CN113817008B - Preparation method and application of novel succinyl hexadecanoic macrolide - Google Patents
Preparation method and application of novel succinyl hexadecanoic macrolide Download PDFInfo
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Abstract
The invention relates to a novel macrolide compound 4 '-O- (6-O-succinyl-glucosyl) -25-methyl ivermectin and 4' -O- (6-O-succinyl-glucosyl) -25-ethyl ivermectin, a microbial conversion preparation method thereof and application thereof in preparing medicaments for preventing and controlling agricultural and forestry pests and mites.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for preparing 4 '-O- (6-O-succinyl-glucosyl) -25-methyl ivermectin and 4' -O- (6-O-succinyl-glucosyl) -25-ethyl ivermectin by microbial transformation and application thereof in preparing medicaments for preventing and controlling agricultural and forestry pests and mites.
Background
25-methyl ivermectin and 25-ethyl ivermectin are novel sixteen-membered macrolide antibiotics, and have the characteristics of broad spectrum, high efficiency, low toxicity, small dosage, safer use and the like, and the chemical structural formulas of the antibiotics are as follows:
compared with avermectin, ivermectin and milbemycin, the active compound has the effect of dispelling and killing internal and external parasitic diseases such as nematodes, mites, ticks, lice, flies and maggots of animals such as pigs, cattle, sheep, horses, rabbits and poultry, and has a good prospect.
Microbial transformation is to modify a complex substrate by microbial cells, that is, to catalyze a reaction on a specific site (group) of the substrate by using a certain or a certain series of enzymes generated in the metabolic process of the microorganism, so that the molecular structure of the substrate is changed into another similar compound. The microbial transformation has the advantages of directional reaction, single product, less side reaction, rapid biomass accumulation, short transformation time, high expression efficiency of invertase, mild reaction condition, safety, environmental protection and the like, and is a convenient means for carrying out structural modification on natural active compounds.
According to the invention, the bacillus subtilis strain Bacillus subtilis ATCC 6633 is utilized to convert 25-methyl/25-ethyl ivermectins, so that 2 novel sixteen-membered macrolide active compounds with novel structures are obtained, the activity of the novel sixteen-membered macrolide active compounds is equivalent to that of a precursor compound 25-methyl/25-ethyl ivermectins, but the crystallinity of the novel sixteen-membered macrolide active compounds is better than that of the precursor compound, and the novel sixteen-membered macrolide active compounds are favorable for quality control of subsequent products and reduction of production cost. In addition, the structure of compounds 1-2 has a carboxyl group compared to the parent compound, indicating that water solubility is increased, which is advantageous for development as a water-based insecticide.
Disclosure of Invention
1. A compound having the structural formula:
wherein r=ch 3 Or CH (CH) 2 CH 3 。
Wherein R is 1 =CH 3 Which is compound 1 of the formula:
or alternatively
R 1 =CH 2 CH 3 Which is compound 2 of the formula:
the invention provides a preparation method of a compound 1-2, which comprises the following steps:
1) Preparing a penicillium griseofulvum seed solution: taking a bacillus subtilis strain Bacillus subtilis with the deposit number of ATCC 6633, streaking and inoculating on a PDA solid culture medium, culturing for 5-7 days in a constant temperature incubator at 20-28 ℃, transferring the activated strain into a soybean meal liquid culture medium, and culturing for 40 hours in a horizontal shaking table at 20-28 ℃ and 250rpm to obtain seed liquid;
2) Preparation of Compounds 1-2: transferring 1mL of seed solution into fresh soybean meal liquid culture medium, culturing at 180rpm/min and 28 ℃ for 24 hours, adding 1mL of 25-methyl/25-ethyl ivermectins (with concentration of 10 mg/mL) dissolved by organic solvent into each bottle, and culturing under the same condition for 96 hours to obtain fermentation liquor; extracting the fermentation liquor by using an extractant, recovering the extractant, and then obtaining a pure product of the compound 1-2 through chromatographic separation.
As a preferable technical scheme, the preparation method of the liquid culture medium in the step 1) comprises the following steps: taking 30g of glucose, 5g of yeast extract, 5g of NaCl and K 2 HPO 4 5g, distilled water is fixed to 1000ml, pH is regulated to 7.0-7.2 by 1mol/l NaOH, 50ml is subpackaged per bottle by adopting a 250ml triangular flask, 0.25-0.30g of soybean meal is weighed and placed into the triangular flask, bandaged, and sterilized for 20min under the conditions of 121 ℃ and 0.15 MPa.
