Benzofuran compounds of marine origin and use thereof in the preparation of anti-crop pathogenic fungi drugs

Abstract

The application provides a kind of marine fungus-derived benzofuran compound and its application in preparing anti-crop pathogenic fungi drugs, and relates to the field of marine natural products.Three novel benzofuran compounds, stribenfuran R, stribenfuran T and stribenfuran U, are disclosed, and the structural formula is shown as formula (I). In formula (I), the compound stribenfuran R is R1=CH3, R2=CH2OH; or the compound stribenfuran T is R1=H, R2=COCH3; or the compound stribenfuran U is R1=CH3, R2=CHO. The compound stribenfuran R, stribenfuran T and stribenfuran U have good inhibitory activity on twelve crop pathogenic fungi, and provide alternative compounds for the development of new anti-crop pathogenic fungi.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to three benzofuran compounds from marine fungi and application of the benzofuran compounds in preparation of anti-crop pathogenic fungus drugs. BACKGROUND

[0002] Marine microorganisms are important resources for discovering new antibiotics. Plant pathogenic fungi infect crops during their growth and storage, causing the crops to generate mycotoxins, which affects the yield, production quality and food safety of crops, and causes huge economic losses. At present, anti-crop pathogenic fungus drugs mainly include synthetic antifungal agents, such as triazole fungicides. However, the overuse or improper use of existing antifungal agents has led to drug resistance of 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 fungus drugs is 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 important lead compounds for the research and development of new anti-crop pathogenic fungus drugs. SUMMARY

[0003] A first object of the present application is to provide three benzofuran compounds with anti-crop pathogenic fungus activity and pharmaceutically acceptable salts thereof, and the chemical structural formula is shown as 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] A second object of the present application is to provide application of the compound stribenfuran R, stribenfuran T or stribenfuran U in preparation of anti-crop pathogenic fungus drugs.

[0007] The anti-crop pathogenic fungus drug is preferably a drug for resisting apple anthracnose fungus, wheat scab fungus, wheat sharp eyespot fungus, corn scab fungus, yam anthracnose fungus, rubber tree anthracnose fungus, peanut sclerotinia fungus, banana wilt fungus, rice blast fungus, apple black spot fungus, pineapple black heart fungus and mango anthracnose fungus.

[0008] A third object of the present application is to provide an anti-crop pathogenic fungi drug, 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 ingredient, prepared into a clinically acceptable pharmaceutical preparation. The content of the anti-crop pathogenic fungi drug is 1-99% w / w based on the total weight of the drug.

[0009] A fourth object of the present application is to provide the use of Striatoncidiurn cinctum SCSIO 41432 (GDMCC NO. 65653) in the preparation of the above-mentioned compounds stribenfuran R, stribenfuran T and / or stribenfuran U.

[0010] A fifth object of the present application is to provide a preparation method of the above-mentioned compounds stribenfuran R, stribenfuran T and / or stribenfuran U, which is isolated from the fermentation culture of Striatoncidiurn cinctum SCSIO 41432.

[0011] Specifically comprising the following steps:

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

[0013] The stribenfuran R, the stribenfuran T and the stribenfuran U of the benzofuran compound of the application are new compounds, the three compounds have different degrees of inhibition on twelve test crop pathogenic fungi, the minimum inhibition concentration is 0.78-50 mu g / mL, wherein the stribenfuran U has the best inhibition effect on the test crop pathogenic fungi, can be used for preparing an antifungal drug, and is used for treating crop pathogenic fungal infection. Therefore, the application provides alternative compounds for developing new antifungal drugs for crop pathogens, and has important significance for developing Chinese marine drug resources.

