Marine-derived benzofuran compounds and application thereof in preparation of drugs for resisting pathogenic fungi of crops

Abstract

The invention provides benzofuran compounds derived from marine fungi and application of the benzofuran compounds in preparation of drugs for resisting pathogenic fungi of crops, and relates to the field of marine natural products. The invention discloses three novel benzofuran compounds, i.e., stribenfuran R, stribenfuran T and stribenfuran U. The structural formula of the benzofuran compounds is as shown in formula (I), in the formula (I), the compounds of the stribenfuran R are as follows: R1 = CH3, R2 = CH2OH; or a compound stribenfuran T: R1 is H, and R2 is COCH3; or a compound stribenfuran U: R1 is equal to CH3, and R2 is equal to CHO. According to the present invention, the compounds of the stribenfuran R, the stribenfuran T and the stribenfuran U have good inhibition activity on twelve types of crop pathogenic fungi, such that the alternative compounds are provided for the development of the new anti-crop pathogenic fungi. # imgabs0 #

Description

Technical field:

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to three benzofuran compounds derived from marine fungi and their application in preparing drugs against pathogenic fungi of crops. Background technology:

[0002] Marine microorganisms are an important resource for discovering new antibiotics. Plant pathogenic fungi infect crops during their growth and storage, causing them to produce mycotoxins, which affects crop yield, production quality and food safety, causing huge economic losses. Currently, anti-crop pathogenic fungi drugs are mainly synthetic antifungal agents, such as triazole fungicides. However, the excessive or improper use of existing antifungal agents has led to drug resistance in plant pathogenic fungi and has had adverse effects on the environment and public health. Therefore, in addition to regulating the use of antifungal agents, the development of new anti-crop pathogenic fungi drugs is also particularly important. Benzofuran is composed of a benzene ring and a furan ring, and its derivatives have a wide range of biological activities, such as antifungal, antibacterial, antitumor and antiviral activities. Therefore, benzofuran compounds are an important source of drug lead compounds for the development of new drugs against crop pathogenic fungi. Summary of the invention:

[0003] The first object of the present invention is to provide three benzofuran compounds and pharmaceutically acceptable salts thereof having anti-pathogenic fungi activity against crops, the chemical structural formula of which is shown in formula (I):

[0004]

[0005] In formula (I), the compound stribenfuran R: R1 = CH3, R2 = CH2OH; or the compound stribenfuran T: R1 = H, R2 = COCH3; or the compound stribenfuran U: R1 = CH3, R2 = CHO.

[0006] The second object of the present invention is to provide the use of the compound stribenfuran R, stribenfuran T or stribenfuran U in the preparation of drugs against pathogenic fungi of crops.

[0007] The anti-crop pathogenic fungi drug is preferably a drug against apple anthracnose, wheat fusarium rust, wheat sheath blight, corn fusarium rust, yam anthracnose, rubber tree anthracnose, peanut sclerotinia, banana wilt, rice blast, apple black spot, pineapple black heart and mango anthracnose.

[0008] The third object of the present invention is to provide an anti-pathogenic fungus drug for crops, characterized in that it comprises an effective amount of stribenfuran R, stribenfuran T or stribenfuran U as shown in formula (I), or a pharmaceutically acceptable salt thereof, as an active ingredient, and a pharmaceutically acceptable carrier or excipient, or a pharmaceutically acceptable adjuvant or auxiliary component prepared into a clinically acceptable pharmaceutical preparation. The content of the anti-pathogenic fungus drug for crops is 1 to 99% w / w based on the total weight of the drug.

[0009] The fourth object of the present invention is to provide the use of Striaticonidium cinctum SCSIO 41432 (GDMCCNO.65653) in the preparation of the above-mentioned compounds stribenfuran R, stribenfuran T and / or stribenfuran U.

[0010] The fifth object of the present invention is to provide a method for preparing the above-mentioned compounds stribenfuran R, stribenfuran T and / or stribenfuran U, which are prepared and isolated from the fermentation culture of Striaticonidium cinctum SCSIO41432.

