2018, Vol. 42
文章信息
- 李莉, 李晓明, 李洪雷, Belma Konuklugil, 李鑫, 王斌贵. 2018.
- LI Li, LI Xiao-ming, LI Hong-lei, BELMA Konuklugil, LI Xin, WANG Bin-gui. 2018.
- 海鞘内生真菌焦曲霉Aspergillus ustus TK-5的化学成分研究
- Chemical constituents of Aspergillus ustus TK-5, an endophytic fungus derived from the ascidian Herdmania momus
- 海洋科学, 42(5): 130-137
- Marina Sciences, 42(5): 130-137.
- http://dx.doi.org/10.11759/hykx20180320002
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文章历史
- 收稿日期：2018-03-20
- 修回日期：2018-04-13
2. 中国科学院大学, 北京100049;
3. Department of Pharmacognosy, Faculty of Pharmacy, Ankara University, 06110 Ankara, Turkey
2. University of the Chinese Academy of Sciences, Beijing 100049, China;
3. Department of Pharmacognosy, Faculty of Pharmacy, Ankara University, 06110 Ankara, Turkey
海洋环境的复杂性和生物竞争的多样性造就了海洋生物独特的代谢途径和适应机制, 其中海洋微生物多与海洋动、植物共附生, 有利于激发其沉默基因从而代谢产生结构新颖、具有多种生物活性的次级代谢产物。越来越多的研究已证实在海洋无脊椎动物中分离到的活性物质的真正生产者是其体内共附生的微生物[1-2]。因此以海洋动物为宿主的微生物代谢产物研究引起了科学家们的广泛关注, 成为活性天然产物的主要来源之一[3-5]。
海鞘作为海洋微生物的重要宿主, 其内生真菌天然产物的研究却鲜有报道。本文报道从采自土耳其海域的海鞘(Pyura momus)组织中分离到一株内生真菌Aspergillus ustus TK-5, 根据文献报道A. ustus主要分离自海绵和海鞘, 但其特征产物都是血苋烷型倍半萜和二倍半萜类化合物, 以此确定以上报道的代谢产物不是来自于其宿主, 而是A. ustus自身的代谢产物[6-10]。从A. ustusTK-5发酵培养物中分离鉴定了17个化合物(图 1), 通过一维、二维核磁共振、质谱等光谱技术鉴定了所有化合物的结构, 与文献报道相符主要是血苋烷型倍半萜类(1-10)和二倍半萜类化合物(11-17), 分别是strobilactone A (1)[11], ustusolate E (2)[6], ustusolate C (3)[6], ustusolate D (4)[6], 11-hydroustusolate E (5)[12], 11, 6’-hydroustusolate E (6)[12], (2’E, 4’E, 6’E)-6-(1’-carboxyocta-2’, 4’, 6’- trie ne)-11, 12-epoxy-9, 11-dihydroxydrim-7-ene (7)[13], 12- hydroxy-6-epi-albrassitriol (8)[14], ustusolate A (9)[6], deoxyuvidin B (10)[14], 6-epi-ophiobolin G (11)[15], (6α)-21, 21-O-dihydroophiobolin G (12)[10], 6-epi-ophiobolin K (13)[16], ophiobolin P (14)[17], ophiobolin H (15)[10], ophiobolin Q (16)[17]和ophiobolin R (17)[17]。对所有化合物进行了神经氨酸酶抑制活性测试。
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| 图 1 化合物的结构(1-17) Fig. 1 Structures of compounds 1-17 |
Bruker Avance 500 MHz核磁共振仪; Dionex分析型和制备型高效液相色谱仪; 薄层色谱硅胶GF254和柱色谱硅胶(200~300目)为青岛海洋化工厂分厂产品; Lobar LiChroprep RP-18硅胶(40~63 μm, Merck); 显色剂为茴香醛硫酸溶液和碘; 所用有机溶剂为重蒸的工业级溶剂。
1.2 菌株发酵 1.2.1 菌株菌株A. ustus分离自海鞘(Pyura momus)的新鲜组织, 该样品于2015年5月27日采自土耳其海域。
1.2.2 菌株发酵菌种以琼脂-麦芽膏培养基4℃保存。发酵培养基采用的是大米培养基, 1000 mL三角瓶作为发酵容器, 每瓶加入大米70 g, 玉米浆0.2 g, 蛋白胨0.3 g, 酵母粉0.5 g, 味精0.6 g, 海水100 mL。规模发酵100瓶, 在121℃下高压灭菌20 min, 待冷却后接种, 28℃静置培养30 d。
1.3 提取与分离发酵产物经乙酸乙酯萃取后减压浓缩得到粗提物107.5 g, 将粗提物进行硅胶真空柱层析, 根据极性从小到大(石油醚/乙酸乙酯到二氯甲烷/甲醇)进行梯度洗脱, 经TLC和HPLC检测, 合并得到10个组分(Fr.1~10)。
其中, Fr.5(8.3 g)经反相硅胶柱层析、正相硅胶柱层析(二氯甲烷︰甲醇=150︰1–50︰1)、凝胶SephadexLH-20(甲醇)柱层析和制备薄层层析分离得到化合物1 (8.8 mg)、2 (10.2 mg)、3 (22.4 mg)、5 (15.3 mg)、13 (8.0 mg); Fr.6(9.5 g)经反相硅胶柱层析、正相硅胶柱层析(二氯甲烷︰丙酮=100︰1~40︰1)、制备薄层层析和凝胶SephadexLH-20(甲醇)柱层析分离得到化合物6 (8.2 mg)、7 (7.9 mg)、11 (26.4 mg)、12 (6.1 mg)、14 (7.3 mg)、15 (6.1 mg); Fr.8(12.4 g)经反相硅胶柱层析、凝胶Sephadex LH-20(甲醇)柱层析、制备薄层层析、高效液相制备分离得到化合物4 (6.8 mg)、8 (11.9 mg)、9 (9.9 mg)、10 (17.2 mg)、16 (21.1 mg)、17 (16.3 mg)。
