Dehydroabietyl quaternary ammonium salt and application thereof

Abstract

The invention discloses a dehydroabietyl quaternary ammonium salt of which the chemical structural formula is shown in the following formula. The dehydroabietyl quaternary ammonium salt can be used for effectively inhibiting the growth of valsa mali, Phytophthora capsici, Fusarium graminearum, Fusarium oxysporum f.sp.niveum and Alternaria solani, and the dehydroabietyl quaternary ammonium salt can be used for effectively inhibiting the growth of valsa mali, Phytophthora capsici, Fusarium graminearum, Fusarium oxysporum f.sp.niveum and Alternaria solani. The method has potential value in preparation of green pesticide bactericides, and can reduce adverse effects of the bactericides on the environment and human beings; # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of fungicides, and particularly relates to a class of dehydroabietyl quaternary ammonium salts with bactericidal activity and their application in inhibiting pathogenic fungi of inhibitors. Background Art

[0002] Plant diseases caused by plant pathogenic fungi have caused huge losses to agricultural production, and these diseases will reduce the yield of crops. Using fungicides to control plant diseases is currently the simplest and most effective method. However, the large-scale use of traditional chemical fungicides not only makes plant pathogenic fungi develop drug resistance, but also has a serious impact on the ecological environment and human health. Therefore, there is an urgent need to develop new green-friendly and highly efficient fungicides.

[0003] Plant-derived fungicides are significantly superior to traditional chemical fungicides in terms of application potential due to their excellent biocompatibility. Rosin is an important natural forest chemical product in China, and the annual output of gum rosin in China is about 400,000 tons. As the main active ingredient of disproportionated rosin, dehydroabietic acid exhibits higher structural stability and significant antioxidant properties compared with other components. Dehydroabietyl derivatives have shown significant effects in the agricultural field, and these derivatives exhibit good inhibitory activities against fungi, weeds and pests. At the same time, quaternary ammonium salt derivatives also have very strong bactericidal activities.

[0004] Currently, most quaternary ammonium salt fungicides are used for disinfection and killing bacteria, and there are few reports on their application in inhibiting plant pathogenic fungi. Summary of the Invention

[0005] The present invention provides a dehydroabietyl quaternary ammonium salt, a preparation method thereof and an application as a fungicide. The chemical structural formula of the dehydroabietyl quaternary ammonium salt is as follows:

[0006]

[0007] The preparation method of the above dehydroabietyl quaternary ammonium salt is as follows:

[0008] 1. Dissolve dehydroabietylamine in formic acid, stir until clear, then add it to formaldehyde and stir for 10 - 60 minutes, and then heat to 65 - 95 °C. After cooling the mixture to room temperature, alkalize it with 1M - 6M alkali solution, and then extract with an organic solvent (such as chloroform, ethyl acetate, acetonitrile, etc.) to obtain a crude product. Finally, separate and purify it by column chromatography to obtain N,N-dimethyl dehydroabietylamine (Compound 1);

[0009] The alkali is selected from sodium carbonate, sodium bicarbonate, sodium hydroxide, potassium hydroxide, etc.;

[0010] 2. Mix N,N-dimethyl dehydroabietylamine (Compound 1) with Compound 2 and dissolve them in an organic solvent (such as chloroform, ethyl acetate, acetonitrile, etc.), and heat the mixture at 50 - 100 °C for 12 - 48 h; after the reaction is completed, remove the organic solvent by vacuum distillation, and wash the residue with an organic solvent to obtain ortho-dehydroabietyl quaternary ammonium salt (o-Rbs), meta-dehydroabietyl quaternary ammonium salt (m-Rbs), and para-dehydroabietyl quaternary ammonium salt (p-Rbs) respectively;

[0011] Compound 2 is 1,2-bis(bromomethyl)benzene (2o), 1,3-bis(bromomethyl)benzene (2m), or 1,4-bis(bromomethyl)benzene (2p).

[0012]

[0013] Another object of the present invention is to apply the above dehydroabietyl quaternary ammonium salt in the preparation of preparations for inhibiting Valsa mali, Phytophthora capsici, Fusarium graminearum, Fusarium oxysporum f. sp. niveum, and Alternaria solani.

[0014] The component (or active ingredient) of the preparation for controlling plant pathogenic bacteria of the present invention is dehydroabietyl quaternary ammonium salt, and one or more excipients acceptable for crop disease control preparations can also be added, or it can be compounded with other active ingredients to play a synergistic bacteriostatic effect; the dehydroabietyl quaternary ammonium salt can be prepared into various dosage forms of pesticides or various forms of chemical fertilizers through existing technical equipment.

