Cyclic quaternary ammonium salt N-hydronopyl tetrahydropyrrole compound as well as synthesis method and antibacterial application thereof

Abstract

The invention discloses a cyclic quaternary ammonium salt N-hydronopyl tetrahydropyrrole compound as well as a synthesis method and antibacterial application thereof, and belongs to the technical field of plant antibiosis. Hydronopyl chloride and tetrahydropyrrole are taken as raw materials, an N-hydronopyl tetrahydropyrrole intermediate is prepared through reaction, and the cyclic quaternary ammonium salt N-hydronopyl tetrahydropyrrole compound is prepared through a one-step reaction. Then, the cyclic quaternary ammonium salt N-hydronopyl tetrahydropyrrole compound and an alpha-bromoacetophenone compound or a benzyl bromide compound are subjected to a quaternization reaction, and the cyclic quaternary ammonium salt N-hydronopyl tetrahydropyrrole compound can be synthesized; the cyclic quaternary ammonium salt N-hydronopyl tetrahydropyrrole compound has excellent broad-spectrum antibacterial activity, and especially has a remarkable inhibition effect on common phytopathogens such as rhizoctonia solani, oxysporum tomentosum, colletotrichum carpopogonum, fusarium oxysporum, tobacco black shank, Fusarium fulvum, coriolus versicolor, fusarium verticillium and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant antibacterial technology, and in particular to a cyclic quaternary ammonium salt N-hydrogenated nopol tetrahydropyrrole compound, a synthesis method thereof, and antibacterial applications. BACKGROUND

[0002] In recent years, plant pathogenic fungi have rapidly proliferated, seriously threatening crop health. Although traditional chemical agents are still the main means of prevention and control at present, the spread of drug-resistant strains has led to a continuous decrease in their efficacy, while also causing environmental pollution problems. Therefore, the development of green fungicides is of great significance in responding to the challenge of fungal drug resistance and reducing environmental pressure.

[0003] Hydrogenated nopol derivatives use beta-pinene in pine oil as a raw material, which is a renewable forest chemical resource, can reduce the dependence on traditional chemical raw materials, and reduce environmental impact. It has a strong inhibitory effect on plant pathogenic fungi and can be used as a fungicide in agriculture to prevent and control diseases, improve yield and quality, while reducing the use of traditional pesticides and reducing environmental pollution. Under this background, tetrahydropyrrole has good application potential as a promising intermediate in the development of fungicides. In the prior art, some of its derivatives are components of many drugs or chemical intermediates, and have a wide range of uses in medicine, food, daily chemicals, pesticides and many other fields.

[0004] In existing research, nopol and hydrogenated nopol were synthesized from beta-pinene; the latter was halogenated to synthesize hydrogenated nopol chloride and hydrogenated nopol bromide, both of which reacted with dimethylamine and diethylamine to obtain tertiary amines of hydrogenated nopol, which further underwent quaternary ammonium reactions with halogenated alkanes and alpha, omega-dihalogenated alkanes to obtain many single quaternary ammonium salts and symmetrical gemini quaternary ammonium salts containing hydrogenated nopol. These compounds have good inhibitory and killing effects on plant pathogenic fungi and common harmful bacteria. However, the four groups connected to the nitrogen atom in these quaternary ammonium salt molecules are separate from each other and do not connect to each other, and can rotate freely around the N-C bond. In existing research, quaternary ammonium salt compounds with a cyclic structure in which the four substituents of the nitrogen atom are connected by covalent bonds (in particular, cyclic quaternary ammonium salts in which the nitrogen atom is a component of the ring system) have not been reported in the literature. In order to further study the relationship between chemical structure and antibacterial activity, the tertiary amine of hydrogenated nopol N atom in the ring was used as a starting point to develop highly efficient and low-toxicity cyclic quaternary ammonium salt fungicides, which have important research value and application prospects. SUMMARY

[0005] The present application aims to provide a cyclic quaternary ammonium salt N-hydrogenated nopol tetrahydropyrrole compound to solve the problems raised in the background.

[0006] To achieve the above-mentioned purpose, the embodiments of the present application provide the following technical solutions: A cyclic quaternary ammonium salt N-hydrogenated norbornyl tetrahydro pyrrole compound, a structural general formula of which is any one of the following structural formulae: ; ; R is independently hydrogen or a substituent; The substituent is any one of -Br, -Cl, -F and -NO2; The substituent is any one of para-substitution, ortho-substitution and meta-substitution.

[0007] Further, a structural general formula of the cyclic quaternary ammonium salt N-hydrogenated norbornyl tetrahydro pyrrole compound is: ; R is any one of hydrogen, para-bromine, meta-bromine, para-chlorine, ortho-fluorine and para-nitro.

[0008] Further, a structural general formula of the cyclic quaternary ammonium salt N-hydrogenated norbornyl tetrahydro pyrrole compound is:

[0009] ; R is any one of hydrogen, para-bromine, meta-chlorine, meta-fluorine, para-nitro and meta-nitro.

[0010] Another object of the embodiment of the present application is to provide a synthesis method of the cyclic quaternary ammonium salt N-hydrogenated norbornyl tetrahydro pyrrole compound, comprising the following steps: The N-hydrogenated norbornyl tetrahydro pyrrole is subjected to quaternary ammonium reaction with an alpha-bromoacetophenone compound or a benzyl bromide compound to obtain the cyclic quaternary ammonium salt N-hydrogenated norbornyl tetrahydro pyrrole compound; A structural general formula of the alpha-bromoacetophenone compound is: ; A structural general formula of the benzyl bromide compound is: ; R is independently hydrogen or a substituent; The substituent is any one of -Br, -Cl, -F and -NO2; The substituent is any one of para-substitution, ortho-substitution and meta-substitution.

[0011] Further, the synthesis method of the N-hydrogenated norbornyl tetrahydro pyrrole comprises the following steps: The hydrogenated norbornyl chloride and the tetrahydro pyrrole are placed in anhydrous ethanol, and sodium hydroxide is added to perform a heating reflux reaction to obtain the N-hydrogenated norbornyl tetrahydro pyrrole.

[0012] Further, the synthesis method of the cyclic quaternary ammonium salt N-hydrogenated nopol tetrahydropyrrole compound specifically comprises the following steps: placing N-hydrogenated nopol tetrahydropyrrole and an alpha-bromoacetophenone compound in ethyl acetate, and heating to 65-75 DEG C to react to obtain the cyclic quaternary ammonium salt N-hydrogenated nopol tetrahydropyrrole compound; or placing N-hydrogenated nopol tetrahydropyrrole and a benzyl bromide compound in ethyl acetate to heat and reflux to react to obtain the cyclic quaternary ammonium salt N-hydrogenated nopol tetrahydropyrrole compound.

[0013] Another object of the embodiment of the present application is to provide an application of the cyclic quaternary ammonium salt N-hydrogenated nopol tetrahydropyrrole compound in inhibiting plant pathogenic fungi.

[0014] Further, the plant pathogenic fungi include one or more of Rhizoctonia solani, Dothiorella gregaria, Guignardia bidwelli, Fusarium oxysporum, Phytophthora parasitica, Botryobasidium pilatum, Physoderma mori and Fusarium sambucinum.

[0015] The present application uses hydrogenated nopol chloride and tetrahydropyrrole as raw materials to prepare N-hydrogenated nopol tetrahydropyrrole intermediates through reaction, and then undergoes quaternary ammonium reaction with an alpha-bromoacetophenone compound or a benzyl bromide compound to synthesize the cyclic quaternary ammonium salt N-hydrogenated nopol tetrahydropyrrole compound. The cyclic quaternary ammonium salt N-hydrogenated nopol tetrahydropyrrole compound has excellent broad-spectrum bacteriostatic activity, especially remarkable inhibitory effect on common plant pathogenic fungi such as Rhizoctonia solani, Dothiorella gregaria, Guignardia bidwelli, Fusarium oxysporum, Phytophthora parasitica, Botryobasidium pilatum, Physoderma mori and Fusarium sambucinum, and can be used for the development and application of green pesticides, as a broad-spectrum low-drug plant antibacterial agent, and helps the green prevention and control of diseases, and provides a new direction for the chemical processing and utilization of beta-pinene and the in-depth study of the relationship between the chemical structure and biological activity of pinene derivatives. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Fig. 2 is a growth curve diagram of Fusarium oxysporum mycelium under the action of compound 3b.

[0017] Figure 2 Fig. 4 is a growth curve diagram of Fusarium oxysporum mycelium under the action of compound 3d.

[0018] Figure 3Scanning electron microscope images of the Fusarium oxysporum f. sp. niveum hyphae treated by different groups: In the figure, a and b are scanning electron microscope images of the Fusarium oxysporum f. sp. niveum hyphae treated by the blank control group at different magnifications; c and d are scanning electron microscope images of the Fusarium oxysporum f. sp. niveum hyphae treated by the compound 3b at different magnifications; e and f are scanning electron microscope images of the Fusarium oxysporum f. sp. niveum hyphae treated by the compound 3d at different magnifications.