1.4 活性测试利用神经氨酸酶抑制剂筛选试剂盒(包括神经氨酸酶检测缓冲液、神经氨酸酶、神经氨酸酶荧光底物、Milli-Q水)进行神经氨酸酶抑制活性测试[18]。
1.4.1 样品的准备将待测化合物和阳性对照奥司他韦用甲醇分别配制成浓度为100 μmol/L的溶液。
1.4.2 样品检测的准备在96孔荧光酶标板内每孔依次加入70 μL神经氨酸酶检测缓冲液、10 μL神经氨酸酶、上述配置的待筛选的神经氨酸酶抑制剂样品或阳性对照奥司他韦溶液(样品和对照分别加入0、1、2、5、7.5、10 μL)及Milli-Q水(10、9、8、5、2.5、0 μL, 使每孔总体积为90 μL), 每个样品做三个平行。
1.4.3 检测振动混匀约1 min; 37℃孵育2 min使抑制剂和神经氨酸酶充分相互作用; 每孔加入10 μL神经氨酸酶荧光底物; 再振动混匀约1 min; 37℃孵育30 min后进行荧光测定, 激发波长为322 nm, 发射波长为450 nm。
1.4.4 计算酶标仪测定每孔的吸光值(OD值)。取三孔平均OD值, 按IR% =(OD空白对照–OD样品)/OD空白对照×100%式计算样品对神经氨酸酶的抑制率(IR%), 并得到IC50。
2 化合物结构鉴定和神经氨酸抑制活性结果 2.1 化合物结构鉴定化合物1:无色油状液体, 13C NMR (125 MHz, DMSO-d6) δC: 175.0 (C, C-11), 132.0 (C, C-8), 127.3 (CH, C-7), 73.4 (C, C-9), 68.3 (CH2, C-12), 63.3 (CH, C-6), 45.2 (CH, C-5), 44.4 (CH2, C-3), 37.0 (C, C-10), 33.7 (C, C-4), 32.3 (CH3, C-15), 29.7 (CH2, C-1), 24.3 (CH3, C-14), 18.4 (CH3, C-13), 17.5 (CH2, C-2); 1H NMR (500 MHz, DMSO-d6) δH: 5.95 (1H, s, 9-OH), 5.80 (1H, s, H-7), 4.84 (1H, d, J=12.2 Hz, H-12), 4.72 (1H, d, J=12.2 Hz, H-12), 4.65 (1H, d, J=5.7 Hz, 6-OH), 4.31 (1H, s, H-6), 1.90 (1H, td, J=13.6, 3.9 Hz, H-1), 1.76 (1H, d, J=13.4 Hz, H-1), 1.65 (1H, d, J=4.9 Hz, H-5), 1.59 (1H, d, J=13.6 Hz, H-2), 1.43 (1H, d, J=13.3 Hz, H-2), 1.28 (1H, d, J=13.5 Hz, H-3), 1.25 (3H, s, H-14), 1.16 (1H, t, J=12.8 Hz, H-3), 1.03(3H, s, H-15), 0.96 (3H, s, H-13)。其波谱数据与strobilactone A[11]的文献报道一致。化合物1的比旋光度为[a]D20–130.0 (c 0.10, CHCl3)与文献报道[a]D20–110.0 (c 0.70, CHCl3)的接近, 说明绝对构型也相同。
化合物2:白色固体, 13C NMR (125 MHz, CDCl3) δC: 192.7 (CH, C-6’), 174.8 (C, C-11), 164.7 (C, C-1’), 146.7 (CH, C-4’), 141.4 (CH, C-3’), 137.6 (CH, C-5’), 135.7 (C, C-8), 129.6 (CH, C-2’), 123.4 (CH, C-7), 74.8 (C, C-9), 69.1 (CH2, C-12), 67.5 (CH, C-6), 45.0 (CH, C-5), 45.0 (CH2, C-3), 38.1 (C, C-10), 34.1 (C, C-4), 32.6 (CH3, C-13), 30.5 (CH2, C-1), 25.0 (CH3, C-14), 18.6 (CH3, C-15), 17.8 (CH2, C-2); 1H NMR (500 MHz, CDCl3) δH: 9.68 (1H, d, J=7.6 Hz, H-6’), 7.40 (1H, dd, J=15.4, 11.2 Hz, H-3’), 7.16 (1H, m, H-4’), 6.44 (1H, dd, J=15.5, 7.6 Hz, H-5’), 6.30 (1H, d, J=15.4 Hz, H-2’), 5.94 (1H, s, H-7), 5.79 (1H, s, H-6), 4.97 (1H, d, J=12.4 Hz, H-12), 4.74 (1H, d, J=12.5 Hz, H-12), 2.13 (1H, d, J=8.1 Hz, H-1), 2.06 (1H, d, J=4.6 Hz, H-5), 1.71 (1H, d, J=8.0 Hz, H-1), 1.60 (1H, m, H-2), 1.45 (1H, d, J=11.6 Hz, H-2), 1.32 (1H, m, H-3), 1.26 (1H, d, J=7.7 Hz, H-3), 1.20 (3H, s, H-13), 1.13 (3H, s, H-15), 1.01 (3H, s, H-14)。其波谱数据与ustusolate E[6]文献报道一致。化合物2的比旋光度为[a]D20–300.0 (c 0.10, MeOH)与文献报道[a]D20–320.0 (c 0.10, MeOH)的接近, 说明绝对构型也相同。