As a preferable technical scheme, the organic solvent in the step 2) comprises one or more of methanol, ethanol and DMSO.
As a preferable technical scheme, the extractant in the step 2) comprises one or more of ethyl acetate, n-butanol, isobutyl acetate, diethyl ether, petroleum ether, dichloromethane or chloroform.
As a preferred embodiment, the purification of step 2) comprises reversed-phase high performance liquid chromatography.
As a preferred embodiment, the purification in step 2) further comprises silica gel column chromatography.
The present invention further provides pesticidal compositions comprising compound 1 and/or compound 2 as described above, said compositions further comprising one or more conventional carriers and/or diluents. The pesticide composition can be in the form of water dispersible granules, emulsifiable concentrates, aqueous suspensions, oil suspensions, microemulsions or tablets.
The invention further provides application of the compound 1-2 in preparing medicines for preventing and controlling crop diseases and insect pests. The crop diseases and insect pests comprise nematodes and red spiders.
Drawings
FIG. 1 high resolution mass spectrum of compound 1 obtained in example 1;
FIG. 2 shows the hydrogen spectrum of compound 1 obtained in example 1;
FIG. 3 carbon spectrum of Compound 1 obtained in example 1;
FIG. 4 DEPT135 spectrum of compound 1 obtained in example 1;
FIG. 5 Compound 1 obtained in example 1 1 H- 1 H COSY carbon profile;
FIG. 6 HMQC spectrum of Compound 1 obtained in example 1;
FIG. 7 is a HMBC spectrum of Compound 1 obtained in example 1;
FIG. 8 high resolution mass spectrum of compound 2 obtained in example 2;
FIG. 9 shows the hydrogen spectrum of compound 2 obtained in example 2;
FIG. 10 carbon spectrum of compound 2 obtained in example 2;
FIG. 11 DEPT135 spectrum of compound 2 obtained in example 2;
FIG. 12 Compound 2 obtained in example 2 1 H- 1 H COSY carbon profile;
FIG. 13 HMQC spectrum of Compound 2 obtained in example 2;
FIG. 14 HMBC spectra of compound 2 obtained in example 2.
Detailed Description
The invention is illustrated below with specific examples, it being understood that the examples are intended to illustrate the invention and not to limit it, the scope and core content of which are defined by the claims.
Example 1
1) Taking a bacillus subtilis strain Bacillus subtilis with the deposit number of ATCC 6633, streaking and inoculating on a PDA solid culture medium, culturing for 5-7 days in a constant temperature incubator with the temperature of 20-28 ℃, transferring the activated strain into a soybean meal liquid culture medium, culturing for 40 hours in a horizontal shaking table with the temperature of 20-28 ℃ and the speed of 250rpm to obtain seed liquid, transferring 1mL of the seed liquid into 100mL of a fresh soybean meal liquid culture medium per bottle, culturing for 24 hours at 180rpm and the temperature of 28 ℃, respectively adding 25-methyl ivermectin (with the concentration of 10 mg/mL) dissolved by 1mL of an organic solvent into each bottle, and culturing for 96 hours under the same condition to obtain a fermentation broth.
2) Fermenting and culturing to obtain 2L fermentation liquor according to the method, extracting the fermentation liquor with equal volume of ethyl acetate for three times to obtain ethyl acetate extract liquid, concentrating the extract liquid to dryness under the condition of reducing pressure at 50 ℃ to obtain 4.5g oily substance.
The obtained oily substance was subjected to column chromatography on a silica gel column (particle size 100 to 200 mesh) with chloroform: gradient elution with methanol=100:0-60:40 (V/V) was performed and detected by thin layer identification (TLC) to give 3 fractions. Component 3 was subjected to gel (Sephadex LH-20) column chromatography to give component 3-1, which was then further purified using semi-preparative column chromatography to give pure 4"-O- (6-O-succinyl-glucosyl) -25-methyl ivermectin (1).
