Application of small molecule compound C607-0736 in preparation of product for treating and / or preventing bacterial infection

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of a small molecule compound C607-0736 in preparation of a product for treating and / or preventing bacterial infection. The small molecule compound C607-0736 can target outer membrane protein OmpV or protein MipA of vibrio cholerae, vibrio parahaemolyticus, vibrio vulnificus, salmonella, klebsiella pneumoniae, vibrio bath, escherichia coli and vibrio anguillarum, so that the colonization ability of pathogenic bacteria in vivo is reduced, and the effect of treating and / or preventing bacterial infection is achieved. The limitation of traditional antibiotic treatment is overcome, a new thought and method are provided for treating and preventing bacterial infection, the action mechanism of C607-0736 is clear, and C607-0736 has the advantages of high selectivity, low side effect, low dosage and high efficiency, and shows a good application prospect in clinical tests.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to the use of a small molecule compound C607-0736 in preparing products for treating and / or preventing bacterial infections. Background Art

[0002] Cholera, caused by Vibrio cholerae, is one of the most threatening diseases to humanity in history, causing millions of deaths. Cholera infection causes massive watery diarrhea and vomiting, which can lead to severe dehydration, hypovolemic shock, and even death if not treated promptly in a short period of time. Vibrio cholerae is the pathogen of cholera, colonizing epithelial cells in the human small intestine and causing the disease by producing a series of virulence factors. Adhesion is the first step in bacterial colonization. Vibrio cholerae can pass through the mucus layer on the intestinal mucosa and adhere to the brush border of the intestinal mucosal epithelial cells in the upper small intestine. The outer membrane protein adhesin plays an important role in this process, and the adhesion mechanism of Vibrio cholerae remains largely unknown.

[0003] Currently, the treatment for Vibrio cholerae infection mainly relies on oral rehydration therapy and antibiotics. However, oral rehydration therapy does not affect the survival and virulence of bacteria, so its effectiveness is limited. The widespread use of antibiotics not only faces the increase of multidrug-resistant Vibrio cholerae, but also may disrupt the balance of intestinal microecology and lead to adverse consequences. Therefore, it is becoming increasingly urgent to develop new, non-antibiotic drugs for the treatment of cholera. Summary of the invention

[0004] In view of this, the purpose of the present invention is to develop a new, non-antibiotic drug for treating cholera. The present invention provides the use of a small molecule compound C607-0736 in the preparation of a product for treating and / or preventing bacterial infection.

[0005] The present invention provides the use of a small molecule compound C607-0736 in the preparation of a product for treating and / or preventing bacterial infection. The molecular structure of the small molecule compound C607-0736 is C 26 H 26 N 2 O 4 Its chemical name is N-{2-[(furan-2-yl)methyl(propan-2-yl)amino]ethyl}-1-oxo-4-phenyl-1H-isochromene-3-carboxamide, and its relative molecular mass is 430.5 g / mol.

[0006] Preferably, the bacteria include pathogenic bacteria containing outer membrane protein OmpV (Outer membrane protein V) or protein MipA (MltA-interacting protein).

[0007] Preferably, the pathogens containing outer membrane protein OmpV (Outer membrane protein V) or protein MipA (MltA-interacting protein) include one or more of Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Salmonella, Klebsiella pneumoniae, Vibrio fusoni, Escherichia coli and Vibrio anguillarum.

[0008] Preferably, the concentration of the small molecule compound C607-0736 is ≥20 μM.

[0009] Preferably, the treatment and / or prevention of bacterial infection comprises providing the small molecule compound C607-0736 to the patient and / or susceptible population via an oral or non-oral route.

[0010] The present invention also provides a product for treating and / or preventing bacterial infection, wherein the active ingredient of the product is a small molecule compound C607-0736, and the molecular structure of the small molecule compound C607-0736 is C 26 H 26 N 2 O 4 Its chemical name is N-{2-[(furan-2-yl)methyl(propan-2-yl)amino]ethyl}-1-oxo-4-phenyl-1H-isochromene-3-carboxamide, and its relative molecular mass is 430.5 g / mol.

[0011] Preferably, the bacteria include pathogenic bacteria containing outer membrane protein OmpV (Outer membrane protein V) or protein MipA (MltA-interacting protein).

