Application of compound 4-(9H-carbazole-9-yl) phenylboronic acid in preparation of medicine for preventing or treating porcine epidemic diarrhea

Abstract

The invention belongs to the technical field of animal epidemic disease prevention and control, and particularly relates to application of a compound 4-(9H-carbazole-9-yl) phenylboronic acid in preparation of a medicine for preventing or treating porcine epidemic diarrhea. It is accidentally found that the compound 4-(9H-carbazole-9-yl) phenylboronic acid can inhibit PEDV proliferation, has a certain inhibition effect on PEDV, can inhibit infection and amplification of PEDV in cells, can be used for preparing the medicine for preventing or treating porcine epidemic diarrhea, lays a certain technical foundation for prevention and control of PED, and has wide application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal disease prevention and control, and particularly relates to the application of a compound 4-(9H-carbazol-9-yl)phenylboronic acid in the preparation of a drug for preventing or treating porcine epidemic diarrhea. Background Art

[0002] Porcine epidemic diarrhea (PED) is a viral infectious disease caused by porcine epidemic diarrhea virus (PEDV). Diseased pigs mainly show clinical symptoms such as diarrhea, vomiting, anorexia, dehydration, and weight loss. Pigs of all ages can be infected by PEDV and show different degrees of disease characteristics. Generally, adult pigs show mild infection symptoms or no symptoms, while piglets are mostly severely infected, and the fatality rate can be as high as 100%, bringing huge economic losses to the live pig breeding industry. Due to the relatively fast mutation of PEDV, the prevention and control effect of vaccines is relatively limited. Therefore, the development of relevant preventive and therapeutic drugs has very important technical significance for the prevention and control of PED.

[0003] The compound 4-(9H-carbazol-9-yl)phenylboronic acid is an organic compound that exerts its functions depending on carbazole and phenylboronic acid, and is mainly used in the fields of medicine and chemical industry. At present, the applications of 4-(9H-carbazol-9-yl)phenylboronic acid are basically in the laboratory research stage, and no large-scale commercial medical applications have been seen.

[0004] The present invention unexpectedly discovers that the compound 4-(9H-carbazol-9-yl)phenylboronic acid can significantly inhibit the replication of PEDV, reduce the PEDV titer, can be used to prepare a drug for anti-PEDV infection, and is used for preventing or treating PED, with broad application prospects. Summary of the Invention

[0005] In view of the above technical problems, the present invention unexpectedly discovers that the compound 4-(9H-carbazol-9-yl)phenylboronic acid can significantly inhibit the replication of PEDV and can be used to prepare a drug for anti-PEDV infection. Specifically, it includes the following contents:

[0006] In a first aspect, the present invention provides the application of a compound 4-(9H-carbazol-9-yl)phenylboronic acid or a pharmaceutically acceptable salt thereof in the preparation of an antiviral drug. The structural formula of the compound 4-(9H-carbazol-9-yl)phenylboronic acid is shown as formula (Ⅰ) below:

[0007]

[0008] Preferably, the virus is porcine epidemic diarrhea virus.

[0009] Preferably, the compound 4-(9H-carbazol-9-yl)phenylboronic acid or a pharmaceutically acceptable salt thereof is combined with a pharmaceutically acceptable carrier and / or excipient to form any pharmaceutically acceptable dosage form.

[0010] Preferably, the dosage form includes tablets, sprays, granules, capsules, oral liquids, injections, and suspensions.

[0011] In a second aspect, the present invention provides the use of the compound 4-(9H-carbazol-9-yl)phenylboronic acid or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing porcine epidemic diarrhea. The structural formula of the compound 4-(9H-carbazol-9-yl)phenylboronic acid is shown as formula (I) below:

[0012]

[0013] Preferably, the compound 4-(9H-carbazol-9-yl)phenylboronic acid or a pharmaceutically acceptable salt thereof is combined with a pharmaceutically acceptable carrier and / or excipient to form any pharmaceutically acceptable dosage form.

[0014] Preferably, the dosage form includes tablets, sprays, granules, capsules, oral liquids, injections, and suspensions.

