A method for inhibiting aflatoxin production using 3-methylmorpholine-pyridine-pyrimidine derivatives

Abstract

This invention discloses a method for inhibiting aflatoxin production using a 3-methylmorpholine-pyridine-pyrimidine derivative. The 3-methylmorpholine-pyridine-pyrimidine derivative is imino-methyl-[1-[6-[(3R)-3-methylmorpholine-4-yl]-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl]cyclopropyl]-oxo-λ6-thionane, with a concentration of 4.8 μM-48 μM. At 48 μM, the inhibitory effect on the production of aflatoxins AFB1 and AFB2 by Aspergillus flavus is optimal. This invention is of great significance for aflatoxin control and food safety protection.

Description

Technical Field

[0001] This invention relates to the field of aflatoxin control, specifically to a method for inhibiting aflatoxin production using a 3-methylmorpholine-pyridine-pyrimidine derivative. Background Technology

[0002] A 3-methylmorpholine-pyridin-pyrimidine derivative: imino-methyl-[1-[6-[(3R)-3-methylmorpholin-4-yl]-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl]cyclopropyl]-oxo-lambda6-sulfane, with the English name imino-methyl-[1-[6-[(3R)-3-methylmorpholin-4-yl]-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl]cyclopropyl]-oxo-lambda6-sulfane, is a complex structure with multiple functional groups including 3-methylmorpholine, pyridine, and pyrimidine.

[0003] The molecular formula of imino-methyl-[1-[6-[(3R)-3-methylmorpholin-4-yl]-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl]cyclopropyl]-oxo-λ6-thion is C 20 H 24 N6O2S, molecular weight: 412.5. SMILES code: C[C@@H]1CO CCN1C2=NC(=NC(=C2)C3(CC3)[S@](=N)(=O)C)C4=C5C=CNC5=NC=C4; Structural formula:

[0004]

[0005] Aflatoxin is a highly toxic and carcinogenic secondary metabolite, primarily synthesized by the Aspergillus flavus and Aspergillus parasiticus fungi. These molds can cause contamination during crop growth, harvesting, storage, and processing, especially under suitable temperature and humidity conditions. Aflatoxin exists in several forms, including AFB1, AFB2, AFG1, and AFG2, with AFB1 being the most common and toxic form, classified as a Group 1 carcinogen by the International Agency for Research on Cancer. Furthermore, consuming food containing aflatoxin can lead to various health problems, such as malabsorption, infertility, endocrine disorders, congenital malformations, and immune system deficiencies, posing a serious threat to the economy and public health. Therefore, preventing aflatoxin accumulation is crucial for ensuring food safety and protecting public health. Summary of the Invention

[0006] The first objective of this invention is to provide a method for inhibiting the production of aflatoxin, specifically by using a 3-methylmorpholine-pyridine-pyrimidine derivative to inhibit aflatoxin production.

[0007] Preferably, the 3-methylmorpholine-pyridine-pyrimidine derivative is imino-methyl-[1-[6-[(3R)-3-methylmorpholine-4-yl]-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl]cyclopropyl]-oxo-λ6-thionane.

[0008] Preferably, the concentration of the imino-methyl-[1-[6-[(3R)-3-methylmorpholin-4-yl]-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl]cyclopropyl]-oxo-λ6-thione is 4.8 μM-48 μM.

[0009] Preferably, the concentration of the imino-methyl-[1-[6-[(3R)-3-methylmorpholin-4-yl]-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl]cyclopropyl]-oxo-λ6-thionane is 48 μM.

[0010] Preferably, the aflatoxin is AFB1 or AFB2.

[0011] Preferably, the aflatoxin is produced by Aspergillus flavus.

[0012] The second objective of this invention is to provide an agent for inhibiting the production of aflatoxin, the active ingredient of which comprises imino-methyl-[1-[6-[(3R)-3-methylmorpholin-4-yl]-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl]cyclopropyl]-oxo-λ6-thionane.

[0013] Preferably, the concentration of the imino-methyl-[1-[6-[(3R)-3-methylmorpholin-4-yl]-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl]cyclopropyl]-oxo-λ6-thione is 4.8 μM-48 μM.

[0014] A second object of the present invention is to provide the use of imino-methyl-[1-[6-[(3R)-3-methylmorpholin-4-yl]-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl]cyclopropyl]-oxo-λ6-thionane, or the methods described above, or the preparations described above, in inhibiting the production of aflatoxin.

[0015] Preferably, the concentration of the imino-methyl-[1-[6-[(3R)-3-methylmorpholin-4-yl]-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl]cyclopropyl]-oxo-λ6-thionane is 48 μM, the aflatoxin is AFB1 or AFB2, and the aflatoxin is produced by Aspergillus flavus.

