Azobenzene Compounds Containing Isopropanolamine Substructures, Preparation Methods Thereof and Applications

Abstract

The present invention relates to the technical field of plant disease resistance, and specifically relates to azobenzene compounds containing an isopropanolamine substructure, and a preparation method and application thereof. The specific technical solution is as follows: The present invention designs and synthesizes a class of azobenzene compounds containing an isopropanolamine substructure, formulates them into a solution with dimethyl sulfoxide, dilutes it with secondary water, then adds β-cyclodextrin, dissolves it by ultrasonic treatment, and obtains nanoparticles after freeze-drying. After the azobenzene compounds are encapsulated by β-cyclodextrin, at room temperature, the solubility in water increases from 3.15×10 ‑6 to 3.43×10 ‑6 , the water solubility is improved, and the higher the temperature, the better the water solubility, and further agricultural application is carried out. The present invention solves the problems that the drug molecules have a short duration of field efficacy and poor leaf wettability, resulting in reduced efficacy, is beneficial to pesticide deposition and leaf absorption, improves bioavailability and biocompatibility, and at the same time provides a new strategy for applying a nano-system for encapsulating drug molecules with β-cyclodextrin to enhance crop disease resistance.

Description

Technical Field

[0001] The present invention relates to the technical field of plant disease resistance, and particularly relates to an azobenzene compound containing an isopropanolamine substructure, a preparation method thereof, and an application thereof. Background Art

[0002] The spread of plant pathogens in the agricultural environment is becoming increasingly serious, which not only threatens agricultural production, but also may affect human health and safety. These mainly include diseases such as rice bacterial blight, pepper bacterial wilt, Chinese cabbage leaf spot, tobacco bacterial wilt, citrus canker, kiwifruit canker, cucumber gray mold, pepper fusarium wilt, rape sclerotinia, wheat scab, potato late blight, blueberry root rot, botryosphaeria dothidea, pitaya anthracnose, rice sheath blight, etc., mainly manifested as necrosis, withering, rot, fruit drop and other symptoms. However, traditional fungicides all have problems such as low utilization rate, rapid release of active ingredients, short effective time, and serious ecological pollution. Moreover, with their overuse, the problem of drug resistance is becoming increasingly severe. Therefore, it is urgent to develop new and efficient pesticides to solve these problems. Summary of the Invention

[0003] Aiming at the deficiencies of the prior art, the present invention provides an azobenzene compound containing an isopropanolamine substructure, a preparation method thereof, and an application thereof, solving the problems that drug molecules are prone to photoinversion in the field and have poor leaf wettability, resulting in reduced drug efficacy.

[0004] To achieve the above object, the present invention is realized through the following technical solutions:

[0005] The present invention discloses an azobenzene compound containing an isopropanolamine substructure, including the compound shown in formula (I) or its stereoisomer, or its salt or its solvate,

[0006]

[0007] wherein, R1 and R2 are respectively one or more of hydrogen, deuterium, halogen, optionally substituted or unsubstituted alkyl, optionally substituted or unsubstituted alkenyl, optionally substituted or unsubstituted alkynyl, optionally substituted or unsubstituted alkoxy, optionally substituted or unsubstituted cycloalkyl, optionally substituted or unsubstituted aryl, optionally substituted or unsubstituted heteroaryl;

[0008] or R1 and R2 are connected to form an optionally substituted 5-10 membered ring or a ring containing a heteroatom, and the heteroatom is one or more of N, O, S.

[0009] Preferably, R1 and R2 are respectively hydrogen, methyl, ethyl, propyl, benzyl, 1-(4-fluorobenzyl)piperazinyl, 1-tert-butylpiperazinyl, 1-isopropylpiperazinyl, 1-(2-fluorobenzyl)piperazinyl, methyl 4-piperidinecarboxylate, ethyl 4-piperidinecarboxylate, 2-methylpiperidinyl, 3-methylpiperidinyl, 4-methylpiperidinyl, imidazolyl, 2-F-benzyl, 3-F-benzyl, 4-F-benzyl, 3-OCH3-benzyl, 2-OCH3-benzyl, 4-methyl-benzyl-2,6-dimethylmorpholinyl, 3,5-dimethylpiperidinyl.

[0010] Preferably, when R1 and R2 are linked to form a ring, they are the following groups:

[0011]

[0012] Correspondingly, a preparation method of an azobenzene compound containing an isopropanolamine substructure has the following reaction equation:

[0013]

[0014] Correspondingly, a composition includes the above-mentioned azobenzene compound containing an isopropanolamine substructure or the azobenzene compound containing an isopropanolamine substructure prepared by the above-mentioned preparation method.

[0015] Preferably, the dosage form of the composition is one or more of emulsifiable concentrate, powder, wettable powder, granule, aqueous solution, suspending agent, ultra-low volume spray, soluble powder, microcapsule, smoke agent, emulsion in water and water dispersible granule.