[0014] The mangrove mud-derived fungus Striatonectria cinctum SCSIO41432 of the application was preserved in the Guangdong Microbial Culture Collection Center (GDMCC) on December 19, 2024, and the address of the GDMCC is No. 59, Building 5, Institute of Microbiology, Guangdong Academy of Sciences, 100, Xianlie Road, Guangzhou, and the postcode is 510070, and the preservation number is GDMCC NO: 65653. BRIEF DESCRIPTION OF DRAWINGS

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

[0016] Figure 2 The single crystal structure diagram of the compound stribenfuran R. 1 The H NMR (500MHz) spectrum diagram, the solvent is DMSO-d6;

[0017] Figure 3 The single crystal structure diagram of the compound stribenfuran R. 13 The C NMR (125MHz) spectrum diagram, the solvent is DMSO-d6;

[0018] Figure 4 The single crystal structure diagram of the compound stribenfuran T. 1 The H NMR (500MHz) spectrum diagram, the solvent is DMSO-d6;

[0019] Figure 5 The single crystal structure diagram of the compound stribenfuran T. 13 The C NMR (125MHz) spectrum diagram, the solvent is DMSO-d6;

[0020] Figure 6 The single crystal structure diagram of the compound stribenfuran U. 1 The H NMR (500MHz) spectrum diagram, the solvent is DMSO-d6;

[0021] Figure 7 stribenfuran U 13 C NMR (125 MHz) spectra in DMSO-d6;

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

[0023] Figure 9 Structure of stribenfuran U and growth curve, scanning electron microscopy, transmission electron microscopy and fluorescence microscopy images of the effect of stribenfuran U on the mycelial growth of C. gloeosporioides. DETAILED DESCRIPTION

[0024] In order to make the purposes, technical solutions and beneficial technical effects of the present application clearer, the present application will be further described in detail below in combination with embodiments. It should be understood that the specific embodiments described in the present specification are only used to explain the present application and do not limit the present application.

[0025] Example 1 Striatonectria cinctum SCSIO 41432

[0026] The fungus Striatonectria cinctum SCSIO 41432 of the present application was isolated from the mangrove sediment collected from Gaoqiao Mangrove Nature Reserve in Zhanjiang City, Guangdong Province, China. The strain was preserved in Guangdong Microbial Culture Collection Center (GDMCC) on December 19, 2024, which is located at No. 59 Building, 5th Floor, Institute of Microbiology, Guangdong Academy of Sciences, 100 Middle Martyrs Road, Guangzhou, Guangdong, China, with a postal code of 510070, and the preservation number is GDMCC NO: 65653.

[0027] Example 2 Fermentation of fungi and isolation and preparation of stribenfuran R, stribenfuran T and stribenfuran U

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

[0029] 2.2 Extraction: After the end of PDB medium fermentation, the mycelium was inactivated with an equal volume of ethyl acetate, ultrasonic for 15 min, then the mycelium and fermentation broth were separated with gauze, and then extracted with ethyl acetate for 3 times until colorless, concentrated by rotary evaporation, and then combined to obtain black crude extract (53.6g).

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

[0031] Structure identification of Stribenfuran R, Stribenfuran T and Stribenfuran U

[0032] The structure analysis test was carried out on the benzofuran compounds isolated in Example 2, and the following physicochemical property data was obtained:

[0033] Stribenfuran R: colorless needle-like crystal (0.15 x 0.10 x 0.05 mm) Figure 1 ); UV (CH3OH) λ max (log ε) 217 (4.06), 251 (3.60), 257 (3.60), 281 (2.91), 289 (2.76) nm; IR (film) v max 3284, 2964, 2918, 2852, 1629, 1587, 1490, 1433, 1375, 1209, 1070, 995, 827 cm -1 ; 1 H and 13 C NMR nuclear magnetic data, see Table 1, Figures 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 x 0.07 x 0.05 mm 3 , monoclinic, α = 90°, β = 93.120 (18) °, γ = 90°, Z = 4, T = 99.8 (7) K, space group P21 / n, μ (Cu Kα) = 0.704 mm -1 , Dcalc= 1.268 g / cm 3 , 2575 reflections collected. The final R1 values were 0.0942 (I > 2σ (I)). The final wR (F 2The final R1values were 0.0942 (all data). The final wR (F 2 )values were 0.2538 (all data). The goodness of fit on F 2 was 1.089. Figure 1 , CCDC 2401501).

