CN113461757B - Preparation method and application of novel sixteen-membered macrolide - Google Patents
Preparation method and application of novel sixteen-membered macrolide Download PDFInfo
- Publication number
- CN113461757B CN113461757B CN202110711042.6A CN202110711042A CN113461757B CN 113461757 B CN113461757 B CN 113461757B CN 202110711042 A CN202110711042 A CN 202110711042A CN 113461757 B CN113461757 B CN 113461757B
- Authority
- CN
- China
- Prior art keywords
- compound
- culturing
- column chromatography
- culture medium
- hours
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
- C07H17/08—Hetero rings containing eight or more ring members, e.g. erythromycins
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/90—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having two or more relevant hetero rings, condensed among themselves or with a common carbocyclic ring system
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
- C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/07—Optical isomers
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to novel macrolide compounds 5 ' -hydroxy-25-methyl-25-methyl ivermectin, 3 ' -hydroxy-25-methyl and 3 ' -hydroxy-25-methyl-nectin and a microbial conversion preparation method thereof and application thereof in preparing medicaments for preventing and controlling agriculture 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 5 '-hydroxy-25-methyl-ether, 3' -hydroxy-25-methyl ivermectin, 5 '-hydroxy-25-ethyl-ether and 3' -hydroxy-25-ethyl-ether through microbial transformation and application thereof in preparing medicaments for preventing and controlling agriculture 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.
The invention uses the Penicillium griseofulvum strain Penicilium griseofulvum to transform 25-methyl/25-ethyl ivermectins to obtain novel 4 sixteen-membered macrolide active compounds with novel structures, the activity of which is equivalent to that of a precursor compound 25-methyl/25-ethyl ivermectins, but the crystallinity of which is better than that of the precursor compound, thereby being beneficial to quality control of subsequent products and reducing the production cost. In addition, the structures of compounds 1-4 have one more hydroxyl group than the parent compound, indicating that the water solubility is increased, which is advantageous for development as a water-based pesticide.
Disclosure of Invention
1. A compound having the structural formula:
wherein R is 1 ＝CH 3 Or CH (CH) 2 CH 3 ；R 2 ＝OCH 3 Or OH; r is R 3 ＝CH 2 OH or CH 3 。
Wherein R is 1 ＝CH 3 ,R 2 ＝OCH 3 ,R 3 ＝CH 2 OH, which is compound 1 of the structural formula:
or alternatively
R 1 ＝CH 3 ,R 2 ＝OH,R 3 ＝CH 3 Which is compound 2 of the formula:
or alternatively
R 1 ＝CH 2 CH 3 ,R 2 ＝OCH 3 ,R 3 ＝CH 2 OH, which is compound 3 of the structural formula:
or alternatively
R 1 ＝CH 2 CH 3 ,R 2 ＝OH,R 3 ＝CH 3 Which is compound 4 of the formula:
the invention provides a preparation method of compounds 1-4, which comprises the following steps:
1) Preparing a penicillium griseofulvum seed solution: taking a Penicillium griseofulvum strain Penicilium griseofulvum with a deposit number of CICC40293, streaking and inoculating the Penicillium griseofulvum strain 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 1-4: 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 pure products of the compounds 1-4 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 and/or compound 3 and/or compound 4 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 compounds 1-4 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 high resolution mass spectrum of compound 2 obtained in example 1;
FIG. 5 shows the hydrogen spectrum of compound 2 obtained in example 1;
FIG. 6 carbon spectrum of compound 2 obtained in example 1;
FIG. 7 is a high resolution mass spectrum of compound 3 obtained in example 2;
FIG. 8 shows the hydrogen spectrum of compound 3 obtained in example 2;
FIG. 9 carbon spectrum of compound 3 obtained in example 2;
FIG. 10 is a high resolution mass spectrum of compound 4 obtained in example 2;
FIG. 11 shows the hydrogen spectrum of compound 4 obtained in example 2;
FIG. 12 carbon spectrum of Compound 4 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 Penicillium griseofulvum strain Penicilium griseofulvum with the deposit number of CICC40293, 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, culturing for 40 hours in a horizontal shaking table at 20-28 ℃ and 250rpm to obtain seed liquid, transferring 1mL of the seed liquid into 100mL of fresh soybean meal liquid culture medium per bottle, culturing for 24 hours at 180rpm/min and 28 ℃, adding 1mL of organic solvent-dissolved 25-methyl ivermectin (with the concentration of 10 mg/mL) 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 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 2 was subjected to gel (Sephadex LH-20) column chromatography to give component 2-1, which was then further purified using semi-preparative column chromatography to give pure 5 "-hydroxy-25-methyl ivermectin (1), 3" -hydroxy-25-methyl ivermectin (2).
