CN108299307B - Compound, preparation method and bacteriostatic application thereof - Google Patents

Abstract

The invention relates to a compound and a preparation method and application thereof, in particular to a preparation method of a novel medicament for inhibiting pestalotiopsis, peronophythora litchi and peanut black rot. The naphthylacetic acid, bromoethanol and carbendazim raw medicines are used as synthetic raw materials and react to obtain the naphthylacetic acid-carbendazim coupling compound, namely the final product. The invention also discloses nuclear magnetic resonance structural data, a hydrogen spectrogram and a carbon spectrogram of the novel compound. The compound has good bacteriostatic action on Pestalotiopsis, Peronophythora litchi and peanut black rot.

Description

Compound, preparation method and bacteriostatic application thereof

Technical Field

The invention belongs to the field of pesticides, and particularly relates to a compound and a preparation method and application thereof.

Background

Research shows that the carbendazim has certain inhibiting effect on Pestalotiopsis, Peronophythora litchi and peanut black rot due to its broad spectrum. The Pestalotiopsis fungus (Pestalotiopsis) is a kind of widely distributed plant pathogenic bacteria, has large harmfulness and high incidence, can parasitize approximately fifty-poly plants, shows symptoms of leaf spot, rot, ulcer and the like after the plants are damaged, and seriously affects the yield and the quality of products. The pathogen exists in parasitic, saprophytic and endogenous species. The host plants of endophytic pestalotiopsis are very wide, including Pinaceae, Cunningaceae, Orchidaceae, Palmaceae, Theaceae, etc. Research shows that carbendazim has good performance on the loquat gray spot disease caused by pestalotiopsis when indoor toxicity is measured, but the field control effect is general, and the reason is probably that the field drug resistance is generated due to long-term application of the carbendazim in the field.

Peronophythoralitchi (Peronophythoralitchi) causes serious litchi downy mildew in China, which occurs from the flowering phase to the fruit picking phase, particularly the fruit is most seriously damaged, and the losses of flower drop, fruit drop and fruit rot can reach 30-70%. During the storage period, the disease can continue to develop, and the storage, transportation and export sales of fresh litchi fruits are seriously influenced. In the face of the increasingly severe peronophythora litchi, fruit growers have to use multiple medicines together, and the control is enhanced by improving the application frequency and concentration, so that the pathogenic drug resistance is improved, the drug effect is reduced, the production cost is increased, the consequences and risks such as pesticide residue and the like are caused, and the healthy development of the litchi industry is restricted. Researches show that carbendazim has systemic and therapeutic effects on the disease, but has only moderate prevention effect, and is a medicament which is very easy to generate resistance after being used for decades.

Peanut black rot is caused by infection with red shell fungus (Calonectria ilicifola Boedijin & Reitsma). It is reported that no suitable medicament for preventing and treating peanut black rot exists at present, and no high-resistance variety can be selected. The peanut black rot pathogen not only causes destructive damage to peanuts, but also has damage to other various crops such as soybeans, blueberries, alfalfa, cowpeas and the like. When the black rot of peanut is harmful to peanut, it will infect underground tissue. The root tip is infected first, and then the main root and the hypocotyl become black and necrotic. Infected young and adult plants will wilt and die. As a broad-spectrum bactericide, the carbendazim has a certain inhibiting effect on the peanut black rot bacteria through indoor research, but the field effect is obviously reduced, and the carbendazim can be related to the resistance generated by long-term application of the pesticide in the field. In view of the characteristics of carbendazim, on the basis of keeping the bactericidal effect of the carbendazim, the carbendazim is coupled with naphthylacetic acid, the structure is modified to obtain a novel compound naphthylacetic acid-carbendazim, and the coupler is transported to the original position, namely the root tip of the peanut injury by utilizing the polar directional conduction characteristic of the naphthylacetic acid, so that the aim of directional action is fulfilled.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, provides a compound with remarkable bacteriostatic effect and economy, and also provides a preparation method and bacteriostatic application thereof.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the first technical scheme is as follows:

in one aspect, the invention provides a compound of formula (I):

the second technical scheme is as follows:

in a further aspect of the invention there is provided a bacteriostatic agent comprising a compound of formula (I):

the third technical scheme is as follows:

in a further aspect the invention provides a process for the preparation of a compound of formula (I):