[0012] Preferably, the pathogens containing outer membrane protein OmpV (Outer membrane protein V) or protein MipA (MltA-interacting protein) include one or more of Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Salmonella, Klebsiella pneumoniae, Vibrio fusoni, Escherichia coli and Vibrio anguillarum.

[0013] Preferably, the concentration of C607-0736 used in the product is ≥10 mg / kg.

[0014] Preferably, the product includes one or more of medicines, cleansers, creams, ointments, pastes, gels, sprays, animal feed additives and vaccines; the dosage form of the medicine includes one or more of solid dosage forms, liquid dosage forms and semi-solid dosage forms; the solid dosage forms include one or more of capsules, tablets, pastes and powders; the liquid dosage forms include one or more of solutions, suspensions and emulsions; the semi-solid dosage forms include one or more of ointments, lotions, creams and gel patches.

[0015] Beneficial effects of the present invention:

[0016] The present invention utilizes computer-aided drug design technology to screen a small molecule compound specific drug C607-0736, which can specifically target the outer membrane protein OmpV of Vibrio cholerae, thereby effectively blocking the adhesion and colonization process of Vibrio cholerae to intestinal epithelial cells. Protein MipA (MltA-interacting protein) is highly homologous to the outer membrane protein OmpV (Outer membrane protein V), belongs to the MipA / OmpV family, and is highly conserved in a variety of pathogens such as Vibrio parahaemolyticus, Vibrio vulnificus, Salmonella, Klebsiella pneumoniae, Vibrio thrombolyticus, Escherichia coli, and Vibrio anguillarum. Therefore, C607-0736 can effectively block the in vivo colonization process of pathogens containing outer membrane protein OmpV or protein MipA, reduce the host load, and resist bacterial infection.

[0017] The small molecule compound C607-0736 used in the present invention complies with Lipinski's five rules, that is, the molecular weight is less than 500; the number of hydrogen bond donors is less than 5; the number of hydrogen bond acceptors is less than 10; the lipid-water partition coefficient is less than 5; the number of rotatable bonds does not exceed 10, and has better pharmacokinetic properties, higher bioavailability during metabolism in the body, and is therefore more likely to become an oral drug.

[0018] The present invention innovatively discovered that the small molecule compound C607-0736 has therapeutic potential in treating infections caused by a variety of pathogens, and can have a therapeutic effect at a low dose (20 μM), effectively reducing the intestinal colonization ability of bacteria, aiming to make up for the shortcomings of existing treatment methods and provide patients with more effective and targeted treatment options.

[0019] In terms of basic research, the molecular mechanism of C607-0736 in preventing and treating infections caused by various pathogens such as Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus will be fully explored, the common targets of C607-0736 in treating bacterial infections will be analyzed, and the function of C607-0736 in other pathogens will be explored. This project is of great significance for further exploring the pathogenicity and evolutionary mechanisms of pathogens such as Vibrio cholerae and developing targeted preventive and therapeutic drugs.

[0020] The development of this small compound specific drug not only provides new ideas and methods for the treatment of Vibrio cholerae infection, but also overcomes many limitations of traditional antibiotic treatment. Its high selectivity, low side effects, low dose, high efficiency and clear mechanism of action make this drug show good application prospects in clinical trials. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the following is a brief introduction to the drawings required for describing the embodiment. Among them:

[0022] Figure 1 is the chemical structure diagram of C607-0736 2D;

[0023] Figure 2 The effect of different concentrations of C607-0736 on the growth of different bacteria;

[0024] Figure 3 The effect of 20 μM C607-0736 on the cell adhesion ability of different bacteria;

[0025] Figure 4 The effect of 20 μM C607-0736 on the in vivo colonization ability of different bacteria;

[0026] Figure 5 The effect of 20 μM C607-0736 on the qRT-PCR expression of virulence genes (tcpP, toxT, ctxA and tcpA) produced by V. cholerae in AKI culture medium;

[0027] Figure 6 These are H&E staining images of the heart, liver, spleen, lung, and kidney of adult mice after two weeks of treatment with 10 mg / kg of C607-0736. DETAILED DESCRIPTION

[0028] The present invention provides the use of a small molecule compound C607-0736 in the preparation of a product for treating and / or preventing bacterial infection. The molecular structure of the small molecule compound C607-0736 is C 26 H 26 N 2 O 4Its chemical name is N-{2-[(furan-2-yl)methyl(propan-2-yl)amino]ethyl}-1-oxo-4-phenyl-1H-isochromene-3-carboxamide, and its relative molecular mass is 430.5 g / mol.