[0011] The specific steps include:

[0012] The fermentation culture of SCSIO 41432 is prepared, the fermentation culture is soaked in ethyl acetate, the mycelium and the fermentation liquid are separated by gauze after ultrasonic treatment, and then extracted and concentrated by ethyl acetate to obtain a black crude extract, the extract is separated by reverse medium pressure C18 column chromatography, the mobile phase is methanol / water from a volume ratio of 10:90 to 100:0 gradient elution, the second elution part Fr.10 of the mobile phase of methanol / water = 80:20 is eluted by a normal phase medium pressure chromatograph, a silica gel separation column, and the mobile phase is dichloromethane / methanol 100:0 to 0:100 gradient elution, the normal phase chromatography 2% methanol washout part Fr.10-2 is separated and purified by high performance liquid chromatography to obtain stribenfuran R; the reverse chromatography methanol / water = 90:10 first elution part Fr.11 is separated and purified by high performance liquid chromatography to obtain stribenfuran T and U. .

[0013] The benzofuran compounds stribenfuran R, stribenfuran T and stribenfuran U of the present invention are new compounds. The three compounds all have different degrees of inhibition on twelve tested crop pathogenic fungi, and the minimum inhibitory concentration is 0.78-50 μg / mL. Among them, stribenfuran U has the best inhibition effect on the tested crop pathogenic fungi, and can be used to prepare antifungal drugs for treating crop pathogenic fungal infections. Therefore, the present invention provides alternative compounds for developing new anti-crop pathogenic fungi drugs, which is of great significance for the development of China's marine drug resources.

[0014] The mangrove sediment-derived fungus Striaticonidium cinctum SCSIO41432 of the present invention was deposited in the Guangdong Microbiological Culture Collection Center (GDMCC) on December 19, 2024, address: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Postal Code: 510070, and the deposit number is GDMCC NO: 65653. Description of the drawings:

[0015] Figure 1 This is the single crystal structure diagram of the compound stribenfuran R.

[0016] Figure 2 for the compound stribenfuran R 1 H NMR (500 MHz) spectrum, the solvent was DMSO-d6;

[0017] Figure 3 for the compound stribenfuran R 13 C NMR (125 MHz) spectrum, the solvent was DMSO-d6;

[0018] Figure 4 For the compound stribenfuran T 1 H NMR (500 MHz) spectrum, the solvent was DMSO-d6;

[0019] Figure 5 For the compound stribenfuran T 13 C NMR (125 MHz) spectrum, the solvent was DMSO-d6;

[0020] Figure 6 For the compound stribenfuran U 1 H NMR (500 MHz) spectrum, the solvent was DMSO-d6;

[0021] Figure 7 For the compound stribenfuran U 13 C NMR (125 MHz) spectrum, the solvent was DMSO-d6;

[0022] Figure 8 The compounds stribenfuran R, stribenfuran T and stribenfuran U 1 H- 1 H COSY and HMBC key 2D NMR data;

[0023] Fig. 9 Shown are the structure diagram of the compound stribenfuran U and the growth curve, scanning electron microscopy, transmission electron microscopy and fluorescence microscopy images of the effect of the compound stribenfuran U on the mycelial growth of C. gloeosporioides. Specific implementation method:

[0024] In order to make the purpose, technical scheme and beneficial technical effect of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described in this specification are only used to explain the present invention and are not used to limit the present invention.

[0025] Example 1 Encircling Striaticonidium cinctum SCSIO 41432

[0026] The fungus Striaticonidium cinctum SCSIO 41432 of the present invention was isolated from the mangrove sediment collected from the Gaoqiao Mangrove Reserve in Zhanjiang City, Guangdong Province, China. The strain was deposited in the Guangdong Microbiological Culture Collection Center (GDMCC) on December 19, 2024, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, Postal Code: 510070, and the collection number is GDMCC NO: 65653.

[0027] Example 2 Fungal fermentation and separation and preparation of stribenfuran R, stribenfuran T and stribenfuran U

[0028] 2.1 Fermentation: First, the strain Striaticonidium cinctum SCSIO 41432 preserved in paraffin oil was inoculated into an MB plate (by mass fraction, including 1.5% malt extract powder, 1.8% agar powder, 2.5% sea salt, pH 7.4-7.8) and cultured until new colonies grew, inoculated into a PDB liquid culture medium, cultured at 28°C in a shaker (180 rpm) for 3 days, and then inoculated into a sterilized PDB culture medium for fermentation, a total of 130 bottles (40 L), and fermented at room temperature for 48 days to prepare a PDB culture medium fermentation product of the fungus Striaticonidium cinctum SCSIO 41432;

[0029] 2.2 Extraction: After the fermentation of PDB medium is completed, the mycelium is inactivated with an equal volume of ethyl acetate, ultrasonicated for 15 minutes, and then the mycelium and fermentation liquid are separated with gauze, and then extracted with ethyl acetate three times until colorless, and then concentrated by rotary evaporation and combined to obtain a black crude extract (53.6g).

