Brominated gamma-arylbutenolide compounds, methods of making and using the same

Abstract

The application discloses a kind of brominated gamma-aryl butenolide compounds and preparation method and application thereof, first from marine-derived microorganism isolation obtain series brominated gamma-aryl butenolide compounds, to five kinds of agricultural economic crops pathogenic bacteria: Colletotrichum gloeosporioides, olive fruit rot fungus Pestalotiopsis microspora, banana wilt fungus Panama disease, Botrytis cinerea and oil gan black rot fungus Diaporthephoenicicola have good antibacterial effect, and to clinical first-line tumor drug camptothecin, etoposide drug resistance enzyme TDP1 and TDP2 have significant inhibitory effect, provide pharmacodynamic material basis for subsequent fruit and vegetable agricultural fungi antibacterial research, and provide solid experimental data for widening the application of marine drug source molecule.

Description

Technical field:

[0001] The present invention relates to the field of pharmaceutical biotechnology, and in particular to a brominated gamma-aryl butene lactone compound, a preparation method and an application thereof. Background technology:

[0002] γ-Arylbutenolides are a class of natural products containing a four-carbon heterocyclic lactone substituted with 3-phenyl and 4-benzyl groups. In terms of habitat / source, γ-arylbutenolides are primarily derived from marine microorganisms, endophytic fungi in plant and animal samples, and environmental fungi isolated from soil. Statistics show that 176 γ-arylbutenolides have been reported, of which 106 compounds, or 54%, originate from marine microorganisms. Fifty-seven compounds, or 29%, originate from plant endophytic fungi. Bioactivity analysis of these 176 compounds revealed that 68.2% (120 compounds) exhibited a wide range of pharmacological activities, including antioxidant (32), antiviral (30), α-glucosidase inhibition (28), antibacterial (25), anti-inflammatory (13), anti-tumor (12), cytotoxic (5), and antifouling (5). The remaining active compounds showed other activities (9 compounds, accounting for 6%), such as enhancing learning and memory activity and protective activity (3 compounds, accounting for 2.4%).

[0003] As the number of new aromatic γ-butenolide compounds continues to increase, reviews of these structures have also been reported. For example, Joe and colleagues have reviewed butenolides from marine microorganisms, but they only focused on three types of butenolides (rubrolides, cadiolides, and prunolides) from marine tunicates. Chatterjee et al. reviewed the synthetic strategies of aromatic γ-butenolides and the research on related natural products from 2010 to 2023, focusing mainly on the synthesis of γ-butenolides, but less research has been conducted on the isolation of their natural products, their structural diversity, and their biological activities.

[0004] The inventors have also previously studied the preparation method of γ-aryl butene lactone compounds and their application in the preparation of cardiovascular protective drugs, see CN116947831A. The formula of the rice culture medium used in this patent is: 100g rice, 115mL water, salinity 3.3%, and natural pH, which requires further study. Summary of the invention:

[0005] The purpose of the present invention is to provide a brominated γ-aryl butene lactone compound and a preparation method and application thereof.

[0006] The present invention is achieved through the following technical solutions:

[0007] Brominated γ-aryl butenolide compounds, the structural formula of which is as follows:

[0008]

[0009] The preparation method of the brominated γ-aryl butenolide compound is separated from the fermentation culture of the fungus Aspergillus terreus EGF7-0-1 symbiotically attached to the marine herbal soft coral (Sinularia sp.), and specifically comprises the following steps:

[0010] 1) Cultivation of the fungus Aspergillus terreus EGF7-0-1, a symbiotic fungus of marine herbal soft coral (Sinularia sp.): The fungus Aspergillus terreus EGF7-0-1, a symbiotic fungus of marine herbal soft coral, was inoculated into a culture flask containing PDA medium and cultured in a shaking incubator at 28°C to obtain an activated seed culture solution. The activated seed culture solution was then inoculated into a culture flask containing rice culture medium and cultured in a static incubator at 28°C for 35 days. The rice culture medium consisted of 100 g of rice, 115 mL of water, 3.0% to 5.0% NaBr, and a natural pH.

[0011] 2) extracting the culture medium of the combined bacteria obtained in step 1) with ethyl acetate, and then concentrating to obtain rice single culture ethyl acetate extract, and then subjecting it to silica gel column chromatography with petroleum ether-ethyl acetate system to V PE :V EtOAc =100:0→0:100 elution, and 8 crude fractions Fr.1 to Fr.8 were obtained based on thin layer TLC results. LC-MS analysis confirmed that Fr.4, Fr.5, Fr.6, and Fr.8 were the target fractions. After secondary separation by silica gel column chromatography, HPLC and chiral separation were performed to obtain compounds 1-14. The specific steps are as follows:

[0012] Fr.4 was purified by silica gel column chromatography using a dichloromethane-methanol system. Elution gave four fractions, Fr.4-1 to Fr.4-4; Fr.4-1 was purified by reverse phase preparative HPLC (RP-HPLC) to As the mobile phase, the mixture I(t R =36.5min); Mixture I was subjected to normal phase semi-preparative HPLC (NP-HPLC) with V 正己烷 :V 异丙醇 =20:80 as the mobile phase for chiral separation to obtain compound 1 (t R =16.0 min) and compound 2 (t R =19.0min); Fr.4-3 was prepared by reverse phase HPLC (RP-HPLC) to Purification with mobile phase gave mixture II (t R =42.0 min) and compound 5 (t R =45.4min); Mixture II was subjected to normal phase semi-preparative HPLC (NP-HPLC) with V 正己烷 :V 异丙醇 =20:80 as the mobile phase for chiral separation to obtain compound 3 (t R =18.0 min) and compound 4 (t R =21.2min);

[0013] Fr.5 was purified by silica gel column chromatography using a dichloromethane-methanol system. Elution gave five fractions: Fr.5-1 to Fr.5-5; Fr.5-3 was purified by RP-HPLC. Purification with mobile phase gave mixture III (t R =25.5min) and compound 8 (t R =36.5min); the mixture III was subjected to NP-HPLC to V 正己烷 :V 异丙醇 =22:78 as the mobile phase for chiral separation to obtain compound 6 (t R =20.0 min) and compound 7 (t R =23.0min);

[0014] Fr.6 was purified by silica gel column chromatography using a dichloromethane-methanol system. Elution gave 4 fractions Fr.6-1 to Fr.6-4; Fr.6-2 was purified by RP-HPLC. The mobile phase was used for purification to obtain compound 9 (t R =14.0 min) and compound 10 (t R =16.0min); Fr.6-3 was analyzed by RP-HPLC with The mobile phase was used for purification to obtain compound 11 (t R =55.0 min) and compound 12 (t R =59.0min);

[0015] Fr.8 was purified by silica gel column chromatography using a dichloromethane-methanol system. Elution gave 6 fractions Fr.8-1 to Fr.8-6; Fr.8-3 was purified by RP-HPLC. Purification with mobile phase gave mixture IV (t R =45.5min); the mixture IV was subjected to NP-HPLC to V 正己烷 :V 异丙醇 =30:70 as the mobile phase for chiral separation to obtain compound 13 (t R=35.3 min) and compound 14 (t R =40.0min).

