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CN114369051B - Pyrrolidinol compound and preparation method and application thereof - Google Patents

Pyrrolidinol compound and preparation method and application thereof Download PDF

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CN114369051B
CN114369051B CN202210069462.3A CN202210069462A CN114369051B CN 114369051 B CN114369051 B CN 114369051B CN 202210069462 A CN202210069462 A CN 202210069462A CN 114369051 B CN114369051 B CN 114369051B
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杜丰玉
肖�琳
于晓凤
牛赡光
曲田丽
周远明
王进田
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Qingdao Agricultural University
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Abstract

The invention relates to the technical field of microbial pesticides, in particular to a pyrrolidinol compound obtained from a liquid fermentation product of a strain Aspergillus sp.TR15 (preserved in China center for type culture Collection, with a preservation date of 2021, 11 and 26 days, and a preservation number of CCTCC NO: M20211402), a preparation method thereof and application thereof in inhibiting plant pathogenic bacteria and killing nematodes. The pyrrolidinol compound is a salt or solvate acceptable in pesticides of compounds 1 and 2 shown in formula (one). Compounds 1 and 2 have remarkable inhibitory activity on hypha growth and spore germination of drug-resistant botrytis cinerea, and IC 50 The value is better than that of azoxystrobin and boscalid which are common commercial bactericides. In addition, compounds 1 and 2 also have nematicidal activity against meloidogyne incognita, LD 50 The values were 311.5 and 162.0. Mu.g/mL, respectively. Thus, compounds 1 and 2 are useful as lead compounds or novel pesticide ingredients having plant pathogenic bacteria inhibiting and nematicidal activity.
Figure DDA0003458431370000011

Description

Pyrrolidinol compound and preparation method and application thereof
Technical Field
The invention relates to the technical field of microbial pesticides, in particular to a pyrrolidinol compound obtained from a liquid fermentation product of Aspergillus sp.TR15 strain, a preparation method thereof and application thereof in the aspects of plant pathogen inhibition and nematode killing.
Background
Botrytis cinerea can harm field growth and fruit storage of various cash crops such as grapes, strawberries, tomatoes and the like, and economic losses exceeding 100 hundred million dollars can be estimated to be caused annually worldwide. Therefore, gray mold is classified as one of ten plant fungal diseases in the world. For example: shandong is the biggest fruit and vegetable production base in China, and gray mold is high due to the humid and warm climatic conditions of fruit and vegetable greenhouses, so that great economic losses are caused each year. At present, gray mold control is still based on chemically synthesized pesticides, for example: azoxystrobin and boscalid, and the like. Although chemical synthesized pesticides can better prevent and treat gray mold, the resistance of the strain is continuously developed due to a relatively single action mechanism, so that the vicious circle of resistance generation, increase of chemical synthesized pesticides use, resistance development, pesticide residue excess and environmental pollution is involved. Except gray mold, the fruit and vegetable greenhouse is also infected by the southern root knot nematode, and the two are often combined, so that the economic loss is increased, and the control difficulty is greatly increased.
The search for active ingredients with plant pathogenic bacteria inhibiting or nematicide effect from biological resources such as microorganisms, plants and the like has become an important source for developing new environment-friendly pesticides. Compared with chemical synthesized pesticides, the microbial secondary metabolite generally only contains carbon, hydrogen, oxygen and nitrogen atoms, and has the advantages of easy fermentation production, relatively low toxicity, good environmental compatibility and the like; in addition, its mechanism of controlling plant diseases and pests often has a "multi-point effect", and has become an important weapon against drug-resistant diseases and pests.
According to the research of the literature, the pyrrolidinol compound has various functions of resisting tumor, immunosuppression, virus, bacteria and the like. The two pyrrolidinols related to the invention are novel compounds which are not reported, the structural characteristics of the structural skeleton, the fatty long-chain substituent and the like are different from those of the known pyrrolidinols, and the pyrrolidinols are found to have comprehensive control effects on drug-resistant botrytis cinerea and meloidogyne incognita for the first time.
