CN113817008B - Preparation method and application of novel succinyl hexadecanoic macrolide - Google Patents

Abstract

The present invention relates to novel macrolide compound 4 "-O-(6-O-succinyl-glucosyl)-25-methyl ivermectin and 4"-O-(6-O-succinyl-glucosyl)-25-ethyl ivermectin and Its microbial transformation preparation method and its application in the preparation of medicines for preventing and controlling agricultural and forestry pests and pest mites.

Description

Preparation method and use of novel succinyl hexadecacyclic lactone

Technical Field

The invention belongs to the field of biotechnology, and specifically relates to 4"-O-(6-O-succinyl-glucosyl)-25-methylivermectin and 4"-O-(6-O-succinyl-glucosyl)-25-ethyl ivermectin and a method for preparing the same through microbial transformation, and an application of the same in preparing drugs for preventing and controlling agricultural and forestry pests and mites.

Background Art

25-methyl ivermectin and 25-ethyl ivermectin are new 16-membered macrolide antibiotics with the characteristics of broad spectrum, high efficiency, low toxicity, small dosage and safer use. Their chemical structures are as follows:

Activity tests have shown that compared with avermectin, ivermectin and milbemycin, it has a killing effect on a variety of parasites such as nematodes, mites, ticks, lice, flies and maggots that cause internal and external parasitic diseases in pigs, cattle, sheep, horses, rabbits and poultry, making it a promising anti-parasitic lead compound.

Microbial transformation is the structural modification of complex substrates by microbial cells, that is, using one or a series of enzymes produced during microbial metabolism to catalyze reactions on specific sites (groups) of the substrate, so that the molecular structure of the substrate changes to another similar compound. Microbial transformation has the advantages of directional reaction, relatively simple products, few side reactions, fast biomass accumulation, short transformation time, high efficiency of invertase expression, mild reaction conditions, safety and environmental protection, etc. It is a convenient means of structural modification of natural active compounds.

The present invention utilizes the Bacillus subtilis ATCC 6633 strain to transform 25-methyl/25-ethyl ivermectins to obtain two new and novel structural 16-membered macrolide active compounds, the activity of which is equivalent to that of the precursor compound 25-methyl/25-ethyl ivermectins, but the crystallinity of which is better than that of the precursor compound, which is beneficial to the quality control of subsequent products and the reduction of production costs. In addition, compared with the parent compound, the structure of compound 1-2 has a carboxyl group, indicating that the water solubility will increase, which is beneficial to the development of a water-based insecticide.

Summary of the invention

1. A compound having the following structural formula:

Wherein, R＝CH ₃ or CH ₂ CH ₃ .

Wherein, R ₁ ＝CH ₃ , which is a compound 1 of the following structural formula:

or

R ₁ =CH ₂ CH ₃ , which is a compound 2 of the following structural formula:

The present invention provides a method for preparing compound 1-2, comprising the following steps:

1) Preparing Penicillium griseofulvum seed solution: taking Bacillus subtilis strain with the deposit number of ATCC 6633, streaking inoculated on PDA solid culture medium, culturing in a constant temperature incubator at 20-28° C. for 5-7 days, transferring the activated strain to soybean meal liquid culture medium, placing in a horizontal shaker at 20-28° C. and 250 rpm for 40 hours to obtain seed solution;

2) Preparation of compound 1-2: 1 ml of the seed solution was transferred to a fresh soybean meal liquid culture medium in each bottle, and cultured at 180 rpm/min and 28°C for 24 h. Then, 1 mL of 25-methyl/25-ethylivermectins (concentration 10 mg/mL) dissolved in an organic solvent was added to each bottle, and after transformation and culture for 96 hours under the same conditions, a fermentation broth was obtained; the fermentation broth was extracted with an extractant, the extractant was recovered, and then chromatographic separation was performed to obtain a pure product of compound 1-2.

As a preferred technical solution, the preparation method of the liquid culture medium in step 1) is: take 30g of glucose, 5g of yeast extract, 5g of NaCl and _5g of _K2HPO4 , dilute to 1000ml with distilled water, adjust the pH to 7.0-7.2 with 1mol/l NaOH, use 250ml conical flasks to dispense 50ml per bottle, weigh 0.25-0.30g of soybean meal into the conical flask, wrap it, and sterilize it at 121℃ and 0.15MPa for 20min.