1) esterification of naphthylacetic acid (II) and bromoethanol (III) gives compounds of the formula (IV):

2) reacting a compound of formula (IV) with carbendazim (V) under basic conditions to obtain a compound of formula (I):

as a further development of the invention, a catalyst is also present in step 1), said catalyst being selected from the group consisting of N, N' -Dicyclohexylcarbodiimide (DCC), EDCI, DIC, DMAP and HOBT.

As a further improvement of the invention, the method specifically comprises the following steps:

1) adding 10mmol of naphthylacetic acid, 10mmol of N, N' -Dicyclohexylcarbodiimide (DCC), catalytic amount of 4-Dimethylaminopyridine (DMAP) and 12mmol of bromoethanol into 30mL of dichloromethane, stirring at room temperature for 2h, adding water after the reaction is finished, extracting, concentrating, and carrying out column chromatography to obtain a colorless intermediate compound (IV);

2) adding 5mmol of carbendazim original drug into 20mL of dimethylformamide, stirring at room temperature, adding 7.5mmol of cesium carbonate, stirring for 30min, cooling to 0 ℃, dropwise adding 30mL of N, N-dimethylformamide solution dissolved with 6mmol of intermediate compound (IV), slowly heating to room temperature, stirring for reaction, after the reaction is finished, adjusting the pH value to be neutral by using 3mol/L HCl solution, extracting with ethyl acetate, and carrying out column chromatography to obtain naphthylacetic acid-carbendazim.

The technical scheme is as follows:

in the last aspect, the invention provides application of the compound shown in the formula (I) in bacteriostasis, sterilization and disinfection of pestalotiopsis, peronophythora litchi and peanut black rot pathogen.

Further, the compound of formula (I) is used for inhibiting.

The basic conditions described herein are selected from inorganic bases, organic bases, or any combination thereof, present in any suitable concentration, wherein the inorganic base is selected from sodium hydroxide, sodium carbonate, potassium carbonate, cesium carbonate, potassium bicarbonate, sodium bicarbonate, and disodium hydrogen phosphate, preferably cesium carbonate, potassium carbonate, or sodium carbonate; the organic base is selected from triethylamine, pyridine, diisopropylethylamine, diisopropylamine, ammonia water, preferably ammonia water.

The inventors of the present application have conducted a great deal of research work in order to find a novel bacteriostatic agent that can replace carbendazim, and have attempted to structurally modify carbendazim in order to overcome the resistance of germs to carbendazim. In addition, considering that the initial disease site of peanut black rot is at the root of the plant, the root tip is the initial site of the damage. The auxin is an important carrier, and the auxin comprises naphthylacetic acid (NAA), indolebutyric acid and the like, and has the effects of directional transmission, plant growth promotion and the like. The inventors have attempted to achieve bacteriostatic effects by incorporating naphthalene acetic acid into roots with directional conductivity, and the results of such attempts have been enjoyable and successful.

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

the compound has obvious inhibition effect on pathogenic bacteria such as pestalotiopsis, peronophythora litchii, peanut black rot and the like. The naphthylacetic acid-carbendazim with the concentration of 100 mu g/mL has better inhibition effect on three germs, the inhibition rate reaches more than 85%, and the inhibition effect of 25 mu g/mL medicament on Pestalotiopsis and Peronospora litchi is more than 90%. Compared with the original carbendazim, the compound keeps the bactericidal property of the carbendazim and has the function of conducting to roots. The invention has the advantages of less raw materials and economy. The compound belongs to a novel medicament, the resistance of the compound is negligible, and the action site of the compound is not only limited to leaves but also can be conducted to roots.