[0016] Preferably, the formula of the PDA culture medium is: 200 g potatoes, 20 g glucose, 15-20 g agar, 1000 mL water, 3.5% salinity, and natural pH.

[0017] Preferably, in the above step 2), the normal phase semi-preparative HPLC (NP-HPLC) uses a Phenylephrine semi-preparative column with a specification of 10 mm × 250 mm, 5 μm (chromatographic column 2); the reverse phase preparative HPLC (RP-HPLC) uses a Kromasil semi-preparative column with a specification of 10 mm × 250 mm, 5 μm (chromatographic column 1);

[0018] Preferably, in the above step 2), the detection wavelengths are 220 and 306 nm, and the flow rate of the mobile phase is 2 mL / min.

[0019] The inventors isolated and obtained the above-mentioned new brominated γ-aromatic ring butene lactone compounds for the first time from the fermentation culture of the fungus Aspergillusterreus EGF7-0-1, which is symbiotically attached to the marine herbal soft coral (Sinularia sp.), and found that it has antibacterial activity against pathogenic fungi of agricultural economic crops, and has a significant inhibitory effect on the clinical first-line tumor drug camptothecin and etoposide resistance enzymes TDP1 and TDP2.

[0020] The present invention also protects the use of the brominated γ-aryl butenolide compounds in the preparation of inhibitors of pathogenic fungi of agricultural economic crops, as well as the use of the brominated γ-aryl butenolide compounds in the preparation of inhibitors of tumor drugs such as camptothecin and etoposide resistance enzymes TDP1 and TDP2.

[0021] The beneficial effects of the present invention are as follows: The inventors isolated the novel brominated γ-aromatic ring butenolide compounds described above from the fermentation culture of the fungus Aspergillus terreus EGF7-0-1, a symbiotic organism of the marine herbal soft coral (Sinularia sp.). Furthermore, the inventors discovered for the first time that this series of compounds exhibited significant antibacterial effects against fungi pathogenic to fruits and vegetables, including papaya anthracnose (Colletotrichum gloeosporioides), olive fruit rot (Pestalotiopsis microspora), banana wilt (Panama disease), gray mold (Botrytis cinerea), and black rot (Diaporthephoenicicola), as well as significant inhibitory effects against the clinically first-line oncology drug camptothecin and the etoposide resistance enzymes TDP1 and TDP2, providing solid experimental data for broadening the application of marine-derived drug molecules.

[0022] The marine herbal soft coral (Sinularia sp.) symbiotic fungus Aspergillus terreus EGF7-0-1 of the present invention has been disclosed in CN116947831A. Description of the drawings:

[0023] Figure 1 It is compound 1-14 1 H- 1 Schematic diagram of H-COSY and key HMBC correlation;

[0024] Figure 2 is the HRESIMS mass spectrum of compound 1;

[0025] Figure 3 is the IR spectrum of compound 1;

[0026] Figure 4 are the measured and calculated ECD spectra of compound 1;

[0027] Figure 5 is compound 1 1 H- 1 H COSY spectrum (CD3OD);

[0028] Figure 6 is the HMBC spectrum (CD3OD) of compound 1;

[0029] Figure 7 is the HR-ESI-MS mass spectrum of compound 2;

[0030] Figure 8 is the IR spectrum of compound 2;

[0031] Figure 9 are the measured and calculated ECD spectra of compound 2;

[0032] Figure 10 is compound 2 1 H- 1 H COSY spectrum (CD3OD);

[0033] Figure 11 is the HMBC spectrum (CD3OD) of compound 2;

[0034] Figure 12 is the HRESIMS mass spectrum of compound 3;

[0035] Figure 13 is the IR spectrum of compound 3;

[0036] Figure 14 are the measured and calculated ECD spectra of compound 3;

[0037] Figure 15 is compound 3 1 H- 1 H COSY spectrum (CD3OD);

[0038] Figure 16 is the HMBC spectrum (CD3OD) of compound 3;

[0039] Figure 17 is the HRESIMS mass spectrum of compound 4;

[0040] Figure 18 is the IR spectrum of compound 4;

[0041] Figure 19 are the measured and calculated ECD spectra of compound 4;

[0042] Figure 20 is compound 4 1 H- 1 H COSY spectrum (CD3OD);

[0043] Figure 21 is the HMBC spectrum (CD3OD) of compound 4;

[0044] Figure 22 is the HRESIMS mass spectrum of compound 5;

[0045] Figure 23 is the IR spectrum of compound 5;

[0046] Figure 24 are the measured and calculated ECD spectra of compound 5;

[0047] Figure 25 is compound 5 1 H- 1 H COSY spectrum (CD3OD);

[0048] Figure 26 is the HMBC spectrum (CD3OD) of compound 5;

[0049] Figure 27 is the HRESIMS mass spectrum of compound 6;

[0050] Figure 28 is the IR spectrum of compound 6;

[0051] Figure 29 are the measured and calculated ECD spectra of compound 6;

[0052] Figure 30 is compound 6 1 H- 1H COSY spectrum;

[0053] Figure 31 is the HMBC spectrum (CD3OD) of compound 6;

[0054] Figure 32 is the HRESIMS mass spectrum of compound 7;

[0055] Figure 33 is the IR spectrum of compound 7;

[0056] Figure 34 are the measured and calculated ECD spectra of compound 7;

[0057] Figure 35 is compound 7 1 H- 1 H COSY spectrum (CD3OD);

[0058] Figure 36 is the HMBC spectrum (CD3OD) of compound 7;

[0059] Figure 37 is the HRESIMS mass spectrum of compound 8;

[0060] Figure 38 is the IR spectrum of compound 8;

[0061] Figure 39 are the measured and calculated ECD spectra of compound 8;

[0062] Figure 40 is compound 8 1 H- 1 H COSY spectrum;

[0063] Figure 41 is the HMBC spectrum (CD3OD) of compound 8;

[0064] Figure 42 is the HRESIMS mass spectrum of compound 9;

[0065] Figure 43 is the IR spectrum of compound 9;

[0066] Figure 44 are the measured and calculated ECD spectra of compound 9;

[0067] Figure 45 is compound 9 1 H- 1 H COSY spectrum (CD3OD);

[0068] Figure 46 is the HMBC spectrum (CD3OD) of compound 9;

[0069] Figure 47 is the HRESIMS mass spectrum of compound 10;

[0070] Figure 48 is the IR spectrum of compound 10;

[0071] Figure 49 are the measured and calculated ECD spectra of compound 10;

[0072] Figure 50 is compound 10 1 H- 1 H COSY spectrum (CD3OD);

[0073] Figure 51 is the HMBC spectrum (CD3OD) of compound 10;

[0074] Figure 52 is the HRESIMS mass spectrum of compound 11;

[0075] Figure 53 is the IR spectrum of compound 11;

[0076] Figure 54 are the measured and calculated ECD spectra of compound 11;

[0077] Figure 55 is compound 11 1 H- 1 H COSY spectrum (CD3OD);

