An indolinedione piperazine compound, a preparation method and application thereof

Abstract

The application discloses an indole dione piperazine compound and a preparation method and application thereof, and belongs to the field of pesticide discovery. The compound is reported for the first time, is a novel indole dione piperazine compound, and is obtained by fermentation and extraction separation of a culture medium inoculated with Aspergillus aculeatus Aa-1. The novel indole dione piperazine compound asperinamide A has good insecticidal activity on Spodoptera exigua 2nd instar larvae.

Description

Technical Field

[0001] This invention belongs to the field of phytochemistry technology, specifically relating to an indole-diketopiperazine compound, its preparation method, and its insecticidal activity against noctuid moths. Background Technology

[0002] Noctuid moths are mostly phytophagous pests with diverse methods of damage. Some burrow underground, biting off plant roots, stems, and seedlings, such as cutworms; others bore into stems or fruits, like the fall armyworm larvae, which can devour large quantities of gramineous crops such as rice, sugarcane, and corn, as well as various crops from the Asteraceae and Brassicaceae families; still others expose themselves on the surface of the host plant, such as armyworms. Noctuid moths are characterized by their wide adaptability, long migration distances, high reproductive capacity, and heavy damage, causing enormous agricultural losses both domestically and internationally. Currently, control of noctuid moths is mainly achieved through chemical pesticides such as chlorantraniliprole, tetrachlorantraniliprole, indoxacarb, lufenuron, and chlorfenapyr. Although chemical pesticides are highly effective, fast-acting, and convenient, long-term use inevitably leads to pesticide resistance in noctuid moths, resulting in a series of ecological and environmental problems and human health issues, negatively impacting the high-quality development of the agricultural economy.

[0003] Biopesticides are pesticide formulations made directly from living organisms or their metabolites for the control of pests, diseases, and weeds. Biopesticides are characterized by high efficiency, safety, and good environmental compatibility, playing a vital role in green agriculture and food safety. Among them, microbial metabolite pesticides, such as jinggangmycin, avermectin, spinosad, huaguangmycin, and liuyangmycin, have seen rapid development in recent years and have become one of the main types of microbial pesticides in my country. Indole-diketopiramate alkaloids are fungal secondary metabolites, mostly isolated from Aspergillus and Penicillium fungi. They are derived from the combination of different amino acids and are widely distributed in nature. These compounds contain two parent nuclei: indole and diketopiramate, and possess good and broad biological activities, such as antitumor, antibacterial, immunomodulatory, and insecticidal effects, providing lead compounds for drug development. Chinese invention patent CN102675293B discloses an indole-diketopiperazine derivative, its preparation method and application. This indole-diketopiperazine compound is obtained by fermentation culture and extraction separation of Eurotium cristatum and has good brine shrimp killing activity.

[0004] Therefore, how to provide a biopesticide for controlling noctuid moths has become a current research hotspot and challenge. Summary of the Invention

[0005] The first objective of this invention is to provide a novel indole-diketopiperazine compound; the second objective is to provide a method for preparing the indole-diketopiperazine compound; and the third objective is to provide the application of the indole-diketopiperazine compound in the control of Noctuidae insects, thus providing a new compound entity for the biological control of Noctuidae insects.

[0006] To achieve the first objective mentioned above, the present invention adopts the following technical solution:

[0007] An indole-diketone piperazine compound, the structural formula of which is shown in the following formula: asperinamide A:

[0008]

[0009] The indole-diketopiperazine compound of this invention was prepared from the tobacco-derived fungus *Aspergillus aculeatus* Aa-1. The novel indole-diketopiperazine compound was named asperinamide A by high-resolution mass spectrometry and superconducting nuclear magnetic resonance spectroscopy. The novel indole-diketopiperazine compound asperinamide A exhibits good insecticidal activity against second-instar larvae of the fall armyworm.

[0010] To achieve the second objective mentioned above, the present invention adopts the following technical solution:

[0011] A method for preparing an indole-dione-piperazine compound with the above-mentioned structure, wherein the indole-dione-piperazine compound is obtained by fermentation, extraction and separation from a culture medium inoculated with Aspergillus echinococcosis Aa-1.