Drawings

FIG. 1 shows the preparation of the compounds of formula (I) according to the invention¹H NMR spectrum;

FIG. 2 shows the preparation of the compounds of formula (I) according to the invention¹³A C NMR spectrum;

in the attached figure 3, 1-a, 1-b and 1-c are respectively 100 mug/mL, 50 mug/mL and 25 mug/mL naphthylacetic acid-carbendazim bacteriostatic diagrams for pestalotiopsis bacteria, and figure 1-d is a blank control for pestalotiopsis bacteria;

in figure 4, 2-a, 2-b and 2-c are respectively 100. mu.g/mL, 50. mu.g/mL and 25. mu.g/mL naphthylacetic acid-carbendazim bacteriostasis diagrams for Phytophthora litchi Fr, and figure 2-d is a blank control for Phytophthora litchi Fr;

in the attached figure 5, 3-a, 3-b and 3-c are respectively 100 mug/mL, 50 mug/mL and 25 mug/mL naphthylacetic acid-carbendazim bacteriostatic diagrams for peanut black rot germs, and figure 3-d is a blank control of peanut black rot germs.

Detailed Description

The present invention will be described in further detail with reference to specific examples.

EXAMPLE 1 preparation of the Compound of formula (I)

10mmol of naphthylacetic acid (II), 10mmol of N, N' -Dicyclohexylcarbodiimide (DCC), a catalytic amount of 4-Dimethylaminopyridine (DMAP) and 12mmol of bromoethanol (III) were added to 30mL of dichloromethane and stirred at room temperature for 2 hours. After the reaction is finished, adding more water for extraction, concentrating, and carrying out column chromatography to obtain a colorless intermediate compound (IV).

Adding 5mmol of carbendazim original drug (V) into 20mL of dimethylformamide, stirring at room temperature, adding 7.5mmol of cesium carbonate, stirring for 30min, cooling to 0 ℃, dropwise adding 30mL of N, N-Dimethylformamide (DMF) solution containing 6mmol of intermediate compound (IV), slowly heating to room temperature, and stirring for 36 h. After the reaction is finished, adjusting the pH value to be neutral by using 3mol/L HCl solution, extracting by using ethyl acetate, and carrying out column chromatography to obtain a white solid compound, namely the naphthylacetic acid-carbendazim, of the formula (I).

Nuclear magnetic data of a compound of formula (I):¹H NMR(500MHz,CDCl₃)δ10.82(s,1H),7.87(d,J＝8.3Hz,1H),7.83(d,J＝7.7Hz,1H),7.74(d,J＝8.2Hz,1H),7.62(t,J＝7.4Hz,1H),7.41-7.48(m,2H),7.36(dd,J＝8.6,7.3Hz,1H),7.33–7.28(m,1H),7.25–7.17(m,3H),4.47(s,4H),3.97(s,2H),3.77(d,J＝14.2Hz,3H).

¹³C NMR(125MHz,CDCl₃)δ171.46,155.12,148.36,140.71,133.75,132.22,132.03,130.13,128.65,128.01,127.97,126.29,125.70,125.36,123.67,122.07,121.83,118.03,110.30,62.20,53.87,44.75,38.90.

effect example 1 indoor bacteriostatic test for Pestalotiopsis bacteria

0.01g of the compound of formula (I) obtained in example 1 was added to 10mL of sterile water (containing 1mL of ethanol) and, after sufficient dissolution, 10 was obtained³mu.g/mL of the mother liquor. Adding 5mL of the mother liquor into 45mL of culture medium, fully shaking until alcohol is volatilized, averagely pouring the mother liquor into three culture dishes, respectively inoculating 5 mm-diameter pestalotiopsis pathogen fungus blocks, measuring the diameters of the bacterial colonies after culturing for 72h, and calculating the average value of the bacteriostasis rate to obtain the effect of 100 mu g/mL of the medicament on the pathogens. 2.39mL, 1.15mL, 0.57mL, 0.28mL, 0.14mL of the mother liquor were added to 45mL of the culture medium, and the effect of the drug on the germs was obtained at 50. mu.g/mL, 25. mu.g/mL, 12.5. mu.g/mL, 6.25. mu.g/mL, 3.125. mu.g/mL, respectively, according to the above test procedures. According to the method, the same amount of carbendazim water solution is respectively added into the fungus blocks, and the inhibition rate of the fungus blocks on pathogenic bacteria is measured. Blank control only 5mL of sterile water was added. 0..