As a preferred technical solution, the organic solvent in step 2) includes one or more of methanol, ethanol, and DMSO.

As a preferred technical solution, the extraction agent in step 2) includes one or more of ethyl acetate, n-butanol, isobutyl acetate, ethyl ether, petroleum ether, dichloromethane or chloroform.

As a preferred technical solution, the purification in step 2) comprises reverse phase high performance liquid chromatography.

As a preferred technical solution, the purification in step 2) further comprises silica gel column chromatography.

The present invention further provides a pesticide composition containing the above-mentioned compound 1 and/or compound 2, wherein the composition further contains one or more conventional carriers and/or diluents. The pesticide composition can be in the form of water dispersible granules, emulsifiable concentrates, water suspensions, oil suspensions, microemulsions or tablets.

The present invention further provides the use of the above-mentioned compound 1-2 in the preparation of a drug for preventing and controlling crop diseases and insect pests. The crop diseases and insect pests include nematodes and red spiders.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a high-resolution mass spectrum of compound 1 obtained in Example 1;

FIG2 is a hydrogen spectrum of compound 1 obtained in Example 1;

FIG3 is the carbon spectrum of compound 1 obtained in Example 1;

FIG4 DEPT135 spectrum of compound 1 obtained in Example 1;

FIG5 ¹ H- ¹ H COSY carbon spectrum of compound 1 obtained in Example 1;

FIG6 HMQC spectrum of compound 1 obtained in Example 1;

FIG7 HMBC spectrum of compound 1 obtained in Example 1;

FIG8 is a high-resolution mass spectrum of compound 2 obtained in Example 2;

FIG9 is a hydrogen spectrum of compound 2 obtained in Example 2;

FIG10 is the carbon spectrum of compound 2 obtained in Example 2;

FIG11 DEPT135 spectrum of compound 2 obtained in Example 2;

FIG12 ¹ H- ¹ H COSY carbon spectrum of compound 2 obtained in Example 2;

FIG13 HMQC spectrum of compound 2 obtained in Example 2;

FIG14 is the HMBC spectrum of compound 2 obtained in Example 2.

DETAILED DESCRIPTION

The present invention is described below with specific examples. It should be understood that the examples are used to illustrate the present invention rather than to limit the present invention. The scope and core content of the present invention are determined according to the claims.

Example 1

1) Take the Bacillus subtilis strain with the deposit number of ATCC 6633, streak inoculate it on a PDA solid culture medium, and culture it in a constant temperature incubator at 20-28° C. for 5-7 days. Transfer the activated strain to a soybean meal liquid culture medium, place it in a horizontal shaker at 20-28° C. and 250 rpm for 40 hours to obtain a seed liquid. Transfer 1 ml of the seed liquid to 100 mL of fresh soybean meal liquid culture medium in each bottle, and culture it at 180 rpm and 28° C. for 24 hours. Then, add 1 mL of 25-methyl ivermectin (concentration 10 mg/mL) dissolved in an organic solvent to each bottle, and transform and culture it under the same conditions for 96 hours to obtain a fermentation liquid.

2) Fermentation was performed according to the above method to obtain 2 L of fermentation broth, and the fermentation broth was extracted three times with equal volumes of ethyl acetate to obtain ethyl acetate extracts, and the extracts were concentrated to dryness under reduced pressure at 50° C. to obtain 4.5 g of oily substance.

The obtained oily substance was subjected to column chromatography on a silica gel column (particle size 100-200 mesh), and gradient elution was performed using chloroform:methanol = 100:0-60:40 (V/V). Three components were obtained by thin layer chromatography (TLC). Component 3 was subjected to gel (Sephadex LH-20) column chromatography to obtain component 3-1, which was then further purified using semi-preparative column chromatography to obtain pure 4"-O-(6-O-succinyl-glucosyl)-25-methyl ivermectin (1).

The thin layer identification method is as follows: the converted sample and blank control are spotted on a silica gel G thin layer plate, developed under the developing agent conditions of chloroform:methanol (8:2), taken out and dried after running, first observed under ultraviolet light at 254nm, then developed with concentrated sulfuric acid, and the conversion results were checked.

