CN114369051A - Pyrrolidine alcohol compound and preparation method and application thereof - Google Patents

Pyrrolidine alcohol compound and preparation method and application thereof Download PDF

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CN114369051A
CN114369051A CN202210069462.3A CN202210069462A CN114369051A CN 114369051 A CN114369051 A CN 114369051A CN 202210069462 A CN202210069462 A CN 202210069462A CN 114369051 A CN114369051 A CN 114369051A
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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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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, the preservation date is 2021, 11 and 26 months, and the preservation number is CCTCC NO: M20211402), a preparation method thereof and application thereof in inhibiting phytopathogen and killing nematodes. Azole compoundsThe alkanol compound is compounds 1 and 2 shown in formula (I) and salts or solvates of the compounds and the compounds which are acceptable in pesticide. The compounds 1 and 2 have significant inhibitory activity on hypha growth and spore germination of drug-resistant botrytis cinerea, IC50The value is superior to that of the common commercial fungicides azoxystrobin and boscalid. In addition, Compounds 1 and 2 also have nematicidal activity against Meloidogyne incognita, LD50Values were 311.5 and 162.0. mu.g/mL, respectively. Therefore, the compounds 1 and 2 can be used as lead compounds or new pesticide components with the activities of inhibiting phytopathogen and killing nematodes.
Figure DDA0003458431370000011

Description

Pyrrolidine alcohol 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 a strain Aspergillus sp.TR15, a preparation method thereof and application thereof in inhibiting phytopathogen and killing nematodes.
Background
Botrytis cinerea (Botrytis cinerea) can jeopardize field growth and fruit storage of many commercial crops such as grapes, strawberries and tomatoes, and is expected to cause economic losses of over $ 100 million each year worldwide. Thus, gray mold is listed as one of the ten major plant fungal diseases in the world. For example: shandong is the largest fruit and vegetable production base in China, and the grey mold is caused to be high due to the humid and warm climatic conditions of the fruit and vegetable greenhouses, so that the great economic loss is caused every year. Up to now, the control of gray mold is still mainly based on chemically synthesized pesticides, such as: azoxystrobin and boscalid and the like. Although the chemical synthetic pesticide can better prevent and treat the gray mold, the relative single action mechanism of the chemical synthetic pesticide causes the continuous development of the drug resistance of strains, thereby falling into a vicious circle of resistance generation, increased use of the chemical synthetic pesticide, resistance development, pesticide residue excess and environmental pollution. Besides gray mold, the fruit and vegetable greenhouses are infected by southern root knot nematode disease, and the two often act together, so that economic loss is increased, and the control difficulty is greatly increased.
The active ingredients with the functions of inhibiting plant pathogenic bacteria or killing nematodes are searched from biological resources such as microorganisms, plants and the like, and become important sources for developing new environment-friendly pesticides. Compared with chemical synthetic 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, the pesticide composition has a 'multi-point action' mechanism for controlling plant diseases and insect pests, and becomes an important weapon for resisting drug-resistant diseases and insect pests.
According to research in literature, the pyrrolidine alcohol compounds have various effects such as anti-tumor, immunosuppression, antivirus and antibacterial. The two related pyrrolidine alcohol compounds are novel compounds which are not reported, the structural skeleton, the fat long-chain substituent and other structural characteristics of the compounds are different from those of the known pyrrolidine alcohol compounds, and the compounds 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 pyrrolidine alcohol compound and a preparation method and application thereof.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a compound 1 and a compound 2 represented by the formula (I) and a pesticidally acceptable salt or solvate of both:
Figure BDA0003458431350000021
the preparation method of the compound comprises the following steps: inoculating Aspergillus sp.TR15 into sterilized liquid culture medium, standing at 28 deg.C for fermentation, and extracting and separating the fermentation culture medium; the preservation number of the strain is CCTCC NO: m20211402.
On the basis of 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 with the pH value of 7.
Preferably, the extraction and separation steps of the fermentation medium are as follows:
(1) extracting the fermentation culture medium with ethyl acetate for 2-3 times, mixing extractive solutions, and concentrating to obtain fermentation crude extract;
(2) performing silica gel column chromatography on the crude fermentation extract, and performing gradient elution by using dichloromethane-methanol with gradient of 100:1 to 1:1(v/v) as a solvent according to the ascending order of the polarity of eluent; collecting dichloromethane-methanol-40: 1 eluate, performing reverse phase silica gel column chromatography, and performing gradient elution with methanol-water as solvent with gradient of 1:9 to 1:0 (v/v);
(3) collecting methanol-water (3: 7(v/v) reversed phase silica gel eluate, purifying by semi-preparative high performance liquid chromatography with mobile phase of 55% methanol-water, detection wavelength of 210nm, flow rate of 3mL/min, and collecting retention time tRThe components with the values of 14.6min and 19.0min obtain the compound 1 and the compound 2 shown in the formula (I).
