Water-soluble organic ionic salt as well as synthesis method and application thereof

Abstract

The invention discloses a water-soluble organic ionic salt and a synthesis method and application thereof, and belongs to the technical field of antibiosis. Comprising the following steps: 1) preparing a first intermediate QBr; (2) preparing a second intermediate YDOBr; and 3) adding YDOBr into pyridine, uniformly stirring, reacting at 75-85 DEG C for 16-20 hours, cooling the system to room temperature, separating out orange red solid, carrying out suction filtration, and washing a filter cake with dichloromethane to obtain orange solid, namely the target product water-soluble organic ion salt YDOPy. The synthesis method of the water-soluble organic ionic salt is simple and convenient, and pyridine ionic salt is introduced based on a core element of indandione to realize the antibacterial property of the water-soluble organic ionic salt.

Description

Technical Field

[0001] The invention belongs to the technical field of antibacterial, and specifically relates to a water-soluble organic ion salt and a synthesis method and application thereof. Background Art

[0002] Staphylococcus aureus (SA) is a Gram-positive pathogen that can be spread through air, water, food and living media. The latest monitoring of China's bacterial resistance monitoring network CHINET shows that SA accounts for the third proportion of bloodstream infection. Gram-positive cocci are the main pathogens of BSI in children, and the detection rate of SA ranks in the top three. Data show that the mortality rate of hospitalization for Staphylococcus aureus bloodstream infection can be as high as 20% to 30%. When conditions are right, it can usually cause a variety of diseases. One is an invasive disease, which is mainly manifested as purulent infection of various organs, such as skin and soft tissue infection, bone and joint infection, urinary tract infection, infective endocarditis, etc., and can also lead to systemic infections such as sepsis, septic syndrome, septic shock, etc.; the other is a toxic disease, which is caused by external toxins, and the main symptoms are acute gastroenteritis, including nausea, vomiting and diarrhea. In addition, the bacteria is closely related to medical device-related infections and deep infections, such as catheter-related infections, osteomyelitis, endocarditis, etc., which increase the patient's mortality rate and risk of poor prognosis, posing a major threat to human health and global public health.

[0003] Disinfection plays an important role in killing pathogenic microorganisms and controlling infections, and is an important means of cutting off the spread of diseases. Long-term use of disinfectants often induces bacterial resistance. Long-term exposure to ultraviolet rays can cause skin damage, and in severe cases, it can cause skin cancer. Due to the defects of traditional disinfectants and disinfection technologies, there is an urgent need to develop new bacterial control methods to eliminate bacterial contamination. Organic small molecules have the characteristics of simple synthesis and flexible and controllable structural design. The physicochemical properties can be adjusted by introducing specific functional groups to achieve their antibacterial properties. Summary of the invention

[0004] In view of the above problems existing in the prior art, the purpose of the present invention is to design and provide a technical solution for a water-soluble organic ion salt and its synthesis method and use. The synthesis method is simple, and pyridinium ion salt is introduced based on the core unit of indanedione to achieve its antibacterial property.

[0005] The water-soluble organic ion salt is characterized by being a compound shown in the following formula:

[0006]

[0007] The method for synthesizing the water-soluble organic ion salt is characterized by comprising the following steps:

[0008] 1) Equimolar amounts of 5-bromo-2-(dimethylamino)benzaldehyde and 1,2-dibromoethane are mixed in acetone, and reflux reaction is carried out under nitrogen protection, and the plate is monitored. After the raw material reaction is complete, purification treatment is performed; the reaction system is extracted with dichloromethane and water, and the organic phase is collected and spin-dried to obtain a crude product, which is then purified by column chromatography, using 200-300 mesh silica gel powder as the stationary phase, and dichloromethane and ethyl acetate are prepared as the mobile phase in a volume ratio of 9-11:1 to obtain the first intermediate 2-(2-bromoethoxy)-4-(dimethylamino)benzaldehyde, referred to as QBr, with a chemical formula of C 11 H 14 BrNO2;