[0019] Figure 4 Relative conductivity curve of the Fusarium oxysporum f. sp. niveum hyphae under the action of the compounds 3b and 3d. DETAILED DESCRIPTION

[0020] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work belong to the scope of protection of the present application.

[0021] As a secondary amine compound, tetrahydropyrrole can react with alkyl halide to generate tertiary amine and further react with alkyl halide to generate quaternary ammonium salt. Quaternary ammonium salt compound is one of cationic surfactants, which has the characteristics of excellent water solubility, chemical stability, strong surface activity, good dispersion and corrosion inhibition performance, high efficiency and low toxicity, and broad-spectrum antibacterial ability. Based on this idea, the hydrogenated nopol compound is synthesized by introducing the tetrahydropyrrole active group through structural modification on the basis of hydrogenated nopol. This provides new basis for the resource utilization of β-pinene and the structure-activity relationship research of its derivatives, and provides a new idea for the research and potential application value of high-efficiency fungicides.

[0022] In the embodiments of the present application, the benzyl bromide compound with high activity of halogen and the alpha-bromoacetophenone compound are selected to react with the hydrogenated nopol tetrahydropyrrole respectively to obtain the cyclic quaternary ammonium salt with N atom in the ring, and the chemical structure is optimized. Since the ring is a five-membered ring and is approximately planar, the stability is good and the steric hindrance is not large. Therefore, the reaction is fast and there is no possibility of elimination reaction, the purity of the obtained quaternary ammonium salt is high, and the product post-treatment is simple. The synthesized quaternary ammonium salt is not only characterized by nuclear magnetic resonance and infrared spectrum analysis for chemical structure, but also is subjected to growth inhibition activity test on the growth of a plurality of plant pathogenic fungi by using the mycelial growth rate method, and the antibacterial mechanism is researched by mycelial growth curve determination, scanning electron microscope observation (SEM) and relative conductivity determination.

[0023] Specifically, in one embodiment of the present application, a cyclic quaternary ammonium salt N-hydrogenated nopol tetrahydropyrrole compound is provided, and the structural general formula is any one of the following structural formulae: wherein R is independently hydrogen or a substituent. Preferably, the substituent is any one of -Br, -Cl, -F, -NO2, and the substituent can be any one of para-substitution, ortho-substitution and meta-substitution; In a preferred embodiment of the present application, the cyclic quaternary ammonium salt N-hydroazepyl tetrahydropyrrole compound has a general structure of: wherein R is any one of hydrogen (H), para-bromine (p-Br), meta-bromine (m-Br), para-chlorine (p-Cl), ortho-fluorine (o-F) and para-nitrogen (p-NO2).

[0024] In a preferred embodiment of the present application, the cyclic quaternary ammonium salt N-hydroazepyl tetrahydropyrrole compound has a general structure of: wherein R is any one of hydrogen (H), para-bromine (p-Br), meta-chlorine (m-Cl), meta-fluorine (m-F), para-nitrogen (p-NO2) and meta-nitrogen (m-NO2).

[0025] In another embodiment of the present application, a synthesis method of the above-mentioned cyclic quaternary ammonium salt N-hydroazepyl tetrahydropyrrole compound is also provided, comprising the following steps: quaternary ammonium reaction of N-hydroazepyl tetrahydropyrrole and α-bromoacetophenone compound or benzyl bromide compound, to obtain the cyclic quaternary ammonium salt N-hydroazepyl tetrahydropyrrole compound; The α-bromoacetophenone compound has a general structure of: The benzyl bromide compound has a general structure of: wherein R is independently hydrogen or a substituent; the substituent is any one of -Br, -Cl, -F, -NO2; the substituent is any one of para-substitution, ortho-substitution and meta-substitution.

[0026] Specifically, the synthesis method of N-hydroazepyl tetrahydropyrrole comprises the following steps: hydroazepyl chloride and tetrahydropyrrole in anhydrous ethanol, and adding sodium hydroxide to perform a heating reflux reaction, to obtain the N-hydroazepyl tetrahydropyrrole.

[0027] The synthesis route is as follows: ​​​​​​ .

[0028] In addition, the synthesis method of the cyclic quaternary ammonium salt N-hydrogenated nopyl tetrahydropyrrole compound specifically comprises: placing N-hydrogenated nopyl tetrahydropyrrole and an alpha-bromoacetophenone compound in ethyl acetate, and heating to 65-75 DEG C to react to obtain the cyclic quaternary ammonium salt N-hydrogenated nopyl tetrahydropyrrole compound; The synthesis route is as follows: ; In the formula, 2a, 3a: R = H; 2b, 3b: R = p-Br; 2c, 3c: R = m-Br; 2d, 3d: R = p-Cl; 2e, 3e: R = o-F; 2f, 3f: R = p-NO2.

[0029] or placing N-hydrogenated nopyl tetrahydropyrrole and a benzyl bromide compound in ethyl acetate to heat and reflux to react to obtain the cyclic quaternary ammonium salt N-hydrogenated nopyl tetrahydropyrrole compound; The synthesis route is as follows: ; In the formula, 4a, 5a: R = H; 4b, 5b: R = P-Br; 4c, 5c: R = m-Cl; 4d, 3d: R = m-F; 4e, 5e: R = p-NO2; 4f, 5f: R = m-NO2.

[0030] In the embodiment of the present application, the cyclic quaternary ammonium salt is synthesized by using beta-pinene of pine oil, a broad-spectrum low-drug plant antibacterial agent is developed, and the green disease prevention and control is assisted. Specifically, N-hydrogenated nopyl tetrahydropyrrole is prepared by using hydrogenated nopyl chloride and tetrahydropyrrole, and is then quaternized with alpha-bromoacetophenone and benzyl bromide compounds respectively, the structure is characterized by IR and NMR, the antibacterial activity is evaluated by the mycelial growth rate method, and the antibacterial mechanism of some compounds on specific plant pathogens is preliminarily explored.

[0031] The following examples are some specific implementation cases of the present application in practical application, but are not limited thereto.

[0032] In the following examples, the reagents and analytical instruments used are as follows: Hydrogenated nopol chloride, purity (GC) 97.6%, provided by the College of Forestry, Jiangxi Agricultural University; tetrahydropyrrole, sodium hydroxide, ethanol, ethyl acetate, petroleum ether (boiling point 60~90 ℃), α-bromoacetophenone (6 kinds), benzyl bromide (6 kinds) compounds and the like are commercially available chemical reagents. FL-9790 gas chromatograph, Wenling Fulai Analytical Instrument Co., Ltd.; Nicolet IR 6700 infrared spectrometer (KBr pressed disc), Thermo Fisher Scientific, USA; Bruker AVANCE 400 type nuclear magnetic resonance instrument (CDCl3 as solvent, TMS as internal standard), Bruker Company, Switzerland; JNM-ECZ600R / S1 600Hz nuclear magnetic resonance spectrometer (CDCl3 as solvent, TMS as internal standard), JEOL Company, Japan; 44X-6T microscopic melting point tester, Shanghai Optical Instrument Factory No. 6.

[0033] The strains used in the following examples and the main equipment for the antibacterial test are as follows: Rhizoctonia solani (K), Sphaeropsis sapinea (S), Colletotrichum fructicola (Cf), Fusarium oxysporum f. sp. Niveum (X), Phytophthora parasitica var. nicotianae (11), Poria vaporaria (H), Coriolus versicolor (V), and Fusarium verticillioides (O) were provided by the Forest Protection Teaching and Research Office of the College of Forestry, Jiangxi Agricultural University. It should be noted that these strains are commonly known. Antibacterial equipment: LDZX-50KBS vertical pressure steam sterilization pot, Shanghai Shen'an Medical Instrument Factory; SW-CJ-10 sterile clean bench, Suzhou Purification Equipment Co., Ltd.; CHP-250 intelligent incubator, Shanghai Sanfa Scientific Instrument Co., Ltd.

[0034] Example 1: The example provides a synthesis method of N-hydrogenated nopol tetrahydropyrrole, which specifically includes the following steps: In a 250 mL reaction flask, 0.2 mol of norbomyl hydride, 0.22 mol of tetrahydropyrrole, 120 mL of anhydrous ethanol, and 8.2 g of sodium hydroxide were placed, stirred, and heated to reflux. After 8 h, a sample was taken for gas chromatography analysis. The results showed that there was no norbomyl hydride, and thus the reaction was stopped. After the reaction solution was cooled, it was filtered. The filtrate was first distilled to recover the ethanol, and then vacuum distilled to distill the product. N-norbomyl tetrahydropyrrole was obtained as a colorless liquid, b.p. 123-123 °C / 1730 Pa, with a yield of 87% and a GC purity of 97%.