[0078] Figure 56 is the HMBC spectrum (CD3OD) of compound 11;

[0079] Figure 57 is the HRESIMS mass spectrum of compound 12;

[0080] Figure 58 is the IR spectrum of compound 12;

[0081] Figure 59 are the measured and calculated ECD spectra of compound 12;

[0082] Figure 60 is compound 12 1 H- 1 H COSY spectrum (CD3OD);

[0083] Figure 61 is the HMBC spectrum (CD3OD) of compound 12;

[0084] Figure 62 is the HRESIMS mass spectrum of compound 13;

[0085] Figure 63 is an IR chart of compound 13;

[0086] Figure 64 is the experimental and calculated ECD spectra of compound 13;

[0087] Figure 65 is the 1 H- 1 H COSY spectra (CD3OD);

[0088] Figure 66 is the HMBC spectra of compound 13 (CD3OD);

[0089] Figure 67 is the HRESIMS spectra of compound 14;

[0090] Figure 68 is an IR chart of compound 14;

[0091] Figure 69 is the experimental and calculated ECD spectra of compound 14;

[0092] Figure 70 is the 1 H- 1 H COSY spectra (CD3OD);

[0093] Figure 71 is the HMBC spectra of compound 14 (CD3OD);

[0094] Figure 72 is the chiral resolution chart of compounds 1 and 2;

[0095] Figure 73 is the chiral resolution chart of compounds 3 and 4;

[0096] Figure 74 is the chiral resolution chart of compounds 6 and 7;

[0097] Figure 75 is the chiral resolution chart of compounds 13 and 14. DETAILED DESCRIPTION:

[0098] The following further describes the present application without limiting the present application.

[0099] Example 1 Preparation of bromo γ-aryl butenolide compounds

[0100] I. Microbial fermentation

[0101] Culture medium composition:

[0102] Rice culture medium: 100 g rice, 115 mL water, 3.0% to 5.0% NaBr, natural pH;

[0103] PDA medium: 200 g potatoes, 20 g glucose, 20 g agar, 1000 mL water, 3.5% salinity, natural pH.

[0104] Activation of the strain: Use an inoculating loop to pick the Aspergillus terreus EGF7-0-1 strain, a symbiotic fungus of the marine herbal soft coral (Sinularia sp.), from the strain tube and transfer it to a culture bottle containing PDA medium (400 mL / 1000 mL). Place the bottle in a constant temperature shaker (165 rpm) at 28°C for 2 days to obtain seed solution.

[0105] Inoculation: Pipette 10 mL of activated seed culture into a 1-L culture flask containing 400 mL of rice culture medium for a single culture of 50 L. Incubate the culture at 28°C for 35 days.

[0106] 2. Separation of brominated γ-arylbutene lactone compounds

[0107] Extraction: After the rice culture of strain EGF7-0-1 was completed, 250 mL of EtOAc was added to each culture flask, and the flask was shaken on a shaker at 165 r / min for 3 times (8 h / time). The EtOAc extracts were filtered and combined, and the rice single culture EtOAc extracts (65 g) were obtained after vacuum concentration. The extracts were then eluted with petroleum ether-ethyl acetate system on a silica gel column (V PE :V EtOAc =100:0→0:100, 1L / bottle), and 8 crude fractions Fr.1 to Fr.8 were combined based on thin-layer TLC results. After obtaining the extract, targeted separation was performed based on the characteristics of halogen and isotopic peaks. After obtaining crude fractions via silica gel column chromatography, LC-MS analysis was performed to obtain UV, MS, and isotopic peak information, identifying Fr.4, Fr.5, Fr.6, and Fr.8 as target fractions. After secondary separation by silica gel column chromatography, HPLC (both 220 and 306 nm detection) and chiral separation were performed to obtain compounds 1-14. Specific separation information is as follows:

[0108] Fr.4 (1.1 g) was purified by silica gel column chromatography using a dichloromethane-methanol system. Elution gave 4 fractions (Fr.4-1 to Fr.4-4). Fr.4-1 (28.3 mg) was purified by RP-HPLC (Kromasil semi-preparative column 10 mm × 250 mm, 5 μm, designated as column 1, 2mL / min) to obtain a mixture I (t R=36.5min, 25.5mg); the mixture I was subjected to NP-HPLC (Phenomen semi-preparative column 10mm×250mm, 5μm, recorded as column 2, V 正己烷 :V 异丙醇 =20:80, 2 mL / min) to obtain compound 1 (t R =16.0 min, 10.5 mg) and compound 2 (t R =19.0min, 10.0mg). Fr.4-3 (0.9g) was purified by RP-HPLC (column 1, 2mL / min) to obtain mixture II (t R =42.0 min, 18.0 mg) and compound 5 (t R =45.4min, 6.5mg); Mixture II was HPLC-ELECTROPHOSPHATE (column 2, V 正己烷 :V 异丙醇 =20:80, 2 mL / min) to give compound 3 (t R =18.0 min, 7.5 mg) and compound 4 (t R =21.2min, 7.8mg).

[0109] Fr.5 (46.5 g) was purified by silica gel column chromatography using a dichloromethane-methanol system. Five fractions (Fr.5-1 to Fr.5-5) were obtained by elution. Fr.5-3 (50.5 mg) was purified by RP-HPLC (column 1, 2mL / min) to obtain mixture III (t R =25.5min, 30.0mg) and compound 8 (t R =36.5min, 5.0mg); Mixture III was chirally separated by NP-HPLC (chromatographic column 2, V 正己烷 :V 异丙醇 =22:78, 2 mL / min) to obtain compound 6 (t R =20.0 min, 12.5 mg) and compound 7 (t R =23.0min, 12.4mg).

[0110] Fr.6 (5.1 g) was purified by silica gel column chromatography using a dichloromethane-methanol system. Four fractions (Fr.6-1 to Fr.6-4) were obtained by elution. Fr.6-2 (1.5 g) was purified by RP-HPLC (column 1, 2mL / min) to obtain compound 9 (t R =14.0 min, 7.5 mg) and compound 10 (t R=16.0min, 7.7mg). Fr.6-3 (0.6g) was purified by RP-HPLC (column 1, 2mL / min) to obtain compound 11 (t R =55.0min, 10.2mg) and compound 12 (t R =59.0min, 10.0mg).

[0111] Fr.8 (8.3 g) was purified by silica gel column chromatography using a dichloromethane-methanol system. Elution gave 6 fractions (Fr.8-1 to Fr.8-6). Fr.8-3 (2.5 g) was purified by RP-HPLC (column 1, 2mL / min) to obtain the mixture IV (t R =45.5min, 17.5mg). Mixture IV was chirally separated by NP-HPLC (chromatographic column 2, V 正己烷 :V 异丙醇 =30:70, 2 mL / min) to obtain compound 13 (t R =35.3 min, 7.0 mg) and compound 14 (t R =40.0 min, 7.0 mg).