[0009] 2) Equimolar amounts of QBr, sodium hydroxide and 1,3-indanedione are mixed in ethanol, and the mixture is refluxed under nitrogen protection for 10-14 hours. The reaction system is extracted with dichloromethane and water, and the organic phase is collected and dried to obtain a crude product. The crude product is then purified by column chromatography, using 200-300 mesh silica gel powder as the stationary phase and dichloromethane and ethyl acetate as the mobile phase in a volume ratio of 4-6:1 to obtain the second intermediate 2-(2-(2-bromoethoxy)-4-(dimethylamino)benzylidene)-1H-indene-1,3(2H)-dione, referred to as YDOBr, with a chemical formula of C 20 H 18 BrNO3;

[0010] 3) Add YDOBr to pyridine and stir evenly, use pyridine as solvent and reactant, react at 75-85°C for 16-20 hours, cool the system to room temperature, precipitate orange-red solid, filter, and rinse the filter cake with dichloromethane to obtain an orange solid, which is the target product, water-soluble organic ion salt (1-(2-(5-(dimethylamino)-2-((1,3-diketo-1,3-dihydro-2H-inden-2-ylidene)methyl)phenoxy)ethyl)pyridin-1-ium, referred to as YDOPy, with a chemical formula of C 25 H 23 N2O3 + Br - ;

[0011] Its synthesis path is:

[0012]

[0013] The method for synthesizing the water-soluble organic ion salt is characterized in that in step 1), the volume ratio of dichloromethane to ethyl acetate is 10:1.

[0014] The synthesis method of the water-soluble organic ion salt is characterized in that in step 2), the reflux reaction time is 12 hours, and the volume ratio of dichloromethane to ethyl acetate is 5:1.

[0015] The method for synthesizing the water-soluble organic ion salt is characterized in that in step 3), the reaction temperature is 80° C. and the reaction time is 18 hours.

[0016] The water-soluble organic ion salt is used in the preparation of antibacterial preparations.

[0017] The water-soluble organic ion salt has antibacterial application to Staphylococcus aureus.

[0018] The water-soluble organic ion salt and its synthesis method and use have a simple synthesis method, and the pyridinium ion salt is introduced based on the core unit of indanedione to achieve its antibacterial properties. The strain inhibition zone size of the water-soluble organic ion salt YDOPy is 10.1 mm; the MIC value of YDOPy is 31.25 μg / mL; YDOPy with a concentration of one times the MIC can reduce the virulence of MRSA infection in vivo and play an anti-MRSA role in vivo; after being treated with YDOPy, the cell wall of Staphylococcus aureus has been destroyed or the synthesis stage of the bacterial cell wall has been inhibited, showing an antibacterial effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of YDOPy obtained in Example 1 of the present invention;

[0020] Figure 2 It is the inhibition zone diagram of Experiment 1 of the present invention;

[0021] Figure 3 This is a graph showing the plate colony count results of Experiment 2 of the present invention;

[0022] Figure 4 This is a survival curve diagram of larvae in different groups in Experiment 3 of the present invention;

[0023] Figure 5 This is the electron microscope image of Experiment 4 of the present invention. DETAILED DESCRIPTION

[0024] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments and drawings. It should be understood by those skilled in the art that the content described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0025] Example 1

[0026] 1) Equimolar amounts of 5-bromo-2-(dimethylamino)benzaldehyde and 1,2-dibromoethane were mixed in acetone, and the reaction was refluxed under nitrogen protection, and the plate was monitored. After the reaction of the raw materials was complete, purification was performed. The reaction system was extracted with dichloromethane and water, and the organic phase was collected and dried to obtain a crude product. Then, column chromatography was used for purification, using 200-300 mesh silica gel powder as the stationary phase, and dichloromethane and ethyl acetate were prepared as the mobile phase in a volume ratio of 10:1 to obtain the first intermediate QBr;

[0027] The molecular formula of QBr with the atomic number and its precise NMR data are as follows:

[0028]

[0029] 1 H NMR (400MHz, Chloroform-d) δ10.22(d,J=0.7Hz,1H,10),7.74(d,J=8.9Hz,1H,1),6.35(ddd,J=8.9,2.3,0. 8Hz,1H,3),6.04(d,J=2.2Hz,1H,5),4.38(t,J=6.2Hz,2H,12),3.68(t,J=6.2Hz,2H,13),3.07(s,6H,8,9).