[0035] Example 2: This example provides a method for synthesizing N-norbomyl-N- benzoylmethyl tetrahydropyrrole bromide. Referring to the above synthesis route, the method specifically includes the following steps: In a 100 mL reaction flask, 0.01 mol of N-norbomyl tetrahydropyrrole (1), 0.01 mol of α-bromoacetophenone (2a), and 25 g of ethyl acetate were placed, stirred, and heated to 70 °C. After 6 h, a sample was taken for gas chromatography analysis. If there was no N-norbomyl tetrahydropyrrole (1), the reaction was completed. The ethyl acetate was evaporated, and the residue was washed with petroleum ether 4-5 times, 5 mL each time. After the petroleum ether was separated, the residue was dried under vacuum (about 10 min, 60 °C heating). N-norbomyl-N-benzoylmethyl tetrahydropyrrole bromide (denoted as compound 3a, 4A4'-H-Br) was obtained and weighed.

[0036] Referring to the above synthesis method, α-bromoacetophenone (2a) was replaced with p-bromo-α-bromoacetophenone (2b) to react with N-norbomyl tetrahydropyrrole (1). N-norbomyl-N-p-bromobenzoylmethyl tetrahydropyrrole bromide (denoted as compound 3b, 4A4'-p-BrBr) was obtained.

[0037] Referring to the above synthesis method, α-bromoacetophenone (2a) was replaced with 2,2'-dibromoacetophenone (2c) to react with N-norbomyl tetrahydropyrrole (1). N-norbomyl-N-m-bromobenzoylmethyl tetrahydropyrrole bromide (denoted as compound 3c, 4A4'-m-BrBr) was obtained.

[0038] Referring to the above synthesis method, α-bromoacetophenone (2a) was replaced with 2-bromo-4'-chloroacetophenone (2d) to react with N-norbomyl tetrahydropyrrole (1). N-norbomyl-N-p-chlorobenzoylmethyl tetrahydropyrrole bromide (denoted as compound 3d, 4A4'-p-ClBr) was obtained.

[0039] Referring to the above synthesis method, the α-bromoacetophenone (2a) is replaced by 2-bromo-2'-fluoroacetophenone (2e) to react with N-hydroxynorbornyl tetrahydro pyrrole (1) to obtain N-hydroxynorbornyl-N-o-fluorobenzoylmethyl tetrahydro pyrrole bromide (denoted as compound 3e, 4A4'-o-FBr).

[0040] Referring to the above synthesis method, the α-bromoacetophenone (2a) is replaced by 2-bromo-2'-fluoroacetophenone (2e) to react with N-hydroxynorbornyl tetrahydro pyrrole (1) to obtain N-hydroxynorbornyl-N-o-fluorobenzoylmethyl tetrahydro pyrrole bromide (denoted as compound 3e, 4A4'-o-FBr).

[0041] Example 3: This example provides a synthesis method of N-hydroxynorbornyl-N- benzyl tetrahydro pyrrole bromide, which specifically comprises the following steps with reference to the above synthesis route: In a 100 mL reaction bottle, 0.01 mol of N-hydroxynorbornyl tetrahydro pyrrole (1), 0.01 mol of benzyl bromide (4a) and 30 mL of ethyl acetate are placed and reacted under reflux. After 8 h, sample for gas chromatography analysis. After the reaction is completed, the ethyl acetate is evaporated, the residue is washed with petroleum ether for 5 times, 5 mL each time, shaken for 3 min, and the petroleum ether is separated after the layers are clear. After the last time of separating the petroleum ether, the residue is heated slightly under vacuum to remove the solvent to obtain the product N-hydroxynorbornyl-N-benzyl tetrahydro pyrrole bromide (denoted as compound 5a, 4A4'-H-BzBr).

[0042] Referring to the above synthesis method, the benzyl bromide (4a) is replaced by p- bromobenzyl bromide (4b) to react with N-hydroxynorbornyl tetrahydro pyrrole (1) to obtain N-hydroxynorbornyl-N-p-bromobenzyl tetrahydro pyrrole bromide (denoted as compound 5b, 4A4'-p-BrBzBr).

[0043] Referring to the above synthesis method, the benzyl bromide (4a) is replaced by p- bromobenzyl bromide (4b) to react with N-hydroxynorbornyl tetrahydro pyrrole (1) to obtain N-hydroxynorbornyl-N-p-bromobenzyl tetrahydro pyrrole bromide (denoted as compound 5b, 4A4'-p-BrBzBr).

[0044] Referring to the above synthesis method, the benzyl bromide (4a) is replaced by p- bromobenzyl bromide (4b) to react with N-hydroxynorbornyl tetrahydro pyrrole (1) to obtain N-hydroxynorbornyl-N-p-bromobenzyl tetrahydro pyrrole bromide (denoted as compound 5b, 4A4'-p-BrBzBr).

[0045] Referring to the above synthesis method, benzyl bromide (4a) is replaced by p-nitrobenzyl bromide (4e) to react with N-hydroxynorbornyl tetrahydropyrrole (1) to obtain N-hydroxynorbornyl-N-p-nitrobenzyl tetrahydropyrrole bromide (denoted as compound 5e, 4A4'-p-NO2BzBr).

[0046] Referring to the above synthesis method, benzyl bromide (4a) is replaced by p-nitrobenzyl bromide (4e) to react with N-hydroxynorbornyl tetrahydropyrrole (1) to obtain N-hydroxynorbornyl-N-p-nitrobenzyl tetrahydropyrrole bromide (denoted as compound 5e, 4A4'-p-NO2BzBr). Experimental Example: I. Bacteriostatic activity test: using the mycelial growth rate method, two series of 12 quaternary ammonium salt compounds synthesized in Example 2-3 were prepared to test the inhibition activity on the growth of 8 plant pathogenic fungi. The calculation formula of the inhibition rate is as follows: Inhibition rate (%) = [(mycelial growth diameter of the control group - mycelial growth diameter of the treatment group) / mycelial growth diameter of the control group - 0.5 mm] x 100%; Using IBM SPSS Statistics 25 software for virulence regression analysis, a linear regression equation (y = a + bx) between the logarithmic value of the mass concentration of the compound (x) and the inhibition rate probability value (y) was established. Through the equation, the concentration value corresponding to y = 50% was calculated, that is, the half maximal inhibitory concentration (IC 50 ) of the compound, which represents the mass concentration of the drug solution required for the compound to produce 50% inhibition effect.

[0047] II. Mycelial growth curve determination: Under sterile conditions, prepare compound 3b (IC 50 = 3.8 mg / L) and 3d (IC 50 = 3.7 mg / L) drug solution. Take equal volume of drug solution and add to 60 mL sterilized potato dextrose (PDW) medium, mix. Treat watermelon fusarium wilt fungus cake with 5 mm puncher and transfer to conical flask, 1 piece per flask. Incubate at 28 ℃, 140 r / min on a shaking table for 9 d, set up a blank control group (without adding drugs). Take 3 flasks every 24 h, rinse the mycelium, vacuum filter, dry at 55 ℃ to constant weight, weigh and record (average of 3 times). Take the incubation time as the abscissa and the mycelial dry weight as the ordinate to draw the growth curve.

[0048] III. Scanning electron microscope observation of mycelium: watermelon fusarium wilt fungus was cultured using potato dextrose agar (PDA) solid culture method. Compound 3b and 3d were prepared into drug solution at IC 50 concentration, equal volume was added to 60 mL sterilized PDA medium, mixed and poured into plates, and a blank control was set up. After the medium solidified, 5 mm fungus cake was inoculated and incubated at 28 ℃ for 5 d. Then 1-2 mm 2The mycelial blocks were fixed with 2.5% glutaraldehyde at 4 ℃ for 12 h, then rinsed with pH=7.0 phosphate buffer for 2-3 times (15 min each time), and dehydrated with 30%, 50%, 70%, 95%, and 100% ethanol gradient (15 min each time). After being frozen and vacuum dried for 24 h, the samples were treated with gold plating, and then the mycelial morphology was observed by using a scanning electron microscope to analyze the effect of the compound on the morphology of the Fusarium oxysporum f. sp. niveum mycelium.

[0049] Four, determination of cell membrane permeability: the compounds 3b and 3d were evaluated for the cell membrane permeability of the Fusarium oxysporum f. sp. niveum. The mycelium of the Fusarium oxysporum f. sp. niveum in the logarithmic growth phase was extracted, and fresh mycelium was obtained after being suction filtered and washed with sterile water. 0.1 g of the Fusarium oxysporum f. sp. niveum mycelium was weighed and placed in a 50 mL centrifuge tube, and then 20 mL of the compound solution with a concentration of IC 50 was added, and a blank control group with the same amount of sterile water, DMSO, and no drug solution was set. The conductivity of the treated mycelium was measured at 0, 15, 30, 60, 90, 120, and 180 min by using a conductivity meter, and then the mycelium was heated in boiling water for 5 min for inactivation, and the conductivity was measured again after cooling. Each sample group was measured in triplicate, and the relative conductivity (η) was used to evaluate the cell membrane permeability. The calculation formula of the relative conductivity (η) is as follows: ; In the formula, A1 is the conductivity after being treated for different times, S / m; A2 is the conductivity at 0 min, S / m; A 灭活处理 is the conductivity after inactivation treatment, S / m.