Disclosure of Invention
The invention aims to provide a pyrrolidinol compound, a preparation method and application thereof.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
compound 1 and compound 2 of formula (one):
Figure BDA0003458431350000021
the preparation method of the compound comprises the following steps: inoculating Aspergillus sp.TR15 strain into sterilized liquid culture medium, standing at 28deg.C for fermentation, and extracting and separating fermentation culture medium; the preservation number of the strain is CCTCC NO: m20211402.
Based on the scheme, the liquid culture medium is prepared from 2% of sucrose, 2% of mannitol, 0.5% of peptone, 0.3% of yeast extract powder and 20% of artificial seawater potato extract, and the pH value is 7.
Preferably, the extraction and separation steps of the fermentation medium are as follows:
(1) Extracting the fermentation medium with ethyl acetate for 2-3 times, mixing the extractive solutions, and concentrating to obtain fermentation crude extract;
(2) Subjecting the above fermented crude extract to silica gel column chromatography, and gradient eluting with dichloromethane-methanol with gradient of 100:1-1:1 (v/v) as solvent according to the polarity increasing sequence of the eluent; collecting dichloromethane-methanol=40:1 eluting component, performing reverse phase silica gel column chromatography, and performing gradient elution with methanol-water with gradient of 1:9 to 1:0 (v/v) as solvent;
(3) Collecting methanol-water=3:7 (v/v) reverse phase silica gel eluting component, semi-preparative high performance liquid chromatography purification, mobile phase 55% methanol-water, detection wavelength 210nm, flow rate 3mL/min, and respectively collecting retention time t R The components with values of 14.6min and 19.0min are obtained to obtain formula (I)) Compound 1 and compound 2 are shown.
Application of pyrrolidinol compounds in inhibiting plant pathogenic bacteria and killing nematode is provided.
Preferably, the pyrrolidinol compound is at least one of the compounds of claim 1; the plant pathogenic bacteria are botrytis cinerea; the nematode is meloidogyne incognita.
The compounds of the present invention may be combined with other drugs in the form of a composition; the composition comprises a compound represented by the formula (I) and a pesticidally acceptable salt and/or solvate as active ingredients, and a pesticidally acceptable carrier, and the compound of the present invention may be used in combination with other active ingredients as long as antagonism does not occur therebetween.
The term "compound of the invention" as used herein means a compound of formula (one) in any form, i.e., any salt or non-salt form (e.g., in free acid or free base form, or in pharmaceutically acceptable salt form thereof), and any physical form thereof (e.g., including non-solid forms (e.g., liquid or semi-solid forms), and solid forms (e.g., amorphous or crystalline forms, specific polymorphic forms, solvates, including hydrates (e.g., mono-, di-, and hemi-hydrates)), and mixtures of various forms thereof.
Solvate compounds
For solvates of the compounds of the present invention or salts thereof in crystalline form, those skilled in the art will appreciate that pesticidally acceptable solvates may be formed wherein the solvent molecules are incorporated into the crystal lattice upon crystallization. The solvates may comprise nonaqueous solvents such as ethanol, isopropanol, DMSO, acetic acid, ethanolamine, and ethyl acetate, or they may comprise water as the solvent (which is incorporated into the lattice). Solvates in which water is the solvent that is incorporated into the crystal lattice are commonly referred to as "hydrates". The hydrate comprises stoichiometric hydrates and a component comprising variable amounts of water. The present invention includes such solvates.
The invention has the advantages that:
(1) The two pyrrolidinyl alcohol compounds related by the invention are novel compounds which are not reported, the structural characteristics of the structural skeleton, the fatty long chain substituent group and the like are different from those of the known pyrrolidinyl alcohol compounds, and the pyrrolidinyl alcohol compounds are found to have comprehensive control effects on drug-resistant botrytis cinerea and meloidogyne incognita for the first time, and can be used as lead compounds or novel pesticide components with plant pathogenic bacteria inhibiting and nematicidal activities.
(2) Compounds 1 and 2 of formula (one) are capable of significantly inhibiting the growth of hyphae of resistant Botrytis cinerea, and IC thereof 50 Values were 27.39 and 40.24 μg/mL, respectively; it also has remarkable inhibitory activity on spore germination of drug-resistant botrytis cinerea, IC 50 Values were 36.58 and 21.99 μg/mL, respectively; the activity is superior to that of azoxystrobin and boscalid which are commonly used chemical synthesis bactericides. In addition, compounds 1 and 2 of formula (one) also have nematicidal activity against meloidogyne incognita, LD 50 The values were 311.5 and 162.0. Mu.g/mL, respectively.