[0030] 2) An equimolar amount of QBr and 1,3-indanedione were mixed in ethanol, 1 equivalent of NaOH was added, and the mixture was refluxed under nitrogen protection for 12 hours. The reaction system was extracted with dichloromethane and water, and the organic phase was collected and dried to obtain a crude product. The crude product was then purified by column chromatography using 200-300 mesh silica gel powder as the stationary phase and dichloromethane and ethyl acetate as the mobile phase in a volume ratio of 5:1 to obtain the second intermediate YDOBr;

[0031] The YDOBr molecular formula with the atomic number and its precise NMR data are as follows:

[0032]

[0033] 1 H NMR(400MHz,Chloroform-d)δ9.32(d,J=9.2Hz,1H,2),8.46(s,1H,20),7.93–7.88(m,2H,17,18),7.74–7.67(m,2H,16,19), 6.50(dd,J=9.2,2.4Hz,1H,5), 6.14(d,J=2.4Hz,1H,7), 4.41(t,J=6.6Hz,2H,22), 3.79–3.73(m,2H,23), 3.16(s,6H,10,11).

[0034] 3) YDOBr was added to pyridine and stirred evenly. Pyridine was used as a solvent and reactant. The reaction was carried out at 80° C. for 18 hours. The system was cooled to room temperature to precipitate an orange-red solid. The solid was filtered and the filter cake was washed with dichloromethane to obtain an orange solid, which was the target product, a water-soluble organic ion salt YDOPy.

[0035] The YDOPy molecular formula with atomic number and its precise NMR data are as follows:

[0036]

[0037] 1 H NMR(400MHz,Methanol-d4)δ9.32–9.22(m,3H,2,26,30),8.71–8.64(m,1H,28) ,8.31(t,J=7.1Hz,2H,27,29),8.12(s,1H,20),7.92–7.84(m,2H,17,18),7.80( dd,J=5.5,3.1Hz,2H,16,19),6.50(dd,J=9.3,2.4Hz,1H,5),6.18(d,J=2.4Hz, 1H,7),5.25(t,J=4.9Hz,2H,22),4.68(t,J=4.8Hz,2H,23),3.17(s,6H,10,11).

[0038] In Example 1, in step 1), the volume ratio of dichloromethane:ethyl acetate is 11:1 or 9:1; in step 2), the reflux reaction time is 10 hours or 14 hours, and the volume ratio of dichloromethane:ethyl acetate is 4:1 or 6:1; in step 3), the reaction temperature is 75°C or 85°C, and the reaction time is 16 hours or 20 hours; the target product, water-soluble organic ion salt YDOPy, can also be obtained.

[0039] The water-soluble organic ion salt obtained by the invention has an antibacterial effect, and in particular has a significant antibacterial effect on Staphylococcus aureus.

[0040] The following corresponding experimental data further demonstrates the antibacterial effect of the water-soluble organic ion salt YDOPy.

[0041] Experiment 1: Paper diffusion method inhibition zone experiment

[0042] 1. Preparation of bacterial solution: Pick a single colony from the Staphylococcus aureus plate and place it in sterile TSB broth, and culture it at 37°C, 280r / min, and shake for 12 hours. The next day, take an appropriate amount of bacterial solution and adjust the concentration to 0.5 McFarland turbidimetric, and dilute it 100 times with fresh MH broth for later use.

[0043] 2. Preparation of antibacterial storage solution: Take 16 mg of YDOPy and fully dissolve it in 2 mL of MH broth to prepare 8 mg / mL storage solution and store it in a sterile centrifuge tube.

[0044] 3. Inoculation: Pipette an appropriate amount of diluted bacterial solution onto the MH agar medium plate and spread it evenly with a sterile bacterial spreader stick to ensure that the bacterial solution is evenly distributed on the surface of the agar plate.