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 (9:1), taken out for airing after running, observed under an ultraviolet lamp of 254nm, developed by concentrated sulfuric acid, and the conversion result is inspected.
Semi-preparative column chromatography conditions: mobile phase: methanol-water (92:8); 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. Peaks with retention times of 11.5min and 15.5min were collected to give 5 "-hydroxyymethyl-25-methyl ivermectin (1, 6.9 mg), 3" -hydroxy-25-methyl ivermectin (2, 61.3 mg).
3) Structural identification of Compounds 1-2.
The structure of compounds 1-2 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 300 Bio 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 45 H 68 O 15 ；
HRESI-MS m/z 871.4500 (calculated value: C) 45 H 68 NaO 15 ,871.4456)；
UVλ max (EtOH)nm(logε):245(4.05)；
IR v max cm -1 ：3371,2927,1734,1456,1384,1261,1040；
Hydrogen spectrum @ 1 H NMR) and carbon spectrum [ ] 13 C NMR) data are shown in tables 1 and 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 44 H 66 O 14 ；
HRESI-MS m/z 841.4350 (calculated value: C) 44 H 66 NaO 14 ,841.4399)；
UVλ max (EtOH)nm(logε):245(4.00)；
IR v max cm -1 ：3466,2932,1722,1451,1380,1183,1064,992；
Hydrogen spectrum @ 1 H NMR) and carbon spectrum [ ] 13 C NMR) data are shown in tables 1 and 2.
Example 2
1) Taking a Penicillium griseofulvum strain Penicilium griseofulvum with the deposit number of CICC40293, 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, culturing for 40 hours in a horizontal shaking table at 20-28 ℃ and 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/min and 28 ℃, adding 1mL of 25-methyl ivermectin dissolved in 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 3L 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 6.5g 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 2 was subjected to gel (Sephadex LH-20) column chromatography to give component 2-1, which was then further purified using semi-preparative column chromatography to give pure 5 "-hydroxy-25-ethyl-ivermectin (3, 11.4 mg), 3" -hydroxy-25-ethyl-ivermectin (4, 98.9 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 (9:1), taken out for airing after running, observed under an ultraviolet lamp of 254nm, developed by concentrated sulfuric acid, and the conversion result is inspected.
Semi-preparative column chromatography conditions: mobile phase: methanol-water (92:8); 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. Peaks with retention times of 12.1min and 16.0min were collected to give 5 "-hydroxy-25-ethyl-ivermectin (3), 3" -hydroxy-25-ethyl-ivermectin (4).
3) Structural identification of Compounds 3-4.
Structure of compound 3:
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 46 H 70 O 15 ；
HRESI-MS m/z 885.4646 (calculated value: C) 46 H 70 NaO 15 ,885.4612)；
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.
Structure of compound 4:
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 45 H 68 O 14 ；
HRESI-MS m/z 855.4514 (calculated value: C) 45 H 68 NaO 14 ,855.4507)；
UVλ max (EtOH)nm(logε):243(4.01)；
IR v max cm -1 ：3451,2921,1737,1455,1377,1003；
Hydrogen spectrum @ 1 H NMR) and carbon spectrum [ ] 13 C NMR) data are shown in tables 1 and 2.
TABLE 1 Compounds 1-4 in CDCl 3 Nuclear magnetic data of hydrogen spectrum (400 MHz)
TABLE 2 Compounds 1-4 in CDCl 3 Nuclear magnetic data of carbon spectrum (100 MHz)
Example 3
Biological Activity of Compounds 1-4 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 3
Table 3: activity of Compounds 1-4 against Tetranychus cinnabarinus
Example 4
Activity assay of Compounds 1-4 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 the virulence regression equation, LC50 values, correlation coefficients and 95% confidence limits were calculated using software SPSS 22.0. Wherein mortality = (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 4.
Table 4: activity of Compounds 1-4 against pine wood nematodes
The invention uses the Penicillium griseofulvum strain Penicilium griseofulvum to transform 25-methyl/25-ethyl ivermectins, and 4 novel sixteen-membered macrolide compounds can be prepared: 5 "-hydroxy-25-methyl ivermectin (1), 3" -hydroxy-25-methyl ivermectin (2), 5 "-hydroxy-25-ethyl-conveyor (3) and 3" -hydroxy-25-ethyl-conveyor (4). The 4 compounds have good effects in preventing and controlling agricultural and forestry pests or mites.
