CN110283140B

CN110283140B - Light-operated plant disease control agent containing 1,3, 4-oxadiazole azobenzene and application thereof

Info

Publication number: CN110283140B
Application number: CN201910548254.XA
Authority: CN
Inventors: 何道航; 帅建波
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2019-06-24
Filing date: 2019-06-24
Publication date: 2022-01-18
Anticipated expiration: 2039-06-24
Also published as: CN110283140A

Abstract

The invention belongs to the field of pesticide bactericides and discloses a light-operated plant disease control agent containing 1,3, 4-oxadiazole azobenzene and application thereof. The control agent has a structure shown as a chemical structural formula I, wherein X is F, Cl or Br; the synthesis method comprises the steps of oxidation cyclization, reduction, diazotization coupling and methylation reaction in sequence. The structure can generate photoisomerization after being irradiated by ultraviolet light, and the trans-configuration with low activity is converted into the cis-configuration with high activity, so that the drug activity can be regulated and controlled as required. After stopping light stimulation, the high-activity cis-structure can automatically weaken the activity along with the prolonging of time, and the accumulation of the active medicine in the environment can be reduced. The invention can overcome the defects of the traditional chemical pesticide bactericide which has high residue and is easy to generate drug resistance.

Description

Light-operated plant disease control agent containing 1,3, 4-oxadiazole azobenzene and application thereof

Technical Field

The invention discloses a light-operated plant disease control agent containing 1,3, 4-oxadiazole azobenzene, a preparation method thereof and application thereof in light-operated resistance to plant pathogenic bacteria, belonging to the field of light-operated pesticide bactericides.

Background

Fungal diseases have always been one of the most important factors limiting agricultural production, with crop yield losses caused by phytopathogenic fungi of up to 20% per year and further losses of 10% after harvest (Science,2018, 360(6390): 739-. The use of chemical drugs is the most important means for controlling fungal diseases of crops at present. Generally, once applied, chemical pesticides have no conscious control of their activity, such that they still accumulate in the environment at high doses of the active ingredient after exerting a bactericidal effect. However, the constant accumulation of active drugs in the environment leads to many adverse side effects, such as resistance, toxicity to beneficial organisms, food safety, environmental pollution, etc. Therefore, how to skillfully design the pesticide bactericide molecules with controllable activity and accurately control the activity of the pesticide bactericide molecules to ensure that the pesticide bactericide molecules do not affect other environments after exerting the pesticide effect is of great significance for safe application of the pesticide molecules and slowing down the generation of resistance.

In the field of medicine, the biological activity of drug molecules is controlled by using an optical switch, so that the environmental toxicity of the drug and the drug resistance of pathogenic bacteria can be reduced, and the precise control of the drug activity can be realized (Nature Chemistry,2013, 5(11): 924-928). The idea has been remarkably developed in the field of medicine, such as light-controlled antibiotics, light-controlled antitumor drugs, light-controlled enzyme inhibitors, and the like. Therefore, the optical switch is introduced into the active group of the pesticide bactericide, and the design of the optical controllable pesticide molecules can certainly promote the development of environment-friendly pesticides, thereby effectively solving the defects of high residue, easy generation of resistance and the like of the existing pesticides.

1,3, 4-oxadiazole is a nitrogen-containing and oxygen-containing heterocyclic group with various biological activities, is an important component in a plurality of pharmaceutically active molecular structures, and is also widely applied to the structural design of pesticide bactericide molecules. Azobenzene and its derivatives have been attracting attention as a photoswitch. Because of the advantages of easy synthesis, high photoisomerization efficiency, rapid reversible conversion between isomers and the like, azobenzene or derivatives thereof are widely applied to the biological function regulation and control of active molecules as photoswitches. However, there is no report on the regulation of the activity of azobenzene-containing derivatives against plant pathogenic fungi.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and introduces a light control thought into the design of agricultural antibacterial molecules. The 1,3, 4-oxadiazole group of the antibacterial active pharmacodynamic fragment is combined with the photoswitch azobenzene group to synthesize the 1,3, 4-oxadiazole azobenzene derivative with a light-operated structure. The natural factor-light is used to control the opening and closing of the activity of the compound, and the precise and controllable activity of the pesticide antibacterial molecules is realized.