[0015] In a third aspect, the present invention provides the use of the compound 4-(9H-carbazol-9-yl)phenylboronic acid or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating porcine epidemic diarrhea. The structural formula of the compound 4-(9H-carbazol-9-yl)phenylboronic acid is shown as formula (I) below:

[0016]

[0017] Preferably, the compound 4-(9H-carbazol-9-yl)phenylboronic acid or a pharmaceutically acceptable salt thereof is combined with a pharmaceutically acceptable carrier and / or excipient to form any pharmaceutically acceptable dosage form.

[0018] Preferably, the dosage form includes tablets, sprays, granules, capsules, oral liquids, injections, and suspensions.

[0019] The beneficial effect of the present invention is that it was unexpectedly found that the compound 4-(9H-carbazol-9-yl)phenylboronic acid can significantly inhibit the replication and proliferation of PEDV, and can be used to prepare drugs against PEDV infection for preventing or treating PED, with broad application prospects. Description of the Drawings

[0020] Figure 1 Effect of pretreatment of PEDV virions with gradient concentrations of the compound 4-(9H-carbazol-9-yl)phenylboronic acid on PEDV N RNA in Vero cells;

[0021] Figure 2 Effect of simultaneous addition of gradient concentrations of compound 4-(9H-carbazol-9-yl)phenylboronic acid and PEDV virus particles on PEDV N RNA in Vero cells;

[0022] Figure 3 Effect of post-treatment of PEDV virus particles with gradient concentrations of compound 4-(9H-carbazol-9-yl)phenylboronic acid on PEDV N RNA in Vero cells.

[0023] Figure 4 Effect of treatment with compound 4-(9H-carbazol-9-yl)phenylboronic acid on the activity of Vero cells. Detailed implementation manners

[0024] 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.

[0025] The PEDV strain is the GI strain CH / SX / 2015, GenBank No. MT783684, which is independently isolated and preserved by the Animal Immunization and Metabolism Innovation Team of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0026] Example 1 Detection of PEDV N RNA after pretreatment of PEDV virus particles with gradient concentrations of 4-(9H-carbazol-9-yl)phenylboronic acid

[0027] 1. Method

[0028] 1) Digest and passage Vero cells, and inoculate them at 1×10 5 cells / mL into a 12-well cell culture plate at 1 mL / well. Culture in an incubator at 37°C and 5% CO2.

[0029] 2) When the cell density reaches about 70%, use compound 4-(9H-carbazol-9-yl)phenylboronic acid with final concentrations of 2 μM, 4 μM, 8 μM, 16 μM, 32 μM, 62.5 μM, 125 μM, 250 μM, 500 μM, 1 mM, and 2 mM respectively, and 10 4 TCID 50 of PEDV virus particles to co-incubate at 37°C for 30 min. At this time, the volume is 10 μL.

[0030] 3) Use the processed PEDV-infected cells for 1 h. During this process, the final concentrations of the compound 4-(9H-carbazol-9-yl)phenylboronic acid are 20 nM, 40 nM, 80 nM, 160 nM, 320 nM, 625 nM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM, respectively. After 1 h, replace the medium with fresh DMEM medium containing 5% serum, and collect the cells 12 h after virus infection.

[0031] 4) Use trizol lysis solution to lyse and extract the RNA in the cells. The detailed operation is carried out according to the trizol instruction manual.

[0032] 5) Reverse transcribe the extracted RNA in an amount of 500 ng / 10 μL. The detailed operation is carried out according to the reverse transcription kit instruction manual.

[0033] 6) Perform real-time fluorescence quantitative PCR detection on the reverse-transcribed samples. The detailed operation is carried out according to the fluorescence quantitative PCR polymerase instruction manual. The information of the fluorescence quantitative primers is as follows:

[0034] PEDV N-F: TGGTGGCTGCTGTCAAGG,

[0035] PEDV N-R: TTTTCGACAAATTCCGCAT;

[0036] Actin-F: ATCGTGCGTGACATTAAG,

[0037] Actin-R: ATTGCCAATGGTGATGAC.