[0050] Five, results and discussion: 1. Reaction: the bromine in the α-bromoacetophenone compound and the benzyl bromide compound is very active and can easily undergo substitution reactions (such as hydrolysis, aminolysis, cyanolysis, etc.). When they react with N-hydroazepine tetrahydro pyrrole (1), a tertiary amine, it is very easy and fast, especially when the α-bromoacetophenone compound is in contact with N-hydroazepine tetrahydro pyrrole (1) in ethyl acetate, insoluble substances are generated. This is because the carbonyl and benzene ring form a π-π conjugated system and are electron-withdrawing, which makes the electron cloud density of the carbon atom connected to bromine even lower, which is more conducive to the approach of the nitrogen atom of the tertiary amine to form a bond. At the same time, when a general halogenated alkane and a tertiary amine react, there will be an elimination reaction and a substitution reaction to generate a quaternary ammonium salt, which will compete with each other, but the above two types of bromides do not have β-hydrogen atoms, and the elimination of HBr will not occur. Therefore, the reaction of N-hydroazepine tetrahydro pyrrole (1) with them is single, the post-treatment method of the product is simple, and the purity of the obtained product is high.

[0051] 2. Structural analysis and characterization: N-hydroazepine tetrahydro pyrrole (1): C25 H 27 N, a colorless and transparent liquid, with a yield of 87% and a pressure of 122~123 °C / 1730 Pa. 1 H NMR (400MHz, CDCl3), δ (ppm): 2.497 (m, 4H, 2 α- CH2), 2.389 (m, 2H, 11- CH2), 2.323 (m, 1H, 2- CH), 1.971~1.863 (m, 6H, 7- CH a , 5- CH, 1- CH, 3- CH a , 10- CH2), 1.776 (m, 4H, 2 β- CH2), 1.631 (m, 2H, 4- CH2), 1.476 (m, 1H, 3- CH b ), 1.179 (s, 3H, 9- CH3), 1.016 (s, 3H, 8- CH3), 0.879 (d, J=9.2Hz, 1H, 7- CH b ); 13 C NMR (100Hz, CDCl3), δ (ppm): 55.47 (C -11 ), 54.26 (2C -α ), 46.66 (C -2 ), 41.48(C -5 ), 39.75 (C -1 ), 38.68 (C -6 ), 37.09 (C -10 ), 33.66 (C -7 ), 28.19 (C -9 ), 26.48(C -4 ), 23.36 (2C -β ), 23.25 (C -8 ), 22.57 (C -3 ); IR (liquid film), ν max (cm -1): 2934, 2907, 2878, 2784, 1468, 1383, 1365, 1139, 877.

[0052] Structural characterization data of quaternary ammonium salt compounds 3a~3f: 3a: N-hydroazepyl-N-benzoylmethyltetrahydropyrrole bromide (4A4'-H-Br) C 23 H 34 NOBr white solid, yield 89%, m.p. 159.8~160.0 ℃. 1 H NMR (600MHz, CDCl3), δ (ppm): 8.174 (d, J=7.2Hz, 2H, 15- CH, 19- CH), 7.612 (d, J=7.8Hz, 1H, 17- CH), 7.489 (t, J=7.8Hz, 2H, 16- CH, 18- CH), 5.760 (m, 2H, 12- CH2),4.380 (m, 2H, α- CH2), 3.867 (m, 2H, 11- CH2), 3.592 (m, 2H, α'- CH2), 2.449 (m, 2H, β- CH2), 2.291 (m, 1H, 2- CH), 2.137 (m, 2H, β'- CH2), 1.938~1.668 (m, 8H, 10- CH2, 7- CH a , 5- CH, 1- CH, 3- CH a , 4- CH2), 1.382 (m, 1H, 3- CH b ), 1.096 (s, 3H, 9- CH3), 0.812(d, J=7.2Hz, 1H, 7- CH b ), 0.794 (s, 3H, 8- CH3); 13 C NMR (150MHz, CDCl3), δ (ppm):191.48 (C -13 ), 134.89 (C-17 ), 133.79 (C -14 ), 129.05 (C -15 , C -19 ), 128.60 (C -16 ,C -18 ), 64.79, 64.73 (C -α , C -α' ), 63.78 (C -12 ), 59.92 (C -11 ), 45.43 (C -2 ), 40.95(C -5 ), 38.43 (C -6 ), 38.20 (C -1 ), 33.13 (C -10 ), 31.11 (C -7 ), 27.80 (C -9 ), 25.98(C -4 ), 22.94 (C -8 ), 22.12 (C -3 ), 22.05, 21.92 (C -β , C -β' ); IR (KBr), ν max (cm -1 ):3105, 3058, 3026, 2938, 2907, 2881, 2867, 1696, 1598, 1582, 1468, 1449, 1398,1385, 1369, 1345, 1232, 1189, 1085, 1000, 990, 977, 920, 758, 690。

[0053] 3b: N-hydroxynorbornyl-N-p-bromobenzoylmethyltetrahydropyrrole bromide (4A4'-p-BrBr) C 23 H 33 NOBr2white solid, yield 91%, m.p. 196.5~196.7 ℃. 1 H NMR (600MHz, CDCl3), δ(ppm): 7.993 (d, J=7.2Hz, 2H, 15- CH, 19- CH), 7.470 (d, J=7.2Hz, 2H, 16- CH, 18- CH), 5.630 (m, 2H, 12-CH2), 4.142 (m, 2H, α- CH2), 3.827 (m, 2H, 11- CH2), 3.546(m, 2H, α'- CH2), 2.286 (m, 2H, β- CH2), 2.158 (m, 1H, 2- CH), 2.079 (m, 2H, β'- CH2), 1.847~1.579 (m, 8H, 10- CH2, 7- CH a , 5- CH, 1- CH, 3- CH a , 4- CH2), 1.212 (m, 1H, 3- CH b ), 0.973 (s, 3H, 9- CH3), 0.675 (s, 4H, 7- CH b , 8- CH3); 13 C NMR (150MHz, CDCl3),δ (ppm): 190.94 (C -13 ), 132.42 (C -15 , C -19 ), 130.45, 130.31 (C -16 , C -18 ), 64.82(C -α ), 64.74 (C -α' ), 64.29 (C -12 ), 59.95 (C -11 ), 45.50 (C -2 ), 41.02 (C -5 ), 38.52(C -6 ), 38.35 (C -1 ), 33.19 (C -10 ), 31.16 (C -7 ), 27.88 (C -9 ), 26.04 (C -4 ), 23.10(C -8 ), 22.22 (C -3 ), 22.13, 22.02 (C -β , C-β' ); IR (KBr), v max (cm -1 ): 3117, 3093,3033, 2978, 2937, 2916, 2867, 1695, 1586, 1566, 1465, 1449, 1412, 1399, 1383,1363, 1334, 1278, 1233, 1225, 1185, 1096, 1074, 1007, 992, 987, 956, 869,843, 809, 796.

[0054] 3c: N-hydroxynorbornyl-N-m-bromobenzoylmethyltetrahydropyrrole bromide (4A4'-m-BrBr) C 23 H 33 NOBr2 white solid, yield 89%, m.p. 89.9~90.2 ℃. 1 H NMR (600MHz, CDCl3), δ (ppm):8.302 (d, J=7.8Hz, 1H, 19- CH), 8.221 (s, 1H, 15- CH), 7.712 (d, J=7.8Hz, 1H, 17- CH), 7.376 (t, J=7.8Hz, 1H, 18- CH), 5.870 (m, 2H, 12- CH2), 4.393 (m, 2H, α- CH2),3.885 (m, 2H, 11- CH2), 3.564 (m, 2H, α'- CH2), 2.465 (m, 2H, β- CH2), 2.303 (m, 1H, 2- CH), 2.125 (m, 2H, β'- CH2), 1.937~1.683 (m, 8H, 10- CH2, 7- CH a , 5- CH, 1- CH, 3- CH a , 4- CH2), 1.335 (m, 1H, 3- CH b ), 1.113 (s, 3H, 9-CH3), 0.827 (s, 3H, 8- CH3), 0.802(d, J=14.4Hz, 1H, 7- CH b ); 13 C NMR (150MHz, CDCl3), δ (ppm): 190.69 (C -13 ),137.65 (C -15 ) 135.54 (C -16 ), 131.08, 130.85 (C -17 , C -19 ), 127.81 (C -18 ), 123.30(C -14 ), 64.85, 64.78 (C -α , C -α' ), 64.33 (C -12 ), 59.96 (C -11 ), 45.54 (C -2 ), 41.02(C -5 ), 38.51 (C -6 ), 38.33 (C -1 ), 33.21 (C -10 ), 31.18 (C -7 ), 27.87 (C -9 ), 26.04(C -4 ), 23.08 (C -8 ), 22.24 (C -3 ), 22.17, 22.00 (C -β , C -β' ); IR (KBr), ν max (cm -1 ):3457, 3410, 3065, 2939, 2917, 1867, 1696, 1567, 1469, 1456, 1424, 1384, 1368,1222, 1071, 995, 926, 896, 847, 785, 747, 681。