[0030] 2.3 Separation: Dissolve the crude extract with a small amount of methanol and mix with silica gel, and perform medium-pressure reverse phase C18 column chromatography. The mobile phase is methanol / water with a volume ratio of 10:90 to 100:0 for gradient elution, and 14 elution parts Fr.1-Fr.14 are obtained in sequence. The second elution part Fr.10 with a mobile phase of methanol / water = 80:20 is passed through a normal phase medium pressure chromatograph, a silica gel separation column, and the mobile phase is dichloromethane / methanol 100:0, 98:2, 95:5, 90:10, 70:30, 40:60, 0:100 v / v gradient elution. The elution parts of 4 column volumes are combined into 1 part, and 7 elution parts Frs.10-1-10-7 are obtained. The 2% methanol washout fraction Fr.10-2 of normal phase chromatography was separated and purified using a semi-preparative Hitachi HITACHI HPLC and a NanoChrom column (ChromCore C18, 5 μm, 10×250 mm) (mobile phase: 85% CH3OH / H2O, 3 mL / min) to obtain stribenfuran R (36.1 mg, t R =18.2min). The first elution fraction Fr.11 of the reverse chromatographic methanol / water = 90:10 was separated and purified using a semi-preparative Hitachi HITACHI high performance liquid chromatography and a NanoChrom column (ChromCore C18, 5μm, 10×250mm) (mobile phase: 93% by volume CH3OH / H2O, 3mL / min) to obtain stribenfuran T (6.4mg, t R =16.3min) and stribenfuran U(23.0mg,tR =22.0min).

[0031] Example 3 Structural Identification of Stribenfuran R, Stribenfuran T and Stribenfuran U

[0032] The benzofuran compound isolated and obtained in Example 2 was subjected to structural analysis and testing to obtain the following physicochemical property data:

[0033] Stribenfuran R: Colorless needle-shaped crystals ( Figure 1 ); UV(CH3OH)λ max (logε)217(4.06),251(3.60),257(3.60),281(2.91),289(2.76)nm; IR(film)ν max 3284,2964,2918,2852,1629,1587,1490,1433,1375,1209,1070,995,827cm -1 ; 1 H and 13 C NMR data, see Table 1. Figure 2-3 and 8; HRESIMS m / z 247.1317 [M+H] + (calcd for C 15 H 19 O3 + ,243.1329).

[0034] Single crystal data of Stribenfuran R: C 15 H 18 O3, Mr=246.29, crystal size 0.23×0.07×0.05mm 3 ,monoclinic, α=90°, β=93.120(18)°, γ=90°, Z=4, T=99.8(7)K, space group P21 / n, μ(CuKα)=0.704mm -1 ,Dcalc=1.268g / cm 3 ,2575reflections collected.The final R1 values ​​were 0.0942(I>2σ(I)).The final wR(F 2)values ​​were 0.0942(I>2σ(I)).The final R1values ​​were 0.1148(all data).The final wR(F 2 )values ​​were 0.2538(all data).Thegoodness of fit on F 2 was 1.089.( Figure 1 ,CCDC 2401501).

[0035] Stribenfuran T: yellow oil; UV(CH3OH)λ max (logε)210(4.04),239(3.50),249(3.43)nm; IR(film)ν max 3415,2960,2929,1651,1589,1548,1392,1321.1209,1139,1053,1026,996cm -1 ; 1 H and 13 C NMR data, see Table 1. Figure 4-5 , 8; HRESIMS m / z 245.1179[M+H] + (calcdfor C 15 H 17 O3 + ,245.1172).

[0036] Stribenfuran U: yellow oil; UV(CH3OH)λ max (logε)214(4.18),242(3.72),251(3.91),273(3.13)nm; IR(film)ν max 3201,2922,2854,1645,1633,1585,1496,1435,1298,1246,1203.1055,1026,968cm -1 ; 1 H and 13 C NMR data, see Table 1. Figure 6-7 and 8; HRESIMS m / z 243.1035 [MH] - (calcd for C 15 H 15 O3 - ,243.1027).

[0037] Table 1. Stribenfuran R, stribenfuran T and stribenfuran U l H and 13 C NMR data (in DMSO-d6)

[0038]

[0039]

[0040] a overlapped signal

[0041] According to the above physical and chemical data analysis, the chemical structures of the compounds stribenfuran R, stribenfuran T and stribenfuran U are shown in formula (I):

[0042]

[0043] In formula (I), the compound stribenfuran R: R1 = CH3, R2 = CH2OH; or the compound stribenfuran T: R1 = H, R2 = COCH3; or the compound stribenfuran U: R1 = CH3, R2 = CHO.