[0015] The beneficial effects of the present invention are as follows:

[0016] 1. Using dehydroabietylamine as a raw material, the present invention connects two dehydroabietyl groups through a benzyl group to obtain 3 kinds of dehydroabietyl quaternary ammonium salts. The antibacterial experiment results show that this compound can inhibit the growth of Valsa mali, Phytophthora capsici, Fusarium graminearum, Fusarium oxysporum f. sp. niveum, and Alternaria solani. It has potential value in the preparation of green pesticide fungicides and can reduce the adverse effects of fungicides on the environment and humans;

[0017] 2. The present invention applies dehydroabietyl quaternary ammonium salt (m-Rbs) to the protection of apple crops. The experimental results show that dehydroabietyl quaternary ammonium salt (m-Rbs) effectively reduces the degree of fruit rot and has a good control effect on Valsa mali on apple fruits. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the bacteriostatic effect diagram of the dehydroabietyl quaternary ammonium salt of the present invention and the positive control on the 2nd day; in the figure, Figure a is Valsa mali, Figure b is Phytophthora capsici, Figure c is Fusarium graminearum, Figure d is Fusarium oxysporum f. sp. niveum, and Figure e is Alternaria solani;

[0019] Figure 2 It is the comparison diagram of the continuous effect of meta-dehydroabietyl quaternary ammonium salt (m-Rbs) in Example 2 of the present invention from 3 to 7 days. In the figure, Figure A is Valsa mali, Figure B is Phytophthora capsici, Figure C is Fusarium graminearum, Figure D is Fusarium oxysporum f. sp. niveum, and Figure E is Alternaria solani;

[0020] Figure 3 It is the protection effect and treatment effect diagram of meta-dehydroabietyl quaternary ammonium salt (m-Rbs) of the present invention on apples;

[0021] Figure 4 It is the schematic diagram of the changes in organ tissues in the acute toxicity experiment of meta-dehydroabietyl quaternary ammonium salt (m-Rbs) of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention will be further described in detail below with reference to the drawings and embodiments. However, the content should not be regarded as a limitation of the present invention. In the present embodiment, the methods are all operated according to conventional methods unless otherwise specified, and the reagents used are conventional reagents or reagents prepared according to conventional methods unless otherwise specified;

[0023] Example 1: Preparation of dehydroabietyl quaternary ammonium salt

[0024] (1) Dissolve 3 g (10.5 mmol) of dehydroabietylamine in 10 mL of formic acid. After stirring until clear, add it to 0.945 g (31.5 mmol) of formaldehyde and stir for 10 minutes. Then heat to 65 °C. After cooling the mixture to room temperature, alkalize it with 2 M sodium hydroxide solution. Add ethyl acetate to extract the alkalized product, collect the ethyl acetate phase, and concentrate it at 45 °C to obtain the crude product. The crude product is purified by chromatographic separation to obtain N,N-dimethyl dehydroabietylamine;

[0025] (2) Dissolve 6.87 g of N,N-dimethyl dehydroabietylamine and 2.64 g of 1,2-bis(bromomethyl)benzene in acetonitrile, heat the reaction at 85 °C for 24 h. After the reaction, remove acetonitrile by vacuum distillation, wash the residue with ethyl acetate, and then purify it by column chromatography to obtain ortho-dehydroabietyl quaternary ammonium salt (o-Rbs);

[0026] (3) Dissolve 6.87 g of N,N-dimethyl dehydroabietylamine and 2.64 g of 1,3-bis(bromomethyl)benzene in acetonitrile, heat the reaction at 85 °C for 24 h. After the reaction, remove acetonitrile by vacuum distillation, wash the residue with ethyl acetate, and then purify it by column chromatography to obtain meta-dehydroabietyl quaternary ammonium salt (m-Rbs);

[0027] (4) Dissolve 6.87 g of N,N-dimethyl dehydroabietylamine and 2.64 g of 1,4-bis(bromomethyl)benzene in acetonitrile, heat the reaction at 85 °C for 24 h. After the reaction, remove acetonitrile by vacuum distillation, wash the residue with ethyl acetate, and then purify it by column chromatography to obtain para-dehydroabietyl quaternary ammonium salt (p-Rbs).