[0112] The structural formulas of Compound 1-Compound 14 are as follows:

[0113]

[0114] 3. Structural identification of brominated γ-arylbutene lactone compounds

[0115] Using the characteristic isotope mass spectrometry peaks of bromine sources, combined with silica gel and HPLC methods, and relying on LC-MS-guided targeted separation, chiral compounds are separated and purified; NMR, MS, UV, ECD / calculated ECD and ORD / calculated ORD techniques are used to determine the absolute configuration of the separated compounds.

[0116] Structural identification of compounds 1 and 2: Terreroide A (compound 1) and 8"-epi-TerreroideA (compound 2) are both light yellow oily colloids. Figure 1 It is compound 1-14 1 H- 1 Schematic diagram of H-COSY and key HMBC correlation; Figure 2 is the HRESIMS mass spectrum of compound 1; Figure 3 is the IR spectrum of compound 1; Figure 4 are the measured and calculated ECD spectra of compound 1; Figure 5 is compound 1 1 H-1 H COSY spectrum (CD3OD); Figure 6 is the HMBC spectrum (CD3OD) of compound 1; Figure 7 is the HR-ESI-MS mass spectrum of compound 2; Figure 8 is the IR spectrum of compound 2; Figure 9 are the measured and calculated ECD spectra of compound 2; Figure 10 is compound 2 1 H- 1 H COSY spectrum (CD3OD); Figure 11 is the HMBC spectrum (CD3OD) of compound 2; Figure 72 This is the chiral separation diagram of compounds 1 and 2.

[0117] HRESIMS gives a set of quasi-molecular ion peaks [MH] - and its isotope peak information [M–H+2] - m / z 501.0540 / 503.0495 (Compound 1: calculated value is C 24 H 22 BrO7, 501.0549) and m / z 501.0542 / 503.0497 (Compound 2: calculated for C 24 H 22 BrO7, 501.0549), and [MH] - :[M–H+2] - The abundance ratio is 1:1, indicating that the molecular formulas of compound 1 and compound 2 are both C 24 H 23 BrO7, with an unsaturation degree of 13. Analysis of the NMR signals of compound 1 and compound 2 (Table 2) showed that the two were very similar except for slight differences around C-8", suggesting that they were diastereomers with different stereo configurations at the C-8 position. The chiral column was successfully separated (the ratio of the two was about 1:1, Figure 75 ) to obtain compound 1 and compound 2. (Compound 1) and (Compound 2) It also proved that Compound 1 and Compound 2 were epimers.

[0118] The data of compound 1 are now analyzed as an example. The NMR spectrum shows the presence of a para-substituted aromatic group (A2B2 system) [δ H 7.53,d,8.8Hz,2H,H-2' / 6'; 6.86d,8.8Hz,2H,H-3' / 5'; δ C130.4 (CH, C-2' / 6'), 116.6 (CH, C-3' / 5'), 123.4 (C, C-1' and 159.4 (C, C-4')], an ABX aromatic ring [δ H 6.48,d,8.4Hz,1H,H-5”; 6.55,dd,8.4,2.1Hz,1H,H-6”; 6.45,d,2.1Hz,1H,H-2”; δ C 126.6(C,C-1”),δ C 132.4(CH,C-2”),δ C 120.0(C,C-3”),δ C 153.0(C,C-4”),δ C 117.4 (CH, C-5") and δ C 130.8 (CH, C-6")], two carbonyl carbons [δ C 170.3 (C, C-2), 171.4 (C, C-7)], a fully substituted five-membered α, β-unsaturated lactone ring [δ C 170.3 (C, C-2), 139.8 (C, C-3), 129.3 (C, C-4) and 86.7 (C, C-5)], 1 methyl ester group [δ H 3.79,s;δ C 171.4 (C, C-7), 54.1 (-OCH3, C-8)], two methylene signals [δ H 3.45,s,H-6 and 2.94,dd,17.2,7.6,H a -7",3.23,dd,17.2,5.4,H b -7”); δ C 39.5 (CH2, C-6) and 35.0 (CH2, C-7")] and a quaternary carbon with oxygen δ C 77.5 (C, C-9"), suggesting that compound 1 is an aromatic substituted γ-butene lactone compound. Combined with the mass spectrometry quasi-molecular ion peak [MH] - and isotope peak [M-H+2] - The abundance ratio of 1:1 suggests that compound 1 is 1-bromosubstituted. Comparing the NMR data of compound 1 with the known compound aspernolide A (Phytochemistry 2009, 70, 128-132), it was found that one methylene group was missing [δ H 1.66(t,1.5,H-8”);δ C 32.5(C,C-8”)], and an additional methine information [δ H 4.29 (dd, 7.6, 5.4, H-8”); δC 53.9 (C, C-8")], and the chemical shift value shifted 21.4 ppm to the downfield due to the heavy atom effect (University Chemistry, 2016, 31, 32-36), which also suggested that its C-8" was substituted by bromine.

[0119] The 2D NMR data were further carefully analyzed and the NMR data of compound 1 were assigned by HSQC (Table 1). 1 H- 1 H-COSY spectrum ( Figure 5 ) have three simple two-carbon connected relationships: H-2'(6') / H-3'(5'), H-5" / H-6", H-7" / H-7b" / H-8". In the HMBC spectrum, H-6 / C-5 / C-7 / C-1" / C-6"; H-7a" / H-7b" / C-3" / C-4" / C-9"; H-8" / C-3" / C-9"; H-11" / C-9" / C-10"; the planar structure of compound 1 was determined ( Figure 1 ).

[0120] Table 1 Measured and calculated ORD data of compounds 1-7 and 9-14

[0121]

[0122] The same method was used to determine that the planar structures of compound 2 and compound 1 were the same. A careful analysis of the NMR data of the two revealed that there was only a slight difference around C-8", and the optical rotation values ​​of the two were (Compound 1) and (Compound 2), it is speculated that the two have different stereo configurations at C-8". The absolute configurations of C-5 of Compound 1 and Compound 2 were determined by the measured ECD / calculated ECD method; while the absolute configuration of C-8" was determined by the measured ORD / calculated ORD method. The absolute configurations of C-5 of Compound 1 and Compound 2 were determined to be R configurations by the measured and calculated ECD method ( Figure 4 、 Figure 9 ). At the same time, by comparing the measured and calculated ORD, it was found (Table 2) that the measured optical rotation value (Compound 1) and (Compound 2) and calculated value [α] D +77.8(5R,8”R-1) and [α] D +20.4(5R,8"S-2), the absolute configurations of compound 1 and compound 2 were determined to be 5R,8"R-Terreroide A and 5R,8"S-8"-epi-Terreroide A, respectively.

[0123] The structures of compounds 3 and 4 were as follows: Figure 12-21 As shown, Figure 12 is the HRESIMS mass spectrum of compound 3; Figure 13 is the IR spectrum of compound 3; Figure 14 are the measured and calculated ECD spectra of compound 3; Figure 15 is compound 3 1 H- 1 H COSY spectrum (CD3OD); Figure 16 is the HMBC spectrum (CD3OD) of compound 3; Figure 17 is the HRESIMS mass spectrum of compound 4; Figure 18 is the IR spectrum of compound 4; Figure 19 are the measured and calculated ECD spectra of compound 4; Figure 20 is compound 4 1 H- 1 H COSY spectrum (CD3OD); Figure 21 is the HMBC spectrum (CD3OD) of compound 4; Figure 73 This is the chiral separation diagram of compounds 3 and 4.