While the use of the compounds 1-4 of the present invention has been described by way of specific examples, those skilled in the art can, in light of the present disclosure, suitably modify the starting materials, process conditions, etc., to accomplish corresponding other objects, all of which are intended to be encompassed by the present invention, as would be apparent to one skilled in the art without departing from 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 (2)
1. A process for producing a macrolide compound, characterized in that the macrolide compound is selected from the group consisting of compound 1Or compound 2->The method comprises the following steps:
1) Taking Penicillium griseum strain Penicillium with the deposit number of CICC40293, 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, culturing for 40 hours in a horizontal shaking table at 20-28 ℃ and 250rpm to obtain seed liquid, transferring 1mL of the seed liquid into 100mL of fresh soybean meal liquid culture medium per bottle, culturing for 24 hours at 180rpm/min and 28 ℃, respectively adding 1mL of 25-methylvermectin with the concentration of 10mg/mL of organic solvent dissolved into each bottle, and culturing for 96 hours under the same condition to obtain fermentation liquor; 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; subjecting the oily substance obtained to column chromatography on a silica gel column with a particle size of 100-200 meshes, and subjecting the oily substance to column chromatography with chloroform with a volume ratio of 100:0-60:40: gradient elution is carried out on methanol, and 3 components are obtained through TLC thin layer identification detection; subjecting the component 2 to Sephadex LH-20 gel column chromatography to obtain a component 2-1, and further purifying by using semi-preparative column chromatography to obtain pure compound 1 and compound 2, wherein the semi-preparative column chromatography conditions are as follows: methanol-water with 92:8 mobile phase; 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, and collecting the peak with retention time of 11.5min to obtain compound 1, and collecting the peak with retention time of 15.5min to obtain compound 2.
2. A process for producing a macrolide compound, characterized in that the macrolide compound is selected from the group consisting of compound 3Or compound 4->The method comprises the following steps:
1) Taking Penicillium griseum strain Penicillium with the deposit number of CICC40293, 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, culturing for 40 hours in a horizontal shaking table at 20-28 ℃ and 250rpm to obtain seed liquid, transferring 1mL of the seed liquid into 100mL of fresh soybean meal liquid culture medium per bottle, culturing for 24 hours at 180rpm/min and 28 ℃, respectively adding 1mL of 25-ethylverin with the concentration of 10mg/mL of organic solvent dissolved into each bottle, and culturing for 96 hours under the same condition to obtain fermentation liquor; fermenting and culturing to obtain 3L 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 6.5g oily substance; subjecting the oily substance obtained to column chromatography on a silica gel column with a particle size of 100-200 meshes, and subjecting the oily substance to column chromatography with chloroform with a volume ratio of 100:0-60:40: gradient elution is carried out on methanol, and 3 components are obtained through TLC thin layer identification detection; subjecting the component 2 to Sephadex LH-20 gel column chromatography to obtain a component 2-1, and further purifying by using semi-preparative column chromatography to obtain pure compound 3 and compound 4, wherein the semi-preparative column chromatography conditions are as follows: methanol-water with 92:8 mobile phase; 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, and collecting the peak with retention time of 12.1min to obtain compound 3, and collecting the peak with retention time of 16.0min to obtain compound 4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110711042.6A CN113461757B (en) | 2021-06-25 | 2021-06-25 | Preparation method and application of novel sixteen-membered macrolide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110711042.6A CN113461757B (en) | 2021-06-25 | 2021-06-25 | Preparation method and application of novel sixteen-membered macrolide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113461757A CN113461757A (en) | 2021-10-01 |
| CN113461757B true CN113461757B (en) | 2023-08-22 |
Family
ID=77872990