In order to achieve the above object, the present invention provides a 1,3, 4-oxadiazole azobenzene-containing light-controlled plant disease control agent, namely, (3, 4-dimethoxy-phenyl) - {4- [5- (4-X-phenyl) - [1,3,4] oxadiazol-2-yl ] -phenyl } -diazene, having a chemical structural formula as shown below:

wherein X is F, Cl or Br.

The invention also provides a preparation method of the control agent, which takes 4-nitro-benzoic acid (4-X-benzylidene) -acylhydrazone as a raw material to prepare the light-operated plant disease control agent containing 1,3, 4-oxadiazole azobenzene through an oxidation cyclization reaction, a reduction reaction, a diazotization coupling reaction and a methylation reaction. The method specifically comprises the following steps:

the light-operated plant disease control agent containing 1,3, 4-oxadiazole azobenzene is used for light-operated control of plant pathogenic fungi and bacterial diseases. Wherein the fungi include Botrytis cinerea, Fusarium graminearum and Magnaporthe grisea; the bacteria include citrus canker bacteria.

The light control conditions used in the invention are as follows: the wavelength of the light source is 365-380 nm; the illumination time is once every 4-6 h, and each illumination time is 2-5 min.

Compared with the prior art, the invention has the following beneficial effects:

1. the control agent of the invention has simple preparation, easily obtained raw materials and mild reaction conditions, and is suitable for popularization and application.

2. The control agent obtained by the invention can generate photoisomerization after being irradiated by ultraviolet light, and is converted from a trans-configuration with low activity into a cis-configuration with high activity, namely, the activity of the medicament can be controlled by the irradiation of the ultraviolet light, so that the control of medicament molecules on the aspect of controlling crop diseases can be realized according to the requirements.

3. The preventing and treating agent prepared by the invention can automatically weaken the activity after stopping light stimulation, and the high-activity cis-structure is spontaneously converted into the low-activity trans-structure along with time, thereby having positive effects on reducing the accumulation of active drugs in the environment and weakening the drug resistance of pathogenic microorganisms. Overcomes the defects of high residue, high pollution, easy generation of drug resistance and the like of the common active bactericide.

Drawings

FIG. 1 is a nuclear magnetic resonance spectrum of the preventive agent of the present invention, wherein (a) a hydrogen spectrum and (b) a carbon spectrum.

FIG. 2 is a high-resolution mass spectrum of the preventive agent of the present invention.

Fig. 3 shows the changes of the ultraviolet-visible spectrum and the hydrogen spectrum of nuclear magnetic resonance before and after the light irradiation of the control agent of the invention, wherein (a) the ultraviolet-visible spectrum and (b) the hydrogen spectrum.

FIG. 4 is an in vitro inhibitory activity of a control agent of the present invention against Botrytis cinerea, wherein (a) a lighted blank, (b) a lighted 200 μ g/mL control agent, (c) a lighted 100 μ g/mL control agent, (d) an unlighted blank, (e) an unlighted 200 μ g/mL control agent, and (f) an unlighted 100 μ g/mL control agent.

FIG. 5 is a graph of the in vivo control efficiency of the inventive control agents against Botrytis cinerea, wherein (a) a blank control of light, (b) no light control agent treatment, and (c) light control agent treatment.

Detailed Description

The invention is further described with reference to the following specific embodiments and the accompanying drawings. It should be noted that the embodiments described below are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the invention without making any inventive step, shall fall within the scope of protection of the claims of this application.

Example 1: preparation of 2- (4-fluoro-phenyl) -5- (4-nitro-phenyl) - [1,3,4] oxadiazole,

oxidation cyclization reaction: in a 150mL two-necked round-bottomed flask, 2g (6.96mmol) of 4-nitro-benzoic acid (4-fluoro-benzylidene) -acylhydrazone as a starting material and 30mL of absolute ethanol were added, and the mixture was stirred at 70 ℃ until dissolved. 9.8g (34.82mmol) of chloramine T (5g +4.8g, 2h apart) were added in two portions and stirred at 80 ℃ under reflux. And (3) monitoring the reaction by a point plate, after the reaction is finished, filtering to remove impurities while the reaction is hot, taking the filtrate, evaporating the solvent under reduced pressure, adding a small amount of distilled water for washing, filtering, washing, recrystallizing the obtained crude product with EtOH, and drying to obtain 1.23g of yellow powdery solid with the yield of 62%.