[0124] Terreroide B (Compound 3) and 8"-epi-Terreroide B (Compound 4) are light yellow oily colloids. HRESIMS gives a group of quasi-molecular ion peaks [MH] - and its isotope peak information [M–H+2] - m / z 519.0670 / 521.0626 (Compound 3: calculated value is C 24 H 24 BrO8, 519.0655) and m / z 519.0650 / 521.0638 (Compound 4: calculated for C 24 H 24 BrO8, 519.0655), and [MH] - :[M–H+2] - The abundance ratio is 1:1, indicating that the molecular formulas of compounds 3 and 4 are both C 24 H 25 BrO8, unsaturation degree is 12. Analysis of the NMR signals of compound 3 and compound 4 (Table 2) showed that except for slight differences around C-8", the two are very similar, and it is speculated that they are diastereomers with different stereo configurations at C-8". The chiral column was successfully separated (the ratio of the two is about 1:1, Figure 73 ) to obtain compound 3 and compound 4. (Compound 3) and (Compound 4) also proves that compound 3 and compound 4 are diastereomers. The data of compound 3 is now analyzed as an example. The NMR data of compound 3 is very similar to that of the known compound aspernolide B (Phytochemistry, 2009, 70, 128-132), suggesting that they may have the same structural fragment. 13 In the C NMR and DEPT spectra, compound 3 lacks a saturated methylene signal δ compared with aspernolide B. H 1.53 (m, 2H) and δ C 44.7 (CH2, C-8"), an additional methine signal δ H 4.21 (dd, 11.1, 2.5, 1H) and δ C 67.3 (CH, C-8”). It is speculated that the bromine substitution of C-8” may be affected by the heavy atom effect, causing its chemical shift to shift to the downfield (△δ C =+22.6). and combined 1 H- 1 The key correlation signals of HCOSY are H-7a” / H-7b” / H-8” and H-7a” / H-7b” / C-3” / C-4” / C-9”; H-8” / C-3” / C-9” in HMBC spectrum, which confirmed the planar structure of compound 3 ( Figure 1 ).

[0125] The same method was used to determine that the planar structures of compound 4 and compound 3 were the same. A careful analysis of their NMR data revealed that there was only a slight difference around C-8", and the optical rotation values ​​of the two were (Compound 3) and (Compound 4), it is speculated that it is a diastereomer with different stereo configuration at C-8". Similarly, the absolute configurations of C-5 and C-8" were determined by the measured ECD and calculated ECD, measured ORD and calculated ORD methods. The absolute configuration of C-5 of compound 3 and compound 4 was determined to be R configuration by measured and calculated ECD ( Figure 14 and Figure 19 ), and by comparing the measured and calculated ORD (Table 1) (Compound 3) and (Compound 4) and calculated value [α] D +60.4(5R,8”R-3) and [α] D +13.4(5R,8”S-4), the absolute configurations of compound 3 and compound 4 were determined to be 5R,8”R-Terreroide B and 5R,8”S-8”-epi-Terreroide B, respectively.

[0126] Structural identification of compound 5: Figure 22-26 As shown, Figure 22 is the HRESIMS mass spectrum of compound 5; Figure 23 is the IR spectrum of compound 5; Figure 24 are the measured and calculated ECD spectra of compound 5; Figure 25 is compound 5 1 H- 1 H COSY spectrum (CD3OD); Figure 26 is the HMBC spectrum (CD3OD) of compound 5.

[0127] Terreroide C (compound 5) is a light yellow oily gel. HRESIMS gives a group of quasi-molecular ion peaks [MH] - and its isotope peak information [M–H+2] - m / z 519.0655 / 521.0642 (calculated value is C 24 H 24 BrO8, 519.0655), and [MH] - :[M–H+2] - The abundance ratio is 1:1, suggesting that the molecular formula is C 24 H 25 BrO8, unsaturation degree is 12. The NMR data of compound 5 are very similar to those of the known compound aspernolide B (Table 2), suggesting that they may have the same structural fragment. 13 In the C NMR and DEPT spectra, compound 5 lacks an aromatic ring signal δ compared with aspernolide B. H 6.49 (d, 8.2), δ C 116.6 (CH, C-5"), adding a quaternary carbon signal δ C 110.0 (C, C-5”). It is speculated that the bromine substitution of C-5” may be affected by the heavy atom effect, causing its chemical shift to shift to the high field (△δ C =+6.6). The planar structure of compound 5 was determined by combining the correlation between H-6" / C-2' / C-5" / C-4"; H-2" / C-3" / C-4" / C-6" in the HMBC spectrum ( Figure 1 ). Further according to the biogenic pathway and quantum chemical ECD calculation ( Figure 24 ). The absolute configuration of compound 5 was determined to be 5R.

[0128] The structures of compounds 6 and 7 were as follows: Figure 27-36 As shown, Figure 27 is the HRESIMS mass spectrum of compound 6; Figure 28 is the IR spectrum of compound 6; Figure 29are the measured and calculated ECD spectra of compound 6; Figure 30 is compound 6 1 H- 1 H COSY spectrum; Figure 31 is the HMBC spectrum (CD3OD) of compound 6; Figure 32 is the HRESIMS mass spectrum of compound 7; Figure 33 is the IR spectrum of compound 7; Figure 34 are the measured and calculated ECD spectra of compound 7; Figure 35 is compound 7 1 H- 1 H COSY spectrum (CD3OD); Figure 36 is the HMBC spectrum (CD3OD) of compound 7; Figure 74 This is the chiral separation diagram of compounds 6 and 7.

[0129] Terreroide D (Compound 6) and 8"-epi-Terreroide D (Compound 7) are light yellow oily colloids. HRESIMS gives a group of quasi-molecular ion peaks [MH] - and its isotope peak information [M–H+2] - m / z 533.0800 / 535.0762 (Compound 6: calculated value is C 25 H 26 BrO8, 533.0811) and m / z 533.0789 / 535.0783 (Compound 7: calculated for C 25 H 26 BrO8, 533.0811), and [MH] - :[M–H+2] - The abundance ratio of compounds 6 and 7 is 1:1, indicating that the molecular formulas of compounds 6 and 7 are both C 25 H 27 BrO8, unsaturation degree is 12. Analysis of the NMR signals of compound 6 and compound 7 (Table 3) showed that except for slight differences around C-8", the two are very similar, and it is speculated that they are diastereomers with different stereo configurations at C-8". The chiral column was successfully separated (the ratio of the two is about 1:1, Figure 74 ) to obtain compound 6 and compound 7. (Compound 6) and (Compound 7) also proved that compound 6 and compound 7 are diastereomers. Now take the data of compound 6 as an example to analyze it. The NMR data of compound 6 and compound 3 are very similar. 13 In the C NMR and DEPT spectra, compound 6 has an additional methoxy signal δ compared to compound 3. H 3.24 (s, 3H) and δC 49.8(-O C H3). Combined with the δ H 3.24 The related information of methoxy group and C-10" and C-11" determines the planar structure of compound 6 ( Figure 1 ).