化合物3:白色固体, 13C NMR (125 MHz, DMSO-d6) δC: 174.3 (C, C-11), 165.4 (C, C-1’), 145.7 (CH, C-3’), 142.7 (CH, C-5’), 136.6 (C, C-8), 129.6 (CH, C-4’), 121.3 (CH, C-7), 119.0 (CH, C-2’), 73.1 (C, C-9), 68.2 (CH2, C-12), 65.7 (CH, C-6), 65.4 (CH, C-7’), 44.4 (CH2, C-3), 44.2 (CH, C-5), 42.5 (CH2, C-6’), 37.2 (C, C-10), 33.3 (C, C-13), 32.9 (C, C-4), 29.5 (CH2, C-1), 24.0 (CH3, C-14), 23.2 (CH3, C-8’), 18.2 (CH3, C-15), 17.4 (CH2, C-2); 1H NMR (500 MHz, DMSO-d6) δH: 7.18 (1H, m, H-3’), 6.31 (1H, dd, J=15.4, 10.4 Hz, H-4’), 6.30 (1H, s, 9-OH), 6.27 (1H, dd, J=15.4, 7.1 Hz, H-5’), 5.88 (1H, d, J=15.4 Hz, H-2’), 5.78 (1H, s, H-7), 5.58 (1H, s, H-6), 4.88 (1H, d, J=12.6 Hz, H-12), 4.78 (1H, d, J=12.7 Hz, H-12), 4.61 (1H, s, 7’-OH), 3.70 (1H, dd, J=11.8, 5.9 Hz, H-7’), 2.21 (1H, t, J=5.3 Hz, H-6’), 2.02 (1H, dd, J=4.8 Hz, H-5), 1.96 (1H, dd, J=13.6, 3.5 Hz, H-1), 1.84 (1H, d, J=13.3 Hz, H-1), 1.60(1H, dd, J=17.8, 9.0 Hz, H-2), 1.47 (1H, d, J=13.3 Hz, H-2), 1.34 (1H, d, J=12.5 Hz, H-3), 1.21 (1H, m, H-3), 1.07 (3H, s, H-15), 1.06 (3H, s, H-13), 1.05 (3H, d, J=6.6 Hz, H-8’), 0.92 (3H, s, H-14)。其波谱数据与ustusolate C[6]的文献报道一致。化合物3的比旋光度为[a]D20–700.0 (c 0.10, MeOH)与文献报道[a]D20–700.0 (c 0.10, MeOH)的完全一致, 说明绝对构型也相同。
化合物4:无色液体, 13C NMR (125 MHz, DMSO-d6) δC: 174.3 (C, C-11), 165.1 (C, C-1’), 143.9 (CH, C-3’), 138.8 (CH, C-5’), 136.7 (C, C-8), 130.4 (CH, C-4’), 122.7 (CH, C-7), 121.2 (CH, C-2’), 101.2 (CH, C-6’), 73.1 (C, C-9), 68.2 (CH2, C-12), 66.0 (CH, C-6), 52.4 (CH3, 6’-OCH3), 52.4 (CH3, 6’-OCH3), 44.4 (CH2, C-3), 44.1 (CH, C-5), 37.2 (C, C-10), 33.3 (C, C-4), 32.1 (CH3, C-14), 29.5 (CH2, C-1), 24.3 (CH3, C-15), 18.3 (CH3, C-13), 17.4 (CH2, C-2); 1H NMR (500 MHz, DMSO-d6) δH: 7.26 (1H, dd, J=15.2, 11.1 Hz, H-3’), 6.54 (1H, dd, J=15.5, 11.1 Hz, H-4’), 6.19 (1H, dd, J=15.5, 4.8 Hz, H-5’), 6.10 (1H, d, J=15.4 Hz, H-2’), 5.79 (1H, s, H-7), 5.59 (1H, s, H-6), 4.90 (1H, d, J=4.0 Hz, H-6’), 4.87 (1H, s, H-12), 4.80 (1H, m, H-12), 3.22 (6H, s, 6’-OCH3), 2.03 (1H, d, J=4.3 Hz, H-5), 1.97 (1H, dd, J=11.7, 7.0 Hz, H-1), 1.83 (1H, d, J=21.2, 8.6 Hz, H-1), 1.48 (1H, m, H-2), 1.45 (1H, m, H-2), 1.34 (1H, d, J=12.3 Hz, H-3), 1.19 (1H, d, J=17.6 Hz, H-3), 1.07 (3H, s, H-13), 1.06 (3H, s, H-15), 0.92 (3H, s, H-14)。其波谱数据与ustusolate D[6]的文献报道一致。化合物4的比旋光度为[a]D20–280.0 (c 0.10, MeOH)与文献报道[a]D25–300.0 (c 0.10, MeOH)的数据接近, 说明绝对构型也相同。
化合物5:黄色油状液体, 13C NMR (125 MHz, DMSO-d6) δC: 194.2 (CH, C-6’), 164.6 (C, C-1’), 147.9 (CH, C-4’), 143.3 (C, C-8), 141.4 (CH, C-3’), 137.3 (CH, C-5’), 129.6 (CH, C-2’), 116.6 (CH, C-7), 97.2 (C, C-11), 76.3 (C, C-9), 67.4 (CH, C-6), 65.6 (CH2, C-12), 44.9 (CH, C-5), 44.3 (CH2, C-3), 37.9 (C, C-10), 33.1 (C, C-4), 32.4 (CH3, C-14), 31.4 (CH2, C-1), 24.3 (CH3, C-15), 18.4 (CH3, C-13), 17.6 (CH2, C-2); 1H NMR (500 MHz, DMSO-d6) δH: 9.63 (1H, d, J=7.9 Hz, H-6’), 7.46 (2H, ddd, J=33.2, 14.6, 11.2 Hz, H-3’, 4’), 6.60 (1H, dd, J=14.8, 7.8 Hz, H-5’), 6.51 (1H, d, J=14.7 Hz, H-2’), 6.34 (1H, d, J=8.2 Hz, 9-OH), 5.51 (1H, s, H-6), 5.21 (1H, d, J=8.2 Hz, H-7), 4.85 (1H, s, 11-OH), 4.39 (1H, d, J=12.8 Hz, H-12), 4.09 (1H, d, J=12.9 Hz, H-12), 2.09 (1H, d, J=4.5 Hz, H-5), 1.87 (1H, t, J=14.0 Hz, H-1), 1.58 (1H, dd, J=26.7, 13.3 Hz, H-1), 1.43 (1H, d, J=12.9 Hz, H-2), 1.33 (1H, d, J=12.3 Hz, H-2), 1.22 (2H, d, J=5.0 Hz, H-3), 1.14 (3H, s, H-13), 1.08 (3H, s, H-15), 0.93 (3H, s, H-14)。其波谱数据与11-hydroustusolate E[12]的文献报道基本一致。文献中没有该化合物的比旋光度报道, 但化合物5的比旋光度为[a]D20–100.0 (c 0.10, MeOH), 与上述相似化合物的符号相同, 提示其绝对构型与上述化合物相同。