[0035] Stribenfuran T: yellow oil; UV (CH3OH) λ max (log ε) 210 (4.04), 239 (3.50), 249 (3.43) nm; IR (film) v max 3415, 2960, 2929, 1651, 1589, 1548, 1392, 1321. 1209, 1139, 1053, 1026, 996 cm -1 ; 1 H and 13 C NMR data, see Table 1, Figures 4-5 8; HRESIMS m / z 245.1179 [M + H] + (calcd for C 15 H 17 O3 + , 245.1172).

[0036] Stribenfuran U: yellow oil; UV (CH3OH) λ max (log ε) 214 (4.18), 242 (3.72), 251 (3.91), 273 (3.13) nm; IR (film) v max 3201, 2922, 2854, 1645, 1633, 1585, 1496, 1435, 1298, 1246, 1203. 1055, 1026, 968 cm -1 ; 1 H and 13 C NMR data, see Table 1, Figures 6-7 8; HRESIMS m / z 243.1035 [M - H] - (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 physicochemical data analysis, the chemical structural formula of the compounds stribenfuran R, stribenfuran T and stribenfuran U is as 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 Bacteriostatic experiment of the benzofuran compounds stribenfuran R, stribenfuran T and stribenfuran U of Example 3.

[0045] The 12 crop pathogenic fungi, apple anthracnose fungus (Colletotrichum gloeosporioides Penz.), wheat Gibberella sanbinetti, wheat Ceratobasidium cornigerum, corn Gibberella zeae, yam anthracnose fungus (Colletotrichum gloeosporioides), rubber tree anthracnose fungus (Colletotrichum acutatum), peanut sclerotium disease fungus (Rhizoctonia solani), banana fusarium wilt fungus (Fusarium oxysporum), rice blast fungus (Pyricularia oryaza), apple black spot fungus (Alternaria alternate), pineapple black heart disease fungus (Curvularia australiensis), and mango anthracnose fungus (Colletotrichum asianum) were used as test fungi, and the 96-well plate pouring method was used to test the activity of 200 μL system of compounds stribenfuran R, stribenfuran T and stribenfuran U. Specifically:

[0046] The 12 experimental fungi were cultured in PDB medium, and the shaker was cultured for 3 days. The OD value method was used to prepare the fungus liquid to a fungus concentration of 10 6 ~ 10 7 CFU / mL. Prepare 500 μg / mL of the test compound dissolved in DMSO and the positive control nystatin. Add PDB medium to the 96-well plate with the gun, 160 μL to the first column, 100 μL to the second to eleventh columns, and 120 μL to the twelfth column. The eleventh and twelfth columns are used as positive and negative controls, respectively. Take 40 μL of the compound solution and add it to the first column. Set the volume of the gun to 100 μL, and carefully suck up and down the test drug in the first column 3 times to mix evenly. Use the gun to suck 100 μL of the mixture from the first column and add it to the corresponding second column, and mix again before sucking 100 μL to the third column. In this way, dilute to the tenth column, and discard 100 μL from the tenth column. Add 100 μL of the diluted fungus liquid to each well of columns 1-12 with the gun. At this time, the compound concentration is 50, 25, 13, 6.3, 3.2, 1.6, 0.78, 0.39, 0.20 and 0.10 μg / mL, respectively. Cover the lid, gently shake, and place in a 28°C incubator for 24-48 hours. The eleventh and twelfth columns are used as positive and negative controls, respectively. Determine the minimum inhibitory concentration value of each compound. Each sample is done in triplicate. The experimental results are shown in Table 2.

[0047] Fungal growth curve analysis: Colletotrichum gloeosporioides strains were cultured overnight in PDB medium. The fungal suspension was adjusted to 10 x 106CFU / 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), respectively. The OD of the culture was recorded at 0, 1, 2, 4, 6, 8, 12, 18, 24, 36 h, respectively, using a 96-well plate. 600nm .

[0048] Scanning electron microscope (SEM) analysis: Colletotrichum gloeosporioides strains were cultured in PDB medium for 2 days, then collected after 12 hours of stimulation with stribenfuran U at 1 MIC. 0.5% DMSO treatment was used as a negative control. The Colletotrichum gloeosporioides strains were fixed in 2.5% glutaraldehyde at 4°C overnight. Subsequently, the samples were gradually dehydrated with ethanol and dried using a Christ Alpha 2-4L SCbasic vacuum freeze dryer (Wetzlar, Germany). After sputtering with gold, the SU8100 scanning electron microscope (accelerating voltage: 5 kV) was used for imaging.