(3) The two pyrrolidinol compounds are produced by fermenting Aspergillus sp.TR15 strain, which is easy to carry out large-scale fermentation production; and compared with chemical synthesized pesticides, the microbial secondary metabolite has good environmental compatibility, and the mechanism of multi-point action is also often provided for preventing and controlling plant diseases and insect pests, so that drug resistance is not easy to generate.
Drawings
FIG. 1 is a diagram of Compound 1 of formula (one) 1 H NMR spectrum.
FIG. 2 is a diagram of Compound 1 of formula (one) 13 C NMR spectrum.
FIG. 3 is a diagram showing Compound 2 of formula (one) 1 H NMR spectrum.
FIG. 4 shows a compound 2 of formula (one) 13 C NMR spectrum.
FIG. 5 shows the inhibitory activity of Compounds 1 and 2 of formula (I) against drug-resistant Botrytis cinerea (filter paper method).
FIG. 6 shows the inhibitory activity of Compound 1 of formula (I) against drug-resistant Botrytis cinerea (Resazurin method).
FIG. 7 shows the inhibitory activity of Compound 2 of formula (I) against drug-resistant Botrytis cinerea (Resazurin method).
Biological preservation information
The Aspergillus TR15 is named as Aspergillus sp.tr15 and is preserved in China center for type culture collection, the preservation address is the eight-path 299-number China center for type culture collection of university of Wuhan in mountain area of Wuhan, hubei province, the preservation number is CCTCC NO: M20211402, and the preservation date is 2021, 11 and 26 days.
Detailed Description
The terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art unless otherwise indicated.
The invention will be described in further detail below in connection with specific embodiments and with reference to the data. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
The invention separates the pyrrolidinol compound pointed out in the following examples from the liquid fermentation product of Aspergillus sp.TR15 strain (preserved in China center for type culture Collection, with a preservation date of 2021, 11, 26, and a preservation number of CCTCC NO: M20211402), wherein the structures of the compound 1 and the compound 2 are as follows (Arabic numerals in the structures are the standard positions of carbon atoms):
Figure BDA0003458431350000051
example 1: process for preparing compound 1 and compound 2
(1) Bacterial strain fermentation culture
The strain Aspergillus sp.TR15 (size 2X 2 cm) grown on the surface of the PDA plate was excised, inoculated into a sterilized conical flask containing a liquid medium, and allowed to stand at 28℃for fermentation for 30 days. The liquid culture medium is prepared from 2% of sucrose, 2% of mannitol, 0.5% of peptone, 0.3% of yeast extract powder and 20% of artificial seawater potato extract, and the pH value is 7. The above percentages are by weight.
Extracting the fermentation medium with ethyl acetate for 3 times, mixing the extracts, and concentrating to obtain a fermentation crude extract.
(2) Preparation of the Compounds
Subjecting the above fermented crude extract to silica gel column chromatography (inner diameter 65mm, length 300mm, glass chromatographic column with sand plate and suction nozzle), and gradient eluting with dichloromethane-methanol with gradient of 100:1-1:1 (v/v, same as below) as solvent according to polarity increasing sequence of eluent; the dichloromethane-methanol=40:1 elution fractions were collected and subjected to reverse phase silica gel column chromatography (inner diameter 30mm, length 600mm, glass chromatography column with standard tetrafluoro-section gate) with gradient elution with methanol-water as solvent at a gradient of 1:9 to 1:0.
Collecting methanol-water=3:7 (v/v) reverse phase silica gel eluting component, semi-preparative high performance liquid chromatography purification, mobile phase 55% methanol-water, detection wavelength 210nm, flow rate 3mL/min, and respectively collecting retention time t R The components with the values of 14.6min and 19.0min are obtained to obtain the compound 1 and the compound 2.