[0038] 2. Results

[0039] The results are as Figure 1 shown. Real-time fluorescence quantitative PCR detection shows that at 12 h after PEDV infection, the PEDV N RNA in the pretreatment group with the compound 4-(9H-carbazol-9-yl)phenylboronic acid is significantly lower than that in the control group, and shows a concentration gradient-dependent inhibition of PEDV infection.

[0040] Detection of PEDV N RNA by simultaneous addition of gradient concentrations of 4-(9H-carbazol-9-yl)phenylboronic acid and PEDV virus particles in Example 2

[0041] 1. Method

[0042] 1) Digest and passage Vero cells, and inoculate them at 1×10 5 cells / mL into a 12-well cell culture plate at 1 mL / well. Culture in a 37 °C, 5% CO2 incubator.

[0043] 2) When the cell density reaches about 70%, use 4-(9H-carbazol-9-yl)phenylboronic acid compounds at 62.5 μM, 125 μM, 250 μM, 500 μM, and 1 mM respectively, and premix with 10 4 TCID 50 PEDV virions, and the volume is 10 μL at this time.

[0044] 3) Add the premixed virus solution directly to the cell supernatant to infect the cells for 1 h. At this time, the final concentrations of 4-(9H-carbazol-9-yl)phenylboronic acid compound are 625 nM, 1.25 μM, 2.5 μM, 5 μM, and 10 μM respectively.

[0045] 4) After 1 h, replace the medium with fresh DMEM medium containing 5% serum, and collect the cells 12 h after virus infection.

[0046] 5) Use trizol lysis solution to lyse and extract RNA from the cells, and the detailed operation is carried out according to the trizol instruction manual.

[0047] 6) Reverse transcribe the extracted RNA according to the amount of 500 ng / 10 μL, and the detailed operation is carried out according to the reverse transcription kit instruction manual.

[0048] 7) Perform real-time fluorescence quantitative PCR detection on the reverse-transcribed samples. The detailed operation is carried out according to the fluorescence quantitative PCR polymerase instruction manual. The information of the fluorescence quantitative primers is as follows:

[0049] PEDV N-F: TGGTGGCTGCTGTCAAGG,

[0050] PEDV N-R: TTTTCGACAAATTCCGCAT;

[0051] Actin-F: ATCGTGCGTGACATTAAG,

[0052] Actin-R: ATTGCCAATGGTGATGAC.

[0053] 2. Results

[0054] The results are as Figure 2 shown. Real-time fluorescence quantitative PCR detection shows that 12 h after PEDV infection, the PEDV N RNA in the treatment group of 4-(9H-carbazol-9-yl)phenylboronic acid compound is significantly lower than that in the control group, and shows concentration-gradient-dependent inhibition of PEDV infection.

[0055] Example 3 Detection of PEDV N RNA by adding 4-(9H-carbazol-9-yl)phenylboronic acid at gradient concentrations at 6 h after PEDV infection 1. Method

[0056] 1) Digest and passage Vero cells, and inoculate them at a density of 1×10 5 cells / mL into a 12-well cell culture plate at 1 mL / well. Culture them in an incubator at 37°C and 5% CO2.

[0057] 2) When the cell density reaches about 70%, infect the cells with 10 4 TCID 50 PEDV virus particles for 6 h.

[0058] 3) After 6 h, add 4-(9H-carbazol-9-yl)phenylboronic acid to final concentrations of 625 nM, 1.25 μM, 2.5 μM, 5 μM, and 10 μM respectively, and harvest the cells 12 h after virus infection.

[0059] 4) Use trizol lysis solution to lyse and extract the RNA in the cells, and perform the detailed operation according to the trizol instruction manual.

[0060] 5) Reverse transcribe the extracted RNA in an amount of 500 ng / 10 μL, and perform the detailed operation according to the reverse transcription kit instruction manual.

[0061] 6) Perform real-time fluorescence quantitative PCR detection on the reverse-transcribed samples. Perform the detailed operation according to the fluorescence quantitative PCR polymerase instruction manual. The information of the fluorescence quantitative primers is as follows:

[0062] PEDV N-F: TGGTGGCTGCTGTCAAGG,

[0063] PEDV N-R: TTTTCGACAAATTCCGCAT;

[0064] Actin-F: ATCGTGCGTGACATTAAG,

[0065] Actin-R: ATTGCCAATGGTGATGAC.