[0029] The 2D chemical structure diagram of the small molecule compound C607-0736 is as follows Figure 1 As shown, the small molecule compound C607-0736 used in the present invention complies with Lipinski's five rules, that is, the molecular weight is less than 500; the number of hydrogen bond donors is less than 5; the number of hydrogen bond acceptors is less than 10; the lipid-water partition coefficient is less than 5; the number of rotatable bonds does not exceed 10, and it has better pharmacokinetic properties and higher bioavailability during metabolism in the body, so it is more likely to become an oral drug. By targeting the OmpV protein of Vibrio cholerae, it can inhibit the ability of Vibrio cholerae to adhere to host cells and OMV to deliver cholera toxin, thereby reducing the accumulation of cholera toxin in the host body, thereby reducing the colonization ability of Vibrio cholerae, and achieving the purpose of treating Vibrio cholerae infection.

[0030] In the present invention, the bacteria preferably include pathogens containing outer membrane protein OmpV (Outer membrane protein V) or protein MipA (MltA-interacting protein), and the pathogens containing outer membrane protein OmpV (Outermembrane protein V) or protein MipA (MltA-interacting protein) preferably include one or more of Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Salmonella, Klebsiella pneumoniae, Vibrio fusoni, Escherichia coli and Vibrio anguillarum; the concentration of the small molecule compound C607-0736 is preferably ≥20 μM. The OmpV (Outer membrane protein V) mentioned in the present invention is highly homologous to the protein MipA (MltA-interacting protein) and belongs to the MipA / OmpV family; the small molecule compound C607-0736 can target OmpV or protein MipA of Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Salmonella, Klebsiella pneumoniae, Vibrio fusoni, Escherichia coli and Vibrio anguillarum, thereby reducing the colonization ability of pathogens in the body, thereby achieving the purpose of treating bacterial infections.

[0031] In the present invention, the treatment and / or prevention of bacterial infection preferably includes providing small molecule compound C607-0736 to patients and / or susceptible people through oral or non-oral routes. The non-oral route is preferably parenteral administration, and it is further preferred that the small molecule compound C607-0736 is used as an additive to hand sanitizer, hand cream and / or sunscreen, and the small molecule compound C607-0736-related preparations are provided to susceptible people through non-oral routes to control the transmission route and protect susceptible people; the oral route preferably also includes preparing the small molecule compound C607-0736 alone or in combination with other drugs in the form of tablets or capsules, and entering the body through oral administration, and providing the small molecule compound C607-0736-related preparations to susceptible people and / or patients to prevent or treat bacterial infections.

[0032] The present invention also provides a product for treating and / or preventing bacterial infection, wherein the active ingredient of the product is a small molecule compound C607-0736, and the molecular structure of the small molecule compound C607-0736 is C 26 H 26 N 2 O 4 Its chemical name is N-{2-[(furan-2-yl)methyl(propan-2-yl)amino]ethyl}-1-oxo-4-phenyl-1H-isochromene-3-carboxamide, and its relative molecular mass is 430.5 g / mol.

[0033] In the present invention, the bacteria preferably include pathogens containing outer membrane protein OmpV (Outer membrane protein V) or protein MipA (MltA-interacting protein), and the pathogens containing outer membrane protein OmpV (Outermembrane protein V) or protein MipA (MltA-interacting protein) preferably include one or more of Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Salmonella, Klebsiella pneumoniae, Vibrio fusoni, Escherichia coli and Vibrio anguillarum.

[0034] In the present invention, the concentration of C607-0736 in the product is preferably ≥10 mg / kg; the product preferably includes one or more of a drug, a cleanser, a cream, an ointment, an ointment, a gel, a spray, an animal feed additive, and a vaccine; the drug preferably includes a human drug and / or a veterinary drug; the dosage form of the drug preferably includes one or more of a solid dosage form, a liquid dosage form, and a semisolid dosage form; the solid dosage form preferably includes one or more of a capsule, a tablet, an ointment, and a powder, the liquid dosage form preferably includes one or more of a solution, a suspension, and an emulsion, and the semisolid dosage form preferably includes one or more of an ointment, a lotion, a cream, and a gel patch. The examples show that after mice took C607-0736 at a dose of 10 mg / kg for 2 consecutive weeks, no obvious damage was caused to the heart, liver, spleen, lung, and kidney tissues, indicating that the small molecule compound C607-0736 has biological safety.