[0012] The *Aspergillus aculeatus* Aa-1 strain is deposited by the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 40424 and classified as *Aspergillus aculeatus*. The deposit date is November 14, 2022, and the deposit location is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The *Aspergillus aculeatus* Aa-1 strain has been disclosed in Chinese Patent CN115851552A regarding *Aspergillus aculeatus* Aa-1 and its application in tobacco quality improvement.

[0013] Furthermore, the preparation method of the above-mentioned indole-diketopiramate compounds includes the following steps:

[0014] (1) The Aspergillus echinospora Aa-1 was inoculated into malt extract medium;

[0015] (2) The culture base after inoculation was allowed to ferment at 28-30℃, and the extract was obtained by ultrasonic extraction with ethyl acetate and vacuum concentration.

[0016] (3) The above extract was subjected to silica gel column chromatography with gradient elution of petroleum ether-ethyl acetate;

[0017] (4) The eluent fraction with a volume ratio of petroleum ether to ethyl acetate of 1:1 in step (3) was subjected to silica gel column chromatography, and the eluent was petroleum ether to ethyl acetate with a volume ratio of 10:3.

[0018] (5) The petroleum ether-ethyl acetate eluent from step (4) is eluted by reverse-phase silica gel column elution, and the eluent is methanol-water with a volume ratio of 30-80%.

[0019] (6) The eluent fraction with a methanol-water volume ratio of 50% from step (5) was purified by thin-layer chromatography to obtain the indole-dione piperazine compound asperinamide A.

[0020] Preferably, the fermentation time in step (2) is 30 to 40 days.

[0021] Preferably, in step (3), the petroleum ether-ethyl acetate elution gradient is 30:1 to 1:1.

[0022] Preferably, the developing system for thin-layer chromatography purification in step (6) is dichloromethane:methanol = 30:1.

[0023] To achieve the third objective mentioned above, the present invention adopts the following technical solution:

[0024] This invention discloses the application of the indole-dione-piperazine compound represented by the above-described formula asperinamide A in the control of noctuid moths.

[0025] This invention also discloses the application of the indole-dione-piperazine compound represented by formula asperinamide A in the preparation of insecticides for agricultural noctuid moths.

[0026] Preferably, the noctuid moth is the fall armyworm.

[0027] The beneficial effects of this invention are as follows:

[0028] (1) The fungal-derived indole-dione piperazine compound asperinamide A was prepared by SciFinder n Database search revealed that the indole-dione-piperazine compound asperinamide A is a novel natural product;

[0029] (2) The method provided by the present invention can quickly locate and accurately separate the indole-diketopiperazine compounds;

[0030] (3) The indole-dione piperazine compound asperinamide A has good insecticidal activity against second-instar larvae of fall armyworm (superior to the positive control drug matrine). Attached Figure Description

[0031] Figure 1 This is a molecular structure diagram of asperinamide A, an indole-dione piperazine compound described in this invention.

[0032] Figure 2 This is a high-resolution mass spectrum of asperinamide A, an indole-dione piperazine compound described in this invention.

[0033] Figure 3 The photon spectrum (500 MHz, methanol-d4) of the indole-diketopiperazine compound asperinamide A described in this invention is shown.

[0034] Figure 4 This is the carbon spectrum (125MHz, methanol-d4) of the indole-diketopiperazine compound asperinamide A described in this invention.

[0035] Figure 5 This is a single-crystal diffraction pattern of asperinamide A, an indole-diketopiperazine compound described in this invention. Detailed Implementation

[0036] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] Example 1:

[0038] A method for preparing an indole-diketone piperazine compound includes the following steps:

[0039] (1) Inoculate Aspergillus echinospora Aa-1 into 1000mL Erlenmeyer flasks, each containing sterilized malt extract culture medium (Qingdao High-tech Park Haibo Biotechnology Co., Ltd., HB4176-3);

[0040] (2) The above-mentioned Erlenmeyer flask was placed at 28°C for static fermentation culture for 30 days. The culture medium of the fermented Aspergillus echinococcosis Aa-1 strain was extracted by ultrasonic extraction with 300 mL of ethyl acetate and concentrated under vacuum to obtain the extract.