[0016] Advantages of this invention:

[0017] Imino-methyl-[1-[6-[(3R)-3-methylmorpholin-4-yl]-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl]cyclopropyl]-oxo-λ6-thione can effectively inhibit the synthesis of aflatoxin by Aspergillus flavus, with the best inhibitory effect at a concentration of 48 μM. This is of great significance for the prevention and control of aflatoxin and the protection of food safety. Attached Figure Description

[0018] Figure 1 It is a thin-layer chromatography analysis of aflatoxin.

[0019] Figure 2 It represents the relative fluorescence intensity of aflatoxin. Detailed Implementation

[0020] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0021] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0022] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0023] Example 1: Imino-methyl-[1-[6-[(3R)-3-methylmorpholin-4-yl]-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl]cyclopropyl]-oxo-λ6-thione inhibits aflatoxin production.

[0024] Aspergillus flavus (NRRL 3357) was obtained from the USDA-ARS. Initiation culture: A small amount of the strain, frozen at -80°C with glycerol, was evenly spread onto PDA medium for activation. After 4 days of aeration in a 37°C incubator, green spores were observed to form.

[0025] The preparation method of PDA medium is as follows: Weigh 200g of potatoes, wash, peel and cut into small pieces, add water and boil until soft (boil for 30 minutes, until it can be pierced by a glass rod), filter with four layers of gauze, add 20g of glucose and 15g of agar, continue to heat and stir to mix well, cool slightly and then add water to 1000ml, seal with a breathable membrane, autoclave (121℃) for 15 minutes, take out and dispense into disposable plastic petri dishes (9 cm in diameter), pour 25ml of sterile medium into each dish, and store in a refrigerator at 4℃ for later use.

[0026] Spore counting of *Aspergillus flavus*: (Turbidimetric method) *Aspergillus flavus* spores were repeatedly washed with 0.05% Tween 20 water until the spores were dispersed particles. After serial dilution, appropriate amounts of the spore suspension were loaded onto a cell counting plate. The spore count was calculated using an automated cell counter (Bio-rad). The initial density was obtained by diluting with Tween 20 (0.05%) water to a value of 0.8 × 10⁻⁶. 6 A spore suspension of 1 spore per milliliter should be prepared and used on the same day.

[0027] 1 liter of GMS liquid synthetic medium was prepared as follows: 50 g glucose, 3 g (NH4)2SO4, 2 g MgSO4·7H2O, 10 g KH2PO4, 700 mg Na2B4O7·10H2O, 680 mg NaMoO4·2H2O, 10 mg FeSO4·7H2O, 300 mg CuSO4·5H2O, 1.76 g ZnSO4·7H2O, and 110 mg MnSO4·H2O were dissolved in water, and the volume was brought up to 1 liter with water. The mixture was then autoclaved (121°C) for 15 minutes to obtain the GMS liquid synthetic medium.

[0028] Prepare a stock solution of imino-methyl-[1-[6-[(3R)-3-methylmorpholin-4-yl]-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl]cyclopropyl]-oxo-λ6-thione compound: Dissolve 0.9 mg of imino-methyl-[1-[6-[(3R)-3-methylmorpholin-4-yl]-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl]cyclopropyl]-oxo-λ6-thione powder (purchased from Shanghai Taoshu Biotechnology Co., Ltd., product catalog number T3338) in 1.8 mL of sterilized GMS liquid synthesis medium, and filter sterilize using a 0.22 μm filter to obtain a 1.21 mM stock solution.

[0029] External application experiment of imino-methyl-[1-[6-[(3R)-3-methylmorpholin-4-yl]-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl]cyclopropyl]-oxo-λ6-thione of Aspergillus flavus:

[0030] 18 mL GMS liquid synthesis medium, 2 mL spore suspension and 80 μL compound stock solution;

[0031] 17.2 mL GMS liquid synthesis medium, 2 mL spore suspension and 0.8 mL compound stock solution;

[0032] 18 mL of GMS liquid synthetic medium and 2 mL of spore suspension were used as controls;

[0033] The three combinations were added to each flask, and the flasks were incubated in a shaker at 28°C and 180 rpm for 6 days. 200 μL of the culture medium from day 2 to day 6 was taken for aflatoxin extraction and detected by thin-layer chromatography (TLC).