[0035] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0036] In an embodiment of the present invention:

[0037] Caco-2 cells were purchased from Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China);

[0038] Five-day-old CD-1 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China);

[0039] TRIzol reagent: 15596026; Invitrogen, Waltham, MA, USA;

[0040] NanoDrop 2000 spectrophotometer was purchased from Thermo Fisher Scientific, MA, USA;

[0041] PrimeScript TM RT Reagent Kit and gDNA Eraser were purchased from Takara, Kusatsu, Japan;

[0042] Unless otherwise specified, other raw materials used are common commercially available products.

[0043] Example 1

[0044] Growth curve experiment

[0045] (1) Vibrio cholerae O1, Vibrio cholerae O139, Salmonella and Klebsiella pneumoniae were inoculated in 20 mL LB medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl) at a volume ratio of 1:1000; Vibrio parahaemolyticus and Vibrio vulnificus were inoculated in 20 mL LBS medium (10 g / L tryptone, 5 g / L yeast extract, 30 g / L NaCl) at a volume ratio of 1:1000, and cultured at 37°C, 180 rpm for 12 h.

[0046] (2) Take 1 mL of overnight bacteria from each bacterial solution, wash it three times with sterile PBS and adjust the OD to 600 =1.0.

[0047] (3) The bacterial solution obtained in step (2) was transferred to LB medium containing 0 μM, 1 μM, 10 μM, 20 μM and 50 μM C607-0736 inhibitor at a volume ratio of 1:1000, mixed and added to a sterile 96-well plate with 200 μL. Each bacterium was cultured in triplicate.

[0048] (4) Place the 96-well plate in the microbial monitor and set the instrument to measure the OD every 30 minutes. 600 The value was tested for a total of 24 hours.

[0049] (5) Collect data and plot the effects of different concentrations of C607-0736 on the growth of different bacteria. Figure 2 As shown, Figure 2 a in the middle represents Vibrio cholerae O1, Figure 2 b in the middle represents Vibrio cholerae O139, Figure 2 c in the middle represents Vibrio parahaemolyticus, Figure 2 The d in the middle represents Vibrio vulnificus, Figure 2 The "e" in the middle represents Salmonella. Figure 2 The f in the middle represents Klebsiella pneumoniae.

[0050] Depend on Figure 2 It can be seen that C607-0736 has no effect on the growth of Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Salmonella and Klebsiella pneumoniae.

[0051] Example 2

[0052] In vitro adhesion assay

[0053] (1) Prepare Caco-2 cells. Caco-2 resuscitation: thaw frozen Caco-2 cells at 37°C and quickly transfer them into a cell bottle. Add 10 mL to 15 mL of DMEM (containing 10% FBS at equilibrium concentration) and culture for 2 days. Take 5 mL of trypsin and place in a CO 2(5%), lyse the cells at 37°C for 5 min, discard the trypsin, add 10 mL to 15 mL of DMEM, and mix by pipetting. 5 Plate cells in 24-well plates.

[0054] (2) Inoculate Vibrio cholerae O1, Vibrio cholerae O139, Salmonella, and Klebsiella pneumoniae in 20 mL LB medium at a volume ratio of 1:1000; inoculate Vibrio parahaemolyticus and Vibrio vulnificus in 20 mL LBS medium at a volume ratio of 1:1000. Incubate at 37°C, 180 rpm for 12 h.

[0055] (3) Use DMEM without FBS to adjust the overnight bacterial concentration to 5×10 6 / mL, infecting Caco-2 cells. Caco-2 cells were divided into Control group and C607-0736 group, each group was set up with 3 replicates, and treated as follows:

[0056] Control group: 1 mL of diluted bacterial solution was added to each well of Caco-2 cells.

[0057] C607-0736 group: 1 mL of diluted bacterial solution was added to each well of Caco-2 cells, and then C607-0736 inhibitor was added at a final concentration of 20 μM.