化合物6:白色固体, 13C NMR (125 MHz, DMSO-d6) δC: 165.5 (C, C-1’), 145.5 (CH, C-4’), 144.9 (CH, C-3’), 142.8 (C, C-8), 133.2 (CH, C-5’), 126.6 (CH, C-2’), 117.0 (CH, C-7), 97.4 (CH, C-11), 76.3 (C, C-9), 75.1 (CH, C-6’), 69.5 (CH, C-6), 65.8 (CH2, C-12), 44.9 (CH, C-5), 44.3 (CH2, C-3), 37.8 (C, C-10), 33.1 (C, C-4), 32.5 (CH3, C-14), 31.4 (CH2, C-1), 24.2 (CH3, C-15), 18.4 (CH3, C-13), 18.2 (CH3, C-7’), 17.6 (CH2, C-2); 1H NMR (500 MHz, DMSO-d6) δH: 7.20 (1H, ddd, J=10.8, 5.8, 3.0 Hz, H-3’), 6.43 (1H, m, H-4’), 6.30 (1H, ddd, J=15.4, 10.1, 4.9 Hz, H-5’), 5.91 (1H, d, J=15.3 Hz, H-2’), 5.57 (1H, s, H-7), 5.48 (1H, s, H-6), 5.21 (1H, s, 11-OH), 4.38 (1H, d, J= 12.6 Hz, H-12), 4.08 (1H, d, J=12.9 Hz, H-12), 2.07 (1H, d, J=4.5 Hz, H-5), 1.87 (1H, dd, J=13.7, 10.0 Hz, H-1), 1.59 (1H, d, J=12.8 Hz, H-1), 1.43 (1H, d, J=13.3 Hz, H-2), 1.32 (1H, d, J=12.1 Hz, H-2), 1.22 (2H, d, J=10.8 Hz, H-3), 1.13 (3H, s, H-14), 1.08 (3H, s, H-15), 1.03 (3H, d, J=6.2 Hz, H-7’), 0.92 (3H, s, H-13)。其波谱数据与11, 6’-hydroustusolate E[12]的报道一致。文献中没有该化合物的比旋光度报道, 但化合物6的比旋光度为[a]D20–60.0 (c 0.10, MeOH), 与上述相似化合物的符号相同, 提示其绝对构型与上述化合物相同。
化合物7:黄色油状液体, 13C NMR (125 MHz, DMSO-d6) δC: 165.4 (C, C-1’), 145.1 (CH, C-4’), 142.6 (CH, C-3’), 141.6 (C, C-8), 135.4 (CH, C-5’), 131.2 (CH, C-6’), 127.4 (CH, C-2’), 120.0 (CH, C-7’), 117.1 (CH, C-7), 97.2 (CH, C-11), 76.3 (C, C-9), 66.4 (CH, C-6), 65.6 (CH2, C-12), 44.9 (CH, C-5), 44.3 (CH2, C-3), 37.8 (C, C-10), 33.1 (C, C-4), 32.5 (CH3, C-14), 31.4 (CH2, C-1), 24.2 (CH3, C-15), 18.4 (CH3, C-13), 18.2 (CH3, C-8’), 17.6 (CH2, C-2); 1H NMR (500 MHz, DMSO-d6) δH: 7.21 (1H, dd, J=15.2, 11.3 Hz, H-3’), 6.69 (1H, dt, J=17.8, 8.9 Hz, H-5’), 6.34 (1H, dd, J=14.8, 11.4 Hz, H-4’), 6.22 (1H, dd, J=27.8, 12.8 Hz, H-6’), 6.01 (1H, dt, J=13.8, 6.8 Hz, H-7’), 5.91 (1H, d, J=15.2 Hz, H-2’), 5.57 (1H, d, J=4.3 Hz, H-7), 5.48 (1H, d, J=1.8 Hz, H-6), 5.20 (1H, s, 9-OH), 4.82 (1H, s, 11-OH), 4.38 (1H, d, J=12.8 Hz, H-12), 4.08 (1H, d, J=12.9 Hz, H-12), 2.07 (1H, d, J=4.6 Hz, H-5), 1.87 (1H, dd, J=13.6, 9.6 Hz, H-1), 1.80 (3H, d, J=6.8 Hz, H-8’), 1.57 (1H, m, H-1), 1.43 (1H, d, J=12.9 Hz, H-2), 1.32 (1H, d, J=12.2 Hz, H-2), 1.21 (2H, m, H-3), 1.13 (3H, s, H-13), 1.08 (3H, s, H-15), 0.92 (3H, s, H-14)。其波谱数据与(2’E, 4’E, 6’E)- 6- (1’-carboxyocta-2’, 4’, 6’-triene)-11, 12-epoxy- 9, 11- dih ydr oxydrim-7-ene[13]的文献报道一致。化合物7的比旋光度为[a]D20–260.0 (c 0.10, MeOH)与文献报道[a]D20–266.0 (c 0.10, MeOH)的数据接近, 说明绝对构型也相同。