[0055] 3d: N-hydroxynorbornyl-N-p-chlorobenzoylmethyltetrahydropyrrole bromide (4A4'-p-ClBr) C 23 H 33 NOClBr White solid, yield 89%, m.p. 190.1~190.3 ℃.1 H NMR (600MHz, CDCl3), δ(ppm): 8.038 (d, J=8.4Hz, 2H, 15- CH, 19- CH), 7.266 (d, J=7.8Hz, 2H, 16- CH, 18- CH), 5.579 (m, 2H, 12- CH2), 4.100 (m, 2H, α- CH2), 3.793 (m, 2H, 11- CH2), 3.533(m, 2H, α'- CH2), 2.250 (m, 2H, β- CH2), 2.120 (m, 1H, 2- CH), 2.052 (m, 2H, β'- CH2), 1.819~1.555 (m, 8H, 10- CH2, 7- CH a , 5- CH, 1- CH, 3- CH a , 4- CH2), 1.178 (m, 1H, 3- CH b ), 0.936 (s, 3H, 9- CH3), 0.636 (s, 4H, 7- CH b , 8- CH3); 13 C NMR (150MHz, CDCl3),δ (ppm): 190.63 (C -13 ), 132.16 (C -17 ) 130.26 (C -15 , C -19 ), 129.38 (C -16 , C -18 ),64.81 (C -α ), 64.74 (C -α' ), 64.16 (C -12 ), 59.99 (C -11 ), 45.46 (C -2 ), 40.98 (C -5 ),38.48 (C -6), 38.30 (C -1 ), 33.15 (C -10 ), 31.15 (C -7 ), 27.84 (C -9 ), 26.00 (C -4 ),23.05 (C -8 ), 22.20 (C -3 ), 22.12, 21.97 (C -β , C -β' ); IR (KBr), ν max (cm -1 ):3177,3053, 3022, 2979, 2922, 2904, 2869, 1685, 1589, 1572, 1466, 1406, 1384, 1368,1282, 1229, 1093, 1011, 980, 934, 912, 825.

[0056] 3e: N-hydroxynorbornyl-N-o-fluorobenzoylmethyltetrahydropyrrole bromide (3e, 4A4'-o-FBr) C 23 H 33 NOFBr White solid, yield 88%, m.p. 101.4~101.6 ℃. 1 H NMR (400MHz, CDCl3), δ(ppm): 8.074 (d, J=8.0Hz, 1H, 19- CH), 7.634 (d, J=8.0Hz, 1H, 16- CH), 7.321 (t,J=8.0Hz, 1H, 18- CH), 7.208 (t, J=92Hz, 1H, 17- CH), 5.498 (m, 2H, 12- CH2), 4.330(m, 2H, α- CH2), 4.004 (m, 2H, α'- CH2), 3.689 (m, 2H, 11- CH2), 2.447 (m, 2H, β- CH2), 2.299 (m, 1H, 2- CH), 2.236 (m, 2H, β'- CH2), 2.008~1.679 (m, 8H, 10- CH2, 7-CH a , 5- CH, 1- CH, 3- CH a , 4- CH2), 1.361 (m, 1H, 3- CH b ), 1.131 (s, 3H, 9- CH3), 0.847(d, J=9.6Hz, 1H, 7- CH b ) 0.835 (s, 3H, 8- CH3); 13 C NMR (100MHz, CDCl3), δ (ppm):189.28 (C -13 ), 163.01 (C -15 ) 136.77 (C -16 ), 125.17 (C -19 ), 130.86 (C -14 ), 122.54(C -18 ), 117.03 (C -17 ), 66.07 (C -12 ), 64.62 (2C -α ), 59.29 (C -11 ), 45.66 (C -2 ),41.00 (C -5 ), 38.49 (C -6 ), 38.29 (C -1 ), 33.19 (C -10 ), 31.04 (C -7 ), 27.84 (C -9 ),26.00 (C -4 ), 22.99 (C -8 ), 21.99 (C -3 , 2C -β ); IR (KBr), ν max (cm -1 ): 3061, 2990,2912, 2867, 2830, 1699, 1611, 1580, 1454, 1387, 1369, 1278, 1221, 1051, 908,765。

[0057] 3f: N-hydroazolyl-N-p-nitrobenzoylmethyltetrahydropyrrole bromide (4A4'-p-NO2Br) C 23 H 33 N2O3Br White solid, yield 91%, m.p. 189.9~190.2 ℃. 1 H NMR (600MHz, CDCl3), δ(ppm): 8.411 (d, J=6.6Hz, 2H, 16- CH, 18- CH), 8.156 (d, J=6.3Hz, 2H, 15- CH, 19- CH), 5.956 (m, 2H, 12- CH2), 4.230 (m, 2H, α- CH2), 3.865 (m, 2H, 11- CH2), 3.576(m, 2H, α'- CH2), 2.375 (m, 2H, β- CH2), 2.181 (m, 1H, 2- CH), 2.101 (m, 2H, β'- CH2), 1.883~1.645 (m, 8H, 10- CH2, 7- CH a , 5- CH, 1- CH, 3- CH a , 4- CH2), 1.259 (m, 1H, 3- CH b ), 1.004 (s, 3H, 9- CH3), 0.726 (s, 3H, 8- CH3), 0.706 (d, J=12.0Hz, 1H, 7- CH b ); 13 C NMR (150MHz, CDCl3), δ (ppm): 190.94 (C -13 ), 151.02 (C -17 ) 138.25(C -14 ), 131.08, 130.33 (C -16 , C -18 ), 124.08 (C -15 , C-19 ), 65.28 (C -12 ), 64.89,64.80 (C -α , C -a' ), 61.33 (C -11 ), 45.54 (C -2 ), 41.03 (C -5 ), 38.55 (C -6 ), 38.45(C -1 ), 33.20 (C -10 ), 31.21 (C -7 ), 27.89 (C -9 ), 26.03 (C -4 ), 23.18 (C -8 ), 22.37(C -3 ), 22.28, 22.05 (C -β , C -β' ); IR (KBr), ν max (cm -1 ):3034, 2988, 2938, 2908,2865, 1689, 1604, 1529, 1467, 1368, 1345, 1285, 1228, 1067, 905, 856, 747。

[0058] Quaternary ammonium salt compounds 5a~5f were characterized by the following data: 5a: N-hydroazepyl-N-benzyltetrahydropyrrole bromide (4A4'-H-BzBr) C 22 H 34 NBr white solid, yield 86%, m.p. 178.8~179.0 ℃. 1 H NMR (600MHz,CDCl3), δ (ppm): 7.425 (m, 5H, Ar- H), 4.785 (m, 2H, 12- CH2), 3.824 (m, 2H, 11- CH2), 3.519 (m, 2H, α- CH2), 3.163 (m, 2H, α'- CH2), 2.177 (m, 3H, 2- CH, β- CH2),2.005 (m, 2H, β'- CH2), 1.870~1.689 (m, 8H, 10- CH2,7- CH a , 5- CH, 1- CH, 3- CH a , 4- CH2),1.307 (m, 1H, 3- CH b ), 1.162 (s, 3H, 9- CH3), 0.908 (s, 3H, 8- CH3), 0.704 (d, J=9.6Hz, 1H, 7- CH b ); 13 C NMR (150MHz, CDCl3), δ (ppm): 134.19 (C -14 ,C -18 ), 132.49(C -15 , C -17 ), 130.47, 127.28 (C -13 ), 125.30 (C -16 ),61.82 (C -12 ), 61.27, 61.02(C -α , C -a' ), 58.54 (C -11 ), 45.93 (C -2 ), 41.09 (C -5 ), 38.83 (C -1 ), 38.59 (C -6 ),33.46 (C -10 ), 30.77 (C -7 ), 28.01 (C -9 ), 26.12 (C -4 ), 23.43 (C -8 ), 22.19 (C -3 ),21.11 (C -β , C -β' ); IR (KBr), ν max (cm -1 ): 3470, 3415, 3071, 2926, 2940, 2910,2878, 1735, 1715, 1612, 1593, 1490, 1468, 1383, 1366, 1252, 1075, 1014, 918,849, 830, 824, 764, 743。