Wherein the thin layer identification method comprises the following steps: the converted sample and blank control point are spread on a silica gel G thin layer plate under the condition of developing agent of chloroform-methanol (8:2), taken out for airing after running, observed under an ultraviolet lamp of 254nm, developed with concentrated sulfuric acid, and the conversion result is inspected.
Semi-preparative column chromatography conditions: mobile phase: methanol-water (containing thousandth of acetic acid) (90:10); column C18,9.4 x 250mm; detection wavelength: 244nm; flow rate: 1.5mL/min; column temperature: 30 ℃; the sample injection amount is as follows: 50uL. The peak with retention time of 14.35min was collected to give 4"-O- (6-O-succinyl-glucosyl) -25-methyl ivermectin (1, 14.5 mg).
3) Structural identification of compound 1.
The structure of compound 1 was determined by 1D and 2D NMR, MS, etc. spectroscopy as follows:
compound identification involves the following instrumentation:
the nuclear magnetic resonance spectrum is measured by a superconducting nuclear magnetic resonance apparatus (ZHONGKE-400) of the middle Ke oxford company;
the mass spectrum and the high-resolution mass spectrum are measured by a Q-TOF Micro LC-MS-MS mass spectrometer of Waters company;
the UV spectrum was measured using a Varian Cary 300Bio spectrophotometer spectrometer from Varian company;
specific data for the compounds are as follows:
structure of compound 1:
property of white crystalline powder;
solubility, namely, the water-insoluble organic matter is easy to dissolve in chloroform, acetone, methanol and ethanol;
molecular formula C 55 H 82 O 22 ;
HRESI-MS m/z:1117.5198[M+Na] + (calculated value: C 55 H 82 O 22 Na,1117.5195);
UVλ max (EtOH)nm(logε):245(4.45);
IR v max cm -1 :3381,2963,1724,1680,1448,1384,1261,1091;
Hydrogen spectrum @ 1 H NMR) and carbon spectrum [ ] 13 C NMR) data are shown in table 1.
Example 2
1) Taking a bacillus subtilis strain Bacillus subtilis with the deposit number of ATCC 6633, streaking and inoculating on a PDA solid culture medium, culturing for 5-7 days in a constant temperature incubator with the temperature of 20-28 ℃, transferring the activated strain into a soybean meal liquid culture medium, culturing for 40 hours in a horizontal shaking table with the temperature of 20-28 ℃ and the speed of 250rpm to obtain seed liquid, transferring 1mL of the seed liquid into 100mL of a fresh soybean meal liquid culture medium per bottle, culturing for 24 hours at 180rpm and the temperature of 28 ℃, respectively adding 1mL of 25-methyl ivermectin (with the concentration of 10 mg/mL) dissolved by an organic solvent into each bottle, and culturing for 96 hours under the same condition to obtain fermentation liquor.
2) Fermenting and culturing to obtain 2L fermentation liquor according to the method, extracting the fermentation liquor with equal volume of ethyl acetate for three times to obtain ethyl acetate extract liquid, concentrating the extract liquid to dryness under the condition of reducing pressure at 50 ℃ to obtain 5.3g oily substance.
The obtained oily substance was subjected to column chromatography on a silica gel column (particle size 100 to 200 mesh) with chloroform: gradient elution with methanol=100:0-60:40 (V/V) was performed and detected by thin layer identification (TLC) to give 3 fractions. Component 3 was subjected to gel (Sephadex LH-20) column chromatography to give component 3-1, which was then further purified using semi-preparative column chromatography to give pure and 4"-O- (6-O-succinyl-glucosyl) -25-ethyl ivermectin (2, 25.8 mg).
Wherein the thin layer identification method comprises the following steps: the converted sample and blank control point are spread on a silica gel G thin layer plate under the condition of developing agent of chloroform-methanol (8:2), taken out for airing after running, observed under an ultraviolet lamp of 254nm, developed with concentrated sulfuric acid, and the conversion result is inspected.
Semi-preparative column chromatography conditions: mobile phase: methanol-water (containing thousandth of acetic acid) (90:10); column C18,9.4 x 250mm; detection wavelength: 244nm; flow rate: 1.5mL/min; column temperature: 30 ℃; the sample injection amount is as follows: 50uL. The peak with retention time of 14.63min was collected to give 4"-O- (6-O-succinyl-glucosyl) -25-ethyl ivermectin (2).