[0066] 2. Results

[0067] The results are as Figure 3 shown. Real-time fluorescence quantitative PCR detection shows that the PEDV N RNA in the treatment groups with 2.5 μM and above concentrations of the compound 4-(9H-carbazol-9-yl)phenylboronic acid 6 h after PEDV infection is significantly lower than that in the control group, and shows a concentration gradient-dependent inhibition of PEDV infection.

[0068] Example 4 Detection of the cytotoxicity of the compound 4-(9H-carbazol-9-yl)phenylboronic acid on Vero cells.

[0069] 1. Method

[0070] 1) Digest and passage Vero cells, and inoculate them at 1×10 5 cells / mL into a 96-well cell culture plate at 0.1 mL / well. Culture in an incubator at 37°C and 5% CO2.

[0071] 2) When the cell density reaches about 70%, treat the cells with 4-(9H-carbazol-9-yl)phenylboronic acid at a final concentration of 10 μM for 24 h.

[0072] 3) At 24 h, use a CCK8 kit to detect the changes in cell proliferation, and perform the detailed operation according to the instructions of the CCK8 kit.

[0073] 2. Results

[0074] The results are as Figure 4 shown. The results detected by an enzyme-linked immunosorbent assay (ELISA) reader show that treating the cells with 10 μM of 4-(9H-carbazol-9-yl)phenylboronic acid for 24 h does not affect the cell viability and has good safety.

[0075] The above results indicate that the compound 4-(9H-carbazol-9-yl)phenylboronic acid of the present invention can significantly inhibit the replication and proliferation of PEDV, can be used to prepare drugs against PEDV infection, and is used for the prevention or treatment of PED, having broad application prospects.

[0076] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather to the broadest scope consistent with the principles and novel features disclosed herein.

Claims

1. Use of the compound 4-(9H-carbazole-9-yl)phenylboronic acid or a pharmaceutically acceptable salt thereof in the preparation of an antiviral drug, wherein the structural formula of the compound 4-(9H-carbazole-9-yl)phenylboronic acid is as shown in the following formula (I):

2. The use according to claim 1, characterized in that The virus is porcine epidemic diarrhea virus.

3. The use according to claim 1 or 2, characterized in that: The compound 4-(9H-carbazole-9-yl)phenylboronic acid or a pharmaceutically acceptable salt thereof is added with a pharmaceutically acceptable carrier and / or auxiliary material to prepare any pharmaceutically acceptable dosage form.

4. The use according to claim 3, characterized in that The dosage forms include tablets, sprays, granules, capsules, oral liquids, injections, and suspensions.

5. Use of the compound 4-(9H-carbazole-9-yl)phenylboronic acid or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing porcine epidemic diarrhea, wherein the structural formula of the compound 4-(9H-carbazole-9-yl)phenylboronic acid is as shown in the following formula (I):

6. The use according to claim 5, characterized in that The compound 4-(9H-carbazole-9-yl)phenylboronic acid or a pharmaceutically acceptable salt thereof is added with a pharmaceutically acceptable carrier and / or auxiliary material to prepare any pharmaceutically acceptable dosage form.

7. The use according to claim 6, characterized in that The dosage forms include tablets, sprays, granules, capsules, oral liquids, injections, and suspensions.

8. Use of the compound 4-(9H-carbazole-9-yl)phenylboronic acid or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating porcine epidemic diarrhea, wherein the structural formula of the compound 4-(9H-carbazole-9-yl)phenylboronic acid is shown in the following formula (I):

9. The use according to claim 8, characterized in that The compound 4-(9H-carbazole-9-yl)phenylboronic acid or a pharmaceutically acceptable salt thereof is added with a pharmaceutically acceptable carrier and / or auxiliary material to prepare any pharmaceutically acceptable dosage form.

10. The use according to claim 9, characterized in that The dosage forms include tablets, sprays, granules, capsules, oral liquids, injections, and suspensions.

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