(3) Structural identification of compounds
TABLE 1 Compounds 1 1 H (500 MHz) and 13 C NMR(125MHz)
spectrogram data (solvent for NMR test: deuterated chloroform)
Figure BDA0003458431350000061
Figure BDA0003458431350000071
TABLE 2 Compounds 2 1 H (500 MHz) and 13 C NMR(125MHz)
spectrogram data (solvent for NMR test: deuterated chloroform)
Figure BDA0003458431350000072
Figure BDA0003458431350000081
Compound 1, colorless powder, HR-ESI-MS m/z 316.2634[ M+H ]] + Prompt molecular formula C 21 H 33 NO, which is 1 H- (FIG. 1) and 13 the C-NMR (FIG. 2) data are shown in Table 1.
Compound 2, colorless powder, HR-ESI-MS m/z 346.31027[ M+H ]] + Prompt molecular formula C 23 H 39 NO, which is 1 H- (FIG. 3) and 13 the C-NMR (FIG. 4) data are shown in Table 2.
Example 2: determination of bacteriostatic Activity
The antibacterial activity is qualitatively determined by a filter paper sheet method and quantitatively determined by a resazurin method. The method comprises the following specific steps:
(1) Test plant pathogenic bacteria
A drug resistant gray mold fungus ZC-8 is isolated from vegetable greenhouses in the Shandong mass, and is a common chemical synthetic bactericide for gray mold: azoxystrobin and boscalid are resistant to drugs.
(2) Preparation of sample solutions
Compound 1 and compound 2 were dissolved in methanol to prepare a mother liquor of 20mg/mL for measurement by a filter paper sheet method.
A volume of mother liquor was drawn up separately and diluted with 50% methanol-water solution to 1920, 1440, 960, 480 and 240mg/L of different concentrations of sample solution for the resazurin assay.
(3) Filter paper sheet process
A bacterial cake with the length of about 5mm is picked up at the edge of a bacterial colony of the drug-resistant botrytis cinerea ZC-8 by a puncher, and is placed at the central position of a 9cm PDA flat plate. 2 sterile filter paper sheets with the length of 5mm are respectively and symmetrically placed on two sides of a bacterial cake, and the distance between the two sterile filter paper sheets and the bacterial cake is about 25mm and is on the same straight line.
mu.L of the compound 1 and compound 2 mother liquor was pipetted onto a filter paper (loading 0.1 mg) and an equal volume of methanol was added as a control. After culturing at 28℃for 5 days, it was observed whether or not Compound 1 and Compound 2 inhibited the growth of drug-resistant Botrytis cinerea ZC-8.
(4) Resazurin method
Experimental methods refer to the following documents: nat.Commun.2020,11 (01): 1608:1-19; lett.Appl. Microbiol.2015,61:238-244, and was partially adjusted according to the plant pathogen tested. The method comprises the following specific steps:
the drug-resistant gray mold bacteria ZInoculating C-8 to PDA plate, culturing at 25deg.C for 7-10d, collecting spores with sterile water, and adjusting concentration to 1×10 5 And each mL. Taking 96-well cell culture plates, and respectively adding 45 mu L of liquid culture medium (35 mu L of 1% glucose sterile water solution and 10 mu L of PDB solution), 5 mu L of sample solution with different concentrations and 10 mu L of spore suspension into each well, wherein an equal volume of solvent is added to serve as a negative control, and azoxystrobin and boscalid are used as positive controls; while equal volumes of liquid medium and sample solution (1920 mg/L) were added to the zeroed wells, no spore suspension was added.
3 replicates are set for each concentration, and after shaking and mixing, 96-well cell culture plates are properly sealed and shake cultured for 24 hours at 25 ℃. Resazurin dye (40. Mu. Mol/L) was added and incubated at 25℃in the dark until the negative control turned light pink. The absorbance of each well was measured by an enzyme-labeled instrument at a wavelength of 570 nm. Calculating bacteriostasis rate and IC by SPSS statistical software 50 Values.
The measurement results of the filter paper sheet method are shown in FIG. 5. Therefore, the compound 1 and the compound 2 can obviously inhibit the hypha growth of the drug-resistant gray mold fungus ZC-8 and generate obvious antibacterial zones. The results of the resazurin method are shown in fig. 6, 7 and table 3. It can be seen that both compounds 1 and 2 are capable of significantly inhibiting the hyphal growth of resistant botrytis cinerea, IC thereof 50 Values were 27.39 and 40.24 μg/mL, respectively; it also has remarkable inhibitory activity on spore germination of drug-resistant botrytis cinerea, IC 50 Values were 36.58 and 21.99 μg/mL, respectively; the activity is superior to that of azoxystrobin and boscalid which are commonly used chemical synthesis bactericides.