[0058] (4) Take another 10 -3 , 10 -4 100 μL of each gradient bacterial solution was spread on LB plates and cultured at 37°C for 12 h. The colonies were counted as input.

[0059] (5) Caco-2 cells in the control group and C607-0736 group were cultured in an incubation chamber containing 5% CO 2 Incubate at 37°C for 120 min. Discard the supernatant and wash three times with PBS.

[0060] (5) Add 200 μL of 1% TritonX-100 to each well and shake at room temperature for 5 min. Add 800 μL of PBS and pipette the bacterial solution to lyse the cells.

[0061] (7) Take 10 -1 , 10 -2 100 μL of each gradient bacterial solution was spread on LB plates and cultured at 37°C for 12 h. The colonies were counted as output.

[0062] (8) Calculate the adhesion rate according to the following formula. The result is as follows: Figure 3 As shown, Figure 3 a in the middle represents Vibrio cholerae O1, Figure 3b in the middle represents Vibrio cholerae O139, Figure 3 c in the middle represents Vibrio parahaemolyticus, Figure 3 The d in the middle represents Vibrio vulnificus, Figure 3 The "e" in the middle represents Salmonella. Figure 3 The f in the middle represents Klebsiella pneumoniae.

[0063] Adhesion rate: Adhesion rate = the percentage of the output colony count to the input colony count

[0064] Depend on Figure 3 It can be seen that C607-0736 can significantly inhibit the adhesion ability of Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Salmonella and Klebsiella pneumoniae.

[0065] Example 3

[0066] Intestinal colonization experiment

[0067] 1. Vibrio cholerae O1, Vibrio cholerae O139

[0068] This study used an intestinal colonization experiment in 3-day-old suckling mice (of either sex) of the CD-1 mouse strain to evaluate the therapeutic effect of C607-0736 against Vibrio cholerae O1 and Vibrio cholerae O139.

[0069] Vibrio cholerae O1 and Vibrio cholerae O139 were inoculated into 20 mL LB medium at a volume ratio of 1:1000 and cultured at 37°C and 180 rpm for 12 h.

[0070] The mice were divided into a Control group and a C607-0736 group, with 6 mice in each group, and treated as follows:

[0071] Control group: LD adjusted overnight bacterial OD 600 The concentration was 1.0, and two gradients were made for dilution. 10 μL of bacterial solution was taken to feed mice;

[0072] C607-0736 group: LD to adjust overnight bacterial OD 600 The concentration of the bacterial suspension was 1.0, and two gradients were diluted. 10 μL of the bacterial suspension was fed to mice. Subsequently, the small molecule inhibitor C607-0736 was orally administered once at a dose of 10 mg / kg.

[0073] After 22 h of feeding the mice with the bacterial solution, the mice were euthanized, and the small intestine of each mouse was removed, weighed, homogenized, and inoculated on LB agar plates containing polymyxin B (30 μg / μl) and cultured at 37°C for 12 h.

[0074] 2. Vibrio parahaemolyticus and Vibrio vulnificus

[0075] In this experiment, intestinal colonization experiments were performed on 6-week-old female C57BL / 6 mice or 4-week-old female ICR mice to evaluate the therapeutic effect of C607-0736 against Vibrio parahaemolyticus or Vibrio vulnificus.

[0076] The mice were divided into a Control group and a C607-0736 group, with 6 mice in each group, and treated as follows:

[0077] Control group: Mice were gavaged with streptomycin (20 mg) and then orally administered with 10 9 CFU of bacteria.

[0078] Group C607-0736: Mice were gavaged with streptomycin (20 mg) and then orally administered with 10 9 CFU of bacteria, followed by oral administration of C607-0736 at a dose of 10 mg / kg. 22 h after infection, the mice were euthanized, and the small intestine of each mouse was removed, weighed, homogenized, and plated on LB agar plates.

[0079] 3. Salmonella

[0080] In this study, the intestinal colonization experiment of 6-week-old female BALB / c mice was used to evaluate the therapeutic effect of C607-0736 against Salmonella.

