]
He F, Sun Y, Liu K, et al. Indole alkaloids from marine- derived fungus Aspergillus sydowii SCSIO 00305[J]. J Antibiot, 2012, 65(2): 109-111.
[11]
Yamazaki M, Fujimoto H, Okuyama E. Structure determination of six tryptoquivaline-related metabolites from Aspergillus fumigatus[J]. Tetrahedron Lett, 1976, 17(33): 2861-2864. DOI:10.1016/S0040-4039(01)85521-0
[12]
Wang F, Fang Y, Zhu T, et al. Seven new prenylated indole diketopiperazine alkaloids from holothurian- derived fungus Aspergillus fumigatus[J]. Tetrahedron, 2008, 64(34): 7986-7991. DOI:10.1016/j.tet.2008.06.013
[13]
Afiyatullov S S, Zhuravleva O I, Chaikina E L, et al. A new spirotryprostatin from the marine isolate of the fungus Aspergillus fumigatus[J]. Chem Nat Comp, 2012, 48(1): 95-98.
[14]
Takahashi C, Matsushita T, Doi M, et al. Fumiquinazolines A-G, novel metabolites of a fungus separated from a Pseudolabrus marine fish[J]. J Chem Soc Perk T, 1995, 18(18): 2345-2353.
[15]
Li X J, Zhang Q, Zhang A L, et al. Metabolites from Aspergillus fumigatus, an endophytic fungus associated with melia azedarach, and their antifungal, antifeedant, and toxic activities[J]. J Agric Food Chem, 2012, 60(13): 3424-3431. DOI:10.1021/jf300146n