化合物8:白色固体, 13C NMR (125 MHz, DMSO-d6) δC: 138.1 (C, C-8), 131.9 (CH, C-7), 74.6 (C, C-9), 67.0 (CH, C-6), 61.9 (CH2, C-11), 61.5 (CH2, C-12), 48.2 (CH, C-5), 43.3 (CH2, C-3), 42.0 (C, C-10), 36.5 (CH3, C-14), 32.8 (C, C-4), 32.3 (CH2, C-1), 22.9 (CH3, C-15), 18.3 (CH2, C-2), 17.4 (CH3, C-13); 1H NMR (500 MHz, DMSO-d6) δH: 5.65 (1H, s, H-7), 4.87 (1H, s, 12-OH), 4.61 (1H, s, 11-OH), 4.25 (1H, d, J=6.9 Hz, 6-OH), 4.07 (1H, s, 9-OH), 4.05 (1H, s, H-12), 4.02 (1H, s, H-6), 3.99 (1H, s, H-12), 3.48 (1H, m, H-11), 3.43 (1H, d, J=11.0 Hz, H-11), 1.72 (1H, d, J=10.2 Hz, H-5), 1.61 (1H, t, J=11.2 Hz, H-1), 1.46 (1H, d, J=12.7 Hz, H-1), 1.36 (2H, m, H-2), 1.25 (2H, t, J=13.1 Hz, H-3), 1.10 (3H, s, H-14), 1.01 (3H, s, H-15), 0.90 (3H, s, H-13)。其波谱数据与12-hydroxy- 6-epi-albrassitriol[14]的文献报道一致。文献中没有该化合物的比旋光度报道, 但化合物8的比旋光度为[a]D20–140.0 (c 0.05, MeOH), 与上述相似化合物的符号相同, 提示其绝对构型与上述化合物相同。
化合物9:无色油状液体, 13C NMR (125 MHz, DMSO-d6) δC: 165.6 (C, C-1’), 144.8 (CH, C-3’), 144.4 (C, C-8), 141.3 (CH, C-5’), 135.3 (CH, C-7’), 131.2 (CH, C-6’), 127.5 (CH, C-4’), 120.4 (CH, C-2’), 119.9 (CH, C-7), 74.1 (C, C-9), 66.2 (CH, C-6), 61.7 (CH2, C-11), 60.6 (CH2, C-12), 44.6 (CH, C-5), 44.0 (CH2, C-3), 40.1 (C, C-10), 33.3 (C, C-4), 32.6 (CH3, C-14), 31.8 (CH2, C-1), 24.5 (CH3, C-15), 18.6 (CH3, C-13), 18.3 (CH3, C-8’), 18.2 (CH2, C-2); 1H NMR (500 MHz, DMSO-d6) δH: 7.19(1H, dd, J=15.2, 11.3 Hz, H-3’), 6.67 (1H, m, H-5’), 6.33 (1H, m, H-4’), 6.22 (1H, dd, J=19.1, 8.1 Hz, H-6’), 6.01 (1H, td, J=14.0, 6.8 Hz, H-7’), 5.89 (1H, d, J=15.2 Hz, H-2’), 5.77 (1H, dd, J=9.3, 5.1 Hz, H-7), 5.54 (1H, m, H-6), 4.87 (1H, s, 12-OH), 4.74 (1H, s, 11-OH), 4.46 (1H, s, 9-OH), 4.43 (2H, s, H-12), 5.32 (1H, d, J=10.1 Hz, H-11), 3.45 (1H, d, J=11.4 Hz, H-11), 1.98 (1H, dd, J=7.0, 4.3 Hz, H-5), 1.87(4H, dd, J=15.8, 7.1 Hz, H-8’, 1), 1.60 (1H, d, J=14.2 Hz, H-2), 1.43 (2H, t, J=15.0 Hz, H-2, 1), 1.29 (1H, d, J=12.3 Hz, H-3), 1.23 (1H, s, H-3), 1.17 (3H, s, H-13), 1.05 (3H, s, H-15), 0.91 (3H, s, H-14)。其波谱数据与ustusolate A[6]的一致。化合物9的比旋光度为[a]D20–70.0 (c 0.10, MeOH)与文献报道[a]D20–68.0 (c 0.10, MeOH)的数据接近, 说明绝对构型也相同。
化合物10:白色固体, 13C NMR (125 MHz, DMSO-d6) δC: 198.8 (C, C-6), 158.9 (C, C-8), 127.8 (CH, C-7), 76.6 (CH, C-3), 61.8 (CH, C-5), 57.8 (CH2, C-11), 57.1 (CH, C-9), 41.4 (C, C-10), 37.3 (C, C-4), 36.5 (CH2, C-1), 28.3 (CH3, C-14), 26.5 (CH2, C-2), 21.4 (CH3, C-12), 15.6 (CH3, C-13), 15.3 (CH3, C-15); 1H NMR (500 MHz, DMSO-d6) δH: 5.70 (1H, s, H-7), 5.62 (1H, s, 11-OH), 4.38 (1H, d, J=4.8 Hz, 3-OH), 3.73 (1H, d, J=11.2 Hz, H-11), 3.60 (1H, m, H-11), 3.01 (1H, m, H-3), 2.24 (1H, s, H-9), 2.12 (1H, s, H-5), 1.65 (1H, d, J=4.9 Hz, H-5), 1.98 (3H, s, H-12), 1.90 (1H, d, J=12.4 Hz, H-2), 1.45 (3H, ddd, J=20.4, 10.3, 6.9 Hz, H-2, 1), 1.11 (3H, s, H-14), 0.99 (3H, s, H-15), 0.83 (3H, s, H-13)。其波谱数据与deoxyuvidin B[15]的文献报道一致。文献中没有该化合物的比旋光度报道, 但化合物10的比旋光度为[a]D20–85.0 (c 0.10, MeOH), 与上述相似化合物的符号相同, 提示其绝对构型与上述化合物相同。