Example 2: preparation of 4- [5- (4-fluoro-phenyl) - [1,3,4] oxadiazole-2-yl ] -aniline,

reduction reaction: 2g (7.01mmol) of the product obtained in example 1 was put in a 150mL flask, 15mL of an aqueous solution of CaCl2 (8g/50mL of water) and a solvent DMF50mL were added and stirred until dissolved, 6.85g (105.2mmol) of zinc powder was added, and the resulting mixture was refluxed at 100 ℃ for 8 hours. The reaction solution was filtered while hot, the filtrate was poured into a beaker containing 100mL of ice water, placed in a refrigerator (5 ℃ C.) and left to stand overnight, filtered to give a crude product, which was then recrystallized from a mixed solvent of DMF/ethanol to give 1.52g of a yellow solid in 85% yield.

Example 3: preparation of (3, 4-dimethoxy-phenyl) - {4- [5- (4-fluoro-phenyl) - [1,3,4] oxadiazol-2-yl ] -phenyl } -diazene,

1. diazotization coupling reaction: 200mg (0.78mmol) of the product obtained in example 2 was taken, 2.5mL of HCl (1M) and 5mL of water were added, and the mixture was stirred at 50 ℃ until dissolved. Then transferring to 0-5 ℃ ice salt bath, slowly dripping NaNO₂Aqueous solution (57mg in 5mL water) and the resulting mixture was allowed to react at this temperature for 1h to complete the diazotization. The resulting diazonium salt was added dropwise to an ethanolic solution containing o-methoxyphenol (107mg,0.86mmol in 5mL ethanol) at 0-5 deg.C. After the dropwise addition, the pH of the solution is adjusted to 8-9 by using 1M NaOH, and the obtained mixed solution is continuously invertedThe coupling reaction should be completed in 2 h. Filtering, washing with water for 2 times, and drying to obtain the azophenol.

2. Methylation reaction: 200mg (0.52mmol) of the azophenol product obtained in step 1 is dissolved in 20mL of THF solvent, and K is added under stirring₂CO₃258mg (1.87mmol) and iodomethane (380 mg,2.67mmol) were reacted at room temperature for 3 days. The solvent is removed by rotary evaporation, the crude product is dissolved by 20mL of DCM, and then is washed by saturated sodium bicarbonate and saturated saline in turn, after being dried by anhydrous magnesium sulfate, the yellow solid 157mg of the final product is obtained by column chromatography separation, and the yield is 75%.¹H NMR(400MHz,DMSO-d₆)δ8.31 (d,J＝8.5Hz,Ph-H,2H),8.22(dd,J＝8.7,5.4Hz,Ph-H,2H),8.05(d,J＝8.5Hz, Ph-H,2H),7.68(dd,J＝8.5,2.1Hz,Ph-H,1H),7.49(t,J＝8.8Hz,Ph-H,3H), 7.21(d,J＝8.6Hz,Ph-H,1H),3.89(d,J＝7.7Hz,OCH₃-H,6H).¹³C NMR(101 MHz,DMSO-d₆)δ164.04,150.02,146.65,128.41,125.13,123.61,121.98,117.08, 111.80,102.33.HRMS(ESI),m/z calcd for C₂₂H₁₈FN₄O₃[M+H]⁺405.1357；found, 405.1354。

Example 4: photoisomerization of the inventive control Agents

1. Ultraviolet spectrum determination: the control agent obtained in example 3 was dissolved in DMSO to prepare a solution to be tested at a concentration of 30 μ M. And 3mL of the prepared solution to be detected is added into a quartz cuvette, and an ultraviolet-visible absorption spectrum of the target molecule is recorded by an ultraviolet spectrophotometer. Then irradiating the solution with ultraviolet light source (365nm) for 2min to isomerize the structure of the preventing and treating agent, and recording the ultraviolet visible absorption spectrum of the solution with an ultraviolet spectrophotometer. And finally, placing the mixture in a room under natural light conditions, and recording the ultraviolet visible absorption spectrum of the control agent molecular solution by using the spectrophotometer after 12 hours.

As shown in fig. 3 (a), after the control agent prepared by the present invention is irradiated by ultraviolet light, the intensity of the pi-pi transition absorption peak at 388nm is obviously reduced, which indicates that the control agent of the present invention is isomerized from trans to cis configuration. Then the composition is placed under the natural light condition, and the pi-pi transition absorption peak at 388nm gradually recovers towards the peak position before the light irradiation along with the time extension, thereby illustrating the reversibility of the photoisomerization process of the control agent.

