水母共附生真菌Aspergillus fumigatus SCSIO41214中生物碱类化合物研究.pdf

从水母共附生真菌 Aspergillus fumigatus SCSIO41214中分离得到 8个已知生物碱类化合物。这些化合物主要采用包括正相硅胶、反相硅胶等常规柱层析技术,以及高效液相色谱进行分离纯化。首次通过核磁共振波谱等多种现代波谱技术确定了化合物 1 的平面结构,通过单晶衍射技术确定了其相对构型,化合物 28 是通过波谱分析并结合文献数据比对确定了化合物的结构。体外抗炎活性研究筛选发现,化合物 4 在 10molL1浓度下显示弱的抑制 RAW264.7 细胞中 LPS 引起的 NO的释放。关键词:水母;Aspergillus fumigatus SCSIO41214;生物碱;fumuquinazoline K 收稿日期：2022-09-16;修订日期：2022-10-08。姚衍桃编辑 基金项目：国家自然科学基金项目(21977102);广东省基础与应用基础研究基金粤深联合基金(2021B1515120046)作者简介：杨斌,男,副研究员,从事海洋药物化学研究。email: 通信作者：刘永宏。email: Received date:2022-09-16;Revised date:2022-10-08.Editor:YAO Yantao Foundation item:National Natural Science Foundation of China(21977102);Guangdong-Joint Foundation of Shenzhen(2021B1515120046)Corresponding author:LIU Yonghong.email: 1 Introduction Marine symbiotic and epiphyte microorganisms are widely recognized as rich sources for the discovery of structurally novel and biologically active natural products.However,only few studies have focused on isolation of small-molecule natural products from jellyfish-associated microoraganisms,which are also recognized as a productive resource of bioactive molecules(Wang et al,2015;Li et al,2021;Li et al,2022).The giant Nemopilema nomurai,which causes serious damage,inducing outbreaks in Chinas coastal waters and even death of tourists for stinging,is one of the most widely distributed venomous jellyfish(Yue et al,2015).In our preliminary study,several fungus strains obtained from giant jellyfish N.nomurai are investigated.Several relevant metabolites,including 196 热 带 海 洋 学 报 Vol.42,No.4/Jul.,2023 1 penicillixanthone A,displayed potent anti-HIV-1 activity by inhibiting infection against CCR5-tropic HIV-1 SF162,CXCR4-tropic HIV-1 NL4-3(Tan et al,2019)and fumigatosides B-D,and represented the first examples of glycosidated fumiquinzoline-type alkaloids from nature(Liu et al,2015),have been isolated.As part of our ongoing investigation into the unique and bioactive natural products from jellyfish derived fungus,a strain identified as Aspergillus fumigatus SCSIO41214 was studied,which led to the isolation of eight known compounds(Fig.1).Their structures were elucidated on the basis of extensive NMR spectroscopic data analysis,and X-ray single-crystal diffraction.Compounds 1 8 were tested for their inhibitory activities against LPS-induced NO production in RAW 264.7 cells.Fig.1 Structures of compounds 1 8 2 Results and discussion Compound 1 was isolated as a white crystal.Its molecular formula was established to be C23H22N4O5 by HRESIMS peak at m/z 457.1484 M+Na+(calcd.for C23H22NaN4O5 457.1482),indicating 15 degree unsaturation.The 1H NMR spectrum of 1 showed a methyl at H 1.61(d,J=6.5Hz,H-12),a methoxyl at H 3.17(s,H-27),three