[0130]

[0131]

[0132]

[0133] The same method was used to determine that the planar structures of compound 7 and compound 6 were the same. Analysis of their NMR data revealed that there was only a slight difference around C-8", and the optical rotation values ​​of the two were (Compound 6) and (Compound 7), it is speculated that it is a diastereomer with different stereo configuration at C-8". The absolute configurations of C-5 and C-8" are determined by the same method of measured ECD and calculated ECD, measured ORD and calculated ORD. The absolute configuration of C-5 of compound 6 and compound 7 is R configuration ( Figure 29 and Figure 34 ), and by comparing the measured and calculated ORD (Table 1) (Compound 6) and (Compound 7) and calculated value [α] D +66.0 (5R, 8"R-6) and [α] D +17.2(5R,8”S-7), the absolute configurations of compound 6 and compound 7 were determined to be 5R,8”R-Terreroide D and 5R,8”S-8”-epi-Terreroide D, respectively.

[0134] Structural identification of compound 8: Figures 37-41 As shown, Figure 37 is the HRESIMS mass spectrum of compound 8; Figure 38 is the IR spectrum of compound 8; Figure 39 are the measured and calculated ECD spectra of compound 8; Figure 40 is compound 8 1 H- 1 H COSY spectrum; Figure 41 This is the HMBC spectrum (CD3OD) of compound 8.

[0135] Terreroide E (Compound 8) is a light yellow oily gel. HRESIMS gives a group of quasi-molecular ion peaks [MH] -and its isotope peak information [M–H+2] - and [M–H+4] - m / z 596.9752 / 598.9735 / 600.9716 (calculated value is C 24 H 23 Br2O8,596.9760) and [MH] - :[M–H+2] - :[M–H+4] - The abundance ratio of is 1:3:1, suggesting that the molecular formula is C 24 H 24 Br2O8, unsaturation degree is 12. The NMR data of compound 8 are very similar to those of compound 5 (see Table 3). Comprehensive analysis 13 CNMR and DEPT spectra showed that compound 8 lacked an aromatic ring signal δ compared to compound 5. H 6.89 (d, 8.8), δ C 116.7 (CH, C-5'), adding a quaternary carbon signal δ C 111.4 (C, C-5'). It is speculated that the bromine substitution at C-5' may have caused the heavy atom effect, causing its chemical shift to shift upfield (△δ C =+5.3). The planar structure of compound 8 was determined by combining the correlation between H-3' / C-1' / C-4' / C-5'; H-6' / C-1' / C-2' / C-5' in the HMBC spectrum ( Figure 1 ). Further according to the biogenic pathway and quantum chemical ECD calculation ( Figure 39 ). The absolute configuration of compound 8 was determined to be 5R.

[0136] The structures of compounds 9 and 10 were as follows: Figures 42-51 As shown, Figure 42 is the HRESIMS mass spectrum of compound 9; Figure 43 is the IR spectrum of compound 9; Figure 44 are the measured and calculated ECD spectra of compound 9; Figure 45 is compound 9 1 H- 1 HCOSY spectrum (CD3OD); Figure 46 is the HMBC spectrum (CD3OD) of compound 9; Figure 47 is the HRESIMS mass spectrum of compound 10; Figure 48 is the IR spectrum of compound 10; Figure 49 are the measured and calculated ECD spectra of compound 10; Figure 50 is compound 10 1 H- 1 H COSY spectrum (CD3OD); Figure 51is the HMBC spectrum of compound 10 (CD3OD).

[0137] Terreroide F (compound 9) and 8"-epi-Terreroide F (compound 10) are yellowish oil gum, HRESIMS gives a set of quasi-molecular ion peaks [M-H] - and isotopic peak information [M-H+2] - and [M-H+4] - m / z 610.9900 / 612.9872 / 614.9841 (compound 9: calculated value for C 25 H 25 Br2O8, 610.9916) and m / z 610.9897 / 612.9904 / 614.9838 (compound 10: calculated value for C 25 H 25 Br2O8, 610.9916) and [M-H] - : [M-H+2] - : [M-H+4] - The abundance ratio is 1:3:1, indicating that the molecular formula of compound 9 and compound 10 is C 25 H 26 Br2O8, and the unsaturation is 12. The NMR signals of compound 9 and compound 10 (Table 3) are very similar except for the slight difference around C-8". It is speculated that they are epimers with different stereochemical configurations at C-8". At the same time, the optical rotation values of (compound 9) and (compound 10) also prove that compound 9 and compound 10 are epimers. Taking the data of compound 9 as an example, it is analyzed. The NMR data of compound 9 and compound 6 are very similar. By analyzing the 13 C NMR and DEPT spectrum, it is found that compared with compound 6, compound 9 lacks an aromatic ring signal δ H 6.51 (d, 8.8), δ C 115.2 (CH, C-5"), and adds a quaternary carbon signal δ C 111.1 (C, C-5"). It is speculated that the C-5" may be substituted by bromine element, and the chemical shift is shifted to high field (△δ C = +4.1) by heavy atom effect. Combined with the key correlation of H- 1 H 1 H COSY H-7a" / H-7b" / H-8", and HMBC spectrum H-7a" / H-7b" / C-3' / C-4" / C-9"; H-8" / C-3" / C-9" of compound 9, the planar structure of compound 9 is determined. Figure 1).

[0138] The same method was used to determine that the planar structures of compound 10 and compound 9 were the same. Analysis of the NMR data of the two revealed that there was only a slight difference around C-8", and the optical rotation values ​​of the two were (Compound 9) and (Compound 10), it is speculated that it is a diastereomer with different stereo configurations at C-8". Similarly, the absolute configurations of C-5 and C-8" were determined based on the measured ECD and calculated ECD, measured ORD and calculated ORD methods. The absolute configurations of C-5 of compounds 9 and 10 were determined to be R configurations by measured and calculated ECDs ( Figure 44 and Figure 49 ), and by comparing the measured and calculated ORD (Table 1) (Compound 9) and (Compound 10) with calculated values ​​[α] + 62.7 (5R, 8″R-9) and [α] D +22.3(5R,8”S-10), the absolute configurations of compound 9 and compound 10 were determined to be 5R,8”R-Terreroide F and 5R,8”S-8”-epi-Terreroide F, respectively.

[0139] The structures of compounds 11 and 12 were as follows: Figures 52-61 As shown, Figure 52 is the HRESIMS mass spectrum of compound 11; Figure 53 is the IR spectrum of compound 11; Figure 54 are the measured and calculated ECD spectra of compound 11; Figure 55 is compound 11 1 H- 1 HCOSY spectrum (CD3OD); Figure 56 is the HMBC spectrum (CD3OD) of compound 11; Figure 57 is the HRESIMS mass spectrum of compound 12; Figure 58 is the IR spectrum of compound 12; Figure 59 are the measured and calculated ECD spectra of compound 12; Figure 60 is compound 12 1 H- 1 HCOSY spectrum (CD3OD); Figure 61 is the HMBC spectrum (CD3OD) of compound 12.