[0034] TLC detection of aflatoxin: Mark the rectangular chromatography plate; one complete plate can be used for 19 spottings. Lightly draw a straight line with a pencil 0.5 cm from the top and bottom edges of the glass plate (the location of the liquid diffusion imprint and the spotting location line); select the smooth edge of the glass plate as the spotting line and mark it, making one dot every 1 cm with a pencil as the spotting location marker. Prepare the developing solvent according to a chloroform to acetone volume ratio of 9:1 (9 mL chloroform and 1 mL acetone), requiring 5 mL of developing solvent per glass plate. Use a pipette tip to transfer 100 μL of bacterial culture to a 1.5 mL centrifuge tube, add 50 μL of chloroform for extraction, vortex thoroughly, centrifuge at 12000 rpm for 6 minutes, and use a pipette tip to collect 10 μL of the lower layer (chloroform layer), perpendicularly contacting the spotting marks (cross-shaped pattern), and slowly displace the liquid. The standard concentration is 2 μg / mL, and 10 μL is used for spotting. First, add 1 mL of developing solvent to the glass developing tank to rinse the groove, then discard the remaining developing solvent, and add another 5 mL of developing solvent. Hold the glass plate vertically in the liquid with one side of the sampled area pointing downwards and the other side upwards, holding it with both hands. Once the plate touches the bottom of the groove, gently release it and lean it against the side of the developing tank. After approximately 4 minutes, remove the glass plate and take a picture using a gel imaging system (Bio-rad). The TLC images were analyzed using GelAnalyzer 19.1 (www.gelanalyzer.com) by Istvan Lazar Jr., PhD and Istvan Lazar Sr., PhD, CSc software to determine the total fluorescence intensity of the corresponding aflatoxin B1 in the fluorescent spots.

[0035] TLC images showing the change in aflatoxin content over time in Aspergillus flavus culture media with different concentrations of imino-methyl-[1-[6-[(3R)-3-methylmorpholin-4-yl]-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl]cyclopropyl]oxo-λ6-thione. Figure 1As shown, the synthesis of aflatoxin was significantly inhibited after culturing samples treated with 48 μM imino-methyl-[1-[6-[(3R)-3-methylmorpholin-4-yl]-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl]cyclopropyl]oxo-λ6-thione for 2, 3, 4, 5 and 6 days.

[0036] The relative fluorescence intensity of TLC images showing the change of aflatoxin content over time in Aspergillus flavus culture media with different concentrations of imino-methyl-[1-[6-[(3R)-3-methylmorpholin-4-yl]-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl]cyclopropyl]oxo-λ6-thione is used for quantification. Figure 2 As shown, after culturing samples treated with 48 μM imino-methyl-[1-[6-[(3R)-3-methylmorpholin-4-yl]-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl]cyclopropyl]-oxo-λ6-thione for 2, 3, 4, 5 and 6 days, the fluorescence intensity of aflatoxin was significantly lower than that of the control sample, proving that toxin synthesis was significantly inhibited. The samples treated with 4.8 μM imino-methyl-[1-[6-[(3R)-3-methylmorpholin-4-yl]-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl]cyclopropyl]-oxo-λ6-thion showed a slight inhibitory effect on aflatoxin. Overall, the aflatoxin inhibition was more pronounced with increasing concentration of imino-methyl-[1-[6-[(3R)-3-methylmorpholin-4-yl]-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl]cyclopropyl]-oxo-λ6-thion.

[0037] The above experiments show that imino-methyl-[1-[6-[(3R)-3-methylmorpholin-4-yl]-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl]cyclopropyl]-oxo-λ6-thionane can effectively inhibit the synthesis of aflatoxin.

Claims

1. A method for inhibiting aflatoxin production for non-disease diagnosis and treatment purposes, characterized in that, Aflatoxin production was inhibited using a 3-methylmorpholine-pyridine-pyrimidine derivative, wherein the 3-methylmorpholine-pyridine-pyrimidine derivative is imino-methyl-[1-[6-[(3R)-3-methylmorpholine-4-yl]-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl]cyclopropyl]-oxo-λ6-thion, and the aflatoxin is produced by Aspergillus flavus (… Aspergillus flavus )produce.

2. The method according to claim 1, characterized in that, The concentration of the imino-methyl-[1-[6-[(3R)-3-methylmorpholin-4-yl]-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl]cyclopropyl]-oxo-λ6-thionane is 4.8 μM-48 μM.

3. The method according to claim 2, characterized in that, The concentration of the imino-methyl-[1-[6-[(3R)-3-methylmorpholin-4-yl]-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl]cyclopropyl]-oxo-λ6-thionane was 48 μM.

4. The method according to claim 1, characterized in that, The aflatoxin is either AFB1 or AFB2.

5. The use of imino-methyl-[1-[6-[(3R)-3-methylmorpholin-4-yl]-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl]cyclopropyl]-oxo-λ6-thionane in the inhibition of aflatoxin production for non-disease diagnostic and therapeutic purposes, wherein the aflatoxin is produced by Aspergillus flavus ( Aspergillus flavus )produce.

6. The application according to claim 5, characterized in that, The concentration of the imino-methyl-[1-[6-[(3R)-3-methylmorpholin-4-yl]-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl]cyclopropyl]-oxo-λ6-thionane is 48 μM, and the aflatoxin is AFB1 or AFB2.

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