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110711042.6A Active CN113461757B (en) | 2021-06-25 | 2021-06-25 | Preparation method and application of novel sixteen-membered macrolide |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113461757B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114931144A (en) * | 2022-06-21 | 2022-08-23 | 台州科技职业学院 | Macrolide compound and application thereof in prevention and treatment of agricultural and forestry pests and pest mites |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1043742A (en) * | 1988-12-19 | 1990-07-11 | 伊莱利利公司 | Macrolides compound |
| EP1632498A1 (en) * | 2004-09-03 | 2006-03-08 | Syngenta Participations AG | Derivatives of avermectin, avermectin monosaccharide and avermectin aglycone |
| CN101679451A (en) * | 2007-05-03 | 2010-03-24 | 梅瑞尔有限公司 | Comprise C-13 alkoxyl group ether macrolides compound and phenyl pyrazoles compound compositions |
| KR20150008725A (en) * | 2013-07-15 | 2015-01-23 | 한국생명공학연구원 | Pyripyropene S and process for preparing the same |
| CN104910228A (en) * | 2014-03-10 | 2015-09-16 | 浙江农林大学 | 16-membered macrolide compound and application thereof |
| CN106167815A (en) * | 2016-05-30 | 2016-11-30 | 浙江农林大学 | The Preparation method and use of it dimension streptozotocin derivative |
-
2021
- 2021-06-25 CN CN202110711042.6A patent/CN113461757B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1043742A (en) * | 1988-12-19 | 1990-07-11 | 伊莱利利公司 | Macrolides compound |
| EP1632498A1 (en) * | 2004-09-03 | 2006-03-08 | Syngenta Participations AG | Derivatives of avermectin, avermectin monosaccharide and avermectin aglycone |
| CN101679451A (en) * | 2007-05-03 | 2010-03-24 | 梅瑞尔有限公司 | Comprise C-13 alkoxyl group ether macrolides compound and phenyl pyrazoles compound compositions |
| KR20150008725A (en) * | 2013-07-15 | 2015-01-23 | 한국생명공학연구원 | Pyripyropene S and process for preparing the same |
| CN104910228A (en) * | 2014-03-10 | 2015-09-16 | 浙江农林大学 | 16-membered macrolide compound and application thereof |
| CN106167815A (en) * | 2016-05-30 | 2016-11-30 | 浙江农林大学 | The Preparation method and use of it dimension streptozotocin derivative |
Non-Patent Citations (1)
| Title |
|---|
| "Biotransformation of Erythrodiol for New Food Supplements with Anti-Inflammatory Properties";Pingping Shen，et al.;《J. Agric. Food Chem.》;20200730;第68卷;第5910-5916页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113461757A (en) | 2021-10-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| RU2139874C1 (en) | Polycyclic compounds, method and strain for their preparing, antiparasitic composition and method of supression of parasitic infections | |
| JPH0335796A (en) | Cyclic depsipeptide substance, its production and anthelmintics containing the same | |
| CN103740606B (en) | Plant raw streptomycete and produce antibiotic promise and irrigate method and the application of nest mycin | |
| CN106167815A (en) | The Preparation method and use of it dimension streptozotocin derivative | |
| CN113461757B (en) | Preparation method and application of novel sixteen-membered macrolide | |
| CN113817008B (en) | Preparation method and application of novel succinyl hexadecanoic macrolide | |
| CN104098585B (en) | Mibemycin analogue, its preparation method and application | |
| CN103242338B (en) | Macrolide compound as well as preparation method and application thereof | |
| FI94262B (en) | Process for preparing macrolide against parasites | |
| CN111153797B (en) | Active substance for killing nematode and its preparing process and application | |
| CN112795617B (en) | Marine fungus secondary metabolite and preparation and application thereof | |
| CN107722032B (en) | Anhydride compound with prodenia litura poisoning activity and preparation method and application thereof | |
| CN101830868B (en) | Oligosporol derivatives and application thereof | |
| JP2805564B2 (en) | New substance PF1101B substance and its manufacturing method | |
| CN106912489A (en) | The preparation method and applications of AVM analog derivative | |
| CN114853629B (en) | Dipeptide compound and preparation method and application thereof | |
| CN111909166B (en) | Two new milbemycins, preparation method and application thereof | |
| CN105462859B (en) | It is a kind of for preventing and treating the branch acremonium bacterial strain of cigarette aphid | |
| CN112010870A (en) | Novel sixteen-membered macrolide compound, preparation method and application thereof | |
| JPS63227590A (en) | Antibiotic milbemycin compound and production thereof | |
| CN113200995A (en) | Compound Sterigmatocystin, preparation method thereof and application thereof in pesticide | |
| CN117844671A (en) | Streptomyces purplish red and alkaloid compounds and application thereof | |
| JP2014037351A (en) | New depsipeptide pf1442 substance and production method thereof | |
| CN117510391A (en) | New tertiary amine alkali compound and preparation method and application thereof | |
| CN101967131B (en) | Oligosporol derivatives and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |