CN105732601B - Coumarin kind compound of the functional group containing imidazoles and its preparation method and application - Google Patents

Abstract

The invention discloses coumarin compounds containing imidazole functional groups and their preparation methods and applications. The specific compounds include 7-[8-imidazole/(2/4-methylimidazole) octyloxy]coumarin, It is prepared by substitution reaction of 7‑(8‑bromooctyloxy)coumarin and corresponding imidazole compounds, and the compounds have good antibacterial and bactericidal effects on aquatic pathogens such as Flavobacterium columnar and Streptococcus agalactiae.

Description

Coumarin compound containing imidazole functional group and its preparation method and application

technical field

The invention relates to the field of antibacterial active substances for aquatic products, relates to coumarin compounds containing imidazole functional groups, in particular to 7-[8-imidazole/(2/4-methylimidazole)octyloxy]coumarin , its preparation method and the application of antibacterial activity.

Background technique

Coumarin compounds are an important class of organic heterocyclic compounds with a wide range of biological activities. They are widely used in the fields of medicine, life sciences and pesticide synthesis. They can be used as antioxidants, anticoagulants, and antitumor agents. It is also widely used in fields such as insecticides and antibacterial agents. At present, the chemical agents used to prevent and control aquatic pathogens in the domestic aquaculture industry are mainly transplanted human and veterinary drugs, mainly including macrolides, tetracyclines, chloramphenicol, sulfonamides, etc. Overdosage and frequent use of these antibiotics will gradually increase the drug resistance of pathogenic bacteria, and will easily cause food and environmental safety problems such as drug residues and water pollution. Therefore, the development of new antibacterial drugs is one of the problems to be solved urgently to ensure the sustainable and high-speed development of the aquatic industry.

Contents of the invention

The object of the present invention is to provide coumarin compounds containing imidazole functional groups and their preparation methods and applications. The compounds can be used in the aquatic industry to prevent and control the diseases of aquatic pathogens such as Flavobacterium columnar and Streptococcus agalactiae.

To achieve the above object, the present invention adopts the following technical solutions:

A coumarin compound containing an imidazole functional group, the structure of which is shown in formula 1:

Wherein, R ₁ is H or -CH ₃ , and R ₂ is H or -CH ₃ .

The preparation method of the above-mentioned coumarin compound containing imidazole functional group may further comprise the steps:

Add 0.6-0.8g of 7-(8-bromooctyloxy)coumarin and 1.3-1.5g of anhydrous potassium carbonate into the solvent and stir at room temperature for 1-2h to obtain material C. Add 0.4-0.5 After g imidazole, 2-methylimidazole or 4-methylimidazole, stir at room temperature for 20-24h, then evaporate the solvent under reduced pressure to obtain material D, add water to material D and extract with chloroform, and extract the organic phase obtained with After drying over anhydrous sodium sulfate and concentrating under reduced pressure to obtain a crude solid product, the crude solid product was separated and purified by column chromatography to obtain 7-(8-imidazole octyloxy)coumarin, 7-[8-(2-methylimidazole) Octyloxy] coumarin or 7-[8-(4-methylimidazole) octyloxy]coumarin, the structure is shown in formula 2, formula 3 or formula 4 respectively:

The preparation method of the 7-(8-bromooctyloxy)coumarin comprises the following steps: adding 4.8-5.0g of 7-hydroxycoumarin and 13.8-14.0g of anhydrous potassium carbonate into the solvent and stirring at room temperature 2-3h to get material A, add 24-28g 1,8-dibromooctane to material A and then reflux reaction at 60-65°C for 20-24h, after reaction, distill off the solvent under reduced pressure to get material B, add to material B After adding water, it was extracted with chloroform, and the organic phase obtained by the extraction was dried with anhydrous sodium sulfate and then concentrated under reduced pressure to obtain a crude solid product, which was separated and purified by column chromatography to obtain the starting reactant 7-(8-bromooctyloxy ) coumarin, the structure is as shown in formula 5:

Application of the above-mentioned coumarin compounds containing imidazole functional groups in the preparation of medicines for preventing and treating aquatic pathogens.

The pathogen is Flavobacterium columnar or Streptococcus agalactiae.

The aquatic product is fish.

Application of the above-mentioned coumarin compounds containing imidazole functional groups in the preparation of drugs with bacteriostatic and bactericidal effects on Flavobacterium columnar.

Application of the above-mentioned coumarin compounds containing imidazole functional groups in the preparation of drugs with antibacterial and bactericidal effects on Streptococcus agalactiae.