[0140] Terreroide G (Compound 11) and 8"-epi-Terreroide G (Compound 12) are light yellow oily colloids. HRESIMS gives a group of quasi-molecular ion peaks [MH] -and its isotope peak information [M–H+2] - , [M–H+4] - m / z 578.9641 / 580.9626 / 582.9613 (Compound 11: calculated value is C 24 H 21 Br2O7, 578.9654) and m / z 578.9624 / 580.9610 / 582.9596 (Compound 12: calculated values ​​for C 24 H 21 Br2O7, 578.9654) and [MH] - :[M–H+2] - :[M–H+4] - The abundance ratio of is 1:3:1, suggesting that the molecular formula is C 24 H 22 Br2O7, the degree of unsaturation is 13. Observation of its 1D-NMR spectrum confirmed that compound 11 and compound 12 are a pair of diastereomers. Since there are only slight differences around C-8", it is speculated that they are diastereomers with different stereo configurations at the C-8 position. At the same time, the optical rotation values (Compound 11) and (Compound 12) also proved that compound 11 and compound 12 are diastereomers. Now take the data of compound 11 as an example to analyze it. The NMR data of compound 11 is very similar to that of compound 1. Comprehensive analysis 13 C NMR and DEPT spectra showed that compound 11 lacked an aromatic ring signal δ compared with compound 1. H 6.86 (d, 8.8), δ C 116.6 (CH, C-5'), adding a quaternary carbon signal δ C 111.3 (C, C-5'). It is speculated that the bromine substitution at C-5' may have caused the heavy atom effect, causing its chemical shift to shift upfield (△δ C =+5.3). The planar structure of compound 11 was determined by combining the H-3' / C-1' / C-4' / C-5' and H-6' / C-1' / C-2' / C-5' correlations in the HMBC spectrum ( Figure 1 ).

[0141] The same method was used to determine that the planar structures of compound 12 and compound 11 were identical. Analysis of their NMR data revealed that there was only a slight difference around C-8", and the optical rotation values ​​of the two were (Compound 11) and (Compound 12), it is speculated that it is a diastereomer with different stereo configuration at C-8". Similarly, the absolute configurations of C-5 and C-8" were determined based on the measured ECD and calculated ECD, measured ORD and calculated ORD methods. The absolute configurations of C-5 of compound 11 and compound 12 were determined to be R configuration ( Figure 54 , Figure 59 ), and by comparing the measured and calculated ORD (Table 1) and and the calculated value [α] D +64.3 (5R, 8” R-11) and [α] D +23.0(5R,8"S-12), the absolute configurations of compound 11 and compound 12 were determined to be 5R,8"R-Terreroide G and 5R,8"S-8"-epi-Terreroide G, respectively.

[0142] The structures of compounds 13 and 14 were as follows: Figures 62-71 As shown, Figure 62 is the HRESIMS mass spectrum of compound 13; Figure 63 is the IR spectrum of compound 13; Figure 64 are the measured and calculated ECD spectra of compound 13; Figure 65 is compound 13 1 H- 1 HCOSY spectrum (CD3OD); Figure 66 is the HMBC spectrum (CD3OD) of compound 13; Figure 67 is the HRESIMS mass spectrum of compound 14; Figure 68 is the IR spectrum of compound 14; Figure 69 are the measured and calculated ECD spectra of compound 14; Figure 70 is compound 14 1 H- 1 HCOSY spectrum (CD3OD); Figure 71 is the HMBC spectrum (CD3OD) of compound 14; Figure 75 This is the chiral separation diagram of compounds 13 and 14.

[0143] Terreroide H (Compound 13) and 8"-epi-Terreroide H (Compound 14) are light yellow oily colloids. HRESIMS gives a group of quasi-molecular ion peaks [MH] - and its isotope peak information [M–H+2] - , [M–H+4] - and [M–H+6] -The values ​​are m / z 688.9009 / 690.8991 / 692.8964 / 694.8929 (Compound 13: calculated values ​​are C 25 H 24 Br3O8, 688.9021) and m / z 688.9015 / 690.8959 / 692.8969 / 694.8971 (Compound 14: calculated values ​​for C 25 H 24 Br3O8, 688.9021) and [MH] - :[M–H+2] - :[M–H+4] - :[M–H+6] - The abundance ratio is 1:3:3:1, indicating that the molecular formula is C 25 H 25 Br3O8, the degree of unsaturation is compound 12. Analysis of the NMR signals of compound 13 and compound 14 (Table 4) shows that except for slight differences around C-8", the two are very similar, and it is speculated that they are diastereomers with different stereo configurations at the C-8 position. The chiral column was successfully separated (the ratio of the two is about 1:1, Figure 75 ) to obtain compound 13 and compound 14. At the same time, the optical rotation value (Compound 13) and (Compound 14) also proved that compound 13 and compound 14 are diastereomers (Table 3). Now we analyze the data of compound 13 as an example. The NMR data of compound 13 and compound 9 are very similar. Comprehensive analysis 13 CNMR and DEPT spectra showed that compound 13 lacked an aromatic ring signal δ compared with compound 9. H 6.89 (d, 8.5), δ C 116.7 (CH, C-5'), adding a quaternary carbon signal δ C 111.4 (C, C-5'). It is speculated that the bromine substitution at C-5' may have caused the heavy atom effect, causing its chemical shift to shift upfield (△δ C =+5.3). The planar structure of compound 13 was determined by combining the H-3' / C-1' / C-4' / C-5'; H-6' / C-1' / C-2' / C-5' correlations in the HMBC spectrum ( Figure 1 ).

[0144] The same method was used to determine that the planar structures of compound 14 and compound 13 were identical. A careful analysis of the NMR data of the two revealed that there was only a slight difference around C-8", and the optical rotation values ​​of the two were and It is speculated that it is a diastereomer with different stereo configurations at C-8". The same method of measured ECD and calculated ECD, measured ORD and calculated ORD is used to determine the absolute configurations of C-5 and C-8". The absolute configurations of C-5 of compound 13 and compound 14 are R configuration ( Figure 64 、 Figure 69 ); By comparing the measured and calculated ORD (Table 1), it is found that the measured (Compound 13) and (Compound 14) and calculated value [α] D The results of [α]+53.7 (5R,8”R-13) and [α]+32.4 (5R,8”S-14) were consistent, and the absolute configurations of compounds 13 and 14 were determined to be 5R,8”R-TerreroideH and 5R,8”S-8”-epi-Terreroide H, respectively.

[0145] Example 2 Application of brominated γ-aryl butenolide compounds in the preparation of inhibitors of pathogenic fungi of agricultural economic crops and in the preparation of inhibitors of tumor drugs such as camptothecin and etoposide resistance enzymes TDP1 and TDP2.