[0045] 4. Sticker sheets: Use sterile tweezers to pick up paper sheets and carefully stick them on the bacterial plates. Stick 6 paper sheets on each plate, ensuring that the distance between them and the edge of the plate is not less than 15 mm, and the distance between the paper sheets is not less than 24 mm. Dilute the antibacterial stock solution to 2 mg / mL, add 10 μL of antibacterial solution to the paper sheets, set up a negative control with 10 μL of MH culture medium and a positive control with 10 μL of penicillin-streptomycin antibiotics.

[0046] 5. Cultivation: Place the agar plate with the paper slip in an incubator and culture at 37°C for 24 hours.

[0047] 6. Read the diameter of the inhibition zone: Use a ruler to measure the diameter of the inhibition zone on the back of the culture dish. If there is no inhibition zone, the diameter of the inhibition zone is the same as that of the paper, that is, 5 mm.

[0048] Results: In this experiment, four antibacterial molecules were subjected to a paper diffusion experiment, where M1 was YDOPy, M5 was MH medium (negative control), and M6 was penicillin-streptomycin antibiotics (positive control). Figure 1 As shown, the size of the inhibition zone of the strain is 10.1 mm.

[0049] Experiment 2: Determination of minimum inhibitory concentration (MIC)

[0050] The MIC value of YDOPY against the Staphylococcus aureus strain used in the experiment was determined by the microbroth dilution method, referring to the CLSL 2022 standard. The specific steps are as follows:

[0051] 1. Preparation of antibiotic stock solution: Refer to the antibacterial instructions to select a suitable solvent to fully dissolve penicillin-streptomycin antibiotics, store in a -80℃ refrigerator, and dilute to the required concentration before use.

[0052] 2. Sample addition: Pipette 200 μL of the prepared probe storage solution (concentration of 8000 μg / mL) into a sterile 96-well plate, and add 200 μL of fresh MH broth to make the concentration of the first well 4000 μg / mL. Use the two-fold dilution method, use a pipette to transfer 200 μL of the mixed solution in the first well to the second well, then add 200 μL of broth, and use a pipette to evenly blow in the well to mix the probe. Then, pipette 200 μL of the drug solution from the second well to the third well, and dilute to the ninth well in sequence. Discard the excess 200 μL of the drug solution in the last well. A drug concentration gradient of 4000 μg / mL, 2000 μg / mL, 1000 μg / mL, 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL, and 15.63 μg / mL was obtained, with 200 μL in each well.

[0053] 3. Inoculation: After shaking and mixing the prepared bacterial solution, 200 μL was drawn and added to wells 1 to 9 respectively (equivalent to the bacterial solution being diluted 1 times), and the final concentration gradient was 2000 μg / mL, 1000 μg / mL, 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL, 15.63 μg / mL, 7.81 μg / mL. The 10th well was a positive control well with 1× penicillin-streptomycin antibiotics added, the 11th well was a negative control well with bacterial solution and an equal volume of culture medium added, and the 12th well was a blank control well with only culture medium added. Each experiment was repeated three times.

[0054] 4. Cultivation: Seal the 96-well plate with sealing film, place it on a micro-oscillator to oscillate to mix evenly, and culture it in a 37°C incubator for 24 hours.

[0055] 5. Bacterial count: The plate colony count method was used to determine the antibacterial activity of YDOPy against Staphylococcus aureus. The solution in the wells that were clear and free of bacterial growth was pipetted and mixed evenly. 100 μL was spread evenly on the culture medium plate and cultured in a 37°C incubator for 24 h.

[0056] Results: The minimum concentration at which no bacterial growth can be seen on the plate is the MIC value of YDOPy against Staphylococcus aureus. The results are valid only when the wells of the positive control group are turbid with bacterial growth and the wells of the negative control group are clear with no bacterial growth. The MIC values ​​of the antibacterial molecules against Staphylococcus aureus strains were determined by the microbroth dilution method. The results are as follows: Figure 2 shown.

[0057] The results of plate colony counts are as follows Figure 3 As shown, the MIC value of YDOPy was 31.25 μg / mL.