oxygenated/nitrogenated methines at H 5.92(s,H-14),5.61(s,H-2),4.63(q,J=7.0Hz,H-11),nine olefinic protons ranging from H 7.26 to 8.29.The 13CNMR,and DEPT spectroscopic data of 1 showed 23 carbon signals,including one methyl,one methoxyl,one methylene,twelve methines(i.e.,nine olefinic carbons and three nitrogenated carbons),and eight quaternary carbons(i.e.,one oxygenated carbon,three carbonyls,and four olefinic carbons).By the HSQC spectrum,all proton resonances were unambiguously assigned to their respective carbons.Its 1D and 2D NMR spectra of 1 were almost the same as those of chaetominine(2)(Jiao et al,2006).The distinction was attributed to the presence of a methoxyl group of 1,as which is evident from its molecular weight,to be 14 amu more than that of the known analogue,as well as in its NMR spectra.Further COSY correlation and HMBC correlations from CH3-27 to C-10,and CH3-12 to C-10,and C-11 confirmed the planar structure of 1,which was confirmed by its single-crystal X-ray crystallographic analysis(Fig.2).The achievable precision of the Flack parameter(0.5(3)for 1 is limited,which could only support the determination of the relative configurations as 2S*,3S*,11R*and 14R*.Consequently,the structure of 1 was determined and named as fumuquinazoline K.Fumuquinazoline K has been reported that it was able to prevent the conformation of the winged-helix domain of the methyl methanesulfonate and ultraviolet-sensitive 81(MUS81)via forming a strong binding affinity to the enzyme via perturbation approach(Ngo et al,2021).However,its structure,including the relative configurations of the stereogenic carbons,was firstly elucidated based on spectroscopic analysis and the X-ray data.杨斌等:水母共附生真菌 Aspergillus fumigatus SCSIO41214 中生物碱类化合物研究 197 1 Fig.2 Selected 1H-1H COSY,HMBC correlations and X-ray crystallographic structure of 1 Additionally,the structures of other known compounds,chaetominine(2)(Jiao et al,2006),3-(4-oxoquinazolin-3-yl)spiro5-oxolane-2,2-dione(3)(Buttachon et al.2012),fumiquinazoline J(4)Zhang et al,2011;Yang et al,2013),fumitremorgin C(5)(Wang et al,2019),spirotryprostatin A(6)(Cui et al,1996;Wang et al,2019),brevianamide F(7)(Son et al,2007),cyclo-(L-Pro-L-Tyr)(8)(Jayatilake et al,1996)were also isolated and identified by using their NMR and MS data,and comparing these data with those reported in the literature.This is the first report of these compounds from the jellyfish associated fungus.The anti-inflammatory activities of the isolated metabolites were also evaluated on the basis of their inhibition effects of NO production in the LPS induced mouse macrophage RAW264.7 cells.At a concentration of 10M,compound 4 showed inhibitory abilities with inhibitory rates at 28.5%,while the positive control LNMMA showed inhibition rate at 31.1%.Other compounds showed no inhibition activities.3 The physicochemical data of the compounds Fumuquinazoline K(1):HR ESI-MS m/z 435.1670 M+H+(calcd.for C23H23N4O5 435.1663);457.1484 M+Na+(calcd.for C23H22NaN4O5 457.1482);25D 3.5(0.1,c,MeOH);UV(MeOH)max(log):210(0.74),230(0.52)nm;IR(film)vmax:3356,1724,1666,1608,1479cm1;1H NMR(500MHz,DMSO-d6):8.29(1H,s,H-25),8.18(1H,s,H-19),7.88(1H,td,J=8.5,1.5Hz,H-21),7.70(1H,d,J=8.0Hz,H-22),7.59(1H,t,J=7.5Hz,H-20),7.51(1H,d,J=8.0Hz,H-8),7.50(1H,d,J=7.5Hz,H-5),7.44(1H,td,J=7.5,1.0Hz,H-7),7.26(1H,td,J=7.5,1.0Hz,H-6),5.92(1H,s,H-14),5.61(1H,s,H-2),4.63(1H,q,J=7.0Hz,H-11),3.17(3H,s,H-27),2.96(1H,t,J=7.5Hz,H-13a),2.54(1H,m,H-13b),1.61(3H,d,J=6.5Hz,H-12).13C NMR(125MHz,DMSO-d6):172.0(C,C-10),165.7(C,C-17),160.0(C,C-15),147.4(C,C-23),146.6(C,C-25),138.7(C,C-9),136.7(C,C-4),134.7(CH,C-21),129.9(CH,C-7),127.3(CH,C-20),127.2(CH,C-22),126.4(CH,C-19),125.5(CH,C-6),124.9(CH,C-5),121.2(C,C-18),114.5(CH,C-8),82.5(CH,C-2),76.4(C,C-3),59.8(CH,C-14),50.4(CH,C-11),48.6(CH3,C-27),39.0(CH2,C-13),14.0(CH3,C-12).X-ray crystal data for compound 1:chemical formulae C23H24N4O6,F.W=434.44,white color crystals,0.150.110.1mm3,trigonal,space group:P3121,a=10.7396(4),b=10.7396(4),c=35.6809(12),=90,=120.V=3564.0(3)3,Z=6,Dc=1.214g/cm3,F(000)=1368.0,Cu K radiation,=1.54184,range for data collection:7.432 to 148.206;8851 reflections collected,4382 unique(Rint=0.0564),the structure was refined by full-matrix least-squares on F2.Final GoF=1.047.Final R indexes I2(I):R1=0.0793,wR2=0.2256.Final R indexes(all data)R1=0.0.0846,wR2=0.2360;Flack Parameters=0.5(3);Largest diff.peak and hole:0.48 and-0.48e3.Data were collected on Agilent Xcalibur Nova single-crystal diffractometer using Cu K radiation.The crystal structure was refined by full-matrix least-squares calculation.Crystallographic data for the structure of 1 have been deposited in the Cambridge Crystallographic Data Centre(deposition number:CCDC 1847139).Chaetominine(2):ESI-MS m/z:403 M+H+;1H NMR(700MHz,CD3OD):8.23(1H,brs,H-24),8.13 198 热 带 海 洋 学 报 Vol.42,No.4/Jul.,2023 1(1H,s,H-19),7.81(1H,t,J=7.7Hz,H-21),7.70(1H,d,J=7.5Hz,H-22),7.52(1H,t,J=7.0Hz,H-20),7.45(1H,d,J=8.4Hz,H-5),7.37(1H,t,J=7.7Hz,H-8),7.20(1H,t,J=7.7Hz,H-6),5.88(1H,s,H-12),5.55(1H,s,H-2),4.55(1H,q,J=14.0,6.3Hz,H-24),2.88(1H,t,J=12.6Hz,H-13a),2.47(1H,d,J=11.9Hz,H-13b),1.55(1H,d,J=2.8Hz,H-26).13C NMR(150MHz,CD3OD):172.0(C,C-25),165.6(C,C-15),160.0(C,C-11),147.4(C,C-23),146.8(C,C-21),138.7(C,C-9),134.7(CH,C-4,C-19),129.9(CH,C-7),127.3(CH,C-17),127.2(CH,C-18),126.4(CH,C-20),125.5(CH,C-6),124.9(CH,C-5),120.9(C,C-16),114.5(CH,C-8),82.5(CH,C-2),76.4(C,C-3),59.6(CH,C-24),50.1(CH,C-14),38.2(CH2,C-13),14.0(CH3,C-12).3-(4-oxoquinazolin-3-yl)spiro5-oxolane-2,2-dione(3):Pale