[0016] Correspondingly, a method for using the above-mentioned azobenzene compound containing an isopropanolamine substructure or the azobenzene compound containing an isopropanolamine substructure prepared by the above-mentioned preparation method or the above-mentioned composition is to act the azobenzene compound or composition containing an isopropanolamine substructure on harmful substances or their living environment, or directly contact with plants.

[0017] Correspondingly, a method for preparing nano-pesticides using the above-mentioned azobenzene compound containing an isopropanolamine substructure or the azobenzene compound containing an isopropanolamine substructure prepared by the above-mentioned preparation method is to prepare a dimethyl sulfoxide solution of the azobenzene compound containing an isopropanolamine substructure, add cyclodextrin, dissolve it by ultrasonic wave, and then freeze-dry to obtain nano-particles.

[0018] Preferably, the cyclodextrin is β-cyclodextrin, and the molar ratio of the cyclodextrin to the azobenzene compound containing an isopropanolamine substructure is 1:1.

[0019] Correspondingly, it includes the nano-particles prepared by the above-mentioned preparation method.

[0020] Correspondingly, the application of the azobenzene compound containing an isopropanolamine substructure as described above, or the azobenzene compound containing an isopropanolamine substructure prepared by the above preparation method, or the above composition, or the nanoparticles prepared by the above preparation method, or the above nanoparticles in the prevention and control of agricultural pests and diseases.

[0021] Preferably, the agricultural pests and diseases controlled by the azobenzene compound containing an isopropanolamine substructure are rice bacterial blight, tobacco bacterial wilt, cucumber bacterial blight, konjac bacterial blight, citrus canker, grape canker, tomato canker, kiwifruit canker, apple canker, cucumber gray mold, pepper fusarium wilt, rape sclerotinia rot, wheat scab, potato late blight, blueberry root rot;

[0022] The agricultural pests and diseases controlled by the nanoparticles are tobacco mosaic virus, cucumber mosaic virus, potato virus X, rice bacterial blight, citrus canker, tobacco bacterial wilt, kiwifruit canker, rice bacterial leaf streak, cucumber gray mold, pepper fusarium wilt, rape sclerotinia rot, wheat scab, potato late blight, blueberry root rot, Botryosphaeria dothidea, pitaya anthracnose, rice sheath blight, green caterpillars, woolly caterpillars, aphids, scale insects, whiteflies, root-knot nematodes, dagger nematodes, stem nematodes, cotton spider mites, citrus red spider mites.

[0023] The present invention has the following beneficial effects:

[0024] 1. Based on the azobenzene structure, the present invention introduces groups such as piperazine, piperidine, and isopropanolamine that may improve the biological activity of the target compound into this system, synthesizes a series of azobenzene compounds containing an isopropanolamine structure, and determines their biological activities. On this basis, a photo-responsive stimulus nanoparticle is constructed by using host-guest interaction and the host molecule β-cyclodextrin (β-CD), and the biological activity of the nanoparticle is explored to achieve controlled release and slow release. At the same time, it is found that the compound and its host-guest complex have good inhibitory effects on pathogenic bacteria, and have good inhibitory effects on pathogenic bacteria [such as Xanthomonas oryzae pv.oryzae (Xoo), Xanthomonas axonopodis pv.citri (Xac), Clavibacter michiganensis subsp.michiganensis (Cmm), etc.], providing an important scientific basis for the research and development of new pesticides.

[0025] 2. The nano-system of the β-cyclodextrin-encapsulated drug molecule azobenzene compound of the present invention has a simple preparation method and high repeatability. Stable and uniform spherical nanoparticles with a diameter of about 1200 nm are obtained.

[0026] 3. The nano-system of β-cyclodextrin-encapsulated drug molecule azobenzene compounds of the present invention has better stability and wettability than the drug molecule azobenzene compounds, which is conducive to pesticide deposition and leaf absorption, improves bioavailability and biocompatibility, and has a long slow-release duration, photo-controlled triggered release of active ingredients, and good anti-plant pathogen activity. It can solve the problems of short field efficacy duration, poor leaf wettability, and low effective utilization rate of drug molecules, is conducive to pesticide deposition and leaf absorption, improves bioavailability and biocompatibility, and at the same time provides a new strategy for applying the nano-system of β-cyclodextrin-encapsulated drug molecules to enhance crop disease resistance.