3) Structure identification of compound 2.
Structure of compound 2:
property of white crystalline powder;
solubility, namely, the water-insoluble organic matter is easy to dissolve in chloroform, acetone, methanol and ethanol;
molecular formula C 56 H 84 O 22 ;
HRESI-MS m/z 1131.5356 (calculated value: C) 56 H 84 O 22 Na,1131.5352);
UVλ max (EtOH)nm(logε):245(4.06);
IR v max cm -1 :3466,2931,1719,1451,1380,1120,1066,993;
Hydrogen spectrum @ 1 H NMR) and carbon spectrum [ ] 13 C NMR) data are shown in tables 1 and 2.
TABLE 1 Compounds 1-2 in CD 3 Nuclear magnetic data in the hydrogen spectrum (400 MHz) and carbon spectrum (100 MHz) in OD
Example 3
Biological Activity of Compounds 1-2 against Tetranychus cinnabarinus
Test organism: tetranychus cinnabarinus (Tetranychus cinnabarinus): under the condition of artificial climate chamber [ (26+ -1) DEGC, RH (70+ -5)%, H/D14], inoculating on broad bean seedling for culturing.
The experimental method comprises the following steps: adopts leaf disc insect immersing method: and selecting adult mites which are fed indoors and have consistent physiological states. Selecting broad bean leaves with consistent growth, making leaf discs with the diameter of 2cm by using a puncher, placing the leaf backs of the leaf discs on absorbent cotton in the center of a plastic dish, picking up 3 leaf discs of each dish by using a small-sized writing brush, inoculating mites on the leaf discs, adding a proper amount of water into 30 heads of each leaf disc, and placing the leaf discs in a culture room with the temperature of 26+/-1 ℃ and the illumination intensity of 3000-4500 lx, 14h/d and RH of 50-75%. After 2 hours, the number of mites is checked under a stereoscopic microscope, and the number of mites on each leaf dish is not less than 20. Dissolving a sample to be tested in a small amount of acetone, diluting with water by taking alkylphenol ethoxylates as an emulsifying agent, preparing medicaments with mass concentrations of 0.005, 0.01, 0.02, 0.04 and 0.08mg/L, placing the medicaments in a beaker, clamping blades by forceps, sequentially soaking the medicaments from low concentration to high concentration for 5 seconds, treating female mites by using distilled water in comparison, wherein each mass concentration is treated, and repeating each treatment for 3 times. And (3) after the medicines on the leaves are dried, placing the treated leaf discs in a climatic chamber with the photoperiod of (26+/-1) DEG C and 14 hours for culturing for 24 hours, and adding a small amount of water into a culture dish for moisturizing. Mites are very active after the drug infusion, slow down the activity after 5-8 hours after treatment, and the insects are static after 12-24 hours. Death criterion: when the test is performed, the mite body is touched by the writing brush, and the dead person is judged to be dead. The test results are shown in Table 2
Table 2: activity of Compounds 1-2 against Tetranychus cinnabarinus
Example 4
Activity assay of Compounds 1-2 on pine wood nematodes
Test organism: takes pine wood nematodes as insect test agents
The test method comprises the following steps: inoculating prepared pine wood nematodes to PDA culture medium full of Botrytis cinerea, culturing in a constant temperature incubator at 25deg.C for 7 days, picking out the culture medium containing pine wood nematodes, soaking in distilled water for 24 hr, filtering to separate out nematodes, and preparing into suspension of 2500 head/mL for use.
The method comprises the steps of dissolving a sample to be detected in a small amount of acetone by adopting an immersion liquid method, diluting with water by taking alkylphenol ethoxylates as an emulsifying agent, and respectively preparing the mixture into 5 mass concentrations of 1, 2, 5, 10 and 20mg/L. Taking 10 mu L of the test agent and 90 mu L of pine wood nematode suspension in a 96-well culture plate, taking the emulsifiable concentrate without the sample to be tested as a control, and culturing in a culture box at 25 ℃. Each mass concentration was 1 treatment, each treatment was repeated 3 times, and after 24 hours microscopic examination, the survival number and death number of pine wood nematodes were counted.