[0049] Transmission electron microscope (TEM) analysis: The Colletotrichum gloeosporioides strains were cultured in PDB medium as described in the SEM analysis, then they were fixed in 2.5% glutaraldehyde at 4°C for 48 hours. Subsequently, they were washed with 0.1M PBS buffer for 3 times, 15 minutes each time, then dehydrated with ethanol gradient (50, 75, 80, 95, and 100%) and 100% acetone, 15 minutes each time. After permeation, embedding, solidification, sectioning, and staining, imaging was performed using TEM (SU8100).

[0050] Fluorescence microscope analysis: Colletotrichum gloeosporioides strains were cultured in PDB medium as described in the SEM analysis. The Colletotrichum gloeosporioides strains were stained using the Annexin V-Cy5 / SYTOX Green Apoptosis Kit. Fluorescence microscopic images were taken with a Leica SP8 confocal microscope (Leica Microsystems, Germany).

[0051] Table 2, Minimum inhibitory concentration values of the compounds against 12 tested fungi (μg / mL)

[0052]

[0053]

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

[0055] In summary, the present application provides a new candidate compound for the development of new anti-crop pathogenic fungi, which has important significance for the development of new drugs with Chinese independent intellectual property rights.

Claims

1. A class of benzofuran compounds, or a pharmaceutically acceptable salt thereof, having a chemical structure as shown in any one of the following compounds of Formula (I): wherein 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 of claim 1 in the preparation of a medicine for resisting crop pathogenic fungi. The medicine for resisting crop pathogenic fungi is a medicine for resisting apple anthracnose fungus, wheat scab fungus, wheat sharp eyespot fungus, corn scab fungus, yam anthracnose fungus, rubber tree anthracnose fungus, peanut sclerotinia fungus, banana wilt fungus, rice blast fungus, apple black spot fungus, pineapple black heart fungus, and mango anthracnose fungus.

3. Use according to claim 2, characterized in that, An effective amount of the benzofuran compound of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient, together with a pharmaceutically acceptable carrier or excipient, or together with a pharmaceutically acceptable adjuvant or auxiliary ingredient, is prepared into a clinically acceptable pharmaceutical preparation.

4. An agricultural crop pathogenic fungus control agent, characterized by comprising the compound of claim 1 or 2. The content of the benzofuran compound is 1-99% w / w based on the total weight of the medicine.

5. The anti-crop pathogenic fungal agent of claim 4, wherein, The isolated stribenfuran R, T and U are prepared from a fermentation culture of Striatoncium cinctum SCSIO 41432, which is deposited under the accession number GDMCC NO. 65653.

6. A production method for producing the benzofuran compound according to claim 1, characterized by The steps include:

7. The production method according to claim 6, wherein The fermentation culture of Striatoncium cinctum SCSIO 41432 is prepared, soaked with ethyl acetate, and separated into mycelium and fermentation broth after ultrasonic treatment and gauze filtration. The mycelium and fermentation broth are extracted with ethyl acetate, concentrated, combined, and separated by reverse medium-pressure C18 column chromatography. The mobile phase is methanol / water, and the gradient elution is performed from 10:90 to 100:0 by volume. The second elution fraction Fr. 10 with a mobile phase of methanol / water = 80:20 is separated by normal-phase medium-pressure chromatography, a silica gel separation column, and a gradient elution with a mobile phase of dichloromethane / methanol 100:0-0:

100. The 2% methanol elution fraction Fr. 10-2 of the normal-phase chromatography is separated and purified by high-performance liquid chromatography to obtain stribenfuran R. The first elution fraction Fr. 11 with a mobile phase of methanol / water = 90:10 of the reverse chromatography is separated and purified by high-performance liquid chromatography to obtain stribenfurans T and U.

8. Use of Striatoncium cinctum SCSIO 41432 in the preparation of the benzofuran compound of claim 1, wherein the Striatoncium cinctum SCSIO 41432 is deposited under the accession number GDMCC NO. 65653. ​

Images