Semi-preparative column chromatography conditions: mobile phase: methanol-water (containing 1/1000 acetic acid) (90:10); chromatographic column C18, 9.4*250mm; detection wavelength: 244nm; flow rate: 1.5mL/min; column temperature: 30℃; injection volume: 50uL. The peak with a retention time of 14.35min was collected to obtain 4"-O-(6-O-succinyl-glucosyl)-25-methyl ivermectin (1, 14.5mg).

3) Structural identification of compound 1.

The structure of compound 1 was determined by 1D and 2D NMR, MS and other spectral analyses as follows:

The instruments involved in compound identification are as follows:

The nuclear magnetic resonance spectra were measured using a superconducting nuclear magnetic resonance instrument (Zhongke-400) from Oxford Zhongke Company;

Mass spectra and high-resolution mass spectra were measured using a Waters Q-TOF Micro LC-MS-MS mass spectrometer;

The UV spectra were measured using a Varian Cary 300 Bio spectrophotometer from Varian;

The specific data of the compound are as follows:

The structure of compound 1:

Properties: White crystalline powder;

Solubility: easily soluble in chloroform, acetone, methanol, ethanol, insoluble in water;

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;

The hydrogen spectrum ( ¹ H NMR) and carbon spectrum ( ¹³ C NMR) data are shown in Table 1.

Example 2

1) Take the Bacillus subtilis strain with the deposit number of ATCC 6633, streak inoculate it on a PDA solid culture medium, and culture it in a constant temperature incubator at 20-28° C. for 5-7 days. Transfer the activated strain to a soybean meal liquid culture medium, place it in a horizontal shaker at 20-28° C. and 250 rpm for 40 hours to obtain a seed liquid. Transfer 1 ml of the seed liquid to 100 mL of fresh soybean meal liquid culture medium in each bottle, and culture it at 180 rpm and 28° C. for 24 hours. Then, add 1 mL of 25-ethyl ivermectin (concentration 10 mg/mL) dissolved in an organic solvent to each bottle, and transform and culture it under the same conditions for 96 hours to obtain a fermentation liquid.

2) Fermentation was performed according to the above method to obtain 2 L of fermentation broth, and the fermentation broth was extracted three times with equal volumes of ethyl acetate to obtain ethyl acetate extracts, and the extracts were concentrated to dryness under reduced pressure at 50° C. to obtain 5.3 g of oily substance.

The obtained oily substance was subjected to column chromatography on a silica gel column (particle size 100-200 mesh), and gradient elution was performed with chloroform:methanol=100:0-60:40 (V/V), and three components were obtained by thin layer chromatography (TLC). Component 3 was subjected to gel (Sephadex LH-20) column chromatography to obtain component 3-1, which was then further purified by semi-preparative column chromatography to obtain pure 4"-O-(6-O-succinyl-glucosyl)-25-ethyl ivermectin (2, 25.8 mg).

The thin layer identification method is as follows: the converted sample and blank control are spotted on a silica gel G thin layer plate, developed under the developing agent conditions of chloroform:methanol (8:2), taken out and dried after running, first observed under ultraviolet light at 254nm, then developed with concentrated sulfuric acid, and the conversion results were checked.

Semi-preparative column chromatography conditions: mobile phase: methanol-water (containing 1/1000 acetic acid) (90:10); chromatographic column C18, 9.4*250mm; detection wavelength: 244nm; flow rate: 1.5mL/min; column temperature: 30℃; injection volume: 50uL. The peak with a retention time of 14.63min was collected to obtain 4"-O-(6-O-succinyl-glucosyl)-25-ethyl ivermectin (2).

3) Structural identification of compound 2.

The structure of compound 2:

Properties: White crystalline powder;

Solubility: easily soluble in chloroform, acetone, methanol, ethanol, insoluble in water;

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;

The hydrogen spectrum ( ¹ H NMR) and carbon spectrum ( ¹³ C NMR) data are shown in Tables 1 and 2.

Table 1 H (400 MHz) and C (100 MHz) NMR data of compound 1-2 in CD ₃ OD

Example 3

Biological activity of compounds 1-2 against Tetranychus cinnabarinus

Test organisms: Tetranychus cinnabarinus: inoculated on broad bean seedlings and cultured in an artificial climate chamber [(26±1)℃, RH(70±5)%, H/D14].