[0146] 1. Antifungal activity results:

[0147] The mycelial growth rate assay method of Shao Xuehua et al. (Plant Protection, 2019, 45, 199) was used to determine the antifungal activity of new compounds 1-14 against the mycelia of five pathogenic fungi of economic crops: papaya anthracnose fungus Colletotrichum gloeosporioides (Cg for short), olive fruit rot fungus Pestalotiopsis microspora (Pm for short), banana wilt fungus Panama disease (Pd for short), gray mold fungus Botrytis cinerea (Bc for short) and black rot fungus Diaporthephoenicicola (Dp for short).

[0148] Comparative Example: Refer to Application Example 1 in CN116947831A. This Example 1 differs from Example 1 of the present application in that the rice culture medium contains 3.0% to 5.0% NaCl salinity, while the present application uses NaBr. Furthermore, the ethyl acetate extract is isolated using a different method. Compounds F1, F2, and F8-F13 obtained in CN116947831A were also tested for their antibacterial activity against the mycelia of five fungi pathogenic to economic crops.

[0149] The results of the antibacterial experiments (Table 5) showed that compounds 1-14 had good antibacterial activity against C. gloeosporioides. 50The values ​​ranged from 1.37 to 70.16 μg / mL. Compounds 1 and 2 had the best effects (EC 50 =3.30, 1.37 μg / mL). Compounds 11 and 12 showed good antibacterial activity against Botrytis cinerea (EC 50 =20.60, 32.93 μg / mL). However, compounds F1, F2, and F8-F13 in the patent "Method for preparing γ-aryl butenolide compounds and their use in preparing cardiovascular protective drugs" (CN116947831A) had no antibacterial effect on five economic crop pathogenic fungi.

[0150] Table 5. Inhibition rate of mycelial growth of five economic crop pathogens by compounds (%, 100 ppm) or EC 50 Value (μg / mL)

[0151]

[0152]

[0153] Note: “NA” means inactive; “—” means not tested; carbendazim is the Yangxin control drug.

Claims

1. A brominated γ-aryl butenolide compound, characterized in that: The structural formula is as follows:

2. A method for preparing a brominated γ-aryl butene lactone compound, characterized in that: The method is isolated from the fermentation culture of Aspergillus terreus EGF7-0-1, a fungus symbiotically associated with marine herbal soft corals, and specifically comprises the following steps: 1) Cultivation of Aspergillus terreus EGF7-0-1, a fungus symbiotically associated with marine herbal soft corals: The fungus symbiotically associated with marine herbal soft corals, Aspergillus terreus EGF7-0-1, was inoculated into a culture flask containing PDA medium and cultured in a shaking incubator at 28°C to obtain an activated seed culture solution. The activated seed culture solution was then inoculated into a culture flask containing rice culture medium and cultured at 28°C indoors for 35 days; the rice culture medium consisted of 100 g of rice, 115 mL of water, 3.0% to 5.0% NaBr, and a natural pH. 2) extracting the culture medium of the combined bacteria obtained in step 1) with ethyl acetate, and then concentrating to obtain rice culture ethyl acetate extract, and then subjecting the extract to silica gel column chromatography with petroleum ether-ethyl acetate system to V PE :V EtOAc =100:0→0:100 elution, and 8 crude fractions Fr.1 to Fr.8 were obtained based on thin layer TLC results. LC-MS analysis confirmed that Fr.4 to Fr.6 and Fr.8 were the target fractions. After secondary separation by silica gel column chromatography, HPLC and chiral separation were performed to obtain compounds 1-14. The specific steps are as follows: Fr.4 was purified by silica gel column chromatography using a dichloromethane-methanol system. Elution gave 4 fractions Fr.4-1 to Fr.4-4; Fr.4-1 was purified by reverse phase preparative HPLC. As the mobile phase, a mixture I with a retention time of 36.5 min was obtained; the mixture I was subjected to normal phase semi-preparative HPLC with V 正己烷 :V 异丙醇 =20:80 as the mobile phase chiral separation to obtain compound 1 with a retention time of 16.0 min and compound 2 with a retention time of 19.0 min; Fr.4-3 was subjected to reverse phase preparative HPLC to The mobile phase was used for purification to obtain mixture II with a retention time of 42.0 min and compound 5 with a retention time of 45.4 min; the mixture II was subjected to normal phase semi-preparative HPLC with V 正己烷 :V 异丙醇 =20:80 is the mobile phase chiral separation to obtain compound 3 with a retention time of 18.0 min and compound 4 with a retention time of 21.2 min; Fr.5 was purified by silica gel column chromatography using a dichloromethane-methanol system. Elution gave five fractions, Fr.5-1 to Fr.5-5; Fr.5-3 was purified by reverse phase preparative HPLC. The mobile phase was used for purification to obtain a mixture III with a retention time of 25.5 min and a compound 8 with a retention time of 36.5 min; the mixture III was purified by normal phase semi-preparative HPLC with V 正己烷 :V 异丙醇 =22:78 is the chiral separation of the mobile phase to obtain compound 6 with a retention time of 20.0 min and compound 7 with a retention time of 23.0 min; Fr.6 was purified by silica gel column chromatography using a dichloromethane-methanol system. Elution gave 4 fractions Fr.6-1 to Fr.6-4; Fr.6-2 was purified by reverse phase preparative HPLC. As the mobile phase for purification, the compound 9 with a retention time of 14.0 min and the compound 10 with a retention time of 16.0 min were obtained; Fr.6-3 was purified by reverse phase preparative HPLC with For mobile phase purification, compound 11 with a retention time of 55.0 min and compound 12 with a retention time of 59.0 min were obtained; Fr.8 was purified by silica gel column chromatography using a dichloromethane-methanol system. Elution gave 6 fractions Fr.8-1 to Fr.8-6; Fr.8-3 was purified by reverse phase preparative HPLC. The mobile phase was used for purification to obtain a mixture IV with a retention time of 45.5 min; the mixture IV was subjected to normal phase semi-preparative HPLC with V 正己烷 :V 异丙醇 =30:70 as the mobile phase for chiral separation to obtain compound 13 with a retention time of 35.3 min and compound 14 with a retention time of 40.0 min; the structural formulas of compound 1-compound 14 are as follows:

3. The preparation method according to claim 2, characterized in that The formula of PDA culture medium is as follows: 200 g potatoes, 20 g glucose, 15-20 g agar, 1000 mL water, 3.5% salinity, and natural pH.

4. The preparation method according to claim 2, characterized in that In step 2), the normal phase semi-preparative HPLC uses a Phenomen semi-preparative column with a specification of 10 mm×250 mm and 5 μm; the reverse phase preparative HPLC uses a Kromasil semi-preparative column with a specification of 10 mm×250 mm and 5 μm.

5. Use of the brominated γ-aryl butenolide compound according to claim 1 in the preparation of an inhibitor of pathogenic fungi of agricultural economic crops.

6. The use according to claim 5, characterized in that The pathogenic fungi of agricultural economic crops are selected from papaya anthracnose, olive fruit rot, banana wilt, gray mold and black rot of oleifera.

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