A coumarin derivative containing an amide bond, and a preparation method and application thereof

Abstract

The application discloses a coumarin derivative containing an amide bond and a preparation method and application thereof, and has the characteristics that the specific compound structure of the coumarin derivative is 4-methyl-7-acetylamino coumarin, and the preparation method steps are as follows: 1) 3-hydroxyphenyl ethyl carbamate is prepared by amidation reaction of m-aminophenol and ethyl chloroformate; 2) 4-methyl-7-ethoxycarbonylamino coumarin is prepared by esterification reaction of 3-hydroxyphenyl ethyl carbamate and ethyl acetoacetate; 3) 4-methyl-7-aminocoumarin is prepared by esterolysis reaction of 4-methyl-7-ethoxycarbonylamino coumarin and a mixture of glacial acetic acid and concentrated sulfuric acid; and 4) 4-methyl-7-acetylamino coumarin is prepared by esterification reaction of 4-methyl-7-aminocoumarin, pyridine and acetic anhydride, and the advantages are that the compound has good antiviral and killing effects on white spot syndrome virus, the synthesis method is simple, and the conversion rate is high.

Description

TECHNICAL FIELD

[0001] The present application relates to a coumarin derivative, in particular to a coumarin derivative containing an amide bond and a preparation method and application thereof. BACKGROUND

[0002] Coumarin compounds are a class of natural organic compounds with benzene α-pyrone lactone ring structure, which have anticoagulation, antibacterial, anti-inflammatory, antioxidant, antitumor, antiviral and enzyme inhibition and other biological activities, and are widely used in the fields of medicine and pesticide. For example, antibacterial drugs such as novobiocin and coumarin; anti-inflammatory drugs such as daphnein. However, there are still some problems, such as (1) the solubility still needs to be improved, which affects the bioavailability to some extent; (2) the target of coumarin compounds after heterocyclic modification is diverse, and the specific mechanism is not completely clear; (3) some reported active molecules have chirality, but the specific configuration has not been proved. The white spot syndrome induced by white spot syndrome virus (WSSV) has the characteristics of wide spread, high mortality and fast death rate, which has seriously threatened the safety of shrimp culture in China for many years. After the shrimp is infected with WSSV, the body color is red with empty stomach, the reaction to the outside world is slow and often static at the bottom of the water, and the white spot appears on the shell in the later stage of the disease, and the mortality rate is 100% within 3-14 days after the onset. SUMMARY

[0003] The technical problem to be solved by the present application is to provide a coumarin derivative containing an amide bond which has excellent effect on resisting white spot syndrome virus, and a preparation method and application thereof.

[0004] The technical scheme adopted by the present application to solve the above technical problem is: a coumarin derivative containing an amide bond, the structure of the coumarin derivative is shown as formula 1:

[0005]

[0006] The preparation method of the above-mentioned coumarin derivative containing an amide bond comprises the following steps:

[0007] (1) preparing 3-hydroxyphenylamino ethyl carbamate by amidation reaction of m-aminophenol and ethyl chloroformate;

[0008] (2) preparing 4-methyl-7-ethoxycarbonylamino coumarin by esterification reaction of 3-hydroxyphenylamino ethyl carbamate and acetyl acetic acid ethyl ester;

[0009] (3) preparing 4-methyl-7-amino coumarin by esterolysis reaction of 4-methyl-7-ethoxycarbonylamino coumarin with a mixture of glacial acetic acid and concentrated sulfuric acid;

[0010] (4) 4-methyl-7-acetamidocoumarin is prepared by esterification of 4-methyl-7-amino coumarin with pyridine and acetic anhydride.

[0011] Further, the preparation of 3-hydroxyphenylcarbamic acid ethyl ester in step (1) is as follows: 101 mmol of aminophenol and 104 mmol of NaHCO3 are placed in a round bottom flask, dissolved in 150 mL of ethyl acetate and 10 mL of distilled water, 94 mmol of ethyl chloroformate is slowly added dropwise, after the dropwise addition is completed, stirring is carried out at 20-25°C for 1 h, 50 mL of distilled water is added and stirring is carried out for 3 h, the water layer and the organic layer are separated by extraction, the organic layer is washed with water, 1 mol / L H2SO4, saturated NaCl solution three times respectively, and then washed with distilled water once, the organic phase is dried with anhydrous magnesium sulfate, and then evaporated to dryness under reduced pressure with a rotary evaporator to obtain 3-hydroxyphenylcarbamic acid ethyl ester. -1 H2SO4, saturated NaCl solution respectively three times, and then washed with distilled water once, the organic phase is dried with anhydrous magnesium sulfate, and then evaporated to dryness under reduced pressure with a rotary evaporator to obtain 3-hydroxyphenylcarbamic acid ethyl ester.