[0028] The characterization data of compound 1, ortho-dehydroabietyl quaternary ammonium salt (o-Rbs), meta-dehydroabietyl quaternary ammonium salt (m-Rbs) and para-dehydroabietyl quaternary ammonium salt (p-Rbs) are as follows:

[0029] Compound 1: 1 1H-NMR (500 MHz, CDCl3) δ 7.17 (d, J = 10 Hz, 1H), 6.96 (d, J = 5 Hz, 1H), 6.87 (s, 1H), 2.88 (t, J = 5 Hz, 2H), 2.83 - 2.78 (m, 1H), 2.28 (s, 6H), 2.24 (s, 2H), 1.77 (t, J = 10 Hz, 2H), 1.72 - 1.68 (m, 2H), 1.66 - 1.61 (m, 2H), 1.57 - 1.51 (t, J = 15 Hz, 2H), 1.44 - 1.40 (t, J = 10 Hz, 1H), 1.22 (s, 3H), 1.21 (d, J = 1 Hz, 6H), 0.84 (s, 3H). 13 13C-NMR (126 MHz, CDCl3) 147.8, 146.3, 134.8, 126.8, 124.2, 123.7, 71.1, 49.2, 44.2, 38.8, 38.4, 37.4, 36.4, 33.5, 30.2, 25.7, 24.0, 19.1, 19.0, 18.9. HRMS (ESI, m / z) = 314.2844 [M+H + .

[0030] o-Rbs: 11H NMR (500 MHz, DMSO-d6) δ 7.85 (d, J = 10 Hz, 1H), 7.71 (d, J = 5 Hz, 1H), 7.13

[0031] (d, J = 5 Hz, 1H), 6.97 (s, 1H), 6.87 (s, 1H), 3.62 (s, 2H), 3.13 (d, J = 29.7 Hz, 6H), 2.91 - 2.84 (m, 1H), 2.79 - 2.77 (t, J = 5 Hz, 2H), 2.26 (s, 2H), 2.04 (d, J = 5 Hz, 2H), 1.93 (d, J = 5 Hz, 2H), 1.70 - 1.68 (t, J = 5 Hz, 2H), 1.62 (d, J = 10 Hz, 2H), 1.39 (d, J = 5 Hz, 1H), 1.27 (s, 3H), 1.19 (s, 3H), 1.16 (d, J = 5 Hz, 6H). 13 13C NMR (126 MHz, DMSO-d6) δ 147.51, 145.65, 136.38, 134.50, 131.14, 130.14, 126.78, 124.23, 75.26, 67.18, 50.89, 48.21, 38.09, 37.67, 37.61, 33.37, 30.03, 25.84, 24.41, 19.63, 19.46, 18.53. HRMS (ESI, m / z) = 365.8104 [M] 2+ / 2.

[0032] m-Rbs: 1 1H NMR (500 MHz, DMSO-d6) δ 7.94 (s, 1H), 7.79 (d, J = 25 Hz, 2H), 7.67 - 7.64 (t,

[0033] J = 10 Hz, 1H), 7.14 (d, J = 5 Hz, 2H), 6.97 (d, J = 10 Hz, 2H), 6.88 (s, 2H), 3.72 (s, 4H), 3.13 (s, 12H), 2.93 - 2.88 (m, 2H), 2.79 - 2.76 (t, J = 5 Hz, 4H), 2.29 (s, 4H), 2.09 (d, J = 5 Hz, 4H), 1.89 (d, J = 5 Hz, 4H), 1.75 - 1.70 (t, J = 10 Hz, 4H), 1.67 (d, J = 10 Hz, 4H), 1.39 (d, J = 10 Hz, 2H), 1.28 (s, 6H), 1.15 (d, J = 5 Hz, 12H). 1313C NMR (126 MHz, DMSO-d6) δ 147.55, 145.71, 138.38, 135.53, 134.45, 129.66, 129.40, 126.80, 124.19, 124.12, 76.78, 69.59, 51.64, 47.75, 38.05, 37.84, 37.63, 33.35, 29.70, 25.72, 24.41, 24.40, 19.63, 19.34, 18.57. HRMS (ESI, m / z) = 365.8161

[0034] [M] 2+ / 2.