Nematodes moving or in the shape of "S", waves, curls, and spirals are considered living worms, while nematodes that are motionless and in the shape of "C", J ", stiff, and have no light-shielding properties in the body wall are considered dead worms. Mortality and corrective mortality were calculated separately and virulence regression equation, LC, was calculated using software SPSS 22.0 50 Values, correlation coefficients, and 95% confidence limits. Wherein die outRate = (number of dead nematodes/number of nematodes tested) ×100%; corrected mortality = (treatment mortality-control mortality)/(1-control mortality) ×100%. The test results are shown in Table 3.
Table 3: activity of Compounds 1-2 against pine wood nematodes
The invention uses bacillus subtilis Bacillus subtilis ATCC 6633 to convert 25-methyl/25-ethyl ivermectins, and can prepare 2 novel sixteen-membered macrolide compounds: 4"-O- (6-O-succinyl-glucosyl) -25-methyl ivermectin (1) and 4" -O- (6-O-succinyl-glucosyl) -25-ethyl ivermectin (2). The 2 compounds have good effects in preventing and controlling agricultural and forestry pests or mites.
The use of the compounds 1-2 according to the present invention has been described by way of specific examples, and those skilled in the art can, with reference to the present disclosure, suitably modify the starting materials, process conditions, etc. to achieve corresponding other objects, all of which do not depart from the spirit of the invention, and all similar substitutions and modifications will be apparent to those skilled in the art and are intended to be included within the scope of the present invention.
The above-described embodiments are merely preferred embodiments of the present invention, and the present invention is not limited in any way, and other variations and modifications may be made without departing from the technical aspects set forth in the claims.
Claims (6)
4. a process for preparing a compound according to claims 1-3, characterized in that the process comprises the steps of:
1) Preparing a penicillium griseofulvum seed solution: taking a bacillus subtilis strain Bacillus subtilis with the deposit number of ATCC 6633, streaking and inoculating on a PDA solid culture medium, culturing for 5-7 days in a constant temperature incubator at 20-28 ℃, transferring the activated strain into a soybean meal liquid culture medium, and culturing for 40 hours in a horizontal shaking table at 20-28 ℃ and 250rpm to obtain seed liquid;
2) Preparation of the compound: transferring 1mL of seed liquid into fresh soybean meal liquid culture medium, culturing for 24 hours at the temperature of 28 ℃ at 180rpm/min, adding 1mL of 25-methyl ivermectin or 25-ethyl ivermectin dissolved by organic solvent into each bottle, and culturing for 96 hours under the same condition at the concentration of 10mg/mL to obtain fermentation liquor;
3) Fermenting and culturing to obtain 2L fermentation liquor according to the method, extracting the fermentation liquor with equal volume of ethyl acetate for three times to obtain ethyl acetate extract liquor, concentrating the extract liquor to dryness under the condition of reducing pressure at 50 ℃ to obtain oily substances; subjecting the oily substance to column chromatography on silica gel column with particle size of 100-200 meshes, gradient eluting with chloroform-methanol with volume ratio of 100:0-60:40, and identifying and detecting by TLC thin layer to obtain 3 components; subjecting the component 3 to Sephadex LH-20 gel column chromatography to obtain a component 3-1, and further purifying by semi-preparative column chromatography to obtain pure compound 1 or 2, wherein the semi-preparative column chromatography conditions are as follows: the mobile phase is 90:10Methanol-water, wherein the water contains one thousandth of acetic acid; column C18,9.4 x 250mm; detection wavelength: 244nm; flow rate: 1.5mL/min; column temperature: 30 ℃; the sample injection amount is as follows: 50uL, collecting a peak with a retention time of 14.35min to obtain a compound 1, and collecting a peak with a retention time of 14.63min to obtain a compound 2, wherein the compound 1 is:compound 2 is->
5. A pharmaceutical composition comprising one or more of the compounds of claims 1-3, together with one or more conventional carriers and/or diluents.
6. Use of a compound according to any one of claims 1 to 3 or a pharmaceutical composition according to claim 5 in the manufacture of a medicament for controlling crop diseases and insect pests, which are nematodes or red arachnids.
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CN1738828A (en) * | 2002-12-20 | 2006-02-22 | 辛根塔参与股份公司 | Avermectin b1 and avermectin b1 monosaccharide derivatives having an alkoxymethyl substituent in the 4''- or 4'-position |
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