The beneficial effects of the present invention are reflected in:

The coumarin compounds containing imidazole functional groups disclosed by the present invention specifically include 7-(8-imidazole octyloxy)coumarin, 7-[8-(2-methylimidazole)octyloxy]coumarin Chlorin and 7-[8-(4-methylimidazole) octyloxy]coumarin can be prepared by substitution reaction between 7-(8-bromooctyloxy)coumarin and corresponding imidazole compounds to produce The rate is high, and this type of compound has good antibacterial effect on aquatic pathogens such as Flavobacterium columnar and Streptococcus agalactiae, and can be used in the aquatic industry to prevent and control diseases of aquatic pathogens such as Flavobacterium columnar.

Description of drawings

Fig. 1 is the synthetic route schematic diagram of the coumarin compound containing imidazole functional group of the present invention;

Fig. 2 is a schematic diagram of the synthetic route of starting reactant 7-(8-bromooctyloxy)coumarin.

Fig. 3 is 7-(8-imidazole octyloxy) coumarin, 7-[8-(2-methylimidazole) octyloxy] coumarin, 7-[8-(4-methylimidazole) octyl Schematic diagram of the synthetic route of oxy]coumarin.

Detailed ways

The present invention will be described in detail below in conjunction with the accompanying drawings and embodiments.

During the research process of the synthesis and screening of 7-substituted coumarin derivatives, the applicant found that some coumarin-imidazole hybrid compounds have a good inhibitory effect on certain aquatic pathogens, and the antibacterial activity of these compounds It is closely related to the type of substituent at the 7-position. Among them, 7-[8-imidazole/(2/4-methylimidazole) octyloxy]coumarin and other compounds (refer to Figure 1 for synthesis) have good inhibitory and killing effects on aquatic pathogens such as Flavobacterium columnar and Streptococcus agalactiae. Inactivation, has the application value of preventing and treating bacterial diseases of aquatic animals.

(1) Compound preparation

(1) Preparation of synthetic intermediate compound 7-(8-bromooctyloxy)coumarin

Referring to Fig. 2, 4.86g (30mmol) of purchased 7-hydroxycoumarin, 13.8g of anhydrous K ₂ CO ₃ (100mmol) and 120mL of anhydrous acetone were added to a 250mL round-bottomed flask, stirred at room temperature for 2h, and then 24.5 g (90 mmol) of 1,8-dibromooctane was added into the round bottom flask, and refluxed at 65° C. for 24 h. The anhydrous acetone in the reaction system was evaporated under reduced pressure (45°C, 0.05MPa negative pressure), and 100mL of distilled water was added to the round bottom flask to obtain a mixed solution, which was extracted with chloroform (150mL×4), and the organic mixture of the lower layer was combined. Mutually. Add 20-30 g of anhydrous sodium sulfate to the organic phase and let stand overnight, remove the solid sodium sulfate by filtration, and concentrate under reduced pressure (45°C, 0.05 MPa negative pressure) to obtain a crude solid compound. The crude product was separated and purified by silica gel column chromatography (V _{petroleum ether} : V _{ethyl acetate} = 4:1), and the target compound was collected by TLC detection, and concentrated under reduced pressure to remove the eluting reagent (55°C, 0.05MPa negative pressure) to obtain 6.63g White needle-like solid.

(2) Preparation of 7-(8-imidazole octyloxy)coumarin (compound 1)

Referring to Figure 3, add 0.72g (2mmol) 7-(8-bromooctyloxy)coumarin and 1.38g (10mmol) anhydrous K ₂ CO ₃ to a 100mL round bottom flask containing 40mL acetonitrile, and stir at room temperature for 1h Add 0.408g (6mmol) imidazole to the round bottom flask, continue to stir at room temperature for 24h, after the reaction is complete, distill acetonitrile (55°C, 0.05MPa negative pressure), add 30mL deionized water to the round bottom flask It was extracted with chloroform (50 mL×3), and the lower organic phases were combined. Add 2-3 g of anhydrous sodium sulfate to the organic phase and let it stand overnight, filter to remove the solid anhydrous sodium sulfate, and concentrate under reduced pressure (55° C., 0.05 MPa negative pressure) to obtain a crude solid compound. The crude product was separated and purified by silica gel column chromatography (V _chloroform : V _methanol = 10:1), the target compound was collected by TLC detection, and concentrated under reduced pressure to remove the eluting reagent (55°C, 0.05MPa negative pressure) to obtain 0.599g white gel solid with the following structure:

(3) Preparation of 7-[8-(2-methylimidazole) octyloxy]coumarin (compound 2)