[0027] 4. The nano-system of β-cyclodextrin-encapsulated drug molecule azobenzene compounds prepared by the present invention has potential application value in agriculture through its ability to inhibit biofilm growth and subsequent bacterial infection. In addition, the risk assessment results show that this nano-system is environmentally friendly and non-toxic to various non-target organisms including human cell lines and aquatic organism Brachydanio reio. By forming a complex, the inclusion compound improves its biocompatibility and bioavailability, and finally realizes the dissociation of the complex through the response to light stimulation, thereby achieving the maximum active effect and slow-controlled pesticide release. Brief Description of the Drawings

[0028] Figure 1 are the nuclear magnetic resonance hydrogen spectrum and ultraviolet spectrum diagrams of two configurations of the drug molecule azobenzene compound 3a (3,3'-((diazene-1,2-diylbis(4,1-phenylene))bis(oxy))bis(1-((3-methoxybenzyl)(methyl)amino)propan-2-ol));

[0029] Figure 2 is the HRMS of β-cyclodextrin-based nanoparticles (3a@β-CD);

[0030] Figure 3 are the titration curve and Jobs curve determination results of β-cyclodextrin-based nanoparticles (3a@β-CD);

[0031] Figure 4 are the transmission electron microscope images (200nm, 500nm) of trans-azobenzene compound 3a, cis-azobenzene compound 3a', β-cyclodextrin-based nanoparticles (3a@β-CD), and after decomposition of β-cyclodextrin-based nanoparticles (UV-3a@β-CD);

[0032] Figure 5 are the water solubility test diagrams and contact angle test results of trans-azobenzene compound 3a, cis-azobenzene compound 3a', β-cyclodextrin-based nanoparticles (3a@β-CD), and after decomposition of β-cyclodextrin-based nanoparticles (UV-3a@β-CD);

[0033] Figure 6 Solubility determination of azobenzene compounds 3a and β-cyclodextrin-based nanoparticles (3a@β-CD);

[0034] Figure 7 Zeta potential test of trans-azobenzene compound 3a, cis-azobenzene compound 3a', β-cyclodextrin-based nanoparticles (3a@β-CD), and after decomposition of β-cyclodextrin-based nanoparticles (UV-3a@β-CD);

[0035] Figure 8 UV spectrum of β-cyclodextrin-based nanoparticles (3a@β-CD) releasing small drug molecules under ultraviolet light;

[0036] Figure 9 Crystal violet test for anti-biofilm activity of trans-azobenzene compound 3a, cis-azobenzene compound 3a, β-cyclodextrin-based nanoparticles (3a@β-CD), and after decomposition of β-cyclodextrin-based nanoparticles (UV-3a@β-CD);

[0037] Figure 10 Quantitative analysis of anti-biofilm activity of trans-azobenzene compound 3a, cis-azobenzene compound 3a, β-cyclodextrin-based nanoparticles (3a@β-CD), and after decomposition of β-cyclodextrin-based nanoparticles (UV-3a@β-CD);

[0038] Figure 11 Control effect of trans-azobenzene compound 3a, cis-azobenzene compound 3a, β-cyclodextrin-based nanoparticles (3a@β-CD), and after decomposition of β-cyclodextrin-based nanoparticles (UV-3a@β-CD) against Xanthomonas oryzae pv. oryzae in vivo. Specific implementation manners

[0039] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0040] Unless otherwise specified, the technical means used in the implementation examples are conventional means well known to those skilled in the art.

[0041] Azobenzene has a broad spectrum of biological activities, such as antibacterial, antifungal, antitumor, anticancer, and antioxidant activities, etc., and has extensive applications in the fields of materials science and biology. At the same time, groups such as piperazine, piperidine, and isopropanolamine also exhibit extensive biological activities in pesticides. In addition, due to the photo-isomerism of azobenzene, it can produce two isomers, which have the advantages of reversible structure and no side reactions under light conditions, and is a commonly used molecular switch. Therefore, the present invention rationally prepares azobenzene derivatives containing piperazine / piperidine-substituted isopropanolamine active groups and co-assembles them with β-cyclodextrin into drug-loaded nanoparticles, and applies them to the prevention and control of agricultural pests and diseases.

[0042] 1. The present invention discloses azobenzene compounds containing an isopropanolamine substructure, including the compounds shown in formula (I) or their stereoisomers, or their salts or their solvates,

[0043]

[0044] wherein R1 and R2 are each independently one or more of hydrogen, deuterium, halogen, optionally substituted or unsubstituted alkyl, optionally substituted or unsubstituted alkenyl, optionally substituted or unsubstituted alkynyl, optionally substituted or unsubstituted alkoxy, optionally substituted or unsubstituted cycloalkyl, optionally substituted or unsubstituted aryl, and optionally substituted or unsubstituted heteroaryl;

[0045] or R1 and R2 are linked together to form an optionally substituted 5- to 10-membered ring or a ring containing a heteroatom, and the heteroatom is one or more of N, O, and S.

[0046] Further, R1 and R2 are each independently hydrogen, methyl, ethyl, propyl, benzyl, 1-(4-fluorobenzyl)piperazinyl, 1-tert-butylpiperazinyl, 1-isopropylpiperazinyl, 1-(2-fluorobenzyl)piperazinyl, methyl 4-piperidinecarboxylate, ethyl 4-piperidinecarboxylate, 2-methylpiperidinyl, 3-methylpiperidinyl, 4-methylpiperidinyl, imidazolyl, 2-F-benzyl, 3-F-benzyl, 4-F-benzyl, 3-OCH3-benzyl, 2-OCH3-benzyl, 4-methyl-benzyl-2,6-dimethylmorpholinyl, 3,5-dimethylpiperidinyl.