[0044] Example 4 Antibacterial experiment on the benzofuran compounds stribenfuran R, stribenfuran T and stribenfuran U of Example 3.

[0045] Twelve crop pathogenic fungi, including Colletottichum gloeosporioides Penz., Gibberella sanbinetti, Ceratobasidium cornigerum, Gibberella zeae, Colletotrichum gloeosporioides, Colletotrichum acutatum, Rhizoctonia solani, Fusarium oxysporum, Pyricularia oryaza, Alternaria alternate, Curvularia australiensis and Colletotrichum asianum, were used as test fungi. The compounds stribenfuran R, stribenfuran T and stribenfuran asianum were tested by 96-well plate pouring method. U was used to test the activity of 200 μL system. Specifically:

[0046] The 12 experimental fungi were cultured in PDB medium and shaken for 3 days. The bacterial solution was adjusted to a fungal concentration of 10 6 ~10 7 CFU / mL. Prepare 500μg / mL of the test compound and the positive control nystatin dissolved in DMSO. Use a pipette to add PDB medium to the 96-well plate, add 160μL to the first column, 100μL to the second to eleventh columns, and 120μL to the twelfth column. Columns 11 and 12 are used as positive and negative controls, respectively. Pipette 40μL of the compound solution and add it to the first column. Set the pipette volume to 100μL and carefully pipette up and down 3 times the test drug in the first column to mix evenly. Use a pipette to pipette 100μL of the mixture from the first column to the corresponding second column, mix well, and then pipette 100μL to the third column. And so on, until the dilution reaches the tenth column, pipette 100 microliters from the tenth column and discard. Use a pipette to add 100μL of the diluted bacterial solution to each well of columns 1-12. At this time, the compound concentrations were 50, 25, 13, 6.3, 3.2, 1.6, 0.78, 0.39, 0.20 and 0.10 μg / mL. Cover the lid, shake gently, and place in a 28°C incubator for 24 to 48 hours. Columns 11 and 12 were used as positive and negative controls, respectively. Determine the minimum inhibitory concentration value of each compound. Each sample was replicated 3 times. The experimental results are shown in Table 2.

[0047] Fungal growth curve analysis: Dioscorea anthracnose strains were cultured overnight in PDB medium. The fungal suspension was adjusted to 10×106 CFU / mL and cultured in PDB medium. Then, the compound stribenfuran U was added to the culture at a series of concentrations (0, 0.25, 0.5, 1, 2, and 4 MIC). The OD of the culture was recorded at 0, 1, 2, 4, 6, 8, 12, 18, 24, and 36 h using a 96-well plate. 600nm .

[0048] Scanning electron microscopy (SEM) analysis: Dioscorea anthracnose strains were cultured in PDB medium for 2 days and then stimulated with stribenfuran U at 1 MIC for 12 hours before collection. 0.5% DMSO treatment was used as a negative control. Dioscorea anthracnose strains were fixed in 2.5% glutaraldehyde overnight at 4°C. Subsequently, the samples were gradually dehydrated with ethanol and dried using a ChristAlpha 2-4LSCbasic vacuum freeze dryer (Wetzlar, Germany). After spraying with gold, SU8100 scanning electron microscope (accelerating voltage: 5 kV) was used for photography.

[0049] Transmission electron microscopy (TEM) analysis: As described for SEM analysis, the yam anthracnose pathogen strains were cultured in PDB medium and then fixed in 2.5% glutaraldehyde at 4°C for 48 hours. Subsequently, they were washed three times with 0.1M PBS buffer for 15 minutes each, and then dehydrated with ethanol gradient (50, 75, 80, 95 and 100%) and 100% acetone for 15 minutes each. After infiltration, embedding, fixation, sectioning and staining, they were imaged using TEM (SU8100).

[0050] Fluorescence microscopy analysis: C. chinensis strains were cultured in PDB medium as described in SEM analysis. C. chinensis was stained using the Annexin V-Cy5 / SYTOX Green apoptosis kit. Fluorescence microscopy images were taken using a Leica SP8 confocal microscope (Leica Microsystems, Germany).