[0058] Experiment 3: Animal experiment to detect the effect of antibacterial molecules on the virulence of Staphylococcus aureus

[0059] The invertebrate wax moth was used to construct an in vivo infection model. The antibacterial effect of YDOPy on Staphylococcus aureus in vivo was studied by detecting the effects of different concentrations of YDOPy on the virulence of Staphylococcus aureus, 125ug / mL, 62.5ug / mL, and 31.25ug / mL. The wax moth larvae weighing between 350-450mg (to ensure that the weight of each larva is equal), about 25-30mm in length, and milky white in color were selected. They were stored in a dark environment at 4℃ and used within 1 week from the date of purchase.

[0060] The specific steps are as follows:

[0061] 1. Preparation of bacterial solution: Take out the Staphylococcus aureus strain from the -80℃ refrigerator and place it in fresh sterile MH medium. Cultivate it at 37℃ overnight with shaking until the logarithmic growth phase. Adjust the concentration of the bacterial solution to 109 CFU / mL for later use.

[0062] 2. Grouping: The larvae were randomly divided into 5 groups (AC group as experimental group, DE group as control group), with 10 larvae in each group. Group A was composed of larvae injected with MIC concentration of YDOPy + bacterial solution, Group B was composed of larvae injected with 1 / 2MIC concentration of YDOPy + bacterial solution, Group C was injected with bacterial solution only, Group D was the control group injected with sterile MH culture medium, and Group E was the blank control group without any treatment.

[0063] 3. Preparation of drug solution: YDOPy was prepared into a 4 mg / mL stock solution. The YDOPy stock solution was added into 5 mL of MH medium to make the final concentrations equal to MIC and 1 / 2MIC respectively.

[0064] 4. Infection: Use a 20μL Hamilton needle or micro-syringe to inject 20μL of the diluted bacterial solution into each wax moth larva in the AC group through the right front leg. Place the injected wax moth in a sterile culture dish and incubate in a 37℃ incubator for 2h. (The used Hamilton needle is washed 3 times with sterile saline and set aside.)

[0065] 5. Injection: After 2 hours, the culture dish was removed, and the larvae in group A were injected with 20 μL YDOPy at MIC concentration, the larvae in group B were injected with 20 μL YDOPy at 1 / 2 MIC concentration, the larvae in groups C and D were injected with an equal volume of MH medium, and the larvae in group E were not treated as a blank control group. The injection process requires fast and accurate, and multiple injections in the same area should be avoided to avoid unnecessary mechanical damage to the larvae.

[0066] 6. Incubation: Place the treated larvae in a plastic culture dish, mark the time of injection, and incubate in a 37°C incubator. Record the survival of the larvae every 12 hours (when the larvae do not respond to physical stimulation, they are considered dead) for 72 hours.

[0067] 7. Result analysis: The obtained data were entered into Graphpad Prism 9 software to draw the survival curves of larvae in different groups. Figure 4 As shown in the data, compared with the control group, the survival rate of the greater wax moth larvae infected by MRSA in the MIC group was significantly prolonged, and the survival rates between the two were statistically significant (P<0.05), while there was no significant difference in the survival rate between the 1 / 2MIC group and the control group (P>0.05). The experimental results show that a concentration of daphnetin at twice the MIC can reduce the virulence of MRSA infection in the body and exert an anti-MRSA effect in the body.

[0068] Experiment 4: Observation of the effect of daphnetin on the ultrastructure of MRSA cells under scanning electron microscope

[0069] 1. Preparation of bacterial solution: Pipette 5 μL of bacterial culture solution into 5 mL of broth and culture at 37°C until the logarithmic growth phase. Use sterile saline to adjust the concentration of the bacterial solution to 0.5 McFarland turbidity and complete the inoculation of the diluted bacterial solution within 15 minutes.

[0070] 2. Bacterial pretreatment: Add YDOPy mother solution to the drug-containing group to make the final concentration MIC (31.25μg / mL), and the control group without YDOPy is cultured in a 37℃ incubator for 24h. Centrifuge the pretreated bacterial solution at 8000×g for 10min, discard the supernatant, fix the precipitate with 2.5% glutaraldehyde for 4h (or overnight at 4℃), and then wash it with 0.2M PBS buffer (PH=6.8) 3 times, each time for 15-20min. Dehydrate it with 30%, 50%, 70%, 85% and 95% ethanol once, each time for 15-20min; then treat it with 100% ethanol twice, each time for 15-20min. Replace it with isoamyl acetate (tert-butyl alcohol) for more than 3h, preferably overnight.