[0012] Further, the preparation of 4-methyl-7-ethoxycarbamoyl coumarin in step (2) is as follows: 47 mmol of 3-hydroxyphenylcarbamic acid ethyl ester and 70 mmol of acetoacetic acid ethyl ester are mixed and placed in a round bottom flask, 60 mL of 70 wt% H2SO4 solution is slowly added dropwise, stirring is carried out at 20-25°C for 2 h, after the reaction is completed, the reaction product is poured into a 300 mL ice water mixture, stirring is carried out until the precipitation is completed, suction filtration is carried out, the precipitate is washed with ice water three times, and the solid is dried to obtain 4-methyl-7-ethoxycarbamoyl coumarin.

[0013] Further, the preparation of 4-methyl-7-ethoxycarbamoyl coumarin in step (2) is as follows: 47 mmol of 3-hydroxyphenylcarbamic acid ethyl ester and 70 mmol of acetoacetic acid ethyl ester are mixed and placed in a round bottom flask, 60 mL of 70 wt% H2SO4 solution is slowly added dropwise, stirring is carried out at 20-25°C for 2 h, after the reaction is completed, the reaction product is poured into a 300 mL ice water mixture, stirring is carried out until the precipitation is completed, suction filtration is carried out, the precipitate is washed with ice water three times, and the solid is dried to obtain 4-methyl-7-ethoxycarbamoyl coumarin.

[0014] Further, the preparation of 4-methyl-7-ethoxycarbamoyl coumarin in step (2) is as follows: 47 mmol of 3-hydroxyphenylcarbamic acid ethyl ester and 70 mmol of acetoacetic acid ethyl ester are mixed and placed in a round bottom flask, 60 mL of 70 wt% H2SO4 solution is slowly added dropwise, stirring is carried out at 20-25°C for 2 h, after the reaction is completed, the reaction product is poured into a 300 mL ice water mixture, stirring is carried out until the precipitation is completed, suction filtration is carried out, the precipitate is washed with ice water three times, and the solid is dried to obtain 4-methyl-7-ethoxycarbamoyl coumarin.

[0015] The above-mentioned coumarin derivative containing an amide bond is used for preparing a drug for inhibiting and / or killing a white spot syndrome virus.

[0016] Compared with the prior art, the coumarin derivative containing an amide bond, the preparation method and application thereof have the advantages that the coumarin derivative containing an amide bond is 4-methyl-7-acetamidocoumarin, a chemically synthesized coumarin derivative is provided, the coumarin derivative has excellent antiviral effect, and the synthesis process is relatively simple, the yield is high, and the conversion is easy. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 A schematic diagram of a total synthesis route of 4-methyl-7-acetamidocoumarin;

[0018] Figure 2 A copy number inhibition graph of WSSV and a mortality rate curve of juvenile shrimp of 4-methyl-7-acetamidocoumarin, wherein A is an inhibition curve of WSSV copy number and a protection rate curve of juvenile shrimp for 72h of 4-methyl-7-acetamidocoumarin at different concentrations, B is a mortality rate curve of juvenile shrimp after the juvenile shrimp is soaked with 4-methyl-7-acetamidocoumarin and WSSV at the same time, and C is a copy number graph of WSSV after the juvenile shrimp is soaked with 4-methyl-7-acetamidocoumarin and WSSV at the same time for 24h, 48h and 72h;

[0019] Figure 3 A copy number inhibition graph of WSSV and a mortality rate curve of juvenile shrimp of 4-methyl-7-acetamidocoumarin, wherein A is a copy number of WSSV at 72h after the juvenile shrimp is infected for 24h or 48h and then treated with 4-methyl-7-acetamidocoumarin, B is a mortality rate graph of the juvenile shrimp after the juvenile shrimp is infected for 24h and then treated with 4-methyl-7-acetamidocoumarin, and C is a mortality rate graph of the juvenile shrimp after the juvenile shrimp is infected for 48h and then treated with 4-methyl-7-acetamidocoumarin;

[0020] Figure 4 A copy number inhibition graph of WSSV and a mortality rate curve of juvenile shrimp of 4-methyl-7-acetamidocoumarin, A is a copy number graph of WSSV after WSSV is pre-incubated with 4-methyl-7-acetamidocoumarin for 1h, 2h and 4h and then infects the juvenile shrimp, and B is a mortality rate graph of the juvenile shrimp after WSSV is pre-incubated with 4-methyl-7-acetamidocoumarin for 1h, 2h and 4h and then infects the juvenile shrimp;