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 is not produced therebetween.
The term "compound of the invention" as used herein means a compound of formula (i) in any form, i.e. in 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, particular polymorphic forms, solvates, including hydrates (e.g. mono-, di-and semi-hydrates)), and mixtures of forms thereof.
Solvates
For solvates of the compounds of the invention or salts thereof in crystalline form, one skilled in the art will appreciate that pesticidally acceptable solvates may be formed in which solvent molecules are incorporated into the crystal lattice upon crystallization. Solvates may comprise non-aqueous 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 crystal lattice). Solvates in which water is the solvent (which is incorporated into the crystal lattice) are commonly referred to as "hydrates". The hydrate comprises a stoichiometric hydrate and a component comprising variable amounts of water. The present invention includes such solvates.
The invention has the advantages that:
(1) the two related pyrrolidine alcohol compounds are novel compounds which are not reported, the structural framework, the fat long-chain substituent and other structural characteristics of the compounds are different from those of the known pyrrolidine alcohol compounds, the compound is found to have a comprehensive control effect on drug-resistant botrytis cinerea and meloidogyne incognita for the first time, and the compound can be used as a lead compound or a novel pesticide component for inhibiting plant pathogenic bacteria and killing nematode activity.
(2) The compounds 1 and 2 shown in the formula I can obviously inhibit the hypha growth of drug-resistant botrytis cinerea, and the IC thereof50Values of 27.39 and 40.24. mu.g/mL, respectively; it also has significant inhibitory activity on spore germination of drug-resistant Botrytis cinerea, IC50Values of 36.58 and 21.99. mu.g/mL, respectively; the activity is superior to that of the common chemically synthesized bactericides azoxystrobin and boscalid. In addition, the compounds 1 and 2 of the formula (I) also have nematicidal activity against Meloidogyne incognita, LD50Values were 311.5 and 162.0. mu.g/mL, respectively.
(3) The two pyrrolidinol compounds related by the invention are produced by fermentation of a strain Aspergillus sp.TR15, and are easy to carry out large-scale fermentation production; secondly, compared with the chemical synthetic pesticide, the microbial secondary metabolite has good environmental compatibility, and the prevention and treatment of plant diseases and insect pests also have a multi-point action mechanism and are not easy to generate drug resistance.
Drawings
FIG. 1 is a drawing showing a formula (I) of a compound 11H NMR spectrum.
FIG. 2 is a drawing showing a formula (I) of Compound 113C NMR spectrum.
FIG. 3 is a drawing showing a scheme for preparing a compound 2 represented by the formula (I)1H NMR spectrum.
FIG. 4 is a drawing showing a scheme for preparing a compound 2 represented by the formula (I)13C NMR spectrum.
FIG. 5 shows the inhibitory activity of compounds 1 and 2 represented by the formula (I) against drug-resistant Botrytis cinerea (filter paper method).
FIG. 6 shows the inhibitory activity of Compound 1 represented by formula (I) against drug-resistant Botrytis cinerea (Resazurin method).
FIG. 7 shows the inhibitory activity of Compound 2 represented by formula (I) against drug-resistant Botrytis cinerea (Resazurin method).
Biological preservation information
Aspergillus oryzae TR15 derived from suaeda glauca, named Aspergillus sp.TR15, is preserved in China center for type culture Collection, with the preservation address of China center for type culture Collection of Wuhan university No. 299 in eight-way in flood and mountain area of Wuhan City, Hubei province, the preservation number is CCTCC NO: M20211402, and the preservation date is 26 months at 11 months in 2021.
Detailed Description
Terms used in the present invention have generally meanings as commonly understood by one of ordinary skill in the art, unless otherwise specified.
The present invention will be described in further detail with reference to the following data in conjunction with specific examples. 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 pyrrolidine alcohol compounds indicated in the following examples from liquid fermentation products of a strain Aspergillus sp.TR15 (preserved in China center for type culture Collection, preservation date: 2021, 11 and 26 months, preservation number: CCTCC NO: M20211402), wherein the structures of the compounds 1 and 2 are as follows (the Arabic numbers in the structures are the mark positions of carbon atoms):
Figure BDA0003458431350000051
。
example 1: process for producing compound 1 and compound 2
(1) Fermenting and culturing the strain
The strain Aspergillus sp.TR15 (size 2X 2cm) grown on the surface of PDA plate was cut out, inoculated into a sterilized Erlenmeyer flask containing a liquid medium, and left to ferment at 28 ℃ 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 has a pH value of 7. The above% are all weight%.
Extracting the fermentation culture medium with ethyl acetate for 3 times, mixing extractive solutions, and concentrating to obtain crude fermentation extract.