2. Nuclear magnetic hydrogen spectrum determination: dissolving the control agent obtained in the embodiment in deuterated DMSO to prepare a solution to be detected of 10mg/mL, recording the nuclear magnetic spectrum of a target molecule by using a nuclear magnetic resonance spectrometer, irradiating the solution for 2min by using an ultraviolet light source (365nm) to enable the solution to reach a cis-form photostability, and recording the nuclear magnetic spectrum again.

As shown in fig. 3 (b), after 2min of ultraviolet irradiation, the intensity of the original signal peak of the compound is weakened, and a new hydrogen atom signal peak appears in the high field direction near the original signal peak, which also indicates that the control agent of the present invention has cis-trans isomerism after being irradiated by ultraviolet light, and a cis-structure of the control agent molecule is generated.

Example 5: the control agent obtained by the invention is used for measuring the light-operated in-vitro activity of plant pathogenic bacteria

In the test example, the inhibitory activity of the control agent on fungi (botrytis cinerea, fusarium graminearum and pyricularia oryzae) and bacteria (citrus canker pathogen) was measured by a hypha growth rate method and a turbidity method, respectively.

1. Fungi

The experimental method comprises the following steps: the control agent of the present invention was dissolved in a small amount of DMSO (final concentration of 0.5%, v/v) and the mother liquor was diluted with 0.1% Tween aqueous solution to a final concentration of 2000, 1000. mu.g/mL. 1mL of the liquid medicine with each concentration is respectively transferred by a liquid transfer gun and added with 9mL of melted PDA culture medium to be uniformly mixed, and the mixture is poured into a disposable culture dish (9cm) to prepare a medicine-containing flat plate. A cake with a diameter of 5mm is taken from the edge of Botrytis cinerea hyphae cultured on PDA for one week, and placed in the center of a solidified drug-containing plate with the hyphae facing downwards. The inoculated plate was transferred to a constant temperature incubator and incubated at 25 ℃. And when the blank control hyphae grow over the whole flat plate, measuring the hyphae growth diameter on each flat plate, and calculating the inhibition rate of each treatment group on the hyphae growth.

Light control conditions: the treatment is repeated in three groups, the first group is treated without light, and the second group is irradiated with 365nm ultraviolet light for 2min before the drug-containing plate is inoculated. The third group was illuminated every 6h (2 min each) on the basis of the second group. The same dose of DMSO was used as a blank control, and the light treatment was the same as the drug treatment.

The test results are shown in fig. 4, and by combining fig. 4 and table 1, it can be concluded that the control agent of the present invention shows obvious photoactivation every 6h of light. Taking Botrytis cinerea as an example, the activity of the light group is improved by about 4 times every 6 hours compared with the activity of the non-light group. Meanwhile, the antibacterial activity of the photoactivatable target molecules is far less than that of target molecules subjected to multiple times of illumination, which shows that the photoactivatable drug molecules can spontaneously convert to prototype molecules with low activity after stopping illumination stimulation, and a thought is provided for controlling the activity of pesticide molecules according to needs. When the drug effect is required, the drug activity can be activated by light. After the drug effect is exerted, the active molecules in the environment can spontaneously weaken the activity, the property can greatly reduce the residue of the active drug molecules in the environment, and the drug has positive effects on solving the problems of microbial drug resistance, food safety and environment.

TABLE 1 light-controlled in vitro antibacterial Activity of the inventive control Agents

2. Bacteria

The experimental method comprises the following steps: after dissolving the control agent of the invention in a small amount of DMSO, the control agent is diluted by NB culture solution containing 0.1% Tween to a final concentration of 200. mu.g/mL and 100. mu.g/mL. Adding 100 μ L of each concentration liquid medicine into 96-well plate, and adding 100 μ L of fresh bacterial liquid (OD)₅₉₅0.1), three replicates per concentration were made. The 96-well plate was placed in a constant temperature incubator and incubated at 28 ℃. After 24 hours of incubation, the final OD was measured and the inhibition at each concentration was calculated.

Light control conditions: three groups of experiments are repeated, wherein one group is not subjected to illumination treatment, and the second group of drug mother liquor is irradiated for 2min by an ultraviolet light source (365nm) before being added into the pore plate and then inoculated with bacteria; the third group was illuminated every 6h (2 min each) on the basis of the second group.