化合物11:白色透明固体, 13C NMR (125 MHz, DMSO-d6) δC: 207.1 (C, C-5), 193.1 (CH, C-21), 177.4 (C, C-3), 157.8 (CH, C-8), 140.3 (C, C-7), 136.6 (C, C-19), 135.9 (CH, C-16), 130.5 (CH, C-4), 124.2 (CH, C-17), 120.2 (CH, C-18), 52.3 (CH, C-14), 50.2 (CH, C-6), 49.3 (CH, C-2), 46.1 (CH2, C-1), 45.6 (C, C-11), 44.5 (CH2, C-12), 44.1 (CH, C-10), 32.8 (CH, C-15), 31.1 (CH2, C-9), 27.9 (CH2, C-13), 26.6 (CH3, C-25), 23.1 (CH3, C-22), 21.4 (CH3, C-23), 18.4 (CH3, C-24), 17.3 (CH3, C-20); 1H NMR (500 MHz, DMSO-d6) δH: 9.27 (1H, s, H-21), 6.80 (1H, d, J=6.2 Hz, H-8), 6.10 (1H, t, J=11.2 Hz, H-17), 6.02 (2H, d, J=15.4 Hz, H-18, 4), 5.12 (1H, t, J=10.0 Hz, H-16), 3.38 (1H, s, H-6), 2.92 (1H, d, J=20.5 Hz, H-9), 2.60 (3H, dd, J=31.9, 12.4 Hz, H-2, 10, 15), 2.21 (1H, m, H-9), 2.07 (3H, s, H-20), 2.03 (2H, d, J=3.4 Hz, H-2, 1), 1.90 (1H, m, H-14), 1.84 (3H, s, H-25), 1.77 (3H, s, H-24), 1.69 (1H, m, H-13), 1.44 (2H, dd, J=11.1, 8.9 Hz, H-12), 1.17 (2H, dd, J=24.1, 14.8 Hz, H-1, 13), 0.98 (3H, d, J=6.7 Hz, H-23), 0.87 (3H, s, H-22)。其波谱数据与6-epi-ophiobolin G[16]的文献报道一致。化合物11的比旋光度为[a]D20+107.0 (c 0.10, MeOH)与文献报道[a]D23+117.0 (c 1.05 MeOH)的数据接近, 说明绝对构型也相同。
化合物12:棕红色透明固体, 13C NMR (125 MHz, DMSO-d6) δC: 210.6 (C, C-5), 181.0 (C, C-3), 136.4 (CH, C-16), 135.8 (C, C-19), 133.4 (C, C-7), 133.1 (CH, C-4), 130.2 (CH, C-8), 123.7 (CH, C-17), 120.4 (CH, C-18), 67.9 (CH2, C-21), 53.5 (CH, C-6), 52.3 (CH, C-14), 51.1 (CH, C-2), 46.6 (CH2, C-1), 45.1 (C, C-11), 44.4 (CH2, C-12), 43.9 (CH, C-10), 32.6 (CH, C-15), 29.3 (CH2, C-13), 28.0 (CH2, C-9), 26.6 (CH3, C-24), 23.0 (CH3, C-23), 21.3 (CH3, C-22), 18.3 (CH3, C-25), 17.6 (CH3, C-20); 1H NMR (500 MHz, DMSO-d6) δH: 6.02 (2H, m, H-18, 17), 5.94 (1H, s, H-8), 5.73 (1H, d, J=5.2 Hz, H-4), 5.11 (1H, t, J= 9.9 Hz, H-16), 4.15 (1H, d, J=11.9 Hz, H-21), 3.87 (1H, d, J=11.8 Hz, H-21), 3.60 (1H, d, J=2.6 Hz, H-6), 2.77 (1H, d, J=12.8 Hz, H-2), 2.51 (3H, m, H-10, 15, 9), 2.07 (3H, s, H-20), 2.03 (1H, dd, J=13.1, 3.4 Hz, H-9), 1.87 (2H, m, H-14, 1), 1.80 (3H, s, H-25), 1.74 (3H, s, H-24), 1.63 (1H, s, H-13), 1.50 (1H, dd, J=11.8, 4.3 Hz, H-12), 1.39 (1H, ddd, J=10.8, 8.9, 4.4 Hz, H-12), 1.27 (1H, m, H-13), 1.12 (1H, t, J=13.0 Hz, H-1), 0.98 (3H, s, H-22), 0.94 (3H, d, J=6.7 Hz, H-23)。其波谱数据与(6α)-21, 21-O-dihydroophiobolin G[10]的文献报道一致。化合物12的比旋光度为[a]D20+35.0 (c 0.05, CHCl3)与文献报道[a]D20 +49.0 (c 0.10, CHCl3)的数据相近, 说明绝对构型与文献一致。
化合物13:白色固体, 13C NMR (125 MHz, DMSO-d6) δC: 216.0 (C, C-5), 194.6 (CH, C-21), 159.7 (CH, C-8), 141.5 (C, C-7), 136.1 (CH, C-16), 135.3 (C, C-19), 123.3 (CH, C-17), 120.1 (CH, C-18), 74.6 (C, C-3), 54.7 (CH2, C-4), 51.4 (CH, C-14), 49.6 (CH, C-2), 48.3 (CH, C-6), 44.9 (CH2, C-12), 44.3 (C, C-11), 43.2 (CH, C-10), 41.0 (CH2, C-1), 31.9 (CH, C-15), 30.0 (CH2, C-9), 27.1 (CH2, C-13), 26.1 (CH3, C-25), 25.2 (CH3, C-20), 22.9 (CH3, C-22), 21.1 (CH3, C-23), 17.9 (CH3, C-24); 1H NMR (500 MHz, DMSO-d6) δH: 9.13 (1H, s, H-21), 6.97 (1H, d, J=5.3 Hz, H-8), 6.11 (2H, m, H-17, 18), 5.27 (1H, m, H-16), 4.68 (1H, s, 3-OH), 3.05 (1H, d, J=10.7 Hz, H-6), 2.77 (1H, d, J=16.1 Hz, H-4), 2.63 (2H, dd, J=28.7, 14.6 Hz, H-9), 2.47 (1H, d, J=10.5 Hz, H-10), 2.29 (2H, m, H-15, 2), 2.21 (1H, d, J=16.0 Hz, H-4), 1.97 (2H, m, H-14, 1), 1.80 (3H, s, H-24), 1.71 (3H, s, H-25), 1.66 (1H, dd, J=13.7, 4.3 Hz, H-13), 1.59 (1H, m, H-1), 1.41 (2H, m, H-12), 1.27 (3H, s, H-20), 1.09 (1H, dd, J=14.9, 7.1 Hz, H-13), 0.93 (3H, d, J=6.6 Hz, H-23), 0.93 (3H, s, H-22)。其波谱数据与6-epi-ophiobolin K[17]的文献报道一致。化合物13的比旋光度为[a]D20+170.0 (c 0.10, MeOH), 与文献报道[a]D23+155.0 (c 0.08 MeOH)的数据接近, 说明绝对构型也相同。
化合物14:无色透明固体, 13C NMR (125 MHz, DMSO-d6) δC: 171.1 (C, C-21), 145.2 (CH, C-8), 136.4 (C, C-19), 135.6 (CH, C-16), 126.2 (C, C-7), 124.1 (CH, C-17), 120.2 (CH, C-18), 112.2 (C, C-5), 80.5 (C, C-3), 54.0 (CH2, C-4), 53.0 (CH, C-2, 6), 52.1 (CH, C-14), 44.8 (CH2, C-12), 44.1 (C, C-11), 42.5 (CH, C-10), 41.7 (CH2, C-1), 33.1 (CH, C-15), 29.7 (CH2, C-9), 28.1 (CH2, C-13), 26.6 (CH3, C-25), 24.9 (CH3, C-20), 23.7 (CH3, C-22), 21.4 (CH3, C-23), 18.3 (CH3, C-24); 1H NMR (500 MHz, DMSO-d6) δH: 6.97 (1H, s, H-8), 6.03 (2H, m, H-17, 18), 5.13 (1H, t, J=9.9 Hz, H-16), 3.33 (1H, m, H-6), 2.58 (1H, d, J=20.7 Hz, H-15), 2.50 (1H, dd, J=16.2, 9.5 Hz, H-9), 2.36 (1H, d, J=12.3 Hz, H-10), 2.30 (1H, d, J=14.6 Hz, H-4), 2.19 (1H, d, J=14.4 Hz, H-4), 2.04 (1H, m, H-9), 1.89 (1H, dd, J=18.7, 7.9 Hz, H-14), 1.83 (3H, s, H-24), 1.69 (3H, s, H-25), 1.69 (2H, dd, J=12.7, 9.0 Hz, H-1, 2), 1.49 (2H, m, H-12, 1), 1.30 (3H, s, H-20), 1.21 (2H, m, H-13), 0.98 (3H, d, J=6.7 Hz, H-23), 0.79 (3H, s, H-22)。其波谱数据与ophiobolin P[18]的文献报道一致。化合物14的比旋光度为[a]D20+100.0 (c 0.05, MeOH)与文献报道[a]D25+93.8 (c 0.67, MeOH)的数据相近, 说明绝对构型与文献一致。
化合物15:白色透明固体, 13C NMR (125 MHz, DMSO-d6) δC: 139.4 (C, C-7), 138.0 (CH, C-16), 135.1 (C, C-19), 123.0 (CH, C-8), 121.9 (CH, C-17), 120.5 (CH, C-18), 119.7 (C, C-5), 79.8 (C, C-3), 71.7 (CH2, C-21), 55.3 (CH, C-10), 52.8 (CH, C-6), 50.1 (CH, C-2), 50.3 (CH2, C-4), 47.4 (CH, C-14), 43.7 (C, C-11), 43.2 (CH2, C-12), 36.2 (CH2, C-1), 35.7 (CH, C-15), 26.8 (CH2, C-13), 26.5 (CH3, C-25), 25.9 (CH3, C-20), 25.2 (CH2, C-9), 20.4 (CH3, C-23), 18.9 (CH3, C-22), 18.2 (CH3, C-24); 1H NMR (500 MHz, DMSO-d6) δH: 5.96 (2H, m, H-18, 17), 5.60 (1H, dd, J=8.2, 6.3 Hz, H-8), 5.20 (1H, m, H-16), 4.47 (2H, s, H-21), 3.01 (1H, d, J=9.8 Hz, H-6), 2.68 (1H, dd, J=15.9, 9.2 Hz, H-15), 2.48 (1H, dd, J=13.8, 8.5 Hz, H-9), 2.24 (1H, d, J=13.3 Hz, H-4), 2.19 (1H, dd, J=16.8, 7.0 Hz, H-2), 2.05 (1H, dt, J=18.4, 9.4 Hz, H-14), 1.91 (1H, d, J=13.3 Hz, H-4), 1.90 (3H, s, H-25), 1.72 (3H, s, H-24), 1.67 (2H, m, H-13, 9), 1.55 (3H, m, H-12, 10, 1), 1.38 (3H, m, H-13, 12, 1), 1.23 (3H, s, H-20), 0.89 (3H, s, H-22), 0.89 (3H, d, J=6.7 Hz, H-23)。其波谱数据与ophiobolin H[10]的文献报道一致。化合物15的比旋光度为[a]D20+17.0 (c 0.10, MeOH)与文献报道[a]D20+25.0 (c 0.10, CHCl3)的数据接近, 说明绝对构型也相同。