[0021] Figure 5Figure of inhibition of WSSV copy number and mortality curve of juvenile shrimp by 4-methyl-7-acetamidocoumarin, wherein A is no replacement of 4-methyl-7-acetamidocoumarin, B is replacement of 4-methyl-7-acetamidocoumarin once, C is replacement of 4-methyl-7-acetamidocoumarin twice, D is replacement of 4-methyl-7-acetamidocoumarin thrice, E is replacement of 4-methyl-7-acetamidocoumarin four times, and F is replacement of 4-methyl-7-acetamidocoumarin four times. DETAILED DESCRIPTION

[0022] The present application is further described in detail by the following examples with reference to the accompanying drawings. Example 1

[0024] A coumarin derivative containing an amide bond, the structure of which is shown in Formula 1:

[0025] Example 2

[0027] The preparation method of the coumarin derivative containing an amide bond in the above-mentioned Example 1, as shown in Formula 1, comprises the following steps: Figure 1

[0028] 1. Preparation of ethyl 3-hydroxyanilinoformate (intermediate compound 1)

[0029] Take 11.040 g (101 mmol) of m-aminophenol and 8.772 g (104 mmol) of NaHCO3 and dissolve them in 150 mL of ethyl acetate and 10 mL of distilled water in a 250 mL round-bottom flask, slowly add 10.263 g (94 mmol) of ethyl chloroformate dropwise, after the dropwise addition is completed, stir at 20-25°C for 1 h, then add 50 mL of distilled water and stir for 3 h, separate the water layer and the organic layer by extraction, wash the organic layer with water, 1 mol·L -1 H2SO4, and saturated NaCl solution three times each, and then wash once with distilled water, dry the organic phase with anhydrous magnesium sulfate, and then evaporate to dryness under reduced pressure with a rotary evaporator to obtain ethyl 3-hydroxyanilinoformate, the structure of which is shown in Formula 2:

[0030]

[0031] ​2. Preparation of 4-methyl-7-carboethoxyamino coumarin (intermediate compound 2) 8.010 g (47 mmol) of ethyl 3-hydroxyanilinecarbamate and 9.05 g (70 mmol) of ethyl acetoacetate were mixed and then added dropwise to 60 mL of 70 wt% H2SO4 solution in a 100 mL round bottom flask. The mixture was stirred at 20-25 °C for 2 h, after which solid was produced. After the reaction was completed, the reaction mixture was poured into 300 mL of ice water mixture and stirred until the precipitation was completed. The precipitate was filtered, washed with ice water three times, and dried to obtain 4-methyl-7-carboethoxyamino coumarin, the structure of which is shown in formula 3:

[0032]

[0033] 3. Preparation of 4-methyl-7-amino coumarin (intermediate compound 3)

[0034] 7.024 g of 4-methyl-7-carboethoxyamino coumarin was weighed into a mixture of 25 g of glacial acetic acid and 25 g of concentrated sulfuric acid, and heated to reflux for 6 h. After the reaction was completed, the reaction mixture was poured into 200 mL of ice water mixture, and the pH was adjusted to 8.5 with 50 wt% NaOH solution. The light yellow precipitate was allowed to stand overnight, filtered, washed with ice water three times, and dried to obtain 4-methyl-7-amino coumarin, the structure of which is shown in formula 4:

[0035]

[0036] 4. Preparation of 4-methyl-7-acetamidocoumarin (target compound, P13) 0.209 g of 4-methyl-7-amino coumarin was weighed into a 100 mL round bottom flask, and 20 mL of dichloromethane, 1 mL of pyridine, and 1 mL of acetic anhydride were added. The mixture was stirred for 12 h, and 20 mL of dichloromethane was added to the reaction mixture. After the mixture was completely mixed, the reaction mixture was filtered, the precipitate was washed with 20 mL of dichloromethane three times, and dried in an oven to obtain 4-methyl-7-acetamidocoumarin, the structure of which is shown in formula 1:

[0037]

[0038] The structures of the intermediate compounds and target compounds are shown in Tables 1 and 2.

[0039] Table 1. Properties of the compounds

[0040]

[0041] Table 2. Properties of the compounds 1 H NMR, 13 C NMR and ESI-MS data

[0042]

[0043]

[0044] From Table 1, it can be seen that the preparation method of the target compound provided by the present application has a high yield, and from Table 2, the structure of the target compound can be determined. In Table 2, the chemical shifts of 10.36 in the hydrogen spectrum and 169.05 in the carbon spectrum of the target compound are those of the amide group, which proves the correctness of the structure of the reaction product. Specific embodiment three

[0046] P13 Anti-White Spot Syndrome Virus Activity Assay

[0047] 1. Experimental materials and methods

[0048] (1) Test materials

[0049] Virus material: White spot syndrome virus (WSSV), from Zhejiang Marine Breeding Research Institute; experimental animals: Pacific white shrimp post-larvae, from the Qingjiang base of Zhejiang Marine Breeding Research Institute.