[0047] Further, when R1 and R2 are linked to form a ring, they are the following groups:

[0048]

[0049] 2. The present invention discloses a preparation method of azobenzene compounds containing an isopropanolamine substructure, and the reaction equation is as follows:

[0050]

[0051] wherein R1 and R2 are as described above.

[0052] Furthermore,

[0053]

[0054] 3. The present invention discloses a composition comprising the azobenzene compound containing an isopropanolamine substructure as described above or the azobenzene compound containing an isopropanolamine substructure prepared by the above preparation method.

[0055] Among them, the dosage form of the composition is one or more of emulsifiable concentrate, powder, wettable powder, granule, aqueous solution, suspension, ultra-low volume spray, soluble powder, microcapsule, smoke agent, emulsion in water and water dispersible granule.

[0056] 4. The present invention discloses a method for using the azobenzene compound containing an isopropanolamine substructure as described above or the azobenzene compound containing an isopropanolamine substructure prepared by the above preparation method or the above composition, which is to act the azobenzene compound containing an isopropanolamine substructure or the composition on harmful substances or their living environment, or directly contact with plants.

[0057] 5. The present invention discloses a method for preparing nano-pesticides using the azobenzene compound containing an isopropanolamine substructure as described above or the azobenzene compound containing an isopropanolamine substructure prepared by the above preparation method. Prepare an N,N-dimethyl sulfoxide solution of the azobenzene compound (or drug molecule) containing an isopropanolamine substructure, add cyclodextrin, dissolve it by ultrasonic wave, and then freeze-dry to obtain nano-particles.

[0058] Among them, the drug molecule can be the azobenzene compound disclosed in the present invention, or at least one of plant resistance inducers, antiviral agents, fungicides, bactericides, insecticides, nematicides and acaricides containing azobenzene compounds with low solubility.

[0059] Furthermore, the cyclodextrin is β-cyclodextrin, and the molar ratio of the cyclodextrin to the azobenzene compound (or drug molecule) containing an isopropanolamine substructure is 1:1.

[0060] Correspondingly, it includes the nano-particles prepared by the above preparation method, that is, a nano-system in which β-cyclodextrin encapsulates drug molecules.

[0061] Correspondingly, the application of the azobenzene compound containing an isopropanolamine substructure as described above or the azobenzene compound containing an isopropanolamine substructure prepared by the above preparation method or the above composition or the nano-particles prepared by the above preparation method or the above nano-particles in the prevention and control of agricultural pests and diseases can be specifically understood as the application in the preparation of drugs for enhancing the disease resistance of host plants against agricultural pests and diseases.

[0062] Furthermore, the agricultural pests and diseases controlled by the azobenzene compounds containing isopropanolamine substructures are bacterial blight of rice, bacterial wilt of tobacco, bacterial blight of cucumber, bacterial blight of konjac, citrus canker, grape canker, tomato canker, kiwifruit canker, apple canker, gray mold of cucumber, fusarium wilt of pepper, sclerotinia sclerotiorum of rapeseed, scab of wheat, late blight of potato, and root rot of blueberry.

[0063] The agricultural pests and diseases controlled by the nanoparticles are tobacco mosaic virus, cucumber mosaic virus, potato virus X, bacterial blight of rice, citrus canker, bacterial wilt of tobacco, kiwifruit canker, bacterial leaf streak of rice, gray mold of cucumber, fusarium wilt of pepper, sclerotinia sclerotiorum of rapeseed, scab of wheat, late blight of potato, root rot of blueberry, botryosphaeria dothidea, anthracnose of pitaya, sheath blight of rice, green worms, caterpillars, aphids, scale insects, whiteflies, root-knot nematodes, dagger nematodes, stem nematodes, cotton spider mites, and citrus red spiders.

[0064] The present invention will be further described below in conjunction with specific embodiments.

[0065] Example 1 Preparation of Intermediate (E)-1,2-bis(4-(oxiran-2-ylmethoxy)phenyl)diazene

[0066] 4,4-Dihydroxyazobenzene (0.93 mmol) was added to a 15 mL pressure-resistant bottle containing 5 mL of N,N-dimethylformamide, and then potassium hydroxide (2.33 mmol) was added. After stirring at room temperature for 30 min, epibromohydrin was added, and the reaction was carried out at room temperature for 8 h. After the reaction was completed by TLC tracking, yellow solid precipitated after adding water, and the intermediate, yellow solid, was obtained by suction filtration with a yield of 95.60%. The structural formula is as follows:

[0067]

[0068] Example 2 Preparation of (E)-3,3'-((diazene-1,2-diylbis(4,1-phenylene))bis(oxy))bis(1-((3-methoxybenzyl)(methyl)amino)propan-2-ol)

[0069] (E)-1,2-Bis(4-(oxiran-2-ylmethoxy)phenyl)diazene (0.61 mmol) and N-methyl-3-methoxybenzylamine (1.29 mmol) were added to 8 mL of isopropanol solution. After the reaction was stopped after 8 h at 65 °C, it was extracted with 40 mL of ethyl acetate, washed with water, dried, concentrated by evaporation, and purified by column chromatography to obtain a yellow solid with a yield of 79.57%.