[0059] 5b: N-hydroazolyl-N-p-bromobenzyl tetrahydropyrrole bromide (4A4'-p-BrBzBr) C 22 H 33 NCIBr white solid, yield 88%, m.p. 188.7~189.0 ℃. 1 H NMR (600MHz, CDCl3), δ (ppm): 7.593 (d,J=8.4Hz, 2H, 15- CH, 17- CH), 7.553 (d, J=8.4Hz, 2H, 14- CH, 18- CH), 4.972 (m, 2H, 12- CH2), 4.026 (m, 2H, α- CH2), 3.305 (m, 2H, 11- CH2), 3.647 (m, 2H, α'- CH2), 2.312(m, 3H, 2- CH, β- CH2), 2.135 (m, 2H, β'- CH2), 2.001~1.836 (m, 8H, 10- CH2, 7- CH a , 5- CH, 1- CH, 3- CH a , 4- CH2), 1.353 (m, 1H, 3- CH b ), 1.213 (s, 3H, 9- CH3), 1.056 (s, 3H, 8- CH3), 0.856 (d, J=9.6Hz, 1H, 7- CH b ); 13 C NMR (150MHz, CDCl3), δ (ppm): 134.14(C -15 , C -17 ), 132.49 (C -14 , C -18 ), 127.25 (C -16 ), 125.31 (C -13 ), 61.88 (C -12), 61.21, 60.98 (C -α , C -a' ), 58.42 (C -11 ), 45.94 (C -2 ), 41.08 (C -5 ), 38.84 (C -1 ),38.57 (C -6 ), 33.43 (C -10 ), 30.74 (C -7 ), 27.98 (C -9 ), 26.08 (C -4 ), 23.39 (C -8 ),22.18 (C -3 ), 21.58 (C -β , C -β' ); IR (KBr), ν max (cm -1 ): 3465, 3409, 2942, 2909,1592, 1487, 1468, 1382, 1368, 1074, 1013, 865, 847, 829, 651, 596。

[0060] 5c: N-hydroxynorbornyl-N-m-chlorobenzyl tetrahydropyrrole bromide (4A4'-m-ClBzBr) C 22 H 34 NBr white solid, yield 86%, m.p. 97.5~97.8 ℃. 1 H NMR (600MHz, CDCl3), δ (ppm): 7.477 (s, 1H, 14- CH), 7.461 (d, J=7.8Hz, 1H, 18- CH), 7.301 (d, J=7.8Hz, 1H, 16- CH), 7.258 (t,J=7.2Hz, 1H, 17- CH), 4.780 (m, 2H, 12- CH2), 3.833 (m, 2H, 11- CH2), 3.559 (m, 2H, α- CH2), 3.169 (m, 2H, α'- CH2), 2.187 (m, 3H, 2- CH, β- CH2), 2.035 (m, 2H,β'- CH2), 1.893~1.688 (m, 8H, 10- CH2, 7- CH a , 5- CH, 1- CH, 3- CH a , 4- CH2), 1.303 (m, 1H, 3- CH b ),1.059 (s, 3H, 9- CH3), 0.917 (s, 3H, 8- CH3), 0.704 (d, J=9.6Hz, 1H, 7- CH b ); 13 CNMR (150MHz, CDCl3), δ (ppm): 135.06 (C -15 ), 132.11 (C -14 ), 130.82 (C -16 , C -18 ),130.71 (C -17 ), 130.26 (C -13 ), 61.63 (C -α , C -a' ), 61.24 (C -12 ), 58.55 (C -11 ), 46.01(C -2 ), 41.10 (C -5 ), 38.86 (C -1 ), 38.59 (C -6 ), 33.46 (C -10 ), 30.82 (C -7 ), 27.98(C -9 ), 26.11 (C -4 ), 23.41 (C -8 ), 22.07 (C -3 ), 21.54 (C -β , C -β' ); IR (KBr), ν max (cm -1): 3456, 3407, 3046, 2956, 2927, 2868, 2848, 1951, 1889, 1706, 1598, 1573,1466, 1435, 1383, 1365, 1221, 1213, 1170, 1102, 1086, 1055, 980, 918, 898,874, 847, 792, 722, 713, 686.

[0061] 5d: N-hydroxynorbornyl-N-m-fluorobenzyl tetrahydropyrrole bromide (4A4'-m-FBzBr) 22 H 33 NFBr white solid, yield 85%, m.p. 82.3~82.5 ℃. 1 H NMR (600MHz, CDCl3), δ (ppm): 7.436 (s, 1H, 14- CH), 7.385 (d, J=9.6Hz, 1H, 18- CH), 7.346 (d, J=9.6Hz, 1H, 16- CH), 7.133 (t,J=7.8Hz, 1H, 17- CH), 4.915 (m, 2H, 12- CH2), 4.032 (m, 2H, 11- CH2), 3.619 (m, 2H, α- CH2), 3.240 (m, 2H, α'- CH2), 2.300 (m, 3H, 2- CH, β- CH2), 2.100 (m, 2H, β'- CH2),1.980~1.774 (m, 8H, 10- CH2, 7- CH a , 5- CH, 1- CH, 3- CH a , 4- CH2), 1.403 (m, 1H, 3- CH b ),1.162 (s, 3H, 9- CH3), 1.014 (s, 3H, 8- CH3), 0.808 (d, J=9.6Hz, 1H, 7- CHb ); 13 CNMR (150MHz, CDCl3), δ (ppm): 161.77 (C -15 ), 131.08 (C -14 ), 130.47 (C -13 ),128.33 (C -18 ),119.25 (C -16 ), 117.66 (C -17 ), 61.71 (C -12 ), 61.53, 61.29 (C -α C -a' ), 58.62 (C -11 ), 45.86 (C -2 ), 41.08 (C -5 ), 38.78 (C -1 ), 38.54 (C -6 ), 33.43(C -10 ), 30.76 (C -7 ), 27.91 (C -9 ), 26.08 (C -4 ), 23.35 (C -8 ), 22.12 (C -3 ), 21.54(C -β C -β' ); IR (KBr), ν max (cm -1 ): 3329, 3080, 3047, 3012, 2980, 2936, 2907,2865, 1730, 1614, 1589, 1490, 1465, 1384, 1363, 1321, 1260, 1156, 1068, 924,887, 877, 794, 757, 693.

[0062] 5e: N-Hydroxynorbutyryl-N-p-nitrobenzyltetrahydropyrrole bromide (4A4'-p-NO2BzBr) C 22 H 33 N2O2Br is a white solid with a yield of 87% and an mp value of 192.5~192.7 °C. 1 H NMR (600MHz, CDCl3), δ (ppm): 8.163(d, J=8.4Hz, 2H, 15- CH, 17-CH), 7.962 (d, J=9.0Hz, 2H, 14- CH, 18- CH), 5.151 (m,2H, 12- CH2), 4.007 (m, 2H, 11- CH2), 3.607 (m, 2H, α- CH2), 3.261 (m, 2H, α'- CH2),2.299 (m, 2H, β- CH2), 2.239 (m, 1H, 2- CH), 2.062 (m, 2H, β'- CH2), 1.943~1.748(m, 8H, 10- CH2, 7- CH a , 5- CH, 1- CH, 3- CH a , 4- CH2), 1.370 (m, 1H, 3- CH b ), 1.125 (s,3H, 9- CH3), 0.971 (s, 3H, 8- CH3), 0.777 (d, J=9.6Hz, 1H, 7- CH b ); 13 C NMR (150MHz,CDCl3), δ (ppm): 148.97 (C -16 ), 135.43 (C -13 ), 134.13 (C -15 , C -17 ), 124.16 (C -14 ,C -18 ), 61.70 (C -12 ), 61.62, 61.46 (C -α , C -α' ), 58.85 (C -11 ), 46.02 (C -2 ), 41.11(C -5 ), 38.91 (C -1 ), 38.65 (C -6 ), 33.49 (C -10 ), 30.79 (C -7), 26.13 (C -9 ), 23.47 (C -4 ), 22.25 (C -8 ), 21.71 -3 ), 21.71 α (C -β , C -β' ); IR (KBr), ν max (cm -1 ): 3472,3402, 3078, 2983, 2939, 2915, 1608, 1527, 1468, 1351, 1220, 1108, 1019, 873,856, 755, 720, 704, 655, 630.