[0081] The mice were divided into a Control group and a C607-0736 group, with 6 mice in each group, and treated as follows:

[0082] Control group: The mice were deprived of water and food for 12 hours and then gavaged with streptomycin (20 mg). Water and food were restored 4 hours after gavage. After water and food were restored for 18 hours, they were deprived of water and food again. After being deprived of water and food again for 2.5 hours, they were given 10 mg of streptomycin orally. 7 CFU of bacteria;

[0083] C607-0736 group: mice were deprived of water and food for 12 hours and then gavaged with streptomycin (20 mg). Water and food were restored 4 hours after gavage. Water and food were deprived again 18 hours after restoration. After deprivation of water and food for 2.5 hours, 10 mg of streptomycin was taken orally. 7 CFU of bacteria. Then C607-0736 was orally administered once at a dose of 10 mg / kg.

[0084] Water and food were restored 4 h after infection with Salmonella , and mice were euthanized 4 days later, and the colon of each mouse was removed, weighed, homogenized, and plated on LB agar plates.

[0085] 4. Klebsiella pneumoniae

[0086] In this study, intestinal colonization experiments were performed in 6-week-old female BALB / c mice to evaluate the therapeutic effect of C607-0736 against Klebsiella pneumoniae.

[0087] The mice were divided into a Control group and a C607-0736 group, with 6 mice in each group, and treated as follows:

[0088] Control group: Mice were gavaged with streptomycin (20 mg) and then orally administered with 10 9 CFU of bacteria;

[0089] Group C607-0736: Mice were gavaged with streptomycin (20 mg) and then orally administered with 10 9 CFU of bacteria, followed by oral administration of C607-0736 once at a dose of 10 mg / kg.

[0090] 22 h after infection with K. pneumoniae , mice were euthanized, and the colon of each mouse was removed, weighed, homogenized, and plated on LB agar plates.

[0091] Determine and calculate the colonization amount of each group of bacteria on the LB agar plate. The results are as follows: Figure 4 As shown, Figure 4 a in the middle represents Vibrio cholerae O1, Figure 4 b in the middle represents Vibrio cholerae O139, Figure 4 c in the middle represents Vibrio parahaemolyticus, Figure 4 The d in the middle represents Vibrio vulnificus, Figure 4 The "e" in the middle represents Salmonella. Figure 4 The f in the middle represents Klebsiella pneumoniae.

[0092] Colonization amount = log10 (total number of colonies / weight of tissue taken),

[0093] The total number of colonies was calculated by multiplying the number of colonies on the plate by the dilution factor.

[0094] Depend on Figure 4 It can be seen that C607-0736 can significantly inhibit the intestinal colonization ability of Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Salmonella and Klebsiella pneumoniae.

[0095] Example 4

[0096] RNA extraction and qRT-PCR experiments

[0097] (1) Inoculate Vibrio cholerae O1 in 20 mL LB medium at a volume ratio of 1:1000 and culture at 37°C and 180 rpm for 12 h to obtain overnight bacteria;

[0098] (2) The overnight bacteria were divided into a WT group and a C607-0736 group, with three replicates in each group and treated as follows:

[0099] WT group: overnight bacteria were transferred to AKI medium without C607-0736 inhibitor (tryptone 15 g / L yeast extract 4 g / L NaCl 5 g / L, NaHCO) at a volume ratio of 1:100. 3 100g / L);

[0100] C607-0736 group: overnight bacteria were transferred to AKI medium containing C607-0736 inhibitor at a final concentration of 20 μM at a volume ratio of 1:100;

[0101] The cells were cultured under anaerobic conditions for 6 h and the bacteria were collected.

[0102] (3) In a liquid nitrogen environment, the sample was quickly ground and Trizol was added to lyse the cells.

[0103] (4) After Trizol lysis, collect the supernatant, add an equal volume of isopropanol, centrifuge and discard the supernatant.

[0104] (5) Wash the precipitate twice with enzyme-free 75% ethanol solution and dry the RNA precipitate at room temperature.

[0105] (6) The precipitate was dissolved in enzyme-free water, and after measuring the concentration, cDNA was synthesized using StarScript III RT MasterMix. SYBR Green fluorescent dye was used for qRT-PCR analysis to analyze the effect of C607-0736 on the expression of Vibrio cholerae virulence genes (tcpP, toxT, ctxA and tcpA).

[0106] To normalize the sample data, rrsA was used as a normalization control and the -ΔΔCT Methods The relative expression levels were calculated as fold change values. The experiment was repeated three times and the results are shown in Figure 5 shown.

[0107] Depend on Figure 5 It can be seen that C607-0736 does not affect the expression ability of Vibrio cholerae virulence genes.