[0070] The structures, 1H NMR and 13C NMR data of other synthesized azobenzene compounds containing isopropanolamine structures are shown in Table 1.

[0071] 1H and 13C NMR data of the compounds in Table 1

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079] Identification of the two configurations of compound 3a in Example 3

[0080] Since azobenzene compounds are photo-isomers, they have two configurations: cis and trans. Therefore, the two configurations of compound 3a were identified by 1H NMR. 1 1H NMR( Figure 1 A) showed that after irradiation with ultraviolet light (λ = 365 nm) for 60 s, multiple peaks at 7.89 - 7.83 (a and b) and 7.02 - 6.98 ppm (c and d) were attenuated. In addition, multiple peaks were enhanced from 6.87 - 6.84 ppm (b') and 6.79 - 6.76 ppm (d'). In addition, various peaks at 4.06 - 4.02 ppm (e and f) were weakened, while new peaks appeared at 3.95 - 3.90 ppm (f'). The chemical shifts of alkyl hydrogens shifted to higher fields (lower chemical shifts) overall, indicating that trans-azobenzene isomerized after ultraviolet irradiation and the structure changed to the cis configuration, with an increase in electron cloud density, resulting in an overall shift of the chemical shift to higher fields. Therefore, these compounds can obtain the cis structure by ultraviolet irradiation.

[0081] The photo-isomerism of 3a was evaluated using a UV-Vis spectrophotometer. As Figure 1 shown in B, 1C, the results indicated that compound 3a was transformed from trans to cis-3a' under ultraviolet irradiation. After 35 s, the configuration inversion was completed. Under visible light irradiation, the conversion of compound 3a' from cis to trans was reversible. Similarly, the configuration inversion was completed after 35 s of exposure. Therefore, 3a has obvious photo-isomerization characteristics, and the configuration was successfully changed from trans to cis under 35 s of ultraviolet irradiation. In addition, under the alternating irradiation of 365 nm and 450 nm, the reversible photo-isomerization process can be switched multiple times without obvious fatigue phenomenon, as Figure 1 shown in D, indicating that compound 3a has good convertibility and great potential as a molecular switch.

[0082] Example 4 Preparation of Drug-Loaded Nanoparticles 3a@β-CD

[0083] Select a representative and highly active target compound 3a, weigh 18.86 mg of drug 3a, dissolve it in 1 mL of dimethyl sulfoxide (DMSO) to prepare a 0.03 M DMSO solution, dilute the solution to 0.15 mM with dd H2O, then add 34 mg of β-CD, dissolve it by ultrasonic treatment, and obtain nanoparticles by freeze-drying.

[0084] Characterize the prepared nanoparticles. The mass spectrum is obtained by a high-resolution mass spectrometer (UitiMate 3000, Thermo Scientific). Transmission electron microscopy (TEM) images are obtained on a FEI Talos F200C electron microscope (voltage: 200 kV) (FEI, U.S.A.). Ultraviolet-visible (UV-vis) spectra are obtained using a UV-1900 spectrophotometer (Shimadzu Co. Company, Japan).

[0085] The mass spectrometry results are as Figure 2 shown. The results show that the present invention successfully prepared 3a@β-CD by an environmentally friendly non-covalent self-assembly method and the binding is 1:1 (molar ratio). The titration curve and Jobs curve are as Figure 3 shown. The results show that the compound 3a and β-CD bind in a 1:1 ratio.

[0086] The transmission electron microscopy results are as Figure 4 shown. The results show that the present invention successfully prepared nanoparticles 3a@β-CD with a spherical structure by an environmentally friendly non-covalent self-assembly method, and the average particle size is about 1200 nm.

[0087] Example 5 Tests on Solubility, Leaf Surface Wettability and Stability of Drug-Loaded Nanoparticles

[0088] To analyze the solubility of the nanoparticles (3a@β-CD), the trans-azobenzene compound 3a, the cis-azobenzene compound 3a, the β-cyclodextrin-based nanoparticles (3a@β-CD) and the decomposed β-cyclodextrin-based nanoparticles (UV-3a@β-CD) were analyzed from a macroscopic perspective. As can be seen from Figure 5 A, the addition of the host molecule β-CD improves the water solubility of the molecule (see Figure 6 and Table 2), and the solution changes from turbid to clear. Under ultraviolet light irradiation, due to the size mismatch, the compound 3a is transformed from trans-3a to cis-3a'. 3a' slips out of the β-CD cavity, and the solution changes from clear in 3a@β-CD to turbid in UV-3a@β-CD.