Experimental method: leaf disc immersion method: select adult mites raised indoors and in the same physiological state. Select broad bean leaves with the same growth, use a hole puncher to make a leaf disc with a diameter of 2 cm, and place the leaf with the back facing up on the absorbent cotton in the center of a plastic dish. There are 3 leaf discs per dish. Use a small brush to pick up adult mites and inoculate them on the leaf discs. There are 30 mites per leaf disc, and add appropriate amount of water. Place in a culture room at (26±1)℃, light intensity of 3000～4500lx, 14h/d, RH 50%～75%. After 2 hours, check the number of adult mites under a stereo microscope. The number of mites on each leaf disc should not be less than 20. The sample to be tested was dissolved in a small amount of acetone, and alkylphenol polyoxyethylene ether was used as an emulsifier. It was diluted with water to prepare agents with mass concentrations of 0.005, 0.01, 0.02, 0.04, and 0.08 mg/L. The agents were placed in a beaker, and the leaves were clamped with tweezers to soak the agents from low concentration to high concentration in sequence. The soaking time was 5s. The female adult mites were treated with distilled water as a control. Each mass concentration was a treatment, and each treatment was repeated 3 times. After the agent on the leaves dried, the treated leaf discs were placed in an artificial climate chamber at (26±1)℃ and a 14h photoperiod for 24h, and a small amount of water was added to the culture dish to keep it moist. After soaking, the mites were very active. They began to slow down their activities 5-8 hours after treatment, and the insect bodies were still after 12-24 hours. Death judgment criteria: During the inspection, the mite body was lightly touched with a brush. Those that did not move at all were 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 test of compound 1-2 against pine wood nematode

Test organisms: Pine wood nematode

Experimental method: inoculate the prepared pine wood nematodes onto the PDA culture medium covered with tomato gray botrytis (Botrytis cinerea), culture in a constant temperature incubator at 25°C for 7 days, pick out the culture medium containing pine wood nematodes, soak it in distilled water, filter and separate the nematodes after 24 hours, and prepare it into a suspension of about 2500 heads/mL for use.

The sample to be tested was dissolved in a small amount of acetone by the immersion method, and alkylphenol polyoxyethylene ether was used as an emulsifier, and diluted with water to prepare 5 mass concentrations of 1, 2, 5, 10, and 20 mg/L. 10 μL of the test agent and 90 μL of pine wood nematode suspension were taken in a 96-well culture plate, and the emulsion without the sample to be tested was used as a control, and the plates were cultured in a 25°C incubator. Each mass concentration was treated as one treatment, and each treatment was repeated 3 times. After 24 hours, the number of pine wood nematodes that survived and died was counted under a microscope.

Nematodes that move or are in an "S" shape, wavy shape, curled shape, or spiral shape are considered alive, and nematodes that are immobile and in a "C" shape, "J" shape, rigid, and have no light-shielding properties on their body walls are considered dead. The mortality rate and corrected mortality rate were calculated separately, and the toxicity regression equation, LC ₅₀ value, correlation coefficient, and 95% confidence limit were calculated using software SPSS22.0. Among them, mortality rate = (number of dead nematodes/number of tested nematodes) × 100%; corrected mortality rate = (mortality rate of treatment group - mortality rate of control group)/(1-mortality rate of control group) × 100%. The test results are shown in Table 3.

Table 3: Activity of compounds 1-2 against pine wood nematodes

The present invention utilizes Bacillus subtilis ATCC 6633 to transform 25-methyl/25-ethyl ivermectins to prepare two novel 16-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 two compounds have good effects in preventing and controlling agricultural and forestry pests or mites.

The uses of compounds 1-2 of the present invention have been described through specific examples. Those skilled in the art can refer to the contents of the present invention and appropriately change the raw materials, process conditions and other links to achieve corresponding other purposes. The relevant changes do not deviate from the contents of the present invention. All similar replacements and modifications are obvious to those skilled in the art and are deemed to be included in the scope of the present invention.

The above-described embodiments are only preferred solutions of the present invention and are not intended to limit the present invention in any form. Other variations and modifications are possible without exceeding the technical solutions described in the claims.

Claims (6)

1. A macrolide compound has the following general structure:

wherein r=ch ₃ Or CH (CH) ₂ CH _3。

2. The macrolide compound according to claim 1, having the structure:

3. the macrolide compound according to claim 1, having the structure:

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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