[0063] 5f: N-hydroxynorbuxine-N-m-nitrobenzyl tetrahydropyrrole bromide (4A4'-m-NO2BzBr) C 22 H 33 N2O2Br white solid, yield 86%, m.p. 123.4~123.7 ℃. 1 H NMR (600MHz, CDCl3), δ (ppm): 8.427(s, 1H, 14- CH), 8.180 (d, J=8.4Hz, 2H, 16- CH, 18- CH), 7.596 (t, J=7.8Hz, 1H, 17- CH), 5.083 (m, 2H, 12- CH2), 3.931 (m, 2H, α- CH2),3.627 (m, 2H, 11- CH2), 3.245 (m,2H, α'- CH2), 2.247 (m, 2H, β- CH2), 2.187 (m, 1H, 2- CH), 2.061~1.669 (m, 8H, 10- CH2, 7- CH a , 5- CH, 1- CH, 3- CH a , 4- CH2), 1.340 (m, 1H, 3- CH b ), 1.064 (s, 3H,9- CH3), 0.935 (s, 3H, 8- CH3), 0.720 (d, J=9.6Hz, 1H, 7- CH b ); 13 C NMR (150MHz, CDCl3), δ(ppm): 148.27 (C -15 ), 139.26 (C -14 ), 130.79 (C -18 ), 130.52 (C -13 ), 127.04 (C -16 ),125.37 (C -17 ), 61.70 (C -12 ), 61.37 (C -α , C -a' ), 58.98 (C -11 ), 46.20 (C -2 ), 41.09(C -5 ), 38.93 (C -1 ), 38.60 (C -6 ), 33.44 (C -10 ), 30.93 (C -7 ), 27.97 (C -9 ), 26.10(C -4 ), 23.37 (C -8 ), 22.07 (C -3 ), 21.66 (C -β , C -β' ); IR (KBr), ν max (cm -1 ): 3511,3420, 3081, 3035, 2993, 2938, 2918, 2866, 1731, 1684, 1610, 1530, 1476, 1462,1386, 1348, 1303, 1218, 1154, 1092, 905, 821, 726, 702, 676。

[0064] Structure analysis: 13 compounds, 1 (N-hydronorbornyl tetrahydropyrrole), 3a~3f, 5a~5f, structure analysis showed that they all contain hydronorbornyl. In the hydrogen spectrum, there are two methyl singlets, each 3H; in the carbon spectrum, 3 3° C (C -2 , C -5 , C-1 ) and 2 1° C (C -9 C -8 ); On the infrared spectrum, there is 1385 cm⁻¹ -1 1365 cm -1 The two absorption peaks indicate the presence of geminal dimethyl [C(CH3)2]. Except for compound 1, the other 12 compounds all have 5 or 4 hydrogen atoms at 7.0 ppm–8.5 ppm in the proton NMR spectrum, and one 4°C and five 3°C (3a, 5a) or two 4°C and four 3°C (3b–3f, 5b–5f) at 120–160 ppm in the carbon NMR spectrum. The 3000 cm⁻¹ infrared spectrum also shows... -1 The CH absorption peak with a benzene ring is located at 1500~1620 cm⁻¹. -1 The presence of benzene ring skeletal peaks indicates that all 12 compounds possess a benzene ring structure; carbonyl groups are present at 190 ppm in carbon spectra 3b-3f. Carbon, also present in infrared spectra. The strong absorption peak (~1700 cm⁻¹) -1 These basic structural fragments indicate that these compounds are the target compounds to be synthesized in this paper, and the number of C and H atoms in the NMR analysis is also completely consistent with the molecular formula of the corresponding compounds.

[0065] 3. The fungistatic activity of the target compounds: In the examples of the present application, the mycelial growth rate method was adopted to preliminarily determine the growth inhibition of the synthesized target compounds on eight plant pathogenic fungi at a concentration of 100 mg / L. The determination results show that these target compounds all exhibit certain inhibition activity on the growth of the above-mentioned eight plant pathogenic fungi, and the specific inhibition rates are shown in Table 1. In general, the 3 series (3a-3f) as a whole are superior to the 5 series (5a-5f). At a concentration of 100 mg / L, the fungistatic rates of compounds 3b, 3c, 3d, 3f, 5a, 5b and 5c on Sclerotinia sclerotiorum (S), Phytophthora parasitica (11), Serpula lacrymans (H) all reach 100%; the fungistatic rates of 3a on 11 and H also reach 100%, and the fungistatic rate of 3a on Rhizoctonia solani (K) is close to 90%; the fungistatic rates of 3b, 3c, 3d, 5a and 5b on K all reach 100%, the fungistatic rates of 3b, 3c, 3d and 5b on Fusarium oxysporum (X) reach or are close to 90%, which are equivalent to the fungistatic rates of chlorothalonil (100% on K and H), even higher than the fungistatic rates of chlorothalonil (78.5% on S, 88.3% on X and 84.2% on 11), and far higher than the fungistatic rates of kresoxim-methyl. Although the fungistatic rates of the twelve compounds on Conidiobolus geosporus (Cf), Fusarium verticillioides (O) and Mycogone rosea (V) are not as high as the inhibition on the above-mentioned five fungi, but from the data listed in Table 1, it can be seen that the fungistatic rates of compounds 3b and 3c on Cf are more than 80%, which are higher than the fungistatic rate of chlorothalonil at the same concentration (76.1%), and the fungistatic rates of 3d and 5b on Cf are 75.0%, close to the fungistatic rate of chlorothalonil. The fungistatic rates of 3b, 3c, 3d and 5b on O are all more than 80%, among which the fungistatic rate of 3c is 91.8%, slightly higher than the fungistatic rate of chlorothalonil (91.3%), the fungistatic rates of 3a, 3f, 5a and 5c on O are all more than 70%, and the inhibition effect is good. The fungistatic rates on V are also higher, and the fungistatic rates of compounds 3c (77.3%), 5b (74.2%), 3b (69.7%) and 3d (66.7%) are higher than the fungistatic rate of chlorothalonil on V (61.0%). In general, the fungistatic effects of compounds 3b, 3c, 3d, 5a and 5b on the above eight plant pathogenic fungi are superior to or close to the fungistatic effects of chlorothalonil and kresoxim-methyl.

[0066] Table 1. The fungistatic rates of the target compounds on eight plant pathogenic fungi at a concentration of 100 mg / L (unit: %)

[0067] In order to more accurately evaluate the antibacterial activity of the target compounds, the IC 50 values of all the compounds on eight fungi were determined in the examples of the present application. The specific data are shown in Table 2.

[0068] Table 2 IC of target compounds on 8 plant pathogenic fungi 50 Value (unit: mg / L)

[0069] As can be seen from the data in Table 2, compounds 3b, 3c, 3d and 5b exhibit relatively significant inhibitory activity on multiple plant pathogenic fungi, and the IC 50 values are relatively low. For example, the IC 50 values of compound 3b on fungi X and 11 are 3.8 mg / L and 8.5 mg / L, respectively; the IC 50 values of compound 3c on S, X, 11, H and O are all ≤10 mg / L; and the IC 50 values of compounds 3d and 5b on S, X and 11 are also all ≤10 mg / L, indicating that these compounds can effectively inhibit the growth of multiple fungi at a lower concentration, and have a wide antibacterial spectrum. Further analysis found that the IC 50 values of compounds 3b and 3d on watermelon fusarium (X) are 3.8 mg / L and 3.7 mg / L, respectively, which are very close to the control agent kresoxim-methyl (IC 50 = 3.4 mg / L), indicating that these two compounds have excellent inhibitory effect on watermelon fusarium, showing good application potential.

[0070] In addition, the mycelial growth rate method was adopted to preliminarily determine the growth inhibition of the target compounds synthesized in the embodiments of the present application and the comparative compounds (comparative compound a: dimethyl dihydrogenated nopyl ammonium chloride, comparative compound b: dimethyl dihydrogenated nopyl ammonium iodide, comparative compound c: N,N-dihydrogenated nopyl piperidine iodide, comparative compound d: N,N-dihydrogenated nopyl morpholine chloride, comparative compound e: N,N-dihydrogenated nopyl morpholine bromide, comparative compound f: N,N-dihydrogenated nopyl morpholine iodide) synthesized in the prior art (CN113754547A) on 8 plant pathogenic fungi at a concentration of 50 mg / L, and the results are shown in Table 3.

[0071] Table 3 Inhibition rate of target compounds on 8 plant pathogenic fungi at a concentration of 50 mg / L (unit: %)

[0072] As can be seen from Table 3, the substituents and their substitution positions of the cyclic quaternary ammonium salt N-hydrogenated nopyl tetrahydro-pyrrole compounds or the different ring structures connected to the N atom all cause obvious differences in the antibacterial performance, wherein the compounds 3b, 3c, 3d and 5b prepared in the embodiments of the present application have significant antibacterial effects on various plant pathogenic fungi.