Referring to Figure 3, add 0.72g (2mmol) 7-(8-bromooctyloxy)coumarin and 1.38g (10mmol) anhydrous K ₂ CO ₃ to a 100mL round bottom flask containing 40mL acetonitrile, and stir at room temperature for 1h Add 0.492g (6mmol) 2-methylimidazole to the round bottom flask, continue stirring at room temperature for 24h, after the reaction is complete, distill off acetonitrile (55°C, 0.05MPa negative pressure), add 30 mL of deionized water and extracted with chloroform (50 mL×3), and combined the lower organic phases. Add 2-3 g of anhydrous sodium sulfate to the organic phase and let it stand overnight, filter to remove the solid anhydrous sodium sulfate, and concentrate under reduced pressure (55° C., 0.05 MPa negative pressure) to obtain a crude solid compound. The crude product was separated and purified by silica gel column chromatography (V _chloroform : V _methanol = 10:1), and the target compound was collected by TLC detection, and concentrated under reduced pressure to remove the eluting reagent (55°C, 0.05MPa negative pressure) to obtain 0.615g white gel solid with the following structure:

(4) Preparation of 7-[8-(4-methylimidazole) octyloxy]coumarin (compound 3)

Referring to Figure 3, add 0.72g (2mmol) 7-(8-bromooctyloxy)coumarin and 1.38g (10mmol) anhydrous K ₂ CO ₃ to a 100mL round bottom flask containing 40mL acetonitrile, and stir at room temperature for 1h Add 0.492g (6mmol) 4-methylimidazole to the round bottom flask, continue to stir at room temperature for 24h, after the reaction is complete, distill off acetonitrile (55°C, 0.05MPa negative pressure), add 30 mL of deionized water and extracted with chloroform (50 mL×3), and combined the lower organic phases. Add 2-3 g of anhydrous sodium sulfate to the organic phase and let it stand overnight, filter to remove the solid anhydrous sodium sulfate, and concentrate under reduced pressure (55° C., 0.05 MPa negative pressure) to obtain a crude solid compound. The crude product was separated and purified by silica gel column chromatography (V _chloroform : V _methanol = 10:1), and the target compound was collected by TLC detection, and concentrated under reduced pressure to remove the eluting reagent (55°C, 0.05MPa negative pressure) to obtain 0.648g white gel solid with the following structure:

(5) The structure-related data of compounds 1-3 are shown in Tables 1 and 2:

Properties of Compounds 1 to 3 in Table 1

Table 2 ¹ H NMR, ¹³ C NMR and ESI-MS data of compounds 1-3

As can be seen from Table 1, the preparation method of compound 1～3 provided by the invention has higher yield, can determine the structure of compound 1～3 from table 2, i.e. 7-(8-bromooctyloxy)coumarin The bromine atom on the side chain of the alkanes is replaced by the corresponding imidazole group to generate the target compound.

(2) The effect of compounds 1-3 on inhibiting aquatic pathogens (flavobacterium columnar and streptococcus agalactiae)

2.1 Test material

Medium:

Brain heart infusion medium (BHI) (dehydrated calf brain infusion powder 12.5g, dehydrated bovine heart infusion powder 5.0g, peptone 10.0g, glucose 2.0g, disodium hydrogen phosphate 2.5g, NaCl 5.0g, distilled water 1000mL, pH Value 7.4±0.2), used for Streptococcus agalactiae culture;

Shieh nutrient broth medium (peptone 5g, yeast extract 0.5g, sodium acetate 0.01g, barium chloride 0.01g, dipotassium hydrogen phosphate 0.1g, potassium dihydrogen phosphate 0.05g, magnesium sulfate 0.3g, calcium chloride 0.0067 g, ferric sulfate 0.001g, sodium bicarbonate 0.05g, distilled water 1000mL, pH value 7.4±0.2), used for the cultivation of Flavobacterium columnar.

Preparation of test solution:

A certain amount of compounds 1, 2 and 3 were accurately weighed and dissolved in dimethyl sulfoxide (DMSO), and the solution was sterilized by filtration with a 0.22 μm filter membrane, and stored at 4°C for future use.

Bacteria solution preparation:

The tested strains were inoculated on the corresponding medium agar plates, cultured at their optimum growth temperature for 18-24 hours, and prepared with sterile saline to prepare a bacterial suspension of 1-2×10 ⁸ cfu/mL for use.

2.2 Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of compounds 1-3

The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were detected by the doubling dilution method. Dilute the bacterial suspension to 1-2×10 ⁵ cfu/mL with the corresponding culture medium, and take the lowest drug concentration that completely inhibits bacterial growth in the test tube as the minimum inhibitory concentration (MIC). Take 10 μL of the culture solution in the test tube without bacterial growth and spread it on the agar plate of the corresponding medium, observe the petri dish after culturing at 28°C for 24 hours, and take the lowest concentration that kills 99.9% of the number of bacteria as the minimum bactericidal concentration (MBC). Three replicates were set up for each strain. The inhibitory effect data of compounds 1-3 on two kinds of aquatic pathogenic bacteria are listed in Tables 3 and 4.