(2) Preparation of the Compounds
Subjecting the crude fermented extract to silica gel column chromatography (glass column chromatography with internal diameter of 65mm and length of 300mm, sand plate and suction nozzle), and gradient eluting with dichloromethane-methanol with gradient of 100:1 to 1:1(v/v, the same below) as solvent according to the ascending order of eluent polarity; the dichloromethane-methanol 40:1 fractions were collected and subjected to reverse phase silica gel column chromatography (glass column chromatography with an inner diameter of 30mm and a length of 600mm, standard tetrafluoro gate) with methanol-water as a solvent in a gradient of 1:9 to 1: 0.
Collecting methanol-water (3: 7(v/v) reversed phase silica gel eluate, purifying by semi-preparative high performance liquid chromatography with mobile phase of 55% methanol-water, detection wavelength of 210nm, flow rate of 3mL/min, and collecting retention time tRFractions with values of 14.6min and 19.0min to obtain compound 1 and compound 2.
(3) Structural identification of compounds
TABLE 1 preparation of Compound 11H (500MHz) and13C NMR(125MHz)
spectrogram data (solvent for NMR measurement: deuterated chloroform)
Figure BDA0003458431350000061
Figure BDA0003458431350000071
TABLE 2 preparation of Compound 21H (500MHz) and13C NMR(125MHz)
spectrogram data (solvent for NMR measurement: deuterated chloroform)
Figure BDA0003458431350000072
Figure BDA0003458431350000081
Compound 1, colorless powder, HR-ESI-MS M/z 316.2634[ M + H]+The molecular formula is shown as C21H33NO, which1H- (FIG. 1) and13the C-NMR (FIG. 2) data are shown in Table 1.
Compound 2, colorless powder, HR-ESI-MS M/z 346.31027[ M + H]+The molecular formula is shown as C23H39NO, which1H- (FIG. 3) and13the C-NMR (FIG. 4) data are shown in Table 2.
Example 2: determination of bacteriostatic Activity
The antibacterial activity is qualitatively determined by adopting a filter paper method and quantitatively determined by adopting a resazurin method. The method comprises the following specific steps:
(1) test plant pathogenic bacteria
Drug-resistant botrytis cinerea ZC-8, isolated from a vegetable greenhouse in Zhu city, which is a commonly used chemically synthesized fungicide for botrytis cinerea: the azoxystrobin and the boscalid have drug resistance.
(2) Preparation of sample solution
Compound 1 and Compound 2 were dissolved in methanol to prepare a 20mg/mL stock solution for use in the filter paper assay.
A certain volume of mother liquor is respectively sucked and diluted by 50 percent methanol-water solution to sample solutions with different concentrations of 1920, 1440, 960, 480 and 240mg/L for the determination of the Resazurin method.
(3) Filter paper sheet method
A puncher is used for punching a fungus cake about 5mm from the edge of a colony of drug-resistant botrytis cinerea ZC-8, and the fungus cake is placed in the center of a PDA (personal digital assistant) plate of 9 cm. 2 sterile filter paper sheets with the thickness of 5mm are respectively and symmetrically placed on two sides of the fungus cake, the distance between the two sterile filter paper sheets and the fungus cake is about 25mm, and the two sterile filter paper sheets are positioned on the same straight line.
mu.L of compound 1 and compound 2 mother liquors were pipetted onto a filter paper sheet (loading 0.1mg) to add an equal volume of methanol as a control. Culturing at 28 deg.C for 5 days, and observing whether Compound 1 and Compound 2 can inhibit the growth of drug-resistant Botrytis cinerea ZC-8.
(4) Resazurin method
The experimental methods are referenced in the following documents: nat, Commun.2020,11(01) 1608: 1-19; Lett.appl.Microbiol.2015,61: 238-. The method comprises the following specific steps:
inoculating drug-resistant Botrytis cinerea ZC-8 to PDA plate, culturing at 25 deg.C for 7-10 days, collecting spores in sterile water, and adjusting concentration to 1 × 105one/mL. Taking a 96-well cell culture plate, respectively adding 45 mu L of liquid culture medium (35 mu L of 1% glucose sterile aqueous solution and 10 mu L of PDB solution), 5 mu L of sample solutions with different concentrations and 10 mu L of spore suspension into each well, taking the addition of an isovolumetric solvent as a negative control, and taking azoxystrobin and boscalid as a positive control; while equal volumes of liquid medium and sample solution (1920mg/L) were added to the zeroing wells, no spore suspension was added.