[0035] p-Rbs: 1 1H NMR (500 MHz, DMSO-d6) δ 7.75 (s, 2H), 7.15 (d, J = 10 Hz, 1H), 6.97 (d, J = 5

[0036] Hz, 1H), 6.87 (s, 1H), 3.72 (s, 2H), 3.11 (s, 6H), 2.93 - 2.88 (m, 1H), 2.79 - 2.76 (t, J = 5 Hz, 2H), 2.29 (s, 2H), 2.08 (d, J = 10 Hz, 2H), 1.90 - 1.86 (t, J = 10 Hz, 2H), 1.75 - 1.70 (t, J = 10 Hz, 2H), 1.65 (d, J = 10 Hz, 2H), 1.34 - 1.30 (t, J = 10 Hz, 1H), 1.27 (s, 3H), 1.20 (s, 3H), 1.15 (d, J = 5 Hz, 6H). 13 13C NMR (126 MHz, DMSO-d6) δ 147.55, 145.72, 134.41, 133.99, 130.77, 126.81, 124.19, 76.72, 69.56, 51.73, 51.64, 47.73, 38.08, 37.80, 37.64, 33.35, 29.79, 25.78, 24.43, 24.39, 19.55, 19.37, 18.55. HRMS (ESI, m / z) = 365.8080 [M] 2+ / 2.

[0037] Example 2: Detection of the Antifungal Activity of Dehydroabietyl Quaternary Ammonium Salt

[0038] The mycelial growth rate method was used to test the antifungal activities of the ortho-dehydroabietyl quaternary ammonium salt (o-Rbs), meta-dehydroabietyl quaternary ammonium salt (m-Rbs), and para-dehydroabietyl quaternary ammonium salt (p-Rbs) prepared in Example 1 against Valsa mali, Phytophthora capsici, Fusarium graminearum, Fusarium oxysporum f. sp. niveum, and Alternaria solani. At the same time, benzalkonium bromide (BAB) was used as the positive control, and deionized water was used as the blank control.

[0039] The dehydroabietyl quaternary ammonium salts and benzalkonium bromide (BAB) were respectively mixed with potato dextrose agar (PDA) medium to finally obtain media containing 200, 100, 50, 25, 12.5, and 6.25 μg / mL of the compounds. The medium without the compound was used as the blank control. Subsequently, a mycelial disc with a diameter of 6 mm was inoculated in the center of the PDA medium. Three parallel experiments were carried out for each compound, and the cultures were incubated at 27 °C for 48 h. The length of the mycelium in each medium was measured using the cross method, and the inhibitory activity of the compound against the fungus was calculated using the following formula. The EC was calculated using SPSS software based on the inhibition rate of the compound against the fungus at different concentrations. 50 At the same time, the inhibition rate of the meta-dehydroabietyl quaternary ammonium salt (m-Rbs) against Valsa mali from the second day to the seventh day was measured to evaluate the antifungal persistence of the compound against the fungus.

[0040] Inhibition rate (%) = (D - d) / (D - 6) × 100

[0041] In the formula: D represents the mycelial length (mm) of the blank control group, and d represents the mycelial length (mm) of the treatment group.

[0042] Table 1 Antibacterial activities and EC of dehydroabietyl quaternary ammonium salts and BAB against 5 plant pathogenic fungi 50

[0043]

[0044]

[0045] The antibacterial effect of the dehydroabietyl quaternary ammonium salt on the second day is shown in Figure 1 , and it can be seen from the results that the dehydroabietyl quaternary ammonium salt has good antifungal activities against 5 plant pathogenic fungi; it can be seen from Table 1 that the EC of the ortho-quaternary ammonium salt o-Rbs against V. mali, P. capsica, F. graminearum, F. oxysporum f. sp. Niveum, and A. solani50 The values were 9.25, 20.984, 16.960, 17.060, 21.719 μg / mL respectively. The EC 50 values of the meta-quaternary ammonium salt m-Rbs against the above five pathogenic fungi were 2.649, 8.998, 7.025, 3.809, 21.848 μg / mL respectively. The EC 50 values of the para-quaternary ammonium salt p-Rbs against the above five pathogenic fungi were 7.374, 19.490, 15.358, 14.734, 12.631 μg / mL respectively. The EC 50 values of the positive control BAB against the above five pathogenic fungi were 5.982, 15.777, 25.545, 32.747, 31.000 μg / mL respectively. The EC 50 values of the compounds o-Rbs and p-Rbs against F.graminearum, F.oxysporum f.sp.niveum and A.solani were lower than those of the positive control BAB. The EC 50 values of the compound m-Rbs against the five plant pathogenic fungi were all lower than those of BAB.

[0046] The antibacterial effects of the meta-dehydroabietyl quaternary ammonium salt on the 3rd - 7th day are shown in Figure 2 , and during the period when the blank control group was covered with the culture medium, the dehydroabietyl quaternary ammonium salt at each concentration had good antifungal persistence against the five plant pathogenic fungi.