[0041] (3) The above extracts were subjected to silica gel column chromatography, and eluted with petroleum ether-ethyl acetate (volume ratios of 30:1, 10:1, 5:1, 2:1 and 1:1) as gradient elution buffers to obtain a total of 5 fractions Fr.1 to Fr.5;

[0042] (4) Fraction Fr.5 (eluted by petroleum ether-ethyl acetate in a volume ratio of 1:1) was subjected to silica gel column chromatography with a petroleum ether-ethyl acetate volume ratio of 10:3 to obtain one subfraction Fr.5.1.

[0043] (5) Subfraction Fr.5.1 was eluted by Lobar LiChroprep RP-18 reversed-phase silica column (elution buffer was methanol-water, volume ratio of 30%, 40%, 50%, 60%, 70%, 80%) to obtain 6 subfractions Fr.5.1.1 to 5.1.6;

[0044] (6) The subfraction Fr.5.1.3 (eluting buffer: 50% methanol-water, v / v) was purified by thin-layer chromatography (developing system: dichloromethane:methanol = 30:1) to obtain the indole-diketopiperazine compound asperinamide A, the molecular structure of which is shown in the figure below. Figure 1 .

[0045] Characterization:

[0046] The molecular structure of the indole-diketopiperazine compound asperinamide A obtained in Example 1 was determined by high-resolution mass spectrometry, superconducting nuclear magnetic resonance spectroscopy, and single-crystal diffraction. The physicochemical properties of the indole-diketopiperazine compound asperinamide A are as follows:

[0047] Properties: Colorless crystals; Solubility: Easily soluble in methanol and acetone, slightly soluble in dichloromethane, insoluble in petroleum ether and ethyl acetate; Molecular formula: C 28 H 31 N3O6; Optical rotation: [α] 20 D +25 (methanol); UV absorption spectrum λ max (Methanol, logε) 247 (3.72), 306 (3.04), 335 (2.78) nm; High-resolution mass spectrometry (HRESI-MS): m / z 504.2144 [MH] - (Theoretical value C) 28 H 30 N3O6, 504.2135); superconducting nuclear magnetic resonance hydrogen and carbon spectra are shown in Table 1.

[0048] Table 1: NMR data of indole-dione piperazine compound asperinamide A

[0049]

[0050] Example 2:

[0051] Application of indole-dione piperazine compound asperinamide A

[0052] This embodiment refers to the People's Republic of China Agricultural Industry Standard for Indoor Bioassay of Pesticides—Artificial Feed Mixture Method (NY / T 1154.10-2008) to determine the insecticidal activity of the indole-dione-piperazine compound asperinamide A against fall armyworm. The results are shown in Table 2. The specific process is as follows:

[0053] The fall armyworm was provided by the Plant Protection Research Center of the Tobacco Research Institute, Chinese Academy of Agricultural Sciences, and was reared indoors using artificial feed. Rearing conditions were: temperature (25±1)℃, relative humidity (RH) (70±5)%, and photoperiod L:D = (16:8)h. Adults were reared in oviposition cages and fed with 10% honey water. To prevent pathogen infection of the artificial feed and the fall armyworm, the rearing equipment was disinfected regularly.