Each concentration is set to be 3 times, after shaking and mixing evenly, the 96-hole cell culture plate is properly sealed, and the shaking culture is carried out for 24 hours at the temperature of 25 ℃. Adding resazurin dye (40 mu mol/L), keeping out of the sun, and culturing at constant temperature of 25 ℃ until the color of the negative control becomes light pink. The microplate reader measures the absorbance of each well at a wavelength of 570 nm. Calculating the antibacterial rate and IC by using SPSS statistical software50The value is obtained.
The 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 botrytis cinerea ZC-8 and generate obviousThe zone of inhibition of bacteria. The results of the resazurin assay are shown in FIGS. 6 and 7 and Table 3. As can be seen, compounds 1 and 2 both significantly inhibited the mycelial growth of drug-resistant Botrytis cinerea, the IC thereof50Values of 27.39 and 40.24. mu.g/mL, respectively; it also has significant inhibitory activity on spore germination of drug-resistant Botrytis cinerea, IC50Values of 36.58 and 21.99. mu.g/mL, respectively; the activity is superior to that of the common chemically synthesized bactericides azoxystrobin and boscalid.
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: determination of nematicidal Activity
The method comprises the following steps of (1) determining the nematicidal activity of a compound 1 and a compound 2 by taking Meloidogyne incognita as a test nematode:
(1) hatching of second instar nematodes
Collecting cucumber root diseases infected with Meloidogyne incognita, picking nematode egg masses, and shaking and dispersing for 4min with 1% NaClO solution. Washing with distilled water, sieving with 200 mesh sieve, transferring the filtrate into culture dish, incubating at 28 deg.C in dark for 2-3d, and collecting second-instar larva (J2 s).
(2) Preparation of sample solution
Compound 1 and Compound 2 were dissolved in dimethyl sulfoxide (DMSO) to make a 40mg/mL stock solution, and diluted with 0.1% Tween 80 aqueous solution to obtain sample solutions of different concentrations for use.
(3) Determination of nematicidal Activity
mu.L of the Meloidogyne incognita suspension containing 40-60 pieces of J2s and different concentrations of the sample solution were pipetted into 48-well plates, and the assay was repeated 3 times for each concentration, with the addition of an equal volume of solvent as a negative control. Culturing the culture plate at 28 deg.C under shaking for 48 hr, observing under microscope, and calculating and correcting mortality and LD with the non-response of J2s stimulated by fine needle as death standard50The value is obtained.
Corrected mortality rate (control nematode survival rate-treatment nematode survival rate) × 100%/control nematode survival rate.
The results show that compounds 1 and 2 have moderate nematicidal activity, LD, against Meloidogyne incognita50Values were 311.5 and 162.0. mu.g/mL, respectively.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only illustrative of the present invention and are not intended to limit the scope of the present invention, and any modifications, equivalent alterations and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims (6)

1. Compounds 1 and 2 of formula (I) and their pesticidally acceptable salts:
Figure FDA0003458431340000011
2. a process for producing a compound 1 and a compound 2 represented by the formula (I) below and a pesticidally acceptable salt thereof: the method is characterized by comprising the following steps: inoculating Aspergillus sp.TR15 into sterilized liquid culture medium, standing at 28 deg.C for fermentation, and extracting and separating the fermentation culture medium; the preservation number of the strain is CCTCC NO: m20211402.
3. The method according to claim 2, wherein the liquid medium is prepared from sucrose 2%, mannitol 2%, peptone 0.5%, yeast extract 0.3%, 20% artificial seawater potato extract, pH 7.
4. The method according to claim 2, wherein the step of extracting and separating the fermentation medium comprises:
(1) extracting the fermentation culture medium with ethyl acetate for 2-3 times, mixing extractive solutions, and concentrating to obtain fermentation crude extract;
(2) performing silica gel column chromatography on the crude fermentation extract, and performing gradient elution by using dichloromethane-methanol with gradient of 100:1 to 1:1(v/v) as a solvent according to the ascending order of the polarity of eluent; collecting dichloromethane-methanol-40: 1 eluate, performing reverse phase silica gel column chromatography, and performing gradient elution with methanol-water as solvent with gradient of 1:9 to 1:0 (v/v);
(3) collecting methanol-water (3: 7(v/v) reversed phase silica gel eluate, purifying by semi-preparative high performance liquid chromatography with mobile phase of 55% methanol-water, detection wavelength of 210nm, flow rate of 3mL/min, and collecting retention time tRThe components with the values of 14.6min and 19.0min obtain the compound 1 and the compound 2 shown in the formula (I).
5. Use of a pyrrolidinol compound to inhibit phytopathogens, wherein said pyrrolidinol compound is at least one of the compounds according to claim 1; the plant pathogenic bacteria are botrytis cinerea.
6. Use of a pyrrolidinol compound in nematicidal applications, wherein said pyrrolidinol compound is at least one of the compounds of claim 1; the nematode is Meloidogyne incognita.
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