[0089] Table 2

[0090]

[0091]

[0092] The wettability of nanoparticles (3a@β-CD) was evaluated by the dynamic contact angle method. Rice leaves cultivated in a separate greenhouse were fixed on a glass slide, and droplets of different solutions were dropped on the leaf surface using a micro syringe. The dynamic contact angle was detected with a JC-2000D contact angle meter after 10 s, and each solution was tested three times on different regions of the leaf.

[0093] The wettability of 3a@β-CD on tobacco leaves was monitored by the contact angle measurement method. In these experiments, a small contact angle indicates a lower probability of the droplet rolling off the leaf surface. As Figure 5 shown, compared with the contact angle of compound 3a dissolved in water (92.0°), the contact angle caused by 3a@β-CD encapsulation was significantly reduced (82.0°). Therefore, the spherical structure of 3a@β-CD improved the wetting effect of the hydrophobic drug molecule compound 3a on rice leaves, potentially promoting drug deposition, leaf absorption, bioavailability, and biocompatibility.

[0094] As Figure 7 shown, the stability of nanoparticles (3a@β-CD) was evaluated, its Zeta potential was tested, and it was found that the stability of nanoparticles (3a@β-CD) was higher than that of compound 3a, indicating that the preparation of nanoparticles (3a@β-CD) improved the stability of the system.

[0095] Example 6 Test on the Photo-responsive Release of Azobenzene Compound 3a from Drug-loaded Nanoparticles

[0096] As Figure 8 shown, with the irradiation of ultraviolet light, 3a@β-CD gradually dissociated to release cis-compound 3a, and the dissociation time was 1 min, which was nearly twice as slow as the inversion of compound 3a' to compound 3a', achieving the effect of sustained and controlled release.

[0097] Example 7 Crystal Violet Test on the Anti-biofilm Activity of the Photo-responsive Release of Azobenzene Compound 3a from Drug-loaded Nanoparticles in vitro

[0098] The formation of biofilm plays a crucial role in the early infection and colonization of Xoo. We conducted a quantitative crystal violet staining experiment to explore the formation of Xoo biofilm under the action of the compound. As Figure 9As shown, the results indicate that the compound and its complexes can significantly inhibit the formation of Xoo biofilm. Without complex treatment, Xoo cells form a dense and abundant biofilm. However, after treatment with different doses of 3a, 3a’, 3a@β-CD, and UV-3a@β-CD, the content of Xoo biofilm decreases. At higher compound concentrations, the formation of Xoo biofilm gradually decreases and shows concentration dependence.

[0099] Example 8 Fluorescence Microscopy Determination of the Anti-biofilm Activity of Azobenzene Compound 3a Released by Drug-loaded Nanoparticles in vitro

[0100] The crystal violet staining experiment shows that the constructed binary complex has good inhibitory activity against the formation of Xoo biofilm at sub-concentrations. As Figure 10 shown, Xoo live cells were stained green by green fluorescence staining method, which further verified this point. In the control group, the biofilm was arranged tightly, showing a dense sheet-like green fluorescence, and only a few free bacteria showed single-point green fluorescence. Samples treated with 3a, 3a’, 3a@β-CD, and UV-3a@β-CD also showed green fluorescence; however, the 50 smaller the EC value, the smaller the dense sheet-like fluorescence band formed by the biofilm, and the smaller the single-point green fluorescence formed by the free live cells. As the compound concentration increases, the dense sheet-like green fluorescence band corresponding to the biofilm gradually weakens, and the single-point green fluorescence band corresponding to the free cells gradually strengthens. This further indicates that the compound and the constructed binary complex can weaken the formation of Xoo biofilm without affecting the normal growth of Xoo.

[0101] Example 9 Pharmacological Experiment 1

[0102] EC 50 (median effective concentration) is an important indicator for evaluating the sensitivity of plant pathogens to compounds, and it is also an important parameter for setting the compound concentration when studying the action mechanism of target compounds. In the concentration gradient experiment, five appropriate concentrations were set by the two-fold dilution method. Finally, the inhibition rate of the medicament on plant pathogens and the medicament concentration were converted into logarithmic values, and the toxicity curve was obtained by regression analysis using SPSS software, and the EC 50 was calculated.