[0050] Preparation of test drug solution: 500 mg of P13 to be tested was accurately weighed into a 10 mL volumetric flask, dissolved with dimethyl sulfoxide (DMSO) and made up to volume, to obtain a test drug solution with a concentration of 50 mg / mL, which was stored in a 4°C refrigerator for standby use.

[0051] (2) WSSV infection concentration detection

[0052] Pacific white shrimp post-larvae were randomly added to six-hole plates, with 6 mL of culture water and 10 post-larvae per hole. The WSSV group was soaked with different concentrations of virus diluent, with concentrations of 1.6×10 4 , 7.5×10 5 , 1.6×10 6 , 7.5×10 7 and 7.5×10 8 copies / μL. The blank control group only had culture water. The temperature was maintained at 28±0.5°C during the experiment. The experiment lasted for 3 days, and the mortality of post-larvae was recorded every 24 hours. According to the experimental results, the WSSV concentration at which the mortality of post-larvae reached 100% within 3 days was selected as the infection concentration for the subsequent experiment.

[0053] (3) Toxicity detection of P13 on Pacific white shrimp post-larvae

[0054] The Litopenaeus vannamei postlarvae were randomly added to the six-hole plate, each containing 6 mL of aquaculture water and 10 postlarvae. The postlarvae were soaked in different concentrations of P13 (0.1-100 mg / L), respectively, and a solvent control group (soaked in 0.2% DMSO) and a blank control group were set. The temperature was kept at 28±0.5℃, and the survival status of the postlarvae was observed and recorded continuously for 72 h.

[0055] (4) Detection of P13 antiviral activity

[0056] a. The Litopenaeus vannamei postlarvae were randomly added to the six-hole plate, each containing 6 mL of aquaculture water and 10 postlarvae. The postlarvae were soaked in WSSV diluent (final concentration 1.6×10 6 copies / μL) and P13 liquid (0.63, 1.25, 25, 5, 10 and 20 mg / L) at the same time. The control group was added with WSSV virus diluent and 0.04% DMSO. The survival of the postlarvae was observed every 12 h;

[0057] b. The postlarvae were first soaked in WSSV diluent (final concentration 1.6×10 6 copies / μL), incubated at 28℃ for 24 or 48 h, then the virus liquid was sucked and discarded, the aquaculture water was rinsed 3 times, 20 mg / L P13 was added, and the control group was added with 0.04% DMSO. The survival of the postlarvae was observed every 12 h;

[0058] c. P13 (20 mg / L) and WSSV diluent (8×10 7 copies / μL) were pre-incubated for 1, 2 or 4 h, and the control group was incubated with 0.04% DMSO. Then the mixed liquid was diluted 50 times to soak the postlarvae, at this time the concentration of P13 was 0.4 mg / L (P13 does not work at this concentration), and the copy concentration of WSSV diluent was 1.6×10 6 copies / μL. The postlarvae were cultured at 28℃ for 72 h, collected, and the DNA was extracted using the marine animal tissue genomic DNA rapid extraction kit (Tiangen), and stored at -80℃ for use.

[0059] d. The Litopenaeus vannamei postlarvae were randomly added to the six-hole plate, each containing 6 mL of aquaculture water and 10 postlarvae, and five experimental groups a, b, c, d and e were set. WSSV diluent (final concentration 1.6×10 6 copies / μL) was added, and after 24 h the solution was sucked and discarded, the aquaculture water was rinsed 3 times, 10 mg / L P13 or 0.02% DMSO was added, and fresh P13 or DMSO was replaced every 24 h. The control group was replaced with aquaculture water. Group a was replaced 0 times, group b was replaced 1 time, group c was replaced 2 times, group d was replaced 3 times, and group e was replaced 4 times. The postlarvae were cultured at 28℃ for 120 h, and the mortality rate of the postlarvae was recorded every 12 h.

[0060] Experimental method as above, four times more drug, 28 ℃ culture to 120 h, every 8 h take three tail shrimp, extraction DNA, -80 ℃ storage for later use. Collection of shrimp, marine animal tissue genomic DNA rapid extraction kit (Tiangen) extraction DNA, -80 ℃ storage for later use.