yellow oil;ESI-MS m/z:348 M+H+;1H NMR(700MHz,CDCl3):8.64(1H,s,H-22),8.29(1H,d,J=7.7Hz,H-16),7.90(1H,t,J=7.7Hz,H-18),7.77(1H,d,J=7.0Hz,H-5),7.76(1H,d,J=8.6Hz,H-19),7.75(1H,d,J=7.7Hz,H-17),7.40(1H,t,J=7.7Hz,H-7),7.19(1H,t,J=7.7Hz,H-6),7.02(1H,d,J=7.7Hz,H-8),5.70(1H,t,J=10.5Hz,H-11),3.25(1H,dd,J=14.0,10.5Hz,H-12a),3.11(1H,dd,J=14.0,4.2Hz,H-12b).13C NMR(150MHz,CDCl3):175.4(C,C-2),171.1(C,C-10),160.1(C,C-14),147.2(CH,C-22),147.2(C,C-20),142.6(C,C-9),134.6(CH,C-18),131.4(CH,C-7),127.6(CH,C-17),127.3(CH,C-19),126.7(C,C-4),126.1(CH,C-16),125.4(CH,C-5),123.1(CH,C-6),121.9(C,C-15),110.5(CH,C-8),80.7(C,C-3),57.2(CH,C-11),33.7(CH2,C-12).Fumiquinazoline J(4):ESI-MS m/z:357 M+H+;1H NMR(700MHz,DMSO-d6):9.58(1H,s,H-19),8.15(1H,dd,J=8.0,1.0Hz,H-10),7.81(1H,td,J=7.8,7.3Hz,H-8),7.64(1H,d,J=8.0Hz,H-7),7.53(1H,td,J=7.8,7.5Hz,H-9),7.41(1H,d,J=8.0Hz,H-24),7.38(1H,d,J=8.5Hz,H-21),7.11(1H,td,J=8.0,7.0Hz,H-22),7.00(1H,td,J=7.5,7.0Hz,H-22),5.71(1H,br,H-14),3.44(1H,dd,J=17.5,3.5Hz,H-15a),3.24(1H,dd,J=18.0,4.5Hz,H-15b),2.12(3H,s,H-16).13C NMR(150MHz,DMSO-d6):169.2(C,C-1),159.4(C,C-12),154.4(C,C-4),146.7(C,C-6),134.9(CH,C-8),134.0(C,C-18,C-20),127.4(CH,C-7,C-9),127.3(C,C-25),126.4(CH,C-10),122.3(CH,C-22),120.2(C,C-11),119.4(CH,C-23),118.2(CH,C-24),111.8(CH,C-21),105.6(C,C-17),54.6(C,C-3),54.2(CH,C-14),25.6(CH2,C-15),18.4(CH3,C-16).Fumitremorgin C(5):ESI-MS m/z:380 M+H+;1H NMR(700MHz,CDCl3):7.94(1H,s,H-1),7.43(1H,d,J=8.6Hz,H-16),6.85(1H,d,J=2.1Hz,H-19),6.82(1H,d,J=9.1Hz,H-17),5.98(1H,d,J=9.5Hz,H-3),4.83(1H,m,H-6),4.18(1H,dd,J=11.6,4.9Hz,H-6),4.05(1H,m,H-12),3.83(3H,s,OCH3-18),3.60(2H,m,H-9),3.44(2H,m,H-15),3.10(dd,J=15.8,11.6Hz,H-13),2.34(1H,m,H-7a),2.23(1H,d,J=1.9Hz,H-7b),2.06(1H,dd,J=7.2,4.1Hz,H-8a),1.94(1H,d,J=8.7Hz,H-8b),1.94(3H,d,J=0.8Hz,H-23),1.64(3H,d,J=0.8Hz,H-24).13C NMR(150MHz,CDCl3):169.6(C,C-5),165.8(C,C-11),156.5(C,C-18),137.1(C,C-20),134.0(C,C-22),132.2(C,C-2),124.2(CH,C-21),120.8(C,C-15),118.9(CH,C-16),109.5(CH,C-17),106.2(C,C-14),95.3(CH,C-19),59.3(CH,C-6),56.8(CH,C-12),55.8(CH3,C-25),51.0(CH,C-3),45.4(CH2,C-9),30.6(CH2,C-7),28.6(CH2,C-7),25.7(CH3,C-23),23.1(CH2,C-8),22.0(CH2,C-13),18.1(CH3,C-24).Spirotryprostatin A(6):ESI-MS m/z:396 M+H+;1H NMR(700MHz,CD3OD):7.04(1H,d,J=8.4Hz,H-4),6.56(1H,dd,J=8.4,2.1Hz,H-5),6.50(1H,d,J=2.1Hz,H-7),5.11(1H,dt,J=9.1,1.4Hz,H-19),5.04(1H,dd,J=12.5,7.7Hz,H-9),4.73(1H,d,J=9.1Hz,H-18),4.45(1H,t,J=7.7Hz,H-12),3.80(3H,s,OMe-6),3.57(1H,m,H-15),2.61(1H,dd,J=13.3,9.5Hz,H-8a),2.35(1H,dd,J=13.3,10.1Hz,H-8b),2.33(1H,m,H-13a),2.14(1H,m,H-13b),2.06(1H,m,H-14a),2.03(1H,m,H-14b),1.67(3H,d,J=0.7Hz,H-21),1.20(3H,d,J=8.4Hz,H-22).13C