[0071] 3. Electron microscopic observation: Pipette the mixed bacterial suspension and drop it on the cover glass and freeze it at -80℃. After the sample is dried and gold-sputtered by an ion sputtering instrument, it is observed and photographed by a scanning electron microscope.

[0072] Results: The Staphylococcus aureus bacteria without YDOPy treatment had complete morphology, smooth and rounded spherical surface, and uniform shape and size. When the concentration of YDOPy was MIC, the number of bacteria decreased, the cell morphology and size varied, some bacterial cells had irregular morphology, and the bacteria began to appear inward and wrinkled. After YDOPy treatment, the cell wall of Staphylococcus aureus had been destroyed or the synthesis stage of the bacterial cell wall had been inhibited, showing an antibacterial effect.

[0073] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A water-soluble organic ion salt, characterized in that: The compound shown in the following formula:

2. The method for synthesizing a water-soluble organic ion salt according to claim 1, characterized in that The following steps are involved: 1) Equimolar amounts of 5-bromo-2-(dimethylamino)benzaldehyde and 1,2-dibromoethane are mixed in acetone, and reflux reaction is carried out under nitrogen protection, and the plate is monitored. After the raw material reaction is complete, purification treatment is performed; the reaction system is extracted with dichloromethane and water, and the organic phase is collected and spin-dried to obtain a crude product, which is then purified by column chromatography, using 200-300 mesh silica gel powder as the stationary phase, and dichloromethane and ethyl acetate are prepared as the mobile phase in a volume ratio of 9-11:1 to obtain the first intermediate 2-(2-bromoethoxy)-4-(dimethylamino)benzaldehyde, referred to as QBr, with a chemical formula of C 11 H 14 BrNO2; 2) Equimolar amounts of QBr, sodium hydroxide and 1,3-indanedione are mixed in ethanol, and the mixture is refluxed under nitrogen protection for 10-14 hours. The reaction system is extracted with dichloromethane and water, and the organic phase is collected and dried to obtain a crude product. The crude product is then purified by column chromatography, using 200-300 mesh silica gel powder as the stationary phase and dichloromethane and ethyl acetate as the mobile phase in a volume ratio of 4-6:1 to obtain the second intermediate 2-(2-(2-bromoethoxy)-4-(dimethylamino)benzylidene)-1H-indene-1,3(2H)-dione, referred to as YDOBr, with a chemical formula of C 20 H 18 BrNO3; 3) Add YDOBr to pyridine and stir evenly, use pyridine as solvent and reactant, react at 75-85°C for 16-20 hours, cool the system to room temperature, precipitate orange-red solid, filter, and rinse the filter cake with dichloromethane to obtain an orange solid, which is the target product, water-soluble organic ion salt (1-(2-(5-(dimethylamino)-2-((1,3-diketo-1,3-dihydro-2H-inden-2-ylidene)methyl)phenoxy)ethyl)pyridin-1-ium, referred to as YDOPy, with a chemical formula of C 25 H 23 N2O3 + Br - ; Its synthesis path is:

3. The method for synthesizing a water-soluble organic ion salt according to claim 1, characterized in that In step 1), the volume ratio of dichloromethane to ethyl acetate is 10:

1.

4. The method for synthesizing a water-soluble organic ion salt according to claim 1, characterized in that In step 2), the reflux reaction time is 12 hours, and the volume ratio of dichloromethane to ethyl acetate is 5:

1.

5. The method for synthesizing a water-soluble organic ion salt according to claim 1, characterized in that In step 3), the reaction temperature is 80° C. and the reaction time is 18 hours.

6. Use of the water-soluble organic ion salt according to claim 1 in the preparation of antibacterial preparations.

7. Antibacterial use of the water-soluble organic ion salt as claimed in claim 6 against Staphylococcus aureus.

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