[0061] (5) Detection of WSSV genome DNA copy number

[0062] After DNA extraction, the concentration and purity were measured using a microspectrophotometer. The DNA was diluted with sterile water to adjust the concentration to 30 ng / μL, which was used as the template for qPCR. The detection primer was VP28 141 (VP28-F: 5'-AAACCTCCGCATTCCTGTGA-3', VP28-R: 5'-TCCGCATCTTCTTCCTTCAT-3'), and the reaction system and reaction procedure of qPCR are shown in Tables 3 and 4. The quantitative PCR results were converted according to the standard curve made by pMD19T-VP28 141 standard, and the viral copy number was obtained.

[0063] Table 3 PCR reaction system

[0064]

[0065] Table 4 PCR reaction procedure

[0066]

[0067] 2. Analysis of experimental results

[0068] P13 has crystal precipitation at 30-100 mg / L, and has no effect on the survival rate of larvae at 20 mg / L. 1.6 x 10 6 copies / μL of WSSV diluent soaked larvae, the mortality rate of larvae within 3 d was 100%, so this viral concentration was selected as the challenge concentration for subsequent experiments.

[0069] The concentration gradient detection results of the drug are shown in Figure 2 A, the anti-WSSV activity of P13 increases with the increase of drug concentration, and the inhibition rate of 20 mg / L P13 on virus replication is more than 90%. In addition, P13 can effectively improve the survival rate of WSSV infected larvae, and the 72 h protection rate of 20 mg / L P13 on larvae is 75% Figure 2 B). The drug and virus were soaked in larvae at the same time, and the WSSV copy number at 24, 48 and 72 h was reduced by 1.6 times, 10.3 times and 8.0 times Figure 2C). P13 can effectively inhibit the proliferation of WSSV in larvae and improve their survival rate.

[0070] Soaking juvenile shrimp in the drug for 24 and 48 hours after WSSV infection showed that P13 could still effectively inhibit viral proliferation 24 hours after WSSV infection. Figure 3 A) Within 24 hours of WSSV infection, the cumulative mortality rate of juvenile shrimp reached 100% within 60 hours; WSSV P13 In the treatment group, the cumulative mortality rate of juvenile shrimp within 60 hours was only 25%, and the survival time was extended to 96 hours. Figure 3 B). WSSV infection 48 hours later, WSSV... P13 The survival time of juvenile shrimp in the treatment group was extended to 60 hours. Figure 3 C). It is evident that P13 can prolong the survival time of WSSV-infected juvenile shrimp and has a certain therapeutic effect.

[0071] Compared with the WSSV control group, the WSSV copy number decreased by 1.6, 2.1, and 2.3 times after 1, 2, and 4 hours of pre-incubation with P13 WSSV virus particles, respectively, thereby reducing the mortality rate of larvae. Figure 4 (AB). It is evident that P13 effectively reduces the infectivity of viral particles, and this weakening effect becomes more pronounced with prolonged incubation time.

[0072] The effect of continuous drug changes on the antiviral efficacy of P13 is as follows: Figure 5 As shown. In the experimental group where the drug was not changed, WSSV DMSO The juvenile shrimp in the group began to die after 12 hours, and the cumulative mortality rate reached 100% within 60 hours. (WSSV) P13 The juvenile shrimp in the group began to die after 36 hours, and the cumulative mortality rate within 60 hours was 30%. Figure 5 A). Subsequently, the medication was changed 1, 2, 3, and 4 times every 24 hours, WSSV. P13 The cumulative mortality rates of the juvenile shrimp at 108 h were 90%, 80%, 60%, and 30%, respectively, and after four consecutive changes of medication, the cumulative mortality rate at 120 h was 45%. Figure 5 BE). After four consecutive changes of medication, live shrimp were sampled every 8 hours for WSSV. DMSO In the treatment groups, the viral load in the larvae gradually increased over time. In the P13-WSSV treatment group, the viral load in the larvae remained consistently lower than that in the WSSV treatment group. DMSO The treatment group showed an overall downward trend. Figure 5 F).

[0073] Therefore, P13 can significantly inhibit WSSV proliferation, improve the survival rate of WSSV infected shrimp, and continuous replacement of the drug can further improve the antiviral effect of P13.

[0074] The above description is not a limitation of the present application, and the present application is not limited to the above examples. Changes, modifications, additions or substitutions made by those skilled in the art within the spirit and scope of the present application should also be within the protection scope of the present application.

Claims

1. Use of a coumarin derivative containing an amide bond for the manufacture of a medicament for inhibiting and / or killing the white spot syndrome virus, characterized in that, The structure of the coumarin derivative is shown in formula 1: Formula 1.

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