TABLE 3 determination of inhibitory Activity of Compound 1 and Compound 2 against drug-resistant Botrytis cinerea ZC-8 by resazurin method
Figure BDA0003458431350000091
Example 3: nematicidal Activity assay
The nematicidal activity of the compound 1 and the compound 2 is measured by taking the meloidogyne incognita as a test nematode, and the specific steps are as follows:
(1) Hatching of second-instar nematodes
Collecting cucumber root diseases infected with meloidogyne incognita, picking up nematode egg masses, and shaking and dispersing for 4min by using 1% NaClO solution. Distilled water is washed to pass through a 200-mesh sieve, the filtrate is transferred into a culture dish, incubated for 2-3d under the dark condition at 28 ℃, and the second-instar larvae (J2 s) are collected for standby.
(2) Preparation of sample solutions
Compound 1 and compound 2 were dissolved in dimethyl sulfoxide (DMSO) to prepare a mother liquor of 40mg/mL, and diluted with 0.1% tween 80 aqueous solution to obtain sample solutions of different concentrations for use.
(3) Nematicidal Activity assay
200. Mu.L of the suspension containing 40-60J 2s of Meloidogyne incognita and the sample solutions with different concentrations were respectively aspirated into a 48-well plate, and an equal volume of solvent was added as a negative control, and the measurement was repeated 3 times for each concentration. Shake culturing the culture plate at 28deg.C for 48 hr, observing under microscope, and calculating and correcting death rate and LD with fine needle stimulated J2s non-reaction as death standard 50 Values.
Corrected mortality = (control nematode survival-treatment nematode survival) ×100%/control nematode survival).
The results show that compounds 1 and 2 have moderate nematicidal activity against meloidogyne incognita, LD 50 The values were 311.5 and 162.0. Mu.g/mL, respectively.
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the scope of the invention, but to limit the invention to the particular embodiments, and any modifications, equivalents, and improvements made thereto, are intended to be included within the spirit and scope of the invention.

Claims (4)

1. Compound 1 and compound 2 of formula (one):
Figure FDA0004146537620000011
2. a process for the preparation of compound 1 and compound 2 of formula (one) as defined in claim 1, and their pesticidally acceptable salts: the method is characterized by comprising the following steps of: inoculating Aspergillus sp.TR15 strain into sterilized liquid culture medium, standing at 28deg.C for fermentation, and extracting and separating fermentation culture medium; the preservation number of the strain is CCTCC NO: m20211402;
wherein the liquid culture medium is prepared from 2% of sucrose, 2% of mannitol, 0.5% of peptone, 0.3% of yeast extract and 20% of artificial seawater potato extract, and has a pH of 7;
wherein, the extraction and separation steps of the fermentation culture medium are as follows:
(1) Extracting the fermentation medium with ethyl acetate for 2-3 times, mixing the extractive solutions, and concentrating to obtain fermentation crude extract;
(2) Subjecting the above fermented crude extract to silica gel column chromatography, and gradient eluting with dichloromethane-methanol with gradient of 100:1-1:1 (v/v) as solvent according to the polarity increasing sequence of the eluent; collecting dichloromethane-methanol=40:1 eluting component, performing reverse phase silica gel column chromatography, and performing gradient elution with methanol-water with gradient of 1:9 to 1:0 (v/v) as solvent;
(3) Collecting methanol-water=3:7 (v/v) reverse phase silica gel eluting component, semi-preparative high performance liquid chromatography purification, mobile phase 55% methanol-water, detection wavelength 210nm, flow rate 3mL/min, and respectively collecting retention time t R The components with the values of 14.6min and 19.0min are obtained to obtain the compound 1 and the compound 2 shown in the formula (I).
3. Use of a pyrrolidinol compound for inhibiting plant pathogens, wherein the pyrrolidinol compound is at least one of the compounds of claim 1; the plant pathogenic bacteria are botrytis cinerea.
4. Use of a pyrrolidinol compound for nematicidal purposes, wherein the pyrrolidinol compound is at least one of the compounds of claim 1; the nematode is meloidogyne incognita.
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