Preparation method of cyclic dipeptide compound and application of cyclic dipeptide compound in inhibition of actinobacillus pleuropneumoniae

Abstract

The invention belongs to the field of veterinary drugs, and discloses a preparation method of a cyclic dipeptide compound and application of the cyclic dipeptide compound in inhibiting actinobacillus pleuropneumoniae, and the chemical structural formula of the compound is # imgabs0 #. The compound is obtained by extracting and purifying honeysuckle stem. The compound has a remarkable effect of inhibiting multi-drug-resistant actinobacillus pleuropneumoniae in vitro, and has a wide prospect when being developed into a veterinary drug for treating porcine contagious pleuropneumonia.

Description

Technical Field

[0001] The invention belongs to the field of veterinary medicine, and particularly relates to a preparation method of a cyclic dipeptide compound and application thereof in inhibiting porcine Actinobacillus pleuropneumoniae. Background Art

[0002] Porcine contagious pleuropneumonia is a highly contagious respiratory infection caused by Actinobacillus pleuropneumoniae. Characterized by acute hemorrhagic pleuropneumonia and high mortality, it has a widespread impact on the global pig industry and is a major threat to intensive pig farming. Actinobacillus pleuropneumoniae is highly susceptible to developing resistance to existing antibiotics, severely impacting the disease's cure rate and causing significant economic losses to pig farmers. Furthermore, food safety issues caused by the overuse of veterinary drugs are already significant. In particular, overuse of antibiotics not only leads to excessive drug residues, impacting meat quality, but also potentially contributing to the emergence of "superbugs." The development of new veterinary drugs is urgent. Traditional Chinese medicine (TCM) offers unique advantages in veterinary drug development (such as low residue levels, multiple targets, and resistance resistance), making it a research hotspot in recent years. TCM holds the potential to become a key solution for antibiotic reduction / replacement.

[0003] Lonicera japonica Lonicera japonica The dried stems and branches of Thunb. It has the effects of clearing heat and detoxifying, dispersing wind and unblocking the meridians. It is clinically used to treat fever caused by febrile diseases, dysentery caused by heat toxins, carbuncles, ulcers, etc. Modern pharmacology shows that honeysuckle vine has multiple activities, including antibacterial, anti-inflammatory, immunomodulatory, and antiviral.

[0004] Cyclic dipeptides are widely found in plants. Their nucleus is a class of structurally stable cyclic peptide compounds formed by the condensation of two amino acids head to tail through a peptide bond. They have multiple biological activities such as antibacterial and anti-tumor activities. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the present invention aims to disclose a preparation method of a cyclic dipeptide compound and its application in inhibiting porcine Actinobacillus pleuropneumoniae.

[0006] The compound of the present invention is characterized by having the following chemical structure: .

[0007] The preparation method of the compound of the present invention comprises the following steps: (1) Extract the honeysuckle vine with 70% ethanol, concentrate the extract until it has no alcohol taste, add ethanol and stir until it is completely dissolved; (2) The solution was added to a XAD-16 macroporous adsorption resin column, eluted with water, discarded the eluate, eluted with 50% ethanol, discarded the eluate, and continued to elute with 68% ethanol, collected the eluate, and concentrated the eluate into an extract; (3) The extract was further separated by silica gel column chromatography, eluted with a mixture of chloroform and methanol in a volume ratio of 50:1, discarded the eluent, and then eluted with a mixture of chloroform and methanol in a volume ratio of 40:1, discarded the eluent, and continued to elute with a mixture of chloroform and methanol in a volume ratio of 35:1. The fractions were combined under the guidance of TLC, and the eluent of the fraction with a dark spot under a UV lamp at 254 nm was collected. The eluent was collected and concentrated to obtain the extract; (4) The extract was further purified by preparative HPLC using C18 as a filler, a detection wavelength of 280 nm, and an acetonitrile-0.1% trifluoroacetic acid solution (64:36) as a mobile phase. The eluate with the main HPLC peak and a peak purity of >98% was collected, concentrated, and lyophilized to obtain the compound of the present invention.

[0008] Beneficial effects: The compound of the present invention has a significant in vitro inhibitory effect on multidrug-resistant Actinobacillus pleuropneumoniae and has the potential to be developed as a drug for treating porcine contagious pleuropneumoniae. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 The nuclear magnetic resonance of the compound of the present invention 1 H-NMR spectrum Figure 2 The nuclear magnetic resonance of the compound of the present invention 13 C-NMR spectrum Figure 3 The NMR HSQC spectrum of the compound of the present invention is Figure 4 The HMBC nuclear magnetic resonance spectrum of the compound of the present invention is Figure 5 High-resolution mass spectrometry of the compounds of the present invention DETAILED DESCRIPTION

[0010] The present invention will be further described below with reference to the examples, but they do not limit the implementation of the present invention.