The minimum inhibitory concentration (MIC) of table 3 compounds to Flavobacterium columnar, Streptococcus agalactiae

The minimum bactericidal concentration (MBC) of table 4 compounds to Flavobacterium columnar, Streptococcus agalactiae

As can be seen from Table 3, the inhibitory activity of compounds 1-3 to Streptococcus agalactiae is equivalent to that of the reference drug enrofloxacin, and the inhibitory activity of compound 2 to Flavobacterium columnar is consistent with that of the reference drug, while compounds 1 and 3 have the same inhibitory activity on Flavobacterium columnar. The inhibitory activity of Bacillus was slightly weaker than that of the control drug; as can be seen from Table 4, the killing activity of compounds 1-3 on Flavobacterium columnar was slightly weaker than that of the control drug, while the killing activity on Streptococcus agalactiae was equivalent to that of the control drug.

(3) Safety evaluation of compounds 1-3

Taking crucian carp as indicator organisms, the safety of compounds 1-3 to aquatic animals was evaluated.

test animal material

Crucian carp (Carassius auratus) is healthy and well-proportioned, with a body weight of 3±0.5g.

Taking crucian carp as indicator organisms, the safety of compounds 1-3 to aquatic animals was evaluated. During the experiment, the death or poisoning of the test fish was observed at any time. The judgment standard of death is that the gill cover stops moving or the tail peduncle of the fish is lightly tapped with a blunt instrument such as a glass rod or tweezers, and the fish is dead without any stress response; slow. During the experiment, the dead fish were fished out in time so as not to affect the water quality.

After 48 hours, the number of surviving fish was recorded, and the fish mortality was calculated by the following formula.

Table 4 shows the median lethal concentration (LC ₅₀ ) of compounds 1-3 on crucian carp.

Table 5 The median lethal concentration (LC50) of compounds 1 and 2 to crucian carp

Comparing the data in Table 4 and 5, it can be seen that at the minimum inhibitory concentration, none of the compounds 1, 2 and 3 will have a toxic effect on crucian carp.

Claims (7)

1. The coumarin compound containing imidazole functional group is characterized in that: the structure of the compound is as shown in formula 1: Wherein, R ₁ is H or -CH ₃ , and R ₂ is H or -CH ₃ . 2. prepare the method for the coumarin compound containing imidazole functional group as claimed in claim 1, it is characterized in that: comprise the following steps: Add 0.6-0.8g of 7-(8-bromooctyloxy)coumarin and 1.3-1.5g of anhydrous potassium carbonate into the solvent and stir at room temperature for 1-2h to obtain material C. Add 0.4-0.5 After g imidazole, 2-methylimidazole or 4-methylimidazole, stir at room temperature for 20-24h, then evaporate the solvent under reduced pressure to obtain material D, add water to material D and extract with chloroform, and extract the organic phase obtained with After drying over anhydrous sodium sulfate and concentrating under reduced pressure to obtain a crude solid product, the crude solid product was separated and purified by column chromatography to obtain 7-(8-imidazole octyloxy)coumarin, 7-[8-(2-methylimidazole) Octyloxy] coumarin or 7-[8-(4-methylimidazole) octyloxy]coumarin, the structure is shown in formula 2, formula 3 or formula 4 respectively: 3. The method according to claim 2, characterized in that: the preparation method of said 7-(8-bromooctyloxy)coumarin comprises the following steps: 4.8-5.0g 7-hydroxycoumarin and 13.8 - Add 14.0g of anhydrous potassium carbonate to the solvent and stir at room temperature for 2-3h to obtain material A, add 24-28g of 1,8-dibromooctane to material A and then reflux at 60-65°C for 20-24h After the reaction, the solvent was evaporated under reduced pressure to obtain material B, water was added to material B and extracted with chloroform, the organic phase obtained by the extraction was dried with anhydrous sodium sulfate and then concentrated under reduced pressure to obtain a solid crude product, which was separated and purified by column chromatography , to obtain 7-(8-bromooctyloxy)coumarin, the structure is as shown in formula 5: 4. The application of the coumarin compounds containing imidazole functional groups in the preparation of medicines for preventing and treating aquatic pathogens as claimed in claim 1, characterized in that: the pathogens are Flavobacterium columnar or Streptococcus agalactiae. 5. The application according to claim 4, characterized in that: the aquatic products are fish. 6. the application of the coumarin compound containing imidazole functional group as claimed in claim 1 in the preparation of the medicine with antibacterial and bactericidal effect on Flavobacterium columnar. 7. the application of the coumarin compound containing imidazole functional group as claimed in claim 1 in the preparation of the medicine with antibacterial and bactericidal effect on Streptococcus agalactiae.