[0054] Asperinamide A, an indole-dione-piperazine compound, was prepared as a stock solution with acetone and added to artificial feed to obtain toxic feeds with final concentrations of 0.1, 0.2, 0.4, 0.6, 0.8, and 1 mg / mL. The feeds were placed in 24-well plates, and two-instar fall armyworm larvae of uniform growth were selected. One larva was inoculated into each well, with each treatment repeated three times, and 20 larvae per replicate. Matrine treatment was used as a positive control, and acetone treatment as a blank control. The final acetone content in each treatment should not exceed 1%. The treated larvae were kept and observed at a temperature of (25±1)℃, relative humidity (RH) of (70±5)%, and a photoperiod of L:D = (16:8) h. After 48 h, the number of dead larvae was counted. Larvae were considered dead if they showed no reaction or only a slight reaction when lightly touched with a fine brush. The mortality rate and corrected mortality rate of fall armyworm were calculated (if the control mortality rate was <5%, no correction was needed; if the control mortality rate was between 5% and 20%, correction was required). The calculation formula is:

[0055] Mortality rate (%) = (Number of dead insects / Total number of insects treated) × 100%

[0056] Corrected mortality rate (%) = (Treatment group mortality rate - Control group mortality rate) / (1 - Control group mortality rate) × 100%

[0057] The results, as shown in Table 2, revealed that the indole-dione-piperazine compound asperinamide A exhibited good insecticidal activity against second-instar larvae of the fall armyworm, with the corrected mortality rate gradually increasing with increasing compound concentration. The median lethal concentration (LC50) of asperinamide A against second-instar larvae of the fall armyworm was also determined. 50 The concentration was 0.299 mg / mL, which was superior to the positive control drug matrine (LC50). 50 The value was 0.348 mg / mL.

[0058] Table 2: Insecticidal activity of indole-dione piperazine compound asperinamide A against second-instar larvae of fall armyworm.

[0059]

[0060]

[0061] The data in the table are mean ± standard error. Different lowercase letters in the same column indicate significant differences as determined by Duncan's new multiple range test (P < 0.05).

[0062] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0063] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An indole-diketopiperazine compound, characterized in that, The structural formula of the compound is shown in the following formula: asperinamide A 。 2. The method for preparing an indole-diketone piperazine compound according to claim 1, characterized in that, Specifically, the following steps are included: (1) Aspergillus echinosporum ( Aspergillus aculeatus Aa-1 was inoculated into malt extract medium; the preservation number of Aspergillus echinospora Aa-1 was CGMCC No. 40424, the preservation date was November 14, 2022, and the preservation location was No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. (2) The culture medium after inoculation was allowed to ferment statically at 28~30℃, and the extract was obtained by ultrasonic extraction with ethyl acetate and vacuum concentration. (3) The above extract was subjected to silica gel column chromatography with gradient elution of petroleum ether-ethyl acetate; (4) The eluent fraction with a volume ratio of petroleum ether to ethyl acetate of 1:1 in step (3) was subjected to silica gel column chromatography, and the eluent was petroleum ether to ethyl acetate with a volume ratio of 10:

3. (5) The petroleum ether-ethyl acetate eluent from step (4) is eluted by reverse-phase silica gel column elution, and the eluent is methanol-water with a volume ratio of 30-80%. (6) The eluent fraction with a methanol-water volume ratio of 50% from step (5) was purified by thin-layer chromatography to obtain the indole-dione piperazine compound asperinamide A.

3. The method for preparing an indole-diketone piperazine compound according to claim 2, characterized in that, Step (2) fermentation time is 30-40 days.

4. The method for preparing an indole-diketone piperazine compound according to claim 2, characterized in that, In step (3), the petroleum ether-ethyl acetate elution gradient is 30:1 to 1:

1.

5. The method for preparing an indole-diketone piperazine compound according to claim 2, characterized in that, The developing system for thin-layer chromatography purification in step (6) is dichloromethane:methanol = 30:

1.

6. The application of the indole-diketone piperazine compound according to claim 1, characterized in that, The application of the indole-dione piperazine compound represented by formula asperinamide A in the control of agricultural noctuid moths.

7. The application of the indole-diketone piperazine compound according to claim 1, characterized in that, The indole-dione piperazine compound represented by formula asperinamide A is used in the preparation of insecticides for agricultural noctuid moths.

8. The application of an indole-dione-piperazine compound according to claim 6 or 7, characterized in that, The insect in question is the fall armyworm (Noctuidae family).

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