[0011] Example 1 In vitro activity of the compounds of the present invention against multidrug-resistant Actinobacillus pleuropneumoniae 1. Materials Strains: Multidrug-resistant Actinobacillus pleuropneumoniae strains were provided by the Institute of Animal Husbandry and Veterinary Medicine, Henan Academy of Agricultural Sciences.

[0012] Reagents: DMSO (McLean), purified water (homemade), tryptone soy agar (TSA) medium (McLean), tryptone soy broth (TSB) medium (Beijing Luqiao Technology Co., Ltd.), nicotinamide adenine dinucleotide (NAD) (Aladdin), and fetal bovine serum (mycoplasma-free) (Zhejiang Tianhang Biotechnology Co., Ltd.).

[0013] Main instruments: electronic balance (Mettler XS105DU), clean bench, CO2 incubator (Shenzhen Reward Life Science Co., Ltd., model D180).

[0014] 2. Test methods Preparation of the compound solution of the present invention: Take the compound of the present invention, dissolve it in DMSO to prepare a 10 mg / mL solution, and then add purified water to dilute it to 100 μg / mL.

[0015] Preparation of bacterial solution: Prepare TSA medium plates and TSB liquid medium, add 0.02% NAD and 5% calf serum, and place in a 4°C refrigerator for later use. Take 0.2 mL of the strain and inoculate it into TSB liquid medium, incubate it in a 5% CO2 incubator at 37°C for 24 hours, and dilute the bacteria to a concentration of 10 8 CFU / mL, set aside.

[0016] Minimum inhibitory concentration (MIC) determination: The test tube doubling dilution method was used. Eight sterile test tubes were numbered, with tubes 1 through 7 serving as the experimental group and the eighth serving as the blank control. 1 mL of TSB liquid medium was added to each of tubes 1 through 8. 1 mL of the drug solution was pipetted into tube 1, mixed, and then 1 mL was pipetted into tube 2, and so on to tube 7. After mixing, 1 mL was discarded. The drug concentrations in the test tubes were now 50.00, 25.00, 12.50, 6.25, 3.125, 1.56, and 0.78 μg / mL, respectively. No drug was added to tube 8, and blank solvent was added instead. Use a micropipette to take 0.1 mL of bacterial solution and add it to tubes 1 to 8. Shake well and place in a 37°C incubator for 18 to 24 hours. Use turbidity as an indicator. If the solution in the test tube is turbid, the bacteria are growing well and there is no antibacterial effect. Otherwise, there is an antibacterial effect. The lowest drug dilution concentration at which the solution in the test tube is not turbid is the minimum inhibitory concentration.

[0017] Minimum bactericidal concentration (MBC) determination: Use a micropipette to draw 0.1 mL of the solution from the test tubes in the test group that were determined to have no bacterial growth in the MIC determination and spread it on a TSA culture medium plate. Incubate at 37°C for 18-24 hours and observe the results. The minimum bactericidal concentration is the drug dilution concentration at which the number of bacterial colonies does not exceed 5.

[0018] The MIC of the compound of the present invention against multidrug-resistant Actinobacillus pleuropneumoniae is 1.56 μg / mL, and the MBC is 3.125 μg / mL, indicating that the compound of the present invention has significant in vitro inhibitory activity against multidrug-resistant Actinobacillus pleuropneumoniae.

[0019] Example 2 The preparation method of the compound of the present invention: (1) 50 kg of honeysuckle vine was extracted with 70% ethanol, and the extract was concentrated until there was no alcohol smell. An appropriate amount of ethanol was added and stirred to dissolve it completely; (2) The solution was added to a XAD-16 macroporous adsorption resin column (medicinal material: macroporous resin = 1:1, W / W), eluted with water until colorless, discarded the eluent, eluted with 50% ethanol for 10 column volumes, discarded the eluent, and continued to elute with 68% ethanol for 10 column volumes. The eluent was collected and concentrated into an extract; (3) The extract was further separated by silica gel column chromatography (medicinal material: silica gel = 1:20), eluted with a mixture of chloroform and methanol in a volume ratio of 50:1 for 5 column volumes, discarded the eluent, and then eluted with a mixture of chloroform and methanol in a volume ratio of 40:1 for 10 column volumes, discarded the eluent, and continued to elute with a mixture of chloroform and methanol in a volume ratio of 35:1. The fractions were combined under the guidance of TLC, and the eluent of the fractions with dark spots under the TLC at 254 nm of the ultraviolet lamp was collected and concentrated to the extract; a small amount of extract was dissolved in methanol, and the main spot at 254 nm of the ultraviolet lamp was detected by TLC. If there was more than one main spot, the above silica gel column chromatography steps were repeated until the TLC detected one main spot at 254 nm of the ultraviolet lamp.