The results show that the bacteriostatic action of the medicaments of 100 mu g/mL, 50 mu g/mL, 25 mu g/mL, 12.5 mu g/mL, 6.25 mu g/mL and 3.125 mu g/mL on pestalotiopsis relative to the blank control is respectively as follows: 100%, 93.2%, 73.7%, 34.2%, 10.2%. The inhibition rate of carbendazim on germs at each concentration is 100%.

Effect example 2 indoor bacteriostatic test on Peronospora litchi

0.01g of the compound of formula (I) obtained in example 1 was added to 10mL of sterile water (containing 1mL of ethanol) and, after sufficient dissolution, 10 was obtained³mu.g/mL of the mother liquor. Adding 5mL of the mother solution into 45mL of culture medium, fully shaking until alcohol is volatilized, averagely pouring the mother solution into three culture dishes, respectively inoculating phytophthora litchi strain blocks with the diameter of 5mm, measuring the diameter of a bacterial colony after culturing for 72h, and calculating the average value of the bacteriostasis rate to obtain the effect of 100 mu g/mL of the medicament on bacteria. 2.39mL, 1.15mL, 0.57mL, 0.28mL, 0.14mL of the mother liquor were added to 45mL of the culture medium, and the effect of the drug on the germs was obtained at 50. mu.g/mL, 25. mu.g/mL, 12.5. mu.g/mL, 6.25. mu.g/mL, 3.125. mu.g/mL, respectively, according to the above test procedures. According to the method, the same amount of carbendazim water solution is respectively added into the fungus blocks, and the inhibition rate of the fungus blocks on pathogenic bacteria is measured. Blank control only 5mL of sterile water was added.

The results show that the bacteriostasis effects of the medicaments of 100 mu g/mL, 50 mu g/mL, 25 mu g/mL, 12.5 mu g/mL, 6.25 mu g/mL and 3.125 mu g/mL on the phytophthora litchi bacteria are respectively as follows compared with the blank control: 100%, 92.8%, 58.6%, 24.0%, 9.2%. The inhibition rate of carbendazim on germs at each concentration is 100%.

Effect example 3 indoor bacteriostatic test on Sophia melanosporum

0.01g of the compound of formula (I) obtained in example 1 was added to 10mL of sterile water (containing 1mL of ethanol) and, after sufficient dissolution, 10 was obtained³mu.g/mL of the mother liquor. Adding 5mL of the mother solution into 45mL of culture medium, fully shaking until alcohol is volatilized, averagely pouring the mother solution into three culture dishes, respectively inoculating peanut black rot fungus blocks with the diameter of 5mm, measuring the diameter of a bacterial colony after culturing for 72h, and calculating the average value of the bacteriostasis rate to obtain the effect of the 100 mug/mL medicament on germs. 2.39mL, 1.15mL, 0.57mL, 0.28mL, 0.14mL of the mother liquor were added to 45mL of the culture medium, and the effect of the drug on the germs was obtained at 50. mu.g/mL, 25. mu.g/mL, 12.5. mu.g/mL, 6.25. mu.g/mL, 3.125. mu.g/mL, respectively, according to the above test procedures. Adding equal amount of aqueous solution of carbendazim into the blocks respectively, and measuring their effect on pathogenic bacteriaThe inhibition rate. Blank control only 5mL of sterile water was added.

The results show that the bacteriostatic action of the medicaments of 100 mu g/mL, 50 mu g/mL, 25 mu g/mL, 12.5 mu g/mL, 6.25 mu g/mL and 3.125 mu g/mL on the alternaria solani is respectively as follows compared with that of a blank control: 85.5%, 61.6%, 24.8%, 14.2%, 8.7%, 5.4%. The inhibition rate of carbendazim on germs at each concentration is 100%.