[0047] Example 3: Experiment on the protective effect and therapeutic effect of the meta-dehydroabietyl quaternary ammonium salt (m-Rbs) on apples after being infected by Valsa mali

[0048] Firstly, V.mali was activated and cultured on PDA medium for 48 hours. Meanwhile, the compound m-Rbs and benzalkonium bromide (BAB) were dissolved in deionized water and diluted stepwise to prepare solutions with concentrations of 50, 100, 200 μg / mL. Intact apple fruits were selected, disinfected with 75% ethanol, and then circular wounds with a diameter of 6 mm were made in the middle of the fruits using a sterile puncher to evaluate the protective effect (PE) and therapeutic effect (CE) of the compound m-Rbs on apples.

[0049] For the protective effect (PE): First, evenly spray the prepared compound solution on the surface of apples. Use the apples sprayed with deionized water as the blank control. Inoculate V. mali after 24 hours. For the curative effect (CE): First, inoculate V. mali on the surface of apples. Evenly spray the compound solution after 24 hours. Use the apples sprayed with deionized water as the blank control. There are 3 parallel samples in each group. Place the apples in an incubator at 25 °C for 9 - 12 days. Evaluate the protective and curative effects of compound m-Rbs and benzalkonium bromide (BAB) on apples after V. mali infection according to the following formula.

[0050] Protective effect / Curative effect (%) = (C - T) / C × 100, where C is the lesion area of apples in the blank control group and T is the lesion area of apples in the experimental group containing the drug.

[0051] The results are shown in Figure 3 , and it can be visually seen from the figure that both m-Rbs and BAB have good protective and curative effects on apples;

[0052] Table 2 Protective and curative effects of m-Rbs and B-B on apples

[0053]

[0054] As shown in Table 2, at concentrations of 50, 100, and 200 μg / mL, the protective effects (PE) of m-Rbs on apples were 52.14%, 67.81%, and 78.04% respectively, slightly lower than those of BAB on apples (61.91%, 74.65%, and 79.04%). At concentrations of 50, 100, and 200 μg / mL, the curative effects (CE) of m-Rbs on apples were 58.61%, 71.48%, and 78.12% respectively, which were basically equal to those of BAB on apples (62.69%, 69.93%, and 75.00%); the results showed that m-Rbs had similar effects to the positive control BAB. Therefore, m-Rbs has good potential for practical application in crop protection.

[0055] Example 4: Acute toxicity experiment of meta-dehydroabietyl quaternary ammonium salt (m-Rbs)

[0056] The acute oral toxicity test of compound m-Rbs was evaluated by the LD50 method. Five concentrations (0, 3125, 6250, 12500, and 25000 μg / mL) of the compound m-Rbs solution were determined in the preliminary experiment. Select 40 ICR mice (20 males and 20 females, weighing 40 ± 2 g) and randomly divide them into 5 groups, with 8 mice in each group (4 males and 4 females), and raise them in separate cages. Administer the drug by gavage once a day at the concentrations determined in the preliminary experiment, 800 μL each time. Record the number of deaths in each group every day, and the experiment lasts for 7 days;

[0057] Table 3 Acute toxicity data of m-Rbs in mice

[0058]

[0059] Table 3 The results show that: m-Rbs has very low toxicity to mice, and its median lethal dose (LD 50 ) is 6484.198 mg / kg. According to the standard of GB 15193.3-2014, the toxicity of m-Rbs is actually non-toxic, and the lethal dose for ordinary humans is estimated to be about 500 g / person.

[0060] All the mice in the experimental group with a concentration of 25000 μg / mL died on the 5th day. The dead mice were immediately dissected, and the organ tissues were collected and photographed. The surviving mice in the experimental group with a concentration of 3125 and the blank group were sacrificed and dissected after the 7th day, and the organ tissues were collected and photographed;

[0061] The results are shown in Figure 4 . The results show that: in the 3125 μg / mL group, only the colors of the liver, spleen and heart became slightly darker, and no obvious color changes were observed in the other organs, and there were no significant lesions; in the 25000 μg / mL group, the colors of all organs except the heart deepened and the color changes were more obvious, but no obvious lesions were observed either. Therefore, the compound m-Rbs is safe and non-toxic and can be applied to agricultural fungicides.

Claims

1. A dehydroabietyl quaternary ammonium salt with a chemical structural formula as shown in the following formula:

2. Use of the dehydroabietyl quaternary ammonium salt according to claim 1 in the preparation of a preparation for inhibiting Valsa mali, Phytophthora capsici, Fusarium graminearum, Fusarium oxysporum f. sp. niveum, and Alternaria solani.

Images