[0020] (4) The extract obtained in step (3) was dissolved in methanol to prepare a 100 μg / mL solution, and further purified by preparative HPLC using C18 as a filler, a detection wavelength of 280 nm, and an acetonitrile-0.1% trifluoroacetic acid solution (64:36) as a mobile phase. The eluate with the main HPLC peak and a peak purity of >98% was collected, concentrated, and lyophilized to obtain the compound of the present invention.

[0021] Example 3 Structural confirmation of the compound described in the present invention Pale yellow amorphous powder, soluble in methanol. High-resolution mass spectrometry yielded m / z 505.1600 [M + Na] + (calculated value 505.1600) molecular ion peak, combined with 1 H-NMR spectra and 13 The molecular formula of the compound was inferred to be C 26 H 22 N6O4. 1In the H-NMR spectrum, a total of 18 proton signals are given, of which δ3.39 (overlapped, 2H, H-10',H-10'') and δ3.43 (overlapped, 2H, H-10',H-10'') are methylene proton signals, δ4.92 (dd, 2H, J = 4.9,9.6 Hz, H-3,H-6) is the proton signal on the carbon connected to the nitrogen in the 2,5-diketopiperazine structure of the cyclic dipeptide, δ6.71 (dd, 1H, J = 1.8,8.5 Hz, H-6') , δ6.93 (d, 1H, J = 1.8 Hz, H-4') , δ7.04 (br.t, 1H, J = 7.4 Hz, H-5'') , δ7.10 (br.t, 1H, J = 7.6 Hz, H-6'') , δ7.17(s,2H, H-2',H2'') , δ7.24 (d, 1H, J = 8.5 Hz, H-7') , δ7.33 (br.d, 1H, J = 8.1Hz, H-7'') , δ7.46(s, 1H, H-6'''), δ7.62(br.d, 1H,J=7.9Hz, H-4'') are aromatic proton signals, δ10.88(s, 1H, H-1''') and δ11.08(s, 1H, H-3''') are active proton signals on nitrogen. 13 In the C-NMR spectrum, a total of 21 carbon signals were given, of which δ24.7 (C-10', C-10''), δ56.7 (C-3, C-6), δ108.9 (C-3', C-3''), δ125.1 (C-2', C-2''), and δ171.8 (C-2, C-5) are overlapping carbon signals. The relevant carbon and hydrogen signals were assigned by HSQC and HMBC spectra, as shown in Table 1. Based on the above information, the chemical structure of the compound described in the present invention was determined to be: .

[0022] Table 1 1 H (600 MHz) and 13 C (150 MHz) NMR Spectroscopic Data in DMSO ( δ inppm, J in Hz). .

Claims

1. A compound, characterized in that The chemical structure is as follows: 。 2. A compound according to claim 1, characterized in that The preparation method comprises the following steps: (1) Extract the honeysuckle vine with 70% ethanol, concentrate the extract until it has no alcohol taste, add ethanol and stir until it is completely dissolved; (2) The solution was added to a XAD-16 macroporous adsorption resin column, eluted with water, discarded the eluate, eluted with 50% ethanol, discarded the eluate, and continued to elute with 68% ethanol, collected the eluate, and concentrated the eluate into an extract; (3) The extract was further separated by silica gel column chromatography, eluted with a mixture of chloroform and methanol in a volume ratio of 50:1, discarded the eluent, and then eluted with a mixture of chloroform and methanol in a volume ratio of 40:1, discarded the eluent, and continued to elute with a mixture of chloroform and methanol in a volume ratio of 35:

1. The fractions were combined under the guidance of TLC, and the eluent of the fraction with a dark spot under a UV lamp at 254 nm was collected. The eluent was collected and concentrated to obtain the extract; (4) The extract was further purified by preparative HPLC using C18 as a filler, a detection wavelength of 280 nm, and an acetonitrile-0.1% trifluoroacetic acid solution (64:36) as a mobile phase. The eluate with the main HPLC peak and a peak purity of >98% was collected, concentrated, and lyophilized to obtain the compound of the present invention.

3. The compound according to claim 1, characterized in that It has significant in vitro inhibitory effect on multidrug-resistant Actinobacillus pleuropneumoniae.

4. The compound according to claim 1, characterized in that Used to treat contagious pleuropneumonia in pigs.

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