化合物16:无色透明液体, 13C NMR (125 MHz, DMSO-d6) δC: 205.8 (C, C-5), 193.1 (CH, C-21), 177.1 (C, C-3), 158.3 (CH, C-8), 139.6 (CH, C-16), 138.6 (C, C-7), 129.4 (CH, C-4), 128.0 (CH, C-17), 73.5 (CH, C-18), 71.4 (C, C-19), 51.2 (CH, C-14), 49.1 (CH, C-6), 48.6 (CH, C-2), 44.9 (C, C-11), 44.8 (CH2, C-1), 43.7 (CH2, C-12), 42.8 (CH, C-10), 32.1 (CH, C-15), 30.2 (CH2, C-9), 27.1 (CH2, C-13), 26.0 (CH3, C-25), 25.2 (CH3, C-24), 22.4 (CH3, C-22), 20.1 (CH3, C-23), 16.6 (CH3, C-20); 1H NMR (500 MHz, DMSO-d6) δH: 9.21 (1H, s, H-21), 6.92 (1H, d, J=4.5 Hz, H-8), 5.93 (1H, s, H-4), 5.32 (2H, m, H-17, 16), 4.48 (1H, d, J=4.9 Hz, H-18), 4.01 (1H, d, J=9.7 Hz, H-6), 3.08 (1H, d, J=20.4 Hz, H-9), 2.68 (1H, dd, J=17.4, 6.9 Hz, H-2), 2.59 (1H, m, H-15), 2.27 (2H, ddd, J=20.7, 14.4, 6.5 Hz, H-9, 10), 2.03 (3H, s, H-20), 1.97 (1H, m, H-1), 1.94 (1H, m, H-14), 1.57 (1H, m, H-13), 1.45 (1H, dd, J=11.5, 4.6 Hz, H-12), 1.38 (1H, td, J=11.1, 4.5 Hz, H-12), 1.27 (1H, m, H-13), 1.16 (1H, m, H-1), 1.06 (3H, s, H-24), 1.04 (3H, s, H-25), 0.92 (3H, d, J= 6.5 Hz, H-23), 0.81 (3H, s, H-22)。其波谱数据与ophiobolin Q[18]的文献报道一致。化合物16的比旋光度为[a]D20+140.0 (c 0.10, MeOH)与文献报道[a]D25+126.0 (c 0.39, MeOH)的数据相近, 说明绝对构型与文献一致。
化合物17:无色透明液体, 13C NMR (125 MHz, DMSO-d6) δC: 205.8 (C, C-5), 193.1 (CH, C-21), 177.0 (C, C-3), 157.8 (CH, C-8), 141.9 (CH, C-16), 139.8 (C, C-7), 129.4 (CH, C-4), 126.4 (CH, C-17), 82.9 (CH, C-18), 71.8 (C, C-19), 56.7 (CH3, 18-OCH3), 51.6 (CH, C-14), 49.1 (CH, C-6), 48.7 (CH, C-2), 44.9 (CH2, C-1), 44.8 (C, C-11), 43.7 (CH2, C-12), 42.7 (CH, C-10), 31.6 (CH, C-15), 31.3 (CH2, C-9), 27.1 (CH2, C-13), 26.8 (CH3, C-25), 24.8 (CH3, C-24), 22.3 (CH3, C-22), 21.1 (CH3, C-23), 16.6 (CH3, C-20); 1H NMR (500 MHz, DMSO-d6) δH: 9.20 (1H, d, J=6.7 Hz, H-21), 6.81 (1H, m, H-8), 5.93 (1H, s, H-4), 5.66 (1H, m, H-16), 5.20 (1H, t, J=10.6 Hz, H-17), 3.67 (1H, d, J=9.9 Hz, H-18), 3.25 (3H, s, 18-OCH3), 3.08 (1H, d, J=20.7 Hz, H-6), 2.74 (1H, t, J=12.4 Hz, H-2), 2.61 (1H, m, H-9), 2.53 (1H, m, H-15), 2.24 (1H, m, H-9), 2.05 (1H, m, H-1), 2.03 (3H, s, H-20), 1.98 (1H, m, H-14), 1.94 (1H, m, H-13), 1.59 (1H, m, H-12), 1.46 (1H, m, H-12), 1.39 (1H, m, H-10), 1.29 (1H, m, H-13), 1.17 (1H, m, H-1), 1.14 (3H, s, H-25), 1.05 (3H, s, H-24), 1.02 (3H, d, J=6.6 Hz, H-23), 0.80 (3H, s, H-22)。其波谱数据与ophiobolin R[18]的文献报道一致。化合物17的比旋光度为[a]D20+70.0 (c 0.10, MeOH)与文献报道[a]D25+60.8 (c 0.24, MeOH)的数据相近, 说明绝对构型与文献一致。
2.2 神经氨酸酶抑制活性结果实验结果显示化合物2、6、7、9、11、13对神经氨酸酶具有明显的抑制活性, 其半数抑制浓度(IC50)分别为31.8、37.3、28.4、36.8、46.6、37.6 μM, 而阳性对照的IC50则为1.2 μM。根据实验结果和化合物结构可知, 对于倍半萜类化合物, 11-OH和C-6所连侧链的不饱和程度可提高其神经氨酸酶抑制活性, 而二倍半萜类化合物则是C-21的醛基取代可提高其神经氨酸酶抑制活性。
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