NMR(150MHz,CD3OD):183.2(C,C-2),169.5(C,C-11),168.9(C,C-17),162.2(C,C-6),144.4(C,C-7a),139.1(C,C-20),128.2(CH,C-4),122.7(CH,C-19),120.1(C,C-3a),107.7(CH,C-5),97.7(CH,C-7),62.3(CH,C-9),61.8(CH,C-12),59.8(CH,C-18),57.1(C,C-3),55.9(C,OCH3-6),46.2(CH2,C-14),35.3(CH2,C-8),28.5(CH2,C-13),25.6(CH3,C-22),24.5(CH2,C-15),18.1(CH3,C-21).Brevianamide F(7):ESI-MS m/z:284 M+H+;25D75.3(c 0.1 MeoH);1H NMR(500MHz,CD3OD):7.75(1H,s,NH-1),7.58(1H,d,J=8.0Hz,H-4),7.34(1H,d,J=8.0Hz,H-7),7.12(1H,d,J=2.0Hz,C-2),7.07 杨斌等:水母共附生真菌 Aspergillus fumigatus SCSIO41214 中生物碱类化合物研究 199 1(1H,t,J=7.5Hz,H-6),6.98(1H,t,J=7.5Hz,H-5),4.32(1H,t,J=5.0Hz,H-9),4.09(1H,t,J=8.5Hz,H-12),3.32(1H,m,H-15a),3.23(2H,m,H-15b,H-8a),3.09(1H,dd,J=14.5,5.5Hz,H-8b),1.99(1H,m,H-14a),1.68(1H,m,H-14b),1.61(1H,m,H-13a),1.40(1H,m,H-13b).13C NMR(125MHz,CD3OD):169.0(C,C-11),165.5(C,C-17),135.9(C,C-7a),127.3(C,C-3a),124.4(CH,C-2),120.8(CH,C-6),118.6(CH,C-4),118.2(CH,C-5),111.2(CH,C-7),109.2(C,C-3),58.4(CH,C-12),55.2(CH,C-9),44.6(CH2,C-15),27.6(CH2,C-14),25.8(CH2,C-8),21.8(CH2,C-13).Cyclo-(L-Pro-L-Tyr)(8):ESI-MS m/z:261 M+H+;25D51.2(c 0.2 MeoH);1H NMR(500MHz,CD3OD):7.28(1H,s,NH-2),7.09(2H,d,J=7.5Hz,H-12,H-16),6.81(2H,d,J=7.5Hz,H-13,H-15),5.89(1H,s,NH-8),4.25(1H,dd,J=9.5,2.0Hz,H-9),4.12(1H,t,J=8.0Hz,H-6),3.67(1H,m,H-3a),3.61(1H,m,H-3b),3.52(1H,dd,J=15.0,4.0Hz,H-10a),2.82(1H,dd,J=14.5,10.0Hz,H-10b),2.38(1H,m,H-5a),2.04(1H,m,H-4a),1.96(1H,m,H-5b),1.94(1H,m,H-4b).13C NMR(125MHz,CD3OD):169.7(C,C-7),165.2(C,C-1),155.6(C,C-14),130.3(CH,C-12,C-16),127.0(C,C-11),116.1(CH,C-13,C-15),59.2(CH,C-6),56.3(CH,C-9),45.5(CH2,C-3),35.9(CH2,C-10),28.4(CH2,C-5),22.5(CH2,C-4).4 Experiment 4.1 General experimental procedures The NMR spectra was measured with a Bruker-AC500 or AVANCE-III-HD700 NMR spectrometer with TMS as an internal standard.HR-ESI-MS data was recorded on a Bruker micro-TOF-QII mass spectrometer.Optical rotations were recorded on an Anton Paar MCP500 polarimeter.CD spectra was recorded with a Chirascan circular dichroism spectrometer(Applied Photophysics).UV spectra was obtained on a Shimadzu UV-2401PC spectrophotometer(Shimadzu Corporation,Kyoto,Japan).Semi-preparative HPLC was performed on HTACHI L2000 and HTACHI L2130 with YMC ODS SERIES(YMC-pack ODS-A,YMC Co.,Ltd.,10mm250mm,5m,Kyoto,Japan).YMC gel(ODS-A,12nm,S-50m)was used for column chromatography.The silica gel GF254 used for TLC was supplied by the Qingdao Marine Chemical Factory,Qingdao,China.4.2 Fungal material Aspergillus fumigatus SCSIO41214(Original Number:J08NF-8)was isolated from the jellyfish Nemopilema nomurai collected off the southern coast of Korea in June 2007 and identified by Dr.K.S.Bae.The specimen was identified by Dr.Xiuping Lin.The strain was deposited in the RNAM Center,South China Sea Institute of Oceanology,Chinese Academy of Sciences.4.3 Extraction and isolation The strain SCSIO41214 was cultured