From table 1, it is understood that the control agent of the present invention exhibits a significant photoactivation phenomenon against citrus canker pathogens. At a concentration of 100. mu.g/mL, the inhibitory activity of the light group (once every 6 h) against the citrus canker pathogen increased by 17.2% compared to the non-light group. The activity of the treatment group irradiated once before inoculation is increased by only 2.7 percent compared with the non-irradiated group, which shows that the activity of the culture medium containing the medicine is gradually reduced after the irradiation is stopped.

Example 6: the control agent obtained by the invention has light-operated in-vivo control efficiency on botrytis cinerea

In the test example, the in vivo antibacterial activity of the control agent of the present invention against botrytis cinerea was measured by a tomato live transfer method.

The experimental method comprises the following steps: according to Standard of Operation Protocol (SOP) for measuring biological activity of pesticides, a control agent is dissolved in a small amount of DMSO, and then 0.1% Tween aqueous solution is used for diluting to obtain a liquid medicine with the concentration of 200 mug/mL. Fresh tomato fruits were disinfected by wiping the surface with 75% alcohol and rinsed clean with sterile water. After the residual flushing liquid on the surface of the fruit is naturally air-dried, the liquid medicine is uniformly sprayed on the surface of the fruit and is air-dried under natural conditions. Pricking epidermis with diameter of 5mm in the middle of the fruit with a sterilized inoculating needle, and clamping the Botrytis cinerea cake with diameter of 5mm on the surface of the wound with sterilized forceps. The inoculated fruit was then incubated in an incubator at 25 ℃ and 90% relative humidity. And after the hyphae of the blank control group fully grow, measuring the growth diameter of the hyphae treated by each medicament, and calculating the control efficiency of each medicament treatment.

Light control conditions: two groups of drug treatments were set for this experiment, one group was irradiated with a 380nm UV light source (5min each time every 4 h) and the other group was not irradiated with light. The blank was treated with the same dose of solvent and with the same light.

TABLE 2 light-controlled in vivo control efficiency of the inventive control agents on Botrytis cinerea

Serial number	Control Effect (%, 200. mu.g/mL) on Botrytis cinerea
		I-no illumination	25.8
I-illumination	83.3

As shown in FIG. 5, it is understood from the results of the tests in combination with FIG. 5 and Table 2 that after 5 days of incubation at constant temperature and humidity, the surface of the tomato of the blank group was almost covered with Botrytis cinerea hyphae under light conditions. The growth of botrytis cinerea hyphae on the surfaces of tomatoes sprayed with the medicine without illumination is slightly weaker than that of blank control, and the control effect on the botrytis cinerea hyphae is 25.8% by calculation of the control effect. However, after the spraying treatment of the medicine is carried out once every 4h of illumination (every 5min), the growth of botrytis cinerea is obviously inhibited, and the control effect reaches 83.3 percent, which shows that the compound has a remarkable light-operated control effect on tomato botrytis cinerea.

In the field of medicine, the study of combining antibacterial active fragment quinolone with photoswitch azobenzene to form photoisomerizable azoquinolone is carried out in the early stage. The structure can obviously improve the in vitro antibacterial activity to human pathogenic bacteria under the stimulation of ultraviolet light and can be gradually inactivated after the stimulation is stopped (Nature Chemistry,2013, 5(11): 924-928). The light-operated control agent can obviously improve the antibacterial activity to plant pathogenic fungi and bacteria under the stimulation of ultraviolet light, and can spontaneously weaken the activity after the stimulation is stopped. The method also provides a new idea for the practical application possibility of the light-operated control agent in the field of pesticide bactericides. The invention is suitable for the greenhouse for planting the fruits and the vegetables, the light source of which can be controlled.

Claims

1. A light-operated plant disease control agent containing 1,3, 4-oxadiazole azobenzene is characterized by having the following chemical structural formula:

wherein X is F, Cl or Br.

2. Use of the controlling agent according to claim 1 for controlling fungal and bacterial diseases of plants.

3. Use according to claim 2, characterized in that the control agent is a light control agent.

4. Use according to claim 3, wherein the light-controlled light source is ultraviolet light.

5. The use according to claim 3, wherein the light source wavelength of the light control comprises 365-380 nm.

6. Use according to claim 3 or 4 or 5, characterized in that the conditions of the light control are: the illumination time is once every 4-6 h, and each illumination time is 2-5 min.

7. The use of claim 6, wherein the plant fungi include Botrytis cinerea, Fusarium graminearum, and Magnaporthe grisea; the plant bacteria comprise citrus canker bacteria.