[0051] Table 2. Minimum inhibitory concentrations of compounds against 12 strains of tested fungi (μg / mL)

[0052]

[0053]

[0054] Results and discussion: As shown in Table 2, the compounds stribenfuran R, stribenfuran T and stribenfuran U have good inhibitory activity against twelve crop pathogenic fungi. In particular, the inhibitory activity of stribenfuran U against Gibberella zeae and Colletotrichum gloeosporioides was 1.6 and 0.78 μg / mL, respectively, which was better than the positive control nystatin. In order to further study the inhibitory effect of stribenfuran U on Colletotrichum gloeosporioides, the growth curve, scanning electron microscopy, transmission electron microscopy and fluorescence microscopy analysis were performed ( Fig. 9 ). The results showed that stribenfuran U could completely inhibit the growth of Dioscorea anthracnose within 24 hours at a concentration of 2MIC. Scanning electron microscopy (SEM) results showed that the mycelium in the control group was smooth and full, while after treatment with stribenfuran U at a concentration of 1MIC for 12 hours, the mycelium surface showed obvious pits and wrinkles. Further transmission electron microscopy (TEM) analysis showed that the cytoplasm distribution of Dioscorea anthracnose in the control group was uniform, while after treatment with stribenfuran U, there were more low-density particles in the cytoplasm, and slight particle wall separation could be observed. Fluorescence microscopy results showed that no obvious fluorescence was detected in the control group, while red and green fluorescence were detected after treatment with stribenfuran U at a concentration of 1MIC, respectively. The above results indicate that stribenfuran U can inhibit the growth of Dioscorea anthracnose by damaging the integrity of the cell membrane and destroying the external structure of the strain. This provides support for the development of fungicides against crop pathogens based on this.

[0055] In summary, the present invention provides new candidate compounds for the development of new resistance to crop pathogenic fungi, which is of great significance to the development of new drugs with independent intellectual property rights in China.

Claims

1. A benzofuran compound, or a pharmaceutically acceptable salt thereof, having a chemical structure as shown in any compound of formula (I): In formula (I), the compound stribenfuran R: R1 = CH3, R2 = CH2OH; or the compound stribenfuran T: R1 = H, R2 = COCH3; or the compound stribenfuran U: R1 = CH3, R2 = CHO.

2. Use of the benzofuran compound according to claim 1 in the preparation of drugs for resisting pathogenic fungi of crops.

3. The use according to claim 2, characterized in that: The anti-crop pathogenic fungi drug is a drug for resisting apple anthracnose, wheat fusarium rust, wheat sheath blight, corn fusarium rust, yam anthracnose, rubber tree anthracnose, peanut sclerotinia, banana wilt, rice blast, apple black spot, pineapple black heart and mango anthracnose.

4. A drug for resisting pathogenic fungi of crops, characterized in that: A clinically acceptable pharmaceutical preparation comprising an effective amount of the benzofuran compound of claim 1 as an active ingredient, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient, or a pharmaceutically acceptable adjuvant or auxiliary component.

5. The anti-crop pathogenic fungus drug according to claim 4, characterized in that: Based on the total weight of the drug, the content of the benzofuran compound is 1 to 99% w / w.

6. A method for preparing the benzofuran compound according to claim 1, characterized in that The invention is prepared and separated from the fermentation culture of Striaticonidium cinctum SCSIO 41432, wherein the deposit number of the Striaticonidium cinctum SCSIO 41432 is GDMCC NO.65653.

7. The preparation method according to claim 6, characterized in that: The following steps are involved: The fermentation culture of SCSIO 41432 is prepared, the fermentation culture is soaked in ethyl acetate, mycelium and fermentation liquid are separated by gauze after ultrasonic treatment, and then extracted and concentrated by ethyl acetate respectively to obtain a black crude extract, the extract is separated by reverse medium pressure C18 column chromatography, the mobile phase is methanol / water from a volume ratio of 10:90 to 100:0 gradient elution, the second elution part Fr.10 of the mobile phase of methanol / water = 80:20 is eluted by a normal phase medium pressure chromatograph, a silica gel separation column, and the mobile phase is dichloromethane / methanol 100:0 to 0:100 gradient elution, the normal phase chromatography 2% methanol washout part Fr.10-2 is separated and purified by high performance liquid chromatography to obtain stribenfuran R; the reverse chromatography methanol / water = 90:10 first elution part Fr.11 is separated and purified by high performance liquid chromatography to obtain stribenfuran T and U.

8. Use of Striaticonidium cinctum SCSIO 41432 in preparing the benzofuran compounds according to claim 1, wherein the Striaticonidium cinctum SCSIO 41432 has a deposit number of GDMCC NO.65653.

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