[0103] The turbidimetric method was used to test the median effective concentration EC 50, the test subjects were Xanthomonas oryzae pv. oryzae (Xoo), Xanthomonas citri subsp. citri (Xac), and Clavibacter michiganensis subsp. michiganensis (Cmm). DMSO was dissolved in the medium as a blank control. Xanthomonas oryzae pv. oryzae (the rice bacterial blight pathogen on M210 solid medium) was placed in NB medium and cultured in a constant temperature shaker at 28 °C and 180 rpm until the logarithmic growth phase for standby; Xanthomonas citri subsp. citri (on M210 solid medium) was placed in NB medium and cultured in a constant temperature shaker at 28 °C and 180 rpm until the logarithmic growth phase for standby; Clavibacter michiganensis subsp. michiganensis was placed in Cmm medium and cultured in a constant temperature shaker at 28 °C and 180 rpm until the logarithmic growth phase for standby. The medicament (compound) was prepared into 5 mL of toxic NB' liquid medium with different concentrations (e.g., 100, 50, 25, 12.5, 6.25 μg / mL) and added to test tubes. 40 μL of NB' liquid medium containing phytopathogenic bacteria was added respectively, and they were shaken in a constant temperature shaker at 28 - 30 °C and 180 rpm. The rice bacterial blight pathogen was cultured for 36 h, and Xanthomonas citri subsp. citri was cultured for 48 h. The OD 595 value of the bacterial liquid at each concentration was measured on a spectrophotometer, and the OD 595 value of the toxic sterile NB liquid medium at the corresponding concentration was also measured.

[0104] Among them, NB medium: water: 1 L, glucose: 10 g, peptone: 5 g, beef extract: 3 g, yeast powder: 1 g.

[0105] M210 solid medium: water: 1 L, glucose: 10 g, peptone: 5 g, beef extract: 3 g, yeast powder: 1 g, agar: 15 g.

[0106] Cmm medium: beef extract: 2 g, peptone: 10 g, yeast powder: 5 g, malt extract: 5 g, glycerol: 2 g, Tween 80: 50 mg, casein amino acids: 5 g, magnesium sulfate heptahydrate: 1 g, agar: 15 g, secondary water: 1 L.

[0107] Corrected OD value = OD value of the bacterial-containing medium - OD value of the sterile medium

[0108] Inhibition rate % = [(OD value of the bacterial liquid in the control medium after correction - OD value of the toxic medium after correction) / OD value of the bacterial liquid in the control medium after correction] × 100%

[0109] The examples of the present invention are used to illustrate the technical solutions of the present invention, but the content of the examples is not limited thereto. The experimental results of the target compounds and the complex 3a@β-CD are shown in Tables 3 - 6.

[0110] Table 3 EC of two configurations of azobenzene compounds containing isopropanolamine structure against Xanthomonas oryzae pv. oryzae 50

[0111]

[0112]

[0113] Table 4 EC of two configurations of azobenzene compounds containing isopropanolamine structure against Xanthomonas citri subsp. citri 50

[0114]

[0115]

[0116] Table 5 EC of two configurations of azobenzene compounds containing isopropanolamine structure against Clavibacter michiganensis subsp. michiganensis 50

[0117]

[0118]

[0119] Table 6 EC of complex 3a@β-CD before and after release and β-CD against Xanthomonas oryzae pv. oryzae 50

[0120]

[0121] It can be seen from Tables 3 - 6 that in the in vitro antibacterial test, both configurations of the target compounds showed good inhibitory activities against plant pathogenic bacteria (Xanthomonas oryzae pv. oryzae, Xanthomonas citri subsp. citri, and Clavibacter michiganensis subsp. michiganensis). The activities of the cis configurations of most compounds were better than those of the trans configurations, and both were better than those of the commercial drugs, bismerthiazol and thiodiazole copper. The lowest EC 50 against Xanthomonas oryzae pv. oryzae was 2.44 μg / mL and 0.95 μg / mL (compounds 3a and 3a'); the lowest EC 50 against Xanthomonas citri subsp. citri was 0.52 μg / mL and 0.39 μg / mL (compounds 3a and 3a'); the lowest EC 50 against Pseudomonas syringae pv. actinidiae was 15.63 μg / mL and 4.99 μg / mL (compounds 3l and 3l'); the in vitro antibacterial activity of complex 3a@β-CD against Xanthomonas oryzae pv. oryzae was slightly lower than that of compound 3a, but its antibacterial activity was still higher than that of thiodiazole copper and bismerthiazol. The above results indicate that azobenzene compounds containing isopropanolamine structure can be used to prepare pesticides against plant pathogenic bacteria.

[0122] Example 10 Pharmacological Experiment 2

[0123] Based on the in vitro antibacterial activity data of the target compounds, compound 3a was selected as the research drug for the in vivo antibacterial experiment on plants. The in vivo antibacterial activities of the target compound 3a, the complex molecule 3a@β-CD, the dissociated complex molecule UV-3a@β-CD, and the positive control drugs bismerthiazol and thiodiazole copper against Xanthomonas oryzae pv. oryzae were tested.

[0124] The leaf clipping method was used to test the biological activity of the drugs against Xanthomonas oryzae pv. oryzae in vivo. The specific method is as follows: Dip a sterilized scissors into the bacterial suspension containing Xanthomonas oryzae pv. oryzae (OD 595 = 0.8) and inoculate it on rice plants (rice variety: Fengyouxiangzhan, cultivation time: 8 weeks). For the protective activity, the 200 μg / mL liquid medicine (target compound 3a, complex molecule 3a@β-CD, UV-3a@β-CD, β-CD, and positive control drugs bismerthiazol and thiodiazole copper) was evenly sprayed on the rice leaves, and then the bacteria were inoculated after 24 hours; for the therapeutic activity, the bacteria were first inoculated on the rice plants, and then the 200 μg / mL liquid medicine was evenly sprayed on the rice leaves after 24 hours. After culturing the treated samples in an artificial climate chamber (temperature: 28°C, humidity: 90%) for 14 days, the corresponding control effects were obtained through the grading standard calculation method.