[0108] Example 5

[0109] Biosafety Experiment

[0110] In this study, intestinal colonization experiments were performed on 8-week-old female C57BL / 6 mice to evaluate the damage of C607-0736 to mice.

[0111] Eight-week-old female C57BL / 6 mice were divided into a Control group and a C607-0736 group, with 6 mice in each group, and treated as follows:

[0112] Control group: gavage with normal saline (10 mg / kg) every day for 2 weeks;

[0113] C607-0736 group: C607-0736 (10 mg / kg) was gavaged daily for 2 weeks.

[0114] The body weight of mice was monitored weekly. The major organs, including heart, liver, spleen, lung, and kidney, were collected after the final treatment and fixed in 10% neutral buffered formalin overnight and embedded in paraffin. The paraffin-embedded sections were dewaxed and stained with hematoxylin-eosin to observe the severity of inflammation in the stained sections. Figure 6 shown.

[0115] Depend on Figure 6 It can be seen that no obvious histological abnormalities were observed in the heart, liver, spleen, lung and kidney tissues of the experimental animals. It can be seen that after mice took C607-0736 at a dose of 10 mg / kg for 2 consecutive weeks, there was no obvious toxic effect on the heart, liver, spleen, lung and kidney tissues, indicating that the small molecule compound C607-0736 has biosafety.

[0116] It should be noted that the description of these embodiments is only used to help understand the present invention, but does not constitute a limitation of the present invention. The above are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

Claims

1. Use of the small molecule compound C607-0736 in the preparation of a product for treating and / or preventing bacterial infection, characterized in that: The molecular structure of the small molecule compound C607-0736 is C 26 H 26 N2O4, chemical name is N-{2-[(furan-2-yl)methyl(propan-2-yl)amino]ethyl}-1-oxo-4-phenyl-1H-isochromene-3-carboxamide, relative molecular mass is 430.5g / mol.

2. The use according to claim 1, characterized in that: The bacteria include pathogenic bacteria containing outer membrane protein OmpV (Outermembrane protein V) or protein MipA (MltA-interacting protein).

3. The use according to claim 2, characterized in that: The pathogenic bacteria containing outer membrane protein OmpV (Outer membrane protein V) or protein MipA (MltA-interacting protein) include one or more of Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Salmonella, Klebsiella pneumoniae, Vibrio fusoni, Escherichia coli and Vibrio anguillarum.

4. The use according to claim 1, characterized in that: The concentration of the small molecule compound C607-0736 is ≥20 μM.

5. The use according to claim 1, characterized in that: The method for treating and / or preventing bacterial infection comprises providing the small molecule compound C607-0736 to patients and / or susceptible people through oral or non-oral routes.

6. A product for treating and / or preventing bacterial infection, characterized in that: The active ingredient of the product is a small molecule compound C607-0736, and the molecular structure of the small molecule compound C607-0736 is C 26 H 26 N2O4, chemical name is N-{2-[(furan-2-yl)methyl(propan-2-yl)amino]ethyl}-1-oxo-4-phenyl-1H-isochromene-3-carboxamide, relative molecular mass is 430.5g / mol.

7. The product according to claim 6, characterized in that The bacteria include pathogenic bacteria containing outer membrane protein OmpV (Outermembrane protein V) or protein MipA (MltA-interacting protein).

8. The product according to claim 7, characterized in that The pathogenic bacteria containing outer membrane protein OmpV (Outer membrane protein V) or protein MipA (MltA-interacting protein) include one or more of Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Salmonella, Klebsiella pneumoniae, Vibrio fusoni, Escherichia coli and Vibrio anguillarum.

9. The product according to claim 6, characterized in that The usage concentration of C607-0736 in the product is ≥10 mg / kg.

10. The product according to any one of claims 6 to 9, characterized in that: The product includes one or more of medicines, cleansers, creams, ointments, pastes, gels, sprays, animal feed additives and vaccines; the dosage form of the medicine includes one or more of solid dosage forms, liquid dosage forms and semi-solid dosage forms; the solid dosage form includes one or more of capsules, tablets, pastes and powders; the liquid dosage form includes one or more of solutions, suspensions and emulsions; the semi-solid dosage form includes one or more of ointments, lotions, creams and gel patches.

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