[0073] 4. Growth curve determination of Fusarium oxysporum f. sp. niveum: The growth trend of Fusarium oxysporum f. sp. niveum mycelium under the action of compounds 3b and 3d was determined by mycelium dry weight method, and the mycelium without adding compounds was taken as the CK group for comparison, and the results are shown in Figure 1 and Figure 2 . From 0 to 1 day, the mycelium of the CK group grew slowly and was in the lag phase; from 1 to 2 days, the mycelium dry weight increased significantly, and the growth rate peaked on the second day, entering the logarithmic growth phase, with active enzyme system and vigorous metabolism; from 2 to 9 days, the growth slowed down and entered the stable phase. Compared with the CK group, the mycelium dry weight treated with 3b and 3d was significantly lower at each stage, and the inhibition effect of 3d was better than that of 3b. The results show that 3b and 3d can effectively inhibit the growth of Fusarium oxysporum f. sp. niveum mycelium, and 3d has more significant inhibition, which can provide important reference for subsequent prevention and control research.

[0074] 5. Effect of compounds 3b and 3d on the morphology of Fusarium oxysporum f. sp. niveum mycelium: The ultrastructure of Fusarium oxysporum f. sp. niveum mycelium treated with compounds 3b and 3d at IC 50 mass concentration was observed in detail by scanning electron microscope (SEM), and the results are shown in Figure 3 . The results show that compared with the untreated control group, the mycelium treated with compounds 3b and 3d has significant differences in morphology. As shown in Figure 3 a and b, the untreated mycelium has a full appearance and complete shape, and the surface is smooth. In contrast, the mycelium treated with compounds 3a and 3d has a rough and concave surface, obvious wrinkles are observed, and part of the mycelium is broken (see Figure 3 c-f). These results show that it is speculated that compounds 3b and 3d can change the morphology of Fusarium oxysporum f. sp. niveum mycelium, thereby possibly damaging the cell wall and cell membrane structure of the mycelium, and ultimately leading to the death of the mycelium.

[0075] 6. Effect of compounds 3b and 3d on cell membrane permeability: The effect of compounds 3b and 3d on the cell membrane of Fusarium oxysporum f. sp. niveum was explored by determining the change of extracellular ion conductivity, and the results are shown in Figure 4 . The integrity and permeability of the cell membrane are crucial, and when it is damaged, electrolytes (such as potassium, sodium, calcium ions) in the cell will penetrate to the outside of the cell to varying degrees due to factors such as concentration gradient, resulting in significant changes in the extracellular ion conductivity. The determination results can indirectly reflect the permeability of the extracellular electrolyte of the mycelium and the damage condition of the cell membrane. The experimental results show that the relative conductivity of the CK group tends to be stable over time; the relative conductivity of the mycelium suspension treated with 3b and 3d increases sharply within 0-30 min, and grows slowly after 60 min, and the relative conductivity at each time point is significantly higher than that of the CK group. This shows that 3b and 3d can significantly change the permeability of the mycelium cell membrane, damage the cell membrane structure, and lead to the leakage of the contents.

[0076] The present application embodiment takes hydrogenated nopol chloride and tetrahydropyrrole as raw materials, to prepare N-hydrogenated nopol tetrahydropyrrole intermediates by reaction, and then to synthesize 12 N-hydrogenated nopol tetrahydropyrrole cyclic quaternary ammonium salt compounds by quaternary ammonium reaction with α-bromoacetophenone and benzyl bromide compounds respectively. The structures of all target compounds are characterized by nuclear magnetic resonance (HNMR, 1 , 13 C NMR) and infrared spectroscopy (IR) and the like. The antibacterial activity results show that at a concentration of 100 mg / L, compounds 3b, 3c, 3d, 5a and 5b exhibit broad-spectrum antibacterial activity against a variety of plant pathogenic fungi (including K, S, 11 and H strains), with an inhibition rate of 100%, and their antibacterial effect is equivalent to or better than the commonly used fungicide chlorothalonil, or even far exceeds the fungicide kresoxim-methyl. In addition, their inhibition rates on other three plant pathogenic fungi (Guignardia bidwellii, Fusarium decemellum, Botryosphaeria dothidea) are also higher or close to chlorothalonil and kresoxim-methyl. Among the two types of cyclic quaternary ammonium salt compounds, the ones with Br or Cl atoms at the para or meta position of the benzene ring have better inhibition effect on the tested plant pathogenic fungi. Further dose-effect analysis shows that the IC 50 of 3b and 3d on X strain is 3.8 mg / L and 3.7 mg / L respectively, which is equivalent to kresoxim-methyl (IC 50 = 3.4 mg / L), showing good application prospect. Mechanism studies show that 3b and 3d can inhibit the growth of Fusarium oxysporum f. sp. niveum through multiple pathways: significantly reducing the mycelial biomass (growth curve analysis), destroying the normal morphology of mycelium (scanning electron microscopy shows surface wrinkles and breaks), and enhancing the cell membrane permeability (conductivity determination). These research results not only add a new series to the chemical processing and utilization of β-pinene, but also provide some new data for the in-depth study of the relationship between the chemical structure and biological activity of pinene derivatives, and provide a new direction and idea for the development and potential application of tetrahydropyrrole type cyclic quaternary ammonium salt containing hydrogenated nopol as a high-efficiency fungicide.

[0077] Based on the above ideal embodiments according to the present application, through the above description, relevant personnel can make various changes and modifications without deviating from the technical idea of the present application. The technical scope of the present application is not limited to the contents in the specification.

Claims

1. A cyclic quaternary ammonium salt N-hydronorbutyryl tetrahydropyrrole compound, characterized in that, The general structural formula of the cyclic quaternary ammonium salt N-hydronorbutyryl tetrahydropyrrole compound is any one of the following structural formulas: ； ； In the formula, R is independently a hydrogen or a substituent; The substituent is any one of -Br, -Cl, -F, and -NO2; The substituent is any one of para-substitution, ortho-substitution, and meta-substitution.

2. The cyclic quaternary ammonium salt N-hydronorbutyryl tetrahydropyrrole compound according to claim 1, characterized in that, The general structural formula of the cyclic quaternary ammonium salt N-hydronorbutyryl tetrahydropyrrole compounds is: ； In the formula, R is any one of hydrogen, para-bromo, meta-bromo, para-chloro, ortho-fluorine, and para-nitro.

3. The cyclic quaternary ammonium salt N-hydronorbutyryl tetrahydropyrrole compound according to claim 1, characterized in that, The general structural formula of the cyclic quaternary ammonium salt N-hydronorbutyryl tetrahydropyrrole compounds is: ； In the formula, R is any one of hydrogen, para-bromo, meta-chloro, meta-fluorine, para-nitro, and meta-nitro.

4. A method for synthesizing cyclic quaternary ammonium salt N-hydronorbutyryl tetrahydropyrrole compounds as described in any one of claims 1-3, characterized in that, Includes the following steps: N-Hydronylnorbutyrate is quaternized with α-bromoacetophenone compounds or benzyl bromide compounds to obtain the cyclic quaternary ammonium salt N-Hydronylnorbutyrate compounds. The general structural formula of the α-bromoacetophenone compounds is: ； The general structural formula of the benzyl bromide compounds is: ； In the formula, R is independently a hydrogen or a substituent; The substituent is any one of -Br, -Cl, -F, and -NO2; The substituent is any one of para-substitution, ortho-substitution, and meta-substitution.

5. The method for synthesizing cyclic quaternary ammonium salt N-hydronorbutyryl tetrahydropyrrole compounds according to claim 4, characterized in that, The method for synthesizing the N-hydronorbutyltetrahydropyrrole includes the following steps: Hydrogenated nobyl chloride and tetrahydropyrrole were placed in anhydrous ethanol, and sodium hydroxide was added. The mixture was then heated under reflux to obtain the N-hydrogenated nobyl tetrahydropyrrole.

6. The method for synthesizing cyclic quaternary ammonium salt N-hydronorbutyryl tetrahydropyrrole compounds according to claim 4, characterized in that, Specifically, it includes: N-Hydronylnorbutyroxene and α-bromoacetophenone compounds were placed in ethyl acetate and heated to 65-75°C to react and obtain the cyclic quaternary ammonium salt N-Hydronylnorbutyroxene compounds. Alternatively, N-hydronomyltetrahydropyrrole and benzyl bromide compounds can be reacted with ethyl acetate under reflux to obtain the cyclic quaternary ammonium salt N-hydronomyltetrahydropyrrole compounds.

7. The application of a cyclic quaternary ammonium salt N-hydronorbutyryl tetrahydropyrrole compound as described in any one of claims 1-3 in the inhibition of plant pathogens.

8. The application of the cyclic quaternary ammonium salt N-hydronorbutyltetrahydropyrrole compound according to claim 7 in inhibiting plant pathogens, characterized in that, The plant pathogens include one or more of the following: rice sheath blight fungus, pine shoot blight fungus, fruit scab fungus, watermelon wilt fungus, tobacco black shank fungus, cottony rot fungus, variegated sclerotium fungus, and verticillium fungus.

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