Effect example 4 conductance test

Accurately weighing a certain amount of naphthylacetic acid-carbendazim, dissolving the naphthylacetic acid-carbendazim in about 5mL of acetone, adding 0.2mL of tween-80 serving as a surfactant, uniformly mixing, diluting with water to obtain a solution with the concentration of 0.5mmol/L, and uniformly spraying the solution on peanut plants with the concentration of 10-15 cm. When spraying, the flowerpot is covered with plastic film paper to prevent the liquid medicine from entering the soil. Taking 10g of peanut roots after 36h, adding the peanut roots into a mixed solution of 90mL of methanol and 10mL of water, mashing the peanut roots by using a tissue homogenizer, and taking out the peanut roots to soak for 10 h. After suction filtration, the residue was poured into a leaching bottle and extracted with 100mL of methanol aqueous solution for 2 times, the filtrates were combined, concentrated and extracted with dichloromethane for 3 times, dried over anhydrous sodium sulfate, and concentrated to dryness in a water bath. Dissolving with 5mL acetone, purifying with glass chromatographic column, concentrating in water bath to obtain sample, dissolving with acetone, diluting to 2mL, and detecting with liquid chromatography to obtain a solution with content of 0.75 + -0.02 μ g/g. No carbendazim was detected in the roots in the same manner as described above.

The liquid chromatograph is equipped with an autosampler and a diode array detector. Chromatographic conditions are as follows: the column was a reverse phase bonding column (250 mm. times.4 mm) of HP C18, U.S.A., at 25 ℃ and an eluent of methanol: water (80: 20, volume ratio), at a flow rate of 1mL/min, at a sample size of 10. mu.L, at a detection wavelength of 250 nm.

The embodiments described above are only preferred embodiments of the invention and are not exhaustive of the possible implementations of the invention. Any obvious modifications to the above would be obvious to those of ordinary skill in the art, but would not bring the invention so modified beyond the spirit and scope of the present invention.

Claims (7)

1. A compound of formula (I):

2. a bacteriostatic agent comprising a compound of formula (I):

3. a process for the preparation of a compound of formula (I) as claimed in claim 1: the method is characterized by comprising the following steps:

1) esterification of naphthylacetic acid (II) and bromoethanol (III) gives compounds of the formula (IV):

2) reacting a compound of formula (IV) with carbendazim (V) under basic conditions to obtain a compound of formula (I):

4. a process according to claim 3 for the preparation of a compound of formula (I): characterized in that a catalyst is also present in step 1), said catalyst being selected from the group consisting of lN, N' -Dicyclohexylcarbodiimide (DCC), EDCI, DIC, DMAP and HOBT.

5. A process for the preparation of a compound of formula (I) according to claim 3 or 4: the method is characterized by comprising the following steps:

1) adding 10mmol of naphthylacetic acid, 10mmol of N, N' -Dicyclohexylcarbodiimide (DCC), catalytic amount of 4-Dimethylaminopyridine (DMAP) and 12mmol of bromoethanol into 30mL of dichloromethane, stirring at room temperature for 2h, adding water after the reaction is finished, extracting, concentrating, and carrying out column chromatography to obtain a colorless intermediate compound (IV);

2) adding 5mmol of carbendazim original drug into 20mL of dimethylformamide, stirring at room temperature, adding 7.5mmol of cesium carbonate, stirring for 30min, cooling to 0 ℃, dropwise adding 30mL of N, N-dimethylformamide solution dissolved with 6mmol of intermediate compound (IV), slowly heating to room temperature, stirring for reaction, after the reaction is finished, adjusting the pH value to be neutral by using 3mol/L HCl solution, extracting with ethyl acetate, and carrying out column chromatography to obtain naphthylacetic acid-carbendazim.

6. Use of a compound of formula (I) as defined in claim 1 for bacteriostasis, sterilization and disinfection.

7. Use of a compound of formula (I) as defined in claim 1 for inhibiting pestalotiopsis, peronophythora litchii, alternaria arachidicola.

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