[0125] The grading standard is as follows:

[0126] First, measure the spot area of each leaf and the entire leaf area, and then calculate the leaf area by measuring the percentage of the entire spot area. Secondly, classify these leaves according to the following grade standards: Grade 1, the lesion area is less than 5% of the entire leaf area. Grade 3, the lesion area accounts for 6 - 10% of the whole leaf area; Grade 5, the lesion area 5 accounts for 11 - 20% of the whole leaf area; Grade 7, the lesion area accounts for 21 - 50% of the whole leaf area; Grade 9, the lesion area accounts for more than 50% of the entire leaf area;

[0127] The calculation method of the disease index is as follows:

[0128] Disease index = ∑(number of leaves at each grade × corresponding grade) / (total number of leaves × highest grade)

[0129] The calculation method of the control effect is as follows:

[0130] Control effect % = (disease index of the control group - disease index of the treatment group) / disease index of the control group × 100%

[0131] The examples of the present invention are used to illustrate the technical solutions of the present invention, but the content of the examples is not limited thereto. The experimental results of the target compounds are shown in Table 7 and Figure 11 as shown.

[0132] Table 7 Control effects of complex 3a@β-CD, compound 3a, β-CD, BT, and TC against Xanthomonas oryzae pv. oryzae

[0133]

[0134] a Negative control group. b Analysis of variance was used for statistical analysis, assuming equal variances (P>0.05) and not assuming equal variances (P<0.05). Different capital letters indicate significant differences (P<0.05) among the treatment groups.

[0135] As can be seen from Table 7: Compounds 3a, 3a', complex 3a@β-CD, and UV-3a@β-CD showed good protective activities (41.54%, 46.98%, 51.22%, 55.84%) and therapeutic activities (36.83%, 43.34%, 48.37%, 52.05%) against Xanthomonas oryzae pv. oryzae, and their control effects were better than those of the commercial drugs bismerthiazol (35.06%, 31.86%) and thiodiazole copper (31.74%, 27.03%); the cis configuration of compound 3a was superior to its trans configuration 3a', and the control effects of complex 3a@β-CD and UV-3a@β-CD were better than those of compounds 3a and 3a', indicating that the introduction of β-cyclodextrin in this system could improve the bioavailability of the drug and thus enhance its in vivo antibacterial activity; in addition, UV-3a@β-CD was superior to complex 3a@β-CD, indicating that the photo-responsive stimulation could dissociate the complex, achieve sustained release and control release, and reach the maximum antibacterial ability, continuously release small drug molecules, and achieve persistent antibacterial activity.

[0136] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. An azobenzene compound containing an isopropanolamine substructure, characterized in that: It is a compound represented by formula (I) or a salt thereof, wherein R1 and R2 are methyl, ethyl, propyl, 2-F-benzyl, 3-F-benzyl, 4-F-benzyl, 3-OCH3-benzyl, 2-OCH3-benzyl, 4-methyl-benzyl respectively; or when R1 and R2 are connected to form a ring, they are the following groups:

2. The preparation method of the azobenzene compound containing an isopropanolamine substructure according to claim 1, characterized in that: The reaction equation is as follows:

3. A composition, characterized in that: It includes the azobenzene compound containing an isopropanolamine substructure described in claim 1 or the azobenzene compound containing an isopropanolamine substructure prepared in claim 2.

4. A method for preparing a nano-pesticide using the azobenzene compound containing an isopropanolamine substructure described in claim 1 or the azobenzene compound containing an isopropanolamine substructure prepared according to claim 2, characterized in that: Prepare a dimethyl sulfoxide solution of the azobenzene compound containing an isopropanolamine substructure, add cyclodextrin, dissolve it by ultrasonic treatment, and then freeze-dry to obtain nanoparticles. The cyclodextrin is β-cyclodextrin.

5. The preparation method according to claim 4, characterized in that: The molar ratio of the cyclodextrin to the azobenzene compound containing an isopropanolamine substructure is 1:

1.

6. The nano-pesticide prepared by the preparation method described in claim 4 or 5.

7. The application of the azobenzene compound containing an isopropanolamine substructure described in claim 1 or the azobenzene compound containing an isopropanolamine substructure prepared in claim 2 or the composition described in claim 3 or the nano-pesticide prepared by the preparation method described in claim 4 or 5 or the nano-pesticide described in claim 6 in the prevention and control of agricultural pests and diseases; the agricultural pests and diseases are rice bacterial blight, citrus canker, and tomato canker.

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